- Volume 128, Issue 8, 1982

Production of the volatile metabolite, isobutyraldoxime O-methyl ether (IBME) by a Moraxella-like organism NCIB 11650 was investigated under a variety of environmental conditions using gas chromatography. Under aerobic conditions up to 10 μg IBME ml−1 was produced on mineral salts media containing 0.5% (w/v) glucose or succinate as sole C source with 0·1% (w/v) NH4Cl as sole N source. Exogenous l-valine further stimulated IBME formation up to 25 μg ml−1 but supplementation of the medium with d-isomer or other amino acids had little effect on IBME production and did not lead to the appearance of analogues of IBME. Trapping experiments using [14C]valine confirmed that IBME was derived from this amino acid. Several other bacterial species examined, e.g. Alcaligenes sp. NCIB 11652, another Moraxella-like organism NCIB 11651 and Pseudomonas sp. NCIB 11653 also produced IBME under similar conditions. The Alcaligenes strain synthesized up to 20 μg ml−1 in the absence of valine and up to 90 μg ml−1 in its presence.

The production of IBME exhibited many features characteristic of the formation of a secondary metabolite. Thus biosynthesis was confined to a narrower range of temperature than cell division, was almost completely suppressed by 300 mm-phosphate and was inhibited by high concentrations of readily utilizable C sources. Although IBME synthesis in the Moraxella-like organism NCIB 11650 appeared to be growth-related, its formation by both the Alcaligenes sp. and the Moraxella-like organism NCIB 11651 was delayed until the late-exponential and early-stationary phases of growth. The biological significance of this novel class of secondary metabolite is discussed and a possible biosynthetic route proposed.

Glutamate synthase (GOGAT), glutamine synthetase (GS), NAD+-dependent glutamate dehydrogenase (GDH) and NAD+-dependent alanine dehydrogenase (AlaDH) activities were detected in cell extracts of Bacillus stearothermophilus PH24, a strain deficient in NADP+-dependent GDH. GDH and GOGAT activities were low under most growth conditions and GOGAT was not detectable in extracts of cells grown with amino acids as carbon and nitrogen source. AlaDH and GS activities were more variable, the former being high in cells grown on l-alanine as carbon and nitrogen source. GS was repressed during growth with high concentrations of NH4C1 as nitrogen source but a corresponding increase in AlaDH activities suggests that this enzyme may replace NADP+-dependent GDH as the main enzyme for ammonia assimilation under these conditions.

GOGAT was purified 40-fold using affinity chromatography on NADPH-Sepharose. The molecular weight of the partially purified enzyme was estimated to be 160000 and K m values for NADPH, 2-oxoglutarate and l-glutamine were 22, 15 and 29 μm, respectively. Glutamine could be replaced by NH4C1 as nitrogen donor (K m 44 mm) but the rate was only 10% to 15% that of the l-glutamine-dependent reaction. The pH optimum for glutamine-dependent activity was 8·0 and the temperature optimum 75 C: the enzyme displayed a discontinuous Arrhenius plot over the range 30 °C to 75 °C. Azaserine, l-methionine sulphone and Cibacron Blue 3GA were all inhibitors and the enzyme was rapidly inactivated in the presence of NADPH when l-glutamine and 2-oxoglutarate were absent.

Pseudomonas putida PP3 grew on dl-2-monochloropropionate (2MCPA) with a release of chloride ions consistent with the dechlorination of both isomers. The organism grew on either d-or l-2MCPA. Dehalogenase activity in cell-free extracts showed that both d-and l-2MCPA were dehalogenated. Pseudomonas putida PP3 contains two dehalogenases, and studies with the separated enzymes revealed that the fraction I enzyme used both d-and l-2MCPA, the rate of dechlorination of l-2MCPA being 80% of the rate of d-2MCPA dechlorination. The product of the reaction, lactate, retained the same optical configuration as the substrate provided. The fraction II dehalogenase also dechlorinated d-and l-2MCPA, with the same difference in rates as for the fraction I dehalogenase, but the lactates produced were of the opposite configuration to their precursors. The two dehalogenases showed further differences with respect to inhibition by two sulphydryl-blocking agents, N-ethylmaleimide and p-chloromercuribenzoate. Fraction I dehalogenase was considerably more sensitive to these two reagents compared with the fraction II dehalogenase. Dithiothreitol partially protected the fraction I dehalogenase from N-ethylmaleimide inhibition. The results are discussed in terms of the possible evolutionary relationships of the two dehalogenases.

Crude cell extracts of the fission yeast Schizosaccharomyces pombe (strains NCYC 132 and 972h−) can phosphorylate dihydroxyacetone but not glycerol; activity for dl-glyceraldehyde is very low. This suggests that a specific dihydroxyacetone kinase is present and catalyses the second step in a pathway for glycerol utilization, in which the initial step is an oxidation by an NAD+-linked glycerol dehydrogenase. In support of this pathway (glycerol→dihydroxyacetone→dihydroxyacetone phosphate), both strains can utilize glycerol or dihydroxyacetone. but not dl-glyceraldehyde, as growth substrates. Both enzymes are subject to catabolite repression and may also be inducible but are not co-ordinately regulated.

Malate dehydrogenase (MDH; EC 1.1.1.37) from Dictyostelium discoideum was purified and characterized. MDH activity from whole cells was purified 275-fold. The mitochondrial cytoplasmic MDH present co-purified through three ion exchange and affinity chromatography steps. The isoenzymes were barely separable by either disc gel electrophoresis or isoelectric focusing. The purified preparation containing both isoenzymes had a single pH optimum (9·3–9·5) and an apparent molecular weight of 70000. It exhibited linear kinetics and responded to known inhibitors of MDH, i.e. thyroxine and hydroxymalonate. Michaelis and dissociation constants obtained with this preparation were similar to those obtained with a 10-fold purified mitochondrial MDH.

Nitrobacter agilis, a chemolithotrophic bacterium, utilizes ammonia as well as nitrite as a nitrogen source for growth. The growth yields were increased about twofold by growing the bacterium in a nitrite medium supplemented with 2 mm-ammonium chloride. Higher concentrations of ammonium chloride, however, competitively inhibited nitrite oxidation and growth of the bacterium. Washed cells readily incorporated 15NH4 +, 15NH2OH, 15NO2 − and 15NO3 − respectively (in decreasing order) into cell nitrogen. Enzyme activities in cell extracts of ammonia-supplemented cultures, compared to those without ammonia, were greater for glutamate dehydrogenase (EC 1.4.1.2) and similar for glutamine synthetase (EC 6.3.1.2), whereas glutamate synthase (EC 1.4.1.14) was barely detectable. Inhibitors of glutamine synthetase (l-methionine dl-sulphoximine) and glutamate synthase (azaserine) did not affect the incorporation of 15NH4 − and 15NO2 − into cell nitrogen of washed cells. The results indicate that glutamate dehydrogenase is a key enzyme in N. agilis for the assimilation of either nitrite or ammonia. Glutamine synthetase, which was also active in cell extracts, is probably required for the production of glutamine. Glutamate synthase, however, does not appear to be an important enzyme for ammonia assimilation.

A total of 46 rhizobial strains were assessed for anaerobic growth in the presence of nitrate, and, using the criteria of nitrate utilization and nitrous oxide and nitrogen production, for their ability to denitrify. Nitrite production was also measured. Half of the strains were denitrifiers: these included all five strains of R. meliloti tested which produced N2 from nitrate and most of the slow-growing rhizobia, but none of the 14 strains of R. trifolii.

Nine strains of Agrobacterium produced extracellular cyclic (1→2)-β-d-Glucan. Most of the strains produced an octasaccharide repeating-unit of succinoglycan. Mutant strains of Agrobacterium sp. and mutant strain 10C3K derived from Alcaligenes faecalis var. myxogenes, producing curdlan without succinoglycan or with a slight amount of succinoglycan, produced only cyclic (1→2)-β-d-Glucan. Cyclic (1→2)-β-d-Glucan was shown by paper chromatography and methylation analysis to be composed of two components without other glucosidic linkages. These were confirmed to be heptadecaose and octadecaose by field desorption mass spectrometric analysis.

The permeability of intact gas vesicles, isolated from the cyanobacterium Anabaena flos-aquae, to percyclofluorobutane (C4F8), has been investigated by two methods. Warburg manometry showed that a suspension of intact gas vesicles exchanges more C4F8 than an equivalent suspension of collapsed ones. Pressure nephelometry showed that increasing the partial pressure of C4F8 in solution caused a corresponding increase in apparent critical collapse pressure of the gas vesicles. From these observations it is concluded that C4F8 is able to permeate freely through the gas vesicle wall. If it permeates via pores, these must be at least as large as the gas molecules, which have a collision diameter of 0·63 nm. Warburg manometry showed that the volume of C4F8 exchanged exceeds the volume of gas-vesicle gas space, and this indicates that, in addition, there is specific absorption of the gas, probably on the inner, gas-facing surface of the gas vesicle wall.

Auxotrophic, morphological and drug-resistant mutants of Cochliobolus heterostrophus (= Bipolaris maydis = Helminthosporium maydis = Drechslera maydis) were isolated after mutagenesis with ethyl methanesulphonate. Exposure of the fungus to other mutagens (N-methyl-N′-nitro-N-nitrosoguanidine, nitrous acid, and UV light) resulted in recovery of morphological but not auxotrophic mutants. Antibiotic enrichment or filtration enrichment in medium containing high levels of sorbose increased the proportion of auxotrophs in the surviving population. All sexually fertile mutants were found to have single gene lesions; those with the same phenotype were allelic in some cases but not in others. Defined minimal and complete media, designed to facilitate genetic and molecular biological manipulations, supported maximal growth rate at 30 °C and maximal conidiation of the fungus at 23 °C with a 16 h long-wave UV light photoperiod. Under all in vitro growth conditions tested, near-isogenic lines of two known races of the fungus were essentially identical. Sexual fertility was improved by a programme of breeding and selection, but not by physical or chemical changes in the medium used for crossing. The fungus can be stored conveniently on silica gel.

A temperature-sensitive DNA replication mutant of Staphylococcus aureus NCTC 8325 has been isolated and characterized. After transfer to the non-permissive temperature (42 °C), DNA synthesis continued for 30 min and the mean DNA content increased by 56%. The amount of residual DNA synthesis was not reduced when the non-permissive temperature was raised, nor when chloramphenicol was added at the time of the temperature shift. During incubation at 42 °C, mutant bacteria accumulated the capacity to synthesize DNA after return to the permissive temperature (30 °C) in the presence of chloramphenicol. This capacity was lost when chloramphenicol was present at 42 °C. The properties of the mutant are consistent with a defect in the initiation of DNA replication at 42 °C.

A UV endonuclease with activity towards UV-irradiated lambda DNA but no activity towards non-irradiated DNA was isolated from a crude lysate of the unicellular cyanobacterium Synechocystis PCC 6308. The enzyme has a molecular weight between 15000 and 18000. ATP was essential for maximum enzyme activity and the optimum pH was 7·5–7·8.

A new class of regulatory mutants in the cysB locus has been isolated by plating cysM strains, under anaerobic conditions, on medium containing 1,2,4-triazole. The isolated cysB mutants are cysteine prototrophs and triazole-resistant, although the levels of cysteine and O-acetyl-l-serine sulphydrylase are not changed. In contrast to the constitutive cysB mutants identified previously, the expression of the cysteine biosynthetic enzymes in the newly isolated mutants is regulated by the same factors as in wild-type strains. In the double mutant cysE cysB2971, the cysteine biosynthetic enzymes are absent with the exception of O-acetyl-l-serine sulphydrylase.

Salmonella typhimurium cysM mutants grow at a normal rate under aerobic conditions, but only after a lag period under anaerobic conditions. No difference in the induction of two cysteine biosynthetic enzymes sulphite reductase and O-acetyl-l-serine sulphydrylase in wild-type, cysK and cysM strains was observed under aerobic conditions. Under anaerobic conditions, however, the cysM strain differed from the others in showing a long delay in the induction of sulphite reductase. These observations are consistent with the assumption that the observed growth delay of the cysM mutant under anaerobic conditions is the result of abnormalities in the regulation of sulphite reductase.

Plasmids pRL1JI and pRL6JI, which carry determinants essential for symbiosis in Rhizobium leguminosarum feld isolates 248 and 128C53, respectively, were both incompatible with two other transmissible plasmids that did not carry symbiotic determinants. When derivatives of these two plasmids (pRL3JI or pRL4JI) were introduced into a strain which already contained pRL1JI or pRL6JI, recombinant replicons were often obtained: these were of uniform size for each pair of incompatible plasmids. Recombinant nodulation plasmids were also observed following infrequent interactions with pRL10JI, a natural nodulation plasmid that does not belong to the same incompatibility group as pRL3JI or pRL4JI.

By the formation of a recombinant nodulation plasmid, it has been possible to transfer the determinants for nodulation ability (Nod+), nitrogen fixation (Fix+) and hydrogen uptake (Hup+) from pRL6JI, a plasmid that was not self-transmissible, on to a replicon that was transmissible at high frequency and carried a selectable drug resistance marker. In the case of pRL10JI, which is also a non-transmissible nodulation plasmid, the formation of a recombinant nodulation plasmid transferred the symbiotic determinants to a transmissible replicon belonging to a different incompatibility group from pRL10JI itself.

Transfer of plasmid pIJ1008, a recombinant of two indigenous Rhizobium leguminosarum plasmids into R. leguminosarum strain 300 produced strain 3960, which reduced significantly more N2 in pea root nodules than did strain 300 itself. Strain 3960 was superior to the field isolate 128C53, from which the symbiotic determinants of pIJ1008 were derived, and transfer of plasmid pIJ1008 into two other genetic backgrounds also improved symbiotic performance compared to the introduction of other nodulation plasmids.

As plasmid pIJ1008 carries genetic determinants for an uptake hydrogenase activity (Hup+) as well as nodulation capability (Nod+) and other determinants for symbiotic nitrogen fixation (Fix+), the increased effectiveness of strains carrying pIJ1008 may result from their capacity to conserve energy by recovering H2 evolved by nitrogenase.

Intact plant studies with 15N showed that the superior N2 fixation capability associated with pIJ1008 was enhanced by 2 mm-NO3 −, a common concentration of soil N. It was also shown that, as plants grew older, the hydrogenase determined by pIJ1008 was not able to recycle all the hydrogen evolved by pea root nodules.