DNA sequencing and analysis of the low-Ca2+-response plasmid pCD1 of Yersinia pestis KIM5

Abstract

The low-Ca2+-response (LCR) plasmid pCD1 of the plague agent Yersinia pestis KIM5 was sequenced and analyzed for its genetic structure. pCD1 (70,509 bp) has an IncFIIA-like replicon and a SopABC-like partition region. We have assigned 60 apparently intact open reading frames (ORFs) that are not contained within transposable elements. Of these, 47 are proven or possible members of the LCR, a major virulence property of human-pathogenic Yersinia spp., that had been identified previously in one or more of Y. pestis or the enteropathogenic yersiniae Yersinia enterocolitica and Yersinia pseudotuberculosis. Of these 47 LCR-related ORFs, 35 constitute a continuous LCR cluster. The other LCR-related ORFs are interspersed among three intact insertion sequence (IS) elements (IS100 and two new IS elements, IS1616 and IS1617) and numerous defective or partial transposable elements. Regional variations in percent GC content and among ORFs encoding effector proteins of the LCR are additional evidence of a complex history for this plasmid. Our analysis suggested the possible addition of a new Syc- and Yop-encoding operon to the LCR-related pCD1 genes and gave no support for the existence of YopL. YadA likely is not expressed, as was the case for Y. pestis EV76, and the gene for the lipoprotein YlpA found in Y. enterocolitica likely is a pseudogene in Y. pestis. The yopM gene is longer than previously thought (by a sequence encoding two leucine-rich repeats), the ORF upstream of ypkA-yopJ is discussed as a potential Syc gene, and a previously undescribed ORF downstream of yopE was identified as being potentially significant. Eight other ORFs not associated with IS elements were identified and deserve future investigation into their functions.

FIG. 1

Genetic organization of the LCR plasmid, pCD1, of Y. pestis KIM5. The map shows significant ORFs and transposable elements as well as replication and partitioning regions. For genes on the outside of the map, transcription proceeds clockwise. Genes on the inside are transcribed from the complementary strand. The oriR and sopC regions are not transcribed. The placement of transposable elements (solid boxes) does not indicate the direction of transcription. A d indicates a defective element, while a p indicates a partial element. Most IS remnants are not denoted on this figure.

FIG. 2

Genetic organization of the replication and partitioning regions of pCD1. (A) The replication region contains four genes (repB, copA, tap, and repA) and an origin of replication (oriR). Black boxes indicate the promoter regions for repB and repA; the promoter region for copA is not shown. (B) The par region possesses two putative trans-acting genes (sopA and sopB) and a putative DNA binding site (sopC). Arrowheads within the promoter region show the locations and orientations of short, imperfect repeats. The six 45-bp direct repeats that compose sopC are shown as open boxes, with the locations of five 16-bp inverted repeat structures designated by arrowheads. RBS, ribosome binding site. For both panels, arrows indicate the directions of transcription of individual genes, while locations within pCD1 are indicated by numbers that follow the numbering system in Fig. 1.

FIG. 3

Diagrammatic representation of four clusters of intact, defective, and partial IS elements in pCD1. A p or −p indicates that only part of the IS element is present. A d indicates an IS element that is likely defective. Arrows show the presence of inverted repeats within IS elements. Each type of IS element is represented by a unique design that is maintained in each of the four panels. In panel D, the yadA′/′yadA pseudogene is represented as yadA. Numbers correspond to nucleotide positions in Fig. 1.