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.
Zhang
Jun Kai
JK
Department of Microbiology, B103 Chemical and Life Science Laboratory, University of Illinois, 601 S. Goodwin Ave., Urbana, IL 61801, USA.
White
Andrea K
AK
Kuettner
H Craig
HC
Boccazzi
Paolo
P
Metcalf
William W
WW
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
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