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. 2002 Mar;184(5):1449-54.
doi: 10.1128/JB.184.5.1449-1454.2002.

Directed mutagenesis and plasmid-based complementation in the methanogenic archaeon Methanosarcina acetivorans C2A demonstrated by genetic analysis of proline biosynthesis

Jun Kai Zhang  1 , Andrea K White, H Craig Kuettner, Paolo Boccazzi, William W Metcalf
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Directed mutagenesis and plasmid-based complementation in the methanogenic archaeon Methanosarcina acetivorans C2A demonstrated by genetic analysis of proline biosynthesis

Jun Kai Zhang et al. J Bacteriol. 2002 Mar.

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.

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Figures

FIG. 1.
FIG. 1.
The M. acetivorans C2A proABC locus. The physical structure of a 5,139-bp fragment of the M. acetivorans chromosome is shown in the center (heavy black line), with the positions of the proA, proB, and proC genes shown as gray arrows. The regions covered by the six plasmids whose DNA sequences were determined are shown as thin black lines at the top, with the names of the respective plasmids shown below each line. The position and relative orientation of each in vitro-constructed insertion mutation are indicated by a flag on the center line. The black flags represent Tn5-407 insertions, the white flags represent Tn5-ileS12 insertions, and the gray flags represent insertions of the pac-ori-aph cassette. The regions contained in several plasmid shuttle vectors used for complementation studies are shown as thin black lines at the bottom. The names of the respective plasmids and antibiotic resistance markers are shown above each line. The endonuclease restriction sites used for construction of these shuttle vectors are indicated.
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