- Volume 147, Issue 4, 2001
Volume 147, Issue 4, 2001
- Review Article
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- Antigens And Immunity
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Antibody response against Escherichia coli heat-stable enterotoxin expressed as fusions to flagellin
More LessThe heat-stable toxin (ST) produced by enterotoxigenic Escherichia coli strains causes diarrhoea by altering the fluid secretion in intestinal epithelial cells. Here, the effectiveness of a flagellin fusion protein of Salmonella containing a 19-amino-acid sequence derived from the ST sequence (FLA–ST) in generating antibodies capable of neutralizing the toxic activity of ST was evaluated. This fusion protein, and an alternative construction where two cysteine residues in the ST sequence were substituted by alanines (STmt), were delivered to the immune system by three distinct strategies: (i) orally, using an attenuated Salmonella strain expressing FLA–ST; (ii) intraperitoneally, by injection of purified FLA–ST; (iii) orally, using attenuated Salmonella carrying a eukaryotic expression plasmid (pCDNA3) with the gene encoding FLA–ST. The results showed that the flagellin system can be used as a carrier to generateST-neutralizing antibodies. However, it should be mentioned that humoral immune response against ST was only obtained when the mutated ST sequence was employed. FLA–ST was found to be non-immunogenic when delivered via the oral route with attenuated Salmonella strains. However, a flagellin antibody response was obtained by immunizing mice with Salmonella carrying pCDNA3/FLA-STmt. Oral immunization with Salmonella carrying the eukaryotic expression plasmid (pCDNA3/FLA–STmt) seems to be a promising method to elicit an appropriate response against fusions to flagellin.
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- Biochemistry
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Clostridium botulinum type A haemagglutinin-positive progenitor toxin (HA+-PTX) binds to oligosaccharides containing Galβ1-4GlcNAc through one subcomponent of haemagglutinin (HA1)
Haemagglutinin (HA) activity of Clostridium botulinum type A 19S and 16S toxins (HA-positive progenitor toxin; HA+-PTX) was characterized. HA titres against human erythrocytes of HA+-PTX were inhibited by the addition of lactose, D-galactose, N-acetyl-D-galactosamine and D-fucose to the reaction mixtures. A direct glycolipid binding test demonstrated that type A HA+-PTX strongly bound to paragloboside and some neutral glycolipids, but did not bind to gangliosides. Type A HA+-PTX also bound to asialoglycoproteins (asialofetuin, neuraminidase-treated transferrin), but not to sialoglycoproteins (fetuin, transferrin). Although glycopeptidase F treatment of asialofetuin abolished the binding of HA+-PTX, endo-α-N-acetylgalactosaminidase treatment did not. Thus these results can be interpreted as indicating that type A HA+-PTX detects and binds to Galβ1-4GlcNAc in paragloboside and the N-linked oligosaccharides of glycoproteins. Regardless of neuraminidase treatment, type A HA+-PTX bound to glycophorin A which is a major sialoglycoprotein on the surface of erythrocytes. Both native glycophorin A and neuraminidase-treated glycophorin A inhibited the binding of erythrocytes to type A HA+-PTX. Since the N-linked oligosaccharide of glycophorin A is di-branched and more than 50% of this sugar chain is monosialylated, type A HA+-PTX probably bound to the unsialylated branch of the N-linked oligosaccharide of glycophorin A and agglutinated erythrocytes. One subcomponent of HA, designated HA1, did not agglutinate native erythrocytes, although it did bind to erythrocytes, paragloboside and asialoglycoproteins in a manner quite similar to that of HA+-PTX. These results indicate that type A HA+-PTX binds to oligosaccharides through HA1.
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- Bioenergetics And Transport
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Intracellular pH regulation by Mycobacterium smegmatis and Mycobacterium bovis BCG
More LessMycobacteria are likely to encounter acidic pH in the environments they inhabit; however intracellular pH homeostasis has not been investigated in these bacteria. In this study, Mycobacterium smegmatis and Mycobacterium bovis [Bacille Calmette–Guérin (BCG)] were used as examples of fast- and slow-growing mycobacteria, respectively, to study biochemical and physiological responses to acidic pH. M. smegmatis and M. bovis BCG were able to grow at pH values of 4·5 and 5·0, respectively, suggesting the ability to regulate internal pH. Both species of mycobacteria maintained their internal pH between pH 6·1 and 7·2 when exposed to decreasing external pH and the maximum ΔpH observed was approximately 2·1 to 2·3 units for both bacteria. The ΔpH of M. smegmatis at external pH 5·0 was dissipated by protonophores (e.g. carbonyl cyanide m-chlorophenylhydrazone), ionophores (e.g. monensin and nigericin) and N,N′-dicyclohexylcarbodiimide (DCCD), an inhibitor of the proton-translocating F1F0-ATPase. These results demonstrate that permeability of the cytoplasmic membrane to protons and proton extrusion by the F1F0-ATPase plays a key role in maintaining internal pH near neutral. Correlations between measured internal pH and cell viability indicated that the lethal internal pH for both strains of mycobacteria was less than pH 6·0. Compounds that decreased internal pH caused a rapid decrease in cell survival at acidic pH, but not at neutral pH. These data indicate that both strains of mycobacteria exhibit intracellular pH homeostasis and this was crucial for the survival of these bacteria at acidic pH values.
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- Development And Structure
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Frozen motion of gliding bacteria outlines inherent features of the motility apparatus
More LessHigh-resolution data of actively gliding wild-type bacteria of four different species and of four different gliding mutants of Myxococcus xanthus were obtained from scanning electron micrographs. By shock freezing and freeze drying, motility-associated surface patterns could be fixed and consequently distinct intermediate states of motion could be observed for the first time. It is shown that these topographic patterns are immediately lost when gliding motility is stopped by blocking the respiratory chain with potassium cyanide or sodium azide. From the surface topography, the mode of action of the gliding apparatus of all four bacterial species examined can be described as a twisted circularly closed ‘band’. During gliding, groups of nodes of the supertwisted apparatus show evidence of travelling like waves along the trichomes. However, the spacing between the nodes is not constant but varies within a certain range. This indicates that they are flexibly modulated as a consequence of the gliding state of the individual trichome.
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Defining the mycoplasma ‘cytoskeleton’: the protein composition of the Triton X-100 insoluble fraction of the bacterium Mycoplasma pneumoniae determined by 2-D gel electrophoresis and mass spectrometry
More LessAfter treating Mycoplasma pneumoniae cells with the nonionic detergent Triton X-100, an undefined, structured protein complex remains that is called the ‘Triton X-100 insoluble fraction’ or ‘Triton shell’. By analogy with eukaryotic cells and supported by ultrastructural analyses it is supposed that this fraction contains the components of a bacterial cytoskeleton-like structure. In this study, the composition of the Triton X-100 insoluble fraction was defined by electron microscopic screening for possible structural elements, and by two-dimensional (2-D) gel electrophoresis and MS to identify the proteins present. Silver staining of 2-D gels revealed about 100 protein spots. By staining with colloidal Coomassie blue, about 50 protein spots were visualized, of which 41 were identified by determining the mass and partial sequence of tryptic peptides of individual proteins. The identified proteins belonged to several functional categories, mainly energy metabolism, translation and heat-shock response. In addition, lipoproteins were found and most of the proteins involved in cytadherence that were previously shown to be components of the Triton X-100 insoluble fraction. There were also 11 functionally unassigned proteins. Based on sequence-derived predictions, some of these might be potential candidates for structural components. Quantitatively, the most prevalent proteins were the heat-shock protein DnaK, elongation factor Tu and subunits α and β of the pyruvate dehydrogenase complex (PdhA, PdhB), but definite conclusions regarding the composition of the observed structures can only be drawn after specific proteins are assigned to them, for example by immunocytochemistry.
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- Environmental Microbiology
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Transcriptional regulation of styrene degradation in Pseudomonas putida CA-3
More LessThe GenBank accession number for the sequence determined in this work is AF257095.
The styrene degradative pathway in Pseudmonas putida CA-3 has previously been shown to be divided into an upper pathway involving the conversion of styrene to phenylacetic acid and a lower pathway for the subsequent degradation of phenylacetic acid. It is reported here that expression of the regulatory genes styS and styR is essential for transcription of the upper pathway, but not for degradation of the lower pathway inducer, phenylacetic acid. The presence of phenylacetic acid in the growth medium completely repressed the upper pathway enzymes even in the presence of styrene, the upper pathway inducer. This repression is mediated at the transcription level by preventing expression of the styS and styR regulatory genes. Finally, an examination was made of the various stages of the diauxic growth curve obtained when P. putida CA-3 was grown on styrene together with an additional carbon source and it is reported that catabolite repression may involve a different mechanism to transcriptional repression by an additional carbon source.
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- Genetics And Molecular Biology
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Feedback control of polyketide metabolism during tylosin production
More LessTylosin is produced by Streptomyces fradiae via a combination of polyketide metabolism and synthesis of three deoxyhexose sugars, of which mycaminose is the first to be added to the polyketide aglycone, tylactone (protylonolide). Previously, disruption of the gene (tylMII) encoding attachment of mycaminose to the aglycone unexpectedly abolished accumulation of the latter, raising the possibility of a link between polyketide metabolism and deoxyhexose biosynthesis in S. fradiae. However, at that time, it was not possible to eliminate an alternative explanation, namely, that downstream effects on the expression of other genes, not involved in mycaminose metabolism, might have contributed to this phenomenon. Here, it is shown that disruption of any of the four genes (tylMI–III and tylB) specifically involved in mycaminose biosynthesis elicits a similar response, confirming that production of mycaminosyl-tylactone directly influences polyketide metabolism in S. fradiae. Under similar conditions, when mycaminose biosynthesis was specifically blocked by gene disruption, accumulation of tylactone could be restored by exogenous addition of glycosylated tylosin precursors. Moreover, certain other macrolides, not of the tylosin pathway, were also found to elicit qualitatively similar effects. Comparison of the structures of stimulatory macrolides will facilitate studies of the stimulatory mechanism.
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The signal transducer (BlaRI) and the repressor (BlaI) of the Staphylococcus aureus β-lactamase operon are inducible
More LessThe precise start points for transcription of the blaZ and of the blaRI/blaI genes of the Staphylococcus aureus β-lactamase operon have been determined by primer extension analysis. Consequently the overlapping promoter sequences were deduced. Northern blots showed that the synthesis of the 2100 nt mRNA from blaRI is inducible and that a blaI probe hybridized to the same mRNA as the blaRI probe. The gene cat, encoding chloramphenicol acetyltransferase, was fused separately to the blaZ and blaRI/blaI promoters, and used to compare their strengths. The promoter for blaZ is about six times stronger than that for blaRI/blaI and the synthesis of chloramphenicol acetyltransferase from both promoters is inducible, supporting the results from the Northern blots.
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Surface exposure and protease insensitivity of Borrelia burgdorferi Erp (OspEF-related) lipoproteins
Borrelia burgdorferi can encode numerous lipoproteins of the Erp family. Although initially described as outer surface proteins, the technique used in that earlier study has since been demonstrated to disrupt bacterial membranes and allow labelling of subsurface proteins. Data are now presented from additional analyses indicating that Erp proteins are indeed surface exposed in the outer membrane. Surface localization of these infection-associated proteins indicates the potential for interactions of Erp proteins with vertebrate tissues. Some Erp proteins were resistant to in situ digestion by certain proteases, suggesting that those proteins fold in manners which hide protease cleavage sites, or that they interact with other protective membrane components. Additionally, cultivation of B. burgdorferi in the presence of antibodies directed against Erp proteins inhibited bacterial growth.
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Type IV O antigen modification genes in the genome of Shigella flexneri NCTC 8296
More LessThe GenBank accession number for the sequence reported in this paper is AF288197.
The genes encoding type IV O antigen glucosylation were characterized from both Escherichia coli and Shigella flexneri. The putative O antigen modification genes from E. coli, o120 o306 o443, were PCR-amplified and introduced into S. flexneri serotype Y strain SFL124. Immunogold labelling and phage sensitivity indicated the presence of both serotype Y and serotype 4a O antigens on the cell surface of the resulting recombinant SFL124 strains, suggesting that only partial serotype conversion was conferred by the E. coli genes. The type IV O antigen modification genes were then isolated and characterized from S. flexneri serotype 4a strain NCTC 8296. A 3·8 kb chromosomal fragment conferred complete conversion to serotype 4a when introduced into SFL124. Sequence analysis of the fragment revealed the presence of three genes, gtrA IV gtrB IV gtrIV Sf. DNAs homologous to bacteriophage int and attP were located upstream of gtrA IV, suggesting that this region of the NCTC 8296 genome may have originated from a bacteriophage; however, a serotype-converting phage could not be induced from this strain nor from other strains used in this study. Comparison of the GtrIVSf and GtrIVEc (o443) proteins revealed that they are 41% identical and 63% similar, which is the highest degree of similarity reported among the S. flexneri O antigen glucosyltransferases.
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Detergent-independent in vitro activity of a truncated Bacillus signal peptidase
The Gram-positive eubacterium Bacillus subtilis contains five chromosomally encoded type I signal peptidases (SPases) for the processing of secretory pre-proteins. Even though four of these SPases, denoted SipS, SipT, SipU and SipV, are homologous to the unique SPase I of Escherichia coli, they are structurally different from that enzyme, being almost half the size and containing one membrane anchor instead of two. To investigate whether the unique membrane anchor of Bacillus SPases is required for in vitro activity, soluble forms of SipS of B. subtilis, SipS of Bacillus amyloliquefaciens and SipC of the thermophile Bacillus caldolyticus were constructed. Of these three proteins, only a hexa-histidine-tagged soluble form of SipS of B. amyloliquefaciens could be isolated in significant quantities. This protein displayed optimal activity at pH 10, which is remarkable considering the fact that the catalytic domain of SPases is located in an acidic environment at the outer surface of the membrane of living cells. Strikingly, in contrast to what has been previously reported for the soluble form of the E. coli SPase, soluble SipS was active in the absence of added detergents. This observation can be explained by the fact that a highly hydrophobic surface domain of the E. coli SPase, implicated in detergent-binding, is absent from SipS.
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The Bacillus subtilis yabQ gene is essential for formation of the spore cortex
More LessAn extensive screening for transcripts with probes specific for the genes in a 108 kb region from rrnO to spo0H of the Bacillus subtilis chromosome led to identification of an operon, yabP–yabQ–divIC–yabR, the expression of which was initiated at the second hour of sporulation and in a σE-dependent manner. Among three y genes in the operon, deletion of the yabQ gene, which is predicted to encode a protein product of 468 residues with five membrane-spanning domains, resulted in a large decrease in numbers of chloroform-, lysozyme- and heat-resistant spores, compared to findings with the wild-type strain. Electron microscopy revealed that development of the spore cortex was blocked in the yabQ mutant. In addition, although the spore coat was visible, the inner coat layer of the mutant seemed partially detached from the outer coat. In sporangia of the strains harbouring an in-frame fusion of the green fluorescent protein gene to yabQ, fluorescence was detected around the forespore. This localization did not depend on SpoIVA or on CotE functions, both of which determine proper localization of coat proteins and cortex formation. The yabQ deletion did not affect expression of genes involved in cortex synthesis. These results suggest that the YabQ protein localizes in the membrane of the forespore and plays an important role in cortex formation.
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New members of the ctrA regulon: the major chemotaxis operon in Caulobacter is CtrA dependent
More LessThe Caulobacter crescentus che promoter region consists of two divergent promoters, directing expression of the major chemotaxis operon and a novel gene cagA (chemotaxis associated gene A). Analyses of start sites by primer extension and alignment of the divergent promoters revealed significant similarities between them at the −35 promoter region. Both mcpA and cagA are differentially expressed in the cell cycle, with maximal activation of transcription in predivisional cells. The main difference between the mcpA and cagA promoters is that, in common with the fljK flagellin, cagA is expressed in swarmer cells. A cagA–lacZ promoter fusion that contains 36 bases of untranslated mRNA has sufficient information to segregate the lacZ transcript to swarmer cells. Expression of mcpA and cagA was dependent on DNA replication. Transcriptional epistasis experiments were performed to identify potential regulators in the flagellar hierarchy. The sigma factor RpoN, which is required for flagellar biogenesis, is not required for mcpA and cagA expression. Mutations in the genes for the MS-ring and the switch complex (flagellar class II mutants) do not affect expression of mcpA and cagA. However, CtrA, an essential response regulator of flagellar gene transcription, is required.
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A new simvastatin (mevinolin)-resistance marker from Haloarcula hispanica and a new Haloferax volcanii strain cured of plasmid pHV2
More LessThe GenBank accession number for the sequence reported in this paper is AF123438.
The mevinolin-resistance determinant, hmg, encodes the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and is a commonly used selectable marker in halobacterial genetics. Plasmids bearing this marker suffer from instability in Haloferax volcanii because the resistance gene was derived from the genome of this species and is almost identical in sequence to the chromosomal copy. In order to reduce the level of homologous recombination between introduced plasmid vectors and the chromosome of Haloferax, a homologue of the hmg determinant was obtained from the distantly related organism, Haloarcula hispanica. The nucleotide sequences of the wild-type genes (hmgA) of these two species are only 78% identical, and the predicted protein sequences show 71% identity. In comparison to the wild-type hmgA gene, the resistance gene from a mutant resistant to simvastatin (an analogue of mevinolin) showed a single base substitution in the putative promoter. Plasmids constructed using the new resistance determinant were stably maintained under selection in Hfx. volcanii and possessed very low recombination rates with the chromosome of this species. In addition, an improved strain of Hfx. volcanii was developed to overcome the plasmid instability and growth reduction observed in the commonly used WFD11 strain.
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The product of the ybdE gene of the Escherichia coli chromosome is involved in detoxification of silver ions
More LessTranscription of the ybcZ–ylcA ylcBCD–ybdE region of the Escherichia coli K38 chromosome was analysed by Northern RNA–DNA hybridization, RT-PCR and primer extension. Transcription of a dicistronic ybcZ–ylcA mRNA and a tetracistronic ylcBCD–ybdE mRNA was induced by silver and was initiated from the sigma-70 promoters ylcAp and ylcBp. Expression of β-galactosidase activity from a Φ(ylcBp–lacZ) operon fusion was also induced by Ag+ and Cu2+, but not by Zn2+. In-frame deletion of ybdE from the chromosome yielded a silver-sensitive E. coli mutant strain which did not differ in its copper resistance from its wild-type strain. On the other hand, deletion of the copA gene for the copper-exporting P-type ATPase CopA resulted in copper sensitivity, but not in silver sensitivity. A ΔybdE ΔcopA double mutant strain behaved towards copper as the ΔcopA strain and towards silver as the ΔybdE strain. Thus, in E. coli, the YlcBCD–YbdE system may be involved in silver- but not in copper resistance, and CopA may be involved in copper- but not in silver resistance.
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Topological investigations of the FomA porin from Fusobacterium nucleatum and identification of the constriction loop L6
More LessThe GenBank accession number for the sequence reported in this paper is X72583.
Porin FomA in the outer membrane of Fusobacterium nucleatum is a trimeric protein, which exhibits permeability properties similar to that of the well-known enterobacterial diffusion porins. The proposed topology model of the FomA monomer depicts the β-barrel motif typical of diffusion porins, consisting of 16 antiparallel β-strands. To investigate the accuracy of the FomA model and assess the topological relationship with other porins, individual deletions of variable size in seven of the eight surface-exposed regions of the porin were genetically engineered. Deletions in the predicted loops L1 to L7 were tolerated by the FomA porins, as judged by a normal assembly in the outer membrane of Escherichia coli and a sustained pore-forming ability. Deletions in the largest proposed external region, loop L6, made the FomA porins considerably more permeable to antibiotics, indicating larger pore channels. The distinctly increased uptake rates and size exclusion limits displayed by the L6 deletion mutant porins, suggest that loop L6 folds back into the β-barrel thereby constricting the native FomA channel. Thus, the position of the channel constriction loop appears to be shifted towards the C terminus in the FomA porin, as compared to the crystal structures of five non-specific diffusion porins.
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The gene yghK linked to the glc operon of Escherichia coli encodes a permease for glycolate that is structurally and functionally similar to L-lactate permease
More LessIn Escherichia coli the glc operon involved in glycolate utilization is located at 67·3 min and formed by genes encoding the enzymes glycolate oxidase (glcDEF) and malate synthase G (glcB). Their expression from a single promoter upstream of glcD is induced by growth on glycolate and regulated by the activator encoded by the divergently transcribed gene glcC. Gene yghK, located 350 bp downstream of glcB, encodes a hydrophobic protein highly similar to the L-lactate permease encoded by lldP. Expression studies have shown that the yghK gene (proposed name glcA) is transcribed from the same promoter as the other glc structural genes and thus belongs to the glc operon. Characterization of a glcA::cat mutant showed that GlcA acts as glycolate permease and that glycolate can also enter the cell through another transport system. Evidence is presented of the involvement of L-lactate permease in glycolate uptake. Growth on this compound was abolished in a double mutant of the paralogous genes glcA and lldP, and restored with plasmids expressing either GlcA or LldP. Characterization of the putative substrates for these two related permeases showed, in both cases, specificity for the 2-hydroxymonocarboxylates glycolate, L-lactate and D-lactate. Although both GlcA and LldP recognize D-lactate, mutant analysis proved that L-lactate permease is mainly responsible for its uptake.
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- Pathogenicity And Medical Microbiology
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Analysis of the role of flagellar activity in virulence gene expression in Vibrio cholerae
More LessExpression of virulence factors and motility of Vibrio cholerae are intimately linked by an as yet uncharacterized mechanism. Several lines of evidence indicate that the activity of the flagellum of V. cholerae might have an impact on virulence gene regulation, as alterations of the motility phenotype, either by mutation or by inhibitory drugs, result in varied levels of virulence factor production. The Na+-driven polar flagella of some Vibrio species are proposed to act as mechanosensors, sensing media viscosity. It has been suggested that the V. cholerae flagellum might act as a ‘voltmeter’, responding to changes in membrane potential, or might sense some environmental conditions that lead to the repression of virulence factors in V. cholerae. To test these hypotheses, β-galactosidase levels of several types of non-motile mutant derivatives of a V. cholerae toxT::lacZ reporter strain were analysed following changes in media viscosity, membrane potential and other environmental conditions. Like the parental strain, the non-motile strain showed increased toxT::lacZ expression in high-viscosity media, suggesting that the sensing of media viscosity does not occur via the flagella. Other molecules that might be able to detect changes in media viscosity could include mechanosensitive (MS) ion channels found in the bacterial membrane. However, a V. cholerae derivative strain mutated in two putative MS channels still showed increased toxT::lacZ expression in high-viscosity media, indicating that these putative ion channels of V. cholerae are not involved in the viscosity effect and suggesting an as yet uncharacterized mechanism for sensing of media viscosity. The flagellum does not appear to act as a voltmeter, as β-galactosidase activities of the non-flagellate derivative strain were found to be similar to those of the parental strain after artificially changing the sodium membrane bioenergetics. Similarly, several environmental conditions known to reduce toxin expression were equally effective in reducing toxT::lacZ expression in the motile or non-motile strains. In conclusion, the flagellum of V. cholerae does not act as a mechanosensor, voltmeter or signal transducer for environmental conditions. Thus, alternative mechanisms for the detection of these conditions must exist that likely do not involve the ToxR molecule, as the sensing of all of the tested parameters occurred when the TcpP/H proteins alone activated the toxT::lacZ reporter gene.
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Antigenic variation of gonococcal pilin expression in vivo: analysis of the strain FA1090 pilin repertoire and identification of the pilS gene copies recombining with pilE during experimental human infection
More LessThe GenBank accession numbers for the sequences reported in this paper are U58840–U58851, U61269, L28978, L28980, L28987 and L28990–L28992.
Antigenic variation of gonococcal pilin involves a family of variable genes that undergo homologous recombination, resulting in transfer of variant sequences from the pilS silent gene copies into the complete pilE expression locus. Little is known about the specific recombination events that are involved in assembling new variant pilin genes in vivo. One approach to understanding pilin variation in vivo is to carry out experimental human infections with a gonococcal strain having a fully characterized repertoire of pilin genes, so that the specific recombination events occurring in vivo can be determined. To this end, the authors cloned, sequenced and mapped the pilin genes of strain FA1090 of Neisseria gonorrhoeae. This strain contains one pilE locus and 19 silent gene copies that are arranged in five pilS loci; the pilE locus and four of the pilS loci are clustered in a 35 kb region of the chromosome. The general features of the pilin loci in FA1090 are similar to those in strain MS11, in which the mechanism of pilin variation has been extensively studied. However, none of the silent copy sequences are identical in the two strains, which emphasizes the extreme variability in this gene family among gonococci. Three male volunteers were inoculated with the same variant of strain FA1090 and developed urethritis within 2–4 d. The pilE gene sequences from a total of 23 colonies cultured from the subjects were analysed, determining which pilS silent copy donated each portion of the expressed pilE genes. There were 12 different pilin variants, one of which was the original inoculum variant, among the in vivo-expressed pilE gene sequences. The pilE of the inoculum variant was derived entirely from a single silent copy (pilS6c1). However, the pilE genes in the majority of the colonies cultured from the infected subjects were chimeras of sequence derived from two or three silent copies. Recombination to generate new pilE sequences involved exchange of single variable minicassettes, multiple minicassettes, entire silent gene copies, or (rarely) recombination within a minicassette.
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Differences in sialic acid density in pathogenic and non-pathogenic Aspergillus species
More LessAspergillus fumigatus is a ubiquitous soil fungus that causes invasive lung disease in the immunocompromised host. The structure of the conidial wall has not been well characterized although it is thought that adhesins present on the surface are involved in attachment of the conidia to host lung cells and proteins, which is a prerequisite for the establishment of infection. Negatively charged carbohydrates on the conidial surface have been previously identified as the molecules responsible for attachment of conidia to extracellular matrix proteins. The aim of this research was to identify carbohydrates on the conidial surface that contribute to its negative charge. Direct chemical analysis and indirect binding assays have demonstrated that A. fumigatus possesses sialic acids on the conidial surface. Pre-treatment of A. fumigatus conidia with sialidase decreased binding of a sialic acid-specific lectin, Limax flavus agglutinin (LFA), to the conidial surface and decreased adhesion of conidia to the positively charged polymer poly L-lysine. Two other sialic acid-specific lectins, Maackia amurensis agglutinin and Sambucus nigra agglutinin, exhibited negligible binding to A. fumigatus conidia indicating that 2,3-α- and 2,6-α-linked sialic acids are not the major structures found on the conidial surface. Mild acid hydrolysis and purification of conidial wall carbohydrates yielded a product that had the same R F as the Neu5Ac standard when analysed by high-performance thin-layer chromatography. A density of 6·7×105 sialic acid residues per conidium was estimated using a colorimetric assay. Conidia grown on a minimal medium lacking sialic acid also reacted with LFA, indicating that sialic acid biosynthesis occurs de novo. Sialic acid biosynthesis was shown to be regulated by nutrient composition: the density of sialic acids on the surface of conidia grown in minimal media was lower than that observed when conidia were grown on rich, complex media. It has previously been shown that pathogenic Aspergillus species adhere to basal lamina proteins to a greater extent than non-pathogenic Aspergillus species. To determine whether the expression of sialic acid on the conidial surface was correlated with adhesion to basal lamina, conidia from other non-pathogenic Aspergillus species were tested for their reactivity towards LFA. Flow cytometric analysis demonstrated that A. fumigatus had a significantly greater sialic acid density than three non-pathogenic Aspergillus species. Sialic acids on the conidial wall may be involved in adhesion to fibronectin, a component of the basal lamina, as binding of A. fumigatus conidia to fibronectin was strongly inhibited in the presence of a sialylated glycoprotein.
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The role and relevance of phospholipase D1 during growth and dimorphism of Candida albicans
More LessThe phosphatidylcholine-specific phospholipase D1 (PLD1) in Saccharomyces cerevisiae is involved in vesicle transport and is essential for sporulation. The gene encoding the homologous phospholipase D1 from Candida albicans (PLD1) was used to study the role of PLD1 in this pathogenic fungus. In vitro and in vivo expression studies using Northern blots and reverse transcriptase-PCR showed low PLD1 mRNA levels in defined media supporting yeast growth and during experimental infection, while enhanced levels of PLD1 transcripts were detected during the yeast to hyphal transition. To study the relevance of PLD1 during yeast and hyphal growth, an essential part of the gene was deleted in both alleles of two isogenic strains. In vitro PLD1 activity assays showed that pld1 mutants produced no detectable levels of phosphatidic acid, the hydrolytic product of PLD1 activity, and strongly reduced levels of diacylglycerol, the product of lipid phosphate phosphohydrolase, suggesting no or a negligible background PLD1 activity in the pld1 mutants. The pld1 mutants showed no growth differences compared to the parental wild-type in liquid complex and minimal media, independent of the growth temperature. In addition, growth rates of pld1 mutants in media with protein as the sole source of nitrogen were similar to growth rates of the wild-type, indicating that secretion of proteinases was not reduced. Chlamydospore formation was normal in pld1 mutants. When germ tube formation was induced in liquid media, pld1 mutants showed similar rates of yeast to hyphal transition compared to the wild-type. However, no hyphae formation was observed on solid Spider medium, and cell growth on cornmeal/Tween 80 medium indicated aberrant morphogenesis. In addition, pld1 mutants growing on solid media had an attenuated ability to invade the agar. In a model of oral candidosis, pld1 mutants showed no attenuation of virulence. In contrast, the mutant was less virulent in two different mouse models. These data suggest that PLD1 is not essential for growth and oral infections. However, they also suggest that a prominent part of the phosphatidic acid and diacylglycerol pools is produced by PLD1 and that the level of these components is important for morphological transitions under certain conditions in C. albicans.
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- Physiology And Growth
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Motions caused by the growth of Bacillus subtilis macrofibres in fluid medium result in new forms of movement of the multicellular structures over solid surfaces
More LessBacillus subtilis macrofibres, highly ordered multicellular structures, undergo twisting and writhing motions when they grow in fluid medium as a result of forces generated by the elongation of individual cells. Macrofibres are denser than the fluid medium in which they are cultured, consequently they settle to the bottom of the growth chamber and grow in contact with it. The ramifications of growth on plastic and glass surfaces were examined. Macrofibres were observed to rotate about a vertical axis near the centre of their length in a chiral-specific direction. Right-handed fibres rotated clockwise on plastic surfaces at approximately 4° min−1, left-handed structures of lower twist rotate anti-clockwise at about half that rate. Very large ball structures produced late in macrofibre formation perched on many small protruding fibres but rotated only when driven by large fibres attached to their periphery. Closer examination showed that fibres made contact with surfaces at only a few points along their length (between 1 and 6 on glass). The regions in contact with the surface changed periodically as a result of rotation of the fibre shaft caused by growth. Every time the weight of a fibre transferred from one contact point to another, each section of the fibre took a small step approximately proportional to its distance from the fibre mid-point. The net result was a rolling of each section over the surface so that the fibre rotation about a vertical axis was produced. Macrofibres also took large steps when part of the structure rose off the floor, swept through an arc in the fluid and then returned to the floor at a new location. The rate of movement during a large step, measured as the change of angle between the moving and stationary portions of the fibre, was 5° s−1. These observations reveal that the forces derived from helical growth that lead to macrofibre formation also cause characteristic macrofibre motion that differs from classical motility.
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Effect of hydrolysable and condensed tannins on growth, morphology and metabolism of Streptococcus gallolyticus (S. caprinus) and Streptococcus bovis
More LessStreptococcus gallolyticus (S. caprinus) was resistant in vitro to at least 7% (w/v) tannic acid and 4% (w/v) acacia condensed tannin, levels 10-fold greater than those tolerated by S. bovis. Growth of S. gallolyticus in liquid medium was characterized by a lag period which increased, and a growth rate which decreased, with increasing tannin concentration. S. gallolyticus was also more tolerant to the presence of simple phenolic acid monomers than was S. bovis, but the lag period was still concentration dependent. Gallate decarboxylase activity in S. gallolyticus was elevated in the presence of tannic acid or gallic acid but not with other phenolic acids. Scanning electron microscopic analysis showed that both the size and shape of S. gallolyticus and S. bovis changed in response to tannin but only S. gallolyticus was surrounded by an extracellular polysaccharide matrix which accumulated in a tannin-concentration-dependent fashion. Washing of the cells to remove extracellular polysaccharide increased the lag period of S. gallolyticus in the presence of 1% (w/v) tannic acid from 4 h to 6 h. In contrast, increasing extracellular polysaccharide synthesis in S. bovis did not increase its tolerance to tannic acid. These data demonstrate that S. gallolyticus has developed a number of mechanisms to reduce the potential effect of tannins on cell growth, and that these mechanisms provide the organism with a selective advantage over S. bovis when grown in the presence of tannins.
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The glucomannokinase of Prevotella bryantii B14 and its potential role in regulating β-glucanase expression
More LessThe SWISS-PROT accession number for the sequence of P. bryantii B14 glucomannokinase reported in this paper is P82680.
Prevotella bryantii B14 has a transport system for glucose and mannose, but β-glucanase expression is only catabolite-repressed by glucose. P. bryantii B14 cell extracts had ATP-dependent gluco- and mannokinase activities, and significant phosphoenolpyruvate- or GTP-dependent hexose phosphorylation was not observed. Mannose inhibited glucose phosphorylation (and vice versa), and activity gels indicated that a single protein was responsible for both activities. Glucose was phosphorylated at a faster rate than was mannose [V max 280 nmol hexose (mg protein)−1 min−1 versus 60 nmol hexose (mg protein)−1 min−1, respectively] and glucose was a better substrate for the kinase (K m 0·12 mM versus 1·2 mM, respectively). The purified glucomannokinase (1250-fold) had a molecular mass of 68 kDa, but SDS-PAGE gels indicated that it was a dimer (monomer 34·5 kDa). The N-terminus (25 residues) had an 8 amino acid segment that was homologous to other bacterial glucokinases. The glucomannokinase was competitively inhibited by the nonmetabolizable glucose analogue 2-deoxyglucose (2DG), and cells grown with glucose and 2DG had lower rates of glucose consumption than did cells given only glucose. When the ratio of 2DG to glucose was increased, the glucose consumption rate decreased and the β-glucanase activity increased. The glucose consumption rate and the glucomannokinase activity of cells treated with 2DG were highly correlated (r 2=0·98). This result suggested that glucomannokinase activity was either directly or indirectly regulating β-glucanase expression.
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- Plant-Microbe Interactions
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Classification of rhizobia based on nodC and nifH gene analysis reveals a close phylogenetic relationship among Phaseolus vulgaris symbionts
More LessThe GenBank accession numbers for the sequences reported in this paper are AF217261 through AF217272 for nodC and AF218126, AF275670 and AF275671 for nifH.
The nodC and nifH genes were characterized in a collection of 83 rhizobial strains which represented 23 recognized species distributed in the genera Rhizobium, Sinorhizobium, Mesorhizobium and Bradyrhizobium, as well as unclassified rhizobia from various host legumes. Conserved primers were designed from available nucleotide sequences and were able to amplify nodC and nifH fragments of about 930 bp and 780 bp, respectively, from most of the strains investigated. RFLP analysis of the PCR products resulted in a classification of these rhizobia which was in general well-correlated with their known host range and independent of their taxonomic status. The nodC and nifH fragments were sequenced for representative strains belonging to different genera and species, most of which originated from Phaseolus vulgaris nodules. Phylogenetic trees were constructed and revealed close relationships among symbiotic genes of the Phaseolus symbionts, irrespective of their 16S-rDNA-based classification. The nodC and nifH phylogenies were generally similar, but cases of incongruence were detected, suggesting that genetic rearrangements have occurred in the course of evolution. The results support the view that lateral genetic transfer across rhizobial species and, in some instances, across Rhizobium and Sinorhizobium genera plays a role in diversification and in structuring the natural populations of rhizobia.
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- Systematics And Evolution
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Hybrid genotypes in the pathogenic yeast Cryptococcus neoformans
Amplified fragment length polymorphism (AFLP) genotyping of isolates of the pathogenic fungus Cryptococcus neoformans suggested a considerable genetic divergence between the varieties C. neoformans var. neoformans and C. neoformans var. grubii on the one hand versus C. neoformans var. gattii on the other. This divergence is supported by additional phenotypic, biochemical, clinical and molecular differences. Therefore, the authors propose the existence of two species, C. neoformans (Sanfelice) Vuillemin and C. bacillisporus Kwon-Chung, which differ in geographical distribution, serotypes and ecological origin. Within each species three AFLP genotypes occur, which differ in geographical distribution and serotypes. Differences in ecological origin (AIDS patients, non-AIDS patients, animals or the environment) were found to be statistically not significant. In C. neoformans as well as in C. bacillisporus one of the genotypes represented a hybrid. The occurrence of hybridization has consequences for the reproductive biology of the species, as new genotypes with altered virulence or susceptibility to antifungal drugs may arise through the exchange of genetic material.
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Rapid phenotypic change and diversification of a soil bacterium during 1000 generations of experimental evolution
More LessEvolutionary pathways open to even relatively simple organisms, such as bacteria, may lead to complex and unpredictable phenotypic changes, both adaptive and non-adaptive. The evolutionary pathways taken by 18 populations of Ralstonia strain TFD41 while they evolved in defined environments for 1000 generations were examined. Twelve populations evolved in liquid media, while six others evolved on agar surfaces. Phenotypic analyses of these derived populations identified some changes that were consistent across all populations and others that differed among them. The evolved populations all exhibited morphological changes in their cell envelopes, including reductions of the capsule in each population and reduced prostheca-like surface structures in most populations. Mean cell length increased in most populations (in one case by more than fourfold), although a few populations evolved shorter cells. Carbon utilization profiles were variable among the evolved populations, but two distinct patterns were correlated with genetic markers introduced at the outset of the experiment. Fatty acid methyl ester composition was less variable across populations, but distinct patterns were correlated with the two physical environments. All 18 populations evolved greatly increased sensitivity to bile salts, and all but one had increased adhesion to sand; both patterns consistent with changes in the outer envelope. This phenotypic diversity contrasts with the fairly uniform increases in competitive fitness observed in all populations. This diversity may represent a set of equally probable adaptive solutions to the selective environment; it may also arise from the chance fixation of non-adaptive mutations that hitchhiked with a more limited set of beneficial mutations.
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Characterization of lactobacilli from Scotch malt whisky distilleries and description of Lactobacillus ferintoshensis sp. nov., a new species isolated from malt whisky fermentations
More LessThe GenBank accession number for the 16S rRNA sequence of strain R7-84 is AF071856.
Sixty-four strains of Lactobacillus were isolated from fermentation samples from 23 malt whisky distilleries located in the major whisky producing regions of Scotland. The strains were assigned to 26 ribotype patterns. Strains of some ribotype patterns were widely distributed and recovered from distilleries throughout Scotland, while strains representing other ribotypes were particular to a specific region or even a certain distillery. Repeated sampling of a single distillery over a 12 month period showed that the range of bacteria present, as indicated by ribotyping, was stable, but was influenced by changes in malt supply and the period of closure for annual maintenance. Partial 16S rDNA sequence analysis of ribotype representatives revealed Lactobacillus brevis, Lactobacillus fermentum, Lactobacillus paracasei and Lactobacillus pentosus as the major species present in the distilleries; however, four isolates could not be identified by this procedure. Determination of the full 16S rDNA gene sequence from one of these isolates (strain R7-84) revealed >98·5% similarity to Lactobacillus buchneri and its phylogenetic neighbours. DNA from two other strains showed greater than 70% hybridization to DNA from R7-84 under non-stringent renaturation conditions and DNA from strain R7-84 shared less than 65% hybridization with members of the L. buchneri group. It is proposed that these three strains should be placed in a new species for which the name Lactobacillus ferintoshensis represented by the type strain R7-84T is suggested.
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