TxtH is a key component of the thaxtomin biosynthetic machinery in the potato common scab pathogen Streptomyces scabies

Abstract

Streptomyces scabies causes potato common scab disease, which reduces the quality and market value of affected tubers. The predominant pathogenicity determinant produced by S. scabies is the thaxtomin A phytotoxin, which is essential for common scab disease development. Production of thaxtomin A involves the nonribosomal peptide synthetases (NRPSs) TxtA and TxtB, both of which contain an adenylation (A-) domain for selecting and activating the appropriate amino acid during thaxtomin biosynthesis. The genome of S. scabies 87.22 contains three small MbtH-like protein (MLP)-coding genes, one of which (txtH) is present in the thaxtomin biosynthesis gene cluster. MLP family members are typically required for the proper folding of NRPS A-domains and/or stimulating their activities. This study investigated the importance of TxtH during thaxtomin biosynthesis in S. scabies. Biochemical studies showed that TxtH is required for promoting the soluble expression of both the TxtA and TxtB A-domains in Escherichia coli, and amino acid residues essential for this activity were identified. Deletion of txtH in S. scabies significantly reduced thaxtomin A production, and deletion of one of the two additional MLP homologues in S. scabies completely abolished production. Engineered expression of all three S. scabies MLPs could restore thaxtomin A production in a triple MLP-deficient strain, while engineered expression of MLPs from other Streptomyces spp. could not. Furthermore, the constructed MLP mutants were reduced in virulence compared to wild-type S. scabies. The results of our study confirm that TxtH plays a key role in thaxtomin A biosynthesis and plant pathogenicity in S. scabies.

Keywords: Streptomyces scabies; MbtH-like protein; adenylation domain; nonribosomal peptide synthetases; potato common scab; thaxtomin A.

Figure 1

(A) Organization of the thaxtomin biosynthetic gene cluster in Streptomyces scabies 87.22. The block arrows represent the genes within the cluster, and the direction of each arrow indicates the direction of transcription. Biosynthetic genes txtA, txtB, txtC, txtD and txtE are represented in black, the regulatory gene txtR is grey, and the MbtH‐like protein (MLP)‐encoding txtH gene is orange. (B) The proposed biosynthetic pathway of thaxtomin A in S. scabies 87.22.

Figure 2

(A) Amino acid alignment of TxtH from Streptomyces scabies with other MbtH‐like protein (MLP) homologues. Highly conserved amino acids are highlighted as follows: black, 100% identity; dark grey, 80–99% identity; grey, 60–79% identity; light grey, <60% identity. The MLP signature sequence is indicated by the black line above the alignment, and the conserved residues subjected to mutation in the S. scabies TxtH are indicated with the asterisks. (B) Phylogenetic analysis of the MLP homologues. The tree was constructed using the maximum likelihood algorithm, and bootstrap values >50% for 1000 repetitions are shown. The scale bar indicates the number of amino acid substitutions per site. The S. scabies TxtH is highlighted in red, while the other MLPs encoded in the S. scabies genome are shown in blue. The non‐cognate MLPs used for the complementation experiments are indicated with the black asterisks. Ssc, Streptomyces scabies; Sac, Streptomyces acidiscabies; Stu, Streptomyces turgidiscabies; Seu, Streptomyces europaeiscabiei.

Figure 3

(A) Western blot analysis of soluble His₆‐TxtA‐A and His_6‐TxtB‐A expressed in the presence and absence of His‐tagged and untagged TxtH. Lanes: 1, His₆‐TxtB‐A co‐expressed with His₆‐TxtH; 2, His₆‐TxtB‐A expressed without His₆‐TxtH; 3, His₆‐TxtA‐A co‐expressed with His₆‐TxtH; 4, His₆‐TxtA‐A expressed without His₆‐TxtH; 5, His₆‐TxtB‐A co‐expressed with TxtH; 6, His₆‐TxtA‐A co‐expressed with TxtH; 7, His₆‐TxtB‐A expressed without TxtH; 8, His₆‐TxtA‐A expressed without TxtH. (B) Western blot analysis of soluble His₆‐TxtA‐A and His_6‐TxtB‐A that was co‐expressed with wild‐type and mutant His₆‐TxtH proteins. The lanes corresponding to the different His₆‐TxtH point mutants are indicated, and lanes containing A‐domain produced in the presence (+) or absence (–) of wild‐type His₆‐TxtH are also shown. (C) Western blot analysis of soluble His₆‐TxtA‐A and His_6‐TxtB‐A expressed in the absence of an MbtH‐like protein (MLP) (lanes 4 and 8) or co‐expressed with His₆‐TxtH (lanes 3 and 7), His₆‐MLP_lipo (lanes 2 and 6) or His₆‐MLP_scab (lanes 1 and 5).

Figure 4

Production of thaxtomin A by Streptomyces scabies strains. Shown are the mean thaxtomin A production levels (ng thaxtomin A/mg dry cell weight) from triplicate cultures of each strain, with error bars representing the standard deviation from the mean. Means with different letters (a, b, c, d) were determined to be significantly different (P ≤ 0.05). (A) Thaxtomin A production levels in S. scabies 87.22 and in the ΔtxtH mutant isolates 1–4. (B) HPLC chromatograms of culture extracts from S. scabies 87.22 (i), ΔtxtH1 (ii), Δmlp_lipo/ΔtxtH (iii), ΔtxtH/Δmlp_scab (iv) and Δmlp_lipo/ΔtxtH/Δmlp_scab (v). The peak corresponding to thaxtomin A in each chromatogram is indicated with the red asterisks, and the peaks corresponding to the thaxtomin B and thaxtomin D intermediates are indicated with ▼ and ∇, respectively. (C) Thaxtomin A production levels in the ΔtxtH1 mutant isolate following complementation with the txtH gene. (D) Thaxtomin A production levels in the Δmlp_lipo/ΔtxtH/Δmlp_scab (ΔΔΔ) mutant following complementation with the txtH, mlp_lipo, mlp_scab, cdaX and clav_p1293 genes. VC, vector control.

Figure 5

RT‐PCR analysis of gene expression in Streptomyces scabies 87.22 and the ΔtxtH1 mutant. Reverse transcription reactions containing (+) or lacking (–) reverse transcriptase enzyme were used as a template for the PCR, while control (C) reactions contained water in place of the cDNA template. The number of cycles used for each set of gene‐specific primers is indicated. The gyrA gene encoding the DNA gyrase subunit A was included as a loading control.

Figure 6

Potato tuber slice assay for assessing the virulence phenotype of Streptomyces scabies strains. Tuber slices were inoculated with wild‐type and mutant S. scabies strains and were incubated for 10 days. Uninoculated medium (YMS_m) was included as a negative control. The bioassay was performed twice in total and representative results are shown.