Journal of general microbiology最新文献

The aetiological agents of Lyme disease form a phylogenetically heterogeneous group, composed of three species, Borrelia burgdorferi, Borrelia garinii, and group VS461. We have compared the sizes of the linear plasmid that carries the genes encoding the major outer-surface proteins OspA and OspB as well as the size and structure of the chromosome among the Lyme disease spirochaetes. We have found differences in the sizes of the ospA-containing plasmids, but not the linear chromosomes among the three species. The ospA-containing plasmid size of 50 kb in B. burgdorferi isolates is significantly smaller than the size of 55 kb in B. garinii isolates and 56 kb in group VS461 isolates. The chromosome was found to be linear in all three Borrelia species, but not significantly different in size.

The two protein exotoxins secreted by Bacillus anthracis are composed of three distinct components: protective antigen (PA), lethal factor (LF), and (o)edema factor (EF). We have developed a genetic strategy that permits us selectively to inactivate each of the genes coding for PA, EF or LF. This strategy involved the deletion of a portion of the structural gene and the insertion of an antibiotic resistance cassette. With this technique, double mutant strains of B. anthracis producing only one toxin component have been constructed. Characterization of the mutant strains indicated that they produced the expected single toxin protein. Using a simple, two-step protocol, we have purified PA, LF and EF to homogeneity from culture supernatants. These three mutant strains are potentially powerful tools for studying the individual effect of each toxin component in vitro and in vivo.

A synthetic gene coding for interleukin-2 (IL-2) was used to produce large amounts of recombinant IL-2 (met-IL-2) in Escherichia coli. Met-IL-2 was found to accumulate in the cytoplasm in an insoluble, aggregated form. Inclusion bodies located at the pole caps of cells were detected using immunogold labelling. Constructs were designed to fuse the IL-2 gene to DNA fragments encoding signal peptides for an outer-membrane protein (OmpA) or for a periplasmic protein (PhoA) of E. coli. No significant maturation was observed with these fusion proteins which were found in an insoluble form in the cytoplasm. The influence of charge disposition at the N-terminus of the mature portion of the protein was investigated by replacing positively charged amino acids with glutamic acid. None of the introduced substitutions had any effect. Various factors that might affect expression, secretion and folding were examined in an attempt to obtain secretion. By fusing IL-2 to the precursor maltose-binding protein (preMBP) a large fraction of the preMBP-IL-2 protein was correctly processed and transported to the periplasmic space. IL-2 derived from MBP-IL-2 after FXa cleavage possessed similar specific activity to recombinant IL-2 produced in Chinese Hamster ovary cells.

Salmonella paratyphi B and Salmonella java are biovars of common serotype 1,4,[5],12:b:1,2 which respectively cause human paratyphoid fever and gastroenteritis. In order to define genotypes and phylogenetic relationships in this group, we examined representative strains for restriction fragment length polymorphisms (RFLPs) in and around the 16S ribosomal RNA (rrn) genes, and the five to eleven insertion sites of the Salmonella-specific DNA insertion sequence IS200. One of four 16S rrn profiles was predominant, and was shared by the majority of strains, irrespective of their designation as S. paratyphi B or S. java. On the other hand, thirteen unique IS200 profiles were found and this technique was able to distinguish, for the first time, distinct genotypes for S. paratyphi B and S. java. One of the S. paratyphi B profiles, Spj-IP1.0, represented a globally-distributed clone. Greater diversity was detected within IS200 profiles of S. java than within those of S. paratyphi B. IS200 profiles described a phylogenetic complex in which strains of both biovars could be placed. They constituted reproducible molecular fingerprints, which could be compared in a band-matching database suitable for molecular epidemiological typing.

The possible functions of two recently described flagellar genes, fliU and fliV, have been examined. Introduction of gene fliC, encoding the bacterial flagellin protein, into a number of flagellin-deficient Salmonella and Escherichia coli strains failed to complement the mutations in these strains, and the FliC flagellin was accumulated in the bacterial cytoplasm. Complementation with fliU and fliV, which map downstream of fliC, restored motility to some of the mutants which became flagellated. After inactivation of either fliU or fliV, such complementation no longer occurred and the flagellin protein accumulated in the cytoplasm, which suggested that both genes are required for the secretion of flagellin and expression of motility. Expression of these genes from high copy number plasmids resulted in the synthesis of exceptionally long flagella and in detection of the FliV protein on polyacrylamide gels.

Nucleic acid sequence-based amplification (NASBA), an isothermal amplification technique for nucleic acids (NA), was investigated for the species-specific identification of mycobacteria. A set of primers was selected from a highly conserved region of the 16S rRNA sequence of mycobacteria sandwiching a variable sequence to perform amplification of mycobacterial RNA. Species-specific probes for the M. tuberculosis complex, M. avium-paratuberculosis, M. intracellulare and M. leprae were hybridized in-solution with the amplified nucleic acids of 10 pathogenic mycobacteria and 11 closely related bacteria, as well as with human-derived NA in an enzyme-linked gel assay (ELGA). Each probe was shown to hybridize specifically to the amplified single-stranded RNA of the corresponding species. Thirty-two clinical isolates of M. tuberculosis strains from different parts of the world were correctly identified by NASBA using the M. tuberculosis-complex-specific probe. In combination with the ELGA, NASBA could identify mycobacteria rapidly, i.e. in less than 6 h. The relative simplicity and rapidity of this technique makes it an attractive tool for species-specific identification of mycobacteria.

Most strains of Helicobacter pylori are naturally competent for uptake of chromosomal DNA. Transformation frequencies for streptomycin resistance or rifampicin resistance markers ranged from 1 x 10(-4) to 1 x 10(-3) per viable cell using a plate transformation procedure. Transformation of a metronidazole resistance marker (MtrR) was demonstrated when either a laboratory-derived mutant or a MtrR clinical isolate were used as the source of donor DNA. MtrR was transformed at a frequency of 3 x 10(-5) per viable cell. All H. pylori strains tested produce large amounts of DNAase, which may reduce DNA available for transformation. Four H. pylori plasmids were isolated. DNA fragments from H. pylori plasmids were deleted or rearranged when cloned in pUC19 and propagated in Escherichia coli DH5 alpha. An H. pylori plasmid, pUOA26 which contained a chloramphenicol resistance determinant from Campylobacter coli, was constructed in H. pylori. This plasmid could be successfully introduced by natural transformation only into H. pylori recipients which contained a homologous resident plasmid. Transformation of pUOA26 into plasmid-free cells of H. pylori was achieved by electroporation. Transformation frequencies were 1 x 10(-4) transformants per viable cell when plasmid DNA was isolated from the same strain; however, introduction of pUOA26 DNA derived from H. pylori 8091 into a different H. pylori strain, NCTC 11639, resulted in transformation at much lower frequencies (< or = 1 x 10(-7) per viable cell).(ABSTRACT TRUNCATED AT 250 WORDS)

Anaplasma marginale is a rickettsial parasite of bovine erythrocytes causing world-wide economic losses in livestock production. Despite its importance, little is known about this rickettsia at a molecular level because it has not been cultured in vitro, and there is no small-animal model. Although several genes have been cloned and sequenced, the gross genome structure of the organism has not yet been well characterized. We separated intact bovine erythrocytes from leucocytes, and determined the genome size of A. marginale by use of restriction endonuclease cleavage and pulsed-field gel electrophoresis (PFGE). A value of 56 mol% G+C was obtained for this genome by spectral analysis. Undigested A. marginale DNA failed to migrate under several different electrophoretic conditions, indicating a circular genome. Digestions of intact A. marginale DNA were performed using restriction endonucleases NotI, SfiI and PacI. Complete digestion with SfiI resulted in 12 distinct bands ranging in size from 14 to 170 kbp. Total size determined by addition of SfiI-digested fragments was approximately 1200 kbp. PacI cleaved the A. marginale genome from three different isolates into just three fragments, of 598, 557 and 97 kbp. Incomplete digestion produced a band measuring 1250 kbp. These results indicate that A. marginale has a circular genome between 1200 and 1260 kbp, with a G+C content of 56 mol%.

Rabbit erythrocytes were agglutinated by 43.4% of group B streptococci isolated from bovines but by none isolated from humans. Haemagglutination was enhanced by cultivation of the bacteria under microaerophilic conditions. Most of the haemagglutinating strains had protein type antigen X, either alone, or in combination with polysaccharide antigens. Heat and proteolytic treatment of the bacteria destroyed the haemagglutination activity. The haemagglutinin could be solubilized from the bacterial surface by mutanolysin treatment and isolated from culture supernatant fluid by ammonium sulphate precipitation. The isolated haemagglutinin did not cause direct agglutination of erythrocytes. However, binding of the haemagglutinin to rabbit erythrocytes could be visualized by agglutination of haemagglutinin-treated erythrocytes by specific antiserum obtained by absorption. Western blotting showed that the haemagglutinin obtained from erythrocyte lysates contained an antibody-reactive band with a molecular mass of 43 kDa. Haemagglutination-positive strains adhered to HeLa cells in higher numbers than did haemagglutination-negative strains. The HeLa cell adherence of Group B streptococci was inhibited in the presence of isolated haemagglutinin or of specific antiserum against the haemagglutinin. These observations suggest that the haemagglutinating adhesins of bovine group B streptococcal isolates are directly involved in the adherence mechanisms of these organisms.

beta-Glucanase synthesis in Bacillus subtilis was repressed by glucose and other substrates of glycolysis. Experiments with different pts mutants showed that the phosphoenolpyruvate: sugar phosphotransferase system is not involved in carbon catabolite repression of beta-glucanase synthesis. Carbon catabolite repression of beta-glucanase synthesis was completely abolished in a ccpA mutant. An operator structure similar to those upstream of amyE and the xyl operon was found and was shown by site-directed mutagenesis to be the target for carbon catabolite repression of beta-glucanase synthesis. The presence of this operator on a multi-copy plasmid resulted in a reduced repression of both beta-glucanase and alpha-amylase synthesis. It seems likely that the gene encoding these enzymes are part of one regulon with respect to catabolite repression.