Table of Contents Author Guidelines Submit a Manuscript
Archaea
Volume 2015 (2015), Article ID 483194, 16 pages
http://dx.doi.org/10.1155/2015/483194
Research Article

Isolation and Molecular Identification of Auxotrophic Mutants to Develop a Genetic Manipulation System for the Haloarchaeon Natrinema sp. J7-2

1State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China
2Henan Academy of Medical and Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450052, China
3Hubei Provincial Cooperative Innovation Center of Industrial Fermentation, Wuhan 430072, China

Received 19 February 2015; Revised 30 March 2015; Accepted 23 April 2015

Academic Editor: Sonja-Verena Albers

Copyright © 2015 Jie Lv et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Our understanding of the genus Natrinema is presently limited due to the lack of available genetic tools. Auxotrophic markers have been widely used to construct genetic systems in bacteria and eukaryotes and in some archaeal species. Here, we isolated four auxotrophic mutants of Natrinema sp. J7-2, via 1-methyl-3-nitro-1-nitroso-guanidin mutagenesis, and designated them as J7-2-1, J7-2-22, J7-2-26, and J7-2-52, respectively. The mutant phenotypes were determined to be auxotrophic for leucine (J7-2-1), arginine (J7-2-22 and J7-2-52), and lysine (J7-2-26). The complete genome and the biosynthetic pathways of amino acids in J7-2 identified that the auxotrophic phenotype of three mutants was due to gene mutations in leuB (J7-2-1), dapD (J7-2-26), and argC (J7-2-52). These auxotrophic phenotypes were employed as selectable makers to establish a transformation method. The transformation efficiencies were determined to be approximately 103 transformants per g DNA. And strains J7-2-1 and J7-2-26 were transformed into prototrophic strains with the wild type genomic DNA, amplified fragments of the corresponding genes, or the integrative plasmids carrying the corresponding genes. Additionally, exogenous genes, bgaH or amyH gene, were expressed successfully in J7-2-1. Thus, we have developed a genetic manipulation system for the Natrinema genus based on the isolated auxotrophic mutants of Natrinema sp. J7-2.