Computational protocol: Complete Genome Sequence of Pseudomonas aeruginosa MTB-1, Isolated from a Microbial Community Enriched by the Technical Formulation of Hexachlorocyclohexane

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Protocol publication

[…] Gamma-hexachlorocylohexane (γ-HCH) (also called gamma-benzenehexachloride or lindane) is a chlorinated organic insecticide that has caused serious environmental problems due to its toxicity and long persistence in upland soils (, ). The technical formulation of HCH (t-HCH) mainly consists of an insecticidal γ-isomer and noninsecticidal α-, β-, and δ-isomers. Our previous use of a microbial community enriched by t-HCH from t-HCH-contaminated soil in India () led to the successful isolation of a γ-HCH-degrading bacterium, Sphingomonas sp. strain MM-1 (, ), and a non-γ-HCH-degrading bacterium, Pseudomonas aeruginosa MTB-1 (). The strains MM-1 and MTB-1 have been deposited in the Japan Collection of Microorganisms (JCM) under the accession numbers JCM 19685 and JCM 19686, respectively.The MTB-1 genome was sequenced using 454 GS FLX+ (Roche) and MiSeq (Illumina) systems. A fragment library and a paired-end library were constructed for the 454 sequencing, and 229,833 reads (179 Mb) and 44,402 reads (19 Mb) were obtained, respectively, while the 151-bp paired-end sequencing with Illumina generated 3,862,256 reads (578 Mb). The reads obtained by the two systems were assembled using Newbler version 2.8 (Roche), which produced 45 contigs and a single scaffold. The finishing was facilitated using the two computer programs GenoFinisher and AceFileViewer () (http://www.ige.tohoku.ac.jp/joho/gf_e/). The scaffold consisted of 15 nonrepeat contigs, and all of the 15 gaps, including those located between the last and the first contigs, were closed by in silico analyses, in which the DNA sequences of each copy of repeats were precisely determined. The finished sequence was confirmed by FinishChecker, an accessory tool of GenoFinisher.The sequence was annotated by the NCBI Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP), and the resulting annotation was subjected to manual curation using the annotation support tool of GenomeMatcher (). In the curation, by referencing annotation data obtained from the Microbial Genome Annotation Pipeline (http://www.migap.org/), we corrected the start codon positions and added several genes that were missing in the PGAAP annotation.The complete sequence of the MTB-1 genome consisted of one circular chromosome with a size of 6,580,038 bp. It carried four copies of rRNA operons, 64 tRNA genes, and 6,078 protein-encoding genes. The six lin genes specific for the conversion of γ-HCH to β-ketoadipate (linA to linF) () were not found in the MTB-1 genome. A detailed comparison of the MTB-1 genome with the genomes of other P. aeruginosa strains may provide us with some insights into the reason(s) why MTB-1 persistently coproliferated with the γ-HCH degrader MM-1 in the liquid culture enriched by t-HCH. […]

Pipeline specifications

Software tools Newbler, PGAP, GenomeMatcher
Application Genome data visualization
Organisms Pseudomonas aeruginosa, Sphingomonas sp. MM-1
Chemicals Lindane