Use of regulated cell lysis in a lethal genetic selection in Escherichia coli: identification of the autoinducer-binding region of the LuxR protein from Vibrio fischeri ATCC 7744.
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abstract
A lethal genetic selection utilizing the bacteriophage lambda lysis genes (S, R, RZ) has been developed and used in conjunction with a luminescence screen to allow the isolation and characterization of six missense mutations and two nonsense mutations in the luxR gene from Vibrio fischeri ATCC 7744. A transcriptional fusion of the lysis genes in operonR downstream of a truncated luxI gene allows control of cell lysis by the addition of synthetic autoinducer to the growth medium. The six missense mutations isolated resulted in changes in the LuxR protein of Asp at position 79 to Asn (hereafter designated as D79N), V82I, V109L, L118F, S123I, and H217Y. Variant LuxR proteins with amino acid changes of D79N, V82I, V82L, and H127Y were shown to require higher concentrations of autoinducer to elicit a certain amplitude response than is required by the wild-type protein. We believe that the clustering of a total of seven randomly generated missense mutations in a 49-amino-acid region of the LuxR primary sequence defines a critical portion of the LuxR protein. The observation that proteins with lesions in this region responded to elevated levels of autoinducer suggests that the autoinducer-binding site is constructed, at least in part, from several amino acid residues within the 79-to-127 region of the LuxR protein.
