Showing papers in "Microbiology in 1989"

Degradation of Fungal Cell Walls by Lytic Enzymes of Trichoderma harzianum

Abstract: SUMMARY: In in vitro tests, two strains of Trichoderma harzianum failed to parasitize colonies of Fusarium oxysporum f. sp. vasinfectum and F. oxysporum f. sp. melonis. However, these strains were strongly mycoparasitic on Rhizoctonia solani and Pythium aphanidermatum. When grown in liquid cultures containing laminarin, chitin or fungal cell walls as sole carbon sources, both strains of T. harzianum released, 3-β-glucanase and chitinase into the medium. Higher levels of these enzymes were induced in strain T-203 than in T-35 by hyphal cell walls of F. oxysporum. When the lytic enzymes produced by T-35 were incubated with hyphal cell walls of the test fungi, more glucose and N-acetyl-d-glucosamine was released from cell walls of R. solani and Sclerotium rolfsii than from those of F. oxysporum. Treatment of F. oxysporum cell walls with 2 m-NaOH, protease or trypsin prior to their incubation with the lytic enzymes of T. harzianum significantly increased the release of glucose and N-acetyl-d-glucosamine. The effect of these treatments on R. solani and S. rolfsii cell walls was much lower. These results suggest that proteins in the cell walls of F. oxysporum may make these walls more resistant than those of R. solani or S. rolfsii to degradation by extracellular enzymes of T. harzianum.

The penetration of antibiotics into aggregates of mucoid and non-mucoid Pseudomonas aeruginosa.

Abstract: Cells of mucoid and non-mucoid Pseudomonas aeruginosa in colonies were at least one-thousandfold less sensitive to the antibiotics tobramycin or cefsulodin than were cells of the same bacteria in dispersed suspension. We did not detect any difference between the mucoid form and the non-mucoid form in the antibiotic sensitivity of colonies, from which we infer that the exopolysaccharide of the mucoid form does not contribute to colony-resistance by forming a barrier to antibiotic diffusion. Mathematical models were constructed in order to estimate time-courses of penetration of tobramycin and cefsolodin into biofilms and microcolonies of mucoid and non-mucoid P. aeruginosa. For tobramyen penetration, adsorption of antibiotic to the exopolysaccharide of the glycocalyx and antibiotic uptake by cells were taken into account in the calculations. The longest time-period for the concentration of tobramycin at the base of a biofilm 100 µm deep to rise to 90% of the concentration outside the biofilm was predicted to be 2·4 h. For cefsulodin penetration, irreversible hydrolysis catalysed by β-lactamase was taken into account, using β-lactamase levels taken from the literature. The calculations predicted that the cefsulodin concentration at the base of antifilm 100 µm deep would rise to 90% of the external concentration in 29 s when the β-lactamase was synthesized at the basal level. For a similar biofilm of bacteria synthesizing enhanced levels of β-lactamase (‘derepressed’), the concentration of cefsulodin at the base was calculated to rise to 41% of the external concentration in about 50s and then remain at that level. This was despite the fact that cefsulodin is a poor substrate for this β-lactamase.

Control of carbon flux to acetate excretion during growth of Escherichia coli in batch and continuous cultures.

Abstract: Summary: During growth of Escherichia coli ML308 on pyruvate in a continuous culture (turbidostat) or batch culture, flux of carbon into the cells exceeds the amphibolic capacity of the central pathways. This is balanced by diversion of carbon flux to acetate excretion which in turn diminishes the efficiency of carbon conversion to biomass [g] dry wt (mol substrate)−1]. However, restriction of carbon supply in a chemostat diminishes flux to acetate excretion and at a dilution rate (D = μ) of 0·35 h−1 or less, no flux to acetate excretion was sustained thus permitting perfect balance between carbon input on the one hand, and the output to biosynthesis and energy generation on the other. This, in turn, improves the efficiency of carbon conversion to biomass. Inclusion of 3-bromopyruvate (an inhibitor of pyruvate dehydrogenase) at a concentration which diminishes growth rate (μ) to 0·35 h−1 or less also prevented flux to acetate excretion. Furthermore, in a family of fluoroacetate-resistant strains, excessive flux of pyruvate was balanced by diversion of carbon flux to lactate excretion rather than acetate and a higher growth rate (μ = 0·63 h−1) was sustained.

Physical and biochemical characterization of the qacA gene encoding antiseptic and disinfectant resistance in Staphylococcus aureus.

Abstract: We have previously cloned a 3·5 kb fragment from the Staphylococcus aureus multiresistance plasmid pSK1 which carries the qacA determinant responsible for linked resistance to acriflavine (Acr), ethidium bromide (Ebr), quaternary ammonium compounds (Qar), propamidinc isethionate (Pir), and diamidinodiphenylamine dihydrochloride (Ddr). This report presents a biochemical and physical analysis of qacA and shows the widespread carriage of this gene on S. aureus resistance plasmids. Tn5 insertion mutagenesis defined the extent of qacA to within 2·40 kb of pSK1 DNA. Examination of the expression of insertion and deletion mutants of the cloned qacA sequences in both maxicells and minicells led to the association of a 50 kDa protein, designated QacA, with the Acr Ebr Qar Pir Ddr phenotype. Based on fluorimetric and isotopic assays used to determine the extent of accumulation of ethidium bromide by S. aureus strains harbouring pSK1, we propose that the basis of Acr Ebr Qar Pir Ddr in S. aureus is a qacA-mediated efflux system.

Staphylococcus aureus bacteriophages mediating the simultaneous lysogenic conversion of beta-lysin, staphylokinase and enterotoxin A: molecular mechanism of triple conversion.

Abstract: A new group of serotype F bacteriophages of Staphylococcus aureus has been found which mediates the simultaneous triple-lysogenic conversion of enterotoxin A, staphylokinase and β-lysin. The phages were recovered from methicillin-resistant strains of S. aureus isolated in Irish hospitals between 1971 and 1988 and from strain PS42-D, which has been used as the propagating strain for the S. aureus typing phage 42D since before 1965. The molecular mechanism of triple conversion mediated by three of these phages was determined by molecular cloning, restriction endonuclease site mapping and hybridization analysis, and compared with the mechanism of β-lysin and staphylokinase conversion mediated by the serotype F, double-converting phage β 13. The genetic determinants mediating expression of enterotoxin A (entA) and staphylokinase (sak) were cloned from the DNA of the triple-converting phage and expression of the cloned determinants detected in Escherchia coli and S. aureus. The entA and sak determinants were closely linked in the phage DNA adjacent to the phage attachment site (attP) in each case and furthermore, the sak determinant of phage o 13 was also located near its attP. The restriction maps of the entA-, sak- and attP-containing DNA regions of the three triple-converting phages were very similar to each other and to the corresponding sak- and attP-containing DNA region of phage o 13. Hybridization analysis using a cloned β-lysin determinant (hlb) and cloned attP-containing DNA fragments as probes demonstrated that β-lysin conversion mediated by the triple-converting phages and phage o13 was caused by insertional inactivation of the chromosomally encoded hlb determinant by orientation-specific integration of phage DNA following lysogenization.

Characterization of Staphylococcus species by ribosomal RNA gene restriction patterns.

Abstract: SUMMARY: The rRNA gene restriction patterns of 110 strains belonging to 12 staphylococcal species have been determined. The strains, isolated from various sources, were epidemiologically unrelated. Total DNA was cleaved with restriction enzymes HindlII and Eco RI, electrophoretically separated and probed with radiolabelled 16S rDNA from Bacillus subtilisinserted in a plasmid vector, pBR322. Fourty-four distinct Hindlll patterns and 44 distinct EcoRl patterns were observed. Strains belonging to different species had different patterns. Although distinct patterns were also observed within some species, a core of common bands could be discerned within each species or subspecies. Analysis of the patterns revealed two taxa in Staphylococcusxylosus which were not evident using phenotypic characteristics. Of 18 strains which were difficult to identify using phenotypic schemes, 15 showed patterns typical of known species. The three remaining atypical strains showed unusual patterns and may belong either to a known species, not included in the study, or to a new species. Since various patterns were observed within some species (e.g. S. aureus and S. epidermidis), rRNA gene restriction patterns may have epidemiological, as well as taxonomic interest.

Determination of the Substrates for Sulphate-reducing Bacteria within Marine and Esturaine Sediments with Different Rates of Sulphate Reduction

Abstract: The substrates used by sulphate-reducing bacteria in sediment slurries from Loch Eil, Loch Etive and the Tay estuary were determined by selectively inhibiting sulphate reduction with 20 mM-molybdate and measuring the resultant substrate accumulation. Substrate accumulation was linear after molybdate addition, and the rate of accumulation closely matched sulphate reduction rates, indicating that metabolic pathways other than those specifically involving sulphate reduction were not affected by the inhibitor. In sediments from all three sites acetate was a major substrate, although the percentage of sulphate reduced due to acetate oxidation varied considerably among the sites (Tay estuary, 35%; Loch Eil, 64%; Loch Etive, 100%). In addition to acctate, 17 individual substrates were shown to be involved in sulphate reduction to varying extents in the Tay estuary and Loch Eil sediments; these included lactate, H2, propionate, iso- and n-butyrate, iso- and n-valerate, 2-methylbutyrate and amino acids. At both sites propionate accounted for between 6 and 12% of sulphate reduction. Butyrate (n- and iso-), iso-valerate and 2-methylbutyrate were of approximately equal importance at each site and together accounted for 13 and 11%, respectively, of the sulphate reduction in the Tay estuary and Loch Eil sediments. Lactate was only importnat in the Tay estuary sediments, where it accounted for 43% of sulphate reduction. The rate of accumulation of amino acids was greatest in the Tay estuary sediments, but the contribution of amino acids to sulphate reduction was higher in the Loch Eil (9%) than in the Tay estuary sediments (2%). Of the 21 individual amino acids that were measured there was a linear increase in nine; the most important of these were serine, glutamate and arginine. In general, when sulphate reduction rates were high the substrates for this process were more varied than when rates were low. Combining the results of two experiments and assuming complete degradation of the individual substrates, almost all the sulphate reduction could be accounted for at each site (Tay estuary, 101%; Loch Eil, 98%; Loch Etive, > 100%).

Taxonomic Differentiation of Bacteriophages of Lactococcus lactis by Electron Microscopy, DNA-DNA Hybridization, and Protein Profiles

Abstract: SUMMARY: Thirty-seven virulent and 19 temperate bacteriophages of Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris were classified in a taxonomic system on the basis of morphology, DNA-DNA hybridization, and protein composition. As judged from electron microscopy and susceptibility to cleavage by restriction endonucleases, the genome of all the bacteriophages investigated is composed of double stranded DNA. Seven virulent phage groups were recognized: types P034 (genome size 18·1 kilobase pairs, kb), P001 (20·2 kb), P008 (29·7 kb), P335 (36·4 kb), P026 (51·5 kb), P107 (51·5 kb), and P087 (54·5 kb). In addition, two temperate phage groups were established: types TP-40-3 (genome size 42·1 kb) and TP-936-1 (37·8 kb). Phages within each group revealed strong DNA homology and similar protein compositions, whereas no significant DNA homology and different proteins were found in phages of different groups. Virulent phages of group P335 exhibited strong DNA homology with the temperate phages of group TP-936-1.

The effect of oxygen on denitrification in Paracoccus denitrificans and Pseudomonas aeruginosa.

Abstract: Denitrification by Paracoccus denitrificans and Pseudomonas aeruginosa was studied using quadrupole membrane-inlet mass spectrometry to measure simultaneously and continuously dissolved gases. Evidence was provided for aerobic denitrification by both species: in the presence of O2, N2O production increased in Pa. denitrificans, while that of N2 decreased; with Ps. aeruginosa, the concentrations of both N2 and N2O increased on introducing O2 into the gas phase. Disappearance of NO-3 was monitored in anaerobically and aerobically grown cells which were maintained either anaerobically or aerobically: the rate and extent of NO-3 utilization by both species depended on growth and maintenance conditions. The initial rate of disappearance was most rapid under completely anaerobic conditions, and lowest rates occurred when cells were grown anaerobically and maintained aerobically. In nitrogen balance experiments both species converted over 87% of the added NO-3 to N2 and N2O under both anaerobic and aerobic maintenance conditions.

L-arabinose and D-xylose catabolism in Aspergillus niger.

Abstract: SUMMARY: A mutant of Aspergillus niger unable to grow on d-xylose and l-arabinose has been isolated. Genetic analysis revealed that the mutation is located on linkage group IV. Enzymic analysis revealed a deficiency in d-xylulose kinase activity. After transfer of growing mycelium to a medium containing either d-xylose or l-arabinose, the mutant accumulates large amounts of arabitol and xylitol, as shown by 13C NMR spectroscopy. These data and an analysis of enzyme activities induced by d-xylose and l-arabinose in the wild-type strain led to the following catabolic pathway for d-xylose: d-xylose - xylitol - d-xylulose - d-xylulose 5-phosphate; and for l-arabinose: l-arabinose - l-arabitol - l-xylulose - xylitol - d-xylulose - d-xylulose 5-phosphate. The reduction steps of the sugars to the corresponding polyols are all NADPH dependent. The oxidation steps of the polyols to the sugars are all NAD+ dependent. Fractionation of cell-free extracts gave information about the specificity of the enzymes and showed that all the reactions are catalysed by different enzymes.

Protein-mediated Adhesion of Lactobacillus fermentum Strain 737 to Mouse Stomach Squamous Epithelium

Abstract: The mechanism of adhesion of Lactobacillus fermentum strain 737 to mouse stomach squamous epithelium was investigated. Adhesion inhibition tests involving chelators, monosaccharides, periodate and concanavalin A and the use of bacteria grown in the presence of tunicamycin failed to clarify the adhesive mechanism. Washed bacterial cells had reduced adhesive capacity, except in the presence of spent broth culture supernatant fraction or cell washings. Spent culture supernatant fractions of erythrosine-supplemented broth did not enhance adhesion of washed cells. The adhesion-promoting factor(s) in the spent broth culture supernatant fractions and cell washings bound to both bacterial and epithelial cell surfaces, but did not promote adhesion of two other Lactobacillus strains which were not of mouse origin, thereby indicating host specificity for the adhesion-promoting activity. Chemical characteristics of the adhesion-promoting factor were determined by pretreatment of the dialysis retentate of spent broth culture supernatant fractions with proteolytic enzymes, concanavalin A-Sepharose or periodate before the adhesion assay. The adhesin was non-dialysable, pronase-sensitive, heat sensitive at 100 degrees C, had no affinity for concanavalin A-Sepharose and contained no carbohydrate groups active in the adhesion process. The protein profiles of dialysis retentates of spent broth culture supernatant fractions after bacterial growth in the absence and presence of erythrosine were determined by 2-dimensional SDS-PAGE. Gel filtration by HPLC was used for purification of an adhesion-promoting fraction. The host-specific adhesion of L. fermentum strain 737 was mediated by a protein, with an Mr of 12-13000, that was not detectable in cells grown in the presence of erythrosine. A model for the mode of binding of the adhesin to host epithelia and bacterial surfaces is proposed.

Ultrastructural Localization of a Cuticle-degrading Protease Produced by the Entomopathogenic Fungus Metarhizium anisopliae during Penetration of Host (Manduca sexto) Cuticle

Abstract: SUMMARY: Gold-labelled rabbit antiserum was used to demonstrate that a cuticle-degrading protease (Pr1) is produced by Metarhizium anisopliae during penetration of host (Manduca sexta) procuticle. The protease was secreted by infection structures (appressoria) on the cuticle surface and by the penetrant hyphae within the cuticle. Penetration of the procuticle was by a combination of enzymic degradation and mechanical pressure. Initially Pr1 was confined to the immediate vicinity of the fungal structures; however the enzyme diffused throughout the cuticle during later stages of pathogenesis. When hyphae were labelled during growth in culture under conditions conducive to rapid synthesis of Pr1, gold particles distributed over the fungal cell wall, indicating binding of Pr1 to hyphae.

Glucose Transport in Crabtree-positive and Crabtree-negative Yeasts

Abstract: SUMMARY: The kinetic parameters of glucose transport in four Crabtree-positive and four Crabtree-negative yeasts were determined. The organisms were grown in aerobic glucose-limited chemostats at a dilution rate of 0·1 h-1. The results show a clear correlation between the presence of high-affinity glucose transport systems and the absence of aerobic fermentation upon addition of excess glucose to steady-state cultures. The presence of these H+-symport systems could be established by determination of intracellular accumulation of 6-deoxy-[3H]glucose and alkalinization of buffered cell suspensions upon addition of glucose. In contrast, the yeasts that did show aerobic alcoholic fermentation during these glucose pulse experiments had low-affinity facilitated-diffusion carriers only. In the yeasts examined the capacity of the glucose transport carriers was higher than the actual glucose consumption rates during the glucose pulse experiments. The relationship between the rate of sugar consumption and the rate of alcoholic fermentation was studied in detail with Saccharomyces cerevisiae. When S. cerevisiae was pulsed with low amounts of glucose or mannose, in order to obtain submaximal sugar consumption rates, fermentation was already occurring at sugar consumption rates just above those which were maintained in the glucose-limited steady-state culture. The results are interpreted in relation with the Crabtree effect. In Crabtree-positive yeasts, an increase in the external glucose concentration may lead to unrestricted glucose uptake by facilitated diffusion and hence, to aerobic fermentation. In contrast, Crabtree-negative yeasts may restrict the entry of glucose by their regulated H+-symport systems and thus prevent the occurrence of overflow metabolism.

Polymerase chain reaction for the detection of Mycobacterium leprae.

Abstract: SUMMARY: A polymerase chain reaction (PCR) using heat-stable Taq polymerase is described for the specific detection of Mycobacterium leprae, the causative agent of leprosy. A set of primers was selected on the basis of the nucleotide sequence of a gene encoding the 36 kDa antigen of M. leprae. With this set of primers in the PCR, M. leprae could be detected specifically with a detection limit approximating one bacterium. This PCR appears to meet the criteria of specificity and sensitivity required for a useful tool in epidemiology and eventually for the control of leprosy.

Molecular analysis of a plasmid-encoded phenol hydroxylase from Pseudomonas CF600.

Abstract: Pseudomonas strain CF600 is able to utilize phenol and 3,4-dimethylphenol as sole carbon and energy source. We demonstrate that growth on these substrates is by virtue of plasmid-encoded phenol hydroxylase and a meta-cleavage pathway. Screening of a genomic bank, with DNA from the previously cloned catechol 2,3-dioxygenase gene of the TOL plasmid pWW0, was used in the identification of a clone which could complement a phenol-hydroxylase-deficient transposon insertion mutant. Deletion mapping and polypeptide production analysis identified a 1.2 kb region of DNA encoding a 39.5 kDa polypeptide which mediated this complementation. Enzyme activities and growth properties of Pseudomonas strains harbouring this fragment on a broad-host-range expression vector indicate that phenol hydroxylase is a multicomponent enzyme containing the 39.5 kDa polypeptide as one component.

Reductive and non-reductive mechanisms of iron assimilation by the yeast Saccharomyces cerevisiae.

Abstract: Iron reduction and uptake was studied in wild-type and haem-deficient strains of Saccharomyces cerevisiae. Haem-deficient strains lacked inducible ferri-reductase activity and were unable to take up iron from different ferric chelates such as Fe(III)-citrate or rhodotorulic acid. In contrast, ferrioxamine B was taken up actively by the mutants as well as by the wild-type strains. At a low extracellular concentration, uptake was insensitive to ferrozine and competitively-inhibited by Ga(III)-desferrioxamine B. Extracellular reductive dissociation of the siderophore occurred at higher extracellular concentrations. Two mechanisms appear to contribute to the uptake of ferrioxamine B by S. cerevisiae: one with high affinity, by which the siderophore is internalized as such and another with lower affinity by which iron is dissociated from the ligand prior to uptake.

Cepabactin from Pseudomonas cepacia, a new type of siderophore

Abstract: In iron-deficient conditions of growth Pseudomonas cepacia ATCC 25416 excreted both pyochelin and a low-molecular-mass compound which strongly chelated iron(III), and facilitated iron translocation as demonstrated by growth and uptake experiments. The name cepabactin is proposed for this new siderophore. Comparisons of UV-visible spectra and chromatographic behaviour, together with 1H-NMR spectra, led to the conclusion that cepabactin is 1-hydroxy-5-methoxy-6-methyl-2(1H)-pyridinone, a compound which can be considered as a cyclic hydroxamate, but also as a heterocyclic analogue of catechol. This pyridinone has already been described by other workers as an antibiotic produced by Pseudomonas alcaligenes, and by a soil isolate closely related to Pseudomonas cepacia. Thus, cepabactin appears to act as a siderophore for more than one species of non-fluorescent pseudomonad.

Increased Cell Wall Porosity in Saccharomyces cerevisiae after Treatment with Dithiothreitol or EDTA

Abstract: SUMMARY: Intact cells of Saccharomyces cerevisiae were able to endocytose FITC-dextrans of 70 kDa, but not of 150 kDa, whereas spheroplasts took up both components. The rate of uptake of 70 kDa dextrans by spheroplasts was about three times higher than that by intact cells. Pretreatment of intact cells with dithiothreitol (DTT) or EDTA increased the rate of uptake of 70 kDa dextrans considerably, but 150 kDa dextrans were still excluded. Release of periplasmic invertase activity into the medium by glucose-derepressed cells was negligible in control cell suspensions, but was strongly stimulated in the presence of DTT. The released invertase had an apparent molecular mass of 320 kDa, indicating that the dimeric form was released. In the presence of EDTA only a slight increase in the release of invertase was observed. Pretreatment with DTT was accompanied by an increased loss of cell wall proteins. This suggests that the loss of mannoproteins, in combination with a more general opening up of the wall by reducing disulphide bridges, increases cell wall porosity. It is argued from the Stokes radius of 70 kDa dextran (5·8 nm) that yeast cell walls are, in principle, permeable to globular proteins with a molecular mass up to 400 kDa.

Bilophila wadsworthia, gen. nov. and sp. nov., a unique gram-negative anaerobic rod recovered from appendicitis specimens and human faeces.

Abstract: Summary: Strongly catalase-positive Gram-negative anaerobic rods were isolated from approximately half of all intra-abdominal specimens received from patients with gangrenous and perforated appendicitis, and subsequently also from normal faecal specimens The organism was originally detected on Bacteroides-bile-aesculin (BBE) agar, and grew slowly on non-selective anaerobic media containing blood It was stimulated by bile and differed from other known genera by being urease- and catalase-positive, and by reducing nitrate It did not reduce sulphate Other anaerobic Gram-negative rods showed no homology by DNA dot-blot hybridization The thermal melting profile of chromosomal DNA showed 39-40 mol% G + C The whole-cell fatty acid methyl ester profile included cyclic and branched long-chain acids, and differed from those of all other anaerobes that have been tested β-Lactamase was not detected The name Bilophila wadsworthia gen nov, sp nov is proposed for this organism

Mutations affecting the cytochrome d-containing oxidase complex of Escherichia coli K12: identification and mapping of a fourth locus, cydD.

Abstract: SUMMARY: A mutant of Escherichia coli K12 has been isolated affected in a gene, designated cydD, distinct from the three previously described loci involved in the synthesis of assembly of the cytochrome bd oxidase complex. The mutant, obtained by nitrosoguanidine mutagenesis, lacks the spectroscopically detectable components of this oxidase, namely cytochromes b 558, b 595 and d. Cytochrome oxidase o is the sole CO-binding cytochrome in membranes of the mutant, but the soluble haemoprotein b-590 and catalase activity appear unaffected. Discrimination between Cyd+ and Cyd- strains is facilitated by the development of a defined low-phosphate medium that allows the inclusion of Zn2+ as well as azide, inhibitors of respiratory electron transfer particularly via cytochrome o. Mapping with F-prime factors and by P1 cotransductional frequencies shows the mutation to map near 19-3 min on the E. coli chromosome, distinct from cydC, which maps at 18·9 min. The gene order in this region was tested in a three-factor cross and demonstrates the order zbj::Tn10(YYC199)–cydD–aroA, consistent with cotransduction frequencies.

Surface Hydiophobicity of Spores of Bacillus spp.

Abstract: Summary: The surface hydrophobicity of 12 strains of Bacillus spp. was examined in a hexadecane-aqueous partition system. Mature and germinated spores of Bacillus megaterium QM B1551 transferred to the hexadecane layer, while vegetative and sporulating cells did not. Wild-type spores were more hydrophobic than spores of an exosporium-deficient mutant of B. megaterium QM B1551, although the mutant spores were shown to be hydrophobic to some extent by using increased volumes of hexadecane. This result suggests that the exosporium is more hydrophobic than the spore coat and that the surface hydrophobicity of spores depends mainly on components of the exosporium. The surface hydrophobicity of spores of nine other species of Bacillus was also examined, and spores having an exosporium were more hydrophobic than those lacking an exosporium. Thus measurement of the hydrophobicity of spores by the hexadecane partition method may provide a simple and rapid preliminary means of determining the presence or absence of an exosporium.

Nucleotide sequence of the neopullulanase gene from Bacillus stearothermophilus

Abstract: The gene (nplT) for a new type of pullulan-hydrolysing enzyme, neopullulanase, from Bacillus stearothermophilus TRS40 was sequenced. The DNA sequence revealed only one large open reading frame, composed of 1764 bases and 588 amino acid residues (M r 69 144). Although the thermostable neopullulanase contained eight cysteine residues, they did not provide conformational stability by disulphide bonds. A comparison was made of the amino acid sequences of α-amylase, neopullulanase, isoamylase, pullulanase and cyclodextrin glucanotransferase. All the enzymes examined contained four highly conserved regions which probably constitute the active centres of the enzymes. The amino acid residues required for the specificity of neopullulanase are compared with those of α-amylase and other amylolytic enzymes.

Degradation of Epichlorohydrin and Halohydrins by Bacterial Cultures Isolated from Freshwater Sediment

Abstract: SUMMARY: Three bacterial cultures, the Gram-negative strain AD1 and the Gram-positive strains AD2 and AD3, were isolated from freshwater sediment after enrichment with epichlorohydrin as sole carbon source. In batch cultures of strain AD1 and strain AD3, epichlorohydrin was rapidly degraded to 3-chloro-l,2-propanediol. Crude extracts of strain AD1 contained epoxide hydrolase activity towards epichlorohydrin, epibromohydrin, glycidol and propylene oxide as substrates. In contrast, strain AD2 did not actively convert epichlorohydrin but utilized 3-chloro-l,2-propanediol produced by slow chemical hydrolysis. No epichlorohydrin epoxide hydrolase was found in extracts of this organism. Crude extracts of strains AD1 and AD2 dehalogenated a number of mono- and dihalogenated alcohols and ketones, such as 1,3-dichloro-2-propanol, 3-chloro-l,2-propanediol, l-chloro-2-propanol, l,3-dibromo-2-propanol, chloro-acetone and 1,3-dichloroacetone. Dehalogenation yielded epoxides as products. The results suggest that epichlorohydrin is converted by strain AD1 via 3-chloro-l,2-propanediol and glycidol by the action of an epoxide hydrolase and a dehalogenase, respectively. The same route for dehalogenation proceeds in strain AD2, which, however, is dependent on chemical hydrolysis of epichlorohydrin rather than enzymic conversion.

A morphology index for characterization of cell shape in Candida albicans.

Abstract: Summary: The morphology of Candida albicans cells was determined from their maximum length, maximum diameter and septal diameter in a mathematical ratio, the morphology index (Mi), which usually ranged from approximately 1 for spherical yeast cells to approximately 4 for true hyphae, with elongated yeast cells and pseudohyphae giving intermediate values. Mi could be determined with high reproducibility for C. albicans grown in a variety of environments. The highest mean Mi was seen with cells grown in serum and Eagle's medium at 37°C, the lowest with cells grown in Sabouraud glucose broth at 26°C. Variant strains of C. albicans gave Mi values that remained constant in a variety of growth environments. The Mi facilitated detection of two variants that grew exclusively in the yeast form, one that grew as elongated yeasts but could be induced to form pseudohyphae in serum, and one consistently pseudohyphal variant. Cells with a mean Mi up to 2·5 could be easily separated at septal junctions by mild ultrasonication, whereas cells with a mean Mi greater than 3·5 tended not to separate under these conditions. The chitin content of C. albicans cells was almost twice as great in cells with a Mi approaching 4 as in cells with a Mi close to 1. The wide range of Mi distributions for a single C. albicans isolate in different environments demonstrates that the fungus does not undergo abrupt changes of morphological phase: rather there are continual changes in morphology between spherical yeasts and true hyphae at the extremes. The study shows that Mi can be used reliably in place of subjective descriptions of morphology to indicate the shape of a C. albicans cell. It should facilitate the detection of molecular and cellular markers specific for morphogenesis in the fungus.

Production of thiol-dependent haemolysins by Listeria monocytogenes and related species.

Abstract: Twenty-six strains belonging to the five main species of the genus Listeria were examined for production of thiol-dependent exotoxins. All strains of L. monocytogenes cultured in charcoal-treated broth secreted a haemolytic factor at a level ranging from 200 to 800 haemolytic units (HU) ml-1, except for the strain EGD (1500 HU ml-1) and the type strain CIP 82110T (10 HU ml-1). The haemolytic activity reached a maximum level by 8-10 h and then rapidly declined as soon as bacterial exponential growth ceased. The titres of haemolytic activity were markedly reduced when bacteria were grown in charcoal-untreated broth. The haemolytic factor produced by L. monocytogenes strains was characterized as listeriolysin O (Mr about 60,000), a member of the group of thiol-dependent exotoxins. Strains of Listeria ivanovii also produced high levels of thiol-dependent exotoxin (about 2500 HU ml-1), in both charcoal-treated and untreated broth. Small amounts of haemolytic factor (about 9-30 HU ml-1) were also produced by Listeria seeligeri in charcoal-treated broth. The haemolysin produced by L. seeligeri was identified for the first time as a thiol-dependent exotoxin of Mr about 60,000, antigenically related to listeriolysin O. As expected, we failed to detect thiol-dependent exotoxin in the two nonhaemolytic species, Listeria innocua and Listeria welshimeri.

A plasmid which can be transferred between Escherichia coli and Pasteurella haemolytica by electroporation and conjugation.

Abstract: Summary: Three broad-host-range plasmids (pRK290, pSa4 and pKT230) and one native Pasteurella haemolytica plasmid (pPH33) were used in transformation experiments with P. haemolytica strains T179 (serotype Al), Y216 (serotype A2)and its capsular-deficient variant Y216/NS1. No transformants were detected with either heat-shock or freeze-thaw techniques. However, by electroporation, all P. haemolytica strains were transformed by pPH33 but not by pRK290 or pSa4. The highest frequency obtained was 91 × 104 transformants per μg of pPH33 DNA with P. haemolytica strain Y216/NS1. Although pPH33 itself was non-transmissible by conjugation, it could be mobilized from Escherichia coli, using the transfer function of the IncP plasmid pRK2013, into P. haemolytica at a frequency of 0·3-2·2 × 10−3 per recipient cell.

D-xylose utilization by Saccharomyces cerevisiae.

Abstract: Summary: Although it is generally accepted that Saccharomyces cerevisiae is unable to assimilate d-xylose, four strains were found to utilize xylose aerobically at different efficiencies in the presence of a mixture of substrates. The degree of d-xylose utilization by S. cerevisiae ATCC 26602 depended upon the presence of other substrates or yeast extract. The greatest amount of xylose (up to 69% over 7 d) was utilized when sugar substrates such as d-ribose were co-metabolized. Much lower degrees of utilization occurred with co-metabolism of organic acids, polyols or ethanol. A mixture of d-glucose, d-ribose, d-raffinose, glycerol and d-xylose resulted in greater xylose utilization than the presence of a single substrate and xylose. The absence of growth on a co-substrate alone did not prevent the utilization of xylose in its presence. Xylose was co-metabolized with ribose under anaerobic conditions but at a much slower rate than under aerobic conditions. When [14C]xylose was utilized in the presence of ribose under anaerobic conditions, the radioactive label was detected mainly in xylitol and not in the small amounts of ethanol produced. Under aerobic conditions the radioactive label was distributed between xylitol (91·3 ± 0·8%), CO2 (2·6 ± 2·3%) and biomass (1·7 ± 0·6%). No other metabolic products were detected. Whereas most xylose was dissimilated rather than assimilated by S. cerevisiae, the organism apparently possesses a pathway which completely oxidizes xylose in the presence of another substrate.

Differentiation of Staphylococcal Species and Strains by Ribosomal RNA Gene Restriction Patterns

Abstract: SUMMARY: Staphylococcal DNA was digested with endonucleases and probed with labelled ribosomal RNA (rRNA) from Escherichia coli. Reproducible restriction patterns containing between seven and 22 bands were obtained for seven different species of staphylococci. These profiles were species-specific with different strains of a particular species sharing an identical or similar restriction pattern. The results reported here indicate that rRNA gene restriction pattern analyses have an application in the taxonomy of staphylococci.

High frequency variation of colony morphology and chromosome reorganization in the pathogenic yeast Candida albicans.

Abstract: A clinical isolate of the pathogenic yeast Candida albicans varied in its colony morphology from smooth (o-smooth) to semi-rough type (SRT) and concomitantly lost its virulence for mice In terms of DNA content, the smooth parent was near triploid when Saccharomyces cerevisiae strains of known ploidy were used as references The SRT variant showed several features characteristic of polyploidy From the SRT variant, revertant-like smooth (r-smooth) variants with recovered virulence were derived at a frequency of 5 × 10-3 The results of pulsed-field gel electrophoresis on chromosomal DNA showed changes in patterns of chromosome-sized DNA bands in the SRT variant as well as in r-smooth variants, which correlated with these variations Correlations between colony morphology, state of ploidy and virulence of this asporogenous yeast are considered