- Volume 141, Issue 4, 1995
Volume 141, Issue 4, 1995
- Review Article
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- Microbiology Comment
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- Biochemistry
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A basic serine protease from Paecilomyces lilacinus with biological activity against Meloidogyne hapla eggs
Scanning electron micrographs of the nematode-egg-parasitic fungus Paecilomyces lilacinus infecting eggs of the root-knot nematode Meloidogyne spp. suggested the involvement of lytic enzymes. When grown on a liquid mineral salts medium, supplemented with different substrates as the sole N-and C-source, the fungus produced an extracellular protease. Colloidal chitin, vitellin and intact eggs of the root-knot nematode Meloidogyne hapla induced proteolytic activity that was repressed by glucose. The protease was partially purified from the culture filtrate by affinity chromatography. It has a molecular mass of 33·5 kDa, a pH optimum of 10·3, a temperature optimum of 60°C and an isoelectric point above pH 10·2. The enzyme was completely inhibited by PMSF. The amino acid sequence, as derived from the nucleotide sequence of a cDNA clone, had high homology with several subtilisin-like serine proteases. It was shown that the purified enzyme degrades vitellin. The protease quantitatively bound to nematode eggs, and eggs incubated with the purified protease eventually floated. Incubation of the purified protease with nematode eggs significantly influenced their development as demonstrated by time-lapse microscopy. Immature eggs were highly vulnerable to protease treatments, whereas those containing a juvenile were more resistant. In addition, hatched larvae were not visibly affected by the protease. It can be concluded that the serine protease might play a role in penetration of the fungus through the egg-shell of nematodes.
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Immunochemical, genetic and morphological comparison of fucosylation mutants of Dictyostelium discoideum
Mutations in three loci in Dictyostelium discoideum which affect fucosylation are described. Mutations in two of these loci resulted in the simultaneous loss of two separate carbohydrate epitopes. The GA-X epitope, which was competed by L-fucose, was absent in strains carrying a modC354, modD352 or modE353 mutation. These strains exposed a new carbohydrate epitope, competed by N-acetylglucosamine, and the size of several glycoproteins was reduced. A second epitope (GA-XII) was also absent in strains carrying the modC354 or modE353 mutations, reducing the size of the glycoprotein which normally expresses it. Fucose content was reduced in the three mutants, suggesting that each mutation affected a separate step in fucosylation. The lesions did not appear to inhibit synthesis of the underlying carbohydrate, because detergent extracts of mutant vesicles were more active than normal vesicles at transferring [14C]fucose from GDP-[14C]fucose to endogenous acceptor species. The modD352 and modE353 mutant strains incorporated exogenous [3H]fucose poorly, suggesting that lesions in the modD and modE genes interfere with the biosynthesis of fucoconjugates downstream from the previously described GDP-fucose synthesis defect of the modC mutation. Intac modE353 mutant vesicles were relatively inefficient in in vitro assays, suggesting a global fucosylation defect (which is consistent with the loss of both glycoantigens, GA-X and GA-XII, in this mutant). Finally, the modC354 mutation led to delayed ac}cumulation of slime sheath in vitro. The three genetic loci define a fucosylation pathway in D. discoideum comprising defined biochemical steps which contribute to multicellular morphogenesis in this organism.
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- Development And Structure
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Characterization of npf mutants identifying developmental genes in Physarum
In Physarum polycephalum , uninucleate haploid amoebae develop into macroscopic multinucleate plasmodia. Wild-type, sexual development is triggered when two amoebae carrying different alleles of matA fuse to form a zygote which develops into a diploid plasmodium. Mutations in the matA genetic region give rise to apogamic strains in which a single haploid amoeba can develop into a haploid plasmodium. An essential stage in both sexual and apogamic plasmodium formation is an extended cell cycle in uninucleate cells, which ends with the formation of a binucleate cell by mitosis without cytokinesis. Using a ‘brute force’ screening method, we have isolated mutants blocked in apogamic plasmodium development. Genetic analysis showed that the mutations we have identified were unlinked to matA , unlike mutations previously identified following an enrichment step. Most of the loci revealed by our screen were represented by only one allele, indicating that further screening should lead to the identification of additional genes required for plasmodium development. Phenotypic analysis showed that different mutants were blocked at different stages of plasmodium formation. Some of the mutations blocking apogamic development at an early stage, close to the start of the long cell cycle, failed to block sexual development in zygotes homozygous for the mutation. Since the two modes of plasmodium formation differ only in the initiation of development, these mutations presumably interfere with the initiation process. In the remaining mutants, in which both sexual and apogamic development were blocked, development first became abnormal towards the end of the long cell cycle. This suggested that the wild-type gene products were required by this time and was consistent with previous evidence that many changes in cellular organization and gene expression occur during the long cell cycle. Each of these mutants showed a different terminal phenotype and some aspects of plasmodium development occurred normally although others were blocked, suggesting that development involves multiple pathways rather than a dependent sequence of events. Phenotypic analysis of double mutants supported this conclusion and also revealed epistatic interactions, presumably due to blocks in the same pathway. In several of the mutants, terminally differentiated cells died by an apoptosis-like mechanism; since this was never observed in vegetative cells, it was presumably triggered by the failure of development. Phenotypic analyses of additional mutants will extend our understanding of the pathways involved in plasmodium development.
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S layer regeneration in Methanococcus voltae protoplasts
More LessThe regeneration of the paracrystalline proteinaceous cell wall (S layer) of the methanogen Methanococcus voltae was examined by electron microscopy. The S layer was removed from the cell surface by exposing the cells to a protoplasting buffer but was completely regenerated by 60-80 min resuspension in a regeneration medium by some repair mechanism (normal cell generation time is 16 h). The protoplast surface appeared in freeze-fracture to be initially smooth and featureless and remained so for the first 20 min in regeneration medium. Thereafter, nascent S layer appeared on the surface in the form of paracrystalline patches of various sizes that were distributed over the entire surface. The patches were closely associated with disturbances of the cell surface curvature which may have been caused by electrostatic interactions. The steps leading to the complete regeneration of the S layer could not be followed due to the sampling times used, but presumably the patches grew large enough to contact neighbouring patches and somehow fused into a coherent layer. Other ultrastructural changes occurred to the cell envelope during S layer regeneration, most notably the recurrence of the intramembrane particles of the concave fracture face of the plasma membrane. Flagella remained attached to the protoplasts and were apparently still functional. The major changes to the protein profile of untreated cells, protoplasts and regenerating protoplasts included the loss and resynthesis of polypeptides at the 76 kDa (S layer polypeptide) and 60 kDa (unknown polypeptide) positions. SDS-PAGE evidence indicated that new S layer polypeptides were synthesized 20 min prior to their appearance as patches on the cell surface. The S layer polypeptide may have been shed from the surface during the early stages of regeneration to form the extracellular sheet-like material observed in large quantity in the regeneration medium. The response of the cell to changes that occur at the cell surface indicates that some feedback mechanism gears the synthesis of S layer and associated envelope components.
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- Genetics And Molecular Biology
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Trichoderma harzianumgenes induced during growth onRhizoctonia solanicell walls
More LessTrichoderma harzianumis a biocontrol agent that attacks a range of economically important phytopathogenic fungi. In an attempt to identify genes specifically expressed by T. harzianum during growth on cell walls of Rhizoctonia solani, we carried out differential screening of an induced cDNA library. In this paper we report the analysis of the sequence and expression of two cDNA clones that encode putative mycoparasitism-related proteins of T. harzianum. One of these clones corresponds to a gene, inda 1, that encodes a protein of 570 amino acids with a predicted molecular mass of 62853 Da. The predictedamino acid sequence of inda1 showed a high degree of similarity with amino acid permeases from several other organisms. The other cDNA clone corresponds to a gene, indc11, that encodes a novel protein of 340 ami acids with a predicted molecular mass of 37010 Da. The use of this methodology should provide specific genetic markers to follow mycoparasitism by Trichoderma spp.
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Expression of the structural mox genes in Paracoccus denitrificans follows wild-type regulation in mutants with a deletion in mxaY, the gene encoding the signal sensor
More LessDuring growth on the C1 substrates methanol or methylamine, Paracoccus denitrificans is able to activate the expression of the genes encoding methanol dehydrogenase. In a previous paper the isolation of an operon containing two regulatory genes, mxaYX (formerly known as moxYX) and a third gene mxaZ (formerly known as moxZ) was described. MxaY and MxaX were shown to have homology with the signal sensors and the response regulators, respectively. Here we describe the isolation and characterization of mutants with marked and unmarked mutations in mxaZ, mxaY and mxaX. Expression of the structural mox genes was analysed by measuring the expression of a mxaF (moxF)-lacZ transcriptional fusion in the presence of mxaZ, mxaY, mxaX or combinations of these genes. Mutants that were unable to express mxaX were impaired for growth on methanol, did not synthesize MDH and could not express a mxaF-lacZ transcriptional fusion. This indicates that the response regulator MxaX is essential for expression of the structural mox genes. Mutants that had a deletion in mxaY or both mxaY and mxaZ were able to grow on methanol and were able to regulate the expression of the mxaF-lacZ fusion just like the wild-type. These findings indicate that mxaY + and mxaZ + are not essential for normal C1 regulation. In addition the results suggest that, at least in the absence of the signal sensor MxaY, MxaX can be activated via a different, but parallel, signal transduction pathway.
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Inducible expression of heterologous genes targeted to a chromosomal platform in the cyanobacterium Synechococcus sp. PCC 7942
More LessHigh-level, inducible expression of heterologous genes in the cyanobacterium Synechococcus sp. strain PCC 7942 was obtained using the Escherichia coli trc promoter and lacl repressor. The petE gene of Anabaena sp. strain PCC 7937 encoding plastocyanin precursor protein and the E. coli uidA gene encoding β-glucuronidase were initially placed under the control of the trc promoter and lacl repressor by cloning into the E. coli pTrc99C expression vector and were introduced into the chromosomal platform for integration in metF (PIM) of the Synechococcus R2-PIM9 recipient strain. These pTrc99C-derived constructs often gave rise to transformants that did not contain a complete insert gene, probably because of gene conversion events. Selection of the desired Synechococcus R2-PIM9 transformants was vastly improved using the new pTrcIS vector that contains the aadA gene encoding streptomycin resistance as an extra antibiotic resistance marker. The influence of IPTG concentration and induction time on gene expression with the E. coli trcllacl system in Synechococcus was determined using β-glucuronidase as a reporter. The Anabaena PCC 7937 petE gene in Synechococcus was expressed to a high level upon induction with IPTG as shown by RNA and immunoblot analysis. The general usability of pTrcIS as a cloning vector for inducible heterologous gene expression in Synechococcus was confirmed by the introduction of several more genes.
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IS292: a novel insertion element from Agrobacterium
More LessA new insertion sequence, IS292, located in the 6b gene of a nopaline-type Agrobacterium strain (X88-292) isolated from poplar was identified and sequenced. IS292 is 2494 bp long, has 21 bp inverted terminal repeats with two mismatches, and generates 10 bp direct repeats upon integration. No sequence similarity was found between IS292 and other insertion elements associated with Agrobacterium, but it shows strong similarity with ISRI1 from Rhizobium leguminosarum bv. viciae. The occurrence of IS292-like sequences in various Agrobacterium isolates, especially different Agrobacterium strains isolated from the same biotope, was demonstrated by DNA hybridization.
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Plasmids plP419 and plP421 from Bacteroides: 5-nitroimidazole resistance genes and their upstream insertion sequence elements
More LessSUMMERY:The genetic organization of two different 5-nitroimidazole (5-Ni) resistance genes was investigated: nimC and nimD from Bacteroides plasmids plP419 and plP421, respectively. The nimC gene (492 bp) and the nimD gene (495 bp) directed the synthesis of polypeptides with deduced molecular masses of 18·37 kDa and 18·48 kDa, respectively. The predicted proteins showed 67-83% identity and 78-91% similarity with the products of two other nimA and nimB genes previously described and could be derived from a common ancestral gene. An insertion sequence element (IS1170) was identified upstream of the nimC gene. IS1170 is 1604 bp in length and is flanked by imperfect inverted repeats (15 bp). IS 1170 is similar to the Bacteroides insertion sequence element IS942 with an identity of 70% at the nucleotide level. The single copy of IS 1170 present on plasmid plP419 is integrated 24 bp upstream of the initiation codon of nimC. Similar genetic organization was found on plasmid plP421. One copy of another insertion sequence (IS1169) was found 4 bp upstream of the first ATG codon of the nimD gene. This element (1325 bp) shows a strong homology at the nucleotide level (70% identity) with IS 1186 and IS 1168 found to be associated with the Bacteroides carbapenem resistance gene cfiA, and the 5-Nir genes nifiA and nimB, respectively. There is strong evidence that, as in the case of the cfiA gene, the transcription of the four nim genes so far studied is directed by outward-oriented promoters, carried on the right ends of the different insertion sequence elements.
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Variation in the size of the repeat region of the fibrinogen receptor (clumping factor) of Staphylococcus aureus strains
More LessSUMMERY:The ability of Staphylococcus aureus to bind to fibrinogen and fibrin is believed to be an important factor in the initiation of foreign body and wound infections. Recently, the gene encoding the fibrinogen receptor (clumping factor, ClfA) of S. aureus strain Newman was cloned and sequenced. The ClfA protein possesses a highly unusual 308 residue dipeptide repeat region composed predominantly of Asp and Ser. Polymerase chain reaction analysis of seven different strains showed that the size of the clfA repeat coding region varies from 580 bp to 1320 bp. In contrast, the clfA region A is the same size in each strain. The size of the clfA repeat region did not correlate with the ability of these strains to form clumps in a solution of fibrinogen. Indeed, the strain with the smallest repeat size of 580 bp clumped almost as well as strain Newman. Each strain of S. aureus examined contained several high molecular mass proteins that reacted with anti-ClfA region A antibody. In some cases the molecular mass of the major protein varied in accordance with the length of the coding sequence for the repeat region R.
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A gene region in Dichelobacter nodosus encoding a lipopolysaccharide epitope
More LessDichelobacter nodosus is a Gram-negative anaerobic bacterium that is the causative organism of footrot in sheep. A D. nodosus locus responsible for a modification of the host lipopolysaccharide (LPS) in Escherichia coli was cloned and sequenced. Genetic studies showed that the modification occurred within the inner-core region of the host LPS, most likely to one or more of the heptose molecules. Antibodies eluted from the modified LPS reacted preferentially with the lipid-A-core region of D. nodosus LPS, suggesting that the cloned epitope was present in this region of the D. nodosus LPS. The gene responsible for the modification, IpsA, potentially encoded a polypeptide of approximately 37 kDa which was highly basic, a characteristic of enzymes which interact with the acidic inner LPS core. The IpsA gene appeared to be arranged in a complex operon with a downstream gene, prfC, which encoded a protein with similarity to E. coli peptide-chain release factor 3.
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Novel phosphotransferase system genes revealed by bacterial genome analysis - a gene cluster encoding a unique Enzyme I and the proteins of a fructose-like permease system
More LessPrevious publications have demonstrated the presence of a cryptic gene encoding a novel Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Recent Escherichia coli genome sequencing revealed a gene (ptsA) encoding a new Enzyme I homologue in the 89.1-89.3 centisome region. We have analysed this region, and here describe and characterize open reading frames (ORFs) encoding (1) a fused PTS Enzyme I-IIAFru homologue, (2) a glycerol dehydrogenase, (3) a transaldolase homologue, (4) two PTS IIBFru homologues, (5) a PTS IICFru homologue, and (6) homologues of pyruvate formate-lyase and its activating enzyme. Binary comparison scores, multiple alignments and phylogenetic trees establish the families of proteins to which each of the relevant ORFs belong. Identification of the putative products of this gene cluster leads to the proposal that several of the proteins encoded in this region function in anaerobic carbon metabolism.
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Identification of a 60 kb region of the chromosome of Pseudomonas fluorescens NCIB 10586 required for the biosynthesis of pseudomonic acid (mupirocin)
Pseudomonic acid (mupirocin) produced by Pseudomonas fluorescens is a polyketide antibiotic which blocks isoleucyl-tRNA synthetase. Knowledge of the biosynthetic pathways leading to pseudomonic acid production may help in engineering related antibiotics with other useful properties. To help define these pathways, we have isolated 13 non-producing mutants using Tn5 and Tn1725 mutagenesis. Seven of the Tn5 insertions mapped within a 55 kb region. The remaining insertions, and one gene encoding resistance to pseudomonic acid, mapped at separate locations. DNA that overlapped the seven clustered Tn5 insertions was isolated on a series of clones, extending over a region in excess of 60 kb. Non-producing mutants generated via gene disruption suggest that the biosynthetic cluster extends throughout this region.
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A Clostridium acetobutylicum regulator gene (regA) affecting amylase production in Bacillus subtilis
More LessPlasmid pMET7C containing a 6.05 kb DNA insert from Clostridium acetobutylicum P262 made Escherichia coli F19 cells sensitive to metronidazole. The nucleotide sequence of the C. acetobutylicum DNA controlling metronidazole sensitivity in E. coli F19 revealed an ORF of 972 bp which encoded a protein of 324 amino acids with a calculated M r of 35000. The amino acid sequence encoded by the ORF contained a helix-turn-helix DNA-binding domain and was homologous to the catabolite control protein, CcpA, from Bacillus subtilis and Bacillus megaterium, a tRNA repressor of E. coli encoded by the shl gene, and the GaIR, LaCI and PurR repressors of E. coli. The C. acetobutylicum ORF, which was termed regA, complemented a B.subtilis ccpA mutant and an E. coli shl mutant, but was unable to complement E. coli galR, lacI or purR mutants. To determine whether the regA gene product was involved in the regulation of amylase gene expression in C. acetobutylicum, a starch-degrading enzyme gene (staA) from C. acetobutylicum NCIMB 8052 was cloned. The RegA protein inhibited the degradation of starch by the C. acetobutylicum staA gene product in E. coli.
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Streptomyces peucetius daunorubicin biosynthesis gene, dnrF: sequence and heterologous expression
The dnrF gene, responsible for conversion of aklavinone to ɛ-rhodomycinone via C-11 hydroxylation, was mapped in the daunorubicin (Dnr) gene cluster of Streptomyces peucetius ATCC 29050, close to drrAB, one of the anthracycline-resistance genes. The dnrF gene was sequenced and should encode a protein of 489 amino acids with a molecular mass of 52 kDa. The deduced DnrF protein shows significant similarities with bacterial FAD- and NADPH-dependent hydroxylases either required to introduce hydroxyl groups into polycyclic aromatic polyketide antibiotics or involved in catabolism of aromatic compounds. Heterologous expression of dnrF in Streptomyces lividans TK23 and in Escherichia coli demonstrated that the gene encodes a NADPH-dependent hydroxylase catalysing the hydroxylation of aklavinone to yield ɛ-rhodomycinone. The enzyme is inactive on anthracyclines glycosylated at position C-7 and its activity decreases to a different extent with other substrate modifications, indicating that DnrF has a significant substrate specificity.
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Lactococcus lactis glyceraldehyde-3-phosphate dehydrogenase gene, gap: further evidence for strongly biased codon usage in glycolytic pathway genes
More LessThe gene gap, encoding glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), was isolated from a genomic library of Lactococcus lactis LM0230 DNA. Plasmids containing the L. lactis gene were able to complement a gap mutant of Escherichia coli. The nucleotide sequence of gap predicted a polypeptide chain of 337 amino acids for the enzyme and a subunit molecular mass of 36043. The codon usage in gap and four other glycolytic genes from L. lactis showed a high degree of bias, when compared with 84 other chromosomal genes. Northern blot analysis of total L. lactis RNA showed that gap hybridized strongly with a 1·3 kb transcript. The 5' end of the transcript was determined by primer extension analysis to be a C located 35 bp upstream from the gap start codon. These transcript analyses, and the orientation of the open reading frames in the DNA flanking gap, indicated that in L. lactis gap is expressed on a monocistronic transcript. Nucleotide sequencing indicated that the DNA adjacent to gap did not encode other glycolytic pathway enzymes. The DNA sequence flanking gap contained two open reading frames (ORF156 and ORF211) of unknown function. The 3' end of a clpA homologue was identified in the sequence upstream of ORF156. The location of gap on the L. lactis DL11 chromosome map was determined to be between map coordinates 0·530 and 0·660.
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Biochemical genetics of a natural population of Escherichia coli: seasonal changes in alleles and haplotypes
More LessThe level of diversity, degree of enzyme polymorphism, effective population size, and the relative roles of drift and selection were examined in a cross-section of a natural Escherichia coli population based on random samples of haplotypes of E. coli isolated from sewage. The population studied contained E. coli strains derived from a human population of approximately 16000 individuals, as well as from other sources. Three sample sets were taken between May and August. Each set consisted of 100 E. coli clones. Six enzyme loci [GPI (5 alleles), GPD (5 alleles), PGD (10 alleles), ADH (8 alleles), IDH (6 alleles), PGM (6 alleles)] were surveyed electrophoretically for each clone; 159 different haplotypes were obtained and it is likely that all possible combinations are present in the population sampled. The large numbers of different haplotypes observed were distributed as a series of four genetically similar families of clones. The large estimated effective population size (N e = 1010) means that the observed large and highly significant changes in allele frequencies with time are not due to genetic drift. Selection, though not necessarily at the loci studied, is considered the only likely explanation.
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Mercury resistance as a selective marker for recombinant mycobacteria
More LessThe use of antibiotic-resistance markers for the selection of recombinant mycobacteria is widespread but questionable considering the development of live recombinant BCG vaccines. In contrast, vector-encoded resistance to heavy metals such as mercury may represent an interesting alternative for the development of live vaccines compatible with use in humans and in animals. The mercury resistance genes (mer) from Pseudomonas aeruginosa and from Serratia marcescens were cloned into the Escherichia coli-Mycobacterium shuttle vector pRR3. The resulting vectors, designated pMR001 and pVN2, were introduced by electroporation into Mycobacterium smegmatis, Mycobacterium bovis BCG and Mycobacterium tuberculosis. The recombinant mycobacteria were stable in vitro and in vivo, and had high-level mercury resistance, thus indicating that the mer genes can be useful as selective markers in mycobacteria.
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- Pathogenicity And Medical Microbiology
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Missense mutations that alter the DNA-binding domain of the MtrR protein occur frequently in rectal isolates of Neisseria gonorrhoeae that are resistant to faecal lipids
More LessSUMMERY:Resistance of Neisseria gonorrhoeae to structurally diverse hydrophobic agents (HAs) has been associated with missense or deletion mutations in the mtrR (m ultiple transferable r esistance R egulator) gene of laboratory-derived strains but their prevalence in clinical isolates was heretofore unknown. Since faecal lipids provide strong selective pressure for the emergence of variants resistant to HAs (HAR), the nucleotide sequence of the mtrR gene from rectal isolates of N. gonorrhoeae, which displayed different levels of HAR, was determined. Compared to the mtrR gene possessed by the HA-sensitive strain FA19, each clinical isolate contained mutations in the coding and/or promoter regions of their mtrR gene. A missense mutation in codon 45 (Gly-45 to Asp) was the most common mutation found in the strains studied and impacted the structure of the helix-turn-helix domain of the MtrR protein thought to be important in DNA-binding activity. Two clinical isolates bearing a missense mutation in codon 45 also contained a single basepair deletion in a 13 bp inverted sequence positioned within the mtrR promoter region. Introduction of mtrR sequences amplified from the clinical isolates into strain FA19 revealed that acquisition of the single basepair deletion was correlated with high level HAR while mutations in the mtrR-coding region provided for an intermediate level of HAR.
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Effect of sialylation of lipopolysaccharide of Neisseria gonorrhoeae on recognition and complement-mediated killing by monoclonal antibodies directed against different outer-membrane antigens
More LessGrowth of gonococci in the presence of CMP-N-acetylneuraminic acid (CMP-NANA) has previously been shown to induce resistance to the bactericidal effect of normal human serum and is accompanied by sialylation of the gonococcal lipopolysaccharide (LPS). We have used monoclonal antibodies (mAbs) to compare the effect of LPS sialylation on recognition of gonococci and complement-mediated killing by antibodies directed either against LPS or against defined epitopes on outer-membrane protein PI. Despite differences in binding to sialylated LPS on Western blots, all three mAbs directed against LPS showed considerably reduced binding to gonococci grown in the presence of CMP-NANA and a concomitant reduction in ability to promote complement-mediated killing. In contrast, mAbs directed against previously defined epitopes on a surface exposed loop of PI showed little difference in binding between sialylated and non-sialylated gonococci and promoted killing of the sialylated gonococci. Similarly a mAb directed against an epitope on a loop of the outer-membrane Rmp protein, which had previously been shown to block killing by antibodies directed against other surface antigens, also exerted a blocking effect with sialylated gonococci. Thus in the present study the continued biological effect of mAbs was correlated with the ability of the antibody to recognize surface-exposed epitopes on sialylated gonococci. Despite the presence of the sialylation which is likely to occur in vivo , it should be possible to induce complement-mediated killing by focusing the immune response to those surface-exposed epitopes which are least susceptible to the potential inhibitory effect of LPS sialylation.
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Characterization of the trypsin-like activity of Bacteroides forsythus
More LessBacteroides forsythus, a bacterial species frequently associated with disease periodontal sites, is known to possess trypsin-like activity. The present study was undertaken to determine the major characteristics of this activity. The trypsin-like activity was mainly found on the surface of the bacteria and could be solubilized with a zwitterionic detergent (Zwittergent 3-14). Using N-a-benzoyl-DL-arginine-p-nitroanilide as substrate, the optimum pH was between 7·5 and 8·5 and the optimum temperature was 35°C. The evidence suggests that the enzyme is a serine protease since it was strongly inhibited by diisopropylfluorophosphate (DFP), N-a-p-tosyl-L-lysine chloromethyl ketone hydrochloride, leupeptin and antipain. The B. forsythus trypsin-like enzyme cleaved numerous chromogenic synthetic peptides containing either an arginine or lysine bond, but could not hydrolyse native proteins including casein, gelatin and BSA. Incubation of a cell envelope extract of B. forsythus the presence of [3H]DFP, which is known to bind irreversibly to serine proteases, labelled two bands at 70 and 81 kDa following SDS-PAGE (under reducing conditions) and fluorography. It is suggested that the B. forsythus trypsin-like enzyme may be mainly involved in the degradation of small peptides resulting from hydrolysis of larger proteins by other oral bacteria.
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The bceT gene of Bacillus cereus encodes an enterotoxic protein
More LessA toxin gene (bceT) on a 2·9 kb DNA fragment of Bacillus cereus B-4ac was cloned and expressed in Escherichia coli, and its nucleotide sequence determined. The DNA fragment contained an open reading frame capable of encoding a polypeptide of 336 amino acids with a molecular mass of 41039 Da. The translated product in E. coli exhibited Vero cell cytotoxicity, and was positive in a vascular permeability assay. It also caused fluid accumulation in a ligated mouse ileal loop and was lethal to mice upon injection. These biological activities are considered characteristic of diarrhoeal enterotoxins. We therefore conclude that this gene, designated bceT, encodes one of the enterotoxic proteins of B. cereus which cause food-borne diarrhoea.
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- Physiology And Growth
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Evidence for cell-density-dependent regulation of catalase activity in Rhizobium leguminosarum bv. phaseoli
More LessPretreatment of Rhizobium leguminosarum bv. phaseoli cultures with low, non-lethal levels of H2O2 led to them becoming more resistant to killing by higher concentrations of this oxidant. The sensitivity of R. leguminosarum to H2O2-mediated oxidative stress varied with the growth phase of the cultures. Stationary phase cells were many times more resistant to killing by 3 mM H2O2 than exponentially growing cultures. Unexpectedly, the catalase activity of cultures was found to rise to a maximum in the early-exponential growth phase and rapidly fall to a minimum during late-exponential growth. Further investigation showed that the induction and subsequent repression of catalase activity in exponential cultures is a cell-density-dependent phenomenon which appears to be controlled by the accumulation of extracellular compound(s) in the growth medium at high cell densities. In this respect, control of catalase R. leguminosarum resembles a number of other cell-density-regulated phenomena in bacteria which are controlled by the accumulation of extracellular molecules: the best studied example of this quorum sensing is the control of bacterial bioluminescence by the lux autoinducer. Preliminary data indicated that this extracellular component is a non-proteinaceous, heat-stable molecule.
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A temperature-compensated ultradian clock ticks in Schizosaccharomyces pombe
More LessSUMMARY:An ultradian oscillation is described for Schizosaccharomyces pombe which meets the criteria for a cellular clock, i.e. timekeeping device. The rhythm can be induced by transfer from circadian conditions (stationary phase or very slow growth) to ultradian conditions (rapid growth). It can also be synchronized by ultradian temperature cycles of 6°C difference. Released to constant temperature, the rhythm persists for 20 h without damping. The period of the free-running rhythm is temperature-compensated and in no experiment did period length fall outside the narrow range between 40 and 44 min. The parameter observed is the septum index, i.e. the percentage of cells occupying the last stage of the cell cycle in wild-type cells before final division. The results suggest control of the cell division processes by the ultradian clock.
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Disruption of the actin cytoskeleton in budding yeast results in formation of an aberrant cell wall
More LessSUMMERY:A temperature-sensitive, conditionally lethal actin mutant of Saccharomyces cerevisiae, DBY 1693, was used to study, using light and electron microscopy, dysfunction of the actin cytoskeleton in the morphogenesis of the cell wall. Cells of this mutant strain survived at least 24 h at the restrictive temperature (37°C). These cells showed isodiametric growth. Mutant cells accumulated vesicles, probably as a consequence of chaotic secretory transport caused by loss of polarity. A conspicuous morphological response to the dysfunction of actin was the formation of an aberrant wall over the whole surface of the isodiametrically-growing cell. This wall was of loose texture with protruding glucan microfibrils incompletely masked with amorphous matrix. It resembled the regenerating cell wall on the surfaces of yeast protoplasts. The localization of wall synthesis over the whole surface of temperature sensitive actin mutant cells was in accordance with an even distribution of submembranous actin in the form of patches (similarly to regenerating protoplasts). Delocalization of finger-like invaginations of the plasma membrane from the bud region to the whole surface of the growing cell was also found in mutant cells.
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Bacillus subtilis levansucrase: the efficiency of the second stage of secretion is modulated by external effectors assisting folding
More LessWe investigated whether the concentration of H+ or metal ions such as Ca2+, or both, on the external side of the cytoplasmic membrane is involved in coupling of folding and secretion of Bacillus subtilis levansucrase by studying the modulation of each isolated event. In vitro at 30°C, in the absence of Ca2+, the equilibrium between the unfolded and the folded states of levansucrase was rapidly and totally displaced toward the folded state by a small pH shift from 7·4 to 6·0. Ca2+ (> 5 mM) acted as a catalyst of folding at pH ≥ 7. In vivo pulse-chase experiments at 30°C showed that, in the absence of Ca2+, the rate and the yield of the second step of levansucrase secretion were strongly dependent on the external pH. In acidic growth medium (pH 5·8), secretion was efficient. In contrast, at pH ≥ 7, the presence of Ca2+ was essential for secretion. In bacteria grown at high temperature (48°C), both external acidic pH and Ca2+ were required for efficient secretion. Moreover, a levansucrase variant altered in its calcium affinity was efficiently secreted only under acidic growth conditions. Depending on the culture conditions, the differences in H+ or Ca2+ concentrations which are maintained between the opposite sides of the energized cytoplasmic membrane could be adequate to catalyse conformational transition which could play a critical role in the second step of the protein release. These environmental parameters could also affect the yield of secretion of some other secretory proteins, leading to the hypothesis that several different secretion mechanisms could coexist in B. subtilis.
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Physiological roles of leupeptin and extracellular proteases in mycelium development of Streptomyces exfoliatus SMF13
More LessStreptomyces exfoliatus SMF13 produced leupeptin, chymotrypsin-like protease (CTP), metalloprotease, and trypsin-like protease (TLP) extracellularly. The activity of TLP was specifically inhibited by leupeptin. Production of leupeptin was closely associated with growth but leupeptin was inactivated by leupeptin-inactivating protein (LIP) when growth reached the stationary phase in submerged cultures, or when aerial mycelia started to form on surface cultures. Autolysis of mycelia after the stationary phase in submerged cultures was apparently retarded by the addition of leupeptin; on surface cultures, aerial mycelium formation was clearly retarded by the addition of leupeptin. We propose that CTP participates primarily in utilization of a proteinaceous nitrogen source, that TLP functions as an essential enzyme involved in the metabolism of mycelial protein, that leupeptin inhibits the activity of TLP and that LIP inactivates leupeptin. The cascade of regulatory actions of the compounds, which are produced sequentially during mycelium development, may provide selective advantages in adverse culture conditions.
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- Plant-Microbe Interactions
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Flavohaemoglobin HmpX: a new pathogenicity determinant in Erwinia chrysanthemi strain 3937
More LessUnlike wild-type Erwinia chrysanthemi strain 3937, which fully macerates inoculated Saintpaulia plants, HmpX- mutants produce necrotic lesions or no symptoms. The hmpX gene was sequenced and the corresponding protein sequence analysed. We show that HmpX belongs to a family of flavohaemoproteins (HMP), previously identified in two yeasts and in Escherichia coli. Comparisons of protein sequences at the secondary structure level by hydrophobic cluster analysis have shown that HmpX possesses two functional regions, a haemoglobin domain in its N-terminal part and a flavin reductase domain in its C-terminal part. In an HmpX- strain, the synthesis of pectate lyases, which are pathogenicity determinants in E. chrysanthemi, was reduced in conditions of low oxygen tension. Using gus fusion in hmpX, it was shown that hmpX transcription was induced in coculture with tobacco cells. A putative function for HmpX is discussed.
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Synergism between Erwinia pectate lyase isoenzymes that depolymerize both pectate and pectin
More LessSUMMERY:Phytopathogenic Erwinia bacteria cause tissue maceration by secretion of pectinolytic enzymes such as pectate lyase (PL). Sequencing of overlapping genomic fragments from Erwinia carotovora subsp. atroseptica established the organization of a 7·5 kbp region encoding PL isoenzymes. Two intergenic regions of 656 and 645 bp separate three enzyme coding regions of 1125 bp exhibiting approximately 80% positional identity. The promoters of each of the three genes contain a segment with high homology to the binding sequence of the E. chrysanthemi KdgR transcription repressor, implying similar mechanisms of gene regulation in the two bacterial species. Separate expression of the pel genes in the Escherichia coli -pT7-7 system and purification of their products yielded PLs at 7-33 mg (I culture)-1 with greater than 95% purity. Availability of the recombinant enzymes allowed determination of the kinetic differences amongst the PL isoforms, PL1, PL2 and PL3. The results show that PL is not strictly confined to depolymerization of pectate since each isoenzyme more readily degrades 31 % esterified pectin. Addition of isoenzyme combinations revealed no synergism with respect to degradation of pectate or 31% esterified pectin. However, addition of enzyme combinations containing PL3 enhanced the activity towards 68% esterified pectin, against which individual PL activities were low, by up to 64%. These data suggest that the combination of PL isoenzymes extends the range of pectic substrates which the bacterium can degrade.
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- Systematics
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Phylogenetic analysis of the ballistosporous anamorphic genera Udeniomyces and Bullera, and related basidiomycetous yeasts, based on 18S rDNA sequence
More LessSUMMERY:The small subunit nuclear ribosomal DNA (18S rDNA) sequence was determined for twelve species of basidiomycetous anamorphic yeasts, i.e. three species of Udeniomyces, seven species of Bullera, Cryptococcus albidus and Phaffia rhodozyma. For phylogenetic analysis, these sequences were aligned with published sequences for 36 other fungal species. Molecular phylogenetic analysis of maximum likelihood and parsimony showed that the 44 species of basidiomycetes analysed were divided into three major lineages. The ballistosporous yeast genera Udeniomyces and Bullera were clearly separated. On the phylogenetic tree, Udeniomyces megalosporus, U. puniceus and U. piricola showed a very close relationship with one another, and composed a lineage with Mrakia frigida, P. rhodozyma and Cystofilobasidium capitatum at high bootstrap confidence level. On the other hand, eight species of Bullera made lineages with selected species of Tremella (Tremellaceae), Filobasidium and Filobasidiella (Filobasidiaceae), Cryptococcus albidus and Trichosporon cutaneum. The molecular phylogeny deduced from the 18S rDNA sequence showed a possibility of heterogeneity among the species of Bullera at the generic level.
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- Genome Analysis
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Filling the gap between hns and adhE in Escherichia coli K12
More LessAs a consequence of the absence of a general coordination process in the sequencing of the Escherichia coli K12 genome, to completely sequence the genome in a reasonable time it is important to fill in gaps between known regions. We report the sequence of the hns-adh region, at 27 min on the genome.
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