Expanding fluorescence imaging tools in Staphylococcus aureus: Optimized expression of red fluorescent proteins.

Fluorescent proteins (FPs) are widely used as molecular imaging tools for visualizing protein localization, gene expression, and bacterial labeling. However, most commercially available FPs are optimized for expression in human cells or Escherichia coli, and often exhibit poor expression in other bacterial species due to differences in codon usage preference, which critically affects translation efficiency. Staphylococcus aureus, a clinically important Gram-positive bacterium, presents additional challenges for heterologous protein expression due to codon usage bias and gene regulatory mechanisms. While our previous study has developed expression vectors that enable robust expression of GFP and its color variants in S. aureus, expression of red fluorescent proteins (RFPs) remain limited. In this study, I improved the expression of red fluorescent proteins (RFPs)-mCherry, mOrange2, E2-Crimson-and the photoconvertible protein Dendra2 in S. aureus. Codon optimization was performed based on S. aureus codon usage preferences, and RNA secondary structures at the 5' region were minimized to enhance translation efficiency. The fully optimized mCherry(Sa2) exhibited strong fluorescence at both colony and single-cell levels. Similarly, mOrange2(Sa), E2-Crimson(Sa), and Dendra2(Sa) showed robust expression following host-adapted codon design, and Dendra2(Sa) retained its photoconvertible functionality. These results demonstrate that codon usage adjustment and RNA structure optimization are effective strategies for achieving high-level expression of diverse fluorescent proteins in S. aureus and provide valuable insights into optimizing heterologous protein expression in non-model bacterial systems.