Employing Site-Specific Recombination for Conditional Genetic Analysis in Sinorhizobium meliloti

Abstract

The ability to remove a genetic function from an organism with good temporal resolution is crucial for characterizing essential genes or genes that act in complex developmental programs. The rhizobium-legume symbiosis involves an elaborate two-organism interaction requiring multiple levels of signal exchange. As an important step toward probing rhizobium genetic functions with temporal resolution, we present the development of a conditional gene deletion system in Sinorhizobium meliloti that employs Cre/loxP site-specific recombination. This system enables chemically inducible and irreversible gene deletion or gene upregulation. Recombinase-mediated excision events can be positively or negatively selected or monitored by a colorimetric assay. The system may be adaptable to various bacterial species, in which recombinase activity may be placed under the control of diverse user-defined promoters. This system also shows promise for uses in promoter trapping and biosensing applications.

Adaptation of the Alphaproteobacterium Rhodobacter sphaeroides to stationary phase

“…This 350 bp fragment contained the RBS, translation start and the following nucleotides of the coding region. Located immediately downstream of this 350 bp fragment in pK18mobII was the rpoC / thrA tandem terminator (Harrison et al ., ). Following conjugation of the plasmid into R .…”

“…This 350 bp fragment contained the RBS, translation start and the following nucleotides of the coding region. Located immediately downstream of this 350 bp fragment in pK18mobII was the rpoC/thrA tandem terminator (Harrison et al, 2011). Following conjugation of the plasmid into R. sphaeroides using the E. coli strain S17-1 (Simon et al, 1983), homologous recombination (single cross-over) between the cloned fragment in the suicide vector and the genomic copy of the target gene resulted in two incomplete copies of the target gene.…”

A novel LuxR‐type solo of Sinorhizobium meliloti, NurR, is regulated by the chromosome replication coordinator, DnaA and activates quorum sensing

“…Promoter‐reporter fusions always included 300–500 bp of the upstream region of each gene (including the ribosomal binding site) plus the start codon along with up to 9 additional codons. For the TRC, the construct reported in Schlüter et al , was modified firstly by the addition of the rpoC / thrA tandem terminator (Harrison et al , ) inserted between the divergently reading genes for cerulean and mCherry, and secondly by modifying the 4 x T1 transcription terminator array between mCherry and mVenus by removing two copies of the T1 terminator. Typically, promoters were assembled into the cassette using the plasmid pK18mobII, a suicide plasmid in S. meliloti .…”

Multidisciplinary approaches for studying rhizobium–legume symbioses

“…Several groups have employed signature-tagged mutagenesis (STM) for genome-wide identification of genes involved in symbiosis or competition for nodule occupancy. Harrison et al 2011;Landeta et al 2011;Heil et al 2012;diCenzo et al 2013;Yurgel et al 2013a;Milunovic et al 2014;diCenzo et al 2016b;Döhlemann et al 2016; diCenzo and Finan 2018…”

“…While transcriptomic, proteomic, and metabolic approaches can help provide insights into the unique developmental stages, genetic and cell biology tools are required to validate conclusions reached from these systems-level analyses. The in-ducible Cre-lox system by Harrison et al (2011) is a step in this direction, as are the lux bioreporters for the in vivo analysis of the spatial and temporal presence of plant metabolites (Pini et al 2017). Similarly, the S. meliloti multiple gene-expression reporter strain for examining the role of plant genes in nodule development helps address this problem (Lang et al 2018).…”