Structural and functional analysis of pt38, a 2.9kb plasmid of Streptococcus thermophilus yogurt strain

The cryptic plasmid pt38 (2911 bp) of Streptococcus thermophilus ST2783, a strain isolated from Bulgarian yogurt, was subcloned and sequenced. Five ORFs (ORF1 to ORF5) were identified, although putative transcription initiation and termination signals, and Shine-Dalgarno sequence could only be localized for three of them (ORF1, ORF2, and ORF5). ORF2 would specify a 142-amino acid protein sharing a high degree of homology with plasmid-born low-molecular-weight heat stress proteins described in a variety of S. thermophilus strains. On the other hand, ORF1 would encode a 311-residue protein, which was found to be almost identical to the putative Rep proteins of previously sequenced S. thermophilus rolling circle-replicating plasmids. Intracellular single-stranded pt38 DNA was detected, showing that, in fact, the plasmid replicates via a rolling circle mechanism. A putative double-strand origin with significant homology to that of pC194, and a ssoA-type single-strand origin were also identified on the nucleotide sequence of pt38. A DNA region that can be transcribed into a small RNA (ctRNA) complementary to the leader segment of the rep (ORF1) mRNA is proposed to be involved in the control of plasmid replication. In vitro synthesis of this ctRNA was observed, and this constitutes the first report on the existence of such antisense RNAs, likely acting as regulatory elements, in S. thermophilus plasmids.