Structural and functional analysis of pCI65st, a 6 center dot 5 kb plasmidfrom Streptococcus thermophilus NDI-6

The 6.5 kb cryptic plasmid pC165st from Streptococcus thermophilus NDI-6, a strain isolated from the Indian fermented milk dahi, was subcloned and seq uenced. Five putative ORFs were identified. ORF1 could encode a 315 aa poly peptide almost identical to the RepA protein of previously sequenced S. the rmophilus plasmids, indicating that pC165st is one of the pC194 group of sm all Gram-positive rolling-circle plasmids. ORFs 2 and 4 were virtually iden tical and could specify proteins of approximately 150 aa with significant s imilarity 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 4 12 aa protein which had high similarity to the HsdS (specificity) proteins of type I restriction-modification systems. Variants of strain NDI-6 which lacked pC165st were readily isolated after subculture of the parent strain at 32 degrees C. The plasmid-bearing parent culture was significantly more resistant to a temperature shift from 42 degrees C to 62 degrees C than its plasmid-free variant and expressed proteins which corresponded with the pr edicted translation products from ORF2 and ORF4. In addition, plasmid-free mutants were lysed in broth by baderiophages to which the parent culture wa s resistant.