Transformation of Multi-Antibiotic Resistant Stenotrophomonas maltophilia with GFP Gene to Enable Tracking its Survival on Pine Trees

Pinus massoniana Lamb., commonly known as Masson Pine, is one of the most important tree species for planted forests in China. This species is, however, threatened by pine wilt disease caused by Bursaphelenchus xylophilus. Stenotrophomonas maltophilia (Palleroni & Bradbury 1993) Smal-007, a bacterium isolated from the body surface of native B. xylophilus, was evidenced to possess the ability to prevent and control this disease. In this study, we focus on exploring effective transformation and green fluorescent protein (GFP)-labeling of Smal-007, in order to facilitate its later investigation. The results indicated that the recombination of antibiotic Tp (trimethoprim), and the uncoupling reagent, CCCP (carbonyl cyanide m-chlorophenyl hydrazine), was effective for the transformation of the multidrug-resistant bacterium. An optimal transformation procedure, including electroporation, was established. To the best of our knowledge, this is the first report where such a method was used for S. maltophilia transformation. Furthermore, Smal-007 was labeled by GFP, allowing the monitoring of its survival ability in pine trees. The labeling was robust and recognizable in isolates recovered from pine needles and bark. In summary, our study indicated that combining uncoupling reagents could be a useful approach to finding operative antibiotic markers for the transformation of multidrug-resistant bacteria. In addition, our successful labeling of Smal-007 with GFP could improve the understanding of its ecological impact, when used as a biocontrol agent.