title: AI-1 influences the kinase activity but not the phosphatase activity of LuxN of Vibrio harveyi.
abstract: The Gram-negative bacterium Vibrio harveyi produces and responds to three autoinducers, AI-1, AI-2, and CAI-1 to regulate cell density dependent gene expression by a process referred to as quorum sensing. The concentration of the autoinducers is sensed by three cognate hybrid sensor kinases, and information is channeled via the HPt protein LuxU to the response regulator LuxO. Here, a detailed biochemical study on the enzymatic activities of the membrane-integrated hybrid sensor kinase LuxN, the sensor for N-(d-3-hydroxybutanoyl)homoserine lactone (AI-1), is provided. LuxN was heterologously overproduced as the full-length protein in Escherichia coli. LuxN activities were characterized in vitro and are an autophosphorylation activity with an unusually high ATP turnover rate, stable LuxU phosphorylation, and a slow phosphatase activity with LuxU approximately P as substrate. The presence of AI-1 affected the kinase but not the phosphatase activity of LuxN. The influence of AI-1 on the LuxN--> LuxU signaling step was monitored, and in the presence of AI-1, the kinase activity of LuxN, and hence the amount of LuxU approximately P produced, were significantly reduced. Half-maximal inhibition of kinase activity by AI-1 occurred at 20 mum. Together, these results indicate that AI-1 directly interacts with LuxN to down-regulate its autokinase activity and suggest that the key regulatory step of the AI-1 quorum sensing system of Vibrio harveyi is AI-1-mediated repression of the LuxN kinase activity.
authors.affiliation: Ludwig-Maximilians-Universität München, Department Biologie I, Bereich Mikrobiologie, Maria-Ward-Strasse 1a, D-80638 München, Germany.
authors.forename: Melanie
authors.initials: M
authors.lastname: Timmen
authors.forename: Bonnie L
authors.initials: BL
authors.lastname: Bassler
authors.forename: Kirsten
authors.initials: K
authors.lastname: Jung
mesh_terms: D015107:4-Butyrolactone / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D015536:Down-Regulation; D004789:Enzyme Activation; D011485:Protein Binding; D011494:Protein Kinases / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D014157:Transcription Factors / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D014733:Vibrio / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D014157:Transcription Factors; C030654:N-(3-oxohexanoyl)-3-aminodihydro-2(3H)-furanone; D011494:Protein Kinases; C084290:luxN protein, Vibrio harveyi; D015107:4-Butyrolactone