The signal recognition particle (SRP)-translocation pathway is conserved in all three domains of life and delivers membrane and secretory proteins to the cytoplasmic membrane or endoplasmic reticulum. We determined the requirement in the cariogenic oral pathogen Streptocococcus mutans of the three universally conserved elements of the SRP pathway: Ffh͞SRP54, scRNA, and FtsY͞SR␣. Previously, we reported that insertional interruption of S. mutans ffh was not lethal, but resulted in acid sensitivity. To test whether S. mutans could survive extensive disruption of the SRP pathway, single and double deletions of genes encoding Ffh, scRNA, and FtsY were generated. Without environmental stressors, all mutant strains were viable, but unlike the wild-type, none could initiate growth at pH 5.0 or in 3.5% NaCl. Survival of challenge with 0.3 mM H2O2 was also diminished without ffh. Members of the YidC͞Oxa1͞Alb3 family are also ubiquitous, involved in the translocation and assembly of membrane proteins, and have been identified in prokaryotes͞mitochondria͞chloroplasts. Two genes encoding YidC homologs, YidC1 and YidC2, are present in streptococcal genomes with both expressed in S. mutans. Deletion of YidC1 demonstrated no obvious phenotype. Elimination of YidC2 resulted in a stress-sensitive phenotype similar to SRP pathway mutants. Mutants lacking both YidC2 and SRP components were severely impaired and barely able to grow, even in the absence of environmental stress. Here, we report the dispensability of the cotranslational SRP protein translocation system in a bacterium. In S. mutans, this pathway contributes to protection against rapid environmental challenge and may overlap functionally with YidC2.protein translocation ͉ streptococcus ͉ Ffh ͉ FtsY ͉ membrane biogenesis

The gene spa P (formerly designated as spa Pl) encoding the M, 185,000 surface antigen (I/II) of Streptococcus mutans, serotype c (NGS), has been sequenced. The gene (4683 bp) encodes a protein of 1561 amino acid residues including putative signal peptide (residues I-38) and transmembrane (residues 1537-1556) sequences. The N-terminal region (6(r550) has alanine-rich repeats and is predicted to be a-helical. However, the C-terminal region (800-1540) is proline-rich and favours an extended structure. Except for a short central variable region the sequences appear to be highly conserved for S. mutuns serotype c. N-Terminal sequencing of separated antigen I and antigen II polypeptides suggests that the former represents the N-terminal and the latter the C-terminal portions of the intact antigen.Antigen I/II; Streptococcal surface antigen; spa PI

New vectors were constructed for efficient transposon Tn917-mediated mutagenesis of poorly transformable strains of Streptococcus mutans(pTV1-OK) and subsequent recovery of interrupted genes in Escherichia coli(pTV21⌬2TetM). In this report, we demonstrate the utility of Tn917 mutagenesis of a poorly transformable strain of S. mutans (JH1005) by showing (i) the conditional replication of pTV1-OK, a repA(Ts) derivative of the broad-host-range plasmid pWVO1 harboring Tn917, in JH1005 at the permissive temperature (30؇C) versus that at the nonpermissive temperature (45؇C); (ii) transposition frequencies similar to those reported for Bacillus subtilis (10 ؊5 to 10 ؊4) with efficient plasmid curing in 90 to 97% of the erythromycin-resistant survivors following a temperature shift to 42 to 45؇C; and (iii) the apparent randomness of Tn917 insertion as determined by Southern hybridization analysis and the ability to isolate nutritional mutants, mutants in acid tolerance, and mutants in bacteriocin production, at frequencies ranging from 0.1 to 0.7%. Recovery of transposon-interrupted genes was achieved by two methods: (i) marker rescue in E. coli with the recovery vector pTV21⌬2TetM, a tetracycline-resistant and ampicillin-sensitive Tn917-pBR322 hybrid, and (ii) "shotgun" cloning of genomic libraries of Tn917 mutants into pUC19. Sequence analyses revealed insertions at five different genetic loci in sequences displaying homologies to Clostridium spp. fhs (66% identity), E. coli dfp (43% identity), and B. subtilis ylxM-ffh (58% identity), icd (citC [69% identity]), and argD (61% identity). Insertions in icd and argD caused nutritional requirements; the one in ylxM-ffh caused acid sensitivity, while those in fhs and dfp caused both acid sensitivity and nutritional requirements. This paper describes the construction of pTV1-OK and demonstrates that it can be efficiently employed to deliver Tn917 into S. mutans for genetic analyses with some degree of randomness and that insertions in the chromosome can be easily recovered for subsequent characterization. This represents the first published report of successful Tn917 mutagenesis in the genus Streptococcus.

The gene (spaP) coding for the Streptococcus mutans major surface protein antigen P1 (or IIH) has been cloned into Escherichia coli (