A. Cebolla et al., EXPRESSION VECTORS FOR THE USE OF EUKARYOTIC LUCIFERASES AS BACTERIALMARKERS WITH DIFFERENT COLORS OF LUMINESCENCE, Applied and environmental microbiology, 61(2), 1995, pp. 660-668
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.