Genetic and Biochemical Characterization of a Gene Operon for trans-Aconitic Acid, a Novel Nematicide from Bacillus thuringiensis

Abstract

Edited by Ruma Banerjeetrans-Aconitic acid (TAA) is an isomer of cis-aconitic acid (CAA), an intermediate of the tricarboxylic acid cycle that is synthesized by aconitase. Although TAA production has been detected in bacteria and plants for many years and is known to be a potent inhibitor of aconitase, its biosynthetic origins and the physiological relevance of its activity have remained unclear. We have serendipitously uncovered key information relevant to both of these questions. Specifically, in a search for novel nematicidal factors from Bacillus thuringiensis, a significant nematode pathogen harboring many protein virulence factors, we discovered a high yielding component that showed activity against the plant-parasitic nematode Meloidogyne incognita and surprisingly identified it as TAA. Comparison with CAA, which displayed a much weaker nematicidal effect, suggested that TAA is specifically synthesized by B. thuringiensis as a virulence factor. Analysis of mutants deficient in plasmids that were anticipated to encode virulence factors allowed us to isolate a TAA biosynthesis-related (tbr) operon consisting of two genes, tbrA and tbrB. We expressed the corresponding proteins, TbrA and TbrB, and characterized them as an aconitate isomerase and TAA transporter, respectively. Bioinformatics analysis of the TAA biosynthetic gene cluster revealed the association of the TAA genes with transposable elements relevant for horizontal gene transfer as well as a distribution across B. cereus bacteria and other B. thuringiensis strains, suggesting a general role for TAA in the interactions of B. cereus group bacteria with nematode hosts in the soil environment. This study reveals new bioactivity for TAA and the TAA biosynthetic pathway, improving our understanding of virulence factors employed by B. thuringiensis pathogenesis and providing potential implications for nematode management applications.

The CRISPR-Cas systems were selectively inactivated during evolution of Bacillus cereus group for adaptation to diverse environments

“…As pathogens for insects and nematodes, B. thuringiensis produces diverse virulence factors [39,[61][62][63][64][65]. Production of multiple virulence factors benefits the populations by facilitating host infection, that represent more resources.…”

Searching Hit Potential Antimicrobials in Natural Compounds Space Against Biofilm Formation

“…In searching for new nematicidal factors from Bacillus thuringiensis against the plant-parasitic nematode Meloidogyne incognita, a component identified as trans-Aconitic acid (TAA) was found. The acid showed high nematicidal activity, suggesting that TAA is specifically synthesized by the bacillus as a virulence factor, TAA acid has shown activity nematicide for Bacillus thuringiensis bacterium [31]. The MM/PBSA calculation brings the information of how strong is the binding between protein and ligand, for this, the algorithm considers the binding free energy equal to the free energy of the 122 complex minus the sum of the free energy of the protein plus the free energy of the ligand, see equation…”

2‐Methylcitrate cycle: a well‐regulated controller of Bacillus sporulation

“…Bacillus thuringiensis cells were cultured at 28 C in GYS medium and sampled at 6 h and 12 h, respectively. Cells were harvested (35 ml cultures) by centrifugation at 4 C. The cell pellets were re-suspended in 10 ml H 2 O and then subjected to ultrasonication for the extraction of intracellular 2-methylcitrate, followed by procedures identical to those used for trans-aconitic acid, another TCArelated metabolite (Du et al, 2017).…”