Insertional inactivation of the gene encoding a 76-kilodalton cell surface polypeptide in Streptococcus gordonii Challis has a pleiotropic effect on cell surface composition and properties.
A library of Streptococcus gordonii DL1-Challis DNA was constructed in lambda gt11. Phage plaques were screened for production of antigens that reacted with antiserum to S. gordonii cell surface proteins. A recombinant phage denoted lambda gt11-cp2 was isolated that carried 1.85 kb of S. gordonii DNA and that expressed an antigen with a molecular mass of 29 kDa in Escherichia coli. Antibodies that reacted with the expression product were affinity purified and were shown to react with a single polypeptide antigen with a molecular mass of 76 kDa in S. gordonii DL1-Challis. A segment (0.85 kb) of the cloned DNA within the transcription unit was ligated into a nonreplicative plasmid carrying an erythromycin resistance determinant and transformed into S. gordonii DL1-Challis. The plasmid integrated onto the chromosome, and expression of the 76-kDa polypeptide antigen was abolished. The gene inactivation had no obvious effect on bacterial growth or on a number of phenotypic properties, including hydrophobicity and adherence. However, it abolished serum-induced cell aggregation, mutant cells had reduced aggregation titers in saliva and in colostrum immunoglobulin A, and it also reduced coaggregation with some Actinomyces species. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of cell envelope proteins from wild-type and mutant strains showed that as well as lacking the surface-exposed 76-kDa polypeptide, mutant cell envelopes were deficient in several other polypeptides, including those that bound to immunoglobulin A. Expression of the gene encoding the 76-kDa polypeptide in S. gordonii appeared to be critical for functional conformation of the cell surface.
Jenkinson
H F
HF
Department of Oral Biology and Oral Pathology, University of Otago, Dunedin, New Zealand.
Easingwood
R A
RA
D000818:Animals; D000954:Antigens, Surface / Q000235:genetics*; D001422:Bacterial Adhesion / Q000235:genetics; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000096:biosynthesis; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D006801:Humans; D007070:Immunoglobulin A / Q000276:immunology; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D008970:Molecular Weight; D009154:Mutation; D010641:Phenotype; D011817:Rabbits; D015183:Restriction Mapping; D013291:Streptococcus / Q000235:genetics*
D000954:Antigens, Surface; D004269:DNA, Bacterial; D007070:Immunoglobulin A; D008565:Membrane Proteins