Cloning and inactivation of the gene responsible for a major surface antigen on Streptococcus mutans

Abstract

To understand more fully the biological function(s) and investigate the reported cross-reactivity with heart tissue of antigen P1 (I/II) of Streptococcus mutans (serotype c), this molecular biological study of the responsible gene, spaP, was undertaken. A 5.2 kb Hin dIII fragment of strain NG5 was cloned into Escherichia coli JM109 by a shotgun procedure with pUC18 as the vector. Recombinant SM2949 expressed a P1 fusion protein under the control of the streptococcal promoter. Southern analysis revealed hybridization of pSM2949 with DNA from Strep. mutans (serotypes c, e, f), Strep. cricetus (a) and Strep. sobrinus (d), but not Strep. sobrinus (g), Strep. rattus (b) or Strep. downei (h). Recombinant (r) antigen was detected in E. coli periplasm, indicating the presence of a signal sequence. This product (of Mr 155K) showed partial identity to the native streptococcal P1 antigen by Ouchterlony double-diffusion analysis. The N-terminal 28 amino acid residues of rP1 were determined by Edman degradation analysis and an end-labelled oligonucleotide probe corresponding to residues 8-13 was used to determine the 5'-3' orientation of spaP by Southern hybridization with restriction enzyme digests of pSM2949. Rabbit antisera made against native and rP1 did not cross-react with human heart tissue. Isogenic mutants of strain NG8 were isolated after transformation with insertionally inactivated spaP. Each mutant was non-reactive with anti-P1 antisera. Selected mutants were shown to have a defective spaP gene incorporated into their chromosomal DNA.(ABSTRACT TRUNCATED AT 250 WORDS)