Characterization of pURB500 from the archaeon Methanococcus maripaludis and construction of a shuttle vector

The complete sequence of the 8,285-bp plasmid pURB500 from Methanococcus maripaludis C5 was deter-mined. Sequence analysis identified 18 open reading frames as well as two regions of potential iterons and complex secondary structures. The shuttle vector, pDLT44, for M. maripaludis JJ was constructed from the entire pURB500 plasmid and pMEB.2, an Escherichia coli vector containing a methanococcal puromycin-resistance marker (P. Gernhardt, O. Possot, M. Foglino, L. Sibold, and A. Klein, Mol. Gen. Genet. 221:273–279, 1990). By using polyethylene glycol transformation, M. maripaludis JJ was transformed at a frequency of 3.3 3 107 transformants per mg of pDLT44. The shuttle vector was stable in E. coli under ampicillin selection but was maintained at a lower copy number than pMEB.2. Based on the inability of various restriction fragments of pURB500 to support maintenance in M. maripaludis JJ, multiple regions of pURB500 were required. pDLT44 did not replicate in Methanococcus voltae. Methanogenic archaea produce methane as their main end product and are abundant in a variety of anaerobic habitats (for a review, see reference 56). In general, molecular and physiological studies of methanogens have been performed without the aid of the genetic tools commonly available fo