Leigh Lab : Genetic Tools
J o h n    L e i g h    L a b

University of Washington Department of Microbiology
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John Leigh, Ph.D.

Genetic tools for the mesophilic methanococci

The mesophilic methanococci have long been attractive candidates for genetic manipulation. These methanogenic Archaea grow relatively rapidly under laboratory conditions and plate for single colonies at high efficiency. In 1987 Bertani and Baresi transformed Methanococcus voltae in the first genetic manipulation of any methanogen. In 1990 A. Klein's lab developed a puromycin resistance marker for positive selection and introduced DNA by transformation and recombination into the chromosome. Using their protocol, J. Leigh's lab transformed Methanococcus maripaludis in 1991, and in 1994 an optimized transformation protocol was developed by W. Whitman's group. Leigh's group produced a transposon insertion mutant in a M. maripaludis nif gene in 1995, and implemented a second selectable marker (neomycin resistance) in 1996. Thus, the first proven and workable genetic system for a methanogen was in place. Recently Whitman's group has developed a series of replicative expression vectors, and Leigh's lab has implemented a negative selection strategy for producing markerless mutations. Leigh's group has produced dozens of mutations in M. maripaludis in their study of nitrogen regulation.
Currently, genetic tools for M. maripaludis include:
  • High efficiency transformation
  • Positive selection (two antibiotic resistance markers)
  • Integrative and replicative vectors
  • Negative genetic selection
  • Ex-situ transposon insertion mutagenesis
  • Reporter genes

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