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Rhodococcus opacus B4 (= NBRC 108011)

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close this sectionAbout this Microorganism

Courtesy of Dr. Kato (Hiroshima univ.)

Rhodococcus opacus B4 was isolated as an organic solvent-tolerant bacterium from gasoline-contaminated soil. Members of the genus Rhodococcus are common in many environmental niches from soil to sea water and show remarkable metabolic versatility, including their ability to degrade a wide variety of organic compounds. They are high G+C Gram-positive bacteria classified into the order Actinomycetales, suborder Corynebacterineae, and family Nocardiaceae. R. opacus B4 utilize many aromatic and aliphatic hydrocarbons including benzene, toluene, xylene, ethylbenzene, propylbenzene, n-octane and n-decane as sole sources of carbon and energy. R. opacus B4 is able to retain their metabolic activity in organic solvents and therefore to mediate biosynthesis as a whole-cell catalyst.

Genome analysis of R. opacus B4 genome revealed six replicons composed of one linear chromosome (7,913,450 bp, 67.9% G+C, 7,252 ORF), two linear plasmids pROB01 (558,192 bp, 65.8% G+C, 593 ORF), pROB02 (244,997 bp, 64.2% G+C, 248 ORF), and three circular plasmids pKNR (111,160 bp, 65.0% G+C, 102 ORF), pKNR01 (4,367 bp, 64.5% G+C, 6 ORF) and pKNR02 (2,773 bp, 63.8% G+C, 2 ORF). R. opacus B4 genome showed overall conservation of synteny with sequenced Rhodococcus jostii RHA1 genome which also possesses a linear chromosome. R. opacus B4 chromosome is more similar in its gene arrangement to the circular chromosome Nocardia farcinica than those of Mycobacterium tuberculosis and Corynebacterium glutamicum or the linear chromosome of Streptomyces coelicolor, being consistent with their taxonomic relationships. On the other hand, telomeric sequences of the three linear replicons were conserved with other actinomycete invertorons. The genome encodes extremely abundant catabolic pathways for aromatic compounds including benzene, benzoate, phenol, 4-nitrophenol, 4-hydroxybenzoate, p-cumate, catechol, protocatechuate, and phenylacetate. The genome further encodes genes involved in the degradation of various organic compounds including naphthalene, indene, nicotine, thiocarbamate herbicide (EPTC), and thiocyanate. Genome analysis of R. opacus B4 thus provides important insights into biochemical and genetic bases of the metabolic versatility of Rhodococcus.

This work was conducted as a part of the project "Development of a Technological Infrastructure for Industrial Bioprocesses" of the New Energy and Industrial Technology Development Organization (NEDO), Japan.

close this sectionProject history

close this date 2013-03-12 ..... 1
2013-03-12 Release of proteome analysis result of Rhodococcus opacus B4
imageWe opened proteome results of Rhodococcus opacus B4 produced by using MALDI-TOFMS, LC-MS/MS and Protein Sequencer.

close this sectionSummary of the genomic data

Genomic size 8,834,939 bp
G+C content 67.62 %
Number of ORFs assigned 8,203
Percentage of the coding regions 90.37 %
Percentage of the intronic regions 0.00 %
Number of rRNA genes 12
Number of tRNA genes 49
Number of other features

close this sectionGeneral Procedure

The nucleotide sequence of the R. opacus B4 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 2-5 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 PRISM3730 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 PRISM3730.
    Raw sequence data corresponding to approximately 10-fold coverage were assembled using PHRED/PHRAP/CONSED software (
    The sequencing of large repeats, including the chromosome terminal inverted repeats, was performed by transposon-mediated method using a Template Generation System II kit (Finnzymes).

  • Gap closing
    Fosmid end sequences were mapped onto the assembled sequence.
    Fosmid clones that link two contigs were selected and sequenced by primer walking to close gaps.
    The sequencing of difficult templates was performed using the CUGA Sequencing Kit (Nippon Genetech).

  • Validation of the assembled sequence data
    Sequence assembly and genomic scaffolds were further validated by Optical Mapping (OpGen) using Nco I restriction endonuclease. No discrepancy was found between the optical maps and the simulated cutting patterns of the R. opacus B4 chromosomes within the resolution of the method.

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.

  • These predicted ORFs were further evaluated using the Frameplot program.

  • The translated sequences of the predicted protein-coding genes were searched against the nonredundant UniProt database (version 14.0) and the protein signature database, InterPro version 18.0.

  • 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