Characterization of the Arn lipopolysaccharide modification system essential for zeamine resistance unveils its new roles in Dickeya oryzae physiology and virulence.

The zeamines produced by Dickeya oryzae are potent polyamine antibiotics and phytotoxins that are essential for bacterial virulence. We recently showed that the RND efflux pump DesABC in D. oryzae confers partial resistance to zeamines. To fully elucidate the bacterial self-protection mechanisms, in this study we used transposon mutagenesis to identify the genes encoding proteins involved in zeamine resistance in D. oryzae EC1. This led to the identification of a seven-gene operon, arnEC1 , that encodes enzyme homologues associated with lipopolysaccharide modification. Deletion of the arnEC1 genes in strain EC1 compromised its zeamine resistance 8- to 16-fold. Further deletion of the des gene in the arnEC1 mutant background reduced zeamine resistance to a level similar to that of the zeamine-sensitive Escherichia coli DH5α. Intriguingly, the arnEC1 mutants showed varied bacterial virulence on rice, potato, and Chinese cabbage. Further analyses demonstrated that ArnBCATEC1 are involved in maintenance of the bacterial nonmucoid morphotype by repressing the expression of capsular polysaccharide genes and that ArnBEC1 is a bacterial virulence determinant, influencing transcriptional expression of over 650 genes and playing a key role in modulating bacterial motility and virulence. Taken together, these findings decipher a novel zeamine resistance mechanism in D. oryzae and document new roles of the Arn enzymes in modulation of bacterial physiology and virulence.

Gram-negative bacteria resist antimicrobial agents by a DzrR-mediated envelope stress response.

BACKGROUND: Envelope stress responses (ESRs) are critical for adaptive resistance of Gram-negative bacteria to envelope-targeting antimicrobial agents. However, ESRs are poorly defined in a large number of well-known plant and human pathogens. Dickeya oryzae can withstand a high level of self-produced envelope-targeting antimicrobial agents zeamines through a zeamine-stimulated RND efflux pump DesABC. Here, we unraveled the mechanism of D. oryzae response to zeamines and determined the distribution and function of this novel ESR in a variety of important plant and human pathogens. RESULTS: In this study, we documented that a two-component system regulator DzrR of D. oryzae EC1 mediates ESR in the presence of envelope-targeting antimicrobial agents. DzrR was found modulating bacterial response and resistance to zeamines through inducing the expression of RND efflux pump DesABC, which is likely independent on DzrR phosphorylation. In addition, DzrR could also mediate bacterial responses to structurally divergent envelope-targeting antimicrobial agents, including chlorhexidine and chlorpromazine. Significantly, the DzrR-mediated response was independent on the five canonical ESRs. We further presented evidence that the DzrR-mediated response is conserved in the bacterial species of Dickeya, Ralstonia, and Burkholderia, showing that a distantly located DzrR homolog is the previously undetermined regulator of RND-8 efflux pump for chlorhexidine resistance in B. cenocepacia. CONCLUSIONS: Taken together, the findings from this study depict a new widely distributed Gram-negative ESR mechanism and present a valid target and useful clues to combat antimicrobial resistance.

A Substrate-Activated Efflux Pump, DesABC, Confers Zeamine Resistance to Dickeya zeae.

Zeamines are a family of polyamino phytotoxins produced by Dickeya zeae EC1. These phytotoxins are also potent antibiotics against a range of microorganisms. To understand how D. zeae EC1 can protect itself from the antimicrobial activity of zeamines, we tested whether the ABC transporter genes within the zms (zeamine synthesis) gene cluster were related to zeamine resistance. Our results ruled out the possible involvement of these ABC transporters in zeamine resistance and instead unveiled an RND (resistance-nodulation-cell division) efflux pump, DesABC, which plays an important role in zeamine resistance in D. zeae EC1. The desAB genes are located next to the zms gene cluster, but desC is at a distant location in the bacterial genome. Null mutation of the desABC genes in a zeamine-minus derivative of strain EC1 led to about an 8- to 32-fold decrease in zeamine tolerance level. This efflux pump was zeamine specific and appeared to be conserved only in Dickeya species, which may explain the high potency of zeamines against a wide range of bacterial pathogens. Significantly, expression of the desAB genes was abolished by deletion of zmsA, which encodes zeamine biosynthesis but could be induced by exogenous addition of zeamines. The results suggest that sophisticated and coordinated regulatory mechanisms have evolved to govern zeamine production and tolerance. Taken together, these findings documented a novel signaling role of zeamines and the first resistance mechanism against zeamines, which is a family of potent and promising antibiotics against both Gram-positive and Gram-negative bacterial pathogens.IMPORTANCE Zeamines are a family of newly identified phytotoxins and potent antibiotics produced by D. zeae EC1. Unlike most bacterial organisms, which are highly sensitive, D. zeae EC1 is tolerant to zeamines, but the mechanisms involved are unknown. Our study showed, for the first time, that a new RND efflux pump, DesABC, is indispensable for D. zeae EC1 against zeamines. We found that the DesABC efflux pump was zeamine specific and appeared to be conserved only in the Dickeya species, which may explain the high potency of zeamines against a wide range of bacterial pathogens. We also showed that expression of DesABC efflux system genes was induced by zeamines. These findings not only provide an answer to why D. zeae EC1 is much more tolerant to zeamines than other bacterial pathogens but also document a signaling role of zeamines in modulation of gene expression.

A nonribosomal peptide synthase containing a stand-alone condensation domain is essential for phytotoxin zeamine biosynthesis.

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.