Robustness encoded across essential and accessory replicons of the ecologically versatile bacterium Sinorhizobium meliloti

Abstract

Bacterial genome evolution is characterized by gains, losses, and rearrangements of functional genetic segments. The extent to which large-scale genomic alterations influence genotype-phenotype relationships has not been investigated in a high-throughput manner. In the symbiotic soil bacterium Sinorhizobium meliloti, the genome is composed of a chromosome and two large extrachromosomal replicons (pSymA and pSymB, which together constitute 45% of the genome). Massively parallel transposon insertion sequencing (Tn-seq) was employed to evaluate the contributions of chromosomal genes to growth fitness in both the presence and absence of these extrachromosomal replicons. Ten percent of chromosomal genes from diverse functional categories are shown to genetically interact with pSymA and pSymB. These results demonstrate the pervasive robustness provided by the extrachromosomal replicons, which is further supported by constraint-based metabolic modeling. A comprehensive picture of core S. meliloti metabolism was generated through a Tn-seq-guided in silico metabolic network reconstruction, producing a core network encompassing 726 genes. This integrated approach facilitated functional assignments for previously uncharacterized genes, while also revealing that Tn-seq alone missed over a quarter of wild-type metabolism. This work highlights the many functional dependencies and epistatic relationships that may arise between bacterial replicons and across a genome, while also demonstrating how Tn-seq and metabolic modeling can be used together to yield insights not obtainable by either method alone.

A Virtual Nodule Environment (ViNE) for modelling the inter-kingdom metabolic integration during symbiotic nitrogen fixation

“…Prior to constructing the integrated plant – bacterium metabolic model, an updated metabolic reconstruction of S. meliloti Rm1021 was prepared as described in the Materials and Methods. Briefly, the highly refined core metabolic network iGD726 (34) was combined with the comprehensive accessory metabolism of the genome-scale metabolic network iGD1575 (33). Most of the reactions were compared against the literature, referenced where possible, and mass and charge balanced.…”

“…Most of the reactions were compared against the literature, referenced where possible, and mass and charge balanced. The updated model consists of 1348 genes (Table S2), and incorporates information from 240 literature sources (listed in the Excel file of File S1) that include published transposon-sequencing (Tn-seq) data (34) and Phenotype MicroArray data (33, 71, 72) for wild-type and mutant strains.…”

“…A new S. meliloti metabolic reconstruction was built using the existing core metabolic reconstruction iGD726 (34) as a starting point. First, the biomass composition was updated as summarized in Table S1.…”

“…The working reconstruction was manually expanded to contain accessory metabolic pathways following our previously reported workflow (34). Briefly, the reconstruction was expanded by the addition of one pathway at a time.…”

“…An automated expansion of the metabolic network was then performed. Using the ‘tncore_expand’ function of the Tn-Core Toolbox, all reactions absent in the working reconstruction but present in the S. meliloti genome-scale metabolic reconstruction iGD1575b (34) were transferred to the working reconstruction. Then, i) all unnecessary ‘source’ reactions were removed, ii) most metabolic reactions associated with an unknown gene were removed, and iii) some newly added reactions likely to be incorrect based on published literature were deleted.…”