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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

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).
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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).

Mentions: To test the hypothesis that rare codons play a role in the regulation of antibiotic production, we chose to focus on the pyoluteorin biosynthesis gene (plt) cluster because it has a large number of rare codons (Figure 2) and has been the subject of intensive study by our group (Nowak-Thompson et al., 1999; Brodhagen et al., 2004, 2005; Kidarsa et al., 2011). The plt cluster consists of genes with regulatory, biosynthetic, and transport functions (Figure 2A; Nowak-Thompson et al., 1999). It has been noted that pltR, which encodes a LysR family regulator required for transcriptional activation of pyoluteorin biosynthetic genes, has a higher frequency of rare codons than do the other genes in the plt gene cluster (Figure 2C; Nowak-Thompson et al., 1999). pltR contains all 61 amino acid-encoding codons except CGT, a preferred Arg codon with a usage frequency of 9.2 codons per 1000 codons (Table 3). To determine if the rare codons in pltR influence pyoluteorin production, we made a derivative of Pf-5 having a modified pltR in which 35 different types of rare codons were substituted with preferred synonymous codons, resulting in alterations to 126 of the 343 codons in pltR (Figure 3A). Among the 35 optimized codons, 23 were completely substituted (Table 3). 12 codons were partially substituted (Table 3) because complete codon modification was predicted to result in complex secondary structures of DNA oligonucleotides that were difficult to synthesize. The resultant codon-modified strain, LK298, produced 15 times more pyoluteorin than did the wild-type strain Pf-5 (Figure 3B). The final cell density of the 24 h culture of LK298 was slightly lower than the wild-type (Figure 3B), which is consistent with our previous observation of an inverse relationship between the amount of pyoluteorin produced in a Pf-5 culture and the culture's final cell density (Kidarsa et al., 2011). However, the slight growth difference did not affect the result that codon optimization in pltR promoted pyoluteorin production.


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)

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).
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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).
Mentions: To test the hypothesis that rare codons play a role in the regulation of antibiotic production, we chose to focus on the pyoluteorin biosynthesis gene (plt) cluster because it has a large number of rare codons (Figure 2) and has been the subject of intensive study by our group (Nowak-Thompson et al., 1999; Brodhagen et al., 2004, 2005; Kidarsa et al., 2011). The plt cluster consists of genes with regulatory, biosynthetic, and transport functions (Figure 2A; Nowak-Thompson et al., 1999). It has been noted that pltR, which encodes a LysR family regulator required for transcriptional activation of pyoluteorin biosynthetic genes, has a higher frequency of rare codons than do the other genes in the plt gene cluster (Figure 2C; Nowak-Thompson et al., 1999). pltR contains all 61 amino acid-encoding codons except CGT, a preferred Arg codon with a usage frequency of 9.2 codons per 1000 codons (Table 3). To determine if the rare codons in pltR influence pyoluteorin production, we made a derivative of Pf-5 having a modified pltR in which 35 different types of rare codons were substituted with preferred synonymous codons, resulting in alterations to 126 of the 343 codons in pltR (Figure 3A). Among the 35 optimized codons, 23 were completely substituted (Table 3). 12 codons were partially substituted (Table 3) because complete codon modification was predicted to result in complex secondary structures of DNA oligonucleotides that were difficult to synthesize. The resultant codon-modified strain, LK298, produced 15 times more pyoluteorin than did the wild-type strain Pf-5 (Figure 3B). The final cell density of the 24 h culture of LK298 was slightly lower than the wild-type (Figure 3B), which is consistent with our previous observation of an inverse relationship between the amount of pyoluteorin produced in a Pf-5 culture and the culture's final cell density (Kidarsa et al., 2011). However, the slight growth difference did not affect the result that codon optimization in pltR promoted pyoluteorin production.

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