- Volume 127, Issue 1, 1981

Fractionated extracts of Propionibacterium shermanii were found to contain the readily dissociable type of fatty acid synthase complex. For optimal activity the partially purified synthase required the presence of acetyl-CoA, malonyl-CoA, NADH and NADPH as well as a cell-free fraction containing the acyl carrier protein (ACP). When ACP-containing fractions obtained from P. shermanii grown in the presence of [1-14C]pantothenate were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis, the radioactivity was located in a peak corresponding to a molecular weight of approx. 8500–9000. The biological activity of the ACP was relatively heat-stable and trypsin-resistant. When the labelled ACP-containing fraction was subjected to gel filtration on a calibrated Sephadex G-75 column, a radioactive peak corresponding to a molecular weight of 22000 was obtained that could participate in the 14CO2-malonyl-CoA exchange reaction in the presence of the other requisite components.

Hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glucose dehydrogenase, glucose oxidase and gluconate dehydrogenase have been demonstrated in mycelial homogenates from a strain of Aspergillus flavus that produces considerable amounts of kojic acid. The enzymes were assayed after different periods of growth under different experimental conditions and their activities were compared with the rate of kojic acid production. Glucose oxidase activity was too low to account for the formation of kojic acid but the other enzymes had sufficient activities. Correlation of the pattern of enzyme activities under different experimental conditions with kojic acid concentrations provided evidence for the involvement of glucose dehydrogenase and gluconate dehydrogenase in kojic acid biosynthesis. Based on these data a possible pathway for the biosynthesis of kojic acid is presented. Gluconic acid-δ-lactone and at least one of the three compounds, 3-ketogluconic acid lactone, 3-ketoglucose and oxykojic acid, are believed to be intermediates in this pathway.

ATP: citrate lyase has been found in 13 strains of yeast (representing six genera) which are capable of accumulating lipid to above 20 % of their biomass. The enzyme is absent in 10 other yeasts which do not accumulate lipid. The presence of the enzyme is therefore directly correlated to the phenomenon of oleaginicity. The enzyme is located in the cytosol fraction of the yeasts and is probably the sole means of producing acetyl-CoA in most oleaginous yeasts. The specific activity of the enzyme correlates with the specific rate of lipid synthesis as determined in nitrogen-limited chemostat cultures of Lipomyces starkeyi, though not with the lipid content of the cells. From this and by calculation, it may be inferred that the enzyme is possibly the rate-limiting reaction for lipid biosynthesis.

A species of the imperfect fungus Monilia produced extracellular, intracellular and mycelial-bound β-glucosidases when grown on cellulose. The extracellular enzyme was induced on cellulose but was repressed by cellobiose and glucose. Derepression of this enzyme occurred when cellobiose and glucose were nearly exhausted from the growth medium. Intracellular and mycelial-bound β-glucosidases were induced on cellulose and cellobiose but appeared to be repressed on glucose and lactose. One extracellular (EG-1) and two intracellular (IG-1, IG-2) β-glucosidases were produced when Monilia sp. was grown on cellulose. The molecular weights of EG-1 and IG-2 were 37500 and that of IG-1 was 480000. The K m of EG-1 and IG-2 was 8 × 10−5 M for p-nitrophenyl-β-d-glucoside (PNPG), and 5·7 × 10−3 M for cellobiose; the K i for glucose, a competitive inhibitor, was 6·7 × 10−4 m. The K m of IG-1 was 1·67 × 10−3 m for PNPG and 2·0 × 10−2 m for cellobiose; the K i for glucose, a non-competitive inhibitor, was 1·63 × 10−3 m. β-Glucosidases EG-1 and IG-2 showed optimal activity at pH4.5 whilst the pH optimum for IG-1 was 4·2. Temperature optima were 50 °C for EG-1 and IG-2, and 60–65 °C for IG-1. Their respective isoelectric points were 8·3, 8·3 and 4·4.

Protoplasts from four different species of Streptomyces regenerated cells efficiently in hypertonic soft agar medium overlaid on partially dehydrated regeneration medium. The efficiencies of regeneration were strongly dependent upon the incubation temperatures for cell growth and for protoplast regeneration. Cell growth temperatures (before protoplast formation) required for efficient protoplast regeneration varied from species to species, and did not necessarily correlate with the optimum temperatures for protoplast regeneration. Under the best conditions, protoplasts from all four species were able to regenerate viable cells at nearly 100% efficiency and also formed confluent lawns of mycelia when plated in high concentrations. The temperatures for cell growth and protoplast regeneration also affected the frequencies of genetic recombinants obtained by protoplast fusion in S. fradiae, and highest recombinant frequencies were obtained under conditions which favoured efficient protoplast regeneration. With the modified procedure described, maximum frequencies of genetic recombinants were obtained by treating parental protoplasts with 40 to 60% polyethylene glycol 1000.

Cultures of a variety of pneumococcal strains maintained competence longer and gave higher yields of transformants when incubated in M1 medium compared with NS medium. This was most probably due to the cells remaining competent for longer in M1 medium. Various parameters controlling the development of competence in M1 medium were investigated. The onset of competence was delayed in M1 medium compared with that in NS medium, probably due to the presence of Casamino acids in the former. Competence developed normally over a pH range of 7·3 to 8·3, with cultures inoculated from the same batch of frozen ‘precultures’ showing consistent characteristics. This was not observed when frozen ‘sensitization’ cultures were revived.

The average cell chain length increased with the development of competence in all the strains tested and, with the exception of cultures which had entered the stationary growth phase, declined after the culture had lost its competence. The extent of the increase in chain length was dependent upon the pH of the medium.

Five conjugative plasmids governing different antibiotic resistance patterns were identified in wild strains of enteric bacteria isolated in Czechoslovakia and the G.D.R between 1976 and 1979. They have been characterized as members of the new incompatibility group IncU (reference plasmid RA3 from Japan). The molecular sizes of the IncU plasmids ranged between 18 and 37 megadaltons: their restriction fragments patterns indicated them to be distinct types.

The effects of oxolinic acid and novobiocin - which respectively inhibit the A and B subunits of DNA gyrase, and therefore inhibit DNA synthesis - have been examined in sporulating cultures of Bacillus subtilis. Although both inhibitors prevent sporulation, this is not due to inhibition of DNA synthesis. Instead, they affect protein synthesis generally, though weakly, but have very marked effects on the formation of individual enzymes. These effects are reproducible, but the type of enzyme that will be affected is not predictable. The results point to an involvement of DNA gyrase in the transcription of some genes. This is suggested as the reason for the effect of the inhibitors on spore formation, which they block mainly at Stage O-1.

The flow of carbon to α-acetolactate in Escherichia coli K12 is shown to involve the endogenous pool of α-ketobutyrate (α-KB). In vivo, the acetohydroxy acid synthase (AHAS) isoenzymes have an affinity for α-KB sufficiently high that α-acetolactate production is severely limited when α-KB is supplied exogenously. The ability of threonine deaminase to make α-KB is correlated with the synthesis of the AHAS isoenzymes. Mutations in ilvA that alter the catalytic and allosteric properties of threonine deaminase affect α-KB production and the expression of the AHAS isoenzymes in a direct way. The ilvA538 mutation results in a feedback-hypersensitive threonine deaminase and slow α-KB and AHAS production. A spontaneous revertant of an ilvA538 strain expressing a feedback-resistant threonine deaminase produces α-KB and AHAS more quickly. A physiological role for the activator (valine) site on threonine deaminase is proposed and valine is shown to increase α-KB production in vivo. Valine can thus regulate its own biosynthetic pathway without jeopardizing the production of isoleucine. The physiological implications of the role of α-KB in the biosynthesis of acetolactate are discussed.

Growth of Erwinia chrysanthemi in a glucose/yeast extract/salts medium, adjusted to various water activities (a w) with NaCl, was not observed below 0·970 a w. Growth rate increased when a w was lowered from 0·998 to 0·990, below which growth rate again declined rapidly. Similar results were obtained using mannose to adjust a w. Growth rate declined linearly between the limits of 0·998 a w and 0·970 a w on a sodium polypectate/yeast extract/salts medium adjusted with mannose. Extracellular pectate lyase (PL) production on the sodium polypectate/yeast extract/salts medium increased when a w was lowered from 0·998 a w to 0·990 a w using NaCl as a w adjuster. However, when either mannose or lactose was used to adjust a w over this range, PL production decreased considerably. PL was more stable in the presence of NaCl than with lactose or mannose. The significance of the differential effects of NaCl as opposed to organic a w adjusters on PL production is discussed.

Asexual sporangiospores of Mucor racemosus produced on a minimal sporulation medium (M-spores) germinated only if glucose, mannose or a complex substrate such as peptone, yeast extract or Casamino acids was present. Once germinated, growth was supported by a wide range of substrates including amino acids, carbohydrates or organic acids. Sporangiospores produced on a nutritionally complex sporulation medium (C-spores) germinated on a wide range of carbon sources. C-spore phenotype was pleiotropic in that sporangiospores capable of germinating on cellobiose could always germinate on glutamate or xylose; but C-spores capable of germinating on xylose or glutamate did not always germinate on cellobiose. There was a hierarchy of substrates capable of initiating germination with glucose = mannose > xylose > glutamate > cellobiose. C-spores also differed from M-spores by initiating germination in the presence of the non-metabolizable glucose analogue 3-O-methylglucose. These results suggest that at least two sporangiospore phenotypes are produced depending upon the concentration and type of ingredients present in the sporulation medium.

5-Fluorouracil powerfully inhibits growth of Candida utilis. Isolates that are resistant to fluorouracil all have a reduced ability to transport uracil but most also have other defects. Their capacity to take up a wide range of amino acids is greatly reduced, as is their ability to alter rates of amino acid transport during nitrogen starvation. These isolates may be defective in the coupling of energy generation to transport systems.

The influence of pH on chitin hydrolysis by Streptomycetes from a range of acidic and neutral soils was studied in vitro in an acid soil. On the basis of activity ranges and optima for chitin hydrolysis, acidophilic, acidoduric and neutrophilic categories of Streptomycetes were distinguished; these categories were broadly related to the pH requirements for growth. Responses of Streptomycetes to chitin amendment of acidic organic and mineral horizons of a pine forest soil were studied. Acidophiles were involved in the decomposition process and the resulting ammonification led subsequently to activity of neutrophiles. This succession was particularly marked in the poorly buffered mineral horizon. Similar, but smaller, responses occurred when the horizons were amended with mycelium from basidiomycete sporocarps. The role of streptomycetes in decomposition of fungal chitin in acidic litters and soils is discussed.

The organization and expression of the pyruvate dehydrogenase complex genes, aceE (E1), aceF (E2) and lpd (E3), were investigated using a series of λ transducing phages carrying different segments of the nadC-aroP-aceE-aceF-lpd region of the chromosome of Escherichia coli. The polypeptides synthesized following the infection of u.v.-irradiated lysogenic and non-lysogenic hosts were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography.

The aceE and lpd gene products were readily identified by their sizes, M r 100000 (pyruvate dehydrogenase. E1 component) and 56500 (lipoamide dehydrogenase. E3 component), but considerable heterogeneity was detected for the aceF gene product (acetyltransferase, E2 component). The main E2 polypeptides, M r 80000 and 83000, were accompanied by a family of minor polypeptides. M r 86000, 89000 and 91000, but no precursor-product relationships were apparent. A very large polypeptide, M r 190000, was also detected and found to contain the E1 component but in uncertain combination. In addition, the product of a gene in the nadC-aroP region was detected as a polypeptide with M r 36500.

The existence of a single promoter for the aceE and ace genes and a separate promoter for the lpd gene was confirmed. The lpd gene was shown to be transcribed with the same polarity as the ace genes (clockwise with respect to the E. coli linkage map). The relative rates of expression of the three genes from the bacterial promoters were estimated as 0·94:1·0:1·4–2·3 (E1:E2:E3) on a molar basis. The excess production of lipoamide dehydrogenase components (E3) indicates that the lpd promoter functions independently, at least in supplying components for the 2-oxoglutarate dehydrogenase complex. When expressed from PL, the powerful phage promoter, the ace and lpd gene products accounted for the greater part of the newly synthesized protein.

Spores produced by a mutant of Bacillus subtilis germinate slowly and are sensitive to lysozyme. The spore coat of the mutant appears to lack a single polypeptide (mol. wt 36000) which normally is deposited in the outer layers of the spore during stage V of sporulation. The mutation maps between leuA and argA on the B. subtilis chromosome and is genotypically and phenotypically distinct from another mutation ger-36 which maps in the same region: consequently it has been assigned a new locus spoVIA. The phenotype of the mutant confirms a previous suggestion that lysozyme resistance is conferred by the outermost layers of the spore coat.

The specific activities of the key enzymes involved in the biosynthesis of the exopolysaccharide alginic acid by Azotobacter vinelandii were determined in extracts of batchcultured organisms grown with different carbon sources in the presence of limited and excess inorganic phosphate. Alginic acid production was also measured. Glucose, fructose, sorbitol, mannitol, glycerol and gluconate resembled sucrose in supporting much greater alginate production in media containing growth-limiting amounts of inorganic phosphate. Mannose supported only poor growth with no alginate formation, and growth did not occur on acetate. Increases in the specific activities of phosphomannose isomerase, GDPmannose pyrophosphorylase and GDPmannose dehydrogenase were accompanied by increased alginic acid production. Our results accord with the suggestion that alginate formation is controlled by derepression of key biosynthetic enzymes.