- Volume 148, Issue 1, 2002

Antisense repression was used as a method to alter gene function in the human-pathogenic fungus Cryptococcus neoformans. The calcineurin A gene (CNA1) and the laccase gene (LAC1) were targeted since disruption of these loci results in phenotypes that are easy to screen (temperature sensitivity and lack of melanin, respectively). Serotype D yeasts were transformed with a plasmid containing the CNA1 cDNA in an antisense orientation under the control of the inducible GAL7 promoter, and serotype A yeasts were transformed with a plasmid containing the LAC1 cDNA in an antisense orientation under the control of the constitutive actin promoter. The calcineurin transformants demonstrated a temperature-sensitive phenotype only when grown on galactose, and the laccase transformants had decreased melanin production. Northern blot analysis of the calcineurin antisense transformants confirmed that the inducible phenotype was associated with a decrease in the native CNA1 transcript levels. Furthermore, it was possible to modestly impair growth of C. neoformans at 37 °C by using a 30 bp antisense oligonucleotide targeting CNA1. Antisense repression is now available as a tool for molecular studies in this organism, and may be applicable to other human-pathogenic fungi that have less amenable genetic systems.

Although chlorophenol (CP) degradation has been studied, no bacterium responsible for degradation of CP under denitrifying conditions has been isolated. Moreover, little substantial evidence for anaerobic degradation of CPs coupled with denitrification is available even for mixed cultures. Degradation of CP [2-CP, 3-CP, 4-CP, 2,4-dichlorophenol (DCP) or 2,6-DCP] under denitrifying conditions was examined in anaerobic batch culture inoculated with activated sludge. Although 3-CP, 4-CP, 2,4-DCP and 2,6-DCP were not stably degraded, 2-CP was degraded and its degradation capability was sustained in a subculture. However, the rate of 2-CP degradation was not significantly enhanced by subculturing. In 2-CP-degrading cultures, nitrate was consumed stoichiometrically and concomitantly during 2-CP degradation, and a dechlorination intermediate was not detected, suggesting that 2-CP degradation was coupled with nitrate reduction. A 2-CP-degrading enrichment culture degraded 2-CP in the presence of nitrate, but did not in the absence of nitrate or the presence of sulfate. This suggests that the enrichment culture strictly requires nitrate for degradation of 2-CP. The apparent specific growth rate of the 2-CP degrading species was 0·0139 d−1. Thus the apparent doubling time of the 2-CP-degrading population in the enrichment culture was greater than 50 d, which may explain difficulty in enrichment and isolation of micro-organisms responsible for CP degradation under denitrifying conditions.

Plasmid pHT128, a derivative of the broad-host-range IncQ vector pGSS33, was successfully introduced into Neisseria meningitidis. Under optimal conditions, pHT128 was transferred from Escherichia coli to N. meningitidis by triparental conjugation at a frequency of 10−5–10−6. The copy number of pHT128 in N. meningitidis was almost the same as in E. coli, in which the copy number of IncQ plasmids per chromosome is estimated to be 10. pHT128 was maintained as an episome in N. meningitidis in the presence of chloramphenicol, a marker of the plasmid. It was also shown that an opc or pilE1 gene cloned on pHT128 could be expressed in N. meningitidis under control of the tac promoter and could complement a mutation of opc or pilE1, respectively. In addition, the conjugational introduction of pHT128 into N. meningitidis was demonstrated to be independent of natural transformation competence. All the results indicate that pHT128 is a useful vector for N. meningitidis as a new genetical tool.

Clostridium perfringens α-toxin, which is one of the main agents involved in the development of gas gangrene, stimulates production in neutrophils. Exposure of rabbit neutrophils to the α-toxin induced firm adhesion of the cells to fibrinogen and fibronectin. Incubation of rabbit neutrophils and neutrophil lysates with α-toxin led to the production of diacylglycerol (DG) and L-α-phosphatidic acid (PA), respectively. The toxin-induced DG and PA formation preceded the toxin-induced adhesion of the neutrophils to fibrinogen and fibronectin, and the production of . Pertussis toxin inhibited the α-toxin-induced formation of PA, the adhesion of the neutrophils to fibrinogen and production. GTPγS stimulated the events induced by the α-toxin, whereas GDPβS inhibited them. The α-toxin stimulated phosphorylation of a protein with a molecular mass of about 40 kDa. In addition, treatment of the cells with 1-oleoyl-2-acetyl-sn-glycerol (OAG) and phorbol-12,13-dibutyrate (PDBu) stimulated cell adhesion, production of and phosphorylation of the 40 kDa protein, but had no effect on the formation of PA. The events induced by the presence of OAG and PDBu were not inhibited by pertussis toxin. Protein kinase C inhibitors, H-7, staurosporine and chelerythrine, blocked α-toxin-induced adhesion, production of and phosphorylation of the 40 kDa protein. These observations suggested that α-toxin-stimulated adhesion to the matrix and production were due to the formation of DG, through activation of phospholipid metabolism by a pertussis-toxin-sensitive GTP-binding protein, followed by activation of protein kinase C by DG.

The sequence of the celO gene from Clostridium thermocellum F7 was determined. The gene product, cellulase CelO (Ct-Cel5F), had a modular structure consisting of a carbohydrate-binding module of the CBM3 family and a catalytic domain of the glycosyl hydrolase family 5. The presence of the dockerin module indicated that the enzyme was a component of the cellulosome complex. The thermostable recombinant gene product was active on cellodextrins, barley β-glucan, carboxymethylcellulose and insoluble cellulose. Cellobiose was the only product released from amorphic and crystalline cellulose, cellotetraose and higher cello-oligosaccharides, identifying CelO as a cellobiohydrolase. The cleavage pattern of p-nitrophenyl β-D-cellotetraoside, blockage of the hydrolysis of NaBH4-reduced cellopentaose and the reduction in substrate viscosity suggested activity from the reducing end in a processive mode after making random cuts. Binding to insoluble, i.e. amorphous, and crystalline cellulose was mediated by the carbohydrate-binding module CBM3b, with a preference for the crystalline substrate.

The design and evaluation of a set of universal primers and probe for the amplification of 16S rDNA from the Domain Bacteria to estimate total bacterial load by real-time PCR is reported. Broad specificity of the universal detection system was confirmed by testing DNA isolated from 34 bacterial species encompassing most of the groups of bacteria outlined in Bergey’s Manual of Determinative Bacteriology. However, the nature of the chromosomal DNA used as a standard was critical. A DNA standard representing those bacteria most likely to predominate in a given habitat was important for a more accurate determination of total bacterial load due to variations in 16S rDNA copy number and the effect of generation time of the bacteria on this number, since rapid growth could result in multiple replication forks and hence, in effect, more than one copy of portions of the chromosome. The validity of applying these caveats to estimating bacterial load was confirmed by enumerating the number of bacteria in an artificial sample mixed in vitro and in clinical carious dentine samples. Taking these parameters into account, the number of anaerobic bacteria estimated by the universal probe and primers set in carious dentine was 40-fold greater than the total bacterial load detected by culture methods, demonstrating the utility of real-time PCR in the analysis of this environment.

X-ray absorption near edge structure (XANES) spectroscopy at the sulfur K-edge was applied to probe the speciation of sulfur of metabolically different sulfur-accumulating bacteria in situ. Fitting the spectra using a least-square fitting routine XANES reveals at least three different forms of sulfur in bacterial sulfur globules. Cyclooctasulfur dominates in the sulfur globules of Beggiatoa alba and the very recently described giant bacterium Thiomargarita namibiensis. A second type of sulfur globules is present in Acidithiobacillus ferrooxidans: here the sulfur occurs as polythionates. In contrast, in purple and green sulfur bacteria the sulfur mainly consists of sulfur chains, irrespective of whether it is accumulated in globules inside or outside the cells. These results indicate that the speciation of sulfur in the sulfur globules reflects the different ecological and physiological properties of different metabolic groups of bacteria.

A phosphatase-overproducing Citrobacter sp. (NCIMB 40259) was grown in an air-lift reactor in steady-state continuous culture under limitation of carbon, phosphorus or nitrogen. Substantial biofilm formation, and the highest phosphatase activity, were observed under lactose limitation. However, the total amount of biofilm wet biomass and the phosphatase specific activity were reduced in phosphorus- or nitrogen-limited cultures or when glucose was substituted for lactose as the limiting carbon source. Scanning electron microscopy (SEM), transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM) showed differences in cell and biofilm morphology in relation to medium composition. Electron microscopy suggested that the differences in biofilm formation may relate to differential expression of fimbriae on the cell surface.

2-Methyladenosine (methyl-ado) has demonstrated selective activity against Mycobacterium tuberculosis, which indicates that differences in the substrate preferences between mycobacterial and human purine metabolic enzymes can be exploited to develop novel drugs for the treatment of mycobacterial diseases. Therefore, in an effort to better understand the reasons for the anti-mycobacterial activity of methyl-ado, its metabolism has been characterized in Mycobacterium smegmatis. In a wild-type strain, methyl-ado was phosphorylated by adenosine kinase to methyl-AMP, which was further converted to methyl-ATP and incorporated into RNA. In contrast, a mutant strain of M. smegmatis was isolated that was resistant to methyl-ado, deficient in adenosine kinase activity and was not able to generate methyl-ado metabolites in cells treated with methyl-ado. These results indicated that phosphorylated metabolites of methyl-ado were responsible for the cytotoxic activity of this compound. Methyl-ado was not a substrate for either adenosine deaminase or purine-nucleoside phosphorylase from M. smegmatis. Treatment of M. smegmatis with methyl-ado resulted in the inhibition of ATP synthesis, which indicated that a metabolite of methyl-ado inhibited one of the enzymes involved in de novo purine synthesis. These studies demonstrated the importance of adenosine kinase in the activation of methyl-ado to toxic metabolites in M. smegmatis.

A nitroreductase with distinct properties that can activate the prodrug 5-aziridinyl-2,4-dinitrobenzamide (CB 1954) was isolated from Bacillus amyloliquefaciens. The encoding gene was identified as a homologue of the ywrO of Bacillus subtilis, and was obtained as a PCR product by reverse genetics, cloned and the entire nucleotide sequence determined. The gene was found to reside between homologues of the B. subtilis alsD and yswB genes; however, the ywrO and yswB genes of B. amyloliquefaciens were not separated by a fourth gene, ywsA. The B. amyloliquefaciens ywrO gene was overexpressed, the recombinant protein purified and its properties were compared with those of two CB 1954-activating enzymes, Escherichia coli B nitroreductase (NTR) and Walker DT-diaphorase (DTD). In common with these enzymes menadione was an electron acceptor (K m 3 μM) and activity with this substrate was inhibited by the presence of dicoumarol (K i 1·0 μM). In contrast, YwrO showed a marked preference for NADPH as a cofactor (K m 40 μM) and therefore could not be classified as a DTD (EC 1.6.99.2). The flavin FMN was an acceptor with high affinity. B. amyloliquefaciens YwrO was shown to be a flavoprotein with a monomeric molecular mass of 21·5 kDa by calculation and SDS-PAGE. The cytotoxic 4-hydroxylamine derivative was the single CB 1954 reduction product, but B. amyloliquefaciens YwrO was inactive with the bischloroethyl analogue of CB 1954, SN 23862. In both of these properties B. amyloliquefaciens YwrO more closely resembles DTD than NTR. Its K m for CB 1954 was lower than that of NTR (617 μM compared to 862 μM). Enhanced in vitro cytotoxicity of CB 1954 was demonstrated on incubation of V79 cells with prodrug, NADPH and B. amyloliquefaciens YwrO. The work has led to the identification of a previously unknown nitroreductase, B. amyloliquefaciens YwrO, with distinct properties which will aid the rational selection of appropriate genes for applications in directed enzyme prodrug therapy (DEPT).

During an infection of a higher eukaryote, dormant spores of a Bacillus species have been previously shown to be present in cells that can generate the toxic agent peroxynitrite (PON). Dormant spores of Bacillus subtilis were much more resistant to killing by PON than were growing cells, and spore-coat alteration or removal greatly decreased PON resistance. Spores were not killed by PON through DNA damage and lost no dipicolinic acid (DPA) during PON treatment. However, PON-killed spores lost DPA during subsequent heat treatments that caused much less DPA release from untreated spores. Although dead, the PON-killed spores germinated and initiated metabolism but never went through outgrowth; the great majority of germinated PON-killed spores also took up propidium iodide, indicating that they had suffered significant membrane damage and were dead. Together these data suggest that spore killing by PON is through some type of damage to the spore’s inner membrane.

Three mutant strains (ntcA, hetR, hetF) of the cyanobacterium Nostoc punctiforme unable to differentiate heterocysts were characterized and examined for their ability to form a symbiotic association with the bryophyte Anthoceros punctatus. Previously unknown characteristics of the N. punctiforme hetR mutant include differentiation of chilling-resistant akinetes, while vegetative cells of the ntcA mutant randomly lysed, yielding short filaments, following ammonium deprivation. Strains with mutations in hetF and hetR infected A. punctatus with similar frequency to that of wild-type N. punctiforme but did not support growth of the plant partner. These results confirm that the infection of A. punctatus by hormogonia leading to the establishment of an association is physiologically uncoupled from the development of a functional diazotrophic association. They also indicate that heterocyst regulatory elements downstream from HetR and HetF are required in both free-living and symbiotic heterocyst differentiation and nitrogenase expression. A strain with a mutation in the global nitrogen regulator ntcA did not infect A. punctatus despite its ability to differentiate hormogonia at a low frequency. When complemented with one or more copies of ntcA, the mutant strain infected A. punctatus at a similar frequency as the wild-type and supported growth of the plant partner in the absence of combined nitrogen. These results established a connection between the presence of a functional copy of ntcA and the magnitude of hormogonium differentiation, and the behaviour of the formed hormogonia.

Oenococcus oeni (formerly Leuconostoc oenos) can perform malolactic fermentation, converting L-malate to L-lactate and carbon dioxide, in wines. The energy and redox potential required to support the growth of the micro-organism are supplied mainly by the consumption of carbohydrates via the heterolactic pathway. In the first steps of hexose metabolism two molecules of NAD(P)+ are consumed, which must be regenerated in later reactions. The aim of this work was to test if aerobic growth of O. oeni promotes higher cell yields than anaerobic conditions, as has been shown for other lactic acid bacteria. O. oeni M42 was found to grow poorly under aerobic conditions with glucose as the only carbohydrate in the medium. It was demonstrated that O2 inactivates the enzymes of the ethanol-forming pathway, one of the two pathways which reoxidizes NAD(P)+ cofactors in the heterolactic catabolism of glucose. These results suggest that the regeneration of cofactors is the limiting factor for the aerobic consumption of glucose. When external electron acceptors, such as fructose or pyruvate, were added to glucose-containing culture medium the growth of O. oeni was stimulated slightly; fructose was converted to mannitol, oxidizing two molecules of NAD(P)H, and pyruvate was transformed to lactate, enabling the regeneration of NAD+. The addition of cysteine seemed to suppress the inactivation of the ethanol-forming pathway enzymes by O2, enabling glucose consumption in aerobic conditions to reach similar rates to those found in anaerobic conditions.

Various chemicals with harmful effects are not themselves toxic, but are metabolized in vivo to produce toxic products. One example is methanol in Dictyostelium, which is lethal to cells containing the acrA gene, but relatively harmless to acrA mutants. This makes methanol resistance one of the tightest genetic selections in Dictyostelium. Loss of acrA also confers cross-resistance to unrelated compounds such as acriflavine and thiabendazole. We have used insertional mutagenesis to demonstrate that the acrA locus encodes the peroxisomal catalase A enzyme. Disruption of the catA gene results in parallel resistance to acriflavine. Molecular and biochemical studies of several previously characterized methanol-resistant strains reveal that each lacks catalase activity. One allele, acrA2, contains a 13 bp deletion which introduces a frameshift in the middle of the gene. The involvement of catalase in methanol resistance in Dictyostelium compares with its role in methanol metabolism in yeast and rodents. However, this is the first study to show that catalase is required for the toxicity of acriflavine. Our results imply that acriflavine and thiabendazole are precursors which must be oxidized to generate biologically active species. The catA/acrA gene is also a potentially invaluable negative selectable marker for Dictyostelium molecular genetics.