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

Department of Microbiology, B103 Chemical and Life Science Laboratory, University of Illinois, 601 S. Goodwin Ave., Urbana, IL 61801, USA.
Journal of Bacteriology (Impact Factor: 2.81). 04/2002; 184(5):1449-54. DOI: 10.1128/JB.184.5.1449-1454.2002
Source: PubMed


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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    • "Multicopy expression can be achieved by using the E. coli/Methanosarcina shuttle vector pWM321 (Metcalf et al., 1997; Zhang et al., 2002). The plasmid offers a large multi cloning site but no promoter to drive the expression of the gene of interest and no RBS. "
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    Frontiers in Microbiology 07/2012; 3:259. DOI:10.3389/fmicb.2012.00259 · 3.99 Impact Factor
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    • "Furthermore, it is possible to generate chromosomal deletion mutants using selection markers [4] or clean deletion systems [5] [6]. However, there are certain limitations: there is only puromycin available as a selectable marker and there is no protein production system allowing affinity purification of recombinant enzymes. "
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    Archaea 07/2012; 2012(4):973743. DOI:10.1155/2012/973743 · 2.71 Impact Factor
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    • "This study pAMG109 φC31-attB vector with M. barkeri mcrB promoter fusion to uidA with a AAA start site This study pJK026A φC31-attB vector with PmcrB promoter fusion to uidA This study pJK027A φC31-attB vector with PmcrB(tetO1) promoter fusion to uidA This study pJK028A φC31-attB vector with PmcrB(tetO3) promoter fusion to uidA This study pJK029A φC31-attB vector with PmcrB(tetO4) promoter fusion to uidA This study pJK031A φC31-attP vector with PmcrB(tetO1) promoter fusion to uidA This study pJK032A φC31-attP vector with PmcrB(tetO3) promoter fusion to uidA This study pJK033A φC31-attP vector with PmcrB(tetO4) promoter fusion to uidA This study pJK200 Fosmid vector encoding chloramphenicol and puromycin resistance with oriV, lattP and fC31-attB This study pWM321 E. coli/Methanosarcina shuttle vector (Metcalf et al. 1997) pWM357 Fosmid cloning vector (Zhang et al. 2002) pGK50A Vector for testing gene essentially using PmcrB(tetO1), encodes kanamycin and puromycin resistance This study pGK51A Vector for testing gene essentially using PmcrB(tetO3), encodes kanamycin and puromycin resistance This study pGK52A Vector for testing gene essentially using PmcrB(tetO4), encodes kanamycin and puromycin resistance This study pGK50B Vector for testing gene essentially using PmcrB(tetO1), encodes kanamycin and puromycin resistance, tetR gene is in opposite orientation to pGK50A "
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    ABSTRACT: A highly efficient method for chromosomal integration of cloned DNA into Methanosarcina spp. was developed utilizing the site-specific recombination system from the Streptomyces phage phiC31. Host strains expressing the phiC31 integrase gene and carrying an appropriate recombination site can be transformed with non-replicating plasmids carrying the complementary recombination site at efficiencies similar to those obtained with self-replicating vectors. We have also constructed a series of hybrid promoters that combine the highly expressed M. barkeri PmcrB promoter with binding sites for the tetracycline-responsive, bacterial TetR protein. These promoters are tightly regulated by the presence or absence of tetracycline in strains that express the tetR gene. The hybrid promoters can be used in genetic experiments to test gene essentiality by placing a gene of interest under their control. Thus, growth of strains with tetR-regulated essential genes becomes tetracycline-dependent. A series of plasmid vectors that utilize the site-specific recombination system for construction of reporter gene fusions and for tetracycline regulated expression of cloned genes are reported. These vectors were used to test the efficiency of translation at a variety of start codons. Fusions using an ATG start site were the most active, whereas those using GTG and TTG were approximately one half or one fourth as active, respectively. The CTG fusion was 95% less active than the ATG fusion.
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