Department of Oncological Sciences,
University of Utah Medical School, Salt Lake City, Utah
84132,1 and The Institute for
Genome Science, Rockville, Maryland 208502
We have analyzed a panel of independent North American
isolates of the Lyme disease agent spirochete, Borrelia
burgdorferi (sensu stricto), for the presence of linear plasmids
with sequence similarities to the 12 linear plasmids present in the
B. burgdorferi type strain, isolate B31. The frequency of
similarities to probes from each of the 12 B31 plasmids varied
from 13 to 100% in the strain panel examined, and these similarities
usually reside on plasmids similar in size to the cognate B31
plasmid. Sequences similar to 5 of the 12 B31 plasmids were found in
all of the isolates examined, and >66% of the panel members
hybridized to probes from 4 other plasmids. Sequences similar to most
of the B. burgdorferi B31 plasmid-derived DNA probes used
were also found on linear plasmids in the related Eurasian Lyme agents
Borrelia garinii and Borrelia afzelii; however,
some of these plasmids had uniform but substantially different sizes
from their B. burgdorferi counterparts.
 |
INTRODUCTION |
The spirochetes that cause Lyme
disease, members of the Borrelia burgdorferi (sensu lato)
group of species, are known to harbor numerous extrachromosomal DNA
elements. For ease of discussion we will refer to these elements as
plasmids, although some may be present in all natural isolates, and
some may carry essential genes, so they should perhaps more correctly
be called "mini-chromosomes" (2). All natural isolates
examined carry multiple linear plasmids in the 5 to 110 kbp size range
and multiple circular plasmids in the 9 to 70 kbp range. Different
isolates have similar but nonidentical linear plasmid band patterns in
electrophoresis gels (e.g., those seen in references 3, 4,
5, and 33). Circular plasmid contents are
more difficult to display, but in the isolates that have been analyzed,
multiple, related plasmid types are always present (e.g., those seen in
references 9, 22, 27, 31, and
36). A number of studies have shown that plasmid
loss correlates with loss of infectivity in mice (15, 25,
34), so it is of interest to understand whether these
plasmids have uniform structures in the wild and to understand
the distribution of these plasmids among natural
isolates.
Only one Borrelia isolate, B. burgdorferi B31,
has been the subject of a comprehensive study that unequivocally
identified all of its plasmids. The analyzed culture of this strain,
B31 MI, carries 12 linear and 9 circular plasmids, and the nucleotide sequence of each is known (8, 12). Over 90% of the genes on
the characterized Lyme agent plasmids have no known homologs outside of
the Borrelia genus (8), and a number of these
genes encode outer surface proteins that are antigenic during infection of mammals (10, 13, 17, 21, 23, 29, 32, 35). We report here
an analysis of plasmids that are related to the 12 known linear B31
plasmids in a panel of Lyme disease borreliae.
| |
MATERIALS AND METHODS |
The B. burgdorferi strains used were previously
described (7); Borrelia garinii and
Borrelia afzelii strains and sources are listed (see Table
4). Contour-clamped homogeneous electric field (CHEF) electrophoresis
and Southern analysis (28) were performed as
previously described (6, 9). Southern probes were prepared
with [32P]dCTP (Amersham) and Pharmacia ReadyToGo random
priming kits. The DNA templates for random priming were either
whole Escherichia coli plasmid DNA clones (cloned DNA
fragments in plasmid pUC18 [12]) or DNA inserts from
such plasmids amplified by PCR using opposing primers outside of the
DNA insertion site. DNA transfer, hybridization, and wash conditions
were as previously described (6), except that membranes were
washed with two final 15-min posthybridization washes of 0.1×
SSC-0.1% sodium dodecyl sulfate (1× SSC is 0.15 M NaCl plus 0.015 M
sodium citrate) at 54°C for high-stringency analysis of B. burgdorferi (sensu stricto) strains; for other Borrelia
species the washes were done at 24°C to maximize hybridization there.
All membranes were only probed once.
| |
RESULTS AND DISCUSSION |
B31-like linear plasmids in North American B. burgdorferi (sensu stricto) isolates.
We first screened
linear plasmid DNA clones from the genome sequencing project (8,
12) to identify ones whose insert DNAs hybridized uniquely or
nearly so to only one of the strain B31 linear plasmids when used as
probes in Southern analyses; substantially more than half of the
plasmids tested were found to be unsuitable. Table 1
indicates where each of the chosen 31 DNA probes lies on the
linear plasmids. These DNAs were then used as probes in Southern
analyses (28) of CHEF electrophoresis gels of whole cellular
DNAs from a panel of 15 geographically diverse North American isolates
of B. burgdorferi (sensu stricto) (data not shown). Table 2
presents the results of probing this panel with the above probes, and
Table 3 summarizes the findings (the B31
linear plasmids are named according to their approximate DNA content in
kilobase pairs [8, 12]).
These data allow a number of conclusions to be drawn.
(i) Of the 465 probe-strain combinations tested, 322 (69%)
showed clear high-stringency hybridization to apparently linear
plasmids in members of the strain panel. Only 16 probe-strain
combinations (3.4%) showed clear hybridization to the chromosome as
well as to a plasmid, and two reacted only with the chromosome. (ii)
When their hybridization targets are present, probes from strain B31 linear plasmids nearly always hybridize with linear plasmids of similar, but often not identical, size in other isolates; only 47 of
the 322 reactive probe-strain combinations (15%) hybridized only to a
linear plasmid not within 15% of the size of the cognate B31 plasmid.
(iii) In most cases (for example, see lp38), all probes from a given
B31 MI plasmid hybridize to the same plasmid (identically sized DNAs)
within individual reactive panel members. It appears that ongoing
exchange of DNA sequences among linear plasmids of different size is
not extremely rapid, although it does occur. (iv) In 47 of 322 reactive
probe-strain combinations (15%), multiple linear plasmids clearly
hybridized to the probe, indicating the presence of probe-like
sequences on two or more linear plasmids in those isolates. This
suggests that the paralogies on plasmids in these strains are at least
partly different from those in the B31 plasmids (8). (v) B31
plasmid lp36 is unusual in that probes derived from it often hybridize
to targets of substantially different size from the B31 MI 36-kbp
plasmid. Although similarity to at least one of the five B31 lp36
probes used is present in all of the 15 isolates, in 10 of the 15 isolates tested they are present on linear plasmids in the 23 to 30 kbp
size range, and five isolates carry them on a 35- to 38-kbp plasmid.
(vi) Plasmids apparently cognate to a number of the B31 MI linear
plasmids are present in all or nearly all members of our strain panel.
Five of the B31 plasmids, lp28-1, lp28-3, lp28-4, lp36, and lp54,
appear to have similarly sized counterparts in all 15 members of the panel (the lp36 counterparts are more variable in size than the other
four [above]); lp25 and lp28-2 have counterparts in 14 of the 15 isolates; and lp17 and lp38 have counterparts in 10 of the isolates.
Plasmids cognate to the lp5, lp21, and lp56 probes are less common,
being present in 3, 2, and 3 of the 15 isolates, respectively. Our
definition of cognate or corresponding plasmid simply means a plasmid
that hybridizes to the B31 plasmid probe; it does not imply that the
plasmids must have completely similar structures.
The presence of corresponding plasmids in most natural isolates tested
strongly suggests that they are important in the natural life of
B. burgdorferi. Our methods could underestimate the plasmids present for the following reasons: some linear plasmids tend to be lost
quite rapidly with passage in culture (15, 25, 34), our
rather stringent hybridization conditions would cause us to miss
divergent but orthologous sequences, and a probe's target could be
missing from an otherwise cognate plasmid. We attempted to minimize the
first problem by using low-passage cultures; 14 of the 15 cultures used
had been passaged fewer than 12 times since isolation. In only a few of
the tested strains did ethidium bromide-stained electrophoresis gels
show a clear linear plasmid band that did not hybridize to the probes
used; these were ~5-kbp apparently linear plasmids in isolates 21579, 21721, 28534, and CA-3-82, which did not react with the B31 lp5 probe.
DNAs that were <7 kbp were not analyzed with the other probes, so it
is not known if these plasmids are related to other probes used in this
study. The above data do not imply that no additional types of linear
plasmids are present in B. burgdorferi, but it does suggest
that there may not be a large number of other, as yet unknown, common
linear plasmid types.
Linear plasmids in non-burgdorferi Lyme agent
spirochetes.
We also analyzed a geographically diverse panel
of 12 B. garinii and 8 B. afzelii isolates with a
subset of the probes listed in Table 1. The results of these
experiments are given in Table 4. A
majority (73%) of the B. garinii and B. afzelii
probe-strain combinations showed some sequence similarity to B31 on
apparently linear plasmids. In some cases, the plasmids may be quite
similar in the three species. For example, B31 plasmid lp54 clone CM64 hybridizes to 53- to 56-kbp plasmids in all three species. This agrees
with the observations that the ospAB and P27 genes have been
found on a plasmid of this size in members of these species that have
been analyzed (5, 18, 23, 24). The hybridization target of
lp17 probe CL47 is universally present on 21- to 28-kbp plasmids in
B. garinii and B. afzelii, but is present in less than half of the B. burgdorferi isolates we examined. In
other cases, the sizes of the plasmids harboring the hybridization
targets in the other species are either variable (e.g., lp28-4
and lp28-2) or are systematically very different from B. burgdorferi. For example, the lp28-3 target is present on a 54-kbp
plasmid in all of the B. garinii and B. afzelii
isolates tested, the lp36 target is usually present on a 21- to 23-kbp
plasmid in B. garinii but is rarely present in B. afzelii, and the lp38 probe DH46 target is usually present on a
22-kbp plasmid in B. garinii and a 25-kbp plasmid in
B. afzelii. A large fraction of B31 plasmid sequences have
similar sequences on plasmids in these other species; however, there
are substantial differences in plasmid structure among the three
species.
Plasmid lp56 appears to have formed by the relatively recent
integration of a member of the 32-kbp circular plasmid (cp32) family
into a 20- to 25-kbp linear plasmid (8). The probe from the
non-cp32-like portion of lp56 hybridized only to ~55-kbp linear plasmids in our panel of B. burgdorferi isolates,
suggesting that the putative linear progenitor of lp56 is not
common in this species. However, most B. garinii and
B. afzelii isolates carry ~20- and ~25-kbp linear
plasmids, respectively, that do hybridize with the EK58 probe. It thus
appears possible that the linear progenitor of B. burgdorferi lp56 (without the integrated cp32) could be one of
these more common 20- or 25-kbp B. garinii or B. afzelii plasmids.
Conclusions.
This is the first study to systematically analyze
the linear plasmid contents of Borrelia isolates from the
perspective gained from knowledge of the complete plasmid content of
B. burgdorferi B31 MI. We find that at least one of the
sequences tested from 5 of the 12 B31 linear plasmids are present in
all 15 of the B. burgdorferi (sensu stricto) isolates
examined, and at least one of the sequences from two additional
plasmids was present in 14 of 15 isolates. Two B31 linear plasmids had
relatives in 10 of the 15 isolates, and only three plasmids appear to
have cognates in fewer than 25% of the isolates examined. Previous,
single-probe studies on B31 lp17, lp28-1, lp25, lp38, and lp54
generally agree with the above conclusions (1, 3, 5, 14, 16,
19-21, 24, 33). Circular plasmids similar to B31 cp9, cp26, and
multiple cp32s have also been found to be present in nearly all
isolates that have been carefully examined (9, 10, 11, 15, 22, 26,
27, 30, 31). In summary, the B31 linear plasmid sequences are
usually present in other B. burgdorferi isolates, and when present they are highly likely to be located on a plasmid of similar size. Thus, there appears to be a substantial uniformity of linear plasmid sequence content among various independent B. burgdorferi isolates. Most probes from the B31 linear plasmids
also hybridized with linear plasmids from B. garinii and
B. afzelii, but in a number of cases they have substantially
different sizes from the cognate B31 plasmid. Such systematic
differences suggest that there may not be free exchange of these
plasmids between species.
How similar are the overall structures of corresponding plasmids of
similar size in different isolates? In general, we do not yet know the
answer to this question; however, the lp54 plasmids from the four
natural Lyme agent isolates examined have similar gene orders and
restriction maps (18; R. van Vugt and S. Casjens, unpublished observations), and the circular cp9's and
cp26's have been shown to each have similar structures in
different isolates (11, 12, 31). We have found that multiple
probes from individual B31 linear plasmids nearly always hybridize to
the same plasmid in other isolates, suggesting that other
plasmids may also have generally conserved genetic structures.
Curiously, in spite of this evidence of uniformity, examination of
linear plasmid sequences has shown considerable evidence of recent,
rather massive genetic instability on the strain B31 linear plasmids in
the form of many past duplicative rearrangements (8). In
addition, observations made here that unique B31 linear plasmid-derived
probes sometimes hybridize to multiple plasmids or to plasmids of
different sizes in other strains supports the idea that the genetic
information on these plasmids is not completely constant (unlike the
B. burgdorferi chromosome, which appears to be very stable
with the exception of the extreme right few kilobase pairs [5,
8]). How can the apparent overall uniformity in plasmid size
observed here exist in the face of evidence for apparently frequent
plasmid rearrangement events? A more detailed analysis of plasmids
present in other strains will be required to resolve this paradox.
The work was supported by a grant from the G. Harold and Leila Y. Mathers Charitable Foundation.
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Abstract
Introduction
Present address: Slotervaart Ziekenhuis, Afdeling Bacteriologie,
1066 EC Amsterdam, The Netherlands.
