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Journal of Bacteriology, July 2005, p. 4822-4829, Vol. 187, No. 14
0021-9193/05/$08.00+0     doi:10.1128/JB.187.14.4822-4829.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Analysis of the ospC Regulatory Element Controlled by the RpoN-RpoS Regulatory Pathway in Borrelia burgdorferi

Xiaofeng F. Yang,1 Meghan C. Lybecker,2 Utpal Pal,3 Sophie M. Alani,1 Jon Blevins,1 Andrew T. Revel,1 D. Scott Samuels,2 and Michael V. Norgard1*

Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390,1 Division of Biological Sciences, The University of Montana, Missoula, Montana 59812,2 Section of Rheumatology, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 065203

Received 16 February 2005/ Accepted 15 April 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Outer surface lipoprotein C (OspC) is a key virulence factor of Borrelia burgdorferi. ospC is differentially regulated during borrelial transmission from ticks to rodents, and such regulation is essential for maintaining the spirochete in its natural enzootic cycle. Recently, we showed that the expression of ospC in B. burgdorferi is governed by a novel alternative sigma factor regulatory network, the RpoN-RpoS pathway. However, the precise mechanism by which the RpoN-RpoS pathway controls ospC expression has been unclear. In particular, there has been uncertainty regarding whether ospC is controlled directly by RpoS ({sigma}s) or indirectly through a transactivator (induced by RpoS). Using deletion analyses and genetic complementation in an OspC-deficient mutant of B. burgdorferi, we analyzed the cis element(s) required for the expression of ospC in its native borrelial background. Two highly conserved upstream inverted repeat elements, previously implicated in ospC regulation, were not required for ospC expression in B. burgdorferi. Using similar approaches, a minimal promoter that contained a canonical –35/–10 sequence necessary and sufficient for {sigma}s-dependent regulation of ospC was identified. Further, targeted mutagenesis of a C at position –15 within the extended –10 region of ospC, which is postulated to function like the strategic C residue important for E{sigma}s binding in Escherichia coli, abolished ospC expression. The minimal ospC promoter also was responsive to coumermycin A1, further supporting its {sigma}s character. The combined data constitute a body of evidence that the RpoN-RpoS regulatory network controls ospC expression by direct binding of {sigma}s to a {sigma}s-dependent promoter of ospC. The implication of our findings to understanding how B. burgdorferi differentially regulates ospC and other ospC-like genes via the RpoN-RpoS regulatory pathway is discussed.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Borrelia burgdorferi, the spirochetal agent of Lyme disease, is maintained in nature via a complex enzootic cycle involving Ixodes scapularis ticks and small rodents (50, 51). During transmission, the spirochete differentially expresses many of its constituent proteins for adaptation to its diverse host environments. Among those differentially regulated in this manner are outer surface lipoproteins A (OspA) and C (OspC) (11, 31, 32, 47, 48). OspA is expressed principally by spirochetes harbored in unfed, flat ticks and functions as an essential adhesion molecule for colonization and survival within the tick midgut (34-36, 62). OspC, which is upregulated in B. burgdorferi at the time of tick engorgement, is essential for the B. burgdorferi infection of mice (21) and for the migration of B. burgdorferi from tick midguts to salivary glands (15, 20, 37).

Given their importance in the life cycle of B. burgdorferi and/or the pathogenesis of Lyme disease, the elucidation of the regulatory networks that govern the differential expression of OspA and OspC has become a central focus for understanding the molecular mechanisms by which B. burgdorferi adapts to its disparate host environments. However, the discernment of the molecular basis of gene regulation in B. burgdorferi generally has been hampered by a lack of systems for genetically manipulating the spirochete, particularly for virulent strains (7, 56). Nonetheless, recent advances in borrelial genetics have led to the development of selectable markers and shuttle vectors (5, 12, 14, 16, 44, 45, 53), targeted gene inactivations (for a review, see reference 41), and identification of B. burgdorferi virulence factors (21, 37, 39, 62). Similar advances also have culminated in the discovery of the first B. burgdorferi genetic regulatory network, the RpoN-RpoS pathway (25, 61). In this pathway, a two-component response regulator, Rrp2, functions as an enhancer-binding protein (EBP), along with the alternative sigma factor RpoN ({sigma}N), to control the expression of another alternative sigma factor, RpoS ({sigma}s). RpoS, in turn, regulates the expression of OspC, other "group I" lipoproteins (e.g., DbpA and the Mlp family) (58, 59), and additional infection-associated immunogens (61).

The discovery of the RpoN-RpoS regulatory network prompts an important question concerning how {sigma}s, in particular, induces the expression of ospC and other virulence-associated genes. One possibility is that {sigma}s controls ospC expression via an unidentified transactivator, which could bind to the regulatory region for the activation of ospC. Relative to this hypothesis, two sets of conserved inverted repeats (IRs) located upstream of the ospC promoter (Fig. 1) have been proposed to be candidate binding sites for a potential transactivator(s) (29, 55). An alternative possibility is that ospC contains a {sigma}s-dependent promoter; in this case, {sigma}s would directly control the transcriptional activation of ospC by binding to the ospC promoter. Along these lines, predicated on determinations of transcriptional initiation, ospC has been predicted to possess a typical –35/–10 {sigma}70 promoter (18, 28, 29, 33). However, sequence information alone is likely insufficient for distinguishing between {sigma}s and {sigma}70 promoters, inasmuch as {sigma}s and {sigma}70 are highly related and recognize the same core promoter elements (19, 24). Recent studies have shown that {sigma}s promoter selectivity is attained by several promoter-specific sequence elements, architectural DNA-binding proteins, or DNA topology (24). For example, in Escherichia coli, {sigma}s preferentially recognizes promoters on a relaxed template (26) and DNA relaxation is required for transcription by the holoenzyme containing {sigma}s (E{sigma}s) during the cellular response to osmotic stress (6). Interestingly, in B. burgdorferi, the regulation of ospC gene expression involves not only the RpoN-RpoS signaling pathway (25) but also DNA supercoiling (1), raising the possibility that ospC utilizes a {sigma}s-dependent promoter. Additional experiments are therefore warranted to define whether the ospC gene utilizes a {sigma}70 or a {sigma}s promoter.



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  		FIG. 1. Upstream regions of the ospC genes of B. burgdorferi strains 297 and B31. Pairs of divergent arrows denote the two putative inverted repeat elements (IR1 and IR2). The –35 and –10 promoter elements, ribosomal-binding site (RBS), and the ATG start codon are shown in boldface type. Filled arrowheads indicate the starting positions of each deletion ({Delta}) described in the legend to Fig. 3A. The –15 C residue (boxed) within the extended –10 region was targeted for mutagenesis. The asterisks mark two previously identified transcriptional initiation sites (28, 29, 33). min, start of deletion made to yield the minimal promoter construct diagrammed in Fig. 3A.

 
Regarding initial efforts to investigate ospC promoter activity, Sohaskey et al. (49) first showed that when transiently expressed in B. burgdorferi, a 551-bp promoter region of ospC was capable of driving the expression of a chloramphenicol acetyltransferase (CAT) reporter gene. Carroll et al. (8) later constructed a stable shuttle vector for B. burgdorferi in which the 179-bp region upstream of ospC (containing the IRs) was fused to a green fluorescent protein (gfp) reporter gene. When subjected to different environmental conditions, the construct in B. burgdorferi regulated the expression of GFP akin to OspC expression (8). More recently, Eggers et al. (13) further analyzed the activity of the ospC promoter in a surrogate E. coli background using another gfp reporter system. Despite these efforts, a direct examination of the ospC promoter element and its influence on OspC expression in the relevant B. burgdorferi background has been lacking. To this end, we previously constructed an ospC mutant complemented with a shuttle vector harboring a complete wild-type ospC gene (37). Herein, we used this construct to generate nested deletions within the upstream regulatory region of ospC; this approach has allowed us to identify the cis element involved in ospC regulation in the native background of B. burgdorferi. The implications of our findings relative to the control of ospC expression by the RpoN-RpoS regulatory pathway in B. burgdorferi are discussed.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains and culture conditions. Low-passage, wild-type clone BbAH130 of B. burgdorferi strain 297, the OspC-deficient mutant, the OspC-deficient mutant complemented with a shuttle vector carrying a wild-type ospC gene, and the RpoS-deficient mutant were described previously (25, 37). High-passage B. burgdorferi strain 297 was obtained by continuously passaging it more than 230 times in vitro, and a high-passage B. burgdorferi strain B31 (B31-A) was provided by Patricia Rosa (5). Spirochetes stored at –70°C were inoculated into Barbour-Stoenner-Kelly-H (BSK-H) medium (Sigma Chemical Co., St. Louis, MO) (38) and were cultivated under various environmental conditions of temperature (23o or 37°C) and pH (7.5 or 8.0) (58). Cultures were harvested for analysis at the late logarithmic phase of growth (5 x 107 cells/ml), as determined by enumerating spirochetes via dark-field microscopy. For coumermycin A1 experiments, E. coli strain DH5{alpha} was grown at 23°C and then was diluted 1:100 in Luria-Bertani broth (with or without 1 µg/ml of coumermycin A1). Cultures were allowed to grow at 23°C for about 2 h (optical density at 600 nm of about 0.4). B. burgdorferi was cultivated in BSK-H medium at 23°C until the mid-logarithmic phase of growth, at which time the culture was diluted 1:100 in BSK-H medium containing or lacking 20 ng/ml of coumermycin A1; the cultures were then allowed to grow at 23°C (for about 2 weeks) to the mid-logarithmic phase. E. coli TOP10 (Invitrogen, Carlsbad, CA) was used as a host for cloning experiments.

Promoter mutagenesis. Construction of the shuttle vector pOspC-wt (pBSV2-OspC) was described previously (37). pOspC-wt contains the complete coding region of ospC as well as the 141-bp sequence upstream of the ATG initiation codon (see Fig. 3A). To construct shuttle vectors with various deletions in the region of the ospC promoter, a series of PCRs using the Expand High Fidelity PCR system (Roche Diagnostics, Indianapolis, IN) were performed. The template was pOspC-wt, and the primer pairs for each construct are listed in Table 1. The resulting PCR fragments were cloned into pCR-XL-TOPO (Invitrogen, Carlsbad, CA). The resulting plasmids and pOspC-wt were then digested with HindIII and XbaI and ligated together to generate pOspC-{Delta}IR1, pOspC-{Delta}IR1+2, pOspC-min, and pOspC-{Delta}-35. To construct a pOspC-C/A point mutation, a QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used with the corresponding primer pairs (Table 1). The template used was pOspC-wt. Sequence analysis was performed to verify the desired mutation. A resultant HindIII-XbaI fragment was excised and then subcloned back into pOspC-wt to ensure that only the desired mutation was present in pOspC-C/A.



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  		FIG. 3. Influence of upstream cis elements on ospC expression. (A) Diagram of a series of shuttle vector constructs containing various versions of the upstream region of ospC. Nucleotide positions are relative to the ATG start codon, where A is position +1. All six constructs are denoted by the prefix pOspC- followed by the pertinent deletion or mutation (indicated at the left). wt, wild-type aspC gene; {Delta}IR1, deletion of IR1; {Delta}IR1+2, deletion of IR1 and IR2; min, minimal promoter for ospC; {Delta}-35, deletion of IR1, IR2, and the –35 sequence; C/A, targeted point mutation of C (within the extended –10 region) to A. (B) Immunoblot of the ospC mutant (OspC–) transformed with the various pOspC shuttle vectors shown in panel A. All cultures were grown at 37°C (pH 7.5) and were harvested at the late logarithmic phase of growth. Antibodies directed against OspC and FlaB were pooled. Numbers at the left denote protein molecular mass markers (in kilodaltons). Densitometry results for OspC are as follows: lane 1, 2.0; lane 2, 0.008; lane 3, 2.3; lane 4, 2.2; lane 5, 2.0; lane 6, 1.7; lane 7, 0.05; and lane 8, 0.2. WT, wild type.

 

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  		TABLE 1. Oligonucleotide primers used in this study

 
B. burgdorferi transformations. The OspC-deficient (streptomycin-resistant) mutant (37) or the RpoS-deficient (erythromycin-resistant) mutant (25) was made electrocompetent and transformed with various shuttle vector constructs. Transformants were selected by kanamycin treatment. The general procedure for transforming B. burgdorferi has been described previously (42, 62). Twenty to 50 µg of plasmid DNA was used in each transformation experiment. After electroporation, the mixture (0.05 ml) was diluted into 100 ml of BSK-H medium and was incubated at 32°C overnight to allow for recovery. Relevant antibiotics were added to the cultures in the following final concentrations: 50 µg/ml for streptomycin, 50 ng/ml for erythromycin, and 200 µg/ml for kanamycin. The cultures were then aliquoted into multiple 96-well tissue culture plates (230 µl/well). Two to 3 weeks after plating, the wells that contained positive cultures were identified by color change of the medium; the presence of viable spirochetes was verified by dark-field microscopy. In general, 10% or less of the wells were positive for growth and therefore were considered to be clonal as a result of limiting dilution (10). To confirm that these clones harbored the desired shuttle vector, whole-cell lysates were used to transform E. coli TOP10. Plasmid DNA was rescued from E. coli transformants, and restriction digestions were performed to verify recovery of the pertinent plasmid.

RT-PCR. Total RNAs from B. burgdorferi were isolated using a NucleoSpin RNA II purification kit (BD Biosciences, Palo Alto, CA) according to instructions provided by the manufacturer. To remove potential DNA contamination, RNA was further treated with DNA-free DNase Treatment and Removal Reagents (Ambion, Austin, TX). The concentration of RNA was determined by UV spectrophotometry using an ND-1000 UV-Vis spectrophotometer (NanoDrop Technologies, Wilmington, DE). RT-PCRs were performed using a Titan One Tube RT-PCR system (Roche Diagnostics, Indianapolis, IN). Conditions for reverse transcriptase (RT)-PCRs were as recommended by the manufacturer; a 50-µl buffered reaction mixture contained 40 ng of bacterial RNA, 0.4 µM concentrations of each of the oligonucleotide primers, 5 mM dithiothreitol, 0.2 mM concentrations of each deoxynucleoside triphosphate, 5 U of RNase inhibitor, and 1 µl of enzyme mixture. Primers used for amplification of ospC and rpoS transcripts were described previously (25). Five microliters of the RT-PCR mixture was used for agarose gel electrophoresis.

SDS-PAGE and immunoblotting. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were carried out as previously described (60). Cells were loaded in gel lanes at 5 x 107 cells per lane. Rat polyclonal antisera against OspC and {sigma}s and monoclonal antibody 8H3-33 against FlaB were described previously (58). For coumermycin A1 experiments, OspC was detected by immunoblotting with monoclonal antibody 4B8F4 (for detection in E. coli) (33) or with rabbit polyclonal antiserum (for detection in B. burgdorferi). Most immunoblots were developed colorimetrically; for some of these, densitometry was used to assess the relative amounts of protein per gel lane using a Kodak Gel Logic 200 instrument with 1D image analysis software (version 3.6; Kodak, Rochester, NY). For other selected immunoblots (e.g., coumermycin A1 experiments and certain experiments involving the detection of {sigma}s), blotted membranes were developed by chemiluminescence using either ECL Plus Western Blotting Detection system (Amersham Biosciences, Piscataway, NJ) or Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Sciences, Boston, MA).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
trans-Complementation for studying the ospC promoter. As an initial approach for assessing how ospC is controlled by RpoS, we exploited a previously constructed ospC mutant of B. burgdorferi (OspC–) complemented with a wild-type version of ospC that was cloned into the shuttle vector pBSV2 (pOspC-wt) (37, 53) (Fig. 2A and B). The advantage of our approach was that ospC expression could be assessed directly via the detection of OspC in B. burgdorferi. To first examine whether regulation of the cloned ospC gene in pOspC-wt was similar to that of a native, endogenous ospC gene, wild-type B. burgdorferi and the complemented OspC mutant (OspC–/pOspC-wt) were cultivated in BSK-H medium under various temperature and pH conditions. In both wild-type B. burgdorferi and the OspC–/pOspC-wt complemented strain, ospC expression was induced at an elevated temperature (Fig. 2C, lanes 2 and 8) and was inhibited by increased culture pH (Fig. 2C, lanes 3 and 9). These results confirmed that the ospC gene located on a shuttle vector responded to environmental stimuli in a manner similar to that of an endogenous ospC gene, indicating the suitability of trans-complementation for assessing ospC gene regulation.



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  		FIG. 2. Approach for studying the regulation of ospC. (A) Structure of the ospC locus in cp26 (top) and ospC disruption by the aadA (streptomycin resistance) marker (16) (bottom). Only the relevant portions of cp26 are shown. WT, wild type. (B) The shuttle vector pBSV2 (53) harboring a wild-type copy of ospC (pOspC-wt) used for genetic complementation of the ospC mutant of B. burgdorferi. In this construct, the kanamycin resistance gene is driven by the constitutive promoter of the borrelial flgB gene (PflgB-Kan). (C) SDS-PAGE (Coomassie blue staining) (top) and immunoblotting (bottom) of whole-cell lysates of B. burgdorferi strains cultivated under various conditions of temperature and pH. M, molecular mass marker (in kilodaltons). WT, parental low-passage B. burgdorferi 297. OspC–, the ospC mutant. OspC–/pOspC-wt, the ospC mutant complemented with the shuttle vector pOspC-wt. Lanes 1, 4, and 7, spirochetes cultivated in BSK-H medium (pH 7.5) incubated at 23°C. Lanes 2, 5, and 8, spirochetes cultivated in BSK-H medium (pH 7.5) at 37°C. Lanes 3, 6 and 9, spirochetes cultivated in BSK-H medium (37°C) adjusted to pH 8.0. The migration of OspC in the Coomassie blue-stained gel (top) is indicated by the arrow. In the immunoblot (bottom), only the relevant portion of the gel is shown.

 
IR elements are not required for the expression of ospC. Previously, two partially overlapping IRs were identified upstream of the ospC gene of B. burgdorferi (29) (Fig. 1). These IRs are highly conserved among ospC homologs of many B. burgdorferi strains (29, 55) and have been hypothesized to be involved in ospC regulation (1, 55). To assess the potential role(s) of the IRs in ospC expression, we first generated complementation constructs lacking either IR1 or both IR1 and IR2 (Fig. 3A). The resulting mutated constructs were then transformed into the OspC– mutant, and OspC expression was monitored by immunoblot analysis. Densitometry revealed that plasmids harboring either deletion construct in the OspC– mutant still promoted the expression of OspC (lanes 4 and 5) at levels close to that for the wild type (lane 3), indicating that neither of the two IRs upstream of ospC is required for ospC expression.

Minimum promoter for ospC expression. Deletion of the two IRs removed the majority of the sequence upstream of the –35/–10 promoter of ospC that was previously noted by others (18, 28, 29, 33) (Fig. 3A). To further examine whether an additional 18 bp remaining immediately upstream of the –35 consensus sequence played a role in ospC regulation, the 18-bp region was deleted. The resulting construct contained only the minimal –35/–10 sequence (pOspC-min) (Fig. 3A). The OspC– mutant of B. burgdorferi complemented with pOspC-min readily expressed OspC (Fig. 3B, lane 6). However, a complementation construct lacking an additional 17 nucleotides extending into the putative –35 consensus sequence had greatly diminished OspC expression in the OspC– mutant (Fig. 3B, lane 7). These results indicate that a minimal –35/–10 ospC promoter sequence is necessary and sufficient for OspC expression in B. burgdorferi.

Recent data demonstrate that although the –35/–10 consensus sequences for {sigma}s and {sigma}70 promoters tend to be indistinguishable (19, 24), some minor sequence differences may exist, especially within the extended –10 element (4). In this regard, a –13 C residue in this extended –10 region is strategic for interacting with E{sigma}s in E. coli (4). We therefore mutagenized a candidate C residue conserved at position –15 within the ospC minimal promoter (Fig. 1) and assessed the influence of this point mutation on ospC expression. As shown in Fig. 3B (lane 8), the OspC– mutant complemented with this construct expressed significantly lower levels of OspC than the mutant transformed with the wild-type ospC gene.

To examine whether the ospC gene driven only by a minimal promoter remained responsive to environmental stimuli, B. burgdorferi strain OspC–/pOspC-min was cultivated under various conditions of temperature and pH. As in the case of either wild-type B. burgdorferi or OspC–/pOspC-wt (Fig. 2C), ospC expression in the OspC–/pOspC-min strain was inhibited by decreased temperature and increased culture pH (Fig. 4, lanes 1 and 3), indicating normal ospC regulation.



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  		FIG. 4. Influence of culture temperature and pH on ospC expression driven by a minimal promoter. Spirochetes were cultivated in BSK-H medium (pH 7.5) at 23°C (lanes 1) or 37°C (lanes 2) or adjusted to pH 8.0 in medium at 37°C (lanes 3). Whole-cell lysates were either stained with Coomassie blue (left) or immunoblotted with pooled antibodies directed against FlaB and OspC (right). Numbers at the left denote protein molecular mass markers (in kilodaltons).

 
{sigma}s controls ospC expression via the minimal promoter. Wild-type ospC expression is controlled via the RpoN-RpoS regulatory network, which culminates in {sigma}s regulating ospC expression (25, 61). To garner evidence that {sigma}s directly controls ospC expression via interaction with the minimal promoter, the complementation construct pOspC-min was transformed into an rpoS-deficient mutant of B. burgdorferi (25). The resulting strain, RpoS–/pOspC-min, no longer expressed ospC at either the RNA or protein level (Fig. 5), demonstrating that {sigma}s controls ospC expression via the minimal promoter.



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  		FIG. 5. ospC expression driven by the minimal promoter is {sigma}s dependent. Top panel, immunoblot of whole-cell lysates of the ospC mutant (OspC–), ospC mutant transformed with pOspC-min (OspC–/pOspC-min), or an rpoS mutant (RpoS–) transformed with pOspC-min. All cultures were grown at 37°C and harvested at the late logarithmic phase of growth. Antisera directed against {sigma}s and OspC were pooled for immunoblotting. Numbers at the left denote protein molecular mass markers (in kilodaltons). Bottom two panels, RT-PCR and agarose gel electrophoresis for the detection of ospC and rpoS transcripts, respectively. Only the relevant portions of the agarose gel are shown.

 
Influence of coumermycin A1 on activity of the ospC promoter. Elevated culture temperature reduces DNA supercoiling in B. burgdorferi that, in turn, induces the expression of ospC (1). This same OspC induction was mimicked by treatment of B. burgdorferi with coumermycin A1, an inhibitor of DNA gyrase that also culminates in decreased DNA supercoiling (43). Relaxation of supercoiling is one of the factors that enhances promoter selectivity by E{sigma}s (24). In addition, the IRs were postulated to play a role in the regulation of ospC by supercoiling (1). To examine which ospC promoter element(s) responds to DNA supercoiling, E. coli (Fig. 6A) and the B. burgdorferi OspC– mutant (Fig. 6B) carrying the complementing plasmids were cultivated at room temperature and treated with coumermycin A1. In the heterologous E. coli strain transformed with a wild-type ospC gene, OspC is not expressed by E. coli cultivated at room temperature, whereas coumermycin A1 treatment induced the expression of OspC in E. coli cultivated under the same conditions (Fig. 6A). This same coumermycin A1 induction effect was obtained in E. coli when IR1 of ospC was absent (Fig. 6A). However, plasmid constructs that lacked both IR1 and IR2 (pOspC-{Delta}IR1+2) or that contained only the minimal promoter (pOspC-min) failed to respond to coumermycin A1 treatment (Fig. 6A). In wild-type B. burgdorferi cultivated at room temperature for 2 weeks, 20 ng/ml of coumermycin A1 also induced the expression of ospC (Fig. 6B). The same coumermycin A1 induction effect was observed with the OspC– strain of B. burgdorferi complemented with either a wild-type copy of ospC (pOspC-wt) or the minimal promoter of ospC (pOspC-min) (Fig. 6B). Treatment of the same complemented strains of B. burgdorferi with 100 ng/ml of coumermycin A1 for 24 h yielded the same results (data not shown). Of note, the minimal promoter of ospC remained responsive to coumermycin A1 treatment and, thus, to a DNA supercoiling effect in B. burgdorferi, but not in E. coli transformed with the same plasmid.



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  		FIG. 6. Influence of coumermycin A1 on ospC expression driven by various promoter constructs. (A) E. coli strain DH5{alpha} carrying various mutant ospC promoters (top of panel A; abbreviations as defined for Fig. 3A) was treated (+) or not treated (–) at 23°C with 1 µg/ml of coumermycin A1. (B) Wild-type (WT) B. burgdorferi or various complemented derivatives of the ospC mutant (OspC–) (top of panel B; abbreviations as in Fig. 3A) were either treated (+) or not treated (–) at 23°C with 20 ng/ml of coumermycin A1. Whole-cell lysates were then immunoblotted with either monoclonal antibody (panel A) or antiserum (panel B) directed against OspC and developed using chemiluminescence.

 
Loss of ospC expression correlates with the loss of {sigma}s in high-passage populations of B. burgdorferi. Continuous serial passage of B. burgdorferi in vitro results in a reduction or loss of OspC expression (30, 47, 57). Inasmuch as our data indicate that the expression of ospC is under the direct control of {sigma}s, we investigated whether the loss of OspC expression in high-passage populations of B. burgdorferi correlated with a concomitant loss of {sigma}s. To examine this, low- and high-passage populations of B. burgdorferi strains 297 and B31 were subjected to SDS-PAGE and immunoblotting for colorimetric detection of OspC and {sigma}s (Fig. 7). OspC was absent in high-passage strain B31, a finding that correlated with an undetectable level of {sigma}s (Fig. 7). A minimal quantity of OspC was detected in high-passage strain 297 even though {sigma}s also was not detectable in the same strain (Fig. 7). However, when similar immunoblots were developed by a more sensitive chemiluminescence method, a small amount of {sigma}s was detected (data not shown). These combined findings suggest that the continuous in vitro passage of B. burgdorferi results in a loss of {sigma}s that, in turn, leads to the loss of ospC expression.



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  		FIG. 7. {sigma}s expression correlates with OspC expression in low- and high-passage strains of B. burgdorferi 297 and B31. Low-passage (LP) and high-passage (HP) B. burgdorferi strains 297 and B31 were cultivated in BSK-H medium at 37°C and were harvested at the late logarithmic stage of growth. Antisera directed against {sigma}s and OspC were pooled for immunoblotting. Numbers at the left denote protein molecular mass markers (in kilodaltons).

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The B. burgdorferi genome encodes about 175 known or putative lipoprotein genes comprising about 10% of the total B. burgdorferi genomic coding capacity (9, 17). This remarkable feature distinguishes B. burgdorferi from virtually all other pathogenic bacteria (17) and has engendered the contention that the membrane lipoproteins likely play an important role in the adaptation of B. burgdorferi to both its arthropod and mammalian hosts (22, 54). In this regard, it is increasingly well documented that a number of these lipoproteins indeed are differentially expressed during the transmission of B. burgdorferi between ticks and mammals (2); recent demonstration of the essential roles for the OspA and OspC in spirochete transmission and mammalian infection further underscores the importance of lipoproteins in the complex life cycle of B. burgdorferi (21, 37, 62). Continuing efforts to elucidate the molecular mechanisms that modulate the expression of borrelial lipoproteins thus likely will hold the key for understanding how B. burgdorferi survives in nature via its complex adaptive responses (22, 41, 46).

Previously, we identified a novel genetic regulatory network, the RpoN-RpoS pathway, that governs the expression of several borrelial lipoproteins, including OspC, DbpA, and the Mlp family (i.e., "group I" lipoproteins) (58). In the present study, we exploited ospC as a model system for further understanding the mechanism(s) governing the expression of lipoprotein genes regulated by the RpoN-RpoS pathway. Regulation by the RpoN-RpoS pathway, however, is predicated on the requirement for a {sigma}s-dependent promoter to drive expression of the downstream target gene. Prior reports, however, have suggested that the ospC promoter was the {sigma}70 type (based on sequence analysis and primer extension studies) (18, 28, 29, 33), and the additional presence of two conserved IR elements upstream of the ospC gene potentially also constituted a binding site(s) for a putative transactivator of ospC regulation. As such, one attractive hypothesis has been that ospC actually is regulated indirectly by the RpoN-RpoS pathway via the induction of a requisite transactivator, followed by expression of ospC via a {sigma}70-like promoter.

By exploiting an ospC mutant of B. burgdorferi and a shuttle vector carrying a wild-type copy of ospC, herein we now have provided several lines of evidence that the ospC gene likely is regulated directly by the binding of {sigma}s to its {sigma}s-dependent promoter. First, the two IRs (potential transactivator-binding site[s]) were dispensable; a minimal –35/–10 promoter sequence was both necessary and sufficient for ospC expression. Second, the minimal ospC promoter defined in our study remained responsive to key environmental stimuli typically associated with the regulation of ospC (e.g., temperature and pH). Third, as shown in complementation studies with a {sigma}s-deficient mutant of B. burgdorferi, the ospC gene containing the minimal promoter remained {sigma}s dependent. Fourth, consistent with the fact that E{sigma}s polymerase binds preferentially to relaxed DNA (26), ospC expression controlled by the minimal promoter increased in response to coumermycin A1.

The alternative sigma factor {sigma}s is a general stress factor that controls the expression of many genes essential for bacterial stationary-phase adaptation (23). Although {sigma}s modulates the expression of a distinct group of genes, its structure and molecular function are very similar to those of the housekeeping sigma factor, {sigma}70 (RpoD), and the consensus promoter sequence for E{sigma}s is similar to that used by E{sigma}70 (19, 24). In fact, a typical {sigma}s-dependent promoter binds to both E{sigma}s and E{sigma}70 in vitro, thereby precluding the use of electrophoretic gel shift assays for distinguishing between {sigma}s and {sigma}70 promoters. As such, there has been a great interest in elucidating the mechanism by which E{sigma}s recognizes and discriminates its cognate promoter in vivo. In E. coli, several factors, such as a high-salt condition, presence of an additional trans regulator, or the local relaxation of target DNA, have been shown to contribute to promoter selectivity for E{sigma}s (24). In addition, certain promoter sequence elements, especially a C nucleotide within the extended –10 region, can play an important role in E{sigma}s selectivity (4). This C nucleotide was suggested to interact with a key Lys173 residue of {sigma}s in E. coli (4). In the present study, we showed that mutation of a candidate –15 C nucleotide within the extended –10 region of the minimal promoter for ospC greatly diminished OspC expression, further suggesting that a C residue within this extended –10 region also is important for {sigma}s-dependent activation in B. burgdorferi. On the other hand, {sigma}s of B. burgdorferi does not possess an obvious Lys residue that corresponds to Lys173 of E coli {sigma}s. Further experiments are therefore warranted to identify the residue(s) in B. burgdorferi {sigma}s involved in interacting with the –15 C nucleotide.

Eggers et al. (13) recently performed an analysis of promoter elements involved in the expression of ospC and other B. burgdorferi genes using a GFP reporter system in E. coli. Although our results regarding ospC expression are largely in agreement with those of Eggers et al. (13), some differences are noteworthy. Eggers et al. (13) showed that deletion of the IR elements upstream of the ospC promoter significantly reduced the level of ospC promoter activity in B. burgdorferi; it therefore was concluded that the upstream (IR) region likely functions as an enhancer-binding site for maximal expression of ospC. In contrast, densitometry performed on immunoblots indicated that deletion of both IRs did not dramatically affect ospC expression in B. burgdorferi. The reason for this discrepancy is unclear but may be grounded in the fact that we assayed for native OspC expression, whereas Eggers et al. (13) used a GFP-based reporter assay.

Much of the work reported by Eggers et al. (13) exploited E. coli as a surrogate system for assessing ospC promoter activity. Whereas using surrogate systems can be valuable, in our studies we noted differences in the regulation of ospC when present in E. coli or its native B. burgdorferi background. For example, IR2 was required for ospC induction by coumermycin A1-induced relaxation of DNA supercoiling in E. coli, but not in B. burgdorferi. This may be due to differences in {sigma}s function, plasmid topology, or other trans-acting factors that exist between the two species. Thus, with continuing advances in borrelial genetics, particularly the applications of targeted mutagenesis, gene inactivation, and improved shuttle vectors, studying B. burgdorferi gene regulation in the relevant native background should be the preferred experimental approach.

A distinguishing feature of B. burgdorferi as a prokaryote is its remarkable plasmid complexity (52). While the extremely large complement of circular and linear plasmids in B. burgdorferi likely offers a genetic plasticity that allows it to adapt readily to its diverse arthropod and mammalian hosts, it also may engender the genetic instability that typifies the population biology of B. burgdorferi (3, 52). The spontaneous loss by B. burgdorferi in culture of lp25, encoding the pncA gene essential for B. burgdorferi infectivity (39), is a prime example of this kind of genetic instability (27, 40, 57). Another genetic phenomenon, yet to be elucidated, is that continuous passage of B. burgdorferi in vitro also results in greatly diminished ospC expression (47, 57). Our data appear to provide the first insights into the loss of ospC expression. Namely, the loss of ospC expression correlated with the apparent loss of (or reduction in) {sigma}S within the B. burgdorferi population; such loss in OspC expression was not attributed to the loss of cp26 or the loss of ospC or rpoS (data not shown). Although the precise mechanism accounting for this phenomenon thus remains unclear, we hypothesize that continuous in vitro passage of B. burgdorferi may adversely influence the ability of Rrp2 to become activated by its cognate histidine kinase, thereby blocking rpoS expression. Alternatively, {sigma}S in B. burgdorferi under continuous in vitro passage may be susceptible to a form of posttranscriptional regulation, such as proteolysis, that has been observed for other bacteria (23).

OspC has been classified with other borrelial lipoproteins, denoted "group I" lipoproteins, such as DbpA, OspF, and the Mlp family, which appear to be regulated by similar environmental cues. Therefore, the fact that ospC is regulated directly by {sigma}s may be applicable to the regulation of other group I lipoprotein genes. On the other hand, such extrapolation to other group I lipoprotein genes will require further experimental corroboration, as presented herein for ospC. This is particularly important given the fact that although dbpA is induced by elevated temperature, its responsiveness to culture pH differs from that of ospC (58). In the case of the mlp genes, they are coordinately regulated in a pattern very similar, but not identical, to that of ospC (59). Thus, it is premature to conclude that dbpA or the mlp lipoprotein genes have E{sigma}s promoters. It is therefore not inconceivable that another layer of gene regulation, yet to be elucidated, remains for the regulation of other group I lipoprotein genes.


   ACKNOWLEDGMENTS
 
We thank Erol Fikrig for helpful discussions, Philip Stewart and Patricia Rosa for supplying pBSV2 and high-passage strain B31-A, Craig Sampson for providing monoclonal antibody 4B8F4, and Sharyl Bundle for generating rabbit antiserum.

Funding for this work was provided by grant AI-59602 (to M.V.N.) and AI-51486 (to D.S.S.) from the Lyme disease program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.


   FOOTNOTES
 
* Corresponding author. Mailing address: Dept. of Microbiology, U.T. Southwestern Medical Center, 6000 Harry Hines Blvd., Dallas, TX 75390-9048. Phone: (214) 648-5900. Fax: (214) 648-5905. E-mail: michael.norgard{at}utsouthwestern.edu. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Journal of Bacteriology, July 2005, p. 4822-4829, Vol. 187, No. 14
0021-9193/05/$08.00+0     doi:10.1128/JB.187.14.4822-4829.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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