A Rieske-Type Oxygenase of Pseudomonas sp. BIOMIG1 Converts

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A Rieske-type Oxygenase of Pseudomonas sp. BIOMIG1 converts Benzalkonium Chlorides to Benzyldimethyl Amine Emine Ertekin, Konstantinos T. Konstantinidis, and Ulas Tezel Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b03705 • Publication Date (Web): 28 Oct 2016 Downloaded from http://pubs.acs.org on October 29, 2016

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A Rieske-type Oxygenase of Pseudomonas sp. BIOMIG1

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converts Benzalkonium Chlorides to Benzyldimethyl Amine

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Emine Ertekin1, Konstantinos T. Konstantinidis3,4 and Ulas Tezel1,2,*

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1

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TURKEY

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TURKEY

Institute of Environmental Sciences, Bogazici University, Bebek 34342 Istanbul,

Center for Life Sciences and Technologies, Bogazici University, Bebek 34342 Istanbul.

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Atlanta GA, 30332-0512, USA

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*Corresponding author. Phone: +90 212-359-4604; fax: +90 212-257-5033;

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e-mail address: [email protected] (U. Tezel)

School of Civil and Environmental Engineering, Georgia Institute of Technology,

School of Biology, Georgia Institute of Technology, Atlanta GA, 30332-0512, USA

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ABSTRACT

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Recently, an array of eight genes involved in the biotransformation of benzalkonium

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chlorides (BACs) – an active ingredient of many disinfectants - to benzyldimethyl amine

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(BDMA) was identified in the genome of Pseudomonas sp. BIOMIG1, which is a

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bacterium present in various environments and mineralizes BACs. In this study, we

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showed that heterologous expression of an oxygenase gene (oxyBAC) present in this gene

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array in E. coli resulted in formation of BDMA from BACs at a rate of 14 µM h-1.

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oxyBAC is phylogenetically classified as a Rieske-type oxygenase (RO) and belongs to a 1 ACS Paragon Plus Environment

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group which catalyzes the cleavage of C-N+ bond between either methyl or alkyl ester

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and a quaternary nitrogen (N) of natural QACs such as stachydrine, carnitine and

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trimethylglycine. Insertion of two glycine into the Rieske domain and substitution of

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tyrosine with leucine in the mononuclear iron center differentiate oxyBAC from other

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ROs that cleaves C-N+, and presumably facilitate the cleavage of saturated alkyl chain

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from quaternary N via N-dealkylation reaction. In addition, unlike other ROs, oxyBAC

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did not require a specific reductase to function. Our results demonstrate that oxyBAC

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represents a new member of RO associated with BAC degradation, and have implications

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for controlling the fate of BACs in the environment.

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INTRODUCTION

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Quaternary ammonium compounds (QACs) are cationic surfactants that are used as

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disinfectants in industrial, domestic and medical applications extensively since 1930s1 .

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Benzalkonium chlorides (BACs) are the most commonly used group of QACs1. Due to

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their unrestricted use since decades, BACs are continuously discharged into the

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environment either directly or through wastewater treatment plants, which are not

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designed to treat such pollutants like BACs. As a result, BACs are present in every

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compartment of the environment at concentrations ranging from 0.05 mg L-1 in surface

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waters 2 to 5 mg L-1 in hospital effluents 2, 3. Contamination of the environment with

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BACs can cause severe ecological and public health problems such as eco-toxicity 1 and

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dissemination of antimicrobial resistance4.

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Despite their antimicrobial properties, some microorganisms in the environment can degrade BACs and convert them to non-toxic end products 5-7. BAC degradation

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commences with an initial attack to the alkyl chain, which cleaves the C-N bond. This N-

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dealkylation reaction results in the formation of benzlydimethyl amine (BDMA) and an

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alkanaldehyde as intermediates. As BDMA is approximately 500 times less toxic than

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BAC8, this is the key step in BAC biotransformation that alleviates the environmental

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impact of BACs.

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Several microorganisms capable of catalyzing this reaction have been

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characterized to date9-11. However, there is relatively limited information on the genetics

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of QAC N-dealkylation reaction achieved by those microorganisms. To date, tetradecyl

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trimethyl ammonium bromide monooxygenase (TTABMO) of Pseudomonas putida

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ATCC 12633 12 and amine oxidase of a Pseudomonas nitroreducens B 13 which

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converted QACs to tertiary amines have been characterized. TTABMO is a typical

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flavoprotein that utilizes nicotinamide adenine dinucleotide phosphate (NADPH) and

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flavin adenine dinucleotide (FAD) as cofactors, whereas the amine oxidase of

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Pseudomonas nitroreducens B requires only FAD. Phylogenetically, amine oxidase is

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more closely related to Pseudomonas sp. pseudooxynicotine amine oxidase14, which

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catalyzes deamination of pseudooxynicotine, rather than TTABMO, although both of

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them are QAC N-dealkylating ezymes. In addition, three genes encoding oxygenases that

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metabolized glycine betaine (gbcA), stachydrine (stc2) and carnitine (cntA), which are

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naturally occurring QACs, have been identified15, 16. Those enzymes are Rieske non-

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heme iron dependent oxygenases that catalyze the N-dealkylation of QACs (Figure S1).

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Among them, stc2 and gbcA removes methyl group from quaternary N (Figure S1A and

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B) whereas cntA cleaves the C-N+ bond between an ester group and the quaternary N

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(Figure S1C). However none of them were shown to attack on C-N+ between a saturated

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alkane and quaternary N that is present in synthetic QACs such as BACs.

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Recently, we isolated a new BAC-degrader, Pseudomonas sp. BIOMIG1, and

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demonstrated that an array of genes including multidrug transporters, a transcriptional

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regulator, a sterol binding domain protein, an integrase and an oxygenase, all encoded on

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the same piece of DNA, were essential for BAC degradation. Moreover, the same array

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of genes was also highly abundant in BAC degrading microbial communities originating

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from four different environments 17. The only catabolic gene present in this array encoded

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a predicted oxygenase (oxyBAC), which was not similar to any other QAC degrading

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enzymes previously identified. In this study, our objective was to elucidate the function

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of the oxygenase gene identified in the BIOMIG1 by genetic manipulations using

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heterologous expression of the gene in E.coli. Therefore our study identified a new gene

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and the corresponding enzyme involved in the QAC degradation, which might be

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widespread in the natural and engineered systems.

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MATERIALS AND METHODS

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Microorganisms. Pseudomonas sp. BIOMIG1BAC (GenBank Accession No:

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MCRS00000000) which has been shown to mineralize BACs and BDMA and

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Pseudomonas sp. BIOMIG1BDMA (MCRT01000000) which converts BACs to BDMA but

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cannot utilize BDMA were isolated from a BAC degrading enrichment community

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developed from sewage taken from a local wastewater treatment plant in Istanbul,

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Turkey17. In addition, Pseudomonas sp. BIOMIG1BD (MCRU01000000) which utilizes

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BDMA but not BACs has been generated from Pseudomonas sp. BIOMIG1BAC by serial

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culturing on a salt medium (SM) containing only BDMA as the carbon source 17.

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Pseudomonas sp. BIOMIG1N (MCRV00000000) that degrades neither BACs nor BDMA

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has been generated by culturing the Pseudomonas sp. BIOMIG1BDMA only in LB. E.coli

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BL21(DE3) pLysS (Invitrogen, Thermo Fisher Scientific, USA) was used in heterologous

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expression experiments. Summary of the strains and their characteristics are given in

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Table 1.

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Bioinformatic identification and classification of BAC oxygenase. In order to identify

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the genes involved in the BAC conversion to BDMA, genomes of aforementioned four

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BIOMIG1 phenotypes were compared as described elsewhere17. Briefly, the gene

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sequences in the genomes were compared in a pairwise manner using “reciprocal best

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match blast”. The set of genes that was present in the genome of the Pseudomonas sp.

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BIOMIG1BAC and Pseudomonas sp. BIOMIG1BDMA but absent in the Pseudomonas sp.

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BIOMIG1BD and Pseudomonas sp. BIOMIG1N was determined as the candidate genes

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involved in BAC to BDMA transformation. An array composed of 8 genes was identified.

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An oxygenase was the only catabolic gene in this array and selected as the candidate gene

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responsible for BAC transformation to BDMA via N-dealkylation reaction. This gene

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was mentioned as oxyBAC here after.

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The amino acid sequence of oxyBAC (Text S1) was annotated using BLASTP in

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the non-redundant protein sequence database of “National Center of Biotechnology

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Information” (NCBI, http://www.ncbi.nlm.nih.gov/protein). For the phylogenetic

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classification of oxyBAC, protein sequences of homolog oxygenases along with

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previously identified QAC degrading enzymes were used. Briefly, protein sequences

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were aligned using ClustalW implemented in Geneious® 7.1 (Amsterdam, Netherlands)

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and Jukes-Cantor neighbor joining method was used for clustering.

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PCR primers for oxyBAC were designed to verify the presence of the gene in the

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BIOMIG1 phenotypes as well as the in the E.coli host used in heterologous expression

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experiments with Primer 3 module in Geneious® 7.1 (Amsterdam, Netherlands) using

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the gene sequences identified by genome comparison. The primer sequences are provided

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in Table 1. The amplification conditions were as follows; denaturation at 95°C for 10

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seconds, annealing at 60°C for 30 seconds and elongation at 72°C for 1 minute, for 30

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cycles.

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BAC biotransformation experiments with Pseudomonas sp. BIOMIG1 phenotypes.

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Pseudomonas sp. BIOMIG1BAC and Pseudomonas sp. BIOMIG1BDMA were grown in LB

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containing 50 mg L-1 BACs (Barquat 80, Lonza Inc., Switzerland). An overnight grown

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culture of each phenotype was resuspended in SM8, 17 and spiked with 150 µM BAC

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containing equimolar amounts of dodecyl benzyl dimethyl ammonium chloride

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(C12BDMA-Cl, TCI Chemicals, Japan) and tetradecyl benzyl dimethyl ammonium

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chloride (C14BDMA-Cl, TCI Chemicals, Japan). After all BAC was consumed, cultures

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were diluted to OD600 of 0.02 AU (~106 cells mL-1) in SM and respiked with 300 µM

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BACs. The cultures were incubated at room temperature on a rotary shaker at 130 rpm.

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Samples were taken intermittently to monitor BAC concentration by HPLC using the

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method described elsewhere17, and cell growth was measured by plate counting. On the

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other hand, BIOMIG1BD and BIOMIG1N were grown in LB with 1000 mg L-1 BDMA or

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only in LB, respectively. The overnight grown cultures were resuspended in SM to an

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OD600 of 0.2 AU, and spiked with 300 µM BAC. BAC concentration and cell density

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were measured as described above.

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Heterologous expression of genes in E. coli. The oxyBAC gene was chemically

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synthesized by GenScript and inserted into pet29a+ vector (Merck Millipore, Germany),

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between the NdeI and NcoI restriction sites, respectively (Figure S2). The E.coli

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BL21(DE3) pLysS strain (Invitrogen, Thermo Fisher Scientific, USA) was transformed

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with plasmid containing oxyBAC (E. colioxyBAC) (Table 1). For protein overexpression,

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E.coli cells were grown in LB broth containing 30 µg mL-1 kanamycin (Sigma-Aldrich,

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Germany) and 34 µg mL-1 chloramphenicol (Sigma-Aldrich, Germany) at 37°C. After the

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turbidity reached at OD600 of 0.5, 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG)

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(Sigma-Aldrich, Germany) was added to initiate induction, which was carried out at room

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temperature for 24 hours. Expression of the 43 kDa oxyBAC protein was confirmed

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using SDS-PAGE of crude protein extracts (Figure S3).

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Following induction, E. colioxyBAC cells were harvested and inoculated into 20 mL

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SM containing 20 µM of C12BDMA-Cl at a final O.D. of 2.0. At determined intervals,

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C12BDMA-Cl utilization and BDMA production were monitored by HPLC as described

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by Ertekin et al. (2016)17. A control assay was performed with E. coli cells transformed

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with the pet29a+ plasmid not containing oxyBAC gene (E. coliN, Table 1). To calculate

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biotransformation rates, experimental data was fitted to Michaelis-Menten growth model

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using Berkeley-Madonna software employing Runge-Kutta 4 integration method, as

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described by Ertekin et al. (2016)17.

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RESULTS AND DISCUSSION

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Phylogenetic analysis and classification of oxyBAC. Analysis of the amino acid

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sequence of oxyBAC confirmed that it encoded a conserved Rieske domain [2Fe-2S] and

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an iron containing catalytic active site, and therefore this protein was identified as a

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Rieske oxygenase (RO). Nam et al. (2001)18 classified ROs into four distinct groups

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according to the amino acid sequence patterns of their oxygenase subunits. Those four

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RO groups (Group I-IV, Figure 1) mainly catalyze cis and trans hydroxylation of a

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benzene ring. However, it was recently reported that some of the newly discovered ROs

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did not cluster with any of these groups, thus representing a novel group. The ROs that

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belonged to the latter group include enzymes like stachydrine demethylase (stc2)15,

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glycine demethylase (gbcA)19 and carnitine oxygenase (cntA)16, which could carry out

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non-ring hydroxylating reactions. More specifically, the latter enzymes attack on C-N+

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bond between a methyl or ester groups and quaternary N of natural QACs resulting in

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removal of these groups from the N atom and formation of a tertiary amine (Figure S1A,

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B and C) In order to dealineate the evolutionary relationship of oxyBAC with the

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previously characterized ROs, a phylogenetic tree was constructed using the amino acid

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sequences of its closest homologs along with two recently identified QAC degrading

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enzymes (Figure 1). Our results demonstrated that oxyBAC belonged to a distinct clade

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including stc2, gbcA and cntA which catalyze N-dealkylation of aforementioned natural

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QACs. This group was further denoted as Group V ROs in this study. The previously

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identified enzymes degrading QAC disinfectants such as the amine oxidase of

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Pseudomonas nitroreducense B6 and tetradecyltrimethylammonium bromide

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monooxygenase of Pseudomonas putida ATCC 12633 (TTBMO) 12 are non-RO enzymes

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and were more closely related to Group II ROs as opposed to Group V enzymes.

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Furthermore, among ROs, Group V was more closely related to monooxygenases

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such as the choline monooxygenase (cmo). On the other hand, cmo cannot catalyze a N-

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dealkylation reaction but can oxidize the terminal C of the alcohol group attached to the

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quaternary N of the choline (Figure S1D) 20. Within this group, oxyBAC had 26% amino

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acid similarity to stc2 which was its closest biochemically characterized homolog. This

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relatively low homology to previously characterized enzymes doing N-dealkylation of

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short saturated and un-saturated alkyl groups suggests that oxyBAC is a novel enzyme,

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potentially involved in the N-dealkylation of a long-chain saturated alkyl group from a

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quaternary N (Figure S1E). The previously identified BAC dealkylating amine oxidase6

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and TTABMO12 showed substantially lower homology to oxyBAC (13% and 17%,

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respectively) compared to cntA, gbcA and stc2.

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The most similar amino acid sequence (~97% amino acid similarity) to oxyBAC

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was present in the draft genome of Novosphingobium sp. B7 (GenBank Accession

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Number: WP_022676119.1). The gene encoding this protein was located on a 3 kbp long

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contig. The sequence of this contig was almost identical to the gene cluster containing

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oxyBAC (98% nucleotide similarity). However, neither Novosphingobium sp. B7, nor the

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aforementioned protein has been previously tested for QAC degradation. The flanking

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regions of oxyBAC in our Pseudomonas sp. BIOMIG1 strain consisted of two MFS

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transporters, a tetR family type transcriptional regulator, a sterol binding domain protein,

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a hypothetical protein, a Lyrs family transcriptional regulator and a phage integrase17.

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This structure was also conserved in the genome of Novosphingobium sp. B7, where the

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homolog oxygenase was flanked with a tetR family transcriptional regulator, a sterol

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binding protein and two hypothetical proteins. In the latter gene cluster, a reductase gene

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next to oxyBAC was not identified which is not usual for ROs. The latter finding

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indicated that oxyBAC could function without a specific reductase or recruit a non-

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specific reductase for electron transfer.

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Pseudomonas sp. BIOMIG1 with oxyBAC grows on BACs. We confirmed that

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oxyBAC gene was present in the genomes of Pseudomonas sp. BIOMIG1BAC and

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BIOMIG1BDMA, but absent in the genomes of Pseudomonas sp. BIOMIG1BD and

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Pseudomonas sp. BIOMIG1N by amplifying a ~700 bp region of the gene by PCR using a

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specific primer set (Figure 2A). Given that BIOMIG1BD and BIOMIG1N were generated

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from BIOMIG1BAC and BIOMIG1BDMA, respectively by selective cultivation in BAC free

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media, it can be assumed that oxyBAC was lost from the genome of the former two

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strains which was also consistent with the sequence comparison of the draft genomes of

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BIOMIG1 phenotypes in our previous study17.

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Both BIOMIG1BAC and BIOMIG1BDMA completely utilized 250 µM BAC within

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50 h (Figure 2B and C). Given the fact that BDMA transformation rate is faster than

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BAC conversion to BDMA8, BDMA was not detected in the flasks inoculated with

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BIOMIG1BAC. Moreover, the cell density increased by approximately 200 folds (Figure

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2B). The results suggested that BIOMIG1BAC transformed BACs to BDMA which was

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quickly utilized as a carbon and energy source promoting cell growth. On the other hand,

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equimolar amount of BDMA was produced when BACs were utilized by BIOMIG1BDMA.

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However, cell density increased only 10 folds which was less than that of BIOMIG1BAC

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(Figure 2C) . This results suggested that oxyBAC was only involved in the BAC to

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BDMA conversion and does not support growth on the BDMA. In contrast, BIOMIG1BD

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and BIOMIG1N, which did not contain oxyBAC, neither utilized BACs nor grew in the

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SM containing BACs (Figure 2D and E). In conclusion, absence of oxyBAC in

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BIOMIG1 resulted in subsequent loss of BAC transformation to BDMA.

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E. coli overexpressing oxyBAC gene converts BACs to BDMA. In order to further

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confirm the function of oxyBAC, biotransformation experiments were conducted with E.

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colioxyBAC cells heterologously expressing the gene. A control assay was prepared with E.

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coliN cells transformed with an empty plasmid. We verified that oxyBAC was present in

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E. colioxyBAC and absent in E. coliN using PCR targeting the oxyBAC (Figure 3A). In the

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biotransformation assay, C12BDMA-Cl was used as the substrate at 20 µM concentration.

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E. coliN.did not utilize C12BDMA-Cl in the course of incubation (Figure 3B). On

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the other hand C12BDMA-Cl was completely converted to BDMA within 1.5 h in the

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flasks inoculated with E. colioxyBAC (Figure 3C). The rate of C12BDMA-Cl

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biotransformation was predicted as 14 µM hr-1. This results confirmed that oxyBAC is

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the enzyme responsible for the conversion of BAC to BDMA via N-dealkylation reaction.

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These rates were very similar with the native enzyme of Pseudomonas sp. BIOMIG117,

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indicating that oxyBAC could function effectively outside of its original host. The high

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efficiency in integrating oxyBAC into a heterologous expression system (e.g., E. coli),

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indicates that the gene might be promiscuous in horizontal gene transfer and easy to

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manipulate in further biotechnological applications.

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To date, two other enzymes capable of biotransforming QAC disinfectants have

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been identified. An amine oxidase of Pseudomonas nitroreducens B13 could convert

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C12BDMA-Cl and C14BDMA-Cl to BDMA with ~50% efficiency and a rate of 20 nmol

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substrate/mg of protein/min and 7 nmol substrate mg of protein-1 min-1, respectively and

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TTABMO of Pseudomonas putida ATCC 12633 could convert

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tetradecyltrimethylammonium bromide with a rate of 128.6 nmol TMA min-1 mg protein-

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1

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enzymes were phylogenetically distant to oxyBAC and groupV ROs. The most closely

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related enzyme to amine oxidase of Pseudomonas nitroreducens B was a

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pseudooxynicotine amine oxidase (pao, GenBank accession number: AFD54463.1)14.

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Both amine oxidases could attact the C-N bond and catalyze a dealkylation reaction.

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Additionaly, the closest homolog of TTABMO was a typeV secretion protein; Rhs of

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Pseudomonas sp. (GenBank accession no: WP_049586647.1) These results imply that

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there might be multiple genetic mechanisms and enzymes capable of biotransforming

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QAC disinfectants in the environment. Yet, oxyBAC appears to be the most efficient

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mechanism known, at least based on the product yields in our experiments.

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oxyBAC is a unique N-dealkylating RO. Rieske type oxygenases are growing group of

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oxygenase enzymes which are important for biodegradation of xenobiotic pollutants 21, 22.

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The presence of a conserved Rieske motif (-CXHX15-17CXXH-) and mononuclear iron

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center qualify oxyBAC as an RO. We demonstrated that oxyBAC cleaves C-N+ bond

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between a long saturated alkyl chain and a quaternary N, which is a unique reaction type

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compared to other N-dealkylating ROs, because the other N-dealkylating ROs either

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targets a methyl group and demethylate an amine 21, 22 or a quaternary ammonium15, 19, or

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an unsaturated short alkyl chain16. In addition, ROs are multicomponent enzymes,

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consisting of an oxygenase reductase and sometimes also a ferredoxin subunit 23, 24. In

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contrast, oxyBAC facilitates without a specific reductase, which suggested that it

. Despite catalyzing similar reactions, those previously identified QAC degrading

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probably recruits a nonspecific reductase present in the host strain. Although this is

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uncommon, multicomponent ROs with promiscuous reductases have been identified 25, 26.

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These facts suggests that oxyBAC is a unique RO.

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In order to understand the unique features of oxyBAC that gives its novel reaction

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capabilities, Rieske domain and the mononuclear iron center of oxyBAC was compare to

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other N-dealkylating ROs (Figure 4). Rieske domain of oxyBAC was 51, 46 and 43%

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similar to those of cntA, gbcA and stc2, respectively. The key difference between the

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Rieske domains of oxyBAC and other N-dealkylating ROs was the presence of 19

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residues, instead of 17 between CXH and CXXH conserved regions. The reason for this

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was the insertion of two glycine (G) into this region (Figure 4A). In addition, adjacent 2

283

amino acids (LR) before and 3 amino acids (RIL) after the two glycine are unique to

284

oxyBAC where as they are VA and KLV in both stc2 and gbcA (Figure 4A). Thus, LR-

285

GG-RIL motif is unique for oxyBAC Rieske domain and might underlie its unique

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biochemical function. Moreover, mononuclear iron center of the oxyBAC is 42, 54 and

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58% similar to those of cntA, gbcA and stc2, respectively. Substitution of bridging

288

aspartate (D) to bridging glutamate (E) is common to all N-dealkylating ROs compared to

289

other ROs including cmo (Figure 4B). Zhu et al (2014) suggested that this substitution is

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the main reason for the attack on C-N+ in the N-dealkylation reaction which can be

291

achieved by all Group V ROs except cmo which cannot attack on C-N+ bond. On the

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other hand, substitution of tyrosine (Y) with leucine (L) adjacent to first conserved

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histidine (H) in mononuclear iron center is unique to oxyBAC (Figure 4B). These unique

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features of oxyBAC peptide sequence may resulted in the N-dealkylation of saturated

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long-chain alky groups from QACs without a need of a specific reductase for the reaction.

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Varification of this hypothesis via genetic experiments should be part of future work.

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ENVIRONMENTAL SIGNIFICANCE AND APPLICATIONS

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In this study, we identified a gene responsible for detoxification of BACs by converting

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BACs to BDMA, confirmed its function genetically and also provide a PCR primer

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specific for the gene. The oxyBAC enzyme can be used to develop advanced wastewater

302

treatment technologies where BACs are problem such as poorly treated wastewater and

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effluents originating from hospitals and pharmaceutical facilities. In addition, oxyBAC

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based biotechnology could have applications in bioremediation of environments

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contaminated with BACs. For instance, the primers developed here can be used to

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evaluate the BAC treatment potential of activated sludge treating domestic, industrial and

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agricultural wastewater as well as of the natural environments contaminated with BACs.

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On the other hand, microorganisms having oxyBAC gene are not desirable in medical

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environments since they would decrease the efficacy of disinfectant most of which have

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BACs as active ingredients and thus could cause proliferation of pathogens. Such threats

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may be minimized by systematically analyzing the abundance of oxyBAC in the medical

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settings and taking necessary precautions to avoid contamination of indoor environment

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by microorganisms harboring oxyBAC gene.

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Protein based experiments are necessary for further characterization of oxyBAC.

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The multicomponent structure can be fully resolved once the reductase and ferredoxin

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subunits of the enzyme are identified and analyzed via crystallography. Additionally,

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Protein characteristics such as substrate specificity and binding efficiency, optimum

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working temperature, pH, etc. should be determined for implementing technology

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applications. Also, the stoichiometry of the N-dealkylation reaction needs to be defined

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by identifying the total repertoire of metabolites. All of these matters are crucial for better

321

understanding of how oxyBAC functions, which is critical for BAC degradation in the

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environment.

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The enzymes degrading BACs and QACs are diverse in terms of structure and

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phylogeny. This suggests that there are possibly a number of enzymes in the environment

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yet to be discovered. Candidate QAC degrader strains and enzymes can be identified and

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tested using ongoing whole genome sequencing projects.

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In summary, the discovery of oxyBAC as a novel RO enzyme expands our

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knowledge on the biotransformation mechanisms of disinfectants in the environment and

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applications and complications it provides.

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331

SUPPORTING INFORMATION

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oxyBAC amino acid sequence; biotransformation reactions of stachydrine, glycine

333

betaine, carnitine, choline and BACs by stc2, gbcA, cntA, cmo and oxyBAC,

334

respectively; pET29a(+) plasmid map showing the locations of oxyBAC and other genes

335

as well as restriction sites and SDS-PAGE image of crude protein extracts of uninduced

336

and induced E. coli cells with pET29a(+) plasmid with oxyBAC gene. This material is

337

available free of charge via the Internet at http://pubs.acs.org.

338

339

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ACKNOWLEDGEMENT

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This study was partially supported by Bogazici University Scientific Research Projects

343

Fund (BAP 7130), EU Research Executive Agency FP7-PEOPLE-MC-CIG

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(ROBODAR-293665), The Scientific and Technological Research Council of Turkey

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(TUBITAK, 113Y528 and BIDEB 2214a).

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TABLES

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Table1. Strains, plasmids and primers used in this study Strains, plasmids and primers Pseudomonas sp. BIOMIG1 ::aBAC ::BDMA

Source

A strain isolated from sewage taken from an urban wastewater treatment plant in Istanbul, TR Can utilize both BACs and BDMA Can convert BACs to BDMA but cannot utilize BDMA Cannot utilize BACs but can utilize BDMA Can utilize neither BACs nor BDMA

17

Escherichia coli BL21 (DE3)pLysS ::oxyBAC ::N

Heterologous expression of oxyBAC under the T7 promoter Contains pET29+ with oxyBAC Contains pET29+ with no oxyBAC

Invitrogen

Plasmids pET29a+

For overexpression of oxyBAC

::BD ::N

349

Description

Primers oxyBAC-F 5’ -TTATATGCAACAGATCCATCCCCT-3’ oxyBAC-R 5’-TTATGTACGCCGTAGGCGGGGCC-3’ a appear as superscrips in the text

17 17

17 17

This study This study

Merc Millipore

This study This study

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FIGURES

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Figure 1. Phylogenetic relationship of oxyBAC, ttmao, BAC amine oxidase, microbial

353

Rieske type terminal oxygenases (Groups I-IV) and eukaryotic Rieske type choline

354

monooxygenases. Benzylsuccinate synthase was used as the outgroup. Amino acid

355

sequences were aligned using CLUSTLW, genetic distances were calculated using Jukes-

356

Cantor model and the tree was built with neighbor-joining model using a bootstrap value

357

of 1000. Reactions show generalized mechanisms of oxidation of substrates by Rieske

358

type oxygenases.

359 360

Figure 2. (A) oxyBAC PCR amplicons of four BIOMIG1 phenotypes on agarose gel.

361

Profile of C12BDMA-Cl utilization, BDMA formation and cell growth by (B)

362

BIOMIG1BAC, (C) BIOMIG1BDMA, (D) BIOMIG1BD and (E) BIOMIG1N (Error bars

363

represent one standard deviation of the means, n = 3).

364 365

Figure 3. (A) oxyBAC PCR amplicons of two E. coli phenotypes on agarose gel. Profile

366

of C12BDMA-Cl utilization and BDMA formation by (B) E. coliN and (C) E. colioxyBAC

367

(Error bars represent one standard deviation of the means, n = 3).

368 369

Figure 4. Multiple sequence alignment of (A) Rieske domain and (B) mononuclear iron

370

center of oxyBAC with other Group V Rieske oxygenases and oxyBAC of

371

Novosphingobium sp. B7. ↑ and ∆ denote amino acid insertion and substitution,

372

respectively.

373

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Figure 1. Phylogenetic relationship of oxyBAC, ttmao, BAC amine oxidase, microbial

377

Rieske type terminal oxygenases (Groups I-IV) and eukaryotic Rieske type choline

378

monooxygenases. Benzylsuccinate synthase was used as the outgroup. Amino acid

379

sequences were aligned using CLUSTLW, genetic distances were calculated using Jukes-

380

Cantor model and the tree was built with neighbor-joining model using a bootstrap value

381

of 1000. Reactions show generalized mechanisms of oxidation of substrates by Rieske

382

type oxygenases.

383

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384 385

Figure 2. (A) oxyBAC PCR amplicons of four BIOMIG1 phenotypes on agarose gel.

386

Profile of C12BDMA-Cl utilization, BDMA formation and cell growth by (B)

387

BIOMIG1BAC, (C) BIOMIG1BDMA, (D) BIOMIG1BD and (E) BIOMIG1N (Error bars

388

represent one standard deviation of the means, n = 3).

389

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Figure 3. (A) oxyBAC PCR amplicons of two E. coli phenotypes on agarose gel. Profile

392

of C12BDMA-Cl utilization and BDMA formation by (B) E. coliN and (C) E. colioxyBAC

393

(Error bars represent one standard deviation of the means, n = 3).

394

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Figure 4. Multiple sequence alignment of (A) Rieske domain and (B) mononuclear iron

397

center of oxyBAC with other Group V Rieske oxygenases and oxyBAC of

398

Novosphingobium sp. B7. ↑ and ∆ denote amino acid insertion and substitution,

399

respectively.

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GRAPHICAL ABSTRACT

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