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

Figure 1

Growth behavior of the auxotrophic strains in different media. The method used to determine the nutrients required for the four auxotrophic mutants is described in Materials and Methods, after which the phenotypes of the mutants were confirmed. We picked out a single colony of each of the four mutants and cultivated it in a liquid MGM medium at 42°C. The cultures of each strain were obtained, centrifuged, and resuspended in MM medium. Then, the suspended cultures were inoculated in liquid media of MGM, MM, and MM with the corresponding nutrients added (as shown in the figure, leucine was added to the MM medium to reconfirm the phenotype of J7-2-1; lysine was added for reconfirming J7-2-26, arginine for J7-2-52, and threonine and arginine for J7-2-22). In corresponding solid media, the suspended cultures were diluted with MM media before spreading on the corresponding media plates. Their growth states were recorded and pictures were taken after storage at 42°C for 7–15 days. MGM: a complete medium that could support the growth of all J7-2 auxotrophic strains; MM: a minimal medium that could only support the growth of the J7-2 prototrophic strain; MM+aa: MM supplied with the relevant single amino acid to a final concentration of 50 μg/mL.