Isolation of cell surface antigen mutants of Myxococcus xanthus by use of monoclonal antibodies.
Monoclonal antibodies (MAbs) with affinities for molecules on the cell surface of the procaryote Myxococcus xanthus were used in a screening strategy for the isolation of mutants lacking particular cell surface molecules. From a large library of independent mutants created by Tn5 transposon mutagenesis, mutants were isolated which lacked reactivities with MAb 1604 (a MAb specific for a cell surface protein) and MAbs 2600, 1733, 1514, 1412, and 783 (MAbs specific for carbohydrate epitopes on the O antigen of lipopolysaccharide [LPS]). The defect in antibody recognition was shown by genetic crosses and DNA hybridization experiments to be caused by the Tn5 transposon acting as a mutation at a single locus. Quantitative enzyme-linked immunosorbent assays showed that particular mutant strains had no detectable affinity for the specific MAb probe. LPS mutants were resistant to myxophage Mx8, and this provided a selection method for isolating a large number of new LPS mutants. A class of Mx8-resistant mutants lacked reactivity with MAb 1514 and therefore was defective in the O antigen of LPS. A class of Mx1-resistant mutants lacked reactivity with MAb 2254, a MAb specific for a carbohydrate epitope on the core of LPS. A comparison of MAb binding to different mutant strains revealed a principle for mapping epitopes and showed that MAbs 1514 and 2254 recognize side-chain carbohydrates rather than backbone carbohydrates within the LPS molecule.
Fink
J M
JM
Department of Microbiology, University of Minnesota Medical School, Minneapolis 55455.
Kalos
M
M
Zissler
J F
JF
D000911:Antibodies, Monoclonal / Q000276:immunology*; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology*; D000954:Antigens, Surface / Q000235:genetics / Q000276:immunology*; D004251:DNA Transposable Elements; D015151:Immunoblotting; D009154:Mutation; D009231:Myxococcales / Q000235:genetics / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D000954:Antigens, Surface; D004251:DNA Transposable Elements