A genetic analysis of the functions of LuxN: a two-component hybrid sensorkinase that regulates quorum sensing in Vibrio harveyi

The bioluminescent marine bacterium Vibrio harveyi controls light productio n using two parallel quorum-sensing systems. V. harveyi produces two autoin ducers (Al-l and AI-2), which are recognized by cognate membrane-bound two- component hybrid sensor kinases called LuxN and LuxQ respectively, Under co nditions of low cell density, in the absence of autoinducer, the hybrid sen sors are kinases, and under conditions of high cell density, in the presenc e of autoinducer, the sensors are phosphatases, These activities allow LuxN and LuxQ to modulate the level of phosphorylation of the response regulato r protein LuxO, LuxO, in turn, controls the transcription of the genes enco ding luciferase, The phosphorelay protein LuxU is required for signalling t o LuxO, In this report, we present a genetic analysis of the activities of the AI-l sensor LuxN. Point mutations and in frame deletions were construct ed in luxN and recombined onto the chromosome of V. harveyi for in vivo phe notypic analysis, We show that the conserved histidine (H471) in the sensor kinase domain of LuxN is required for kinase activity but not for phosphat ase activity, In contrast, the conserved aspartate (D771) in the response r egulator domain of LuxN is required for both activities. Furthermore, the L uxN phosphatase activity is localized to the response regulator domain, Our results indicate that the LuxN kinase activity is regulated by the presenc e of Al-l, whereas the LuxN phosphatase activity is constitutive, We also s how that signalling from the two V. harveyi quorum-sensing systems is not e quivalent. Al-l and LuxN have a much greater effect on the level of LuxO ph osphate and therefore Lux expression than do AI-2 and LuxQ.