Directed Mutagenesis and Plasmid-Based Complementation in the Methanogenic Archaeon Methanosarcina acetivorans C2A Demonstrated by Genetic Analysis of Proline Biosynthesis

We report here the first use of directed mutagenesis in Methanosarcina acetivorans C2A. The method employs homologous recombination-mediated gene replacement and was used to construct a variety of proline auxotrophs with mutations in the proABC locus. Each mutation was also complemented in trans with autonomously replicating Methanosarcina-Escherichia plasmid shuttle vectors.