- Volume 154, Issue 7, 2008
Volume 154, Issue 7, 2008
- Review
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Diffusible signals and interspecies communication in bacteria
More LessMany bacteria use cell–cell communication mediated by diffusible signal molecules to monitor their population density or confinement to niches and to modulate their behaviour in response to these aspects of their environment. Work on signalling systems within individual species has formed a platform for studies of interspecies interactions that can occur within polymicrobial communities in nature. In addition to signalling between organisms that synthesize the same or related signal molecules, it is becoming evident that bacteria can sense signal molecules that they do not synthesize, thereby eavesdropping on signalling by other organisms in their immediate environment. Furthermore, molecules such as antibiotics that are considered not to be signals for the producing species can have effects on gene expression in other bacteria that indicate a signalling function. Interspecies signalling can lead to alteration in factors contributing to the virulence or persistence of bacterial pathogens as well as influencing the development of beneficial microbial communities. Here we review our current understanding of interspecies signalling in bacteria and the signals involved, what is known of the underlying signal transduction mechanisms and their influences on bacterial behaviour.
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- Mini-Review
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Transcription factor dynamics
More LessGene expression is a fundamental process that is highly conserved from humans to bacteria. The first step in gene expression, transcription, is performed by structurally conserved DNA-dependent RNA polymerases (RNAPs), which results in the synthesis of an RNA molecule from a DNA template. In bacteria, a single species of RNAP is responsible for transcribing both stable RNA (i.e. t- and rRNA) and protein-encoding genes (i.e. mRNA), unlike eukaryotic systems, which use three distinct RNAP species to transcribe the different gene classes (RNAP I transcribes most rRNA, RNAP II transcribes mRNA, and RNAP III transcribes tRNA and 5S rRNA). The versatility of bacterial RNAP is dependent on both dynamic interactions with co-factors and the coding sequence of the template DNA, which allows RNAP to respond appropriately to the transcriptional needs of the cell. Although the majority of the research on gene expression has focused on the initiation stage, regulation of the elongation phase is essential for cell viability and represents an important topic for study. The elongation factors that associate with RNAP are unique and highly conserved among prokaryotes, making disruption of their interactions a potentially important target for antibiotic development. One of the most significant advances in molecular biology over the last decade has been the use of green fluorescent protein (GFP) and its spectral variants to observe the subcellular localization of proteins in live intact cells. This review discusses transcription dynamics with respect to RNAP and its associated transcription elongation factors in the two best-studied prokaryotes, Escherichia coli and Bacillus subtilis.
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- Cell And Developmental Biology
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Two GDP-mannose transporters contribute to hyphal form and cell wall integrity in Aspergillus nidulans
In order to identify novel genes affecting cell wall integrity, we have generated mutant strains of the filamentous fungus Aspergillus nidulans that show hypersensitivity to the chitin-binding agent Calcofluor White (CFW). Affected loci are designated cal loci. The phenotype of one of these alleles, calI11, also includes shortened hyphal compartments and increased density of branching in the absence of CFW, as well as reduced staining of cell walls by the lectin FITC–Concanavalin A (ConA), which has strong binding affinity for mannosyl residues. We have identified two A. nidulans genes (AN8848.3 and AN9298.3, designated gmtA and gmtB, respectively) that complement all aspects of the phenotype. Both genes show strong sequence similarity to GDP-mannose transporters (GMTs) of Saccharomyces and other yeasts. Sequencing of gmtA from the calI11 mutant strain reveals a G to C mutation at position 943, resulting in a predicted alanine to proline substitution at amino acid position 315 within a region that is highly conserved among other fungi. No mutations were observed in the mutant strain's allele of gmtB. Meiotic mapping demonstrated a recombination frequency of under 1 % between the calI locus and the phenA locus (located ∼9.5 kb from AN8848.3), confirming that gmtA and calI are identical. A GmtA–GFP chimera exhibits a punctate distribution pattern, consistent with that shown by putative Golgi markers in A. nidulans. However, this distribution did not overlap with that of the putative Golgi equivalent marker CopA–monomeric red fluorescent protein (mRFP), which may indicate that the physically separated Golgi-equivalent organelles of A. nidulans represent physiologically distinct counterparts of the stacked cisternae of plants and animals. These findings demonstrate that gmtA and gmtB play roles in cell wall metabolism in A. nidulans similar to those previously reported for GMTs in yeasts.
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- Biochemistry And Molecular Biology
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Synthesis and biological evaluation of NAS-21 and NAS-91 analogues as potential inhibitors of the mycobacterial FAS-II dehydratase enzyme Rv0636
More LessThe identification of potential new anti-tubercular chemotherapeutics is paramount due to the recent emergence of extensively drug-resistant strains of Mycobacterium tuberculosis (XDR-TB). Libraries of NAS-21 and NAS-91 analogues were synthesized and evaluated for their whole-cell activity against Mycobacterium bovis BCG. NAS-21 analogues 1 and 2 demonstrated enhanced whole-cell activity in comparison to the parental compound, and an M. bovis BCG strain overexpressing the dehydratase enzyme Rv0636 was resistant to these analogues. NAS-91 analogues with ortho-modifications gave enhanced whole-cell activity. However, extension with biphenyl modifications compromised the whole-cell activities of both NAS-21 and NAS-91 analogues. Interestingly, both libraries demonstrated in vitro activity against fatty acid synthase II (FAS-II) but not FAS-I in cell-free extracts. In in vitro assays of FAS-II inhibition, NAS-21 analogues 4 and 5 had IC50 values of 28 and 19 μg ml−1, respectively, for the control M. bovis strain, and the M. bovis BCG strain overexpressing Rv0636 showed a marked increase in resistance. In contrast, NAS-91 analogues demonstrated moderate in vitro activity, although increased resistance was again observed in FAS-II activity assays with the Rv0636-overexpressing strain. Fatty acid methyl ester (FAME) and mycolic acid methyl ester (MAME) analysis of M. bovis BCG and the Rv0636-overexpressing strain revealed that the effect of the drug was relieved in the overexpressing strain, further implicating and potentially identifying Rv0636 as the target for these known FabZ dehydratase inhibitors. This study has identified candidates for further development as drug therapeutics against the mycobacterial FAS-II dehydratase enzyme.
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The proper ratio of GrpE to DnaK is important for protein quality control by the DnaK–DnaJ–GrpE chaperone system and for cell division
More LessA balance of the intracellular concentrations of molecular chaperones in response to environmental conditions is of considerable importance for cellular homeostasis. Here, the physiological consequences of overexpression of GrpE in wild-type Escherichia coli MC4100 were examined. Overexpression of GrpE resulted in defects in cell division and growth, but overexpression of GrpE-G122D, which carries the G122D point mutation resulting in impaired interaction with DnaK, did not; this indicated that the effect of GrpE overexpression could be related to the DnaK chaperone function. Phase-contrast and fluorescence micrographs suggested that the N-terminal GFP-fused GrpE was colocalized with DnaK on the surface of inclusion bodies. An in vitro luciferase-refolding activity assay using purified DnaK, DnaJ and GrpE proteins demonstrated that high concentrations of GrpE significantly inhibited DnaK-mediated refolding. Furthermore, cell-free extracts from wild-type cells and GrpE-G122D-overexpressing cells significantly enhanced the refolding of luciferase. In the GrpE-overexpressing cells, abnormal localization of the cell-division protein FtsZ was observed by indirect immunofluorescence microscopy. In conclusion, the overexpression of GrpE caused a defect in the functionality of the DnaK chaperone system; this would result in filamentous morphology via abnormalities in the cell-division machinery.
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Deletion of the msdS/AfmsdC gene induces abnormal polarity and septation in Aspergillus fumigatus
More Lessα-Mannosidases play an important role in the processing of mannose-containing glycans in eukaryotes. A deficiency in α-mannosidase is lethal in humans and cattle. In contrast to mammals, Saccharomyces cerevisiae does not require the endoplasmic reticulum α-mannosidase gene for growth. However, little is known of the consequence of loss of function of class I α-mannosidases in filamentous fungi. In this study, the msdS/AfmsdC gene was identified to encode 1,2-α-mannosidase MsdS in Aspergillus fumigatus. Soluble MsdS expressed in Escherichia coli was characterized as a typical class I α-mannosidase. The msdS gene was deleted by replacement of the msdS gene with a pyrG gene. Although the mutant showed a defect in N-glycan processing, as well as a reduction of cell wall components and a reduced ability of conidiation, it appeared that the rate of hyphal growth was not affected. Morphology analysis revealed abnormal polarity and septation at the stages of germination, hyphal growth and conidiation. Although the mechanism by which the N-glycan processing affects polarity and septation is unclear, our results show that msdS is involved in polarity and septation in A. fumigatus.
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Colutellin A, an immunosuppressive peptide from Colletotrichum dematium
Colletotrichum dematium is an endophytic fungus recovered from a Pteromischum sp. growing in a tropical forest in Costa Rica. This fungus makes a novel peptide antimycotic, colutellin A, with a MIC of 3.6 μg ml−1 (48 h) against Botrytis cinerea and Sclerotinia sclerotiorum. Collutellin A has a mass of 1127.7 Da and contains residues of Ile, Val, Ser, N-methyl-Val and β-aminoisobutryic acid in nominal molar ratios of 3 : 2 : 1 : 1 : 1, respectively. Independent lines of evidence suggest that the peptide is cyclic and sequences of Val-Ile-Ser-Ile and Ile-Pro-Val have been deduced by MS/MS as well as Edman degradation methods. Colutellin A inhibited CD4+ T-cell activation of interleukin 2 (IL-2) production with an IC50 of 167.3±0.38 nM, whereas cyclosporin A in the same test yielded a value of 61.8 nM. Inhibition of IL-2 production by collutellin A at such a low concentration indicates the potential immunosuppressive activity of this compound. In repeated experiments, cyclosporin A at or above 8 μg ml−1 exhibited high levels of cytotoxicity on human peripheral blood mononuclear cells, whereas collutellin A or DMSO (carrier) alone, after 24 and 48 h of culture, exhibited no toxicity. Because of these properties collutellin A has potential as a novel immunosuppressive drug.
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Sulfur oxidation of Paracoccus pantotrophus: the sulfur-binding protein SoxYZ is the target of the periplasmic thiol–disulfide oxidoreductase SoxS
More LessThe periplasmic thiol–disulfide oxidoreductase SoxS is essential for chemotrophic growth of Paracoccus pantotrophus with thiosulfate. To trap its periplasmic partner, the cysteine residues of the CysXaaXaaCys motif of SoxS (11 kDa) were changed to alanine by site-directed mutagenesis. The disrupted soxS gene of the homogenote mutant G ΩS was complemented with plasmids carrying the mutated soxS[C13A] or soxS[C16A] gene. Strain G ΩS(pRD179.6[C16A]S) displayed a marginal thiosulfate-oxidizing activity, suggesting that Cys13S binds the target protein. Evidence is presented that SoxS specifically binds SoxY. (i) Immunoblot analysis using non-reducing SDS gel electrophoresis and anti-SoxS and anti-SoxYZ antibodies identified the respective antigens of strain G ΩS(pRD179.6[C16A]S) at the 25 kDa position, suggesting an adduct of about 14 kDa, close to the value expected for SoxY migration. (ii) A mutant unable to produce SoxYZ, such as strain G ΩX(pRD187.7[C16A]S), did not form a SoxS(C16A) adduct, while addition of homogeneous SoxYZ resulted in the 25 kDa adduct. (iii) The SoxY and SoxZ subunits were distinguished by site-directed mutagenesis of the cysteine residue in SoxZ. SoxYZ(C53S) formed the 25 kDa adduct with SoxS(C16A). These results demonstrate that the target of SoxS is the sulfur-binding protein SoxY of the SoxYZ complex. As SoxYZ is reversibly inactivated, SoxS may activate SoxYZ as a crucial function for chemotrophy of P. pantotrophus.
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The Bacillus subtilis ABC transporter EcsAB influences intramembrane proteolysis through RasP
More LessThe Bacillus subtilis σ W regulon is induced by different stresses that most probably affect integrity of the cell envelope. The activity of the extracytoplasmic function (ECF) sigma factor σ W is modulated by the transmembrane anti-sigma factor RsiW, which undergoes stress-induced degradation in a process known as regulated intramembrane proteolysis, finally resulting in the release of σ W and the transcription of σ W-controlled genes. Mutations in the ecsA gene, which encodes an ATP binding cassette (ABC) of an ABC transporter of unknown function, block site-2 proteolysis of RsiW by the intramembrane cleaving protease RasP (YluC). In addition, degradation of the cell division protein FtsL, which represents a second RasP substrate, is blocked in an ecsA-negative strain. The defect in σ W induction of an ecsA-knockout strain could be partly suppressed by overproducing RasP. A B. subtilis rasP-knockout strain displayed the same pleiotropic phenotype as an ecsA knockout, namely defects in processing α-amylase, in competence development, and in formation of multicellular structures known as biofilms.
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Point mutations within the streptococcal regulator of virulence (Srv) alter protein–DNA interactions and Srv function
More LessGroup A Streptococcus (GAS) possesses a complex regulatory system enabling the organism to colonize a range of physiologically distinct host sites. Within this network of regulators is the streptococcal regulator of virulence (Srv). Srv is a member of the CRP/FNR family of transcriptional regulators and is most similar to pleiotropic regulatory factor A (PrfA), a positive regulator of virulence in Listeria monocytogenes. Members of this family possess a characteristic C-terminal helix–turn–helix motif (HTH) that facilitates binding to DNA targets. Genome scanning identified four targets in GAS that were similar to the consensus DNA target recognized by PrfA. Furthermore, previous amino acid sequence alignments identified conserved residues within the Srv HTH which are necessary for function in PrfA and CRP. Here we investigated the ability of Srv to interact with DNA and evaluated the role of the HTH in this interaction. Purified recombinant Srv (rSrv) was found to co-purify with an untagged form of Srv. Glutaraldehyde cross-linking and gel-filtration chromatography indicated that this co-purification is likely due to the ability of Srv to oligomerize. Electrophoretic mobility shift assays (EMSAs) demonstrated that rSrv retarded the mobility of DNA targets and a supershift analysis confirmed the observation was rSrv-dependent. Competition EMSA indicated that rSrv had a higher relative affinity for the DNA targets studied than non-specific DNA. Site-directed mutagenesis of residues predicted to be in or near the HTH resulted in a decrease or abrogation of DNA binding. Complementation of MGAS5005Δsrv with one of these site-directed mutants failed to restore wild-type SpeB activity. Taken together, these data suggest that the Srv HTH is necessary for DNA binding and Srv function.
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Autoregulated expression of the gene coding for the leucine-responsive protein, Lrp, a global regulator in Salmonella enterica serovar Typhimurium
More LessIn this study, the lrp gene encoding the leucine-responsive regulatory protein (Lrp) in Salmonella enterica serovar Typhimurium was found to be negatively autoregulated. Its transcription start site was determined by primer extension analysis, showing that the lrp promoter is located at a different site to that inferred previously from the S. Typhimurium genome sequence. Chromosomal DNA fragments that include the promoter region were bound by purified Lrp protein in vitro, producing up to four distinct protein–DNA complexes. DNase I footprinting identified regions that were protected by the protein in vitro as well as bases that became hypersensitive to DNase I treatment following Lrp binding. A clear pattern of periodic hypersensitivity was detected between positions −130 and +15 that was consistent with wrapping of the DNA around Lrp in a nucleoprotein complex that includes the putative promoter region. Lrp–DNA interaction in this region was fully consistent with the observed repression of lrp transcription by this protein. Leucine was found to modulate Lrp-mediated autorepression by remodelling the Lrp–DNA nucleoprotein complex.
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Cellulose modulates biofilm formation by counteracting curli-mediated colonization of solid surfaces in Escherichia coli
In enterobacteria, the CsgD protein activates production of two extracellular structures: thin aggregative fimbriae (curli) and cellulose. While curli fibres promote biofilm formation and cell aggregation, the evidence for a direct role of cellulose as an additional determinant for biofilm formation is not as straightforward. The MG1655 laboratory strain of Escherichia coli only produces limited amounts of curli and cellulose; however, ectopic csgD expression results in strong stimulation of curli and cellulose production. We show that, in a csgD-overexpressing derivative of MG1655, cellulose production negatively affects curli-mediated surface adhesion and cell aggregation, thus acting as a negative determinant for biofilm formation. Consistent with this observation, deletion of the bcsA gene, necessary for cellulose production, resulted in a significant increase in curli-dependent adhesion. We found that cellulose production increased tolerance to desiccation, suggesting that the function of cellulose might be related to resistance to environmental stresses rather than to biofilm formation. Production of the curli/cellulose network in enterobacteria typically takes place at low growth temperature (<32 °C), but not at 37 °C. We show that CsgD overexpression can overcome temperature-dependent control of the curli-encoding csgBA operon, but not of the cellulose-related adrA gene, suggesting very tight temperature control of cellulose production in E. coli MG1655.
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The pst operon of enteropathogenic Escherichia coli enhances bacterial adherence to epithelial cells
More LessEnteropathogenic Escherichia coli (EPEC) adheres in vivo and in vitro to epithelial cells. Two main adhesins, the bundle-forming pilus and intimin, encoded by the bfp operon and eae, respectively, are responsible for the localized and the intimate adherence phenotypes. Deletion of the pst operon of EPEC abolishes the transport of inorganic phosphate through the phosphate-specific transport system and causes the constitutive expression of the PHO regulon genes. In the absence of pst there is a decrease in the expression of the main EPEC adhesins and a reduction in bacterial adherence to epithelial cells in vitro. This effect is not related to PHO constitutivity, because a Δpst phoB double mutant that is defective in the transcription of the PHO genes also displayed low levels of adherence and expression of adhesins. Likewise, a PHO-constitutive phoR mutation did not affect bacterial adherence. The expression of the per operon, which encodes the bfp and ler regulators PerA and PerC, is also negatively affected by the pst deletion. Overall, the data presented here demonstrate that the pst operon of EPEC plays a positive role in the bacterial adherence mechanism by increasing the expression of perA and perC and consequently the transcription of bfp and eae.
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- Biodiversity And Evolution
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The mosaic structure of the mcyABC operon in Microcystis
More LessAn extensive study of the mcyABC genes and regions flanking the mcy gene cluster was performed in naturally occurring Microcystis strains. Lack of methylation in strains producing only desmethyl7-microcystin was found to be associated with point mutations in substrate-binding sequence motifs of the N-methyltransferase (NMT) domain in McyA. Multiple recombination events giving rise to ‘phylogenetic mosaics’ were detected within the NMT-domain-encoding mcyA sequences and the adenylation (A) domain sequences of mcyB and mcyC. Recombination leading to exchanges between the mcyB and mcyC regions encoding A domains in modules McyB1 and McyC was also detected. A previously reported replacement of the A domain in McyB1 was found to involve the region between the conserved motifs A3 and A8/A9. In all microcystin-producing strains the mcy gene cluster was flanked by the genes uma1 and dnaN. Clear indications of recombination, an insertion element and footprints of IS elements were found in the dnaN–mcyJ intergenic region. Among the non-microcystin producers, uma1 and dnaN were linked in some, but not all strains. Most non-producing strains lacked all mcy genes, while one strain possessed a partially deleted mcy operon. Our results show that frequent horizontal gene transfer events in addition to point mutations and insertions/deletions contribute to variation in the mcy gene cluster.
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Staphylococcus aureus host specificity: comparative genomics of human versus animal isolates by multi-strain microarray
More LessStaphylococcus aureus is a commensal and pathogen of several mammalian species, particularly humans and cattle. We aimed to (i) identify S. aureus genes associated with host specificity, (ii) determine the relatedness of human and animal isolates, and (iii) identify whether human and animal isolates typically exchanged mobile genetic elements encoding virulence and resistance genes. Using a well-validated seven-strain S. aureus microarray, we compared 56 UK S. aureus isolates that caused infection in cows, horses, goats, sheep and a camel with 161 human S. aureus isolates from healthy carriers and community acquired infections in the UK. We had previously shown that human isolates are clustered into ten dominant and a few minor lineages, each with unique combinations of surface proteins predicted to bind to human proteins. We found that the animal-associated S. aureus clustered into ten lineages, with 61 % assigned to four lineages, ST151, ST771, ST130 and ST873, that were unique to animals. The majority of bovine mastitis was caused by isolates of lineage ST151, ST771 and ST97, but a few human lineages also caused mastitis. S. aureus isolated from horses were more likely to cluster into human-associated lineages, with 54 % of horse-associated S. aureus assigned to the human clusters CC1, CC8 and CC22; along with the presence of some multi-drug resistant strains, this suggests a human origin. This is the most comprehensive genetic comparison of human versus animal S. aureus isolates conducted, and because we used a whole-genome approach we could estimate the key genes with the greatest variability that are associated with host specificity. Several genes conserved in all human isolates were variable or missing in one or more animal lineages, including the well-characterized lineage specific genes fnbA, fnbB and coa. Interestingly, genes carried on mobile genetic elements (MGEs) such as chp, scn and sak were less common in animal S. aureus isolates, and bap was not found. There was a lot of MGE variation within lineages, and some evidence that exchange of MGEs such as bacteriophage and pathogenicity islands between animal and human lineages is feasible, but there was less evidence of antibiotic resistance gene transfer on the staphylococcal cassette chromosomes (SCC) or plasmids. Surprisingly, animal lineages are closely related to human lineages and only a handful of genes or gene combinations may be responsible for host specificity.
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- Environmental Microbiology
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The new group of non-pathogenic plant-associated nitrogen-fixing Burkholderia spp. shares a conserved quorum-sensing system, which is tightly regulated by the RsaL repressor
More LessA novel group of nitrogen-fixing plant-associated Burkholderia species has emerged in the last few years. The purpose of this investigation was to determine if these species possess an N-acylhomoserine lactone (AHL) quorum-sensing (QS) cell–cell signalling system, and whether it is important for nitrogen fixation and other phenotypic features in Burkholderia kururiensis. It was determined that B. kururiensis, and other members of this Burkholderia species cluster, contain at least one highly conserved system, designated BraI/R, which produces and responds to N-dodecanoyl-3-oxo-homoserine lactone (C12-3-oxo-AHL). The BraI/R AHL QS is not involved in the regulation of nitrogen fixation or in several other important phenotypes, indicating that it may not be a global regulatory system. The BraI/R system is similar to LasI/R of Pseudomonas aeruginosa and, as with lasI/R, there is a repressor gene, rsaL, between the braI/R genes. B. kururiensis normally synthesizes very low levels of C12-3-oxo-AHL, but the situation dramatically changes when RsaL is missing since an rsaL mutant displays a marked increase in AHL production. This unique stringent regulation indicates that RsaL could be an on/off switch for AHL QS in B. kururiensis and the ability to produce very high levels of AHL also questions the role of this molecule in the novel group of Burkholderia. The presence of a well-conserved and distinct AHL QS system among all the diazotrophic Burkholderia is a further indication that they are closely related, and that this system might play an important and conserved role in the lifestyle of this novel group of bacterial species.
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Regulation of autoinducer 2 production and luxS expression in a pathogenic Edwardsiella tarda strain
More LessEdwardsiella tarda is a bacterial pathogen that can infect both humans and animals. TX1, an Ed. tarda strain isolated from diseased fish, was found to produce autoinducer 2 (AI-2)-like activity that was growth phase dependent and modulated by growth conditions. The gene coding for the AI-2 synthase was cloned from TX1 and designated luxSEt . LuxSEt was able to complement the AI-2 mutant phenotype of Escherichia coli strain DH5α. Expression of luxSEt correlated with AI-2 activity and was increased by glucose and decreased by elevated temperature. The effect of glucose was shown to be mediated through the cAMP-CRP complex, which repressed luxSEt expression. Overexpression of luxSEt enhanced AI-2 activity in TX1, whereas disruption of luxSEt expression by antisense RNA interference (i) reduced the level of AI-2 activity, (ii) impaired bacterial growth under various conditions, (iii) weakened the expression of genes associated with the type III secretion system and biofilm formation, and (iv) attenuated bacterial virulence. Addition of exogenous AI-2 was able to complement the deficiencies in the expression of TTSS genes and biofilm production but failed to rescue the growth defects. Our results (i) demonstrated that the AI-2 activity in TX1 is controlled at least in part at the level of luxSEt expression, which in turn is regulated by growth conditions, and that the temporal expression of luxSEt is essential for optimal bacterial infection and survival; and (ii) suggested the existence in Ed. tarda of a LuxS/AI-2-mediated signal transduction pathway that regulates the production of virulence-associated elements.
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Genetic and functional characterization of the gene cluster directing the biosynthesis of putisolvin I and II in Pseudomonas putida strain PCL1445
More LessPseudomonas putida PCL1445 secretes two cyclic lipopeptides, putisolvin I and putisolvin II, which possess a surface-tension-reducing ability, and are able to inhibit biofilm formation and to break down biofilms of Pseudomonas species including Pseudomonas aeruginosa. The putisolvin synthetase gene cluster (pso) and its surrounding region were isolated, sequenced and characterized. Three genes, termed psoA, psoB and psoC, were identified and shown to be involved in putisolvin biosynthesis. The gene products encode the 12 modules responsible for the binding of the 12 amino acids of the putisolvin peptide moiety. Sequence data indicate that the adenylation domain of the 11th module prioritizes the recognition of Val instead of Leu or Ile and consequently favours putisolvin I production over putisolvin II. Detailed analysis of the thiolation domains suggests that the first nine modules recognize the d form of the amino acid residues while the two following modules recognize the l form and the last module the l or d form, indifferently. The psoR gene, which is located upstream of psoA, shows high similarity to luxR-type regulatory genes and is required for the expression of the pso cluster. In addition, two genes, macA and macB, located downstream of psoC were identified and shown to be involved in putisolvin production or export.
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Diversity and spatial distribution of sediment ammonia-oxidizing crenarchaeota in response to estuarine and environmental gradients in the Changjiang Estuary and East China Sea
More LessAmmonia-oxidizing archaea (AOA) have recently been found to be potentially important in nitrogen cycling in a variety of environments, such as terrestrial soils, wastewater treatment reactors, marine waters and sediments, and especially in estuaries, where high input of anthropogenic nitrogen is often experienced. The sedimentary AOA diversity, community structure and spatial distribution in the Changjiang Estuary and the adjacent East China Sea were studied. Multivariate statistical analysis indicated that the archaeal amoA genotype communities could be clustered according to sampling transects, and the station located in an estuarine mixing zone harboured a distinct AOA community. The distribution of AOA communities correlated significantly with the gradients of surface-water salinity and sediment sorting coefficient. The spatial distribution of putative soil-related AOA in certain sampling stations indicated a strong impact of the Changjiang freshwater discharge on the marine benthic microbial ecosystem. Besides freshwater, nutrients, organic matter and suspended particles, the Changjiang Diluted Water might also contribute to the transport of terrestrial archaea into the seawater and sediments along its flow path.
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- Genes And Genomes
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An extra-cytoplasmic function sigma factor and anti-sigma factor control carotenoid biosynthesis in Azospirillum brasilense
Strains Sp7 and Cd of Azospirillum brasilense, a plant growth-promoting rhizobacterium, differ in synthesis of carotenoids. While colonies of strain Sp7 have a white–cream colour on plates, colonies of strain Cd are orange–pink coloured because of the synthesis of carotenoids. Screening of a mini-Tn5 mutant library of A. brasilense Sp7 revealed two orange–pink-coloured mutants that produced carotenoids. Cloning and sequencing of the Tn5 flanking region in both the carotenoid-producing mutants of Sp7 revealed insertion of Tn5 in an ORF encoding anti-σ factor, a ChrR-like protein. The upstream region of the Tn5-mutated ORF contained another ORF that encoded an extra-cytoplasmic function (ECF)-class σ factor (σ E, RpoE). When the nucleotide sequences of the corresponding ORFs from the carotenoid-producing strain Cd were analysed, the sequence of the Cd σ E was identical to that of the carotenoid non-producing strain Sp7, but the Cd anti-σ E ORF had a deletion that caused frame shifting and creation of a stop codon. This resulted in the premature termination of the protein, which was about 7 kDa smaller than the Sp7 anti-σ E. Cloning of Sp7 anti-σ E in a broad-host-range expression vector and expression in A. brasilense Cd and in the anti-σ E knockout mutant of A. brasilense Sp7 resulted in the inhibition of carotenoid synthesis. Similarly, cloning and overexpression of A. brasilense Sp7 σ E in A. brasilense Sp7 resulted in the production of carotenoids. These observations clearly indicate that carotenoid synthesis in A. brasilense is controlled by σ E with its cognate anti-σ E.
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- Pathogens And Pathogenicity
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A novel cell wall-anchored peptidoglycan hydrolase (autolysin), IspC, essential for Listeria monocytogenes virulence: genetic and proteomic analysis
Linru Wang and Min LinWe have recently concluded that a Listeria monocytogenes 86 kDa immunogenic surface protein, IspC, is a cell wall-anchored peptidoglycan hydrolase (autolysin), capable of degrading the cell wall peptidoglycan of the bacterium itself. To determine if this enzyme has any biological functions and/or plays a role in virulence, we in-frame-deleted the ispC gene from the L. monocytogenes chromosome. This ΔispC mutant exhibited complete abrogation of expression of IspC and displayed no defects in in vitro growth, colony and microscopic morphologies, or biochemical characteristics. Lack of IspC led to attenuated virulence in mice, evidenced by a significant reduction in bacterial counts in livers and brains and no mortality compared with the wild-type. Furthermore, the data from assays using various eukaryotic cells for adhesion, invasion, actin tail formation, plaque formation and intracellular growth indicated that the mutant was severely attenuated in virulence in a cell culture model in a cell type-dependent manner. The findings that (i) the mutant was impaired for adhesion to certain eukaryotic cells, and (ii) both purified IspC and its C-terminal cell wall-binding domain were capable of binding sheep choroid plexus (SCP) epithelial cells and Vero cells, supported the role of IspC as an adhesin in virulence. The ΔispC mutant exhibited a marked defect in adhesion to and invasion of SCP cells but not human brain microvascular endothelial cells (HBMEC), suggesting that IspC is necessary for crossing the blood–cerebrospinal fluid barrier. Proteomic and immunological analysis showed a reduced surface expression of some known or putative virulence factors (e.g. ActA, InlC2 and a flagellin homologue, FlaA) due to IspC deficiency. Altogether, this study demonstrates that IspC, expressed as a minor autolysin in vitro, is not important for cell division or separation but is essential for full virulence of L. monocytogenes in vivo.
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Interaction of Salmonella enterica serovar Typhi with cultured epithelial cells: roles of surface structures in adhesion and invasion
More LessIn this study we investigate the ability of Salmonella enterica serovar Typhi (S. Typhi) surface structures to influence invasion and adhesion in epithelial cell assay systems. In general, S. Typhi was found to be less adherent, invasive and cytotoxic than S. enterica serovar Typhimurium (S. Typhimurium). Culture conditions had little effect on adhesion of S. Typhi to cultured cells but had a marked influence on invasion. In contrast, bacterial growth conditions did not influence S. Typhi apical invasion of polarized cells. The levels of S. Typhi, but not S. Typhimurium, invasion were increased by application of bacteria to the basolateral surface of polarized cells. Expression of virulence (Vi) capsule by S. Typhi resulted in a modest reduction in adhesion, but profoundly reduced levels of invasion of non-polarized cells. However, Vi capsule expression had no affect on invasion of the apical or basolateral surfaces of polarized cells. Mutation of the staA, tcfA or pilS genes did not affect invasion or adhesion in either the presence or the absence of Vi capsule.
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Characterization of a mouse model of plague after aerosolization of Yersinia pestis CO92
Yersinia pestis is a Gram-negative bacterium, and the causative agent of bubonic plague and pneumonic plague. Because of its potential use as a biological warfare weapon, the plague bacterium has been placed on the list of category A select agents. The dynamics of pneumonic infection following aerosolization of the highly virulent Y. pestis CO92 strain have been poorly studied; therefore, the purpose of this study was to determine the LD50 dose, bacterial dissemination, cytokine/chemokine production and tissue damage in Swiss-Webster mice over a 72 h course of infection. We exposed mice in a whole-body Madison chamber to various doses of Y. pestis CO92 aerosolized by a Collison nebulizer, and determined that the LD50 presented dose (Dp) of the bacterium in the lungs was 2.1×103 c.f.u. In a subsequent study, we infected mice at a Dp of 1.3×104 c.f.u., and harvested organs and blood at 1, 24, 48 and 72 h post-infection. Histopathological examination, in addition to measurement of bacterial dissemination and cytokine/chemokine analysis, indicated progressive tissue injury, and an increased number of animals succumbing to infection over the course of the experiment. Using these data, we were able to characterize the mouse plague model following aerosolization of Y. pestis CO92.
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Functional genomics of PycR, a LysR family transcriptional regulator essential for maintenance of Pseudomonas aeruginosa in the rat lung
The human opportunistic pathogen Pseudomonas aeruginosa is the major cause of morbidity and mortality of cystic fibrosis patients and is responsible for a variety of infections in compromised hosts. Using PCR-based signature-tagged mutagenesis, we identified a P. aeruginosa STM5437 mutant with an insertion into the PA5437 gene (called pycR for putative pyruvate carboxylase regulator). PycR inactivation results in 100 000-fold attenuation of virulence in the rat lung in vivo. PycR has the signature of a transcriptional regulator with a predicted helix–turn–helix motif binding to a typical LysR DNA binding site in the PA5436 (pycA)–PA5437 (pycR) intercistronic region. Two pyruvate carboxylase subunits (pycA and pycB) are divergently transcribed upstream of pycR. Transcriptional start sites of pycR and pycA are located at −127 and −88 bp upstream of their initiation codons with Shine–Dalgarno and putative promoter sequences containing −10 and −35 sequences. The DNA binding of PycR was confirmed by DNA mobility shift assay. Genome-wide transcriptional profiling and quantitative real-time PCR (qRT-PCR) indicated that the genes differentially regulated by PycR include two pyruvate carboxylase genes and genes necessary for lipid metabolism, lipolytic activity, anaerobic respiration and biofilm formation. PycR is a regulator with pleiotropic effects on virulence factors, such as lipase and esterase expression and biofilm formation, which are important for maintenance of P. aeruginosa in chronic lung infection.
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ClpXP proteases positively regulate alginate overexpression and mucoid conversion in Pseudomonas aeruginosa
More LessOverproduction of the exopolysaccharide alginate and conversion to a mucoid phenotype in Pseudomonas aeruginosa are markers for the onset of chronic lung infection in cystic fibrosis (CF). Alginate production is regulated by the extracytoplasmic function (ECF) σ factor AlgU/T and the cognate anti-σ factor MucA. Many clinical mucoid isolates carry loss-of-function mutations in mucA. These mutations, including the most common mucA22 allele, cause C-terminal truncations in MucA, indicating that an inability to regulate AlgU activity by MucA is associated with conversion to the mucoid phenotype. Here we report that a mutation in a stable mucoid strain derived from the parental strain PAO1, designated PAO581, that does not contain the mucA22 allele, was due to a single-base deletion in mucA (ΔT180), generating another type of C-terminal truncation. A global mariner transposon screen in PAO581 for non-mucoid isolates led to the identification of three regulators of alginate production, clpP (PA1801), clpX (PA1802), and a clpP paralogue (PA3326, designated clpP2). The PAO581 null mutants of clpP, clpX and clpP2 showed decreased AlgU transcriptional activity and an accumulation of haemagglutinin (HA)-tagged N-terminal MucA protein with an apparent molecular mass of 15 kDa. The clpP and clpX mutants of a CF mucoid isolate revert to the non-mucoid phenotype. The ClpXP and ClpP2 proteins appear to be part of a proteolytic network that degrades the cytoplasmic portion of truncated MucA proteins to release the sequestered AlgU, which drives alginate biosynthesis.
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Induction of innate immunity by lipid A mimetics increases survival from pneumonic plague
This study analysed the effect of priming the innate immune system using synthetic lipid A mimetics in a Yersinia pestis murine pulmonary infection model. Two aminoalkyl glucosaminide 4-phosphate (AGP) Toll-like receptor 4 (TLR4) ligands, delivered intranasally, extended time to death or protected against a lethal Y. pestis CO92 challenge. The level of protection was dependent upon the challenge dose of Y. pestis and the timing of AGP therapy. Protection correlated with cytokine induction and a decreased bacterial burden in lung tissue. AGP protection was TLR4-dependent and was not evidenced in transgenic TLR4-deficient mice. AGP therapy augmented with subtherapeutic doses of gentamicin produced dramatically enhanced survival. Combined, these results indicated that AGPs may be useful in protection of immunologically naive individuals against plague and potentially other infectious agents, and that AGP therapy may be used synergistically with other therapies.
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Characterization of the Francisella tularensis subsp. novicida type IV pilus
More LessFrancisella tularensis causes the disease tularaemia. Type IV pili (Tfp) genes are present in the genomes of all F. tularensis subspecies. We show that the wild-type F. tularensis subsp. novicida expresses pilus fibres on its surface, and mutations in the Tfp genes pilF and pilT disrupt pilus biogenesis. Mutations in other Tfp genes (pilQ and pilG) do not eliminate pilus expression. A mutation in pilE4 eliminates pilus expression, whereas mutations in the other pilin subunits pilE1–3 and pilE5 do not, suggesting that pilE4 is the major pilus structural subunit. The virulence regulator MglA is required for pilus expression, and it regulates the transcription of a putative Tfp glycosylation gene (FTN0431). However, MglA does not regulate transcription of pilF, pilT or pilE4, and a strain lacking FTN0431 still expresses pili; thus, it is unclear how MglA regulates pilus expression. Only pilF was also required for protein secretion, while pilE4 and pilT were not, indicating that there is very little overlap of the protein secretion/Tfp functions of the pil genes. The protein secretion component pilE1 was more important for in vitro intramacrophage growth and mouse virulence than the Tfp component pilE4. Our results provide the first genetic characterization of the novel Tfp system of F. tularensis.
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- Physiology
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Diazotrophy under continuous light in a marine unicellular diazotrophic cyanobacterium, Gloeothece sp. 68DGA
More LessNitrogenase is extremely sensitive to molecular oxygen (O2), and unicellular diazotrophic cyanobacteria separate nitrogen (N2)-fixation and photosynthesis to protect nitrogenase from O2 produced by photosynthesis. When grown under 12 h light/12 h dark cycles (LD), the marine unicellular diazotrophic cyanobacterium Gloeothece sp. 68DGA expressed the nitrogenase protein and its activity (acetylene reduction activity) only during the dark phase. However, this strain was able to grow diazotrophically under continuous light (CL). To determine whether nitrogenase synthesis and N2-fixation are temporally separated from photosynthesis in the Gloeothece cells that have fully acclimated to CL, the proportion of cells containing nitrogenase (the Fe-protein of nitrogenase) in the culture was measured using an immunocytochemical technique. Cells were grown in a continuous-culture device to maintain constant cell density. Under LD, the cells showed diurnal oscillation of nitrogenase activity, photosynthesis, respiration and the expression and the abundance of the Fe-protein. The oscillation was gradually reduced after the transfer of the cells to CL, and was lost after 23–25 days of cultivation under CL. In CL-acclimated cultures, the Fe-protein was always detected in about 94 % of the cells, although the nitrogenase activity was about one-third of the maximum activity in LD-acclimated cultures. These results suggest that synthesis of nitrogenase proceeds without diurnal oscillation in the CL-acclimated cells of Gloeothece sp. 68DGA. As the respiration rate in CL-acclimated culture was as high as the maximum rate observed in LD-acclimated culture, O2-uptake mechanism(s) may have been upregulated to maintain low intracellular pO2.
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Oral bacteria in biofilms exhibit slow reactivation from nutrient deprivation
More LessThe ability of oral bacteria to enter a non-growing state is believed to be an important mechanism for survival in the starved micro-environments of the oral cavity. In this study, we examined the reactivation of nutrient-deprived cells of two oral bacteria in biofilms, Streptococcus anginosus and Lactobacillus salivarius. Non-growing cells were generated by incubation in 10 mM potassium phosphate buffer for 24 h and the results were compared to those of planktonic cultures. When both types of cells were shifted from a rich, peptone–yeast extract–glucose (PYG) medium to buffer for 24 h, dehydrogenase and esterase activity measured by the fluorescent dyes 5-cyano-2,3-ditolyl-tetrazolium chloride (CTC) and fluorescein diacetate (FDA), respectively, was absent in both species. However, the membranes of the vast majority of nutrient-deprived cells remained intact as assessed by LIVE/DEAD staining. Metabolic reactivation of the nutrient-deprived biofilm cells was not observed for at least 48 h following addition of fresh PYG medium, whereas the non-growing planktonic cultures of the same two strains were in rapid growth in less than 2 h. At 72 h, the S. anginosus biofilm cells had recovered 78 % of the dehydrogenase activity and 61 % of the esterase activity and the biomass mm−2 had increased by 30–35 %. With L. salivarius at 72 h, the biofilms had recovered 56 % and 75 % of dehydrogenase and esterase activity, respectively. Reactivation of both species in biofilms was enhanced by removal of glucose from PYG, and S. anginosus cells were particularly responsive to yeast extract (YE) medium. The data suggest that the low reactivity of non-growing biofilm cells to the introduction of fresh nutrients may be a survival strategy employed by micro-organisms in the oral cavity.
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The role of FIS protein in the physiological control of the expression of the Escherichia coli meta-hpa operon
More LessExpression from the Escherichia coli W meta-hpa operon promoter (Pg) is under a strict catabolic repression control mediated by the cAMP-catabolite repression protein (CRP) complex in a glucose-containing medium. The Pg promoter is also activated by the integration host factor (IHF) and repressed by the specific transcriptional regulator HpaR when 4-hydroxyphenylacetate (4HPA) is not present in the medium. Expression from the hpa promoter is also repressed in undefined rich medium such as LB, but the molecular basis of this mechanism is not understood. We present in vitro and in vivo studies to demonstrate the involvement of FIS protein in this catabolic repression. DNase I footprinting experiments show that FIS binds to multiple sites within the Pg promoter. FIS-site I overlaps the CRP-binding site. By using an electromobility shift assay, we demonstrated that FIS efficiently competes with CRP for binding to the Pg promoter, suggesting an antagonist/competitive mechanism. RT-PCR showed that the Pg repression effect is relieved in a FIS deleted strain. The repression role of FIS at Pg was further demonstrated by in vitro transcription assays. These results suggest that FIS contributes to silencing the Pg promoter in the exponential phase of growth in an undefined rich medium when FIS is predominantly expressed.
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Inactivation of spkD, encoding a Ser/Thr kinase, affects the pool of the TCA cycle metabolites in Synechocystis sp. strain PCC 6803
More LessThe inactivation of sll0776 (spkD), a gene encoding a protein Ser/Thr kinase in Synechocystis PCC 6803, led to a pleiotropic phenotype of the SpkD null mutant. This mutant is impaired in its growth ability under low concentration of inorganic carbon (Ci), though its Ci-uptake system is not affected. Addition of glucose, phosphoglyceraldehyde or pyruvate does not allow the mutant to grow under low-Ci conditions. In contrast, this growth defect can be restored when the low-Ci culture medium is supplemented with metabolites of the TCA cycle. Growth of the mutant is also inhibited when ammonium is provided as nitrogen source, whatever the carbon regime of the cells, due to the high demand for 2-oxoglutarate, which is the carbon skeleton for ammonium assimilation. When mutant cells are cultured under standard growth conditions, the intracellular concentration of 2-oxoglutarate is 20 % lower than is observed in the wild-type strain. However, this decrease of 2-oxoglutarate level only slightly affects the phosphorylation state of PII, a protein that regulates nitrogen and carbon metabolism according to the intracellular levels of 2-oxoglutarate. Properties of the SpkD mutant suggest that the Ser/Thr kinase SpkD could be involved in adjusting the pool of the TCA cycle metabolites according to Ci supply in the culture medium.
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- Corrigendum
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