Genetic Methods and Construction of Chromosomal Mutations in Methanogenic Archaea

Genetic manipulation through markerless exchange enables the modification of several genomic regions without leaving a selection marker in the genome. Here, a method using hpt coding for hypoxanthine phosphoribosyltransferase as a counter selectable marker is described. For Methanosarcina species a chromosomal deletion of the hpt gene is firstly generated, which confers resistance to the purine analogue 8-aza-2,6-diaminopurine (8-ADP). In a second step, the reintroduction of the hpt gene on a plasmid leads to a selectable loss of 8-ADP resistance after a homologous recombination event (pop-in). A subsequent pop-out event restores the 8-ADP resistance and can generate chromosomal mutants with frequencies of about 50%.