title: Cloning-free genome engineering in Sinorhizobium meliloti advances applications of Cre/loxP site-specific recombination.
abstract: The soil-dwelling α-proteobacterium Sinorhizobium meliloti serves as model for studies of symbiotic nitrogen fixation, a highly important process in sustainable agriculture. Here, we report advancements of the genetic toolbox accelerating genome editing in S. meliloti. The hsdMSR operon encodes a type-I restriction-modification (R-M) system. Transformation of S. meliloti is counteracted by the restriction endonuclease HsdR degrading DNA which lacks the appropriate methylation pattern. We provide a stable S. meliloti hsdR deletion mutant showing enhanced transformation with Escherichia coli-derived plasmid DNA and demonstrate that using an E. coli plasmid donor, expressing S. meliloti methyl transferase genes, is an alternative strategy of increasing the transformation efficiency of S. meliloti. Furthermore, we devise a novel cloning-free genome editing (CFGE) method for S. meliloti, Agrobacterium tumefaciens and Xanthomonas campestris, and demonstrate the applicability of this method for intricate applications of the Cre/lox recombination system in S. meliloti. An enhanced Cre/lox system, allowing for serial deletions of large genomic regions, was established. An assay of lox spacer mutants identified a set of lox sites mediating specific recombination. The availability of several non-promiscuous Cre recognition sites enables simultaneous specific Cre/lox recombination events. CFGE combined with Cre/lox recombination is put forward as powerful approach for targeted genome editing, involving serial steps of manipulation to expedite the genetic accessibility of S. meliloti as chassis.
authors.affiliation: LOEWE Center for Synthetic Microbiology and Faculty of Biology, Philipps-Universität Marburg, Marburg, Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Döhlemann
authors.affiliation: LOEWE Center for Synthetic Microbiology and Faculty of Biology, Philipps-Universität Marburg, Marburg, Germany.
authors.forename: Meike
authors.initials: M
authors.lastname: Brennecke
authors.affiliation: LOEWE Center for Synthetic Microbiology and Faculty of Biology, Philipps-Universität Marburg, Marburg, Germany. Electronic address: anke.becker@synmikro.uni-marburg.de.
authors.forename: Anke
authors.initials: A
authors.lastname: Becker
mesh_terms: D018274:Electroporation; D004926:Escherichia coli / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D016680:Genome, Bacterial / Q000235:genetics*; D019426:Integrases / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics*; D016962:Sinorhizobium meliloti / Q000235:genetics* / Q000378:metabolism; D014169:Transformation, Bacterial
chemical_list: C045073:Cre recombinase; D019426:Integrases
keywords: Cre/loxP recombination; Electroporation; Genome editing; Sinorhizobium meliloti