Polychlorinated Biphenyl Rhizoremediation by <i>Pseudomonas fluorescens</i> F113 Derivatives, Using a <i>Sinorhizobium meliloti nod</i> System To Drive <i>bph</i> Gene Expression

Rhizoremediation of organic chemicals requires high-level expression of biodegradation genes in bacterial strains that are excellent rhizosphere colonizers. Pseudomonas fluorescens F113 is a biocontrol strain that was shown to be an excellent colonizer of numerous plant rhizospheres, including alfalfa. Although a derivative of F113 expressing polychlorinated biphenyl (PCB) biodegradation genes (F113pcb) has been reported previously, this strain shows a low level of bph gene expression, limiting its rhizoremediation potential. Here, a high-level expression system was designed from rhizobial nod gene regulatory relays. Nod promoters were tested in strain F113 by using beta-galactosidase transcriptional fusions. This analysis showed that nodbox 4 from Sinorhizobium meliloti has a high level of expression in F113 that is dependent on an intact nodD1 gene. A transcriptional fusion of a nodbox cassette containing the nodD1 gene and nodbox 4 fused to a gfp gene was expressed in the alfalfa rhizosphere. The bph operon from Burkholderia sp. strain LB400 was cloned under the control of the nodbox cassette and was inserted as a single copy into the genome of F113, generating strain F113L::1180. This new genetically modified strain has a high level of BphC activity and grows on biphenyl as a sole carbon and energy source at a growth rate that is more than three times higher than that of F113pcb. Degradation of PCBs 3, 4, 5, 17, and 25 was also much faster in F113L::1180 than in F113pcb. Finally, the modified strain cometabolized PCB congeners present in Delor103 better than strain LB400, the donor of the bph genes used.