Genome-Wide Transposon Screen of a Pseudomonas syringae mexB Mutant 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 with mutations 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 of 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 performed in a hypersusceptible mutant strain revealed numerous transporters that would otherwise be dispensable under 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 with respect to toleration of novel toxicants. IMPORTANCE Bacteria use protein pumps to remove toxic compounds from the cell interior, enabling survival in diverse environments. These protein pumps can be highly redundant, making their targeted examination difficult. In this study, we exposed mutant populations of Pseudomonas syringae to diverse toxicants to identify pumps that contributed to survival in those conditions. In parallel, we examined pump redundancy by testing mutants of a population lacking the primary efflux transporter responsible for toxin tolerance. We identified partial substrate overlap for redundant transporters, as well as several pumps that appeared more substrate specific. For bacteria that are found in diverse environments, having multiple, partially redundant efflux pumps likely allows flexibility in habitat colonization.

Distinctiveness of genes contributing to growth ofPseudomonas syringaein diverse host plant species

“…A randomly-barcoded mariner transposon library of P. syringae B728a described previously [13,14] was inoculated by vacuum infiltration into the leaves of the three plant species to identify genes contributing to host colonization. This mutant library contains 281,417 total strains having insertions mapped in the B728a genome.…”

“…RB-TnSeq is a powerful method that has been used in a variety of bacterial species to identify those genes that contribute to fitness in diverse in vitro conditions [11,14,15]. In one TnSeq study, about 100 genes were found to either increase or decrease the fitness of Dickeya dadantii in chicory [16], highlighting the importance of the biosynthetic pathways for leucine, cysteine, and lysine for bacterial growth within the plant.…”

Distinctiveness of genes contributing to growth of Pseudomonas syringae in diverse host plant species

“…RB-TnSeq is a powerful method that has been used in a variety of bacterial species to identify those genes that contribute to fitness in diverse in vitro conditions [12,15,19]. In one TnSeq study, about 100 genes were found to either increase or decrease the fitness of Dickeya dadantii in chicory [20], highlighting the importance of the biosynthetic pathways for leucine, cysteine, and lysine for bacterial growth within the plant.…”

“…A randomly-barcoded mariner transposon library of P. syringae B728a described previously [14,15] was inoculated by vacuum infiltration into the leaves of the three plant species to identify genes contributing to host colonization. This mutant library contains 281,417 total strains having insertions mapped in the B728a genome.…”

Differential virulence contributions of the efflux transporter MexAB-OprM inPseudomonas syringaeinfecting a variety of host plants

“…The efflux transporter MexAB-OprM may be required to tolerate the presence of these plant defense compounds as they accumulate to high levels in the apoplast. As different plant species produce different defensive compounds (Tegos et al 2002), despite its wide substrate range MexB is not capable of excluding all potential antimicrobial molecules (Helmann et al 2019). It is tempting to speculate, therefore, that those common bean varieties and other plant species in which strain B728a achieves the lowest population size and for which MexB was required for maximal growth produce inhibitory compounds and that these compounds are also substrates for MexB.…”