- Volume 128, Issue 3, 1982

The content of glycogen phosphorylase (1,4-α-d-glucan:orthophosphate α-d-glucosyl-transferase, EC 2.4.1.1) in yeast (Saccharomyces carlsbergensis) cells depended on the growth phase. Cells of the early exponential phase under carbohydrate-limited conditions showed low, but significant, phosphorylase activity; the activity markedly increased in the late exponential growth phase, concomitant with the appearance of measurable phosphorylase antigen. This pointed to an induction of the enzyme. During the interexponential phase (the slow proliferation phase during the diauxic growth of the culture, when the cells switch to utilization of accumulated ethanol) and the stationary growth phase, phosphorylase concentration remained constant while its specific molecular activity increased further, probably caused by conversion of the enzyme to an active form. During transition of stationary phase cells to growth, phosphorylase activity and concentration slowly decreased in the cells at a rate compatible with dilution by newly synthesized proteins. A residual activity always remained, which could be attributed to the presence of active phosphorylase, detectable by activity staining after gel electrophoresis in the presence of glycogen. No direct correlation could be detected between the specific molecular activity of phosphorylase and glycogen metabolism. This indicated that covalent modification of the enzyme regulated the total capacity of the enzyme available to the cell, rather than the actual activity limiting glycogen breakdown.

A cell-bound β-glucosidase (β-d-glucoside glucohydrolase; EC 3.2.1.21) from Clostridium thermocellum was purified to apparent homogeneity. A molecular weight of about 43000 gel fluration of the native enzyme on Ultrogel AcA34. A constant ratio of aryl-β-glucosidase and cellcouse throughout purification, similar heat stabilities, pH profiles and sensitivity to different hiibitor and competitive inhibition of the aryl-β-gluosidase and the β suggest that the same enzyme accounts for the aryl-β-glucosidase and the cellobiase activities. However, the affinity for cellobiose was very much lower than for p-β-d-glucoside. The β-glucosidase had maximum rates at pH 6·0 to 6·5 for both activities. The enzyme was specific for substrates with the both activities. β-configuration. particulary and β-1,3 and β-1,2 linkages. The enzyme did not hydrolyse carboxymethylcellulose or cellulose, but hydrolysed cello-oligosaccharides. It was strongly inhibited by d-glucono-δ-lactone was sensitive to thiol reagents. When preparations of C. thermocellm cellulase were supplemented with purified β-glucosidase, glucose was the predominant product of cellulose and the rate of saccharification was increased.

Histidinol dehydrogenase has been purified from a derepressed mutant of Escherichia coli B. A molecular weight of about 91000 was estimated by gel filtration. The native enzyme seems to be composed of two similar subunits which have a molecular weight of 52000 as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The pI of the enzyme as determined by isoelectric focusing is 4·75. The enzyme is maximally active at pH 9·5. It is highly specific for NAD+ and histidinol, with a Km (NAD+) of 0.57 mM and a Km (histidinol) of 14 m. Mn2+ is required for maximal activity. The enzyme is completely inactivated by 8 m-urea but regains its activity very quickly upon removal of the urea. Mn2+ and histidinol protect the enzyme from heat inactivation.

Enzymes involved in the synthesis of teichoic acid and its linkage to the wall in Bacillus subtilis W23 were measured in chemostat cultures growing at equilibrium at a dilution rate of 0·2 h−1 in different concentrations of inorganic phosphate. All the enzymes, except teichoic acid glucosyl transferase, which was insensitive to changes in phosphate concentration, were almost undetectable at 0·5 mm-phosphate. At higher phosphate concentrations the changes in activity of the enzymes of linkage unit synthesis were sufficient to account for the changes in the rate of incorporation of teichoic acid into the wall in vivo. Between 3·5 and 4·5 mm-phosphate the amount of teichoic acid synthesized in vivo increased, but no increase in the ability of toluenized bacteria to synthesize teichoic acid could be detected. Allosteric regulation might therefore be important at high phosphate concentrations. Bacteria maintained a constant ATP content and a constant adenylate energy charge during chemostat growth at all phosphate concentrations.

Treatments leading to the initiation of germination of Mucor racemosus sporangiospores were examined. The results support the hypothesis that glucose is a specific trigger molecule for the initiation of Mucor racemosus sporangiospores. Glucose and some of the glucose analogues tested could initiate germination, mannose, 3-O-methyl-d-glucose, 5-thio-d-glucose and 6-deoxy-d glucose being the most effective. The initiation event appeared to depend on the concentration of the initiator, with glucose and 3-O-methyl-d-glucose exhibiting nearly identical kinetic constants. Spores accumulated not only glucose and 3-O-methyl-d-glucose, but also the 1-O-methyl-d-glucose analogue, which did not initiate germination. The accumulated 3-O-methyl-d-glucose was not metabolized. The initiation sequence appeared to require the continued presence of the initiator as well as protein synthesis.

The role of amino acids as triggers of Mucor racemosus sporangiospore germination was investigated. No single amino acid was as effective as glucose or peptone at triggering germination induced by glucose or peptone was pH-independent, whereas germination induced by glutamate was pH-dependent. The composition of the free amino acid pool of M-spores (those unable to germinate on glutamate) was qualitatively and quantitatively similar to that of C-spores (those capable of germinating on glutamate) with the exceptions of hydroxyproline and methionine whose concentrations were several-fold higher in C-spores. Glutamate and leucine were taken up by germinating and non-germinating spores; however, significant protein synthesis occurred only in germinating spores. Spores not triggered by 3-O-methyl-d-glucose (M-spores) contained about one-half the protein of those triggered by 3-O-methyl-d-glucose (C-spores). C-spores initiated to germinate by 3-O-methyl-d-glucose decreased in total organic carbon and protein over a 6 h period: removal of the 3-O-methyl-d-glucose resulted in an immediate halt of protein degradation and spore swelling. These results suggest that protein is a major endogenous reserve in M. racemosus sporangiospores and that its turnover is a necessary event for glucose-triggered germination.

Strains of Escherichia coli deficient in post-replication recombination repair were more sensitive to bleomycin than wild-type, repair-proficient strains. Mutants lacking excision repair functions were no more sensitive to bleomycin than the wild-type strains, indicating that this pathway is not involved in the repair of bleomycin-damaged DNA. Plasmid R46 not only protected repair-proficient strains but also those with recB, recC, uvrA or lig genotypes, suggesting that R46 protection against bleomycin is independent of these host repair functions. However, R46 protection was abolished in recA or polA strains, indicating that these gene functions are necessary for plasmid-mediated protection. It is suggested that protection may be due to a recA +-dependent interaction of a plasmid-encoded product with host DNA polymerase I, resulting in an increase in the DNA repair capacity of cells.

Galactitol-positive strains of Escherichia coli K12 are inhibited by the galactitol analogues l-fucitol and 2-deoxy-d-galactitol, but not by d-fucitol; Salmonella typhimurium LT2 is not inhibited by these compounds. Most mutants selected as resistant to either toxic compound are unable to utilize galactitol as carbon source, but a relatively rare class is inducible for the Enzyme II of the galactitol: phosphoenolpyruvate phosphotransferase system, the product of which is d-galactitol 6-phosphate. The lesion in one such mutant maps near metG at about min 45 on the E. coli genome.

A specialized transducing phage for the srlA gene, specifying the sorbitol-specific Enzyme II of the phosphoenolpyruvate: sugar phosphotransferase system, was constructed and its DNA was analysed by restriction endonuclease digestion. Phage construction involved four steps: (1) integration of λ into the srlA gene; (2) selection of phage carrying (a) the left and (b) the right end of the srlA gene by means independent of the function of the new DNA acquired; (3) reconstitution of the srlA gene in a dilysogen of these two phage; and (4) the excision, using the heteroimmune lambdoid phage 21, of a plaque-forming srlA + phage from the dilysogenic chromosome. Comparison of the DNA restriction digests of the transducing phage with those of its parents and of wild-type λ revealed fragments consisting partly of λ and partly of Escherichia coli DNA. The junction points in the intermediate phage define a site that must lie within the reconstituted gene of the final phage. This technique should be of general application in relating genes, cloned by our method, to DNA sequences.

When Escherichia coli carrying a thermosensitive mutation in DNA ligase was grown at the restrictive temperature, several functions associated with the SOS system were induced. These included λ prophage induction, W-reactivation and W-mutagenesis of ultraviolet-irradiated λ phage, and recA protein synthesis, all of which were lexA + recA + recB + dependent and chloramphenicol sensitive, and lexA +-dependent filamentation. These results indicate that ligase-deficient growth leads to the induction of the SOS system, and that all the above functions may respond to common induction signals.

Antibodies were detected by an enzyme-linked immunosorbent assay (ELISA) in sera from rabbits immunized with outer membranes from colonial opacity variants of Neisseria gonorrhoeae P9. ELISA-inhibition experiments with purified antigens revealed approximately equal proportions of antibodies directed against each of the three major surface antigens, lipopolysaccharide. the major outer membrane protein (protein I) and protein II, the variable protein associated with colonial opacity. Inhibition experiments with intact gonococci showed considerable surface antigenic diversity which could be correlated with differences between the protein II species present. Despite their considerable structural homology, different protein II species from colonial variants of the same strain showed little cross-reactivity with specific anti-protein II sera, thus demonstrating the considerable variation in that part of the antigen which is exposed on the surface of the gonococcus and is closely involved in pathogenic mechanisms.

Chlamydia trachomatis causes common infections of the eyes and genital tract in man. The mechanism by which this obligate intracellular bacterium is taken into epithelial cells is unclear. The results described here support the concept that chlamydial infection of HeLa cells is under bidirectional cyclic nucleotide control, with guanosine 3′:5′-cyclic monophosphate (cGMP) acting as a stimulator, and adenosine 3′:5′-cyclic monophosphate (cAMP) as an inhibitor. Treatment of the HeLa cells with the divalent cation ionophore A23187, with carbamoylcholine, or with prostaglandins known to increase the concentration of endogenous cGMP, also increased host cell susceptibility to chlamydial infection. Cyclic GMP was only effective if added at or before chlamydial inoculation, suggesting that its main effect was on chlamydial uptake. The stimulatory effect of cGMP, but not antagonism by cAMP, was abolished if the cells were first treated with any of four different inhibitors of prostaglandin synthesis, suggesting a critical role for endogenous prostaglandin synthesis. Centrifugation of chlamydiae on to host cells was followed by a rapid increase in the mobility of Ca2+ across the cell membrane. The interrelationships of these observations and the possibility that chlamydiae and other intracellular pathogens might evoke alterations in host cell prostaglandin and cyclic nucleotide concentrations to aid their own uptake are discussed.

Quantitative analysis of the staining of cholera enterotoxin on the surface of cells with specific antibodies against each subunit of cholera toxin, using a Fluorescence-Activated Cell Sorter, showed that not only subunit A but also subunit B penetrates the cell membrane. The detection of each subunit inside the cell was facilitated by the use of saponin, an agent which increases membrane permeability.

Populations of Saccharomyces cerevisiae NCYC 366, grown anaerobically under conditions that lead to enrichment of membranes with specific sterols and fatty-acyl residues, were subjected to freeze-thaw cycles and cold-shock stress. One freeze-thaw cycle caused considerable loss in viability, the loss being greater with organisms enriched in campesterol or cholesterol rather than ergosterol or stigmasterol. Retention of viability was greater in populations enriched in linoleyl rather than oleyl residues. Organisms enriched in ergosterol and either oleyl or cetoleyl residues were equally susceptible to death following freeze-thaw cycles, but less so than organisms enriched in palmitoleyl residues. Speed of freezing had little effect on retention of viability in organisms enriched in ergosterol and linoleyl residues, but an effect was observed in populations enriched in cholesterol and linoleyl residues. In populations enriched in ergosterol and linoleyl residues, sodium chloride protected against loss of viability, whereas in organisms enriched in cholesterol and linoleyl residues the salt rendered populations more susceptible to death following freezing and thawing. Resistance to cold-shock stress was also greater in populations enriched in ergosterol or stigmasterol rather than campesterol or cholesterol, and with organisms enriched in linoleyl rather than oleyl residues.