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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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Principal Component Analysis of genes involved in HCH degradation pathway. (A) PCA individual factor plot showing the grouping of the nine Sphingobium strains based upon the sequence divergence and copy number of the set of lin genes. Principle components 1 (accounting for 30.87% of the variation of the strains) and 2 (14.47%) were chosen as the separation of the strains by these PCs demonstrated the highest fidelity to known HCH degradation ability. (B) PCA variable factor plot using principle component 1 and 2, showing the contribution of the lin genetic sequences (s.linA, s.linB…) and copy number (c.linA, c.linB…) to the variation of the nine Sphingobium strains. HCH degradation ability was plotted as a supplementary categorical variable (not factored into the PCs), with non-HCH degraders coded as 0, partial degraders as 1, and complete degraders as 2.
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Fig9: Principal Component Analysis of genes involved in HCH degradation pathway. (A) PCA individual factor plot showing the grouping of the nine Sphingobium strains based upon the sequence divergence and copy number of the set of lin genes. Principle components 1 (accounting for 30.87% of the variation of the strains) and 2 (14.47%) were chosen as the separation of the strains by these PCs demonstrated the highest fidelity to known HCH degradation ability. (B) PCA variable factor plot using principle component 1 and 2, showing the contribution of the lin genetic sequences (s.linA, s.linB…) and copy number (c.linA, c.linB…) to the variation of the nine Sphingobium strains. HCH degradation ability was plotted as a supplementary categorical variable (not factored into the PCs), with non-HCH degraders coded as 0, partial degraders as 1, and complete degraders as 2.

Mentions: In literature, the degradation ability of these strains under consideration is in the order: HDIPO4>IP26>RL3. In order to further substantiate the relationship between lin system diversity and degradation efficiency, principle component analysis (PCA) was performed on the copy number and sequence divergence for these strains, with HCH degradation plotted as a supplementary variable to visualize correlation. The analysis demonstrated a close grouping of the four lin-deficient strains, i.e. SYK6, DS20, LL03, and P25 under PCA 1 and 2 (accounting for 32.57 and 24.99% of the variation in these strains, respectively) (Figure 9A). HDIPO4 and IP26 were colocalized in quadrant 4, opposite from the non-degrader cluster in terms of both dimension 1 and dimension 2, which is appropriate given that these two strains have degradation rates documented to be faster than archetypal strains UT26S and B90A [14] (Figure 9A). While copy number variation for linF, linDER, and linB were mapped most closely to the HCH degradation vector (Figure 9B), the copy numbers for linC, linGHIJ and again linB played a more important role in differentiating these strains, as they correlated significantly to PCA1 (p values 5.897964e-05, 2.998361e-02, and 1.206315e-02, respectively). In terms of sequence, linA and linR showed high correlation to degradation ability (Figure 9B), while linL, linM, and linK were significantly correlated to PCA1 (p values 1.340825e-04, 2.934465e-04, 4.192410e-04, respectively) and linI, linH, linC, linB, and linF were the most significant contributors to PCA2 (p values 0.0007954298, 0.0010696278, 0.0105835905, 0.0107931486, 0.0222238515, respecitvely). This suggests that while variation can be seen throughout the lin pathway and the copy number of linF, linDER and linB sequence for linA and R might have the most impact in optimizing the efficacy of an HCH enzymatic bioremediation system.Figure 9


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)

Principal Component Analysis of genes involved in HCH degradation pathway. (A) PCA individual factor plot showing the grouping of the nine Sphingobium strains based upon the sequence divergence and copy number of the set of lin genes. Principle components 1 (accounting for 30.87% of the variation of the strains) and 2 (14.47%) were chosen as the separation of the strains by these PCs demonstrated the highest fidelity to known HCH degradation ability. (B) PCA variable factor plot using principle component 1 and 2, showing the contribution of the lin genetic sequences (s.linA, s.linB…) and copy number (c.linA, c.linB…) to the variation of the nine Sphingobium strains. HCH degradation ability was plotted as a supplementary categorical variable (not factored into the PCs), with non-HCH degraders coded as 0, partial degraders as 1, and complete degraders as 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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Fig9: Principal Component Analysis of genes involved in HCH degradation pathway. (A) PCA individual factor plot showing the grouping of the nine Sphingobium strains based upon the sequence divergence and copy number of the set of lin genes. Principle components 1 (accounting for 30.87% of the variation of the strains) and 2 (14.47%) were chosen as the separation of the strains by these PCs demonstrated the highest fidelity to known HCH degradation ability. (B) PCA variable factor plot using principle component 1 and 2, showing the contribution of the lin genetic sequences (s.linA, s.linB…) and copy number (c.linA, c.linB…) to the variation of the nine Sphingobium strains. HCH degradation ability was plotted as a supplementary categorical variable (not factored into the PCs), with non-HCH degraders coded as 0, partial degraders as 1, and complete degraders as 2.
Mentions: In literature, the degradation ability of these strains under consideration is in the order: HDIPO4>IP26>RL3. In order to further substantiate the relationship between lin system diversity and degradation efficiency, principle component analysis (PCA) was performed on the copy number and sequence divergence for these strains, with HCH degradation plotted as a supplementary variable to visualize correlation. The analysis demonstrated a close grouping of the four lin-deficient strains, i.e. SYK6, DS20, LL03, and P25 under PCA 1 and 2 (accounting for 32.57 and 24.99% of the variation in these strains, respectively) (Figure 9A). HDIPO4 and IP26 were colocalized in quadrant 4, opposite from the non-degrader cluster in terms of both dimension 1 and dimension 2, which is appropriate given that these two strains have degradation rates documented to be faster than archetypal strains UT26S and B90A [14] (Figure 9A). While copy number variation for linF, linDER, and linB were mapped most closely to the HCH degradation vector (Figure 9B), the copy numbers for linC, linGHIJ and again linB played a more important role in differentiating these strains, as they correlated significantly to PCA1 (p values 5.897964e-05, 2.998361e-02, and 1.206315e-02, respectively). In terms of sequence, linA and linR showed high correlation to degradation ability (Figure 9B), while linL, linM, and linK were significantly correlated to PCA1 (p values 1.340825e-04, 2.934465e-04, 4.192410e-04, respectively) and linI, linH, linC, linB, and linF were the most significant contributors to PCA2 (p values 0.0007954298, 0.0010696278, 0.0105835905, 0.0107931486, 0.0222238515, respecitvely). This suggests that while variation can be seen throughout the lin pathway and the copy number of linF, linDER and linB sequence for linA and R might have the most impact in optimizing the efficacy of an HCH enzymatic bioremediation system.Figure 9

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