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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

Functional classification of the 917 AGA-containing genes in the genome of Pf-5. The percentage of AGA-containing genes in each JCVI functional role category is shown. Numbers in parentheses indicate the total number of AGA-containing genes in each JCVI functional role category for the Pf-5 genome.
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Figure 6: Functional classification of the 917 AGA-containing genes in the genome of Pf-5. The percentage of AGA-containing genes in each JCVI functional role category is shown. Numbers in parentheses indicate the total number of AGA-containing genes in each JCVI functional role category for the Pf-5 genome.

Mentions: The observation that the AGA rare codon regulates pyoluteorin production prompted us to investigate the distribution of the AGA codon in the genome of Pf-5. Strain Pf-5 has 917 AGA-containing genes, which comprise ca. 15% of the protein-coding genes in the genome (Table 2). These 917 AGA-containing genes fall into all of the JCVI functional role categories (Figure 6, Supplemental file 2), indicating that AGA-containing genes collectively have broad functions in the physiology of the bacterial cell. In the genome of Pf-5, the functional groups “Regulatory functions” and “Transcription” have 91 and 14 AGA-containing genes, respectively. Many genes predicted to encode sigma factors, sigma factor-associated regulators, and other regulatory proteins were among the AGA-containing genes within these two functional groups. Examples include rpoN (sigma-54, σ54) and genes predicted to encode four sigma-54 dependent transcriptional regulators (PFL_1636, 5041, 5309, 5468), five sigma-70 factors (PFL_1373, 2746, 3156, 4041, 5720), and seven GGDEF-containing proteins (PFL_0087, 2458, 3325, 3596, 4322, 4715, 5054). The “Cell envelope” functional group contains 86 AGA-containing genes. This functional group contains large numbers of genes encoding proteins involved in type I or IV pilus formation and polysaccharide synthesis. For example, three AGA-containing genes (PFL_3592, 3593, 3594) encode type I pilus proteins, and six AGA-containing genes (pilCLNOV, PFL_5311) encode type IV pilus proteins. In addition, at least 14 AGA-containing genes (pslDJC, wbpML, lptC, PFL_0526, 3082, 5099, 5100, 5101, 5102, 5103, 5104) are involved in polysaccharide synthesis. Representatives of the “Cellular processes” functional group include genes encoding catalase KatE and KatG, and the multidrug resistance protein PmpM. Overall, our analyses revealed that the small fraction of genes containing the AGA rare codon participate in diverse functions in P. protegens Pf-5, including but not restricted to antibiotic 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)

Functional classification of the 917 AGA-containing genes in the genome of Pf-5. The percentage of AGA-containing genes in each JCVI functional role category is shown. Numbers in parentheses indicate the total number of AGA-containing genes in each JCVI functional role category for the Pf-5 genome.
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Related In: Results  -  Collection

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Figure 6: Functional classification of the 917 AGA-containing genes in the genome of Pf-5. The percentage of AGA-containing genes in each JCVI functional role category is shown. Numbers in parentheses indicate the total number of AGA-containing genes in each JCVI functional role category for the Pf-5 genome.
Mentions: The observation that the AGA rare codon regulates pyoluteorin production prompted us to investigate the distribution of the AGA codon in the genome of Pf-5. Strain Pf-5 has 917 AGA-containing genes, which comprise ca. 15% of the protein-coding genes in the genome (Table 2). These 917 AGA-containing genes fall into all of the JCVI functional role categories (Figure 6, Supplemental file 2), indicating that AGA-containing genes collectively have broad functions in the physiology of the bacterial cell. In the genome of Pf-5, the functional groups “Regulatory functions” and “Transcription” have 91 and 14 AGA-containing genes, respectively. Many genes predicted to encode sigma factors, sigma factor-associated regulators, and other regulatory proteins were among the AGA-containing genes within these two functional groups. Examples include rpoN (sigma-54, σ54) and genes predicted to encode four sigma-54 dependent transcriptional regulators (PFL_1636, 5041, 5309, 5468), five sigma-70 factors (PFL_1373, 2746, 3156, 4041, 5720), and seven GGDEF-containing proteins (PFL_0087, 2458, 3325, 3596, 4322, 4715, 5054). The “Cell envelope” functional group contains 86 AGA-containing genes. This functional group contains large numbers of genes encoding proteins involved in type I or IV pilus formation and polysaccharide synthesis. For example, three AGA-containing genes (PFL_3592, 3593, 3594) encode type I pilus proteins, and six AGA-containing genes (pilCLNOV, PFL_5311) encode type IV pilus proteins. In addition, at least 14 AGA-containing genes (pslDJC, wbpML, lptC, PFL_0526, 3082, 5099, 5100, 5101, 5102, 5103, 5104) are involved in polysaccharide synthesis. Representatives of the “Cellular processes” functional group include genes encoding catalase KatE and KatG, and the multidrug resistance protein PmpM. Overall, our analyses revealed that the small fraction of genes containing the AGA rare codon participate in diverse functions in P. protegens Pf-5, including but not restricted to antibiotic 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