- Volume 157, Issue 4, 2011
Volume 157, Issue 4, 2011
- Review
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Major players on the microbial stage: why archaea are important
As microbiology undergoes a renaissance, fuelled in part by developments in new sequencing technologies, the massive diversity and abundance of microbes becomes yet more obvious. The Archaea have traditionally been perceived as a minor group of organisms forced to evolve into environmental niches not occupied by their more ‘successful’ and ‘vigorous’ counterparts, the bacteria. Here we outline some of the evidence gathered by an increasingly large and productive group of scientists that demonstrates not only that the Archaea contribute significantly to global nutrient cycling, but also that they compete successfully in ‘mainstream’ environments. Recent data suggest that the Archaea provide the major routes for ammonia oxidation in the environment. Archaea also have huge economic potential that to date has only been fully realized in the production of thermostable polymerases. Archaea have furnished us with key paradigms for understanding fundamentally conserved processes across all domains of life. In addition, they have provided numerous exemplars of novel biological mechanisms that provide us with a much broader view of the forms that life can take and the way in which micro-organisms can interact with other species. That this information has been garnered in a relatively short period of time, and appears to represent only a small proportion of what the Archaea have to offer, should provide further incentives to microbiologists to investigate the underlying biology of this fascinating domain.
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- Mini-Review
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Parallel evolution and local differentiation in quinolone resistance in Pseudomonas aeruginosa
Alex Wong and Rees KassenThe emergence and spread of antibiotic resistance in pathogens is a major impediment to the control of microbial disease. Here, we review mechanisms of quinolone resistance in Pseudomonas aeruginosa, an important nosocomial pathogen and a major cause of morbidity in cystic fibrosis (CF) patients. In this quantitative literature review, we find that mutations in DNA gyrase A, the primary target of quinolones in Gram-negative bacteria, are the most common resistance mutations identified in clinical samples of all origins, in keeping with previous observations. However, the identities of non-gyrase resistance mutations vary systematically between samples isolated from CF patients and those isolated from acute infections. CF-derived strains tend to harbour mutations in the efflux pump regulator nfxB, while non-CF strains tend to bear mutations in the efflux regulator mexR or in parC, which encodes one of two subunits of DNA topoisomerase IV. We suggest that differences in resistance mechanisms between CF and non-CF strains result either from local adaptation to different sites of infection or from differences in mutational processes between different environments. We further discuss the therapeutic implications of local differentiation in resistance mechanisms to a common antibiotic.
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- Cell And Molecular Biology Of Microbes
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Yeast response and tolerance to polyamine toxicity involving the drug : H+ antiporter Qdr3 and the transcription factors Yap1 and Gcn4
The yeast QDR3 gene encodes a plasma membrane drug : H+ antiporter of the DHA1 family that was described as conferring resistance against the drugs quinidine, cisplatin and bleomycin and the herbicide barban, similar to its close homologue QDR2. In this work, a new physiological role for Qdr3 in polyamine homeostasis is proposed. QDR3 is shown to confer resistance to the polyamines spermine and spermidine, but, unlike Qdr2, also a determinant of resistance to polyamines, Qdr3 has no apparent role in K+ homeostasis. QDR3 transcription is upregulated in yeast cells exposed to spermine or spermidine dependent on the transcription factors Gcn4, which controls amino acid homeostasis, and Yap1, the main regulator of oxidative stress response. Yap1 was found to be a major determinant of polyamine stress resistance in yeast and is accumulated in the nucleus of yeast cells exposed to spermidine-induced stress. QDR3 transcript levels were also found to increase under nitrogen or amino acid limitation; this regulation is also dependent on Gcn4. Consistent with the concept that Qdr3 plays a role in polyamine homeostasis, QDR3 expression was found to decrease the intracellular accumulation of [3H]spermidine, playing a role in the maintenance of the plasma membrane potential in spermidine-stressed cells.
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The soxRS response of Escherichia coli can be induced in the absence of oxidative stress and oxygen by modulation of NADPH content
More LessThe soxRS regulon protects Escherichia coli cells against superoxide and nitric oxide. Oxidation of the SoxR sensor, a [2Fe–2S]-containing transcriptional regulator, triggers the response, but the nature of the cellular signal sensed by SoxR is still a matter of debate. In vivo, the sensor is maintained in a reduced, inactive state by the activities of SoxR reductases, which employ NADPH as an electron donor. The hypothesis that NADPH levels affect deployment of the soxRS response was tested by transforming E. coli cells with genes encoding enzymes and proteins that lead to either build-up or depletion of the cellular NADPH pool. Introduction of NADP+-reducing enzymes, such as wheat non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase or E. coli malic enzyme, led to NADPH accumulation, inhibition of the soxRS regulon and enhanced sensitivity to the superoxide propagator methyl viologen (MV). Conversely, expression of pea ferredoxin (Fd), a redox shuttle that can oxidize NADPH via ferredoxin-NADP(H) reductase, resulted in execution of the soxRS response in the absence of oxidative stress, and in higher tolerance to MV. Processes that caused NADPH decline, including oxidative stress and Fd activity, correlated with an increase in total (NADP++NADPH) stocks. SoxS expression can be induced by Fd expression or by MV in anaerobiosis, under conditions in which NADPH is oxidized but no superoxide can be formed. The results indicate that activation of the soxRS regulon in E. coli cells exposed to superoxide-propagating compounds can be triggered by depletion of the NADPH stock rather than accumulation of superoxide itself. They also suggest that bacteria need to finely regulate homeostasis of the NADP(H) pool to enable proper deployment of this defensive response.
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A unique Coxiella burnetii lipoprotein involved in metal binding (LimB)
More LessCoxiella burnetii is the bacterial agent of Q fever in humans. Here, we describe a unique, ∼7.2 kDa, surface-exposed lipoprotein involved in metal binding which we have termed LimB. LimB was initially identified as a potential metal-binding protein on far-Western (FW) blots containing whole-cell lysate proteins when probed with nickel-coated horseradish peroxidase (Ni-HRP) and developed with a chemiluminescent HRP substrate. The corresponding identity of LimB as CBU1224a was established by matrix-assisted laser desorption ionization-tandem time-of-flight mass spectrometry. blast analyses with CBU1224a showed no significant similarity to sequences outside strains of C. burnetii. Additional in silico analyses revealed a putative 20 residue signal sequence with the carboxyl end demarcated by a potential lipobox (LSGC) whose Cys residue is predicted to serve as the N-terminal, lipidated Cys of mature LimB. The second residue of mature LimB is predicted to be Ala, an uncharged envelope localization residue. These features suggest that CBU1224a is synthesized as a prolipoprotein which is subsequently lipidated, secreted and anchored in the outer membrane. Mature LimB is predicted to contain 45 aa, of which there are 10 His and 5 Cys; both amino acids are frequently involved in binding transition metal cations. Recombinant LimB (rLimB) was generated and its Ni-HRP-binding activity demonstrated on FW blots. Ni-HRP binding by rLimB was inhibited by >95 % on FW blots done in the presence of EDTA, imidazole, Ni2+ or Zn2+, and roughly halved in the presence of Co2+ or Fe3+. The limB gene was maximally expressed at 3–7 days post-infection in Coxiella-infected Vero cells, coinciding with exponential phase growth. Two isoforms of LimB were detected on FW and Western blots, including a smaller (∼7.2 kDa) species that was the predominant form in small cell variants and a larger isoform (∼8.7 kDa) in large cell variants. LimB is Sarkosyl-insoluble, like many omps. The predicted surface location of LimB was verified by immunoelectron and immunofluorescence microscopy using anti-rLimB antibodies. Overall, the results suggest that LimB is a unique Coxiella lipoprotein that serves as a surface receptor for divalent metal cations and may play a role in acquiring at least one of these metals during intracellular growth.
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T-box-mediated control of the anabolic proline biosynthetic genes of Bacillus subtilis
More LessBacillus subtilis possesses interlinked routes for the synthesis of proline. The ProJ–ProA–ProH route is responsible for the production of proline as an osmoprotectant, and the ProB–ProA–ProI route provides proline for protein synthesis. We show here that the transcription of the anabolic proBA and proI genes is controlled in response to proline limitation via a T-box-mediated termination/antitermination regulatory mechanism, a tRNA-responsive riboswitch. Primer extension analysis revealed mRNA leader transcripts of 270 and 269 nt for the proBA and proI genes, respectively, both of which are synthesized from SigA-type promoters. These leader transcripts are predicted to fold into two mutually exclusive secondary mRNA structures, forming either a terminator or an antiterminator configuration. Northern blot analysis allowed the detection of both the leader and the full-length proBA and proI transcripts. Assessment of the level of the proBA transcripts revealed that the amount of the full-length mRNA species strongly increased in proline-starved cultures. Genetic studies with a proB–treA operon fusion reporter strain demonstrated that proBA transcription is sensitively tied to proline availability and is derepressed as soon as cellular starvation for proline sets in. Both the proBA and the proI leader sequences contain a CCU proline-specific specifier codon prone to interact with the corresponding uncharged proline-specific tRNA. By replacing the CCU proline specifier codon in the proBA T-box leader with UUC, a codon recognized by a Phe-specific tRNA, we were able to synthetically re-engineer the proline-specific control of proBA transcription to a control that was responsive to starvation for phenylalanine.
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An extracytoplasmic function sigma factor cotranscribed with its cognate anti-sigma factor confers tolerance to NaCl, ethanol and methylene blue in Azospirillum brasilense Sp7
More LessAzospirillum brasilense, a plant-growth-promoting rhizobacterium, is exposed to changes in its abiotic environment, including fluctuations in temperature, salinity, osmolarity, oxygen concentration and nutrient concentration, in the rhizosphere and in the soil. Since extra-cytoplasmic function (ECF) sigma factors play an important role in stress adaptation, we analysed the role of ECF sigma factor (also known as RpoE or σ E) in abiotic stress tolerance in A. brasilense. An in-frame rpoE deletion mutant of A. brasilense Sp7 was carotenoidless and slow-growing, and was sensitive to salt, ethanol and methylene blue stress. Expression of rpoE in the rpoE deletion mutant complemented the defects in growth, carotenoid biosynthesis and sensitivity to different stresses. Based on data from reverse transcriptase-PCR, a two-hybrid assay and a pull-down assay, we present evidence that rpoE is cotranscribed with chrR and the proteins synthesized from these two overlapping genes interact with each other. Identification of the transcription start site by 5′ rapid amplification of cDNA ends showed that the rpoE–chrR operon was transcribed by two promoters. The proximal promoter was less active than the distal promoter, whose consensus sequence was characteristic of RpoE-dependent promoters found in alphaproteobacteria. Whereas the proximal promoter was RpoE-independent and constitutively expressed, the distal promoter was RpoE-dependent and strongly induced in response to stationary phase and elevated levels of ethanol, salt, heat and methylene blue. This study shows the involvement of RpoE in controlling carotenoid synthesis as well as in tolerance to some abiotic stresses in A. brasilense, which might be critical in the adaptation, survival and proliferation of this rhizobacterium in the soil and rhizosphere under stressful conditions.
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Transcriptional repressor CopR acts by inhibiting RNA polymerase binding
More LessCopR is a transcriptional repressor encoded by the broad-host-range streptococcal plasmid pIP501, which also replicates in Bacillus subtilis. It acts in concert with the antisense RNA, RNAIII, to control pIP501 replication. CopR represses transcription of the essential repR mRNA about 10- to 20-fold. In previous work, DNA binding and dimerization constants were determined and the motifs responsible localized. The C terminus of CopR was shown to be required for stability. Furthermore, SELEX of the copR operator revealed that in vivo evolution was for maximal binding affinity. Here, we elucidate the repression mechanism of CopR. Competition assays showed that CopR–operator complexes are 18-fold less stable than RNA polymerase (RNAP)–pII complexes. DNase I footprinting revealed that the binding sites for CopR and RNAP overlap. Gel-shift assays demonstrated that CopR and B. subtilis RNAP cannot bind simultaneously, but compete for binding at promoter pII. Due to its higher intracellular concentration CopR inhibits RNAP binding. Additionally, KMnO4 footprinting experiments indicated that prevention of open complex formation at pII does not further contribute to the repression effect of CopR.
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Mapping of the interactions between partition proteins Delta and Omega of plasmid pSM19035 from Streptococcus pyogenes
More LessFormation of the segrosome, a nucleoprotein complex crucial for proper functioning of plasmid partition systems, involves interactions between specific partition proteins (ParA-like and ParB-like), ATP and specific DNA sequences (the centromeric sites). Although partition systems have been studied for many years, details of the segrosome formation are not yet clear. Organization of the pSM19035-encoded partition system is unique; in contrast with other known par systems, here, the δ and ω genes do not constitute an operon. Moreover, Omega [a ParB-like protein which has a Ribbon-Helix-Helix (RHH) structure] recognizes multiple centromeric sequences located in the promoters of δ, ω and copS (copy-number control gene). The ParA-like protein Delta is a Walker-type ATPase. In this work, we identify the interaction domains and requirements for dimerization and hetero-interactions of the Delta and Omega proteins of pSM19035 plasmid. The RHH structures are involved in Omega dimerization in vivo and its N-terminal unstructured part is indispensable for association with Delta, both in vivo and in vitro. Omega does not need to form dimers to interact with Delta. ATP binding is not required for Delta dimerization but is important for interaction with Omega in vivo. The in vitro interaction between Delta and Omega depends on ATP but does not require the presence of specific DNA segments (the centromere) recognized by Omega. The C-terminal part of the Delta protein (aa 198–284) is indispensable for interaction with Omega. Delta most probably interacts with Omega as a dimer since two amino acid substitutions in a conserved region between the A′ and B motifs abolish both the dimerization of Delta and its interaction with Omega.
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Dissection of the relative contribution of the Schizosaccharomyces pombe Ctr4 and Ctr5 proteins to the copper transport and cell surface delivery functions
More LessThe Ctr1 family of proteins mediates high-affinity copper (Cu) acquisition in eukaryotic organisms. In the fission yeast Schizosaccharomyces pombe, Cu uptake is carried out by a heteromeric complex formed by the Ctr4 and Ctr5 proteins. Unlike human and Saccharomyces cerevisiae Ctr1 proteins, Ctr4 and Ctr5 are unable to function independently in Cu acquisition. Instead, both proteins physically interact with each other to form a Ctr4–Ctr5 heteromeric complex, and are interdependent for secretion to the plasma membrane and Cu transport activity. In this study, we used S. cerevisiae mutants that are defective in high-affinity Cu uptake to dissect the relative contribution of Ctr4 and Ctr5 to the Cu transport function. Functional complementation and localization assays show that the conserved Met-X3-Met motif in transmembrane domain 2 of the Ctr5 protein is dispensable for the functionality of the Ctr4–Ctr5 complex, whereas the Met-X3-Met motif in the Ctr4 protein is essential for function and for localization of the hetero-complex to the plasma membrane. Moreover, Ctr4/Ctr5 chimeric proteins reveal unique properties found either in Ctr4 or in Ctr5, and are sufficient for Cu uptake on the cell surface of Sch. pombe cells. Functional chimeras contain the Ctr4 central and Ctr5 carboxyl-terminal domains (CTDs). We propose that the Ctr4 central domain mediates Cu transport in this hetero-complex, whereas the Ctr5 CTD functions in the regulation of trafficking of the Cu transport complex to the cell surface.
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Emw1p/YNL313cp is essential for maintenance of the cell wall in Saccharomyces cerevisiae
More LessThere are six essential genes in the Saccharomyces cerevisiae genome which encode proteins bearing the tetratricopeptide repeat (TPR) domain that mediates protein–protein interaction. Thus far, the function of one of them, YNL313c, remains unknown. Our conditional mutants of YNL313c display osmoremedial temperature sensitivity, hypersensitivity to both Calcofluor White and low concentrations of SDS, and osmoremedial caffeine sensitivity. These are hallmarks of mutants that display cell wall defects. Accordingly we rename the gene as EMW1 (essential for maintenance of the cell wall). Loss of Emw1p function is not associated with abrogation of the cell wall integrity (CWI) MAP kinase cascade. Instead, emw1ts mutants activate this cascade even at permissive temperature, indicating that loss of Emw1p function does not cause a defect in sensors and effectors of cell wall signalling, but leads to a cell wall defect directly. Constitutive activation of the CWI cascade is reflected by the overproduction of chitin by emw1ts mutants, a compensatory response frequently displayed by cell wall mutants. Growth is restored to emw1ts mutants incubated at otherwise non-permissive temperature when GFA1 is overexpressed. GFA1 encodes the hexosephosphate aminotransferase that catalyses the rate-limiting step in the pathway that synthesizes the chitin precursor UDP-GlcNAc. The possibility that Emw1p is required for function of Gfa1p was ruled out, because the emw1ts phenotype persists when the requirement for Gfa1p is bypassed. Furthermore, if loss of Emw1p function leads to loss of function of Gfa1p, then chitin synthesis would be diminished. Instead, a stimulation of the synthesis of this polymer is detected. Consequently, the defect associated with emw1ts mutants may be associated with compromise in one of the remaining processes that depend on UDP-GlcNAc, namely N-glycosylation or glycosylphosphatidylinositol (GPI)-anchor synthesis.
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A novel role for SarX in Staphylococcus epidermidis biofilm regulation
More LessBiofilm production by staphylococci is an important virulence determinant mediated by the icaADBC-encoded polysaccharide intercellular adhesin (PIA) or by surface and extracellular proteins. Deletion of the Staphylococcus accessory regulator sarX significantly reduced biofilm-forming capacity in Staphylococcus epidermidis CSF41498, whereas multicopy sarX complemented the sarX mutant and increased wild-type biofilm production. In Staphylococcus aureus, SarX negatively regulates the accessory gene regulator (Agr) system, which in turn has strain-specific effects on biofilm regulation. Here we found that purified S. epidermidis SarX protein bound specifically to the agr P3 promoter. However RT-PCR analysis revealed that both mutation of sarX and multicopy sarX activated RNAIII transcription, making it difficult to correlate sarX-mediated biofilm regulation with altered agr activity. In contrast, RT-PCR and immunoblot analysis revealed that icaA transcription and PIA expression were decreased in the sarX mutant, whereas multicopy sarX increased ica and PIA expression. Furthermore, multicopy sarX did not promote biofilms in an icaC mutant. Finally, purified SarX protein bound specifically to the ica operon promoter. Taken together, these data reveal that the S. epidermidis SarX protein regulates the transcriptional activity of the agr and ica loci and controls the biofilm phenotype, primarily by regulating icaADBC transcription and PIA production.
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- Environmental And Evolutionary Microbiology
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Increased level of intragenomic 16S rRNA gene heterogeneity in commensal strains closely related to Haemophilus influenzae
More LessThe 16S rRNA gene sequence of strains closely related to, but excluded from, Haemophilus influenzae was investigated and a conspicuously high number of polymorphic nucleotide positions due to intragenomic 16S rRNA gene heterogeneity was observed. The average frequency of 16S rRNA gene polymorphic nucleotide positions in 31 variant strains was 7.0×10−3, which is approximately ten times the level observed in validated strains of H. influenzae. Sixty-seven polymorphic nucleotide positions in seven strains most likely originated from the simultaneous presence of two distinct types of helix 18 as a consequence of prior recombinatorial events. The increased level of 16S rRNA gene polymorphism in commensal taxa excluded from the pathogenic species H. influenzae is unexplained. The heterogeneity imposes difficulties on rRNA gene-based classification and systematics.
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- Genes And Genomes
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Phenotypic and molecular characterization of Salmonella enterica serovar Sofia, an avirulent species in Australian poultry
More LessSalmonella enterica serovar Sofia (S. Sofia) is often isolated from chickens in Australia. However, despite its high frequency of isolation from chicken and chicken meat products, S. Sofia is rarely associated with animal or human salmonellosis, presumably because this serovar is avirulent in nature. The objective of this work was to investigate the phenotypic and molecular properties of S. Sofia in order to assess its pathogenic potential. Our in vivo studies support the observation that this serovar can colonize tissues, but does not cause disease in chickens. This was further confirmed with tissue culture assays, which showed that the ability of S. Sofia to adhere, invade and survive intracellularly is significantly diminished compared with the pathogenic Salmonella enterica serovar Typhimurium (S. Typhimurium) 82/6915. Molecular analysis of Salmonella pathogenicity islands (SPIs) showed that most of the differences observed in SPI1 to SPI5 of S. Sofia could be attributed to minor changes in the sequences, as indicated by a loss or gain of restriction cleavage sites within these regions. Sequence analysis demonstrated that the majority of virulence genes identified were predicted to encode proteins sharing a high identity (75–100 %) with corresponding proteins from S. Typhimurium. However, a number of virulence genes in S. Sofia have accumulated mutations predicted to affect transcription and/or translation. The avirulence of this serovar is probably not the result of a single genetic change but rather of a series of alterations in a large number of virulence-associated genes. The acquisition of any single virulence gene will almost certainly not be sufficient to restore S. Sofia virulence.
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Introgression in the genus Campylobacter: generation and spread of mosaic alleles
More LessHorizontal genetic exchange strongly influences the evolution of many bacteria, substantially contributing to difficulties in defining their position in taxonomic groups. In particular, how clusters of related bacterial genotypes – currently classified as microbiological species – evolve and are maintained remains controversial. The nature and magnitude of gene exchange between two closely related (approx. 15 % nucleotide divergence) microbiologically defined species, Campylobacter jejuni and Campylobacter coli, was investigated by the examination of mosaic alleles, those with some ancestry from each population. A total of 1738 alleles from 2953 seven-locus housekeeping gene sequence types (STs) were probabilistically assigned to each species group with the model-based clustering algorithm structure. Alleles with less than 75 % assignment probability to one of the populations were confirmed as mosaics using the structure linkage model. For each of these, the putative source of the recombinant region was determined and the allele was mapped onto a clonalframe genealogy derived from concatenated ST sequences. This enabled the direction and frequency of introgression between the two populations to be established, with 8.3 % of C. coli clade 1 alleles having acquired C. jejuni sequence, compared to 0.5 % for the reciprocal process. Once generated, mosaic genes spread within C. coli clade 1 by a combination of clonal expansion and lateral gene transfer, with some evidence of erosion of the mosaics by reacquisition of C. coli sequence. These observations confirm previous analyses of the exchange of complete housekeeping alleles and extend this work by describing the processes of horizontal gene transfer and subsequent spread within recipient species.
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Nucleoid-associated protein HU controls three regulons that coordinate virulence, response to stress and general physiology in Salmonella enterica serovar Typhimurium
The role of the HU nucleoid-associated proteins in gene regulation was examined in Salmonella enterica serovar Typhimurium. The dimeric HU protein consists of different combinations of its α and β subunits. Transcriptomic analysis was performed with cultures growing at 37 °C at 1, 4 and 6 h after inoculation with mutants that lack combinations of HU α and HU β. Distinct but overlapping patterns of gene expression were detected at each time point for each of the three mutants, revealing not one but three regulons of genes controlled by the HU proteins. Mutations in the hup genes altered the expression of regulatory and structural genes in both the SPI1 and SPI2 pathogenicity islands. The hupA hupB double mutant was defective in invasion of epithelial cell lines and in its ability to survive in macrophages. The double mutant also had defective swarming activity and a competitive fitness disadvantage compared with the wild-type. In contrast, inactivation of just the hupB gene resulted in increased fitness and correlated with the upregulation of members of the RpoS regulon in exponential-phase cultures. Our data show that HU coordinates the expression of genes involved in central metabolism and virulence and contributes to the success of S. enterica as a pathogen.
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Genomic characterization of asymptomatic Escherichia coli isolated from the neobladder
The replacement of the bladder with a neobladder made from ileal tissue is the prescribed treatment in some cases of bladder cancer or trauma. Studies have demonstrated that individuals with an ileal neobladder have recurrent colonization by Escherichia coli and other species that are commonly associated with urinary tract infections; however, pyelonephritis and complicated symptomatic infections with ileal neobladders are relatively rare. This study examines the genomic content of two E. coli isolates from individuals with neobladders using comparative genomic hybridization (CGH) with a pan-E. coli/Shigella microarray. Comparisons of the neobladder genome hybridization patterns with reference genomes demonstrate that the neobladder isolates are more similar to the commensal, laboratory-adapted E. coli and a subset of enteroaggregative E. coli than they are to uropathogenic E. coli isolates. Genes identified by CGH as exclusively present in the neobladder isolates among the 30 examined isolates were primarily from large enteric isolate plasmids. Isolations identified a large plasmid in each isolate, and sequencing confirmed similarity to previously identified plasmids of enteric species. Screening, via PCR, of more than 100 isolates of E. coli from environmental, diarrhoeagenic and urinary tract sources did not identify neobladder-specific genes that were widely distributed in these populations. These results taken together demonstrate that the neobladder isolates, while distinct, are genomically more similar to gastrointestinal or commensal E. coli, suggesting why they can colonize the transplanted intestinal tissue but rarely progress to acute pyelonephritis or more severe disease.
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- Microbial Pathogenicity
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Enhanced recovery of Salmonella Typhimurium DT104 from exposure to stress at low temperature
More LessSalmonella enterica serovar Typhimurium (S. Typhimurium) remains an important cause of food-borne infection in the developed world. In order to establish infection within a host, Salmonella must survive and recover from a range of environmental stresses. S. Typhimurium strain SL1344 is among the most extensively studied pathogenic Salmonella strains, while S. Typhimurium phage type DT104 is an important type that has been associated with pandemic spread and a high number of food-borne disease outbreaks over the last two decades. In this study, we have compared the abilities of these two S. Typhimurium types to recover from stress exposures commonly encountered in food production, including 685 mM NaCl, pH 3.8, low temperature (6 °C) and combinations thereof. Following removal from prolonged (8 days) stress, DT104 cultures that had been exposed to low temperature, with or without additional stress, resumed exponential growth more rapidly than SL1344 cultures exposed to the same conditions. SL1344 showed higher levels of filamentation than DT104 in response to NaCl exposure at low temperature. Further, SL1344 incurred higher levels of membrane damage in response to elevated NaCl and pH 3.8 at both temperatures compared with DT104. However, both strains recovered normal cell division and membrane integrity within 6 h when all stresses were removed. Expression of the Salmonella pathogenicity island 1 gene prgH, the first gene in the prg/org operon, was monitored using a chromosomal reporter in which gfp+ expression was driven by the prgH promoter. Recovery of prgH expression was comparable for SL1344 and DT104 exposed to stress at 22 °C. However, DT104 cultures exposed to pH 3.8 or combined NaCl and low-pH stress at low temperature resumed prgH expression more rapidly than SL1344. Both strains recovered maximal levels of prgH expression after 6 h recovery from all stresses and, interestingly, maximal levels of prgH expression were significantly higher in SL1344, consistent with prgH expression in late-exponential, non-stressed SL1344 and DT104 cultures. Together, these data show that S. Typhimurium is capable of rapid recovery from environmental and food-related stresses, and give insight into the enhanced ability of DT104 compared with SL1344 to adapt to such stresses, which may contribute to the success of this globally disseminated pathogenic phage type.
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Yersinia pseudotuberculosis mntH functions in intracellular manganese accumulation, which is essential for virulence and survival in cells expressing functional Nramp1
Manganese has an important yet undefined role in the virulence of many bacterial pathogens. In this study we confirm that a null mutation in Yersinia pseudotuberculosis mntH reduces intracellular manganese accumulation. An mntH mutant was susceptible to killing by reactive oxygen species when grown under manganese-limited conditions. The mntH mutant was defective in survival and growth in macrophages expressing functional Nramp1, but in macrophages deficient in Nramp the bacteria were able to survive and replicate. In Galleria mellonella, the mntH mutant was attenuated. Taken together, these data suggest a role for manganese in Y. pseudotuberculosis during macrophage intracellular survival, protecting the bacteria from the antimicrobial products released during the respiratory burst.
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Ler of pathogenic Escherichia coli forms toroidal protein–DNA complexes
Enteropathogenic and enterohaemorrhagic Escherichia coli are related pathotypes of bacteria that cause acute watery diarrhoea and haemorrhagic colitis, respectively, and enterohaemorrhagic E. coli can lead to a serious complication known as haemolytic uraemic syndrome. In both bacteria the global regulatory protein Ler controls virulence. The ler gene is found within the locus of enterocyte effacement, or LEE, encoding a type III secretion system necessary for injecting effector proteins into intestinal epithelial cells and causing net secretory diarrhoea. The nucleoid-associated protein H-NS silences, whereas Ler serves as an anti-silencer of, multiple LEE operons. Although Ler has a higher affinity for DNA than does H-NS, the precise molecular mechanism by which Ler increases LEE transcription remains to be determined. In this report we investigate the oligomerization activity of Ler. In solution, Ler forms dimers and soluble aggregates of up to 5000 kDa molecular mass, and appears to oligomerize more readily than the related protein H-NS. An insertional mutation into the Ler linker region diminished oligomerization activity. Despite being proteins of similar mass and having homologous DNA-binding domains, Ler and H-NS complexed to DNA migrated to distinct locations, as determined by an electrophoretic mobility shift assay, implying that the related proteins form different 3D shapes in the presence of DNA. Lastly, we present electron microscopy images of toroidal Ler–DNA structures that are predicted to be involved in stimulating gene expression.
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Chlamydia trachomatis secretion of hypothetical protein CT622 into host cell cytoplasm via a secretion pathway that can be inhibited by the type III secretion system inhibitor compound 1
More LessUsing antibodies raised with C. trachomatis fusion proteins, we localized a hypothetical protein encoded by the ORF ct622 in the cytoplasm of C. trachomatis-infected mammalian cells. The detection was specific since the antibody labelling of CT622 protein was removed by preabsorption with CT622 but not other fusion proteins. We similarly confirmed that CT621, a known secretion protein encoded by a hypothetical ORF downstream of ct622, was secreted into host cell cytosol. Proteins CT622 and CT621 displayed a similar secretion pattern, with both intra-inclusion and host cell cytosol localization, that was distinct from that of CPAF (chlamydial protease/proteasome-like activity factor). However, the expression and secretion kinetics differed significantly between CT622 and CT621: CT622 mRNA was detected at 2 h, protein at 6 h and secretion of protein into host cell cytoplasm at 36 h post-infection, while CT621 mRNA was detected at 8 h, protein at 16 h and secretion at 24 h. The secretion of both CT622 and CT621 was blocked by N′-(3,5-dibromo-2-hydroxybenzylidene)-4-nitrobenzohydrazide (compound 1), an inhibitor known to target the type III secretion system of bacteria. These results suggest that CT621 and CT622 may fulfil different functions during chlamydial intracellular growth. Further characterization of these proteins may generate important information for understanding chlamydial pathogenesis.
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The muramidase EtgA from enteropathogenic Escherichia coli is required for efficient type III secretion
Enteropathogenic Escherichia coli (EPEC) is an important cause of infectious diarrhoea. It colonizes human intestinal epithelial cells by delivering effector proteins into the host cell cytoplasm via a type III secretion system (T3SS) encoded within the chromosomal locus of enterocyte effacement (LEE). The LEE pathogenicity island also encodes a lytic transglycosylase (LT) homologue named EtgA. In the present work we investigated the significance of EtgA function in type III secretion (T3S). Purified recombinant EtgA was found to have peptidoglycan lytic activity in vitro. Consistent with this function, signal peptide processing and bacterial cell fractionation revealed that EtgA is a periplasmic protein. EtgA possesses the conserved glutamate characteristic of the LT family, and we show here that it is essential for enzymic activity. Overproduction of EtgA in EPEC inhibits bacterial growth and induces cell lysis unless the predicted catalytic glutamate is mutated. An etgA mutant is attenuated for T3S, red blood cell haemolysis and EspA filamentation. BfpH, a plasmid-encoded putative LT, was not able to functionally replace EtgA. Overall, our results indicate that the muramidase activity of EtgA is not critical but makes a significant contribution to the efficiency of the T3S process.
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UafB is a serine-rich repeat adhesin of Staphylococcus saprophyticus that mediates binding to fibronectin, fibrinogen and human uroepithelial cells
Staphylococcus saprophyticus is an important cause of urinary tract infection (UTI), particularly among young women, and is second only to uropathogenic Escherichia coli as the most frequent cause of UTI. The molecular mechanisms of urinary tract colonization by S. saprophyticus remain poorly understood. We have identified a novel 6.84 kb plasmid-located adhesin-encoding gene in S. saprophyticus strain MS1146 which we have termed uro-adherence factor B (uafB). UafB is a glycosylated serine-rich repeat protein that is expressed on the surface of S. saprophyticus MS1146. UafB also functions as a major cell surface hydrophobicity factor. To characterize the role of UafB we generated an isogenic uafB mutant in S. saprophyticus MS1146 by interruption with a group II intron. The uafB mutant had a significantly reduced ability to bind to fibronectin and fibrinogen. Furthermore, we show that a recombinant protein containing the putative binding domain of UafB binds specifically to fibronectin and fibrinogen. UafB was not involved in adhesion in a mouse model of UTI; however, we observed a striking UafB-mediated adhesion phenotype to human uroepithelial cells. We have also identified genes homologous to uafB in other staphylococci which, like uafB, appear to be located on transposable elements. Thus, our data indicate that UafB is a novel adhesin of S. saprophyticus that contributes to cell surface hydrophobicity, mediates adhesion to fibronectin and fibrinogen, and exhibits tropism for human uroepithelial cells.
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N-Octanoylhomoserine lactone signalling mediated by the BpsI–BpsR quorum sensing system plays a major role in biofilm formation of Burkholderia pseudomallei
More LessThe genome of Burkholderia pseudomallei encodes three acylhomoserine lactone (AHL) quorum sensing systems, each comprising an AHL synthase and a signal receptor/regulator. The BpsI–BpsR system produces N-octanoylhomoserine lactone (C8HL) and is positively auto-regulated by its AHL product. The products of the remaining two systems have not been identified. In this study, tandem MS was used to identify and quantify the AHL species produced by three clinical B. pseudomallei isolates – KHW, K96243 and H11 – three isogenic KHW mutants that each contain a null mutation in an AHL synthase gene, and recombinant Escherichia coli heterologously expressing each of the three B. pseudomallei AHL synthase genes. BpsI synthesized predominantly C8HL, which accounted for more than 95 % of the extracellular AHLs produced in stationary-phase KHW cultures. The major products of BpsI2 and BpsI3 were N-(3-hydroxy-octanoyl)homoserine lactone (OHC8HL) and N-(3-hydroxy-decanoyl)homoserine lactone, respectively, and their corresponding transcriptional regulators, BpsR2 and BpsR3, were capable of driving reporter gene expression in the presence of these cognate lactones. Formation of biofilm by B. pseudomallei KHW was severely impaired in mutants lacking either BpsI or BpsR but could be restored to near wild-type levels by exogenous C8HL. BpsI2 was not required, and BpsI3 was partially required for biofilm formation. Unlike the bpsI mutant, biofilm formation in the bpsI3 mutant could not be restored to wild-type levels in the presence of OHC8HL, the product of BpsI3. C8HL and OHC8HL had opposite effects on biofilm formation; exogenous C8HL enhanced biofilm formation in both the bpsI3 mutant and wild-type KHW while exogenous OHC8HL suppressed the formation of biofilm in the same strains. We propose that exogenous OHC8HL antagonizes biofilm formation in B. pseudomallei, possibly by competing with endogenous C8HL for binding to BpsR.
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Deletion of the mmpL4b gene in the Mycobacterium abscessus glycopeptidolipid biosynthetic pathway results in loss of surface colonization capability, but enhanced ability to replicate in human macrophages and stimulate their innate immune response
More LessMycobacterium abscessus is considered to be the most virulent of the rapidly growing mycobacteria. Generation of bacterial gene knockout mutants has been a useful tool for studying factors that contribute to virulence of pathogenic bacteria. Until recently, the optimal genetic approach to generation of M. abscessus gene knockout mutants was not clear. Based on the recent identification of genetic recombineering as the preferred approach, a M. abscessus mutant was generated in which the gene mmpL4b, critical to glycopeptidolipid synthesis, was deleted. Compared to the previously well-characterized parental strain 390S, the mmpL4B deletion mutant had lost sliding motility and the ability to form biofilm, but acquired the ability to replicate in human macrophages and stimulate macrophage Toll-like receptor 2. This study demonstrates that deletion of a gene associated with expression of a cell-wall lipid can result in acquisition of an immunostimulatory, invasive bacterial phenotype and has important implications for the study of M. abscessus pathogenesis at the cellular level.
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Comparative analysis and mutation effects of fpp2–fpp1 tandem genes encoding proteolytic extracellular enzymes of Flavobacterium psychrophilum
Flavobacterium psychrophilum is a very significant fish pathogen that secretes two biochemically characterized extracellular proteolytic enzymes, Fpp1 and Fpp2. The genes encoding these enzymes are organized as an fpp2–fpp1 tandem in the genome of strain F. psychrophilum THC02/90. Analysis of the corresponding encoded proteins showed that they belong to two different protease families. For gene function analysis, new genetic tools were developed in F. psychrophilum by constructing stable isogenic fpp1 and fpp2 mutants via single-crossover homologous recombination. RT-PCR analysis of wild-type and mutant strains suggested that both genes are transcribed as a single mRNA from the promoter located upstream of the fpp2 gene. Phenotypic characterization of the fpp2 mutant showed lack of caseinolytic activity and higher colony spreading compared with the wild-type strain. Both characteristics were recovered in the complemented strain. One objective of this work was to assess the contribution to virulence of these proteolytic enzymes. LD50 experiments using the wild-type strain and mutants showed no significant differences in virulence in a rainbow trout challenge model, suggesting instead a possible nutritional role. The gene disruption procedure developed in this work, together with the knowledge of the complete genome sequence of F. psychrophilum, open new perspectives for the study of gene function in this bacterium.
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- Physiology And Biochemistry
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Biochemical and immunological characterization of a cpn60.1 knockout mutant of Mycobacterium bovis BCG
Pathogenic mycobacteria possess two homologous chaperones encoded by cpn60.1 and cpn60.2. Cpn60.2 is essential for survival, providing the basic chaperone function, while Cpn60.1 is not. In the present study, we show that inactivation of the Mycobacterium bovis BCG cpn60.1 (Mb3451c) gene does not significantly affect bacterial growth in 7H9 broth, but that this knockout mutant (Δcpn60.1) forms smaller colonies on solid 7H11 medium than the parental and complemented strains. When growing on Sauton medium, the Δcpn60.1 mutant exhibits a thinner surface pellicle and is associated with higher culture filtrate protein content and, coincidentally, with less protein in its outermost cell envelope in comparison with the parental and complemented strains. Interestingly, in this culture condition, the Δcpn60.1 mutant is devoid of phthiocerol dimycocerosates, and its mycolates are two carbon atoms longer than those of the wild-type, a phenotype that is fully reversed by complementation. In addition, Δcpn60.1 bacteria are more sensitive to stress induced by H2O2 but not by SDS, high temperature or acidic pH. Taken together, these data indicate that the cell wall of the Δcpn60.1 mutant is impaired. Analysis by 2D gel electrophoresis and MS reveals the upregulation of a few proteins such as FadA2 and isocitrate lyase in the cell extract of the mutant, whereas more profound differences are found in the composition of the mycobacterial culture filtrate, e.g. the well-known Hsp65 chaperonin Cpn60.2 is particularly abundant and increases about 200-fold in the filtrate of the Δcpn60.1 mutant. In mice, the Δcpn60.1 mutant is less persistent in lungs and, to a lesser extent, in spleen, but it induces a comparable mycobacteria-specific gamma interferon production and protection against Mycobacterium tuberculosis H37Rv challenge as do the parental and complemented BCG strains. Thus, by inactivating the cpn60.1 gene in M. bovis BCG we show that Cpn60.1 is necessary for the integrity of the bacterial cell wall, is involved in resistance to H2O2-induced stress but is not essential for its vaccine potential.
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The nitrogen interaction network in Synechococcus WH5701, a cyanobacterium with two PipX and two PII-like proteins
More LessNitrogen regulation involves the formation of different types of protein complexes between signal transducers and their transcriptional or metabolic targets. In oxygenic phototrophs, the signal integrator PII activates the enzyme N-acetyl-l-glutamate kinase (NAGK) by complex formation. PII also interacts with PipX, a protein with a tudor-like domain that mediates contacts with PII and with the transcriptional regulator NtcA, to which it binds to increase its activity. Here, we use a combination of in silico, yeast two-hybrid and in vitro approaches to investigate the nitrogen regulation network of Synechococcus WH5701, a marine cyanobacterium with two PII (GlnB_A and GlnB_B) and two PipX (PipX_I and PipX_II) proteins. Our results indicate that GlnB_A is functionally equivalent to the canonical PII protein from Synechococcus elongatus. GlnB_A interacted with PipX and NAGK proteins and stimulated NAGK activity, counteracting arginine inhibition. GlnB_B had only a slight stimulatory effect on NAGK activity, but its potential to bind effectors and form heterotrimers in Synechococcus WH5701 indicates additional regulatory functions. PipX_II, and less evidently PipX_I, specifically interacted with GlnB_A and NtcA, supporting a role for both Synechococcus WH5701 PipX proteins in partner swapping with GlnB_A and NtcA.
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Sulfur globule oxidation in green sulfur bacteria is dependent on the dissimilatory sulfite reductase system
More LessGreen sulfur bacteria (GSB) oxidize sulfide and thiosulfate to sulfate, with extracellular globules of elemental sulfur as an intermediate. Here we investigated which genes are involved in the formation and consumption of these sulfur globules in the green sulfur bacterium Chlorobaculum tepidum. We show that sulfur globule oxidation is strictly dependent on the dissimilatory sulfite reductase (DSR) system. Deletion of dsrM/CT2244 or dsrT/CT2245, or the two dsrCABL clusters (CT0851–CT0854, CT2247–2250), abolished sulfur globule oxidation and prevented formation of sulfate from sulfide, whereas deletion of dsrU/CT2246 had no effect. The DSR system also seems to be involved in the formation of thiosulfate, because thiosulfate was released from wild-type cells during sulfide oxidation, but not from the dsr mutants. The dsr mutants incapable of complete substrate oxidation oxidized sulfide and thiosulfate about twice as fast as the wild-type, while having only slightly lower growth rates (70–80 % of wild-type). The increased oxidation rates seem to compensate for the incomplete substrate oxidation to satisfy the requirement for reducing equivalents during growth. A mutant in which two sulfide : quinone oxidoreductases (sqrD/CT0117 and sqrF/CT1087) were deleted exhibited a decreased sulfide oxidation rate (∼50 % of wild-type), yet formation and consumption of sulfur globules were not affected. The observation that mutants lacking the DSR system maintain efficient growth suggests that the DSR system is dispensable in environments with sufficiently high sulfide concentrations. Thus, the DSR system in GSB may have been acquired by horizontal gene transfer as a response to a need for enhanced substrate utilization in sulfide-limiting habitats.
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Identification and characterization of the Streptomyces globisporus 1912 regulatory gene lndYR that affects sporulation and antibiotic production
Here, we report the identification and functional characterization of the Streptomyces globisporus 1912 gene lndYR, which encodes a GntR-like regulator of the YtrA subfamily. Disruption of lndYR arrested sporulation and antibiotic production in S. globisporus. The results of in vivo and in vitro studies revealed that the ABC transporter genes lndW–lndW2 are targets of LndYR repressive action. In Streptomyces coelicolor M145, lndYR overexpression caused a significant increase in the amount of extracellular actinorhodin. We suggest that lndYR controls the transcription of transport system genes in response to an as-yet-unidentified signal. Features that distinguish lndYR-based regulation from other known regulators are discussed.
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