Journal of Bacteriology

Auto-regulation of the Synthesis of the MobM Relaxase Encoded by the Promiscuous Plasmid pMV158.

Publication Date: 2012 Jan 27 PMID: 22287528
Authors: Lorenzo-Diaz, F. - Solano-Collado, V. - Lurz, R. - Bravo, A. - Espinosa, M.
Journal: J Bacteriol

The streptococcal promiscuous plasmid pMV158 (5540-bp) replicates by the rolling circle mechanism and can be mobilized among a wide number of Gram-positive and -negative bacteria. The plasmid region involved in its conjugative transfer includes the mobM gene, which encodes the MobM relaxase, and the cis-acting origin of transfer (oriT). MobM initiates transfer by cleavage of supercoiled pMV158 DNA at a specific dinucleotide within oriT. In the present work, we have performed a detailed transcriptional analysis to assess the role of MobM in the control of its own gene expression. By in vivo and in vitro approaches, we demonstrated that mobM transcription in Escherichia coli was mostly initiated from a promoter (Pmob2) different from the one (Pmob1) used in Lactococcus lactis. Whereas promoter Pmob1 was embedded within the oriT sequence, promoter Pmob2 was placed apart but adjacent to oriT. Further, MobM was able to repress the expression of its own gene from both promoters. Given the promiscuity of pMV158, the organization of the mobM promoter region suggests a strategy of the plasmid to cope with different transcription machineries of the hosts it colonizes.

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Global transcriptional control by NsrR in Bacillus subtilis.

Publication Date: 2012 Jan 27 PMID: 22287527
Authors: Kommineni, S. - Lama, A. - Popescu, B. - Nakano, M. M.
Journal: J Bacteriol

The NO-sensitive NsrR repressor of Bacillus subtilis, which carries a [4Fe-4S] cluster, controls transcription of nasD and hmp (class I regulation) under anaerobic conditions. Here we describe another class of NsrR regulation (class II regulation) that controls a more diverse collection of genes. Base substitution analysis showed that [4Fe-4S]-NsrR recognizes a partial dyad symmetry within the class I cis-acting sites, whereas NO-insensitive interaction of NsrR with an A+T-rich class II regulatory site showed relaxed sequence specificity. Genome-wide transcriptome studies identified genes that are under the control of the class II NsrR regulation. The class II NsrR regulon includes genes controlled by both AbrB and Rok repressors, which also recognize A+T-rich sequences, and by the Fur repressor. Transcription of class II genes was elevated in an nsrR mutant during anaerobic fermentative growth with pyruvate. Although NsrR binding to the class II regulatory sites was NO insensitive in vitro, transcription of class II genes was moderately induced by NO, which involved reversal of NsrR-dependent repression, suggesting that class II repression is also NO sensitive. In all NsrR-repressed genes tested, the loss of NsrR repressor activity was not sufficient to induce transcription, as induction required the ResD response regulator. The ResD-ResE signal transduction system is essential for activation of genes involved in aerobic and anaerobic respiration. This study indicated coordinated regulation between ResD and NsrR and uncovered a new role of ResD and NsrR in transcriptional regulation during anaerobiosis of B. subtilis.

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Reduced Aeration Affects the Expression of the NorB Efflux Pump of S. aureus by Post-translational Modification of MgrA.

Publication Date: 2012 Jan 27 PMID: 22287526
Authors: Truong-Bolduc, Q. C. - Liao, C. H. - Villet, R. - Bolduc, G. R. - Estabrooks, Z. - Taguezem, G. F. - Hooper, D. C.
Journal: J Bacteriol

We previously showed that at acid pH, the transcription of norB encoding the NorB efflux pump increases due to a reduction in the phosphorylation level of MgrA, which in turn leads to a reduction in bacterial killing by moxifloxacin, a substrate of the NorB efflux pump.In this study, we demonstrated that reduced oxygen levels did not affect the transcript levels of mgrA, but modified the dimerization of the MgrA protein, which remained mostly in its monomeric form. Under reduced aeration, we also observed a 21.7-fold increase in the norB transcript levels after 60 min of growth that contributed to a fourfold increase in the MICs of moxifloxacin and sparfloxacin of S. aureus RN6390.The relative proportion of MgrA in monomeric and dimeric forms was altered by treatment with H(2)O(2), but incubation of purified MgrA with extracts of cells grown under reduced but not normal aeration prevented MgrA from being converted to its dimeric DNA-binding form. This modification was associated with cleavage of a fragment of the dimerization domain of MgrA without change in MgrA phosphorylation and an increase in transcript levels of genes encoding serine proteases in cells incubated at reduced aeration.Taken together, these data suggest that modification of MgrA by proteases underlies the reversal of its repression of norB and increased resistance to NorB substrates in response to reduce aeration conditions, illustrating a third mechanism of post-translational modification, in addition to oxidation and phosphorylation, that modulates the regulatory activities of MgrA.

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The ChrA Response Regulator in Corynebacterium diphtheriae Controls Hemin-Regulated Gene Expression through Binding to the hmuO and hrtAB Promoter Regions.

Publication Date: 2012 Jan 27 PMID: 22287525
Authors: Burgos, J. M. - Schmitt, M. P.
Journal: J Bacteriol

Corynebacterium diphtheriae, the etiologic agent of diphtheria, utilizes heme and hemoglobin (Hb) as iron sources for growth. Heme-iron utilization involves HmuO, a heme oxygenase that degrades cytosolic heme, resulting in the release of heme associated iron. Expression of the hmuO promoter is under dual regulation, in which transcription is repressed by DtxR and iron and activated by a heme source, such as hemin or Hb. Hemin-dependent activation is primarily mediated by the ChrAS two-component system, in which ChrS is a putative heme-responsive sensor kinase while ChrA is proposed to serve as a response regulator that activates transcription. It was recently shown that the ChrAS system similarly regulates the hrtAB genes, which encode an ABC transporter involved in the protection of C. diphtheriae from hemin toxicity. In this study, we characterized the phospho-relay mechanism in the ChrAS system and provide evidence for the direct regulation of the hmuO and hrtAB promoters by ChrA. A fluorescence staining method was used to show that ChrS undergoes auto-phosphorylation and that the phosphate moiety is subsequently transferred to ChrA. Promoter fusion studies identified regions upstream of the hmuO and hrtAB promoters that are critical for the heme-dependent regulation by ChrA. Electrophoretic mobility shift assays revealed that ChrA specifically binds at the hmuO and hrtAB promoter regions and that binding is phosphorylation-dependent. A phosphorylation defective mutant of ChrA (ChrAD50A) exhibited significantly diminished binding to the hmuO promoter region relative to wild type ChrA. DNase I footprint analysis further defined the sequences in the hmuO and hrtAB promoters that are involved in ChrA binding and this analysis revealed that the DtxR binding site at the hmuO promoter partially overlaps the binding site for ChrA. DNase I protection studies as well as promoter fusion analysis suggest that ChrA and DtxR compete for binding at the hmuO promoter. Collectively, these data demonstrate that the ChrA response regulator directly controls the expression of hmuO and the hrtAB genes and the binding activity of ChrA is dependent on phosphorylation by its cognate sensor kinase ChrS.

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A Clp/Hsp100 chaperone functions in Myxococcus xanthus sporulation and self-organization.

Publication Date: 2012 Jan 27 PMID: 22287524
Authors: Yan, J. - Garza, A. G. - Bradley, M. D. - Welch, R. D.
Journal: J Bacteriol

The Clp/Hsp100 proteins are chaperones that play a role in protein degradation and re-activation. In bacteria, they exhibit a high degree of pleiotropy, effecting both individual and multicellular phenotypes. In this article we present the first characterization of a Clp/Hsp100 homolog in Myxococcus xanthus (MXAN_4832). Deletion of MXAN_4832 causes defects in both swarming and aggregation related to cell motility and the production of fibrils, which are an important component of the extracellular matrix of a swarm. The deletion also effects the formation of myxospores during development, causing them to become heat-sensitive. The protein product of MXAN_4832 can act as a chaperone in vitro, providing biochemical evidence in support of our hypothesis that MXAN_4832 is a functional Clp/Hsp100 homolog. There are a total of twelve Clp/Hsp100 homologs in M. xanthus including MXAN_4832, and based on its mutational and biochemical characterization they may well represent an important group.

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Inactivation of Haemophilus influenzae LPS biosynthesis genes interferes with outer membrane localization of the Hap autotransporter.

Publication Date: 2012 Jan 27 PMID: 22287523
Authors: Spahich, N. A. - Hood, D. W. - Moxon, E. R. - St Geme, J. W. 3rd
Journal: J Bacteriol

Nontypeable Haemophilus influenzae is a major cause of localized respiratory tract disease and initiates infection by colonizing the nasopharynx. Colonization requires adherence to host epithelial cells, mediated by surface proteins such as the Hap adhesin. In this study, we identified a relationship between Hap levels in the outer membrane and lipopolysaccharide (LPS) biosynthesis enzymes. We found that mutation of the rfaF, pgmB, lgtC, kfiC, orfE, rfbP, lsgB, and lsgD genes involved in the synthesis of the LPS oligosaccharide core in H. influenzae strain Rd/HapS243A resulted in loss of Hap in the bacterial outer membrane and a decrease in hap transcript. In contrast, the same mutations had no effect on outer membrane localization of H. influenzae P5 and IgA1 protease or levels of the p5 or iga1 transcripts, suggesting a Hap-specific effect. Elimination of the HtrA periplasmic protease resulted in a return of Hap to the outer membrane and restoration of hap transcript levels. Consistently, in lgtC phase off bacteria Hap was absent from the outer membrane and hap transcript was reduced. Hap localization and hap transcript levels were not related to LPS size but to the functions of the LPS biosynthesis enzymes themselves. We speculate that the lack of certain LPS biosynthesis enzymes causes Hap to mislocalize and accumulate in the periplasm, where it is degraded by HtrA. This degradation then leads to a decrease in hap transcript. Together, the data highlight a novel interplay between Hap and LPS biosynthesis that can influence H. influenzae interactions with the host.

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Transcriptional and metabolomic consequences of luxS inactivation reveal a metabolic rather than quorum sensing role for LuxS in Lactobacillus reuteri 100-23.

Publication Date: 2012 Jan 27 PMID: 22287522
Authors: Wilson, C. M. - Aggio, R. B. - O'Toole, P. W. - Villas Boas, S. - Tannock, G. W.
Journal: J Bacteriol

Autoinducer-2 (AI-2)-mediated quorum sensing has been extensively studied in relation to the regulation of microbial behaviour. There are, however, two potential roles for the AI-2 synthase (LuxS). The first is in the production of AI-2 and the second is as an enzyme in the activated methyl cycle where it catalyses the conversion of S-ribosylhomocysteine to homocysteine. The by-product of the reaction catalysed by LuxS is (S)-4,5-dihydroxy-2,3-pentanedione (DPD), which spontaneously forms the furanones known collectively as AI-2. The mammalian gut contains a complex collection of bacterial species so a method of interspecies communication might influence community structure and function. Lactobacillus reuteri 100-23 is an autochthonous inhabitant of the rodent forestomach where it adheres to the non-secretory epithelium forming a biofilm. Microarray comparisons of gene expression profiles of the L. reuteri 100-23 wild type and a luxS mutant under different culture conditions revealed altered transcription of genes encoding proteins associated with cysteine biosynthesis/oxidative stress response, urease activity, and sortase-dependent proteins. Metabolomic analysis showed that the luxS mutation affected cellular levels of fermentation products, fatty acids and amino acids. Cell density-dependent changes (log phase versus stationary phase growth) in gene transcription were not detected, indicating that AI-2 was unlikely to be involved in gene regulation mediated by quorum-sensing in L. reuteri 100-23.

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Fur-Mediated Activation of Gene Transcription in the Human Pathogen Neisseria gonorrhoeae.

Publication Date: 2012 Jan 27 PMID: 22287521
Authors: Yu, C. - Genco, C. A.
Journal: J Bacteriol

It is well established that the Ferric uptake regulatory protein (Fur) functions as a transcriptional repressor in diverse microorganisms. Recent studies demonstrated that Fur also functions as a transcriptional activator. In this study we defined Fur-mediated activation of gene transcription in the sexually transmitted disease pathogen Neisseria gonorrhoeae. Analysis of 37 genes previously determined to be iron-induced and which contained putative Fur boxes revealed that only 30 of these genes exhibited reduced transcription in a gonococcal fur mutant strain. Fur-mediated activation was established by examining binding of Fur to the putative promoter regions of 16 Fur-activated genes with variable binding affinities observed. Only approximately 50% of the newly identified Fur-regulated genes bound Fur in vitro, suggesting that additional regulatory circuits exist which may function through a Fur-mediated indirect mechanism. The gonococcal Fur-activated genes displayed variable transcription patterns in a fur mutant strain which correlated with the position of the Fur box in each (promoter) region. These results suggest that Fur-mediated direct transcriptional activation is fulfilled by multiple mechanisms involving either competing with a repressor or recruiting RNA polymerase. Collectively, our studies have established that gonococcal Fur functions as an activator of gene transcription through both direct and indirect mechanisms.

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Transformation-induced SOS regulation and carbon catabolite control of the V. cholerae integron integrase: connecting environment and genome plasticity.

Publication Date: 2012 Jan 27 PMID: 22287520
Authors: Baharoglu, Z. - Krin, E. - Mazel, D.
Journal: J Bacteriol

The human pathogen V. cholerae carries a chromosomal superintegron (SI). The SI contains an array of hundreds of gene cassettes organized in tandem which are stable in conditions where no particular stress is applied to bacteria (such as during laboratory growth). Rearrangements of these cassettes are catalyzed by the activity of the associated integron integrase. Understanding the regulation of integrase expression is pivotal to fully comprehend the role played by this genetic reservoir for bacterial adaptation and its connection with the development of antibiotic resistance. Our previous work established that the integrase is regulated by the bacterial SOS response and that it is induced during bacterial conjugation. Here we show that transformation, another horizontal gene transfer (HGT) mechanism, also triggers integrase expression through SOS induction, underlining the importance of HGT in genome plasticity. Moreover, we report a new cAMP-CRP dependent regulation mechanism of the integrase, highlighting the influence of the extracellular environment on chromosomal gene content. Altogether, our data suggest interplay between different stress responses and regulatory pathways for the modulation of the recombinase expression, thus showing how the SI remodeling mechanism is merged into bacterial physiology.

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Phosphatidylglycerol::prolipoprotein diacylglyceryl transferase (Lgt) of E. coli has seven transmembrane segments and its essential residues are embedded in the membrane.

Publication Date: 2012 Jan 27 PMID: 22287519
Authors: Pailler, J. - Aucher, W. - Pires, M. - Buddelmeijer, N.
Journal: J Bacteriol

Lgt of Escherichia coli catalyses the transfer of an sn-1,2-diacylglyceryl group from phosphatidylglycerol to prolipoproteins. The enzyme is essential for growth as demonstrated here by analysis of an lgt depletion strain. Cell fractionation demonstrated that Lgt is an inner membrane protein. Its membrane topology was determined by fusing Lgt to beta-galactosidase and alkaline phosphatase and by substituted cysteine accessibility method (SCAM) studies. The data show that Lgt is embedded in the membrane by seven transmembrane segments, that its N-terminus faces the periplasm and that its C-terminus faces the cytoplasm. Highly conserved amino acids in Lgt of both Gram-negative and Gram-positive bacteria were identified. Lgt enzymes are characterized by a so-called Lgt signature motif in which four residues are invariant. Ten conserved residues were substituted by alanine and the activity of these Lgt variants was analysed by their ability to complement the lgt depletion strain. Residues Y26, N146 and G154 are absolutely required for Lgt function, and R143, E151, R239 and E243 are important. The results demonstrate that the majority of the essential residues of Lgt are located in the membrane and that the Lgt signature motif faces the periplasm.

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Mutational analyses reveal overall topology and functional regions of NilB, a bacterial outer membrane protein required for host-association in a model animal-bacterial mutualism.

Publication Date: 2012 Jan 27 PMID: 22287518
Authors: Bhasin, A. - Chaston, J. M. - Goodrich-Blair, H.
Journal: J Bacteriol

The gammaproteobacterium Xenorhabdus nematophila is a mutualistic symbiont that colonizes the intestine of the nematode Steinernema carpocapsae. nilB (nematode intestine localization) is essential for X. nematophila colonization of nematodes and is predicted to encode an integral outer membrane beta-barrel protein, but evidence supporting this prediction has not been reported. The function of NilB is not known, but when expressed with two other factors encoded by nilA and nilC, it confers upon non-cognate Xenorhabdus spp. the ability to colonize S. carpocapsae nematodes. We present evidence that NilB is a surface-exposed outer membrane protein whose expression is repressed by NilR and growth in nutrient rich medium. Bioinformatic analyses reveal that NilB is the only characterized member of a family of proteins distinguished by N-terminal region tetratricopeptide repeats (TPR) and a conserved C-terminal domain of unknown function (DUF560). Members of this family occur in diverse bacteria, and are prevalent in the genomes of mucosal pathogens. Insertion and deletion mutational analyses support a beta-barrel structure model with an N-terminal globular domain, fourteen transmembrane strands, and seven extracellular surface loops, and reveal critical roles for the globular domain and surface loop 6 in nematode colonization. Epifluorescence microscopy of these mutants demonstrates that NilB is necessary at early stages of colonization. These findings are an important step in understanding the function of NilB, and, by extension, its homologs in mucosal pathogens.

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Asymmetric disposal of individual protein aggregates in Escherichia coli, one aggregate at a time.

Publication Date: 2012 Jan 27 PMID: 22287517
Authors: Lloyd-Price, J. - Hakkinen, A. - Kandhavelu, M. - Marques, I. J. - Chowdhury, S. - Lihavainen, E. - Yli-Harja, O. - Ribeiro, A. S.
Journal: J Bacteriol

Escherichia coli employ an asymmetric strategy at division, segregating unwanted substances in older poles, which has been associated to aging in these organisms. The kinetics of this process is still poorly understood. Using the MS2 coat protein fused to green fluorescent protein (MS2-GFP) and a reporter construct with multiple MS2 binding sites, we track individual RNA-MS2-GFP complexes in E. coli cells from the time that they are produced. Analysis of the kinetics and brightness of spots shows that these appear in the midcell region, are composed of a single RNA-MS2-GFP complex, and reach a pole before another target RNA is formed, typically remaining there thereafter. The choice of pole is probabilistic and heavily biased towards one pole, similar to what was observed in studies regarding protein aggregates. Additionally, this mechanism is found to act independently on each disposed molecule. Finally, while the RNA-MS2-GFP complexes are disposed, the MS2-GFP tagging molecules alone are not. We conclude that this asymmetric mechanism to segregate damage at the expense of aging individuals acts probabilistically on individual molecules, and is capable of accurate classification of molecules for disposal.

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Differentiation of function among the RsbR paralogs in the general stress response of Bacillus subtilis with regard to light perception.

Publication Date: 2012 Jan 27 PMID: 22287516
Authors: van der Steen, J. B. - Avila-Perez, M. - Knippert, D. - Vreugdenhil, A. - van Alphen, P. - Hellingwerf, K. J.
Journal: J Bacteriol

The general stress response of Bacillus subtilis can be activated by a wide range of signals, including low intensities of visible light. It is regulated by a dedicated sigma factor via a complex signal transduction pathway that makes use of stressosomes: hetero-oligomeric complexes that include one or more of the RsbR proteins (RsbRA, RsbRB, RsbRC and RsbRD). The response to blue light is mediated by the photoreceptor YtvA. Here we show which of the four RsbR proteins are necessary for the activation of the sigma(B) response by blue light. Experiments performed with single, double and triple-deletion strains in the rsbR genes show that RsbRB and RsbRA function antagonistically, with the former being a negative regulator and the latter a positive regulator of the YtvA-dependent light activation of the stress response. A strain with RsbRB as the only RsbR protein is unable to respond to light-activation of sigma(B). Furthermore, RsbRC and RsbRD can replace RsbRA's function only in the absence of RsbRB. This differentiation of function is confined to light stress, as strains with RsbRA or RsbRB as the only RsbR protein behave similar in our experimental conditions in response to physicochemical stresses. Interestingly, RsbRB's absence is sufficient to result in light-activation of the general stress response at wild type expression levels of ytvA, while it was previously reported that YtvA could only activate sigma(B) when overproduced, or when cells are supplemented with an additional environmental stress.

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Functional analysis of the CpsA protein of Streptococcus agalactiae.

Publication Date: 2012 Jan 27 PMID: 22287515
Authors: Hanson, B. R. - Runft, D. L. - Streeter, C. - Kumar, A. - Carion, T. W. - Neely, M. N.
Journal: J Bacteriol

Streptococcal pathogens such as Streptococcus agalactiae (GBS) are an important cause of systemic disease, which is facilitated in part by the presence of a polysaccharide capsule. The CpsA protein is a putative transcriptional regulator of the capsule locus, but its exact contribution to regulation is unknown. To address the role of CpsA in regulation, full-length GBS CpsA and two truncated forms of the protein were purified and analyzed for DNA binding ability. Assays demonstrated that CpsA is able to bind specifically to two putative promoters within the capsule operon with similar affinity, and full-length protein is required for specificity. Functional characterization of CpsA confirmed that the DeltacpsA strain produced less capsule than wild type, and demonstrated that production of full length CpsA or the DNA-binding region of CpsA resulted in increased capsule levels. In contrast, production of a truncated form of CpsA lacking the extracellular LytR domain (CpsA-245) in the wild type background resulted in a dominant negative decrease in capsule production. GBS expressing CpsA-245, but not the DeltacpsA strain, were attenuated in human whole blood. However, the DeltacpsA strain showed significant attenuation in a zebrafish infection model. Furthermore, chain length was observed to be variable in a CpsA-dependent manner, but could be restored to wild type levels when grown with lysozyme. Taken together, these results suggest that CpsA is a modular protein influencing multiple regulatory functions that may include not only capsule synthesis, but also cell wall associated factors.

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Transcriptional and post-transcriptional events control copper-responsive expression of a Rhodobacter capsulatus multicopper oxidase.

Publication Date: 2012 Jan 27 PMID: 22287514
Authors: Rademacher, C. - Moser, R. - Lackmann, J. W. - Klinkert, B. - Narberhaus, F. - Masepohl, B.
Journal: J Bacteriol

The copper-regulated Rhodobacter capsulatus cutO (multicopper oxidase) gene confers copper tolerance and is encoded in the tri-cistronic orf635-cutO-cutR operon. Transcription of cutO strictly depends on the promoter upstream of orf635 as demonstrated by lacZ reporter fusions to nested promoter fragments. Remarkably, orf635 expression was not affected by copper availability, whereas cutO and cutR were only expressed in the presence of copper. Differential regulation was abolished by site-directed mutations within the orf635-cutO intergenic region suggesting that this region encodes a copper-responsive mRNA element. Bioinformatic predictions and RNA structure probing experiments revealed an intergenic stem-loop structure as the candidate mRNA element. This is the first reported post-transcriptional copper response mechanism in bacteria.

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The adaptor protein MecA is a negative regulator of the expression of late competence genes in Streptococcus thermophilus.

Publication Date: 2012 Jan 27 PMID: 22287513
Authors: Boutry, C. - Wahl, A. - Delplace, B. - Clippe, A. - Fontaine, L. - Hols, P.
Journal: J Bacteriol

In Streptococcus thermophilus, the ComRS regulatory system governs the transcriptional level of comX expression, and hence controls the early stage of competence development. The present work focuses on the post-translational control of the activity of the sigma factor ComX and therefore on the late stage of competence regulation. In silico analysis performed on the S. thermophilus genome revealed the presence of a homologue of mecA (mecA(St)), which codes for the adaptor protein that is involved in ComK degradation by ClpCP in Bacillus subtilis. Using reporter strains and microarrays experiments, we showed that MecA(St) represses late competence genes without affecting the early competence stage in conditions that are not permissive for competence development. In addition, this repression mechanism was not only found to act downstream of comX expression but to be fully dependent on the presence of a functional comX gene. This negative control was similarly released in strains deleted for clpC, mecA, and clpC-mecA. Using artificial conditions of comX expression, we next showed that the abundance of ComX is higher in absence of MecA or ClpC. Finally, bacterial two-hybrid assays strongly suggested that MecA interacts with both ComX and ClpC. Based on these results, we proposed that ClpC and MecA act together in the same regulatory circuit to control the abundance of ComX in S. thermophilus.

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Complete Genome Sequence of Brucella suis VBI22, Isolated from Bovine Milk.

Publication Date: 2012 Feb PMID: 22275106
Authors: Tae, H. - Shallom, S. - Settlage, R. - Hawkins, G. N. - Adams, L. G. - Garner, H. R.
Journal: J Bacteriol

Brucella suis is the causative agent of swine brucellosis and is known to be able to infect several different hosts, including cattle, dogs, and horses, without causing disease symptoms. Here we report the complete genome sequence of Brucella suis VBI22, which was isolated from raw milk from an infected cow.

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Genome sequences of six pseudoalteromonas strains isolated from arctic sea ice.

Publication Date: 2012 Feb PMID: 22275105
Authors: Bian, F. - Xie, B. B. - Qin, Q. L. - Shu, Y. L. - Zhang, X. Y. - Yu, Y. - Chen, B. - Chen, X. L. - Zhou, B. C. - Zhang, Y. Z.
Journal: J Bacteriol

Yu et al. (Polar Biol. 32:1539-1547, 2009) isolated 199 Pseudoalteromonas strains from Arctic sea ice. We sequenced the genomes of six of these strains, which are affiliated to different Pseudoalteromonas species based on 16S rRNA gene sequences, facilitating the study of physiology and adaptation of Arctic sea ice Pseudoalteromonas strains.

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Genome Sequence of Benzo(a)pyrene-Degrading Bacterium Novosphingobium pentaromativorans US6-1.

Publication Date: 2012 Feb PMID: 22275104
Authors: Luo, Y. R. - Kang, S. G. - Kim, S. J. - Kim, M. R. - Li, N. - Lee, J. H. - Kwon, K. K.
Journal: J Bacteriol

Novosphingobium pentaromativorans US6-1 showed a good ability to degrade high-molecular-weight polycyclic aromatic hydrocarbons. We report the draft genome sequence of strain US6-1, which contains a main chromosome (5,096,413 bp, G+C content of 63.1%) and two plasmids (188,476 and 60,085 bp). The majority of the aromatic-hydrocarbon-degrading genes are encoded in the larger plasmid.

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Genome Sequence for "Candidatus Mycoplasma haemominutum," a Low-Pathogenicity Hemoplasma Species.

Publication Date: 2012 Feb PMID: 22275103
Authors: Barker, E. N. - Darby, A. C. - Helps, C. R. - Peters, I. R. - Hughes, M. A. - Radford, A. D. - Novacco, M. - Boretti, F. S. - Hofmann-Lehmann, R. - Tasker, S.
Journal: J Bacteriol

We present the genome sequence of "Candidatus Mycoplasma haemominutum" strain Birmingham 1, a low-pathogenicity feline hemoplasma strain.

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Genome Sequence of the Marine Bacterium Vibrio campbellii DS40M4, Isolated from Open Ocean Water.

Publication Date: 2012 Feb PMID: 22275102
Authors: Dias, G. M. - Thompson, C. C. - Fishman, B. - Naka, H. - Haygood, M. G. - Crosa, J. H. - Thompson, F. L.
Journal: J Bacteriol

Vibrio sp. strain DS40M4 is a marine bacterium that was isolated from open ocean water. In this work, using genomic taxonomy, we were able to classify this bacterium as V. campbellii. Our genomic analysis revealed that V. campbellii DS40M4 harbors genes related to iron transport, virulence, and environmental fitness, such as those encoding anguibactin and vanchrobactin biosynthesis proteins, type II, III, IV, and VI secretion systems, and proteorhodopsin.

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Genome Sequence of the Arsenite-Oxidizing Strain Agrobacterium tumefaciens 5A.

Publication Date: 2012 Feb PMID: 22275101
Authors: Hao, X. - Lin, Y. - Johnstone, L. - Liu, G. - Wang, G. - Wei, G. - McDermott, T. - Rensing, C.
Journal: J Bacteriol

Microbial transformations of arsenic influence its mobility and toxicity. We report the draft genome sequence of the arsenite-oxidizing strain Agrobacterium tumefaciens 5A isolated from an As-contaminated soil in the Madison River Valley, MT. A large number of metal (or metalloid) resistance genes, especially contributing to arsenite oxidation, were identified.

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Draft Genome Sequence of Probiotic Strain Lactobacillus rhamnosus R0011.

Publication Date: 2012 Feb PMID: 22275100
Authors: Tompkins, T. A. - Barreau, G. - de Carvalho, V. G.
Journal: J Bacteriol

Lactobacillus rhamnosus R0011 is a commercially available probiotic that is widely used in human dietary supplements and pharmaceutical products. We prepared a draft genome sequence consisting of 10 contigs totaling 2,900,620 bases and a G+C content of 46.7% for this strain.

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Draft Genome Sequence of Probiotic Strain Pediococcus acidilactici MA18/5M.

Publication Date: 2012 Feb PMID: 22275099
Authors: Barreau, G. - Tompkins, T. A. - de Carvalho, V. G.
Journal: J Bacteriol

Pediococcus acidilactici MA18/5M is a commercially available probiotic that is widely used in swine, poultry, aquaculture feeds, and human dietary supplements. We prepared a genome sequence for this strain consisting of 2 scaffolds totaling 1,992,928 bases including gaps for a total of 3,346 bases and a G+C content of 42%.

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Genome Sequence of Deep-Sea Manganese-Oxidizing Bacterium Marinobacter manganoxydans MnI7-9.

Publication Date: 2012 Feb PMID: 22275098
Authors: Wang, H. - Li, H. - Shao, Z. - Liao, S. - Johnstone, L. - Rensing, C. - Wang, G.
Journal: J Bacteriol

Here we report the draft genome of Marinobacter manganoxydans MnI7-9, isolated from a deep-sea hydrothermal vent in the Indian Ocean and capable of oxidizing manganese even when there is a very high concentration of Mn(2+). The strain also displayed high resistance and adsorption ability toward many metal(loid)s.

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Genome Sequence of Enterobacter cloacae subsp. dissolvens SDM, an Efficient Biomass-Utilizing Producer of Platform Chemical 2,3-Butanediol.

Publication Date: 2012 Feb PMID: 22275097
Authors: Xu, Y. - Wang, A. - Tao, F. - Su, F. - Tang, H. - Ma, C. - Xu, P.
Journal: J Bacteriol

Enterobacter cloacae subsp. dissolvens SDM has an extraordinary characteristic of biomass utilization for 2,3-butanediol production. Here we present a 4.9-Mb assembly of its genome. The key genes for regulation and metabolism of 2,3-butanediol production were annotated, which could provide further insights into the molecular mechanism of high-yield production of 2,3-butanediol.

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Complete Genome Sequence of the Fenitrothion-Degrading Burkholderia sp. Strain YI23.

Publication Date: 2012 Feb PMID: 22275096
Authors: Lim, J. S. - Choi, B. S. - Choi, A. Y. - Kim, K. D. - Kim, D. I. - Choi, I. Y. - Ka, J. O.
Journal: J Bacteriol

Burkholderia species are ubiquitous in soil environments. Many Burkholderia species isolated from various environments have the potential to biodegrade man-made chemicals. Burkholderia sp. strain YI23 was isolated from a golf course soil and identified as a fenitrothion-degrading bacterium. In this study, we report the complete genome sequence of Burkholderia sp. strain YI23.

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Genome Sequence of Pseudomonas stutzeri SDM-LAC, a Typical Strain for Studying the Molecular Mechanism of Lactate Utilization.

Publication Date: 2012 Feb PMID: 22275095
Authors: Jiang, T. - Gao, C. - Su, F. - Zhang, W. - Hu, C. - Dou, P. - Zheng, Z. - Tao, F. - Ma, C. - Xu, P.
Journal: J Bacteriol

Pseudomonas stutzeri SDM-LAC is an efficient lactate utilizer with various applications in biocatalysis. Here we present a 4.2-Mb assembly of its genome. The annotated four adjacent genes form a lactate utilization operon, which could provide further insights into the molecular mechanism of lactate utilization.

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Expression of multidrug resistance efflux pump gene norA is iron-responsive in Staphylococcus aureus.

Publication Date: 2012 Jan 20 PMID: 22267518
Authors: Deng, X. - Sun, F. - Ji, Q. - Liang, H. - Missiakas, D. - Lan, L. - He, C.
Journal: J Bacteriol

Staphylococcus aureus utilizes efflux transporter NorA to pump out a wide range of structurally dissimilar drugs, conferring low-level multidrug resistance. The regulation of norA expression has yet to be fully understood although past studies have revealed that this gene is under the control of the global transcriptional regulator MgrA and the two-component system ArlRS. To identify additional regulators of norA, we screened a transposon library in strain Newman expressing the transcriptional fusion norA-lacZ for altered beta-galactosidase activity. We identify a transposon insertion in fhuB, a gene that encodes a ferric hydroxamate uptake system permease and propose that the norA transcription is iron-responsive. In agreement with this observation, addition of FeCl(3) repressed the induction of norA-lacZ, suggesting that bacterial iron uptake plays an important role in regulating norA transcription. In addition, a fur (ferric uptake regulator) deletion exhibited compromised norA transcription and reduced resistance to quinolone as compared to the wild-type strain, indicating fur as a positive regulator of norA. A putative Fur box identified in the promoter region of norA was confirmed by electrophoretic mobility shift (EMSA) and DNase I footprint assays. Finally, by employing a siderophore secretion assay, we reveal that NorA may contribute to the export of siderophores. Collectively, our experiments uncover some novel interactions between cellular iron level and norA regulation in S. aureus.

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Dynamics of speB mRNA Transcripts in Streptococcus pyogenes.

Publication Date: 2012 Jan 20 PMID: 22267517
Authors: Chen, Z. - Itzek, A. - Malke, H. - Ferretti, J. J. - Kreth, J.
Journal: J Bacteriol

Streptococcus pyogenes (Group A streptococcus, GAS) is a human-specific pathogen that causes a variety of diseases ranging from superficial infection to life-threatening diseases. SpeB, a potent extracellular cysteine proteinase, plays an important role in the pathogenesis of GAS infections. Previous studies show that SpeB expression and activity are controlled at the transcriptional and posttranslational levels, though it had been unclear whether speB was also regulated at the post-transcriptional level. In this study, we examined the growth phase dependent speB mRNA abundance and decay using qRT-PCR and Northern blot analyses. We observed that the speB mRNA accumulated rapidly during exponential growth, which occurred concomitantly with an increase of speB mRNA stability. A closer observation revealed that the increased speB mRNA stability was mainly due to progressive acidification. Inactivation of RNase Y, a recently identified endoribonuclease, revealed a role in processing and degradation of speB mRNA. We conclude that the increased speB mRNA stability contributes to the rapid accumulation of speB transcript during growth.

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Bacillus subtilis RapA phosphatase domain interaction with its substrate Spo0F~P and inhibitor PhrA peptide.

Publication Date: 2012 Jan 20 PMID: 22267516
Authors: Diaz, A. R. - Core, L. J. - Jiang, M. - Morelli, M. - Chiang, C. H. - Szurmant, H. - Perego, M.
Journal: J Bacteriol

Rap proteins in Bacillus subtilis regulate the phosphorylation level or the DNA-binding activity of response regulators such as Spo0F, involved in sporulation initiation, or ComA, regulating competence development. Rap proteins can be inhibited by specific peptides generated by the export-import processing pathway of the Phr proteins. Rap proteins have a modular organization comprising an amino terminal alpha-helical domain connected to a domain formed by six tetratricopeptide (TPR) repeats. In this study, the molecular basis for the specificity of the RapA phosphatase for its substrate Spo0F approximately P and its inhibitor pentapeptide PhrA was analyzed in part by generating chimeric proteins with RapC that targets the DNA-binding domain of ComA, rather than Spo0F approximately P, and is inhibited by the PhrC pentapeptide. In vivo analysis of sporulation efficiency or competence-induced gene expression as well as in vitro biochemical assays allowed the identification of the amino terminal sixty amino acids as sufficient to determine Rap specificity for its substrate and the central TPR3-5 repeats as providing binding specificity toward the Phr peptide inhibitor. The results allowed the prediction and testing of key residues in RapA that are essential for PhrA binding and specificity, thus demonstrating how the widespread structural fold of the TPR repeat is highly versatile to use a common interaction mechanism for a variety of functions in eukaryotic and prokaryotic organisms.

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Francisella tularensis RipA protein topology and identification of functional domains.

Publication Date: 2012 Jan 20 PMID: 22267515
Authors: Mortensen, B. L. - Fuller, J. R. - Taft-Benz, S. - Collins, E. J. - Kawula, T. H.
Journal: J Bacteriol

Francisella tularensis is a Gram-negative coccobacillus and is the etiological agent of the disease tularemia. Expression of the cytoplasmic membrane protein RipA is required for Francisella replication within macrophages and other cell types; however, the function of this protein remains unknown. RipA is conserved among all sequenced Francisella species, and RipA-like proteins are present in a number of individual strains of a wide variety of species scattered throughout the prokaryotic kingdom. Crosslinking studies revealed that RipA forms homoligomers. Using a panel of RipA-GFP and RipA-PhoA fusion constructs, we determined that RipA has a unique topology within the cytoplasmic membrane with the N- and C- termini in the cytoplasm and periplasm, respectively. RipA has two significant cytoplasmic domains, one comprised roughly of amino acids 1 - 50, and the second flanked by the second and third transmembrane domains and comprising amino acids 104 - 152. RipA functional domains were identified by measuring the effects of deletion mutations, amino acid substitution mutations and spontaneously arising intragenic suppressor mutations on intracellular replication, induction of IL-1beta secretion by infected macrophages, and oligomer formation. Results from these experiments demonstrated that each of the cytoplasmic domains and specific amino acids within these domains were required for RipA function.

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Suppression of a dnaKJ deletion by multicopy dksA results from nonfeedback regulated transcripts that originate upstream of the major dksA promoter.

Publication Date: 2012 Jan 20 PMID: 22267514
Authors: Chandrangsu, P. - Wang, L. - Choi, S. H. - Gourse, R. L.
Journal: J Bacteriol

DksA is an RNA polymerase (RNAP) binding transcription factor that controls expression of a large number of genes in concert with the small molecule "alarmone" ppGpp. DksA also aids in the resolution of conflicts between RNAP and DNA polymerase (DNAP) during genome replication. DksA was originally identified as a multicopy suppressor of the temperature-sensitivity caused by deletion of the genes coding for the DnaKJ chaperone system. Here, we address a longstanding question regarding the role of DksA in DeltadnaKJ suppression. We demonstrate that DksA expression from a multicopy plasmid is necessary and sufficient for suppression, that overexpression occurs despite the fact that the major dksA promoter is feedback-regulated in wild-type cells, and that weak, nonfeedback-regulated transcription originating upstream of the major promoter for the dksA gene accounts for overexpression. We tentatively rule out three potential explanations for suppression related to known functions of DnaKJ. Because a determinant in DksA needed for the regulation of transcription initiation, but not for resolution of RNAP-DNAP conflicts, is needed to bypass the need for DnaKJ, we suggest that suppression results from an unidentified product whose promoter is directly or indirectly regulated by DksA.

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CarF mediates signaling by singlet oxygen, generated via photoexcited protoporphyrin IX, in Myxococcus xanthus light-induced carotenogenesis.

Publication Date: 2012 Jan 20 PMID: 22267513
Authors: Galbis-Martinez, M. - Padmanabhan, S. - Murillo, F. J. - Elias-Arnanz, M.
Journal: J Bacteriol

Blue light triggers carotenogenesis in the non-phototrophic bacterium Myxococcus xanthus by inducing inactivation of an anti-sigma factor, CarR, and the consequent liberation of the cognate extracytoplasmic function (ECF) sigma, CarQ. CarF, the protein implicated earliest in the response to light, does not resemble any known photoreceptor. It interacts physically with CarR and is required for its light-driven inactivation but the mechanism is unknown. Blue light sensing in M. xanthus has been attributed to the heme precursor, protoporphyrin IX (PPIX), which can generate the highly reactive singlet oxygen species ((1)O(2)) by energy transfer to oxygen. However, (1)O(2) involvement in M. xanthus light-induced carotenogenesis remains to be established. Here, we present genetic evidence on the involvement of PPIX as well as (1)O(2) inlight-induced carotenogenesis in M. xanthus, and on how these are linked to CarF in the signal transduction pathway. Response to light was examined in carF-bearing or carF-deficient M. xanthus strains lacking endogenous PPIX due to deletion of hemB, or accumulating PPIX due to deletion of hemH (hemB and hemH are early- and late-acting heme biosynthesis genes, respectively). This demonstrated that light-induction of the CarQ-dependent promoter, P(QRS), correlated directly with cellular PPIX levels. Furthermore, we show that P(QRS) activation is triggered by (1)O(2) and is inhibited by exogenously supplied hemin, and that CarF is essential for the action of (1)O(2). Thus, our findings indicate that blue light interaction with PPIX generates (1)O(2), which must be transmitted via CarF to trigger the transcriptional response underlying light-induced carotenogenesis in M. xanthus.

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Regulatory Linkages Between Flagella and Surfactant During Swarming Behavior: Lubricating the Flagellar Propeller?

Publication Date: 2012 Jan 20 PMID: 22267512
Authors: Xu, J. - Platt, T. G. - Fuqua, C.
Journal: J Bacteriol

Many bacteria explore their immediate environment using flagellar locomotion, and on semi-solid surfaces this type of flagella-driven motility is known as swarming (22)....

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Resistance Phenotypes mediated by Aminoacyl-Phosphatidylglycerol Synthases.

Publication Date: 2012 Jan 20 PMID: 22267511
Authors: Arendt, W. - Hebecker, S. - Jager, S. - Nimtz, M. - Moser, J.
Journal: J Bacteriol

The specific aminoacylation of the phospholipid phosphatidylglycerol (PG) with alanine or with lysine catalyzed by aminoacyl-phosphatidylglycerol synthases (aaPGS) was shown to render various organisms less susceptible to antibacterial agents. This study makes use of Pseudomonas aeruginosa chimeric mutant strains producing lysyl-phosphatidylglycerol (L-PG) instead of the naturally occurring alanyl-phosphatidylglycerol (A-PG) to study the resulting impact on bacterial resistance. Consequences of such artificial phospholipid composition were studied in the presence of an overall of seven antimicrobials (beta-lactams, a lipopeptide antibiotic, CAMPs) to quantitatively assess the effect of A-PG substitution (with L-PG, L-PG and A-PG, increased A-PG levels). For the employed Gram negative P. aeruginosa model system an exclusive 'charge repulsion mechanism' does not explain the attenuated antimicrobial susceptibility due to PG modification. Additionally, the specificity of nine orthologous aaPGS enzymes was experimentally determined. The newly characterized protein sequences allowed to establish a significant group of A-PG synthase sequences which were bioinformatically compared to the related group of L-PG synthesizing enzymes. The analysis revealed a diverse origin for the evolution of A-PG and L-PG synthases as the specificity of an individual enzyme is not reflected in terms of a characteristic sequence motif. This finding is relevant for future development of potential aaPGS inhibitors.

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Perturbing the oxidizing environment of the periplasm stimulates the PhoQ/PhoP system in E. coli.

Publication Date: 2012 Jan 20 PMID: 22267510
Authors: Lippa, A. M. - Goulian, M.
Journal: J Bacteriol

The PhoQ/PhoP two-component system is repressed by divalent cations such as Mg(2+) and Ca(2+) in the growth medium and stimulated by low pH, and certain cationic antimicrobial peptides. In E. coli, it has recently been shown that the histidine kinase PhoQ is also modulated by at least two additional factors, the small membrane proteins SafA and MgrB. This raises the possibility that the PhoQ/PhoP circuit has additional regulatory components and integrates additional input signals. We screened E. coli transposon insertion mutants to look for proteins that modulate the activity of the PhoQ/PhoP system and uncovered a role for DsbA, a periplasmic oxidant that facilitates the formation of disulfide bonds. Deletion of dsbA or dsbB, which maintains a pool of oxidized DsbA, leads to increased transcription of at least two PhoP-regulated genes. Addition of the reducing agent DTT to wild-type cells has a similar effect and treatment of a dsbA null strain with the oxidant Cu(2+)rescues the reporter gene expression phenotype. We also demonstrate that expression of an MgrB mutant that lacks cysteines blocks the effect of a dsbA null mutation on PhoQ/PhoP activity, suggesting that MgrB acts downstream of DsbA in this pathway. Taken together, these results demonstrate that a decrease in the oxidizing activity of the periplasm stimulates PhoQ/PhoP and may reveal a new input stimulus for this important two-component system.

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The genome of Halomonas strain GFAJ-1: blueprint to fame or business as usual.

Publication Date: 2012 Jan 20 PMID: 22267509
Authors: Kim, E. H. - Rensing, C.
Journal: J Bacteriol

"evidence for arsenate in macromolecules that normally contain phosphate, most notably nucleic acids" (18)....

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Group X aldehyde dehydrogenases of Pseudomonas aeruginosa PAO1 degrade hydrazones.

Publication Date: 2012 Jan 20 PMID: 22267508
Authors: Taniyama, K. - Itoh, H. - Takuwa, A. - Sasaki, Y. - Yajima, S. - Toyofuku, M. - Nomura, N. - Takaya, N.
Journal: J Bacteriol

Hydrazones are natural and synthetic compounds containing a C=N-N moiety. Here we found that the opportunistic pathogen Pseudomonas aeruginosa PAO1 produced NAD(+)- or NADP(+)-dependent hydrazone dehydrogenase (Hdh), which converts hydrazones to the corresponding hydrazides and acids rather than to the simple hydrolytic product aldehydes. Gene cloning indicated that the Hdh is part of the group X aldehyde dehydrogenase (Aldh) family, which is distributed among bacteria although their physiological roles remain unknown. The PAO1 strain up-regulated Hdh in the presence of the hydrazone, adipic acid bis-(ethylidene hydrazide) (AEH). Gene disruption of the Hdh-encoding hdhA (PA4022) decreased growth rates in culture medium containing AEH as the sole carbon source, and this effect was more obvious in the double-gene disruptant of hdhA and its orthologous exaC (PA1984), indicating that these genes are responsible for hydrazone utilization. Recombinant proteins of Group X Aldh from Escherichia coli, Paracoccus denitrificans and Ochrobactrum anthropi also acted as an Hdh in that they produced Hdh activity in the cells and degraded hydrazones. These findings indicated the physiological roles of Group X Aldh in bacteria and that they comprise a distinct Aldh subfamily.

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OnpA, an unusual flavin-dependent monooxygenase containing a cytochrome b5 domain.

Publication Date: 2012 Jan 20 PMID: 22267507
Authors: Xiao, Y. - Liu, T. T. - Dai, H. - Zhang, J. J. - Liu, H. - Tang, H. - Leak, D. J. - Zhou, N. Y.
Journal: J Bacteriol

Ortho-nitrophenol 2-monooxygenase (EC 1.14.13.31) from Alcaligenes sp. strain NyZ215 catalyzes monooxygenation of ortho-nitrophenol to form catechol via ortho-benzoquinone. Sequence analysis of this onpA-encoded enzyme revealed that it contained a flavin-binding monooxygenase domain and a heme-binding cytochrome b5 domain. OnpA was purified to homogeneity as a His-tagged protein, and was considered to be a monomer as determined by gel filtration. FAD and heme were identified by HPLC (high-performance liquid chromatography) and HPLC-MS (mass spectrometry) as cofactors in this enzyme, and quantitative analysis indicated 1 mole of the purified recombinant OnpA contained 0.66 mole of FAD and 0.20 mole of heme. However, the enzyme activity of OnpA was increased by 60% and 450% after addition of FAD and hemin, respectively, suggesting that the optimal stoichiometry was 1:1:1. In addition, site-directed mutagenesis experiments confirmed that two highly conserved histidines located in the cytochrome b5 domain were associated with binding of the heme, and the cytochrome b5 domain was involved in the OnpA activity. These results indicate that OnpA is an unusual FAD-dependent monooxygenase containing a fused cytochrome b5 domain that is essential for its activity. Therefore, we here link cytochrome b5 to flavin-dependent monooxygenases.

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Insights into the gene expression profile of the not cultivable haemotrophic Mycoplasma suis during acute infection using proteome analysis.

Publication Date: 2012 Jan 20 PMID: 22267506
Authors: Felder, K. M. - Carranza, P. M. - Gehrig, P. M. - Roschitzki, B. - Barkow-Oesterreicher, S. - Hoelzle, K. - Riedel, K. - Kube, M. - Hoelzle, L. E.
Journal: J Bacteriol

Haemotrophic mycoplasmas, cell-wall-less bacteria whose niche is the erythrocyte of their hosts, have never been cultivated in vitro. Therefore, knowledge of their pathogenesis is fundamental. Mycoplasma suis infects pigs, causing either acute fatal haemolytic anaemia or chronic low-grade anaemia, growth retardation and immune suppression. Recently, the complete genomes of two haemotrophic mycoplasma species, M. suis and M. haemofelis, were sequenced, offering new strategies for the analysis of their pathogenesis.In this study we implemented a proteomic approach to identify M. suis proteins during acute infection by tandem mass spectrometry. Twenty-two per cent of the predicted proteins encoded in M. suis str. KI_3806 were identified. These included nearly all encoded proteins of the glycolysis and the nucleotide metabolism. The lipid metabolism however is underrepresented. A high proportion of the detected proteins are involved in information storage and processing (72.6%). In addition, several proteins of different functionality, i.e. posttranslational modification, membrane genesis, signal transduction, intracellular trafficking, inorganic ion transport and defence mechanisms were identified. In its reduced genome M. suis harbours 65.3% (str. Illinois) and 65.9% (str. KI_3806) of genes encoding hypothetical proteins. Of these only 6.3% were identified at proteome level. All proteins identified in this study are present in both M. suis strains and are encoded in higher conserved regions of the genome sequence. In conclusion, our proteome approach is a further step towards the elucidation of pathogenesis and life cycle of M. suis as well as the establishment of an in vitro cultivation system.

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Complete Genome Sequence of Salmonella enterica Serovar Pullorum RKS5078.

Publication Date: 2012 Feb PMID: 22247537
Authors: Feng, Y. - Xu, H. F. - Li, Q. H. - Zhang, S. Y. - Wang, C. X. - Zhu, D. L. - Cao, F. L. - Li, Y. G. - Johnston, R. N. - Zhou, J. - Liu, G. R. - Liu, S. L.
Journal: J Bacteriol

Salmonella enterica serovar Pullorum is a chicken-adapted pathogen, causing pullorum disease. Its strict host adaptation has been suspected to result in gene decay. To validate this hypothesis and identify the decayed genes, we sequenced the complete genome of S. Pullorum RKS5078. We found 263 pseudogenes in this strain and conducted functional analyses of the decayed genes.

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Genome Sequence of Corynebacterium glutamicum ATCC 14067, Which Provides Insight into Amino Acid Biosynthesis in Coryneform Bacteria.

Publication Date: 2012 Feb PMID: 22247536
Authors: Lv, Y. - Liao, J. - Wu, Z. - Han, S. - Lin, Y. - Zheng, S.
Journal: J Bacteriol

We report the genome sequence of Corynebacterium glutamicum ATCC 14067 (once named Brevibacterium flavum), which is useful for taxonomy research and further molecular breeding in amino acid production. Preliminary comparison with those of the reported coryneform strains revealed some notable differences that might be related to the difficulties in molecular manipulation.

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Genome Sequence of Edwardsiella ictaluri 93-146, a Strain Associated with a Natural Channel Catfish Outbreak of Enteric Septicemia of Catfish.

Publication Date: 2012 Feb PMID: 22247535
Authors: Williams, M. L. - Gillaspy, A. F. - Dyer, D. W. - Thune, R. L. - Waldbieser, G. C. - Schuster, S. C. - Gipson, J. - Zaitshik, J. - Landry, C. - Banes, M. M. - Lawrence, M. L.
Journal: J Bacteriol

Edwardsiella ictaluri is the cause of extensive mortalities and economic losses to the channel catfish industry of the southeast United States. Here we report the complete genome of Edwardsiella ictaluri 93-146. Whole-genome sequence analysis of E. ictaluri provides a tool for understanding the genomic regions specific to the species and the Edwardsiella genus.

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Genome Sequence of Corynebacterium casei UCMA 3821, Isolated from a Smear-Ripened Cheese.

Publication Date: 2012 Feb PMID: 22247534
Authors: Monnet, C. - Loux, V. - Bento, P. - Gibrat, J. F. - Straub, C. - Bonnarme, P. - Landaud, S. - Irlinger, F.
Journal: J Bacteriol

Corynebacterium casei is one of the most prevalent species present on the surfaces of smear-ripened cheeses, where it contributes to the production of the desired organoleptic properties. Here, we report the draft genome sequence of Corynebacterium casei UCMA 3821 to provide insights into its physiology.

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Draft Genome Sequence of Plant Growth-Promoting Rhizobium Mesorhizobium amorphae, Isolated from Zinc-Lead Mine Tailings.

Publication Date: 2012 Feb PMID: 22247533
Authors: Hao, X. - Lin, Y. - Johnstone, L. - Baltrus, D. A. - Miller, S. J. - Wei, G. - Rensing, C.
Journal: J Bacteriol

Here, we describe the draft genome sequence of Mesorhizobium amorphae strain CCNWGS0123, isolated from nodules of Robinia pseudoacacia growing on zinc-lead mine tailings. A large number of metal(loid) resistance genes, as well as genes reported to promote plant growth, were identified, presenting a great future potential for aiding phytoremediation in metal(loid)-contaminated soil.

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Draft Genome Sequences of the Biocontrol Bacterium Mitsuaria sp. Strain H24L5A.

Publication Date: 2012 Feb PMID: 22247532
Authors: Rong, X. - Gurel, F. B. - Meulia, T. - McSpadden Gardener, B. B.
Journal: J Bacteriol

Mitsuaria sp. strain H24L5A is a plant-associated bacterium with proven capacities to suppress plant pathogens. Here, we report the draft genome sequences and automatic annotation of H24L5A. Comparative genomic analysis indicates H24L5A's similarity to the Leptothrix and Methylibium species, as well as several genes potentially contributing to its biocontrol activities.

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Genome Sequence of Strain HIMB30, a Novel Member of the Marine Gammaproteobacteria.

Publication Date: 2012 Feb PMID: 22247531
Authors: Huggett, M. J. - Rappe, M. S.
Journal: J Bacteriol

Strain HIMB30 was isolated from coastal Hawaii seawater by extinction culturing in seawater-based oligotrophic medium. It is a phylogenetically unique member of the class Gammaproteobacteria that is only distantly related to its closest cultured relatives. Here we present the genome sequence of strain HIMB30, including genes for proteorhodopsin-based phototrophy and the Calvin-Benson-Bassham cycle.

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Complete Genome Sequence of Leuconostoc mesenteroides subsp. mesenteroides Strain J18, Isolated from Kimchi.

Publication Date: 2012 Feb PMID: 22247530
Authors: Jung, J. Y. - Lee, S. H. - Lee, S. H. - Jeon, C. O.
Journal: J Bacteriol

Leuconostoc mesenteroides subsp. mesenteroides is one of the most predominant lactic acid bacterial groups during kimchi fermentation. Here, we report the complete genome sequence of L. mesenteroides subsp. mesenteroides J18, which was isolated from kimchi. The genome of the strain consists of a 1,896,561-bp chromosome and five plasmids.

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Draft Genome Sequence of Pantoea ananatis B1-9, a Nonpathogenic Plant Growth-Promoting Bacterium.

Publication Date: 2012 Feb PMID: 22247529
Authors: Kim, H. J. - Lee, J. H. - Kang, B. R. - Rong, X. - McSpadden Gardener, B. B. - Ji, H. J. - Park, C. S. - Kim, Y. C.
Journal: J Bacteriol

Pantoea ananatis B1-9 is an endophytic Gram-negative rhizobacterium that was isolated for its ability to promote plant growth and improve crop yield in the field. Here we report the draft genome sequence of P. ananatis B1-9. Comparison of this sequence to the sequenced genome of a plant-pathogenic P. ananatis strain, LMG20103, indicated that the pathogenesis-related genes were absent, but a subset of gene functions that may be related to its plant growth promotion were present.

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Complete genome sequence of strain 1860, a crenarchaeon of the genus pyrobaculum able to grow with various electron acceptors.

Publication Date: 2012 Feb PMID: 22247528
Authors: Mardanov, A. V. - Gumerov, V. M. - Slobodkina, G. B. - Beletsky, A. V. - Bonch-Osmolovskaya, E. A. - Ravin, N. V. - Skryabin, K. G.
Journal: J Bacteriol

Strain 1860, a novel member of the genus Pyrobaculum, is a hyperthermophilic organotrophic crenarchaeon growing anaerobically with various electron acceptors. The complete genome sequence reveals genes for several membrane-bound oxidoreductases, the Embden-Meyerhof and Entner-Doudoroff pathways for glucose metabolism, the tricarboxylic acid cycle, the glyoxylate cycle, and the dicarboxylate/4-hydroxybutyrate cycle.

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Genome Sequence of Lactobacillus rhamnosus ATCC 8530.

Publication Date: 2012 Feb PMID: 22247527
Authors: Pittet, V. - Ewen, E. - Bushell, B. R. - Ziola, B.
Journal: J Bacteriol

Lactobacillus rhamnosus is found in the human gastrointestinal tract and is important for probiotics. We became interested in L. rhamnosus isolate ATCC 8530 in relation to beer spoilage and hops resistance. We report here the genome sequence of this isolate, along with a brief comparison to other available L. rhamnosus genome sequences.

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Draft Genome Sequences of the Pseudomonas fluorescens Biocontrol Strains Wayne1R and Wood1R.

Publication Date: 2012 Feb PMID: 22247526
Authors: Rong, X. - Baysal Gurel, F. - Meulia, T. - McSpadden Gardener, B. B.
Journal: J Bacteriol

Pseudomonas fluorescens strains Wayne1R and Wood1R have proven capacities to improve plant health. Here we report the draft genome sequences and automatic annotations of both strains. Genome comparisons reveal similarities with P. fluorescens strain Pf-5, reveal the novelty of Wood1R, and indicate some genes that may be related to biocontrol.

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Draft Genome Sequence of the Virulent Strain 01-B526 of the Fish Pathogen Aeromonas salmonicida.

Publication Date: 2012 Feb PMID: 22247525
Authors: Charette, S. J. - Brochu, F. - Boyle, B. - Filion, G. - Tanaka, K. H. - Derome, N.
Journal: J Bacteriol

Aeromonas salmonicida is an important fish pathogen, mainly of salmonids. This bacterium causes a disease named furunculosis, which is particularly detrimental for the aquaculture industry. Here, we present the draft genome sequence of A. salmonicida 01-B526, a strain isolated from a brook trout that is more virulent than A. salmonicida reference strain A449, for which a genome sequence is available.

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Nitric oxide stress resistance in Porphyromonas gingivalis is mediated by a putative hydroxylamine reductase.

Publication Date: 2012 Jan 13 PMID: 22247513
Authors: Boutrin, M. C. - Wang, C. - Aruni, W. - Li, X. - Fletcher, H. M.
Journal: J Bacteriol

Porphyromonas gingivalis, the causative agent of adult periodontitis, must maintain nitric oxide (NO) homeostasis and surmount nitric oxide stress from host immune response or other oral bacteria to survive in the periodontal pocket. To determine the involvement of a putative hydroxylamine reductase (PG0893) and a putative nitrite reductase-related protein (PG2213) in P. gingivalis W83 NO stress resistance, genes encoding those proteins were inactivated by allelic exchange mutagenesis. Isogenic mutants P. gingivalis FLL455 (PG0893::ermF) and FLL456 (PG2213::ermF) were black-pigmented and showed similar growth rates, gingipain and hemolytic activities compared to the wild-type strain. In contrast to the wild-type strain, P. gingivalis FLL455 was more sensitive to NO. Complementation of P. gingivalis FLL455 with the wild-type gene restored the level of NO sensitivity similar to the parent strain. P. gingivalis FLL455 and FLL456 showed similar sensitivity to oxidative stress as the wild-type strain. DNA microarray analysis showed that PG0893 and PG2213 were up regulated 1.4- and 2-fold respectively in cells exposed to NO. In addition, 178 genes were up regulated and 201 genes down regulated more than 2-fold. The majority of these modulated genes was hypothetical or of unknown function. PG1181, a predicted transcriptional regulator was up regulated 76-fold. Transcriptome in silico analysis of the microarray data showed major metabolomic variations in key pathways. Collectively, these findings indicate that PG0893 and several other genes may play an important role in P. gingivalis NO stress resistance.

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J-Western forms of Helicobacter pylori cagA constitute a distinct phylogenetic group with a widespread geographic distribution.

Publication Date: 2012 Jan 13 PMID: 22247512
Authors: Duncan, S. S. - Valk, P. L. - Shaffer, C. L. - Bordenstein, S. R. - Cover, T. L.
Journal: J Bacteriol

Chronic infection with Helicobacter pylori strains expressing the bacterial oncoprotein CagA confers an increased risk of gastric cancer. While much is known about the ancestry and molecular evolution of Western, East Asian, and Amerindian cagA sequences, relatively little is understood about a fourth group, known as "J-Western", which has been detected mainly in strains from Okinawa, Japan. We show here that J-Western cagA sequences have a more widespread global distribution than previously recognized, occur in strains with multiple different ancestral origins (based on MLST analysis), and did not arise recently. When comparing Western and J-Western forms of CagA, there are 45 fixed or nearly fixed amino acid differences, and J-Western forms contain a unique 4-amino-acid insertion. The mean nucleotide diversity of synonymous sites (pi(s)) is slightly lower in the J-Western group than in Western or East Asian groups (0.066, 0.086, and 0.083 respectively), which suggests that the three groups have comparable, but not equivalent, effective population sizes. The reduced pi(s) of the J-Western group is attributable to ancestral recombination events within the 5' region of cagA. Population genetic analyses suggest that within the cagA region encoding EPIYA motifs, the East Asian group underwent a marked reduction in effective population size compared to the Western and J-Western groups, in association with positive selection. Finally, we show that J-Western cagA sequences are found mainly in strains producing m2 forms of the secreted VacA toxin, and propose that these functionally interacting proteins co-evolved to optimize the gastric colonization capacity of H. pylori.

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The SPI-7 Family of Integrative and Conjugative Elements within Enterobacteriaceae: Structure, Diversity and Mobility.

Publication Date: 2012 Jan 13 PMID: 22247511
Authors: Seth-Smith, H. M. - Fookes, M. C. - Okoro, C. K. - Baker, S. - Harris, S. R. - Scott, P. - Pickard, D. - Quail, M. A. - Churcher, C. - Sanders, M. - Harmse, J. - Dougan, G. - Parkhill, J. - Thomson, N. R.
Journal: J Bacteriol

Integrative and Conjugative Elements (ICEs) are self-mobile genetic elements found in the genomes of some bacteria. These elements may confer a fitness advantage upon their host bacteria through the cargo genes they carry. Salmonella Pathogenicity Island 7 (SPI-7), found within some pathogenic strains of Salmonella enterica, possesses features indicative of an ICE, and carries genes implicated in virulence. We aimed to identify and fully analyse ICEs related to SPI-7 within the genus Salmonella, and other Enterobacteriaceae. We report the sequence of two novel SPI-7-like elements, found within strains of Salmonella bongori, which share 97% nucleotide identity over conserved regions with SPI-7 and with each other. Although SPI-7 within Salmonella Typhi appears to be fixed within the chromosome, we present evidence that these novel elements are capable of excision and self-mobility. Phylogenetic analyses show that these Salmonella mobile elements share a common ancestor which existed approximately 3.6-15.8 million years ago. Additionally, we identified more distantly related ICEs, with distinct cargo regions, within other strains of Salmonella as well as within Citrobacter, Erwinia, Escherichia, Photorhabdus and Yersinia species. In total we report on a collection of 17 SPI-7 related ICEs within enterobacterial species, of which six are novel. Using comparative and mutational studies, we have defined a core of 27 genes essential for conjugation. We present a growing family of SPI-7 related ICEs whose mobility, abundance and cargo variability indicate that these elements may have had a large impact on the evolution of the Enterobacteriaceae.

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Mutations at several loci cause increased expression of ribonucleotide reductase in Escherichia coli.

Publication Date: 2012 Jan 13 PMID: 22247510
Authors: Feeney, M. A. - Ke, N. - Beckwith, J.
Journal: J Bacteriol

Production of deoxyribonucleotides for DNA synthesis is an essential and tightly regulated process. The class Ia ribonucleotide reductase (RNR), the product of the nrdAB genes, is required for aerobic growth of Escherichia coli. In catalyzing the reduction of ribonucleotides, two of the cysteines of RNR become oxidized forming a disulfide bond. To regenerate active RNR, the cell uses thioredoxins and glutaredoxins to reduce the disulfide bond. Strains that lack thioredoxins 1 and 2 and glutaredoxin 1 do not grow because RNR remains in its oxidized, inactive form. However, suppressor mutations that lead to RNR overproduction allow glutaredoxin 3 to reduce sufficient RNR for growth of these mutant strains. We previously described suppressor mutations in the dnaA and dnaN genes that had such effects. Here we report the isolation of new mutations that lead to increased levels of RNR. These include mutations that were not known to influence production of RNR previously, such as a mutation in the hda gene and insertions in the nrdAB promoter region of insertion elements IS1 and IS5. Bioinformatic analysis raises the possibility that IS element insertion in this region represents an adaptive mechanism in nrdAB regulation in E. coli and closely related species. We also characterize mutations altering different amino acids in DnaA and DnaN than those isolated before.

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Outer Membrane Targeting, Ultrastructure and Single Molecule Localization of the Enteropathogenic Escherichia coli Type IV Pilus Secretin BfpB.

Publication Date: 2012 Jan 13 PMID: 22247509
Authors: Lieberman, J. A. - Frost, N. A. - Hoppert, M. - Fernandes, P. J. - Vogt, S. L. - Raivio, T. L. - Blanpied, T. A. - Donnenberg, M. S.
Journal: J Bacteriol

Type IV Pili (T4P) are filamentous surface appendages required for tissue adherence, motility, aggregation, and transformation in a wide array of bacteria and archaea. The bundle-forming pilus (BFP) of enteropathogenic Escherichia coli (EPEC) is a prototypical T4P and confirmed virulence factor. T4P fibers are assembled by a complex biogenesis machine that extrudes pili through an outer membrane (OM) pore formed by the secretin protein. Secretins constitute a superfamily of proteins that assemble into multimers and support the transport of macromolecules by four evolutionarily ancient secretion systems: T4Ps, type II secretion, type III secretion, and phage assembly. Here, we determine that the lipoprotein transport pathway is not required for targeting the BfpB secretin protein of the EPEC T4P to the OM and describe the ultrastructure of the single particle averaged structures of the assembled complex by transmission electron microscopy (TEM). Furthermore, we use Photo-activated Localization Microscopy to determine the distribution of single BfpB molecules fused to photo-activated mCherry. Contrary to findings in other T4P systems, we find that BFP components predominantly have an uneven distribution through the cell envelope and are only found at one or both poles in a minority of cells. Additionally, we report that concurrent mutation of both the T4bP secretin and retraction ATPase can result in viable cells and find that these cells display paradoxically low levels of cell envelope stress response activity. These results imply that secretins can direct their own targeting, have complex distributions and provide feedback information on the state of pilus biogenesis.

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An AMP-forming Acetyl-CoA Synthetase in the outermost membrane of the hyperthermophilic Crenarchaeon Ignicoccus hospitalis.

Publication Date: 2012 Jan 13 PMID: 22247508
Authors: Mayer, F. - Kuper, U. - Meyer, C. - Daxer, S. - Muller, V. - Rachel, R. - Huber, H.
Journal: J Bacteriol

Ignicoccus hospitalis, a hyperthermophilic, chemolithoautotrophic Crenarchaeon was found to possess a new CO(2)-fixation pathway, the dicarboxylate/4-hydroxybutyrate cycle. The primary acceptor molecule for this pathway is acetyl-CoA, which is regenerated in the cycle via the characteristic intermediate 4-hydroxybutyrate. In the presence of acetate, acetyl-CoA can alternatively be formed in a one-step mechanism via an AMP-forming acetyl-CoA synthetase (ACS). This enzyme was identified after membrane preparation by 2D native PAGE/SDS-PAGE, followed by MALDI-TOF MS/MS and N-terminal sequencing. The ACS of I. hospitalis exhibits a molecular mass of approximately 690 kDa with a monomeric molecular mass of 77 kDa. Activity tests on isolated membranes and bioinformatic analyses indicated that the ACS is a constitutive membrane-associated (but not an integral) protein complex. Unexpectedly, immuno-labeling on cells of I. hospitalis and the other described Igniccoccus species revealed that its ACS is localized at the outermost membrane. This is in perfect coincidence to recent results that the ATP synthase and the H(2):sulfur oxidoreductase complexes are located in the outermost membrane of I. hospitalis, too. These results imply that the intermembrane compartment of I. hospitalis is not only the site of ATP synthesis but may also be involved in the primary steps of CO(2)-fixation.

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The O-methyltransferase SrsB catalyzes the decarboxylative methylation of alkylresorcylic acid during phenolic lipid biosynthesis by Streptomyces griseus.

Publication Date: 2012 Jan 13 PMID: 22247507
Authors: Nakano, C. - Funa, N. - Ohnishi, Y. - Horinouchi, S.
Journal: J Bacteriol

Streptomyces griseus contains the srs operon required for phenolic lipid biosynthesis. The operon consists of srsA, srsB and srsC, which encode a type III polyketide synthase, an O-methyltransferase and a flavoprotein hydroxylase, respectively. We previously reported that the recombinant SrsA protein synthesized 3-(13' -methyltetradecyl)-4-methylresorcinol, using iso-C(16) fatty acyl-CoA as a starter substrate, and malonyl-CoA and methylmalonyl-CoA as extender substrates. An in vitro SrsA reaction using [(13)C(3)]malonyl-CoA confirmed that the order of extender substrate condensation was methylmalonyl-CoA, followed by two extensions with malonyl-CoA. Furthermore, SrsA was revealed to produce an alkylresorcylic acid as its direct product, rather than an alkylresorcinol as previously proposed. Functional SrsB protein was produced in the membrane fraction in Streptomyces lividans and used for the in vitro SrsB reaction. When the SrsA reaction was coupled, SrsB produced alkylresorcinol methyl ether in the presence of S-adenosyl-L-methionine (SAM). SrsB was incapable of catalyzing O-methylation of alkylresorcinol, indicating that alkylresorcylic acid was the substrate of SrsB and that SrsB catalyzed the conversion of alkylresorcylic acid to alkylresorcinol methyl ether, namely by both O-methylation of the hydroxyl group (C-6) and decarboxylation of the neighboring carboxyl group (C-1). O-Methylated alkylresorcylic acid was not detected in the in vitro SrsAB reaction, though it was presumably stable, indicating that O-methylation did not precede decarboxylation. We therefore postulated that O-methylation was coupled with decarboxylation and proposed that SrsB catalyzed the feasible SAM-dependent decarboxylative methylation of alkylresorcylic acid. To our best of knowledge, this is the first report of a methyltransferase that catalyzes decarboxylative methylation.

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Further unravelling the regulatory twist - metabolic coinducer-mediated CbbR-cbbI promoter interactions in Rhodopseudomonas palustris CGA010.

Publication Date: 2012 Jan 13 PMID: 22247506
Authors: Joshi, G. S. - Zianni, M. - Bobst, C. E. - Tabita, F. R.
Journal: J Bacteriol

The cbb(I) region of Rhodopseudomonas palustris contains the cbbLS genes encoding form I ribulose -1, 5 -bisphosphate (RuBP) carboxylase oxygenase (RubisCO) along with a divergently transcribed regulator gene, cbbR. Juxtaposed between cbbR and cbbLS are the cbbRRS genes encoding an unusual three protein two-component (CbbRRS) system that modulates the ability of CbbR to influence cbbLS expression. The nature of the metabolic signals that Rps. palustris CbbR perceives to regulate cbbLS transcription is not known. Thus, in this study, the CbbR binding region was first mapped within the cbbLS promoter using gel mobility shift assays and DNase I footprinting. In addition, potential metabolic coinducers (metabolites) were tested for their ability to alter the cbbLS promoter binding properties of CbbR. Gel mobility shift assays and surface plasmon resonance analyses together indicated that biosynthetic intermediates such as RuBP, ATP, fructose 1,6-bisphosphate and NADPH enhanced DNA binding by CbbR. These coinducers did not yield identical CbbR-dependent DNase I footprints indicating that the coinducers caused significant changes in DNA structure. These in vitro studies suggest that cellular signals such as fluctuating metabolite concentrations are perceived and transduced to the cbbLS promoter via the master regulator CbbR.

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Characterization of Escherichia coli dinJ-yafQ toxin-antitoxin system: insights from mutagenesis data.

Publication Date: 2012 Jan 13 PMID: 22247505
Authors: Armalyte, J. - Jurenaite, M. - Beinoraviciute, G. - Teiserskas, J. - Suziedeliene, E.
Journal: J Bacteriol

Escherichia coli dinJ-yafQ operon codes for a functional toxin-antitoxin system. YafQ toxin is a ribonuclease, which upon overproduction specifically inhibits translation process by cleaving cellular mRNA at specific sequences. DinJ is an antitoxin and counteracts YafQ-mediated toxicity by forming a strong protein complex. In the present study we used site-directed mutagenesis of YafQ to determine the amino acids important for its catalytic activity. The His50Ala, His63Ala, Asp67Ala, Trp68Ala, Trp68Phe, Arg83Ala, His87Ala, Phe91Ala substitutions of the predicted active site residues of YafQ, abolished mRNA cleavage in vivo, whereas Asp61Ala and Phe91Tyr mutations inhibited YafQ ribonuclease activity only moderately. We show, that upon overexpression, YafQ cleaved mRNAs preferably 5' to A, between the second and the third nucleotide in the codon, in vivo. YafQ also showed ribonuclease activity against mRNA, tRNA and 5S rRNA molecules in vitro, albeit with no strong specificity. The endoribonuclease activity of YafQ was inhibited in the complex with DinJ antitoxin in vitro. DinJ-YafQ protein complex and DinJ antitoxin alone selectively bind to one of the two palindromic sequences present in the intergenic region upstream the dinJ-yafQ operon suggesting the autoregulation mode of this TA system.

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An Evolutionary link between the mycobacterial plasmid pAL5000 replication protein RepB and Extra Cytoplasmic Function (ECF) family of sigma factors.

Publication Date: 2012 Jan 13 PMID: 22247504
Authors: Basu, A. - Chatterjee, S. - Chatterjee, S. - Das Gupta, S. K.
Journal: J Bacteriol

Mycobacterial plasmid pAL5000 represents a family of plasmids found mostly in Actinobacteria. It replicates using two plasmid encoded proteins RepA and RepB. While BLAST searches indicate that RepA is a Replicase family protein, the evolutionary connection of RepB cannot be established as no significant homologous partner (E < 10(-3)) outside the RepB family can be identified. To obtain insight into the structure-function and evolutionary connections of RepB, an investigation was undertaken using homology modeling, phylogenetic and mutational analysis methods. The results indicate that although synthesized from the same operon phylogenetic affinities of RepA and RepB differ. The operon thus may have evolved through random breaking and joining events. Homology modeling predicted the presence of a three-helical Helix-Turn-Helix domain characteristic of the region 4 of Extra Cytoplasmic Function (ECF) sigma factors, in the C-teminal region of RepB. At the N terminal region there is a helical stretch, which may be distantly related to region 3 of sigma factors. Mutational analysis identified two arginines indispensible for RepB activity, one each located within the C and N terminal conserved regions respectively. Apart from analyzing the domain organization of the protein, the significance of the presence of a highly conserved A/T rich element, within the RepB binding site, was investigated. Mutational analysis revealed, that although this motif does not bind RepB, its integrity is important for efficient DNA-protein interaction and replication to occur. The present investigation unravels the possibility that RepB like proteins and their binding sites represent ancient DNA-protein interaction modules.

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CtsR Regulation in mcsAB Deficient Gram-positive Bacteria.

Publication Date: 2012 Jan 13 PMID: 22247503
Authors: Tao, L. - Chattoraj, P. - Biswas, I.
Journal: J Bacteriol

CtsR is an important repressor that modulates the transcription of class III stress genes in gram-positive bacteria. In Bacillus subtilis, a model gram-positive organism, the DNA binding activity of CtsR is regulated by McsAB mediated phosphorylation of the protein where the phosphorylated CtsR is a substrate for degradation by ClpCP complex. Surprisingly, in many gram-positive bacteria including streptococci the mcsAB genes are absent; therefore, how the CtsR activity is modulated in those bacteria remains unknown. Here we show that the post-translational modulation of CtsR activity is different in Streptococcus mutans, a dental pathogen. We observed that among all the Clp-related proteins, only ClpL is involved in the degradation of CtsR. Neither ClpP nor ClpC had any effect on the degradation of CtsR. We also found that phosphorylation of CtsR on a conserved arginine residue within the winged Helix-turn-Helix domain is necessary for modulation of repressor activity of CtsR as demonstrated by both in vitro and in vivo assays. We speculate that CtsR is regulated post-translationally by a different mechanism in S. mutans and possibly in other streptococci.

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Regulation of bacteriocin production and cell death by the VicRK signaling system in Streptococcus mutans.

Publication Date: 2012 Jan 6 PMID: 22228735
Authors: Senadheera, D. B. - Cordova, M. - Ayala, E. A. - Chavez de Paz, L. E. - Singh, K. - Downey, J. S. - Svensater, G. - Goodman, S. D. - Cvitkovitch, D. G.
Journal: J Bacteriol

The VicRK two component signaling system modulates biofilm formation, genetic competence and stress tolerance in Streptococcus mutans. Here, we show that the VicRK modulates bacteriocin production and cell viability, in part by direct modulation of the competence stimulating peptide (CSP) production in S. mutans. Global transcriptome and real-time transcriptional analysis of the VicK-deficient mutant (SmuvicK) revealed significant modulation of several bacteriocin-related loci, including nlmA/B, nlmC and nlmD (p<0.001), suggesting a role for the VicRK in producing mutacins IV, V and VI. Bacteriocin overlay assays revealed an altered ability of the vic mutants to kill related species. Since a well-conserved VicR binding site (TGTWAH-N(5)-TGTWAH) was identified within the comC coding region, we confirmed VicR binding to this sequence using DNA footprinting. Overexpression of the vic operon caused growth-phase dependent repression of comC, comDE, and comX. In the vic mutants, transcription of nlmC/cipB encoding mutacin V, previously linked to CSP-dependent cell lysis, as well as expression of its putative immunity factor encoded by immB, were significantly affected relative to wild type (p<0.05). In contrast to previous reports that proposed a hyperresistant phenotype for the VicK mutant in cell viability, release of extracellular genomic DNA was significantly enhanced in SmuvicK (p<0.05), likely as a result of increased autolysis compared with the parent. The drastic influence of VicRK on cell viability was also demonstrated using vic mutant biofilms. Taken together, we have identified a novel regulatory link between the VicRK and ComDE systems to modulate bacteriocin production and cell viability of S. mutans.

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The role of long and short replication initiation proteins in the fate of IncP-1 plasmids.

Publication Date: 2012 Jan 6 PMID: 22228734
Authors: Yano, H. - Deckert, G. E. - Rogers, L. M. - Top, E. M.
Journal: J Bacteriol

Broad-host-range IncP-1 plasmids generally encode two replication initiation proteins, TrfA1 and TrfA2. TrfA2 is produced from an internal translational start site within trfA1. While TrfA1 was previously shown to be essential for replication in Pseudomonas aeruginosa, its role in other bacteria within its broad host range has not been established. To address the role of TrfA1 and TrfA2 in other hosts, efficiency of transformation, plasmid copy number (PCN), and plasmid stability were first compared between a mini-IncP-1beta plasmid and its trfA1(-) variant in four phylogenetically distant hosts: Escherichia coli, Pseudomonas putida, Sphingobium japonicum, and Cupriavidus necator. TrfA2 was sufficient for replication in these hosts, but the presence of TrfA1 enhanced transformation efficiency and PCN. However, TrfA1 did not contribute to, or even negatively affected, long-term plasmid persistence. When trfA genes were cloned under a constitutive promoter in the chromosomes of the four hosts, strains expressing either both TrfA1 and TrfA2, or TrfA1 alone, again generally elicited a higher PCN of an IncP-1beta replicon than strains expressing TrfA2 alone. When a single species of TrfA was produced at different concentrations in E. coli cells, TrfA1 maintained a three- to four-fold higher PCN than TrfA2 at the same TrfA concentrations, indicating that replication mediated by TrfA1 is more efficient than that by TrfA2. These results suggest that the broad-host-range properties of IncP-1 plasmids are essentially conferred by TrfA2 and the intact replication origin alone, but that TrfA1 is nonetheless important to efficiently establish plasmid replication upon transfer into a broad range of hosts.

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Three-Dimensional Structures of Pathogenic and Saprophytic Leptospira Revealed by Cryo-Electron Tomography.

Publication Date: 2012 Jan 6 PMID: 22228733
Authors: Raddi, G. - Morado, D. R. - Yan, J. - Haake, D. A. - Yang, X. F. - Liu, J.
Journal: J Bacteriol

Leptospira interrogans is the primary causative agent of the most widespread zoonotic disease, leptospirosis. An in-depth structural characterization of L. interrogans is needed to understand its biology and pathogenesis. In this study, cryo-electron tomography (cryo-ET) was used to compare pathogenic and saprophytic species and examine the unique morphological features of this group of bacteria. Specifically, our study revealed a structural difference between the cell envelopes of L. interrogans and L. biflexa involving variations in the lipopolysaccharide (LPS) layer. Through cryo-ET and subvolume averaging, we determined the first three-dimensional (3-D) structure of the flagellar motor of leptospira, with novel features in the flagellar C ring, export apparatus and stator. Together with direct visualization of chemoreceptor arrays, DNA packing, periplasmic filaments, spherical cytoplasmic bodies, and a unique "cap" at the cell end, this report provides structural insights into these fascinating Leptospira.

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Physiology of resistant Deinococcus geothermalis bacterium aerobically cultivated in low manganese medium.

Publication Date: 2012 Jan 6 PMID: 22228732
Authors: Liedert, C. - Peltola, M. - Bernhardt, J. - Neubauer, P. - Salkinoja-Salonen, M.
Journal: J Bacteriol

This dynamic proteome study describes the physiology of growth and survival of Deinococcus geothermalis, in conditions simulating paper machine waters being aerobic, warm, and low in carbon and manganese. Industrial environment of this species differ from its natural habitats, geothermal springs and deep ocean subsurfaces, by being highly exposed to oxygen. Quantitative proteome analysis using two-dimensional gel electrophoresis and bioinformatic tools showed expression change for 165 proteins from which 47 were assigned to a function. We propose that D. geothermalis grew and survived in aerobic conditions by channelling central carbon metabolism to pathways where mainly NADPH rather than NADH was retrieved from the carbon source. Major part of carbon substrate was converted into succinate which was not a fermentation product but likely served combating reactive oxygen species (ROS). Transition from growth to nongrowth resulted in downregulation of the oxidative phosphorylation observed as reduced expression of V-type ATPase responsible for ATP synthesis in D. geothermalis. The battle against oxidative stress was seen as upregulation of superoxide dismutase (Mn-dependent) and catalase as well as several protein repair enzymes including FeS cluster assembly proteins of the Suf system, peptidylprolyl isomerase and chaperones. Addition of soluble Mn reinitiated respiration and proliferation with concomitant acidification indicating that aerobic metabolism was restricted by access to manganese. We conclude that D. geothermalis prefers to combat ROS using manganese dependent enzymes but when manganese is not available central carbon metabolism is employed to produce ROS neutralising metabolites at the expense of high utilisation of carbon substrate.

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Legionella pneumophila LidA Affects Nucleotide Binding and Activity of the Host GTPase Rab1.

Publication Date: 2012 Jan 6 PMID: 22228731
Authors: Neunuebel, M. R. - Mohammadi, S. - Jarnik, M. - Machner, M. P.
Journal: J Bacteriol

Legionella pneumophila, the causative agent of a severe pneumonia known as Legionnaires' disease, intercepts material from host cell membrane transport pathways to create a specialized vacuolar compartment that supports bacterial replication. Delivery of bacterial effector proteins into the host cell requires the Dot/Icm type IV secretion system. Several effectors, including SidM, SidD, and LepB, were shown to target the early secretory pathway by manipulating the activity of the host guanosine triphosphatase (GTPase) Rab1. While the function of these effectors has been well characterized, the role of another Rab1-interacting protein from L. pneumophila, the effector protein LidA, is poorly understood. Here, we show that LidA binding to Rab1 stabilized the Rab1-guanosine nucleotide complex, protecting it from inactivation by GTPase activating proteins (GAPs) and from nucleotide extraction. The protective effect of LidA on the Rab1-guanine nucleotide complex was concentration-dependent, consistent with a 1:1 stoichiometry of the LidA-Rab1 complex. The central coiled-coil region of LidA was sufficient for Rab1 binding and to prevent GAP-mediated inactivation or nucleotide extraction from Rab1. In addition, the central region mediated binding to phosphatidylinositol 3-phosphate and other phosphoinositides. When bound to Rab1, LidA interfered with the covalent modification of Rab1 by phosphocholination or AMPylation, and it also blocked de-AMPylation of Rab1 by SidD and de-phosphocholination by Lem3. Based on these findings we propose a role for LidA in bridging the membrane of the LCV with that of secretory transport vesicles surrounding the LCV.

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Characterization and Functional Analysis of Atl, a Novel Gene Encoding Autolysin in Streptococcus suis.

Publication Date: 2012 Jan 6 PMID: 22228730
Authors: Ju, C. X. - Gu, H. W. - Lu, C. P.
Journal: J Bacteriol

Streptococcus suis serotype 2 (S. suis 2) is an important swine and human pathogen responsible for septicemia and meningitis. A novel gene, designated Atl and encoding a major autolysin of S. suis 2 virulent strain HA9801, was identified and characterized in this study. The Atl protein contains 1,025 amino acids with a predicted molecular weight of 113 kDa and has a conserved N-acetylmuramoyl-L-alanine amidase domain. Recombinant Atl was expressed in Esherichia coli, and its bacteriolytic and fibronectin-binding activities were confirmed by zymography and Western affinity blotting. Two bacteriolytic bands were shown in the sodium dodecyl sulfate extracts of HA9801, while both were absent from the Atl inactivated mutant. Cell chains of the mutant strain became longer than that of the parental strain. In the autolysis assay, HA9801 decreased to 20% of the initial OD value, while the mutant strain had almost no autolytic activity. The biofilm capacity of the Atl mutant was reduced approximately 30% compared to the parental strain. In the zebrafish infection model, the 50% lethal dose of the mutant strain was increased up to 5-fold. Furthermore, the adherence to HEp-2 cells of the Atl mutant was 50% less than that of the parental strain. Based on the functional analysis of the recombinant Atl and observed effects of Atl inactivation on HA9801, we conclude that Atl is a major autolysin of HA9801. It takes part in cell autolysis, separation of daughter cells, biofilm formation, fibronectin-binding activity, cell adhesion and pathogenesis of HA9801.

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Helicobacter pylori Relies Primarily on the Purine Salvage Pathway for Purine Nucleotide Biosynthesis.

Publication Date: 2012 Feb PMID: 22194455
Authors: Liechti, G. - Goldberg, J. B.
Journal: J Bacteriol

Helicobacter pylori is a chronic colonizer of the gastric epithelium and plays a major role in the development of gastritis, peptic ulcer disease, and gastric cancer. In its coevolution with humans, the streamlining of the H. pylori genome has resulted in a significant reduction in metabolic pathways, one being purine nucleotide biosynthesis. Bioinformatic analysis has revealed that H. pylori lacks the enzymatic machinery for de novo production of IMP, the first purine nucleotide formed during GTP and ATP biosynthesis. This suggests that H. pylori must rely heavily on salvage of purines from the environment. In this study, we deleted several genes putatively involved in purine salvage and processing. The growth and survival of these mutants were analyzed in both nutrient-rich and minimal media, and the results confirmed the presence of a robust purine salvage pathway in H. pylori. Of the two phosphoribosyltransferase genes found in the H. pylori genome, only gpt appears to be essential, and an Deltaapt mutant strain was still capable of growth on adenine, suggesting that adenine processing via Apt is not essential. Deletion of the putative nucleoside phosphorylase gene deoD resulted in an inability of H. pylori to grow on purine nucleosides or the purine base adenine. Our results suggest a purine requirement for growth of H. pylori in standard media, indicating that H. pylori possesses the ability to utilize purines and nucleosides from the environment in the absence of a de novo purine nucleotide biosynthesis pathway.

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Involvement of T6 Pili in Biofilm Formation by Serotype M6 Streptococcus pyogenes.

Publication Date: 2012 Feb PMID: 22155780
Authors: Kimura, K. R. - Nakata, M. - Sumitomo, T. - Kreikemeyer, B. - Podbielski, A. - Terao, Y. - Kawabata, S.
Journal: J Bacteriol

The group A streptococcus (GAS) Streptococcus pyogenes is known to cause self-limiting purulent infections in humans. The role of GAS pili in host cell adhesion and biofilm formation is likely fundamental in early colonization. Pilus genes are found in the FCT (fibronectin-binding protein, collagen-binding protein, and trypsin-resistant antigen) genomic region, which has been classified into nine subtypes based on the diversity of gene content and nucleotide sequence. Several epidemiological studies have indicated that FCT type 1 strains, including serotype M6, produce large amounts of monospecies biofilm in vitro. We examined the direct involvement of pili in biofilm formation by serotype M6 clinical isolates. In the majority of tested strains, deletion of the tee6 gene encoding pilus shaft protein T6 compromised the ability to form biofilm on an abiotic surface. Deletion of the fctX and srtB genes, which encode pilus ancillary protein and class C pilus-associated sortase, respectively, also decreased biofilm formation by a representative strain. Unexpectedly, these mutant strains showed increased bacterial aggregation compared with that of the wild-type strain. When the entire FCT type 1 pilus region was ectopically expressed in serotype M1 strain SF370, biofilm formation was promoted and autoaggregation was inhibited. These findings indicate that assembled FCT type 1 pili contribute to biofilm formation and also function as attenuators of bacterial aggregation. Taken together, our results show the potential role of FCT type 1 pili in the pathogenesis of GAS infections.

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Mutation of the Sensor Kinase chvG in Rhizobium leguminosarum Negatively Impacts Cellular Metabolism, Outer Membrane Stability, and Symbiosis.

Publication Date: 2012 Feb PMID: 22155778
Authors: Vanderlinde, E. M. - Yost, C. K.
Journal: J Bacteriol

Two-component signal transduction systems (TCS) are a main strategy used by bacteria to sense and adapt to changes in their environment. In the legume symbiont Rhizobium leguminosarum biovar viciae VF39, mutation of chvG, a histidine kinase, caused a number of pleiotropic phenotypes. ChvG mutants are unable to grow on proline, glutamate, histidine, or arginine as the sole carbon source. The chvG mutant secreted smaller amounts of acidic and neutral surface polysaccharides and accumulated abnormally large amounts of poly-ss-hydroxybutyrate. Mutation of chvG caused symbiotic defects on peas, lentils, and vetch; nodules formed by the chvG mutant were small and white and contained only a few cells that had failed to differentiate into bacteroids. Mutation of chvG also destabilized the outer membrane of R. leguminosarum, resulting in increased sensitivity to membrane stressors. Constitutive expression of ropB, the outer membrane protein-encoding gene, restored membrane stability and rescued the sensitivity phenotypes described above. Similar phenotypes have been described for mutations in other ChvG-regulated genes encoding a conserved operon of unknown function and in the fabXL genes required for synthesis of the lipid A very-long-chain fatty acid, suggesting that ChvG is a key component of the envelope stress response in Rhizobium leguminosarum. Collectively, the results of this study demonstrate the important and unique role the ChvG/ChvI TCS plays in the physiology, metabolism, and symbiotic competency of R. leguminosarum.

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BpaB and EbfC DNA-Binding Proteins Regulate Production of the Lyme Disease Spirochete's Infection-Associated Erp Surface Proteins.

Publication Date: 2012 Feb PMID: 22155777
Authors: Jutras, B. L. - Verma, A. - Adams, C. A. - Brissette, C. A. - Burns, L. H. - Whetstine, C. R. - Bowman, A. - Chenail, A. M. - Zuckert, W. R. - Stevenson, B.
Journal: J Bacteriol

Vector-borne pathogens regulate their protein expression profiles, producing factors during host infection that differ from those produced during vector colonization. The Lyme disease agent, Borrelia burgdorferi, produces Erp surface proteins throughout mammalian infection and represses their synthesis during colonization of vector ticks. Known functions of Erp proteins include binding of host laminin, plasmin(ogen), and regulators of complement activation. A DNA region immediately 5' of erp operons, the erp operator, is required for transcriptional regulation. The B. burgdorferi BpaB and EbfC proteins exhibit high in vitro affinities for erp operator DNA. In the present studies, chromatin immunoprecipitation (ChIP) demonstrated that both proteins bind erp operator DNA in vivo. Additionally, a combination of in vivo and in vitro methods demonstrated that BpaB functions as a repressor of erp transcription, while EbfC functions as an antirepressor.

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The translocation domain in trimeric autotransporter adhesins is necessary and sufficient for trimerization and autotransportation.

Publication Date: 2012 Feb PMID: 22155776
Authors: Mikula, K. M. - Leo, J. C. - Lyskowski, A. - Kedracka-Krok, S. - Pirog, A. - Goldman, A.
Journal: J Bacteriol

Trimeric autotransporter adhesins (TAAs) comprise one of the secretion pathways of the type V secretion system. The mechanism of their translocation across the outer membrane remains unclear, but it most probably occurs by the formation of a hairpin inside the beta-barrel translocation unit, leading to transportation of the passenger domain from the C terminus to the N terminus through the lumen of the beta-barrel. We further investigated the phenomenon of autotransportation and the rules that govern it. We showed by coexpressing different Escherichia coli immunoglobulin-binding (Eib) proteins that highly similar TAAs could form stochastically mixed structures (heterotrimers). We further investigated this phenomenon by coexpressing two more distantly related TAAs, EibA and YadA. These, however, did not form heterotrimers; indeed, coexpression was lethal to the cells, leading to elimination of one or another of the genes. However, substituting in either protein the barrel of the other one so that the barrels were identical led to formation of heterotrimers as for Eibs. Our work shows that trimerization of the beta-barrel, but not the passenger domain, is necessary and sufficient for TAA secretion while the passenger domain is not.

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Genes Involved in Immunity to and Secretion of Aureocin A53, an Atypical Class II Bacteriocin Produced by Staphylococcus aureus A53.

Publication Date: 2012 Feb PMID: 22155775
Authors: Nascimento Jdos, S. - Coelho, M. L. - Ceotto, H. - Potter, A. - Fleming, L. R. - Salehian, Z. - Nes, I. F. - Bastos Mdo, C.
Journal: J Bacteriol

Aureocin A53 is an antimicrobial peptide produced by Staphylococcus aureus A53. The genetic determinants involved in aureocin A53 production and immunity to its action are organized in at least four transcriptional units encoded by the 10.4-kb plasmid pRJ9. One transcriptional unit carries only the bacteriocin structural gene, aucA. No immunity gene is found downstream of aucA, as part of the same transcriptional unit. Further downstream of aucA is found an operon which contains the three genes aucEFG, whose products seem to associate to form a dedicated ABC transporter. When aucEFG were expressed in RN4220, an aureocin A53-sensitive S. aureus strain, this strain became partially resistant to the bacteriocin. A gene disruption mutant in aucE was defective in aureocin A53 externalization and more sensitive to aureocin A53 than the wild-type strain, showing that aucEFG are involved in immunity to aureocin A53 by active extrusion of the bacteriocin. Full resistance to aureocin A53 was exhibited by transformants carrying, besides aucEFG, the operon formed by two genes, aucIB and aucIA, located between aucA and aucEFG and carried in the opposite strand. AucIA and AucIB share similarities with hypothetical proteins not found in the gene clusters of other bacteriocins. A gene disruption mutant in orf8, located upstream of aucA and whose product exhibits about 50% similarity to a number of hypothetical membrane proteins found in many Gram-positive bacteria, was strongly affected in aureocin A53 externalization but resistant to aureocin A53, suggesting that Orf8 is also involved in aureocin A53 secretion.

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EspD Is Critical for the Virulence-Mediating ESX-1 Secretion System in Mycobacterium tuberculosis.

Publication Date: 2012 Feb PMID: 22155774
Authors: Chen, J. M. - Boy-Rottger, S. - Dhar, N. - Sweeney, N. - Buxton, R. S. - Pojer, F. - Rosenkrands, I. - Cole, S. T.
Journal: J Bacteriol

ESAT-6 system 1 (ESX-1)-mediated secretion in Mycobacterium tuberculosis is dependent on proteins encoded by the cotranscribed espA-espC-espD gene cluster. While the roles of EspA and EspC with respect to the ESX-1 secretion system have been actively investigated, the function of EspD remains unknown. We show that EspD is secreted by M. tuberculosis, but unlike EspA and EsxA, its export does not exclusively require the ESX-1 system. Evidence for stabilization of cellular levels of EspA and EspC by EspD is presented, and depletion of EspD results in loss of EsxA secretion. Site-directed mutagenesis of EspD reveals that its role in the maintenance of cellular levels of EspA in M. tuberculosis is distinct from its facilitation of EsxA secretion. The same mutagenesis experiments have also shown that secretion of EspD is not required for the secretion of EsxA. Our findings highlight a critical and complex role for EspD in modulating the ESX-1 secretion system in M. tuberculosis.

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A Single-Domain FlgJ Contributes to Flagellar Hook and Filament Formation in the Lyme Disease Spirochete Borrelia burgdorferi.

Publication Date: 2012 Feb PMID: 22155773
Authors: Zhang, K. - Tong, B. A. - Liu, J. - Li, C.
Journal: J Bacteriol

FlgJ plays a very important role in flagellar assembly. In the enteric bacteria, flgJ null mutants fail to produce the flagellar rods, hooks, and filaments but still assemble the integral membrane-supramembrane (MS) rings. These mutants are nonmotile. The FlgJ proteins consist of two functional domains. The N-terminal rod-capping domain acts as a scaffold for rod assembly, and the C-terminal domain acts as a peptidoglycan (PG) hydrolase (PGase), which allows the elongating flagellar rod to penetrate through the PG layer. However, the FlgJ homologs in several bacterial phyla (including spirochetes) often lack the PGase domain. The function of these single-domain FlgJ proteins remains elusive. Herein, a single-domain FlgJ homolog (FlgJ(Bb)) was studied in the Lyme disease spirochete Borrelia burgdorferi. Cryo-electron tomography analysis revealed that the flgJ(Bb) mutant still assembled intact flagellar basal bodies but had fewer and disoriented flagellar hooks and filaments. Consistently, Western blots showed that the levels of flagellar hook (FlgE) and filament (FlaB) proteins were substantially decreased in the flgJ(Bb) mutant. Further studies disclosed that the decreases of FlgE and FlaB in the mutant occurred at the posttranscriptional level. Microscopic observation and swarm plate assay showed that the motility of the flgJ(Bb) mutant was partially deficient. The altered phenotypes were completely restored when the mutant was complemented. Collectively, these results indicate that FlgJ(Bb) is involved in the assembly of the flagellar hook and filament but not the flagellar rod in B. burgdorferi. The observed phenotype is different from that of flgJ mutants in the enteric bacteria.

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Isolation and Characterization of the Prochlorococcus Carboxysome Reveal the Presence of the Novel Shell Protein CsoS1D.

Publication Date: 2012 Feb PMID: 22155772
Authors: Roberts, E. W. - Cai, F. - Kerfeld, C. A. - Cannon, G. C. - Heinhorst, S.
Journal: J Bacteriol

Cyanobacteria, including members of the genus Prochlorococcus, contain icosahedral protein microcompartments known as carboxysomes that encapsulate multiple copies of the CO(2)-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) in a thin protein shell that enhances the catalytic performance of the enzyme in part through the action of a shell-associated carbonic anhydrase. However, the exact mechanism by which compartmentation provides a catalytic advantage to the enzyme is not known. Complicating the study of cyanobacterial carboxysomes has been the inability to obtain homogeneous carboxysome preparations. This study describes the first successful purification and characterization of carboxysomes from the marine cyanobacterium Prochlorococcus marinus MED4. Because the isolated P. marinus MED4 carboxysomes were free from contaminating membrane proteins, their protein complement could be assessed. In addition to the expected shell proteins, the CsoS1D protein that is not encoded by the canonical cso gene clusters of alpha-cyanobacteria was found to be a low-abundance shell component. This finding and supporting comparative genomic evidence have important implications for carboxysome composition, structure, and function. Our study indicates that carboxysome composition is probably more complex than was previously assumed based on the gene complements of the classical cso gene clusters.

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Surface-Localized Spermidine Protects the Pseudomonas aeruginosa Outer Membrane from Antibiotic Treatment and Oxidative Stress.

Publication Date: 2012 Feb PMID: 22155771
Authors: Johnson, L. - Mulcahy, H. - Kanevets, U. - Shi, Y. - Lewenza, S.
Journal: J Bacteriol

Extracellular DNA acts as a cation chelator and induces the expression of antibiotic resistance genes regulated by Mg(2+) levels. Here we report the characterization of novel DNA-induced genes in Pseudomonas aeruginosa that are annotated as homologs of the spermidine synthesis genes speD (PA4773) and speE (PA4774). The addition of sublethal concentrations of DNA and membrane-damaging antibiotics induced expression of the genes PA4773 to PA4775, as shown using transcriptional lux fusions and quantitative RT-PCR. Exogenous polyamine addition prevented DNA- and peptide-mediated gene induction. Mutation of PA4774 resulted in an increased outer membrane (OM) susceptibility phenotype upon polymyxin B, CP10A, and gentamicin treatment. When the membrane-localized fluorescent probe C(11)-BODIPY(581/591) was used as an indicator of peroxidation of membrane lipids, the PA4774::lux mutant demonstrated an increased susceptibility to oxidative membrane damage from H(2)O(2) treatment. Addition of exogenous polyamines protected the membranes of the PA4774::lux mutant from polymyxin B and H(2)O(2) treatment. Polyamines from the outer surface were isolated and shown to contain putrescine and spermidine by using high-performance liquid chromatography and mass spectrometry. The PA4774::lux mutant did not produce spermidine on the cell surface, but genetic complementation restored surface spermidine production as well as the antibiotic and oxidative stress resistance phenotypes of the membrane. We have identified new functions for spermidine on the cell surface and propose that polyamines are produced under Mg(2+)-limiting conditions as an organic polycation to bind lipopolysaccharide (LPS) and to stabilize and protect the outer membrane against antibiotic and oxidative damage.

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Proline Utilization by Bacillus subtilis: Uptake and Catabolism.

Publication Date: 2012 Feb PMID: 22139509
Authors: Moses, S. - Sinner, T. - Zaprasis, A. - Stoveken, N. - Hoffmann, T. - Belitsky, B. R. - Sonenshein, A. L. - Bremer, E.
Journal: J Bacteriol

l-Proline can be used by Bacillus subtilis as a sole source of carbon or nitrogen. We traced l-proline utilization genetically to the putBCP (ycgMNO) locus. The putBCP gene cluster encodes a high-affinity proline transporter (PutP) and two enzymes, the proline dehydrogenase PutB and the Delta(1)-pyrroline-5-carboxylate dehydrogenase PutC, which jointly catabolize l-proline to l-glutamate. Northern blotting, primer extension, and putB-treA reporter gene fusion analysis showed that the putBCP locus is transcribed as an l-proline-inducible operon. Its expression was mediated by a SigA-type promoter and was dependent on the proline-responsive PutR activator protein. Induction of putBCP expression was triggered by the presence of submillimolar concentrations of l-proline in the growth medium. However, the very large quantities of l-proline (up to several hundred millimolar) synthesized by B. subtilis as a stress protectant against high osmolarity did not induce putBCP transcription. Induction of putBCP transcription by external l-proline was not dependent on l-proline uptake via the substrate-inducible PutP or the osmotically inducible OpuE transporter. It was also not dependent on the chemoreceptor protein McpC required for chemotaxis toward l-proline. Our findings imply that B. subtilis can distinguish externally supplied l-proline from internal l-proline pools generated through de novo synthesis. The molecular basis of this regulatory phenomenon is not understood. However, it provides the B. subtilis cell with a means to avoid a futile cycle of de novo l-proline synthesis and consumption by not triggering the expression of the putBCP l-proline catabolic genes in response to the osmoadaptive production of the compatible solute l-proline.

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Role of ArlRS in Autolysis in Methicillin-Sensitive and Methicillin-Resistant Staphylococcus aureus Strains.

Publication Date: 2012 Feb PMID: 22139508
Authors: Memmi, G. - Nair, D. R. - Cheung, A.
Journal: J Bacteriol

Autolysis plays an essential role in bacterial cell division and lysis with beta-lactam antibiotics. Accordingly, the expression of autolysins is tightly regulated by several endogenous regulators, including ArlRS, a two component regulatory system that has been shown to negatively regulate autolysis in methicillin-sensitive Staphylococcus aureus (MSSA) strains. In this study, we found that inactivation of arlRS does not play a role in autolysis of methicillin-resistant S. aureus (MRSA) strains, such as community-acquired (CA)-MRSA strains USA300 and MW2 or the hospital-acquired (HA)-MRSA strain COL. This contrasts with MSSA strains, including Newman, SH1000, RN6390, and 8325-4, where autolysis is affected by ArlRS. We further demonstrated that the striking difference in the roles of arlRS between MSSA and MRSA strains is not due to the methicillin resistance determinant mecA. Among known autolysins and their regulators, we found that arlRS represses lytN, while no effect was seen on atl, lytM, and lytH expression in both CA- and HA-MRSA strains. Transcriptional-fusion assays showed that the agr transcripts, RNAII and RNAIII, were significantly more downregulated in the arlRS mutant of MW2 than the MSSA strain Newman. Importantly, provision of agr RNAIII in trans to the MW2 arlRS mutant via a multicopy plasmid induced autolysis in this MRSA strain. Also, the autolytic phenotype in the arlRS mutant of MSSA strain Newman could be rescued by a mutation in either atl or lytM. Together, these data showed that ArlRS impacts autolysis differently in MSSA and MRSA strains.

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Synthetic Lethality of the lytE cwlO Genotype in Bacillus subtilis Is Caused by Lack of D,L-Endopeptidase Activity at the Lateral Cell Wall.

Publication Date: 2012 Feb PMID: 22139507
Authors: Hashimoto, M. - Ooiwa, S. - Sekiguchi, J.
Journal: J Bacteriol

Bacterial peptidoglycan acts as an exoskeleton to protect the bacterial cell. Although peptidoglycan biosynthesis by penicillin-binding proteins is well studied, few studies have described peptidoglycan disassembly, which is necessary for a dynamic structure that allows cell growth. In Bacillus subtilis, more than 35 genes encoding cell wall lytic enzymes have been identified; however, only two d,l-endopeptidases (lytE and cwlO) are involved in cell proliferation. In this study, we demonstrated that the d,l-endopeptidase activity at the lateral cell wall is essential for cell proliferation. Inactivation of LytE and CwlO by point mutation of the catalytic residues caused cell growth defects. However, the forced expression of LytF or CwlS, which are paralogs of LytE, did not suppress lytE cwlO synthetic lethality. Subcellular localization studies of these d,l-endopeptidases showed LytF and CwlS at the septa and poles, CwlO at the cylindrical part of the cell, and LytE at the septa and poles as well as the cylindrical part. Furthermore, construction of N-terminal and C-terminal domain-swapped enzymes of LytE, LytF, CwlS, and CwlO revealed that localization was dependent on the N-terminal domains. Only the chimeric proteins that were enzymatically active and localized to the sidewall were able to suppress the synthetic lethality, suggesting that the lack of d,l-endopeptidase activity at the cylindrical part of the cell leads to a growth defect. The functions of LytE and CwlO in cell morphogenesis were discussed.

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Genome-Scale Metabolic Reconstruction and Hypothesis Testing in the Methanogenic Archaeon Methanosarcina acetivorans C2A.

Publication Date: 2012 Feb PMID: 22139506
Authors: Benedict, M. N. - Gonnerman, M. C. - Metcalf, W. W. - Price, N. D.
Journal: J Bacteriol

Methanosarcina acetivorans strain C2A is a marine methanogenic archaeon notable for its substrate utilization, genetic tractability, and novel energy conservation mechanisms. To help probe the phenotypic implications of this organism's unique metabolism, we have constructed and manually curated a genome-scale metabolic model of M. acetivorans, iMB745, which accounts for 745 of the 4,540 predicted protein-coding genes (16%) in the M. acetivorans genome. The reconstruction effort has identified key knowledge gaps and differences in peripheral and central metabolism between methanogenic species. Using flux balance analysis, the model quantitatively predicts wild-type phenotypes and is 96% accurate in knockout lethality predictions compared to currently available experimental data. The model was used to probe the mechanisms and energetics of by-product formation and growth on carbon monoxide, as well as the nature of the reaction catalyzed by the soluble heterodisulfide reductase HdrABC in M. acetivorans. The genome-scale model provides quantitative and qualitative hypotheses that can be used to help iteratively guide additional experiments to further the state of knowledge about methanogenesis.

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Lag phase is a distinct growth phase that prepares bacteria for exponential growth and involves transient metal accumulation.

Publication Date: 2012 Feb PMID: 22139505
Authors: Rolfe, M. D. - Rice, C. J. - Lucchini, S. - Pin, C. - Thompson, A. - Cameron, A. D. - Alston, M. - Stringer, M. F. - Betts, R. P. - Baranyi, J. - Peck, M. W. - Hinton, J. C.
Journal: J Bacteriol

Lag phase represents the earliest and most poorly understood stage of the bacterial growth cycle. We developed a reproducible experimental system and conducted functional genomic and physiological analyses of a 2-h lag phase in Salmonella enterica serovar Typhimurium. Adaptation began within 4 min of inoculation into fresh LB medium with the transient expression of genes involved in phosphate uptake. The main lag-phase transcriptional program initiated at 20 min with the upregulation of 945 genes encoding processes such as transcription, translation, iron-sulfur protein assembly, nucleotide metabolism, LPS biosynthesis, and aerobic respiration. ChIP-chip revealed that RNA polymerase was not "poised" upstream of the bacterial genes that are rapidly induced at the beginning of lag phase, suggesting a mechanism that involves de novo partitioning of RNA polymerase to transcribe 522 bacterial genes within 4 min of leaving stationary phase. We used inductively coupled plasma mass spectrometry (ICP-MS) to discover that iron, calcium, and manganese are accumulated by S. Typhimurium during lag phase, while levels of cobalt, nickel, and sodium showed distinct growth-phase-specific patterns. The high concentration of iron during lag phase was associated with transient sensitivity to oxidative stress. The study of lag phase promises to identify the physiological and regulatory processes responsible for adaptation to new environments.

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Role of the Umo Proteins and the Rcs Phosphorelay in the Swarming Motility of the Wild Type and an O-Antigen (waaL) Mutant of Proteus mirabilis.

Publication Date: 2012 Feb PMID: 22139504
Authors: Morgenstein, R. M. - Rather, P. N.
Journal: J Bacteriol

Proteus mirabilis is a Gram-negative bacterium that exists as a short rod when grown in liquid medium, but during growth on surfaces it undergoes a distinct physical and biochemical change that culminates in the formation of a swarmer cell. How P. mirabilis senses a surface is not fully understood; however, the inhibition of flagellar rotation and accumulation of putrescine have been proposed to be sensory mechanisms. Our lab recently isolated a transposon insertion in waaL, encoding O-antigen ligase, that resulted in a loss of swarming but not swimming motility. The waaL mutant failed to activate flhDC, the class 1 activator of the flagellar gene cascade, when grown on solid surfaces. Swarming in the waaL mutant was restored by overexpression of flhDC in trans or by a mutation in the response regulator rcsB. To further investigate the role of the Rcs signal transduction pathway and its possible relationship with O-antigen surface sensing, mutations were made in the rcsC, rcsB, rcsF, umoB (igaA), and umoD genes in wild-type and waaL backgrounds. Comparison of the swarming phenotypes of the single and double mutants and of strains overexpressing combinations of the UmoB, UmoD, and RcsF proteins demonstrated the following: (i) there is a differential effect of RcsF and UmoB on swarming in wild-type and waaL backgrounds, (ii) RcsF inhibits UmoB activity but not UmoD activity in a wild-type background, and (iii) UmoD is able to modulate activity of the Rcs system.

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The Sulfur Oxygenase Reductase from the Mesophilic Bacterium Halothiobacillus neapolitanus Is a Highly Active Thermozyme.

Publication Date: 2012 Feb PMID: 22139503
Authors: Veith, A. - Botelho, H. M. - Kindinger, F. - Gomes, C. M. - Kletzin, A.
Journal: J Bacteriol

A biochemical, biophysical, and phylogenetic study of the sulfur oxygenase reductase (SOR) from the mesophilic gammaproteobacterium Halothiobacillus neapolitanus (HnSOR) was performed in order to determine the structural and biochemical properties of the enzyme. SOR proteins from 14 predominantly chemolithoautotrophic bacterial and archaeal species are currently available in public databases. Sequence alignment and phylogenetic analysis showed that they form a coherent protein family. The HnSOR purified from Escherichia coli after heterologous gene expression had a temperature range of activity of 10 to 99 degrees C with an optimum at 80 degrees C (42 U/mg protein). Sulfite, thiosulfate, and hydrogen sulfide were formed at various stoichiometries in a range between pH 5.4 and 11 (optimum pH 8.4). Circular dichroism (CD) spectroscopy and dynamic light scattering showed that the HnSOR adopts secondary and quaternary structures similar to those of the 24-subunit enzyme from the hyperthermophile Acidianus ambivalens (AaSOR). The melting point of the HnSOR was approximately 20 degrees C lower than that of the AaSOR, when analyzed with CD-monitored thermal unfolding. Homology modeling showed that the secondary structure elements of single subunits are conserved. Subtle changes in the pores of the outer shell and increased flexibility might contribute to activity at low temperature. We concluded that the thermostability was the result of a rigid protein core together with the stabilizing effect of the 24-subunit hollow sphere.

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A Transposon Site Hybridization Screen Identifies galU and wecBC as Important for Survival of Yersinia pestis in Murine Macrophages.

Publication Date: 2012 Feb PMID: 22139502
Authors: Klein, K. A. - Fukuto, H. S. - Pelletier, M. - Romanov, G. - Grabenstein, J. P. - Palmer, L. E. - Ernst, R. - Bliska, J. B.
Journal: J Bacteriol

Yersinia pestis is able to survive and replicate within murine macrophages. However, the mechanism by which Y. pestis promotes its intracellular survival is not well understood. To identify genes that are important for Y. pestis survival in macrophages, a library comprised of approximately 31,500 Y. pestis KIM6+ transposon insertion mutants (input pool) was subjected to negative selection in primary murine macrophages. Genes underrepresented in the output pool of surviving bacteria were identified by transposon site hybridization to DNA oligonucleotide microarrays. The screen identified several genes known to be important for survival of Y. pestis in macrophages, including phoPQ and members of the PhoPQ regulon (e.g., pmrF). In addition, genes predicated to encode a glucose-1-phosphate uridylyltransferase (galU), a UDP-N-acetylglucosamine 2-epimerase (wecB) and a UDP-N-acetyl-d-mannosamine dehydrogenase (wecC) were identified in the screen. Viable-count assays demonstrated that a KIM6+ galU mutant and a KIM6+ wecBC mutant were defective for survival in murine macrophages. The galU mutant was studied further because of its strong phenotype. The KIM6+ galU mutant exhibited increased susceptibility to the antimicrobial peptides polymyxin B and cathelicidin-related antimicrobial peptide (CRAMP). Polyacrylamide gel electrophoresis demonstrated that the lipooligosaccharide (LOS) of the galU mutant migrated faster than the LOS of the parent KIM6+, suggesting the core was truncated. In addition, the analysis of LOS isolated from the galU mutant by mass spectrometry showed that aminoarabinose modification of lipid A is absent. Therefore, addition of aminoarabinose to lipid A and complete LOS core (galU), as well as enterobacterial common antigen (wecB and wecC), is important for survival of Y. pestis in macrophages.

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Validation of the Essential ClpP Protease in Mycobacterium tuberculosis as a Novel Drug Target.

Publication Date: 2012 Feb PMID: 22123255
Authors: Ollinger, J. - O'Malley, T. - Kesicki, E. A. - Odingo, J. - Parish, T.
Journal: J Bacteriol

Mycobacterium tuberculosis is a pathogen of major global importance. Validated drug targets are required in order to develop novel therapeutics for drug-resistant strains and to shorten therapy. The Clp protease complexes provide a means for quality control of cellular proteins; the proteolytic activity of ClpP in concert with the ATPase activity of the ClpX/ClpC subunits results in degradation of misfolded or damaged proteins. Thus, the Clp system plays a major role in basic metabolism, as well as in stress responses and pathogenic mechanisms. M. tuberculosis has two ClpP proteolytic subunits. Here we demonstrate that ClpP1 is essential for viability in this organism in culture, since the gene could only be deleted from the chromosome when a second functional copy was provided. Overexpression of clpP1 had no effect on growth in aerobic culture or viability under anaerobic conditions or during nutrient starvation. In contrast, clpP2 overexpression was toxic, suggesting different roles for the two homologs. We synthesized known activators of ClpP protease activity; these acyldepsipeptides (ADEPs) were active against M. tuberculosis. ADEP activity was enhanced by the addition of efflux pump inhibitors, demonstrating that ADEPs gain access to the cell but that export occurs. Taken together, the genetic and chemical validation of ClpP as a drug target leads to new avenues for drug discovery.

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The Polyketide Pks1 Contributes to Biofilm Formation in Mycobacterium tuberculosis.

Publication Date: 2012 Feb PMID: 22123254
Authors: Pang, J. M. - Layre, E. - Sweet, L. - Sherrid, A. - Moody, D. B. - Ojha, A. - Sherman, D. R.
Journal: J Bacteriol

Infections caused by biofilms are abundant and highly persistent, displaying phenotypic resistance to high concentrations of antimicrobials and modulating host immune systems. Tuberculosis (TB), caused by Mycobacterium tuberculosis, shares these qualities with biofilm infections. To identify genetic determinants of biofilm formation in M. tuberculosis, we performed a small-scale transposon screen using an in vitro pellicle biofilm assay. We identified five M. tuberculosis mutants that were reproducibly attenuated for biofilm production relative to that of the parent strain H37Rv. One of the most attenuated mutants is interrupted in pks1, a polyketide synthase gene. When fused with pks15, as in some M. tuberculosis isolates, pks1 contributes to synthesis of the immunomodulatory phenolic glycolipids (PGLs). However, in strains such as H37Rv with split pks15 and pks1 loci, PGL is not produced and pks1 has no previously defined role. We showed that pks1 complementation restores biofilm production independently of the known role of pks1 in PGL synthesis. We also assessed the relationship among biofilm formation, the pks15/1 genotype, and M. tuberculosis phylogeography. A global survey of M. tuberculosis clinical isolates revealed surprising sequence variability in the pks15/1 locus and substantial variation in biofilm phenotypes. Our studies identify novel M. tuberculosis genes that contribute to biofilm production, including pks1. In addition, we find that the ability to make pellicle biofilms is common among M. tuberculosis isolates from throughout the world, suggesting that this trait is relevant to TB propagation or persistence.

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LytF, a Novel Competence-Regulated Murein Hydrolase in the Genus Streptococcus.

Publication Date: 2012 Feb PMID: 22123253
Authors: Berg, K. H. - Ohnstad, H. S. - Havarstein, L. S.
Journal: J Bacteriol

Streptococcus pneumoniae and probably most other members of the genus Streptococcus are competent for natural genetic transformation. During the competent state, S. pneumoniae produces a murein hydrolase, CbpD, that kills and lyses noncompetent pneumococci and closely related species. Previous studies have shown that CbpD is essential for efficient transfer of genomic DNA from noncompetent to competent cells in vitro. Consequently, it has been proposed that CbpD together with the cognate immunity protein ComM constitutes a DNA acquisition mechanism that enables competent pneumococci to capture homologous DNA from closely related streptococci sharing the same habitat. Although genes encoding CbpD homologs or CbpD-related proteins are present in many different streptococcal species, the genomes of a number of streptococci do not encode CbpD-type proteins. In the present study we show that the genomes of nearly all species lacking CbpD encode an unrelated competence-regulated murein hydrolase termed LytF. Using Streptococcus gordonii as a model system, we obtained evidence indicating that LytF is a functional analogue of CbpD. In sum, our results show that a murein hydrolase gene is part of the competence regulon of most or all streptococcal species, demonstrating that these muralytic enzymes constitute an essential part of the streptococcal natural transformation system.

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HopX1 in Erwinia amylovora Functions as an Avirulence Protein in Apple and Is Regulated by HrpL.

Publication Date: 2012 Feb PMID: 22123252
Authors: Bocsanczy, A. M. - Schneider, D. J. - Declerck, G. A. - Cartinhour, S. - Beer, S. V.
Journal: J Bacteriol

Fire blight is a devastating disease of rosaceous plants caused by the Gram-negative bacterium Erwinia amylovora. This pathogen delivers virulence proteins into host cells utilizing the type III secretion system (T3SS). Expression of the T3SS and of translocated and secreted substrates is activated by the alternative sigma factor HrpL, which recognizes hrp box promoters upstream of regulated genes. A collection of hidden Markov model (HMM) profiles was used to identify putative hrp boxes in the genome sequence of Ea273, a highly virulent strain of E. amylovora. Among potential virulence factors preceded by putative hrp boxes, two genes previously known as Eop3 and Eop2 were characterized. The presence of functionally active hrp boxes upstream of these two genes was confirmed by beta-glucuronidase (GUS) assays. Deletion mutants of the latter candidate genes, renamed hopX1(Ea) and hopAK1(Ea), respectively, did not differ in virulence from the wild-type strain when assayed in pear fruit and apple shoots. The hopX1(Ea) deletion mutant of Ea273, complemented with a plasmid overexpressing hopX1(E)(a), suppressed the development of the hypersensitivity response (HR) when inoculated into Nicotiana benthamiana; however, it contributed to HR in Nicotiana tabacum and significantly reduced the progress of disease in apple shoots, suggesting that HopX1(Ea) may act as an avirulence protein in apple shoots.

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Homologues and Bioengineered Derivatives of LtnJ Vary in Ability to Form D-Alanine in the Lantibiotic Lacticin 3147.

Publication Date: 2012 Feb PMID: 22123251
Authors: Suda, S. - Lawton, E. M. - Wistuba, D. - Cotter, P. D. - Hill, C. - Ross, R. P.
Journal: J Bacteriol

Ltnalpha and Ltnbeta are individual components of the two-peptide lantibiotic lacticin 3147 and are unusual in that, although ribosomally synthesized, they contain d-amino acids. These result from the dehydration of l-serine to dehydroalanine by LtnM and subsequent stereospecific hydrogenation to d-alanine by LtnJ. Homologues of LtnJ are rare but have been identified in silico in Staphylococcus aureus C55 (SacJ), Pediococcus pentosaceus FBB61 (PenN), and Nostoc punctiforme PCC73102 (NpnJ, previously called NpunJ [P. D. Cotter et al., Proc. Natl. Acad. Sci. U. S. A. 102:18584-18589, 2005]). Here, the ability of these enzymes to catalyze d-alanine formation in the lacticin 3147 system was assessed through heterologous enzyme production in a DeltaltnJ mutant. PenN successfully incorporated d-alanines in both peptides, and SacJ modified Ltnalpha only, while NpnJ was unable to modify either peptide. Site-directed mutagenesis was also employed to identify residues of key importance in LtnJ. The most surprising outcome from these investigations was the generation of peptides by specific LtnJ mutants which exhibited less bioactivity than those generated by the DeltaltnJ strain. We have established that the reduced activity of these peptides is due to the inability of the associated LtnJ enzymes to generate d-alanine residues in a stereospecific manner, resulting in the presence of both d- and l-alanines at the relevant locations in the lacticin 3147 peptides.

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Effects of Cortex Peptidoglycan Structure and Cortex Hydrolysis on the Kinetics of Ca2+-Dipicolinic Acid Release during Bacillus subtilis Spore Germination.

Publication Date: 2012 Feb PMID: 22123250
Authors: Zhang, P. - Thomas, S. - Li, Y. Q. - Setlow, P.
Journal: J Bacteriol

The kinetic parameters of the release of Ca(2+)-dipicolinic acid (CaDPA) during germination of spore populations and multiple individual spores of Bacillus subtilis strains with major alterations in the structure of the spore peptidoglycan (PG) cortex or lacking one or both of the two redundant enzymes involved in cortex hydrolysis (cortex-lytic enzymes [CLEs]) were determined. The lack of the CLE CwlJ greatly slowed CaDPA release with a germinant receptor (GR)-dependent germinant, l-valine, or a non-GR-dependent germinant, dodecylamine. The absence of the cortex-specific PG modification muramic acid-delta-lactam also increased the time needed for full CaDPA release during germination with both types of germinants. In contrast, increased cortex PG cross-linking was associated with faster times for initiation of CaDPA release with both l-valine and dodecylamine but not with faster CaDPA release once this release had been initiated. These data suggest that the precise structure of the spore cortex plays a significant role in determining the timing and the rate of CaDPA release during B. subtilis spore germination and, further, that this effect is independent of effects of GRs.

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In Vitro Recombinants of Antibiotic-Resistant Chlamydia trachomatis Strains Have Statistically More Breakpoints than Clinical Recombinants for the Same Sequenced Loci and Exhibit Selection at Unexpected Loci.

Publication Date: 2012 Feb PMID: 22123249
Authors: Srinivasan, T. - Bruno, W. J. - Wan, R. - Yen, A. - Duong, J. - Dean, D.
Journal: J Bacteriol

Lateral gene transfer (LGT) is essential for generating between-strain genomic recombinants of Chlamydia trachomatis to facilitate the organism's evolution. Because there is no reliable laboratory-based gene transfer system for C. trachomatis, in vitro generation of recombinants from antibiotic-resistant strains is being used to study LGT. However, selection pressures imposed on in vitro recombinants likely affect statistical properties of recombination relative to naturally occurring clinical recombinants, including prevalence at particular loci. We examined multiple loci for 16 in vitro-derived recombinants of ofloxacin- and rifampin-resistant L(1) and D strains, respectively, grown with both antibiotics, and compared these with the same sequenced loci among 11 clinical recombinants. Breakpoints and recombination frequency were examined using phylogenetics, bioinformatics, and statistics. In vitro and clinical isolates clustered perfectly into two groups, without misclassification, using Ward's minimum variance based on breakpoint data. As expected, gyrA (confers ofloxacin resistance) and rpoB (confers rifampin resistance) had significantly more breakpoints among in vitro recombinants than among clinical recombinants (P < 0.0001 and P = 0.02, respectively, using the Wilcoxon rank sum test). Unexpectedly, trpA also had significantly more breakpoints for in vitro recombinants (P < 0.0001). There was also significant selection at other loci. The strongest bias was for ompA in strain D (P = 3.3 x 10(-8)). Our results indicate that the in vitro model differs statistically from natural recombination events. Additional genomic studies are needed to determine the factors responsible for the observed selection biases at unexpected loci and whether these are important for LGT to inform approaches for genetically manipulating C. trachomatis.

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Biochemical Disclosure of the Mycolate Outer Membrane of Corynebacterium glutamicum.

Publication Date: 2012 Feb PMID: 22123248
Authors: Marchand, C. H. - Salmeron, C. - Bou Raad, R. - Meniche, X. - Chami, M. - Masi, M. - Blanot, D. - Daffe, M. - Tropis, M. - Huc, E. - Le Marechal, P. - Decottignies, P. - Bayan, N.
Journal: J Bacteriol

Corynebacterineae is a specific suborder of Gram-positive bacteria that includes Mycobacterium tuberculosis and Corynebacterium glutamicum. The cell wall of these bacteria is composed of a heteropolymer of peptidoglycan (PG) linked to arabinogalactan (AG), which in turn is covalently associated with an atypical outer membrane, here called mycomembrane (M). The latter structure has been visualized by cryo-electron microscopy of vitreous sections, but its biochemical composition is still poorly defined, thereby hampering the elucidation of its physiological function. In this report, we show for the first time that the mycomembrane-linked heteropolymer of PG and AG (M-AG-PG) of C. glutamicum can be physically separated from the inner membrane on a flotation density gradient. Analysis of purified M-AG-PG showed that the lipids that composed the mycomembrane consisted almost exclusively of mycolic acid derivatives, with only a tiny amount, if any, of phospholipids and lipomannans, which were found with the characteristic lipoarabinomannans in the plasma membrane. Proteins associated with or inserted in the mycomembrane were extracted from M-AG-PG with lauryl-dimethylamine-oxide (LDAO), loaded on an SDS-PAGE gel, and analyzed by tandem mass spectrometry or by Western blotting. Sixty-eight different proteins were identified, 19 of which were also found in mycomembrane fragments released by the terminal-arabinosyl-transferase-defective DeltaAftB strain. Almost all of them are predicted to contain a signal sequence and to adopt the characteristic beta-barrel structure of Gram-negative outer membrane proteins. These presumed mycomembrane proteins include the already-known pore-forming proteins (PorA and PorB), 5 mycoloyltransferases (cMytA, cMytB, cMytC, cMytD, and cMytF), several lipoproteins, and unknown proteins typified by a putative C-terminal hydrophobic anchor.

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Mur Regulates the Gene Encoding the Manganese Transporter MntH in Brucella abortus 2308.

Publication Date: 2012 Feb PMID: 22101848
Authors: Menscher, E. A. - Caswell, C. C. - Anderson, E. S. - Roop, R. M. 2nd
Journal: J Bacteriol

MntH is the only high-affinity manganese transporter identified in Brucella. A previous study showed that MntH is required for the wild-type virulence of Brucella abortus 2308 in mice (Anderson ES, et al., Infect. Immun. 77:3466-3474, 2009) and indicated that the mntH gene is regulated in a manganese-responsive manner in this strain by a Mur homolog. In the study presented here, the transcriptional start site for mntH in B. abortus 2308 was determined by primer extension analysis. Specific interactions between Mur and the mntH promoter region were demonstrated in an electrophoretic mobility shift assay (EMSA), and a Mur binding site was identified in the -55 to -24 region of the mntH promoter by DNase I footprint analysis. The specificity of the interaction of Mur with the putative Mur box was further evaluated by EMSA employing oligonucleotides in which the consensus nucleotides in this region were substituted. These studies not only confirm a direct role for Mur in the Mn-responsive regulation of mntH expression in Brucella abortus 2308 but also identify the cis-acting elements upstream of mntH that are responsible for this regulation.

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Sequential Closure of the Cytoplasm and Then the Periplasm during Cell Division in Escherichia coli.

Publication Date: 2012 Feb PMID: 22101847
Authors: Skoog, K. - Soderstrom, B. - Widengren, J. - von Heijne, G. - Daley, D. O.
Journal: J Bacteriol

To visualize the latter stages of cell division in live Escherichia coli, we have carried out fluorescence recovery after photobleaching (FRAP) on 121 cells expressing cytoplasmic green fluorescent protein and periplasmic mCherry. Our data show conclusively that the cytoplasm is sealed prior to the periplasm during the division event.

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The Vibrio cholerae Mannitol Transporter Is Regulated Posttranscriptionally by the MtlS Small Regulatory RNA.

Publication Date: 2012 Feb PMID: 22101846
Authors: Mustachio, L. M. - Aksit, S. - Mistry, R. H. - Scheffler, R. - Yamada, A. - Liu, J. M.
Journal: J Bacteriol

Vibrio cholerae continues to pose a health threat in many developing nations and regions of the world struck by natural disasters. It is a pathogen that rapidly adapts to aquatic environments and the human small intestine. Small regulatory RNAs (sRNAs) may contribute to this adaptability. Specifically, the mannitol operon sRNA (MtlS sRNA; previously designated the IGR7 sRNA) is transcribed antisense to the 5' untranslated region of the mtl operon, encoding the mannitol-specific phosphotransferase system. Mannitol is a six-carbon sugar alcohol that accumulates in the human small intestine, the primary site of V. cholerae colonization. To better understand the V. cholerae mtl operon at a molecular level, we investigated mtlA expression in the presence of various carbon sources and the role of the MtlS sRNA. We observed that MtlA protein is present only in cells grown on mannitol sugar, whereas MtlS sRNA is expressed during growth on all sugars other than mannitol. In contrast, mtlA mRNA is expressed in similar amounts regardless of the carbon source used for bacterial growth. These observations suggest that the regulation of MtlA protein expression is a posttranscriptional event. We further demonstrate that MtlS sRNA overexpression repressed MtlA synthesis without affecting the stability of the messenger and that this process is largely independent of Hfq. We propose a model in which, when carbon sources other than mannitol are present, MtlS sRNA is transcribed, base pairs with the 5' untranslated region of the mtlA mRNA, occluding the ribosome binding site, and inhibits the synthesis of the mannitol-specific phosphotransferase system.

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Unveiling Unusual Features of Formation of Septal Partition and Constriction in Mycobacteria--an Ultrastructural Study.

Publication Date: 2012 Feb PMID: 22101845
Authors: Vijay, S. - Anand, D. - Ajitkumar, P.
Journal: J Bacteriol

The ultrastructural functions of the electron-dense glycopeptidolipid-containing outermost layer (OL), the arabinogalactan-mycolic acid-containing electron-transparent layer (ETL), and the electron-dense peptidoglycan layer (PGL) of the mycobacterial cell wall in septal growth and constriction are not clear. Therefore, using transmission electron microscopy, we studied the participation of the three layers in septal growth and constriction in the fast-growing saprophytic species Mycobacterium smegmatis and the slow-growing pathogenic species Mycobacterium xenopi and Mycobacterium tuberculosis in order to document the processes in a comprehensive and comparative manner and to find out whether the processes are conserved across different mycobacterial species. A complete septal partition is formed first by the fresh synthesis of the septal PGL (S-PGL) and septal ETL (S-ETL) from the envelope PGL (E-PGL) in M. smegmatis and M. xenopi. The S-ETL is not continuous with the envelope ETL (E-ETL) due to the presence of the E-PGL between them. The E-PGL disappears, and the S-ETL becomes continuous with the E-ETL, when the OL begins to grow and invaginate into the S-ETL for constriction. However, in M. tuberculosis, the S-PGL and S-ETL grow from the E-PGL and E-ETL, respectively, without a separation between the E-ETL and S-ETL by the E-PGL, in contrast to the process in M. smegmatis and M. xenopi. Subsequent growth and invagination of the OL into the S-ETL of the septal partition initiates and completes septal constriction in M. tuberculosis. A model for the conserved sequential process of mycobacterial septation, in which the formation of a complete septal partition is followed by constriction, is presented. The probable physiological significance of the process is discussed. The ultrastructural features of septation and constriction in mycobacteria are unusually different from those in the well-studied organisms Escherichia coli and Bacillus subtilis.

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Decreased Transport Restores Growth of a Salmonella enterica apbC Mutant on Tricarballylate.

Publication Date: 2012 Feb PMID: 22101844
Authors: Boyd, J. M. - Teoh, W. P. - Downs, D. M.
Journal: J Bacteriol

Mutants of Salmonella enterica lacking apbC have nutritional and biochemical properties indicative of defects in iron-sulfur ([Fe-S]) cluster metabolism. An apbC mutant is unable to grow on tricarballylate as a carbon source. Based on the ability of ApbC to transfer an [Fe-S] cluster to an apoprotein, this defect was attributed to poor loading of the [Fe-S] cluster-containing TcuB enzyme. Consistent with these observations, a previous study showed that overexpression of iscU, which encodes an [Fe-S] cluster molecular scaffold, suppressed the tricarballylate growth defect of an apbC mutant (J. M. Boyd, J. A. Lewis, J. C. Escalante-Semerena, and D. M. Downs, J. Bacteriol. 190:4596-4602, 2008). In this study, tcuC mutations that suppress the growth defect of an apbC mutant by decreasing the intracellular concentration of tricarballylate are described. Collectively, the suppressor analyses support a model in which reduced TcuB activity prevents growth on tricarballylate by (i) decreasing catabolism and (ii) allowing levels of tricarballylate that are toxic to the cell to accumulate. The apbC tcuC mutant strains described here reveal that the balance of the metabolic network can be altered by the accumulation of deleterious metabolites.

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CitA/CitB Two-Component System Regulating Citrate Fermentation in Escherichia coli and Its Relation to the DcuS/DcuR System In Vivo.

Publication Date: 2012 Feb PMID: 22101843
Authors: Scheu, P. D. - Witan, J. - Rauschmeier, M. - Graf, S. - Liao, Y. F. - Ebert-Jung, A. - Basche, T. - Erker, W. - Unden, G.
Journal: J Bacteriol

Citrate fermentation by Escherichia coli requires the function of the citrate/succinate antiporter CitT (citT gene) and of citrate lyase (citCDEFXG genes). Earlier experiments suggested that the two-component system CitA/CitB, consisting of the membrane-bound sensor kinase CitA and the response regulator CitB, stimulates the expression of the genes in the presence of citrate, similarly to CitA/CitB of Klebsiella pneumoniae. In this study, the expression of a chromosomal citC-lacZ gene fusion was shown to depend on CitA/CitB and citrate. CitA/CitB is related to the DcuS/DcuR two-component system which induces the expression of genes for fumarate respiration in response to C(4)-dicarboxylates and citrate. Unlike DcuS, CitA required none of the cognate transporters (CitT, DcuB, or DcuC) for function, and the deletion of the corresponding genes showed no effect on the expression of citC-lacZ. The citAB operon is preceded by a DcuR binding site. Phosphorylated DcuR bound specifically to the promoter region, and the deletion of dcuS or dcuR reduced the expression of citC. The data indicate the presence of a regulatory cascade consisting of DcuS/DcuR modulating citAB expression (and CitA/CitB levels) and CitA/CitB controlling the expression of the citCDEFXGT gene cluster in response to citrate. In vivo fluorescence resonance energy transfer (FRET) and the bacterial two-hybrid system (BACTH) showed interaction between the DcuS and CitA proteins. However, BACTH and expression studies demonstrated the lack of interaction and cross-regulation between CitA and DcuR or DcuS and CitB. Therefore, there is only linear phosphoryl transfer (DcuS-->DcuR and CitA-->CitB) without cross-regulation between DcuS/DcuR and CitA/CitB.

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Substrate Recognition Mechanism and Substrate-Dependent Conformational Changes of an ROK Family Glucokinase from Streptomyces griseus.

Publication Date: 2012 Feb PMID: 22101842
Authors: Miyazono, K. - Tabei, N. - Morita, S. - Ohnishi, Y. - Horinouchi, S. - Tanokura, M.
Journal: J Bacteriol

Carbon catabolite repression (CCR) is a widespread phenomenon in many bacteria that is defined as the repression of catabolic enzyme activities for an unfavorable carbon source by the presence of a preferable carbon source. In Streptomyces, secondary metabolite production often is negatively affected by the carbon source, indicating the involvement of CCR in secondary metabolism. Although the CCR mechanism in Streptomyces still is unclear, glucokinase is presumably a central player in CCR. SgGlkA, a glucokinase from S. griseus, belongs to the ROK family glucokinases, which have two consensus sequence motifs (1 and 2). Here, we report the crystal structures of apo-SgGlkA, SgGlkA in complex with glucose, and SgGlkA in complex with glucose and adenylyl imidodiphosphate (AMPPNP), which are the first structures of an ROK family glucokinase. SgGlkA is divided into a small alpha/beta domain and a large alpha+beta domain, and it forms a dimer-of-dimer tetrameric configuration. SgGlkA binds a beta-anomer of glucose between the two domains, and His157 in consensus sequence 1 plays an important role in the glucose-binding mechanism and anomer specificity of SgGlkA. In the structures of SgGlkA, His157 forms an HC3-type zinc finger motif with three cysteine residues in consensus sequence 2 to bind a zinc ion, and it forms two hydrogen bonds with the C1 and C2 hydroxyls of glucose. When the three structures are compared, the structure of SgGlkA is found to be modified by the binding of substrates. The substrate-dependent conformational changes of SgGlkA may be related to the CCR mechanism in Streptomyces.

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Iron Storage Proteins Are Essential for the Survival and Pathogenesis of Mycobacterium tuberculosis in THP-1 Macrophages and the Guinea Pig Model of Infection.

Publication Date: 2012 Feb PMID: 22101841
Authors: Reddy, P. V. - Puri, R. V. - Khera, A. - Tyagi, A. K.
Journal: J Bacteriol

Iron is one of the crucial elements required for the growth of Mycobacterium tuberculosis. However, excess free iron becomes toxic for the cells because it catalyzes the production of reactive oxygen radicals, leading to oxidative damage. Hence, it is essential for the pathogen to have the ability to store intracellular iron in an iron-rich environment and utilize it under iron depletion. M. tuberculosis has two iron storage proteins, namely BfrA (Rv1876; a bacterioferritin) and BfrB (Rv3841; a ferritin-like protein). However, the demonstration of biological significance requires the disruption of relevant genes and the evaluation of the resulting mutant for its ability to survive in the host and cause disease. In this study, we have disrupted bfrA and bfrB of M. tuberculosis and demonstrated that these genes are crucial for the storage and supply of iron for the growth of bacteria and to withstand oxidative stress in vitro. In addition, the bfrA bfrB double mutant (H37Rv DeltabfrA DeltabfrB) exhibited a marked reduction in its ability to survive inside human macrophages. Guinea pigs infected with H37Rv DeltabfrA DeltabfrB exhibited a marked diminution in the dissemination of the bacilli to spleen compared to that of the parental strain. Moreover, guinea pigs infected with H37Rv DeltabfrA DeltabfrB exhibited significantly reduced pathological damage in spleen and lungs compared to that of animals infected with the parental strain. Our study clearly demonstrates the importance of these iron storage proteins in the survival and pathogenesis of M. tuberculosis in the host and establishes them as attractive targets for the development of new inhibitors against mycobacterial infections.

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