- Volume 120, Issue 1, 1980

The activity of enzymes involved in polyol metabolism was investigated in uredospores and mycelial cultures of the flax rust fungus, Melampsora lini. Mycelial cultures displayed polyol dehydrogenase activity with a range of sugars using either NADH or NADPH as a cofactor. Highest activity was found with fructose and NADPH. The relative pattern with different substrates was unaffected by the carbohydrate used in the culture medium. Highest absolute activity was detected with culture media containing d-glucitol or d-mannitol. Mannitol and d-arabitol were the polyols oxidized by mycelial extracts. Flax rust uredospores could use aldohexoses, ketohexose and aldopentoses as substrates for polyol synthesis. Uredospore extracts were also capable of oxidizing d-arabitol with NAD+ as cofactor.

A micro-organism capable of growth on cyclohexane as the sole carbon source has been isolated from soil of an ash wood; the organism has been identified as a pseudomonad. Growth, respiration and enzymic studies with the organism are consistent with an oxidation route for cyclohexane proceeding via cyclohexanol, cyclohexanone, ε-caprolactone, 6-hydroxycaproate and adipate. Cell-free extracts of the organism grown on cyclohexane demonstrated cyclohexane hydroxylase activity which was found to be very labile, dependent on molecular oxygen and specific for NADH; the product of this reaction was identified as cyclohexanol.

Viomycin and capreomycin, antibiotics produced by Streptomyces vinaceus and S. capreolus respectively, are potent inhibitors of bacterial protein synthesis. Although these organisms are highly tolerant of their own products in vivo, their ribosomes are fully sensitive to the action of the drugs in vitro. However, they possess novel, antibiotic-inactivating enzymes (viomycin phosphotransferase, capreomycin phosphotransferase, capreomycin acetyl-transferase) which, in addition to possible biosynthetic roles, may contribute to the resistances observed in vivo.

Acetone precipitates from culture filtrates of three isolates of Botrytis cinerea were resolved by isoelectric focusing on polyacrylamide gels to detect differences in the polygalacturonase and protein patterns. Only a few bands – four in the protein patterns and two in the polygalacturonase patterns – were common to all the isolates. Differences were also detected in polygalacturonase and protein patterns of the same isolate at different ages of culture (7, 14 and 21 d). Identical polygalacturonase patterns were obtained when isoelectric focusing was applied to an acetone precipitate either directly or after further purification by ion-exchange chromatography.

The extractability of carotenoid pigments from whole cells by sequential extraction at room temperature with polar and non-polar organic solvents, Triton X-100 and sodium dodecyl sulphate was studied in 40 pigmented, non-photosynthetic bacteria from 15 genera. The bacteria segregated into four groups according to the extractability of their pigments with detergent and methyl alcohol. There was no correlation between extractability and taxonomic status. Solvent and detergent extractions were effective with only 50% and 35% of the bacteria, respectively. Lyophilization of cells prior to extraction did not enhance extract-ability with solvents. Complete removal of the pigments from selected organisms with diethyl ether (following pretreatment with acetone) and with Triton X-100 could be obtained from cell wall and membrane fragments after disruption of the cells. A single extraction of wall and membrane fragments with methyl alcohol was effective in all but one case. The evidence indicates that the pigments are probably associated with protein and that the cell wall or intact membrane is the barrier to their extraction from whole cells.

A method has been developed for the continuous spectrophotometric assay of dihydroxy-acetone synthase present in methanol-grown yeasts. This enzyme catalyses the condensation of formaldehyde with a pentose phosphate (most probably xylulose 5-phosphate) to give dihydroxyacetone and glyceraldehyde 3-phosphate. The assay is based on the NADH-and ATP-linked formation of l-glycerol 3-phosphate from dihydroxyacetone via the added coupling enzymes glycerol kinase and glycerol-3-phosphate dehydrogenase, and the reaction was followed by the decrease in absorbance at 340 nm. Using this assay, the pH optimum of the dihydroxyacetone synthase was shown to be 7.4 to 7.6. The enzyme, which catalyses a transketolase-like reaction, is a separate enzyme from the classical transketolase because their specific activities varied independently in Candida boidinii and Hansenula polymorpha grown on different substrates and the two enzyme activities could be separated by ion-exchange chromatography.

Between 30 s and 5 min after transfer from light to darkness, the rate of acetylene reduction by cultures of the unicellular cyanobacterium Gloeocapsa sp. 1430/3 decreased rapidly. During the same period, there was a rapid disappearance of storage glucan. There was also a sharp fall in the intracellular concentration of ATP followed by a slower recovery. It is concluded that, in the dark, the catabolism of storage glucan is the only potential source of ATP and/or reductant and that the low rate of N2 fixation in the dark is not due to limitations in the supply of ATP.

Thirteen mutants unable to grow on pyruvate have been isolated from the facultative methylotroph Pseudomonas AM1. Five of these lacked 2-oxoglutarate dehydrogenase and were thus obligate methylotrophs; one lacked the E1 (decarboxylase) component and the others probably lacked the E2 (transsuccinylase) component, but this could not be confirmed because transacylases could not be measured in the oxo-acid dehydrogenases of Pseudomonas AM1. Six mutants lacked pyruvate dehydrogenase (probably the E2, trans-acetylase, component) and one lacked both oxo-acid dehydrogenases. Although unable to oxidize pyruvate, one of the pyruvate dehydrogenase mutants was able to catalyse a coenzyme A-independent decarboxylation of pyruvate. The growth properties of the pyruvate dehydrogenase mutants confirmed that the loss of this enzyme is sufficient to confer on a previously facultative methylotroph the properties of a restricted facultative methyltroph similar to the hyphomicrobia. One mutant was only able to grow on C1, C2 and C3 compounds when a supplement of glyoxylate was added but was able to grow on other multicarbon compounds such as succinate. This confirms that there is a common reaction in the pathways for assimilation of C1, C2 and C3 compounds in this organism.

Pseudomonas AM1 grows on 1,2-propanediol with a mean doubling time of 7 h. The growth responses of a wide range of mutants suggest that propanediol is assimilated into cell material by way of pyruvate and acetyl-CoA. The normal enzymes for the initial metabolism of propanediol [NAD(P)+-linked dehydrogenase, propanediol oxidase and propanediol dehydratase] were absent. The initial oxidation of propanediol was catalysed by methanol dehydrogenase but only in the presence of a large molecular weight (possibly protein) stimulatory factor that seemed to change the substrate specificity of the enzyme; the product of the oxidation was lactate.

The asymmetry of the DNA duplex due to polynucleotide strand complementarity could be the molecular basis of cell polarity in spore-forming bacteria. To test this possibility, the relationship of DNA strand segregation to the spore location pattern in chains of sporangia was investigated in Bacillus megaterium. Spores containing one chromosome labelled in one of the complementary strands were formed from cells that had been allowed to segregate pulse-labelled chromosomes in minimal medium at 30°C. A second crop of spores was then formed from cells originating from the labelled spore population. The second generation spores inherited labelled strands from the first spore population by random segregation. In contrast, the patterns of spore positions in sporangial chains were non-random. Furthermore, the non-randomness of patterning was stable and was unaffected by growth temperature (15 to 37 °C) or by enrichment of the minimal medium used in the segregation experiments. Since the pattern of DNA strand segregation is random and the spore location pattern in chains of sporangia is non-random, the asymmetry of the DNA duplex cannot be the determinant of cell polarity.

The formation of the glyoxylate cycle enzymes isocitrate lyase and malate synthase is strongly regulated in Aspergillus nidulans and the enzymes are induced at high levels in mycelium grown on acetate and found at low levels in mycelium grown on hexose. A search was made for constitutive mutants forming the enzymes when grown on hexose by the use of a pyruvate carboxylaseless (pycA) strain. This strain does not grow on hexose since it requires a source of C4 tricarboxylic acid cycle intermediates and it was hoped that among revertants selected for growth on hexose some may effect C4 synthesis by the constitutive formation of the glyoxylate cycle enzymes.

Four constitutive strains were isolated. Three had no pyruvate carboxylase activity and each was found to contain two new mutations: a suppressor mutation su + of the pyruvate carboxylase lesion, and a mutation icl c which caused low constitutive isocitrate lyase activity. The greater activities in the constitutive strains were due to interactions between the icl c and the su + and pycA mutations.

The four low-level constitutive mutations icl c defined two genes affecting isocitrate lyase formation. Recessive mutations in icl c A (linkage group IV; three alleles) caused 10-fold increased activities in sucrose-grown mycelium, as did a semi-dominant mutation in icl c B (linkage group I). The icl c genes are not linked to acuD (structural gene for isocitrate lyase) and did not detectably affect the formation of either malate synthase or acetyl-CoA synthase, the structural gene (acuA) for which is tightly linked to acuD. There was a synergistic interaction in the double mutant icl c A1; icl c B1. Two alternative interpretations of the icl c genes remain open: one is a model for genetic regulation with negative (icl c A gene product) and positive (icl c B gene product) elements; the other is endogenous induction due to the accumulation of metabolites resulting from the unidentified metabolic lesions. We did not find any icl c B mutations uninducible for isocitrate lyase amongst acetate non-utilizing mutants.

A fifth isocitrate lyase constitutive mutant was isolated in the wild-type strain. The mutant contained another recessive mutation (A4) in the icl c A gene.

The high constitutive isocitrate lyase activities in icl c A1; icl c B1 double mutants did not provide replacement of the C4 requirement in a pyruvate carboxylaseless strain since the triple mutant pycA3; icl c A1; icl c B1 did not grow on sucrose. Revertants of this strain selected for growth on sucrose again contained suppressor mutations su + of the pycA lesion and the high constitutive activities for malate synthase decreased as known mutant genes were replaced. The nature of the suppressor gene su + function(s) providing an alternative source of C4 intermediates for growth is not known.

The assignment to linkage group VII of the coumarin sensitivity mutation couA351, which leads to a loss of colony-forming ability on agar containing coumarin, was established using the linkage group VII markers cobA1 and tsgK21. Complementation of the couA351 and bsgA5 mutations was an effective method of selecting heterozygous diploids at 211C, i.e. without requiring a temperature sensitivity mutation. A morphological mutation, frtB353, which affects the distribution of fruiting bodies was also assigned to linkage group VII. Both the couA351 and frtB353 mutations were discovered in the tsgK21 strain NP187. Eight independently isolated, recessive mutations leading to resistance to 300 g CoCl2 ml-1 were allelic with the cob A1 mutation. The partially dominant cob-353 mutation was shown to map in linkage group VII on the basis of its segregation relative to the couA351 and tsgK21 mutations and is almost certainly an allele of the cobA locus. These results are consistent with there being only a single locus at which mutations can lead to resistance to high concentrations of CoCl2. Two temperature sensitivity mutations tsgM357 and tsgG4 were also assigned to linkage group VII.

The labelling of DNA by pulse/chase experiments in Staphylococcus aureus has been investigated, analysing the products by alkaline sucrose velocity centrifugation. In S. aureus NCTC 8325 a short (60 s) pulse of [3H]thymidine labels both small (10 to 20 S) fragments and DNA that co-sediments with long-term label. In a thymidine-requiring derivative, 8325thy, most pulse label is incorporated into small fragments. In both bacterial strains small fragments can be chased into high molecular weight DNA. Thymidine starvation of 8325thy prior to pulse labelling results in smaller fragments (4 to 10S) being labelled. In a subsequent chase with unlabelled thymidine this label is incorporated into high molecular weight DNA, although more slowly than in the absence of thymidine starvation. The fact that nalidixic acid, an antibiotic which specifically inhibits DNA replication in S. aureus, does not inhibit the [3H]thymidine incorporation immediately after thymidine starvation and that nalidixic acid slows down the increase in size of pulse-labelled fragments through inhibition of DNA synthesis suggests that thymidine starvation results in changes at the replication fork. The possible nature of these changes is discussed. It is proposed that one of the results of thymidine starvation is to cause a long-lived gap between DNA synthesized before starvation and DNA synthesized after starvation.

Optimum conditions of pH, temperature and multiplicity of bacteriophage infection for the production of phage-associated lysin from Group C streptococci were defined. Lysin obtained by the optimum method was used to make protoplast membranes of Group A type 12 streptococci. These membranes consistently contained very low concentrations of rhamnose, indicating minimal cell wall contamination. The development of such a reproducible technique for the preparation of protoplast membranes will permit more meaningful studies on the involvement of these membranes in the pathogenesis of acute post-streptococcal glomerulonephritis.