- Volume 90, Issue 2, 1975

SUMMARY: Chitin synthase of Mortierella vinacea was present in the ‘microsomal’ fraction (100000 g precipitate), the ‘cell-wall’ fraction (2000 g precipitate) and the ‘mitochondrial’ fraction (10000 g precipitate). The properties of the ‘microsomal’ enzyme were investigated. The pH optimum was between 5·8 and 6·2, and the temperature optimum was between 31 and 33 °C. The K m for UDP N -acetyl-d-glucosamine was 1·8 mm. The enzyme was stimulated by Mg2+ and a slight stimulation was also effected by N-acetyl-d-glucosamine. Soluble chitodextrins were inhibitory. A pH-dependent, heat-stable inhibitor of chitin synthase activity was present in the soluble cytoplasm from the mycelium. The effects of aeration and glucose concentration on enzyme production in growing cultures were also investigated; maximum specific activity of chitin synthase was associated with the cessation of exponential growth.

SUMMARY: Four strains of Desulfovibrio each, excreted pyruvate to a constant level during growth; it was re-absorbed when the substrate (lactate) was exhausted. Malate, succinate, fumarate and malonate also accumulated during growth.

One of the strains (Hildenborough) excreted α-ketoglutarate as well as pyruvate when incubated in nitrogen-free medium; the former was re-absorbed on addition of NH4Cl. In a low-lactate nitrogen-free medium, strain Hildenborough rapidly re-absorbed the pyruvate initially excreted, but did not re-absorb the α-keto-glutarate. Arsenite (1 mm) prevented the accumulation of α-ketoglutarate; 1 mm-malonate did not affect the accumulation of keto acids.

Isocitrate dehydrogenase activity (NAD-specific) in all strains was lower than NADP-specific glutamate dehydrogenase activity. α-Ketoglutarate dehydrogenase could not be detected in any strain. NADPH oxidase activity was demonstrated.

This and previous work indicate that a tricarboxylic acid pathway from citrate to α-ketoglutarate exists in Desulfovibrio spp., and that succinate can be synthesized via malate and fumarate; however, an intact tricarboxylic acid cycle is evidently not present. The findings are compared with observations on biosynthetic pathways in Clostridia, obligate lithotrophs, phototrophs, and methylotrophs, and various facultative bacteria.

SUMMARY: A study has been made of the levels of 6-methylaminopurine and 5-methyl-cytosine in the DNA of Bacillus subtilis during thymine deprivation. While DNA synthesis was inhibited by thymine deprivation, DNA methylation continued. Base analysis indicated that this aberrant methylation involved an increase solely in the amount of 5-methylcytosine. These aberrant 5-methylcytosine residues were removed from the DNA during continued growth of bacteria in medium lacking thymine. In contrast, 5-methylcytosine residues synthesized during normal growth were relatively unaffected by thymine deprivation. The results are interpreted to indicate that the extensive DNA damage which occurs during thymine deprivation is due in part to exonuclease digestion of regions of DNA containing aberrant 5-methylcytosine residues.

SUMMARY: A new method is described for the efficient conversion of Schizosaccharomyces pombe cells into protoplasts. The following parameters of guanine uptake determined in whole cells were unchanged in protoplasts: K m value, requirement for an energy source, sensitivity to competitive inhibitors, pH optimum, as well as the typical variation of the initial velocity of uptake observed during the growth phase.

SUMMARY: Mitotic mapping in the cellular slime mould Dictyostelium discoideum was investigated by analysing the gene order and map distances of four genetic markers on linkage group II: whi, acrA, tsgD (previously reported) and a new spore shape marker sprB. The previously suggested gene order has been revised to centromere, whi, acrA, tsgD/sprB, on the basis of the analysis of four different diploids. One class of diploid which was previously thought to arise by mitotic crossing-over between tsgD and acrA probably arose by mutation at the acrA locus or by reversion of the tsgD locus followed by mitotic crossing-over at another interval. These and other problems associated with mitotic mapping are discussed. Evidence is presented that for a particular class of cross-over diploid (white, temperature-resistant, methanol-resistant) an origin from a single cross-over event is likely rather than a multiple cross-over origin. It is suggested that multiple mitotic cross-overs, on the same arm of a chromosome, are rare in D. discoideum.

SUMMARY: When yeast cells were incubated for 4 to 8 h in yeast extract-peptone-glucose medium, pH 6, containing 8 mm-manganese, and then plated on selective media, there was a strong induction of antibiotic-resistant mutations. Indirect evidence suggests that practically all resistant mutants selected were of independent origin. The analysis of manganese-induced resistant mutants showed that most were extranuclear, while those tested showed recombination with known mitochondrial markers.

Our results suggest that manganese can be considered as a mutagen which specifically induces mitochondrial mutations in Saccharomyces cerevisiae.

SUMMARY: The isolation of Escherichia coli chromosomal mutants that increased, the level of resistance of a partially tetracycline-sensitive mutant of r100-1 is described. Plasmid-less derivatives of these moderately resistant mutants were phenotypically similar to the cmlB mutants described by Reeve (1966 , 1968) , and also mapped in the same region. The level of intrinsic resistance to both chloramphenicol and tetracycline was increased about twofold. Also, the levels of R factor-determined resistance to these drugs were increased by this host mutation and tetracycline resistance was expressed constitutively. A cmlB mutant accumulated tetracycline at a threefold] lower rate than the wild-type strain, and it is proposed that the mutants have an altered permeability to the drugs and that this acts syner-gistically with the products of the R factor chloramphenicol and tetracycline resistance genes.

SUMMARY: The isolation and properties of a mutant of Escherichia coli k12 that is totally unable to take up and utilize gluconate are described. Genetical analysis shows this phenotype to be associated with two lesions. One phenotype, designated GntM−, is the result of a mutation in a gene co-transducible with malA; the other, designated GntS−, is the result of a mutation in a gene (gntS) co-transducible with fdp. The GntS−-phenotype differs little from that of wild-type cells, but GntM− GntS− organisms grow on gluconate only after a prolonged lag and form a gluconate uptake system that is strongly repressed by pyruvate. Moreover, such GntM− mutants readily give rise to further mutants that form a gluconate uptake system, gluconate kinase and 6-phosphogluconate dehydratase constitutively; in partial diploids, this constitutivity is recessive to the inducible character. It is postulated that the GntM− phenotype is due to malfunction of a negative control gene gntR, and that gntS + specifies the activity of a gluconate uptake system.

SUMMARY: To test the hypothesis that chloramphenicol production in Streptomyces venezuelae depends on the presence of a plasmid, mapping analysis was carried out by using eight markers in addition to chloramphenicol production and melanoid pigment formation. The sequence of the eight markers was determined on a circular linkage map as follows: -his-ade-str-leu-lys-met-ilv-pro-(his-). This sequence resulted in the frequency of quadruple crossover (q.c.o.) recombinants having the lowest value, 3·2 to 4·9 %. However, the character of chloramphenicol non-production, which was obtained by incubating mycelia with acriflavin, was not located on this linkage map; more than 15 % q.c.o. recombinants would have been required to explain the results. From these results and other tests, it is concluded that chloramphenicol production is controlled by a plasmid. This plasmid appeared to be non-transferable in conjugation.

SUMMARY: The particulate fraction of disrupted Chromobacterium violaceum grown under cyanide-evolving conditions was unable to oxidize ascorbate plus N,N,N′,N′-tetra-methyl-p-phenylenediamine (TMPD), but oxidized NADH and succinate by a linear respiratory pathway which was very resistant to inhibition by cyanide. When the bacteria were grown under conditions where little cyanide evolution occurred, particulate fractions developed the ability to oxidize ascorbate-TMPD by a pathway highly sensitive to cyanide inhibition; respiratory activity with NADH and succinate proceeded via both the cyanide-sensitive and -resistant pathways. Studies with respiratory inhibitors, and the cytochrome compositions of the fractions derived from cultures grown under both conditions, are presented. A soluble, carbon monoxide-binding cytochrome c was found, and this appears similar to those found recently in Beneckea natriegens, methylotrophic bacteria and the marine pseudomonad B16.

SUMMARY: The incorporation of [U-14C] glucose, -maltose and -maltotriose into the polysaccharides of a brewer’s yeast, Saccharomyces cerevisiae (NCYC240), in synchronous culture was followed during the fermentation of brewer’s wort. The synchrony was induced by repeated subculture in brewer’s wort at regular intervals, using cells skimmed from the surface of previous brews. The 14C-labelled sugars were assimilated simultaneously by the yeast and small but significant amounts of each incorporated into cellular polysaccharides. The low molecular weight acid-soluble fraction exhibited a cyclic fluctuation attributable to changes in enzymic activity associated with the synchronous behaviour of the yeast. Cyclic changes were also observed in the incorporation of labelled sugars into the mannan fraction of the walls, the uptake rising to successive maxima at the same stage of each budding cycle, followed by a rapid loss as the next cycle began. The incorporation of 14C into glycogen exhibited a stepwise character, in which brief periods of glycogen buildup alternated with longer periods of stability. The periods of glycogen biosynthesis coincided with the periods of most intensive budding. Incorporation of 14C into the glucan of the walls was a continuous process and could not be correlated with any particular phase of the cell cycle. Incorporation of 14C into glycogen and glucan was stable.

SUMMARY: The unidirectional K+ fluxes across the mycelial surface of Neocosmospora vasinfecta were determined using 42K. Influx was mediated by at least two kinetically distinct systems, one having an apparent Km of 6·5 μ-equiv. K+/l and the other of about 1·0 m-equiv. K+/1. The Vmax for both systems was in the range 18 to 22 μ-equiv. K+/100 mg mycelial dry matter/h (1· to 1·2 m-equiv. K+/1 cell-water/min). Influx was strongly inhibited by 2,4-dinitrophenol, sodium azide, sodium arsenate and anaerobiosis. K+ efflux was dependent on the external K+concentration and ranged from 3 to 10% of mycelial K+/h. The maximum efflux rate was always considerably less than the initial influx rate for the K+concentrations examined. During incubation in dilute KCl solutions, K+ influx decreased to a value approaching the K+ efflux rate. It is considered that equilibrium with external K+ is attained primarily by the regulation of K+influx, and that this may be the principal mechanism controlling cytoplasmic K+ levels.

Adsorption of K+was also observed throughout the K+concentration range examined and can be attributed to two distinct K+-binding entities at the mycelial surface, half-saturating at approximately 0·1 mm- and 4·4 mm-KCl respectively.