Structural and functional analysis of pCI65st, a 6{middle dot}5 kb plasmid from Streptococcus thermophilus NDI-6

• Published in: Microbiology (Society for General Microbiology) 1999-01, Vol.145 (1), p.127-134
• Main Authors: O'Sullivan, Tadhg, van Sinderen, Douwe, Fitzgerald, Gerald
• Format: Article
• Language: English
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/13500872-145-1-127

Department of Microbiology, University College, Cork, Ireland Author for correspondence: Gerald Fitzgerald. Tel: [phone]. Fax: [phone]. e-mail: [e-mail] ABSTRACT Summary: The 6·5 kb cryptic plasmid pCI65st from Streptococcus thermophilus NDI-6, a strain isolated from the Indian fermented milk dahi, was subcloned and sequenced. Five putative ORFs were identified. ORF1 could encode a 315 aa polypeptide almost identical to the RepA protein of previously sequenced S. thermophilus plasmids, indicating that pCI65st is one of the pC194 group of small Gram-positive rolling-circle plasmids. ORFs 2 and 4 were virtually identical and could specify proteins of approximately 150 aa with significant similarity to the small heat-shock proteins described from a variety of Gram-positive bacteria. ORF3 could encode a 415 aa protein similar to enolase, an enzyme involved in glycolysis and gluconeogenesis. ORF5 could encode a 412 aa protein which had high similarity to the HsdS (specificity) proteins of type I restriction-modification systems. Variants of strain NDI-6 which lacked pCI65st were readily isolated after subculture of the parent strain at 32 °C. The plasmid-bearing parent culture was significantly more resistant to a temperature shift from 42 °C to 62 °C than its plasmid-free variant and expressed proteins which corresponded with the predicted translation products from ORF2 and ORF4. In addition, plasmid-free mutants were lysed in broth by bacteriophages to which the parent culture was resistant. Keywords: Streptococcus thermophilus , plasmid, heat-shock genes, enolase, type I HsdS The GenBank accession number for the sequence reported in this paper is AF027167.