Effects of codon bias on heterologous expression of fluorescent proteins in Xylella fastidiosa
The xylem pathogenic bacterium Xylella fastidiosa is characterized by slow growth, a small genome, and low codon usage bias in protein-coding genes. In most organisms, genes have bias in codon usage that reflects availability of cognate tRNAs charged with amino acids. Codon usage mismatch between a genome and a heterologous gene can reduce protein expression. Although a constitutive GFP-expressing X. fastidiosa strain was created by expressing the gfp gene from a strong ribosomal promoter, previous attempts to create moderately-expressed fluorescent biosensors in X. fastidiosa have been unsuccessful. Therefore, we hypothesize that a codon-harmonized fluorescent protein expresses better in X. fastidiosa. To test this hypothesis, we adapted the fluorescent protein mNeonGreen. After normalization to X. fastidiosa codon usage the gene was synthesized and the codon-harmonized and wildtype mNeonGreen alleles were fused to the hxfA promoter and transformed into X. fastidiosa Temecula1 to generate biosensors that are specific for the X. fastidiosa quorum sensing signal diffusible signal factor (DSF). The effect of codon normalization on protein expression and fluorescence output in bacteria will be discussed.