ToxB encodes a canonical GTP cyclohydrolase II in toxoflavin biosynthesis and ribA expression restored toxoflavin production in a ΔtoxB mutant

Abstract

Burkholderia glumae synthesizes toxoflavin, a phytotoxin that contributes the virulence of this phytopathogen. The toxoflavin biosynthetic gene cluster contains a tox operon composed of five genes, toxABCDE, and toxB is predicted to encode a GTP cyclohydrolase II, which is the first enzyme (RibA) in riboflavin biosynthesis. ToxE is also homologous to RibD, the bifunctional deaminase/reductase acting on the RibA product. This suggests that toxoflavin and riboflavin biosynthesis share the first two steps in their respective biosyntheses. In this study, we demonstrated that ToxB and B. glumae RibA (Bglu-RibA) both displayed GTP cyclohydrolase II activity with comparable kinetic parameters. When toxB was inactivated, toxoflavin production was abolished, and introduction of a plasmid copy of Bglu-ribA restored toxoflavin production in a ΔtoxB mutant. ToxB and Bglu-RibA can thus be defined as GTP cyclohydrolase II isozymes, even though Bglu-ribA is not a genetic equivalent of toxB because the chromosomal copy of Bglu-ribA is unable to support toxoflavin production in the absence of toxB. In LB agar culture, toxAB is incapable of complementing ΔtoxB though toxABC induced toxoflavin accumulation in the ΔtoxB mutant up to 80 % of the WT level. This indicates that toxBC co-expression is a critical factor for toxoflavin biosynthesis in this condition, suggesting that the WD repeat protein ToxC acts as a scaffolding protein in a toxoflavin biosynthetic metabolon. In LB liquid culture, toxAB successfully restored toxoflavin production, suggesting that a role of toxBC co-expression is dependent upon growth condition.