Cif Is Negatively Regulated by the TetR Family Repressor CifR

Abstract

We previously reported that the novel Pseudomonas aeruginosa toxin Cif is capable of decreasing apical membrane expression of the cystic fibrosis transmembrane conductance regulator (CFTR). We further demonstrated that Cif is capable of degrading the synthetic epoxide hydrolase (EH) substrate S-NEPC [(2S,3S)-trans-3-phenyl-2-oxiranylmethyl 4-nitrophenol carbonate], suggesting that Cif may be reducing apical membrane expression of CFTR via its EH activity. Here we report that Cif is capable of degrading the xenobiotic epoxide epibromohydrin (EBH) to its vicinal diol 3-bromo-1,2-propanediol. We also demonstrate that this epoxide is a potent inducer of cif gene expression. We show that the predicted TetR family transcriptional repressor encoded by the PA2931 gene, which is immediately adjacent to and divergently transcribed from the cif-containing, three-gene operon, negatively regulates cif gene expression by binding to the promoter region immediately upstream of the cif-containing operon. Furthermore, this protein-DNA interaction is disrupted by the epoxide EBH in vitro, suggesting that the binding of EBH by the PA2931 protein product drives the disassociation from its DNA-binding site. Given its role as a repressor of cif gene expression, we have renamed PA2931 as CifR. Finally, we demonstrate that P. aeruginosa strains isolated from cystic fibrosis patient sputum with increased cif gene expression are impaired for the expression of the cifR gene.

Signature Motifs Identify an Acinetobacter Cif Virulence Factor with Epoxide Hydrolase Activity

“…Interestingly, we were unable to detect significant aCif catalysis of epibromohydrin, a substrate hydrolyzed by P. aeruginosa Cif and originally used to characterize its EH activity (27,46). Direct side-by-side epibromohydrin hydrolysis assays confirmed the discrepancy (data not shown).…”

“…Gene expression was monitored by qRT-PCR in WT A. nosocomialis cultured in the absence or presence of three epoxide compounds. One of these was epibromohydrin, a known activator of cif transcription in P. aeruginosa (46). However, because our substrate profiling indicated that aCif has a strong preference for (S)-styrene oxide (Fig.…”

“…6A). CifR binds to the intergenic sequence between the cifR and morB genes and blocks transcription in both directions; repression is relieved in the presence of various epoxides (21,46). Based on its 50% sequence identity to cifR, we hypothesized that the WP_004707394 locus encodes a functionally equivalent Acinetobacter CifR ("aCifR") protein that can regulate cif expression in A. nosocomialis by the same mechanisms observed previously in P. aeruginosa.…”

“…Next, we tested the ability of epoxides to increase transcription of both cif and cifR genes, which are coordinately repressed by CifR in P. aeruginosa (46). Gene expression was monitored by qRT-PCR in WT A. nosocomialis cultured in the absence or presence of three epoxide compounds.…”

LapD is a bis-(3′,5′)-cyclic dimeric GMP-binding protein that regulates surface attachment by Pseudomonas fluorescens Pf0–1

“…E. coli expressing PleD R148A was obtained from Urs Jenal (University of Basel, Switzerland), and the protein was purified as described (15,31). Purification of LapD6H and variants from E. coli was performed as previously described (32), with the addition of 1% Triton to all buffers. Protein purifications ranged from 79% to 96% purity, as determined by SDS PAGE gel staining and densitometry, and molar concentrations of each protein were corrected according to relative purity before all assays.…”