- Volume 131, Issue 5, 1985
Volume 131, Issue 5, 1985
- Biochemistry
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Mercuric Reductase Enzymes from Streptomyces Species and Group B Streptococcus
Mercury volatilization (Hg2+ reductase) activity has been found with Hg2+-resistant isolates of three Streptomyces species and with three Hg2+-resistant strains of group B Streptococcus from clinical sources in Japan. Hg2+ reductase activities in crude cell extracts showed the temperature sensitivity, the requirement for an added thiol compound and the characteristic dependence on NAD(P)H cofactors of similar enzymes isolated from other bacteria.
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Glutamine Synthetase from Pseudomonas syringae pv. tabaci: Properties and Inhibition by Tabtoxinine-β-lactam
More LessGlutamine synthetase from Pseudomonas syringae pv. tabaci was purified 500-fold. Maximum activity was observed with 10 mm-glutamate, 20 mm-ATP and 4 mm-NH4Cl. The enzyme exhibited substrate inhibition; higher levels of glutamate, Mg. ATP or NH4Cl decreased its activity. The γ-glutamyltransferase activity was inhibited by Mg2+ (75% at 10mm-Mg2+). The enzyme was heat stable and there appeared to be only one form present. Tabtoxinine-β-lactam, a hydrolytic product of tabtoxin produced by pv. tabaci, inactivated the enzyme. This inhibition was linear with respect to the concentration of the inhibitor, and enzyme activity could not be recovered by dialysis, acetone precipitation or incubation with crude cell lysate. Mg. ATP and ammonium ions were required for binding of the inhibitor: incubation of tabtoxinine-β-lactam with the enzyme in the presence of both Mg. ATP and ammonium ions resulted in a greater decrease in synthetase activity than incubation with either one or neither component. Tabtoxinine-β-lactam did not inhibit the γ-glutamyltransferase activity of the enzyme if ADP was used in the assay, but did when ATP was used.
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Preliminary Characterization of an Antibiotic Produced by Xanthomonas albilineans Which Inhibits DNA Synthesis in Escherichia coli
More LessChlorosis-inducing isolates of Xanthomonas albilineans, the sugarcane leaf scald pathogen, produced a mixture of antibacterial compounds in culture. The antibiotic mixture, which eluted as a single strongly retarded peak from Sephadex LH-20 in methanol, was bactericidal to Escherichia coli. Inhibition of E. coli was not reversed by added nutrients, and affected cells were not lysed but many accumulated polyphosphate granules. The major antibacterial component, isolated in crystalline form after HPLC, is given the trivial name albicidin. Near the minimum inhibitory concentration, albicidin caused a complete block to DNA synthesis, followed by partial inhibition of RNA and protein synthesis, as assessed by incorporation of radioactive precursors. Spontaneous antibiotic-resistant mutants of E. coli showed no cross-resistance between albicidin and inhibitors of either subunit of DNA gyrase. Mixing albicidin with purified DNA from E. coli did not alter the thermal denaturation behaviour of the DNA, or the absorption spectrum of the antibiotic. PolA+ and PolA– strains of E. coli were equally sensitive to albicidin. indicating that the antibiotic does not bind to or modify DNA. Selective inhibition of DNA synthesis without evidence of DNA binding suggests a specific interaction of albicidin with an essential replication protein.
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- Development And Structure
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Immunogold Localization of p55-Fibril Protein and p25-Spiralin in Spiroplasma Cells
More LessTransmission electron microscopy of thin sectioned cells of the honeybee spiroplasma BC3 revealed evidence of a helically twisted ribbon closely associated with the cytoplasmic surface of the plasma membrane. The cellular distribution of p55-fibril protein as demonstrated by immunogold staining with anti-p55 antibody was consistent with the presence of p55 in this ribbon. We conclude that spiroplasma fibrils are arranged in a single helically twisted ribbon rather than forming a contractile sheath or branched axial fibre. Immunogold staining with anti-p25-spiralin antibody confirmed that this protein is localized in the plasma membrane and also showed that it occurs in extracellular strands. These strands may be antigenically distinct from the integral form of the protein.
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Induction of Spheroplasts in Capnocytophaga ochracea
More LessA gentle technique for preparing spheroplasts of Capnocytophaga ochracea strain 25 is described. Cells in the exponential phase were washed with 1·0 m-NaCl, agitated in 1·0 m-NaCl for 2 h at 30°C and exposed to lysozyme in a Tris/salts buffer, pH 7·0. This procedure resulted in 98% spheroplast formation with complete removal of the peptidoglycan layer as detected by both phase-contrast and electron microscopy in combination with chemical analysis.
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Ultrastructure of Bacilli and the Bacillary Origin of the Macrophagic Inclusions in Whipple’s Disease
More LessAn electron microscopic and cytochemical study of the Whipple bacillus in jejunal biopsies from three untreated patients was made using fixation procedures developed for the satisfactory preservation of bacterial ultrastructure. The envelopes of the normal- looking bacilli present free in the lamina propria consisted of the following layers. (i) A cytoplasmic membrane with a triple-layered profile and a mean thickness (peak-to- peak distance) of 6.08 nm. (ii) A thick (20 nm) cell wall containing peptidoglycan; the wall had a hitherto undescribed inner layer that contained polysaccharides, possibly teichoic acids. (iii) Surrounding the cell wall, a surface membrane with a symmetric profile and a mean peak-to-peak distance of 4.74 nm. The ultrastructural pattern of the Whipple bacillus wall corresponds to that of Gram- positive bacteria, but with an additional surface membrane. This membrane is different from the outer membrane of Gram- negative bacteria because it has a symmetric profile, is thinner and has no periodic acid-Schiff (PAS)-positive components.
Normal-looking bacilli were seen very rarely inside jejunal macrophages, but degenerating bacteria were abundant in these phagocytes. Electron microscopy and ultrastructural cytochemistry of Whipple bacilli inside jejunal macrophages of the three untreated patients showed that the degenerative process is a sequence that leads to the loss of bacillary forms and to the accumulation of bacterial remnants resistant to degradation by the macrophage. These remnants correspond to the innermost, polysaccharide-containing portion of the bacillus wall. The progressive accumulation of these PAS-positive wall remnants is the origin of the intramacrophagic inclusions that are important in the histological diagnosis of Whipple’s disease. The reported results indicate that in the three patients studied, the Whipple bacillus multiplies extracellularly, the bacteria that are phagocytosed by macrophages being degraded.
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- Ecology
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The Effects of Wall Populations on Coexistence of Bacteria in the Liquid Phase of Chemostat Cultures
More LessWe have examined the effects of wall populations on coexistence between strains of Escherichia coli in the liquid phase of mixed (two-strain) chemostats. The wall populations of the two competing strains became established soon after the start of the cultures and, although the relative abundance of the strains in the liquid phase could change over time by several orders of magnitude, the composition of an established wall population did not change markedly. The bacterial strains examined could not displace an established wall population of a competing strain. The presence of a permanent wall population allowed a strain that was less fit in the liquid phase to coexist with a superior strain. The resulting coexistence did not require that the inferior strain attached to the vessel wall better than the superior strain. We believe that the coexistence developed because the inferior strain survived and reproduced on the vessel wall. The progeny from that wall population then provided replacements for the bacteria that the inferior strain lost through a selective disadvantage in the liquid phase of the culture. By replacing the chemostat vessel, hence eliminating the wall populations, we could distinguish between cases where the coexistence depended on the presence of a wall population and where it resulted from some alternative mechanism.
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The Barrier-Ring Plate Technique for Studying Extracellular Enzyme Diffusion and Microbial Growth in Model Soil Environments
More LessA novel technique has been developed to study complex spatial interactions between microorganisms, enzymes and their substrates in soil using barriers composed of soil and soil components inserted into agar plates. This has allowed the investigation of extracellular enzyme diffusion and microbial growth through soil-like but carefully controlled environments. Using ‘barrier-ring plates’ the effects of small quantities of soil and various soil components on endoglucanase and β-d-glucosidase diffusion was shown. Bentonite, with a relatively high unit surface area and a high cation exchange capacity, reduced the distance diffused by both enzymes. Kaolinite, a clay with a relatively low unit surface area and a low cation exchange capacity, had no effect while the colloidal-size (<2 μm) clay-humic fraction separated from a silt loam soil reduced the distance diffused by endoglucanase by an amount intermediate between that of kaolinite and bentonite. The same barrier-ring plate technique was used to demonstrate how soil components differentially affect the radial growth of a cellulolytic Streptomyces sp. and Trichoderma υiride, T. koningii and Botryotrichum piluliferum.
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Agrobacterium rhizogenes Promotes the Initial Growth of Bare Root Stock Almond
More LessBare root stock almond trees treated with Agrobacterium rhizogenes 232 had a larger root number and root mass after 90 d than those treated with autoclaved or filter-sterilized bacterial suspensions, or with medium alone. Treatment with A. rhizogenes 232 also led to significant increases in leaf number, stem diameter and shoot elongation during the first growing season after treatment. The bacterium was recoverable from both the rhizosphere and the bulk soil around the treated roots, but never from uninoculated control roots. Thus, the reaction of almond to A. rhizogenes was not a pathological one even though this bacterium is classified as a pathogen.
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- Genetics And Molecular Biology
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Nucleotide Sequence and Complementation Analysis of a Polycistronic Sporulation Operon, spoVA, in Bacillus subtilis
P. FORT and J. ERRINGTONWe have determined the nucleotide sequence of a 3706 bp stretch of Bacillus subtilis chromosomal DNA that complements all known spoVA mutations. The sequence contains five consecutive large open reading frames capable of encoding proteins of molecular weights ranging from approximately 15000 to 36000. Analysis using integrational plasmids suggests that the region is likely to be transcribed as a single mRNA. A novel form of complementation analysis, based on derivatives of bacteriophage ϕ105 carrying the cloned spoVA locus, has been used to define four distinct complementation groups among the eight previously characterized spoVA mutations. The spoVA locus is the largest polycistronic sporulation operon yet characterized.
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Lysis of Escherichia coli by Cloned ϕX174 Gene E Depends on its Expression
U. BLÄSI, B. HENRICH and W. LUBITZThe lysis gene E of bacteriophage ϕX174 was cloned under transcriptional control of the lefthanded lambda promoter, giving rise to plasmid pSB12. Plasmid pSB22, identical to pSB12 except for an amber mutation in gene E, was constructed in the same way. Induction of the cloned wild-type gene by heat inactivation of the thermosensitive λ cI857 repressor resulted in lysis of the host bacteria. With plasmid pSB22 only amber suppressor strains of Escherichia coli lysed after heat inactivation of λ cI857. Lysis of E. coli was shown to depend on the rate of gene E translation and on the growth phase of the bacteria. Stationary cells could not be lysed by the gene E product (gpE), even if present in sufficient amounts to lyse growing cells. By isotopic labelling gpE could be detected among the proteins synthesized in normal E. coli as well as in minicells. Determination of gene E expression suggested that gpE synthesis is translationally regulated.
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Phage pilHα: a Phage Which Adsorbs to IncHI and IncHII Plasmid-coded Pili
Phage pilHα was specific for bacterial strains, of various genera, harbouring plasmids of the HI and HII incompatibility groups. Plaque formation was temperature sensitive in that plaques formed at 26°C but not at 37°C. Plaques were fairly clear, irregular in outline and varied from pin point to about 2 mm in diameter on all hosts where plaques were detected. The phage had an isometric hexagonal outline with a diameter of 25 nm. It contained RNA but differed from all but one other plasmid-dependent RNA phage by being sensitive to chloroform. It adsorbed along the length of the shafts of IncHI and HII plasmid-coded pili.
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Evolution of Tn21-related Transposons: Isolation of Tn2425, which Harbours IS161
More LessThe isolation of two multi-resistance transposons, Tn2425 and Tn1831, and their relation to Tn21 and Tn2424, is described. A 1·7 kb segment present in Tn2424 and Tn2425 was identified as an IS element by rec-independent transposition, resulting in a Cointegrate structure that carries two direct repeated copies of the IS element. By the isolation of this IS element we demonstrated that transposition is one mechanism leading to sequence variations in Tn21-like structures, especially in the region between the mer operon and the sul gene.
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Plasmid Fusions Mediated by One End of TnA
More LessWe have observed plasmid fusions in a recA background mediated by a single end of TnA. These occur when transposase is provided either in cis or in trans. Insertions of the plasmid carrying the TnA inverted repeat sequence occur at many sites in the target plasmid. The point of fusion on the plasmid carrying TnA sequences always appears to be located in the region which carries the TnA inverted repeat sequence. In contrast to the transposition of an intact TnA element, plasmid fusions mediated by one end of TnA are very rare events. The implications of our results for models of transposition are discussed.
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Mutagenesis of a Rhizobium Plasmid Carrying Hydrogenase Determinants
More LessTransposon Tn5-mob, a Tn5 derivative containing the mobilization site of plasmid RP4, was introduced into Rhizobium leguminosarum strain 128C53 on the suicide vector pSup5011. Transposon insertions into the plasmids of strain 128C53 were selected after mating the mutagenized population with a non-nodulating R. leguminosarum recipient. In this way, a tenfold enrichment was effected for plasmid-linked mutations. Transconjugants that had received mutagenized symbiotic plasmids were applied individually to the roots of pea seedlings and, after three weeks, the resulting nodules were screened for the absence of hydrogenase activity. Eight hydrogenase-deficient (Hup–) mutants of R. leguminosarum strain 128C53 were isolated. Physical and genetic analyses suggest that each Hup– mutant was due to a single insertion of Tn5-mob into the symbiotic plasmid, pRL6JI. All mutations were suppressible by pHU1, a cosmid clone carrying some of the hup genes of Rhizobium japonicum.
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Inhibitory Reactions between Natural Isolates of Streptomyces
More LessNine different strains of Streptomyces (‘S. scabies’) have been isolated from scab lesions on potato tubers. These strains were obtained from different cultivars grown in different geographical areas. Four different types of inhibitory reactions were observed when these strains were tested in pairs with each other. Three of these inhibitory reactions resembled the lethal zygosis phenotype that has been reported for other species of Streptomyces. All of the strains exhibited at least two of these inhibitory reactions and were sensitive to the inhibitory reactions of at least six of the other isolates. Five of these isolates were shown to harbour plasmid DNA.
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Structural Organization of a Hypermethylated Nuclear DNA Component in Physarum polycephalum
More LessDigestion of Physarum polycephalum nuclear DNA using the restriction endonuclease HpaII generates two components, distinguishable on the basis of their molecular size. The high-molecular-weight, HpaII-resistant component, which accounts for 20% of the DNA, contains a fivefold greater concentration of 5-methylcytosine residues than the low-molecular-weight HpaII-digested fraction. Segments of hypermethylated (M+) DNA are largely composed of a single, long, highly repeated sequence, and this major element is sometimes associated with other less highly repetitive sequences in the M+ DNA fraction. Restriction mapping of cloned Physarum M+ DNA segments, and Southern blot analysis of genomic DNA using subcloned segments of M+ DNA as a probe, provide evidence for sequence variation within different copies of the dominant highly repeated element, and possibly the other associated repeats in M+ DNA, and additionally that almost complete tandemly repeated copies of the major repeat are found in some M+ DNA segments.
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The Effect of Ploidy on the Stability of Plasmodial Heterokaryons in Physarum polycephalum
More LessThe stability of actively growing heterokaryons made by fusing haploid and diploid plasmodia of Physarum polycephalum has been investigated with the aid of genetic markers affecting plasmodial colour and amino acid requirements. All heterokaryons initially expressed the dominant alleles present in both component plasmodia, but after a few subcultures every heterokaryon synthesized from a haploid plus a diploid plasmodium changed to express the recessive alleles carried by the haploid component. In contrast, heterokaryons synthesized from combinations of haploid plasmodia remained stable throughout many subcultures. No evidence was found for the segregation of incompatibility alleles responsible for heterokaryon instability, although the possibility could not be excluded that a gene closely linked to mat A was involved. A direct test of the effect of ploidy on heterokaryon stability was carried out, in which the use of isogenic haploid and diploid plasmodia allowed the effect of incompatibility genes to be eliminated. Again the phenotype of every haploid plus diploid heterokaryon changed to that of the haploid component, whereas haploid plus haploid heterokaryons remained stable. Variations in the relative sizes of the haploid and diploid plasmodia altered the speed, but not the direction, of the changes observed. Analysis of progeny indicated that the changes in phenotype were due to loss of diploid nuclei from the heterokaryons.
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Genetic Analysis in Streptomyces chrysomallus
More LessA circular linkage map was developed for Streptomyces chrysomallus, a producer of actinomycin C. The map order of various marker loci was deduced from matings and to a minor extent from protoplast fusions. The map strongly resembles that of Streptomyces coelicolor A3(2). The recombination frequencies were low and highly variable (from 10–9 to 5 × 10–6). Plasmid pIJ303 expressed its thiostrepton resistance gene in S. chrysomallus but did not promote chromosomal transfer or induce the Ltz+ phenotype. The data provide a background of genetics for investigations of antibiotic synthesis in this strain.
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Stability of a Catabolic Plasmid in Continuous Culture
More LessA wild-type strain of Pseudomonas putida PPK1, carrying a non-conjugative TOL plasmid, was grown in continuous culture under carbon-limitation with m-toluate as growth substrate. When the medium was changed to benzoate a plasmid-free strain appeared after about 100 h. After this event the proportion of plasmid-containing bacteria declined rapidly but in several experiments this was followed by oscillations in the relative frequencies of the plasmid-free and plasmid-containing populations. About 1% of the total population retained the plasmid after 600 h growth under benzoate-limitation. When the input medium was returned to m-toluate the plasmid-free bacteria disappeared and the TOL+ population recovered to 100%. The plasmid in the wild-type strain was stably maintained during 500 h of chemostat growth under succinate-limitation. TOL+ strains re-isolated from continuous culture under benzoate-limitation retained their TOL plasmids for longer periods. It is suggested that plasmid loss is related to a failure in the control of partitioning at cell division but that this is not absolute and allows the maintenance of a residual low level of plasmid-containing bacteria in the population.
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