NRSub: a non-redundant database for Bacillus subtilis.

In the context of the international project aimed at sequencing the whole genome of Bacillus subtilis we have developed a non-redundant, fully annotated database of sequences from this organism. Starting from the B.subtilis sequences available in the EMBL, GenBank and DDBJ collections we have removed all encountered duplications and then added extra annotations to the sequences (e.g. accession numbers for the genes, locations on the genetic map, codon usage, etc.) We have also added cross-references to the EMBL, MEDLINE, SWISS-PROT and ENZYME data banks. The present system results from merging of the NRSub and SubtiList databases and the sequence contigs used in the two systems are identical. NRSub is distributed as a flatfile in EMBL format (which is supported by most sequence analysis software packages) and as an ACNUC database, while SubtiList is distributed as a relational database under 4th Dimension. It is possible to access the data through two dedicated World Wide Web servers located in France and Japan.     

NRSub: a non-redundant data base for the Bacillus subtilis genome.

We have organized the DNA sequences of Bacillus subtillis from the EMBL collection to build the NRSub data base. This data base is free from duplications and all detected overlapping sequences are merged into contigs. Data on gene mapping and codon usage are also included. NRSub is publically available through anonymous FTP in flat file format or structured on the form of an ACNUC data base. Under this format, it is possible to use NRSub with the retrieval program Query--win. This program integrates a graphical interface and may be installed on any kind of UNX computer under X Window and on which the Vibrant and Motif libraries are available.     

The NRSub database: update 1997.

In the context of the international project aiming at sequencing the whole genome of Bacillus subtilis we have developed NRSub, a non-redundant database of sequences from this organism. Starting from the B.subtilis sequences available in the repository collections we have removed all encountered duplications, then we have added extra annotations to the sequences (e.g. accession numbers for the genes, locations on the genetic map, codon usage index). We have also added cross-references with EMBL/GenBank/DDBJ, MEDLINE, SWISS-PROT and ENZYME databases. NRSub is distributed through anonymous FTP as a text file in EMBL format and as an ACNUC database. It is also possible to access the database through two dedicated World Wide Web servers located in France (http://acnuc.univ-lyon1.fr/nrsub/nrsub.++ +html ) and in Japan (http://ddbjs4h.genes.nig.ac.jp/ ).     

OWL--a non-redundant composite protein sequence database.

A comprehensive, non-redundant composite protein sequence database is described. The database, OWL, is an amalgam of data from six publicly-available primary sources, and is generated using strict redundancy criteria. The database is updated monthly and its size has increased almost eight-fold in the last six years: the current version contains > 76,000 entries. For added flexibility, OWL is distributed with a tailor-made query language, together with a number of programs for database exploration, information retrieval and sequence analysis, which together form an integrated database and software resource for protein sequences.     

SubtiList: the reference database for the Bacillus subtilis genome

SubtiList is the reference database dedicated to the genome of Bacillus subtilis 168, the paradigm of Gram-positive endospore-forming bacteria. Developed in the framework of the B.subtilis genome project, SubtiList provides a curated dataset of DNA and protein sequences, combined with the relevant annotations and functional assignments. Information about gene functions and products is continuously updated by linking relevant bibliographic references. Recently, sequence corrections arising from both systematic verifications and submissions by individual scientists were included in the reference genome sequence. SubtiList is based on a generic relational data schema and a World Wide Web interface developed for the handling of bacterial genomes, called GenoList. The World Wide Web interface was designed to allow users to easily browse through genome data and retrieve information according to common biological queries. SubtiList also provides more elaborate tools, such as pattern searching, which are tightly connected to the overall browsing system. SubtiList is accessible at http://genolist.pasteur.fr/SubtiList/. Similar bacterial databases are accessible at http://genolist.pasteur.fr/.     

The Bacillus subtilis ureABC operon.

The Bacillus subtilis ureABC operon encodes homologs of the three subunits of urease enzymes of the family Enterobacteriaceae. Disruption of ureC prevented utilization of urea as a nitrogen source and resulted in a partial growth defect in minimal medium containing limiting amounts of arginine or allantoin as the sole nitrogen source.     

Insertion mutagenesis in Bacillus subtilis (Marmur)

A method for obtaining mutations in Bacillus subtilis using integration into the chromosome of the plasmid pHV60 ligated to chromosomal DNA fragments was developed. Auxotrophic mutants acquire, in addition, chloramphenicol-resistance, due to insertion of appropriate plasmid determinants. Chromosomal localization was established and the properties of several mutants were studied.     

Bacteria-shaped Gymnoplasts (Protoplasts) of Bacillus subtilis

Addition of glucose to the medium in which Bacillus subtilis was grown lowered the pH and increased the amount of lysylphosphatidylglycerol relative to the phosphatidylglycerol content of the membrane fraction. This change in phospholipid composition was accompanied by changes in the shape and behavior of the gymnoplasts obtained by cell wall removal with lysozyme. These gymnoplasts appeared to retain most of their original cell shape and internal organization, often with preservation of the mesosomes. Cells harvested from neutral growth medium gave the usual spherical gymnoplasts. In a hypotonic medium, the spherical gymnoplasts lysed rapidly, whereas the rod-like gymnoplasts lost only part of their cell content while showing a tendency to preserve the original shape. This type of gymnoplast could not be produced from cells grown in neutral medium by simply raising the magnesium concentration. When this was done the gymnoplasts assumed bizarre shapes; they became compact and susceptible to the tonicity of the medium. Gymnoplasts or protoplasts, produced from bacilli exposed to low pH values, were found not to conform to the formulations on "protoplasts" given in 1958 by 13 authors. Cells exposed to a low environmental pH during growth seemed to possess a more rigid membrane structure than cells grown at neutral pH.     

The spoIIN279(ts) mutation affects the FtsA protein of Bacillus subtilis.

The spo-279(ts) mutation, originally thought to be located in the spoIIG operon of Bacillus subtilis, has been mapped in close proximity but outside of the spoIIG locus. This mutation defines a new gene, spoIIN, located midway between the spoIIG and the spoVE loci, and whose product is required for successful completion of the asymmetric septation step. The spoIIN locus was cloned using a combination of 'walking steps' upstream from the spoIIG region and hybridization screening of a bacteriophage lambda library. Sequencing of DNA fragments able to rescue the spoIIN279(ts) mutation revealed that the spoIIN locus is identical with the B subtilis counterpart of the Escherichia coli ftsA gene. After cloning the ftsA region from a strain containing the spoIIN279(ts) mutation we found that this mutation converts the ninth residue of the FtsA protein from serine to asparagine. The spoIIN279(ts) mutation, which is recessive, leads to filamentation during growth at 42 degrees C and causes defective formation of the sporulation septum at this non-permissive temperature. The FtsA protein is therefore required for proper cell septation, both during vegetative growth and sporulation. Possible additional roles of FtsA during sporulation are discussed.     

SPiD: a subtilis protein interaction database.

MOTIVATION: Protein-protein interactions are a potential source of valuable clues in determining the functional role of as yet uncharacterized gene products in metabolic pathways. Graph-like structures emerging from the accumulation of interaction data make it difficult to maintain a consistent and global overview by hand. Bioinformatics tools are needed to perform this graph visualization while maintaining a link to the experimental data. RESULTS: "SPiD" is an online database for exploring networks of interacting proteins in Bacillus subtilis characterized by the two-hybrid system. Graphical displays of interaction networks are created dynamically as users interactively navigate through these networks. Third party applications can interface the database through a Common Object Request Broker Architecture (CORBA) tier. AVAILABILITY: SPiD is available through its web site at http://www-mig.versailles.inra.fr/bdsi/SPiD, and through an Interoperable Object Reference (IOR) and its associated Interface Definition Language (IDL). CONTACT: hoebeke@versailles.inra.fr     

The divIVA minicell locus of Bacillus subtilis.

The Bacillus subtilis divIVA1 mutation causes misplacement of the septum during cell division, resulting in the formation of small, circular, anucleate minicells. This study reports the cloning and sequence analysis of 2.4 kb of the B. subtilis chromosome including the divIVA locus. Three open reading frames were identified: orf, whose function is unknown; divIVA; and isoleucyl tRNA synthetase (ileS). We identified the point mutation in the divIVA1 mutant allele. Inactivation of divIVA produces a minicell phenotype, whereas overproduction of DivIVA results in a filamentation phenotype. Mutants with mutations at both of the minicell loci of B. subtilis, divIVA and divIVB, possess a minicell phenotype identical to that of the DivIVB- mutant. The DivIVA-mutants, but not the DivIVB- mutants, show a decrease in sporulation efficiency and a delay in the kinetics of endospore formation. The data support a model in which divIVA encodes the topological specificity subunit of the minCD system. The model suggests that DivIVA acts as a pilot protein, directing minCD to the polar septation sites. DivIVA also appears to be the interface between a sporulation component and MinCD, freeing up the polar septation sites for use during the asymmetric septation event of the sporulation process.     

Aminopeptidases of Bacillus subtilis.

Three enzymes with L- and one enzyme with D-aminopeptidase (EC 3.4.11; alpha-aminoacyl peptide hydrolase) activity have been separated from each other and partially purified from Bacillus subtilis 168 W.T., distinguished with respect to their molecular weights and catalytic properties, and studied in relation to the physiology of this bacterium. One L-aminopeptidase, designated aminopeptidase I, has a molecular weight of 210,000 +/- 20,000, is produced early in growth, and hydrolyzes L-alanyl-beta-naphthylamide most rapidly. Another, designated aminopeptidase II, molecular weight 67,000 +/- 10,000, is also produced early in growth and hydrolyzes L-lysyl-beta-naphthylamide most rapidly. A third, aminopeptidase III, molecular weight 228,000 +/- 20,000, is produced predominantly in early stationary phase and most efficiently utilizes L-alpha-aspartyl-beta-naphthylamide as substrate. The synthesis of aminopeptidase III in early stationary phase suggests that selective catabolism of peptides occurs at this time, perhaps related to the cessation of growth or the onset of early sporulation-associated events. A D-aminopeptidase which hydrolyzes the carboxyl-blocked dipeptide D-alanyl-D-alanyl-beta-naphthylamide (as well as D-alanyl-beta-naphthylamide and D-alanyl-D-alanyl-D-alanine) has also been identified, separated from aminopeptidase II, and purified 170-fold. D-Aminopeptidase, molecular weight 220,000 +/- 20,000, is localized predominantly in the cell wall and periplasm of the organism. This evidence and the variation of the activity during the growth cycle suggest an important function in cell wall or peptide antibiotic metabolism.