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

Abstract

Summary 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.

Functional Cross-Talk of MbtH-Like Proteins During Thaxtomin Biosynthesis in the Potato Common Scab Pathogen Streptomyces scabiei

“…Liquid cultures were grown with shaking (200-250 rpm) in Luria-Bertani (LB) Lennox medium (Fisher Scientific, Ottawa, ON, Canada), low salt LB broth (1% w/v tryptone; 0.5% w/v yeast extract; and 0.25% w/v NaCl), super optimal broth (SOB) or super optimal broth with catabolite repression (SOC) medium (New England Biolabs, Whitby, ON, Canada), while solid cultures were grown on LB Lennox (or low salt LB) medium containing 1.5% w/v agar (NEOGEN, Michigan, United States). When required, the solid or liquid growth media were supplemented with antibiotics as described before (Li et al, 2019a). E. coli strains were maintained at 4 • C for short-term storage or at −80 • C in 20% v/v glycerol for long-term storage (Sambrook and Russell, 2001).…”

“…Liquid cultures were typically grown with shaking (200 rpm) in trypticase soy broth (TSB; BD Biosciences, Mississauga, ON, Canada) medium with stainless steel springs. S. scabiei cultures for analysis of thaxtomin production were prepared by inoculating oat bran broth containing 0.35% w/v cellobiose (OBBC) with TSB seed cultures of each strain and then incubating at 25 • C for 7 days as described before (Li et al, 2019a). Plate cultures were grown on potato mash agar (PMA; Fyans et al, 2016), International Streptomyces Project Medium 4 (ISP-4; BD Biosciences), nutrient agar (BD Biosciences, 1.5% w/v agar), and soy flour mannitol agar (SFMA; Kieser et al, 2000).…”

“…Construction of the expression plasmids pACYCDuet-1/HIS 6 -txtA A , pACYCDuet-1/HIS 6 -txtB A , and pET28b/HIS 6 -txtH was described in Li et al (2019a). The MLP-encoding genes CGL27_RS10110 and CGL27_RS02360 from Streptomyces sp.…”

“…Thaxtomins were extracted from S. scabiei OBBC cultures and were detected by reverse phase HPLC as described before (Li et al, 2019a). Briefly, each strain was cultured in triplicate, and in the case of the MLP overexpression strains, two different isolates per strain were cultured in triplicate for a total of six cultures.…”

“…The arrangement of the core domains is unusual when compared to most other NRPSs, which typically have a C-A-PCP domain arrangement (Süssmuth and Mainz, 2017). Immediately downstream of txtB is the txtH gene, which encodes an MLP that is required for the soluble expression of the TxtA and TxtB A-domains (referred to herein as TxtA A and TxtB A ) in E. coli, suggesting that it exhibits a chaperone function in S. scabiei (Li et al, 2019a). Deletion of txtH in S. scabiei significantly reduced thaxtomin A production levels, though some production could still occur.…”