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The Rare Codon AGA Is Involved in Regulation of Pyoluteorin Biosynthesis in Pseudomonas protegens Pf-5.

Yan Q, Philmus B, Hesse C, Kohen M, Chang JH, Loper JE - Front Microbiol (2016)

Bottom Line: The resultant mutant produced pyoluteorin at levels 15 times higher than that of the wild-type Pf-5.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[Formula: see text], the cognate tRNA for the AGA codon, significantly increased pyoluteorin production by Pf-5.

View Article: PubMed Central - PubMed

Affiliation: Department of Botany and Plant Pathology, Oregon State University Corvallis, OR, USA.

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[Formula: see text], 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.

No MeSH data available.


Related in: MedlinePlus

Substitution of the AGA rare codon of gfp with a synonymous preferred codon increased GFP activity in Pf-5. (A) Diagram to show the modification of AGA rare codons in plasmid pPROBE'-gfp(tagless). Only part of the plasmid is shown. The black bars in the above diagram above show the five AGA rare codons in the transcriptional fusion prnA::gfp(AGA). The empty bars in the lower diagram show the synonymous codons that were substituted for the AGA codons in prnA::gfp(CGC). (B) The relative GFP activity of Pf-5 containing the AGA codon-modified transcriptional fusion prnA::gfp(CGC), denoted with an asterisk, was significantly higher (student t-test, p < 0.01) than Pf-5 containing the unmodified prnA::gfp(AGA). No obvious growth (OD600) difference was observed between strains (data not shown). Values represent the average of four replicates and error bars show the standard deviation.
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Figure 5: Substitution of the AGA rare codon of gfp with a synonymous preferred codon increased GFP activity in Pf-5. (A) Diagram to show the modification of AGA rare codons in plasmid pPROBE'-gfp(tagless). Only part of the plasmid is shown. The black bars in the above diagram above show the five AGA rare codons in the transcriptional fusion prnA::gfp(AGA). The empty bars in the lower diagram show the synonymous codons that were substituted for the AGA codons in prnA::gfp(CGC). (B) The relative GFP activity of Pf-5 containing the AGA codon-modified transcriptional fusion prnA::gfp(CGC), denoted with an asterisk, was significantly higher (student t-test, p < 0.01) than Pf-5 containing the unmodified prnA::gfp(AGA). No obvious growth (OD600) difference was observed between strains (data not shown). Values represent the average of four replicates and error bars show the standard deviation.

Mentions: To further investigate the importance of the AGA codon in gene expression of P. protegens Pf-5 in a system independent of pyoluteorin production, we compared GFP activity from a native gfp, which has five AGA codons, to that from a modified gfp, in which the AGA codons were substituted with the preferred synonymous codon CGC (usage frequency 36.6, Table 2). Both the wild-type gfp and the AGA-codon modified gfp genes were fused with the promoter of the pyrrolnitrin biosynthesis gene prnA (Figure 5A). The GFP activities expressed from the resultant transcriptional fusions prnA::gfp(CGC) and prnA::gfp(AGA) in wild-type Pf-5 were assayed in 24 h cultures grown in NBGly. As shown in Figure 5B, Pf-5 harboring prnA::gfp(CGC) expressed significantly higher levels of GFP fluorescence than the strain harboring prnA::gfp(AGA), although both transcriptional fusions were expressed from the same promoter. These results indicated that optimizing the AGA rare codon of gfp promoted GFP expression, thereby providing a second line of evidence for the importance of the AGA rare codon in gene expression in P. protegens Pf-5.


The Rare Codon AGA Is Involved in Regulation of Pyoluteorin Biosynthesis in Pseudomonas protegens Pf-5.

Yan Q, Philmus B, Hesse C, Kohen M, Chang JH, Loper JE - Front Microbiol (2016)

Substitution of the AGA rare codon of gfp with a synonymous preferred codon increased GFP activity in Pf-5. (A) Diagram to show the modification of AGA rare codons in plasmid pPROBE'-gfp(tagless). Only part of the plasmid is shown. The black bars in the above diagram above show the five AGA rare codons in the transcriptional fusion prnA::gfp(AGA). The empty bars in the lower diagram show the synonymous codons that were substituted for the AGA codons in prnA::gfp(CGC). (B) The relative GFP activity of Pf-5 containing the AGA codon-modified transcriptional fusion prnA::gfp(CGC), denoted with an asterisk, was significantly higher (student t-test, p < 0.01) than Pf-5 containing the unmodified prnA::gfp(AGA). No obvious growth (OD600) difference was observed between strains (data not shown). Values represent the average of four replicates and error bars show the standard deviation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4836200&req=5

Figure 5: Substitution of the AGA rare codon of gfp with a synonymous preferred codon increased GFP activity in Pf-5. (A) Diagram to show the modification of AGA rare codons in plasmid pPROBE'-gfp(tagless). Only part of the plasmid is shown. The black bars in the above diagram above show the five AGA rare codons in the transcriptional fusion prnA::gfp(AGA). The empty bars in the lower diagram show the synonymous codons that were substituted for the AGA codons in prnA::gfp(CGC). (B) The relative GFP activity of Pf-5 containing the AGA codon-modified transcriptional fusion prnA::gfp(CGC), denoted with an asterisk, was significantly higher (student t-test, p < 0.01) than Pf-5 containing the unmodified prnA::gfp(AGA). No obvious growth (OD600) difference was observed between strains (data not shown). Values represent the average of four replicates and error bars show the standard deviation.
Mentions: To further investigate the importance of the AGA codon in gene expression of P. protegens Pf-5 in a system independent of pyoluteorin production, we compared GFP activity from a native gfp, which has five AGA codons, to that from a modified gfp, in which the AGA codons were substituted with the preferred synonymous codon CGC (usage frequency 36.6, Table 2). Both the wild-type gfp and the AGA-codon modified gfp genes were fused with the promoter of the pyrrolnitrin biosynthesis gene prnA (Figure 5A). The GFP activities expressed from the resultant transcriptional fusions prnA::gfp(CGC) and prnA::gfp(AGA) in wild-type Pf-5 were assayed in 24 h cultures grown in NBGly. As shown in Figure 5B, Pf-5 harboring prnA::gfp(CGC) expressed significantly higher levels of GFP fluorescence than the strain harboring prnA::gfp(AGA), although both transcriptional fusions were expressed from the same promoter. These results indicated that optimizing the AGA rare codon of gfp promoted GFP expression, thereby providing a second line of evidence for the importance of the AGA rare codon in gene expression in P. protegens Pf-5.

Bottom Line: The resultant mutant produced pyoluteorin at levels 15 times higher than that of the wild-type Pf-5.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[Formula: see text], the cognate tRNA for the AGA codon, significantly increased pyoluteorin production by Pf-5.

View Article: PubMed Central - PubMed

Affiliation: Department of Botany and Plant Pathology, Oregon State University Corvallis, OR, USA.

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[Formula: see text], 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.

No MeSH data available.


Related in: MedlinePlus