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J Bacteriol. 2000 Jul; 182(13): 3858–3862.
PMCID: PMC94563
Note

Phosphorelay as the Sole Physiological Route of Signal Transmission by the Arc Two-Component System of Escherichia coli

Abstract

The Arc two-component system, comprising a tripartite sensor kinase (ArcB) and a response regulator (ArcA), modulates the expression of numerous genes involved in respiratory functions. In this study, the steps of phosphoryl group transfer from phosphorylated ArcB to ArcA were examined in vivo by using single copies of wild-type and mutant arcB alleles. The results indicate that the signal transmission occurs solely by His-Asp-His-Asp phosphorelay.

The ArcB-ArcA two-component signal transduction system of Escherichia coli regulates the expression of more than 30 operons depending on the redox conditions of growth (12, 17, 18). This system comprises ArcB as the membrane-bound sensor kinase and ArcA as the cognate response regulator (Fig. (Fig.1).1). The ArcB protein has three cytoplasmic domains: a primary transmitter domain (H1) containing a conserved His292, a receiver domain (D1) containing a conserved Asp576, and a secondary transmitter domain (H2) containing a conserved His717 (10, 13, 15, 27). ArcB thus belongs to the tripartite hybrid sensor kinase subfamily (23), which also includes BarA (21), EvgS (29), and TorS (14) of E. coli, BvgS of Bordetella pertussis (1), LemA of Pseudomonas syringae pv. syringae (9), and RteA of Bacteroides thetaiotaomicron (25).

FIG. 1
Schematic representation of ArcB and ArcA. (Top) The N-terminal transmembrane domain of ArcB was determined by alkaline phosphatase fusions (16). A putative leucine zipper (6) and a PAS domain (26) were predicted on the basis of amino acid sequence homology. ...

In vitro studies showed that the primary transmitter domain of ArcB is autophosphorylated at His292 at the expense of ATP (8, 11). The phosphoryl group is then sequentially transferred to Asp576 and His717 and from there to Asp54 of ArcA. However, the phosphoryl group on His292 could also be directly transferred in vitro to ArcA at a very low rate (8). An in vivo study utilizing ArcB domains borne by a low-copy-number plasmid led to the conclusion that the phosphoryl group from His292 could be transferred to ArcA and that this transfer was regulated by the nature of the carbon source. On the other hand, the phosphoryl group from His717 could also be transferred to ArcA, but this transfer was regulated by redox conditions (19). Possible misleading results caused by multiple gene dosage effect and different degrees of catabolite repression during utilization of various carbon sources, however, were not discussed. In yet another study, it was suggested that His717 received the phosphoryl group from an unknown sensor kinase (10). Here, we address the questions of whether ArcA can be phosphorylated by both H1 and H2 and whether H2 can be phosphorylated by a noncognate sensor kinase, under in vivo conditions in cells bearing a single copy of an arcB allele on the chromosome.

Strategy for the in vivo study with modified arcB.

The strains, phage, and plasmids used in this study are listed in Table Table1.1. To determine the sequence of phosphotransfer in the Arc system, the arcB+ allele on the chromosome was replaced by various mutant sequences (Fig. (Fig.2).2). The strategy of single-copy replacement circumvents possible complementation, epistatic, and dosage effects. The phenotypic consequences of ArcB modification were analyzed by changes in the in vivo levels of phosphorylated ArcA (ArcA-P), as indicated by expressions of target operons. We employed a λΦ(cydA′-lacZ) operon fusion as an ArcA-P-activable reporter and a λΦ(lldP′-lacZ) operon fusion as an ArcA-P-repressible reporter. A Δfnr::Tn9(Cmr) allele was incorporated into the λΦ(cydA′-lacZ)-harboring strains to avoid its repression by Fnr (3).

TABLE 1
E. coli K-12 strains, phage, and plasmids used in this study
FIG. 2
Allele replacement strategy. (A) Construction of the ΔarcB::Tetr strains. The 5′- and 3′-flanking DNA fragments of arcB (fragments 1 and 2) were prepared by PCR, using chromosomal DNA from strain MC4100 as the template and, respectively, ...

Requirement of all three conserved residues of ArcB for its ArcA-phosphorylating activity.

To verify the importance of His292, Asp576, and His717 in the phosphotransfer pathway leading to the formation of ArcA-P, we replaced the chromosomal arcB+ allele by arcBH292Q, arcBD576A, or arcBH717Q in a reporter strain bearing λΦ(cydA′-lacZ) or λΦ(lldP′-lacZ). The cells were grown aerobically or anaerobically and their β-galactosidase activity levels were assayed. It was found that all three mutants exhibited phenotypes indistinguishable from that of the ΔarcB mutant, suggesting that ArcB phosphorylates ArcA exclusively by the relay pathway (Fig. (Fig.3).3). Western analysis showed that all point mutants did produce wild-type levels of ArcB (Fig. (Fig.4).4). To confirm that H1 cannot mediate the phosphorylation of ArcA without H2, we replaced arcB+ by arcB1–520, arcB1–661, arcB1–661, D576A, or arcBD576A, H717Q. All of the tested alleles gave a null phenotype (Table (Table2).2).

FIG. 3
Effects of mutations in the conserved amino acid residues of ArcB on the expressions of λΦ(cydA′-lacZ) or a λΦ(lldP′-lacZ). The cydAB operon encodes cytochrome d oxidase (5), and the lldPRD operon encodes ...
FIG. 4
Western blot analysis. A 1-ml sample of cultures grown aerobically in Luria-Bertani broth was harvested at an optical density at 600 nm of 0.5. The pelleted cells were washed with 1 ml of 10 mM Tris-HCl (pH 8.0) and solubilized by incubation at 95°C ...
TABLE 2
Effects of various mutant arcB alleles on the expressions of λΦ(cydA′-lacZ) or λΦ (lldP′-lacZ)a

His717 of H2 derives its phosphoryl group exclusively from the relay.

To test whether His717 can be phosphorylated by a noncognate sensor kinase(s), we replaced arcB+ by arcBH292Q, D576A. The mutant showed an arcB-null phenotype, despite the presence of His717 in the ArcB protein (Table (Table2).2). Furthermore, when arcB638–778 (H2) was expressed from a low-copy-number plasmid in an ΔarcB background, an arcB-null phenotype was also found. On the other hand, when the same plasmid was tested in an arcB1–661 background, an arcB+ phenotype was obtained (data not shown). This latter result indicated that the phosphorylation of ArcA via His717 depended on the presence of His292 and Asp576 and that the phosphorelay involved an intermolecular reaction between different ArcB domains, in agreement with the results of our previous in vitro study (8).

Discussion and conclusion.

We were prompted to undertake this study not only because in vitro enzymatic data (8) and in vivo properties of cells with multiple gene dosage (19) may be misleading, but also because of certain conflicting results. For instance, in a study of purified proteins, the rate of ArcA phosphorylation catalyzed by ArcB78–661 (H1-D1) was less than an order of magnitude smaller than that catalyzed by a mixture of ArcB78–661 (H1-D1) and ArcB638–778 (H2), indicating a predominant role of the phosphorelay (8). By contrast, in a study of everted vesicles, the rate of ArcA phosphorylation catalyzed by ArcBD576Q was almost as high as that catalyzed by wild-type ArcB, indicating a predominant role of His292 as a direct phosphoryl group donor to ArcA (27). However, in a third study, the same ArcBD576Q mutant protein (encoded by a low-copy-number plasmid) was inactive in vivo as a phosphoryl group donor to ArcA (19). Paradoxically, in that same study, ArcBH717L apparently was able to serve as a phosphoryl group donor to ArcA (19).

The results from the present study, based on single-copy arcB alleles, indicate that the sole route of phosphotransfer from ArcB to ArcA is by a relay involving His292, Asp576, and His717 of the sensor kinase (Fig. (Fig.5).5). In particular, there is no evidence for direct phosphoryl group transfer from His292 to ArcA or for the phosphorylation of ArcA by an unknown kinase via the H2 domain of ArcB. Thus, the mode of signal transmission in the Arc system seems to be no more elaborate than that proposed for the Bvg (28) and Tor (14) systems.

FIG. 5
Model for signal transduction by the Arc system. Heavy solid arrows indicate the forward phosphotransfer reactions leading to formation of ArcA-P. Hatched arrows indicate the reverse phosphotransfer reactions leading to signal decay (7). Arrows with crosses ...

Acknowledgments

This work was supported by U. S. Public Health Service grant GM40993 from NIGMS of the National Institutes of Health.

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