EXPRESSION VECTORS FOR THE USE OF EUKARYOTIC LUCIFERASES AS BACTERIALMARKERS WITH DIFFERENT COLORS OF LUMINESCENCE

An easy way to identify microorganisms is to provide them with gene ma rkers that confer a unique phenotype. Several genetic constructions we re developed to use eukaryotic luciferase genes for bacterial tagging. The firefly and click beetle luciferase genes, luc and lucOR, respect ively, were cloned under constitutive control and regulated control fr om different transcriptional units driven by P1, lambda P-R, and Ptrc promoters. Comparison of the expression of each gene in Escherichia co li cells from identical promoters showed that bioluminescence produced by luc could be detected luminometrically in a more sensitive manner. In contrast, luminescence from intact lucOR-expressing cells was much more stable and resistant to high temperatures than that from luc-exp ressing cells. To analyze the behavior of these constructions in other gram-negative bacteria, gene fusions with luc genes were cloned on br oad-host-range vectors. Measurements of light emission from Rhizobium meliloti, Agrobacterium tumefaciens, and Pseudomonas putida cells indi cated that both luciferases were poorly expressed from P1 in most bact erial hosts. In contrast, the lambda promoter P-R yielded constitutive ly high levels of luciferase expression in all bacterial species teste d. P-R activity was not regulated by temperature when the thermosensit ive repressor cI857 was present in the bacterial species tested, excep t for E. coli. In contrast, the regulated lacl(q)-PtrculcOR fusion exp ression system behaved in a manner similar to that observed in E. coli cells. After IPTG (isopropyl-beta-D-thiogalactopyranoside) induction, this system produced the highest levels of lucOR expression in all ba cterial species tested. As proof of the utility of these constructions , we were able to identify P. putida colonies with fusions of either l uc or lucOR to P-R in a mixed population.