- Volume 133, Issue 8, 1987

SUMMARY: The hydrolysis of lactose in Streptococcus bovis was shown to be catalysed by β-D-galactosidase. To isolate lactose catabolic genes, a gene library of S. bovis DNA was constructed in bacteriophage λ47.1 and recombinants expressing β-D-galactosidase activity were detected using 5-bromo-4-chloro-indoyl-β-D-galactoside (X-Gal). The gene was cloned on a 7·8 kb DNA restriction fragment into pBR322 to generate the recombinant plasmid pHG1, which also encoded S. bovis lactose permease, thiogalactoside transacetylase and lactose repressor protein. The position and orientation of the four genes were determined by subcloning and transposon mutagenesis. The β-D-galactosidase, lactose permease and thiogalactoside transacetylase genes constitute an operon controlled by the repressor protein. β-D-Galactosides induced expression of the S. bovis lac genes in Escherichia coli while glucose had no effect. The nucleotide sequence of the presumptive regulatory region of the S. bovis lac operon was determined and compared with the corresponding E. coli sequence. The operators and the 5’ end of the lacZ genes showed strong identity. The catabolite activator protein binding sequence, present in the E. coli promoter, was absent from the corresponding S. bovis region.

SUMMARY: A Vibrio alginolyticus SDS-resistant, Ca2+-dependent serine exoprotease gene, cloned on a 7-1 kb DNA fragment in the recombinant plasmid pVP100, was expressed from its own promoter in Escherichia coli. Although active exoprotease was produced by late stationary phase E. coli(pVP100) cultures after 15 h incubation in proteinaceous medium containing Ca2+, transcription and translation of the exoprotease occurred before 6 h, during exponential growth. The cloned exoprotease was synthesized as a pool of inactive precursor molecules during exponential growth, and released as active exoprotease 8 h later by a process which did not require protein synthesis or involve cell lysis. Release of the exoprotease by E. coli(pVP100) was not inhibited by o-phenanthroline, quinacrine or cerulenin. Supernatant samples from E. coli(pVP100) cultures contained two SDS-resistant exoproteases with apparent M r values of approximately 54000 and 39000. The cloned exoprotease activity was inhibited by EDTA and a serine protease inhibitor, but was not affected by an inhibitor of trypsin-like enzymes.

SUMMARY: Pseudomonas aeruginosa region II alginate genes are involved in the biosynthesis of the uronic acid containing exopolysaccharide, alginic acid. We have subcloned and overexpressed various DNA fragments contained within region II in an attempt to further characterize and more precisely localize the genes involved in alginate production. Overexpression of the genes controlling alginate biosynthesis within region II was accomplished by placing various cloned restriction fragments under the transcriptional control of the hybrid trp-lac (tac) promoter, and plasmid encoded proteins were examined in a maxicell expression system. We correlated various region II plasmid constructions with the ability to complement specific alginate negative (alg) mutants and code for polypeptides. Several proteins suspected of being involved in alginate production were encoded by sequences within region II. The results of this study further reveal that the transcriptional orientation of the alg loci within region II appears to be in the direction from argF to pmi. The specific activities of phosphomannomutase (PMM) and GDP-mannose pyrophosphorylase (GMP), two enzymes involved in the formation of the alginate precursor GDP-mannuronic acid, were measured in region II alg mutants and in cells overexpressing cloned segments from region II. Based on these enzyme measurements, we conclude that the remaining region II alg genes do not encode either PMM or GMP. These results support the suggestion that the remaining alg genes in region II are likely to be involved in post GDP-mannuronic acid processing events such as mannuronic acid transport, polymerization, secretion, epimerization and acetylation.

SUMMARY: Xanthine oxidase with acetaldehyde as substrate (the XOA system) generated superoxide anion and hydrogen peroxide, but this system had only weak bactericidal activity. Addition of Fe2+ and EDTA to the XOA system (XOA-Fe-EDTA system) increased bactericidal activity against Staphylococcus aureus, Escherichia coli, Listeria monocytogenes and Salmonella typhimurium, although both Mycobacterium tuberculosis and Candida albicans remained highly resistant. Catalase (H2O2 scavenger) and mannitol (OH scavenger) almost completely inhibited the bactericidal activity of the XOA-Fe-EDTA system whereas SOD (O2 - scavenger) was less inhibitory, Azide (1O2 scavenger) caused no such inhibition. The results suggest the possible role of OH, H2O2 and O2 - in the XOA-Fe-EDTA-mediated antimicrobial system, as effector molecules. There was no correlation between resistance of a given bacterium to active oxygen and the level of endogenous active oxygen-scavengers.

SUMMARY: The susceptibilities of six micro-organisms to active oxygen species generated in the xanthine oxidase-mediated bactericidal system were as follows: Escherichia coli 81 ≥ Listeria monocytogenes EGD ≥ Salmonella typhimurium HKB-1 ≥ Staphylococcus aureus Smith ≥ Mycobacterium tuberculosis H37Rv ≥ Candida albicans NIH A207 (the last two organisms were essentially resistant to this system). The H2O2-Fe-mediated halogenation system exhibited a higher microbicidal activity. When the micro-organisms were compared for their sensitivity to bactericidal activity of resident mouse peritoneal macrophages (Møs), C. albicans, Staph, aureus and E. coli were killed rapidly, whereas M. tuberculosis, L. monocytogenes and S. typhimurium were more resistant. In tests for the ability to trigger an oxidative burst in mouse peritoneal Møs (as measured by chemiluminescence), Staph, aureus showed the highest activity followed by the other organisms in the following order: C. albicans > E. coli > L. monocytogenes ⋍ M. tuberculosis. S. typhimurium exhibited no triggering activity. The high susceptibility of Staph, aureus and E. coli to Mø bactericidal activity, and the partial resistance of L. monocytogenes and M. tuberculosis, correlated with their susceptibility to active oxygen and the H2O2-Fe-mediated halogenation reaction.

SUMMARY: The amino-terminal end of the 168 kDa adherence protein from the membrane of Mycoplasma pneumoniae was sequenced up to 12 amino acids. A synthetic peptide containing nine amino acids of this sequence was used to study the antigenicity of the amino-terminus of the 168 kDa protein and the involvement of the homologous sequence of the protein in the adherence process. The synthetic peptide when coupled to ovalbumin was immunogenic in rabbits. Antibodies against this peptide epitope could be demonstrated in sera taken during natural M. pneumoniae infection in humans. The structural domain of the 168 kDa protein homologous with the synthetic peptide did not appear to be involved in adherence, as the synthetic peptide or its homologous antibody failed to inhibit adherence of M. pneumoniae.

SUMMARY: We have investigated the presence of the aerobactin system and the location of the aerobactin genes in enteroinvasive strains of Escherichia coli. Also, we cloned the aerobactin region and its flanking sequences from the chromosome of a strain of Shigella flexneri and compared the molecular organization of the aerobactin genes in the two genera. Of the 11 enteroinvasive E. coli strains studied, 5 possessed the aerobactin genes, which were located on the chromosome in each case. These strains produced and utilized aerobactin and also were susceptible to the bacteriocin cloacin-DF13. Restriction endonuclease mapping and hybridization experiments showed that the regions corresponding to the aerobactin-specific sequences were very similar in both enteroinvasive E. coli and S. flexneri. However, differences were found in the region corresponding to the aerobactin receptor gene. The regions flanking the aerobactin system in enteroinvasive E. coli and S. flexneri exhibited some similarities but were different from those in pColV-K30. Under iron-limiting conditions, aerobactin-producing enteroinvasive E. coli and S. flexneri synthesized outer-membrane proteins of 76 and 77 kDa, respectively, which cross-reacted immunologically with rabbit antiserum raised against the 74 kDa pCoIV-K 30-encoded ferric aerobactin receptor.

SUMMARY: The extracellular haemolysin produced by Vibrio vulnificus strain FCC was partially purified from the culture supernate by sequential ammonium sulphate precipitation, gel filtration with Sepharose 4B, and DEAE-Sephacel ion-exchange column chromatography. Using this semi-purified haemolysin as the antigen, several monoclonal antibodies (MAbs) were established; they were all of the IgG2b class with lambda light chains. One representative MAb, 6F8D, completely neutralized the haemolytic activity and mouse lethal activity of extracellular toxin(s). In immunoblotting analysis of the peptides of the semi-purified haemolysin separated by SDS-PAGE, this MAb reacted, in particular, with a 36 kDa peptide. These findings suggest that the haemolysin is probably identical to the lethal toxin in the culture supernate of V. vulnificus strain FCC, which contained the 36 kDa peptide.

SUMMARY: Saccharomyces cerevisiae was unable to grow in media containing above about 1·5 mm free sulphite at pH 4·0, whereas Saccharomycodes ludwigii grew at the same pH value in the presence of 7·8 mm free sulphite. Expressed in terms of μl of intracellular water, the initial velocity of sulphite accumulation by S'codes ludwigii was approximately twice that of S. cerevisiae, although the former yeast accumulated at equilibrium only about one-third of the amount of sulphite accumulated by S. cerevisiae. A Woolf-Hofstee plot for accumulation of SO2 by S'codes ludwigii at pH 3·0 and 30°C gave a vertical line. Incorporation of sulphite in growth media induced excretion of acetaldehyde by both yeasts, the rate being greater by S'codes ludwigii than S. cerevisiae. Acetaldehyde excretion was accompanied by release of lower concentrations of pyruvate. Excretion of 2-oxoglutarate was barely detectable. It is suggested that the greater resistance of S'codes ludwigii to sulphite, compared with S. cerevisiae, may be explained partly by its decreased capacity to accumulate the compound, and partly by its ability to produce more acetaldehyde.

SUMMARY: Fourteen yeast strains from six genera were analysed for the presence of long-chain alcohols. Six strains from three genera contained long-chain alcohols, highest levels being found in Candida albicans. The alcohols were identified and determined by TLC, GLC and GLC-MS. The major long-chain alcohols synthesized by these organisms were saturated, primary alcohols with C14, C16 or C18 chain length. Unsaturated long-chain alcohols were not detected. In all strains that produced long-chain alcohols, the relative proportions were C16 > C18 > C14. Long-chain alcohol contents were higher in organisms from anaerobically, as compared with aerobically, grown cultures reaching about 650 μg (g dry wt organisms)-1 in stationary-phase cultures of C. albicans. In cultures of C. albicans, synthesis of long-chain alcohols occurred only after the end of exponential growth. The alcohols were predominantly present as free alcohols. The fatty-acyl chain-length profile of the triacylglycerol and to a lesser extent the sterol/wax ester fractions from C. albicans reflected that of the long-chain alcohols produced by this yeast.

SUMMARY: Addition of plasma membrane depolarizing agents, such as dinitrophenol (DNP) and azide, to cells of Saccharomyces cerevisiae under aerobic conditions, is known to cause an increase in the cAMP level within 15 s. We found that both compounds lowered the intracellular pH (measured by in vivo 3P-NMR) drastically within the same time period. Plasma membrane depolarization, however, was much slower: DNP and azide had no effect on the membrane potential during, respectively, the first 2 min and the first 10 min after addition. Apparently, the intracellular pH of yeast is much more sensitive to perturbation than the membrane potential. The effect of both compounds on the cAMP level was highly dependent on the extracellular pH: when the latter was raised, the effect disappeared completely between pH 6 and 7. A similar dependence on the extracellular pH was observed for the lowering of intracellular pH. Addition of organic acids, such as acetate and butyrate, at low pH and under aerobic conditions, also caused an immediate increase in the cAMP level and an immediate drop in the intracellular pH. These results suggest that agents such as DNP and azide do not raise the cAMP level in yeast cells because of their membrane depolarizing properties but because they lower the intracellular pH. Under anaerobic conditions, DNP. azide and organic acids were much less effective in increasing the cAMP level. Addition of a small amount of glucose, however, restored their capacity to enhance the cAMP level. This suggests that under anaerobic conditions and in the absence of glucose the ATP level is a limiting factor for cAMP synthesis.

SUMMARY: Addition of glucose to derepressed cells of the yeast Saccharomyces cerevisiae is known to cause a rapid, transient increase in the cAMP level, which lasts for 1–2 min and induces a cAMP-dependent protein phosphorylation cascade. The glucose-induced cAMP signal cannot be explained solely on the basis of an increased ATP level. Transient membrane depolarization and transient intracellular acidification have been suggested as possible triggers for the cAMP peak. Addition of glucose to cells in which the plasma membrane had been depolarized still produced the increase in the cAMP level excluding membrane depolarization as the possible trigger. Using in vivo 31 P NMR-spectroscopy we followed phosphate metabolism and the time course of the drop in the intracellular pH after addition of glucose with a time resolution of 15 s. Under aerobic conditions the initial pH and ATP level were high. On addition of glucose, they both showed a rapid, transient drop, which lasted for about 30 s. Under anaerobic conditions, the initial pH and ATP level were low and on addition of glucose they both increased relatively slowly compared to aerobic conditions. Several conditions were found in which the pH drop which occurs under aerobic conditions could be blocked completely without effect on the cAMP signal or without completely preventing it: addition of NH4Cl together with glucose at high extracellular pH and addition of a low concentration of glucose before a high concentration. Also, when glucose was added twice to the same cells no consistent relationship was observed between the pH drop and the cAMP peak. These results appear to exclude transient intracellular acidification as the trigger for the cAMP signal. Hence, we conclude that the effect of glucose cannot be explained on the basis of effects known to be caused by the membrane depolarizing compounds which cause increases in the cAMP level. A new, more specific kind of interaction appears to be involved.

SUMMARY: Cells of Clostridium pasteurianum grown on glucose lacked enzyme systems specific for sorbitol metabolism: the phosphoenolpyruvate (PEP)-dependent phosphotransferase (PTS) and sorbitol-6-phosphate dehydrogenase. These activities were induced by growth on sorbitol, but were partially repressed by glucose. During growth on excess glucose and sorbitol, the sorbitol PTS was absent until the growth rate declined at the onset of stationary phase. Synthesis of at least two PTS proteins, one soluble and one membrane-bound, was repressed in exponentially growing cells. In contrast to the PTS, sorbitol-6-phosphate dehydrogenase was present during the exponential phase.

Glucose and methyl α-glucoside inhibited the activity of the sorbitol PTS in permeabilized cells, and this inhibition was competitive at the level of PEP. This implies that in intact cells sorbitol is excluded by glucose, at least in part due to competition between the glucose and sorbitol phosphotransferases for a common pool of phosphate and energy. Exclusion of sorbitol may be an important factor in the repression of sorbitol metabolism by glucose.

SUMMARY: Incorporation of polysaccharides into the walls of regenerating protoplasts of Candida albicans was followed in the presence of papulacandin B, tunicamycin and nikkomycin. With the first drug, chitin was incorporated normally whereas incorporation of glucans and mannoproteins was significantly decreased. Tunicamycin decreased incorporation of all wall polymers when added at the beginning of the regeneration process but blocked only mannan and alkali-insoluble glucan incorporation when added after 5 h. Nikkomycin inhibited chitin synthesis, and the walls formed by the protoplasts were enriched in alkali-soluble glucan. Pulse-chase experiments suggested that a precursor-product relationship between the alkali-soluble and alkali-insoluble glucans existed in the wall. The results obtained with the antibiotics were confirmed and extended by cytological studies using wheat-germ agglutinin labelled with colloidal gold and concanavalin A-ferritin as specific markers of chitin and mannoproteins respectively. The results support the idea that regeneration of walls by protoplasts occurs in two steps: firstly, a chitin microfibrillar skeleton is formed, and in a later step glucan-mannoprotein complexes are added to the growing structure. The chitin skeleton probably allows the orderly spatial arrangement of the other polymers giving rise to the regenerated cell wall.

SUMMARY: The effect of phosphate on the different stages of the life cycle of Myxococcus coralloides D has been examined. A high concentration of phosphate inhibited the process of fructification, but glycerol-induced myxospore formation and germination were independent of phosphate concentration. Acid and alkaline phosphatases were detected and both were released into liquid medium during the exponential growth phase. On solid medium, phosphatase activities showed different patterns according to the stage of the life cycle. The increase of these two activities was transient during glycerol-induced myxospore formation. Differences in phosphatase activities during germination of glycerol-induced myxospores and fruiting-body myxospores were found.

SUMMARY: In order to explore the function of each of the four enzymes involved in ammonium assimilation in Neurospora crassa [NADP-dependent glutamate dehydrogenase (GDH), NADH-dependent glutamate synthase (GOGAT) and the α and β isozymes of glutamine synthetase (GS)] growth curves, enzymic activities and intracellular pools of ammonium, glutamate and glutamine were determined in the wild-type as well as in mutants lacking one or two of these biosynthetic enzymes. These parameters were measured in ammonium-limited chemostat-type cultures, in which conditions resembling a continuous culture were achieved for the first time for a filamentous fungus, and in ammonium-excess batch cultures. NADP-dependent GDH appeared to be the main provider of cellular glutamate in the presence of limited and excess ammonium but could be partially replaced by NADH-dependent GOGAT depending upon the presence of GSβ. GOGAT appeared to have the main role in the recycling of glutamine to glutamate, particularly in ammonium-limited conditions. GSβ was the main provider of cellular glutamine but GSα could substitute for this activity if GDH was also present. In conditions of ammonium excess GDH and GOGAT were repressed by glutamate and GS was repressed by glutamine, as previously found. However, in conditions of ammonium limitation these enzymes appeared to escape nitrogen repression.

SUMMARY: Mutants of the filamentous fungus Neurospora crassa which grow well only in a medium in which the concentration of calcium is lower than in the medium used for culture of the wild-type strain were isolated by use of the ionophore A23187 and medium which contained a high concentration of calcium. These mutants showed a significantly lower uptake of calcium into their vacuoles than the wild-type, while other activities associated with the transport of calcium across several other types of membrane were not affected. The results indicated an essential role for the vacuoles in the regulation of the cytosolic concentration of calcium.