Journal of Bacteriology

Release of Small Molecules During Germination of Spores of Bacillus Species.

Publication Date: 2008 May 9 PMID: 18469112
Authors: Setlow, B. - Wahome, P. G. - Setlow, P.
Journal: J Bacteriol

Free amino acids, dipicolinic acid and unidentified small molecules were released early in Bacillus spore germination before hydrolysis of the peptidoglycan cortex, but adenine nucleotides and 3-phosphoglycerate were not. These results indicate that early in germination there is a major selective change in the permeability of the spore's inner membrane.

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Modulation of covR expression in Streptococcus mutans UA159.

Publication Date: 2008 May 9 PMID: 18469111
Authors: Chong, P. - Drake, L. - Biswas, I.
Journal: J Bacteriol

The biofilm-forming Streptococcus mutans is a Gram-positive bacterium that resides in the human oral cavity, and is considered to be the primary etiological agent in the formation of dental caries. The global response regulator CovR, which lacks a cognate sensor kinase, is essential for pathogenesis and biofilm-formation of this bacterium, but it is not clear how covR expression is regulated in S. mutans. In this communication, we present the results of our studies examining various factors that regulate the expression of covR in S. mutans UA159. Southern hybridization and PCR analysis indicated that CovR is an orphan response regulator in various isolates of S. mutans. The transcriptional start site for covR was found to be 221 base pairs upstream of the ATG start codon, and sited-directed mutagenesis of the upstream TATAAT box confirmed our findings. Expression of covR is growth phase dependent, with maximal expression observed during exponential growth phase. While changes to the growth temperature did not significantly affect expression of covR, increasing the pH or the concentration of Mg(2+) in the growth medium leads to an increase in covR expression. The results of semiquantitative RT-PCR analysis and in vivo transcriptional fusion reporter assays indicated that CovR autoregulates its own expression; this was verified by EMSA and DNase I protection assays, which demonstrated direct binding of CovR to the promoter region. Apparently, regulation by Mg(2+) and autoregulation of covR are not linked. A detailed analysis of the regulation of CovR may lead to a better understanding of pathogenesis of S. mutans as well as further insight in the prevention of dental caries.

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Clusters of Charged Residues at the C-terminus of MotA and N-terminus of MotB Are Important for Function of the E. coli Flagellar Motor.

Publication Date: 2008 May 9 PMID: 18469110
Authors: Hosking, E. R. - Manson, M. D.
Journal: J Bacteriol

MotA contains a conserved C-terminal cluster of negatively charged residues, and MotB contains a conserved N-terminal cluster of positively charged residues. Charge-altering mutations affecting these residues impair motility but do not diminish Mot protein levels. The motility defects are reversed by second-site mutations targeting the same or partner protein.

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Bacterial development in the fast lane.

Publication Date: 2008 May 9 PMID: 18469109
Authors: Kroos, L.
Journal: J Bacteriol

Timing is everything; in love, in war, in sports, and in politics, but what about during multicellular development of Myxococcus xanthus bacteria? On p. ? of this issue, Higgs et al. ...

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Involvement of a Novel Efflux System in Biofilm-specific Resistance to Antibiotics.

Publication Date: 2008 May 9 PMID: 18469108
Authors: Zhang, L. - Mah, T. F.
Journal: J Bacteriol

Bacteria growing in biofilms are more resistant to antibiotics when compared to their planktonic counterparts. How this transition occurs is unclear, but it is likely there are multiple mechanisms of resistance that act together in order to provide an increased overall level of resistance to the biofilm. We have identified a novel efflux pump in Pseudomonas aeruginosa that is important for biofilm-specific resistance to a subset of antibiotics. Complete deletion of the genes encoding this pump, PA1874-1877, in a PA14 background results in an increase in sensitivity to tobramycin, gentamicin and ciprofloxacin, specifically when this mutant strain is growing in a biofilm. This efflux pump is more highly expressed in biofilm cells than planktonic cells, providing an explanation for why these genes are important for biofilm but not planktonic resistance to antibiotics. Furthermore, expression of these genes in planktonic cells increases their resistance to antibiotics. We have previously shown that ndvB is important for biofilm-specific resistance (Mah, T. F., B. Pitts, B. Pellock, G. C. Walker, P. S. Stewart, and G. A. O'Toole, Nature 426:306-310, 2003). Our discovery that combining the ndvB mutation with the PA1874-1877 deletion results in a mutant strain that is more sensitive to antibiotics than either single mutant strain suggests that ndvB and PA1874-1877 contribute to two different mechanisms of biofilm-specific resistance to antibiotics.

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Lactobacillus reuteri DSM 20016 produces cobalamin-dependent diol dehydratase in metabolosomes and metabolises 1,2-propanediol by disproportionation.

Publication Date: 2008 May 9 PMID: 18469107
Authors: Sriramulu, D. D. - Liang, M. - Hernandez-Romero, D. - Raux-Deery, E. - Lunsdorf, H. - Parsons, J. B. - Warren, M. J. - Prentice, M. B.
Journal: J Bacteriol

A Lactobacillus reuteri strain isolated from sourdough is known to produce the vitamin cobalamin. The organism requires this for glycerol co-fermentation by a cobalamin-dependent enzyme usually termed glycerol dehydratase in the synthesis of the antimicrobial substance reuterin. We show that the cobalamin-synthesizing capacity of another L. reuteri strain (20016, the type strain, isolated from the human gut and recently sequenced as F275) is genetically and phenotypically linked, as in Enterobacteriaceae, to the production of a cobalamin-dependent enzyme which is associated with a bacterial microcompartment (metabolosome) and known as diol dehydratase. We show that this enzyme allows L. reuteri to carry out a disproportionation reaction converting 1, 2-propanediol to propionate and propanol. The wide distribution of this operon suggests it is adapted to horizontal transmission between bacteria. However, significant genetic and phenotypic differences are noted in a Lactobacillus background compared to Enterobacteriaceae. Electron microscopy reveals that the bacterial microcompartment in L. reuteri occupies a smaller percentage of the cytoplasm than in Gram-negative bacteria. DNA sequence data shows evidence of a different regulatory control mechanism from that in Gram-negative bacteria with the presence of a catabolite responsive element (cre sequence) immediately upstream of the pdu operon encoding diol dehydratase and metabolosome structural genes in L. reuteri. The metabolosome-associated diol dehydratase we describe is the only candidate glycerol dehydratase present on inspection of the L. reuteri F275 genome sequence.

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Lipoprotein signal peptides are processed by Lsp and Eep of Streptococcus uberis.

Publication Date: 2008 May 9 PMID: 18469106
Authors: Denham, E. L. - Ward, P. N. - Leigh, J. A.
Journal: J Bacteriol

Lipoprotein signal peptidase (lsp) is responsible for cleaving the signal peptide sequence of lipoproteins in Gram positive bacteria. Investigation of the role of Lsp in S. uberis, a common cause of bovine mastitis, was undertaken using the lipoprotein MtuA (a protein essential for virulence) as a marker. The S. uberis lsp mutant phenotype displayed novel lipoprotein processing. Not only was full length (uncleaved) MtuA detected by Western blot, but during late log phase, a lower molecular weight derivative of MtuA was evident. Similar analysis of a S. uberis double mutant, containing insertions disrupting both lsp and eep (a homologue of the Enterococcus faecalis "enhanced expression of pheromone") indicated a role for eep in cleavage of lipoproteins in the absence of Lsp. Such a function may indicate a role for eep in maintenance of secretion pathways during disruption of normal lipoprotein processing.

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SO-LAAO, a novel L-amino acid oxidase that enables Streptococcus oligofermentans to over-compete Streptococcus mutans by generating H2O2 from peptone.

Publication Date: 2008 May 9 PMID: 18469105
Authors: Tong, H. - Chen, W. - Shi, W. - Qi, F. - Dong, X.
Journal: J Bacteriol

We previously demonstrated that Streptococcus oligofermentans suppressed the growth of Streptococcus mutans, the primary cariogenic pathogen, by producing hydrogen peroxide (H2O2) through lactate oxidase (Lox) activity. In this study, we found that the lox mutant of S. oligofermentans regained the inhibition while growing on peptone rich plates. Further studies demonstrated that the H2O2 produced on peptone by S. oligofermentans was mainly derived from seven L-amino acids, i.e. L-aspartic acid, L-tryptophan, L-lysine, L-isoleucine, L-arginine, L-asparagine and L-glutamine, indicating the possible existence of L-amino acids oxidase (L-AAO) that can produce H2O2 from L-amino acids. Through searching S. oligofermentans genome for ORFs with a conserved FAD binding motif that exists in the known L-AAOs including those of snake venom, fungi and bacteria, a putative L-amino acids oxidase gene, assigned as aaoso, was cloned and over expressed in Escherichia coli. The purified protein SO-LAAO showed a molecular weight of 43 KDa on SDS-PAGE, and catalyzed H2O2 formation from the seven L-amino acids determined above, thus confirming its L-amino acid oxidase activity. The SO-LAAO identified in S. oligofermentans differed evidently from the known L-AAOs in both substrate profile and sequence, suggesting that it could represent a novel L-amino acid oxidase. An aaoso mutant of S. oligofermentans did lose H2O2 formation from the seven L-amino acids, further verifying its function as an L-AAO. Furthermore the inhibition of S. oligofermentans over S. mutans in a peptone rich mix-species biofilm was greatly reduced in the aaoso mutant, indicating its importance in interspecies competition.

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Characterization of Clostridium perfringens spores that lack SpoVA proteins and dipicolinic acid.

Publication Date: 2008 May 9 PMID: 18469104
Authors: Paredes-Sabja, D. - Setlow, B. - Setlow, P. - Sarker, M. R.
Journal: J Bacteriol

Spores of Clostridium perfringens possess high heat-resistance and when these spores germinate and return to active growth they can cause gastrointestinal disease. Work with Bacillus subtilis has shown that the spore's dipicolinic acid (DPA) level can markedly influence both spore germination and resistance, and that the proteins encoded by the spoVA operon are essential for DPA uptake by the developing spore during sporulation. We now find that proteins encoded by the spoVA operon are also essential for the uptake of Ca(2+) and DPA into the developing spore during C. perfringens sporulation. Spores of a spoVA mutant had little if any Ca(2+) and DPA, and their core water content was approximately 2-fold higher than that of wild-type spores. These DPA-less spores did not germinate spontaneously, as do DPA-less B. subtilis spores. Indeed, wild-type and spoVA C. perfringens spores germinated similarly with a mixture of L-asparagine and KCl (AK), KCl alone or a 1:1 chelate of Ca(2+) and DPA (Ca-DPA). However, the C. perfringens spoVA spores had 20-fold lower viability than wild-type spores. Decoated wild-type and spoVA spores exhibited little if any germination with AK, KCl, or exogenous Ca-DPA, and had 10(3)- to 10(4)-fold lower colony forming efficiency than intact spores. However, lysozyme treatment rescued these decoated spores. Although the level of DNA protective alpha/beta-type small, acid-soluble spore proteins in spoVA spores was similar to that in wild-type spores, spoVA spores exhibited markedly lower resistance to moist heat, formaldehyde, HCl, hydrogen peroxide, nitrous acid and UV radiation. In sum, these results suggest that: (i) SpoVA proteins are essential for Ca-DPA uptake by developing spores during C. perfringens sporulation; (ii) SpoVA proteins and Ca-DPA release are not required for C. perfringens spore germination; and (iii) a low core water content is essential for full resistance of C. perfringens spores to moist heat, UV radiation and chemicals.

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FliZ is a post-translational activator of FlhD4C2-dependent flagellar gene expression.

Publication Date: 2008 May 9 PMID: 18469103
Authors: Saini, S. - Brown, J. D. - Aldridge, P. D. - Rao, C. V.
Journal: J Bacteriol

Flagellar assembly proceeds in a sequential manner beginning at the base and concluding with the filament. A critical aspect of assembly is that gene expression is coupled to assembly. When cells transition from a non-flagellated to a flagellated state, gene expression is sequential, reflecting the manner in which the flagellum is made. A key mechanism for establishing this temporal hierarchy is the sigma(28)-FlgM checkpoint, which couples the expression of late flagellar (Pclass3) genes to the completion of the hook-basal body. In this work, we investigate the role of FliZ in coupling middle flagellar (Pclass2) gene expression to assembly in Salmonella enterica serovar Typhimurium. We demonstrate that FliZ is an FlhD4C2-dependent activator of Pclass2/middle gene expression. Our results suggest that FliZ regulates the concentration of FlhD4C2 post-translationally. We also demonstrate that FliZ functions independently of the flagellar-specific sigma factor sigma(28) and the filament-cap chaperone/FlhD4C2-inhibitor FliT. Furthermore, we show that the previously described ability of sigma(28) to activate Pclass2/middle gene expression is, in fact, due to FliZ, as both are expressed from the same overlapping Pclass2 and Pclass3 promoters at the fliAZY locus. We conclude by discussing the role of FliZ regulation with respect to flagellar biosynthesis based on our characterization of gene expression and FliZ's role in swimming and swarming motility.

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Switching control of the expression of ptsG from the Mlc regulon to the NagC regulon.

Publication Date: 2008 May 9 PMID: 18469102
Authors: El Qaidi, S. - Plumbridge, J.
Journal: J Bacteriol

The Mlc and NagC transcriptional repressors bind to similar 23 bp operators. The sequences are weakly palindromic with just 4 positions totally conserved. There is no cross regulation observed between the repressors in vivo but there are no obvious bases which could be responsable for operator site discrimination. To investigate the basis for operator recognition and to try to understand what differentiates NagC sites from Mlc sites we have undertaken mutagenesis experiments to convert the ptsG gene from being regulated by Mlc into a gene regulated by NagC. There are two Mlc operators upstream of ptsG and to switch ptsG to the NagC regulon, it was necessary to change two different characteristics of both operators. Firstly, we replaced the A-T bp, at position +/-11 from the center of symmetry of the operators, for G-C bp. Secondly, we changed the sequence of CG bp in the central region of the operator (positions -4 to +4 around the center of symmetry). Our results show that changes at either of these locations are sufficient to lose regulation by Mlc but that both types of changes in both operators are necessary to convert ptsG to a gene regulated by NagC. In addition these experiments confirmed that two operators are necessary for regulation by NagC. We also show that regulation of ptsG by Mlc involves some co-operative binding of Mlc to the two operators.

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The Francisella Pathogenicity Island Protein PdpD is required for full virulence and associates with homologues of the type VI secretion system.

Publication Date: 2008 May 9 PMID: 18469101
Authors: Ludu, J. S. - de Bruin, O. M. - Duplantis, B. N. - Schmerk, C. L. - Chou, A. Y. - Elkins, K. L. - Nano, F. E.
Journal: J Bacteriol

Francisella tularensis is a highly infectious, facultative intracellular bacterial pathogen that is the causative agent of tularemia. Nearly a century ago researchers observed that tularemia was often fatal in North America, but almost never fatal in Europe and Asia. The chromosomes of F. tularensis strains carry two identical copies of the Francisella pathogenicity island (FPI), and the FPI of North American-specific biotypes contain two genes, anmK and pdpD, that are not found in biotypes that are distributed over the entire Northern Hemisphere. In this work we have studied the virulence contribution of anmK and pdpD using F. novicida, which is very closely related to F. tularensis but which carries only one copy of the FPI. We showed that anmK and pdpD are necessary for full virulence but not for intracellular growth. This is in sharp contrast to most other FPI genes that have been studied to date, which are required for intracellular growth. We also showed that PdpD is localized to the outer membrane. Further, over-expression of PdpD affects the cellular distribution of FPI-encoded proteins IglA, IglB and IglC. Finally, deletions of FPI genes encoding proteins that are homologues of known components of type VI secretion systems abolished the altered distribution of IglC and the outer membrane localization of PdpD.

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VirB3-VirB6 and VirB8-VirB11, but not VirB7, are essential for mediating persistence Brucella in the reticuloendothelial system.

Publication Date: 2008 May 9 PMID: 18469100
Authors: den Hartigh, A. B. - Rolan, H. G. - de Jong, M. F. - Tsolis, R. M.
Journal: J Bacteriol

The Brucella abortus virB locus contains 12 open reading frames, termed virB1 through virB12, which encode a type IV secretion system (T4SS). Polar mutations in the virB locus markedly reduce the ability of B. abortus to survive in cultured macrophages or to persist in organs of mice. While a non-polar deletion of the virB2 gene reduces survival in cultured macrophages and in organs of mice, a non-polar deletion of virB1 only reduces macrophage survival, while virB12 is dispensable for either virulence trait. Here we investigated the role of the remaining genes in the virB locus during macrophage survival and mouse virulence. Mutants carrying non-polar deletions of the virB3, virB4, virB5, virB6, virB7, virB8, virB9, virB10 or virB11 gene were constructed and characterized. All mutations reduced the ability of B. abortus to survive in J774A.1 mouse macrophage-like cells to a degree similar to that caused by a deletion of the entire virB locus. Deletion of virB3, virB4, virB5, virB6, virB8, virB9, virB10 or virB11 markedly reduced the ability of B. abortus to persist in the spleens of mice at eight weeks after infection. Interestingly, deletion of virB7 did not reduce the ability of B. abortus to persist in spleens of mice. We conclude that virB2, virB3, virB4, virB5, virB6, virB8, virB9, virB10 or virB11 are essential for mouse virulence of B. abortus while functions encoded by the virB1, virB7 and virB12 genes are not required for organ persistence in this animal model.

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Role of Dipicolinic Acid in the Germination, Stability and Viability of Spores of Bacillus subtilis.

Publication Date: 2008 May 9 PMID: 18469099
Authors: Magge, A. - Granger, A. C. - Wahome, P. G. - Setlow, B. - Vepachedu, V. R. - Loshon, C. A. - Peng, L. - Chen, D. - Li, Y. Q. - Setlow, P.
Journal: J Bacteriol

Spores of Bacillus subtilis spoVF strains that cannot synthesize dipicolinic acid (DPA) but take it up during sporulation were prepared in medium with various DPA concentrations and the germination and viability of these spores were measured as well as the DPA content in individual spores. Levels of some other small molecules were also measured in DPA-less spores. These studies have allowed the following conclusions. 1) Spores with no or low DPA levels that lack either the cortex-lytic enzyme (CLE) SleB or the receptors that respond to nutrient germinants could be isolated, but were unstable and spontaneously initiated early steps in spore germination. 2) Spores that lacked SleB and nutrient germinant receptors and also had low DPA levels were more stable. 3) Spontaneous germination of spores with no or low DPA levels was at least in part via activation of SleB. 4) The other redundant CLE, CwlJ, was activated only by the release of high levels of DPA from spores. 5) Low levels of DPA were sufficient for the viability of spores that lacked most alpha/beta-type small, acid-soluble spore proteins. 6) DPA levels accumulated in spores prepared in low DPA-containing media varied greatly between individual spores, in contrast to more homogeneous DPA levels in individual spores made in media with high DPA concentrations. 7) At least the great majority of spores of several spoVF strains that contained no DPA also lacked other major spore small molecules, and had gone through some of the early reactions in spore germination.

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Functional analysis of GlnE, an essential adenylyl transferase in Mycobacterium tuberculosis.

Publication Date: 2008 May 9 PMID: 18469098
Authors: Carroll, P. - Pashley, C. A. - Parish, T.
Journal: J Bacteriol

Glutamine synthetase (GS) plays an important role in nitrogen assimilation. The major GS of Mycobacterium tuberculosis is GlnA1, a type I GS whose activity is controlled by post-transcriptional modification by GlnE. GlnE is an adenylyl-transferase comprised of an adenylylating domain and a deadenylylating domain which modulate GS activity. We previously demonstrated that GlnE is essential in M. tuberculosis in normal growth medium. In this study, we further show that GlnE is required under multiple medium conditions, including in nitrogen-limited medium. We demonstrate that adenylylation is the critical activity for M. tuberculosis survival, since we were able to delete the deadenylylation domain with no apparent effect on growth or GS activity. Furthermore, we identified a critical aspartate residue in the proposed nucleotidyltransferase motif. Temperature sensitive mutants of GlnE were generated and shown to have a defect in growth and GS activity in nitrogen-limited medium. Finally, we were able to generate a GlnE null mutant in the presence of L-methionine sulfoximine, a GS inhibitor, and glutamine supplementation. In the presence of these supplements, the null mutant was able to grow similarly to wild-type. Surprisingly, the GlnE mutant was able to survive and grow for extended periods in liquid medium, but not solid medium, in the absence of GS inhibition. Thus we have confirmed that the unusual requirement of M. tuberculosis for GlnE adenylylation activity is linked to the activity of GS in the cell.

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CpcM post-translationally methylates asparagine-71/72 of phycobiliprotein beta subunits in Synechococcus sp. strain PCC 7002 and Synechocystis sp. strain PCC 6803.

Publication Date: 2008 May 9 PMID: 18469097
Authors: Shen, G. - Leonard, H. S. - Schluchter, W. M. - Bryant, D. A.
Journal: J Bacteriol

Cyanobacteria produce phycobilisomes, macromolecular light-harvesting complexes mostly assembled from phycobiliproteins. Phycobiliprotein beta subunits contain a highly conserved, gamma-N-methylasparagine residue, which results from the post-translational modification of Asn71/72. Through comparative genomic analyses, a gene, denoted cpcM, was identified that (1) encodes a protein with sequence similarity to other S-adenosylmethionine-dependent methyltransferases; (2) is found in all sequenced cyanobacterial genomes; and (3) often occurs near genes encoding phycobiliproteins in cyanobacterial genomes. The cpcM genes of Synechococcus sp. strain PCC 7002 and Synechocystis sp. strain PCC 6803 were insertionally inactivated. Mass spectrometric analyses of phycobiliproteins isolated from the mutants confirmed that the CpcB, ApcB and ApcF were 14 Da lighter than their wild-type counterparts. Trypsin digestion and mass analyses of phycobiliproteins isolated from the mutants showed that tryptic peptides from phycocyanin that included Asn72 were also 14 Da lighter than the equivalent peptides from wild-type strains. Thus, CpcM is the methyltransferase that modifies the amide nitrogen of Asn71/72 of CpcB, ApcB and ApcF. When cells were grown at low light intensity, the cpcM mutants were phenotypically similar to the wild-type strains. However, the mutants were sensitive to high-light stress, and the cpcM mutant of Synechocystis sp. strain PCC 6803 was unable to grow at moderately high light intensities. Fluorescence emission measurements showed that the ability to perform state transitions was impaired in the cpcM mutants and suggested that energy transfer from phycobiliproteins to the photosystems was also less efficient. The possible functions of asparagine N-methylation of phycobiliproteins are discussed.

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Substrate specificity of the 3-Methylcrotonyl-CoA and Geranyl-CoA Carboxylases from Pseudomonas aeruginosa.

Publication Date: 2008 May 9 PMID: 18469096
Authors: Aguilar, J. A. - Diaz-Perez, C. - Diaz-Perez, A. L. - Rodriguez-Zavala, J. S. - Nikolau, B. J. - Campos-Garcia, J.
Journal: J Bacteriol

Biotin-containing 3-methylcrotonyl-coenzyme A carboxylase (MCCase) and geranyl-coenzyme A carboxylase (GCCase) from Pseudomonas aeruginosa were expressed as His-tagged recombinant proteins in Escherichia coli. Both native and recombinant MCCase and GCCase showed pH and temperature optima of 8.5 and 37 degrees C. The apparent K0.5 values of MCCase for 3-methylcrotonyl-CoA (MC-CoA), ATP, and bicarbonate were 9.8 microM, 13 microM, and 0.8 microM, respectively. MCCase activity showed sigmoidal kinetics for all the substrates and did not carboxylate geranyl-CoA (G-CoA). In contrast, GCCase catalyzed the carboxylation of both G-CoA and MC-CoA. GCCase also showed a sigmoidal kinetic behavior for G-CoA, and bicarbonate, but Michaelis-Menten kinetics for MC-CoA and the co-substrate ATP. The apparent kinetic constant values of GCCase were K05 8.8 microM and 1.2 microM for G-CoA and bicarbonate, respectively; Km 10 microM for ATP, and 14 microM for MC-CoA. The catalytic efficiencies of GCCase for G-CoA and MC-CoA were 56 and 22, respectively, indicating that G-CoA is preferred over MC-CoA as a substrate. The enzymatic properties of GCCase suggest that it may substitute for MCCase in leucine catabolism and that both MCCase and GCCase enzymes play an important role in the leucine and acyclic terpenes catabolic pathways.

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The CI repressors of Shiga toxin-converting prophages are involved in co-infection of Escherichia coli strains, which causes a down regulation in the production of Shiga toxin 2.

Publication Date: 2008 May 9 PMID: 18469095
Authors: Serra-Moreno, R. - Jofre, J. - Muniesa, M.
Journal: J Bacteriol

Shiga toxins (Stx) are the main virulence factors associated with a form of Escherichia coli known as STEC (Shiga toxin-producing E. coli). They are encoded in temperate lambdoid phages located on the chromosome of STEC. STEC strains can carry more than one prophage. Consequently, toxin and phage production might have been influenced by the presence of more than one Stx prophage on the bacterial chromosome. To examine the effect of the number of prophages on Stx production, we produced E. coli K-12 strains carrying either one Stx2 prophage or two different Stx2 prophages. We used recombinant phages in which an antibiotic resistance gene (aph, cat or tet) was incorporated in the middle of the Shiga toxin operon. Shiga toxin was quantified by immunoassay and by cytotoxicity assay on Vero cells (CD50). When two prophages were inserted in the host chromosome, Shiga toxin production as the rate of lytic cycle activation fell. cI repressor seems to be involved in the second prophage incorporation. Incorporation and establishment of lysogenic state of the two prophages, which lowers toxin production, could be regulated by the CI repressors of both prophages operating in trans. Although sequence of the cI genes of the phages studied differed, the CI protein conformation was conserved. Results indicate that the presence of more than one prophage in the host chromosome could be regarded as a mechanism to allow genetic retention in the cell, by reducing the activation of lytic cycle and hence the pathogenicity of the strains.

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The putative hybrid sensor kinase SypF coordinates biofilm formation in Vibrio fischeri by acting upstream of two response regulators, SypG and VpsR.

Publication Date: 2008 May 9 PMID: 18469094
Authors: Darnell, C. L. - Hussa, E. A. - Visick, K. L.
Journal: J Bacteriol

Colonization of the Hawaiian squid Euprymna scolopes by the marine bacterium Vibrio fischeri requires the symbiosis polysaccharide (syp) gene cluster, which contributes to symbiotic initiation by promoting biofilm formation on the surface of the symbiotic organ. We previously described roles for the syp-encoded response regulator, SypG, and an unlinked gene encoding the sensor kinase, RscS, in controlling syp transcription and inducing syp-dependent cell-cell aggregation phenotypes. Here, we report the involvement of an additional syp-encoded regulator, the putative sensor kinase SypF, in promoting biofilm formation. Through the isolation of an increased activity allele, sypF1, we determined that SypF can function to induce syp transcription as well as a variety of biofilm phenotypes, including wrinkled colony formation, adherence to glass, and pellicle formation. SypF1-mediated transcription of the syp cluster was entirely dependent on SypG. However, the biofilm phenotypes were reduced, not eliminated, in the sypG mutant. These phenotypes were also reduced in a mutant deleted for sypE, another syp-encoded response regulator. However, SypF1 still induced phenotypes in a sypG sypE double mutant, suggesting that SypF1 might activate another regulator(s). Our subsequent work revealed that the residual SypF1-induced biofilm formation depended on VpsR, a putative response regulator, and cellulose biosynthesis. These data support a model in which a network of regulators and at least two polysaccharide loci contribute to biofilm formation in V. fischeri.

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TtOmp85 from Thermus thermophilus HB27: An ancestral type of the Omp85 protein family.

Publication Date: 2008 May 2 PMID: 18456816
Authors: Nesper, J. - Brosig, A. - Ringler, P. - Patel, G. J. - Muller, S. A. - Kleinschmidt, J. H. - Boos, W. - Diederichs, K. - Welte, W.
Journal: J Bacteriol

Proteins belonging to the Omp85 family are involved in the assembly of beta-barrel outer membrane proteins or in the translocation of proteins across the outer membrane in bacteria, mitochondria and chloroplasts. The cell envelope of the thermophilic bacterium Thermus thermophilus HB27 is multilayered including an outer membrane that is not well characterized. Neither the precise lipid composition nor much about integral membrane proteins is known. The genome of HB27 encodes one Omp85-like protein, TtOmp85, representing an ancestral type of this family. We overexpressed TtOmp85 in T. thermophilus and purified it from the native outer membranes. In the presence of detergent, purified TtOmp85 existed mainly as a monomer, composed of two stable protease resistant modules. Circular dichroism spectroscopy indicated beta-sheet secondary structure. Electron microscopy of negatively stained lipid embedded TtOmp85 revealed ring-like structures with a central cavity of approximately 1.5 nm in diameter. Single channel conductance recordings indicated that TtOmp85 forms ion channels with two different conducting states, characterized by conductances of approximately 0.4 nS and approximately 0.65 nS, respectively.

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Temperature Sensitivity and Cell Division Defects in an Escherichia coli yghB/yqjA Double Mutant, Encoding Related and Conserved Inner Membrane Proteins.

Publication Date: 2008 May 2 PMID: 18456815
Authors: Thompkins, K. - Chattopadhyay, B. - Xiao, Y. - Henk, M. C. - Doerrler, W. T.
Journal: J Bacteriol

Ludox density gradients were used to enrich for Escherichia coli mutants with conditional growth defects and alterations in membrane composition. A temperature sensitive mutant named Lud135 was isolated with mutations in two related, nonessential genes: yghB and yqjA. YghB harbors a single missense mutation (G203D) and yqjA contains a nonsense mutation (W92TGA) in Lud135. Both mutations are required for the temperature sensitive phenotype: targeted deletion of both genes in a wild type background results in a strain with a similar phenotype and expression of either gene from a plasmid restores growth at elevated temperatures. The mutant has altered membrane phospholipid levels, with elevated levels of acidic phospholipids, when grown under permissive conditions. Growth of Lud135 under nonpermissive conditions is restored by the presence of millimolar concentrations of divalent cations Ca(2+), Ba(2+), Sr(2+) or Mg(2+) or 300-500 mM NaCl but not 400 mM sucrose. Microscopic analysis of Lud135 demonstrates a dramatic defect at a late stage of cell division when cells are grown under permissive conditions. YghB and yqjA belong to the conserved and widely distributed dedA gene family for which no function has been reported. The two ORFs encode predicted polytopic inner membrane proteins with 61% amino acid identity. It is likely that YghB and YqjA play redundant but critical roles in membrane biology that are essential for completion of cell division in E. coli.

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Acyl chain specificity of the acyltransferases lpxA and lpxD and substrate availability contributes to lipid A fatty acid heterogeneity in Porphyromonas gingivalis.

Publication Date: 2008 May 2 PMID: 18456814
Authors: Bainbridge, B. W. - Karimi-Naser, L. - Reife, R. - Blethen, F. - Ernst, R. - Darveau, R. P.
Journal: J Bacteriol

The lipid A of Porphyromonas gingivalis is heterogeneous with regard to the number, type, and placement of fatty acids. Analysis of lipid A by MALDI-TOF mass spectrometry reveals clusters of peaks differing by 14 mass units indicative of an altered distribution of the fatty acids generating different lipid A structures. To examine whether the transfer of hydroxy fatty acids with different chain lengths could account for the clustering of lipid A structures, lpxAPg and lpxDPg were cloned and expressed in Escherichia coli strains mutant for the homologous gene. Lipid A from strains expressing either of the P. gingivalis transferases was found to contain sixteen carbon hydroxy fatty acids in addition to the normal E. coli fourteen carbon hydroxy fatty acids demonstrating that these acyltranferases display a relaxed acyl chain length specificity. Both lpxA and lpxD, from either E. coli or P. gingivalis, were also able to incorporate odd chain fatty acids into lipid A when grown in the presence of 1% propionic acid. This indicates that E. coli lipid A acyltransferases do not have an absolute specificity for fourteen carbon hydroxy fatty acids, but can transfer fatty acids differing by one carbon unit if the fatty acid substrates are available. We conclude that the relaxed specificity of the P. gingivalis lipid A acyltransferases and the substrate availability account for the lipid A structural clusters that differ by 14 mass units observed in P. gingivalis lipopolysaccharide preparations.

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Role of the DksA-like protein in the pathogenesis and diverse metabolic activity of Campylobacter jejuni.

Publication Date: 2008 May 2 PMID: 18456813
Authors: Yun, J. - Jeon, B. - Barton, Y. W. - Plummer, P. - Zhang, Q. - Ryu, S.
Journal: J Bacteriol

DksA is well-known for its regulatory role in the transcription of ribosomal RNA and genes involved in amino acid synthesis in many bacteria. DksA is also reported to control expression of virulence genes in pathogenic bacteria. Here, we elucidated the roles of the DksA-like protein (CJJ81176_0160, Cj0125c) in the pathogenesis of Campylobacter jejuni. Like in other bacteria, transcription of stable RNA was repressed by the DksA-like protein under stressful conditions in C. jejuni. Transcriptomic and proteomic analyses of C. jejuni 81-176 and its isogenic mutant of the DksA-like protein showed differential expression of many genes involved in amino acid metabolism, iron-related metabolism, and other metabolic reactions. Also the C. jejuni mutant of the DksA-like protein demonstrated a decreased ability to invade intestinal cells and induce release of interleukin-8 from intestinal cells. These results suggest the DksA-like protein plays an important regulatory role in diverse metabolism and virulence of C. jejuni.

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Functional analysis of the essential GTP-binding protein coding gene cgtA of Vibrio cholerae.

Publication Date: 2008 May 2 PMID: 18456812
Authors: Shah, S. - Das, B. - Bhadra, R. K.
Journal: J Bacteriol

The cgtA gene, coding for the conserved G protein CgtA, is essential in bacteria. In contrast to a previous report, here we show by genetic analysis that cgtA is essential in Vibrio cholerae even in a relA negative background. Depletion of CgtA affected the growth of V. cholerae and rendered the cells highly sensitive to replication inhibitor hydroxyurea. Overexpression of V. cholerae CgtA caused distinct elongation of Escherichia coli cells. Deletion analysis indicated that the C-terminal end of the CgtA plays a critical role for its proper function.

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Two-state allosteric modeling suggests protein equilibrium as an integral component for cAMP specificity in the cAMP receptor protein of Escherichia coli.

Publication Date: 2008 May 2 PMID: 18456811
Authors: Youn, H. - Koh, J. - Roberts, G. P.
Journal: J Bacteriol

Activation of the cAMP receptor protein (CRP) from Escherichia coli is highly specific to its allosteric ligand, cAMP. Ligands such as adenosine and cGMP, which are structurally similar to cAMP, fail to activate wild-type CRP. However, several cAMP-independent CRP variants (termed CRP*) exist that can be further activated by both adenosine and cGMP, as well as by cAMP. This has remained a puzzle because the substitutions in many of these CRP* variants lie far from the cAMP-binding pocket (>10 A) and therefore should not directly affect that pocket. Here we show a surprising similarity in the altered ligand specificity of four CRP* variants with a single substitution in D53S, G141K, A144T, or L148K, and we propose a common basis for this phenomenon. The increased active protein population caused by an equilibrium shift in these variants is hypothesized to preferentially stabilize ligand binding. This explanation is completely consistent with the cAMP specificity in the activation of wild-type CRP. The model also predicts that WT CRP should be activated even by the lower affinity ligand, adenosine, which we experimentally confirmed. The study demonstrates that protein equilibrium is an integral factor for ligand specificity in an allosteric protein, in addition to the direct effects of ligand pocket residues.

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The effect of growth temperature on Crl-dependent regulation of {sigma}S activity in Salmonella enterica serovar Typhimurium.

Publication Date: 2008 May 2 PMID: 18456810
Authors: Robbe-Saule, V. - Carreira, I. - Kolb, A. - Norel, F.
Journal: J Bacteriol

The small regulatory protein Crl favors association of the stationary phase sigma factor sigma(S) (RpoS) with the core enzyme polymerase, and thereby increases sigma(S) activity. Crl has a major physiological impact at low levels of sigma(S). Here, we report that Crl effects on sigma(S)-dependent gene expression, the H2O2 resistance of Salmonella enterica serovar Typhimurium and its resistance to acidic pH, are larger at 28 degrees C than at 37 degrees C. Immunoblot experiments revealed a negative correlation between sigma(S) and Crl levels: production of Crl was slightly higher at 28 degrees C than at 37 degrees C whereas sigma(S) levels were about 2-fold lower at 28 degrees C than at 37 degrees C. At both temperatures, Crl was present in excess of sigma(S), and increasing Crl levels further did not increase the H2O2 resistance level of Salmonella and the expression of the sigma(S) -dependent gene katE encoding the stationary phase catalase. In contrast, increasing sigma(S) level rendered Salmonella more resistant to H2O2 at 28 degrees C, increased the expression of katE and reduced the magnitude of Crl activation. In addition, the effect of Crl on katE transcription in vitro was not dependent on temperature. These results suggest that the effect of temperature on Crl-dependent regulation of the katE gene and the H2O2 resistance are mediated mainly via an effect on sigma(S) levels. In addition, our results revealed that sigma(S) exerts a negative effect in the production of Crl in stationary phase when the cells contain high levels of sigma(S).

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The active site cysteinyls and hydrophobic cavity residues of ResA are important for cytochrome c maturation in Bacillus subtilis.

Publication Date: 2008 May 2 PMID: 18456809
Authors: Hodson, C. T. - Lewin, A. - Hederstedt, L. - Le Brun, N. E.
Journal: J Bacteriol

ResA is an extra-cytoplasmic membrane-bound thiol-disulfide oxidoreductase required for cytochrome c maturation in Bacillus subtilis. Previous biochemical and structural studies have revealed that the active site cysteinyls cycle between oxidised and reduced states with a low reduction potential, and that, upon reduction, a hydrophobic cavity forms close to the active site. Here we report in vivo studies of ResA-deficient B. subtilis complemented with a series of ResA variants. Using a range of methods to analyse the cellular cytochrome c content, we demonstrate: (i) that the N-terminal trans-membrane segment of ResA serves principally to anchor the protein to the cytoplasmic membrane, but also plays a role in mediating the activity of the protein; (ii) that the active site cysteines are important for cytochrome c maturation activity; (iii) that Pro141, which forms part of the hydrophobic cavity, and which adopts a cis-conformation, plays an important role in protein stability; (iv) that Glu80, which lies at the base of the hydrophobic cavity, is important for cytochrome c maturation activity; and, finally, (v) that Pro141 and Glu80 ResA mutant variants promote selective maturation of low levels of one c-type cytochrome, subunit II of the cytochrome c oxidase caa3, indicating that this apo-cytochrome is distinct from the other three endogenous c-type cytochromes of B. subtilis.

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Identification of the L,D-Transpeptidases for Peptidoglycan Cross-linking in Escherichia coli.

Publication Date: 2008 May 2 PMID: 18456808
Authors: Magnet, S. - Dubost, L. - Marie, A. - Arthur, M. - Gutmann, L.
Journal: J Bacteriol

Three active site cysteine L,D-transpeptidases can individually anchor the Braun lipoprotein to the Escherichia coli peptidoglycan. Here, we show that two additional enzymes of the same family form peptide bonds between the third residues of peptidoglycan stems generating meso-DAP(3)-->meso-DAP(3) unusual cross-links. This activity partially replaces the D,D-transpeptidase activity of penicillin-binding proteins.

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Cortex peptidoglycan lytic activity in germinating Bacillus anthracis spores.

Publication Date: 2008 May 2 PMID: 18456807
Authors: Dowd, M. M. - Orsburn, B. - Popham, D. L.
Journal: J Bacteriol

Bacterial endospore dormancy and resistance properties depend on the relative dehydration of the spore core, which is maintained by the spore membrane and its surrounding cortex peptidoglycan wall. During spore germination, the cortex peptidoglycan is rapidly hydrolyzed by lytic enzymes packaged into the dormant spore. The peptidoglycan structures in both dormant and germinating Bacillus anthracis Sterne spores were analyzed. B. anthracis dormant spore PG was similar to that found in other species. During germination, B. anthracis released peptidoglycan fragments into the surrounding medium more quickly than some other species. A major lytic enzymatic activity was a glucosaminidase, probably YaaH, that cleaved between N-acetylglucosamine and muramic-delta-lactam. An epimerase activity previously proposed to function on spore peptidoglycan was not detected, and it is newly proposed that glucosaminidase products were previously misidentified as epimerase products. Spore cortex lytic enzymes and their regulators are attractive targets for development of germination inhibitors, to kill spores, and activators, to cause loss of resistance properties, for decontamination of sites of spore release.

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Cell-Cell Communication in Bacteria: United We Stand.

Publication Date: 2008 May 2 PMID: 18456806
Authors: von Bodman, S. B. - Willey, J. M. - Diggle, S. P.
Journal: J Bacteriol

The field of cell-cell signaling and coordinated microbial group behavior arose from two independent discoveries reported about 40 years ago. ...

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The two-component system CesRK controls the transcriptional induction of cell envelope-related genes in Listeria monocytogenes in response to cell wall-acting antibiotics.

Publication Date: 2008 May 2 PMID: 18456805
Authors: Gottschalk, S. - Bygebjerg-Hove, I. - Bonde, M. - Nielsen, P. K. - Nguyen, T. H. - Gravesen, A. - Kallipolitis, B. H.
Journal: J Bacteriol

The two-component system CesRK of Listeria monocytogenes responds to cell wall-acting antibiotics. Here, we show that CesRK controls the transcription of several cell-envelope related genes. The CesRK-dependent induction of these genes may be viewed as an attempt by L. monocytogenes to protect itself against the damaging effects of cell wall-acting antibiotics.

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Integration Host Factor positively regulates virulence gene expression in Vibrio cholerae.

Publication Date: 2008 May 2 PMID: 18456804
Authors: Stonehouse, E. - Kovacikova, G. - Taylor, R. K. - Skorupski, K.
Journal: J Bacteriol

Virulence gene expression in Vibrio cholerae is dependent upon a complex transcriptional cascade that is influenced by both specific and global regulators in response to environmental stimuli. Here we report that the global regulator IHF positively affects virulence gene expression in V. cholerae. Inactivation of ihfA or ihfB, the genes encoding the IHF subunits, decreased expression of the two main virulence factors tcpA and ctx and prevented TCP and CT production. IHF was found to directly bind to and bend the tcpA promoter region at an IHF consensus site centered at -162 using gel mobility shift assays and DNase I footprinting experiments. Deletion or mutation of the tcpA IHF consensus site resulted in the loss of IHF binding and additionally disrupted the binding of the repressor H-NS. DNase I footprinting revealed that H-NS protection overlaps with both the IHF and ToxT binding sites at the tcpA promoter. In addition, disruption of ihfA in an hns or toxT mutant background had no effect on tcpA expression. These results suggest that IHF may function at the tcpA promoter to alleviate H-NS repression.

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Transcription Factors CysB and SfnR Constitute the Hierarchical Regulatory System for the Sulfate Starvation Response in Pseudomonas putida.

Publication Date: 2008 May 2 PMID: 18456803
Authors: Kouzuma, A. - Endoh, T. - Omori, T. - Nojiri, H. - Yamane, H. - Habe, H.
Journal: J Bacteriol

Pseudomonas putida DS1 is able to utilize dimethyl sulfone as a sulfur source. Expression of the sfnFG operon responsible for dimethyl sulfone oxygenation is directly regulated by a sigma(54)-dependent transcriptional activator, SfnR, which is encoded within the sfnECR operon. We investigated the transcription mechanism for the sulfate starvation-induced expression of these sfn operons. Using an in vivo transcription assay and in vitro DNA-binding experiments, we revealed that SfnR negatively regulates the expression of sfnECR by binding to the downstream region of the transcription start point. Additionally, we demonstrated that a LysR-type transcriptional regulator, CysB, directly activates the expression of sfnECR by binding to its upstream region. CysB is a master regulator that controls the sulfate starvation response of the sfn operons, as is the case for the sulfonate utilization genes of Escherichia coli, although CysBDS1 appeared to differ from that of E. coli CysB in terms of the effect of O-acetylserine on DNA binding ability. Furthermore, we investigated what effector molecules repress the expression of sfnFG and sfnECR in vivo using the disruptants of the sulfate assimilatory genes, cysNC and cysI. The measurements of mRNA levels of the sfn operons in these gene disruptants suggested the expression of sfnFG is repressed by sulfate itself, while the expression of sfnECR is repressed by the downstream metabolites in the sulfate assimilatory pathway, such as sulfide and cysteine. These results indicate that SfnR plays a role independent of CysB in the sulfate starvation-induced expression of the sfn operons.

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Mutagenesis of the Shigella flexneri autotransporter IcsA reveals novel functional regions involved in IcsA biogenesis and recruitment of host N-WASP.

Publication Date: 2008 May 2 PMID: 18456802
Authors: May, K. L. - Morona, R.
Journal: J Bacteriol

The IcsA (VirG) protein of S. flexneri is a polarly localised, outer membrane protein that is essential for virulence. Within host cells, IcsA activates the host actin regulatory protein, N-WASP, which in turn recruits the Arp2/3 complex that nucleates host actin to form F-actin comet tails and initiate bacterial motility. Linker-insertion mutagenesis was undertaken to randomly introduce 5 amino acid in-frame insertions within IcsA. Forty-seven linker-insertion mutants were isolated and expressed in S. flexneri DeltaicsA. Mutants were characterised for IcsA protein production, cell surface-expression and localisation, intercellular spreading, F-actin comet tail formation, and N-WASP recruitment. Using this approach we have identified a putative auto-chaperone region required for IcsA biogenesis and our data suggests an additional region, not previously identified, is required for N-WASP recruitment.

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Extracytoplasmic processes impaired by inactivation of trxA (thioredoxin gene) in Bacillus subtilis.

Publication Date: 2008 May 2 PMID: 18456801
Authors: Moller, M. C. - Hederstedt, L.
Journal: J Bacteriol

The trxA gene is regarded essential in Bacillus subtilis but the roles of the TrxA protein in this gram-positive bacterium are largely unknown. Inactivation of trxA results in deoxyribonucleoside and cysteine or methionine auxotrophy. This phenotype is as expected if the TrxA protein is important for activity of the class Ib ribonucleotide reductase and adenosine-5'-phosphosulfate/3'-phosphoadenosine-5'-phophosulfate reductase. We demonstrate that TrxA-deficiency in addition causes defects in endospore and cytochrome c synthesis. These effects were suppressed by BdbD-deficiency indicating that TrxA in the cytoplasm is the primary electron donor to several different thiol-disulfide oxidoreductases active on the outer side of the B. subtilis cytoplasmic membrane.

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Signal pathway in the salt-activated expression of the SPI1/type III secretion system in Salmonella enterica serovar Typhimurium.

Publication Date: 2008 Apr 25 PMID: 18441068
Authors: Mizusaki, H. - Takaya, A. - Yamamoto, T. - Aizawa, S. I.
Journal: J Bacteriol

Salmonella enterica serovar Typhimurium secretes virulence factors for invasion called Sip proteins or Sips into its hosts through a type III secretion system (T3SS). In the absence of its host, S. enterica induces Sip secretion in response to sucrose or simple salts such as NaCl. We have analyzed induction of host-independent Sip secretion by monitoring protein secretion by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), assembly of needle complexes by electron microscopy, and transcription of virulence regulatory genes by quantitative reverse transcriptase PCR (real-time PCR). SDS-PAGE showed that the addition of sucrose or simple salts such as NaCl to the growth medium induced Sip secretion without altering flagellar protein secretion, which requires a distinct T3SS. Electron microscopy confirmed that the amount of secreted Sips increased as the number of assembled needle complexes increased. Real-time PCR revealed that added sucrose or NaCl enhanced transcription of hilA, hilC, and hilD, which encode known regulators of Salmonella virulence. However, epistasis analysis implicated HilD and HilA, but not HilC, in the direct pathway from the salt stimulus to the Sip secretion response. Further analyses showed that the two-component signal transduction pathway BarA/SirA, but not the two-component sensor kinase EnvZ, directly activated hilD and hilA transcription and, thus, Sip secretion, in response to either sucrose or NaCl. Finally, real-time PCR showed that salt does not influence transcription of the BarA/SirA-dependent csrB and csrC genes. A model is proposed for the major pathway through which sucrose or salt signal to enhance virulence gene expression.

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Salmonella enterica requires ApbC function for growth on tricarballylate: Evidence of functional redundancy between ApbC and IscU.

Publication Date: 2008 Apr 25 PMID: 18441067
Authors: Boyd, J. M. - Lewis, J. A. - Escalante-Semerena, J. C. - Downs, D. M.
Journal: J Bacteriol

Mutants of Salmonella enterica lacking apbC have nutritional and biochemical properties indicative of defects in [Fe-S] cluster metabolism. Here we show that apbC is required for S. enterica to use tricarballylate as carbon and energy source. Tricarballylate catabolism requires three gene products, TcuA, B and C. Of relevance to this work is the TcuB protein, which has two [4Fe-4S] clusters required for function, making it a logical target for the apbC effect. TcuB activity was 100-fold lower in an apbC mutant than the isogenic apbC(+) strain. Genetic data show that de-repression of the iscRSUAhscABfdxorf3 operon or over-expression of iscU from a plasmid compensates for the lack of ApbC during growth on tricarballylate. The studies described herein provide evidence that the scaffold protein IscU has functional overlap with ApbC, and that ApbC function is involved in the synthesis of active TcuB.

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Response regulator DegU of Listeria monocytogenes controls temperature-responsive flagellar gene expression in its unphosphorylated state.

Publication Date: 2008 Apr 25 PMID: 18441066
Authors: Mauder, N. - Williams, T. - Fritsch, F. - Kuhn, M. - Beier, D.
Journal: J Bacteriol

We demonstrate that in Listeria monocytogenes temperature-responsive transcriptional control of flagellar genes does not rely on the phosphorylation of the conserved phosphorylation site (D55) in the receiver domain of response regulator DegU. Furthermore, proper control of DegU-regulated genes involved in ethanol tolerance and virulence is independent of receiver phosphorylation.

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Evidence for an elongated dimeric structure of heparin-binding haemagglutinin from M. tuberculosis.

Publication Date: 2008 Apr 25 PMID: 18441065
Authors: Esposito, C. - Pethoukov, M. V. - Svergun, D. I. - Ruggiero, A. - Pedone, C. - Pedone, E. - Berisio, R.
Journal: J Bacteriol

Heparin-binding haemagglutinin, HBHA, is a virulence factor of tuberculosis which is responsible for extra-pulmonary dissemination of this disease. A thorough biochemical characterisation of HBHA has provided experimental evidence of a coiled coil nature of HBHA. These data, together with the low resolution structure of both a full-length and a truncated form of HBHA by Small Angle X-ray Scattering have unambiguously indicated that HBHA has a dimeric structure with an elongated shape.

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A genomic islet mediates flagellar phase variation in Escherichia coli strains carrying flagellin-specifying locus flk.

Publication Date: 2008 Apr 25 PMID: 18441064
Authors: Feng, L. - Liu, B. - Liu, Y. - Ratiner, Y. A. - Hu, B. - Li, D. - Zong, X. - Xiong, W. - Wang, L.
Journal: J Bacteriol

The occurrence of unilateral flagellar phase variation was previously demonstrated in Escherichia coli strains carrying the non-fliC flagellin-specifying locus flk. In this study, we investigated the mechanism involved in this process. By sequencing and sequence analysis, the flk region between chromosomal genes yhaC and rnpB was characterized in all described flk-positive E. coli strains including H35 identified in this study (the others are H3, H36, H47, and H53), and found to contain a putative integrase gene and flanking direct repeats in addition to the flk-specifying flagellin gene flkA and a fliC repressor gene flkB, indicating a typical genomic islet, named the flk GI. The horizontal transfer potential of the flk GI was indicated by the detection of the excised extrachromosomal circular form of the flk GI. By generating the fliC expressing variants of H3 and H47, unilateral flagellar phase variation in flk-positive strains was shown to be mediated by the excision of the flk GI. The function of the proposed integrase gene was confirmed by deletion and complementation test. The potential integration sites of the flk GI were identified. A general model for flagellar phase variation in flk-positive E. coli strains can be shown as fliC(off) + flkA(on) --> fliC(on) + flkA(-). This is the first time that a molecular mechanism for flagellar phase variation has been reported in E. coli.

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Chromosomal toxin-antitoxin systems may act as anti-addiction modules.

Publication Date: 2008 Apr 25 PMID: 18441063
Authors: Saavedra De Bast, M. - Mine, N. - Van Melderen, L.
Journal: J Bacteriol

Toxin-antitoxin (TA) systems are widespread among bacterial chromosomes and mobile genetic elements. Although in plasmids TA systems have a clear role in their vertical inheritance by selectively killing plasmid-free daughter-cells (post-segregational killing or addiction phenomenon), the physiological role of chromosomally-encoded ones still remains under debate. The assumption that chromosomally-encoded TA systems are part of stress response networks and/or programmed cell death machinery has been questioned recently by the observation that none of the 5 canonical chromosomally-encoded TA systems in the E. coli chromosome seems to confer any selective advantage under stressful conditions (Tsilibaris et al., J. Bacteriol., 2007). Their prevalence in bacterial chromosomes indicates that they might have been acquired through horizontal gene transfer. Once integrated in chromosomes, they might in turn interfere with their homologues encoded by mobile genetic elements. In this work, we show that the chromosomally-encoded ccdEch system indeed protects the cell against post-segregational killing mediated by its F-plasmid ccdFhomologue. Moreover, competition experiments have shown that this system confers a fitness advantage under post-segregational condition mediated by the ccdF system. We propose that ccdEch acts as an anti-addiction module and, more generally, that the integration of TA systems in bacterial chromosomes could drive the evolution of plasmid-encoded ones and select toxins that are no longer recognised by the anti-addiction module.

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The hybrid sensor kinase RscS integrates positive and negative signals to modulate biofilm formation in Vibrio fischeri.

Publication Date: 2008 Apr 25 PMID: 18441062
Authors: Geszvain, K. - Visick, K. L.
Journal: J Bacteriol

Overexpression of the Vibrio fischeri sensor kinase RscS induces expression of the symbiosis polysaccharide (syp) gene cluster and promotes biofilm phenotypes such as wrinkled colony morphology, pellicle formation and surface adherence. RscS is predicted to be a hybrid sensor kinase with a histidine kinase/ATPase domain (HATPase), a receiver-like domain (Rec) and a histidine phosphotransferase domain (Hpt). Bioinformatic analysis also revealed three potential signal detection domains within RscS: two transmembrane helices forming a transmembrane region (TMR), a large periplasmic (PP) domain and a cytoplasmic PAS domain. In this work, we genetically dissected the contribution of these domains to RscS function. Substitutions within the carboxy-terminal domain supported the identification of RscS as a hybrid sensor kinase: disruption of both HATPase and Rec domains abolished induction of syp transcription, wrinkled colony morphology, pellicle formation and surface adherence, while disruption of Hpt resulted in decreased activity. The PAS domain was also critical for RscS activity; substitutions in PAS resulted in a loss of activity. Generation of a cytoplasmic, N-terminal deletion derivative of RscS resulted in partial loss of activity, suggesting a role for localization to the membrane and/or sequences within the TMR and PP domain. Finally, substitutions within the first transmembrane helix of the TMR and deletions within the PP domain both resulted in increased activity. Thus, RscS integrates both inhibitory and stimulatory signals from the environment to regulate biofilm formation by V. fischeri.

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Spontaneous Amplification of the Actinorhodin Gene Cluster in Streptomyces coelicolor Involving Native Insertion Sequence IS466.

Publication Date: 2008 Apr 25 PMID: 18441061
Authors: Widenbrant, E. M. - Tsai, H. H. - Chen, C. W. - Kao, C. M.
Journal: J Bacteriol

We observed a spontaneous amplification in the Streptomyces coelicolor chromosome including genes encoding biosynthetic enzymes of the antibiotic, actinorhodin. A new junction of two tandem segments has, inserted within it, a third copy of a transposable element existing twice elsewhere in the chromosome, suggesting its involvement in the amplification mechanism.

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Glucomannan-mediated attachment of Rhizobium leguminosarum to pea root hairs is required for competitive nodule infection.

Publication Date: 2008 Apr 25 PMID: 18441060
Authors: Williams, A. - Wilkinson, A. - Krehenbrink, M. - Russo, D. - Zorreguieta, A. - Downie, J. A.
Journal: J Bacteriol

The Rhizobium leguminosarum biovar viciae genome contains several genes predicted to determine surface polysaccharides. Mutants predicted to affect the initial steps of polysaccharide synthesis were identified and characterised. In addition to the known cellulose (cel) and acidic exopolysaccharide (EPS) (pss) genes, we mutated three other loci; one (gmsA) determines glucomannan synthesis, one (gelA) determines a gel-forming polysaccharide, but the role of the other (an exoY-like gene) was not identified. Mutants were tested for attachment and biofilm formation in vitro and on root hairs; the mutant lacking the EPS was defective for both, but mutation of gelA or the exoY-like gene had no effect on either type of attachment. The cellulose (celA) mutant attached and formed normal biofilms in vitro, but did not form a biofilm on root hairs, although attachment did occur. The cellulose-dependent biofilm on root hairs appears not to be critical for nodulation, because the celA mutant competed similarly with the wild-type for nodule infection. The glucomannan (gmsA) mutant attached and formed normal biofilms in vitro, but was defective for attachment and biofilm formation on root hairs. Although this mutant formed nodules on peas, it was very strongly outcompeted by the wild-type in mixed inoculations, showing that glucomannan is critical for competitive nodulation. The polysaccharide synthesis genes around gmsA are highly conserved among other rhizobia and agrobacteria, but absent from closely-related bacteria (such as Brucella spp.) that are not normally plant associated, suggesting that these genes may play a wide role in bacterial-plant interactions.

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RscS functions upstream of SypG to control the syp locus and biofilm formation in Vibrio fischeri.

Publication Date: 2008 Apr 25 PMID: 18441059
Authors: Hussa, E. A. - Darnell, C. L. - Visick, K. L.
Journal: J Bacteriol

Two component signal transduction systems, composed of sensor kinase (SK) and response regulator (RR) proteins, allow bacterial cells to adapt to changes, such as environmental flux or the presence of a host. RscS is a SK required for Vibrio fischeri to initiate a symbiotic partnership with the Hawaiian squid Euprymna scolopes, likely due to its role in controlling the symbiosis polysaccharide (syp) genes and thus biofilm formation. To determine which RR(s) functions downstream of RscS, we performed epistasis experiments with a library of 35 response regulator mutants. We found that several RRs contributed to RscS-mediated biofilm formation in V. fischeri. However, only the syp-encoded symbiosis regulator SypG was required for both biofilm phenotypes and syp transcription induced by RscS. These data support the hypothesis that RscS functions upstream of SypG to induce biofilm formation. In addition, this work also revealed a role for the syp-encoded RR SypE in biofilm formation. To our knowledge, no other study has used a large-scale epistasis approach to elucidate two component signaling pathways. Therefore, this work both contributes to our understanding of regulatory pathways important for symbiotic colonization by V. fischeri and establishes a paradigm for evaluating two component pathways in the genomics era.

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Roles of pneumococcal DivIB in cell division.

Publication Date: 2008 Apr 25 PMID: 18441058
Authors: Le Gouellec, A. - Roux, L. - Fadda, D. - Massidda, O. - Vernet, T. - Zapun, A.
Journal: J Bacteriol

DivIB is a division protein conserved in most eubacteria, also known as FtsQ in Gram negative organisms. DivIB is localized at the division site and forms a complex with two other division proteins, FtsL and DivIC/FtsB. The precise function of these three bitopic membrane proteins that are central to division process remains unknown. We report here the characterization of a divIB deletion mutant in Streptococcus pneumoniae, which is a coccus that divides with parallel planes. Unlike its homologue FtsQ in Escherichia coli, pneumococcal DivIB is not required for growth in rich medium, but the DeltadivIB mutant forms chains of diplococci and a small fraction of enlarged cells with defective septa. However, the deletion mutant does not grow in a chemically defined medium. In the absence of DivIB and protein synthesis, the partner FtsL is rapidly degraded, whereas other division proteins are not affected, pointing to a role of DivIB in stabilizing FtsL. This is further supported by the finding that an additional copy of ftsL restores growth of the DeltadivIB mutant in defined medium. Functional mapping of the three distinct alpha-, beta-, and gamma-domains of the extracellular region of DivIB revealed that a complete beta-domain is required to fully rescue the deletion mutant. DivIB with a truncated beta-domain reverts only the chaining phenotype, indicating that DivIB has distinct roles early and late in the division process. Most importantly, the deletion of divIB increases the susceptibility to beta-lactams, more evidently in a resistant strain, suggesting a function in cell wall synthesis.

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The genome of Heliobacterium modesticaldum, a phototrophic representative of the Firmicutes containing the simplest photosynthetic apparatus.

Publication Date: 2008 Apr 25 PMID: 18441057
Authors: Sattley, W. M. - Madigan, M. T. - Swingley, W. D. - Cheung, P. C. - Clocksin, K. M. - Conrad, A. L. - Dejesa, L. C. - Honchak, B. M. - Jung, D. O. - Karbach, L. E. - Kurdoglu, A. - Lahiri, S. - Mastrian, S. D. - Page, L. E. - Taylor, H. L. - Wang, Z. T. - Raymond, J. - Chen, M. - Blankenship, R. E. - Touchman, J. W.
Journal: J Bacteriol

Despite being the only phototrophic representatives of the bacterial phylum Firmicutes, genomic analyses of heliobacteria have yet to be reported. Here we present the complete sequence and analysis of the genome of Heliobacterium modesticaldum, a thermophilic species of this unique family of phototrophs. The genome is a single 3.1 Mb circular chromosome containing 3138 open reading frames. As suspected from physiological studies of heliobacteria that have failed to show photoautotrophic growth, genes encoding enzymes of known autotrophic pathways in other phototrophic organisms, including ribulose bisphosphate carboxylase (RuBisCO-Calvin cycle), citrate lyase (reverse citric acid cycle), and malyl-CoA lyase (3-hydroxypropionate pathway), are not present in the H. modesticaldum genome. Thus, heliobacteria appear to be the only known anaerobic anoxygenic phototrophs incapable of autotrophy. Although some cellular activities, such as nitrogen fixation, have a full complement of genes in H. modesticaldum, other processes, including carbon metabolism and endosporulation, are more genetically streamlined than in most other low G+C Gram-positive bacteria. Moreover, several genes encoding photosynthetic functions in phototrophic purple bacteria are absent from the heliobacteria. In contrast to the nutritional flexibility of many anoxygenic phototrophs, the complete genome sequence of H. modesticaldum reveals an organism with a notable degree of metabolic specialization and genomic reduction.

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Regulatory network controlling extracellular proteins in Erwinia carotovora ssp. carotovora: FlhDC, the master regulator of flagellar genes, activates rsmB regulatory sRNA production by affecting gacA and hexA (lrhA) expression.

Publication Date: 2008 Apr 25 PMID: 18441056
Authors: Cui, Y. - Chatterjee, A. - Yang, H. - Chatterjee, A. K.
Journal: J Bacteriol

Erwinia carotovora ssp. carotovora (Ecc) produces an array of extracellular proteins (= exoproteins) including plant cell wall-degrading enzymes and Harpin, an effector responsible for eliciting hypersensitive reaction. Exoprotein genes are co-regulated by the quorum-sensing signal, N-acyl homoserine lactone, plant signals, an assortment of transcriptional factors/regulators (GacS/A, ExpR1, ExpR2, KdgR, RpoS, HexA, RsmC) and post-transcriptional regulators (RsmA, rsmB RNA). rsmB RNA production is positively regulated by GacS/A, a two-component system, and negatively regulated by HexA (= PecT in Erwinia chrysanthemi; LrhA [LysR homolog A] in E. coli), and RsmC, a putative transcriptional adaptor. While free RsmA, an RNA-binding protein, promotes decay of mRNAs of exoprotein genes, binding of RsmA with rsmB RNA neutralizes the RsmA effect. In the course of studies of GacA regulation, we discovered that a locus, bearing strong homology to the flhDC operon of E. coli, also controls extracellular enzyme production. A Tn-insertion FlhDC(-) mutant produces very low levels of pectate lyase, polygalacturonase, cellulase, protease and HarpinEcc and is severely attenuated in its plant virulence. The production of these exoproteins is restored in the mutant carrying an FlhDC(+) plasmid. Sequence analysis and transcript assays disclosed that the flhD operon of Ecc, like those of other enterobacteria, consists of flhD and flhC. Complementation analysis revealed that the regulatory effect requires functions of both flhD and flhC products. The data presented here show that FlhDC positively regulates gacA, rsmC and fliA, and negatively regulates hexA (lrhA). Evidence shows that FlhDC controls extracellular protein production through cumulative effects on hexA and gacA. Reduced levels of GacA and elevated levels of HexA in the FlhDC(-) mutant are responsible for inhibition of rsmB RNA production, a condition conducive to accumulation of free RsmA. Indeed, studies with an RsmA(-) FlhDC(-) double mutant and multiple copies of rsmB(+) DNA establish that the negative effect of FlhDC deficiency is exerted via RsmA. The FlhDC-mediated regulation of fliA has no bearing on exoprotein production in Ecc. Our observations for the first time establish a regulatory connection between FlhDC, HexA, GacA and rsmB RNA in the context of exoprotein production and virulence of Ecc.

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Streptococcal Antagonism In Oral Biofilms: Streptococcus sanguinis and Streptococcus gordonii interference with Streptococcus mutans.

Publication Date: 2008 Apr 25 PMID: 18441055
Authors: Kreth, J. - Zhang, Y. - Herzberg, M. C.
Journal: J Bacteriol

Biofilms are polymicrobial, with diverse bacterial species competing for limited space and nutrients. Under healthy conditions, the different species in biofilms maintain an ecological balance. This balance can be disturbed by environmental factors and interspecies interactions. These perturbations can enable dominant growth of certain species leading to disease. To model clinically relevant interspecies antagonism, we studied three well-characterized and closely related oral species, Streptococcus gordonii, Streptococcus sanguinis and cariogenic Streptococcus mutans. S. sanguinis and S. gordonii used oxygen availability and the differential production of hydrogen peroxide (H2O2) to compete effectively against S. mutans. Interspecies antagonism was influenced by glucose with reduced production of H2O2. Furthermore, aerobic conditions stimulated the competence system and the expression of the bacteriocin mutacin IV of S. mutans, as well as the H2O2-dependent release of heterologous DNA from mixed cultures of S. sanguinis and S. gordonii. These data provide new insights into ecological factors that determine the outcome of competition between pioneer colonizing oral streptococci and the survival mechanisms of S. mutans in the oral biofilm.

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The ArgP protein stimulates the Klebsiella pneumoniae gdhA promoter in a lysine-sensitive manner.

Publication Date: 2008 Apr 18 PMID: 18424527
Authors: Goss, T. J.
Journal: J Bacteriol

The lysine-sensitive factor that binds to the upstream region of the Klebsiella pneumoniae gdhA promoter and stimulates gdhA transcription during growth in minimal medium has been proposed to be the K. pneumoniae ArgP protein (M. R. Nandineni, R. S. Laishram, and J. Gowrishankar. 2004. J. Bacteriol. 186:6391-6399.). A knockout mutation of the K. pneumoniae argP gene was generated and used to assess the roles of exogenous lysine and argP in the regulation of the gdhA promoter. Disruption of argP reduced the strength and the lysine-dependent regulation of the gdhA promoter. Electrophorectic mobility shift assays using crude extracts prepared from wild type and argP-defective strains indicted the presence of an argP-dependent factor whose ability to bind the gdhA promoter was lysine-sensitive. DNase I footprinting studies using purified K. pneumoniae ArgP protein indicated that ArgP bound the region that lies approximately 50 to 100 base pairs upstream of the gdhA transcription start site in a manner that was sensitive to the presence of lysine. Substitutions within the region bound by ArgP affected the binding of ArgP to the gdhA promoter region in vitro and the argP-dependent stimulation of the gdhA promoter in vivo. These observations suggest that elevated intracellular levels of lysine reduce the affinity of ArgP for its binding site at the gdhA promoter, preventing ArgP from binding to and stimulating transcription from the promoter in vivo.

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The critical role of embC in Mycobacterium tuberculosis.

Publication Date: 2008 Apr 18 PMID: 18424526
Authors: Goude, R. - Amin, A. G. - Chatterjee, D. - Parish, T.
Journal: J Bacteriol

Arabinan polymers are major components of the cell wall in Mycobacterium tuberculosis and are involved in maintaining its structure, as well as playing a role in host-pathogen interactions. In particular, lipoarabinomannan (LAM) has multiple immuno-modulatory effects. In the non-pathogenic species, Mycobacterium smegmatis, EmbC has been identified as a key arabinosyl transferase involved in the incorporation of arabinose into LAM and an embC mutant is viable, but lacks LAM. In contrast, we demonstrate here, that in M. tuberculosis, embC is an essential gene under normal growth conditions, suggesting a more crucial role for LAM in the pathogenic mycobacteria. M. tuberculosis EmbC has a similar activity to M. smegmatis EmbC, since we were able to complement an embC mutant of M. smegmatis with embCMtb, confirming that it encodes a functional arabinosyltransferase. In addition, we observed that the size of LAM produced in M. smegmatis was dependent on the level of expression of embCMtb. Northern analysis revealed that embC is expressed as part of a polycistronic message encompassing embC and three upstream genes. The promoter region for this transcript was identified and found to be up-regulated in stationary phase, but down-regulated during hypoxia-induced non-replicating persistence. In conclusion, we have identified one of the key genes involved in LAM biosynthesis in M. tuberculosis and confirmed its essential role in this species.

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UpaG, a new member of the trimeric autotransporter family of adhesins in uropathogenic Escherichia coli.

Publication Date: 2008 Apr 18 PMID: 18424525
Authors: Valle, J. - Mabbett, A. N. - Ulett, G. C. - Toledo-Arana, A. - Wecker, K. - Totsika, M. - Schembri, M. A. - Ghigo, J. M. - Beloin, C.
Journal: J Bacteriol

The ability of Escherichia coli to colonize both intestinal and extra-intestinal sites is driven by the presence of specific virulence factors, among which are the autotransporter (AT) proteins. Members of the trimeric AT adhesin family are important virulence factors for several Gram-negative pathogens and mediate adherence to eukaryotic cells and extracellular matrix proteins. In this study, we characterized a new trimeric AT adhesin (UpaG) from uropathogenic E. coli. Molecular analysis of UpaG revealed that it is translocated to the cell-surface and adopts a multimeric conformation. We demonstrated that UpaG is able to promote cell aggregation and biofilm formation on abiotic surfaces in CFT073 and various UPEC strains. In addition, UpaG expression resulted in the adhesion of CFT073 to human bladder epithelial cells, with specific affinity to fibronectin and laminin. Prevalence analysis revealed that upaG is strongly associated with E. coli strains from the B2 and D phylogenetic groups, while deletion of upaG had no significant effect on the ability of CFT073 to colonize the mouse urinary tract. Thus, UpaG is a novel trimeric AT adhesin from E. coli that mediates aggregation, biofilm formation and adhesion to various ECM proteins.

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Conserved Residues Asp16 and Pro24 of TnaC-tRNAPro Participate in Tryptophan Induction of tna Operon Expression.

Publication Date: 2008 Apr 18 PMID: 18424524
Authors: Cruz-Vera, L. R. - Yanofsky, C.
Journal: J Bacteriol

In E. coli, interactions between the nascent TnaC-tRNA(Pro) peptidyl-tRNA and the translating ribosome create a tryptophan binding site in the ribosome where bound tryptophan inhibits TnaC-tRNA(Pro) cleavage. This inhibition delays ribosome release, thereby inhibiting Rho factor binding and action, resulting in increased tna operon transcription. Replacing Trp12 of TnaC by any other amino acid residue was shown previously to prevent tryptophan binding and induction of tna operon expression. Genome-wide comparisons of TnaC amino acid sequences identify Asp16 and Pro24, as well as Trp12, as highly conserved TnaC residues. Replacing these residues by other residues was previously shown to influence tryptophan induction of tna operon expression. In this study in vitro analyses were performed to examine the potential roles of Asp16 and Pro24 in tna operon induction. Replacing Asp16 and Pro24 of TnaC of E. coli by other amino acids established that these residues are essential for free tryptophan binding and inhibition of TnaC-tRNA(Pro) cleavage - at the peptidyl transferase center. Asp16 and Pro24 are in fact located in spatial positions corresponding to critical residues of AAP, another ribosome regulatory peptide. Sparsomycin-methylation protection studies further suggested that segments of 23S RNA were arranged differently in ribosomes bearing TnaC's with either the Asp16Ala or the Pro24Ala change. Thus features of the amino acid sequence of TnaC of the nascent TnaC-tRNA(Pro) peptidyl-tRNA, in addition to the presence of Trp12, are necessary for the nascent peptide to create a tryptophan binding/inhibition site in the translating ribosome.

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Mutations in the tacF Gene of Clinical Strains and Laboratory Transformants of Streptococcus pneumoniae: Impact on Choline Auxotrophy and Growth Rate.

Publication Date: 2008 Apr 18 PMID: 18424523
Authors: Gonzalez, A. - Llull, D. - Morales, M. - Garcia, P. - Garcia, E.
Journal: J Bacteriol

The nutritional requirement that Streptococcus pneumoniae has for the aminoalcohol choline as a component of teichoic and lipoteichoic acids appears to be exclusive to this prokaryote. A mutation in the tacF gene, which putatively encodes an integral membrane protein (possibly, a teichoic acid repeat unit transporter), has been recently identified as responsible for generating a choline-independent phenotype of S. pneumoniae (Damjanovic, M., A. S. Kharat, A. Eberhardt, A. Tomasz, and W. Vollmer. 2007. J. Bacteriol. 189:7105-7111). We now report that Streptococcus mitis can grow in choline-free medium as previously illustrated for Streptococcus oralis. While we confirmed the finding by Damjanovic et al. of the involvement of TacF in the choline dependence of the pneumococcus, the genetic transformation of S. pneumoniae R6 using S. mitis SK598 DNA and several PCR-amplified tacF fragments, suggested that a minimum of two mutations were required to confer improved fitness to choline-independent S. pneumoniae mutants. This conclusion is supported by sequencing results also reported here indicating that a spontaneous mutant of S. pneumoniae (strain JY2190) able to proliferate in the absence of choline (or analogs) is also a double mutant for the tacF gene. Microscopic observations and competition experiments during co-cultivation of choline-independent strains confirmed that a minimum of two amino acid changes were required to confer improved fitness to choline-independent pneumococcal strains when growing in media lacking any aminoalcohol. Our results suggest complex relationships among the different regions of the TacF teichoic acid repeat unit transporter.

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Transcriptional interference and repression modulates conjugative ability of the symbiotic plasmid of Rhizobium etli.

Publication Date: 2008 Apr 18 PMID: 18424522
Authors: Sepulveda, E. - Perez-Mendoza, D. - Ramirez-Romero, M. A. - Soto, M. J. - Lopez-Lara, I. M. - Geiger, O. - Sanjuan, J. - Brom, S. - Romero, D.
Journal: J Bacteriol

Bacteria of the order Rhizobiales are able to establish nitrogen-fixing symbioses with legumes. Commonly, genes for symbiosis are harboured on large symbiotic plasmids. Although transfer of symbiotic plasmids is commonly detected in nature, there are few experimentally characterized examples. In Rhizobium etli, the product of rctA inhibits conjugation of the symbiotic plasmid by reducing transcription of the virB operon. rctA is transcribed divergently from this operon and its product is predicted to have a DNA binding domain. Here, using DNAase I footprinting and binding assays, we demonstrate specific binding of RctA to the virB operon promoter. A nine-bp motif in the spacer region of this promoter (the rbm box), as well as the presence of a functional -10 region, are critical elements for RctA binding. Transcriptional fusion analyses revealed that elimination of either element provokes a relief of RctA-mediated repression. These data support a model in which RctA inhibits access of the RNA polymerase to the virB promoter. Interestingly, rctA expression levels are modulated by transcriptional interference with transcripts emanating from the virB promoter. This phenomenon adds another level of regulation for this system, thus revealing a novel mechanism of plasmid transfer regulation in the Rhizobiales.

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Identification of a novel trimeric autotransporter adhesin in the cryptic genospecies of Haemophilus.

Publication Date: 2008 Apr 18 PMID: 18424521
Authors: Sheets, A. J. - Grass, S. A. - Miller, S. E. - Geme, J. W. 3rd
Journal: J Bacteriol

Haemophilus biotype IV strains belonging to the recently recognized Haemophilus cryptic genospecies are an important cause of maternal genital tract and neonatal systemic infections and initiate infection by colonizing the genital or respiratory epithelium. To gain insight into the mechanism of Haemophilus cryptic genospecies colonization, we began by examining prototype strain 1595 and three other strains for adherence to genital and respiratory epithelial cell lines. Strain 1595 and two of the three other strains demonstrated efficient adherence to all cell lines. Using a stably adherent variant of strain 1595, we generated a Mariner transposon library and identified 16 non-adherent mutants. All of these mutants lacked surface fibers and contained an insertion in the same ORF, encoding a 157-kDa protein designated Cha for cryptic Haemophilus adhesin. Analysis of the predicted amino acid sequence of Cha revealed the presence of an N-terminal signal peptide and a C-terminal domain bearing homology to YadA-like and Hia-like trimeric autotransporters. Examination of the C-terminal 120 amino acids of Cha demonstrated mobility as a trimer on an SDS-PAGE gel and the capacity to present the passenger domain of the Hia trimeric autotransporter on the bacterial surface. Southern analysis revealed that the gene encoding Cha is conserved among clinical isolates of the Haemophilus cryptic genospecies and is absent from the closely related H. influenzae. We speculate that Cha is important in the pathogenesis of disease due to the Haemophilus cryptic genospecies and is in part responsible for the apparent tissue tropism of these organisms.

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Functional analysis of the protein machinery required for the transport of lipopolysaccharide to the outer membrane of Escherichia coli.

Publication Date: 2008 Apr 18 PMID: 18424520
Authors: Sperandeo, P. - Lau, F. K. - Carpentieri, A. - De Castro, C. - Molinaro, A. - Deho, G. - Silhavy, T. J. - Polissi, A.
Journal: J Bacteriol

Lipopolysaccharide (LPS) is an essential component of the outer membrane (OM) in most Gram negative bacteria and its structure and biosynthetic pathway are well known. Nevertheless, the mechanisms of transport and assembly of this molecule to the cell surface are poorly understood. The inner membrane (IM) transport protein MsbA, is responsible for flipping LPS across the IM. Additional components of the LPS transport machinery downstream of MsbA have been identified, namely the OM protein complex LptD/LptE (formerly Imp/RlpB, respectively), the periplasmic LptA protein, the IM associated cytoplasmic ABC protein LptB, and LptC (formerly YrbK), a new essential IM component of the LPS transport machinery characterized in this work. Here we show that depletion of any of the above proteins leads to common phenotypes i) appearance of abnormal membrane structures in the periplasm; ii) accumulation of de novo synthesized LPS in two membrane fractions with lower density than the OM; iii) accumulation of a modified LPS, which is ligated to repeating units of colanic acid in the outer leaflet of IM. Our results suggest that LptA, LptB, LptC, LptD and LptE operate in the LPS assembly pathway and, together with other as yet unidentified components, could be part of a complex devoted to the transport of LPS from the periplasmic surface of the IM to the OM. Moreover, the location of at least one of these five proteins in each and every cellular compartment suggests a model for how the LPS assembly pathway is organized and ordered in space.

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Formate acts as a diffusible signal to induce Salmonella invasion.

Publication Date: 2008 Apr 18 PMID: 18424519
Authors: Huang, Y. - Suyemoto, M. - Garner, C. D. - Cicconi, K. M. - Altier, C.
Journal: J Bacteriol

To infect an animal host, Salmonella must penetrate the intestinal epithelial barrier. This process of invasion requires a type III secretion system encoded within Salmonella pathogenicity island I (SPI1). We found that a mutant of acetate kinase and phosphotransacetylase (ackA-pta) was deficient in invasion and SPI1 expression, but that invasion gene expression was completely restored by supplying media conditioned by growth of the wild type strain, suggesting that a signal produced by the wild type but not by the ackA-pta mutant was required for invasion. This mutant also excreted 68-fold less formate into culture medium, and the addition of sodium formate to cultures restored both the expression of SPI1 and the invasion of cultured epithelial cells by the mutant. The effect of formate was pH-dependent, requiring a pH below neutrality, and studies in mice showed that the distal ileum, the preferred site of Salmonella invasion in this species, had the appropriate formate concentration and pH to elicit invasion, while the cecum contained no detectable formate. Furthermore, we found that formate affected the major regulators of SPI1, hilA and hilD, but that the primary routes of formate metabolism played no role in its activity as a signal.

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YscP and YscU Switch the Substrate Specificity of the Yersinia Type III Secretion System by Regulating Export of the Inner Rod Protein YscI.

Publication Date: 2008 Apr 18 PMID: 18424518
Authors: Wood, S. - Jin, J. - Lloyd, S. A.
Journal: J Bacteriol

Pathogenic yersiniae utilize a type III secretion system to inject antihost factors, called Yops, directly into the cytosol of eukaryotic cells. The Yops are injected via a needle-like structure, comprised of the YscF protein, on the bacterial surface. While the needle is being assembled, Yops cannot be secreted. YscP and YscU switch the substrate specificity of the secretion system to enable Yop export once the needle attains its proper length. Here we demonstrate that the inner rod protein YscI plays a critical role in substrate specificity switching. We show that YscI is secreted by the type III secretion system and that YscI secretion is abnormally elevated in a yscP mutant. Furthermore, we show that mutations in the cytoplasmic domain of YscU reduce YscI secretion by the yscP null strain. We also demonstrate that three YscI mutants (Q84A, L87A and L96A) secrete substantial amounts of Yops, yet exhibit severe defects in needle formation. In the absence of YscP, these same mutants assemble needles but are unable to secrete Yops. Together, these results suggest that formation of the inner rod, not the needle, is critical for substrate specificity switching and that YscP and YscU exert their effects on substrate export by controlling the secretion of YscI.

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ApoFnr binds as a monomer to promoters regulating the expression of enterotoxin genes of Bacillus cereus.

Publication Date: 2008 Apr 18 PMID: 18424517
Authors: Esbelin, J. - Jouanneau, Y. - Armengaud, J. - Duport, C.
Journal: J Bacteriol

Bacillus cereus Fnr is a member of the Crp/Fnr (cAMP-binding protein/fumarate nitrate reduction regulatory protein) family of helix-turn-helix transcriptional regulators. It is essential for the expression of hbl and nhe enterotoxin genes independently of the oxygen tension in the environment. We studied aerobic Fnr binding to target sites in promoters regulating the expression of enterotoxin genes. B. cereus Fnr was overexpressed and purified as either a C-terminal His-tagged (FnrHis) or an N-terminal Strep-tagged (StrepFnr) fusion protein. Both recombinant Fnrs were produced as apoforms (clusterless) and occurred as mixtures of monomer and oligomers in solution. However, apoFnrHis was mainly monomeric, while apoStrepFnr was mainly oligomeric, suggesting that the His-tagged C-terminal extremity may interfere with oligomerization. The oligomeric state of apoStrepFnr was dithiothreitol-sensitive, underlining the importance of a disulphide bridge for apoFnr oligomerization. Electrophoretic mobility shift assays showed that monomeric, but not oligomeric apoFnr, bound to specific sequences located in the promoter regions of the enterotoxin regulators fnr, resDE and plcR and the structural genes hbl and nhe. The question of whether apoFnr binding is regulated in vivo by redox-dependent oligomerization is discussed.

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Bacteriophage P22 Antitermination BoxB Sequence Requirements Are Complex and Overlap with Those of {lambda}

Publication Date: 2008 Apr 18 PMID: 18424516
Authors: Cocozaki, A. I. - Ghattas, I. R. - Smith, C. A.
Journal: J Bacteriol

Transcription antitermination in phages lambda and P22 uses N proteins that bind to similar boxB RNA hairpins in regulated transcripts. In contrast to the lambda N-boxB interaction, the P22 N-boxB interaction has not been extensively studied. An NMR structure of the P22 N peptide boxBleft complex and limited mutagenesis have been reported, but do not reveal a consensus sequence for boxB. We have used a plasmid-based antitermination system to screen boxBs with random loops and to test boxB mutants. We find P22 N requires boxB to have a GNRA-like loop with no simple requirements on remaining sequences in the loop or stem. U:A or A:U base pairs are strongly preferred adjacent to the loop, and appear to modulate N binding in cooperation with the loop and distal stem. A few GNRA-like hexaloops have moderate activity. Some boxB mutants bind P22 and lambda N, indicating that the requirements imposed on boxB by P22 N overlap those imposed by lambda N. Point mutations can dramatically alter boxB specificity between P22 and lambda N. A boxB specific for P22 N can be mutated to lambda N specificity by a series of single mutations via a bifunctional intermediate, as predicted by neutral theories of evolution.

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Glucosamine found as a substituent of both phosphate groups in Bordetellae lipid A backbones: role of a BvgAS-activated ArnT ortholog.

Publication Date: 2008 Apr 18 PMID: 18424515
Authors: Marr, N. - Tirsoaga, A. - Blanot, D. - Fernandez, R. - Caroff, M.
Journal: J Bacteriol

Endotoxins are amphipathic lipopolysaccharides (LPS), major constituents of the outer membrane of Gram-negative bacteria. They consist of a lipid region covalently linked to a core oligosaccharide to which may be linked a repetitive glycosidic chain carrying antigenic determinants. Most of their biological activities have been associated with the lipid moiety of the molecule: unique to Gram-negative bacteria, it is a ligand of the mammalian TLR4-MD2-CD14 pathogen recognition receptor complex. Lipid A preparations are often heterogeneous, both with respect to the number and length of fatty acids, and the nature of substituents on the phosphate groups, when present. The variants can significantly affect host immune responses. Nine species have been described in the Bordetella genus and the lipopolysaccharide fine structures of seven of them have been published. In this report, lipids A from B. pertussis Tohama I and B. bronchiseptica strain 4650 were further characterized and revealed to have a glucosamine substituting both lipid A phosphate groups of the diglucosamine backbone. These substitutions have not been previously described in bordetellae. Moreover, a B. pertussis transposon mutation that maps within a gene encoding a Bordetella ArnT (formerly PmrK) glycosyl transferase ortholog does not carry this substitution, thus providing a genetic basis for the modification. RT-PCR of this locus showed that it is Bvg-regulated, suggesting that the ability of Bordetella to modify lipid A via this glucosamine modification is a potential virulence trait.

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Reciprocal expression of ospA and ospC in single cells of Borrelia burgdorferi.

Publication Date: 2008 May PMID: 18359818
Authors: Srivastava, S. Y. - de Silva, A. M.
Journal: J Bacteriol

Outer surface proteins (Osp) A and C of the Lyme disease spirochete (Borrelia burgdorferi) are selectively produced and of functional significance in the tick vector and mammalian host, respectively. Some studies indicate a simple, reciprocal relationship where the signals and pathways that turn on ospC also turn off ospA. Other studies indicate a more complex regulation where many spirochetes produce both proteins and others produce one of the proteins or neither protein. Here, we have used flow cytometry to characterize ospA and ospC transcript and protein levels in individual bacterial cells grown in culture. The results support a simple, reciprocal model where, at the level of single cells, the transcription of ospC is linked to the repression of ospA. We also demonstrate that under conditions conducive for OspC production, spirochetes display an "all or none" response, with some cells displaying high levels of ospC transcription and others demonstrating little or no transcription. Despite the reciprocal regulation of ospA and ospC at the single-cell level, we propose that spirochetes display an array of phenotypes due to stochasticity in the pathways that regulate osp expression and the slow turnover of outer surface proteins.

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