%0 Journal Article
%T Establishing a Markerless Genetic Exchange System for Methanosarcina mazei Strain G£¿1 for Constructing Chromosomal Mutants of Small RNA Genes
%A Claudia Ehlers
%A Dominik J ger
%A Ruth A. Schmitz
%J Archaea
%D 2011
%I Hindawi Publishing Corporation
%R 10.1155/2011/439608
%X 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 sRNA154. Characterizing M. mazei ¦¤154 under nitrogen limiting conditions demonstrated differential expression of at least three cytoplasmic proteins and reduced growth strongly arguing for a prominent role of sRNA154 in regulation of nitrogen fixation by posttranscriptional regulation.
%U http://www.hindawi.com/journals/arch/2011/439608/