- Volume 143, Issue 4, 1997
Volume 143, Issue 4, 1997
- Systematics
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Use of multilocus enzyme electrophoresis to examine genetic relationships amongst isolates of Mycobacterium intracellulare and related species
More LessAs part of a larger study investigating diversity and distribution of Mycobacterium spp. in Australia, multilocus enzyme electrophoresis was used to assess genetic relationships at 17 enzyme loci amongst a collection of reference strains and isolates initially identified on biochemical and other grounds as M. intracellulare (70), ‘X’ mycobacteria (10), M. scrofulaceum (7), M. avium (8) and M. avium subsp. paratuberculosis (2). Two of the isolates initially identified as M. intracellulare were shown to be quite distinct from the others. Both gave negative results in a species-specific DNA probe test, whilst one was positive by PCR. These results emphasize the uncertainties involved in identifying members of this group. The other M. intracellulare isolates formed a cohesive but diverse group, being divided into 48 electrophoretic types (ETs), with a mean genetic diversity of 0∙38. Forty-three of these ETs contained only single isolates. There was no clear relationship between the serovar and ET designation. The index of association calculated for M. intracellulare was significantly different from zero, suggesting that it is a clonal species. PFGE was also applied to selected isolates from the ETs containing multiple isolates, and some of these could be differentiated further. The strains of M. scrofulaceum and ‘X’ mycobacteria were distinct from M. intracellulare, but themselves were highly heterogeneous, with mean genetic diversities of 0∙66 and 0∙65, respectively. Each of these groups may represent more than one species. M. avium strains were distinct from the two M. avium subsp. paratuberculosis strains, as well as from the other mycobacteria studied.
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Genomic relationships between selected phage types of Salmonella enterica subsp. enterica serotype typhimurium defined by ribotyping, IS200 typing and PFGE
More LessThe genomic relationship between isolates representing 17 definitive phage types (DTs) of Salmonella enterica subsp. enterica serotype typhimurium (S. typhimurium) were analysed using three different typing methods: IS200 typing using the restriction enzymes EcoRI and Pvull, ribotyping using Smal and EcoRI, and PFGE using Xbal. These methods were used to study four DTs in greater detail; in all 18 (DT 49), 10 (DT 110), five (DT 120) and seven (DT 135) isolates were studied. The combined data generated two large clusters, which could be divided into five groups. Within the first cluster, a close similarity was indicated between isolates of the following phage types: group A – DTs 44, 49, 135 and 204c, with DT 9 distantly related; group B – DTs 95 and 99; and group C – DTs 104a, 110 and 120. The other large cluster contained group D – DTs 10, 20 and 146, with DT 12 distantly related, and group E – DTs 69, 103 and 153. The same grouping was observed by principal component analysis, but a minimum spanning tree linked DT 12 to group E and not group D in this analysis. Among the typing methods used, IS200 gave the best representation of the overall similarity between the S. typhimurium isolates. Five different IS200 profiles were obtained among isolates belonging to DT 49. Only one profile was observed within each of the phage types DT 110, 120 and 135. All isolates within each of these four phage types were of one ribotype. Isolates of DT 49 showed four PFGE patterns, while one pattern was present within isolates of the three other phage types. Members of these four phage types were found to be clonally related as they formed tight subclusters separated from isolates of other phage types.
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- Genome Analysis
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A 23.4 kb segment at the 69°-70° region of the Bacillus subtilis genome
More LessWithin the framework of the international project aimed at the sequencing of the Bacillus subtilis genome, a 23 911 bp chromosome segment has been cloned and sequenced. This region (23433 bp; 69°-70° of the genetic map) contains 17 complete ORFs and a partial one. A homology search for the products deduced from the 18 ORFs revealed that twelve of them had significant similarity to known proteins, including the quinolone-resistance protein, ABC transporter, aldehyde dehydrogenase, amino acid transporter, fosmidomycin-resistance protein, CDP-glucose 4,6-dehydratase, glucose-1-phosphate cytidyltransferase and cytochrome P450/NADPH-cytochrome P450 reductase.
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A 23 911 bp region of the Bacillus subtilis genome comprising genes located upstream and downstream of the Iev operon
Within the framework of the European project to sequence the whole Bacillus subtilis 168 genome, a 23 911 bp long chromosomal DNA fragment located around 233° on the B. subtilis genetic map was cloned and sequenced. From the generated sequencing data and the results of the homology search, the primary structure of this region was determined. In addition to the whole Iev operon, the region contains putative genes for an amino acid permease, two different alcohol dehydrogenases, a chitosanase, a protein belonging to the LysR family of transcriptional regulators, a protein related to the MerR transcriptional regulator, up to four proteins related to the product of the spoF gene, and genes coding for nine more inferred proteins of unknown function.
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The signal peptidase II (lsp) gene of Bacillus subtilis
More LessThe gene encoding the type II signal peptidase (SPase II) of Bacillus subtilis was isolated by screening a genomic DNA library of this bacterium for the ability to increase the levels of globomycin resistance in Escherichia coli, and to complement the growth deficiency at the non-permissive temperature of E. coli strain Y815 carrying a temperature-sensitive mutation in its lsp gene for SPase II. The deduced amino acid sequence of the B. subtilis SPase II showed significant similarity with those of other known SPase II enzymes. Activity of the B. subtilis SPase II was demonstrated by a pulse-labelling experiment in E. coli. In B. subtilis, the lsp gene is flanked by the isoleucyl-tRNA synthetase (ileS) gene and the pyrimicline biosynthetic (pyr) gene cluster, which is known to map at 139° of the chromosome. In the Gram-positive bacteria studied thus far, lsp appears to be the first gene in an operon. The promoter-distal gene (orf4) of this operon specifies a hypothetical protein in bacteria and yeast.
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Analysis of the genetic polymorphism between three Streptococcus thermophilus strains by comparing their physical and genetic organization
More LessThe physical maps of Streptococcus thermophilus CNRZ368 and NST2280 strain: were constructed by analysing PFGE patterns obtained with the low-frequency cutting enzymes SmaI, BssHU and SfiI. Their chromosomes are 1864 and 1840 kb circular molecules, respectively. Comparison of their physical maps with that of the reference A054 strain revealed a relatively conserved organization of the restriction sites. Three variable regions were detected with the map of CNRZ368 whereas 15 were found with the map of NST2280. To construct the genetic maps, probes corresponding to 10 single-copy genes, the rrn genes and the insertion sequences IS1191, IS981 and ISS1 were hybridized to Southern blots of chromosomal DNA digested with the different mapping enzymes. Comparison of the genetic maps of the three strains showed a conserved location of the mapped single-copy genes. However, six rrn loci were present in the chromosome of A054 and CNRZ368 whereas five were present in the NST2280 chromosome. A polymorphism was also found in the copy number of the insertion sequences between the three strains.
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Directed introduction of DNA cleavage sites to produce a high-resolution genetic and physical map of the Acinetobacter sp. strain ADP1 (BD413UE) chromosome
More LessThe natural transformability of the soil bacterium Acinetobacter sp. ADP1 (BD413UE), formerly classified as A. calcoaceticus, has facilitated previous physiological and biochemical investigations. In the present studies, the natural transformation system was exploited to generate a physical and genetic map of this strain's 3780·191 kbp circular chromosome. Previously isolated Acinetobacter genes were modified in vitro to incorporate a recognition sequence for the restriction endonuclease Not l. Following transformation of the wild-type strain by the modified DNA, homologous recombination placed each engineered Not l cleavage site at the chromosomal location of the corresponding gene. This allowed precise gene localization and orientation of more than 40 genes relative to a physical map which was constructed with transverse alternating field electrophoresis (TAFE) and Southern hybridization methods. The positions of Not l, Asc l and l-Ceu l recognition sites were determined, and the latter enzyme identified the presence of seven ribosomal RNA operons. Multiple chromosomal copies of insertion sequence IS 1236 were indicated by hybridization. Several of these copies were concentrated in one region of the chromosome in which a spontaneous deletion of approximately 100 kbp occurred. Moreover, contrary to previous reports, ColE1-based plasmids appeared to replicate autonomously in Acinetobacter sp. ADP1.
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