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

An easy way to identify microorganisms is to provide them with gene markers that confer a unique phenotype. Several genetic constructions were developed to use eukaryotic luciferase genes for bacterial tagging. The firefly and click beetle luciferase genes, luc and lucOR, respectively, were cloned under constitutive control and regulated control from different transcriptional units driven by P1, lambda P-R, and Ptrc promoters. Comparison of the expression of each gene in Escherichia coli 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-expressing cells. To analyze the behavior of these constructions in other gram-negative bacteria, gene fusions with luc genes were cloned on broad-host-range vectors. Measurements of light emission from Rhizobium meliloti, Agrobacterium tumefaciens, and Pseudomonas putida cells indicated that both luciferases were poorly expressed from P1 in most bacterial hosts. In contrast, the lambda promoter P-R yielded constitutively high levels of luciferase expression in all bacterial species tested. P-R activity was not regulated by temperature when the thermosensitive repressor cI857 was present in the bacterial species tested, except for E. coli. In contrast, the regulated lacl(q)-PtrculcOR fusion expression 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 bacterial species tested. As proof of the utility of these constructions, we were able to identify P. putida colonies with fusions of either luc or lucOR to P-R in a mixed population.