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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.

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Phylogenic constructions ofSphingobiumspp.Sphingobium baderi LL03, S. lactosutens DS20, S. chinhatense IP26, S. quisquilarium P25, S. ummariense RL3, Sphingobium sp. HDIPO4, S. indicum B90A, S. japonicum UT26S and Sphingobium sp. SYK6. (A) 16S rRNA gene sequence-based phylogeny was performed with the neighbor joining method using TreeconW 1.3b (bootstrap value = 1000) (B) The amino acid sequences of 28 single copy genes were concatenated for all ten genomes and phylogeny performed with the neighbor joining method using TreeconW 1.3b (bootstrap value =1000) (C) &(D) Whole genome based tetranucleotide correlation and ANI based pairwise comparisions between the genomes was carried out for all the genomes. A pearson correlation matrix was constructed on the basis of their tetranucleotide correlation and ANI values, followed by hierarchical clustering on the resultant matrix using MEV4.9.0, and visualized on MEGA4.
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Fig3: Phylogenic constructions ofSphingobiumspp.Sphingobium baderi LL03, S. lactosutens DS20, S. chinhatense IP26, S. quisquilarium P25, S. ummariense RL3, Sphingobium sp. HDIPO4, S. indicum B90A, S. japonicum UT26S and Sphingobium sp. SYK6. (A) 16S rRNA gene sequence-based phylogeny was performed with the neighbor joining method using TreeconW 1.3b (bootstrap value = 1000) (B) The amino acid sequences of 28 single copy genes were concatenated for all ten genomes and phylogeny performed with the neighbor joining method using TreeconW 1.3b (bootstrap value =1000) (C) &(D) Whole genome based tetranucleotide correlation and ANI based pairwise comparisions between the genomes was carried out for all the genomes. A pearson correlation matrix was constructed on the basis of their tetranucleotide correlation and ANI values, followed by hierarchical clustering on the resultant matrix using MEV4.9.0, and visualized on MEGA4.

Mentions: Four different phylogenetic methods (16S rRNA gene sequence, single copy gene sequences, tetranucleotide frequency based correlation, and average nucleotide identity (ANI)) were used to analyze the relationships of the nine strains under study (Figure 3). The consensus tree topology obtained by these methods clustered S. indicum B90A, S. japonicum UT26S, S. chinhatense IP26, and Sphingobium sp. HDIPO4, with the exception of the single copy gene approach. Notably, these four strains were the only ones with an entirely complete lin pathway, thus suggesting convergent evolution through HCH selection pressure. Furthermore, ANI topology supported the grouping of Sphingobium sp. HDIPO4 and S. chinhatense IP26 as subspecies (≥99.34%) (ANI values within the subspecies >98%) [18]. The other five strains i.e., LL03, DS20, RL3, P25 and SYK6 did not produce a consensus phylogeny, with relationships differing between these approaches; in short, strains with the complete lin pathway formed a closed group whereas, the others have diverged. In addition, 16S rRNA and single copy gene approaches may be problematic for differentiation among highly related strains (as these methodologies do not consider the influence of HGT). However, ANI based pairwise comparison has clustered LL03 and RL3 (partial lin gene deficient but HCH degraders) in a monophyletic clade with P25 (partial lin gene deficient and slow HCH degrader) forming a close relationship. Moreover, DS20 and SYK6 (non-HCH degraders) were clustered together. This suggests that ANI based phylogeny is more appropriate and mirrors their relationship with respect to HCH degradation.Figure 3


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)

Phylogenic constructions ofSphingobiumspp.Sphingobium baderi LL03, S. lactosutens DS20, S. chinhatense IP26, S. quisquilarium P25, S. ummariense RL3, Sphingobium sp. HDIPO4, S. indicum B90A, S. japonicum UT26S and Sphingobium sp. SYK6. (A) 16S rRNA gene sequence-based phylogeny was performed with the neighbor joining method using TreeconW 1.3b (bootstrap value = 1000) (B) The amino acid sequences of 28 single copy genes were concatenated for all ten genomes and phylogeny performed with the neighbor joining method using TreeconW 1.3b (bootstrap value =1000) (C) &(D) Whole genome based tetranucleotide correlation and ANI based pairwise comparisions between the genomes was carried out for all the genomes. A pearson correlation matrix was constructed on the basis of their tetranucleotide correlation and ANI values, followed by hierarchical clustering on the resultant matrix using MEV4.9.0, and visualized on MEGA4.
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Related In: Results  -  Collection

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Fig3: Phylogenic constructions ofSphingobiumspp.Sphingobium baderi LL03, S. lactosutens DS20, S. chinhatense IP26, S. quisquilarium P25, S. ummariense RL3, Sphingobium sp. HDIPO4, S. indicum B90A, S. japonicum UT26S and Sphingobium sp. SYK6. (A) 16S rRNA gene sequence-based phylogeny was performed with the neighbor joining method using TreeconW 1.3b (bootstrap value = 1000) (B) The amino acid sequences of 28 single copy genes were concatenated for all ten genomes and phylogeny performed with the neighbor joining method using TreeconW 1.3b (bootstrap value =1000) (C) &(D) Whole genome based tetranucleotide correlation and ANI based pairwise comparisions between the genomes was carried out for all the genomes. A pearson correlation matrix was constructed on the basis of their tetranucleotide correlation and ANI values, followed by hierarchical clustering on the resultant matrix using MEV4.9.0, and visualized on MEGA4.
Mentions: Four different phylogenetic methods (16S rRNA gene sequence, single copy gene sequences, tetranucleotide frequency based correlation, and average nucleotide identity (ANI)) were used to analyze the relationships of the nine strains under study (Figure 3). The consensus tree topology obtained by these methods clustered S. indicum B90A, S. japonicum UT26S, S. chinhatense IP26, and Sphingobium sp. HDIPO4, with the exception of the single copy gene approach. Notably, these four strains were the only ones with an entirely complete lin pathway, thus suggesting convergent evolution through HCH selection pressure. Furthermore, ANI topology supported the grouping of Sphingobium sp. HDIPO4 and S. chinhatense IP26 as subspecies (≥99.34%) (ANI values within the subspecies >98%) [18]. The other five strains i.e., LL03, DS20, RL3, P25 and SYK6 did not produce a consensus phylogeny, with relationships differing between these approaches; in short, strains with the complete lin pathway formed a closed group whereas, the others have diverged. In addition, 16S rRNA and single copy gene approaches may be problematic for differentiation among highly related strains (as these methodologies do not consider the influence of HGT). However, ANI based pairwise comparison has clustered LL03 and RL3 (partial lin gene deficient but HCH degraders) in a monophyletic clade with P25 (partial lin gene deficient and slow HCH degrader) forming a close relationship. Moreover, DS20 and SYK6 (non-HCH degraders) were clustered together. This suggests that ANI based phylogeny is more appropriate and mirrors their relationship with respect to HCH degradation.Figure 3

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