Integration of Foreign DNA in an Intergenic Region of the ArchaeonMethanosarcina mazeiWithout Effect on Transcription of Adjacent Genes

Macario, Everly Conway de; Guerrini, Massimo; Dugan, Charles B.; Macario, Alberto J. L.

doi:10.1006/jmbi.1996.0494

Cited by 17 publications

(11 citation statements)

References 16 publications

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“…For example, there is no information available about origins of DNA replication, and our understanding of mechanisms governing gene expression in these organisms is rudimentary. Systems for genetic transformation have been described for some methanogens (Gernhardt et al ., 1990; Conway de Macario et al ., 1996; Metcalf et al ., 1997; Whitman et al ., 1997), but not for Methanococcus jannaschii or Methanobacterium thermoautotrophicum ΔH whose complete genome sequences are known (Bult et al ., 1996; Smith et al ., 1997). The need for systems to enable studies on the effects of genetic manipulation in vivo has led to an intensive search for extrachromosomal elements with potential for use as cloning vectors.…”

Section: Introductionmentioning

confidence: 99%

Molecular analysis of Methanobacterium phage ΨM2

Pfister

Wasserfallen

Stettler

et al. 1998

Molecular Microbiology

133

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SummaryThe methanogenic archaeon Methanobacterium thermoautotrophicum Marburg is infected by the doublestranded DNA phage ⌿M2. The complete phage genome sequence of 26 111 bp was established. Thirty-one open reading frames (orfs), all of them organized in the same direction of transcription, were identified. On the basis of comparison of the deduced amino acid sequences to known proteins and by searching for conserved motifs, putative functions were assigned to the products of six orfs. These included three proteins involved in packaging DNA into the capsid, two putative phage structural proteins and a protein related to the Int family of site-specific recombinases. Analysis of the N-terminal amino acid sequences of three phageencoded proteins led to the identification of two genes encoding structural proteins and of peiP, the structural gene of pseudomurein endoisopeptidase. This enzyme is involved in the lysis of host cells, and it appears to belong to a novel enzyme family. peiP was overexpressed in Escherichia coli, and its product was shown to catalyse the in vitro lysis of M. thermoautotrophicum cells. Comparison of the phage ⌿M2 DNA sequence with parts of the sequence of the wild-type phage ⌿M1 suggests that ⌿M2 is a deletion derivative, which formed by homologous recombination between two copies of a direct repeat.

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Section: Introductionmentioning

confidence: 99%

Molecular analysis of Methanobacterium phage ΨM2

Pfister

Wasserfallen

Stettler

et al. 1998

Molecular Microbiology

133

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“…These tools include plasmid shuttle vectors (Metcalf et al 1997), very high efficiency transformation (Metcalf et al 1997), random in vivo transposon mutagenesis , directed mutagenesis of specific genes , multiple selectable markers (Boccazzi et al 2000), reporter gene fusions (M. Pritchett and W. Metcalf, unpubl. ), integration vectors (Conway de Macario et al 1996), and anaerobic incubators for large-scale growth of methanogens on solid media (Metcalf et al 1998). Furthermore, and in contrast to other known methanogens, genetic analysis can be used to study the process of methanogenesis: Because Methanosarcina species are able to utilize each of the three known methanogenic pathways, mutants in a single pathway are viable (M. Pritchett and W. Metcalf, unpubl.).…”

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confidence: 99%

The Genome of M. acetivorans Reveals Extensive Metabolic and Physiological Diversity

Galagan¹,

Nusbaum²,

Roy³

et al. 2002

Genome Res.

Self Cite

578

456

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Methanogenesis, the biological production of methane, plays a pivotal role in the global carbon cycle and contributes significantly to global warming. The majority of methane in nature is derived from acetate. Here we report the complete genome sequence of an acetate-utilizing methanogen, Methanosarcina acetivorans C2A. Methanosarcineae are the most metabolically diverse methanogens, thrive in a broad range of environments, and are unique among the Archaea in forming complex multicellular structures. This diversity is reflected in the genome of M. acetivorans.

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“…In the methanogenic archaea, shuttle vectors and gene disruption systems have been developed in Methanococcus maripaludis based on puromycin and the puromycin N-acetyltransferase gene (pac) (25,48,61) or neomycin and aminoglycoside phosphotransferase genes (2). The puromycin-pac system has also been applied for genetic manipulation in Methanococcus voltae (6,7,60), as well as in various Methanosarcina species (19,44). Genetic transformation has also been observed in the thermophilic methanogen Methanobacterium thermoautotrophicum (now Methanothermobacter thermautotrophicus) (64).…”

mentioning

confidence: 99%

Disruption of a Sugar Transporter Gene Cluster in a Hyperthermophilic Archaeon Using a Host-Marker System Based on Antibiotic Resistance

et al. 2007

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We have developed a gene disruption system in the hyperthermophilic archaeon Thermococcus kodakaraensis using the antibiotic simvastatin and a fusion gene designed to overexpress the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene (hmg Tk ) with the glutamate dehydrogenase promoter. With this system, we disrupted the T. kodakaraensis amylopullulanase gene (apu Tk ) or a gene cluster which includes apu Tk and genes encoding components of a putative sugar transporter. Disruption plasmids were introduced into wild-type T. kodakaraensis KOD1 cells, and transformants exhibiting resistance to 4 M simvastatin were isolated. The transformants exhibited growth in the presence of 20 M simvastatin, and we observed a 30-fold increase in intracellular HMG-CoA reductase activity. The expected gene disruption via double-crossover recombination occurred at the target locus, but we also observed recombination events at the hmg Tk locus when the endogenous hmg Tk gene was used. This could be avoided by using the corresponding gene from Pyrococcus furiosus (hmg Pf ) or by linearizing the plasmid prior to transformation. While both gene disruption strains displayed normal growth on amino acids or pyruvate, cells without the sugar transporter genes could not grow on maltooligosaccharides or polysaccharides, indicating that the gene cluster encodes the only sugar transporter involved in the uptake of these compounds. The ⌬apu Tk strain could not grow on pullulan and displayed only low levels of growth on amylose, suggesting that Apu Tk is a major polysaccharide-degrading enzyme in T. kodakaraensis.

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Integration of Foreign DNA in an Intergenic Region of the ArchaeonMethanosarcina mazeiWithout Effect on Transcription of Adjacent Genes

Cited by 17 publications

References 16 publications

Molecular analysis of Methanobacterium phage ΨM2

Molecular analysis of Methanobacterium phage ΨM2

The Genome of M. acetivorans Reveals Extensive Metabolic and Physiological Diversity

Disruption of a Sugar Transporter Gene Cluster in a Hyperthermophilic Archaeon Using a Host-Marker System Based on Antibiotic Resistance

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