- Volume 153, Issue 9, 2007
Volume 153, Issue 9, 2007
- Pathogens And Pathogenicity
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Intracellular type III secretion by cytoplasmic Shigella flexneri promotes caspase-1-dependent macrophage cell death
More LessThe Gram-negative bacterium Shigella flexneri triggers pro-inflammatory apoptotic cell death in macrophages, which is crucial for the onset of an acute inflammatory diarrhoea termed bacillary dysentery. The Mxi-Spa type III secretion system promotes bacterial uptake and escape into the cytoplasm, where, dependent on the translocator/effector protein IpaB, caspase-1 [interleukin (IL)-1β-converting enzyme] and its substrate IL-1β are activated. Here, we show that in the course of a macrophage infection, IpaB is secreted intracellularly for more than 1 h post-infection and progressively accumulates in aggregates on the bacterial surface. Concomitantly, the bacterial pool of IpaB is gradually depleted. The protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) dose-dependently inhibited the Mxi-Spa-dependent secretion of IpaB triggered by the dye Congo red in vitro and abolished translocation of IpaB into the host-cell cytoplasm of S. flexneri-infected macrophages. CCCP specifically inhibited S. flexneri-triggered macrophage death in a dose-dependent manner, even if added up to 60 min post-infection. Addition of CCCP 15 min after infection blocked macrophage cell death, the activation of caspase-1 and the maturation of IL-1β, without affecting uptake or escape of S. flexneri from the phagosome. By contrast, CCCP used at the same concentration had no effect on ATP-induced caspase-1 activation or staurosporine-induced apoptosis. Our results indicate that under the conditions used, CCCP rapidly and specifically blocks bacterial type III secretion, and thus, intracellular type III secretion promotes cytotoxicity of S. flexneri.
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The Helicobacter pylori CagF protein is a type IV secretion chaperone-like molecule that binds close to the C-terminal secretion signal of the CagA effector protein
More LessType IV secretion systems are common bacterial macromolecule transporters that have been adapted to various functions, such as effector protein translocation to eukaryotic cells, nucleoprotein transfer to bacterial or eukaryotic cells, and DNA transport into and out of bacterial cells. Helicobacter pylori, the causative agent of bacterial gastritis, peptic ulcers, gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma, uses the Cag type IV secretion system to inject the CagA protein into host cells, thereby altering gene expression profiles and the host cell cytoskeleton. The molecular mechanism of CagA recognition as a type IV substrate is only poorly understood, but seems to be more complex than that of other type IV secretion systems. Apart from 14 essential components of the secretion apparatus, CagA translocation specifically requires the presence of four additional Cag proteins. Here we show that the CagA-binding protein CagF is a secretion chaperone-like protein that interacts with a 100 aa region that is adjacent to the C-terminal secretion signal of CagA. The interaction between CagA and CagF takes place at the bacterial cytoplasmic membrane, and is independent of a functional type IV secretion apparatus and other cag-encoded factors. Our data indicate that CagF binding precedes recognition of the C-terminal CagA translocation signal, and that both steps are required to recruit CagA to the type IV translocation channel.
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TGF-β1 induces transendothelial migration of the pathogenic fungus Sporothrix schenckii by a paracellular route involving extracellular matrix proteins
More LessSporotrichosis, a mycosis caused by Sporothrix schenckii, is characterized by lymphocutaneous lesions. In immunocompromised hosts, this fungus may invade the bloodstream and disseminate to other tissues, such as lung and bone. Our group previously showed that S. schenckii yeasts adhere to endothelial monolayers and that this interaction is modulated by cytokines. Using 3.0 μm-pore culture inserts, the present work shows that transforming growth factor (TGF)-β1 led to a 80±26 % increase in fungal migration across endothelial monolayers and inhibited fungus internalization by 55±23.5 %, when compared to untreated cells. The major surface endothelial molecules recognized by S. schenckii were not modulated by TGF-β1. These data suggested that a paracellular route is preferentially used by S. schenckii during the transmigration of cultured endothelial cells. It was further observed that TGF-β1 increased the subendothelial matrix exposure and that anti-fibronectin (anti-FN) and anti-laminin (anti-LM) antibodies abolished the increase in S. schenckii association with endothelial monolayers induced by TGF-β1. These antibodies also inhibited (38.2±4.29 % and 50.8±17.3 %, respectively) the adhesion of S. schenckii to freshly prepared native endothelial matrices. Furthermore, transendothelial migration of S. schenckii was blocked by anti-FN and anti-LM antibodies. These data indicate that TGF-β1-induced S. schenckii adhesion to endothelial monolayers results from the increased exposure of the subendothelial extracellular matrix and that this event may contribute to the enhancement of transendothelial migration.
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Phenotypic characterization of OmpX, an Ail homologue of Yersinia pestis KIM
The goal of this study was to characterize the Yersinia pestis KIM OmpX protein. Yersinia spp. provide a model for studying several virulence processes including attachment to, and internalization by, host cells. For Yersinia enterocolitica and Yersinia pseudotuberculosis, Ail, YadA and Inv, have been implicated in these processes. In Y. pestis, YadA and Inv are inactivated. Genomic analysis of two Y. pestis strains revealed four loci with sequence homology to Ail. One of these genes, designated y1324 in the Y. pestis KIM database, encodes a protein designated OmpX. The mature protein has a predicted molecular mass of 17.47 kDa, shares approximately 70 % sequence identity with Y. enterocolitica Ail, and has an identical homologue, designated Ail, in the Y. pestis CO92 database. The present study compared the Y. pestis KIM6+ parental strain with a mutant derivative having an engineered disruption of the OmpX structural gene. The parental strain (and a merodiploid control strain) expressed OmpX at 28 and 37 °C, and the protein was detectable throughout all phases of growth. OmpX was required for efficient adherence to, and internalization by, cultured HEp-2 cell monolayers and conferred resistance to the bactericidal effect of human serum. Deletion of ompX resulted in a significantly reduced autoaggregation phenotype and loss of pellicle formation in vitro. These results suggest that Y. pestis OmpX shares functional homology with Y. enterocolitica Ail in adherence, internalization into epithelial cells and serum resistance.
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Strain-dependent induction of epithelial cell oncosis by Campylobacter jejuni is correlated with invasion ability and is independent of cytolethal distending toxin
More LessInduction of host cell death is thought to play an important role in bacterial pathogenesis. Campylobacter jejuni is a prevalent cause of bacterial enteritis; however, its effects on enterocytes remain unclear. The present study indicates for the first time that C. jejuni induces oncotic, rather than apoptotic death of T84 enterocytes. C. jejuni-treated enterocytes exhibited extensive cytoplasmic vacuolation, rapid (3–6 h) loss of plasma membrane integrity (‘cytotoxicity’), loss of mitochondrial transmembrane potential, and ATP depletion. Enterocytes also exhibited increased oligonucleosomal DNA fragmentation, a feature characteristic of apoptosis. However, consistent with a non-apoptotic process, DNA fragmentation and cytotoxicity were not caspase dependent. During apoptosis, caspases mediate cleavage of poly(ADP-ribose) polymerase; however, cleavage was not observed in C. jejuni-treated monolayers. Cytotoxicity, ATP depletion and DNA fragmentation were not prevented by the deletion of the cytolethal distending toxin (CDT) gene, indicating that C. jejuni causes enterocyte oncosis via a mechanism that is CDT independent. The ability to cause oncosis was significantly decreased in a FlaAFlaB mutant (CDT+) that was defective in the ability to adhere and invade enterocytes. Analysis of clinical isolates revealed that oncosis was strain dependent and correlated with increased invasive ability. These observations offer new insights into the pathogenesis of C. jejuni infection.
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The cyclic AMP receptor protein modulates quorum sensing, motility and multiple genes that affect intestinal colonization in Vibrio cholerae
More LessVibrio cholerae is the causative agent of cholera, which continues to be a major public health concern in Asia, Africa and Latin America. The bacterium can persist outside the human host and alternates between planktonic and biofilm community lifestyles. Transition between the different lifestyles is mediated by multiple signal transduction pathways including quorum sensing. Expression of the Zn-metalloprotease haemagglutinin (HA)/protease is subject to a dual regulation which involves the quorum-sensing regulator HapR and the cAMP receptor protein. In a previous study, we observed that a mutant defective in the cAMP-receptor protein (CRP) expressed lower levels of HapR. To further investigate the role of CRP in modulating HapR and other signal transduction pathways, we performed global gene expression profiling of a Δcrp mutant of El Tor biotype V. cholerae. Here we show that CRP is required for the biosynthesis of cholera autoinducer 1 (CAI-1) and affects the expression of multiple HapR-regulated genes. As expected, the Δcrp mutant produced more cholera toxin and enhanced biofilm. Expression of flagellar genes, reported to be affected in ΔhapR mutants, was diminished in the Δcrp mutant. However, an epistasis analysis indicated that cAMP–CRP affects motility by a mechanism independent of HapR. Inactivation of crp inhibited the expression of multiple genes reported to be strongly induced in vivo and to affect the ability of V. cholerae to colonize the small intestine and cause disease. These genes included ompU, ompT and ompW encoding outer-membrane proteins, the alternative sigma factor σ E required for intestinal colonization, and genes involved in anaerobic energy metabolism. Our results indicate that CRP plays a crucial role in the V. cholerae life cycle by affecting quorum sensing and multiple genes required for survival of V. cholerae in the human host and the environment.
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Transcriptome analysis of Yersinia pestis in human plasma: an approach for discovering bacterial genes involved in septicaemic plague
Yersinia pestis is the aetiologic agent of plague. Without appropriate treatment, the pathogen rapidly causes septicaemia, the terminal and fatal phase of the disease. In order to identify bacterial genes which are essential during septicaemic plague in humans, we performed a transcriptome analysis on the fully virulent Y. pestis CO92 strain grown in either decomplemented human plasma or Luria–Bertani medium, incubated at either 28 or 37 °C and harvested at either the mid-exponential or the stationary growth phase. Y. pestis genes involved in 12 iron-acquisition systems and one iron-storage system (bfr, bfd) were specifically induced in human plasma. Of these, the ybt and tonB genes (encoding the yersiniabactin siderophore virulence factor and the siderophore transporter, respectively) were induced at 37 °C, i.e. under conditions mimicking the mammalian environment. Growth in human plasma also upregulated genes involved in the synthesis of five fimbrial-like structures (including the Psa virulence factor), and in purine/pyrimidine metabolism (the nrd genes). Genes known to play a role in the virulence of several bacterial pathogens (such as those encoding the Lpp lipoprotein and non-iron metal-uptake proteins) were induced in human plasma, during either the exponential or the stationary phase. Finally, 120 genes encoding proteins of unknown function were upregulated in human plasma. Eleven of these genes were specifically transcribed at 37 °C and may thus represent new virulence factors that are important during the septicaemic phase of human plague.
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Nanomechanical properties of glucans and associated cell-surface adhesion of Streptococcus mutans probed by atomic force microscopy under in situ conditions
This study used atomic force microscopy (AFM) to probe the local cell-surface interactions associated with the glucan polymers of Streptococcus mutans, the macromolecules most commonly attributed to the virulence of this microbe. In situ force spectroscopy was used to quantitatively probe and correlate cell-surface adhesion and dynamics with S. mutans UA140 wild-type and five glucosyltransferase mutants. Adhesion between the tooth surface and S. mutans is largely mediated by glucan production from sucrose via three glucosyltransferases (Gtfs; GtfB, GtfC and GtfD). To monitor the contribution of these particular Gtfs, isogenic mutants of S. mutans were constructed by specific gene inactivation and compared to the wild-type under sucrose and non-sucrose conditions. We report direct measurement of the mechanical properties associated with glucan macromolecules demonstrating that the local adhesion strength increases in a time-dependent process, with a decrease in the average number of rupture events. This finding suggests that S. mutans attaches mainly through glucans to surfaces in the presence of sucrose. In addition, a possible role of the Gtf proteins in sucrose-independent attachment is supported by the decreased adhesion properties of the GtfBCD mutant compared to the wild-type.
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Selection of transposon mutants of Mycobacterium tuberculosis with increased macrophage infectivity identifies fadD23 to be involved in sulfolipid production and association with macrophages
More LessAlterations to the composition or architecture of the mycobacterial cell envelope can affect the macrophage infectivity of the bacillus. To further characterize the mycobacterial gene products that modulate the interaction with host cells, we employed transposon mutagenesis and screened for mutants that demonstrated an enhanced binding affinity toward macrophages. After successive rounds of mutant selection and enrichment, a total of five mutants were isolated that harboured gene disruptions within loci involved in lipid synthetic pathways as well as genes coding for putative hypothetical proteins. One mutant in particular, with a disruption in the Rv3826 gene (fadD23), was repeatedly isolated during library screening. Analysis of the cell envelope constituents of the Tn : : fadD23 strain revealed a lack of sulfolipid production which was restored following complementation with the wild-type gene.
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Attenuation and protective efficacy of an O-antigen-deficient mutant of Francisella tularensis LVS
Francisella tularensis is a zoonotic, Gram-negative coccobacillus that causes tularemia in humans and animals. F. tularensis subspecies tularensis (type A) and F. tularensis subspecies holarctica (type B) are antigenically similar and more virulent than Francisella novicida in humans. The genetic locus that encodes the LPS O antigen was found to be substantially different between the type B live vaccine strain (LVS) and F. novicida. One LVS-specific gene with homology to a galactosyl transferase was selected for allelic replacement using a sacB–chloramphenicol expression suicide plasmid, and recombinants were screened for colony morphology on Congo red agar that matched that of F. novicida. Two mutants (WbtIS187Y and WbtIG191V) were isolated that contained substitutions in conserved motifs in the sugar transamine/perosamine synthetase (WbtI) of the O-antigen locus, and the latter mutant was extensively tested and characterized. WbtIG191V grew at the same rate as the parent strain in Chamberlain's defined medium, completely lacked O antigen, was serum-sensitive but could grow in a mouse macrophage cell line, had increased resistance to sodium deoxycholate, and was highly attenuated in mice. Complementation of WbtIG191V with the wild-type wbtI gene in trans restored normal LPS synthesis, phenotypic properties similar to the parent, and virulence in mice. Immunization with WbtIG191V protected mice against a relatively low-dose intraperitoneal challenge with LVS, but was less protective against a high-dose challenge. These results indicate that complete loss of O antigen alters the surface phenotype and abrogates virulence in F. tularensis, but also compromises the induction of full protective immunity against F. tularensis infection in mice.
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- Physiology
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Sodium regulates Escherichia coli acid resistance, and influences GadX- and GadW-dependent activation of gadE
More LessEnteric bacteria must survive the extreme acid of the stomach (pH 2 or less) before entering the intestine where they can colonize and cause disease. Escherichia coli is superior to most other Enterobacteriaceae in surviving pH 2 acid stress because it has four known acid-resistance systems, the most studied of which depends on glutamic acid. Glutamate-dependent acid resistance requires glutamate decarboxylase isozymes GadA and GadB, as well as a glutamate/γ-aminobutyric acid antiporter encoded by gadC. The regulatory protein GadE is the essential activator of the gadA and gadBC genes. The transcription of gadE, however, is controlled by numerous proteins. Two of these proteins, GadX and GadW, are AraC-family regulators whose sensory input signals are not known. Since Na+ and K+ play important roles in pH homeostasis, the contribution of these ions toward the regulation of this acid-resistance system was examined. The results indicated that a decrease in Na+, but not K+, concentration coincided with diminished acid resistance, and decreased expression of the gadE, gadA and gadBC genes. However, Na+-dependent regulation of these genes dissipated in the absence of GadX and GadW. Since Na+ levels did not regulate gadX or gadW transcription, it is proposed that GadX and GadW sense intracellular Na+ concentration or some consequence of altered Na+ levels.
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Role for dnaK locus in tolerance of multiple stresses in Staphylococcus aureus
Heat-shock proteins are essential for stress tolerance and allowing organisms to survive conditions that cause protein unfolding. The role of the Staphylococcus aureus DnaK system in tolerance of various stresses was studied by disruption of dnaK by partial deletion and insertion of a kanamycin gene cassette. Deletion of dnaK in S. aureus strain COL resulted in poor growth at temperatures of 37 °C and above, and reduced carotenoid production. The mutant strain also exhibited increased susceptibility to oxidative and cell-wall-active antibiotic stress conditions. In addition, the mutant strain had slower rates of autolysis, suggesting a correlation between DnaK and functional expression of staphylococcal autolysins. Deletion of dnaK also resulted in a decrease in the ability of the organism to survive in a mouse host during a systemic infection. In summary, the DnaK system in S. aureus plays a significant role in the survival of S. aureus under various stress conditions.
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PimM, a PAS domain positive regulator of pimaricin biosynthesis in Streptomyces natalensis
Sequencing of the DNA region on the left fringe of the pimaricin gene cluster revealed the presence of a 579 bp gene, pimM, whose deduced product (192 aa) was found to have amino acid sequence homology with bacterial regulatory proteins. Database comparisons revealed that PimM combines an N-terminal PAS domain with a C-terminal helix–turn–helix (HTH) motif of the LuxR type. Gene replacement of pimM from the Streptomyces natalensis chromosome with a mutant version lacking the HTH DNA-binding domain resulted in complete loss of pimaricin production, suggesting that PimM is a positive regulator of pimaricin biosynthesis. Complementation of the ΔpimM mutant with a single copy of pimM integrated into the chromosome restored pimaricin production. The insertion of a single copy of pimM, with its own promoter, into the S. natalensis wild-type strain boosted pimaricin production. Gene expression analyses in S. natalensis wild-type and ΔpimM by reverse transcriptase PCR (RT-PCR) of the pimaricin gene cluster revealed the targets for the PimM regulatory protein. According to these analyses, the genes responsible for initiation and first elongation cycles of polyketide chain extension are among the major targets for regulation. Other pim genes are differentially affected. Interestingly, our results indicate that PimM plays its regulatory role independently of PimR, the first pathway-specific regulator of pimaricin biosynthesis.
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- Plant-Microbe Interactions
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Fusarium verticillioides GAP1, a gene encoding a putative glycolipid-anchored surface protein, participates in conidiation and cell wall structure but not virulence
More LessFusarium verticillioides is an important pathogen of maize that causes ear rot and produces the mycotoxins known as fumonisins. To date, knowledge of pathogenicity and the regulation of fumonisin biosynthesis in F. verticillioides is limited. Here, the molecular characterization of GAP1, a gene encoding a putative 540 aa protein that belongs to a glycolipid-anchored surface (GAS) protein family, is presented. F. verticillioides GAP1 was identified as an expressed sequence tag (EST) upregulated in a culture condition conducive to conidiation and fumonisin B1 (FB1) production. GAP1 null mutants GAM126 (Δgap1 : : HYG) and GAG8 (Δgap1 : : GEN) exhibited restricted growth, with more aerial hyphae than their wild-type progenitor on solid media. No defect in mycelial mass or filamentous growth was observed when the GAM126 and GAG8 strains were grown in liquid media under shaking conditions. When grown in suspended conditions, GAM126 and GAG8 strains produced significantly fewer conidia and produced comparatively densely branched hyphae. Concanavalin A staining indicated that the GAP1 deletion altered the cell wall carbohydrate composition/deposition process. Deletion of GAP1 did not affect the production level of FB1 or F. verticillioides virulence on maize seedlings and stalks. Complementation of GAM126 with the wild-type GAP1 gene restored growth, conidiation and cell wall abnormality phenotypes. The results suggest that GAP1 is associated with growth, development and conidiation in F. verticillioides, but not with pathogenicity or regulation of FB1.
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Host-specific regulation of symbiotic nitrogen fixation in Rhizobium leguminosarum biovar trifolii
More LessStrains of Rhizobium leguminosarum bv. trifolii (Rlt) able to form effective nodules on Trifolium ambiguum (Caucasian clover, CC) form ineffective nodules on Trifolium repens (white clover, WC), whereas strains that form effective nodules on WC usually do not nodulate CC. Here, we investigate the genetic basis of the host-specific nitrogen-fixation phenotype of CC rhizobia. A cosmid library of the symbiotic plasmid from the WC rhizobium strain Rlt NZP514 was introduced into the CC rhizobium strain Rlt ICC105. An 18 kb Asp718 fragment containing the nifABHDKEN and fixABCX genes of NZP514 that imparted the Fix+ phenotype was identified. Tn5 mutagenesis of this region revealed that the nifHDKEN, fixABC and nifB genes were required for the Fix+ phenotype, but that the nifA gene was not. Introduction of several plasmids containing NZP514 nif/fix genes into an ICC105 nifA mutant strain demonstrated that the NifA protein of ICC105 was able to activate expression of the NZP514 nif/fix genes but not the ICC105 nif/fix genes in WC nodules. Reporter gene fusion studies showed that the host-specific regulation of the nif/fix genes depended on the DNA region between the promoters of the divergently transcribed nifH and fixA genes. We hypothesize that a protein acting either in response to a host-specific signal or in the absence of such a signal is able to bind upstream of the NifA-binding sites and interact with NifA to prevent it activating nif/fix gene expression.
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Role of sdhA and pfkA and catabolism of reduced carbon during colonization of cucumber roots by Enterobacter cloacae
We have been using a mutational approach to determine how plant-beneficial bacteria such as Enterobacter cloacae 501R3 obtain carbon and energy for colonization of subterranean portions of cucumber and other plants. Reduced carbon detected in cucumber root exudate consisted of 73.3 % amino acids, 22.2 % organic acids and 4.4 % carbohydrate. Ent. cloacae M2, a mini-Tn5 Km transposon mutant of strain 501R3, was severely reduced in in vitro growth relative to strain 501R3 on the mixture of amino acids and organic acids detected in cucumber root exudate when these compounds were supplied as the sole source of carbon and energy, but was similar in growth on the mixture of carbohydrates detected in this exudate. Molecular and biochemical characterization of Ent. cloacae M2 indicated that the transposon was inserted in sdhA, which encodes a subunit of succinate dehydrogenase. Ent. cloacae A-11, a mutant of strain 501R3 with a mini-Tn5 Km insertion in pfkA, was severely reduced in in vitro growth relative to strain 501R3 on the mixture of carbohydrates detected in cucumber root exudate, but similar in growth on the mixture of amino acids and organic acids. When strains A-11 and M2 were coapplied with strain 501R3 to cucumber seeds above carrying capacity in competitive root colonization assays, populations of strains A-11 and M2 were roughly one order of magnitude lower than those of strain 501R3 in cucumber rhizosphere, while populations of strains A-11 and M2 were similar to one other when coapplied to cucumber seeds. When Ent. cloacae strains were coapplied to cucumber seeds below carrying capacity, populations of A-11 and M2 were roughly two to three orders of magnitude lower than those of 501R3 in cucumber rhizosphere, and populations of A-11 were significantly lower than those of M2 when these two strains were coapplied to cucumber seed. The experiments reported here indicate an important role for pfkA and sdhA and the catabolism of carbohydrates, and of amino acids and organic acids, respectively, in the colonization of cucumber roots by Ent. cloacae. The results reported here also indicate that catabolism of carbohydrates by this bacterium is more important than catabolism of amino acids and organic acids at lower population densities, despite the much higher relative quantities of amino acids and organic acids detected in cucumber root exudate.
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- Theoretical Microbiology
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Host gastro-intestinal dynamics and the frequency of colicin production by Escherichia coli
More LessThe production of antimicrobial compounds known as colicins has been shown to be an important mediator of competitive interactions among Escherichia coli genotypes. There is some understanding of the forces responsible for determining the frequency of colicin production in E. coli populations; however, this understanding cannot explain all of the observed variation. A survey of colicin production in E. coli isolated from native Australian mammals revealed that the frequency of colicin production in strains isolated from carnivores was significantly lower than the frequency of production in strains recovered from herbivores or omnivores. The intestine of Australian carnivores is tube-like and gut turnover rates are rapid compared with the turnover rates of the intestinal tracts of herbivores and omnivores, all of which possess a hindgut fermentation chamber. A mathematical model was developed in order to determine if variation in gut turnover rates could determine if a host was more likely to harbour a colicin-producing strain or a non-producer. The model predicted that a colicin producer was more likely to dominate in the gut of a host with lower gut turnover rates, and a non-producer to dominate in hosts with rapid gut turnover rates.
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