Helicobacter pylori FlgR is an enhancer-independent activator of sigma54-RNA polymerase holoenzyme

Abstract

Helicobacter pylori FlgR activates transcription with sigma54-RNA polymerase holoenzyme (sigma54-holoenzyme) from at least five flagellar operons. Activators of sigma54-holoenzyme generally bind enhancer sequences located >70 bp upstream of the promoter and contact sigma54-holoenzyme bound at the promoter through DNA looping to activate transcription. H. pylori FlgR lacks the carboxy-terminal DNA-binding domain present in most sigma54-dependent activators. As little as 42 bp of DNA upstream of the flaB promoter and 26 bp of DNA sequence downstream of the transcriptional start site were sufficient for efficient FlgR-mediated expression from a flaB'-'xylE reporter gene in H. pylori, indicating that FlgR does not use an enhancer to activate transcription. Other examples of sigma54-dependent activators that lack a DNA-binding domain include Chlamydia trachomatis CtcC and activators from the other Chlamydia spp. whose genomes have been sequenced. FlgR from Helicobacter hepaticus and Campylobacter jejuni, which are closely related to H. pylori, appear to have carboxy-terminal DNA-binding domains, suggesting that the loss of the DNA-binding domain from H. pylori FlgR occurred after the divergence of these bacterial species. Removal of the amino-terminal regulatory domain of FlgR resulted in a constitutively active form of the protein that activated transcription from sigma54-dependent genes in Escherichia coli. The truncated FlgR protein also activated transcription with E. coli sigma54-holoenzyme in an in vitro transcription assay.

FIG. 1.

Comparison of a partial sequence of H. pylori FlgR with those of other σ⁵⁴-dependent activators. The conserved Walker A, Walker B, and sensor II motifs within the AAA+ domains of selected σ⁵⁴-dependent activators are indicated. X, any amino acid residue, with the number that follows indicating the spacing between the motifs. The sequences from σ⁵⁴-dependent activators that are shown are those of S. meliloti DctD (Sm DctD); H. pylori FlgR (Hp FlgR); H. hepaticus FlgR (Hh FlgR); Campylobacter jejuni FlgR (Cj FlgR); C. trachomatis CtcC (Ct CtcC); and activators from C. muridarum (Cm act), C. pneumoniae (Cp act), and Chlamydophila caviae (Cc act). Helix 4 indicates the last helix of the α-helical subdomain of the DctD AAA+ domain, which was predicted by threading the DctD sequence onto the “A. aeolicus” NtrC1 AAA+ domain structure (27). The serine residue underlined in helix 4 of the DctD sequence is Ser-390, which is the carboxy terminus of the DctD AAA+ domain used in the studies described here. The predicted helix-turn-helix motifs of DctD, H. hepaticus FlgR, and C. jejuni FlgR are indicated. The four amino acid residues of the carboxy termini of the FlgR proteins are shown for comparison.

FIG. 2.

Immunoblot of cell extracts of wild-type H. pylori and flgR and flgS mutant strains. Each lane contained cell extracts (50 μg of protein) from one of the H. pylori strains. The blot was probed with antiserum directed against the H. pylori flagellum. Lane 1, wild-type H. pylori ATCC 43504; lane 2, MGD1 (flgR:aphA3 mutant strain); lane 3, MGD2 (flgS:aphA3 mutant strain). FlgE, FlaB, and FlaA bands are indicated.

FIG. 3.

Immunoblot of FlgR in H. pylori cell extracts. The immunoblot was probed with an antibody directed against the histidine-tagged FlgR protein. Arrow, band corresponding to FlgR. Different amounts of cell extract of wild-type H. pylori were loaded onto lanes 1 to 7 (lane 1, 1 × 10⁹ cells; lane 2, 5 × 10⁸ cells; lane 3, 2 × 10⁸ cells; lane 4, 1 × 10⁸ cells; lane 5, 5 × 10⁷ cells; lane 6, 2 × 10⁷ cells; lane 7, 1 × 10⁷ cells). In lane 8, 10⁹ cells of the mutant H. pylori flgR:cat strain were loaded. Different amounts of purified MBP-FlgR were loaded onto lanes 9 to 14 (lane 9, 200 ng; lane 10, 100 ng; lane 11, 50 ng; lane 12, 25 ng; lane 13, 12.5 ng; lane 14, 6 ng).

FIG. 4.

In vivo activity of AAA+ domains of FlgR and DctD. Activity of full-length FlgR, the FlgR AAA+ domain, and the DctD AAA+ domain on a dctA′-′lacZ reporter gene in E. coli. Gray bars, activity for culture in which the expression of the FlgR or DctD proteins was induced with IPTG; black bars, activity for cultures in which the expression of the proteins was not induced with IPTG. Lanes 1 and 2, activity for full-length FlgR; lanes 3 and 4, activity for the FlgR AAA+ domain; lanes 5 and 6, activity for the DctD AAA+ domain.

FIG. 5.

In vitro transcriptional activation with the FlgR AAA+ domain. Reaction mixtures contained FlgR AAA+ domain (monomer) at the indicated concentrations. Arrow, transcript of the expected size (∼155 nucleotides) from the glnA promoter.