JBC Invitrogen Ultrasensitive Cytokine Assays

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Originally published In Press as doi:10.1074/jbc.M411799200 on November 29, 2004

J. Biol. Chem., Vol. 280, Issue 6, 4360-4366, February 11, 2005
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A Role for SlyD in the Escherichia coli Hydrogenase Biosynthetic Pathway*

Jie Wei Zhang{ddagger}, Gareth Butland§, Jack F. Greenblatt§, Andrew Emili§, and Deborah B. Zamble{ddagger}

From the {ddagger}Department of Chemistry, University of Toronto, Toronto, Ontario M5S 3H6, Canada and the §Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G 1L6, Canada

Received for publication, October 18, 2004 , and in revised form, November 23, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The [NiFe] centers at the active sites of the Escherichia coli hydrogenase enzymes are assembled by a team of accessory proteins that includes the products of the hyp genes. To determine whether any other proteins are involved in this process, the sequential peptide affinity system was used. The analysis of the proteins in a complex with HypB revealed the peptidyl-prolyl cis/trans-isomerase SlyD, a metal-binding protein that has not been previously linked to the hydrogenase biosynthetic pathway. The association between HypB and SlyD was confirmed by chemical cross-linking of purified proteins. Deletion of the slyD gene resulted in a marked reduction of the hydrogenase activity in cell extracts prepared from anaerobic cultures, and an in-gel assay was used to demonstrate diminished activities of both hydrogenase 1 and 2. Western analysis revealed a decrease in the final proteolytic processing of the hydrogenase 3 HycE protein, indicating that the metal center was not assembled properly. These deficiencies were all rescued by growth in medium containing excess nickel, but zinc did not have any phenotypic effect. Experiments with radioactive nickel demonstrated that less nickel accumulated in {Delta}slyD cells compared with wild type, and overexpression of SlyD from an inducible promoter doubled the level of cellular nickel. These experiments demonstrate that SlyD has a role in the nickel insertion step of the hydrogenase maturation pathway, and the possible functions of SlyD are discussed.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The production of metalloenzymes frequently requires dedicated auxiliary proteins to assemble the functional metallocenters (1, 2). In the case of an enzyme with a single ion bound to unmodified protein ligands, maturation usually involves just one partner protein (1, 3). These factors, referred to as metallochaperones (3), deliver the correct metal ion to the target protein via protein-protein interactions (1). For the biosynthesis of more complex metallocenters, multiple accessory proteins are often required (2). These factors control a cascade of events that can include gathering and insertion of all of the inorganic and organic components, partial construction of the metal center, posttranslational modifications, electron transfer, protein folding, and/or hydrolysis of nucleotide triphosphates to drive the whole process forward (2). These molecular factories generate enzymes that are essential for a variety of fundamental cellular processes, but many of the protein components have not yet been identified or fully characterized.

The hydrogenase enzymes, which catalyze the reversible formation of dihydrogen (H2) from two protons and two electrons, contain several different types of active sites (4, 5). In Escherichia coli the hydrogenases are all members of the [NiFe] class of enzymes that have nickel, iron, and three non-protein diatomic ligands in a deeply buried active site (6, 7). The outline of the general sequence of events during hydrogenase metallocenter assembly in E. coli has been largely derived from studies of the hydrogenase 3 large subunit (HycE), although the overall process is similar for the other isoenzymes (for recent reviews see Refs. 2 and 7–9). The main auxiliary proteins are encoded by the hypA–F genes (10–12), and the general purpose folding chaperones GroEL and GroES may also be required for optimal maturation (13). During the first steps of the pathway the iron and its diatomic ligands are prepared and inserted into the hydrogenase 3 precursor protein HycE by HypCDEF. Next, HypC remains associated with HycE while HypA and the GTPase HypB facilitate insertion of the nickel ion. The nickel serves as part of the recognition motif for the isoenzyme-specific protease HycI, and the processing of the [NiFe]-containing protein is completed following cleavage of a C-terminal fragment.

Many of the details of the hydrogenase biosynthetic pathway are not yet understood, and it is possible that not all of the individual components are known. For example, a nickel metallochaperone for E. coli hydrogenase 3 has not been identified. To search for other factors involved in this process we are using a proteomics approach to characterize native multiprotein complexes (14). These experiments led to the isolation of SlyD, an E. coli protein of unknown function that has not been previously associated with this pathway (see Fig. 1). SlyD is a member of the FK506-binding protein (FKBP)1 family of peptidyl-prolyl isomerases (PPIases) that was originally cloned because genetic mutations provide resistance to lysis induced by the phage {phi}X174 (sensitivity to lysis) (15). In addition to the PPIase domain at the N terminus, SlyD also has a 50-residue C-terminal domain that is rich in potential metal-binding residues (Fig. 1), containing 15 histidines, 6 cysteines, and 7 glutamates/aspartates (15, 16). Previous studies demonstrated that SlyD can bind a variety of different metals including up to three nickel ions (16, 17). The metal-binding domain of SlyD is not required for PPIase activity (17), although metal-ion binding inhibits the PPIase activity of the full-length protein (17) and in practice it allows for purification by immobilized metal affinity chromatography (16).



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  		FIG. 1.
Primary sequence and putative domain organization of E. coli SlyD (15–17, 61).

 

In this report we show that SlyD interacts with the hydrogenase accessory protein HypB and that it has a role in hydrogenase metallocenter assembly. Knocking out the gene for SlyD results in reduced hydrogenase activity and the detection of unprocessed HycE protein, indicating that the maturation pathway is blocked. This deficiency can be complemented by the addition of nickel to the growth medium. Furthermore, the {Delta}slyD strain accumulates less nickel then the wild-type strain when grown under anaerobic conditions. How SlyD might participate in hydrogenase metallocenter assembly, including a possible role as a nickel source, is discussed.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Restriction endonucleases were purchased from New England Biolabs. DNA oligonucleotides were purchased from Sigma. DNA ligase was purchased from MBI Fermentas. Pfu DNA polymerase was from Stratagene. All water was eluted from a Milli-Q water system (Millipore). The anti-HypB (18) and anti-HycE (19) rabbit polyclonal antibodies were a generous gift from Prof. A. Böck (University of Munich, Germany). Expression and purification of HypB will be described elsewhere.2

Bacterial Strains—The slyD-SPA (ST395) and hypB-SPA (ST713) strains (see Table I) were constructed from E. coli DY330 (wild type) as described previously (14). The {Delta}slyD strain was prepared as described previously (20). The {Delta}hycI (HD709) and wild-type MC4100 strains of E. coli (21, 22) were from the laboratory of Prof. A. Böck (University of Munich, Germany).


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  		TABLE I
Bacterial strains

 
Sequential Peptide Affinity (SPA) Purification of Protein Complexes and Protein Identification—Cells were grown anaerobically for 16 h as described below. Extracts were prepared and SPA purification was performed as described (14). Tandem MS analysis of the samples was performed by microcapillary liquid chromatography electrospray tandem MS (liquid chromatography-electrospray ionization-MS/MS). Briefly, the protein mixture was resuspended in 100 mM NH4HCO3, 1 mM CaCl2, pH 8.5, and digested by trypsin overnight at 37 °C with 2 µl of immobilized trypsin Poros beads (PerSeptive). The digested peptides were fractionated on a 7.5-cm (100 µm ID) reverse phase C18 capillary column attached inline to a ThermoFinnigan LCQ-Deca ion trap mass spectrometer. The entire digested sample was loaded and analyzed as described (23). All tandem mass spectra were searched using the SEQUEST computer algorithm against an E. coli protein sequence data base compiled in-house. Each spectral match was validated by using the STATQUEST probability algorithm and was significant at a 99% confidence level (24).

Growth Conditions and Preparation of Crude Cell Extracts for Biochemical Assays—Cells were grown anaerobically following inoculation with 1% (v/v) overnight culture at 37 °C in sealed flasks of buffered TGYEP medium containing 10 g of tryptone, 5 g of yeast extract, 69 mM K2HPO4, and 22 mM KH2PO4/liter (25) for the indicated times. The medium was supplemented with 1 µM sodium molybdate, 1 µM sodium selenite, 30 mM sodium formate, and 0.5% glucose, except for in the analysis of hydrogenase 1 and 2 or induction of the pBAD vectors, in which case the glucose and formate were replaced with 0.8% glycerol and 15 mM sodium fumarate (26). NiSO4 or ZnCl2 salts were added to the medium at the indicated concentrations. When required, the antibiotics kanamycin (50 µg/ml) or chloramphenicol (34 µg/ml) were included in the growth medium. Cells were harvested by centrifugation and washed with 50 mM potassium phosphate buffer, pH 7.0, and resuspended in the same buffer containing 1 mM dithiothreitol, 0.5 mM benzamidine, 0.2 mM phenylmethylsulfonyl fluoride, and trace amount of DNase I. Crude cell extracts were prepared by sonication and subsequent centrifugation at 15,000 x g for 20 min at 4 °C. The supernatant was quickly frozen in N2(l) and stored at –80 °C. The protein concentrations of crude cell extracts were determined by the BCA protein assay system (Pierce). The addition of extra nickel to the extracts did not affect any of the activities monitored (data not shown).

Plasmids—To clone into the pET24b plasmid (Novagen), the slyD gene was amplified by PCR from DH5{alpha} E. coli by using the forward 5'-CAGGAGATCATATGAAAGTAGCAAAAGACCTGGTG-3' and reverse 5'-GTCACTTCTCGAGTATTAGTGGCAACCGCAAC-3' primers. The PCR product was purified by using the QIAquick PCR purification kit (Qiagen) and digested with NdeI and XhoI. Following elution from an agarose gel with the QIAquick kit, the fragment was ligated with pET24b digested with the same enzymes and then transformed into XL-2 Blue E. coli (Stratagene). Plasmid DNA was isolated from cultures grown in kanamycin-supplemented LB by using a Qiagen HiSpeed Plasmid Miniprep kit. The pET24-SlyD construct was verified by dideoxynucleotide sequencing (ACGT, Toronto). The same procedure was used to clone slyD into the pBAD24 vector (American Type Culture Collection) to make pBAD24-SlyD except that the forward 5'-CTATTCTTCGCTAGCTCAGGAGATATCATGAAAG-3' and reverse 5'-GTCACTTTCTAGATATTAGTGGCAACCGCAAC-3' primers were used, the PCR product and plasmid DNA were cut with NheI and XbaI, and ampicillin was used in the medium.

Purification of SlyD—BL21(DE3) E. coli transformed with pET24-SlyD were grown in kanamycin-containing LB to an A600 of 0.7, induced by the addition of 0.35 mM isopropyl 1-thio-{beta}-D-galactopyranoside, and incubated for an additional 3 h at 37 °C. The bacteria were harvested by centrifugation (30 min at 4600 rpm), and the cell pellet was resuspended in 25 ml of lysis buffer/liter of culture (50 mM Tris, pH 8, 0.1 mM phenylmethylsulfonyl fluoride) and sonicated on ice. All subsequent steps were performed at 4 °C. The lysate was centrifuged for 30 min at 18,000 rpm in an SS34 rotor, and the supernatant was loaded onto a Ni2+-nitrilotriacetic acid-agarose column (Qiagen) pre-equilibrated in lysis buffer containing 100 mM NaCl. The column was washed with 10 column volumes of lysis buffer containing 500 mM NaCl, the proteins were eluted in lysis buffer containing 100 mM imidazole, pH 7.4, and the eluate was dialyzed against 20 mM Tris, pH 8, 1 mM EDTA. The solution was loaded onto a Mono-Q HR 5/5 column (Amersham Biosciences) equilibrated with 20 mM Tris, pH 7.5, and eluted with linear gradient of 200–400 mM NaCl over 75 ml. The fractions containing SlyD were identified by SDS-PAGE, pooled, concentrated in a Amicon Ultra centrifugal filter, and loaded onto a Superdex 75 column (Amersham Biosciences) equilibrated in 10 mM HEPES, 100 mM KCl, pH 7.6. The fractions containing pure SlyD were identified by SDS-PAGE, pooled, treated with 100 mM TCEP and stored in the anaerobic glove box at 4 °C.

Chemical Cross-linking—SlyD and/or HypB were incubated in 100 mM potassium phosphate, pH 6.5, for 30 min at room temperature. A final concentration of 12.5 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) (EDC, Pierce) was added to the reactions, followed by a 1-h incubation at room temperature and analysis on 12.5% SDS-polyacrylamide gels that were stained with Coomassie dye.

Western Analysis—Cell extracts were resolved on SDS-polyacrylamide gels and transferred onto nitrocellulose or polyvinylidene difluoride (HycE Western) membranes. The blots were probed with the appropriate primary antibody at a 1:3000 dilution (mouse monoclonal anti-FLAG antibody, Sigma) or a 1:1000 dilution (anti-HycE, anti-HypB). The 2° antibody used, diluted 1:30,000, was either the goat anti-rabbit or goat anti-mouse horseradish peroxidase conjugate (Bio-Rad). The enhanced chemiluminescence technique (Pierce) was used for detection.

Hydrogenase Activity Assays—Total hydrogenase activity of crude cell extracts was measured by hydrogen-dependent reduction of benzyl viologen according to the procedure of Ballantine and Boxer (27). Reactions were prepared in a septum-sealed cuvette in an anaerobic glove box (95% N2 and 5% H2). One unit of activity is defined as 1 µmol of benzyl viologen reduced/min and an extinction coefficient of 7400 M–1 cm–1 was used (27). To measure the activities of hydrogenases 1 and 2 an in-gel assay was performed (28). Crude cell extracts were resolved on 10 or 12.5% polyacrylamide gels run at 4 °C for 4 h at 200 V. All buffers and gels contained 0.1% SDS. The gels were incubated in 100 mM potassium phosphate buffer, pH 7, containing 0.5 mM benzyl viologen (Sigma) and 1 mM triphenyltetrazolium chloride (Sigma) in the anaerobic glove box at room temperature for 16 h. The data were analyzed on a Fluorochem 8800 gel documentation system (Alpha Innotech).

Cellular Nickel Accumulation—The indicated concentrations of 63Ni (specific activity 420 mCi/mmol; PerkinElmer life Sciences) were added to cells grown anaerobically. BL21(DE3) transformed with pET24-SlyD or pET24b were grown in LB, and DY330 and {Delta}slyD cells were grown in TGYEP supplemented with glucose and formate. The cells were harvested and extensively washed with 50 mM potassium phosphate, pH 7.0, then crude extracts were prepared by sonication in the same buffer and subsequent centrifugation at 15,000 x g for 20 min. The radioactivity was measured by scintillation counting in 5 ml of UltimaGold scintillation fluid with a Liquid Scintillation Analyzer Tri-Carb 2100TR (Packard Instruments).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
SlyD Directly Interacts with HypB—The SPA system is a method to identify the components of multiprotein complexes in bacterial cells under native conditions (14). The protein of interest is genomically tailored with a tag that encodes three modified FLAG sequences as well as a calmodulin-binding peptide. The target protein and any associated proteins are then isolated by two affinity chromatography steps. The identity of each protein is determined either by tryptic digestion of the whole eluent followed by liquid chromatography-electrospray ionization-MS/MS or resolution on SDS-PAGE followed by matrix-assisted laser desorption ionization MS/MS of individual gel bands. We are using this procedure to map out protein-protein interactions between the Hyp factors and other E. coli proteins3 in bacteria grown under conditions that favor hydrogenase expression (11, 25). Upon SPA purification of tagged HypB from ST713, both the HypB protein and the histidine-rich protein called SlyD (Fig. 1) were identified (data not shown). Furthermore, inspection of data collected for another study revealed that HypB was identified in the purified SlyD-SPA complex isolated from strain ST395 (63). SlyD was not detected in a complex with any of the other accessory proteins investigated to date, including HypA, HypC, HypD, or HybG.3

To confirm the HypB and SlyD protein-protein interaction, chemical cross-linking of purified recombinant proteins was performed (Fig. 2). Upon incubation of SlyD with EDC, a slightly faster mobility band was observed, possibly because of an internal cross-link. In the cross-linking reaction containing both HypB and SlyD, a new band was observed at the mobility of a 1:1 HypB-SlyD complex. The presence of HypB in this covalent complex was confirmed by Western analysis with an anti-HypB antibody (18) (data not shown). A faint band at a larger molecular weight than the heterodimer was also observed in the HypB reactions both with or without SlyD (Fig. 2, lanes 5 and 6), and it is most likely a HypB homodimer (18).



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  		FIG. 2.
SlyD associates with HypB. Purified HypB (5 µM) and/or SlyD (10 µM) were incubated in the presence or absence of EDC. The reactions were resolved on a 12.5% SDS-polyacrylamide gel, and the proteins were stained with Coomassie dye. The HypB-SlyD cross-link is indicated by the arrow.

 
Expression of slyD—It is not known what factors control the expression of slyD, although complementation experiments indicated uninduced constitutive expression driven by a cryptic promoter (15), and homology to the promoters that utilize {sigma}70 was noted (16). The hydrogenases are regulated by various metabolites, but expression of all of the hydrogenase isoenzymes is repressed by oxygen (29, 30), and the hyp operon is regulated by the anaerobic transcription factor FNR (31). Furthermore, nickel regulates the expression of the nickel uptake transporter in E. coli and various nickel-containing enzymes in other organisms (Ref. 7 and references therein). To determine whether these growth conditions also affect SlyD production, Western analysis was performed on extracts from strain ST395 with an anti-FLAG antibody. The amount of SlyD-SPA detected was not affected by excess nickel (up to 300 µM) in the growth medium whether cells were grown open to the atmosphere instead of in sealed anaerobic flasks or whether LB medium was used instead of TGYEP containing formate (data not shown).

SlyD Contributes to Hydrogenase Activity—Many auxiliary proteins for metallocenter assembly in enzymes were originally identified, because genetic mutants are deficient in the enzyme activity (9). To test whether SlyD plays a role in hydrogenase maturation, the slyD gene was disrupted (20). This deletion resulted in a noticeably slower growth rate under our anaerobic conditions (data not shown) to a similar degree as that previously reported for {Delta}slyD strains grown aerobically (32), and this difference was not investigated further. Wild-type and {Delta}slyD cells were grown under anaerobic conditions for 6 or 17 h, and hydrogenase activity was measured in cell extracts by monitoring anaerobic benzyl viologen reduction in a solution containing hydrogen gas (27). The activity detected in the wild-type extracts prepared after 6 h of growth was similar to that previously reported (29, 33), and in either strain relatively less hydrogenase activity was routinely detected after the longer growth period (Fig. 3 and data not shown). At both time points, however, hydrogenase activity was significantly abrogated by the slyD deletion (Fig. 3, A and B). To confirm that this phenotype was a result of the slyD deletion and not a downstream polar effect, slyD was expressed in-trans from a pBAD24 vector (34) transformed into the {Delta}slyD strain. The PBAD promoter is induced with arabinose and inhibited by glucose (34), so for these experiments cells were grown in TGYEP supplemented with glycerol and fumarate instead of glucose and formate. Arabinose titration of the {Delta}slyD strain transformed with pBAD-SlyD produced increasing amounts of hydrogenase activity (data not shown), with wild-type levels restored upon exposure to 100 µM arabinose (Fig. 3C). An increase in hydrogenase activity was not observed if the cells were transformed with the empty pBAD24 plasmid (Fig. 3C).



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  		FIG. 3.
{Delta}slyD cells are deficient in soluble hydrogenase activity and complemented with nickel. A, cultures were anaerobically grown in TGYEP medium containing glucose, formate, and the indicated concentrations of added nickel for 6 h. Cell extracts were prepared from wild-type strain DY330 (solid bars) and the {Delta}slyD deletion strain (striped bars) and tested for hydrogenase activity with the anaerobic benzyl viologen solution assay. B, same as A except the cells were grown for 17 h. C, cell extracts were prepared from wild-type DY330, {Delta}slyD transformed with the pBAD vector, or {Delta}slyD transformed with pBAD-SlyD vector, and tested for hydrogenase activity with the anaerobic benzyl viologen solution assay. Cultures were anaerobically grown in TGYEP media supplemented with glycerol and fumarate as well as 100 µM arabinose. The results represent the average of two independent measurements, and error bars indicate ± 1 S.D.

 
The decrease of hydrogenase activity in the {Delta}slyD strain is a similar phenotype, although not as extreme, as that observed following deletion of the individual hyp genes (11). However, the spectrophotometric hydrogenase assay of crude cell extracts is not specific for any particular hydrogenase isoenzyme (35), so it does not necessarily reflect whether a protein is required for the maturation of all three expressed hydrogenases. To investigate whether SlyD affects the activity of hydrogenases 1 and 2, fumarate and glycerol were added to the growth media (26), cell extracts were resolved on a polyacrylamide gel, and the activity of these two enzymes were analyzed with an in-gel benzyl viologen assay (28). In this strain of E. coli (DY330) the activity of hydrogenase 2 was much stronger than hydrogenase 1, so 50 µg of cell extracts were loaded to quantitate the activity for hydrogenase 1, and only 2 µg of cell extracts were used for the analysis of hydrogenase 2 (Fig. 4). The disruption of slyD caused a reduction in the activity of both hydrogenase 1 and 2 (Fig. 4), and the activity was restored by induction of slyD from the pBAD-SlyD vector (data not shown).



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  		FIG. 4.
Hydrogenase 1 and 2 deficiency in the {Delta}slyD mutant is complemented with nickel. A, activity staining of hydrogenase 1 and 2 in extracts prepared from wild-type (WT) or {Delta}slyD cells. Either 50 or 2 µg of crude cell extracts (CE) were analyzed from cells grown anaerobically with or without 500 µM nickel added to the TGYEP media containing glycerol and fumarate. B, quantitation of two experiments such as that shown in A. The activities detected for each enzyme in cell extracts from wild-type (solid bars) and {Delta}slyD (striped bars) cells were normalized in each experiment to that of the wild-type strain grown in the absence of added nickel. Error bars indicate ± 1 S.D.

 
Complementation of {Delta}slyD with Nickel—HypB is implicated in the nickel insertion step of hydrogenase biosynthesis, because the hydrogenase deficiency of hypB mutants is complemented by excess nickel in the growth medium (10, 11, 36). Of the other Hyp factors, only hypA lesions are partially complemented with nickel (26). The fact that SlyD can bind nickel ions (17)4 and that it interacts directly with HypB (Fig. 2) suggested that SlyD may also be involved at this step of the pathway. To test this hypothesis, NiSO4 was added to the media of {Delta}slyD and wild-type cells, and hydrogenase activity was analyzed in cell extracts by using the solution benzyl viologen assay (Fig. 3). The concentrations of nickel used did not have a significant affect on the activity detected in the wild-type extracts. The addition of 1 µM nickel did not have a noticeable affect on the hydrogenase activity of the {Delta}slyD extracts (data not shown), but an increase in activity was observed at higher concentrations of metal. Wild-type levels of activity were restored to the {Delta}slyD cells grown for either 6 or 17 h in the presence of 500 µM nickel (Fig. 3). Similarly, excess nickel partially complemented the hydrogenase 1 and 2 deficiency of the {Delta}slyD strain (Fig. 4). This effect is specific for nickel, because the addition of the same amounts of zinc to the growth medium did not complement the {Delta}slyD deficiency (data not shown).

In one of the final steps of hydrogenase 3 maturation, the protease HycI cleaves 32 residues from the C terminus of the HycE precursor protein (37), and the processed and unprocessed forms of HycE can be resolved by Western analysis (37). Nickel insertion is a prerequisite for this proteolysis step so processing the C-terminal tail is diagnostic of whether the metal center has been correctly incorporated (37). To determine whether SlyD affects the processing of hydrogenase 3, the HycE protein was examined in both the wild-type and {Delta}slyD cells. Cell extracts from a {Delta}hycI strain of E. coli and the corresponding wild-type strain were also examined to verify the mobility of the processed and unprocessed HycE proteins (data not shown) (21, 22). In the absence of SlyD, the HycE protein migrates at a slightly larger molecular weight (Fig. 5). This observation that the C-terminal fragment of the protein has not been processed in the {Delta}slyD strain indicates incomplete metallocenter assembly. Growth in the presence of excess nickel complements this deficiency and restores processing (Fig. 5) in agreement with the results from the hydrogenase activity assays. The mobility of the HycE protein in the wild-type strain was not affected by nickel in the medium (data not shown). No significant difference in the total amount of HycE protein was detected in the SlyD mutant extracts when compared with wild-type extracts.



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  		FIG. 5.
C-terminal processing of HycE. The {Delta}slyD mutant strain was grown anaerobically in TGYEP media containing glucose and formate either in the absence or presence of 500 µM added NiSO. Crude cell extracts were resolved on a 10% SDS-polyacrylamide gel 4followed by Western analysis with the anti-HycE antibody. The wild-type (WT) DY330 extract used in the experiment shown was prepared from cells grown with 500 µM nickel in the media, but nickel did not affect the mobility of HycE in the wild-type strain.

 
SlyD Increases Nickel Accumulation—Finally, experiments with the radioactive isotope 63Ni were performed to determine whether SlyD influences the nickel content of E. coli cells grown under anaerobic conditions. Cells were exposed to radioactive nickel at the start of anaerobic growth and at each time point aliquots were removed, washed, and the radioactivity was measured in either whole cells (data not shown) or in crude cell extracts (Fig. 6A). Most of the observed uptake occurred in the first 2 h. The time course of nickel uptake in the {Delta}slyD cells was similar to wild type, but the final level of nickel accumulation was only about half that detected in the wild-type cells. Similarly, the nickel content was monitored in anaerobically grown BL21(DE3) cells transformed with pET24-SlyD. Induction of SlyD with isopropyl 1-thio-{beta}-D-galactopyranoside resulted in overexpression of the protein, visibly detectable in cell extracts on a Coomassie-stained SDS-polyacrylamide gel (data not shown), and an increase in nickel accumulation compared with cells transformed with the control pET24 vector (Fig. 6B).



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  		FIG. 6.
SlyD influences cellular nickel accumulation. A, DY330 (filled circles) and {Delta}slyD (open circles) cells were grown under anaerobic conditions in TGYEP media in the presence of 0.25 µM63Ni. At the indicated time points crude cell extracts were prepared, and nickel accumulation was measured by scintillation counting. Data from individual experiments were normalized to the wild-type sample taken at 4 h. B, BL21(DE3) transformed with the pET24b (solid bars) or pET24b-SlyD plasmid (striped bars) were grown anaerobically in LB supplemented with 0.1 mM isopropyl 1-thio-{beta}-D-galactopyranoside and the indicated concentrations of 63Ni. After 20 h, crude cell extracts were prepared, and nickel accumulation was measured by scintillation counting. Data were normalized to the pET24b, 0.25 µM 63Ni control. Values represent the average of two separate experiments, and error bars represent ± 1 S.D.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Three [NiFe] hydrogenase enzymes are expressed in E. coli as components of various types of anaerobic metabolism (35). There is a fourth encoded isoenzyme (38), but this operon is silent in wild-type cells (30). SlyD is a protein that was not previously linked with these systems, but the experiments described in this report demonstrate that it is required for optimal activity of all three hydrogenases. The only aspect of production that is common to all isoenzymes is metallocenter biosynthesis by the accessory proteins, a subset of which act pleiotropically (7–9). Thus the influence of SlyD on the three isoenzymes suggests that the protein is involved in metallocenter assembly. Furthermore, complementation of {Delta}slyD by growth in excess nickel indicates that this protein has a role in nickel insertion and does not influence the preceding steps of diatomic ligand preparation, iron incorporation, holding the protein in a competent state for Ni2+ insertion, or the subsequent proteolysis of the C-terminal tail and internalization of the complete metal center. Exactly how SlyD participates in nickel insertion is not clear. It is possible that SlyD affects the nickel uptake or export mechanisms of E. coli and thus indirectly influences hydrogenase production, but the specific interaction with HypB suggests that SlyD is more directly involved.

As with many members of the PPIase superfamily (39, 40), a cellular function has not been identified for SlyD. It has been suggested that PPIases are important for protein folding and stability, for the formation of multiprotein complexes, or as switch mechanisms that regulate the activity of the substrate proteins and contribute to cell signaling (39–41). Furthermore, the actual PPIase activity of these factors is not always required (39, 41). None of the other known members of the FKBP family has a metal-binding domain similar to the C-terminal sequence of SlyD, but several have been implicated in metal homeostasis pathways. For example, a recent study demonstrated that FKBP52 interacts with the copper metallochaperone Atox1 and has a role in copper efflux (42). Mouse FKBP23 contains two Ca(II)-binding EF-hand motifs, and the ER-localized protein may act as a Ca(II)-dependent chaperone (43). Furthermore, mutant mice deficient in another homologue, FKBP12, had symptoms that mimic human congenital heart disorder, and it was suggested that the phenotype was because of an effect on the calcium release activity of muscle receptors (44).

The only E. coli hydrogenase accessory protein that has been shown to bind nickel is HybF (28), a protein that functions in the maturation of hydrogenases 1 and 2 and is replaced by HypA in the hydrogenase 3 pathway (26). The Helicobacter pylori HypA protein binds stoichiometric nickel and forms a heterodimer with HypB (45), but this activity has not yet been demonstrated for the E. coli protein. The E. coli HypA and HypB are implicated in the nickel insertion step because, like SlyD, mutations produce a hydrogenase-deficient phenotype that is complemented by growth in high nickel concentrations (10, 11, 26), and mutants that decrease the GTPase activity of HypB inhibit nickel incorporation into HycE (36). HypB homologues from many organisms have histidine-rich regions and in vitro studies of HypB proteins from Bradyrhizobium japonicum (46) and Rhizobium leguminosarum (47) established that they bind multiple nickel ions. Further examination of the B. japonicum protein established that the polyhistidine sequence is involved in nickel storage (48), but a mutant lacking the His-rich region could still bind a single nickel ion and support hydrogenase production (48, 49). Similar polyhistidine stretches are observed in accessory proteins for other nickel enzymes, such as UreE and CooJ from the urease and carbon monoxide dehydrogenase biosynthetic pathways, respectively (7). Again the His-rich regions appear to be for nickel storage and can be separated from the nickel insertion activity (7). The fact that neither HypB nor any of the other E. coli hydrogenase proteins contain a similar region led to the suggestion that an alternative protein could fulfill the role of a nickel supplier for hydrogenase biosynthesis, and such a function was proposed for SlyD (17).

Thus one potential role for SlyD is that of a nickel source for the hydrogenase metallocenter assembly pathway. Previous studies have clearly shown that SlyD can bind multiple nickel ions (16, 17), and the inherent metal-binding activity is sufficiently strong to cause SlyD to be a common contaminant on immobilized metal affinity chromatography of E. coli extracts (16, 50, 51). Furthermore, an estimated 104 molecules of SlyD are expressed in normally growing cells (32), the same order of magnitude as the 25,000 molecules/cell of HypB synthesized under anaerobic conditions (18). A nickel source for the hydrogenase enzymes would mean that production does not rely on the availability of freely diffusible metal ions and that cellular exposure to potentially toxic ions is minimized. A recent study of the E. coli transcription factor NikR, which regulates expression of the nickel membrane transporter (52, 53), demonstrated that it responds to nickel concentrations corresponding to only a few free ions (54), suggesting that it would not allow uncomplexed metal to accumulate in the cell. It will be interesting to examine whether SlyD plays a role in production of Glyoxylase I, the only other known nickel enzyme in E. coli (55). It is also possible that SlyD is not specific for nickel but plays a more general role in cellular metal homeostasis, in analogy with the eukaryotic and cyanobacterial metallothioneins (56–58).

The lack of SlyD results in diminished but not negligible total cellular nickel, which could indicate a reduction in the level of available metal. However, this result could also be due to less nickel incorporation into the hydrogenase enzymes. If nickel insertion is deficient when SlyD is not expressed, the cellular demand for nickel will be met and the uptake will be turned off at much lower levels of accumulation. Thus it is possible that another property of SlyD participates in hydrogenase biosynthesis to promote optimal activity of HypB and facilitate nickel insertion, working either in cooperation with or instead of the metal binding activity. This hypothesis is supported by the observation that the levels of hydrogenase activity in the {Delta}slyD strain can be restored to wild-type levels when grown in the presence of excess nickel, whereas the {Delta}hypB mutation is only partially complemented (36). The fact that metal binding inhibits the PPIase activity of SlyD (17) makes it tempting to speculate that the two functions are coupled together in some type of switch mechanism, but additional experiments are required to test this hypothesis. SlyD sensitizes E. coli to the bacteriophage {phi}X174 lysis protein E by stabilizing the protein (59), possibly through the isomerization of an essential proline residue (60), so perhaps SlyD stabilizes HypB or one of the other proteins in the hydrogenase biosynthetic pathway. It is interesting to note that SlyD belongs to a subfamily of FKBPs with an extra domain (Fig. 1), named an IF domain (61). This domain is required for a chaperone-like folding activity in a homologous FKBP from Methanococcus thermolithotrophicus (62), an activity that is independent of the PPIase activity (62). Finally, some FKBPs modulate signal transduction pathways as architectural factors that bind and correctly orient interacting components of multiprotein complexes (40). In a similar fashion, SlyD may interact with the hydrogenase accessory proteins and be responsible for transiently anchoring and orienting them during metal transfer.

In summary, this study reveals that SlyD is a new component of the hydrogenase metallocenter assembly pathway in E. coli. This protein interacts specifically with HypB and influences the nickel insertion step to promote optimal hydrogenase production. At this time, the function of this metal-binding protein is not clear; however there are four putative non-exclusive properties of SlyD that could act in hydrogenase metallocenter assembly, metal binding, PPIase, folding chaperone, and architectural assembly. Experiments are now underway to characterize the role of SlyD in the maturation pathway. SlyD homologues are encoded in a variety of prokaryotic genomes including H. pylori, Haemophilus influenzae, and Pseudomonas aeruginosa, although with variable amounts of the C-terminal metal-binding domain. It will be interesting to address whether or not SlyD is also involved in metallocenter biosynthesis in these organisms.


   FOOTNOTES
 
* This work was supported in part by grants from the Natural Sciences and Engineering Research Council of Canada and the Canada Research Chairs Program (to D. B. Z.) and the Protein Engineering Network of Centers of Excellence and Genome Canada (to J. F. G. and A. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed. Tel./Fax: 416-978-3568; E-mail: dzamble{at}chem.utoronto.ca.

1 The abbreviations used are: FKBP, FK506-binding protein; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride); MS, mass spectrometry; PPIase, peptidyl-prolyl cis/trans-isomerase; SPA, sequential peptide affinity; TCEP, Tris(2-carboxyethyl)phosphine hydrochloride. Back

2 M. R. Leach, S. Sandal, H. Sun, and D. B. Zamble, manuscript in preparation. Back

3 G. Butland, J. W. Zhang, J. F. Greenblatt, A. Emili, and D. B. Zamble, unpublished data. Back

4 J. W. Zhang and D. B. Zamble, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank S. C. Wang for the pET24-SlyD vector, Dr. M. R. Leach for HypB and a critical reading of the manuscript, and X. Yang, W. Yang, and A. Starostine for technical assistance. We alsothank Prof. A. Böck for the generous donation of the anti-HycE and anti-HypB antibodies as well as E. coli strains MC4100 and HD709, and we thank Dr. M. Blokesch for advice.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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