Codon-optimized fluorescent proteins designed for expression in low-GC gram-positive bacteria

Abstract

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.

FIG. 1.

Clustal W sequence alignment of all four codon-optimized FPs. Amino acid changes performed with respect to GFPopt are highlighted in gray.

FIG. 2.

Visualization of FP production in B. anthracis vegetative bacteria and spores by fluorescence microscopy. Strains carrying different FP genes were grown to stationary phase overnight in LB and analyzed by fluorescence microscopy: B. anthracis Ames 33 harboring pSW4-GFPmut1 (A), pSW4-GFPopt (B), pSW4-CFP (D), pSW4-CFPopt (E), pSW4-YFP (G), pSW4-YFPopt (H), or pSW4-PAGFP before (J) or after (K) photoactivation with a 413-nm laser. For spore images, bacteria were grown on low-nutrient sporulating plates for multiple days, heat inactivated in order to kill remaining vegetative bacteria, and analyzed by fluorescence microscopy: B. anthracis Ames 33 spores expressing GFPopt (C), CFPopt (F), or YFPopt (I) or plasmid-free (L).

FIG. 3.

FACS analysis of bacteria expressing original and codon-optimized FPs. B. anthracis Ames 33 carrying pSW4 plasmids with the respective fluorescent genes were grown overnight in LB and subjected to analysis by flow cytometry. (A) Comparison of MFIs of bacteria. All differences were statistically significant (P = 0.0309, GFP versus GFPopt; P < 0.0001, CFP versus CFPopt; P = 0.0005, YFP versus YFPopt). Error bars indicate standard deviations. (B) FACS detection of B. anthracis vegetative cells expressing FPs.

FIG. 4.

(A) RT-PCR analysis of mRNA isolated from B. anthracis vegetative cells expressing FP genes. B. anthracis carrying pSW4 plasmids with FP genes was grown in LB to mid-logarithmic phase, and RNA was extracted as outlined in Materials and Methods. The level of gyrase A (gyrA) mRNA served as an internal control. Transcriptional expression levels of all FPs were normalized and expressed as fold changes with respect to the gyrA control. (B) Western blot analysis of FPs expressed in B. anthracis. Bacteria were grown as described above and lysed, and equal amounts of protein were subjected to SDS-PAGE and Western blotting using GFP-specific polyclonal antibodies.

FIG. 5.

pagA promoter analysis using GFPopt as a reporter. (A) Growth curves (broken lines) and fluorescence intensities (solid lines) of B. anthracis expressing GFPopt under the control of the pagA promoter. GFPopt-harboring bacteria containing virulence plasmid pXO1 (strain A35) or plasmid free (strain A33) were grown in NBY broth supplemented with 10% FBS in the presence or absence of sodium bicarbonate (0.9%, wt/vol) and a 5% CO₂ atmosphere. At different time points, samples were analyzed for fluorescence in a fluorimeter. CPS, counts per second. Error bars indicate standard deviations. (B) Western blotting of bacteria grown to stationary phase. Lysates were processed as described for Fig. 4.

FIG. 6.

Fluorescence microscopic analysis of S. aureus strain RN4220(pTetON) (A) and RN4220 constitutively expressing GFPopt (B), CFPopt (C), or YFPopt (D) from the P_xyl/tetO promoter. Bacteria were grown in tryptic soy broth to stationary phase and microscopically analyzed for fluorescence.