- Volume 142, Issue 11, 1996
Volume 142, Issue 11, 1996
- Review Article
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- Microbiology Comment
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- Antigens And Immunity
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Sequence variability of FrpB, a major iron-regulated outer-membrane protein in the pathogenic neisseriae
More LessThe FrpB protein from pathogenic neisseriae is a 77 kDa iron-regulated outer-membrane protein that belongs to the family of TonB-dependent receptors and may have potential as a vaccine component. Comparison between the frpB gene from three different meningococcal strains and a published gonococcal one revealed that the region from residues 350 to 390 displays pronounced sequence variability. In a model for the topology of FrpB in the outer membrane, this region corresponds to loop 7, the longest of the predicted 13 surface-exposed loops. Binding of four out of a total of eight bactericidal monoclonal antibodies to synthetic peptides corresponding to loop 7 showed that their epitopes are located here. The frpB genes from five additional meningococcal strains were cloned and sequenced in this region. Pairwise comparisons showed different degrees of similarity.
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- Biochemistry
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Kinetic analysis and substrate specificity of Escherichia coli dimethyl sulfoxide reductase
More LessWe have characterized the substrate specificity of dimethyl sulfoxide reductase (DmsABC) of Escherichia coli by determining K m and k cat values for 22 different substrates. The enzyme has a very broad substrate specificity. The K m values varied 470-fold, while k cat values varied only 20-fold, implicating K m as the major determinant of k cat/K m values. Sulfoxides and pyridine N-oxide exhibited the lowest K m values, followed by aliphatic N-oxides. The k cat values for these compounds also followed the same pattern. Substitution at the 2 or 3 position of the pyridine N-oxide ring had little effect on K m’ while substitution at the 4 position had a greater effect, and increased K m. Negatively charged substrates were poorly accepted. A few compounds that are not S- or N-oxides were also reduced by the enzyme. Most compounds reduced by DmsABC were not toxic to E. coli under anaerobic growth conditions, and E. coli was able to use many of these compounds anaerobically as terminal electron acceptors in the presence of glycerol. Anaerobic growth on sulfoxides is solely due to DmsABC expression. However, there appears to be another as yet unidentified terminal reductase capable of using pyridine N-oxides as terminal electron acceptors.
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- Environmental Microbiology
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Studies on the isopropylbenzene 2,3-dioxygenase and the 3-isopropylcatechol 2,3-dioxygenase genes encoded by the linear plasmid of Rhodococcus erythropolis BD2
More LessThe enzymes responsible for the degradation of isopropylbenzene (IPB) and co-oxidation of trichloroethene (TCE) by Rhodococcus erythropolis BD2 are encoded by the linear plasmid pBD2. Fragments containing IPB catabolic genes were cloned from pBD2 and the nucleotide sequence was determined. By means of database searches and expression of the cloned genes in recombinant strains, we identified five clustered genes, ipbA1A2A3A4C, which encode the three components of the IPB 2,3-dioxygenase system, reductaseIPB (ipbA4), ferredoxinIPB (ipbA3) and the two subunits of the terminal dioxygenase (ipbA1A2), as well as the 3-isopropylcatechol (IPC) 2,3-dioxygenase (ipbC). The protein sequences deduced from the ipbA1A2A3A4C gene cluster exhibited significant homology with the corresponding proteins of analogous degradative pathways in Gram-negative and Gram-positive bacteria, but the gene order differed from most of them. IPB 2,3-dioxygenase and 3-IPC 2,3-dioxygenase could both be expressed in Escherichia coli, but the IPB 2,3-dioxygenase activities were too low to be detected by polarographic and TCE degradative means. However, inhibitor studies with the R. erythropolis BD2 wild-type are in accordance with the involvement of the IPB 2,3-dioxygenase in TCE oxidation.
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- Genetics And Molecular Biology
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Immunochemical structure of the OmpD porin from Salmonella typhimurium
More LessThe OmpD porin was isolated and purified from Salmonella typhimurium strain SH 7454 (ompC::Tn10), digested with cyanogen bromide (CNBr) and the peptide fragments were separated by SDS-PAGE. N-terminal sequencing identified a total of 96 residues from four distinct peptides. The sequence showed that OmpD is homologous to NmpC (75% identity), Lc (75%) and OmpC (70%) from Escherichia coli, and OmpC (68%) from S. typhimurium. The sequence was essentially identical to the translated sequence of an nmpC-like gene of S. typhimurium, currently placed at 38·6 centisomes of the chromosome. Our results and other data suggest, however, that this gene is actually the ompD gene, which is more correctly placed in the 34 centisome region of the chromosome. The CNBr-generated peptides were also screened with 16 anti-S. typhimurium OmpD monoclonal antibodies by Western blotting. These results, in conjunction with the prediction of the OmpD folding pattern based on the known three-dimensional structure of E. coli OmpF, showed a close immunological relationship among S. typhimurium OmpD and E. coli NmpC and Lc, and a strong conservation of sequences within the transmembrane β strands of these porins and E. coli OmpC, PhoE and OmpF, and Salmonella typhi OmpC.
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hymA (hypha-like metulae), a new developmental mutant of Aspergillus nidulans
More LessAsexual fruiting body development in Aspergillus nidulans requires a precise spatial and temporal coordinated expression of many genes. Insertional mutagenesis was used to isolate and characterize a new mutant of A. nidulans in which hyphal growth was slightly reduced and conidiophore development was specifically blocked at the metula stage. In contrast to the uninucleate metulae of the wild-type, in the mutant these structures were elongated, multinucleate and septate. Further differentiation and production of phialides by a budding-like process was not observed. The mutant metulae thus resembled hyphae rather than metulae and the gene was therefore named hypha-like metulae (hymA). The hymA gene was mapped to linkage group VI. The integrated vector was rescued with border sequences from the integration site. The border sequences were used to isolate a cosmid from a wild-type library which was subcloned to a 5 kb fragment able to complement the mutation in trans. This fragment encoded a 1·8 kb transcript expressed in hyphae and throughout development. It is proposed that hymA is involved in budding processes, and is required for the formation of metulae and for their further differentiation.
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The purB gene of Escherichia coli K-12 is located in an operon
More LessThe structural gene (purB) for succinyl-AMP (S-AMP) lyase and three additional ORFs are on the same DNA strand of the chromosome of Escherichia coli. Cassette mutagenesis and primer extension mapping demonstrated that purB is co-transcribed with an upstream gene (ORF23, or ycfC) encoding a 22·9 kDa membrane-associated protein of non-essential, but unknown, function unrelated to purine biosynthesis. The purB operon lies between phoP and an ORF expressing an essential function which may correspond to asuE (trmU). S-AMP lyase was purified to near homogeneity. The purified enzyme is a homotetramer of 50 kDa subunits, has a K m for S-AMP of 3·7 μM and a pH optimum of 7·4–7·6.
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Proline biosynthesis in Streptococcus thermophilus: characterization of the proBA operon and its products
More LessThe presence of proline in the medium was not essential for growth of Streptococcus thermophilus, indicating that there is a proline biosynthetic pathway in this organism. Genetic and biochemical analysis identified and characterized this pathway. Two genes, designated proB and proA, were cloned, sequenced and characterized. Biochemical analysis of the proB- and proA-encoded enzymes showed that the proline biosynthetic pathway of S. thermophilus is similar to the one previously described in Escherichia coli. The deduced amino acid sequence of a 2·408 kb DNA region containing the genes revealed the similarity of the S. thermophilus gene products to ProB and ProA of E. coli and Serratia marcescens, and to the corresponding N- and C-terminal domains of the bifunctional plant enzyme Δ1-pyrroline-5-carboxylate synthetase of Vigna aconitifolia. Northern blot analysis showed that the two genes in S. thermophilus are organized in a single operon with proB proximal and proA distal to the promoter; primer extension analysis indicated that proBA transcription is not under repressive control by exogenously supplied proline.
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RP4::Mu3A-mediated in vivo cloning and transfer of a chlorobiphenyl catabolic pathway
Chromosomal DNA fragments encoding the ability to utilize biphenyl as sole carbon source (Bph+) were mobilized by means of plasmid RP4::Mu3A from strain JB1 (tentatively identified as Burkholderia sp.) to Alcaligenes eutrophus CH34 at a frequency of 10−8 per transferred plasmid. The mobilized DNA integrated into the recipient chromosome or was recovered as catabolic prime plasmids. Three Bph+ prime plasmids were transferred from A. eutrophus to Escherichia coli and back to A. eutrophus without modification of the phenotype. The transferred Bph+ DNA segments allowed metabolism of biphenyl, 2-, 3- and 4-chlorobiphenyl, and diphenylmethane. Genes involved in biphenyl degradation were identified on the prime plasmids by DNA-DNA hybridization and by gene cloning. Bph+ prime plasmids were transferred to Burkholderia cepacia, Pseudomonas aeruginosa, Comamonas testosteroni and A. eutrophus and the catabolic genes were expressed in those hosts. Transfer of the plasmid to the 3-chlorobenzoate-degrading bacterium Pseudomonas sp. B13 allowed the recipient to mineralize 3-chlorobiphenyl. Other catabolic prime plasmids were obtained from JB1 by selection on m-hydroxybenzoate and tyrosine as carbon sources. 16S rRNA sequence data demonstrated that the in vivo transfer of bph was achieved between bacteria belonging to two different branches of the β-Proteobacteria.
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Complete sequence and organization of the Serratia marcescens biotin operon
More LessThe nucleotide sequence of the biotin (bio) operon of wild-type Serratia marcescens Sr41 was determined. Five ORFs were identified to encode BioA (7,8-diaminopelargonic acid aminotransferase), BioB (biotin synthase), BioF (7-keto-8-aminopelargonic acid synthase), BioC (an enzyme catalysing the synthesis of pimeloyl-CoA) and BioD (dethiobiotin synthase), in this order. The operon was deduced to be transcribed divergently to the left into bioA and to the right into the bioBFCD genes. The promoters and a common predicted operator for both bioA and bioBFCD genes were located between the bioA and bioB genes. The predicted amino acid sequences of these enzymes were similar to the sequences of the corresponding enzymes of Escherichia coli. Analysis of expression of the lacZ structural gene fused with the bioA and bioB promoters revealed that the biotin operon was subject to biotin-mediated feedback repression.
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KatP, a novel catalase-peroxidase encoded by the large plasmid of enterohaemorrhagic Escherichia coli O157:H7
More LessA gene coding for a catalase-peroxidase activity was identified on a 9·7 kb Smal DNA fragment derived from the large plasmid p0157 of enterohaemorrhagic Escherichia coli (EHEC) 0157:H7 strain EDL 933. Nucleotide sequencing revealed an ORF of 2208 bp and predicted a 736 amino acid polypeptide with a molecular mass of 81·8 kDa. This putative protein was found to be highly homologous to members of the bacterial bifunctional catalase-peroxidase family. Analysis of its amino acid sequence revealed the presence of characteristic peroxidase 1 and 2 motifs. In addition, an N-terminal signal sequence was found, suggesting that the catalase-peroxidase is transported through the cytoplasmic membrane. EHEC catalase-peroxidase activities were investigated in cytoplasmic and periplasmic crude extracts as well as in culture supernatants from wild-type and recombinant E. coli strains. EHEC-specific catalase-peroxidase activity was detected primarily in the periplasm in strain EDL 933. The newly discovered enzyme was designated KatP, to indicate its plasmid origin. PCR analysis of representative strains of all enteric E. coli pathogroups (i.e. enterohaemorrhagic, enterotoxigenic, enteropathogenic, enteroaggregative and enteroinvasive E. coli) revealed a close association between the occurrence of EHEC-haemolysin and the katP gene in Shiga-like-toxin-producing E. coli 0157 strains.
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- Pathogenicity And Medical Microbiology
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Role of the Bordetella pertussis P.69/pertactin protein and the P.69/pertactin RGD motif in the adherence to and invasion of mammalian cells
The role of the Bordetella pertussis P.69/pertactin protein in mammalian cell adhesion and invasion was investigated. Salmonella strains expressing surface-associated P.69/pertactin from a chromosomally located prn gene were significantly more invasive than isogenic parental strains. This effect was most pronounced in the poorly invasive, semi-rough S. typhimurium strain LB5010. Escherichia coli K-12 strain HB101 harbouring the plasmid p41869D, which encodes the full-length prn gene under the control of the tac promoter on the broad-host-range plasmid pMMB66EH, was significantly more adhesive to HEp-2 and Chinese Hamster Ovary (CHO) cells growing in culture than E. coli HB101(pMMB66EH). However, the ability of E. coli to invade mammalian cells was not affected by P.69/pertactin expression. P.69/pertactin-mediated adhesiveness of HB101 to HEp-2 and CHO cells was not influenced by the viability of the bacterial cells. However, adherence was markedly reduced when assays were performed for less than 3 h, at 4°C or in the presence of cycloheximide, suggesting the active participation of the eukaryotic cell in bacterial adhesion. Site-directed mutagenesis was used to mutate Asp to Glu in an Arg-Gly-Asp (RGD→RGE) sequence present in mature P.69/pertactin and the mutated gene was cloned in the same broad-host-range vector (plasmid p41869E). This mutation had no detectable influence on the ability of P.69/pertactin to mediate adhesion of HB101 to HEp-2 or CHO cells. Plasmids p41869D and p41869E were introduced into the bvg-negative B. pertussis strain BP347. Expression of P.69RGD or P.69RGE did not enhance the adhesiveness of BP347 for epithelial (HEp-2 and CHO) cells.
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- Physiology And Growth
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Phosphate-starvation-inducible proteins in Bacillus subtilis: a two-dimensional gel electrophoresis study
More LessA two-dimensional (2-D) gel electrophoresis study of Bacillus subtilis strain 168 identified 20 proteins that are strongly induced in response to phosphate starvation. The induction of nine of these phosphate-starvation-induced (Psi) proteins was dependent on a functional PhoR protein. PhoR is the histidine sensor-kinase component of a phosphate-concentration-sensing two-component regulatory system which, together with its partner response regulator PhoP, controls the expression of genes in the Pho regulon. Genes encoding PhoR-dependent Psi proteins are therefore likely to be members of the Pho regulon. SpoOA ~ P, the response regulator of the signal transduction pathway required for the induction of sporulation, has previously been shown to negatively affect the induction of the Pho regulon by repressing the phoP-phoR operon. The induction pattern of some PhoR-dependent Psi proteins was altered in a spoOA mutant such that their synthesis continued for longer than was found with the wild-type. The most abundant Psi protein, Psi1–3, was characterized by N-terminal sequencing of internal peptide fragments and shown to have a high similarity to an Escherichia coli protein which is involved in phosphate uptake during phosphate starvation.
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Changes in cell morphology and carnitine acetyltransferase activity in Candida albicans following growth on lipids and serum and after in vivo incubation in mice
More LessCandida albicans C316, maintained in the yeast form, showed a proliferation of peroxisomes when grown on triolein or serum as sole carbon source but these structures were absent from glucose-grown cells. Peroxisomes were also apparent in C. albicans obtained after injection into mice and recovery from intraperitoneal washings and kidneys; they may therefore be useful markers to assess a potential in vivo response in cells that are growing in vitro. Trans-cell-wall structures also occurred in C. albicans grown on triolein or serum, and in cells cultured in vivo, but were not seen in cells grown on glucose. These structures consisted of electron-dense fibrillar material penetrating through the cell wall from the plasmalemma side and protruded out to the exterior of the cell. Endoplasmic reticulum, located at the periphery of the cell, was found to be in close proximity with these cell wall structures. Carnitine acetyltransferase (CAT; EC 2.3.1.7), the key enzyme for the translocation of acetyl units between intracellular compartments, was present in low activities in glucose-grown cells; its activity was increased some 100-fold in triolein-grown cells but only 4-fold in serum-grown cells. It was not possible to assess this activity in the in vivo-cultured cells. Two separate CAT proteins, partially purifed from isolated microchondria and peroxisomes, respectively, were identified, with different specificities and kinetic properties.
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Organic acid excretion by Streptomyces lividans TK24 during growth on defined carbon and nitrogen sources
More LessCultures of Streptomyces lividans TK24 grown in defined media containing certain rapidly used carbon and nitrogen sources excreted high levels of organic acids. These were identified by HPLC and enzymic assays as pyruvic acid and 2-oxoglutaric acid. Acidification occurred only with glucose as the principal carbon source, and depended on the nitrogen source used. With nitrate as the sole nitrogen source, high levels of pyruvate and small amounts of 2-oxoglutarate were produced. Carbon from D-[U-14C]glucose was converted into both organic acids. Combining glucose with a selection of amino acids as primary nitrogen/secondary carbon sources yielded less pyruvate and more 2-oxoglutarate. Carbon from both 14C-labelled glucose and amino acids was metabolized to both organic acids. Adding nitrate to this combination caused a reversion of the acid production pattern to that of the glucose-nitrate combination, as if the amino acids were absent. Addition of ammonium salts to any combination of carbon and nitrogen sources completely prevented organic acid formation.
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Ferric-reductase activities in whole cells and cell fractions of Vibrio (Listonella) anguillarum
More LessThe ability of Vibrio (Listonella) anguillarum strains from serotype groups O1 and O2 to reduce Fe3+ in the form of different chelates was investigated. All strains, grown in M9 minimal medium supplemented with 0·2% Casamino acids, reduced Fe3+ complexed by citrate, nitrilotriacetic acid and EDTA. In whole cells, the degree of reduction was dependent on the Fe3+ ligand and on the strain, with the greatest values corresponding to ferric dicitrate and serotype group O1 strains, respectively. The ferric-reductase activity increased, over the basal levels, when the cells were grown with iron added as ferric dicitrate, haemin or haemoglobin. All strains also reduced ferricyanide, a compound that is not transported into the bacterial cells. Ferricyanide reduction was also increased when the cells were grown in the presence of an iron source. All of the cell fractions (periplasm, membranes and cytoplasm) showed Fe3+-reducing activity, with the highest values observed in the presence of Mg2+, NADH and FAD in the assay buffer. Cytoplasmic ferric-reductase could be visualized using native polyacrylamide or starch gel electrophoresis, whereas the periplasmic and membrane reductase(s) could only be detected on starch gels. The results indicate the presence of different ferric-reductase activities in V. anguillarum, which could be involved in the different iron-acquisition systems present in this micro-organism, i.e. siderophore-mediated systems and siderophore-independent mechanisms.
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The acid tolerance response of Salmonella typhimurium provides protection against organic acids
More LessSalmonella typhimurium encounters a variety of acid stress situations during pathogenesis and in the natural environment. These include the extreme low pH encountered in the stomach and a less acidic intestinal environment containing large amounts of organic weak acids (volatile fatty acids). The acid tolerance response (ATR) is a complex defence system that can minimize the lethal effects of extreme low pH (pH 3). The data presented illustrate that the ATR can also defend against weak acids such as butyric, acetic or propionic acids. Although an acid shock of pH 4·4 induced the ATR, growth in subinhibitory concentrations of weak acids did not. Various mutations shown to affect tolerance to extreme acid conditions (pH 3) were tested for their effects on tolerance to weak acids. An rpoS mutant lacking the alternative sigma factor sS failed to protect cells against weak acids as well as extreme acid pH. The fur (ferric uptake regulator) and atp (Mg2+-dependent ATPase) mutants defective in extreme acid tolerance showed no defects in their tolerance to weak acids. Curiously, the atbR mutant that exhibits increased tolerance to extreme acid pH proved sensitive to weak acids. Several insertions that rendered cells sensitive to organic acids were isolated, all of which proved to be linked to the rpoS locus.
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- Plant-Microbe Interactions
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Evidence for the association of the enteric bacterium Ewingella americana with internal stipe necrosis of Agaricus bisporus
More LessInternal stipe necrosis of Agaricus bisporus is recognized as an emerging and potentially serious disease in the UK mushroom industry. Symptoms are visible only on harvest and appear as a variable browning reaction in the centre of stipes, which may be accompanied by limited collapse of the internal tissues. The hypothesis that this problem is of bacterial origin was investigated, initially by an extensive bacteriological examination of affected mushrooms. The enteric bacterium Ewingella americana was isolated from at least 93% of symptomatic mushrooms. Various strains of Pseudomonas fluorescens are also usually present in diseased mushrooms, but no single P. fluorescens biovar was consistently associated with the disease. Typical symptoms were reproduced following infection trials with isolates of E. americana derived from diseased mushrooms. In addition, strains recovered from diseased mushrooms following such trials were shown by RFLP studies to be identical to those applied, thereby confirming Koch's postulates for these strains. The possible contribution of other bacterial species to symptom development is discussed.
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- Genome Analysis
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Physical mapping of Mycobacterium bovis BCG Pasteur reveals differences from the genome map of Mycobacterium tuberculosis H37Rv and from M. bovis
A Dral restriction map of the ~ 4·35 Mb circular chromosome of the vaccine strain Mycobacterium bovis BCG Pasteur was constructed by linking all 21 Dral fragments, ranging in size from 6 to 820 kb, using specific clones that spanned the Dral recognition sites as hybridization probes. The positions of 20 known genes were also established. Comparison of the resultant genome map with that of the virulent tubercle bacillus Mycobacterium tuberculosis H37Rv revealed extensive global conservation of the genomes of these two members of the M. tuberculosis complex. Possible sites of evolutionary rearrangements were localized on the chromosome of M. bovis BCG Pasteur by comparing the Asnl restriction profile with that of M. tuberculosis H37Rv. When selected cosmids from the corresponding areas of the genome of M. tuberculosis H37Rv were used as hybridization probes to examine different BCG strains, wild-type M. bovis and M. tuberculosis H37Rv, a number of deletions up to 10 kb in size, insertions and other polymorphisms were detected. In addition to the known deletions covering the genes for the protein antigens ESAT-6 and mpt64, other genetic loci exhibiting polymorphisms or rearrangements were detected in M. bovis BCG Pasteur.
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Gene arrangement and organization in a ~ 76 kb fragment encompassing the oriC region of the chromosome of Mycobacterium leprae
A continuous 75627 bp segment of the Mycobacterium leprae chromosome spanning the oriC region was sequenced. The gene order at this locus was similar to that found in the replication origin region of many other prokaryotes, particularly Mycobacterium tuberculosis and Streptomyces coelicolor. As in the case of several Gram-positive bacteria, essential genes involved in basic cellular functions, such as DNA or RNA metabolism (dnaA, dnaB, dnaN, gyrB, gyrA, pcnB, recF, rnpA, ssb), cell wall synthesis (ponA, pbpA) and probably cell division (gidB, rodA) were found. Strikingly, the gidA gene was absent from this part of the genome and there was no rRNA operon near oriC. The gyrA gene harbours an intein coding sequence indicating that protein splicing is required to produce the mature A subunit of DNA gyrase. Among the many other noteworthy features were ORFs encoding putative serine/threonine protein kinases and a protein phosphatase, three tRNA genes, one M. leprae-specific repetitive element and a glnQ pseudogene.
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- Bacillus Subtilis Genome Sequencing Project
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The Bacillus subtilis genes for ribonucleotide reductase are similar to the genes for the second class I NrdE/NrdF enzymes of Enterobacteriaceae
More LessWe have cloned and sequenced the nrd (nucleotide reductase) locus of Bacillus subtilis. The locus seems to be organized in an operon comprising four ORFs. The first three encode polypeptides highly similar to the product of the coding sequences characterizing the nrdEF operons of Enterobacteriaceae. The sequencing of the conditional lethal mutation ts-A13, localized in the nrdE cistron, and the lethality of insertional mutations targeted in the internal region of nrdE and nrdF, demonstrated the essential role of this locus. The fourth ORF, ymaB, part of the putative operon, which is not similar to any known protein, is also essential. The regulation of expression of the operon, monitored by lacZ transcriptional fusions, is similar to the regulation of the functionally relevant nrdAB operon of Escherichia coli. The operon was induced by thymidine starvation and its expression was directly or indirectly affected by RecA function. Genetic and functional analysis strongly indicates that in B. subtilis the class I ribonucleotide reductase encoded by this nrd operon is evolutionary distant from the homologous class I enzyme of Enterobacteria.
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Organization of the Bacillus subtilis 168 chromosome between kdg and the attachment site of the SPβ prophage: use of Long Accurate PCR and yeast artificial chromosomes for sequencing
More LessWithin the Bacillus subtilis genome sequencing project, the region between lysA and ilvA was assigned to our laboratory. In this report we present the sequence of the last 36 kb of this region, between the kdg operon and the attachment site of the SPβ prophage. A two-step strategy was used for the sequencing. In the first step, total chromosomal DNA was cloned in phage M13-based vectors and the clones carrying inserts from the target region were identified by hybridization with a cognate yeast artificial chromosome (YAC) from our collection. Sequencing of the clones allowed us to establish a number of contigs. In the second step the contigs were mapped by Long Accurate (LA) PCR and the remaining gaps closed by sequencing of the PCR products. The level of sequence inaccuracy due to LA PCR errors appeared to be about 1 in 10000, which does not affect significantly the final sequence quality. This two-step strategy is efficient and we suggest that it can be applied to sequencing of longer chromosomal regions. The 36 kb sequence contains 38 coding sequences (CDSs), 19 of which encode unknown proteins. Seven genetic loci already mapped in this region, xpt, metB, ilvA, ilvD, thyB, dfrA and degR were identified. Eleven CDSs were found to display significant similarities to known proteins from the data banks, suggesting possible functions for some of the novel genes: cspD may encode a cold shock protein; bcsA, the first bacterial homologue of chalcone synthase; exol, a 5′ to 3′ exonuclease, similar to that of DNA polymerase I of Escherichia coli; and bsaA, a stress-response-associated protein. The protein encoded by ypIP has homology with the transcriptional NifA-like regulators. The arrangement of the genes relative to possible promoters and terminators suggests 19 potential transcription units.
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Mapping of the 150 kb spoIIIC-pheA region of the Bacillus subtilis chromosome using Long Accurate PCR and three yeast artificial chromosomes
More LessWe constructed a PCR map of the 150 kb spoIIIC-pheA region of the Bacillus subtilis chromosome. It was established using known sequences of the spoIIIC, blt, aadK, sacC, spoVB and pheA loci and eight random sequence tags. The tags were generated using PFGE-purified DNA of yeast artificial chromosome (YAC) 11–17 from the yeast clone which carries the major part of this region. The ends of two other YACs were positioned on the map using total DNA extracted from yeast cells carrying them. The procedure allowed the placement of precisely known and new (putative) genes on the physical chromosome map and the generation of sufficient amounts of DNA for sequencing this region. Apart from allowing correction of the genetic map in this region, these results demonstrate how a collection of long segments of bacterial chromosome and Long Accurate PCR can be used for reliable high-resolution physical mapping of an extended chromosome area.
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A 22 kb DNA sequence in the cspB-glpPFKD region at 75° on the Bacillus subtilis chromosome
More LessA 21808 bp nucleotide sequence at 75° on the genetic map of the Bacillus subtilis chromosome was determined. The sequence of this region is adjacent to the glpPFKD operon involved in glycerol utilization. Twenty-six ORFs were identified, one of which corresponds to the cspB gene, encoding a cold-shock protein. Seventeen of the deduced protein sequences of these ORFs displayed significant homology to known proteins in the data banks. One putative operon was identified, consisting of five ORFs, that is probably involved in the uptake and processing of copper. The location of cspB in this sequence does not confirm the genetic mapping data, indicating that the gene is closely linked to comK, which is located at 80° on the B. subtilis chromosome.
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The 52°-55° segment of the Bacillus subtilis chromosome: a region devoted to purine uptake and metabolism, and containing the genes cotA, gabP and guaA and the pur gene cluster within a 34960 bp nucleotide sequence
More LessWithin the framework of the international project for sequencing the entire Bacillus subtilis genome, we have determined the complete sequence of the segment flanking the purE-D gene cluster (55°) as far as cotA (52°). This segment (34960 bp) contains, as well as 12 genes already identified as part of the pur operon, 17 putative ORFs and one partial one. Two of them (gabP and guaA) are known B. subtilis genes. The gene product of cotA (formerly pig) shows significant similarity to oxidoreductases (phenoxazine synthase and bilirubin oxidase). The putative products of ORFs yeaB (Czd protein), yeaC (MoxR), yebA (CNG-channel and cGMP-channel proteins from eukaryotes), yebB (hypothetical 32·9 kDa protein of Escherichia coli), yecA (amino acid permease) and yecB (adenine deaminase) were similar to proteins in data banks.
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The ampS-nprE (124°–-127°) region of the Bacillus subtilis 168 chromosome: sequencing of a 27 kb segment and identification of several genes in the area
More LessA stretch of DNA approximately 27 kb in length, adjacent to the nprE gene of Bacillus subtilis, has been sequenced. The sequenced fragment carries a total of 23 ORFs. Of these, 15 could be assigned probable functions based on homologies to characterized genes either in B. subtilis or in other organisms. The sequencing of this region has also allowed us to assign to this area adeC and strB, previously located on the other side of nprE, between nprE and the pyr operon.
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Sequence analysis of a 50 kb region between spoOH and rrnH on the Bacillus subtilis chromosome
The 49630 bp spoOH-rrnH region of the Bacillus subtilis genome has been fully sequenced. The sequence contains one partial and 62 complete ORFs, one partial and three complete rRNA genes and a cluster of six tRNA genes. The direction of the transcription and translation of 61 ORFs is the same as that of the movement of the replication fork. A homology search of 40 ORFs in newly determined sequence revealed that 27 of them had significant similarity to known proteins such as elongation factor G, elongation factor Tu, pseudouridine synthase I and ribosomal proteins. Two adjacent genes, ybaD and ybaE, appeared to encode proteins belonging to the ATP-binding cassette (ABC) family.
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The 25°–36° region of the Bacillus subtilis chromosome: determination of the sequence of a 146 kb segment and identification of 113 genes
More LessWe determined a 146 kb contiguous sequence at the 25°–36° region of the Bacillus subtilis chromosome containing the amyE-srfA segment. Among the 113 ORFs identified, 33 are already known. Functions were assigned to 38 ORFs by a search of non-redundant protein sequence data banks and those of 16 ORFs were suggested through significant similarity with reported sequences. The amino acid sequences of 13 of the ORFs were similar to proteins of unknown function of Escherichia coli, Haemophilus influenzae and other species. We did not find similarities for 29 ORFs to any known proteins. The 146 kb region is rich in enzymes (35 ORFs) related to the metabolism of low molecular mass compounds and five genes for surfactin production occupy about 26 kb of the region.
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Cloning and sequencing of a 40·6 kb segment in the 73°–76° region of the Bacillus subtilis chromosome containing genes for trehalose metabolism and acetoin utilization
More LessIn the framework of the international project aimed at the sequencing of the Bacillus subtilis genome, a 40·6 kb chromosome segment, which contains the tre locus, has been cloned and sequenced. This region (40601 bp; 73°–76° on the genetic map) contains 38 complete ORFs and one partial one. Three ORFs, the closest to the hsdC locus, correspond to the treP, treA and treR genes encoding enzyme IITre, trehalose-6-phosphate hydrolase and the repressor of the tre operon, respectively. A homology search for the products deduced from the 39 ORFs revealed that 23 exhibit significant similarity to known proteins, e.g. proteins involved in acetoin utilization, deoxyribonuclease, methyladenine glycosidase, hydroxyisobutyrate dehydrogenase, multidrug resistance proteins, protein phosphatase, cyclic-nucleotide phosphodiesterase, 5′-nucleotidase and NADP(H)-flavin oxidoreductase. Based on the gene organization and the results of the homology search, it is predicted that YfjG, YfjH, YfjI, YfjJ and YfjK form an acetoin dehydrogenase system (acetoin regulatory protein, and acetoin dehydrogenase components/subunits E3, E2, E1β and E1α, respectively). yfkN, an extremely large ORF comprising 4386 nucleotides, seems to correspond to the fusion of the genes for 2′,3′-cyclic-nucleotide 2′-phosphodiesterase and 5′-nucleotidase precursor.
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The dnaB-pheA (256°–240°) region of the Bacillus subtilis chromosome containing genes responsible for stress responses, the utilization of plant cell walls and primary metabolism
Within the framework of the international programme to sequence the genome of Bacillus subtilis strain 168, we were allocated the region between dnaB (256°) and pheA (240°). The sequencing of this region is now complete and we report our primary analysis of the 114 kb region containing 114 ORFs. In addition to previously characterized genes, we have identified genes involved in the utilization of plant cell wall polysaccharides, stress responses and the metabolism of amino acids, cell walls, DNA and fatty acids. We also discuss various structural and physical features, including the orientation of genes with respect to replication, putative start and stop codons, ribosome binding sites and ρ-independent transcription terminators.
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Sequence of the 305°–307° region of the Bacillus subtilis chromosome
More LessThe nucleotide sequence of the Bacillus subtilis 168 chromosomal segment located between yvhJ (307°) and secA (305°) was determined. This 20·3 kb region encompasses 23 ORFs, 17 of which have been sequenced previously. Comparison of sequences obtained here with the previously obtained ones revealed seven discrepancies. The products of the sequenced genes are involved in the regulation of degradative enzymes, competence, flagellar motility and protein secretion. Putative functions of newly identified genes are based on sequence homologies.
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Integrated mapping and sequencing of a 115 kb DNA fragment from Bacillus subtilis: sequence analysis of a 21 kb segment containing the sigL locus
More LessA sequence strategy which combines a low redundancy shotgun approach and directed sequencing has been elaborated. Essentially, the sequences, as well as the size of the fragments utilized for a low coverage shotgun approach, were exploited for the construction of a physical map of the region to be sequenced. The latter considerably simplified the subsequent directed sequencing steps. We report the physical mapping of a 115 kb segment which covers nearly 100 kb of the hisA-cysB region of the Bacillus subtilis chromosome and contains previously sequenced genes sigL and sacB. Sequencing and analysis of a 21305 bp segment, which includes the sigL locus, revealed 21 ORFs, apparently belonging to at least seven transcription units. This segment has a G+C content greater than 47%, compared to 43% characteristic of the flanking regions, and mainly consists of genes whose products seem to be involved in the synthesis of an exopolysaccharide. These observations leave open the possibility that the analysed fragment has been acquired through horizontal transfer.
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New genes in the 170° region of the Bacillus subtilis genome encode DNA gyrase subunits, a thioredoxin, a xylanase and an amino acid transporter
More LessA DNA contig of 26·2 kb covering the 170° region of the Bacillus subtilis strain 168 genome was isolated and sequenced. For DNA isolation, suitable restriction sites at the end of previously known genes were chosen to amplify adjacent unknown DNA regions by inverse PCR. On the basis of the DNA sequence, 26 ORFs were identified of which egIS and ccdA, as well as parts of citB and tkt have been described previously. Here we report the complete sequences of the aconitase (citB) and transketolase (tkt) genes. Of the other proteins encoded on the 26·2 kb fragment, eight revealed similarities to previously described proteins. These included a pair of newly identified DNA gyrase subunits A (grIA) and B (grIB), a sodium/proton-dependent alanine carrier (alsT), a member of the thioredoxin family (tlpA), an endo-1,4-β-xylanase (xynD) and a response regulator protein. Comparison of the physical and the genetic maps revealed several differences. According to its flanking sequences the lexA (dinR) gene which was previously mapped at 162° was found to be adjacent to yneA localized at 170°. Genes citB and egIS were located the opposite way round and closer together than expected from the genetic map (citB at 173° and egIS at 170°). The prkA gene, which was mapped at 169°, was not present on the respective fragment. Sequence comparison actually showed that prkA is located close to 70° on the B. subtilis genome.
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Systematic sequencing of the 283 kb 210°-232° region of the Bacillus subtilis genome containing the skin element and many sporulation genes
As part of the Bacillus subtilis genome sequencing project, we have determined a 283 kb contiguous sequence from 210° to 232° of the B. subtilis genome. This region contains the 48 kb skin element which is excised during sporulation by a site-specific recombinase. In this region, 310 complete ORFs and one tRNA gene were identified: 66 ORFs have been sequenced and characterized previously by other workers, e.g. acc, ans, bfm, blt, bmr, comE, comG, dnaK, rpoD and sin operons; cwIA, gpr and lysA genes; many sporulation genes and operons, spoOA, spoIIA, spoIIM, spoIIP, spoIIIA, spoIIIC, spoIVB, spoIVCA, spoIVCB and spoVA, etc. The products of 84 ORFs were found to display significant similarity to proteins with known function in data banks, e.g., proteins involved in nucleotide metabolism, lipid biosynthesis, amino acid transport (ABC transporter), phosphate-specific transport, the glycine cleavage system, the two-component regulatory system, cell wall autolysis, ferric uptake and sporulation. However, the functions of more than half of the ORFs (52%, 160 ORFs) are still unknown. In the skin element containing 60 ORFs, 32 ORFs (53%) encode proteins which have significant homology to gene products of the B. subtilistemperate phage ø105 and/or the defective phage PBSX.
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Sequencing of a 65 kb region of the Bacillus subtilis genome containing the lic and cel loci, and creation of a 177 kb contig covering the gnt-sacXY region
Within the framework of an international project for the sequencing of the entire Bacillus subtilis genome, this paper communicates the sequencing of a chromosome region containing the lic and cel loci (65 kb), which creates a 177 kb contig covering the region from gnt to sacXY. This 65 kb region contains 64 ORFs (62 complete and two partial genes). The 14th, 15th and 17th genes correspond to licT, licS and katE, encoding the antiterminator for licS transcription, β-glucanase (lichenase) and catalase 2, respectively. The 11th, 30th, 36th, 39th, 41st, 45th–48th, 51st and 58th genes are designated deaD, pepT, gaIE, aldY, msmX, cydABCD, sigY and katX because their products probably encode ATP-dependent RNA helicase, tripeptidase, UDP-glucose-4-epimerase, aldehyde dehydrogenase, multiple sugar-binding transport ATP-binding protein, the respective components of cytochrome d ubiquinol oxidase and ATP-binding cassette transporter, σ-factor of RNA polymerase and catalase, respectively. The 60th–64th genes are celRABCD, which are probably involved in cellobiose utilization. Gene organization and gene features in the gnt-sacXY region are discussed.
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