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Division of Bacteriology and Parasitology, Tulane National Primate Research Center, Tulane University Health Sciences Center, Covington, LA 70433, USA
Correspondence
Ramesh Ramamoorthy
rramesh{at}tulane.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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A supplementary table listing the reagents and settings used for confocal microscopy is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The genome of B. burgdorferi strain B31 is composed of a linear chromosome, nine circular plasmids and 12 linear plasmids (Casjens et al., 2000
; Fraser et al., 1997). One of the genetic elements that display prolific differential expression in response to environmental signals is linear plasmid 54 (lp54) (Brooks et al., 2003; Carroll et al., 2000; Clifton et al., 2006; Ojaimi et al., 2003; Revel et al., 2002; Tokarz et al., 2004). lp54 of B. burgdorferi B31 consists of 76 ORFs that include lipoproteins such as OspA and OspB (Barbour & Garon, 1987) and decorin-binding proteins A (DbpA) and B (DbpB) (Hagman et al., 1998). In addition to these immunogenic proteins, lp54 also carries eight out of the 14 members of gene family 54. Paralogues of this gene family exhibit significant intrafamily sequence divergence, with amino acid similarity and identity values as low as 7.35 and 5.4 %, respectively (McDowell et al., 2005). Two members of this family, BBA64 (Gilmore et al., 1997) and BBA66, have been localized to the surface of the spirochaete (Brooks et al., 2006).
Members of gene family 54 display distinct expression patterns. Some members (bba64 and bba66) of the family are silent during the unfed-tick phase (Gilmore et al., 2001; Tokarz et al., 2004) but are turned on during tick feeding (Tokarz et al., 2004). Several members (bba64, bba65, bba66, bba73 and bbi36/38) are expressed in the vertebrate host (Gilmore et al., 1997, 2007; Anguita et al., 2000; Liang et al., 2002; Brooks et al., 2006; Clifton et al., 2006; Nowalk et al., 2006). Although the functions of most of the paralogues remain unknown, one member, bba68, is known to bind to human factor H (Kraiczy et al., 2004; Wallich et al., 2005) and impart resistance (Brooks et al., 2005). Recent data indicate that bba68 is not expressed during infection, as inferred from real-time RT-PCR analyses and the absence of an antibody (Ab) response to the protein in infected animals. Moreover, bba68 expression is not dependent on RpoS (McDowell et al., 2006). The differential expression of these genes may be reproduced in culture under conditions that mimic the unfed tick (pH 8.0, 23 °C) or the feeding tick (pH 7.0, 35 °C). In general, the expression of bba64, bba65, bba66, bba71 and bba73 is upregulated while that of bba69, bba70, bbi36/38 and bbi39/41 is down-regulated under culture conditions that resemble the tick feeding process (Carroll et al., 2000; Clifton et al., 2006; Ojaimi et al., 2003; Ramamoorthy & Scholl-Meeker, 2001; Revel et al., 2002). The effect of inclusion of blood in the culture medium was largely similar to the effect observed under feeding-tick-like conditions (Tokarz et al., 2004). However, with respect to gene family 54, spirochaetes cultured in implanted dialysis membrane chambers (DMCs) display an expression pattern that resembles neither the flat (unfed) nor the feeding tick (Revel et al., 2002; Brooks et al., 2003).
The regulation of expression of two members of the gbb54 family, bba64 and bba66, has recently been investigated. The expression of bba66 was shown to require the presence of a sequence motif that is the binding site for a sequence-specific DNA-binding protein (Clifton et al., 2006). The expression of bba64 has also been shown to be associated with a sequence-specific DNA-binding activity (Indest & Philipp, 2000). However, based on their sequence specificities, these two paralogues appear to recruit distinct DNA-binding proteins (Clifton et al., 2006). In the case of bba64, the binding site has been localized to a 43 nt region (designated k2) immediately upstream of the –35 element (Indest & Philipp, 2000). The k2 region harbours two features that may comprise the DNA-binding site, an inverted repeat sequence (IRS) and a downstream poly-T tract. Poly-T tracts have been speculated to be involved in regulating gene expression in B. burgdorferi (Sohaskey et al., 1999; Caimano et al., 2005). In a recent study, the expression of bba64 was found to be down-regulated in an rpoS mutant as compared to its isogenic wild-type parent (Fisher et al., 2005). However, a subsequent study found the expression of bba64 to be uniquely constitutive as compared to other paralogues of this gene family with respect to both culture temperature and culture medium pH (Clifton et al., 2006). Therefore, the role of RpoS in the expression of bba64 remains somewhat uncertain.
In this study, we critically examined the role of RpoS in the expression of bba64 by complementing the B31 A3rpoS mutant with a wild-type copy of the rpoS gene inserted into the chromosome. We also investigated the role of the upstream sequence, specifically the IRS and the poly-T tract within the k2 region, in the expression of bba64 using gfp as a reporter. The importance of the k2 region in bba64 expression was examined using a combination of mutations and deletion.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and B31 5A4NP1 (cp9– bbe02 : : kanr) (Kawabata et al., 2004), as well as the B31 A3 rpoS mutant (Elias et al., 2002) were used in the current study. The Escherichia coli strains Top 10 (Invitrogen) and XL1 Blue MR (Stratagene) were used in the generation of constructs and for the preparation of plasmids for the transformation of B. burgdorferi. E. coli transformants were selected by plating on Luria agar (1.3 %) supplemented with 100 µg ampicillin ml–1, 10 µg gentamicin ml–1 or 100 µg spectinomycin ml–1. B. burgdorferi strains and transformants were grown in BSK II+6 % rabbit serum (Sigma) or in BSK-H complete media (Sigma). Spirochaetes were cultured in 5 % CO2, 3 % O2 and 92 % N2 at 34 °C. The cultures were set up at an initial density of 1x105 organisms ml–1 and harvested at stationary phase (1–2x108 organisms ml–1). Spirochaetal cultures for confocal microscopy were harvested at late exponential phase. Enumeration of cells in culture was performed by dark-field microscopy.
Generation of B31 A3rpoS/rpoS+clones.
The rpoS mutant was complemented with a wild-type copy of strain B31 rpoS that was targeted to the chromosome at the BB0472–BB0473 intergenic site simply because this presented a large region of sequence with no known function. The first step in the assembly of the complementation construct was the construction of a hybrid bmpA promoter–aadA gene for positive selection of transformants in B. burgdorferi. The bmpA promoter (bmpAp) region was amplified with primers T79 and B83 and cloned into pQE30. The aadA coding sequence (conferring streptomycin resistance in B. burgdorferi) (Frank et al., 2003) was amplified from plasmid pAM34 [American Type Culture Collection (ATCC) catalogue no. 77185] using primers T227 and B237, and cloned downstream of the bmpAp. The bmpAp–aadA gene was then transferred to pBR322 by PCR using primers T228 and B237. Next, a 1.3 kb DNA fragment containing the wild-type rpoS gene and 5' flanking sequence, including the RpoN promoter, was amplified from B31 by PCR using primers T267 and B274. This fragment was cloned downstream of the bmpAp–aadA sequence. To target the rpoS gene to the BB0472–BB0473 intergenic locus on the chromosome, BB0472 and BB0473 sequences were cloned upstream of bmpAp–aadA and downstream of rpoS, respectively. The primers are all listed in Table 1. The resulting plasmid, designated p472ApSrpoS473, was used to transform the B31 A3rpoS mutant by electroporation, as described elsewhere (Samuels, 1995). After overnight recovery, the electroporated spirochaetes were plated on semisolid BSK-H containing streptomycin (50 µg ml–1) and kanamycin (100 µg ml–1) (Sung et al., 2000). The plates were incubated at 35 °C in a candle jar container. Colonies usually appeared 2 weeks after plating. The colonies were transferred to liquid media and subsequently expanded. The integration of the wild-type rpoS was confirmed by Western blotting and PCR analysis. Two clones were chosen for further characterization.
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) to yield pQE30-A64p–gfp. To generate pQE30-A64p5'm, the bba64 promoter region was amplified in two parts using primer sets T81 and B86 and T82 and B87. The T81/B86 and T82/B87 amplicons were assembled in pQE30/XhoI/EcoRI in a three-way ligation to yield pQE30-A64p5'm–gfp. To generate pQE30-A64p5'3'm–gfp, the promoter region was amplified from pQE30-A64p5'm using primers T81 and B121 and cloned into the XhoI/EcoRI sites of pQE30-A64p5'm–gfp.
The shuttle vector derivatives of these constructs were generated as follows. For cloning into pBSV2G (Elias et al., 2003), the fragments (A64p–gfp, A64p5'm–gfp and A64p5'3'm–gfp) were amplified using primers T188 and B199. A minimal promoter construct, A64pmin–gfp, was generated from pQE30-A64p–gfp using primers T239 and B199. These fragments were all cloned into pBSV2G at the KpnI/PvuI sites, resulting in plasmids pBSV2G-A64p–gfp, pBSV2G-A64p5'm–gfp, pBSV2G-A64p5'3'm–gfp and pBSV2G-A64pmin–gfp. Finally, a promoterless gfp construct was also generated for use as a control. The promoter sequences of all constructs were confirmed by sequencing using the T88 primer.
Transformation of B. burgdorferi.
Plasmid DNAs for electroporation were produced under sterile conditions using Qiagen Tip100 columns. The cells were prepared for electroporation as described elsewhere (Samuels, 1995). Electrocompetent B. burgdorferi was transformed as described elsewhere (Samuels, 1995) with the different promoter–gfp fusion plasmids, with a minor modification. Ten micrograms of DNA was electroporated into 90 µl of cells. Immediately following electroporation, the cells were resuspended in 10 ml liquid BSK-H media and incubated overnight at 34 °C to allow the cells to recover. The transformants were selected according to the limiting-dilution method (Yang et al., 2004). After overnight recovery, the cultures were supplemented with 40 ml fresh BSK-H containing gentamicin (40 µg ml–1) and kanamycin (100 µg ml–1), and distributed into 96-well tissue-culture plates (200 µl per well). Two to three weeks after plating, wells that were positive for dividing spirochaetes were identified by a colour change in the medium, and the presence of viable spirochaetes was verified by dark-field microscopy. The antibiotic-resistant clones were inoculated into 1 ml complete BSK-H medium containing the relevant antibiotics. After 3 days, the transformants were expanded into 15 ml BSK-H complete media. The 15 ml culture was used for the preparation of freezer stocks and to inoculate fresh cultures for analysis of gene expression.
Generation of rat polyclonal anti-RpoS Ab.
To assess RpoS expression, a rat polyclonal anti-RpoS Ab was generated. Briefly, B. burgdorferi rpoS was cloned into the pQE30 expression vector (Qiagen) and expressed as a hexahistidine fusion protein in E. coli. Overexpression resulted in an insoluble fusion protein that was purified under denaturing conditions, dialysed to remove urea and then used for the preparation of rat anti-RpoS Ab (Genemed Synthesis). The specificity of the anti-RpoS Ab was verified in E. coli using whole-cell lysates prepared from uninduced and IPTG-induced cells carrying the pQE30-his6rpoSBb plasmid. Whereas the Ab showed strong reactivity to a band of 
33 kDa in the induced sample, consistent with the expected size of the fusion protein, there was no reactivity with the uninduced sample (data not shown). The Ab was then titrated to determine the highest dilution of the Ab that provided the best signal in Western blots (data not shown). A dilution of 1 : 200 provided the best signal.
RNA isolation and RT-PCR.
DNA-free RNA was isolated from B31 A3, B31 A3rpoS and B31 A3rpoS/rpoS+ as previously described (Ramamoorthy et al., 1996). Furthermore, the integrity and concentration of each RNA sample were verified as described previously (Ramamoorthy et al., 1996). About 200 ng total RNA was converted to cDNA in a 10 µl volume using Taqman reverse transcription reagents (Applied Biosystems) following the manufacturer's instructions. cDNA synthesis was primed with random hexamers and carried out under the following conditions: 26 °C for 10 min followed by 48 °C for 30 min. The enzyme was inactivated at 95 °C for 5 min prior to PCR. PCR was performed with 2 ng of each cDNA using ProofStart polymerase (Qiagen) in a volume of 30 µl. To rule out amplification from DNA, reactions containing RNA without reverse transcriptase were also included with the bba64 primer set. The primers used were as follows: T253 and T306 (bba64), and FlaBF and FlaBR (flaB). The reaction conditions consisted of a 5 min, 95 °C denaturation step, followed by 40 cycles of 95 °C for 30 s, 45 °C for 30 s, and 72 °C for 1 min, and then a final extension step at 72 °C for 10 min.
Western blotting.
Whole-cell lysates were prepared from stationary-phase cultures and normalized to an OD600 of 5, as described previously (Ramamoorthy & Philipp, 1998). For the analysis of protein expression, 10 µl (unless specified otherwise) of each sample was electrophoresed through a 12.5 % SDS–polyacrylamide gel and the proteins were transferred to nitrocellulose. Following Ab incubations, protein bands were visualized using the chromogen 4-chloro-1-naphthol. The following Abs were used: mAb specific for BBA64 (Indest et al., 1997), rabbit polyclonal anti-GFP Ab (Santa-Cruz Biotechnology), anti-FlaB mAb H9724 (University of Texas Health Sciences Center, San Antonio), anti-OspC mAb B5 mAb (Mbow et al., 1999) and rat polyclonal anti-RpoS Ab (this study). For quantitative analysis of protein expression, the Western blots were digitized and the intensity of individual bands was quantified by densitometry using Kodak Molecular Imaging Software, version 4.0. All experiments were repeated at least once and the analyses of the pooled data are presented.
Immunofluorescence staining and confocal microscopy.
The spirochaetal cultures were spun down and the resulting pellets were washed twice with PBS (Invitrogen) to remove the culture medium. The pellets were resuspended in PBS at a density of 2x108 cells ml–1. A 50 µl volume of borrelial suspension containing 1x107 cells was applied to Superfrost Plus slides (Fisher). Smears were air-dried, taking care to protect them from exposure to direct light. Slides were fixed in methanol for 10 min. Bacterial smears were blocked for 1 h in blocking buffer [PBS containing 10 % normal goat serum (Invitrogen), 0.2 % fish skin gelatin (FSG; Sigma) and 0.02 % sodium azide (Sigma)]. The blocking solution was removed by gently flicking the slides before addition of the primary Abs. Primary Abs were diluted to the desired concentration in a PBS–FSG buffer (PBS, 0.2 % FSG, 0.02 % sodium azide) (see Supplementary Table S1). Isotype Ab controls (Dako) in combination with the corresponding secondary-Ab–fluorochrome conjugates were also included in the analyses. The slides were washed with PBS buffer after the application of each Ab. All incubations were performed in a dark humidified slide chamber at room temperature. Finally, slides were mounted in anti-quenching medium (Sigma) with premium coverslips (Surgipath) and sealed. The stained and mounted slides were stored in the dark at 4 °C until imaging. Imaging was performed using a Leica TCS SP2 true confocal laser-scanning microscope, DMIRE2 (Leica), equipped with three lasers (Ar, Ar–Kr, He–Ne) that span from the visible to the far-red region of the spectrum. Using Leica software, the fluorescence of individual fluorochromes was captured separately in sequential mode after optimization to reduce bleed through between the channels (photomultiplier tubes). Images of individual channels were also merged to obtain composite images containing all channels.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Therefore, we set out to complement the rpoS mutation to definitively ascertain the dependence of bba64 expression on RpoS. For complementation, a construct containing a wild-type copy of the rpoS (bb0771) gene inserted into the bb0472–bb0473 intergenic site (Fig. 1) was used to transform B31 A3rpoS (Elias et al., 2002), the same strain used in the earlier study. The complemented clone, B31 A3rpoS/rpoS+, was characterized by PCR using total DNA. The PCR amplification patterns were consistent with the expected genotype (data not shown). Plasmid profile analysis confirmed the presence of all plasmids that were present in the parental strain, B31A3 (Elias et al., 2002). Moreover, the complemented clones displayed normal growth kinetics in BSK-H and BSK II media.
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, RpoS). To further confirm RpoS expression in the complemented clones, we also tested the samples for the presence of OspC, a known RpoS-dependent protein (Hübner et al., 2001). As expected, the presence of RpoS in the complemented clones restored the expression of OspC (Fig. 2a, panel OspC). Finally, we examined the samples for the presence of BBA64 using an anti-BBA64 mAb (Gilmore et al., 1997). Whereas no BBA64 expression was detected in the absence of RpoS (Fig. 2a, panel BBA64, lane 2), this protein was clearly present in the two complemented clones at a level similar to the wild-type level (Fig. 2a, panel BBA64, lanes 3 and 4). The dependence of bba64 expression on RpoS was further verified at the mRNA level by RT-PCR. The bba64 sequence could be amplified from RNA derived from the wild-type and the complemented strains, but not from the rpoS mutant (Fig. 2b, panel bba64). In contrast, the constitutively expressed flaB transcript was present in all samples examined (Fig. 2b, panel flaB). These results conclusively establish the dependence of BBA64 expression on the alternative sigma factor RpoS under conditions of high cell density.
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). This region is characterized by an IRS terminating in a poly-T tract. Presumably, the IRS is the site of interaction with the DNA-binding protein. Therefore, the IRSs were mutated, either singly (A64p5'm–gfp) or in combination (A64p5'3'm–gfp) (Fig. 3). To further assess the importance of the k2 region and any other potential regulatory sequences upstream of the promoter, another construct was generated in which the sequence upstream of the –35 was entirely deleted (A64pmin–gfp) (Fig. 3). The expression of the marker gfp gene from these constructs was compared to the expression of gfp from a wild-type construct (A64p–gfp) and a promoterless construct (gfp) (Fig. 3). For the assay of gene expression, the promoter–gfp fusion constructs were assembled in the E. coli–B. burgdorferi shuttle vector pBSV2G. The shuttle vector constructs were introduced into B. burgdorferi B31 5A4NP1, a highly transformable and infectious strain (Kawabata et al., 2004). Moreover, the presence of a kanamycin-resistance determinant on linear plasmid 25 (lp25) provides positive selection for the presence of this plasmid in transformants. Determinants on the lp25 plasmid have been shown to be essential for virulence (Grimm et al., 2004; Labandeira-Rey & Skare, 2001; Purser & Norris, 2000). Therefore, all transformants were selected with kanamycin to ensure the presence of lp25.
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, panel GFP) and normalized to the corresponding FlaB bands (Fig. 4a, panel FlaB). The normalized values were then expressed relative to the wild-type promoter construct (A64p–gfp) (Fig. 4b). As expected, no GFP expression was detectable in the absence of the bba64 promoter (Fig. 4a, panel GFP, lane 1). In contrast, the GFP band was evident for all of the promoter constructs (lanes 2–4). However, surprisingly, the expression of GFP from the two mutant constructs A64p5'm–gfp and A64p5'3'm–gfp, as well as from the minimal promoter construct A64pmin–gfp, was similar to the level of expression derived from the wild-type promoter construct (A64p–gfp) (Fig. 4b). To further ensure that the levels reflected the true transcription potential of these fusions and were not the consequence of other determinants, these samples were also screened with Abs specific for BBA64 and RpoS. All samples were positive for both proteins, and more importantly, with the exception of the wild-type construct, which exhibited slightly lower levels (75 % of that of the other samples) of both RpoS and BBA64, the levels of these two proteins were similar in all other samples, including the promoterless gfp fusion (Fig. 4a, panels RpoS and BBA64).
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; Fisher et al., 2005; Yang et al., 2005; this study). Second, only a proportion of cultured spirochaetes stain positive for expression from an ospC promoter (Carroll et al., 2003) or OspC (our unpublished observations). We speculated that the expression of RpoS in cultured spirochaetes, for unknown reasons, is limited to a subpopulation, and consequently results in the selective expression of OspC. We therefore examined the populations of transformed spirochaetes for the expression of GFP, OspC and BBA64 proteins by confocal microscopy.
We first examined the relationship between BBA64, GFP and OspC expression at the population level using pBSV2G-A64p–gfp-transformed B31 5A4NP1 spirochaetes. Slides containing these spirochaetes were stained with an anti-OspC mAb (Mbow et al., 1999) followed by a rabbit polyclonal anti-B. burgdorferi Ab, and subjected to confocal microscopy. The Abs, dilutions and wavelengths used are listed in Supplementary Table S1. The expression of both GFP and OspC was found to be limited to a subpopulation of cells. The green fluorescence of GFP was noticeable in only some spirochaetes (Fig. 5, compare panel GFP and panel Bb) against a teeming background of spirochaetes that appeared negative for GFP [panel Bb+GFP; the overlap of GFP (green) and Bb (blue) appears as sea green]. Similarly, the expression of OspC was also restricted [panels OspC (red) versus Bb (blue), and Bb+OspC; overlap appears pink]. Most notably, cells with the OspC+ phenotype congregated with cells that exhibited a GFP+ phenotype (panel Bb+GFP+OspC; the overlap of the three colours appears as yellow staining). In the second experiment, we examined the relationship between OspC and BBA64 in spirochaetal populations. Slides were stained first with the mouse anti-BBA64 Ab followed by the anti-OspC Ab. Again, only a limited number of spirochaetes appeared positive for BBA64 (panel BBA64, red) or OspC (panel OspC, blue), but more importantly, these two subpopulations were the same (panel BBA64+OspC; the overlap appears as pink staining). Taken together, these results indicate that the same subpopulation of spirochaetes express all three proteins, GFP, BBA64 and OspC.
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GFP or 1GFP+OspC). In contrast, GFP fluorescence was clearly visible in numerous spirochaetes transformed with all of the other bba64 promoter constructs (Fig. 6, columns 2–5). Most notably, in all four cases, the same subpopulations stained positive for both GFP and OspC (panels GFP+OspC). These results suggest that the expression of GFP and OspC shares a common feature that is maintained in all the GFP-expressing clones analysed in this study.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). A subsequent report found bba64 to be constitutively expressed in the same strain with respect to the two culture variables tested, pH and temperature (Clifton et al., 2006), both of which conditions influence the expression of RpoS (Hübner et al., 2001; Yang et al., 2000, 2003). Therefore, it was crucial to complement the RpoS defect for an unambiguous assessment of its role in the expression of bba64. Complementation of the rpoS mutant restored the expression of bba64 to a level comparable to the wild-type level, thereby definitively establishing a requirement for RpoS for expression. Incidentally, to our knowledge, this is the first report of complementation of a B. burgdorferi rpoS mutant with a chromosomal copy of the wild-type gene. Using this strategy, we were fortunate to restore RpoS to nearly the same level as that observed in the wild-type parental strain. Although chromosomal integration may be challenging as compared to shuttle-vector transformation, it may be the ideal choice in certain cases by circumventing problems associated with plasmid maintenance and/or copy numbers. Finally, the bb0472–bb0473 intergenic chromosomal target should prove useful for targeting other genes for complementation studies.
In addition to RpoS, one other factor may be involved in the expression of bba64. This factor is the putative DNA-binding protein previously demonstrated to specifically bind to the k2 sequence upstream of the gene (Indest & Philipp, 2000). Surprisingly, however, mutations of the IRS, the most prominent feature within the k2 region, failed to evoke any response vis-à-vis protein expression. Similarly, deletion of the entire upstream sequence beginning with the k2 region also proved to have no effect. Therefore, the expression of GFP in culture appears to utilize just the bba64 basal promoter. It is essential to note that in all cases, the expression of GFP was limited to the same subpopulation of cells that also expressed OspC. Phenotypic heterogeneity of OspC has been previously observed in spirochaetal populations during tick feeding (Schwan et al., 1995; Schwan & Piesman, 2000) and in culture (Earnhart et al., 2007). Since both OspC and BBA64 require RpoS for expression, it is tantalizing to speculate that in culture only a limited number of spirochaetes express RpoS, or alternatively express higher levels of RpoS, resulting in the observed phenotypic heterogeneity at the population level.
The passivity of the sequence upstream of the bba64 basal promoter in cultured spirochaetes is similar to that reported for the ospC gene. In the case of ospC, a deletion of the sequence upstream of the promoter, which features an IRS, results in no effect on gene expression in vitro (Yang et al., 2005; Xu et al., 2007). Nonetheless, the IRS, subsequently dubbed the operator, assumes functional significance in vivo, wherein its presence is crucial for the suppression of OspC expression post-infection (Xu et al., 2007). It is very likely that a DNA-binding protein is responsible for this suppression of ospC, although no such protein has yet been reported. In contrast to ospC, a DNA-binding protein specific to bba64 has been shown to be present in cultured spirochaetes (Indest & Philipp, 2000). However, the lack of any response from the k2 region suggests that the reported bba64-specific DNA-binding protein is inactive in cultured spirochaetes under the conditions tested. Alternatively, the expression of the bba64-specific DNA-binding protein may be very low or absent in strain B31 5A4NP1, the focus of this study. Notwithstanding, based on its location downstream of the stop codon of bba65, it is very likely that the k2 region with its IR element functions as a transcription terminator for bba65.
Two reports that are pertinent to the discussion of bba64 regulation must be highlighted. Anguita et al. (2000) noted that the high-passage but infectious strain N40-P75 failed to express bba64 and several other genes now known to be RpoS-regulated in vivo (Fisher et al., 2005), despite a vigorous synthesis of OspC (Anguita et al., 2000) and BBA64 (our unpublished observations) in vitro. The failure to induce gene expression appears to be unrelated to any gross loss of genetic material (Anguita et al., 2000). Therefore, the simplest explanation for these observations is that the in vivo expression of RpoS or some other common factor is defective in this high-passage variant, leading to a broader loss of gene expression. A more recent investigation of gene expression during persistent infection of mice has revealed the down-regulation of bba64 mRNA expression in the ear relative to that in cultured spirochaetes at all time points tested (Gilmore et al., 2007), although importantly, unlike N40-P75, the down-regulation of bba64 mRNA appears in this case to be specific, as the same tissue sample(s) exhibited an upregulation of bba65 and bba66, two other RpoS-dependent genes (Fisher et al., 2005). However, this loss of expression in the ear was countered by the expression of bba64 elsewhere in the body, as these mice continued to harbour anti-BBA64 Abs throughout the course of infection. If these observations hold true, it suggests that bba64 expression in the ear, and perhaps other organs, is repressed. Such repression may well involve the k2 region and the putative bba64-specific DNA-binding protein.
The pattern of expression of bba64 in culture in response to different environmental conditions and during infection of the vertebrate host points to a complex mode of regulation of bba64. Moreover, its expression pattern suggests an important function in establishing and maintaining infection in the vertebrate host. Given this importance, it is crucial to continue to explore the function and regulation of bba64 expression and assess its role in virulence and pathogenesis. Finally, understanding the function and regulation of this molecule may also shed light on the orchestration of regulation of the other members of the gbb54 gene family and their contribution to the overall molecular strategies of this pathogen.
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ACKNOWLEDGEMENTS |
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Portions of this work were presented at the American Society of Microbiology meeting in Atlanta, June 2005, and at the Gordon Conference on the Biology of Spirochaetes, Il Ciocco, Italy, April 2006.
Edited by: R. J. Lamont
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Received 13 July 2007;
revised 21 September 2007;
accepted 4 October 2007.
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