title: Establishing a markerless genetic exchange system for Methanosarcina mazei strain Gö1 for constructing chromosomal mutants of small RNA genes.
abstract: A markerless genetic exchange system was successfully established in Methanosarcina mazei strain Gö1 using the hpt gene coding for hypoxanthine phosphoribosyltransferase. First, a chromosomal deletion mutant of the hpt gene was generated conferring resistance to the purine analog 8-aza-2,6-diaminopurine (8-ADP). The nonreplicating allelic exchange vector (pRS345) carrying the pac-resistance cassette for direct selection of chromosomal integration, and the hpt gene for counterselection was introduced into this strain. By a pop-in and ultimately pop-out event of the plasmid from the chromosome, allelic exchange is enabled. Using this system, we successfully generated a M. mazei deletion mutant of the gene encoding the regulatory non-coding RNA sRNA(154). Characterizing M. mazei ΔsRNA(154) under nitrogen limiting conditions demonstrated differential expression of at least three cytoplasmic proteins and reduced growth strongly arguing for a prominent role of sRNA(154) in regulation of nitrogen fixation by posttranscriptional regulation.
authors.affiliation: Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität zu Kiel, Kiel, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Ehlers
authors.forename: Dominik
authors.initials: D
authors.lastname: Jäger
authors.forename: Ruth A
authors.initials: RA
authors.lastname: Schmitz
mesh_terms: D019847:Chromosomes, Archaeal; D017353:Gene Deletion; D022761:Gene Transfer, Horizontal; D005827:Genetics, Microbial / Q000379:methods*; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D017020:Methanosarcina / Q000235:genetics*; D010957:Plasmids; D019642:RNA, Archaeal / Q000235:genetics*; D022661:RNA, Untranslated / Q000235:genetics*; D011995:Recombination, Genetic; D012641:Selection, Genetic
chemical_list: D019642:RNA, Archaeal; D022661:RNA, Untranslated; D007041:Hypoxanthine Phosphoribosyltransferase