Fig 4. VerA is an AraC-like transcriptional activator that positively regulates aerB and the vc0513-vc0515 operon.
(A) Overnight cultures of wild-type N16961 carrying either an IPTG-inducible copy of verA (+ verA) or empty vector (control) were diluted 1:100 in fresh LB containing kanamycin and IPTG (1 mM). RNA was isolated from cells at mid-log phase and prepared for RNA-seq (see Methods and Materials). Heat map shows normalized expression values for differentially expressed genes across three biological replicates. (B-C) Overnight cultures of strains carrying lacZ transcriptional reporters were diluted 1:100 in LB and grown for 3 h at 37°C. Kanamycin and inducer IPTG (500 μM) were included in the growth medium for retention and trans expression (+) of verA or zur from an IPTG-inducible promoter. Promoter activity (in Miller Units) was measured via β-galactosidase assays (See Methods and Materials). (B) PverA-lacZ activity was measured in wild-type, Δzur, Δvsp-II, and Δzur Δvsp-II strains carrying a plasmid-borne, IPTG-inducible copy of verA (+, striped bars) or an empty vector control (-, solid bars). (C) Activity from a PaerB-lacZ reporter was measured in wild-type, Δzur, and Δzur verA::STOP backgrounds harboring a plasmid-borne, IPTG-inducible copy of verA or zur (+, striped bars) or an empty vector control (-, solid bars). (D) Proposed model for Zur repression of the verA promoter (solid line, red) via a conserved Zur binding site (black box) and subsequent VerA-dependent activation (dashed arrow, green) of the aerB and verA promoters.