Streptococcus mutans Genes That Code for Extracellular Proteins in Escherichia coli K-12

Abstract

Chromosomal DNA from Streptococcus mutans 6715 (serotype g) was cloned into Escherichia coli K-12 by using the cosmid pJC74 cloning vector and a bacteriophage X in vitro packaging system. Rabbit antiserum against S. mutans extracellular proteins was used for immunological screening of the clone bank. Twenty-one clones produced weak to strong precipitin bands around the colonies, but only after the X c1857 prophage was induced by being heated to lyse the E. coli cells. None of the clones expressed enzyme activity for several known S. mutans extracellular enzymes. One of these clones contained a 45-kilobase recombinant plasmid designated pYA721. An 8.5-kilobase fragment of S. mutans DNA from pYA721 was isolated and recloned into the BamHI restriction site of the plasmid vector pACYC184 to construct pYA726. pYA726 contained all, or nearly all, of the gene for a surface protein antigen (the spaA protein) of S. mutans 6715. This was deduced from immunological studies in which extracts of cells harboring pYA726 reacted with antisera against both purified 6715 spaA protein (about 210,000 daltons) and the immunologically similar antigen I/II of serotype c strains of S. mutans. In addition, the S. mutans spaA protein was found to possess at least one antigenic determinant not present on the protein specified by pYA726. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of E. coli clone extracts revealed that pYA726 produced a polypeptide with a molecular mass of about 180,000 daltons which was predominantly found in the periplasmic space of E. coli cells. Antisera to the spaA protein of S. mutans reacted with extracellular protein from representative strains of S. mutans serotypes a, c, d, e, f, and g, but not b.

Immunolocalization of Streptococcus mutans sr gene product in recombinant Escherichia coli

“…Both proteins were principally found in the cytoplastn or associated with the cytoplasmic membrane, indicating that the proteins are not secreted into the periplasmic space. However, cloned protein PI and Spa A were able to penetrate the cytoplasmic metnbrane and were predotninantly found in the periplastnic space (6,7). Two different explanations can be given to these observations: either the protein is synthesized in E. coli in high amounts resulting in an aggregation of the protein which prevents the passage through the cytoplastnic tnembrane or the lack of a post-transcriptional procedure in E. eoli.…”

Signal sequence and alanine-rich region of streptococcal protein antigen A of Streptococcus sobrinus can direct localization of alkaline phosphatase to the periplasm of Escherichia coli

“…Previously, we have shown that recombinant SpaA protein expressed by the S. sobrinus spaA gene cloned into E. coli cells was predominantly localized to the periplasm of the E. coli cells [7]. In this communication, we analyze using TnphoA the sequences needed to mediate this periplasmic localization of the S. sobrinus SpaA protein.…”

“…The virulence of the mutans streptococci depends on extracellular expression of virulence determinants including glucosyltransferases and surface protein antigens. A family of proteins designated by di¡erent laboratories as antigen B [3], antigen I/II [4], SpaP1 [5] or PAc [6] expressed by Streptococcus mutans and SpaA [7] or PAg [8] protein expressed by Streptococcus sobrinus are important virulence determinants for the mutans streptococci. Both of these proteins as well as a similar protein produced by Streptococcus sanguis [9] have been shown to interact with salivary agglutinins to facilitate colonization of enamel surfaces [10] or interact with £uid phase agglutinin to mediate cell to cell aggregation [11].…”

Localization on the cell surface of streptococcal protein antigen A

“…Proteins closely associated with the cell wall appear to facilitate the colonization of enamel surfaces by S. sobrinus and other members of the mutans streptococci. S. sobrinus cells express a major high molecular mass protein called SpaA [2,3]. The SpaA protein also has been referred to as PAg [4].…”

“…Proteins antigenicaUy similar to the SpaA protein have been shown to be produced by all of the serotypes of the mutans streptococci with the exception of serotype b [2,7,8] and also by Streptococcus sanguis [9]. Sequence analysis of the spaA gene indicates that the mature SpaA protein has a M r of 160500 [10].…”