- Volume 138, Issue 1, 1992
Volume 138, Issue 1, 1992
- Review Article
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- Biochemistry
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Identification of indigo-related pigments produced by Escherichia coli containing a cloned Rhodococcus gene
More LessSummary: Pigments produced by Escherichia coli containing a cloned piece of DNA from Rhodococcus sp. ATCC 21145 were extracted in chloroform and separated into blue and pink components. Evidence from TLC, NMR spectroscopy, absorption spectrum analysis and solubility behaviour suggested that the blue pigment was indigo and the pink pigment was indirubin, a structural isomer of indigo. The proposed pathway for pigment production on LB agar involves the conversion of tryptophan to indole by tryptophanase of E. coli and the oxidation of indole to indigo by the product of the cloned Rhodococcus DNA insert.
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- Biotechnology
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Quantitative separation of bacteria in saline solution using lanthanide Er(III) and a magnetic field
More LessSummary: A trivalent lanthanide ion, erbium (Er3+), has been used in combination with a magnetic separation technique to isolate seven bacterial species from suspensions in 0·9% saline. Erbium has an exceptionally high atomic magnetic moment of 9·3 Bohr magnetons, and following addition as ErCl3 (final concentration 5 mM) to bacterial suspensions, it imparts the magnetic moment to the bacterial cells by ionic binding to the cell surface. Strains of Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Psuedomonas aeruginosa, Staphylococcus epidermidis, Staphylococcus saprophyticus and Enterococcus faecalis were obtained from the Quality Control Depository of The Cleveland Clinic Foundation, Cleveland, Ohio, USA as suspensions in 0·9% NaCl, in concentrations ranging from 102 to 108 c.f.u. ml−1. Bacteria were separated from solution inside a capillary flow cell exposed to a highly non-homogeneous magnetic field (maximum field intensity was 0·4 T) and quantified by a light scattering method. The quantity of cellular deposition in the magnetic field was correlated with the initial concentration of cells in the suspension, expressed in c.f.u. ml−1, and sample volume (1·5 and 3·0 ml), sample pH (prior to ErCl3 addition), affinity to Gram stain (negative vs positive) and species. Magnetic deposition was observed for concentrations as low as 102 and 103 c.f.u. ml−1, and a significant correlation between average scattered light intensity and initial cell concentration (correlation coefficient ≥ 0·98) was established in the range 104 to 108 c.f.u. ml−1 for all but one species (P. aeruginosa). Magnetic deposition increased with increasing pH from 7·0 to 10·0 which is consistent with the prevailing view on the mechanism of the lanthanide ion binding to bacterial cells (ionic, to the cell surface). No significant difference in magnetic deposition was observed between individual strains, or between Gram-positive and -negative bacteria. Magnetic isolation of cells may find application in rapid total cell count determination, such as rapid urine screening.
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Biolistic transformation of prokaryotes: factors that affect biolistic transformation of very small cells
More LessSummary: Five bacterial species were transformed using particle gun-technology. No pretreatment of cells was necessary. Physical conditions (helium pressure, target cell distance and gap distance) and biological conditions (cell growth phase, osmoticum concentration, and cell density) were optimized for biolistic transformation of Escherichia coli and these conditions were then used to successfully transform Agrobacterium tumefaciens, Erwinia amylovora, Erwinia stewartii and Pseudomonas syringae pv. syringae. Transformation rates for E. coli were 104 per plate per 0·8 μg DNA. Although transformation rates for the other species were low (<102 per plate per 0·8 μg DNA), successful transformation without optimization for each species tested suggests wide utility of biolistic transformation of prokaryotes. E. coli has proven to be a useful model system to determine the effects of relative humidity, particle size and particle coating on efficiency of biolistic transformation.
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- Development And Structure
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Preferential expression of one β-tubulin gene during flagellate development in Physarum
Summary: The microbial eukaryote Physarum polycephalum displays several distinct cell types in its life cycle, including amoebae, flagellates and plasmodia. Despite its relative simplicity, Physarum has a tubulin gene family of complexity comparable to that of Drosophila. We have identified β-tubulin cDNAs from Physarum that are derived from the betA β-tubulin locus and encode β1A tubulin. We have also identified a partial cDNA for the unlinked betB β-tubulin gene, which encodes β1B tubulin. The polypeptide sequences encoded by betA and betB show 99% identity, but the nucleotide sequences show only 85% identity, consistent with an ancient duplication of these genes. The betB gene is expressed in amoebae, flagellates and plasmodia, whereas betA is expressed only in amoebae and flagellates. During the amoeba-flagellate transition the level of betA transcript increases over 100-fold, while the level of betB transcript changes very little. Thus Physarum has a mechanism for regulating the level of discrete β-tubulin transcripts differentially during flagellate development. A need for this differential regulation could account for the maintenance of the virtually isocoding betA and betB β-tubulin genes.
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- Ecology
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Rate of plasmid transfer among Escherichia coli strains isolated from natural populations
More LessSummary: The conjugative plasmid R1 was introduced into ten strains of Escherichia coli isolated from natural populations. Spontaneous nalidixic-acid-resistant mutants of the ten strains served as recipients. The ten donor and recipient strains were mated in all combinations and the rate at which R1 transferred between the strains was determined. The rate of transfer ranged from 5·2 × 10−11–1·1 × 10−18 ml per cell h−1, and averaged 1·3 − 10−15 ml per cell h−1. The results of these experiments suggest that the rates of conjugative transfer are far too low for plasmids to be maintained as parasites in their host populations. Infectious transfer is insufficient; plasmids must confer a selective advantage to their host to be maintained.
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- Genetics And Molecular Biology
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Identification of expression signals of the mycobacteriophages Bxb1, L1 and TM4 using the Escherichia–Mycobacterium shuttle plasmids pYUB75 and pYUB76 designed to create translational fusions to the lacZ gene
More LessSummary: Mycobacterial expression signals were cloned using specially constructed gene fusion shuttle plasmid probes carrying a truncated Escherichia coli lacZ (β-galactosidase) gene which lacked a promoter, a ribosome binding site, and an ATG start codon. Libraries of mycobacteriophage Bxb1, L1 and TM4 DNAs were constructed, and introduced by electroporation into Mycobacterium smegmutis and the ‘bacille Calmette-Guérin’ (BCG). Clones carrying mycobacterial expression sequences were detected by their blue colour or characteristic fluorescence when plated on media containing chromogenic or fluorogenic substrates. Varying degrees of β-galactosidase expression were observed, and one Bxb1 expression signal was identified where β-galactosidase expression is repressed in phage lysogens.
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Recombination and UV resistance of Escherichia coli with the cloned recA and recBCD genes of Serratia marcescens and Proteus mirabilis: evidence for an advantage of intraspecies combination of P. mirabilis RecA protein and RecBCD enzyme
More LessSummary: In Escherichia coli, constituents of the main recombination pathway are provided by the genes recA (RecA protein) and recBCD (RecBCD enzyme). Recombination in conjugation experiments and repair of UV damage of E. coli mutants deleted for recA, for recBCD or for recA plus recBCD were restored, although to different degrees, by the cloned recA and recBCD genes from Serratia marcescens or Proteus mirabilis. When both recombination enzymes were from the same species, repair and recombination efficiencies had the order E. coli > S. marcescens > P. mirabilis. However, the P. mirabilis recA plus recBCD genes resulted in higher levels of repair and recombination than those obtained with one component from P. mirabilis (recA or recBCD) and the other from E. coli or S. marcescens. The data provide evidence for the similarity of RecABCD pathways of recombination among enteric bacteria and suggest an in vivo advantage of an intraspecies combination of P. mirabilis RecA protein and RecBCD enzyme over interspecies combinations. This could point to a cooperation between these basic recombination enzymes. The molecular processes which could be involved are discussed.
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DNA sequence variability at the rplX locus of Bacillus subtilis
More LessSummary: The pattern and extent of DNA sequence variability at the rplX locus (encoding ribosomal protein L24) has been investigated in nine strains of Bacillus subtilis. Overall, there is a very low level of nucleotide diversity, even at silent sites, which is probably due to selection among synonymous codons. By analogy with Escherichia coli, there may also be some effect of the relative proximity of rplX to the chromosomal origin of replication. The small number of nucleotide substitutions are non-randomly distributed: all of the synonymous changes are in valine codons. From the sequence differences the strains can be divided into two groups, which are not coincident with their previous classification; this observation is consistent with recombination among strains.
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Cloning of dapD, aroD and asd of Leptospira interrogans serovar icterohaemorrhagiae, and nucleotide sequence of the asd gene
More LessSummary: Metabolites such as diaminopimelate and some aromatic derivatives, not synthesized in mammalian cells, are essential for growth of bacteria. As a first step towards the design of a new human live vaccine that uses attenuated strains of Leptospira interrogans, the asd, aroD and dapD genes, encoding aspartate β-semialdehyde dehydrogenase, 3-dehydroquinase and tetrahydrodipicolinate N-succinyltransferase, respectively, were cloned by complementation of Escherichia coli mutants. The complete nucleotide sequence of the asd gene was determined and found to contain an open reading frame capable of encoding a protein of 349 amino acids with a calculated M r of 38007. Comparison of this deduced L. interrogans aspartate β-semialdehyde dehydrogenase amino acid sequence with those of the same enzyme from Saccharomyces cerevisiae and Corynebacterium glutamicum revealed 46% and 36% identity, respectively. By contrast, the identity between the L. interrogans enzyme and the Streptococcus mutans or E. coli enzymes was less than 31%. Highly conserved sequences within aspartate semialdehyde dehydrogenase from the five organisms were observed at the amino and carboxyl termini, and around the cysteine of the active site.
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Identification of an insecticidal crystal protein from Bacillus thuringiensis DSIR517 with significant sequence differences from previously described toxins
More LessSummary: The nucleotide (nt) sequence of a DNA segment containing the majority of a gene cloned from Bacillus thuringiensis DSIR517 encoding a 130 kDa insecticidal crystal protein has been determined. Sequence analysis reveals an open reading frame (ORF) of 3453 nt. The ATG initiation codon, which is preceded by a potential ribosome-binding site sequence, was confirmed by N-terminal amino acid sequencing. The ORF extends beyond the 3” terminus of the cloned fragment; however, the high degree of homology between the deduced amino acid sequence of this ORF and other Cry proteins suggests the clone lacks only five C-terminal amino acids. Making this assumption, the ORF of 3468 nt encodes a protein of 1156 amino acids with an estimated molecular mass of 129700 Da. Analysis of the deduced amino acid sequence reveals a number of features characteristic of Cry proteins. Alignment of the Cry 517 protein sequence with other Cry proteins suggests it is most closely related to the crylA-E genes but sufficiently different to form a new cryI gene subclass.
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IS902, an insertion element of the chronic-enteritis-causing Mycobacterium avium subsp. silvaticum
Summary: An insertion sequence element of Mycobacterium avium subsp. silvaticum was isolated and its complete nucleotide squence determined. IS902 is 1470 bp in size and is repeated 10-12 times per genome. An open reading frame of 1200 bp was identified, encoding a protein product of M r 43932. This protein is highly similar to the predicted proteins of IS900 of Mycobacterium paratuberculosis, IS116 of Streptomyces clavuligerus and IS110 of Streptomyces coelicolor. IS902 lacks terminal inverted repeats and flanking direct repeats but displays insertion site specificity.
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Physical mapping of the mec region of an Australian methicillin-resistant Staphylococcus aureus lineage and a closely related American strain
More LessSummary: Methicillin-resistant (Mcr) staphylococci contain chromosomal DNA that is absent from Mcs cells. This extra DNA harbours the methicillin resistance determinant mec and often other resistance determinants. The mec region can differ substantially in structure among different isolates. We present studies on the mec region of a group of Staphylococcus aureus isolates prevalent in Australia and London. Southern hybridization analyses of a prototype Australian isolate, ANS46, and an isogenic Mcs deletion mutant, ANS62, allowed the physical map of the region to be extended to 55 kb. The DNA corresponding to the deletion, which includes mec and resistance determinants for mercury, cadmium (Cd) and tetracycline, amounted to 41 kb. It was bounded precisely at one end by the macrolides-lincosamides-streptogramin B (MLS)-resistance transposon, Tn554. Near the other end was an element with homology to Tn554, ΨTn554, which carried the Cdr determinant. The mec region of an American Mcr isolate, R35, was found to be virtually the same as that of ANS46, except that it lacked Tn554. Another class of American Mcr isolates, prevalent since 1987, differs markedly from ANS46 in mec region organization. However, this other American class also contains an insertion of Tn554 in the mec region, and the attachment site for this insertion was found to have significant homology to attachment sites for the Tn554 and ΨTn554 insertions in the mec region of the Australian strain. These results suggest possible roles of Tn554 and Tn554-like elements in the evolutionary variation of the mec region.
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DNA homology between siderophore genes from fluorescent pseudomonads
More LessSummary: Many species of pseudomonads produce fluorescent siderophores involved in iron uptake. We have investigated the DNA homology between the siderophore synthesis genes of an opportunist animal pathogen, Pseudomonas aeruginosa, and three plant-associated species Pseudomonas syringae, Pseudomonas putida and Pseudomonas sp. B10. There is extensive homology between the DNA from the different species, consistent with the suggestion that the different siderophore synthesis genes have evolved from the same ancestral set of genes. The existence of DNA homology allowed us to clone some of the siderophore synthesis genes from P. aeruginosa, and genetic mapping indicates that the cloned DNA lies in a locus previously identified as being involved in siderophore production.
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Diversity of cleavage patterns of Salmonella 23S rRNA
More LessSummary: The recent discovery of the phenomenon that some prokaryotes fragment their 23S rRNA during post-transcriptional processing of precursor rRNA has been shown to be particularly prevalent among strains and species of Salmonella. Some strains of Salmonella cleaved 23S rRNA at multiple sites producing several fragments. The cleavage patterns of 23S rRNA differed among Salmonella strains and sometimes among the rRNA operons in the same strain. Fragmentation of 23S rRNA was not observed in strains of the closely related species Escherichia coli. Fragmentation of 23S rRNA occurred in Brucella and Agrobacterium but the cleavage pattern was not as diverse as that demonstrated in Salmonella. Introduction of cleavage sites into precusor 23S rRNA of Salmonella is probably a recent evolutionary event.
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Construction of an insertional-inactivation cloning vector for Escherichia coli using a Rhodococcus gene for indigo production
More LessSummary: pSLH8, an insertional-inactivation cloning vector for Escherichia coli has been constructed by inserting a pigment gene (probably encoding an indole dioxygenase) from Rhodococcus sp. ATCC 21145 into pUC18. Wild-type E. coli colonies containing pSLH8 produce insoluble indigo and turn dark blue on unsupplemented LB agar. Insertion of DNA fragments into the unique BamHI, EcoRI, EcoRV, HindIII, PstI, SphI and SstI polylinker cloning sites disrupts the reading frame of the fully sequenced pigment gene and results in unpigmented colonies. pSLH8 may be an attractive alternative to pUC18 and similar plasmids because it does not require specifically mutated host strains or an expensive substrate for colour development.
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Intergeneric hybrids between Saccharomycopsis fibuligera and Yarrowia lipolytica
More LessSummary: Saccharomycopsis fibuligera can utilize starch but not tributyrin as the sole carbon source, whereas Yarrowia lipolytica can utilize tributyrin but not starch. S. fibuligera mutant strain 193 met was crossed to Y. lipolytica A his1 to produce hybrids. The intergeneric hybrids were able to utilize both starch and tributyrin as sole carbon source. They were mitotically unstable and gave segregants during prolonged vegetative culture. The majority of these segregants had the phenotype of the S. fibuligera parent, with a very low number of genetic recombinants. A more stable hybrid was produced by protoplast fusion between a mutant, ade met, obtained from UV treatment of a Met− mitotic segregant of the hybrid 14i and Y. lipolytica B lys5 leu2 ade1 xpr2. Sixteen of the 40 mitotic segregants of this hybrid were recombinants. Furthermore when a haploid mitotic segregant of S. fibuligera 193 met, met glu, was crossed to Y. lipolytica B leu2 ade1 xpr2 more stable hybrids were also obtained. Eleven of the 35 mitotic segregants of the hybrid SA11 were recombinants. Such high frequencies of recombinant sectors from these hybrids should provide a system to establish genetic mapping analysis for the novel Saccharomycopsis-Yarrowia recombinant strains.
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- Immunology
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Purification and characterization of a tryptic peptide of Borrelia burgdorferi flagellin, which reduces cross-reactivity in immunoblots and ELISA
More LessSummary: In man the early immune response in Lyme disease is primarily directed against the endoflagellin antigen. Isolated flagellar protein of Borrelia burgdorferi suggests itself as a suitable test antigen. However, cross-reactivity between flagellins of B. burgdorferi, Escherichia coli, Bacillus subtilis, Proteus mirabilis and Salmonella typhimurium was demonstrated by immunoblotting and ELISA with polyclonal rabbit-hyperimmune-sera. Tryptic cleavage of recombinant B. burgdorferi 41 kDa flagellin, expressed in E. coli, produced a peptide fragment which was recognized exclusively by antisera to Borrelia species. This peptide was designated as the 14 kDa fragment due to its migratory behaviour in SDS-PAGE. The fragment is part of the variable region of the flagellin, as proven by amino acid sequencing. The flagellin peptide was employed as an antigen in ELISA and immunoblot assays, testing the polyclonal sera mentioned above. The specificity was superior to that obtained with the intact recombinant flagellin.
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Localization of conserved B-cell epitopes among encapsulated and non-encapsulated Haemophilus influenzae P2 porin proteins using synthetic peptides
More LessThe P2 outer membrane protein of Haemophilus influenzae belongs to a class of apparently ubiquitous proteins in Gram-negative bacteria that function as porins. Murine hybridomas raised to the P2 protein and synthetic peptides were used to investigate the structural and antigenic relationships among P2 proteins of encapsulated and non-encapsulated H. influenzae. Three monoclonal antibodies (mAbs), P2-17, P2-18 and P2-19, recognizing epitopes on the P2 protein, as shown by Western immunoblotting of outer membrane preparations, and purified and recombinant P2 proteins are described. The epitopes reactive with the mAbs were widely distributed among H. influenzae strains since 70-100% of strains of encapsulated and non-encapsulated isolates collected worldwide were recognized by individual mAbs. None of the mAbs reacted with H. parainfluenzae or other bacterial species. The peptide composition of P2 epitopes was determined by analysis of mAb reactivity with a series of overlapping synthetic peptides that covered the amino acid sequences of H. influenzae type b. The domains recognized by these mAbs were completely distinct. mAb P2-18, reactive with an epitope conserved among all H. influenzae P2 porin molecules which were screened, recognized a peptide corresponding to the N-terminal segment (residues 1-14). The P2-17- and P2-19-specific epitopes were located between residues 28 and 55, and 101 and 129, respectively. None of the epitopes were exposed on the cell surface since no mAbs bound to intact live bacteria. The identification of epitopes that are shared among P2 proteins from different strains of encapsulated and non-encapsulated H. influenzae strains suggests that the P2 porin protein from all H. influenzae strains has been highly conserved and that these epitopes may represent amino acid residues critical to the porin's structure.
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- Pathogenicity And Molecular Microbiology
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The phenolic mycoside of Mycobacterium ulcerans: structure and taxonomic implications
More LessSummary: Mycobacterium ulcerans and some pathogenic mycobacterial species elaborate wax A consisting of related longchain β-diol components (phthiocerol and related compounds) esterified by multimethyl-branched fatty acids. With the exception of M. ulcerans, wax A-containing mycobacteria also synthesize glycosylated phenol phthiocerol diester and related compounds: the so-called phenolic mycosides. In a deliberate effort to characterize this latter class of compounds in M. ulcerans, 20 strains were examined. Phenolic mycosides were found in two strains. Application of chemical analyses, including one- and two-dimensional NMR spectroscopy, allowed the structural elucidation of glycolipids identified as 3-O-methyl-α-l-rhamnosyl phenol phthiocerol diphthioceranate and related compounds which correspond to mycoside G, previously characterized in M. marinum by other investigators. As phenolic mycosides are highly species-specific molecules, this finding stresses the close phylogenetic link between M. marinum and M. ulcerans. Incidentally, a survey of the mycolate content of M. ulcerans showed that methoxymycolate could not be detected in three strains.
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Immunogenic outer-membrane proteins of Haemophilus influenzae type b in infection
More LessSummary: Outer-membrane proteins (OMPs) from Haemophilus influenzae type b (strain Eagan), grown both in vitro (broth) and in vivo (rat intra-peritoneal), were separated by SDS-PAGE. The major OMPs were present in both growth conditions although the amounts of OMP a and OMP d were reduced in rat-grown organisms. There were strong additional bands in in-vivo-grown organisms at 51 and 92 kDa. Antiserum was raised in rabbits against in-vivo-grown bacteria, and absorbed with lysates of in-vitro-grown bacteria. This serum was used in Western blot analysis of OMPs from in-vitro- and in-vivo-grown cells to identify immunogenic proteins present in infection. These infection-associated OMPs had apparent molecular masses of 43 kDa, 48 kDa, 81 kDa and > 200 kDa. Bands of reactivity, of the same molecular mass as some of these, were found on immunoblots when rat and human convalescent sera were used as the source of primary antibody. In particular, a band of 81 kDa was recognized by pooled rat and three human convalescent sera.
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Salmonella plasmids of the pre-antibiotic era
More LessSummary: In order to provide a profile of the plasmid gene pool in Salmonella prior to the clinical use of antibiotics, a molecular genetic analysis was made of plasmids in strains collected by E. D. G. Murray between 1917 and 1954. These pre-antibiotic era (PAE) salmonellae contain conjugative plasmids of the same incompatibility groups as contemporary enterobacterial plasmids. Upon analysis of total plasmid content, 42 plasmids, sized between 23 and 72 MDa, were found. We defined and investigated six groups of these PAE Salmonella plasmids in terms of three groups of genes; those involved in plasmid maintenance and incompatibility, DNA repair and virulence. Of the five groups, three were replicon-typed to groups IncI1, IncX and IncFII; one group exhibited no homology to contemporary Inc/Rep probes, and one group represented virulence plasmids containing a common plasmid-partitioning locus. The results indicated that most of the PAE groups were progenitors of contemporary R-plasmids, except for the virulence plasmids, which have generally not evolved as vectors of antibiotic resistance.
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- Physiology And Growth
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Replacement of cellular potassium by caesium in Chlorella emersonii: differential sensitivity of photoautotrophic and chemoheterotrophic growth
More LessSummary: Photoautotrophic growth of Chlorella emersonii in the presence of 1 mM-CsCl resulted in a 34% increase in cell doubling time and an 83% reduction in the final cell yield as compared to growth in the absence of Cs+. In contrast, the presence of 1 mM-Cs+ had no effect on chemoheterotrophic growth in the dark with glucose. These observations were correlated with the stage of growth at which Cs+-induced cellular K+ loss was evident. For photoautotrophically growing cells this occurred during the exponential growth phase (after 4 d); for chemoheterotrophically growing cells this was during stationary phase (after 10 d). Inhibition of chemoheterotrophic cell division occurred after 2 d in 50 mM-Cs+ or 5 d in 20 mM-Cs+, and coincided with a decline in intracellular K+ to ∼2 nmol (106 cells)−1. Accumulation of Cs+ ceased after 2 d in both of these cases. Cell doubling times during chemoheterotrophic growth remained approximately constant at internal K+ levels between 7 and 28 nmol (106 cells)−1. In contrast, a decline in intracellular K+, from 42 to 19 nmol (106 cells)−1, after 4 d photoautotrophic growth in the presence of Cs+, was concurrent with the 34% increase in cell doubling times. Photosynthesizing cells accumulated approximately 2-fold more Cs+ than respiring cells after incubation for 12 h in HEPES buffer, pH 8. Culture age and intracellular K+ levels had little effect on the ability of C. emersonii to accumulate Cs+. Externally supplied K+ inhibited Cs+ accumulation to a greater extent in photosynthesizing cells (75% inhibition at 10 mM- K+, 1 mM-Cs+) than in respiring cells (50% inhibition at 10 mM-K+, 1 mM-Cs+). Greatly elevated Na+ light in the absence and presence of 10 mM-Cs+ accumulation in both cases. Incubation of cells in buffer in the light in the absence and presence of 10 mM-Cs+ resulted in decreases in cellular K+ of 44% and 77% respectively and a concomitant 66% reduction in the rate of photosynthesis in both cases. A Cs+-induced K+ loss of 71% from respiring cells had no effect on the rate of respiration. These results indicate that it is not the presence of Cs+ in cells that is growth inhibitory, but rather the resulting loss of K+ and that photosynthesis and photoautotrophic growth are more sensitive to this K+ loss than respiration and chemoheterotrophic growth.
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Calcium regulation of growth and differentiation in Streptococcus pneumoniae
More LessSummary: Streptococcus pneumoniae requires 0·15 mm-Ca2+ in the medium for optimal growth. Increasing the Ca2+ concentration to 1 mm triggers either a differentiative state, competence for genetic transformation during exponential growth, or partial lysis as soon as the cultures enter stationary phase. Genetic and physiological data both suggest that these responses are under the control of activator(s), excreted in the presence of high Ca2+ concentrations. 45Ca2+ transport is also stimulated by the activator(s). The amiloride derivative 2”,4′-dimethylbenzamil (DMB) inhibits 45Ca2+ transport and prevents lysis and competence development. This provides evidence in favour of the involvement of Ca2+ transport in competence and culture lysis. On the other hand, addition of DNA to a competent culture prevents lysis of wild-type bacteria while a mutant, defective for DNA uptake, is not protected from lysis by exogenous DNA. An hypothesis is proposed for competence induction as a global metabolic response to Ca2+, under the control of competence factor.
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Excretion of anthranilate and 3-hydroxyanthranilate by Saccharomyces cerevisiae: relationship to iron metabolism
E. Lesuisse, M. Simon, R. Klein and P. LabbeSummary: Resting suspensions of cells of Saccharomyces cerevisiae grown in iron-rich or iron-deficient conditions were studied by following the fluorescence emission changes (λem 400-460 nm, λexc. 300-340 nm) occurring in these suspensions upon addition of glucose and ferric iron. The results show that, in addition to NAD(P)H, metabolites of the aromatic amino acid pathway interfere with the fluorescence measurements, and that they could be involved in ferric iron reduction. Wild-type strains of S. cerevisiae are known to excrete anthranilic acid and 3-hydroxyanthranilic acid in response to glucose. The major fluorescing compound excreted by a chorismate-mutase-deficient mutant strain of S. cerevisiae was identified as anthranilic acid. The excretion of anthranilic and 3-hydroxyanthranilic acids was correlated with the ferric-reducing capacity of the extracellular medium. Excretion during growth was much greater by cells cultured in iron-rich medium than by cells grown in iron-deficient medium. The possibility was examined that a link could exist between the biosynthesis of aromatics and the ferri-reductase activity of the cells, via chorismate synthase and its putative diaphorase-associated activity. Two ferri-reductase-deficient mutants excreted much less 3-hydroxyanthranilate than did the parental wild-type strains. However, the ferri-reductase activity of a chorismate-synthase-deficient mutant was comparable to that of the parental strain.
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Effect of salt stress on lipid composition and membrane fluidity of the salttolerant yeast Zygosaccharomyces rouxii
More LessSummary: When the salt-tolerant yeast Zygosaccharomyces rouxii was grown in YPD medium containing 15% (w/v) NaCl, the relative amounts of C16:1 and C18:1 fatty acids in acyl lipids increased and those of C18:0 and C18:2 acids decreased both in whole cells and in crude plasma membrane preparations, as compared with cells grown in YPD medium alone. The proportions of C12:0 and C14:0 acids, which are minor components of yeast lipids, decreased in whole cells and markedly increased in plasma membranes when 15% NaCl was included in the growth medium. The degree of unsaturation of fatty acids in the membranes and in whole cells decreased in the presence of 15% NaCl in the culture medium. The amount of free ergosterol in the membranes of cells grown in 15% NaCl increased to 2·9 times that of control cells. The ratio of ergosterol to phospholipid increased to 5 times that of control cells, whereas the ratio of phospholipid to protein in the membranes of cells grown in 15% NaCl decreased to less than half that of control cells. The fluorescence polarization value of DPH (1,6-diphenyl-1,3,5-hexatriene) in membranes of cells grown in 15% NaCl was 1·2 times higher than that for membranes from control cells, indicating a decrease in membrane fluidity in the presence of a high concentration of NaCl.
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Chitinase and chitin synthase 1: counterbalancing activities in cell separation of Saccharomyces cerevisiae
More LessSummary: Previous results [E. Cabib, A. Sburlati, B. Bowers & S. J. Silverman (1989) Journal of Cell Biology 108, 1665-1672] strongly suggested that the lysis observed in daughter cells of Saccharomyces cerevisiae defective in chitin synthase 1 (Chs1) was caused by a chitinase that partially degrades the chitin septum in the process of cell separation. Consequently, it was proposed that in wild-type cells, Chs1 acts as a repair enzyme by replenishing chitin during cytokinesis. The chitinase requirement for lysis has been confirmed in two different ways: (a) demethylallosamidin, a more powerful chitinase inhibitor than the previously used allosamidin, is also a much better protector against lysis and (b) disruption of the chitinase gene in chs1 cells eliminates lysis. Reintroduction of a normal chitinase gene, by transformation of those cells with a suitable plasmid, restores lysis. The percentage of lysed cells in strains lacking Chs1 was not increased by elevating the chitinase level with high-copy-number plasmids carrying the hydrolase gene. Furthermore, the degree of lysis varied in different chs1 strains; lysis was abolished in chs1 mutants containing the scs1 suppressor. These results indicate that, in addition to chitinase, lysis requires other gene products that may become limiting.
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Influence of pH on the growth and leaf-maceration ability of fungi involved in the decomposition of floating leaves of Nymphaea alba in an acid water
More LessSummary: Ten species of fungi were isolated from floating leaves cut from plants of Nymphaea alba in various initial stages of decay, which were collected from an acidic moorland pool. None of the fungal species isolated belonged to the aquatic Hyphomycetes sensu Ingold or the aero-aquatic fungi sensu Van Beverwijk. Growth experiments were conducted with five of the species on media containing glucose, polysaccharide or isolated cell walls of N. alba, each at three pH values. The fungi investigated were capable of growth on a variety of structural polysaccharides, indicating their potential importance in leaf degradation. Low pH inhibited growth on media containing glucose, pectin or cell wall fraction. Utilization of carboxymethylcellulose and crystalline cellulose did not differ much within the pH range studied. The fungi isolated were grown on N. alba leaf strips at three pH values to study the influence of pH on leaf maceration. All the fungi investigated could develop at low pH (4·0), but maceration was only observed at pH 5·5 or 7·5. It is likely that inhibition of pectin degradation is an important factor causing suppression of leaf fragmentation at low pH. This may contribute to the inhibition of the decomposition of macrophyte remains in acid aquatic systems.
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The regulation of fatty acid biosynthesis in some estuarine strains of Flexibacter
More LessSummary: The mechanism of unsaturated fatty acid biosynthesis in two estuarine strains of Flexibacter was investigated. Addition of cyclic AMP (cAMP) inhibited the incorporation of radiolabeled acetate into fatty acids of lateexponential phase cultures of strain Inp2. Cerulenin selectively inhibited the incorporation of radioactive acetate into the major fatty acid, 16:1. When Flexibacter strain Inp3 was grown in a medium of high osmotic strength, polyunsaturated fatty acids were present, but they were absent from cultures grown in media with an osmotic strength close to that of sea water. Addition of cAMP to cultures growing at high osmotic strength suppressed the formation of polyunsaturated fatty acids; the uncoupler 2,4-dinitrophenol had a similar effect. The findings are discussed in the context of the suggestion made by Intriago and Floodgate (Journal of General Microbiology 137, 1503–1509, 1991) that Flexibacter strains possess both the anaerobic and aerobic pathways for unsaturated fatty acid biosynthesis, the activity of the latter being modulated by the intracellular concentration of cAMP.
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Growth of the marine luminous bacterium Vibrio fischeri on 3′:5′-cyclic AMP: correlation with a periplasmic 3′:5′-cyclic AMP phosphodiesterase
More LessSummary: The marine luminous bacterium Vibrio fischeri, the species-specific light-organ symbiont of monocentrid (pinecone) fish, grew on 3′:5′-cyclic adenosine monophosphate (cAMP) as a sole source of carbon and energy, a capability not previously described in bacteria. In a minimal, chemically defined medium containing cAMP as the sole source of carbon and energy, V. fischeri cells grew more slowly than on glucose or ribose, but as quickly as on 5′-AMP. Expression of luminescence, which is dependent on cAMP in V. fischeri, was stimulated in cells grown on cAMP compared to cells grown on glucose or ribose. All strains of V. fischeri tested (MJ-1, B-61, ATCC 7744, MJ-A1, CG-A1) grew on cAMP, as did strains of two other marine luminous bacteria, V. logei and Photobacterium phosphoreum, and strains of the terrestrial enteric bacterium Serratia marcescens. Other tested species of marine luminous bacteria (P. leiognathi, Shewanella [Alteromonas] hanedai, V. harveyi, V. orientalis, V. splendidus) and terrestrial enteric bacteria (Escherichia coli, Salmonella typhimurium), which grew on 5′-AMP or ribose, did not grow on cAMP. Assays on intact cells and periplasmic extracts revealed that V. fischeri MJ-1 produced a periplasmic 3′:5′-cAMP phosphodiesterase (cAMP phosphodiesterase) of very high specific activity [9·0 μmol phosphate released (mg protein)−1 min−1] and narrow substrate specificity (3′:5′-cAMP and 3′:5′-cGMP were attacked). The novel periplasmic location and unusually high activity of cAMP phosphodiesterase appear to account for the ability of this species to grow on cAMP. The periplasmic cAMP phosphodiesterase of V. fischeri night play a role in degrading free cAMP in seawater or in a cAMP-mediated aspect of the light organ symbiosis with monocentrid fish.
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A re-examination of the pathway for ornithine biosynthesis in a thermophilic and two mesophilic Bacillus species
Summary: The expression of Bacillus stearothermophilus genes complementing arginine auxotrophs of Escherichia coli was studied. The activity responsible for the formation of ornithine in B. stearothermophilus was identified as a repressible ornithine acetyltransferase (genetic symbol argJ) encoded by the same DNA fragment as the argC, orgA and argB genes. Bacillus subtilis and Bacillus licheniformis displayed the same pattern of enzyme activities as B. stearothermophilus. In contrast to previous reports, these organisms consequently use the cyclic pathway of ornithine biosynthesis. B. stearothermophilus also possesses a broad specificity aminoacylase which exhibits low affinity towards N 2-acetyl-l-ornithine.
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Purification and properties of methyl mercaptan oxidase from Thiobacillus thioparus TK-m
More LessSummary: Methyl mercaptan (MM)-oxidase was purified to near homogeneity from Thiobacillus thioparus TK-m grown on dimethyl sulphide. The enzyme was a monomer with an M r value of approximately 40000. It oxidized MM stoichiometrically to formaldehyde, S2- and H2O2. The enzyme had a K m of 31·3 μM for MM. It was also shown to oxidize ethyl mercaptan. MM-oxidase from T. thioparus was shown to have some different characteristics from MM-oxidase of Hyphomicrobium EG, such as a higher K m, the absence of inhibition by S2- and the inability to catalyse the oxidation of S2-.
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