Methylation-independent aerotaxis mediated by the Escherichia coli Aer protein

Abstract

Aer is a membrane-associated protein that mediates aerotactic responses in Escherichia coli. Its C-terminal half closely resembles the signaling domains of methyl-accepting chemotaxis proteins (MCPs), which undergo reversible methylation at specific glutamic acid residues to adapt their signaling outputs to homogeneous chemical environments. MCP-mediated behaviors are dependent on two specific enzymes, CheR (methyltransferase) and CheB (methylesterase). The Aer signaling domain contains unorthodox methylation sites that do not conform to the consensus motif for CheR or CheB substrates, suggesting that Aer, unlike conventional MCPs, might be a methylation-independent transducer. Several lines of evidence supported this possibility. (i) The Aer protein was not detectably modified by either CheR or CheB. (ii) Amino acid replacements at the putative Aer methylation sites generally had no deleterious effect on Aer function. (iii) Aer promoted aerotactic migrations on semisolid media in strains that lacked all four of the E. coli MCPs. CheR and CheB function had no influence on the rate of aerotactic movements in those strains. Thus, Aer senses and signals efficiently in the absence of deamidation or methylation, methylation changes, methylation enzymes, and methyl-accepting chemotaxis proteins. We also found that chimeric transducers containing the PAS-HAMP sensing domain of Aer joined to the signaling domain and methylation sites of Tar, an orthodox MCP, exhibited both methylation-dependent and methylation-independent aerotactic behavior. The hybrid Aear transducers demonstrate that methylation independence does not emanate from the Aer signaling domain but rather may be due to transience of the cellular redox changes that are thought to trigger Aer-mediated behavioral responses.

FIG. 1.

Absence of CheR- and CheB-dependent Aer band shifts in SDS-PAGE. Protein extracts of strains containing pPA144 (Tsr), pSB20 (Aer), or both plasmids were analyzed on 10 to 20% gradient gels, as described in Materials and Methods. Tsr and Aer were visualized by Western blotting with a polyclonal antiserum directed against residues 290 to 470 of the Tsr signaling domain (2). Host strains were UU1250 (CheR⁺ CheB⁺), UU1535 (CheR⁻ CheB⁻), and UU1249 (CheR⁻ CheB⁺).

FIG. 2.

Methylation-independent aerotaxis on tryptone semisolid agar plates. Host strains were UU1250 (CheR⁺ CheB⁺), UU1535 (CheR⁻ CheB⁻), and UU1249 (CheR⁻ CheB⁺); plasmids were pJC3 (Tsr), pCJ30 (vector), pSB20 (Aer), and pSB137 (Aer-D60N). Plates in the upper row contained 20 μM IPTG and were incubated at 30°C for 15 h. Plates in the lower row contained 50 μM IPTG and were incubated at 30°C for 17 h. All plates contained 50 μg of ampicillin/ml.

FIG. 3.

Methylation regions of Tsr, Tar, Trg, and Aer. Known methylation sites in Tsr, Tar, and Trg are shown in open boxes marked with their residue number(s). The Tar and Trg sites shown in dashed boxes correspond in position and in sequence motif to the fifth methylation site of Tsr but are not known to be methylated. Black boxes and white residue letters indicate the corresponding sites in Aer, below which are shown the Aer residue numbers, the Aer methylation site designations, and the mutational replacements made at each site.

FIG. 4.

Aerotaxis promoted by Aer methylation-site mutants. (A) Plasmid-containing UU1250 colonies on tryptone soft agar plates containing 50 μM IPTG and incubated for 16 h at 30°C. (B) Plasmid-containing UU1117 colonies on minimal succinate soft agar (with no IPTG) and incubated for 22 h at 30°C. Both plates contained 50 μg of ampicillin/ml. Plasmids in both strain backgrounds were as follows (reading from left to right): top row, pSB20 (Aer) and pCJ30 (vector control); middle row, pDM6 (Aer-1*), pDM7 (Aer-2*), and pDM8 (Aer-3*); bottom row, pDM5 (Aer-4*) and pDM9 (Aer-5*).

FIG. 5.

Aerotaxis promoted by a pseudorevertant of the Aer 1*/2*/4* triple methylation site mutant. Plasmid-containing UU1535 (CheR⁻ CheB⁻) colonies were on a tryptone soft agar plate containing 50 μM IPTG and 50 μg of ampicillin/ml. The plate was incubated at 30°C for 17 h. Plasmids were pSB20 (Aer⁺), pDM13 (Aer-1*/2*/4*), and pKG128 (Aer-1*/2*/4*/T431 M).

FIG. 6.

Domain organization and functional features of Aer and Tar and the positions of join points in Aer-Tar chimeras. The primary structures of the molecules are drawn to the same scale and aligned at the chimera join points. In the Aear-260 chimera, Aer residues 1 to 260 were joined to Tar residues 269 to 553. In the Aear-269 chimera, Aer residues 1 to 269 were joined to Tar residues 278 to 553.

FIG. 7.

Aerotactic signaling by Aer-Tar chimeras. Plasmid-containing colonies were on tryptone soft agar plates containing 50 μM IPTG and 50 μg of ampicillin/ml. Plates were incubated at 30°C for 15 h. Strains were UU1535 (CheR⁻ CheB⁻) and UU1250 (CheR⁺ CheB⁺). Plasmids were pSB20 (Aer), pKG121 (Aear-269), and pKG119 (Aear-260).

FIG. 8.

Current working model of Aer signal transduction. Aer is anchored to the cytoplasmic side of the inner membrane and probably monitors respiratory status via redox changes in a component of the electron transport system (ETS). Transience of the redox signal could account for methylation-independent sensory adaptation during aerotaxis, as discussed in the text.