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Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec

Identification, cloning and heterologous expression of active [NiFe]-hydrogenase 2 from Citrobacter sp. SG in Escherichia coli

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Johannes A.H. Maier, Sergey Ragozin, Albert Jeltsch ∗ Institute of Biochemistry, Stuttgart University, 70569 Stuttgart, Germany

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Article history: Received 3 December 2014 Received in revised form 27 January 2015 Accepted 29 January 2015 Available online xxx

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Keywords: Hydrogen production Type 2 [NiFe]-hydrogenase Citrobacter Hydrogen measurement

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1. Introduction

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Hydrogen (H2 ) is a potential alternative energy carrier which only produces water and heat upon combustion. Today, industrial hydrogen production mainly uses thermochemical processes based on fossil fuels or electrolysis of water. Therefore, biotechnological approaches to produce H2 from biomass are an interesting alternative. We introduce here a novel direct hydrogen measurement system using a semiconducting device specific for hydrogen detection. Using this device, a bacterium producing considerable amounts of hydrogen under aerobic cultivation was isolated and identified by 16S ribosomal DNA sequencing as Citrobacter sp. The enzyme responsible for the observed hydrogenase activity was partially purified by 3 chromatographic purification steps and could be identified by peptide mass fingerprinting to be a type 2 [NiFe]-hydrogenase. Expression of the [NiFe]-hydrogenase 2 containing operon from Citrobacter sp. SG in Escherichia coli allowed recombinant hydrogen production. The [NiFe]-hydrogenase 2 identified here may be useful for biotechnological hydrogen production. We speculate that the expression of the hydrogenase in Citrobacter may be an adaptation to growth in acidic conditions. © 2015 Published by Elsevier B.V.

Hydrogen is a potential alternative pollution-free energy carrier, because its combustion products are only water and heat and it has a large energy content per mass (2H2 + O2 → 2H2 O with H = −143,000 kJ/kg). Today, industrial ways to produce hydrogen are, for example, the multistep steam-methane reforming process (Yi and Harrison, 2005) or electrolysis of water (Armaroli and Balzani, 2011). For these processes energy in form of fossil fuels or electricity is needed. Hydrogen production via microbial biotechnology provide the benefits of using biomass as an energy source or even sunlight directly. In nature, hydrogen is produced by microorganisms either in the process of nitrogen fixation (Hoffman et al., 2013), fermentative processes (Kothari et al., 2012) or by photosynthetic microorganisms under certain conditions (Basak and Das, 2007). Hydrogen converting enzymes, called hydrogenases, can be classified by their active sites into [NiFe]-, [FeFe]- and [Fe]hydrogenases (Lubitz et al., 2014). The most common and widest studied ones are [NiFe]-hydrogenases (Vignais and Billoud, 2007).

∗ Corresponding author at: Institute of Biochemistry, Faculty of Chemistry, University Stuttgart, Pfaffenwaldring 55, D-70569 Stuttgart, Germany. Tel.: +49 711 685 64390; fax: +49 711 685 64392. E-mail address: [email protected] (A. Jeltsch).

The core enzyme of [NiFe]-hydrogenases consists of a small subunit and a catalytically active large subunit (Forzi and Sawers, 2007; Volbeda et al., 2013). Electrons are carried either to, or away from the active site via iron-sulfur centers in the small subunit (Forzi and Sawers, 2007). Hydrogenases undergo a complex maturation process that depends on the activity of additional enzymes (Shomura and Higuchi, 2012). This includes, for example, the synthesis of diatomic ligands (CN− and CO) for the active sites of [NiFe] and [FeFe]-hydrogenases, the coordination of the active site iron, nickel insertion and proteolytic maturation (Böck et al., 2006; FontecillaCamps et al., 2007). Most hydrogenases are sensitive to molecular oxygen. [FeFe]-hydrogenases are inactivated irreversibly by oxygen and [NiFe]-hydrogenases are transiently inactivated by the formation of oxidized inactive states (Abou Hamdan et al., 2013; De Lacey et al., 2007). Oxygen has also been shown to regulate expression of certain hydrogenases (Richard et al., 1999). Here we report the application of a semiconducting device as an alternative for gas chromatography for fast and quantitative detection of hydrogen in the gas phase. Using this device we discovered a bacterium which is able to produce hydrogen under aerobic cultivation. We isolated the bacterial strain and show from ribosomal DNA (rDNA) sequences that it is closely related to Citrobacter freundii. Biochemical investigations were undertaken to identify the active hydrogenase which finally could be partially purified by applying different chromatographic steps and identified by mass spectrometry. The complete operon of the identified [NiFe]-hydrogenase 2

http://dx.doi.org/10.1016/j.jbiotec.2015.01.025 0168-1656/© 2015 Published by Elsevier B.V.

Please cite this article in press as: Maier, J.A.H., et al., Identification, cloning and heterologous expression of active [NiFe]-hydrogenase 2 from Citrobacter sp. SG in Escherichia coli. J. Biotechnol. (2015), http://dx.doi.org/10.1016/j.jbiotec.2015.01.025

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of Citrobacter sp. SG was cloned and overexpressed in E. coli BL21CodonPlusTM (DE3) leading to recombinant hydrogen production. The [NiFe]-hydrogenase 2 identified here may be useful for biotechnological hydrogen production.

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2. Materials and methods

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2.1. Strains and growth conditions

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Citrobacter sp. SG was cultured in Erlenmeyer flasks at 30 ◦ C with shaking. Cells were grown overnight in lysogeny broth (LB) medium supplemented with 57 ␮M ferric ammonium citrate, 46.3 mM H3 BO3 , 9.2 mM MnCl2 , 1.6 mM Na2 MoO4 , 0.77 mM ZnSO4 0.17 mM Co(NO3 )2 and 1 ␮M NiCl2 . For in vivo hydrogen measurements with air perfusion, 1 L overnight cultures of Citrobacter sp. SG and untransformed E. coli BL21-CodonPlusTM (DE3) were transferred to 2 L round bottom flasks which were flushed with air at a constant flow rate of 75 mL/min. 25 mmol of sterile glucose was added to the cultures and H2 production was measured for 3 h. For hydrogenase purification experiments Citrobacter sp. SG overnight cultures were harvested by centrifugation at 4,400 × g for 20 min at 4 ◦ C and washed with 50 mL STE buffer (100 mM NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM ethylenediaminetetraacetic acid (EDTA)). The pellets were frozen at -20 ◦ C until further use. Recombinant E. coli BL21-CodonPlusTM (DE3) transformed with pET-28a(+) vector containing Citrobacter sp. SG hydrogenase 2 operon were grown in LB medium supplemented with 57 ␮M ferric ammonium citrate, 46.3 mM H3 BO3 , 9.2 mM MnCl2 , 1.6 mM Na2 MoO4 , 0.77 mM ZnSO4 , 0.17 mM Co(NO3 )2 , NiCl2 (1 ␮M or 500 ␮M) and 25 ␮g/mL Kanamycin. Cells were cultured in Erlenmeyer flasks at 37 ◦ C until mid-exponential phase (OD600 0.5-0.6) and recombinant protein expression was induced by addition of 0.5 mM IPTG. Induction was performed at 18 ◦ C overnight and cells were used immediately for hydrogen measurements.

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2.2. Genomic DNA preparation

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For preparation of genomic DNA, the proteinase K/sodium dodecyl sulfate (SDS)/phenol extraction method was used (Herrmann and Frischauf, 1987). 5 mL LB overnight culture of Citrobacter sp. SG was grown at 30 ◦ C while shaking. Cells were harvested using a benchtop centrifuge at 12,000 × g for 2 min at room temperature. The resulting pellet was resuspended in 450 ␮L 50 mM Tris-HCl, pH 8, 10 mM EDTA, 50 ␮g RNAseA (Qiagen), 10 mg proteinase K (Qiagen) and 0.6% SDS and incubated for 1 h at 37 ◦ C. The sample was transferred into an 1.5 mL Eppendorf tube containing 500 ␮L of Phase Lock GelTM (5 Prime). 500 ␮L of phenol:chloroform:isoamyl alcohol (25:24:1) was added, mixed well by inverting and then centrifuged at 12,000 × g for 2 min at room temperature. Under the Phase Lock GelTM the organic phase is trapped allowing decanting the aqueous phase easily. The organic extraction step was performed twice. The aqueous supernatant was transferred to a fresh Eppendorf tube and 50 ␮L of 3 M sodium acetate, pH 5.2 and 300 ␮L isopropanol were added. The tube was mixed carefully by inverting until the DNA precipitated. The DNA was spooled with a sterile glass rod and dipped into 70% ethanol for 30 s for washing. Finally the DNA was dried, dissolved in 1 mL 10 mM Tris-HCL, pH 8, 1 mM EDTA and the concentration was measured. Isolated genomic DNA was stored at +4 ◦ C.

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2.3. 16S rDNA sequencing

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16S rDNA was amplified by PCR from isolated genomic DNA, 121 cloned using TA cloning (StrataClone PCR Cloning Kit, Agilent) (Ji 122 et al., 2006), and sequenced. The obtained 16S rDNA sequence 123 Q3 has been deposited to GenBank [GenBank:?????????]. Primers for 120

amplifying 16S rDNA region were 5 -CCG AAT TCG TCG ACA ACA GAG TTT GAT CCT GGC TCAG-3 and 5 -CCC GGG ATC CAA GCT TAC GGC TAC CTT GTT ACG ACTT-3 . The obtained DNA sequence was subjected to the BLAST web server (Altschul et al., 1997), hosted by the NCBI (National Center for Biotechnology Information). 16S rDNA sequences of close relatives Citrobacter freundii [GenBank: AF025365.1], Citrobacter braakii [GenBank: HQ288930.1], Citrobacter murliniae [GenBank: AF025369.1], Citrobacter sp. S77 [GenBank: AB668058.1] and of E. coli [GenBank: LM993812.1] were aligned using ClustalW2 (Larkin et al., 2007) using default alignment parameters. The alignment was visualized with BioEdit (http://www.mbio.ncsu.edu/bioedit/bioedit.html). Using the neighbor-joining algorithm, a phylogenetic tree was constructed. Dendroscope (Huson and Scornavacca, 2012) was used for the visualization of the tree. 2.4. Membrane fraction preparation A cell pellet of 1 L Citrobacter sp. SG overnight culture (approximately 4 g wet weight) was thawed on ice and suspended in 20 mL sonication buffer consisting of 20 mM Tris-HCl pH 8, 20 mM NaCl, 5% glycerol, 1 mM dithiothreitol (DTT). The cells were lysed by sonication using a Bandelein SONOPLUS HD 2200 device (15 cycles of 15 seconds with 40% pulsation and 30% power). The lysate was centrifuged at 7,600 × g for 40 min at 4 ◦ C. The resulting extract was ultracentrifuged at 84,400 × g for 1 h at 4 ◦ C. The membrane fraction was washed with 8 mL of wash buffer consisting of 20 mM Tris-HCl pH 8, 500 mM NaCl, 5% glycerol 1 mM DTT and centrifuged again at 126,000 × g for 1 h at 4 ◦ C. The resulting pellet was solubilized with agitation at 4 ◦ C for 1 h in 2.5 mL of a buffer consisting of 20 mM Tris-HCl pH 8, 20 mM NaCl, 5% glycerol, 1 mM DTT, 1 mM EDTA, 1% Triton X-100. 2.5. Chromatographic purification steps The solubilized membrane fraction was concentrated to approximately 1 mL using Amicon Ultra-15 (100,000 nominal molecular weight limit (NMWL); Millipore) and loaded onto a hydroxyapatite column (1.6 × 6 cm, Sigma-Aldrich). The column was equilibrated in a buffer containing 10 mM potassium phosphate, pH 8, 2% glycerol, 1 mM DTT and 0.05% Triton X-100. Proteins were eluted with two linear gradients of potassium phosphate ranging from 10 mM to 90 mM and from 90 mM to 170 mM using a flow rate of 0.5 mL/min. Fractions of 1 mL were collected and tested for hydrogenase activity (50 ␮L sample volume). Fractions eluting at conductivity values ranging from 4 mS/cm to 13 mS/cm were pooled and buffer exchanged to MonoQ loading buffer (20 mM Tris-HCl, pH 8, 20 mM NaCl, 5% glycerol, 1 mM DTT, 1 mM EDTA and 0,05% Triton X-100) and concentrated to 750 ␮L using Amicon Ultra-15 (100,000 NMWL; Millipore). The sample was loaded onto a MonoQ column (Mono Q 5/50 GL, GE Healthcare). Protein was eluted with a linear NaCl gradient ranging from 20 mM to 400 mM NaCl over a volume of 45 mL. Fractions of 1 mL were collected. Hydrogenase containing fractions eluted at conductivity values between 15 mS/cm and 25 mS/cm, were pooled and applied to Amicon Ultra-15 (100,000 NMWL). As the hydrogenase activity was detectable in the flow-through, the retained liquid (250 ␮L) was washed 3 times with 2 mL of buffer containing 20 mM Tris-HCl, pH 8, 500 mM NaCl, 5% glycerol, 1 mM DTT, 1 mM EDTA and 0.05% Triton X-100. The flow-through was collected, buffer exchanged to 20 mM Tris-HCl, pH 8, 500 mM NaCl, 5% glycerol, 1 mM DTT, 1 mM EDTA and 0.05% Triton X-100 and concentrated with Amicon Ultra-15 (30,000 NMWL) to 500 ␮L. 100 ␮L of the sample was applied consecutively four times onto a gelfiltration column (Superdex 200 10/300). The column was operated at a flow rate of 0.5 mL/minute and fractions of 0.5 mL were collected. Fractions

Please cite this article in press as: Maier, J.A.H., et al., Identification, cloning and heterologous expression of active [NiFe]-hydrogenase 2 from Citrobacter sp. SG in Escherichia coli. J. Biotechnol. (2015), http://dx.doi.org/10.1016/j.jbiotec.2015.01.025

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containing hydrogenase activity were pooled and concentrated to 500 ␮L using Amicon Ultra-15 (30,000 NMWL). The sample was TCA precipitated and analyzed by SDS-PAGE (12% acrylamide gel, stained with Coomassie Brilliant Blue R-250).

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Different protein bands from the partially purified hydrogenase protein sample obtained by the described chromatographic purification steps were analyzed by peptide mass fingerprinting. Samples were prepared by in-gel digestion using an modified protocol of (Shevchenko et al., 2007). After electrophoresis, the gel was stained with Coomassie Brilliant Blue R-250 and rinsed with water for several hours. Protein bands to be analyzed were excised and cut into 1 mm3 cubes. Gel pieces were transferred into 1.5 mL Eppendorf tubes and destained 3 times with 300 ␮L of 50 mM ammonium bicarbonate:acetonitrile (1:1 [v/v]) for 10 min with occasional vortexing. 100 ␮L of acetonitrile was added and samples were incubated at room temperature with occasional vortexing until gel pieces became white and shrunk. Acetonitrile was removed by pipetting and subsequently drying. Then, 30 ␮L of trypsin buffer consisting of 50 mM ammonium bicarbonate, 10% acetonitrile and 5 ng/␮L trypsin were added to the dry gel pieces and the digestion reactions were incubated overnight at 37 ◦ C. The liquid was transferred into fresh Eppendorf tubes and dried using an Eppendorf Concentrator plus at 30 ◦ C for 2 h. The dried peptides were solubilized in 0.1% trifluoroacetic acid (TFA) and 1 ␮L was spotted onto the target (AchorChip Standard (800 ␮m)). After drying of the spotted sample, 1 ␮L of matrix solution was added on top (matrix solution: 0.7 mg/mL HCCA (alpha-Cyano-4hydroxycinnamic acid) suspended in a solution of 85% acetonitrile, 15% H2 O, 0.1% TFA and 1 mM NH4 H2 PO4 ). Preparations were dried at room temperature. Peptide profiles were analyzed with matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) using a Bruker Autoflex Speed spectrometer. For external calibration, Peptide Calibration Standard II (Bruker Daltonics, Cat. number #222570) was dissolved in 125 ␮L of TA30 (30% acetonitrile, 70% 0.1% TFA) and premixed with HCCA matrix solution in a ratio of 1:200. 1 ␮L was spotted and dried. Peptides in a range between 400 and 3000 Da were detected. With the obtained mass spectra the Mascot search engine (http://www.matrixscience.com/) was used to identify proteins. SwissProt database was used as sequence database, up to two missed cleavages were allowed and the taxonomy parameter was set to ‘Proteobacteria’. 2.7. Hydrogen measurements BL21-CodonPlusTM

Hydrogen production of E. coli (DE3), E. coli XL1-Blue, Citrobacter sp. SG and E. coli BL21-CodonPlusTM (DE3) recombinantly expressing the hydrogenase 2 operon of Citrobacter sp. SG were assayed in 10 mL of induced bacterial culture cultivated at 20 ◦ C in sealed Lenz flat-bottomed flasks without shaking. 0.56 mmol of glucose was added just before starting the measurement. The hydrogen concentration in the 40 mL headspace of the flask was measured with the Hydrogen Leak Detector H2000 (Sensistor) every hour for up to 9 h. In hydrogen production assays with air perfusion, 1 L overnight cultures of Citrobacter sp. SG and untransformed E. coli BL21CodonPlusTM (DE3) were cultivated as described above and the H2 concentrations in the outlet gas flows were measured with the Hydrogen Leak Detector H2000 (Sensistor). The measured H2 concentrations were used to calculate the H2 production rates. Hydrogenase activity of partially purified protein samples from Citrobacter sp. SG were determined by the production of H2 in an assay with methyl viologen (MV) and sodium dithionite (Na2 S2 O4 )

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Fig. 1. Gas chromatography analysis of gas phase of a Citrobacter sp. SG culture. (A) Gas chromatography analysis of reference gases. The orange line corresponds to pure nitrogen, green to hydrogen with a slight contamination with air (indicated by the shoulder) and blue to a mixture of hydrogen and nitrogen (1:3, v/v). Nitrogen and oxygen gas cannot be separated under the given conditions and both run at an apparent retention time of 1.53 min. (B) Gas chromatography analysis of the gas phase of a Citrobacter sp. SG culture. The first peak with a retention time of 1.48 min corresponds to hydrogen, the second peak with a retention time of 1.53 min corresponds to a contamination with air.

(NaDT). MV is reduced by sodium dithionite and then is able to transfer electrons onto metal containing proteins (Peck and Gest, 1956). Hydrogenase activity was measured in 1 mL of buffer A1 (20 mM Tris-HCl, pH 8, 20 mM NaCl, 5% glycerol, 1 mM DTT, 1 mM EDTA) containing 5 mM MV and 50 mM Na2 S2 O4 . After incubation for 15 min at room temperature the concentration of H2 was measured with the Hydrogen Leak Detector H2000 (Sensistor). 2.8. Cloning of hydrogenase 2 operon of Citrobacter sp. SG The operon coding for Citrobacter sp. SG hydrogenase 2 (Fig. 4A) was amplified from genomic DNA by PCR using Phusion DNA Polymerases (Thermo Scientific) and cloned into pET-28a(+) vector using the following primers (5 -GGA ATA ACT ATG CTA GCA CTG GAG ATA ACT CTC TCA TCA ATTC-3 and 5 -ATC CGG CGG ATC CTC ACG CGC TGG TGAC-3 ). The N-terminal His-tag in pET was removed afterwards by site-directed mutagenesis (Lanio and Jeltsch, 2002). The DNA sequence of the operon sequenced and it has been deposited to GenBank [GenBank:????????]. Gene expression was carried out in E. coli BL21-CodonPlusTM (DE3) cells. 2.9. GC analysis To exclude cross reactivity of the Hydrogen Leak Detector H2000 (Sensistor) hydrogen measurement device, samples of gas produced by the Citrobacter sp. SG were also analyzed by gas chromatography. An Agilent 7890 gas chromatography system was used equipped with a thermal conductivity detector and a 30 m × 0.32 mm × 20.0 ␮m Hewlett Packard Plot Q

Please cite this article in press as: Maier, J.A.H., et al., Identification, cloning and heterologous expression of active [NiFe]-hydrogenase 2 from Citrobacter sp. SG in Escherichia coli. J. Biotechnol. (2015), http://dx.doi.org/10.1016/j.jbiotec.2015.01.025

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Fig. 2. Comparison of H2 production rates of Citrobacter sp. SG and Escherichia coli. (A) Hydrogen evolution of Citrobacter sp. SG, Escherichia coli BL21-CodonPlusTM (DE3) and Escherichia coli XL1-Blue. 10 mL overnight cultures were supplemented with 560 ␮mol glucose and hydrogen evolution was measured in the 40 mL headspace of the culture flask. The figure shows the averages of 2–4 independent biological experiments, error bars indicate the SEM. (B) Example of the hydrogen production of Citrobacter sp. SG and E. coli BL21-CodonPlusTM (DE3) determined under aerobic cultivation conditions. 1 L overnight cultures were flushed with a constant air flow of 75 mL/min and the H2 production rate was determined for up to 3.5 h after the initial addition of glucose (25 mmol). (C) Average H2 production rate of Citrobacter sp. SG and E. coli BL21-CodonPlusTM (DE3) determined after 180 min in two independent experiments arried out as described in panel B. The error bar corresponds to the SEM.

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3.1. Hydrogen detection with semiconducting Hydrogen Leak Detector In this study we demonstrate for the first time the potential of a Hydrogen Leak Detector H2000 (Sensistor) for biotechnological research. This semi-conducting detector is used by engineers to prove the integrity of machines by detecting very small leakage of hydrogen gas. We applied this detector to test microbiological samples for possible bio-hydrogen production. The detector reliably responded to hydrogen concentrations ranging from few parts per million (ppm) to several thousand ppm. Calibrating gas with 10 ppm hydrogen was provided by the manufacturer. The response time is in the range of seconds and measurement could be done with several seconds interval. To exclude possible cross reactivity to any substance other than hydrogen and validate the detection of hydrogen, we analysed gas samples produced by microorganisms also by gas chromatography to confirm the presence of hydrogen (Fig. 1).

3.2. Citrobacter sp. SG identification and hydrogen production rate We fortuitously discovered a bacterium which was able to produce unusual large amounts of molecular hydrogen even when cultivated under aerobic conditions. The bacterium was classified by 16S rDNA sequencing and revealed to be closely related to Citrobacter freundii [GenBank: AF025365.1] and was then called Citrobacter sp. SG (for Stuttgart, Germany) (Fig. 3A). As a reference we also determined hydrogen production of Escherichia coli BL21-CodonPlusTM (DE3) and Escherichia coli XL1-Blue under the same conditions (Fig. 2A). For hydrogen measurement an aerobically grown over-night culture was transferred to a sealed flask and glucose was added. Then, hydrogen was measured in the head space of the culture in regular intervals. In case of Citrobacter sp. SG, hydrogen production was immediately detectable and it increased over time. Escherichia coli BL21-CodonPlusTM (DE3) showed negligible hydrogen production (below 10 ppm) throughout the whole experiment. This result was expected as E. coli BL21 lacks hydrogenase activity due to a mutation in the FNR gene (Pinske et al., 2011). In Escherichia coli XL1-Blue this gene is unaltered and this strain started producing small amounts of hydrogen after 8 h of incubation (90 ppm after 9 h), indicating that hydrogenase activity appeared when the cultures became increasingly anaerobic. In

Please cite this article in press as: Maier, J.A.H., et al., Identification, cloning and heterologous expression of active [NiFe]-hydrogenase 2 from Citrobacter sp. SG in Escherichia coli. J. Biotechnol. (2015), http://dx.doi.org/10.1016/j.jbiotec.2015.01.025

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Fig. 3. Identification of Citrobacter sp. SG and partial purification of its hydrogenase. (A) Phylogenetic tree of a multiple sequence alignment of 16S rDNA sequences of the isolated Citrobacter sp. SG, some Citrobacter species using Escherichia coli as an outgroup (bar: 0.01 substitutions per site). The isolated Citrobacter sp. SG is closely related to Citrobacter freundii. (B) Partial purification of the Citrobacter sp. SG hydrogenase. Coomassie blue stained SDS-PAGE (12%) gel of hydrogenase fractions after gel filtration, which were used for peptide mass fingerprinting. The protein bands labelled by asterisks 1 and 2 were identified to be the large and small subunit of hydrogenase 2, respectively (marker: 170, 130, 100, 70, 55, 40, and 35 kDa).

Table 1 Partial purification of Citrobacter sp. SG hydrogenase. Purification step

Clear lysate Membran fraction Hydroxy apatite column MonoQ column Superdex 200 column

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0.02 0.09 0.37 1.78 2.41

contrast, Citrobacter sp. SG showed hydrogen production activity already after 1-2 h (200 ppm) and the hydrogen contents of the gas phase over the culture increased steadily to about 2,700 ppm after 8 h. Hence, Citrobacter sp. SG showed much stronger hydrogen production than E. coli and the hydrogen production was not restricted to anaerobic growth conditions. To measure the hydrogen production capabilities of Citrobacter sp. SG and E. coli BL21-CodonPlusTM (DE3) more precisely and under aerobic cultivation, we perfused 1 L overnight cultures (OD600 approximately 3.5) with a constant air flow of 75 mL/min. Glucose was added at the beginning of the experiment. The hydrogen concentration in the carrier gas was measured over 3 h and the hydrogen production rates were calculated (Fig. 2B). Citrobacter sp. SG showed 100 times higher H2 production rates compared to E. coli BL21-CodonPlusTM (DE3). After 3 h of incubation a maximum H2 production rate of 1.1 ␮mol/min was reached. In comparison, E. coli BL21-CodonPlusTM (DE3) showed a constant H2 production rate of approximately 10 nmol/min. 3.3. Purification and identification of Citrobacter sp. SG hydrogenase To identify the enzyme responsible for the observed oxygen tolerant hydrogen production of Citrobacter sp. SG different purification steps were applied. First, membranes were isolated from cleared lysate by ultracentrifugation and subsequently solubilized in Triton X-100. This purification step led to an increase in specific activity of almost 5 fold (Table 1). The solubilized membranes were further purified by hydroxyapatite column chromatography which increased the specific activity by another 4 fold. Anion exchange chromatography (MonoQ column) enriched the specific activity by

almost 5 fold and gelfiltration by another 1.3 fold (Table 1). Overall the hydrogenase activity could be enriched by about 120 fold. The gelfiltration fractions containing hydrogenase activity were precipitated by trichloroacetic acid (TCA) and analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. 3B). Several protein bands from the gel were extracted and further analyzed by mass spectrometry. The proteins with the apparent sizes of 60 kDa and 39 kDa could be identified by the peptide mass fingerprint search program Mascot to be the large and small subunits of the [NiFe]-hydrogenase 2 complex from Citrobacter freundii. Both proteins were unique matches to the peptide fingerprint pattern with Molecular Weight Search (mowse) scores for small and large subunits of 176 and 253, respectively (typically scores >67 are considered significant with p < 0.05). Based on the enzymatic activity of the most active fraction and the protein amount of hydrogenase 2 in the fraction, the specific hydrogen production activity of the hydrogenase 2 from Citrobacter sp. SG could be estimated to be 20 ␮mol/min/mg.

3.4. Cloning and expression of [NiFe]-hydrogenase 2 of Citrobacter sp. SG The E.coli hydrogenase 2 operon consists of 8 open reading frames (hybC, hybA, hybB, hybC, hybD, hybE and hybG) (Menon et al., 1994; Sargent et al., 1998). The same genomic architecture is present in the genome of Citrobacter freundii [GenBank: CP007557.1]. The proteins encoded by the operon include the small and large subunits of the catalytically active hydrogenase heterodimer (hybO and hybC, respectively). The hybA ORF codes for a 4Fe-4S ferredoxin-type component and participates in electron transfer to or from the catalytic site. hybB codes for a cytochrome b subunit. hybD, hybE, hybF and hybG are involved in processing the hydrogenase by metal insertion and maturation. The complete operon coding for the identified [NiFe]hydrogenase 2 from Citrobacter sp. SG was amplified by PCR from genomic DNA and cloned into the pET-28(a)+ vector (Fig. 4A). Subsequently the N-terminal histidine-tag provided in the vector was removed by site-directed mutagenesis and the entire sequence was validated by DNA sequencing. The recombinant operon was overexpressed in E. coli BL21-CodonPlusTM (DE3) cells (Fig. 4B). The protein bands corresponding to the small and large subunits of

Please cite this article in press as: Maier, J.A.H., et al., Identification, cloning and heterologous expression of active [NiFe]-hydrogenase 2 from Citrobacter sp. SG in Escherichia coli. J. Biotechnol. (2015), http://dx.doi.org/10.1016/j.jbiotec.2015.01.025

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Fig. 4. Cloning and recombinant activity of Citrobacter sp. SG hydrogenase in E. coli BL21-CodonPlusTM (DE3) cells. (A) Plasmid map of the hydrogenase 2 operon from Citrobacter sp. SG cloned into pET-28(a)+. The operon consists of 8 open reading frames (hybO, hybA, hybB, hybC, hybD, hybE, hybF and hybG). (B) Heterologous expression of cloned hydrogenase 2 from Citrobacter sp. SG in E. coli BL21-CodonPlusTM . Coomassie blue stained SDS-PAGE (15%) gel of whole cell extracts with and without IPTG induction. Indicated are the protein bands occurring after induction (marker: 170, 130, 100, 70, 55, 40, 35 and 25 kDa). (C) H2 evolution of E. coli BL21-CodonPlusTM (DE3) expressing the recombinant hydrogenase 2 operon from Citrobacter sp. SG (pETHyb). Untransformed E. coli BL21-CodonPlusTM (DE3) were used as control. H2 production was determined in 10 mL batch cultures containing 1 ␮M NiCl2 in the medium. Bacteria expressing pETHyb were also cultivated in media containing 500 ␮M NiCl2 . Addition of increased amounts of NiCl2 has been shown previously not to stimulate hydrogen production in BL21 (Pinske et al., 2011). The figure shows the averages of 2-4 independent biological experiments, error bars indicate the SEM.

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the [NiFe]-hydrogenase 2 complex appear in the isopropyl-␤-D1thiogalactopyranoside (IPTG) induced sample at around 39 kDa and around 60 kDa.

producing proteins from Citrobacter sp. and it shows that this enzyme complex is able to produce hydrogen when recombinantly expressed in E. coli.

3.5. Hydrogenase activity measurements of recombinantly expressed [NiFe]-hydrogenase 2 of Citrobacter sp. SG

4. Discussion

Hydrogenase activity of E. coli BL21-CodonPlusTM (DE3) cells expressing the [NiFe]-hydrogenase 2 complex from Citrobacter sp. SG was assessed in 10 mL of induced bacterial culture. Hydrogenase activity was measured every hour after addition of glucose (0.56 mmol) for 9 hs in total (Fig. 4C). Since Ni2+ has been reported to be limiting for hydrogenase activity in BL21(Pinske et al., 2011), these experiments were conducted in medium supplemented with either 1 ␮M NiCl2 or 500 ␮M NiCl2 . Hydrogen production could be measured after 5 h of incubation when a hydrogen content of 250 ppm was determined in the headspace of the culture supplemented with 1 ␮M NiCl2 . Supplementing the growth media with 500 ␮M NiCl2 resulted in a hydrogen concentration of 1000 ppm. This result confirms the correct identification of the hydrogen

In this work, we introduce an easy to use, specific and highly sensitive semiconducting device for hydrogen detection, which to our knowledge was not reported so far to be used in biotechnological research. Using this device, we discovered a hydrogen producing bacterium and identified it by 16S rDNA sequencing to be closely related to Citrobacter freundii and called it Citrobacter sp. SG. Hydrogen production by Citrobacter sp. SG was 30-100 fold higher than production by different E. coli strains under the given conditions which included the cultivation with a constant air flow. Comparison of the H2 production rate of Citrobacter sp. SG to literature is difficult, due to the fact that in reports on hydrogen producing bacteria cultivation conditions usually are strictly anaerobic (Fabiano and Perego, 2002; Kotay and Das, 2007; Oh et al., 2003; Pinske et al., 2011).

Please cite this article in press as: Maier, J.A.H., et al., Identification, cloning and heterologous expression of active [NiFe]-hydrogenase 2 from Citrobacter sp. SG in Escherichia coli. J. Biotechnol. (2015), http://dx.doi.org/10.1016/j.jbiotec.2015.01.025

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The proteins responsible for the hydrogen production of Citrobacter sp. SG were identified by partial chromatographic purification followed by SDS-PAGE and peptide mass fingerprinting to be [NiFe]-hydrogenase 2. The bacteria used as the starting material for the purification process were aerobically cultivated in shaking cultures. This indicates that Citrobacter sp. SG expresses [NiFe]hydrogenase 2 not only under anaerobic conditions, and suggests an unusual regulation of hydrogenase 2 in Citrobacter sp. SG, considering reports on hydrogenase 2 being repressed under aerobic conditions (Giel et al., 2006; Richard et al., 1999). During the hydrogenase purification process, ambient oxygen probably inactivated the enzyme but by providing a strongly reducing environment (like sodium dithionite), the activity can be restored, which is in accordance with literature (Vincent et al., 2005). The specific H2 production activity of hydrogenase 2 from Citrobacter sp. SG of about 20 ␮mol/min/mg is comparable to 18.7 ␮mol/min/mg what was reported earlier for an unidentified O2 -stable hydrogenase isolated from a Citrobacter sp. (Eguchi et al., 2012) and higher than what was reported for the corresponding Escherichia coli enzyme (2.5 ␮mol/min/mg) (Lukey et al., 2010). We have cloned and expressed the hydrogenase 2 operon from Citrobacter sp. SG in E. coli BL21-CodonPlusTM (DE3) cells. By providing important maturation enzymes encoded within the hyb operon (HybE, HybF and HybG) it was possible to overcome the limitations for hydrogenase maturation set by E. coli BL21(DE3) and obtain active hydrogenase. We observed a significant hydrogen production by the recombinantly expressed protein. Supplementing bacterial cultures with higher concentrations of NiCl2 further increased the hydrogen production. To our knowledge this is the first report of heterologously expressed active type 2 [NiFe]hydrogenase from any Citrobacter sp. For technical application the hydrogen yield of the system has to be increased. Possible adjustments could be the introduction of further maturation proteins (Jacobi et al., 1992) or the optimization of culture conditions. One known metabolic role of hydrogenases is in fermentative metabolism under anaerobic conditions, where proton reduction serves as final electron acceptor. In addition, hydrogenases are involved in oxidation of hydrogen to provide reduction equivalents (Greening and Cook, 2014; Vignais and Billoud, 2007). Surprisingly, the Citrobacter sp. identified here shows hydrogen production under aerobic conditions leading to the question of the biological role of this process. We speculate that the hydrogen production might play a role in the adaptation of the bacteria for growth under acidic conditions, because it allows to increase the intracellular pH by reduction of protons followed by their release as hydrogen gas as described in other systems (Hayes et al., 2006; Noguchi et al., 2010). 5. Conclusions We show here that Citrobacter sp. SG produces hydrogen under aerobic cultivation conditions. Expression of the [NiFe]-hydrogenase 2 operon from Citrobacter sp. SG allows the recombinant hydrogen production in E. coli. This observation may pave the way for a direct biological hydrogen production from biomass or sunlight. Competing interests The authors declare that they have no competing interests. Authors’ contributions J.A.H.M., S.R. and A.J. devised the study and planed the experiments. J.A.H.M. and S.R. performed all experiments. J.A.H.M., S.R.

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and A.J. analyzed the data and interpreted the results. J.A.H.M. and A.J. wrote the manuscript. All authors read and approved the final manuscript. Acknowledgements We acknowledge the help by Dennis Wan Hussin from the Institut für Technische Chemie, Stuttgart University, 70569 Stuttgart, Germany, who operated the gas chromatograph and analyzed the results. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jbiotec. 2015.01.025. References Abou Hamdan, A., Burlat, B., Gutiérrez-Sanz, O., Liebgott, P.-P., Baffert, C., De Lacey, A.L., Rousset, M., Guigliarelli, B., Léger, C., Dementin, S., 2013. O2-independent formation of the inactive states of NiFe hydrogenase. Nat. Chem. Biol. 9, 15–17, http://dx.doi.org/10.1038/nchembio.1110. Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402, http://dx.doi.org/10.1093/nar/25.17.3389. Armaroli, N., Balzani, V., 2011. The hydrogen issue. ChemSusChem 4, 21–36, http://dx.doi.org/10.1002/cssc.201000182. Basak, N., Das, D., 2007. The prospect of purple non-sulfur (PNS) photosynthetic bacteria for hydrogen production: the present state of the art. World J. Microbiol. Biotechnol. 23, 31–42, http://dx.doi.org/10.1007/s11274-006-9190-9. Böck, A., King, P.W., Blokesch, M., Posewitz, M.C., 2006. Maturation of Hydrogenases. In: Poole, R.K. (Ed.), Advances in Microbial Physiology. Academic Press, pp. 1–225. De Lacey, A.L., Fernandez, V.M., Rousset, M., Cammack, R., 2007. Activation and inactivation of hydrogenase function and the catalytic cycle: spectroelectrochemical studies. Chem. Rev. 107, 4304–4330, http://dx.doi.org/10.1021/cr0501947. Eguchi, S., Yoon, K.-S., Ogo, S., 2012. O2-stable membrane-bound [NiFe]hydrogenase from a newly isolated Citrobacter sp. S-77. J. Biosci. Bioeng. 114, 479–484, http://dx.doi.org/10.1016/j.jbiosc.2012.05.018. Fabiano, B., Perego, P., 2002. Thermodynamic study and optimization of hydrogen production by Enterobacter aerogenes. Int. J. Hydrog. Energy 27, 149–156, http://dx.doi.org/10.1016/S0360-3199(01)00102-1. Fontecilla-Camps, J.C., Volbeda, A., Cavazza, C., Nicolet, Y., 2007. Structure/function relationships of [NiFe]- and [FeFe]-Hydrogenases. Chem. Rev. 107, 4273–4303, http://dx.doi.org/10.1021/cr050195z. Forzi, L., Sawers, R.G., 2007. Maturation of [NiFe]-hydrogenases in Escherichia coli. BioMetals 20, 565–578, http://dx.doi.org/10.1007/s10534-006-9048-5. Giel, J.L., Rodionov, D., Liu, M., Blattner, F.R., Kiley, P.J., 2006. IscR-dependent gene expression links iron-sulphur cluster assembly to the control of O2-regulated genes in Escherichia coli. Mol. Microbiol. 60, 1058–1075, http://dx.doi.org/10.1111/j.1365-2958.2006.05160.x. Greening, C., Cook, G.M., 2014. Integration of hydrogenase expression and hydrogen sensing in bacterial cell physiology. Curr. Opin. Microbiol., Cell regulation 18, 30–38, http://dx.doi.org/10.1016/j.mib.2014.02.001. Hayes, E.T., Wilks, J.C., Sanfilippo, P., Yohannes, E., Tate, D.P., Jones, B.D., Radmacher, M.D., BonDurant, S.S., Slonczewski, J.L., 2006. Oxygen limitation modulates pH regulation of catabolism and hydrogenases, multidrug transporters, and envelope composition in Escherichia coli K-12. BMC Microbiol. 6, 89, http://dx.doi.org/10.1186/1471-2180-6-89. Herrmann, B.G., Frischauf, A.-M., 1987. [15] Isolation of genomic DNA. In: Shelby, L., Berger, A.R.K. (Eds.), Methods in Enzymology. Academic Press, pp. 180–183. Hoffman, B.M., Lukoyanov, D., Dean, D.R., Seefeldt, L.C., 2013. Nitrogenase: a draft mechanism. Acc. Chem. Res. 46, 587–595, http://dx.doi.org/10.1021/ar300267m. Huson, D.H., Scornavacca, C., 2012. Dendroscope 3: An Interactive Tool for Rooted Phylogenetic Trees and Networks. Syst. Biol. sys062. doi:10.1093/sysbio/sys062. Jacobi, A., Rossmann, R., Böck, A., 1992. The hyp operon gene products are required for the maturation of catalytically active hydrogenase isoenzymes in Escherichia coli. Arch. Microbiol. 158, 444–451. Ji, H., Greener, A., Sorge, J.A., Bauer, J., Gibbs, R., Carstens, C.-P., 2006. Method for Ligating Nucleic Acids and Molecular Cloning, 7109178. Kotay, S.M., Das, D., 2007. Microbial hydrogen production with Bacillus coagulans IIT-BT S1 isolated from anaerobic sewage sludge. Bioresour. Technol. 98, 1183–1190, http://dx.doi.org/10.1016/j.biortech.2006.05.009. Kothari, R., Singh, D.P., Tyagi, V.V., Tyagi, S.K., 2012. Fermentative hydrogen production – An alternative clean energy source. Renew. Sustain. Energy Rev. 16, 2337–2346, http://dx.doi.org/10.1016/j.rser.2012.01.002.

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