Biochemical study of multiple CheY response regulators of the chemotactic pathway of Rhodobacter sphaeroides

Abstract

The six copies of the response regulator CheY from Rhodobacter sphaeroides bind to the switch protein FliM. Phosphorylation by acetyl phosphate (AcP) was detected by tryptophan fluorescence quenching in three of the four CheYs that contain this residue. Autophosphorylation with Ac(32)P was observed in five CheY proteins. We also show that all of the cheY genes are expressed simultaneously; therefore, in vivo all of the CheY proteins could bind to FliM to control the chemotactic response. Consequently, we hypothesize that in this complex chemotactic system, the binding of some CheY proteins to FliM, does not necessarily imply switching of the flagellar motor.

FIG. 1.

Multiple alignment of the six CheY proteins from R. sphaeroides and CheY from S. enterica serovar Typhimurium. Sequence alignment was performed by using PILEUP (GCG-Wisconsin Package, version 9.1). Secondary structure prediction was carried out by using the program PSIPRED (8, 14). Identical residues are shaded in tones of black. Asterisks indicate relevant residues for the function of CheY_St.

FIG. 2.

RT-PCR of the six R. sphaeroides cheY transcripts. Total RNA was isolated from WS8 wild-type cells. One-fifth of each of the PCRs was loaded onto a 7.5% polyacrylamide gel. Lanes 1 to 6, show the amplification products of cheY1-6, respectively, and the expected sizes of these products are for 207 bp for cheY1, 354 bp for cheY2, 360 bp for cheY3, 360 bp for cheY4, 363 bp for cheY5, and 399 bp for cheY6. Lane 7 shows a control, with the oligonucleotides for amplification of cheY6, lacking avian myeloblastosis virus reverse transcriptase in the reaction.

FIG. 3.

Intrinsic fluorescence of the R. sphaeroides CheY proteins. Purified CheY proteins were mixed with 25 mM AcP never exceeding 4% of the initial volume, and kinetic determinations were performed as described previously (13). Given that each CheY displays a different fluorescence intensity, emission was normalized for each protein to an arbitrary intensity of 1.0.

FIG. 4.

CheY phosphorylation by AcP. A phosphorimage of the six CheY proteins incubated with Ac³²P for 1 min is shown. The reaction was stopped by the addition of 5 μl of 5× sample buffer for SDS-PAGE. Ac³²P was synthesized as described previously (24).

FIG. 5.

Binding of CheY and phospho-CheY to FliM. The six CheY proteins immobilized to Sepharose beads were assayed for binding to FliM, FliMΔ13, and FliMΔ20 in the presence or absence of AcP. Control lane shows nonspecific binding of FliM to bovine serum albumin-Sepharose beads. Immunoblots were probed with anti-FliM_Rs antibodies. The added purified FliM, FliMΔ13, and FliMΔ20 proteins are shown for reference.