Articles Microbiology

January 2010 Vol.55 No.1: 32−37 doi: 10.1007/s11434-009-0706-1

SPECIAL TOPICS:

Hydrogen metabolic pathways of Rhodospirillum rubrum under artificial illumination ZHU RuiYan1,2,3 & LI JiLun1* 1

College of Biological Sciences, State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100193, China; College of Environmental and Chemical Engineering, Yanshan University, Qinhuangdao 066004, China; 3 Engineering Research Center for Bioenergy of Hebei Province, Qinhuangdao 066004, China 2

Received March 25, 2009; accepted July 8, 2009

Multiple enzymes involved in hydrogen metabolism have been detected in photosynthetic bacterium Rhodospirillum rubrum under various growth conditions. To investigate hydrogen metabolic pathways and the contribution of each pathway to hydrogen photoproduction in R. rubrum under the artificial illumination condition, three mutants were constructed, including nifHanfDG double mutant lacking both Fe-nitrogenase and Mo-nitrogenase, anfDG mutant lacking Fe-nitrogenase and nifHhupL double mutant (uptake hydrogenase deficient mutant). Comparison of the hydrogen production of these 3 mutants with R. rubrum wild type and the uptake hydrogenase deficient mutant showed that there was a third pathway involved in hydrogen production besides Mo-nitrogenase and Fe-nitrogenase, which mainly contributed to hydrogen evolution. Only a small portion of hydrogen was generated by the third pathway. The hydrogen produced by Mo-nitrogenase, Fe-nitrogenase and the third pathway was about 93.5%, 4.9% and 1.5%, respectively, while the hydrogen consumed by uptake hydrogenase was about 13.3%. The investigation of the formate-linked hydrogenase activity indicated that the third pathway for hydrogen production was not mediated by the formate-linked hydrogenase, but probably by some unknown enzyme. Rhodospirillum rubrum, hydrogen metabolic pathways, Mo-nitrogenase, Fe-nitrogenase, uptake hydrogenas Citation:

Zhu R Y, Li J L. Hydrogen metabolic pathways of Rhodospirillum rubrum under artificial illumination. Chinese Sci Bull, 2010, 55: 32―37, doi: 10.1007/s11434-009-0706-1

Rhodospirillum rubrum, a purple nonsulfur photosynthetic bacterium, has been used extensively for the study of nitrogen fixation [1–3], and it was identified as the first photosynthetic bacterium that could produce hydrogen from various organic acids [3–5]. The existence of multiple enzymes involved in hydrogen metabolism among various photosynthetic bacteria has been reported [6–8]. Among them, R. rubrum seems to be the most versatile in its modes of hydrogen metabolism. Nitrogenase has been reported to be the key enzyme involved in hydrogen evolution in R. rubrum [9,10]. The reduction of protons to hydrogen, catalyzed by Mo-nitrogenase, accompanies the reduction of nitrogen to ammonia. Hydrogen production occurs at a lower rate than nitrogen fixation since only 25% of the *Corresponding author (email: [email protected]) © Science China Press and Springer-Verlag Berlin Heidelberg 2010

electrons would be allocated for proton reduction in a pure nitrogen atmosphere [11]. The general reaction for biological nitrogen fixation catalyzed by Mo-nitrogenase can be written as follows (the reaction catalyzed by either Mo-nitrogenase or Fe-nitrogenase requires metabolic energy in the form of ATP): N2+8H++8e−+16MgATP→2NH3+H2+16MgADP+16Pi Two kinds of nitrogenases have been identified in R. rubrum: Mo-nitrogenase (encoded by nifHDK) and Fe-nitrogenase (encoded by anfHDGK) according to the atom in the active center [12–14]. Fe-nitrogenase differs from conventional Mo-nitrogenase chemically, physically and by its catalytic properties. Fe-nitrogenase seems to allocate a higher proportion of electrons to the reduction of protons than Mo-nitrogenase does. In a pure nitrogen at mosphere, csb.scichina.com www.springerlink.com/scp

ZHU RuiYan, et al. Chinese Sci Bull

approximately 75% of the electrons would be allocated for proton reduction and 25% for nitrogen reduction. The proposed electron distribution of Fe-nitrogenase for reduction of proton and nitrogen is: N2+24e−+24H+ →2NH3+9H2. However, all electrons supplied to either Mo-nitrogenase or Fe-nitrogenase would contribute to proton reduction in the absence of nitrogen gas. Another H2-evolution activity has been detected when R. rubrum was cultivated in the presence of CO and hydrogen is produced according to the net reaction: CO + H2O → CO2 + H2 [15,16]. Hydrogen evolution activity was also reported when R. rubrum was cultured under darkness with pyruvate as the carbon source with a proposed role of dissipating excess reducing power [16–18]. An uptake hydrogenase, with the evident function of catalyzing the consumption of hydrogen, has been observed in R. rubrum [1,19]. Uptake hydrogenase in R. rubrum is composed of two dissimilar subunits, in which the large subunit (encoded by hupL) functions as the active center and the small subunit (encoded by hupS) mediates electron transfer from the active center to the external electron receptor [20]. During photoheterotrophic growth with a limited supply of bound nitrogen, the uptake hydrogenase is expressed along with the N2 fixation system [10]. Even though multiple enzymes involved in hydrogen metabolism have been detected under various growth conditions in R. rubrum, there is no evidence to ascertain the types of enzymes taking part in hydrogen metabolism and the contribution of each enzyme to hydrogen yield under the artificial illumination condition. In this report, we present evidence that there are at least three pathways involved in hydrogen evolution and one in hydrogen consumption under the artificial illumination condition through disrupting main hydrogen metabolic pathways.

1 Materials and methods 1.1 Bacterial strains and growth conditions Bacterial strains and plasmids used in this study are listed in Table 1. Precultures of R. rubrum were grown in rich SMN medium aerobically at 30°C as described previously [21].

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Nitrogenase derepression malate-glutamate medium (MG) for hydrogen photoproduction is minimal medium with 7 mmol/L sodium glutamate and 30 mmol/L malate [13]. Four milliliter of SMN-grown culture in the exponential phase was inoculated into 125 mL glass vials containing 120 mL MG medium. Then, the glass vials were sealed with rubber stoppers and exposed to the artificial illumination provided by superlux lamps with light intensity of approximately 2000−2500 lux. The cultivation temperature was controlled at 30−32°C. One milliliter of air was left in the headspace of the glass vial for the convenience of hydrogen collection to avoid vials’ breaking due to strait space and high gas pressure. The 50 mL gas-tight syringe was inserted into the rubber stopper to collect hydrogen after hydrogen evolution began. Escherichia coli DH5α as a cloning host for all plasmid-cloning experiments and E. coli S17-1 as a mating donor were grown in LB medium at 37°C with vigorous shaking. Antibiotics were used at the following concentrations (μg/mL): for R. rubrum, streptomycin sulfate (Sm), 100; gentamicin (Gm), 10; kanamycin sulfate (Km), 12.5; nalidixic acid (Nx), 20; chloramphenicol (Cm), 5 and for E. coli, ampicillin (Ap), 100; chloramphenicol, 25; gentamicin, 5; kanamycin, 50 and tetracycline hydrochloride (Tc), 12.5. 1.2 Construction of anfDG deletion mutants In-frame deletions of R. rubrum genes were performed by allelic exchange strategy. The 1.2-kb of 5′ flanking of anfDG containing engineered BamH I and Sac I sites (Two primers were: 5′-CGGATCCGCGCTTCGTACCAAACGG-3′, 5′-CG AGCTCTGGTTGCGTGCTCCTTAACG-3′) and 3′ flanking of anfDG containing Sac I and Sph I sites at the both ends (Two primers were: 5′-CGAGCTCAATCGCCTCGTCCTCG G-3′, 5′-GGCATGCTCCATGTCGGTGAACAGGG-3′) was amplified by using genomic DNA as the template, respectively. The two sequenced fragments were inserted into pSUP202 at BamH I and Sph I sites, generating the vector pRYZA. Finally, aacC1 (encoding Gmr) from pUCGM was inserted into pRYZA at the Sac I site, yielding suicide vector pRYZB (Figure 1). After being transformed into E. coli S17-1, pRYZB was conjugated into R. rubrum UR2 (wild

Table 1 Bacterial strains and plasmids Strains or plasmids R. rubrum UR2 UR801 UR802 UR206 UR803 UR804 Plasmids pRYZ1 pRYZB

Relevant genotype and description

Reference

Wild type; Smr ∆hupL::aacC1, Smr Gmr ∆anfDG::aacC1, Smr Gmr nifH::kan, Smr Kmr nifH::kan ∆hupL::aacC1, Smr Kmr Gmr nifH::kan ∆anfDG::aacC1, Smr Kmr Gmr

[21] [23] this study [22] this study this study

2.7-kb BamH I-Sal I of ΔhupL::aacC1 was cloned into pSUP202, Apr Cmr Gmr 3.3-kb BamH I-Sph I of ΔanfDG::aacC1 was cloned into pSUP202, Apr Cmr Gmr

[23] this study

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2 Results 2.1 Nitrogenase activities of R. rubrum wild type and its derivatives

Figure 1

Suicide vector to inactivate Fe-nitrogenase.

type) and UR206 (nifH) as described previously [22]. NxrGmr R. rubrum colonies were isolated after 7−10 d and replica-printed to identify Cms colonies resulting from a double-crossover recombination event (Cmr is encoded by pSUP202). All mutations were verified by colony PCR. The two anfDG mutants were designated UR802 (ΔanfDG:: aacC1) and UR804 (nifH:: kanΔanfDG::aacC1), respectively. pRYZ1 [23] containing ΔhupL::aacC1 was transformed into R. rubrum nifH mutant (UR206), then hupLnifH double mutant was obtained and designated UR803. 1.3 Assays of nitrogenase activity Whole cell nitrogenase activity was assayed by the method of acetylene reduction as described previously [9]. One milleliter of R. rubrum culture was taken out from the glass vessel and injected into a 9-mL serum bottle filled with oxygen-free argon. Subsequently, 1 mL acetylene was injected into the serum bottle to start the reaction. The reaction lasted 5 min and was terminated by adding 0.2 mL 30% (w/v) trichloroacetic acid. The ethylene gas mixture in the headspaces of the serum bottle was withdrawn and injected into a gas chromatograph equipped with a flame ionization detector and separated by GDX-502 using nitrogen as carrier gas.

To identify whether deletion of uptake hydrogenase or Fe-nitrogenase affects nitrogenase activity, acetylene reduction catalyzed by R. rubrum wild type and its derivatives was measured in the course of fermentation. Nitrogenase activity in the R. rubrum wild type appeared at the 18th−20th hour and reached the maximum (760 nmol C2H4mL−1h−1A600−1) during cultivation 40−60 h. Similar nitrogenase activities were observed in R. rubrum uptake hydrogenase deficient mutant (UR801) and anfDGhupL double mutant (UR802), and the maximal nitrogenase activity of these two strains reached 790 and 760 nmol C2H4m−1h−1A600−1 at the same time, respectively (Figure 2). It indicates that deletion of hupL or anfDG has no significant effect on nitrogenase activity in R. rubrum. However, low nitrogenase activities were detected in Mo-nitrogenase deficient mutants (nifH and nifHhupL), and the maximal nitrogenase activity decreased to 5.8% and 5.5% of that in the wild type, respectively. R. rubrum mutant deficient of both Mo-nitrogenase and Fe-nitrogenase exhibited low nitrogenase activity below detectable limits (<10). The investigation on the relationship between nitrogenase activities and hydrogen yields of R. rubrum wild type and its derivatives (Figures 2 and 3) indicated that hydrogen yield was closely related to nitrogenase activity.

1.4 Assays of formate-linked hydrogenase Formate-linked hydrogenase was assayed by measuring the rates of H2 production based on sodium formate as substrate. The assay was carried out in a 10 mL rubber stoppered vial in the light or in the dark for 10 min. The stoppered vial containing the reaction mixture (100 μL, 50 mmol/L pH 7.0 potassium phosphate buffers, 20 mmol/L sodium formate) was degassed and flushed with argon, and then 900 μL cultures was injected into the vial anaerobically. The reactions were started by addition of cultures and terminated by 0.2 mL of 30% (w/v) trichloroacetic acid. Hydrogen samples in the gas phase were withdrawn and injected into a gas chromatograph equipped with a thermal conductivity detector and separated by a 5 Å molecular sieve using argon as carrier gas.

Figure 2 Time course of nitrogenase activities of R. rubrum wild type and its derivatives.

Figure 3 Hydrogen yields of R. rubrum wild type and its derivatives under artificial illumination condition.

ZHU RuiYan, et al. Chinese Sci Bull

2.2 Hydrogen production by R. rubrum deficient of both Mo-nitrogenase and Fe-nitrogenase To further explore whether there is another pathway involved in hydrogen production in R. rubrum besides nitrogenases, R. rubrum mutant deficient of both nitrogenase systems was constructed and its hydrogen production was investigated under the same condition as for the wild type (Figure 3). R. rubrum lacking both Mo- and Fe-nitrogenase could evolve approximately 9 mL/L gas mixture under the artificial illumination. The gas mixture was measured and the results showed that hydrogen could be detected besides oxygen and nitrogen in the gas phase evolved by R. rubrum nifHanfDG mutant. One milliliter of air was left in the headspace of the glass vial when R. rubrum UR804 was cultivated, so the total hydrogen produced by nifHanfDG mutant (UR804) was 8 mL/L (9 mL minus 1 mL of air) which was significantly lower than that by the wild type or Fe-nitrogenase deficient strain. The presence of oxygen and nitrogen in the gas mixture produced by R. rubrum nifHanfDG mutant was due to low hydrogen yield with the air not completely replaced with hydrogen (only hydrogen could be detected in the biogas produced by R. rubrum wild type, because 1 mL of air was replaced by a large amount of hydrogen completely). That R. rubrum nifHanfDG mutant could produce hydrogen indicated that there was a third pathway involved in hydrogen production in R. rubrum besides the two kinds of nitrogenase systems. R. rubrum lacking both nitrogenase systems with intact uptake hydrogenase activity could produce hydrogen suggesting that R. rubrum nifHanfDG mutant probably could produce a large volume of hydrogen in the absence of uptake hydrogenase activity. Thus, we have tried to construct the triplicate mutant deficient of Mo-nitrogenase, Fe-nitrogenase and uptake hydrogenase. Unexpectedly, the triplicate mutant was not obtained presumably due to lethality. 2.3 Hydrogen metabolic pathways in R. rubrum under the artificial illumination condition As shown in Figure 3, hydrogen yield of R. rubrum UR2 and UR801 under the artificial illumination with light intensity of approximately 2000−2500 lux was (2251±80) and (2598±92) mL/L, respectively, indicating that 13.3% of hydrogen was consumed by the uptake hydrogenase. PreTable 2

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vious experiments have reported that deletion of uptake hydrogenase could improve hydrogen yield 1.56-fold that of R. rubrum wild type with light intensity of approximately 40000−60000 lux, indicating uptake hydrogenase consumed lower hydrogen with light intensity of about 2000−2500 lux than that with 40000−60000 lux. Because hydrogen yield was improved with increased light intensity (below saturation light intensity) in hupL deletion mutant but not in R. rubrum UR2, hydrogen yield of UR801 with light intensity of about 2000−2500 lux was lower than that with 40000− 60000 lux [23]. R. rubrum UR206 (nifH) and UR803 (nifHhupL) produced hydrogen at about (128±17) and (167±9) mL/L in MG medium containing molybdenum, respectively, indicating that the synthesis of Fe-nitrogenase in R. rubrum was not inhibited by molybdate. The contribution of each pathway to hydrogen yield is shown in Table 2. The hydrogen produced by Mo-nitrogenase, Fe-nitrogenase and the third pathway was approximately 93.5%, 4.9% and 1.5%, respectively; while the hydrogen consumed by uptake hydrogenase was about 13.3% of the total hydrogen yield under our experimental conditions. 2.4 Formate-linked hydrogenase activity in R. rubrum Although we have known that there is a third pathway involved in hydrogen production besides the two nitrogenase systems, more data are needed to identify the type of this enzyme. Three distinct hydrogenase activities have been reported in R. rubrum and each hydrogenase is independent of the others [16]. CO-induced hydrogenase functions only in the presence of CO, thus it was inactive under this cultivation condition due to the absence of CO in the system. We proposed that formate-linked hydrogenase might be the major enzyme involved in hydrogen production in R. rubrum deficient of both nitrogenase systems. To investigate whether formate-linked hydrogenase functions when R. rubrum is cultivated in MG medium, formate-linked hydrogenase activities in the R. rubrum wild type and UR804 were measured both in the light/dark and in the presence/absence of sodium formate. As shown in Figure 4(a), activity of for mate-linked hydrogenase in R. rubrum UR804 could not be detected either in the presence or in the absence of formate. It indicated that hydrogen production of R. rubrum UR804 in MG medium was not mediated by the formate-linked hydrogenase. The result suggested that formate-linked hy-

Hydrogen metabolic pathways of R. rubrum under artificial illumination and the contribution of each pathway to hydrogen yield

Formula Y (mL/L) Percentage

Hup YUR2−YUR801 −347 −13.3%

Hydrogen produced or consumed by Mo-N2ase Fe-N2ase Y UR801−YUR803 YUR2−YUR802 +2430 +128 +93.5% +4.9%

Other pathway YUR801−YMo-N2ase−YFe-N2ase +40 +1.5%

Total H2 yield YUR801 2598 100%

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drogenase did not function in MG medium. Interestingly, formate-linked hydrogenase activities of the R. rubrum wild type under these conditions were not the same as those of R. rubrum UR804 (Figure 4(a)). The presence or absence of formate has no significant effect on the formate-linked hydrogenase activity of R. rubrum UR2 in the light, but the effect is obvious in the dark. Formate-linked hydrogenase activity of R. rubrum UR2 in the presence of formate was 6.4-fold that in the absence of formate in the dark. Because hydrogen production in R. rubrum was closely related to nitrogenase activity, nitrogenase activities of R. rubrum UR2 and UR804 were then measured both in the presence/absence of formate and in the light/dark (Figure 4(b)). The presence or absence of formate had no significant effect on nitrogenase activities of R. rubrum wild type in the light. Nitrogenase activity of R. rubrum UR2 in the absence of sodium formate in the dark reached (35±5) nmol C2H4mL−1h−1A600−1 (7%―8% that of the wild type under illumination), however, it decreased to zero in the presence of sodium formate, indicating that sodium formate affects nitrogenase activity. Relationship comparison between nitrogenase activity and hydrogen production of the wild type in the dark showed that hydrogen produced by the wild type in the presence of formate in the dark was not catalyzed by nitrogenase, but probably by the formate-linked hydrogenase; however, the same result was not obtained in R. rubrum UR804, probably because nitrogenase inactivation affects formate-linked hydrogenase activity.

Figure 4 Formate-linked hydrogenase activities (a) and nitrogenase activities (b) of R. rubrum wild type and nifHanfDG mutant in the presence/absence of sodium formate with/without light. L, In the light; D, in the dark; F, in the presence of formate.

3 Discussion This study demonstrated that there are at least three enzymes involved in hydrogen evolution: Mo-nitrogenase, Fe-only nitrogenase and the third enzyme. Each enzyme makes different contributions to hydrogen evolution, and Mo-nitrogenase is the key enzyme involved in hydrogen evolution under the artificial illumination condition. It indicates that the increase of Mo-nitrogenase activity would effectively improve hydrogen photoproduction. Fe-nitrogenase has been reported as the key enzyme involved in hydrogen production [14,24]. Rhodobacter capsulatusnif HDKhupL double mutant produces a larger amount of hydrogen than the R. capsulatus wild type does [24]; however, R. rubrum nifHhupL mutant produced hydrogen only 7.4% and 6.4% that of the wild type and R. rubrum hupL mutant, respectively, probably due to the weak expression of Fenitrogenase. In spite of the low hydrogen yield, Fe-nitrogenase still plays an important role in hydrogen evolution. This opinion was further evidenced by the study of nitrogenase activities and hydrogen yields of R. rubrum nifH mutant and nifHanfDG mutant (Figures 2 and 3). Both nitrogenase activity and hydrogen yield of nifH mutant are higher than those of nifHanfDG mutant, indicating the existence of Fe-nitrogenase in hydrogen metabolic pathway and its contribution to hydrogen yield. Capacity to produce hydrogen of R. rubrum nifHanf DG mutant indicates that at least another pathway is involved in hydrogen evolution besides Mo- and Fe-nitrogenase. The simultaneous presence of multiple enzymes involved in hydrogen production under the artificial illumination condition complicates investigation of the third enzyme. Three hydrogenase activities involved in hydrogen production have been identified in R. rubrum including CO-induced hydrogenase, formate-linked hydrogenase and Fe-only hydrogenase [8,16,25]. Hydrogen produced by R. rubrum deficient of both Mo-nitrogenase and Fe-nitrogenase is not from either CO-induced hydrogenase which functions only in the presence of CO or Fe-only hydrogenase which functions only in the presence of pyruvate as the electron donor [25]. Even though we predict that the third enzyme involved in hydrogen evolution would be formate-linked hydrogenase, the result seems not to support this opinion. Low hydrogen yield of R. rubrum nifHanfDG mutant increases the difficulty to analyze the third pathway. Triplicate mutant deficient of Mo-nitrogenase, Fe-nitrogenase and uptake hydrogenase has been tried to be constructed to improve hydrogen yield of the third pathway, however, we failed. Thus, more data are needed to identify the key enzyme in the third hydrogen production pathway. Once the enzyme in the third pathway would be identified, the increase of its activity would mean the improvement of hydrogen photoproduction of R. rubrum. In addition, uptake hydrogenase functions as consump-

ZHU RuiYan, et al. Chinese Sci Bull

tion of hydrogen evolved by the nitrogenase systems or other pathways. Inactivation of the uptake hydrogenase in R. rubrum leads to increased nitrogenase-dependent hydrogen photoproduction [10,23]. Kern had described that R. rubrum hup– strain increased nitrogenase-dependent hydrogen photoproduction by 200% over the period of 28 d [10], however, R. rubrum UR801 in this study only increased by 15% due to different cultivation conditions. Increased light intensity (under saturation light intensity) could improve hydrogen yield of R. rubrum hupL deletion mutant rather than that of the wild type (unpublished data); therefore, hydrogen yield of uptake hydrogenase deletion mutant with low light intensity is lower than that with saturation light intensity. In conclusion, at least 3 enzymes are involved in hydrogen evolution under the artificial illumination: Mo-nitrogenase, Fe-nitrogenase, the third unknown enzyme; and uptake hydrogenase is involved in hydrogen consumption.

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This work was supported by the National High Technology Research and Development Program of China (Grant No. 2006AA05z108).

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