- Volume 146, Issue 9, 2000
Volume 146, Issue 9, 2000
- Microbiology Comment
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- Biochemistry
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Binding of glycoglycerolipid derived from membranes of Acholeplasma laidlawii PG8 and synthetic analogues to lymphoid cells
A component that binds to human lymphoid cells was isolated from the membranes of Acholeplasma laidlawii PG8. The component was extracted using the Bligh–Dyer method and purified using a silica-gel column and TLC. The active component was identified as 3-O-[2′-O-(α-D-glucopyranosyl)- 6′-O-acyl-α-D-glucopyranosyl]-1,2-di-O- acyl-sn-glycerol (GAGDG) using 1H- and 13C-NMR and GC-MS. The compositions of the major saturated fatty acids were nC 14 (17·8%), isoC14 (10·7%) and nC 16 (34·9%) as determined by GC-MS. The amounts of unsaturated species were less than 10% of those of the corresponding saturated acids. GAGDGs which have three tetradecanoyl groups were synthesized. These synthetic GAGDGs, as well as GAGDGs derived from A. laidlawii membranes, had a high binding affinity for MOLT-4 and HUT-78 (human T cell lines), Raji (a B cell line), HL-60 (a monoblastoid cell line) and primary cultured human T cells. The binding affinities of GAGDGs with an isoC14 acyl group was higher than those with nC14 and nC16 acyl groups. The binding to lymphoid cells reveals a novel biological activity of GAGDGs.
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Evidence that the rate-limiting step for the biosynthesis of arachidonic acid in Mortierella alpina is at the level of the 18:3 to 20:3 elongase
More LessMortierella alpina, a fungus used commercially as a source of arachidonic acid, 20:4(n-6), has been examined to see if growth on lipid-based carbon sources leads to repression of either fatty acid biosynthesis and/or fatty acid desaturation and elongation. Changes in the activities of ATP:citrate lyase, isocitrate lyase, carnitine acetyltransferase, malic enzyme, glucose-6-phosphate dehydrogenase and pyruvate kinase when the fungus was grown on fatty-acid-based (Tween) carbon sources were consistent with (i) the cells using the fatty acyl portion of the substrate as the sole carbon source, (ii) pyruvate kinase being the source of pyruvate for biosynthesis under these conditions and (iii) malic enzyme’s major function being as a provider of NADPH for lipid biosynthesis. The abolition of fatty acid synthase activity when cells were grown on Tweens indicated the cessation of de novo fatty acid biosynthesis under these conditions. The fatty acyl composition of the lipid accumulated by the fungus grown on Tweens 20, 40 and 80 showed that desaturation and elongation of the substrate lipid still occurred. The absolute amount of arachidonic acid synthesized by Tween-grown cells was the same as for cells grown on glucose. The transformation of incorporated fatty acids into 20:4(n-6) was, it appeared, limited at the elongation of 18:3(n-6) to 20:3(n-6) as, in every case, 18:1, 18:2 and 18:3(n-6) increased in amount in the Tween-grown cells. These data show for the first time that fatty acid synthesis is regulated separately from fatty acid desaturation/elongation and that the latter reactions are not repressed by growth of the fungus on simple fatty acids. Furthermore, the data strongly implicate the elongation of 18:3(n-6) to 20:3(n-6) as the limiting step in arachidonic acid biosynthesis by Mort. alpina.
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Regulation of the glucose-specific phosphotransferase system (PTS) of Staphylococcus carnosus by the antiterminator protein GlcT
The GenBank accession number for the complete ORF of the gene encoding GlcT is Y14029.
The ptsG operon of Staphylococcus carnosus consists of two adjacent genes, glcA and glcB, encoding glucose- and glucoside-specific enzymes II, respectively, the sugar permeases of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). The expression of the ptsG operon is glucose-inducible. Putative RAT (ribonucleic antiterminator) and terminator sequences localized in the promoter region of glcA suggest regulation via antitermination. The glcT gene was cloned and the putative antiterminator protein GlcT was purified. Activity of this protein was demonstrated in vivo in Escherichia coli and Bacillus subtilis. In vitro studies led to the assumption that phosphoenolpyruvate-dependent phosphorylation of residue His105 via the general PTS components enzyme I and HPr facilitates dimerization of GlcT and consequently activation. Because of the high similarity of the two ptsG-RAT sequences of B. subtilis and S. carnosus, in vivo studies were performed in B. subtilis. These indicated that GlcT of S. carnosus is able to recognize ptsG-RAT sequences of B. subtilis and to cause antitermination. The specific interaction between B. subtilis ptsG-RAT and S. carnosus GlcT demonstrated by surface plasmon resonance suggests that only the dimer of GlcT binds to the RAT sequence. HPr-dependent phosphorylation of GlcT facilitates dimer formation and may be a control device for the proper function of the general PTS components enzyme I and HPr necessary for glucose uptake and phosphorylation by the corresponding enzyme II.
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- Genetics And Molecular Biology
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Allele-specific gene targeting in Candida albicans results from heterology between alleles
More LessThe GenBank accession numbers for the sequences reported in this paper are AF247189 and AF247190 for PHR1-1 and PHR1-2, respectively.
The opportunistic fungal pathogen Candida albicans is asexual and diploid. Thus, introduction of recessive mutations requires targeted gene replacement of two alleles to effect expression of a recessive phenotype. This is often performed by recycling of a URA3 marker gene that is flanked by direct repeats of hisG. After targeting to a locus, recombination between the repeats excises URA3 leaving a single copy of hisG in the disrupted allele. The remaining functional allele is targeted in a second transformation with the same URA3 marked construct. Replacement can be highly biased toward one allele. At the PHR1 locus, there was an approximately 50-fold preference for replacement of the disrupted versus the functional allele in a heterozygous mutant. This preference was reduced six- to eightfold when the transforming DNA lacked the hisG repeats. Nonetheless, there remained a sixfold preference for targeting a particular allele of PHR1 and this was evident even in transformations of the parental strain containing two wild-type alleles of PHR1. Both wild-type alleles were cloned and nucleotide sequence comparison revealed 24 heterologies over a 2 kb region. Using restriction site polymorphisms to distinguish alleles, it was observed that transformation with the cloned DNA of allele PHR1-1 preferentially targeted allele 1 of the genome. Transformations with PHR1-2 exhibited the reciprocal specificity. In both these instances, heterology was present in the flanking regions of the transforming DNA. When the transforming DNA was chosen from a region 100% identical in both alleles, alleles 1 and 2 were targeted with equal frequency. It is concluded that sequence heterology between alleles results in an inherent allele specificity in targeted recombination events.
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Cell wall perturbation in yeast results in dual phosphorylation of the Slt2/Mpk1 MAP kinase and in an Slt2-mediated increase in FKS2–lacZ expression, glucanase resistance and thermotolerance
The protein kinase C (PKC1) pathway is essential for maintaining cell integrity in yeast. Here it is shown that various forms of cell wall damage result in activation of the downstream MAP kinase Slt2/Mpk1. Several cell wall mutants displayed enhanced FKS2–lacZ expression, a known output of Slt2 activation. A similar response was obtained with wild-type cells grown in the presence of the cell wall perturbants Calcofluor white and Zymolyase. Upregulation of FKS2–lacZ in response to sublethal concentrations of these agents fully depended on the presence of Slt2. The same cell wall stress conditions resulted in dual threonine and tyrosine phosphorylation of Slt2. Both Slt2 phosphorylation and FKS2–lacZ induction could be largely prevented by providing osmotic support to the plasma membrane. Interestingly, Slt2 phosphorylation in response to cell wall damage required the putative plasma-membrane-located sensor Mid2 but not Hcs77/Wsc1. Finally, cell wall perturbation gave rise to cells with increased resistance to glucanase digestion and heat shock. These responses depended on the presence of Slt2. These results indicate that weakening of the cell wall activates the Slt2/Mpk1 MAP kinase pathway and results in compensatory changes in the cell wall.
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Suppression of sorbitol dependence in a strain bearing a mutation in the SRB1/PSA1/VIG9 gene encoding GDP-mannose pyrophosphorylase by PDE2 overexpression suggests a role for the Ras/cAMP signal-transduction pathway in the control of yeast cell-wall biogenesis
Complementation studies and allele replacement in Saccharomyces cerevisiae revealed that PSA1/VIG9, an essential gene that encodes GDP-mannose pyrophosphorylase, is the wild-type SRB1 gene. Cloning and sequencing of the srb1-1 allele showed that it determines a single amino acid change from glycine to aspartic acid at residue 276 (srb1 D276). Genetic evidence is presented showing that at least one further mutation is required for the sorbitol dependence of srb1 D276. A previously reported complementing gene, which this study has now identified as PDE2, is a multi-copy suppressor of sorbitol dependence and is not, as was previously suggested, the SRB1 gene. srb and pde2 mutants share a number of phenotypes, including lysis upon hypotonic shock and enhanced transformability. These data are consistent with the idea that the Ras/cAMP pathway might modulate cell-wall construction.
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Each peptide of the two-component lantibiotic lacticin 3147 requires a separate modification enzyme for activity
More LessThe genetic determinants for production and immunity to the two-component lantibiotic lacticin 3147 are encoded by a 12·6 kb region of the plasmid pMRC01. This region contains ten genes arranged in two divergent clusters; these include the structural genes and a number of genes whose products show significant similarity to proteins involved in the biosynthesis of other lantibiotics. Using a strategy of deletion and mutational analysis, the effect of disruption of a number of these genes was investigated. Inactivation of either of the structural genes, ltnA1 or ltnA2, resulted in mutants that were incapable of producing active lacticin 3147; however, the combination of the cell-free supernatant from both mutants resulted in a restoration of bacteriocin activity, confirming that processing and export of the structural peptides can occur independently. An unusual feature of the lacticin 3147 gene cluster is the presence of two lanM homologues, whose gene products are proposed to be involved in the dehydration and thioether-forming reactions which result in lanthionine bridge formation. Mutants created in the ltnM1 and ltnM2 genes were also incapable of lantibiotic production, confirming an essential role for these enzymes in the lacticin 3147 biosynthetic pathway and supporting the assertion that these proteins are modification enzymes. Interestingly, addition of purified LtnA1, but not purified LtnA2, to the cell-free supernatant of the ltnM1 mutant restored bacteriocin activity; in contrast, only purified LtnA2 could complement the cell-free supernatant of the ltnM2 mutant. Creation of a number of double mutants supported these findings, and confirmed that LtnM1 is required to produce mature LtnA1, while LtnM2 is required to produce mature LtnA2.
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The synthesis of the bacteriocin sakacin A is a temperature-sensitive process regulated by a pheromone peptide through a three-component regulatory system
More LessSakacin A is a bacteriocin produced by Lactobacillus sakei Lb706. The gene cluster (sap) encompasses a regulatory unit composed of three consecutive genes, orf4 and sapKR. sapKR encode a histidine protein kinase and a response regulator, while orf4 encodes the putative precursor of a 23-amino-acid cationic peptide (termed Sap-Ph). The authors show that Sap-Ph serves as a pheromone regulating bacteriocin production. Lb706 produced bacteriocin when the growth temperature was kept at 25 or 30 °C, but production was reduced or absent at higher temperatures (33·5–35 °C). Production was restored by lowering the growth temperature to 30 °C, but at temperatures of 33–34 °C also by adding exogenous Sap-Ph to the growth medium. A knock-out mutation in orf4 abolished sakacin A production. Exogenously added Sap-Ph complemented this mutation, unambiguously showing the essential role of this peptide for bacteriocin production. Another sakacin A producer, Lactobacillus curvatus LTH1174, had a similar response to temperature and exogenously added Sap-Ph.
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Study of the bldG locus suggests that an anti-anti-sigma factor and an anti-sigma factor may be involved in Streptomyces coelicolor antibiotic production and sporulation
More LessThe GenBank accession numbers for the sequences reported in this paper are AF134889 and AL035636
A cloned 2·5 kb DNA fragment that can restore antibiotic production and sporulation to a bldG mutant encodes a 113 aa protein showing similarity to a family of anti-anti-sigma factors from Bacillus and Staphylococcus; and the deduced product of a closely spaced downstream ORF, designated ORF3, shows similarity to cognate anti-sigma factors. The homologues in Bacillus regulate the activity of sporulation- and stress-response-specific sigma factors. However, there is no sigma factor gene near bldG and ORF3. bldG is transcribed both as a monocistronic and a polycistronic mRNA, the latter including the downstream ORF3 gene. The two transcripts were present at all time points during growth and both were upregulated when aerial mycelium and pigmented antibiotics were seen. At all time points, the monocistronic bldG transcript was two- to threefold more abundant than the polycistronic transcript. Mapping of the mRNA 5′ ends indicated that bldG transcription is initiated from two transcription start sites located 82 and 123 bp upstream of the bldG translation start. A constructed bldG null mutant had the same phenotype as previously isolated bldG point mutations, some of which were shown to have potentially significant base changes within bldG. When compared to the wild-type strain, the null mutant showed no differences in the levels of transcription from the two bldG promoters. These results suggest that bldG is not involved in autoregulation.
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ISBst12, a novel type of insertion-sequence element causing loss of S-layer-gene expression in Bacillus stearothermophilus ATCC 12980
More LessThe GenBank accession number for the sequence reported in this paper is AF162268.
The cell surface of the surface layer (S-layer)-carrying strain of Bacillus stearothermophilus ATCC 12980 is completely covered with an oblique lattice composed of the S-layer protein SbsC. In the S-layer-deficient strain, theS-layer gene sbsC was still present but was interrupted by a novel type of insertion sequence (IS) element designated ISBst12. The insertion site was found to be located within the coding region of the sbsC gene, 199 bp downstream from the translation start of SbsC. ISBst12 is 1612 bp long, bounded by 16 bp imperfect inverted repeats and flanked by a directly repeated 8 bp target sequence. ISBst12 contains an ORF of 1446 bp and is predicted to encode a putative transposase of 482 aa with a calculated theoretical molecular mass of 55562 Da and an isoelectric point of 9·13. The putative transposase does not exhibit a typical DDE motif but displays aHis-Arg-Tyr triad characteristic of the active site of integrases from the bacteriophage λ Int family. Furthermore, two overlapping leucine-zipper motifs were identified at the N-terminal part of the putative transposase. As revealed by Southern blotting, ISBst12 was present in multiple copies in the S-layer-deficient strain as well as in the S-layer-carrying strain. Northern blotting indicated that S-layer gene expression is already inhibited at the transcriptional level, since no sbsC-specific transcript could be identified in the S-layer-deficient strain. By using PCR, ISBst12 was also detected in B. stearothermophilus PV72/p6, in its oxygen-induced strain variant PV72/p2 and in the S-layer-deficient strain PV72/T5.
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Characterization of IS900 loci in Mycobacterium avium subsp. paratuberculosis and development of multiplex PCR typing
More LessThe GenBank accession numbers for the sequences reported in this paper are AJ011838, AJ250015–AJ250023 and AJ251434–AJ251437.
Mycobacterium avium subsp. paratuberculosis is a pathogen that causes chronic inflammation of the intestine in many animals, including primates, and is implicated in Crohn’s disease in humans. It differs from other members of the M. avium complex in having 14–18 copies of IS900 inserted into conserved loci in its genome. In the present study, genomic DNA flanking 14 of these insertions was characterized and homologues in the Mycobacterium tuberculosis and M. avium subsp. avium genomes were identified. These included regions encoding a sigma factor (sigJ) at locus 3, a nitrate reductase (nirA) at locus 4, a transcription regulator (tetR) and polyketide synthase at locus 6, and a 6-O-methylguanine methyltransferase at locus 9. In addition, locus numbers were assigned to 9 of 15 RFLP bands previously described. IS900 insertion at 7 of the 14 characterized loci was into the RBS of a gene substituting an RBS encoded by IS900 sited two bases closer to the initiation codon. IS900 insertion at five loci interrupted an ORF at the target site, one of which encoded a homologue of the immunodominant mycobacterial DesA1 protein. Eleven of eighty-one M. avium subsp. paratuberculosis isolates lacked the insertion site at locus 6 together with flanking genomic DNA. This region was also absent from seven reference strains of M. avium subsp. avium, from one M. avium subsp. silvaticum and from six other mycobacterial species. A multiplex PCR of IS900 loci (MPIL) typing method was developed which was able to discriminate 10 different types of M. avium subsp. paratuberculosis from the panel of 81 isolates with consistent differences between those of bovine and ovine origin. Nine MPIL types corresponded with a single PstI/BstEII RFLP type, suggesting that this method may be applicable to typing of M. avium subsp. paratuberculosis directly from a sample without the need for culture. The remaining MPIL type corresponded with seven PstI/BstEII RFLP types. Further resolution of these may come from sequencing the remaining four uncharacterized IS900 loci.
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Inhibition of chromosome replication in Mycobacterium smegmatis: effect of the rpmH–dnaA promoter region
More LessIn a previous study a functional mycobacterial origin of replication, oriC, was isolated on a plasmid. However, it was found that origin function was inhibited by the presence of the adjacent dnaA gene or its regulatory region, so that plasmids containing both of these regions next to the origin did not yield transformants. This inhibition could be due either to overexpression of dnaA on a plasmid being toxic, the transcription of dnaA into the downstream origin topologically inhibiting its function, or to the DnaA boxes upstream of dnaA somehow interacting with the DnaA boxes in the origin to prevent its function. To distinguish between these possibilities, plasmids were constructed lacking different parts of the dnaA gene: the promoter, the DnaA boxes, or both. Additionally, the putative dnaA promoter region was replaced by mycobacterial sequences that exhibit weaker or null promoter activity. The results indicate that the rpmH–dnaA promoter region, but not the DnaA boxes, is the principal cause of the incompatibility observed and suggest that this region could be playing a role in the inhibition of chromosome replication.
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Molecular basis for resistance to the anticancer drug cisplatin in Dictyostelium
More LessThe GenBank accession numbers for the sequences reported in this paper are AF233610 (S-1-P lyase), AF233612 (PIP5K), AF233611 (P2Y purine receptor 1), AF233613 (CAAX prenyl protease) and AF233614 (unidentified gene).
The efficacy of the widely used chemotherapeutic drug cisplatin is limited by the occurrence of drug-resistant tumour cells. To fully exploit the potential of this drug in cancer therapy, it is imperative to understand the molecular basis of cisplatin resistance. Using an insertional mutagenesis technique in cells of Dictyostelium discoideum, we have identified six genes which are involved in cisplatin resistance. None of these genes has been previously linked to resistance to this drug. Several of these genes encode proteins that are involved in signal transduction pathways which regulate cell death, cell proliferation or gene regulation. The resistance of these mutant strains is specific for cisplatin, since deletion of these genes does not confer resistance to other DNA-damaging agents. Significantly, the disruption of three of these genes, encoding the sphingosine-1-phosphate lyase, the RegA cAMP phosphodiesterase and a phosphatidylinositol-4-phosphate 5-kinase, also results in abnormalities in the multicellular development of this organism, although there is no change in the rate of mitotic cell growth. This study has identified previously unsuspected molecular pathways which function in the cellular response to cisplatin and are required for normal morphogenesis, and underscores the complexity of the cellular response to cisplatin. These pathways provide potential targets for modulating the response to this important drug.
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Fructose operon mutants of Spiroplasma citri
More LessThe GenBank accession number for the sequence reported in this paper is AF202665.
Fructose-negative mutants of Spiroplasma citri wild-type strain GII-3 were selected by two methods. The first method is based on the selection of spontaneous xylitol-resistant mutants, xylitol being a toxic fructose analogue. Five such mutants were obtained, but only one, xyl3, was unable to use fructose and had no phosphoenolpuryvate:fructose phosphotransferase system (fructose-PTS) activity. Amplification and sequencing of the fructose permease gene of mutant xyl3 revealed the presence of an adenylic insertion leading to a truncated permease. The second method is based on inactivation of fruA and/or fruK by homologous recombination involving one crossing-over between the chromosomal genes and inactivated genes carried by replicative plasmids. Fructose-negative mutants were obtained at a frequency of about 10%. Fructose-PTS activity and 1-phosphofructokinase activity were not detected in four representative mutants that were characterized (H31, H45, E38 and E53). In strain H31, Southern blot analysis and PCR showed that the result of homologous recombination was, as expected, the presence in the chromosome of two mutated fruA–fruK copies with the plasmid sequence in between. Only the mutated copy, under control of the fructose operon promoter, was transcribed. This work describes for the first time the use of two methods to obtain fructose-auxotrophic mutants of S. citri. The method involving homologous recombination is a general procedure for gene disruption in S. citri.
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The Sinorhizobium meliloti ExpE1 protein secreted by a type I secretion system involving ExpD1 and ExpD2 is required for biosynthesis or secretion of the exopolysaccharide galactoglucan
More LessIn Sinorhizobium meliloti the biosynthesis of the exopolysaccharide galactoglucan (EPS II) is directed by the exp genes. The expD1 and expD2 gene products are homologous to components of type I secretion systems. ExpE1, the gene of which is located adjacent to expD1 and expD2, was detected in S. meliloti cells and culture supernatants. ExpD1 and ExpD2 were required for the secretion of ExpE1, indicating that ExpE1 is secreted by a type I secretion system involving ExpD1 and ExpD2. ExpE1 contains 15 aspartate- and glycine-rich nonapeptide repeats that were suggested to bind Ca2+. The ability to bind Ca2+ was demonstrated for a recombinant ExpE1 protein. Extracellular EPS II was not detected in cultures of non-polar expD1, expD2 and expE1 deletion mutants implying that these three genes are required for biosynthesis or secretion of galactoglucan.
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The replication and stable-inheritance functions of IncP-9 plasmid pM3
More LessThe GenBank accession number for the sequence reported in this paper is AF078924.
Little is known of the transfer and maintenance machinery of the IncP-9 plasmids, which are found in Pseudomonas spp. and include both degradative and resistance plasmids. One such plasmid, pM3, which confers resistance to streptomycin and tetracycline, was found repeatedly in Pseudomonas species from numerous locations in Belarus. pM3 has a broad host range, but is unable to replicate in enterobacteria at 37 °C and above. A mini derivative, pMT2, was constructed by partial PstI digestion and ligation with a fragment encoding KmR. The complete sequence of pMT2 was determined. Analysis of its 8526 bp of pM3 DNA revealed several ORFs whose predicted polypeptide products were found to have similarity to previously analysed proteins involved in plasmid replication (rep gene), transfer (mpf; mating-pair formation gene) and stable maintenance (par, mrs genes). The organization of these genes showed similarity to several plasmid systems including the Ti and pSYM plasmids as well as IncP-1 plasmids. Subcloning narrowed down the region required for replication, and identified the putative rep gene and putative par promoter region as able to express incompatibility. rep deletion mutants were lost from the cell line, and expression of the rep gene was shown to be controlled by negative autoregulation. A pMT2 derivative with an insertion between the rep and par genes showed very weak, if any, ability to replicate autonomously, suggesting that plasmid maintenance may depend on a close interaction of rep and par functions.
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Conjugal transfer of plasmid pMV158: uncoupling of the pMV158 origin of transfer from the mobilization gene mobM, and modulation of pMV158 transfer in Escherichia coli mediated by IncP plasmids
More LessThe streptococcal plasmid pMV158 encodes a gene cassette involved in its mobilization by large conjugative plasmids. Two elements compose this region: i) the mobM gene, encoding the MobM protein that initiates transfer, and ii) the origin of transfer, oriT, which is the target of MobM. In vitro, MobM protein introduces a specific nick within the pMV158-oriT region on supercoiled pMV158 DNA. This paper reports the uncoupling of the oriT and the mobM gene, the latter being placed under the control of an inducible promoter. Upon induction, the vector containing pMV158-oriT was transferred in Escherichia coli matings at a moderate frequency whereas, in vitro, purified MobM protein efficiently cleaved the vector harbouring the pMV158-oriT. Transfer of this vector, as well as transfer of pMV158 in E. coli, required the presence of either the IncW R388 or the IncP RP4 plasmids as auxiliary plasmids. Dissection of the functions encoded by RP4 showed that the traG and traF genes were essential for pMV158 mobilization.
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Mosaic plasmids and mosaic replicons: evolutionary lessons from the analysis of genetic diversity in IncFII-related replicons
More LessThe EMBL accession numbers for the sequences reported in this paper are AJ009980 (pGSH500 alpha replicon) and AJ009981 (pLV1402 alpha replicon).
The alpha replicons of the multi-replicon plasmids pGSH500 and pLV1402 have been characterized by DNA sequence analysis. Analysis of the DNA sequence of a 3672 bp HindIII fragment from pFDT100, which contains the pGSH500 alpha replicon, revealed similarity to a number of replicons belonging to, or related to, those of the IncFII family. The replicon region contains copA, tapA, repA and oriR, and replication initiation and termination sites are related to those from the IncFII replicon of R1. A copB gene was found to lie upstream of the HindIII site in the parental plasmid pGSH500. Downstream of oriR, a 707 bp region shows 72·6% identity to a region of the Escherichia coli chromosome at 43·3′, suggesting this region of pGSH500 may have been incorporated into the plasmid during a past chromosomal recombination event. Oligonucleotide primers homologous to consensus regions in the copB and repA genes, and the oriR regions from a number of IncFII-related replicons were used to amplify replication regions from pLV1402. Analysis of the amplified regions has shown the presence of copB, copA, tapA and repA genes. Phylogenetic analysis of Rep protein sequences from the RepFIIA family of antisense-control-regulated replicons revealed the presence of three distinct subgroups of Rep proteins. Comparative analysis of DNA and protein sequences from members of the RepFIIA family provides evidence supporting the roles of both non-selective divergence in co-integrate (multi-replicon) plasmids and Chi-mediated-recombination in replicon evolution, and in particular, that such processes may have been widespread in the evolution of the RepFIIA family.
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Metal-ion tolerance in Escherichia coli: analysis of transcriptional profiles by gene-array technology
More LessEscherichia coli was adapted to grow in medium containing substantially elevated concentrations of either Zn(II), Cd(II), Co(II) or Ni(II). Whole-genome transcriptional profiles were generated from adapted strains and analysed for significant alteration in transcript abundance with reference to a wild-type strain. Similar alterations in specific message levels were observed for strains adapted to the four metal ions. One unexpected trend was the increase in transcript level of genes involved in transposition of IS elements, particularly insA. Subsequent expression of insA-7 from a heterologous promoter in E. coli conferred tolerance to Zn(II).
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- Pathogenicity And Medical Microbiology
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Effect of salivary secretory IgA on the adhesion of Candida albicans to polystyrene
Attachment of Candida albicans to plastic materials of dental prostheses or to salivary macromolecules adsorbed on their surface is believed to be a critical event in the development of denture stomatitis. In an earlier study, it was shown that adhesion of C. albicans to polystyrene, a model system to study the adhesion of C. albicans to plastic materials, can be partially inhibited with an mAb directed against cell wall polysaccharides of C. albicans. In the present study, the role of whole saliva in the adhesion of C. albicans to polystyrene has been investigated, and three mAbs directed against epitopes of cell wall mannoproteins have been used to mimic the inhibitory effect observed with salivary secretory IgA (sIgA) on the adhesion of C. albicans to polystyrene. In the absence of whole saliva, adherence of C. albicans 3153 increased with germination. However, the presence of whole saliva enhanced the adhesion to polystyrene of C. albicans 3153 yeast cells but decreased the adhesion of germinated cells. The enhancement of adhesion of yeast cells to polystyrene mediated by saliva was confirmed with an agerminative mutant of C. albicans 3153. The inhibition of the adhesion of C. albicans 3153 germ tubes to polystyrene was due to the salivary sIgA since sIgA-depleted saliva enhanced the adhesion of C. albicans 3153 to polystyrene. The inhibitory effect mediated by sIgA was not related to the inhibition of germination but to the blockage of adhesins expressed on the cell wall surface of the germ tubes. The three mAbs studied reduced the adhesion of C. albicans 3153 to polystyrene at levels equivalent to those for purified sIgA. The highest reduction in the adhesion was obtained with the IgA mAb N3B. The best results were obtained when the three mAbs were combined. The results suggest that whole saliva plays a different role in the adhesion of C. albicans to polystyrene depending on the morphological phase of C. albicans. These results may give new insights into the conflicting role of saliva in the adhesion of C. albicans to plastic materials of dental prostheses.
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- Physiology And Growth
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Hyperphosphorylation of Msn2p and Msn4p in response to heat shock and the diauxic shift is inhibited by cAMP in Saccharomyces cerevisiae
In response to various stresses, as well as during the diauxic transition, the Msn2p and Msn4p transcription factors of Saccharomyces cerevisiae are activated and induce a large set of genes. This activation is inhibited by the Ras/cAMP/PKA (cAMP-dependent protein kinase) pathway. Here we show by immunoblotting experiments that Msn2p and Msn4p are phosphorylated in vivo during growth on glucose, and become hyperphosphorylated at the diauxic transition and upon heat shock. This hyperphosphorylation is correlated with activation of Msn2/4p-dependent transcription. An increased level of cAMP prevents and reverses these hyperphosphorylations, indicating that kinases other than PKA are involved. These results suggest that PKA and stress-activated kinases control Msn2/4p activity by antagonistic phosphorylation. It was also noted that Msn4p is transiently increased at the diauxic transition. Msn2p and Msn4p present different hyperphosphorylation patterns in response to different stresses.
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Mutants of Mycobacterium smegmatis impaired in stationary-phase survival
More LessThe GenBank accession numbers for the sequences determined in this work are: AJ277088 (mutant 272A), AJ277089 (mutant 272E), AJ27790 (mutant 317C), AJ277152 (mutant 492A) and AJ276883 (mutant 3910D).
A bank of 600 insertional mutants of Mycobacterium smegmatis was screened for mutants defective in stationary-phase survival. Of 74 mutants picked by the initial screen, 21 had stationary-phase survival defects and 7 of these were studied in more detail. In general, mutants survived stationary phase significantly less well in rich medium than under carbon-starvation conditions. In all cases the loss of viability in stationary phase was not complete even after prolonged incubation. All mutants showed an initial decrease in viability, during the first 40 d in stationary phase, followed by an increase in viable counts that returned viability close to the levels of the wild-type. Southern hybridization experiments showed that recovery of viability was not a consequence of precise excision or movement of the transposon. Two of the survival mutants differed from the wild-type in their colony morphology, and recovery of their viability in stationary phase was coincident with the return of wild-type colony morphology. It is possible that second-site suppressor mutations accumulate that alleviate the effects of the original mutation. For five of the mutants the DNA flanking the site of transposition was amplified by ligation-mediated PCR and sequenced to identify the disrupted locus. In each case, homologous genes were identified in the Mycobacterium tuberculosis genome, three of which have clearly predicted functions in M. tuberculosis as a penicillin-binding protein, in biotin biosynthesis and as a polyketide synthase. This is the first identification of genes implicated in the stationary-phase survival of mycobacteria.
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- Systematics And Evolution
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Concerted evolution of duplicate fla genes in Campylobacter
More LessCampylobacters have two similar copies (flaA and flaB) of their flagellin gene. It has been hypothesized that the two copies can serve for antigenic phase variation. Analysis of polymorphisms within aligned multiple DNA sequences of the Campylobacter flagellin genes revealed high pairwise homoplasy indexes between flaB/flaB pairs that were not observed between any flaA/flaA pairings or flaA/flaB pairings. Thus it seems there are constraints on the sequence of flaB that distinguish it from flaA. Nevertheless, segments of the two genes that are highly variable between strains are conserved between the flaA and flaB copies of the genes within a strain. The patterns of synonymous and non-synonymous differences suggest that one segment of the flagellin sequence is under selective pressure at the amino acid sequence level. Another segment of the protein is maintained within a strain by conversion or recombination. Comparisons of strict consensus amino acid sequences did not reveal any motifs that are uniquely FlaA or FlaB, but there are differences between FlaA and FlaB in those amino acids available for post-translational modification. The observed pattern of concerted evolution of portions of a structural gene is an unusual finding in bacteria and should be searched for with other duplicated genes. Concerted evolution was unexpected for genes involved in phase variation since it minimizes the antigenic repertoire that can be expressed by a single clone in the face of the host immune response.
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Genetic variation of dTDP-l-rhamnose pathway genes in Salmonella enterica
More LessThe GenBank accession numbers for the sequences reported in this paper are AF279615–AF279625 for the rml gene sets and AF279626–AF279648 for the rmlB gene fragments.
The genetic variation in the dTDP-L-rhamnose pathway gene set (rmlB, rmlD, rmlA, rmlC) in Salmonella enterica was examined after sequencing the four genes from 11 rml-containing gene clusters encoding seven O antigens, and a 903 bp rmlB segment from another 23 strains representing the seven subspecies. There was considerable sequence variation and strong polarity in the nature and level of variation among rml genes. The 5′ end of the rml gene set, including rmlB, rmlD and most of rmlA, is in general subspecies specific. In contrast, the 3′ end, including part of rmlA and all of rmlC, is O antigen specific. The G+C content of the 3′ end is lower than that of the 5′ end. The variation in the 3′ end of the gene set is much greater than that of the 5′ end. It is apparent that the rml gene set of S. enterica includes genes with two different evolutionary histories. In addition, there has been extensive recombination in the gene set, probably related to O antigen transfer between subspecies. These findings provide evidence for the lateral transfer of O antigen genes between species and among subspecies of S. enterica. The results have also shown that conserved genes at the end of an O antigen gene cluster play a major role in mediating exchange of the central serogroup-specific regions.
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A deeply branched novel phylotype found in Japanese paddy soils
The GenBank/EMBL/DDBJ accession numbers for the sequences of the novel soil clones and their aligned data set are D88480–D88489 and ds36901, respectively.
Novel 16S rDNA clones which possibly constitute a sister clade from the two known archaeal lineages, Crenarchaeota and Euryarchaeota, were found in paddy soil environments. Overall signature sequences showed that the clone sequences shared a majority of signature sequence features with the Archaea and Eukarya. However, there were at least nine nucleotides which distinguished the novel clones from the domains Archaea and Eukarya. Phylogenetic trees, drawn by maximum-parsimony, neighbour-joining and maximum-likelihood methods, also supported the unique phylogenetic position of the clones. Both signature sequence and phylogenetic analyses strongly suggest that the novel organisms constitute a new group and their phylogenetic positions are distant from the Crenarchaeota and Euryarchaeota. A specific primer set was designed to detect the presence of the novel group of organisms in terrestrial environments. Specific DNA fragments were amplified from all paddy soil DNAs, suggesting that the novel organisms are widely distributed in rice paddy fields in Japan.
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