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.

Download full-text


Available from: Paolo Boccazzi
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The discovery of the third domain of life, the Archaea, is one of the most exciting findings of the last century. These remarkable prokaryotes are well known for their adaptations to extreme environments; however, Archaea have also conquered moderate environments. Many of the archaeal biochemical processes, such as methane production, are unique in nature and therefore of great scientific interest. Although formerly restricted to biochemical and physiological studies, sophisticated systems for genetic manipulation have been developed during the last two decades for methanogenic archaea, halophilic archaea and thermophilic, sulfur-metabolizing archaea. The availability of these tools has allowed for more complete studies of archaeal physiology and metabolism and most importantly provides the basis for the investigation of gene expression, regulation and function. In this review we provide an overview of methods for genetic manipulation of Methanosarcina spp., a group of methanogenic archaea that are key players in the global carbon cycle and which can be found in a variety of anaerobic environments.
    Preview · Article · Jul 2012 · Frontiers in Microbiology
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Methanosarcina mazei is one of the model organisms for the methanogenic order Methanosarcinales whose metabolism has been studied in detail. However, the genetic toolbox is still limited. This study was aimed at widening the scope of utilizable methods in this group of organisms. (i) Proteins specific to methanogens are oftentimes difficult to produce in E. coli. However, a protein production system is not available for methanogens. Here we present an inducible system to produce Strep-tagged proteins in Ms. mazei. The promoter p1687, which directs the transcription of methyl transferases that demethylate methylamines, was cloned into plasmid pWM321 and its activity was determined by monitoring β-glucuronidase production. The promoter was inactive during growth on methanol but was rapidly activated when trimethylamine was added to the medium. The gene encoding the β-glucuronidase from E. coli was fused to a Strep-tag and was cloned downstream of the p1687 promoter. The protein was overproduced in Ms. mazei and was purified in an active form by affinity chromatography. (ii) Puromycin is currently the only antibiotic used as a selectable marker in Ms. mazei and its relatives. We established neomycin resistance as a second selectable marker by designing a plasmid that confers neomycin resistance in Ms. mazei.
    Full-text · Article · Jul 2012 · Archaea
  • Source
    • "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 "
    [Show abstract] [Hide abstract]
    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.
    Full-text · Article · Jan 2009 · Archaea (Vancouver, B.C.)
Show more