Limits...
Comparative genomic analysis of nine Sphingobium strains: insights into their evolution and hexachlorocyclohexane (HCH) degradation pathways.

Verma H, Kumar R, Oldach P, Sangwan N, Khurana JP, Gilbert JA, Lal R - BMC Genomics (2014)

Bottom Line: Genes associated with nitrogen stress response and two-component systems were found to be enriched.Further, in HDIPO4, linA was found as a hybrid of two natural variants i.e., linA1 and linA2 known for their different enantioselectivity.The bacteria isolated from HCH dumpsites provide a natural testing ground to study variations in the lin system and their effects on degradation efficacy.

View Article: PubMed Central - PubMed

Affiliation: Room No, 115, Molecular Biology Laboratory, Department of Zoology, University of Delhi, Delhi 110007, India. ruplal@gmail.com.

ABSTRACT

Background: Sphingobium spp. are efficient degraders of a wide range of chlorinated and aromatic hydrocarbons. In particular, strains which harbour the lin pathway genes mediating the degradation of hexachlorocyclohexane (HCH) isomers are of interest due to the widespread persistence of this contaminant. Here, we examined the evolution and diversification of the lin pathway under the selective pressure of HCH, by comparing the draft genomes of six newly-sequenced Sphingobium spp. (strains LL03, DS20, IP26, HDIPO4, P25 and RL3) isolated from HCH dumpsites, with three existing genomes (S. indicum B90A, S. japonicum UT26S and Sphingobium sp. SYK6).

Results: Efficient HCH degraders phylogenetically clustered in a closely related group comprising of UT26S, B90A, HDIPO4 and IP26, where HDIPO4 and IP26 were classified as subspecies with ANI value >98%. Less than 10% of the total gene content was shared among all nine strains, but among the eight HCH-associated strains, that is all except SYK6, the shared gene content jumped to nearly 25%. Genes associated with nitrogen stress response and two-component systems were found to be enriched. The strains also housed many xenobiotic degradation pathways other than HCH, despite the absence of these xenobiotics from isolation sources. Additionally, these strains, although non-motile, but posses flagellar assembly genes. While strains HDIPO4 and IP26 contained the complete set of lin genes, DS20 was entirely devoid of lin genes (except linKLMN) whereas, LL03, P25 and RL3 were identified as lin deficient strains, as they housed incomplete lin pathways. Further, in HDIPO4, linA was found as a hybrid of two natural variants i.e., linA1 and linA2 known for their different enantioselectivity.

Conclusion: The bacteria isolated from HCH dumpsites provide a natural testing ground to study variations in the lin system and their effects on degradation efficacy. Further, the diversity in the lin gene sequences and copy number, their arrangement with respect to IS6100 and evidence for potential plasmid content elucidate possible evolutionary acquisition mechanisms for this pathway. This study further opens the horizon for selection of bacterial strains for inclusion in an HCH bioremediation consortium and suggests that HDIPO4, IP26 and B90A would be appropriate candidates for inclusion.

Show MeSH

Related in: MedlinePlus

Genome recruitment plot mapping onSphingomonassp MM-1 plasmid A) pISP3 and B) pISP4: the raw reads of the six novely sequenced strains were mapped on plasmids ofSphingomonassp. MM1. The orange bars are depicting IS6100 while the lin genes were marked with grey and yellow bars showing their position in reference.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4289293&req=5

Fig6: Genome recruitment plot mapping onSphingomonassp MM-1 plasmid A) pISP3 and B) pISP4: the raw reads of the six novely sequenced strains were mapped on plasmids ofSphingomonassp. MM1. The orange bars are depicting IS6100 while the lin genes were marked with grey and yellow bars showing their position in reference.

Mentions: Genome recruitment plots were created to map the raw reads of the six novel-sequenced strains to Sphingobium plasmid sequences to investigate the possibility of these plasmids playing a role in transfer of the lin genes. MM-1 plasmids pISP3 and pISP4 in particular were found to have a high percentage of coverage which was maximum with S. ummariense RL3 (Figure 6). As pISP3 houses linDER, it is highly probable that plasmid uptake and duplication may explain the triplication of linDER in RL3. The recent metagenomics analysis of the HCH dumpsite also reflected the enrichment of pISP3, suggesting its availability for other sphingomonads strains present at the HCH dumpsite [10]. Furthermore, pISP4 encodes linB, linC, and linF, and similarly shows a high degree of coverage by RL3. Consistent with the absence of linC from the RL3 draft genome, which was confirmed by PCR amplification by using the primer 5′-GCGGATCCGCATGTCTGATTTGAGCGGC-3′ and 3′-GCCTCGAGTCAGATCGCGGTAAAGCCGCCGTC-5′, there is a gap in the coverage seen in the plasmid region containing linC (11,370 to 12,122 bp), which is a region flanked by two IS6100 elements in MM-1 (Figure 6). This points to the possibility that the plasmid has undergone either acquisition in MM-1 or looping out from RL3 of the linC gene during the course of evolution, mediated directly by IS6100. Mapping the raw reads of the six newly-sequenced Sphingobium strains to the plasmid sequences for MM-1 and UT26S, several of the MM-1 plasmids, but none of the UT26S plasmids demonstrated a high degree of coverage. Additionally, the proportionally higher presence of lin genes on plasmids in MM-1 than in UT26S suggests that strain MM-1 acts as a reservoir for plasmids allowing for the effective spread of the lin system, and thus may be an important strain to include in the consortium development as a potential disseminator of the lin system. Also, strains sharing similar arrangement profile of lin genes with MM-1 i.e., RL3, IP26 and HDIPO4, should be included into designing a consortium.Figure 6


Comparative genomic analysis of nine Sphingobium strains: insights into their evolution and hexachlorocyclohexane (HCH) degradation pathways.

Verma H, Kumar R, Oldach P, Sangwan N, Khurana JP, Gilbert JA, Lal R - BMC Genomics (2014)

Genome recruitment plot mapping onSphingomonassp MM-1 plasmid A) pISP3 and B) pISP4: the raw reads of the six novely sequenced strains were mapped on plasmids ofSphingomonassp. MM1. The orange bars are depicting IS6100 while the lin genes were marked with grey and yellow bars showing their position in reference.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4289293&req=5

Fig6: Genome recruitment plot mapping onSphingomonassp MM-1 plasmid A) pISP3 and B) pISP4: the raw reads of the six novely sequenced strains were mapped on plasmids ofSphingomonassp. MM1. The orange bars are depicting IS6100 while the lin genes were marked with grey and yellow bars showing their position in reference.
Mentions: Genome recruitment plots were created to map the raw reads of the six novel-sequenced strains to Sphingobium plasmid sequences to investigate the possibility of these plasmids playing a role in transfer of the lin genes. MM-1 plasmids pISP3 and pISP4 in particular were found to have a high percentage of coverage which was maximum with S. ummariense RL3 (Figure 6). As pISP3 houses linDER, it is highly probable that plasmid uptake and duplication may explain the triplication of linDER in RL3. The recent metagenomics analysis of the HCH dumpsite also reflected the enrichment of pISP3, suggesting its availability for other sphingomonads strains present at the HCH dumpsite [10]. Furthermore, pISP4 encodes linB, linC, and linF, and similarly shows a high degree of coverage by RL3. Consistent with the absence of linC from the RL3 draft genome, which was confirmed by PCR amplification by using the primer 5′-GCGGATCCGCATGTCTGATTTGAGCGGC-3′ and 3′-GCCTCGAGTCAGATCGCGGTAAAGCCGCCGTC-5′, there is a gap in the coverage seen in the plasmid region containing linC (11,370 to 12,122 bp), which is a region flanked by two IS6100 elements in MM-1 (Figure 6). This points to the possibility that the plasmid has undergone either acquisition in MM-1 or looping out from RL3 of the linC gene during the course of evolution, mediated directly by IS6100. Mapping the raw reads of the six newly-sequenced Sphingobium strains to the plasmid sequences for MM-1 and UT26S, several of the MM-1 plasmids, but none of the UT26S plasmids demonstrated a high degree of coverage. Additionally, the proportionally higher presence of lin genes on plasmids in MM-1 than in UT26S suggests that strain MM-1 acts as a reservoir for plasmids allowing for the effective spread of the lin system, and thus may be an important strain to include in the consortium development as a potential disseminator of the lin system. Also, strains sharing similar arrangement profile of lin genes with MM-1 i.e., RL3, IP26 and HDIPO4, should be included into designing a consortium.Figure 6

Bottom Line: Genes associated with nitrogen stress response and two-component systems were found to be enriched.Further, in HDIPO4, linA was found as a hybrid of two natural variants i.e., linA1 and linA2 known for their different enantioselectivity.The bacteria isolated from HCH dumpsites provide a natural testing ground to study variations in the lin system and their effects on degradation efficacy.

View Article: PubMed Central - PubMed

Affiliation: Room No, 115, Molecular Biology Laboratory, Department of Zoology, University of Delhi, Delhi 110007, India. ruplal@gmail.com.

ABSTRACT

Background: Sphingobium spp. are efficient degraders of a wide range of chlorinated and aromatic hydrocarbons. In particular, strains which harbour the lin pathway genes mediating the degradation of hexachlorocyclohexane (HCH) isomers are of interest due to the widespread persistence of this contaminant. Here, we examined the evolution and diversification of the lin pathway under the selective pressure of HCH, by comparing the draft genomes of six newly-sequenced Sphingobium spp. (strains LL03, DS20, IP26, HDIPO4, P25 and RL3) isolated from HCH dumpsites, with three existing genomes (S. indicum B90A, S. japonicum UT26S and Sphingobium sp. SYK6).

Results: Efficient HCH degraders phylogenetically clustered in a closely related group comprising of UT26S, B90A, HDIPO4 and IP26, where HDIPO4 and IP26 were classified as subspecies with ANI value >98%. Less than 10% of the total gene content was shared among all nine strains, but among the eight HCH-associated strains, that is all except SYK6, the shared gene content jumped to nearly 25%. Genes associated with nitrogen stress response and two-component systems were found to be enriched. The strains also housed many xenobiotic degradation pathways other than HCH, despite the absence of these xenobiotics from isolation sources. Additionally, these strains, although non-motile, but posses flagellar assembly genes. While strains HDIPO4 and IP26 contained the complete set of lin genes, DS20 was entirely devoid of lin genes (except linKLMN) whereas, LL03, P25 and RL3 were identified as lin deficient strains, as they housed incomplete lin pathways. Further, in HDIPO4, linA was found as a hybrid of two natural variants i.e., linA1 and linA2 known for their different enantioselectivity.

Conclusion: The bacteria isolated from HCH dumpsites provide a natural testing ground to study variations in the lin system and their effects on degradation efficacy. Further, the diversity in the lin gene sequences and copy number, their arrangement with respect to IS6100 and evidence for potential plasmid content elucidate possible evolutionary acquisition mechanisms for this pathway. This study further opens the horizon for selection of bacterial strains for inclusion in an HCH bioremediation consortium and suggests that HDIPO4, IP26 and B90A would be appropriate candidates for inclusion.

Show MeSH
Related in: MedlinePlus