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

ABSTRACT: 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, arn_EC1 , that encodes enzyme homologues associated with lipopolysaccharide modification. Deletion of the arn_EC1 genes in strain EC1 compromised its zeamine resistance 8- to 16-fold. Further deletion of the des gene in the arn_EC1 mutant background reduced zeamine resistance to a level similar to that of the zeamine-sensitive Escherichia coli DH5α. Intriguingly, the arn_EC1 mutants showed varied bacterial virulence on rice, potato, and Chinese cabbage. Further analyses demonstrated that ArnBCAT_EC1 are involved in maintenance of the bacterial nonmucoid morphotype by repressing the expression of capsular polysaccharide genes and that ArnB_EC1 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.

SUBMITTER: Liang Z

PROVIDER: S-EPMC10632790 | biostudies-literature | 2023 Dec

REPOSITORIES: biostudies-literature

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

Liang Zhibin Z Huang Luhao L Liu Huidi H Zheng Ying Y Feng Jiani J Shi Zurong Z Chen Yufan Y Lv Mingfa M Zhou Jianuan J Zhang Lian-Hui LH Chen Shaohua S

Molecular plant pathology 20230922 12

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, arn<sub>EC1</ ...[more]

PMID: 37740253

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Project description: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.

Project description:UNLABELLED:Burkholderia cenocepacia causes opportunistic infections in plants, insects, animals, and humans, suggesting that "virulence" depends on the host and its innate susceptibility to infection. We hypothesized that modifications in key bacterial molecules recognized by the innate immune system modulate host responses to B. cenocepacia. Indeed, modification of lipopolysaccharide (LPS) with 4-amino-4-deoxy-L-arabinose and flagellin glycosylation attenuates B. cenocepacia infection in Arabidopsis thaliana and Galleria mellonella insect larvae. However, B. cenocepacia LPS and flagellin triggered rapid bursts of nitric oxide and reactive oxygen species in A. thaliana leading to activation of the PR-1 defense gene. These responses were drastically reduced in plants with fls2 (flagellin FLS2 host receptor kinase), Atnoa1 (nitric oxide-associated protein 1), and dnd1-1 (reduced production of nitric oxide) null mutations. Together, our results indicate that LPS modification and flagellin glycosylation do not affect recognition by plant receptors but are required for bacteria to establish overt infection. IMPORTANCE:Virulence and pathogenicity are properties ascribed to microbes, which actually require careful consideration of the host. Using the term "pathogen" to define a microbe without considering its host has recently been debated, since the microbe's capacity to establish a niche in a given host is a critical feature associated with infection. Opportunistic bacteria are a perfect example of microbes whose ability to cause disease is intimately related to the host's ability to recognize and respond to the infection. Here, we use the opportunistic bacterium Burkholderia cenocepacia and the host plant Arabidopsis thaliana to investigate the role of bacterial surface molecules, namely, lipopolysaccharide and flagellin, in contributing to infection and also in eliciting a host response. We reveal that both molecules can be modified by glycosylation, and although the modifications are critical for the bacteria to establish an infection, they do not impact the host's ability to recognize the pathogen.

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

Publications

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

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