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Applied Microbiology and Biotechnology

, Volume 103, Issue 17, pp 7161–7175 | Cite as

High throughput quantification of the functional genes associated with RDX biodegradation using the SmartChip real-time PCR system

  • J. M. Collier
  • B. Chai
  • J. R. Cole
  • M. M. Michalsen
  • Alison M. CupplesEmail author
Methods and protocols
  • 98 Downloads

Abstract

The explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a contaminant at many military sites. RDX bioremediation as a clean-up approach has been gaining popularity because of cost benefits compared to other methods. RDX biodegradation has primarily been linked to six functional genes (diaA, nfsI, pnrB, xenA, xenB, xplA). However, current methods for gene quantification have the risk of false negative results because of low theoretical primer coverage. To address this, the current study designed new primer sets using the EcoFunPrimer tool based on sequences collected by the Functional Gene Pipeline and Repository and these were verified based on residues and motifs. The primers were also designed to be compatible with the SmartChip Real-Time PCR system, a massively parallel singleplex PCR platform (high throughput qPCR), that enables quantitative gene analysis using 5,184 simultaneous reactions on a single chip with low volumes of reagents. This allows multiple genes and/or multiple primer sets for a single gene to be used with multiple samples. Following primer design, the six genes were quantified in RDX-contaminated groundwater (before and after biostimulation), RDX-contaminated sediment, and uncontaminated samples. The final 49 newly designed primer sets improved upon the theoretical coverage of published primer sets, and this corresponded to more detections in the environmental samples. All genes, except diaA, were detected in the environmental samples, with xenA and xenB being the most predominant. In the sediment samples, nfsI was the only gene detected. The new approach provides a more comprehensive tool for understanding RDX biodegradation potential at contaminated sites.

Keywords

RDX xplA xenA xenB diaA nfsI pnrB High throughout qPCR 

Notes

Acknowledgements

Thanks to Tiffany Stedfeldt for providing the Red Cedar River water samples and thanks to Santosh Gunturu for his assistance with the RDP tools. Thanks to Vidhya Ramalingam and Jean-Rene Thelusmond for providing the agricultural soils. Thanks to Aaron King and Jeffrey Weiss (US Army Corps of Engineers) and Malcolm Gander (Naval Facilities Engineering Command) for providing the RDX-contaminated groundwater and sediment samples. The research was partially supported through an Interdisciplinary Team Building Initiative Grant (MSU) and an Academic Achievement Graduate Assistantship (MSU).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2019_10022_MOESM1_ESM.pdf (4.2 mb)
ESM 1 (PDF 4333 kb)

References

  1. Adrian NR, Chow T (2001) Identification of hydroxylamino-dinitroso-1,3,5-triazine as a transient intermediate formed during the anaerobic biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine. Environ Toxicol Chem 20(9):1874–1877Google Scholar
  2. Andeer PF, Stahl DA, Bruce NC, Strand SE (2009) Lateral transfer of genes for hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) degradation. Appl Environ Microbiol 75(10):3258–3262Google Scholar
  3. Beller HR (2002) Anaerobic biotransformation of RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) by aquifer bacteria using hydrogen as the sole electron donor. Water Res 36(10):2533–2540Google Scholar
  4. Bernstein A, Adar E, Nejidat A, Ronen Z (2011) Isolation and characterization of RDX-degrading Rhodococcus species from a contaminated aquifer. Biodegrad 22(5):997–1005Google Scholar
  5. Bhushan B, Halasz A, Spain JC, Hawari J (2002) Diaphorase catalyzed biotransformation of RDX via N-denitration mechanism. Biochem Biophys Res Commun 296(4):779–784.  https://doi.org/10.1016/s0006-291x(02)00874-4 Google Scholar
  6. Blehert DS, Fox BG, Chambliss GH (1999) Cloning and sequence analysis of two Pseudomonas flavoprotein xenobiotic reductases. J Bacteriol 181(20):6254–6263Google Scholar
  7. Boyle B, Dallaire N, MacKay J (2009) Evaluation of the impact of single nucleotide polymorphisms and primer mismatches on quantitative PCR. BMC Biochem 9:75.  https://doi.org/10.1186/1472-6750-9-75 Google Scholar
  8. Chakraborty S, Sakka M, Kimura T, Sakka K (2008) Cloning and expression of a Clostridium kluyveri gene responsible for diaphorase activity. Biosci Biotechnol Biochem 72(3):735–741.  https://doi.org/10.1271/bbb.70606 Google Scholar
  9. Clausen J, Robb J, Curry D, Korte N (2004) A case study of contaminants on military ranges: Camp Edwards, Massachusetts, USA. Environ Pollut 129(1):13–21.  https://doi.org/10.1016/j.envpol.2003.10.002 Google Scholar
  10. Cole JR, Wang Q, Fish JA, Chai B, McGarrell DM, Sun Y, Brown CT, Porras-Alfaro A, Kuske CR, Tiedje JM (2014) Ribosomal database project: data and tools for high throughput rRNA analysis. Nucleic Acids Res 42(D1):D633–D642.  https://doi.org/10.1093/nar/gkt1244 Google Scholar
  11. Coleman NV, Nelson DR, Duxbury T (1998) Aerobic biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) as a nitrogen source by a Rhodococcus sp., strain DN22. Soil Biol Biochem 30(8–9):1159–1167Google Scholar
  12. Crocker F, Indest K, Jung C, Hancock D, Fuller M, Hatzinger P, Vainberg S, Istok J, Wilson E, Michalsen M (2015) Evaluation of microbial transport during aerobic bioaugmentation of an RDX-contaminated aquifer. Biodegradation 26(6):443–451.  https://doi.org/10.1007/s10532-015-9746-1 Google Scholar
  13. Fish JA, Chai B, Wang Q, Sun Y, Brown CT, Tiedje JM, Cole JR (2013) FunGene: the functional gene pipeline and repository. Front Microbiol 4:291.  https://doi.org/10.3389/fmicb.2013.00291 Google Scholar
  14. Fox J, Weisberg S (2011) An {R} companion to applied regressionGoogle Scholar
  15. Fuller ME, Hatzinger PB, Condee CW, Andaya C, Rezes R, Michalsen MM, Crocker FH, Indest KJ, Jung CM, Blakeney G, Istok JD, Hammett SA (2017) RDX degradation in bioaugmented model aquifer columns under aerobic and low oxygen conditions. Appl Microbiol Biotechnol 101(13):5557–5567.  https://doi.org/10.1007/s00253-017-8269-6 Google Scholar
  16. Fuller ME, McClay K, Hawari J, Paquet L, Malone TE, Fox BG, Steffan RJ (2009) Transformation of RDX and other energetic compounds by xenobiotic reductases xenA and xenB. Appl Microbiol Biotechnol 84(3):535–544Google Scholar
  17. Fuller ME, McClay K, Higham M, Hatzinger PB, Steffan RJ (2010) Hexahydro-1, 3, 5-trinitro-1, 3, 5-triazine (RDX) bioremediation in groundwater: are known RDX-degrading bacteria the dominant players? Bioremed J 14(3):121–134Google Scholar
  18. Gunturu S, Ouyang Y, Carvalho TD, Cole J (2018) EcoFunPrimer tool: pipeline to design primers for ecofunctional genes for high-throughput qPCR platforms. Paper presented at the poster number 750 ASM microbe, AtlantaGoogle Scholar
  19. Hatzinger PB (2014) Passive biobarrier for treating comingled perchlorate and RDX in groundwater at an active range. ESTCP fact sheet ER-201028Google Scholar
  20. Hatzinger PB, Lippincott D (2012) In situ bioremediation of energetic compounds in groundwater. ESTCP Final Report ER-200425Google Scholar
  21. Indest KJ, Crocker FH, Athow R (2007) A TaqMan polymerase chain reaction method for monitoring RDX-degrading bacteria based on the xplA functional gene. J Microbiol Methods 68(2):267–274Google Scholar
  22. Jacobsen CS, Nielsen TK, Vester JK, Stougaard P, Nielsen JL, Voriskova J, Winding A, Baldrian P, Liu BB, Frostegard A, Pedersen D, Tveit AT, Svenning MM, Tebbe CC, Ovreas L, Jakobsen PB, Blazewicz SJ, Hubablek V, Bertilsson S, Hansen LH, Cary SC, Holben WE, Ekelund F, Baelum J (2018) Inter-laboratory testing of the effect of DNA blocking reagent G2 on DNA extraction from low-biomass clay samples. Sci Rep-Uk 8:ARTN 5711.  https://doi.org/10.1038/s41598-018-24082-y Google Scholar
  23. Kanitkar YH, Stedtfeld RD, Hatzinger PB, Hashsham SA, Cupples AM (2017) Development and application of a rapid, user-friendly, and inexpensive method to detect Dehalococcoides reductive dehalogenase genes from groundwater. Appl Microbiol Biotechnol 101(11):4827–4835.  https://doi.org/10.1007/s00253-017-8203-y Google Scholar
  24. Kitts CL, Cunningham DP, Unkefer PJ (1994a) Isolation of 3 hexahydro-1,3,5-trinitro-1,3,5-triazine-degrading species of the family Enterobacteriaceae from nitramine explosive-contaminated soil. Appl Environ Microbiol 60(12):4608–4611Google Scholar
  25. Kitts CL, Cunningham DP, Unkefer PJ (1994b) Isolation of three hexahydro-1, 3, 5-trinitro-1, 3, 5-triazine-degrading species of the family Enterobacteriaceae from nitramine explosive-contaminated soil. Appl Environ Microbiol 60(12):4608–4611Google Scholar
  26. Kitts CL, Green CE, Otley RA, Alvarez MA, Unkefer PJ (2000) Type I nitroreductases in soil enterobacteria reduce TNT (2, 4, 6-trinitrotoluene) and RDX (hexahydro-1, 3, 5-trinitro-1, 3, 5-triazine). Can J Microbiol 46(3):278–282Google Scholar
  27. Koncevicius K (2018) Fast statistical hypothesis tests on rows and columns of matrices. 0.1.0 ednGoogle Scholar
  28. Lane D (1991) 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematics. John Wiley and Sons, Chichester, pp 115–175Google Scholar
  29. Lee B-U, Choi M-S, Kim D-M, Oh K-H (2017) Genome shuffling of Stenotrophomonas maltophilia OK-5 for improving the degradation of explosive RDX (Hexahydro-1,3,5-trinitro-1,3,5-triazine). Curr Microbiol 74(2):268–276.  https://doi.org/10.1007/s00284-016-1179-5 Google Scholar
  30. Lee B-U, Choi M-S, Oh K-H (2013) Comparative analysis of explosive RDX-induced proteomes in the Pseudomonas sp HK-6 wild-type strain and its rpoH mutant strain. Biotechnol Bioprocess Eng 18(6):1224–1229.  https://doi.org/10.1007/s12257-013-0249-9 Google Scholar
  31. Li RW, Giarrizzo JG, Wu S, Li W, Duringer JM, Craig AM (2014) Metagenomic insights into the RDX-degrading potential of the ovine rumen microbiome. PLoS One 9(11):e110505.  https://doi.org/10.1371/journal.pone.0110505 Google Scholar
  32. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods 25(4):402–408Google Scholar
  33. Looft T, Johnson TA, Allen HK, Bayles DO, Alt DP, Stedtfeld RD, Sul WJ, Stedtfeld TM, Chai B, Cole JR (2012) In-feed antibiotic effects on the swine intestinal microbiome. PNAS 109(5):1691–1696Google Scholar
  34. Mestdagh P, Lefever S, Volders P-J, Derveaux S, Hellemans J, Vandesompele J (2016) Long non-coding RNA expression profiling in the NCI60 cancer cell line panel using high-throughput RT-qPCR. Sci Data 3:160052Google Scholar
  35. Michalsen MM, King AS, Rule RA, Fuller ME, Hatzinger PB, Condee CW, Crocker FH, Indest KJ, Jung CM, Istok JD (2016) Evaluation of biostimulation and bioaugmentation to stimulate hexahydro-1,3,5-trinitro-1,3,5,-triazine degradation in an aerobic groundwater aquifer. Environ Sci Technol 50(14):7625–7632.  https://doi.org/10.1021/acs.est.6b00630 Google Scholar
  36. Michalsen MM, King SA, Istok JD, Crocker FH, Fuller ME, Kucharzyk KH, Gander MJ (Submitted) Degradation rates following field-scale bioaugmentation for RDX-contaminated groundwater remediationGoogle Scholar
  37. Michalsen MM, Weiss R, King A, Gent D, Medina VF, Istok JD (2013) Push-pull tests for estimating RDX and TNT degradation rates in groundwater. Ground Water Monit Remidiat 33(3):61–68.  https://doi.org/10.1111/gwmr.12016 Google Scholar
  38. Nejidat A, Kafka L, Tekoah Y, Ronen Z (2008) Effect of organic and inorganic nitrogenous compounds on RDX degradation and cytochrome P-450 expression in Rhodococcus strain YH1. Biodegrad 19(3):313–320Google Scholar
  39. Newell C (2008) Treatment of RDX and HMX plumes using mulch biowalls. ESTCP Final Report ER-200426Google Scholar
  40. Paulin MM, Nicolaisen MH, Jacobsen CS, Gimsing AL, Sorensen J, Baelum J (2013) Improving Griffith's protocol for co-extraction of microbial DNA and RNA in adsorptive soils. Soil Biol Biochem 63:37–49.  https://doi.org/10.1016/j.soilbio.2013.02.007 Google Scholar
  41. R Core Team (2018) R: a language and environment for statistical computingGoogle Scholar
  42. Ritalahti KM, Amos BK, Sung Y, Wu Q, Koenigsberg SS, Löffler FE (2006) Quantitative PCR targeting 16S rRNA and reductive dehalogenase genes simultaneously monitors multiple Dehalococcoides strains. Appl Environ Microbiol 72(4):2765–2774Google Scholar
  43. Rutledge RG, Stewart D (2008) A kinetic-based sigmoidal model for the polymerase chain reaction and its application to high-capacity absolute quantitative real-time PCR. BMC Biotechnol 8:8.  https://doi.org/10.1186/1472-6750-8-47 Google Scholar
  44. Rutledge RG, Stewart D (2010) Assessing the performance capabilities of LRE-based assays for absolute quantitative real-time PCR. PLoS One 5(3):e9731.  https://doi.org/10.1371/journal.pone.0009731 Google Scholar
  45. Rylott EL, Jackson RG, Sabbadin F, Seth-Smith HMB, Edwards J, Chong CS, Strand SE, Grogan G, Bruce NC (2011) The explosive-degrading cytochrome P450 XplA: biochemistry, structural features and prospects for bioremediation. Bba-Proteins Proteom 1814(1):230–236.  https://doi.org/10.1016/j.bbapap.2010.07.004 Google Scholar
  46. Seth-Smith HMB, Edwards J, Rosser SJ, Rathbone DA, Bruce NC (2008) The explosive-degrading cytochrome P450 system is highly conserved among strains of Rhodococcus spp. Appl Environ Microbiol 74(14):4550–4552Google Scholar
  47. Seth-Smith HMB, Rosser SJ, Basran A, Travis ER, Dabbs ER, Nicklin S, Bruce NC (2002) Cloning, sequencing, and characterization of the hexahydro-1,3,5-trinitro-1,3,5-triazine degradation gene cluster from Rhodococcus rhodochrous. Appl Environ Microbiol 68(10):4764–4771Google Scholar
  48. Stedtfeld RD, Williams MR, Fakher U, Johnson TA, Stedtfeld TM, Wang F, Khalife WT, Hughes M, Etchebarne BE, Tiedje JM, Hashsham SA (2016) Antimicrobial resistance dashboard application for mapping environmental occurrence and resistant pathogens. FEMS Micro Ecol 92(3):fiw020.  https://doi.org/10.1093/femsec/fiw020 Google Scholar
  49. Takada-Hoshino Y, Matsumoto N (2004) An improved DNA extraction method using skim milk from soils that strongly adsorb DNA. Microbes Environ 19(1):13–19Google Scholar
  50. Thompson KT, Crocker FH, Fredrickson HL (2005) Mineralization of the cyclic nitramine explosive hexahydro-1,3,5-trinitro-1,3,5-triazine by Gordonia and Williamsia spp. Appl Environ Microbiol 71(12):8265–8272.  https://doi.org/10.1128/Aem.71.12.8265-8272.2005 Google Scholar
  51. USGAO (2004) United States General Accounting Office. Department of Defense Operational Ranges. More reliable cleanup cost estimates and a proactive approach to identifying contamination are needed. Report to Congressional Requesters http://www.dtic.mil/cgi-bin/GetTRDoc?AD=ADA435939&Location=U2&doc=GetTRDocpdf. vol Report to Congressional Requesters. http://www.dtic.mil/cgi-bin/GetTRDoc?AD=ADA435939&Location=U2&doc=GetTRDoc.pdf, Report to Congressional Requesters. http://www.dtic.mil/cgi-bin/GetTRDoc?AD=ADA435939&Location=U2&doc=GetTRDoc.pdf. Accessed 01 Jan 2018
  52. Wang F-H, Qiao M, Su J-Q, Chen Z, Zhou X, Zhu Y-G (2014) High throughput profiling of antibiotic resistance genes in urban park soils with reclaimed water irrigation. Environ Sci Technol 48(16):9079–9085Google Scholar
  53. Wang F, Stedtfeld RD, Kim O-S, Chai B, Yang L, Stedtfeld TM, Hong SG, Kim D, Lim HS, Hashsham SA, Tiedje JM, Sul WJ (2016) Influence of soil characteristics and proximity to Antarctic research stations on abundance of antibiotic resistance genes in soils. Environ Sci Technol 50(23):12621–12629.  https://doi.org/10.1021/acs.est.6b02863 Google Scholar
  54. Warnes GR, Bolker B, Bonebakker L, Gentleman R, Liaw WHA, Lumley T, Maechler M, Magnusson A, Moeller S, Schwartz M, Venables B (2016) gplots: various R programming tools for plotting dataGoogle Scholar
  55. Wickham H (2016) ggplot2: elegant graphics for data analysisGoogle Scholar
  56. Wilson FP, Cupples AM (2016) Microbial community characterization and functional gene quantification in RDX-degrading microcosms derived from sediment and groundwater at two naval sites. Appl Microbiol Biotechnol 100(16):7297–7309Google Scholar
  57. Young DM, Unkefer PJ, Ogden KL (1997) Biotransformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by a prospective consortium and its most effective isolate Serratia marcescens. Biotechnol Bioeng 53(5):515–522Google Scholar
  58. Zhao JS, Greer CW, Thiboutot S, Ampleman G, Hawari J (2004a) Biodegradation of the nitramine explosives hexahydro-1,3,5-trinitro-1,3,5-triazine and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine in cold marine sediment under anaerobic and oligotrophic conditions. Can J Microbiol 50(2):91–96Google Scholar
  59. Zhao JS, Halasz A, Paquet L, Beaulieu C, Hawari J (2002) Biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine and its mononitroso derivative hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine by Klebsiella pneumoniae strain SCZ-1 isolated from an anaerobic sludge. Appl Environ Microbiol 68(11):5336–5341Google Scholar
  60. Zhao JS, Paquet L, Halasz A, Hawari J (2003) Metabolism of hexahydro-1,3,5-trinitro-1,3,5-triazine through initial reduction to hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine followed by denitration in Clostridium bifermentans HAW-1. Appl Microbiol Biotechnol 63(2):187–193Google Scholar
  61. Zhao JS, Spain J, Thiboutot S, Ampleman G, Greer C, Hawari J (2004b) Phylogeny of cyclic nitramine-degrading psychrophilic bacteria in marine sediment and their potential role in the natural attenuation of explosives. FEMS Micro Ecol 49(3):349–357Google Scholar

Copyright information

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

Authors and Affiliations

  • J. M. Collier
    • 1
  • B. Chai
    • 2
  • J. R. Cole
    • 2
  • M. M. Michalsen
    • 3
  • Alison M. Cupples
    • 1
    Email author
  1. 1.Department of Civil and Environmental EngineeringMichigan State UniversityEast LansingUSA
  2. 2.Department of Plant, Soil and Microbial SciencesEast LansingUSA
  3. 3.U.S. Army Engineer Research Development CenterSeattleUSA

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