- Volume 146, Issue 9, 2000
Volume 146, Issue 9, 2000
- Genetics And Molecular Biology
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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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