Isolation and molecular identification of auxotrophic mutants to develop a genetic manipulation system for the haloarchaeon natrinema sp. J7-2

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 μ 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.