A Two-component Signal Transduction Pathway Regulates Manganese Homeostasis in Synechocystis 6803, a Photosynthetic Organism*

Elemental manganese is essential for the production of molecular oxygen by cyanobacteria, plants, and algae. In the cyanobacterium Synechocystis sp. PCC 6803, transcription of the mntCAB operon, encoding a high affinity Mn transporter, occurs under Mn starvation (nm Mn) conditions but not in Mn-sufficient (μm Mn) growth medium. Using a strain in which the promoter of this operon directs the transcription of the luxAB reporter genes, we determined that inactivation of the slr0640 gene, which encodes a histidine kinase sensor protein component of a two-component signal transduction system, resulted in constitutive high levels oflux luminescence. Systematic targeted inactivation mutagenesis also identified slr1837 as the gene encoding the corresponding response regulator protein. We have named these two genes manS (manganese-sensor) andmanR (manganese-regulator), respectively. A polyhistidine-tagged form of the ManS protein was localized in the Synechocystis 6803 cell membrane. Directed replacement of the conserved catalytic His-205 residue of this protein by Leu abolished its activity, although the mutated protein was present in cyanobacterial membrane. This mutant also showed suboptimal rates of Mn uptake under either Mn-starved or Mn-sufficient growth condition. These data suggest that the ManS/ManR two-component system plays a central role in the homeostasis of manganese inSynechocystis 6803 cells.

Manganese is an essential transition metal in almost all organisms. It plays a critical role for the photoautotrophic life style of cyanobacteria, algae, and plants. During oxygenic photosynthesis, a cluster of four Mn atoms in the photosystem II complex in thylakoid membranes catalyzes photolysis of water to produce molecular oxygen (1). Despite this importance of Mn in the biosphere, the regulatory details of cellular Mn homeostasis remain poorly understood (2,3).
We have identified previously MntABC, an ABC-type permease that mediates high affinity Mn transport in the cyanobacterium Synechocystis sp. PCC 6803 (4,5). The protein components of this transporter are encoded by three neighboring genes, mntA, mntB, and mntC, organized in an operon mntCAB. This transporter functions when Synechocystis 6803 cells are grown under Mn starvation conditions. A second high affinity Mn transporter functions in Synechocystis 6803 cells grown under Mn-sufficient conditions (5). Furthermore, a low affinity Mn transporter also operates in these cells. It is evident that Mn uptake in these cyanobacterial cells is controlled via a carefully modulated regulatory process.
The maintenance of homeostasis in a cell often requires complex sets of components that regulate the balance of metabolism. Such a regulatory cascade of events is initiated with the perception of the status of the environment with regard to specific metabolites or nutrients. Almost all cells utilize signaling cascades to respond to both positive and negative environmental stimuli. During recent years, two-component signal transduction has been recognized as a widely used strategy by which cells adapt and respond to their environment (6 -10). This means of sensing the environment is utilized by bacteria, as well as plants, and involves at least two separate protein components. At the beginning of the signal transduction chain is a protein containing a sensor domain that is typically a histidine kinase with a His residue that is essential to a phosphorylation cascade. The second component is the response regulator that contains a receiver regulatory domain with a critical aspartic acid residue, an acceptor of the phosphate group from the His group in the histidine kinase. Often, a second domain of the response regulator protein binds directly to DNA and interacts with the transcription machinery to regulate the expression of a set of genes (7)(8)(9). Recent analysis has shown that among the various bacterial species with completely sequenced genomes, cyanobacteria have the largest numbers of two-component sensor regulator pairs (10,11). In Synechocystis 6803 there are 43 proteins containing the canonical histidine kinase sensor domains and 40 proteins containing the response regulator signature (11). In the recently sequenced genome of the filamentous N 2 -fixing cyanobacterium Anabaena sp. PCC 7120, 195 genes encode components of such two-component signal transduction systems (12). To date, functional roles have been determined for only a limited number of such proteins in cyanobacteria. These include two-component systems for responses to extreme environmental conditions such as general nutrient limitation and high light stress (13), phosphate limitation (14), and cold stress (15,16). One of the first two-component sensor-regulator pairs to be identified in Synechocystis 6803 was the Cph1/Rcp1 proteins that are in-volved in light-regulation (17,18). The Cph1 protein was identified originally as a homolog of phytochrome (19). Recently, a two-component pair has been described that is involved in regulating the stoichiometry between photosystem I and photosystem II complexes by sensing changes in the redox state of the plastoquinone pool in Synechocystis 6803 membranes (20).
To understand the mechanism by which cyanobacteria sense the status of the available pool of manganese, we have engineered a reporter strain in which the expression of bacterial luciferase genes is controlled by Mn concentration in the growth medium. Using this strain, we have discovered a twocomponent sensor protein, ManS, which is involved in the sensing of Mn in Synechocystis 6803. We have also identified ManR, the cognate response regulator protein. We discuss the critically important role of the ManS/ManR system for the regulation of the overall Mn homeostasis in these cyanobacterial cells.

MATERIALS AND METHODS
Bacterial Growth Conditions-Wild-type and mutant cells of Synechocystis 6803 were grown at 30°C in BG11 medium (21), buffered at pH 8.0, and bubbled with air. Solid medium for cyanobacterial growth was BG11, supplemented with 1.5% agar and 5 mM sodium thiosulfate. Continuous illumination was provided by fluorescent lamps at 50 mol photons⅐m Ϫ2 s Ϫ1 . For Mn-uptake assays, Synechocystis 6803 cells were conditioned in Mn-free liquid BG11 medium, in which ferric ammonium citrate was replaced by ferric nitrate, and Mn was omitted from the trace element components. To starve Synechocystis 6803 for Mn, cells were grown in liquid BG11, washed, and then grown in Mn-free BG11 for 36 h prior to the uptake assays.
Escherichia coli cells for the propagation of plasmids and manipulation of DNA were grown in Luria Broth medium. Plasmids were propagated in E. coli strain DH5␣. Procedures for the growth of E. coli strains and for the manipulation of DNA were as described (22).
Construction of a pmntlux Reporter Strain and Isolation of Mutants-The construction and chromosomal insertion of the luxAB reporter fusion gene were as described previously (23). Specifically, a DNA fragment corresponding to the Ϫ6 to Ϫ139 nucleotides upstream of the translation initiation codon of the mntC gene (4), representing the promoter region (pmnt) of the mntCAB operon of Synechocystis 6803, was fused to the promoterless bacterial luxAB genes that encode the Lux reporter protein (see Fig. 1). This reporter construct was inserted at a site downstream of the ndhB gene in Synechocystis 6803, and the resulting strain (pmntlux) was used in the screening procedure outlined below. A genomic priming system (New England Biolabs) was used to mobilize a transposon containing a chloramphenicol-resistance (Cm R ) gene for random insertion into the DNA of 110 different cosmids, which contained chromosomal fragments of Synechocystis 6803 used previously for genome sequencing (24). The pmntlux strain of Synechocystis 6803 was transformed with this transposon inactivation library, and Cm R mutants that constitutively expressed the pmnt::luxAB reporter gene were isolated. For this purpose, the lux luminescence from Cm R colonies was assayed using a VIM camera system (model C-1400-47; Hamamatsu Photonics Co., Hamamatsu, Japan) and processed on an Argus 50 image analyzer (Hamamatsu Photonics Co). Genomic DNA isolated from each mutant strain was digested with HhaI, and after self-ligation it was used as the template for inverse PCR with outward primers corresponding to the 5Ј-and 3Ј-terminal regions of the Cm R cassette. The exact positions of the cassette in the mutant genomes were determined by sequencing the respective PCR products (see Fig.  2). To identify the corresponding regulator protein, four response regulator genes that are not localized in the 110 cosmids mentioned above were inactivated individually in the pmntlux reporter strain by inserting a kanamycin-resistance (Km R ) cassette in the respective coding regions, essentially as described previously (24).
Modification of the Histidine Kinase Gene manS-The coding region of the manS gene was fused translationally to a DNA segment encoding a hexahistidyl domain followed by a c-Myc epitope (see Fig. 3) in the pTYE007 plasmid (25). In addition, the Km R cassette and a 500-bp DNA fragment downstream of the manS gene were inserted distal to the c-myc gene. This construct (pTYE007-manS) was used to modify the manS gene. First, a previously described PCR-based method (26) was used to change the nucleotide A604 in the manS gene to a T, resulting in the replacement of His-205 in the ManS protein to a leucine residue.
Second, a periplasmic loop region (amino acids 60 to 132) of the ManS protein was removed by deleting a StuI/EcoRI DNA fragment from pTYE007-manS. These constructs were then used to transform the pmntlux reporter strain to produce two Km R mutant strains, pmntlux-manS⌬60 -132 and pmntlux-manSH205L, respectively.

SDS-PAGE and Western Blot Analysis of ManS Protein-Membranes from
Synechocystis 6803 cells were prepared as described earlier (27). SDS-PAGE was performed as described in Ref. 28. Polypeptides were electrotransferred to nitrocellulose membranes, which were then incubated with anti c-Myc antibodies (Santa Cruz Biotechnology). Goat anti-rabbit IgG conjugated to peroxidase was used to amplify the signals, and the reacting bands were visualized using enhanced chemiluminescence reagents (Amersham Biosciences).
Kinetics of Luminescence from Cells Containing the pmnt::luxAB Reporter Gene-The strains containing pmnt::luxAB as a reporter gene were grown in BG11. Before and after exposure to Mn-deficient medium the cells were adjusted to a density of OD 750 ϭ 0.1 or 0.01. A 300-l aliquot of the cell suspension was placed in the reaction tube in a Lumi-counter (model 2500; Microtech-Nichion, Chiba, Japan). The luminescence intensity was recorded in the absence of decanal for 1 min after which 10 l of 5% decanal was added. The luminescence intensity rose rapidly to attain the maximum within 10 s and then declined gradually. The maximum luminescence intensity was taken as a measure of the expression level of the pmnt::luxAB reporter gene.
Manganese Uptake Assays-Mn 2ϩ uptake assays were performed essentially as described earlier (5). Synechocystis 6803 cells were washed and resuspended in Mn-free BG11. After the addition of radioactive 54 Mn, 100-l samples were collected at specific time points and quickly dispersed in 5 ml of BG11 containing 10 mM cold Mn. The samples were collected on nitrocellulose membrane filters (BA83; Schleicher & Schuell) by vacuum filtration, and the filters were immersed in scintillation fluid and counted on an LS 5000 TD scintillation counter (Beckman Instruments).
RT-PCR Analysis of Expression of Metal Transporter Genes-The relative amounts of transcripts from various genes were evaluated by the RT-PCR method (29). Total RNA from Synechocystis 6803 cells cultured in normal or Mn-free BG11 medium was extracted according to Ref. 30, treated with RNase-free DNase I (Roche Molecular Biochemicals), and then purified by phenol-chloroform extraction and ethanol precipitation. A reverse transcription reaction was performed using Superscript II enzyme (Invitrogen). The products were amplified by PCR and then analyzed by electrophoresis on 0.8% agarose gels. Primers were designed so that the amplified products would be internal to the coding region of each gene. All of the forward primers were designed for sequences downstream of the translation initiation codon, and the reverse primers were designed to obtain ϳ350-bp-long PCR products from each gene. The RNaseP gene was used as a control template with constitutive expression levels (31). To identify factors that mediate such Mn-mediated regulation, a pmntlux reporter strain was constructed ( Fig. 1A; also see "Materials and Methods"). In this strain, the promoter of the mntCAB operon directs the transcription of the luxAB reporter genes. It is noteworthy that in this strain the endogenous mntCAB operon has not been modified. In these cells, the expression of the reporter gene and that of the mntCAB operon were similarly regulated by Mn (data not shown). This reporter strain was mutagenized randomly by transformation with a transposon inactivation library (see "Materials and Methods"), and the resultant Cm R colonies were screened for mutants that exhibited expression of the reporter gene under Mn-sufficient conditions (Fig. 1, B and C). Of nearly 20,000 Cm R colonies, we identified two such colonies. They were called manS-1 and manS-2, respectively.

Isolation of Mutants with Unregulated
Identification of a Genetic Locus That Regulates Expression of the pmnt Promoter-Analysis of the manS-1 and manS-2 strains showed that in both mutants, the Cm R gene was inserted in the same open reading frame, slr0640 (also termed hik27) (Cyanobase; www.kazusa.or.jp/cyano/cyano.html), although at two different positions ( Fig. 2A). This gene encodes a histidine kinase sensor protein that belongs to a two-component signal transduction system in Synechocystis 6803 cells (11). Because this protein is involved in manganese sensing (see below), we have named it ManS, and we have named the corresponding gene manS. The ManS protein has 441 residues with a predicted molecular mass of 49.2 kDa. COG (www. ncbi.nlm.nih.gov/COG/) and SMART (smart.embl-heidelberg. de/) analysis indicated that ManS is a histidine kinase sensor protein (10), with two transmembrane domains, a HisKA domain that includes the conserved and catalytically important His-205 residue, a HAMP dimerization domain, and a HAT-Pase domain (Fig. 2B).

Detection of ManS and Analysis of Expression of the pmnt::luxAB Reporter
Gene-To detect the ManS protein, we added both a polyhistidine tag and a c-Myc epitope tag at the C-terminal end of this protein (Fig. 3A). Using monospecific antibodies against the c-Myc protein, we were able to detect a 54-kDa protein (Fig. 3B) in the pmntlux reporter cells that have been transformed with the WT 1 /His/Myc version of the manS gene. The predicted molecular mass of this tagged protein is 52.9 kDa. To determine the role of His-205 in ManS in the predicted autophosphorylation and phosphotransfer events in such a two-component signal transduction system, this res- 1 The abbreviation used is: WT, wild-type. idue was changed to Leu. Such a modification still allowed the accumulation of the ManS protein in cell membrane, although at a reduced level (Fig. 3B). Finally, to evaluate the potential role of the periplasmic domain of this protein in binding Mn, a conserved loop (residues 60 to 132) was deleted from the protein. However, such a mutation resulted in the absence of the protein in the membrane (Fig. 3B). Fig. 3C shows the time-dependent changes in the lux luminescence intensity from the pmntlux cells, harboring various forms of the ManS protein. When grown in Mn-supplemented medium, luminescence from the WT, as well as the WT/His/ Myc strains, were extremely low. Incubation in Mn-depleted medium increased the level of luminescence from these cells by nearly 10,000-fold. In contrast, both the ⌬60 -132 (with no MntS protein) and the H205L strains exhibited very high levels of luminescence even when grown in the presence of Mn. These data demonstrated that the ManS protein is a strong determinant in Mn-mediated regulation of transcription of the pmnt promoter, and the His-205 residue plays an important role in this process.
Identification of the Cognate Response Regulator ManR-As described above, extensive mutagenesis of the pmntlux reporter strain using the cosmid inactivation library identified the ManS sensor but not the corresponding response regulator protein. The known response regulator genes in Synechocystis 6803 (11) that are not represented in this cosmid library were inactivated systematically in the reporter strain. Among such inactivation mutants, only the slr1837 (Fig. 2C) mutant cells exhibited high levels of lux luminescence, grown under either Mn-sufficient or Mn-depleted conditions (data not shown), similar to the observations with the mntS mutant cells described above. We concluded that the slr1837 gene encodes the response regulator that interacts with ManS and named it ManR. The ManR protein is 234 residues long with a predicted molecular mass of 25.6 kDa. COG and SMART analysis (see above) indicated that ManR is a member of the OmpR subfamily of response regulators and has an N-terminal CheY-like receiver domain (Fig. 2D) that includes the conserved phosphate acceptor Asp-52 residue (10). ManR also has a C-terminal winged-helix-type DNA binding domain, suggesting that it interacts directly with the pmnt promoter.
Manganese Uptake Activities in the manS Mutant Cells-Synechocystis 6803 cells have at least two high affinity Mnuptake systems (3,5). Among them, the MntABC transporter is present and active when cells are grown under Mn starvation conditions. As a consequence, wild-type cells had high Mnuptake activities when grown under both Mn-sufficient and Mn-deficient conditions (Fig. 4). As reported previously (5), the ⌬mntC mutant lacking any functional MntABC transporter showed poor uptake activity under Mn-deprived conditions. However, it had normal Mn-uptake activity when grown in Mn-sufficient condition. In contrast, the Mn-uptake activity of the H205L mutant, as well as in the original manS-1 mutant (data not shown), was unaffected by the presence or absence of the Mn in the growth medium. Notably, this activity was significantly less than that in wild-type cells, as well as in the ⌬mntC cells grown in the presence of Mn. Evidently, the MntS sensor protein controls not only the expression of the mntCAB operon but also the cellular Mn status in Synechocystis 6803.
Transcription of Other Metal Transporter Genes in the manS Mutant Cells-As mentioned earlier, the level of the mntC transcript was undetectable in wild-type cells grown in BG11 medium and increased significantly under Mn-starvation conditions (Fig. 5). In contrast, mntC was expressed at a high level in the H205L mutant cells, both in the presence and absence of Mn in the growth medium. Interestingly, transcript levels of feoB (slr1392) and znuA (slr2043), two genes whose expression is repressed in complete BG11 medium, were increased slightly in the H205L mutant strain. It is noteworthy that feoB encodes an iron transporter (32), and znuA (formerly called zntC) encodes a component of an ABC transporter for zinc (3). DISCUSSION The data presented in this paper demonstrate that the twocomponent sensor protein ManS and the response regulator protein ManR are required for the regulation of transcription of the mntCAB operon encoding an uptake Mn permease in Synechocystis 6803 cells. In a canonical bacterial two-component system, the sensor His kinase protein is often localized in the cell membrane. As shown in Fig. 2B, the ManS protein is predicted to have two transmembrane domains. Cyanobacterial cells have two internal membrane systems, the plasma membrane and the thylakoid membrane (33). Because the ManS sensor is presumably involved in the perception of extracellular Mn concentration, we have reasoned that this integral membrane protein is localized in the plasma membrane (Fig. 2B). It is tempting to speculate that the periplasmic domain of ManS is involved in the physical interaction with the Mn 2ϩ cation.
To date, the ManS/ManR pair constitutes the only known two-component signal transduction system for manganese. Among various transition metals, such two-component systems have been identified for copper and silver. Two different copper-responsive two-component systems are present in E. coli, namely, CusS/CusR (34), and PcoS/PcoR (35). In Pseudomonas syringae, a plant pathogen, the CopS/CopR two-component system provides copper resistance (36), whereas in Salmonella, the SilS/SilR system provides resistance to silver ions (37). During the preparation of this manuscript, Reyes and co-workers (38) reported that in Synechocystis 6803 cells, the RppA/ RppB two-component system identified originally by Li and Sherman (20) as a redox regulation system also has a role in nickel sensing. In all of these examples, the genes encoding the sensor His kinase and the response regulator are organized in operons. In contrast, the manS gene is located far away from the manR gene in the chromosome of Synechocystis 6803 (www.kazusa.or.jp/cyano/cyano.html).
During recent years, homologs of the MntABC transporter have been implicated to have significant roles in various bacterial infectious processes (39,40). A number of regulator proteins for bacterial Mn homeostasis have also been identified. Notably, Que and Helmann (41) (43). Interestingly, in E. coli cells, both Fur, an iron-dependent regulator, and MntR, a manganese-dependent regulator, control the expression of the mntH gene (44). It is noteworthy that none of these organisms has any known twocomponent signal transduction system for Mn.
As shown in Fig. 5, the ManS His kinase sensor protein appears to have some regulatory effect on the expression of the feoB and znuA genes. In other bacterial systems, expression of the iron transporter FeoB is known to be regulated by the well known regulator Fur. In Synechocystis 6803 cells, expression of the znuA gene is transcriptionally regulated by the Zur repressor protein, encoded by the sll1938 gene. 2 It is known that the Mn 2ϩ cation can bind to Fur (and presumably Zur) (45). It is possible that in the absence of ManS activity, an unregulated supply of Mn inside the cyanobacterial cells may lead to binding of this metal to Fur and Zur, with consequent transcriptional repression of the feoB and znuA genes. However, the dominant effect of ManS is on the pmnt promoter (Fig. 3C), indicating that the primary function of this protein is in sensing Mn.
Because of their oxygenic photosynthetic lifestyle, cyanobacterial cells must monitor carefully the available levels of Mn. The data presented in this manuscript demonstrate that the ManS/ManR two-component system in Synechocystis 6803 cells is an important determinant in the sensing of external Mn concentration. An additional interesting finding during this  2) and H205L (3, 4) strains. Transcript abundance in cells grown in the presence (1,3) or absence (2,4) of added Mn in the BG11 medium was determined by the RT-PCR method. The transcript level of RNaseP in each sample is shown as a control. The absence of contamination of DNA was confirmed by PCR without reverse transcriptase (ϪRTase) reaction. See "Results" for further details. study is that in the H205L mutant strain, Mn-uptake activity is suboptimal under both Mn-sufficient and Mn-deficient conditions (Fig. 4), raising the question whether pmnt is the only promoter in Synechocystis 6803 cells that is regulated by ManS. It is possible that the Mn-responsive signal transduction pathway initiating with the ManS His kinase has more than one cognate response regulator, one of which (ManR) acts on pmnt, whereas the other(s) may control the expression of the second high affinity Mn transporter, as well as that of the Mn-efflux system(s) in these cyanobacterial cells. Which other promoters are regulated by the ManS sensor, as well as how and where Mn binds to this protein, are being investigated currently.