Domain organization and flavin adenine dinucleotide-binding determinants in the aerotaxis signal transducer Aer of Escherichia coli

Abstract

Aerotactic responses in Escherichia coli are mediated by the membrane transducer Aer, a recently identified member of the superfamily of PAS domain proteins, which includes sensors of light, oxygen, and redox state. Initial studies of Aer suggested that it might use a flavin adenine dinucleotide (FAD) prosthetic group to monitor cellular redox changes. To test this idea, we purified lauryl maltoside-solubilized Aer protein by His-tag affinity chromatography and showed by high performance liquid chromatography, mass spectrometry, and absorbance spectroscopy that it bound FAD noncovalently. Polypeptide fragments spanning the N-terminal 290 residues of Aer, which contains the PAS motif, were able to bind FAD. Fusion of this portion of Aer to the flagellar signaling domain of Tsr, the serine chemoreceptor, yielded a functional aerotaxis transducer, demonstrating that the FAD-binding portion of Aer is sufficient for aerosensing. Aerotaxis-defective missense mutants identified two regions, in addition to the PAS domain, that play roles in FAD binding. Those regions flank a central hydrophobic segment needed to anchor Aer to the cytoplasmic membrane. They might contact the FAD ligand directly or stabilize the FAD-binding pocket. However, their lack of sequence conservation in Aer homologs of other bacteria suggests that they play less direct roles in FAD binding. One or both regions probably also play important roles in transmitting stimulus-induced conformational changes to the C-terminal flagellar signaling domain to trigger aerotactic behavioral responses.

Figure 1

Sequence features and working model of Aer.

Figure 2

Absorbance spectra of wild-type Aer and an FAD-binding mutant. His-tagged Aer proteins were solubilized in lauryl maltoside and were purified as detailed in Materials and Methods. Absorbance measurements were made with an Hitachi U-3300 UV/VIS spectrophotometer connected to a microcomputer. uv solutions software (Hitachi Instruments, San Jose, CA) was used to obtain the differential spectrum shown at the bottom of the figure. The vertical dashed lines mark the two local absorbance maxima of FAD.

Figure 3

Aerotaxis assays on succinate semisolid agar. Plates were photographed after incubation at 35°C for 18 h. (A) Colony morphology of Aer⁻ (UU1117) and Aer⁺ (RP437) strains at different succinate concentrations. (B) Effect of Tar function on colony morphology of aerotaxis-defective strains, UU1117 [Δ(aer) (tar⁺)], and UU1259 [Δ(aer) Δ(tar)]. Plates contained 30 mM succinate. (C) Colony morphology of UU1117 containing pSB20 (Aer) or pSB100 (Aesr) at comparable levels of expression. Plates contained 30 mM succinate and 50 μg/ml ampicillin.

Figure 4

FAD-binding properties of Aer missense mutants, deletions, and fragments. Residue conservation: The primary structures of Aer homologs from P. putida, Tn1721, Y. pestis, V. cholerae (three ORFs), S. putrefaciens, and S. typhimurium were aligned with that of E. coli. At each residue position, the height of the bar reflects the number of homologs with an amino acid identical to that of E. coli Aer. Missense mutations: The indicated amino acid replacements were found among mutD-induced, aerotaxis-defective mutants of pSB20. FAD binding by the mutant proteins was assessed by the overexpression test. FAD⁻/FAD⁺ fragments: The shaded and solid lines indicate the segment of the Aer protein made in nonsense mutants [oc, ochre (UAA); am, amber (UAG)] or from subcloned portions of the aer coding region. Capital letters at the ends of the fragments indicate vector-encoded amino acids; numbers indicate initial and final codons from aer. Hydrophobic segment deletions: Numbers give the aer codons at either end of the deletion; letters within the deletion gaps indicate amino acids of the joining linker. Neither deletion protein binds FAD or supports aerotaxis. Cysteine mutations: The positions of the three cysteine residues in Aer and their replacement mutations are shown. The triply mutant protein binds FAD and supports aerotaxis. Aer/Tsr hybrid: Domain schematic of the Aesr transducer, which has the signaling domain of Tsr and supports aerotaxis (see Fig. 3C).

Figure 5

Possible roles of the F1 and F2 segments of Aer. The shading conventions are the same ones used in Figs. 1 and 4: , NifL/PAS similarity segment; IIII, F1 region; ▨, hydrophobic segment; IIII, F2 region; ▨, MCP similarity segment. (A) F1 and F2 directly comprise the FAD-binding pocket. (B) F1 and F2 stabilize the FAD-binding pocket through interactions with the NifL/PAS domain. (C) F1 and F2 indirectly stabilize the FAD-binding pocket. In all three models, input stimuli sensed by the FAD ligand, ostensibly via redox changes, are transmitted to the output signaling domain through the F2 region. The hydrophobic segment anchors Aer to the inner membrane, but otherwise may play no role in input-output communication.