- Volume 124, Issue 2, 1981

SUMMARY: The passive permeation and facilitated diffusion of glycerol into Escherichia coli K1060, an unsaturated fatty acid auxotroph, were studied as a function of temperature and membrane lipid fatty acid composition using a stopped-flow spectrophotometric assay of glycerol permeation. The relative rates of glycerol passive and mediated entry were both significantly influenced by the fluidity of the membrane lipids, increasing as the gel to liquid-crystalline phase transition midpoint temperature of the membrane lipids decreased. The rate of passive glycerol permeation, but not the rate of glycerol facilitated diffusion, decreased as the membrane lipids were converted to the gel state. The apparent activation energies for passive and facilitated diffusion of glycerol, measured in cells whose membrane lipids were in the liquid-crystalline state, were 15–16 and 10–11 kcal mol−1, respectively, and neither value was significantly influenced by the fatty acid composition or fluidity of the membrane lipids. The mechanistic implications of these observations for the function of the glycerol facilitated diffusion system of E. coli are discussed.

SUMMARY: Crude extracts of Candida boidinii grown on glucose, xylose or ethanol gave single peaks of classical transketolase activity following chromatography on columns of hydroxylapatite; the enzyme was heat-stable and showed no appreciable activity with formaldehyde as acceptor in place of ribose 5-phosphate. Extracts of methanol-grown cells showed two peaks of transketolase activity following chromatography on both hydroxylapatite and DEAE-cellulose. One peak was identified with that found for the cells grown on substrates other than methanol; the other peak showed dihydroxyacetone synthase activity in addition to transketolase activity. Both activities in the latter peak were very unstable and have been ascribed to one enzyme on the basis of identical rates of denaturation at all temperatures tested between 0 and 40 ¼C. It is suggested that this enzyme is a special transketolase synthesized only during methylotrophic growth of the yeast and, in contrast to classical transketolase, is capable of using equally well either formaldehyde or ribose 5-phosphate as glycolaldehyde acceptor. A method based on heat treatment has been suggested for the simultaneous assay of both transketolases present in crude extracts of a methylotrophically grown yeast.

An alcohol dehydrogenase of broad specificity was purified 43-fold from extracts of Mycobacterium tuberculosis var. bovis (BCG) grown on Sauton medium. It was also present in cells grown in Dubos medium with Tween 80 and bovine serum albumin. The enzyme, which appeared to be soluble, acted as an oxidoreductase in the system butan-1-ol–NADP. It was eluted from Sephadex G-200, hydroxylapatite and DEAE-cellulose in a single peak. The molecular weight, as determined by gel filtration on Sephadex G-200, was about 75 000. Results of electrophoresis in sodium dodecyl sulphate–polyacrylamide gels were compatible with the existence of two subunits each of molecular weight 37 500. The optimum pH was about 8·5 when the enzyme catalysed the oxidation of butan-1-ol, and about 8·2 for the reverse reaction. The apparent K m was 0·125 mm for butyraldehyde and 0·22 m for butan-1-ol. The dehydrogenase activity was maintained after heat treatment (40 min at 55 °C) in the presence of 30% (w/v) glycerol, but was abolished by heating (40 min at 55 °C) in the presence of 0·1 m-EDTA. The activity of enzyme inactivated by heat and EDTA could be fully restored at room temperature in the presence of 2 mm-Zn2+.

Three methods for the purification of Staphylococcus aureus delta haemolysin were compared ( Kantor et al., 1972 ; Kreger et al., 1971 ; Heatley, 1971 ). The products of these purifications from the culture supernatant of S. aureus strain RN25 were compared by electrophoresis, amino acid analysis, amino-terminal sequence analysis and thin-layer chromatography. The method of Heatley (1971) was found to be superior in terms of recovery and purity of the product. Delta haemolysin prepared by the method of Kreger et al. (1971) could not be sequenced successfully prior to treatment aimed at the removal of the N-formyl group at the amino-terminus. Delta haemolysin appears to exist in two distinct molecular forms, one with N-formylmethionine and the other with un-formylated methionine in the amino-terminal position. The former polypeptide species is purified preferentially by the method of Kreger et al. (1971) . Thin-layer chromatography of the products of each method of purification revealed that they were all heterogeneous, although the major component from the product of the method of Heatley (1971) represented not less than 70% of the product.

Glucan synthase activity in cell-free extracts of Saccharomyces cerevisiae was partially stabilized when cells were broken in the presence of sucrose. Under these conditions a significant amount of enzyme activity remained in the supernatant after high-speed centrifugation. When this supernatant fraction was incubated with UDPglucose, microfibrils were synthesized. Microfibrils were insoluble in water, ethanol and acid, and soluble in alkali. Under the electron microscope they appeared more or less uniform with an average length of about 0·5 μm. Alkali-insoluble residue appeared in the form of densely-packed longer microfibrils. After acidification of alkali-solubilized glucan, shorter microfibrils were reprecipitated. Microfibrils were digested by both endo- and exo-1,3-β-glucanase. In the latter case, glucose was the only product indicating that the microfibrils consist of 1,3-β-glucan with no detectable branches.

Mutagenesis of Xanthomonas campestris yielded two major classes of mutant, both having cell surface polysaccharides fundamentally different from the wild-type. The wild-type bacterium produced copious amounts of extracellular slime polysaccharide containing glucose, mannose and glucuronic acid in a ratio of 2:2:1. ‘Non-mucoid’ mutants produced trace amounts of exopolysaccharide identical to the wild-type product; ‘crenated’ mutants produced material with an unusual composition containing sugars normally found in the lipopolysaccharide. Analysis of lipopolysaccharide fractions from these strains showed that the wild-type polysaccharide fraction contained predominantly glucose. Polysaccharides from the two classes of mutant bacteria were similar and contained rhamnose, galactose and smaller amounts of glucose.

SUMMARY: Lipase from Propionibacterium acnes has been purified 4800-fold from crude culture supernatant. The purified enzyme preparation had no assayable protease, hyaluronate lyase or acid phosphatase activities. The molecular weight of the lipase was 46 770 as determined by gel filtration. Sodium dodecyl sulphatepolyacrylamide gel electrophoresis revealed a major protein component (mol. wt 41 190) together with two minor protein components (mol. wt 67 000 and 125 900). The lipase had a pH optimum of 68, was most stable in the pH range 5·0 to 6·0 and was completely inactivated after 30 min at 60 °C. The lipase hydrolysed trilaurin, triolein, trimyristin and tripalmitin at decreasing rates and did not exhibit phospholipase activity. Analysis of the reaction products from the hydrolysis of triolein by P. acnes lipase did not demonstrate an accumulation of 2-monoolein which suggested that the enzyme did not exhibit a positional specificity for the 1-position of the triacylglycerol.

Crude lipase preparations contained an aggregated high molecular weight form of the enzyme which was eluted with the void volume from Sephadex G-200. This aggregated form was dissociated to produce the lower molecular weight lipase species by subsequent dialysis and elution from Sephadex G-200 using buffer with a higher ionic strength.

Glucose-6-phosphate isomerase was partially purified from cells of Dictyostelium discoideum in the culmination stage of development. The enzyme had a pH optimum of about 8·5 in Tris/HCl buffer. Activity was not inhibited by p-chloromercuribenzoate or iodoacetate. The enzyme exhibited MichaelisMenten kinetics and the K m values for glucose 6-phosphate and fructose 6-phosphate were 2 mm and 0·1 mm, respectively. Erythrose 4-phosphate was a strong competitive inhibitor of the enzyme with a K i value of 3·8 μm, whereas 6-phospho-gluconate was less effective with a K i of 0·1 mm.

Summary: A mutation, gerj50, causing defective spore germination has been located between lys-1 and trpC2 on the Bacillus subtilis map and is 92% cotransduced with trpC2 by phage PBS1. Spores of strains containing gerJ50 will respond to germinants but the fall in absorbance is only 60% of that of the wild-type. During germination, mutant spores reach only an intermediate phase-grey stage, in contrast to those of the wild-type which become dark. The germination response, measured by the release of dipicolinic acid and the loss of resistance to heat, chloroform, octanol or toluene, is normal. The release of hexosamine-containing fragments from mutant spores is incomplete but has similar kinetics to that of wild-type spores. The mutant spores are more sensitive to heating at 90 °C which may point to a structural abnormality, but there is no evidence of a spore coat deficiency from electron microscopy of thin sections or freeze fractures or from the chemical resistances of the spores. Thus, the germination of mutant spores is initiated normally, but blocked at a late stage.

Summary: Six mutations in the spoIIA locus have been mapped by transformation crosses. The Recombination Index between mutations at opposite ends of the locus was about 0·17; all six mutations are likely to be allelic. A mutation leading to oligosporogeny was found to be closely linked on both sides to mutations leading to asporogeny. For two pairs of mutants, crosses between the members of each pair yielded no wild-type recombinants, but the mutations were found not be identical. Another of the mutations was found to be partly suppressed by the general nonsense suppressor su + 3, and is therefore presumably a chain-terminating mutation.

Antibiotic non-producing mutants were isolated from an oxytetracycline (OTC) producing strain of Streptomyces rimosus. Cosynthesis tests and feeding known intermediates of the OTC pathway allowed the classification of the mutants into several groups. The biosynthetic lesion was determined for several of the mutants. Some of the mutants deficient in the reduction of 5a, 11a-dehydrooxytetracycline, the final step in the pathway, were unable to synthesize a cofactor (CSF1) essential for this reaction. Mutations apparently in genes for enzymes of the main OTC pathway were found for three of a possible four steps between 4-aminodedimethylaminoanhydrotetracycline (4-amino-ATC) and OTC. Mutations affecting three other steps of a possible five before 4-amino-ATC were found, but unambiguous identification of these was not possible. The genes for OTC production, including those for CSF1 synthesis, were located in two diametrically opposite clusters on the S. rimosus chromosomal linkage map. No evidence for plasmid-borne genes was obtained.

The properties of various mutants showed that, in the presence of an inorganic nitrogen source, agar utilization by Streptomyces coelicolor requires the action of at least three groups of enzymes: a diffusible or cell-free extracellular agarase, some further degradative step(s), and the enzymes of galactose metabolism. Two unselected mutations (dag), both leading to loss of diffusible agarase and an undefined second agar utilization function, were detected among a small number of S. coelicolor derivatives from a culture collection, and mapped to the 9 o’clock ‘silent’ region of the genetic map. One of these mutations (dagA1) had apparently resulted, directly or indirectly, from the insertion of the plasmid SCP1 in this region during formation of the NF fertility type. The other (dag-2) was 100% linked to dagA1 and the integrated SCP1, but only 98% linked to the site of insertion of SCP1 in an ‘NF-like’ but Dag+ strain. SCP1-chromosome interactions in other regions of the genetic map had no effect on the agar-solubilizing phenotype, nor had three other unstable genetic phenomena in S. coelicolor.

Genetic transfer has been studied between wild-type Nostoc muscorum and a multiply marked mutant strain. Experiments conducted with heat-killed donor and live recipient cells indicated that the transfer occurred in both directions and may have been the result of transformation rather than conjugation. This conclusion was supported by the sensitivity of the process to DNAase. There was some indication of linkage between het and eth.

Mycobacterium tuberculosis var. bovis (BCG) grown on Sauton medium normally forms a pellicle; in the absence of added Zn2+, however, the pellicle sank during incubation and the yield was only about 20% of normal. The Zn2+-starved bacteria were morphologically similar to normal bacteria and were still acid-fast at 7 d as well as 14 d. The Zn2+-starved bacteria had slightly higher free lipid and phospholipid contents than normal; the content of hexoses was lower and proteins slightly lower. The deficient culture medium became opalescent and alkaline. Aspartate and ammonium ions accumulated. There was twice as much protein in deficient as in normal medium; moreover, a class of proteins precipitable at pH 4·5, which was hardly detectable in normal medium, was present in appreciable amounts in deficient medium. The content of aldehydes, measured with yeast alcohol dehydrogenase, was also doubled in deficient medium. Fractionation of acid-soluble aldehydes obtained from deficient medium after acid treatment of a bisulphite precipitate suggested the presence of several complex molecules bearing aldehyde groups. The need for Zn2+ in the medium may be explained by the presence in normal BCG of a Zn2+-requiring NADP-dependent alcohol dehydrogenase activity whose affinity for aldehydes is especially high.