The ColRS signal transduction system responds to the excess of external zinc, iron, manganese, and cadmium

Abstract

Background: The ColRS two-component system has been shown to contribute to the membrane functionality and stress tolerance of Pseudomonas putida as well as to the virulence of Pseudomonas aeruginosa and plant pathogenic Xanthomonas species. However, the conditions activating the ColRS pathway and the signal(s) sensed by ColS have remained unknown. Here we aimed to analyze the role of the ColRS system in metal tolerance of P. putida and to test whether ColS can respond to metal excess.

Results: We show that the ColRS system is necessary for P. putida to tolerate the excess of iron and zinc, and that it also contributes to manganese and cadmium tolerance. Excess of iron, zinc, manganese or cadmium activates ColRS signaling and as a result modifies the expression of ColR-regulated genes. Our data suggest that the genes in the ColR regulon are functionally redundant, as several loci have to be deleted to observe a significant decrease in metal tolerance. Site-directed mutagenesis of ColS revealed that excess of iron and, surprisingly, also zinc are sensed by a conserved ExxE motif in ColS's periplasmic domain. While ColS is able to sense different metals, it still discriminates between the two oxidation states of iron, specifically responding to ferric and not ferrous iron. We propose a signal perception model involving a dimeric ColS, where each monomer donates one ExxE motif for metal binding.

Conclusions: Several transition metals are essential for living organisms in certain amounts, but toxic in excess. We show that ColRS is a sensor system which detects and responds to the excess of physiologically important metals such as zinc, iron and manganese. Thus, the ColRS system is an important factor for metal homeostasis and tolerance in P. putida.

Figure 1

Metal tolerances of different P. putida strains.P. putida wild-type strain PaW85 (wt), the colS-deficient strain (colS), colS-deficient strain complemented with the colS gene under the control of the inducible P_tac promoter (StacS), colR-deficient strain (colR), colR-deficient strain complemented with the colR gene under the control of the inducible P_tac promoter (RtacR) and colR-deficient strain complemented with the D51A mutant colR gene under the control of the inducible P_tac promoter (RtacR_D51A) were grown on solid LB medium containing different metal salts for 20 hours at 30°C. ColS and ColR expression was induced with 0.5 mM IPTG indicated by “+”. Approximately 5000 cells were inoculated per spot.

Figure 2

ColR-regulated genes respond to excess of zinc. β-galactosidase activities measured in P. putida wild-type (wt), colR- and colS-deficient strains (colR and colS, respectively) carrying the transcriptional fusions of PP0268, PP0737, PP0035, PP0900, PP0903, PP1636, PP2579 or PP5152 promoters with lacZ in the plasmid p9TT_BlacZ. P. putida wild-type was grown in LB medium or LB where 0.6 mM or 1.7 mM ZnSO₄ was added. colR- and colS-deficient strains were grown in LB or LB supplemented with 0.6 mM ZnSO₄. Data (means with 95% confidence intervals) of at least three independent experiments are presented. Asterisks indicate statistically significant differences (p < 0.05, two-way ANOVA with post-hoc Tukey’s Unequal N HSD test) between values obtained in LB and in LB supplemented with ZnSO₄.

Figure 3

ColR-regulated genes respond to excess of zinc, iron, manganese and cadmium. β-galactosidase activities measured in P. putida wild-type (wt) and colS-deficient strain (colS) carrying the transcriptional fusions of PP0268 or PP0903 promoters with lacZ in the plasmid p9TT_BlacZ. Bacteria were grown in LB medium and in LB containing either 0.6 mM ZnSO₄, 0.15 mM FeSO₄, 0.5 mM MnCl₂, 0.1 mM CoCl₂, 2 mM CuSO₄, 0.5 mM NiSO₄ or 0.2 mM CdSO₄. Data (means with 95% confidence intervals) of at least three independent experiments are presented. Asterisks indicate statistically significant differences (p < 0.05, two-way ANOVA with post-hoc Tukey’s Unequal N HSD test) between values obtained in LB and in LB supplemented with metal salt.

Figure 4

Expression of ColR is not induced by metal stress. (A) β-galactosidase activities measured in P. putida wild-type PaW85 strain carrying the transcriptional fusion of the colRS operon promoter with lacZ in the plasmid p9TT_BlacZ. Bacteria were grown in LB medium and in LB containing 0.6 mM ZnSO₄ or 0.15 mM FeSO₄. Data (means with 95% confidence intervals) of at least four independent experiments are presented. (B) Western blot showing ColR expression in P. putida wild-type (wt) and colR-deficient strain (colR). Location of ColR is indicated with an arrow. Proteins were extracted from bacteria grown in LB medium and in LB containing 0.6 mM ZnSO₄ or 0.15 mM FeSO₄. All lanes contain 3 μg of total protein extract.

Figure 5

Sequence analysis of the periplasmic domain of ColS. (A) Localization of the ColS protein in the inner membrane. Numbers correspond to the amino acid residues in ColS sequence showing the first and the last amino acid of ColS, its transmembrane domains and the periplasmic domain. (B) Amino acid sequence of the periplasmic domain of P. putida ColS. Glutamic acids of the putative iron binding motif are underlined. Asterisks indicate the amino acid residues mutated in this study. (C) Conservation of ColS’s periplasmic domain. Sequence logo for ColS periplasmic domain was created with the WebLogo server using 47 ColS sequences annotated in the Pseudomonas Genome Database. The acidic and basic amino acids are indicated in black and dark grey, respectively. Other amino acids are presented in light grey. The degree of sequence conservation at each position is indicated as the total height of a stack of letters, measured in arbitrary “bit” units, with a theoretical maximum of 4.3 bits at each position.

Figure 6

Conserved glutamic acids of the ExxE motif in ColS are necessary for metal-promoted activation of a ColR-regulated promoter. β-galactosidase activities measured in P. putida colS-deficient strain complemented with either the wild-type colS (StacS) or the colS variants carrying single substitutions of H35A, E38Q, D57N, H95A, E96Q, H105A, E126Q, E129Q or the double substitutions of E126Q and E129Q under the control of the inducible P_tac promoter. All strains carry the transcriptional fusion of the PP0903 promoter with lacZ in the plasmid p9TT_BlacZ. Bacteria were grown in LB medium containing 0.1 mM IPTG or 0.15 mM FeSO₄ or 0.1 mM IPTG and 0.15 mM FeSO₄ or 0.1 mM IPTG and 0.6 mM ZnSO₄. Data (means with 95% confidence intervals) of at least six independent experiments are presented. Asterisks indicate a statistically significant difference (p < 0.01, Student’s t-test) between the StacS strain and a strain carrying a mutant ColS in a particular medium.

Figure 7

ColS responds to ferric iron. β-galactosidase activities measured in P. putida wild-type PaW85 strain carrying the transcriptional fusion of the PP0903 promoter with lacZ in the plasmid p9TT_BlacZ. Bacteria were grown in LB medium and in LB containing 0.15 mM FeSO₄ or 0.075 mM Fe₂(SO₄)₃ with and without 0.5 mM Na-ascorbate. Data (means with 95% confidence intervals) of at least six independent experiments are presented. Asterisks indicate a statistically significant difference (p < 0.05, two-way ANOVA with post-hoc Bonferroni’s multiple comparison test) between values obtained in media containing no Na-ascorbate and media supplemented with Na-ascorbate.

Figure 8

Model of signal recognition and activation of the ColRS system. When Zn²⁺ or Fe³⁺ concentration is low, metal ions are not bound by the periplasmic domain of ColS and ColR is not phosphorylated. When P. putida experiences metal excess, a Zn²⁺ or Fe³⁺ ion binds with four glutamic acids of two ExxE motifs from two ColS proteins. Ion binding changes ColS conformation and the conserved histidine (H) in the dimerization and histidine phosphotransfer domain (DHp) is autophosporylated by the catalytic domain (CA) of ColS. Both in cis and in trans phosphorylation mechanisms are presented. Phosphate group is subsequently transferred from ColS to ColR and as a result ColR becomes active as a transcription regulator.