- Volume 135, Issue 12, 1989

The pili expressed by all isolates of Neisseria gonorrhoeae react with two monoclonal antibodies, SM1 and SM2. In contrast, although many isolates of Neisseria meningitidis also express pili (class I) which react with antibodies SM1 and SM2, a proportion express pili (class II) which fail to react. In order to define the epitopes recognized by these antibodies, a series of overlapping peptides corresponding to the amino acid sequence of conserved regions of gonococcal pili have been synthesized. The minimum epitope recognized by antibody SM1 was found to comprise a linear peptide EYYLN, corresponding to residues 49–53 of mature pilin. In contrast, antibody SM2 reacted with a number of peptides from around the cysteine residue (Cys 1) at position 120, suggesting that an extended region may contribute to a conformational epitope recognized by this antibody in the native protein. The identification of the two epitopes defines structural differences between the classes of pili expessed by meningococci. In order to determine the distribution of pilin gene sequences in Neisseria we used as hybridization probes an oligonucleotide (PS1) with the sequence 5′-GAGTATTACCTGAATCA-3' which spans the coding region for the SM1 epitope, and a fragment of the 3′ end of the gonococcal pilE gene which contains conserved sequences flanking the two Cys codons and encodes the SM2 epitope. All strains of N. gonorrhoeae and N. meningitidis tested, regardless of piliation phenotype, harboured DNA sequences homologous to those encoding the carboxy-terminus of meningococcal class I pilin. Furthermore, all gonococci and all meningocococci producing class I pili hybridized with oligonucleotide probe PS1. Non-reverting non-piliated derivatives of previously class I pilus-producing strains showed reduced hybridization signals with this probe, but nevertheless retained sequences homologous to the coding sequence for the SM1 epitope. However, meningococci producing class II pili could be divided into two groups on the basis of their reaction with the PS1 probe: half the strains tested failed to react, which is consistent with our previous analysis of silent class I pilin sequences; the remainder reacted (relatively weakly) with the probe, suggesting that the silent pil sequences in these strains extend further towards the 5′ end of the pilin gene than in strains studied previously. Some strains of Neisseria lactamica reacted weakly with both types of probe but failed to produce SM1-reactive pili. In contrast,

A small plasmid of 3·95 kb, encoding resistance to chloramphenicol (Cm) was detected in three of 33 Staphylococcus hyicus strains. The plasmid in each of the three strains was indistinguishable by Southern-blot hybridization and restriction enzyme analysis. It was shown by curing and by transformation to specify resistance to Cm. A preliminary restriction map of the plasmid, designated pSC2, is presented. Chloramphenicol acetyltransferase was demonstrated by enzyme assay and by SDS-PAGE of cell-free lysates of pSC2 transformants.

Twenty ‘commensal’ oral or ‘pathogenic’ vaginal isolates of Candida albicans were examined for colony morphology on malt/yeast-extract and serum-based agar media. Diverse and variable colony morphology was seen on serum agar. In 17 strains, selective subculture of morphologically atypical colonies produced progeny which had reverted to the morphology of the majority of parental colonies. However, in one strain, a highly stable colony variant was isolated which did not revert on subculture. In two further strains, variants were isolated which could be maintained with at least 99 % homogeneous colony type by selective colony subculture, but reversion to the parental type or switching to other morphologies occurred at rates of 10−2 to 10−4: a rapid switching phenomenon. The relative proportions of mycelial or yeast forms were the main determinants of colony morphology. The variants were biotyped using a selection of biochemical tests. The stable variant differed from its parent in several characters, including rate of production of a proteinase enzyme. The pathogenicity of variants was compared in mice, and both stable and switching variants differed in virulence from their parental strains. Colony-type variation on suitable media is thus a powerful tool in the isolation of mutants or variants of C. albicans which differ from ‘isogenic’ parents in significant biological properties. Such variants may aid identification and characterization at the molecular level of determinants of, for example, pathogenicity and morphogenesis.

A laboratory yeast isolate identified as Exophiala jeanselmei synthesized melanin and showed polyphenol oxidase activity with dihydroxyphenylalanine (DOPA) and pyrogallol. Electrophoresis on native and denaturing gels, and activity staining with DOPA revealed two immunologically distinguishable bands of Mr about 100000 and 120000, which could be separated by (NH4)2SO4 fractionation. The enzyme isolated from this yeast gave optimal activity at higher pH (8·5–10) and temperature (37–40 °C) than other known melanin-synthesizing activities.

The growth of the dematiaceous hyphomycetes (darkly pigmented microfungi) Alternaria phragmospora, Alternaria chlamydospora and Ulocladium chartarum was altered by increasing concentrations of NaCl in the growth medium. U. chartarum demonstrated the greatest sensitivity to salinity changes: chlamydospora-like structures were common at NaCl concentrations > 5 %. Lipid analyses of these fungi are presented. Steryl esters and fatty acids were the main lipid classes in the apolar fractions. Phosphatidylethanolamine, phosphatidylgly-cerol and phosphatidylcholine were the main polar compounds. Most phospholipids decreased at higher salinity. The major fatty acid was oleic acid (18:1) which decreased at 10 % NaCl. A similar pattern was observed for palmitic acid (16:0). Myristic acid (14:0) and myristoleic acids (14:2, 14:1) were detected in all isolates cultured at 10 % NaCl.

Colicins A, E1, E2 and E3 belong to the BtuB group of colicins. The NH2-terminal region of colicin A is required for translocation, and defects in this region cannot be overcome by osmotic shock of sensitive cells. In addition to BtuB, colicin A requires OmpF for efficient uptake by sensitive cells. The roles of BtuB and OmpF in translocation and binding to the receptor of the colicins A, E1, E2 and E3 were compared. The results suggest that for colicin A OmpF is used both as a receptor and for translocation across the outer membrane. In contrast, for colicin E1, OmpF is used neither as a receptor nor for translocation. For colicins E2 and E3, the situation is intermediate: only BtuB is used as a receptor but both BtuB and OmpF are involved in the translocation step.

The effects of growth conditions on the production of 987P fimbriae by the enterotoxigenic Escherichia coli strain 1592 were examined in steady state chemostat experiments at different specific growth rates. The amount of fimbriae produced by fimbriate cells (P+) was dependent on the specific growth rate (μ). Under aerobic growth conditions fimbriae production increased with higher μ values till μ = 0·40 h−1and decreased again at μ values close to μ max (0·48 h−1). Under anaerobic growth conditions the maximal production was comparable to that under aerobic growth conditions, and was also maximal close to μ max (0·16 h−1). Phase variation, measured as the percentage of fimbriate cells in a particular population, was independent of μ. The composition of the growth medium influenced both phase variation and overall production of fimbriae. A shift from minimal to a complex medium induced a rapid reduction in the amount of fimbriae per P+cell and a slower reduction in the percentage of P+cells. A shift from complex to minimal medium resulted in an increase in the percentage of P+cells and a constant amount of fimbriae per P+cell. The frequency of the phase switch was calculated for different growth conditions. The frequency of the P+ → P−switch between two steady states was 2·7 × 10−2. In batch culture the frequency of the P− → P+switch was minimally 2·9 x 10−2. The results indicate that phase variation and the production of 987P fimbriae by fimbriate cells are under independent physiological control.

A mutant (D6-13) of Trichoderma reesei that produced low levels of extracellular β-glucosidase was physiologically and biochemically characterized. The mutant was unable to grow and produced very little cellulase activity on Solka-Floc, Avicel, micro-crystalline cellulose and acid-swollen cellulose. Whereas growth was nearly normal on cellobiose, lactose and glucose, cellulase production was slightly decreased on cellobiose and lactose. The mutant produced nearly normal levels of FPA (filter-paper activity) and endoglucanase on sophorose. A distinct lag and a lowered specific sugar consumption rate was observed for the mutant on 0·3% cellobiose; this appeared to be due to lowered wall-bound β-glucosidase activity. The multiplicity and role of different β-glucosidases in T. reesei is discussed.

Analysis of periplasmic enzymes of Bradyrhizobium japonicum or Rhizobium leguminosarum biovar phaseoli was hampered by the fact that only small amounts of marker enzyme activities were released from cells by osmotic shock or by lysozyme/EDTA treatment. However, up to 95 % of total activity of certain periplasmic marker enzymes could be measured in intact cells by including 003% Triton X-100 in enzyme assays. Less than 5% of the cytoplasmic marker enzyme -hydroxybutyrate dehydrogenase could be measured under these conditions, indicating that treatment with dilute detergent does not significantly perturb the cytoplasmic membrane. Various lines of evidence indicate that the dilute detergent treatment does not significantly alter wall structure but, instead, permits the assay of periplasmic enzymes by facilitating diffusion of substrates and products to and from the periplasmic space. For pyrophosphatase and phosphodiesterase, the proportion of total enzyme activity which could be measured in intact cells was higher in bacteroids than in bacteria at all Triton concentrations, suggesting that the outer membrane of bacteroids may be more permeable than the outer membrane of cultured bacteria. The effect of detergent on the hydrolysis of four disaccharides sucrose, α,α-trehalose, maltose and lactose was studied using intact cultured R. leguminosarum biovar phaseoli. The response of lactose hydrolysis to the addition of detergent was four-fold greater than the response of the other disaccharidase activities, indicating that a rhizobial lactase may be present in the periplasmic space.

The effect of bicarbonate on the growth and product formation by a periodontopathic bacterium, Actinobacillus actinomycetemcomitans, was examined in an anaerobic chemostat culture with fructose as the limiting nutrient. The chemostat cultures were run at dilution rates between 0·04 and 0·25 h−1and the maximum growth yield (Y fructose max) was estimated to be 40·3 and 61·7 g dry wt (mol fructose)−1in the absence and presence of bicarbonate, respectively. The major fermentation products in the absence of bicarbonate were formate, acetate, ethanol and succinate, with small amounts of lactate. The addition of bicarbonate to the medium resulted in a marked decrease in ethanol production and in a significant increase in succinate production. Washed cells possessed activity for the cleavage of formate to CO2 and H2, which seemed to play a role in supplying CO2 for the synthesis of succinate in the absence of bicarbonate. The study of enzyme activities in cell-free extracts suggested that fructose was fermented by the Embden-Meyerhof-Parnas pathway. The values of Y ATP maxand the efficiency of ATP generation (ATP-Eff) during fructose catabolism were estimated and higher values were obtained for the culture in the presence of bicarbonate: 20·2 g dry wt (mol ATP)−1and 30 mol ATP (mol fructose)−1, respectively, versus Y ATP max = 15·1 and ATP-Eff = 2·7 in the absence of bicarbonate.

The response of the yeast Hansenula anomala to a reduction in water activity (a w) from 0·998 to 0·925 (adjusted with glucose or NaCl) was monitored. Natural abundance 13C NMR spectroscopy and HPLC analysis revealed that the type of carbon source determined which polyols were present intracellularly at 0·998 a w. At 0·95 a w (NaCl), glycerol was accumulated in all instances irrespective of the type of carbon source indicating the primary role of glycerol in osmoregulation. The carbon source had a bearing only on which other polyol(s) were accumulated. During growth on glucose at 0·95 a w (NaCl or glucose), glycerol was accumulated intracellularly in the exponential growth phase with a concentration ratio (intra-/extracellular) as high as 10000-fold whereas during the stationary phase arabitol accumulation occurred to a lower concentration ratio while the glycerol concentration decreased. The specific growth rate and cell volume decreased with increasing NaCl or glucose concentrations. This indicated that the yeast had no specific requirement for these compounds for optimum growth but tolerated high concentrations. Reducing the a w to 0·95 resulted in increasing intracellular concentrations of glycerol and arabitol whereas below 0·95 a W (NaCl or glucose), the intracellular polyol concentration decreased while the polyol concentration ratios across the cell membrane increased. During the early exponential growth phase at 0·95 a w, glycerol was accumulated in sufficiently high concentrations to achieve an osmotic balance across the membrane whereas in the stationary phase the arabitol and glycerol concentration was insufficient to maintain the osmotic balance.