In vivo analysis of the proteolytic activity and effects of "sequestering" and negative domination of Escherichia coli lon-mutants using the lux regulon of Vibrio fischeri
Zavil'gel'skiĭ, G.B.; Eroshnikov, G.E.; Manukhov, I.V.; Rasulova, F.S.; Ginodman, L.M.; Mel'nikov, E.E.; Starkova, N.N.; Rotanova, T.V.
Molekuliarnaia Biologiia 33(5): 797-802
1999
ISSN/ISBN: 0026-8984 PMID: 10579184 Document Number: 498036
Bioluminescent method was used for in vivo assessment of the activity of Lon-protease and its mutant forms from Escherichia coli. The method is based on the ability of Lon-protease to decompose protein LuxR, the activator of right operon luxICDABE from Vibrio fisheri. AB1157 lon+ and AB1889 lon1 strain cells were transformed by recombinant plasmid pAC16 containing DNA fragment with complete lux-regulon of V. fisheri in vector pACY184. Close correlation was observed between the proteolytic activity of mutant lon-proteases determined by standard method and of that measured by the bioluminescent method. The effects of presence of various plasmids on the level of bioluminescence were described. The effect of negative predominance was manifested to the greatest extent in strain AB1157 cells with plasmids pBRK362Q and pGEX-AP. Apparently, polypeptide domains A and P are mainly responsible for interaction between Lon-protease subunits forming active tetramer.