Mutational analysis of a conserved signal-transducing element: the HAMP linker of the Escherichia coli nitrate sensor NarX

Abstract

The HAMP linker, a predicted structural element observed in sensor proteins from all domains of life, is proposed to transmit signals between extracellular sensory input domains and cytoplasmic output domains. HAMP (histidine kinase, adenylyl cyclase, methyl-accepting chemotaxis protein, and phosphatase) linkers are located just inside the cytoplasmic membrane and are projected to form two short amphipathic alpha-helices (AS-1 and AS-2) joined by an unstructured connector. The presumed helices are comprised of hydrophobic residues in heptad repeats, with only three positions exhibiting strong conservation. We generated missense mutations at these three positions and throughout the HAMP linker in the Escherichia coli nitrate sensor kinase NarX and screened the resulting mutants for defective responses to nitrate. Most missense mutations in this region resulted in a constitutive phenotype mimicking the ligand-bound state, and only one residue (a conserved Glu before AS-2) was essential for HAMP linker function. We also scanned the narX HAMP linker with an overlapping set of seven-residue deletions. Deletions in AS-1 and the connector resulted in constitutive phenotypes. Two deletions in AS-2 resulted in a novel reversed response phenotype in which the response to ligand was the opposite of that seen for the narX(+) strain. These observations are consistent with the proposed HAMP linker structure, show that the HAMP linker plays an active role in transmembrane signal transduction, and indicate that the two amphipathic alpha-helices have different roles in signal transduction.

FIG. 1.

HAMP linkers from Escherichia coli K-12 sensory proteins. Sequences of HAMP linkers from five E. coli MCPs, 15 sensor kinases, two putative diguanylate cyclase/phosphodiesterases (YfiN and YhjK), and the S. enterica MCP Tar are shown. The approximate limits of AS-1, the connector, and AS-2 are indicated. Heptad repeats of hydrophobic residues are denoted by letters A through G; positions usually occupied by hydrophobic residues in the amphipathic sequences are indicated by capital letters and, when present, are boxed in the sequences. Positions in AS-1 are numbered relative to the conserved Pro at position +1 in AS-1, and positions in AS-2 are numbered relative to the conserved Glu immediately before AS-2 (see text). Conserved residues Pro at position +1 in AS-1, Glu in the connector, and Met/Leu at position +11 in AS-2 are indicated with bold boxes. Shaded residues are those at which missense mutations have been identified previously. Numbers above the NarX sequence denote residues at which missense mutations were isolated in this study.

FIG. 2.

Missense mutations and deletions in the NarX HAMP linker. NarX amino acid sequence, heptad repeats, and the positions of AS-1 and AS-2 are displayed as in Fig. 1. Missense mutations are indicated above the NarX amino acid sequence; impaired-induction and constitutive phenotypic classifications are described in the text. Positions indicated with an asterisk are loci of multiple substitutions, isolated by both directed and random mutagenesis. Deletions in the NarX HAMP linker are represented by solid boxes under the wild-type NarX amino acid sequence; the deleted residues are indicated in the solid boxes. The deletion Pro-181 through Ser-220 replaces the indicated residues with a Cys residue. β-Galactosidase specific activity was measured as described in Materials and Methods and is expressed in Miller units. Mutant narX alleles were expressed from derivatives of plasmid pHG165 transformed into strain VJS5054 Φ(narG-lacZ) narX narPQ pcnB. Cultures were grown anaerobically in the absence (−NO₃⁻) or presence (+NO₃⁻) of 40 mM NaNO₃. Activity for narX⁺ was 20 Miller units in the absence of nitrate and approximately 2,000 Miller units with added nitrate; activity for the vector only was about 10 Miller units irrespective of added nitrate.