- Volume 123, Issue 2, 1981

The germination of blastospores of Candida albicans is accompanied by a rise in the intracellular concentration of adenosine 3′:5′-cyclic monophosphate (cyclic AMP). Germination was induced either by peptides isolated from seminal plasma or by an amino acid mixture, and both germination and the rise in cyclic AMP required a temperature of 37 °C. The rise occurred during the first hour of incubation, but full germination required a temperature of 37 °C for 4 h. Germination and the rise in cyclic AMP, in the presence of suboptimal concentrations of inducers, were stimulated by theophylline. Pre-incubation of cells with dithiothreitol in the absence of inducers inhibited subsequent germination but not the rise in cyclic AMP. Germination and the rise in cyclic AMP were inhibited if dithiothreitol or N-succinimidyl-3-(4-hydroxyphenyl)propionate was present during the induction period.

Escherichia coli 5′-nucleotidase was purified to apparent homogeneity as judged by polyacrylamide gel electrophoresis. Its molecular weight was estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, by gel exclusion chromatography, and by membrane filtration; values of 66000, 48500, and 15000 to 30000, respectively, were obtained. The enzyme was completely released from bacteria by osmotic shock treatment. The apparently anomalous behaviour of 5′-nucleotidase in terms of the molecular sieving hypothesis for the release of enzymes by osmotic shock proposed by Smith & Wyatt (1974) and extended by Broad & Smith (1979) is discussed.

Glutamate synthase, an enzyme involved in glutamate biosynthesis in nitrogen-deficient environments, has been purified 200-fold from the photosynthetic bacterium Rhodospirillum rubrum. The in vivo association of this NADPH-linked glutamate synthase with the photosynthetic membranes was suggested by its co-sedimentation with the chromatophore membranes and its ability to couple glutamate formation to light-generated reducing power. Although it was not homogeneous, the enzyme co-chromatographed with iron after three successive column chromatography steps and had the spectrum of a flavoprotein which suggests it may be an iron-sulphur flavoprotein similar to the enzyme from other prokaryotes. The Km values of glutamate synthase for NADPH, glutamine and 2-oxoglutarate were 15 µm, 130 µm and 35 µm, respectively. The molecular weight of glutamate synthase was salt-dependent in that the enzyme isolated in 0·4 m-NaCl showed a molecular weight of 840000, whereas in the absence of salt the molecular weight was about 260000. This phenomenon may be common to other glutamate synthases, since the molecular weight of this enzyme from Klebsiella pneumoniae was also influenced in a similar manner by NaCl.

Mutant strains of Escherichia coli male cells defective in Ca2+,Mg2+-dependent ATPase (unc) were constructed and tested for their ability to form a complex between sex pili and the filamentous phage fd under conditions where either the membrane potential or the cellular concentration of ATP was lowered. The uncoupler carbonyl cyanide m-chlorophenyl-hydrazone and the respiratory inhibitor cyanide, as well as valinomycin-K+ and colicin El, all markedly diminished complex formation, indicating that the maintenance of a membrane potential, but probably not the pH gradient, is essential for the formation of the complex. Since complex formation with freshly centrifuged cells (which initially lacked sex pili) as well as with preincubated cells (in which pre-existing pili were available for complex formation) was inhibited by exposure to the inhibitors, energy seems to be required for both the reappearance (probably assembly) and the maintenance of sex pili on the cell surface. Brief exposure of freshly centrifuged cells to arsenate resulted in only partial inhibition of complex formation. However, marked inhibition of complex formation was observed following exposure to arsenate of preincubated cells possessing sex pili. This indicates that compounds such as ATP may also be required for maintenance of sex pili on the cell surface.

An investigation of sediments from the littoral (shallow water) and profundal (deep water) zones of Blelham Tarn, a shallow eutrophic lake, showed marked differences in the microbial decomposition processes. These differences were due largely to differences in the degree of oxygenation, supply of electron acceptors, and mean summer temperature at the two sites. The changes in the hypolimnion (the deep water zone formed on thermal stratification, which may be treated essentially as a closed system) could be used to calculate profundal rates of aerobic respiration, NO− 3 and SO2− 4 reduction, and methanogenesis, relative to the accumulation of CO2. Laboratory measurements demonstrated that NH+ 4 accumulation, SO2− 4 reduction and methanogenesis were more intense in the profundal than in the littoral zone. Anaerobic processes that occurred in the littoral sediments did so at greater depths than in the profundal sediments. The release of CH4 and N2 bubbles also provided estimates of the importance of these processes at the two sites. At both sites aerobic respiration was the most important component (about 50%) of carbon mineralization; SO2− 4 reduction was the least important, accounting for only a small percentage of carbon turnover. Pathways of NO− 3 reduction and methanogenesis accounted for approximately equal proportions (varying between 15 and 25%) of the carbon mineralized. When the results were adjusted to account for the relative areas of the profundal and littoral zones, the former was the more important site of methanogenesis and SO2− 4 reduction, whereas aerobic respiration and NO− 3 reduction were greater in the littoral zone. The major end-product of NO− 3 reduction was NH+ 4 in the profundal and N2 in the littoral zone. The higher and continued levels of nitrification, which recycled the NH+ 4 in the littoral sediments, were thought to contribute to this.

Anaerobic bacterial activity was detected in wetwoods of living Populus deltoides Bartr. and Ulmus americana L. by analysis of chemical, structural and microbiological parameters. Wetwood contained significant quantities (⩾1·0 mm) of bacterial fermentation products including acetate, butyrate, propionate, ethanol, isobutyrate, isopropanol and methane. Oxygen was not detected in wetwood and the ammonia concentration was low (⩽5 μm). Scanning electron microscopy showed that vessel-to-ray pit membranes of wetwood xylem tissue were almost completely destroyed and were associated with dense bacterial populations. Bacterial nitrogenase activity was detected in wetwood samples. Anaerobic bacterial populations in wetwood were as large or larger than populations capable of aerobic growth, and contained (in total cell numbers g−1) heterotrophic (106 to 107), nitrogen-fixing (105 to 106) and methanogenic (103 to 104) species. Anaerobic bacteria were 104 times more numerous in wetwood than in sapwood. Thirteen strains of anaerobic heterotrophic bacteria were isolated and characterized from wetwood and included Clostridium, Bacteroides, Erwinia, Edwardsiella, Klebsiella and Lactobacillus species. Only two strains, a pectindegrading Clostridium species and a nitrogen-fixing Erwinia species, were prevalent in wetwoods of all the trees examined. The metabolic features of the strains examined correlated to the described chemical, structural and microbiological properties of wetwood.

The temperate actinophage SH10 has a wide host-range. In size and morphology it is similar to phage λ. It has a latent period of 40 to 50 min. The average burst size depends on the host strain and varies between 60 and 100 at 28°C. At 37°C the burst size is diminished by a factor of ten. The adsorption constant varies between 1·55 × 10−9 and 8·8 × 10−10 ml min−1 depending on the host strain. After mutagenesis by combined u.v.- and X-irradiation, clear and virulent mutants of SH10 were isolated. In addition, thermosensitive mutants of SH10 were obtained which fall into three complementation groups. The thermosensitive character was host-dependent. Phage SH10 is sensitive to sodium pyrophosphate and viable deletion mutants were isolated by virtue of their increased pyrophosphate resistance.

Several chlorate-resistant mutants of Penicillium chrysogenum were isolated and analysed; all were affected in nitrate assimilation. Nine loci were recognized by complementation analysis and these appear to be equivalent to the niaD (nitrate reductase structural gene), nirA (control locus) and seven cnx loci (responsible for the biosynthesis of a cofactor for nitrate reductase) of Aspergillus nidulans. The organization of the nitrate assimilation genes appears to be similar in both organisms, even to the extent of having contiguous genes coding for the nitrate and nitrite reductase enzymes.

The contribution of phagocytes and antibody to protection against Salmonella typhimurium during the early phase of infection in mice was analysed. Following intravenous injection, most of the bacteria were trapped in the liver and spleen within 10 to 60 min and killed within 6 h; surviving organisms began to multiply in these tissues after 24 h and reached a maximum at 5 to 7 d. The transient killing phase was abrogated by treatment with carrageenan, a macrophage blocker, but not by whole-body X-irradiation. These observations suggest that carrageenan-sensitive, but radio-resistant macrophages play an important role in the early phase of the infection. Actively immunized mice showed accelerated trapping and killing; the protection observed at the early stage of infection in immunized mice could be passively transferred to normal mice, whereas carrageenan-treated mice did not kill the bacteria even after receiving immune serum. It seems that the synergistic action of macrophages and antibody provides the main initial primary defence in immune animals.

When Dictyostelium discoideum amoebae were harvested from nutrient medium and suspended in a starvation buffer to initiate development, approximately 30% of the total cellular β-N-acetylglucosaminidase activity was secreted into the extracellular fluid within 4 h. During this same period, only 10% of the total cellular acid phosphatase and acid protease activities were secreted. When the cells were pretreated overnight with 5 mg sodium hadacidin ml−1 and then suspended in starvation buffer, 60% of the glucosaminidase and 30% of the acid phosphatase activities were secreted, while the level of acid protease secretion remained at 10%. The secretory behaviour of hadacidin-treated cells was, however, identical to that of untreated cells when 0·1 m-sucrose was added to the starvation buffer to enhance lysosomal enzyme secretion.

Treatment with hadacidin also affected the intracellular content of these enzyme activities. After 16 h exposure to 5 mg hadacidin ml−1, the cellular levels of glucosaminidase and acid protease activity were decreased by 50% and 30%, respectively, while acid phosphatase activity remained unchanged. All of the changes observed upon hadacidin treatment were time dependent and were not evident if the cells were exposed to the drug for only 4 h. These results suggest that hadacidin treatment affects the lysosomal system of D. discoideum.

A class of chlorate-resistant mutants of Rhizobium japonicum 110 has been isolated which is devoid of nitrate reductase activity but forms normal amounts of this enzyme when the medium is supplemented with 100 μm-sodium molybdate. Addition of molybdate to the mutants in the presence of chloramphenicol results in the formation of active nitrate reductase detected by nitrite secretion in the culture supernatant.

Pseudomonas aeruginosa lectins interact with Escherichia coli strains O86B7 and O128B12, which possess B and H (O) blood group determinants, respectively. The interaction could be demonstrated by specific agglutination of the bacteria, by haemagglutination inhibition tests and by lectin-mediated peroxidase binding to the bacteria. The agglutination of E. coli O86B7 by the Pseudomonas galactose-binding lectin was inhibited by d-galactose and by the lipopolysaccharide extracted from E. coli O86B7. Similarly, the specific agglutination of E. coli O128B12 by the Pseudomonas mannose-binding lectin (which also binds l-fucose, l-galactose and d-fructose) was inhibited by d-mannose, l-fucose, l-galactose and d-fructose, as well as by the lipopolysaccharide extracted from E. coli O128B12. The interaction between E. coli O128B12 and the Pseudomonas mannose-binding lectin was also demonstrated by lectin-mediated peroxidase binding to the bacterial surface. Peroxidase binding was also inhibited by the above-mentioned sugars and E. coli O128B12 lipopolysaccharide.

Treatment of cells of the two E. coli strains with protein-denaturing agents did not reduce their agglutination by the Pseudomonas lectins. On the other hand, oxidation of the cell surface sugars by sodium metaperiodate or boiling the cells in the presence of 1 % acetic acid for 1 h abolished their agglutination by the two lectins. It is, therefore, suggested that the Pseudomonas lectins interact with the B and H (O) blood group determinant sugars (d-galactose in E. coli O86B7 and l-fucose in E. coli O128B12) residing in the lipopoly-saccharides of these E. coli strains.

A theoretical analysis has been made of five different media which have been used for growth of the methylotrophic yeasts Hansenula polymorpha and Candida boidinii. The media compositions were found to differ significantly in their content of trace elements. Chemostat studies revealed that low growth yields, particularly at high dilution rates, were probably due to unexpected limitations in trace elements.