In vivo analysis of the proteolytic activity, sequestration, and negative dominance of Escherichia coli Lon mutants using the Vibrio fischeri lux regulon

The activity of Escherichia coil Lon protease and its mutant forms was assa yed in vivo taking advantage of the Lon ability to hydrolyze LuxR, an activ ator of the Vibrio fischeri luxICDABE rightward operon. E. coil strains AB1 157 lon(+) and AB1899 lon1 were transformed with pAC16 consisting of vector pACYC184 and the entire lux regulon of V. fischeri. Introduced in AB1899 l on1 (pAC16) cells, recombinant plasmids with the full-length wild-type lonA (pBRlon, pGEX-NAP), lonA with missense mutations (pBRlonS679A, pBRlonK326Q , etc,), and deletion derivatives of the gene (pGEX-AP and pGEX-P) varied i n effect on their luminescence. The bioluminescence and standard methods yi elded similar estimates of the proteolytic activity of mutant Lon proteases . Plasmid pBRlonS679A directed synthesis of Lon lacking proteolytic activit y and significantly decreased luminescence of AB1899 lon1 cells. This was e xplained by the ability of Lon-S679A to bind LuxR (sequestration), This eff ect was not detected with pGEX-AP and pGEX-P, suggesting that substrate bin ding is due to the N domain of Lon. Plasmids pBRK362Q, pGEX-AP, and, to a l esser extent, pGEX-P conferred properties of a lon(-) mutant to AB1157 lon( +) (pAC16) cells, thus displaying negative dominance. This indicated that t he A domain is necessary for the interaction of Lon subunits resulting in a ctive tetramer.