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Volume 145,
Issue 5,
1999
Volume 145, Issue 5, 1999
- Genetics And Molecular Biology
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Structural and putative regulatory genes involved in cellulose synthesis in Rhizobium leguminosarum bv. trifolii
More LessSix genes involved in cellulose synthesis in Rhizobium leguminosarum bv. trifolii were identified using Tn5 mutagenesis. Four of them displayed homology to the previously cloned and sequenced Agrobacterium tumefaciens cellulose genes celA, celB, celC and celE. These genes are organized similarly in R. leguminosarum bv. trifolii. In addition, there were strong indications that two tandemly located genes, celR1 and celR2, probably organized as one operon, are involved in the regulation of cellulose synthesis. The deduced amino acid sequences of these genes displayed a high degree of similarity to the Caulobacter crescentus DivK and PleD proteins that belong to the family of two-component response regulators. This is to our knowledge the first report of genes involved in the regulation of cellulose synthesis. Results from attachment assays and electron microscopic studies indicated that cellulose synthesis in R. leguminosarum bv. trifolii is induced upon close contact with plant roots during the attachment process.
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Functional analysis of the O antigen glucosylation gene cluster of Shigella flexneri bacteriophage SfX
More LessPrevious studies have shown that Shigella flexneri bacteriophage X (SfX) encodes a glucosyltransferase (GtrX, formerly Gtr), which is involved in O antigen modification (serotype Y to serotype X). However, GtrX alone can only mediate a partial conversion. More recently, a three-gene cluster has been identified next to the attachment site in the genome of two other S. flexneri bacteriophages (i.e. SfV and SfII). This gene cluster was postulated to be responsible for a full O antigen conversion. Here it is reported that besides the gtrX gene, the other two genes in the gtr locus of SfX were also involved in the O antigen modification process. The first gene in the cluster (gtrA) encodes a small highly hydrophobic protein which appears to be involved in the translocation of lipid-linked glucose across the cytoplasmic membrane. The second gene in the cluster (gtrB) encodes an enzyme catalysing the transfer of the glucose residue from UDP-glucose to a lipid carrier. The third gene (gtrX) encodes a bacteriophage-specific glucosyltransferase which is largely responsible for the final step, i.e. attaching the glucosyl molecules onto the correct sugar residue of the O antigen repeating unit. A three-step model for the glucosylation of bacterial O antigen has been proposed.
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Molecular analysis of the recA gene and SOS box of the purple non-sulfur bacterium Rhodopseudomonas palustris no. 7
More LessThe recA gene of the purple non-sulfur bacterium Rhodopseudomonas palustris no. 7 was isolated by a PCR-based method and sequenced. The complete nucleotide sequence consists of 1089 bp encoding a polypeptide of 363 amino acids which is most closely related to the RecA proteins from species of Rhizobiaceae and Rhodospirillaceae. A recA-deficient strain of R. palustris no. 7 was obtained by gene replacement. As expected, this strain exhibited increased sensitivity to DNA-damaging agents. Transcriptional fusions of the recA promoter region to lacZ confirmed that the R. palustris no. 7 recA gene is inducible by DNA damage. Primer extension analysis of recA mRNA located the recA gene transcriptional start. A sequential deletion of the fusion plasmid was used to delimit the promoter region of the recA gene. A gel mobility shift assay demonstrated that a DNA-protein complex is formed at this promoter region. This DNA-protein complex was not formed when protein extracts from cells treated with DNA-damaging agents were used, indicating that the binding protein is a repressor. Comparison of the minimal R. palustris no. 7 recA promoter region with the recA promoter sequences from other α-Proteobacteria revealed the presence of the conserved sequence GAACA-N6-G(A/T)AC. Site-directed mutations that changed this consensus sequence abolished the DNA-damage-mediated expression of the R. palustris recA gene, confirming that this sequence is the SOS box of R. palustris and probably plays the same role in other α-Proteobacteria.
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- Genomics
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Nucleotide sequence of the Bacillus subtilis temperate bacteriophage SPβc2
The Bacillus subtilis 168 chromosomal region extending from 184° to 195°, corresponding to prophage SPβ, has been completely sequenced using DNA of the thermoinducible SPβc2 mutant. This 134416 bp segment comprises 187 putative ORFs which, according to their orientation, were grouped into three clusters. Compared to its host, SPβc2 is characterized by a lower G+C content, shorter mean ORF length, as well as a different usage of start codons. Nearly 75% of predicted ORFs do not share significant homologies to sequences in available databases. The only highly similar proteins to SPβc2-encoded ones are host paralogues. SPβc2 promoter regions contain SOS box consensus sequences and a repeated motif, designated SPβ repeated element (SPBRE), that is absent from the host genome. Gene sspC, encoding the small acid-soluble protein C, that has been previously sequenced and mapped to the vicinity of the SPβ region, was found to be part of the prophage.
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- Pathogenicity And Medical Microbiology
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Proteolytic cleavage of the A subunit is essential for maximal cytotoxicity of Escherichia coli 0157:H7 Shiga-like toxin-1
More LessMembers of the bacterial Shiga toxin family consist of a single A subunit that is non-covalently associated with a pentamer of B subunits. These toxins bind to receptors on susceptible mammalian cells and enter the cells by endocytic uptake. During cell entry, the 32 kDa A subunit is cleaved by the membrane-anchored protease furin to generate a catalytically active, 27·5 kDa A1 fragment and a 4·5 kDa A2 fragment. Previous studies have shown that mutating the furin site to prevent cleavage did not significantly affect toxin potency, suggesting that cleavage is not required for toxin activity. Here it is confirmed that preventing cleavage at the usual processing site does not prevent proteolytic processing of the Escherichia coli Shiga-like toxin-1 A subunit. However, simultaneous mutation of both the primary furin-recognition site and a nearby putative furin cleavage site did prevent intracellular processing of the A subunit. Comparison of the cytotoxicities of purified recombinant toxins to cultured mammalian cells demonstrated that even on prolonged incubation with toxin, the unprocessed mutant was 60-fold less toxic than the wild-type protein or other mutants still capable of being proteolytically processed during cell entry.
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The large plasmids of Shiga-toxin-producing Escherichia coli (STEC) are highly variable genetic elements
More LessShiga-toxin-producing Escherichia coli (STEC) of different serotypes are known to harbour large plasmids. The aim of this study was to investigate, using the example of the plasmid-encoded serine protease EspP, whether these plasmids are a uniform genetic element present in STEC. Examination of 201 diarrhoeagenic E. coli strains using a newly developed espP-specific PCR showed that espP is specific for STEC and present in 57% of STEC belonging to 16 different serotypes. The espP genes of the 16 STEC serotypes varied to a certain extent, as shown by nucleotide sequence and restriction enzyme analyses, but the DNA regions adjacent to the espP gene were completely different. When two further STEC-plasmid markers, the catalase-peroxidase gene katP and the enterohaemorrhagic E. coli-haemolysin gene EHEC-hlyA were included, many combinations of the three markers were found, depending in part on the serotype. In addition, strains possessing none of the three markers still harboured large plasmids. In the most prevalent STEC serogroup, O157, it was observed that the plasmid of sorbitol-fermenting STEC O157:H- lacks the espP and katP genes although both genes are present in the plasmid of the non-sorbitol-fermenting STEC O157:H7. The EHEC-hlyA gene, however, is present in both. In conclusion, this study shows that the large plasmids of STEC are not uniform genetic elements but heterogeneous in both their gene composition and arrangement.
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Fimbriae- and flagella-mediated association with and invasion of cultured epithelial cells by Salmonella enteritidis
More LessSalmonella enteritidis expresses flagella and several finely regulated fimbriae, including SEF14, SEF17 and SEF21 (type 1). A panel of mutants was prepared in three strains of S. enteritidis to elucidate the role of these surface appendages in the association with and invasion of cultured epithelial cells. In all assays, the naturally occurring regulatory-defective strain 27655R associated with tissue culture cells significantly more than wild-type progenitor strains LA5 and S1400/94. Compared with wild-type strains, SEF14 mutants had no effect on association and invasion, whereas SEF17, SEF21 and aflagellate mutants showed significant reductions in both processes. Histological examination suggested a role for SEF17 in localized, aggregative adherence, which could be specifically blocked by anti-SEF17 sera and purified SEF17 fimbriae. SEF21-mediated association was neutralized by mannose and a specific monoclonal antibody, although to observe enhanced association it was necessary for the bacteria to be in fimbriate phase prior to infection. Additionally, aflagellate mutants associated and invaded less than motile bacteria. This study demonstrated the potential for multifactorial association and invasion of epithelial cells which involved SEF17 and SEF21 fimbriae, and flagella-mediated motility.
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- Physiology And Growth
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Varying division planes of secondary constrictions in spheroidal Escherichia coli cells
More LessPlanes of successive divisions in Escherichia coli have been proposed to be either parallel or perpendicular to each other, restricted to one or two dimensions. To test the hypothesis that divisions can occur in planes alternating in three dimensions, a method was developed to generate cells with secondary constrictions during growth in suspension. The method involves a combination of thymine limitation (to manipulate chromosome replication rate) and mecillinam treatment (to inhibit penicillin-binding protein 2). The former modifies timing of terminations, the latter results in spheroidal cells. Such cells displayed secondary constrictions after adding deoxyguanosine (accelerating replication rate), thus temporarily enhancing division signals. The successive constrictions were seen to develop in planes that were tilted relative to each other, and in positions related to those of the nucleoids, visualized by staining with DAPI (4',6-diamidino-2-phenylindole dihydrochloride hydrate). Visualizing cell envelopes with FM 4–64 by confocal scanning laser microscopy supported the conclusion that planes of successive divisions can alternate in three dimensions.
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Anaerobic oxidations of cysteate: degradation via L-cysteate: 2-oxoglutarate aminotransferase in Paracoccus pantotrophus
More LessAnoxic, fresh-water enrichment cultures to oxidize different organosulfonates were set up with nitrate, ferric iron or sulfate as electron acceptors. Pure cultures were easily obtained with two naturally occurring sulfonates, cysteate (2-amino-3-sulfopropionate) and taurine (2-aminoethanesulfonate), under nitrate-reducing conditions. These two sulfonates were also oxidized during reduction of iron(III), though isolation of pure cultures was not successful. One nitrate-reducing cysteate-oxidizing bacterium, strain NKNCYSA, was studied in detail. It was identified as Paracoccus pantotrophus. Eighteen sulfonates were tested, and the organism degraded cysteate, taurine, isethionate (2-hydroxyethanesulfonate), sulfoacetate or 3-amino-propanesulfonate with concomitant reduction of nitrate, presumably to molecular nitrogen. The carbon skeleton of these substrates was converted to cell material and, presumably, CO2. The amino group was released as ammonia and the sulfono moiety was recovered as sulfate. Cell-free extracts of P. pantotrophus NKNCYSA contained constitutive L-cysteate:2-oxoglutarate aminotransferase (EC 2.6.1.-) and glutamate dehydrogenase (EC 1.4.1.4). Taurine:pyruvate aminotransferase, in contrast, was inducible.
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Possible involvement of cAMP in aerial mycelium formation and secondary metabolism in Streptomyces griseus
More LessIn Streptomyces griseus, A-factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone) triggers secondary metabolism and morphogenesis by binding a repressor protein (ArpA) and dissociating it from DNA. UV-mutagenesis of the A-factor-deficient mutant HH1 generated strain HO2, defective in the synthesis of ArpA and therefore able to form aerial mycelium, spores and streptomycin. Shotgun cloning of chromosomal DNA from wild-type S. griseus in strain HO2 yielded a gene that suppressed aerial mycelium formation and streptomycin production. Nucleotide sequencing and subcloning revealed that the gene encoded a eukaryotic-type adenylate cyclase (CyaA). In mutant HO2 production of cAMP was growth-dependent until the middle of the exponential growth stage; the production profile was the same as in the wild-type strain. However, the amount of cAMP produced was five times larger when mutant HO2 harboured cyaA on the high-copy-number plasmid plJ486. Consistent with this, supplying cAMP exogenously at a high concentration to mutant HO2 suppressed formation of both aerial mycelium and streptomycin. On the other hand, some lower concentrations of cAMP stimulated or accelerated aerial mycelium formation. No effects of exogenous cAMP on morphogenesis and secondary metabolism were apparent in the wild-type strain. In addition, disruption of the chromosomal cyaA gene in the wild-type strain had almost no effect. Introducing cyaA cloned in either a low- or a high-copy-number plasmid suppressed morphogenesis and secondary metabolism not only in mutant HO2 but also in other arpA mutants, implying that the effects of cAMP became apparent in the arpA-defective background. When mutant HO2 carried cyaA on a plasmid, synthesis of the stringent response factor ppGpp was greatly reduced; this may account for the observed suppression by cAMP of morphogenesis and secondary metabolism. cAMP also affected protein tyrosine phosphorylation, as determined with anti-phosphotyrosine antibody.
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Butane metabolism by butane-grown ‘Pseudomonas butanovora’
More LessThe pathway of butane metabolism by butane-grown ‘Pseudomonas butanovora' was determined to be butane → 1-butanol → butyraldehyde → butyrate. Butane was initially oxidized at the terminal carbon to produce 1-butanol. Up to 90% of the butane consumed was accounted for as 1-butanol when cells were incubated in the presence of 5 mM 1-propanol (to block subsequent metabolism of 1-butanol). No production of the subterminal oxidation product, 2-butanol, was detected, even in the presence of 5 mM 2-pentanol (an effective inhibitor of 2-butanol consumption). Ethane, propane and pentane, but not methane, were also oxidized. Butane-grown cells consumed 1-butanol and other terminal alcohols. Secondary alcohols, including 2-butanol, were oxidized to the corresponding ketones. Butyraldehyde was further oxidized to butyrate as demonstrated by blocking butyrate metabolism with 1 mM sodium valerate. Butyrate also accumulated from butane when cells were incubated with 1 mM sodium valerate. The pathway intermediates (butane, 1-butanol, butyraldehyde and butyrate) and 2-butanol stimulated O2 consumption by butane-grown cells. 1-Butanol, butyraldehyde and butyrate supported growth of ‘P. butanovora’, as did 2-butanol and lactate.
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- Plant-Microbe Interactions
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A unique eukaryotic β-xylosidase gene from the phytopathogenic fungus Cochliobolus carbonum
More LessThe plant-pathogenic fungus Cochliobolus carbonum secretes one major β-xylosidase (Xyp1) when grown on xylan or maize cell walls. cDNA and genomic DNA encoding Xyp1 were isolated using PCR primers based on peptide sequences from the purified protein. XYP1 contains three introns, has 5' and 3' untranslated regions of 74 and 145 bp, respectively, and is predicted to encode a protein of 328 amino acids (M r 36 700) with four N-glycosylation sites. Although it is secreted, Xyp1 has no predicted signal peptide. Furthermore, Xyp1 appears not to be processed at the N-terminus because one of the peptides isolated from the mature protein is located only six amino acids downstream of the translational start methionine. The primary sequence of Xyp1 is unrelated to any known eukaryotic β-xylosidase but has 35% overall identity to two bacterial bifunctional β-xylosidase/α-arabinosidases. Mutation of XYP1 by targeted gene replacement resulted in the loss of the major β-xylosidase activity corresponding to the product of XYP1, but a significant amount of secreted β-xylosidase activity (25% of wild-type) remained in the culture filtrates. The xyp1 mutant was still fully pathogenic on maize.
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- Systematics And Evolution
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Towards understanding the evolution of the human commensal yeast Candida albicans
More LessAllelic frequencies and relationships for one dimorphic locus and three unlinked polymorphic loci have been determined for 114 unrelated isolates of Candida albicans, including 14 laboratory reference strains and 50 strains from each of two geographic regions. Although there was no indication of geographical partitioning, there were significant correlations for specific allelic pairs among loci and little evidence that any alleles were in Hardy-Weinberg equilibrium. This gives additional support for the concept that the primary mode of genetic inheritance in this species is clonal, with other intracellular genetic events playing a lesser role in the creation of genomic diversity. Through inference of this and other known attributes of closely related Candida species, such as sequence analysis of IS1 and the ITS2 (internal transcribed spacer 2) region of the rDNA cistron, the deduced phylogeny suggests an evolutionarily recent origin for many frequently isolated strains. This finding will be of interest in the context of understanding pathogenicity and drug resistance in this human commensal yeast.
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