The Leucine-Responsive Regulatory Protein Lrp Participates in Virulence Regulation Downstream of Small RNA ArcZ in Erwinia amylovora

Abstract

Erwinia amylovora causes the devastating fire blight disease of apple and pear trees. During systemic infection of host trees, pathogen cells must rapidly respond to changes in their environment as they move through different host tissues that present distinct challenges and sources of nutrition. Growing evidence indicates that small RNAs (sRNAs) play an important role in disease progression as posttranscriptional regulators. The sRNA ArcZ positively regulates the motility phenotype and transcription of flagellar genes in E. amylovora Ea1189 yet is a direct repressor of translation of the flagellar master regulator, FlhD. We utilized transposon mutagenesis to conduct a forward genetic screen and identified suppressor mutations that increase motility in the Ea1189ΔarcZ mutant background. This enabled us to determine that the mechanism of transcriptional activation of the flhDC mRNA by ArcZ is mediated by the leucine-responsive regulatory protein, Lrp. We show that Lrp contributes to expression of virulence and several virulence-associated traits, including production of the exopolysaccharide amylovoran, levansucrase activity, and biofilm formation. We further show that Lrp is regulated posttranscriptionally by ArcZ through destabilization of lrp mRNA. Thus, ArcZ regulation of FlhDC directly and indirectly through Lrp forms an incoherent feed-forward loop that regulates levansucrase activity and motility as outputs. This work identifies Lrp as a novel participant in virulence regulation in E. amylovora and places it in the context of a virulence-associated regulatory network.IMPORTANCE Fire blight disease continues to plague the commercial production of apples and pears despite more than a century of research into disease epidemiology and disease control. The causative agent of fire blight, Erwinia amylovora coordinates turning on or off specific virulence-associated traits at the appropriate time during disease development. The development of novel control strategies requires an in-depth understanding of E. amylovora regulatory mechanisms, including regulatory control of virulence-associated traits. This study investigates how the small RNA ArcZ regulates motility at the transcriptional level and identifies the transcription factor Lrp as a novel participant in the regulation of several virulence-associated traits. We report that ArcZ and Lrp together affect key virulence-associated traits through integration of transcriptional and posttranscriptional mechanisms. Further understanding of the topology of virulence regulatory networks can uncover weak points that can subsequently be exploited to control E. amylovora.

Keywords: FlhDC; Hfq; fire blight.

FIG 1

Swimming motility of suppressor Tn mutants. Shown are mutants resulting from Tn5 mutagenesis of the E. amylovora Ea1189ΔarcZ mutant that were selected as motility suppressors and for which the Tn insertion site was successfully identified by sequencing. Blue bar, wild-type strain (wt); orange bar, ΔarcZ mutant. Green bars represent Tn mutants with a significant (P < 0.05) increase in motility compared to that of the ΔarcZ mutant by Student’s t test. Error bars represent standard deviations, and the experiment was repeated 4 times.

FIG 2

Lrp is a motility regulator epistatic to ArcZ. Swimming (A) and swarming (B) motility of indicated strains grown in or on the surface of soft agar medium, respectively. Error bars represent standard deviations, and groups with shared uppercase letter designations do not differ significantly (P > 0.05) from each other by Tukey’s honestly significant difference (HSD) test. (C) Representative images of swarming colonies after 48 h of incubation at 28°C. Scale bars, 3 mm.

FIG 3

Lrp regulates abundance of flagellar transcripts epistatic to ArcZ. Relative abundance of flhDC (A) or fliC (B) transcripts as determined by quantitative real-time PCR using recA as an endogenous control. The experiment was repeated three times, and error bars represent standard deviations. Groups with shared uppercase letter designations do not differ significantly (P > 0.05) from each other by Tukey’s HSD test.

FIG 4

ArcZ regulates Lrp posttranscriptionally by destabilizing lrp mRNA. (A) Relative fluorescence of strains carrying the Lrp translational fusion construct. (B) Relative abundance of lrp mRNA as determined by quantitative real-time PCR. (C) Lrp transcript stability following addition of rifampin at time zero. All experiments were conducted at least three times, and error bars represent standard deviations. *, P < 0.05 compared to wild-type by Student’s t test.

FIG 5

Lrp affects levansucrase activity epistatic to ArcZ. Levansucrase activity was assayed from overnight cultures by mixing culture supernatants in a 1:1 ratio with levansucrase assay buffer (phosphate-buffered 2 M sucrose) and incubating at 37°C for 24 h. Groups with shared uppercase letter designations do not differ significantly (P > 0.05) by Tukey’s HSD test. The experiment was repeated four times, and error bars represent standard deviations.

FIG 6

Lrp affects production of amylovoran epistatic to ArcZ. Amylovoran was quantified from supernatants of cultures grown in MBMA medium for 24 h by addition of cetylpyridinium chloride. Groups with shared uppercase letter designations do not differ significantly (P > 0.05) by Tukey’s HSD test. The experiment was repeated four times, and error bars represent standard deviations.

FIG 7

Lrp reverses high crystal violet staining of the E. amylovora Ea1189ΔarcZ mutant. Cells were grown in 96-well plates. After removal of planktonic cells, adherent cells were stained with crystal violet. Following rinsing of unbound crystal violet and drying, stain bound to adherent cells was solubilized by an ethanol-acetone solution, and the 590-nm absorbance was measured and normalized to the OD₆₀₀ of the cultures. *, P < 0.05 compared to wild-type by Student’s t test.

FIG 8

Lrp participates in overall virulence regulation. (A) Immature pears were inoculated with 10³ cells of E. amylovora strains and incubated at 28°C under high humidity conditions. Diameters of disease lesions on pears were measured every 24 h. (B) Representative pictures of inoculated immature pears. (C) Apple shoots on potted trees were cut inoculated with scissors dipped in a bacterial suspension of 5 × 10⁸ CFU/ml. Lesion length from the point of inoculation was measured at indicated time points. *, P < 0.05 compared to wild-type by Student’s t test. Experiments were repeated at least twice, with at least six replicates per experiment.

FIG 9

Complementation of the E. amylovora Ea1189ΔarcZ mutant with flhDC and its effects on amylovoran (A), levansucrase (B), and crystal violet staining (C) phenotypes. Experiments were repeated 4 times. *, P < 0.05 relative to wild-type phenotypes by Student’s t test.

FIG 10

Proposed model of ArcZ and Lrp regulation of virulence-associated traits in E. amylovora. ArcZ regulates flhDC directly and indirectly through Lrp in an incoherent feed-forward loop (blue box). As the target of the loop, FlhDC regulates motility and levansucrase activity as outputs. ArcZ affects amylovoran production through Lrp, but amylovoran production is not affected by FlhDC. Lrp modulation of amylovoran and indirect effects on motility and levansucrase activity through FlhDC result in Lrp playing a role in general virulence regulation (orange box).