Opening the s-triazine ring and biuret hydrolysis during conversion of atrazine by Frankia sp. strain EuI1c Medhat Rehan a, c, * , Gomaah El Fadly a , Mona Farid a , Aml El sharkawy a , Stefan Fr € anzle b , Ren  e Ullrich b , Harald Kellner b , Martin Hofrichter b a Department of Genetics, Kafrelsheikh University, 33516, Kafr El-Sheikh, Egypt b Department of Bio- and Environmental Sciences, International Institute Zittau, TU Dresden, Markt 23, 02763 Zittau, Germany c Department of Plant Production and Protection, College of Agriculture and Veterinary Medicine, Qassim University, Saudi Arabia article info Article history: Received 16 September 2016 Received in revised form 24 October 2016 Accepted 4 November 2016 Keywords: Frankia s-triazine ring-cleavage Reverse genetics qRT PCR abstract Three genes have been identified through sequence analysis to encode putative AtzD/TrzD and AtzE enzymes and a TrzR transcriptional regulator via deduced amino acid sequences of functional AtzD/TrzD and/or AtzE published in the literature via a query sequence for homologues at the protein level using BLASTP. The operon was predicted to encode an s-triazine ring-opening amidohydrolase, TrzD (FraEuI1c_3137) and a GntR family transcriptional regulator, TrzR (FraEuI1c_3136), which may regulate the expression of ring-cleavage enzyme whereas the putative atzE (FraEuI1c_1007) gene encodes aspartyl/glutamyl-tRNA (Asn/Gln) amidotransferase subunit A in the course of s-triazine degradation by Frankia strain EuI1c. LC-MS analysis of Frankia sp EuI1c culture filtrates grown in the presence of atrazine or desethyl-desisopropylatrazine revealed a metabolite with a molecular ion (major peak) of m/z 102.7, which was identified as biuret. The trzD (FraEuI1c_3137) gene expression increased up to 4.7-fold in its abundance under 2 mM atrazine exposure when qRT-PCR was applied. Moreover, the mRNA level of the putative trzR (FraEuI1c_3136) gene that is proposed to regulate the gene function of FraEuI1c_3137 exhibited a dose-response and peaked at 2 mM atrazine with a 6.5 fold-increased mRNA level. The putative atzE (FraEuI1c_1007) mRNA level exhibited dose-respond and upregulated up to 10-fold change under the same stress dose. © 2016 Elsevier Ltd. All rights reserved. 1. Introduction Most of the industrial xenobiotic chemicals that cause envi- ronmental hazards can be degraded by microorganisms, which thereby contribute to the balance of the global carbon cycle. It is nature's way of recycling wastes that it can break down organic substance to smaller compounds by enzymes produced in bacteria, fungi or algae (Reineke, 2001). S-Triazines are a class of xenobiotic compounds that have been widely applied as herbicides and were formerly used worldwide to control broadleaf and grassy weeds in cereal, corn, sorghum and sugarcane growing and thus helped to gain high yields in agricul- ture. Atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5- triazine) is the most frequently applied s-triazine herbicide and still in use in a few advanced and many developing countries for crop protection. Atrazine in the environment behaves as a moder- ately persistent chemical in concentrations exceeding the maximum tolerable level recommended by the U.S. Environmental Protection Agency (3 mgL 1 )(Mandelbaum et al., 1993). Cyanuric acid is a hydroxylated intermediate in the degradation of several s- triazine herbicides and melamine. It can be further converted by the amidohydrolases AtzDEF or TrzDEF to eventually end up with carbon dioxide and ammonia. AtzD and/or TrzD hydrolyze cyanuric acid to release carboxybiuret, which spontaneously decarboxylates to biuret. A variety of gram-positive and gram-negative bacteria (e.g. Pseudomonas sp. strain NRRL B-12227, Pseudomonas sp. Strain ADP, Moorella thermoacetica ATCC 39073, Microbacterium Abbreviations: atzD, Atrazine D; trzD, Triazine D; atzE, Atrazine E; trzE, Triazine E; LC-MS, Liquid Chromatography-Mass Spectrometry; AtzDEF, Atrazine gene D, E and F; TrzDEF, Tirazine gene D, E and F; atzR, Atrazine R; trzN, Triazine N; qRT-PCR, Quantitative real Time-polymerase chain reaction; mRNA, Messenger ribonucleic acid; MOPS, 3-(N-morpholino)propanesulfonic acid; DEA, Desethylatrazine; DEDIA, Desethyl-desisopropylatrazine. * Corresponding author. Department of Genetics, Kafrelsheikh University, 33516, Kafr El-Sheikh, Egypt. Tel.: þ20 1006976664; fax: þ20 479102930. E-mail address: medhat.rehan@agr.kfs.edu.eg (M. Rehan). Contents lists available at ScienceDirect International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod http://dx.doi.org/10.1016/j.ibiod.2016.11.013 0964-8305/© 2016 Elsevier Ltd. All rights reserved. International Biodeterioration & Biodegradation 117 (2017) 14e21