- Volume 154, Issue 9, 2008
Volume 154, Issue 9, 2008
- Review
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Anti-silencing: overcoming H-NS-mediated repression of transcription in Gram-negative enteric bacteria
More LessThe H-NS nucleoid-associated DNA-binding protein is an important global repressor of transcription in Gram-negative bacteria. Recently, H-NS has been implicated in the process of xenogeneic silencing, where it represses the transcription of foreign genes acquired by horizontal transfer. This raises interesting questions about the integration of the horizontally acquired genes into the existing gene regulatory networks of the microbe. In particular, how do bacteria derepress silenced genes in order to benefit from their expression without compromising competitive fitness through doing so inappropriately? This article reviews current knowledge about the derepression of genes that are transcriptionally silenced by H-NS. It describes a variety of anti-silencing mechanisms involving (i) protein-independent processes that operate at the level of local DNA structure, (ii) DNA-binding proteins such as Ler, LeuO, RovA, SlyA, VirB, and proteins related to AraC, and (iii) modulatory mechanisms in which H-NS forms heteromeric protein–protein complexes with full-length or partial paralogues such as StpA, Sfh, Hha, YdgT, YmoA or H-NST. The picture that emerges is one of apparently ad hoc solutions to the problem of H-NS-mediated silencing, suggesting that microbes are capable of evolving anti-silencing methods based on the redeployment of existing regulatory proteins rather than employing dedicated, bespoke antagonists. There is also evidence that in a number of cases more sophisticated regulatory processes have been superimposed on these rather simple anti-silencing mechanisms, broadening the range of environmental signals to which H-NS-repressed genes respond.
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- Biochemistry And Molecular Biology
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Effect of upstream curvature and transcription factors H-NS and LRP on the efficiency of Escherichia coli rRNA promoters P1 and P2 – a phasing analysis
More LessTo study the influence of DNA curvature and DNA-binding proteins, which interact with curved DNA on bacterial promoters, we constructed two sets of promoter variants in which a synthetic DNA-bending module was fused at defined distances and angular orientations with respect to the transcription start sites. The distance between the synthetic binding site centre and the transcription start site of the different constructs varied by up to 20 bp, corresponding to almost two complete helical B-DNA turns. The rRNA promoters rrnB P1 and rrnB P2 were selected as target promoters. While in its natural context P1 depends on upstream curved DNA and several transcription factors that bind to this region, promoter P2 is not preceded by curved DNA, nor is it believed to be directly regulated by transcription factors. In vitro transcription measurements of both promoters in the absence of transcription factors varied with the phase of the curved upstream DNA element, underlining the importance of DNA conformation to promoter efficiency. Specific binding of H-NS and LRP to the curved DNA element was demonstrated by gel shift and footprint analysis. Binding affinity was not notably altered for the different distance variants. We demonstrated that the two proteins acted as repressors for both promoters. The extent of H-NS-mediated repression for both promoters did not vary strongly with the phasing of the upstream binding module. In contrast, LRP-dependent repression showed a clear dependence on the angular orientation of the constructs. Phasing-dependent repression is very distinct for P2 but only rudimentary for the P1 promoter.
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Genetic and physiological diversity of Tetragenococcus halophilus strains isolated from sugar- and salt-rich environments
Tetragenococcus halophilus is known to flourish in extreme salt environments. Recently, this halophilic bacterium also appeared as the dominant microflora during storage of sugar thick juice, an intermediate product of beet sugar production. Although T. halophilus can cause degradation of thick juice, dominance of this bacterium does not always result in degradation. In this study T. halophilus strains from high-salt and high-sugar environments, and in particular from degraded and non-degraded thick juice, were compared in detail. Both physiological and genetic characterization using Biolog, repetitive PCR fingerprinting (rep-PCR) and random amplified polymorphic DNA (RAPD) technology, revealed clear differences between T. halophilus strains isolated from salt- and sugar-rich environments. However, no strain pattern could be specifically and systematically associated with degraded or non-degraded thick juice. Remarkably, halophilic T. halophilus strains were not able to grow in sugar thick juice. Irrespective of the differences between the strains from high-salt or high-sugar environments, DNA–DNA hybridization grouped all strains within the species T. halophilus, except one isolate from sugar thick juice that showed different physiological and genetic characteristics, and that may represent a new species of Tetragenococcus.
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Changes in membrane lipid composition in ethanol- and acid-adapted Oenococcus oeni cells: characterization of the cfa gene by heterologous complementation
Cyclopropane fatty acid (CFA) synthesis was investigated in Oenococcus oeni. The data obtained demonstrated that acid-grown cells or cells harvested in the stationary growth phase showed changes in fatty acid composition similar to those of ethanol-grown cells. An increase of the CFA content and a decrease of the oleic acid content were observed. The biosynthesis of CFAs from unsaturated fatty acid phospholipids is catalysed by CFA synthases. Quantitative real-time-PCR experiments were performed on the cfa gene of O. oeni, which encodes a putative CFA synthase. The level of cfa transcripts increased when cells were harvested in stationary phase and when cells were grown in the presence of ethanol or at low pH, suggesting transcriptional regulation of the cfa gene under different stress conditions. In contrast to Escherichia coli, only one functional promoter was identified upstream of the cfa gene of O. oeni. The function of the cfa gene was confirmed by complementation of a cfa-deficient E. coli strain. Nevertheless, the complementation remained partial because the conversion percentage of unsaturated fatty acids into CFA of the complemented strain was much lower than that of the wild-type strain. Moreover, a prevalence of cycC19 : 0 was observed in the membrane of the complemented strain. This could be due to a specific affinity of the CFA synthase from O. oeni. In spite of this partial complementation, the complemented strain of E. coli totally recovered its viability after ethanol shock (10 %, v/v) whereas its viability was only partly recovered after an acid shock at pH 3.0.
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Production of curcuminoids by Escherichia coli carrying an artificial biosynthesis pathway
More LessCurcuminoids, which are produced specifically by plants of the order Zingiberales, have long been used as food additives because of their aromatic, stimulant and colouring properties and as traditional Asian medicines because of their anti-tumour, antioxidant and hepatoprotective activities. Curcuminoids are therefore attractive targets for metabolic engineering. An artificial curcuminoid biosynthetic pathway, including reactions of phenylalanine ammonia-lyase (PAL) from the yeast Rhodotorula rubra, 4-coumarate : CoA ligase (4CL) from Lithospermum erythrorhizon and curcuminoid synthase (CUS) from rice (Oryza sativa), a type III polyketide synthase, was constructed in Escherichia coli for the production of curcuminoids. Cultivation of the recombinant E. coli cells in the presence of tyrosine or phenylalanine, or both, led to production of bisdemethoxycurcumin, dicinnamoylmethane and cinnamoyl-p-coumaroylmethane. Another E. coli system carrying 4CL and CUS genes was also used for high-yield production of curcuminoids from exogenously supplemented phenylpropanoid acids: p-coumaric acid, cinnamic acid and ferulic acid. The yields of curucminoids were up to ∼100 mg l−1. Furthermore, this system gave approximately 60 mg curcumin l−1 from 10 g rice bran pitch, an industrial waste discharged during rice edible oil production, as a source of ferulic acid.
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Lactobacillus plantarum response to inorganic carbon concentrations: PyrR2-dependent and -independent transcription regulation of genes involved in arginine and nucleotide metabolism
More LessLactobacillus plantarum susbp. plantarum is a capnophilic Gram-positive heterotroph with optimal growth in 4 % CO2-enriched air. At low inorganic carbon (Ci) concentrations, the pyr genes encoding the enzymes of the pyrimidine biosynthetic pathway were overexpressed, in agreement with a previous study showing that these genes are regulated at the transcription level in response to Ci via a PyrR2-mediated mechanism. A previous study of high-CO2-requiring (HCR) mutants revealed an unknown genetic link between arginine regulation and Ci-dependent nutritional needs. To better understand L. plantarum's adaptation to Ci availability, additional Ci-responsive genes were sought in the arginine biosynthetic pathway (arg and car genes) using slot-blot hybridization and a proteomic differential 2D gel electrophoresis (DIGE) global approach. Besides the nine pyr-encoded proteins, 16 new Icr (inorganic-carbon-regulated) proteins accumulated differentially in response to Ci availability, suggesting that the Ci response involves several metabolic pathways and adaptation processes. Among these Icr proteins only argininosuccinate lyase, encoded by argH, was involved in arginine biosynthesis. Three proteins involved in the purine biosynthetic pathway and nucleotide conversion, adenylate kinase (Adk), GMP synthase (GuaA), and IMP dehydrogenase (GuaB), accumulated differentially in response to changes in Ci levels. Expression of the Icr protein-encoding genes argH and guaB was regulated at the transcription level or by RNA stability in response to Ci availability, as previously demonstrated for the pyr genes. However, PyrR2 was not essential for the Ci-regulated transcription of argH and guaB, demonstrating that PyrR2 modulates only a subset of Ci-regulated genes. These results suggest that the Ci response may involve at least two regulatory mechanisms in L. plantarum.
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Functional role of Trp-105 of Enterococcus faecalis azoreductase (AzoA) as resolved by structural and mutational analysis
More LessEnterococcus faecalis azoreductase (AzoA) is a very active enzyme with a broad spectrum of substrate specificity and is capable of degrading various azo dyes. The enzyme has an absolute requirement for reduced FMN, which delivers a total of four electrons from NADH to the substrate, resulting in the cleavage of the nitrogen double bond. In this study, we report the identification of amino acid residues critical for FMN binding in AzoA. FMN is stabilized by 22 amino acid residues, eight of which, Trp-105, Asn-106, Leu-107, Gly-150, Gly-151, Tyr-153, Asn-121 and Tyr-129, are involved in binding the FMN isoalloxazine ring. In silico analysis of the amino acid residues revealed that the Trp residue at position 105 of AzoA is the most likely significant contributor to the binding of FMN to the enzyme and is involved in FMN stabilization and destabilization. Site-directed mutagenesis analysis of Trp-105 was performed to determine the role of this amino acid residue in FMN binding and azo dye reductive activity. The mutant proteins were overexpressed in Escherichia coli and purified by anion-exchange and size-exclusion chromatography. The replacement of Trp-105 by the small side-chain amino acids Ala and Gly caused complete loss of both affinity for FMN and enzyme activity. Substitution of Tyr for Trp-105 did not significantly decrease the V max of the enzyme (22 % reduction). Substitutions with three bulky side-chain amino acids, Gln, Phe and His, produced enzymes with lower V max values (decreases of 68.2, 30.6 and 8.2-fold, respectively). However, these mutated enzymes maintained K m values similar to the wild-type enzyme. This study provides an insight into the catalytic properties of AzoA in FMN stabilization and enzyme activity.
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The Salmonella SPI-2 effector SseJ exhibits eukaryotic activator-dependent phospholipase A and glycerophospholipid : cholesterol acyltransferase activity
More LessIntracellular replication of Salmonella enterica serovar Typhimurium within membrane-bound compartments, called Salmonella-containing vacuoles, depends on the activities of several effector proteins translocated by the Salmonella pathogenicity island 2 (SPI-2)-encoded type III secretion system. The SPI-2 effector protein SseJ shows similarity at the amino acid level to several GDSL lipases with glycerophospholipid : cholesterol acyltransferase (GCAT) activity. In this study, we show that catalytic serine-dependent phospholipase A (PLA) and GCAT activity of recombinant SseJ is potentiated by factor(s) present in HeLa cells, RAW macrophages and Saccharomyces cerevisiae. SseJ activity was enhanced with increasing amounts of, or preincubation with, eukaryotic cell extracts. Analysis of the activating factor(s) shows that it is soluble and heat- and protease-sensitive. We conclude that PLA and GCAT activities of SseJ are potentiated by proteinaceous eukaryotic factor(s).
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Two distinct arabinofuranosidases contribute to arabino-oligosaccharide degradation in Bacillus subtilis
More LessBacillus subtilis produces α-l-arabinofuranosidases (EC 3.2.1.55; AFs) capable of releasing arabinosyl oligomers and l-arabinose from plant cell walls. Here, we show by insertion-deletion mutational analysis that genes abfA and xsa(asd), herein renamed abf2, encode AFs responsible for the majority of the intracellular AF activity in B. subtilis. Both enzyme activities were shown to be cytosolic and functional studies indicated that arabino-oligomers are natural substrates for the AFs. The products of the two genes were overproduced in Escherichia coli, purified and characterized. The molecular mass of the purified AbfA and Abf2 was about 58 kDa and 57 kDa, respectively. However, native PAGE gradient gel analysis and cross-linking assays detected higher-order structures (>250 kDa), suggesting a multimeric organization of both enzymes. Kinetic experiments at 37 °C, with p-nitrophenyl-α-l-arabinofuranoside as substrate, gave an apparent K m of 0.498 mM and 0.421 mM, and V max of 317 U mg−1 and 311 U mg−1 for AbfA and Abf2, respectively. The two enzymes displayed maximum activity at 50 °C and 60 °C, respectively, and both proteins were most active at pH 8.0. AbfA and Abf2 both belong to family 51 of the glycoside hydrolases but have different substrate specificity. AbfA acts preferentially on (1→5) linkages of linear α-1,5-l-arabinan and α-1,5-linked arabino-oligomers, and is much less effective on branched sugar beet arabinan and arabinoxylan and arabinogalactan. In contrast, Abf2 is most active on (1→2) and (1→3) linkages of branched arabinan and arabinoxylan, suggesting a concerted contribution of these enzymes to optimal utilization of arabinose-containing polysaccharides by B. subtilis.
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GDP-mannose pyrophosphorylase is essential for cell wall integrity, morphogenesis and viability of Aspergillus fumigatus
More LessGDP-mannose pyrophosphorylase (GMPP) catalyses the synthesis of GDP-mannose, which is the precursor for the mannose residues in glycoconjugates, using mannose 1-phosphate and GTP as substrates. Repression of GMPP in yeast leads to phenotypes including cell lysis, defective cell wall, and failure of polarized growth and cell separation. Although several GMPPs have been isolated and characterized in filamentous fungi, the physiological consequences of their actions are not clear. In this study, Afsrb1, which is a homologue of yeast SRB1/PSA1/VIG9, was identified in the Aspergillus fumigatus genome. The Afsrb1 gene was expressed in Escherichia coli, and recombinant AfSrb1 was functionally confirmed as a GMPP. By the replacement of the native Afsrb1 promoter with an inducible Aspergillus nidulans alcA promoter, the conditional inactivation mutant strain YJ-gmpp was constructed. The presence of 3 % glucose completely blocked transcription of P alcA –Afsrb1, and was lethal to strain YJ-gmpp. Repression of Afsrb1 expression in strain YJ-gmpp led to phenotypes including hyphal lysis, defective cell wall, impaired polarity maintenance, and branching site selection. Also, rapid germination and reduced conidiation were documented. However, in contrast to yeast, strain YJ-gmpp retained the ability to direct polarity establishment and septation. Our results showed that the Afsrb1 gene is essential for cell wall integrity, morphogenesis and viability of Aspergillus fumigatus.
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Mitochondrial involvement in aspirin-induced apoptosis in yeast
More LessWe have previously reported that aspirin induces apoptosis in manganese superoxide dismutase (MnSOD)-deficient Saccharomyces cerevisiae cells when cultivated on the non-fermentable carbon source ethanol. Here, we investigated the role of mitochondria in aspirin-induced apoptosis. We report that aspirin had an inhibitory effect on cellular respiration, and caused the release of most of the mitochondrial cytochrome c and a dramatic drop in the mitochondrial membrane potential (ΔΨm). Also, aspirin reduced the intracellular cytosolic pH in the MnSOD-deficient cells growing in ethanol medium, but this did not seem to be the initial trigger that committed these cells to aspirin-induced apoptosis. Furthermore, loss of ΔΨm was not required for aspirin-induced release of cytochrome c, since the initial release of cytochrome c occurred prior to the disruption of the ΔΨm. It is thus possible that cytochrome c release does not involve the early onset of the mitochondrial permeability transition, but only an alteration of the permeability of the outer mitochondrial membrane.
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Candida albicans UPC2 is transcriptionally induced in response to antifungal drugs and anaerobicity through Upc2p-dependent and -independent mechanisms
More LessMany genes in the Candida albicans ergosterol biosynthetic pathway are controlled by the transcriptional activator Upc2p, which is upregulated in the presence of azole drugs and has been suggested to regulate its own transcription by an autoregulatory mechanism. The UPC2 promoter was cloned upstream of a luciferase reporter gene (RLUC). UPC2–RLUC activity was induced in response to ergosterol biosynthesis inhibitors and in response to anaerobicity. Under both conditions, induction correlates with the magnitude of sterol depletion. Azole inducibility in the parental strain was approximately 100-fold, and in a UPC2 homozygous deletion strain was 17-fold, suggesting that, in addition to autoregulation, UPC2 transcription is controlled by a novel, Upc2p-independent mechanism(s). Curiously, basal UPC2–RLUC activity was fivefold higher in the deletion strain, which may be an indirect consequence of the lower sterol level in this strain, or a direct consequence of repression by an autoregulatory mechanism. These results suggest that transcriptional regulation of UPC2 expression is important in the response to antifungal drugs, and that this regulation occurs through Upc2p-dependent as well as novel Upc2p-independent mechanisms.
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Biochemical characterization of a mitochondrial-like organelle from Blastocystis sp. subtype 7
More LessA mitochondrion-like organelle (MLO) was isolated from isotonic homogenates of Blastocystis. The organelle sedimented at 5000 g for 10 min, and had an isopycnic density in sucrose of 1.2 g ml−1. Biochemical characterization enabled the demonstration of several key enzymes that allowed the construction of a metabolic pathway consisting of an incomplete Krebs cycle linked to the oxygen-sensitive enzymes pyruvate : NADP+ oxidoreductase (PNO), acetate : succinate CoA transferase (ASCT) and succinate thiokinase (STK), which cumulatively are responsible for recycling CoA and generating ATP. The organelle differs from typical aerobic mitochondria in possessing an oxygen-sensitive PNO that can use FAD+ or FMN+ as electron acceptor but is inactive with NAD+, Spinacia oleracea ferredoxin or Clostridium pasteurianum ferredoxin. A gene with 77 % sequence similarity to the PNO mitochondrion precursor cluster from Euglena gracilis sp[Q941N5] was identified in the Blastocystis genome database. A second cluster with 56 % sequence similarity to the pyruvate : ferredoxin oxidoreductase (PFOR) from Trichomonas vaginalis was also identified, which is in agreement with the concept that the PNO gene arose through the fusion of a eubacterial gene for PFOR with the gene for NADPH : cytochrome p450 reductase. Hydrogenase activity was not detected under the conditions used in this study. The Blastocystis oranelle therefore demonstrates significant biochemical differences from traditional mitochondria and hydrogenosomes, but possesses features of both. Based upon the results of this study, the Blastocystis organelle falls into the category of a MLO.
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Moonlighting function of glutamate racemase from Mycobacterium tuberculosis: racemization and DNA gyrase inhibition are two independent activities of the enzyme
More LessGlutamate racemase (MurI) provides d-glutamate, a key building block in the peptidoglycan of the bacterial cell wall. Besides having a crucial role in cell wall biosynthesis, MurI proteins from some bacteria have been shown to act as an inhibitor of DNA gyrase. Mycobacterium tuberculosis and Mycobacterium smegmatis MurI exhibit these dual characteristics. Here, we show that the two activities of M. tuberculosis MurI are unlinked and independent of each other. The racemization function of MurI is not essential for its gyrase-inhibitory property. MurI–DNA gyrase interaction influences gyrase activity but has no effect on the racemization activity of MurI. Overexpression of MurI in vivo provides resistance to the action of ciprofloxacin, suggesting the importance of the interaction in gyrase modulation. We propose that the moonlighting activity of MurI has evolved more recently than its racemase function, to play a transient yet important role in gyrase modulation.
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Membrane-association determinants of the ω-amino acid monooxygenase PvdA, a pyoverdine biosynthetic enzyme from Pseudomonas aeruginosa
The l-ornithine N δ -oxygenase PvdA catalyses the N δ -hydroxylation of l-ornithine in many Pseudomonas spp., and thus provides an essential enzymic function in the biogenesis of the pyoverdine siderophore. Here, we report a detailed analysis of the membrane topology of the PvdA enzyme from the bacterial pathogen Pseudomonas aeruginosa. Membrane topogenic determinants of PvdA were identified by computational analysis, and verified in Escherichia coli by constructing a series of translational fusions between PvdA and the PhoA (alkaline phosphatase) reporter enzyme. The inferred topological model resembled a eukaryotic reverse signal-anchor (type III) protein, with a single N-terminal domain anchored to the inner membrane, and the bulk of the protein spanning the cytosol. According to this model, the predicted transmembrane region should overlap the putative FAD-binding site. Cell fractionation and proteinase K accessibility experiments in P. aeruginosa confirmed the membrane-bound nature of PvdA, but excluded the transmembrane topology of its N-terminal hydrophobic region. Mutational analysis of PvdA, and complementation assays in a P. aeruginosa ΔpvdA mutant, demonstrated the dual (structural and functional) role of the PvdA N-terminal domain.
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The pathway by which the yeast protein kinase Snf1p controls acquisition of sodium tolerance is different from that mediating glucose regulation
More LessIt recently became apparent that the highly conserved Snf1p protein kinase plays roles in controlling different cellular processes in the yeast Saccharomyces cerevisiae, in addition to its well-known function in glucose repression/derepression. We have previously reported that Snf1p together with Gis4p controls ion homeostasis by regulating expression of ENA1, which encodes the Ena1p Na+ extrusion system. In this study we found that Snf1p is rapidly phosphorylated when cells are exposed to NaCl and this phosphorylation is required for the role of Snf1p in Na+ tolerance. In contrast to activation by low glucose levels, the salt-induced phosphorylation of Snf1p promoted neither phosphorylation nor nuclear export of the Mig1p repressor. The mechanism that prevents Mig1p phosphorylation by active Snf1p under salt stress does not involve either hexokinase PII or the Gis4p regulator. Instead, Snf1p may mediate upregulation of ENA1 expression via the repressor Nrg1p. Activation of Snf1p in response to glucose depletion requires any of the three upstream protein kinases Sak1p, Tos3p and Elm1p, with Sak1p playing the most prominent role. The same upstream kinases were required for salt-induced Snf1p phosphorylation, and also under these conditions Sak1p played the most prominent role. Unexpectedly, however, it appears that Elm1p plays a dual role in acquisition of salt tolerance by activating Snf1p and in a presently unknown parallel pathway. Together, these results indicate that under salt stress Snf1p takes part in a different pathway from that during glucose depletion and this role is performed together as well as in parallel with its upstream kinase Elm1p. Snf1p appears to be part of a wider functional network than previously anticipated and the full complexity of this network remains to be elucidated.
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Bacterial partitioning proteins affect the subcellular location of broad-host-range plasmid RK2
More LessIt has been demonstrated that plasmids are not randomly distributed but are located symmetrically in mid-cell, or ¼, ¾ positions in bacterial cells. In this work we compared the localization of broad-host-range plasmid RK2 mini-replicons, which lack an active partitioning system, in Escherichia coli and Pseudomonas putida cells. In E. coli the location of the plasmid mini-replicon cluster was at the cell poles. In contrast, in Pseudomonas cells, as a result of the interaction of chromosomally encoded ParB protein with RK2 centromere-like sequences, these mini-derivatives were localized in the proximity of mid-cell, or ¼, ¾ positions. The expression of the Pseudomonas parAB genes in E. coli resulted in a positional change in the RK2 mini-derivative to the mid-cell or ¼, ¾ positions. Moreover, in a P. putida parAB mutant, both RK2 mini-derivatives and the entire RK2 plasmid exhibited disturbances of subcellular localization. These observations raise the possibility that in certain bacteria chromosomally encoded partitioning machinery could affect subcellular plasmid positioning.
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Roles of c-type cytochromes in respiration in Neisseria meningitidis
More LessThree c-type cytochromes were identified in Neisseria meningitidis, based on predictions from genome sequences, that were hypothesized to be involved in electron transport to terminal electron acceptor reductases for oxygen (the cytochrome cbb 3 oxidase) and nitrite (the nitrite reductase, AniA). Mutants were generated by allelic exchange with disrupted copies of the genes encoding these cytochromes and the phenotypes of the resultant mutants analysed. It was found that cytochrome c 5 is required for in vivo nitrite reductase activity, whereas cytochromes c x and c 4 are both required for efficient growth using oxygen as an electron acceptor. Mutants in c x, c 4, and c x+c 4 have a decreased capacity to reduce oxygen, but there is a background oxygen-reduction activity, indicating that there may be other routes for electron transfer from the cytochrome bc 1 complex to the cytochrome cbb 3 oxidase, whereas cytochrome c 5 appears to be the sole route of electrons to the nitrite reductase in N. meningitidis. Interestingly, cytochrome c x is highly similar to a domain of copper nitrite reductases from various proteobacteria, whereas cytochrome c 5 has high identity with a domain of the cytochrome cbb 3 oxidase of Neisseria gonorrhoeae, yet these two proteins function in oxygen respiration and nitrite respiration, respectively. This highlights a limitation of predicting protein function from similarity to known proteins, i.e. very closely related protein domains in different organisms can have different redox partners.
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Non-ribosomal peptide synthetase module fusions to produce derivatives of daptomycin in Streptomyces roseosporus
Genetic engineering has been applied to reprogramme non-ribosomal peptide synthetases (NRPSs) to produce novel antibiotics, but little is known about what determines the efficiency of production. We explored module exchanges at nucleotide sequences encoding interpeptide linkers in dptD, a gene encoding a di-modular NRPS subunit that incorporates 3-methylglutamic acid (3mGlu12) and kynurenine (Kyn13) into daptomycin. Mutations causing amino acid substitutions, deletions or insertions in the inter-module linker had no negative effects on lipopeptide yields. Hybrid DptD subunits were generated by fusing the 3mGlu12 module to terminal modules from calcium-dependent antibiotic (CDA) or A54145 NRPSs, and recombinants produced daptomycin analogues with Trp13 or Ile13 at high efficiencies. A recombinant expressing DptD with a hybrid Kyn13 module containing a di-domain from a d-Asn module caused the production of a new daptomycin analogue containing Asn13.
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Strain variation in ppGpp concentration and RpoS levels in laboratory strains of Escherichia coli K-12
More LessLaboratory strains and natural isolates of Escherichia coli differ in their level of stress resistance due to strain variation in the level of the sigma factor σ S (or RpoS), the transcriptional master controller of the general stress response. We found that the high level of RpoS in one laboratory strain (MC4100) was partially dependent on an elevated basal level of ppGpp, an alarmone responding to stress and starvation. The elevated ppGpp was caused by two mutations in spoT, a gene associated with ppGpp synthesis and degradation. The nature of the spoT allele influenced the level of ppGpp in both MC4100 and another commonly used K-12 strain, MG1655. Introduction of the spoT mutation into MG1655 also resulted in an increased level of RpoS, but the amount of RpoS was lower in MG1655 than in MC4100 with either the wild-type or mutant spoT allele. In both MC4100 and MG1655, high ppGpp concentration increased RpoS levels, which in turn reduced growth with poor carbon sources like acetate. The growth inhibition resulting from elevated ppGpp was relieved by rpoS mutations. The extent of the growth inhibition by ppGpp, as well as the magnitude of the relief by rpoS mutations, differed between MG1655 and MC4100. These results together suggest that spoT mutations represent one of several polymorphisms influencing the strain variation of RpoS levels. Stress resistance was higher in strains with the spoT mutation, which is consistent with the conclusion that microevolution affecting either or both ppGpp and RpoS can reset the balance between self-protection and nutritional capability, the SPANC balance, in individual strains of E. coli.
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- Biodiversity And Evolution
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Simultaneous presence of two different copies of the 16S rRNA gene in Bartonella henselae
More LessBartonella henselae is an emerging pathogen of increasing medical significance. Previous investigations have revealed two different 16S rRNA gene variants among B. henselae isolates, resulting in delineation of the B. henselae population into 16S RNA type I and type II isolates. While studying 191 B. henselae isolates by multi-locus sequence typing (MLST) we detected three isolates that could not be assigned to a distinct 16S RNA type upon direct sequencing because of ambiguous nucleotides in a distinct region of the 16S rRNA gene. Cloning and sequencing of the target region of the 16S rRNA gene suggested that these atypical isolates contained different 16S rRNA gene copies. Southern blot and hybridization experiments confirmed the presence of two different 16S RNA gene copies in each isolate. The isolates were further analysed by 16S RNA type-specific PCR, which assigned them to both 16S RNA types I and II. These results suggest that a small percentage of B. henselae isolates may harbour two different 16S rRNA gene copies. These isolates, which accounted for 1.6 % of the isolates in our study, have probably emerged by horizontal gene transfer. The implications of these findings for identification and genotyping studies on B. henselae are discussed.
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- Environmental Microbiology
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Highly conserved genes in Geobacter species with expression patterns indicative of acetate limitation
More LessAnalysis of the genome of Geobacter sulfurreducens revealed four genes encoding putative symporters with homology to ActP, an acetate transporter in Escherichia coli. Three of these genes, aplA, aplB and aplC, are highly similar (over 90 % identical) and fell within a tight phylogenetic cluster (Group I) consisting entirely of Geobacter homologues. Transcript levels for all three genes increased in response to acetate limitation. The fourth gene, aplD, is phylogenetically distinct (Group II) and its expression was not influenced by acetate availability. Deletion of any one of the three genes in Group I did not significantly affect acetate-dependent growth, suggesting functional redundancy. Attempts to recover mutants in which various combinations of two of these genes were deleted were unsuccessful, suggesting that at least two of these three transporter genes are required to support growth. Closely related Group I apl genes were found in the genomes of other Geobacter species whose genome sequences are available. Furthermore, related genes could be detected in genomic DNA extracted from a subsurface environment undergoing in situ uranium bioremediation. The transporter genes recovered from the subsurface were most closely related to Group I apl genes found in the genomes of cultured Geobacter species that were isolated from contaminated subsurface environments. The increased expression of these genes in response to acetate limitation, their high degree of conservation among Geobacter species and the ease with which they can be detected in environmental samples suggest that Group I apl genes of the Geobacteraceae may be suitable biomarkers for acetate limitation. Monitoring the expression of these genes could aid in the design of strategies for acetate-mediated in situ bioremediation of uranium-contaminated groundwater.
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The role of σ B in persistence of Staphylococcus epidermidis foreign body infection
Staphylococcal biofilm formation depends on the transcription factor σ B. We further investigated the role of σ B in biofilm formation and persistence in vitro and in vivo in a subcutaneous rat model. As expected, expression of all σ B operon genes was transiently higher in the first 6 h of biofilm formation compared to planktonic bacteria, concurrent with a temporary upregulation of icaA and aap expression. However, we also observed a second upregulation of sigB expression in biofilm more than 2 days old without upregulation of icaA or aap. Biofilm formation by Staphylococcus epidermidis strains 8400 and 1457 was compared to that of isogenic mutants with inactivation of rsbU, of rsbUVW and of the entire σ B operon. Both wild-type strains and the constitutively sigB-expressing rsbUVW mutant showed a strong biofilm-positive phenotype. The rsbUVW mutant biofilm was, however, thinner and more evenly spread than the wild-type biofilm. Inactivation of SigB in the rsbUVWsigB mutant or mutation of the positive regulator RsbU reduced both the number of sessile bacteria and polysaccharide intercellular adhesin (PIA) synthesis. These differences between the wild-types and their respective mutants appeared after 6 h in in vitro biofilms but only after 4 days in in vivo biofilms. Our results provide additional evidence for a role for σ B in biofilm formation. They also suggest a role for σ B in biofilm maturation and stability that is independent of PIA or accumulation-associated protein (Aap) and point to significant differences in the temporal development between in vitro and in vivo biofilms.
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Long-term persistence of virulent Yersinia pestis in soil
Plague is characterized by geographical foci from which it re-emerges after decades of silence, a fact currently explained by enzootic and epizootic cycles between plague-susceptible and plague-resistant rodents. To assess the potential role of soil in plague epidemiology, we experimentally investigated whether Yersinia pestis could persist alive and virulent in soil. Sterilized soil inoculated with virulent Y. pestis biotype Orientalis was regularly sampled for 40 weeks in duplicate. Each sample was observed by acridine orange staining and immunofluorescence using an anti-Y. pestis polyclonal antibody, and DNA was extracted for PCR amplification and sequencing of the Y. pestis ureD, caf1 and pla genes. All samples were inoculated onto selective agar, and samples from soil that had been incubated for 10, 60, 165, 210 and 280 days were also inoculated into each of two BALB/c female mice. The mouse experiment was performed in triplicate. Non-inoculated, sterilized soil samples were used as negative controls. Micro-organisms fluorescing orange and detected by immunofluorescence were identified as Y. pestis in all samples. They were recovered in pure agar cultures for up to 30 weeks but thereafter were contaminated with Pseudomonas spp. Soil that had been inoculated with Y. pestis proved to be fully virulent in mice, which died with Y. pestis septicaemia and multiple organ involvement. Negative control mice showed no signs of disease. These data indicate that Y. pestis biotype Orientalis can remain viable and fully virulent after 40 weeks in soil. This study is a first step on which to base further investigations of a potential telluric reservoir for Y. pestis, which could represent an alternative mechanism for the maintenance of plague foci.
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- Genes And Genomes
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An analysis of initiation codon utilization in the Domain Bacteria – concerns about the quality of bacterial genome annotation
More LessUsing custom software (Inidon) we have examined the initiation codon utilization in 620 complete bacterial genomes downloaded from the National Center for Biotechnology Information (NCBI). The mean utilization of ATG, GTG and TTG codons is 80.1, 11.6 and 7.8 %, respectively. In most cases in which similar species or strains have been analysed the utilization percentages of the three initiation codons are remarkably similar, but in certain cases the results exhibit significant differences.
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Introduction of marker-free deletions in Bacillus subtilis using the AraR repressor and the ara promoter
More LessWe have developed a system for the induction of marker-free mutation of Bacillus subtilis. The system features both the advantages of the use of antibiotic-resistance markers for mutant selection, and the ability to efficiently remove the markers, leaving unmarked mutations in the genome. It utilizes both a selective marker cassette and a counter-selective marker cassette. The selective marker cassette contains a chloramphenicol-resistance gene and the araR gene, which encodes the repressor for the arabinose operon (ara) of B. subtilis. The counter-selective marker cassette consists of a promoterless neomycin (Nm)-resistance gene (neo) fused to the ara promoter. First, the chromosomal araR locus is replaced with the counter-selective marker cassette by double-crossover homologous recombination and positive selection for Nm resistance. The selective marker cassette is connected with upstream and downstream sequences from the target locus, and is integrated into the upstream region of the target locus by a double-crossover event. This integration is also positively selected for, using chloramphenicol resistance. In the resultant strain, AraR, encoded by araR on the selective marker cassette, represses the expression of neo in the absence of l-arabinose. Finally, the eviction of the selective marker cassette together with the target locus is achieved by an intra-genomic single-crossover event between the two downstream regions of the target locus, and can be selected for based on Nm resistance, because of the excision of araR. The counter-selective marker cassette remaining in the genome, whose expression is switched on or off based on the excision or introduction of the selective marker cassette, is used again for the next round of deletion. Using this system, the 3.8 kb iolS–csbC region and the 41.8 kb hutM–csbC region have been efficiently and successfully deleted, without leaving markers in the target loci. The positive selection and simple procedure will make it a useful tool for the construction of multiple mutations.
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Development of a replicable oriC plasmid for Mycoplasma gallisepticum and Mycoplasma imitans, and gene disruption through homologous recombination in M. gallisepticum
More LessThe genome of Mycoplasma gallisepticum strain Rlow has been sequenced completely, but subsequent genetic studies have been limited by the lack of a replicable vector system. In this study, replicable plasmids were constructed for M. gallisepticum and Mycoplasma imitans using the oriC region upstream from the soj gene. The oriC plasmids of M. gallisepticum (pGTLori) and M. imitans (pMIori) replicated in both species, but Mycoplasma pneumoniae could not support replication of pGTLori. A 180 bp section of the oriC region of M. gallisepticum was found to be the minimal region required for plasmid replication in M. gallisepticum strain S6, the shortest oriC region defined for mycoplasmas. Targeted gene disruption of vlhA1.1 of M. gallisepticum S6 was attempted using these oriC plasmids. Constructs made in pPLoriC7 integrated into the M. gallisepticum genomic oriC region, not into the targeted gene, whereas those made in pMIori disrupted the vlhA1.2 gene, which has 97 % DNA sequence identity with the vlhA1.1 gene. During in vitro passages, antimicrobial selection pressure did not influence the rate of chromosomal integration. These oriC plasmids will thus be useful for genetic studies, including inactivation or expression of selected genes, in M. gallisepticum and M. imitans, and will lead to a better understanding of their molecular biology. They are, to our knowledge, the first replicable plasmids developed for the Pneumoniae phylogenetic group of mycoplasmas.
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- Pathogens And Pathogenicity
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Global transcriptional analysis of Mycoplasma hyopneumoniae following exposure to norepinephrine
More LessMycoplasma hyopneumoniae, a component of the porcine respiratory disease complex, colonizes the respiratory tract of swine by binding to the cilia of the bronchial epithelial cells. Mechanisms of pathogenesis are poorly understood for M. hyopneumoniae, but previous work has indicated that it responds to the environmental stressors heat shock, iron deprivation and oxidative compounds. For successful infection, M. hyopneumoniae must effectively resist host responses to the colonization of the respiratory tract. Among these are changes in hormonal levels in the mucosal secretions. Recent work in the stress responses of other bacteria has included the response to the catecholamine norepinephrine. The idea that M. hyopneumoniae can respond to a host hormone, however, is novel and has not previously been demonstrated. To test this, organisms in the early exponential phase of growth were exposed to 100 μM norepinephrine for 4 h, and RNA samples from these cultures were collected and compared to RNA samples from control cultures using two-colour PCR-based M. hyopneumoniae microarrays. The M. hyopneumoniae response included slowed growth and changes in mRNA transcript levels of 84 genes, 53 of which were upregulated in response to norepinephrine. A larger proportion of the genes upregulated than those downregulated were involved with transcription and translation. The downregulated genes were mostly involved with metabolism, which correlated with the reduction in growth of the mycoplasma. Approximately 51 % of the genes were hypothetical with no known function. Thus, in response to norepinephrine, M. hyopneumoniae appears to upregulate protein expression while downregulating general metabolism.
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Transcriptional interplay among the regulators Rrp2, RpoN and RpoS in Borrelia burgdorferi
More LessThe RpoN–RpoS alternative sigma factor pathway is essential for key adaptive responses by Borrelia burgdorferi, particularly those involved in the infection of a mammalian host. A putative response regulator, Rrp2, is ostensibly required for activation of the RpoN-dependent transcription of rpoS. However, questions remain regarding the extent to which the three major constituents of this pathway (Rrp2, RpoN and RpoS) act interdependently. To assess the functional interplay between Rrp2, RpoN and RpoS, we employed microarray analyses to compare gene expression levels in rrp2, rpoN and rpoS mutants of parental strain 297. We identified 98 genes that were similarly regulated by Rrp2, RpoN and RpoS, and an additional 47 genes were determined to be likely regulated by this pathway. The substantial overlap between genes regulated by RpoS and RpoN provides compelling evidence that these two alternative sigma factors form a congruous pathway and that RpoN regulates B. burgdorferi gene expression through RpoS. Although several known B. burgdorferi virulence determinants were regulated by the RpoN–RpoS pathway, a defined function has yet to be ascribed to most of the genes substantially regulated by Rrp2, RpoN and RpoS.
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Isolation and characterization of α-enolase, a novel fibronectin-binding protein from Streptococcus suis
More LessStreptococcus suis is an important swine pathogen that causes meningitis, endocarditis, arthritis and septicaemia. As a zoonotic agent, S. suis also causes similar diseases in humans. Binding of pathogenic bacteria to extracellular matrix components enhances their adhesion to and invasion of host cells. In the present study we isolated and identified a novel fibronectin-binding protein from S. suis. The native protein (designated SsEno) possessed not only high homology with other bacterial enolases but also enolase activity. We cloned, expressed and purified SsEno and showed that it is ubiquitously expressed by all S. suis serotypes and we identified its surface localization using immunoelectron microscopy. ELISA demonstrated that SsEno binds specifically to fibronectin and plasminogen in a lysine-dependent manner. Additional surface plasmon resonance assays demonstrated that SsEno binds to fibronectin or plasminogen with low nanomolar affinity. Inhibition experiments with anti-SsEno antibodies also showed that bacterial SsEno is important for the adhesion to and invasion of brain microvascular endothelial cells by S. suis. Overall, the present work is the first study, to our knowledge, to demonstrate a fibronectin-binding activity of a bacterial enolase, and shows that, similar to other bacterial fibronectin-binding proteins, SsEno may contribute to the virulence of S. suis.
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Threonine biosynthetic genes are essential in Cryptococcus neoformans
More LessWe identified and attempted to disrupt the Cryptococcus neoformans homoserine and/or threonine biosynthetic genes encoding aspartate kinase (HOM3), homoserine kinase (THR1) and threonine synthase (THR4); however, each gene proved recalcitrant to disruption. By replacing the endogenous promoters of HOM3 and THR1 with the copper-repressible CTR4-1 promoter, we showed that HOM3 and THR1 were essential for the growth of C. neoformans in rich media, when ammonium was the nitrogen source, or when threonine was supplied as an amino acid instead of a dipeptide. Moreover, the severity of the growth defect associated with HOM3 or THR1 repression increased with increasing incubation temperature. We believe this to be the first demonstration of threonine biosynthetic genes being essential in a fungus. The necessity of these genes for C. neoformans growth, particularly at physiologically relevant temperatures, makes threonine biosynthetic genes ideal anti-cryptococcal drug targets.
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The LysR-type transcriptional regulator Hrg counteracts phagocyte oxidative burst and imparts survival advantage to Salmonella enterica serovar Typhimurium
More LessLysR-type transcriptional regulators (LTTRs) are one of the key players that help bacteria adapt to different environments. We have designated STM0952, a putative LTTR in Salmonella enterica serovar Typhimurium (S. Typhimurium), as hydrogen peroxide resistance gene (hrg). A hrg knockout mutant of S. Typhimurium was sensitive to oxidative products of the respiratory burst, specifically to H2O2. The hrg mutant is profoundly attenuated in a murine model of infection and showed decreased intracellular proliferation in macrophages. It also induced increased amounts of reactive oxygen species and co-localization with gp91phox in the macrophage cell line, when compared to the wild-type. A strain overexpressing the hrg gene showed a survival advantage over the wild-type Salmonella under H2O2-induced stress. Microarray analysis suggested the presence of an Hrg regulon, which is required for resistance to the toxic oxidative products of the reticuloendothelial system.
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- Physiology
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Identification and functional characterization of NifA variants that are independent of GlnB activation in the photosynthetic bacterium Rhodospirillum rubrum
More LessThe activity of NifA, the transcriptional activator of the nitrogen fixation (nif) gene, is tightly regulated in response to ammonium and oxygen. However, the mechanisms for the regulation of NifA activity are quite different among various nitrogen-fixing bacteria. Unlike the well-studied NifL–NifA regulatory systems in Klebsiella pneumoniae and Azotobacter vinelandii, in Rhodospirillum rubrum NifA is activated by a direct protein–protein interaction with the uridylylated form of GlnB, which in turn causes a conformational change in NifA. We report the identification of several substitutions in the N-terminal GAF domain of R. rubrum NifA that allow NifA to be activated in the absence of GlnB. Presumably these substitutions cause conformational changes in NifA necessary for activation, without interaction with GlnB. We also found that wild-type NifA can be activated in a GlnB-independent manner under certain growth conditions, suggesting that some other effector(s) can also activate NifA. An attempt to use Tn5 mutagenesis to obtain mutants that altered the pool of these presumptive effector(s) failed, though much rarer spontaneous mutations in nifA were detected. This suggests that the necessary alteration of the pool of effector(s) for NifA activation cannot be obtained by knockout mutations.
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Expression of copA and cusA in Shewanella during copper stress
More LessCopper homeostasis is tightly regulated in all living cells as a result of the necessity and toxicity of this metal in free cationic form. In Gram-negative bacteria CPx-type ATPases (e.g. CopA in Escherichia coli) and heavy-metal efflux RND proteins (e.g. CusA in E. coli) play an important role in transport of copper across the cytoplasmic and outer membrane. We investigated the expression of CusA- and CopA-like proteins in Shewanella oneidensis MR1 and Shewanella strain MB4, a Mn(IV)-reducing isolate from a metal-polluted harbour sediment. Q-PCR analysis of total mRNA extracted from cultures grown under aerobic conditions with 25 μM copper showed significantly increased expression of cusA (Student's t-test: MR1, P<0.0001; MB4, P=0.0006). This gene was also induced in the presence of 100 μM copper and 10 or 25 μM cadmium in both tested strains. In the absence of oxygen, with fumarate as final electron acceptor and 100 μM copper, a prolonged lag phase (5 h) was observed and general fitness decreased as evidenced by twofold lower copy numbers of 16S rRNA compared to aerobic conditions. cusA expression in cells grown under these conditions remained at comparable levels (MR1) or was slightly decreased (MB4), compared to aerobic copper challenges. A gene homologous to the copA gene of S. oneidensis was not detected in strain MB4. Although low copA copy numbers were observed in strain MR1 under conditions with 25 and 100 μM copper, copA was not detected in mRNA from cultures grown in the presence of 10 μM cadmium, or in the absence of added heavy metals. However, copA was highly induced under anaerobic conditions with 100 μM copper (P=0.0011). These results suggest essentially different roles for the two proteins CopA and CusA in the copper response in S. oneidensis MR1, similar to findings in more metal-resistant bacteria such as Escherichia coli and Cupriavidus metallidurans.
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Important role of the nucleotide excision repair pathway in Mycobacterium smegmatis in conferring protection against commonly encountered DNA-damaging agents
More LessMycobacteria are an important group of human pathogens. Although the DNA repair mechanisms in mycobacteria are not well understood, these are vital for the pathogen's persistence in the host macrophages. In this study, we generated a null mutation in the uvrB gene of Mycobacterium smegmatis to allow us to compare the significance of the nucleotide excision repair (NER) pathway with two important base excision repair pathways, initiated by uracil DNA glycosylase (Ung) and formamidopyrimidine DNA glycosylase (Fpg or MutM), in an isogenic strain background. The strain deficient in NER was the most sensitive to commonly encountered DNA-damaging agents such as UV, low pH, reactive oxygen species, hypoxia, and was also sensitive to acidified nitrite. Taken together with previous observations on NER-deficient M. tuberculosis, these results suggest that NER is an important DNA repair pathway in mycobacteria.
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The alternative sigma factor SigF of Mycobacterium smegmatis is required for survival of heat shock, acidic pH and oxidative stress
More LessThe alternative sigma factor SigF of Mycobacterium tuberculosis has been characterized in detail as a general-stress, stationary-phase sigma factor involved in the virulence of the bacterium. While a homologous gene has been annotated in the genome of the fast-growing Mycobacterium smegmatis, little experimental evidence is available on the function of this gene. Here, we demonstrate that SigF of M. smegmatis is required for resistance to hydrogen peroxide, heat shock and acidic pH, but not for survival in human neutrophils. No difference in sensitivity to isoniazid was observed between the wild-type strain and the ΔsigF mutant, suggesting that SigF-mediated resistance to hydrogen peroxide was via a pathway independent of KatG or AhpC. RT-PCR and 5′-RACE (rapid amplification of cDNA ends) analyses showed that sigF of M. smegmatis was co-transcribed with rsbW (thought to encode an anti-sigma factor for SigF) and MSMEG_1802 (unknown function) and was expressed from two promoters, one upstream of MSMEG_1802 and the second upstream of rsbW. Analysis of transcriptional lacZ fusion constructs in the sigF-deletion background revealed that the MSMEG_1802 promoter was dependent on SigF for expression. Moreover, MSMEG_1802-lacZ was induced twofold upon entry into stationary phase, while exposure of exponentially growing cultures to various stress conditions (e.g. heat, cold, ethanol, hydrogen peroxide or different pH values) did not lead to induction of MSMEG_1802-lacZ. Expression of rsbW-lacZ was independent of SigF and remained constant throughout the growth cycle and under various stress conditions unless the bacteria were challenged with d-cycloserine.
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- Plant-Microbe Interactions
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Component and protein domain exchange analysis of a thermoresponsive, two-component regulatory system of Pseudomonas syringae
Two closely related phytopathogenic bacterial strains, Pseudomonas syringae pv. glycinea PG4180 and P. syringae pv. tomato DC3000, produce the chlorosis-inducing phytotoxin coronatine (COR) in a remarkably divergent manner. PG4180 produces COR at the virulence-promoting temperature of 18 °C, but not at 28 °C. In contrast, temperature has no effect on COR synthesis in DC3000. A modified two-component system consisting of the histidine protein kinase (HPK), CorS, the response regulator (RR), CorR, and a third component, CorP, governs COR biosynthesis in both strains. A plasmid-based component and domain swapping approach was used to introduce different combinations of RRs, HPKs and hybrid HPKs into corS mutants of both strains. Subsequently, expression levels of the COR biosynthetic cma operon were determined using RNA dot-blot analysis, suggesting that CorRSP of PG4180 mediates a thermoresponsive phenotype dependent on the genomic background of each strain. The reciprocal experiment demonstrated a loss of temperature dependence in the corS mutant of PG4180. The presence of corR from PG4180 led to more pronounced cma expression in DC3000 and was associated with thermoresponsiveness, while corS of PG4180 did not mediate a temperature-dependent phenotype in the DC3000 mutant containing native corR and corP. These findings were substantiated by RT-PCR experiments. The C-terminal domain of CorS of PG4180 mediated thermosensing, while the N terminus did not respond to temperature changes, suggesting cytosolic perception of the temperature signal.
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Volumes and issues
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Volume 170 (2024)
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Volume 169 (2023)
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Volume 168 (2022)
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Volume 167 (2021)
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Volume 166 (2020)
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Volume 165 (2019)
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Volume 164 (2018)
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Volume 158 (2012)
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Volume 2 (1948)
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Volume 1 (1947)