title: Directed mutagenesis and plasmid-based complementation in the methanogenic archaeon Methanosarcina acetivorans 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.
authors.affiliation: Department of Microbiology, B103 Chemical and Life Science Laboratory, University of Illinois, 601 S. Goodwin Ave., Urbana, IL 61801, USA.
authors.forename: Jun Kai
authors.initials: JK
authors.lastname: Zhang
authors.forename: Andrea K
authors.initials: AK
authors.lastname: White
authors.forename: H Craig
authors.initials: HC
authors.lastname: Kuettner
authors.forename: Paolo
authors.initials: P
authors.lastname: Boccazzi
authors.forename: William W
authors.initials: WW
authors.lastname: Metcalf
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000235:genetics; D019843:Archaeal Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D019766:Genes, Archaeal; D005816:Genetic Complementation Test / Q000379:methods*; D050698:Glutamate-5-Semialdehyde Dehydrogenase; D017020:Methanosarcina / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods*; D017851:Phosphotransferases (Carboxyl Group Acceptor) / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D011392:Proline / Q000096:biosynthesis*; D011762:Pyrroline Carboxylate Reductases / Q000235:genetics; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA
chemical_list: D019843:Archaeal Proteins; D011392:Proline; D000445:Aldehyde Oxidoreductases; D050698:Glutamate-5-Semialdehyde Dehydrogenase; D011762:Pyrroline Carboxylate Reductases; D017851:Phosphotransferases (Carboxyl Group Acceptor); C020650:glutamate 5-kinase