title: Construction of phoE-caa, a novel PCR- and immunologically detectable marker gene for Pseudomonas putida.
abstract: In this paper we describe the construction and use in Pseudomonas putida WCS358 of phoE-caa, a novel hybrid marker gene, which allows monitoring both at the protein level by immunological methods and at the DNA level by PCR. The marker is based on the Escherichia coli outer membrane protein gene phoE and 75 bp of E. coli caa, which encode a nonbacteriocinic fragment of colicin A. This fragment contains an epitope which is recognized by monoclonal antibody (MAb) 1C11. As the epitope is contained in one of the cell surface-exposed loops of PhoE, whole cells 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 starvation, and single cells could be detected by immunofluorescence microscopy with MAb 1C11. Using a wide-host-range vector the phoE-caa gene was introduced into P. putida WCS358. The gene appeared to be expressed under phosphate limitation in this species, and the gene product was present in the membrane fraction and reacted with MAb 1C11. The hybrid PhoE-ColA protein could be detected on whole cells of WCS358 mutant strains lacking (part of) the O-antigen of the lipopolysaccharide but not on wild-type WCS358 cells, unless these cells had previously been washed with 10 mM EDTA. In addition to immunodetection, the phoE-caa marker gene could be specifically detected by PCR with one primer directed to a part of the phoE sequence and a second primer that annealed to the caa insert.
authors.affiliation: Leiden University, Institute of Molecular Plant Sciences, The Netherlands.
authors.forename: S A
authors.initials: SA
authors.lastname: Zaat
authors.forename: K
authors.initials: K
authors.lastname: Slegtenhorst-Eegdeman
authors.forename: J
authors.initials: J
authors.lastname: Tommassen
authors.forename: V
authors.initials: V
authors.lastname: Geli
authors.forename: C A
authors.initials: CA
authors.lastname: Wijffelman
authors.forename: B J
authors.initials: BJ
authors.lastname: Lugtenberg
mesh_terms: D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000235:genetics*; D005819:Genetic Markers / Q000235:genetics; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D016958:Pseudomonas putida / Q000235:genetics*; D014162:Transfection
chemical_list: D005819:Genetic Markers