Journal of Bacteriology

Complete Genome Sequence of Methanothermobacter marburgensis, a methanoarchaeon model organism (07-08-10).

Publication Date: 2010 Aug 27 PMID: 20802048
Authors: Liesegang, H. - Kaster, A. K. - Wiezer, A. - Goenrich, M. - Wollherr, A. - Seedorf, H. - Gottschalk, G. - Thauer, R. K.
Journal: J Bacteriol

The circular genome sequence of the chemolithoautotrophic euryarchaeon Methanothermobacter marburgensis with 1,639,135 bp was determined and compared with that of M. thermautotrophicus. The genomes of the two model methanogens differ substantially in protein coding sequences, in insertion-sequence (IS) like elements, and in clustered regularly interspaced short palindromic repeats (CRISPR) loci.

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The Draft Genome Sequences of Actinobacillus pleuropneumoniae Serotypes 2 and 6.

Publication Date: 2010 Aug 27 PMID: 20802047
Authors: Zhan, B. - Angen, O. - Hedegaard, J. - Bendixen, C. - Panitz, F.
Journal: J Bacteriol

Actinobacillus pleuropneumoniae is a bacterial pathogen that causes highly contagious respiratory infection in pigs and has serious impact on production economy and animal welfare. As clear differences in virulence between serotypes have been observed, the genetic basis should be investigated at the genomic level. Here we present the draft genome sequences of the A. pleuropneumoniae serotypes 2 (strain 4226) and 6 (strain Femo).

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Complete Genome Sequence of Staphylococcus aureus strain JKD6008, a ST239 clone of Methicillin-Resistant Staphylococcus aureus with intermediate level vancomycin resistance.

Publication Date: 2010 Aug 27 PMID: 20802046
Authors: Howden, B. P. - Seemann, T. - Harrison, P. F. - McEvoy, C. R. - Stanton, J. A. - Rand, C. J. - Mason, C. W. - Jensen, S. O. - Firth, N. - Davies, J. K. - Johnson, P. D. - Stinear, T. P.
Journal: J Bacteriol

We report here the complete 2.92 Mb genome sequence of a clinical isolate of methicillin resistant Staphylococcus aureus subsp. aureus demonstrating intermediate level vancomycin resistance. The strain, named JKD6008, belongs to multi-locus sequence type 239 and was isolated from the blood stream of a patient in New Zealand in 2003.

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Comparative genomic characterization of Actinobacillus pleuropneumoniae.

Publication Date: 2010 Aug 27 PMID: 20802045
Authors: Xu, Z. - Chen, X. - Li, L. - Li, T. - Wang, S. - Chen, H. - Zhou, R.
Journal: J Bacteriol

The Gram-negative bacterium Actinobacillus pleuropneumoniae is the etiologic agent of porcine contagious pleuropneumoniae, a lethal respiratory infectious disease causing great economic losses in the swine industry worldwide. In order to better interpret the genetic background of serotypic diversity, nine genomes of A. pleuropneumoniae reference strains of serovars 1, 2, 4, 6, 9, 10, 11, 12 and 13 were sequenced using rapid high-throughput approach. Based on twelve genomes of corresponding serovar reference strains including three publicly available complete genomes (serovars 3, 5b and 7) of this bacterium, we performed a comprehensive analysis of comparative genomics and first reported a global genomic characterization for this pathogen. Clustering of 26,012 predicted protein-coding genes showed that the pan genome of A. pleuropneumoniae consists of 3,303 gene clusters, which contain 1,709 core genome genes, 822 distributed genes and 772 strain-specific genes. The genome components involved in the biogenesis of capsular polysaccharide and lipopolysaccharide O-antigen relative to serovar diversity were compared and their genetic diversity was depicted. Our findings shed more light on genomic features associated with serovar diversity of A. pleuropneumoniae, and provide broader insight into both pathogenesis research and clinical/epidemiological application against the severe disease caused by this swine pathogen.

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Loss of compartmentalization of {sigma}E activity need not prevent formation of spores by Bacillus subtilis.

Publication Date: 2010 Aug 27 PMID: 20802044
Authors: Chary, V. K. - Xenopoulos, P. - Eldar, A. - Piggot, P. J.
Journal: J Bacteriol

Compartmentalization of the activities of RNA polymerase sigma factors is a hallmark of formation of spores by Bacillus subtilis. It is initiated soon after the asymmetrically located sporulation division with the activation of sigma(F) in the smaller cell, the prespore. sigma(F) then directs a signal via the membrane protease SpoIIGA to activate sigma(E) in the larger mother cell by processing of pro-sigma(E). Here we show that sigma(E) can be activated in the prespore with little effect on sporulation efficiency, implying that complete compartmentalization of sigma(E) activity is not essential for spore formation. sigma(E) activity in the prespore can be obtained by inducing transcription in the prespore of spoIIGA, or of sigE*, which encodes a constitutively active form of sigma(E), but not of spoIIGB, which encodes pro-sigma(E). We infer that sigma(E) compartmentalization is partially attributed to a competition between the compartments for the activation signaling protein SpoIIR. Normally SpoIIGA is predominantly located in the mother cell and as a consequence confines sigma(E) activation to it. In addition, we find that CsfB, previously shown to inhibit sigma(G), is independently inhibiting sigma(E) activity in the prespore. CsfB thus appears to serve as gatekeeper function in blocking the action of two sigma factors in the prespore: it prevents sigma(G) from becoming active before completion of engulfment, and helps prevent sigma(E) from becoming active at all.

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Biogenesis of Salmonella enterica serovar Typhimurium Membrane Vesicles Provoked by Induction of PagC.

Publication Date: 2010 Aug 27 PMID: 20802043
Authors: Kitagawa, R. - Takaya, A. - Ohya, M. - Mizunoe, Y. - Takade, A. - Yoshida, S. I. - Isogai, E. - Yamamoto, T.
Journal: J Bacteriol

Gram-negative bacteria ubiquitously release membrane vesicles (MVs) into the extracellular milieu. Although MVs are the product of growing bacteria, not of cell lysis or death, the regulatory mechanisms underlying MV formation remained unknown. We have found that MV biogenesis is provoked by the induction of PagC, a Salmonella specific protein whose expression is activated by conditions that mimic acidified macrophage phagosomes. PagC is a major constituent of Salmonella MVs and increased expression accelerates vesiculation. Expression of PagC is regulated at the post-transcriptional and/or post-translational level in a sigmaS (RpoS)-dependent manner. Serial quantitative analysis has demonstrated that MV formation can accelerate when quantity of the MV constituents, OmpX as well as PagC, rises. Overproduction of PagC dramatically impacts the difference in the relative amount of vesiculation, but the corresponding overproduction of OmpX was less pronounced. Quantitative examination of the ratios of PagC and OmpX in the periplasm, outer membrane and MVs demonstrates that PagC is preferentially enriched in MVs released from Salmonella cells. This suggests that specific protein sorting mechanisms operate when MVs are formed. The possible role(s) of PagC-MV in host cells is discussed.

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The Aldehyde Dehydrogenase (ALDH) of Gluconacetobacter diazotrophicus is a Quinohemeprotein containing PQQ, Cytochrome b and Cytochrome c. Molecular and Catalytic Properties.

Publication Date: 2010 Aug 27 PMID: 20802042
Authors: Gomez-Manzo, S. - Chavez-Pacheco, J. L. - Contreras-Zentella, M. - Sosa-Torres, M. E. - Arreguin-Espinosa, R. - Perez de la Mora, M. - Membrillo-Hernandez, J. - Escamilla, J. E.
Journal: J Bacteriol

Several aldehyde dehydrogenase (ALDH) complexes have been purified from membranes of acetic acid bacteria. The enzyme structures and the chemical nature of the prosthetic groups associated with these enzymes remain a matter of debate. Here we report on the molecular and catalytic properties of the membrane-bound ALDH complex of the diazotrophic bacterium Gluconacetobacter diazotrophicus. The purified ALDH complex is a heterodimer comprising two subunits of 79.7 and 50 kDa respectively. Reverse phase HPLC and EPR spectroscopy led us to demonstrate, for the first time, the unequivocal presence of pyrroloquinoline quinone (PQQ) prosthetic group associated to an ALDH complex from acetic acid bacteria. Additionally, heme b was detected by UV/Vis spectroscopy and confirmed by reverse phase HPLC. The smaller subunit bears three cytochromes c. Aliphatic aldehydes, but not formaldehyde, were suitable substrates. Using ferricyanide as electron acceptor, the enzyme showed a optimum pH of 3.5 that shifted to pH 7.0 when phenazine methosulphate plus 2,6-dichlorophenolindophenol were the electron acceptors. Acetaldehyde did not reduce measurable levels of cytochrome b and c centres; however, the dithionite-reduced hemes were conveniently oxidized by ubiquinone-1; this finding suggests that cytochrome b and the cytochromes c constitute an intramolecular redox sequence that delivers electrons to the membrane ubiquinone.

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The Elemental Sulfur Responsive Protein (SipA) from the Hyperthermophilic Archaeon Pyrococcus furiosus is Regulated by Sulfide in an Iron-Dependent Manner.

Publication Date: 2010 Aug 27 PMID: 20802041
Authors: Clarkson, S. M. - Newcomer, E. C. - Young, E. G. - Adams, M. W.
Journal: J Bacteriol

The gene (sipA) encoding the sulfur-induced protein A (PF2025) is highly up-regulated during growth of Pyrococcus furiosus on elemental sulfur (S degrees ). Expression of sipA is regulated by sulfide, the product of S degrees reduction, but in an iron-dependent manner. SipA is proposed to play a role in intracellular iron sulfide detoxification.

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Biosynthetic Pathway for {gamma}-cyclic Sarcinaxanthin in Micrococcus luteus: Heterologous Expression and Evidence for Diverse and Multiple Catalytic Functions of C50 Carotenoid Cyclases.

Publication Date: 2010 Aug 27 PMID: 20802040
Authors: Netzer, R. - Stafsnes, M. H. - Andreassen, T. - Goksoyr, A. - Bruheim, P. - Brautaset, T.
Journal: J Bacteriol

We report the cloning and characterization of the biosynthetic gene cluster (crtE, crtB, crtI, crtE2, crtYg, crtYh, and crtX) of the gamma-cyclic C50 carotenoid sarcinaxanthin in Micrococcus luteus NCTC2665. Expression of the complete and partial gene cluster in Escherichia coli hosts revealed that sarcinaxanthin biosynthesis from the precursor molecule farnesyl pyrophosphate (FPP) goes via C40 lycopene, C45 nonaflavuxanthin, C50 flavuxanthin and C50 sarcinaxanthin. Glucosylation of sarcinaxanthin was accomplished by the crtX gene product. This is the first report describing the biosynthetic pathway of a gamma-cyclic C50 carotenoid. Expression of the corresponding genes from the marine M. luteus isolate Otnes7 in a lycopene producing E. coli host resulted in the production of up to 2.5 mg/g cell dry weight sarcinaxanthin in shake flasks. In an attempt to experimentally understand the specific difference between the biosynthetic pathways of sarcinaxanthin and its structurally related epsilon-cyclic decaprenoxanthin, we constructed a hybrid gene cluster with the gamma-cyclic C50 carotenoid cyclase genes crtYg and crtYh from M. luteus substituted with the analogous epsilon-cyclic C50 carotenoid cyclase genes crtYe and crtYf from the natural decaprenoxanthin producer Corynebacterium glutamicum. Surprisingly, expression of this hybrid gene cluster in E. coli host resulted in accumulation of not only decaprenoxanthin, but also sarcinaxanthin and the asymmetric epsilon- and gamma-cyclic C50 carotenoid sarprenoxanthin described for the first time in this work. Together, these data contributed to new insight into the diverse and multiple functions of bacterial C50 carotenoid cyclases as key catalysts for the synthesis of structurally different carotenoids.

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AglJ adds the first sugar of the N-linked pentasaccharide decorating the Haloferax volcanii S-layer glycoprotein.

Publication Date: 2010 Aug 27 PMID: 20802039
Authors: Kaminski, L. - Abu-Qarn, M. - Guan, Z. - Naparstek, S. - Ventura, V. V. - Raetz, C. R. - Hitchen, P. G. - Dell, A. - Eichler, J.
Journal: J Bacteriol

Like Eukarya and Bacteria, Archaea also perform N-glycosylation. Using the haloarchaeon Haloferax volcanii as a model system, a series of Agl proteins involved in the archaeal version of this post-translational modification have been identified. In the present study, the participation of HVO_1517 in N-glycosylation was considered, given its homology to a known component of the eukaryal N-glycosylation pathway and because of the genomic proximity of HVO_1517 to agl genes encoding known elements of the Hfx. volcanii N-glycosylation process. By combining deletion of HVO_1517 together with mass spectrometric analysis of both dolichol phosphate monosaccharide-charged carriers and the S-layer glycoprotein, evidence was obtained showing the participation of HVO_1517, renamed AglJ, in adding the first hexose of the N-linked pentasaccharide decorating this reporter glycoprotein. Deletion of aglJ, however, did not fully prevent the attachment of a hexose residue to the S-layer glycoprotein. Moreover, in the absence of AglJ, the level of only one of the three monosaccharide-charged dolichol phosphate carriers detected in the cell was reduced. Nonetheless, in cells lacking AglJ, no further sugar subunits were added to the remaining monosaccharide-charged dolichol phosphate carriers or to the monosaccharide-modified S-layer glycoprotein, pointing to the importance of the sugar added through the actions of AglJ for proper N-glycosylation. Finally, while aglJ can be deleted, Hfx. volcanii surface layer integrity is compromised in the absence of the encoded protein.

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Mechanism of Positive Regulation by DsrA and RprA sRNAs: Pairing increases Translation and Protects rpoS mRNA From Degradation.

Publication Date: 2010 Aug 27 PMID: 20802038
Authors: McCullen, C. - Benhammou, J. - Majdalani, N. - Gottesman, S.
Journal: J Bacteriol

Small, non-coding RNAs (sRNAs) regulate gene expression in Escherichia coli by base pairing with mRNAs and modulating translation and mRNA stability. The sRNAs DsrA and RprA stimulate the translation of the stress response transcription factor RpoS by base pairing with the 5' untranslated region of the rpoS mRNA. In this study, we found that the rpoS mRNA was unstable in the absence of DsrA and RprA and that expression of these sRNAs increased both the accumulation and the half-life of the rpoS mRNA. Mutations in dsrA, rprA, or rpoS that disrupt the predicted pairing sequences and reduce translation of RpoS also destabilize the rpoS mRNA. We found that the rpoS mRNA accumulates in an RNase E mutant strain in the absence of sRNA expression and, therefore, is degraded by an RNase E-mediated mechanism. DsrA expression is required, however, for maximal translation even when rpoS mRNA is abundant. This suggests that DsrA protects rpoS mRNA from degradation by RNase E and that DsrA has a further activity in stimulating RpoS protein synthesis. rpoS mRNA is subject to degradation by an additional pathway, mediated by RNase III, which, in contrast to the RNase E-mediated pathway, occurs in the presence and absence of DsrA or RprA. rpoS mRNA and RpoS protein levels are increased in an RNase III mutant strain with or without the sRNAs, suggesting that the role of RNase III in this context is to reduce the translation of RpoS even when the sRNAs are acting to stimulate translation.

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A novel six-rhodopsin system in a single archaeon.

Publication Date: 2010 Aug 27 PMID: 20802037
Authors: Fu, H. Y. - Lin, Y. C. - Chang, Y. N. - Tseng, H. - Huang, C. C. - Liu, K. C. - Huang, C. S. - Su, C. W. - Weng, R. R. - Lee, Y. Y. - Ng, W. V. - Yang, C. S.
Journal: J Bacteriol

Microbial rhodopsins, a diverse group of photoactive proteins found in Archaea, Bacteria, and Eukarya, function in photosensing and photoenergy harvesting and may have been present in the resource-limited early global environment. Four different physiological functions have been identified and characterized for nearly 5000 retinal-binding photoreceptors, these being ion transporters that transport proton or chloride and sensory rhodopsins that mediate light-attractant and/or repellent responses. The greatest number of rhodopsins previously observed in a single archaeon had been four. Here, we report a newly discovered six-rhodopsin system in a single archaeon, Haloarcula marismortui, showing a more diverse absorbance spectral distribution than any previously known rhodopsin system, and for the first time, two light-driven proton transporters that respond to the same wavelength. All six rhodopsins were first shown to be expressed in H. marismortui, the greatest number ever identified in a single archaeon, and these were then overexpressed in E. coli. The proteins were purified for absorption spectra and photocycle determination, followed by measurement of ion transportation and phototaxis. The results clearly indicated the existence of a two-isochromatic proton transporter system and a new type of sensory rhodopsin-like transducer in H. marismortui.

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A comparative analysis of the biochemical and functional properties of C-terminal domains of autotransporters.

Publication Date: 2010 Aug 27 PMID: 20802036
Authors: Marin, E. - Bodelon, G. - Fernandez, L. A.
Journal: J Bacteriol

Autotransporters (ATs) are the largest group of proteins secreted by Gram-negative bacteria and include many virulence factors from human pathogens. ATs are synthesized as large precursors with a C-domain that is inserted in the outer membrane (OM) and is essential for translocation of a N-terminal passenger domain to the extracellular milieu. Several mechanisms have been proposed for ATs secretion. Self-translocation models suggest transport across a hydrophilic channel formed by an internal pore of the beta-barrel or by oligomerization of C-domains. Alternatively, an assisted-translocation model suggests that transport employs a conserved machinery of the bacterial OM such as the Bam-complex. In this work we have investigated ATs secretion by carrying out a comparative study to analyze the conserved biochemical and functional features of different C-domains selected from ATs of gamma, beta, alpha and epsilon class of proteobacteria. Our results indicate that C-domains having an N-terminal alpha-helix and a beta-barrel constitute functional transport units for translocation of peptides and immunoglobulin domains with disulfide bonds. In vivo and in vitro analysis show that multimerization is not a conserved feature in AT C-domains. Further, we demonstrate that deletion of the conserved alpha-helix severely impairs beta-barrel folding and OM insertion and thereby blocks passenger domain secretion. These observations suggest that AT beta-barrel without its alpha-helix cannot form a stable hydrophilic channel in the OM for protein translocation. The implications of our data for understanding AT secretion are discussed.

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A commensal gone bad: complete genome sequence of the prototypical enterotoxigenic Escherichia coli strain H10407.

Publication Date: 2010 Aug 27 PMID: 20802035
Authors: Crossman, L. C. - Chaudhuri, R. R. - Beatson, S. A. - Wells, T. J. - Desvaux, M. - Cunningham, A. F. - Petty, N. K. - Mahon, V. - Brinkley, C. - Hobman, J. L. - Savarino, S. J. - Turner, S. M. - Pallen, M. J. - Penn, C. W. - Parkhill, J. - Turner, A. K. - Johnson, T. J. - Thomson, N. R. - Smith, S. G. - Henderson, I. R.
Journal: J Bacteriol

In most cases Escherichia coli exists as a harmless commensal organism but may on occasion cause intestinal and/or extraintestinal disease. Enterotoxigenic E. coli are the predominant cause of E. coli-mediated diarrhea in the developing world and are responsible for a significant portion of paediatric deaths. In this study we determined the complete genomic sequence of E. coli H10407, a prototypical strain of enterotoxigenic E. coli, which reproducibly elicits diarrhea in human volunteer studies. We performed genomic and phylogenetic comparisons with other E. coli strains revealing that the chromosome is closely related to the non-pathogenic commensal strain E. coli HS and to the laboratory strains E. coli K-12 and C. Furthermore, these analyses demonstrated that there were no chromosomally-encoded factors unique to any sequenced ETEC strains. Comparison of the E. coli H10407 plasmids with those from several ETEC strains revealed the plasmids had a mosaic structure but that several loci were conserved amongst ETEC strains. This study provides a genetic context for the vast amount of experimental and epidemiological data published thus far.

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Radiation Desiccation Response Motif (RDRM) like sequences are involved in transcriptional activation of deinococcal ssb gene by ionizing radiation, but not by desiccation.

Publication Date: 2010 Aug 27 PMID: 20802034
Authors: Ujaoney, A. K. - Potnis, A. A. - Kane, P. - Mukhopadhyaya, R. - Apte, S. K.
Journal: J Bacteriol

Single stranded DNA binding protein (SSB) levels during post-stress recovery of Deinococcus radiodurans were significantly enhanced by (60)Co gamma rays or Mitomycin-C treatment, but not upon exposure to ultraviolet rays (UV), hydrogen-peroxide (H2O2) or desiccation. Addition of rifampicin prior to post irradiation recovery blocked such induction. In silico analysis of ssb promoter region revealed a 17 bp palindromic Radiation/Desiccation Response Motif (RDRM1) at -114 to -98 bps and a somewhat similar sequence (RDRM2) at -213 to -197 bps, upstream of the ssb open reading frame. Involvement of these cis-elements in radiation responsive ssb gene expression was assessed by constructing transcriptional fusions of edited versions of ssb promoter region with a non-specific acid phosphatase encoding reporter gene, phoN. Recombinant D. radiodurans strains carrying such constructs clearly revealed (a) transcriptional induction of ssb promoter upon irradiation and mitomycin-C treatment, but not by UV or H2O2 treatments, and (b) involvement of both RDRM like sequences in such activation of SSB expression, in an additive manner.

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A periplasmic LolA derivative with a lethal disulfide bond activates the Cpx stress response system.

Publication Date: 2010 Aug 27 PMID: 20802033
Authors: Tao, K. - Watanabe, S. - Narita, S. I. - Tokuda, H.
Journal: J Bacteriol

LolA accommodates the acyl chains of lipoproteins in its hydrophobic cavity, and shuttles between the inner and outer membranes through the hydrophilic periplasm to place lipoproteins in the outer membrane. The LolA(I93C/F140C) derivative, in which Cys replaces Ile at position 93 and Phe at position 140, strongly inhibited growth in the absence of a reducing agent because of the lethal intramolecular disulfide bond between the two Cys residues. Expression of I93C/F140C was found to activate the Cpx two-component system, which responds to cell envelope stress. The inhibition of growth by I93C/F140C was partly suppressed by overproduction of LolCDE, which is an ATP-binding cassette transporter, and mediates the transfer of lipoproteins from the inner membrane to LolA. A substantial portion of the oxidized form, but not the reduced one, of I93C/F140C expressed on LolCDE overproduction was recovered in the membrane fraction whereas wild-type LolA was localized in the periplasm even when LolCDE was overproduced. Moreover, LolCDE overproduction stabilized I93C/F140C and therefore caused an increase in its level. Taken together, these results indicate that oxidized I93C/F140C stably binds to LolCDE, which causes strong envelope stress.

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Complete Genome Sequence of Mycoplasma hyorhinis Strain HUB-1.

Publication Date: 2010 Aug 27 PMID: 20802032
Authors: Liu, W. - Fang, L. - Li, S. - Li, Q. - Zhou, Z. - Feng, Z. - Luo, R. - Shao, G. - Wang, L. - Chen, H. - Xiao, S.
Journal: J Bacteriol

Mycoplasma hyorhinis is generally considered a swine pathogen, yet is most commonly found infecting laboratory cell lines. An increasing body of evidence suggests that chronic infections of M. hyorhinis may cause oncogenic transformation. Here, we announce the complete genome sequence of M. hyorhinis strain HUB-1.

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Genome sequence of the solvent-producing bacterium Clostridium carboxidivorans strain P7T.

Publication Date: 2010 Aug 20 PMID: 20729368
Authors: Paul, D. - Austin, F. W. - Arick, T. - Bridges, S. M. - Burgess, S. C. - Dandass, Y. S. - Lawrence, M. L.
Journal: J Bacteriol

Clostridium carboxidivorans strain P7(T) is a strictly anaerobic acetogenic bacterium that produces acetate, ethanol, butanol, and butyrate. The C. carboxidivorans genome contains all the genes for the carbonyl branch of the Wood-Ljungdahl pathway for CO2 fixation, and it encodes enzymes for conversion of acetyl CoA into butanol and butyrate.

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Genetics and Regulation of the Major Enzymes of Alanine Synthesis in Escherichia coli.

Publication Date: 2010 Aug 20 PMID: 20729367
Authors: Kim, S. H. - Schneider, B. L. - Reitzer, L.
Journal: J Bacteriol

Genetic analysis of alanine synthesis in the model genetic organism Escherichia coli has implicated avtA, the still uncharacterized alaA and alaB, and probably other genes. We identified alaA as yfbQ. We then transferred mutations in several transaminase genes into a yfbQ mutant, and isolated a mutant that required alanine for optimal growth. For cells grown with carbon sources other than pyruvate the major alanine-synthesizing transaminases are AvtA, YfbQ (AlaA), and YfdZ (which we designate as AlaC). Growth with pyruvate as the carbon source and multicopy suppression suggest that several other transaminases can contribute to alanine synthesis. Expression studies showed that alanine modestly repressed avtA and yfbQ, but had no effect on yfdZ. The leucine-responsive regulatory protein (Lrp) mediated control by alanine. We purified YfbQ and YfdZ and showed that both are dimers with Kms for pyruvate within the intracellular range of pyruvate concentration.

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Characterization of the RNA degradosome of Pseudoalteromonas haloplanktis: conservation of the RNase E-RhlB interaction in the {gamma}-Proteobacteria.

Publication Date: 2010 Aug 20 PMID: 20729366
Authors: Ait-Bara, S. - Carpousis, A. J.
Journal: J Bacteriol

The degradosome is a multienzyme complex involved in mRNA degradation in Escherichia coli. The essential endoribonuclease RNase E contains a large non-catalytic region necessary for protein-protein interactions with other components of the RNA degradosome. Interacting proteins include the DEAD-box RNA helicase RhlB, the glycolytic enzyme enolase and the exoribonuclease PNPase. Pseudoalteromonas haloplanktis, a psychrotolerant gamma-Proteobacterium distantly related to E. coli, encodes homologs of each component of the RNA degradosome. In P. haloplanktis, RNase E associates with RhlB and PNPase, but not enolase. Plasmids expressing P. haloplanktis RNase E (Ph-RNase E) can complement E. coli strains lacking Ec-RNase E. Ph-RNase E, however, does not confer a growth advantage to E. coli at low temperature. Ph-RNase E makes a heterologous protein-protein interaction with Ec-RhlB, but not with Ec-enolase or Ec-PNPase. The Ph-RNase E binding sites for RhlB and PNPase were mapped by deletion analysis. The PNPase binding site is located at the C-terminal end of Ph-RNase E at the same position as in Ec-RNase E, but the sequence of the site is not conserved. The sequence of the RhlB binding site in Ph-RNase E is related to the sequence in Ec-RNase E. Together with the heterologous interaction between Ph-RNase E and Ec-RhlB, our results suggest that the underlying structural motif for the RNase E-RhlB interaction is conserved. Since the activity of Ec-RhlB requires its physical interaction with Ec-RNase E, conservation of the underlying structural motif over a large evolutionary distance could be due to constraints involved in the control of RhlB activity.

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The Enterococcus faecalis rnjB is required for pilin gene expression and biofilm formation.

Publication Date: 2010 Aug 20 PMID: 20729365
Authors: Gao, P. - Pinkston, K. L. - Nallapareddy, S. R. - van Hoof, A. - Murray, B. E. - Harvey, B. R.
Journal: J Bacteriol

Pili in Gram-positive bacteria play a major role in the colonization of host tissue and in the development of biofilms. They are promising candidates for vaccines or drug targets since they are highly immunogenic, and share common structural and functional features among various Gram-positive pathogens. Numerous publications have helped build a detailed understanding of pilus surface assembly, yet regulation of pilin gene expression has not been well defined. Utilizing a monoclonal antibody developed against the Enterococcus faecalis major pilus protein EbpC, we identified mutants from a transposon (Tn) insertion library which lack surface exposed Ebp pili. In addition to insertions in the ebp regulon, an insertion in ef1184 (dapA) significantly reduced levels of EbpC. Analysis of in-frame deletion mutants of dapA and the downstream gene rnjB further demonstrated that rnjB was responsible for the deficiency of EbpC. Sequence analysis revealed that rnjB encodes a putative RNase J2. Subsequent qRT-PCR and Northern blot analysis demonstrated that the ebpABC mRNA transcript level was significantly decreased in the rnjB deletion mutant. In addition, using a reporter gene assay, we confirmed that rnjB affects expression of the ebpABC operon. Functionally, the rnjB deletion mutant was attenuated in its ability to produce biofilm, similar to that of an ebpABC deletion mutant which lacks Ebp pili. Together, these results demonstrate the involvement of rnjB in E. faecalis pilin gene expression and provide insight into a novel mechanism of regulation of pili production in Gram-positive pathogens.

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Sigma Factor F does not Prevent Rifampin Inhibition of RNA Polymerase or Cause Rifampin Tolerance in Mycobacterium tuberculosis.

Publication Date: 2010 Aug 20 PMID: 20729364
Authors: Hartkoorn, R. C. - Sala, C. - Magnet, S. J. - Chen, J. M. - Pojer, F. - Cole, S. T.
Journal: J Bacteriol

The tolerance of Mycobacterium tuberculosis to anti-tuberculosis drugs is a major reason for the lengthy therapy needed to treat a tuberculosis infection. Rifampin is a potent inhibitor of RNA polymerase (RNAP) in vivo, but has been shown to be less effective against stationary phase bacteria. Sigma factor F is associated with bacteria entering stationary phase and has been proposed to impact rifampin activity. Here we investigate whether RNAP containing SigF is more resistant to rifampin inhibition in vitro, and whether over-expression of sigF renders M. tuberculosis more tolerant to rifampin. Real-time and radiometric in vitro transcription assays revealed that rifampin equally inhibits transcription by RNAP containing sigma factors SigA and SigF, therefore ruling out the hypothesis that SigF may be responsible for increased resistance of the enzyme to rifampin in vitro. In addition, over-expression or deletion of sigF did not alter rifampin susceptibility in axenic cultures of M. tuberculosis, indicating that SigF does not affect rifampin tolerance in vivo.

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Inactivation of a heterocyst-specific invertase indicates a principal role of sucrose catabolism in the heterocysts of Anabaena sp.

Publication Date: 2010 Aug 20 PMID: 20729363
Authors: Lopez-Igual, R. - Flores, E. - Herrero, A.
Journal: J Bacteriol

Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that carries out N2 fixation in specialized cells called heterocysts, which exchange nutrients and regulators with the filament's vegetative cells that perform the photosynthetic fixation of CO2. The Anabaena genome carries two genes coding for alkaline/neutral invertases, invA and invB. As shown by Northern analysis, both genes were expressed monocistronically and induced under nitrogen deprivation, although induction was stronger for invB than for invA. Whereas expression of an InvA-N-GFP fusion was homogeneous along the cyanobacterial filament, consistent with lack of dependence on HetR, expression of an InvB-N-GFP fusion upon combined nitrogen deprivation took place mainly in differentiating and mature heterocysts. In a hetR genetic background, the InvB-N-GFP fusion was strongly expressed all along the filament. An insertional mutant of invA could grow diazotrophically but was impaired in nifHDK induction and exhibited an increased frequency of heterocysts suggesting a regulatory role of the invertase-mediated carbon flux in vegetative cells. In contrast, an invB mutant was strongly impaired in diazotrophic growth showing a crucial role of sucrose catabolism mediated by the InvB invertase in the heterocysts.

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THE N-TERMINAL DOMAIN OF THE Aliivibrio fischeri LuxR IS A TARGET OF THE GroEL CHAPERONIN.

Publication Date: 2010 Aug 20 PMID: 20729362
Authors: Manukhov, I. V. - Melkina, O. E. - Goryanin, I. I. - Baranova, A. V. - Zavilgelsky, G. B.
Journal: J Bacteriol

Here we show that the C-terminal domain of the LuxR activates the transcription of Aliivibrio fischeri luxICDABEG in both Escherichia coli SKB178 gro(+) and E. coli OFB1111 groEL673 strains to the same level. Using affine chromatography we showed that GroEL binds to N-termonal domain of LuxR pointing at the GroEL/GroES requirement for the folding of the N terminal domain of LuxR.

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Molecular basis of vancomycin-dependence in VanA-type Staphylococcus aureus VRSA-9.

Publication Date: 2010 Aug 20 PMID: 20729361
Authors: Meziane-Cherif, D. - Saul, F. A. - Moubareck, C. - Weber, P. - Haouz, A. - Courvalin, P. - Perichon, B.
Journal: J Bacteriol

Vancomycin-resistant Staphylococcus aureus VRSA-9 clinical isolate was partially dependent on glycopeptide for growth. The responsible vanA operon had the same organization as that of Tn1546 and was located on a plasmid. The chromosomal D-Ala:D-Ala ligase (ddl) gene had two point mutations that led to Q260K and A283E substitutions, resulting in a 200-fold decrease in enzymatic activity compared to that of the wild-type strain VRSA-6. To gain insight into the mechanism of enzyme impairment, we determined the crystal structure of VRSA-9 Ddl and showed that the A283E mutation induces new ion-pair/hydrogen bond interactions leading to an asymmetric rearrangement of side chains in the dimer interface. The Q260K substitution is located in an exposed external loop and did not induce any significant conformational change. The VRSA-9 strain was susceptible to oxacillin due to synthesis of pentadepsipeptide precursors ending in D-alanyl-D-lactate which are not substrates for the beta-lactam resistant penicillin binding protein PBP2'. Comparison with the vancomycin partially-dependent VRSA-7, whose Ddl is 5-fold less efficient than that of VRSA-9, indicated that the levels of vancomycin dependence and susceptibility to beta-lactams correlate with the degree of Ddl impairment. Ddl drug targeting could therefore be an effective strategy against vancomycin-resistant S. aureus.

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An extreme thermophile, Thermus thermophilus, is a polyploid bacterium.

Publication Date: 2010 Aug 20 PMID: 20729360
Authors: Ohtani, N. - Tomita, M. - Itaya, M.
Journal: J Bacteriol

An extremely thermophilic bacterium, Thermus thermophilus HB8, is one of the model organisms for systems biology. Its genome consists of a chromosome (1.85 Mb), a megaplasmid (0.26 Mb) designated pTT27, and a plasmid (9.3 kb) designated pTT8, and the complete sequence is available. We show here that T. thermophilus is a polyploid organism, which harbors multiple genomic copies in a cell. In the case of the HB8 strain, the copy number of the chromosome was estimated to be four or five, and the copy number of the pTT27 megaplasmid seemed to be equal to that of the chromosome. It has never been discussed whether T. thermophilus is haploid or polyploid. However, the finding that it is polyploid is not surprising, as Deinococcus radiodurans, an extremely radioresistant bacterium closely related to Thermus, is well known to be a polyploid cell. As is the case for D. radiodurans in the radiation environment, the polyploidy in T. thermophilus might allow for genomic DNA protection, maintenance, and repair at elevated growth temperatures. The polyploidy often complicates the recognition of an essential gene in T. thermophilus as a model organism for systems biology.

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DEFICIENCY IN L-SERINE DEAMINASE INTERFERES WITH ONE-CARBON METABOLISM AND CELL WALL SYNTHESIS IN E. coli K-12.

Publication Date: 2010 Aug 20 PMID: 20729359
Authors: Zhang, X. - El-Hajj, Z. W. - Newman, E.
Journal: J Bacteriol

E. coli K-12 provided with glucose and a mixture of amino acids depletes L-serine more quickly than any other amino acid even in the presence of ammonium sulfate. A mutant without three 4Fe4S L-serine deaminases (SdaA, SdaB and TdcG) of E. coli K-12 is unable to do this. The high L-serine that accumulates when such a mutant is exposed to amino acid mixtures starves the cells for C1 units, and interferes with cell wall synthesis. We suggest that at high concentrations, L-serine decreases synthesis of UDP-N-acetylmuramate-L-alanine by the murC-encoded ligase, weakening the cell wall and producing misshapen cells and lysis. The inhibition by high L-serine is overcome in several ways: by a large concentration of L-alanine, by overproducing MurC together with low concentration of L-alanine, and by overproducing FtsW, thus promoting septal assembly- and also by overexpression of the glycine cleavage operon. S-adenosylmethionine reduces lysis and allows extensive increase in biomass without improving cell division. This suggests that E. coli has a metabolic trigger for cell division. Without that reaction, if no other inhibition occurs, other metabolic functions can continue and cells can elongate, and replicate their DNA, reaching at least 180x their usual length, but cannot divide.

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Genome sequences of Pelagibaca bermudensis HTCC2601T and Maritimibacter alkaliphilus HTCC2654T, the type strains of two marine Roseobacter genera.

Publication Date: 2010 Aug 20 PMID: 20729358
Authors: Thrash, J. C. - Cho, J. C. - Ferriera, S. - Johnson, J. - Vergin, K. L. - Giovannoni, S. J.
Journal: J Bacteriol

Pelagibaca bermudensis HTCC2601(T) and Maritimibacter alkaliphilus HTCC2654(T) represent two marine genera in the globally significant Roseobacter clade of the Alphaproteobacteria. Here we present the genome sequences of these organisms, isolated from the Sargasso Sea using dilution-to extinction culturing, which offer insight into the genetic basis for the metabolic and ecological diversity of this important group.

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Mutational analysis of Bacillus megaterium QM B1551 cortex-lytic enzymes.

Publication Date: 2010 Aug 20 PMID: 20729357
Authors: Christie, G. - Ustok, F. I. - Lu, Q. - Packman, L. C. - Lowe, C. R.
Journal: J Bacteriol

Molecular-genetic and muropeptide analysis techniques have been applied to examine the function in vivo of the B. megaterium QM B1551 SleB and SleL proteins. In common with B. subtilis and B. anthracis, the presence of anhydro-muropeptides in B. megaterium germination exudates, which is indicative of lytic transglycosylase activity, is associated with an intact sleB structural gene. B. megaterium sleB cwlJ double mutant strains complemented with engineered SleB variants in which the predicted N- or C- terminal domains have been deleted (SleB-DeltaN and SleB-DeltaC), efficiently initiate and hydrolyse the cortex, generating anhydro-muropeptides in the process. Additionally, sleB cwlJ strains complemented with SleB-DeltaN or SleB-DeltaC, in which glutamate and aspartate residues have individually been changed to alanine, all retain the ability to hydrolyse the cortex to varying degrees during germination, with concomitant release of anhydro-muropeptides to the surrounding medium. These data indicate that while the presence of either the N- or C-terminal domain of B. megaterium SleB is sufficient for initiation of cortex hydrolysis and the generation of anhydro-muropeptides, the perceived lytic transglycosylase activity may be derived from an enzyme(s), perhaps exclusively, or in addition to SleB, that has yet to be identified. B. megaterium SleL appears to be associated with the epimerase-type activity observed previously in B. subtilis, differing from the glucosaminidase function that is apparent in B. cereus/B. anthracis.

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Complete Genome Sequence of Staphylococcus aureus strain JKD6159, a Unique Australian clone of ST93-IV Community Methicillin-Resistant Staphylococcus aureus.

Publication Date: 2010 Aug 20 PMID: 20729356
Authors: Chua, K. - Seemann, T. - Harrison, P. F. - Davies, J. K. - Coutts, S. J. - Chen, H. - Haring, V. - Moore, R. - Howden, B. P. - Stinear, T. P.
Journal: J Bacteriol

Community methicillin-resistant Staphylococcus aureus (cMRSA) is an emerging issue that has resulted in multiple worldwide epidemics. We report the first complete genome sequence of an ST93-MRSA-IV clinical isolate that caused severe invasive infection and a familial outbreak of skin infection. This isolate is a representative of the most common Australian clone of cMRSA that is unique and unrelated to the previously sequenced genomes of S. aureus.

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The Roles of Multiple Acetoacetyl-CoA Reductases in Polyhydroxybutyrate Biosynthesis in Ralstonia eutropha H16.

Publication Date: 2010 Aug 20 PMID: 20729355
Authors: Budde, C. F. - Mahan, A. E. - Lu, J. - Rha, C. - Sinskey, A. J.
Journal: J Bacteriol

The bacterium Ralstonia eutropha H16 synthesizes polyhydroxybutyrate (PHB) from acetyl-CoA through reactions catalyzed by a beta-ketothiolase (PhaA), an acetoacetyl-CoA reductase (PhaB), and a polyhydroxyalkanoate synthase (PhaC). An operon of three genes encoding these enzymatic steps was discovered in R. eutropha and has been well studied. Sequencing and analysis of the R. eutropha genome revealed putative isologs for each of the PHB biosynthetic genes, many of which had never been characterized. In addition to the previously identified phaB1, the genome contains the isologs phaB2, phaB3, as well as 15 other potential acetoacetyl-CoA reductases. We have investigated the roles of the three phaB isologs by deleting them from the genome individually and in combination. It was discovered that the gene products of both phaB1 and phaB3 contribute to PHB biosynthesis in fructose minimal medium, while in plant oil minimal medium and rich medium phaB3 seems to be unexpressed. This raises interesting questions concerning the regulation of phaB3 expression. Deletion of the gene phaB2 did not result in an observable phenotype in the conditions tested, although this gene does encode an active reductase. Addition of the individual reductase genes to the genome of the DeltaphaB1,2,3 strain restored PHB production, and in the course of our complementation experiments we serendipitously created a PHB hyper-producing mutant. Measurement of PhaB and PhaA activities of the mutant strains indicated that the thiolase reaction is the limiting step in PHB biosynthesis in R. eutropha H16 during nitrogen limited growth on fructose.

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Characterization of the CDP-2-glycerol biosynthetic pathway in Streptococcus pneumoniae.

Publication Date: 2010 Aug 20 PMID: 20729354
Authors: Wang, Q. - Xu, Y. - Perepelov, A. V. - Xiong, W. - Wei, D. - Shashkov, A. S. - Knirel, Y. A. - Feng, L. - Wang, L.
Journal: J Bacteriol

Capsule polysaccharide (CPS) plays an important role in the virulence of Streptococcus pneumoniae, and is usually used as the pneumococcal vaccine target. Glycerol-2-phosphate is found in the CPS of S. pneumoniae types 15A and 23F and is rarely found in the polysaccharides of other bacteria. The biosynthetic pathway of the nucleotide-activated form of glycerol-2-phosphate (NDP-2-glycerol) has never been identified. In this study, three genes (gtp1, gtp2, and gtp3) from S. pneumoniae 23F that have been proposed to be involved in the synthesis of NDP-2-glycerol were cloned and the enzyme products were expressed, purified, and assayed for their respective activities. Capillary electrophoresis was used to detect novel products from the enzyme-substrate reactions, and the structure of the product was elucidated using electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. Gtp1 was identified as a reductase, catalyzing the conversion of 1,3-dihydroxyacetone to glycerol; Gtp3 was identified as a glycerol-2-phosphotransferase, catalyzing the conversion of glycerol to glycerol-2-phosphate; and Gtp2 was identified as a cytidylyltransferase, transferring CTP to glycerol-2-phosphate to form CDP-2-glycerol as the final product. The kinetic parameters of Gtp1 and Gtp2 were characterized in depth, and the effects of temperature, pH, and cations on these two enzymes were analyzed. This is the first time that the biosynthetic pathway of CDP-2-glycerol has been identified biochemically, which provides a method to enzymatically synthesize this compound.

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A mutation within the {beta} subunit of Escherichia coli RNA polymerase impairs transcription from bacteriophage T4 middle promoters.

Publication Date: 2010 Aug 20 PMID: 20729353
Authors: James, T. D. - Cashel, M. - Hinton, D. M.
Journal: J Bacteriol

During infection of Escherichia coli, bacteriophage T4 usurps the host transcriptional machinery, redirecting it to the expression of early, middle, and late phage genes. Middle genes, whose expression begins about 1 minute post-infection, are transcribed both from the extension of early RNA into middle genes and by the activation of T4 middle promoters. Middle promoter activation requires the T4 transcriptional activator MotA and co-activator AsiA, which are known to interact with sigma(70), the specificity subunit of RNA polymerase. T4 motA amber (am) or asiAam phage grows poorly in wild type E. coli. However, previous work has found that T4 motAam does not grow in the E. coli mutant strain, TabG. We show here that the RNA polymerase in TabG contains two mutations within its beta subunit gene: rpoB E835K and rpoB G1249D. We find that the G1249D mutation is responsible for restricting the growth of either T4 motAam or asiAam and impairing transcription from MotA/AsiA-activated middle promoters in vivo. With one exception, transcription from tested T4 early promoters is either unaffected or in some cases, even increases, and there is no significant growth phenotype for the rpoB E835K/G1249D strain in the absence of T4 infection. In reported structures of thermophilic RNA polymerase, the G1249 residue is located immediately adjacent to a hydrophobic pocket, called the Switch 3 loop. This loop is thought to aid in the separation of the RNA from the DNA-RNA hybrid as RNA enters the RNA exit channel. Our results suggest that the presence of MotA and AsiA may impair the function of this loop or that this portion of beta may influence interactions among MotA, AsiA, and RNA polymerase.

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Orientations of the Bacteroides fragilis capsular polysaccharide biosynthesis loci promoters during symbiosis and infection.

Publication Date: 2010 Aug 20 PMID: 20729352
Authors: Troy, E. B. - Carey, V. J. - Kasper, D. L. - Comstock, L. E.
Journal: J Bacteriol

Orientations of the seven invertible polysaccharide biosynthesis loci promoters of B. fragilis were quantified from bacteria grown in vitro, from feces of monoassociated and complex colonized mice, and from B. fragilis-induced murine abscesses. Bacteria grown in vivo have greater variability in orientations of polysaccharide locus promoters than culture grown organisms.

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The flagellar basal-body associated protein, FlgT, essential for a novel ring structure in sodium-driven Vibrio motor.

Publication Date: 2010 Aug 20 PMID: 20729351
Authors: Terashima, H. - Koike, M. - Kojima, S. - Homma, M.
Journal: J Bacteriol

In Vibrio alginolyticus, the flagellar motor can rotate at remarkably high speed 3 approximately 4 times faster than the E. coli or Salmonella motor. Here we found a Vibrio-specific protein, FlgT, in the purified flagellar basal body fraction. Defects of FlgT resulted in partial Fla(-) and Mot(-) phenotypes, suggesting that FlgT is involved in formation of the flagellar structure and generating flagellar rotation. Electron microscopic observation of the basal body of DeltaflgT cells revealed a smaller LP ring structure compared to wild type, and most of the T ring was lost. His6-tagged FlgT could be co-isolated with MotY, the T-ring component, suggesting that FlgT may interact with the T ring composed of MotX and MotY. From these lines of evidence, we conclude that FlgT associates with the basal body and is responsible to form an outer ring of the LP ring, named the H ring, which can be distinguished from the LP ring formed by FlgH and FlgI. Vibrio-specific structures, e.g., the T ring and H ring might contribute the more robust motor structure compared to that of E. coli and Salmonella.

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AmrZ beta-sheet residues are essential for DNA-binding and transcriptional control of Pseudomonas aeruginosa virulence genes.

Publication Date: 2010 Aug 13 PMID: 20709902
Authors: Waligora, E. A. - Ramsey, D. M. - Pryor, E. E. Jr - Lu, H. - Hollis, T. - Sloan, G. P. - Deora, R. - Wozniak, D. J.
Journal: J Bacteriol

AmrZ is a putative ribbon-helix-helix (RHH) transcriptional regulator. RHH proteins utilize residues within the beta-sheet for DNA binding while the alpha-helices promote oligomerization. AmrZ is of interest due to its dual roles as a transcriptional activator and repressor, regulating genes encoding virulence factors associated with both chronic and acute P. aeruginosa infection. In this study, crosslinking revealed that AmrZ forms oligomers in solution but the amino terminus, containing an unordered region and beta-sheet, were not required for oligomerization. The first 12 unordered residues (extended amino terminus) contributed minimally to DNA-binding. Mutagenesis of the AmrZ beta-sheet demonstrated that residues 18, 20 and 22 were essential for DNA binding at both activation and repressor sites, suggesting that AmrZ utilizes a similar mechanism for binding these sites. Mice infected with amrZ mutants exhibited reduced bacterial burden, morbidity, and mortality. Direct in vivo competition assays showed a 5-fold competitive advantage for wild type over an isogenic amrZ-mutant. Finally, the reduced infection phenotype of the amrZ-null strain was similar to a strain expressing a DNA-binding-deficient AmrZ variant, indicating DNA binding and transcriptional regulation by AmrZ is responsible for in the in vivo virulence defect. This recent infection data, along with the previously identified AmrZ-regulated virulence factors, suggest the necessity of AmrZ transcriptional regulation for optimal virulence during acute infection.

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Brochothrix thermosphacta bacteriophages feature heterogeneous and highly mosaic genomes and utilize unique prophage insertion sites.

Publication Date: 2010 Aug 13 PMID: 20709901
Authors: Kilcher, S. - Loessner, M. J. - Klumpp, J.
Journal: J Bacteriol

Brochothrix belongs to the low GC branch of Gram-positive bacteria (Firmicutes), closely related to Listeria, Staphylococcus, Clostridium and Bacillus. B. thermosphacta is a non-proteolytic food spoilage organism, adapted to growth in vacuum-packaged meats. We report the first genome sequences and characterization of Brochothrix bacteriophages. Phage A9 is a myovirus with 89 nm capsid diameter and a 171 nm contractile tail; it belongs to the Spounavirinae subfamily and shares significant homologies with Listeria phage A511, Staphylococcus phage Twort, and others. The A9 unit genome is 127 kb long with 11 kb terminal redundancy; it encodes 198 proteins and 6 tRNAs. Phages BL3 and NF5 are temperate Siphoviruses with a head diameter of 55-59 nm. The BL3 tail is 270 nm long, whereas NF5 features a short tail of only 94 nm. The NF5 genome (36.95 kb) encodes 57 gene products, and BL3 (41.52 kb) encodes 65 products, and both are arranged in lifecycle-specific modules. Surprisingly, BL3 and NF5 show little relatedness to Listeria phages, but rather to lactococcal phages. Peptide mass fingerprinting of viral proteins indicate programmed -1 translational frameshifts in the NF5 capsid and the BL3 major tail protein. Both NF5 and BL3 feature circularly permuted, terminally redundant genomes, packaged by a headful mechanism, and integrases of the serine (BL3) and tyrosine (NF5) types. They utilize unique target sequences not previously described: BL3 inserts into the 3'-end of a RNA methyltransferase, whereas NF5 integrates into the 5'-terminal part of a putative histidinol-phosphatase. Interestingly, both genes are reconstituted by phage sequence.

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Small Genes under Sporulation Control in the Bacillus subtlis genome.

Publication Date: 2010 Aug 13 PMID: 20709900
Authors: Schmalish, M. - Maiques, E. - Nikolov, L. - Camp, A. H. - Chevreux, B. - Muffler, A. - Rodriguez, S. - Perkins, J. - Losick, R.
Journal: J Bacteriol

Using an oligonucleotide microarray, we searched for previously unrecognized transcription units in intergenic regions in the genome of Bacillus subtilis, with an emphasis on identifying small genes activated during spore formation. Nineteen transcription units were identified eleven of which were shown to depend on one or more sporulation regulatory proteins for their expression. A high proportion of the transcription units contained small, functional ORFs. One such newly identified ORF is a member of a family of six structurally similar genes that are transcribed under the control of the sporulation transcription factors sigma(E) or sigma(K). A multiple mutant lacking all six genes was found to sporulate with slightly higher efficiency than the wild type, suggesting that under standard laboratory conditions the expression of these genes imposes a small cost on the production of heat-resistant spores. Finally, three of the transcription units specified small, non-coding RNAs, one of which was under the control of the sporulation transcription factor sigma(E) and another under the control of the motility sigma factor sigma(D).

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Identification, source and metabolism of N-ethylglutamate in Escherichia coli.

Publication Date: 2010 Aug 13 PMID: 20709899
Authors: White, R. H.
Journal: J Bacteriol

N-Ethyl-glutamate (NEG) was detected in Escherichia coli BL-21 cells grown on LB broth, where it was found to occur at a concentration of approximately 4 mM in these cells. The same cells grown on M9 glucose medium contained no detectable amount of NEG. Analysis of the LB broth showed the presence of NEG, a compound never before reported as a natural product. Isotope dilution analysis showed it occurred at a concentration of 160 muM in LB broth. Analysis of yeast extract and tryptone, the organic components of LB broth, both showed the presence NEG. It was demonstrated that NEG can be generated during the autolysis of the yeast used in the preparation of the yeast extract. Growth of these cells in LB broth prepared in deuterated water, showed no incorporation of deuterium into the NEG, demonstrating that E. coli cells did not generate the NEG. Cell growth rates were not effected by the addition of 5 mM NEG to either LB or M9 glucose medium. [(2)H4-ethyl]-L-NEG was found to be readily incorporated into the cells and metabolized by the cells. From these results it is concluded that all of the NEG present in the cells was taken up from the medium. NEG could serve a sole nitrogen source for E. coli when grown on M9 glucose medium in the presence of glucose but could not serve as the sole carbon source on M9 medium in the absence of glucose.

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Metabolic Network Analysis of Pseudomonas aeruginosa during Chronic Cystic Fibrosis Lung Infection.

Publication Date: 2010 Aug 13 PMID: 20709898
Authors: Oberhardt, M. A. - Goldberg, J. B. - Hogardt, M. - Papin, J. A.
Journal: J Bacteriol

Systems level modeling is beginning to be used to decipher high throughput data in the context of disease. In this study, we present an integration of expression microarray data with a genome-scale metabolic model of P. aeruginosa in the context of a chronic Cystic Fibrosis (CF) lung infection. A genome-scale model of P. aeruginosa metabolism was tailored to represent the metabolic states of two clonally related lineages of P. aeruginosa isolated from the lungs of a CF patient at different points over a 44 month timecourse, giving a mechanistic glimpse into how the bacterial metabolism adapts over time in the CF lung. Metabolic capacities were analyzed to determine how tradeoffs between growth and other important cellular processes shift during the disease progression. Genes whose knockouts were either significantly growth reducing or lethal in silico were also identified for each timepoint, and serve as hypotheses for future drug targeting efforts specific to the stages of disease progression.

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Integration of Metabolism and Virulence by Clostridium difficile CodY.

Publication Date: 2010 Aug 13 PMID: 20709897
Authors: Dineen, S. S. - McBride, S. M. - Sonenshein, A. L.
Journal: J Bacteriol

CodY, a global regulatory protein that monitors the nutrient sufficiency of the environment by responding to the intracellular levels of GTP and the branched-chain amino acids, was previously shown to be a potent repressor of toxin gene expression in Clostridium difficile during growth in rich medium. In the intestinal tract, such derepression of toxin synthesis would lead to destruction of epithelial cells and the liberation of potential nutrients for the bacterium. CodY is likely to play an important role in regulating overall cellular physiology as well. In this study, DNA microarray analysis and affinity purification of CodY-DNA complexes were used to identify and distinguish the direct and indirect effects of CodY on global gene transcription. A codY null mutation resulted in >4-fold overexpression of 146 genes (organized in 82 apparent transcription units) and underexpression of 19 genes. In addition to the toxin genes, genes for amino acid biosynthesis, nutrient transport, fermentation pathways, membrane components and surface proteins were overexpressed in the codY mutant. Genome-wide analysis identified more than 350 CodY binding regions, many of which are likely to correspond to sites of direct CodY-mediated regulation. About 60% of the CodY-repressed transcription units were associated with binding regions. Several of these genes were confirmed to be direct targets of CodY by gel mobility shift and DNase I footprinting assays.

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CztR, a LysR-type transcriptional regulator involved in zinc homeostasis and oxidative stress defense in Caulobacter crescentus.

Publication Date: 2010 Aug 13 PMID: 20709896
Authors: Braz, V. S. - da Silva Neto, J. F. - Italiani, V. C. - Marques, M. V.
Journal: J Bacteriol

Caulobacter crescentus is a free-living Alphaproteobacterium that has eleven predicted LysR-type transcriptional regulators (LTTRs). Previously, a C. crescentus mutant strain with a miniTn5lacZ transposon inserted into a gene encoding an LTTR was isolated, which was sensitive to cadmium. In this work, a mutant strain with a deletion was obtained and the role of this LTTR (called here CztR) was evaluated. The transcriptional start site of this gene was determined by primer extension analysis and its promoter was cloned in front of a lacZ reporter gene. beta-Galactosidase activity assays, performed in the wild type and mutant strains, indicated that this gene is twofold induced when cells enter stationary phase and that it is negatively autoregulated. Moreover, this regulator is essential for the expression of the divergent cztA gene at stationary phase, in minimal medium and in response to zinc depletion. This gene encodes a hypothetical protein containing ten predicted transmembrane segments, and its expression pattern suggests that it encodes a putative zinc transporter. The cztR strain also showed to be sensitive to superoxide (generated by paraquat), and to hydrogen peroxide but not to tert-butyl hydroperoxide. Expression of katG and ahpC, but not of the superoxide dismutases genes, was increased in the cztR mutant. A model is proposed to explain how CztR binding to the divergent regulatory regions could activate cztA expression and repress its own transcription.

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Comparative genome biology of a serogroup B carriage and disease strain supports a polygenic nature of meningococcal virulence.

Publication Date: 2010 Aug 13 PMID: 20709895
Authors: Joseph, B. - Schneiker-Bekel, S. - Schramm-Gluck, A. - Blom, J. - Claus, H. - Linke, B. - Schwarz, R. F. - Becker, A. - Goesmann, A. - Frosch, M. - Schoen, C.
Journal: J Bacteriol

Neisseria meningitidis serogroup B strains are responsible for most meningococcal cases in the industrialized countries, and strains belonging to the clonal complex ST-41/44 are among the most prevalent serogroup B strains in carriage and disease. Here we report the first genome and transcriptome comparison of a serogroup B carriage strain from the clonal complex ST-41/44 with the serogroup B disease strain MC58 form the clonal complex ST-32. Both genomes are highly co-linear with only three major genome rearrangements that are associated with the integration of mobile genetic elements. They further differ in about 10% of their gene content with the highest variability in gene presence as well as gene sequence found for proteins involved in host cell interactions comprising Opc, NadA, TonB-dependent receptors, RTX toxin and two-partner secretion system proteins. Whereas housekeeping genes coding for metabolic functions were highly conserved, there were considerable differences in their expression pattern upon adhesion to human nasopharyngeal cells between both strains including differences in energy metabolism and stress response. In line with these genomic and transcriptomic differences, both strains also showed marked differences in their in vitro infectivity and in serum resistance. Together, these data support the concept of a polygenic nature of meningococcal virulence comprising differences in the repertoire of adhesins as well as in the regulation of metabolic genes and suggest a prominent role for immune selection and genetic drift in shaping the meningococcal genome.

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Mn2+ dependent in vitro activity of the Staphylococcus aureus lipoteichoic acid synthase enzyme using fluorescently labeled lipid substrate.

Publication Date: 2010 Aug 13 PMID: 20709894
Authors: Karatsa-Dodgson, M. - Wormann, M. E. - Grundling, A.
Journal: J Bacteriol

Lipoteichoic acid is an important cell wall component of Gram-positive bacteria. The key enzyme responsible for polyglycerolphosphate lipoteichoic acid synthesis in the Gram-positive pathogen Staphylococcus aureus is the membrane embedded lipoteichoic acid synthase enzyme LtaS. It is presumed that LtaS hydrolyzes the glycerolphosphate head group of the membrane lipid phosphatidylglycerol (PG) and catalyzes the formation of the polyglycerolphosphate LTA backbone chain. Here, we describe an in vitro assay for this new class of enzyme using PG with a fluorescently labeled fatty acid chain (NBD-PG) as substrate and the recombinant soluble C-terminal enzymatic domain of LtaS (eLtaS). Thin layer chromatography and mass spectrometry analysis of the lipid reaction products revealed that eLtaS is sufficient to cleave the glycerolphosphate head group from NBD-PG resulting in the formation of NBD-diacylglycerol. An excess of soluble glycerolphosphate could not compete with the hydrolysis of fluorescently labeled PG lipid substrate contrary to the addition of unlabeled PG. This indicates that the enzyme recognizes and binds, besides the glycerolphosphate head group, other parts of the lipid substrate. Furthermore, eLtaS activity was Mn(2+) ion dependent, Mg(2+) and Ca(2+) supported only weak enzyme activity. Addition of Zn(2+) or EDTA inhibited enzyme activity even in the presence of Mn(2+). The pH optimum of the enzyme was 6.5, characteristic for an enzyme that functions extracellularly. Lastly, we show that the in vitro assay can be used to study the enzyme activity of other members of the lipoteichoic acid synthase enzyme family.

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Domain analysis of a modular {alpha}-L-arabinofuranosidase with a unique carbohydrate binding strategy from the fiber degrading bacterium Fibrobacter succinogenes S85.

Publication Date: 2010 Aug 13 PMID: 20709893
Authors: Yoshida, S. - Hespen, C. W. - Beverly, R. L. - Mackie, R. I. - Cann, I. K.
Journal: J Bacteriol

Family 43 glycoside hydrolases (GH43s) are known to exhibit various activities involved in hemicellulose hydrolysis. Thus, these enzymes contribute to efficient plant cell wall degradation, a topic of much interest to biofuel production. In this study, we characterized a unique GH43 protein from F. succinogenes S85. The recombinant protein showed alpha-L-arabinofuranosidase activity, specifically with arabinoxylan. The enzyme is, therefore, an arabinoxylan arabinofuranohydrolase (AXH). The F. succinogenes AXH (FSUAXH1) is a modular protein that is composed of a signal peptide, a GH43 catalytic module, a unique beta-sandwich module (XX-domain), a family 6 carbohydrate-binding module (CBM6), and a F. succinogenes-specific paralogous module-1 (FPm-1). Truncational analysis and site-directed mutagenesis of the protein revealed that the GH43 domain/XX-domain constitute a new form of carbohydrate binding module, and that residue Y484 in the XX-domain is essential for binding to arabinoxylan, although protein structural analyses may be required to confirm some of the observations. Kinetic studies demonstrated that the Y484A mutation leads to a higher kcat for a truncated derivative of FSUAXH1 composed of only the GH43 catalytic module and the XX-domain. However, an increase in the Km for arabinoxylan led to a three-fold decrease in catalytic efficiency. Based on the knowledge that most XX-domains are found only in GH43 proteins, the evolutionary relationships within the GH43 family were investigated. These analyses showed that in GH43 members with a XX-domain, the two modules have co-evolved, and that the length of a loop within the XX-domain may serve as an important determinant of substrate specificity.

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Elucidation of {beta}-oxidation Pathways in Ralstonia eutropha H16 by Examination of Global Gene Expression.

Publication Date: 2010 Aug 13 PMID: 20709892
Authors: Brigham, C. J. - Budde, C. F. - Holder, J. W. - Zeng, Q. - Mahan, A. E. - Rha, C. - Sinskey, A. J.
Journal: J Bacteriol

Ralstonia eutropha H16 is capable of growth and polyhydroxyalkanoate production on plant oils and fatty acids. However, little is known about the triacylglycerol and fatty acid degradation pathways of this bacterium. We compare whole-cell gene expression of R. eutropha H16 during growth and polyhydroxyalkanoate production on trioleate and fructose. Trioleate is a triacylglycerol that serves as a model for plant oils. Among the genes of note, two potential fatty acid beta-oxidation operons and two putative lipase genes were shown to be upregulated in trioleate cultures. The genes of the glyoxylate bypass also exhibit increased expression during growth on trioleate. We observed that single beta-oxidation operon deletion mutants of R. eutropha could grow using palm oil or crude palm kernel oil as the sole carbon source, regardless of which operon was present in the genome, but a double mutant was unable to grow under these conditions. A lipase deletion mutant did not exhibit a growth defect in emulsified oil cultures, but did exhibit a phenotype in cultures containing non-emulsified oil. Mutants of the glyoxylate shunt gene isocitrate lyase were able to grow in the presence of oils, while a malate synthase (aceB) deletion mutant grew more slowly than wild-type. Gene expression under polyhydroxyalkanoate storage conditions was also examined. Many findings of this analysis confirm results from previous studies by our group and others. This work represents the first examination of global gene expression involving triacylglycerol and fatty acid catabolism genes in R. eutropha.

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Diversity of monomers in nonribosomal peptides: towards the prediction of origin and biological activity.

Publication Date: 2010 Aug 6 PMID: 20693331
Authors: Caboche, S. - Leclere, V. - Pupin, M. - Kucherov, G. - Jacques, P.
Journal: J Bacteriol

Nonribosomal peptides (NRPs) are molecules produced by micro-organisms and presenting a broad spectrum of biological activities and pharmaceutical applications (e.g. antibiotic, immuno-modulating, anti-tumor). One particularity of the NRPs is the biodiversity of their monomers extending far beyond the 20 proteogenic amino acid residues. Norine, a comprehensive database of NRPs, allowed us to overview for the first time the main characteristics of the NRPs and especially their monomer biodiversity. Our analysis highlighted a significant similarity relationship between NRPs synthesized by bacteria and those isolated from metazoa, especially from sponges, supporting the hypothesis that some NRPs isolated from sponges are actually synthesized by symbiotic bacteria rather than by the sponges themselves. A comparison of peptide monomeric compositions as a function of biological activity showed that some monomers are specific to a class of activities. An analysis of monomer composition of peptide products predicted from genomic information (metagenomics and high-throughput genome sequencing) or of new peptides detected by mass spectrometry analysis applied to a culture supernatant can provide indications of the origin of the peptide and/or its biological activity.

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The Caulobacter Tol-Pal complex is essential for outer membrane integrity and the positioning of a polar localization factor.

Publication Date: 2010 Aug 6 PMID: 20693330
Authors: Yeh, Y. C. - Comolli, L. R. - Downing, K. H. - Shapiro, L. - McAdams, H. H.
Journal: J Bacteriol

Cell division in Caulobacter crescentus involves constriction and fission of the inner membrane (IM) followed about 20 min later by fission of the outer membrane (OM) and daughter cell separation. In contrast to Escherichia coli, the Caulobacter Tol-Pal complex is essential. Cryo-electron microscope images of the Caulobacter cell envelope exhibited outer membrane disruption, and cells failed to complete cell division in TolA, TolB, or Pal mutant strains. In wild type cells, components of the Tol-Pal complex localize to the division plane in early predivisional cells and remain predominantly at the new pole of swarmer and stalked progeny upon completion of division. The Tol-Pal complex is required to maintain the position of the transmembrane TipN polar marker, and indirectly the PleC histidine kinase, at the cell pole, but it is not required for the polar maintenance of other transmembrane and membrane associated polar proteins tested. Co-immunoprecipitation experiments show that both TolA and Pal interact directly or indirectly with TipN. We propose that disruption of the trans-envelope Tol-Pal complex releases TipN from its subcellular position. The Caulobacter Tol-Pal complex is thus a key component of cell envelope structure and function, mediating OM constriction at the final step of cell division, as well as the positioning of a protein localization factor.

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Actin-based motility of Burkholderia thailandensis requires a central acidic domain of BimA that recruits and activates the cellular Arp2/3 complex.

Publication Date: 2010 Aug 6 PMID: 20693329
Authors: Sitthidet, C. - Stevens, J. M. - Field, T. R. - Layton, A. N. - Korbsrisate, S. - Stevens, M. P.
Journal: J Bacteriol

Burkholderia species use BimA for intracellular actin-based motility. Uniquely, B. thailandensis BimA harbors a central and acidic (CA) domain. The CA domain was required for actin-based motility, binding to the cellular Arp2/3 complex and Arp2/3-dependent polymerization of actin monomers. Our data reveal distinct strategies for actin-based motility among Burkholderia species.

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Bright mutants of Vibrio fischeri ES114 reveal conditions and regulators that control bioluminescence and expression of the lux operon.

Publication Date: 2010 Aug 6 PMID: 20693328
Authors: Lyell, N. L. - Dunn, A. K. - Bose, J. L. - Stabb, E. V.
Journal: J Bacteriol

Vibrio fischeri ES114, an isolate from the Euprymna scolopes light organ, produces little bioluminescence in culture but is approximately 1000-fold brighter when colonizing the host. Cell-density dependent regulation alone cannot explain this phenomenon, because cells within colonies on solid media are much dimmer than symbiotic cells despite their similar cell density. To better understand this low luminescence in culture, we screened approximately 20,000 mini-Tn5 mutants of ES114 for increased luminescence and identified twenty-eight independent 'luminescence-up' mutants with insertions in fourteen loci. Mutations affecting the Pst phosphate uptake system led to the discovery that luminescence is up-regulated under low-phosphate conditions by PhoB, and we also found that ainS, which encodes an autoinducer synthase, mediates repression of luminescence during growth on plates. Other novel 'luminescence-up' mutants had insertions in acnB, topA, tfoY, phoQ, guaB and two specific tRNA genes. Two loci, hns and lonA, were previously described as repressors of bioluminescence in transgenic Escherichia coli carrying the light-generating lux genes, and mutations in arcA and arcB were consistent with our report that Arc represses lux. Our results reveal a complex regulatory web governing luminescence and show how certain environmental conditions are integrated into regulation of the pheromone-dependent lux system.

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Genetic analysis of the Nitrogen Assimilation Control protein (NAC) from Klebsiella pneumoniae.

Publication Date: 2010 Aug 6 PMID: 20693327
Authors: Rosario, C. J. - Janes, B. K. - Bender, R. A.
Journal: J Bacteriol

The Nitrogen Assimilation Control protein (NAC) from Klebsiella pneumoniae is a typical LysR-type transcriptional regulator (LTTR) in many ways. However the lack of a physiologically relevant coeffector for NAC and the fact that NAC can carry out many of its functions as a dimer make NAC unusual among the LTTRs. In the absence of a crystal structure for NAC, we analyzed the effects of amino acid substitutions with a variety of phenotypes in an attempt to identify functionally important features of NAC. A substitution that changes the glutamine at amino acid 29 to alanine (Q29A) resulted in a NAC that was seriously defective in binding to DNA. The H26D substitution resulted in a NAC that could bind and repress transcription, but not activate transcription. The I71A substitution resulted in a NAC polypeptide that remained monomeric. NAC tetramers can bind to both long and shorter binding sites (like other LTTRs). However the absence of a coeffector to induce the conformational change needed for the switch from the former to the latter raised a question. Are there two conformations of NAC analogous to the other LTTRs? The G217R substitution resulted in a NAC that could bind to the longer sites but had difficulty in binding to the shorter sites, and the I222R and A230R substitutions resulted in a NAC that could bind to the shorter sites but had difficulty in binding properly to the longer sites. Thus there appear to be two conformers of NAC that can freely interconvert in the absence of a coeffector.

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Conserved motifs involved in ATP hydrolysis by MalT, a signal transduction ATPase with numerous domains (STAND) from Escherichia coli.

Publication Date: 2010 Aug 6 PMID: 20693326
Authors: Marquenet, E. - Richet, E.
Journal: J Bacteriol

The signal transduction ATPases with numerous domains (STAND) are sophisticated, signaling proteins that are related to AAA+ proteins and control various biological processes, including apoptosis, gene expression and innate immunity. They function as tightly regulated switches, with the off and on positions corresponding to an ADP-bound, monomeric form and an ATP-bound, multimeric form, respectively. Protein activation is triggered by inducer binding to the sensor domain. ATP hydrolysis by the nucleotide-binding oligomerization domain (NOD) ensures the generation of the ADP-bound form. Here, we use MalT, an Escherichia coli transcription activator, as a model system to identify STAND conserved motifs involved in ATP hydrolysis besides the catalytic acidic residue. Alanine substitution of the conserved polar residue (H131) that is located two residues downstream from the catalytic residue (D129) blocks ATP hydrolysis and traps MalT in an active, ATP-bound, multimeric form. This polar residue is also conserved in AAA+. Based on AAA+ X-ray structures, we proposed that it is responsible for the proper positioning of the catalytic and the sensor I residues for the hydrolytic attack. Alanine substitution of the putative STAND sensor I (R160) abolished MalT activity. Substitutions of R171 impaired both ATP hydrolysis and multimerization, which is consistent with an arginine finger function and provides further evidence that ATPhydrolysis is primarily catalyzed by MalT multimers.

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A multitask ATPase serving different ABC-type sugar importers in Bacillus subtilis.

Publication Date: 2010 Aug 6 PMID: 20693325
Authors: Ferreira, M. J. - de Sa-Nogueira, I.
Journal: J Bacteriol

Bacillus subtilis is able to utilize arabino-polysaccharides derived from plant biomass. Here, by combining genetic and physiological analyses we characterize the AraNPQ importer and identify primary and secondary transporters of B. subtilis involved in the uptake of arabino-saccharides. We show that the ABC-type importer AraNPQ is involved in the uptake of alpha-1,5-arabino-oligosaccharides, at least up to four L-arabinosyl units. Although this system is the key transporter for alpha-1,5-arabinotriose and alpha-1,5-arabinotetraose, the results indicate that alpha-1,5-arabinobiose is also translocated by the secondary transporter AraE. This broad specificity proton symporter is the major transporter for arabinose and also accountable for the uptake of xylose and galactose. In addition, MsmX is shown to be the ATPase that energizes the incomplete AraNPQ importer. Furthermore, the results suggest the existence of at least one more unidentified MsmX-dependent ABC importer responsible for the uptake of non-linear alpha-1,2- and alpha-1,3-arabino-oligosaccharides. This study assigns MsmX as a multipurpose B. subtilis ATPase required to energize different saccharide transporters, the arabino-oligosaccharide-specific AraNPQ-MsmX system, a putative MsmX-dependent ABC transporter specific for non-linear arabino-oligosaccharides, and the previously characterized maltodextrins-specific MdxEFG-MsmX system.

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Sit and stay a while: how BfiSR controls irreversible attachment in Pseudomonas aeruginosa biofilms.

Publication Date: 2010 Aug 6 PMID: 20693324
Authors: Goodman, A. L.
Journal: J Bacteriol

In this issue of Journal of Bacteriology, Petrova and Sauer demonstrate that the two-component system BfiSR regulates the transition from initial to irreversible surface attachment in Pseudomonas aeruginosa via ribonuclease G-dependent post-transcriptional regulation of the small RNA rsmZ. This study provides a new mechanism for an intermediate checkpoint in biofilm development.

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Regulation of autotrophic CO2 fixation in the archaeon Thermoproteus neutrophilus.

Publication Date: 2010 Aug 6 PMID: 20693323
Authors: Ramos-Vera, W. H. - Labonte, V. - Weiss, M. - Pauly, J. - Fuchs, G.
Journal: J Bacteriol

Thermoproteus neutrophilus, a hyperthermophilic, chemolithoautotrophic, anaerobic crenarchaeon, uses a novel autotrophic CO2 fixation pathway, the dicarboxylate/hydroxybutyrate cycle. The regulation of the central carbon metabolism was studied on the level of whole cells, enzyme activity, proteome, transcription, and gene organization. The organism proved to be a facultative autotroph, which prefers organic acids as carbon sources that can easily feed into the metabolite pools of this cycle. Addition of the preferred carbon sources acetate, pyruvate, succinate and 4-hydroxybutyrate to cultures resulted in a stimulation of the growth rate and a diauxic growth response. The characteristic enzyme activities of the carbon fixation cycle, fumarate hydratase, fumarate reductase, succinyl-CoA synthetase and enzymes catalyzing the conversion of succinyl-CoA to crotonyl-CoA, were differentially down-regulated in the presence of acetate and, to a lesser extent, in the presence of other organic substrates. This regulation pattern correlated well with the differential expression profile of the proteome as well as with the transcription of the encoding genes. The genes encoding PEP carboxylase, fumarate reductase and four enzymes catalyzing the conversion of succinyl-CoA to crotonyl-CoA are clustered. Two putative operons, one comprising succinyl-CoA reductase plus 4-hydroxybutyrate-CoA ligase genes, the other 4-hydroxybutyryl-CoA dehydratase plus fumarate reductase genes, were divergently transcribed into leaderless mRNAs. The promoter regions were characterized and used for isolating DNA binding proteins. Besides an Alba protein a 18 kDa protein characteristic for autotrophic Thermoproteales was identified that specifically bound to the promoter region. This system may be suitable for molecular analysis of transcription regulation of autotrophy related genes.

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Dynamic polar sequestration of excess MurG may regulate enzymatic function.

Publication Date: 2010 Sep PMID: 20644141
Authors: Michaelis, A. M. - Gitai, Z.
Journal: J Bacteriol

Advances in bacterial cell biology have demonstrated the importance of protein localization for protein function. In general, proteins are thought to localize to the sites where they are active. Here we demonstrate that in Escherichia coli, MurG, the enzyme that mediates the last step in peptidoglycan subunit biosynthesis, becomes polarly localized when expressed at high cellular concentrations. MurG only becomes polarly localized at levels that saturate MurG's cellular requirement for growth, and E. coli cells do not insert peptidoglycan at the cell poles, indicating that the polar MurG is not active. Fluorescence recovery after photobleaching (FRAP) and single-cell biochemistry experiments demonstrate that polar MurG is dynamic. Polar MurG foci are distinct from inclusion body aggregates, and polar MurG can be remobilized when MurG levels drop. These results suggest that polar MurG represents a temporary storage mechanism for excess protein that can later be remobilized into the active pool. We investigated and ruled out several candidate pathways for polar MurG localization, including peptidoglycan biosynthesis, the MreB cytoskeleton, and polar cardiolipin, as well as MurG enzymatic activity and lipid binding, suggesting that polar MurG is localized by a novel mechanism. Together, our results imply that inactive MurG is dynamically sequestered at the cell poles and that prokaryotes can thus utilize subcellular localization as a mechanism for negatively regulating enzymatic activity.

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Discontinuity and limited linkage in the homologous recombination system of a hyperthermophilic archaeon.

Publication Date: 2010 Sep PMID: 20644140
Authors: Grogan, D. W. - Rockwood, J.
Journal: J Bacteriol

Genetic transformation of Sulfolobus acidocaldarius by a multiply marked pyrE gene provided a high-resolution assay of homologous recombination in a hyperthermophilic archaeon. Analysis of 100 Pyr(+) transformants revealed that this recombination system could transfer each of 23 nonselected base pair substitutions to the recipient chromosome along with the selected marker. In 30% of the recombinants, donor markers were transferred as multiple blocks. In at least 40% of the recombinants, donor markers separated by 5 or 6 bp segregated from each other, whereas similar markers separated by 2 bp did not segregate. Among intermarker intervals, the frequency of recombination tract endpoints varied 40-fold, but in contrast to other recombination systems, it did not correlate with the length of the interval. The average length of donor tracts (161 bp) and the frequent generation of multiple tracts seemed generally consistent with the genetic properties observed previously in S. acidocaldarius conjugation. The efficiency with which short intervals of diverged pyrE sequence were incorporated into the genome raises questions about the threat of ectopic recombination in Sulfolobus spp. mediated by this apparently efficient yet permissive system.

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Characterization of the structure and biological functions of a capsular polysaccharide produced by Staphylococcus saprophyticus.

Publication Date: 2010 Sep PMID: 20639341
Authors: Park, S. - Kelley, K. A. - Vinogradov, E. - Solinga, R. - Weidenmaier, C. - Misawa, Y. - Lee, J. C.
Journal: J Bacteriol

Staphylococcus saprophyticus is a common cause of uncomplicated urinary tract infections in women. S. saprophyticus strain ATCC 15305 carries two staphylococcal cassette chromosome genetic elements, SCC(15305RM) and SCC(15305cap). The SCC(15305cap) element carries 13 open reading frames (ORFs) involved in capsular polysaccharide (CP) biosynthesis, and its G+C content (26.7%) is lower than the average G+C content (33.2%) for the whole genome. S. saprophyticus strain ATCC 15305 capD, capL, and capK (capD(Ssp), capL(Ssp), and capK(Ssp)) are homologous to genes encoding UDP-FucNAc biosynthesis, and gtaB and capI(Ssp) show homology to genes involved in UDP-glucuronic acid synthesis. S. saprophyticus ATCC 15305 CP, visualized by immunoelectron microscopy, was extracted and purified using anionic-exchange and size exclusion chromatography. Analysis of the purified CP by (1)H and (13)C nuclear magnetic resonance (NMR) spectroscopy and gas-liquid chromatography revealed two types of branched tetrasaccharide repeating units composed of the following: -4)-beta-Glc-(1-3)-Sug-(1-4)-beta-GlcA-(1- | beta-GlcNAc-(1-2) Sug represents two stereoisomers of 2-acetamido-2,6-dideoxy-hexos-4-ulose residues, one of which has an arabino configuration. The encapsulated ATCC 15305 strain was resistant to complement-mediated opsonophagocytic killing by human neutrophils, whereas the acapsular mutant C1 was susceptible. None of 14 clinical isolates reacted with antibodies to the ATCC 15305 CP. However, 11 of the 14 S. saprophyticus isolates were phenotypically encapsulated based on their resistance to complement-mediated opsonophagocytic killing and their failure to hemagglutinate when cultivated aerobically. Ten of the 14 clinical strains carried homologues of the conserved staphylococcal capD gene or the S. saprophyticus gtaB gene, or both. Our results suggest that some strains of S. saprophyticus are encapsulated and that more than one capsular serotype exists.

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Aromatic acid metabolites of Escherichia coli K-12 can induce the marRAB operon.

Publication Date: 2010 Sep PMID: 20639340
Authors: Chubiz, L. M. - Rao, C. V.
Journal: J Bacteriol

MarR is a key regulator of the marRAB operon involved in antibiotic resistance and solvent stress tolerance in Escherichia coli. We show that two metabolic intermediates, 2,3-dihydroxybenzoate and anthranilate, involved in enterobactin and tryptophan biosynthesis, respectively, can activate marRAB transcription. We also found that a third intermediate involved in ubiquinone biosynthesis, 4-hydroxybenzoate, activates marRAB transcription in the absence of TolC. Of the three, however, only 2,3-dihydroxybenzoate directly binds MarR and affects its activity.

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In-depth profiling of the LiaR response of Bacillus subtilis.

Publication Date: 2010 Sep PMID: 20639339
Authors: Wolf, D. - Kalamorz, F. - Wecke, T. - Juszczak, A. - Mader, U. - Homuth, G. - Jordan, S. - Kirstein, J. - Hoppert, M. - Voigt, B. - Hecker, M. - Mascher, T.
Journal: J Bacteriol

The Lia system, a cell envelope stress response module of Bacillus subtilis, is comprised of the LiaRS two-component system and a membrane-anchored inhibitor protein, LiaF. It is highly conserved in the Firmicutes bacteria, and all orthologs investigated so far are activated by cell wall antibiotics. In response to envelope stress, the systems in Firmicutes cocci induce the expression of a number of genes that are involved in conferring resistance against its inducers. In contrast, a complete picture of the LiaR regulon of B. subtilis is still missing and no phenotypes could be associated with mutants lacking LiaRS. Here, we performed genome-wide transcriptomic, proteomic, and in-depth phenotypic profiling of constitutive "Lia ON" and "Lia OFF" mutants to obtain a comprehensive picture of the Lia response of Bacillus subtilis. In addition to the known targets liaIH and yhcYZ-yhdA, we identified ydhE as a novel gene affected by LiaR-dependent regulation. The results of detailed follow-up gene expression studies, together with proteomic analysis, demonstrate that the liaIH operon represents the only relevant LiaR target locus in vivo. It encodes a small membrane protein (LiaI) and a phage shock protein homolog (LiaH). LiaH forms large oligomeric rings reminiscent of those described for Escherichia coli PspA or Arabidopsis thaliana Vipp1. The results of comprehensive phenotype studies demonstrated that the gene products of the liaIH operon are involved in protecting the cell against oxidative stress and some cell wall antibiotics. Our data suggest that the LiaFSR system of B. subtilis and, presumably, other Firmicutes bacilli coordinates a phage shock protein-like response.

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Genetic and functional analyses of the mob operon on conjugative transposon CTn341 from Bacteroides spp.

Publication Date: 2010 Sep PMID: 20639338
Authors: Peed, L. - Parker, A. C. - Smith, C. J.
Journal: J Bacteriol

Bacteroides are Gram-negative anaerobes indigenous to the intestinal tract of humans, and they are important opportunistic pathogens. Mobile genetic elements, such as conjugative transposons (CTns), have contributed to an increase in antibiotic resistance in these organisms. CTns are self-transmissible elements that belong to the superfamily of integrative and conjugative elements (ICEs). CTn341 is 52 kb; it encodes tetracycline resistance and its transfer is induced by tetracycline. The mobilization region of CTn341 was shown to be comprised of a three-gene operon, mobABC, and the transfer origin, oriT. The three genes code for a nicking accessory protein, a relaxase, and a VirD4-like coupling protein, respectively. The Mob proteins were predicted to mediate the formation of the relaxosome complex, nick DNA at the oriT, and shuttle the DNA/protein complex to the mating-pore apparatus. The results of mutational studies indicated that the three genes are required for maximal transfer of CTn341. Mob gene transcription was induced by tetracycline, and this regulation was mediated through the two-component regulatory system, RteAB. The oriT region of CTn341 was located within 100 bp of mobA, and a putative Bacteroides consensus nicking site was observed within this region. Mutation of the putative nick site resulted in a loss of transfer. This study demonstrated a role of the mobilization region for transfer of Bacteroides CTns and that tetracycline induction occurs for the mob gene operon, as for the tra gene operon(s), as shown previously.

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The omptins of Yersinia pestis and Salmonella enterica cleave the reactive center loop of plasminogen activator inhibitor 1.

Publication Date: 2010 Sep PMID: 20639337
Authors: Haiko, J. - Laakkonen, L. - Juuti, K. - Kalkkinen, N. - Korhonen, T. K.
Journal: J Bacteriol

Plasminogen activator inhibitor 1 (PAI-1) is a serine protease inhibitor (serpin) and a key molecule that regulates fibrinolysis by inactivating human plasminogen activators. Here we show that two important human pathogens, the plague bacterium Yersinia pestis and the enteropathogen Salmonella enterica serovar Typhimurium, inactivate PAI-1 by cleaving the R346-M347 bait peptide bond in the reactive center loop. No cleavage of PAI-1 was detected with Yersinia pseudotuberculosis, an oral/fecal pathogen from which Y. pestis has evolved, or with Escherichia coli. The cleavage and inactivation of PAI-1 were mediated by the outer membrane proteases plasminogen activator Pla of Y. pestis and PgtE protease of S. enterica, which belong to the omptin family of transmembrane endopeptidases identified in Gram-negative bacteria. Cleavage of PAI-1 was also detected with the omptins Epo of Erwinia pyrifoliae and Kop of Klebsiella pneumoniae, which both belong to the same omptin subfamily as Pla and PgtE, whereas no cleavage of PAI-1 was detected with omptins of Shigella flexneri or E. coli or the Yersinia chromosomal omptins, which belong to other omptin subfamilies. The results reveal a novel serpinolytic mechanism by which enterobacterial species expressing omptins of the Pla subfamily bypass normal control of host proteolysis.

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Separate DNA Pol II- and Pol IV-dependent pathways of stress-induced mutation during double-strand-break repair in Escherichia coli are controlled by RpoS.

Publication Date: 2010 Sep PMID: 20639336
Authors: Frisch, R. L. - Su, Y. - Thornton, P. C. - Gibson, J. L. - Rosenberg, S. M. - Hastings, P. J.
Journal: J Bacteriol

Previous work showed that about 85% of stress-induced mutations associated with DNA double-strand break repair in carbon-starved Escherichia coli result from error-prone DNA polymerase IV (Pol IV) (DinB) and that the mutagenesis is controlled by the RpoS stress response, which upregulates dinB. We report that the remaining mutagenesis requires high-fidelity Pol II, and that this component also requires RpoS. The results identify a second DNA polymerase contributing to stress-induced mutagenesis and show that RpoS promotes mutagenesis by more than the simple upregulation of dinB.

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Complete genome sequence of Lactobacillus fermentum CECT 5716, a probiotic strain isolated from human milk.

Publication Date: 2010 Sep PMID: 20639335
Authors: Jimenez, E. - Langa, S. - Martin, V. - Arroyo, R. - Martin, R. - Fernandez, L. - Rodriguez, J. M.
Journal: J Bacteriol

Lactobacillus fermentum is a heterofermentative lactic acid bacterium and is frequently isolated from mucosal surfaces of healthy humans. Lactobacillus fermentum CECT 5716 is a well-characterized probiotic strain isolated from human milk and, at present, is used in commercial infant formulas. Here, we report the complete and annotated genome sequence of this strain.

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The heat shock protein YbeY is required for optimal activity of the 30S ribosomal subunit.

Publication Date: 2010 Sep PMID: 20639334
Authors: Rasouly, A. - Davidovich, C. - Ron, E. Z.
Journal: J Bacteriol

The highly conserved bacterial ybeY gene is a heat shock gene whose function is not fully understood. Previously, we showed that the YbeY protein is involved in protein synthesis, as Escherichia coli mutants with ybeY deleted exhibit severe translational defects in vivo. Here we show that the in vitro activity of the translation machinery of ybeY deletion mutants is significantly lower than that of the wild type. We also show that the lower efficiency of the translation machinery is due to impaired 30S small ribosomal subunits.

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Complete genome sequence of Croceibacter atlanticus HTCC2559T.

Publication Date: 2010 Sep PMID: 20639333
Authors: Oh, H. M. - Kang, I. - Ferriera, S. - Giovannoni, S. J. - Cho, J. C.
Journal: J Bacteriol

Here we announce the complete genome sequence of Croceibacter atlanticus HTCC2559(T), which was isolated by high-throughput dilution-to-extinction culturing from the Bermuda Atlantic Time Series station in the Western Sargasso Sea. Strain HTCC2559(T) contained genes for carotenoid biosynthesis, flavonoid biosynthesis, and several macromolecule-degrading enzymes. The genome confirmed physiological observations of cultivated Croceibacter atlanticus strain HTCC2559(T), which identified it as an obligate chemoheterotroph.

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Roles of minor pilin subunits Spy0125 and Spy0130 in the serotype M1 Streptococcus pyogenes strain SF370.

Publication Date: 2010 Sep PMID: 20639332
Authors: Smith, W. D. - Pointon, J. A. - Abbot, E. - Kang, H. J. - Baker, E. N. - Hirst, B. H. - Wilson, J. A. - Banfield, M. J. - Kehoe, M. A.
Journal: J Bacteriol

Adhesive pili on the surface of the serotype M1 Streptococcus pyogenes strain SF370 are composed of a major backbone subunit (Spy0128) and two minor subunits (Spy0125 and Spy0130), joined covalently by a pilin polymerase (Spy0129). Previous studies using recombinant proteins showed that both minor subunits bind to human pharyngeal (Detroit) cells (A. G. Manetti et al., Mol. Microbiol. 64:968-983, 2007), suggesting both may act as pilus-presented adhesins. While confirming these binding properties, studies described here indicate that Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role as a wall linker. Pili were localized predominantly to cell wall fractions of the wild-type S. pyogenes parent strain and a spy0125 deletion mutant. In contrast, they were found almost exclusively in culture supernatants in both spy0130 and srtA deletion mutants, indicating that the housekeeping sortase (SrtA) attaches pili to the cell wall by using Spy0130 as a linker protein. Adhesion assays with antisera specific for individual subunits showed that only anti-rSpy0125 serum inhibited adhesion of wild-type S. pyogenes to human keratinocytes and tonsil epithelium to a significant extent. Spy0125 was localized to the tip of pili, based on a combination of mutant analysis and liquid chromatography-tandem mass spectrometry analysis of purified pili. Assays comparing parent and mutant strains confirmed its role as the adhesin. Unexpectedly, apparent spontaneous cleavage of a labile, proline-rich (8 of 14 residues) sequence separating the N-terminal approximately 1/3 and C-terminal approximately 2/3 of Spy0125 leads to loss of the N-terminal region, but analysis of internal spy0125 deletion mutants confirmed that this has no significant effect on adhesion.

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Regulation of ciaXRH operon expression and identification of the CiaR regulon in Streptococcus mutans.

Publication Date: 2010 Sep PMID: 20639331
Authors: Wu, C. - Ayala, E. A. - Downey, J. S. - Merritt, J. - Goodman, S. D. - Qi, F.
Journal: J Bacteriol

The ciaRH operon in Streptococcus mutans contains 3 contiguous genes, ciaXRH. Unlike the CiaRH system in other streptococci, only the ciaH-null mutant displays defective phenotypes, while the ciaR-null mutant behaves like the wild type. The objective of this study was to determine the mechanism of this unusual property. We demonstrate that the ciaH mutation caused a >20-fold increase in ciaR transcript synthesis. A ciaRH double deletion reversed the ciaH phenotype, suggesting that overexpressed ciaR might be responsible for the observed ciaH phenotypes. When ciaR was forced to be overexpressed by a transcriptional fusion to the ldh promoter in the wild-type background, the same ciaH phenotypes were restored, confirming the involvement of overexpressed ciaR in the ciaH phenotypes. The ciaH mutation and ciaR overexpression also caused transcriptional alterations in 100 genes, with 15 genes upregulated >5-fold. Bioinformatics analysis identified a putative CiaR regulon consisting of 8 genes/operons, including the ciaXRH operon itself, all of which were upregulated. In vitro footprinting on 4 of the 8 promoters revealed a protected region of 26 to 28 bp encompassing two direct repeats, NTTAAG-n5-WTTAAG, 10 bp upstream of the -10 region, indicating direct binding of the CiaR protein to these promoters. Taken together, we conclude that overexpressed CiaR, as a result of either ciaH deletion or forced expression from a constitutive promoter, is a mediator in the CiaH-regulated phenotypes.

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Massive gene duplication event among clinical isolates of the Mycobacterium tuberculosis W/Beijing family.

Publication Date: 2010 Sep PMID: 20639330
Authors: Domenech, P. - Kolly, G. S. - Leon-Solis, L. - Fallow, A. - Reed, M. B.
Journal: J Bacteriol

As part of our effort to uncover the molecular basis for the phenotypic variation among clinical Mycobacterium tuberculosis isolates, we have previously reported that isolates belonging to the W/Beijing lineage constitutively overexpress the DosR-regulated transcriptional program. While generating dosR knockouts in two independent W/Beijing sublineages, we were surprised to discover that they possess two copies of dosR. This dosR amplification is part of a massive genomic duplication spanning 350 kb and encompassing >300 genes. In total, this equates to 8% of the genome being present as two copies. The presence of IS6110 elements at both ends of the region of duplication, and in the novel junction region, suggests that it arose through unequal homologous recombination of sister chromatids at the IS6110 sequences. Analysis of isolates representing the major M. tuberculosis lineages has revealed that the 350-kb duplication is restricted to the most recently evolved sublineages of the W/Beijing family. Within these isolates, the duplication is partly responsible for the constitutive dosR overexpression phenotype. Although the nature of the selection event giving rise to the duplication remains unresolved, its evolution is almost certainly the result of specific selective pressure(s) encountered inside the host. A preliminary in vitro screen has failed to reveal a role of the duplication in conferring resistance to common antitubercular drugs, a trait frequently associated with W/Beijing isolates. Nevertheless, this first description of a genetic remodeling event of this nature for M. tuberculosis further highlights the potential for the evolution of diversity in this important global pathogen.

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Genome sequence of Fulvimarina pelagi HTCC2506T, a Mn(II)-oxidizing alphaproteobacterium possessing an aerobic anoxygenic photosynthetic gene cluster and Xanthorhodopsin.

Publication Date: 2010 Sep PMID: 20639329
Authors: Kang, I. - Oh, H. M. - Lim, S. I. - Ferriera, S. - Giovannoni, S. J. - Cho, J. C.
Journal: J Bacteriol

Fulvimarina pelagi is a Mn(II)-oxidizing marine heterotrophic bacterium in the order Rhizobiales. Here we announce the draft genome sequence of F. pelagi HTCC2506(T), which was isolated from the Sargasso Sea by using dilution-to-extinction culturing. The genome sequence contained a xanthorhodopsin gene as well as a photosynthetic gene cluster, which suggests the coexistence of two different phototrophic mechanisms in a single microorganism.

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The paralogous MarR/DUF24-family repressors YodB and CatR control expression of the catechol dioxygenase CatE in Bacillus subtilis.

Publication Date: 2010 Sep PMID: 20639328
Authors: Chi, B. K. - Kobayashi, K. - Albrecht, D. - Hecker, M. - Antelmann, H.
Journal: J Bacteriol

The redox-sensing MarR/DUF24-type repressor YodB controls expression of the azoreductase AzoR1 and the nitroreductase YodC that are involved in detoxification of quinones and diamide in Bacillus subtilis. In the present paper, we identified YodB and its paralog YvaP (CatR) as repressors of the yfiDE (catDE) operon encoding a catechol-2,3-dioxygenase that also contributes to quinone resistance. Inactivation of both CatR and YodB is required for full derepression of catDE transcription. DNA-binding assays and promoter mutagenesis studies showed that CatR protects two inverted repeats with the consensus sequence TTAC-N(5)-GTAA overlapping the -35 promoter region (BS1) and the transcriptional start site (TSS) (BS2). The BS1 operator was required for binding of YodB in vitro. CatR and YodB share the conserved N-terminal Cys residue, which is required for redox sensing of CatR in vivo as shown by Cys-to-Ser mutagenesis. Our data suggest that CatR is modified by intermolecular disulfide formation in response to diamide and quinones in vitro and in vivo. Redox regulation of CatR occurs independently of YodB, and no protein interaction was detected between CatR and YodB in vivo using protein cross-linking and mass spectrometry.

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Complete genome sequence of the diesel-degrading Acinetobacter sp. strain DR1.

Publication Date: 2010 Sep PMID: 20639327
Authors: Jung, J. - Baek, J. H. - Park, W.
Journal: J Bacteriol

The genus Acinetobacter is ubiquitous in soil, aquatic, and sediment environments and includes pathogenic strains, such as A. baumannii. Many Acinetobacter species isolated from various environments have biotechnological potential since they are capable of degrading a variety of pollutants. Acinetobacter sp. strain DR1 has been identified as a diesel degrader. Here we report the complete genome sequence of Acinetobacter sp. DR1 isolated from the soil of a rice paddy.

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Pmr, a histone-like protein H1 (H-NS) family protein encoded by the IncP-7 plasmid pCAR1, is a key global regulator that alters host function.

Publication Date: 2010 Sep PMID: 20639326
Authors: Yun, C. S. - Suzuki, C. - Naito, K. - Takeda, T. - Takahashi, Y. - Sai, F. - Terabayashi, T. - Miyakoshi, M. - Shintani, M. - Nishida, H. - Yamane, H. - Nojiri, H.
Journal: J Bacteriol

Histone-like protein H1 (H-NS) family proteins are nucleoid-associated proteins (NAPs) conserved among many bacterial species. The IncP-7 plasmid pCAR1 is transmissible among various Pseudomonas strains and carries a gene encoding the H-NS family protein, Pmr. Pseudomonas putida KT2440 is a host of pCAR1, which harbors five genes encoding the H-NS family proteins PP_1366 (TurA), PP_3765 (TurB), PP_0017 (TurC), PP_3693 (TurD), and PP_2947 (TurE). Quantitative reverse transcription-PCR (qRT-PCR) demonstrated that the presence of pCAR1 does not affect the transcription of these five genes and that only pmr, turA, and turB were primarily transcribed in KT2440(pCAR1). In vitro pull-down assays revealed that Pmr strongly interacted with itself and with TurA, TurB, and TurE. Transcriptome comparisons of the pmr disruptant, KT2440, and KT2440(pCAR1) strains indicated that pmr disruption had greater effects on the host transcriptome than did pCAR1 carriage. The transcriptional levels of some genes that increased with pCAR1 carriage, such as the mexEF-oprN efflux pump genes and parI, reverted with pmr disruption to levels in pCAR1-free KT2440. Transcriptional levels of putative horizontally acquired host genes were not altered by pCAR1 carriage but were altered by pmr disruption. Identification of genome-wide Pmr binding sites by ChAP-chip (chromatin affinity purification coupled with high-density tiling chip) analysis demonstrated that Pmr preferentially binds to horizontally acquired DNA regions. The Pmr binding sites overlapped well with the location of the genes differentially transcribed following pmr disruption on both the plasmid and the chromosome. Our findings indicate that Pmr is a key factor in optimizing gene transcription on pCAR1 and the host chromosome.

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A putrescine-inducible pathway comprising PuuE-YneI in which gamma-aminobutyrate is degraded into succinate in Escherichia coli K-12.

Publication Date: 2010 Sep PMID: 20639325
Authors: Kurihara, S. - Kato, K. - Asada, K. - Kumagai, H. - Suzuki, H.
Journal: J Bacteriol

Gamma-aminobutyrate (GABA) is metabolized to succinic semialdehyde by GABA aminotransferase (GABA-AT), and the succinic semialdehyde is subsequently oxidized to succinate by succinic semialdehyde dehydrogenase (SSADH). In Escherichia coli, there are duplicate GABA-ATs (GabT and PuuE) and duplicate SSADHs (GabD and YneI). While GabT and GabD have been well studied previously, the characterization and expression analysis of PuuE and YneI are yet to be investigated. By analyzing the amino acid profiles in cells of DeltapuuE and/or DeltagabT mutants, this study demonstrated that PuuE plays an important role in GABA metabolism in E. coli cells. The similarity of the amino acid sequences of PuuE and GabT is 67.4%, and it was biochemically demonstrated that the catalytic center of GabT is conserved as an amino acid residue important for the enzymatic activity in PuuE as Lys-247. However, the regulation of expression of PuuE is significantly different from that of GabT. PuuE is induced by the addition of putrescine to the medium and is repressed by succinate and low aeration conditions; in contrast, GabT is almost constitutive. Similarly, YneI is induced by putrescine, while GabD is not. For E. coli, PuuE is important for utilization of putrescine as a sole nitrogen source and both PuuE and YneI are important for utilization of putrescine as a sole carbon source. The results demonstrate that the PuuE-YneI pathway was a putrescine-inducible GABA degradation pathway for utilizing putrescine as a nutrient source.

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The ABC transporter HrtAB confers resistance to hemin toxicity and is regulated in a hemin-dependent manner by the ChrAS two-component system in Corynebacterium diphtheriae.

Publication Date: 2010 Sep PMID: 20639324
Authors: Bibb, L. A. - Schmitt, M. P.
Journal: J Bacteriol

Corynebacterium diphtheriae, the causative agent of the severe respiratory disease diphtheria, utilizes hemin and hemoglobin as iron sources for growth in iron-depleted environments. Because of the toxicity of high levels of hemin and iron, these compounds are often tightly regulated in bacterial systems. In this report, we identify and characterize the C. diphtheriae hrtAB genes, which encode a putative ABC type transporter involved in conferring resistance to the toxic effects of hemin. Deletion of the hrtAB genes in C. diphtheriae produced increased sensitivity to hemin, which was complemented by a plasmid harboring the cloned hrtAB locus. The HrtAB system was not involved in the uptake and use of hemin as an iron source. The hrtAB genes are located on the C. diphtheriae genome upstream from the chrSA operon, which encodes a previously characterized two-component signal transduction system that regulates gene expression in a heme-dependent manner. The hrtB promoter is activated by the ChrAS system in the presence of hemin or hemoglobin, and mutations in the chrSA genes abolish heme-activated expression from the hrtB promoter. It was also observed that transcription from the hrtB promoter is reduced in a dtxR deletion mutant, suggesting that DtxR is required for optimal expression of hrtAB. Previous studies proposed that the ChrS sensor kinase may be responsive to an environmental signal, such as hemin. We show that specific point mutations in the ChrS N-terminal transmembrane domain result in a reduced ability to activate the hrtB promoter in the presence of a heme source, suggesting that this putative sensor region is essential for the detection of a signal produced in response to hemin exposure. This study shows that the HrtAB system is required for protection from hemin toxicity and that expression of the hrtAB genes is regulated by the ChrAS two-component system. This study demonstrates a direct correlation between the detection of heme or a heme-associated signal by the N-terminal sensor domain of ChrS and the transcriptional activation of the hrtAB genes.

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H-NS silences gfp, the green fluorescent protein gene: gfpTCD is a genetically Remastered gfp gene with reduced susceptibility to H-NS-mediated transcription silencing and with enhanced translation.

Publication Date: 2010 Sep PMID: 20639321
Authors: Corcoran, C. P. - Cameron, A. D. - Dorman, C. J.
Journal: J Bacteriol

The bacterial nucleoid-associated protein H-NS, which preferentially targets and silences A+T-rich genes, binds the ubiquitous reporter gene gfp and dramatically reduces local transcription. We have redesigned gfp to reduce H-NS-mediated transcription silencing and simultaneously improve translation in vivo without altering the amino acid sequence of the GFP protein.

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Posttranscriptional regulation of glutamine synthetase in the filamentous Cyanobacterium Anabaena sp. PCC 7120: differential expression between vegetative cells and heterocysts.

Publication Date: 2010 Sep PMID: 20639319
Authors: Galmozzi, C. V. - Saelices, L. - Florencio, F. J. - Muro-Pastor, M. I.
Journal: J Bacteriol

Genes homologous to those implicated in glutamine synthetase (GS) regulation by protein-protein interaction in the cyanobacterium Synechocystis sp. strain PCC 6803 are conserved in several cyanobacterial sequenced genomes. We investigated this GS regulatory mechanism in Anabaena sp. strain PCC 7120. In this strain the system operates with only one GS inactivation factor (inactivation factor 7A [IF7A]), encoded by open reading frame (ORF) asl2329 (gifA). Following addition of ammonium, expression of gifA is derepressed, leading to the synthesis of IF7A, and consequently, GS is inactivated. Upon ammonium removal, the GS activity returns to the initial level and IF7A becomes undetectable. The global nitrogen control protein NtcA binds to the gifA promoter. Constitutive high expression levels of gifA were found in an Anabaena ntcA mutant (CSE2), indicating a repressor role for NtcA. In vitro studies demonstrate that Anabaena GS is not inactivated by Synechocystis IFs (IF7 and IF17), indicating the specificity of the system. We constructed an Anabaena strain expressing a second inactivating factor, containing the amino-terminal part of IF17 from Synechocystis fused to IF7A. GS inactivation in this strain is more effective than that in the wild type (WT) and resembles that observed in Synechocystis. Finally we found differential expression of the IF system between heterocysts and vegetative cells of Anabaena.

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Multiple promoters contribute to swarming and the coordination of transcription with flagellar assembly in Salmonella.

Publication Date: 2010 Sep PMID: 20639318
Authors: Wozniak, C. E. - Chevance, F. F. - Hughes, K. T.
Journal: J Bacteriol

In Salmonella, there are three classes of promoters in the flagellar transcriptional hierarchy. This organization allows genes needed earlier in the construction of flagella to be transcribed before genes needed later. Four operons (fliAZY, flgMN, fliDST, and flgKL) are expressed from both class 2 and class 3 promoters. To investigate the purpose for expressing genes from multiple flagellar promoters, mutants were constructed for each operon that were defective in either class 2 transcription or class 3 transcription. The mutants were checked for defects in swimming through liquids, swarming over surfaces, and transcriptional regulation. The expression of the hook-associated proteins (FlgK, FlgL, and FliD) from class 3 promoters was found to be important for swarming motility. Both flgMN promoters were involved in coordinating class 3 transcription with the stage of assembly of the hook-basal body. Finally, the fliAZY class 3 promoter lowered class 3 transcription in stationary phase. These results indicate that the multiple flagellar promoters respond to specific environmental conditions and help coordinate transcription with flagellar assembly.

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ArgR-regulated genes are derepressed in the Legionella-containing vacuole.

Publication Date: 2010 Sep PMID: 20622069
Authors: Hovel-Miner, G. - Faucher, S. P. - Charpentier, X. - Shuman, H. A.
Journal: J Bacteriol

Legionella pneumophila is an intracellular pathogen that infects protozoa in aquatic environments and when inhaled by susceptible human hosts replicates in alveolar macrophages and can result in the often fatal pneumonia called Legionnaires' disease. The ability of L. pneumophila to replicate within host cells requires the establishment of a specialized compartment that evades normal phagolysosome fusion called the Legionella-containing vacuole (LCV). Elucidation of the biochemical composition of the LCV and the identification of the regulatory signals sensed during intracellular replication are inherently challenging. L-Arginine is a critical nutrient in the metabolism of both prokaryotic and eukaryotic organisms. We showed that the L. pneumophila arginine repressor homolog, ArgR, is required for maximal intracellular growth in the unicellular host Acanthamoeba castellanii. In this study, we present evidence that the concentration of L-arginine in the LCV is sensed by ArgR to produce an intracellular transcriptional response. We characterized the L. pneumophila ArgR regulon by global gene expression analysis, identified genes highly affected by ArgR, showed that ArgR repression is dependent upon the presence of L-arginine, and demonstrated that ArgR-regulated genes are derepressed during intracellular growth. Additional targets of ArgR that may account for the argR mutant's intracellular multiplication defect are discussed. These results suggest that L-arginine availability functions as a regulatory signal during Legionella intracellular growth.

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Functional identification of the Proteus mirabilis core lipopolysaccharide biosynthesis genes.

Publication Date: 2010 Sep PMID: 20622068
Authors: Aquilini, E. - Azevedo, J. - Jimenez, N. - Bouamama, L. - Tomas, J. M. - Regue, M.
Journal: J Bacteriol

In this study, we report the identification of genes required for the biosynthesis of the core lipopolysaccharides (LPSs) of two strains of Proteus mirabilis. Since P. mirabilis and Klebsiella pneumoniae share a core LPS carbohydrate backbone extending up to the second outer-core residue, the functions of the common P. mirabilis genes was elucidated by genetic complementation studies using well-defined mutants of K. pneumoniae. The functions of strain-specific outer-core genes were identified by using as surrogate acceptors LPSs from two well-defined K. pneumoniae core LPS mutants. This approach allowed the identification of two new heptosyltransferases (WamA and WamC), a galactosyltransferase (WamB), and an N-acetylglucosaminyltransferase (WamD). In both strains, most of these genes were found in the so-called waa gene cluster, although one common core biosynthetic gene (wabO) was found outside this cluster.

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Systems-level metabolic flux profiling elucidates a complete, bifurcated tricarboxylic acid cycle in Clostridium acetobutylicum.

Publication Date: 2010 Sep PMID: 20622067
Authors: Amador-Noguez, D. - Feng, X. J. - Fan, J. - Roquet, N. - Rabitz, H. - Rabinowitz, J. D.
Journal: J Bacteriol

Obligatory anaerobic bacteria are major contributors to the overall metabolism of soil and the human gut. The metabolic pathways of these bacteria remain, however, poorly understood. Using isotope tracers, mass spectrometry, and quantitative flux modeling, here we directly map the metabolic pathways of Clostridium acetobutylicum, a soil bacterium whose major fermentation products include the biofuels butanol and hydrogen. While genome annotation suggests the absence of most tricarboxylic acid (TCA) cycle enzymes, our results demonstrate that this bacterium has a complete, albeit bifurcated, TCA cycle; oxaloacetate flows to succinate both through citrate/alpha-ketoglutarate and via malate/fumarate. Our investigations also yielded insights into the pathways utilized for glucose catabolism and amino acid biosynthesis and revealed that the organism's one-carbon metabolism is distinct from that of model microbes, involving reversible pyruvate decarboxylation and the use of pyruvate as the one-carbon donor for biosynthetic reactions. This study represents the first in vivo characterization of the TCA cycle and central metabolism of C. acetobutylicum. Our results establish a role for the full TCA cycle in an obligatory anaerobic organism and demonstrate the importance of complementing genome annotation with isotope tracer studies for determining the metabolic pathways of diverse microbes.

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Localization and cellular amounts of the WalRKJ (VicRKX) two-component regulatory system proteins in serotype 2 Streptococcus pneumoniae.

Publication Date: 2010 Sep PMID: 20622066
Authors: Wayne, K. J. - Sham, L. T. - Tsui, H. C. - Gutu, A. D. - Barendt, S. M. - Keen, S. K. - Winkler, M. E.
Journal: J Bacteriol

The WalRK two-component regulatory system coordinates gene expression that maintains cell wall homeostasis and responds to antibiotic stress in low-GC Gram-positive bacteria. Phosphorylated WalR (VicR) of the major human respiratory pathogen Streptococcus pneumoniae (WalR(Spn)) positively regulates transcription of several surface virulence genes and, most critically, pcsB, which encodes an essential cell division protein. Despite numerous studies of several species, little is known about the signals sensed by the WalK histidine kinase or the function of the WalJ ancillary protein encoded in the walRK(Spn) operon. To better understand the functions of the WalRKJ(Spn) proteins in S. pneumoniae, we performed experiments to determine their cellular localization and amounts. In contrast to WalK from Bacillus subtilis (WalK(Bsu)), which is localized at division septa, immunofluorescence microscopy showed that WalK(Spn) is distributed throughout the cell periphery. WalJ(Spn) is also localized to the cell surface periphery, whereas WalR(Spn) was found to be localized in the cytoplasm around the nucleoid. In fractionation experiments, WalR(Spn) was recovered from the cytoplasmic fraction, while WalK(Spn) and the majority of WalJ(Spn) were recovered from the cell membrane fraction. This fractionation is consistent with the localization patterns observed. Lastly, we determined the cellular amounts of WalRKJ(Spn) by quantitative Western blotting. The WalR(Spn) response regulator is relatively abundant and present at levels of approximately 6,200 monomers per cell, which are approximately 14-fold greater than the amount of the WalK(Spn) histidine kinase, which is present at approximately 460 dimers (920 monomers) per cell. We detected approximately 1,200 monomers per cell of WalJ(Spn) ancillary protein, similar to the amount of WalK(Spn).

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Overlapping repressor binding sites result in additive regulation of Escherichia coli FadH by FadR and ArcA.

Publication Date: 2010 Sep PMID: 20622065
Authors: Feng, Y. - Cronan, J. E.
Journal: J Bacteriol

Escherichia coli fadH encodes a 2,4-dienoyl reductase that plays an auxiliary role in beta-oxidation of certain unsaturated fatty acids. In the 2 decades since its discovery, FadH biochemistry has been studied extensively. However, the genetic regulation of FadH has been explored only partially. Here we report mapping of the fadH promoter and document its complex regulation by three independent regulators, the fatty acid degradation FadR repressor, the oxygen-responsive ArcA-ArcB two-component system, and the cyclic AMP receptor protein-cyclic AMP (CRP-cAMP) complex. Electrophoretic mobility shift assays demonstrated that FadR binds to the fadH promoter region and that this binding can be specifically reversed by long-chain acyl-coenzyme A (CoA) thioesters. In vivo data combining transcriptional lacZ fusion and real-time quantitative PCR (qPCR) analyses indicated that fadH is strongly repressed by FadR, in agreement with induction of fadH by long-chain fatty acids. Inactivation of arcA increased fadH transcription by >3-fold under anaerobic conditions. Moreover, fadH expression was increased 8- to 10-fold under anaerobic conditions upon deletion of both the fadR and the arcA gene, indicating that anaerobic expression is additively repressed by FadR and ArcA-ArcB. Unlike fadM, a newly reported member of the E. coli fad regulon that encodes another auxiliary beta-oxidation enzyme, fadH was activated by the CRP-cAMP complex in a manner similar to those of the prototypical fad genes. In the absence of the CRP-cAMP complex, repression of fadH expression by both FadR and ArcA-ArcB was very weak, suggesting a possible interplay with other DNA binding proteins.

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Molecular characterization of GrlA, a specific positive regulator of ler expression in enteropathogenic Escherichia coli.

Publication Date: 2010 Sep PMID: 20622062
Authors: Jimenez, R. - Cruz-Migoni, S. B. - Huerta-Saquero, A. - Bustamante, V. H. - Puente, J. L.
Journal: J Bacteriol

Enteropathogenic Escherichia coli (EPEC) infections are characterized by the formation of attaching and effacing (A/E) lesions on the surfaces of infected epithelial cells. The genes required for the formation of A/E lesions are located within the locus of enterocyte effacement (LEE). Ler is the key regulatory factor controlling the expression of LEE genes. Expression of the ler gene is positively regulated by GrlA, which is encoded by the LEE. Here, we analyze the mechanism by which GrlA positively regulates ler expression and show that in the absence of H-NS, GrlA is no longer essential for ler activation, further confirming that GrlA acts in part as an H-NS antagonist on the ler promoter. Single-amino-acid mutants were constructed to test the functional significance of the putative helix-turn-helix (HTH) DNA binding motif found in the N-terminal half of GrlA, as well as at the C-terminal domain of the protein. Several mutations within the HTH motif, but not all, completely abolished GrlA activity, as well as specific binding to its target sequence downstream from position -54 in the ler regulatory region. Some of these mutants, albeit inactive, were still able to interact with the negative regulator GrlR, indicating that loss of activity was not a consequence of protein misfolding. Additional residues in the vicinity of the HTH domain, as well as at the end of the protein, were also shown to be important for GrlA activity as a transcriptional regulator, but not for its interaction with GrlR. In summary, GrlA consists of at least two functional domains, one involved in transcriptional activation and DNA binding and the other in heterodimerization with GrlR.

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Identification and characterization of a phosphodiesterase that inversely regulates motility and biofilm formation in Vibrio cholerae.

Publication Date: 2010 Sep PMID: 20622061
Authors: Liu, X. - Beyhan, S. - Lim, B. - Linington, R. G. - Yildiz, F. H.
Journal: J Bacteriol

Vibrio cholerae switches between free-living motile and surface-attached sessile lifestyles. Cyclic diguanylate (c-di-GMP) is a signaling molecule controlling such lifestyle changes. C-di-GMP is synthesized by diguanylate cyclases (DGCs) that contain a GGDEF domain and is degraded by phosphodiesterases (PDEs) that contain an EAL or HD-GYP domain. We constructed in-frame deletions of all V. cholerae genes encoding proteins with GGDEF and/or EAL domains and screened mutants for altered motility phenotypes. Of 52 mutants tested, four mutants exhibited an increase in motility, while three mutants exhibited a decrease in motility. We further characterized one mutant lacking VC0137 (cdgJ), which encodes an EAL domain protein. Cellular c-di-GMP quantifications and in vitro enzymatic activity assays revealed that CdgJ functions as a PDE. The cdgJ mutant had reduced motility and exhibited a small decrease in flaA expression; however, it was able to produce a flagellum. This mutant had enhanced biofilm formation and vps gene expression compared to that of the wild type, indicating that CdgJ inversely regulates motility and biofilm formation. Genetic interaction analysis revealed that at least four DGCs, together with CdgJ, control motility in V. cholerae.

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Transcriptional regulation of the heterocyst patterning gene patA from Anabaena sp. strain PCC 7120.

Publication Date: 2010 Sep PMID: 20622060
Authors: Young-Robbins, S. S. - Risser, D. D. - Moran, J. R. - Haselkorn, R. - Callahan, S. M.
Journal: J Bacteriol

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 forms a periodic pattern of nitrogen-fixing heterocysts when grown in the absence of combined nitrogen. PatA is necessary for proper patterning of heterocysts along filaments. In this study, apparent transcriptional start points (tsps) were identified at nucleotides -305, -614, and -645 relative to the translational start site (-305, -614, and -645 tsps). Transcriptional reporter fusions were used to show that transcription from the -305 tsp was induced in all cells of filaments in response to nitrogen deprivation, required hetR for induction, and increased in a patA mutant. Transcription from -614/-645 tsp reporter fusions was spatially regulated and occurred primarily in cells that would become heterocysts. Complementation of a patA mutant strain by alleles encoding substitutions in, or deletion of, the putative phosphoacceptor C-terminal domain indicates that the PATAN domain can function independently of the C-terminal domain of PatA. Localization of a ring of PatA-GFP at sites of cell division, as well as the formation of enlarged cells with altered cell morphology when patA was overexpressed, suggests that PatA may participate in cell division.

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A novel heme a insertion factor gene cotranscribes with the Thermus thermophilus cytochrome ba3 oxidase locus.

Publication Date: 2010 Sep PMID: 20622059
Authors: Werner, C. - Richter, O. M. - Ludwig, B.
Journal: J Bacteriol

Studying the biogenesis of the Thermus thermophilus cytochrome ba(3) oxidase, we analyze heme a cofactor insertion into this membrane protein complex. Only three proteins linked to oxidase maturation have been described for this extreme thermophile, and in particular, no evidence for a canonical Surf1 homologue, required for heme a insertion, is available from genome sequence data. Here, we characterize the product of an open reading frame, cbaX, in the operon encoding subunits of the ba(3)-type cytochrome c oxidase. CbaX shares no sequence identity with any known oxidase biogenesis factor, and CbaX homologues are found only in the Thermaceae group. In a series of cbaX deletion and complementation experiments, we demonstrate that the resulting ba(3) oxidase complexes, affinity purified via an internally inserted His tag located in subunit I, are severely affected in their enzymatic activities and heme compositions in both the low- and high-spin sites. Thus, CbaX displays typical features of a generic Surf1 factor essential for binding and positioning the heme a moiety for correct assembly into the protein scaffold of oxidase subunit I.

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Dual two-component regulatory systems are involved in aromatic compound degradation in a polychlorinated-biphenyl degrader, Rhodococcus jostii RHA1.

Publication Date: 2010 Sep PMID: 20622058
Authors: Takeda, H. - Shimodaira, J. - Yukawa, K. - Hara, N. - Kasai, D. - Miyauchi, K. - Masai, E. - Fukuda, M.
Journal: J Bacteriol

A Gram-positive polychlorinated-biphenyl (PCB) degrader, Rhodococcus jostii RHA1, degrades PCBs by cometabolism with biphenyl. A two-component BphS1T1 system encoded by bphS1 and bphT1 (formerly bphS and bphT) is responsible for the transcription induction of the five gene clusters, bphAaAbAcAdC1B1, etbAa1Ab1CbphD1, etbAa2Ab2AcD2, etbAdbphB2, and etbD1, which constitute multiple enzyme systems for biphenyl/PCB degradation. The bphS2 and bphT2 genes, which encode BphS2 and BphT2, virtually identical to BphS1 (92%) and BphT1 (97%), respectively, were characterized. BphS2T2 induced the activation of the bphAa promoter in a host, Rhodococcus erythropolis IAM1399, in the presence of a variety of aromatics, including benzene, toluene, ethylbenzene, xylenes, isopropylbenzene, and chlorinated benzenes, as effectively as BphS1T1. The substrate spectrum of BphS2T2 was the same as that of BphS1T1, except for biphenyl, which is a substrate only for BphS1T1. BphS2T2 activated transcription from the five promoters of biphenyl/PCB degradation enzyme gene clusters as effectively as BphS1T1. The targeted disruptions of the bphS1, bphS2, bphT1, and bphT2 genes indicated that all these genes are involved in the growth of RHA1 on aromatic compounds. The hybrid system with bphS1 and bphT2 and that with bphS2 and bphT1 were constructed, and both systems conducted induced activation of the bphAa promoter, indicating cross-communication. These results indicated that RHA1 employs not only multiple enzyme systems, but also dual regulatory systems for biphenyl/PCB degradation. Comparison of the sequences, including bphS2T2, with the bphS1T1-containing sequences and the corresponding sequences in other rhodococcal degraders suggests that bphS2T2 might have originated from bphS1T1.

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Genome sequence of the oligotrophic marine Gammaproteobacterium HTCC2143, isolated from the Oregon Coast.

Publication Date: 2010 Sep PMID: 20601481
Authors: Oh, H. M. - Kang, I. - Ferriera, S. - Giovannoni, S. J. - Cho, J. C.
Journal: J Bacteriol

Strain HTCC2143 was isolated from Oregon Coast surface waters using dilution-to-extinction culturing. Here we present the genome of strain HTCC2143 from the BD1-7 clade of the oligotrophic marine Gammaproteobacteria group. The genome of HTCC2143 contains genes for carotenoid biosynthesis and proteorhodopsin and for proteins that have potential biotechnological significance: epoxide hydrolases, Baeyer-Villiger monooxygenases, and polyketide synthases.

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Complete genome sequence of Halalkalicoccus jeotgali B3(T), an extremely halophilic archaeon.

Publication Date: 2010 Sep PMID: 20601480
Authors: Roh, S. W. - Nam, Y. D. - Nam, S. H. - Choi, S. H. - Park, H. S. - Bae, J. W.
Journal: J Bacteriol

Halalkalicoccus jeotgali B3(T), isolated from salt-fermented seafood from South Korea, is an extremely halophilic archaeon belonging to the family Halobacteriaceae. Here, we present the complete genome sequence of the type strain H. jeotgali B3(T) (3,698,650 bp, with a G+C content of 62.5%), which consists of one chromosome and six plasmids. This is the first complete genome sequence of the Halalkalicoccus species.

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Expression of tropodithietic acid biosynthesis is controlled by a novel autoinducer.

Publication Date: 2010 Sep PMID: 20601479
Authors: Geng, H. - Belas, R.
Journal: J Bacteriol

The interactions between marine prokaryotic and eukaryotic microorganisms are crucial to many biological and biogeochemical processes in the oceans. Often the interactions are mutualistic, as in the symbiosis between phytoplankton, e.g., the dinoflagellate Pfiesteria piscicida and Silicibacter sp. TM1040, a member of the Roseobacter taxonomic lineage. It is hypothesized that an important component of this symbiosis is bacterial production of tropodithietic acid (TDA), a biologically active tropolone compound whose synthesis requires the expression of tdaABCDEF (tdaA-F), as well as six additional genes (cysI, malY, paaIJK, and tdaH). The factors controlling tda gene expression are not known, although growth in laboratory standing liquid cultures drastically increases TDA levels. In this report, we measured the transcription of tda genes to gain a greater understanding of the factors controlling their expression. While the expression of tdaAB was constitutive, tdaCDE and tdaF mRNA increased significantly (3.7- and 17.4-fold, respectively) when cells were grown in standing liquid broth compared to their levels with shaking liquid culturing. No transcription of tdaC was detected when a tdaCp::lacZ transcriptional fusion was placed in 11 of the 12 Tda(-) mutant backgrounds, with cysI being the sole exception. The expression of tdaC could be restored to 9 of the remaining 11 Tda(-) mutants-tdaA and tdaH failed to respond-by placing wild-type (Tda(+)) strains in close proximity or by supplying exogenous TDA to the mutant, suggesting that TDA induces tda gene expression. These results indicate that TDA acts as an autoinducer of its own synthesis and suggest that roseobacters may use TDA as a quorum signal.

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Analysis of the role of the type III effector inventory of Pseudomonas syringae pv. phaseolicola 1448a in interaction with the plant.

Publication Date: 2010 Sep PMID: 20601478
Authors: Zumaquero, A. - Macho, A. P. - Rufian, J. S. - Beuzon, C. R.
Journal: J Bacteriol

In Pseudomonas syringae, the type III secretion system (T3SS) is essential for disease in compatible hosts and for eliciting the hypersensitive response in incompatible hosts. P. syringae pathovars secrete a variable number of type III effectors that form their secretomes. The secretome of Pseudomonas syringae pv. phaseolicola 1448a (Pph1448a) currently includes 22 experimentally validated effectors, one HrpL-regulated candidate for which translocation results have been inconsistent, two translocated candidates for which in planta expression has not been established, one bioinformatically identified candidate, and six candidates that have been experimentally discarded. We analyzed the translocation and/or expression of these and other candidates to complete the Pph1448a effector inventory, bringing this inventory to 27 bona fide effectors, including a new one that does not belong to any of the previously described effector families. We developed a simple process for rapidly making single and double knockout mutants and apply it to the generation of an effector mutant collection that includes single knockouts for the majority of the Pph1448a effector inventory. We also generated two double mutant strains containing effectors with potentially redundant functions and analyzed the virulence of the single and double mutant strains as well as strains expressing each of the effectors from a plasmid. We demonstrate that AvrB4-1 and AvrB4-2, as well as HopW1-1 and HopW1-2, are fully redundant and contribute to virulence in bean plants, thus validating this approach for dissecting the contribution of the Pph1448a type III effector inventory to virulence. We also analyzed the effect that the expression of these four effectors from Pseudomonas syringae pv. tomato DC3000 (PtoDC3000) has during its interaction with Arabidopsis thaliana, establishing that AvrB4-1, but not the others, determines a restriction of bacterial growth that takes place mostly independently of the salicylic acid (SA)-signaling pathway.

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Structural study of the Serratia entomophila antifeeding prophage: three-dimensional structure of the helical sheath.

Publication Date: 2010 Sep PMID: 20601477
Authors: Sen, A. - Rybakova, D. - Hurst, M. R. - Mitra, A. K.
Journal: J Bacteriol

The sheath of the Serratia entomophila antifeeding prophage, which is pathogenic to the New Zealand grass grub Costelytra zealandica, is a 3-fold helix formed by a 4-fold symmetric repeating motif disposed around a helical inner tube. This structure, determined by electron microscopy and image processing, is distinct from that of the other known morphologically similar bacteriophage sheaths.

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A monoacylglycerol lipase from Mycobacterium smegmatis Involved in bacterial cell interaction.

Publication Date: 2010 Sep PMID: 20601476
Authors: Dhouib, R. - Laval, F. - Carriere, F. - Daffe, M. - Canaan, S.
Journal: J Bacteriol

MSMEG_0220 from Mycobacterium smegmatis, the ortholog of the Rv0183 gene from M. tuberculosis, recently identified and characterized as encoding a monoacylglycerol lipase, was cloned and expressed in Escherichia coli. The recombinant protein (rMSMEG_0220), which exhibits 68% amino acid sequence identity with Rv0183, showed the same substrate specificity and similar patterns of pH-dependent activity and stability as the M. tuberculosis enzyme. rMSMEG_0220 was found to hydrolyze long-chain monoacylglycerol with a specific activity of 143 +/- 6 U mg(-1). Like Rv0183 in M. tuberculosis, MSMEG_0220 was found to be located in the cell wall. To assess the in vivo role of the homologous proteins, an MSMEG_0220 disrupted mutant of M. smegmatis (MsDelta0220) was produced. An intriguing change in the colony morphology and in the cell interaction, which were partly restored in the complemented mutant containing either an active (ComMsDelta0220) or an inactive (ComMsDelta0220S111A) enzyme, was observed. Growth studies performed in media supplemented with monoolein showed that the ability of both MsDelta0220 and ComMsDelta0220S111A to grow in the presence of this lipid was impaired. Moreover, studies of the antimicrobial susceptibility of the MsDelta0220 strain showed that this mutant is more sensitive to rifampin and more resistant to isoniazid than the wild-type strain, pointing to a critical structural role of this enzyme in mycobacterial physiology, in addition to its function in the hydrolysis of exogenous lipids.

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Complete genome sequence of Beijerinckia indica subsp. indica.

Publication Date: 2010 Sep PMID: 20601475
Authors: Tamas, I. - Dedysh, S. N. - Liesack, W. - Stott, M. B. - Alam, M. - Murrell, J. C. - Dunfield, P. F.
Journal: J Bacteriol

Beijerinckia indica subsp. indica is an aerobic, acidophilic, exopolysaccharide-producing, N(2)-fixing soil bacterium. It is a generalist chemoorganotroph that is phylogenetically closely related to facultative and obligate methanotrophs of the genera Methylocella and Methylocapsa. Here we report the full genome sequence of this bacterium.

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Whole genome sequences of two Xylella fastidiosa strains (M12 and M23) causing almond leaf scorch disease in California.

Publication Date: 2010 Sep PMID: 20601474
Authors: Chen, J. - Xie, G. - Han, S. - Chertkov, O. - Sims, D. - Civerolo, E. L.
Journal: J Bacteriol

Xylella fastidiosa is a Gram-negative plant-pathogenic bacterium causing many economically important diseases, including almond leaf scorch disease (ALSD) in California. Genome information greatly facilitates research on this nutritionally fastidious organism. Here we report the complete genome sequences of two ALSD strains of this bacterium, M12 and M23.

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Central role of manganese in regulation of stress responses, physiology, and metabolism in Streptococcus pneumoniae.

Publication Date: 2010 Sep PMID: 20601473
Authors: Ogunniyi, A. D. - Mahdi, L. K. - Jennings, M. P. - McEwan, A. G. - McDevitt, C. A. - Van der Hoek, M. B. - Bagley, C. J. - Hoffmann, P. - Gould, K. A. - Paton, J. C.
Journal: J Bacteriol

The importance of Mn(2+) for pneumococcal physiology and virulence has been studied extensively. However, the specific cellular role(s) for which Mn(2+) is required are yet to be fully elucidated. Here, we analyzed the effect of Mn(2+) limitation on the transcriptome and proteome of Streptococcus pneumoniae D39. This was carried out by comparing a deletion mutant lacking the solute binding protein of the high-affinity Mn(2+) transporter, pneumococcal surface antigen A (PsaA), with its isogenic wild-type counterpart. We provide clear evidence for the Mn(2+)-dependent regulation of the expression of oxidative-stress-response enzymes SpxB and Mn(2+)-SodA and virulence-associated genes pcpA and prtA. We also demonstrate the upregulation of at least one oxidative- and nitrosative-stress-response gene cluster, comprising adhC, nmlR, and czcD, in response to Mn(2+) stress. A significant increase in 6-phosphogluconate dehydrogenase activity in the psaA mutant grown under Mn(2+)-replete conditions and upregulation of an oligopeptide ABC permease (AppDCBA) were also observed. Together, the results of transcriptomic and proteomic analyses provided evidence for Mn(2+) having a central role in activating or stimulating enzymes involved in central carbon and general metabolism. Our results also highlight the importance of high-affinity Mn(2+) transport by PsaA in pneumococcal competence, physiology, and metabolism and elucidate mechanisms underlying the response to Mn(2+) stress.

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DNA uptake sequence-mediated enhancement of transformation in Neisseria gonorrhoeae is strain dependent.

Publication Date: 2010 Sep PMID: 20601472
Authors: Duffin, P. M. - Seifert, H. S.
Journal: J Bacteriol

Natural transformation is the main means of horizontal genetic exchange in the obligate human pathogen Neisseria gonorrhoeae. Neisseria spp. have been shown to preferentially take up and transform their own DNA by recognizing the nonpalindromic 10- or 12-nucleotide sequence 5'-ATGCCGTCTGAA-3' (additional semiconserved nucleotides are underlined), termed the DNA uptake sequence (DUS10 or DUS12). Here we investigated the effects of the DUS on transformation and DNA uptake for several laboratory strains of N. gonorrhoeae. We found that all strains showed efficient transformation of DUS containing DNA (DUS10 and DUS12) but that the level of transformation with DNA lacking a DUS (DUS0) was variable in different strains. The DUS-enhanced transformation was 20-fold in two strains, FA1090 and FA19, but was approximately 150-fold in strains MS11 and 1291. All strains tested provide some level of DUS0 transformation, and DUS0 transformation was type IV pilus dependent. Competition with plasmid DNA revealed that transformation of MS11 was enhanced by the addition of excess plasmid DNA containing a DUS while FA1090 transformation was competitively inhibited. Although FA1090 was able to mediate much more efficient transformation of DNA lacking a DUS than was MS11, DNA uptake experiments showed similar levels of uptake of DNA containing and lacking a DUS in FA1090 and MS11. Finally, DNA uptake was competitively inhibited in both FA1090 and MS11. Taken together, our data indicate that the role of the DUS during DNA transformation is variable between strains of N. gonorrhoeae and may influence multiple steps during transformation.

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CrfA, a small noncoding RNA regulator of adaptation to carbon starvation in Caulobacter crescentus.

Publication Date: 2010 Sep PMID: 20601471
Authors: Landt, S. G. - Lesley, J. A. - Britos, L. - Shapiro, L.
Journal: J Bacteriol

Small noncoding regulatory RNAs (sRNAs) play a key role in the posttranscriptional regulation of many bacterial genes. The genome of Caulobacter crescentus encodes at least 31 sRNAs, and 27 of these sRNAs are of unknown function. An overexpression screen for sRNA-induced growth inhibition along with sequence conservation in a related Caulobacter species led to the identification of a novel sRNA, CrfA, that is specifically induced upon carbon starvation. Twenty-seven genes were found to be strongly activated by CrfA accumulation. One-third of these target genes encode putative TonB-dependent receptors, suggesting CrfA plays a role in the surface modification of C. crescentus, facilitating the uptake of nutrients during periods of carbon starvation. The mechanism of CrfA-mediated gene activation was investigated for one of the genes predicted to encode a TonB-dependent receptor, CC3461. CrfA functions to stabilize the CC3461 transcript. Complementarity between a region of CrfA and the terminal region of the CC3461 5'-untranslated region (5'-UTR) and also the behavior of a deletion of this region and a site-specific base substitution and a 3-base deletion in the CrfA complementary sequence suggest that CrfA binds to a stem-loop structure upstream of the CC3461 Shine-Dalgarno sequence and stabilizes the transcript.

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The gene ssl3076 encodes a protein mediating the salt-induced expression of ggpS for the biosynthesis of the compatible solute glucosylglycerol in Synechocystis sp. strain PCC 6803.

Publication Date: 2010 Sep PMID: 20601470
Authors: Klahn, S. - Hohne, A. - Simon, E. - Hagemann, M.
Journal: J Bacteriol

Acclimation to high salt concentrations involves concerted changes in gene expression. For the majority of salt-regulated genes, the mechanism underlying the induction process is not known. The gene ggpS (sll1566), which encodes the glucosylglycerol-phosphate synthase responsible for the synthesis of the compatible solute glucosylglycerol (GG), is specifically induced by salt in the cyanobacterial model strain Synechocystis sp. strain PCC 6803. To identify mechanisms mediating this salt-specific gene regulation, the ggpS promoter was analyzed in more detail. 5' rapid amplification of cDNA ends (5'-RACE) experiments revealed that the adjacent open reading frame (ORF), which is annotated as unknown protein Ssl3076, overlaps with the transcriptional start site of the ggpS gene. Reporter gene expression analyses indicated an essential role for the intact ssl3076 gene in the salt-regulated transcription of a gfp reporter gene. Promoter fragments containing a mutated ssl3076 lost the salt regulation; similarly, a frameshift mutation in ssl3076 resulted in a high level of ggpS expression under low-salt conditions, thereby establishing this small ORF, named ggpR, as a negative regulator of ggpS. Interestingly, small ORFs were also found adjacent to ggpS genes in the genomes of other GG-accumulating cyanobacteria. These results suggest that the GgpR protein represses ggpS expression under low-salt conditions, whereas in salt-shocked and salt-acclimated cells a stress-proportional ggpS expression occurs, leading to GG accumulation.

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Developments in Defining dif.

Publication Date: 2010 Sep PMID: 20601469
Authors: Campodonico, E. M. - Zusman, D. R.
Journal: J Bacteriol

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Analysis of RuvABC and RecG involvement in the escherichia coli response to the covalent topoisomerase-DNA complex.

Publication Date: 2010 Sep PMID: 20601468
Authors: Sutherland, J. H. - Tse-Dinh, Y. C.
Journal: J Bacteriol

Topoisomerases form a covalent enzyme-DNA intermediate after initial DNA cleavage. Trapping of the cleavage complex formed by type IIA topoisomerases initiates the bactericidal action of fluoroquinolones. It should be possible also to identify novel antibacterial lead compounds that act with a similar mechanism on type IA bacterial topoisomerases. The cellular response and repair pathways for trapped topoisomerase complexes remain to be fully elucidated. The RuvAB and RecG proteins could play a role in the conversion of the initial protein-DNA complex to double-strand breaks and also in the resolution of the Holliday junction during homologous recombination. Escherichia coli strains with ruvA and recG mutations are found to have increased sensitivity to low levels of norfloxacin treatment, but the mutations had more pronounced effects on survival following the accumulation of covalent complexes formed by mutant topoisomerase I defective in DNA religation. Covalent topoisomerase I and DNA gyrase complexes are converted into double-strand breaks for SOS induction by the RecBCD pathway. SOS induction following topoisomerase I complex accumulation is significantly lower in the ruvA and recG mutants than in the wild-type background, suggesting that RuvAB and RecG may play a role in converting the initial single-strand DNA-protein cleavage complex into a double-strand break prior to repair by homologous recombination. The use of a ruvB mutant proficient in homologous recombination but not in replication fork reversal demonstrated that the replication fork reversal function of RuvAB is required for SOS induction by the covalent complex formed by topoisomerase I.

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A systems biology approach to modeling vibrio cholerae gene expression under virulence-inducing conditions.

Publication Date: 2010 Sep PMID: 20601467
Authors: Kanjilal, S. - Citorik, R. - LaRocque, R. C. - Ramoni, M. F. - Calderwood, S. B.
Journal: J Bacteriol

Vibrio cholerae is a Gram-negative bacillus that is the causative agent of cholera. Pathogenesis in vivo occurs through a series of spatiotemporally controlled events under the control of a gene cascade termed the ToxR regulon. Major genes in the ToxR regulon include the master regulators toxRS and tcpPH, the downstream regulator toxT, and virulence factors, the ctxAB and tcpA operons. Our current understanding of the dynamics of virulence gene expression is limited to microarray analyses of expression at selected time points. To better understand this process, we utilized a systems biology approach to examine the temporal regulation of gene expression in El Tor V. cholerae grown under virulence-inducing conditions in vitro (AKI medium), using high-resolution time series genomic profiling. Results showed that overall gene expression in AKI medium mimics that of in vivo studies but with less clear temporal separation between upstream regulators and downstream targets. Expression of toxRS was unaffected by growth under virulence-inducing conditions, but expression of toxT was activated shortly after switching from stationary to aerating conditions. The tcpA operon was also activated early during mid-exponential-phase growth, while the ctxAB operon was turned on later, after the rise in toxT expression. Expression of ctxAB continued to rise despite an eventual decrease in toxT. Cluster analysis of gene expression highlighted 15 hypothetical genes and six genes related to environmental information processing that represent potential new members of the ToxR regulon. This study applies systems biology tools to analysis of gene expression of V. cholerae in vitro and provides an important comparator for future studies done in vivo.

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Analyzing the regulatory role of the HigA antitoxin within Mycobacterium tuberculosis.

Publication Date: 2010 Sep PMID: 20585061
Authors: Fivian-Hughes, A. S. - Davis, E. O.
Journal: J Bacteriol

Bacterial chromosomally encoded type II toxin-antitoxin (TA) loci may be involved in survival upon exposure to stress and have been linked to persistence and dormancy. Therefore, understanding the role of the numerous predicted TA loci within the human pathogen Mycobacterium tuberculosis has become a topic of great interest. Antitoxin proteins are known to autoregulate TA expression under normal growth conditions, but it is unknown whether they have a more global role in transcriptional regulation. This study focuses on analyzing the regulatory role of the M. tuberculosis HigA antitoxin. We first show that the M. tuberculosis higBA locus is functional within its native organism, as higB, higA, and Rv1957 were successfully deleted from the genome together while the deletion of higA alone was not possible. The effects of higB-Rv1957 deletion on M. tuberculosis global gene expression were investigated, and a number of potential HigA-regulated genes were identified. Transcriptional fusion and protein-DNA-binding assays were utilized to confirm the direct role of HigA in Rv1954A-Rv1957 repression, and the M. tuberculosis HigA DNA-binding motif was defined as ATATAGG(N(6))CCTATAT. As HigA failed to bind to the next-most-closely related motif within the M. tuberculosis genome, HigA may not directly regulate any other genes in addition to its own operon.

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Identification and characterization of a new ferric enterobactin receptor, CfrB, in Campylobacter.

Publication Date: 2010 Sep PMID: 20585060
Authors: Xu, F. - Zeng, X. - Haigh, R. D. - Ketley, J. M. - Lin, J.
Journal: J Bacteriol

The ferric enterobactin (FeEnt) receptor CfrA is present in the majority of Campylobacter jejuni isolates and is responsible for high-affinity iron acquisition. Our recent work and that of others strongly suggested the existence of another FeEnt uptake system in Campylobacter. Here we have identified and characterized a new FeEnt receptor (designated CfrB) using both in vitro and in vivo systems. CfrB, a homolog of C. jejuni NCTC 11168 Cj0444, shares approximately 34% of amino acid identity with CfrA. Alignment of complete CfrB sequences showed that the CfrB is highly conserved in Campylobacter. Immunoblotting analysis using CfrB-specific antiserum demonstrated that CfrB was dramatically induced under iron-restricted conditions and was produced in the majority of Campylobacter coli (41 out of 45) and in some C. jejuni (8 out of 32) primary strains from various sources and from geographically diverse areas. All of the CfrB-producing C. coli strains also produced CfrA, which was rarely observed in the tested C. jejuni strains. Isogenic cfrB, cfrA, and cfrA cfrB double mutants were constructed in 43 diverse Campylobacter strains. Growth promotion assays using these mutants demonstrated that CfrB has a major role in FeEnt iron acquisition in C. coli. Chicken colonization experiments indicated that inactivation of the cfrB gene alone greatly reduced and even abolished Campylobacter colonization of the intestines. A growth assay using CfrB-specific antiserum strongly suggested that specific CfrB antibodies could block the function of CfrB and diminish FeEnt-mediated growth promotion under iron-restricted conditions. Together, this work reveals the complexity of FeEnt systems in the two closely related Campylobacter species and demonstrates the important role of the new FeEnt receptor CfrB in Campylobacter iron acquisition and in vivo colonization.

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Relatedness of Vibrio cholerae O1/O139 isolates from patients and their household contacts, determined by multilocus variable-number tandem-repeat analysis.

Publication Date: 2010 Sep PMID: 20585059
Authors: Kendall, E. A. - Chowdhury, F. - Begum, Y. - Khan, A. I. - Li, S. - Thierer, J. H. - Bailey, J. - Kreisel, K. - Tacket, C. O. - LaRocque, R. C. - Harris, J. B. - Ryan, E. T. - Qadri, F. - Calderwood, S. B. - Stine, O. C.
Journal: J Bacteriol

The genetic relatedness of Vibrio cholerae O1/O139 isolates obtained from 100 patients and 146 of their household contacts in Dhaka, Bangladesh, between 2002 and 2005 was assessed by multilocus variable-number tandem-repeat analysis. Isolate genotypes were analyzed at five loci containing tandem repeats. Across the population, as well as within households, isolates with identical genotypes were clustered in time. Isolates from individuals within the same household were more likely to have similar or identical genotypes than were isolates from different households, but even within a household, isolates from different individuals often had different genotypes. When household contacts were sampled regularly for 3 weeks after the illness of the household index patient, isolates with genotypes related to the index patient appeared in contacts, on average, approximately 3 days after the index patient, while isolates with unrelated genotypes appeared in contacts approximately 6 days after. Limited data revealed that multiple isolates from the same individual collected within days of each other or even from a single stool sample may have identical, similar, or unrelated genotypes as well. Our results demonstrate that genetically related V. cholerae strains cluster in local outbreaks but also suggest that multiple distinct strains of V. cholerae O1 may circulate simultaneously within a household.

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Regulation of galactose metabolism through the HisK:GalR two-component system in Thermoanaerobacter tengcongensis.

Publication Date: 2010 Sep PMID: 20581213
Authors: Qian, Z. - Wang, Q. - Tong, W. - Zhou, C. - Wang, Q. - Liu, S.
Journal: J Bacteriol

Thermoanaerobacter tengcongensis could utilize galactose as a carbon source via the enzymes encoded by a novel gal operon, whose regulation mechanism has yet to be elucidated. We propose here that the gal operon in T. tengcongensis is regulated through a HisK:GalR two-component system. By using radioactive isotope assay and genetic analysis, we found that the kinase of this system, HisK, is phosphorylated by ATP, and the regulator, GalR, accepts a phosphoryl group during phosphorelay, in which the phosphoryl group at HisK-His-259 is transferred to GalR-Asp-56. Two-dimensional electrophoresis, followed by Western blotting, revealed that phosphorylation status of GalR is uniquely dependent on the galactose stimulus in vivo. Furthermore, DNA pulldown assays demonstrated that the phosphorylated GalR prefers binding to the operator DNA O(2), whereas the unphosphorylated GalR to O(1). A model of HisK:GalR is proposed to explain how galactose mediates the expression of the gal operon in T. tengcongensis.

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Geobacter uraniireducens NikR displays a DNA binding mode distinct from other members of the NikR family.

Publication Date: 2010 Sep PMID: 20581212
Authors: Benanti, E. L. - Chivers, P. T.
Journal: J Bacteriol

NikR is a nickel-responsive ribbon-helix-helix transcription factor present in many bacteria and archaea. The DNA binding properties of Escherichia coli and Helicobacter pylori NikR (factors EcNikR and HpNikR, respectively) have revealed variable features of DNA recognition. EcNikR represses a single operon by binding to a perfect inverted repeat sequence, whereas HpNikR binds to promoters from multiple genes that contain poorly conserved inverted repeats. These differences are due in large part to variations in the amino acid sequences of the DNA-contacting beta-sheets, as well as residues preceding the beta-sheets of these two proteins. We present here evidence of another variation in DNA recognition by the NikR protein from Geobacter uraniireducens (GuNikR). GuNikR has an Arg-Gly-Ser beta-sheet that binds specifically to an inverted repeat sequence distinct from those recognized by Ec- or HpNikR. The N-terminal residues that precede the GuNikR beta-sheet residues are required for high-affinity DNA binding. Mutation of individual arm residues dramatically reduced the affinity of GuNikR for specific DNA. Interestingly, GuNikR tetramers are capable of binding cooperatively to the promoter regions of two different genes, nik(MN)1 and nik(MN)2. Cooperativity was not observed for the closely related G. bemidjiensis NikR, which recognizes the same operator sequence. The cooperative mode of DNA binding displayed by GuNikR could affect the sensitivity of transporter gene expression to changes in intracellular nickel levels.

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(p)ppGpp inhibits polynucleotide phosphorylase from streptomyces but not from Escherichia coli and increases the stability of bulk mRNA in Streptomyces coelicolor.

Publication Date: 2010 Sep PMID: 20581211
Authors: Gatewood, M. L. - Jones, G. H.
Journal: J Bacteriol

ppGpp regulates gene expression in a variety of bacteria and in plants. We proposed previously that ppGpp or its precursor, pppGpp [referred to collectively as (p)ppGpp], or both might regulate the activity of the enzyme polynucleotide phosphorylase in Streptomyces species. We have examined the effects of (p)ppGpp on the polymerization and phosphorolysis activities of PNPase from Streptomyces coelicolor, Streptomyces antibioticus, and Escherichia coli. We have shown that (p)ppGpp inhibits the activities of both Streptomyces PNPases but not the E. coli enzyme. The inhibition kinetics for polymerization using the Streptomyces enzymes are of the mixed noncompetitive type, suggesting that (p)ppGpp binds to a region other than the active site of the enzyme. ppGpp also inhibited the phosphorolysis of a model RNA substrate derived from the rpsO-pnp operon of S. coelicolor. We have shown further that the chemical stability of mRNA increases during the stationary phase in S. coelicolor and that induction of a plasmid-borne copy of relA in a relA-null mutant increases the chemical stability of bulk mRNA as well. We speculate that the observed inhibition in vitro may reflect a role of ppGpp in the regulation of antibiotic production in vivo.

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Rickettsia prowazekii uses an sn-glycerol-3-phosphate dehydrogenase and a novel dihydroxyacetone phosphate transport system to supply triose phosphate for phospholipid biosynthesis.

Publication Date: 2010 Sep PMID: 20581209
Authors: Frohlich, K. M. - Roberts, R. A. - Housley, N. A. - Audia, J. P.
Journal: J Bacteriol

Rickettsia prowazekii is an obligate intracellular pathogen that possesses a small genome and a highly refined repertoire of biochemical pathways compared to those of free-living bacteria. Here we describe a novel biochemical pathway that relies on rickettsial transport of host cytosolic dihydroxyacetone phosphate (DHAP) and its subsequent conversion to sn-glycerol-3-phosphate (G3P) for synthesis of phospholipids. This rickettsial pathway compensates for the evolutionary loss of rickettsial glycolysis/gluconeogenesis, the typical endogenous source of G3P. One of the components of this pathway is R. prowazekii open reading frame RP442, which is annotated GpsA, a G3P dehydrogenase (G3PDH). Purified recombinant rickettsial GpsA was shown to specifically catalyze the conversion of DHAP to G3P in vitro. The products of the GpsA assay were monitored spectrophotometrically, and the identity of the reaction product was verified by paper chromatography. In addition, heterologous expression of the R. prowazekii gpsA gene functioned to complement an Escherichia coli gpsA mutant. Furthermore, gpsA mRNA was detected in R. prowazekii purified from hen egg yolk sacs, and G3PDH activity was assayable in R. prowazekii lysed-cell extracts. Together, these data strongly suggested that R. prowazekii encodes and synthesizes a functional GpsA enzyme, yet R. prowazekii is unable to synthesize DHAP as a substrate for the GpsA enzymatic reaction. On the basis of the fact that intracellular organisms often avail themselves of resources in the host cell cytosol via the activity of novel carrier-mediated transport systems, we reasoned that R. prowazekii transports DHAP to supply substrate for GpsA. In support of this hypothesis, we show that purified R. prowazekii transported and incorporated DHAP into phospholipids, thus implicating a role for GpsA in vivo as part of a novel rickettsial G3P acquisition pathway for phospholipid biosynthesis.

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Complete nucleotide sequence of TOL plasmid pDK1 provides evidence for evolutionary history of IncP-7 catabolic plasmids.

Publication Date: 2010 Sep PMID: 20581207
Authors: Yano, H. - Miyakoshi, M. - Ohshima, K. - Tabata, M. - Nagata, Y. - Hattori, M. - Tsuda, M.
Journal: J Bacteriol

To understand the mechanisms for structural diversification of Pseudomonas-derived toluene-catabolic (TOL) plasmids, the complete sequence of a self-transmissible plasmid pDK1 with a size of 128,921 bp from Pseudomonas putida HS1 was determined. Comparative analysis revealed that (i) pDK1 consisted of a 75.6-kb IncP-7 plasmid backbone and 53.2-kb accessory gene segments that were bounded by transposon-associated regions, (ii) the genes for conjugative transfer of pDK1 were highly similar to those of MOB(H) group of mobilizable plasmids, and (iii) the toluene-catabolic (xyl) gene clusters of pDK1 were derived through homologous recombination, transposition, and site-specific recombination from the xyl gene clusters homologous to another TOL plasmid, pWW53. The minireplicons of pDK1 and its related IncP-7 plasmids, pWW53 and pCAR1, that contain replication and partition genes were maintained in all of six Pseudomonas strains tested, but not in alpha- or betaproteobacterial strains. The recipient host range of conjugative transfer of pDK1 was, however, limited to two Pseudomonas strains. These results indicate that IncP-7 plasmids are essentially narrow-host-range and self-transmissible plasmids that encode MOB(H) group-related transfer functions and that the host range of IncP-7-specified conjugative transfer was, unlike the situation in other well-known plasmids, narrower than that of its replication.

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Genome sequence of the milbemycin-producing bacterium Streptomyces bingchenggensis.

Publication Date: 2010 Sep PMID: 20581206
Authors: Wang, X. J. - Yan, Y. J. - Zhang, B. - An, J. - Wang, J. J. - Tian, J. - Jiang, L. - Chen, Y. H. - Huang, S. X. - Yin, M. - Zhang, J. - Gao, A. L. - Liu, C. X. - Zhu, Z. X. - Xiang, W. S.
Journal: J Bacteriol

Streptomyces bingchenggensis is a soil-dwelling bacterium producing the commercially important anthelmintic macrolide milbemycins. Besides milbemycins, the insecticidal polyether antibiotic nanchangmycin and some other antibiotics have also been isolated from this strain. Here we report the complete genome sequence of S. bingchenggensis. The availability of the genome sequence of S. bingchenggensis should enable us to understand the biosynthesis of these structurally intricate antibiotics better and facilitate rational improvement of this strain to increase their titers.

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The pdh operon is expressed in a subpopulation of stationary-phase bacteria and is important for survival of sugar-starved Streptococcus mutans.

Publication Date: 2010 Sep PMID: 20581205
Authors: Busuioc, M. - Buttaro, B. A. - Piggot, P. J.
Journal: J Bacteriol

Streptococcus mutans is a facultative member of the oral plaque and is associated with dental caries. It is able to survive long periods of sugar starvation. We show here that inactivation of pdhD, putatively encoding a subunit of the pyruvate dehydrogenase complex, impairs survival of both batch cultures and biofilms. We show that pdhD and the downstream genes pdhA, pdhB, and pdhC form an operon that is predominantly transcribed in stationary phase. Analysis with fluorescent reporters revealed a bimodal expression pattern for the pdh promoter, with less than 1% of stationary-phase populations expressing pdh. When it was first detected, after 1 day of sugar starvation in batch culture, expression was mostly in individual bacteria. At later times, expressing bacteria were often in chains. The lengths of the chains increased with time. We infer that the pdh-expressing subpopulation is able grow and divide and to persist for extended times in stationary phase.

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Identification of a receptor subunit and putative ligand-binding residues involved in the Bacillus megaterium QM B1551 spore germination response to glucose.

Publication Date: 2010 Sep PMID: 20581204
Authors: Christie, G. - Gotzke, H. - Lowe, C. R.
Journal: J Bacteriol

The molecular basis for the recognition of glucose as a germinant molecule by spores of Bacillus megaterium QM B1551 has been examined. A chromosome-located locus (BMQ_1820, renamed gerWB) is shown to encode a receptor B-protein subunit that interacts with the GerUA and GerUC proteins to form a receptor that is cognate for both glucose and leucine. GerWB represents the third receptor B protein that binds to glucose in this strain. Site-directed mutagenesis (SDM) experiments conducted on charged proline and aromatic residues predicted to reside in the transmembrane domains of a previously characterized receptor B protein, GerVB, reveal the importance to receptor function of a cluster of residues predicted to reside in the middle of the transmembrane 6 (TM6) domain. Reductions in the region of 70- to 165-fold in the apparent affinity of receptors for glucose in which Glu196, Tyr191, and Phe192 are individually replaced by SDM indicate that some or all of these residues may be directly involved in the binding of glucose and perhaps other germinants to the germinant receptor.

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Size dependence of protein diffusion in the cytoplasm of Escherichia coli.

Publication Date: 2010 Sep PMID: 20581203
Authors: Nenninger, A. - Mastroianni, G. - Mullineaux, C. W.
Journal: J Bacteriol

Diffusion in the bacterial cytoplasm is regarded as the primary method of intracellular protein movement and must play a major role in controlling the rates of cell processes. A number of recent studies have used green fluorescent protein (GFP) tagging and fluorescence microscopy to probe the movement and distribution of proteins in the bacterial cytoplasm. However, the dynamic behavior of indigenous proteins must be controlled by a complex mixture of specific interactions, combined with the basic physical constraints imposed by the viscosity and macromolecular crowding of the cytoplasm. These factors are difficult to unravel in studies with indigenous proteins. To what extent the addition of a GFP tag might affect the movement of a protein through the cytoplasm has also remained unknown. To resolve these problems, we have carried out a systematic study of the size dependence of protein diffusion coefficients in the Escherichia coli cytoplasm, using engineered GFP multimers (from 2 to 6 covalently linked GFP molecules). Diffusion coefficients were measured using confocal fluorescence recovery after photobleaching (FRAP). At least up to 110 kDa (four linked GFP molecules), the diffusion coefficient varies with size roughly as would be predicted from the Einstein-Stokes equation for a classical (Newtonian) fluid. Thus, protein diffusion coefficients are predictable over this range. GFP tagging of proteins has little impact on the diffusion coefficient over this size range and therefore need not significantly perturb protein movement. Two indigenous E. coli proteins were used to show that their specific interactions within the cell are the main controllers of the diffusion rate.

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Compartmentalized glucose metabolism in Pseudomonas putida is controlled by the PtxS repressor.

Publication Date: 2010 Sep PMID: 20581202
Authors: Daddaoua, A. - Krell, T. - Alfonso, C. - Morel, B. - Ramos, J. L.
Journal: J Bacteriol

Metabolic flux analysis revealed that in Pseudomonas putida KT2440 about 50% of glucose taken up by the cells is channeled through the 2-ketogluconate peripheral pathway. This pathway is characterized by being compartmentalized in the cells. In fact, initial metabolism of glucose to 2-ketogluconate takes place in the periplasm through a set of reactions catalyzed by glucose dehydrogenase and gluconate dehydrogenase to yield 2-ketogluconate. This metabolite is subsequently transported to the cytoplasm, where two reactions are carried out, giving rise to 6-phosphogluconate, which enters the Entner-Doudoroff pathway. The genes for the periplasmic and cytoplasmic set of reactions are clustered in the host chromosome and grouped within two independent operons that are under the control of the PtxS regulator, which also modulates its own synthesis. Here, we show that although the two catabolic operons are induced in vivo by glucose, ketogluconate, and 2-ketogluconate, in vitro we found that only 2-ketogluconate binds to the regulator with an apparent K(D) (equilibrium dissociation constant) of 15 muM, as determined using isothermal titration calorimetry assays. PtxS is made of two domains, a helix-turn-helix DNA-binding domain located at the N terminus and a C-terminal domain that binds the effector. Differential scanning calorimetry assays revealed that PtxS unfolds via two events characterized by melting points of 48.1 degrees C and 57.6 degrees C and that, in the presence of 2-ketogluconate, the unfolding of the effector binding domain occurs at a higher temperature, providing further evidence for 2-ketogluconate-PtxS interactions. Purified PtxS is a dimer that binds to the target promoters with affinities in the range of 1 to 3 muM. Footprint analysis revealed that PtxS binds to an almost perfect palindrome that is present within the three promoters and whose consensus sequence is 5'-TGAAACCGGTTTCA-3'. This palindrome overlaps with the RNA polymerase binding site.

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Functional relationships between the AcrA hairpin tip region and the TolC aperture tip region for the formation of the bacterial tripartite efflux pump AcrAB-TolC.

Publication Date: 2010 Sep PMID: 20581201
Authors: Kim, H. M. - Xu, Y. - Lee, M. - Piao, S. - Sim, S. H. - Ha, N. C. - Lee, K.
Journal: J Bacteriol

Tripartite efflux pumps found in Gram-negative bacteria are involved in antibiotic resistance and toxic-protein secretion. In this study, we show, using site-directed mutational analyses, that the conserved residues located in the tip region of the alpha-hairpin of the membrane fusion protein (MFP) AcrA play an essential role in the action of the tripartite efflux pump AcrAB-TolC. In addition, we provide in vivo functional data showing that both the length and the amino acid sequence of the alpha-hairpin of AcrA can be flexible for the formation of a functional AcrAB-TolC pump. Genetic-complementation experiments further indicated functional interrelationships between the AcrA hairpin tip region and the TolC aperture tip region. Our findings may offer a molecular basis for understanding the multidrug resistance of pathogenic bacteria.

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Xer1-mediated site-specific DNA inversions and excisions in Mycoplasma agalactiae.

Publication Date: 2010 Sep PMID: 20562305
Authors: Czurda, S. - Jechlinger, W. - Rosengarten, R. - Chopra-Dewasthaly, R.
Journal: J Bacteriol

Surface antigen variation in Mycoplasma agalactiae, the etiologic agent of contagious agalactia in sheep and goats, is governed by site-specific recombination within the vpma multigene locus encoding the Vpma family of variable surface lipoproteins. This high-frequency Vpma phase switching was previously shown to be mediated by a Xer1 recombinase encoded adjacent to the vpma locus. In this study, it was demonstrated in Escherichia coli that the Xer1 recombinase is responsible for catalyzing vpma gene inversions between recombination sites (RS) located in the 5'-untranslated region (UTR) in all six vpma genes, causing cleavage and strand exchange within a 21-bp conserved region that serves as a recognition sequence. It was further shown that the outcome of the site-specific recombination event depends on the orientation of the two vpma RS, as direct or inverted repeats. While recombination between inverted vpma RS led to inversions, recombination between direct repeat vpma RS led to excisions. Using a newly developed excision assay based on the lacZ reporter system, we were able to successfully demonstrate under native conditions that such Xer1-mediated excisions can indeed also occur in the M. agalactiae type strain PG2, whereas they were not observed in the control xer1-disrupted VpmaY phase-locked mutant (PLMY), which lacks Xer1 recombinase. Unless there are specific regulatory mechanisms preventing such excisions, this might be the cost that the pathogen has to render at the population level for maintaining this high-frequency phase variation machinery.

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Genomic identification of a novel mutation in hfq that provides multiple benefits in evolving glucose-limited populations of Escherichia coli.

Publication Date: 2010 Sep PMID: 20543067
Authors: Maharjan, R. - Zhou, Z. - Ren, Y. - Li, Y. - Gaffe, J. - Schneider, D. - McKenzie, C. - Reeves, P. R. - Ferenci, T. - Wang, L.
Journal: J Bacteriol

Beneficial mutations in diversifying glucose-limited Escherichia coli populations are mostly unidentified. The genome of an evolved isolate with multiple differences from that of the ancestor was fully assembled. Remarkably, a single mutation in hfq was responsible for the multiple benefits under glucose limitation through changes in at least five regulation targets.

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Phosphorylation and dephosphorylation among Dif chemosensory proteins essential for exopolysaccharide regulation in Myxococcus xanthus.

Publication Date: 2010 Sep PMID: 20543066
Authors: Black, W. P. - Schubot, F. D. - Li, Z. - Yang, Z.
Journal: J Bacteriol

Myxococcus xanthus social gliding motility, which is powered by type IV pili, requires the presence of exopolysaccharides (EPS) on the cell surface. The Dif chemosensory system is essential for the regulation of EPS production. It was demonstrated previously that DifA (methyl-accepting chemotaxis protein [MCP]-like), DifC (CheW-like), and DifE (CheA-like) stimulate whereas DifD (CheY-like) and DifG (CheC-like) inhibit EPS production. DifD was found not to function downstream of DifE in EPS regulation, as a difD difE double mutant phenocopied the difE single mutant. It has been proposed that DifA, DifC, and DifE form a ternary signaling complex that positively regulates EPS production through the kinase activity of DifE. DifD was proposed as a phosphate sink of phosphorylated DifE (DifE approximately P), while DifG would augment the function of DifD as a phosphatase of phosphorylated DifD (DifD approximately P). Here we report in vitro phosphorylation studies with all the Dif chemosensory proteins that were expressed and purified from Escherichia coli. DifE was demonstrated to be an autokinase. Consistent with the formation of a DifA-DifC-DifE complex, DifA and DifC together, but not individually, were found to influence DifE autophosphorylation. DifD, which did not inhibit DifE autophosphorylation directly, was found to accept phosphate from autophosphorylated DifE. While DifD approximately P has an unusually long half-life for dephosphorylation in vitro, DifG efficiently dephosphorylated DifD approximately P as a phosphatase. These results support a model where DifE complexes with DifA and DifC to regulate EPS production through phosphorylation of a downstream target, while DifD and DifG function synergistically to divert phosphates away from DifE approximately P.

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Remembering Malcolm J. Casadaban.

Publication Date: 2010 Sep PMID: 20511498
Authors:
Journal: J Bacteriol

Malcolm J. Casadaban died on 13 September 2009 from an infection and was found to have a weakened strain of the bacterium Yersinia pestis in his blood. This tragic event took the life of one of the most creative and influential geneticists of our time. In the late 1970s and '80s, Malcolm invented novel approaches which changed the way many of us did science. Jon Beckwith, Tom Silhavy, and Olaf Schneewind have chronicled his scientific life from graduate school to his death and give us insight into Malcolm's genius. Philip Matsumura Editor in Chief, Journal of Bacteriology.

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