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PubMed1995

SubtiList: a relational database for the Bacillus subtilis genome.

In the framework of the international collaborative project aiming to sequence the whole Bacillus subtilis chromosome, we have created a relational database for managing and analysing information associated with the molecular genetics of this bacterium: SubtiList. It allows recovery of non-redundant DNA sequences of the B. subtilis genome, as well as related information, i.e. genes, proteins, etc. A logical structure has been designed with appropriate links between the different objects, and a set of procedures has been implemented for data updating and management. The database is organized around a core constituted by all known contigs of B. subtilis, i.e. sets of non-redundant sequences created from original entries in the EMBL data library. A user-friendly interface has been developed to make the database easy to consult. Sequence analysis tools have been integrated into the database, such as a program for rapid similarity searching of protein data banks, and a powerful DNA pattern searching program. Thanks to the consistency of SubtiList, we have performed a codon usage analysis by Factorial Correspondence Analysis, and a study of the distribution of the isoelectric points of known proteins of B. subtilis. The SubtiList database is available through anonymous ftp (address 'ftp.pasteur.fr' or IP number 157.99.64.12, directory '/pub/GenomeDB/SubtiList').

Algorithms↗
PubMed2005

ArrayOme: a program for estimating the sizes of microarray-visualized bacterial genomes.

ArrayOme is a new program that calculates the size of genomes represented by microarray-based probes and facilitates recognition of key bacterial strains carrying large numbers of novel genes. Protein-coding sequences (CDS) that are contiguous on annotated reference templates and classified as 'Present' in the test strain by hybridization to microarrays are merged into ICs (ICs). These ICs are then extended to account for flanking intergenic sequences. Finally, the lengths of all extended ICs are summated to yield the 'microarray-visualized genome (MVG)' size. We tested and validated ArrayOme using both experimental and in silico-generated genomic hybridization data. MVG sizing of five sequenced Escherichia coli and Shigella strains resulted in an accuracy of 97-99%, as compared to true genome sizes, when the comprehensive ShE.coli meta-array gene sequences (6239 CDS) were used for in silico hybridization analysis. However, the E.coli CFT073 genome size was underestimated by 14% as this meta-array lacked probes for many CFT073 CDS. ArrayOme permits rapid recognition of discordances between PFGE-measured genome and MVG sizes, thereby enabling high-throughput identification of strains rich in novel genes. Gene discovery studies focused on these strains will greatly facilitate characterization of the global gene pool accessible to individual bacterial species.

Computational Biology↗
PubMedDate not supplied

From ribosome to riboswitch: control of gene expression in bacteria by RNA structural rearrangements.

Structural elements in the 5' region of a bacterial mRNA can have major effects on expression of downstream coding sequences. Folding of the nascent RNA into the helix of an intrinsic transcriptional terminator results in premature termination of transcription and in failure to synthesize the full-length transcript. Structure in the translation initiation region of an mRNA blocks access of the translation initiation complex to the ribosome binding site, thereby preventing protein synthesis. RNA structures can also affect the stability of an RNA by altering sensitivity to ribonucleases. A wide variety of mechanisms have been uncovered in which changes in mRNA structure in response to a regulatory signal are used to modulate gene expression in bacteria. These systems allow the cell to recognize an impressive array of signals, and to monitor those signals in many different ways.

DNA-Directed RNA Polymerases↗
PubMed1984

Multiple joined genes prevent product degradation in Escherichia coli.

A method is described that allows the expression of a stable human proinsulin product in Escherichia coli as encoded by either a fused or an unfused gene construction. In the fused system, the human proinsulin coding sequence is joined to the 3' side of a fragment containing the lac promoter and the coding sequence for a small part of the NH2 terminus of beta-galactosidase. In the unfused system, the proinsulin coding sequence is linked directly to a fragment containing the Tac promoter followed by a bacterial Shine-Dalgarno sequence. In both systems, the human proinsulin product is too unstable to be detected by NaDodSO4/polyacrylamide gel electrophoresis or even pulse-chase analysis. However, when multiple copies of the proinsulin coding sequence are tandemly linked such that the resultant protein product contains multiple copies of the proinsulin domain, the stability of the product is markedly increased in both the fused and the unfused expression systems. In the unfused system, three tandemly linked proinsulin polypeptide domains are required for stabilization, whereas two proinsulin domains plus the bacterial leader protein enhance stability in the fused system. The polypeptide product of a multiple copy proinsulin gene can be cleaved into single proinsulin units by cyanogen bromide treatment.

Cloning, Molecular↗
PubMed1987

Cis-acting sequences sufficient for correct tissue and temporal specificity of larval serum protein 1 genes of Drosophila.

We have constructed hybrid genes in which the coding region of the bacterial gene chloramphenicol acetyl transferase (CAT) has been linked to varying lengths of upstream sequences of Drosophila genes for larval serum sequence 1 (LSP1). These have been inserted into a P-element transformation vector and subsequently transferred into the germ-line of recipient flies. Transformants carrying the CAT gene linked to 1650 bp, 570 bp or 377 bp of upstream LSP1alpha sequences, or 745 bp or 471 bp of upstream beta sequences express CAT with the same developmental and tissue specificity as the endogenous LSP1 genes. Constructs having only 66 bp of upstream LSP1beta sequences, however, show extremely low levels of CAT expression in tissues and at developmental stages in which LSP1 is not expressed. We discuss the significance of short regions of homology between the DNA upstream of the alpha and beta genes, which lie within the regions identified by the transformation experiments as being required for the cis-regulation of LSP1 synthesis.

Journal Article↗
PubMed1981

Factors governing the expression of a bacterial gene in mammalian cells.

Cultured monkey kidney cells transfected with simian virus 40 (SV40)-pBR322-derived deoxyribonucleic acid (DNA) vectors containing the Escherichia coli gene (Ecogpt, or gpt) coding for the enzyme xanthine-guanine phosphoribosyltransferase (XGPRT) synthesize the bacterial enzyme. This paper describes the structure of the messenger ribonucleic acids (mRNA's) formed during the expression of gpt and an unexpected feature of the nucleotide sequence in the gpt DNA segment. Analyses of the gpt-specific mRNA's produced during infection of CV1 cells indicate that in addition to the mRNA's expected on the basis of known simian virus 40 RNA splicing patterns, there is a novel SV40-gpt hybrid mRNA. The novel mRNA contains an SV40 leader segment spliced to RNA sequences transcribed from the bacterial DNA segment. The sequence of the 5'-proximal 345 nucleotides of the gpt DNA segment indicates that the only open translation phase begins with an AUG about 200 nucleotides from the end of the gpt DNA. Two additional AUGs as well as translation terminator codons in all three phases precede the XGPRT initiator codon. Deletion of the two that are upstream of the putative start codon increases the level of XGPRT production in transfected cells; deletion of sequences that contain the proposed XGPRT initiator AUG abolishes enzyme production. Based on the location of the XGPRT coding sequence in the recombinants and the structure of the mRNA's, we infer that the bacterial enzyme can be translated from an initiator AUG that is 400 to 800 nucleotides from the 5' terminus of the mRNA and preceded by two to six AUG triplets.

Animals↗
PubMed1973

Nucleotide sequences from tryptophan messenger RNA of Escherichia coli: the sequence corresponding to the amino-terminal region of the first polypeptide specified by the operon.

Mutants with internal deletions that terminate near the operator end of the tryptophan operon of E. coli were used in studies on the nucleotide sequence at the 5' end of the messenger RNA transcribed on this operon. The findings obtained permitted identification of a sequence of 43 nucleotides corresponding to the aminoterminal 11 amino acids of anthranilate synthetase component I, the polypeptide specified by the operator-proximal structural gene of the operon. It was also shown that the translation initiation codon for this polypeptide is preceded on the messenger by a "leader" sequence of unknown function, at least 150 nucleotides in length.

Amino Acid Sequence↗
PubMed1992

Tagging of plant potyvirus replication and movement by insertion of beta-glucuronidase into the viral polyprotein.

Infectious RNA transcripts were generated from full-length cDNA clones of the tobacco etch potyvirus genome containing an insertion of the bacterial beta-glucuronidase (GUS) gene between the polyprotein-coding sequences for the N-terminal 35-kDa proteinase and the helper component-proteinase. The recombinant virus was able to spread systemically in plants and accumulated to a level comparable with wild-type tobacco etch potyvirus. Proteolytic processing mediated by the 35-kDa proteinase and helper component-proteinase resulted in production of an enzymatically active GUS-helper component-proteinase fusion protein. A virus passage line that retained the GUS insert after numerous plant-to-plant transfers, as well as a line that sustained a deletion of the GUS sequence, was recovered. Use of an in situ histochemical GUS assay in time-course experiments allowed the visualization of virus activity in single, mechanically inoculated leaf epidermal cells, in neighboring epidermal and mesophyll cells, in phloem-associated cells after long-distance transport, and in cells surrounding vascular tissues of organs above and below the site of inoculation. This system represents a powerful tool to study plant virus replication, short- and long-distance virus movement, and virus-host interactions. Additionally, we show that potyviruses may serve as highly efficient, autonomously replicating vectors for the expression of foreign genes in plants.

Endopeptidases↗
PubMed2004

Isolation of mak1 from Actinoplanes missouriensis and evidence that Pep2 from Streptomyces coelicolor is a maltokinase.

The gene mak1FN coding for maltokinase from Actinoplanes missouriensis is located in a cluster similar to glycogen metabolism clusters identified in Streptomyces coelicolor. Sequence comparisons demonstrate that mak1-related genes coding for homologous proteins are present in many bacterial genomes including taxonomic distantly related groups such as Rhodospirillales or green sulfur bacteria. More than 50% of the aligned sequences are longer than the mak1 gene from A. missouriensis, and the N-terminal portion of these putative maltokinases exhibit high sequence homologies with trehalose synthases. A more detailed sequence comparison indicates a relationship of maltokinases to aminoglycoside phospho-transferases and protein kinases. Transformation of S. lividans with plasmid vectors containing either the mak1 gene from A. missouriensis or the pep2 gene from S. coelicolor resulted in recombinant strains, which produced measurable amounts of maltokinase activity. The proteins Pep2 and Mak1 were over expressed with Streptomyces lividans 66 as a heterologous host and further characterized. The possible physiological function of maltokinases is discussed.

Amino Acid Sequence↗
PubMed1982

Nucleotide sequence of Escherichia coli purF and deduced amino acid sequence of glutamine phosphoribosylpyrophosphate amidotransferase.

The Escherichia coli gene purF, coding for 5-phosphoribosylamine:glutamine pyrophosphate phosphoribosyltransferase (amidophosphoribosyltransferase) was subcloned from a ColE1-purF plasmid into pBR322. Amidophosphoribosyltransferase levels were elevated more than 5-fold in the ColE1-purF plasmid-bearing strain compared to the wild type control, and a further 10- to 13-fold elevation was observed in several pBR322 derivatives. The nucleotide sequence of a 2478-base pair PvuI-HinfI fragment encoding purF was determined. The purF45 structural gene codes for a 56,395 Mr protein chain having 504 amino acid residues. Methionine-1 is removed by processing in vivo leaving cysteine as the NH2-terminal residue. The deduced amino acid sequence was confirmed by comparisons with the NH2-terminal amino acid sequence determined by automated Edman degradation (Tso, J. Y., Hermodson, M. A., and Zalkin, H. (1982) J. Biol. Chem. 257, 3532-3536) and amino acid analyses of CNBr peptides including a 4-residue peptide from the CO2H terminus of the enzyme. Nucleotide sequences characteristic of bacterial promoter-operator regions were identified in the 5' flanking region. The coding region appears to be preceded by a 277-297 nucleotide mRNA leader. A deletion removing the putative promoter-operator region results in defective purF expression.

Amidophosphoribosyltransferase↗
PubMed1990

Bacterial production and purification of recombinant human prolactin.

Escherichia coli cells transformed with a recombinant plasmid (pT7L) containing the coding sequence of human prolactin (hPrl) expressed a new protein. This protein, comigrating with human Prl on sodium dodecyl sulfate (SDS)-polyacrylamide gels, represented 50% of the total bacterial extract. Immunoprecipitation of [35S]methionine-labeled bacterial lysate with a rabbit antiserum to hPrl followed by SDS-polyacrylamide gel electrophoresis (PAGE) analysis showed that the major component had a Mr identical to that of standard hPrl. The majority of the recombinant hPrl (r-hPrl) accumulated in inclusion bodies. Analysis of these inclusion bodies by SDS-PAGE under nonreducing conditions showed that they are composed mostly of fully reduced monomers. Solubilization of the inclusion bodies and protein denaturation were performed in 8 M urea. Refolding during the renaturation procedure was confirmed by SDS-PAGE under nonreducing conditions. r-hPrl was further purified by gel permeation chromatography on a fast protein liquid chromatography column. More than 95% of the molecules were recovered as oxidized monomeric forms. The refolded molecule was tested for its bioactivity in the Nb2 lymphoma mitogenic assay. The dose-response curves obtained with either r-hPrl or pituitary-derived hPrl showed a complete parallelism. Furthermore, Nb2 cell proliferation was completely blocked by addition of hPrl antiserum to both preparations. Recombinant hPrl is identical to natural hPrl except for an additional methionine group at the amino terminal end.

Amino Acid Sequence↗
PubMed1982

Location and nucleotide sequence of the gene for the proton-translocating subunit of wheat chloroplast ATP synthase.

The proton-translocating subunit of wheat chloroplast ATP synthase is encoded by a chloroplast gene that has been accurately mapped and whose nucleotide sequence has been determined. The predicted sequence of 81 amino acids has been confirmed in part by determination of the sequence of the first 40 amino acids from the NH(2) terminus of the protein, and it shows 100% homology with the known amino acid sequence of the spinach protein but no more than 35% homology with the amino acid sequences of bacterial and mitochondrial proteins. The gene shows no deviation from the "universal" genetic code and is not split. A potential ribosome binding site is located 12 nucleotides upstream from the initiation codon, but sequences homologous to prokaryotic promotors and transcription terminators are not apparent.

Journal Article↗
PubMed1999

In vivo emergence of multidrug-resistant mutants of Pseudomonas aeruginosa overexpressing the active efflux system MexA-MexB-OprM.

During a 6-month period, 21 pairs of Pseudomonas aeruginosa isolates susceptible (pretherapy) and resistant (posttherapy) to antipseudomonal beta-lactam antibiotics were isolated from hospitalized patients. In vivo emergence of beta-lactam resistance was associated with the overexpression of AmpC beta-lactamase in 10 patients. In the other 11 patients, the posttherapy isolates produced only low, basal levels of beta-lactamase and had increased levels of resistance to a variety of non-beta-lactam antibiotics (e.g., quinolones, tetracyclines, and trimethoprim) compared with the levels of beta-lactamase production and resistance of their pretherapy counterparts. These data suggested the involvement of the MexA-MexB-OprM active efflux system in the multidrug resistance phenotype of the posttherapy strains. Immunoblotting of the outer membrane proteins of these 11 bacterial pairs with a specific polyclonal antibody raised against OprM demonstrated the overexpression of OprM in all the posttherapy isolates. To determine whether mutations in mexR, the regulator gene of the mexA-mexB-oprM efflux operon, could account for the overproduction of the efflux system, sequencing experiments were carried out with the 11 bacterial pairs. Eight posttherapy isolates were found to contain insertions or deletions that led to frameshifts in the coding sequences of mexR. Two resistant strains had point mutations in mexR that yielded single amino acid changes in the protein MexR, while another strain did not show any mutation in mexR or in the promoter region upstream of mexR. Introduction of a plasmid-encoded wild-type mexR gene into five posttherapy isolates partially restored the susceptibility of the bacteria to selected antibiotics. These results indicate that in the course of antimicrobial therapy multidrug-resistant active efflux mutants overexpressing the MexA-MexB-OprM system may emerge as a result of mutations in the mexR gene.

Anti-Bacterial Agents↗
PubMed1985

Nucleotide sequence and mutational analysis of an immunity repressor gene from Bacillus subtilis temperate phage phi 105.

We have identified and sequenced a bacteriophage phi 105 gene encoding an immunity repressor, the first to be characterized from a temperate phage infecting a Gram-positive host. Using superinfection immunity as an assay for repressor function, the phi 105 repressor gene was located within a 740-bp PvuII-HindIII subfragment near the left end of the phi 105 EcoRI-F fragment. We show that the repressor is specified by the 5'-proximal coding sequence of a translationally overlapping gene pair, transcribed from right to left on the conventional phi 105 map. Comparison of its amino acid sequence (146 residues) with that of a large number of Gram-negative bacterial and phage repressors revealed a putative DNA-binding region between positions 20 and 39. The coding region is preceded by a strong Shine-Dalgarno sequence 5' AAAGGAG 3'. Deletion analysis of the 5'-flanking DNA allowed to identify transcriptional control elements. Their structure, 5' TTGTAT 3' at -35 and 5' TATAAT 3' at -10, strongly suggests that the phi 105 repressor gene is transcribed by the major vegetative form of B. subtilis RNA polymerase, as would be expected for an early phage gene.

Amino Acid Sequence↗
PubMed1997

Characteristic enrichment of DNA repeats in different genomes.

Using computer programs developed for this purpose, we searched for various repeated sequences including inverted, direct tandem, and homopurine-homopyrimidine mirror repeats in various prokaryotes, eukaryotes, and an archaebacterium. Comparison of observed frequencies with expectations revealed that in bacterial genomes and organelles the frequency of different repeats is either random or enriched for inverted and/or direct tandem repeats. By contrast, in all eukaryotic genomes studied, we observed an overrepresentation of all repeats, especially homopurine-homopyrimidine mirror repeats. Analysis of the genomic distribution of all abundant repeats showed that they are virtually excluded from coding sequences. Unexpectedly, the frequencies of abundant repeats normalized for their expectations were almost perfect exponential functions of their size, and for a given repeat this function was indistinguishable between different genomes.

Algorithms↗
PubMed1996

Tetrameric malate dehydrogenase from a thermophilic Bacillus: cloning, sequence and overexpression of the gene encoding the enzyme and isolation and characterization of the recombinant enzyme.

The gene encoding the tetrameric malate dehydrogenase (MDH) in a thermophilic Bacillus species (BI) has been cloned in an Escherichia coli plasmid. The nucleotide sequence of the gene, the first to be elucidated for a tetrameric MDH, shows the MDH subunit to contain 312 amino acids and have a molecular mass of 33648 Da, which confirms the experimentally determined value of about 35 kDa. Like the genomic DNA of BI, the MDH gene is relatively AT-rich; this contrasts with the generally GC-rich nature of the DNA of thermophilic Bacillus species. Comparison of amino acid sequences reveals that BI MDH bears greater structural similarity to lactate dehydrogenases (LDHs) than to other (dimeric) MDHs. MDHs and LDHs resemble each other in catalytic mechanism and several other respects. However, whereas MDHs in the majority of organisms are dimers, the tetrameric structure is favoured among LDHs. The stronger structural resemblance that BI MDH has to LDHs than to the dimeric MDHs provides some explanation as to why Bacillus MDH, unlike most other MDHs, is tetrameric. A 1 kb fragment containing the BI MDH gene, produced in a PCR, has been cloned into a high-expression E. coli plasmid vector. BI MDH synthesized from this clone constitutes about 47% of the total protein in cell extracts of the E. coli strain carrying the clone. MDH purified from BI and that purified from the E. coli strain carrying the MDH gene clone appear to be identical proteins by several criteria. A number of characteristics of the MDH have been elucidated, including the molecular masses of the native enzyme and the subunit, N-terminal amino acid sequence, isoelectric point, pH optimum for activity, thermostability, stability to pH, urea and guanidinium chloride and several kinetic parameters. Whereas the MDH is a stable tetramer in the pH range 5-7, it appears to be converted into a stable dimer at pH 3.5. This suggests that the dimer is a stable intermediate in the dissociation of the tetramer to monomers at low pH.

Amino Acid Sequence↗
PubMed1988

A mycoplasma high-affinity transport system and the in vitro invasiveness of mouse sarcoma cells.

FS9 mouse sarcoma cells were previously shown to be highly invasive when confronted with chicken heart fibroblasts using Abercrombie's confronted explant technique. This invasion could be inhibited by addition to the assay of Fab fragments of a monoclonal antibody directed against p37, a protein associated with the surface of FS9 cells. We have cloned and sequenced the gene for p37. We show that it originates from Mycoplasma hyorhinis and that UGA is a tryptophan codon in this organism. We present evidence that the p37 gene is part of an operon encoding two additional proteins which are highly similar to components of the periplasmic binding-protein-dependent transport systems of Gram-negative bacteria, and we suggest that p37 is part of a homologous, high-affinity transport system in M. hyorhinis, a Gram-positive bacterium. We discuss the influence of p37 and M. hyorhinis on contact inhibition of locomotion of mammalian cells.

Amino Acid Sequence↗