Patent ID: 7390620

Claim:
Method for the identification of obligatory essential microbial nucleic acid sequences comprising the steps (a) providing a conditionally replicating vector, comprising (i) a conditionally replicating origin of replication which can be activated or inactivated by changing the conditions under which a host cell is cultivated (ii) a selectable marker (iii) nucleic acid sequences of a microorganism (b) transforming microbial host cells with said conditionally replicating vector, wherein the host cells is selected such that the nucleic acid sequence of (a) (iii) is homologous to an endogenous nucleic acid sequence of the host cell in order to allow homologous recombination, (c) subjecting the transformed host cells to insertional duplication mutagenesis by homologous recombination, resulting in viable and non viable (lethal) integrants, (d) directly identifying lethal integrants by separating viable and nonviable integrants of step (c) under permissive conditions versus non-permissive conditions for replication of the conditionally replicating vector of (a), (e) characterizing nucleic acid sequence from lethal integrants and/or polypeptides encoded thereby which are obligatory essential for the viability of said microorganism.