Molecular characterization of two-component systems of Helicobacter pylori

Abstract

Two-component systems are frequently involved in the adaptation of bacteria to changing environmental conditions at the level of transcriptional regulation. Here we report the characterization of members of the two-component systems of the gastric pathogen Helicobacter pylori deduced from the genome sequence of strain 26695. We demonstrate that the response regulators HP166, HP1043, and HP1021 have essential functions, as disruption of the corresponding genes is lethal for the bacteria, irrespective of the fact that HP1043 and HP1021 have nonconserved substitutions in crucial amino acids of their receiver domains. An analysis of the in vitro phosphorylation properties of the two-component proteins demonstrates that HP244-HP703 and HP165-HP166 are cognate histidine kinase-response regulator pairs. Furthermore, we provide evidence that the variability of the histidine kinase HP165 caused by a poly(C) tract of variable length close to the 3' end of open reading frame 165/164 does not interfere with the kinase activity of the transmitter domain of HP165.

FIG. 1

Relationship of response regulators HP166, HP1043, and HP1365 to the output domain of OmpR of E. coli. The residues building the hydrophobic core of the OmpR output domain (23) are marked by black shading. Gaps introduced to maximize the alignment are indicated by dots. Amino acid positions are given on the right.

FIG. 2

Alignment of the receiver domains of the H. pylori response regulators HP166, HP1365, HP703, HP1021, and HP1043 and comparison with the receiver domain consensus sequence. Gaps introduced to maximize the alignment are indicated by dots. Positions where the indicated amino acids appear with a probability of more than 90% according to a comparison of 79 two-component response regulators (39) are highlighted by black shading. Positions considered to be invariant are indicated by arrowheads.

FIG. 3

Comparison of the 2D protein maps of the H. pylori mutants G27/HP244::km (B) and G27[flgR⁻] (C) and the wild-type strain G27 (A). Whole-cell lysates of H. pylori were prepared as described in Materials and Methods, and 100 μg of protein extract was loaded onto the isoelectric focusing gels over a pH range from pH 4 to pH 8. The protein spots missing in the 2D maps of the mutants are indicated by arrows and were identified by LC-mass spectrometry. The positions of molecular weight (MW) standards (in thousands) are given on the right.

FIG. 4

Autophosphorylation and phosphotransfer reactions of histidine kinases GST-HP244 and GST-HP165. GST-HP244 (lanes 1 to 5) and GST-HP165 (lanes 6 to 10) were incubated in individual reactions with [γ-³³P]ATP and the respective response regulators indicated above the lanes. For HP1021, the purified receiver domain, indicated as HP1021R, was used in the phosphorylation assay. The positions of the phosphorylated (-P) proteins are marked by arrows.

FIG. 5

Autophosphorylation and phosphotransfer reactions of histidine kinases GST-HP165, GST-HP165(C13), and GST-HP165(C9). (A) Alignment of the different C-terminal sequences of histidine kinase HP165 due to frameshifting as a consequence of the various lengths of a poly(C) tract close to the 3′ end of the coding region. (B) Length of the poly(C) tract in ORF HP165/164 in different isolates of H. pylori. (C) Autophosphorylation of fusion proteins GST-HP165, GST-HP165(C13), and GST-HP165(C9) representing reading frames A, C, and B, respectively, in the presence of [γ-³³P]ATP (lanes 1 to 3) and phosphotransfer reaction of fusion proteins GST-HP165, GST-HP165(C13), and GST-HP165(C9) in the presence of [γ-³³P]ATP and the cognate response regulator HP166 (lanes 4 to 6). The positions of phosphorylated (-P) proteins are marked by arrows.