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 ␤-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.

Degradation of PCB congeners by bacterial strains

“…LB400 (Bopp 1986), (which expresses bph upon induction with biphenyl) the rifampicin resistant root-colonising nondegrader Pseudomonas fluorescens F113rif (Shanahan et al 1992), and the genetically modified F113 derivatives Pseudomonas fluorescens F113L::1180 (Villacieros et al 2005) and Pseudomonas fluorescens F113rifpcb (Brazil et al 1995) (which express bph constitutively under the control of different promoters).…”

“…Growing roots may transport inoculated bacteria through soil (Karlson et al 4 1995, Villacieros et al 2005. Besides forming a habitat and transport system for microorganisms, plant roots also provide substrate supporting the growth of PCB-degrading bacteria Hedge 1995, Olson et al 2001).…”

“…the use of microbes in conjunction with plants, is a promising bioremediation strategy (Trapp and Karlson 2001;Leigh et al 2006;Yee et al 1998). Consequently, efforts were undertaken to expand the degradation capacities of rhizospherecompetent bacteria (Villacieros et al 2005;Brazil et al 1995;Yee et al 1998). A number of plantmicrobe consortia have been tested recently (Ryslava et al 2003;Demnerova et al 2005).…”

“…In a first step, genes encoding the bph pathway were cloned from LB400 and inserted into a transposon (Dowling et al 1993). Then the bphcassette was chromosomally integrated into F113 to generate F113rifpcb (Brazil et al 1995) and F113L::1180(Villacieros et al 2005.…”

“…Different from earlier studies (e.g. Gibson et al 1993;Villacieros et al 2005), we quantified the time-scale of bacterial breakdown of PCB in the rhizosphere of contaminated soils, and for commercial mixtures of PCBs (Aroclor 1016, 1221 and 1232). As known by the authors, this is the first publication of Monod parameters for bacterial strains metabolising a wide range of PCBs.…”