- Volume 137, Issue 12, 1991
Volume 137, Issue 12, 1991
- Physiology And Growth
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Phenotypic variation of Pseudomonas putida and P. tolaasii affects the chemotactic response to Agaricus bisporus mycelial exudate
More LessThe chemotactic response of wild-type Pseudomonas putida and P. tolaasii, and a phenotypic variant of each species, to Agaricus bisporus mycelial exudate was examined. Both P. putida, the bacterium responsible for initiating basidiome development of A. bisporus, and P. tolaasii, the causal organism of bacterial blotch disease of the mushroom, displayed a positive chemotactic response to Casamino acids and to A. bisporus mycelial exudate. The response was both dose- and time-dependent and marked differences were observed between the response time of the wild-type strains and their phenotypic variants. Phenotypic variants responded rapidly to both attractants and reached a maximum response after 10–20 min, whereas the wild-types took 45–60 min. The differences are partly explained by the more rapid swimming speed of the phenotypic variants. Both variants responded maximally to similar concentrations of Casamino acids and mycelial exudates. Investigations into the nature of the attractants contained in the mycelial exudate indicated that they are predominantly small (M r < 2000) thermostable compounds. Sugars present in the exudate did not elicit a chemotactic response in any isolate, but a mixture of 14 amino acids detected in the exudate accounted for between 50 and 75% of the chemotactic response of the fungal exudate.
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Phenotypic variation of Pseudomonas putida and P. tolaasii affects attachment to Agaricus bisporus mycelium
More LessThe effect of phenotypic variation on attachment of Pseudomonas tolaasii and P. putida to Agaricus bisporus mycelium was investigated. Quantitative studies demonstrated the ability of each isolate to attach rapidly and firmly to A. bisporus mycelium and significant differences in attachment of wild-type and phenotypic variant strains were observed. This was most pronounced in P. tolaasii, where the percentage attachment of the wild-type form was always greater than that of the phenotypic variant. The medium upon which the bacteria were cultured, prior to conducting an attachment assay, had a significant effect on their ability to attach. Attachment of the wild-type form of P. putida was enhanced when the assay was performed in the presence of CaCl2, suggesting the involvement of electrostatic forces. No correlation was observed between bacterial hydrophobicity and ability to attach to A. bisporus mycelium. Scanning electron microscopy confirmed the results obtained from the quantitative studies and provided further evidence for marked differences in the ability of the pseudomonads to attach to mycelium. Fibrillar structures and amorphous material were frequently associated with attached cells and appeared to anchor bacteria to each other and to the hyphal surface. A time-course study of attachment using transmission electron microscopy revealed the presence of uneven fibrillar material on the surface of cells. This material stained positive for polysaccharide and may be involved in ensuring rapid, firm attachment of the cells.
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Behaviour of Azospirillum brasilense in a spatial gradient of oxygen and in a ‘redox’ gradient of an artificial electron acceptor
More LessAzospirillum brasilense responds to a spatial gradient of oxygen by formation of a band in the zone of optimal oxygen concentration. The same behaviour was observed in a ‘redox’ gradient of tetramethyl-p-phenylenediamine (TMPD) oxidized by ferricyanide, under conditions where oxygen uptake was inhibited by cyanide. Inhibitors of the bc 1-complex of the redox chain did not inhibit aerotaxis. Cells inhibited by a high cyanide concentration still showed slow motility and weak attraction to oxygen, possibly as a result of cyanide-resistant respiration (3–5% of the initial level). It is suggested that electron flow through the redox chain (and/or consequent changes in protonmotive force), rather than binding of oxygen to terminal oxidases, may be a signal in the aerotactic response of A. brasilense.
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Age-dependent changes in extracellular proteins, aminopeptidase and proteinase activities in Frankia isolate BR
More LessTo investigate protein secretion by the nitrogen-fixing actinomycete Frankia isolate BR, we designed a rapid DEAE adsorption, salt elution and Biogel P6DG desalination method to concentrate protein from the growth medium. Secreted proteins reached a maximum concentration (5·6 mgl–1) in the medium at growth arrest. Analysis by SDS-PAGE detected up to 63 extracellular polypeptides when Frankia cells were grown under stirred conditions in BAP medium supplemented with phosphatidylcholine and MES buffer and 65 proteins in stirred BAP media alone. The pattern of extracellular polypeptides changed during growth. Several extracellular proteolytic activities were detected and compared with intracellular ones. The substrate specificity of the extracellular and intracellular aminopeptidase activities were the same. Also, the electrophoretic migration patterns of secreted and intracellular aminopeptidases could not be distinguished. Secretion of the proline-specific aminopeptidase FAP 3.BR appeared to be the only one that arrested at the end of the exponential phase of growth. At least 15 proteinases (PF) were secreted: 10 had the same electrophoretic mobility as their intracellular counterparts after SDS-gelatine-PAGE while five (PF-39·5, PF-38·5, PF-36·5, PF-25·5 and PF-20·5 kDa) had a different electrophoretic mobility and, therefore, appeared to be exclusively extracellular. At least seven extracellular proteinases appeared to increase coordinately in activity shortly before growth arrest.
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Escherichia coli metabolism in space
More LessSUMMARY: Cultures of the bacterium Escherichia coli were grown in the orbiting Biocosmos 2044 satellite in order to evaluate the effects of the space environment – weightlessness and heavy particle radiation – on growth parameters and energy metabolism, which have previously been reported to be affected, and on induction of the SOS response, which reflects DNA damage to the cell. We found no differences between the flight samples and control ground cultures in the growth yield per gram of carbon, in mean cell mass (from which we deduce that the growth rate was unaltered) or in the level of expression of the SOS response. These observations indicate that free-growing bacterial cells do not expend significant energy fighting gravity and that cosmic radiation within a space capsule does not produce significant levels of DNA damage.
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Effects of alcohols on the respiration and fermentation of aerated suspensions of baker’s yeast
More LessThe immediate effects of externally added alcohols on CO2 production and O2 consumption of suspensions of washed, aerated baker’s yeast were studied by stopped-flow membrane inlet mass spectrometry. Glucose-supported fermentation was progressively inhibited by increasing ethanol concentration (0–20%, v/v). The inhibition by ethanol was quite different from that observed for acetaldehyde; thus it is unlikely that toxicity of the latter can account for the observed effects. For five different alkanols (methanol, ethanol, 1-propanol, 2-propanol and 1-butanol) increasing inhibition of anaerobic fermentation was correlated with increased partition coefficients into a hydrophobic milieu. This suggests that the action of ethanol is primarily located at a hydrophobic site, possibly at a membrane. Results for respiratory activities were not as definite as for those for anaerobic metabolism because some alkanols act as respiratory substrates as well as giving inhibitory effects.
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Examination of the role of Na+ in the physiology of the Na+-dependent soil bacterium Azotobacter salinestris
More LessThe growth of the Na+-dependent soil bacterium Azotobacter salinestris strain 184 was inhibited only 36% by 50 μm-carbonyl cyanide m-chlorophenylhydrazone (CCCP) at alkaline pH, whereas other species of this genus were inhibited 80–90% under the same conditions. Growth of strain 184 at alkaline pH was inhibited 66% by 50 μm-monensin and 100% by monensin plus CCCP. The majority of the ATPase activity on everted membrane vesicles prepared from strain 184 grown at alkaline pH was sensitive to azide and N,N′-dicyclohexylcarbodiimide (DCCD), but ATPase was less sensitive to these inhibitors when Na+ was present. The respiratory activity of strain 184 was neither dependent on nor activated by Na+ and was unaffected by the antagonistic Na+-analogues K+ and Rb+. A Na -dependent, 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) supersensitive NADH oxidase was not present in strain 184. Na+ was required in the growth medium to promote optimal cell yields. Limiting the amount of Na+ available caused a lag phase in which cell viability was lost. Viability was maintained by the addition of Li+ or Mg2+ to Na+-limited medium, but only Li+ appeared to promote growth. K+ appeared to be a competitive inhibitor of a Na+/Li+ site required for cell growth. Rb+ was a more complex competitor and affected the final yield and the growth rate of strain 184. Rb+-tolerant mutants of strain 184 were selected and the majority of these were found to be defective in the Na+-dependent acid excretion normally observed with A. salinestris. Analysis of an acid over-producing strain showed that Rb+ appeared to be an uncompetitive inhibitor of Na+-dependent growth and in competition with Na+ as a promoter of acidification.
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Isolation and characterization of Pseudomonas putida mutants affected in arginine, ornithine and citrulline catabolism: function of the arginine oxidase and arginine succinyltransferase pathways
C. Tricot, V. STALON and C. LEGRAINPseudomonas putida mutants impaired in the utilization of arginine are affected in either the arginine succinyltransferase pathway, the arginine oxidase route, or both. However, mutants affected in one of the pathways still grow on arginine as sole carbon source. Analysis of the products excreted by both wild-type and mutant strains suggests that arginine is mainly channelled by the oxidase route. Proline non-utilizing mutants are also affected in ornithine utilization, confirming the role of proline as an intermediate in ornithine catabolism. Mutants affected in ornithine cyclodeaminase activity still grow on proline and become unable to use ornithine. Both proline non-utilizing mutants and ornithine-cyclodeaminase-minus mutants are unable to use citrulline. These results, together with induction of ornithine cyclodeaminase when wild-type P. putida is grown on citrulline, indicate that utilization of citrulline as a carbon source proceeds via proline with ornithine as an intermediate. Thus in P. putida, the aerobic catabolism of arginine on the one hand and citrulline and ornithine on the other proceed by quite different metabolic segments.
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- Plant-Microbe Interactions
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Metabolism of glutamate and aspartate in bacteroids isolated from soybean root nodules
More LessBacteroids were isolated anaerobically from root nodules of soybean (Glycine max L. Merr.) and supplied with 14C- or 15N-labelled glutamate or aspartate under microaerobic conditions where bacteroids retained a high activity of N2 fixation. Glutamate and aspartate significantly stimulated N2 fixation (C2H2 reduction) and respiration (CO2 evolution) of isolated bacteroids. They were both utilized as substrates for bacteroid respiration and their nitrogen was rapidly released from the bacteroids as NH3. Amino-oxyacetate (AOA) strongly inhibited glutamate utilization, but aspartate utilization was only slightly affected by AOA. Substantial activity of aspartate ammonia-lyase (aspartase) was detected in the cell-free extract of bacteroids. These results suggest that the major pathway of glutamate utilization in bacteroids is transamination to form aspartate followed by direct deamination of aspartate by aspartase. It is also suggested that the 4-aminobutyrate pathway (GABA-shunt) is partly responsible for the catabolism of glutamate by soybean nodule bacteroids. The results are discussed in terms of the possible relationships between C4-dicarboxylates and glutamate in carbon and nitrogen metabolism in soybean nodule bacteroids.
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- Systematics
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Epidemiological analysis of a methicillin-resistant Staphylococcus aureus outbreak using restriction fragment length polymorphisms of genomic DNA
More LessThe genomic DNA of 58 isolates of methicillin-resistant Staphylococcus aureus (MRSA) obtained during an infection outbreak at two major Canberra hospitals was analysed for restriction fragment length polymorphism (RFLP) by digestion with the endonuclease SmaI and resolution of the fragments by pulsed-field gel electrophoresis. Based on the fraction of common fragments generated by the endonuclease, DNA similarities among the isolates were estimated. Distance matrix analysis showed that the MRSA isolates could be divided into two major clusters (RFLP types I and II) and one minor one (type 46). A fourth group of miscellaneous isolates was found to be heterogeneous in terms of DNA sequence similarity. The epidemiological data indicated that RFLP type I was most common in the intensive care units in the two hospitals, with particular subtypes of RFLP type I concentrated in individual units. RFLP type II and the miscellaneous group were more generally distributed. Type 46 isolates appear to be related to a group which was present in epidemics in Melbourne hospitals in the early 1980s. Using the standard phage set, the RFLP type I group was largely untypable. However, type II isolates were all phage typable, with a shared susceptibility to phages 29/85/95/90; type 46 isolates had a shared susceptibility to phages 85/90. The miscellaneous isolates were of variable phage types.
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