A genetic system for Archaea of the genus
            <i>Methanosarcina</i>
            : Liposome-mediated transformation and construction of shuttle vectors

et al. 2008

Archaea

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116

179

Summary 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 φC31. Host strains expressing the φC31 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.

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

New methods for tightly regulated gene expression and highly efficient chromosomal integration of cloned genes for Methanosarcina species

Guss

Rother

et al. 2008

Archaea

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116

179

“…In vivo testing of these putative functional roles for Ech has not previously been possible because of a lack of methods for genetic analysis of Methanosarcina species; however, recent advances in the field now make such a study feasible (17)(18)(19)(20). In this report, we describe construction and characterization of an M. barkeri mutant lacking Ech.…”

mentioning

confidence: 99%

Genetic analysis of the archaeon Methanosarcina barkeri Fusaro reveals a central role for Ech hydrogenase and ferredoxin in methanogenesis and carbon fixation

Meuer

Kuettner

Proc. Natl. Acad. Sci. U.S.A.

et al. 2002

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166

174

(ii) Under autotrophic growth conditions, the enzyme catalyzes the energetically unfavorable reduction of ferredoxin by H 2, most probably driven by reversed electron transport, and the reduced ferredoxin thus generated functions as low potential electron donor for the synthesis of pyruvate in an anabolic pathway. (iii) Reduced ferredoxin in addition provides the reducing equivalents for the first step of methanogenesis from H 2͞CO2, the reduction of CO2 to formylmethanofuran. Thus, in vivo genetic analysis has led to the identification of the electron donor of this key initial step of methanogenesis.

“…This problem has been alleviated somewhat in recent years by the development of genetic tools for use in members of the two distantly related genera, Methanococcus and Methanosarcina. These tools include selectable markers for genetic crosses (4-7), methods for high efficiency transformation (8,9), plasmid shuttle vectors (8,10), and methods for gene replacement (4,11,12) (the latter only in Methanococcus species). Despite these advances there continues to be a need for development of new tools for genetic analysis of methanoarchaea.…”

mentioning

confidence: 99%

In vivo transposon mutagenesis of the methanogenic archaeon Methanosarcina acetivorans C2A using a modified version of the insect mariner -family transposable element Himar1

Proc. Natl. Acad. Sci. U.S.A.

Pritchett

Lampe

et al. 2000

We present here a method for in vivo transposon mutagenesis of a methanogenic archaeon, Methanosarcina acetivorans C2A, which because of its independence from host-specific factors may have broad application among many microorganisms. Because there are no known Methanosarcina transposons we modified the mariner transposable element Himar1, originally found in the insect Hematobia irritans, to allow its use in this organism. This element was chosen because, like other mariner elements, its transposition is independent of host factors, requiring only its cognate transposase. Modified mini-Himar1 elements were constructed that carry selectable markers that are functional in Methanosarcina species and that express the Himar1 transposase from known Methanosarcina promoters. These mini-mariner elements transpose at high frequency in M. acetivorans to random sites in the genome. The presence of an Escherichia coli selectable marker and plasmid origin of replication within the mini-mariner elements allows facile cloning of these transposon insertions to identify the mutated gene. In preliminary experiments, we have isolated numerous mini-mariner-induced M. acetivorans mutants, including ones with insertions that confer resistance to toxic analogs and in genes that encode proteins involved in heat shock, nitrogen fixation, and cell-wall structures.