Robustness encoded across essential and accessory replicons in an ecologically versatile bacterium

Abstract

Bacterial genome evolution is characterized by gains, losses, and rearrangements of functional genetic segments. The extent to which genotype-phenotype relationships are influenced by large-scale genomic alterations 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 contributions of chromosomal genes to 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 modelling. 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 misses over a quarter of wild type metabolism. This work highlights the strong functional dependencies and epistatic relationships that may arise between bacterial replicons and across a genome, while also demonstrating how Tn-seq and metabolic modelling can be used together to yield insights not obtainable by either method alone.

Tn-Core: A Toolbox for Integrating Tn-seq Gene Essentiality Data and Constraint-Based Metabolic Modeling

“…In the case of S. meliloti, the core models contain an average of 434 genes, of which 286 genes (~ 66%) are invariably present and the rest are from a set of 777 variably present genes. In other words, a third of core S. meliloti metabolic genes can be functionally replaced by alternative genes or pathways, consistent with recent experimental work [27]. The variable and invariable core genes were mapped to KEGG pathways [35] using eggNOG-mapper [36] to identify functional biases.…”

“…Two core models were produced, each using a growth threshold of 50% the full model, with 50,000 iterations, and with Tn-seq essential genes pre-identified. In one Tn-Core run, only Tn-seq data [27] was used; in the second run, the same Tn-seq data plus RNA-seq data [28] was included. The sizes of both models are summarized in Table 1, and the inclusion of RNA-seq data resulted in a somewhat larger core model.…”

“…The ability of the core models to capture context-specific core metabolism was examined by predicting the essentiality of central carbon metabolic genes (Figure 3). Results were compared to both the full iGD1575 model and to the manually constructed S. meliloti iGD726 core metabolic reconstruction [27].…”

“…Additionally, the GIMME model and Tn-Core model produced with Tn-seq and RNA-seq data (FBA implementation) share > 87% of their genes. Thus, at least in S. meliloti where essential genes tend to be highly expressed [27], both Tn-Core and GIMME perform similarly and the choice of algorithm would be driven primarily by the type of data being incorporated with the GSMR.…”

Chromids Aid Genome Expansion and Functional Diversification in the Family Burkholderiaceae

“…Thus, secondary replicons may be more available for integration of horizontally acquired DNA through recombination. Secondary replicons, especially in their early evolution, contain few housekeeping genes (Harrison et al 2010); for example, Tn-seq screens with S. meliloti and B. cenocepacia have identified 10-fold to 50-fold enrichments of essential genes on the chromosome relative to the secondary replicons (Higgins et al 2017;diCenzo et al 2018). As such, integration of new DNA within secondary replicons have a lower probability of disrupting an important gene.…”