- Volume 130, Issue 8, 1984

Spheroplasts were prepared from the filamentous cyanobacterium Anabaena variabilis by lysozyme treatment followed by mild sonication. The rate of photosynthesis and the initial rate of accumulation of inorganic carbon within the spheroplasts were very close to those observed in intact cells. The steady-state intracellular inorganic carbon pool, however, was smaller in spheroplasts than in intact cells. It is suggested that the cell wall does not play an essential role in the process of HCO3 − transport (leading to accumulation of inorganic carbon internally) but may have a significant impact on the diffusional dissipation of the intracellular pool of inorganic carbon.

The bactericidal activity of Tinopal AN [1,1-bis(3,N-5-dimethyl-benzoxazol-2-yl)-methine p-toluene sulphonate] was shown to be due to a mechanism entirely independent of its inhibitory effects upon NADH dehydrogenase which were reported previously. Whereas the compound had no significant effect upon DNA synthesis in Escherichia coli D22, RNA and protein synthesis were immediately and markedly inhibited. In confirmation, Tinopal AN caused an immediate cessation in inducible β-galactosidase synthesis in the same organism. An in vitro assay of the transcription of calf-thymus DNA by purified E. coli RNA polymerase showed that this process was inhibited by Tinopal AN.

An arginine auxotrophic mutant was obtained from Streptomyces griseoflavus (bicozamycin-producing strain). The mutant grew on synthetic agar supplemented with either arginine, ornithine, citrulline or argininosuccinate, but produced massive aerial mycelium and bicozamycin only with citrulline. In liquid culture, citrulline also completely restored the ability of the mutant to produce bicozamycin. Culture with arginine or ornithine markedly changed intracellular pools of these ornithine-cycle amino acids, but did not affect the other amino acid pools. The ability to produce antibiotic (but not that to form aerial mycelium) was partially restored by certain mutations to ethionine resistance (Eth-1 and Eth-2). These mutations caused decreased or increased S-adenosylmethionine synthetase activity, but both resulted in a 4·5–8-fold increase in the intracellular S-adenosylmethionine pool. Exogenous addition of S-adenosylmethionine (0·5–3 mM) also partially restored the antibiotic-producing ability of the arginine auxotroph. No difference in the S-adenosylmethionine pool was observed in organisms grown with arginine and citrulline. It was suggested that citrulline and S-adenosylmethionine are somehow involved in the initiation of differentiation and secondary metabolism of S. griseoflavus.

The rate of accumulation of streptomycin by streptomycin-sensitive strains of Escherichia coli and Bacillus megaterium, grown in chemostats, was related to the growth rate prior to addition of the antibiotic. For E. coli the length of the lag period that preceded accumulation was also growth rate-dependent. Thus faster growing cultures accumulated streptomycin more rapidly and with a shorter lag than slower growing cultures. The rate of efflux of streptomycin from bacteria that had accumulated streptomycin was not greatly influenced by growth rates of the cultures. At a particular growth rate, accumulation of streptomycin was found to be faster at higher concentrations of the antibiotic. Rapid accumulation of streptomycin was not observed with continuous cultures of a streptomycin-resistant strain of E. coli. Accumulation of streptomycin was abolished when growth was inhibited by either terminating the flow of fresh medium to a chemostat or by adding inhibitors that block protein synthesis. These results suggest that the rate of accumulation of streptomycin is related to the concentration of streptomycin-sensitive ribosomes that are actively engaged in protein synthesis within the bacterial cells.

Microthrix parvicella, cultivated in a medium with Tween 80 and Casamino acids, utilized only the oleic acid moiety of Tween 80 as carbon and energy source. The cell yield from Tween 80 was about 0·32 g dry weight of cells per g of Tween 80 consumed. As only the oleic acid moiety of Tween 80 was utilized, the cell yield from oleic acid was 1·3 g dry weight of cells per g oleic acid consumed. The amount of carbon produced as CO2 was less than 30% of the oleic acid-carbon and this low value was in agreement with the high cell yield. In batch culture M. parvicella stored large amounts of lipid material during the early growth phase. The fatty acids of the lipid globules were similar to the fatty acids supplied as carbon source. The percentage composition of the biomass changed to give C/N percentage ratios of about 15 during the early growth phase due to the high concentration of internal lipids and the low concentration of protein. The growth rate in batch culture was about 0·016 h−1 but was affected by the concentration of Casamino acids in the medium.

Increasing the inorganic phosphate concentration of the medium from 0·01% to 1% (w/v) resulted in 4·5-fold higher production of carotene in Blakeslea trispora. The pattern of β-carotene synthesis in B. trispora remained essentially the same in both high (1%) and low (0·01%) phosphate grown cultures. Higher concentrations of intracellular orthophosphate and lower activities of acid and alkaline phosphatase were found in the high phosphate grown cells as compared to the low phosphate grown cultures. The intracellular acid and alkaline phosphatase activities of B. trispora were competitively inhibited by inorganic phosphate. One or more forms of the acid and the alkaline phosphatases were apparently repressed during active carotenogenesis in the high phosphate grown B. trispora as compared to low phosphate grown cells. An inverse relationship was found between carotenogenesis and the intracellular phosphatase activities and it is suggested that inorganic phosphate concentration affects β-carotene synthesis in B. trispora by regulating the intracellular phosphatase levels in the culture.

Chitin synthetase preparations from cell walls and chitosomes of the fungus Mucor rouxii were tested for their ability to synthesize chitosan when incubated with uridine diphosphate N-acetyl-D-glucosamine in the presence of chitin deacetylase. The most effective chitin synthetase preparation was one dissociated from cell walls with digitonin. The rate of chitosan synthesis by the wall-dissociated chitin synthetase was about three times that of an equivalent amount of cell walls. The chitosan-synthesizing ability of chitosomes was relatively low, but was more than tripled by treatment with digitonin. Presumably, digitonin improves chitosan yields by dissociating chitin synthetase. The dissociated enzyme would produce dispersed chitin chains that could be attacked by chitin deacetylase before they have had time to crystallize into microfibrils. The regulation of chitin and chitosan syntheses in vivo may be determined by the organization of chitin synthetase molecules at the cell surface. Those molecules that remain organized as a complex, similar if not identical to that found in chitosomes, would produce mainly chitin. Chitosan would be preferentially produced by chitin synthetase molecules which are dispersed upon reaching the cell surface.

API 20E, API ZYM and eight other enzymic API systems were tested on 123 strains belonging to 18 Erwinia species, six Enterobacter agglomerans strains and 22 reference strains belonging to other phytopathogenic genera and other enterobacterial species. The data obtained, from a total of 130 tests, were subjected to numerical analysis. Test reproducibility within the API 20E system varied from 88 to 100%. The numerical analysis revealed 12 phenons; in six of these phenons two or three subphenons could be differentiated. Several of these (sub)phenons corresponded to established Erwinia species and could be differentiated from each other by 25 characters. No clearcut distinction could be made between the ‘amylovora’, ‘carotovora’ and ‘herbicola’ groups. Seven phenons were further analysed with the API 50CHE system. The results provided evidence for the retention of Er. quercina, Er. nigrifluens, Er. salicis, Er. amylovora, Er. rubrifaciens, Er. mallotivora, Er. stewartii, Er. cypripedii and Er. chrysanthemi as separate taxa and supported the synonymy within the pairs Er. ananas and Er. uredovora, Er. dissolvens and Enterobacter cloacae, Er. carotovora subsp. atroseptica and Er. carotovora subsp. carotovora, Er. milletiae and one of the Er. herbicola clusters. The inadequacy of the present classification of several Erwinia species, such as Er. herbicola and Er. rhapontici, is highlighted. The results show that API systems are a useful and rapid alternative to conventional phenotypical testing for the classification and identification of Erwinia species.

Deep-sea bacteria were isolated from the digestive tract of animals inhabiting depths of 5900 m in the Puerto Rico Trench and 4300 m near the Walvis Ridge. Growth of two bacterial strains was measured in marine broth and in solid media under a range of pressures and temperatures. Both strains were barophilic at 2 °C (± 1 °C) with an optimal growth rate of 0·22 h-1 at a pressure 30% lower than that encountered in situ. At 1 atm they grew at temperatures ranging from 10·2 to 180·2 °C (± 0·3 °C), while in situ pressures increased the upper temperature limit to 230·3 °C. Both strains were identified as members of the genus Vibrio, based on standard taxonomic tests and mol % G + C values (470·0 and 470·1). Ribonucleotide sequences determined for 5S ribosomal RNA from each strain confirmed relationship to the Vibrio-Photobacterium group, as represented by V. harveyi and P. phosphoreum, but the barophiles were clearly distinct from these species. Secondary structure conformed to the established model for eubacterial 5S rRNA.

A numerical classification of the yeast genus Schizosaccharomyces was undertaken using 60 strains and 100 characters. Three distinct clusters were observed, corresponding to S. pombe (SSM = 83%), both varieties of S. japonicus (SSM = 78%), and S. octosporus(SSM = 75%). Schizosaccharomyces malidevorans and S. slooffiae, each of which is available only as the type, could not be differentiated from S. pombe and S. octosporusrespectively.