Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2000 Mar;182(6):1481-91.
doi: 10.1128/JB.182.6.1481-1491.2000.

Functional analysis of PvdS, an iron starvation sigma factor of Pseudomonas aeruginosa

L Leoni  1 , N Orsi, V de Lorenzo, P Visca
Affiliations

Functional analysis of PvdS, an iron starvation sigma factor of Pseudomonas aeruginosa

L Leoni et al. J Bacteriol. 2000 Mar.

Abstract

In Pseudomonas aeruginosa, iron modulates gene expression through a cascade of negative and positive regulatory proteins. The master regulator Fur is involved in iron-dependent repression of several genes. One of these genes, pvdS, was predicted to encode a putative sigma factor responsible for the transcription of a subset of genes of the Fur regulon. PvdS appears to belong to a structurally and functionally distinct subgroup of the extracytoplasmic function family of alternative sigma factors. Members of this subgroup, also including PbrA from Pseudomonas fluorescens, PfrI and PupI from Pseudomonas putida, and FecI from Escherichia coli, are controlled by the Fur repressor, and they activate transcription of genes for the biosynthesis or the uptake of siderophores. Evidence is provided that the PvdS protein of P. aeruginosa is endowed with biochemical properties of eubacterial sigma factors, as it spontaneously forms 1:1 complexes with the core fraction of RNA polymerase (RNAP, alpha(2)betabeta' subunits), thereby promoting in vitro binding of the PvdS-RNAP holoenzyme to the promoter region of the pvdA gene. These functional features of PvdS are consistent with the presence of structural domains predicted to be involved in core RNAP binding, promoter recognition, and open complex formation. The activity of pyoverdin biosynthetic (pvd) promoters was significantly lower in E. coli overexpressing the multicopy pvdS gene than in wild-type P. aeruginosa PAO1 carrying the single gene copy, and pvd::lacZ transcriptional fusions were silent in both pfrI (the pvdS homologue) and pfrA (a positive regulator of pseudobactin biosynthetic genes) mutants of P. putida WCS358, while they are expressed at PAO1 levels in wild-type WCS358. Moreover, the PvdS-RNAP holoenzyme purified from E. coli lacked the ability to generate in vitro transcripts from the pvdA promoter. These observations suggest that at least one additional positive regulator could be required for full activity of the PvdS-dependent transcription complex both in vivo and in vitro. This is consistent with the presence of a putative activator binding site (the iron starvation box) at variable distance from the transcription initiation sites of promoters controlled by the iron starvation sigma factors PvdS, PfrI, and PbrA of fluorescent pseudomonads.

PubMed Disclaimer

Figures

FIG. 1
FIG. 1
Alignment of the PvdS protein with Fur-controlled putative ECF sigma factors (PbrA, PfrI, PupI, and FecI), stress response sigma factors (AlgU and RpoE), and the primary sigma (RpoD). The sequences were translated from the GenBank entries given in parentheses: P. aeruginosa PvdS (U128919) and AlgU (L021119), P. fluorescens PbrA (X79908), P. putida PfrI (X82038) and PupI (X77918), and E. coli FecI (U14003), RpoE (U37089), and RpoD (J016887). For RpoD, the N-terminal 85 residues and a 254-residue nonconserved sequence (nc) between regions 1 and 2 are not shown. Regions 2, 3, and 4 of RpoD, conserved among the ς70 family (23), are shown below the alignment. Conserved residues among the entire data set are highlighted in reverse type. Shaded areas indicate conserved residues among the ECF family. Conserved residues among the Fur-controlled ECF sigma factors are boxed. Identical residues and the following sets of residues are considered matched: DE; NQ; RK; ST; FYW; ILVM. Plus signs and asterisks indicate amino acid positions selected for construction of maximum-likelihood trees.
FIG. 2
FIG. 2
Maximum-likelihood tree inferred from the primary structure alignment shown in Fig. 1. The analysis was limited to the 145 positions marked by the plus symbols in Fig. 1. The quartet puzzling method was used with a gamma-distributed model of site-to-site variation using eight rate categories. The gamma distribution parameter α estimated from the data set was 2.03, and the log-likelihood of the tree was −1,973.18. The scale bar represents 1.0 amino acid substitution per site. Numbers above nodes are quartet puzzling reliability values. Bootstrap confidence levels were comparable to quartet puzzling reliability values.
FIG. 3
FIG. 3
Overexpression of the His6-tagged PvdS protein (PvdS6H) in E. coli M15(pDMI,1) carrying the pPvdS6H expression plasmid. The PvdS6H protein was purified under denaturing conditions from the insoluble cell fraction. SDS-PAGE analysis of protein samples from key steps of the purification are shown: lane 1, uninduced whole cell extract; lane 2, induced whole cell extract; lane 3, soluble cell extract; lane 4, insoluble cell extract; lane 5, supernatant of DNP-treated inclusion bodies; lane 6, precipitate of detergent-treated inclusion bodies; lane 7, renatured fraction of the eluted PvdS6H protein; M, protein size standards of 200.0, 116.2, 97.4, 66.2, 45.0, 31.0, and 21.0 kDa (high and medium range; Bio-Rad). The arrow indicates the PvdS6H protein.
FIG. 4
FIG. 4
Copurification of PvdS with the core fraction of E. coli RNAP. The FLAG-tagged PvdS protein (PvdSF) was overexpressed in E. coli M15(pDMI,1) carrying the pPvdSF expression plasmid and purified under nondenaturing conditions from the soluble cell fraction. Lane 1, whole cell extracts of the uninduced culture; lane 2, whole cell extracts of the induced culture; lane 3, PvdS-RNAP complex (subunits α2ββ′), eluted from the FLAG affinity column; lane 4, commercial vegetative RNAP (subunits ς70α2ββ′; Epicentre Technologies). Protein size markers (high and medium range; Bio-Rad) are shown on the left. (A) SDS-PAGE analysis of protein samples. (B) Western blot analysis with anti-FLAG antibodies. (C) Western blot analysis with antibodies against the α subunit of the E. coli RNAP.
FIG. 5
FIG. 5
Expression of pvd::lacZ fusions (plasmids pPV51, pMP190::PpvdD, and pMP190::PpvdE) in E. coli MC4100(pDMI,1) harboring alternatively plasmids pPvdSWT, pPvdS6H, and pPvdSF. Cultures in LB medium (A600 = ≅0.4) were induced with 1 mM IPTG and incubated for additional 3 h at 37°C prior to testing for β-galactosidase activity (expressed in Miller units [28]). Uninduced cultures (0 mM IPTG) were used as controls.
FIG. 6
FIG. 6
Gel retardation assay for binding of PvdS and RNAP to the pvdA promoter in the presence of heparin (0.05 mg/ml). A 32P-labeled DNA fragment of 177 bp encompassing the pvdA promoter was used as the DNA probe. Lane 1, negative control (free DNA probe); lanes 2 to 4, vegetative E. coli RNAP holoenzyme (subunits ς70α2ββ′), 0.08 to 0.32 pmol; lanes 5 to 7, core RNAP (subunits α2ββ′), 0.08 to 0.32 pmol; lanes 8 to 10, PvdS6H protein, 0.25 to 1 pmol; lanes 11 to 15, PvdSF-core RNAP complex, 0.06 to 1 pmol; lanes 16 to 20, PvdS6H (0.06 to 1 pmol) and core RNAP (0.16 pmol). The amount of probe was 0.1 pmol for each sample. Arrows indicate the PpvdA probe.
FIG. 7
FIG. 7
Proposed regulatory cascade for pyoverdin (pvd) biosynthetic genes. In the presence of iron, the Fur protein binds the Fur box on the pvdS promoter, thereby repressing PvdS expression. In the absence of iron, Fur repression is relieved and pvdS is transcribed. The PvdS protein is an alternative sigma factor which confers to the core RNAP (cRNAP) specificity for pvd promoters. Additional positive regulator(s) may also be involved in the regulatory network.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Angerer A, Enz S, Ochs M, Braun V. Transcriptional regulation of ferric citrate transport in Escherichia coli K12. FecI belongs to a new subfamily of ς70-type factors that respond to extracytoplasmatic stimuli. Mol Microbiol. 1995;18:163–174. - PubMed
    1. Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem. 1976;72:248–254. - PubMed
    1. Brutsche S, Braun V. SigX of Bacillus subtilis replaces the ECF sigma factor FecI of Escherichia coli and is inhibited by RsiX. Mol Gen Genet. 1997;256:416–425. - PubMed
    1. Callanan M, Sexton R, Dowling D N, O'Gara F. Regulation of the iron uptake genes in Pseudomonas fluorescens M114 by pseudobactin M114: the pbrA sigma factor does not mediate the siderophore regulatory response. FEMS Microbiol Lett. 1996;144:61–66. - PubMed
    1. Casabadan M J, Cohen S N. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol. 1980;138:179–207. - PubMed

Publication types

MeSH terms

LinkOut - more resources