Abstract
Novosphingobium lindaniclasticum LE124T is a hexachlorocyclohexane (HCH)-degrading bacterium isolated from a high-dosage-point HCH dumpsite (450 mg HCH/g soil) located in Lucknow, India (27°00′N and 81°09′E). Here, we present the annotated draft genome sequence of strain LE124T, which has an estimated size of 4.86 Mb and is comprised of 4,566 coding sequences.
GENOME ANNOUNCEMENT
Sphingomonads represent a naturally selected taxon that can degrade and/or assimilate a wide range of xenobiotic compounds, including mono- and polycyclic aromatic compounds, chlorinated compounds, and pesticides (1). Members of this taxon are also known to be efficient degraders of hexachlorocyclohexane (HCH) isomers (1–3). In conformity with our primary objective, i.e., to elucidate the pangenomic variations across HCH-degrading genotypes, we have already sequenced two sphingomonads (4, 5). Both of their genomes represent species belonging to the genus Sphingobium (4, 5). We have sequenced the genome of yet another sphingomonad, belonging to the genus Novosphingobium, i.e., N. lindaniclasticum strain LE124T, isolated from an HCH dumpsite (6).
The genomic DNA of strain LE124T was sequenced by the Illumina genome analyzer IIx platform (paired-end library, 2 kb, n = 4.96 × 108, 90 bp/read) and the 454 GS FLX Titanium platform (single reads, n = 70,343,174, >350 bp). The draft genome sequence (4.86 Mb) of strain LE124T was assembled (150× coverage) into 156 contigs (>500 bp ± 10 bp) using ABySS v.1.3.3 assembler (7), set at a k-mer size of 57. The final validated (based on paired-end criterion) assembly (N50 of contigs, 37.4 kb) (8) was annotated using RAST v.4.0 (9) and the NCBI Prokaryotic Genomes Automatic Annotation Pipeline v.2.1 (PGAAP) (http://www.ncbi.nlm.nih.gov/genomes/static/Pipeline.html), which predicted 4,566 coding sequences (average G+C percentage, 64.6).
RAST annotation (9) predicted 414 subsystems and 30 putative metal resistance proteins. Nine rRNAs, 54 tRNAs, and 28 pseudogenes were found using PGAAP. Additionally, 52 transposases, 40 ABC transporters, and 90 transcriptional regulators were found in the genome. BLASTp-based (10) comparison with the ISfinder database (11) reported 10 insertion sequence (IS) elements (IS3/21) assigned to Sphingobium spp. (4). Plasmid-related genes, i.e., repA, par, and conjugative transfer genes, were detected on contig 113 (77,158 bp). Average nucleotide identity (ANI) (12) analysis revealed that the draft genome of N. lindaniclasticum LE124T is phylogenetically related to those of Erythrobacter litoralis (77.82%), Novosphingobium aromaticivorans (77.72%), and Sphingopyxis alaskensis (76.6%).
The HCH-degrading lin genes (13, 14) were found scattered throughout the draft genome assembly. A single copy of linA (dehydrochlorinase) was represented in the sequenced genome. Additionally, linH, linK, linL, linM, and two copies of linG were also present. In contrast to Sphingobium indicum B90A (4), linB (haloalkane dehalogenase) and linDER were absent from the genome of strain LE124T; this was also confirmed by PCR amplification. We have recently reported that HCH selection pressure is responsible for bringing lin genes through horizontal gene transfer (HGT) into sphingomonads (15), and these findings reflect that strain LE124T has yet to acquire these genes through HGT.
Interestingly, the draft genome of strain LE124T also showed the presence of a benzoate-degrading gene cluster with the presence of 4-hydroxybenzoate 3-monooxygenase, benzoate transporter proteins, and genes for the chloroaromatic catechol branch of the β-ketoadipate pathway. Additionally, genes encoding ortho-halobenzoate 1,2-dioxygenase alpha- and beta-intracellular serine protease (ISP) protein (ohbA, ohbB), known to be involved in xenobiotic and benzoate degradation, were also found. The genetic compendium required to resolve the intergenus-level genetic divergence of the lin genes and degradation pathway can be analyzed by doing comparative analyses of genomes of sphingomonads that have now been sequenced.
Nucleotide sequence accession numbers.
This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession no. ATHL00000000. The version described in this paper is version ATHL01000000.
ACKNOWLEDGMENTS
The work was supported by grants from the Department of Biotechnology (DBT), Government of India, under project no. BT/PR3301/BCE/8/875/11, the University of Delhi/Department of Science and Technology Promotion of University Research and Scientific Excellence du DST—PURSE grant and the National Bureau of Agriculturally Important Microorganisms (NBAIM) AMASS/2006–07/NBAIM/CIR, and the All India Network Project Soil Biodiversity-Biofertilizer (ICAR). A.S., N.N., N.S., and R.K. gratefully acknowledge the University Grants Commission (UGC) and the Council for Scientific and Industrial Research (CSIR), New Delhi, for providing research fellowships. This paper was finalized during the renewed visit under the Alexander von Humbolt fellowship (at the University of Freiburg, Germany) awarded to R.L.
Footnotes
Citation Saxena A, Nayyar N, Sangwan N, Kumari R, Khurana JP, Lal R. 2013. Genome sequence of Novosphingobium lindaniclasticum LE124T, isolated from a hexachlorocyclohexane dumpsite. Genome Announc. 1(5):e00715-13. doi:10.1128/genomeA.00715-13.
REFERENCES
- 1. Lal R, Pandey G, Sharma P, Kumari K, Malhotra S, Pandey R, Raina V, Kohler HP, Holliger C, Jackson C, Oakeshott JG. 2010. Biochemistry of microbial degradation of hexachlorocyclohexane and prospects for bioremediation. Microbiol. Mol. Biol. Rev. 74:58–80 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2. Lal R, Dadhwal M, Kumari K, Sharma P, Singh A, Kumari H, Jit S, Gupta SK, Nigam A, Lal D, Verma M, Kaur J, Bala K, Jindal S. 2008. Pseudomonas sp. to Sphingobium indicum: a journey of microbial degradation and bioremediation of hexachlorocyclohexane. Indian J. Microbiol. 48:3–18 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3. Lal R, Dogra C, Malhotra S, Sharma P, Pal R. 2006. Diversity, distribution and divergence of lin genes in hexachlorocyclohexane-degrading sphingomonads. Trends Biotechnol. 24:121–130 [DOI] [PubMed] [Google Scholar]
- 4. Anand S, Sangwan N, Lata P, Kaur J, Dua A, Singh AK, Verma M, Kaur J, Khurana JP, Khurana P, Mathur S, Lal R. 2012. Genome sequence of Sphingobium indicum B90A, a hexachlorocyclohexane-degrading bacterium. J. Bacteriol. 194:4471–4472 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5. Niharika N, Sangwan N, Ahmad S, Singh P, Khurana JP, Lal R. 2013. Draft genome sequence of Sphingobium chinhatense IP26T, isolated from a hexachlorocyclohexane dumpsite. Genome Announc. 1(4):e00680-13. 10.1128/genomeA.00680-13 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6. Saxena A, Anand S, Dua A, Sangwan N, Khan F, Lal R. 2013. Novosphingobium lindaniclasticum sp. nov., a hexachlorocyclohexane (HCH)-degrading bacterium isolated from an HCH dumpsite. Int. J. Syst. Evol. Microbiol. 63:2160–2167 [DOI] [PubMed] [Google Scholar]
- 7. Simpson JT, Wong K, Jackman SD, Schein JE, Jones SJ, Birol I. 2009. ABySS: a parallel assembler for short read sequence data. Genome Res. 19:1117–1123 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8. Li H, Durbin R. 2010. Fast and accurate long-read alignment with Burrows-Wheeler Transform. Bioinformatics 26:589–595 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ, Olson R, Osterman AL, Overbeek RA, McNeil LK, Paarmann D, Paczian T, Parrello B, Pusch GD, Reich C, Stevens R, Vassieva O, Vonstein V, Wilke A, Zagnitko O. 2008. The RAST server: rapid annotations using subsystems technology. BMC Genomics 9 10.1186/1471-2164-9-7575 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403–410 [DOI] [PubMed] [Google Scholar]
- 11. Siguier P, Perochon J, Lestrade L, Mahillon J, Chandler M. 2006. ISfinder: the reference centre for bacterial insertion sequences. Nucleic Acids Res. 34:D32–D36. 10.1093/nar/gkj014 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12. Konstantinidis KT, Tiedje JM. 2005. Towards a genome-based taxonomy for prokaryotes. J. Bacteriol. 187:6258–6264 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13. Dogra C, Raina V, Pal R, Suar M, Lal S, Gartemann KH, Holliger C, Van der Meer JR, Lal R. 2004. Organization of lin genes and IS6100 among different strains of hexachlorocyclohexane-degrading Sphingomonas paucimobilis: evidence for horizontal gene transfer. J. Bacteriol. 186:2225–2235 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14. Malhotra S, Sharma P, Kumari H, Singh A, Lal R. 2007. Localization of HCH catabolic genes (lin genes) in Sphingobium indicum B90A. Indian J. Microbiol. 47:271–275 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15. Sangwan N, Verma H, Kumar R, Negi V, Lax S, Khurana P, Khurana JP, Gilbert JA, Lal R. Reconstructing an ancestral genotype of two hexachlorocyclohexane degrading Sphingobium species using metagenomic sequence data. ISME J., in press [DOI] [PMC free article] [PubMed] [Google Scholar]