- Volume 136, Issue 11, 1990

N-Acetyl-d-glucosamine-induced germ tube formation in Candida albicans at 37 °C was accompanied by an increase in the rate of protein phosphorylation. The calmodulin antagonist trifluoperazine and the Ca2+ ionophore A23187, which inhibited germ tube formation, also reduced the rate of phosphorylation. The rate of phosphorylation was also reduced when cells were incubated at 25 °C, which favoured yeast-phase growth. Two-dimensional SDS-PAGE analysis of phosphoproteins from germ-tube-forming and yeast cells revealed two germ-tube-specific and three yeast-specific phosphoproteins. Germ tubes and hyphae had more calmodulin activity than yeast cells, irrespective of the germ-tube-inducing condition used. As a first step towards understanding the inhibitory effect of trifluoperazine on germ tube formation, calmodulin from C. albicans was purified to homogeneity. It was heat stable, and displayed a pronounced Ca2+-induced shift in electrophoretic mobility.

Schizosaccharomyces pombe cell walls contain two major glycoprotein species, I and II, with molecular masses of 2 × 106 and 5 × 105 Da respectively, as determined by gel filtration chromatography and PAGE. The ratio of sugar to protein is higher in species I than in species II. Much of the sugar in both glycoproteins (about 85% in wild-type cells) is O-linked to the peptide moiety. The morphological sph1 mutant is resistant to papulacandin B, and its cell wall contains less glycoprotein II (but not less glycoprotein I) than the parental wild-type strain, although glycoprotein II is still synthesized and released into the growth medium. Papulacandin B largely reverses the morphological alteration of the mutant, and returns the ratio between species I and II to about that found in the parental strain, although the absolute amount of species II is still lower in the mutant. The results point to the importance of the relative amounts of the different wall polymers in determining cell morphology.

The major part of the wall of Schizosaccharomyces pombe consists of (1→3)-α-glucan and (1→3)-β-glucan with some (1→6)-β-linkages. Although in hydrolysed samples only a minute amount of glucosamine could be detected, this amino sugar may play an essential role as an integral part of a glucosaminoglycan/glucan complex. Treatment of the wall with either nitrous acid or chitinase changed the solubility properties of the β-glucan, which suggests that the glucosaminoglycan/glucan complex is essentially similar to that found in walls of other fungi. An enzyme with properties similar to that of chitin synthase of other fungi, and probably responsible for the synthesis of the glucosaminoglycan, was detected in a mixed-membrane fraction.

The basidiomycete Schizophyllum commune produces an extracellular bacteriolytic enzyme when grown on heat-killed cells of Bacillus subtilis as sole C, N and P source. The enzyme catalyses the dissolution of isolated B. subtilis cell walls at an optimum pH of 3·2–3·4, releasing muramyl reducing groups, which indicates that it is a muramidase. Although low levels of enzyme activity are present when the fungus is grown in the absence of bacteria, full enzyme production appears to be induced by bacterial cells and repressed by glucose. Whole bacteria are not lysed by the enzyme at pH 3·3, but are rendered osmotically fragile, and lyse when the pH is raised to 7 or higher. The muramidase is effective against several Gram-positive bacteria but did not lyse any of the Gramnegative species tested.

Activities of the polyol dehydrogenases of Puccinia graminis f. sp. tritici were surveyed by measuring polyol-dependent rates of reduction of NAD+ and NADP+ in cell-free extracts of axenically-grown mycelia. Seven of the eight polyols tested caused NADP+ reduction, with highest activity for d-glucitol, followed by l-arabitol, xylitol, erythritol, galactitol and ribitol, and low activity with d-arabitol; only d-mannitol failed to support activity. Inactivation rates were consistent with at least three separate enzymes, specific for d-glucitol, xylitol and l-arabitol respectively, with apparent K m values of 170–198 mm for xylitol and d-glucitol (K m for NADP+ 36–55 µM), and 34 mm for l-arabitol (K m for NADP+ 1·2 µM). The NADP+-dependent activities were almost completely inhibited by 2 mm-dithiothreitol, in contrast to the NAD+-dependent activities, which were stimulated. NAD+-dependent activity was highest with d-mannitol, followed by successively lower activities with d-arabitol, xylitol and d-glucitol, with no activity with any of the other polyols; each of the four active polyols appeared to be oxidized by a different enzyme. All four NAD+-dependent activities were rapidly lost after Sephadex treatment of crude extracts.

The utilization of guanidino and ureido compounds was studied in several Pseudomonas species. Multiple routes of agmatine catabolism were found. All members of the homology group I of Pseudomonas use the initial deamination of agmatine to carbamoylputrescine which is subsequently converted to putrescine. In Pseudomonas indigofera, the catabolism of agmatine can also occur via an initial hydrolysis of the amidino group to putrescine catalyzed by an agmatine amidinohydrolase. A third pathway was found in Pseudomonas cepacia, namely oxidative deamination producing guanidinobutyraldehyde catalyzed by agmatine dehydrogenase, followed by formation of guanidino-butyrate and removal of urea by guanidinobutyrate amidinohydrolase to produce 4-aminobutyrate. Novel amidino-hydrolases were characterized in P. putida for the utilization of arcaine and audouine, and in P. cepacia for arcaine, homoarginine and guanidinovalerate. Guanidinovalerate amidinohydrolase was also detected in P. doudoroffii. Some of these amidinohydrolases accept more than one substrate, e.g. guanidinobutyrate and guanidinovalerate utilization by P. doudoroffii and P. cepacia, the catabolism of arcaine and audouine by P. putida, and the degradation of arcaine and homoarginine by P. cepacia.

The glycolipid of the halophilic archaeobacterium Halobacterium sodomense has been analysed by spectroscopic methods including 13C NMR to establish structural details, and has been shown to be a sulphated C20, C20 mannosyl glucosyl diether glycerol. This structure differs from all other known halobacterial glycolipids in having a sulphate substitution at C-2 of the mannose residue, and in being an α-d-(1→4)-linked disaccharide.

Streptococcus mutans and Streptococcus sobrinus are the major causative organisms of human dental caries. Pulsed-field gel electrophoresis (PFG) showed that the restriction enzyme NotI produced ten and six DNA fragments from the genomes of S. mutans strain MT8148 and S. sobrinus strain 6715, respectively. The sizes of the chromosomes of S. mutans and S. sobrinus were each estimated to be about 2200 kb. The NotI restriction map of S. mutans MT8148 genome was constructed by Southern blot analysis with probes that overlapped two adjacent NotI fragments. Several virulence-associated genes of S. mutans were placed on the NotI restriction map. In addition, unique ‘fingerprints’ of S. mutans chromosomal DNA digested with NotI were produced by PFG, and these may be useful for epidemiological studies.

The genes encoding fructosebisphosphatase and phosphoribulokinase present on a 2·5 kb SalI fragment from Xanthobacter flavus H4-14 were sequenced. Two large open reading frames (ORFs) were identified, preceded by plausible ribosome-binding sites. The ORFs were transcribed in the same direction and were separated by 39 base pairs. They encoded proteins of 364 and 291 amino acids, with molecular masses of 38739 and 33409 Da, respectively. The ORFs were identified as the genes encoding FBPase and PRK, respectively, on the basis of similarity with FBPase and PRK sequences from other sources.

Variants of a methicillin-resistant Staphylococcus aureus showing loss of or reduced resistance to the antibiotic were isolated at frequencies of 0·1–100% from cultures which had been starved, grown at elevated temperature, or given small doses of UV radiation. Three types of variant were identified on the basis of population distribution of resistance to the antibiotic, and field-inversion gel electrophoresis of digests of the chromosome cut with the rarecutting restriction endonuclease SmaI. Type I variants are methicillin-sensitive and have a deletion in the mec region of the chromosome. Type II varinats show reduced methicillin resistance and rearranged DNA elsewhere in the chromosome. Type III variants show reduced methicillin resistance and no detectable change in the chromosome. Type I deletions were mapped using cloned fragments from the mec region. In 13 of the 16 independently isolated deletion mutants, one of the deletion endpoints appears to correlate with the positions of insertion sequences or transposons found in this region of the staphylococcal chromosome.

A bacterial isolate (O-1) which degrades different substituted benzenesulphonates was identified as a strain of Alcaligenes sp. using chemotaxonomic, DNA:DNA hybridization and serolgical methods. Its ability to mineralize orthanilic acid (2-aminobenzenesulphonate) is correlated with the presence of a 117 MDa plasmid, designated pSAH. Selective curing of pSAH resulted in the abolition of the orthanilic acid minieralization phenotype, which could be fully restored by re-establishment of pSAH. A second plasmid of 6·8 MDa, designated pME1702, with no detectable functions, is also present in Alcaligenes sp. O-1. Upon conjugation with Alcaligenes sp. O-1 as donor, the plasmid-free recipient Pseudomonas putida PaW130 acquird the ability to utilize orthanilic Alcalignes sp. O-1. Growth characteristics, the inducibility of enzyme activity (desulphonation), and expression of characteristic proteins during mineralization of orthanilic acid indicate that a plasmid-encoded cluster of genes is involed in the biodegradation of orthanilic acid. Southern blot hybridizations did not reeal any relationship between pSAH and the archetypal TOL plasmid pWW0, which also mediates degradation of substituted aromatic compounds.

We describe a method for determining the rate parameter of conjugative plasmid transfer that is based on single estimates of donor, recipient and transconjugant densities and the growth rate in exponential phase of the mating culture. The formula for estimating the plasmid transfer rate, γ, was derived from a mathematical model describing cell growth and plasmid transfer in batch culture. Computer simulations were used to explore the sensitivity of this method to the realities of bacterial life, such as growth rate differences, plasmid segregation and transitory derepression of pilus synthesis. As predicted by the theory, mating experiments with the plasmid R1 in Escherichia coli K12 demonstrated that the estimate γ is unaffected by cell density, donor:recipient ratio and mating time. Unlike previous techniques, our method allows us to investigate the effect of environmental factors on plasmid transfer rates when these factors also influence population growth rates. To illustrate this, we examined the effect of temperature on the rate of plasmid transfer.

Comparison of the inferred amino acid sequence of outer-membrane protein PIB from gonococcal strain P9 with those from other serovars reveals that sequence variations occur in two discrete regions of the molecule centred on residues 196 (Var1) and 237 (Var2). A series of peptides spanning the amino acid sequence of the protein were synthesized on solid-phase supports and reacted with a panel of monoclonal antibodies (mAbs) which recognize either type-specific or conserved antigenic determinants on PIB. Four type-specific mAbs reacted with overlapping peptides in Var1 between residues 192-198. Analysis of the effect of amino acid substitutions revealed that the mAb specificity is generated by differences in the effect of single amino acid changes on mAb binding, so that antigenic differences between strains are revealed by different patterns of reactivity within a panel of antibodies. The variable epitopes in Var1 recognized by the type-specific mAbs lie in a hydrophilic region of the protein exposed on the gonococcal surface, and are accessible to complement-mediated bactericidal lysis. In contrast, the epitope recognized by mAb SM198 is highly conserved but is not exposed in the native protein and the antibody is non-bactericidal. However, the conserved epitope recognized by mAb SM24 is centred on residues 198-199, close to Var1, and is exposed for bactericidal killing.

Bacterial exotoxins may contribute to the pathogenic potential of micro-organisms through interactions with cells of the host defence system as well as by directly damaging host tissue. The present studies were designed to explore mechanisms of interaction between bovine granulocytes and the leucotoxin produced by Pasteurella haemolytica, a major cause of bovine respiratory disease. Leucotoxin-containing supernatant from P. haemolytica A1 caused rapid cell death in isolated bovine granulocytes that was close to half-maximal by 5 min and nearly 90% complete after 30 min at 37 °C. Maintaining granulocytes at ice-water temperature markedly attenuated or prevented the toxic effect; furthermore, if exposed to supernatants at ice-water temperature and then washed, most cells remained viable even after rewarming to room temperature. However, even a very brief exposure (about 5 s) at 37 °C led to extensive cell death even after immediate cold dilution and washing. Granule enzymes such as arylsulphatase were released far more slowly than cytosol contents. Leucotoxin purified by column chromatography showed temperature dependence and divergence between cytosol and granule marker release similar to those observed with the crude supernatant preparation. These findings indicate that irreversible interaction between P. haemolytica leucotoxin and bovine granulocytes is initiated very rapidly at 37 °C but markedly impeded at low temperature, while granule enzyme release follows cytosol marker release over a much longer period. The results suggest either a requirement for target cell metabolic activity to initiate toxin effects or a temperature-dependent receptor conformation, with granule enzyme release following as a secondary consequence of granulocyte death.

The effect of fixation on the activity of malate dehydrogenase (decarboxylating) and pyruvate synthase was investigated in Trichomonas vaginalis. Subsequently a cytochemical staining method was developed for the demonstration of malate dehydrogenase activity in hydrogenosomes. After fixation of cells in low concentrations of glutaraldehyde and incubation in the presence of malate and the tetrazolium compound 2-(2′-benzothiazolyl)-5-styryl-3-(4′-phthalhydrazidyl) tetrazolium chloride, an electron-dense deposit was produced in the hydrogenosomes. During the whole procedure strictly anaerobic conditions were required. Attempts to develop an analogous procedure for pyruvate synthase failed because even low concentrations of glutaraldehyde strongly inhibited enzyme activity. When cells were fixed in low concentrations of glycolaldehyde and acetaldehyde, a high enzyme activity was retained, but no staining could be achieved. Application of both staining methods to the sapropelic ciliates Trimyema compressum and Plagiopyla nasuta gave negative results.

The decay rate of the potential to synthesize proteins after inhibition of transcription with rifampicin (Rif) was analysed at different times of energy and nutrient starvation for the marine Vibrio sp. S14. The decline of protein synthesis following Rif treatment is due to the instability of mRNA and permits an estimate of the functional mRNA half-life. The half-life of the mRNA pool was found to increase 6-fold (from 1·7 to 10·3 min) during a period of 24 h of total energy and nutrient starvation. To resolve whether the increase in the mean mRNA half-life was a result of the stabilization of the entire pool or if proteins expressed during starvation were translated from very stable mRNAs, the half-lives of specific mRNAs were measured. It was found that the half-lives of mRNAs common to both growing and starving cells were increased between 2- and 3-fold during a period of 24 h starvation, and that some starvation-specific proteins were encoded by extremely long-lived mRNAs (up to 70 min). The possible role of stabilization of mRNA as a mechanism to economize protein synthesis during starvation conditions is discussed