CONSTRUCTION OF PHOE-CAA, A NOVEL PCR AND IMMUNOLOGICALLY DETECTABLE MARKER GENE FOR PSEUDOMONAS-PUTIDA

In this paper we describe the construction and use in Pseudomonas puti da WCS358 of phoE-caa, a novel hybrid marker gene, which allows monito ring both at the protein level by immunological methods and at the DNA level by PCR. The marker is based on the Escherichia coli outer membr ane protein gene phoE and 75 bp of E. coli can, which encode a nonbact eriocinic fragment of colicin A. This fragment contains an epitope whi ch is recognized by monoclonal antibody (MAb) 1C11. As the epitope is contained in one of the cell surface-exposed loops of PhoE, whole cell s of bacteria expressing the protein can be detected by using the MAb. The marker gene contains only E. coli sequences not coding for toxins and therefore can be considered environmentally safe. The hybrid PhoE -ColA protein was expressed in E. coli under conditions of phosphate s tarvation, and single cells could be detected by immunofluorescence mi croscopy with MAb 1C11. Using a wide-host-range vector the phoE-caa ge ne was introduced into P. putida WCS358. The gene appeared to be expre ssed under phosphate limitation in this species, and the gene product was present in the membrane fraction and reacted with MAb 1C11. The hy brid PhoE-ColA protein could be detected on whole cells of WCS358 muta nt strains lacking (part of) the O-antigen of the lipopolysaccharide b ut not on wild-type WCS358 cells, unless these cells had previously be en washed with 10 mM EDTA. In addition to immunodetection, the phoE-ca a marker gene could he specifically detected by PCR with one primer di rected to a part of the phoE sequence and a second primer that anneale d to the caa insert.