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∼P as substrate. The presence of AI-1 the kinase the phosphatase activity of The of AI-1 on the LuxU was and in the presence of AI-1, the kinase activity of LuxN, and the of LuxU∼P were of kinase activity by AI-1 AI-1 with LuxN to activity and the of the AI-1 quorum of Vibrio harveyi is of the LuxN kinase 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∼P as substrate. The presence of AI-1 the kinase the phosphatase activity of The of AI-1 on the LuxU was and in the presence of AI-1, the kinase activity of LuxN, and the of LuxU∼P were of kinase activity by AI-1 AI-1 with LuxN to activity and the of the AI-1 quorum of Vibrio harveyi is of the LuxN kinase Vibrio harveyi is a bacterium and and quorum sensing. harveyi three quorum AI-1, an sensed by LuxN is for AI-2, a sensed by is in is is sensed by and is to for three hybrid sensor is via to the response regulator with the HPt protein LuxU as a response of with the for the the regulator for quorum gene expression is as the for harveyi quorum gene is cell the to and the are a concentration of is is to of the and a the hybrid sensor to high concentration and high cell density of quorum in biochemical of the is The of LuxU was by LuxU a with the to the of and the of is to of the of of in the harveyi to AI-2, of to a is the enzymatic activities characterized enzymatic activities and to LuxN an activity an high ATP turnover AI-1 an on LuxN kinase the LuxN phosphatase activity is by and and were were in and in in a the was with of was by of for of and gene was by the and The was with and with to in is of the of a in to was by a with of a and and the The was with and and the was in The was by and of a for of a of by of with and was of the and was by the with the were with the and was by the The was with and and to was by with the and and in the was to to a of were by of was with and LuxN, with a by an of were and were in were and in and a cell the was by an to and were in and in of was the of were in and with for were and as The LuxU was the by with protein was with the process the was to and was with LuxN in with and in The was by of and The was and the was by the of by on were on were to a for and a of were in to an LuxU was the of and the of LuxN and were by and ATP and were by of LuxU∼P was by of and LuxN in were to and to a was by the of as were by were with the by with to the of the 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were in the phosphatase The phosphatase activity was the and LuxU∼P was was and is for the phosphatase activity of AI-1 on the phosphatase activity of the in the presence of AI-1 were to the in and to the of sensor are with and as the of with of LuxN with the of for the of the in the and in the of the of and of harveyi to the LuxN three enzymatic activities a autophosphorylation and to LuxN to a and of the enzymatic activities of LuxN and the of AI-1 on were in was and in as a full-length protein the in vitro LuxN were with of LuxN was to an unusually and the of LuxN was autophosphorylation was high ATP of the the ATP and LuxN and the and sensor kinase hybrid sensor kinase characterized to The LuxN autophosphorylation a for the quorum in by sensor vitro with the for and in LuxN and in LuxU the of to the and autophosphorylation of LuxN, the is the in the presence of the of LuxU was to is in the of the for sensor regulator The of is to the ATP concentration in in vitro were in vitro for the of a of the ATP LuxU to as a LuxN to of the as a were LuxU to in the presence of the in were of ATP to LuxN and of LuxU was and The of LuxU in a is the of LuxN enzymatic are is the of activity to the of ATP the of of is in with were the and with as and is the were in the presence of the response and the cognate to the response the presence of the the response regulator the of a response regulator to in the are for the of of the unusually high turnover of LuxN kinase the ATP in LuxN a high LuxU∼P phosphatase a phosphatase activity of LuxN with LuxU∼P as activity was with activity of LuxN and the of LuxN in the to the are in LuxN is for the of biochemical are in with the of the harveyi expression in harveyi and harveyi in a density dependent of was in the presence of AI-1 the a phosphatase activity to expression for the in kinase and phosphatase activity was for the hybrid sensor and the the on sensor is of in LuxU∼P were with full-length LuxN and the of LuxN the protein was in for of the in to the enzymatic activities of LuxN in vitro and to the the AI-1 on of the of to the phosphatase activity of LuxN is of AI-1 on the of LuxN the presence of AI-1 the of LuxU∼P in the LuxU is in with the the concentration of LuxU∼P and of of AI-1 in the of LuxU∼P in the presence of AI-1 with the AI-1 concentration in of harveyi the kinase activity of LuxN was as the AI-1 concentration was the of kinase activity was to the as the of LuxU∼P in a LuxU the LuxN activity was by an AI-1 kinase and an AI-1 phosphatase activity of the hybrid sensor kinase The of enzymatic activities are The of autophosphorylation was to the of was LuxN was LuxU∼P of the kinase activity with the the a to to the high of the a the LuxU in the the the of was for the response regulator of the of LuxN, the of is for the a phosphatase activity the of LuxU and the of the response regulator LuxO. the for of gene expression in in harveyi is by sensor kinases, and the activities are for and was the three was LuxN in vitro of the kinase and phosphatase activities is of the enzymatic activities of the in the presence of cognate an of and in the harveyi quorum LuxN to the of sensor are with and as the of with of LuxN with the of for the of the in the and in the of the of and of harveyi to the LuxN three enzymatic activities a autophosphorylation and to LuxN to a and of the enzymatic activities of LuxN and the of AI-1 on were in was and in as a full-length protein the in vitro LuxN were with of LuxN was to an unusually and the of LuxN was autophosphorylation was high ATP of the the ATP and LuxN and the and sensor kinase hybrid sensor kinase characterized to The LuxN autophosphorylation a for the quorum in by sensor vitro with the for and in LuxN and in LuxU the of to the and autophosphorylation of LuxN, the is the in the presence of the of LuxU was to is in the of the for sensor regulator The of is to the ATP concentration in in vitro were in vitro for the of a of the ATP LuxU to as a LuxN to of the as a were LuxU to in the presence of LuxO. the in were of ATP to LuxN and of LuxU was and The of LuxU in a is the of LuxN enzymatic are is the of activity to the of ATP the of of is in with were the and with as and is the were in the presence of the response and the cognate to the response the presence of the the response regulator the of a response regulator to in the are for the of of the unusually high turnover of LuxN kinase the ATP in LuxN a high LuxU∼P phosphatase a phosphatase activity of LuxN with LuxU∼P as activity was with activity of LuxN and the of LuxN in the to the are in LuxN is for the of biochemical are in with the of the harveyi expression in harveyi and harveyi in a density dependent of was in the presence of AI-1 the a phosphatase activity to expression for the in kinase and phosphatase activity was for the hybrid sensor and the the on sensor is of in LuxU∼P The were with full-length LuxN and the of LuxN the protein was in for of the in to the enzymatic activities of LuxN in vitro and to the the AI-1 on of the of to the phosphatase activity of LuxN is of AI-1 on the of LuxN the presence of AI-1 the of LuxU∼P in the LuxU is in with the the concentration of LuxU∼P and of of AI-1 in the of LuxU∼P in the presence of AI-1 with the AI-1 concentration in of harveyi the kinase activity of LuxN was as the AI-1 concentration was the of kinase activity was to the as the of LuxU∼P in a LuxU the LuxN activity was by The an AI-1 kinase and an AI-1 phosphatase activity of the hybrid sensor kinase The of enzymatic activities are The of autophosphorylation was to the of was LuxN was LuxU∼P of the kinase activity with the the a to to the high of the a the LuxU in the the the of was for the response regulator of the of LuxN, the of is for the a phosphatase activity the of LuxU and the of the response regulator LuxO. the for of gene expression in in harveyi is by sensor kinases, and the activities are for and was the three was LuxN in vitro of the kinase and phosphatase activities is of the enzymatic activities of the in the presence of cognate an of and in the harveyi quorum