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Journal of Bacteriology, December 2008, p. 7885-7891, Vol. 190, No. 24
0021-9193/08/$08.00+0     doi:10.1128/JB.00324-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

In Vitro CpG Methylation Increases the Transformation Efficiency of Borrelia burgdorferi Strains Harboring the Endogenous Linear Plasmid lp56

Qiang Chen,^1, Joshua R. Fischer,^1, Vivian M. Benoit,¹ Nicholas P. Dufour,¹ Philip Youderian,² and John M. Leong^1*

Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, Massachusetts 01655,¹ Casilla 385, Ancud, Chile²

Received 4 March 2008/ Accepted 12 September 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Borrelia burgdorferi is the causative agent of Lyme disease, the most common vector-borne illness in the Northern hemisphere. Low-passage-number infectious strains of B. burgdorferi exhibit extremely low transformation efficiencies—so low, in fact, as to hinder the genetic study of putative virulence factors. Two putative restriction-modification (R-M) systems, BBE02 contained on linear plasmid 25 (lp25) and BBQ67 contained on lp56, have been postulated to contribute to this poor transformability. Restriction barriers posed by other bacteria have been overcome by the in vitro methylation of DNA prior to transformation. To test whether a methylation-sensitive restriction system contributes to poor B. burgdorferi transformability, shuttle plasmids were treated with the CpG methylase M.SssI prior to the electroporation of a variety of strains harboring different putative R-M systems. We found that for B. burgdorferi strains that harbor lp56, in vitro methylation increased transformation by at least 1 order of magnitude. These results suggest that in vitro CpG methylation protects exogenous DNA from degradation by an lp56-contained R-M system, presumably BBQ67. The utility of in vitro methylation for the genetic manipulation of B. burgdorferi was exemplified by the ease of plasmid complementation of a B. burgdorferi B31 A3 BBK32 kanamycin-resistant (B31 A3 BBK32::Kan^r) mutant, deficient in the expression of the fibronectin- and glycosaminoglycan (GAG)-binding adhesin BBK32. Consistent with the observation that several surface proteins may promote GAG binding, the B. burgdorferi B31 A3 BBK32::Kan^r mutant demonstrated no defect in the ability to bind purified GAGs or GAGs expressed on the surfaces of cultured cells.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Borrelia burgdorferi is the causative agent of Lyme disease, the most common vector-borne illness in the United States (32, 33). The genome of this bacterium is extremely complex, consisting of a linear chromosome and as many as 23 linear and circular plasmids (lp and cp, respectively), the largest number among any of the bacteria that have been genome sequenced to date (2, 8, 17, 34). Plasmids such as lp25, lp28-1, lp28-4, and lp36 have documented importance for infection of the tick or the mammalian host, and the study of the function of endogenous plasmids is integral to an understanding of the pathogenesis of B. burgdorferi (9, 10, 14, 23, 25, 35, 37).

Although this bacterium can be cultured in vitro, the efficiency of transformation is extremely low, so low, in fact, as to significantly hinder the rigorous investigation of the pathogenesis of Lyme disease through the mutagenesis of putative virulence genes and subsequent complementation (27). It has been suggested that the poor transformability of B. burgdorferi is due to the presence of multiple restriction-modification (R-M) systems that degrade exogenous DNA. Indeed, B. burgdorferi contains several genes that are homologous to known R-M genes of other bacteria, such as BBE02 on lp25, BBQ67 on lp56, and BBH09 on lp28-3 (8, 26). Strains lacking lp25 or lp56 are 10- to 30-fold more transformable, and strains lacking both are 500- to 1,000-fold more transformable (15). Moreover, specific inactivation of BBE02 results in a strain with higher transformability (11). Finally, as one might predict, on the occasions that strains harboring lp25 are successfully transformed, transformants are often found to have lost this plasmid (15), which, particularly given its important functions in the biology of B. burgdorferi, complicates genetic analysis of this organism.

If restriction of exogenous DNA contributes to the poor transformability of B. burgdorferi, prior in vitro methylation may stabilize transformed DNA and boost transformation efficiency. For example, the transformation of Helicobacter pylori, which contains multiple R-M systems, has been enhanced by in vitro modification of foreign DNA by methylases present in H. pylori extracts (3), and the treatment of plasmids with several commercially available methylases significantly increases the transformation efficiency of Streptomyces griseus (13). In this study, we show that in vitro CpG methylation of a shuttle plasmid by a commercially available methylase dramatically increases the transformation efficiency of B. burgdorferi strains and that this increase depends on the presence of lp56, suggesting that methylation protects foreign DNA from degradation by an R-M system, presumably BBQ67, located on that plasmid. The utility of in vitro methylation for the genetic manipulation of B. burgdorferi was exemplified by the relative ease of plasmid complementation of a B. burgdorferi B31 A3 BBK32 mutant, which was deficient in the expression of the fibronectin (Fn)- and glycosaminoglycan (GAG)-binding adhesin BBK32.

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Bacteria. B. burgdorferi B31 A3 (a gift from P. A. Rosa, National Institute of Allergy and Infectious Diseases, Hamilton, MT) is a low-passage-number B31 clone containing all 21 endogenous plasmids except for cp9 (4). Strains 5A4, 5A1, and 5A18 (18; gifts from S. J. Norris, University of Texas Health Science Center, Houston, TX) are B31 derivatives and their plasmid profiles are shown below (see Table 2). 5A4NP1 and 5A18NP1 (gifts from H. Kawabata and S. J. Norris) are BBE02 mutants derived from 5A4 and 5A18, respectively (11).

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TABLE 2. Methylation-mediated increase in transformation efficiency is largely dependent on lp56a

DNA methods. Routine DNA manipulations were performed as described previously (28). All constructs were generated in Escherichia coli DH10B and confirmed by partial sequencing to ensure that no mutation was introduced into the BBK32 coding region by PCR. Plasmid DNA of E. coli was isolated using a plasmid mega kit (Qiagen, Valencia, CA). Borrelia DNA was isolated as described previously (20). The amount of DNA was spectrophotometrically quantitated by measuring the absorbance at 260 nm.

In vitro methylation. Plasmid DNA was methylated in vitro by M.SssI (New England Biolabs, Beverly, MA) as recommended by the manufacturer. Briefly, DNA was incubated with the M.SssI (1 unit/µg DNA) in the presence of 1x NEBuffer 2 (New England Biolabs) and 160 µM S-adenosylmethionine at 37°C for 4 h. M.SssI was heat inactivated at 65°C for 20 min and removed by phenol-chloroform-isoamyl alcohol extraction as described previously (28). To test the efficacy of M.SssI, methylated DNA was digested with enzymes either blocked by M.SssI (such as SalI) or not blocked by M.SssI (such as BamHI). Sensitivity to SalI and resistance to BamHI indicated that the DNA was efficiently methylated by M.SssI.

Transformation studies. Electroporation of B. burgdorferi was performed as described previously (29) with minor modifications. B. burgdorferi was grown to mid-log phase (5 x 10⁷/ml) in Barbour-Stoenner-Kelly II (BSK II) medium, harvested by centrifugation, and washed with electroporation solution (0.27 M sucrose, 15% glycerol). Competent cells (2.5 x 10⁹ in 100 µl) were electroporated with 10 to 120 µg of methylated or mock-treated DNA. After electroporation, bacterial cells were immediately resuspended in 15 ml BSK II medium and allowed to recover for 18 to 24 h at 33°C. Transformants were isolated by limiting dilution in BSK II medium supplemented with an appropriate antibiotic (200 µg/ml kanamycin [Kan] or 100 µg/ml streptomycin [Str]) in addition to the antibiotic mixture for Borrelia (Sigma-Aldrich, St. Louis, MO). Briefly, recovered bacterial cells were subjected to fivefold serial dilution, and from each dilution 100-µl aliquots were plated onto 96-well tissue culture plates with low-evaporation lids (Becton Dickinson, Franklin Lakes, NJ). Wells were evaluated for the presence of live spirochetes by dark-field microscopy after 10 days or by assessment of the color change of the medium after 15 days. To confirm that positive wells represented true transformants, spirochetes from a random sampling of 15 positive wells were expanded in 5 ml of fresh BSK II medium with selection and grown to log phase. One milliliter of B. burgdorferi culture was harvested by centrifugation in a microcentrifuge. Bacterial pellets were washed with 0.2% bovine serum albumin in phosphate-buffered saline three times, resuspended in 10 µl water, and boiled for 10 min to obtain a crude extract of B. burgdorferi DNA. PCR was performed using Taq polymerase (Invitrogen, Houston, TX), 0.5 µl of DNA crude extract as the template, and primers specific for the Kan cassette (kanF [5'-ATGAGCCATATTCAACGG-3'] and kanR [5'-CTCATCGAGCATCAAATG-3']) or the aadA cassette (aadAF [5'-GCCGGCTAATACCCGAGC-3'] and aadAR [5'-TTACATGATATATCTCCCAAT-3']) to detect the presence of pBSV2 or pKFSS1 (and its derivative). Clones that resulted in a PCR product of the predicted size were deemed true transformants. The rate of PCR-negative, Kan^r clones was typically less than 25%, whereas PCR-negative Str^r clones were detected at a higher frequency, sometimes greater than 50%. To further confirm that antibiotic-resistant clones were true transformants, DNA was isolated from a random sampling of clones that demonstrated PCR evidence of the transforming plasmid and transformed into E. coli, selecting for the antibiotic marker on the input plasmid. This plasmid was recovered from the E. coli transformant and the identity of the plasmid was confirmed by restriction analysis.

Previous transformation studies have found an association between transformants and the loss of endogenous plasmids that contain putative R-M systems (11, 15), so we tested subsets of (usually 10) transformants for the presence of these plasmids by PCR as described previously (23). In all but one experiment, the loss of these plasmids was not correlated with the methylation state of the transforming DNA and averaged less than 10% (data not shown), which, for reasons that are unclear, is less than previously observed (11, 15). In the single experiment in which transformants were found to have lost lp25 frequently, this loss also was not correlated with methylation (data not shown), and this experiment was excluded from our analysis.

To estimate the total number of transformants, the number of true transformants from a microtiter plate was multiplied by the dilution factor for that plate. When more than one dilution gave positive wells, plates with very high or very low percentages of positive wells were excluded from the analysis to minimize inaccuracies due to the presence of multiple clones per well or due to extremely low sampling numbers, respectively. The transformation efficiency was calculated by dividing the total estimated transformants by the amount of DNA transformed. The transformation frequency was calculated by dividing the total estimated transformants by the number of viable spirochetes subject to transformation.

Insertional inactivation of the B31 A3 BBK32 mutant. A suicide vector was constructed for the targeted disruption of BBK32. First, a 2-kb sequence from BBK26 to the first 47 bp of BBK32 was cloned into pCR-XL TOPO (Invitrogen) by PCR using primers K26F (5'-GGAAAGGTATGGAGCTTATATGAT-3') and K32R8 (5'-GCACCGGTCTAGGCTACCTAGGCCAAAAAGTAATCCCAAAGCCAAATATTTA-3') containing an AgeI site and an AvrII site (underlined) to generate pCR-TOPO k26-k32. Next, a 1.5-kb sequence from BBK32 to BBK34 was amplified using primers K32F (5'-GCCCTAGGTTTATAAGTTGTGATTTATTCAT-3') containing a novel AvrII site and K34R (5'-GCACCGGTTATCAAGGGGCCATTATTCTTCAT-3') containing a novel AgeI site (underlined). This amplicon was cloned into pCR-TOPO k26-k32 by use of the engineered AvrII and AgeI restriction sites to generate pCR-TOPO k26-k34. A Kan^r cassette with a flaB promoter sequence was amplified by PCR with primers PflaBkanF (5'-GCCCTAGGGCCGGCTGTCTGTCGCCT-3') and PflaBkanR (5'-GCCCTAGGTTAGGCGAATGAGCTAGCGCC-3') containing novel AvrII sites (underlined), and this cassette was cloned into pCR-TOPO k26-k34 by use of the engineered AvrII sites to generate pCR-TOPO k26-k34::PflaB kan^R. Electrocompetent B31 A3 spirochetes were transformed with 30 µg of pCR-TOPO k26-k34::PflaB kan^R plasmid DNA and cultured in BSK H complete medium at 37°C for 24 h as previously described (29). Aliquots of the culture were mixed with 1.8% analytical-grade agarose (Bio-Rad, Hercules, CA) and plated onto a solidified BSK II medium-agarose layer in sterilized 100- by 20-mm tissue culture dishes (Corning Incorporated, Corning, NY) in the presence of Kan (200 µg/ml). Plates were incubated at 37°C in 5% CO₂ for 18 days. Six Kan^r colonies were obtained and expanded at 37°C in liquid BSK H complete medium containing Kan, followed by genomic DNA preparation (20). PCR was performed with primers specific for Kan and for endogenous plasmids of B. burgdorferi (23). Two transformants retained the full plasmid contents as the parental strain and carried an insertion of the appropriate size of Kan at the BBK32 locus, determined by PCR with primers flanking the insertion sites. One of them was chosen as the B31 A3 BBK32 mutant in the following experiments.

Complementation of the B31 A3 BBK32 mutant. A 1.6-kb sequence containing BBK32 and its upstream region (0.6 kb) was cloned into pCR2.1 TOPO (Invitrogen) by PCR using primers K30F (5'-AGTCAAAGCTTCTGCAATATCACTAAATTCTAG-3'), containing a HindIII site, and K32R13 (5'-CCGGGATCCTTAGTACCAAACGCCATTC-3'), containing a BamHI site (sites are underlined). The 1.6-kb fragment containing BBK32 was cloned into pKFSS1 by use of the engineered HindIII and BamHI sites to generate pKFSS1k32. The complement plasmid pKFSS1k32 and the vector pKFSS1 were in vitro methylated as described above. The methylated plasmids or mock-treated plasmids were transformed into the B31 A3 BBK32::Kan^r mutant and transformants were isolated by Hind III and Bam HI limiting dilution as described above. For a sampling of B31 A3 BBK32::Kan^r clones that had been transformed with methylated pKFSS1k32 or pKFSS1, PCR was performed with primers specific for aadA and for all endogenous plasmids of B. burgdorferi (23). One pKFSS1k32 and one pKFSS1 transformant containing the full endogenous plasmid content were chosen and were designated as B31 A3 BBK32::Kan^r/pKFSS1k32 and B31 A3 BBK32::Kan^r/pKFSS1, respectively.

Western and far-Western blotting. B. burgdorferi whole-cell lysates were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1 x 10⁸ bacteria per lane). Proteins were then transferred to polyvinylidene difluoride membranes and detected with either a polyclonal antibody against MBP-BBK32 or a monoclonal antibody, CB1 (a gift from J. Benach, Stony Brook University, Stony Brook, NY) against FlaB. For far-Western blotting, membranes were incubated with 30 µg/ml bovine Fn (Sigma-Aldrich) for 1 h and probed with rabbit serum against Fn (Sigma). Anti-mouse or anti-rabbit immunoglobulin G conjugated with alkaline phosphatase was used as the secondary antibody. XP (5-bromo-4-chloro-3-indolylphosphate) was used as the substrate for signal detection.

Bacterial binding assays. B. burgdorferi cells were radiolabeled with [³⁵S]methionine and incubated with purified GAGs or HEp-2 human epithelial cells to determine the percentage of binding as described previously (5). HEp-2 cells were treated with heparinase I (formerly known as heparinase), heparinase III (formerly known as heparitinase), chondroitinase ABC (Sigma-Aldrich), or a combination of the three lyases prior to bacterial binding as described previously (5).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Pretreatment of plasmids with M.SssI significantly increases the transformation efficiency of the low-passage-number infectious B. burgdorferi strain B31. The commercially available CpG methylase M.SssI, which methylates both unmethylated and hemimethylated DNA and requires only a 2-nucleotide sequence (CG) for recognition (24), is capable of blocking a wide range of restriction enzymes. CpG methylation confers sensitivity to some restriction enzymes, such as the E. coli McrA, McrBC, and Mrr restriction systems (12, 38), but identifiable homologs of these enzymes are not present in the B. burgdorferi genome (8; http://cmr.tigr.org). The widely used Kan^r B. burgdorferi shuttle plasmid pBSV2 (36), a pBSV2-derived Str^r plasmid, pKFSS1 (7), and its derivative, pKFSS1k32 (see Table 3 below), were modified using M.SssI. Following methylation, the plasmids were treated with SalI, a restriction enzyme that recognizes an M.SssI-sensitive cleavage site (GTCGAC), to assess the efficacy of the methylase reactions. Each of the methylase-treated plasmids was resistant to SalI cleavage following treatment, exhibiting the same electrophoretic pattern (supercoiled and open circular forms of plasmids) as uncut plasmid, whereas mock-methylated plasmids were cleaved by SalI, resulting in a single (linear) species (Fig. 1).

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TABLE 3. Complementation of B31 A3 BBK32::Kan mutant by use of in vitro CpG-methylated plasmidsa

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FIG. 1. Methylation of shuttle plasmids. The indicated plasmids were mock treated or treated with the methylase M.SssI prior to B. burgdorferi transformation. To assess methylation status, plasmids ("Input") were mock or SalI digested. DNA isolated from B. burgdorferi transformants was transformed into E. coli and then recovered ("Rec") and mock or SalI digested. Supercoiled, open circular, and linearized plasmids are indicated by arrows.

Strain B31 A3, a low-passage-number infectious derivative of B. burgdorferi strain B31 which retains a nearly complete set of endogenous B31 plasmids (lacking only cp9) and can infect both ticks and mammals (4) was transformed with methylated or mock-methylated pBSV2. Pilot experiments revealed that the efficiency of transformation was considerably higher for methylated pBSV2, so B. burgdorferi was electroporated with two times more mock-methylated plasmid than methylated plasmid. When methylated and mock-methylated plasmids were compared in three experiments, in vitro methylation resulted in an average 39-fold increase in the number of true transformants per µg of plasmid (4.9 versus 0.13 clones/µg) (Table 1). The presence of pBSV2 in a random sampling of Kan^r clones was confirmed by PCR, and this plasmid was recovered upon the transformation of E. coli with total DNA from these clones, selecting for Kan^r. As expected, pBSV2 recovered in this manner was sensitive to SalI, indicating that after propagation in E. coli, the plasmid returned to an unmethylated state (Fig. 1 and data not shown).

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TABLE 1. CpG methylation with M.SssI increases transformation efficiency of B. burgdorferi B31 A3

To determine if the increase in transformation was specific to the B31 A3 clone of B. burgdorferi, methylated or mock-methylated pBSV2 was transformed into the B. burgdorferi B31 derivative strain 5A4, which contains a full complement of endogenous plasmids (23). As observed for B31 A3, transformation was increased dramatically (by >32-fold) upon methylation (Table 2, first three rows). This increase in transformation was also not specific for Kan^r plasmids, because M.SssI methylation of pKFSS1, a Str^r plasmid derived from pBSV2, also resulted in a dramatic (>60-fold) increase in 5A4 transformation (Table 2, fourth row; Fig. 1).

In vitro CpG methylation of DNA increases the efficiency of B. burgdorferi transformation in an apparently lp56-dependent manner. To explore whether a putative B. burgdorferi R-M system may be responsible for the transformation barrier eliminated by CpG methylation, methylated or unmethylated plasmids were electroporated into various 5A4-related B. burgdorferi strains lacking putative R-M systems (Table 2). Strain 5A1 is a moderately transformable derivative of strain B31 that differs from 5A4 by lacking lp56 (and the putative restriction enzyme BBQ67 encoded on that plasmid [18]), and we confirmed that 5A1 was in most experiments transformed at moderate efficiency with unmethylated pBSV2 or pKFSS1 (Table 2, fifth through seventh rows). However, in contrast to what was seen for strain 5A4, prior CpG methylation did not result in a dramatic and convincing increase in 5A1 transformation efficiency. (In two experiments, the increase was less than twofold, and in the third, a calculated ninefold increase was based on an exceedingly small number of transformants [Table 2, fourth through seventh rows]). Similarly, strain 5A18, which lacks lp56 as well as lp28-4 and lp5 (18), showed a moderately high transformation efficiency that was increased less than twofold by methylation.

The above findings suggest that lp56 encodes the major transformation barrier bypassed by CpG methylation. To more directly investigate the hypothesis that methylation also bypasses the transformation barrier due to the putative restriction enzyme BBE02 encoded on lp25, we transformed 5A4 NP1, a 5A4 BBE02 mutant that retains lp56 (11). The transformation of 5A4 NP1 with methylated pKFSS1 resulted in 1,200 transformants per µg, a 1,600-fold increase from what was seen for the unmethylated pKFSS1 (Table 2, 10th row), indicating that BBE02 is not required for CpG methylation to dramatically increase transformation efficiency. In contrast, the high transformation efficiency of 5A18 NP1, which lacks lp56 in addition to BBE02 (11), was essentially unchanged by prior methylation (Table 2, 11th row). These results indicate that lp56, but not BBE02, is required for in vitro CpG methylation to have a dramatic effect on transformation efficiency and suggest that an lp56-encoded restriction enzyme, presumably BBQ67, is responsible for the transformation barrier abrogated by treatment with M.SssI.

Analysis of a B. burgdorferi mutant deficient in BBK32 indicates redundancy in GAG-binding pathways by strain B31 A3. The BBK32 protein was first identified as a B. burgdorferi surface lipoprotein that promotes bacterial binding to Fn (21) and was more recently shown to also promote binding of the high-passage-number, nonadhesive B. burgdorferi strain B314 to the GAGs heparin and dermatan sulfate (5). Several other B. burgdorferi surface proteins, including DbpA, DbpB, and Bgp, have been shown to bind GAGs, and the expression of DbpA or DbpB is sufficient to confer GAG binding by strain B314 (6, 20). To assess the relative importance of BBK32 in GAG binding by strain B31 A3, we inserted a Kan cassette (conferring Kan^r) at the 5' end of BBK32, resulting in the B31 A3 BBK32::Kan^r mutant, which was found to retain all of the endogenous plasmids of B31 A3 (Fig. 2A and data not shown; also see Materials and Methods). Initial attempts to generate a plasmid-complemented derivative of the B31 A3 BBK32::Kan^r mutant by electroporation with (unmethylated) pKFSS1k32, a derivative of pKFSS1 carrying the BBK32 gene and its upstream sequence, were unsuccessful (data not shown). To test whether in vitro methylation can be utilized to assist the genetic assessment of GAG binding by B. burgdorferi, pKFSS1k32 was methylated (or mock treated) prior to the electroporation of the B31 A3 BBK32::Kan^r mutant. As we found for other derivatives of strain B31, the transformation efficiency of methylated pKFSS1k32 was (in this case 10-fold) higher than that for mock-treated plasmid, and 10 Str^r clones were isolated (Table 3). Similarly, the transformation efficiency of the vector control, pKFSS1, was increased (122-fold) upon the electroporation of the B31 A3 BBK32::Kan^r mutant. Several B31 A 3 BBK32::Kan^r clones that had been transformed with methylated pKFSS1k32 or pKFSS1 were tested by PCR for the presence of all endogenous plasmids (see Materials and Methods), and transformants carrying the full plasmid complement were selected for further analysis.

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FIG. 2. Generation of the B31 A3 BBK32::Kanr mutant. (A) A BBK32::Kanr insertion at codon 15 of BBK32 was crossed onto plasmid lp36 by homologous recombination to generate the B31 A3 BBK32::Kanr mutant. (B) The B31 A3 BBK32::Kanr mutant does not bind Fn. Whole-cell lysates from B31 A3 WT, the B31 A3 BBK32::Kanr mutant ("bbk32"), and the B31 A3 BBK32::Kanr mutant complemented with BBK32 on a shuttle plasmid ("bbk32/pk32") were immunoblotted with anti-MBP-BBK32 or anti-FlaB antiserum (as a loading control) or subjected to a gel overlay assay with purified Fn, followed by detection with a monoclonal anti-Fn antibody. -, anti-.

To confirm that the disruption of BBK32 with the Kan^r cassette resulted in the loss of BBK32 production, whole-cell lysates of B31 A3, the B31 A3 BBK32::Kan^r mutant, or the B31 A3 BBK32::Kan^r/pKFSS1k32 mutant (Fig. 2B, top) were subjected to immunoblotting with antiserum raised against an MBP-BBK32 fusion protein. As predicted, BBK32 was produced by B31 A3 and the B31 A3 BBK32::Kan^r/pKFSS1k32 mutant but not by the B31 A3 BBK32::Kan^r mutant. The B31 A3 BBK32::Kan^r/pKFSS1k32 mutant produced BBK32 at a level higher than that seen for B31 A3, presumably due to a copy number effect. Functional BBK32 production was also confirmed by Fn binding in gel overlay assays using purified Fn and detection with a monoclonal anti-Fn antibody (Fig. 2B, middle). A vector control strain, the B31 A3 BBK32::Kan^r/pKFSS1 mutant, was indistinguishable from the B31 A3 BBK32 mutant in both assays (data not shown).

To test if GAG binding of B31 A3 is diminished by the inactivation of BBK32, the wild-type (WT) B31 A3, the B31 A3 BBK32::Kan^r mutant, and the B31 A3 BBK32::Kan^r/pKFSS1k32 mutant were tested for binding to the GAGs heparin, dermatan sulfate, and chondroitin-6-sulfate immobilized in microtiter wells. As expected, none of the strains bound to chondroitin-6-sulfate, which is not efficiently recognized by B. burgdorferi (Table 4). The B31 A3 BBK32::Kan^r mutant bound to heparin and dermatan sulfate at levels indistinguishable from those seen for the parental strain B31 A3 and the B31 A3 BBK32/pKFSS1k32 complemented mutant. Similarly, when we measured binding to HEp-2 epithelial cells, which do not produce Fn at detectable levels (5), the binding of the B31 A3 BBK32::Kan^r mutant was indistinguishable from that of the WT (data not shown). As expected, binding by both WT B31 A3 and the B31 A3 BBK32::Kan^r mutant was diminished by the enzymatic removal of GAGs (data not shown). Together, these results indicate that BBK32-independent pathways are capable of promoting binding to purified GAGs or to GAGs expressed on the surfaces of mammalian cells.

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TABLE 4. B31 A3 BBK32::Kan mutant retains GAG-binding activitya

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The extremely low transformation efficiency of infectious strains of B. burgdorferi imposes enormous challenges to the genetic study of the pathogenesis of Lyme disease (27). At least part of this transformation barrier is mediated by R-M systems, because specific inactivation of the lp25-contained BBE02, which is homologous to known restriction enzyme genes, results in an approximately 10- to 50-fold increase in transformation efficiency (11). In addition, the absence of lp56, which encodes BBQ67, a putative restriction enzyme, is also associated with an 30-fold increase in transformation efficiency of shuttle plasmid pBSV2 (15).

In this study, we show that CpG methylation by the commercially available methylase M.SssI increased the transformation of B. burgdorferi strain B31 A3 approximately 40-fold. The enhancement of the transformation rate was not specific for a given antibiotic resistance marker, as the methylation of pKFSS1 or its derivative, pKFSS1k32, which confer Str^r, also resulted in a dramatic boost in transformation efficiency. Similarly, increases in transformation efficiency upon CpG methylation were observed for four B31-related strains. However, it is not clear how conserved R-M systems are among B. burgdorferi isolates (19), and further studies are required to determine if M.SssI will enhance the transformation of diverse B. burgdorferi strains. It should be noted that we found considerable experiment-to-experiment variation in transformation efficiency in this study, consistent with previous observations (11, 15). In addition, the extremely low number of transformants (<10) often obtained when unmethylated plasmid was used resulted in considerable variation in the calculated relative increase in transformation rate upon methylation. Although more extensive titration of the amount of DNA used in each condition might have resulted in a smaller range of transformation efficiencies, such a result would have been unlikely to alter our central finding that CpG methylation results in a dramatic increase in the transformation efficiency of B. burgdorferi.

To test the utility of in vitro methylation in the genetic analysis of a putative virulence factor, we utilized M.SssI treatment to generate a plasmid-complemented version of a B31 A3 BBK32 mutant. Given that at least three other surface proteins, namely, DbpA, DbpB, and Bgp, bind to GAGs, we tested whether the inactivation of BBK32 results in a discernible GAG-binding defect. Indeed, the B31 A3 BBK32::Kan^r mutant bound to purified heparin and dermatan sulfate and to GAGs expressed on cultured mammalian cells in a manner that was indistinguishable from that seen for the parental WT and the plasmid-complemented strains, suggesting that the GAG-binding activities of BBK32 can be compensated for by other GAG-binding adhesins. When we tested an independently derived B31 derivative harboring an insertion in BBK32 (30), we found a mild (35%) defect in GAG binding (Q. Chen, unpublished data), suggesting that depending on the particular strain background, a BBK32 mutant may display a subtle GAG-binding phenotype.

The loss of lp56 is associated with an 30-fold increase in transformation efficiency (15), which roughly correlates with the 40-fold increase in pBSV2 transformation efficiency after CpG methylation (Table 1). In addition, M.SssI treatment of the plasmid did not dramatically increase the transformation efficiencies of strains that lack lp56 (Table 2). Although we cannot rule out that the marginal increase in transformation efficiencies sometimes associated with the methylation of plasmids prior to the electroporation of strains lacking lp56 reflects protection from other putative R-M systems (8, 26), the results overall are consistent with the hypothesis that the most important effect of CpG methylation is the bypass of an lp56-dependent restriction barrier. In contrast, although the presence of BBE02 diminished transformation rates, as previously observed (11), reinforcing the notion that BBE02 represents a bona fide restriction barrier, the increase in transformation associated with CpG methylation was not dependent on its presence. Because BBQ67 on lp56 encodes a putative restriction enzyme, we speculate that BBQ67 encodes an enzyme that cleaves foreign DNA in a manner that can be inhibited by CpG methylation. Further investigation is required to test this.

Strains lacking lp25, lp56, or both have been used in genetic studies of B. burgdorferi because their elevated transformation rates compared to those of WT B. burgdorferi facilitate genetic manipulation (1, 16, 30, 31, 39). Strains lacking lp25 are noninfectious due to the absence of pncA (BBE22), encoding a nicotinamidase that is essential for infectivity, but such strains have been used for genetic studies of pathogenicity because their infectivity can be restored by the introduction of pncA on a shuttle plasmid (22, 30). Nevertheless, plasmid-deficient strains that have been complemented with a single plasmid-contained gene may not completely reflect WT bacteria, because endogenous B. burgdorferi plasmids may encode multiple factors required for full infectivity. For example, lp25 contains not only pncA but also BBE16, which is required for the efficient retention of B. burgdorferi in larval ticks and contributes to infectivity in mice (25). In vitro methylation may be a means to more easily perform genetic studies on B. burgdorferi strains that carry all endogenous plasmids. The BBE02 mutant strain 5A4NP1 retains all endogenous plasmids and is moderately transformable (11). We showed here that CpG methylation dramatically boosts even the elevated transformation rate of 5A4NP1, a feature that may make the generation of comprehensive plasmid libraries in B. burgdorferi feasible. Finally, the restriction of electroporated DNA may be a limiting factor for methods of genetic manipulation besides simple plasmid transformation, e.g., transposon mutagenesis or allelic exchange, and therefore methylation may have the potential for widespread utility in the study of the biology and pathogenesis of B. burgdorferi.

 
We thank Steve Norris, Hiroki Kawabata, Patti Rosa, Scott Samuels, Jorge Benach, and Jon Skare for strains, reagents, and/or helpful discussion, Kayla Hagman and Brian Skehan for technical advice, and Jenifer Coburn for both advice and training.

This work was supported by NIH R01 AI37601 to J.M.L.

 
* Corresponding author. Mailing address: Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655. Phone: (508) 856-4059. Fax: (508) 856-3355. E-mail: John.Leong{at}umassmed.edu

Published ahead of print on 10 October 2008.

Present address: Monsanto Company, 325 Vassar St., Cambridge, MA 02139.

Present address: Monsanto Company, 800 N. Lindbergh Ave., St. Louis, MO 63167.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Journal of Bacteriology, December 2008, p. 7885-7891, Vol. 190, No. 24
0021-9193/08/$08.00+0     doi:10.1128/JB.00324-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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