CHARACTERIZATION OF A NOVEL STREPTOCOCCUS-THERMOPHILUS ROLLING-CIRCLEPLASMID USED FOR VECTOR CONSTRUCTION

The complete nucleotide sequence of pER371, a native plasmid in Strept ococcus thermophilus ST137, was determined. A putative open reading fr ame coding for a replication protein, Rep371, was identified. A charac teristic promoter sequence and ribosome-binding site were found upstre am of Rep371. Rep371 (247 amino acid residues) does not show homology with RepA and RepS of the small S. thermophilus cryptic plasmids pST1- No.29 and pST1 respectively. The plus-origin sequence and Rep371 are h ighly homologous to the corresponding elements of the Staphylococcus a ureus plasmids pC194 and pSK89. A novel 140-nucleotide palindromic min us-origin sequence, which is structurally similar but does not show se quence homology to the palA region of pC194, was identified in pER371. A palindromic sequence capable of forming a putative hairpin structur e was identified and subsequently recognized as being highly conserved among several lactococcal rolling-circle plasmids. Cloning vectors de rived from pER371 should provide valuable gene-delivery vehicles for t he genetic engineering of lactic acid bacteria.