Draft Genome Sequence of Ralstonia sp. Strain GA3-3, Isolated from Australian Suburban Soil

Abstract

Ralstonia sp. strain GA3-3 is a hexachlorocyclohexane (HCH)-degrading bacterial strain isolated from suburban soil in Canberra, Australia. The genome of strain GA3-3 was sequenced to investigate its ability to degrade α-HCH. Here, we report the annotated genome sequence of this strain.

GENOME ANNOUNCEMENT

Several bacterial strains were isolated during a hexachlorocyclohexane (HCH) degradation study in Australia. Two strains, including Ralstonia sp. strain GA3-3, were chosen to be sequenced based on their HCH degradation phenotype. GA3-3 was isolated from suburban soil with no known history of HCH exposure in Canberra, Australia, after three cycles of enrichment with 50 ppm α-HCH as the sole carbon source. Further study of GA3-3 revealed that it was able to degrade α-HCH, albeit incompletely and very slowly compared to other known HCH degraders (1).

The genomic DNA of GA3-3 was prepared using the Qiagen Genomic-tip 20/G kit for bacteria, according to the manufacturer’s instructions. Fragments of 500-bp length were then sequenced using Illumina HiSeq 2000 technology at the John Curtin School of Medical Research, Australian National University. The Ray assembler was used to assemble 8,533,778 100-bp paired-end reads using a k-mer length of 63 (2). This assembly generated 62 contigs of >500 bp in length, with an N₅₀ value of 193,594 bp. The total size of the assembly was 6.7 Mb, with a G+C content of 66.7%. The paired-end information was able to combine 26 of the contigs into 11 scaffolds, making a total of 47 scaffolds or contigs for the assembly.

Annotation of the genome using the NCBI Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP) predicted 6,174 protein-coding sequences in GA3-3, with 1,439 (23.4%) annotated only as hypothetical proteins. The assembly was also predicted to contain 55 tRNA and 14 rRNA sequences. A BLAST search of the GA3-3 16S rRNA sequence against the NCBI database revealed 99.7% (1,403-nucleotide alignment) identity between GA3-3 and other Ralstonia spp.; however, further investigation is needed to determine the true identity of GA3-3.

All previously studied HCH-degrading bacteria share the same pathway for HCH degradation, which requires the linA-linF genes (3–6). A BLAST search was performed on the genome of GA3-3 using the protein sequences encoded by the linA-linF genes (accession no. YP_003545302, BAI96793, YP_003544005, YP_003547114, YP_003547119, and BAI98845, for linA, linB, linC, linD, linE, and linF, respectively) of Sphingobium japonicum UT26S from the NCBI database. However, no linA-linF genes or their homologues were detected in GA3-3 (1). Another α-HCH degradation study that was performed using GA3-3 at this stage revealed no degradation activity (S. Pearce, unpublished data). The absence of these genes, coupled with the knowledge that the lin genes are normally associated with the transposable insertion element IS6100 (5, 7, 8), may explain the loss of activity since the first degradation experiment.

Nucleotide sequence accession numbers.

This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession no. AQPZ00000000. The version described in this paper is version AQPZ01000000.