Indole Biodegradation in Acinetobacter sp. Strain O153: Genetic and Biochemical Characterization

Abstract

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 Iif-mediated 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: Acinetobacter; bacterial metabolism; bacterial signaling; biodegradation; cofactor-independent oxygenases; indole.

FIG 1

Growth kinetics of Acinetobacter sp. strain O153. (A) Growth in minimal medium with different carbon and nitrogen sources. (B) Growth in minimal medium supplemented with different concentrations of indole as a nitrogen source and succinate as a carbon source. Filled squares, NH₄Cl (5 g/liter) and succinate (5 mM) (positive control); filled triangles, indole (1 mM) and succinate; filled rhombus, NH₄Cl and indole (1 mM); filled circles, indole (1 mM); open squares, no N source and succinate (negative control); open circles, 0.1 mM indole; crosses, 0.5 mM indole; open triangles, 0.75 mM indole; open rhombus, 1.5 mM indole; striped squares, 2 mM indole. Dashed lines represent trend lines using a moving average data approximation (period = 2).

FIG 2

Whole-cell bioconversion assays using different substrates and cells of Acinetobacter sp. O153. (A) Substrates without cells (negative control). (B) Bioconversion with uninduced (overnight culture grown without indole) cells. (C) Bioconversion with induced (overnight culture grown in the presence of 1 mM indole) cells. Solid lines indicate spectra of initial and final products; dashed lines indicate spectra of intermediate products during a 6-h bioconversion cycle.

FIG 3

Organization and distribution of iif and ant genes in different microbial genomes. Genes are represented by arrows (drawn to scale as indicated). Homologous genes are highlighted in the same pattern according to the scheme in the top line for Acinetobacter sp. O153. White arrows, indole degradation unrelated/unknown. Strains and genomic fragments boxed in solid lines indicate reported activity of certain Iif-homologous proteins; dashed boxes highlight strains for which indole biodegradation activity was reported at the genus level. Identities (percent) of amino acid sequences between Iif proteins of strain O153 and homologs are indicated under the corresponding ORFs.

FIG 4

Stacked HPLC chromatograms (211 nm) of enzymatic reaction products and standards. Top three curves, standards; middle four curves, reaction products of indole and Iif proteins as indicated on the right; bottom curve, reaction product of 3-hydroxyindolin-2-one and IifA. Numbers indicate peaks of three major compounds: peak 1, 3-hydroxyindolin-2-one; peak 2, anthranilic acid; peak 3, indole.

FIG 5

Functional characterization of IifA. (A) Oxygen consumption by IifA. Red, green, and blue indicate different substrate concentrations as indicated. Reactions were initiated at 90 s. (B) UV spectra of the transition between 3-hydroxyindolin-2-one and anthranilic acid, catalyzed by IifA. The inset shows the spectral difference between the substrate and the product.

FIG 6

Proposed indole biodegradation pathway in Acinetobacter sp. O153: step 1, indole; step 2, indole-2,3-dihydrodiol; step 3, 3-hydroxyindolin-2-one; step 4, anthranilic acid; step 5, isatin; step 6, 1H-indol-3-ol (indoxyl); step 7, indigo. Brackets indicate an intermediate not experimentally detected.