- Volume 139, Issue 11, 1993

(—)-Verrucosan-2β-ol (C20H34O), a rare diterpene with a 3,6,6,5-tetracyclic ring system, has been isolated and identified for the first time from the phototrophic bacterium Chloroflexus aurantiacus. Furthermore, an unsaturated diterpenoid hydrocarbon (C20H32) with a similar carbon skeleton was found in the same organism. This prokaryote, naturally occurring in hot spring microbial mats, is considered to be one of the oldest bacterial life forms on earth. Verrucosane-type diterpenoids had previously been detected only in some liverworts (Hepaticae), forming a unique group in the plant kingdom.

Glucose-6-phosphate dehydrogenase (G6PD; d-glucose 6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49) has been purified from Aspergillus nidulans and Aspergillus niger by a combination of affinity and anion exchange chromatography. A 500–1000-fold purification was obtained and the final enzyme preparations were shown to be pure but not homogeneous. For both fungi the purified enzyme preparation gave two bands on native and denaturing gels. The catalytically active form is a multimer. The molecular mass of the monomers is 60 and 57 kDa for A. nidulans and 55 and 53 kDa for A. niger. Both enzymes exhibited strict specificity towards both substrates glucose 6-phosphate and NADP+. The A. nidulans and A. niger G6PD enzymes catalyse the conversion of glucose 6-phosphate via a random order mechanism. Inhibition studies provided evidence for the physiological role of G6PD as producer of NADPH in both fungi.

The mobilizable plasmid pMD101 (ampR, ampC) was constructed by inserting cloned ampC, the structural gene for the chromosomal AmpC β-lactamase of Citrobacter freundii, and the closely linked ampR encoding the transcriptional regulator essential for enzyme induction, into the broad host-range plasmid pKT231. Plasmid pMD101 was transconjugated into Proteus mirabilis VI and its isogenic, cell-wall-less protoplast L-form LVI. AmpC β-lactamase was expressed constitutively from cloned ampR and ampC in bacteria and in some L-form protoplasts. However, induction of the enzyme by β-lactam antibiotics occurred only in bacterial cells and not in the cell-wall- and peptidoglycan-deficient L-form. In agreement with current models, induction of AmpC β-lactamase is thought to be initiated by an induction signal arising from the metabolic disturbance of cell-wall peptidoglycan.

Cell walls were prepared from four strains belonging to group 2 alkaliphilic Bacillus spp. Non-peptidoglycan components were extracted, with trichloroacetic acid, from the cell wall preparations and isolated by DEAE-cellulose column chromatography. All the components were acidic, and were composed of amino acids and sugars. Several components with different compositions were detected. The cell walls commonly contained teichuronopeptide, composed of polyglucuronic acid and a polypeptide of acidic amino acids.

Acetobacter xylinum strain NRRL B42 and its derivative RCGr1 produce a complex exopolysaccharide, acetan, containing glucose, mannose, glucuronic acid and rhamnose in a 4:1:1:1 molar ratio. The in vitro synthesis of acetan, employing electroporated cells as the enzyme system and the respective 14C-labelled sugar nucleotide precursors, is described. The synthesis of the prenyl-linked heptasaccharide repeat unit, already observed in EDTA-treated cells, was confirmed, as well as the formation of other saccharides not related to acetan biosynthesis, including a high molecular mass glucan. The acetan formed was characterized by gel filtration, specific radioactive labelling with each precursor and permethylation analysis. It was also shown that acetan contains acetyl residues and that using [14C]acetyl CoA as donor, radioactivity was detected both at the polysaccharide and at the prenyl-linked oligosaccharide stage.

(-)-Verrucosan-2β-ol (C20H34O), a rare diterpene with a 3,6,6,5-tetracyclic ring system, has been isolated and identified for the first time from the phototrophic bacterium Chloroflexus aurantiacus. Furthermore, an unsaturated diterpenoid hydrocarbon (C20H32) with a similar carbon skeleton was found in the same organism. This prokaryote, naturally occurring in hot spring microbial mats, is considered to be one of the oldest bacterial life forms on earth. Verrucosane-type diterpenoids had previously been detected only in some liverworts (Hepaticae), forming a unique group in the plant kingdom.

Catabolism of isonicotinate by Mycobacterium sp. INA1 has been shown to proceed via 2-hydroxyisonicotinate, 2,6-dihydroxyisonicotinate (citrazinate), citrazyl-CoA and 2,6-dioxopiperidine-4-carboxyl-CoA. An extended pathway involving propane-1,2,3-tricarboxylate as a further intermediate is presented in this paper. Propane-1,2,3-tricarboxylate was oxidized stepwise to 2-oxoglutarate involving an oxidase, aconitase and isocitrate dehydrogenase. Isonicotinate dehydrogenase catalyses the first step of isonicotinate metabolism in Mycobacterium sp. INA1. The enzyme was purified to apparent homogeneity by a three-step procedure. Enrichment was accompanied by partial loss in specific activity. The native enzyme had a molecular mass of either 125 kDa or 250 kDa, when estimated by native gradient PAGE or gel filtration, respectively. SDS-gel electrophoresis revealed three types of subunits with molecular masses of approximately 83, 31 and 19 kDa. N-Terminal amino acid sequences of all three subunits have been determined. Molybdenum, iron, acid-labile sulphur and FAD were present at molar ratios of 1, 4, 4, 1 per protomer (125 kDa). The molybdenum-complexing cofactor was shown to be molybdopterin cytosine dinucleotide. Besides isonicotinate, only quinoline-4-carboxylate was found to be oxidized at appreciable rates.

Cell extracts of Streptomyces tendae grown in nikkomycin production media contained an enzyme (HisAT) that transaminated l-histidine as the sole amino substrate with pyruvate as the amino group acceptor. HisAT was purified about 190-fold from the crude extract of S. tendae. The enzyme was determined by gel filtration and SDS-PAGE to be a homodimer with a subunit molecular mass of approximately 45 kDa. The aminotransferase had maximum activity at pH 7·0 and 37 °C. The enzyme was highly specific for l-histidine; pyruvate, 2-oxobutyrate, 2-oxovalerate and 2-oxocaproate were used as keto acceptors to about the same extent. The reaction mechanism was ping-pong. The K m values for l-histidine and pyruvate, determined from Lineweaver-Burk plots, were 25 mm and 10 mm, respectively. Neither cell extracts of non-producing S. tendae mutants nor extracts of Streptomyces lividans, a species that does not synthesize nikkomycins, showed transaminating activity with a narrow substrate specificity for l-histidine as the amino donor. This strongly suggests that the formation of HisAT is essential for nikkomycin production.

Glucose-6-phosphate dehydrogenase (G6PD; D-glucose 6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49) has been purified from Aspergillus nidulans and Aspergillus niger by a combination of affinity and anion exchange chromatography. A 500-1000-fold purification was obtained and the final enzyme preparations were shown to be pure but not homogeneous. For both fungi the purified enzyme preparation gave two bands on native and denaturing gels. The catalytically active form is a multimer. The molecular mass of the monomers is 60 and 57 kDa for A. nidulans and 55 and 53 kDa for A. niger. Both enzymes exhibited strict specificity towards both substrates glucose 6-phosphate and NADP+. The A. nidulans and A. niger G6PD enzymes catalyse the conversion of glucose 6-phosphate via a random order mechanism. Inhibition studies provided evidence for the physiological role of G6PD as producer of NADPH in both fungi.

Multiple forms of β-glucosidase (EC 3.2.1.21) of Sporotrichum thermophile were produced when the fungus was grown in a cellulose medium. One β-glucosidase was purified 16fold from 6-d-old culture filtrates by ion-exchange and gel-filtration chromatography. The purified enzyme was free of cellulase activity. It hydrolysed aryl β-d-glucosides and β-d-linked diglucosides. It was optimally active at pH 5·4, at 65 °C. The apparent K m values for p-nitrophenyl β-d-glucoside (PNPG) and cellobiose were 0·29 and 0·83 mm, respectively. Glucose, fucose, nojirimycin and gluconolactone inhibited β-glucosidase competitively. At high (> 1 mm) substrate concentration, β-glucosidase catalysed a parallel transglycosylation reaction. The transglycosylation product formed from cellobiose appeared to be a β-linked tetramer of glucose. Admixtures of β-glucosidase and cellulase components showed that the concept of cellobiose inhibition of cellulases was not valid for all components of the cellulase system of S. thermophile. β-Glucosidase supplementation also stimulated cellulose hydrolysis by cellulases when there was no accumulation of cellobiose in reaction mixture.

The filamentous fungus Botrytis cinerea was found to produce four 1,3-β-glucanases (Glu I, II, III, IV). These enzymes were visualized by activity-staining after separation by native polyacrylamide gel electrophoresis (PAGE). During growth on glucose as the single carbon source, only Glu III was detectable in the culture supernatant of B. cinerea. After glucose was exhausted from the medium, the extracellular (1,3)(1,6)-β-d-glucan (cinerean) capsule of the fungus was degraded. In this phase the other three enzymes became detectable and the amount of all four enzymes increased. The enzyme with the greatest activity was Glu II, which was purified to homogeneity by SDS-PAGE. Its M r was 75000 and its isoelectric point (pI) was 5·2. Glycosylation of Glu II was shown by the periodic acid/Schiff reaction after SDS-PAGE. Glu II cleaved cinerean and laminarin. Both substrates were degraded in an exo-manner as shown by product characterization, and by studying the viscosity decrease in comparison with the liberation of reducing groups. The concentration of substrate that gave half-maximal velocity (S0·5) for Glu II was 580μg ml−1 for cinerean and 152 μg ml−1 for laminarin. For Glu III, also purified to homogeneity by SDS-PAGE, an M r of 84000 and a pI of 3·6 were determined. Glu III cleaved laminarin (S0·5 119 μg ml−1) but not cinerean. Glu I and Glu IV were purified by activity-stained native PAGE. Both enzymes cleaved cinerean and laminarin in an exo-manner. Glu I focused at pI 4·9; its S0·5 was 275 μg ml−1 with cinerean and 138 μg ml−1 with laminarin. The pI of Glu IV was 3.4; its S0·5 was 171 μg ml−1 for cinerean and 27 μg ml−1 for laminarin.

A polysaccharide of molecular mass at least 300000 Da was isolated from walls of Bacillus megaterium NCIB 7581. Three sugars, N-lactyl-3-amino-3,6-dideoxyhexose, N-acetylglucosamine and glucose were present in equimolar proportions, and accounted for 90% of the carbon of the polysaccharide. The remainder of the polymer was an unidentified acidic molecule, with a hydrophobic nature.

This study had two objectives: to determine the number of (phenotypic) ciliate species co-existing in 1 m2 of sandy river sediment at a maximum temperature of 4 °C; and to determine the ecological mechanism(s) facilitating their co-existence. The ciliate community was diverse (65 species [8 of which are new], belonging to 50 genera, from 17 orders). The sediment supported a superficial mat of diatoms (> 30 species). These served as food for at least 16 ciliate species. The size frequency distribution of ingested diatoms was almost identical to that for the diatoms in the sediment: thus the probability of a diatom being ingested appears to be a simple function of its relative abundance. Two factors were probably important for the co-existence of ciliate species: wide variation in cell size and shape enabled them to occupy most habitats; and they deployed a variety of feeding mechanisms to consume the variety of microbial food types. Taken as a whole, the ciliate community was capable of feeding on all microbes, including other protozoa, up to a size of about 80 μm. Considering the broad diversity of ciliate habitats available within 1 m2, the importance of physical transport processes in the river basin, and the known cosmopolitan distribution of many ciliate species, it is believed likely that the species richness we recorded is representative of the expanse of sandy sediment in this river, on this occasion.

Marker components of the phospholipids of Ruminococcus flavefaciens and Fibrobacter succinogenes were identified for studies on the degradation of forage by these bacteria growing in mixed culture. The principal fatty acid methyl esters and dimethyl acetals detected varied between strains and were influenced by the addition of a mixture of higher volatile fatty acids and vitamins to the medium, but these effects were small compared to the differences between the species. When two strains of R. flavefaciens were grown on a mixture of clover and ryegrass, and on barley straw in the presence or absence of two strains of F. succinogenes, the solubilization of plant material tended to be lowered by the presence of F. succinogenes. R. flavefaciens was the predominant bacterium among colonies recovered from roll tubes, and the phospholipids were primarily those of R. flavefaciens. Analysis of the culture supernatant liquids showed that F. succinogenes produced greater amounts of free and bound xylose from both clover and straw than did R. flavefaciens. With both forages, cultures containing the two species produced more soluble free arabinose, and less soluble-bound arabinose, than either species grown alone.

In wild-type Streptomyces ambofaciens ATCC 23877, pigment-defective (Pig−) mutants arise at a frequency of about 0·5%; this genetic instability is related to genomic rearrangements such as deletions and/or amplifications of DNA sequences. On media containing oxolinic acid and novobiocin, which interact with the A and B subunits of DNA gyrase, respectively, the frequency of variants increased dramatically. The Pig− mutant frequency was increased to almost 100% on a medium containing oxolinic acid at a concentration allowing 55% survival. On solid medium containing either oxolinic acid or novobiocin at subinhibitory concentrations, most colonies exhibited a ‘patchwork’ phenotype, characterized by the presence of numerous Pig− sectors. Similar phenomena were not observed on media containing the transcriptional inhibitor rifampicin or the translational inhibitor streptomycin. Many of the Pig− mutants exhibited a pleiotropic phenotype and were affected in aerial mycelium formation, colony growth and/or prototrophy. Moreover, the same kinds of rearrangements (deletions and/or amplifications of DNA sequences) were found in both induced and spontaneous Pig− mutants. The results suggest either that DNA gyrase is directly involved in genetic instability or that an SOS-like system is implicated.