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Volume 160,
Issue 9,
2014
Volume 160, Issue 9, 2014
- Review
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Mycobacterium tuberculosis adhesins: potential biomarkers as anti-tuberculosis therapeutic and diagnostic targets
More LessAdhesion to host cells is a precursor to host colonization and evasion of the host immune response. Conversely, it triggers the induction of the immune response, a process vital to the host’s defence against infection. Adhesins are microbial cell surface molecules or structures that mediate the attachment of the microbe to host cells and thus the host–pathogen interaction. They also play a crucial role in bacterial aggregation and biofilm formation. In this review, we discuss the role of adhesins in the pathogenesis of the aetiological agent of tuberculosis, Mycobacterium tuberculosis. We also provide insight into the structure and characteristics of some of the characterized and putative M. tuberculosis adhesins. Finally, we examine the potential of adhesins as targets for the development of tuberculosis control strategies.
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- Cell and Molecular Biology of Microbes
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Sulfate assimilation pathway intermediate phosphoadenosine 5′-phosphosulfate acts as a signal molecule affecting production of curli fibres in Escherichia coli
More LessThe enterobacterium Escherichia coli can utilize a variety of molecules as sulfur sources, including cysteine, sulfate, thiosulfate and organosulfonates. An intermediate of the sulfate assimilation pathway, adenosine 5′-phosphosulfate (APS), also acts as a signal molecule regulating the utilization of different sulfur sources. In this work, we show that inactivation of the cysH gene, leading to accumulation of phosphoadenosine 5′-phosphosulfate (PAPS), also an intermediate of the sulfate assimilation pathway, results in increased surface adhesion and cell aggregation by activating the expression of the curli-encoding csgBAC operon. In contrast, curli production was unaffected by the inactivation of any other gene belonging to the sulfate assimilation pathway. Overexpression of the cysH gene downregulated csgBAC transcription, further suggesting a link between intracellular PAPS levels and curli gene expression. In addition to curli components, the Flu, OmpX and Slp proteins were also found in increased amounts in the outer membrane compartment of the cysH mutant; deletion of the corresponding genes suggested that these proteins also contribute to surface adhesion and cell surface properties in this strain. Our results indicate that, similar to APS, PAPS also acts as a signal molecule, albeit with a distinct mechanism and role: whilst APS regulates organosulfonate utilization, PAPS would couple availability of sulfur sources to remodulation of the cell surface, as part of a more global effect on cell physiology.
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Alternaria alternata transcription factor CmrA controls melanization and spore development
More LessMelanin is a black pigment widely distributed across the kingdoms, from bacterial to human. The filamentous fungus Alternaria alternata is a typical ‘black fungus’, which produces melanin in its hyphal and especially its asexual spore cell walls. Its biosynthesis follows the dihydroxynaphthalene (DHN) pathway with 1,8-DHN as an intermediate. Two genes, encoding a polyketide synthase (pksA) and a 1,3,8-trihydroxynaphthalene (THN) reductase (brm2), along with a putative transcription factor, CmrA, comprise a small gene cluster. Here we show that CmrA controls the expression of pksA and brm2, but that it also controls the expression of a scytalone dehydratase encoding gene (brm1) located elsewhere in the genome. The regulatory function of CmrA was shown in a reporter assay system. Al. alternata CmrA was expressed in the filamentous fungus Aspergillus nidulans where it was able to induce the expression of a reporter construct under the control of the putative pksA promoter. This suggests direct binding of CmrA to the promoter of pksA in the heterologous system. Likewise, silencing of cmrA in Al. alternata led to white colonies due to the lack of melanin. In addition, hyphal diameter and spore morphology were changed in the mutant and the number of spores reduced. Silencing of brm2 and inhibition of melanin biosynthesis by tricyclazole largely phenocopied the effects of cmrA silencing, suggesting a novel regulatory function of melanin in morphogenetic pathways.
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Genetic and mutational characterization of the small alarmone synthetase gene relV of Vibrio cholerae
More LessIn Vibrio cholerae, the causative agent of cholera, products of three genes, relA, spoT and relV, govern nutritional stress related stringent response (SR). SR in bacteria is critically regulated by two intracellular small molecules, guanosine 3′-diphosphate 5′-triphosphate (pppGpp) and guanosine 3′,5′-bis(diphosphate) (ppGpp), collectively called (p)ppGpp or alarmone. Evolution of relV is unique in V. cholerae because other Gram-negative bacteria carry only relA and spoT genes. Recent reports suggest that RelV is needed for pathogenesis. RelV carries a single (p)ppGpp synthetase or RelA-SpoT domain (SYNTH/RSD) and belongs to the small alarmone synthetase (SAS) family of proteins. Here, we report extensive functional characterizations of the relV gene by constructing several deletion and site-directed mutants followed by their controlled expression in (p)ppGpp0 cells of Escherichia coli or V. cholerae. Substitution analysis indicated that the amino acid residues K107, D129, R132, L150 and E188 of the RSD region of RelV are essential for its activity. While K107, D129 and E188 are highly conserved in RelA and SAS proteins, L150 appears to be conserved in the latter group of enzymes, and the R132 residue was found to be unique in RelV. Extensive progressive deletion analysis indicated that the amino acid residues at positions 59 and 248 of the RelV protein are the functional N- and C-terminal boundaries, respectively. Since the minimal functional length of RelV was found to be 189 aa, which includes the 94 aa long RSD region, it seems that the flanking residues of the RSD are also important for maintaining the (p)ppGpp synthetase activity.
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H-NS regulates the Vibrio parahaemolyticus type VI secretion system 1
More LessThe marine bacterium Vibrio parahaemolyticus, a major cause of food-borne gastroenteritis, employs a type VI secretion system 1 (T6SS1), a recently discovered protein secretion system, to combat competing bacteria. Environmental signals such as temperature, salinity, cell density and surface sensing, as well as the quorum-sensing master regulator OpaR, were previously reported to regulate T6SS1 activity and expression. In this work, we set out to identify additional transcription regulators that control the tightly regulated T6SS1 activity. To this end, we determined the effect of deletions in several known virulence regulators and in two regulators encoded within the T6SS1 gene cluster on expression and secretion of the core T6SS component Hcp1 and on T6SS1-mediated anti-bacterial activity. We report that VP1391 and VP1407, transcriptional regulators encoded within the T6SS1 gene cluster, are essential for T6SS1 activity. Moreover, we found that H-NS, a bacterial histone-like nucleoid structuring protein, which mediates transcription silencing of horizontally acquired genes, serves as a repressor of T6SS1. We also show that activation of surface sensing and high salt conditions alleviate the H-NS-mediated repression. Our results shed light on the complex network of environmental signals and transcription regulators that govern the tight regulation over T6SS1 activity.
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Transcriptional dynamics of developmental genes assessed with an FMN-dependent fluorophore in mature heterocysts of Anabaena sp. strain PCC 7120
More LessAnabaena sp. strain PCC 7120 is a filamentous cyanobacterium that differentiates nitrogen-fixing heterocysts when available combined nitrogen is limiting. Growth under diazotrophic conditions results in a mixture of ‘new’ (recently differentiated) and ‘old’ (mature) heterocysts. The microoxic environment present in heterocysts makes the interpretation of gene expression using oxygen-dependent fluorophores, including GFP, difficult. The work presented here evaluates the transcriptional dynamics of three developmental genes in mature heterocysts utilizing EcFbFP, a flavin mononucleotide-dependent fluorophore, as the reporter. Expression of both GFP and EcFbFP from the heterologous petE promoter showed that, although GFP and EcFbFP fluoresced in both vegetative cells and new heterocysts, only EcFbFP fluoresced in old heterocysts. A transcriptional fusion of EcFbFP to the late-stage heterocyst-specific nifB promoter displayed continued expression beyond the cessation of GFP fluorescence in heterocysts. Promoter fusions of the master regulator of differentiation, hetR, and its inhibitors, patS and hetN, to GFP and EcFbFP were visualized to determine their role(s) in heterocyst function after morphogenesis. The expression of hetR and hetN was found to persist beyond the completion of development in most heterocysts, whereas patS expression ceased. These data are consistent with a model of heterocyst patterning in which patS is involved in de novo pattern formation, hetN is required for pattern maintenance, and hetR is needed for all stages of development.
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The lack of OmpF, but not OmpC, contributes to increased antibiotic resistance in Serratia marcescens
More LessThe environmental organism Serratia marcescens is one of the primary causes of numerous nosocomial outbreaks and opportunistic infections. Multi-drug resistance is now a common feature among S. marcescens clinical isolates, complicating the efficacy of treatment. Recent reports have attributed antibiotic resistance to altered porin expression as well as perturbation of the intrinsic AmpC beta-lactamase production pathway. In this study, we aimed to genetically correlate the absence of OmpF and OmpC classical porins with increased antibiotic resistance. In generating isogenic porin mutant strains, we avoided incorporating additional resistance through the use of antibiotic cassettes in gene replacement and adopted an alternative strategy in creating clean unmarked mutant strains. We found that lack of OmpF, but not OmpC, significantly increased antibiotic MIC values to the beta-lactam drugs such as ampicillin and cefoxitin as well as to nitrofurantoin. Furthermore, we found that cefoxitin did not induce intrinsic AmpC beta-lactamase production, indicating that the increased MIC values were a result of reduced permeability of cefoxitin due to the lack of OmpF. Genetic deletion of both ompF and ompC did not compromise the integrity of the bacterial cell envelope in optimal growth conditions, suggesting that other outer-membrane porins may function in a compensatory role to facilitate nutrient uptake and cell envelope integrity. Taken together, to our knowledge this is the first study that genetically correlates increased antibiotic resistance with altered porin expression in S. marcescens.
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Characterization of stipe elongation of the mushroom Coprinopsis cinerea
Previously, we observed an acid-induced short-term wall extension in Flammulina velutipes apical stipes during a 15 min period after a change from a neutral to an acidic pH. This acid-induced stipe wall extension was eliminated by heating and reconstituted by a snail expansin-like protein, although we failed to isolate any endogenous expansin-like protein from F. velutipes because of its limited 1 mm fast elongation region. In this study, we report that Coprinopsis cinerea stipes possess a 9 mm fast elongation apical region, which is suitable as a model material for wall extension studies. The elongating apical stipe showed two phases of acid-induced wall extension, an initial quick short-term wall extension during the first 15 min and a slower, gradually decaying long-term wall extension over the subsequent 2 h. After heating or protein inactivation pretreatment, apical stipes lost the long-term wall extension, retaining a slower short-term wall extension, which was reconstituted by an expansin-like snail protein. In contrast, the non-elongating basal stipes showed only a weaker short-term wall extension. We propose that the long-term wall extension is a protein-mediated process involved in stipe elongation, whereas the short-term wall extension is a non-protein mediated process not involved in stipe elongation.
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Expression levels of transcription factors in Escherichia coli: growth phase- and growth condition-dependent variation of 90 regulators from six families
More LessThe expression pattern of the genome in Escherichia coli is controlled by regulating the utilization of a limited number of RNA polymerases between a total of 4600 genes on its genome. The distribution pattern of RNA polymerase on the genome changes after two steps of protein–protein interaction with seven sigma subunits and about 300 transcription factors (TFs). Based on a systematic search for the regulation target promoters recognized by each TF, we propose two novel concepts: each TF regulates a number of target promoters; and each promoter is regulated by many TFs. In parallel, attempts have been made to determine the intracellular concentrations of all TFs using two systems: quantitative immunoblot analysis using TF-specific antibodies; and reporter assay of TF promoter activities. The direct measurement of TF protein level has so far been published for a set of 60 regulators with known functions. This study describes the determination of growth phase-dependent expression levels of 90 TFs using the reporter assay system. The translational fusion vector was constructed from the TF promoter sequence including an N-terminal proximal TF segment and the reporter GFP. At the beginning of cell growth, high-level expression was observed only for a small number of TFs. In the exponential phase, approximately 80 % TFs are expressed, but the expressed TF species change upon transfer to the stationary phase. Significant changes in the pattern of TF expression were observed between aerobic and anaerobic conditions. The list of intracellular levels of TFs provides further understanding to the transcription regulation of the E. coli genome under various stressful conditions.
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Control of chitin and N-acetylglucosamine utilization in Saccharopolyspora erythraea
Chitin degradation and subsequent N-acetylglucosamine (GlcNAc) catabolism is thought to be a common trait of a large majority of actinomycetes. Utilization of aminosugars had been poorly investigated outside the model strain Streptomyces coelicolor A3(2), and we examined here the genetic setting of the erythromycin producer Saccharopolyspora erythraea for GlcNAc and chitin utilization, as well as the transcriptional control thereof. Sacch. erythraea efficiently utilize GlcNAc most likely via the phosphotransferase system (PTSGlcNAc); however, this strain is not able to grow when chitin or N,N′-diacetylchitobiose [(GlcNAc)2] is the sole nutrient source, despite a predicted extensive chitinolytic system (chi genes). The inability of Sacch. erythraea to utilize chitin and (GlcNAc)2 is probably because of the loss of genes encoding the DasABC transporter for (GlcNAc)2 import, and genes for intracellular degradation of (GlcNAc)2 by β-N-acetylglucosaminidases. Transcription analyses revealed that in Sacch. erythraea all putative chi and GlcNAc utilization genes are repressed by DasR, whereas in Strep. coelicolor DasR displayed either activating or repressing functions whether it targets genes involved in the polymer degradation or genes for GlcNAc dimer and monomer utilization, respectively. A transcriptomic analysis further showed that GlcNAc not only activates the transcription of GlcNAc catabolism genes but also activates chi gene expression, as opposed to the previously reported GlcNAc-mediated catabolite repression in Strep. coelicolor. Finally, synteny exploration revealed an identical genetic background for chitin utilization in other rare actinomycetes, which suggests that screening procedures that used only the chitin-based protocol for selective isolation of antibiotic-producing actinomycetes could have missed the isolation of many industrially promising strains.
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Role of the yakA gene in morphogenesis and stress response in Penicillium marneffei
More LessPenicillium marneffei is a thermally dimorphic fungus and a highly significant pathogen of immunocompromised individuals living in or having travelled in south-east Asia. At 25 °C, P. marneffei grows filamentously. Under the appropriate conditions, these filaments (hyphae) produce conidiophores bearing chains of conidia. Yet, when incubated at 37 °C, or upon infecting host tissue, P. marneffei grows as a yeast that divides by binary fission. Previously, an Agrobacterium-mediated transformation system was used to randomly mutagenize P. marneffei, resulting in the isolation of a mutant defective in normal patterns of morphogenesis and conidiogenesis. The interrupted gene was identified as yakA. In the current study, we demonstrate that the yakA mutant produced fewer conidia at 25 °C than the wild-type and a complemented strain. In addition, disruption of the yakA gene resulted in early conidial germination and perturbation of cell wall integrity. The yakA mutant exhibited abnormal chitin distribution while growing at 25 °C, but not at 37 °C. Interestingly, at both temperatures, the yakA mutant possessed increased chitin content, which was accompanied by amplified transcription of two chitin synthase genes, chsB and chsG. Moreover, the expression of yakA was induced during post-exponential-phase growth as well as by heat shock. Thus, yakA is required for normal patterns of development, cell wall integrity, chitin deposition, appropriate chs expression and heat stress response in P. marneffei.
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Characterization of a novel two-partner secretion system implicated in the virulence of Pseudomonas aeruginosa
More LessPseudomonas aeruginosa is an opportunistic human pathogen implicated in nosocomial infection and infecting people with compromised immune systems such as cystic fibrosis patients. Although multiple genes involved in P. aeruginosa pathogenesis have been characterized, the overall mechanism of virulence is not fully understood. In this study, we identified a functional two-partner secretion (TPS) system, composed of the PdtA exoprotein and its cognate pore-forming β-barrel PdtB transporter, which is implicated in the virulence of P. aeruginosa. We found that the predicted PdtA exoprotein is related to the HMW-like adhesins subfamily TPS systems. We demonstrate here that limitation of inorganic phosphate (Pi) allows the production of PdtA protein. We show that PdtA is processed during its outer-membrane translocation, with an N-terminal domain released into the extracellular environment and a C-terminal domain associated with the outer membrane of the cell. We also obtained evidence that the transport of PdtA is strictly dependent on the production of PdtB, a result confirming that these proteins constitute a functional TPS system. Furthermore, using the Caenorhabditis elegans model of infection, we show that a pdtA mutant is less virulent than the wild-type strain.
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- Environmental and Evolutionary Microbiology
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Sequence and expression divergence of an ancient duplication of the chaperonin groESEL operon in Vibrio species
Heat-shock proteins are molecular chaperones essential for protein folding, degradation and trafficking. The human pathogen Vibrio vulnificus encodes a copy of the groESEL operon in both chromosomes and these genes share <80 % similarity with each other. Comparative genomic analysis was used to determine whether this duplication is prevalent among Vibrionaceae specifically or Gammaproteobacteria in general. Among the Vibrionaceae complete genome sequences in the database (31 species), seven Vibrio species contained a copy of groESEL in each chromosome, including the human pathogens Vibrio cholerae, Vibrio parahaemolyticus and V. vulnificus. Phylogenetic analysis of GroEL among the Gammaproteobacteria indicated that GroESEL-1 encoded in chromosome I was the ancestral copy and GroESEL-2 in chromosome II arose by an ancient gene duplication event. Interestingly, outside of the Vibrionaceae within the Gammaproteobacteria, groESEL chromosomal duplications were rare among the 296 genomes examined; only five additional species contained two or more copies. Examination of the expression pattern of groEL from V. vulnificus cells grown under different conditions revealed differential expression between the copies. The data demonstrate that groEL-1 was more highly expressed during growth in exponential phase than groEL-2 and a similar pattern was also found in both V. cholerae and V. parahaemolyticus. Overall these data suggest that retention of both copies of groESEL in Vibrio species may confer an evolutionary advantage.
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Adherence to abiotic surface induces SOS response in Escherichia coli K-12 strains under aerobic and anaerobic conditions
During the colonization of surfaces, Escherichia coli bacteria often encounter DNA-damaging agents and these agents can induce several defence mechanisms. Base excision repair (BER) is dedicated to the repair of oxidative DNA damage caused by reactive oxygen species (ROS) generated by chemical and physical agents or by metabolism. In this work, we have evaluated whether the interaction with an abiotic surface by mutants derived from E. coli K-12 deficient in some enzymes that are part of BER causes DNA damage and associated filamentation. Moreover, we studied the role of endonuclease V (nfi gene; 1506 mutant strain) in biofilm formation. Endonuclease V is an enzyme that is involved in DNA repair of nitrosative lesions. We verified that endonuclease V is involved in biofilm formation. Our results showed more filamentation in the xthA mutant (BW9091) and triple xthA nfo nth mutant (BW535) than in the wild-type strain (AB1157). By contrast, the mutant nfi did not present filamentation in biofilm, although its wild-type strain (1466) showed rare filaments in biofilm. The filamentation of bacterial cells attaching to a surface was a consequence of SOS induction measured by the SOS chromotest. However, biofilm formation depended on the ability of the bacteria to induce the SOS response since the mutant lexA Ind− did not induce the SOS response and did not form any biofilm. Oxygen tension was an important factor for the interaction of the BER mutants, since these mutants exhibited decreased quantitative adherence under anaerobic conditions. However, our results showed that the presence or absence of oxygen did not affect the viability of BW9091 and BW535 strains. The nfi mutant and its wild-type did not exhibit decreased biofilm formation under anaerobic conditions. Scanning electron microscopy was also performed on the E. coli K-12 strains that had adhered to the glass, and we observed the presence of a structure similar to an extracellular matrix that depended on the oxygen tension. In conclusion, it was proven that bacterial interaction with abiotic surfaces can lead to SOS induction and associated filamentation. Moreover, we verified that endonuclease V is involved in biofilm formation.
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- Genes and Genomes
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Genome comparison of three serovar 5 pathogenic strains of Haemophilus parasuis: insights into an evolving swine pathogen
More LessHaemophilus parasuis is the causative agent of Glässer’s disease, a systemic disorder characterized by polyarthritis, polyserositis and meningitis in pigs. Although it is well known that H. parasuis serovar 5 is the most prevalent serovar associated with the disease, the genetic differences among strains are only now being discovered. Genomes from two serovar 5 strains, SH0165 and 29755, are already available. Here, we present the draft genome of a third H. parasuis serovar 5 strain, the formal serovar 5 reference strain Nagasaki. An in silico genome subtractive analysis with full-length predicted genes of the three H. parasuis serovar 5 strains detected 95, 127 and 95 strain-specific genes (SSGs) for Nagasaki, SH0165 and 29755, respectively. We found that the genomic diversity within these three strains was high, in part because of a high number of mobile elements. Furthermore, a detailed analysis of large sequence polymorphisms (LSPs), encompassing regions ranging from 2 to 16 kb, revealed LSPs in virulence-related elements, such as a Toll-IL receptor, the AcrA multidrug efflux protein, an ATP-binding cassette (ABC) transporter, lipopolysaccharide-synthetizing enzymes and a tripartite ATP-independent periplasmic (TRAP) transporter. The whole-genome codon adaptation index (CAI) was also calculated and revealed values similar to other well-known bacterial pathogens. In addition, whole-genome SNP analysis indicated that nucleotide changes tended to be increased in membrane-related genes. This analysis provides further evidence that the genome of H. parasuis has been subjected to multiple lateral gene transfers (LGTs) and to fine-tuning of virulence factors, and has the potential for accelerated genome evolution.
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Transcriptomic analysis of the role of Rim101/PacC in the adaptation of Ustilago maydis to an alkaline environment
More LessAlkaline pH triggers an adaptation mechanism in fungi that is mediated by Rim101/PacCp, a zinc finger transcription factor. To identify the genes under its control in Ustilago maydis, we performed microarray analyses, comparing gene expression in a wild-type strain versus a rim101/pacC mutation strain of the fungus. In this study we obtained evidence of the large number of genes regulated mostly directly, but also indirectly (probably through regulation of other transcription factors), by Rim101/PacCp, including proteins involved in a large number of physiological activities of the fungus. Our analyses suggest that the response to alkaline conditions under the control of the Pal/Rim pathway involves changes in the cell wall and plasma membrane through alterations in their lipid, protein and polysaccharide composition, changes in cell polarity, actin cytoskeleton organization, and budding patterns. Also as expected, adaptation involves regulation by Rim101/PacC of genes involved in meiotic functions, such as recombination and segregation, and expression of genes involved in ion and nutrient transport, as well as general vacuole functions.
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- Microbial Pathogenicity
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Salmonella methylglyoxal detoxification by STM3117-encoded lactoylglutathione lyase affects virulence in coordination with Salmonella pathogenicity island 2 and phagosomal acidification
More LessIntracellular pathogens such as Salmonella enterica serovar Typhimurium (S. Typhimurium) manipulate their host cells through the interplay of various virulence factors. A multitude of such virulence factors are encoded on the genome of S. Typhimurium and are usually organized in pathogenicity islands. The virulence-associated genomic stretch of STM3117–3120 has structural features of pathogenicity islands and is present exclusively in non-typhoidal serovars of Salmonella. It encodes metabolic enzymes predicted to be involved in methylglyoxal metabolism. STM3117-encoded lactoylglutathione lyase significantly impacts the proliferation of intracellular Salmonella. The deletion mutant of STM3117 (Δlgl) fails to grow in epithelial cells but hyper-replicates in macrophages. This difference in proliferation outcome was the consequence of failure to detoxify methylglyoxal by Δlgl, which was also reflected in the form of oxidative DNA damage and upregulation of kefB in the mutant. Within macrophages, the toxicity of methylglyoxal adducts elicits the potassium efflux channel (KefB) in the mutant which subsequently modulates the acidification of mutant-containing vacuoles (MCVs). The perturbation in the pH of the MCV milieu and bacterial cytosol enhances the Salmonella pathogenicity island 2 translocation in Δlgl, increasing its net growth within macrophages. In epithelial cells, however, the maturation of Δlgl-containing vacuoles were affected as these non-phagocytic cells maintain less acidic vacuoles compared to those in macrophages. Remarkably, ectopic expression of Toll-like receptors 2 and 4 on epithelial cells partially restored the survival of Δlgl. This study identified a novel metabolic enzyme in S. Typhimurium whose activity during intracellular infection within a given host cell type differentially affected the virulence of the bacteria.
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Dual role of MsRbpA: transcription activation and rescue of transcription from the inhibitory effect of rifampicin
More LessMsRbpA is an RNA polymerase (RNAP) binding protein from Mycobacterium smegmatis. According to previous studies, MsRbpA rescues rifampicin-induced transcription inhibition upon binding to the RNAP. Others have shown that RbpA from Mycobacterium tuberculosis (MtbRbpA) is a transcription activator. In this study, we report that both MsRbpA and MtbRbpA activate transcription as well as rescue rifampicin-induced transcription inhibition. Transcription activation is achieved through the increased formation of closed RNAP–promoter complex as well as enhanced rate of conversion of this complex to a stable transcriptionally competent RNAP–promoter complex. When a 16 aa peptide fragment (Asp 58 to Lys 73) was deleted from MsRbpA, the resulting protein showed 1000-fold reduced binding with core RNAP. The deletion results in abolition of transcription activation and rescue of transcription from the inhibitory effect of rifampicin. Through alanine scanning of this essential region of MsRbpA, Gly 67, Val 69, Pro 70 and Pro 72 residues are identified to be important for MsRbpA function. Furthermore, we report here that the protein is indispensable for M. smegmatis, and it appears to help the organism grow in the presence of the antibiotic rifampicin.
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Plasticity of fimbrial genotype and serotype within populations of Bordetella pertussis: analysis by paired flow cytometry and genome sequencing
The fimbriae of Bordetella pertussis are required for colonization of the human respiratory tract. Two serologically distinct fimbrial subunits, Fim2 and Fim3, considered important vaccine components for many years, are included in the Sanofi Pasteur 5-component acellular pertussis vaccine, and the World Health Organization recommends the inclusion of strains expressing both fimbrial serotypes in whole-cell pertussis vaccines. Each of the fimbrial major subunit genes, fim2, fim3, and fimX, has a promoter poly(C) tract upstream of its −10 box. Such monotonic DNA elements are susceptible to changes in length via slipped-strand mispairing in vitro and in vivo, which potentially causes on/off switching of genes at every cell division. Here, we have described intra-culture variability in poly(C) tract lengths and the resulting fimbrial phenotypes in 22 recent UK B. pertussis isolates. Owing to the highly plastic nature of fimbrial promoters, we used the same cultures for both genome sequencing and flow cytometry. Individual cultures of B. pertussis contained multiple fimbrial serotypes and multiple different fimbrial promoter poly(C) tract lengths, which supports earlier serological evidence that B. pertussis expresses both serotypes during infection.
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- Physiology and Biochemistry
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Structure of the Zymomonas mobilis respiratory chain: oxygen affinity of electron transport and the role of cytochrome c peroxidase
The genome of the ethanol-producing bacterium Zymomonas mobilis encodes a bd-type terminal oxidase, cytochrome bc 1 complex and several c-type cytochromes, yet lacks sequences homologous to any of the known bacterial cytochrome c oxidase genes. Recently, it was suggested that a putative respiratory cytochrome c peroxidase, receiving electrons from the cytochrome bc 1 complex via cytochrome c 552, might function as a peroxidase and/or an alternative oxidase. The present study was designed to test this hypothesis, by construction of a cytochrome c peroxidase mutant (Zm6-perC), and comparison of its properties with those of a mutant defective in the cytochrome b subunit of the bc 1 complex (Zm6-cytB). Disruption of the cytochrome c peroxidase gene (ZZ60192) caused a decrease of the membrane NADH peroxidase activity, impaired the resistance of growing culture to exogenous hydrogen peroxide and hampered aerobic growth. However, this mutation did not affect the activity or oxygen affinity of the respiratory chain, or the kinetics of cytochrome d reduction. Furthermore, the peroxide resistance and membrane NADH peroxidase activity of strain Zm6-cytB had not decreased, but both the oxygen affinity of electron transport and the kinetics of cytochrome d reduction were affected. It is therefore concluded that the cytochrome c peroxidase does not terminate the cytochrome bc 1 branch of Z. mobilis, and that it is functioning as a quinol peroxidase.
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Volumes and issues
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Volume 74 (1973)
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Volume 73 (1972)
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Volume 72 (1972)
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Volume 71 (1972)
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Volume 70 (1972)
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Volume 69 (1971)
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Volume 68 (1971)
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Volume 67 (1971)
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Volume 66 (1971)
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Volume 65 (1971)
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Volume 64 (1970)
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Volume 63 (1970)
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Volume 62 (1970)
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Volume 61 (1970)
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Volume 60 (1970)
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Volume 59 (1969)
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Volume 58 (1969)
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Volume 57 (1969)
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Volume 56 (1969)
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Volume 55 (1969)
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Volume 54 (1968)
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Volume 53 (1968)
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Volume 52 (1968)
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Volume 51 (1968)
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Volume 50 (1968)
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Volume 49 (1967)
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Volume 48 (1967)
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Volume 47 (1967)
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Volume 46 (1967)
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Volume 45 (1966)
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Volume 44 (1966)
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Volume 43 (1966)
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Volume 42 (1966)
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Volume 41 (1965)
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Volume 40 (1965)
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Volume 39 (1965)
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Volume 38 (1965)
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Volume 37 (1964)
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Volume 36 (1964)
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Volume 35 (1964)
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Volume 34 (1964)
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Volume 33 (1963)
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Volume 32 (1963)
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Volume 31 (1963)
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Volume 30 (1963)
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Volume 29 (1962)
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Volume 28 (1962)
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Volume 27 (1962)
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Volume 26 (1961)
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Volume 25 (1961)
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Volume 24 (1961)
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Volume 23 (1960)
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Volume 22 (1960)
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Volume 21 (1959)
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Volume 20 (1959)
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Volume 19 (1958)
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Volume 18 (1958)
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Volume 17 (1957)
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Volume 16 (1957)
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Volume 15 (1956)
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Volume 14 (1956)
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Volume 13 (1955)
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Volume 12 (1955)
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Volume 11 (1954)
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Volume 10 (1954)
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Volume 9 (1953)
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Volume 8 (1953)
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Volume 7 (1952)
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Volume 6 (1952)
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Volume 5 (1951)
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Volume 4 (1950)
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Volume 3 (1949)
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Volume 2 (1948)
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Volume 1 (1947)
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