ORIGINAL ARTICLE Genetic labelling and application of the isoproturon- mineralizing Sphingomonas sp. strain SRS2 in soil and rhizosphere K.E. Kristensen 1,2 , C.S. Jacobsen 1,3 , L.H. Hansen 2 , J. Aamand 1 , J.A.W. Morgan 4 , C. Sternberg 5 and S.R. Sørensen 1 1 Department of Geochemistry, Geological Survey of Denmark and Greenland (GEUS), Copenhagen K, Denmark 2 Department of Microbiology, Institute of Biology, University of Copenhagen, Copenhagen K, Denmark 3 Department of Natural Science and Department of Ecology, Royal Veterinary and Agricultural University, Copenhagen F, Denmark 4 Warwick HRI, University of Warwick, Wellesbourne, Warwick, UK 5 BioCentrum, Center for Biomedical Microbiology, Technical University of Denmark, Lyngby, Denmark Introduction The phenylurea herbicide isoproturon (N-(4-isopropyl- phenyl)-N¢,N¢-dimethylurea) is one of the most heavily used pesticides in the northern part of Europe, and due to its widespread and extensive usage, isoproturon has been detected as a contaminant of rivers, streams, lakes, marine and ground waters across Europe (Sørensen et al. 2003). One promising way of efficiently and cost-effect- ively cleaning up polluted soils and groundwater resources is bioremediation using pesticide-degrading bac- teria. This approach has been used for the treatment of soils and water contaminated with other xenobiotics such as polycyclic aromatic hydrocarbons and methyl tert- buthyl ether (Salanitro et al. 2000; Samanta et al. 2002; Zanardini et al. 2002). However, the utilization of this strategy for the treatment of isoproturon-polluted envi- ronments has not yet been reported. Earlier studies sug- gested that Sphingomonas sp. strain SRS2 was responsible for the degradation of isoproturon within the Deep Slade Field (Bending et al. 2003) from where the strain was ori- ginally isolated (Sørensen et al. 2001). During metabolic degradation, two successive N-demethylations are fol- lowed by a cleavage of the urea side chain after which the ring structure is metabolized by unknown mechanisms (Sørensen et al. 2001). Previous studies have shown that strain SRS2 is capable of inducing mineralization of iso- proturon when inoculated into artificially contaminated Keywords bacteria, degradation, lux, pesticides, soil, survival. Correspondence Sebastian R. Sørensen, Department of Geochemistry, Geological Survey of Denmark and Greenland (GEUS), Øster Voldgade 10, DK-1350 Copenhagen K, Denmark. E-mail: srs@geus.dk 2005/0041: received 17 January 2005, revised 10 March 2006 and accepted 12 April 2006 doi:10.1111/j.1472-765X.2006.01956.x Abstract Aims: To construct a luxAB-labelled Sphingomonas sp. strain SRS2 maintaining the ability to mineralize the herbicide isoproturon and usable for monitoring the survival and distribution of strain SRS2 on plant roots in laboratory sys- tems. Methods and Results: We inserted the mini-Tn5-luxAB marker into strain SRS2 using conjugational mating. In the transconjugant mutants luciferase was produced in varying levels. The mutants showed significant differences in their ability to degrade isoproturon. One luxAB-labelled mutant maintained the abil- ity to mineralize isoproturon and was therefore selected for monitoring colon- ization of barley roots. Conclusions: We successfully constructed a genetically labelled isoproturon- mineralizing-strain SRS2 and demonstrated its ability to survive in soil and its colonization of rhizosphere. Significance and Impact of the Study: The construction of a luxAB-labelled strain SRS2 maintaining the degradative ability, provides a powerful tool for ecological studies serving as the basis for evaluating SRS2 as a bioremediation agent. Letters in Applied Microbiology ISSN 0266-8254 280 Journal compilation ª 2006 The Society for Applied Microbiology, Letters in Applied Microbiology 43 (2006) 280–286 ª 2006 The Authors