Fig 1.
AltR represses its own promoter and the thiosulfinate-tolerance (alt) gene cluster in Pantoea ananatis.
(A) Schematic of the altR promoter bioluminescent reporter construct. The intergenic region between altG and altR was cloned as the altR promoter (PaltR) upstream of the luxCDABE operon. Predicted AltR binding boxes and the inverted-repeat 2 [IR2] motif, the σ⁷⁰ promoter region (–35/ –10), and the ribosome-binding site (RBS) are indicated. Alignment of the predicted AltR and IR2 boxes is shown next to the alt cluster diagram, with AltR box colored in blue and IR2 box colored in orange in both the diagram and the sequence alignment. (B) Zone-of-inhibition (ZOI) assay showing PaltRLuxK6 activity in P. ananatis PNA 97-1R wild-type (WT), Δalt, and ΔaltR strains. The WT strain displays a distinct ring of bioluminescence at the ZOI border, indicating AltR de-repression in response to allicin exposure. (C) Quantification of relative ZOI area among WT, Δalt, and ΔaltR strains. Values represent mean ± SD of three biological replicates each with three technical replicates of the ratio of the ZOI area relative to WT. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test (p < 0.001). The Δalt mutant exhibited a significantly larger inhibition zone than WT, whereas ΔaltR did not differ significantly from WT. (D) Volcano plot of differential gene expression from RNA-seq comparing WT and ΔaltR strains (three biological replicates each). Genes that are up-regulated are highlighted in red, while down regulated genes are blue, with specific significant genes labelled. Deletion of altR resulted in strong up-regulation of altA–altJ and down-regulation of the YchH-like gene (B9Q16_RS12865). (E) Normalized RNA-seq read-coverage maps across the alt gene cluster in WT (red) and ΔaltR (blue). Plots show mean log-scaled coverage for three biological replicates, with individual replicate tracks below. In ΔaltR, coverage increases uniformly across altA–altJ, confirming AltR-mediated repression of the gene cluster.
Fig 2.
Thiosulfinates specifically trigger de-repression of the altR promoter in Pantoea ananatis.
(A–B) Zone-of-inhibition (ZOI) assays showing PaltR::lux reporter activity in Pantoea ananatis PNA 97-1R following treatment with the thiosulfinates allicin (A) and dimethyl thiosulfinate (“methicin”) (B). Both compounds induced a distinct ring of bioluminescence at the periphery of the inhibition zone, indicating AltR de-repression under sub-inhibitory concentrations. (C–D) Treatment with other thiol-reactive or oxidative compounds, including N-ethylmaleimide (NEM) (C) and diamide (D), no luminescent induction was observed, showing that AltR is unresponsive to these generic thiol oxidants. (E–G) The disulfide precursors diallyl disulfide (DADS) (E) and dimethyl disulfide (DMDS) (G), as well as hydrogen peroxide (H₂O₂) (F), also failed to trigger AltR de-repression. The absence of a luminescent ring in these treatments confirms that AltR activation is specific to thiosulfinates and not to disulfides or general oxidative stress.
Fig 3.
altR promoter de-repression coincides with necrosis during onion infection.
Red onion scale assays were performed using bioluminescently labeled Pantoea ananatis PNA 97-1R PaltRLuxK6 to visualize altR promoter activity during infection. Necrotic symptoms and corresponding bioluminescence (colored yellow) became evident at 3 days post-inoculation (dpi) and intensified at 4 dpi, coinciding with tissue collapse. The luminescence signal remained confined within necrotic tissue, consistent with thiosulfinate production from damaged onion cells. Note that signal intensities are not normalized across time points because images were captured independently on different days.
Fig 4.
The predicted altR box is required for promoter repression.
(A) Diagram of the altR promoter region showing the predicted altR box (CAATCTAC[N6]GTAGATTG). Inverted repeat (IR) half-sites are highlighted, with transversion mutations introduced in mod1 (one half-site) and mod2 (both half-sites). (B) Zone-of-inhibition (ZOI) assays of P. ananatis PNA 97-1R strains carrying PaltRmod1LuxK6 and PaltRmod2LuxK6 reporter constructs. Mutation of one or both IR half-sites led to constitutive bioluminescence across the plate, indicating complete loss of AltR-mediated repression.
Fig 5.
Cysteine residues of AltR are required for thiosulfinate-dependent de-repression.
(A) Zone-of-inhibition (ZOI) assays showing bioluminescence from PNA 97-1R PaltRLuxK6 strains carrying different altR cysteine-to-serine substitution alleles following treatment with synthesized allicin. Strains retaining Cys100 (e.g., CCC, CSS, CSC, CCS) displayed a luminescent ring at the edge of the inhibition zone, whereas all Cys100Ser (C100S) mutants lacked a luminescent ring, indicating loss of AltR responsiveness to allicin. (B) Quantitative analysis of relative ZOI area for wild-type and mutant strains in response to allicin. Data represents three biological replicates, each with three technical replicates of the ratio of the ZOI area relative to WT, analyzed by one-way ANOVA followed by Tukey’s multiple-comparison test (p < 0.001). (C) ZOI assays of the same altR cysteine mutants exposed to methicin (dimethyl thiosulfinate). As with allicin, only strains retaining Cys100 exhibited promoter induction, confirming that this residue mediates AltR redox sensing of thiosulfinates. (D) Relative ZOI area measurements for methicin assays analyzed as described in (B).
Fig 6.
Quantitative analysis of cysteine-to-serine substitutions on AltR-mediated de-repression and reduced repression in response to thiosulfinates.
(A) Zone-of-inhibition (ZOI) assay imaged using the Newton 7.0 (Vilber Smart Imaging) bioluminescence detection system. Six regions of interest (ROIs) were selected from both the induction ring (wt1 ~ wt6) and the background region (wt1b~wt6b) to measure photon counts, as illustrated. (B) Quantification of bioluminescence for P. ananatis PaltRLuxK6 altR cysteine mutants exposed to allicin. Mean maximum photon counts (blue bars) within the inhibition zone and mean background photon counts (orange bars) are shown for each allele. Strains retaining Cys100 exhibited significant de-repression compared to their background levels, while C100S mutant alleles did not. (C) Quantification of bioluminescence for the same mutant panel exposed to methicin. Similar trends were observed, confirming that Cys100 is essential for thiosulfinate-dependent induction. Error bars represent standard deviation of three biological replicates, each with three technical replicates. Significance was determined by two-sample t-tests: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).
Fig 7.
AltR cysteine mutations alter bacterial proliferation in onion tissue.
(A) Bacterial population sizes of PNA 97-1R PaltRLuxK6 strains carrying non-C100S altR alleles (CCC, CSS, CSC, CCS) on red onion scales at 4 days post-inoculation (dpi). The CSC mutant exhibited significantly reduced bacterial loads, comparable to the Δalt strain. (B) Bacterial populations of C100S mutant strains (SCC, SCS, SSC, SSS) at 4 dpi. All C100S alleles showed decreased proliferation relative to wild type but maintained higher populations than Δalt. Each data point represents a single biological replicate (six biological replicates, five technical repeats per biological replicates). Data were log₁₀-transformed prior to analysis. Statistical significance was assessed using one-way ANOVA followed by Tukey’s multiple-comparison test (p < 0.001). Pairwise comparisons against the WT control were evaluated by t-test.