- Volume 153, Issue 9, 2007
Volume 153, Issue 9, 2007
- Mini-Review
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Sialic acid utilization by bacterial pathogens
More LessSialic acid occupies the terminal position within glycan molecules on the surfaces of many vertebrate cells, where it functions in diverse cellular processes such as intercellular adhesion and cell signalling. Pathogenic bacteria have evolved to use this molecule beneficially in at least two different ways: they can coat themselves in sialic acid, providing resistance to components of the host's innate immune response, or they can use it as a nutrient. Sialic acid itself is either synthesized de novo by these bacteria or scavenged directly from the host. In this mini-review we will summarize recent findings relating to sialic acid transport, modification of sialic acid by O-acetylation, and the mechanisms of sialic acid-mediated complement resistance.
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- Biochemistry And Molecular Biology
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Conjugative DNA transfer in Streptomyces: SpdB2 involved in the intramycelial spreading of plasmid pSVH1 is an oligomeric integral membrane protein that binds to dsDNA
More LessIn the current model of conjugal plasmid transfer in mycelium-forming streptomycetes, plasmid transfer by the FtsK-like TraB protein is followed by the subsequent spreading of the newly transferred plasmid within the neighbouring mycelial compartments. Several plasmid-encoded Spd proteins are involved in the plasmid spreading by an unknown mechanism. spdB2 of the conjugative pSVH1 plasmid of Streptomyces venezuelae was heterologously expressed in Escherichia coli and Streptomyces lividans, with a C-terminal His-tag-encoding sequence. Induction of spdB2-His expression affected viability in both species. The integral membrane protein SpdB2-His was eluted from the membrane fraction of S. lividans with Triton X-100, and purified as a soluble protein by Ni-NTA affinity chromatography. Cross-linking experiments with glutaraldehyde showed that SpdB2-His formed oligomers. SpdB2-His had a nonspecific DNA-binding activity: while all types of dsDNA were bound, single-stranded M13-DNA was not recognized. The spd genes of the spdB3–spd79–spdB2 operon of pSVH1 were simultaneously expressed in E. coli with different affinity tags. While expression of StrepII-SpdB3 was not detected, Spd79-flag and SpdB2-His were localized in the membrane fraction of E. coli. In the absence of SpdB2, most of the Spd79-flag protein was found in the cytoplasmic fraction, indicating that SpdB2 affects localization of Spd79. Pulldown assays with StrepII-TraB protein of pSVH1 demonstrated that TraB interacted with SpdB2, suggesting that the septal DNA translocator TraB is also involved in intramycelial plasmid spreading.
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A generalized transducing phage for the murine pathogen Citrobacter rodentium
A virulent phage (φCR1) capable of generalized transduction in Citrobacter rodentium was isolated from the environment and characterized. C. rodentium is a natural pathogen of mice, causing transmissible murine colonic hyperplasia. Sequencing of its genome has recently been completed and will soon be fully annotated and published. C. rodentium is an important model organism for infections caused by the human pathogens enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC). φCR1 uses a lipopolysaccharide receptor, has a genome size of approximately 300 kb, and is able to transduce a variety of markers. φCR1 is the first reported transducing phage for C. rodentium and will be a useful tool for functional genomic analysis of this important natural murine pathogen.
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Dissecting the Salmonella response to copper
More LessIntracellular copper homeostasis in bacteria is maintained as the result of a complex ensemble of cellular processes that in Escherichia coli involve the coordinated action of two systems, cue and cus. In contrast, the pathogenic bacterium Salmonella harbours only the cue regulon, including copA, which is shown here to be transcriptionally controlled by CueR. Mutant strains in the CueR-regulated genes were constructed to characterize the response of Salmonella enterica serovar Typhimurium to high concentrations of extracellular copper under both aerobic and anaerobic conditions. Unlike its counterpart in E. coli, inactivation of cuiD displays the most severe phenotype and is also required for copper tolerance under anaerobic conditions. Deletion of copA has a mild effect in aerobiosis, but strongly impairs survival in the absence of oxygen. In a ΔcopA strain, a second Salmonella-specific P-type ATPase, GolT, can substitute the copper transporter, diminishing the effect of its deletion. The overall results highlight the importance of the cue system for controlling intracellular copper stress. The observed differences between Salmonella and E. coli in handling copper excess may contribute to our understanding of the distinct capability of these related pathogenic bacteria to survive outside the host.
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Analysis of functional domains present in the N-terminus of the SipB protein
More LessSipB (593 aa), one of the Salmonella invasion proteins (Sips), is secreted via the Salmonella pathogenicity island 1 (SPI-1) type III secretion system (T3SS). Here, we report the delineation of several functional regions present in the SipB protein. Our data show that residues 3–8 of the SipB protein are essential for its secretion from the bacterial cell and that the SicA chaperone, which is important to ensure stability of SipB and SipC in the bacterial cytosol, binds to SipB somewhere between amino acids 80 and100 of the SipB N-terminal region. Interestingly, the N-terminal region (residues 1–160) of SipB (SipB160) cannot be secreted via the SPI-1 T3SS, but fusion of the C-terminal amphipathic region (residues 300–593) to SipB160 can restore secretion via this system.
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Transcriptional linkage of Haloferax volcanii proteasomal genes with non-proteasomal gene neighbours including RNase P, MOSC domain and SAM-methyltransferase homologues
More LessComparative genomics reveals a common theme of 20S proteasome and proteasome-activating nucleotidase genes dispersed throughout archaeal genomes yet arranged in conserved linkages with gene homologues of translation and/or transcription machineries. To provide biological evidence for these linkages as well as insight into proteasome operon organization, transcripts of the five proteasomal genes of the halophilic archaeon Haloferax volcanii were analysed by Northern (RNA) blotting, RT-PCR and primer extension. These included psmA, psmB and psmC, encoding the 20S proteasomal subunits α1, β and α2, as well as panA and panB, encoding the PanA and PanB proteasome-activating nucleotidase proteins, respectively. All five of these genes are dispersed throughout the H. volcanii genome. For each proteasomal gene, a distinct transcript was detected by Northern blotting that was similar in size to the respective coding region. For both psmA and psmC, an additional transcript was detected that was 1.34 and 0.85 kb greater, respectively, than the coding region. Further analysis by Northern blotting and RT-PCR revealed that psmA was co-transcribed with genes encoding a Pop5 homologue of the RNase P endoRNase as well as an S-adenosylmethionine (SAM)-dependent methyltransferase. Likewise, psmC was co-transcribed with a downstream gene encoding a molybdenum cofactor sulfurase C-terminal (MOSC) domain protein. Additional proteasomal and neighbouring gene-specific transcriptional linkages were detected by RT-PCR. These results provide the first evidence that proteasome and tRNA modification genes are co-transcribed, reveal that a number of additional enzymes including those predicted to facilitate metal–sulfur cluster assembly are co-regulated with proteasomes at the transcriptional level, and provide further insight into proteasome gene transcription in archaea.
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Expression analysis of extracellular proteins from Phanerochaete chrysosporium grown on different liquid and solid substrates
More LessWhite-rot fungi secret a large number of hydrolytic and oxidative enzymes for degradation of lignocellulosic material. The sequencing of the genome of the white-rot fungus Phanerochaete chrysosporium has facilitated the characterization of its complete extracellular proteome. P. chrysosporium was grown on liquid medium, containing glucose, cellulose or wood chips as the carbon source, and also in solid substrate fermentation bags. For liquid-grown cultures, the extracellular protein fraction was separated by 2D gel electrophoresis. Protein spots were analysed by in-gel digestion and liquid chromatography (LC)/MS/MS. A total of 18 additional protein spots from the 2D gels yielded hits from blast searches. From solid substrate cultures in which the fungus was grown in bags, the proteins were resolved by SDS-PAGE, subjected to in-gel digestion and then identified by LC/MS/MS. An additional 16 proteins yielded hits on blast searches. Enzymes involved in cellulose, hemicellulose, lignin and protein degradation were identified. Expression patterns were very similar between cellulose-grown cultures and wood-grown cultures. In addition to enzymes which act on lignocellulosic material, proteases were also found, indicating the need of fungi to scavenge for nitrogen in wood.
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Cloning and characterization of two K+ transporters of Debaryomyces hansenii
More LessTwo genes from the halotolerant yeast Debaryomyces hansenii were cloned, DhTRK1 and DhHAK1. These genes encode K+ transporters with sequence similarities to the TRK and HAK transporters from Debaryomyces occidentalis and Candida albicans. The DhHAK1p transporter was only expressed in K+-starved cells, as shown by Northern blot analysis. Both DhTRK1p and DhHAK1p were expressed in a trk1Δ trk2Δ mutant of Saccharomyces cerevisiae, unable to grow at low K+. This expression resulted in partial recovery of growth and ability to retain K+ at low concentrations. In liquid media, 0.5 M NaCl affected growth of these S. cerevisiae transformants as it does in D. hansenii, resulting in a much less deleterious effect than in wild-type S. cerevisiae. Kinetics of Rb+ uptake in the transformants suggest that DhTRK1p and DhHAK1p code for moderate-affinity K+ transporters exhibiting a sigmoid response against Rb+ concentration and presenting a deviation from classic Michaelis–Menten kinetics at low substrate concentrations. Rb+ uptake by the DhTRK1p transporter was stimulated by millimolar concentrations of Na+ at pH 4.5. The good performance of DhTRK1p in the presence of NaCl may be a key feature in the halotolerance of D. hansenii.
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Ethanol production from xylose by recombinant Saccharomyces cerevisiae expressing protein-engineered NADH-preferring xylose reductase from Pichia stipitis
A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis (PsXR and PsXDH, respectively) has the ability to convert xylose to ethanol together with the unfavourable excretion of xylitol, which may be due to intercellular redox imbalance caused by the different coenzyme specificity between NADPH-preferring XR and NAD+-dependent XDH. In this study, we focused on the effect(s) of mutated NADH-preferring PsXR in fermentation. The R276H and K270R/N272D mutants were improved 52- and 146-fold, respectively, in the ratio of NADH/NADPH in catalytic efficiency [(k cat/K m with NADH)/(k cat/K m with NADPH)] compared with the wild-type (WT), which was due to decrease of k cat with NADPH in the R276H mutant and increase of K m with NADPH in the K270R/N272D mutant. Furthermore, R276H mutation led to significant thermostabilization in PsXR. The most positive effect on xylose fermentation to ethanol was found by using the Y-R276H strain, expressing PsXR R276H mutant and PsXDH WT: 20 % increase of ethanol production and 52 % decrease of xylitol excretion, compared with the Y-WT strain expressing PsXR WT and PsXDH WT. Measurement of intracellular coenzyme concentrations suggested that maintenance of the of NADPH/NADP+ and NADH/NAD+ ratios is important for efficient ethanol fermentation from xylose by recombinant S. cerevisiae.
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The DUF81 protein TauE in Cupriavidus necator H16, a sulfite exporter in the metabolism of C2 sulfonates
More LessThe degradation of taurine, isethionate and sulfoacetate in Cupriavidus necator (Ralstonia eutropha) H16 was shown by enzyme assays to be inducible, and each pathway involved sulfoacetaldehyde, which was subject to phosphatolysis by a common sulfoacetaldehyde acetyltransferase (Xsc, H16_B1870) to yield acetyl phosphate and sulfite. The neighbouring genes encoded phosphate acetyltransferase (Pta, H16_B1871) and a hypothetical protein [domain of unknown function (DUF)81, H16_B1872], with eight derived transmembrane helices. RT-PCR showed inducible transcription of these three genes, and led to the hypothesis that H16_B1872 and orthologous proteins represent a sulfite exporter, which was named TauE.
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Involvement of a chromomycin ABC transporter system in secretion of a deacetylated precursor during chromomycin biosynthesis
More LessChromomycin A3 is an antitumour antibiotic that acts by inhibiting transcription and replication of DNA. The producer micro-organism Streptomyces griseus subsp. griseus is highly resistant to chromomycin A3 and to the structurally related compound mithramycin upon induction with chromomycin A3. The biosynthetic gene cluster of chromomycin contains three genes involved in self-resistance to chromomycin in S. griseus: cmrA and cmrB encode a type I ATP-binding cassette (ABC) transporter, and cmrX encodes a UvrA-like protein of ABC excision nuclease systems. These genes are linked in the chromosome, together with a gene encoding a transcriptional repressor (cmmRII). Involvement of these genes in chromomycin resistance was determined through gene inactivation, and heterologous expression in Streptomyces albus. Inactivation of cmrX produced a chromomycin-sensitive low-producer strain, while inactivation of cmmRII generated a high-chromomycin-producer strain, which was resistant to chromomycin, and also to mithramycin. Expression of either cmrA and cmrB, or cmrX, in S. albus generated strains with low chromomycin resistance; it was therefore necessary to co-express the three genes to achieve high levels of resistance. However, the CmrAB ABC transporter conferred a high level of resistance to the biosynthesis intermediate 4A,4E-O-dideacetyl-chromomycin A3. A model is proposed for the biosynthesis of, and self-resistance to, chromomycin A3 in S. griseus subsp. griseus.
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Isolation and characterization of putative Pseudobutyrivibrio ruminis promoters
More LessNovel plasmids were constructed for the analysis of DNA fragments from the rumen bacterium Pseudobutyrivibrio ruminis. Five previously unidentified promoters were characterized using a novel primer extension method to identify transcription start sites. The genes downstream of these promoters were not identified, and their activity in expression of genomic traits in wild-type P. ruminis remains putative. Comparison with promoters from this and closely related species revealed a consensus sequence resembling the binding motif for the RNA polymerase σ 70-like factor complex. Consensus −35 and −10 sequences within these elements were TTGACA and ATAATATA respectively, interspaced by 15–16 bp. The consensus for the −10 element was extended by one nucleotide upstream and downstream of the standard hexamer (indicated in bold). Promoter strengths were measured by reverse transcription quantitative PCR and β-glucuronidase assays. No correlation was found between the composition and context of elements within P. ruminis promoters, and promoter strength. However, a mutation within the −35 element of one promoter revealed that transcriptional strength and choice of transcription start site were sensitive to this single nucleotide change.
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- Environmental Microbiology
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A novel bacterial disease of the European shore crab, Carcinus maenas – molecular pathology and epidemiology
Several rickettsia-like diseases have been reported in arthropods (insects and crustaceans), some of which result in significant losses of economically important species such as shrimp and crabs. This study reports on the molecular pathology of a recently emerged disease of the European shore crab, Carcinus maenas, termed milky disease – named as a result of the unusual milky appearance of the haemolymph (blood). This disease was more prevalent (>26 %) during summer months when the water temperature in a pilot crab farm was approximately 19 °C. The putative causative agent of the disease was a Gram-negative bacterium that could not be cultured on a range of agar-based growth media. Diseased crabs showed significant reductions in free blood cell numbers and total serum protein. Such animals also displayed raised levels of glucose and ammonium in blood. Ultrastructural and in situ hybridization studies revealed that the causative agent associated with milky disease multiplied in the fixed phagocytes of the hepatopancreas (digestive gland), ultimately to be released into the haemolymph, where the circulating blood cells showed little response to the presence of these agents. Attempts to induce the infection by short-term temperature stress failed, as did transmission experiments where healthy crabs were fed infected tissues from milky disease affected individuals. Sequence analysis of the 16S rRNA gene from the milky disease bacteria indicated that they are a previously undescribed species of α-proteobacteria with little phylogenetic similarity to members of the order Rickettsiales.
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- Genes And Genomes
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The morphogenetic regulator Czf1p is a DNA-binding protein that regulates white–opaque switching in Candida albicans
More LessCzf1p has been demonstrated to regulate the switch between the yeast-cell morphology and filamentous morphologies of the human fungal pathogen Candida albicans. The predicted amino acid sequence of Czf1p contains a zinc-cluster motif similar to the DNA-binding domains of proteins such as Saccharomyces cerevisiae Gal4p, suggesting that Czf1p is a DNA-binding protein. Czf1p also demonstrates genetic interaction and a two-hybrid interaction with a second regulator of C. albicans cellular morphology, Efg1p. During growth in contact with an agar matrix, Efg1p has a negative effect on filamentation and Czf1p antagonizes this effect. In addition to regulating cellular morphology, Efg1p plays a role in regulating the cell-type switch between the commonly observed white phase of C. albicans and the opaque, mating-competent phase. While overexpression of EFG1 stimulates the switch from opaque to white, the results reported here demonstrate that overexpression of CZF1 promotes the reverse switch, from white to opaque. We also demonstrate that Czf1p binds CZF1 promoter DNA in vitro. Therefore, for the regulation of both contact-dependent filamentation and white–opaque switching, Czf1p and Efg1p have opposing functions.
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Genome sequence comparison and superinfection between two related Pseudomonas aeruginosa phages, D3112 and MP22
More LessA temperate transposable bacteriophage (MP22) was isolated from a Korean clinical isolate of Pseudomonas aeruginosa. It has a coliphage λ-like morphology and a double-stranded DNA genome. The complete nucleotide sequence and annotation of the MP22 genome and its characteristics are presented. The MP22 genome is 36 409 bp long with a G+C content of 64.2 mol%. The genome contains 51 proposed ORFs, of which 48 (94 %) display synteny and significant nucleotide and protein sequence similarity to the corresponding ORFs of the closely related phage, D3112. Three of the predicted ORFs are unique proteins, whose functions are yet to be revealed. The phage c repressors exhibit striking dissimilarities and, when present as a single gene, did not show cross-immunity. In contrast, although an MP22 lysogen could be productively infected with D3112, MP22 could not grow on a D3112 lysogen, indicating a role of other D3112 genes in superinfection exclusion.
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Effects of Fis on Escherichia coli gene expression during different growth stages
More LessFis is a nucleoid-associated protein in Escherichia coli that is abundant during early exponential growth in rich medium but is in short supply during stationary phase. Its role as a transcriptional regulator has been demonstrated for an increasing number of genes. In order to gain insight into the global effects of Fis on E. coli gene expression during different stages of growth in rich medium, DNA microarray analyses were conducted in fis and wild-type strains during early, mid-, late-exponential and stationary growth phases. The results uncovered 231 significantly regulated genes that were distributed over 15 functional categories. Regulatory effects were observed at all growth stages examined. Coordinate upregulation was observed for a number of genes involved in translation, flagellar biosynthesis and motility, nutrient transport, carbon compound metabolism, and energy metabolism at different growth stages. Coordinate down-regulation was also observed for genes involved in stress response, amino acid and nucleotide biosynthesis, energy and intermediary metabolism, and nutrient transport. As cells transitioned from the early to the late-exponential growth phase, different functional categories of genes were regulated, and a gradual shift occurred towards mostly down-regulation. The results demonstrate that the growth phase-dependent Fis expression triggers coordinate regulation of 15 categories of functionally related genes during specific stages of growth of an E. coli culture.
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Co-regulation of the nitrogen-assimilatory gene cluster in Clostridium saccharobutylicum
More LessNitrogen assimilation is important during solvent production by Clostridium saccharobutylicum NCP262, as acetone and butanol yields are significantly affected by the nitrogen source supplied. Growth of this bacterium was dependent on the concentration of organic nitrogen supplied and the expression of the assimilatory enzymes, glutamine synthetase (GS) and glutamate synthase (GOGAT), was shown to be induced in nitrogen-limiting conditions. The regions flanking the gene encoding GS, glnA, were isolated from C. saccharobutylicum genomic DNA, and DNA sequencing revealed that the structural genes encoding the GS (glnA) and GOGAT (gltA and gltB) enzymes were clustered together with the nitR gene in the order glnA-nitR-gltAB. RNA analysis showed that the glnA-nitR and the gltAB genes were co-transcribed on 2.3 and 6.2 kb RNA transcripts respectively, and that all four genes were induced under the same nitrogen-limiting conditions. Complementation of an Escherichia coli gltD mutant, lacking a GOGAT small subunit, was achieved only when both the C. saccharobutylicum gltA and gltB genes were expressed together under anaerobic conditions. This is believed to be the first functional analysis of a gene cluster encoding the key enzymes of nitrogen assimilation, GS and GOGAT. A similar gene arrangement is seen in Clostridium beijerinckii NCIMB 8052, and based on the common regulatory features of the promoter regions upstream of the glnA operons in both species, we suggest a model for their co-ordinated regulation by an antitermination mechanism as well as antisense RNA.
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Transcriptional regulation of the sulfate-starvation-induced gene sfnA by a σ 54-dependent activator of Pseudomonas putida
More LessThe σ 54-dependent transcriptional regulator SfnR is essential for the use of dimethyl sulfone (DMSO2) as a sulfur source by Pseudomonas putida DS1. SfnR binds three SfnR-binding sites (sites 1, 2 and 3) within an intergenic region of the divergently transcribed sfnAB and sfnFG gene clusters. The site 1 region, proximal to the sfnF gene, is indispensable for the expression of the sfnFG operon, which encodes components of DMSO2 monooxygenase. We investigated the transcriptional regulation of the sfnAB operon and possible functions of the sfnA gene. RT-PCR analysis revealed that the sfnAB gene cluster, which is similar to homologues of the acyl-CoA dehydrogenase family, was transcribed as an operon, and its expression was regulated by SfnR under conditions of sulfate starvation. Deletion analyses using lacZ as a reporter demonstrated that the region up to at least −138 bp from the transcription start point of sfnA (containing sites 2 and 3) was necessary for the expression of the sfnAB operon. A growth test of the sfnA-disrupted mutant revealed the possibility that sfnA may be involved in the use of methanethiol as a sulfur source.
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Deletion of a previously uncharacterized flagellar-hook-length control gene fliK modulates the σ 54-dependent regulon in Campylobacter jejuni
A previously unannotated, putative fliK gene was identified in the Campylobacter jejuni genome based on sequence analysis; deletion mutants in this gene had a ‘polyhook’ phenotype characteristic of fliK mutants in other genera. The mutants greatly overexpressed the σ 54-dependent flagellar hook protein FlgE, to form unusual filamentous structures resembling straight flagella in addition to polyhooks. The genome sequence reveals only one gene predicted to encode an orthologue of the NtrC-family activator required for σ 54-dependent transcription. Hence, all σ 54-dependent genes in the genome would be overexpressed in the fliK mutant together with flgE. Microarray analysis of genome-wide transcription in the mutant showed increased transcription of a subset of genes, often downstream of σ 54-dependent promoters identified by a quality-predictive algorithm applied to the whole genome. Assessment of genome-wide transcription in deletion mutants in rpoN, encoding σ 54, and in the σ 54-activator gene flgR, showed reciprocally reduced transcription of genes that were overexpressed in the fliK mutant. The fliA (σ 28)-dependent regulon was also analysed. Together the data clearly define the roles of the alternative sigma factors RpoN and FliA in flagellar biogenesis in C. jejuni, and identify additional putative members of their respective regulons.
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- Pathogens And Pathogenicity
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Intoxication of epithelial cells by plasmid-encoded toxin requires clathrin-mediated endocytosis
More LessIt has been shown that the autotransporter plasmid-encoded toxin (Pet) of enteroaggregative Escherichia coli (EAEC) produces cytotoxic and enterotoxic effects. Both effects can be explained by the proteolytic activity of Pet on its intracellular target α-fodrin (αII spectrin). In addition, Pet cytotoxicity and enterotoxicity depend on Pet serine protease activity, and on its internalization into epithelial cells. However, the mechanisms of Pet uptake by epithelial cells are unknown. Here, we show that Pet interacts with the plasma membrane of epithelial cells, and afterwards is detected inside the cells. Furthermore, Pet was internalized via clathrin-mediated endocytosis, since its internalization was inhibited by monodansylcadaverine and sucrose, but not by filipin or methyl-β-cyclodextrin, which are drugs that interfere with protein entry via a clathrin-independent pathway. Additionally, Pet was immunoprecipitated by anti-clathrin antibodies, but not by anti-caveolin antibodies. Moreover, small interfering RNA (siRNA), designed to knock out clathrin gene expression in HEp-2 cells, prevented Pet internalization, and thereby the Pet-induced cytotoxic effect. However, the use of siRNA to knock out caveolin expression had no effect on Pet internalization, and the cytotoxic effect was clearly observed. Together, these data indicate that Pet secreted by EAEC binds to the cell surface via an unknown receptor, to be taken up by clathrin-mediated endocytosis, and exert its toxic effect in the cytoplasm.
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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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Volumes and issues
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Volume 170 (2024)
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