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Brevibacillus brevis NBRC 100599

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close this sectionAbout this Microorganism

Brevibacillus brevis NBRC 100599 (= 47), which was formerly known as Bacillus brevis 47, is a strictly aerobic, gram-positive, spore-forming bacterium isolated from soil. B. brevis NBRC 100599 has a characteristic three-layered cell wall composed of a peptidoglycan-layer and the outer two S-layers, each consisting of single protein species, and is known to secrete large amounts of proteins into the culture media. The proteins secreted by this strain are mainly derived from the outer two layers of the cell wall, which are shed into the medium with the progress of cell growth. In contrast to its ability of efficient production and secretion of cell wall proteins, this bacterium secretes little protease, which enables easy recovery of the secreted proteins with minimal degradation. Taking advantage of these features, B. brevis NBRC 100599 has been used as a good host for production of heterologous proteins of both prokaryotic and eukaryotic origin.

Sequencing and annotation of the genome of B. brevis NBRC 100599 revealed a single circular chromosome (6,296,436 bp; G+C content of 47.3%) containing 5,949 predicted protein-coding genes, about 30% of which had orthologs in each of the Bacillus genomes. Significant synteny was observed only in the vicinity of the oriC region between B. brevis NBRC 100599 and the Bacillus species. The genome seemed to lack several sugar metabolism genes found in Bacillus subtilis, being in good agreement with the results in other strains showing that B. brevis is unable to utilize L-arabinose, D-mannose and D-xylose. The abundance of genes involved in response to environmental stress, such as those encoding sigma factors and chemotaxis receptors, suggests that this bacterium has a great capacity of adaptation to diverse environments.

close this sectionProject history

close this date 2009-05-09 ..... 1
2009-05-09 Brevibacillus brevis NBRC 100599 database was updated (We changed EC number of several ORFs)
imageList of ORFs updated in annotation
EC
IDoldnew
BBR47_01180 2.7.1.37 2.7.11.1
BBR47_01710 6.3.5.8 2.6.1.85
BBR47_01720 6.3.5.8 / 4.1.3.27 2.6.1.85 / 4.1.3.27
BBR47_05010 2.7.1.37 2.7.11.1
BBR47_18030 4.3.1.8 2.5.1.61
BBR47_18040 4.1.2.75 4.2.1.75
BBR47_22040 1.7.99.5 1.5.1.20
BBR47_23950 2.7.1.37 2.7.11.1
BBR47_37250 2.7.1.37 2.7.11.1

close this sectionSummary of the genomic data

BBR
Genomic size 6,296,436 bp
G+C content 47.27 %
Number of ORFs assigned 5,949
Percentage of the coding regions 88.44 %
Percentage of the intronic regions 0.00 %
Number of rRNA genes 44
5S16S23S
141515
Number of tRNA genes 127
AlaArgAsnAspCysGln
795724
GluGlyHisIleLeuLys
71045146
MetPheProSerThrTrp
846872
TyrVal
48
Number of other features
(misc_RNA,misc_feature,repeat)
0

close this sectionGeneral Procedure

The nucleotide sequence of the B. brevis 47 genome was determined by the whole genome shotgun sequencing method as in the case of other organisms analyzed at NITE-DOB.


General Procedure
  • DNA shotgun library
    DNA shotgun library with inserts of 1-8 kb in pUC118 vector (TAKARA) was constructed.

  • Fosmid library
    A Fosmid library with inserts of 40 kb in the pCC1FOS fosmid vector was constructed using the CopyControl Fosmid Library Production Kit (Epicentre).

  • Nucleotide sequencing
    Plasmid clones were end-sequenced using dye-terminator chemistry on an ABI PRISM3700 sequencer (ABI).
    Fosmid DNA was extracted from E. coli transformants using the Montage BAC96 MiniPrep Kit (Millipore) and end-sequencing was carried out using dye-terminator chemistry on ABI PRISM3700.
    Raw sequence data corresponding to approximately 16-fold coverage were assembled using PHRED/PHRAP/CONSED software (http://www.phrap.org).

  • Gap closing
    Long sequence gaps between contigs were filled up by sequencing corresponding cosmid clones or DNA fragments obtained by PCR using the B. brevis genomic DNA as a template and primers placed at the ends of contigs flanking the gaps, while short sequence gaps were filled up by sequencing appropriate bridging WGS clones if available.

  • Validation of the assembled sequence data
    From the final nucleotide sequence, PCR primer sequences were generated at appropriate intervals throughout the genome which were then used to amplify the corresponding genomic regions. The restriction enzyme digestion patterns of each of the PCR fragments thus obtained were accordingly compared with those deduced from the sequence data of the regions to validate the correctness of the assembled sequence data.

Genome analysis and annotation
  • Putative nontranslated genes were identified using the Rfam and tRNAscan-SE programs, whereas rRNA genes were identified using the BLASTN program.

  • For the identification of protein-coding genes, the genome sequence was translated in six frames to generate potential protein products of open reading frames (ORFs) longer than 90 bp, with ATG, GTG and TTG considered as potential initial codons.

  • The potential protein sequences were compared with the UniProt databases using the BLASTP program.

  • Potential protein sequences that showed significant similarities to known protein sequences in the database were selected.

  • The start sites were manually inspected and altered in comparison to the prediction obtained by GLIMMER and GeneHacker.

  • The translated sequences of the predicted protein-coding genes were searched against the nonredundant UniProt database (version 13.0) and the protein signature database, InterPro version 16.0.

  • The KEGG database was used for pathway reconstruction.

  • Signal peptides in proteins were predicted using SignalP, whereas transmembrane helices were predicted using TMHMM.

close this sectionRelated links to external databases