The Use of Transposon Insertion Sequencing to Interrogate the Core Functional Genome of the Legume Symbiont Rhizobium leguminosarum

Abstract

The free-living legume symbiont Rhizobium leguminosarum is of significant economic value because of its ability to provide fixed nitrogen to globally important leguminous food crops, such as peas and lentils. Discovery based research into the genetics and physiology of R. leguminosarum provides the foundational knowledge necessary for understanding the bacterium's complex lifestyle, necessary for augmenting its use in an agricultural setting. Transposon insertion sequencing (INSeq) facilitates high-throughput forward genetic screening at a genomic scale to identify individual genes required for growth in a specific environment. In this study we applied INSeq to screen the genome of R. leguminosarum bv. viciae strain 3841 (RLV3841) for genes required for growth on minimal mannitol containing medium. Results from this study were contrasted with a prior INSeq experiment screened on peptide rich media to identify a common set of functional genes necessary for basic physiology. Contrasting the two growth conditions indicated that approximately 10% of the chromosome was required for growth, under both growth conditions. Specific genes that were essential to singular growth conditions were also identified. Data from INSeq screening on mannitol as a sole carbon source were used to reconstruct a metabolic map summarizing growth impaired phenotypes observed in the Embden-Meyerhof-Parnas pathway, Entner-Doudoroff pathway, pentose phosphate pathway, and tricarboxylic acid cycle. This revealed the presence of mannitol dependent and independent metabolic pathways required for growth, along with identifying metabolic steps with isozymes or possible carbon flux by-passes. Additionally, genes were identified on plasmids pRL11 and pRL12 that are likely to encode functional activities important to the central physiology of RLV3841.

Modulating Pathogenesis with Mobile-CRISPRi

“…Essential genes are typically defined as genes that are indispensable for growth in rich culture media, whereas CE genes are required only in specific conditions, such as maintenance in a host niche (2). Next-generation sequencing of bacterial transposon (Tn)-mutant libraries (e.g., transposon sequencing [Tn-Seq] [3] and insertion sequencing [INSeq] [4]) from infected animals has enabled the comprehensive identification of essential and CE genes in a single experiment, rapidly increasing our knowledge of which genes are required for pathogenesis (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)). There are two major limitations of using Tn-Seq to study CE genes, both arising from the complete loss of function usually caused by Tn mutagenesis.…”

Multidisciplinary approaches for studying rhizobium–legume symbioses

“…A mariner transposon was used to examine the core genome of R. leguminosarum bv. viciae (Perry and Yost 2014;Perry et al 2016), as well as to investigate the effects of carbon source and O 2 levels, a key signal during symbiosis, on the fitness of gene disruptions (Wheatley et al 2017). Similarly, a Tn5-derived transposon was used to interrogate the core genome of S. meliloti and to inspect how chromosomal gene phenotypes differ when the extrachromosomal replicons are removed (diCenzo et al 2018a).…”

Application of Transposon Insertion Sequencing to Agricultural Science

“…Most of what is currently known about genes involved in beneficial plant interactions has been determined by comparing genomes and individually testing genes that correlate with a phenotype of interest (Levy et al, 2018). A high throughput method of gathering gene functional information and relating genotype to phenotype is required to rapidly advance our understanding of the complex molecular interactions that occur between microbes and plants (van Opijnen and Camilli, 2013;Perry et al, 2016) and move beyond cataloging microbial community members toward a mechanistic understanding of agricultural microbiomes (Poole et al, 2018).…”