Abstract
The tssABC1 locus is part of the Hcp secretion island I (HSI-I) type VI secretion system (T6SS) in Pseudomonas aeruginosa Previous work implicated the tssC1 gene in P. aeruginosa biofilm-specific antibiotic resistance, and tssC1 is preferentially expressed in biofilms compared to planktonic cells. Using a DNA-dependent protein pulldown approach, we discovered that PA3225, an uncharacterized LysR-type transcriptional regulator, specifically bound to the tssABC1 upstream regulatory region. The deletion of PA3225 led to a 2-fold decrease in tssA1 expression levels in planktonic cells compared to the wild type, and tssA1 expression was slightly reduced in ΔPA3225 biofilms compared to wild-type biofilms. Intriguingly, further investigations revealed that the ΔPA3225 mutant was less susceptible to multiple, structurally unrelated antibiotics with various mechanisms of action when grown planktonically. The ΔPA3225 mutant was additionally more resistant to ciprofloxacin when grown in a biofilm. The decreased antibiotic susceptibility of the ΔPA3225 strain was linked to the transcriptional upregulation of the MexAB-OprM efflux pump. By using transcriptome sequencing (RNA-seq), other PA3225-regulated genes were identified, and the products of these genes, such as the putative ABC transporter PA3228, may also contribute to antibiotic resistance.