- Volume 98, Issue 1, 1977

Summary: Compact-colony forming active substance (CCFAS), the material responsible for the compact colonies of Staphylococcus aureus observed in serum soft agar, was found to be an alkaline-stable, associated polysaccharide containing galactose, N-acetylglucosamine, ribitol, phosphorus and a small quantity of alanine. This substance, when extracted from strains unable to produce protein A and clumping factor, was able to absorb the serum-reacting factor whereas a teichoic acid preparation of one strain could not. The formation of CCFAS was unaffected by the age of the cells, whereas when staphylococci were cultured at alkaline pH, young cells produced more clumping factor than old ones. Both fibrinogen and its degradation products were capable of inducing compact colonies in a strain of S. aureus. The ability of human sera to interact in compact-colony formation was independent of the immunoglobulin content. Thus neither protein A, clumping factor, nor teichoic acid participate in the CCFAS reaction.

Summary: The value of the adenylate energy charge, i.e. ([ATP] + [ADP])/([ATP] + [ADP] + [AMP]), during batch culture of Beneckea natriegens remained relatively constant during the exponential and early stationary phases of the growth cycle. During exponential growth the intracellular ATP content remained constant, the amount of ATP in the culture increasing proportionally with growth; these conditions were unaltered during growth in the presence of added cyclic AMP. On cessation of growth, significant variation in bacterial ATP content was observed depending on whether growth of the cultures terminated due to exhaustion of carbon or nitrogen from the medium, and on the presence or absence of added cyclic AMP.

SUMMARY: Solid media containing carboxymethylcellulose (CMC) were developed to detect Cx cellulase-producing micro-organisms. Hydrolysis of CMC was seen as a clear zone around colonies after flooding plates with 1% aqueous hexadecyltrimethyl-ammonium bromide. Tests with ten bacterial and four fungal species showed that the degree of substitution (DS) of the CMC affects both growth and enzyme production. Most of the organisms produced more Cx cellulase on CMC with a DS of 0·9, but CMC with a DS of 0·4 was better for one fungus. A qualitative measure of cellulase production may be obtained by calculating the ratio of zone size to colony diameter. Solid media containing CMC provided a more rapid assay of Cx cellulase production than a medium containing native cellulose.

Summary: Induction of ethanolamine ammonia-lyase formation in Escherichia coli required both ethanolamine and vitamin B12, and was gratuitous during growth on glycerol. Ethanolamine analogues inhibited enzyme activity and inhibited growth with ethanolamine as the nitrogen source, but did not act as inducers. Enzyme formation was more rapid when ethanolamine was added to cultures containing vitamin B12 rather than the reverse. Enzyme formation was subject to catabolite repression, glucose and acetate being particularly effective. Chloramphenicol, 1-aminopropan-2-ol and 1,3-diaminopropan-2-ol prevented enzyme induction. Ethanolamine ammonia-lyase, resolved from its cobamide coenzyme, was purified 35-fold. The apoenzyme was stable for several days in the presence of ethanolamine, dithio-threitol, glycerol and K+ ions.

Enzyme formation therefore requires both substrate and cobamide coenzyme to be present simultaneously as inducers.

Summary: The growth rate of Saccharomyces cerevisiae was increased by adding a mixture of amino acids to cultures containing proline as the sole nitrogen source. The transition from balanced growth in the basal medium (doubling time 4 h) to balanced growth in the enriched medium (doubling time 2 h) took about 2·5 h. The rate of RNA accumulation increased soon after the enrichment to almost its final value. This increase began after a short lag of 10 to 15 min, therefore synthesis of new RNA polymerase molecules may be required before stable RNA production can increase. The different stable RNA species were not stimulated at different times after the enrichment, but all increased continuously throughout the transition. The rRNA species accumulated in a co-ordinate fashion at a rate faster than the rate of tRNA accumulation.

Summary: The alginate depolymerase associated with bacteriophage infection of Azotobacter vinelandii has been used in the analysis of sodium alginate. The enzyme degraded the polysaccharide to a series of oligouronides each containing a terminal 4-deoxy-α-l-erythro-hex-4-enopyranuronosyl residue. Analysis of these oligouronides, together with kinetic information, indicated that the enzyme was specific for mannuronic acid-containing regions of the polyuronide. The specificity of the enzyme made it possible to determine the primary structure of the macromolecule.

The phage-induced enzyme was shown to be distinct from the alginate lyase elaborated by the host organisms by its pH optimum, molecular weight, Michaelis constant and stability.

Summary: A technique is described by which both oxygen and nitrate (or nitrite or chlorate) levels were continuously monitored during bacterial respiration. Paracoccus (Micrococcus) denitrificans and Escherichia coli oxidizing succinate rapidly ceased to reduce nitrate when oxygen was available, and equally rapidly commenced nitrate reduction when all the oxygen had been consumed. By contrast, membrane vesicles isolated from P. denitrificans reduced oxygen and nitrate simultaneously. The respiratory nitrate reductase in intact cells of P. denitrificans appeared to be inaccessible to chlorate present in the reaction medium, and it is suggested that the nitrate reductase is orientated on the plasma membrane so that nitrate gains access from the inner (cytosolic) face.

Summary: Abortion of oogonia of Phytophthora cactorum grown on media containing cholestanol occurs at or just before early meiotic prophase. Meiosis is possibly controlled by a steroid hormone, for which cholesterol is an effective precursor but cholestanol is not.

Summary: The structure of mitochondria and of vacuoles in Candida utilis and Schizo-saccharomyces pombe has been studied by electron microscopy of serial thin sections and subsequent model building. The models of the two cells of C. utilis which were studied confirmed our earlier findings, made by high voltage electron microscopy of thick sections, that there is a single, branched and continuous mitochondrial network in the cell ( Davison & Garland, 1975 ). A model of a S. pombe cell showed that the mitochondrial structure was far more continuous than expected from inspection of thin sections, there being but two large and two small mitochondria. The models demonstrated that the few large vacuoles in C. utilis were interconnected into a single cluster, whereas in S. pombe there were two separate complexes of interconnected vacuoles towards each pole of the cell.

Summary: The effect of the blue and near ultraviolet reversible photoreaction on conidial development in Botrytis cinerea was studied by observing microscopically selected conidiophores. Conidiophore development was divided into six stages: when the developing conidiophores from stage 2 (i.e. a mature conidiophore) to stage 5 (i.e. a conidiophore with conidium initial) were exposed to blue light for a short time, conidiation was suppressed; the conidiophores already formed de-differentiated to ‘sterile’ conidiophores with sharply pointed tips. The suppression of conidial development by blue light could be reversed by subsequent exposure to near ultraviolet light, and conidia then developed normally. This mycochrome system functioned reciprocally within the range of identified conidiophore developmental stages and near ultraviolet light acted only at the same developmental stage as was inhibited by blue light.

Summary: The ‘relaxed particles’ formed during methionine starvation of Escherichia coli a19 (Hfr rel met rns) have been isolated by large-scale rate-zonal density gradient ultracentrifugation. The proteins and rRNA species associated with these particles have been examined.

The rRNA species present are precursor and mature forms of 16S and 23S rRNA. The bulk of the rRNA which accumulates during starvation is found within the particles. The proteins prepared directly from the particles give strong multiple immunoprecipitates with antisera specific to 30S and 50S ribosomal proteins. The soluble proteins, prepared and examined in the same manner, do not give this immunological reaction. Two-dimensional electrophoresis patterns of the proteins from the particles show that the proteins co-migrate with proteins from 30S and 50S ribosomes and are entirely dissimilar to the proteins prepared by the same methods from the soluble fraction of the cells. On the basis of these and other observations, it is concluded that the ‘relaxed particles’ are not artefacts but are arrested ribosome precursors containing both rRNA and certain ribosomal proteins.

The free pool of ribosomal proteins is low in exponential-phase cells and is not significantly increased by a 2 h period of starvation for glucose.

The implications of these observations concerning the proteins associated with ‘relaxed’ and ‘chloramphenicol particles’ are discussed in relation to ribosome biogenesis and the stabilization of rRNA.

Summary: The R factor pMG2 protects Pseudomonas aeruginosa against the lethal effects of ultraviolet (u.v.) and gamma irradiation, and methyl methanesulphonate and N-methyl-N′-nitro-N-nitrosoguanidine treatment. Enhanced survival occurs in strains of uvr + rec + (wild-type) genotype and a variety of uvr rec + type mutants. No protection occurs in a recA-type mutant. The plasmid also enhances u.v.-induced mutagenesis. These effects appear to be due to host-cell controlled plsmid-determined DNA repair function(s). Studies on P. aeruginosa strains deficient in DNA polymerase I (polA) suggest that a plasmid-determined repair resynthesis function may be responsible for increased u.v.-survival and enhanced u.v.-mutability in pMG2-containing bacteria.

Summary: Recombinants between Streptomyces coelicolor a3(2) and Streptomyces griseus Kr.15 were obtained using methods of hybrid construction. Recombinant Rcgi, obtained from a cross between S. griseus and a S. coelicolor uf (SCPI−) strain, phenotypically resembled S. coelicolor uf strains and in crosses with a S. coelicolor NF donor strain produced recombinant progeny at a frequency of 100 %. Recombinant Rcg3, like SCPI-carrying S. coelicolor strains, inhibited SCPI− strains of S. coelicolor and in crosses with a uf recipient strain of S. coelicolor generated recombinants at high frequency. In crosses between S. griseus and RcgI the frequency of recombinant formation was increased about 100-fold relative to crosses between S. griseus and S. coelicolor. Effective transfer of S. griseus and Rcg3 chromosomal markers into RcgI and S. coelicolor, respectively, indicated that S. griseus had donor properties.

Studies of the ability of recombinants to support phage growth indicated that parental chromosomal fragments containing genes involved in control of phage-receptor formation and intracellular growth were present in the hybrids. Grisin-producing recombinants, capable of restricting phages attacking S. coelicolor and S. griseus, were obtained.

Summary: Evidence is presented that genes determining the pathway of methylenomycin A synthesis are carried on the SCP1 plasmid. All 16 mutations (mmy) leading to lack of antibiotic synthesis were SCP1-linked. Phenotypic classification, by co-synthesis and other criteria, suggested that they fell into at least five classes. When the wild-type SCP1 plasmid was transferred to Streptomyces lividans or Streptomyces parvulus, material that was chromatographically and biologically indistinguishable from methylenomycin A was produced. Recombination between some pairs of mmy mutations was detected. In crosses of mmy mutants of NF (integrated SCP1 donor) strains with SCP1−, a very high frequency of chromosomal recombination occurred; thus methylenomycin production appears not to be an important cause of the ultra-fertility normally associated with nf × SCP1− crosses.

Summary: Phage Mu-1 and a thermoinducible derivative, Mu-1 cts 62 were inserted into the broad host range R factor RP4. These hybrid plasmids were transferred by conjugation to a phytopathogenic bacterium Pseudomonas solanacearum GM11000 and a legume-root nodule bacterium Rhizobium meliloti 2011. The Mu genome is transcribed and translated in these new hosts: P. solanacearum (RP4::Mu cts) cultures have a spontaneous production of about 5 × 105 plaque-forming units ml−1 which is similar to the frequency of spontaneous Mu production in E. coli; the Mu production of R. meliloti is lower (about 102 plaque-forming units ml−1).

SUMMARY: Several mutants have been isolated from the facultative methylotroph, Methylobacterium organophilum, using either N-methyl-N′-nitro-N-nitrosoguanidine or ultraviolet light as mutagens. One of these isolates, a glutamate auxotroph lacking isocitrate dehydrogenase, has been transformed to prototrophy, using wild-type DNA, at a frequency of 0·5%. Competence and DNA uptake occur only in cultures which are near the end of exponential growth, and maximal transformation requires a DNA concentration of 100 μg ml−1.