Polychlorinated biphenyl rhizoremediation by Pseudomonas fluorescens F113 derivatives, using a Sinorhizobium meliloti nod system to drive bph gene expression

Abstract

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.

FIG. 1.

(A) Confocal laser scanning microscopy image of P. fluorescens F113rif(pBG1157) in the alfalfa rhizosphere in a gnotobiotic system (Leonard jar). Expression of the gfp gene under the nodbox system resulted in green fluorescence visible in individual cells and microcolonies. (B) Epifluorescence image of P. fluorescens F113L::1180(pHC60) in the alfalfa rhizosphere in a PCB-spiked soil microcosm. Expression of the gfp gene from a constitutive lac promoter resulted in green fluorescence observed in bacteria colonizing the root surface (toward the root tip) 14 days after inoculation.

FIG. 2.

Construction of nod system-bph fusions introduced into derivatives of P. fluorescens F113 in a replicative vector (pBG1197) or through transposition to a genomic location (pBG1180). The solid triangles represent mini-Tn5 inverted repeats. N, NsiI; P, PstI. Only relevant restriction sites are shown. For a description of the plasmids see Table 1.

FIG. 3.

Colonization of M. sativa rhizosphere by P. fluorescens strains. Solid symbols, inoculation with P. fluorescens F113LacZY; open symbols, inoculation with P. fluorescens F113L::1180. Symbols: ♦, F113LacZY counts per gram of root; ▪, total viable counts per gram of soil; Δ, F113L::1180 counts per gram of root; □, total viable counts per gram of soil. All weights are fresh weights. Arithmetic means and standard deviations for three replicates are shown.

FIG. 4.

Cometabolism of PCB congeners from Delor 103 by P. fluorescens F113L::1180 and Burkholderia sp. strain LB400. Cells were grown for 14 days at 28°C in minimal medium supplemented with 0.5 g/liter biphenyl and 50 mg/liter Delor 103. After incubation, hexane extracts were analyzed using gas chromatography. The bars indicate the percentages of PCB congeners remaining after incubation, normalized by comparison to abiotic controls. Arithmetic means for three independent experiments analyzed in duplicate and standard errors are shown. The peak numbers refer to congeners, as shown in Table 2.