Genome-wide transposon screen of aPseudomonas syringae mexBmutant reveals the substrates of efflux transporters

Abstract

Bacteria express numerous efflux transporters that confer resistance to diverse toxicants present in their environment. Due to a high level of functional redundancy of these transporters, it is difficult to identify those that are of most importance in conferring resistance to specific compounds. The resistance-nodulation-division (RND) protein family is one such example of redundant transporters that are widespread among Gram-negative bacteria. Within this family, the MexAB-OprM protein complex is highly-expressed and conserved among Pseudomonas species. We exposed barcoded transposon mutant libraries in isogenic wild-type and ΔmexB backgrounds in P. syringae B728a to diverse toxic compounds in vitro to identify mutants with increased susceptibility to these compounds. Mutants in genes encoding both known and novel redundant transporters, but with partially overlapping substrate specificities were observed in a ΔmexB background. Psyr_0228, an uncharacterized member of the Major Facilitator Superfamily of transporters, preferentially contributes to tolerance of acridine orange and acriflavine. Another transporter located in the inner membrane, Psyr_0541, contributes to tolerance to acriflavine and berberine. The presence of multiple redundant, genomically encoded, efflux transporters appears to enable bacterial strains to tolerate a diversity of environmental toxins. This genome-wide screen in a hyper-susceptible mutant strain revealed numerous transporters that would otherwise be dispensable in these conditions. Bacterial strains such as P. syringae that likely encounter diverse toxins in their environment such as in association with many different plant species, probably benefit from possessing multiple redundant transporters that enable versatility to tolerate novel toxicants.

Application of Transposon Insertion Sequencing to Agricultural Science

“…Identified and used a hyper-susceptible mutant to identify the substrates of redundant transporters. Identified genes that contribute to tolerance of acridine orange, acriflavine and berberine Helmann et al, 2019b Herbaspirillum seropedicae SmR1…”

“…This study recorded phenotypes for thousands of genes and proposed specific functions for transporter proteins, catabolic enzymes, and domains of unknown function. Transporter substrates (Helmann et al, 2019b) and abiotic and biotic stress tolerance genes (Calero et al, 2017) can also be investigated using these techniques. A Tn-Seq study in P. putida KT2440 revealed that genes related to membrane stability are important for p-coumaric acid tolerance (Calero et al, 2017).…”