- Volume 111, Issue 1, 1979

Mutants of Streptomyces parvulus that are blocked in the synthesis of the phenoxazinone-containing antibiotic, actinomycin, were isolated by the ‘agar piece’ method (after ultra-violet irradiation or treatment with 8-methoxypsoralen plus near-ultraviolet light). Radio-labelling experiments in conjunction with paper, thin-layer and column chromatography revealed that 4-methyl-3-hydroxyanthranilic acid (MHA) is a major metabolite accumulated by these mutants. Studies in vitro and in vivo provided evidence that MHA is a precursor of the phenoxazinone chromophore, actinocin. Normally MHA does not accumulate during growth or antibiotic synthesis by the parental strains. Protoplasts derived from the mutant strain AM5 synthesized MHA in significant amounts. A scheme is proposed for the biosynthesis of actinomycin D that accounts for the accumulation of MHA by the mutants.

A bacterium capable of growth on 5-hydroxyisophthalate was isolated from soil. Both 4,5-dihydroxyisophthalate and protocatechuate were found in the growth medium and were also oxidized by bacterial extracts. These extracts catalysed the decarboxylation of 4,5-dihydroxyisophthalate to give protocatechuate under anaerobic conditions. Extracts oxidized 5-hydroxyisophthalate only when reduced pyridine nucleotide was present, and did so at a faster rate with NADPH than with NADH. When the decarboxylase was removed by DEAE-cellulose chromatography, 4,5-dihydroxyisophthalate was identified as the product of 5-hydroxyisophthalate oxidation. A pathway for catabolism of 5-hydroxyisophthalate involving hydroxylation to 4,5-dihydroxyisophthalate followed by decarboxylation to protocatechuate, the ring-fission substrate, is proposed. Further oxidation of protocatechuate is by the ortho pathway.

Glutamine synthetase has been purified to homogeneity from two N2-fixing cyanobacteria, Anabaena cylindrica and a species of Nostoc (the phycobiont of Peltigera canina). The activities of the A. cylindrica enzyme in the biosynthetic and transferase assays were, respectively, 9.4 and 32 μmol product formed min−1 (mg protein)−1; the corresponding values for the Nostoc sp. enzyme were 6.5 and 20. Stabilization of the enzyme required Mg2+, glutamate, EDTA and a thiol reagent to be present during purification. The molecular weight of the A. cylindrica enzyme was 591000 as estimated by sedimentation analysis, 660000 by gel filtration and 565000 by polyacrylamide gel electrophoresis; the Nostoc sp. enzyme gave values of 630000 by gel filtration and 575000 by electrophoresis. The molecular weights of the sub-units of each enzyme were approximately 49000 to 50 000. Electron microscopy revealed that each molecule was composed of 12 sub-units arranged in two superimposed hexagonal rings. The maximum diameter of the rings was 13.6 nm and the distance between the centres of adjacent sub-units was 4.9 nm. When dialysed in the absence of stabilizing ligands the A. cylindrica enzyme lost activity and the protein band characteristic of the native enzyme was replaced by three bands with approximate molecular weights of 510000, 310000 and 130000. These sub-species re-associated and activity was restored by adding 2-mercaptoethanol and substrates. A similar reversible deactivation has been observed with glutamine synthetase from photosynthetic eukaryotes and yeast but no similar data have been reported for a N2-fixing prokaryote.

Exopolysaccharides and lipopolysaccharides were prepared from a number of myxobacterial strains representing the more complex types. The exopolysaccharides were isolated from fruiting bodies and from liquid and solid cultures. The polysaccharides secreted by the bacillary forms in solid or liquid media closely resembled the material obtained from fruiting bodies, the monosaccharides present being in the same approximate molar ratios. Many of the sugars present in the exopolysaccharides were also detected in the lipopoly-saccharides, suggesting an economic use of sugar nucleotide synthetic systems. Several, but not all, lipopolysaccharides contained material resembling 3-O-methylxylose in its chromatographic mobility. In addition, a faster-moving spot, as yet unidentified, was noted in some hydrolysates. The commonest monosaccharide components of the lipo-polysaccharide were rhamnose, mannose, glucose and galactose. Small quantities of amino sugars, particularly glucosamine and galactosamine, were also detected.

Attachment of washed Mycoplasma gallisepticum cells to glass was quantified with organisms in which membrane lipids were labelled with 3H. Siliconization of the test tubes decreased attachment, while centrifugation increased it. Attachment increased with temperature, decreased with increasing pH and ionic strength of the attachment mixture, but was un-affected by Ca2+, Mg2+ and EDTA. This suggests that ionic bonds, but not salt bridges, participate in the attachment process. Glycophorin, the major receptor responsible for M. gallisepticum attachment to erythrocytes, partially inhibited the attachment of the organisms to glass. However, bovine serum albumin also decreased attachment. Extensive pre-treatment of the organisms with trypsin decreased their ability to attach to glass by about 35 to 40%. Trypsin and pronase failed to detach the organisms already bound to glass, suggesting that external mycoplasma cell components, other than membrane proteins, also participate in attachment of the organisms to glass.

Wild isolates of Botrytis cinerea were analysed to assess their suitability for genetic studies. When compared on synthetic and artificial media under laboratory conditions, the isolates differed in rate of growth, conidiation, sclerotia production and resistance to benomyl. Asexual progeny of individual cultures were sometimes phenotypically diverse, indicating that they could be heterokaryotic or heteroplasmic. Attempts to derive auxotrophic, nystatin-resistant and benomyl-resistant mutants from mutagen-treated conidia were not successful: only morphological and spore colour variants were recovered, and most of the morphological variants were phenotypically unstable. Cultures produced both macro-conidiophores and microconidiophores. Macroconidia were about 10 μm long × 8 μm wide, contained 1 to 12 nuclei (mean 3.4) and usually germinated readily on agar media. Microconidia were about 3 μm diameter, uninucleate and rarely germinated on agar media. The perfect state, Botryotinia fuckeliana, was not recovered from mixed inocula of the wild strains. The significance of the results is discussed in relation to the use of B. cinerea for laboratory studies and the possible genetic basis of natural variations.

The optimum concentration of polyethylene glycol 1000 (PEG) for the production of recombinants through protoplast fusion in Streptomyces coelicolor was about 50% (w/v). The addition of 14% (v/v) dimethyl sulphoxide to the fusion mixture enhanced recombination frequencies, but only at sub-optimal PEG concentrations. After treatment of protoplasts with 50% PEG for 1 min, the frequency of recombinants in a multi-factor ‘cross’ sometimes exceeded 20% of the total progeny. The frequency of recombinants in the progeny could be significantly enhanced by ultraviolet irradiation of the parental protoplast suspensions immediately before fusion.

The isolation and characterization of 29 new germination (Ger) mutants of Bacillus subtilis 168 is described. These were classified, along with previously described mutants, into seven groups according to map location. The mutations in 26 GerA mutants mapped between cysB and thr; detailed mapping of two of these has located them very close to citG. These mutants were deficient in germination in alanine, but responded to the germinative combination of asparagine, glucose, fructose and KCl. One GerB mutant mapped on the origin-proximal side of hisA; it was normal in germination in alanine, but deficient in germination in a mixture of asparagine, glucose, fructose and KCl. Two GerC mutants were linked to lys, but were separable from a temperature-sensitive growth deficiency mapping between lys and trp. The GerC mutants had a similar germination phenotype to the GerA mutants. Three GerD mutants did not germinate in either of the above germinants or in Penassay Broth. They were located on the side of ery distal to cysA. The GerE mutant, which did not germinate in any of the three germinants, was located very close to citF and possessed an altered spore coat. The two GerF mutants were defective in germination in all three germinants and mapped on the origin proximal-side of hisA, but much closer to his than did the GerB mutant. A phosphoglycerate kinase-negative mutant altered in germination mapped between cysB and hisA (GerG). These mutants have established a minimum of seven locations important to germination, and will be useful in the development and appraisal of theories of spore germination.

Mutants of Escherichia coli K12 which accumulated the haem precursor porphobilinogen are described. The mutants grew very slowly on carbon and energy sources which K12 uses only oxidatively, and they had low catalase activities, suggesting that they were deficient in haem. Extracts had one-tenth of the parental activity of the enzyme porphobilinogen deaminase. In transduction, the mutation mapped close to genes ilvD and metE at minute 84. The gene was tentatively identified as hemC, coding for porphobilinogen deaminase. The gene symbol hemC replaces the earlier and temporary symbol popE.

The ampicillin-resistant Haemophilus influenzae strain Ve445 which caused purulent meningitis and septicaemia in a newborn child in Germany contained a 4.4 megadalton (Mdal) plasmid (pVe445) and produced a TEM type β-lactamase. The transformation to ampicillin resistance of a sensitive Escherichia coli strain with isolated pVe445 DNA proved that the structural gene for the β-lactamase resided on this plasmid genome. Molecular DNA-DNA hybridization studies and electron microscope DNA heteroduplex analysis indicated that pVe445 probably contained 38 to 41% of the ampicillin translocation DNA segment (TnA) found on R factors of enteric origin. The TnA fragment present in pVe445 most likely does not contain both of the inverted repeat sequences of TnA. DNA-DNA polynucleotide sequence studies indicated that the 4.4 Mdal plasmid pVe445 was unrelated to the 30 to 38 Mdal H. influenzae R plasmids but was closely related to the 4.1 Mdal ampicillin resistance specifying H. influenzae plasmid RSF0885 isolated in the U.S.A. The H. influenzae plasmid pVe445 shared 91% of its base sequences with the β-lactamase specifying Neisseria gonorrhoeae plasmid pMR0360 (4.4 Mdal) and had 85% of its base sequences in common with the β-lactamase specifying N. gonorrhoeae plasmid pMR0200 (3.2 Mdal). All of the four 3.2 to 4.4 Mdal β-lactamase specifying R plasmids of H. influenzae and N. gonorrhoeae investigated probably have a common evolutionary origin.

The influence of specific growth rate on the ammonia-assimilating enzymes glutamine synthetase, NADP-linked glutamate synthase and NADP-linked glutamate dehydrogenase was studied in ammonia-limited and glucose-limited chemostat cultures of Erwinia carotovora var. carotovora, E. nigrifluens and E. amylovora. The response of these organisms, representatives of various ‘enzyme groups’ within the genus Erwinia, showed considerable variation and confirmed the heterogeneity of the genus with respect to ammonia-assimilating enzymes. The overall significance of the glutamine synthetase/glutamate synthase pathway in any organism where the synthesis of these enzymes is not constitutive is questioned.

A mathematical model for hyphal growth and branching is described which relates cytological events within hyphae to mycelial growth kinetics. Essentially the model quantifies qualitative theories of hyphal growth in which it is proposed that vesicles containing wall precursors and/or enzymes required for wall synthesis are generated at a constant rate throughout a mycelium and travel to the tips of hyphae where they fuse with the plasma membrane, liberating their contents into the wall and increasing the surface area of the hypha to give elongation. The hypothesis that there% a duplication cycle in hyphae which is equivalent to the cell cycle observed in unicellular micro-organisms is also included in the model. Predictions from the model are compared with experimentally observed growth kinetics of mycelia of Geotrichum candidurn and Aspergillus nidulans. The finite difference model which was constructed is capable of predicting changes in hyphd length and in the number and positions of branches and septa on the basis of changes in vesicle and nuclear concentration. Predictions were obtained using the model which were in good agreement with experimentally observed data.