Nondisruptive detection of activity of catabolic promoters of Pseudomonas putida with an antigenic surface reporter system.
A simple procedure to detect the switching on and off of catabolic promoters of Pseudomonas putida, at the level of single cells based on the immunodetection of a reporter epitope expressed on the surface of bacterial cells, has been developed. To do this, the antigenic sequence Asp-Leu-Pro-Pro-Asn-Ser-Asp-Val-Val-Asp, from a coronavirus, was inserted genetically in the permissive site around amino acid position 153 of the LamB protein (maltose and lambda phage receptor) of Escherichia coli. When the hybrid lamB gene is transcribed, the epitope becomes presented on the surface of the bacterial cells in a configuration available to specific antibodies. To validate this notion in nonenteric bacteria, the expression and correct processing of LamB were confirmed by coupling the lamB gene to the salicylate-responsive Psa1 promoter of the NAH7 (naphthalene degradation) plasmid in Pseudomonas putida. Subsequently, a hybrid lamB gene carrying the sequence of the coronavirus antigen was placed downstream of the m-toluate-responsive Pm promoter of the TOL (toluene degradation) plasmid. Exposure of the epitope on the Pseudomonas cell surface was monitored through fluorescence of whole cells treated with a monoclonal antibody against the heterologous antigen. Fluorescence emission was dependent on the presence of m-toluate in the medium, thus permitting detection of the Pm promoter switching on by simple optical inspection of individual cells, even in situations when these are a very minor component of a complex bacterial community.
Cebolla
A
A
Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
Guzmán
C
C
de Lorenzo
V
V
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000956:Antigens, Viral / Q000096:biosynthesis / Q000235:genetics; D001425:Bacterial Outer Membrane Proteins; D001483:Base Sequence; D001565:Benzoates / Q000494:pharmacology; D017934:Coronavirus / Q000235:genetics; D000939:Epitopes / Q000032:analysis / Q000096:biosynthesis*; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D017930:Genes, Reporter / Q000502:physiology*; D005822:Genetic Vectors / Q000235:genetics*; D008562:Membrane Glycoproteins / Q000235:genetics; D008969:Molecular Sequence Data; D009281:Naphthalenes / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D018272:Porins; D011401:Promoter Regions, Genetic; D016958:Pseudomonas putida / Q000235:genetics* / Q000378:metabolism; D011991:Receptors, Virus / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D064370:Spike Glycoprotein, Coronavirus; D014050:Toluene / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000235:genetics
D000911:Antibodies, Monoclonal; D000956:Antigens, Viral; D001425:Bacterial Outer Membrane Proteins; D001565:Benzoates; D000939:Epitopes; D008562:Membrane Glycoproteins; D009281:Naphthalenes; D018272:Porins; D011991:Receptors, Virus; D011993:Recombinant Fusion Proteins; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; C016532:maltoporins; C009503:3-toluic acid; C031721:naphthalene; D014050:Toluene