- Volume 144, Issue 5, 1998
Volume 144, Issue 5, 1998
- Review Article
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Bacterial alginate biosynthesis – recent progress and future prospects
More LessThe extracellular polysaccharide alginate has been widely associated with chronic Pseudomonas aeruginosa infections in the cystic fibrosis lung. However, it is clear that alginate biosynthesis is a more widespread phenomenon. Alginate plays a key role as a virulence factor of plant-pathogenic pseudomonads, in the formation of biofilms and with the encystment process of Azotobacter spp.
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- Microbiology Comment
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- Antigens And Immunity
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Characterization of novel immunodominant antigens of Mycobacterium tuberculosis
More LessSeven novel antigens of Mycobacterium tuberculosis, which had previously been identified based on reactivity to sera from patients with tuberculosis, were characterized. Nucleotide sequence analysis of the genes encoding these seven antigens identified one of them as the FtsH and a second as the aminoimidazole ribotide synthase of M. tuberculosis. Antisera raised to the recombinant forms of each of these seven antigens were used to study the distribution of these proteins within mycobacterial species as well as to determine their subcellular localization and hydrophobicity. Four of the seven antigens were conserved only among pathogenic strains of mycobacteria. Of the seven proteins studied, FtsH and a second protein of unknown identity were localized in membranes. Two were cytosolic, while two others, which had a high proline content, weretightly associated with the cell wall. One protein was secreted. This secreted protein could be identified by serum from a majority of tuberculosis patients but not BCG-vaccinated individuals, suggesting its potential use in the immunodiagnosis of tuberculosis.
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- Biochemistry
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The Streptomyces reticuli α-chitin-binding protein CHB2 and its gene
More LessWhen co-cultivated with chitin-containing fungi, Strepfomyces reticuli secretes the chitin-binding protein CHB2. Microscopical and immunological investigations revealed that CHBZ acts like a glue to mediate the contact between the fungal and the Streptomyces hyphae. CHBZ was purified to homogeneity, and the sequence of its N-terminal amino acids was determined and used to deduce an oligonucleotide, which was then used to probe a subgenomic library. The ch62 gene was cloned, sequenced and overexpressed. The deduced mature protein has a molecular mass of 18.6 kDa, and a large number of its amino acids are identical to those of CHBl from Streptomyces oliwaceowiridis. CHB2 effectively targets different types of α-chitin, but no other polysaccharide. The dissociation constant (Kd) for binding to purified crab shell chitin is 0 27 μM. Immunological studies suggest that homologues of CHBl and CHBZ are secreted by streptomycetes while growing in the presence of a-chitin-containing substrates.
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Sequence and structural relationships of leucocins A-, B- and C-TA33a from Leuconostoc mesenteroides TA33a
Amino acid sequences of two of the three bacteriocins from Leuconostoc mesenteroides TA33a were determined and their sequence-structure relationships investigated. Leucocin B-TA33a consists of 31 amino acid residues, with a molecular mass of 3466 Da. Leucocin B-TA33a does not belong to the pediocin family of bacteriocins, but shares 62% homology with mesenterocin 52B. A partial sequence of 36 amino acids of leucocin C-TA33a (4598 Da) was determined. Leucocin C-TA33a belongs to the class II bacteriocins having the consensus YGNGV motif. The third bacteriocin, leucocin A-TA33a, is identical to leucocin A-UAL 187. Circular dichroism spectra of the leucocins in aqueous solution and micellar SDS indicated that they undergo a structural transition when in a membrane-mimicking environment. Theoretical predictions from circular dichroism data suggest that leucocins A-, B- and C-TA33a adopt a β-structure (48%) in membrane-mimicking environments. Sequence alignments and secondary structure predictions for the N-terminus of leucocins A- and C-TA33a predicted that Cys-9 and Cys-14 are connected by a disulfide bridge and form two β-strands.
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Structure of asparagine-linked oligosaccharides of an aspartic proteinase from the zygomycete fungus Rhizomucor pusillus
More LessThe zygomycete fungus Rhizomucor pusillus (previously called Mucor pusillus) secretes an aspartic proteinase containing two asparagine-linked, high-mannose type oligosaccharide chains at Asn79 and Asn188. For structural elucidation of the carbohydrate moieties, the protein was divided into two portions, an N-terminal portion containing Asn79 and a C-terminal portion containing Asn188, by a specific autocatalytic cleavage under alkaline conditions. Each of the asparagine-linked oligosaccharides was then released by peptide-N-glycosidase F digestion and pyridylaminated with a fluorescent reagent, 2-aminopyridine, at the reducing end. High-performance liquid chromatography analyses showed that the structure of the asparagine-linked oligosaccharide chain attached to residue Asn79 was Man5 GlcNAc2, and that bound to residue Asn188 was Man5 GlcNAc2 and Man5GlcNAc2. These observations suggest that the processing of mannose residues in asparagine-linked oligosaccharides in the Golgi apparatus of Rhizomucor resembles that in mammalian cells.
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- Biotechnology
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Metabolism of ferulic acid via vanillin using a novel CoA-dependent pathway in a newly-isolated strain of Pseudomonas fluorescens
More LessA soil bacterium, designated Pseudomonas fluorescens AN103, was isolated based on its ability to grow on ferulic acid as a sole source of carbon and energy. In addition, this strain was found to metabolize a number of related phenolic substrates which contained a hydroxyl group at the para position of the aromatic ring. During growth on ferulic acid, transient accumulation of vanillic acid and trace amounts of protocatechuic acid were detected in the culture medium. Washed cells grown on ferulic acid readily oxidized vanillin, vanillic acid and protocatechuic acid, the three putative intermediates of the metabolic pathway. The side-chain cleavage of ferulic acid to produce vanillin was demonstrated in vitro for the first time and this enzyme-catalysed reaction was shown to have an essential requirement for CoASH, ATP and MgCl2. This conversion involved a two-step process involving a CoA ligase followed by the side-chain cleavage. The addition of NAD increased the oxidation of vanillin to vanillic acid and had an overall effect of increasing the rate of ferulic acid cleavage. The application of 13C-NMR studies in vitro revealed acetyl-CoA as the C2 side-chain cleavage product. High levels of inducible ferulate-CoA ligase and NAD-linked vanillin dehydrogenase were detected and a novel pathway for ferulic acid metabolism in this organism is proposed.
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Accumulation and effects of cadmium on sulphate-reducing bacterial biofilms
More LessBiofilms comprising a pure and a mixed culture of sulphate-reducing bacteria (SRB) were grown in continuous culture. When exposed to 20 or 200 μM Cd, both cultures accumulated Cd but the mixed culture accumulated more and continued to accumulate Cd during the experiment, whereas accumulation by the pure cultures ceased after 4-6 d. Unlike the pure culture, the mixed culture also accumulated both protein and carbohydrate throughout the experiment proportionally to Cd which showed that accumulation required the production of biofilm material. Electron microscopy showed the presence of polysaccharide and particulates in both pure and mixed cultures, irrespective of the presence of Cd. However, energy-dispersive X-ray analysis (EDXA) showed that accumulation of Cd in the form of CdS occurred in biofilms exposed to Cd while back-scattered electron imaging of sections indicated that the accumulation of Cd was localized in a superficial layer of the biofilm. The mechanism of uptake, therefore, appeared to be entrapment and/or precipitation of CdS at the biofilm surface. The relatively low Cd uptake by the pure culture biofilm was attributed to its less efficient growth and polysaccharide production. These results indicate that mixed SRB cultures are more effective than pure cultures for metal removal and underlines significant differences between the biology of pure and mixed cultures.
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- Development And Structure
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Identification of two glycosylated components of Mycoplasma penetrans: a surface-exposed capsular polysaccharide and a glycolipid fraction
Among the wall-less mycoplasmas only a few species have been identified with a capsule at their cell surface. Mycoplasma penetrans is a recently identified mycoplasma with unique morphology, isolated from HIV-infected patients. Using transmission electron microscopy, it was found that M. penetrans is surrounded by capsular material 11 nm (strain GTU-54-6A1) to 30 nm (strain HF-2) thick, which can be stained with ruthenium red and labelled with cationized ferritin. The polysaccharide composition of this capsule was indicated by its staining with periodic acid-thiocarbohydrazide silver proteinate and the abolition of ruthenium red staining of the cell surface by neuraminidase treatment. In addition, proteinase K treatment of the M. penetrans cells resulted in removal of the capsule, suggesting that polypeptides may contribute in anchoring it to the membrane or in its stability. Two different types of glycosylated material were detected in mycoplasma extracts by SDS-PAGE and periodic acid-Schiff staining. The first component was a high-molecular-mass material, which was heat- and proteinase-K-labile and which probably constitutes the capsular polymer. The other component was a low-molecular-mass glycolipid fraction, which was proteinase-K-, heat- and EDTA-resistant. The identification of a capsule at the M. penetrans cell surface is of particular interest for a mycoplasma which has been shown to adhere to various host cells and to penetrate into their intracellular compartments. The capsule may have significance in the pathogenesis of disease associated with infection by this organism.
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Environmentally controlled dimorphic cycle in a fission yeast
More LessThe fission yeast Schizosaccharomyces pombe shows bipolar growth and is a convenient model for studying cell polarity and polar growth. This paper shows that the related Schiz. japonicus var. japonicus can switch to unipolar growth and can exist in both yeast and mycelial phases. On solid media, the yeast phase is unstable and prone to switch to the mycelial form, which shows unipolar growth by tip elongation. The hyphae can colonize the body of the substrate (true mycelium) or just its surface (pseudo-mycelium). The yeast-to-mycelium transition and the growth of the mycelium are regulated by a nutritional gradient and are associated with extensive vacuolation. The mycelium can convert into arthroconidia or return to the yeast phase in response to environmental changes. These environmentally controlled morphological transitions make Schiz. japonicus var. japonicus an attractive model for the investigation of cell polarity and morphogenesis.
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Structural characteristics of halobacterial gas vesicles
More LessGas vesicle formation in halophilic archaea is encoded by a DNA region (the vac region) containing 14 different genes: gvpACNO and gvpDEFGHIJKLM. In Halobacterium salinarum PHH1 (which expresses the p-vac region from plasmid pHH1), gas vesicles are spindle shaped, whereas predominantly cylindrical gas vesicles are synthesized by the chromosomal c-vac region of H. salinarum PHH4 and the single chromosomal mc-vac region of Haloferax mediterranei. Homologous complementation of gvp gene clusters derived from the chromosomal c-vac region led to cylindrical gas vesicles in transformants and proved that the activity of the c-gvpA promoter depended on a gene product from the c-gvpE-M DNA region. Heterologous complementation experiments with transcription units of different vac regions demonstrated that the formation of chimeric gas vesicles was possible. Comparison of micrographs of wild-type and chimeric gas vesicles indicated that the shape was not exclusively determined by GvpA, the major structural protein of the gas vesicle wall. More likely, a dynamic equilibrium of several gvp gene products was responsible for determination of the shape. Transmission electron microscopy of frozen hydrated, wild-type gas vesicles showed moiré patterns due to the superposition of the front and back parts of the ribbed gas vesicle envelope. Comparison of projections of model helices with the moiré pattern seen on the cylindrical part of the gas vesicles provided evidence that the ribs formed a helix of low pitch and not a stack of hoops.
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- Genetics And Molecular Biology
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Genetic diversity in the Mycobacterium tuberculosis complex based on variable numbers of tandem DNA repeats
More LessGenetic loci containing variable numbers of tandem repeats (VNTR loci) form the basis for human gene mapping and identification, forensic analysis and paternity testing. The variability of bacterial tandem repeats has not been systematically studied. Eleven tandem repeat loci in the M. tuberculosis genome were analysed. Five major polymorphic tandem repeat (MPTR) loci contained 15-bp repeats with substantial sequence variation in adjacent copies. Six exact tandem repeat (ETR) loci contained large DNA repeats with identical sequences in adjacent repeats. These 11 loci were amplified in 48 strains to determine the number of tandem repeats at each locus. The strains analysed included 25 wild-type strains of M. tuberculosis, M. bovis, M. africanum and M. microti and 23 substrains of the attenuated M. bovis BCG vaccine. One of the five MPTR loci and all six ETR loci had length polymorphisms corresponding to insertions or deletions of tandem repeats. Most ETR loci were located in intergenic regions where copy number may influence expression of downstream genes. Each ETR locus had multiple alleles in the panel. Combined analysis identified 22 distinct allele profiles in 25 wild-type strains of the M. tuberculosis complex and five allele profiles in 23 M. bovis BCG substrains. Allele profiles were reproducible and stable, as demonstrated by analyses of multiple isolates of particular reference strains obtained from different laboratories. VNTR typing may be generally useful for strain differentiation and evolutionary studies in bacteria.
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An antigenic protein gene of a phytoplasma associated with sweet potato witches’ broom
More LessA gene encoding the major antigenic protein of phytoplasma associated with sweet potato witches’ broom (SPWB) was cloned and analysed by screening the genomic library of SPWB phytoplasma with monoclonal antibodies for SPWB phytoplasma. The entire predicted structural gene encoded an antigenic protein composed of 172 amino acids with a computed molecular mass of 19.15 kDa and a pl value of 9.78. The -10 region of the promoter and the terminator region of the gene were identified and found to be similar to those of prokaryotes. The hydropathy profile of the deduced amino acid sequence consisted of two distinct regions, a strongly hydrophobic N-terminus and a highly hydrophilic C-terminus. This major antigenic protein was also present in phytoplasma associated with peanut witches’ broom (PNWB) and the two showed homology based on the results of Western blot analysis. Southern hybridization. Northern hybridization, primer extension analysis and PCR. The homologous genes of the antigenic protein of SPWB phytoplasma arid PNWB phytoplasma were not found in other phytoplasmas tested.
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Characterization of plasmid pAW63, a second self-transmissible plasmid in Bacillus thuringiensis subsp. kurstaki HD73
More LessBacillus thuringiensis subspecies kurstaki HD73, toxic for lepidopteran larvae, contains two large self-transmissible plasmids of approximately 75 kb, pHT73 and pAW63. The conjugative plasmid pHT73 has been studied extensively and has been shown to harbour the toxin gene cry1Ac, the transposon Tn4430 and several insertion sequences. In this study it was demonstrated that the minor plasmid pAW63 is also self-transmissible and about 10-30 times more efficient in mobilizing plasmid pBC16. To facilitate direct selection for pAW63 transfer, the plasmid was tagged with the tetracycline resistance transposon Tn5401 and in intraspecies matings it was found that after 2 h, all recipients had acquired a copy of the plasmid. Mating experiments demonstrated that pAW63 could be transferred to Bacillus thuringiensis subsp. israelensis. Bacillus cereus, Bacillus licheniformis, Bacillus subtilis and Bacillus sphaericus, and that the conjugative functions were expressed in these hosts. Hybridization studies showed that the replicons of pAW63 and pHT73 were distinct from one another. Sequences homologous to transposon Tn4430 and several insertion sequences were, however, shown to reside on both plasmids.
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The terminal structures of linear plasmids from Rhodococcus opacus
More LessThe telomers of several linear plasmids of Rhodococcus opacus (formerly Nocardia opaca) were studied. The plasmids pHG201, pHG204 and pHG205 carry proteins bound to their ends, as shown by gel retardation experiments. A sequence hybridizing with the terminal sequence of pHG207, a recombinant linear plasmid consisting of the left part of pHG204 and the right part of pHG205, which was analysed in a previous study by the authors, could be detected in all linear plasmids of the wild-type R. opacus strains MR11 and MR22. However, only pHG204 and pHG206 carry terminal inverted repeats (TIRs) like pHG207. Cloning and sequencing of the terminal fragment of pHG204 revealed a nearly perfect TIR of 1016 bp. In contrast, the termini of pHG201 and pHG205 share little homology. Sequence analysis of the two end fragments of pHG201 revealed a similarity of only 65% within the terminal 34/32 bp and a perfect TIR of only 3 bp. The results support the assumption that long TIRs are not absolutely necessary for replication and maintenance of linear plasmids.
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Structural elements of the Streptomyces oriC region and their interactions with the DnaA protein
Streptomycetes differ from other prokaryotic organisms in their mycelial life cycle and in possessing a large, linear, GC-rich chromosome. To deduce structural features of the Streptomyces origin of chromosomal replication, the oriC sequences of three Streptomyces species (S. antibioticus, S. chrysomallus and S. lividans) were compared. In Streptomyces, the oriC region contains 19 DnaA boxes whose location, orientation and spacing are conserved. The consensus sequence of the DnaA box identified within Streptomyces oriC is (T/C)(T/C)(G/A/C)TCCACA (preferred bases underlined). The interactions of DnaA with DNA fragments containing single, two or three DnaA boxes were studied using surface plasmon resonance. The dissociation constant (K D) for specific binding of individual DnaA boxes varied between 12 and 78 nM. Streptomyces oriC does not contain the three AT-rich 13-mer direct repeats present in the 5′ part of the Escherichia coli oriC region. However, short AT-rich sequences are distributed among the DnaA boxes of Streptomyces oriC. Repeated attempts to unwind Streptomyces oriC have been unsuccessful. It remains to be elucidated whether DnaA interacts with putative accessory proteins which help in unwinding Streptomyces oriC.
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A chitin-binding domain in a marine bacterial chitinase and other microbial chitinases: implications for the ecology and evolution of 1,4-β-glycanases
More LessTo examine the ecology and evolution of microbial chitinases, especially the chitin-binding domain, one of the chitinase genes (chiA) from the marine bacterium Vibrio harveyi was analysed. The deduced amino acid sequence of ChiA is not very similar overall to other proteins, except for two regions, the putative catalytic and chitin-binding domains. Among all bacterial chitinases sequenced to date, there is no relationship between percentage similarity of catalytic domains and chitin-binding domains in pairwise comparisons, suggesting that these two domains have evolved separately. The chitin-binding domain appears to be evolutionarily conserved among many bacterial chitinases and is also somewhat similar to cellulose-binding domains found in microbial cellulases and xylanases. To investigate the role of the chitin-binding domain, clones producing versions of ChiA with or without this domain were examined. One version with the domain (ChiA1) bound to and hydrolysed chitin, whereas a truncated ChiA without the putative chitin-binding domain (ChiA2) did not bind to chitin but it could hydrolyse chitin, although not as well. ChiA1 diffused more slowly in agarose containing colloidal chitin than ChiA2, but diffusion of the Two proteins in agarose without colloidal chitin was similar. These results indicate that the chitin-binding domain helps determine the movement of chitinase along N-acetylglucosamine strands and within environments containing chitin.
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Implications of the xylQ gene of TOL plasmid pWW102 for the evolution of aromatic catabolic pathways
More LessPseudomonas putida strain O2C2 is able to grow on toluene, m-xylene and p-xylene through benzoate and the corresponding methylbenzoates (toluates). The catabolic genes are encoded on a large TOL plasmid, pWW102, of >220 kb. The complete catabolic genes were cloned on four large overlapping restriction fragments covering a total of 28 kb of the plasmid, which was carefully mapped by restriction enzyme analysis. The presence of the xyl genes on the cloned DNA was confirmed by assay of representative enzymes of both operons. Virtually all the genes were located on the cloned DNA by hybridization of Southern blots with gene-specific probes from related pathways of other catabolic plasmids. Within the limitations of available restriction sites, the analysis showed that the genes are in two blocks. The major block carries the meta pathway operon xylXYZLTEGFJQKIH with the two regulatory genes xylSR immediately downstream. The upper pathway operon xylUWCMAB(N) is about 2-3 kb downstream of the regulatory genes and transcribed in the same direction as the meta pathway operon. Within each operon the gene order appears to be identical to that found in other TOL plasmids, but the relative location of the operons most closely resembles that found on plasmid pWW53, although there is no evidence of any xyl duplications on pWW102. The nucleotide sequence of the xylQ gene for the acetaldehyde dehydrogenase (acylating; ADA), together with the 3-end of the upstream xylJ (for 2-oxopent-4-enoate hydratase) and the 5-end of the downstream xylJ (for 4-hydroxy-2-oxovalerate aldolase), was determined. The xylQ gene was ligated into expression vector pTrc99a and high levels of XylQ protein were detected by enzyme assay and by SDS-PAGE. All three genes xylJQK showed a high degree of homology with genes encoding isofunctional proteins from other Pseudomonas meta pathways, the highest being with the naphthalene catabolic genes nahLOM from the plasmid of Pseudomonas sp. NCIB 9816. The implications of the sequence homologies to the evolution of these pathways are discussed.
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Spacing and orientation requirements of GcvA-binding sites 3 and 2 and the Lrp-binding region for gcvT::lacZ expression in Escherichia coli
More LessBoth GcvA and Lrp are required for normal regulation of the gcv operon. Moving the GcvA-binding sites 3 and 2 and the Lrp-binding region either closer to, or further away from, the gcv promoter by approximately one helical turn of DNA resulted in a less than twofold decrease in glycine-mediated activation or inosine-mediated repression of a gcvT::lacZ fusion. Moving these sites approximately two helical turns of DNA away from the gcv promoter resulted in a further loss of both activation and repression; moving these sites approximately three helical turns of DNA from the gcv promoter resulted in an essentially complete loss of both glycine-mediated activation and inosinemediated repression. However, when these sites were moved by approximately 1.5 and 2.5 helical turns of DNA away from the gcv promoter, there was a complete loss of both glycine-mediated activation and inosine-mediated repression of the gcvT::lacZ fusion. The flexibility in the absolute distance of the GcvA- and Lrp-binding sites relative to the gcv promoter, but strict orientation dependence of these sites is consistent with a possible protein-protein interaction of either GcvA, Lrp, or both of these proteins with RNA polymerase. Because of the location of these target sites relative to the gcv promoter, it is also likely that DNA looping is required for this mechanism of regulation.
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ppc, the gene for phosphoenolpyruvate carboxylase from an extremely thermophilic bacterium, Rhodothermus obamensis: cloning, sequencing and overexpression in Escherichia coli
More LessThe ppc gene, which encodes phosphoenolpyruvate carboxylase (PEPC) of an extremely thermophilic bacterium, Rhodothermus obamensis, was directly sequenced by the thermal asymmetric interlaced (TAIL) PCR method. An ORF for a 937 amino acid polypeptide was found in the gene. The ppc gene had a high G+C content (66.2 mol%) and the third position of the codon exhibited strong preference for G or C usage (85.0 mol%). The calculated molecular mass was 107 848 Da, which was consistent with the molecular mass of the enzyme as determined by SDS-PAGE (100 kDa). The amino acid sequence of R. obamensis PEPC was closely related to that of PEPC from another thermophile, a Thermus sp., and from a mesophile. Corynebacterium glutamicum, exhibiting 45.3% or 37.7% identity and 61.5% or 56.5% similarity, respectively. By Southern analysis, the ppc gene was found to be present in a single copy in the genomic DNA of this organism. The cloned gene was expressed in Escherichia coli using a pET expression vector system and a thermostable recombinant PEPC was obtained. Comparison of the deduced amino acid sequences of the thermophilic and mesophilic PEPCs revealed distinct or common preferences for specific amino acid composition and substitutions in the two thermophilic enzymes.
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Aspartate carbamoyltransferase from a psychrophilic deep-sea bacterium, Vibrio strain 2693: properties of the enzyme, genetic organization and synthesis in Escherichia coli
The aspartate carbamoyltransferase (ATCase) genes of psychrophilic Vibrio strain 2693 were cloned by complementation in Escherichia coli and the enzyme was partly characterized. The genes constitute a pyrBl operon homologous to the cognate structure in E. coli where pyrB and pyrl respectively encode the catalytic and the regulatory chains of ATCase. The strong sequence similarities noted between Vibrio and E. coli ATCases include extensive conservation of residues involved in interactions between subunits, suggesting that the two enzymes have very similar tertiary and quaternary structures. Vibrio ATCase is, however, not activated by ATP and not synergistically inhibited by CTP and UTP. It is also much more thermolabile than E. coli ATCase. With respect to Pyrococcus abyssi and E. coli ATCases, Vibrio ATCase presents marked differences in composition which could be related to its psychrophilic character. The results of these structural and functional comparisons indicate that Vibrio 2693 ATCase is a suitable model for biochemical studies on structure-function relationships in a ‘cold’ allosteric enzyme. The operon is expressed from a promoter which is immediately followed by a pyrimidine-rich leader ORF terminating within a putative transcription attenuator. These genetic and enzymic data strengthen the evolutionary relationship already noted between Vibrionaceae and Enterobacteriaceae.
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Comparison of PhoP binding to the tuaA promoter with PhoP binding to other Pho-regulon promoters establishes a Bacillus subtilis Pho core binding site
More LessThe phosphate-deficiency response in Bacillus subtilis is regulated by PhoP PhoR, a pair of two-component regulatory proteins. PhoR is a histidine kina and PhoP is a response regulator. Genetic evidence indicates that the Pho-regulon genes, which are induced or repressed under phosphate starvation conditions, are regulated by PhoP and PhoR at the transcriptional level. It has previously been shown that PhoP binds to four Pho-regulon promoters in both unphosphorylated and phosphorylated forms. This study demonstrates that another Pho-regulon gene promoter, the tuaA promoter preceding the operon which is responsible for cell wall teichuronic acid synthesis, is also transcriptionally regulated and is bound by PhoP. The binding affinity for phosphorylated PhoP was about 10-fold higher than that for unphosphorylated PhoP. Both unphosphorylated and phosphorylated PhoP bound upstream of the -20 region in the tuaA promoter. By aligning the Phop binding sites within the Pho-regulon promoters, a consensus core PhoP-binding region composed of four TT(A/T)ACA direct repeats, each separated by 5.2 non-conserved nucleotides was identified. PhoP, phosphorylated or unphosphorylated, binds to such a sequence in all Pho-regulon promoters studied. Phosphorylated PhoP binds to the core binding region with high affinity and to additional regions surrounding this region with similar or lower affinity.
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- Pathogenicity And Medical Microbiology
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Characterization of a haemolysin from Mycobacterium tuberculosis with homology to a virulence factor of Serpulina hyodysenteriae
Scrutiny of sequence data from the Mycobacterium leprae genome sequencing project identified the presence of a gene encoding a 268-amino-acid polypeptide which is highly similar to a pore-forming haemolysin/cytotoxin virulence determinant, TlyA, from the swine pathogen Serpulina hyodysenteriae. Using degenerate oligonucleotide primers based on the TlyA sequences, the Mycobacterium tuberculosis homologue was amplified and this product was used to obtain the clone and sequence a 2.5 kb fragment containing the whole M. tuberculosis tlyA gene. tlyA encodes a 267-amino-acid protein with a predicted molecular mass of 28 kDa. TlyA homologues were identified by PCR in M. leprae, Mycobacterium avium and Mycobacterium bovis BCG, but appeared absent in Mycobacterium smegmatis, Mycobacterium vaccae, Mycobacterium kansasii, Mycobacterium chelonae and Mycobacterium phlei. The M. tuberculosis gene appeared to be the first gene in an operon containing at least two other genes. Introduction of the M. tuberculosis tlyA gene into M. smegmatis using a mycobacterial shuttle expression plasmid converted non-haemolytic cells into those exhibiting significant haemolytic activity. Similarly, inducible haemolytic activity was observed in sonicated bacteria when tlyA was expressed as a His6-tagged fusion protein in Escherichia coli. tlyA mRNA was detected in both M. tuberculosis and M. bovis BCG using RT-PCR, confirming that this gene is expressed in organisms cultured in in vitro.
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Recombination between rRNA operons created most of the ribotype variation observed in the seventh pandemic clone of Vibrio cholerae
More LessIndividual rrn operons and their flanking regions have been analysed in a study of the molecular basis of ribotype variation in the seventh pandemic clone of Vibrio cholerae. The genome of an early isolate of the seventh pandemic clone had nine rrn operons of which two were in tandem with other rrn operons. The site for Bgll, the most discriminatory enzyme used for ribotyping, was found to be present in the 16S sequence of three of the operons of the earliest isolate. This site was observed to be gained or lost in specific operons in many later isolates, presumably by recombination, and this gave most of the ribotype variation. Additional rrn recombination events were uncovered by analysis of the 16S-23S intergenic spacers associated with each operon. Spacers of 431, 509, 607 and 711 bp were found. A total of at least eight rrn recombination events were detected. Three rrn loci were primarily involved in this recombination, with four new forms generated from that in the early strains for operon B and two new forms each for operons C and G. In addition there was variation due to deletion of tandem operons. The frequency of recombination between rrn operons was very high as there were nine new ribotypes found among 47 isolates sampled over the 33 year period of study. This means that any variation could undergo precise reversion by the same recombination event within the time frame covered by the study. Recombination between rrn operons may be a factor in ribotype variation in all systems. The recombination observed is thought to be that which results in concerted evolution and the data give an indication of the rate involved.
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Identification and analysis of a gene (abpA) encoding a major amylase-binding protein in Streptococcus gordonii
Oral streptococci such as Streptococcus gordonii bind the abundant salivary enzyme x-amylase. This interaction may be important in dental plaque formation and metabolism, thus contributing to the initiation and progression of dental caries and periodontal disease, the two most common plaque-mediated diseases. The conjugative transposon Tn916 was used to insertionally inactivate gene(s) essential to the expression of amylase-binding components of S. gordonii Challis, and a mutant deficient in amylase-binding (Challis Tn1) was identified. While wild-type strains of S. gordonii released both 20 kDa and 82 kDa amylase-binding proteins into culture supernatants, Challis Tn1 expressed the 82 kDa but not the 20 kDa protein. The 20 kDa amylase-binding protein was isolated from culture supernatants of S. gordonii Challis by hydroxyapatite chromatography. A partially purified, functionally active 20 kDa protein was sequenced from blots, and the N-terminal sequence obtained was found to be DEP (A) TDAAT(R)NND. A novel strategy, based on the single-specific-primer polymerase chain reaction technique, enabled the gene inactivated by Tn916 to be cloned. Analysis of the resultant nucleotide sequence revealed an open reading frame of 585 bp, designated amylase-binding protein A (abpA), encoding a protein of 20 kDa (AbpA), immediately downstream from the insertion site of Tn916. This protein possessed a potential signal peptide followed by a region having identity with the N-terminal sequence of the 20 kDa amylase-binding protein. These results demonstrate the role of the 20 kDa protein in the binding of amylase to S. gordonii. Knowledge of the nature of amylase-binding proteins may provide a better understanding of the role of these proteins in the colonization of S. gordonii in the oral cavity.
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- Physiology And Growth
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Biological functions of UDP-glucose synthesis in Streptococcus mutans
A gene encoding glucose-1-phosphate uridylyltransferase (EC 2.7.7.9) was isolated from Streptococcus mutans. A cell extract of Escherichia coli expressing the cloned gene exhibited glucose-1-phosphate uridylyltransferase activity. The enzyme catalyses the conversion of D-glucose 1-phosphate and UTP into UDP-D-glucose. Rabbit antiserum against the serotype-c-specific antigen did not react with autoclaved extracts from mutant cells in which the cloned gene was insertionally inactivated. The glucose content of the cell-wall preparation purified from the mutant was very much lowered, whereas there was no observable decrease in the content of rhamnose. When the mutant strain was grown in an acidic environment, its cell viability was much lower than that of the wild-type. These results suggest that UDP-D-glucose functions not only as an immediate precursor of the serotype-c-specific antigen of S. mutans (as a glucose donor for side-chain formation), but is also important for the organism’s viability in environmental conditions of low pH.
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Fused nucleoids resegregate faster than cell elongation in Escherichia coli pbpB(Ts) filaments after release from chloramphenicol inhibition
More LessThe course of nucleoid movement during and upon release from protein synthesis inhibition by chloramphenicol in filaments of Escherichia coli pbpB(Ts) was analysed. Cells were grown at 42 °C in glucose minimal medium for two mass doublings and were treated with chloramphenicol to generate fusion (coalescence) of the nucleoids. Upon release from protein synthesis inhibition, the large distance between the border of the fused nucleoids and the cell poles immediately decreased, before full recovery of the rates of mass growth and length increase at 30 °C. This indicates that nucleoids can reoccupy the DNA-free cell ends independently of cell elongation. During filamentation at 42 °C, the pbpB cells established initial constrictions at midcell and at one-quarter and three-quarter positions. Nevertheless, divisions only started 75 min after chloramphenicol removal at 30 °C, when most nucleoids had moved back into the vacated cell ends. No ‘guillotine-like’ constrictions at the site of the nucleoids occurred.This suggests that segregating nucleoids postpone division recovery at previously established sites. The results are discussed in the light of a working model for transcription/translation-mediated chromosome segregation and nucleoid occlusion of cell division.
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Antibacterial action of dipeptides containing an inhibitor of glucosamine-6-phosphate isomerase
More LessSeveral dipeptides, containing the N 3-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid (FMDP) moiety linked to protein and non-protein amino acids, exhibited a strong growth-inhibitory and bactericidal effect against Bacillus subtilis. FMDP-dipeptides were efficiently transported into bacterial cells by a di-tripeptide permease and subsequently cleaved by intracellular Mn2+/Co2+-dependent peptidases. Cleavage rates [0.1-5.6 μmol min-1 (mg protein)-1] were about two orders of magnitude lower than transport rates [40-200 μmol min-1 (mg dry wt)-1]. The released FMDP inactivated glucosamine-6-phosphate (GlcN-6-P) isomerase, an enzyme catalysing the first committed step in a biosynthetic pathway leading to amino sugar-nucleotide precursors of bacterial peptidoglycan. Inhibition of GlcN-6-P isomerase precluded peptidoglycan biosynthesis and resulted in a strong bacteriolytic effect. Results of the studies on consequences of GlcN-6-P isomerase inhibition upon the action of FMDP-dipeptides provided evidence demonstrating that the lack of endogenous GlcN-6-P could be a reason for the triggering of bacterial autolysis. Peptides containing the inhibitors of GlcN-6-P isomerase are one of the very few antimicrobial agents known that exhibit both bactericidal and fungicidal effects.
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Molecular characterization of an autolytic amidase of Listeria monocytogenes EGD
More LessThe gene encoding a 102 kDa autolysin has been cloned from an expression library of Listeria monocytogenes EGD genomic DNA, using a direct screening protocol. The encoded protein has two domains, an N-terminal enzymic domain showing a high level of homology to the amidase domain of the major autolysin (atl) of Staphylococcus aureus, and a C-terminal, putative cell-wall-binding domain containing four imperfect direct repeats. In order to examine the role of the enzyme, the autolysin-encoding gene was insertionally inactivated by site-specific integration of a temperature sensitive plasmid. The enzyme accounts for 66% of the total lytic enzyme activity when L. monocytogenes walls are used as substrate and several of the major autolytic bands are missing on renaturing gels when compared to the wild-type. The enzyme does not appear to be directly involved in cell separation but has a role in motility. Characterization of the recombinant enzyme expressed in Escherichia coli has revealed it to be an amidase and to be able to hydrolyse a range of peptidoglycan substrates.
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Regulation of the sulfate starvation response in Pseudomonas aeruginosa: role of cysteine biosynthetic intermediates
Pseudomonas aeruginosa PAO1 grew in defined synthetic medium with any of a broad variety of single sulfur sources, including sulfate, cysteine, thiocyanate, alkanesulfonates, organosulfate esters and methionine, but not with aromatic sulfonates, thiophenols or organothiocyanates or isothiocyanates. During growth with any of these compounds except sulfate, cysteine or thiocyanate, a set of 10 sulfate starvation-induced (SSI) proteins was strongly up-regulated, as observed by two-dimensional protein electrophoresis of total cell extracts. A comparable level of up-regulation was found for the hydrolytic enzyme arylsulfatase, which has previously been used as a marker enzyme for the sulfate starvation response. One of the SSI proteins was identified by N-terminal sequencing as a high-affinity periplasmic sulfate-binding protein, and another was related to thiol-specific antioxidants, but the N-terminal sequences of the other SSI proteins revealed no similarity to N-termini of proteins of known function, and they probably represent uncharacterized enzymes involved in sulfur scavenging when preferred sulfur sources are absent. To study the role that cysteine biosynthetic intermediates play in the synthesis of these proteins in vivo, we isolated mini-Tn5 transposon mutants of P. aeruginosa with insertions in the cysN and cysl genes, which encode subunits of ATP-sulfurylase and sulfite reductase, respectively. These two genes were cloned and sequenced. cysl showed high similarity to the cognate gene in Escherichia coli, whereas cysN encoded a 69.3 kDa protein with two domains corresponding to the E. coli CysN and CysC proteins. Sulfate no longer repressed synthesis of the SSI proteins in cysN mutants, but repression was restored by sulfite; in the cysl mutant, sulfate, sulfite and sulfide all led to repression of SSI protein synthesis. This suggests that there are at least two independent corepressors of the sulfate starvation response in this species.
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Repression of nitrogen catabolic genes by ammonia and glutamine in nitrogen-limited continuous cultures of Saccharomyces cerevisiae
Growth of Saccharomyces cerevisiae on ammonia and glutamine decreases the expression of many nitrogen catabolic genes to low levels. To discriminate between ammonia- and glutamine-driven repression of GAP1, PUT4, GDH1 and GLN1, a gln1-37 mutant was used. This mutant is not able to convert ammonia into glutamine. Glutamine-limited continuous cultures were used to completely derepress the expression of GAP1, PUT4, GDH1 and GLN1. Following an ammonia pulse, the expression of GAP1, PUT4 and GDH1 decreased while the intracellular glutamine concentration remained constant, both in the cytoplasm and in the vacuole. Therefore, it was concluded that ammonia causes gene repression independent of the intracellular glutamine concentration. The expression of GLN1 was not decreased by an ammonia pulse but solely by a glutamine pulse. Analysis of the mRNA levels of ILV5 and HIS4 showed that the response of the two biosynthetic genes, GDH1 and GLN1, to ammonia and glutamine in the wild-type and gln1-37 was not due to changes in general transcription of biosynthetic genes. Ure2p has been shown to be an essential element for nitrogen-regulated gene expression. Deletion of URE2 in the gln1-37 background prevented repression of gene expression by ammonia, showing that the ammonia-induced repression is not caused by a general stress response but represents a specific signal for nitrogen catabolite regulation.
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- Systematics
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Rapid identification of urinary tract infection bacteria using hyperspectral whole-organism fingerprinting and artificial neural networks
Three rapid spectroscopic approaches for whole-organism fingerprinting-pyrolysis mass spectrometry (PyMS), Fourier transform infra-red spectroscopy (FT-IR) and dispersive Raman microscopy - were used to analyse a group of 59 clinical bacterial isolates associated with urinary tract infection. Direct visual analysis of these spectra was not possible, highlighting the need to use methods to reduce the dimensionality of these hyperspectral data. The unsupervised methods of discriminant function and hierarchical cluster analyses were employed to group these organisms based on their spectral fingerprints, but none produced wholly satisfactory groupings which were characteristic for each of the five bacterial types. In contrast, for PyMS and FT-IR, the artificial neural network (ANN) approaches exploiting multi-layer perceptrons or radial basis functions could be trained with representative spectra of the five bacterial groups so that isolates from clinical bacteriuria in an independent unseen test set could be correctly identified. Comparable ANNs trained with Raman spectra correctly identified some 80% of the same test set. PyMS and FT-IR have often been exploited within microbial systematics, but these are believed to be the first published data showing the ability of dispersive Raman microscopy to discriminate clinically significant intact bacterial species. These results demonstrate that modern analytical spectroscopies of high intrinsic dimensionality can provide rapid accurate microbial characterization techniques, but only when combined with appropriate chemometrics.
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Division of the genus Enterococcus into species groups using PCR-based molecular typing methods
More LessBroad-range 16S rDNA PCR (BR-PCR) applied to DNA from 32 clinical enterococcal isolates and 12 other enterococci from a clinical reference collection followed by species-specific hybridization analysis identified 25 strains of Enterococcus faecalis and 19 Enterococcus species. Randomly amplified polymorphic DNA (RAPD)analysis using upgma clustering on the same material revealed four different clusters at a similarity level of 49%. Based on partial 16S rDNA sequence analysis of variable regions V4 and V9, it was possible to divide the 19 type strains specifying the genus Enterococcus into 12 different 16S rDNA species groups. The type strain distribution then served as a template for the analysis of the other 44 strains which were assigned to four different species groups (a-d) based on their 16S rDNA motifs. There was good agreement with the RAPD clusters. Species group a was an individual species line containing 25 strains that were identified as E. faecalis. Group b also represented an individual species line of 12 strains identified as E. faecium. The remaining seven strains that formed species groups c and d could not be fully identified to species by this analysis. It was concluded that BR-PCR of 16S rDNA followed by partial sequence analysis of the PCR products is a reliable technique for the identification and classification of enterococci. Further division of unresolved species groups should be achievable if regions other than V4 and V9 of 16S rDNA are also analysed.
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Analysis of lipids reveals differences between ‘Mycobacterium habana’ and Mycobacterium simiae
More LessFatty and mycolic acids and the pattern of glycolipids were studied in a collection of 34 strains of ‘Mycobacterium habana’ and in two strains of Mycobacterium simiae. Major glycolipids of these micro-organisms were assigned to the glycopeptidolipid (GPL) structural type, but both mycobacteria differed in the patterns obtained by TLC. The strains of ‘M. habana’ were separated into four groups (A-D), taking into account the presence or absence of several polar GPLs: group A contained GPL-I, GPL-II and GPL-III; group B contained GPL-I, GPL-II’, GPL-II and GPL-III; group C contained GPL-II’, GPL-II and GPL-III; group B did not contain any of these compounds. Fatty acids of both bacteria were similar, and ranged from 14 to 26 carbon atoms, hexadecanoic, octadecenoic and tuberculostearic acids being predominant. Mycolic acids were also similar by TLC and HPLC, and consisted of α-, α- and ketomycolates. Partial structural analysis by MS carried out in strains ‘M. habana’ TMC 5135 and M. simiae ATCC 25275T revealed that α- and ketomycolates ranged, in general, from 79 to 87 carbon atoms, and α-mycolates from 58 to 67 carbon atoms. The α- and ketomycolates belonged to several structural series, and minor variations were found between the two strains examined. The data obtained justified the synonymy between ‘M. habana’ and M. simiae but indicated, in turn, that the former can be distinguished on the basis of GPL analysis. Most strains of ‘M. habana’ can be defined by the presence of GPL-II and GPL-III, a finding that could be useful in the quality control of potential vaccine strains.
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- Genome Analysis
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Physical map of the genome of Oenococcus oeni PSU-1 and localization of genetic markers
More LessA physical map of the chromosome of Oenocccus oeni PSU-1 was constructed. This represents the first map for a strain of this species. A total of 37 restriction sites for the rare-cutting endonucleases Ascl, Fsel, Notl and Sfil were mapped on the chromosome, which was found to be circular with an estimated size of 1857 kb. Fragment order was determined using several approaches: analysis of partial and double digestions, two-dimensional pulsed-field gel electrophoresis, isolation of linking clones, and Southern hybridization with labelled restriction fragments both from PSU-1 and from O. oeni strain GM. Oenococcal genes alsS/alsD, mleA and mir, two phage attachment sites and recurrent sequences such as IS 1165-like elements and rrn loci were located on the physical map. Specific fragments hybridizing with gene probes from Lactococcus lactis, Leuconostoc mesenteroides and Bacillus subtilis were also identified. The two ribosomal operons have been precisely located and their transcription direction determined.
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Volumes and issues
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