Evidence that RpoS ( S ) in Borrelia burgdorferi Is Controlled Directly by RpoN ( 54 / N )

Lyme disease, caused by Borrelia burgdorferi, is the most commonly reported arthropod-borne disease in both the United States and Europe (32). At the molecular level, certain membrane lipoproteins of B. burgdorferi are vital for maintaining the spirochete in its zoonotic transmission cycle between ticks and mammals. For example, the reciprocal regulation of outer surface (lipo)proteins A (OspA) and C (OspC) is the best-studied paradigm of the dramatic alterations in protein expression patterns that ensue as the spirochete transitions from the arthropod vector into mammalian tissues (8, 22, 30, 31). Our lab determined previously that expression of OspC is regulated by the alternative sigma factor RpoS ( ) (15, 40). RpoS, though, must first be activated by another alternative transcription factor, RpoN ( / ), resulting in an RpoNRpoS regulatory network (10, 15). However, the precise mechanism(s) by which RpoN activates rpoS has not been determined. RpoN could control rpoS expression directly by binding to a region near the rpoS open reading frame (ORF). Alternatively, another transactivator induced by RpoN might activate rpoS expression. We have been most attracted to the notion that RpoN binds directly to a region upstream of the rpoS ORF to facilitate RpoS expression in B. burgdorferi. This is because nucleotides 78 to 63 upstream of the rpoS ORF comprise a theoretical, RpoN-dependent consensus 24/ 12 promoter (33, 34). Many studies with various bacteria have indicated close contact of RpoN with such a 24/ 12 promoter region (5, 6, 21, 33). However, as yet, there have been no experimental data to substantiate this prediction for B. burgdorferi. The purpose of this study was to garner additional evidence that would either substantiate or refute the hypothesis that rpoS is regulated directly by RpoN. Identification of rpoS initiation of transcription. Determination of the initiation of transcription for a given gene can provide strategic information for identifying a gene’s nearby promoter. rpoS transcripts of B. burgdorferi BbAH130 (41) were reverse transcribed in BD SMART-RACE (switching mechanism at 5 end of RNA transcript-rapid amplification of cDNA ends) reactions (BD Biosciences, San Jose, CA) using two rpoS-specific primers (rpoSR422 and rpoSR232) (Table 1), which are 422 and 232 bases, respectively, downstream of the rpoS translation start site. The BD SMART IIA primer, which anneals to the BD SMART IIA oligonucleotide linked to the 3 end of the first-strand cDNA, and an rpoS-specific primer upstream of the gene-specific primers used in first-strand cDNA synthesis (rpoSR125) (Table 1) were used to amplify the resulting cDNAs. PCR-amplified inserts cloned into the pGEM-T Easy vector (Promega, Madison, WI) were digested with the restriction endonuclease DraI (New England Biolabs, Ipswich, MA) to verify that they represented rpoS. Seventeen of 25 clones contained rpoS-specific sequences, as determined by the DraI restriction pattern (not shown). Eight of these 17 clones were selected for further analysis. For five of these eight clones, residue 50 upstream of the rpoS ORF represented the transcript start site by RACE analysis, whereas there was no consensus residue for the other three sequences. The identified transcription start site was 14 nucleotides downstream of the conserved GC dinucleotide (Fig. 1). This result correlates well with the putative 24/ 12 region representing the promoter for rpoS, inasmuch as initiation of transcription from a 24/ 12 promoter occurs between 8 and 21 nucleotides downstream of the GC dinucleotide (2). Assessing RpoN binding to the rpoS promoter. Ideally, direct binding of the B. burgdorferi RpoN protein to the rpoS promoter sequence would provide the most compelling evidence that the rpoS promoter in B. burgdorferi is RpoN dependent. However, numerous attempts to produce soluble recombinant forms of B. burgdorferi RpoN were unsuccessful. This was true for either the full-length protein or a truncated recombinant that preserved the predicted DNA-binding domain. We also employed lysates generated from B. burgdorferi cultures, grown under conditions in which RpoN is active (15, 41), in electrophoretic mobility shift assays (EMSAs). We were unable to demonstrate an interaction of rpoS sequences with borrelial cell lysates in EMSAs. Previous studies have shown functional homology between RpoN proteins from different bacterial species. For example, * Corresponding author. Mailing address: Department of Microbiology, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd., Dallas, TX 75390-9048. Phone: (214) 648-5900. Fax: (214) 648-5905. E-mail: [email protected]. Published ahead of print on 8 December 2006.