Identification and functional study of an iif2 gene cluster for indole degradation in Burkholderia sp. IDO3

Burkholderia sp. IDO3 is an indole-degrading bacterium isolated from activated sludge. A previous genomic clone library assay identified an iif1 gene cluster for indole metabolism in strain IDO3. To further explore the underlying indole degradation mechanisms, the complete genome of strain IDO3 was sequenced (8,003,806 bp). The genome contained three circular chromosomes and one plasmid, and 7550 genes were predicted. Interestingly, in addition to iif1 on chromosome 3, bioinformatic analyses identified a second indole oxygenase gene cluster, iif2, on chromosome 1. Both iif clusters were up-regulated in response to indole. Heterologous expression of iifC1D1 and iifC2D2 in Escherichia coli BL21(DE3) demonstrated that these genes were capable of oxidizing indole to indigo. Gene knockout assays provided additional evidence that iifC2 played crucial roles in indole metabolism. In addition, we identified a novel gene (iifF) in the iif2 cluster. This gene was shown to encode an isatin hydrolase. IifF was expressed in E. coli, and a purified his-tagged enzyme preparation was obtained. IifF converted isatin to isatinate with K m of 4.4 ± 0.7 μM and k cat of 95.5 ± 4 s−1. This is the first study to show that indole can be degraded by two iif gene clusters, and that isatin hydrolase is involved in indole metabolism, improving our understanding of indole metabolic processes.