Submitted to: Journal of Physical Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 24, 1996
Publication Date: N/A
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Interpretive Summary: Streptococcus thermophilus (ST) is a microorganism employed in starter cultures for production of yoghurt and certain cheeses. A quick and convenient way to change the properties of this bacterium for use in the development of new dairy products is through genetic engineering. For this purpose, small and circular DNA molecules called plasmids are needed for transporting new genes among microorganisms. Furthermore, special short DNA stretches called promoters must be present in front of the genes for them to become active. The present study describes the isolation of several promoters from ST. The sequences of their building blocks called nucleic acids were also determined. By comparing these sequences with those of other bacteria, the important regions of the promoters were mapped. This study further shows that one of the promoters is involved in the expression of a DNA repair enzyme in ST. These promoters, along with the information they contain, should provide valuable leads for making plasmids that efficiently express their genes, and remain stable, i.e., continually functioning, in genetically modified dairy fermentation streptococci and possibly lactobacilli.
Technical Abstract: Streptococcus thermophilus (ST) chromosomal DNA fragments generated by partial Sau3A digestion were cloned into the unique BamHI site upstream from the promoterless chloramphenicol acetyltransferase (cat) gene of the Escherichia coli (EC) promoter-probe vector pKK520-3. Recombinant plasmids containing ST sequences with transcription-activation activity were isolat- -ed from chloramphenicol resistant (CmR) EC transformants. A promoterless Streptomyces antibioticus melanin biosynthesis operon (melC) was inserted immediately downstream from the ST sequence to identify DNA with strong promoter activity. Several ST transcription-activation sequences, termed STP's, were isolated and subcloned, and their nucleic acid sequences determined. The -10 and -35 consensus sequences were identified in these putative ST promoters. Detailed analysis of STP3306 sequence data revealed two partial open-reading-frames (ORF) with high degrees of homology to procaryotic GTP-binding protein and DNA repair enzyme, thus providing valuable information for further study on DNA maintenance in this important lactic acid bacterium.