A dual-signaling mechanism mediated by the ArcB hybrid sensor kinase containing the histidine-containing phosphotransfer domain in Escherichia coli

Abstract

The two components ArcB and ArcA play a crucial role in the signal transduction implicated in the complex transcriptional regulatory network that allows Escherichia coli to sense various respiratory growth conditions. ArcB is a hybrid sensor kinase having multiple phosphorylation sites in its primary amino acid sequence, including a transmitter, a receiver, and a histidine-containing phosphotransfer (HPt) domain. ArcA is a DNA-binding transcriptional regulator with a receiver domain. Results of recent in vitro studies revealed multistep His-to-Asp phosphotransfer circuitry in the ArcB-ArcA signaling system. For this report we conducted a series of in vivo experiments using a set of crucial ArcB mutants to evaluate the regulation of the sdh operon. The results suggested that the phosphorylated His-717 site in the HPt domain of ArcB is essential for anaerobic repression of sdh. Nonetheless, the ArcB mutant lacking this crucial His-717 site does not necessarily exhibit a null phenotype with respect to ArcB-ArcA signaling. The HPt mutant appears to maintain an ability to signal ArcA, particularly under aerobic conditions, which results in a significant repression of sdh. Based on these and other in vivo results, we propose a model in which ArcB functions in its own right as a dual-signaling sensor that is capable of propagating two types of stimuli through two distinct phosphotransfer pathways.

FIG. 1

β-Galactosidase activity expressed by the sdh-lacZ transcriptional fusion gene. Strains OG903 (bars denoted by “Wild” [arcB⁺ sdh-lacZ]) and DAC903 (bars denoted by “ΔarcB” [arcB::Cm^r sdh-lacZ]) were grown in either Luria (L) broth (A) or M9-glucose minimal medium (B), under both aerobic (+ O₂) and anaerobic (−O₂) conditions. DAC903 carrying plasmid pLIA001 containing the wild-type arcB gene was also grown under the same conditions as those described above (bars denoted by “ΔarcB” and “W”). The harvested cells were assayed for β-galactosidase, according to the method of Miller (17). The same experiments were repeated more than three times, and the means were plotted with Miller units. Note that error bars are omitted for clarity (the deviations were less than 7%). The horizontal lines are placed to indicate the highest levels of β-galactosidase activities that were observed in the presence of O₂ for the type I and type II repression phenomena.

FIG. 2

β-Galactosidase activity expressed by the sdh-lacZ transcriptional fusion gene. Strain DAC903 (arcB::Cm^r sdh-lacZ) was transformed with the following plasmids: the vector (bars denoted by “ΔarcB”), pLIA001 (bars denoted by “W”), pILA004 (bars denoted by “ΔH1”), and pILA002 (bars denoted by “ΔH2”). These transformants were grown in either Luria (L) broth (A) or M9-glucose minimal medium (B) under both aerobic (+ O₂) and anaerobic (−O₂) conditions. The harvested cells were assayed for β-galactosidase, according to the method of Miller (17), as described for Fig. 1.

FIG. 3

β-Galactosidase activity expressed by the sdh-lacZ transcriptional fusion gene. Strain DAC903 (arcB::Cm^r sdh-lacZ) was transformed with the following plasmids: the vector (bars denoted by “−”), pLIA001 (bars denoted by “W”), pILA004 (bars denoted by “ΔH1”), pLIA003 (bars denoted by “ΔD”), and pILA002 (bars denoted by “ΔH2”). These transformants were grown in either Luria (L) broth (A) or M9-based minimal media, each containing the indicated carbon and energy source (B to H) under the aerobic conditions. The harvested cells were assayed for β-galactosidase, according to the method of Miller (17), as described for Fig. 1.

FIG. 4

Proposed model to explain the dual-signaling mechanism underlying signal transduction in ArcB-ArcA multistep phosphotransfer. This model is based on the in vivo findings of this study, together with those of previous in vivo and in vitro studies (2, 13, 15, 25). Previous in vitro studies suggested that an unprecedented phosphotransfer from His-292 to His-717 may also occur (2, 24). Other details are given in the text.