Phosphonate natural products from host-associated enterobacteria

Polidore, Alexander

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https://hdl.handle.net/2142/120562 Copy

Description

Title

Phosphonate natural products from host-associated enterobacteria

Author(s)

Polidore, Alexander

Issue Date

2023-04-27

Director of Research (if dissertation) or Advisor (if thesis)

Metcalf, William W

Doctoral Committee Chair(s)

Metcalf, William W

Committee Member(s)

• Kuzminov, Andrei
• Mitchell, Douglas
• van der Donk, Wilfred

Department of Study

Microbiology

Discipline

Microbiology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Pantoea
• pantaphos
• natural products
• herbicide
• phosphonate
• biosynthesis
• onion
• center rot
• virulence factor

Abstract

Natural products produced from microorganisms are an invaluable source of therapeutics and pesticides. The renewed interest in bacterial natural products has revealed the potential for natural product discovery among symbiotic microorganisms. Proteobacteria have an incredible potential for production of bioactive natural products including phosphonic acids. The phosphonic acids, defined by the presence of one or more stable phosphorus-carbon bond(s), have a proven track record applications in medicine and agriculture. The plant and insect-associated enterobacteria, Pantoea, produce various antimicrobial compounds with a benefit towards plant health. However, the genus of Pantoea also includes several pathogenic species. Of these, P. ananatis has been shown to cause disease in a wide range of essential food crops including maize, rice, onion, pineapple, honeydew, cantaloupe, and wheat. Pathogenicity in onion has been of particular concern as infection by P. ananatis can cause up to 100% crop loss. Recently, a study on pathogenic P. ananatis strains revealed a biosynthetic gene cluster (HiVir) that was required for onion virulence and was predicted to encode the proteins responsible for production of a phosphonate small molecule. The identification and characterization of this novel phosphonate virulence factor produced from the HiVir gene cluster is the focus of Chapter 2. Here, I show that P. ananatis produces at least three phosphonates, two of which were purified and structural characterized. The first was identified as 2-[hydroxy(phosphono)methyl]maleate, designated pantaphos, and the second molecule was 2-(phosphonomethyl)maleate, a likely biosynthetic precursor. Treatment of onions with purified pantaphos resulted in the hallmark lesions of onion center rot. Moreover, pantaphos exhibited phytotoxic effects on mustard seedlings comparable to the widely used herbicides glyphosate and phosphinothricin. The biosynthetic steps for pantaphos production are described in Chapter 3. Here, I show that the HvrDE dehydratase catalyzes the formation of 2-(phosphonomethyl)maleate from the common phosphonate intermediate, 2-phosphonomethylmalate. This forward reaction only occurred when coupled to methylation by the SAM-methyltransferase, HvrF, which generated a 2-(phosphonomethyl)maleate methyl ester intermediate. The methyl ester intermediate is hydroxylated by a flavin-dependent monooxygenase, HvrB, to form a pantaphos methyl ester, which is then hydrolyzed to form pantaphos either non-enzymatically or through the activity of a predicted carboxyl esterase, HvrJ. The non-enzymatic hydrolysis of methyl-pantaphos made bioactivity testing impossible. However, the lack of bioactivity against plants and microorganisms for 2-(phosphonomethyl)maleate suggests the addition of the hydroxyl moiety is required for bioactivity. Investigation into other host-associated enterobacteria may reveal other bioactive phosphonates. To that end, Chapter 4 focuses on the identification and characterization of phosphonate gene clusters from enterobacterial plant and insect pathogens. A unique gene cluster from the maize pathogen, P. ananatis BD442, which lacks the HiVir gene cluster, contains genes related to amino acid metabolism as well as SAM-dependent proteins and an LDH-family oxidoreductase. Induction of the gene cluster produced at least one phosphonate which is predicted to contain a phosphonolactate backbone structure that may exhibit herbicidal activity. I also show that the insect pathogen, Xenorhabdus bovienii, harbors a homologous HiVir operon (XbHvr) except for an additional argininosuccinate-lyase-like protein (HvrX) not found in P. ananatis HiVir. Induction of XbHvr produced at least four different phosphonates, one of which was identified as 2-(phosphonomethyl)maleate and two are probable pantaphos pathway intermediates. However, the third compound has lost the distinct pantaphos vinyl moiety and may be a new insecticidal molecule formed through Michael-type addition chemistry catalyzed by HvrX.

Graduation Semester

2023-05

Type of Resource

Thesis

Copyright and License Information

Copyright 2023 Alexander Polidore

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