Mutational analysis of the Vibrio fischeri LuxI polypeptide: critical regions of an autoinducer synthase.
Synthesis of the Vibrio fischeri autoinducer, a signal involved in the cell density-dependent activation of bioluminescence, is directed by the luxI gene product. The LuxI protein catalyzes the synthesis of N-acyl-homoserine lactones from S-adenosylmethionine and acylated-acyl carrier protein. We have gained an appreciation of the LuxI regions and amino acid residues involved in autoinducer synthesis by isolating and analyzing mutations generated by random and site-specific mutagenesis of luxI. By random mutagenesis we isolated 13 different single amino acid substitutions in the LuxI polypeptide. Eleven of these substitutions resulted in no detectable autoinducer synthase activity, while the remaining two amino acid substitutions resulted in reduced but detectable activity. The substitutions that resulted in no detectable autoinducer synthase activity mapped to two small regions of LuxI. In Escherichia coli, wild-type luxI showed dominance over all of the mutations. Because autoinducer synthesis has been proposed to involve formation of a covalent bond between an acyl group and an active-site cysteine, we constructed site-directed mutations that altered each of the three cysteine residues in LuxI. All of the cysteine mutants retained substantial activity as an autoinducer synthase in E. coli. Based on the analysis of random mutations we propose a model in which there are two critical regions of LuxI, at least one of which is an intimate part of an active site, and based on the analysis of site-directed mutations we conclude that an active-site cysteine is not essential for autoinducer synthase activity.
Hanzelka
B L
BL
Department of Microbiology, University of Iowa, Iowa City 52242, USA.
Stevens
A M
AM
Parsek
M R
MR
Crone
T J
TJ
Greenberg
E P
EP
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D003545:Cysteine / Q000378:metabolism; D004790:Enzyme Induction; D004926:Escherichia coli / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D008163:Luminescent Measurements; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000235:genetics; D010957:Plasmids; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D014733:Vibrio / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; C085427:LuxI protein, Bacteria; D010455:Peptides; D014157:Transcription Factors; D003545:Cysteine