Abstract
trans-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.
Highlights
Bacillus thuringiensis is an important entomopathogen that belongs to the Bacillus cereus group along with the human
Using genetic and biochemical techniques, we determined the trans-Aconitic acid (TAA) biosynthetic gene operon consisting of TAA biosynthesis-related gene A (tbrA) and tbrB genes in strain CT-43 as well as the isomerism and transportation processes mediated by aconitate isomerase TbrA and TAA transporter TbrB in the TAA biosynthesis of B. thuringiensis
Thuringiensin is a nematicidal factor of strain CT-43, with a gene located on the pCT127 plasmid [21, 22]
Summary
CT-A Is a Nematicidal Compound of B. thuringiensis CT43—B. thuringiensis CT-43 (non-flagellum and previously classified as B. thuringiensis subsp. chinensis) [35] harbors 10 native plasmids carrying diverse toxic genes against nematode and insect targets [34]. TbrB copy number was increased in recombinant BMB2451tbrB, which was constructed based on the BMB2451 strain This strain showed a substantially higher level of TAA in the culture supernatant than BMB2451 (Fig. 7C), in agreement with reports that the introduction of additional transporter genes into metabolite-producing bacteria could significantly promote product yield [49]. Together, these results demonstrated a TAA membrane transporter role for the TbrB protein of B. thuringiensis. Two transposase genes were found at both ends of the TAA gene cluster (Fig. 4A), indicating possible acquisition of the TAA biosynthetic gene cluster by B. cereus and B. thuringiensis bacteria through horizontal gene transfer
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