Comparative genome analysis reveals the evolution of chloroacetanilide herbicide mineralization in Sphingomonas wittichii DC-6

  • Minggen Cheng
  • Xin Yan
  • Jian He
  • Jiguo QiuEmail author
  • Qing ChenEmail author
Original Paper


The environmental fate of the extensively used chloroacetanilide herbicides (CH) has been a cause of increasing concern in the past decade because of their carcinogenic properties. Although microbes play important roles in CH degradation, Sphingomonas wittichii DC-6 was the first reported CH-mineralizing bacterium. In this study, the complete genome of strain DC-6 was sequenced and comparative genomic analysis was performed using strain DC-6 and other three partial CH-degrading bacteria, Sphingobium quisquiliarum DC-2, Sphingobium baderi DE-13, and Sphingobium sp. MEA3-1. 16S rDNA phylogenetic analysis indicated that strain DC-2, MEA3-1, and DE-13 are closely related and DC-6 has relatively distant genetic relationship with the other three strains. The identified CH degradation genes responsible for the upstream and downstream pathway, including cndA, cmeH, meaXY, and meaAB, were all located in conserved DNA fragments (or genetic islands) in the vicinity of mobile element proteins. Protein BLAST in the NCBI database showed that cndA and cmeH were present in the genomes of other sequenced strains isolated from various habitats; however, the gene compositions in these host strains were completely different from those of other sphingomonads, and codon usage of genes for upstream pathway were also different from that of downstream pathway. These results showed that the upstream and downstream pathways of CH degradation in strain DC-6 have evolved by horizontal gene transfer and gene combination. In addition, the genes of the ring-cleavage pathway were not conserved and may have evolved directly from bacterial degradation of hydroxyquinol. The present study provides insights into the evolutionary strategy and microbial catabolic pathway of CH mineralization.


Chloroacetanilide herbicide Genome comparison Mineralizing pathway Sphingomonads Sphingomonas wittichii DC-6 



This work was fund supported by the Fundamental Research Funds for the Central Universities (Grants no. KJQN201940), the National Natural Science Foundation of China (Grants numbers 31600080 and 31800095), the Natural Science Foundation of Jiangsu Province (Grants no. BK20180541).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Informed consent

Informed consent was obtained from all individual participants included in the study.


  1. Armengaud J, Timmis KN, Wittich RM (1999) A functional 4-hydroxysalicylate/hydroxyquinol degradative pathway gene cluster is linked to the initial dibenzo-p-dioxin pathway genes in Sphingomonas sp. strain RW1. J Bacteriol 181(11):3452–3461Google Scholar
  2. Ashby J, Kier L, Wilson AG, Green T, Lefevre PA, Tinwell H, Willis GA, Heydens WF, Clapp MJ (1996) Evaluation of the potential carcinogenicity and genetic toxicity to humans of the herbicide acetochlor. Hum Exp Toxicol 15(9):702–735. Google Scholar
  3. Bai Y, Muller DB, Srinivas G, Garrido-Oter R, Potthoff E, Rott M, Dombrowski N, Munch PC, Spaepen S, Remus-Emsermann M, Huttel B, McHardy AC, Vorholt JA, Schulze-Lefert P (2015) Functional overlap of the Arabidopsis leaf and root microbiota. Nature 528(7582):364–369. Google Scholar
  4. Bian H, Chen J, Cai X, Liu P, Wang Y, Huang L, Qiao X, Hao C (2009) Dechlorination of chloroacetanilide herbicides by plant growth regulator sodium bisulfite. Water Res 43(14):3566–3574. Google Scholar
  5. Chen Q, Wang CH, Deng SK, Wu YD, Li Y, Yao L, Jiang JD, Yan X, He J, Li SP (2014) Novel three-component Rieske non-heme iron oxygenase system catalyzing the N-dealkylation of chloroacetanilide herbicides in sphingomonads DC-6 and DC-2. Appl Environ Microb 80(16):5078–5085. Google Scholar
  6. Cheng M, Li Y, Ma Y, Qiu J, Yan X, He J (2017a) Complete genome sequence of Sphingobium baderi DE-13, an alkyl-substituted aniline-mineralizing bacterium. Curr Microbiol 75(1):27–31. Google Scholar
  7. Cheng M, Meng Q, Yang Y, Chu C, Chen Q, Li Y, Cheng D, Hong Q, Yan X, He J (2017b) The two-component monooxygenase MeaXY initiates the downstream pathway of chloroacetanilide herbicide catabolism in sphingomonads. Appl Environ Microb 83(7):e03241-16. Google Scholar
  8. Chitsaz H, Yee-Greenbaum JL, Tesler G, Lombardo MJ, Dupont CL, Badger JH, Novotny M, Rusch DB, Fraser LJ, Gormley NA, Schulz-Trieglaff O, Smith GP, Evers DJ, Pevzner PA, Lasken RS (2011) Efficient de novo assembly of single-cell bacterial genomes from short-read data sets. Nat Biotechnol 29(10):915–921. Google Scholar
  9. Daubaras DL, Saido K, Chakrabarty AM (1996) Purification of hydroxyquinol 1,2-dioxygenase and maleylacetate reductase: the lower pathway of 2,4,5-trichlorophenoxyacetic acid metabolism by Burkholderia cepacia AC1100. Appl Environ Microb 62(11):4276–4279Google Scholar
  10. Dierickx PJ (1999) Glutathione-dependent cytotoxicity of the chloroacetanilide herbicides alachlor, metolachlor, and propachlor in rat and human hepatoma-derived cultured cells. Cell Biol Toxicol 15(5):325–332Google Scholar
  11. Dong W, Chen Q, Hou Y, Li S, Zhuang K, Huang F, Zhou J, Li Z, Wang J, Fu L, Zhang Z, Huang Y, Wang F, Cui Z (2015) Metabolic pathway involved in 2-methyl-6-ethylaniline degradation by Sphingobium sp. strain MEA3-1 and cloning of the novel flavin-dependent monooxygenase system meaBA. Appl Environ Microb 81(24):8254–8264. Google Scholar
  12. Dwivedi S, Singh BR, Al-Khedhairy AA, Alarifi S, Musarrat J (2010) Isolation and characterization of butachlor-catabolizing bacterial strain Stenotrophomonas acidaminiphila JS-1 from soil and assessment of its biodegradation potential. Lett Appl Microbiol 51(1):54–60. Google Scholar
  13. Furukawa S, Harada T, Thake D, Iatropoulos MJ, Sherman JH (2014) Consensus diagnoses and mode of action for the formation of gastric tumors in rats treated with the chloroacetanilide herbicides alachlor and butachlor. Toxicol Pathol 42(2):386–402. Google Scholar
  14. Gao Y, Jin L, Shi H, Chu Z (2015) Characterization of a novel butachlor biodegradation pathway and cloning of the debutoxylase (Dbo) gene responsible for debutoxylation of butachlor in Bacillus sp. hys-1. J Agr Food Chem 63(38):8381–8390. Google Scholar
  15. Hatta T, Nakano O, Imai N, Takizawa N, Kiyohara H (1999) Cloning and sequence analysis of hydroxyquinol 1,2-dioxygenase gene in 2,4,6-trichlorophenol-degrading Ralstonia pickettii DTP0602 and characterization of its product. J Biosci Bioeng 87(3):267–272Google Scholar
  16. Hayes RP, Green AR, Nissen MS, Lewis KM, Xun L, Kang C (2013) Structural characterization of 2,6-dichloro-p-hydroquinone 1,2-dioxygenase (PcpA) from Sphingobium chlorophenolicum, a new type of aromatic ring-cleavage enzyme. Mol Microbiol 88(3):523–536. Google Scholar
  17. Hou Y, Dong W, Wang F, Li J, Shen W, Li Y, Cui Z (2014) Degradation of acetochlor by a bacterial consortium of Rhodococcus sp.T3-1, Delftia sp.T3-6 and Sphingobium sp.MEA3-1. Lett Appl Microbiol 59(1):35–42. Google Scholar
  18. Kim NH, Kim DU, Kim I, Ka JO (2013) Syntrophic biodegradation of butachlor by Mycobacterium sp. J7A and Sphingobium sp. J7B isolated from rice paddy soil. FEMS Microbiol Lett 344(2):114–120. Google Scholar
  19. Kolvenbach BA, Helbling DE, Kohler HP, Corvini PF (2014) Emerging chemicals and the evolution of biodegradation capacities and pathways in bacteria. Curr Opin Biotech 27:8–14. Google Scholar
  20. Krzywinski M, Schein J, Birol I, Connors J, Gascoyne R, Horsman D, Jones SJ, Marra MA (2009) Circos: an information aesthetic for comparative genomics. Genome Res 19(9):1639–1645. Google Scholar
  21. Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33(7):1870–1874. Google Scholar
  22. Li Y, Chen Q, Wang CH, Cai S, He J, Huang X, Li SP (2013) Degradation of acetochlor by consortium of two bacterial strains and cloning of a novel amidase gene involved in acetochlor-degrading pathway. Bioresource Technol 148:628–631. Google Scholar
  23. Liu W, Gan J, Papiernik SK, Yates SR (2000) Structural influences in relative sorptivity of chloroacetanilide herbicides on soil. J Agr Food Chem 48(9):4320–4325Google Scholar
  24. Liu HM, Cao L, Lu P, Ni H, Li YX, Yan X, Hong Q, Li SP (2012) Biodegradation of butachlor by Rhodococcus sp. strain B1 and purification of its hydrolase (ChlH) responsible for N-dealkylation of chloroacetamide herbicides. J Agr Food Chem 60(50):12238–12244. Google Scholar
  25. Mattes TE, Alexander AK, Coleman NV (2010) Aerobic biodegradation of the chloroethenes: pathways, enzymes, ecology, and evolution. FEMS Microbiol Rev 34(4):445–475. Google Scholar
  26. McCarthy A (2010) Third generation DNA sequencing: pacific biosciences’ single molecule real time technology. Chem Biol 17(7):675–676. Google Scholar
  27. Murakami S, Okuno T, Matsumura E, Takenaka S, Shinke R, Aoki K (1999) Cloning of a gene encoding hydroxyquinol 1,2-dioxygenase that catalyzes both intradiol and extradiol ring cleavage of catechol. Biosci Biotech Bioch 63(5):859–865. Google Scholar
  28. Nigam A, Jit S, Lal R (2010) Sphingomonas histidinilytica sp. nov., isolated from a hexachlorocyclohexane dump site. Int J Syst Evol Micr 60(Pt 5):1038–1043. Google Scholar
  29. Nordin K, Unell M, Jansson JK (2005) Novel 4-chlorophenol degradation gene cluster and degradation route via hydroxyquinol in Arthrobacter chlorophenolicus A6. Appl Environ Microb 71(11):6538–6544. Google Scholar
  30. Novembre JA (2002) Accounting for background nucleotide composition when measuring codon usage bias. Mol Biol Evol 19(8):1390–1394. Google Scholar
  31. Overbeek R, Olson R, Pusch GD, Olsen GJ, Davis JJ, Disz T, Edwards RA, Gerdes S, Parrello B, Shukla M, Vonstein V, Wattam AR, Xia F, Stevens R (2014) The SEED and the Rapid Annotation of microbial genomes using Subsystems Technology (RAST). Nucleic Acids Res 42(Database issue):D206–D214. Google Scholar
  32. Quail MA, Smith M, Coupland P, Otto TD, Harris SR, Connor TR, Bertoni A, Swerdlow HP, Gu Y (2012) A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers. BMC genomics 13:341. Google Scholar
  33. Roberts SJ, Walker A, Cox L, Welch SJ (1998) Isolation of isoproturon-degrading bacteria from treated soil via three different routes. J Appl Microbiol 85(2):309–316Google Scholar
  34. Seffernick JL, Wackett LP (2016) Ancient evolution and recent evolution converge for the biodegradation of cyanuric acid and related triazines. Appl Environ Microb 82(6):1638–1645. Google Scholar
  35. Stamper DM, Tuovinen OH (1998) Biodegradation of the acetanilide herbicides alachlor, metolachlor, and propachlor. Crit Rev Microbiol 24(1):1–22. Google Scholar
  36. Supek F, Vlahovicek K (2004) INCA: synonymous codon usage analysis and clustering by means of self-organizing map. Bioinformatics 20(14):2329–2330. Google Scholar
  37. Tiedje JM, Duxbury JM, Alexander M, Dawson JE (1969) 2,4-D metabolism: pathway of degradation of chlorocatechols by Arthrobacter sp. J Agr Food Chem 17(5):1021–1026. Google Scholar
  38. Travkin VM, Solyanikova IP, Golovleva LA (2006) Hydroxyquinol pathway for microbial degradation of halogenated aromatic compounds. J Environ Sci Heal B 41(8):1361–1382. Google Scholar
  39. Wright F (1990) The ‘effective number of codons’ used in a gene. Gene 87(1):23–29. Google Scholar
  40. Yan X, Gu T, Yi Z, Huang J, Liu X, Zhang J, Xu X, Xin Z, Hong Q, He J, Spain JC, Li S, Jiang J (2016) Comparative genomic analysis of isoproturon-mineralizing sphingomonads reveals the isoproturon catabolic mechanism. Environ Microbiol 18(12):4888–4906. Google Scholar
  41. Yang W, Holmen BA (2008) Relative effects of surfactants and humidity on soil/air desorption of chloroacetanilide and dinitroaniline herbicides. Environ Sci Technol 42(18):6843–6848Google Scholar
  42. Yanze-Kontchou C, Gschwind N (1994) Mineralization of the herbicide atrazine as a carbon source by a Pseudomonas strain. Appl Environ Microb 60(12):4297–4302Google Scholar
  43. Ye CM, Wang XJ, Zheng HH (2002) Biodegradation of acetanilide herbicides acetochlor and butachlor in soil. J Environ Sci-China 14(4):524–529Google Scholar
  44. Zhang J, Sun JQ, Yuan QY, Li C, Yan X, Hong Q, Li SP (2011a) Characterization of the propanil biodegradation pathway in Sphingomonas sp. Y57 and cloning of the propanil hydrolase gene prpH. J Hazard Mater 196:412–419. Google Scholar
  45. Zhang J, Zheng JW, Liang B, Wang CH, Cai S, Ni YY, He J, Li SP (2011b) Biodegradation of chloroacetamide herbicides by Paracoccus sp. FLY-8 in vitro. J Agr Food Chem 59(9):4614–4621. Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Key Laboratory of Microbiological Engineering of Agricultural Environment, Ministry of AgricultureLife Sciences College of Nanjing Agricultural UniversityNanjingChina
  2. 2.College of Life SciencesZaozhuang UniversityZaozhuangChina

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