title: Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker.
abstract: Most bacteria can only be transformed with circular plasmids, so robust DNA integration methods for these rely upon selection of single-crossover clones followed by counter-selection of double-crossover clones. To overcome the limited availability of heterologous counter-selection markers, here we explore novel DNA integration strategies that do not employ them, and instead exploit (i) activation or inactivation of genes leading to a selectable phenotype, and (ii) asymmetrical regions of homology to control the order of recombination events. We focus here on the industrial biofuel-producing bacterium Clostridium acetobutylicum, which previously lacked robust integration tools, but the approach we have developed is broadly applicable. Large sequences can be delivered in a series of steps, as we demonstrate by inserting the chromosome of phage lambda (minus a region apparently unstable in Escherichia coli in our cloning context) into the chromosome of C. acetobutylicum in three steps. This work should open the way to reliable integration of DNA including large synthetic constructs in diverse microorganisms.
authors.affiliation: Clostridia Research Group, BBSRC Sustainable BioEnergy Centre, School of Molecular Medical Sciences, The University of Nottingham, University Park, Nottingham NG7 2RD, UK.
authors.forename: John T
authors.initials: JT
authors.lastname: Heap
authors.forename: Muhammad
authors.initials: M
authors.lastname: Ehsaan
authors.forename: Clare M
authors.initials: CM
authors.lastname: Cooksley
authors.forename: Yen-Kuan
authors.initials: YK
authors.lastname: Ng
authors.forename: Stephen T
authors.initials: ST
authors.lastname: Cartman
authors.forename: Klaus
authors.initials: K
authors.lastname: Winzer
authors.forename: Nigel P
authors.initials: NP
authors.lastname: Minton
mesh_terms: D000426:Alcohol Dehydrogenase / Q000235:genetics / Q000378:metabolism; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D002876:Chromosomes, Bacterial; D046969:Clostridium acetobutylicum / Q000235:genetics; D004247:DNA / Q000737:chemistry; D005819:Genetic Markers; D016679:Genome, Viral; D008969:Molecular Sequence Data; D009962:Orotate Phosphoribosyltransferase / Q000235:genetics; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014169:Transformation, Bacterial
chemical_list: D005819:Genetic Markers; D004247:DNA; D000426:Alcohol Dehydrogenase; D009962:Orotate Phosphoribosyltransferase