Construction of a model secretion system for oral streptococci.
A DNA fragment corresponding to the secretory domain from the Streptococcus mutans GS-5 gtfB gene, which encodes the putative 38-amino-acid signal peptide of the glucosyltransferase I (GTF-I) enzyme product, has been constructed. This fragment was fused with the alpha-amylase structural gene from alkalophilic Bacillus sp. strain 707. This hybrid gene as well as the intact amylase gene were introduced into an Escherichia coli-streptococcus shuttle vector consisting of three components: the E. coli replicon p15Aori from pACYC177, an erythromycin resistance gene from pAM beta-1, and the streptococcal replicon from pVA838. Transformation of the oral noncariogenic bacterium Streptococcus gordonii with the chimeric plasmid harboring the hybrid amylase gene resulted in strong extracellular amylase production. By contrast, transformants containing the intact amylase gene exhibited only trace amounts of amylase activity in culture fluids. Since the two signal peptide structures of the GTF-I enzyme and the Bacillus amylase are distinct from each other, these differences might result from the inability of S. gordonii to correctly process the Bacillus signal peptide. Furthermore, culture fluids from transformants of S. mutans as well as Streptococcus milleri harboring the hybrid amylase gene showed only weak amylase activity. Deletion of the gtfB, gtfC, or ftf gene from S. mutans GS-5 did not increase amylase secretion following transformation with the hybrid amylase gene. These results suggest that in contrast to S. gordonii, the inability of S. mutans and S. milleri to secrete hybrid amylase molecules could result from incorrect interaction of the secretory components of these organisms with amylase precursor molecules.
Shiroza
T
T
Department of Pediatric Dentistry, University of Texas Health Science Center, San Antonio 78284-7888.
Kuramitsu
H K
HK
D000595:Amino Acid Sequence; D000681:Amylases / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D015152:Blotting, Northern; D017353:Gene Deletion; D005964:Glucosyltransferases / Q000235:genetics; D006602:Hexosyltransferases / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009055:Mouth / Q000382:microbiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013291:Streptococcus / Q000201:enzymology* / Q000235:genetics; D014169:Transformation, Bacterial; D001426:Bacterial Proteins
D000681:Amylases; D005964:Glucosyltransferases; D006602:Hexosyltransferases; D011993:Recombinant Fusion Proteins; C021451:inulosucrase; D001426:Bacterial Proteins