Design of the Gaussia luciferase as reporter gene for Chlamydomonas
Bioluminescent proteins are widely used as reporter genes to measure gene expression, determine subcellular protein localization, and study protein–protein interactions. Luciferases are nontoxic, bioluminescent reporter proteins suitable to monitor gene expression quantitatively. Unfortunately, the previously constructed Chlamydomonas-specific Renilla luciferase (Fuhrmann et al. 2004) suffers from low sensitivity, presumably due to low expression levels and/or low protein stability. As in mammalian cells, the luciferase from the marine copepod G. princeps proved to be a much more sensitive reporter than firefly and Renilla luciferases (Tannous et al. 2005); we set out to test this luciferase as a reporter gene in C. reinhardtii.
Previous work had shown that, in Chlamydomonas, adaptation of the codon usage of trangenes significantly improves expression levels (Fuhrmann et al. 1999, 2004). As the codon usage in the native luciferase from G. princeps deviated strongly from that in nuclear genes of C. reinhardtii, we adjusted all codons to the most preferred triplets in Chlamydomonas (according to the codon usage table for Chlamydomonas: http://www.kazusa.org.jp/codon; Fig. 1a). Following this codon optimization in silico, the gene was resynthesized and will be subsequently referred to as G-Luc standing for Gaussia luciferase gene (GenBank accession number EU372000). In all subsequent experiments, G-Luc was compared side-by-side to the Renilla luciferases (Fuhrmann et al. 2004), referred to as R-Luc.
Both luciferase genes were cloned into two different expression cassettes: (1) the PsaD cassette (Fischer and Rochaix 2001), whose promoter is constitutively active at least under photosynthetic conditions and (2) an inducible expression cassette driven by the Hsp70A promoter fused to the 5′ region of the Hsp70B gene (Shao et al. 2007; Fig. 1b). This heat shock gene promoter was shown previously to positively respond to a variety of inducing signals, including heat stress, light, retrograde signals from the plastid, and reactive oxygen species (von Gromoff et al. 1989; Kropat et al. 1997; Kropat and Beck 1998; Schroda et al. 2000; Shao et al. 2007). All gene constructs were introduced into arginine-auxotroph Chlamydomonas cells by glass bead-mediated co-transformation followed by selection for arginine prototrophy.
Expression and heat inducibility of the Gaussia luciferase reporter
To compare the sensitivity of the Chlamydomonas-specific G-Luc as a reporter gene with that of the previously designed R-Luc, 24 transgenic clones from each construct with the constitutive PsaD promoter were randomly chosen and the six best-expressing ones were assayed for their luciferase activities. While only three out of six best R-Luc clones had significant luciferase activity, all six G-Luc clones showed high activity (Fig. 2a). Moreover, when the activities were compared quantitatively, the G-Luc clones displayed, on average, more than 7-fold higher bioluminescence signal intensity than the R-Luc clones (Fig. 2a), indicating that the new G-Luc is more sensitive and more efficient than previously established reporter genes for Chlamydomonas. The very high luciferase activities measured indicate that G-Luc will also be suitable for reporting expression from promoters that are considerably weaker than the PsaD promoter.
Next we wanted to compare the two luciferase genes when expressed under the control of the inducible Hsp70A promoter. To this end, inducibility was determined by measuring luciferase activities in the uninduced and induced states for the four best-expressing co-transformants from each construct (identified among 32 randomly picked clones). Transgene expression was induced by shifting the growth temperature of the algal culture from 23 to 40°C for 1 h. While background expression under non-inducing conditions was comparably low in R-Luc and G-Luc transformants, the G-Luc transformants showed much higher bioluminescence under inducing conditions (on average more than 7-fold; Fig. 2b). This confirms the higher sensitivity of the G-Luc reporter for another expression cassette (Hsp70A promoter + RbcS terminator) and, moreover, indicates that G-Luc can be used as a highly sensitive reporter gene for measuring inducible gene expression in C. reinhardtii.
Luciferase imaging and assessment of protein stability
One of the most powerful applications of luminescent reporter proteins is their use in genetic screens for mutants in cellular signal transduction pathways. This usually requires detection of the reporter gene activity by imaging techniques to facilitate high-throughput screening of mutagenized organisms. Unfortunately, due to the lack of sufficiently sensitive reporters, this has not been possible in Chlamydomonas to date. We, therefore, were interested in testing whether the sensitivity of our new G-Luc reporter gene was sufficiently high to allow visualization of gene expression by luciferase imaging.
To this end, we assayed luciferase activity from both the constitutive and the inducible expression constructs in vivo using a photon-counting camera. Even the best-expressing clone with R-Luc controlled by the PsaD expression cassette (Fig. 2a) did not show enough luminescence to be detectable by luciferase imaging (Fig. 3a). In contrast, G-Luc activity was sufficiently strong to be readily detectable (Fig. 3a). Similar results were obtained for inducible expression from the Hsp70A promoter. While R-Luc activity was barely above the detection limit, inducibility of G-Luc expression was detected with high sensitivity (Fig. 3b).
Different possible explanations can account for the much better performance of G-Luc compared to R-Luc: higher expression rates, higher enzymatic activity or higher stability of the Gaussia enzyme. To distinguish between these possibilities, we performed stability assays by measuring luciferase activities in dependence on the temperature. To this end, algal cultures were subjected to 30 min of high temperature incubation followed by a 30 min recovery phase at room temperature prior to measurement of luciferase activity. If the Gaussia enzyme were indeed more stable than the Renilla enzyme, its activity should decline less sharply with temperature. This was indeed the case (Fig. 4a): while the Renilla luciferase suffered a strong temperature-dependent decline in activity, the Gaussia enzyme was much less affected, suggesting that higher enzyme stability contributes substantially to the superior performance of G-Luc.
To explore the heat inducibility of G-Luc under the control of the Hsp70A promoter in somewhat greater detail, we sought to identify optimum experimental conditions for conducting genetic screens for signaling mutants. We, therefore, tested different combinations of temperatures of the heat shock and recovery times and also included a control construct, in which the heat-shock elements (HSE) were deleted from the Hsp70A promoter (Shao et al. 2007). As expected, this deletion completely abolished heat inducibility under all conditions tested (Fig. 4b and data not shown). Efficient heat induction of the G-Luc reporter was achieved in a wide temperature range, from 40 to 47°C (cp. Figs. 3b, 4b). However, heat shock at higher temperatures required longer recovery times at room temperature before luciferase activity could be visualized by imaging. Whereas following heat shock at 40°C, maximum bioluminescence was measured after 1 h recovery, a recovery phase of 3 h was required to obtain similarly high bioluminescence after a heat shock at 47°C (Fig. 4c).
Next we wanted to confirm that heat induction of luciferase activity parallels G-Luc mRNA accumulation. This was clearly the case upon both induction at 40°C and induction at 47°C (Fig. 4d). At both temperatures, mRNA levels peaked at about the same time as enzyme activities (cp. Fig. 4c, d). Moreover, the kinetics of G-Luc mRNA accumulation correlated, by and large, with heat induction of the endogenous Hsp70A gene (Fig. 4d), ultimately confirming that the luciferase reporter faithfully mirrors promoter activity.
Having established that G-Luc expression can be readily monitored by luminescence imaging, we finally wanted to provide a quantitative assessment of the superior performance of the G-Luc reporter by direct luciferase imaging of primary transformants. To this end, Petri dishes with transformed Chlamydomonas colonies were exposed to the substrate and analyzed by luminescence imaging (Fig. 5). While transformation with G-Luc produced a high number of brightly luminescing colonies, R-Luc luminescence was much lower and barely detectable (Fig. 5). These data ultimately confirm the much higher sensitivity of the G-Luc reporter and its suitability for luminescence imaging.