In vitro phosphorylation study of the arc two-component signal transduction system of Escherichia coli

Abstract

The ArcB and ArcA proteins constitute a two-component signal transduction system that plays a broad role in transcriptional regulation. Under anoxic or environmentally reducing conditions, the sensor kinase (ArcB) is stimulated to autophosphorylate at the expense of ATP and subsequently transphosphorylates the response regulator (ArcA). ArcB is a complex, membrane-bound protein comprising at least three cytoplasmic domains, an N-terminal transmitter domain with a conserved His292 residue (H1), a central receiver domain with a conserved Asp576 residue (D1), and a C-terminal alternative transmitter domain with a conserved His717 residue (H2). To study the phosphoryl transfer pathways of the Arc system, we prepared the following His-tagged proteins: H1, D1, H2, H1-D1, D1-H2, H1-D1-H2, and ArcA. Incubations of various combinations of Arc proteins with [␥-32 P]ATP indicated that H1, but not D1 or H2, catalyzes autophosphorylation; that H1-P transfers the phosphoryl group to D1 much more rapidly than to ArcA; and that D1 accelerates the transphosphorylation of H2. Finally, ArcA is phosphorylated much more rapidly by H2-P than by H1-P. Available data are consistent with a signal transduction model in which (i) reception of a membrane signal(s) triggers autophosphorylation of H1 at His292, (ii) the phosphoryl group can migrate to D1 at Asp576 and subsequently to H2 at His717, and (iii) ArcA receives the phosphoryl group from either His292 or His717, the relative contribution of which is regulated by cytosolic effectors.

A two-component phosphotransfer network involving ArcB, ArcA, and RssB coordinates synthesis and proteolysis of σS (RpoS) in E. coli

“…The latter can actually be seen in the kinetic experiment (Fig. 5F,G), where intially rapidly accumulating phoshorylated ArcA is reduced again over time as was also observed previously (Tsuzuki et al 1995;Georgellis et al 1997). …”

“…Immunoblots are shown in E and G, the corresponding quantification is given in F and H, respectively. lacking the first 77 amino acids, which contain the membrane-spanning domains of wild-type ArcB (this Nterminally truncated form of ArcB was previously shown to be active in vitro) (Georgellis et al 1997). Purified ArcB 78-778 , RssB, ArcA, and [␥ 32 P]ATP were coincubated in various combinations and phosphorylated proteins were visualized by SDS-PAGE and PhosphorImager analysis.…”

“…To obtain the overexpression plasmid pQE30ArcB 78-778 , the arcB gene, excluding the membrane spanning domains (located in amino acids 1-77), was amplified with the primers 5Ј-CTG GATCCCATATGGAGCAACTGGAGGAGT-3Ј (BamHI) and 5Ј-CATAGTCGACCATATCGCGCACCCCGGTCT-3Ј (SalI) (Georgellis et al 1997), digested with BamHI and SalI, and cloned between the corresponding restriction sites of pQE30. Plasmids were verified by DNA sequencing.…”

Tuning of the porin expression under anaerobic growth conditions by His‐to‐Asp cross‐phosphorelay through both the EnvZ‐osmosensor and ArcB‐anaerosensor in Escherichia coli

“…1) (Ishige et al 1994;Tsuzuki et al 1995;Kato et al 1997). A multistep His-to-Asp phosphorelay mechanism was proposed for the ArcB-to-ArcA phosphotransfer pathway, in which the newly uncovered HPt domain plays a crucial role (Tsuzuki et al 1995;Georgellis et al 1997). Nevertheless, the importance of ArcB's complex structure has not yet been fully understood.…”

The ArcA/ArcB two‐component regulatory system of Escherichia coli is essential for Xer site‐specific recombination at psi

“…2C). Mutant 5.4 may, therefore, express a truncated form of ArcB that retains His-292, one of two catalytic histidine residues able to phosphorylate ArcA at Asp-54 (Georgellis et al, 1997). Phosphorylation of ArcA by this truncated ArcB protein could explain the partial ability of mutant 5.4 to carry out Xer recombination at psi.…”