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ABSTRACT: BACKGROUND: The saprophytic pathogen Listeria monocytogenes has to cope with a variety of acidic habitats during its life cycle. The impact of low-temperature coupled with pH decrease for global gene expression and subsequent virulence properties, however, has not been elucidated. RESULTS: qRT-PCR revealed for the first time a transient, acid triggered prfA induction of approximately 4-fold, 5.7-fold, 7-fold and 9.3-fold 60 to 90 min after acid shock of L. monocytogenes at 37[degree sign]C, 25[degree sign]C, 18[degree sign]C, and 10[degree sign]C, respectively. Comparable data were obtained for seven different L. monocytogenes strains, demonstrating that prfA induction under these conditions is a general response of L. monocytogenes. Transcriptome analysis revealed that the in vivo-relevant genes bsh, clpP, glpD, hfq, inlA, inlB, inlE, lisR, and lplA1 as well as many other genes with a putative role during infection are transiently induced upon acid shock conducted at 25[degree sign]C and 37[degree sign]C. Twenty-five genes repressed upon acid shock are known to be down regulated during intracellular growth or by virulence regulators. These data were confirmed by qRT-PCR of twelve differentially regulated genes and by the identification of acid shock-induced genes influenced by sigmaB. To test if up regulation of virulence genes at temperatures below 37[degree sign]C correlates with pathogenicity, the capacity of L. monocytogenes to invade epithelial cells after acid shock at 25[degree sign]C was measured. A 12-fold increased number of intracellular bacteria was observed (acid shock, t = 60 min) that was reduced after adaptation to the level of the unshocked control. This increased invasiveness was shown to be in line with the induction of inlAB. Using a nematode infection assay, we demonstrated that Caenorhabditis elegans fed with acid-shocked L. monocytogenes exhibits a shorter time to death of 50% (TD50) of the worms (6.4 days) compared to infection with unshocked bacteria (TD50 = 10.2 days). CONCLUSIONS: PrfA and other listerial virulence genes are induced by an inorganic acid in a temperature-dependent manner. The data presented here suggest that low pH serves as a trigger for listerial pathogenicity at environmental temperatures.
BMC Genomics 04/2013; 14(1):285. · 4.07 Impact Factor
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ABSTRACT: Listeria monocytogenes is a foodborne human pathogen that can cause invasive infection in susceptible animals and humans. For proliferation within hosts, this facultative intracellular pathogen uses a reservoir of specific metabolic pathways, transporter, and enzymatic functions whose expression requires the coordinated activity of a complex regulatory network. The highly adapted metabolism of L. monocytogenes strongly depends on the nutrient composition of various milieus encountered during infection. Transcriptomic and proteomic studies revealed the spatial-temporal dynamic of gene expression of this pathogen during replication within cultured cells or in vivo. Metabolic clues are the utilization of unusual C(2)- and C(3)-bodies, the metabolism of pyruvate, thiamine availability, the uptake of peptides, the acquisition or biosynthesis of certain amino acids, and the degradation of glucose-phosphate via the pentose phosphate pathway. These examples illustrate the interference of in vivo conditions with energy, carbon, and nitrogen metabolism, thus affecting listerial growth. The exploitation, analysis, and modeling of the available data sets served as a first attempt to a systemic understanding of listerial metabolism during infection. L. monocytogenes might serve as a model organism for systems biology of a Gram-positive, facultative intracellular bacterium.
Frontiers in microbiology. 01/2012; 3:23.
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ABSTRACT: Most bacteria pathogenic for humans have closely related nonpathogenic counterparts that live as saprophytes, commensals or even symbionts (mutualists) in similar or different habitats. The knowledge of how these bacteria adapt their metabolism to the preferred habitats is critical for our understanding of pathogenesis, commensalism and symbiosis, and - in the case of bacterial pathogens - could help to identify targets for new antimicrobial agents. The focus of this review is on the metabolic potentials and adaptations of three different groups of human extra- and intracellular bacterial pathogens and their nonpathogenic relatives. All bacteria selected have the potential to reach the interior of mammalian host cells. However, their ability to replicate intracellularly differs significantly. The question therefore arises whether there are specific metabolic requirements that support stable intracellular replication. Furthermore, we discuss - whenever relevant data for the pathogenic representatives are available - the possible effect of the metabolism on the expression of virulence genes.
FEMS microbiology reviews 08/2011; 36(2):435-62. · 10.96 Impact Factor
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ABSTRACT: Yersinia enterocolitica strains responsible for mild gastroenteritis in humans are very diverse with respect to their metabolic and virulence properties. Strain W22703 (biotype 2, serotype O:9) was recently identified to possess nematocidal and insecticidal activity. To better understand the relationship between pathogenicity towards insects and humans, we compared the W22703 genome with that of the highly pathogenic strain 8081 (biotype1B; serotype O:8), the only Y. enterocolitica strain sequenced so far.
We used whole-genome shotgun data to assemble, annotate and analyse the sequence of strain W22703. Numerous factors assumed to contribute to enteric survival and pathogenesis, among them osmoregulated periplasmic glucan, hydrogenases, cobalamin-dependent pathways, iron uptake systems and the Yersinia genome island 1 (YGI-1) involved in tight adherence were identified to be common to the 8081 and W22703 genomes. However, sets of ~550 genes revealed to be specific for each of them in comparison to the other strain. The plasticity zone (PZ) of 142 kb in the W22703 genome carries an ancient flagellar cluster Flg-2 of ~40 kb, but it lacks the pathogenicity island YAPI(Ye), the secretion system ysa and yts1, and other virulence determinants of the 8081 PZ. Its composition underlines the prominent variability of this genome region and demonstrates its contribution to the higher pathogenicity of biotype 1B strains with respect to W22703. A novel type three secretion system of mosaic structure was found in the genome of W22703 that is absent in the sequenced strains of the human pathogenic Yersinia species, but conserved in the genomes of the apathogenic species. We identified several regions of differences in W22703 that mainly code for transporters, regulators, metabolic pathways, and defence factors.
The W22703 sequence analysis revealed a genome composition distinct from other pathogenic Yersinia enterocolitica strains, thus contributing novel data to the Y. enterocolitica pan-genome. This study also sheds further light on the strategies of this pathogen to cope with its environments.
BMC Genomics 03/2011; 12:168. · 4.07 Impact Factor
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ABSTRACT: Biofilm formation by Salmonella is a serious concern in the food-processing industry and the persistence of the organism in biofilms becomes a constant source of contamination. Since there is zero tolerance for Salmonella in foods, it is important to understand the mechanism of biofilm formation and to prevent the formation. Therefore, this study aimed at investigating the biofilm-forming ability of seafood isolates of Salmonella enterica serovar Weltevreden (S. Weltevreden) under two different nutrient conditions (normal strength trypticase soy broth (TSB) and 1:100 diluted TSB). The role of cellulose production in biofilm formation and in the expression of multicellular behavior (rough, dark, red morphotype: rdar) was investigated. Fourteen isolates of seafood associated S. Weltevreden were studied for biofilm production in polystyrene microtitre plates. Only one (SW49) of 14 was a strong biofilm former on polystyrene template and was able to produce biofilm in both undiluted TSB and 1:100 diluted TSB at 24h. All others produced moderate or weak biofilms which was higher in 1:100 diluted TSB compared to undiluted medium. All the isolates except one were positive by PCR for the three genes, gcpA (stm1987), adrA (yaiC) and csgD. Gene expression of gcpA, adrA and csgD was studied by real-time PCR with the one strong (SW49) and one representative weak (SW30) biofilm former. In SW49 at 24h of incubation, the expression of gcpA from biofilm cells was 33 and 36 times higher than from planktonic cells grown in TSB and diluted TSB respectively and at 72h the expression from biofilm cells was 57 and 61 times higher than that from planktonic cells. Quantification of gene expression did not reveal any significant difference in the expression of csgD and adrA gene. Deletion of gcpA in SW49 resulted in its inability to produce cellulose and consequent inability to bind calcoflour, inability to form rdar colony on Congo Red-agar plates and failure to produce biofilm on polystyrene substrate. The data indicated that, in case of S. Weltevreden, gcpA is critical for activating cellulose synthesis and biofilm formation both in undiluted and diluted TSB. The results of this study suggest the existence of an alternative biofilm regulatory pathway in S. Weltevreden. Role of adrA in cellulose production in nutrient rich medium is known but role of gepA in the above phenomenon is proved in this study. An understanding of the genes involved would help in looking at strategies of repression of the gene to control formation of biofilm.
Microbial Pathogenesis 02/2011; 50(2):114-22. · 1.94 Impact Factor
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ABSTRACT: The capability of Salmonella enterica serovar Typhimurium strain 14028 (S. Typhimurium 14028) to utilize myo-inositol (MI) is determined by the genomic island GEI4417/4436 carrying the iol genes that encode enzymes, transporters, and a repressor responsible for the MI catabolic pathway. In contrast to all bacteria investigated thus far, S. Typhimurium 14028 growing on MI as the sole carbon source is characterized by a remarkable long lag phase of 40 to 60 h. We report here that on solid medium with MI as the sole carbon source, this human pathogen exhibits a bistable phenotype characterized by a dissection into large colonies and a slow-growing bacterial background. This heterogeneity is reversible and therefore not caused by mutation, and it is not observed in the absence of the iol gene repressor IolR nor in the presence of at least 0.55% CO(2). Bistability is correlated with the activity of the iolE promoter (P(iolE)), but not of P(iolC) or P(iolD), as shown by promoter-gfp fusions. On the single-cell level, fluorescence microscopy and flow cytometry analysis revealed a gradual switch of P(iolE) from the "off" to the "on" status during the late lag phase and the transition to the log phase. Deletion of iolR or the addition of 0.1% NaHCO(3) induced an early growth start of S. Typhimurium 14028 in minimal medium with MI. The addition of ethoxyzolamide, an inhibitor of carboanhydrases, elongated the lag phase in the presence of bicarbonate. The positive-feedback loop via repressor release and positive induction by bicarbonate-CO(2) might allow S. Typhimurium 14028 to adapt to rapidly changing environments. The phenomenon described here is a novel example of bistability in substrate degradation, and, to our knowledge, is the first demonstration of gene regulation by bicarbonate-CO(2) in Salmonella.
Journal of bacteriology 01/2011; 193(6):1427-35. · 3.94 Impact Factor
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ABSTRACT: The human pathogen Listeria monocytogenes resides and proliferates within the cytoplasm of epithelial cells. While the virulence factors essentially contributing to this step of the infection cycle are well characterized, the set of listerial genes contributing to intracellular replication remains to be defined on a genome-wide level.
A comprehensive library of L. monocytogenes strain EGD knockout mutants was constructed upon insertion-duplication mutagenesis, and 1491 mutants were tested for their phenotypes in rich medium and in a Caco-2 cell culture assay. Following sequencing of the plasmid insertion site, 141 different genes required for invasion of and replication in Caco-2 cells were identified. Ten in-frame deletion mutants were constructed that confirmed the data. The genes with known functions are mainly involved in cellular processes including transport, in the intermediary metabolism of sugars, nucleotides and lipids, and in information pathways such as regulatory functions. No function could be ascribed to 18 genes, and a counterpart of eight genes is missing in the apathogenic species L. innocua. Mice infection studies revealed the in vivo requirement of IspE (Lmo0190) involved in mevalonate synthesis, and of the novel ABC transporter Lmo0135-0137 associated with cysteine transport. Based on the data of this genome-scale screening, an extreme pathway and elementary mode analysis was applied that demonstrates the critical role of glycerol and purine metabolism, of fucose utilization, and of the synthesis of glutathione, aspartate semialdehyde, serine and branched chain amino acids during intracellular replication of L. monocytogenes.
The combination of a genetic screening and a modelling approach revealed that a series of transporters help L. monocytogenes to overcome a putative lack of nutrients within cells, and that a high metabolic flexibility contributes to the intracellular replication of this pathogen.
BMC Genomics 10/2010; 11:573. · 4.07 Impact Factor
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ABSTRACT: Only three pathogenic bacterial species, Salmonella enterica, Clostridium perfringens, and Listeria monocytogenes, are able to utilize both ethanolamine and 1,2-propanediol as a sole carbon source. Degradation of these substrates, abundant in food and the gut, depends on cobalamin, which is synthesized de novo only under anaerobic conditions. Although the eut, pdu, and cob-cbi gene clusters comprise 40 kb, the conditions under which they confer a selection advantage on these food-borne pathogens remain largely unknown. Here we used the luciferase reporter system to determine the response of the Salmonella enterica serovar Typhimurium promoters P(eutS), P(pocR), P(pduF), and P(pduA) to a set of carbon sources, to egg yolk, to whole milk, and to milk protein or fat fractions. Depending on the supplements, specific inductions up to 3 orders of magnitude were observed for P(eutS) and P(pduA), which drive the expression of most eut and pdu genes. To correlate these significant expression data with growth properties, nonpolar deletions of pocR, regulating the pdu and cob-cbi genes, and of eutR, involved in eut gene activation, were constructed in S. Typhimurium strain 14028. During exponential growth of the mutants 14028ΔpocR and 14028ΔeutR, 2- to 3-fold-reduced proliferation in milk and egg yolk was observed. Using the Caenorhabditis elegans infection model, we could also demonstrate that the proliferation of S. Typhimurium in the nematode is supported by an active ethanolamine degradation pathway. Taking these findings together, this study quantifies the differential expression of eut and pdu genes under distinct conditions and provides experimental evidence that the ethanolamine utilization pathway allows salmonellae to occupy specific metabolic niches within food environments and within their host organisms.
Applied and environmental microbiology 10/2010; 77(1):281-90. · 3.69 Impact Factor
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ABSTRACT: A novel luxCDABE plasmid for the analysis of promoter elements by site-specific integration into the genome of Yersinia enterocolitica was constructed. The versatility of this reporter system was demonstrated by comparing the activity of the inv promoter in the Y. enterocolitica high-pathogenic serotype O : 8 (strain WA-314) with that of the low pathogenic serotype O : 9 (strain Y127). The luciferase activity of a transcriptional fusion between the inv promoter of serotype O : 8 and luxCDABE was about fourfold lower than the activity of the respective O : 9 promoter. This correlated with lower invasin production by Y. enterocolitica serotype O : 8 compared with serotypes O : 9, O : 3 and O : 5,27. However, Y. enterocolitica of serotype O : 8 revealed higher invasiveness than serotype O : 9. When both invasins were expressed in trans at similar levels in the Y. enterocolitica O : 8 Deltainv background strain, cell invasion assays showed a slightly higher invasiveness of the strain producing Inv(O : 8) than the strain producing Inv(O : 9). We provide experimental evidence that this might be due to a higher binding capacity of Inv(O : 8) for cells expressing beta1 integrins compared with Inv(O:9). The Y. enterocolitica O : 8 strain harbouring the P(inv)((O : 8)) : : luxCDABE fusion was then successfully used to follow inv expression in a mouse infection model. These experiments showed for the first time that the inv promoter is active in infected living mice, especially in Peyer's patches of the ileum, the caecal lymph follicle, and the lymph nodes, liver and spleen. The production of invasin in the spleen was demonstrated by Western blot analysis. In conclusion, the presented reporter system enables stable genomic integration of the luxCDABE operon into the chromosome of Yersinia, facilitates in vitro quantification of promoter activities under different bacterial growth conditions, and enables detection of promoter activities in a mouse model.
Microbiology 09/2010; 156(Pt 9):2734-45. · 3.06 Impact Factor
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ABSTRACT: Caenorhabditis elegans is a validated model to study bacterial pathogenicity. We report that Yersinia enterocolitica strains W22703 (biovar 2, serovar O:9) and WA314 (biovar 1B, serovar O:8) kill C. elegans when feeding on the pathogens for at least 15 min before transfer to the feeding strain Escherichia coli OP50. The killing by Yersinia enterocolitica requires viable bacteria and, in contrast to that by Yersinia pestis and Yersinia pseudotuberculosis strains, is biofilm independent. The deletion of tcaA encoding an insecticidal toxin resulted in an OP50-like life span of C. elegans, indicating an essential role of TcaA in the nematocidal activity of Y. enterocolitica. TcaA alone is not sufficient for nematocidal activity because E. coli DH5alpha overexpressing TcaA did not result in a reduced C. elegans life span. Spatial-temporal analysis of C. elegans infected with green fluorescent protein-labeled Y. enterocolitica strains showed that Y. enterocolitica colonizes the nematode intestine, leading to an extreme expansion of the intestinal lumen. By low-dose infection with W22703 or DH5alpha followed by transfer to E. coli OP50, proliferation of Y. enterocolitica, but not E. coli, in the intestinal lumen of the nematode was observed. The titer of W22703 cells within the worm increased to over 10(6) per worm 4 days after infection while a significantly lower number of a tcaA knockout mutant was recovered. A strong expression of tcaA was observed during the first 5 days of infection. Y. enterocolitica WA314 (biovar 1B, serovar O:8) mutant strains lacking the yadA, inv, yopE, and irp1 genes known to be important for virulence in mammals were not attenuated or only slightly attenuated in their toxicity toward the nematode, suggesting that these factors do not play a significant role in the colonization and persistence of this pathogen in nematodes. In summary, this study supports the hypothesis that C. elegans is a natural host and nutrient source of Y. enterocolitica.
Applied and environmental microbiology 09/2010; 76(18):6277-85. · 3.69 Impact Factor
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ABSTRACT: The human pathogen L. monocytogenes is a facultatively intracellular bacterium that survives and replicates in the cytosol of many mammalian cells. The listerial metabolism, especially under intracellular conditions, is still poorly understood. Recent studies analyzed the carbon metabolism of L. monocytogenes by the (13)C isotopologue perturbation method in a defined minimal medium containing [U-(13)C(6)]glucose. It was shown that these bacteria produce oxaloacetate mainly by carboxylation of pyruvate due to an incomplete tricarboxylic acid cycle. Here, we report that a pycA insertion mutant defective in pyruvate carboxylase (PYC) still grows, albeit at a reduced rate, in brain heart infusion (BHI) medium but is unable to multiply in a defined minimal medium with glucose or glycerol as a carbon source. Aspartate and glutamate of the pycA mutant, in contrast to the wild-type strain, remain unlabeled when [U-(13)C(6)]glucose is added to BHI, indicating that the PYC-catalyzed carboxylation of pyruvate is the predominant reaction leading to oxaloacetate in L. monocytogenes. The pycA mutant is also unable to replicate in mammalian cells and exhibits high virulence attenuation in the mouse sepsis model.
Journal of bacteriology 04/2010; 192(7):1774-84. · 3.94 Impact Factor
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ABSTRACT: Thiamine pyrophosphate is an essential cofactor involved in central metabolism and amino acid biosynthesis and is derived from thiamine (vitamin B(1)). The extent to which this metabolite is available to bacterial pathogens replicating within host cells is still little understood. Growth studies using modified minimal Welshimer's broth (mMWB) supplemented with thiamine or the thiamine precursor hydroxymethylpyrimidine (HMP) showed that Listeria monocytogenes, in agreement with bioinformatic prediction, is able to synthesize thiamine only in the presence of HMP. This appears to be due to a lack of ThiC, which is involved in HMP synthesis. The knockout of thiD (lmo0317), which probably catalyzes the phosphorylation of HMP, inhibited growth in mMWB supplemented with HMP and reduced the replication rate of L. monocytogenes in epithelial cells. Mutation of a predicted thiamine transporter gene, lmo1429, led to reduced proliferation of L. monocytogenes in mMWB containing thiamine or thiamine phosphates and also within epithelial cells but had no influence on the expression of the virulence factors Hly and ActA. The toxic thiamine analogue pyrithiamine inhibited growth of wild-type strain EGD but not of the transporter mutant EGDDeltathiT. We also demonstrated that ThiT binds thiamine, a finding compatible with ThiT acting as the substrate-binding component of a multimeric thiamine transporter complex. These data provide experimental evidence that Lmo1429 homologs including Bacillus YuaJ are necessary for thiamine transport in gram-positive bacteria and are therefore proposed to be annotated "ThiT." Taken together, these data indicate that concurrent thiamine uptake and biosynthesis of thiamine precursors is a strategy of L. monocytogenes and possibly other facultative intracellular pathogens to enable proliferation within the cytoplasm.
Journal of bacteriology 02/2009; 191(7):2218-27. · 3.94 Impact Factor
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ABSTRACT: Knockout mutation of STM4432 resulted in a growth-deficient phenotype of Salmonella enterica serovar Typhimurium in the presence of myo-inositol (MI) as the sole carbon source. STM4432 is part of a 22.6-kb genomic island which spans STM4417 to STM4436 (genomic island 4417/4436) and is responsible for MI degradation. Genome comparison revealed the presence of this island in only six Salmonella strains and a high variability of the iol gene organization in gram-negative bacteria. Upon nonpolar deletion of 11 island loci, the genes involved in six enzymatic steps of the MI pathway were identified. The generation time of S. enterica serovar Typhimurium in minimal medium with MI decreases with higher concentrations of this polyol. Reverse transcriptase PCR showed five separate transcriptional units encompassing the genes iolA-iolB, iolE-iolG1, iolC1-iolC2, iolD1-iolD2-iolG2, and iolI2-iolH. Luciferase reporter assays revealed a strong induction of their promoters in the presence of MI but not glucose. The main regulator, IolR, was identified due to a reduced lag phase of a strain mutated in STM4417 (iolR). Deletion of iolR resulted in stimulation of the iol operons, indicating its negative effect on the iol genes of S. enterica serovar Typhimurium in rich medium at a transcriptional level. Bandshift assays demonstrated the binding of this putative repressor to promoter sequences of iolA, iolC1, and iolD1. Binding of IolR to its own promoter and induced iolR expression in an IolR-negative background demonstrate that its transcription is autoregulated. This is the first characterization of MI degradation in a gram-negative bacterium, revealing a complex transcriptional organization and regulation of the S. enterica serovar Typhimurium iol genes.
Journal of bacteriology 12/2008; 191(2):545-54. · 3.94 Impact Factor
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ABSTRACT: Twelve Yersinia enterocolitica mutants carrying luxCDABE-transposon insertions in motility and chemotaxis genes were isolated on the basis of strong low-temperature induction. Two transposons were located within an 11.2 kb enteric flagellar cluster 2 (Flag-2) of Y. enterocolitica biotype 2, serotype O : 9 strain W22703. The Flag-2 gene cluster is absent from the corresponding genomic location of the sequenced strain Y. enterocolitica biotype 1B, serotype O : 8 strain 8081. Evidence for the functionality of the O : 9 Flag-2 genes, probably located within the plasticity zone of the genome, is provided by swarming assays. PCR analysis of 49 strains revealed the presence of Flag-2 genes in biotypes 2-5, but not in biotypes 1A or 1B. Bioluminescence, measured between 6 and 37 degrees C, showed that the expression of all genes located in Flag-2 and in the known flagellar cluster, Flag-1, was highest at approximately 20 degrees C, and that expression of two Flag-2 genes is FlhC-dependent. In a motility assay, a non-motile and a hyper-motile phenotype resulted from knockout mutations of the Flag-1 genes fliS1 and fliT, respectively. Complemented strains validated these results, confirming the regulatory role of FliT.
Microbiology 02/2008; 154(Pt 1):196-206. · 3.06 Impact Factor
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ABSTRACT: Photorhabdus luminescens and Yersinia enterocolitica are both enteric bacteria which are associated with insects. P. luminescens lives in symbiosis with soil nematodes and is highly pathogenic towards insects but not to humans. In contrast, Y. enterocolitica is widely found in the environment and mainly known to cause gastroenteritis in men, but has only recently been shown to be also toxic for insects. It is expected that both pathogens share an overlap of genetic determinants that play a role within the insect host.
A selective genome comparison was applied. Proteins belonging to the class of two-component regulatory systems, quorum sensing, universal stress proteins, and c-di-GMP signalling have been analysed. The interorganismic synopsis of selected regulatory systems uncovered common and distinct signalling mechanisms of both pathogens used for perception of signals within the insect host. Particularly, a new class of LuxR-like regulators was identified, which might be involved in detecting insect-specific molecules. In addition, the genetic overlap unravelled a two-component system that is unique for the genera Photorhabdus and Yersinia and is therefore suggested to play a major role in the pathogen-insect relationship. Our analysis also highlights factors of both pathogens that are expressed at low temperatures as encountered in insects in contrast to higher (body) temperature, providing evidence that temperature is a yet under-investigated environmental signal for bacterial adaptation to various hosts. Common degradative metabolic pathways are described that might be used to explore nutrients within the insect gut or hemolymph, thus enabling the proliferation of P. luminescens and Y. enterocolitica in their invertebrate hosts. A strikingly higher number of genes encoding insecticidal toxins and other virulence factors in P. luminescens compared to Y. enterocolitica correlates with the higher virulence of P. luminescens towards insects, and suggests a putative broader insect host spectrum of this pathogen.
A set of factors shared by the two pathogens was identified including those that are involved in the host infection process, in persistence within the insect, or in host exploitation. Some of them might have been selected during the association with insects and then adapted to pathogenesis in mammalian hosts.
BMC Genomics 02/2008; 9:40. · 4.07 Impact Factor
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Torsten Hain,
Som S Chatterjee,
Rohit Ghai,
Carsten Tobias Kuenne,
André Billion,
Christiane Steinweg,
Eugen Domann,
Uwe Kärst,
Lothar Jänsch,
Jürgen Wehland, [......],
Siegfried Scherer,
Michel Doumith,
Christine Jacquet,
Paul Martin,
Pascale Cossart,
Christophe Rusniock,
Philippe Glaser,
Carmen Buchrieser,
Werner Goebel,
Trinad Chakraborty
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ABSTRACT: This review provides an overview of recent progress in the exploration of genomic, transcriptomic, and proteomic data in Listeria spp. to understand genome evolution and diversity, as well as physiological aspects of metabolism utilized by the bacteria when growing in diverse and varied environments.
International Journal of Medical Microbiology 12/2007; 297(7-8):541-57. · 4.17 Impact Factor
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ABSTRACT: Salmonella enterica serovar Typhimurium (S. typhimurium) survives and proliferates within macrophage cells. A mutant library of strain ATCC 14028 based on gene disruption by homologous recombination was screened in order to identify genes that are required for wild-type-like intracellular replication. Randomly generated chromosomal fragments from the genome of S. typhimurium were cloned into a temperature-sensitive vector, and approximately 8000 individual mutant clones were obtained by insertional-duplication mutagenesis (IDM) upon selection at non-permissive temperature. Large-scale screening for replication defects in mouse macrophages, but not during growth in rich or minimal medium, revealed a set of attenuated mutants that were further characterized by PCR amplification and sequencing of the mutagenic fragments. Following analysis of a Salmonella genome map with the annotated positions of vector insertions, an accumulation of 33 attenuating insertions within genes of ten non-collinear regions was found. Insertions in virK, gipA and five SPI-2 genes as well as seven non-polar deletions validated the screen. No invasion deficiencies of the mutants were observed. The cob-cbi-pdu cluster containing the genes for cobalamin synthesis and 1,2-propanediol degradation was shown to be required for Salmonella replication within macrophages. These data gave rise to a model of eukaryotic glycoconjugates and phospholipids as alternative carbon, nitrogen and energy sources for intracellularly replicating bacteria. The contribution of as yet unknown components of SPI-6 and the Gifsy-1 and Gifsy-2 prophage islands to intracellular replication is reported, as well as the fivefold reduced intracellular growth rate of a mutant with a deletion of STM1677, which probably encodes a LysR-like transcriptional regulator. The intracellular replication rate of three double mutants, each lacking two gene products of the cob-cbi-pdu cluster or the Gifsy-1 prophage, was shown to be lower than that of the respective single mutants, suggesting that additive effects of subtle intracellular advantages contribute to Salmonella fitness in vivo.
Microbiology 05/2007; 153(Pt 4):1207-20. · 3.06 Impact Factor
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ABSTRACT: To analyze the transcriptional response of Yersinia enterocolitica cells to prolonged growth at low temperature, a collection of luxCDABE transposon mutants was cultivated in parallel at optimal (30 degrees C) and suboptimal (10 degrees C) temperatures and screened for enhanced promoter activities during growth until entering stationary phase. Among 5,700 Y. enterocolitica mutants, 42 transcriptional units were identified with strongly enhanced or reduced promoter activity at 10 degrees C compared to 30 degrees C, and changes in their transcriptional levels over time were measured. Green fluorescent protein fusions to 10 promoter regions confirmed the data. The temporal order of induction of the temperature-responsive genes of Y. enterocolitica was deduced, starting with the expression of cold shock genes cspA and cspB and the elevated transcription of a glutamate-aspartate symporter. Subsequently, cold-adapted cells drastically up-regulated genes encoding environmental sensors and regulators, such as UhpABC, ArcA, and methyl-accepting chemotaxis protein I (MCPI). Among the most prominent cold-responsive elements that were transcriptionally induced during growth in early and middle exponential phase are the insecticidal toxin genes tcaA and tcaB, as well as genes involved in flagellar synthesis and chemotaxis. The expression pattern of the late-exponential- to early-stationary-growth phase is dominated by factors involved in biodegradative metabolism, namely, a histidine ammonia lyase, three enzymes responsible for uptake and utilization of glycogen, the urease complex, and a subtilisin-like protease. Double-knockout mutants and complementation studies demonstrate inhibitory effects of MCPI and UhpC on the expression of a putative hemolysin transporter. The data partially delineate the spectrum of gene expression of Y. enterocolitica at environmental temperatures, providing evidence that an as-yet-unknown insect phase is part of the life cycle of this human pathogen.
Journal of Bacteriology 05/2006; 188(8):2945-58. · 3.83 Impact Factor
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ABSTRACT: A successful transition of Listeria monocytogenes from the extracellular to the intracellular environment requires a precise adaptation response to conditions encountered in the host milieu. Although many key steps in the intracellular lifestyle of this gram-positive pathogen are well characterized, our knowledge about the factors required for cytosolic proliferation is still rather limited. We used DNA microarray and real-time reverse transcriptase PCR analyses to investigate the transcriptional profile of intracellular L. monocytogenes following epithelial cell infection. Approximately 19% of the genes were differentially expressed by at least 1.6-fold relative to their level of transcription when grown in brain heart infusion medium, including genes encoding transporter proteins essential for the uptake of carbon and nitrogen sources, factors involved in anabolic pathways, stress proteins, transcriptional regulators, and proteins of unknown function. To validate the biological relevance of the intracellular gene expression profile, a random mutant library of L. monocytogenes was constructed by insertion-duplication mutagenesis and screened for intracellular-growth-deficient strains. By interfacing the results of both approaches, we provide evidence that L. monocytogenes can use alternative carbon sources like phosphorylated glucose and glycerol and nitrogen sources like ethanolamine during replication in epithelial cells and that the pentose phosphate cycle, but not glycolysis, is the predominant pathway of sugar metabolism in the host environment. Additionally, we show that the synthesis of arginine, isoleucine, leucine, and valine, as well as a species-specific phosphoenolpyruvate-dependent phosphotransferase system, play a major role in the intracellular growth of L. monocytogenes.
Journal of Bacteriology 02/2006; 188(2):556-68. · 3.83 Impact Factor
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ABSTRACT: We constructed a novel temperature-sensitive vector as a tool for gene disruption by insertion-duplication mutagenesis (IDM) in Salmonella enterica and related species. A phoN insertion mutant was proven highly stable during growth in LB medium and during infection of macrophage cells in the absence of selection pressure. By progressive shortening of a phoN fragment, the minimal length for effective insertional mutagenesis driven by homologous recombination was determined to be 50 bp, allowing to disrupt even short genes that could not yet be subjected to site-specific IDM. We also showed that plasmid excision from the chromosome restores the wild-type genotype with a reliability of 98%. Intracellular recovery of the excised vector provides the option to switch between two genotypes and thus to rapidly attribute the observed mutant phenotype to the targeted gene. In addition, a fragment library was used to measure the integration rate at various chromosomal sites that varies greatly by at least 2.5 magnitudes, independently from the length of the cloned fragment.
Plasmid 02/2006; 55(1):39-49. · 1.52 Impact Factor
