Abstract
Gene essentiality studies have been performed on numerous bacterial pathogens, but essential gene sets have been determined for only a few plant-associated bacteria. Pseudomonas protegens Pf-5 is a plant-commensal, biocontrol bacterium that can control disease-causing pathogens on a wide range of crops. Work on Pf-5 has mostly focused on secondary metabolism and biocontrol genes, but genome-wide approaches such as high-throughput transposon mutagenesis have not yet been used for this species. In this study, we generated a dense P. protegens Pf-5 transposon mutant library and used transposon-directed insertion site sequencing (TraDIS) to identify 446 genes essential for growth on rich media. Genes required for fundamental cellular machinery were enriched in the essential gene set, while genes related to nutrient biosynthesis, stress responses, and transport were underrepresented. The majority of Pf-5 essential genes were part of the P. protegens core genome. Comparison of the essential gene set of Pf-5 with those of two plant-associated pseudomonads, P. simiae and P. syringae, and the well-studied opportunistic human pathogen P. aeruginosa PA14 showed that the four species share a large number of essential genes, but each species also had uniquely essential genes. Comparison of the Pf-5 in silico-predicted and in vitro-determined essential gene sets highlighted the essential cellular functions that are over- and underestimated by each method. Expanding essentiality studies into bacteria with a range of lifestyles may improve our understanding of the biological processes important for bacterial survival and growth.IMPORTANCE Essential genes are those crucial for survival or normal growth rates in an organism. Essential gene sets have been identified in numerous bacterial pathogens but only a few plant-associated bacteria. Employing genome-wide approaches, such as transposon insertion sequencing, allows for the concurrent analyses of all genes of a bacterial species and rapid determination of essential gene sets. We have used transposon insertion sequencing to systematically analyze thousands of Pseudomonas protegens Pf-5 genes and gain insights into gene functions and interactions that are not readily available using traditional methods. Comparing Pf-5 essential genes with those of three other pseudomonads highlights how gene essentiality varies between closely related species.
Highlights
Gene essentiality studies have been performed on numerous bacterial pathogens, but essential gene sets have been determined for only a few plant-associated bacteria
We created a saturated transposon mutant library and used transposon-directed insertion site sequencing (TraDIS) to successfully identify 446 genes that were essential for P. protegens Pf-5, a plant-associated bacterium, to survive and grow on rich media
The essential gene set showed enrichment of genes required for fundamental cellular machinery, which is consistent with the composition of essential gene sets in other bacteria
Summary
Gene essentiality studies have been performed on numerous bacterial pathogens, but essential gene sets have been determined for only a few plant-associated bacteria. As observed for other bacterial taxa, each pseudomonad is expected to have a number of common essential genes together with an additional set of strain-specific essential genes, which may reflect differences in lifestyle [4, 5] Essential genes are those crucial for survival or normal growth rates in an organism [6, 7]. While gene essentiality studies have been performed on numerous bacterial pathogens, essential gene sets have been determined for only a few plant-associated bacteria, including Herbaspirillum seropedicae SmR1, a plant growth-promoting endophyte [11], Pseudomonas simiae WCS417, isolated from the wheat rhizosphere [12], Pseudomonas syringae pv. While gene essentiality studies have been performed on numerous bacterial pathogens, essential gene sets have been determined for only a few plant-associated bacteria, including Herbaspirillum seropedicae SmR1, a plant growth-promoting endophyte [11], Pseudomonas simiae WCS417, isolated from the wheat rhizosphere [12], Pseudomonas syringae pv. syringae B728a, a foliar plant pathogen [13], and three nitrogen-fixing root endosymbionts [14]
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