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

Modified nucleotides are present in many RNA species in all Domains of Life. While the biosynthetic pathways of such nucleotides are well studied, much less is known about the degradation of RNAs and the return to the metabolism of modified nucleotides, their respective nucleosides or heterocyclic bases. Using an E. coli uracil auxotroph, we screened the metagenomic libraries for genes, which would allow the conversion of 2-thiouracil to uracil and thereby lead to the growth on a defined synthetic medium. We show that a gene encoding a protein consisting of previously uncharacterized Domain of Unknown Function 523 (DUF523) is responsible for such phenotype. We have purified this recombinant protein and demonstrated that it contains a FeS cluster. The substitution of cysteines, which have been predicted to form such clusters, with alanines abolished the growth phenotype. We conclude that DUF523 is involved in the conversion of 2-thiouracil into uracil in vivo.

Rhodococcus rhodochrous PY11 (DSM 101666) is able to use 2-hydroxypyridine as a sole source of carbon and energy. By investigating a gene cluster (hpo) from this bacterium, we were able to reconstruct the catabolic pathway of 2-hydroxypyridine degradation. Here, we report that in Rhodococcus rhodochrous PY11, the initial hydroxylation of 2-hydroxypyridine is catalyzed by a four-component dioxygenase (HpoBCDF). A product of the dioxygenase reaction (3,6-dihydroxy-1,2,3,6-tetrahydropyridin-2-one) is further oxidized by HpoE to 2,3,6-trihydroxypyridine, which spontaneously forms a blue pigment. In addition, we show that the subsequent 2,3,6-trihydroxypyridine ring opening is catalyzed by the hypothetical cyclase HpoH. The final products of 2-hydroxypyridine degradation in Rhodococcus rhodochrous PY11 are ammonium ion and ␣-ketoglutarate. Pyridine and its derivatives are ubiquitous in nature. The pyridine ring is found in alkaloids (e.g., nicotine, actinidine), coenzymes [NAD(P)H, pyridoxal], and man-made solvents, pesticides, and herbicides (e.g., paraquat). Hydroxypyridines are common intermediate metabolites produced during microbial biodegradation of various N-heterocycles (pyridine, nicotine, picoline, 2,6-dipicolinic acid) (1-3).It has previously been reported that Arthrobacter crystallopoietes, Arthrobacter pyridinolis, and Arthrobacter viridescens (4), Achromobacter sp. strain G2 (5), and Nocardia sp. strain PNO (6) use 2-hydroxypyridine (2HP) as a sole carbon and energy source. Through more than 50 years of investigation of pyridine ring metabolism, many intermediates have been identified and metabolic pathways have been proposed. However, the genes and enzymes responsible for 2HP biodegradation have seldom been reported.In Achromobacter sp. G2, 2HP is metabolized via the maleamate pathway (5) (Fig. 1). No enzymes responsible for the initial hydroxylation step of 2HP leading to the formation of 2,5-dihydroxypyridine (2,5DHP) have been reported to date. Nevertheless, the degradation of 2,5DHP, an intermediate of nicotinic acid metabolism, has been fully investigated by Jiménez et al. (7), and all genes encoding the enzymes involved in the maleamate pathway have been identified and characterized (7).Arthrobacter crystallopoietes, A. pyridinolis, and A. viridescens (4) and Arthrobacter sp. strain PY22 (8) produce a blue pigment (nicotine blue) in the medium when grown on 2HP. The nicotine blue has been shown to be a 4,5,4=,5=-tetrahydroxy-3,3=-diazadiphenoquinone-(2,2=) (9) that is an autoxidation product of 2,3,6-trihydroxypyridine (THP). THP can be synthesized via hydroxylation of 2,5DHP, 2,3-dihydroxypyridine (2,3DHP), or 2,6-dihydroxypyridine (2,6DHP); however, only the 2,6DHP 3-hydroxylase, which is involved in the biodegradation of nicotine by Arthrobacter nicotinovorans, has been identified to date (10).We have previously reported that HpyB monooxygenase from Arthrobacter sp. PY22 is sufficient for the conversion of 2HP to THP (8). Since no reaction intermediates have been detected, a consecutive two-st...

A bacterial strain 5HP capable of degrading and utilizing 5-hydroxypicolinic acid as the sole source of carbon and energy was isolated from soil. In addition, the isolate 5HP could also utilize 3-hydroxypyridine and 3-cyanopyridine as well as nicotinic, benzoic and p-hydroxybenzoic acids for growth in the basic salt media. On the basis of 16S rRNA gene sequence analysis, the isolate 5HP was shown to belong to the genus Pusillimonas. Both the bioconversion analysis using resting cells and the enzymatic assay showed that the degradation of 5-hydroxypicolinic acid, 3-hydroxypyridine and nicotinic acid was inducible and proceeded via formation of the same metabolite, 2,5-dihydroxypyridine. The activity of a novel enzyme, 5-hydroxypicolinate 2-monooxygenase, was detected in the cell-free extracts prepared from 5-hydroxypicolinate-grown cells. The enzyme was partially purified and was shown to catalyze the oxidative decarboxylation of 5-hydroxypicolinate to 2,5-dihydroxypyridine. The activity of 5-hydroxypicolinate 2-monooxygenase was dependent on O2, NADH and FAD.

A cryptic plasmid from Arthrobacter rhombi PRH1, designated as pPRH, was sequenced and characterized. It was 5000 bp in length with a G+C content of 66 mol%. The plasmid pPRH was predicted to encode six putative open reading frames (ORFs), in which ORF2 and ORF3 formed the minimal replicon of plasmid pPRH and shared 55-61% and 60-69% homology, respectively, with the RepA and RepB proteins of reported rhodococcal plasmids. Sequence analysis revealed a typical ColE2-type ori located 45 bp upstream of the gene repA. Sequence and phylogenetic analysis led to the conclusion that pPRH is a representative of a novel group of pAL5000 subfamily of ColE2 family plasmids. Three shuttle vectors pRMU824, pRMU824Km and pRMU824Tc, encoding chloramphenicol resistance, were constructed. The latter two harboured additional antibiotic resistance genes kan and tet, respectively. All vectors successfully replicated in Escherichia coli, Arthrobacter and Rhodococcus spp. The vector pRMU824Km was employed for functional screening of 2-hydroxypyridine catabolism encoding genes from Arthrobacter sp. PY22. Sequence analysis of the cloned 6-kb DNA fragment revealed eight putative ORFs, among which hpyB gene encoded a putative monooxygenase.