Versatile Vectors for Efficient Mutagenesis of Bradyrhizobium diazoefficiens and Other Alphaproteobacteria

Abstract

Analysis of bacterial gene function commonly relies on gene disruption or replacement followed by phenotypic characterization of the resulting mutant strains. Deletion or replacement of targeted regions is commonly achieved via two homologous recombination (HR) events between the bacterial genome and a nonreplicating plasmid carrying DNA fragments flanking the region to be deleted. The counterselection of clones that have integrated the entire plasmid in their genome via a single HR event is crucial in this procedure. Various genetic tools and well-established protocols are available for this type of mutagenesis in model bacteria; however, these methods are not always efficiently applicable in less established systems. Here we describe the construction and application of versatile plasmid vectors pREDSIX and pTETSIX for marker replacement and markerless mutagenesis, respectively. Apart from an array of restriction sites optimized for cloning of GC-rich DNA fragments, the vector backbone contains a constitutively expressed gene for mCherry, enabling the rapid identification of clones originating from single or double HR events by fluorescence-assisted cell sorting (FACS). In parallel, we constructed a series of plasmids from which gene cassettes providing resistance against gentamicin, kanamycin, hygromycin B, streptomycin and spectinomycin, or tetracycline were excised for use with pREDSIX-based marker replacement mutagenesis. In proof-of-concept mutagenesis experiments, we demonstrated the potential for the use of the developed tools for gene deletion mutagenesis in the nitrogen-fixing soybean symbiont Bradyrhizobium diazoefficiens (formerly Bradyrhizobium japonicum) and three additional members of the alphaproteobacteria. IMPORTANCEMutation and phenotypic analysis are essential to the study of gene function. Efficient mutagenesis protocols and tools are available for many bacterial species, including various model organisms; however, genetic analysis of less-well-characterized organisms is often impaired by the lack of efficient methods. Here we describe a set of novel genetic tools for facilitated mutagenesis of the nitrogen-fixing soybean symbiont Bradyrhizobium diazoefficiens and related alphaproteobacteria. We demonstrated their usefulness by generating several mutant strains lacking defined genes. Isolation of both antibiotic resistance gene-containing and markerless deletion mutants is greatly facilitated because undesired clones which contain the entire mutagenic plasmid integrated in the genome can be identified on the basis of their fluorescent phenotype derived from the mCherry gene carried by the vector backbone. The possibility to generate markerless mutants assists with the isolation of strains carrying multiple deletions, which can be crucial while studying functionally redundant genes. Many functional studies of biological phenomena rely on the isolation of mutants and the availability of the respective genetic tools. Mutants are preferably constructed in a targeted manner, which traditi...

Dynamic character displacement among a pair of bacterial phyllosphere commensals in situ

“…The list of primers used is provided in Supplementary Data 6 . For the generation of deletion mutants of Leaf68 the flanking regions of ~650 bp were amplified using the upstream (HR1) and downstream (HR2) primers for each mutant respectively (Supplementary Data 6 ) followed by digestion with MunI/KpnI (upstream) and KpnI/Nsil (downstream) and inserted into the pREDSIX vector 102 . The construct was cleaved with KpnI between the two flanking regions of the respective gene and a kanamycin-resistances cassette (KmR) was inserted that has been cut from pRGD-KmR 102 with KpnI.…”

“…For the generation of deletion mutants of Leaf68 the flanking regions of ~650 bp were amplified using the upstream (HR1) and downstream (HR2) primers for each mutant respectively (Supplementary Data 6 ) followed by digestion with MunI/KpnI (upstream) and KpnI/Nsil (downstream) and inserted into the pREDSIX vector 102 . The construct was cleaved with KpnI between the two flanking regions of the respective gene and a kanamycin-resistances cassette (KmR) was inserted that has been cut from pRGD-KmR 102 with KpnI. The orientation of the cassette was confirmed by PCR with primers binding the flanking region (OR/OF, this study) and kanamycin cassette (Kan-2/-4 described previously by Ledermann et al, 2016 102 ).…”

Co‐catabolism of arginine and succinate drives symbiotic nitrogen fixation

“…Sinorhizobium meliloti deletion mutants were generated by replacing the native gene with the aacC1 gene conferring gentamycin resistance using a one-step double homologous recombination procedure as detailed in Ledermann et al (2016). Flanking DNA regions covering 750 bp upstream and downstream of a target gene were PCR amplified (Dataset EV5) and subsequently fused to a central gentamicin resistance gene using splicing by overlapping extension PCR or Gibson assembly (Gibson et al, 2009) to produce gene replacement cassettes.…”

Bradyrhizobium diazoefficiens Requires Chemical Chaperones To Cope with Osmotic Stress during Soybean Infection

“…Plasmid and strain constructions. For deletion mutant generation, the pREDSIX system was used as described previously (77). In short, flanking regions (650-to 900-bp up-and downstream DNA of the region to be deleted) were PCR amplified (see Table S1B for primers and oligonucleotides used in this study) and cloned in tandem orientation into vector pREDSIX.…”

“…Clones that underwent double crossovers were selected by their nonfluorescent phenotype and double checked by PCR using primers binding to the antibiotic resistance cassette (oriented outwards) in combination with primers binding to the genomic region outside the flanking regions used for homologous recombination (oriented toward the deleted region) (see Table S1B ). For markerless deletions, a similar method was used, and isolation and verification was done as described ( 77 ). For detailed cloning and mutagenesis procedures, see Text S1.…”