Establishing a Markerless Genetic Exchange System forMethanosarcina mazeiStrain Gö1 for Constructing Chromosomal Mutants of Small RNA Genes

[摘要] A markerless genetic exchange system was successfully established inMethanosarcina mazeistrain Gö1 using thehptgene coding for hypoxanthine phosphoribosyltransferase. First, a chromosomal deletion mutant of thehptgene was generated conferring resistance to the purine analog 8-aza-2,6-diaminopurine (8-ADP). The nonreplicating allelic exchange vector (pRS345) carrying thepac-resistance cassette for direct selection of chromosomal integration, and thehptgene 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 aM. mazeideletion mutant of the gene encoding the regulatory non-coding RNA sRNA154. CharacterizingM. mazeiΔsRNA154under nitrogen limiting conditions demonstrated differential expression of at least three cytoplasmic proteins and reduced growth strongly arguing for a prominent role of sRNA154in regulation of nitrogen fixation by posttranscriptional regulation.

[学科分类] 微生物学和免疫学