Whole genome analysis of six organophosphate-degrading rhizobacteria reveals putative agrochemical degradation enzymes with broad substrate specificity
Six organophosphate-degrading bacterial strains collected from farm and ranch soil rhizospheres across the Houston-metropolitan area were identified as strains of Pseudomonas putida (CBF10-2), Pseudomonas stutzeri (ODKF13), Ochrobactrum anthropi (FRAF13), Stenotrophomonas maltophilia (CBF10-1), Achromobacter xylosoxidans (ADAF13), and Rhizobium radiobacter (GHKF11). Whole genome sequencing data was assessed for relevant genes, proteins, and pathways involved in the breakdown of agrochemicals. For comparative purposes, this analysis was expanded to also include data from deposited strains in the National Center for Biotechnology Information’s (NCBI) database. This study revealed Zn-dependent metallo-β-lactamase (MBL)-fold proteins similar to OPHC2 first identified in P. pseudoalcaligenes as the likely agents of organophosphate (OP) hydrolysis in A. xylosoxidans ADAF13, S. maltophilia CBF10-1, O. anthropi FRAF13, and R. radiobacter GHKF11. A search of similar proteins within NCBI identified over 200 hits for bacterial genera and species with a similar OPHC2 domain. Taken together, we conclude from this data that intrinsic low-level OP hydrolytic activity is likely prevalent across the rhizosphere stemming from widespread OPHC2-like metalloenzymes. In addition, P. stutzeri ODKF13, P. putida CBF10-2, O. anthropi FRAF13, and R. radiobacter GHKF11 were found to harbor glycine oxidase (GO) enzymes that putatively possess low-level activity against the herbicide glyphosate. These bacterial GOs are reported to catalyze the degradation of glyphosate to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and suggest a possible link to AMPA that can be found in glyphosate-contaminated agricultural soil. The presence of aromatic degradation proteins were also detected in five of six study strains, but are attributed primarily to components of the widely distributed β-ketoadipate pathway found in many soil bacteria.
KeywordsAgrochemicals Bioremediation Organophosphates Rhizosphere
Methyl parathion hydrolase
National Center for Biotechnology Information
Organophosphorus acid Anhydrolase
World Health Organization
The authors wish to thank Annette Frenk, a graduate student at the University of Houston, for her help in compiling background information on relevant agrochemical pathways for this project.
Funding for whole genome sequencing of study strains was provided by the National Science Foundation (NSF) (award no. 1505403). The NSF had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
RI was the principal investigator of this research and all research work was conducted in her laboratory space. RI designed the project parameters, directed research activity inside the laboratory, submitted compiled sequence data to NCBI, and proofread the manuscript. BI helped with drafting the manuscript and with sequence alignment. JK participated in sample preparation for whole genome sequencing and construction of the phylogenetic trees from compiled sequence data. AD assembled DNA sequence data into contigs for submission to NCBI and helped contribute to bioinformatics analysis of sample strains for aromatic biodegradation.
Compliance with ethical standards
This study did involve animals or human participants.
The authors declare that they have no conflict of interest.
- Aho AV, Kernighan BW, Weinberger PJ. (1987). The AWK programming language. Addison-Wesley Longman Publishing Co., Inc.Google Scholar
- Aoki KF, Kanehisa M. (2005). Using the KEGG database resource. Curr Protoc bioinformatics chapter 1 (unit 1): 12Google Scholar
- Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV (2012) SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19(5):455–477. https://doi.org/10.1089/cmb.2012.0021 CrossRefGoogle Scholar
- Bioinformatics B. (2011). FastQC: a quality control tool for high throughput sequence data. Babraham Institute, Cambridge. http://www.bioinformatics.babraham.ac.uk/projects/fastqc
- Bushnell B. BBMap short read aligner. (2016). University of California, Berkeley, California. https://sourceforge.net/projects /bbmap/files
- Dereeper A*, Guignon V*, Blanc G, Audic S, Buffet S, Chevenet F, Dufayard JF, Guindon S, Lefort V, Lescot M, Claverie JM, Gascuel O (2008) Phylogeny.fr: robust phylogenetic analysis for the non-specialist. NucleicAcids Res 36(Web Server issue):W465–469. * joint first authorsGoogle Scholar
- Giesy JP, Solomon KA. (Eds.) (2014). Ecological risk assessment for chlorpyrifos in terrestrial and aquatic systems in North America. Springer Vol 231Google Scholar
- Greim H, Saltmiras D, Mostert V, Strupp C (2015) Evaluation of carcinogenic potential of the herbicide glyphosate, drawing on tumor incidence data from fourteen chronic/carcinogenicity rodent studies. Crit Rev Toxicol 45(3):185–208. https://doi.org/10.3109/10408444.2014.1003423 CrossRefGoogle Scholar
- Harper LL, McDaniel CS, Miller CE, Wild JR (1988) Dissimilar plasmids isolated from Pseudomonas diminuta MG and a Flavobacterium sp. (ATCC 27551) contain identical opd genes. Appl Environ Microbiol 54(10):2586–2589Google Scholar
- Iyer R, Damania A (2016e) Draft genome sequence of the broad-spectrum xenobiotic degrader Achromobacter xylosoxidans ADAF13. Genome Announc 4(2):e00203–e00216Google Scholar
- Iyer R, Damania A (2016f) Draft genome sequence of organophosphate-degrading Ochrobactrum anthropi FRAF13. Genome Announc 4(2):e00295–e00216Google Scholar
- Iyer RS, Wales ME (2012) Integrating interdisciplinary research-based experiences in biotechnology laboratories. Adv Eng Edu 3(1):1–35Google Scholar
- Kumar SV, Fareedullah M, Sudhakar Y, Venkateswarlu B, Kumar EA (2010) Current review on organophosphorus poisoning. Arch Appl Sci Res 2(4):199–215Google Scholar
- Myers JP, Antoniou MN, Blumberg B, Carroll L, Colborn T, Everett LG, Hansen M, Landrigan PJ, Lanphear BP, Mesnage R, Vandenberg LN (2016) Concerns over use of glyphosate-based herbicides and risks associated with exposures: a consensus statement. Environ Health 15(1):19. https://doi.org/10.1186/s12940-016-0117-0 CrossRefGoogle Scholar
- Pandeeti EV, Chakka D, Pandey JP, Siddavattam D (2011) Indigenous organophosphate-degrading (opd) plasmid pCMS1 of Brevundimonas diminuta is self-transmissible and plays a key role in horizontal mobility of the opd gene. Plasmid 65(3):226–231. https://doi.org/10.1016/j.plasmid.2011.02.003 CrossRefGoogle Scholar
- Pandeeti EV, Longkumer T, Chakka D, Muthyala VR, Parthasarathy S, Madugundu AK, Ghanta S, Medipally SR, Pantula SC, Yekkala H, Siddavattam D (2012) Multiple mechanisms contribute to lateral transfer of an organophosphate degradation (opd) island in Sphingobium fuliginis ATCC 27551. G3: Genes| Genomes| Genetics 2(12):1541–1554CrossRefGoogle Scholar
- RCore Team. (2016). A language and environment for statistical computing. Vienna: R Foundation for Statistical Computing. https://www.R-project.org/
- RStudio Team. (2015). RStudio: integrated development environment for R. Boston, Massachusetts. RStudio, Inc. http://www.rstudio.com/
- Segers P, Vancanneyt M, Pot B, Torck U, Hoste B, Dewettinck D, Falsen E, Kersters K, De Vos P (1994) Classification of Pseudomonas diminuta Leifson and Hugh 1954 and Pseudomonas vesicularis Büsing, Döll, and Freytag 1953 in Brevundimonas gen. nov. as Brevundimonas diminuta comb. nov. and Brevundimonas vesicularis comb. nov., respectively. Int J Sys Bacteriol 44(3):499–510. https://doi.org/10.1099/00207713-44-3-499 CrossRefGoogle Scholar
- Singh B, Singh K (2016) Microbial degradation of herbicides. Crit Rev Toxicol 42(2):245–261Google Scholar
- Skinner J (2016) Sublime text: the text editor you’ll fall in love with. Sublime Text [Online]. Available: http://www.sublimetextcom/
- Wickham H, Francois R. (2016a). Dplyr: a grammar of data manipulation, 2013. URL https://github.com/hadley/dplyr.com/hadley/dplyr. Version 0.1.[p 1]
- Wickham H, Francois R. (2016b). Readr: read tabular data. URL https://github.com/hadley/readr. R package version 0.1. 2015;1:2
- Wu N, Deng M, Shi X, Liang G, Yao B, Fan Y (2004) Isolation, purification and characterization of a new organphosphorus hydrolase OPHC2. Chin Sci Bull 49(3):268–272Google Scholar
- Zhang R, Cui Z, Zhang X, Jiang J, Gu JD, Li S (2006) Cloning of the organophosphorus pesticide hydrolase gene clusters of seven degradative bacteria isolated from a methyl parathion contaminated site and evidence of their horizontal gene transfer. Biodegradation 17(5):465–472. https://doi.org/10.1007/s10532-005-9018-6 CrossRefGoogle Scholar
- Zhang Y, An J, Ye W, Yang G, Qian ZG, Chen HF, Cui L, Feng Y (2012) Enhancing the promiscuous phosphotriesterase activity of a thermostable lactonase (GkaP) for the efficient degradation of organophosphate pesticides. Appl Environ Microbiol 78(18):6647–6655. https://doi.org/10.1128/AEM.01122-12 CrossRefGoogle Scholar