- Volume 113, Issue 2, 1979

Mutants of Escherichia coli with altered resistance to low molecular weight organic solvents were isolated. Solvent-resistant mutants showed a decrease in the ratio of phosphatidyl-ethanolamine to the anionic phospholipids (phosphatidylglycerol and cardiolipin) relative to the wild-type, whereas solvent-sensitive strains showed an increase. Reversion studies on representative mutants demonstrated that the phenotypic response to solvents and the changes in phospholipid composition were genetically associated. The fatty acid and lipo-polysaccharide compositions of the various mutants showed no significant differences from the parental strain. The lesions in two of the solvent-sensitive mutants (DC7 and DC9) and one of the resistant mutants (DC11) were mapped by cotransduction with phage P1 and shown to lie very close to the pss locus at 56 min on the Escherichia coli map.

Summary: The proteolytic activation and inactivation of chitin synthase (EC 2.4.1.16) from Neurospora crassa was studied. Chitin synthase was found mostly in an inactive form which could be activated either endogenously or by the addition of exogenous proteases. The addition of protease inhibitors prevented the endogenous activation of the enzyme. The stability of the inactive enzyme improved when buffers of pH values between 8·2 and 9·5 were employed. The inactive enzyme was mainly associated with membranous fractions.

Summary: Two distinct forms of fructose 1,6-bisphosphatase, designated form A and form B, have been partially purified from autotrophically grown cells of the hydrogen bacterium Nocardia opaca 1b. Form A was present when the organism was grown under autotrophic conditions but was not synthesized during heterotrophic growth. Form B was detected in both autotrophically and heterotrophically grown organisms, suggesting that it is a constitutive enzyme. The molecular weights were estimated to be 140000 for form A and 190000 for form B. Both purified enzymes showed fructose bisphosphatase as well as sedoheptulose bisphosphatase activity, with pH optima of 8·2 and 8·8 to 9·0 for forms A and B, respectively. The ratio of fructose bisphosphatase to sedoheptulose bisphosphatase activity was 0·6 for form A and about 4·5 for form B. The enzymes required Mg2+ for activity; Mn2+ could only partially replace Mg2+. Form A was inhibited by ATP and ribulose bisphosphate. AMP exerted a strong allosteric inhibition on form B which was also inhibited by NAD+ and NADH. Phospho eno/pyruvate activated form B. On the basis of these results, different physiological roles for the two forms of fructose bisphosphatase are suggested.

Cell-free extracts of Dictyostelium discoideum contained the enzymes necessary for both oxidative and non-oxidative pentose phosphate metabolism. The specific activities of these enzymes changed little during differentiation. The properties of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were studied with respect to K m values for substrates and cofactor NADP+. The two dehydrogenases were relatively unstable in extracts prepared from early stages of development. The K m value of phospho-glucose isomerase for glucose 6-phosphate was approximately 30-fold higher than that of glucose-6-phosphate dehydrogenase. Measurements of pentose phosphate pathway intermediates were made throughout development. All measured intermediates except fructose 1,6-bisphosphate appeared to accumulate between aggregation and culmination. Fructose 1,6-bisphosphate concentrations remained constant until culmination, then dropped 3-fold during sorocarp construction. Calculation of mass-action ratios for the pentose phosphate reactions suggested that glucose-6-phosphate dehydrogenase was the only reaction greatly displaced from equilibrium. These results are discussed in relation to factors controlling pentose phosphate metabolism during development in D. discoideum.

Summary: When sucrose or acetate was added to washed suspensions of a number of aerobic bacteria, exogenous ferricytochrome c 3+ was reduced, luminol chemiluminescence was enhanced and the rate of adrenaline auto-oxidation was increased. Superoxide dismutase from bovine erythrocytes inhibited all of there reactions, suggesting that superoxide radicals (O− 2) are formed during aerobic bacterial metabolism. Superoxide radicals were probably generated during respiratory chain activity because O− 2 production by intact bacteria was stimulated by antimycin A and the highest O− 2-producing activity was observed with membrane fractions with a high concentration of cytochromes. Exogenous superoxide dismutase partially inhibited NADH-dependent oxgyen consumption by membrane particles of Brevibacterium flavum 22LD.

Summary: Prior to formation of oosphere initials in septum-delimited oogonia of Saprolegnia diclina, the volume density (volume fraction) of central vacuoles containing characteristic clusters of electron-opaque, granular material increased from 6·0 to 54·2% whereas dense-body vesicles decreased in volume density from 30·2 to 9·0%. During this time, dense-body vesicles were reduced in number by 50%, their mean diameter decreased from 0·861 to 0·697 μm and the percentage of vesicles with more than one dense-body granule increased from 5·4 to 20·2. The volume density of lipid bodies did not change, whilst that of the cytoplasmic matrix decreased from 40·0 to 18·2%. Volume densities of mitochondria and nuclei remained constant relative to the peripheral oogonial protoplasm until central vacuoles had enlarged, but then decreased as oosphere initials formed. Small cytoplasmic vesicles were associated both with Golgi dictyosomes and with dense-body vesicles having blebbed membranes. Similar small vesicles were present close to both central vacuole membranes and oogonial walls.

Oogonia grown in calcium-deficient medium showed a high level of oogonium and oosphere abortion. Aborted oogonia showed extreme protoplast disorganization. In unaborted oogonia grown under calcium-deficient conditions, central vacuoles contained crystalline inclusions, and some oogonia did not form secondary wall layers. Organelles were of normal appearance but calcium deficiency increased the volume densities of mitochondria and nuclei at early stages of oogonial development and of dense-body vesicles and peripheral vacuoles at later stages; the volume density of lipid bodies was reduced at all stages. The numerical density of dense-body vesicles progressively increased during oogonial development and was greater at all developmental stages than in oogonia grown under conditions of calcium sufficiency. At late stages of oogonial development, calcium deficiency reduced both the mean diameter of dense-body vesicles and the fraction of them having two or more dense-body granules.

Summary: Recurrent mutation and selection has been used to increase penicillin titre in two closely related strains of Aspergillus nidulans. A selection programme was initiated from each of the two strains (programmes A and B) and continued through six cycles of mutation and selection. Near-ultraviolet light in conjuction with 8-methoxypsoralen was employed as the sole mutagen throughout programme A and ethyl methanesulphonate as the sole mutagen throughout programme B. Excluding the first cycle of A, where only 50 strains were assayed, the selection programmes were identical. In each programme, 100 survivors were assayed for penicillin titre after each mutagenic treatment and, on the basis of a single yield test, the best five strains were picked and carried forward to the next cycle. In both selection programmes, a near 300% increase in penicillin titre was achieved. This yield advance illustrates the effectiveness for strain development of experimental designs involving successive cycles of mutagenesis with a single-stage screen and the selection of the top few percent survivors in each cycle.

Summary: Bacteriocin production by 97 isolates of Rhizobium leguminosarum was investigated and two types of bacteriocins were identified and designated small and medium on the basis of their size. Three isolates appeared to carry determinants of medium bacteriocin production which were self-transmissible at frequencies of 10−1 to 10−2, suggesting the presence of bacteriocinogenic plasmids in these strains. The mobilization of chromosomal genes was associated with transfer of the bacteriocinogenic plasmids. Indirect evidence that the production of small bacteriocins may be determined by plasmids in three other strains is presented.

Summary: The use of a modified procedure for the isolation of covalently closed circular DNA of high molecular weight, followed by agarose gel electrophoresis of the crude extracts, provides a simple screening method for detecting plasmids with molecular weights of more than 250× 106 from Agrobacterium tumefaciens, Pseudomonas putida and Rhizobium species. This method was used for a survey of plasmids in 25 symbiotically effective strains of Rhizobium meliloti from various geographical origins. Of these, 22 strains were found to carry at least one large plasmid. By electron microscopy and measurement of electrophoretic mobility in gels, the molecular weights of most of the plasmids were estimated to range from 90×106 to 200×106.

Evidence for genetic transformation in Neurospora crassa is based on the observations that allo-DNA has a specific effect in producing transformants which is abolished by DNAase treatment and that iso-DNA is not effective in transformation. Here, unambiguous evidence for genetic transformation is provided by transfer of a temperature-sensitive inositol requirement from a donor to a recipient strain. Data provided also suggest the role of growth conditions and the involvement of a nuclease gene in the DNA uptake and transformation of N. crassa.

Summary: A new type of haem-deficient mutant was isolated in Escherichia coli K12 by neomycin selection. The mutant, designated SASX38, accumulated uroporphyrin, coproporphyrin and protoporphyrin. Since it possessed normal ferrochelatase activity, it was assumed to be deficient in protoporphyrinogen oxidase activity. The gene affected in the mutant was designated hemG. Mapping of the hemG gene by phage P1-mediated transduction showed that it was located very close to the chlB gene (frequency of cotransduction 78.7%), between the metE and rha markers. This location is distinct from the other known hem loci in E. coli K12.

Summary: Crosses were made between strains of Dictyostelium discoideum involving two drug resistance markers and the mating-type locus. Over 6000 progeny from 263 individual germinated macrocysts from four single-factor crosses, five two-factor crosses and one three-factor cross were characterized. In most cases the progeny from a single macrocyst were of one genotype, although in the population of macrocysts from any two-factor cross all possible parental and recombinant genotypes were recovered. There was no evidence of linkage between any of the markers examined. No selection against progeny carrying the methanol or the cycloheximide resistance markers was found in two-factor crosses, but selection against progeny carrying both resistance markers was found in the three-factor cross. Germination of macrocysts in all crosses was poor, only once exceeding 2·5 % of the total macrocyst population. A variety of crosses and back-crosses with different parental strains indicated that germination might be influenced by both extrinsic (environmental) and multiple genetic factors. About 10 % of the macrocysts yielded progeny spores that were ambivalent in their mating reactions. After extensive recloning these populations could be resolved to the normal mat A (formerly A1) and mata (formerly A2) mating-types and might therefore have represented aneuploids. The results obtained with D. discoideum macrocysts differ from those obtained with other cellular slime moulds - Dictyostelium mucoroides, Dictyostelium giganteum and Polysphondylium pallidum - and are reminiscent of the results reported for germinated zygospores of Phycomyces blakes-leeanus.

The calcium requirement for optimal growth of Physarum polycephalum is between 0·05 and 2·5 mm. Media without Ca2+ will not support growth though Ca2+-chelating agents, such as EDTA and EGTA, do not have any marked effect on Ca2+ availability at the low pH used to culture this organism. The intracellular Ca2+ concentration varies approximately 10-fold when the external Ca2+ concentration is varied between 0·05 and 25 mm. Ca2+ is concentrated in the mitochondrial and microsomal fractions, but the high concentration found in the cytosol suggests that considerable redistribution of Ca2+ occurs during disruption and fractionation of the plasmodia. 45Ca2+ uptake by intact microplasmodia occurs at rates which are comparable to those required for Ca2+ uptake during growth and generation of new cell mass. This uptake process has two components: one non-saturable with Ca2+, and the other saturable with an apparent maximum velocity of 67 nmol Ca2+ h−1 (mg protein)−1 and an apparent K m of 1·9 mm-Ca2+. As judged by [3H]inulin uptake, part of the 45Ca2+ uptake may be endocytotic.

Ionophores A23187 and Br-X537A stimulate 45Ca2+ uptake. Uptake is inhibited by KCl, Mn2+ and Sr2+ at high concentrations, but is not sensitive to La3+ at concentrations similar to Ca2+ in the uptake medium. KCN and iodoacetic acid inhibit the transport, while cyto-chalasin B, verapamil, tetracaine and procaine have little or no effect.

Summary: The size of the amino acid pool in Bacteroides amylophilus, a rumen bacterium, was limited by the availability of ammonia in the growth medium. Alanine was the major pool constituent irrespective of the growth-limiting nutrient. In steady-state ammonia-limited chemostat cultures, glutamine synthetase (GS) activity was tenfold higher and glutamate dehydrogenase (GDH) activity up to fivefold lower than in maltose-limited cultures. No glutamate synthase (GOGAT) activity was demonstrated. When excess ammonia was pulsed into an ammonia-limited chemostat culture, the glutamine pool expanded rapidly and GS was inactivated. This was consistent with the observation that, in a number of bacteria, GS functions to assimilate ammonia when the prevailing concentration is low. However, GDH was always very active in extracts of B. amylophilus and its Michaelis constant for ammonia was relatively low (1 to 2 mm). This suggested that GDH could continue to function bio-synthetically when GS was derepressed.

Summary: Recent improvements in the optics and electronics of flow cytometry systems, as well as in staining techniques, permit the assay of such minute cellular constituents as the DNA and protein contents of micro-organisms. To assess the usefulness of this technique, DNA and protein content distributions were determined in Escherichia coli, Lactobacillus brevis, Lactobacillus casei, Chlorella kessleri 8k, Saccharomyces cerevisiae, Candida utilis, Schizosaccharomyces pombe and Euglena gracilis.Investigations of the DNA content distributions of polyploid strains of Saccharomyces cerevisiae indicated that the method can be used to determine ploidy. The rapidity of flow cytometry measurements allows accurate determinations in large populations.