title: Development of a pyrE-based selective system for Thermotoga sp. strain RQ7.
abstract: To fully unlock the biotechnological potentials of Thermotoga species, this study aimed to expand the genetic toolbox of Thermotoga by developing a new selective system. The developed system was composed of two components: a recipient strain bearing a deletion in its orotate phosphoribosyltransferase gene pyrE and a shuttle vector expressing a heterologous pyrE as the selectable marker. A spontaneous uracil auxotroph, T. sp. strain RQ7-15, was isolated at 70 °C with 2 mg/ml 5-fluoroorotic acid. The mutant carried a 112 bp deletion in pyrE and was a suitable recipient strain. To avoid homologous recombination, the pyrE gene from another thermophilic bacterium Caldicellulosiruptor saccharolyticus was used as the selectable marker. The gene was cloned into two Thermotoga-E. coli shuttle vectors, controlled by different promoters: the promoter of Thermus S-layer protein (PslpA) in pDH25 and the promoter of the pyrimidine synthesis operon of T. sp. strain RQ7 (PRQ7.pyr) in pDH28. After being introduced into the mutant strain RQ7-15 through natural transformation, both vectors allowed the host to thrive in a minimal medium. Single colonies of transformants were isolated and confirmed by polymerase chain reactions and restriction digestions. In summary, a pyrE-based selective system has been established in T. sp. strain RQ7.
authors.affiliation: Department of Biological Sciences, Bowling Green State University, Bowling Green, OH, 43403, USA.
authors.forename: Dongmei
authors.initials: D
authors.lastname: Han
authors.affiliation: Department of Biological Sciences, Bowling Green State University, Bowling Green, OH, 43403, USA. zxu@bgsu.edu.
authors.forename: Zhaohui
authors.initials: Z
authors.lastname: Xu
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D005819:Genetic Markers; D016965:Gram-Negative Anaerobic Straight, Curved, and Helical Rods / Q000235:genetics*; D009876:Operon; D009962:Orotate Phosphoribosyltransferase / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D005819:Genetic Markers; D009962:Orotate Phosphoribosyltransferase
keywords: 5-FOA; RQ7; RQ7-15; Thermotoga; Uracil auxotroph; pyrE