Codon-Optimized Fluorescent Proteins Designed for Expression in Low-GC Gram-Positive Bacteria

Fluorescent proteins have wide applications in biology. However, not all of these proteins are properly expressed in bacteria, especially if the codon usage and genomic GC content of the host organism are not ideal for high expression. In this study, we analyzed the DNA sequences of multiple fluorescent protein genes with respect to codons and GC content and compared them to a low-GC gram-positive bacterium, Bacillus anthracis. We found high discrepancies for cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and the photoactivatable green fluorescent protein (PAGFP), but not GFP, with regard to GC content and codon usage. Concomitantly, when the proteins were expressed in B. anthracis, CFP- and YFP-derived fluorescence was undetectable microscopically, a phenomenon caused not by lack of gene transcription or degradation of the proteins but by lack of protein expression. To improve expression in bacteria with low genomic GC contents, we synthesized a codon-optimized gfp and constructed optimized photoactivatable pagfp, cfp, and yfp, which were in contrast to nonoptimized genes highly expressed in B. anthracis and in another low-GC gram-positive bacterium, Staphylococcus aureus. Using optimized GFP as a reporter, we were able to monitor the activity of the protective antigen promoter of B. anthracis and confirm its dependence on bicarbonate and regulators present on virulence plasmid pXO1.