Site-Directed Mutagenesis Identifies a Molecular Switch Involved in Copper Sensing by the Histidine Kinase CinS in Pseudomonas putida KT2440

Abstract

In the presence of copper, Pseudomonas putida activates transcription of cinAQ via the two-component system CinS-CinR. The CinS-CinR TCS was responsive to 0.5 μM copper and was specifically activated only by copper and silver. Modeling studies of CinS identified a potential copper binding site containing H37 and H147. CinS mutants with H37R and H147R mutations had an almost 10-fold reduced copper-dependent induction of cinAQ compared to the wild type.

Two-component systems (TCSs) are widely used by bacteria to regulate gene expression in response to environmental stimuli. The typical prokaryotic TCS is constituted by a membrane-bound histidine kinase (HK) and by a response regulator (RR). Canonical HKs contain a variable sensing domain and a conserved kinase domain. Upon sensing a stimulus, the HK autophosphorylates at a conserved histidine and then influences gene expression by phosphorylating its cognate RR at a conserved aspartate. HKs are homodimers in which each monomer phosphorylates the conserved histidine in the kinase core of the other monomer (for a review, see reference 40). RRs are usually DNA binding transcription factors, which undergo conformational changes upon phosphorylation that alter their affinity for target promoters, activating or repressing downstream genes.

The prototypical HK, such as EnvZ, PhoQ, TorS, and VirA, contains two transmembrane helices and a periplasmic sensory region (26), in addition to the conserved cytoplasmic domains. The sensory regions have a great deal of sequence diversity, reflecting the myriad of signals that are detected by these domains. The mechanisms by which these sensors recognize their stimulus are understood only for a few examples (15, 19, 33, 35), and no metal responsive HK has been characterized.

Copper is an essential micronutrient found in most organisms. It is found as a cofactor in many metalloenzymes, such as plastocyanin, azurin, cytochrome c oxidase, and hemocyanin. The main role of copper within copper proteins consists either in mediating electron transfers reactions (in electron shuttles or in many oxidases) or in carrying oxygen (hemocyanin). Nearly all bacterial copper proteins are either extracellular or are located in the periplasm or in cell membranes (13, 37). Compartmentalization of copper requiring enzymes outside of the cytoplasm seems to be a strategy evolved in bacteria to keep cytoplasmic concentrations of copper at a minimum, in order to prevent the highly reactive Cu(I) from entering metal binding sites of noncopper metalloproteins (44). When present in excess, copper damages the cells by metal-catalyzed oxidation of proteins and by causing oxidative damage through the generation of reactive oxygen species (2, 20). Recently, copper has been shown to directly damage the iron-sulfur cluster of dehydratase (23), suggesting that Cu(I) is able to ligate the sulfur atoms causing displacement of iron. Cells have therefore developed systems to detect and respond to copper stress. Several systems known in prokaryotes have been characterized, such as the Escherichia coli CopA P-type ATPase (10, 36), CueO multicopper oxidase (16), the CusCFBA system (12), and the Pseudomonas syringae CopABCD (7). Both E. coli copA and cueO are regulated by CueR, a MerR-like activator, while expression of cusCFBA and copABCD systems are under the control of copper-responsive TCSs CusRS and CopRS, respectively (30, 31, 39). Evidence suggests that the periplasm is the location in which copper exercises much of its toxic effect under aerobic conditions, thus bacteria necessitate copper protective systems in the periplasm (22).

Pseudomonas putida is a common saprophytic bacterium found in soil that has been well studied for biotechnological purposes (42). The sequenced genome of P. putida KT2440 reveals the presence of several potential Cu homeostasis systems, including homologues to genes encoding two putative P-type ATPases similar to CopA of E. coli, two homologues to the P. syringae CopA multicopper oxidase and CopB outer membrane proteins, and a CusCBAF system similar to E. coli CusCFBA (6). The P. putida cinAQ operon is also induced in the presence of copper (34). CinA (NCBI accession number NP_744308) is a member of the azurin-plastocyanin family with high redox potential (456 + 4 mV), while CinQ (NCBI accession number NP_744309) is a nitrile oxidoreductase involved in the nucleoside queuosine biosynthetic pathway (34). Transcribed divergently to cinAQ, the cinRS operon encodes a typical bacterial TCS comprising of a histidine sensor kinase (CinS [NCBI accession number NP_744306]) and a DNA binding RR (CinR [NCBI accession number NP_744307]). P. putida KT2440 cinA and cinQ mutants did not show an increased copper sensitivity (34). In contrast, cinA, cinS, and cinR mutants of P. aeruginosa PAO1 showed greater copper sensitivity in plate assays (41). P. aeruginosa PAO1 cinA and cinR mutants also showed reduced survival on copper surfaces (9), while the cinQ mutant was only slightly more sensitive to copper surfaces than was the wild type.

Bioinformatic analysis of sequenced genomes shows that genes encoding CinA homologues are nearly always found associated with genes encoding other copper homeostatic systems, such as homologues of Pseudomonas CopA multicopper oxidase, or homologues of efflux systems similar to E. coli CusCFBA or the P-type ATPase CopA. This suggests a role of CinA in copper resistance. Other times, cinA genes are found associated with genes encoding cytochrome c variants, suggesting a role of this protein in copper utilization as an alternative component of the electron transfer chain. CinQ is found associated with CinA only among the pseudomonads. Production of queuosine containing tRNA is known to be necessary for the correct identification of the UAG stop codon by the tRNA^Tyr (4, 14); however, the correlation between queuosine and copper homeostasis remains to be elucidated.

CinS is predicted to have two transmembrane helices and a periplasmic sensing domain (Fig. 1B). Analysis of the periplasmic region of CinS shows that its sequence is variable among the pseudomonads and is significantly divergent from the histidine sensor kinase CusS from E. coli (Fig. 1A), though a few residues are conserved between all putative periplasmic copper-sensing HKs (Fig. 1A; also see Fig. S1 in the supplemental material). Histidines, methionines, and cysteines are residues found coordinating copper in the metal binding sites of copper-containing proteins (13). Oxygen from aspartate and glutamate side chains are also involved in the coordination of other soft metals, like zinc, and more rarely, also in the coordination of copper.

FIG. 1.

(A) Alignment of Pseudomonas putida KT2440 CinS residues 1 to 190 with CinS homologues from other pseudomonads and CusS from Shigella and E. coli. Highlighted in dark gray and indicated with white text are the conserved histidines (two of which, H37 and H147 from P. putida KT2440, are also identified) in the periplasmic domain. Horizontal bars indicate the predicted transmembrane helices of CinS from P. putida KT2440 (prediction from the HMMTOP server [43]). Other conserved residues are highlighted; the level of darkness of the gray highlights is indicative of the percentage of identity. (B) Diagram of the HK CinS with two transmembrane helices and a periplasmic sensing domain. Residues in the periplasmic region targeted by site-directed mutagenesis are labeled. Conserved domains in the cytoplasm are also identified, alongside with putative phosphorylation site at the conserved histidine H242. (C) Position of H37 and H147 in the putative Cu binding site of CinS. The prediction of tertiary structure of the periplasmic domain of CinS was obtained by modeling the CinS by using SWISS-MODEL using E. coli Dcus (Protein Data Bank code 1ojg) (33) as a template.

In this study, we show that the CinS-CinR TCS is involved specifically in sensing copper and silver and in activating transcription of the cinAQ operon. Furthermore, we have identified residues likely to be involved in signal sensing in the periplasmic region of CinS and in forming a copper-dependent molecular switch.

Identification of conserved residues in the periplasmic domain of CinS.

A multiple sequence alignment of CinS homologues (Fig. 1A; also see Fig. S1 in the supplemental material) showed that the periplasmic sensory domain is highly variable but that a few residues (corresponding to P. putida KT2440 H37, F38, D42, H147, and H149) are always conserved among phylogenetically distant bacteria. These residues are also conserved in the periplasmic sensing HK CusS from E. coli W3110, as part of the CusR-CusS TCS involved in the copper-dependent activation of cusCFBA, which encodes an efflux system that pumps excess copper out of the periplasm. A prediction of the tertiary structure of the periplasmic domain of CinS from P. putida KT2440 was generated with SWISS-MODEL (1) using the periplasmic sensor domain of E. coli DcuS (Protein Data Bank code 1ojg) (33). The model indicated that H37 and H147 were located in two distinct helices, but in close proximity to each other (Fig. 1C). H146, along with H79, H115, and M133 and M154, is conserved among all the CinS homologues in pseudomonads (see Fig. S1 in the supplemental material), with the exception of a second putative copper-sensing HK CusS (NP_747485) from P. putida KT2440.

In order to investigate a possible role that these residues play in detecting the presence of copper in the periplasm, the cinRS operon from P. putida KT2440 was cloned along with its adjacent and divergently located cinAQ promoter into the pKT2CM-GFP vector containing a promoterless lacZ (24, 29) (see Fig. S2 in the supplemental material). Transcription of lacZ, in the resulting plasmid pKTcinRS, is under the control of the cinAQ promoter (transcriptional fusion PcinAQ::lacZ) (Table 1), permitting the study of the activation of the cinAQ promoter by CinRS with a β-galactosidase assay (28). Mutagenesis of selected amino acids located in the predicted periplasmic region of CinS (Fig. 1B and Table 1) was performed using Stratagene's QuikChange II XL site-directed mutagenesis kit, using a PCR product containing the entire cinRS operon and cinAQ promoter cloned in the pGEM-T Easy vector system (Promega) as a template (pGCin). Mutations were verified by sequencing, and each construct was then subcloned into the pKT2CM-GFP vector (Table 1). The effect of mutations in CinS could be then evaluated by analyzing the transcription of lacZ after activation of the cinAQ promoter.

TABLE 1.

Bacterial strains and plasmids used in this study

Strain or plasmid	Relevant feature(s)	Reference or source
Strains
E. coli XL-1		Stratagene
E. coli W3110	Derived from K-12 λ− F−	3
E. coli W3110 ΔcusS	W3110 strain carrying a disrupted cusS; Kanr	This study
Pseudomonas putida KT2440		ATCC 47054
PAO2810::ISphoA/hah	P. aeruginosa PAO1 with gene disruption cinS::Tetr	University of Washington
PAO2809::ISlacZ/hah	P. aeruginosa PAO1 with gene disruption cinR::Tetr	University of Washington
Plasmids
pGEM-T Easy	Cloning vector; Ampr	Promega
pKT2CM-GFP	pVS1 replicon, p15a origin or replication, containing a BamHI-HindIII fragment carrying a promoterless lacZ gene; Kmr Cmrlac+gfp+	24
pGcin	pGEM-T containing a 2.4-kb BamHI frament with cinRS genes and the cinAQ promoter	This study
pKTcinRS	pTK2CM-GFP containing a 2.4-kb BamHI frament with cinRS genes and the cinAQ promoter that drives expression of lacZ	This study
pKT-C18S	pTKcinRS with cinS mutation C18S	This study
pTK-H37R	pTKcinRS with cinS mutation H37R	This study
pKT-H79R	pTKcinRS with cinS mutation H79R	This study
pKT-H115R	pTKcinRS with cinS mutation H115R	This study
pKT-M133A	pTKcinRS with cinS mutation M133R	This study
pKT-H146R	pTKcinRS with cinS mutation H146R	This study
pKT-H147R	pTKcinRS with cinS mutation H147R	This study
pKT-H146R/H147R/H149R	pTKcinRS with cinS mutations H146R, H147R, and H149R	This study
pKT-H147R/H149R	pTKcinRS with cinS mutations H147R and H149R	This study
pKT-H149R	pTKcinRS with cinS mutation H149R	This study
pKT-M154A	pTKcinRS with cinS mutation M154A	This study
pKT-RRD51N	pTKcinRS with cinR mutation D51N	This study
pKTcinRS-Tag	pKTcinRS containing a Strep-TagII at the C terminus of CinS	This study
pTK-H37R-Tag	pTK-H37R containing a Strep-TagII at the C terminus of CinS	This study
pKT-H147R-Tag	pKT-H147R containing a Strep-TagII at the C terminus of CinS	This study
pKT-H147R/H149R Tag	pKT-H147R/H149R containing a Strep-TagII at the C terminus of CinS	This study
pKTprom	pTK2CM-GFP containing a 200-bp BamHI fragment with the cinAQ promoter that drives expression of lacZ	This study

Low concentrations of copper induce transcription of cinAQ.

Induction assays were performed with P. aeruginosa to reduce the possible interference of overlapping copper-sensing systems present in P. putida KT2440 (6). Plasmids containing the modified cinS alleles were transformed into the P. aeruginosa PAO1 derivative transposon cinS mutant PA2810::ISphoA/hah (obtained from the University of Washington Genome Center) (18). PAO2810::ISphoA/hah cells carrying the reporter plasmid were grown in minimal salts medium, prepared with no copper and with 20 mM of glucose (27), to early log phase (optical density of ∼0.3) at 37°C and then challenged with additions of 0 μM, 0.5 μM, 1 μM, 5 μM, and 10 μM CuSO₄. β-Galactosidase assays were performed after 45 min of copper induction, according to Miller (28).

CinS (expressed in pKTcinRS) showed an 8-fold induction of lacZ at 0.5 μM Cu, compared to the control with no copper (Table 2 and Fig. 2A). These data show that transcription from PcinAQ was very sensitive to low copper concentrations. Although a mathematical comparison of sensitivity of copper induction of the PcinAQ with other copper-inducible promoters is not possible due to the different experimental setups, PcinAQ was activated at concentrations lower than those reported for the CueR-dependent activation of cueO and copA and those reported for the CusR-dependent activation of cusC in E. coli (32). The cinAQ promoter could therefore be of interest for utilization in Cu bioavailability biosensors (for a review, see reference 25). Similar to E. coli CusS-CusR (31), the CinS-CinR TCS was also able to detect silver (see Fig. S5 in the supplemental material).

TABLE 2.

Results of β-galactosidase assay obtained with different cinS_KT2440 alleles in PAO2810::ISphoA/hah (cinS_PAO transposon mutant)

Protein	Result (Miller units) at indicated CuSO4 concn (μM)a
	0	0.5	1	5	10
Wild-type CinS	184 ± 8	1,554 ± 51	2,453 ± 100	6,147 ± 435	5,610 ± 43
CinS-C18S	265 ± 40	2,316 ± 178	4,004 ± 653	5,639 ± 346	6,357 ± 310
CinS-H37R	204 ± 15	194 ± 4	215 ± 13	300 ± 19	289 ± 20
CinS-H79R	212 ± 8	768 ± 50	1,300 ± 31	5,513 ± 143	6,779 ± 129
CinS-H115R	170 ± 10	1,303 ± 144	1,841 ± 140	6,433 ± 175	6,329 ± 189
CinS-M133A	195 ± 10	1,587 ± 79	2,334 ± 189	6,397 ± 344	6,693 ± 267
CinS-H146R	172 ± 7	2,790 ± 233	4,550 ± 502	6,324 ± 402	6,186 ± 267
CinS-H147R	196 ± 14	166 ± 12	180 ± 11	248 ± 16	219 ± 19
CinS-H146R/H147R/H149R	311 ± 40	274 ± 40	307 ± 39	314 ± 42	448 ± 58
CinS-H147R/H149R	175 ± 11	171 ± 16	184 ± 13	252 ± 22	294 ± 18
CinS-H149R	188 ± 9	1,147 ± 87	1,731 ± 107	6,840 ± 382	6,762 ± 274
CinS-M154A	192 ± 72	1,553 ± 129	2,856 ± 310	5,717 ± 214	5,010 ± 468

^aData are averages of the results for three independent induction experiments, with each experiment performed in duplicates. Values are expressed in Miller units with the standard deviation.

FIG. 2.

β-Galactosidase activity (in Miller units) for pKTcinRS expressing the wild-type CinS (A) and the single mutants CinS-H37R (B) and CinS-H147R (C). The plasmids were assayed in PA2810::ISphoA/hah, a PAO1 mutant strain with a disrupted cinS. Cells were grown in minimal salts medium to an optical density of ∼0.3 and then exposed for 45 min at various copper concentrations. (D) Effect of different metals in the activation of PcinAQ, measured as lacZ induction in pKTprom; assay was performed in the ΔcinR::kan PA2809::ISlacZ/hah. In these cells, there is not a functional copper-dependent RR CinR that can activate the promoter in the presence of copper.

Activation of the cinAQ promoter by CinR.

In PA2810::ISphoA/hah, cinS is disrupted by a transposon insertion at position 520, interrupting CinS after the 173 residues after the second transmembrane helix. This mutant still has an intact cinR. In this strain, CinS from P. putida KT2440 (CinS_KT2440) recognizes the copper signal in the periplasm and phosphorylates an RR, which in turns activates transcription of cinAQ_KT2440. To verify that the transcription of the lacZ system was under the control of cinRS _KT2440, the entire cinAQ promoter was cloned into the same vector, yielding pKTprom. On this plasmid, transcription of lacZ is under the control of the cinAQ promoter.

When transformed into PAO2810::ISphoA/hah (PAO1 CinS mutant), pKTprom showed strong induction of lacZ, even in the absence of copper (Table 3). To see whether the promoter was constitutively activated in the absence of CinR_KT2440, or whether CinR_PAO was interfering with its activation, pKTprom was transformed into PA2809::ISlacZ/hah (PAO1 in which cinR had been disrupted by the transposon insertion). β-Galactosidase activity was very low (269 Miller units with no copper added), and no copper dependence was detected (Table 3). These results indicate that phosphorylation of CinR_KT2440 upon copper detection by CinS_KT2440 led to activation of the cinAQ promoter. When CinR_KT2440 was not present (pTKprom), cinAQ transcription was initiated by CinR_PAO, in a copper-independent fashion (Table 3). According to this model, CinR_KT2440 bound to the cinAQ promoter regardless of its phosphorylation status, but it activated transcription only after being phosphorylated by the HK. When the cinAQ promoter was not occupied by CinR_KT2440 (when pKTprom was transformed into PA2810::ISphoA/hah background containing an intact CinR_PAO), then CinR_PAO could bind and activate its expression.

TABLE 3.

Results of β-galactosidase assay obtained for CinR(D51N) and the cinAQ promoter in different PAO1 transposon mutants

Plasmid	PAO1 transposon background	Result (Miller units) at indicated CuSO4 concn (μM)a
		0	0.5	1	5	10
pKTRR-D51N	ΔcinR	376 ± 27	430 ± 11	361 ± 51	359 ± 8	348 ± 10
pKTprom	ΔcinR	269 ± 15	279 ± 33	276 ± 19	289 ± 26	275 ± 24
	ΔcinS	16,043 ± 1,158	15,663 ± 966	13,520 ± 1,144	13,964 ± 1,272	1,695 ± 1,445

^aData are averages of the results for three independent induction experiments, with each experiment performed in duplicates. Values are expressed in Miller units with the standard deviation. The cinR PAO1 transposon mutant is PAO2809::ISlacZ/hah. The cinS PAO1 transposon mutant is PAO2810::ISphoA/hah.

To provide evidence for this model, a pTKcinRS plasmid was mutagenized to introduce a point mutation in the putative phosphorylation site of the RR. Sequence alignment with known TCS DNA binding RR (not shown) identified Asp51 of CinR_KT2440 as the possible site of phosphorylation by an HK. The cinR allele carrying a mutation in Asp51 (plasmid pKTRR-D51N) was assayed in PA2809::ISlacZ/hah (PAO1 strain with a transposon insertion in cinR) and showed very low lacZ induction that was not copper dependent (Table 3).

pKTprom was also assayed in PA2809::ISlacZ/hah to verify whether other metals could determine the activation of the cinAQ promoter in the absence of CinR (Fig. 2D). Of the different metals tested, only Zn was able to activate transcription of lacZ. However, the transcriptional regulator that determined the activation of PcinAQ was not known. P. aeruginosa genomes contains putative genes for 63 HKs and 64 RRs (38) including a putative CzcRS, which has been shown to be responsive to Zn (17). Furthermore, a correlation between copper and zinc resistance has been reported previously for P. aeruginosa PAO1 (5).

The promoter region of cinAQ presents a “cop box” similar to those found in the promoters of several copper and silver homeostasis genes (30, 31). Such “cop boxes” are believed to be recognized by the RR. A “cop box” is also found in the promoter of E. coli W3110 cusCFBA (12). Induction experiments using pKTcinRS transformed into an E. coli W3110 background, also resulted in a copper-dependent activation of cinAQ, but only at higher copper concentrations. Induction experiments using an E. coli W3110 ΔcusS background strain showed no induction of lacZ in the presence of copper, revealing that cinAQ activation was mediated by CusRS_E.coli, not CinRS_KT2440 (data not shown).

His37 and His147 are required for copper-mediated induction.

CinS-H37R and CinS-H147R resulted in minimal induction compared to the wild-type CinS at all tested copper concentrations (Table 2 and Fig. 2A to C). These results suggest that H37 and H147 may be involved in binding of copper in the periplasmic loop of CinS or in the relay of the detection of the environmental stimulus to the cytoplasmic domains of CinS. Sequence alignment (Fig. 1A; also see Fig. S1 in the supplemental material) revealed that both H37 and H147 are always conserved among CinS homologues, indicating the importance of these residues, while tertiary structure predictions indicate that H37 and H147 are located in close proximity (Fig. 1C). The double mutant CinS-H147R/H149R and triple mutant CinS-H146R/H147R/H149R also presented minimal copper inducibility compared to wild-type CinS (Table 2). These results confirm that H147 is an essential residue for the function of CinS.

To verify that CinS-H37R and CinS-H147R were actually translated by the cells, Stratagene's QuikChange II XL site-directed mutagenesis kit was also used to insert a Strep-TagII (WSHPQFEK) between CinS residues D449 and the C-terminal R450 to obtain Strep-tagged versions of CinS alleles from pKTcinRS and pKT-H37R in pKT-H147R and in pKT-H146R/H147R. The Strep-TagII epitopes were detected from membrane extracts (see Fig. S4 in the supplemental material), indicating that these HKs were produced.

H146R and H149R mutations do not affect the ability to sense Cu.

Sequence alignment (Fig. 1A; also see Fig. S1 in the supplemental material) shows that an HHQH cluster (corresponding to residues 146 to 149 in P. putida CinS) is very conserved among the pseudomonads. H146 is conserved mainly among the pseudomonads, while H147 and H149 are conserved among more-diverse groups of bacteria.

The tertiary structure model indicated that H37 and the HHQH sequence are located on two distinct alpha helices (Fig. 1C), but are in close proximity to each other. The proximity of these residues in conjunction with the mutagenesis results implies that H37 and H147 could form a potential copper binding site. Furthermore, the side chains of H146, H149, and the conserved F150 are oriented away from the putative copper binding site and could play a role in modulating the activation of the HK upon copper binding. However, CinS containing a single mutation from H to R at position 146 (CinS-H146R) or at position 149 (CinS-H149R) did not show a reduction in copper inducibility of lacZ compared to the wild-type protein, indicating that mutations at these residues do not affect the ability of the periplasmic domain to relay the signal to the cytoplasmatic domains of the sensor kinase.

Other conserved residues were also mutated (C18S, H79R, H115R, M133A, and M154A) and analyzed to examine the effect of such mutation on the ability of CinS to elicit activation of the cinAQ promoter. Although all these mutants responded similarly to 10 μM of Cu, greater variability was observed in the response to 0.5 and 1 uM of Cu, with some mutants being less responsive than the wild type (H79R and H149 mutants) and others being more responsive (C18S and H146R mutants) (Table 2). C18 is located in the first predicted transmembrane region (Fig. 1B), while three-dimensional modeling locates H146 and H149 in close proximity to the membranes. Mutations in these regions might interfere with the modulation of the activation of the cytoplasmic domains of the sensor kinase.

Possible Cu(I) and Ag(I) molecular switch.

Tertiary structure predictions of the periplasmic region of P. putida CinS shows the residues 146-HHQH-149 at the beginning of a helix (Fig. 1C). This cluster could form a binding site also containing H37, which is predicted to be part of a different helix. The HHQH clusters in CinS homologues are always located in close proximity to a conserved phenylalanine (F150 in P. putida CinS) (Fig. 1A; also see Fig. S1 in the supplemental material).

Sequence alignment shows that one aspartate, D42, is also always conserved, while a second aspartate is always found in close proximity of the HQHH cluster (D143 in CinS). Both aspartates are located in sequence proximity to the hypothetical copper binding site comprising H37 and 146-HHQH-150. As oxygens from aspartates and glutamates are known ligands for soft metals such as Zn in metalloproteins, we could speculate that in the absence of copper, zinc (or another soft metal) occupies the binding site and is coordinated by the two oxygens of the aspartate. Once copper is present in the periplasm, zinc is displaced, the coordination geometry of the site changes, and this elicits the activation of the HK. The CinS model shows that D143 and D42 are too far away from each other to jointly coordinate a metal, and this does not support this model. However, when challenged with both Cu and Zn, the presence of 5 μM of Zn could reduce the induction of lacZ by CinS (see Fig. S3 in the supplemental material). The effective reduction of β-galactosidase activity might be even greater, since Zn was shown to induce the cinAQ promoter (in pTKprom), in a CinRS-independent way (Fig. 2D).

In conclusion, P. putida KT2440 transcribes cinAQ in the presence of copper. We have shown here that activation of cinAQ promoter was mediated by the TCS CinS-CinR. In the periplasmic domain of CinS, we identified two histidines, H37 and H147, that were essential to induce the transcription from the cinAQ promoter, and we propose that these two histidines identify a copper sensing site in the periplasmic domain of periplasmic copper sensing HKs. Mutagenic studies, along with tertiary structure prediction and multiple sequence analysis, support the conclusion that these two histidines, corresponding to P. putida KT2440 H37 and H147 are involved in sensing copper in the periplasmic copper-sensing HKs. Bidentate copper coordination is also found in CueR. In this MerR-like RR, Cu(I) is coordinated by thiolate groups from two cysteines, which confer high affinity (in the zeptomolar range) and high specificity for copper (8).

The low concentrations of copper detected in the periplasm by the TCS CinR-CinS in Pseudomonas putida KT2440 also suggest that this TCS may be involved in not only the activation of resistance systems in response to toxic copper levels, but also the detection of physiological levels of this metal, in order to activate copper utilization pathways. cinA, activated in the presence of copper by the TCS CinR-CinS, may be more involved in copper utilization than in controlling copper homeostasis; however, cinA mutants of P. aeruginosa PAO1 are very sensitive to copper.

It is generally assumed that in aerobically grown bacterial cells, copper is found as Cu(I) in the cytoplasm and as Cu(II) in the periplasm. Experimental data indicate that the biologically active form of copper in the cytoplasm is represented by Cu(I). Cu(I) is sensed by DNA binding regulators such as E. coli CueR, Enterococcus hirae CopY, and Mycobacterium tuberculosis CsoR. Copper is also transported across membranes as Cu(I) by P-type ATPases and shuttled across the cytoplasm by eukaryotic copper chaperons or by the cyanobacterial Atx1 and by E. hirae CopZ. Cu(I) is also responsible for directly damaging fumarase A in vitro (23).

However, several evidences also suggest that Cu(I) is not only available but that it is also a biologically relevant oxidation state in the periplasm. For example, the periplasmic copper chaperon CusF was shown to coordinate Cu(I) (21), indicating that Cu(I) is present in detectable amounts even under aerobic conditions in the periplasm. Furthermore, both P. putida CinS and E. coli CusS respond to Ag(I) (see Fig. S5 in the supplemental material) (11), which is redox inactive and chemically similar to Cu(I), but not to Cu(II) (36). Finally, an important mechanism of copper homeostasis is conferred by periplasmic multicopper oxidases (such as Pseudomonas CopA and E. coli CueO) that catalyzes the oxidation of Cu(I) to the less toxic Cu(II) and, therefore, recognizes the periplasmic Cu(I).