AI-1 Influences the Kinase Activity but Not the Phosphatase Activity of LuxN of Vibrio harveyi

Melanie Timmen,Bonnie L Bassler,Kirsten Jung

doi:10.1074/jbc.m604108200

Melanie Timmen, Bonnie L Bassler + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m604108200

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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.

Highlights

Phorelay to the response regulator LuxO with the HPt protein LuxU acting as intermediate (Fig. 1)
Amino acid replacements of residues located in the LuxP::LuxQ interface sensitize V. harveyi to AI-2, implying that binding of AI-2 to LuxP causes a conformational change within LuxQ that is transmitted across the membrane, presumably affecting LuxQ enzymatic activities [9]
AI-1 has an inhibitory effect on LuxN kinase activity, whereas the LuxN phosphatase activity is unaffected by AI-1

Summary

Introduction

Phorelay to the response regulator LuxO with the HPt protein LuxU acting as intermediate (Fig. 1). Phosphorylated LuxO, a ␴54-dependent response regulator, activates transcription of genes encoding four small regulatory RNAs, which together with Hfq, destabilize the transcript for the LuxR protein, the master transcriptional regulator required for quorum sensing gene expression (4 – 6). Genetic studies indicated that LuxO is phosphorylated at low cell density, causing the luciferase operon to be repressed, and the cells are dark. Despite numerous genetic studies of quorum sensing in V. harveyi, biochemical characterization of the corresponding proteins is rare. Amino acid replacements of residues located in the LuxP::LuxQ interface sensitize V. harveyi to AI-2, implying that binding of AI-2 to LuxP causes a conformational change within LuxQ that is transmitted across the membrane, presumably affecting LuxQ enzymatic activities [9]. AI-1 has an inhibitory effect on LuxN kinase activity, whereas the LuxN phosphatase activity is unaffected by AI-1

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Conclusion