Motility of Helicobacter pylori is coordinately regulated by the transcriptional activator FlgR, an NtrC homolog - PubMed (original) (raw)

Motility of Helicobacter pylori is coordinately regulated by the transcriptional activator FlgR, an NtrC homolog

G Spohn et al. J Bacteriol. 1999 Jan.

Abstract

sigma54 is the subunit of bacterial RNA polymerase that transcribes from promoters with enhancer elements bound by enhancer-binding proteins. By computer searches of Helicobacter pylori genomic sequences, chromosomal gene disruption, and RNA analyses, we have identified sigma54-recognized promoters that regulate transcription of flagellar basal body and hook genes, as well as the enhancer-binding protein FlgR (flagellum regulator), a transactivating protein of the NtrC family. We demonstrate that FlgR is required for bacterial motility and transcription of five promoters for seven basal body and hook genes. In addition, FlgR acts as a repressor of transcription of the sigma28-regulated flaA flagellin gene promoter, while changes in DNA topology repress transcription of the sigma54-regulated flaB flagellin gene promoter. Our data indicate that regulation of flagellar gene expression in H. pylori shows similarities with that in enterobacteriaceae and Caulobacter.

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Figures

FIG. 1

Structural organization of the indicated genes and operons. Dark gray bars indicate flagellar genes; open boxes indicate ORFs of unknown functions and are marked as reported by Tomb and coworkers (33); light gray boxes represent regulatory genes. The kanamycin cassette is indicated by boxes with vertical bars. (A) Genes transcribed by ς54-regulated promoters. flgD and flgE′ encode proteins with 25.5 and 30.5% amino acid identity to the flagellar hook assembly protein FlgD of Salmonella choleraesuis (18) and the structural hook protein FlgE of H. pylori (26), respectively. flgB and flgC constitute an operon encoding two proteins with 31 and 46% amino acid identity to the proximal flagellar rod proteins FlgB of S. choleraesuis (16) and FlgC of Borrelia burgdorferi (13), respectively. flgK encodes for a protein with 27.6% amino acid identity to the flagellar hook-associated protein FlgK (HAP1) of S. choleraesuis (15). (B) Construction of the G27[_flgR_] mutant strain. To study the regulatory protein involved in transcriptional activation of the putative ς54-dependent genes, we searched for NtrC homologs in the genome sequence of H. pylori (33). One protein (HP703) with significant amino acid sequence homology to a series of NtrC-like regulators was detected. The highest degree of amino acid similarity (44.2% identity, 69.1% similarity) was with FleQ, an activator of mucin adhesion and flagellum expression in Pseudomonas aeruginosa (3).

FIG. 2

Bacterial motility assay. The indicated strains were stabbed into semisolid agar medium and incubated at 37°C for 72 h.

FIG. 3

Primer extension analyses of H. pylori RNAs. The elongated primers are indicated by arrows and labeled “P.” Subscript numbers refer to the downstream ORF as designated by Tomb et al. (33). Total RNA was extracted from wild-type H. pylori G27 (lanes +) or from the flgR isogenic mutant (lanes −), annealed to specific primers, and elongated with reverse transcriptase as detailed in Materials and Methods.

FIG. 4

Nucleotide sequences of H. pylori ς54 promoters. Shown is alignment of the sequence from +1 to −60 of the indicated promoter genes and a derived consensus sequence. The promoter region is shaded; identical bases are shown in boldface.

FIG. 5

Transcriptional regulation of flaA by FlgR. The RNA used was from the same preparation as that used in the experiment shown in Fig. 3. Symbols are as in Fig. 3. Control RNAs, cagA and ureAB.

FIG. 6

Novobiocin affects transcription of flaB but not flaA. Total RNA extracted from H. pylori G27 before addition of novobiocin (time zero [lane 0’]) and after 15 and 30 min (lanes 15’ and 30’).

FIG. 7

Proposed model for flagellar gene expression in H. pylori. Dependence of transcription on the specified ς factors is shown for genes and operons indicated in boldface. Genes and operons whose promoters have been deduced on the basis of nucleotide sequence analysis are enclosed by gray boxes.

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Motility of Helicobacter pylori is coordinately regulated by the transcriptional activator FlgR, an NtrC homolog - PubMed (original) (raw)