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

Abstract

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.