Downregulation of , encoding a dihydroxyacid dehydratase, affects growth of and in mice Free

Abstract

Dihydroxyacid dehydratase (DHAD), a key enzyme involved in branched-chain amino acid (BCAA) biosynthesis, catalyses the synthesis of 2-ketoacids from dihydroxyacids. In , DHAD is encoded by gene , and it shares 40 % amino acid sequence identity and conserved motifs with DHAD of encoded by . In this study, was overexpressed in and the resultant protein was characterized as a homodimer (∼155 kDa). Functional characterization of was established by biochemical testing and by genetic complementation of an intron-disrupted -auxotrophic mutant of to prototrophy. Growth of , BL21(DE3) and recombinant BL21(DE3) Δ carrying was inhibited by transient nitric oxide (NO) exposure in minimal medium but growth was restored if the medium was supplemented with BCAA (isoleucine, leucine and valine). This suggested that inactivation of by NO probably inhibited bacterial growth. The role of in was elucidated by antisense and sense RNA constructs. Growth of transformed with a plasmid encoding antisense mRNA was markedly poor in the lungs of infected mice and in Middlebrook 7H9 broth compared to that of sense and vector-alone transformants, but growth was normal when the medium was supplemented with BCAA. Upregulation of was observed during the early exponential phase of growth, under acid stress and , suggesting that has a role in the survival of during normal and stress conditions. It may be concluded that the DHAD encoded by is essential for the survival of and could be a potential drug/vaccine target, as it is absent in mammals.

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2011-01-01
2024-03-29
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