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Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky College of Medicine, MS415 Chandler Medical Center, Lexington, KY 40536-0298, USA
Correspondence
Brian Stevenson
brian.stevenson{at}uky.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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70 subunit for RNA polymerase holoenzyme function. All four genes are co-expressed, and mRNA levels are growth-rate dependent, being produced during the exponential phase. Thus, high metabolic activity is accompanied by increased cellular levels of the only known borrelial methyl donor, enhanced detoxification of methylation by-products, and increased production of DPD. Therefore, production of DPD is directly correlated with cellular metabolism levels, and may thereby function as an extracellular and/or intracellular signal of bacterial health.
Present address: BioVitesse, West Lafayette, IN 47906, USA.
Present address: Department of Oral Biology, University of Florida College of Dentistry, Gainesville, FL 32610, USA.
A supplementary figure showing the results of Q-RT-PCR analysis of luxS transcription by wild-type (wt), rpoN and rpoS B. burgdorferi and two supplementary tables of raw data for the Q-RT-PCR are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Throughout the processes of infecting these two distinct host types, and transmission between each, B. burgdorferi regulates expression of a large number of both metabolic and host-interface proteins. Deciphering the mechanisms by which B. burgdorferi controls gene and protein expression patterns is providing insights into both the physiology of this bacterium and its pathogenic abilities.
Some species of bacteria utilize the metabolic by-product 4,5-dihydroxy-2,3-pentanedione (DPD) in intercellular (and possibly intracellular) signalling. Several different, spontaneously derived forms of DPD have been identified that can function as signals, which are collectively termed ‘autoinducer-2’ (AI-2) (Chen et al., 2002; Miller et al., 2004). DPD/AI-2 is produced from the toxic end product of transmethylation reactions, S-adenosylhomocysteine (SAH), in a two-step reaction catalysed by the enzymes Pfs and LuxS. Some bacteria, such as the syphilis spirochaete Treponema pallidum, possess Pfs but lack LuxS, and therefore detoxify SAH only to the non-toxic S-ribosylhomocysteine (SRH) (Fraser et al., 1998; von Lackum et al., 2006). Other bacteria, B. burgdorferi included, further degrade SRH to DPD and homocysteine via the LuxS enzyme (Babb et al., 2005; Stevenson & Babb, 2002; Stevenson et al., 2003; Sun et al., 2004; Xavier & Bassler, 2003). The majority of bacteria salvage homocysteine for regeneration of methionine, for use in additional transmethylation reactions or protein synthesis (Winzer et al., 2002). B. burgdorferi is among the minority of bacteria which lack methionine synthase, and are therefore unable to utilize the homocysteine produced by LuxS (Babb et al., 2005). Several lines of evidence indicate that B. burgdorferi instead uses the LuxS enzyme to synthesize DPD/AI-2 as a means to control gene expression. Addition of either in vitro-synthesized DPD or supernatant from an Escherichia coli strain producing B. burgdorferi LuxS to cultured B. burgdorferi results in altered expression levels of a number of borrelial proteins (Babb et al., 2005; Stevenson & Babb, 2002; von Lackum et al., 2007). A B. burgdorferi luxS mutant, which is unable to synthesize DPD, likewise exhibits altered protein expression patterns as compared with its wild-type parent (Babb et al., 2005; Stevenson & Babb, 2002; von Lackum et al., 2007). luxS mutant B. burgdorferi are still able to infect mice and ticks, indicating that DPD synthesis is not absolutely essential for infection (Blevins et al., 2004; Hübner et al., 2003). Consistent with those results, luxS mutants can produce all of the proteins identified as being affected by DPD, although mutants produce different levels of those proteins than do wild-type B. burgdorferi (Babb et al., 2005; von Lackum et al., 2006, 2007). Taken together, these data suggest that B. burgdorferi uses DPD as a signal to fine tune gene expression levels, as opposed to a simple on/off switch.
Previous genetic and biochemical analyses identified the pfs and luxS genes of B. burgdorferi on the main chromosome, adjacent to two other genes that are also transcribed in the same direction (Fig. 1) (Babb et al., 2005; Stevenson & Babb, 2002). The genes on either side of this four-gene cluster are transcribed in opposite directions. The gene flanked by pfs and luxS, open reading frame (ORF) BB0376 (ORF numbering system for B. burgdorferi strain B31 as used by Fraser et al., 1997), bears homology to genes of other species that encode MetK [methionine adenosyltransferase/S-adenosylmethionine (SAM) synthase], which catalyses the production of SAM from methionine and ATP (Fraser et al., 1997). SAM serves as a methyl group donor for cellular methylation reactions, with substrates including DNA, tRNA and chemotaxis proteins (Lu, 2000). Due to widespread requirements for methylation reactions, cellular levels of activated methyl donors affect a broad range of metabolic processes. As an example, the metK gene is indispensable for growth of E. coli (Greene et al., 1973; Newman et al., 1998; Wang et al., 2005; Wei & Newman, 2002). The other uncharacterized gene, ORF BB0374, lacks any complete homologues outside the genus Borrelia (BLASTP and BLASTN searches of GenBank performed 4 November 2006). The encoded protein does, however, contain an amino acid sequence similar to that found in the HD-GYP subfamily of HD family protein domains (Galperin et al., 1999). To date, all characterized HD domain-containing proteins have metal-dependent phosphohydrolase activity, with substrates that include cyclic di-GMP, guanosine tetraphosphate and tRNA (Aravind & Koonin, 1998; Galperin et al., 2001; Ryan et al., 2006a). For example, HD-GYP domain proteins of Xanthomonas spp. degrade the small signalling molecule cyclic di-GMP and are linked to both intercellular signalling and pathogenicity (Andrade et al., 2006; Ryan et al., 2006a, b, 2007; Slater et al., 2000). A response regulator protein of B. burgdorferi, Rrp1, synthesizes cyclic di-GMP (Ryjenkov et al., 2005), but the phosphohydrolase responsible for degrading cyclic di-GMP has yet to be identified.
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). To better understand the genetics of the B. burgdorferi Pfs-LuxS system, we examined promoter location, transcriptional linkage and regulation of all four of the adjacent genes, in addition to biologically characterizing enzymic activity of the ORF BB0376 protein product. |
METHODS |
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. B. burgdorferi were cultivated at 34 °C in modified Barbour–Stoenner–Kelly medium (BSK-II, Barbour, 1984). For growth related experiments, BSK-II was inoculated with a 1 : 500 dilution of mid-exponential-phase B31-MI-16 (
107 bacteria ml–1) and grown at 34 °C for 2 (5x106 bacteria ml–1), 4 (1x108 bacteria ml–1), 6 (1.5x108 bacteria ml–1) or 8 days (2x108 bacteria ml–1). In all other cases, bacteria were harvested when in mid-exponential phase.
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). A DNA segment of B. burgdorferi B31-MI-16, encompassing ORF BB0376 from the ribosome-binding site to 3' of the stop codon, was amplified by PCR using oligonucleotide primers METK-1 and METK-2 (Table 1
). The resulting amplicon was cloned into pCR2.1 (Invitrogen). One recombinant plasmid, pBLS602, contains the complete ORF BB0376 under the transcriptional control of the plasmid's lac promoter. The insert of pBLS602 was completely sequenced on both strands to ensure that errors had not been introduced during the cloning processes. E. coli MEW402 was transformed with pBLS602 to create E. coli SPRM1. Maintenance of pBLS602 was ensured by the addition of 50 µg kanamycin ml–1 to all cultures. MEW402 and SPRM1 were each inoculated into M9 medium supplemented with 0.2 % glucose, 2 mg serine ml–1 and 200 µg glycine ml–1, with or without 50 µg leucine ml–1, and cultured at 37 °C (Newman et al., 1998). Bacteria were diluted 1 : 100 daily into fresh media for three successive days. After the third passage, IPTG was added to a final concentration of 1 mM to both cultures, inducing transcription of the B. burgdorferi ORF BB0376 in SPRM1, and acting as a control for the parental strain. Wet mounts of bacteria from each culture were examined by darkfield microscopy using a Nikon Optiphot, and were photographed at 40x magnification using a Nikon Coolpix5700 digital camera with 8x optical zoom.
Production of recombinant ORF BB0374 protein.
The complete ORF BB0374 and the internal fragment encoding the HD-GYP domain were PCR amplified from pRW1 (Babb et al., 2005) using primers SRE5+6B or SRE-HD1+2 respectively (Table 1), and cloned into pET100 (Invitrogen) to produce plasmids pSR1E3 and pSRE-HD. Plasmids were isolated from E. coli and inserts were sequenced. Plasmids were individually transformed into E. coli BL21star(DE3) (Invitrogen), cultured until early exponential phase (OD600 0.5), then production of recombinant proteins was induced by addition of IPTG to 1 mM. Aliquots were taken hourly for OD600 readings, then bacteria were pelleted by centrifugation for protein analysis. Induction of protein production was assessed by SDS-PAGE followed by either Coomassie brilliant blue staining or transfer and immunoblotting using anti-Xpress antibody (Invitrogen).
RNA isolation and cDNA preparation.
Total RNA was extracted from each B. burgdorferi culture described above using TRIzol (Life Technologies), resuspended in RNasecure resuspension reagent (Ambion), and treated with DNase I (Ambion) to remove contaminating DNA. Single 1 µg aliquots of each DNA-free RNA preparation were reverse transcribed using a First Strand cDNA synthesis kit (Roche) with random hexamers and Avian Myeloblastosis Virus (AMV) reverse transcriptase enzyme (RTase) at 55 °C for 30 min. The RTase was inactivated for 10 min at 85 °C followed by 10 min at 4 °C.
Primer extension.
RNA was isolated from a mid-exponential phase culture of B. burgdorferi strain B31-MI-16 as described above. Primer extensions were performed using the Primer Extension system/AMV Reverse Transcriptase system (Promega) with [
-32P]ATP. Briefly, oligonucleotide primers SRB2, SRA2, SRA22C and SRA6 (Table 1) were individually labelled using T4 polynucleotide kinase, then annealed to the RNA by heating to 58 °C followed by cooling to room temperature. Primer extension was performed with AMV RTase for 30 min at 42 °C, and the mixture was immediately heated to 70 °C with the addition of loading buffer (Promega). DNA markers for size determination were also labelled with [-32P]ATP using T4 polynucleotide kinase. The pSRBA plasmid, which contains the DNA sequence 5' of ORF BB0374, was constructed by PCR amplification of B. burgdorferi DNA using the primer pair SRB3+4 and cloned into pCR2.1 (Invitrogen). DNA sequencing was performed on pSRBA (Table 1) using Sequenase version 2.0 (USB) and 
-33P-labelled nucleotides. Primer extension and sequencing products, and labelled size markers were simultaneously separated by urea-PAGE, and labelled DNAs were detected by autoradiography.
Linkage analysis.
To examine transcriptional linkage, cDNA from mid-exponential-phase cultures of B. burgdorferi B31-MI-16 were subjected to PCR with primers SRA1+2, 5+6, 7+8, 9+10, 11+12, 13+14 or 21+22C, to amplify cDNA segments within and spanning the intergenic regions for each of the ORF BB0374, pfs, BB0376 (metK) and luxS open reading frames (Table 1 and Fig. 1). Reaction conditions were 94 °C for 30 s, 50 °C for 30 s, and 68 °C for 30 s for 35 cycles. A RTase-free RNA sample was examined using each primer set, to test for DNA contamination. PCR products were resolved in native 6 % polyacrylamide gels stained with ethidium bromide, followed by observation under UV illumination. Each product was also cloned into pCR2.1, and plasmid inserts were sequenced to verify specific amplification.
Quantitative RT-PCR (Q-RT-PCR).
cDNA samples were amplified in a LightCycler (Roche Applied Science) and data were analysed as described previously (Miller, 2006). Briefly, cDNA was added to a master mix containing 1xPCR buffer, enzyme diluent, dNTPs (Idaho Technology), Platinum Taq (Invitrogen), SYBR green (Molecular Probes), nuclease-free water, and oligonucleotide primers (0.4 µM final concentration). Primers used for amplification of flaB, ORF BB0374, pfs, ORF BB0376, and luxS were FLA3+4, SRA7+8, SRA9+10, SRA11+12 and SRA13+14, respectively (Table1). Reaction conditions consisted of a 2 min initial 94 °C denaturation followed by 50 cycles of 94 °C for 5 s, 55 °C (FLA3+4, SRA11+12, 13+14) or 52 °C (SRA7+8, 9+10) for 10 s and 72 °C for 30 s. LightCycler software v. 3.5.3 was used for quantification and melting curve analysis (Gilmore et al., 2001). All products were separated by electrophoresis through a 1 % agarose gel and stained with ethidium bromide to verify predicted band size. The cDNA was produced from three independently grown cultures for each time point. Expression values obtained from quadruplicate runs of each cDNA sample for the four genes were calculated relative to the mean quadruplicate value for the B. burgdorferi housekeeping gene flaB from the same cDNA preparation.
To test sigma factor selectivity, DNA-free RNA samples from mid-exponential-phase cultures of B. burgdorferi A3, A3ntrA, A34rpoS were isolated and cDNAs were synthesized. Transcript levels for luxS, flaB and ospC were assessed by Q-RT-PCR as above. The same primers as above were used for luxS and flaB, while ospC was examined using OSPC7+8 (Table 1). Reaction conditions for ospC consisted of a 2 min initial 94 °C denaturation followed by 50 cycles of 94 °C for 5 s, 52 °C for 10 s, and 72 °C for 30 s. All analyses were performed a minimum of three times.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Stevenson & Babb, 2002). The previously uncharacterized ORF BB0376 is located between pfs and luxS, with all three genes overlapping. ORF BB0376 encodes a potential protein with a 49.4 % identical and 67.8 % similar amino acid sequence to E. coli K-12 MG1655 metK (data not shown). The product of MetK, SAM, serves as a methyl-group donor for biological processes. Upon methyl-group transfer to a substrate, SAM is converted to SAH, the substrate for the Pfs-catalysed reaction. If ORF BB0376 is indeed a metK homologue, it would play a role in both borrelial methylation reactions and production of DPD. Since it is inappropriate to assume that genes with similar sequences must necessarily encode enzymically identical proteins, we tested the functionality of the ORF BB0376-encoded protein.
Production of MetK is essential for normal growth and cell division of E. coli (Newman et al., 1998; Wang et al., 2005; Wei & Newman, 2002). E. coli strain MEW402 produces wild-type levels of MetK when grown in medium containing leucine (Fig. 2A). Depletion of leucine leads to inhibition of MetK synthesis, resulting in a septation defect and formation of long filaments (Fig. 2B) (Newman et al., 1998; Wang et al., 2005). Plasmid pBLS602, which contains B. burgdorferi ORF BB0376 under control of the lac promoter, was introduced into MEW402, producing strain SPRM1. Cultures of SPRM1 in media lacking leucine formed filaments similar to those of the parent strain (Fig. 2C). Induction of the lac promoter by addition of IPTG restored normal cell division, and the filamentous phenotype was not observed (Fig. 2D). IPTG had no such effect on the parental strain MEW402 (data not shown). This complementation indicated that ORF BB0376 is indeed a metK homologue, and it will be referred to as such through the remainder of this report.
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ORF BB0374, pfs, metK and luxS form an operon
The four genes in question are each transcribed in the same direction and are flanked by genes that are transcribed in opposite directions. There are very few noncoding nucleotides between the four genes: between ORF BB0374 and pfs there are 23 bp, and there are no bases between pfs and metK or metK and luxS. These observations suggest that all four genes are possibly co-transcribed. Indeed, a previous RT-PCR study indicated that pfs and metK are included on the same mRNA, as are metK and luxS (Hübner et al., 2003). We extended that earlier work by demonstrating by RT-PCR that ORF BB0374 and pfs are also transcribed on the same mRNA (Fig. 1). The tight spacing of the four genes would prevent Rho-dependent termination prior to the end of luxS. Examination of the sequence spanning ORF BB0374 through luxS did not reveal any inverted repeats that could serve as Rho-independent terminators. Northern blot analysis using probes derived from ORF BB0374 or luxS revealed only a broad RNA smear (data not shown).
Recognizing the possibility that this four-gene locus could have multiple promoters, we performed primer extension analysis on purified B. burgdorferi RNA. A product was obtained only from extensions originating within ORF BB0374 (data not shown). Control analyses without addition of RNA confirmed that the band observed was due to extension from mRNA. This transcriptional start site was finely mapped using high-resolution gels and simultaneous di-deoxy sequencing of a DNA template using the same primer. Two transcription start sites were detected, 23 and 25 bp 5' of the ORF BB0374 translation initiation codon (Fig. 3). Potential –10 and –35 sequences are located at appropriate distances 5' of the transcription start sites, as is also a consensus extended –10 sequence.
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). Q-RT-PCR analysis of each gene in the ORF BB0374-pfs-metK-luxS operon was performed to determine the connection between gene expression and production of DPD, as well as to examine for evidence of independent regulation. To that end, cultures were inoculated at low densities and harvested after 2 days (5x106 cells ml–1, early exponential phase, approximately four doublings), 4 days (1x108 cells ml–1 mid-exponential phase, approximately 8 doublings), 6 days (1.5x108 cells ml–1, early stationary phase), and 8 days (2x108 cells ml–1, well into stationary phase) (Babb et al., 2005). Total RNA was extracted and transcript levels of each gene were determined (see Supplementary Table S1, available with the online version of this paper). Due to the constitutive expression of flaB, Q-RT-PCR results for ORF BB0374, pfs, metK and luxS were standardized relative to the flaB results. Similar relative levels of expression were observed for all four genes of the operon (Fig. 4). Moreover, expression levels of each gene were at least fivefold higher during the early exponential phase of growth than during the later stationary phase.
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70 and homologues of S and N. Several studies have implicated the importance of RNA polymerase subunits S and N in B. burgdorferi transitional events, including transmission from tick to mammal (Caimano et al., 2004; Elias et al., 2000; Yang et al., 2000). Since the ORF BB0374-pfs-metK-luxS operon is induced during tick-to-mammal transmission (Narasimhan et al., 2002), we examined whether either S or N played a role in regulation of operon transcription. The S subunit is induced as cultures approach stationary phase, and S levels are dependent on N (Caimano et al., 2004; Elias et al., 2000; Yang et al., 2000). B. burgdorferi with specific deletions of either rpoS (S) or rpoN (N) produced luxS transcripts at levels comparable to wild-type bacteria (Supplementary Fig. S1, Supplementary Table S2), indicating that neither alternative sigma factor is directly responsible for transcription of this operon. The control gene, ospC, was not transcribed by either mutant, consistent with previous analyses of that gene (Hübner et al., 2001; Yang et al., 2003). These data indicate that the housekeeping factor 70 interacts with the RNA polymerase holoenzyme 5' of ORF BB0374 to direct transcription of this operon. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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70 RNA polymerase holoenzyme. All four genes were co-expressed, being produced during the exponential phase of growth, and repressed during the stationary phase. These data are consistent with previous observations that B. burgdorferi produces maximal levels of DPD/AI-2 during exponential phase, and increased expression of luxS during the period of rapid growth accompanying transmission from feeding tick vectors to mammalian hosts (Babb et al., 2005; Narasimhan et al., 2002). Thus, high metabolic states are accompanied by increases in three enzymic activities: (1) MetK, which increases cellular levels of the only known borrelial methyl donor, SAM; (2) Pfs, which detoxifies the product of SAM-dependent methylation reactions; and (3) LuxS, which produces DPD and homocysteine. As described above, B. burgdorferi lacks the ability to use homocysteine; the Pfs product, SAH, is not toxic; and the addition of DPD to Lyme disease spirochaetes causes alterations in protein expression patterns. It is likely, therefore, that production of DPD is directly correlated with cellular fitness, and, reciprocally, that DPD may function as a signal of bacterial fitness (Xavier & Bassler, 2003). Such a signal may function extracellularly, to influence the entire population, intracellularly as an alarmone to modify expression levels of that bacterium's own genes, or as a combination of these two possibilities (Beeston & Surette, 2002; Camilli & Bassler, 2006; Redfield, 2002; Xavier & Bassler, 2003).
Since the product of the MetK-catalysed reaction is converted to the Pfs substrate by cellular methyltransferases, MetK makes a direct contribution to AI-2 synthesis, as well as to regulation of the total cellular methyl pool. In many bacteria, a second major methyl-group donor, methylenetetrahydrofolate (THF), is involved in nucleic and amino acid biosynthesis, but B. burgdorferi lacks homologues of most enzymes required for production of THF (Fraser et al., 1997). B. burgdorferi encodes numerous postulated methyltransferases which have potential substrates including tRNA, rRNA, DNA and chemotaxis proteins, with SAM likely to be the only molecule capable of donating activated methyl groups. Therefore, we hypothesize that production of MetK is directly proportional to the rate of total methylation reactions in the cell.
In conclusion, the ORF BB0374-pfs-metK-luxS operon encodes three enzymic activities that directly connect cellular methylation and bacterial growth to production of the signalling molecule DPD. ORF BB0374 encodes a probable phosphohydrolase. The interrelatedness of pathways suggests that regulation of the BB0374-pfs-metK-luxS operon plays a role in coordinating physiological adaptations of B. burgdorferi. Elucidating the regulatory mechanisms controlling transcription of this operon and the effects of each gene product will illuminate further our understanding of the biology of B. burgdorferi and the mechanisms by which the Lyme disease spirochaete infects its diverse hosts.
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ACKNOWLEDGEMENTS |
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Edited by: R. J. Lamont
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Received 15 November 2006;
revised 19 March 2007;
accepted 20 March 2007.
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