- Volume 126, Issue 1, 1981

Catechol methyltransferase (S-adenosyl-l-methionine: catechol O-methyltransferase; EC 2.1.1.6) was localized immunocytochemically in the yeast Candida tropicalis by the unlabelled antibody enzyme method, involving soluble peroxidase-antiperoxidase complex. Immunoreactivity was detected by light and electron microscopy in the outer layer of the cell wall and at the plasma membrane. The possible function of the methyltransferase in C. tropicalis is discussed.

Properties of the Caulobacter penicillin-binding proteins (PBPs) were studied. Several Caulobacter strains possessed at least five major PBPs, as shown previously for C. crescentus CB15, namely PBP1A, PBP1Bs, PBP2, PBP3 and PBP4. PBP4 was not detected in the C. crescentus strain CB13. None of the strains examined possessed the major PBPs of low molecular weight which have been found in other species of bacteria. The biochemical properties of the Caulobacter PBPs were studied further with two strains, C. crescentus CB13 and C. crescentus CB15. In these strains, PBPs of similar molecular weight were similar in properties such as affinity for β-lactam antibiotics and heat sensitivity. These properties were markedly different from those of PBPs of Escherichia coli and other bacteria. To elucidate the functions of the Caulobacter PBPs, the effects of several β-lactam antibiotics on cell morphology and their affinities for different PBPs were examined. The relationship between the antibiotic effect and its affinity for PBPs suggests that PBP3 is involved in the process of cell division.

Penicillium islandicum produced an inducible extracellular chitosanase when grown on chitosan. Large-scale production of the enzyme was obtained using Rhizopus rhizopodiformis hyphae as substrate. Chitosanase was purified 38-fold to homogeneity by ammonium sulphate fractionation and sequential chromatography on DEAE-Biogel A, Biogel P60 and hydroxyl-apatite. Crude enzyme was unstable at 37 °C, but was stabilized by 1·0 mm-Ca2+. The pH optimum for activity was broad and dependent on the solubility of the chitosan substrate. Various physical and chemical properties of the purified enzyme were determined.

Penicillium islandicum chitosanase cleaved chitosan in an endo-splitting manner with maximal activity on polymers of 30 to 60% acetylation. No activity was found on chitin (100% acetylated chitosan) or trimers and tetramers of N-acetylglucosamine. The latter two oligomers and all small oligomers of glucosamine inhibited the activity of chitosanase on 30% acetylated chitosan. The pentamer of N-acetylglucosamine and glucosamine oligomers were slowly cleaved by the enzyme. Analysis of the reaction products from 30% acetylated chitosan indicated that the major oligomeric product was a trimer; with 60% acetylated chitosan as substrate a dimer was also found. The new terminal reducing groups produced by chitosanase hydrolysis of 30% acetylated chitosan were reduced by sodium boro[3H]hydride. The new end residues were found to be N-acetylglucosamine. The analyses strongly indicated that P. islandicum chitosanase cleaved chitosan between N-acetylglucosamine and glucosamine. Both residues were needed for cleavage, and polymers containing equal proportions of acetylated and non-acetylated sugars were optimal for chitosanase activity. The products of reaction depended on the degree of acetylation of the polymer.

Respiration of the soil amoeba Acanthamoeba castellanii is stimulated by low concentrations of H2S (< 17 Pa in the gas phase); consumption of H2S accompanies respiratory stimulation. Inhibition of the main respiratory chain by 1 mm-NaN3 prevents H2S consumption. At higher concentrations, H2S inhibits respiration incompletely; the inhibition is enhanced by 1 mm-salicylhydroxamic acid. Thus this organism has two means of protection against the potentially toxic gas: the ability to oxidize H2S, and the presence of alternative pathways of electron transport which are not blocked by this respiratory inhibitor.

Ubiquinol-15 can be used as an electron donor for both the conventional cytochrome chain and the chloramphenicol-induced alternative pathway of Neurospora crassa mitochondria. The oxidation of ubiquinol-15 through the chloramphenicol-induced cyanide-insensitive pathway is not inhibited by antimycin A and is strongly inhibited by salicylhydroxamic acid. In contrast, the oxidation of duroquinol by N. crassa mitochondria is sensitive to antimycin A under all circumstances, except for some portion which is believed to be a non-enzymic autoxidation reaction. 2-Thenoyltrifluoroacetone, a specific inhibitor of the succinate-ubiquinone oxidoreductase activity of the respiratory chain, was found to be a potent inhibitor of the chloramphenicol-induced alternative oxidase-mediated NADH and ubiquinol-15 oxidation in N. crassa mitochondria. It may be concluded that 2-thenoyltrifluoroacetone interacts with a specific electron carrier of the alternative oxidase, possibly an iron-sulphur centre associated with an ubisemiquinone.

Both a penicillinase and a cephalosporinase were purified from a strain of Serratia marcescens (GN7647) resistant to β-lactam antibiotics. The penicillinase was identical to the type I penicillinase, mediated by Rms212 and R-TEM. The purified cephalosporinase, a typical chromosomally mediated enterobacterial β-lactamase, gave a single protein band on polyacrylamide gel electrophoresis and immunoelectrophoresis; the pI was 9·56 and its molecular weight was approximately 37000. It contained tryptophan but not cysteine. The specific activity was 374 units (mg protein)−1 for the hydrolysis of cephaloridine, and the optimal pH was 8·5. Rabbit antisera raised against the purified cephalosporinase showed no cross-reaction in a neutralization test with cephalosporinases produced by other species of Enterobacteriaceae.

The mode of action of nosiheptide (multhiomycin) on bacterial protein synthesis is closely similar to that of thiostrepton. Both antibiotics inhibit functions of elongation factors Tu and G and greatly reduce the synthesis of guanosine penta- and tetraphosphates in response to stringent factor. Furthermore, the actinomycetes which produce these antibiotics defend themselves against their products in similar fashion. This involves specific pentose-methylation of 23S ribosomal RNA.

Various chemical reagents known to extract spore coat protein were used to extract spore-lytic enzyme (SLE) from intact and germinated spores of Clostridium perfringens. Of the reagents tested, 7·2m-urea plus 10% (v/v) mercaptoethanol, pH2·85, solubilized the most SLE activity per mg spores. The quantity of SLE extracted was dependent on the initial pH of the reagent, with a maximum between pH 2·7 and 3·0. Germinated spores yielded more SLE than non-germinated spores upon urea/mercaptoethanol extraction. SLE release during spore germination probably utilizes a trigger mechanism not satisfied by germination alone. Significant amounts of SLE were released during germination when spores were suspended in potassium chloride or a complex germinant mixture containing brain-heart infusion, yeast extract and chloramphenicol, but not during germination with sodium nitrite, which non-enzymically lysed the cortical peptidoglycan. Greater solubilization of SLE activity was obtained by urea/mercaptoethanol extraction of spores germinated with nitrite than of spores germinated with either potassium chloride or the complex germinant.

The rates of cell division and of protein, DNA and RNA synthesis upon transition of Mycobacterium avium to and from rich medium were examined. The changes in cell morphology (elongation) were also examined by optical and electron microscopy. Upon transfer from poor to rich medium, the rate of synthesis of RNA increased rapidly, followed by an increase in protein synthesis within 3 h and by an increase in DNA synthesis within 7 h; cell division began after a lag of about 10 h. Upon transfer from rich to poor medium, the preshift rates for protein and DNA synthesis changed to postshift rates after 3 h and 7 h, respectively; RNA synthesis stopped immediately, there was a transient fall in total RNA, and synthesis was resumed at a new rate only after 24 h. After the period of adjustment to new medium, the bacteria entered the postshift growth in which cell size, the increase in cell mass (absorbance at 650 nm) and viable counts, and the rates of synthesis of protein, DNA and RNA were constant. Ultrastructural examination of elongated cells during the adjustment period showed that they had septa at different stages of formation, but no evidence of fragmentation was found. It was concluded that cell division in M. avium was by binary fission, and that the notion of a life-cycle was not supported by the present findings.

Four gnotobiotic lambs were fed, after weaning, on a pelleted concentrate diet containing 90% barley. They were inoculated with a defined flora of 11 species of bacteria designed to reproduce the feed digestion of the conventional lamb. Cultural assessments of the growth of the bacteria and analysis of rumen fermentation products showed that rumen function was near normal, and the lambs grew steadily. However, at an age of about 115 to 130 d, rumen function seemed to fail in three of the lambs and they rapidly lost weight and died. The fourth lamb was removed from isolation at about the same time, when there was some slight indication of loss of rumen function. Kept among conventional lambs it then continued to gain weight as did the conventional animals, but its rumen flora changed. The main species in the defined flora remained as major components of the rumen population after the lamb had been exposed to other animals, but in addition a mixed population of other bacteria, like that in conventional animals, quickly developed.

Other experiments showed that gnotobiotic lambs could be routinely weaned on to a concentrate feed containing 45% barley and 50% grass and that, when the lambs were inoculated with a defined flora similar to that given to the lambs in the first experiment, an adequate rumen fermentation could be established and the animals would grow. On changing to an all-grass feed, however, rumen function declined and the lambs ceased to grow. A change back to the barley/grass diet restored rumen fermentation and lamb growth, but whereas amylolytic bacteria were present in normal numbers, cellulolytic bacteria were absent and there appeared to be little, if any, fibre digestion. However, the rumen function again appeared to fail at about the same age of lamb as in the first experiment.

Protoplasts of Streptomyces coelicolor showed the same ultraviolet killing kinetics as spores. Irradiated protoplasts gave rise to recombinants when they were fused with unirradiated protoplasts of a strain carrying complementary genetic markers. The decline with u. v. fluence in the capacity of irradiated protoplasts to yield recombinants inheriting individual markers was some six time less steep than that of the survival of unfused protoplasts; thus, for example, protoplasts reduced to only 0·01% survival still yielded 10% as many recombinants as unirradiated protoplasts. Each of six widely separated markers of the irradiated parent was inherited independently of the others, with a frequency falling exponentially with u. v. fluence.

Complementable spo mutants of Bacillus subtilis could be ‘rescued’ to form heat-resistant spores by fusing them with sporulating cells of the wild-type strain ( Dancer & Mandelstam, 1981 ). The latter strain acted as a ‘rescuer’. Rescuer protoplasts had to be derived from cells grown in a medium that allowed rapid and synchronous sporulation subsequently. Even protoplasts made from vegetative cells growing in such a medium (t 0 protoplasts) acted as rescuers. Therefore, it was not necessary for the rescuer cells to have initiated sporulation before they were converted to protoplasts.

The genetic requirement for t 0 rescuer protoplasts was that they were spo + for the gene to be complemented. Strains in the same complementation group did not rescue one another. Protoplasts of spo strains blocked at stages O, IV and V of sporulation acted equally well as rescuers whether they were converted to protoplasts at t 0 or t 3 (i.e. 3 h after transfer to sporulation medium). However, t 3 protoplasts of stage II and stage III mutants were much less effective rescuers than their t 0 counterparts. This difference was not due to a dominance effect expressed only in t 3 protoplasts.

In Escherichia coli a single copy of Tn10 confers high-level resistance to tetracycline. Resistance itself results from expression of three distinct mechanisms which normally act together ( Shales et al., 1980 ). In cells containing two copies of Tn10, the level of resistance to tetracycline was reduced. This was not due to overproduction of the repressor which controls the resistance genes, because strains diploid for an operator-constitutive allele of Tn10 also exhibited reduced expression of resistance. The negative gene dosage effect resulted from decreased expression of two mechanisms (1 and 2) consequent on enhanced expression of the third mechanism. The net result of increasing the copy number was a decrease in resistance because mechanism 3 was less efficient than mechanisms 1 and 2 in protecting the cell against tetracycline. The DNA sequence responsible for the reduced expression of resistance was contained in the internal Bgl II fragment of Tn10. This sequence, which is probably unique to Tn10, may encode the protein which mediates mechanism 3. Elevated levels of this protein probably cause decreased expression of mechanisms 1 and 2.

Synthesis of fructokinase of Streptomyces violaceoruber has been obtained with a preincubated S-30 fraction from Escherichia coli and RNA from S. violaceoruber. The fructokinase formed was identified by its exceptional catalytic activity at pH 10 and by the similar specificity properties of the native enzyme and the enzyme synthesized in vitro. Its presence was confirmed among the radioactive products of protein synthesis by its transition of molecular weight from 20000 to 80000 in the presence of MgATP and by electrophoresis of purified preparations. The presumptive mRNA of fructokinase contained poly(A) sequences of variable length. Fructokinase mRNA was present in S. violaceoruber when grown with fructose but not with glucose, indicating that the induction of fructokinase is controlled at the level of transcription.

Guinea-pig subcutaneous chambers were infected with a mixture of gonococcal variants of defined outer membrane protein profile. Survival within the chambers was a two-stage process. The initial advantage conferred by the lack of opacity-related outer membrane protein was transient and survivors were replaced by opaque colonial variants. Amongst these survivors were variants which produced opacity-related proteins (IId, IIe and IIf) not present in the initial inoculum. Thus, outer membrane protein composition is an important factor in survival in vivo.

Electron microscopic observations of acutely infected rabbit testes showed that the majority of Treponema pallidum were extracellular, and confined to interstitial regions of the tissue. Organisms were often adjacent to small blood vessels, where they should be freely accessible to non-specific humoral and cellular defence mechanisms. However, there was no accumulation of leucocytes in blood vessels or infiltration of inflammatory cells into infected areas. Inoculation of live treponemes into perforated plastic chambers which had been implanted subcutaneously in rabbits or guinea-pigs did not incite significant infiltration into the chamber fluid of inflammatory cells, in contrast to that seen after inoculation of Neisseria gonorrhoeae. Interaction of antibody from infected rabbits with live treponemes freshly extracted from rabbit testes could not be detected by an indirect fluorescent antibody method. These observations suggest that T. pallidum escapes recognition by host defences.

The cell surface of Bacteroides vulgatus was examined by electron microscopy. The outer membrane complex was removed by EDTA and mild sonication and the antigens of this complex were characterized by enzyme-linked immunosorbent assay and crossed immunoelectrophoresis. The species-specific antigen was identified and was shown to be the major outer membrane protein with a molecular weight of 100000.

The pleiotropic character of the envC chain-forming mutant of Escherichia coli was found to include leakage of periplasmic enzymes and an abnormal tendency to autolyse. Washed suspensions of envC cells released murein fragments into the supernatant, and cell extracts from the mutant were richer than those of the wild type in exo-β-N-acetylglucosaminidase (187% of the wild-type value) and in soluble endopeptidase (256%) activities, but N-acetylmuramoylamidase, d,d-carboxypeptidase, l,d-carboxypeptidase and transglycosylase were not markedly different. When envC cells were grown in medium containing 0·58 m-sucrose, the chains broke up into rods, the l,d-carboxypeptidase activity increased about sixfold and d,d-carboxypeptidase 1B about twofold. It is suggested that l,d-carboxypeptidase is involved in septum splitting. The results suggest that the triggering of autolysis in E. coli envC depends on the alteration of envelope constituents rather than on an enhanced activity of murein hydrolases.