Expression Vectors Using Eukaryotic Luciferases as Bacterial Markers

The eukaryotic nature and presumed absence of firefly and click beetle luciferase genes may provide a unique genotype to bacteria and the variation in wavelength of emitted light provides an additional advantage. To exploit this difference in luciferase assays, luc and lucOR genes, which encode emission of light at 560 nm (yellow-green) and 595 nm (orange), respectively, have been used to develop marker genes for bacteria employing three transcriptional units (Fig. 5.3). The first, an antitetracycline PI promoter, provides considerable constitutive expression when P1::luc and P1::lucOR are borne on ColEl plasmids, but luciferase activity decreases when both fusions are borne on a RK2 de*ivative plasmid. This reduction is due to the presence of a tetR gene in the parental plasmid pRK293 or to differences in copy number. A second system, cI857-XPR, provides a highly regulated promoter which is useful for marking cells for environmental release, as it may be silenced in the natural environment, avoiding the use of nutrients or energy in marker synthesis. In the presence of the cI857 repressor gene, 'kPR is a well-regulated promoter,29 as found when the PI::luc fusion is expressed in E. coli in either ColEl or RK2 replicons. Detection of marked organisms requires induction of the marker gene, allowing identification on solid media or in enrichment broth. In the presence of the cI857 gene, 'kPR is not efficiently regulated in some gram-negative bacteria but constitutive expression of either luc or lucOR from 'kPR can be achieved efficiently in a range of bacteria.30 'kPR expresses high levels of luciferase activity, which are easily detected constitutively. The third system involves a strong promoter, Ptrc, which provides higher and better-regulated luminescence in bacteria that express lucOR and leads to greater in vivo luciferase activity in E. coli and other gram-negative bacteria. Ptrc is effective in a range of bacterial hosts and, following induction, yields

Fig. 5.3. Plasmids constructed to study the expression of eukaryotic luciferases under the control of different promoters in gram-negative bacteria. All of the plasmids were based on the RK2 derivative pRK293. Fragments with the represented fusions of luc and lucOR were cloned in the sites of pRK293 indicated. B, BamHI; Bg, BglII; C, ClaI; H, HindlU S, Salí; X, Xhol. Reprinted with permission from Cebolla A, Vázquez ME, Palomares AJ. Appl Environ Microbiol 1995; 61:660-668.

Fig. 5.3. Plasmids constructed to study the expression of eukaryotic luciferases under the control of different promoters in gram-negative bacteria. All of the plasmids were based on the RK2 derivative pRK293. Fragments with the represented fusions of luc and lucOR were cloned in the sites of pRK293 indicated. B, BamHI; Bg, BglII; C, ClaI; H, HindlU S, Salí; X, Xhol. Reprinted with permission from Cebolla A, Vázquez ME, Palomares AJ. Appl Environ Microbiol 1995; 61:660-668.

higher levels of luminescence than other constructs.31 Use of kPR::luc and XPR::lucOR enables distinction between Pseudomonas putida colonies emitting light of different wavelengths30 (Fig. 5.4).

5.4. Methodology

Luminescence-marked organisms can be monitored using several techniques. Lux and luc genes may be detected by gene probing, but the major advantages of luminescence marker systems lie in the ability to detect light.

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