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Title: The rare codon AGA is involved in regulation of pyoluteorin biosynthesis in Pseudomonas protegens Pf-5

Author
item YAN, QING - Oregon State University
item PHILMUS, BENJAMIN - Oregon State University
item Hesse, Cedar
item KOHEN, MAX - Oregon State University
item CHANG, JEFF - Oregon State University
item Loper, Joyce

Submitted to: Frontiers in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/21/2016
Publication Date: 4/19/2016
Citation: Yan, Q., Philmus, B., Hesse, C.N., Kohen, M., Chang, J.H., Loper, J.E. 2016. The rare codon AGA is involved in regulation of pyoluteorin biosynthesis in Pseudomonas protegens Pf-5. Frontiers in Microbiology. 7:497. doi: 10.3389/fmicb.2016.00497.

Interpretive Summary: Biological control provides a promising strategy for managing plant diseases, but has not yet been utilized widely in agriculture due, in part, to unexplained variation in its success in managing disease. Our research goals are to identify sources of variation in biological control, and devise ways to make it more reliable. We focus on Pseudomonas protegens Pf-5, a bacterial strain that occurs naturally in the soil and on plant roots and produces many antibiotics that are toxic to plant pathogenic fungi, oomycetes and bacteria. In this study, we evaluated the role of codon usage in the regulation of pyoluteorin, an antibiotic toxic to oomycetes. We show that the codon AGA, which is the rarest codon in the Pf-5 genome, is prevalent in the pyoluteorin biosynthesis gene cluster of Pf-5 including the regulatory gene pltR. Substituting AGA with a synonymous preferred codon in pltR increases pyoluteorin production and the expression of pyoluteorin biosynthesis genes. These results add to the information about factors influencing the production of an antibiotic that is required for the biological control of certain plant diseases by Pseudomonas spp.

Technical 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. This capacity to suppress disease is attributed in part 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 atypical trinucleotides are overrepresented in several secondary metabolite gene clusters. For example, pltR, which encodes a transcriptional regulator in the LysR family that controls the expression of pltL and other pyoluteorin biosynthetic genes, has many rare codons. To test the possibility that the rare codons in pltR influence pyoluteorin production, we synthesized a variant of pltR in which 23 rare codons were substituted with synonymous preferred codons. This pltR variant was introduced into Pf-5 through allelic exchange and the resulting strain, LK298, produced a higher concentration of pyoluteorin than the wild-type. Accordingly, the expression of pltL, assessed by using a pltL::gfp transcriptional fusion, was 20 times higher in LK298 than in wild-type Pf-5. To pinpoint the specific rare codon(s) important in the pltR-mediated regulation of pyoluteorin production, we created another four Pf-5 derivatives, each containing a variant of pltR having five to six substitutions of preferred codons for synonymous rare codons. Two resulting derivatives showed higher pyoluteorin production and pltL::gfp transcription than the wild-type. Among the altered codons, AGA drew our interest because it is the rarest codon in the Pf-5 genome. The AGA rare codon of pltR was substituted with a synonymous preferred codon and the resulting strain LK365 overproduced pyoluteorin and overexpressed pltL relative to wild-type Pf-5. Our results indicate that the rare codon AGA of pltR is involved in the regulation of pyoluteorin production in Pf-5.