Complete DNA Sequence and Detailed Analysis of the Yersinia pestis KIM5 Plasmid Encoding Murine Toxin and Capsular Antigen

Abstract

Yersinia pestis, the causative agent of plague, harbors at least three plasmids necessary for full virulence of the organism, two of which are species specific. One of the Y. pestis-specific plasmids, pMT1, is thought to promote deep tissue invasion, resulting in more acute onset of symptoms and death. We determined the entire nucleotide sequence of Y. pestis KIM5 pMT1 and identified potential open reading frames (ORFs) encoded by the 100,990-bp molecule. Based on codon usage for known yersinial genes, homology with known proteins in the databases, and potential ribosome binding sites, we determined that 115 of the potential ORFs which we considered could encode polypeptides in Y. pestis. Five of these ORFs were genes previously identified as being necessary for production of the classic virulence factors, murine toxin (MT), and the fraction 1 (F1) capsule antigen. The regions of pMT1 encoding MT and F1 were surrounded by remnants of multiple transposition events and bacteriophage, respectively, suggesting horizontal gene transfer of these virulence factors. We identified seven new potential virulence factors that might interact with the mammalian host or flea vector. Forty-three of the remaining 115 putative ORFs did not display any significant homology with proteins in the current databases. Furthermore, DNA sequence analysis allowed the determination of the putative replication and partitioning regions of pMT1. We identified a single 2,450-bp region within pMT1 that could function as the origin of replication, including a RepA-like protein similar to RepFIB, RepHI1B, and P1 and P7 replicons. Plasmid partitioning function was located ca. 36 kb from the putative origin of replication and was most similar to the parABS bacteriophage P1 and P7 system. Y. pestis pMT1 encoded potential genes with a high degree of similarity to a wide variety of organisms, plasmids, and bacteriophage. Accordingly, our analysis of the pMT1 DNA sequence emphasized the mosaic nature of this large bacterial virulence plasmid and provided implications as to its evolution.

Characterization of IS1541-like elements in Yersinia enterocolitica and Yersinia pseudotuberculosis

“…As expected from previous studies [3,6,7], IS1541A, IS1541B, IS1541C and IS1541D were inserted in an AT-rich region but none of these four elements were located within an ORF. Sequence analysis of the IS £anking regions revealed that (i) IS1541A was present downstream of an ORF highly homologous (90% sequence identity) with the Serratia marcescens gene himA encoding the integration host factor K subunit [8], (ii) IS1541C was found 4 bp downstream of a DNA segment displaying 86% identity with IS1328, an element previously described in another O:8 strain, WA [9].…”

Antigen engineering can play a critical role in the protective immunity elicited by Yersinia pestis DNA vaccines

“…Sequence analysis for the Y. pestis gene coding for V protein, lcrV, as part of the 70-kb low calcium response (lcr) plasmid, showed a "G+C" content of only 37.71%. Because codon usage is directly linked to preferred tRNA usage in a given cell type, it is conceivable that codon optimization for LcrV gene is important for its high level expression of this bacterial protein in mammalian cells [38]. However, in a previous report, codon optimization for the V gene based on murine codon preference did not make any difference in a DNA vaccine vector pVAX1-CMV-TE when it was tested in mice and the peak anti-V antibody titers were measured [22].…”

Chromosome dynamics in bacteria: triggering replication at opposite location and segregation in opposite direction

“…To determine the cellular localization of ori, we engineered a fluorescent tag to be inserted near ori using the Yersinia pestis parS(pMT1) chromosomal sequence and its corresponding gene-encoding ParB(pMT1) fluorescently tagged [32]. The parS-ParB(pMT1) system from Yersinia has been previously used in C. crescentus and shown not to interfere with the activity of the native C. crescentus ParABS partitioning system [24].…”