Redox signal transduction by the ArcB sensor kinase of Haemophilus influenzae lacking the PAS domain

Abstract

The Arc (anoxic redox control) two-component signal transduction system of Escherichia coli, which comprises the tripartite ArcB sensor kinase and the ArcA response regulator, modulates the expression of numerous operons in response to redox conditions of growth. We demonstrate that the arcA and arcB genes of Haemophilus influenzae specify a two-component system. The Arc proteins of the two bacterial species sufficiently resemble each other that they can participate in heterologous transphosphorylation in vitro. Moreover, the Arc system of H. influenzae mediates transcriptional control according to the redox condition of growth both autologously in its own host and homologously in E. coli, indicating a high degree of functional conservation of the signal transduction system. The H. influenzae ArcB, however, lacks the PAS domain present in the region of E. coli ArcB linking the transmembrane to the cytosolic catalytic domains. Because the PAS domain participates in signal reception in a variety of sensory proteins, including sensors of molecular oxygen and redox state, a similar role was previously ascribed to it in ArcB. Our results demonstrate that the ArcB protein of H. influenzae mediates signal transduction in response to redox conditions of growth despite the absence of the PAS domain.

FIG. 1

Schematic representation of the ArcB sensor kinase. (A) The E. coli protein; (B) the H. influenzae protein. The two N-terminal transmembrane segments (TM) were predicted on the basis of a hydrophobicity plot. The linker region of the E. coli protein (residues 78 to 267) contains a PAS domain (residues 177 to 267) (27). Black boxes represent sequences absent in H. influenzae ArcB. H1 (the primary transmitter domain) is shown with the catalytic determinants H, N, and G (29). The conserved site of autophosphorylation at His292 of the E. coli ArcB sequence corresponds to a conserved His131 of the H. influenzae ArcB sequence. G resembles the nucleotide-binding motif. In the receiver domain (D1), the conserved transphosphorylation site at Asp576 in E. coli ArcB corresponds to the conserved Asp407 of H. influenzae ArcB. In the secondary transmitter domain (H2), the conserved transphosphorylation site at His717 of E. coli ArcB corresponds to the conserved His541 of H. influenzae ArcB.

FIG. 2

Predicted ArcB proteins in other bacterial species. Partial CLUSTALW alignment of the E. coli (EC) ArcB to homologues in S. enterica serovar Typhimurium (ST), Y. pestis (YP), V. cholerae (VC), and H. influenzae (HI) is shown. Regions corresponding to amino acids 93 to 134 and 153 to 271 in E. coli are absent in H. influenzae but are present in the other three bacteria.

FIG. 3

Phosphorylation of H. influenzae ArcB and ArcA in vitro. (A) Kinetics of autophosphorylation of ArcB_Hi and transphosphorylation of ArcA_Hi in the presence of [γ-³²P]ATP. ArcA was added immediately after the 5-min time point. At each time point, samples were withdrawn and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. (B) Quantitation of the relative amounts of radioactivity incorporated in the Arc proteins by using a PhosphorImager.

FIG. 4

Interspecies phosphorylation by Arc proteins of E. coli and H. influenzae. The kinetics of phosphorylation of purified ArcA_Hi by ‵ArcB_Ec (A) and ArcA_Ec by ‵ArcB_Hi (B) were monitored in reaction mixtures containing [γ-³²P]ATP. ArcA was added immediately after 0 min.

FIG. 5

Aerobic and anaerobic expression levels of Φ(lldP-lacZ) in different genetic backgrounds. Solid bars, aerobic levels of β-galactosidase activity; hatched bars, anaerobic levels of β-galactosidase activity.

FIG. 6

Comparison of the responses of ArcB_Ec and ArcB_Hi sensors to various external electron acceptors. E. coli strains carrying Φ(lldP-lacZ) and arcB genes from either E. coli (closed squares) or H. influenzae (open circles) were grown aerobically (E⁰′ = 880 mV) or anaerobically in the presence or absence of nitrate (E⁰′ = 420 mV), dimethyl sulfoxide (E⁰′ = 160 mV), and trimethylamine N-oxide (E⁰′ = 130 mV). β-Galactosidase activity is plotted against the midpoint potential of the electron acceptors. Cultures and assays were performed with three independent samples per point, and variation was less than 10% between replicate samples.

FIG. 7

Expression of lctD mRNA in H. influenzae. A Northern blot containing total RNA extracted from aerobically (+O₂) or anaerobically (−O₂) grown H. influenzae cultures was hybridized with an lctD-specific probe (upper panels). The filter was reprobed with a 23S rRNA specific-probe (lower panels). Lanes 2 to 4 contain RNA samples from H. influenzae strains bearing the following arc alleles: ΔarcA::Kan^r (strain RAA6) (lanes 2); ΔarcA::Kan^r and ΔxylA::Tet^r (strain RAA6V) (lanes 3); and ΔarcA::Kan^r, ΔxylA::Tet^r, and arcA⁺ (strain RAA6C) (lanes 4). Lanes 1 contain RNA samples from the wild-type strain H. influenzae Rd.