The fur-regulated gene encoding the alternative sigma factor PvdS is required for iron-dependent expression of the LysR-type regulator ptxR in Pseudomonas aeruginosa

Abstract

We previously identified a novel regulator of the exotoxin A gene (toxA) in Pseudomonas aeruginosa, PtxR, that belongs to the LysR family of prokaryotic regulatory proteins. Preliminary data also suggest that PtxR affects the expression of siderophores in P. aeruginosa. Because toxA expression and siderophore production in this organism are coordinately regulated by the ferric uptake regulator (Fur) and the Fur-regulated alternative sigma factor PvdS, regulation of ptxR itself in the context of these regulators was examined. RNase protection analyses of ptxR transcription revealed that there are two independent transcription initiation sites (T1 and T2). While transcription from the promoter of T1 is constitutive throughout the growth cycle of PAO1, transcription from the second promoter (P2) is negatively affected by iron. Transcription from the P2 promoter is constitutive in a fur mutant under microaerobic conditions but still iron regulated during aerobic growth. High concentrations (>100 nM) of the ferric uptake regulatory protein (Fur) failed to bind to either of the promoter regions of ptxR in either gel mobility shift assays or DNase I footprint experiments. These results indicate that Fur indirectly regulates the iron-dependent expression of ptxR. Iron-regulated transcription of ptxR from the P2 promoter, but not constitutive expression from the P1 promoter, was dependent on the Fur-regulated alternative sigma factor gene pvdS, even under aerobic conditions. Consequently, there are two levels of iron-regulated expression of ptxR. The iron-regulated expression of ptxR under microaerobic conditions from the P2 promoter of ptxR is mediated indirectly by Fur through the iron-regulated expression of pvdS. In contrast, pvdS-mediated iron regulation of ptxR under aerobic conditions is Fur independent.

FIG. 1

RNase protection assays of ptxR transcription. (A) RNase protection analysis of ptxR transcription in the PAO1 wild type and the Fur mutant PAO1C6 grown under aerobic conditions as previously described (1). RNA was isolated at 12 h from cells grown under low (L)- or high (H)-iron conditions (1). The riboprobe used in these experiments encompasses the 5′ region of ptxR plus its translational initiation codon. This probe detects both the T1 and T2 transcripts. (B) RNase protection analysis of ptxR transcription in the PAO1 wild type and the Fur mutant PAO1C6 grown under microaerobic conditions as previously described (1). (C) RNase protection analysis of ptxR mRNA in the PAO1 wild type and its pvdS mutant, PAO1ΔpvdS. The ptxR internal RNA probe was used in these experiments. The higher band in each lane represents an excess of undigested probe. The lower band in each panel represents ptxR mRNA, including both T1 and T2 transcripts. The cultures were grown aerobically with (H) and without (L) iron. Samples were obtained at the times indicated. The RI for each band was determined by using NIH Image software, version 1.55. An RNA size standard ladder is on the left of each panel. The lanes labeled probe contain the labeled probe used in these studies that was not digested with the RNase cocktail. WT, wild type; C6, PAO1C6.

FIG. 2

RNase protection analysis in the PAO1 wild type (PAO1WT) and its pvdS mutant, PAO1ΔpvdS. (A) The probe from the promoter region of the ptxR gene described in the text was used in these experiments. Included are the probe without any added mRNA, digested with the RNase cocktail, and the undigested probe. The cultures were grown aerobically with (H) and without (L) iron. All samples were obtained at 12 h. (B) The RNA samples were examined with the probe generated from the constitutively expressed lipoprotein gene (oprX) described in the text and elsewhere (1) and entered in GenBank under accession no. AF050676. Although included in the analysis, the undigested probe is not shown in this figure. The RI for each band was determined by using NIH Image software, version 1.55. MW, molecular size standards (measured in bases).