A Nonribosomal Peptide Synthase Containing a Stand-Alone Condensation Domain Is Essential for Phytotoxin Zeamine Biosynthesis

Abstract

Dickeya zeae is the causal agent of rice foot rot and maize stalk rot diseases, which could cause severe economic losses. The pathogen is known to produce two phytotoxins known as zeamine and zeamine II which are also potent antibiotics against both gram-positive and gram-negative bacteria pathogens. Zeamine II is a long-chain aminated polyketide and zeamine shares the same polyketide structure as zeamine II, with an extra valine derivative moiety conjugated to the primary amino group of zeamine II. In this study, we have identified a gene designated as zmsK encoding a putative nonribosomal peptide synthase (NRPS) by screening of the transposon mutants defective in zeamine production. Different from most known NRPS enzymes, which are commonly multidomain proteins, ZmsK contains only a condensation domain. High-performance liquid chromatography and mass spectrometry analyses showed that the ZmsK deletion mutant produced only zeamine II but not zeamine, suggesting that ZmsK catalyzes the amide bond formation by using zeamine II as a substrate to generate zeamine. We also present evidence that a partially conserved catalytic motif within the condensation domain is critical for zeamine production. Furthermore, we show that deletion of zmsK substantially decreased the total antimicrobial activity and virulence of D. zeae. Our findings provide a new insight into the biosynthesis pathway of zeamines and the virulence mechanisms of the bacterial pathogen D. zeae.

A Sfp-type phosphopantetheinyl transferase ZmsO is essential for zeamines production and the virulence of Dickeya zeae

“…The both flanking sequences of Tn5 were amplified and sequenced, and the ORF of zmsO was determined following the methods described previously (Cheng et al 2013;Liu and Chen 2007).…”

“…Since zeamines will inhibit the E. coli growth by formation an inhibition zone on bioassay plate (Zhou et al 2011;Cheng et al 2013;Liao et al 2014;Masschelein et al 2013), we can use the same bioassay plate to compare the antimicrobial activity between different mutants by the size of their inhibition zones on plates. To further study the biosynthesis mechanisms of zeamines in D. zeae, a Tn5 insertion mutant library was screened and over 100 candidate mutants with a decreased antimicrobial activity were collected.…”

“…In addition to its broad antibacterial activity (Wu et al 2010), zeamine is also an important virulence factor of D. zeae (Zhou et al 2011;Cheng et al 2013). To test the impact of zmsO deletion on the bacterial virulence, we challenged tobacco leaves and potato tubes with the null mutant of zmsO, wild type strain EC1 and the complementary strain.…”

“…In addition, zeamines are also important phythotoxins which inhibit rice seeds germination, and accelerate disease symptom development in potato and Chinese cabbages (Zhou et al 2011). Disruption of zeamines production significantly decreases the pathogenicity of D. zeae on rice seeds, Chinese cabbage and potato (Zhou et al 2011;Cheng et al 2013). Two genes associated with the biosynthesis of zeamines in D. zeae have been characterized recently with zmsA encoding a polyketide synthase (Zhou et al 2011), and zmsK encoding a nonribosomal peptide synthase (Cheng et al 2013).…”

“…Disruption of zeamines production significantly decreases the pathogenicity of D. zeae on rice seeds, Chinese cabbage and potato (Zhou et al 2011;Cheng et al 2013). Two genes associated with the biosynthesis of zeamines in D. zeae have been characterized recently with zmsA encoding a polyketide synthase (Zhou et al 2011), and zmsK encoding a nonribosomal peptide synthase (Cheng et al 2013). Zeamines have also been identified from Serratia plymuthica RVH1, and a gene cluster containing 18 ORFs has been designated responsible for zeamines production (Masschelein et al 2013).…”