- Volume 144, Issue 4, 1998
Volume 144, Issue 4, 1998
- Review Article
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- Microbiology Comment
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- Antigens And Immunity
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Affinity purification and characterization of a fibrinogen-binding protein complex which protects mice against lethal challenge with Streptococcus equi subsp. equi
More LessCell-wall-associated proteins from Streptococcus equi subsp. equi, the causative agent of strangles, were analysed with a view to identifying a potential protective antigen. Preparations of these proteins, isolated from mutanolysin extracts of cell walls, were shown to contain one major high-M r protein species (apparent M r 220000 and 550000 when analysed by SDS-PAGE and gel-filtration chromatography, respectively). The high-M r protein bound horse fibrinogen and was purified under non-denaturing conditions using fibrinogen affinity chromatography. The fibrinogen-binding protein (FgBP) reacted with serum taken from horses recovering from strangles and protected mice against lethal challenge from S. equi subsp. equi. The sequence of the corresponding gene (fbp) was determined and shown to encode a mature protein (M r 54597) with predicted coiled-coil structure. An FgBP truncate, lacking the C-terminal cell wall/membrane anchor domain, was overexpressed in and purified from Escherichia coli and was shown to behave in an analogous fashion to the wild-type product in terms of M r estimation, fibrinogen binding and seroreactivity.
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- Biochemistry
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Citrate synthase and 2-methylcitrate synthase: structural, functional and evolutionary relationships
More LessFollowing the complete sequencing of the Escherichia coli genome, it has been shown that the proposed second citrate synthase of this organism, recently described by the authors, is in fact a 2-methylcitrate synthase that possesses citrate synthase activity as a minor component. Whereas the hexameric citrate synthase is constitutively produced, the 2-methylcitrate synthase is induced during growth on propionate, and the catabolism of propionate to succinate and pyruvate via 2-methylcitrate is proposed. The citrate synthases of the psychrotolerant eubacterium DS2-3R, and of the thermophilic archaea Thermoplasma acidophilum and Pyrococcus furiosus, are approximately 40% identical in sequence to the Escherichia coli 2-methylcitrate synthase andalso possess 2-methylcitrate synthase activity. The data are discussed with respect tothe structure, function and evolution of citrate synthase and 2-methylcitrate synthase.
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Use of resistant mutants to characterize the target of mycobacillin in Aspergillus niger membranes
More LessThe mycobacillin-sensitive Aspergillus niger strain G3Br and resistant mutants of it did not show any differences in their total lipid content, although the amounts of phospholipids and sterols, particularly phosphatidylcholine and cholesterol, were lower in resistant cells. Mycobacillin resistance was accompanied by an increase in the phase-transition temperature of plasma membrane preparations. When exposed to mycobacillin, resistant and sensitive cells did not differ qualitatively with respect to most released materials (lysine, proline, Pi, Na+, K+, Ca2+); however, the release of ATP was completely inhibited in resistant cells unless they were exposed to concentrations of mycobacillin exceeding their respective MIC value. Resistant cells, under steady-state conditions, displayed greater uptake and release of the same specific materials - except ATP - as sensitive cells did under similar conditions. Thus release and uptake of those materials except ATP are not implicated in the mode of action of mycobacillin. The inhibiting action of mycobacillin (at concentrations higher than the MIC) on sensitive or resistant cells was completely antagonized by ATP (which did not form any complex with mycobacillin) but not by any of the releasable components, either alone or in combination. This observation, coupled with the authors’ recent findings on ATP release, indicates that the fungistatic action of mycobacillin is due to excessive ATP release, leading to energy starvation. Interestingly, ATP release during the first 2 h of incubation with mycobacillin was minimal, but increased to over 96% during the next 48 h. Release and uptake of ATP via liposomes, prepared with lipid and protein isolated from membranes of the mycobacillin-sensitive parent and resistant mutants, showed that mycobacillin action could be inhibited either by resistant protein or by resistant lipid. The mycobacillin target appears to be a lipid-protein site on the membrane of sensitive A. niger G3Br.
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- Bioenergetics And Transport
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Physiological function and regulation of flavocytochrome c3, the soluble fumarate reductase from Shewanella putrefaciens NCIMB 400
More LessShewanella putrefaciens produces a soluble flavocytochrome c under anaerobic growth conditions. This protein shares sequence similarity with the catalytic subunits of membrane-bound fumarate reductases from Escherichia coli and other bacteria and the purified protein has fumarate reductase activity. It is shown here that this enzyme, flavocytochrome c 3, is essential for fumarate respiration in vivo since disruption of the chromosomal fccA gene, which encodes flavocytochrome c 3, leads to a specific loss of the ability to grow with fumarate as terminal electron acceptor. Growth with nitrate, trimethylamine N-oxide (TMAO) and other acceptors was unaffected. The fccA gene is transcribed as a 2 kb monocistronic mRNA. An adjacent reading frame that bears limited sequence similarity to one of the membrane anchor subunits of E. coli fumarate reductase is not co-transcribed with fccA. Expression of the fccA gene is regulated by anaerobiosis and by the availability of alternative electron acceptors, particularly nitrate and TMAO. DNA sequences have been identified that are required for this regulation.
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Fructose and mannose metabolism in Aeromonas hydrophila: identification transport systems and catabolic pathways
More LessAeromonas hydrophila was examined for fructose and mannose transport systems. A. hydrophila was shown to possess a phosphoenolpyruvate (PEP): fructose phosphotransferase system (fructose-PTS) and a mannose-specific PTS, both induced by fructose and mannose. The mannose-PTS of A. hydrophila exhibited cross-reactivity with Escherichia coli mannose-PTS proteins. The fructose-PTS proteins exhibited cross-reactivities with E. coli and Xanthomonas campestris fructose-PTS proteins. In A. hydrophila grown on mannose as well as on fructose, the phosphorylated derivative accumulated from fructose was fructose 1-phosphate. Identification of fructose 1-phosphate was confirmed by 13C-NMR spectroscopy. 1-Phosphofructokinase (1-PFK), which converts the product of the PTS reaction to fructose 1,6-diphosphate, was present in A. hydrophila grown with fructose but not on mannose. An inducible phosphofructomutase (PFM) activity, an unusual enzyme converting fructose 1-phosphate to fructose 6-phosphate, was detected in extracts induced by mannose or fructose. These results suggest that in cells grown on fructose, fructose 1-phosphate could be converted to fructose 1,6-diphosphate either directly by the 1-PFK activity or via fructose 6-phosphate by the PFM and 6-phosphofructokinase activities. In cells grown on mannose, the degradation of fructose 1-phosphate via PFM and the Embden-Meyerhof pathway appeared to be a unique route.
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- Biotechnology
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Efficient conversion of 5-substituted hydantoins to D-α-amino acids using recombinant Escherichia coli strains
D-Amino acids, important intermediates in the production of semisynthetic penicillins and cephalosporins, are currently prepared from the corresponding hydantoins using bacterial biomass containing two enzymes, hydantoinase and carbamylase. These enzymes convert the hydantoins first into carbamyl derivatives and then into the corresponding D-amino acids. In an attempt to select more efficient biocatalysts, the hydantoinase and carbamylase genes from Agrobacterium tumefaciens (formerly A. radiobacter) were cloned in Escherichia coli. The genes were assembled to give two operon-type structures one having the carbamylase gene preceding the hydantoinase gene and the other with the carbamylase gene following the hydantoinase gene. The recombinant strains stably and constitutively produced the two enzymes and efficiently converted the corresponding hydantoins into p-hydroxyphenyl-glycine and phenylglycine. The order of the genes within the operon and the growth temperature of the strains turned out to be important for both enzyme and D-amino acid production. The configuration with the carbamylase gene preceding the hydantoinase gene was the most efficient one when the biomass was grown at 25°C rather than 37°C. This biomass produced D-amino acid twice as efficiently as the industrial strain of A. tumefaciens. The efficiency was found to be correlated with the level of carbamylase produced, indicating that the concentration of this enzyme is the rate-limiting factor in D-amino acid production under the conditions used on an industrial scale.
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- Environmental Microbiology
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Growth of green sulphur bacteria in experimental benthic oxygen, sulphide, pH and light gradients
More LessThe green sulphur bacterium Prosthecochloris aestuarii (strain CE 2401) was cultured in a benthic gradient chamber to study its growth and photosynthetic activity in experimental gradients of oxygen, sulphide and light. An axenic biofilm was obtained within evenly inoculated artificial sediment after 5 weeks of incubation. The phototrophic biofilm was located 2.2-3.5 mm below the sediment surface, i.e. below the maximal penetration depth of oxygen, thus confirming that growth of P. aestuarii was restricted to strictly anoxic sediment layers. The activity was limited by the diffusive flux of sulphide, showing the role of molecular diffusion in growth of this benthic species. Scalar irradiance was attenuated strongly in the biofilm, with distinct attenuation maxima at 750 nm corresponding to bacteriochlorophyll c (Bchl c) absorption and at 800 nm corresponding to bacteriochlorophyll a (Bchl a) absorption. Using radiance attenuation data as a proxy for photopigment contents it was shown that the ratio Bchl a/Bchl c changed with depth. This indicates chromatic adaptation to changing light climates in the sediment. Total sulphide oxidation was estimated from the sulphide fluxes from below into the reaction zone. Measurements of sulphide oxidation as a function of scalar irradiance in the reaction zone showed that anoxygenic photosynthesis of the biofilm was saturated at a scalar irradiance (430-830 nm)>2 μmol photons m-2 s-1.
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- Genetics And Molecular Biology
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A Candida albicans 37 kDa polypeptide with homology to the laminin receptor is a component of the translational machinery
More LessA cDNA encoding a 37 kDa protein was isolated from an expression library using antibodies raised against mycelial cell walls from Candida albicans. The 37 kDa protein has over 60% sequence identity with the 37 kDa laminin-binding protein (LBP) from humans and over 80% identity with the Yst proteins of Saccharomyces cerevisiae. The C. albicans protein was named CaYst1. It was found in membrane and ribosome fractions but surprisingly, was not found in cell walls. Unlike the human LBP, CaYst1p does not bind laminin. These data indicate that CaYst1p is not a cell-surface receptor for laminin as has been proposed for the human LBP. Instead, like the S. cerevisiae Yst proteins, it appears to be a ribosomal protein. This conclusion is supported by the finding that CaYST1-cDNA complements the lethal phenotype linked to the disruption of both YST genes in S. cerevisiae.
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Repetitive sequences (RPSs) in the chromosomes of Candida albicans are sandwiched between two novel stretches, HOK and RB2, common to each chromosome
A novel sequence designated HOK, which is next to the RPS, a repetitive sequence specific to Candida albicans, was cloned and sequenced. HOK hybridized with all of the chromosomes on which the RPSs were located, but did not hybridize with chromosome 3, which does not harbour any RPSs. Sequence determination revealed that a portion of HOK has significant homology with the B and C1 fragments of Ca3, which is used as a molecular epidemiological probe. A homology search of the deduced amino acids of HOK against the protein database showed partial homology with an isocitrate dehydrogenase of Saccharomyces cerevisiae, although an ORF large enough to encode the enzyme was not detected. To verify the existence of other sequences homologous with HOK, a portion of the HOK sequence was amplified using PCR. Sequence determination of the 41 clones from the PCR products resulted in at least six HOK-homologous clones. Another RPS-containing clone, RB2, was isolated from the Pstl-digested chromosome R or 1. It was determined that RB2a, one of the subclones from RB2, hybridized with all of the chromosomes, including chromosome 3, with which neither HOK nor RPS hybridized. The hybridization profile also showed that RPS is located between HOK and RB2a on chromosomes other than chromosome 3.
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The 172 kb prkA-addAB region from 83° to 97° of the Bacillus subtilis chromosome contains several dysfunctional genes, the glyB marker, many genes encoding transporter proteins, and the ubiquitous hit gene
A 171 812 bp nucleotide sequence betweenprkA andaddAB(83° to 97°) on the genetic map of theBacillus subtilis168 chromosome was determined and analysed. An accurate physical/genetic map of this previously poorly described chromosomal region was constructed. One hundred and seventy open reading frames (ORFs) were identified on this DNA fragment. These include the previously described genes cspB, glpPFKD, spoVR, phoAIV, papQ, citRA, sspB, prsA, hpr, pbpF, hemEHY, aprE, comKandaddAB. ORF yhaFin this region corresponds to the glyB marker. Among the striking features of this region are: an abundance of genes encoding (putative) transporter proteins, several dysfunctional genes, the ubiquitous hit gene, and five multidrug-resistance-like genes. These analyses have also revealed the existence of numerous paralogues of ORFs in this region: about two-thirds of the putative genes seem to have at least one paralogue in theB. subtilis genome
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The N-acetylmuramoyl-L-alanine amidase encoded by the Bacillus subtilis 168 prophage SPβ
More LessHeat shock of Bacillus subtilis CU1147, a strain lysogenic for SPβc2, a prophage with a thermosensitive repressor, results in phage induction and subsequent cell lysis. Cloning in Escherichia coli and sequencing of a DNA fragment of prophage SPβ led to the identification of blyA, the gene encoding a 367 amino acid polypetide with a molecular mass of 39.6 kDa. Purified BlyA obtained from the E. coli clone exhibited an N-acetylmuramoyl-L-alanine amidase activity. Insertional mutagenesis confirmed that the latter enzyme was associated with SPβ-phage-mediated ceil lysis. Analysis of the neighbouring sequence suggested that the two ORFs immediately downstream of blyA and belonging to the same operon encode polypeptides which may be involved in the release of the endolysin. The presence on the chromosomes of B. subtilis or related Bacillus spp. of other, similar genes, and their possible relationship, is discussed.
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An operon encoding three glycolytic enzymes in Lactobacillus delbrueckii subsp. bulgaricus: glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and triosephosphate isomerase
More LessThe structural genes gap, pgk and tpi encoding three glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 3-phosphoglycerate kinase (PGK) and triosephosphate isomerase (TPI), respectively, have been cloned and sequenced from Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus). The genes were isolated after screening genomic sublibraries with specific gap and pgk probes obtained by PCR amplification of chromosomal DNA with degenerate primers corresponding to amino acid sequences highly conserved in GAPDHs and PGKs. Nucleotide sequencing revealed that the three genes were organized in the order gap-pgk-tpi. The translation start codons of the three genes were identified by alignment of the N-terminal sequences. These genes predicted polypeptide chains of 338, 403 and 252 amino acids for GAPDH, PGK and TPI, respectively, and they were separated by 96 bp between gap and pgk, and by only 18 bp between pgk and tpi. The codon usage in gap, pgk, tpi and three other glycolytic genes from L. bulgaricus differed noticeably from that in other chromosomal genes. The site of transcriptional initiation was located by primer extension, and a probable promoter was identified for the gap-pgk-tpi operon. Northern hybridization of total RNA with specific probes showed two transcripts, an mRNA of 1.4 kb corresponding to the gap gene, and a less abundant mRNA of 3.4 kb corresponding to the gap-pgk-tpi cluster. The absence of a visible terminator in the 3′-end of the shorter transcript and the location of this 3′-end inside the pgk gene indicated that this shorter transcript was produced by degradation of the longer one, rather than by an early termination of transcription after the gap gene.
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Pyruvate carboxylase from Corynebacterium glutamicum: characterization, expression and inactivation of the pyc gene
In addition to phosphoenolpyruvate carboxylase (PEPCx), pyruvate carboxylase (PCx) has recently been found as an anaplerotic enzyme in the amino-acid-producing bacterium Corynebacterium glutamicum. Using oligonucleotides designed according to conserved regions of PCx amino acid sequences from other organisms, a 200 bp fragment central to the C. glutamicum PCx gene (pyc) was amplified from genomic DNA by PCR. This fragment was then used to identify and to subclone the entire C. glutamicum pyc gene. The cloned pyc gene was expressed in C. glutamicum, as cells harbouring the gene on plasmid showed four- to fivefold higher specific PCx activities when compared to the wild-type (WT). Moreover, increased PCx protein levels in the pyc-plasmid-carrying strain were readily detected after SDS-PAGE of cell-free extracts. DNA sequence analysis of the pyc gene, including its 5’ and 3’ flanking regions, and N-terminal sequencing of the pyc gene product predicts a PCx polypeptide of 1140 amino acids with an M r of 123070. The amino acid sequence of this polypeptide shows between 62% and 45% identity when compared to PCx enzymes from other organisms. Transcriptional analyses revealed that the pyc gene from C. glutamicum is monocistronic (3.5 kb mRNA) and that its transcription is initiated at an A residue 55 bp upstream of the translational start. Inactivation of the chromosomal pyc gene in C. glutamicum WT led to the absence of PCx activity and to negligible growth on lactate, indicating that PCx is essential for growth on this carbon source. Inactivation of both the PCx gene and the PEPCx gene in C. glutamicum led additionally to the inability to grow on glucose, indicating that no further anaplerotic enzymes for growth on carbohydrates exist in this organism.
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Cloning of new Rhodococcus extradiol dioxygenase genes and study of their distribution in different Rhodococcus strains
Four extradiol dioxygenase genes which encode enzymes active against catechol and substituted catechols were cloned from two different Rhodococcus strains, and their nucleotide sequences were determined. A catechol 2,3-dioxygenase gene (edoC) was shown to be identical to the previously described ipbC gene from the isopropylbenzene operon of Rhodococcus erythropolis. Amino acid sequences deduced from the three other genes (edoA, edoB and edoD) were shown to have various degrees of homology to different extradiol dioxygenases. The EdoA and EdoB dioxygenases were classified as belonging to the third family of type I oxygenases and represented two new subfamilies, whereas the EdoD dioxygenase was a type II enzyme. Analysis of six Rhodococcus strains revealed a wide distribution of the above dioxygenase genes. Rhodococcus sp. I1 was shown to harbour all four of the analysed dioxygenase genes. Nucleotide sequences homologous to the edoB gene were present in all of the strains, including R. erythropolis NCIMB 13065, which did not utilize any of the aromatic compounds analysed. The latter finding points to the existence of a silent pathway(s) for degradation of aromatic compounds in this Rhodococcus strain.
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Genetics of ferulic acid bioconversion to protocatechuic acid in plant-growth-promoting Pseudomonas putida WCS358
More LessTransposon Tn5 genomic mutants of plant-growth-promoting Pseudomonas putida strain WCS358 have been isolated which no longer utilize ferulic and coumaric acids as sole sources of carbon and energy. Genetic studies confirmed previous biochemical data showing that ferulic acid is degraded via vanillic acid, and coumaric acid via hydroxybenzoic acid. The genes involved in these enzymic steps were cloned and characterized. Two proteins designated Fca (26.5 kDa) and Vdh (50.3 kDa) were identified as responsible for the conversion of ferulic acid to vanillic acid; the proteins are encoded by the fca and vdh genes which are organized in an operon structure in the chromosome. The Vdh protein is 69% identical at the amino acid level to the Vdh protein recently identified in Pseudomonas sp. strain HR199 and converts vanillin to vanillic acid. Homology studies revealed that the Vdh proteins exhibited significant identity to aldehyde dehydrogenases from different organisms whereas Fca belonged to the enoyl-CoA hydratase family of proteins. Two proteins, designated VanA (39.9 kDa) and VanB (34.3 kDa), encoded by two genes, vanA and vanB, are organized in an operon in the chromosome. They were found to be responsible for the demethylation of vanillic acid to protocatechuic acid. The VanA proteins showed no homology to any other known protein, while VanB belonged to the ferredoxin family of proteins. This two-component enzyme system demethylated another phenolic monomer, veratric acid, thus indicating broad specificity. Studies of the regulation of the vanAB operon demonstrated that the genes were induced by the substrate, vanillic acid; however, the strongest induction was observed when cells were grown in the presence of the product of the reaction, protocatechuic acid.
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Comparative analysis of Pseudomonas aeruginosa penicillin-binding protein 7 in the context of its membership in the family of low-molecular-mass PBPs
More LessThe Pseudomonas aeruginosa pbpG gene encoding penicillin-binding protein 7, a homologue of the Escherichia coli gene encoding a DD-endopeptidase, was cloned and sequenced. pbpG was located immediately downstream of the phenylalanine hydroxylase (phh) operon. DNA sequencing revealed an open reading frame of 936 bp (starting with a GTG codon) which encodes a protein of 34115 Da. N-terminal amino acid sequencing confirmed the presence of a cleavable N-terminal signal peptide of 23 amino acids. Verification that the protein is a penicillin-binding protein was directly demonstrated by labelling with 125l-labelled penicillin X. Inactivation of P. aeruginosa pbpG by interposon mutagenesis resulted in no obvious phenotypic changes, but when P. aeruginosa PbpG was overexpressed in E. coli using a T7 expression system, cell lysis resulted. P. aeruginosa PbpG resembled E. coli PbpG in being associated with the membrane fraction. Two additional members of the PbpG subfamily were identified in the database. P. aeruginosa PbpG shows 63% identity with E. coli penicillin-binding protein 7 (PbpG) and 60% identity with Vibrio cholerae PbpG, but only 23% identity with Haemophilus influenzae PbpG. The PbpG subfamily and three other subfamilies constituting the low-molecular-mass PBP protein family were analysed by multiple alignment of 26 sequences. PbpG exhibited the consensus motifs of other penicillin-binding proteins. Ten anchor residues were identified that are conserved at the family level within the superfamily of serine-active-site penicillin-interacting proteins.
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A second IgG-binding protein in Staphylococcus aureus
More LessMost strains of Staphylococcus aureus express IgG-binding activity and this binding has been considered to be solely mediated by protein A. However, the existence of a second gene in S. aureus strain 8325-4 encoding an IgG-binding polypeptide was recently reported. This novel IgG-binding polypeptide was found after panning a shotgun phage display library, made from chromosomal DNA, against immobilized human IgG. The complete gene (sbi) encoding this novel IgG-binding protein, denoted protein Sbi, has now been cloned and sequenced. Analysis of other S. aureus strains showed that this gene is not unique for strain 8325-4. The protein consists of 436 amino acids and exhibits an immunoglobulin-binding specificity similar to protein A. Furthermore, it is shown that Sbi is highly expressed in strain Newman 4, which shows that IgG-binding activity in S. aureus can be mediated by proteins other than protein A.
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Oligopeptide permease in Borrelia burgdorferi: putative peptide-binding components encoded by both chromosomal and plasmid loci
More LessTo elucidate the importance of oligopeptide permease for Borrelia burgdorferi, the agent of Lyme disease, a chromosomal locus in B. burgdorferi that encodes homologues of all five subunits of oligopeptide permease has been identified and characterized. B. burgdorferi has multiple copies of the gene encoding the peptide-binding component, OppA; three reside at the chromosomal locus and two are on plasmids. Northern analyses indicate that each oppA gene is independently transcribed, although the three chromosomal oppA genes are also expressed as bi- and tri-cistronic messages. Induction of one of the plasmid-encoded oppA genes was observed following an increase in temperature, which appears to be an important cue for adaptive responses in vivo. The deduced amino acid sequences suggest that all five borrelial OppA homologues are lipoproteins, but the protease-resistance of at least one of them in intact bacteria is inconsistent with outer-surface localization. Insertional inactivation of a plasmid-encoded oppA gene demonstrates that it is not essential for growth in culture.
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Tandem organization and highly disparate expression of the two laccase genes lcc1 and lcc2 in the cultivated mushroom Agaricus bisporus
More LessTwo non-allelic laccase genes (lcc1 and lcc2) in Agaricus bisporus have been mapped to the same cosmid clone and are close together, in tandem. The intergenic region consists of 1562 bp between the stop codon of lcc1 and the start codon of lcc2. Differences between the 5′ non-coding regions of the two genes suggest the potential for their differential regulation. By employing competitive RT-PCR and specific primer pairs that discriminate between lcc1 and lcc2, it has been shown that the level of lcc2 mRNA is approximately 300 times higher than that of lcc1 mRNA in malt extract liquid cultures; in compost cultures lcc2 mRNA is almost 7000 times more abundant than lcc1 mRNA.
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Molecular analysis of Physarum haemagglutinin l: lack of a signal sequence, sulphur amino acids and post-translational modifications
More LessThe cDNAs encoding haemagglutinin I from plasmodia of Physarum polycephalum have been cloned using PCR protocols. The composite haemagglutinin I cDNA sequence, derived from several overlapping clones from PCR fragments, spans 408 nt and the 315 bp ORF encodes a polypeptide of 104 aa without a typical signal sequence. The putative molecular mass deduced from the amino acid sequence (10760.76 Da) corresponds exactly to that determined by electrospray ionization MS (10759.86±.15 Da), suggesting that haemagglutinin I is not subject to post-translational modification. Haemagglutinin I lacks sulphur amino acids and has a β-sheet as the major secondary structure. Expression of the coding sequence in Escherichia coli yielded a product that exhibits the same sugar-binding specificity as natural haemagglutinin I. The deduced amino acid sequence shows little similarity to that of any known lectins and thus apparently represents a novel type of lectin.
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Identification of a CAP (adenylyl-cyclase-associated protein) homologous gene in Lentinus edodes and its functional complementation of yeast CAP mutants
The adenylyl-cyclase-associated protein, CAP, was originally identified in yeasts as a protein that functions in both signal transduction and cytoskeletal organization. This paper reports the identification of a cDNA and genomic DNA that encodes a CAP homologue from the mushroom Lentinus edodes. The L. edodes cap gene contains eight introns and an ORF encoding a 518 amino acid protein. The L. edodes CAP is 35.5% and 40.9% identical at the amino acid level with Saccharomyces cerevisiae CAP and Schizosaccharomyces pombe CAP, respectively. The C-terminal domain shows greater homology (39-46% identity) with yeast CAPs than does the N-terminal domain (27-35% identity). Southern blotting and Northern blotting results suggest that L. edodes cap is a sinμle-copy gene and uniformly expressed. Expression of the L. edodes CAP in both Schiz. pombe and Sacch. cerevisiae complemented defects associated with the loss of the C-terminal domain function of the endogenous CAP. By using a yeast two-hybrid assay, an interaction was demonstrated between the L. edodes CAP and Schiz. pombe actin. This result and the functional complementation test indicate that CAP from L. edodes has a conserved C-terminal domain function.
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D-Amino-acid oxidase gene from Rhodotorula gracilis (Rhodosporidium toruloides) ATCC 26217
The complete nucleotide sequence of the DAO1 gene encoding D-amino-acid oxidase (DAAO) in the yeast Rhodotorula gracilis (Rhodosporidium toruloides) ATCC 26217 has been determined. The primary structure of DAAO was deduced from the nucleotide sequence of a cDNA clone that covered the entire amino acid coding sequence. Comparison of cDNA and genomic sequences of DAO1 revealed the presence of five introns. Because this is the first gene of strain ATCC 26217 that has been cloned so far, the nucleotide sequences of these introns were compared to those from other fungi. Upstream of the structural gene there was a stretch of C + T-rich DNA similar to that found in the promoter region of a number of yeast genes. The cDNA gene, which encoded a protein of 368 amino acids (molecular mass 40 kDa), was overexpressed in Escherichia coli under the control of the strong lipoprotein promoter. Interestingly, a significant fraction (13-62%) of the total DAAO activity was recovered in its apoenzyme form, the percentage depending on the culture conditions. This fact allowed a rapid purification of the recombinant DAAO by affinity chromatography. The high level of expression achieved in E. coli and the possibility of modifying its catalytic properties by protein engineering provide a new model for the study of this enzyme.
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- Pathogenicity And Medical Microbiology
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Candida dubliniensis: phylogeny and putative virulence factors
More LessCandida dubliniensis is a recently identified species which is implicated in oral candidosis in HIV-infected and AIDS patients. The species shares many phenotypic characteristics with, and is phylogenetically closely related to, Candida albicans. In this study the phylogenetic relationship between these two species was investigated and a comparison of putative virulence factors was performed. Four isolates of C. dubliniensis from different clinical sources were chosen for comparison with two reference C. albicans strains. First, the distinct phylogenetic position of C. dubliniensis was further established by the comparison of the sequence of its small rRNA subunit with representative Candida species. The G dubliniensis isolates formed true unconstricted hyphae under most induction conditions tested but failed to produce true hyphae when induced using N-acetylglucosamine. Oral G dubliniensis isolates were more adherent to human buccal epithelial cells than the reference C. albicans isolates when grown in glucose and equally adherent when grown in galactose. The G dubliniensis isolates were sensitive to fluconazole, itraconazole, ketoconazole and amphotericin B. Homologues of seven tested G albicans secretory aspartyl proteinase (SAP) genes were detected in G dubliniensis by Southern analysis. In vivo virulence assays using a systemic mouse model suggest that G dubliniensis is marginally less virulent than C. albicans. These data further confirm the distinct phenotypic and genotypic nature of G dubliniensis and suggest that this species may be particularly adapted to colonization of the oral cavity.
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The Staphylococcus aureus and Staphylococcus epidermidis transferrin-binding proteins are expressed in vivo during infection
More LessStaphylococci express a 42 kDa cell-wall-associated protein which functions as a receptor for the mammalian iron-binding glycoprotein transferrin. To determine whether this transferrin-binding protein (TBP) is expressed during infection, Staphylococcus aureus and Staphylococcus epidermidis were grown in vivo in chambers implanted intraperitoneally in rats. SDS-PAGE and Western blotting of cell wall proteins prepared from staphylococci recovered directly from the chambers revealed the presence of both the TBP and bacterial-surface-associated rat transferrin. To obtain evidence for the in vivo expression of the staphylococcal TBPs in humans, sera and human peritoneal dialysate (HPD) from non-infected patients undergoing continuous ambulatory peritoneal dialysis (CAPD) and sera from healthy human volunteers were screened for anti-TBP antibodies. Western immunoblots revealed that three out of ten samples from the latter group, seven out of ten HPD samples and ten of ten CAPD patient serum samples contained antibodies to the TBP of both S. aureus and S. epidermidis. To gain further insights into the appearance of TBP antibodies, HPD samples were collected over time from CAPD patients whose HPD samples taken immediately after catheter insertion lacked anti-TBP antibodies. In two of these patients, each of whom experienced an episode of peritonitis due to S. epidermidis or Staphylococcus hominis, antibodies to the TBP appeared in the HPD collected immediately post-infection. To determine whether such TBP antibodies were capable of blocking interactions between transferrin and its staphylococcal receptor, HPD immunoglobulin fractions were purified using protein A-Sepharose beads. In competition assays, these immunoglobulins blocked the binding of 125l-labelled transferrin both to whole bacteria and to the isolated 42 kDa TBPs of S. aureus and S. epidermidis. These provide evidence to show that staphylococcal TBPs are expressed in vivo during infection.
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The rrs (16S)-rrl (23S) ribosomal intergenic spacer region as a target for the detection of Haemophilus ducreyi by a heminested-PCR assay
More LessThe intergenic spacer region between the rrs and rrl ribosomal RNA genes of Haemophilus ducreyi was analysed and the DNA sequence was used for the selection of specific PCR primers. A highly sensitive and specific heminested-PCR assay for the identification of H. ducreyi was developed. The assay showed a sensitivity of 96% on genital ulcer specimens from patients with clinically diagnosed chancroid, compared with a sensitivity of 56% for culture methods. These results indicate that this PCR assay has the potential to become an accurate and easy reference method for the detection of H. ducreyi.
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Carbapenems as inhibitors of OXA-13, a novel, integron-encoded β-lactamase in Pseudomonas aeruginosa
More LessA clinical Pseudomonas aeruginosa strain, PAe391, was found to be resistant to a number of antibiotics including ticarcllin, piperacillin, cefsulodin and amikacin, and a disk diffusion assay showed evidence of pronounced synergy between imipenem and various β;-lactam antibiotics. Cloning and nucleotide sequence analysis revealed the dicistronic arrangement of an aac(6’)-lb variant and a novel bla OXA-type gene between the intl and qacEδ1 genes typical of integrons. In PAe391, this integron was apparently chromosome-borne. The β;-lactamase, named OXA-13, displayed nine amino acid changes with respect to OXA-10: I in position 10 of OXA-10 to T (I10T), G20S, D55N, N73S, T107S, Y174F, E229G, S245N and E259A. OXA-13 (plapp = 8.0) showed poor catalytic activity against penicillins as well as cephalosporins, but was efficient in hydrolysing some penicillinase-resistant β;-lactams, such as cefotaxime and aztreonam. It was efficiently inhibited by imipenem (K iapp = 11 nM), and formed a stable complex. While the K iapp value of meropenem was similar (16 nM), the corresponding complex was less stable.
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- Physiology And Growth
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Effects on Bacillus subtilis of conditional expression of the accBC operon encoding subunits of acetyl coenzyme A carboxylase, the first enzyme of fatty acid synthesis
More LessA Bacillus subtilis strain was constructed in which the operon accBC, encoding the biotin carboxyl carrier protein (BCCP) and biotin carboxylase (BC) subunits of acetyl-CoA carboxylase (ACC), was placed under the control of the IPTG-inducible promoter spac. A reduction in the levels of BCCP resulted in a decrease of de novo fatty acid synthesis and in the total content of membrane fatty acids. This strain was dependent upon the presence of IPTG for a normal growth phenotype. Growth was specifically restored by supplying exogenous long branched-chain fatty acids in the medium, indicating that the inducer-dependent phenotype was specifically related to a conditional block in fatty acid biosynthesis. The strain showed a strong decrease in sporulation frequency when it was induced to sporulate in an IPTG-free medium. Germination and outgrowth were both delayed in spores of the mutant obtained in the absence of IPTG.
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The cadmium-stress stimulon of Escherichia coli K-12
More LessThe influence of cadmium on stress protein production in Escherichia coli K-12 (strain MG1655) was analysed using two-dimensional polyacrylamide gel electrophoresis and the gene-protein database of E. coli K-12. Cadmium (273 μM) caused complete but transient inhibition of growth accompanied by the synthesis of cadmium-induced proteins (CDPs). It was found that some CDPs induced during the growth-arrested phase belong to the heat-shock, oxidation stress, SOS and stringent response regulons, while others are general stress inducible proteins (e.g. H-NS, UspA). In addition, trigger factor, adenylate kinase, W-protein, the cold shock protein G041.2, and seven unknown proteins whose synthesis is not known to be controlled by a global regulator, were identified as immediate responders to cadmium exposure. The rate of synthesis of most of the immediate responders to cadmium exposure decreased when the growth of the cells resumed. However, seven CDPs, including those encoded by argl, tyrA and xthA, maintained a high production rate during growth in the presence of cadmium. Two of the unidentified proteins were N-terminally sequenced by Edman degradation. The N-terminal amino acid sequence of one of these proteins (designated F023.3) matches the E. coli open reading frame o216. This ORF is similar to the N-terminal third of the copper-binding protein amine oxidases (encoded by maoA) of both E. coli and Klebsiella pneumoniae (K. aerogenes). The other N-terminally sequenced protein (designated C044.6) matches perfectly the product of the metK gene, S-adenosylmethionine synthetase I. In comparison to untreated cells, cadmium-stressed cells were found to recover more rapidly during subsequent stress conditions, such as ethanol, osmotic, heat shock, and nalidixic acid treatment. The role of the CDPs is discussed in view of their physiological assignments in the cell.
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Phytophthora cactorum can synthesize substances needed for sexual reproduction but requires a stress factor to trigger the process
More LessPhytophthora cactorum grown on basal agarose medium or in liquid basal medium produced oospores after being transferred to water agarose. The numbers of oospores produced under such conditions depended on the age of the culture prior to exposure to nutrient deprivation. When the concentration of basal medium used for cultivation of P. cactorum was increased, the numbers of oospores produced after being transferred to water agarose was also increased. P. cactorum grown on basal agarose medium also produced oospores when its mycelial growth was restricted after reaching the edge of Petri plates. In 5 cm plates oospore formation occurred in the third week, whereas in 9 cm and 14 cm plates oospores appeared in the fourth week. Most oospores were formed near the edge of the plates. The non-saponifiables extracted from mycelia of P. cactorum grown in liquid basal medium were stimulatory to oospore formation by P. cactorum and Phytophthora parasitica, whereas the saponifiables were stimulatory to P. cactorum only. Extracts from culture filtrate and basal medium were not stimulatory to oospore formation by either fungus. When the non-saponifiables were fractionated by Florisil columm chromatography, only fraction 1 was not active. Fractions 2, 3 and 4 were stimulatory to oospore formation by both P. cactorum and P. parasitica. These results support the hypothesis that P. cactorum, and possibly other pythiaceous fungi as well, can synthesize substances needed for sexual reproduction but requires a stress factor to trigger the process.
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Lack of correlation between trehalose accumulation, cell viability and intracellular acidification as induced by various stresses in Saccharomyces cerevisiae
More LessA pma1-1 mutant of Saccharomyces cerevisiae with reduced H+-ATPase activity and the isogenic wild-type strain accumulated high levels of trehalose in response to a temperature upshift to 40 éC and after addition of 10% ethanol, but only modest levels in response to a rapid drop in external pH and after addition of decanoic acid. There was, however, no correlation between the absolute levels of trehalose in the stressed cells and their viability. All these treatments induced a significant decrease in intracellular pH, and surprisingly, this decrease was very similar in both strains, indicating that intracellular acidification could not be the triggering mechanism for trehalose accumulation in response to stress. A careful investigation of metabolic parameters was carried out to explain how trehalose accumulated under the four different stress conditions tested. No single and common mechanism for trehalose accumulation could be put forward and the transcriptional activation of TPS1 was not unequivocally related to trehalose accumulation. Another finding was that a pma1-1 mutant exhibited a two- to threefold greater capacity to accumulate trehalose than the isogenic wild-type. This enhanced disaccharide synthesis could be attributed to a twofold higher trehalose-6-phosphate synthase activity, together with a fourfold higher content of intracellular UDP-Glc. In addition, this mutant showed 1.5-fold higher levels of ATP compared to the wild-type. The various stress treatments studied showed that a drop in intracellular pH does not correlate with trehalose accumulation. It is suggested that plasma membrane alteration could be the physiological trigger inducing trehalose accumulation in yeast.
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- Genome Analysis
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A 35.7 kb DNA fragment from the Bacillus subtilis chromosome containing a putative 12.3 kb operon involved in hexuronate catabolism and a perfectly symmetrical hypothetical catabolite-responsive element
The Bacillus subtilis strain 168 chromosomal region extending from 109† to 112° has been sequenced. Among the 35 ORFs identified, cotT and rapA were the only genes that had been previously mapped and sequenced. Out of ten ORFs belonging to a single putative transcription unit, seven are probably involved in hexuronate catabolism. Their sequences are homologous to Escherichia coli genes exuT, uidB, uxaA, uxaB, uxaC, uxuA and uxuB, which are all required for the uptake of free D-glucuronate, D-galacturonate and β-glucuronide, and their transformation into glyceraldehyde 3-phosphate and pyruvate via 2-keto-3-deoxygluconate. The remaining three ORFs encode two dehydrogenases and a transcriptional regulator. The operon is preceded by a putative catabolite-responsive element (CRE), located between a hypothetical promoter and the RBS of the first gene. This element, the longest and the only so far described that is fully symmetrical, consists of a 26 bp palindrome matching the theoretical B. subtilis CRE sequence. The remaining predicted amino acid sequences that share homologies with other proteins comprise: a cytochrome P-450, a glycosyltransferase, an ATP-binding cassette transporter, a protein similar to the formate dehydrogenase α-subunit (FdhA), a protein similar to NADH dehydrogenases, and three homologues of polypeptides that have undefined functions.
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