- Volume 138, Issue 6, 1992
Volume 138, Issue 6, 1992
- Genetics And Molecular Microbiology
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Physical and genetic mapping of the catA region of Pseudomonas aeruginosa
More LessA prime plasmid has been used as the basis for the construction of a physical and genetic map of a 125 kb segment of the Pseudomonas aeruginosa PAO chromosome. Using pMO1811, a prime plasmid selected for the catA region, a series of Tn5 insertions were obtained which identified two new markers gcu (glycine utilization) and oap (organic acids and alcohols permeability) in the 125 kb region and located them in relation to other known markers of this region. A cosmid bank was constructed from the prime plasmid and an ordered array of cosmid clones for this region identified by restriction endonuclease mapping with EcoRI, HindIII and KpnI, as well as complementation mapping and chromosome walking. By Southern hybridization analyses, it was confirmed that the chromosomal insert carried by pMO1811 was flanked by single, tandemly arranged copies of IS 21 and the orientation of the insert on this prime was determined. This cosmid bank provides a resource for the further analysis of this region of the P. aeruginosa genome.
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Nucleotide sequence of the rhaR−sodA interval specifying rhaT in Escherichia coli
More LessThe precise location of the rhaT gene, encoding rhamnose permease, has been established between sodA and rhaC at 3605–3607 kb of Kohara's physical map, which corresponds to 88·4 min on the Escherichia coli chromosomal map. The dependence of the activity of the rhaT product on the function of rhaC, the rhamnose operon regulatory gene, was established by measuring rhamnose transport in wild-type and rhaC-deficient strains. The sequence of the sodA−rhaC interval displayed a single ORF corresponding to rhaT, which is transcribed counterclockwise on the E. coli chromosome. The ORF was shown to be preceded by a ribosome binding consensus sequence and a catabolite repression protein consensus sequence. The derived amino acid sequence displayed very low homology with any other permease and was clearly dissimilar to the homologous group formed by the xylose, arabinose, galactose and several glucose transporters. Analysis of the rhaT primary sequence identified potential membrane-spanning regions, possibly defining a protein structure model different from the one corresponding to the above-mentioned homologous group.
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Mutations that affect the regulation of phs in Salmonella typhimurium
More LessThe regulation of phs [production of hydrogen sulphide (H2S)] in Salmonella typhimurium is complex. Previous studies have shown that expression is dependent upon the presence of reduced sulphur and anaerobiosis and is modulated by carbon source and growth stage. Transposon mutagenesis failed to find any potential trans-acting factors effective in the regulation of phs in relation to oxygen. Spontaneous mutants capable of expressing phs−lac aerobically were isolated and characterized. These mutations are closely linked to phs and affect not only oxygen regulation but also the requirement for cyclic AMP and reduced sulphur. Analysis of merodiploid strains indicates that these mutations are cis-acting and that phs is not subject to autoregulation.
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Molecular cloning and sequencing of a non-haem bromoperoxidase gene from Streptomyces aureofaciens ATCC 10762
More LessA bromoperoxidase gene (bpoT), recently cloned from Streptomyces aureofaciens Tü24, was used as a probe in Southern blot hybridization of total DNA from S. aureofaciens ATCC 10762. A single SstI fragment of 5·4 kb was detected, which was cloned via an enriched gene library into Escherichia coli. The functional bromoperoxidase gene was located on a 2.1 kb BamHI-HindIII fragment by subcloning into S. lividans TK64, using the multicopy plasmid pIJ486. The enzyme was overproduced in S. lividans TK64 (up to 30000 times compared to S. aureofaciens ATCC 10762) and showed the same electrophoretic and immunological properties as the bromoperoxidase BPO-A2 purified from S. aureofaciens ATCC 10762. DNA sequence analysis revealed an open reading frame encoding a predicted polypeptide with the same M r and N-terminal amino acid sequence as the purified subunit of BPO-A2.
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Characterization of a plasmid from moderately halophilic eubacteria
A plasmid has been isolated for the first time from moderately halophilic eubacteria. Halomonas elongata, Halomonas halmophila, Deleya halophila and Vibrio costicola were found to harbour an 11·5 kbp plasmid (pMH1). The plasmid was isolated and characterized after transformation into Escherichia coli JM101 cells. A restriction map was constructed, and unique restriction sites for EcoRI, EcoRV and ClaI were detected. The occurrence of such a plasmid in the original halophilic strains was confirmed by Southern hybridization. The plasmid carries genetic determinants that mediate resistance to kanamycin, tetracycline, and neomycin. This property, together with its relatively small size, its stability in E. coli cells, and the presence of unique restriction sites, makes pMH1 a good candidate for the development of a cloning vector for moderate halophiles.
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Characterization of a temperate bacteriophage of Lactobacillus delbrueckii subsp. bulgaricus and its interactions with the host cell chromosome
More LessThe temperate bacteriophage mv4 is representative of a widespread phage genetic group of Lactobacillus delbrueckii subsp. lactis or bulgaricus. The genome of this phage is circularly permuted and terminally redundant, as shown by mature mv4 homoduplexes, Southern hybridization and restriction enzyme analysis. A circular map of the mv4 genome was established, with unique sequences totalling 36 kb. The genomic location of the pac site (packaging site of mv4 DNA into phage heads) and the att site (phage integration site into the host cell chromosome) was determined. The genes coding for the two main structural phage proteins and for a phage-associated lysin were also mapped. Phage mv4 is capable of transducing a limited set of pieces of bacterial DNA and several specific chromosomal attachment sites of phage mv4 were identified. Bacteria lysogenic for phage mv4 were shown to be immune to infection by L. delbrueckii virulent phages related to mv4.
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Gram-positive bacteria with a high DNA G+C content are characterized by a common insertion within their 23S rRNA genes
More LessAn insertion of about 100 bases within the central part of the 23S rRNA genes was found to be a phylogenetic marker for the bacterial line of descent of Gram-positive bacteria with a high DNA G+C content. The insertion was present in 23S rRNA genes of 64 strains representing the major phylogenetic groups of Gram-positive bacteria with a high DNA G+C content, whereas it was not found in 23S rRNA genes of 55 (eu)bacteria representing Gram-positive bacteria with a low DNA G+C content and all other known (eu)bacterial phyla. The presence of the insertion could be easily demonstrated by comparative gel electrophoretic analysis of in vitro-amplified 23S rDNA fragments, which contained the insertion. The nucleotide sequences of the amplified fragments were determined and sequence similarities of at least 44% were found. The overall similarity values are lower than those of 16S and 23S rRNA sequences of the particular organism. Northern hybridization experiments indicated the presence of the insertion within the mature 23S rRNA of Corynebacterium glutamicum.
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Clonal variation of chromosome size derived from the rDNA cluster region in Candida albicans
More LessOf the eight Candida albicans chromosomes, chromosome 2, assigned by the MGL1 probe, is more variable in size than the other chromosomes among strains. We found that the clonal variation of chromosome 2, which carries a rDNA gene, occurred at a frequency of up to 10% of the progeny clones. After total chromosomal digestion with XhoI, which has no recognition sites within the rDNA repeat unit, the fragments containing the rDNA cluster were detected by Southern hybridization. The difference in fragment sizes corresponded to the clonal size variation of chromosome 2. The intensity of hybridization with rDNA also correlated with the difference in size. In addition, there was no size change in the non-rDNA region as detected by NotI digestion of chromosome 2, and there was no observed change in the individual rDNA basic repeat unit size. From these lines of evidence, we confirmed that the clonal size variation of chromosome 2 which occurs at high frequency is derived from the size change of the rDNA cluster.
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Distinct plasmid profiles of Pasteurella haemolytica serotypes and the characterization and amplification in Escherichia coli of ampicillin-resistance plasmids encoding ROB-1 β-lactamase
More LessThirty-five isolates of Pasteurella haemolytica from cattle or sheep were screened for the presence of plasmids and for resistance to a range of antibiotics. Eight strains (four of serotype A1, three of serotype A2 and one untypable) contained plasmid DNA and isolates of the same serotype had similar plasmid profiles, which were different from those of the other serotypes. All but one of the plasmid-bearing strains were isolated from pneumonic animals or from animals in contact with pneumonic cattle or sheep. In A2 and untypable strains, there was no obvious correlation between antibiotic resistance and the presence of a specific plasmid. In contrast, all plasmid-bearing A1 strains exhibited ampicillin resistance (ApR), which was shown by transfer studies to be plasmid-mediated. Plasmid DNA prepared from E. coli transformants was not routinely detected on ethidium-bromide-stained agarose gels, but could be amplified to detectable levels by treatment of cultures with chloramphenicol (Cm) or by modifying the growth conditions. The ApR plasmids from P. haemolytica were identical by restriction enzyme analysis. Restriction analysis and hybridization data indicated that these plasmids were closely related to the prototype ROB-1 β-lactamase-encoding plasmid, originally isolated from Haemophilus influenzae. From substrate profiles and isoelectric focusing data, the β-lactamases encoded by the P. haemolytica plasmids were indistinguishable from the ROB-1 β-lactamase.
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- Physiology And Growth
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Effect of Ca2+ and K+ on the intracellular pH of an Escherichia coli L-form
More LessThe L-form NC7, derived from Escherichia coli K12, grew in a complex medium containing 0·2 M-CaCl2 as osmotic stabilizer, but not at pH values above 7·8. The cessation of growth at alkaline pH was not due to cell death. In complex media containing K+ or Na+, the L-form grew over a wide pH range. Growth at alkaline pH was inhibited by 1 mM-amiloride, indicating that Na+/H+ antiport activity was required for growth at alkaline pH. The internal pH (pHi) of the L-form in media containing K+, Na+ or Ca2+ was constant at about 7·8 to 8·0 at external pH (pHo) values of 7·2 and 8·2. The rates of O2 consumption by intact cells, lactate oxidation by membrane vesicles from cells grown in Ca2+-containing medium, and cell division were all strongly repressed under alkaline conditions.
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Roles of K+ and Na+ in pH homeostasis and growth of the marine bacterium Vibrio alginolyticus
More LessThe marine bacterium Vibrio alginolyticus, containing 470 mM-K+ and 70 mM-Na+ inside its cells, was able to regulate the cytoplasmic pH (pHin) in the narrow range 7.6–7.8 over the external pH (pHout) range 6.0–9.0 in the presence of 400 mM-Na+ and 10 mM-K+. In the absence of external K+, however, pHin was regulated only at alkaline pHout values above 7.6. When the cells were incubated in the presence of unusually high K+ (400 mM) and 4 mM Na+, the pHin was regulated only at acidic pHout values below 7.6. These results could be explained by postulating a K+/H+ antiporter as the regulator of pHin over the pHout range 6.0–9.0. When Na+-loaded/K+-depleted cells were incubated in 400 mM-Na+ in the absence of K+, an inside acidic ΔpH was generated at pHout values above 7.0. After addition of diethanolamine the inside acidic ΔpH collapsed transiently and then returned to the original value concomitant with the extrusion of Na+, suggesting the participation of a Na+/H+ antiporter for the generation of an inside acidic ΔpH. In the presence of 400 mM-K+, at least 5 mM-Na+ was required to support cell growth at pHout below 7.5. An increase in Na+ concentration allowed the cells to grow at a more alkaline pHout. Furthermore, cells containing more Na+ inside could more easily adapt to grow at alkaline pHout. These results indicated the importance of Na+ in acidification of the cell interior via a Na+/H+ antiporter in order to support cell growth at alkaline pHout under conditions where the activity of a K+/H+ antiporter is marginal.
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