- Volume 142, Issue 7, 1996
Volume 142, Issue 7, 1996
- Review Article
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- Antigens And Immunity
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Immunoelectron microscopic localization of variable proteins on the surface of Mycoplasma bovis
More LessThe ultrastructural distribution and immunological accessibility of the variable surface proteins VspA, VspB, VspC and VspD were determined by immunoelectron microscopy on the surface of negatively stained cells of Mycoplasma bovis PG45 and 18 subclones, expressing either one or two of the Vsps. The variable proteins VspA, VspB, VspC and VspD, recognized by two monoclonal antibodies (mAb 1E5 and mAb 87-2) and visualized by goat-anti-murine-lgM labelled with gold particles, showed identical distribution patterns on the surfaces of the cells of all M. bovis clones investigated. Gold particles were distributed over the whole cell surface, arranged in clusters. The cell form seemed not to have an influence on the decoration pattern. Gold particles were also observed in irregular distributions around the cells. All clones showed unlabelled cells as well as strongly and weakly labelled cells. There were in general, however, no significant differences in the percentages of unlabelled, weakly labelled and strongly labelled cells, either between clones expressing different Vsps or between individual clones. No correlations were found between the numbers of labelled cells in immunoelectron microscopy and the numbers of labelled colonies in immunobinding assay (IBA) originating from the same broth cultures. The percentage of positive colonies in IBA was generally much higher than the percentage of positive cells in immunoelectron microscopy. The results show that the cells of the M. bovis clones are not identical, but differ in their surface antigens, and reveal the high variable potential of this species.
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- Biochemistry
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Comparative studies of chitinases A and B from Serratia marcescens
More LessSerratia marcescens produces several chitinolytic enzymes, including chitinase A (ChiA) and chitinase B (ChiB). In this study, ChiB was purified to homogeneity using a newly developed protocol based on hydrophobic interaction chromatography. Subsequently, characteristics of ChiB and of the hitherto only partly characterized ChiA were determined and compared. Pure ChiA and ChiB shared several characteristics such as a broad pH optimum around pH 5.0-6.0, and a temperature optimum between 50 and 60 0C. Both enzymes were fairly stable, with half-lives of more than 10 d at 37 0C, pH 6.1. Analyses of the degradation of various N-acetylglucosamine oligomers, fluorogenic substrates and colloidal chitin showed that both enzymes cleave chitobiose [(GlcNAc)2] from (GlcNAc) n and thus possess an exo-N,N’-diacetylchitobiohydrolase activity. Both enzymes were also capable of producing monomers from longer (GlcNAc) n substrates, indicating that they also have an endochitinase (ChiA) or exo-N,N’,N’-triacetylchitotriohydrolase (ChiB) activity. Kinetic analyses with 4-methylumbel-β-D-N,N’-diacetylchitobioside, an analogue of (GlcNAc)3, showed cooperative kinetics for ChiA, whereas for ChiB normal hyperbolic kinetics were observed. ChiA had a higher specific activity towards chitin than ChiB and synergistic effects on the chitin degradation rate were observed upon combining the two enzymes. These results, together with the results of sequence comparisons and previous studies of the cellular localization of the two chitinases in S. marcescens indicate possible roles for ChiA and ChiB in chitin breakdown.
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The Bacillus stearothermophilus NUB36 surA gene encodes a thermophilic sucrase related to Bacillus subtilis SacA
More LessThe complete nucleotide sequence of the surA gene, encoding a sucrase from Bacillus stearothermophilus NUB36, was determined. surA was composed of 1338 bp and encoded 445 amino acid residues. The deduced polypeptide of M r 51 519 showed strong sequence similarity to sucrose and sucrose phosphate hydrolases from Bacillus subtilis, Klebsiella pneumoniae and Vibrio alginolyticus, and contained the ‘sucrose box’ residues thought to be important for catalysis of the transfer of fructose from sucrose. The enzyme was partially purified using affinity chromotography from extracts of Escherichia coli containing the cloned surA. SurA displayed an optimum temperature for sucrose hydrolysis of 55 0C and high stability. The M r of SurA determined by gel filtration was 105000, which suggested that the active form of the enzyme is a dimer. SurA exhibited an apparent K m of 40 mM for sucrose but, unlike the homologous B. subtilis enzyme, had no detectable sucrose phosphate hydrolase activity.
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A reappraisal of the diversity and class distribution of aspartate transcarbamoylases in Gram-negative bacteria
More LessRecently, the subunit composition of class A aspartate transcarbamoylases (ATCases) in fluorescent pseudomonads has been clarified. We present evidence that distribution of this type of ATCase may be more widespread than at first suspected. Bacterial ATCases exist in three forms: class A (molecular mass # 450-500 kDa); class B, typified by Escherichia coli ATCase (# 300 kDa); and class C, typified by Bacillus subtilis ATCase (# 100 kDa). Using gradient gel electrophoresis with activity-staining to scan bacterial sonicates, we report the existence of six more class A ATCases. We have purified one of these, Acinetobacter calcoaceticus ATCase, and found its subunit composition to be similar to that of the pseudomonad ATCases. Two of these ATCases come from bacteria outside the γ-subgroup of the Proteobacteria, one from the α-subgroup and one from Deinococcus radiophilus, a species phylogenetically remote from the Proteobacteria. Unexpectedly, three bacterial species, closely related to the fluorescent pseudomonads and acinetobacters, have ATCases of 100 kDa (class C). One of these, Stenotrophomonas (formerly Xanthomonas) maltophilia has been purified and found to be a homotrimer of 35 kDa polypeptide chains. We believe this is the first time that class C ATCases have been reported in Gram-negative bacteria. A distinctive cluster in the γ-3 subgroup of the Proteobacteria is formed by the enteric bacteria and their relatives. So far only class B ATCases have been reported in this group. The evolutionary implications of these findings are discussed.
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- Bioenergetics And Transport
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Kinetics and regulation of fructose and glucose transport systems are responsible for fructophily in Zygosaccharomyces bailii
More LessA strain of Zygosaccharomyces bailii was selected for studies on fructose and glucose transport to determine the basis of the fructophilic behaviour of this species. Fructose was transported by a specific low-affinity, high-capacity transport system with a K m of 65.6 mM and a V max of 6.7 mmol g-1 h-1 for cells grown on 2% (w/v) fructose, while the transport of glucose showed a K m of 7 mM and a V max of 1.7 mmol g-1 h-1 for cells grown on 2% (w/v) glucose. The transporter of glucose also fructose as a substrate. Fructose inactivated the glucose transporter; inactivation was faster at higher concentrations. Both transporters were partially inductive. Measurements of metabolic fluxes and respiration and fermentation rates supported the general features identified by transport measurements. The kinetics and regulation of transport of the two sugars confirm the fructophilic behaviour previously described by other authors.
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Binding-protein-dependent arginine transport in Pasteurella haemolytica
More LessA periplasmic arginine transport system that is a member of the ATP-dependent transport superfamily was identified in Pasteurella haemolytica. The gene encoding the periplasmic binding protein (lapT) was cloned and the protein overexpressed in Escherichia coli. LapT was purified to homogeneity using a modified osmotic shock procedure and anion-exchange column chromatography. Filter-binding assays established that LapT is an t-arginine-binding protein. Various amino acids were tested for their ability to inhibit L-arginine binding to LapT. When present in 100-fold excess, only L-arginine, D-arginine and citrulline competed with L-arginine for binding. Arginine transport in P. haemolytica whole cells was competitively inhibited by the same amino acids, suggesting that the LapT permease specifically transports L-arginine. The dissociation constant for the L-arginine-LapT complex was 170 nM and the stoichiometry of binding was approximately 0.8 mol L-arginine (mol LapT)-1. A polyclonal antibody raised against the purified protein permitted detection of LapT in P. haemolytica periplasmic fractions.
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Kinetics, energetics and specificity of a general amino acid transporter from the ectomycorrhizal fungus Paxillus involutus
More LessThe kinetics, energetics and specificity of a general amino acid transporter were studied in the ectomycorrhizal fungus Paxillus involutus (Batsch) Fr. The uptake of amino acids showed features characteristic of active transport. After correction for a non-mediated transport component, the kinetics of glutamate, glutamine, alanine and aspartate uptake measured over a wide concentration range followed the simple Michaelis-Menten saturation curves. The apparent K m derived from the Eadie-Hofstee plots ranged from 7 μM for alanine to 27 μM for glutamate. Dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone and NaN3 strongly inhibited amino acid uptake, whereas dicyclohexylcarbodiimide, vanadate and the ionophores monensin and nonactin had no effect on the uptake. Both pH dependence and inhibition by protonophores are consistent with a proton symport mechanism for amino acid uptake by P. involutus. Competition studies indicated a broad substrate recognition by the uptake system, which resembles the general amino acid permease of yeast. Dixon plots of the inhibition of glutamate uptake by alanine, lysine and methionine sulfoximine showed that inhibitions were competitive. The physiological importance of this transporter for the exchange of nitrogenous compounds between fungal and host plant cells in ectomycorrhizal associations is discussed.
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A db-type cytochrome-c oxidase terminates the respiratory chain in Helicobacter pylori
More LessA Helicobacter pylori membrane fraction oxidized yeast and equine cytochrome c, and N,N,N’,N’-tetramethyl-p-phenylenediamine (TMPD). When ascorbate was used as reductant, the V max and apparent K m values were 612 nmol electron min-1 (mg protein)-1 and 14 μM for yeast, and 419 nmol electron min-1 (mg protein)-1 and 19 μM for equine cytochrome c, respectively. For TMPD oxidation, the V max and K m values were 640 nmol electron min-1 (mg protein)-1 and 182 μM, respectively. These oxidase activities showed a high affinity for oxygen. Inhibition of both cytochrome-c and TMPD oxidase activities by 50% was caused by about 4 μM cyanide and about 0.5 mM azide. Redox difference spectra of the membrane solubilized with Triton X-100 showed b- or c-type cytochromes but not aa 3-type cytochromes. c-type and a part of some b-type cytochromes were reduced with ascorbate plus TMPD. A CO difference spectrum revealed that protohaem, but not an aa 3-type cytochrome, may be interacting with CO/oxygen. Only protohaem was detected in the haem fraction extracted from the membrane. Three polypeptides (60, 38 and 29 kDa) were found to be bearing haem c after SDS-PAGE of the membrane. From these results, it was suggested that the cbb 3-type cytochrome-c oxidase, having a haem-copper binuclear centre like the cytochrome aa 3-type oxidase, but differing in a few other properties, functions as a terminal oxidase in the respiratory chain of H. pylori.
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- Development And Structure
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The localization of chitin synthase in membranous vesicles (chitosomes) in Neurospora crassa
More LessPolyclonal anti-chitin synthase antibodies raised against the Saccharomyces cerevisiae CHS2 gene product were used to identify and localize chitin synthase in the filamentous ascomycete Neurospora crassa. A single band of approximately 110 kDa was observed in Western blots of total protein extracts of N. crassa, probed with these antibodies. However, several additional bands were labelled when membrane fraction proteins (microsomes) were probed. Histo-immunochemical localization of chitin synthase confirmed that the polypeptide is compartmentalized in membranous vesicles (chitosomes), which are abundant in the vicinity of the hyphal tip. TEM analysis did not reveal chitin synthase in the plasma membrane. However, dense labelling of membrane-associated chitin synthase was observed by light-microscopic analysis of N. crassa protoplasts and at young hyphal tips.
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Cell wall protein and glycoprotein constituents of Aspergillus fumigatus that bind to polystyrene may be responsible for the cell surface hydrophobicity of the mycelium
More LessCell surface hydrophobicity (CSH) of Aspergillus fumigatus grown both in complex medium (yeast extract/peptone/dextrose; YPD) and minimal (Vogel's N) medium was monitored by assessing attachment of polystyrene microspheres to the cell surface. It was found that mature mycelium was hydrophobic. Treatment of intact mycelium with β-mercaptoethanol (βME) abolished binding of the microspheres to hyphal elements, and coating of the microspheres with βME extracts from mycelium inhibited their attachment to intact mycelial cells. A. fumigatus mycelium was tagged in vivo with biotin and treated with βME. The βME extracts were analysed by SDS-PAGE and Western blotting with both peroxidase-conjugated-ExtrAvidin and concanavalin A (ConA). This procedure allowed identification of cell wall surface proteins and glycoproteins. Rabbit polyclonal antisera were raised against βME extracts obtained from cells grown in YPD and Vogel's N media. These antisera defined some major cell-wall-bound antigens. SDS-PAGE and Western blotting analysis of the cell wall material released by βME and adsorbed on polystyrene microspheres revealed about 19 protein species with apparent molecular masses ranging from 20 to 70 kDa, and two high-molecular-mass glycoproteins of 115 and 210 kDa. Treatment of cells grown in YPD, but not those grown in Vogel's N medium, with βME released a 55 kDa polypeptide able to adsorb to polystyrene microspheres that was detectable with the antisera. The ability to bind to polystyrene particles exhibited by several protein and glycoprotein species released by βME treatment suggested that these cell wall moieties possess exposed hydrophobic domains that could be responsible for the CSH of mycelium.
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- Genetics And Molecular Biology
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Sequence analysis and regulation of a gene encoding a cuticle-degrading serine protease from the nematophagous fungus Arthrobotrys oligospora
More LessThe nematode trapping fungus Arthrobotrys oligospora produces an extracellular serine protease (designated PII) that immobilizes free-living nematodes in bioassays and hydrolyses proteins of the nematode cuticle. Peptides were isolated from PII and partly sequenced. Three internal peptide sequences were used to design synthetic oligonucleotides, which allowed the subsequent isolation of the gene encoding PII from a genomic library. The deduced amino acid sequence indicated that PII is synthesized as a pre-proenzyme containing the mature enzyme, a signal sequence and a propeptide that are removed before the enzyme is secreted into the medium. The primary sequence of PII displayed a high degree of similarity with several other serine proteases of ascomycetes belonging to the subtilisin family. Northern analysis demonstrated that PII was expressed when the fungus was starved of nitrogen and carbon and that the expression was significantly stimulated by the addition to the medium of various soluble and insoluble proteins, including fragments of nematode cuticle. The levels of the mRNA as well as the proteolytic activity of PII were repressed in the presence of more easily metabolized forms of nitrogen (including ammonia, nitrate and amino acids) or glucose. The activity of the enzyme was almost completely inhibited by the peptide Phe-Val, as well as by the amino acid Phe, without a corresponding decrease in mRNA level. Notably, peptides with similar structures are known to be secreted by the host (nematode) and to stimulate the production of infection structures (traps) of the fungus.
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Mutagenesis of three surface-exposed loops of a Bacillus thuringiensis insecticidal toxin reveals residues important for toxicity, receptor recognition and possibly membrane insertion
More LessInformation on the molecular determinants of receptor recognition, membrane insertion and toxin pore-formation was sought by making 42 single and multiple substitutions of residues 312-314 (GYY), 367-370 (YRRP) and 438-441 (SGFS) in the Bacillus thuringiensis insecticidal CrylAc δ-endotoxin by site-directed mutagenesis. These three regions correspond to three putative surface-exposed loops (loops 1, 2 and 3, respectively) in domain II of the δ-endotoxin, forming the molecular apex of the structure. All except mutants GFY (loop 1), YKRA, SRRA, YRKA (loop 2) and TGFS (loop 3) expressed δ-endotoxin protein at wild-type levels which was stable upon activation by Pieris brassicae gut extract or trypsin. Toxicity assays for all the fully stable mutants using Manduca sexta larvae showed that G312, Y367, R368, R369, S438 and G439 are important for activity. Wild-type toxin was then labelled in vivo with [35S]methionine and heterologous competition binding assays were carried out for all the mutants using brush border membrane vesicles prepared from Manduca sexta midgut. Most and least conservative mutations of G439 and least conservative substitutions of Y367, R368 and R369 reduced the ability of the toxin to bind competitively. The most conservative mutation, S441T, gave significantly increased binding. These results suggested that these four residues play a role in the initial receptor binding step in the toxin mechanism. As no significant effect on binding affinity was observed in relatively non-toxic mutants in which residues G312 and S438 were mutated, we suggest that these residues are involved in the subsequent steps of membrane insertion and pore-formation.
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Lysine-induced premature transcription termination in the lysC operon of Bacillus subtilis
More LessThe expression of the Bacillus subtilis lysC operon, which encodes the first specific enzyme of lysine biosynthesis, is controlled by the availability of the end product, lysine. The question of whether lysine exerts its control by inducing premature termination of transcription was addressed using Northern blot analysis. Whereas lys-C-specific RNA from lysine-starved B. subtilis consisted primarily of the expected full-length mRNA (1.6 kb), that from bacteria grown with an excess of lysine consisted of a truncated 0.27 kb RNA in place of the full-length 1.6 kb transcript. On the other hand, a B. subtilis aecA mutant, in which the lysC operon was derepressed owing to a single nucleotide substitution in the region corresponding to the lysC leader transcript, produced full-length lysC mRNA, but no 0.27 kb RNA, even during growth with excess lysine. Mapping of the truncated 0.27 kb lysC RNA by hybridization with oligonucleotide probes showed that it corresponded to the upstream portion of the lysC leader transcript, extending from the transcription initiation site to a putative rho-independent terminator element. Quantitative transcript analysis by hybridization with specific oligonucleotides showed that lysine did not affect the number of lysC-specific RNA molecules but promoted the stoichiometric replacement of full-length mRNA with truncated 0.27 kb molecules. These results indicate that lysine regulates the expression of the lysC operon by effecting the premature termination of transcription at a rho-independent terminator site in the lysC leader region and that the site of the aecA mutation, far upstream of the putative terminator element, must play an essential role in premature transcription termination by a mechanism which is not yet understood.
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Partial deletion of the Saccharomyces cerevisiae GDH3 gene results in novel starvation phenotypes
More LessA small-scale functional analysis screen has revealed several new phenotypes associated with a large deletion of GDH3, one of two Saccharomyces cerevisiae genes known to encode NADP-linked glutamate dehydrogenase. Diploids heterozygous for the deletion are able to sporulate in rich media, while haploid deletants produce dark, wrinkled colonies containing pseudohyphal cells. The haploid cells rapidly lose viability upon starvation.
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Analysis of heat shock gene expression in Lactococcus lactis MG1363
More LessThe induction of the heat shock response in Lactococcus lactis subsp. cremoris strain MG1363 was analysed at the RNA level using a novel RNA isolation procedure to prevent degradation. Cloning of the dnaJ and groEL homologues was carried out. Northern blot analysis showed a similar induction pattern for dnaK, dnaJ and groELS after transfer from 30 0C to 43 0C when MG1363 was grown in defined medium. The dnaK gene showed a 100-fold induction level 15 min after temperature shifting. Induction of the first two genes in the dnaK operon, orf1 and grpE, resembled the pattern observed for the above genes, although maximum induction was observed earlier for orf1 and grpE. Novel transcript sizes were detected in heat-shocked cells. The induction kinetics observed for ftsH suggested a different regulation for this gene. Experimental evidence for a pronounced transcriptional regulation being involved in the heat shock response in L. lactis MG1363 is presented. A gene located downstream of the dnaK operon in strain MG1363, named orf4, was shown not to be regulated by heat shock.
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Diversity of repC plasmid-replication sequences in Rhizobium leguminosarum
More LessHomologues of the plasmid replicator gene repC were detected and characterized in a sample of Rhizobium leguminosarum strains. Conserved PCR primers were designed from published sequences of repC; they amplified a fragment of about 750 bp from 39 out of 41 strains tested, and also from several Sinorhizobium strains, including S. meliloti. Restriction endonuclease digestion showed that the PCR product from individual strains, though uniform in size, was often heterogeneous in sequence. PCR products from 24 field isolates of R. leguminosarum from France, Germany and the UK were cloned and partially sequenced from both ends. Phylogenies constructed from the 5′ and 3′ ends (200 bp each) were largely congruent and demonstrated four clearly defined groups plus several unique strains. Published Agrobacterium repC sequences fall within the phylogeny of R. leguminosarum sequences, though not within any of the four groups. Specific pairs of PCR primers were designed for each of the four groups; 29 out of 41 R. leguminosarum strains gave a PCR product of the expected size with more than one group-specific primer pair. We hypothesize that the sequence groups correspond to incompatibility groups of Rhizobium plasmids.
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Influence of salt on the transcription of the gas-vesicle genes of Haloferax mediterranei and identification of the endogenous transcriptional activator gene
More LessThe transcription of the 14 gvp genes of the gas-vesicle-encoding mc-vac region was investigated, using RNA from 25% and 15% (w/v) salt cultures of the moderately halophilic archaeon Haloferax mediterranei. Transcription occurred only from two promoters, located in front of the mc-gvpA and mc-gvpD genes. In both cultures transcripts spanning the entire mc-gvpDEFGHIJKLM transcription unit were formed only during the exponential growth phase. Amounts of these transcripts were larger in the 25% salt culture, in which the 2.0 kb mc-gvpD mRNA and large amounts of 1.3 kb and 0.45 kb partial mc-gvpD transcripts were also synthesized during the stationary phase. The levels of the mc-gvpD transcripts and of the 324 nt mc-gvpA mRNA increased in parallel during the stationary phase of the 25% salt culture. Only under these conditions were mRNAs spanning the entire mc-gvpACNO transcription unit observed, and gas-vesicles were formed. Investigation of the influence of the mc-gvpDE genes on both mc-vac promoters in transformants revealed that by themselves they were nearly inactive. The addition of mc-gvpE, however, resulted in a high level of constitutively produced mc-gvpA and mc-gvpD mRNA, indicating a transcriptional activator function for the mc-gvpE product.
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RNA polymerase, PurR and MetR interactions at the glyA promoter of Escherichia coli
More LessIn Escherichia coli, the MetR and PurR proteins positively and negatively regulate glyA gene expression, respectively. A DNase I footprint analysis showed that both proteins bind independently to the glyA control region. The PurR protein blocks RNA polymerase (RNAP) from binding to the glyA promoter. The presence of hypoxanthine, the co-repressor of PurR, increases the ability of PurR to prevent RNAP binding, providing a model for repression of the glyA gene by PurR. In contrast, MetR alters the RNAP footprint pattern of the glyA control region. In addition, the MetR footprint is increased in the presence of RNAP, suggesting that the two proteins might interact.
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IS 1236, a newly discovered member of the IS3 family, exhibits varied patterns of insertion into the Acinetobacter calcoaceticus chromosome
More LessAnalysis of spontaneous mutations in Acinetobacter calcoaceticus revealed a 1237 bp insertion sequence named IS 1236 and possessing a nucleotide sequence resembling those of members of the IS3 family. The chromosome of A. calcoaceticus strain ADP1 contains seven copies of IS 1236 which appears to insert preferentially into pobR, the transcriptional activator of the structural gene for p-hydroxybenzoate hydroxylase. IS 1236 creates tandem 3 bp DNA duplications flanking the sites of its insertion in pobR. Different duplication patterns are found following insertion of IS 1236 into pcaH, a structural gene for protocatechuate 3,4-dioxygenase. Therefore the insertion properties of IS 1236 appear to be influenced by its DNA target. Amino acid sequences associated with the apparent transposase function have been conserved in ORFB of IS 1236 whereas the presumed DNA-binding helix-turn-helix region of IS 1236 ORFA exhibits substantial amino acid sequence divergence from its IS3 counterparts. IS 1236 ORFA and ORFB coding sequences overlap considerably, and sequence evidence indicates mechanisms for ORFB expression in IS 1236 may resemble those employed by other members of the IS3 family. Portions of the IS 1236 terminal repeats exhibit substantial sequence divergence from other members of the IS3 family, but evolution appears to have conserved a mechanism preventing expression of the insertion sequence genes as a consequence of transcriptional readthrough.
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Transcriptional regulation of the Escherichia coli rhaT gene
More LessTranscriptional regulation of the rhaT gene, one of the operons forming the rhamnose regulon in Escherichia coli, was studied by fusing its complete or deleted promoter to the reporter gene lacZ. Analysis of β-galactosidase activities induced in these constructions grown under different conditions predicted the presence of two putative control elements: one for the RhaS regulatory protein and activating the gene not only by L-rhamnose but also by L-lyxose or L-mannose, the other for cAMP-catabolite repression protein and activating this gene in the absence of glucose. Anaerobiosis increased the promoter function two- to threefold with respect to the aerobic condition. Experiments involving complementation of strains containing the rhaT-promoter fusion and carrying a deletion in the rhaS and/or rhaR genes with plasmids bearing the rhamnose regulatory genes showed that rhaT is controlled by a regulatory cascade, in which RhaR induces rhaSR and the accumulated RhaS directly activates rhaT.
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Complementation of the hha mutation in Escherichia coli by the ymoA gene from Yersinia enterocolitica: dependence on the gene dosage
The Hha protein from Escherichia coli is highly similar (82%) to the YmoA protein from Yersinia enterocolitica. Both are members of a new class of proteins that modulates gene expression, probably by influencing DNA topology. In this paper, complementation of the hha mutation in E. coli by the ymoA gene from Y. enterocolitica has been studied. We show that the ymoA gene complements one of the phenotypic properties of hha mutants (high level of haemolysin production when they carry the recombinant plasmid pANN202-312) when cloned in a medium-copy-number plasmid but not when carried in a low-copy-number plasmid. Western blot analysis of the expression of YmoA in E. coli rules out inefficient expression of the protein. Surprisingly, the hha gene itself fails to complement the hha mutation when cloned in a medium-copy-number vector and causes genetic rearrangements of the E. coli chromosome as a consequence of insertion sequences mobilization.
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Analysis of putative ABC transporter genes in Mycoplasma hyopneumoniae
More LessA previously described DNA probe specific for Mycoplasma hyopneumoniae (l-141) was fully sequenced and found to consist of 1618 bp and to contain two tandemly repeated ORFs. The deduced amino acid sequence of the two ORFs showed significant homologies with ATP-binding cassette (ABC) transporter proteins, particularly those of the eukaryotic multidrug resistance (MDR) protein family (up to 21 % identity and 47% similarity). A somewhat lower homology was evident with the secretion protein HlyB of the RTX-haemolysin from Escherichia coli. The location of the two ORFs on the M. hyopneumoniae chromosome was downstream of the rrl gene encoding the 23S rRNA, but transcribed in the opposite direction. PCR amplification and subsequent chromosomal analysis by Southern blot hybridization of several M. hyopneumoniae strains showed that all field strains contained the two putative ABC transporter genes. However, some culture collection strains derived from strain J had lost these genes as the result of a 2221 bp deletion.
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- Pathogenicity And Medical Microbiology
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An aroA mutant of Yersinia pestis is attenuated in guinea-pigs, but virulent in mice
More LessThis study describes a PCR-based approach for the production of a rationally attenuated mutant of Yersinia pestis. Degenerate primers were used to amplify a fragment encoding 91.45% of the aroA gene of Y. pestis MP6 which was cloned into pUC18. The remainder of the gene was isolated by inverse PCR. The gene was sequenced and a restriction map was generated. The Y. pestis aroA gene had 75.9% identity with the aroA gene of Yersinia enterocolitica. The cloned gene was inactivated in vitro and reintroduced into Y. pestis strain GB using the suicide vector pGP704. A stable aro-defective mutant, Y. pestis GBΔaroA, was isolated and its virulence was examined in vivo. The mutant was attenuated in guinea-pigs and capable of inducing a protective immune response against challenge with the virulent Y. pestis strain GB. Unusually for an aro-defective mutant, the Y. pestis aroA mutant was virulent in mice, with a median dose which induced morbidity or death similar to that of the wild-type, although time to death was significantly prolonged.
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- Physiology And Growth
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Analysis of starvation conditions that allow for prolonged culturability of Vibrio vulnificus at low temperature
More LessThe response of the estuarine human pathogen Vibrio vulnificus to starvation for carbon, nitrogen or phosphorus, or all three nutrients simultaneously (multiple-nutrient), was examined with respect to the maintenance of culturability during incubation at low temperature. V. vulnificus showed similar survival patterns during starvation for the individual nutrients when kept at 24 0C. On the other hand, cultures prestarved at 24 0C and then shifted to 5 0C maintained culturability at low temperature in a starvation-condition-dependent manner. Carbon and multiple-nutrient starvation were indistinguishable in their ability to mediate maintenance of culturability in the cold. Prolonged starvation for phosphorus had a similar effect, but nitrogen starvation did not allow for maintenance of culturability. Extracellular factors produced during starvation were not observed to have an effect on the culturability of cells incubated at low temperature. Protein synthesis during starvation for individual nutrients was analysed by two-dimensional PAGE of pulse-labelled proteins. Carbon and multiple-nutrient starvation gave nearly identical protein induction patterns involving at least 34 proteins, indicating that carbon starvation determines both responses. Nitrogen starvation for 1 h induced 24 proteins, while phosphorus starvation induced a set of 10 proteins after 1 h and about 40 proteins after 18 h. It is suggested that starvation for carbon or phosphorus induces maintenance of culturability of V. vulnificus incubated at low temperature via the synthesis of distinct sets of starvation-specific proteins.
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Acid tolerance in Rhizobium meliloti strain WSM419 involves a two-component sensor-regulator system
More LessAn acid-sensitive mutant TG5-46, derived from Rhizobium meliloti WSM419 by Tn5 mutagenesis, fails to grow below pH 6.0 whereas the parent strain grows at pH 5.7. The DNA sequence of a 2.2 kb rhizobial DNA region flanking Tn5 in TG5-46 contains two open reading frames, ORF1 (designated actS) and ORF2 (designated actR), having high similarity to the sensor-regulator pairs of the two-component systems involved in signal transduction in prokaryotes. Insertion of an omega interposon into actS in R. meliloti WSM419 resulted in an acid-sensitive phenotype. A DNA fragment from the wild-type complemented the acid-sensitive phenotype of RT295 (ActS-) and TG5-46 (ActR-), while fragments containing only actR or actS complemented TG5-46 and RT295, respectively. The presence of multiple copies of actR complementes not only TG5-46 but also RT295. Cloning DNA upstream from actR and actS into a broad-host-range lacZ expression vector and measuring β-galactosidase activities showed that both genes are constitutively expressed regardless of the external pH. Genomic DNA from all strains of R. meliloti, but no other bacteria tested, hybridized with an actRS probe at high stringency. These data implicate a two-component sensor-regulator protein pair in acid tolerance in R. meliloti and suggest their involvement in pH sensing and/or response by these bacteria.
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Active transport of glucosylglycerol is involved in salt adaptation of the cyanobacterium Synechocystis sp. strain PCC 6803
More LessAn active-transport system for the osmoprotective compound glucosylglycerol (GG) was found in the cyanobacterium Synechocystis sp. strain PCC 6803. Uptake assays with 14C-labelled GG showed that the GG transport was enhanced in cells adapted to increasing concentrations of NaCl. Kinetic studies indicated a Michaelis-Menten relationship. The uptake of GG was energy dependent and occurred against a steep concentration gradient. It was inhibited by uncouplers as well as by a combination of darkness and KCN. The affinity of the transporter seems to be restricted to osmoprotective compounds of cyanobacteria; from a variety of compounds tested only sucrose and trehalose competed with GG for uptake. A salt-sensitive mutant of Synechocystis 6803 unable to synthesize GG could be complemented to salt resistance by exogenous GG. Accumulation of GG from the medium was essential for the restoration of photosynthesis and growth in mutant cells under high-salt conditions. In wild-type cells, the GG transporter probably serves to prevent GG leaking out of salt-stressed cells.
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The Cdc25 protein of Saccharomyces cerevisiae is required for normal glucose transport
More LessThe essential CDC25 gene product of Saccharomyces cerevisiae is the most upstream known component of the RAS/adenylate cyclase pathway. Cdc25 is a GTP-exchange protein involved in activating RAS in response to fermentable carbon sources. In this paper it is reported that the Cdc25 protein, in addition to its stimulatory role in the RAS/adenylate cyclase pathway, regulates glucose transport. Continuous culture studies and glucose uptake experiments showed that the cdc25-1 and the cdc25-5 temperature-sensitive mutants exhibit decreased glucose uptake activity at the restrictive temperature under both repressed and derepressed conditions as compared to the wild-type strain. Because the cdc25-1 mutant is not impaired in its cAMP metabolism, it is concluded that this effect on glucose transport is independent of cAMP levels. Furthermore, it is shown that the decrease in glucose uptake activity is not due to a decrease in protein synthesis or to an arrest in the G1 phase of the cell cycle. In addition to a defect in glucose uptake, the cdc25-5 mutant strain exhibited differences in glucose metabolism, probably due to the decreased cAMP level and hence decreased protein kinase A activity. Because the Cdc25 protein is localized at the membrane, these results indicate that Cdc25 is directly involved in glucose transport and may be in direct contact with the glucose transporters.
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Glucose-triggered signalling in Saccharomyces cerevisiae: different requirements for sugar phosphorylation between cells grown on glucose and those grown on non-fermentable carbon sources
Addition of glucose or fructose to cells of the yeast Saccharomyces cerevisiae grown on a nonfermentable carbon source triggers within a few minutes post-translational activation of trehalase, repression of the CTT1 (catalase) and SSA3 (Hsp70) genes, and induction of the ribosomal protein genes RPL1, RPL25 and RPS33. By using appropriate sugar kinase mutants, it was shown that rapid glucose- or fructose-induced activation of trehalase requires phosphorylation of the sugar. On the other hand, partial induction of RPL1, RPL25 and RPS33 as well as partial repression of CTT1 and SSA3 were observed in the absence of sugar phosphorylation. In glucose-grown nitrogen-starved yeast cells re-addition of a nitrogen source triggers activation of trehalase in a glucose- or fructose-dependent way, but with no apparent requirement for phosphorylation of the sugar. Repression of CTT1 and SSA3 under the same conditions was also largely dependent on the presence of the sugar and also in these cases there was a strong effect when the sugar could not be phosphorylated. Nitrogen induction of RPL1, RPL25 and RPS33 was much less dependent on the presence of the sugar, and only phosphorylated sugar caused a further increase in expression. These results show that two glucose-dependent signalling pathways, which can be distinguished on the basis of their requirement for glucose phosphorylation, appear to be involved in activation of trehalase, repression of CTT1 and SSA3 and induction of ribosomal protein genes. They also show that nutrient-induced repression of CTT1 and SSA3 is not a response to improvement of the growth conditions because the addition of nonmetabolizable sugar does not ameliorate the growth conditions. Similarly, the upshift in ribosomal protein synthesis cannot be a response to increased availability of energy or biosynthetic capacity derived from glucose, but it is apparently triggered to a significant extent by specific detection of glucose as such.
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Effect of hyphal micromorphology on bioreactor performance of antibiotic-producing Saccharopolyspora erythraea cultures
More LessWhen Saccharopolyspora erythraea biomass from submerged culture was filtered (100-120 μn sintered glass filter) the antibiotic yield of the retentate (mean hyphal particle diameter 103 μm) was significantly higher [1.740 mg (mg biomass)-1] than that of the filtrate [1.286 mg (mg biomass)-1; mean hyphal particle diameter 88 μn]. A hypothesis to explain this is that there is a critical hyphal particle diameter, below which the particle is incapable of producing antibiotic. This would be a consequence of the site of antibiotic production occurring at a fixed distance from the growing hyphal tip. A protocol is proposed for calculation of the hypothetical critical hyphal diameter (88 μm in this case). The proportion of retentate (more productive) fraction of biomass varied between 60% and 30% during the course of a batch culture. Bioreactor stirrer speed significantly affected mean hyphal particle diameter (70 fim at 1500 r.p.m.; 124 μm at 750 r.p.m.) and antibiotic productivity [0.867 mg (mg biomass)-1 at 1500 r.p.m.; 0.913 mg (mg biomass)-1 at 750 r.p.m.].
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Guanosine 5′-diphosphate 3′-diphosphate (ppGpp), guanosine 5′-diphosphate 3′-monophosphate (ppGp) and antibiotic production in Streptomyces clavuligerus
More LessStreptomyces clavuligerus produced cephamycin C under all nutrient limitations investigated in batch culture, whilst clavulanic acid was produced under phosphate and carbon limitations. Guanosine 5′-diphosphate 3′-diphosphate (ppGpp) was produced at very low levels in all fermentations, prior to the detection of cephamycin C in the fermentation broth. Guanosine 5′-diphosphate 3′-monophosphate (ppGp) was observed at high levels in all fermentations; it appeared following a downturn in nutrient levels and immediately prior to detection of isopenicillin N synthase (IPNS). ppGp was detected following nutritional shiftdown by amino acid depletion and it was not produced via degradation of ppGpp. The results point to a possible role for ppGp in the regulation of cephamycin production in S. clavuligerus.
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Trypsin-like protease of Streptomyces exfoliatus SMF13, a potential agent in mycelial differentiation
More LessStreptomyces exfoliatus SMF13 sequentially produced leupeptin, leupeptin-inactivating enzyme (LIE) and trypsin-like protease (TLP). TLP was produced upon exhaustion of glucose. Autolysis of mycelium was accompanied by an increase in TLP activity. However, in three bld mutants isolated from S. exfoliatus SMF13 after UV-mutagenesis, mycelium autolysis did not occur, and neither LIE nor TLP was produced, although leupeptin was produced. Production of both LIE and TLP was restored in a spontaneous Spo+ revertant of a bld mutant. In contrast, two whi mutants sequentially produced leupeptin, LIE and TLP. The molecular mass of TLP produced during morphological differentiation was estimated to be 31.8 kDa by SOS-PAGE. The N-terminal amino acid sequence was RVGGTxAAQGNFPFQQxLSM. TLP was competitively inhibited by leupeptin; the inhibition constant was 0.015 μM. TLP effectively hydrolysed the mycelial protein extract of S. exfoliatus SMF13, but the hydrolytic activity was inhibited by leupeptin. It was concluded that morphological differentiation and production of TLP are coordinately regulated, that TLP may function as an enzyme in the metabolism of mycelial proteins, and that the hydrolytic activity of TLP is regulated by autogenous leupeptin in S. exfoliatus SMF13.
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Flux limitations in the ortho pathway of benzoate degradation of Alcaligenes eutrophus: metabolite overflow and induction of the meta pathway at high substrate concentrations
More LessThe growth behaviour of Alcaligenes eutrophus using various concentrations of benzoate was investigated. In batch culture, growth was exponential and growth rate (μ) and yields (Y) were high [μ = 0.51 h-1 and Y X/benzoate = 0.56 mol carbon (mol carbon)-1] when low concentrations of benzoate (< 5 mM) were used. These kinetic parameters were close to the maxima determined in a benzoate-limited chemostat [μmax = 0.55 h-1 and Y max X/benzoate = 0.57 mol carbon (mol carbon)-1] and the part of the energy for maintenance was limited (m ATP = 4.3 ± 2.2 mmol ATP g-1 h-1). When higher concentrations of benzoate were used (up to 40 mM), several metabolic limitations appeared. The specific rate of benzoate consumption was not altered, whereas growth was inhibited [Ki (benzoate) # 27 mM]. Furthermore, high concentrations of catechol together with some 1,2-dihydro-1,2-dihydroxybenzoate (DHB) transiently accumulated in the medium. The accumulation of catechol was attributed to limiting flux through catechol 1,2-dioxygenase estimated to be 5-2 mmol g-1 h-1, whereas that of DHB was provoked by an imbalance in the NADH/NAD- intracellular content. The direct consequence of DHB accumulation was the induction of the meta pathway for the degradation of catechol, and this pathway contributed up to 20% of the total flux of catechol to the central metabolism. Finally, when very high concentrations of benzoate were used (55 mM), both growth and the specific rate of benzoate degradation were diminished due to a strong decrease in benzoate 1,2-dioxygenase specific activity.
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- Systematics
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Evaluation of the DNA fingerprinting method AFLP as a new tool in bacterial taxonomy
We investigated the usefulness of a novel DNA fingerprinting technique, AFLP, which is based on the selective amplification of genomic restriction fragments by PCR, to differentiate bacterial strains at the subgeneric level. In total, 147 bacterial strains were subjected to AFLP fingerprinting: 36 Xanthomonas strains, including 23 pathovars of Xanthomonas axonopodis and six pathovars of Xanthomonas vasicola, one strain of Stenotrophomonas, 90 genotypically characterized strains comprising all 14 hybridization groups currently described in the genus Aeromonas, and four strains of each of the genera Clostridium, Bacillus, Acinetobacter, Pseudomonas and Vibrio. Depending on the genus, total genomic DNA of each bacterium was digested with a particular combination of two restriction endonucleases and the resulting fragments were ligated to restriction halfsite-specific adaptors. These adaptors served as primer-binding sites allowing the fragments to be amplified by selective PCR primers that extend beyond the adaptor and restriction site sequences. Following electrophoretic separation on 5% (w/v) polyacrylamide/8.3 M urea, amplified products could be visualized by autoradiography because one of the selective primers was radioactively labelled. The resulting banding patterns, containing approximately 30-50 visualized PCR products in the size range 80-550 bp, were captured by a high-resolution densitoscanner and further processed for computer-assisted analysis to determine band-based similarity coefficients. This study reveals extensive evidence for the applicability of AFLP in bacterial taxonomy through comparison of the newly obtained data with results previously obtained by well-established genotypic and chemotaxonomic methods such as DNA-DNA hybridization and cellular fatty acid analysis. In addition, this study clearly demonstrates the superior discriminative power of AFLP towards the differentiation of highly related bacterial strains that belong to the same species or even biovar (i.e. to characterize strains at the infrasubspecific level), highlighting the potential of this novel fingerprinting method in epidemiological and evolutionary studies.
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Intra-specific diversity and host specificity within Pasteurella haemolytica based on variation of capsular polysaccharide, lipopolysaccharide and outer-membrane proteins
More LessIntra-specific diversity within Pasteurella haemolytica was assessed by analysing variation in the capsular polysaccharide (serotypes), lipopolysaccharide (LPS) and outer-membrane proteins (OMPs) of 184 isolates recovered from cattle and sheep. Four of 12 serotypes comprised 83% of the total number of isolates, including A1 and A2 as the most frequently recovered serotypes from cattle and sheep, respectively. Nine distinct LPS profiles were identified. Four different core-oligosaccharide patterns were present, each of which occurred alone as rough LPS and also in association with a single O-antigen profile as smooth LPS; the ninth LPS type was also smooth but had a different O-antigen profile. The capsular serotypes could be divided into four groups based on the dominant LPS profile within each serotype: (1) A1, A6, A9, A12 and A5; (2) A2, A8, A14 and A16; (3) A7 and A13; and (4) A11. Smooth LPS of type 1A, which was found only in the first group, was associated primarily with bovine disease isolates, whereas rough LPS of types 1B and 3B were associated primarily with the group 2 serotypes and ovine disease isolates. Similarly, the variation of OMP profiles generated three groups: (1) A1, A6, A9, A12, A5 and A8; (2) A2, A14 and A16; and (3) A7, A11 and A13. Isolates belonging to groups 2 and 3 exhibited greater diversity in their OMP profiles than those belonging to group 1. Although the majority of group 3 isolates possessed profiles unique to that group, a smaller number of A7 isolates possessed profiles with similarities to those of serotypes A1 or A2. OMP profiles clearly differentiated bovine from ovine isolates of the same serotypes. The association both of specific LPS and OMP profiles with bovine or ovine disease isolates suggested a correlation between specific cell-surface structures and host specificity. The combined analysis of capsular serotypes, LPS types and OMP profiles identified seven major groups within P. haemolytica which were responsible for 59% of the disease cases, suggesting a clonal structure for this species. Overall, comparison of the capsular serotypes, LPS types and OMP profiles proved extremely useful for assessing diversity within P. haemolytica.
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- Genome Analysis
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Genomic organization of the entomopathogenic bacterium Bacillus thuringiensis subsp. berliner 1715
More LessA physical and genetic map of the Bacillus thuringiensis subsp. berliner 1715 (serotype 1) chromosome was constructed by pulsed field gel electrophoresis using three restriction enzymes, Ascl, Notl and Sfil. A total of 24 restriction enzyme sites and 28 probes were located on the map. The chromosome size was 5.7 Mb. Consistent with previous mapping of Bacillus cereus chromosomes, the genes were nonrandomly distributed. Genes which are often plasmid-encoded were located on one half of the chromosome, whereas the other half contained rRNA genes and the origin region. Hybridization with macro-restriction fragments showed that the region containing rRNA genes and the origin was similar to that of the B. cereus type strain, ATCC 14579, and confirmed that the region was conserved between the B. cereus and B. thuringiensis chromosomes. The insecticidal toxin probe crylA or the transposon probe Tn4430 hybridized to six extrachromosomal elements of 60, 60, 100, 130, 270 and 600 kb, indicating that the genome size was at least 6.9 Mb.
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The genes lepA and hemN form a bicistronic operon in Bacillus subtilis
More LessThe lepA operon of Bacillus subtilis was found to be bicistronic and to consist of the two genes lepA and hemN, which encode a putative GTP-binding protein and an oxygen-independent coproporhyrinogen III oxidase, respectively. The lepA operon is located immediately upstream of the dnaK operon. Both operons are transcribed in the same direction and are not separated by an obvious transcription-terminator-like structure. The lepA operon is preceded by a potential vegetative promoter, and there is a putative strong intergenic terminator between lepA and hemN. Northern blot experiments revealed only a transcript corresponding to lepA, but expression of hemN was demonstrated in slot-blot and immunoblot experiments using antibodies raised against Histagged HemN. The data suggest that most of the transcripts originating at the potential vegetative promoter are terminated at the intergenic terminator. Readthrough transcription into the downstream dnaK operon was not found.
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The bgIX gene located at 47.8 min on the Escherichia coll chromosome encodes a periplasmic β-glucosidase
More LessA new Escherichia coli gene, bgIX, encoding a β-D-glucosidase (EC 3.2.1.21) has been characterized. The bgIX gene is located adjacent to the dld gene at 47.8 min or 2225 kb on the E. coli chromosome. The sequence of a 2.6 kb DNA fragment from this region revealed a large open reading frame encoding a protein of 765 amino acids. The BgIX protein was purified from the peripiasm, and amino-terminal sequencing suggests that the mature protein is derived from cleavage of a 20 residue signal peptide. A search of the sequence databases revealed that BgIX is a member of a large family of β-glucosidases from a variety of bacteria and fungi, and the plant Arabidopsis thaliana. It differs from the other four E. coli phospho-β-glucosidases in sequence and in its periplasmic rather than cytoplasmic location. The BgIX enzyme has a K m of 18 ± 1 mM and a V max of 3 ± 0.7 μmol min-1 for the colorimetric substrate o-nitrophenyl β-D-glucopyranoside.
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- Corrigendum
- Guidelines For Authors
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