Structural organization of the pFra virulence-associated plasmid of rhamnose-positive Yersinia pestis

Abstract

The 137,036-bp plasmid pG8786 from rhamnose-positive Yersinia pestis G8786 isolated from the high mountainous Caucasian plague focus in Georgia is an enlarged form of the pFra virulence-associated plasmid containing genes for synthesis of the antigen fraction 1 and phospholipase D. In addition to the completely conserved genes of the pFra backbone, pG8786 contains two large regions consisting of 4,642 and 32,617 bp, designated regions 1 and 2, respectively. Region 1 retains a larger part of Salmonella enterica serovar Typhi plasmid pHCM2 resembling the backbone of pFra replicons, while region 2 contains 25 open reading frames with high levels of similarity to the transfer genes of the F-like plasmids. Surprisingly, region 1 is also present in the pFra plasmid of avirulent Y. pestis strain 91001 isolated in Inner Mongolia, People's Republic of China. Despite the fact that some genes typically involved in conjugative transfer of the F-like replicons are missing in pG8786, we cannot exclude the possibility that pG8786 might be transmissive under certain conditions. pG8786 seems to be an ancient form of the pFra group of plasmids that were conserved due to the strict geographical isolation of rhamnose-positive Y. pestis strains in the high mountainous Caucasian plague locus.

FIG. 1.

Map of the pG8786 plasmid. The inner circle shows region 1, region 2, and the pFra-like backbone. The outer circle shows ORFs and their orientation, which are designated on the basis of their positions; the arrows and boxes outside the ring indicate clockwise transcription, and the arrows and boxes inside the ring indicate counterclockwise transcription. The map was derived from the annotated DNA sequence by using the Vector NTI (InforMax) computer program and was edited in CorelDRAW.

FIG. 2.

G+C content and graphic map of pG8786. The plot showing the G+C content was derived by using the Vector NTI program (InforMax). The diagram at the top shows selected ORFs and some other annotated features at the correct scale. The scale below the G+C plot indicates the size of the plasmid. IS285*, IS285 insertion sequence which appeared to be a nonfunctional remnant.

FIG. 3.

Graphic comparison of different pFra plasmids with pG8786 derived by using the Artemis Comparison Tool program (The Wellcome Trust Sanger Institute, Cambridge, United Kingdom). (a) pG8786 and pMT-1 of Y. pestis KIM10+; (b) pG8786 and pMT1 of Y. pestis CO92; (c) pG8786 and pMT1 of Y. pestis KIM5; (d) pG8786 and pMT1 of Y. pestis 91001. Areas of pG8786 that are not present in the other pFra plasmids are labeled region 1 and region 2. Vertical lines indicate similar parts of the plasmids.

FIG. 4.

(a) Phylogenetic tree for the transfer regions from various sources. (b) Phylogenetic tree for the replication origins from different microorganisms. The trees were constructed by using the neighbor-joining method of Saitou and Nei and the Vector NTI program (InforMax). Using AlignX, we determined the calculated distance values (which are indicated in parentheses following the molecule designations on the trees).

FIG. 5.

Alignment of the tra genes of pG8786 (lower diagram) with the tra genes of the pED208 plasmid from S. enterica serovar Typhi (upper diagram). The solid arrows represent ORFs which are equally represented in the two plasmids. The open arrows represent ORFs which are absent in either of the two transfer regions. IS285*, IS285 insertion sequence which appeared to be a nonfunctional remnant. The vertical lines between arrows indicate similar ORFs.