Microbiology (Microbiology)

Publications in this journal

• Article: Mechanism of Intrinsic Resistance to Antimicrobial Peptides of Edwardsiella ictaluri and its Influence on Fish Virulence and Gut Inflammation. Microbiology

  Javier Santander, Taylor Martin, Amanda Loh, Camilo Pohlenz, Delbert M Gatlin, Roy Curtiss
  Microbiology 05/2013;
• Article: RaoN, a Small RNA Encoded within Salmonella Pathogenicity Island-11, Confers Resistance to Macrophage-Induced Stress.

  Yong Heon Lee, Sinyeon Kim, John D Helmann, Bae-Hoon Kim, Yong Keun Park
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  ABSTRACT: Bacterial small noncoding RNAs act as important regulators that control numerous cellular processes. Here we identified RaoN, a novel small RNA encoded in the cspH-envE intergenic region on Salmonella pathogenicity island-11. RaoN contributes to survival under conditions of acid and oxidative stress combined with nutrient limitation, which partially mimic the intramacrophage environment. Indeed, inactivation of raoN reduces the intramacrophage replication of Salmonella enterica serovar Typhimurium. Genome-wide transcriptome analysis revealed that the lactate dehydrogenase gene ldhA is upregulated in the raoN knockout mutant. Notably, both inactivation and overexpression of ldhA in the wild-type strain render Salmonella more sensitive to oxidative stress, particularly when combined with nutrient limitation. However, ldhA is not the sole determinant of RaoN function in facilitating intramacrophage survival of Salmonella. Together, our data suggest that balanced regulation of ldhA expression by RaoN is necessary for survival under in vitro stress conditions and contributes to the intramacrophage growth of Salmonella.
  Microbiology 05/2013;
• Article: Phosphate starvation relayed by PhoB activates the expression of the Pseudomonas aeruginosa σvreI ECF factor and its target genes.

  Laura Faure, María Llamas, Karlijn Bastiaansen, Sophie de Bentzmann, Sarah Bigot
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  ABSTRACT: The cell-surface signalling (CSS) system represents an important regulatory mechanism by which Gram-negative bacteria respond to the environment. Gene regulation by CSS systems is specially present and important in the human opportunistic pathogen Pseudomonas aeruginosa. In this bacterium, these mechanisms mainly regulate the uptake of iron, but also virulence functions. The latter is the case for the P. aeruginosa PUMA3 CSS system formed by the putative VreA receptor, the σVreI extracytoplasmic function (ECF) sigma factor and the VreR anti-sigma factor. A role of this system in P. aeruginosa virulence has been previously demonstrated. However, the condition by which this system is expressed and thus activated has not been elucidated so far. In this work, we have identified and characterized the global regulatory cascade activating the expression of the PUMA3 system. We show that the PhoB transcriptional regulator, part of the PhoB-PhoR two-component signaling system, can sense a limitation of inorganic phosphate (Pi) to turn on the expression of the vreA, vreI and vreR genes, which constitute an operon. Upon expression of these genes in this condition, σVreI factor mediates transcription of most, but not all, of the previously identified σVreI-regulated genes. Indeed, we found new σVreI-targeted genes and we show that σVreI-regulon genes are all located immediately downstream to the vreAIR gene cluster.
  Microbiology 05/2013;
• Article: Comparative proteomic analysis reveals new components of the PhoP regulon and highlights a role for PhoP in the regulation of genes encoding the F1F0 ATP synthase in Edwardsiella tarda.

  Yuanzhi Lv, Kaiyu Yin, Shuai Shao, Qiyao Wang, Yuanxing Zhang
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  ABSTRACT: Edwardsiella tarda is an important cause of haemorrhagic septicaemia in fish and also of gastro- and extra-intestinal infections in humans. We have recently demonstrated that the PhoP-PhoQ two-component regulatory system plays important roles in both virulence and stress tolerance in E. tarda. In this study the proteomes of the wild-type and phop mutant strains were compared to define components of the PhoP regulon in E. tarda EIB202. Overall, 18 proteins whose expression levels exhibited a 2-fold or greater change were identified; 13 of these proteins were found to require the presence of PhoP for full expression, while 5 were expressed at a higher level in the phoP mutant background. Identified proteins were representing diverse functional categories including energy production, amino acid metabolism, and oxidative stress defense. Quantitative real-time PCR (qRT-PCR) analysis of the mRNA levels for the identified proteins confirmed the proteomics data. Interestingly, β subunit of the F1F0 ATP synthase (AtpD) playing an important role in growth and virulence of E. tarda, was listed as one of the proteins whose expression was greatly dependent on PhoP. The F1F0 ATP synthase was encoded in a gene cluster (atpIBEFHAGDC) and the 9 genes were transcribed as an operon. PhoP positively regulated the transcription of the 9 ATP synthase genes and exerted this effect through direct binding to the promoter of atpI. Overall, the results provide new insights into the PhoP regulon and unravel a novel role for PhoP in the regulation of the F1F0 ATP synthase.
  Microbiology 05/2013;
• Article: Biotin sulfoxide reductase contributes to oxidative stress tolerance and virulence in Salmonella enterica serovar Typhimurium.

  Luisa Anna Denkel, Mikael Rhen, Franz-Christoph Bange
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  ABSTRACT: Oxidative stress converts sulfur residues of molecules like biotin and methionine into its oxidized forms. Here we show that the biotin sulfoxide reductase BisC of Salmonella enterica serovar Typhimurium (S. Typhimurium) repairs both oxidized biotin and oxidized methionine. Exposure to H2O2 in vitro reduced survival of a S. Typhimurium ΔbisC mutant. Furthermore, replication of the ΔbisC mutant inside IFN-γ activated macrophages was reduced. In vitro tolerance of the mutant to H2O2 was restored by plasmids carrying either bisC or msrA, the later of which encodes a methioinine sulfoxide reductase. In contrast, the proliferation defect inside IFN-γ activated macrophages was rescued by bisC but not by msrA. Thus growth of the ΔbisC mutant in IFN-γ activated macrophages required repair of oxidized biotin. Both the ΔbisC and a biotin auxotrophic (ΔbioB ) mutant were attenuated in mice, suggesting that besides biotin biosynthesis, biotin repair was essential for virulence of S. Typhimurium in vivo. Attenuation of the ΔbisC mutant was more pronounced in 129 mice that produce a stronger oxidative response. These results show that BisC is essential for full virulence of Salmonella by contributing to the defence of S. Typhimurium against host-derived stress, and provides an attractive drug target since BisC it is not present in mammals.
  Microbiology 05/2013;
• Article: YgfX (CptA) is a multimeric membrane protein that interacts with the succinate dehydrogenase assembly factor SdhE (YgfY).

  Matthew B McNeil, Marina Iglesias Cans, James S Clulow, Peter Fineran
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  ABSTRACT: Serratia sp. strain ATCC 39006 produces the red-pigmented antibiotic prodigiosin. Prodigiosin biosynthesis is regulated by a complex hierarchy that includes the uncharacterised protein, YgfX (DUF1434). The ygfX gene is co-transcribed with sdhE, an FAD assembly factor essential for the flavinylation and activation of the SdhA subunit of succinate dehydrogenase, a central enzyme in the tricarboxylic acid cycle and electron transport chain. The sdhEygfX operon is highly conserved within the Enterobacteriaceae, suggesting that SdhE and YgfX function together. We performed an extensive mutagenesis to gain molecular insights into the uncharacterised protein, YgfX, and have investigated the relationship between YgfX and SdhE. YgfX localised to the membrane, interacted with itself, forming dimers or larger multimers, and interacted with SdhE. The transmembrane helices of YgfX were critical for protein function and the formation of YgfX multimers. Site-directed mutagenesis of residues conserved in DUF1434 proteins revealed a periplasmic tryptophan and a cytoplasmic aspartate that were crucial for YgfX activity. Both of these amino acids were required for the formation of YgfX multimers and interactions with SdhE but not membrane localisation. YgfX also interacted with multiple cell division proteins and had effects on cell morphology. These findings represent an important step in understanding the function of DUF1434 proteins and potential mechanisms used to regulate SdhE. In contrast to a recent report, we found no evidence that YgfX and SdhE form a toxin-antitoxin system. In summary, YgfX functions as a multimeric membrane-bound protein that interacts with SdhE, an important FAD assembly factor that controls SDH activity.
  Microbiology 05/2013;
• Article: Occurrence of mutations impairing sigma factor B (SigB) function upon inactivation of Listeria monocytogenes genes encoding surface proteins.

  Juan J Quereda, María Graciela Pucciarelli, Laura Botello-Morte, Enrique Calvo, Filipe Carvalho, Christiane Bouchier, Ana Vieira, Javier F Mariscotti, Trinad Chakraborty, Pascale Cossart, Torsten Hain, Didier Cabanes, Francisco García-Del Portillo
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  ABSTRACT: Bacteria of the genus Listeria contain the largest family of LPXTG surface proteins covalently anchored to the peptidoglycan. The extent at which these proteins may function or be regulated cooperatively is at present unknown. Because of their unique cellular location, we reasoned that distinct LPXTG proteins could act as elements contributing to cell wall homeostasis or influencing the stability of other surface proteins bound to peptidoglycan. To test this hypothesis, we analysed by proteomics mutants of the intracellular pathogen L. monocytogenes lacking distinct LPXTG proteins implicated in pathogen-host interactions as InlA, InlF, InlG, InlH, InlJ, LapB and Vip. Changes in the cell wall proteome were found in inlG and vip mutants, which exhibited reduced levels of the LPXTG proteins InlH, Lmo0610, Lmo0880 and Lmo2085, all regulated by the stress-related sigma factor SigB. The ultimate basis of this alteration was uncovered by genome sequencing, which revealed that these inlG and vip mutants carried loss-of-function mutations in the rsbS, rsbU and rsbV genes encoding regulatory proteins that control SigB activity. Attempts to recapitulate this negative selection of SigB in large series of new inlG or vip mutants constructed for this purpose were however unsuccessful. These results indicate that inadvertent secondary mutations affecting SigB functionality can randomly arise in L. monocytogenes when using widely used genetic procedures or during sub-culturing. Testing of SigB activity could be therefore valuable when manipulating genetically L. monocytogenes prior to any subsequent phenotypic analysis. This test may be even more justified when generating deletions affecting cell envelope components.
  Microbiology 05/2013;
• Article: MalF is essential for persistence of Mycoplasma gallisepticum in vivo.

  Chi-Wen Tseng, Anna Kanci, Christine Citti, Renate Rosengarten, Chien-Ju Chiu, Zheng-Hong Chen, Steven J Geary, Glenn Browning, Philip F Markham
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  ABSTRACT: There is limited understanding of the molecular basis of virulence in the important avian pathogen Mycoplasma gallisepticum. To define genes that may be involved in colonisation of chickens, a collection of mutants of the virulent Ap3AS strain of M. gallisepticum were generated by signature-tagged transposon mutagenesis. The collection included mutants with single insertions in the genes encoding the adhesin GapA and the cytadherence-related protein CrmA, and Western blotting confirmed that these mutants did not express these proteins. In two separate in vivo screenings, two GapA-deficient mutants (ST mutants 02-1 and 06-1) were occasionally recovered from birds, suggesting that GapA expression may not always be essential for persistence of strain Ap3AS. CrmA-deficient ST mutant 33-1 colonised birds poorly and had reduced virulence, indicating that CrmA was a significant virulence factor, but was not absolutely essential for colonisation. ST mutant 04-1 contained a single transposon insertion in malF, a predicted ABC sugar transport permease, and was unable to be reisolated even when inoculated by itself into group of birds, suggesting that expression of MalF was essential for persistence of M. galliseptium strain Ap3AS in infected birds.
  Microbiology 05/2013;
• Article: Characterization of Salmonella enterica serovar Typhimurium aconitase A.

  Othman Baothman, Matthew Rolfe, Jeffrey Green
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  ABSTRACT: Aconitases (Acn) are iron-sulfur proteins that catalyze the reversible isomerisation of citrate and isocitrate via the intermediate cis-aconitate in the Krebs cycle. Some Acn proteins are bi-functional and under conditions of iron-starvation and oxidative stress lose their iron-sulfur clusters and become post-transcriptional regulators by binding specific mRNA targets. Many bacterial species possess two genetically distinct aconitase proteins, AcnA and AcnB. Current understanding of the regulation and functions of AcnA and AcnB in dual Acn bacteria is based on a model developed in Escherichia coli. Thus, AcnB is the major Krebs cycle enzyme expressed during exponential growth, whereas AcnA is a more stable, stationary phase- and stress- induced enzyme, and both E. coli Acns are bi-functional. Here a second dual Acn bacterium, Salmonella enterica serovar Typhimurium (S. Typhimurium), has been analyzed. Phenotypic traits of S. Typimurium acn mutants were consistent with AcnB acting as the major Acn protein. Promoter fusion experiments indicated that transcription of acnB was ~10-fold greater than that of acnA and that acnA expression was regulated by the cyclic-AMP receptor protein (CRP, glucose-starvation), the fumarate nitrate reduction regulator (FNR, oxygen-starvation), the ferric uptake regulator (Fur, iron-starvation) and the superoxide response protein (SoxR, oxidative stress). In contrast to E. coli, S. Typhimurium acnA was not induced in stationary phase. Furthermore, expression of acnA was enhanced in an acnB mutant presumably to partially compensate for the lack of AcnB activity. Isolated S. Typhimurium AcnA protein had kinetic and mRNA-binding properties similar to those described for E. coli AcnA. Thus, the work reported here provides a second exemplar of the regulation and function of AcnA and AcnB proteins in a dual Acn bacterium.
  Microbiology 05/2013;
• Article: Multiple alcohol dehydrogenases but no functional acetaldehyde dehydrogenase causing excessive acetaldehyde production from ethanol by oral streptococci.

  Sylvia I Pavlova, Ling Jin, Stephen Gasparovich, Lin Tao
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  ABSTRACT: Ethanol consumption and poor oral hygiene are risk factors for oral and esophageal cancers. Although oral streptococci have been found to produce excessive acetaldehyde from ethanol, little is known about the mechanism by which this carcinogen is produced. By screening 52 strains of diverse oral streptococcal species, we identified Streptococcus gordonii V2016 that produced the most acetaldehyde from ethanol. We then constructed gene deletion mutants in this strain and analyzed them for alcohol and acetaldehyde dehydrogenases by zymograms. The results showed that S. gordonii V2016 expressed three primary alcohol dehydrogenases, AdhA, AdhB and AdhE, which all oxidize ethanol to acetaldehyde, but their preferred substrates were 1-propanol, 1-butanol, and ethanol, respectively. Two additional dehydrogenases, S-AdhA and TdhA, were identified with specificities to the secondary alcohol 2-propanol and threonine, respectively, but not to ethanol. S. gordonii V2016 did not show a detectable acetaldehyde dehydrogenase even though its adhE gene encodes a putative bifunctional acetaldehyde/alcohol dehydrogenase. Mutants with adhE deletion showed greater tolerance to ethanol in comparison with the wild type and mutant with adhA or adhB deletion, indicating that AdhE is the major alcohol dehydrogenase in S. gordonii. Analysis of 19 additional strains of S. gordonii, S. mitis, S. oralis, S. salivarius, and Streptococcus sanguinis all showed expressions of up to three alcohol dehydrogenases, but none showed detectable acetaldehyde dehydrogenase, except one strain that showed a novel ALDH. Therefore, expression of multiple alcohol dehydrogenases but no functional acetaldehyde dehydrogenase may contribute to excessive production of acetaldehyde from ethanol by certain oral streptococci.
  Microbiology 05/2013;
• Article: A novel approach to generate a recombinant toxoid vaccine against C. difficile.

  Robert Gk Donald, Mike Flint, Narender Kalyan, Erik Johnson, Susan E Witko, Cheryl Kotash, Ping Zhao, Shakuntala Megati, Irina Yurgelonis, Phillip Kwok Lee, Yury V Matsuka, Elena Severina, Anne Deatly, Mini Sidhu, Kathrin U Jansen, Nigel P Minton, Annaliesa S Anderson
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  ABSTRACT: The Clostridium difficile toxins A and B are primarily responsible for symptoms of C. difficile associated disease and are prime targets for vaccine development. We describe a plasmid-based system for the production of genetically modified toxins in a non-sporulating strain of C. difficile that lacks the toxin genes tcdA and tcdB. TcdA and TcdB mutations targeting established glucosyltransferase cytotoxicity determinants were introduced into recombinant plasmids and episomally expressed toxin mutants purified from C. difficile transformants. TcdA and TcdB mutants lacking glucosyltransferase and autoproteolytic processing activities were ~10,000-fold less toxic to cultured human IMR-90 cells than corresponding recombinant or native toxins. However, both mutants retained residual cytotoxicity that could be prevented by preincubating the antigens with specific antibodies or by formalin treatment. Such non-toxic formalin-treated mutant antigens were immunogenic and protective in a hamster model of infection. The remaining toxicity of untreated TcdA and TcdB mutant antigens was associated with cellular swelling, a phenotype consistent with pore-induced membrane leakage. TcdB substitution mutations previously shown to block vesicular pore formation and toxin translocation substantially reduced residual toxicity. We discuss the implications of these results for the development of a C. difficile toxoid vaccine.
  Microbiology 04/2013;
• Article: Role of MerH in Mercury Resistance in the Archaeon Sulfolobus solfataricus.

  James Schelert, Deepak Rudrappa, Tyler Johnson, Paul Blum
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  ABSTRACT: Crenarchaeota include extremely thermoacidophilic organisms that thrive in geothermal environments dominated by sulfidic ores and heavy metals such as mercury. Mercuric ion, Hg(II), inactivates transcription in the crenarchaeote, Sulfolobus solfataricus, and simultaneously derepresses transcription of a resistance operon, merHAI, through interaction with the MerR transcription factor. While mercuric reductase (MerA) is required for metal resistance, the role of MerH, an adjacent small open reading frame has not been explored. Inactivation of MerH either by nonsense mutation or by in-frame deletion reduced Hg(II) resistance of mutant cells. Promoter mapping studies indicated that Hg(II) sensitivity of the merH nonsense mutant arose through transcriptional polarity and its metal resistance was restored partially by single copy merH complementation. Since MerH was not required in vitro for MerA catalyzed Hg(II) reduction, MerH may play an alternative role in metal resistance. ICP-MS analysis of the MerH deletion strain following metal challenge indicated there was prolonged retention of intracellular Hg(II). Finally, a reduced rate of mer operon induction in the merH deletion mutant suggested that the requirement for MerH could result from metal trafficking to the MerR transcription factor.
  Microbiology 04/2013;
• Article: A novel gene, GEA1, is required for ascus cell wall development in the ascomycete fungus, Fusarium graminearum.

  Hokyoung Son, Jungkwan Lee, Yin-Won Lee
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  ABSTRACT: The ascomycete fungus Fusarium graminearum is a devastating plant pathogen for major cereal crops. Ascospores are produced via sexual reproduction and forcibly discharged from mature perithecia, which function as the primary inocula. Perithecium development involves complex cellular processes and is under polygenic control. In this study, a novel gene, GEA1, was found to be required for ascus wall development in F. graminearum. GEA1 deletion mutants produced normal-shaped perithecia and ascospores, yet ascospores were observed to precociously germinate inside of perithecium. Moreover, GEA1 deletions resulted in abnormal ascus walls that collapsed prior to ascospore discharge. Based on localization of GEA1 to plasma membrane, GEA1 may be directly involved in the ascus wall biogenesis. This is the first report to identify a unique gene required for ascus wall development in F. graminearum.
  Microbiology 04/2013;
• Article: Putative synchronized nuclear divisions without cytokinesis in Rhinosporidium seeberi parasitic life cycle.

  Leonel Mendoza, Raquel Vilela
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  ABSTRACT: Despite numerous studies on Rhinosporidum seeberi parasitic phase, the stages of its nuclear cycle leading to the formation of endoconidia, have yet to be properly described. R. seeberi resists culture and can only be investigated on histological preparations. We have evaluated tissue sections collected from 35 host species with rhinosporidosis searching for the presence of mitotic figures during sporangia development. This study found that soon after endoconidia release, the prominent reddish vesicles typical of this stage vanished leading to the development of 12-70 μm in diameter juvenile sporangia (JS). This stage possesses granular cytoplasm, a thick cell wall, and a central reddish nucleus with a conspicuous nucleolus. The first nuclear division takes place in the JS. It is a rarely encountered event characterized by the development of a distorted nucleus leading to the formation of two nuclei without cytokinesis. The finding of multiple nuclear divisions at prophase, metaphase, and telophase like-stages without cytokinesis was detected in intermediate sporangia (IS). IS with multiple dividing nuclei seem to be at the same stage of nuclear partitioning, suggesting synchronized nuclear division. In these sporangia, the nuclei continue divisions without cytokinesis until the sporangia reached ≥300 μm in diameter. The last nuclear division prior to cytokinesis appears to take place in very large sporangia with thousands of nuclei. The buildup of cytoplasm around each nucleus and the formation of a thin cell wall lead to the formation of endoconidia. This study revealed the presence of several mechanisms of pathogenesis in R. seeberi that deserved further investigation.
  Microbiology 04/2013;
• Article: Role of oxyRKP, a novel LysR-family transcriptional regulator in antimicrobial resistance and virulence in Klebsiella pneumoniae.

  Vijaya Bharathi Srinivasan, Amitabha Mondal, Manjunath Venkataramaiah, Neeraj Kumar Chauhan, Govindan Rajamohan
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  ABSTRACT: Klebsiella pneumoniae is a Gram negative bacillus that causes serious infections in immunocompromised human hosts and exhibits significant degrees of multidrug resistance. In this study, we identified a novel lysR-family regulator (designated as oxyRKP), in the genome of K. pneumoniae NTUH-K2044 whose functions have remained enigmatic so far. In this study, functional characterization of the putative lysR regulator oxyRKP with respect to cellular physiology and antimicrobial susceptibility was evaluated by generating an isogenic mutant, oxyRKP in a hyper virulent clinical isolate of K. pneumoniae. The K. pneumoniae oxyRKP mutant was sensitive to hyper osmotic and bile conditions. Disruption of oxyRKP increased the susceptibility of K. pneumoniae to oxidative (0.78947mM hydrogen peroxide) and nitrosative (30mM acidified nitrite) stress by ~ 1.4 fold and ~ 10 fold respectively. The loss of the Klebsiella regulator led to a decrease in the minimum inhibitory concentrations for chloramphenicol {10 fold}, erythromycin {6 fold}, nalidixic acid {>50 fold}, trimethoprim {10 fold} which could be restored following complementation. The relative change in expression of resistance-nodulation-cell division super family (RND) efflux gene acrB was decreased by ~5 fold in oxyRKP mutant as evidenced by qRT-PCR. In Caenorhabditis elegans model, the oxyRKP mutant exhibited significantly ((P < 0.01) lower virulence. Overall results displayed in this report reflect the pleiotropic role of the oxyRKP signaling system and diversity of the resistance determinants in hyper virulent K1 serotype K. pneumoniae NTUH-K2044.
  Microbiology 04/2013;
• Article: Pseudomonas putida F1 has multiple chemoreceptors with overlapping specificity for organic acids.

  Rebecca E Parales, Rita A Luu, Grischa Y Chen, Xianxian Liu, Victoria Wu, Pamela Lin, Jonathan G Hughes, Vasyl Nesteryuk, Juanito V Parales, Jayna L Ditty
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  ABSTRACT: Previous studies have demonstrated that Pseudomonas putida strains are not only capable of growth on a wide range of organic substrates, but are also chemotactic towards many of these compounds. However, in most cases the specific chemoreceptors that are involved have not been identified. The complete genome sequences of P. putida strains F1 and KT2440 revealed that each strain is predicted to encode 27 methyl-accepting chemotaxis proteins (MCPs) or MCP-like proteins, 25 of which are shared by both strains. It was expected that orthologous MCPs in closely related strains of the same species would be functionally equivalent. We demonstrate here that this is not always the case. Deletion of the gene encoding the P. putida F1 ortholog (locus tag Pput_4520, designated mcfS) of McpS, a known receptor for organic acids in P. putida KT2440, did not result in an obvious chemotaxis phenotype. Therefore, we constructed individual markerless MCP gene deletion mutants in P. putida F1 and screened for defective sensory responses to succinate, malate, fumarate and citrate. This screen resulted in the identification of a receptor, McfQ (locus tag Pput_4894), which responds to citrate and fumarate. An additional receptor, McfR (locus tag Pput_0339), which detects succinate, malate and fumarate, was found by individually expressing each of the 18 genes encoding canonical MCPs from strain F1 in a KT2440 ΔmcpS deletion mutant. Expression of mcfS in the same ΔmcpS deletion mutant demonstrated that, like McfR, McfS responds to succinate, malate, citrate and fumarate. Therefore, at least three receptors, McfR, McfS, and McfQ, work in concert to detect organic acids in P. putida F1.
  Microbiology 04/2013;
• Article: Refinement of the Listeria monocytogenes σB regulon through quantitative proteomic analysis.

  Sana Mujahid, Renato Orsi, Pajau Vangay, Kathryn Boor, Martin Wiedmann
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  ABSTRACT: σB is an alternative σ factor that regulates stress response and virulence genes in the foodborne pathogen Listeria monocytogenes. To gain further insight into σB-dependent regulatory mechanisms in L. monocytogenes, we (i) performed quantitative proteomic comparisons between the L. monocytogenes parent strain 10403S and an isogenic ΔsigB mutant and (ii) conducted a meta-analysis of published microarray studies on the 10403S σB regulon. A total of 134 genes were found to be significantly positively regulated by σB at the transcriptomic level with > 75% of these genes preceded by putative σB-dependent promoters; 21 of these 134 genes were also found to be positively regulated by σB through proteomics. In addition, 15 proteins were only found to be positively regulated by σB through proteomics analyses, including Lmo1349, a putative glycine cleavage system protein. The lmo1349 gene is preceded by a 5' UTR that functions as a glycine riboswitch, which suggests regulation of glycine metabolism by σB in L. monocytogenes. Herein, we propose a model where σB upregulates pathways that facilitate biosynthesis and uptake of glycine, which may then activate this riboswitch. Our data also (i) identified a number of σB-dependent proteins that appear to be encoded by genes that are co-regulated by multiple transcriptional regulators, in particular PrfA, and (ii) found σB-dependent genes and proteins to be overrepresented in the "energy metabolism" role category, highlighting contributions of the σB regulon to L. monocytogenes energy metabolism as well as a role of PrfA and σB interaction in regulating aspects of energy metabolism in L. monocytogenes.
  Microbiology 04/2013;
• Article: A single domain response regulator involved in the general stress response of Methylobacterium extorquens.

  Lisa C Metzger, Anne Francez-Charlot, Julia A Vorholt
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  ABSTRACT: The general stress response of Alphaproteobacteria is regulated by a partner-switching mechanism that involves the alternative sigma factor σEcfG, the anti-sigma factor NepR and the anti-sigma factor antagonist PhyR. To address the question of how the PhyR-NepR-σEcfG cascade is activated and modulated in Methylobacterium extorquens, a forward genetic screen was applied. The screen identified the single domain response regulator Mext_0407 as a novel regulatory element in the general stress response of M. extorquens. Analysis of phenotypes and of transcriptional fusions of PhyR-dependent genes shows that the mext_0407 deletion mutant fails to respond to various stresses. Mext_0407 requires the putative phosphorylatable aspartate-64 for its activity in vivo and genetic evidences suggest that Mext_0407 operates upstream of the PhyR-NepR-σEcfG cascade.
  Microbiology 04/2013;
• Article: Identification of multiple putative S-layer genes partly expressed by Lysinibacillus sphaericus JG-B53.

  Franziska L Lederer, Ulrike Weinert, Tobias J Guenther, Johannes Raff, Stephan Weiß, Katrin Pollmann
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  ABSTRACT: Lysinibacillus sphaericus JG-B53 was isolated from the uranium mining waste pile Haberland near Johanngeorgenstadt, Germany. Previous studies have shown that many bacteria that have been isolated from these heavy metal contaminated environments possess surface layer proteins (S-layers) which enable the bacteria to survive by binding metals with high affinity. Conversely, essential trace elements are able to cross the filter layer and reach the interior of the cell. This is especially true of the S-layer of Lysinibacillus sphaericus JG-B53 which is therefore of high interest for both environmental studies and technical applications. Particularly the latter due to the high amounts isolatable from biomass and the outstanding re-crystallisation and metal binding properties. In this study, S-layer protein gene sequences encoded in the genome of L. sphaericus JG-B53 were identified using next generation sequencing (NGS) technology followed by bioinformatic analyses. The genome of L. sphaericus JG-B53 encodes at least 8 putative S-layer protein genes with distinct differences. Using mRNA analyses the expression of the putative S-layer protein genes was studied. The functional S-layer protein B53 Slp1 was identified as dominantly expressed S-layer protein in Lysinibacillus sphaericus JG-B53 by mRNA studies, SDS PAGE and N-terminal sequencing. B53 Slp1 is characterised by square lattice symmetry and a molecular weight of 116 kDa. The S-layer protein B53 Slp1 shows a high similarity to the functional S-layer protein of Lysinibacillus sphaericus JG-A12, being isolated from the same uranium mining waste pile Haberland described by previous research. These similarities indicate horizontal gene transfer and DNA rearrangements between these bacteria. The presence of multiple S-layer gene copies may enable the bacterial strains to quickly adapt to changing environments.
  Microbiology 04/2013;
• Article: Novel insight into the expression and function of the multicopper oxidases in Candida albicans.

  Xinxin Cheng, Ning Xu, Qilin Yu, Kefan Qian, Xiaohui Ding, Qiang Zhao, Yuzhou Wang, Biao Zhang, Laijun Xing, Mingchun Li
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  ABSTRACT: Iron is an essential element required for most organisms. The high-affinity iron uptake (HAIU) systems in the opportunistic pathogen Candida albicans are activated under iron-limited conditions and are also required for virulence. Here one component of HAIU systems, the multicopper oxidase (MCO) genes, was characterized. We examined the expression of five MCO genes and demonstrated that CaFET3 and CaFET34 were the major MCO genes in response to iron deficiency. Complementation of the Saccharomyces cerevisiae fet3 mutant showed that CaFET34 could effectively rescue the growth phenotype in iron-limited medium. Deletion of CaFET33 and CaFET34 in C. albicans decreased cellular iron content and iron acquisition during iron starvation. However, the fet33/ and fet34/ mutants exhibited no obvious growth defect in solid iron-limited medium while the fet34/ mutant showed a slight growth defect in liquid medium. Further analysis showed that other members of the five MCO genes, especially CaFET3, would compensate for the absence of CaFET33 and CaFET34. Furthermore, we for the first time provided evidence that CaFET34 was implicated in hyphal development in an iron-independent manner and was required for C. albicans virulence in a mouse model of system infection. Together, our results not only expand our understanding about the expression of the MCO genes in C. albicans, but also provide a novel insight into the role of CaFET34 in iron metabolism, hyphal development and virulence.
  Microbiology 04/2013;