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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

Overexpression of the tRNA encoding gene PFL_3991 increased pyoluteorin production by P. protegens Pf-5. PFL_3991 was expressed from a constitutive promoter in plasmid p6010-Arg. Pyoluteorin production of the wild-type strain containing p6010-Arg, denoted with an asterisk, was significantly higher (student t-test, p < 0.01) than the wild-type containing the empty vector pME6010. Values represent the average of three replicates and error bars show the standard deviation.
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Figure 4: Overexpression of the tRNA encoding gene PFL_3991 increased pyoluteorin production by P. protegens Pf-5. PFL_3991 was expressed from a constitutive promoter in plasmid p6010-Arg. Pyoluteorin production of the wild-type strain containing p6010-Arg, denoted with an asterisk, was significantly higher (student t-test, p < 0.01) than the wild-type containing the empty vector pME6010. Values represent the average of three replicates and error bars show the standard deviation.

Mentions: 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. Of the five genes encoding tRNAArg in Pf-5, PFL_3991 encodes the tRNA deciphering the AGA rare codon. Our repeated efforts to delete PFL_3991 from the chromosome of Pf-5 failed (data not shown), possibly due to an essential role of the encoded tRNA in this bacterium. As an alternative approach, we overexpressed PFL_3991 by cloning it downstream of a constitutive promoter in plasmid pME6010. Overexpression of PFL_3991 significantly increased pyoluteorin production by Pf-5 (Figure 4). These data, in line with the result that optimizing Arg codons in pltR promoted the pyoluteorin production (Figure 3B), indicated that biased Arg codon usage regulates pyoluteorin biosynthesis.


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)

Overexpression of the tRNA encoding gene PFL_3991 increased pyoluteorin production by P. protegens Pf-5. PFL_3991 was expressed from a constitutive promoter in plasmid p6010-Arg. Pyoluteorin production of the wild-type strain containing p6010-Arg, denoted with an asterisk, was significantly higher (student t-test, p < 0.01) than the wild-type containing the empty vector pME6010. Values represent the average of three replicates and error bars show the standard deviation.
© Copyright Policy
Related In: Results  -  Collection

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Figure 4: Overexpression of the tRNA encoding gene PFL_3991 increased pyoluteorin production by P. protegens Pf-5. PFL_3991 was expressed from a constitutive promoter in plasmid p6010-Arg. Pyoluteorin production of the wild-type strain containing p6010-Arg, denoted with an asterisk, was significantly higher (student t-test, p < 0.01) than the wild-type containing the empty vector pME6010. Values represent the average of three replicates and error bars show the standard deviation.
Mentions: 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. Of the five genes encoding tRNAArg in Pf-5, PFL_3991 encodes the tRNA deciphering the AGA rare codon. Our repeated efforts to delete PFL_3991 from the chromosome of Pf-5 failed (data not shown), possibly due to an essential role of the encoded tRNA in this bacterium. As an alternative approach, we overexpressed PFL_3991 by cloning it downstream of a constitutive promoter in plasmid pME6010. Overexpression of PFL_3991 significantly increased pyoluteorin production by Pf-5 (Figure 4). These data, in line with the result that optimizing Arg codons in pltR promoted the pyoluteorin production (Figure 3B), indicated that biased Arg codon usage regulates pyoluteorin biosynthesis.

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