The pla gene, encoding plasminogen activator, is not specific to Yersinia pestis
© Hänsch et al. 2015
Received: 9 July 2015
Accepted: 24 September 2015
Published: 5 October 2015
Here we present evidence to show that the pla gene, previously thought to be specific to Yersinia pestis, occurs in some strains of Citrobacter koseri and Escherichia coli. This means that detection of this gene on its own can no longer be taken as evidence of detection of Y. pestis.
Molecular assays aimed at detecting traces of the etiological agent of plague, Yersinia pestis, have focused primarily—and sometimes solely—on the plasminogen activator/coagulase (pla) gene [1, 2]. This gene is located on the pPCP1 plasmid and has been considered the target of choice for plague detection due to its assumed specificity to Y. pestis and its occurrence in multiple copies [3–5]. However, a recent paper about the amplification of the pla gene from tissues from European rats has raised doubts over whether this gene is indeed specific to Y. pestis . We can now confirm this suspicion.
We screened archaeological samples from Italy (6th, 14th and 17th centuries CE), amplifying a 70-base-pair fragment from the pla gene. Full protocols are described in a previous publication , but in brief we performed the work in a dedicated clean laboratory, with physically separated areas for extraction and amplification, following the most stringent criteria for ancient DNA analysis, such as the use of mock extractions and PCR blanks. We used the previously described pla primer pair (Forward primer: GACTGGGTTCGGGCACATGC—Reverse primer: CGGATGTCTTCTCACGGA). Cycling conditions started with an initial activation step at 95 °C for 15 min. This was followed by 50 cycles at 94 °C for 30 s, an assay specific annealing temperature at 60 °C for 30 s, and 72 °C for 1 min, ending with a final elongation step at 72 °C for 10 min. Final cooling was carried out at 8 °C until analysis.
The presence of pla sequences from outside Y. pestis, each derived from a distinct geographical or taxonomic setting, confirms beyond doubt that this gene can no longer be considered specific to Y. pestis. Although there appear to be some potentially informative sequence differences between the pla sequences from Y. pestis and those from other taxa, these findings call into question any results—whether in contemporary diagnostic microbiology or in an ancient DNA setting—that rely on detection of PCR products from this gene alone. Instead, as many researchers in the field already recognise, it is important to obtain sequences from PCR products and detection or identification of Y. pestis should rely on sequences from at least two independent molecular targets. More generally, our observations call into question the wisdom of relying on genes from mobile elements as species-specific markers, given the likelihood that such sequences are able to move from one taxon to another. Interestingly, the roles of the sequence differences between the pla genes, some of which are non-synonymous, in the function and evolution of the pla gene product remain to be determined.
EC and GC performed laboratory experiments. SH, GC and MJP performed bioinformatics analyses. RB, GG, NCS and BB conceived of the study, and participated in its design and coordination and helped to draft the manuscript. EC, BB and MJP wrote the manuscript. All authors read and approved the final manuscript.
Compliance with ethical guidelines
Competing interests The authors declare that they have no competing interests.
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