title: Genetic manipulation of Bacillus methanolicus, a gram-positive, thermotolerant methylotroph.
abstract: We report the fist genetic transformation system, shuttle vectors, and integrative vectors for the thermotolerant, methylotrophic bacterium Bacillus methanolicus. By using a polyethylene glycol-mediated transformation procedure, we have successfully transformed B. methanolicus with both integrative and multicopy plasmids. For plasmids with a single BmeTI recognition site, dam methylation of plasmid DNA (in vivo or in vitro) was found to enhance transformation efficiency from 7- to 11-fold. Two low-copy-number Escherichia coli-B, methanolicus shuttle plasmids, pDQ507 and pDQ508, are described. pDQ508 caries the replication origin cloned from a 17-kb endogenous B. methanolicus plasmid, pBM1. pDQ507 carries a cloned B. methanolicus DNA fragment, pmr-1, possibly of chromosomal origin, that supports maintenance of pDQ507 as a circular, extrachromosomal DNA molecule. Deletion analysis of pDQ507 indicated two regions required for replication, i.e., a 90-bp AT-rich segment containing a 46-bp imperfect, inverted repeat sequence and a second region 65% homologous to the B. subtilis dpp operon. We also evaluated two E. coli-B. subtilis vectors, pEN1 and pHP13, for use as E. coli-B. methanolicus shuttle vectors. The plasmids pHP13, pDQ507, and pDQ508 were segregationally and structurally stable in B. methanolicus for greater than 60 generations of growth under nonselective conditions; pEN1 was segregationally unstable. Single-stranded plasmid DNA was detected in B. methanolicus transformants carrying either pEN1, pHP13, or pDQ508, suggesting that pDQ508, like the B. subtilis plasmids, is replicated by a rolling-circle mechanism. These studies provide the basic tools for the genetic manipulation of B. methanolicus.
authors.affiliation: Biological Process Technology Institute, University of Minnesota, St. Paul 55108, USA.
authors.forename: D
authors.initials: D
authors.lastname: Cue
authors.forename: H
authors.initials: H
authors.lastname: Lam
authors.forename: R L
authors.initials: RL
authors.lastname: Dillingham
authors.forename: R S
authors.initials: RS
authors.lastname: Hanson
authors.forename: M C
authors.initials: MC
authors.lastname: Flickinger
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000235:genetics*; D001483:Base Sequence; D005818:Genetic Engineering; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D016415:Sequence Alignment; D014162:Transfection