- Volume 116, Issue 1, 1980

Extracellular nuclease activity in Staphylococcus aureus was enhanced about fourfold by 1% (w/v) NaCl, KCl, CsCl or LiCl. The pH and concentration of Ca2+ for optimum activity varied with the NaCl concentration; with an increased NaCl concentration, a higher Ca2+ concentration and a lower pH were required. V max, but not K max, varied with the concentration of NaCl. The addition of 3% (w/v) NaCl to growing cultures of S. aureus increased nuclease production fivefold.

The effect of carbon growth substrate on the level of ribulose-1,5-bisphosphate (RuBP) carboxylase in crude cell-free extracts of Rhodomicrobium vannielii (RM5) was investigated. The highest specific activity followed growth with sodium hydrogen malate as carbon source. Cells grown autotrophically or on the reduced carbon substrates, butyrate and ethanol, gave extracts of lower activity. RuBP carboxylase was purified from R. vannielii (RM5) grown on a mixture of pyruvate and malate as carbon source. The enzyme was homogeneous as judged by electrophoresis on polyacrylamide gels of several acrylamide concentrations and had an apparent molecular weight of 430000 as measured by gel filtration. Sodium dodecyl sulphate polyacrylamide gel electrophoresis suggested the presence of three subunit types of molecular weights 56200, 53300 and 15700. Mg2+ was required for maximum enzyme activity although this could be completely replaced by Ni2+ and, to a lesser extent, by Mn2+ or Co2+. As with other high molecular weight RuBP carboxylases, the enzyme was sensitive to inhibition by 6-phosphogluconate.

The rates of synthesis and breakdown of phospholipids in growing cultures of Nocardia polychromogenes were investigated by means of a pulse-labelling technique using 32PO4 3−. The results indicated that phospholipids were broken down and phosphatidylinositol mannosides had a high turnover rate. The other two main components, cardiolipin and phosphatidylethanolamine, had a relatively low rate of turnover.

Cell-free extracts were prepared from sphaeroplasts of aerobically and anaerobically grown, glucose-derepressed and glucose-repressed Saccharomyces carlsbergensis. The activities of the enzymes of the tricarboxylic acid cycle and of related enzymes were measured, together with their distributions after differential centrifugation. Glucose repression lowered activities of enzymes of the tricarboxylic acid cycle by 60 to 89 % under aerobic conditions. Activities were still further decreased under anaerobic glucose-derepressed conditions. 2-Oxoglutarate decarboxylase activity was not detected after anaerobic growth; malate synthase and isocitrate lyase activities were not detected in organisms grown either aerobically or anaerobically. Glucose had little effect on the activities of tricarboxylic acid cycle enzymes under anaerobic conditions, with the exception of citrate synthase whose activity increased under anaerobic glucose-repressed conditions. Differential centrifugation of cell-free extracts showed different distribution patterns of the enzymes under the four growth conditions. The distribution patterns and enzyme activities reflected the differential effects of glucose repression and/or anaerobiosis.

Cell envelope fractions of Moraxella nonliquefaciens were isolated by a slight modification of Osborn’s method. Two main membrane fractions were characterized chemically and morphologically. The density of the fraction containing cytoplasmic membrane material was 1·17 to 1·18 g cm−3 compared with 1·24 to 1·27 g cm−3 for the outer membrane fraction. Lipopolysaccharide was detected almost exclusively in the outer membrane fraction and sodium dodecyl sulphate polyacrylamide gel electrophoresis of this fraction revealed one dominant protein band with an apparent molecular weight of 45000. Cross-contamination of the fractions was estimated to be about 10%, as calculated on the basis of the lipopolysaccharide fatty acid 3-hydroxydodecanoic acid and on the relative activities of d-lactate dehydrogenase and succinate dehydrogenase.

An unspecialized strain of Rhizoctonia solani (FC895) produced in vitro seven isoenzymes (four major and three minor peaks) which were separated by isoelectric focusing over a narrow pH range. The same isoenzyme pattern was detected when hypocotyls of cauliflower seedlings inoculated with the fungus were used as the enzyme source. A specialized strain (FC1241) produced two isoenzymes (one major and one minor peak). The four major isoenzymes of FC895 and the major isoenzyme of FC1241 had the same pH optimum and mode of action on sodium polypectate.

Mycoplasma-like organisms (MLO) associated with lethal yellowing disease of coconut palms exhibited a range of different morphologies even within the same sieve element. The morphologies were revealed by graphic reconstruction from ribbons of serial ultrathin sections containing 45 sections. Five different morphotypes were recognized amongst the 120 organisms reconstructed. MLO could be saccate, erythrocyte-like, cylindrical, monili-form or filiform. The different morphotypes showed differences in linear dimensions apart from their particular morphological characteristics. Saccate MLO were generally the smallest, the maximum length being 1 μm, whilst the filiform organisms could be more than 16 μm long. The morphologies of the MLO were analogous to those now recognized as usual for Mycoplasma and Acholeplasma species in vitro during the later stages of development. Comparison of the individual profile shapes used in the reconstructions with those of MLO from other diseased plants suggest that many previous interpretations of MLO morphology may have been too simple. With the insight provided by the understanding of the relationship between profile shape and three-dimensional form, the ultrastructure of individual profiles is shown to be an important indicator of form.

Antigenic components at the outer surface membranes of seven serotypes of Mycoplasma hominis were analysed by the mycoplasmacidal reaction and the agglutination during growth reaction. Antibody absorbing capacities of the mycoplasma cells were compared with the absorbing capacities of membranes. It was shown that serologically active membrane antigens were mainly heat-labile proteins. No major antigens common to all seven serotypes were detected and each strain had its own specific antigens at the cell surface. Results of analysis indicate that there is a complex antigenic structure exposed in M. hominis and that 7 to 14 cross-reacting antigens may be present at the outer surface in the different serotypes examined. Additional cross-reacting antigens, presumably inner membrane in origin and not exposed at the cell surface, were also demonstrable.

Isolated colonies of Clostridium acetobutylicum, grown anaerobically for 2 d before exposure to aerobic conditions, developed into unique elongated fruiting body-like structures which reached a height of > 10 mm. The elongated structures were surrounded by an extracellular fibrous substance which formed a tough pliable sheath on exposure to air. Vegetative and sporulating cells were restricted to the basal region of the structure; the trunk region contained mainly free spores and a small number of granulated or lysed cells. Colonies maintained under anaerobic conditions did not produce the elongated macroscopic structures.

Lipopolysaccharides, extracted by phenol/chloroform/petroleum ether, from two rough mutants of Salmonella typhimurium of class rfaH were studied by passive haemagglutination inhibition and by methylation analysis. The structural and immunochemical analyses showed that (i) formation of the galactose I unit of the core is defective, but the defect is not complete, and (ii) of those core chains which do receive the galactose I residue, many are not continued to form complete core, but instead terminate at intermediate points. This suggests that the rfaH gene, though involved in formation of the galactose I unit, is not the structural gene for the galactosyltransferase which adds this unit. The rfaH product may be a positive regulator for several rfa genes specifying glycosyltransferases, or it may be a protein needed for the efficient action of several such transferases.

A naturally occurring transmissible plasmid, designated pAVl, has been isolated in Acinetobacter calcoaceticus. It specifies resistance to sulphonamides and is capable of mobilizing two non-transmissible resistance determinants for tetracycline and neomycin, respectively, within strains of A. calcoaceticus. It is incompatible with the P class R factors RP4 and R751 in A. calcoaceticus. On this basis we conclude that pAVl is a member of the P incompatibility group. However, unlike most other P group R factors, pAVl is not transmissible to strains of Escherichia coli, Pseudomonas aeruginosa, Klebsiella or Proteus mirabilis.

Spores of Bacillus cereus mutants selected for slow response to germinants and sensitivity to lysozyme were found to be deficient in coat, but were heat-resistant and contained the same quantity of dipicolinic acid as the wild-type. While the average coat protein content of the spores was 25% of the wild-type, many spores were coatless with large whorls of coat deposited in the cytoplasm. These coat deposits were isolated in Renografin gradients and found to cross-react immunologically with wild-type coat. The proteins extractable from these deposits were virtually identical to those extracted from wild-type spores. The morphology of the coat deposits was very similar to coats of wild-type spores, but with a deficiency of the outermost cross-patched layer. The sites of formation and deposition were altered. Since the mutant reverted to a phenotype identical to the parental strain with a frequency consistent with an initial point mutation, apparently a single defect resulted in alteration of the deposition of the spore coats on to the outer forespore membrane. Despite this defect, mutant cells were able to synthesize and process spore coat precursors into an array of morphological layers very similar to the wild-type. There are apparently distinct morphogenetic pathways for the formation of the spore body and coat layers.

Triethyllead and tripropyllead cations affected growth, energy metabolism and ion transport in Escherichia coli K12. The tripropyllead compound was more liposoluble than the triethyl analogue and was also more effective in inhibiting cell growth and the oxygen uptake of both intact cells and membrane particles. Triethyllead acetate (5 μ m) inhibited growth on non-fermentable carbon sources, such as glycerol and succinate, more markedly than on glucose. At higher concentrations, triethyllead caused significant inhibition of respiration rates of intact cells; the concentration giving 50 % inhibition was 60 μ m for glycerol-grown cells and 158 m for glucose-grown cells. Oxidation of succinate by membrane particles was less sensitive to inhibition by the tripropyl- or triethyllead compounds than were the oxidations of dl-lactate or NADH. Triethyllead acetate [1·9 mol (mg membrane protein)−1] inhibited the reduction by NADH of cytochromes; evidence for more than one site of inhibition in the respiratory chain was obtained. Membrane-bound ATPase activity was strongly inhibited by triethyllead acetate in the absence or presence of Cl−. The concentration of inhibitor giving 50 % inhibition [0·02 μmol (mg membrane protein)−1] was about two orders of magnitude lower than that required for 50 % inhibition of substrate oxidation rates in membranes. Triethyllead acetate (1 μ m) induced swelling of spheroplasts in iso-osmotic solutions of either NH4Cl or NH4Br, presumably as a result of the mediation by the organolead compound of Cl−/OH− and Br−/OH− antiports across the cytoplasmic membrane. Similar exchanges of OH− for F−, NO3 − or SO4 2− or the uniport of H+ could not be demonstrated. Comparisons are drawn between the effects of trialkyllead compounds and those of the more widely studied trialkyltin compounds.

An assay was developed to measure the rate of exopolysaccharide formation by washed non-proliferating suspensions of Pseudomonas NCIB 11264 grown under a range of controlled environmental conditions. The specific activities of certain of the enzymes involved in the formation of the sugar nucleotide precursors of polysaccharide biosynthesis were also measured in steady-state populations. The level of enzyme activity did not reflect either the amount of extracellular polysaccharide produced or the rate at which glucose was incorporated into exopolysaccharide, which was dependent on medium composition, environmental factors, and the rate and stage of growth of the organism. The specific activities were not affected by either cultural conditions or the separate addition of actinomycin D and chloramphenicol, indicating a constitutive biosynthetic system.

Comparison of the pools of glutamic acid and glutamine and of the specific activities of glutamine synthetase and glutamate dehydrogenases in sporulating a/α and non-sporulating α/α cells of Saccharomyces cerevisiae revealed a difference in their nitrogen metabolism. Glutamine synthetase and glutamine appeared to be necessary for the sporulation process, glutamine playing, at least, a catabolic role. However, exogenous glutamine as well as ammonia inhibited sporulation while glutamic acid did not. Glutamine seemed to act through its amino group. Both inhibitors had at least two sites of action, one effective early in sporulation and related to DNA synthesis and the other acting later and not related to it.