Combined use of different Gfp reporters for monitoring single-cell activities of a genetically modified PCB degrader in the rhizosphere of alfalfa

Abstract

Single-cell localization and activity of Pseudomonas fluorescens F113, colonizing alfalfa roots, were monitored using fusions of the Escherichia coli rrnBP1 ribosomal promoter and gfp genes encoding green fluorescent protein (Gfp) of different stability. The monitoring systems permitted non-destructive in situ detection of F113rifpcb cells on the entire root system grown in both the presence and absence of 3-chlorobiphenyl (PCB-2). The root tip and sites of lateral root emergence were found to be hotspots for fast-growing cells. In addition, a reporter strain of P. fluorescens F113rifpcb for degradation of chlorinated biphenyl was constructed, using another gfp fusion with the meta-pathway Pm promoter from Pseudomonas putida (TOL plasmid). Expression of this promoter, which is strongly induced by the PCB-2 degradation product, 3-chlorobenzoate, was tested in vitro and subsequently monitored in vivo on alfalfa roots using the P. fluorescens F113rifpcb reporter. A small but distinct fraction of the introduced bacteria activated the Pm promoter and thus appeared to sense a PCB-2 degradation product in the alfalfa rhizosphere. The degrading cells, which by design were identical to the sensing cells, were located in distinct microcolonies on the root surface or in intercellular crevices between the root epidermal cells. However, PCB-degrading cells were not observed in the root areas containing fast-growing cells, indicating that PCB degradation was not linked to high cellular activity.