Indole is a molecule of considerable biochemical significance, acting as both an interspecies signal molecule and a building block of biological elements. Bacterial indole degradation has been demonstrated for a number of cases; however, very little is known about genes and proteins involved in this process. This study reports the cloning and initial functional characterization of genes (iif and ant cluster) responsible for indole biodegradation in Acinetobacter sp. strain O153. The catabolic cascade was reconstituted in vitro with recombinant proteins, and each protein was assigned an enzymatic function. Degradation starts with oxidation, mediated by the IifC and IifD flavin-dependent two-component oxygenase system. Formation of indigo is prevented by IifB, and the final product, anthranilic acid, is formed by IifA, an enzyme which is both structurally and functionally comparable to cofactor-independent oxygenases. Moreover, the iif cluster was identified in the genomes of a wide range of bacteria, suggesting the potential of widespread Iifmediated indole degradation. This work provides novel insights into the genetic background of microbial indole biodegradation.IMPORTANCE The key finding of this research is identification of the genes responsible for microbial biodegradation of indole, a toxic N-heterocyclic compound. A large amount of indole is present in urban wastewater and sewage sludge, creating a demand for an efficient and eco-friendly means to eliminate this pollutant. A common strategy of oxidizing indole to indigo has the major drawback of producing insoluble material. Genes and proteins of Acinetobacter sp. strain O153 (DSM 103907) reported here pave the way for effective and indigo-free indole removal. In addition, this work suggests possible novel means of indole-mediated bacterial interactions and provides the basis for future research on indole metabolism. KEYWORDS indole, biodegradation, bacterial metabolism, Acinetobacter, bacterial signaling, cofactor-independent oxygenases I ndole is an N-heterocyclic aromatic compound derived mainly by TnaA tryptophanase from L-tryptophan in Escherichia coli (1) and is widely found in natural environments. Indole acts as cell-to-cell signaling molecule that regulates the expression of several virulence genes (2-4), promotes biofilm formation (5-7), and mediates complex predator-prey interactions (8, 9). At high concentrations, indole and its derivatives exhibit toxic activity to both prokaryotic cells and animals and are even considered mutagens (10). Toxic indole concentrations reportedly vary for different microorganisms in the range of 0.5 to 5 mM (11). The main mechanisms of indole toxicity are reported to be an alteration of membrane potential with subsequent inhibition of cell division (12), depletion of ATP levels (13), and an inhibition of acyl-homoserine lactone (AHL)-based quorum sensing by regulator misfolding (14).In order to utilize aromatic compounds as an energy source, microorganisms have