Project description:Many bacteria, often associated with eukaryotic hosts and of relevance for biotechnological applications, harbor a multipartite genome composed of more than one replicon. Biotechnologically relevant phenotypes are often encoded by genes residing on the secondary replicons. A synthetic biology approach to developing enhanced strains for biotechnological purposes could therefore involve merging pieces or entire replicons from multiple strains into a single genome. Here we report the creation of a genomic hybrid strain in a model multipartite genome species, the plant-symbiotic bacterium Sinorhizobium meliloti. We term this strain as cis-hybrid, since it is produced by genomic material coming from the same species' pangenome. In particular, we moved the secondary replicon pSymA (accounting for nearly 20% of total genome content) from a donor S. meliloti strain to an acceptor strain. The cis-hybrid strain was screened for a panel of complex phenotypes (carbon/nitrogen utilization phenotypes, intra- and extracellular metabolomes, symbiosis, and various microbiological tests). Additionally, metabolic network reconstruction and constraint-based modeling were employed for in silico prediction of metabolic flux reorganization. Phenotypes of the cis-hybrid strain were in good agreement with those of both parental strains. Interestingly, the symbiotic phenotype showed a marked cultivar-specific improvement with the cis-hybrid strains compared to both parental strains. These results provide a proof-of-principle for the feasibility of genome-wide replicon-based remodelling of bacterial strains for improved biotechnological applications in precision agriculture.
Project description:We show that the RNA leves of many genes in the S. meliloti overexpressing strain (OE) 2011 (pRK-RcsR1) are more similar to the levels of in wild type (WT) strain 2011 than in the empty vector control (EVC) strain 2011(pRK4352). Since the plasmid-containing strains were grown in the presence of 10 µg/ml tetracycline (Tc), while the WT was grown without Tc, the results suggest that the intrecellular Tc concentration in the OE strain are lower than in the EVC. This is explained by an increased efflux pump activity in the OE strain.
Project description:Investigation of whole genome gene expression level changes in a Sinorhizobium meliloti 1021 rpoH1 rpoH2 double mutant, compared to the wild-type strain. The mutations engineered into this strain render it deficient in symbiotic nitrogen fixation. The mutants analyzed in this study are further described in Mitsui, H, T. Sato, Y. Sato, and K. Minamisawa. 2004. Sinorhizobium meliloti RpoH1 is required for effective nitrogen-fixing symbiosis with alfalfa. Mol Gen Genomics 271:416-425.