Identification of another surface protein antigen I/II gene, paaB, and a putative transcriptional regulator gene, par, from Streptococcus cricetus

Abstract

The flanking region of the antigen I/II gene, paaA , in Streptococcus cricetus was examined using the gene-walking technique. In the region downstream of the paaA gene, another antigen I/II gene designated as paaB was found. The paaB gene was disrupted at the alanine-rich region (A region) by a novel insertion sequence element, IS Scr1. IS Scr1 is a member of the IS 982 family and is composed of a 962-bp sequence and duplicated target DNA (the sequence 5'-TAGCTAAAT-3') resulting from its insertion. To clarify the structural divergence of the two antigen I/II proteins (PAaA and PAaB), computational analysis of the paaB gene was performed and the two structures were compared. The amino acid sequence homology indicated that PAaB resembled PAaA, but the middle region showed little similarity to that of PAaA. Phylogenetic analysis showed that PAaB was better classified in a major group with S. mutans PAc and S. gordonii SspA and SspB than with PAaA. The transcriptional expression of paaA and paaB was demonstrated by reverse transcription (RT)-PCR. In the region upstream of the paaA gene, three genes homologous to the genes located in the region upstream of the S. sobrinus antigen I/II gene ( pag ) were found. Of the three genes, ORF3 showed homology to the par gene encoding a transcriptional repressor for the pag gene in S. sobrinus . Therefore, ORF3 was designated the par gene of S. cricetus . Southern hybridization revealed that the par gene of S. cricetus was not found in other oral streptococci examined in this study.

Characterization of <i>Streptococcus criceti</i> insertion sequence IS<i>Scr1</i>

“…The PCR products were gel-purified and cloned at the SrfI site of a pPCR-Script Amp SK(+) cloning vector (Agilent Technologies). DNA hybridization and detection were performed using the DIG High Prime DNA Labeling and Detection Starter Kit I (Roche Diagnostics, Mannheim, Germany) as described previously (Tamura et al, 2004). Briefly, hybridization was performed at 42°C for 16 h, followed by washing according to the manufacturer's instructions (Roche Diagnostics), and hybridized fragments were visualized with a chromogenic alkaline phosphatase substrate NBT/BCIP after 16-h incubation.…”

“…The virulence-associated genes are targets for IS elements (Spellerberg et al, 2000;Tamura et al, 2004). Streptococcus mutans and S. sobrinus are etiologically considered as cariogenic agents in humans (Hamada and Slade, 1980;Loesche, 1986).…”

“…Regarding IS, ISScr1, a member of the IS982 family, has only been identified from S. criceti E49. The 962-bp ISScr1 was observed to inactivate the paaB gene encoding a cell-surface protein of antigen I/II family protein (Tamura et al, 2004) (Fig. 1).…”

“…Herein we report two discrete insertion sites of ISScr1 in S. criceti E49 and the distribution of ISScr1 in S. criceti strains. IPCR and gene walking To amplify flanking regions of ISScr1 in S. criceti E49, IPCR was first achieved with two outward-pointing primers P1 (5'-AAGAAGTTGGC-CGCAAGGA-3') and P2 (5'-GCGTCAAAACATGGCAG-GAG-3') designed on the basis of the nucleotide sequence of the putative transposase gene, tnp, of the 962-bp ISScr1 (Tamura et al, 2004) (Fig. 1), and Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA).…”

“…The resultant products were directly sequenced. The flanking regions were identified using the gene-walking method as described previously (Tamura et al, 2004).…”