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Title: FDB2 encodes a member of the arylamine N-acetyltransferase family and is necessary for biotransformation of benzoxazolinones by Fusarium verticillioides

item Glenn, Anthony - Tony
item Bacon, Charles

Submitted to: Journal of Applied Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/5/2009
Publication Date: 7/10/2009
Citation: Glenn, A.E., Bacon, C.W. 2009. FDB2 encodes a member of the arylamine N-acetyltransferase family and is necessary for biotransformation of benzoxazolinones by Fusarium verticillioides. Journal of Applied Microbiology. 107:657-671.

Interpretive Summary: Corn plants produce chemical compounds within the plant cells that act as insect feeding deterrents and antimicrobials against plant pathogens (bacteria and fungi). These compounds are commonly referred to as DIMBOA, DIBOA, MBOA, and BOA. Despite the presence of these compounds, essentially all corn grown throughout the world is infected with the fungus Fusarium verticillioides. Infection of corn plants by this fungus is of concern because it produces a class of mycotoxins called fumonisins that are detrimental to the health of animals and potentially humans. Fumonisins can accumulate and contaminate corn and corn products. Thus, it is important that we know more about how this fungus infects and colonizes corn plants and how it responds to the plant’s defensive responses. Concurrent with this, we need to know more about how F. verticillioides persists and dominates the fungal community in cornfield environments. Prior research showed F. verticillioides has the genetic and physiological capacity to detoxify, or biotransform, the plant’s antimicrobials into non-toxic compounds. In this study we expand upon the previous work and identify a critical gene that is necessary for the transformation of 2-aminophenol (an intermediate metabolite in the biotransformation pathway) into the non-toxic metabolite N-(2-hydroxyphenyl)malonamic acid. This gene (FDB2) encodes a member of the arylamine N-acetyltransferase (NAT) family of enzymes. Such NAT enzymes prototypically catalyze the transfer of an acetyl group from acetyl-CoA to the terminal nitrogen of various aromatic and heterocyclic amines, thus inactivating such drugs and harmful natural compounds. We postulate that the F. verticillioides NAT utilizes malonyl-CoA instead of acetyl-CoA to modify such amines. The significance of this modification pathway is discussed relative to ecological fitness and effects on competitive interactions between F. verticillioides and other fungi and bacteria. The impact of biotransformation also is discussed in relation to efficacy of biocontrol applications toward F. verticillioides.

Technical Abstract: Maize produces the benzoxazinones DIMBOA and DIBOA, which naturally transform into the more stable benzoxazolinones MBOA and BOA, respectively. These compounds are implicated in allelopathic weed suppression, insect feeding deterrence, and microbial disease resistance. The mycotoxigenic fungus Fusarium verticillioides has the physiological capacity to biotransform benzoxazolinones into the non-toxic metabolites N-(2-hydroxy-4-methoxyphenyl)malonamic acid (HMPMA) and N-(2-hydroxyphenyl)malonamic acid (HPMA), respectively. The loci FDB1 and FDB2 are required for biotransformation, and the fungus cannot grow on BOA-amended medium if either locus is mutated. Suppression subtractive hybridization was used to identify F. verticillioides genes up-regulated in response to BOA, and a putative arylamine N-acetyltransferase (NAT) was of particular interest since FDB2 was previously postulated to encode transferase activity. The gene was subcloned from a cosmid that complemented an fdb2 mutant strain. The subcloned gene also complemented the mutant. Disruption of the gene eliminated the ability of F. verticillioides to metabolize BOA, and the mutant did not grow. We therefore functionally associate FDB2 as the gene encoding this putative NAT activity. The branch metabolite N-(2-hydroxyphenyl)acetamide was produced at low concentrations in fdb2 mutants suggesting acetylation of an intermediate 2-aminophenol occurred independently of the putative malonylation activity of FDB2. Other genes flanking FDB2 were assessed using a complementation assay, and we briefly discuss FDB3, which encodes a putative Zn(II)2Cys6 transcription factor adjacent to FDB2. Biotransformation of benzoxazolinones by F. verticillioides may enhance its ecological fitness in a maize field environment, and these genetic studies provide greater understanding of metabolic activity that may modulate such ecological interactions.