- Volume 120, Issue 2, 1980

Measurements were made of the rates at which continuously cultured Streptococcus sanguis NCTC 7868 cells accumulated on the inside surfaces of narrow glass capillaries from suspensions of the bacteria flowing down the capillaries at different velocities. Initially, the rate of accumulation of bacteria on the clean walls of the capillary was rapid. The deposition rate decreased with time, however, resulting in a saturation coverage of the glass surface which was considerably less than a monolayer. Multilayer coverage of the tube surface by bacteria was only achieved when fresh nutrient was pumped over deposited cells. This was attributed to cell growth.

Although theoretical considerations of the deposition of small particles on to the walls of a tube suggest that the initial deposition rate should increase with flow rate, this was not the case with cells grown at dilution rates of 0·2 and 0·5 h−1. It is suggested that this can be explained by a polymer bridging mechanism of attachment.

A species of the imperfect fungus Monilia produced cellobiose dehydrogenase extracellularly when grown on cellulose. The inducible enzyme was both bound to the mycelium and released into the growth medium. The enzyme showed a high degree of specificity for cellobiose, but also oxidized lactose and 4-β-glucosylmannose. The specificity of the electron acceptor was restricted to those compounds having a redox potential of +0·22 V. p-Benzoquinone and several other quinones, however, were not reduced. Oxygen was not consumed nor was hydrogen peroxide produced by cellobiose dehydrogenase oxidation of cellobiose. The enzyme had a molecular weight of 48000 and an isoelectric point of 5·3 to 5·5. A new zymogram technique was developed for the detection of cellobiose dehydrogenase in polyacrylamide gels following electrophoresis and isoelectric focusing.

The pyruvate dehydrogenase complex of Pseudomonas aeruginosa PAO was purified by affinity chromatography on ethanol-Sepharose 2B followed by sucrose density gradient centrifugation. The overall purification was 130-fold based on enzyme activity. The purified complex contained three major and one minor polypeptide components when analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. These were identified by heat treatment, limited proteolysis and peptide mapping as pyruvate dehydrogenase (E1; Mr 92500), acetyltransferases (E2; major component, Mr 76000, and minor component, Mr 77 800), and lipoamide dehydrogenase (E3; Mr 58000). The purified complex had a sedimentation coefficient of 48S and the specific activity for the overall reaction of the complex was 6·5 μ substrate transformed (mg protein)−1 min−1 at the optimum pH (7·8) and 25 °C.

The lesions in four ace mutants lacking overall pyruvate dehydrogenase complex activity were identified after partial purification of the corresponding cell-free extracts. Three strains, designated aceA mutants, lacked pyruvate dehydrogenase activity (E1 component) and one strain, an aceB mutant, lacked the activity of the acetyltransferase (E2 component).

A glycopeptide and a peptide have been isolated from bovine seminal plasma which together will induce germination of Candida albicans blastospores at 37 °C and in the presence of glucose and Mn2+. They have molecular weights of 2000 to 3000. Both peptides contain appreciable amounts of aspartic and glutamic acids, only the glycopeptide contains threonine, lysine, histidine and arginine, while only the peptide contains proline. Acid hydrolysates are fully active in inducing germination and a mixture of aspartic acid, lysine, histidine, threonine, proline and β-alanine can replace them. Mn2+ is not then required. Amino acid mixtures are required to be present throughout the whole period in the incubation medium for full germination to take place, but the peptides can be removed after 1 h incubation and if the cells are resuspended in a buffered glucose medium full germination occurs after a further 3 h incubation.

A mutant of Pseudomonas aeruginosa PAO1 originally isolated on the basis of its sensitivity to methyl methanesulphonate was found to be (i) sensitive to u.v.- and γ-irradiation, (ii) deficient in recombination as assayed by transduction and conjugation and (iii) deficient in an ATP-dependent deoxyribonuclease activity. Its marker (mms-13) is cotransducible with argB and pyrE which are mapped at approximately 22 min on the P. aeruginosa chromosome.

Four independent ace mutants of Pseudomonas aeruginosa PAO lacking the activity of the pyruvate dehydrogenase complex have been isolated. They resembled ace mutants of Escherichia coli and Salmonella typhimurium in requiring acetate as an essential supplement for aerobic growth on glucose, succinate or lactate and in their ability to utilize acetate as sole carbon and energy source. Assays for the individual components of the pyruvate dehydrogenase complex indicated that they lacked the pyruvate dehydrogenase component (E1) or the pyruvate dehydrogenase and dihydrolipoamide acetyltransferase components (E1 and E2) but not the lipoamide dehydrogenase component (E3). Genetic studies with plasmid R68.45-mediated conjugation and phage F116L-mediated transduction indicated that the ace mutations are located at approximately 15 min in the P. aeruginosa PAO linkage map.

Plasmids obtained from four field isolates of Rhizobium leguminosarum were visualized following electrophoresis on agarose gels. The relative mobilities of the bands observed corresponded to plasmids with molecular weights of about 100 × 106 and greater. Each field isolate examined had a different pattern of plasmids.

Lysates from R. leguminosarum strain 300 normally produced three plasmid bands, although in some preparations two much larger plasmids were also visible. The smallest plasmid band seen in strain 300 probably contains two co-migrating plasmids, because in one derivative of strain 300 it was replaced by a doublet, presumably as a result of the presence of a small deletion in one of the co-migrating plasmids. No apparent symbiotic defects were associated with the presence of this deletion. However, a non-nodulating derivative of strain 300 (strain 6015) was found to have suffered a deletion in the third-smallest plasmid of this strain.

Transfer of the determinant of medium-sized bacteriocin production pRL1JI (from isolate 248) was correlated with the appearance of an extra plasmid with a molecular weight of about 130 × 106. Another determinant of medium bacteriocinogenicity, pRL4JI (from isolate 309), was correlated with the presence of an extra plasmid of the same size (about 160 × 106) as one seen in the donor strain 309. The third determinant, pRL3JI (from isolate 306), could not be correlated with the presence of an extra plasmid of the same size as any in strain 306, and it appears that recombination occurred between pRL3JI and plasmids resident in strain 300.

Transfer of nodulation ability (Nod+) from derivatives of Rhizobium leguminosarum strain TOM to strain 16015, a non-nodulating mutant of strain 300, occurred at frequencies of 10−5 to 10−6 per recipient. All 39 Nod+ derivatives of strain 16015 examined had the host-range specificity of the donor strain TOM, i.e. the ability to nodulate the pea cultivar ‘Afghanistan’ and the primitive pea line JI 241 in addition to the commercial variety ‘Wisconsin Perfection’ which can be nodulated by strain 300. The transfer of Nod+ was associated with the appearance in strain 16015 of a new plasmid band on agarose gels (mol. wt about 160 × 106) which corresponded in size to one of the three plasmid bands found in strain TOM: we term this new plasmid pRL5JI. In addition to genes for Nod+ and host-range, the plasmid pRL5JI carries genes for nodule function (Fix+) that are absent from strain 16015. Although strain TOM produces a medium-sized bacteriocin, this property was not co-transferred with pRL5JI. The introduction of pRL5JI did not result in the elimination of any of the resident plasmids of strain 16015. The plasmid pRL5JI differs in many respects from the transmissible bacteriocinogenic plasmids pRL1JI, pRL3JI and pRL4JI previously found, and it appears to belong to a different incompatibility group.

Rhizobium phaseoli strain 1233 forms nitrogen-fixing nodules on Phaseolus vulgaris and produces a brown pigment (probably melanin) in plate culture. This strain contains two plasmids of molecular weight about 200 × 106. Spontaneous deletions in the smaller plasmid abolished pigment production and the ability to nodulate Phaseolus beans, suggesting that genes involved in the determination of both these phenotypes are on this plasmid.

The conjugative Rhizobium leguminosarum plasmid pJB5JI, which carries genes that determine the ability to nodulate peas, was transferred from R. leguminosarum strain T3 into R. phaseoli strain 1233. The majority (about 97%) of transconjugants produced pigment and nodulated both peas and Phaseolus beans poorly. The others (about 3%) had received not only pJB5JI but also an extra plasmid present in strain T3 and they had lost the seller of the two plasmids of R. phaseoli strain 1233, suggesting incompatibility between the plasmid transferred from T3 and that lost from the recipient. These strains did not produce pigment nor did they form nitrogen-fixing nodules on Phaseolus beans, but they nodulated peas as well as did R. leguminosarum strain T3.

Most (about 96%) of the bacteria isolated from nodules following inoculation of peas by the pigmented transconjugants failed to produce pigment. These strains contained the larger plasmid from strain 1233, and pJB5JI, but not the smaller plasmid of strain 1233; they behaved like R. leguminosarum in inducing profuse nitrogen-fixing nodules on peas. The few pigment-producing bacteria isolated from pea nodules had suffered a deletion in the smaller plasmid of strain 1233; they nodulated peas poorly but could not form nitrogen-fixing nodules on Phaseolus beans.

Mutants of Escherichia coli K12 defective in the nirB gene lack NADH-dependent nitrite reductase activity and reduce nitrite slowly during anaerobic growth. With one exception, these mutants require cysteine for growth. Cytochrome c 552 synthesis and the assimilation of ammonia are unaffected by the nirB mutation. The defective gene is located between the crp and aroB genes at minute 73 on the E. coli chromosome. Mapping and reversion studies indicate that nirB is identical to the previously described cysG gene. It is suggested that the product of the cysG + (nirB +) gene is an enzyme required for the synthesis of sirohaem, a prosthetic group of enzymes which catalyse the six-electron reduction of nitrite to ammonia and sulphite to sulphide.

A Mycobacterium sp. which grows on ethylene and ethylene glycol was isolated from ditch water. Growth of the organism, respiration of washed organisms, experiments on excretion of metabolic intermediates and enzyme studies were consistent with a new route for the degradation of ethylene glycol via acetaldehyde and acetate. Cell-free extracts of organisms grown on ethylene glycol contained a diol dehydratase which required K+ or NH4 + for activity as well as coenzyme B12 (5′-deoxyadenosyl cobalamin). Ethylene glycol was not an intermediate in ethylene metabolism.

A detailed radiorespirometric and enzymological analysis was made of the wild-type and GFI strains of Thiobacillus A2 grown on numerous sugars. The wild-type used the Embden-Meyerhof, Entner-Doudoroff and pentose phosphate pathways for glucose oxidation only after culture on glucose. The fast-growing strain GFI used all three pathways after growth on glucose, maltose or fructose. The wild-type grown on a wide range of pentoses, hexoses, disaccharides, a trisaccharide and a mixture of glucose and maltose oxidized glucose by means of the Entner-Doudoroff pathway (68 to 90%) and pentose phosphate pathway (10 to 32%). The key enzyme determining the presence or absence of the Embden-Meyerhof pathway was 6-phosphofructokinase. Other mechanisms, such as a phosphoketolase pathway, were shown to be unimportant, even during growth on pentoses. Arabinose was metabolized via hexose phosphate synthesis. The mechanisms of regulation of sugar metabolism and the energetic significance of alternative pathways are discussed.

Phytophthora palmivora zoospores are repelled by many low molecular weight cations. This negative chemotactic response occurs at a different threshold concentration for each cation, that for the most effective ion, H+, being 150 μ m. The effectiveness of different cations parallels their ionic conductivities, the most active cations having the highest conductivities. It is suggested that the close approach of cations to the cell surface reduces the negative charge at the surface and hence changes the transmembrane potential, altering flagellum activity in such a way as to cause turning and negative chemotaxis.

The amount of major proteins per unit of surface area in the outer membrane (OM) of Salmonella typhimurium LT2 remained constant during steady-state growth in different media. Between growth rates of 2·40 doublings h−1 and 0·31 doublings h−1, the surface density of major OM proteins varied between 0·9 × 105 and 1·2 × 105 molecules per μm2, while surface area per cell more than halved. The accumulation of molecules of the major OM proteins was not affected by addition of cyclic AMP to the growth medium. When exponentially growing cells were subjected to shift-up transitions, cell dimensions began to increase after a lag period of 20 min. Accumulation of major OM proteins followed the same pattern as total protein; this created a transitory imbalance of major OM protein density in the shift from acetate minimal medium to LB medium, before the steady situation was reached. After shift-down transitions, cell dimensions began to decrease immediately, cells eventually becoming shorter than in steady-state conditions. No fluctuations in major OM protein density were observed during the shift-down, although final stable levels differed from those in steady-state conditions. All these results indicate that bacteria adapt the accumulation of major proteins into the OM according to the amount of surface. Thus, no large differences exist at different cell sizes, although transitions between media can lead to transitory or stable changes in the composition of the OM.

Intracellular concentrations of ammonia and amino acids, and the specific activity of the acidic amino acid transport system responsible for transport of low concentrations of glutamate in Aspergillus nidulans have been measured during germination on glutamate and ammonia and during various subsequent incubations of the germinated conidia. It is concluded that intracellular glutamine (and not ammonia) is involved in repression of synthesis of the transport system for low concentrations of glutamate and that intracellular ammonia (but not glutamine) is able to inhibit the preformed permease. This conclusion supports our suggestion that glutamine synthetase may well have a more significant role in the regulation of some aspects of nitrogen metabolism than does glutamate dehydrogenase as was previously proposed ( Pateman et al., 1973 ).

Hyphomicrobium X possesses two different enzymes for the oxidation of dimethylamine, namely dimethylamine dehydrogenase and dimethylamine mono-oxygenase. During growth of the organism in batch culture at dissolved oxygen tensions (DOT) in excess of 30 mmHg in media containing trimethylamine as the carbon and energy source, dimethylamine mono-oxygenase was the only enzyme involved in dimethylamine oxidation. The apparent K m of the mono-oxygenase for oxygen was relatively high (23.2 μ m). The enzyme was less sensitive to inhibition by trimethylamine (K i 4·2 mm) than was dimethylamine dehydrogenase (K i 7·1 μ m) and therefore dimethylamine did not accumulate in the culture medium under these conditions. This was in contrast to observations made during anaerobic growth on trimethylamine. During growth of the organism in dimethylamine-limited chemostat cultures, the specific activities of the mono-oxygenase and the dehydrogenase were dependent on the DOT in the culture. When the DOT in the culture growing at a dilution rate of 0.10 h−1 was decreased below 30 mmHg, the activity of the mono-oxygenase also decreased. In contrast, the activity of dimethylamine dehydrogenase increased, indicating that this enzyme gradually took over at the lower DOT. Below values of 5 mmHg the culture became oxygen-limited and below 3 mmHg it was washed out. When the organism was grown at low DOT in medium supplemented with nitrate, essentially the same results were obtained, except that wash-out of the culture under anaerobic conditions did not occur. The organism was able to carry out denitrification under ‘partly aerobic’ conditions (DOT 0 to 20 mmHg at a dilution rate of 0·10 h−1). Over this range of DOT, the mono-oxygenase hardly played a role in dimethylamine oxidation in vivo because the enzyme was inhibited by the nitrite which accumulated in the culture to a concentration of 3 mm. The potential of Hyphomicrobium X to denitrify in the presence of oxygen was dependent on the growth rate of the organism. At low growth rates (0·01 h−1) synthesis of nitrate reductase, which largely determined the rate of nitrate reduction in the culture, was already significant at relatively high DOT (up to 50 mmHg). At high growth rates (0·15 h−1) nitrate reductase activity became apparent only at DOT values below 6 mmHg. The kinetics and the possible ecological significance of this ‘aerobic’ denitrification process are discussed.