Molecular characterization of Borrelia burgdorferi erp promoter/operator elements

Abstract

Many Borrelia burgdorferi Erp outer surface proteins have been demonstrated to bind the host complement regulator factor H, which likely contributes to the ability of these organisms to evade the host innate immune system. B. burgdorferi controls Erp protein synthesis throughout the bacterial infectious cycle, producing the proteins during mammalian infections but repressing their synthesis during tick infections. Defining the mechanism by which B. burgdorferi regulates the expression of these virulence determinants will provide important insight into the biological and pathogenic properties of the Lyme disease spirochete. The present study demonstrates that two highly conserved DNA sequences located 5' of erp operons specifically bind bacterial proteins. Analyses with B. burgdorferi of transcriptional fusions between erp promoter/operator DNAs and the gene for green fluorescent protein indicated that the expression of these operons is regulated at the level of transcriptional initiation. These analyses also indicated significant differences in the promoter strengths of various erp operons, which likely accounts for reported variations in expression levels of different Erp proteins. Mutagenesis of promoter-gfp fusions demonstrated that at least one of the proteins which bind erp operator DNA functions as a repressor of transcription.

FIG. 1.

Alignment of the DNA sequences located 5′ of the 10 erp operons of strain B31, the bbk2.10 operon of strain 297, and the p21erp22 operon of strain N40. The complete sequence 5′ of the B31 erpIJ operon has not been determined (12, 13). Identical nucleotides found in 50% or more of the sequences are boxed and shaded. Start codons (ATG) of the first gene of each erp operon are indicated in bold at the lower right. The start codon of each divergently transcribed bppC gene (CAT) is indicated in bold at the upper left. The identified start of erp transcription (24) and probable −10 and −35 sequences are indicated. Nucleotides are numbered above each line relative to the start of transcription. The5′-most nucleotides of DNAs used in initial EMSA competition experiments are indicated by circled numbers as follows: the 5′ end of G10-AC is labeled “10,” the 5′ end of G11-AC is marked “11,” and so on. All of the competitors extended 3′ beyond the sequences illustrated. Competitor G9-AC was composed of DNA sequences entirely within the erpG gene and therefore is not indicated in this figure. Heavily lined boxes indicate the boundaries of protein-binding sites 1 and 2. The asterisk above bp −412 indicates the 5′ ends of full-length promoter/operator fragments used for the construction of most Perp::gfp fusions, the black square above bp −215 indicates the 5′ ends of fragments used to produce pEAP1 and pGPB, and the diamond above bp −93 indicates the 5′ ends of fragments used for the construction of pEAP2 and pGPL.

FIG. 2.

Construction of Perp::gfp fusion plasmids. A nonessential region of the B. burgdorferi-E. coli shuttle vector pBSV2 was removed to produce pJAH2. The mutant 3 allele of gfp was then cloned into pJAH2 to produce pBLS590. To the 5′ side of gfp are KpnI and BamHI sites that facilitate the easy cloning of any promoter element, with the GG bases of the BamHI site oriented to comprise the 3′ end of any inserted ribosome binding site (consensus sequence, 5′-GGAGG-3′).

FIG. 3.

EMSAs identifying the two protein-binding sites of erp operator DNAs. All assays used protein extracts of B. burgdorferi cultured at 34°C. Experimental data shown in panels A, B, and C were obtained with a labeled probe extending from +18 to −330, while those for panel D were obtained with a probe spanning −31 to −89. For all panels, lanes contained either free DNA or DNA, poly(dI-dC), protein extract, and the DNA competitor noted above the lane. (A and B) Short and long exposures of the same EMSA indicating DNA competition of protein binding to site 2 (A) and site 1 (B). (C) Identification of maximal boundary of site 1. (D) Identification of maximal boundary of site 2. As previously observed (8), complex 1 was less abundant than complex 2, and longer exposure times were required to detect complex 1 on X-ray film. As a result, signals from complex 2 often saturated films that were used to detect complex 1.

FIG. 4.

Determination of relative abundance of the site 2 binding protein. Cell extracts from B. burgdorferi cultured at either 23 or 34°C were assayed by EMSA using labeled erpG promoter DNA. The total amounts of protein extract incubated with the DNA are indicated above each lane.

FIG. 5.

Representative GFP expression by untransformed B. burgdorferi B31-e2 or bacteria carrying a promoterless gfp (pBLS590), gfp with a 5′ ribosome-binding site only (pBLS603), PerpAB::gfp (pBLS591), or PerpG::gfp (pBLS592). Bacteria were either grown to mid-exponential phase at a constant 23°C (shaded) or were shifted from 23 to 34°C after reaching mid-exponential phase (unshaded).

FIG. 6.

Representative analyses of B. burgdorferi carrying deletions in the operators of PerpAB::gfp and PerpG::gfp. Bacteria were either grown to mid-exponential phase at a constant 23°C (shaded) or were shifted from 23 to 34°C after reaching mid-exponential phase (unshaded).

FIG. 7.

Representative analyses of GFP expression by B. burgdorferi carrying chimeric PerpAB/PerpG::gfp transcriptional fusions. Bacteria were either grown to mid-exponential phase at a constant 23°C (shaded) or were shifted from 23 to 34°C after reaching mid-exponential phase (unshaded). Diagrams below each panel illustrate regions of the two erp promoter/operator regions contained in each construct.

FIG. 8.

Representative GFP analyses of B. burgdorferi carrying Pbbk2.10::gfp (pBLS595) or Pp21erp22::gfp (pBLS600). Bacteria were either grown to mid-exponential phase at a constant 23°C (shaded) or were shifted from 23 to 34°C after reaching mid-exponential phase (unshaded).