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Microbiology (2008), 154, 2641–2658

DOI 10.1099/mic.0.2008/019992-0

Transcriptional interplay among the regulators Rrp2, RpoN and RpoS in Borrelia burgdorferi Zhiming Ouyang,3 Jon S. Blevins3 and Michael V. Norgard Correspondence Michael V. Norgard

Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA

[email protected] utsouthwestern.edu

Received 25 April 2008 Revised 3 June 2008 Accepted 6 June 2008

The RpoN–RpoS alternative sigma factor pathway is essential for key adaptive responses by Borrelia burgdorferi, particularly those involved in the infection of a mammalian host. A putative response regulator, Rrp2, is ostensibly required for activation of the RpoN-dependent transcription of rpoS. However, questions remain regarding the extent to which the three major constituents of this pathway (Rrp2, RpoN and RpoS) act interdependently. To assess the functional interplay between Rrp2, RpoN and RpoS, we employed microarray analyses to compare gene expression levels in rrp2, rpoN and rpoS mutants of parental strain 297. We identified 98 genes that were similarly regulated by Rrp2, RpoN and RpoS, and an additional 47 genes were determined to be likely regulated by this pathway. The substantial overlap between genes regulated by RpoS and RpoN provides compelling evidence that these two alternative sigma factors form a congruous pathway and that RpoN regulates B. burgdorferi gene expression through RpoS. Although several known B. burgdorferi virulence determinants were regulated by the RpoN–RpoS pathway, a defined function has yet to be ascribed to most of the genes substantially regulated by Rrp2, RpoN and RpoS.

INTRODUCTION Lyme disease, caused by the spirochaetal bacterium Borrelia burgdorferi (Burgdorfer et al., 1982; Steere et al., 1983), remains the most common arthropod-borne illness in the United States (CDC, 2007). B. burgdorferi has a complex enzootic life cycle that involves an arthropod (Ixodes tick) vector and a mammalian host (most typically small rodents) (Fikrig & Narasimhan, 2006; Hovius et al., 2007). For B. burgdorferi to maintain its presence in nature, it must transit between these two dramatically different environments, and adapt to them efficiently by altering its gene expression patterns (Fikrig & Narasimhan, 2006; Hovius et al., 2007; Rosa et al., 2005; Singh & Girschick, 2004). Many studies have now shown that certain environmental signals, such as temperature, pH, cell density, nutrient availability and other mammal-specific signals, modulate the temporal expression 3These authors contributed equally to this work. Abbreviations: DMC, dialysis membrane chamber; qRT-PCR, real-time quantitative RT-PCR. The array data discussed in this paper have been deposited in the NCBI Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO series accession number GSM284306–284322. A supplementary table listing the oligonucleotide primers used in this study and two supplementary figures showing representative scanned microarray images of 1723 B. burgdorferi ORFs are available with the online version of this paper.

2008/019992 G 2008 SGM Printed in Great Britain

of a number of borrelial membrane (lipo)proteins (Caimano et al., 2007; Ojaimi et al., 2003; Revel et al., 2002; Stevenson et al., 2006; Yang et al., 2000, 2003b). Along these lines, our laboratory and others have previously reported the existence in B. burgdorferi of an alternative sigma factor (RpoN/s54–RpoS/sS) regulatory cascade that governs the expression of outer surface (lipo)protein C (OspC), decorin-binding protein A (DbpA), multicopy lipoprotein-8 (Mlp8), fibronectinbinding protein BBK32, and other potential virulenceassociated proteins (Caimano et al., 2004, 2005, 2007; Fisher et al., 2005; He et al., 2007; Hubner et al., 2001; Smith et al., 2007; Yang et al., 2003a, b, 2005). In this regulatory pathway, Rrp2, a putative response regulator (enhancer-binding protein) of a two-component sensory transduction system, is likely activated via phosphorylation through a histidine kinase (Hk2) (Fraser et al., 1997; Yang et al., 2003a). Activated Rrp2 then ostensibly interacts with the RpoN holoenzyme, and allows open complex formation for RpoN-dependent transcription of rpoS (Burtnick et al., 2007; Yang et al., 2003a). RpoS is then available for the transcription of those genes under its control, such as ospC, dbpA, mlp8 and bbk32. In addition, RpoS also is activated by the small non-coding RNA DsrABb in response to changes in temperature (Lybecker & Samuels, 2007). Although recognition of the RpoN–RpoS cascade was an important first step in elucidating regulatory networks in 2641

Z. Ouyang, J. S. Blevins and M. V. Norgard

virulent B. burgdorferi (Hubner et al., 2001), its discovery has engendered many important questions. For example, how extensive is this pathway in B. burgdorferi; how many genes are affected? Is every RpoN-dependent gene influenced solely via RpoN control over rpoS, or does RpoN control genes independent of its interaction with rpoS? Are there indirect effects; does the pathway regulate other regulators? Are there genes also downregulated by the pathway, as has been implied by the reciprocal regulation of OspC and OspA (Caimano et al., 2005; Schwan et al., 1995; Schwan & Piesman, 2000)? Does Rrp2 activation serve only to allow RpoN-dependent transcription, or does the putative DNA-binding domain of Rrp2 serve some other function(s) in B. burgdorferi? To begin to address some of these questions, Fisher et al. (2005) employed microarray analyses and concluded that there are three patterns of distinct and overlapping regulation in B. burgdorferi, including 254 genes regulated by RpoN alone, 94 genes regulated by RpoS alone, and 51 genes regulated by both RpoN and RpoS. That study raised a number of paradoxes, however, not the least of which was the notion that a majority of the genes regulated by RpoN were independent of its activation of rpoS. More recently, Caimano et al. (2007) performed transcriptional profiling of B. burgdorferi cultivated in dialysis membrane chambers (DMCs) implanted into the peritoneal cavities of rats or rabbits; they observed significant upregulation of rpoS and many RpoS-dependent genes, as well as RpoS-mediated repression of genes in response to mammalian host-specific signals. In the current study, to analyse the regulatory interrelationships between Rrp2, RpoN and RpoS more closely, we took the approach of implementing gene microarrays to compare the transcriptional expression profiles of rrp2, rpoN and rpoS mutants cultivated in vitro under conditions conducive to activation of the RpoN–RpoS pathway. We found that the vast majority of genes had overlapping patterns of regulation by Rrp2, RpoN and RpoS. Moreover, the functions of many of the genes under the regulatory control of Rrp2, RpoN and RpoS have yet to be defined. The combined results substantiate the close interplay between Rrp2, RpoN and RpoS, and thus further confirm their importance in virulence expression by the Lyme disease spirochaete.

METHODS Bacterial strains and culture conditions. Low-passage (not more

than three passages) virulent B. burgdorferi wild-type strain 297 (Hughes et al., 1992) and rrp2, rpoN and rpoS mutants were cultivated in Barbour–Stoenner–Kelly (BSK-H) Complete medium (SigmaAldrich) (Pollack et al., 1993) at 37 uC and 5 % CO2. After the pH of the medium had been adjusted to 6.8 using 1M HCl, BSK-H medium was inoculated with spirochaetes to a final concentration of 16103 cells ml21. Spirochaetes were enumerated by dark-field microscopy, and bacteria were harvested when the culture reached stationary phase (~16108 bacteria ml21; 9 days post-inoculation). To ensure activation of the RpoN–RpoS pathway in the cultures, RpoS and OspC expression was monitored as described below. 2642

Generation of B. burgdorferi mutants employed in this study.

The rpoN mutant BbJSB18-B2 used in this study has been described previously (Smith et al., 2007). Prior to use of this clone in microarray comparisons, genetic complementation of the rpoN mutation was performed on BbJSB18-B2 to ensure that this mutant, which was non-infectious, contained all the plasmids necessary for mammalian infection. To achieve this, a 1.7 kb region of B. burgdorferi strain 297 DNA encoding the rpoN gene and 375 bp upstream of the ORF was PCR-amplified using the primers priAH125 and priAH144 (Hubner et al., 2001) (see Supplementary Table S1) and cloned into pGEM-T easy (Promega). Following confirmation by sequence analysis, the insert was excised by digestion with BamHI and BclI and ligated into the borrelial shuttle vector pJD55, which had been linearized with BamHI. pJD55 is a derivative of pJD44 (Revel et al., 2005) in which the aph[39]-IIIa marker is replaced with PflgB-Kan of pBSV2 (Stewart et al., 2001). BbJSB18-B2 was transformed with the resulting construct, designated pJSB208B, as described previously by Yang et al. (2005), and transformants were selected using kanamycin at a concentration of 160 mg ml21. Clones were confirmed by plasmid recovery, as described by Blevins et al. (2007). The rpoS mutant was constructed using an approach similar to that described by Hubner et al. (2001), except that the erythromycinresistance marker in the mutagenesis construct pALH386 was replaced with the streptomycin-resistance marker from pKFSS1 (Frank et al., 2003). To achieve this, the PflgB-aadA marker was first PCR-amplified from pKFSS1 (Frank et al., 2003) using the primers PflgB-Bam-59 and Sp/Sm-Bam-39 (Supplementary Table S1). The PCR fragment then was cloned into pCR-XL-TOPO (Invitrogen) to generate pXY205. Following confirmation of pXY205 by DNA sequence analysis, the PflgB–aadA marker was excised using BamHI and treated with T4 DNA polymerase to form a blunt-end fragment. pALH362, a suicide vector containing a 4.6 kb region of strain 297 DNA encompassing rpoS (Hubner et al., 2001), was digested with BbsI and also treated with T4 DNA polymerase to blunt the ends. The fragments were then ligated to generate pJSB19, and the resulting clones were confirmed by restriction digestion and PCR analysis. B. burgdorferi transformed with pJSB19 were selected using streptomycin at a concentration of 150 mg ml21, and resulting clones were confirmed by PCR analysis as described by Hubner et al. (2001). For genetic complementation of the rpoS mutant, the 4.2 kb region of B. burgdorferi strain 297 DNA in pXY240 (Smith et al., 2007), which includes 1.9 kb upstream and 1.5 kb downstream of the rpoS ORF, was cloned into the shuttle vector pJD44 (Revel et al., 2005). This was achieved by removing the 4.2 kb region from pXY240 by digestion with KpnI and XbaI and ligating it into pJD44 digested with the same enzymes. The resulting construct, designated pJSB259, was transformed into BbJSB19-A7B, as described previously (Yang et al., 2005). The rrp2 mutant was generated by transforming B. burgdorferi strain 297 with the suicide vector pXY201A (Yang et al., 2003a), which introduced a point mutation (G239C) within the putative C4 ATPbinding motif of Rrp2; this mutation abolishes the ATPase activity that is essential for RpoN-dependent activation of RpoS. Transformants, selected using 60 ng erythromycin ml21, were confirmed as described by Yang et al. (2003a). Complementation of the rrp2 mutant was achieved by transforming the mutant with the suicide vector pXY206B (Yang et al., 2003a); transformants were selected using 150 mg streptomycin ml21. Plasmid profiling was performed on all mutants and complemented strains as described previously (Hubner et al., 2001; Yang et al., 2003a). All mutants and complemented strains contained the same plasmid profile as the B. burgdorferi parental strain 297 (data not shown). The infectivity of the mutants and complemented strains was assessed using the murine needle-challenge model of Lyme borreliosis (Hagman et al., 1998). University of Texas (UT) Southwestern is accredited by the International Association for Assessment and Microbiology 154

Transcriptional interplay of B. burgdorferi regulators Accreditation of Laboratory Animals Care (AAALAC), and all animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at UT Southwestern Medical Center. Prior to infection, the density of bacteria in each culture was determined carefully using dark-field microscopy. Groups of 3- to 5-week-old C3H/HeJ (Jackson Laboratory) or C3H/HeN (Charles River Laboratories) mice were infected via intradermal injection. At 2 or 6 weeks post-infection, ear punch biopsies were recovered from mice and placed in BSK-II media containing borrelia antibiotic mixture (BAM; Sigma-Aldrich), and the outgrowth of spirochaetes in each of these cultures was assessed using dark-field microscopy. SDS-PAGE and immunoblot analysis. RpoS and OspC expression

was assessed by SDS-PAGE and immunoblot analysis, as described previously (Yang et al., 1999). Briefly, spirochaetes were harvested from stationary-phase cultures and washed twice in sterile 0.9 % (w/v) saline solution. A volume of whole-cell lysate equivalent to 46107 bacteria was loaded to each lane on a 12.5 % acrylamide gel. Resolved proteins were either stained with Coomassie Brilliant Blue or transferred to nitrocellulose membranes for immunoblot analysis. A mAb directed against RpoS, designated 6A7-101, was produced in collaboration with the Antibody Production Core Facility at UT Southwestern Medical Center. OspC was detected using anti-OspC mAb 1B2-105 (Smith et al., 2007). To confirm equal loading of bacteria in each lane, immunoblotting for the flagellar core protein (FlaB) was performed using a chicken IgY anti-FlaB antibody (a gift from Dr Kayla Hagman, UT Southwestern Medical Center). Immunoblots for RpoS were developed by chemiluminescence using the ECL Plus Western Blotting Detection System (Amersham Biosciences), whereas membranes for OspC and FlaB were developed colorimetrically using 4-chloro-1-naphthol as the substrate. B. burgdorferi microarray construction. Oligonucleotides representing 1723 putative ORFs of B. burgdorferi B31MI and 19 randomsequence 70-mer negative controls were synthesized as described elsewhere (Terekhova et al., 2006). The oligonucleotides were resuspended in 150 mM sodium phosphate, pH 8.5 (Microarrays Inc.), to a concentration of 40 mM and printed on CodeLink activated slides (Amersham Biosciences), using a Custom arrayer (Microarrays Inc.). Each oligonucleotide was printed in quadruplicate on each array. Arrays were blocked post-printing as per the CodeLink instructions (Amersham Biosciences) with 50 mM ethanolamine. Attachment of probe DNA was confirmed by Microarrays Inc. proprietary Veriprobe assay. Prior to hybridization, arrays were stored under desiccation. Comparative genomic DNA hybridization. Bacterial genomic

DNA was isolated using a Wizard genomic DNA purification kit (Promega) according to the manufacturer’s instructions. DNA was quantified using a NanoDrop ND-100 spectrophotometer (NanoDrop Technologies). Samples (4 mg) of genomic DNA from strains 297 (test) and B31 (reference) were labelled with Alexa Fluor 555 and 647, respectively, using the Bioprimer Plus Array CGH Indirect Genomic Labelling System (Invitrogen) according to the manufacturer’s protocol. Prior to hybridization, slides were prehybridized in a solution of 56SSC (16SSC50.15 M sodium chloride/0.015 M sodium citrate, pH 7), 0.1 % SDS, and 0.1 % (w/v) BSA for 30 min at 55 uC. Following pre-hybridization, slides were washed five times in distilled water, dipped in 2-propanol, dried by centrifugation, and used immediately in the hybridization. After purification of labelled DNA using the Qiaquick PCR purification kit (Qiagen), equal amounts of labelled 297 and B31 DNA were combined and applied to arrays. Slides were covered with lifter coverslips (Erie Scientific), placed into a humidified hybridization chamber (Ambion), and the chamber was placed into a hybridization oven set at 50 uC. After hybridization overnight, slides were washed twice with 26SSC/0.1 % SDS for 15 min at http://mic.sgmjournals.org

42 uC, twice with 0.16SSC/0.1 % SDS for 15 min at room temperature, and twice with 0.16SSC for 1 min at room temperature. Washed slides were then dried by centrifugation and scanned immediately. Microarray scanning and data analysis were performed as described previously (Terekhova et al., 2006). Extraction of RNA from B. burgdorferi. RNA was extracted from

three biological replicates of wild-type parental strain B. burgdorferi 297 and the rpoS, rpoN and rrp2 mutants using Trizol reagent (Invitrogen) according to the manufacturer’s protocol. An additional phenol/chloroform extraction was performed, after which the RNA was precipitated using 2-propanol. Digestion of contaminating genomic DNA in the RNA samples was performed using RNasefree DNase I (GenHunter Technology) and removal of DNA was confirmed by PCR amplification using flaB1 primers specific for the B. burgdorferi flaB gene (Supplementary Table S1). RNA quality was determined using the Agilent Bioanalyser 2100 (Agilent Technologies) in the Microarray Core Facility in the UT Southwestern Medical Center. Synthesis and labelling of cDNA. cDNA was synthesized from

extracted RNA and labelled with Cy3 or Cy5 using the Amersham postlabelling kit according to the manufacturer’s instructions (Amersham Biosciences), with minor modifications. Briefly, 10 mg total RNA was converted to cDNA using CyScript reverse transcriptase, in the presence of 1 ml random nonamers (Amersham Biosciences) and 4.5 mg random hexamers (Invitrogen). The resulting cDNA was labelled with Cy3 or Cy5. Cy3- or Cy5-labelled cDNA from RNA extracted from parental strain 297 was then combined with the corresponding Cy5- or Cy3labelled cDNA generated from RNA derived from the mutants; e.g. Cy5labelled 297 cDNA and Cy3-labelled mutant cDNA or Cy3-labelled 297 cDNA and Cy5-labelled mutant cDNA. Labelled probes were purified using the Qiaquick PCR purification kit (Qiagen) and used in subsequent microarray experiments. Microarray scanning and data analysis. For comparative

transcriptional microarray analysis, slides were hybridized with labelled probes as described above. Hybridized slides were scanned on an Axon 4000B microarray scanner using GenePix Pro 6.1 (Molecular Devices). The image (two representative scans are shown, to give an indication of their quality, in Supplementary Figs S1 and S2) was analysed using the GenePix program, and data then were analysed with Acuity 4.0 microarray informatics software according to the manufacturer’s instructions (Molecular Devices), using a ratiobased normalization method and a cutoff value of a twofold change. Briefly, raw data were first normalized using a ratio-based normalization method to equalize the means and medians of the features to 1. Additionally, the features that were flagged by the software as ‘bad’, ‘absent’ or ‘not found’ were also excluded from further analysis. Statistical analyses were performed using the oneand two-sample significance test (P ,0.05) in the Acuity program, which is a one-sample t test. Differentially expressed genes were identified by both fold change and statistical significance. Quantitative RT-PCR analysis. Real-time quantitative RT-PCR

(qRT-PCR) was employed to validate selected data from the microarray experiments. Specific primers (listed in Supplementary Table S1) for 17 B. burgdorferi genes were designed by using Primer Express software (Applied Biosystems) and validated using 10-fold dilutions (80–0.0008 ng) of B. burgdorferi genomic DNA in an absolute quantification test on an ABI 7500 qRT-PCR system (Applied Biosystems). Standard curves created for all primers had a slope of 23.3±0.3 (data not shown). For measuring gene expression, cDNA was generated from 1 mg of the parental and mutant B. burgdorferi RNAs used in microarray experiments using the SuperScript III Platinum Two-Step qRT-PCR kit according to the manufacturer’s protocol (Invitrogen). qRT-PCR (in quadruplicate) 2643

Z. Ouyang, J. S. Blevins and M. V. Norgard using Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen) was then performed and the relative quantification method (DDCT) was used to calculate the variation of gene expression between B. burgdorferi strain 297 and corresponding mutants. The borrelial flaB gene was used as an endogenous control to normalize all qRTPCR data.

RESULTS Evaluation of rrp2, rpoN and rpoS mutants for mouse infectivity To determine whether rrp2, rpoN and rpoS are required by B. burgdorferi for mouse infectivity, mice were needle-inoculated intradermally with 104, 105 or 106 bacteria of the wildtype 297, mutant, or complemented strains (Hagman et al., 1998). At 2 or 6 weeks post-infection, ear-punch biopsies were collected from mice and placed in BSK-II medium. As shown in Table 1, spirochaetes were recovered from samples of all mice inoculated with wild-type strain 297, or the complemented strains. In contrast, no spirochaetes were recovered from samples of mice inoculated with the rrp2, rpoN or rpoS mutants. Notably, whereas rpoN and rpoS mutants of B. burgdorferi have been reported to be noninfectious for mice (Caimano et al., 2004; Fisher et al., 2005), this is the first study, to our knowledge, to show that an rrp2 mutant also lacks the ability to infect mice. These results indicate that all three regulators are required by B. burgdorferi to infect mammals.

under conditions known to activate the RpoN–RpoS regulatory pathway. To induce expression of rpoS, parental strain 297 and its corresponding mutants were grown to a high cell density (~16108 bacteria ml21) in BSK-H Complete medium at a reduced pH (pH 6.8) and 37 uC (Yang et al., 2000). Consistent with previous reports (Hubner et al., 2001; Smith et al., 2007; Yang et al., 2003a), both RpoS and OspC were expressed at high levels in wildtype strain 297, but not in the rrp2, rpoN or rpoS mutant (Fig. 1). When induction of the RpoN–RpoS pathway by strain 297 was confirmed in a set of cultures, bacteria were collected for the purpose of isolating total RNA for use in subsequent microarray experiments. Validation of a strain B31-based microarray for strain 297 The 70-mer oligonucleotide microarray used in this study contains quadruplicates of probes specific for 1723 ORFs of B. burgdorferi strain B31MI (Terekhova et al., 2006). Although the oligonucleotides employed in this array have been used by others for transcriptional profiling of B. burgdorferi strain 297 (Caimano et al., 2007), we assessed further the suitability of this newly printed microarray for

Borrelial growth conditions that activate the RpoN–RpoS pathway Before transcriptional profiling was undertaken, it was necessary to confirm that borreliae were being cultivated Table 1. Experimental mouse infection with B. burgdorferi strains Strain*

Dose (bacteria) 4

Mouse infectivityD at: 2 weeks p.i.d

6 weeks p.i.

ND§

297 rpoN2

10 105

4/4 0/6

rpoN2/pJSB208B rpoS2 rpoS2/pJSB259 rrp22 rrp2wt-strepr

106 106 106 104 104

6/6 0/4 10/10 0/4 4/4

4/4 ND ND ND

0/4 4/4

*Strains: rpoN2 (rpoN mutant), rpoS2 (rpoS mutant), rrp22 (rrp2 mutant harbouring a rrp2 point mutation of G239C), rrp2wt-strepr (a wild-type rrp2 allele was restored to rrp2G239C using pXY206B). DNumber culture-positive in ear-punch biopsies/total number of mice tested. dp.i., post-infection. §ND, Not determined. 2644

Fig. 1. RpoS and OspC expression assessed by SDS-PAGE (a) and immunoblot (b) analysis. Wild-type 297 and rpoN, rpoS and rrp2 mutants were cultivated in BSK-H Complete medium at pH 6.8 and 37 6C. Cells were harvested when they reached stationary phase (~1¾108 spirochaetes ml”1), and a volume of whole-cell lysate equivalent to 4¾107 cells was loaded in each gel lane. Approximate molecular masses (in kDa) are indicated at the left. Assessment of FlaB via immunoblot was used as an internal control to ensure equal numbers of cells were represented in each gel lane. Microbiology 154

Transcriptional interplay of B. burgdorferi regulators

analysing gene expression in strain 297. Comparative DNA hybridization was performed using genomic DNA extracted from strains 297 and B31. Consistent with earlier reports (Caimano et al., 2007; Terekhova et al., 2006), ~95 % of ORFs from B. burgdorferi 297 were detected using this B31based microarray. These results corroborated the suitability of using a microarray designed from B31 sequence information for transcriptional profiling of strain 297. It should be noted, however, that due to sequence variation between the ospC genes of strains B31 and 297, expression of OspC was not detected in strain 297 upon transcriptional profiling (below) or via genomic hybridization.

Genes regulated by Rrp2, RpoN and RpoS When comparing the gene expression profiles of mutants deficient in Rrp2, RpoN or RpoS, 98 genes were found to be regulated from 2.3- to 633-fold by all three of these regulators (Table 3). Among these genes, 97 were upregulated more than twofold in wild-type 297 (compared with expression levels in the corresponding mutant). Although ospC was not identified in this group of 98 genes due to the variation of ospC sequence between B. burgdorferi strains 297 and B31 (Caimano et al., 2007), qRT-PCR analysis (using 297-specific primers for ospC) showed that ospC expression was upregulated 1848-, 500- and 1408-fold, by Rrp2, RpoN and RpoS, respectively (Table 2). One gene, bba62, was upregulated 6-, 5- or 3-fold in the rrp2, rpoN or rpoS mutant, respectively. Earlier studies have shown that bba62, which encodes the Lp6.6 lipoprotein, is strongly downregulated in an RpoS-dependent manner when B. burgdorferi is cultivated in DMCs implanted into the peritoneal cavities of rats (i.e. under mammalian infection-like conditions) (Caimano et al., 2005, 2007). In agreement with these findings, bba62 was downregulated in B. burgdorferi by RpoS in our study, even under in vitro culture conditions. Comparing gene expression profiles of the rrp2, rpoN and rpoS mutants, 44, 34 and 25 genes, respectively, were upregulated more than 10-fold in wildtype 297. As shown in Fig. 3, most of the highly induced genes were located either on the chromosome (34/98) or on plasmids lp54 (21/98) and cp32s (16/98).

Correlation between microarray data and qRT-PCR To validate the results of the microarray analysis, qRT-PCR was performed on 17 genes from various categories of gene expression profiling (see below and Table 2). In an initial assessment of data correlation, the ratio of transcripts from each strain, as determined by qRT-PCR and microarray, was compared; a correlation coefficient (r) of 0.61 was observed (Fig. 2a). When the log-transformed ratios were compared, an r value of 0.89 was observed (Fig. 2b). Irrespective of these r values, when examining the absolute levels of gene expression for each given gene, similar trends were observed between these experimental conditions (Fig. 2, Table 2). These combined results indicated that the differences observed in mRNA expression levels obtained by qRT-PCR correlated well with those obtained from DNA microarray analyses.

Although a majority (59/98) of genes in this category encode hypothetical proteins and conserved hypothetical

Table 2. Validation of microarray results using quantitative RT-PCR ID

BB0771 BBA05 BBA62 BBA72 BBB19 BBP29 BBA74 BBH01 BB0702 BB0403 BB0673 BB0834 BB0160 BB0519 BB0554 BB0835 BBA61

Gene product (name)

RNA polymerase sigma factor (rpoS) Antigen, S1 Lipoprotein Hypothetical protein Outer surface protein C (ospC) Conserved hypothetical protein Outer-membrane porin (oms28) Conserved hypothetical protein Lipopolysaccharide biosynthesisrelated protein (kdtB) Hypothetical protein Conserved hypothetical protein ATP-dependent Clp protease, subunit C (clpC) Alanine racemase (alr) GrpE protein (grpE) Hypothetical protein Phosphomannomutase (cpsG) Conserved hypothetical protein

Microarray* WT/rpoS”

WT/rpoN”

WT/rrp2”

WT/rpoS”

WT/rpoN”

12.566 98.139 0.193 81.984

50.18 128.012 0.346 67.394

40.439 354.863 0.164 132.358

NA

WT/rrp2”

19.23 34.88 0.25 53.57 500 5.14 0.45 2.53 1.61

61.22 111.11 0.12 166.67 1848.43 7.52 0.31 4.61 1.95

NA

NA

NA

9.93 0.36 45.504 2.361

14.972 0.515 11.608 1.583

11.168 0.287 19.579 1.923

51.72 0.19 88.24 1408.45 5.73 0.42 2.58 2.12

0.648 0.987 1.013

0.363 0.251 0.458

0.521 0.591 1.215

0.66 1.02 0.95

0.53 0.69 0.57

0.55 1.3 1.09

0.842 1.126 1.951 1.125 0.583

0.487 1.281 1.492 2.33 0.74

0.29 5.786 2.616 4.684 0.317

1.06 1.21 2.13 1.46 0.59

0.69 0.91 1.22 0.95 0.41

0.83 5.7 2.57 1.38 0.32

*rpoS2, rpoS mutant; rpoN2, rpoN mutant; rrp22, rrp2 mutant; http://mic.sgmjournals.org

qRT-PCR*

NA,

not applicable; WT, wild-type. 2645

Z. Ouyang, J. S. Blevins and M. V. Norgard

Fig. 2. Correlation between microarray and qRT-PCR data. Original (a) or log-transformed (b) fold changes for 17 differentially expressed genes were compared between wild-type 297 and each of the three (rpoS, rpoN and rrp2) mutants.

proteins, several genes, such as those encoding decorinbinding proteins A and B (DbpA, DbpB) and RpoS, are particularly pertinent to borrelial virulence and survival in the host (Caimano et al., 2004; Hagman et al., 1998; Shi et al., 2008). In addition, eight genes encoding proteins related to chemotaxis were found to be induced by Rrp2, RpoN and RpoS. These include the chemotaxis protein methyltransferase-encoding gene bb0040 (cheR-1), two purine-binding chemotaxis protein-encoding genes, bb0565 (cheW-2) and bb0670 (cheW-3), the chemotaxis histidine kinase-encoding gene bb0567 (cheA-1), the chemotaxis response regulator-encoding gene bb0672 (cheY-3), bb0671 (cheX) and two methyl-accepting chemotaxis protein-encoding genes, bb0680 (mcp-4) and bb0681 (mcp-5). For B. burgdorferi, chemotaxis and motility are presumed to be important for colonization, dissemination into deeper tissues, and for the transition between different hosts (Fraser et al., 1997; Motaleb et al., 2005; Shi et al., 1998). Three lipoprotein-encoding genes, bba36, bba62 and bbi42, as well as two predicted outer-membrane proteinencoding genes, bbq03 and bbk53, came within the highly regulated category (Brooks et al., 2006; Setubal et al., 2006). These putative outer surface proteins may contribute to interactions between B. burgdorferi and its hosts. In addition, a few polypeptides annotated as antigens, including five putative P35-encoding genes (bba73, bba64, bba66, bbk32 and bbh32), one S1 antigen-encoding gene (bba05), and one S2 antigen-encoding gene (bba04), were also found to be induced by Rrp2, RpoN and RpoS. The genome of B. burgdorferi contains one linear chromosome and numerous linear and circular plasmids (Fraser et al., 1997). Several plasmids, such as lp25, lp28-1 and lp36, are essential for borrelial infectivity (Jewett et al., 2007; Purser & Norris, 2000; Stewart et al., 2005). Plasmid lp54 has been implicated as being important for the survival of B. burgdorferi in both tick and mammalian hosts, largely because of the presence of the ospAB and 2646

dbpBA operons (Caimano et al., 2005; Hagman et al., 1998; Neelakanta et al., 2007; Shi et al., 2008; Yang et al., 2004). Many other genes encoded on lp54 have been found to be differentially regulated in response to temperature, pH and other mammalian-derived signals, which are presumed to be mediated, at least in part, by RpoS (Caimano et al., 2005; Clifton et al., 2006; Ojaimi et al., 2003; Revel et al., 2002). Consistent with these previous findings, our data provided further evidence that 22 lp54-encoded genes are regulated by RpoS. In addition, lp36 has been reported to be vital for B. burgdorferi infection of mammal hosts (Jewett et al., 2007). In this regard, studies have shown that B. burgdorferi infectivity in mice was attenuated by the inactivation of lp36-encoded bbk32 (encoding a fibronectin-binding protein) (Seshu et al., 2006) or bbk17 (encoding an adenine deaminase, adeC, which converts adenine to hypoxanthine) (Jewett et al., 2007), thereby underscoring the importance of lp36 in B. burgdorferi pathogenesis. A previous study also found that bbk32 and bbk17 are regulated in response to mammalian signals (Revel et al., 2002). More specifically, He et al. (2007) found that bbk32 is regulated by Rrp2, RpoN and RpoS. Consistent with all of these reports, our data showed that the lp36-encoded bbk32, bbk52.1, bbk53, bbk07 and bbk17 were all positively regulated by Rrp2, RpoN and RpoS (Table 3). In Escherichia coli, RpoS controls many genes involved in cellular metabolism and the stress response (Dong et al., 2008; Farewell et al., 1998; Loewen et al., 1998). Similarly, many B. burgdorferi genes encoding proteins with various putative physiological functions were induced by Rrp2, RpoN and RpoS; these genes included bb0251 (leuS), bb0777 (apt), bbk17 (adeC), bb0257 (cell division), bb0842 (arcB), bb0329 (oppA-2), bba34 (oppAV), bb0313 (ftsJ), bb0797 (mutS), bb0637 (nhaC-1), bbd20 (transposase), bb0646 (hydrolase), bb0728 (nox) and bb0729 (gltP) (Fraser et al., 1997). Among these genes, bb0329 and bba34 encode Microbiology 154

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Table 3. B. burgdorferi genes (n598) regulated by Rrp2, RpoN and RpoS See footnote to Table 2 for explanation of headings. Function

BBD24 BBD07 BBA71 BBA65 BBA37 BBA05 BBA66 BB0844 BBA72 BBA25 BBA24 BBA07 BBA36 BBA06 BBA73 BBA64 BBA34

Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Antigen, S1 Antigen, P35, putative Hypothetical protein Hypothetical protein Decorin-binding protein B Decorin-binding protein A chpAI protein, putative Lipoprotein Hypothetical protein Antigen, P35, putative Antigen, P35 Oligopeptide ABC transporter, periplasmic oligopeptide-binding protein Chemotaxis protein methyltransferase Outer-membrane protein, putative Hypothetical protein RNA polymerase sigma factor Hypothetical protein Hypothetical protein Conserved hypothetical protein Hypothetical protein Conserved hypothetical protein Outer-membrane protein, putative Conserved hypothetical protein Hypothetical protein Immunogenic protein P35 Methyl-accepting chemotaxis protein Conserved hypothetical protein Conserved hypothetical protein Hypothetical protein Conserved hypothetical protein Hypothetical protein Antigen, S2

BB0040 BBQ03 BBD19 BB0771 BBQ02 BBJ02 BBL29 BB0404 BBP29 BBI42 BBR29 BBA35 BBK32 BB0680 BBI31 BBM29 BBA57 BBD001 BBA32 BBA04

Gene

dbpB dbpA

oppAV cheR-1

rpoS

mcp-4

Replicon

Ratio WT : rpoS”

P value

Ratio WT : rpoN”

P value

Ratio WT : rrp2”

P value

lp17 lp17 lp54 lp54 lp54 lp54 lp54 Chromosome lp54 lp54 lp54 lp54 lp54 lp54 lp54 lp54 lp54

136.67 133.01 130.699 112.449 110.036 98.139 97.636 82.36 81.984 71.11 61.382 55.534 49.106 38.742 37.706 35.605 28.672

6.80610205 8.56610204 2.30610205 3.17610204 1.90610205 4.61610203 3.60610205 1.00610206 4.30610205 4.07610204 3.70610204 5.20610205 1.54610204 1.93610203 8.40610205 4.65610204 1.32610203

198.668 118.574 47.931 111.209 103.58 128.012 178.822 99.297 67.394 74.755 60.625 50.984 61.466 54.266 52.739 41.347 633.047

6.34610204 1.48610203 1.15610203 3.12610203 1.83610204 2.15610203 7.81610204 2.72610204 3.43610204 9.76610204 1.65610203 1.40610203 2.36610203 4.46610202 1.94610204 2.73610203 1.09610202

334.061 163.951 195.967 278.807 197.988 354.863 228.09 228.032 132.358 185.967 146.41 99.543 110.551 192.269 86.034 65.42 553.801

0.0061000 9.00610206 2.90610205 1.00610206 1.00610206 5.90610205 8.60610205 0.0061000 2.00610206 0.0061000 0.0061000 1.00610206 1.00610206 5.61610203 0.0061000 2.40610204 3.02610204

Chromosome lp56 lp17 Chromosome lp56 lp38 cp32-8 Chromosome cp32-1 lp28-4 cp32-4 lp54 lp36 Chromosome lp28-4 cp32-6 lp54 lp17 lp54 lp54

16.302 12.814 12.734 12.566 12.501 11.173 10.849 10.376 9.93 9.64 9.557 9.471 9.307 9.179 8.801 8.71 8.444 8.335 7.719 7.61

9.70610205 9.69610204 3.39610203 1.72610203 3.79610202 3.00610206 5.99610204 7.89610203 4.64610204 5.97610204 8.60610205 1.90610204 5.86610204 3.90610205 2.45610204 5.47610204 4.90610205 1.32610202 5.34610204 1.76610203

12.829 14.655 10.014 50.18 6.947 13.813 14.748 7.434 14.972 13.734 13.968 14.648 10.427 7.063 10.494 10.183 6.058 7.186 6.903 9.794

6.31610204 6.23610203 2.30610202 2.84610202 6.04610203 3.10610203 4.50610203 2.90610202 4.05610203 4.43610203 1.46610203 5.86610203 1.05610202 2.17610204 2.45610202 8.68610203 9.28610203 2.58610202 9.27610203 6.70610203

28.602 27.142 10.934 40.439 26.124 29.726 11.217 11.176 11.168 16.475 10.006 18.539 15.082 19.11 10.433 8.082 10.797 14.148 8.006 20.633

0.0061000 0.0061000 1.30610205 0.0061000 9.48610203 6.00610206 5.00610206 1.40610205 3.80610205 1.00610206 1.00610206 3.00610206 2.00610206 0.0061000 6.88610204 0.0061000 4.00610206 8.62610203 6.00610205 7.39610204

Transcriptional interplay of B. burgdorferi regulators

2647

Gene ID

Gene ID

Function

BBA33 BB0689 BB0418 BBD20

Hypothetical protein Hypothetical protein Hypothetical protein Transposase-like protein, authentic frameshift Conserved hypothetical protein, pseudogene Hypothetical protein Purine-binding chemotaxis protein Conserved hypothetical protein Methyl-accepting chemotaxis protein Conserved hypothetical protein Purine-binding chemotaxis protein Hypothetical protein, paralogous family 161, authentic point mutation Outer-membrane protein Conserved hypothetical protein Conserved hypothetical protein Conserved hypothetical protein Hypothetical protein Conserved hypothetical protein Conserved hypothetical protein Conserved hypothetical protein Conserved hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Leucyl-tRNA synthetase Conserved hypothetical protein Hypothetical protein Chemotaxis histidine kinase Hypothetical protein Hypothetical protein Conserved hypothetical protein

BBK52.1 BB0563 BB0565 BBN36 BB0681 BBL36 BB0670 BBN29

Microbiology 154

BBK53 BBR36 BBM36 BBM35 BBJ01 BBQ43 BBO36 BBS38 BBN35 BB0555 BBJ28 BB0776 BB0251 BBQ44 BBH10 BB0567 BBK07 BB0566 BBS31 BB0853.2 BB0777 BBK17 BBO29 BBD05

Adenine phosphoribosyltransferase Adenine deaminase Hypothetical protein Hypothetical protein, paralogous family 84

Gene

cheW-2 mcp-5 cheW-3

leuS

cheA-1

apt adeC

Replicon

Ratio WT : rpoS”

P value

Ratio WT : rpoN”

P value

Ratio WT : rrp2”

P value

lp54 Chromosome Chromosome lp17

7.501 7.423 7.35 7.263

5.00610206 5.23610204 2.81610204 2.32610203

15.13 6.845 5.729 10.243

1.70610203 5.29610203 1.14610203 9.97610203

16.723 12.534 11.367 8.492

2.52610204 0.0061000 0.0061000 1.49610204

lp36

6.711

8.73610203

12.105

8.41610203

5.455

1.74610202

Chromosome Chromosome cp32-9 Chromosome cp32-8 Chromosome cp32-9

6.59 6.403 6.274 6.225 5.756 5.67 5.65

3.65610204 4.20610204 4.66610203 1.37610204 1.45610203 1.64610203 2.88610204

6.459 5.284 7.831 5.361 6.456 4.868 10.087

2.17610203 4.36610203 2.06610202 1.69610203 1.18610202 2.25610203 3.45610203

12.58 11.255 11.752 11.498 9.481 10.179 7.883

3.00610206 0.0061000 7.70610205 0.0061000 0.0061000 0.0061000 5.70610205

lp36 cp32-4 cp32-6 cp32-6 lp38 lp56 cp32-7 cp32-3 cp32-9 Chromosome lp38 Chromosome Chromosome lp56 lp28-3 Chromosome lp36 Chromosome cp32-3 Chromosome Chromosome lp36 cp32-7 lp17

5.595 5.59 5.574 5.496 5.44 5.433 5.432 5.422 5.173 5.144 5.118 5.065 5.004 4.707 4.56 4.452 4.42 4.39 4.136 4.014 3.959 3.878 3.815 3.726

3.69610203 1.37610203 2.68610203 1.08610203 9.49610203 1.77610203 1.60610203 8.70610204 1.49610203 1.17610203 6.69610203 1.80610203 9.40610205 2.99610203 1.21610203 3.52610203 9.56610204 1.39610203 2.37610203 1.62610202 3.61610203 1.62610203 1.53610203 2.29610202

8.44 6.41 6.218 6.262 4.206 5.903 6.241 6.105 5.572 4.455 4.372 5.322 3.862 5.974 5.747 4.008 5.572 4.184 5.937 6.505 4.005 4.738 7.354 3.313

5.93610203 1.05610202 4.29610202 9.96610203 1.07610202 1.45610202 1.15610202 9.50610203 1.32610202 1.69610203 4.28610202 1.08610202 4.75610204 2.45610202 9.78610203 1.97610202 2.10610202 6.60610203 3.55610202 4.43610202 5.86610203 1.26610202 6.40610203 4.92610202

6.951 9.939 8.169 9.379 12.837 8.162 9.299 9.082 7.733 9.31 3.32 5.425 3.056 7.211 5.852 6.594 9.379 8.399 3.229 3.837 4.384 4.794 6.066 6.706

2.45610204 0.0061000 4.40610205 0.0061000 2.30610202 5.00610206 0.0061000 0.0061000 0.0061000 0.0061000 4.47610204 1.40610205 3.00610205 0.0061000 1.16610202 1.40610205 1.10610205 0.0061000 7.40610205 8.40610205 4.00610206 1.95610204 1.91610204 5.59610204

Z. Ouyang, J. S. Blevins and M. V. Norgard

2648

Table 3. cont.

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Table 3. cont. Function

Gene

Replicon

BB0728 BBB09 BB0729 BB0265 BB0257 BB0842

NADH oxidase, water-forming Hypothetical protein Glutamate transporter Hypothetical protein Cell division protein, putative Ornithine carbamoyltransferase, catabolic Hypothetical protein Conserved hypothetical protein Antigen, P35, putative Oligopeptide ABC transporter, periplasmic oligopeptide-binding protein Cell division protein Conserved hypothetical protein Hypothetical protein Hypothetical protein, pseudogene Conserved hypothetical protein, authentic frameshift DNA mismatch repair protein Na+/H+ antiporter Chemotaxis response regulator Hypothetical protein Hydrolase, alpha/beta fold family Hypothetical protein Chemotaxis operon protein Hypothetical protein Conserved hypothetical protein, authentic frameshift Lipoprotein

nox

arcB

Chromosome cp26 Chromosome Chromosome Chromosome Chromosome

3.699 3.634 3.508 3.377 3.35 3.348

2.61610204 5.07610204 2.43610203 1.16610202 7.97610204 1.39610203

3.517 3.68 4.202 3.771 4.237 2.659

3.21610203 3.68610203 6.80610203 3.88610202 1.35610202 3.51610203

8.095 6.393 8.587 3.927 5.218 4.116

7.00610206 1.00610206 0.0061000 8.60610205 0.0061000 2.00610206

oppA-2

Chromosome lp54 lp28-3 Chromosome

3.22 3.192 3.107 3.102

4.50610205 5.21610204 1.02610204 3.39610203

2.822 3.001 3.037 2.335

4.01610204 2.42610203 5.11610203 4.90610203

2.945 4.005 4.116 3.052

3.10610205 1.10610205 9.00610206 6.30610205

ftsJ

Chromosome Chromosome Chromosome Chromosome lp21

2.982 2.899 2.866 2.844 2.775

7.01610203 6.21610204 4.65610204 5.39610204 4.48610203

3.127 3.609 2.984 2.439 3.423

1.18610202 1.02610202 8.92610203 7.19610204 2.74610202

3.637 4.674 3.45 3.025 2.624

4.50610205 5.30610205 1.69610204 7.00610206 4.05610203

mutS nhaC-1 cheY-3

Chromosome Chromosome Chromosome cp32-7 Chromosome lp54 Chromosome lp28-3 lp28-2

2.634 2.593 2.575 2.557 2.535 2.483 2.476 2.358 2.265

2.15610204 6.00610205 3.83610203 1.13610203 1.46610204 2.05610202 1.48610202 1.40610205 9.55610203

2.899 2.613 2.179 2.565 2.535 5.307 3.45 3.717 6.603

2.63610202 3.52610203 3.14610203 3.05610202 2.62610203 1.78610202 3.93610202 3.04610202 6.03610203

3.403 3.406 3.143 2.377 3.252 3.973 4.547 2.717 5.31

1.00610206 1.19610204 1.00610206 2.22610204 4.00610206 1.05610204 7.30610205 1.99610204 3.00610206

lp54

0.193

1.68610203

0.346

2.06610202

0.164

1.17610204

BB0846 BBA01 BBH32 BB0329 BB0313 BB0041 BB0509 BB0845.2 BBU12 BB0797 BB0637 BB0672 BBO43 BB0646 BBA26 BB0671 BBH06 BBG03 BBA62

gltP

cheX

Ratio WT : rpoS”

P value

Ratio WT : rpoN”

P value

Ratio WT : rrp2”

P value

2649

Transcriptional interplay of B. burgdorferi regulators

Gene ID

Z. Ouyang, J. S. Blevins and M. V. Norgard

Fig. 3. Genomic distribution of genes (see Table 3) differentially regulated by Rrp2, RpoN and RpoS. Black bars indicate the numbers of genes that are activated by the Rrp2–RpoN– RpoS pathway, whereas white bars indicate the number of genes downregulated. The total number of genes differentially expressed on each genetic element is noted above or below each bar.

two putative periplasmic oligopeptide-binding proteins, which facilitate the transport of small peptides and essential amino acids (Fraser et al., 1997; Medrano et al., 2007). In addition, bb0728 and bb0729 are presumed to form an operon involved in the uptake of the amino acid glutamate (Fraser et al., 1997). Genes likely regulated by Rrp2, RpoN and RpoS A second subset of 47 genes was most likely regulated by all three of Rrp2, RpoN and RpoS (Table 4). This conclusion derives from the fact that genes in this category were regulated by at least two of the three regulators active in the Rrp2–RpoN–RpoS pathway, and trends favoured regulation by all three members of the pathway. For example, 21 genes were regulated by Rrp2 and RpoN, 24 were regulated by Rrp2 and RpoS, and two (bbq37 and bb0076) were regulated by both RpoN and RpoS. Except for gene bbh01 and the pseudogene bbh09.1, the fold changes of expression for all of the other genes were less substantial (,10-fold) than those genes that were characterized above as being regulated by Rrp2, RpoN and RpoS. When the expression data for all 47 genes in this category were reexamined in an analysis in which the twofold cutoff value and the P value threshold of 0.05 constraints were relaxed, the trends in gene expression changes for all of these genes were similar in all three of the mutants (Table 4). For example, if a gene was repressed or induced by two regulators (e.g. Rrp2 and RpoN), it also was repressed or induced by the third regulator (e.g. RpoS). To further analyse this trend, qRT-PCR was employed to examine the expression of two representative genes in this category: bba74 (oms28) and bbh01. In the microarray experiments, bba74 and bbh01 were not influenced significantly by RpoN and RpoS, respectively. However, qRT-PCR showed that both bba74 and bbh01 were indeed regulated more than twofold by RpoS, RpoN and Rrp2 (Table 2), further substantiating the high probability that genes placed in this category are, in fact, likely influenced by all three of Rrp2, RpoN and RpoS. Among the genes in this category, most have unknown functions, but some genes [bb0012 (hisT), 2650

bb0076 (ftsY), bb0254 (recJ), bb0312 (cheW-1), bb0344 (uvrD), bb0386 (rpsG), bb0461 (dnaX), bb0588 (pfs-2), bba74 (oms28), and the putative plasmid partition proteins bbo32 and bbu05] likely encode physiological functions (Fraser et al., 1997). bba16 (ospB), which belongs to the ospAB operon that is essential for B. burgdorferi colonization and survival within tick midguts (Neelakanta et al., 2007; Yang et al., 2004), was downregulated 3.6-, 2.1 and 1.8-fold, respectively, by Rrp2, RpoN and RpoS. In our test, we also found that bba15 (ospA) was downregulated 1.8-, 1.3- and 1.3-fold, respectively, by Rrp2, RpoN and RpoS. Although the fold change of ospA was just below the detection threshold (twofold) in our microarray data analysis, both ospA and ospB exhibited a similar trend of change for all three mutants. Genes potentially influenced by RpoN or RpoS alone A third set of genes was potentially regulated by either RpoN or RpoS alone. Twelve genes were identified that were regulated in the range of 2.2–4-fold by RpoN alone (Table 5). These findings are inconsistent with the hypothesis that RpoN modulates borrelial gene expression via rpoS. However, qRT-PCR analysis of the expression of three of these RpoN-regulated genes (bb0403, bb0673 and bb0834) showed that all three genes were only moderately affected by the mutation of RpoN (less than twofold) (Table 2). Furthermore, additional analysis of the microarray results revealed marked similarity in the overall patterns of regulatory effect. For example, in all three mutants, the relative trends and fold changes in expression were approximately equivalent for each of these 12 genes potentially regulated by RpoN alone (Table 5). An additional three genes were identified during the transcriptional comparison that were predicted to be regulated by RpoS alone: bbk33, bbm41 and bb0702 (kdtB) were upregulated 3.4-, 3.1- and 2.4-fold, respectively. However, validation of the microarray via qRT-PCR for one of these genes, bb0702, showed that expression of bb0702 was similarly upregulated by RpoS (2.1-fold), RpoN (1.6-fold) Microbiology 154

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Table 4. B. burgdorferi genes (n547) likely regulated by Rrp2, RpoN and RpoS See footnote to Table 2 for explanation of headings. Gene ID BBH01 BBH09.1 BBF14.1 BBR37 BBS39 BBQ49 BBL37 BBN13 BBK52 BB0766 BBO37 BBQ37 BBN42 BBM42 BBK05 BB0843 BB0461

2651

BBS44 BBU05 BBA63 BBU09 BBQ46 BBH33 BBJ23 BB0323 BB0798 BBS27 BB0344 BB0001

Gene

Conserved hypothetical protein Conserved hypothetical protein, pseudogene Hypothetical protein, paralogous family 65, authentic frameshift Conserved hypothetical protein Conserved hypothetical protein Hypothetical protein Conserved hypothetical protein Hypothetical protein, paralogous family 154, authentic frameshift Protein p23 Colicin V production protein, putative Conserved hypothetical protein Conserved hypothetical protein Hypothetical protein Conserved hypothetical protein Hypothetical protein Conserved hypothetical integral membrane protein DNA polymerase III, subunits gamma dnaX and tau Purine-binding chemotaxis protein cheW-1 Conserved hypothetical protein 5-Methylthioadenosine/S-adenosylpfs-2 homocysteine nucleosidase, putative Hypothetical protein Plasmid partition protein, putative Hypothetical protein Conserved hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Competence protein F, putative Hypothetical protein DNA helicase uvrD Hypothetical protein

Replicon

Ratio WT : rpoS”

P value

Ratio WT : rpoN”

P value

Ratio WT : rrp2”

P value

lp28-3 lp28-3

45.504 10.401

1.32610201 1.42610201

11.608 4.356

3.13610202 8.61610203

19.579 10.848

1.31610203 2.61610202

lp28-1

7.098

7.55610202

3.705

4.39610202

2.542

2.59610202

cp32-4 cp32-3 lp56 cp32-8 cp32-9

5.583 4.512 4.078 3.992 3.838

1.24610202 2.80610203 1.04610202 1.18610202 7.09610203

4.448 4.242 2.557 2.684 2.135

1.25610201 1.15610201 1.10610201 6.85610202 1.49610201

7.56 6.894 2.118 4.014 2.724

5.92610204 1.00610206 1.64610203 3.50610205 2.46610203

lp36 Chromosome

3.835 3.259

5.64610202 5.85610203

8.535 1.467

1.80610202 5.22610202

5.064 2.424

2.00610205 2.50610205

cp32-7 lp56 cp32-9 cp32-6 lp36 Chromosome

3.103 3.011 2.673 2.537 2.468 2.456

5.40610203 3.72610203 1.15610203 1.08610203 7.87610204 9.51610204

2.269 3.338 2.01 1.657 2.216 1.718

1.14610201 4.69610202 5.23610202 8.57610202 1.91610201 6.20610202

3.411 1.641 2.189 2.953 3.417 2.387

2.60610204 5.94610203 1.70610205 6.60610203 9.60610205 3.60610205

chromosome

2.417

2.17610203

1.678

9.37610203

2.528

2.00610206

Chromosome cp32-3 Chromosome

2.355 2.031 2.023

7.91610204 1.70610202 3.12610203

1.682 1.62 1.688

5.47610202 9.06610202 2.44610202

2.761 2.411 2.157

6.70610205 6.21610204 1.00610206

cp32-3 lp21 lp54 lp21 lp56 lp28-3 lp38 Chromosome Chromosome cp32-3 Chromosome Chromosome

2.013 1.986 1.922 1.861 1.86 1.844 1.833 1.832 1.82 1.694 1.557 1.048

7.43610203 2.82610202 3.67610202 8.57610203 6.36610202 5.74610203 3.83610202 5.17610202 2.19610202 4.81610202 3.08610202 8.83610201

2.097 2.435 4.961 2.627 3.411 2.795 4.749 2.669 3.934 5.413 2.264 0.379

5.43610202 1.17610202 2.46610203 1.10610202 3.33610202 3.40610202 1.21610202 1.32610202 1.52610202 3.57610202 5.85610203 1.84610203

2.281 3.131 4.111 2.556 2.58 2.604 3.153 2.988 3.228 2.629 2.461 0.49

9.20610205 1.59610203 8.50610205 6.90610205 7.10610204 3.70610203 1.44610202 6.80610205 1.20610205 7.85610204 4.24610204 7.34610203

Transcriptional interplay of B. burgdorferi regulators

BB0312 BBS45 BB0588

Function

Gene ID BBA52 BBR34 BBA03 BBE01 BBF20 BBA16 BB0076 BB0254 BBA22 BBO32 BBI11 BBC12 BB0012 BB0386 BBA74

Function

Gene

Outer-membrane protein Conserved hypothetical protein Outer-membrane protein Hypothetical protein Conserved hypothetical protein Outer surface protein B ospB Signal recognition particle-docking ftsY protein FtsY Single-stranded-DNA-specific exonu- recJ clease Hypothetical protein Plasmid partition protein, putative Hypothetical protein Conserved hypothetical protein Pseudouridylate synthase I hisT Ribosomal protein S7 rpsG Outer-membrane porin oms28

Replicon

Ratio WT : rpoS”

P value 201

Ratio WT : rpoN”

P value

Ratio WT : rrp2”

P value

204

lp54 cp32-4 lp54 lp25 lp28-1 lp54 Chromosome

0.798 0.694 0.641 0.638 0.603 0.558 0.496

2.23610 5.88610202 4.67610202 3.87610201 4.43610202 1.62610202 1.12610202

0.474 0.414 0.351 0.375 0.451 0.477 0.366

3.69610 2.62610202 1.11610202 5.31610203 2.52610203 1.19610203 1.55610202

0.432 0.486 0.366 0.17 0.368 0.282 0.596

7.59610204 7.62610204 7.80610205 8.80610203 6.00610206 6.82610204 1.43610203

Chromosome

0.484

2.86610202

0.542

1.53610201

0.386

7.60610205

lp54 cp32-7 lp28-4 cp9 Chromosome Chromosome lp54

0.473 0.429 0.409 0.407 0.4 0.385 0.36

7.17610204 1.88610204 1.44610204 6.45610204 8.23610204 1.66610203 4.16610203

0.609 0.777 0.616 0.608 0.551 0.695 0.515

1.73610202 5.20610202 2.82610202 1.66610204 3.38610202 8.33610202 2.25610202

0.39 0.33 0.243 0.488 0.405 0.477 0.287

3.90610203 3.00610206 2.07610202 2.33610203 1.65610203 5.44610204 6.40610205

Table 5. B. burgdorferi genes (n512) regulated by RpoN alone See footnote to Table 2 for explanation of headings. Gene ID

Function

BBF26.1

Conserved hypothetical protein, pseudogene Hypothetical protein Conserved hypothetical integral membrane protein Hypothetical protein ATP-dependent Clp protease, subunit C Conserved hypothetical protein Lipoprotein Lipoprotein, putative Replicative DNA helicase Hypothetical protein Hypothetical protein Hypothetical protein

BBD25 BB0234

Microbiology 154

BBQ10 BB0834 BB0673 BBM28 BB0758 BB0111 BBB27 BB0403 BB0077

Gene

clpC lp dnaB

Replicon

Ratio WT : rpoS”

P value

Ratio WT : rpoN”

P value

Ratio WT : rrp2”

P value

lp28-1

2.042

3.55610201

0.292

3.79610203

1.515

4.13610201

lp17 Chromosome

1.527 1.106

1.64610201 7.42610201

2.897 0.47

1.21610202 7.53610204

1.537 0.88

1.27610201 1.84610201

lp56 Chromosome Chromosome cp32-6 Chromosome Chromosome cp26 Chromosome Chromosome

1.096 1.013 0.987 0.925 0.913 0.798 0.772 0.648 0.52

7.34610201 9.07610201 9.65610201 5.75610201 6.65610201 5.82610201 3.25610201 5.25610202 2.74610201

2.408 0.458 0.251 2.412 0.439 0.367 0.379 0.363 0.462

9.36610203 4.62610204 2.10610202 7.35610203 6.88610203 1.83610202 1.52610202 3.34610202 6.44610203

1.223 1.215 0.591 1.387 0.779 0.927 0.555 0.521 0.866

4.45610201 1.61610202 4.28610203 4.67610201 1.31610202 8.25610201 5.33610202 8.91610203 5.85610201

Z. Ouyang, J. S. Blevins and M. V. Norgard

2652

Table 4. cont.

Transcriptional interplay of B. burgdorferi regulators

and Rrp2 (2.0-fold) (Table 2). Taken together, these data trends suggest that these genes are not uniquely influenced by RpoN or RpoS. Genes potentially regulated by Rrp2 only We proposed previously that Rrp2 is likely an enhancerbinding protein (response regulator) (Rappas et al., 2007; Yang et al., 2003a) that, upon activation, ostensibly activates the alternative sigma factor RpoN; activated RpoN then promotes the transcription of rpoS (Hubner et al., 2001; Yang et al., 2003a). Experimental testing of this cascade, however, has been hampered in part by the fact that we and others have been unable to inactivate rrp2 by insertional or other mutagenesis approaches (Burtnick et al., 2007; Yang et al., 2003a). As a way of partially circumventing this obstacle, a point mutation (G239C) was introduced in the putative ATP-binding domain of Rrp2, thereby abolishing RpoN-dependent rpoS expression (Yang et al., 2003a). Comparative microarray analyses then were performed to determine the gene expression profiles of B. burgdorferi strain 297 and the rrp2 mutant. Our data showed that 37 and 69 genes were either upregulated (2.2–5.8- fold) or downregulated (2.1–5.9-fold), respectively, in the wild-type B. burgdorferi strain 297 (Table 6). Several of these genes are particularly noteworthy. First, three genes encoding putative plasmid partition proteins, bbs35 (5.9-fold) on cp32-3, bbr33 (2.6-fold) on cp32-4, and bbk21 (2.6-fold) on lp36, appeared to be repressed by Rrp2. These plasmid partition proteins may be essential for maintaining the sensitive balance among the numerous endogenous B. burgdorferi plasmids. Furthermore, the virulence-associated gene bbe22 (pncA), encoding a nicotinamidase (Purser et al., 2003), was also repressed by Rrp2, as were the putative glycerol uptake and metabolism genes glpA, glpF and glpK (Fraser et al., 1997). Finally, our data imply that Rrp2 may contribute to a general stress-like response in B. burgdorferi, inasmuch as the heatshock protein-encoding genes bb0518 (dnaK-2) and bb0264 (dnaK-1) were regulated by Rrp2. To validate the results of microarray comparisons, qRT-PCR was performed to examine the expression of five genes, bb0160 (alr), bb0519 (grpE), bb0554, bb0835 (cpsG) and bba61. Although the microarray results indicated that all five genes were regulated more than twofold (P ,0.05) by Rrp2 (Table 6), qRT-PCR data revealed that bb0160 and bb0835 were regulated 0.83- and 1.38-fold, respectively, by Rrp2 (Table 2). The qRT-PCR data also indicated that, with the exception of bb0519, these genes were regulated at a similar level by RpoS, RpoN and Rrp2 (Table 2). These data suggest that it is difficult to draw definitive conclusions about regulatory events based on microarray data that exhibit only slight fold changes in individual gene expression patterns.

DISCUSSION Discovery of the Rrp2–RpoN (s54)–RpoS (sS) alternative sigma factor regulatory cascade (Hubner et al., 2001; Yang http://mic.sgmjournals.org

et al., 2003a) was an important advance in understanding borrelial gene regulation, but represented only a first step towards deciphering key adaptive responses needed by B. burgdorferi to sustain itself in nature. It is now clear that this regulatory pathway is vital for modulating virulence expression by B. burgdorferi (Caimano et al., 2004, 2005, 2007; Fisher et al., 2005; He et al., 2007; Neelakanta et al., 2007; Yang et al., 2004), but its recognition has prompted many new questions and has spawned a number of controversies. For example, the results of Fisher et al. (2005) have called into question the linearity of the RpoN– RpoS pathway. From comparative microarray studies with rpoN and rpoS mutants generated in strain B31, those authors concluded that three patterns of regulation exist in B. burgdorferi: 254 genes were regulated by RpoN alone (but not by RpoS), 94 genes were regulated by RpoS alone (but not by RpoN), and 51 genes were regulated by both RpoN and RpoS. The most surprising of these observations was the very high number of genes seemingly regulated by RpoN or RpoS alone (254 and 94 genes, respectively). There were a number of potential caveats, however, in these findings. First, quantitative RT-PCR analysis revealed that rpoS expression decreased in the rpoN mutant by only 2.63-fold (Fisher et al., 2005), a level inordinately low given the well-documented dependence of rpoS expression on RpoN (Hubner et al., 2001). Second, globally, only slight changes in gene expression were observed by Fisher et al. (2005) in their comparative microarray experiments; thus, changes that were considered significant were, at best, modest. For example, for the genes regulated by RpoN or RpoS alone, changes in gene expression had ranges from only 0.49- to 2.28-fold and 0.55- to 2.64-fold, respectively (Fisher et al., 2005). Even for those genes categorized as being regulated by both RpoN and RpoS, the relative changes in gene expression were slight. Many genes that were shown to be significantly induced by RpoS, including bba25 (dbpB), bba66 and bb0844 (Caimano et al., 2007; Clifton et al., 2006; Hubner et al., 2001), were regulated only moderately (less than fourfold) by RpoS in their microarray experiments (Fisher et al., 2005). In addition, bba24 (dbpA) was found to be induced 1.6-fold by RpoS alone, but not by RpoN, which is inconsistent with previous reports (Hubner et al., 2001; Yang et al., 2003a). We contend that it is unwise to draw conclusions about gene regulation from microarray data displaying such relatively small changes. This is particularly true for B. burgdorferi, which is sensitive to many as yet unknown culture variables (Callister et al., 1990; Nelson et al., 1991; Picken et al., 1997; Wang et al., 2004; Yang et al., 2001). As noted earlier, the minimal change in the expression of rpoS mRNA (2.63-fold) by the rpoN mutant used in the study by Fisher et al. (2005) was in marked contrast to our observed change of 19-fold in an analogous rpoN mutant, suggesting that the culture conditions employed by Fisher et al. (2005) were not optimal for induction of the Rrp2– RpoN–RpoS pathway. For the experiments described in the current study, we optimized our in vitro cultivation 2653

Z. Ouyang, J. S. Blevins and M. V. Norgard

Table 6. B. burgdorferi genes (n5106) regulated by Rrp2 alone Gene ID

Function

Gene

Replicon

Ratio WT : rrp2”

P value

BB0519 BB0835 BB0516 BBG01 BBE22 BB0207 BBU02 BB0843.1 BB0297 BB0343 BB0311 BB0667 BB0400 BBH35 BB0554 BB0828 BBK30 BB0518 BB0406 BB0759 BB0693 BBP41 BBP23 BB0415 BBQ30 BB0264 BB0668 BBR23 BB0462 BB0036 BB0453 BBS23 BBK54 BB0836 BBM23 BBL23 BBN23 BB0370 BB0790 BB0192 BB0536 BB0385 BB0072 BB0144

GrpE protein Phosphomannomutase rRNA methylase Hypothetical protein Pyrazinamidase/nicotinamidase UTP-glucose-1-phosphate uridylyltransferase Hypothetical protein Hypothetical protein, pseudogene smg protein Glu-tRNA(Gln) amidotransferase, subunit C Conserved hypothetical protein Conserved hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein DNA topoisomerase I Hypothetical protein Heat-shock protein 70 Hypothetical protein Hypothetical protein Xylose operon regulatory protein Hypothetical protein Pore-forming haemolysin Protein-glutamate methylesterase Pore-forming haemolysin Heat-shock protein 70 Flagellar filament outer layer protein Pore-forming haemolysin Conserved hypothetical protein DNA topoisomerase IV Conserved hypothetical protein Pore-forming haemolysin Conserved hypothetical protein Excinuclease ABC, subunit B Pore-forming haemolysin Pore-forming haemolysin Pore-forming haemolysin Tyrosyl-tRNA synthetase Hypothetical protein Hypothetical protein Zinc protease, putative Basic membrane protein D Hypothetical protein Glycine betaine, L-proline ABC transporter, glycine/betaine/L-proline-binding protein Glutamate transporter, putative Conserved hypothetical protein Glycerol-3-phosphate dehydrogenase, anaerobic Neutrophil-activating protein Conserved hypothetical protein Conserved hypothetical protein Conserved hypothetical protein Hypothetical protein Conserved hypothetical protein, authentic point mutation

grpE cpsG yacO

Chromosome Chromosome Chromosome lp28-2 lp25 Chromosome lp21 Chromosome Chromosome Chromosome Chromosome Chromosome Chromosome lp28-3 Chromosome Chromosome lp36 Chromosome Chromosome Chromosome Chromosome cp32-1 cp32-1 Chromosome lp56 Chromosome Chromosome cp32-4 Chromosome Chromosome Chromosome cp32-3 lp36 Chromosome cp32-6 cp32-8 cp32-9 Chromosome Chromosome Chromosome Chromosome Chromosome Chromosome Chromosome

5.786 4.684 3.432 3.301 3.226 2.977 2.819 2.811 2.792 2.762 2.706 2.686 2.674 2.634 2.616 2.592 2.587 2.497 2.491 2.49 2.466 2.425 2.404 2.402 2.399 2.399 2.376 2.316 2.285 2.273 2.272 2.271 2.259 2.237 2.228 2.183 2.161 0.477 0.473 0.468 0.464 0.453 0.452 0.449

2.00610206 1.37610203 1.16610203 2.00610202 2.33610203 5.45610204 2.85610202 1.13610203 1.20610204 2.40610204 6.44610204 2.36610203 6.42610204 6.17610203 1.60610205 2.56610203 2.54610203 1.30610205 4.90610205 4.36610204 3.96610204 2.61610203 8.99610204 2.95610203 1.06610203 1.00610206 5.80610205 1.27610204 1.40610205 2.00610205 1.90610205 6.20610205 1.90610204 0.00610+00 7.00610206 5.00610205 1.30610205 0.00610+00 2.80610205 1.14610204 3.30610205 2.18610204 2.31610204 2.60610205

Chromosome lp36 Chromosome Chromosome cp26 cp32-7 cp32-6 Chromosome cp32-4

0.446 0.433 0.43 0.428 0.426 0.425 0.422 0.419 0.412

1.80610205 4.00610206 3.80610205 2.82610204 6.20610204 3.30610205 7.74610204 3.00610206 2.32610204

BB0401 BBK22 BB0243 BB0690 BBB11 BBO33 BBM34 BB0816 BBR35

2654

pncA gtaB

gatC

topA dnaK-2

xylR-1 blyA cheB-1 blyA dnaK-1 flaA blyA parE blyA uvrB blyA blyA blyA tyrS

bmpD proX

glpA napA

Microbiology 154

Transcriptional interplay of B. burgdorferi regulators

Table 6. cont. Gene ID BBA47 BB0354 BBF02 BB0847 BBA12 BB0241 BB0352 BBQ34 BBN33 BB0242 BBA54 BBR33 BBA69 BBK21 BBH08 BBS37 BBD11 BBI13 BBD18 BB0127 BBH13 BBI33 BBA42 BBA45 BBB03 BBQ42 BBO27 BB0240 BBK15 BBA43 BBA53 BBK02.1 BBI39 BBJ41 BB0212 BBU03 BBA61 BBF001 BBR31 BBA44 BBI23 BBK02 BB0160 BBA28 BBI22 BBA59 BBI15 BBF14 BBI29 BBI14 BB0762 BBI12 BBS35

Function Hypothetical protein Hypothetical protein Conserved hypothetical protein Hypothetical protein Conserved hypothetical protein Glycerol kinase Hypothetical protein Conserved hypothetical protein Conserved hypothetical protein Hypothetical protein Hypothetical protein Plasmid partition protein, putative Hypothetical protein Plasmid partition protein, putative Hypothetical protein Conserved hypothetical protein Conserved hypothetical protein Conserved hypothetical protein Hypothetical protein Ribosomal protein S1 Conserved hypothetical protein Transposase-like protein, pseudogene Conserved hypothetical protein Conserved hypothetical protein Hypothetical protein Conserved hypothetical protein Conserved hypothetical protein Glycerol uptake facilitator Antigen, P35, putative Conserved hypothetical protein Hypothetical protein Conserved hypothetical protein, pseudogene Hypothetical protein Antigen, P35, putative Hypothetical protein Hypothetical protein Conserved hypothetical protein Conserved hypothetical protein, pseudogene Conserved hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Alanine racemase Hypothetical protein Conserved hypothetical protein Lipoprotein Hypothetical protein Conserved hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Plasmid partition protein, putative

http://mic.sgmjournals.org

Gene

glpK

rpsA

glpF

alr

Replicon

Ratio WT : rrp2”

P value

lp54 Chromosome lp28-1 Chromosome lp54 Chromosome Chromosome lp56 cp32-9 Chromosome lp54 cp32-4 lp54 lp36 lp28-3 cp32-3 lp17 lp28-4 lp17 Chromosome lp28-3 lp28-4 lp54 lp54 cp26 lp56 cp32-7 Chromosome lp36 lp54 lp54 lp36 lp28-4 lp38 Chromosome lp21 lp54 lp28-1 cp32-4 lp54 lp28-4 lp36 Chromosome lp54 lp28-4 lp54 lp28-4 lp28-1 lp28-4 lp28-4 Chromosome lp28-4 cp32-3

0.41 0.41 0.407 0.407 0.4 0.398 0.396 0.396 0.395 0.39 0.385 0.385 0.384 0.381 0.381 0.378 0.375 0.372 0.371 0.371 0.371 0.37 0.37 0.368 0.366 0.36 0.359 0.353 0.35 0.35 0.348 0.334 0.33 0.33 0.33 0.328 0.317 0.309 0.305 0.301 0.299 0.296 0.29 0.286 0.271 0.255 0.253 0.245 0.207 0.194 0.193 0.188 0.169

4.42610203 9.07610204 3.38610203 8.81610204 9.08610204 7.90610205 2.02610204 4.80610205 9.69610204 1.40610205 2.29610204 1.24610203 2.78610203 6.70610205 5.51610203 1.81610204 1.95610203 6.40610204 1.47610204 2.40610204 2.90610204 4.10610203 1.14610203 1.03610203 1.13610204 1.89610204 1.11610203 1.43610203 8.50610203 8.35610204 3.32610204 6.09610203 3.23610203 2.71610203 1.17610204 1.62610202 2.06610203 1.17610203 1.17610203 8.65610204 2.66610202 2.72610203 2.36610202 1.88610202 3.03610202 4.11610203 5.17610203 3.86610203 8.74610204 8.85610203 1.00610206 7.17610203 3.43610202

2655

Z. Ouyang, J. S. Blevins and M. V. Norgard

conditions to maximize induction of the Rrp2–RpoN– RpoS pathway and minimize variability. First, BSK-H complete medium was used in the present study, whereas Fisher et al. (2005) used BSK-II. This is relevant because it has been reported that BSK-H medium is superior to BSKII for use in gene expression studies (Yang et al., 2001). Second, in every experiment, bacteria were cultivated using the same lot of BSK-H medium that was prescreened for the capacity to promote borrelial gene regulation (Smith et al., 2007). Third, the medium used in our study was adjusted to pH 6.8, an important condition for maximizing induction of the Rrp2–RpoN–RpoS pathway (Yang et al., 2000). Fourth, care was taken to ensure that all cultures were grown to similar high spirochaete densities (~16108 bacteria ml21). Fifth, SDS-PAGE and immunoblot analysis was used to confirm induction of RpoS and OspC by wild-type 297 in each set of cultures. Only after it was determined that the Rrp2–RpoN–RpoS pathway was induced significantly was the RNA isolated from these bacteria and then used in microarray experiments. Efforts such as these to ensure the robust induction of the Rrp2– RpoN–RpoS pathway were not implemented in the study of Fisher et al. (2005). The intention of our study was twofold. First, we wished to investigate further the interrelationships among Rrp2, RpoN and RpoS, with emphasis on their functional interplay. As such, this is the first study, to our knowledge, to exploit rrp2, rpoN and rpoS mutants of B. burgdorferi in gene microarray experiments to study the transcriptional influences of all three major components of the pathway. Although microarray data generated from single observations (single time points) can be subject to indirect effects, the slow growth of B. burgdorferi in vitro would serve to minimize such effects. In our comparative transcriptional profiling experiments with the rrp2, rpoN and rpoS mutants, it was clear that 98 genes were under the combined regulation of Rrp2, RpoN and RpoS. Caimano et al. (2007) examined the RpoS regulon in B. burgdorferi cultivated in either DMCs or under more typical in vitro growth conditions. When comparing our data with the microarray data of Caimano et al. (2007), an excellent correlation was observed: 68 (67 induced genes and one repressed gene, bba62) of the 98 genes in our group of targets that were definitively regulated by Rrp2–RpoN– RpoS were also present in the microarray data of those authors. We believe that this level of concordance serves to validate our data, as well as those of Caimano et al. (2007), particularly given the tendency for microarray data to vary between laboratories. In addition to the 98 genes under the control of Rrp2, RpoN and RpoS, an additional group of 47 genes were identified whose expression is also most likely modulated by these three regulators. This conclusion is warranted based on the similarity in the changes in the expression of each given gene among all three mutants. In other words, if one particular gene was regulated by RpoS, it was also found to be regulated by RpoN, although at a less than 2656

twofold change or with a P value of .0.05. Finally, microarray data identified a third group of three and 12 genes, which were regulated by RpoS or RpoN alone, respectively. However, the relative changes in expression of these genes were modest (less than fourfold). Considering that there was substantial overlap in the genes regulated by both RpoN and RpoS, and the fact that there were only a few genes regulated by RpoN or RpoS individually (all of which exhibited only slight differences in expression levels), our data provide strong evidence that RpoN regulates the RpoS-mediated adaptive response in B. burgdorferi. In B. burgdorferi, the putative response regulator Rrp2 has been shown to be required for the RpoN-dependent transcription of rpoS (Burtnick et al., 2007; Yang et al., 2003a). To further confirm the linearity of the pathway, we compared herein the transcriptomes of the wild-type strain 297 and the rrp2 mutant. In addition to the 98 genes known to be regulated by Rrp2–RpoN–RpoS, our data showed that 106 genes were likely regulated by Rrp2 alone in B. burgdorferi. However, for each gene in the group regulated by Rrp2 alone, the changes in gene expression were modest (i.e. less than sixfold). Unfortunately, further investigation into this Rrp2-mediated branch of gene regulation, which ostensibly is independent of the RpoN– RpoS sigma factor cascade, represents a formidable challenge, because all efforts to date to completely disrupt rrp2 (via insertion or deletion) in B. burgdorferi have been unsuccessful (Burtnick et al., 2007; Yang et al., 2003a). Given the fact that mutations in rpoN and rpoS are not lethal for in vitro growth of B. burgdorferi (Caimano et al., 2004; Fisher et al., 2005; Hubner et al., 2001), the inability to totally inactivate rrp2 has led to the supposition that rrp2 likely serves some other essential role that is independent of its activation of RpoN-dependent transcription. Thus, it is hypothesized that, in addition to its presumed enhancerbinding activity (for RpoN), Rrp2 downregulates the expression of other genes whose products are toxic when they are aberrantly expressed in B. burgdorferi. The rrp2 mutant used in this study was theoretically deficient only in its capacity to hydrolyse ATP and thus unable to activate RpoN-dependent transcription, but its putative DNAbinding capability should have been left intact. Therefore, it is reasonable that we would not have been able to identify the toxic targets repressed by Rrp2 by employing the ATP-binding site mutant utilized in the current study. The second major focus of this study was to identify B. burgdorferi ORFs differentially regulated by the RpoN– RpoS pathway that potentially contribute to B. burgdorferi virulence and pathogenesis. To date, only relatively few virulence factors in B. burgdorferi, such as OspAB, OspC, DbpBA and BBK32, have been determined definitively to be under the regulatory control of this pathway (Grimm et al., 2004; Hagman et al., 1998; Neelakanta et al., 2007; Pal et al., 2004; Seshu et al., 2006; Shi et al., 2008; Yang et al., 2004). As such, continued efforts are warranted to elucidate other potential B. burgdorferi virulence factors. In this regard, our data confirmed that the RpoN–RpoS pathway Microbiology 154

Transcriptional interplay of B. burgdorferi regulators

is central to B. burgdorferi, in that it controls the differential expression of many predicted outer surface proteins, cell envelope constituents and putative metabolic genes. It is likely that some of these gene products facilitate the successful colonization and survival of B. burgdorferi within a mammalian host, and ultimately progression to disease. Further research will focus on the identification of new potential virulence factors in the infective life cycle of B. burgdorferi, with the goal of providing further insights into the molecular pathogenesis of this important arthropodborne bacterial pathogen.

ACKNOWLEDGEMENTS We thank Kaiping Deng, Wei Wang, Wei Liu and Jinchun Zhou for many helpful discussions and technical assistance. We also thank Xiaofeng (Frank) Yang for helpful discussions, and the UT Southwestern Microarray Core Facility for suggestions and guidance. This work was supported by Public Health Service Grant AI-059602. J. S. B. was supported by the National Institutes of Health Training Grant (T32-AI07520) and a Ruth L. Kirschstein National Research Service Award (F32-AI058487).

REFERENCES

Dong, T., Kirchhof, M. G. & Schellhorn, H. E. (2008). RpoS regulation

of gene expression during exponential growth of Escherichia coli K12 Mol Genet Genomics 279, 267–277. Farewell, A., Kvint, K. & Nystrom, T. (1998). Negative regulation

by RpoS: a case of sigma factor competition. Mol Microbiol 29, 1039–1051. Fikrig, E. & Narasimhan, S. (2006). Borrelia burgdorferi – traveling

incognito? Microbes Infect 8, 1390–1399. Fisher, M. A., Grimm, D., Henion, A. K., Elias, A. F., Stewart, P. E., Rosa, P. A. & Gherardini, F. C. (2005). Borrelia burgdorferi s54 is

required for mammalian infection and vector transmission but not for tick colonization. Proc Natl Acad Sci U S A 102, 5162–5167. Frank, K. L., Bundle, S. F., Kresge, M. E., Eggers, C. H. & Samuels, D. S. (2003). aadA confers streptomycin resistance in Borrelia

burgdorferi. J Bacteriol 185, 6723–6727. Fraser, C. M., Casjens, S., Huang, W. M., Sutton, G. G., Clayton, R., Lathigra, R. & other authors (1997). Genomic sequence of a Lyme

disease spirochaete, Borrelia burgdorferi. Nature 390, 580–586. Grimm, D., Tilly, K., Byram, R., Stewart, P. E., Krum, J. G., Bueschel, D. M., Schwan, T. G., Policastro, P. F., Elias, A. F. & Rosa, P. A. (2004).

Outer-surface protein C of the Lyme disease spirochete: a protein induced in ticks for infection of mammals. Proc Natl Acad Sci U S A 101, 3142–3147. Hagman, K. E., Lahdenne, P., Popova, T. G., Porcella, S. F., Akins, D. R., Radolf, J. D. & Norgard, M. V. (1998). Decorin-binding protein

Blevins, J. S., Revel, A. T., Smith, A. H., Bachlani, G. N. & Norgard, M. V. (2007). Adaptation of a luciferase gene reporter and lac expression

of Borrelia burgdorferi is encoded within a two-gene operon and is protective in the murine model of Lyme borreliosis. Infect Immun 66, 2674–2683.

system to Borrelia burgdorferi. Appl Environ Microbiol 73, 1501–1513.

He, M., Boardman, B. K., Yan, D. & Yang, X. F. (2007). Regulation of

Brooks, C. S., Vuppala, S. R., Jett, A. M. & Akins, D. R. (2006).

expression of the fibronectin-binding protein BBK32 in Borrelia burgdorferi. J Bacteriol 189, 8377–8380.

Identification of Borrelia burgdorferi outer surface proteins. Infect Immun 74, 296–304. Burgdorfer, W., Barbour, A. G., Hayes, S. F., Benach, J. L., Grunwaldt, E. & Davis, J. P. (1982). Lyme disease – a tick-borne

Hovius, J. W., van Dam, A. P. & Fikrig, E. (2007). Tick–host–pathogen

interactions in Lyme borreliosis. Trends Parasitol 23, 434–438.

spirochetosis? Science 216, 1317–1319.

Hubner, A., Yang, X., Nolen, D. M., Popova, T. G., Cabello, F. C. & Norgard, M. V. (2001). Expression of Borrelia burgdorferi OspC and

Burtnick, M. N., Downey, J. S., Brett, P. J., Boylan, J. A., Frye, J. G., Hoover, T. R. & Gherardini, F. C. (2007). Insights into the complex

DbpA is controlled by a RpoN–RpoS regulatory pathway. Proc Natl Acad Sci U S A 98, 12724–12729.

regulation of rpoS in Borrelia burgdorferi. Mol Microbiol 65, 277–293.

Hughes, C. A., Kodner, C. B. & Johnson, R. C. (1992). DNA analysis

Caimano, M. J., Eggers, C. H., Hazlett, K. R. & Radolf, J. D. (2004).

of Borrelia burgdorferi NCH-1, the first northcentral U.S. human Lyme disease isolate. J Clin Microbiol 30, 698–703.

RpoS is not central to the general stress response in Borrelia burgdorferi but does control expression of one or more essential virulence determinants. Infect Immun 72, 6433–6445. Caimano, M. J., Eggers, C. H., Gonzalez, C. A. & Radolf, J. D. (2005).

Alternate sigma factor RpoS is required for the in vivo-specific repression of Borrelia burgdorferi plasmid lp54-borne ospA and lp6.6 genes. J Bacteriol 187, 7845–7852. Caimano, M. J., Iyer, R., Eggers, C. H., Gonzalez, C., Morton, E. A., Gilbert, M. A., Schwartz, I. & Radolf, J. D. (2007). Analysis of the RpoS

regulon in Borrelia burgdorferi in response to mammalian host signals provides insight into RpoS function during the enzootic cycle. Mol Microbiol 65, 1193–1217. Callister, S. M., Case, K. L., Agger, W. A., Schell, R. F., Johnson, R. C. & Ellingson, J. L. (1990). Effects of bovine serum albumin on the

Jewett, M. W., Lawrence, K., Bestor, A. C., Tilly, K., Grimm, D., Shaw, P., VanRaden, M., Gherardini, F. & Rosa, P. A. (2007). The

critical role of the linear plasmid lp36 in the infectious cycle of Borrelia burgdorferi. Mol Microbiol 64, 1358–1374. Loewen, P. C., Hu, B., Strutinsky, J. & Sparling, R. (1998). Regulation

in the rpoS regulon of Escherichia coli. Can J Microbiol 44, 707–717. Lybecker, M. C. & Samuels, D. S. (2007). Temperature-induced

regulation of RpoS by a small RNA in Borrelia burgdorferi. Mol Microbiol 64, 1075–1089. Medrano, M. S., Ding, Y., Wang, X. G., Lu, P., Coburn, J. & Hu, L. T. (2007). Regulators of expression of the oligopeptide permease A

proteins of Borrelia burgdorferi. J Bacteriol 189, 2653–2659.

ability of Barbour–Stoenner–Kelly medium to detect Borrelia burgdorferi. J Clin Microbiol 28, 363–365.

Motaleb, M. A., Miller, M. R., Li, C., Bakker, R. G., Goldstein, S. F., Silversmith, R. E., Bourret, R. B. & Charon, N. W. (2005). CheX is a

CDC (2007). Lyme disease – United States, 2003–2005. MMWR Morb

Mortal Wkly Rep 56, 573–576.

phosphorylated CheY phosphatase essential for Borrelia burgdorferi chemotaxis. J Bacteriol 187, 7963–7969.

Clifton, D. R., Nolder, C. L., Hughes, J. L., Nowalk, A. J. & Carroll, J. A. (2006). Regulation and expression of bba66 encoding an immuno-

Neelakanta, G., Li, X., Pal, U., Liu, X., Beck, D. S., DePonte, K., Fish, D., Kantor, F. S. & Fikrig, E. (2007). Outer surface protein B is

genic infection-associated lipoprotein in Borrelia burgdorferi. Mol Microbiol 61, 243–258.

critical for Borrelia burgdorferi adherence and survival within Ixodes ticks. PLoS Pathog 3, e33.

http://mic.sgmjournals.org

2657

Z. Ouyang, J. S. Blevins and M. V. Norgard

Nelson, J. A., Bouseman, J. K., Kitron, U., Callister, S. M., Harrison, B., Bankowski, M. J., Peeples, M. E., Newton, B. J. & Anderson, J. F. (1991). Isolation and characterization of Borrelia burgdorferi from

Shi, Y., Xu, Q., McShan, K. & Liang, F. T. (2008). Both decorin-

Illinois Ixodes dammini. J Clin Microbiol 29, 1732–1734.

Singh, S. K. & Girschick, H. J. (2004). Molecular survival strategies of

Ojaimi, C., Brooks, C., Casjens, S., Rosa, P., Elias, A., Barbour, A., Jasinskas, A., Benach, J., Katona, L. & other authors (2003).

the Lyme disease spirochete Borrelia burgdorferi. Lancet Infect Dis 4, 575–583.

Profiling of temperature-induced changes in Borrelia burgdorferi gene expression by using whole genome arrays. Infect Immun 71, 1689–1705.

Smith, A. H., Blevins, J. S., Bachlani, G. N., Yang, X. F. & Norgard, M. V. (2007). Evidence that RpoS (sS) in Borrelia burgdorferi is controlled directly by RpoN (s54/sN). J Bacteriol 189, 2139–2144.

Pal, U., Yang, X., Chen, M., Bockenstedt, L. K., Anderson, J. F., Flavell, R. A., Norgard, M. V. & Fikrig, E. (2004). OspC facilitates

Steere, A. C., Grodzicki, R. L., Kornblatt, A. N., Craft, J. E., Barbour, A. G., Burgdorfer, W., Schmid, G. P., Johnson, E. & Malawista, S. E. (1983). The spirochetal etiology of Lyme disease. N Engl J Med 308,

binding proteins A and B are critical for overall virulence of Borrelia burgdorferi. Infect Immun 76, 1239–1246.

Borrelia burgdorferi invasion of Ixodes scapularis salivary glands. J Clin Invest 113, 220–230.

733–740.

Picken, M. M., Picken, R. N., Han, D., Cheng, Y., Ruzic-Sabljic, E., Cimperman, J., Maraspin, V., Lotric-Furlan, S. & Strle, F. (1997). A

Stevenson, B., von Lackum, K., Riley, S. P., Cooley, A. E., Woodman, M. E. & Bykowski, T. (2006). Evolving models of Lyme disease

two year prospective study to compare culture and polymerase chain reaction amplification for the detection and diagnosis of Lyme borreliosis. Mol Pathol 50, 186–193.

spirochete gene regulation. Wien Klin Wochenschr 118, 643–652.

Pollack, R. J., Telford, S. R., III & Spielman, A. (1993). Standardization

of medium for culturing Lyme disease spirochetes. J Clin Microbiol 31, 1251–1255. Purser, J. E. & Norris, S. J. (2000). Correlation between plasmid

content and infectivity in Borrelia burgdorferi. Proc Natl Acad Sci U S A 97, 13865–13870. Purser, J. E., Lawrenz, M. B., Caimano, M. J., Howell, J. K., Radolf, J. D. & Norris, S. J. (2003). A plasmid-encoded nicotinamidase (PncA)

is essential for infectivity of Borrelia burgdorferi in a mammalian host. Mol Microbiol 48, 753–764. Rappas, M., Bose, D. & Zhang, X. (2007). Bacterial enhancer-binding proteins: unlocking s54-dependent gene transcription. Curr Opin

Struct Biol 17, 110–116. Revel, A. T., Talaat, A. M. & Norgard, M. V. (2002). DNA microarray

analysis of differential gene expression in Borrelia burgdorferi, the Lyme disease spirochete. Proc Natl Acad Sci U S A 99, 1562–1567. Revel, A. T., Blevins, J. S., Almazan, C., Neil, L., Kocan, K. M., de la Fuente, J., Hagman, K. E. & Norgard, M. V. (2005). bptA (bbe16) is

essential for the persistence of the Lyme disease spirochete, Borrelia burgdorferi, in its natural tick vector. Proc Natl Acad Sci U S A 102, 6972–6977. Rosa, P. A., Tilly, K. & Stewart, P. E. (2005). The burgeoning

molecular genetics of the Lyme disease spirochaete. Nat Rev Microbiol 3, 129–143. Schwan, T. G. & Piesman, J. (2000). Temporal changes in outer

surface proteins A and C of the Lyme disease-associated spirochete, Borrelia burgdorferi, during the chain of infection in ticks and mice. J Clin Microbiol 38, 382–388. Schwan, T. G., Piesman, J., Golde, W. T., Dolan, M. C. & Rosa, P. A. (1995). Induction of an outer surface protein on Borrelia burgdorferi

during tick feeding. Proc Natl Acad Sci U S A 92, 2909–2913. Seshu, J., Esteve-Gassent, M. D., Labandeira-Rey, M., Kim, J. H., Trzeciakowski, J. P., Hook, M. & Skare, J. T. (2006). Inactivation of

the fibronectin-binding adhesin gene bbk32 significantly attenuates the infectivity potential of Borrelia burgdorferi. Mol Microbiol 59, 1591–1601. Setubal, J. C., Reis, M., Matsunaga, J. & Haake, D. A. (2006).

Lipoprotein computational prediction in spirochaetal genomes. Microbiology 152, 113–121.

Stewart, P. E., Thalken, R., Bono, J. L. & Rosa, P. (2001). Isolation of a

circular plasmid region sufficient for autonomous replication and transformation of infectious Borrelia burgdorferi. Mol Microbiol 39, 714–721. Stewart, P. E., Byram, R., Grimm, D., Tilly, K. & Rosa, P. A. (2005).

The plasmids of Borrelia burgdorferi: essential genetic elements of a pathogen. Plasmid 53, 1–13. Terekhova, D., Iyer, R., Wormser, G. P. & Schwartz, I. (2006).

Comparative genome hybridization reveals substantial variation among clinical isolates of Borrelia burgdorferi sensu stricto with different pathogenic properties. J Bacteriol 188, 6124–6134. Wang, G., Iyer, R., Bittker, S., Cooper, D., Small, J., Wormser, G. P. & Schwartz, I. (2004). Variations in Barbour–Stoenner–Kelly culture

medium modulate infectivity and pathogenicity of Borrelia burgdorferi clinical isolates. Infect Immun 72, 6702–6706. Yang, X., Popova, T. G., Hagman, K. E., Wikel, S. K., Schoeler, G. B., Caimano, M. J., Radolf, J. D. & Norgard, M. V. (1999). Identification,

characterization and expression of three new members of the Borrelia burgdorferi Mlp (2.9) lipoprotein gene family. Infect Immun 67, 6008– 6018. Yang, X., Goldberg, M. S., Popova, T. G., Schoeler, G. B., Wikel, S. K., Hagman, K. E. & Norgard, M. V. (2000). Interdependence of

environmental factors influencing reciprocal patterns of gene expression in virulent Borrelia burgdorferi. Mol Microbiol 37, 1470– 1479. Yang, X., Popova, T. G., Goldberg, M. S. & Norgard, M. V. (2001).

Influence of cultivation media on genetic regulatory patterns in Borrelia burgdorferi. Infect Immun 69, 4159–4163. Yang, X. F., Alani, S. M. & Norgard, M. V. (2003a). The response

regulator Rrp2 is essential for the expression of major membrane lipoproteins in Borrelia burgdorferi. Proc Natl Acad Sci U S A 100, 11001–11006. Yang, X. F., Hubner, A., Popova, T. G., Hagman, K. E. & Norgard, M. V. (2003b). Regulation of expression of the paralogous Mlp family

in Borrelia burgdorferi. Infect Immun 71, 5012–5020. Yang, X. F., Pal, U., Alani, S. M., Fikrig, E. & Norgard, M. V. (2004).

Essential role for OspA/B in the life cycle of the Lyme disease spirochete. J Exp Med 199, 641–648. Yang, X. F., Lybecker, M. C., Pal, U., Alani, S. M., Blevins, J., Revel, A. T., Samuels, D. S. & Norgard, M. V. (2005). Analysis of the ospC

regulatory element controlled by the RpoN–RpoS regulatory pathway in Borrelia burgdorferi. J Bacteriol 187, 4822–4829.

Shi, W., Yang, Z., Geng, Y., Wolinsky, L. E. & Lovett, M. A. (1998).

Chemotaxis in Borrelia burgdorferi. J Bacteriol 180, 231–235.

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Microbiology 154

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