Construction and characterization of isogenic mutants of Streptococcus mutans deficient in major surface protein antigen P1 (I/II).
The gene (spaP) coding for the Streptococcus mutans major surface protein antigen P1 (or I/II) has been cloned into Escherichia coli (S. F. Lee, A. Progulske-Fox, and A. S. Bleiweis, Infect. Immun. 56:2114-2119, 1988). In the present study, this gene has been disrupted in vitro by insertional inactivation with pVA981, which carries a Tcr marker, and transformed into S. mutans NG8 (serotype c) by electroporation. Upon homologous recombination, the defective spaP was integrated into the genome as demonstrated by Southern hybridization analysis. One Tcr mutant, designated 834, selected by its nonreactivity with anti-P1 monoclonal antibodies, was found to lack the cell surface fuzzy layer which was clearly present on the parent cells. Analysis of extracellular fluids, sodium dodecyl sulfate-solubilized membranes, and cytoplasmic fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that 834 had protein profiles identical to the parent. However, a 185-kilodalton protein which reacts with anti-P1 antibodies was missing from the wall of 834, suggesting that spaP has been specifically inactivated. This mutant displayed levels of glucosyltransferase and fructosyltransferase activities similar to those of the parent. It was much less hydrophobic than the parent. S. mutans NG8 aggregated readily in the presence of clarified whole saliva or a high-molecular-weight salivary agglutinin. This strain also adhered to agglutinin-coated hydroxyapatite. The P1-negative mutants, however, did not display these two properties, suggesting that P1 may play a role in saliva-mediated aggregation and adherence.
Lee
S F
SF
Department of Oral Biology, University of Florida, Gainesville 32610.
Progulske-Fox
A
A
Erdos
G W
GW
Piacentini
D A
DA
Ayakawa
G Y
GY
Crowley
P J
PJ
Bleiweis
A S
AS
D000371:Agglutination; D000942:Antigens, Bacterial / Q000235:genetics*; D000954:Antigens, Surface / Q000235:genetics; D001422:Bacterial Adhesion; D001426:Bacterial Proteins / Q000235:genetics*; D015139:Blotting, Southern; D015153:Blotting, Western; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000235:genetics; D003910:Dextranase / Q000378:metabolism; D005798:Genes, Bacterial; D006602:Hexosyltransferases / Q000378:metabolism; D006882:Hydroxyapatites; D007150:Immunohistochemistry; D012463:Saliva / Q000382:microbiology; D012995:Solubility; D013295:Streptococcus mutans / Q000201:enzymology / Q000235:genetics* / Q000276:immunology
D000942:Antigens, Bacterial; D000954:Antigens, Surface; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D006882:Hydroxyapatites; D006602:Hexosyltransferases; D003910:Dextranase