Supporting Information Margos et al. 10.1073/pnas.0800323105 SI Materials and Methods DNA Extraction. DNA of tick-derived samples and cultures was either prepared by alkaline hydrolysis (1) or extracted by using the DNeasy tissue and blood kit (Qiagen) as described previously (2). Purified DNA of cultured isolates was diluted to a concen- tration of 10 pg/l. Gene Selection and Primer Design. Chromosomal housekeeping genes were selected based on the following criteria: that (i) they are single-copy genes, (ii) they display no more than 5% allelic divergence as assessed by microarray analyses of clinical isolates (3), and (iii) they are, if possible, not adjacent to genes encoding outer surface proteins or hypothetical proteins. Based on the sequenced genomes of B. burgdorferi (B31) (accession no. NC001318) (4), B. garinii (PBi) (accession number NC006156), and B. afzelii (Pko) (accession number 008277) (5), primers were designed to highly conserved regions of eight housekeeping genes that fulfilled these criteria (i.e., clpA, clpX, nifS, pepX, pyrG, recG, rplB, and uvrA) by using OligoExplorer 1.2 (Gene Link). The rationale for this strategy was the future application of this MLST scheme to other species of the Lyme borreliosis group. The primers designed for clpA, clpX, pepX, recG, rplB, and uvrA were nested, and those for nifS and rplB were seminested (Table S6). PCR. HotstarTaq Mastermix (Qiagen), 25 pmol of each primer, forward and reverse, and 2.5 l of template DNA (purified DNA of isolates or tick lysates) were used for the first set of amplifi- cation cycles (25-l final reaction volume). For PCR on tick- derived material, the MgCl 2 concentration was adjusted to 2.5 mM. Bioline Immomix Red, 50 pmol of each primer and 5 l of product derived from the primary set of cycles were used for the second set of amplification cycles (50-l final reaction volume). The PCR conditions for the housekeeping genes, except for recG, were as follows: for the first set of cycles, touchdown PCR was used with annealing temperatures starting from 55°C and decreasing 1°C each cycle. Specific conditions were 95°C for 15 min, 94°C for 30 s, annealing temperature from 55°C to 48°C for 30 s, and an extension step of 72°C for 30 s. An additional 20 cycles were run at 94°C for 30 s, annealing temperature of 48°C, and extension at 72°C for 30 s. After a final extension step for 5 min at 72°C, the samples were kept at 15°C until further analysis. The conditions for the second set of 35 cycles were 95°C for 7 min, 94°C for 30 s, 50°C for 30 s, 72°C for 30 s. After a final extension step for 5 min at 72°C, the samples were kept at 15°C. For recG, the PCR conditons for the first set of cycles were 95°C for 15 min, 94°C for 30 s, 55°C for 30 s, 72°C for 30 s, 30 cycles, and extension at 72°C for 5 min. The conditions for the second set of cycles were identical, except for an initial dena- turing step at 95°C for 7 min. To put the results obtained by this MLST scheme into context with previous studies, the IGS locus and ospC were amplified by using primers and conditions as described previously (6–8). Accession numbers of ospC and IGS sequences obtained in this study and of sequences downloaded from the GenBank database (9, 10) are shown in Table S7. Sequencing. PCR products of all loci were sequenced in forward and reverse directions by using the internal primers (Table S6). Forward and reverse sequences of individual housekeeping genes were compared using the Seqman module of Lasergene version 7 (DNASTAR). For ambiguous sequences, samples were either resequenced or reamplified and resequenced. Mixed infections detected by the presence of double peaks in sequence traces were discarded from further analysis. Sequence Analyses. Version 4.10.9 of DnaSP (11) was used to determine the GC content of the genes. Values for the overall nonsynonymous and synonymous substitutions (dN/dS) of genes were determined in MEGA version 3.1 (12) by using the modified Nei–Gojobori method and the Jukes–Cantor model. For ospC, dN/dS (omega) values for individual codons were determined in Phylemon (http://phylemon.bioinfo.cipf.es/cgi- bin/tools.cgi) using the sitewise likelihood-ratio (SLR) method described by Massingham and Goldman (13). For this, one strain per major ospC group was used for sequence alignment and to construct a neighbor joining tree (1,000 bootstrap repeats) in MEGA 3.1. Table S5 shows the omega values for the individual codons; minus signs in the Results column indicate purifying selection whereas plus signs indicate positive selection. /indicates 95% confidence; /indicates 99% confidence, and /indicates corrected 99% confidence. P values and adjusted P values are given in the corresponding columns. Strains used for SLR analysis included B31, 297, B156, B500, B509, BL522, BL538, MR616, MR623, MR661, MR662, MR640, ca92–1096, IPT2, IPT23, ITP39, IPT58, NE49, Z41293, and Z41493. MLST and Phylogeny. Sequences of individual housekeeping genes that differed by one or more nucleotides were assigned allele numbers by using the nonredundant database program available at www.mlst.net, followed by defining STs for the spirochete samples. In addition to analyzing the STs, the sequences of the house- keeping genes were concatenated. All sequences were aligned by using the ClustalW algorithm of MEGA 3.1 (12), and phyloge- netic trees were constructed. Trees of the concatenated house- keeping genes, the IGS locus, and ospC were generated by using MrBayes 3.1.2 software (14). To choose an appropriate evolu- tionary model for analyses using MrBayes, MrModeltest (Ny- lander 2004, www.abc.se/nylander/) was conducted in Paup 4.0 (15). The concatenated sequences of the housekeeping genes were partitioned into first, second, and third codon positions. Partitions were unlinked for the gamma distribution to allow sites for each codon position to evolve under rates from a gamma distribution with a different shape parameter for each position/ partition. For coding genes, the General Time Reversible model with gamma-distributed rate variation across sites and a propor- tion of invariable sites was used for tree construction, whereas for the IGS sequences, the General Time Reversible model with gamma-distributed rate variation across sites was used. Analyses were continued for 2 10 7 generations or until the average standard deviation of split frequencies was 0.01. Data were sampled every 100th generation. In addition, distance matrices and neighbor joining bootstrap (1,000) trees for individual loci and the concatenated housekeeping genes were obtained by using the Kimura two-parameter model in MEGA. All trees were rooted by using the sequences of the European B. garinii strain PBi obtained from the GenBank database (accession no. NC006156) as an outgroup. Statistics. To test whether the topologies of the MLST tree and the IGS tree differed significantly, a partition homogeneity test (16) based on the incongruence-length difference test (17), Margos et al. www.pnas.org/cgi/content/short/0800323105 1 of 22
Transcript
Supporting InformationMargos et al. 10.1073/pnas.0800323105SI Materials and MethodsDNA Extraction. DNA of tick-derived samples and cultures waseither prepared by alkaline hydrolysis (1) or extracted by usingthe DNeasy tissue and blood kit (Qiagen) as described previously(2). Purified DNA of cultured isolates was diluted to a concen-tration of 10 pg/�l.
Gene Selection and Primer Design. Chromosomal housekeepinggenes were selected based on the following criteria: that (i) theyare single-copy genes, (ii) they display no more than 5% allelicdivergence as assessed by microarray analyses of clinical isolates(3), and (iii) they are, if possible, not adjacent to genes encodingouter surface proteins or hypothetical proteins. Based on thesequenced genomes of B. burgdorferi (B31) (accession no.NC�001318) (4), B. garinii (PBi) (accession number NC�006156),and B. afzelii (Pko) (accession number 008277) (5), primers weredesigned to highly conserved regions of eight housekeepinggenes that fulfilled these criteria (i.e., clpA, clpX, nifS, pepX,pyrG, recG, rplB, and uvrA) by using OligoExplorer 1.2 (GeneLink). The rationale for this strategy was the future applicationof this MLST scheme to other species of the Lyme borreliosisgroup. The primers designed for clpA, clpX, pepX, recG, rplB, anduvrA were nested, and those for nifS and rplB were seminested(Table S6).
PCR. HotstarTaq Mastermix (Qiagen), 25 pmol of each primer,forward and reverse, and 2.5 �l of template DNA (purified DNAof isolates or tick lysates) were used for the first set of amplifi-cation cycles (25-�l final reaction volume). For PCR on tick-derived material, the MgCl2 concentration was adjusted to 2.5mM. Bioline Immomix Red, 50 pmol of each primer and 5 �l ofproduct derived from the primary set of cycles were used for thesecond set of amplification cycles (50-�l final reaction volume).
The PCR conditions for the housekeeping genes, except forrecG, were as follows: for the first set of cycles, touchdown PCRwas used with annealing temperatures starting from 55°C anddecreasing 1°C each cycle. Specific conditions were 95°C for 15min, 94°C for 30 s, annealing temperature from 55°C to 48°C for30 s, and an extension step of 72°C for 30 s. An additional 20cycles were run at 94°C for 30 s, annealing temperature of 48°C,and extension at 72°C for 30 s. After a final extension step for 5min at 72°C, the samples were kept at 15°C until further analysis.The conditions for the second set of 35 cycles were 95°C for 7min, 94°C for 30 s, 50°C for 30 s, 72°C for 30 s. After a finalextension step for 5 min at 72°C, the samples were kept at 15°C.
For recG, the PCR conditons for the first set of cycles were95°C for 15 min, 94°C for 30 s, 55°C for 30 s, 72°C for 30 s, 30cycles, and extension at 72°C for 5 min. The conditions for thesecond set of cycles were identical, except for an initial dena-turing step at 95°C for 7 min.
To put the results obtained by this MLST scheme into contextwith previous studies, the IGS locus and ospC were amplified byusing primers and conditions as described previously (6–8).Accession numbers of ospC and IGS sequences obtained in thisstudy and of sequences downloaded from the GenBank database(9, 10) are shown in Table S7.
Sequencing. PCR products of all loci were sequenced in forwardand reverse directions by using the internal primers (Table S6).Forward and reverse sequences of individual housekeepinggenes were compared using the Seqman module of Lasergeneversion 7 (DNASTAR). For ambiguous sequences, samples were
either resequenced or reamplified and resequenced. Mixedinfections detected by the presence of double peaks in sequencetraces were discarded from further analysis.
Sequence Analyses. Version 4.10.9 of DnaSP (11) was used todetermine the GC content of the genes. Values for the overallnonsynonymous and synonymous substitutions (dN/dS) of geneswere determined in MEGA version 3.1 (12) by using themodified Nei–Gojobori method and the Jukes–Cantor model.
For ospC, dN/dS (omega) values for individual codons weredetermined in Phylemon (http://phylemon.bioinfo.cipf.es/cgi-bin/tools.cgi) using the sitewise likelihood-ratio (SLR) methoddescribed by Massingham and Goldman (13). For this, one strainper major ospC group was used for sequence alignment and toconstruct a neighbor joining tree (1,000 bootstrap repeats) inMEGA 3.1. Table S5 shows the omega values for the individualcodons; minus signs in the Results column indicate purifyingselection whereas plus signs indicate positive selection. �/�indicates 95% confidence; ��/�� indicates 99% confidence,and ���/��� indicates corrected 99% confidence. P valuesand adjusted P values are given in the corresponding columns.Strains used for SLR analysis included B31, 297, B156, B500,B509, BL522, BL538, MR616, MR623, MR661, MR662, MR640,ca92–1096, IPT2, IPT23, ITP39, IPT58, NE49, Z41293, andZ41493.
MLST and Phylogeny. Sequences of individual housekeeping genesthat differed by one or more nucleotides were assigned allelenumbers by using the nonredundant database program availableat www.mlst.net, followed by defining STs for the spirochetesamples.
In addition to analyzing the STs, the sequences of the house-keeping genes were concatenated. All sequences were aligned byusing the ClustalW algorithm of MEGA 3.1 (12), and phyloge-netic trees were constructed. Trees of the concatenated house-keeping genes, the IGS locus, and ospC were generated by usingMrBayes 3.1.2 software (14). To choose an appropriate evolu-tionary model for analyses using MrBayes, MrModeltest (Ny-lander 2004, www.abc.se/�nylander/) was conducted in Paup 4.0(15). The concatenated sequences of the housekeeping geneswere partitioned into first, second, and third codon positions.Partitions were unlinked for the gamma distribution to allowsites for each codon position to evolve under rates from a gammadistribution with a different shape parameter for each position/partition. For coding genes, the General Time Reversible modelwith gamma-distributed rate variation across sites and a propor-tion of invariable sites was used for tree construction, whereasfor the IGS sequences, the General Time Reversible model withgamma-distributed rate variation across sites was used. Analyseswere continued for 2 � 107 generations or until the averagestandard deviation of split frequencies was �0.01. Data weresampled every 100th generation. In addition, distance matricesand neighbor joining bootstrap (1,000) trees for individual lociand the concatenated housekeeping genes were obtained byusing the Kimura two-parameter model in MEGA. All treeswere rooted by using the sequences of the European B. gariniistrain PBi obtained from the GenBank database (accession no.NC�006156) as an outgroup.
Statistics. To test whether the topologies of the MLST tree andthe IGS tree differed significantly, a partition homogeneity test(16) based on the incongruence-length difference test (17),
Margos et al. www.pnas.org/cgi/content/short/0800323105 1 of 22
implemented in Paup 4.0, was applied. For this, IGS sequenceswere added to MLST sequences. The data were partitioned inMLST and IGS and used for parsimony analysis (100 bootstraprepeats, heuristic search) with random addition sequences (10repeats) and tree bisection–reconnection branch swapping.
To test whether there is a different distribution of the allelicprofiles of B. burgdorferi from Europe and North America, apermutation test was performed (Table S3 and Fig. S2). For this,a dissimilarity matrix was calculated from the allelic profiles ofB. burgdorferi by counting the number of genes at which each pairof strains differ (Table S3). Only one isolate/sample of B.burgdorferi per ST was taken to control for possible effects oflocal clonal expansion. The pairwise difference between strain iand strain j was denoted by dij, where dij equals the number ofgenes at which strains i and j differ. D refers to the symmetricdistance matrix whose ith row and jth column entry is dij. Fromthis matrix, a dissimilarity test statistic was calculated as theweighted sum (¥) of the mean dissimilarities within the tworegions compared in this test (i.e., the United States andEurope), where the weights are the relative numbers of strainsin each region (n1 are strains from the United States, and n2 arestrains from Europe), i.e.,
D1 � ��i�j, with i and j in region1�
dij
n1�n1 � 1 /2
and D2 �[i�j, with i and j in region2]
dij
n2�n2 � 1 /2.
The test statistic is a weighted average of the two means
T �n1
n1 � n2D1 �
n2
n1 � n2D2,
with n1 � n2 equaling the total number of STs. Under the nullhypothesis that there is no difference between the allelic profilesof B. burgdorferi strains from the two regions, and under thealternative hypothesis that there is a difference, a permutationtest was performed using one million random permutations.That is, the strains are repeatedly randomly divided into twogroups of the same size as before, and the dissimilarity teststatistic is recalculated.
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4. Casjens S, et al. (2000) A bacterial genome in flux: The twelve linear and nine circularextrachromosomal DNAs in an infectious isolate of the Lyme disease spirochete Bor-relia burgdorferi. Mol Microbiol 35:490–516.
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6. Brisson D, Dykhuizen DE (2004) ospC diversity in Borrelia burgdorferi: Different hostsare different niches. Genetics 168:713–722.
7. Bunikis J, et al. (2004) Sequence typing reveals extensive strain diversity of the Lymeborreliosis agents Borrelia burgdorferi in North America and Borrelia afzelii in Europe.Microbiology 150:1741–1755.
8. Liveris D, Gazumyan A, Schwartz I (1995) Molecular typing of Borrelia burgdorferisensu lato by PCR-restriction fragment length polymorphism analysis. J Clin Microbiol33:589–595.
9. Attie O, et al. (2007) Co-evolution of the outer surface protein C gene (ospC) andintraspecific lineages of Borrelia burgdorferi sensu stricto in the northeastern UnitedStates. Infect Genet Evol 7:1–12.
10. Wang G, et al. (2002) Disease severity in a murine model of lyme borreliosis is associatedwith the genotype of the infecting Borrelia burgdorferi sensu stricto strain. J Infect Dis186:782–791.
11. Rozas J, Sanchez-DelBarrio JC, Messeguer X, Rozas R (2003) DnaSP, DNA polymorphismanalyses by the coalescent and other methods. Bioinformatics 19:2496–2497.
12. Kumar S, Tamura K, Nei M (2004) Integrated software for Molecular EvolutionaryGenetics Analysis and sequence alignment. Brief Bioinform 5:150–163.
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14. Huelsenbeck JP, Ronquist F (2005) in Statistical Methods in Molecular Evolution, edNielsen R (Springer, New York), pp 183–232.
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Fig. S1. Venn diagram of shared alleles and sequence types (STs) between B. burgdorferi strains. Individual circles represent strains from the United States (US)and from Europe (EU). Values inside the circles give the number of alleles/STs found exclusively in these populations, and the number in the overlap indicatesthe number of shared alleles or STs. Only seven alleles of 113 total alleles were shared between strains from the two regions.
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Fig. S2. Permutation test. The distribution of the dissimilarity statistic under the null hypothesis of no difference between B. burgdorferi populations fromEurope and the United States. The bars represent the relative frequencies of the dissimilarity statistic T, which is calculated from the pairwise allelic differencematrix and is dimensionless. The value of the observed statistic was 6.68 (indicated as a dashed line), which is distant from the main body of the histogram (highervalues of the statistic indicating larger within-group differences). This provides significant evidence to reject the null hypothesis (P 0.00285).
Margos et al. www.pnas.org/cgi/content/short/0800323105 4 of 22
Fig. S3. Neighbor joining trees of housekeeping gene sequences. For the concatenated housekeeping genes, both the MrBayes algorithm (Fig. 3) and theneighbor joining tree building method (A) gave rise to the same terminal clades, and bootstrap support for the terminal nodes was high. The trees based onthe two methods were highly similar, rendering the neighbor joining algorithm a convenient approach in the analysis of very large datasets. Rooted neighborjoining trees for the individual housekeeping genes are shown in B–I. As expected, trees of the individual housekeeping genes showed less resolution than thetrees based on the concatenated genes. Branch support values were obtained by a bootstrap procedure (1,000 replications). Only bootstrap values of 50% ormore are shown. Symbols refer to the major IGS genotypes as defined by Bunikis et al. (7). Non-color-coded strains are from the Northeast and Midwest UnitedStates. Blue labels indicate European strains, and yellow labels indicate Californian strains. The branch length of the outgroup B. garinii is not according to scale(indicated by slashes). (Scale bar: 1% divergence.)
Margos et al. www.pnas.org/cgi/content/short/0800323105 5 of 22
Table S1. B. burgdorferi strains used in this study
Strain Biological source Geographic source Collector Provided by
B31 I. scapularis Shelter Island, NY A. Barbour I.S.297 Human patient Connecticut A. Steere I.S.JD1 I. scapularis Ipswich, MA J.P. I.S.N40 I. scapularis Westchester County, NY S. Barthold I.S.BL206 Human patient Westchester County LDP I.S.B515 Human patient Westchester County LDP I.S.B504 Human patient Westchester County LDP I.S.B509 Human patient Westchester County LDP I.S.MR623 Human patient Westchester County LDP I.S.B373 Human patient Westchester County LDP I.S.B156 Human patient Westchester County LDP I.S.MR661 Human patient Westchester County LDP I.S.MR654 Human patient Westchester County LDP I.S.BL538 Human patient Westchester County LDP I.S.BL515 Human patient Westchester County LDP I.S.BL522 Human patient Westchester County LDP I.S.B356 Human patient Westchester County LDP I.S.MR616 Human patient Westchester County LDP I.S.B331 Human patient Westchester County LDP I.S.B361 Human patient Westchester County LDP I.S.B500 Human patient Westchester County LDP I.S.B485 Human patient Westchester County LDP I.S.MR607 Human patient Westchester County LDP I.S.MR662 Human patient Westchester County LDP I.S.B418 Human patient Westchester County LDP I.S.MR640 Human patient Westchester County LDP I.S.B348 Human patient Westchester County LDP I.S.47703UT* I. scapularis Cass County, MN M. Johnson A.G.G.51405UT I. scapularis Cass County, MN M. Johnson A.G.G.16812UT I. scapularis Cumberland County, ME C. Lubelczyk A.G.G.15903UT I. scapularis Cumberland County C. Lubelczyk A.G.G.15506UT I. scapularis Cumberland County C. Lubelczyk A.G.G.15912UT I. scapularis Cumberland County C. Lubelczyk A.G.G.519014UT I. scapularis Van Buren County, MI E. Dobrowolski A.G.G.48102UT I. scapularis Hubbard County, MN M. Johnson A.G.G.498801UT I. scapularis Suffolk County, NY A.G.G. A.G.G.114311UT I. scapularis Cecil County, MD C. Abadam A.G.G.Ca4 I. pacificus California R.S. Lane J.P.Ca5 I. pacificus California R.S. Lane J.P.Ca6 I. pacificus California R.S. Lane J.P.Ca92–0953 Human patient California R.S. Lane J.P.Ca92–1096 Human patient California R.S. Lane J.P.Ca92–1337 Human patient California R.S. Lane J.P.Ca�WTB27 I. pacificus California J.P. J.P.Ca�WTB32 I. pacificus California J.P. J.P.IPT2 I. ricinus Alsace, France CNRB M.C.IPT19 I. ricinus Alsace CNRB M.C.IPT23 I. ricinus Alsace CNRB M.C.IPT39 I. ricinus Alsace CNRB M.C.IPT58 I. ricinus Alsace CNRB M.C.IPT69 I. ricinus Alsace CNRB M.C.IPT135 I. ricinus Auvergne, France CNRB M.C.IPT137 I. ricinus Alsace, France CNRB M.C.IPT190 I. ricinus Normandy, France CNRB M.C.IPT191 I. ricinus Normandy CNRB M.C.IPT193 I. ricinus Normandy CNRB M.C.IPT198 I. ricinus Normandy CNRB M.C.NE49 I. ricinus Switzerland L. Gern M.C.Z41293 I. ricinus Germany M. Wittenbrink M.C.Z41493 I. ricinus Germany M. Wittenbrink M.C.21509LT* I. ricinus Riga district, Babite, Latvia A.B. M.D.20111LT I. ricinus Babite A.B. M.D.22521LT I. ricinus Babite A.B. M.D.20604LT I. ricinus Jurmala, Kemeri, Latvia A.B. M.D.
LDP, human isolates from patients presenting at the Lyme Disease Practice of the Westchester Medical Center, Valhalla, NY; CNRB, Centre National deRéférence des Borrelia.*Spirochete samples carrying the suffix �LT� (Latvia tick) or �UT� (U.S. tick) were not isolated but were amplified by PCR from DNA extracted from ticks.
Margos et al. www.pnas.org/cgi/content/short/0800323105 14 of 22
Table S4. New IGS genotypes among European isolates of B. burgdorferi as determined according to the IGS type definitionpublished by Bunikis et al. (7)
11 Z41293 A C y (6) C T G G G12 Z41493 A C y C T G G A
Strain Z41293: the deletion at position 79 (from the start of the analyzed sequence fragment) is 6 bp instead of 7 bp. It has additional deletions at positions106–117 (from the start of the fragment) (12 bp) and 742–751 (10 bp) as well as insertions of one base at position 308 and two bases at positions 711–712.
Strain Z41493: this strain has an additional insertion at position 308 (from the start of the fragment) and additional deletions at positions 670–671 and779–782.*Positions shown in parentheses are from the start of the sequence fragment analyzed in this study.
Margos et al. www.pnas.org/cgi/content/short/0800323105 17 of 22
B31 NC�001903 4 AY275189 7297 AY275214 7 AY275192 7JD1 DQ437462.1 9 DQ437438 9N40 AY275221 7 AY275211 7BL206 AF467867 10 AF467855 10B515 AF467872 10 AF467860 10B504 EU377780 This study EU377796 This studyB509 EU377781 This study EU377797 This studyMR623 EU377778 This study EU377794 This studyB373 EU377779 This study EU377795 This studyB156 EU377776 This study EU377792 This studyMR661 EU377775 This study EU377791 This studyMR654 EU377777 This study EU377793 This studyBL538 EU377773 This study EU377789 This studyBL515 EU377774 This study EU377790 This studyBL522 EU377772 This study EU377788 This studyB356 AF467873 10 AF467861 10MR616 EU377771 This study EU377787 This studyB331 AF467874 10 AF467862 10B361 EU377770 This study EU377786 This studyB500 AF467878 10 AF467866 10B485 EU377769 This study EU377785 This studyMR607 EU377768 This study EU377784 This studyMR662 EU377767 This study EU377783 This studyB418 AF467877 10 AF467877 10MR640 EU377766 This study EU377782 This studyB348 AF467875 10 AF467863 1047703UT EU375832 This study EU375821 This study51405UT EU375825 This study EU375815 This study16812UT EU375826 This study EU375820 This study15903UT EU375828 This study EU375819 This study15506UT EU375833 This study EU375818 This study15912UT EU375827 This study EU375816 This study519014UT EU375831 This study EU375823 This study48102UT EU375829 This study EU375822 This study498801UT EU375830 This study EU375814 This study114311UT EU375824 This study EU375817 This studyCa4 EU377746 This study EU377801 This studyCa5 EU377747 This study EU377802 This studyCa6 EU377748 This study EU377803 This studyCa92-0953 EU377751 This study EU377806 This studyCa92-1096 EU377752 This study EU377807 This studyCa92-1337 EU377753 This study EU377808 This studyCa�WTB27 EU377749 This study EU377804 This studyCa�WTB32 EU377750 This study EU377805 This studyIPT2 EU377754 This study EU377809 This studyIPT19 EU377755 This study EU377810 This studyIPT23 EU377756* This study EU377811 This studyIPT39 EU377757 This study EU377812 This studyIPT58 EU377758 This study EU377813 This studyIPT69 EU377759 This study EU377814 This studyIPT135 EU377760 This study EU377815 This studyIPT137 EU377761 This study EU377816 This studyIPT190 EU377762* This study EU377817 This studyIPT191 EU377763 This study EU377818 This studyIPT193 EU377764* This study EU377819 This studyIPT198 EU377765* This study EU377820 This studyNE49 EU377743 This study EU377798 This studyZ41293 EU377744* This study EU377799 This studyZ41493 EU377745* This study EU377800 This study21509LT ND ND20111LT ND EU377821 This study22521LT ND ND20604LT ND EU377822 This study
*Alleles identical to these novel European ospC groups were found as unpublished GenBank submissions in 2007 by B. Luft and colleagues, presumably derivedfrom North American B. burgdorferi samples (GenBank accession nos.: EF537408/EF537407, EF537427, and EF537414 corresponding to EU377756/EU377762,EU377764/EU377765/EU377745, and EU377744, respectively).
Margos et al. www.pnas.org/cgi/content/short/0800323105 22 of 22
