- Volume 111, Issue 2, 1979

Halobacterium cutirubrum, an extremely halophilic bacterium, lacks a normal peptidoglycan cell wall. Nevertheless, bacitracin, a known inhibitor of peptidoglycan synthesis, has been shown to completely suppress the growth of this organism (Mescher & Strominger, 1975) In an attempt to clarify this observation, the effect of bacitracin on DNA, RNA, protein and lipid biosynthesis in H. cutirubrum was monitored. Lipid carbohydrate and lipid phosphate were the most affected. These observations were confirmed by uptake studies of [14glycerol into the membrane lipids. The possibility that bacitracin acts against H. cutirubrum through inhibition of dephosphorylation of the isoprenoid pyrophosphate precursors of this organism's diether lipids is discussed.

In a haploid cell culture of Saccharomycodes ludwigii, sporogenous diploid cells homozygous for all the genetic markers including mating type were effectively discriminated on a dye plate consisting of 15 μg trypan blue and 10 μg phloxine B ml−l in synthetic nutrient medium. The diploid cells appeared at a frequency of about 5 × 103 that of the haploid cells and those tested bore the same alleles as the original haploid cells. The low frequency of prototrophic colonies (approximately 1 × 10−8 in a mass mating culture of two different haploid strains of identical mating type and complementary auxotrophic traits, and the morphological changes observed during the suspected diploidizing process suggest that the homozygous diploids originate by direct diploidization in haploid cells. From the diploid cells homozygous for mating type, triploid and tetraploid cells were easily constructed. As with diploid cells, tetrad data from these polyploid clones suggested the absence of crossing-over at meiosis.

Intrastrand self-complementary sequences have been isolated from the DNA of Bacillus subtilis by hydroxyapatite (HA) chromatography following thermal renaturation of strands separated by chromatography on methylated albumin-kieselguhr (MAK). The intrastrand structures derived from the MAK H strand (HA HII) were biologically active showing transforming activity for a wide variety of markers, as well as hybridization to both pulse-labelled and ribosomal RNA. Removal of regions of single-strand DNA with S1 nuclease did not significantly alter the biological activity of the self-annealed molecules. The overall efficiency of transformation and hybridization of the intrastrand self-annealing DNA was low suggesting that many sequences in the population are neither active in transformation to prototrophy nor transcribed into RNA.

Mutants that at one time were thought to be specifically defective in taxis toward aspartate and related amino acids (tar mutants) or specifically defective in taxis toward serine and related amino acids (tsr mutants) are now shown to be pleiotropic in their defects. The tar mutants also lack taxis toward maltose and away from Co2+ and Ni2+. The tsr mutants are altered in their response to a variety of repellents. Double mutants (tar tsr) fail in nearly all chemotactic responses. The tar and tsr mutants provide evidence for two complementary, converging pathways of information flow: certain chemoreceptors feed information into the tar pathway and others into the tsr pathway. The tar and tsr products have been shown to be two different sets of methylated proteins.

Preparations of protoplasm, nuclei and mitochondria were introduced into living Verticillium cells. Most of the hyphae of auxotrophs injected with protoplasm obtained from complementary auxotrophs survived micro-injection (80%). Of these, 21% formed ‘artificial’ heterokaryons from which both parental genotypes were recovered by conidial analysis. Reciprocal injections of protoplasm were made between auxotrophic strains with darkly-pigmented resting structures (Hyl+) and hyaline strains lacking resting structures (Hyl+. Conidial analysis resulted in the recovery of all auxotrophic markers. The majority of phenotypes having darkly-pigmented resting structures were of the Hyl+ type (92 to 96%), but a few were partially or completely hyaline. Reciprocal injections between Hyl+ or Hyl+ 'sooty’ (Sot) strains and Hyl or Hyl Sot+ strains (hyaline strains induced by ultraviolet-irradiation from Sot auxotrophs) revealed the cytoplasmic inheritance of the Hyl marker in contrast to the nuclear inheritance of the Sot marker. The survival of recipient cells injected with preparations of nuclei was 30 to 40% on complete medium but conidial analyses indicated that heterokaryons were not produced. The survival of hyphae injected with mitochondrial preparations was 40 to 45%, and these colonies showed considerable morphological instability which was overcome during 2 to 3 weeks incubation. Conidial analyses of Hyl recipients injected with Hyl+ mitochondrial preparations resulted in 13 to 21% darkly-pigmented variants. These results suggest that one or more factors controlling darkly-pigmented structures were present in the mitochondrial preparations from the Hyl+ donor cytoplasm.

Minicells segregated from Escherichia coli x925 carrying a drug-resistance plasmid were separated from nucleated cells by differential centrifugation and purified by rate-zonal centrifugation in sucrose gradients. Minicells purified in this way were capable of donating the plasmid to nucleated cells. They also incorporated thymidine, uridine and methionine into macromolecules. Methods are described for purification of plasmid-containing mini-cells on a scale large enough to allow isolation of DNA, DNA polymerase and RNA polymerase in sufficient quantities for studies of enzymes involved in replication and transcription of plasmid DNA.

Pseudomonas putida, which can use lysine as a carbon and energy source, has two active transport systems for lysine – a high affinity system, inhibited by arginine and ornithine, and a low affinity system. Lysine-grown organisms had a higher transport activity than succinate-grown organisms but this higher activity was probably not the result of induction by lysine. Pseudomonas acidovorans, a species unable to degrade lysine, has a single high affinity active transport system specific for lysine; this transport system has a physiological role in the maintenance of the internal concentration of free lysine.

Methyl viologen (10 μM) markedly inhibited acetylene reduction (nitrogen fixation) by old but not young cultures of Gloeocapsa sp. LB795, apparently by causing the alga to produce H2O2. H2O2 inhibited acetylene reduction when added to cultures at concentrations greater than 10 μM. As catalase (EC 1.11.1.6) is not present in Gloeocapsa sp. LB795, H2O2 is probably removed by a non-enzymic reaction with ascorbate and also by an enzyme-catalysed reaction with glutathione. Enzymes catalysing the decomposition of H2O2, were most active in young cells which were therefore better able than old cells to metabolize H2O2 produced in the presence of methyl viologen. The maximum activities of these enzymes coincided with maximum nitrogenase activity during the growth of batch cultures, and may provide a protective mechanism for nitrogenase.

Populations of Saccharomyces cerevisiae with plasma membranes enriched in phosphatidyl-ethanolamine lost viability and released cations at a greater rate when suspended in buffered sodium dodecyl sulphate than did populations with membranes enriched in phosphatidylcholine. However, when suspended in buffered sorbitol (1.2 M) containing sodium dodecyl sulphate, sphaeroplasts from organisms with phosphatidylcholine-enriched membranes released cations faster than did sphaeroplasts with membranes enriched in phosphatidylethanolamine. Liposomes prepared in potassium chloride from mixtures of phospholipids from organisms enriched in phosphatidylcholine or phosphatidylethanol-amine lost potassium ions at the same rate when suspensions were supplemented with sodium dodecyl sulphate. Organisms enriched in phosphatidylcholine or phosphatidyl-ethanolamine did not differ in appearance in scanning electron micrographs, in electrophoretic mobilities over the pH range 2 to 9, in the ease with which they were converted into sphaero-plasts by β-glucanase, or in permeability to a range of polyethylene glycols. Walls from phosphatidylcholine- and phosphatidylethanolamine-enriched organisms had the same contents of β-glucans, α-mannan and protein.

The transition from the fermentative mode of growth on glucose to the oxidative mode of growth on ethanol by Saccharomyces cerevisiae is accompanied by many changes collectively known as catabolite derepression. Catabolite derepression caused a progressive increase in the total content of cellular sterols, amounting to a fourfold increase after 8 h; the additional sterols existed almost entirely as esters of monoenoic fatty acids. The increase in sterol content could be explained by the large increase in the activity of β-hydroxymethylglutaryl-CoA reductase (EC 1.1.1.34), but a secondary factor in sterol accumulation may have been the slower rate of cell growth during ethanol oxidation. Subsequent anaerobic growth of derepressed cells resulted in the hydrolysis of steryl esters to free sterols, and a progressive depletion of cellular sterol content.

Incorporation of [3H]leucine in the bacteria of 18 to 48 h-old colonies of Pseudomonas aeruginosa, Pseudornonas putida, Bacillus thuringiensis, Staphylococcus aureus and Escherichia coli enabled the localization of bacterial multiplication sites by means of autoradiography of sagittal sections. In colonies where fast diameter expansion occurred, all the bacteria from the peripheral corona contributed to peripheral growth; in colonies where the expansion was slower, the growth rate of the bacteria in this region was heterogeneous. Besides this peripheral growth, a central region of bacterial multiplication was always found, but with variable localization and extension. In aerobic species, such as P. aeruginosa and P. putida, the central growth site was limited to the zone of oxygen penetration into the bacterial mass. However, in facultatively anaerobic species, bacterial multiplication depended on nutrient supply. For 48 h-old colonies of S. aureus, a more complex localization of growth seemed to be affected simultaneously by nutrient penetration and accumulation of toxic substances.

Tyrosine-sulphate sulphohydrolase was synthesized by actively growing cultures and by resting suspensions of Comamonas terrigena. Polyacrylamide gel electrophoresis revealed that bacteria exposed to exogenous tyrosine sulphate synthesized two forms of this enzyme. Only one of these was detected in extracts of bacteria which had not been exposed to the ester. Both enzymes differed from aryl-sulphate sulphohydrolase in electrophoretic mobility. The synthesis of the induced form was regulated by tyrosine as well as by most intermediates of SO4 2− assimilation. L-Cysteine had no effect on the synthesis of the induced form but stimulated in vitro activity by about 85%. These effectors did not influence either the synthesis or the activity of the other electrophoretic form which was tentatively considered as being constitutive.