Figure 3.

Substitutions of rare codons of pltR with preferred synonymous codons increase pyoluteorin production and transcription of the pyoluteorin biosynthetic gene pltL. (A) The number of types of codons and the number of total codons modified in each strain. Detailed information on the specific substitutions in pltR for each strain is provided in Table 3. ^§ indicates the substituted codons of these strains include AGA, which occurs six times in pltR of Pf-5. Four AGA codons were replaced with CGC and two were replaced with CGG in the modified pltR genes of LK298, LK364, and LK365. Fold change refers to the concentration of pyoluteorin produced by each strain relative to wild-type Pf-5 as shown in (B). (B) Production of pyoluteorin by wild-type Pf-5 and derivative strains having pltR genes with modifications in specific codons. The antibiotic production (black bars) and the growth (OD₆₀₀) (blue line) of each strain are shown. Values represent the average of at least three replicates and error bars show the standard deviation. Asterisks denote strains that produce levels of pyoluteorin that differ significantly from that of wild-type strain Pf-5 by a student t-test analysis (p < 0.01). (C). Substitution of the AGA rare codon with common synonymous codons of pltR increased the promoter activity of pltL assessed with a pltL::gfp transcriptional fusion. Inset, the location of pltR (open arrow) relative to pltL is shown for reference, but pltR was not included in the construct. The promoter activity of pltL in derivatives of Pf-5 containing the wild-type and the codon-modified pltR genes and the pltL::gfp transcriptional fusion was assessed by measuring GFP fluorescence normalized by growth (OD₆₀₀); Values represent the average of three replicates and error bars show the standard deviation. Asterisks denote strains in which the promoter activity of pltL is significantly higher than in wild-type Pf-5, as determined by a Student's t-test (p < 0.05).