Abstract
The soil bacterium Pseudomonas protegens Pf-5 can colonize root and seed surfaces of many plants, protecting them from infection by plant pathogenic fungi and oomycetes. The capacity to suppress disease is attributed to Pf-5's production of a large spectrum of antibiotics, which is controlled by complex regulatory circuits operating at the transcriptional and post-transcriptional levels. In this study, we analyzed the genomic sequence of Pf-5 for codon usage patterns and observed that the six rarest codons in the genome are present in all seven known antibiotic biosynthesis gene clusters. In particular, there is an abundance of rare codons in pltR, which encodes a member of the LysR transcriptional regulator family that controls the expression of pyoluteorin biosynthetic genes. To test the hypothesis that rare codons in pltR influence pyoluteorin production, we generated a derivative of Pf-5 in which 23 types of rare codons in pltR were substituted with synonymous preferred codons. The resultant mutant produced pyoluteorin at levels 15 times higher than that of the wild-type Pf-5. Accordingly, the promoter activity of the pyoluteorin biosynthetic gene pltL was 20 times higher in the codon-modified stain than in the wild-type. pltR has six AGA codons, which is the rarest codon in the Pf-5 genome. Substitution of all six AGA codons with preferred Arg codons resulted in a variant of pltR that conferred increased pyoluteorin production and pltL promoter activity. Furthermore, overexpression of tRNA, the cognate tRNA for the AGA codon, significantly increased pyoluteorin production by Pf-5. A bias in codon usage has been linked to the regulation of many phenotypes in eukaryotes and prokaryotes but, to our knowledge, this is the first example of the role of a rare codon in the regulation of antibiotic production by a Gram-negative bacterium.
Highlights
Intensive efforts have been made in the past decades to elucidate the regulatory mechanisms of antibiotic biosynthesis because of the important roles of antibiotics in agriculture and medicine
The presence of rare codons in the antibiotic gene clusters of Pf-5 raised the possibility that rare codons may be involved in the regulation of antibiotic production in this bacterium
We have shown that the codon AGA in pltR is involved in pyoluteorin production, so it is reasonable to propose that the abundance of the cognate tRNA deciphering the AGA codon will influence the pltR-mediated regulation of pyoluteorin production
Summary
Intensive efforts have been made in the past decades to elucidate the regulatory mechanisms of antibiotic biosynthesis because of the important roles of antibiotics in agriculture and medicine. UUA, the rarest codon in the genome of the Grampositive bacterium Streptomyces coelicolor, is present in several antibiotic biosynthesis gene clusters and has a well-established role in the regulation of antibiotic production (Chandra and Chater, 2008). A mutation in bldA, which encodes the cognate tRNA for the UUA codon in S. coelicolor (Lawlor et al, 1987), has no obvious effect on vegetative growth but abolishes expression of antibiotic biosynthetic genes and the production of antibiotics such as actinorhodin, undecylprodigiosin, methylenomycin, and a calcium-dependent antibiotic (Guthrie and Chater, 1990). The dependence of antibiotic production on bldA can be relieved by substituting the UUA rare codon with a synonymous codon in genes regulating the biosynthesis of these antibiotics. The role of codon usage in regulation of secondary metabolism in Gram-negative bacteria is virtually unknown
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