The Trk Potassium Transporter Is Required for RsmB-Mediated Activation of Virulence in the Phytopathogen Pectobacterium wasabiae

Abstract

Pectobacterium wasabiae (previously known as Erwinia carotovora) is an important plant pathogen that regulates the production of plant cell wall-degrading enzymes through an N-acyl homoserine lactone-based quorum sensing system and through the GacS/GacA two-component system (also known as ExpS/ExpA). At high cell density, activation of GacS/GacA induces the expression of RsmB, a noncoding RNA that is essential for the activation of virulence in this bacterium. A genetic screen to identify regulators of RsmB revealed that mutants defective in components of a putative Trk potassium transporter (trkH and trkA) had decreased rsmB expression. Further analysis of these mutants showed that changes in potassium concentration influenced rsmB expression and consequent tissue damage in potato tubers and that this regulation required an intact Trk system. Regulation of rsmB expression by potassium via the Trk system occurred even in the absence of the GacS/GacA system, demonstrating that these systems act independently and are both required for full activation of RsmB and for the downstream induction of virulence in potato infection assays. Overall, our results identified potassium as an essential environmental factor regulating the Rsm system, and the consequent induction of virulence, in the plant pathogen P. wasabiae.

Importance: Crop losses from bacterial diseases caused by pectolytic bacteria are a major problem in agriculture. By studying the regulatory pathways involved in controlling the expression of plant cell wall-degrading enzymes in Pectobacterium wasabiae, we showed that the Trk potassium transport system plays an important role in the regulation of these pathways. The data presented further identify potassium as an important environmental factor in the regulation of virulence in this plant pathogen. We showed that a reduction in virulence can be achieved by increasing the extracellular concentration of potassium. Therefore, this work highlights how elucidation of the mechanisms involved in regulating virulence can lead to the identification of environmental factors that can influence the outcome of infection.

FIG 1

Trk mutants have low rsmB expression. Expression of P_rsmB::gfp promoter fusion from pRSV206 in P. wasabiae WT and RSV108 (trkH::Tn5), RSV141 (trkH::Tn5), RSV154 (trkH::Tn5), RSV124 (trkA::Tn5), or RSV236 (trkA::Tn5) mutant strains was measured by fluorescence flow cytometry of cells grown for 24 h in MM. Error bars represent standard deviations (SD); n = 3.

FIG 2

Complementation of the trkH and trkA mutants. (A) Expression of a P_rsmB::gfp promoter fusion was measured in WT bacteria harboring the control vector (pOM18), the trkH::Tn5 mutant carrying either the pOM18 empty vector or the vector expressing trkH, p(trkH⁺), and the trkA::Tn5 mutant carrying either the pOM18 empty vector or the vector expressing trkA, p(trkA⁺). Fluorescence was measured in cells collected from cultures grown to an OD₆₀₀ of 0.4 in MM. (B) Pel activity was measured in cell-free supernatants from cultures of the bacterial strains indicated above grown in LB supplemented with PGA at an OD₆₀₀ of 0.4. Error bars represent standard errors of the means (SEM); n = 6.

FIG 3

Mutants in the Trk system are impaired in virulence. The virulence of WT bacteria and trkH::Tn5 and trkA::Tn5 mutants was measured by quantification of the mass of potato tuber maceration induced by these bacteria 48 h after inoculation of the potato tubers. Potatoes were inoculated with approximately 3 × 10⁵ cells of the respective strain grown overnight in LB. **, P < 0.01; n = 7. This is a representative experiment from three independent experiments.

FIG 4

Effect of extracellular potassium on rsmB expression and growth in P. wasabiae. (A) The expression of the rsmB promoter fusion (P_rsmB::gfp) was measured by flow cytometry of cultures of WT bacteria and trkA::Tn5, trkH::Tn5, gacA, gacA trkA::Tn5 mutants grown to an OD₆₀₀ of 0.3 in minimal potassium medium supplemented with a final potassium concentration of 0.25 mM, 2.5 mM, 25 mM, or 250 mM. NG, no growth. Error bars represent SEM; n = 6. (B to E) The OD₆₀₀ was measured throughout growth for WT bacteria and trkH::Tn5 and trkA::Tn5 mutants in minimal potassium medium supplemented with 0.25, 2.5, 25, or 250 mM KCl (for growth rates, see Table S4 in the supplemental material).

FIG 5

Regulation of virulence by extracellular potassium concentration. The virulence of WT bacteria and trkH::Tn5 and trkA::Tn5 mutants was measured by quantification of the mass of macerated tissue 48 h after inoculation of potato tubers. Cells were cultured overnight in LB (approximately 8 mM potassium) and were harvested and resuspended in potassium-free PBS (WT only) or PBS supplemented with a final concentration of either 4.5 mM or 250 mM potassium. Potatoes were inoculated with approximately 3 × 10⁵ cells of the respective strain at the different potassium concentrations. Error bars represent SEM; n = 6; **, P < 0.01; ns, not significant. This is a representative experiment from three independent experiments.

FIG 6

Induction of virulence by extracellular potassium. Virulence of WT bacteria was measured by determining the mass of damaged tissue 48 h after the infection of potato tubers. Potatoes were inoculated with approximately 3 × 10⁵ cells of the respective strain grown overnight in minimal potassium medium supplemented with a final potassium concentration of 0.25 mM and resuspended in potassium-free PBS (0 mM), in PBS (4.5 mM potassium), or in PBS supplemented with potassium to a final concentration of 250 mM. Error bars represent SEM; n = 6; **, P < 0.01. This is a representative experiment from two independent experiments.