species have the largest complement of extrachromosomal replicons of any characterized bacterium (9
). Genes required for in vitro
growth and/or the infectious cycle are located across both the chromosome and the plasmids, and loss of plasmids during in vitro
manipulation can complicate the analysis of the roles of borrelial genes in infection and other biological functions. Here we describe a high-throughput method for determining the plasmid content of B. burgdorferi
by utilizing multiplex PCR with replicon-specific primers followed by detection using Luminex xTAG technology.
The primer set utilized in this study was based on the B. burgdorferi
B31-MI genome sequence and thus permits identification of the plasmids present in that sequence. Two additional 32-kb replicons (cp32-2 and cp32-5) and an additional cp9 replicon (cp9-2) have been described for B31-derived clones (8
). Sequences corresponding to these plasmids were not detected in the B31-MI genome sequence, most likely because those replicons were not present in that B31 derivative. Limited sequences are available for cp32-2, cp32-5, and cp9-2 (10
), so in future iterations of the LPAA we will attempt to include the detection of these plasmids. The results we obtained with other Lyme disease Borrelia
strains verified that the LPAA in its current format would not be suitable for assessing plasmid content in strains other than B31; primers need to be chosen carefully for each B. burgdorferi
strain to obtain an accurate assessment of replicon content. Our analysis of the plasmid content of a large set of B. burgdorferi
clones provided precise statistics on the frequency of plasmid loss. The B31 clones were obtained after five in vitro
passages of the uncloned B31 strain, originally cultured from a tick from Shelter Island, NY (5
). The consistency of genomic sequencing results (18
) suggests that B31 represents the progeny of a single B. burgdorferi
lineage. It is unclear, however, to what degree the observed plasmid content differences and other genetic heterogeneity (such as OspB truncation [15
]) were present in the initial culture inoculum from the tick midgut. In the case of our study of B31 5A18NP1 transposon transformants, 5A18NP1 was a single clone isolated by colony formation after targeted mutagenesis of bbe02
with a suicide vector (29
). Therefore, the observed plasmid content variation occurred at some point following the initial outgrowth of the colony. In both the B31 clones and the STM library, the clones had undergone five in vitro
passages and ~10 doublings per passage, or an estimated 50 generations, prior to testing for plasmid content. Given that 1 or 2 plasmids had been lost in both populations, on average, plasmid loss must be a high-frequency event occurring at a rate of roughly 0.02 plasmid lost per cell-generation.
As observed previously (15
), plasmids vary widely in stability. The smallest plasmid, lp5, appears to derive from one or more fragments of lp21, along with sequences homologous to lp56. Its high sequence similarity to other plasmids makes effective PCR primer design challenging, so the presence of lp5 was not examined in most prior plasmid content analyses. In this report, lp5 was the plasmid that was lost with the highest frequency (39% of B31 clones and 43% of STM transformants); a high frequency of lp5 loss was also observed in a previous study by Elias et al. (15
). Loss of lp5 has no apparent effect on mouse infectivity (unpublished results), but its role in tick colonization or transmission has not been reported.
lp25 and lp28-1 are readily lost during in vitro
culture but are required for full infectivity of B. burgdorferi
). Consistent with this prior work, we found that lp25 was absent from 23% of the B31 clones and that lp28-1 was missing from 27% and 7% of the B31 clone and STM library populations, respectively. lp56, cp9, lp28-4, lp28-2, and lp21 were also lost at relatively high rates (Fig. ). The absence of these plasmids does not result in any apparent growth defect, so they do not appear to be required for in vitro
culture in BSK II medium. The remaining plasmids (the seven cp32 plasmids, lp28-3, lp36, and lp38) were each absent in small numbers of clones (ranging from 4 to 135 clones) in the STM library, which because of the large number of clones provided a more sensitive measure of low rates of plasmid loss.
These findings reinforce previously described results indicating that cp26 and lp54 are extremely stable (8
). cp32 plasmids are also retained in high-passage B31 clones (Fig. ; also see Fig. S3 in the supplemental material); B313 has also been reported to contain cp32-2 (8
), which was not assayed in our study. cp32s are thought to represent closely related prophages of the lysogenic bacteriophage
). Packaging and transduction of cp32 DNA have been confirmed (13
), but thus far, lytic infection has not been demonstrated reproducibly. The long-term retention of cp32s indicates that these plasmids are required for in vitro
growth, are unusually stable, or (potentially) are maintained in the bacterial population by transduction. Because the clones B312, B313, and B314 were all derived from the same parent culture, it is not known whether cp32-1, cp32-3, and cp32-4 are preferentially retained in comparison to the other cp32s; it is possible that a particular subset of plasmids was retained by chance in the high-passage culture (10
In a comprehensive article, Grimm et al. (22
) determined the effects of in vitro
passage and cryogenic storage on plasmid retention in two low-passage, infectious B. burgdorferi
B31 clones in which 21 plasmids were initially detected. The borreliae were cultured at either 35°C or 23°C and were examined at 3 passage (~27 generations) intervals for up to 25 passages. The plasmids that were absent following culture at 35°C were (in order of decreasing frequency) cp9, lp28-4, lp25, lp21, lp17, and lp28-1. These results are similar to those reported for our B31 clone population (Fig. ), although our clones were derived from a passage 5, uncloned B31 culture rather than from a single isogenic progenitor. We additionally observed a high rate of lp5 and lp56 loss and a higher frequency of lp28-1 loss (Fig. ).
The histogram in Fig. underscores the instability of B. burgdorferi plasmids during in vitro culture. Only 14% of the B31 clones and 40% of the STM library had retained all plasmids. The apparently lower rate of plasmid loss for the STM group was due in part to the lack of lp28-4 and lp56 in the 5A18NP1 parent clone (and hence the exclusion of these plasmids from the calculation) as well as to positive selection for lp25 via kanamycin resistance. The difference in plasmid loss between the two groups was not statistically significant. It is a positive sign that over 91% of the STM library clones contained lp25, lp28-1, lp36, and lp54, plasmids known to be required for the infectious cycle. Nevertheless, the results emphasize the need for close scrutiny of plasmid content.
The primer pairs used here were designed to maximize specificity for replicon identification. However, it would be possible to develop primer sets with different criteria, for example, to assay for virulence factors or to identify B. burgdorferi
species in clinical samples. A number of studies have indicated that different B. burgdorferi
species and strains produce distinct clinical manifestations (6
) and that coinfections with more than one strain can occur (17
). A Luminex-based assay may be useful in rapid strain identification.
Luminex-based approaches should prove useful in evaluating the plasmid loss and genetic content of Lyme disease Borrelia and other organisms with unstable genomes, as well as the relationship between these genetic changes and pathogenesis and other biological properties.