Acetyladenylate or its derivative acetylates the chemotaxis protein CheY in vitro and increases its activity at the flagellar switch.
CheY, a key protein in the mechanism of bacterial chemotaxis, is known to interact with the flagellar switch and thereby cause clockwise rotation. This activity of CheY was significantly increased by producing acetyladenylate (AcAMP) within cytoplasm-free bacterial envelopes containing purified CheY. This was achieved by including in the envelopes the enzyme acetyl-CoA synthetase (ACS) and ATP, and adding acetate externally. The fraction of clockwise-rotating envelopes, tethered to glass by their flagella, increased from 14% to 58% by the presence of AcAMP (or its derivative). In parallel experiments carried out with [14C]acetate under similar conditions, CheY became acetylated: [1-14C]acetate was as effective as [2-14C]acetate in labeling CheY, and ACS-dependent labeling of CheY by [alpha-32P]ATP was not detected. The switch proteins, FliG, FliM, and FliN, isolated to purity, were not acetylated. The acetylation was specific for CheY and dependent on its native conformation. The acetylated form the CheY was estimated to be more active than its nonacetylated form by 4-5 orders of magnitude. Acetylated CheY was stable in the presence of the strong nucleophiles hydroxylamine or ethanolamine, indicative of N-acetylation. There was a correlation between the activity of CheY in vivo and its ability to be acetylated in vitro. Thus, proteins with a single substitution at their active site, CheY57DE and CheY109KR, are not active in vivo and accordingly were not acetylated in vitro; in contrast, the protein CheY13DK is active in vivo and was normally acetylated in vitro. The possibility that CheY acetylation plays a role in bacterial chemotaxis is discussed.
Barak
R
R
Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, Israel.
Welch
M
M
Yanovsky
A
A
Oosawa
K
K
Eisenbach
M
M
D000107:Acetylation; D000249:Adenosine Monophosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D001426:Bacterial Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism* / Q000648:ultrastructure; D029968:Escherichia coli Proteins; D005407:Flagella / Q000187:drug effects / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism* / Q000502:physiology; D000072236:Methyl-Accepting Chemotaxis Proteins; D012486:Salmonella typhimurium / Q000378:metabolism* / Q000648:ultrastructure
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C077389:FliN protein, Bacteria; C075660:Flig protein, Bacteria; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; C000607606:cheY protein, E coli; C017987:acetyl adenylate; C066821:FliM protein, Bacteria; D000249:Adenosine Monophosphate