- Volume 110, Issue 1, 1979

Summary: Taxonomic and ecological studies were done on 553 bacterial strains isolated in late summer 1975 from the Arctic Beaufort Sea. Numerical taxonomic analyses were employed using 300 features, assessing similarity by the Jaccard coefficient (S J ) and clustering the strains using single linkage. Bacteria isolated at 4 °C (14 clusters) and 20 °C (13 clusters) were compared with previously described genera. The dominant bacteria in the Beaufort Sea appear to be different from those found in temperate marine environments. Orange-pigmented bacteria, which appear to be Flavobacterium species, were dominant in surface water. Several taxonomic clusters of presumed Vibrio species were found. Many of the Gram-negative rods were highly pleomorphic and could not be identified. Some isolates resembled morphologically the genus Microcyclus. Feature analyses showed evidence of adaptation to the environment. All bacterial isolates were psychrophilic or psychrotrophic. Nutritionally, most of the organisms required organic growth factors such as vitamins.

Summary: A new species of yeast, Candida fusiformata, is described. The strains isolated from cabbages and cauliflowers differ from all other known species of Candida on the basis of the conventional substrate utilization tests. In addition, the strains form curved or spiral-shaped mycelium and branching chains of oval to fusiform cells on which buds are often formed from acicular or ampulliform spicules.

Summary: Octopine-utilizing agrobacteria could be distinguished from non-utilizers on a solid medium containing octopine as the only added nitrogen source if a pH indicator such as bromothymolblue were present. On green bromothymolblue plates, octopine-utilizing strains formed orange-yellow colonies, while those of non-utilizers were translucent. Strains could be tested for utilization of other nitrogen sources such as nopaline by substituting these for octopine in the same medium.

Using bromothymolblue plates, it was found that TI plasmids of octopine-utilizing strains of Agrobacterium tumefaciens were transferable in the presence of octopine, octopinic acid or lysopine, but not in the presence of the analogues nor-octopine, homo-octopine or desmethylhomo-octopine. Transfer in the presence of “inducer” was not detected when bacteria were mated at 37 °C instead of 29 °C, or when either methionine, cysteine or cystine was present in the mating medium. Mutant plasmids were obtained that were conjugative in the absence of an inducer; these were insensitive to methionine and cysteine but, like the wild-type plasmid, were thermosensitive for transfer.

The TI plasmid of an octopine-utilizing strain was introduced into a strain that carried a deleted nopaline TI plasmid. These plasmids were found to be compatible. The properties of this transconjugant showed that the octopine oxidase does not accept nopaline as a substrate. Tumours induced on Kalanchoë daigremontiana by this bacterium were rough and contained octopine. Exclusion of phage S18 was found to be a marker specific for the TI(Kerr14) plasmid.

Summary: The lipopolysaccharide of free-living Rhizobium leguminosarum was isolated and purified, and its homogeneity was determined by gel electrophoresis and, after mild acid degradation, by gel filtration. On electrophoresis, two molecular species were observed. After acid degradation and gel filtration, three components could be isolated: one was very rich in glucose, one contained 2-O-methylfucose, fucose, mannose, galactose, glucose, l-glycero-d-manno-heptose, 2-keto-3-deoxyoctonic acid, l-alanine, quinovosamine and uronic acids, and the third component consisted of low molecular weight material. In the lipid A fraction, d-glucosamine, β-hydroxymyristic acid, β-hydroxypalmitic acid and β-hydroxystearic acid were detected as major components. The phosphorus content was low. No major chemical differences were observed in the neutral sugar and fatty acid compositions of the lipopolysaccharides isolated from bacteria and bacteroids. The lipopolysaccharide of bacteroids was rapidly lost during isolation from the nodules and on dialysis, and behaved anomalously during ultracentrifugation.

Summary: Ferrichrome-promoted iron uptake in Escherichia coli k12 is strictly dependent upon the tonA gene product, a “minor” outer membrane protein. By selection for mutants of E. coli resistant to phages which require “major” outer membrane proteins as receptors, strains with pronounced protein deficiencies were constructed. Such strains were tested for anomalous behaviour of ferrichrome transport. No significant differences in iron uptake were detected in E. coli k12 strains with markedly reduced amounts of protein I. However, a reduction in the initial velocity (up to 40%) was observed in E. coli deficient in outer membrane protein II*. This difference was only evident when cells were grown under iron-starvation conditions; it was abolished when cells were grown in rich medium. Kinetic parameters for ferrichrome transport were determined for maximum velocity but not for K m; double reciprocal plots showed a biphasic nature, probably attributable to a limited number of outer membrane binding sites and to the multi-component nature of the ferrichrome-iron transport system.

Summary: The variation in penicillin titre within populations of cultures of Aspergillus nidulans derived from untreated conidia and from conidia treated with ethyl methanesulphonate (EMS), near-ultraviolet light in the presence of 8-methoxypsoralen (8MOP) or N-methyl-N” -nitro-N-nitrosoguanidine (NTG), each at several dose levels, was determined. Both mutagen-treated and untreated populations showed a continuous distribution of penicillin titres. The population mean titre of the mutagenized populations was decreased and the range of titres was increased relative to those of the control populations. No differences between sister cultures could be detected in three untreated populations, but nine out of ten populations derived from mutagenized conidia showed significant variation for penicillin titre. In general the magnitude of this induced variation increased with increasing dosage of the mutagen. Comparisons at fixed survival levels indicate that 8MOP mutagenesis is less effective for the induction of variation in penicillin titre than EMS or NTG mutagenesis. A statistical procedure was adopted to classify the survivors as unchanged cultures (“0”), itre-increasing mutants (“+”) or titre-decreasing mutants (“-”). The frequency of both “+” and “-” mutants increased following mutagenesis, with NTG being the most active of the three mutagens. Over all treatments, these two mutant classes were recovered with equal frequency. The frequency of “+” mutants was largely independent of mutagen dose, within the ranges used, and moderate treatments (around 10% survival) gave as high or higher frequencies than more extreme doses. All three mutagens, and in particular NTG, produced morphological mutants. These contained an increased frequency of titre-decreasing mutants, but increases in titre appeared to be independent of changes in colony morphology. Estimates based on the observed frequencies of penicillin titre mutants suggest that several hundred genes are potentially capable of affecting this continuous variable.

Summary: One-step mutants of Rhizobium sp. strain 32h1 resistant to d-cycloserine showed substantial (≥ 90%) loss of asymbiotic (agar culture) nitrogenase activity but only partial (25 to 50%) loss of symbiotic (root nodule) nitrogenase activity. Two- and three-step resistant mutants showed a further decline or complete loss of both forms of nitrogenase activity. Since d-cycloserine inhibited the uptake of alanine by the parent strain and since the mutants possessed a defective transport system for alanine, it is suggested that the mutants are unable to concentrate d-cycloserine. Agar cultures of a one-step resistant mutant contained the three morphological types of Rhizobium present in the parent culture. In contrast, only one morphological type of Rhizobium was found in cultures of two- and three-step resistant mutants. Root nodules formed by a one-step resistant mutant on cowpea contained fewer bacteroid-filled plant cells than were present in nodules formed by the parent strain. Root nodules formed by two- and three-step resistant mutants contained only a small number of infected plant cells and in many of these rhizobia had not been released from infection threads. It is suggested that changes in the permeability of the Rhizobium cell wall, resulting in resistance to d-cycloserine, are responsible for the morphological changes and loss of nitrogenase activity in these mutants.

Summary: Ribonuclease, deoxyribonuclease, acid phosphatase and phenoloxidase were detected in preparations of extracellular melanoprotein isolated from cultures of Venturia inaequalis. The high initial levels of activity produced per g fungus declined within a few days of inoculation, to an approximately constant level. About six times as much activity was released by conidia germinating in medium enriched with wood extract than was released in basal medium. After 5 d incubation, the rate of production of enzymically active melanoprotein reflected the rate of growth of the fungus. Isolated melanoprotein stored at 4 °C for 3 months showed up to 190% more hydrolase activity than was measured originally, but longer storage caused a subsequent decrease in activity. Only phenoloxidase decreased continuously during storage. The apparent stability of the bond between melanin and protein under dissociating conditions contrasted with the reversible formation of complexes between the fungal product and polyelectrolytes.

A set of re-isolates of different ages produced randomly variable levels of hydrolase activity when cultured repeatedly over a period of 16 months. On each occasion, however, the specific and total activity of each hydrolase was (with one marginal exception) lower in cultures of the oldest re-isolate than in those of the later re-isolates. In contrast, the specific activity of phenoloxidase was highest in cultures of the oldest re-isolate. These changes were associated with the decline in virulence observed in stored re-isolates of clone E1 of V. inaequalis.

Summary: The O8- and O9-specific lipopolysaccharides of Escherichia coli lost their serological activity during liberation of the polysaccharide moieties (α-mannans) by mild acid hydrolysis, as tested by passive haemagglutination and haemagglutination inhibition. The serological activities and specificities were restored by substitution of the polysaccharides with 1 to 2 stearoyl groups per polysaccharide chain. The mannans obtained by biosynthesis in vitro were serologically active only when bound to the membrane-associated hydrophobic carrier molecule. Liberation of the polysaccharides from the carrier by treatment with aqueous phenol resulted in loss of the serological activity. The O8- and O9-specific mannans of E. coli are thus serologically active when they are part of an amphiphilic molecule and not as free polysaccharides.

Summary: Menaquinones were the only isoprenoid quinones found in 85 of the 95 coryneform bacteria examined. Dihydromenaquinones having nine isoprene units were the main components isolated from Corynebacterium bovis, from other glutamic acid-producing strains, and from Arthrobacter globiformis and related species. Dihydromenaquinones with eight isoprene units were found in Brevibacterium linens, the remaining Corynebacterium species and strains probably belonging to the genus Rhodococcus. Tetrahydromenaquinones with eight isoprene units were found in Arthrobacter simplex and Arthrobacter tumescens, and with nine isoprene units in Cellulomonas and Oerskovia. Kurthia and Curtobacterium were characterized by menaquinones with seven and nine isoprene units, respectively, and Microbacterium lacticum and Corynebacterium aquaticum had comparable amounts of menaquinones with 10 and 11 isoprene units. Strains received as Brevibacterium leucinophagum, Corynebacterium autotrophicum, Corynebacterium nephridii, Mycobacterium flavum, Mycoplana rubra and Protaminobacter ruber contained ubiquinones as their sole isoprenoid quinones. The isoprenoid quinone data correlate well with major trends in coryneform taxonomy and are of value in the classification of coryneform and related bacteria.

Summary: Mutants deficient in several enzymes of methylamine oxidation and assimilation have been obtained by treating Pseudomonas aminovorans with ultraviolet light. One of these mutants was studied in detail and was shown to lack the enzymes of trimethylamine and dimethylamine oxidation. In addition, activities of the following enzymes, all postulated as being involved in C1 metabolism, were lost: hydroxypyruvate reductase, serine-glyoxylate aminotransferase, isocitrate lyase, phosphoeno/pyruvate carboxylase, N-methylglutamate dehydrogenase, γ-glutamylmethylamide synthetase, formate dehydrogenase and dye-linked formaldehyde dehydrogenase. In contrast, three enzymes - NAD+,glutathione-linked formaldehyde dehydrogenase, N-methylalanine dehydrogenase and N-methylglutamate synthase - were retained. This phenotype suggests either a lesion in a regulatory gene, a deletion in an early structural gene of an operon or the loss of a plasmid. Results obtained indicated that P. aminovorans possesses an isocitrate lyase-serine pathway.

Summary: An Ectothiorhodospira sp. was isolated from an alkaline mud sample from Lake Hannington, Kenya. It closely resembled Ectothiorhodospira shaposhnikovii in its ultrastructure, GC content, photosynthetic pigments, flagella position and mode of sulphur deposition, but differed in exhibiting more extreme alkalophily, the pH optimum being 9·0 to 9·5, and in being obligately phototrophic.

Summary: Production of enzymes degrading plant cell walls was studied using media containing cellobiose or ammonium ions (NH4 +) as limiting nutrients. Carboxymethylcellulase (CM-cellulase), xylanase and pectin lyase were primarily cell-associated during exponential growth in batch culture but accumulated in the supernatant during the stationary phase. Activities of CM-cellulase and xylanase were higher in cellobiose-limited than in NH4 +-limited continuous cultures, were inversely related to the growth rate and became progressively more cell-associated as the growth rate increased. The proportion of fermentation products in cellobiose-limited continuous cultures was dependent on the growth rate and the calculated cell yields per mol ATP (Y ATP) varied between 11·92 and 16·39. Glutamate dehydrogenase, an ammonia-assimilating enzyme, was most active in NH4 +-limited continuous cultures. These results are discussed in relation to the growth and metabolism of Ruminococcus flavefaciens in vivo.

Summary: The T incompatibility group plasmid R394 can mobilize the chromosome of Proteus mirabilis strain pm5006. It transferred relatively large segments, corresponding to at least 20 min on the D plasmid chromosomal map of the organism. The frequency of recombination for a large number of selected markers was nearly constant at 5 × 10−6 per donor cell and it is concluded that mobilization takes place from a number of chromosomal sites. All recombinants were R+ and displayed all properties of the plasmid. By analysing crosses for co-inheritance frequencies of unselected markers, a number of chromosomal loci were assembled in linear array. Linkage between markers at the ends of this linkage group was established to markers at the respective termini of the existing D plasmid linkage group. This established a composite circular linkage map of genes of the P. mirabilis strain pm5006 chromosome.

Summary: A new type of haem-deficient mutant was isolated in Escherichia coli K12 by neomycin selection. The mutant was deficient in uroporphyrinogen III cosynthase activity as indicated by the accumulation of uroporphyrin I and coproporphyrin I. The mapping of the corresponding hemD gene by P1-mediated transduction showed that the new gene was located between ilv and cya, at min 83 on the chromosomal map of Escherichia coli K12.

Summary: Intracellular ATP concentrations and energy charge were monitored during the growth of Spiroplasma citri and related to the number of colony-forming units, to the pH of the medium and to incorporation of radioactive precursors. Of three different methods used for extracting nucleotides - trichloroacetic acid, perchloric acid and boiling Tris/H2SO4 buffer - the trichloroacetic acid treatment was the most effective. During the active growth phase the ATP concentration increased exponentially, in the same way as other growth parameters. However, as soon as the metabolic activity slowed down, there was a rapid drop of ATP concentration and of total adenylate content, which was followed much later by a parallel decrease in the number of colony-forming units. During active growth, energy charge remained at around 0·9 and decreased only slowly during the stationary phase. Incorporation of labelled thymidine or phenylalanine followed a different pattern during the late phase of the culture and gave less information concerning the viability of the organisms.