- Volume 128, Issue 4, 1982

The phenotypic resistance to amphotericin methyl ester (AME) of stationary phase cultures of Candida albicans was decreased by alkaline pH values and by treatment with 2-mercaptoethanol or glucanase preparations, and was increased by acid pH values, increased aeration, treatment with N-ethylmaleimide, or the presence of inhibitors of protein synthesis such as trichodermin. The effects of such treatments on endogenous glucanase activity and on the incorporation of glucose residues into the glucan fraction of the organism were studied. The changes in the endogenous levels of lytic activities on laminarin [as a measure of the total (1→3)-β-d-glucanase] and on p-nitrophenyl-β-d-glucoside [reflecting the exo-(1→3)--d-glucanase] were followed in C. albicans cells under a variety of conditions. Treatments which increased AME sensitivity stimulated both total and exo-(13)--d-glucanase activities, while treatments which promoted resistance decreased the levels of both (1→3)--d-glucanases. Changes in the glucan fraction were followed by incubating suspensions of organisms in the presence of trace amounts of [U-14C]glucose. The rate of incorporation of radioactivity fell during the first 2–3 d of stationary phase culture and then rose to high values by 7–8 d; AME resistance increased throughout this period. The rate of incorporation was markedly stimulated by prior treatment of the organisms with 2-mercaptoethanol or glucanase and inhibited by trichodermin or treatment with N-ethylmaleimide.

The addition in the concentration range 0·3–3 mm of the glucose analogues β-d-allose, 3-O-methyl-d-glucose, 2-deoxy-d-glucose or 5-thio-d-glucose to cultures 24 h after inoculation prevented any further increase in AME resistance for the next 2–3 d and resulted in a decrease in the level of resistance established at the time of addition. Radioactivity from 14C- or 3H-labelled analogues added, 24 h after inoculation, to stationary phase cultures was incorporated into the glucan fraction of the organisms.

The incorporation of glucose residues into the glucan fraction is controlled by the activity of glucanases in producing glucose acceptor sites. The results reported confirm that there is a correlation between glucan metabolism, glucanase activity and resistance to AME, in that any factor leading to increased glucanase action also results in decreased resistance and vice versa, while incorporation of certain glucose analogues into the glucan fraction delays the further increase in resistance.

Klebsiella pneumoniae had low levels of nitrogenase activity and antigenic cross reacting material to both nitrogenase components when grown, or when the nif operons were derepressed, under Mo-deficient conditions. When molybdate (100 μm) was added to Mo-deprived cultures shortly after initiating derepression of nitrogenase, activity was rapidly restored and reached up to 60% of that of control cultures supplied with molybdate throughout derepression. Restoration of activity was prevented by chloramphenicol (200 μg ml −1) but not by tetracycline (100 μg ml −1), although both compounds inhibited general protein synthesis at these concentrations. Expression of the nif structural genes was not rapidly stimulated by added molybdate, so the rapid stimulation of activity was due to the activation of a low level of apoprotein present in Mo-deprived cultures. After 16 h of Mo-deprivation, suspensions no longer responded rapidly to added molybdate, consistent with the finding that, when further protein synthesis was inhibited by the addition of chloramphenicol after 4 h Mo-deprivation, polypeptides of the MoFe protein (Kp1) but not the Fe protein (Kp2) of nitrogenase were degraded within the following 18 h.

Mycobacterium microti walls contained three types of mycolic acids, very similar to those found in Mycobacterium tuberculosis. An α-mycolate with two cyclopropane rings, a methoxymycolate with one cyclopropane ring and a methoxyl group, and a ketomycolate with one cyclopropane ring and a keto group were partially characterized. The mycolates made up 34% (by weight) of the peptidoglycan-arabinogalactan-mycolate wall skeleton. Young exponential phase cultures and organisms harvested from mouse lungs contained high proportions of ketomycolates; older cultures had roughly equal proportions of keto- and methoxymycolates. The proportion of α-mycolates increased slightly with age of culture, but was always less than one-third of the total.

The mode of action of berninamycin on bacterial protein synthesis is related to that of thiostrepton, a dissimilar compound. Both antibiotics bind to the complex of 23S RNA with protein L11 and both affect various functions of the ribosomal A site. Also, Streptomyces bernensis and Streptomyces azureus (which, respectively, produce berninamycin and thiostrepton) possess similar ribosomal RNA methylases capable of rendering ribosomes resistant to these compounds. Resistance involves specific pentose-methylation of 23S ribosomal RNA.

In Gloeocapsa, NH4 + formed by N2 fixation was assimilated by the glutamine synthetase-glutamate synthase pathway. The inhibition of acetylene reduction following addition of NH4 + to N2-fixing cultures was not caused by NH4 + itself but was, most probably, related to increased intracellular levels of glutamine.

Analogues of several amino acids inhibited acetylene reduction. Apart from glutamine analogues, which interfered with NH4 + assimilation, these analogues probably acted by inhibiting nitrogenase synthesis. Certain analogues of tryptophan and phenylalanine, which have been reported to overcome NH4 + inhibition of cell differentiation and, possibly, nitrogenase synthesis in heterocystous cyanobacteria growing on combined nitrogen, did not prevent NH4 + from inhibiting acetylene reduction in cultures of Gloeocapsa. In contrast, l-methionine-dl-sulphoximine, which similarly counteracts the effect of NH4 + on heterocyst differentiation, also prevented NH4 + from inhibiting acetylene reduction in Gloeocapsa.

A large number of spontaneous conditional revertants of Bacillus subtilis glutamine auxotrophs were isolated by plating cells on minimal medium and incubating them at various temperatures. No heat-sensitive revertants were found among 2000 scored; however, revertants with a cold-sensitive glutamine requirement were quite common. Growth of the latter class ceased immediately upon transfer to the restrictive temperature implying that glutamine synthetase activity was affected. Mapping data are consistent with a genetic location in or near glnA, the structural gene for glutamine synthetase, for all cold-sensitive revertants tested. These revertants are likely to have second site suppressor mutations in glnA since many have altered levels or kinetics of glutamine synthetase activity or a changed pattern of feedback inhibition.

In yeast, fuchsin resistance at the mitochondrial level is mainly controlled by a nuclear gene (fub). Among the sensitive revertants obtained from a resistant strain of Saccharomyces cerevisiae, different suppression situations have been analysed. A cytoplasmic control of fuchsin resistance was found in one revertant. In two others, two nuclear suppressors have been identified; one of them was linked to the fub gene and exhibited a peculiar cis-trans effect.

The ability of four genetically distinct plasmid-located tetracycline resistance determinants (Tet A, B, C and D) to confer decreased accumulation of tetracycline and some of its analogues by Escherichia coli K12 was examined. Accumulation of oxytetracycline, tetracycline, demethylchlorotetracycline, 6-demethyl-6-deoxy-5-hydroxy-6-methylene-tetracycline, chlorotetracycline, doxycycline and 6-demethyl-6-deoxytetracycline was examined by fluorescence spectroscopy. The determinants varied in their ability to promote decreased accumulation of tetracyclines, defined as an R+/R− fluorescence ratio of < 0·85. Plasmid pIP7 (TetA) caused reduced accumulation of only oxytetracycline, tetracycline and chlorotetracycline, but plasmid pDU301 (TetB) promoted reduced accumulation of all the compounds tested except 6-demethyl-6-deoxytetracycline. The TetC determinant of pBR322 caused decreased uptake of five derivatives, but not doxycycline or 6-demethyl-6-deoxytetra-cycline. Plasmid RA1 (TetD) encoded reduced accumulation of oxytetracycline, tetracycline, 6-demethyl-6-deoxy-5-hydroxy-6-methylenetetracycline and chlorotetracycline. In general, the resistance determinants were more efficient in promoting decreased accumulation of hydrophilic tetracyclines. These accumulation studies provide a satisfactory method for the phenotypic identification of Tet resistance determinants.

Bacteroides nodosus isolates from ovine virulent footrot and ovine benign footrot and bovine isolates of low virulence for sheep were distinguishable from each other by their proteinase isoenzyme patterns after polyacrylamide gel electrophoresis. Variants of low virulence were not always distinguishable from their virulent parent strains. The molecular weights of the isoenzymes ranged from 70000 to 129000. The relationship of isoenzyme patterns to virulence is discussed.

Escherichia coli was grown in chemostat culture under glycerol-limited and ammonium-limited conditions at growth rates between 0·1 and 0·5 h−1. At steady state, the concentrations of cyclic AMP and cyclic GMP and the activities of four constitutive enzymes (glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, NADH oxidase and cyclic phosphodiesterase) were determined in the organism. Addition of exogenous cyclic AMP, cyclic GMP or phencyclidine perturbed the steady state and caused inhibition or stimulation of synthesis of phosphodiesterase and isocitrate dehydrogenase. A novel hypothesis is proposed to account for the ability of bacteria to regulate the synthesis of constitutive enzymes with cyclic nucleotides and possibly other small molecules.

Rhizobium meliloti MVII-1 and Rhizobium lupini H13–3, two strains with five to ten peritrichously inserted complex flagella, were studied with respect to motility and chemotaxis. Cells of both these strains move rapidly with speeds up to 40 μm s−1 (R. meliloti) and 60 μm s−1 (R. lupini) respectively. Increasing viscosity causes little reduction in their swimming velocities as compared with Salmonella typhimurium propelled by plain flagella. It is suggested that complex flagella possess a high ‘flexural rigidity’, which serves to maintain a helix conformation favourable for propulsive efficiency at increased viscosities. Chemotaxis in R. meliloti MVII-1 and R. lupini H13–3 was studied and the conditions required have been defined using the capillary tube assay. All 20 common l-amino acids and l-homoserine were shown to be attractive to R. meliloti MVII-1 with thresholds varying from 10−6 m (proline) to 10−4 m (aspartate). Leucine, proline and lysine elicited optimal responses. Rhizobium lupini H13–3 was also attracted by l-amino acids except for leucine, which elicited no response. Aspartate was a significantly better attractant of R. lupini H13–3 than of R. meliloti MVII-1, and glycine, isoleucine, homoserine, serine, threonine, cysteine, glutamine and glutamate were poorer attractants. Chemotaxis towards various carbohydrates was generally better in R. lupini H13–3 than in R. meliloti MVII-1. It is concluded that bacteria with complex flagella are efficient swimmers in viscous environments and are capable of responding to chemotactic stimuli.

SUMMARY: The fungus Monilinia fructigena grown in a liquid medium containing protein secreted a protease with many similarities to one extractable from apple fruits infected with this pathogen but absent from healthy fruit. Enzymes from both sources degraded several protein substrates optimally at pH 3·4. At this pH, haemoglobin was degraded optimally at 47 °C, while at 37 °C both enzymes were most stable at pH 5. The pI values for the enzymes were close to 4·0, and their action was inhibited by pepstatin and diazoacetyl-norleucine methyl ester plus copper ions but unaffected by p-hydroxymercuribenzoate, EDTA or trypsin inhibitors. Mercaptoethanol was essential for the recovery of enzyme activity from acrylamide gels in which the in vitro and in vivo enzymes were electrophoretically homogeneous. The in vitro enzyme gave a positive reaction for glycoproteins after purification with a Sepharose-haemoglobin affinity medium. Both enzymes were single units with a molecular weight of 37000–40000 on sodium dodecyl sulphate (SDS)-poryacrylamide gels; gel filtration indicated a molecular weight of 34000–38000. The purified in vitro enzyme had no aminopeptidase or carboxypeptidase activity; the products of haemoglobin digestion were peptides with molecular weights on SDS-polyacrylamide gels of 2000–12000. Purified in vitro protease did not damage apple fruit tissue or cultured apple cells, lyse plant protoplasts or release proteins from isolated fruit cell walls. The protease had no effect on active fungal or host enzymes that might have a role in host/pathogen interactions, and did not induce phaseollin formation in bean cotyledons. The plant proteins vicilin, legumin and heat-denatured peroxidase were hydrolysed at pH 3·5 and spinach nitrate reductase was inactivated. No natural protease inhibitors were detected in healthy or infected apples. We suggest that in vivo the protease might be involved in the nutrition of the pathogen.

Bacillus licheniformis was cultivated in a range of defined media varying in both the nature of the growth-limiting component and the concentration of excess nutrients. The compositions of the media were such as to ensure that the final absorbance (A 430) of the culture was the same in each case. Samples taken during the stationary phase were assayed for their content of extracellular serine protease and bacitracin. The nature of the growth-limiting nutrient had a profound effect on the amounts of these products formed while those components which were present in excess also exerted an influence in proportion to their concentration. Thus, for example, a four-fold increase in serine protease production occurred when ammonium replaced glucose as the growth-limiting nutrient. Serine protease and bacitracin production responded differently to these varying cultural conditions suggesting they are subject to separate control mechanisms. The results are discussed in relation to the need for rigorously controlled cultural conditions during physiological studies of this nature.

Amino acid utilization by Euglena gracilis strains z and SM-ZK (a streptomycin-bleached mutant) was studied in the presence or absence of ethanol as a carbon source. Among 21 amino acids examined, glutamate was the most effective nutrient for both strains under all conditions. Methionine, cysteine, threonine, leucine and phenylalanine inhibited the growth of E. gracilis on glutamate as the carbon and nitrogen source, but neither glutamate transport nor respiration was inhibited by these amino acids. The growth inhibition by methionine, threonine and phenylalanine were specifically relieved by glycine, valine and tryptophan, respectively.

The induction by A2 strains of Phytophthora parasitica and Phytophthora palmivora of sexual reproduction in an A1 strain of P. parasitica has been studied using synthetic medium with and without sterol, the strains being separated by a polycarbonate membrane. Production of the inducing α2 hormone by the A2 strain does not appear to require sterol, but sterol greatly enhances the response to the inducing hormone by the A1 strain as well as being required for subsequent oospore development.