Cloning and DNA sequencing of the surface protein antigen I/II (PAa) of Streptococcus cricetus

Tamura, Haruki

doi:10.1016/s0378-1097(01)00077-5

Cited by 8 publications

(20 citation statements)

References 22 publications

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“…Chromosomal DNA and adaptor-ligated DNA libraries of S. criceti E49 for gene-walking were prepared as described previously (31). PCR was performed with AmpliTaq Gold R DNA polymerase (Applied Biosystems, Foster City, Calif., U.S.A.) and degenerate primers based on the deduced amino acid sequences of dextranase genes from S. mutans, S. sobrinus, S. downei, and S. ratti (DDBJ database accession nos.…”

Section: Methodsmentioning

confidence: 99%

“…The conditions for PCR were as follows: 10 min at 95 C, followed by 25 cycles of 30 sec at 94 C, 30 sec at 42 C, and 2 min at 72 C, with a final 5-min elongation at 72 C. Of two amplified fragments (approximately 650-and 440-bp), the fragment of expected size was cloned into the pGEM-T vector (Promega, Madison, Wisc., U.S.A.) and sequenced. The region flanking of the sequenced segment was determined by the genewalking method as described previously (31) with the first internal primers (5'-ACAAAACAGCCACTAACC-CATTCCCAA-3' and 5'-CATCGGTTGTCCCTTG-GCACCATAAC-3') and sequenced on both strands for authenticity. The primers used in this study were synthesized by FASMAC (Kanagawa, Japan).…”

Section: Methodsmentioning

confidence: 99%

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Identification and Characterization of a Dextranase Gene of Streptococcus criceti

Tamura

Yamada

Kato

2007

Microbiology and Immunology

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The dextranase gene, dex, was identified in Streptococcus criceti strain E49 by degenerate PCR and sequenced completely by the gene‐walking method. A sequence of 3,960 nucleotides was determined. The dex gene encodes a 1,200‐amino acid protein, which has a calculated molecular mass of 128,129.91 and pI of 4.15 and is predicted to be a cell‐surface protein. The deduced amino acid sequence of dex showed homology to S. downei dextranase (63.9% identity). Phylogenetic analysis revealed the similarity of the deduced amino acid sequence of dextranases in S. criceti, S. sobrinus, and S. downei. A recombinant form of the protein with six histidine residues tagged in the C‐terminus was partially purified and showed dextranase activity on blue‐dextran sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (BD‐SDS‐PAGE) followed by renaturation. We also detected dextranase activity in S. criceti cell extracts and culture supernatant by renatured BD‐SDS‐PAGE, whereas no dextranase activity of the cells was observed on blue‐dextran brain heart infusion (BD‐BHI) agar plates. Furthermore, PCR‐based mutations of dextranase indicated that a deletion mutant of the C‐terminal region could hydrolyze blue dextrans and that the D453E mutation, W793L mutation, and double mutations (W793L and deletion of the C‐terminal region) resulted in a loss of dextranase activity. These findings suggest that Asp‐453 and Trp‐793 residues of S. criceti dextranase are critical to the enzyme's activity.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Identification and Characterization of a Dextranase Gene of Streptococcus criceti

Tamura

Yamada

Kato

2007

Microbiology and Immunology

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“…The region surrounding the paaA gene of S. cricetus E49 was sequenced using the gene-walking technique as described previously (Tamura et al, 2001). In brief, a Universal Genome Walker Kit (Clontech, Palo Alto, USA) was used to construct a series of adaptor-ligated genomic DNA fragments.…”

Section: Methodsmentioning

confidence: 99%

“…Antigen I/II homologs appear to be preserved in many oral streptococci and the nucleotide sequences of the antigen I/II homologs have been reported for S. sobrinus (LaPolla et al, 1991;Tokuda et al, 1991) and S. cricetus (Tamura et al, 2001). In addition, two copies of antigen I/II homologous genes ( sspA and sspB ) have been sequenced in S. gordonii (Demuth et al, 1996).…”

Section: Introductionmentioning

confidence: 94%

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

et al. 2004

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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.

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Differential binding specificities of oral streptococcal antigen I/II family adhesins for human or bacterial ligands

Jakubovics¹,

Strömberg²,

Dolleweerd³

et al. 2005

Molecular Microbiology

141

191

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SummaryThe antigen I/II (AgI/II) family polypeptides, ranging from 1310 to 1653 amino acid (aa) residues, are cell wall anchored adhesins expressed by most indigenous species of oral streptococci. The polypeptides interact with a wide range of host molecules, in particular salivary agglutinin glycoprotein (SAG or gp340), and with ligands on other oral bacteria. To determine the receptor recognition properties of six different AgI/II family polypeptides from strains of Streptococcus gordonii , Streptococcus intermedius and Streptococcus mutans , the genes were cloned and expressed on the surface of the surrogate host Lactococcus lactis . The S. gordonii SspA and SspB polypeptides mediated higher binding levels of L. lactis cells to surface immobilized gp340 than did S. intermedius Pas protein, or S. mutans SpaP or PAc proteins. However, the AgI/II proteins were all similar in their abilities to mediate aggregation of lactococci by fluid phase gp340. The SpaP I polypeptide from S. mutans Ingbritt, which was C-terminally truncated by approximately 400 aa residues, did not bind gp340. Lactococci expressing AgI/II proteins, including SpaP I , were aggregated by a synthetic 16 aa residue peptide SRCRP2 derived from the aa repeat block sequences within gp340. In coaggregation assays, SspB from S. gordonii was unique in mediating coaggregation with only group A and group E strains of Actinomyces naeslundii . All the other AgI/II polypeptides mediated coaggregation with group C and group D strains of A. naeslundii . Analysis of chimeric protein constructs revealed that coaggregation specificity was determined by sequences within the Nterminal half of AgI/II protein. A synthetic peptide (20 aa residues), which defines a putative adhesion epitope within the C-terminal region of polypeptide, inhibited AgI/II-mediated aggregation by gp340 but did not affect coaggregation with A. naeslundii . These results suggest that different mechanisms operate in interactions of AgI/II family polypeptides with native gp340, gp340 SRCR domain peptide, and A. naeslundii . Specificity of these interactions appears to be determined by discontinuous but interacting regions of the polypeptides, thus providing flexibility in receptor recognition for streptococcal colonization of the human host.

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Cloning and DNA sequencing of the surface protein antigen I/II (PAa) of Streptococcus cricetus

Cited by 8 publications

References 22 publications

Identification and Characterization of a Dextranase Gene of Streptococcus criceti

Identification and Characterization of a Dextranase Gene of Streptococcus criceti

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

Differential binding specificities of oral streptococcal antigen I/II family adhesins for human or bacterial ligands

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