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Regulation of coronafacoyl phytotoxin production by the PAS-LuxR family regulator CfaR in the common scab pathogen Streptomyces scabies.

Cheng Z, Bown L, Tahlan K, Bignell DR - PLoS ONE (2015)

Bottom Line: In this study, we show that CfaR activates coronafacoyl phytotoxin production by binding to a single site located immediately upstream of the putative -35 hexanucleotide box within the promoter region for the biosynthetic genes.The binding activity of CfaR was shown to require both the LuxR and PAS domains, the latter of which is involved in protein homodimer formation.Furthermore, we provide evidence that CfaR is a novel member of the PAS-LuxR family of regulators, members of which are widely distributed among actinomycete bacteria.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Memorial University of Newfoundland, St. John's, NL A1B 3X9, Canada.

ABSTRACT
Potato common scab is an economically important crop disease that is characterized by the formation of superficial, raised or pitted lesions on the potato tuber surface. The most widely distributed causative agent of the disease is Streptomyces scabies, which produces the phytotoxic secondary metabolite thaxtomin A that serves as a key virulence factor for the organism. Recently, it was demonstrated that S. scabies can also produce the phytotoxic secondary metabolite coronafacoyl-L-isoleucine (CFA-L-Ile) as well as other related metabolites in minor amounts. The expression of the biosynthetic genes for CFA-L-Ile production is dependent on a PAS-LuxR family transcriptional regulator, CfaR, which is encoded within the phytotoxin biosynthetic gene cluster in S. scabies. In this study, we show that CfaR activates coronafacoyl phytotoxin production by binding to a single site located immediately upstream of the putative -35 hexanucleotide box within the promoter region for the biosynthetic genes. The binding activity of CfaR was shown to require both the LuxR and PAS domains, the latter of which is involved in protein homodimer formation. We also show that CFA-L-Ile production is greatly enhanced in S. scabies by overexpression of both cfaR and a downstream co-transcribed gene, orf1. Our results provide important insight into the regulation of coronafacoyl phytotoxin production, which is thought to contribute to the virulence phenotype of S. scabies. Furthermore, we provide evidence that CfaR is a novel member of the PAS-LuxR family of regulators, members of which are widely distributed among actinomycete bacteria.

No MeSH data available.


Related in: MedlinePlus

CfaRfull-HIS6 binds to a single site within the cfaR—cfa1 intergenic region.(A) Map of the cfaR—cfa1 intergenic region showing the location of the DNA fragments (indicated by the black bars and labeled a—f) used for EMSAs. The position of the 16 bp palindrome identified upstream of cfa1 is indicated with the white triangle. (B) EMSA s for CfaRfull—HIS6 with the DNA fragments a—f. Reactions contained 50 ng of DNA with (result+) and without (-) CfaRfull—HIS6 protein (3.7 pmol). DNA-protein complexes observed are indicated with *. (C) Sequence of the 40 bp oligonucleotide P1 probe used for EMSAs. The 16 bp palindromic sequence identified upstream of cfa1 is shown in bold. (D) EMSA results for CfaRfull—HIS6 with the P1 oligonucleotide probe. Reactions contained 0.1 pmol of biotin-labeled probe with (+) and without (-) CfaRfull—HIS6 protein (2 pmol). Negative control reactions contained the 40 bp biotin-labeled oligonucleotide P2 probe in place of P1. In addition, competition assays were performed in which an excess (10×) of unlabelled (cold) probe (P1 or P2) was included in the reaction. DNA-protein complexes observed are indicated with *.
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pone.0122450.g002: CfaRfull-HIS6 binds to a single site within the cfaR—cfa1 intergenic region.(A) Map of the cfaR—cfa1 intergenic region showing the location of the DNA fragments (indicated by the black bars and labeled a—f) used for EMSAs. The position of the 16 bp palindrome identified upstream of cfa1 is indicated with the white triangle. (B) EMSA s for CfaRfull—HIS6 with the DNA fragments a—f. Reactions contained 50 ng of DNA with (result+) and without (-) CfaRfull—HIS6 protein (3.7 pmol). DNA-protein complexes observed are indicated with *. (C) Sequence of the 40 bp oligonucleotide P1 probe used for EMSAs. The 16 bp palindromic sequence identified upstream of cfa1 is shown in bold. (D) EMSA results for CfaRfull—HIS6 with the P1 oligonucleotide probe. Reactions contained 0.1 pmol of biotin-labeled probe with (+) and without (-) CfaRfull—HIS6 protein (2 pmol). Negative control reactions contained the 40 bp biotin-labeled oligonucleotide P2 probe in place of P1. In addition, competition assays were performed in which an excess (10×) of unlabelled (cold) probe (P1 or P2) was included in the reaction. DNA-protein complexes observed are indicated with *.

Mentions: Previous transcriptional studies showed that CfaR is required for expression of several coronafacoyl phytotoxin biosynthetic genes [19], and overexpression of CfaR has been demonstrated to enhance phytotoxin production [16]. Given that the biosynthetic genes are expressed as a large polycistronic transcript [19], it was hypothesized that CfaR may control gene activation from the promoter region upstream of cfa1, which is the first gene in the operon (Fig 1A). To investigate this further, CfaR was overexpressed and purified from E. coli as a C-terminal 6 × histidine tagged protein (CfaRfull –HIS6), after which it was used in EMSAs along with six DNA fragments covering different parts of the intergenic region between cfaR and cfa1 (Fig 2A). As shown in Fig 2B, the CfaRfull –HIS6 could only bind to two of the DNA fragments (a and e), both of which covered a 264 bp region immediately upstream of the predicted cfa1 start codon (Fig 2A). Within this region, a 16 bp imperfect palindromic DNA sequence was identified manually (positions -94 to -79 relative to the cfa1 translation start codon; Fig 2A and 2C), and the sequence was found to be highly similar to the previously described PimM binding site consensus sequence CTVGGGAWWTCCCBAG (Fig 3) [23, 37]. EMSAs using DNA fragments lacking this palindrome confirmed that it is essential for binding of CfaRfull –HIS6 to DNA (Fig 2B). Furthermore, CfaRfull –HIS6 could readily bind to a 40 bp labeled oligonucleotide probe (P1) containing only the palindrome and some DNA flanking sequence (Fig 2C and 2D) whereas it did not bind to a control 40 bp probe (P2; Fig 2D) corresponding to the cfaR coding region (see Materials and Methods). Finally, binding to the labeled P1 probe was abolished when an excess of unlabeled P1, but not P2, was included in the reaction mixture (Fig 2D), indicating that the interaction between CfaRfull –HIS6 and P1 is highly specific.


Regulation of coronafacoyl phytotoxin production by the PAS-LuxR family regulator CfaR in the common scab pathogen Streptomyces scabies.

Cheng Z, Bown L, Tahlan K, Bignell DR - PLoS ONE (2015)

CfaRfull-HIS6 binds to a single site within the cfaR—cfa1 intergenic region.(A) Map of the cfaR—cfa1 intergenic region showing the location of the DNA fragments (indicated by the black bars and labeled a—f) used for EMSAs. The position of the 16 bp palindrome identified upstream of cfa1 is indicated with the white triangle. (B) EMSA s for CfaRfull—HIS6 with the DNA fragments a—f. Reactions contained 50 ng of DNA with (result+) and without (-) CfaRfull—HIS6 protein (3.7 pmol). DNA-protein complexes observed are indicated with *. (C) Sequence of the 40 bp oligonucleotide P1 probe used for EMSAs. The 16 bp palindromic sequence identified upstream of cfa1 is shown in bold. (D) EMSA results for CfaRfull—HIS6 with the P1 oligonucleotide probe. Reactions contained 0.1 pmol of biotin-labeled probe with (+) and without (-) CfaRfull—HIS6 protein (2 pmol). Negative control reactions contained the 40 bp biotin-labeled oligonucleotide P2 probe in place of P1. In addition, competition assays were performed in which an excess (10×) of unlabelled (cold) probe (P1 or P2) was included in the reaction. DNA-protein complexes observed are indicated with *.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4380410&req=5

pone.0122450.g002: CfaRfull-HIS6 binds to a single site within the cfaR—cfa1 intergenic region.(A) Map of the cfaR—cfa1 intergenic region showing the location of the DNA fragments (indicated by the black bars and labeled a—f) used for EMSAs. The position of the 16 bp palindrome identified upstream of cfa1 is indicated with the white triangle. (B) EMSA s for CfaRfull—HIS6 with the DNA fragments a—f. Reactions contained 50 ng of DNA with (result+) and without (-) CfaRfull—HIS6 protein (3.7 pmol). DNA-protein complexes observed are indicated with *. (C) Sequence of the 40 bp oligonucleotide P1 probe used for EMSAs. The 16 bp palindromic sequence identified upstream of cfa1 is shown in bold. (D) EMSA results for CfaRfull—HIS6 with the P1 oligonucleotide probe. Reactions contained 0.1 pmol of biotin-labeled probe with (+) and without (-) CfaRfull—HIS6 protein (2 pmol). Negative control reactions contained the 40 bp biotin-labeled oligonucleotide P2 probe in place of P1. In addition, competition assays were performed in which an excess (10×) of unlabelled (cold) probe (P1 or P2) was included in the reaction. DNA-protein complexes observed are indicated with *.
Mentions: Previous transcriptional studies showed that CfaR is required for expression of several coronafacoyl phytotoxin biosynthetic genes [19], and overexpression of CfaR has been demonstrated to enhance phytotoxin production [16]. Given that the biosynthetic genes are expressed as a large polycistronic transcript [19], it was hypothesized that CfaR may control gene activation from the promoter region upstream of cfa1, which is the first gene in the operon (Fig 1A). To investigate this further, CfaR was overexpressed and purified from E. coli as a C-terminal 6 × histidine tagged protein (CfaRfull –HIS6), after which it was used in EMSAs along with six DNA fragments covering different parts of the intergenic region between cfaR and cfa1 (Fig 2A). As shown in Fig 2B, the CfaRfull –HIS6 could only bind to two of the DNA fragments (a and e), both of which covered a 264 bp region immediately upstream of the predicted cfa1 start codon (Fig 2A). Within this region, a 16 bp imperfect palindromic DNA sequence was identified manually (positions -94 to -79 relative to the cfa1 translation start codon; Fig 2A and 2C), and the sequence was found to be highly similar to the previously described PimM binding site consensus sequence CTVGGGAWWTCCCBAG (Fig 3) [23, 37]. EMSAs using DNA fragments lacking this palindrome confirmed that it is essential for binding of CfaRfull –HIS6 to DNA (Fig 2B). Furthermore, CfaRfull –HIS6 could readily bind to a 40 bp labeled oligonucleotide probe (P1) containing only the palindrome and some DNA flanking sequence (Fig 2C and 2D) whereas it did not bind to a control 40 bp probe (P2; Fig 2D) corresponding to the cfaR coding region (see Materials and Methods). Finally, binding to the labeled P1 probe was abolished when an excess of unlabeled P1, but not P2, was included in the reaction mixture (Fig 2D), indicating that the interaction between CfaRfull –HIS6 and P1 is highly specific.

Bottom Line: In this study, we show that CfaR activates coronafacoyl phytotoxin production by binding to a single site located immediately upstream of the putative -35 hexanucleotide box within the promoter region for the biosynthetic genes.The binding activity of CfaR was shown to require both the LuxR and PAS domains, the latter of which is involved in protein homodimer formation.Furthermore, we provide evidence that CfaR is a novel member of the PAS-LuxR family of regulators, members of which are widely distributed among actinomycete bacteria.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Memorial University of Newfoundland, St. John's, NL A1B 3X9, Canada.

ABSTRACT
Potato common scab is an economically important crop disease that is characterized by the formation of superficial, raised or pitted lesions on the potato tuber surface. The most widely distributed causative agent of the disease is Streptomyces scabies, which produces the phytotoxic secondary metabolite thaxtomin A that serves as a key virulence factor for the organism. Recently, it was demonstrated that S. scabies can also produce the phytotoxic secondary metabolite coronafacoyl-L-isoleucine (CFA-L-Ile) as well as other related metabolites in minor amounts. The expression of the biosynthetic genes for CFA-L-Ile production is dependent on a PAS-LuxR family transcriptional regulator, CfaR, which is encoded within the phytotoxin biosynthetic gene cluster in S. scabies. In this study, we show that CfaR activates coronafacoyl phytotoxin production by binding to a single site located immediately upstream of the putative -35 hexanucleotide box within the promoter region for the biosynthetic genes. The binding activity of CfaR was shown to require both the LuxR and PAS domains, the latter of which is involved in protein homodimer formation. We also show that CFA-L-Ile production is greatly enhanced in S. scabies by overexpression of both cfaR and a downstream co-transcribed gene, orf1. Our results provide important insight into the regulation of coronafacoyl phytotoxin production, which is thought to contribute to the virulence phenotype of S. scabies. Furthermore, we provide evidence that CfaR is a novel member of the PAS-LuxR family of regulators, members of which are widely distributed among actinomycete bacteria.

No MeSH data available.


Related in: MedlinePlus