- Volume 131, Issue 6, 1985

Hygromycin B, an aminocyclitol antibiotic that strongly inhibits both 70S and 80S ribosomes, is synthesized by Streptomyces hygroscopicus. Ribosomes from this Gram-positive mycelial bacterium are inhibited in vitro by the antibiotic. In contrast, the streptomycete is highly resistant to the drug in vivo since it possesses hygromycin B phosphotransferase activity. This enzyme has been shown by gel filtration to have a molecular weight of 42000, and to modify its antibiotic substrate to produce 7″-O-phosphoryl-hygromycin B which totally lacks biological activity both in vivo and in vitro.

Novozym 234, a commercially available enzyme from Trichoderma harzianum, has been used to prepare protoplasts from Trichoderma reesei QM 9414. Optimal conditions were : 20 h old mycelium, 0·9 m-KCl as the osmotic stabilizer. 0·1% (w/v) Novozym 234 and 18 h incubation at 28°C. To prevent damage to the protoplasts during isolation the incubation mixtures were agitated by vibration, so avoiding shaking or stirring. More than 95% of the protoplasts were viable and were also metabolically intact as shown by their ability to take up [14C]leucine and incorporate it into cellular protein. Freshly prepared protoplasts could be induced by sophorose to produce and secrete carboxymethylcellulase and β-glucosidase. Optimal conditions were: 0·9 m-KCl as osmotic stabilizer, phosphate buffer pH 6·0, 7 mm-sophorose and 107 protoplasts ml–1. Separation of the secreted proteins by fast protein liquid chromatography revealed at least seven peaks of carboxymethylcellulase activity and β-glucosidase activity, indicating that some multiplicity of the forms of these enzymes is already present at the secretion stage.

The carnitine-responsive mutant yeast Torulopsis bovina ATCC 26014, when grown on medium supplemented with carnitine, contained acetyl- and propionylcarnitines, consistent with the presence of carnitine acetyltransferase, which catalyses the reaction carnitine + acyl-CoA ⇌ acylcarnitine + CoA. Two hypotheses for the metabolic function of carnitine were tested. (1) That by reacting with acyl-CoA and releasing CoA, carnitine decreases the requirement for CoA. The effect of carnitine on the growth of the organism was measured in medium containing limiting amounts of pantothenate, which is a component of CoA. Carnitine did not increase the cell yield, suggesting that it did not exert a pantothenate-sparing effect. (2) That carnitine facilitates utilization of short-chain fatty acids. Carnitine reversed the growth inhibition which occurred when acetate was present in the medium, and also increased the respiration rate when acetate was the substrate, results consistent with the hypothesis that the carnitine acetyltransferase system was the limiting factor in transport of acetyl groups into mitochondria of T. bovina ATCC 26014.

The properties of the carnitine-responsive strain were compared with those of the wild-type, T. bovina ATCC 22987. Growth rate of the wild-type was rapid and was not stimulated by carnitine, nor was it inhibited by acetate in the growth medium. Acetate increased the respiration rate of the wild-type to the same extent in the presence or absence of carnitine. It is postulated that the mutant lacks a mechanism for handling acetate which is present in the wild-type, and that addition of carnitine to the medium enables the carnitine acetyltransferase system of the mutant to increase the rate of transport of acetyl groups into the mitochondria.

Triterpenoids belonging to the hopane family are widespread among prokaryotes. The two major hopanoids isolated from the methylotrophic bacterium ‘Methylosinus trichosporium’ grown on methane or methanol were identified by spectroscopic methods as the aminotriol 35-aminobacteriohopane-32,33,34-triol and a novel aminotetrol, 35-aminobacteriohopane-31,32,33,34-tetrol. The former hopanoid has been previously isolated from the purple non-sulphur bacterium Rhodomicrobium vannielii.

Analysis of the lipids of Mycobacterium tuberculosis, by thin-layer chromatography, revealed the presence of two families of novel glycolipids each having two closely-related members but differing widely in polarity. The least and most polar families of lipids were characterized from M. tuberculosis strains C and H37Rv, respectively; all were based on trehalose, the least polar pair of glycolipids having more long-chain substituents than the more polar pair. The acyl substituents of the least polar of the four glycolipids were mainly straight-chain C16 and C18 acids and 2,4,6-trimethyltetracos-2-enoic (C27-mycolipenic) acid, and the second least polar glycolipid contained major amounts of 3-hydroxy-2,4,6-trimethyltetracosanoic (C27-mycolipanolic) acid in addition to these non-hydroxylated acids. The relatively polar pair of glycolipids were analysed together and released mainly straight-chain C16 and C18 acids, C27-mycolipanolic acid, minor amounts of C25-and C27-mycolipenic acids and major proportions of an acid having the chromatographic properties of 2,4-dimethyldocosanoic acid. The most polar pair of glycolipids co-chromatographed with glycolipid antigens previously detected in Mycobacterium bovis BCG.

The activity of glutamate dehydrogenase (NADP+) (EC 1.4.1.4; NADP-GDH) of Saccharomyces cerevisiae is decreased under conditions in which intracellular ammonia concentration increases. A high internal ammonia concentration can be obtained (a) by increasing the ammonium sulphate concentration in the culture medium, and (b) by growing the yeast either in acetate + ammonia media, where the pH of the medium rises during growth, or in heavily buffered glucose + ammonia media at pH 7·5. Under these conditions cellular oxoglutarate concentrations do not vary and changes in NADP-GDH activity appear to provide a constant rate of oxoglutarate utilization. The following results suggest that the decrease in NADP-GDH activity in ammonia-accumulating yeast cells is brought about by repression of synthesis: (i) after a shift to high ammonium sulphate concentrations, the number of units of activity per cell decreased as the inverse of cell doubling; and (ii) the rate of degradation of labelled NADP-GDH was essentially the same in ammonia-accumulating yeast cells and in controls, whereas the synthesis constant was much lower in the ammonia-accumulating cells than in the controls.

Rhizobium sp. ANU289 lacked glutamate dehydrogenase (GDH) but grew rapidly on ammonia in a well-buffered medium. The properties of glutamine synthetase I (GSI) differed from those of GS from enteric bacteria in a manner which appeared to compensate for the lack of GDH. Adenylylated and unadenylylated GSI had the same biosynthetic activity, and there was also no detectable difference between the two forms with respect to affinities for substrates. GSII, but not GSI, was repressed by growth on ammonia, further suggesting that GSI is important in ammonia assimilation in the presence of high ammonia concentrations. Methylamine was found to exert similar regulatory effects to ammonia and was therefore used to determine the extent to which utilization of various nitrogen sources was controlled by ammonia in strain ANU289.

Spores of Clostridium perfringens contain at least two spore-lytic enzymes active in hydrolysing cortical peptidoglycan. One enzyme has been purified 1800-fold and has a molecular weight of 17400 determined from chromatography on Sephadex G-75. Two protein bands were apparent after SDS-PAGE. The isolated enzyme was investigated for response to temperature, pH, ionic strength and enzyme inhibitors, and for mode of action. A second enzyme activity, differing from the first in apparent molecular weight (29800) as determined by gel exclusion chromatography, and also in its pH optimum and activity on cortical substrate, was also isolated, although not purified to the same extent.

Labelling of the hyphal walls of basidiospore germlings of Schizophyllum commune with radioactive glucose resulted in immediate labelling of the glucans but labelling of chitin was delayed for about 20 min. Radioactive glucose could thus be used as a specific label for wall glucans in a 10 min labelling pulse. Special attention was given to the alkali-insoluble glucans containing (1→3)-β and (1→6)-β linkages and occurring in close association with chitin. Using [3-3H]glucose and treating the labelled glucans with periodate, a distinction could be made between glucose residues incorporated in (1→3) and in (1→6) linkages and the spatial distribution of these synthetic activities could be followed by microautoradiography. The results indicated that in growing hyphae the alkali-insoluble glucan synthesized at the apex was very rich in (1→3) linkages and that synthesis of (1→6) linkages increased subapically from about 10% at the apex to about 60% just outside the extension zone. Inhibition of hyphal growth with cycloheximide or by low glucose concentrations inhibited glucan synthesis at the apex but not in subapical parts. At the same time a shift towards synthesis of (1→6) linkages was observed at the apex.

Vegetative cells of Azotobacter vinelandii were induced to encyst in the presence of β-hydroxybutyrate. Although modal cell volume fluctuated (2·7 to 4 μm3) markedly during exponential growth, the final modal volume attained by mature cysts (1·13 to 1·3 μm3) was relatively constant. Respiratory activity of encysting cultures fell rapidly 3 h after initiation of encystment. This was accompanied by decreased inhibition of respiration by KCN. NADH oxidase activity of membrane preparations fell from approximately 450 to 50 nmol O2 consumed min–1 (mg protein)–1 during differentiation and was also less sensitive to KCN. Difference spectra of cytochromes in membrane preparations revealed a number of changes. Amounts of cytochromes b, c and a 1 all fell during encystment; cytochrome o levels increased whilst cytochrome oxidase d was gradually lost from the membrane during the first 22 h of morphogenesis.

The infectious yeast Candida albicans is capable of growing in either a budding or mycelium form, depending upon the pH of the supporting medium. By monitoring the position of polylysine-coated beads firmly attached to the wall of growing cells, the zones of expansion for the surface of the cell wall have been mapped for the alternative growth forms. Both spatial and temporal differences are demonstrated to exist. During roughly the first two-thirds of bud growth, a very small, highly active apical zone accounts for roughly 70% of surface expansion. The remaining 30% is due to general expansion. When a bud reaches approximately two-thirds of its final surface area, the apical zone shuts down, and subsequent expansion is completed by the general mechanism. During mycelial growth, at least 90% of expansion is due to a small, highly active apical growth zone, and less than 10% is due to the general mechanism. In contrast to budding cells, the apical zone of the growing mycelium never shuts down as long as growth continues in the mycelial form. These distinct temporal and spatial differences in expansion are considered in terms of the regulation of alternative phenotypes in Candida.

The exclusion relationship between the IncI plasmids R144, R64 and ColIb was studied in such a way that incompatibility interference was avoided. Genetic crosses with an R144-derived Hfr donor, crosses with recipient strains carrying R144-derived exclusion genes on a recombinant plasmid compatible with R144, and measurement of transmission frequencies of a recombinant plasmid compatible with IncI plasmids after mobilization by R144 revealed that R144, R64 and ColIb belong to one exclusion group.

Two classes of pyridine nucleotide uptake mutants isolated previously in a strain of Salmonella typhimurium defective in both de novo NAD biosynthesis (nad) and pyridine nucleotide recycling (pncA) were analysed in terms of their genetic relationship to each other and their roles in the transport of nicotinamide mononucleotide as a precursor to NAD. The first class of uptake mutants, pnuA (99 units), failed to grow on nicotinamide mononucleotide (NMN) as a precursor for NAD. The second class, pnuB, grew on lower than normal levels of NMN and suppressed pnuA mutations. A third class of uptake mutant, pnuC, isolated in a nadB pncA pnuB background, also failed to grow on NMN. Transport studies and enzyme analyses confirmed these strains as defective in NMN uptake. A fourth locus, designated pnuD, was found to diminish NMN utilization in a nad pncA + background. Tn10 insertions near pnuA, pnuC and pnuD were isolated and utilized in mapping studies. pnuA was found to map between thr and serB near trpR. The pnuC locus was cotransducible with nadA at 17 units while pnuD mapped at approximately 60 units. The biochemical and genetic data suggest that the pnuA and pnuC gene products cooperate in the utilization of NMN under normal conditions. A pnuB mutant, however, does not require the pnuA gene product for NMN uptake but does rely on the pnuC product. Fusion studies indicate that pnuC is regulated by internal NAD concentrations.

Three classes of Hup‒ mutants of Azotobacter chroococcum were obtained by N-methyl-N′-nitro-N-nitrosoguanidine mutagenesis and screening by H3H uptake: (1) those with no discernible H2-uptake or H2-evolving activity, (2) those showing no uptake but some H2 production and (3) those leaky for both activities. One mutant strain, MCD-124, expressed hydrogenase activity similar to the solubilized wild-type enzyme in O2 sensitivity, sedimenting behaviour and pH optimum. All the other mutants were probably mutated in the hydrogenase structural or processing (methylene blue) genes rather than in genes for hydrogenase-linked respiratory proteins. Four mutants chosen from the first category were complemented for hydrogenase activity by conjugation with Escherichia coli carrying plasmid pHU1 containing Rhizobium japonicum hydrogenase genes. A pHU1 transconjugant of strain MCD-124, on the other hand, did not express any additional hydrogenase activity.

IncHl plasmids and the F plasmid of Escherichia coli display one-way compatibility. An entering IncHl plasmid is incompatible with a resident F plasmid, but is compatible when it is the resident plasmid. There is little molecular homology between IncH1 plasmids and the F plasmid. A single 5 MDal EcoRI restriction enzyme fragment from digests of several IncHl plasmids hybridizes with probes constructed from the primary replication region of F. Homology can be demonstrated only with the gene for the essential replication protein of F (gene E), but the expression of incompatibility behaviour appears to be associated with the presence of the secondary replicon of the F plasmid. Thus R27 and F are compatible under growth conditions allowing replication and maintenance of F by the secondary replicon. However, a mutant F plasmid which lacks the secondary replicon of F is incompatible with R27 in both directions, irrespective of the growth conditions used.

Streptococcus lactis ME2 exhibits at least three mechanisms which confer resistance to virulent bacteriophage. These include plasmid-induced interference with phage adsorption, host-controlled restriction and modification activities, and a heat-sensitive mechanism which suppresses development of virulent phage. Conjugal mating experiments were done with S. lactis ME2 to determine if phage-defence mechanisms present in this strain could be mobilized, associated with plasmid DNA elements and phenotypically characterized in transconjugants. Agar-surface matings of S. lactis ME2 with S. lactis LM0230 demonstrated that lactose-fermenting ability (Lac+) was transferred in a conjugation-like process at frequencies of 10–6 per donor cell and was associated with a 40 MDal plasmid designated pTR1040. Resistance to nisin (Nisr) was acquired or lost simultaneously with Lac+, indicating that pTR1040 carried determinants for both phenotypes. Lac+ Nisr transconjugants that carried a 30 MDal plasmid (pTR2030) exhibited a heat-sensitive phage-defence mechanism (Hsp+) which limited the burst size and plaque size of phage c2 without altering the efficiency of plaquing (e.o.p.) or the level of adsorption. The ability of phage c2 to initiate plaquing at an e.o.p. of 1·0 indicated that DNA injection and early viral gene expression are not affected in the Hsp+ transconjugants. We suggest, therefore, that the Hsp+ phenotype may result from plasmid-induced abortive infection of phage dependent on the presence of pTR2030. Hsp+ transconjugants carrying pTR2030 also promoted high-frequency conjugal transfer of Lac+ Nisr associated with pTR1040 (> 10–1 per donor cell). It was concluded that Hsp+ and determinants for conjugal transfer ability (Tra+) are located on pTR2030.