- Volume 140, Issue 11, 1994
Volume 140, Issue 11, 1994
- Review Article
-
- Antigens And Immunity
-
-
-
Immunization with a multiple antigen peptide containing defined B-and T-cell epitopes: production of bactericidal antibodies against group B Neisseria meningitidis
More LessPrevious analysis of the class 1 outer-membrane (OM) protein of Neisseria meningitidis has identified discrete epitopes to be potential targets for immune attack. The conformation of these epitopes is important for inducing antibodies which can react with the native protein and promote complement-mediated lysis of the meningococcus. The multiple antigen peptide (MAP) system, which consists of an oligomeric branching lysine core to which are attached dendritic arms of defined peptide antigens, confers some conformational stability and also allows for the preparation of immunogens containing both B-cell and T helper (Th)-cell epitopes. In this study, MAPs were synthesized to contain (i) the subtype P1.16b meningococcal class 1 protein B-cell epitope (B-MAP), and (ii) the P1.16b epitope in tandem with a defined Th-cell epitope, chosen from tetanus toxin (BT-MAP). The B-MAP was non-immunogenic in animals. In contrast, incorporation of the Th-cell epitope into BT-MAP induced a strong humoral response towards the class 1 protein B-cell epitope. Antisera from immunized mice and rabbits reacted in ELISA with synthetic peptides containing the B-cell epitope, and also cross-reacted with meningococcal OMs from strains of subtype P1.16b and P1.16a. Murine and rabbit antisera showed similar reactivity and epitope specificity, but did not react with denatured class 1 protein in Western blotting, indicating the predominance of antibodies directed towards conformational epitopes. The antisera from rabbits immunized with BT-MAP promoted complement-mediated bactericidal killing not only of the homologous meningococcal subtype P1.16b strain but also of subtype P1.16a.
-
-
- Biochemistry
-
-
-
Biodegradation of sulphosuccinate: direct desulphonation of a secondary sulphonate
More LessThe bacterial biodegradation of a secondary sulphonate, sulphosuccinate, has been shown to occur by direct desulphonation. A bacterium, designated Pseudomonassp. BS1, was isolated from activated sewage sludge, for its capacity to grow on sulphosuccinate as the sole source of carbon and energy. Cultures grown on sulphosuccinate were able to convert this substrate to sulphite which was subsequently oxidized rapidly to sulphate. The sequence of desulphonation and carbon-chain catabolism of sulphosuccinate was determined from measurements of the kinetics of sulphite and 14CO2release from specifically radiolabelled sulpho[1,4-14C]succinate and sulpho[2,3-14C]succinate, which were synthesized from the corresponding maleic anhydrides. When each radiolabelled compound was incubated separately with washed-cell suspensions of PseudomonasBS1, sulphite was released before 14CO2, as shown by chemical assay and radiorespirometry, respectively. Differences in the kinetics and extent of 14CO2release from the 1,4-and 2,3-labelled substrates were consistent with entry of the intact C4chain into the citric acid cycle. When carrier oxaloacetate was added to incubation mixtures containing resting-cell suspensions and radiolabelled sulphosuccinate, a radiolabelled metabolite with the same HPLC retention time as oxaloacetate accumulated. No radioactive metabolites accumulated when carrier oxaloacetate was replaced with succinate, fumarate or malate. Collectively, the data indicated co-production of sulphite and oxaloacetate from sulphosuccinate, which is interpreted in terms of an oxidative desulphonation mechanism.
-
-
-
-
Gellan lyases-novel polysaccharide lyases
More LessA number of bacterial strains capable of degrading the bacterial exopolysaccharide gellan® have been isolated by standard enrichment procedures. They include several pink-pigmented Gram-negative rod-shaped bacteria. A red-pigmented Gram-positive bacillus earlier found to degrade the exopolysaccharide xanthan fromXanthomonas campestrisalso showed slight gellanase activity. All the Gram-negative bacteria are non-fermentative, motile and amylase-producing. The gellan degradation in each case is due to eliminase-type enzymes(lyases) which appear to be extracellular enzymes cleaving the sequence … β-D-glucosyl-(1→4)-β-D-glucuronosyl… in the tetrasaccharide repeat unit of the substrate polysaccharides. Although in some isolates these enzymes appear to be exo-acting, it appears from the loss in viscosity of the alternative substrate deacetylated rhamsan that they are predominantly endoenzymes. The enzyme activity is inducible: it is almost absent from glucose-grown cells. Associated with the ‘gellanase’ activity, all the Gram-negative bacterial isolates possess intracellular α-L-rhamnosidase and β-D-glucosidase activities apparently located in the periplasm. The enzymes are highly specific and fail to cause significant degradation of most of the other bacterial exopolysaccharides which have been shown to be structurally related to gellan. As well as acting on gellan, they exert similar degradative activity against the chemically deacylated form of polysaccharide S194 (rhamsan gum), which is effectively a gentiobiosylated form of gellan. The enzymes only have relatively slight activity against the natural, acylated gellan-like polysaccharides from the bacteria now designated as strains of Sphingomonas paucimobilis.
-
-
-
Ca2+-ATPase-driven calcium accumulation in Ustilago maydisplasma membrane vesicles
More LessCa2+transport has been measured across plasma membrane vesicles isolated from cells of Ustilago maydis. This transport was found to be ATP- (or to a lesser extent GTP) and Mg2+-dependent. Inconsistent release of Ca2+from intact vesicles was obtained using the calcium ionophore A23187. However, Ca2+was released by Triton X-100 in a concentration-dependent manner. Transport was inhibited by vanadate (>50%) and erythrosin B (about 50%), I 50being about 10 μM for both inhibitors. In the presence of the protonophores CCCP or gramicidin, partial inhibition of Ca2+transport (about 20%) was observed, but the Ca2+-channel blockers, nifedipine, diltiazem and verapamil had no effect, although the latter inhibited proton transport. The results indicate that Ca2+transport in U. maydisis regulated by a P-type ATPase with similar properties to that found in higher plants.
-
-
-
Is the solubilized product from the degradation of lignocellulose by actinomycetes a precursor of humic substances?
More LessThree actinomycetes (Streptomycessp. EC22, Streptomyces viridosporusT7A and Thermomonospora fuscaBD25) were assessed for their ability to degrade ball-milled wheat straw. All gave maximum levels of solubilized lignocellulose products (APPL) at the beginning of the stationary phase of growth (72-96 h). Low-molecular-mass aromatic compounds extracted from the APPL were analysed by reverse-phase and gas chromatography. Although the number of chromatographic peaks detected made identification of the products difficult, p-coumaric acid (4-hydroxycinnamic acid), protocatechuic acid (3,4-dihydroxybenzoic acid), gallic acid (3,4,5-trihydroxybenzoic acid), gallic acid methyl ester (methyl-3,4,5-trihydroxybenzoate) and 4-methoxyphenol were recognized. The infrared spectra of the three strains were similar to the spectra of humic acids, with all APPL extracts showing carbonyl, amino, carboxyl, aliphatic and aromatic group vibrations. Also detected were peptide linkages of proteins. The results suggest a role for actinomycetes in the formation of humic substances in soils and composts.
-
-
-
Partial characterization of specific inducers of a cuticle-degrading protease from the insect pathogenic fungus Metarhizium anisopliae
More LessThe insect pathogenic fungus Metarhizium anisopliaeproduces several extracellular cuticle-degrading proteases and evidence is consistent with one of these, PR1, which is a chymoelastase, being a determinant of pathogenicity. We have shown previously that PR1 production is regulated by both carbon catabolite and nitrogen metabolite repression and also by specific induction under derepressed conditions by insect cuticle. In the present work we have established that an enzymically released proteinaceous component(s) of insect cuticle is capable of inducing PR1 (based on appearance of extracellular activity). Cuticle of the desert locust Schistocerca gregariatreated with KOH to remove protein failed to induce PR1 production, whereas cuticle treated with either chloroform or ether to remove lipids still induced PR1. Cuticle digested with either PR1 or the trypsin-like PR2 of M. anisopliaereleased peptides mainly in the range 150-2000 Da; addition of these peptides generated by PR1 or PR2 at 3 μg alanine equivalents ml-1induced PR1 production to a level similar (75%) to that obtained with untreated insect cuticle. Several amino acids and peptides which are abundant in insect cuticular protein (Ala, Gly, Ala-Ala, Ala-Ala-Ala, Ala-Pro and Pro-Ala) were tested at a range of concentrations and in restricted cultures for their ability to induce PR1. None induced the protease to the levels seen with cuticle or peptides enzymically released from cuticle, although some dimers and notably the monomers Ala and Gly gave 2-2-7-fold enhanced PR1 activity above derepressed basal levels (up to 48-57% of that achieved with induced synthesis on cuticle). There was evidence for more efficient uptake and/or catabolism by M. anisopliaeof alanine di-and tripeptides than of monomer amino acids.
-
- Development And Structure
-
-
-
Endogenous ADP-ribosylation during development of the prokaryote Myxococcus xanthus
More LessWe examined endogenous ADP-ribosylation of proteins during the development of the prokaryote Myxococcus xanthus. In vivoand in vitroendogenous ADP-ribosylation of M. xanthusproteins was detected and the profile of modified proteins changed during development. Adenosine and nicotinamide inhibited ADP-ribosylation. Nicotinamide stimulated cells at low density to develop, in a manner similar to that previously observed with adenosine. Higher concentrations of nicotinamide inhibited aggregation. The in vivoeffects of nicotinamide on developing M. xanthuscells correlate with its in vitroeffects on ADP-ribosylation and the developmental profile of putative ADP-ribosylation substrates. These results suggest that ADP-ribosylation may regulate developmental proteins in M. xanthus.
-
-
- Environmental Microbiology
-
-
-
SDS-degrading bacteria attach to riverine sediment in response to the surfactant or its primary biodegradation product dodecan-1-ol
More LessA laboratory-scale river microcosm was used to investigate the effect of the anionic surfactant sodium dodecyl sulphate (SDS) on the attachment of five Pseudomonasstrains to natural river-sediment surfaces. Three of the Pseudomonasstrains were chosen for their known ability to express alkylsulphatase enzymes capable of hydrolysing SDS, and the other two for their lack of such enzymes. One strain from each category was isolated from the indigenous bacterial population present in the river sediment used; other isolates were from soil or sewage. The alkylsulphatase phenotypes were confirmed by gel zymography of cell extracts. Addition of SDS to mixed suspensions of river sediment with any one of the biodegradation-competent strains stimulated the attachment of bacteria to the sediment particles. In contrast, the attachment of biodegradation-incompetent strains was weak and, moreover, was unaffected by SDS. The SDS-stimulated attachment for competent organisms coincided with rapid biodegradation of the surfactant. The primary intermediate of SDS biodegradation, dodecan-1-ol, accumulated transiently, and the numbers of attached bacteria correlated closely with the amount of dodecan-1-ol present. Direct addition of dodecan-1-ol also stimulated attachment but the effect was more immediate compared with SDS, when there was a lag period of approximately 2 h. To account for these observations, a model is proposed in which SDS stimulates the attachment of biodegradation-competent bacteria through its conversion to dodecan-1-ol, and it is hypothesized that the observed reversibility of the attachment is due to the subsequent removal of dodecan-1-ol by further bacterial metabolism.
-
-
- Genetics And Molecular Biology
-
-
-
Differential activity of Rickettsia rickettsii ompAand ompBpromoter regions in a heterologous reporter gene system
More LessThe outer membrane of the Gram-negative obligate intracellular parasite Rickettsia rickettsiicontains two large surface protein antigens with approximate molecular masses of 200 and 135 kDa termed rOmpA and rOmpB, respectively. rOmpB is the most abundant protein in the outer membrane, while rOmpA is a relatively minor constituent. Densitometry of intrinsically radiolabelled protein profiles from R. rickettsii-infected Vero cells indicated a molar ratio of approximately 1:9 between rOmpA and rOmpB. The putative promoter-5' untranslated regions (5' UTR) from their recently characterized genes (rompAand rompB) were placed in the promoter assay vector pKK232-8 to test whether these elements conserve aspects of differential expression in a heterologous host-reporter system. Primer extension analysis of RNA from Escherichia coliclones containing the constructs indicated that E. coliRNA polymerase faithfully utilizes rompAand rompBtranscription start sites identified previously in R. rickettsii.The rompBinsert directs 28-fold higher levels of chloramphenicol acetyl transferase activity than the rompAinsert.
-
-
-
-
Regulation of transfer genes of promiscuous IncPα plasmid RK2: repression of Tra1 region transcription both by relaxosome proteins and by the Tra2 regulator TrbA
More LessThe Tra1 region of broad host range IncPα plasmid RK2 encodes proteins essential for its promiscuous conjugative transfer and includes oriT, the site at which nicking occurs to initiate transfer replication. Unregulated expression of the Tra1 region genes would be likely to place a major burden on the host. To investigate the control of these genes the three transcriptional promoters from this region were cloned by PCR and inserted into xylEpromoter probe vectors. The strength of traJpand traKpwas estimated to be six to eightfold less than the strong trfApromoter which is required for expression of genes for vegetative replication of RK2. The traGpromoter was about one-tenth the strength of the other two. These promoters are not repressed by products of the central control operon of RK2. However, traJpand traKp, which are arranged as back to back divergent promoters in the oriTregion, are repressed by TraK which constitutes part of the relaxosome necessary for nicking at oriT.A second relaxosome protein, TraJ, represses traJp. traGpis not repressed by any relaxosome proteins. All three promoters are repressed by TrbA, which is encoded at the start of the trboperon containing the rest of the transfer genes (the Tra2 region). These circuits provide: (i) an autoregulatory way of ensuring production of enough relaxosome proteins without overburdening the host; and (ii) a means of coordinating expression of both blocks of transfer genes.
-
-
-
Induction of major heat-shock proteins of Saccharomyces cerevisiae, including plasma membrane Hsp30, by ethanol levels above a critical threshold
Many of the changes induced in yeast by sublethal yet stressful amounts of ethanol are the same as those resulting from sublethal heat stress. They include an inhibition of fermentation, increased induction of petites and stimulation of plasma membrane ATPase activity. Ethanol, at concentrations (4-10%, v/v) that affect growth and fermentation rates, is also a potent inducer of heat-shock proteins including those members of the Hsp70 protein family induced by heat shock. This induction occurs above a threshold level of about 4% ethanol, although different heat-shock proteins and heat-shock gene promoters are optimally induced at different higher ethanol levels. In addition ethanol (6-8%) causes the same two major changes to integral plasma-membrane protein composition that result from a sublethal heat stress, reduction in levels of the plasma membrane ATPase protein and acquisition of the plasma membrane heat-shock protein Hsp30.
-
-
-
áU2, a temperate bacteriophage specific for ‘Arthrobacter aureus’, whose integrative functions work in other corynebacteria
More LessThe temperate bacteriophage AAU2 of ‘Arthrobacter aureus’ C70, isolated from a soil sample, has been purified and characterized. The phage belongs morphologically to the Siphoviridaefamily. Its genome consists of a linear double-stranded DNA of 45.4 kb with cohesive ends. The attPsite and the phage-encoded functions essential for integration were cloned in Escherichia colias a 5.25 kb BglII-EcoRV fragment. The resulting plasmid vector, non-replicating in ‘A. aureus’, is able to integrate at high frequency into the chromosomes of ‘A. aureus’and the industrially used coryneform bacteria Brevibacterium lactofermentumand Corynebacterium glutamicum.
-
-
-
Analysis of the expression and regulation of the gerB spore germination operon of Bacillus subtilis168
More LessThe gerBspore germination operon of Bacillus subtilis168 is a homologue of the gerAspore germination operon. The expression and regulation of the gerBoperon has been examined using a lacZtranscriptional fusion and the transcriptional start defined. The gerBoperon is expressed during sporulation under the control of RNA polymerase containing the forespore-specific sigma factor, σG. This is a further homology to the gerAoperon, which is similarly regulated. It is predicted from the localization of expression and the encoded primary sequences that the GerB proteins are located at the inner spore membrane.
-
-
-
Genetic recombination after cell fusion of protoplasts from the facultative alkaliphile Bacillussp. C-125
More LessProtoplasts were prepared from two auxotrophic and antibiotic-resistant strains (Met-Nalrand Thr-Strr, respectively) of the facultative alkaliphile Bacillussp. C-125 by treatment with lysozyme. The protoplasts fused effectively in the presence of polyethylene glycol. Fusants obtained between two parental protoplasts were regenerated on solid medium containing the two antibiotics, nalidixic acid (Nal) and streptomycin (Str). Parental protoplasts were regenerated at high frequency (43-97%) on non-selective medium but not on selective medium. The NalrStrrfusants had the form of bacilli. Met and Thr markers segregated among the fusants with a predominantly Met+Thr+phenotype. The exfusants seemed to restore the parental ploidy.
-
-
-
Transposon Tn917PF1mutagenesis in Bacillus licheniformis
More LessThe plasmid pTnPF1 containing the transposon Tn917PF1was introduced into the protoplasts of two Bacillus licheniformisstrains in the presence of polyethylene glycol. Transpositions were produced at high temperature which inhibited plasmid replication and kanamycin was used for selection. Transposon Tn917PF1was inserted randomly into the bacterial chromosome, producing different auxotrophic, prophage BLF and bacitracin-non-producing mutants. The auxotrophic mutant phenotypes were characterized by the Holliday-test and some mutations by hybridization with a transposon DNA probe. Insertions for the entire chromosome or for the prophage genophore were found at random, but preferred target sites were detected within limited regions, like the bacitracin synthetase or sulphate reductase genes. The partial physical map of the chromosomal region of bacitracin synthetase was constructed based on the hybridization patterns of insertion mutants.
-
-
-
Malate synthase from Corynebacterium glutamicum: sequence analysis of the gene and biochemical characterization of the enzyme
More LessMalate synthase is one of the key enzymes of the glyoxylate cycle and is essential for growth on acetate as sole carbon source. The aceBgene from Corynebacterium glutamicum, encoding malate synthase, was isolated, subcloned and expressed in Escherichia coliand C. glutamicum. Sequencing of a 3024 bp DNA fragment containing the aceBgene revealed that it is located close to the isocitrate lyase gene aceA. The two genes are separated by 597 bp and are transcribed in divergent directions. The predicted aceBgene product consists of 739 amino acids with an M rof 82362. Interestingly, this polypeptide shows only weak identity with malate synthase polypeptides from other organisms and possesses an extra N-terminal sequence of about 170 amino acid residues. Inactivation of the chromosomal aceBgene led to the absence of malate synthase activity and to the inability to grow on acetate, suggesting that only one malate synthase is present in C. glutamicum. The malate synthase was purified from an aceB-overexpressing C. glutamicumstrain and biochemically characterized. The native enzyme was shown to be a monomer migrating at an M rof about 80000. By sequencing the N-terminus of malate synthase the predicted translational start site of the enzyme was confirmed. The enzyme displayed K mvalues of 30 μM and 12 μM for the substrates glyoxylate and acetyl CoA, respectively. Oxalate, glycolate and ATP were found to be inhibitors of malate synthase activity. The present study provides evidence that the malate synthase from C. glutamicumis functionally similar to other malate synthase enzymes but is different both in size and primary structure.
-
-
-
Cloning and nucleotide sequence of the carboxynorspermidine decarboxylase gene from Vibrio alginolyticus
More LessThe gene (nspC) encoding carboxynorspermidine decarboxylase (CANS DC), the last enzyme in norspermidine biosynthesis, in Vibrio alginolyticuswas isolated by immuno-screening and its complete nucleotide sequence was determined. Sequence analysis of the subcloned fragment (2.0 kb) revealed an ORF of 1131 bp encoding a protein of 377 amino acids with a calculated molecular mass of 42008 Da. The sequence of 20 N-terminal amino acids of purified CANS DC was found to be identical to that predicted from the nspCgene. A putative ribosome binding sequence was observed 8 bp upstream from the translation start site (ATG), and promoter- and terminator-like sequences were detected upstream and downstream of the ORF, respectively. Database searches identified no similar proteins, but the deduced amino acid sequence contained a putative pyridoxal 5′-phosphate binding region similar to those of the bacterial meso-2,6-diaminopimelate decarboxylases and eukaryotic ornithine decarboxylases. Another full ORF was found on the opposite strand downstream from the nspCgene. It encoded a protein of 69 amino acids with a calculated molecular mass of 7441 Da, which exhibited some weak similarity to ScrR,a repressor protein of V. alginolyticus, in the helix-turn-helix DNA binding domain, but did not appear to be expressed in the host cells.
-
- Pathogenicity And Medical Microbiology
-
-
-
Candida albicansexpresses a fibronectin receptor antigenically related to α5β1 integrin
Cell adhesion molecules, by regulating host-micro-organism interaction, play major role in the pathogenesis of infectious diseases. The present study was undertaken to investigate the expression of the fibronectin (FN) receptor prototype, α5β1 integrin, on Candida albicansand its involvement in the adhesion to FN. By immunofluorescence and fluorescence activated cell sorter (FACS) analysis, several monoclonal antibodies (mAbs) directed against human α5 or β1 integrin subunits, or two different antisera to FN receptor positively stained C. albicansyeast and germ tube phases, this immunoreactivity increasing upon germ tube transition. Twenty-five to thirty per cent of [3H]glucose-labelled Candidayeasts specifically adhered to FN and this adhesion was increased upon germ tube transition. C. albicansyeast and gerr tube forms bound to an RGD-containing 120 kDa tryptic fragment of FN and adhesion to FN was markedly inhibited by GRGDSP, but not GRGESP peptides. Moreover, binding of both C. albicansphases to FN was strongly inhibited by anti-α5 SAM-1 mAb, or both anti-fibronectin receptor (FNr) antisera. Overall these results indicate that C. albicansyeast and germ tube phases express a receptor antigenically related to α5β1 integrin which mediates their adhesion to FN. The α5β1 integrin-like receptor expression on C. albicanscould be relevant for fungus-host interaction and in the dissemination process of Candidainfection.
-
-
-
-
Characterization of a 38 kDa penicillin-binding protein and its possible involvement in maintaining stationary-phase cells of Shigella dysenteriae
More LessThis paper reports the first attempt to characterize the penicillin-binding proteins (PBPs) of Shigella dysenteriae,an important human pathogen. The PBP pattern of the membranes of S. dysenteriaeclosely resembles that of Escherichia colimembranes. A 38 kDa PBP which is an important target for the penem SCH34343, the cephamycin cefoxitin and the oxacephem moxalactam, has been purified. This PBP is immunologically related to a PBP of similar molecular mass in E. coliand is present at high levels in stationary-phase cells of S. dysenteriae.
-
- Physiology And Growth
-
-
-
The effect of nutrient limitation on glycerol uptake and metabolism in continuous cultures of Pseudomonas aeruginosa
More LessPseudomonas aeruginosaNM48, a non-mucoid derivative of an alginate-producing strain isolated from a cystic fibrosis patient, was grown in batch culture with glycerol, glucose or succinate as carbon source, and in continuous culture (D0.05 h-1) under glycerol or glucose limitation. Glycerol uptake, glycerol kinase and glycerol-3-phosphate dehydrogenase were induced by glycerol, but not by glucose or succinate. Linear uptake of [14C]glycerol by washed cells (K m≤ 2 μM) was inhibited by unlabelled glycerol and glyceraldehyde, but not by cyanide or the uncoupling agent carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), and was accompanied by substantial intracellular accumulation of glycerol-3-phosphate and/or dihydroxyacetone phosphate but not glycerol. Prolonged growth under glycerol limitation led to substantial increases in the activities and/or concentrations of the enzymes catalysing glycerol uptake and metabolism, together with a 48000 M router-membrane protein which was also over-expressed following prolonged growth under glucose limitation. The N-terminal amino acid sequence (AEAFSPN-) and electrophoretic properties of this protein were the same as those of the previously characterized glucose porin (OprB) from P. aeruginosa, indicating that this porin is active with both glucose and glycerol. It is concluded that during growth under glycerol limitation, glycerol is transported into P. aeruginosaNM48 via OprB and a high-affinity, binding-protein-independent facilitated-diffusion system.
-
-
-
-
Use of a series of chemostat cultures to isolate ‘improved’ variants of the Quorne myco-protein fungus, Fusarium graminearumA3/5
More LessVariants (designated A23-S and A24-S) of the Quorne myco-protein fungus, Fusarium graminearumA3/5 were isolated from a series of glucose-limited cultures grown at a dilution rate of 0.18 h-1for a combined total of 109 d. These variants had unchanged mycelial morphologies but, when grown in mixed culture with the parental strain (A3/5) in glucose-limited chemostat culture at 0.18 h-1, A23-S and A24-S had selection coefficients of 0.013 and 0.017 h-1, respectively, and supplanted A3/5. When a monoculture of A23-S was grown in a glucose-limited culture at a dilution rate of 0.18 h-1, the appearance of highly branched (so-called colonial) mutants was delayed compared with their appearance in chemostat cultures of the parental strain. Furthermore, when a monoculture of A24-S was grown in glucose-limited culture at 0.18 h-1, the appearance of colonial mutants was delayed even further. Thus, it is possible to isolate advantageous (relative to A3/5) variants of F. graminearumA3/5 which have unchanged mycelial morphologies, but in which the appearance of colonial mutants is delayed.
-
-
-
Evolution of Fusarium graminearumA3/5 grown in a glucose-limited chemostat culture at a slow dilution rate
More LessThe evolution of Fusarium graminearum A3/5 grown in a glucose-limited chemostat at a dilution rate of 0.05 h-1(doubling time of 13.9 h) was followed for 957 h or 69 generations. Periodic selection of advantageous mutants was monitored in the culture by determining increases and decreases in the concentration of cycloheximide-resistant macroconidia in the population. Six peaks in the concentration of cycloheximide-resistant macroconidia were observed representing five adaptive changes in the population; on average, an adaptive change occurred once every 148±22 h (mean±SE). The selection coefficient of strains present at the start of each increase in the concentration of cycloheximide-resistant macroconidia (i.e. after the establishment of a new advantageous strain) was determined relative to A3/5 and was found to increase progressively with time. When grown at a dilution rate of 0.05 h-1, the strain (A28-S) isolated from the last adaptive peak had a selection coefficient of 0.023 h-1relative to A3/5, but A28-S lost its selective advantage when grown at a dilution rate of about 0.11 h-1and was at a selective disadvantage when grown at a dilution rate higher than 0.11 h-1. The K mvalue (12±5 μM) for uptake of glucose by A28-S was significantly lower than that for A3/5. The spontaneous mutation rate from cycloheximide sensitivity to cycloheximide resistance was estimated to be 1.8 (±0.2) × 10-6h-1or 2.5 × 10-5generation-1. The culture initially contained about 1 × 106macroconidia ml-1but this decreased with time until, at about 800 h, the culture contained only about 1 × 104macroconidia ml-1. No highly branched (colonial) mutants were observed in glucose-limited cultures at dilution rates of 0.05 h-1even though the evolution of the population was followed for a further 1345 h in a second chemostat, making a total evolutionary period of 2207 h or 159 generations.
-
-
-
Involvement of mitochondria in the assimilatory metabolism of anaerobic Saccharomyces cerevisiaecultures
The possible physiological role of mitochondria in anaerobically grown Saccharomyces cerevisiaewas investigated via enzyme localization and inhibitor studies. Almost all of the activity of citrate synthase (EC 4.1.3.7) was recovered in the mitochondrial fraction after differential centrifugation of spheroplast lysates. The enzyme exhibited a high degree of latency which was demonstrated by sonication of the mitochondrial fractions. Since citrate synthase is an important enzyme in anabolic reactions, a consequence of this localization is the requirement for transport of metabolites across the mitochondrial membranes. Such transport is likely to require energy which, as a result of anaerobiosis, cannot be supplied by respiration. It was therefore investigated whether ATP translocation into the mitochondria by an ADP/ATP translocase might be involved in anaerobic mitochondrial energy metabolism. It was shown that addition of the ADP/ATP translocase inhibitor bongkrekic acid to anaerobic cultures indeed inhibited growth, although only partially. It is concluded that mitochondria of S. cerevisiaefulfil a vital role in anaerobic sugar metabolism.
-
-
-
Motility and chemosensory behaviour of the sulphur bacterium Thiovulum majus
More LessThe swimming track of the sulphur/sulphide-oxidizing bacterium Thiovulum majusis a left-handed helix. The cells modulate swimming speed by changing the tangential speed and/or the pitch and radius of the helix. Whether attached (to a mucous thread) or swimming, the spherical cells rotate around their anterior-posterior axis in a counter-clockwise direction when observed from the posterior pole. Swimming speeds may exceed 600 μm s-1, which is 5-6 times faster than recorded for any other bacterium. Thiovulumcells congregate at oxygen tensions of about 4% atmospheric saturation (0.85 kPa). Cells which accidentally leave the optimum zone make a U-turn within 150-200 μm, thus returning to where they came from. This represents a type of phobic response in which the eventual swimming direction is correlated with the initial direction; it is not a true chemotactic response in the sense that the cells can orient themselves in O2-gradients. The 180°-bend of the swimming path is probably accomplished by changes in the rotational velocity component which take place when the cells swim into an adverse environment. The U-turn response allows the bacteria to maintain the characteristic 100-200 μm thick veils which separate the sulphidic and the oxygenated zone in or above sediments. Evidence for a chemosensory response to sulphide could not be found.
-
-
-
Growth temperature regulates the induction of β-lactamase in Pseudomonas fluorescensthrough modulation of the outer membrane permeation of a β-lactam-inducing antibiotic
More LessThe psychrotrophic bacterium, Pseudomonas fluorescensstrain MFO, is more sensitive to the β-lactam mezlocillin at a low growth temperature (i.e. 8°C) than at a higher growth temperature (28°C). An early effect of this antibiotic at all temperatures is bacterial filamentation, but this occurs later at 8°C than at 28 °C, which suggests a lower permeability of the cell envelopes to mezlocillin at low growth temperature. β-Lactamase production is later induced by mezlocillin, but the level of this induction also depends on the growth temperature, the overall induction being much less efficient at 8 °C. It is hypothesized that the periplasmic concentration of the drug might be too low at 8 °C to allow efficient β-lactamase induction; this hypothesis was confirmed by the demonstration that β-lactamase production is drastically enhanced in cells cultivated at 8°C permeabilized for 10 min by Na-EDTA. In addition, induction kinetic curves display a marked dependence upon growth temperature. A rapid saturation was evident when mezlocillin concentrations were increased at 8°C; this was not seen at 28 T°C at up to 1000 μg mezlocillin ml-1. The results are discussed in terms of two different routes of drug permeation, depending on the growth temperature.
-
-
-
Glycine betaine transport by Staphylococcus aureus: evidence for feedback regulation of the activity of the two transport systems
More LessThe regulation of glycine betaine accumulation by Staphylococcus aureuswas investigated. The accumulation of glycine betaine was regulated by the osmotic pressure of the medium and the low affinity transport system played the major role in this regulation. Mutants were isolated that lack the low affinity, osmotically activated glycine betaine/proline transport system. Such mutants accumulated glycine betaine via the high affinity system but the glycine betaine pool was smaller and responded poorly to osmotic pressure changes. The regulation of glycine betaine transport has revealed that at the steady state net influx is reduced and that this is achieved by inhibition of both the low affinity and the high affinity transport systems. Cells pre-loaded with glycine betaine exhibited a reduced V maxfor both systems: the low affinity system was reduced in activity fivefold and the high affinity system was reduced 10-fold and became virtually undetectable. Although glycine betaine transport at the steady state is reduced, retention of the compatible solute is an active process since addition of an uncoupler provokes rapid release of the accumulated material. These data suggest that feedback regulation of the activity of the uptake systems is a major mechanism for controlling the level of compatible solute accumulation.
-
-
-
Evidence for feedback (trans) regulation of, and two systems for, glycine betaine transport by Staphylococcus aureus
More LessPrevious reports are in conflict as to the number of transport systems for glycine betaine in Staphylococcus aureus.Cells grown in complex medium exhibited a single transport system of moderate affinity. Cells grown in defined medium in the absence of glycine betaine showed a high affinity and a low affinity transport system. Cells grown in the presence of glycine betaine in the presence of osmotic stress in either complex or defined media accumulated large pools of internal glycine betaine. Smaller, but still significant, amounts of glycine betaine were accumulated by cells grown in its presence in either complex or defined media in the absence of osmotic stress. Cells grown in defined medium in the presence of glycine betaine in the presence or absence of osmotic stress showed lower rates of glycine betaine transport than cells grown in its absence. This suggests that glycine betaine transport is subject to feedback or transinhibition by internal glycine betaine. This can explain the difference in observed kinetics in cells grown in complex or defined media, the high affinity system being predominantly inhibited in cells grown in complex medium.
-
-
-
Monitoring of peroxisome induction and degradation by flow cytometric analysis of Hansenula polymorphacells grown in methanol and glucose media: cell volume, refractive index and FITC retention
More LessCell refractive index has been used to monitor peroxisome behaviour in the yeast Hansenula polymorphaby means of flow cytometry. Peroxisomes are inducible organelles which may occupy a large fraction of the cell volume when yeast cells are growing in methanol media. These organelles harbour a catalase that decomposes the hydrogen peroxide produced in methanol oxidation by alcohol oxidase, a peroxisomal enzyme whose subunits are arranged to form a regular crystalloid. Peroxisomes undergo a degradation process mediated by vacuoles whenever they and their enzymes become metabolically redundant (e.g. during growth on glucose). Flow cytometric analyses of side scattered light (depending on cell volume, morphology and structure) and fluorescein isothiocyanate retention (due to the vacuole) were made on two wild-type strains of H. polymorphaduring exponential growth in glucose and methanol media and during nutritional shifts from one carbon source to the other. The same parameters were also analysed for a mutant strain only partially repressed by glucose. We show that both the parameters are substrate-dependent and appear to reflect peroxisome development in the cells. The data reported correlate well with the known cytological and biochemical data, showing the possibility of using flow cytometry, a fast and sensitive technique, to analyse the dynamics of peroxisome proliferation and degradation in response to environmental as well as genetic factors.
-
-
-
Characterization of a brown Nostocspecies from Java that is resistant to high light intensity and UV
More LessA brown Nostocspecies was isolated from a soil sample collected in Java, Indonesia. It has three physiological types of light-absorbing compounds: the photosynthetic pigments (chlorophyll a, carotenoids, phycocyanin, phycoerythrin and allophycocyanin), a brown pigment(s) and three UV-absorbing compounds. The organism undergoes complementary chromatic adaptation by varying its phycobilin content. It is resistant to photobleaching at high intensities of white light, and also to UV-C radiation, under which conditions a green-coloured Nostocspecies (ATCC 27895) was killed. Synthesis of the brown pigment and UV-absorbing compounds was not observed at low oxygen levels. A brown pigment was released suddenly from cells during exponential growth, resulting in a deep brown-coloured medium. In addition, the absorption spectrum of the medium after pigment release had maxima in the UV region, at 256 nm, 314 nm and 400 nm. The appearance of the UV-absorbing compounds coincided with the brown pigment release and with the disappearance from the vegetative cells of peripheral granules. The Nostocspecies reduced acetylene, a measure of nitrogen fixation, in both the light and the dark periods when grown on a 12/12 h light/dark cycle. On a 4/20 h light/dark cycle fixation occurred predominantly in the light period. When grown on this restricted light regime, or at very low light intensity, the cells did not produce the brown pigment or the UV-absorbing compounds.
-
- Genome Analysis
-
-
-
Conservation of gene arrangement and an unusual organization of rRNA genes in the linear chromosomes of the Lyme disease spirochaetes Borrelia burgdorferi, B. gariniiand B. afzelii
More LessPhysical maps of the chromosomes of the Lyme disease spirochaetes Borrelia gariniiand Borrelia afzeliihave been elucidated for the enzymes Cspl, SgrAI, I-CeuI, SmaI, EagI, BssHII, MluI and Apal by two-dimensional pulsed-field gel electrophoresis techniques. The maps contain 42 sites for B. gariniiand 32 for B. afzelii.The mapping studies showed that the two chromosomes are linear DNA molecules of 953 and 948 kbp, respectively. A comparison of the physical maps of B. gariniiand B. afzeliiand the published map of the other Lyme disease spirochaete, Borrelia burgdorferi[Davidson, B. E., MacDougall, J. & Saint Girons, I. (1992) J Bacteriol174, 3766-3774] revealed that the three chromosomes have few endonuclease sites in common, apart from a cluster in rrl(encoding 23S rRNA) and rrs(encoding 16S rRNA). Cloned borrelial genes were used as specific hybridization probes to construct genetic maps, using the physical maps as a basis. The resulting maps contain 41 genetic loci for B. burgdorferi, 39 for B. garinii, and 33 for B. afzelii.In contrast to the physical maps, the three genetic maps are closely related, with no detectable differences in gene order along the entire length of the chromosome. It is concluded that the chromosomes of these three borrelial species have undergone no major rearrangements, deletions or insertions during their evolution from a common ancestor. Detailed mapping of the region of the B. gariniiand B. afzeliichromosomes that encodes rRNA revealed that each chromosome contains one copy of rrsseparated by 5 kbp from two copies each of rrland rrf(encoding 5S rRNA). The gene order is rrs rrIA rrfA rrIB rrfB. B. burgdorferiis the only other member of the eubacteria for which this particular rRNA gene arrangement has been observed. A DNA length polymorphism in the region of the borrelial rRNA genes was shown to be due to the presence of 2.2 kbp more DNA between rrsand rrIAin B. gariniiand B. afzeliithan in B. burgdorferi.
-
-
-
-
Mosaic structure of large regions of the Lactococcus lactissubsp. cremorischromosome
More LessLactococcus lactissubsp. lactisand Lactococcus lactissubsp. cremorisare closely related phenotypically and genetically. Here we report that certain regions of their chromosomes diverge considerably more than others. Conserved regions differ by less than 20%, whilst variable regions differ by more than 60%. This mosaic structure may have arisen by horizontal gene transfer from distantly related bacteria since in a particular region of the L. lactissubsp. cremorischromosome the G + C content and the codon bias are not typical for lactococci. Such an exchange, which conserves the function of the gene and cannot be achieved under selective pressure, may be of considerable importance in the evolution of bacteria.
-
-
-
Sequence and organization of the lactococcal prolate-headed bIL67 phage genome
More LessbIL67 is a broad-host-range prolate-headed phage that is active against Lactococcuscells. The complete phage genome sequence of 22195 bp was established. Thirty-seven open reading frames (ORFs) organized in two clusters were identified. Functions were assigned to the putative products of six of the ORFs on the basis of comparison of the deduced amino acid sequences to known proteins, analysis of structural features of the proteins and search for conserved motifs. These were a DNA polymerase, a protein involved in recombination, a lysin, a terminase subunit, a structural protein and a holin.
-
Volumes and issues
-
Volume 170 (2024)
-
Volume 169 (2023)
-
Volume 168 (2022)
-
Volume 167 (2021)
-
Volume 166 (2020)
-
Volume 165 (2019)
-
Volume 164 (2018)
-
Volume 163 (2017)
-
Volume 162 (2016)
-
Volume 161 (2015)
-
Volume 160 (2014)
-
Volume 159 (2013)
-
Volume 158 (2012)
-
Volume 157 (2011)
-
Volume 156 (2010)
-
Volume 155 (2009)
-
Volume 154 (2008)
-
Volume 153 (2007)
-
Volume 152 (2006)
-
Volume 151 (2005)
-
Volume 150 (2004)
-
Volume 149 (2003)
-
Volume 148 (2002)
-
Volume 147 (2001)
-
Volume 146 (2000)
-
Volume 145 (1999)
-
Volume 144 (1998)
-
Volume 143 (1997)
-
Volume 142 (1996)
-
Volume 141 (1995)
-
Volume 140 (1994)
-
Volume 139 (1993)
-
Volume 138 (1992)
-
Volume 137 (1991)
-
Volume 136 (1990)
-
Volume 135 (1989)
-
Volume 134 (1988)
-
Volume 133 (1987)
-
Volume 132 (1986)
-
Volume 131 (1985)
-
Volume 130 (1984)
-
Volume 129 (1983)
-
Volume 128 (1982)
-
Volume 127 (1981)
-
Volume 126 (1981)
-
Volume 125 (1981)
-
Volume 124 (1981)
-
Volume 123 (1981)
-
Volume 122 (1981)
-
Volume 121 (1980)
-
Volume 120 (1980)
-
Volume 119 (1980)
-
Volume 118 (1980)
-
Volume 117 (1980)
-
Volume 116 (1980)
-
Volume 115 (1979)
-
Volume 114 (1979)
-
Volume 113 (1979)
-
Volume 112 (1979)
-
Volume 111 (1979)
-
Volume 110 (1979)
-
Volume 109 (1978)
-
Volume 108 (1978)
-
Volume 107 (1978)
-
Volume 106 (1978)
-
Volume 105 (1978)
-
Volume 104 (1978)
-
Volume 103 (1977)
-
Volume 102 (1977)
-
Volume 101 (1977)
-
Volume 100 (1977)
-
Volume 99 (1977)
-
Volume 98 (1977)
-
Volume 97 (1976)
-
Volume 96 (1976)
-
Volume 95 (1976)
-
Volume 94 (1976)
-
Volume 93 (1976)
-
Volume 92 (1976)
-
Volume 91 (1975)
-
Volume 90 (1975)
-
Volume 89 (1975)
-
Volume 88 (1975)
-
Volume 87 (1975)
-
Volume 86 (1975)
-
Volume 85 (1974)
-
Volume 84 (1974)
-
Volume 83 (1974)
-
Volume 82 (1974)
-
Volume 81 (1974)
-
Volume 80 (1974)
-
Volume 79 (1973)
-
Volume 78 (1973)
-
Volume 77 (1973)
-
Volume 76 (1973)
-
Volume 75 (1973)
-
Volume 74 (1973)
-
Volume 73 (1972)
-
Volume 72 (1972)
-
Volume 71 (1972)
-
Volume 70 (1972)
-
Volume 69 (1971)
-
Volume 68 (1971)
-
Volume 67 (1971)
-
Volume 66 (1971)
-
Volume 65 (1971)
-
Volume 64 (1970)
-
Volume 63 (1970)
-
Volume 62 (1970)
-
Volume 61 (1970)
-
Volume 60 (1970)
-
Volume 59 (1969)
-
Volume 58 (1969)
-
Volume 57 (1969)
-
Volume 56 (1969)
-
Volume 55 (1969)
-
Volume 54 (1968)
-
Volume 53 (1968)
-
Volume 52 (1968)
-
Volume 51 (1968)
-
Volume 50 (1968)
-
Volume 49 (1967)
-
Volume 48 (1967)
-
Volume 47 (1967)
-
Volume 46 (1967)
-
Volume 45 (1966)
-
Volume 44 (1966)
-
Volume 43 (1966)
-
Volume 42 (1966)
-
Volume 41 (1965)
-
Volume 40 (1965)
-
Volume 39 (1965)
-
Volume 38 (1965)
-
Volume 37 (1964)
-
Volume 36 (1964)
-
Volume 35 (1964)
-
Volume 34 (1964)
-
Volume 33 (1963)
-
Volume 32 (1963)
-
Volume 31 (1963)
-
Volume 30 (1963)
-
Volume 29 (1962)
-
Volume 28 (1962)
-
Volume 27 (1962)
-
Volume 26 (1961)
-
Volume 25 (1961)
-
Volume 24 (1961)
-
Volume 23 (1960)
-
Volume 22 (1960)
-
Volume 21 (1959)
-
Volume 20 (1959)
-
Volume 19 (1958)
-
Volume 18 (1958)
-
Volume 17 (1957)
-
Volume 16 (1957)
-
Volume 15 (1956)
-
Volume 14 (1956)
-
Volume 13 (1955)
-
Volume 12 (1955)
-
Volume 11 (1954)
-
Volume 10 (1954)
-
Volume 9 (1953)
-
Volume 8 (1953)
-
Volume 7 (1952)
-
Volume 6 (1952)
-
Volume 5 (1951)
-
Volume 4 (1950)
-
Volume 3 (1949)
-
Volume 2 (1948)
-
Volume 1 (1947)