- Volume 150, Issue 1, 2004
Volume 150, Issue 1, 2004
- Microbiology Comment
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- Cell And Developmental Biology
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Metronidazole induces programmed cell death in the protozoan parasite Blastocystis hominis
More LessPrevious studies by the authors have shown that the protozoan parasite Blastocystis hominis succumbed to a cytotoxic monoclonal antibody with a number of cellular and biochemical features characteristic of apoptosis in higher eukaryotes. The present study reports that apoptosis-like features are also observed in growing cultures of axenic B. hominis upon exposure to metronidazole, a drug commonly used for the treatment of blastocystosis. Upon treatment with the drug, B. hominis cells displayed key morphological and biochemical features of programmed cell death (PCD), viz. nuclear condensation and nicked DNA in nucleus, reduced cytoplasmic volume, externalization of phosphatidylserine and maintenance of plasma membrane integrity with increasing permeability. This present study also supports the authors' previously postulated novel function for the B. hominis central vacuole in PCD; it acts as a repository where apoptotic bodies are stored before being released into the extracellular space. The implications and possible roles of PCD in B. hominis are discussed.
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Functional relationship between SpoVIF and GerE in gene regulation during sporulation of Bacillus subtilis
More LessThe sporulation-specific SpoVIF (YjcC) protein of Bacillus subtilis is essential for the development of heat-resistant spores. The GerE protein, the smallest member of the LuxR-FixJ family, contains a helix–turn–helix (HTH) motif and is involved in the expression of various sporulation-specific genes. In this study, the gene expression and protein composition of sporulating spoVIF-negative cells were analysed. CgeA, CotG and CotS, which are GerE-dependent coat proteins, were not expressed in the spoVIF-negative cells. Northern blotting showed that SpoVIF regulated the transcription of cgeA, cotG and cotS in a manner similar to that of GerE. In spoVIF-negative cells, gerE mRNA was transcribed normally, but immunoblot analysis using anti-GerE antiserum showed that the quantity of GerE protein was considerably less than that in wild-type controls. Using GFP (green fluorescent protein) fusion proteins, the localization of SpoVIF and GerE was observed by fluorescence microscopy. SpoVIF-GFP was detectable in the mother cell compartment, as was GerE-GFP. These results suggest that SpoVIF directly or indirectly controls the function of the GerE protein, and that SpoVIF is required for gene regulation during the latter stages of sporulation.
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- Biochemistry And Molecular Biology
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Development of an insertional expression vector system for Methylobacterium extorquens AM1 and generation of null mutants lacking mtdA and/or fch
More LessOver the past few years, the genetic ‘toolkit’ available for use with Methylobacterium extorquens AM1 has expanded significantly. Here a further advance is presented and demonstrated, an insertional expression system that allows expression of genes from a stable, unmarked chromosomal locus. This system has been used to better understand the role of the tetrahydrofolate (H4F) pathway in methylotrophy. Previously, it has not been possible to generate null mutants lacking either mtdA (encoding an NADP-dependent methylene-H4F/methylene-tetrahydromethanopterin dehydrogenase) or fch (encoding methenyl-H4F cyclohydrolase). An unmarked strain was generated that expressed the analogous folD gene (encoding a bifunctional NADP-dependent methylene-H4F dehydrogenase/methenyl-H4F cyclohydrolase) from Methylobacterium chloromethanicum CM4T. In this strain, null mutants could be obtained that grew normally on multicarbon substrates but were defective for growth on C1 substrates. Additionally, null mutants of mtdA and/or fch could also be generated in the wild-type by supplementing the succinate medium with formate. These strains were unable to grow on C1 compounds but were not methanol-sensitive. These approaches have demonstrated that the apparent essentiality of mtdA and fch is due to the need for formyl-H4F for biosynthesis of purines and other compounds, and have provided clear genetic evidence that the H4F pathway is required for methylotrophy.
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The importance of the Tat-dependent protein secretion pathway in Streptomyces as revealed by phenotypic changes in tat deletion mutants and genome analysis
More LessStreptomyces are Gram-positive soil bacteria that are used industrially, not only as a source of medically important natural compounds, but also as a host for the secretory production of a number of heterologous proteins. A good understanding of the different secretion processes in this organism is therefore of major importance. The functionality of the recently discovered bacterial twin-arginine translocation (Tat) pathway has already been shown in Streptomyces lividans. Here, the aberrant phenotype of S. lividans ΔtatB and ΔtatC single mutants is described. Both mutants are characterized by a dispersed growth in liquid medium, an impaired morphological differentiation on solid medium and growth retardation. To reveal the extent to which the Tat pathway is used in Streptomyces, putative Tat-dependent precursor proteins of Streptomyces coelicolor, a very close relative of S. lividans, and of Streptomyces avermitilis, of which the genomes have been completely sequenced, were identified by a modified version of the tatfind computer program designed by Rose and colleagues [ Rose, R. W., Brüser, T., Kissinger, J. C. & Pohlschröder, M. (2002). Mol Microbiol 45, 943–950 ]. A list of 230 precursor proteins was obtained; this is the highest number of putative Tat substrates found in any genome so far. In addition to the Streptomyces antibioticus tyrosinase, it was also demonstrated that the secretion of the S. lividans xylanase C is Tat-dependent. The predicted Tat substrates belong to a variety of protein classes, with a high number of proteins functioning in degradation of macromolecules, in binding and transport, and in secondary metabolism. Only a minor fraction of the proteins seem to bind a cofactor. The aberrant phenotype of the ΔtatB and ΔtatC mutants together with the high number of putative Tat-dependent substrates suggests that the Streptomyces Tat pathway has a distinct and more important role in protein secretion than in most other bacteria.
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Characterization of the Staphylococcus aureus mprF gene, involved in lysinylation of phosphatidylglycerol
More LessLysylphosphatidylglycerol (LPG) is a basic phospholipid in which l-lysine from lysyl-tRNA is transferred to phosphatidylglycerol (PG). This study examined whether the Staphylococcus aureus mprF gene encodes LPG synthetase. A crude membrane fraction prepared from wild-type S. aureus cells had LPG synthetase activity that depended on PG and lysyl-tRNA, whereas the membrane fraction from an mprF deletion mutant did not. When S. aureus MprF protein was trans-expressed in wild-type Escherichia coli cells, LPG synthesis was induced, whereas it was not observed in E. coli pgsA3 mutant cells in which the amount of PG is significantly reduced. In addition, LPG synthetase activity and a 93 kDa protein whose molecular size corresponded to that of MprF protein were co-induced in the crude membrane fraction prepared from E. coli cells expressing MprF protein. The K m values of the LPG synthetase activity for PG and for lysyl-tRNA were 56 μM and 6·9 μM, respectively, consistent with those of S. aureus membranes. These results suggest that the MprF protein is LPG synthetase.
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Characterization of a glycosylphosphatidylinositol-bound cell-wall protein (GPI-CWP) in Yarrowia lipolytica
More LessThe structure and composition of the cell wall of yeast has so far been studied mainly in Saccharomyces cerevisiae. It is basically made up of three components: β-glucans, chitin and mannose-containing glycoproteins, also called mannoproteins. Most covalently bound cell-wall mannoproteins belong to the so-called glycosylphosphatidylinositol cell-wall protein (GPI-CWP) family, cell-wall proteins that are bound through the remnant of a GPI residue to 1,6-β-glucan. The non-conventional yeast Yarrowia lipolytica shares Generally Regarded As Safe (GRAS) status with S. cerevisiae, has some industrial applications and is increasingly being proposed as a host for the production of recombinant proteins and as a model in the study of dimorphism. However, very little information on cell-wall structure and composition is available for this organism. Here is described the isolation and characterization of YlCWP1, a homologue of the CWP1 gene from S. cerevisiae, which encodes a GPI-CWP, and the identification of its gene product. YlCWP1 encodes a 221 aa protein that contains a putative signal peptide and a putative GPI-attachment site. It shows 28·5 % overall identity with Cwp1 of S. cerevisiae and a hydropathy profile characteristic of GPI-CWPs. Disruption of YlCWP1, both in the wild-type and in an mnn9 glycosylation-deficient background, led to the identification of Ylcwp1 as a 60 kDa polypeptide present in cell-wall extracts. To the authors' knowledge, this is the first report of a GPI-CWP in Y. lipolytica, and it suggests that the cell-wall organization of Y. lipolytica is similar to that of S. cerevisiae.
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Importance of mycoloyltransferases on the physiology of Corynebacterium glutamicum
Mycoloyltransferases (Myts) play an essential role in the biogenesis of the cell envelope of members of the Corynebacterineae, a group of bacteria that includes the mycobacteria and corynebacteria. While the existence of several functional myt genes has been demonstrated in both mycobacteria and corynebacteria (cmyt), the disruption of any of these genes has at best generated cell-wall-defective but always viable strains. To investigate the importance of Myts on the physiology of members of the Corynebacterineae, a double mutant of Corynebacterium glutamicum was constructed by deleting cmytA and cmytB, and the consequences of the deletion on the viability of the mutant, the transfer of corynomycoloyl residues onto its cell-wall arabinogalactan and trehalose derivatives, and on its cell envelope ultrastructure were determined. The double mutant strain failed to grow at 34 °C and exhibited a growth defect and formed segmentation-defective cells at 30 °C. Biochemical analyses showed that the double mutant elaborated 60 % less cell-wall-bound corynomycolates and produced less crystalline surface layer proteins associated with the cell surface than the parent and cmytA-inactivated mutant strains. Freeze-fracture electron microscopy showed that the ΔcmytA ΔcmytB double mutant, unlike the wild-type and cmytA-inactivated single mutant strains, frequently exhibited an additional fracture plane that propagated within the plasma membrane and rarely exposed the S-layer protein. Ultra-thin sectioning of the double mutant cells showed that they were totally devoid of the outermost layer. Complementation of the double mutant with the wild-type cmytA or cmytB gene restored completely or partially this phenotype. The data indicate that Myts are important for the physiology of C. glutamicum and reinforce the concept that these enzymes would represent good targets for the discovery of new drugs against the pathogenic members of the Corynebacterineae.
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Biosynthesis of the dichloroacetyl component of chloramphenicol in Streptomyces venezuelae ISP5230: genes required for halogenation
More LessFive ORFs were detected in a fragment from the Streptomyces venezuelae ISP5230 genomic DNA library by hybridization with a PCR product amplified from primers representing a consensus of known halogenase sequences. Sequencing and functional analyses demonstrated that ORFs 11 and 12 (but not ORFs 13–15) extended the partially characterized gene cluster for chloramphenicol (Cm) biosynthesis in the chromosome. Disruption of ORF11 (cmlK) or ORF12 (cmlS) and conjugal transfer of the insertionally inactivated genes to S. venezuelae gave mutant strains VS1111 and VS1112, each producing a similar series of Cm analogues in which unhalogenated acyl groups replaced the dichloroacetyl substituent of Cm. 1H-NMR established that the principal metabolite in the disrupted strains was the α-N-propionyl analogue. The sequence of CmlK implicated the protein in adenylation, and involvement in halogenation was inferred from biosynthesis of analogues by the cmlK-disrupted mutant. A role in generating the dichloroacetyl substituent was supported by partial restoration of Cm biosynthesis when a cloned copy of cmlK was introduced in trans into VS1111. Complementation of the mutant also indicated that inactivation of cmlK rather than a polar effect of the disruption on cmlS expression had interfered with dichloroacetyl biosynthesis. The deduced CmlS sequence resembled sequences of FADH2-dependent halogenases. Conjugal transfer of cmlK or cmlS into S. venezuelae cml-2, a chlorination-deficient strain with a mutation mapped genetically to the Cm biosynthesis gene cluster, did not complement the cml-2 lesion, suggesting that one or more genes in addition to cmlK and cmlS is needed to assemble the dichloroacetyl substituent. Insertional inactivation of ORF13 did not affect Cm production, and the products of ORF14 and ORF15 matched Streptomyces coelicolor A3(2) proteins lacking plausible functions in Cm biosynthesis. Thus cmlS appears to mark the downstream end of the gene cluster.
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Organization of the teicoplanin gene cluster in Actinoplanes teichomyceticus
The glycopeptide teicoplanin is used for the treatment of serious infections caused by Gram-positive pathogens. The tcp gene cluster, devoted to teicoplanin biosynthesis in the actinomycete Actinoplanes teichomyceticus, was isolated and characterized. From sequence analysis, the tcp cluster spans approximately 73 kb and includes 39 ORFs participating in teicoplanin biosynthesis, regulation, resistance and export. Of these, 34 ORFs find a match in at least one of the five glycopeptide gene clusters previously characterized. Putative roles could be assigned for most of the tcp genes. The two glycosyltransferases responsible for attaching amino sugars to amino acids 4 and 6 of the teicoplanin aglycon were overexpressed in Escherichia coli and characterized. They both recognize N-acetylglucosamine as the substrate. tGtfA can add a sugar residue in the presence or absence of N-acetylglucosamine at amino acid 4, while tGtfB can only glycosylate the teicoplanin aglycon.
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Bovicin HJ50, a novel lantibiotic produced by Streptococcus bovis HJ50
More LessA bacteriocin-producing strain was isolated from raw milk and named Streptococcus bovis HJ50. Like most bacteriocins produced by lactic acid bacteria, bovicin HJ50 showed a narrow range of inhibiting activity. It was sensitive to trypsin, subtilisin and proteinase K. Bovicin HJ50 was extracted by n-propanol and purified by SP Sepharose Fast Flow, followed by Phenyl Superose and Sephadex G-50. Treatment of Micrococcus flavus NCIB8166 with bovicin HJ50 revealed potassium efflux from inside the cell in a concentration-dependent manner. The molecular mass of bovicin HJ50 was determined to be 3428·3 Da. MS analysis of DTT-treated bovicin HJ50 suggested that bovicin HJ50 contains a disulfide bridge. The structural gene of bovicin HJ50 was cloned by nested PCR based on its N-terminal amino acid sequence. Sequence analysis showed that it encodes a 58 aa prepeptide consisting of an N-terminal leader sequence of 25 aa and a C-terminal propeptide domain of 33 aa. Bovicin HJ50 shows similarity to type AII lantibiotics. Chemical modification using an ethanethiol-containing reaction mixture showed that two Thr residues are modified.
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Aspirin commits yeast cells to apoptosis depending on carbon source
More LessThe effect of aspirin on the growth of a wild-type Saccharomyces cerevisiae strain (EG103), containing both copper,zinc superoxide dismutase (CuZnSOD) and manganese superoxide dismutase (MnSOD), a strain deficient in MnSOD (EG110) and a strain deficient in CuZnSOD (EG118) was measured in media containing different carbon sources. Aspirin inhibited the fermentative growth of all three strains in glucose medium. It inhibited the non-fermentative growth of the MnSOD-deficient strain very drastically in ethanol medium and had no effect on this strain in glycerol or acetate medium. The non-fermentative growth of the other two strains was not affected by aspirin. The growth inhibition of strain EG110 was associated with early necrosis in glucose medium and late apoptosis in ethanol medium. The apoptosis was preceded by a pronounced loss of cell viability. The growth inhibitory effect of aspirin was not reversed by the antioxidants N-acetylcysteine and vitamin E. Furthermore, aspirin itself appeared to act as an antioxidant until the onset of overt apoptosis, when a moderate increase in the intracellular oxidation level occurred. This suggested that reactive oxygen species probably do not play a primary role in the apoptosis of cells exposed to aspirin.
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Azorhizobium caulinodans electron-transferring flavoprotein N electrochemically couples pyruvate dehydrogenase complex activity to N2 fixation
More LessAzorhizobium caulinodans thermolabile point mutants unable to fix N2 at 42 °C were isolated and mapped to three, unlinked loci; from complementation tests, several mutants were assigned to the fixABCX locus. Of these, two independent fixB mutants carried missense substitutions in the product electron-transferring flavoprotein N (ETFN) α-subunit. Both thermolabile missense variants Y238H and D229G mapped to the ETFN α interdomain linker. Unlinked thermostable suppressors of these two fixB missense mutants were identified and mapped to the lpdA gene, encoding dihydrolipoamide dehydrogenase (LpDH), immediately distal to the pdhABC genes, which collectively encode the pyruvate dehydrogenase (PDH) complex. These two suppressor alleles encoded LpDH NAD-binding domain missense mutants G187S and E210G. Crude cell extracts of these fixB lpdA double mutants showed 60–70 % of the wild-type PDH activity; neither fixB lpdA double mutant strain exhibited any growth phenotype at the restrictive or the permissive temperature. The genetic interaction between two combinations of lpdA and fixB missense alleles implies a physical interaction of their respective products, LpDH and ETFN. Presumably, this interaction electrochemically couples LpDH as the electron donor to ETFN as the electron acceptor, allowing PDH complex activity (pyruvate oxidation) to drive soluble electron transport via ETFN to N2, which acts as the terminal electron acceptor. If so, then, the A. caulinodans PDH complex activity sustains N2 fixation both as the driving force for oxidative phosphorylation and as the metabolic electron donor.
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The Clostridium perfringens TetA(P) efflux protein contains a functional variant of the Motif A region found in major facilitator superfamily transport proteins
The Clostridium perfringens tetracycline resistance protein, TetA(P), is an inner-membrane protein that mediates the active efflux of tetracycline from the bacterial cell. This protein comprises 420 aa and is predicted to have 12 transmembrane domains (TMDs). Comparison of the TetA(P) amino acid sequence to that of several members of the major facilitator superfamily (MFS) identified a variant copy of the conserved Motif A. This region consists of the sequence E59xPxxxxxDxxxRK72 and is located within the putative loop joining TMDs 2 and 3 in the predicted structural model of the TetA(P) protein. To study the functional importance of the conserved residues, site-directed mutagenesis was used to construct 17 point mutations that were then analysed for their effect on tetracycline resistance and their ability to produce an immunoreactive TetA(P) protein. Changes to the conserved Phe-58 residue were tolerated, whereas three independent substitutions of Pro-61 abolished tetracycline resistance. Examination of the basic residues showed that Arg-71 is required for function, whereas tetracycline resistance was retained when Lys-72 was substituted with arginine. These results confirm that the region encoding this motif is important for tetracycline resistance and represents a distant version of the Motif A region found in other efflux proteins and members of the MFS family. In addition, it was shown that Glu-117 of the TetA(P) protein, which is predicted to be located in TMD4, is important for resistance although a derivative with an aspartate residue at this position is also functional.
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Protein kinase A is involved in the control of morphology and branching during aerobic growth of Mucor circinelloides
The cAMP signal transduction pathway controls many processes in fungi. The Mucor circinelloides pkaR and pkaC genes, encoding the regulatory (PKAR) and catalytic (PKAC) subunit of the cAMP-dependent protein kinase A (PKA), have been cloned recently. Expression analysis during the dimorphic shift and colony morphology suggested a role for PKAR in the control of morphology and branching. Here strain KFA121, which overexpresses the M. circinelloides pkaR gene, was used to quantify growth and branching under different aerobic growth conditions in a flow-through cell by computerized image analysis. An inverse relationship between the pkaR expression level in KFA121 and the hyphal growth unit length was observed in KFA121, suggesting a central role for PKAR in branching. A biochemical analysis of PKAR using antibodies and enzyme assay demonstrated that the level of PKAR is higher in KFA121 under inducing conditions, i.e. in the presence of high glucose, than in the vector control strain KFA89. Measurement of cAMP binding demonstrated a significant increase (two- to threefold) in PKAR level for KFA121 at the time of germ-tube emission in medium containing 10 g glucose l−1. The level of PKA activity was determined using kemptide in the same crude cell extracts used to determine cAMP binding. Strain KFA121 showed a twofold increase in PKA activity. An excess of free PKAR subunit over PKA holoenzyme was determined using sucrose gradient centrifugation of extracts from KFA89 and KFA121. The data indicate that cAMP-dependent PKA in M. circinelloides might be down-regulated during hyphal-tube emergence and that an increase in PKAR levels results in increased branching.
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- Biodiversity And Evolution
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Analysis of geospecific markers for Helicobacter pylori variants in patients from Japan and Nigeria by triple-locus nucleotide sequence typing
More LessHuman migrations and geographical separation over long periods may have resulted in ecologically distinct populations of Helicobacter pylori infecting individuals in different continents. This study used nucleotide sequence analysis with the aim of defining population-specific genomic motifs in isolates from East Asian and African dyspeptic patients. Sequences of internal fragments (542–627 bp) of three housekeeping genes (ureI, ahpC and atpA) were analysed for 85 isolates from individuals in Japan and China (30 isolates), Nigeria and South Africa (14 isolates), the United Kingdom (32 isolates), and nine miscellaneous reference strains. Phylogenetic analyses showed a high degree of intra-set relatedness amongst sequences from the Japanese and Nigerian isolates, with each robustly segregated as distinct lineages irrespective of cagA presence and vacA allelic type. All strains had unique combined sequence types except for identical paired (antrum/corpus) isolates. Population-specific polymorphisms were identified within each gene which were combined to provide unique motifs defining the Japanese and Nigerian regional populations. The alleles were present at variable frequencies in UK and South African isolates. The findings provide unique evidence of positive selection for conserved nucleotide sites linked to the geographical separation in Japan of a strain subpopulation for which we propose the designation H. pylori geovar ‘orientalis’.
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- Environmental Microbiology
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Demonstration of antifreeze protein activity in Antarctic lake bacteria
More LessAntifreeze proteins (AFPs) are a structurally diverse group of proteins that have the ability to modify ice crystal structure and inhibit recrystallization of ice. AFPs are well characterized in fish and insects, but very few bacterial species have been shown to have AFP activity to date. Thirty eight freshwater to hypersaline lakes in the Vestfold Hills and Larsemann Hills of Eastern Antarctica were sampled for AFPs during 2000. Eight hundred and sixty six bacterial isolates were cultivated. A novel AFP assay, designed for high-throughput analysis in Antarctica, demonstrated putative activity in 187 of the cultures. Subsequent analysis of the putative positive isolates showed 19 isolates with significant recrystallization inhibition (RI) activity. The 19 RI active isolates were characterized using ARDRA (amplified rDNA restriction analysis) and 16S rDNA sequencing. They belong to genera from the α- and γ-Proteobacteria, with genera from the γ-subdivision being predominant. The 19 AFP-active isolates were isolated from four physico-chemically diverse lakes. Ace Lake and Oval Lake were both meromictic with correspondingly characteristic chemically stratified water columns. Pendant Lake was a saline holomictic lake with different chemical properties to the two meromictic lakes. Triple Lake was a hypersaline lake rich in dissolved organic carbon and inorganic nutrients. The environments from which the AFP-active isolates were isolated are remarkably diverse. It will be of interest, therefore, to elucidate the evolutionary forces that have led to the acquisition of functional AFP activity in microbes of the Vestfold Hills lakes and to discover the role the antifreezes play in these organisms.
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Temperature and growth-phase effects on Aeromonas hydrophila survival in natural seawater microcosms: role of protein synthesis and nucleic acid content on viable but temporarily nonculturable response
More LessThe behaviour of Aeromonas hydrophila in nutrient-poor filter-sterilized seawater was investigated at 23 and 5 °C with respect to its growth phase. At both temperatures, the culturable A. hydrophila population declined below the detection level (0·1 c.f.u. ml−1) after 3–5 weeks, depending on the initial physiological state of the cells. During the first week, starved A. hydrophila cells appeared more resistant to the seawater stress at 5 °C than cells initially in the exponential growth phase. This difference was not observed at 23 °C, where de novo protein synthesis seemed to be required for long-term adaptation of cells from the exponential growth phase. Over the duration of the experiments, intact and total cell concentrations were not significantly affected, indicating that bacteria had entered a so-called viable but nonculturable state (VBNC). However, the incubated bacteria rapidly became heterogeneous with respect to their nucleic acid content, and their cell size decreased faster at 23 than at 5 °C. Resuscitation of VBNC cells was attempted by a temperature shift from 5 to 23 °C without exogenous nutrient addition. Comparison of the growth rates of the stressed population and of the untreated bacteria growing in the same autoclaved initial cell suspension showed significantly faster growth for the stressed cells, suggesting that in addition to growth of the few culturable stressed cells, a proportion of injured cells became culturable.
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- Genes And Genomes
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GadE (YhiE): a novel activator involved in the response to acid environment in Escherichia coli
In several Gram-positive and Gram-negative bacteria glutamate decarboxylases play an important role in the maintenance of cellular homeostasis in acid environments. Here, new insight is brought to the regulation of the acid response in Escherichia coli. Overexpression of yhiE, similarly to overexpression of gadX, a known regulator of glutamate decarboxylase expression, leads to increased resistance of E. coli strains under high acid conditions, suggesting that YhiE is a regulator of gene expression in the acid response. Target genes of both YhiE (renamed GadE) and GadX were identified by a transcriptomic approach. In vitro experiments with GadE purified protein provided evidence that this regulator binds to the promoter region of these target genes. Several of them are clustered together on the chromosome and this chromosomal organization is conserved in many E. coli strains. Detailed structural (in silico) analysis of this chromosomal region suggests that the promoters of the corresponding genes are preferentially denatured. These results, along with the G+C signature of the chromosomal region, support the existence of a fitness island for acid adaptation on the E. coli chromosome.
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The csp operon of Streptococcus salivarius encodes two predicted cell-surface proteins, one of which, CspB, is associated with the fimbriae
More LessA Tn917 mutant library was generated to identify genes involved in the biogenesis of Streptococcus salivarius fimbriae. A fimbria-deficient mutant was isolated by negative selection using an immunomagnetic separation technique with specific anti-fimbriae polyclonal antibodies (pAbs). The transposon was inserted in an ORF, called orf176, which encoded a protein of unknown function. The transposon prevented the transcription of orf176 as well as two genes located downstream, which are designated cspA and cspB and which form the csp operon. Sequence analyses of CspA and CspB revealed that both proteins possessed the classic cell-wall-anchoring motif (LPXTG) of Gram-positive bacterial surface proteins. Recombinant CspA (rCspA) and CspB (rCspB) proteins were generated in Escherichia coli and used to produce pAbs. Immunolocalization experiments showed that anti-rCspB, but not anti-rCspA antibodies specifically recognized S. salivarius fimbriae. Our results suggested that the csp operon encoded predicted cell-surface proteins, one of which, CspB, was associated with the fimbriae.
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A polymorphic tandem repeat potentially useful for typing in the chromosome of Yersinia enterocolitica
More LessThe hexanucleotide CCAGCA was found repeated 15 times in tandem on the 5′ side of the virginiamycin acetyl transferase gene of Yersinia enterocolitica strain Y56. The corresponding region was analysed by PCR from 54 clinical strains belonging to the same biotype and serotype, and others from this laboratory collection belonging to different biotypes and serotypes. Each strain produced a single amplification product whose size was variable among strains, revealing that the locus was polymorphic. Nucleotide sequence determination of selected PCR products showed that the polymorphism was due to the precise expansion or reduction in the number of hexanucleotide repeats. Analysis of this locus in a few strains showing the same PFGE pattern showed that it was also polymorphic. These results suggest that this method could be valuable to increase the discriminatory power of current Y. enterocolitica typing schemes.
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Chlamydia trachomatis σ 28 recognizes the fliC promoter of Escherichia coli and responds to heat shock in chlamydiae
More LessThe rpsD gene of Chlamydia trachomatis encodes the alternative σ factor σ 28, which bears strong homology to many bacterial σ factors, including Escherichia coli σ 28 and Bacillus subtilis σ B and σ D. Recently, a σ 28 promoter was identified upstream of the late-cycle-expressed gene hctB, which encodes the Chlamydia-histone-like protein 2 ( Yu & Tan, 2003 ). In this study it is shown that the product of chlamydial rpsD is an E. coli σ 28 homologue. It was found that recombinant chlamydial σ 28, in combination with E. coli core RNA polymerase, initiates transcription in vitro from the E. coli σ 28-dependent promoter of fliC. It was also demonstrated that the recombinant chlamydial σ 28 does not recognize major σ factor σ 70-consensus-like sequences in vitro. In C. trachomatis-infected cells, two rpsD transcripts were detected with 5′ ends located 18 (transcript I) and 54 bp (transcript II) upstream of the translational initiation codon at 16 and 30 h post-infection. When the temperature of cultures infected with C. trachomatis was shifted from 35 to 42 °C, the rpsD transcript I increased dramatically. The levels of chlamydial σ 28, relative to EF-Tu, were greater throughout the exponential growth phase of the reticulate body, but lower late in the developmental cycle. These data support the hypothesis that σ 28 plays a role in the regulatory network that allows chlamydiae to survive changes in its environment, enabling it to complete its unique developmental cycle.
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- Pathogens And Pathogenicity
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The role and regulation of the extracellular proteases of Staphylococcus aureus
More LessStaphylococcus aureus has several extracellular proteases with proposed roles in virulence. SspA (serine protease), SspB (cysteine protease) and Aur (metalloprotease) have been characterized previously and SspA and SspB were found to be cotranscribed. The coding region for the cysteine protease ScpA has been identified and characterized. It is in a probable bi-cistronic operon with scpA located immediately upstream of a coding region for a 108 aa protein that is a specific inhibitor of ScpA. Using primer extension analysis promoters have been mapped and it was found that σ A is the only sigma factor involved in the transcription of scpA, sspABC and aur. The transcription of all the genes occurs maximally at post-exponential phase, being positively regulated by agr (accessory gene regulator) and negatively regulated by sarA (staphylococcal accessory regulator). Furthermore σ B represses transcription from the aur and scp operons similarly to the previously shown effect on ssp [ Horsburgh, M., Aish, J., White, I., Shaw, L., Lithgow, J. & Foster, S. (2002). J Bacteriol 184, 5457–5467 ]. Using mutations in each protease gene the proteolytic cascade of activation has been analysed. Aur, SspA, SspB and ScpA are all produced as zymogens, activated by proteolytic cleavage. Although the metalloprotease, Aur, does catalyse activation of the SspA zymogen, it is not the sole agent capable of conducting this process. Site-directed mutagenesis revealed that Aur is not capable of undergoing auto-proteolysis to achieve activation. The cysteine protease, ScpA, appears to reside outside this cascade of activation, as mature ScpA was observed in the aur, sspA and sspB mutant strains. Using a mouse abscess model, it has been shown that insertional inactivation of sspA or sspB results in significant attenuation of virulence, whilst mutations in aur or scpA do not. It is likely the attenuation observed in the sspA strain is due to polarity on the sspB gene.
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Deletion of the NOT4 gene impairs hyphal development and pathogenicity in Candida albicans
More LessThe Candida albicans NOT4 gene was disrupted in order to investigate the role of Not4p in growth, morphogenesis and pathogenicity. Heterozygote (NOT4/not4), null (not4/not4) and reconstructed heterozygote ([NOT4]/not4) strains of C. albicans, as well as CAF2-1, the parental strain, were grown under conditions that promote hyphal formation. When cultured in liquid medium 199 the heterozygote, reconstructed and wild-type strains began the yeast-to-hyphal transition within 3 h and continued hyphal growth for the duration of experiments. The null mutant also began hyphal growth within 3–5 h but hyphae tended to be shorter and distorted. Subsequently, hyphal growth was arrested and growth returned predominantly to the yeast form. Similar differences were observed when strains were grown on solid Spider medium and medium 199. The parental, heterozygote and reconstructed strains formed normal filamentous networks emanating from colonies. In contrast, the null mutant failed to form hyphae on all solid media tested. The ability of the NOT4 null strain to form biofilms was also investigated, and it was observed that biofilm development does not readily occur for this strain. Virulence of each strain was examined utilizing the mouse model of systemic candidiasis. Mice infected with CAF2-1 succumbed to infection within 3–7 days. All mice infected with the null strain survived for the duration of experiments, while the heterozygote and reconstructed heterozygote strains showed an intermediate level of virulence. These findings suggest that NOT4 may play a role in affecting strain pathogenicity, possibly by regulating expression of certain genes that effect cellular morphogenesis and virulence.
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Intracellular autoregulation of the Mycobacterium tuberculosis PrrA response regulator
More LessTwo-component systems are major regulatory systems for bacterial adaptation to environmental changes. During the infectious cycle of Mycobacterium tuberculosis, adaptation to an intracellular environment is critical for multiplication and survival of the micro-organism within the host. The M. tuberculosis prrA gene, encoding the regulator of the two-component system PrrA–PrrB, has been shown to be induced upon macrophage phagocytosis and to be transiently required for the early stages of macrophage infection. In order to study the mechanisms of regulation of the PrrA–PrrB two-component system, PrrA and the cytoplasmic part of the PrrB histidine kinase were produced and purified as hexahistidine-tagged recombinant proteins. Electrophoretic mobility shift assays indicated that PrrA specifically binds to the promoter of its own operon, with increased affinity upon phosphorylation. Moreover, induction of fluorescence was observed after phagocytosis of a wild-type M. tuberculosis strain containing the gfp reporter gene under the control of the prrA–prrB promoter, while this induction was not seen in a prrA/B mutant strain containing the same construct. These results indicate that the early intracellular induction of prrA depends on the autoregulation of this two-component system.
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- Physiology
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Aggregation of heat-shock-denatured, endogenous proteins and distribution of the IbpA/B and Fda marker-proteins in Escherichia coli WT and grpE280 cells
Submission of wild-type Escherichia coli to heat shock causes an aggregation of cellular proteins. The aggregates (S fraction) are separable from membrane fractions by ultracentrifugation in a sucrose density gradient. In contrast, no protein aggregation was detectable in an E. coli grpE280 mutant either by this technique or by electron microscopy. In search of an explanation for this observation at a molecular level, two kinds of marker proteins were used: Fda (fructose-1,6-biphosphate aldolase), the previously identified S fraction component, and IbpA/B, small heat-shock proteins abundantly associated with the S fraction proteins. Both types of marker proteins, normally never found in the outer-membrane (OM) fraction of WT cells, were present in the OM fraction from grpE cells after heat shock. This pointed to the presence of aggregates smaller than those in WT cells that cosedimented with the OM fraction. The OM fraction was enlarged in grpE cells. Although not proven directly, the presence of still smaller aggregates, not exceeding the solubility level and containing inactive Fda, was noted in the soluble CP fraction containing the cytoplasmic and periplasmic proteins. Therefore, aggregation occurred in both strains, but in a different way. The autoregulation of the heat-shock response causes a greater increase of DnaK/DnaJ and IbpAB levels in grpE cells than in WT after temperature elevation. This may explain the prevalence of the small-sized aggregates in the grpE cells. Estimation of total Fda protein before and after heat shock did not show any loss. This indicated that renaturation rather than proteolysis underlies the final disappearance of the aggregates. Though surprising at first, this is not contradictory with the participation of heat-shock proteases in removal of protein components of the S fraction as shown before, since proteins that are irreversibly denatured are probably substrates for the proteases.
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- Plant-Microbe Interactions
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Secretion of immunodominant membrane protein from onion yellows phytoplasma through the Sec protein-translocation system in Escherichia coli
A gene that encodes a putative SecE protein, which is a component of the Sec protein-translocation system, was cloned from the onion yellows phytoplasma (OY). The identification of this gene and the previously reported genes encoding SecA and SecY provides evidence that the Sec system exists in phytoplasma. In addition, a gene encoding an antigenic membrane protein (Amp) (a type of immunodominant membrane protein) of OY was cloned and sequenced. The OY amp gene consisted of 702 nt encoding a protein of 233 aa which was highly similar to Amp of aster yellows phytoplasma (AY). Part of OY Amp was overexpressed in Escherichia coli, purified, and used to raise an anti-Amp polyclonal antibody. The anti-Amp antibody reacted specifically with an OY-infected plant extract in Western blot analysis and was therefore useful for the detection of OY as well as Amp. Amp has a conserved protein motif that is known to be exported by the Sec system of E. coli. A partial OY Amp protein expressed in E. coli was localized in the periplasm as a shorter, putatively processed form of the protein. It had probably been exported from the cytoplasm to the periplasm through the Sec system. Moreover, OY Amp protein expressed in OY and detected in OY-infected plants was apparently also processed. Because phytoplasmas cannot be cultured or transformed, little information is available regarding their protein secretion systems. This study suggests that the Sec system operates in this phytoplasma to export OY Amp.
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