title: A novel inducible protein production system and neomycin resistance as selection marker for Methanosarcina mazei.
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.
authors.affiliation: Institute for Microbiology and Biotechnology, University of Bonn, 53115 Bonn, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Mondorf
authors.forename: Uwe
authors.initials: U
authors.lastname: Deppenmeier
authors.forename: Cornelia
authors.initials: C
authors.lastname: Welte
mesh_terms: D000345:Affinity Labels / Q000378:metabolism; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D015415:Biomarkers / Q000378:metabolism; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003470:Culture Media / Q000378:metabolism; D004352:Drug Resistance, Microbial; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D000432:Methanol / Q000378:metabolism; D017020:Methanosarcina / Q000187:drug effects / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D008744:Methylamines / Q000378:metabolism; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism; D009355:Neomycin / Q000494:pharmacology*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011691:Puromycin / Q000494:pharmacology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation
chemical_list: D000345:Affinity Labels; D019843:Archaeal Proteins; D015415:Biomarkers; D003470:Culture Media; D008744:Methylamines; D011994:Recombinant Proteins; D011691:Puromycin; D008780:Methyltransferases; D005966:Glucuronidase; D009355:Neomycin; C023336:trimethylamine; D000432:Methanol