Abstract
Plants produce toxic secondary metabolites as defense mechanisms against phytopathogenic microorganisms and predators. L-azetidine-2-carboxylic acid (AZC), a toxic proline analogue produced by members of the Liliaceae and Agavaciae families, is part of such a mechanism. AZC causes a broad range of toxic, inflammatory and degenerative abnormalities in human and animal cells, while it is known that some microorganisms have evolved specialized strategies for AZC resistance. However, the mechanisms underlying these processes are poorly understood. Here, we identify a widespread mechanism for AZC resistance in fungi. We show that the filamentous ascomycete Aspergillus nidulans is able to not only resist AZC toxicity but also utilize it as a nitrogen source via GABA catabolism and the action of the AzhA hydrolase, a member of a large superfamily of detoxifying enzymes, the haloacid dehalogenase-like hydrolase (HAD) superfamily. This detoxification process is further assisted by the NgnA acetyltransferase, orthologue of Mpr1 of Saccharomyces cerevisiae. We additionally show that heterologous expression of AzhA protein can complement the AZC sensitivity of S. cerevisiae. Furthermore, a detailed phylogenetic analysis of AzhA homologues in Fungi, Archaea and Bacteria is provided. Overall, our results unravel a widespread mechanism for AZC resistance among microorganisms, including important human and plant pathogens.
Highlights
L-Azetidine-2-carboxylic acid (AZC) is a toxic analogue of L-proline (L-Pro) produced by members of the Liliaceae, Agavaceae, Asparagaceae, Fabaceae families and by several Beta vulgaris cultivars, such as beetroot or sugar b eet[1,2,3]
Strains on AZC as sole nitrogen source is highly reduced compared to the WT strain, supporting that AZC is assimilated through the GABA metabolic pathway
Given that growth with AZC as sole nitrogen source is NgnAindependent (Fig. 1), we investigated whether an AZC-hydrolase, similar to the AZC-assimilating hydrolase of Pseudomonas A2C strain (AC-hydrolase), is present in A. nidulans
Summary
L-Azetidine-2-carboxylic acid (AZC) is a toxic analogue of L-proline (L-Pro) produced by members of the Liliaceae, Agavaceae, Asparagaceae, Fabaceae families and by several Beta vulgaris cultivars, such as beetroot or sugar b eet[1,2,3]. Growth of ngnAΔ single deletion strain in the presence of AZC either as sole nitrogen source (AZC) or with urea (AZC + U) is similar to that of the WT strain. Growth of azhAΔ single deletion strain in the presence of AZC as sole nitrogen is severely impaired, while in AZC + U is similar to that of the WT, indicating that AzhA is involved in AZC assimilation. Growth of prnBΔ and prnA- strains on AZC as sole nitrogen source is similar to that of the WT strain, supporting that AZC is not assimilated through the proline catabolic pathway. GabAΔ, prnBΔ, gabAΔ prnBΔ mutant strains show no growth defect in the presence of AZC as sole nitrogen source, suggesting that AZC is not transported through the major proline transporter PrnB or the GABA transporter GabA. It has been suggested that AZC-producing plants protect themselves from AZC auto-toxicity, by avoiding the loading of AZC on L-Pro-tRNA via at least three mechanisms: (a) their L-Pro-tRNA activating enzymes are highly specific and discriminate between L-pro and AZC (b) AZC is subcellularly excluded from protein-synthesis s ites[16] and (c) some plants possess an efficient tRNA synthetase editing system, able to remove AZC loaded on L-Pro-tRNA9
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