Re: Borrelia genetics

a_weisman_at_yahoo.com
Date: 02/27/05 

Date: 27 Feb 2005 06:36:00 -0800

eugeneshapiroisapig wrote:
> Hey guys, I could spend a lot of time at the library looking this up
on
> my own, or I could save time and ask here. What I am interested in
now
> are basic facts about the molecular biology of Borrelia species other
> than Borellia burgdorferi. By which I mean hermsii, etc., you know
the
> stuff that causes relapsing fever and things like this. In
particular,
> I am interested now in the genetic organization of these bugs.
> Burgdorferi has I believe 9 circular plasmids, 12 linear plasmids,
and
> a linear chromosome. What about other borrelia? Also interested in
> worldwide distribution of hermsii. Any other information about
> comparative cell biology or evolution would also be appreciated.
Thanks.

Some more material you might be interested in, again, better to read it
at the site (link provided) so you can view tables, images,
illustrations and access references and supplementary materials:

Expanded Diversity among Californian Borrelia Isolates and Description
of Borrelia bissettii sp. nov. (Formerly Borrelia Grou..
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=105228

J Clin Microbiol. 1998 December; 36(12): 3497-3504.
Copyright © 1998, American Society for Microbiology

Expanded Diversity among Californian Borrelia Isolates and Description
of Borrelia bissettii sp. nov. (Formerly Borrelia Group DN127)
D. Postic,1* N. Marti Ras,1 R. S. Lane,2 M. Hendson,2 and G. Baranton1

Unité de Bactériologie Moléculaire et Médicale, Institut Pasteur,
75724 Paris Cedex 15, France,1 and Department of Environmental Science,
Policy and Management, Division of Insect Biology, University of
California, Berkeley, Berkeley, California 947202

*Corresponding author. Mailing address: Unité de Bactériologie
Moléculaire et Médicale, Institut Pasteur, 28 rue du Docteur Roux,
75724 Paris Cedex 15, France. Phone: 33 1 45 68 83 37. Fax: 33 1 40 61
30 01. E-mail: dpostic@pasteur.fr.

Received July 2, 1998; Revisions requested August 27, 1998; Accepted
September 15, 1998.

This article has been cited by other articles in PMC.

ABSTRACT:

Up to now, the only species in the complex Borrelia burgdorferi sensu
lato known to cause Lyme borreliosis in the United States has been B.
burgdorferi sensu stricto. However, some atypical strains closely
related to the previously designated genomic group DN127 have been
isolated in the United States, mostly in California. To explore the
diversity of B. burgdorferi sensu lato group DN127, we analyzed the
nucleotide sequences of the rrf-rrl intergenic spacer regions from 19
atypical strains (18 from California and one from New York) and 13
North American B. burgdorferi sensu stricto strains (6 from
California). The spacer region sequences from the entire B. burgdorferi
sensu lato complex available in data banks were used for comparison.
Phylogenetic analysis of sequences shows that the main species of the
B. burgdorferi sensu lato complex (B. afzelii, B. garinii, B.
andersonii, B. japonica, B. burgdorferi sensu stricto, B. valaisiana,
and B. lusitaniae) each form a coherent cluster. A heterogeneous group
comprising strains belonging to the previously designated group DN127
clustered separately from B. burgdorferi sensu stricto. Within this
cluster, the deep branches expressing the distances between the rrf-rrl
sequences reflect a high level of divergence. This unexpected diversity
contrasts with the monomorphism exhibited by B. burgdorferi sensu
stricto. To clarify the taxonomic status of this highly heterogeneous
group, analysis of the rrs sequences of selected strains chosen from
deeply separated branches was performed. The results show that these
strains significantly diverge at a level that is compatible with
several distinct genomic groups. We conclude that the taxonomy and
phylogeny of North American B. burgdorferi sensu lato should be
reevaluated. For now, we propose that the genomic group DN127 should be
referred to as a new species, B. bissettii sp. nov., and that other
related but distinct strains, which require further characterization,
be referred to as Borrelia spp.

In Eurasia, seven species of the complex Borrelia burgdorferi sensu
lato have been reported. Only three of these species are associated
with Lyme borreliosis. It has also been shown that each pathogenic
species is associated predominantly with a given clinical presentation;
Borrelia burgdorferi sensu stricto is associated with arthritis, B.
garinii is associated with neuroborreliosis, and B. afzelii is
associated with late cutaneous symptoms (2, 39). Up to now, B.
burgdorferi sensu stricto is the only species associated with Lyme
borreliosis in North America. However, two other B. burgdorferi sensu
lato genospecies coexist in the United States, B. andersonii (22) and
the genomic group DN127 (3, 32). B. andersonii seems to be restricted
to a limited ecosystem involving cottontail rabbits and Ixodes dentatus
ticks. In contrast, the genomic group DN127 appears to be involved in
several enzootic transmission cycles (6, 29). A recent study
demonstrated substantial genetic heterogeneity among Californian and
other American strains (24). We took advantage of the unique structure
of ribosomal genes in B. burgdorferi sensu lato to analyze the
polymorphism of some strains isolated in California. A single copy of
the rrs gene is separated by a large spacer (rrs-rrl; 3,000 to 5,000
bp) from two tandemly duplicated copies of rrl and rrf genes (13, 36).
These two copies are separated by a small spacer, rrf-rrl, which is
approximately 250 bp long. The genetic heterogeneity of the group DN127
was first evidenced by analysis of the restriction patterns of the
rrf-rrl spacer (32). However, the results of DNA-DNA hybridization on a
limited number of strains (32) allowed us to place them in a single
genomic group. To clarify the genetic relationships between diverse
North American strains, 20 atypical strains were compared with 13 B.
burgdorferi sensu stricto strains. Identification procedures involved
restriction polymorphism and sequencing studies of both the variable
rrf-rrl spacer and the conserved rrs gene. Sequences of the rrf-rrl
spacer and the rrs gene were used in a phylogenetic analysis. Some
Californian strains are closely related to the genomic group DN127, for
which we propose the name of B. bissettii sp. nov. Other atypical
strains which do not fall into this group are designated merely as
Borrelia spp. in this study. The latter strains cannot be assigned to
specific genomic groups until more isolates representative of each
group are available for further characterization.

MATERIALS AND METHODS:

Bacterial strains and DNA preparation. The designations and origins of
the Borrelia strains used in this study are given in Table 1. The
uncloned strains were grown in BSK II medium at 30°C. DNA was
extracted by using the Dynabeads DNA direct kit (Dynal), a method based
on DNA separation by biomagnetic beads as previously described (17).
DNA samples were stored at -20°C until use for PCRs.

Analysis of restriction patterns of rrf-rrl spacer and sequencing. The
restriction pattern analysis of amplified rrf-rrl spacer was performed
by using primers 1 and 2 as described previously (32). MseI and DraI
restriction patterns were used to compare the strains. The rrf-rrl
spacer was sequenced by a solid-phase approach, using the Cy5-AutoRead
sequencing kit with an ALF express automatic sequencer (Pharmacia)
(17). To amplify the rrf-rrl spacer, we used primers A
(5'-ATTACCCGTATCTTTGGC-3') and D (5'-TCAATAAATGTTTGCTTCTC-3'),
with one of the two primers biotinylated at the 5' end. The
biotinylated strand was then immobilized through the interaction
between biotin and streptavidin by using the Dynabeads M-280
streptavidin kit (Dynal) according to the manufacturer's
instructions. Sequencing was performed by using the sequencing primers
INS1 (5'-GAAAAGAGGAAACACCTGTT-3') in the rrf gene and INS4
(5'-AGCTCCTAGGCATTCACCAT-3') at the 5' end of the rrl gene.

rrs sequencing. Amplification and sequencing of the rrs gene were done
as previously described (17).

Sequence alignments and phylogenetic analysis. Sequences were aligned
both manually on VSM software V.2.0 written by B. Lafay and R. Christen
(33) as described recently (10) and by using the multisequence
alignment program Clustal V (10). Phylogenetic trees were constructed
with distance matrix data (calculated by the method of Jukes of Cantor
[14]) and both the neighbor-joining (NJ) method (34) and the unweighted
pair group with mathematical average (UPGMA) (37) methods in MEGA
software (15). A parsimony method in MEGA was used to analyze the
rrf-rrl intergenic spacer sequences of select strains.

Characterization of the rrs-rrl spacer. The size of the rrs-rrl spacer
was determined after amplification by using primer S15
(5'-GGGCCTTGTACACACCGCCC-3') at the 3' end of rrs and primer INS4
as given above. The PCR mixture (50 µl) contained 10 ng of DNA in 5
µl, 50 mM Tris-HCl, 1.5 mM MgCl2, 20 mM NH4SO4, 200 µM each of the
four deoxynucleoside triphosphates, 10 pmol of each primer, and 0.45 U
of Hot Tub DNA polymerase (Amersham Life Science). The PCR was carried
out for 30 cycles with an amplification profile of denaturation at
93°C for 15 s and then simultaneous annealing and extension at 60°C
for 8 min, with a final extension step at 60°C for 10 min.

PCR products (10 µl) were digested with 5 U of HinfI (Biolabs) in a
total volume of 20 µl. Digested fragments were analyzed by
electrophoresis on a 1.2% agarose gel stained with ethidium bromide.

Nucleotide sequence accession numbers. The nucleotide sequences of
rrf-rrl spacer regions or rrs genes from B. burgdorferi sensu lato
isolates have been deposited in EMBL data bank and assigned accession
nos. AJ006359 to AJ006375, AJ006503 to AJ006512, and AJ224130 to
AJ224141 (see Table 1).

 RESULTS

Individualization of B. bissettii deduced from the analysis of the
rrf-rrl spacer. Analysis of MseI restriction patterns of amplification
products of the spacer between the two tandem copies of the rrl-rrf
ribosomal genes from North American strains revealed 11 different
patterns (Table 2). These patterns were not identical to any of the
patterns recorded previously for B. burgdorferi sensu lato species and
genomic groups. However, they were very similar to the pattern of
strains belonging to B. burgdorferi sensu stricto (Fig. 1). The
analysis of patterns obtained after restriction by DraI confirmed this
heterogeneity (Table 2).

Some strains (CA27, CA372, CA378, and CA394) exhibited exactly the same
pattern as strain DN127, the type strain of the previous genomic group
DN127.

The rrf-rrl spacer region (32) of strains tested ranged from 216 to 257
bp. The percent identity in pairwise alignments of sequences from
Borrelia spp. strains ranged from 85.3 to 100 (data not shown).
However, all strains with identical MseI or DraI patterns exhibited
100% sequence identity, except strain CA404 which differed by one
nucleotide from strains CA31, CA443, and CA446. To compare the
polymorphism, the spacer regions from 10 American strains previously
identified as B. burgdorferi sensu stricto were sequenced. In contrast
with the extreme diversity in Borrelia spp., considerable homogeneity
characterized the sequences of B. burgdorferi sensu stricto strains, as
nucleotide substitutions or deletions occurred in only five positions.

The NJ and UPGMA distance methods were used to construct phylogenetic
trees from sequences obtained in this study. An example of a tree drawn
by the UPGMA distance method is shown in Fig. 2. Each previous species,
namely, B. garinii, B. afzelii, B. valaisiana, B. lusitaniae, B.
tanukii, B. turdi, B. andersonii, and B. japonica, clustered
separately. One large and heterogeneous cluster of 32 sequences
comprises B. burgdorferi sensu stricto, the strains identified as
belonging to B. bissettii sp. nov. (formerly the genomic group DN127,
DN127, CA128, CA55, and 25015), and all strains with atypical MseI and
DraI patterns (Borrelia spp.). Within this cluster, all strains
belonging to B. burgdorferi sensu stricto are closely related, which
contrasts with the strains of Borrelia spp. that are scattered on
several branches. Among the atypical strains, two strains (CA19 and
CA423), despite slight differences in their MseI patterns, fell into
the B. burgdorferi sensu stricto cluster. Given the large diversity
exhibited by the Borrelia spp., segregation of some strains did not
correlate precisely with different trees drawn by phenetic (Fig. 2) or
cladistic methods (data not shown). For example, the placement of
strain CA2 was uncertain, as was the placement of strains CA29 and CA8,
because they constituted a separate cluster comprising four distinct
branches together with Californian strains CA404, CA443, CA446, and
CA31 and B. andersonii in the tree drawn with the NJ distance method
(data not shown).

However, the branch consisting of strains previously placed in the
group DN127 (DN127, CA55, CA128, and 25015), and six additional strains
(CA27, CA370, CA372, CA378, CA394, and CA395) was constant,
irrespective of the method used to construct the trees.

New genomic groups deduced from rrs sequences. To provide an
alternative assessment of phylogenetic relationships between such
divergent strains, we sequenced the entire rrs gene from strains
representing each of the main branches in the rrf-rrl trees. A
phylogenetic tree showing the result of the NJ analysis of sequences is
shown in Fig. 3. The assignment of strain CA19 in B. burgdorferi sensu
stricto was confirmed. The results showed that strains of Borrelia spp.
significantly diverge at a level compatible with distinct genomic
groups. Strains CA31 and CA446 constituted one group. Strains CA8 and
CA29 comprised another group. Strains CA2 and CA28 were located on two
different branches according to the rrf-rrl sequence. They clustered
together by rrs sequence, but their genetic distance is relatively
large. The status of strain CA2 was not clear on the basis of DNA-DNA
hybridization data (32). However, our present results suggest that
strains CA2 and CA28 should constitute a new genomic group. Strains
CA128, CA55, and 25015 segregated with strain DN127. Whether strain
CA13 belong to the latter genomic group remains unknown. The clustering
of strains of Borrelia spp. was consistent with that obtained by the
UPGMA analysis (data not shown).

Polymorphism of the rrs-rrl spacer. It has been shown previously that
the size of the spacer between the rrs gene and the first copy of the
rrl gene varied among different Borrelia species (28). The size of the
rrs-rrl spacer allowed Borrelia species to be distinguished by
decreasing size order from 5,000 bp for B. afzelii to 3,000 bp for B.
burgdorferi sensu stricto and B. andersonii (Fig. 4). As shown in Fig.
4, strains evaluated in this work exhibited PCR products of two
different sizes. All strains assigned to B. bissettii sp. nov. (DN127,
CA55, 25015, CA27, CA394, CA395, CA370, CA372, and CA378), as well as
four strains of Borrelia spp. (CA31, CA404, CA443, and CA446) exhibited
a rrs-rrl spacer with an identical size of approximately 500 bp larger
than that of B. burgdorferi sensu stricto. Other strains of Borrelia
spp. had a rrs-rrl spacer whose size was the same as that of B.
burgdorferi sensu stricto. The analysis of the HinfI restriction
pattern of the rrs-rrl spacer PCR product has been proposed for typing
of B. burgdorferi sensu stricto (18, 19). As described earlier (18), we
also found two DNA fragment patterns among strains of B. burgdorferi
sensu stricto. In contrast to these findings, a strong polymorphism was
observed among atypical strains (Fig. 5). Ten distinctive patterns
recorded from 17 atypical strains are shown in Fig. 5. Notably, the
analyses of polymorphism of both the large rrs-rrl spacer and the small
rrf-rrl spacer produced comparable groupings of strains.

 DISCUSSION

Since the first description of B. burgdorferi in 1982 (7), it has been
assumed that strains in the United States were more homogeneous than
the European strains (8, 9). However, an increasing number of atypical
strains have been recognized in the United States, particularly in the
1990s (1, 5, 9, 12, 16, 21, 24, 35, 41, 42). Some of these strains were
identified as belonging to the species B. andersonii (22), whereas
others constituted a new genomic group called group DN127 (3, 32).
Considerable phenotypic heterogeneity was found among strains described
from California and Colorado (26, 35), and substantial genetic
diversity was reported among a large number of North American strains
(24) on the basis of genomic macrorestriction analysis and ospA and rrl
gene sequencing. Our study emphasizes that the genetic diversity among
American strains is much greater than previously thought.

Phylogenetic analyses of rrs gene sequences have often been used to
evaluate the taxonomic relatedness of B. burgdorferi sensu lato strains
(11, 17, 20, 40). The results of these analyses correlated well with
data from DNA-DNA hybridizations (32). The rrs gene sequence analysis
of atypical strains confirmed the foregoing results and revealed at
least four groups which appear to represent heretofore undescribed
genospecies. CA19 belongs to B. burgdorferi sensu stricto. Three new
genomic groups were CA29-CA8, CA2-CA28, and CA31-CA446, respectively.
The taxonomic position of strain CA13 remains unclear. A polymorphism
was observed in the rrf-rrl restriction patterns of the strains within
the genomic group DN127. Also, some differences were previously
reported for the physical maps of strains DN127 and CA55, which were
classified in two separate groups (8). However, data originated from
the single gene locus hbb (38), as well as information acquired by
multilocus enzyme electrophoresis, involving the whole genome (4) were
consistent with data from DNA-DNA hybridizations showing that DN127 and
CA55 do belong to the same species (32). Moreover, the rrs sequences of
these two strains are 100% identical. To clarify the taxonomic status
of B. burgdorferi sensu lato in the United States, we propose the name
B. bissettii sp. nov. for the genomic group DN127 in honor of Marjorie
L. Bissett, who with her coworker Warren Hill, first described a member
of this group in 1987 (5). We refrain from naming the other genomic
groups until hybridization data are available for genetic
characterization. Instead, we lump them here as Borrelia spp. The
noncoding region between the two copies of rrf and rrl genes was shown
previously to reflect the taxonomic status of strains (32). The
phylogenetic study of this region showed that strains from each B.
burgdorferi sensu lato species clustered as expected taxonomically, and
each cluster clearly diverged from others. In contrast with the high
conservation of sequences within the B. burgdorferi sensu stricto
cluster, rrf-rrl sequences from Borrelia strains exhibited an
unexpected broad diversity. This region is not constrained genetically,
so the number of mutational events found should reflect the relative
antiquity of the members of this bacterial complex. Californian strains
were found scattered in different clusters. Within each cluster, the
deep branches expressing the distances between the rrf-rrl sequences
reflect a high divergence level. According to previous studies (23,
32), these results strongly suggest that these strains should
constitute new genomic groups. Despite some discrepancies between the
different trees, all are consistent with the placement of strains
belonging to B. bissettii sp. nov. in a distinct cluster. Within this
cluster, only strain 25015 was located on a separate branch.

In opposition with what is usually thought, more than two B.
burgdorferi sensu lato species (B. burgdorferi sensu stricto and B.
andersonii) seem to occur in the United States. B. burgdorferi sensu
stricto is transmitted primarily by Ixodes scapularis and Ixodes
pacificus ticks, whereas the new genomic groups described here are
associated with these two ticks plus Ixodes neotomae (now Ixodes
spinipalpis [27]) and some of its rodent hosts. Moreover, I.
spinipalpis also can harbor Borrelia from other genomic groups, since
strains CA2 and CA13 represent two distinct groups. Thus, there are a
variety of ticks and reservoir host for B. burgdorferi sensu lato in
the United States. It is not known whether B. bissettii sp. nov. and
the other novel genomic groups can infect humans. All the strains used
in this study were isolated from ticks or small mammals. As stated by
Oliver (29), clinical manifestations of Lyme borreliosis in the
southern United States are mild and some cases may be asymptomatic.
Thus, the roles of B. bissettii sp. nov. and Borrelia spp. in producing
Lyme borreliosis remain to be demonstrated. In addition, Picken et al.
(30, 31) recently described human strains from Slovenia related to
strain 25015, which belongs to B. bissettii sp. nov. on the basis of
large restriction fragment pattern, protein, and plasmid profile
analyses. If the strains from Slovenia are true members of B. bissettii
sp. nov., the pathogenicity of this species should be evaluated there
as well as in the United States. In Europe, three species are known to
be pathogenic for humans; in the United States, all strains isolated
from humans so far belong to B. burgdorferi sensu stricto. This could
mean that I. spinipalpis does not transmit Borrelia to humans or that
these new genomic groups are nonpathogenic for humans. This latter
hypothesis seems more likely, since I. pacificus and I. scapularis
frequently bite humans and occasionally harbor such strains. Moreover,
I. spinipalpis primarily infests rodents and lagomorphs and rarely
attaches to humans (25). However, despite the great number of strains
isolated from I. scapularis in the United States, 25015 is the only
strain recovered from this tick that belongs to a species other than B.
burgdorferi sensu stricto. Also, this strain is genetically distant
from other strains within B. bissettii sp. nov. This fact could reflect
genetic adaptation to an unusual vector. The heterogeneity encountered
among B. burgdorferi sensu lato in the United States might be compared
to that described in Europe and Asia. On the latter two continents, B.
garinii comprises a more heterogenous collection than B. afzelii and B.
burgdorferi sensu stricto. Aside from the three pathogenic species,
nonpathogenic species, such as B. valaisiana, B. lusitaniae, or B.
japonica, coexist. More data are needed to understand the significance
of the diversity of B. burgdorferi sensu lato and their role in human
Lyme borreliosis.

  ACKNOWLEDGMENTS

We thank T. G. Schwan and R. N. Brown for supplying some of the
strains, I. Saint Girons for critical reading of the manuscript, and E.
Bellenger for technical assistance.

We thank the Pasteur Institute for supporting this work. Also, many of
the Californian strains were acquired during ecologic studies supported
in part by funding to R.S.L. from the U.S. National Institutes of
Health (grant AI22501) and the Centers for Disease Control and
Prevention (cooperative agreement U50/CCU906594).

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  Figures and Tables

 FIG. 1
Mse I restriction polymorphism of the amplified rrf-rrl spacer from
Californian strains. DNA was electrophoresed on a 16% acrylamide gel,
stained with ethidium bromide, and UV illuminated. The species
assignment of strains is given in Table 1 . (more ...)

 FIG. 2
Phylogenetic tree based on a comparison of the rrf-rrl sequences of B.
burgdorferi sensu lato. The branching pattern was generated by the
UPGMA method. The bar represents 1% divergence.

 FIG. 3
Phylogenetic tree based on a comparison of the rrs sequences of B.
burgdorferi sensu lato. The branching pattern was generated by the NJ
method. The bar represents 0.1% divergence.

 FIG. 4
PCR products of rrs-rrl spacer from B. burgdorferi sensu lato strains.
The species assignment of strains is given in Table 1 . Amplification
was carried out by using the S15-INS4 primer set. DNAs were
electrophoresed on a 0.6% agarose gel, stained (more ...)

 FIG. 5
Restriction patterns of Californian strains. The species assignment of
strains is indicated in Table 1 . DNAs from amplified rrs-rrl spacers
were digested by Hin fI. DNAs were electrophoresed on a 1.2% agarose
gel, stained with ethidium bromide, (more ...)

 TABLE 1
B. burgdorferi sensu lato isolates evaluated in this study

 TABLE 2
Mse I and Dra I restriction fragments of amplified rrf-rrl spacer

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