Obligately aerobic Gram-negative rod-shaped bacteria isolated from soil, sewage and river water grew using ethan-1,2-diol (MEG), 2-(2′-hydroxyethoxy)ethanol (DEG) or poly(ethylene glycol)s of up to 1500 molecular weight as sole carbon and energy source, though no single strain used the whole range. Only organisms that grew using MEG could grow on glycine or glycollate. An Acinetobacter strain (S8) degraded DEG and short poly(ethylene glycol)s by non-oxidative removal of ethylene oxide units as acetaldehyde, using a membrane-bound oxygen-sensitive enzyme of a novel type - DEG lyase - and leaving an unusable MEG residue. The C2 units could be used for energy generation by the tricarboxylic acid cycle and for anabolic and anaplerotic functions via the glyoxylate cycle, key enzymes of which were induced by growth of the bacteria on DEG or short poly(ethylene glycol)s.
A strain of Propionibacterium acnes (type I; Marples & McGinley, 1974), isolated from a blackhead acne lesion, produced an acid phosphatase which was present in the culture supernatant in the late-exponential and early-stationary phases of growth. This acid phosphatase was purified more than 45000-fold (4.5% yield). The purified enzyme gave two protein bands on sodium dodecyl sulphate-polyacrylamide gel electrophoresis corresponding to molecular weights of 155000 and 87100. The enzyme had a single peak of activity on Sephadex G-100, with a molecular weight corresponding to 93000. The highly purified acid phosphatase had an optimum activity at pH 5.8, was stable from pH 4.0 to 5.5 and was totally inactivated after 30 min at 55 °C. The enzyme did not show an absolute requirement for metal ions, but was stimulated by Mg2+, Ca2+, Zn2+ and K+ at concentrations between 0.1 and 1 mm. The acid phosphatase was active against a number of monophosphate esters.
Bacillus megaterium sensu stricto (NCIB 7581) produced amylase throughout exponential growth and during the early-stationary phase. Enzyme synthesis occurred in the absence of α-glucans but the yield was maximal when malt extract or starch was supplied as carbon source. Of the nitrogen sources examined, soya flour stimulated the highest yield of amylase. The enzyme was susceptible to reagents that react with thiol groups and had an exo-action on starch yielding maltose with a β-anomeric configuration. It is concluded that the principal starch-hydrolysing enzyme from B. megaterium NCIB 7581 is a (1-4)-α-D-glucan maltohydrolase similar in its properties to other Bacillus and plant β-amylases.
Capillary gas-liquid chromatography (g.l.c.) and g.l.c./mass spectrometry (g.l.c./m.s.) were used to study the fatty acid composition of the lipids of the following 23 species of proteolytic clostridia grown in a Trypticase/yeast extract medium: Clostridium bifermentans, C. cadaveris, C. caloritolerans, C. cochlearium, C. difficile, C. ghoni, C. glycolicum, C. histolyticum, C. lentoputrescens, C. limosum, C. lituseburense, C. malenominatum, C. mangenotii, C. propionicum, C. putrefaciens, C. putrificum, C. scatologenes, C. sordellii, C. sporogenes, C. sticklandii, C. subterminale, C. tetani, C. tetanomorphum. These species contained a total of 55 fatty acids in the range C12 to C18. The methyl esters were separated on a 152 m stainless steel capillary column coated with poly(1,4-butanediol succinate) and characterized by their equivalent chain lengths and by g.l.c./m.s. The predominant acids were n-C14:0 and n-C16:0. There were two groups of organisms: those which contained fatty acids of the n, iso and anteiso series and those which contained only those of the n series. All the organisms which contained iso and anteiso acids also oxidized valine, leucine and isoleucine to the corresponding branched-chain volatile fatty acids. The iso acids found had both even and odd numbers of carbon atoms and were probably derived from valine and leucine, respectively; the anteiso acids all had odd numbers of carbon atoms and were probably derived from isoleucine via 2-methylbutyric acid. These organisms also produced small amounts of monoenoic iso acids. Of the seven species which contained only acids of the n series, C. histolyticum produces acetic acid as its only end-product whereas the other six ferment glutamic acid and produce acetic and n-butyric acids; they also form propionic acid from threonine.
When Pseudomonas stutzeri was grown in iron-deficient conditions it excreted a colourless compound which was identified as nocardamine, a trihydroxamate, which is produced in similar conditions by actinomycetes. The production of this compound (which is related to the ferric iron content of the culture medium), as well as its ability to form very stable Fe(III) complexes, suggest that it is a desferri-siderophore.
Anaerobically grown Staphylococcus epidermidis fermented glucose with the production of lactate and trace amounts of acetate, formate and CO2. Isotopic and inhibitor studies, assays for key enzymes of different metabolic pathways, and fermentation balances, all indicated that glucose was metabolized principally via glycolysis and to a very limited extent by the hexose monophosphate oxidative pathway. Serine fermentation proceeded via deamination and dismutation yielding NH3 and equimolar amounts of lactate, acetate and CO2; small amounts of formate arose by the operation of pyruvate-formate lyase. Incorporation of 0.5% (w/v) glucose in the growth medium depressed serine metabolism by repressing the activities of serine dehydratase and pyruvate dehydrogenase but, conversely, enhanced the activities of phosphofructokinase and lactate dehydrogenase. Glucose-grown organisms at various stages of anaerobic batch growth showed an inverse relationship between the rates of fermentation of serine and glucose. l-Lactate dehydrogenase activity in crude extracts depended on fructose 1,6-bisphosphate, and fructose 1,6-bisphosphate aldolase was found to be a class I aldolase. Despite the presence of ribokinase, d-ribose-5-phosphate isomerase, transaldolase and transketolase, the organisms utilized ribose only after growth aerobically in basal medium, and then at a slow rate after an initial lag period.
Four Gram-positive bacteria have been isolated from separate soil samples by enrichment culture with acetone as sole source of carbon. Whole cells of all strains grown on acetone rapidly oxidized acetone, acetol and methylglyoxal, and three of the four also oxidized isopropanol. The patterns of induced enzymes in cell extracts are compatible with the oxidation sequence: isopropanol → acetone → acetol → methylglyoxal → pyruvate. Although an enzyme system capable of converting acetone into acetol has not been detected, the inclusion of acetol in the pathway is supported by the results of studies with whole cells and [14C]acetone. The proposed pathway of acetone metabolism is contrasted with evidence for an alternative, but not fully understood, pathway used by Mycobacterium vaccae JOB5.
Cerulenin inhibited the secretion of extracellular proteins by washed cell suspensions of Bacillus amyloliquefaciens. α-Amylase and protease secretion were inhibited by 80% and 75%, respectively, over 3 h. Cerulenin at 100 μg ml-1 inhibited incorporation of [1,2-14C]- acetate into intracellular lipid by about 75% without affecting cell growth, total protein synthesis or membrane protein synthesis. The inhibitory effect of cerulenin on α-amylase and protease secretion could be partially reversed if cell suspensions were supplemented with either fatty acids prepared from the lipids extracted from B. amyloliquefaciens or various individual pure fatty acids. Cerulenin significantly altered the ratio of lipid to protein in isolated membranes. However, this alteration was not affected by adding fatty acids which restored enzyme secretion. These results suggest that cerulenin may affect the availability of lipid directly concerned with the secretion process. The differential effect of cerulenin on the production of extracellular proteins and membrane proteins also suggests that the synthesis of these two classes of proteins occurs via mechanisms that differ.
Extracellular endo-(1→3)-α-glucanase (EC 220.127.116.11) was optimally induced in Bacillus circulans WL-12 when grown in a mineral medium containing intact cells of Schizosaccharomyces pombe or extensively purified (1→3)-α-glucan as the carbon source. The enzyme activity was separated from most other proteins, including the β-glucanases, by affinity adsorption of the enzyme on water-insoluble colloidal (1→3)-α-glucan. The enzyme was released by allowing it to hydrolyse this substrate and purified further by DEAE-agarose chromatography and polyacrylamide P-150 gel filtration. Passage over DEAE-agarose apparently also separated a minor (1→3)-α-glucanase component. The principal (1→3)-α-glucanase was specific for the (1→3)-α-glucosidic bond and hydrolysed (1→3)-α-glucan endolytically to a mixture of nigerose and glucose with a transient accumulation of nigerotetraose. The reaction proceeded at a constantly declining rate with either colloidal (1→3)-α-glucan or with the soluble carboxymethyl-(1→3)-α-glucan as substrate. Nigeran, containing alternating (1→3)-α- and (1→4)-α-linkages, was not hydrolysed. Substrate dependence showed Michaelis-Menten kinetics. There was a pH optimum of 7.5 to 8.5 with a pronounced shoulder at pH 5 to 7. The molecular weight was estimated by slab gel electrophoresis in sodium dodecyl sulphate to be 134000. The enzyme did not appear to require divalent metal ions because its activity was stimulated by EDTA. There was evidence for essential thiol groups. Carbohydrate was not detected in the enzyme.