Cu-NirK from Haloferax mediterranei as an example of metalloprotein maturation and exportation via Tat system

Biochimica et Biophysica Acta 1834 (2013) 1003–1009

Contents lists available at SciVerse ScienceDirect

Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbapap

Cu-NirK from Haloferax mediterranei as an example of metalloprotein maturation and exportation via Tat system J. Esclapez, B. Zafrilla, R.M. Martínez-Espinosa, M.J. Bonete ⁎ División de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Alicante, Ap. 99, E-03080 Alicante, Spain

a r t i c l e

i n f o

Article history: Received 30 May 2012 Received in revised form 28 February 2013 Accepted 4 March 2013 Available online 14 March 2013 Keywords: Copper nitrite reductase Haloferax mediterranei Tat system Homologous expression Protein exportation

a b s t r a c t The green Cu-NirK from Haloferax mediterranei (Cu-NirK) has been expressed, refolded and retrieved as a trimeric enzyme using an expression method developed for halophilic Archaea. This method utilizes Haloferax volcanii as a halophilic host and an expression vector with a constitutive and strong promoter. The enzymatic activity of recombinant Cu-NirK was detected in both cellular fractions (cytoplasmic fraction and membranes) and in the culture media. The characterization of the enzyme isolated from the cytoplasmic fraction as well as the culture media revealed important differences in the primary structure of both forms indicating that Hfx. mediterranei could carry out a maturation and exportation process within the cell before the protein is exported to the S-layer. Several conserved signals found in Cu-NirK from Hfx. mediterranei sequence indicate that these processes are closely related to the Tat system. Furthermore, the N-terminal sequence of the two Cu-NirK subunits constituting different isoforms revealed that translation of this protein could begin at two different points, identifying two possible start codons. The hypothesis proposed in this work for halophilic Cu-NirK processing and exportation via the Tat system represents the first approximation of this mechanism in the Halobacteriaceae family and in Prokarya in general. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Denitrification is one of the major pathways of the nitrogen cycle [1]. Through this route, the oxidized nitrogen present in the biosphere is sequentially reduced by microorganisms upon reaching the atmosphere. Denitrifying organisms use this mechanism as an alternative source of energy in environments where oxygen becomes the limiting electron acceptor. One of the most important steps in this pathway involves the reduction of nitrite (NO2−) to nitric oxide (NO): it represents a return to the gaseous state of this element leading to a significant loss of fixed nitrogen from the terrestrial environment [2]. Two types of enzymes, copper-containing nitrite reductases (NirK) or cytochrome-c-dependent nitrite reductases (NirS), are responsible for catalyzing this reaction in denitrifying organisms [3]. Both the kinetics and/or the structure of Cu-NirK from microorganisms belonging to Bacteria and Archaea as Paraccocus denitrificans [1], Achromobacter cycloclastes, Rhodobacter sphaeroides, Alcaligenes faecalis, Haloarcula marismortui and Haloferax denitrificans [4–8] have been reported. These Cu-NirKs are homotrimers, in which a monomer (~35 kDa) contains one type I Cu and one type II Cu site. Due to the type I Cu site, the protein has a strong band near to 600 nm arising

Abbreviations: Cu-NirK, copper dependent nitrite reductase; Tat, twin arginine translocation; Cu I, type I copper center; Cu II, type II copper center; LepB, membrane protease related to Tat system ⁎ Corresponding author. Tel.: +34 96 5903524; fax: +34 96 5909955. E-mail address: [email protected] (M.J. Bonete). 1570-9639/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbapap.2013.03.002

from (Cys) S-CuII charge transfer [9]. The ratio of the intensity of this band to a second charge-transfer absorption band near to 460 nm determines the classification of Cu-NirKs as blue or green Cu-NirKs. Thus, the green enzymes have maxima absorbance at 460, 595, and 700–750 nm, while blue enzymes show little absorbance in the 460 nm range [10]. The analysis of the nirK gene sequences available to date reveals that these proteins show a Tat signal in the N-terminal region indicating a possible translocation of the enzyme through the cytoplasmic membrane [11,12], which is a poorly understood process in the Archaea Domain at the time of writing this paper. There is a lot of evidence of Cu-NirK exportation in some prokaryotic species such as R. sphaeroides [13]. However, no information is available about the possible translocation of Cu-NirK in members of the Archaea Domain. Under these conditions, it is assumed that NirK is involved in the energy production process (through its interaction with respiratory nitrate reductase membrane complex) and the genesis of electronic gradients during oxidative phosphorylation [14]. Periplasmic space in archaea is a porous structure delimited by S-layers [15]. This structure can vary greatly between different species of extremophiles ranging from more or less complex configurations to their total absence. For example, Haloferax volcanii presents a hexagonally packed surface (S) layer composed of glycosylated polypeptides anchored to the cytoplasmic membrane [16]. In this sense, it is likely that extracellular enzymatic systems of these microorganisms might be embedded in this matrix of polysaccharides with the objective of avoiding the dispersion of each one of their components in the growth medium.

1004

J. Esclapez et al. / Biochimica et Biophysica Acta 1834 (2013) 1003–1009

In the present study, we carried out the controlled expression of nirK gene from Haloferax mediterranei using Hfx. volcanii as a halophilic host. This microorganism presents a well characterized glycoprotein layer, so it provides a good model for studies of protein exportation out of the cell. The location and characterization of the heterologously overexpressed Cu-NirK represents a breakthrough in knowledge about the protein maturation and protein translocation processes of the enzymes involved in N-cycle in halophilic archaea and by extension in prokaryotic microorganisms. 2. Experimental 2.1. Materials Oligonucleotide primers (Hm-nirkF 5′-GGGAGACGACCATGGTATCA ACAA C-3′ and Hm-nirkR 5′-TCACTGCCCGGTACCGGCGGTTGTC-3′) were provided by Bonsai Technologies containing two restriction targets for NcoI and KpnI, respectively. pGEM T-easy vector from Promega was used as T/A cloning vector, restriction enzymes and molecular weight markers for SDS-PAGE were purchased from Fermentas and Promega and Novagen supplied T4 DNA ligase and Taq polymerase, respectively. KNO2, methyl viologen (MV) and novobiocin were obtained from Sigma. 2.2. Bacterial and archaeal strains E. coli JM109 (Stratagene) was used for cloning methods. Hfx. mediterranei strain R4 (ATCC 33500) was the source of genomic DNA and Hfx. volcanii strain WFD11 was used as an expression host. 2.3. Cultivation of microorganisms Hfx. mediterranei R4 was grown in a complex medium described by Rodríguez-Varela [17]. Hfx. volcanii was grown in a complex medium defined by Dassarma and Fleischman [18]. During protein expression assays, Hfx. volcanii medium was supplemented with novobiocin 40 μg/ml. 2.4. Bioinformatic analysis Hfx. mediterranei Cu-NirK protein sequence was compared with database using the NCBI BLAST platform. Alignments with different scores were chosen in order to obtain a distance matrix for different NirK proteins. Selected NirK proteins were: UniProt ID: Q877G9 (Hfx. denitrificans), UniProt ID: Q5UYQ0 (Har. marismortui), UniProt ID: D0MHZ0 (Rhodothermus marinus), UniProt ID: Q3ISJ6 (Natronomonas pharaonis), UniProt ID: E4M4B8 (Thermaerobacter subterraneus), UniProt ID: D1C654 (Sphaerobacter thermophilus), UniProt ID: D5VLV7 (Caulobacter segnis), UniProt ID: D8NF14 (Ralstonia solanacearum), UniProt ID: D3A0A1 (Neisseria mucosa), UniProt ID: Q67RG7 (Symbiobacterium thermophilum), UniProt ID: A9WB08 (Chloroflexus aurantiacus), UniProt ID: P38501 (A. faecalis), UniProt ID: Q53239 (R. sphaeroides), UniProt ID: A4YMK7 (Bradyrhizobium sp.). The PeptideCutter program was employed to predict potential cleavage sites cleaved by proteases in the protein sequence. Finally, the TreeView program was used for the analysis of the phylogenetic tree. 2.5. Cloning and expression of Cu-NirK gene DNA from Hfx. mediterranei was isolated as previously described by Dyall-Smith and Doolittle [19]. Standard methods were used for restriction digestions, agarose gel electrophoresis, and ligations [20]. PCR products were cloned into a pGEM T-easy vector in order to carry out an enzymatic restriction of nirK gene with NcoI and KpnI. The gene, with cohesive ends, was correctly inserted in pJAS vector [21]. This halophilic vector contains a strong and constitutive ferredoxin promoter from Halobacterium salinarum and confers resistance

to novobiocin. Hfx. volcanii WFD11 was transformed with the new construction, termed pJAS-Cu-NirK, as described by Cline [22]. The colonies obtained were grown in complex media in order to get a sufficient amount of biomass to carry out the purification of intracellular enzyme. 2.6. Activity assay Nitrite reductase activity was measured as follows: standard enzymatic reactions were carried out following the method previously developed by Martínez-Espinosa and co-workers [23]. After an appropriate reaction time, the concentration of the remaining nitrite was determined by colorimetry [24]. Cu-NirK activity stains of native PAGE gels were achieved following the method described by Vega and Kamin [25]. 2.7. Purification of the recombinant protein Hfx. volcanii cells from expression assay cultures were suspended in 50 mM phosphate buffer (pH 7.5) containing 2.5 M (NH4)2SO4. The cells were disrupted by treating them with the ultrasonic oscillating device Virsonic 475 (Virtis). The resulting solution was centrifuged at 30,000 ×g for 30 min with a L5-65B ultracentrifuge (Beckman & Coulter) and the cell-free supernatant was used as the starting material for subsequent purification of recombinant Cu-NirK. Enzymatic extract was loaded in a Sepharose 4B-CL column (2.6 × 35 cm) and the (NH4)2SO4 concentration was linearly reduced to 0.5 M. Thus, the initial pool of proteins was resolved on the basis of their hydrophobic properties. Active fractions were pooled and loaded in a HiPrep DEAE FF column (1.6 × 10 cm). Unlike most of the proteins contained in the sample, the recombinant enzyme was eluted with a single wash replacing (NH4)2SO4 buffer by 0.2 M of NaCl buffer (50 mM phosphate, pH 7.5). The active pool was concentrated and loaded through a 5 ml HiPrep Q-Sepharose column and the retained proteins were separated with an ion exchange chromatography, raising the NaCl concentration from 0.2 to 0.8 M. The last step of purification was carried out by gel filtration using a HiPrep Sephacryl S-200 column (1.6 × 60 cm) in the presence of 2 M of NaCl without variations of pH. The active fractions were used for subsequent studies. For the analysis of extracellular Cu-NirK, the total protein present within the growth media was concentrated (20:1) by means of the tangential filtration system VivaFlow 200 (Vivascience) with a cut off of 30 kDa. 2.8. Physical and kinetic measurements Quantification of protein was carried out with the Bradford method [26] using bovine serum albumin as standard. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) was carried out by the standard method [27]. Native PAGE electrophoresis was achieved avoiding SDS and mercaptoethanol from each buffer. Spectroscopic measurements in the UV–visible regions were performed using a DU 800 Spectrophotometer (Beckman & Coulter) with a 1 cm-long light path cuvette. The molecular weight of the purified enzyme was determined using the elution volume from the size exclusion chromatography (Sephacryl S-200), and previous calibration of the column by using the gel filtration marker kit for protein molecular weights 12– 200 kDa (Sigma). Physical and kinetic parameters for the recombinant Cu-NirK activity were determined by varying temperature, salt concentration, pH value and the concentration of substrates in the enzymatic assays. 2.9. Protein sequencing The purified Cu-NirK was subjected to SDS-PAGE, and the protein bands were digested with trypsin in order to obtain single peptides. The sequence of these peptides was determined using a Nano-ESI

J. Esclapez et al. / Biochimica et Biophysica Acta 1834 (2013) 1003–1009

HPLC–MS/MS system (Agilent). These sequences were compared with NCBI Protein database using free Mascot software. 2.10. N-terminal amino acid sequencing After native and SDS-PAGE of extra and intracellular fractions, the Cu-NirK isoforms were electrophoretically transferred to PVDF membrane (Immobilon P, Millipore) and stained with Coomassie Blue R-250. Two stained bands were excised and their N-terminal amino acid sequence determined by Edman's sequential degradation using a Procise 494 (Perkin Elmer) protein sequencer. 2.11. Database submission The nirK gene from Hfx. mediterranei has been assigned with the accession number FN555205 in the European Molecular Biology Laboratory (EMBL) nucleotide sequence database. 3. Results and discussion 3.1. Phylogenetic relationships Hfx. mediterranei Cu-NirK protein sequence (Fig. 1) was aligned with their homologues from different denitrifying microorganisms. The phylogenetic tree derived from this alignment (Fig. 1) shows the Hfx. mediterranei Cu-NirK inside the halophilic cluster and relatively far from the other clusters generated. Relationships detected agree with results proposed by Bartossek [28] about the diversity of NirK enzymes. However, it is noteworthy that Cu-NirK from R. marinus (not included in the above reference) appears enclosed in the halophilic NirK cluster indicating a solid connection between this species and halophilic archaea. R. marinus is a thermophilic and slightly halophilic bacterium, included in Sphingobacteriales and, at the moment, it seems to be the closest microorganism relative to the extreme halophilic bacterium

1005

Salinibacter ruber [29]. Despite this, no nirK gene has been found in S. ruber genome. This finding supports the presence of a complete or partial gene machinery which is involved in nitrate respiration occurring as a result of lateral gene transfer, between ancestors of this Bacteroidetes order and halophilic archaea, possibly in a primordial and thermophilic environment [30]. If that is the case, different species from each group evolved, with or without a nitrate respiratory chain, in order to colonize a wide diversity of hypersaline environments, currently inhabited by S. ruber and Halobacteriaceae family. 3.2. Sequence and structural analysis of copper centers Regarding the presence of conserved domains, four significant regions inside the Hfx. mediterranei Cu-NirK sequence have been detected. The first of them (Fig. 2a, continuous line) involves the presence of a region similar to the distinctive Tat motif: SRRxFLK [31]. This region, placed at five residues from the N-terminal extreme, is included in the conserved consensus region TRRR(T/V)L(Q/E) established by the genera Haloferax, Halogeometricum, Halomicrobium, Halorabdus and Haloarcula and obtained by multiple sequence alignment. Consequently, it is probable that this region is acting as the Tat motif for the protein to be exported via the Tat system [11]. The second conserved domain shows the presence of two possible cutting targets for proteases located in positions 27 and 34 from the N-terminal end (Fig. 2a, dotted line). The presence of this sequence is commonly associated with the presence of Tat signals since the mature protein exportation through the cellular membrane requires the removal of the signal peptide. Finally, seven residues in copper binding have been identified (Fig. 2a, marked residues without line) sited in a central position inside the chain. Residues located in these positions may coordinate type I and type II copper centers proposed for this class of enzymes (Fig. 2b). Regarding the structural analysis, a three-dimensional model for halophilic Cu-NirK was obtained using as a template the well-resolved structure of nitrite reductase from Pseudoalteromonas

Fig. 1. Unrooted phylogenetic tree of the Cu-NirK proteins of different microorganisms. The plot of distance matrix obtained from the ClustalW alignment shows the phylogenetic relationships between Rhodothermus marinus (Sphingobacteriales order) and halophilic archaea (Halobacteriaceae family).

1006

J. Esclapez et al. / Biochimica et Biophysica Acta 1834 (2013) 1003–1009

Fig. 2. Conserved domains and active metallic centers found in NirK sequence. In panel a) the export Tat signal (green continuous line), the two conserved cleavage sites for proteases (orange dotted line) and the residues involved in coordination of type I and II copper centers (marked residues without line). Panel b) shows a model with the probable three-dimensional disposition of the two copper centers located in a single subunit (type I copper center) and between two adjacent subunits (type II copper center).

haloplantakis (Q3IGF7). From this model, it is possible to conclude that four residues (His 129, Cys 170, His 178 and Met 183), located in a single subunit, are in the right position to coordinate the type I copper center (Cu I). While the other five residues (His 134, His 169, Asp 132 and His 278, His 329) are related to the type II copper center (Cu II). This second center is placed in the interface of two neighboring subunits so His 134, His 169 and Asp 132 (catalytic residue) belong to the first subunit, whereas His 278 and His 329 belong to the next subunit. The Cu-ligand residues described here have their counter partners in the enzyme characterized from Har. marismortui [7]. 3.3. Cloning, expression and purification of intracellular Cu-NirK The 1080 bp DNA fragment containing the nirK ORF was correctly inserted and expressed in pJAS vector. The recombinant enzyme accounted for about 15% of the total protein of the host. Purification was achieved following nitrite reductase activity in fractions obtained from each of the four chromatographic steps. Fractions of each chromatographic step were analyzed via SDS-PAGE with the aim of ensuring electrophoretic homogeneity (Fig. 3a). The purification process gave a yield of 60% and a purification factor of 9, relative to the initial protein. The two protein bands visualized in line 6 and not present in control extracts were associated with the recombinant protein. Trypsin digestion and identification of each protein band showed a 28 and 23% coverage over Cu-NirK, suggesting that the recombinant protein had been modified at some point during protein expression. In order to elucidate the composition of a native enzyme, a native PAGE of pure enzyme followed by activity NiR staining was carried out (Fig. 4). Results revealed that the intracellular pool of Cu-NirK is composed of at least six different

Fig. 3. Features of purified Cu-NirK. In panel a) SDS-PAGE of Cu-NirK purification stages. Lane 1: molecular weight standards (Fermentas); lane 2: total cellular fraction and protein pool after chromatography in Sepharose 4B-CL (lane 3), DEAE-Sepharose (lane 4), Q-Sepharose (lane 5) and Sephacryl S-200 (lane 6). In panel b) the UV–vis of the purified Cu-NirK is shown. In 300–700 nm zoom, two different peaks are visible (459 and 587 nm, respectively), representatives of copper nitrite reductases. The ratio 459/587 includes the Cu-NirK in the green NirK group.

isoforms of the enzyme. The SDS-PAGE of each of the six bands showed that each one presents a different combination of two isoforms of 44.3 and 39.8 kDa apparent band sizes, the smaller form being the predominant isoform protein in this cellular fraction (Fig. 4). Taking into account the two cleavage sites present in Hfx. mediterranei Cu-NirK sequence, it is possible to propose that the expression of recombinant protein could conclude with the maturation of the initial polypeptide through a cut in one of the two targets present at its N-terminal extreme. Finally, the two possible isoforms could combine to form a pool of active trimers. This maturation mechanism could also explain why it is possible to observe two bands with slightly different masses to those NirK purifications carried out in Har. marismortui [7] or Hfx. denitrificans [8]. 3.4. Physicochemical, kinetics and spectral dependences of Cu-NirK activity The optimal salt concentration and temperature for the enzymatic activity were determined in a single experiment as both parameters exhibit a joint effect on the structural stability of the enzyme. The optimum value for salt concentration (KCl or NaCl) and temperature were obtained with 2 M for both salts tested and around 70 °C. As expected, these findings define Cu-NirK as a thermohalophilic enzyme [32–34]. In terms of pH, the recombinant enzyme shows a moderate acidophility

J. Esclapez et al. / Biochimica et Biophysica Acta 1834 (2013) 1003–1009

Fig. 4. Different isoforms of intracellular Cu-NirK. In panel a), native PAGE of purified Cu-NirK from the intracellular fraction. On the right lane the Coomassie Blue staining of the gel is shown while on the left lane the nitrite reductase activity developed by the same sample is shown. Note the presence of at least six active isoforms. In panel b), SDS-PAGE for each of the five isoforms of the intracellular Cu-NirK. Lanes 1 to 5: Isoforms detected in the native PAGE for the intracellular Cu-NirK (from the highest to the lowest native migration coefficient, respectively); lane 6: molecular weight standards (Fermentas).

reaching its optimum activity value at 5.5 units. Finally, the native mass of the Cu-NirK was calculated using the elution volume of gel filtration chromatography, which was previously calibrated under identical conditions to these experiments, giving a value of 103 ± 5 kDa. Calculation from this data confirmed that the native enzyme acquired a trimeric conformation and suggested that the elution active peak obtained from gel filtration is a mixture of different isoforms of the enzyme, unresolved with this technique. No detection of ammonia traces in buffer reaction after incubation time confirmed that this enzyme is not related to assimilative metabolism (data not shown). Although NO is the product of this reaction, purified Cu-NirK does not produce NO in significant quantities to be detected without high experimental error as has also been described so far [35,36]. Basic kinetics from Cu-NirK for its substrates were obtained. Km was 4.04 ± 0.33 mM and 0.41 ± 0.06 mM for nitrite and MV, respectively. In both determinations, Vmax was established to be 11.5 ± 1.1 U/mg. Intracellular and extracellular pools of recombinant Cu-NirK showed nearly the same kinetic constants (data not shown). In addition, UV–vis spectrum (Fig. 3b) showed two different maxima of absorption at 453 nm and 587 nm suggesting that the enzyme belongs to the green copper NirK group [10]. Spectral differences found between Cu-NirK and Fd-Nir from Hfx. mediterranei [23] confirm that both nitrite-catalyzing enzymes are involved in different metabolic roles.

1007

Fig. 5. Different isoforms of extracellular Cu-NirK. In panel a), native PAGE of purified Cu-NirK from extracellular fraction. On the right lane the Coomassie Blue staining of the gel is shown while on the left lane the nitrite reductase activity developed by the same sample is shown. Note the presence of at least six active isoforms. In panel b), SDS-PAGE for each of the five isoforms of the extracellular Cu-NirK. Lanes 1 to 5: Isoforms detected in the native PAGE for the extracellular Cu-NirK (from the highest to the lowest native migration coefficient, respectively); lane 6: molecular weight standards (Fermentas).

fractions both isoforms have been identified in different concentrations, the 44.3 kDa isoform being the predominant or even the only one present in lanes 4 and 5 (Fig. 5). These data suggest that the halophilic Cu-NirK is involved in a maturation process and exportation via the Tat system, although the size of the isoforms is smaller than expected. 3.6. N-terminal amino acid sequence and cellular location In order to elucidate the maturation process of the protein and its exportation via the Tat system, the first eight amino acids of the two isoforms that appear in the SDS-PAGE have been sequenced. N-terminal amino acid sequences of the 44.3 kDa and 39.8 kDa isoforms were determined to be SLDQTEEP and MEQVAANP, respectively. The 44.3 kDa isoform is obtained as result of the cleavage between the 33rd and 34th residues. Therefore, these data suggest that the 44.3 kDa isoform is exported via the Tat system, being cleaved by the twin arginine signal sequence after its translocation to extracellular medium. The sequence of the small isoform, 39.8 kDa, starts in the 52nd position. No cutting target is predicted in this location so that, it seems more likely that this isoform could be obtained as a result of an alternative translation mechanism [37] or mRNA processing rather than as a cleavage process. 4. Discussion

3.5. Extracellular recombinant Cu-NirK Though high amounts of recombinant Cu-NirK were recovered from the intracellular fraction, a strong nitrite reductase activity was detected in growth media, mainly during the early logarithmic phase of growth. Subsequently, the extracellular enzyme was concentrated by means of a tangential filtration device. Native PAGE (Fig. 5) analysis of this concentrate also revealed the presence of different isoforms of the enzyme. Like the results obtained with the intracellular protein, the SDS-PAGE of each band (Fig. 5) showed the two different size isoforms. However, the comparison of the isoform expression pattern of both samples, intracellular and extracellular fractions, reveals remarkable differences. In the intracellular fraction, the 39.8 kDa isoform is predominant and the 44.3 kDa isoform appears slightly, while in the extracellular

Recombinant Cu-NirK from Hfx. mediterranei has been successfully synthesized and refolded in Hfx. volcanii by means of a new expression method developed for halophilic archaea. Halophilic Cu-NirK is a green copper-dependent nitrite reductase involved in the reduction of nitrite to nitric oxide with UV–vis spectrum similar to those reported from NirK isolated from Hfx. denitrificans [8]. Evidence reported in this work suggests that this enzyme is active in the cytoplasmic fraction but it also carries out its metabolic role outside the cell, possibly embedded in the exopolysaccharide matrix that fills the porous S-layer. The trimeric Cu-NirK protein isolated from the cytoplasm and extracellular medium are composed of a mixture of the two isoforms with 44.3 kDa and 39.8 kDa apparent band sizes. These isoform sizes, which have been determined by SDS-PAGE, could be overestimated

1008

J. Esclapez et al. / Biochimica et Biophysica Acta 1834 (2013) 1003–1009

up to 25% due to the increase of acidic residues in halophilic proteins [38]. The 44.3 kDa isoform predominates in the extracellular fraction and starts from the one of the predicted cutting target (SLDQTEEP). This fact indicates that recognition and cleavage of a complete peptide at this level facilitates the protein transport across the membrane and its exit outside the cell. The 39.8 kDa isoform (small isoform) is the majority of the cytoplasmic isoform. That isoform lacks the signal peptide and could be obtained as a result of an alternative translation mechanism, because of its first codon in the sequence, GUG, could be also used as start codon in Hfx. volcanii. Translation initiation studies carried out in haloarchaea have revealed that these microorganisms use three different mechanisms simultaneously, the largest fraction of transcripts is leaderless, one-third of the transcripts have a 5′-leader devoid of a Shine–Dalgarno sequence and a very small fraction of transcripts include Shine–Dalgarno sequences [37]. In hyperthermophilic archaeon, it has been observed that if GUG works as a translation initial codon, it directs the incorporation of methionine as the N-terminal residue [39]. This fact could explain the presence of methionine as the first amino acid in the small isoform N-terminal sequence (MEQVAANP) although the codon codifies valine when it appears inside the gene. Therefore, the Cu-NirK transcript could be translated in two different ways. More studies are needed to confirm this theory and analyze the cell conditions that induce the translation of one isoform or the other. Once the two possible transcripts are translated, a random trimerization occurs between these two possible isoforms. This process originates the pool of possible isoforms found both inside and outside the cell. Finally, the Tat system of Hfx. volcanii facilitates the exportation of recombinant Cu-NirK active trimers whenever any of the three contain the signal peptide (the large isoform). In the process of exportation through the membrane, the signal peptides of the large isoform are cleaved. Thus, outside the cell we find a mixture of the cleaved and signal-avoided Cu-NirK, prevailing over the large isoform. In contrast only the trimers remain inside the cell exclusively composed by untargeted peptides that not are able to cross the membrane and go outside the cell. This discrimination between targeted and nontargeted peptides looks like a mechanism for regulating the system and final Cu-NirK location. The location of recombinant Cu-NirK outside the cell agrees with and complements the results obtained by Martínez-Espinosa [40] where the extracellular location of membrane-associated NarGH from Hfx. mediterranei is reported. For this reason, there is increasing evidence that the complete reduction of NO3− to N2 could take place through an extracellular enzymatic complex which is part of the machinery associated with the outer face of the cytoplasmic membrane while the rest of soluble enzymes and metabolites (NO3−, NO2−, electron donors and gasses) are embedded in the porous S-layer. In this sense, Hfx. mediterranei Cu-NirK seems to have been adapted to work in this acidic rich-nitrite environment slightly decreasing the optimal pH for the enzymatic activity [41]. Electron donors for NirK such as halocyanin, which is the code for an ORF located upstream to Hfx. mediterranei nirK gene, could also be located within the S-layer making the electron transfer possible (data not shown). This atypical respiratory complex orientation offers advantages to these microorganisms in oxygen-poor environments such as hypersaline ecosystems. With this modification, the presence of NO3− transporters is not required and the electron acceptor can be reduced directly in the growth media increasing the efficiency of the process. Finally, the mobilization of the proteins involved in NO3− respiration appears to be regulated by the Tat system so that they are folded and loaded with metallic cofactors inside the cell before being exported out of the cell where they will take part in their physiological role. This enzyme could be related to environmental nitrite detoxification by eliminating and preventing the harmful effects associated with their accumulation due to its own toxicity and other effects such as pH culture media decrease [40]. The pH obtained for the maximum Cu-NirK activity (5.5– 6.0 units) and the kinetic parameters determined are consistent with the possibility that the enzyme performs a detoxification function. The Km of this enzyme for nitrite is almost 100-fold greater than for other enzymes

NirK characterized. This data suggests that the Hfx. mediterranei Cu-Nirk is adapted to work at high concentrations of nitrite, which could explain the extraordinary tolerance of this microorganism to high concentrations of this compound [41,42]. Funding This work was financially supported by research grants from the Ministerio de Ciencia e Innovación and FEDER funds from Spain (BIO2008-00082), Generalitat Valenciana (GV/2011/038) and University of Alicante (GRE09-25). Acknowledgements The authors would like to thank Dr. David J. Richardson (University of East Anglia, Norwich, UK) for useful discussions. Also, the authors wish to thank Dra. Felicitas Pfeifer (Technische Universität Darmstadt, Darmstadt, Germany) which generously provided us the expression vector pJAS. References [1] R.J.M. Van Spanning, A.P.N. de Boer, W.N.M. Reijnders, S. Spiro, H.V. Westerhoff, A.H. Stouthamer, J. Van der Oost, Nitrite and nitric oxide reduction in Paracoccus denitrificans is under the control of NNR, a regulatory protein that belongs to the FNR family of transcriptional activators, FEBS Lett. 360 (1995) 151–154. [2] W. Zumft, Cell biology and molecular basis of denitrification, Microbiol. Mol. Biol. Rev. 61 (1997) 533–616. [3] K. Heylen, D. Gevers, B. Vanparys, L. Wittebolle, J. Geets, N. Boon, P. De Vos, The incidence of nirS and nirK and their genetic heterogeneity in cultivated denitrifiers, Environ. Microbiol. 8 (2006) 2012–2021. [4] K. Yamaguchi, K. Shuta, S. Suzuki, Roles of Trp144 and Tyr203 in copper-containing nitrite reductase from Achromobacter cycloclastes, Biochem. Biophys. Res. Commun. 336 (2005) 210–214. [5] K. Olesen, A. Veselov, Y. Zhao, Y. Wang, B. Danner, C.P. Scholes, J.P. Shapleigh, Spectroscopic, kinetic, and electrochemical characterization of heterologously expressed wild-type and mutant forms of copper-containing nitrite reductase from Rhodobacter sphaeroides 2.4.3, Biochemistry 37 (1998) 6086–6094. [6] M. Nishiyama, J. Suzuki, M. Kukimoto, T. Ohnuki, S. Horinuchi, T. Beppu, Cloning and characterization of a nitrite reductase gene from Alcaligenes faecalis and its expression in Escherichia coli, J. Gen. Microbiol. 139 (1993) 725–733. [7] H. Ichiki, Y. Tanaka, K. Mochizuki, K. Yoshimatsu, T. Sakurai, T. Fujiwara, Purification, characterization, and genetic analysis of Cu-containing dissimilatory nitrite reductase from a denitrifying halophilic archaeon, Haloarcula marismortui, J. Bacteriol. 183 (2001) 4149–4156. [8] K. Inatomi, L.I. Hochstein, The purification and properties of a copper nitrite reductase from Haloferax denitrificans, Curr. Microbiol. 32 (1996) 72–76. [9] Y. Xie, T. Inoue, N. Seike, H. Matsumura, K. Kanbayashi, Deligeer, K. Itoh, K. Kataoka, K. Yamaguchi, S. Suzuki, Y. Kai, Crystallization and preliminary X-ray crystallographic studies of dissimilatory nitrite reductase isolated from Hyphomicrobium denitrificans A3151, Acta Crystallogr. 60 (2004) 2383–2386. [10] M. Prudencio, R.R. Eady, G. Sawers, The blue copper-containing nitrite reductase from Alcaligenes xylosoxidans: cloning of the nirA gene and characterization of the recombinant enzyme, J. Bacteriol. 181 (1999) 2323–2329. [11] J. Maillard, C.A. Spronk, G. Buchanan, V. Lyall, D.J. Richardson, T. Palmer, G.W. Vuister, F. Sargent, Structural diversity in twin-arginine signal peptide-binding proteins, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 15641–15646. [12] F. Sargent, The twin-arginine transport system: moving folded proteins across membranes, Biochem. Soc. Trans. 35 (2007) 835–847. [13] I.E. Tosques, A.V. Kwiatkowski, J. Shi, J.P. Shapleigh, Characterization and regulation of the gene encoding nitrite reductase in Rhodobacter sphaeroides, J. Bacteriol. 179 (1997) 1090–1095. [14] R.M. Martínez-Espinosa, J.A. Cole, D.J. Richardson, N.J. Watmough, Enzymology and ecology of the nitrogen cycle, Biochem. Soc. Trans. 39 (2011) 175–178. [15] A.F. Ellen, B. Zolghadr, A.M. Driessen, S.V. Albers, Shaping the archaeal cell envelope, Archaea (2010) 608243. [16] M. Sumper, E. Berg, R. Mengele, I. Strobel, Primary structure and glycosylation of the S-layer protein of Haloferax volcanii, J. Bacteriol. 172 (1990) 7111–7118. [17] F. Rodríguez-Valera, F. Ruiz-Berraquero, A. Ramos-Cormenzana, Isolation of extremely halophilic bacteria able to grow in defined inorganic media with single carbon sources, J. Gen. Microbiol. 119 (1980) 535–538. [18] S. Dassarma, E.M. Fleischman, F. Rodriguez-Valera, Media for halophiles, Archaea, A Laboratory Manual Halophiles, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1995, pp. 225–230. [19] M.L. Dyall-Smith, W.F. Doolittle, Construction of composite transposons for halophilic Archaea, Can. J. Microbiol. 40 (1994) 922–929. [20] J. Sambrook, D. Rusell, Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, New York, 2001.

J. Esclapez et al. / Biochimica et Biophysica Acta 1834 (2013) 1003–1009 [21] F. Pfeifer, S. Offner, K. Krüger, P. Ghahraman, C. Englert, Transformation of halophilic archaea and investigation of gas–vesicle synthesis, Syst. Appl. Microbiol. 16 (1994) 569–577. [22] S.W. Cline, L.C. Schalkwyk, W.F. Doolittle, Transformation of the archaebacterium Halobacterium volcanii with genomic DNA, J. Bacteriol. 171 (1989) 4987–4991. [23] R.M. Martínez-Espinosa, F.C. Marhuenda-Egea, M.J. Bonete, Purification and characterisation of a possible assimilatory nitrite reductase from the halophile archaeon Haloferax mediterranei, FEMS Microbiol. Lett. 196 (2001) 113–118. [24] F.D. Snell, C.T. Snell, Colorimetric Methods of Analysis, 2, Van Nostrand, New York, 1949, pp. 804–805. [25] J.M. Vega, H. Kamin, Spinach nitrite reductase. Purification and properties of a siroheme-containing iron–sulfur enzyme, J. Biol. Chem. 252 (1977) 896–909. [26] M.M. Bradford, A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein–dye binding, Anal. Biochem. 72 (1976) 248–254. [27] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685. [28] R. Bartossek, G.W. Nicol, A. Lanzen, H.P. Klenk, C. Schleper, Homologues of nitrite reductases in ammonia-oxidizing archaea: diversity and genomic context, Environ. Microbiol. 12 (2010) 1075–1088. [29] E.F. Mongodin, K.E. Nelson, S. Daugherty, R.T. Deboy, J. Wister, H. Khouri, J. Weidman, D.A. Walsh, R.T. Papke, G. Sanchez Perez, A.K. Sharma, C.L. Nesbø, D. MacLeod, E. Bapteste, W.F. Doolittle, R.L. Charlebois, B. Legault, F. Rodriguez-Valera, The genome of Salinibacter ruber: convergence and gene exchange among hyperhalophilic bacteria and archaea, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 18147–18152. [30] L. Álvarez, C. Bricio, M.J. Gómez, J. Berenguer, Lateral transfer of the denitrification pathway genes among Thermus thermophilus strains, Appl. Environ. Microbiol. 77 (2010) 1352–1358. [31] B.C. Berks, A common export pathway for proteins binding complex redox cofactors, Mol. Microbiol. 22 (1996) 393–404. [32] C. Pire, J. Esclapez, J. Ferrer, M.J. Bonete, Heterologous overexpression of glucose dehydrogenase from the halophilic archaeon Haloferax mediterranei, an enzyme

[33]

[34]

[35]

[36] [37]

[38]

[39]

[40]

[41]

[42]

1009

of the medium chain dehydrogenase/reductase family, FEMS Microbiol. Lett. 200 (2001) 221–227. B. Lledó, R.M. Martínez-Espinosa, F.C. Marhuenda-Egea, M.J. Bonete, Respiratory nitrate reductase from haloarchaeon Haloferax mediterranei: biochemical and genetic analysis, Biochim. Biophys. Acta 1674 (2004) 50–59. B. Zafrilla, R.M. Martínez-Espinosa, J. Esclapez, F. Pérez-Pomares, M.J. Bonete, SufS protein from Haloferax volcanii involved in Fe–S cluster assembly in haloarchaea, Biochim. Biophys. Acta, Proteins Proteomics 1804 (2010) 1476–1482. E. Sawada, T. Satoh, H. Kitamura, Purification and properties of a dissimilatory nitrite reductase of a denitrifying phototrophic bacterium, Plant Cell Physiol. 19 (1978) 1339–1351. C.H. Kim, T.C. Hollocher, Catalysis of nitrosyl transfer reactions by a dissimilatory nitrite reductase (cytochrome c,d1), J. Biol. Chem. 259 (1984) 2092–2099. O. Hering, M. Brenneis, J. Beer, B. Suess, J. Soppa, A novel mechanism for translation initiation in haloarchaea, Mol. Microbiol. 71 (6) (2009) 1451–1463. M.J. Bonete, C. Pire, F.I. Llorca, M. Camacho, Glucose dehydrogenase from the halophilic Archaeon Haloferax mediterranei: enzyme purification, characterisation and N-terminal sequence, FEBS Lett. 383 (1996) 227–229. T.J. Santangelo, L. Cubonová, J.N. Reeve, Shuttle vector expression in Thermococcus kodakaraensis: contributions of cis elements to protein synthesis in a hyperthermophilic archaeon, Appl. Environ. Microbiol. 10 (2008) 3099–3104. R.M. Martinez-Espinosa, E.J. Dridge, M.J. Bonete, J.N. Butt, C.S. Butler, F. Sargent, D.J. Richardson, Look on the positive side! The orientation, identification and bioenergetics of ‘Archaeal’ membrane-bound nitrate reductases), FEMS Microbiol. Lett. 276 (2) (2007) 129–139. R.M. Martínez-Espinosa, B. Zafrilla, M. Camacho, M.J. Bonete, Nitrate and nitrite removal from salted water by Haloferax mediterranei, Biocatal. Biotransform. 25 (2–4) (2007) 295–300. C. Najera-Fernández, B. Zafrilla, M.J. Bonete, R.M. Martínez-Espinosa, Role of the denitrifying Haloarchaea in the treatment of nitrite-brines, Int. Microbiol. 15 (2012) 111–119.