STRUCTURAL AND FUNCTIONAL-ORGANIZATION OF THE YERSINIA-PESTIS BACTERIOCIN PESTICIN GENE-CLUSTER

The primary structure of a 2671 bp DNA fragment between the pla gene ( encoding plasminogen activator) and the origin of replication of the w ild-type Yersinia pestis plasmid pYP358 was determined. Two ORFs of 10 74 and 426 bp with opposite transcription polarities were identified o n both strands. They encode a 357 aa pesticin activity protein (Pst) a nd a 141 aa pesticin immunity polypeptide (Pim). A GC-rich palindromic structure located between pst and pim can form a hairpin loop and ser ve as a rho-independent transcription terminator sequence for both gen es. The site for the interaction with the LexA repressor of the SOS sy stem was found in another palindromic structure preceding the pst stru ctural gene. A deduced 39.9 kDa Pst polypeptide is devoid of a signal peptide, indicating a Sec-independent mode of export. Pst carries a pe ntapeptide typical of TonB-dependent colicins (TonB box) that is neces sary for the interaction with the yersiniabactin/pesticin receptor and for active energy-dependent transport through the outer membrane. The substitution of the last five C-terminal amino acids did not signific antly influence the bactericidal activity of the truncated pesticin. T he pesticin lost its ability to kill sensitive bacteria and to bind to a pesticin receptor after deletion of the last 57 C-terminal amino ac ids. A deduced 16 kDa Pim protein has an N-terminal hydrophobic amino acid stretch with features typical of prokaryotic signal peptides. Pim is a slightly hydrophilic protein with a basic pi. The immunity prote in was localized in the periplasmic space and in the outer-membrane fr action after its overexpression under the polymerase T7 promoter. Seve ral other ORFs were identified on the sequenced 2671 bp fragment, but none of them seemed to encode a typical lysis peptide, which is necess ary for the release of the pesticin. In the promoter region and in the regions preceding and following the pst operon, the DNA sequence has high (> 70 %) identity with other colicin genes. The DNA sequence loca ted 284 bp upstream of the pim gene showed more than 90 % similarity t o antisense RNA I of the ColE1 replicon. This defined the location of the pYP358 origin of ColE1-type replication.