Formation of active bacterial luciferase between interspecific subunits in vivo.
Interspecific complementation between luxAs and luxBs from Vibrio harveyi, Vibrio fischeri, Photobacterium leiognathi and Xenorhabdus luminescens was examined in vivo. The individual genes from these species were cloned on different compatible plasmids or amplified by PCR and brought together to yield cis combinations without extraneous DNA. The beta subunits from V. harveyi and X. luminescens form active enzyme only with alpha subunits from one of these species. All other combinations yield active enzymes. The lack of activity of the V. harveyi and X. luminescens beta subunits with the alpha subunits from V. fischeri and P. leiognathi results from a lack of association. This was shown by in vivo competition in which these beta subunits were overproduced in comparison with the beta and alpha of V. fischeri. No reduction in light was found. Overall, the in vivo results parallel those found in vitro using isolated denatured subunits and renaturation by removal of the denaturant.
Almashanu
S
S
Faculty of Biology, Technion, Israel Institute of Technology, Haifa.
Tuby
A
A
Hadar
R
R
Einy
R
R
Kuhn
J
J
D010582:Bacteriophage lambda; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004755:Enterobacteriaceae / Q000201:enzymology / Q000235:genetics; D004926:Escherichia coli; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D008156:Luciferases / Q000096:biosynthesis* / Q000235:genetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D015183:Restriction Mapping; D013045:Species Specificity; D014733:Vibrio / Q000201:enzymology* / Q000235:genetics
D017931:DNA Primers; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D008156:Luciferases