Fig 3. Several VPS-II genes are upregulated in a Δzur mutant.
(A) A predicted Zur-binding site (TGTTATGTTATAACA) located approximately 200 bp upstream of the verA open reading frame was aligned with the Vibrionacae Zur binding consensus sequence [35]. The predicted start codon (ATG) is indicated in bold. (B) PverA-lacZ transcriptional reporters were introduced into a wild-type and Δzur background, paired with either an empty vector or IPTG-inducible copy of zur (+ zur). Strains were grown overnight and diluted 1:100 into LB with kanamycin and inducer (IPTG, 200 μM). After 3 hours of growth at 37°C (to mid/late exponential phase), promoter activity was quantified in Miller Units by measuring β-galactosidase (LacZ) activity against an ONPG chromogenic substrate (see Methods for more details). (C) Wild-type, Δzur, and ΔznuABC mutants carrying the PverA-lacZ reporter were grown in M9 minimal medium in the presence (+) and absence (-) of exogenous zinc (ZnSO4, 1 μM). After overnight growth (~16 h), promoter activity was measured in Miller units. For bar graphs, raw data points represent biological replicates, error bars represent standard deviation, and asterisks denote statistical difference via Ordinary one-way ANOVA (****, p < 0.0001; **, p < 0.01; *, p < 0.05; and n.s., not significant). (D-E) RNA was isolated from wild-type and Δzur cells at mid-log phase and prepared for RNA-seq (see Methods and Materials). Genes with significant differential expression in Δzur (log 2-fold change > 1, adjusted p-value <0.05) relative to wild-type N16961 are shown. (D) The heat map indicates increased (orange) or decreased (purple) expression relative to the wild-type strain. Black circles represent putative Zur binding sites and lines correspond to likely operons. (E) Log 2-fold expression changes for all VSP-II genes (vc0490-vc0516) are shown alongside a schematic of VSP-II open reading frames. Significantly upregulated genes are shown in orange.