FEMS Microbiology Letters, 366, 2019, fnz233 https://doi.org/10.1093/femsle/fnz233 Advance Access Publication Date: 20 November 2019 Research Letter R E S E A R C H L E T T E R – Environmental Microbiology Assembly of an atrazine catabolic operon and its introduction to Gram-negative hosts for robust and stable degradation of triazine herbicides Alfredo Lazarini-Mart´ınez 1,† , Abigail P ´ erez-Valdespino 1,† , Fernando Hern ´ andez Mart´ınez 1 , Nora Ruiz Ordaz 2 , Juvencio Gal´ındez-Mayer 2 , Cleotilde Ju ´ arez-Ram´ırez 2 and Everardo Curiel-Quesada 1, * 1 Department of Biochemistry, Escuela Nacional de Ciencias Biol´ ogicas del Instituto Polit ´ ecnico Nacional, Prolongaci ´ on de Carpio y Plan de Ayala S/N, Col. Santo Tom´ as. CP11340 Mexico City, Mexico and 2 Department of Biochemical Engineering, Escuela Nacional de Ciencias Biol ´ ogicas del Instituto Polit ´ ecnico Nacional. Av. Wilfrido Massieu 399, Unidad Adolfo L ´ opez Mateos, CP07738 Mexico City, Mexico ∗ Corresponding author: Department of Biochemistry, Escuela Nacional de Ciencias Biolgicas. Prolongacion de Carpio y Plan de Ayala. CP 53100. Mexico City, Mexico. Tel: +5255-5729-6000; E-mail: ecqmixcoacdf@gmail.com One sentence summary: Synthetic operon for highly efficient atrazine degradation. † These authors contributed equally to this work. Editor: Hermann Heipieper ABSTRACT In 1995, Pseudomonas sp. ADP, capable of metabolizing atrazine, was isolated from contaminated soil. Genes responsible for atrazine mineralization were found scattered in the 108.8 kb pADP-1 plasmid carried by this strain, some of them flanked by insertion sequences rendering them unstable. The goal of this work was to construct a transcriptional unit containing the atz operon in an easy to transfer manner, to be introduced and inherited stably by Gram-negative bacteria. atz genes were PCR amplified, joined into an operon and inserted onto the mobilizable plasmid pBAMD1–2. Primers were designed to add efficient transcription and translation signals. Plasmid bearing the atz operon was transferred to different Gram-negative strains by conjugation, which resulted in Tn5 transposase-mediated chromosomal insertion of the atz operon. To test the operon activity, atrazine degradation by transposants was assessed both colorimetrically and by high-performance liquid chromatography (HPLC). Transposants mineralized atrazine more efficiently than wild-type Pseudomonas sp. ADP and did not accumulate cyanuric acid. Atrazine degradation was not repressed by simple nitrogen sources. Genes conferring atrazine-mineralizing capacities were stable and had little or null effect on the fitness of different transposants. Introduction of catabolic operons in a stable fashion could be used to develop bacteria with better degrading capabilities useful in bioremediation. Keywords: atz operon; transferable unit; atrazine degradation INTRODUCTION The continued increase in the world’s population demands agricultural technologies that enable farmers around the world to produce crops with higher yields, while also tending to a fragile environment and preserving the valuable resources of soil and water. Modern agriculture relies more and more on the use of products that help to control undesired plants. Starting in the 1940s, chemical industry developed a variety of Received: 19 August 2019; Accepted: 19 November 2019 C  The Author(s) 2019. Published by Oxford University Press on behalf of FEMS. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com 1 Downloaded from https://academic.oup.com/femsle/article/366/19/fnz233/5634263 by guest on 06 September 2022