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Nitrogen and sulfur metabolisms of Pseudomonas sp. C27 under mixotrophic growth condition
Bioresource Technology 293 (2019) 122169
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Short Communication
Nitrogen and sulfur metabolisms of Pseudomonas sp. C27 under mixotrophic growth condition Hongliang Guoa, Chuan Chenb, Duu-Jong Leec,d,e,f,
T
⁎
a
College of Food Engineering, Harbin University of Commerce, Harbin 150076, China State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, China c Department of Chemical Engineering, National Taiwan University, Taipei 10617, Taiwan d Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei 10607, Taiwan e College of Technology and Engineering, National Taiwan Normal University, Taipei 10610, Taiwan f College of Engineering, Tunghai University, Taichung 40070, Taiwan b
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: Denitrification Ammonia cycle Metabolism Pseudomonas sp. C27
Pseudomonas sp. C27 is a facultative autotrophic bacterium that can grow mixotrophically to undergo denitrifying sulfide removal (DSR) reactions with both organic matters and sulfide as electron donors. A detailed understanding of how the C27 strain simultaneously removes nitrogen, sulfur and carbon from water is critical for optimal DSR process design and implementation. This study is the first to reveal the pathways of nitrogen and sulfur metabolisms, identifying a total of 47 proteins that are related to the nitrogen metabolism and seven proteins to the sulfur metabolism of strain C27 using iTRAQ and LC-MS/MS techniques. The proposed pathway of nitrogen metabolism for strain C27 from external nitrate to nitrogen gas and phosphate with a coupled ammonia cycle is based on the identified proteins, and suggests that nitrate was not essential for nitrogen metabolism and could be replaced by nitrite as the sole nitrogen source for C27.
1. Introduction
and carbon from water by the so-called denitrifying sulfide removal (DSR) process was originally proposed to be carried out with synergetic collaboration between autotrophic and heterotrophic denitrifiers (Chen et al., 2008). Reactor breakdown is expected during long-term operation because the growth rates of heterotrophic and autotrophic
Nitrogen and sulfur wastes in industrial wastewaters must be adequately treated before being safely disposed of (Sun et al., 2019; Zhang et al., 2018). The simultaneous biological removal of sulfur, nitrogen
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Corresponding author at: Department of Chemical Engineering, National Taiwan University, Taipei 10617, Taiwan. E-mail address: [email protected] (D.-J. Lee).
https://doi.org/10.1016/j.biortech.2019.122169 Received 18 August 2019; Received in revised form 17 September 2019; Accepted 18 September 2019 Available online 20 September 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
Accession
gi|514413035 gi|431926551 gi|397687170 gi|426407035 gi|339492866 gi|330809998 gi|472327025 gi|152987611 gi|472326021 gi|330809997 gi|472327024 gi|392422508 gi|472326022 gi|146283849 gi|397685765 gi|431926008 gi|386022204 gi|397685763 gi|146283847 gi|146283839 gi|152986459 gi|330808901 gi|339495599 gi|397685739 gi|116051410 gi|104782449 gi|167036087 gi|116051794 gi|170723354 gi|28868657 gi|386057961 gi|512616562 gi|333901906 gi|170723670 gi|146281320 gi|397688175 gi|392419887 gi|26991407 gi|472327387 gi|28872106 gi|66045921 gi|146305623 gi|146283850 gi|472326202 gi|152985575 gi|397687174 gi|146281278 gi|26988726 gi|431928758 gi|378949840 gi|146282584 gi|512686500 gi|170722737 gi|71733680
NO.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54
MFS transporter nitrate ABC transporter substrate-binding protein nitrate transporter nitrate transport system permease respiratory nitrate reductase alpha chain nitrate reductase, respiratory nitrate reductase 1 subunit alpha nitrate reductase A subunit alpha nitrate reductase subunit alpha nitrate reductase A subunit alpha nitrate reductase, respiratory nitrate reductase 2 subunit beta nitrate reductase A subunit beta nitrate reductase A subunit beta nitrate reductase A subunit beta cytochrome cd1 nitrite reductase cytochrome cd1 nitrite reductase cytochrome c551 denitrification system component cytochrome c-552 denitrification system component cytochrome c-552 denitrification system component cytochrome c-552 nitric-oxide reductase subunit C Cytochrome P450 nitrous-oxide reductase nitrous-oxide reductase nitrous-oxide reductase nitrous-oxide reductase ferredoxin-nitrite reductase glutamine synthetase, type I GMP synthase GMP synthase GMP synthase NAD-dependent glutamate dehydrogenase glutamate dehydrogenase glutamate dehydrogenase glutamate dehydrogenase glutamate dehydrogenase carbamoyl phosphate synthase large subunit carbamoyl phosphate synthase large subunit carbamoyl phosphate synthase small subunit carbamoyl phosphate synthase small subunit carbamoyltransferase family protein ornithine carbamoyltransferase carbamoyltransferase denitrification regulatory protein nirQ transcriptional regulator Anr transcriptional regulator Dnr two-component response regulator NarL two-component sensor NarX O-succinylhomoserine sulfhydrylase O-acetylhomoserine sulfhydrolase O-acetylhomoserine sulfhydrylase sulfite reductase NADPH-sulfite reductase hemoprotein beta-component cbb3-type cytochrome c oxidase subunit II cytochrome c oxidase, cbb3-type subunit III
Description
Table 1 Proteins related to nitrogen metabolism of strain C27.
Pseudomonas aeruginosa RP73 Pseudomonas stutzeri RCH2 Pseudomonas stutzeri DSM 10,701 Pseudomonas sp. UW4 Pseudomonas stutzeri ATCC 17,588 Pseudomonas brassicacearum subsp. NFM421 Pseudomonas denitrificans ATCC 13,867 Pseudomonas aeruginosa PA7 Pseudomonas denitrificans ATCC 13,867 Pseudomonas brassicacearum subsp. NFM421 Pseudomonas denitrificans ATCC 13,867 Pseudomonas stutzeri CCUG 29,243 Pseudomonas denitrificans ATCC 13,867 Pseudomonas stutzeri A1501 Pseudomonas stutzeri DSM 10,701 Pseudomonas stutzeri RCH2 Pseudomonas stutzeri DSM 4166 Pseudomonas stutzeri DSM 10701 Pseudomonas stutzeri A1501 Pseudomonas stutzeri A1501 Pseudomonas aeruginosa PA7 Pseudomonas brassicacearum subsp. NFM421 Pseudomonas stutzeri ATCC 17588 Pseudomonas stutzeri DSM 10701 Pseudomonas aeruginosa UCBPP-PA14 Pseudomonas entomophila L48 Pseudomonas putida GB-1 Pseudomonas aeruginosa UCBPP-PA14 Pseudomonas putida W619 Pseudomonas syringae pv. tomato str. DC3000 Pseudomonas aeruginosa M18 Pseudomonas resinovorans NBRC 106553 Pseudomonas fulva 12-X Pseudomonas putida W619 Pseudomonas stutzeri A1501 Pseudomonas stutzeri DSM 10701 Pseudomonas stutzeri CCUG 29243 Pseudomonas putida KT2440 Pseudomonas denitrificans ATCC 13867 Pseudomonas syringae pv. tomato str. DC3000 Pseudomonas syringae pv. syringae B728a Pseudomonas mendocina ymp Pseudomonas stutzeri A1501 Pseudomonas denitrificans ATCC 13867 Pseudomonas aeruginosa PA7 Pseudomonas stutzeri DSM 10701 Pseudomonas stutzeri A1501 Pseudomonas putida KT2440 Pseudomonas stutzeri RCH2 Pseudomonas fluorescens F113 Pseudomonas stutzeri A1501 Pseudomonas putida NBRC 14164 Pseudomonas putida W619 Pseudomonas syringae pv. phaseolicola 1448A
Species 28 20 133 21 3973 3624 2322 2023 1547 1246 1691 862 529 2010 1945 26 178 127 34 627 14 637 492 360 183 55 1015 225 138 150 21 2076 1540 1481 1428 1600 1439 325 81 216 38 421 1119 74 20 41 45 24 39 24 58 55 59 25
Score 44.4 41.3 58.9 34.5 161.4 161.1 162.8 159.4 159.1 68.6 70.0 69.1 68.8 77.4 76.5 14.3 36.2 39.4 33.5 19.0 88.8 86.6 85.3 84.9 83.4 69.1 62.0 65.9 65.8 66.4 205.1 54.5 54.5 55.7 51.1 136.3 137.2 45.3 45.4 77.5 42.9 80.1 28.4 30.4 28.5 27.9 77.8 48.0 50.1 48.3 69.4 68.6 26.9 40.1
Mass(kDa) 1.4 2.2 4.6 3.1 14.5 12.1 15.9 12.5 4.9 11.5 10 17.6 7.7 9.3 12.2 15.4 3.1 3.2 3.3 12.3 3.3 6.5 6.9 4.4 4.1 1.6 12.6 6.3 5 6.1 0.7 21.2 14.6 15.9 18.2 20.7 19.9 4.2 2.6 7.2 2.4 10.4 21.7 10.2 2.6 4.1 1.2 1.7 3.8 1.7 2.2 1.6 10.9 5.5
Cov(%) 1 1 5 1 70 42 33 15 27 30 15 2 4 33 9 1 8 5 2 43 1 16 5 3 27 1 52 4 3 6 1 13 20 12 2 11 3 14 5 3 2 6 11 4 2 2 1 1 6 1 1 1 3 2
Unique Spectrum 1 1 2 1 1 1 4 2 2 1 2 1 2 2 2 1 1 1 1 3 1 1 2 1 1 1 5 1 1 1 1 2 2 1 1 2 1 2 1 1 1 2 1 3 1 1 1 1 2 1 1 1 2 1
Unique Peptide
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(Matrix Science, London, UK) against database containing Pseudomonas sequences. Other details for protein identification were also available in Guo et al. (2014).
denitrifiers in a single reactor differ greatly (Show et al., 2013; Chen et al., 2018). Proteomic information is critical to understand the reaction networks of a complicated biological system (Guo et al., 2018a,b). Facultative autotrophic bacteria (FAB) can undergo mixotrophic growth with the simultaneous removal of sulfur, nitrogen and carbon from water (Lee et al., 2013; Sorokin et al., 2003). The growth rates of most FAB are low, making their field applications impractical. Chen et al. (2013) Facultative autotrophic bacteria (FAB) can undergo mixotrophic growth with the simultaneous removal of sulfur, nitrogen and carbon from water (Lee et al., 2013; Sorokin et al., 2003). Chen et al. (2013) isolated the first highly efficient FAB, the Pseudomonas sp. C27, from an anaerobic DSR reactor that could support both autotrophic and heterotrophic denitrification reactions so that balanced growth of autotrophic and heterotrophic denitrifiers in that reacted was unlimited. They performed proteomic studies of C27 with mixotrophic growth, anaerobic and micro-aeration growth, and growth with various C/N ratios have been conducted. Based on these works, they proposed pathways of coupled carbon, nitrogen and sulfide cycles and a molecular mechanism of denitrifying sulfide removal for C27. The aforementioned studies loosely described the function of C27, which is a typical FAB. However, the key cycles, which are the nitrogen and sulfur pathways, are regarded as a black box since those works revealed only the overall reactions (NO3− → NO2− → N2; S2− → S0). This work supplements those studies by providing a complete quantitative proteomics analysis of the mixotrophic growth of C27, to provide fundamental reaction frameworks for nitrogen and sulfur cycles.
3. Results and discussion 3.1. Proteins involved in nitrogen/sulfur metabolism of strain C27 DSR reactions were performed with noted concentrations of sulfide as an indicator (Supplementary Materials). Clearly, C27 underwent the DSR reactions and the enzymes in the biological cells were representative of that for a typical FAB. A total of 47 proteins that were involved in the nitrogen metabolism of C27 were identified by iTRAQ and LC-MS/MS and provided in Table 1; they include transport proteins (Nos. 1–4), denitrifying proteins (Nos. 5–15, 20 and 22–25), cytochrome c551, C552 and P450 (Nos. 16–19 and 21), ammonia cycle proteins (Nos. 26, 27, 28–35), phosphate-converting proteins (Nos. 36–39 and 40–42) and regulatory proteins (Nos. 43–47). A total of seven proteins that were involved in sulfur metabolisms of C27 were identified and provided in Table 1; they include sulfhydrylase (Nos. 48–50), sulfite reductase (Nos. 51 and 52), and cytochrome c oxidase (Nos. 53 and 54). 3.2. Functions of identified proteins Four transport proteins of C27 were identified. MFS transporters (No. 1 in Table 1) constitute one of the largest groups of secondary active transporters, and selectively transport a wide range of substrates across biomembranes (Yan, 2013). ABC transporter (No. 2) couples the hydrolysis of ATP to solute transport across biological membranes, and is critical for nitrate respiration (Wanner et al., 1999). Nitrate transporter (No. 3) and nitrate transport system permease (No. 4) are responsible for nitrate transporting. Nitrate permease can also transport cyanate. Two forms of nitrate reductases of the strain C27 were identified – the alpha subunit (Nos. 5–9) and the beta subunit (Nos. 10–13). Nitrate reductase is a membrane-bound anaerobic electron transport enzyme, which comprises the alpha subunit and the beta subunit (Edwards et al., 1983). The alpha subunit is the actual site of nitrate reduction and the beta chain is an electron transfer unit. Identified cytochrome cd1 nitrite reductases (Nos. 14–15) are homodimeric proteins that contain one ctype heme and one d1-type heme per subunit. The c-type heme is an electron entry site, which receives electrons from c-type cytochromes, before d1-type heme uses those electrons to reduce nitrite. Therefore, identified cytochrome c551 (No. 16) and C552 (Nos. 17–19) donate electrons to cytochrome cd1 nitrite reductases (cd1NiR). C552 is a physiological redox partner of cd1NiR, and can regulate intermolecular electron transfer and increase the catalytic activity of cd1NiR. Nitric oxide reductase (No. 20) is an integral membrane component of the anaerobic respiratory chain that catalyzes the reduction of NO to N2O (Zumft et al., 2010). Cytochrome P450 (No. 21) was identified in our earlier research. Cytochrome P450 also exhibits nitric oxide reductase activity, and can transform nitric oxide to nitrous oxide using NADH as the sole electron donor (Nakahara et al., 1993). Nitrous oxide reductase (Nos. 22–25) catalyzes the final step of the denitrification pathway (from N2O to N2) (Pauleta et al., 2013). Ferredoxin-nitrite reductase (No. 26) catalyzes the reduction of nitrite to ammonia. Identified glutamine synthetase (No. 27), involved in nitrogen metabolism via ammonium assimilation, catalyzes the biosynthesis of glutamine from ammonia. GMP synthase (Nos. 28–30) catalyzes the conversion of L-glutamine to L-glutamate. Glutamate dehydrogenase (Nos. 31–35) catalyzes the oxidative deamination of Lglutamate to 2-oxoglutarate and ammonia, and has an important role at the intersection of the carbon and nitrogen metabolic pathways (Wakamatsu et al., 2013).
2. Materials and methods 2.1. Bacterial strain and protein extraction Pseudomonas sp. C27 was isolated from an expanded granular suspended bed reactor in which DSR reactions had been performed for more than half of a year (Chen et al., 2013). This strain is a Gramnegative rod-shaped bacterium, whose 16S rDNA sequence was deposited in GenBank under accession number GQ241351. In this investigation, the C27 strain was activated and cultivated in an anaerobic bottle at 30 °C, which was filled with a medium of 7.5 mM KNO3 or KNO2, 0.64 g/L NaAC·3H2O, 1.0 g/L NH4Cl, 0.5 g/L of NaHCO3, 1.8 g/L of KH2PO4, 3.0 g/L of Na2HPO4·12H2O, 0.5 g/L of MgSO4·7H2O, 0.39 g/ L of Na2S and 0.1 g trace elements at pH 8 (50 g/L of EDTA; 0.14 g/L of CuCl2·2H2O; 0.5 g/L of (NH4)6Mo7O24·4H2O; 0.5 g/L of CoCl2·6H2O; 1.0 g/L ZnCl2; 2.5 g/L of MnCl2·2H2O; 3.6 g/L of FeCl2·4H2O; 7.3 g/L of CaCl2·2H2O). After 18 h of cultivation, as described in Sec 2.1, the bacterial cells were collected by centrifugation at 10,000g for 10 min at 4 °C, and then washed using pH 7.4 PBS buffer. The cells were ground to a powder in liquid N2, and then extracted using Lysis buffer at pH 8.5. Further details can be found in Guo et al. (2014). 2.2. iTRAQ labeling and LC-MS/MS analysis The Trypsin Gold (Promega, Madison, WI, USA) was applied to digest protein collected in Sec. 2.1 at protein to trypsin ratio of 30 at 37 °C for 16 h. The digested peptides were dried by vacuum centrifugation and were processed according to the manufacture for 8-plex iTRAQ reagent (Applied Biosystems, Foster City, CA, USA). The labeled peptide mixtures were then dried again by vacuum centrifugation. The labeled proteins were resuspended in 5% CAN with 0.1% FA and was centrifuged at 20,000 × g for 10 min. The 10 μL of so-yielded supernatant was loaded using an auto-sampler to a C18 trap column installed in LC-20AD nanoHPLC (Shimadzu, Kyoto, Japan) with the elution condition stated in Guo et al. (2014). The MS data were processed using Proteome Discoverer software (Version 1.2.0.208) (Thermo Scientific, CA, USA) to generate peak list. The identification of proteins was conducted using Mascot search engine; version 2.3.02 3
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Fig. 1. The pathway of carbon, nitrogen and sulfur metabolism for Pseudomonas sp. C27. No. 1-54 corresponded to proteins in Table 1.
3.3. Nitrogen/sulfur metabolism of strain C27
Carbamoyl phosphate synthase (CPS) comprises two polypeptide chains, called the large subunit (Nos. 36, 37) and the small subunit (Nos. 38, 39), and it plays an important role in both arginine and pyrimidine biosynthesis by providing carbamoyl phosphate. CPS can catalyze the production of carbamoyl phosphate from L-glutamine, which can be replaced by ammonia as a nitrogen donor. Carbamoyltransferase (Nos. 40–42) catalyzes the conversion of carbamoyl phosphate and L-ornithine to phosphate and L-citrulline. Denitrification regulatory protein nirQ (No. 43) activates nitrite reductase and nitric oxide reductase. Transcriptional regulator Anr (No. 44) and Dnr (No. 45) are required for the denitrification of Pseudomonas, and act together as a regulator of protein nirQ (Arai et al., 1997). Transcriptional regulator Anr regulates the transcription of the Dnr gene, arginine deiminase activity and the production of cyanide by Pseudomonas (Arai et al., 1997). Dnr is a heme-binding transcription factor that is involved in the regulation of nitric-oxide reductase and nitrite reductase in Pseudomonas. Two-component response regulator NarL (No. 46) mediates the synthesis of nitrate reductase in the presence of nitrate. Two-component sensor NarX (No. 47) acts as a sensor of nitrate, and transduces the signal of nitrate availability to the NarL protein. NarX activates NarL by phosphorylation in the presence of nitrate, and plays a negative role in controlling NarL activity by dephosphorylation in the absence of nitrate (Noriega et al., 2010). O-succinylhomoserine sulfhydrylase (No. 48 in Table 1) and Oacetylhomoserine sulfhydrylase (Nos. 49 and 50) are sulfide-utilizing enzymes of various enteric bacteria and fungi. The former catalyzes the reaction of sulfide and O-succinyl-L-homoserine to yield L-homocysteine and succinate (Foglino et al., 1995), and the latter catalyzes the reaction of sulfide and O-acetyl-L-homoserine to yield L-homocysteine and acetate (Tran et al., 2011). Sulfite reductase (No. 51) catalyzes the reversible reaction between sulfide and sulfite, and NADPH hemoprotein (No. 52) is the component of the sulfite reductase complex that catalyzes the six -electron reduction of sulfite to sulfide (Gruez et al., 2000). Cytochrome c oxidase (Nos. 53 and 54) is sulfite oxidase that catalyzes the reaction from sulfite to sulfate (Sugio et al., 2010).
7.5 mM NO3− or NO2− was added as a nitrogen source for C27. Fig. 1 displays a proposed pathway for DSR reactions that is based on the protein functions that were identified in Sec. 3.2 and earlier works. First, extracellular nitrate was transported into C27 cells by transport proteins (Nos. 1–4). Then, intracellular nitrate was converted to nitrite, catalyzed by nitrate reductases (Nos. 5–13). The synthesis of nitrate reductase was regulated by the two-component response regulator NarL (No. 46), which was itself regulated by the two-component sensor NarX (No. 47). The generated nitrite was converted to nitric oxide, nitrous oxide and nitrogen gas in that order, catalyzed by cytochrome cd1 nitrite reductases (Nos. 14–15), nitric oxide reductase (No. 20) and nitrous oxide reductases (Nos. 22–25), respectively. Cytochrome cd1 nitrite reductases were regulated by electron donor cytochrome C551 (No. 16) and redox partner denitrification system component cytochrome C552 (Nos. 17–19). Cytochrome cd1 nitrite reductase and nitric oxide reductase were regulated by the denitrification regulatory protein nirQ (No. 43). Protein nirQ, which was in turn regulated by transcriptional regulator Dnr (No. 45), which was regulated by transcriptional regulator Anr (No. 44). As well as being involved in the denitrifying pathway, the produced nitrite was also converted to ammonia, catalyzed by ferredoxin-nitrite reductase (No. 26). Catalyzed by glutamine synthetase (No. 27), GMP synthase (Nos. 28–30) and glutamate dehydrogenase (Nos. 31–35), the cycle of ammonia can be realized by produced intermediate L-glutamine and L-glutamate. Ammonia was also converted to phosphate via intermediate carbamoyl phosphate, catalyzed by carbamoyl phosphate synthase (Nos. 36–39), HERE and carbamoyltransferase (Nos. 40–42). The added S2− ions can be converted by C27 via four pathways. Sulfide can be converted to elemental sulfur via intermediate L-cysteine, cysteine synthase and cysteine desulfurase, or to elemental sulfur directly by sulfide oxidase. Sulfide can react with O-succinyl-Lhomoserine and O-acetyl-L-homoserine to form succinate and acetate, respectively, catalyzed by O-succinylhomoserine sulfhydrylase (No. 48) 4
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and O-acetylhomoserine sulfhydrylase (Nos. 49 and 50). Sulfide also can be converted to sulfite and then converted to sulfate, catalyzed by sulfite reductase (Nos. 51 and 52) and cytochrome c oxidase (Nos. 53 and 54). Extracellular nitrate was transported across the cytomembrane, and converted to nitrite, and then the nitrogen metabolism of C27 was activated (Jia and Cole, 2005). In the first 2 h of the reaction, 53.8 and 46.5 mg/L sulfide were oxidized in the nitrite and nitrate medium, respectively (Supplementary Materials). Restated, the transport of extracellular nitrate to intracellular nitrate and its subsequent conversion to intracellular nitrite took some time, delayed the reaction about 14% conversion rate. Based on these observations, the strain C27 can be applied to a nitrate-containing or a nitrite-containing medium for denitrification, and since nitrite is a better nitrogen source for C27 than is nitrate, this FAB strain has a niche in competition among various nitrite processes, including partial nitrification, comammox and anammox processes. Further metabolic flux analyses can be performed to verify these claims. The reported data herein can be used as a basis to synthesize biological processes combined with other cycles such as P or Fe cycles.
Pseudomonas aeruginosa. Mol. Microbiol. 25, 1141–1148. Chen, C., Ren, N.Q., Wang, A.J., Yu, Z.G., Lee, D.J., 2008. Simultaneous biological removal of sulfur, nitrogen and carbon using EGSB reactor. Appl. Microbiol. Biotechnol. 78, 1057–1063. Chen, C., Ho, K.L., Liu, F.C., Ho, M., Wang, A.J., Ren, N.Q., Lee, D.J., 2013. Autotrophic and heterotrophic denitrification by a newly isolated strain Pseudomonas sp. C27. Bioresour. Technol. 145, 351–356. Chen, C., Shao, B., Zhang, R.C., Xu, X.J., Zhou, X., Yuan, Y., Ren, N.Q., Lee, D.J., 2018. Mitigating adverse impacts of varying sulfide/nitrate ratios on denitrifying sulfide removal process performance. Bioresour. Technol. 267, 782–788. Edwards, E.S., Rondeau, S.S., Demoss, J.A., 1983. chlC (nar) operon of Escherichia coli includes structural genes for alpha and beta subunits of nitrate reductase. J. Bacteriol. 153, 1513–1520. Foglino, M., Borne, F., Bally, M., Ball, M., Patte, J.C., 1995. A direct sulfhydrylation pathway is used for methionine biosynthesis in Pseudomonas aeruginosa. Microbiolgy 141, 431–439. Gruez, A., Pignol, D., Zeghouf, M., Coves, J., Fontecave, M., Ferrer, J.L., FontecillaCamps, J.C., 2000. Four crystal structures of the 60 kDa flavoprotein monomer of the sulfite reductase indicate a disordered flavodoxin-like module. J. Mol. Biol. 299, 199–212. Guo, H.L., Chen, C., Lee, D.J., Wang, A.J., Ren, N.Q., 2014. Coupled carbon, sulfur and nitrogen cycles of mixotrophic growth of Pseudomonas sp. C27 under denitrifying sulfide removal conditions. Bioresour. Technol. 171, 120–126. Guo, H.L., Chang, Y.J., Lee, D.J., 2018a. Enzymatic saccharification of lignocellulosic biorefinery: research focuses. Bioresour. Technol. 252, 198–215. Guo, H.L., Wang, X.D., Lee, D.J., 2018b. Proteomic researches for lignocellulose-degrading enzymes: a mini-review. Bioresour. Technol. 265, 532–541. Jia, W., Cole, J.A., 2005. Nitrate and nitrite transport in Escherichia coli. Biochem. Soc. Trans. 33, 159–161. Lee, D.J., Pan, X.L., Wang, A.J., Ho, K.L., 2013. Facultative autotrophic denitrifiers in denitrifying sulfide removal granules. Bioresour. Technol. 132, 356–360. Nakahara, K., Tanimoto, T., Hatano, K., Usuda, K., Shoun, H., 1993. Cytochrome P-450 55A1 (P-450dNIR) acts as nitric oxide reductase employing NADH as the direct electron donor. J. Biol. Chem. 268, 8350–8355. Noriega, C.E., Lin, H.Y., Chen, L.L., Williams, S.B., Stewart, V., 2010. Asymmetric crossregulation between the nitrate-responsive NarX-NarL and NarQ-NarP two-component regulatory systems from Escherichia coli K-12. Mol. Microbiol. 75, 394–412. Pauleta, S.R., Dell’Acqua, S., Moura, I., 2013. Nitrous oxide reductase. Coord. Chem. Rev. 257, 332–349. Sorokin, D.Y., 2003. Oxidation of inorganic sulfur compounds by obligatively organotrophic bacteria. Microbiology 72, 641–653. Show, K.Y., Lee, D.J., Pan, X.L., 2013. Simultaneous biological removal of nitrogen-sulfurcarbon: recent advances and challenges. Biotechnol. Adv. 31, 409–420. Sugio, T., Ako, A., Takeuchi, F., 2010. Sulfite oxidation catalyzed by aa3-Type cytochrome c oxidase in Acidithiobacillus ferrooxidans. Biosci. Biotechnol. Biochem. 74, 2242–2247. Sun, C., Yuan, J., Xu, H., Huang, S., Wen, X., Tong, N., Zhang, Y., 2019. Simultaneous removal of nitric oxide and sulfur dioxide in a biofilter under micro-oxygen thermophilic conditions; Removal performance, competitive relationship and bacterial community structure. Bioresour. Technol. 290, 121768. Tran, T.H., Krishnamoorthy, K., Begley, T.P., Ealick, S.E., 2011. A novel mechanism of sulfur transfer catalyzed by O-acetylhomoserine sulfhydrylase in the methioninebiosynthetic pathway of Wolinella succinogenes. Biol. Crystallogr. 67, 831–838. Wanner, C., Soppa, J., 1999. Genetic identification of three ABC transporters as essential elements for nitrate respiration in Haloferax volcanii. Genetics 152, 1417–1428. Wakamatsu, T., Higashi, C., Ohmori, T., Doi, K., Ohshima, T., 2013. Biochemical characterization of two glutamate dehydrogenases with different cofactor specificities from a hyperthermophilic archaeon Pyrobaculum calidifontis. Extremophiles 17, 379─389. Yan, H.C., Huang, W.Y., Yan, C.Y., 2013. Structure and mechanism of a nitrate transporter. Cell Rep. 3, 716–723. Zhang, R.C., Xu, X.J., Chen, C., Xing, D.F., Shao, B., Liu, W.Z., Wang, A.J., Lee, D.J., Ren, N.Q., 2018. Interactions of functional bacteria and their contributions to the performance in integrated autotrophic and heterotrophic denitrification. Water Res. 143, 355–366. Zumft, W.G., Braun, C., Cuypers, H., 2010. Nitric-oxide reductase from PseudomonasStutzeri – primary structure and gene organization of a novel bacterial cytochrome bc complex. FEBS J. 219, 481–490.
4. Conclusion By a quantitative proteomic analysis, 47 proteins that are involved in the nitrogen metabolism and seven proteins that are involved in the sulfur metabolism of C27 in mixotrophic growth with nitrate or nitrite as a sole nitrogen source were identified using iTRAQ and LC-MS/MS. The proteins, including transport proteins, denitrifying proteins, denitrifying protein regulators, ammonia cycle proteins, phosphate-converting proteins and others, were identified for the first time. The nitrogen metabolism pathway from external nitrate to nitrogen gas and phosphate with coupled ammonia cycles and the sulfur metabolism pathway of strain C27 were proposed based on the identified proteins. Acknowledgments The authors gratefully acknowledge funding supported by University Nursing Program for Young Scholars with Creative Talents in Heilongjiang Province (UNPYSCT-2018137) and Scientific Research Project of Harbin University of Commerce (No. 18XN044), Open Project of State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology (No. HC201710), and Harbin Applied Technology Research and Development Project (2017RAXXJ025). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biortech.2019.122169. References Arai, H., Kodama, T., Igarashi, Y., 1997. Cascade regulation of the two CRP/FNR-related transcriptional regulators (ANR and DNR) and the denitrification enzymes in
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Nitrogen and sulfur metabolisms of Pseudomonas sp. C27 under mixotrophic growth condition Hongliang Guoa, Chuan Chenb, Duu-Jong Leec,d,e,f,
T
⁎
a
College of Food Engineering, Harbin University of Commerce, Harbin 150076, China State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, China c Department of Chemical Engineering, National Taiwan University, Taipei 10617, Taiwan d Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei 10607, Taiwan e College of Technology and Engineering, National Taiwan Normal University, Taipei 10610, Taiwan f College of Engineering, Tunghai University, Taichung 40070, Taiwan b
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: Denitrification Ammonia cycle Metabolism Pseudomonas sp. C27
Pseudomonas sp. C27 is a facultative autotrophic bacterium that can grow mixotrophically to undergo denitrifying sulfide removal (DSR) reactions with both organic matters and sulfide as electron donors. A detailed understanding of how the C27 strain simultaneously removes nitrogen, sulfur and carbon from water is critical for optimal DSR process design and implementation. This study is the first to reveal the pathways of nitrogen and sulfur metabolisms, identifying a total of 47 proteins that are related to the nitrogen metabolism and seven proteins to the sulfur metabolism of strain C27 using iTRAQ and LC-MS/MS techniques. The proposed pathway of nitrogen metabolism for strain C27 from external nitrate to nitrogen gas and phosphate with a coupled ammonia cycle is based on the identified proteins, and suggests that nitrate was not essential for nitrogen metabolism and could be replaced by nitrite as the sole nitrogen source for C27.
1. Introduction
and carbon from water by the so-called denitrifying sulfide removal (DSR) process was originally proposed to be carried out with synergetic collaboration between autotrophic and heterotrophic denitrifiers (Chen et al., 2008). Reactor breakdown is expected during long-term operation because the growth rates of heterotrophic and autotrophic
Nitrogen and sulfur wastes in industrial wastewaters must be adequately treated before being safely disposed of (Sun et al., 2019; Zhang et al., 2018). The simultaneous biological removal of sulfur, nitrogen
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Corresponding author at: Department of Chemical Engineering, National Taiwan University, Taipei 10617, Taiwan. E-mail address: [email protected] (D.-J. Lee).
https://doi.org/10.1016/j.biortech.2019.122169 Received 18 August 2019; Received in revised form 17 September 2019; Accepted 18 September 2019 Available online 20 September 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
Accession
gi|514413035 gi|431926551 gi|397687170 gi|426407035 gi|339492866 gi|330809998 gi|472327025 gi|152987611 gi|472326021 gi|330809997 gi|472327024 gi|392422508 gi|472326022 gi|146283849 gi|397685765 gi|431926008 gi|386022204 gi|397685763 gi|146283847 gi|146283839 gi|152986459 gi|330808901 gi|339495599 gi|397685739 gi|116051410 gi|104782449 gi|167036087 gi|116051794 gi|170723354 gi|28868657 gi|386057961 gi|512616562 gi|333901906 gi|170723670 gi|146281320 gi|397688175 gi|392419887 gi|26991407 gi|472327387 gi|28872106 gi|66045921 gi|146305623 gi|146283850 gi|472326202 gi|152985575 gi|397687174 gi|146281278 gi|26988726 gi|431928758 gi|378949840 gi|146282584 gi|512686500 gi|170722737 gi|71733680
NO.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54
MFS transporter nitrate ABC transporter substrate-binding protein nitrate transporter nitrate transport system permease respiratory nitrate reductase alpha chain nitrate reductase, respiratory nitrate reductase 1 subunit alpha nitrate reductase A subunit alpha nitrate reductase subunit alpha nitrate reductase A subunit alpha nitrate reductase, respiratory nitrate reductase 2 subunit beta nitrate reductase A subunit beta nitrate reductase A subunit beta nitrate reductase A subunit beta cytochrome cd1 nitrite reductase cytochrome cd1 nitrite reductase cytochrome c551 denitrification system component cytochrome c-552 denitrification system component cytochrome c-552 denitrification system component cytochrome c-552 nitric-oxide reductase subunit C Cytochrome P450 nitrous-oxide reductase nitrous-oxide reductase nitrous-oxide reductase nitrous-oxide reductase ferredoxin-nitrite reductase glutamine synthetase, type I GMP synthase GMP synthase GMP synthase NAD-dependent glutamate dehydrogenase glutamate dehydrogenase glutamate dehydrogenase glutamate dehydrogenase glutamate dehydrogenase carbamoyl phosphate synthase large subunit carbamoyl phosphate synthase large subunit carbamoyl phosphate synthase small subunit carbamoyl phosphate synthase small subunit carbamoyltransferase family protein ornithine carbamoyltransferase carbamoyltransferase denitrification regulatory protein nirQ transcriptional regulator Anr transcriptional regulator Dnr two-component response regulator NarL two-component sensor NarX O-succinylhomoserine sulfhydrylase O-acetylhomoserine sulfhydrolase O-acetylhomoserine sulfhydrylase sulfite reductase NADPH-sulfite reductase hemoprotein beta-component cbb3-type cytochrome c oxidase subunit II cytochrome c oxidase, cbb3-type subunit III
Description
Table 1 Proteins related to nitrogen metabolism of strain C27.
Pseudomonas aeruginosa RP73 Pseudomonas stutzeri RCH2 Pseudomonas stutzeri DSM 10,701 Pseudomonas sp. UW4 Pseudomonas stutzeri ATCC 17,588 Pseudomonas brassicacearum subsp. NFM421 Pseudomonas denitrificans ATCC 13,867 Pseudomonas aeruginosa PA7 Pseudomonas denitrificans ATCC 13,867 Pseudomonas brassicacearum subsp. NFM421 Pseudomonas denitrificans ATCC 13,867 Pseudomonas stutzeri CCUG 29,243 Pseudomonas denitrificans ATCC 13,867 Pseudomonas stutzeri A1501 Pseudomonas stutzeri DSM 10,701 Pseudomonas stutzeri RCH2 Pseudomonas stutzeri DSM 4166 Pseudomonas stutzeri DSM 10701 Pseudomonas stutzeri A1501 Pseudomonas stutzeri A1501 Pseudomonas aeruginosa PA7 Pseudomonas brassicacearum subsp. NFM421 Pseudomonas stutzeri ATCC 17588 Pseudomonas stutzeri DSM 10701 Pseudomonas aeruginosa UCBPP-PA14 Pseudomonas entomophila L48 Pseudomonas putida GB-1 Pseudomonas aeruginosa UCBPP-PA14 Pseudomonas putida W619 Pseudomonas syringae pv. tomato str. DC3000 Pseudomonas aeruginosa M18 Pseudomonas resinovorans NBRC 106553 Pseudomonas fulva 12-X Pseudomonas putida W619 Pseudomonas stutzeri A1501 Pseudomonas stutzeri DSM 10701 Pseudomonas stutzeri CCUG 29243 Pseudomonas putida KT2440 Pseudomonas denitrificans ATCC 13867 Pseudomonas syringae pv. tomato str. DC3000 Pseudomonas syringae pv. syringae B728a Pseudomonas mendocina ymp Pseudomonas stutzeri A1501 Pseudomonas denitrificans ATCC 13867 Pseudomonas aeruginosa PA7 Pseudomonas stutzeri DSM 10701 Pseudomonas stutzeri A1501 Pseudomonas putida KT2440 Pseudomonas stutzeri RCH2 Pseudomonas fluorescens F113 Pseudomonas stutzeri A1501 Pseudomonas putida NBRC 14164 Pseudomonas putida W619 Pseudomonas syringae pv. phaseolicola 1448A
Species 28 20 133 21 3973 3624 2322 2023 1547 1246 1691 862 529 2010 1945 26 178 127 34 627 14 637 492 360 183 55 1015 225 138 150 21 2076 1540 1481 1428 1600 1439 325 81 216 38 421 1119 74 20 41 45 24 39 24 58 55 59 25
Score 44.4 41.3 58.9 34.5 161.4 161.1 162.8 159.4 159.1 68.6 70.0 69.1 68.8 77.4 76.5 14.3 36.2 39.4 33.5 19.0 88.8 86.6 85.3 84.9 83.4 69.1 62.0 65.9 65.8 66.4 205.1 54.5 54.5 55.7 51.1 136.3 137.2 45.3 45.4 77.5 42.9 80.1 28.4 30.4 28.5 27.9 77.8 48.0 50.1 48.3 69.4 68.6 26.9 40.1
Mass(kDa) 1.4 2.2 4.6 3.1 14.5 12.1 15.9 12.5 4.9 11.5 10 17.6 7.7 9.3 12.2 15.4 3.1 3.2 3.3 12.3 3.3 6.5 6.9 4.4 4.1 1.6 12.6 6.3 5 6.1 0.7 21.2 14.6 15.9 18.2 20.7 19.9 4.2 2.6 7.2 2.4 10.4 21.7 10.2 2.6 4.1 1.2 1.7 3.8 1.7 2.2 1.6 10.9 5.5
Cov(%) 1 1 5 1 70 42 33 15 27 30 15 2 4 33 9 1 8 5 2 43 1 16 5 3 27 1 52 4 3 6 1 13 20 12 2 11 3 14 5 3 2 6 11 4 2 2 1 1 6 1 1 1 3 2
Unique Spectrum 1 1 2 1 1 1 4 2 2 1 2 1 2 2 2 1 1 1 1 3 1 1 2 1 1 1 5 1 1 1 1 2 2 1 1 2 1 2 1 1 1 2 1 3 1 1 1 1 2 1 1 1 2 1
Unique Peptide
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(Matrix Science, London, UK) against database containing Pseudomonas sequences. Other details for protein identification were also available in Guo et al. (2014).
denitrifiers in a single reactor differ greatly (Show et al., 2013; Chen et al., 2018). Proteomic information is critical to understand the reaction networks of a complicated biological system (Guo et al., 2018a,b). Facultative autotrophic bacteria (FAB) can undergo mixotrophic growth with the simultaneous removal of sulfur, nitrogen and carbon from water (Lee et al., 2013; Sorokin et al., 2003). The growth rates of most FAB are low, making their field applications impractical. Chen et al. (2013) Facultative autotrophic bacteria (FAB) can undergo mixotrophic growth with the simultaneous removal of sulfur, nitrogen and carbon from water (Lee et al., 2013; Sorokin et al., 2003). Chen et al. (2013) isolated the first highly efficient FAB, the Pseudomonas sp. C27, from an anaerobic DSR reactor that could support both autotrophic and heterotrophic denitrification reactions so that balanced growth of autotrophic and heterotrophic denitrifiers in that reacted was unlimited. They performed proteomic studies of C27 with mixotrophic growth, anaerobic and micro-aeration growth, and growth with various C/N ratios have been conducted. Based on these works, they proposed pathways of coupled carbon, nitrogen and sulfide cycles and a molecular mechanism of denitrifying sulfide removal for C27. The aforementioned studies loosely described the function of C27, which is a typical FAB. However, the key cycles, which are the nitrogen and sulfur pathways, are regarded as a black box since those works revealed only the overall reactions (NO3− → NO2− → N2; S2− → S0). This work supplements those studies by providing a complete quantitative proteomics analysis of the mixotrophic growth of C27, to provide fundamental reaction frameworks for nitrogen and sulfur cycles.
3. Results and discussion 3.1. Proteins involved in nitrogen/sulfur metabolism of strain C27 DSR reactions were performed with noted concentrations of sulfide as an indicator (Supplementary Materials). Clearly, C27 underwent the DSR reactions and the enzymes in the biological cells were representative of that for a typical FAB. A total of 47 proteins that were involved in the nitrogen metabolism of C27 were identified by iTRAQ and LC-MS/MS and provided in Table 1; they include transport proteins (Nos. 1–4), denitrifying proteins (Nos. 5–15, 20 and 22–25), cytochrome c551, C552 and P450 (Nos. 16–19 and 21), ammonia cycle proteins (Nos. 26, 27, 28–35), phosphate-converting proteins (Nos. 36–39 and 40–42) and regulatory proteins (Nos. 43–47). A total of seven proteins that were involved in sulfur metabolisms of C27 were identified and provided in Table 1; they include sulfhydrylase (Nos. 48–50), sulfite reductase (Nos. 51 and 52), and cytochrome c oxidase (Nos. 53 and 54). 3.2. Functions of identified proteins Four transport proteins of C27 were identified. MFS transporters (No. 1 in Table 1) constitute one of the largest groups of secondary active transporters, and selectively transport a wide range of substrates across biomembranes (Yan, 2013). ABC transporter (No. 2) couples the hydrolysis of ATP to solute transport across biological membranes, and is critical for nitrate respiration (Wanner et al., 1999). Nitrate transporter (No. 3) and nitrate transport system permease (No. 4) are responsible for nitrate transporting. Nitrate permease can also transport cyanate. Two forms of nitrate reductases of the strain C27 were identified – the alpha subunit (Nos. 5–9) and the beta subunit (Nos. 10–13). Nitrate reductase is a membrane-bound anaerobic electron transport enzyme, which comprises the alpha subunit and the beta subunit (Edwards et al., 1983). The alpha subunit is the actual site of nitrate reduction and the beta chain is an electron transfer unit. Identified cytochrome cd1 nitrite reductases (Nos. 14–15) are homodimeric proteins that contain one ctype heme and one d1-type heme per subunit. The c-type heme is an electron entry site, which receives electrons from c-type cytochromes, before d1-type heme uses those electrons to reduce nitrite. Therefore, identified cytochrome c551 (No. 16) and C552 (Nos. 17–19) donate electrons to cytochrome cd1 nitrite reductases (cd1NiR). C552 is a physiological redox partner of cd1NiR, and can regulate intermolecular electron transfer and increase the catalytic activity of cd1NiR. Nitric oxide reductase (No. 20) is an integral membrane component of the anaerobic respiratory chain that catalyzes the reduction of NO to N2O (Zumft et al., 2010). Cytochrome P450 (No. 21) was identified in our earlier research. Cytochrome P450 also exhibits nitric oxide reductase activity, and can transform nitric oxide to nitrous oxide using NADH as the sole electron donor (Nakahara et al., 1993). Nitrous oxide reductase (Nos. 22–25) catalyzes the final step of the denitrification pathway (from N2O to N2) (Pauleta et al., 2013). Ferredoxin-nitrite reductase (No. 26) catalyzes the reduction of nitrite to ammonia. Identified glutamine synthetase (No. 27), involved in nitrogen metabolism via ammonium assimilation, catalyzes the biosynthesis of glutamine from ammonia. GMP synthase (Nos. 28–30) catalyzes the conversion of L-glutamine to L-glutamate. Glutamate dehydrogenase (Nos. 31–35) catalyzes the oxidative deamination of Lglutamate to 2-oxoglutarate and ammonia, and has an important role at the intersection of the carbon and nitrogen metabolic pathways (Wakamatsu et al., 2013).
2. Materials and methods 2.1. Bacterial strain and protein extraction Pseudomonas sp. C27 was isolated from an expanded granular suspended bed reactor in which DSR reactions had been performed for more than half of a year (Chen et al., 2013). This strain is a Gramnegative rod-shaped bacterium, whose 16S rDNA sequence was deposited in GenBank under accession number GQ241351. In this investigation, the C27 strain was activated and cultivated in an anaerobic bottle at 30 °C, which was filled with a medium of 7.5 mM KNO3 or KNO2, 0.64 g/L NaAC·3H2O, 1.0 g/L NH4Cl, 0.5 g/L of NaHCO3, 1.8 g/L of KH2PO4, 3.0 g/L of Na2HPO4·12H2O, 0.5 g/L of MgSO4·7H2O, 0.39 g/ L of Na2S and 0.1 g trace elements at pH 8 (50 g/L of EDTA; 0.14 g/L of CuCl2·2H2O; 0.5 g/L of (NH4)6Mo7O24·4H2O; 0.5 g/L of CoCl2·6H2O; 1.0 g/L ZnCl2; 2.5 g/L of MnCl2·2H2O; 3.6 g/L of FeCl2·4H2O; 7.3 g/L of CaCl2·2H2O). After 18 h of cultivation, as described in Sec 2.1, the bacterial cells were collected by centrifugation at 10,000g for 10 min at 4 °C, and then washed using pH 7.4 PBS buffer. The cells were ground to a powder in liquid N2, and then extracted using Lysis buffer at pH 8.5. Further details can be found in Guo et al. (2014). 2.2. iTRAQ labeling and LC-MS/MS analysis The Trypsin Gold (Promega, Madison, WI, USA) was applied to digest protein collected in Sec. 2.1 at protein to trypsin ratio of 30 at 37 °C for 16 h. The digested peptides were dried by vacuum centrifugation and were processed according to the manufacture for 8-plex iTRAQ reagent (Applied Biosystems, Foster City, CA, USA). The labeled peptide mixtures were then dried again by vacuum centrifugation. The labeled proteins were resuspended in 5% CAN with 0.1% FA and was centrifuged at 20,000 × g for 10 min. The 10 μL of so-yielded supernatant was loaded using an auto-sampler to a C18 trap column installed in LC-20AD nanoHPLC (Shimadzu, Kyoto, Japan) with the elution condition stated in Guo et al. (2014). The MS data were processed using Proteome Discoverer software (Version 1.2.0.208) (Thermo Scientific, CA, USA) to generate peak list. The identification of proteins was conducted using Mascot search engine; version 2.3.02 3
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Fig. 1. The pathway of carbon, nitrogen and sulfur metabolism for Pseudomonas sp. C27. No. 1-54 corresponded to proteins in Table 1.
3.3. Nitrogen/sulfur metabolism of strain C27
Carbamoyl phosphate synthase (CPS) comprises two polypeptide chains, called the large subunit (Nos. 36, 37) and the small subunit (Nos. 38, 39), and it plays an important role in both arginine and pyrimidine biosynthesis by providing carbamoyl phosphate. CPS can catalyze the production of carbamoyl phosphate from L-glutamine, which can be replaced by ammonia as a nitrogen donor. Carbamoyltransferase (Nos. 40–42) catalyzes the conversion of carbamoyl phosphate and L-ornithine to phosphate and L-citrulline. Denitrification regulatory protein nirQ (No. 43) activates nitrite reductase and nitric oxide reductase. Transcriptional regulator Anr (No. 44) and Dnr (No. 45) are required for the denitrification of Pseudomonas, and act together as a regulator of protein nirQ (Arai et al., 1997). Transcriptional regulator Anr regulates the transcription of the Dnr gene, arginine deiminase activity and the production of cyanide by Pseudomonas (Arai et al., 1997). Dnr is a heme-binding transcription factor that is involved in the regulation of nitric-oxide reductase and nitrite reductase in Pseudomonas. Two-component response regulator NarL (No. 46) mediates the synthesis of nitrate reductase in the presence of nitrate. Two-component sensor NarX (No. 47) acts as a sensor of nitrate, and transduces the signal of nitrate availability to the NarL protein. NarX activates NarL by phosphorylation in the presence of nitrate, and plays a negative role in controlling NarL activity by dephosphorylation in the absence of nitrate (Noriega et al., 2010). O-succinylhomoserine sulfhydrylase (No. 48 in Table 1) and Oacetylhomoserine sulfhydrylase (Nos. 49 and 50) are sulfide-utilizing enzymes of various enteric bacteria and fungi. The former catalyzes the reaction of sulfide and O-succinyl-L-homoserine to yield L-homocysteine and succinate (Foglino et al., 1995), and the latter catalyzes the reaction of sulfide and O-acetyl-L-homoserine to yield L-homocysteine and acetate (Tran et al., 2011). Sulfite reductase (No. 51) catalyzes the reversible reaction between sulfide and sulfite, and NADPH hemoprotein (No. 52) is the component of the sulfite reductase complex that catalyzes the six -electron reduction of sulfite to sulfide (Gruez et al., 2000). Cytochrome c oxidase (Nos. 53 and 54) is sulfite oxidase that catalyzes the reaction from sulfite to sulfate (Sugio et al., 2010).
7.5 mM NO3− or NO2− was added as a nitrogen source for C27. Fig. 1 displays a proposed pathway for DSR reactions that is based on the protein functions that were identified in Sec. 3.2 and earlier works. First, extracellular nitrate was transported into C27 cells by transport proteins (Nos. 1–4). Then, intracellular nitrate was converted to nitrite, catalyzed by nitrate reductases (Nos. 5–13). The synthesis of nitrate reductase was regulated by the two-component response regulator NarL (No. 46), which was itself regulated by the two-component sensor NarX (No. 47). The generated nitrite was converted to nitric oxide, nitrous oxide and nitrogen gas in that order, catalyzed by cytochrome cd1 nitrite reductases (Nos. 14–15), nitric oxide reductase (No. 20) and nitrous oxide reductases (Nos. 22–25), respectively. Cytochrome cd1 nitrite reductases were regulated by electron donor cytochrome C551 (No. 16) and redox partner denitrification system component cytochrome C552 (Nos. 17–19). Cytochrome cd1 nitrite reductase and nitric oxide reductase were regulated by the denitrification regulatory protein nirQ (No. 43). Protein nirQ, which was in turn regulated by transcriptional regulator Dnr (No. 45), which was regulated by transcriptional regulator Anr (No. 44). As well as being involved in the denitrifying pathway, the produced nitrite was also converted to ammonia, catalyzed by ferredoxin-nitrite reductase (No. 26). Catalyzed by glutamine synthetase (No. 27), GMP synthase (Nos. 28–30) and glutamate dehydrogenase (Nos. 31–35), the cycle of ammonia can be realized by produced intermediate L-glutamine and L-glutamate. Ammonia was also converted to phosphate via intermediate carbamoyl phosphate, catalyzed by carbamoyl phosphate synthase (Nos. 36–39), HERE and carbamoyltransferase (Nos. 40–42). The added S2− ions can be converted by C27 via four pathways. Sulfide can be converted to elemental sulfur via intermediate L-cysteine, cysteine synthase and cysteine desulfurase, or to elemental sulfur directly by sulfide oxidase. Sulfide can react with O-succinyl-Lhomoserine and O-acetyl-L-homoserine to form succinate and acetate, respectively, catalyzed by O-succinylhomoserine sulfhydrylase (No. 48) 4
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and O-acetylhomoserine sulfhydrylase (Nos. 49 and 50). Sulfide also can be converted to sulfite and then converted to sulfate, catalyzed by sulfite reductase (Nos. 51 and 52) and cytochrome c oxidase (Nos. 53 and 54). Extracellular nitrate was transported across the cytomembrane, and converted to nitrite, and then the nitrogen metabolism of C27 was activated (Jia and Cole, 2005). In the first 2 h of the reaction, 53.8 and 46.5 mg/L sulfide were oxidized in the nitrite and nitrate medium, respectively (Supplementary Materials). Restated, the transport of extracellular nitrate to intracellular nitrate and its subsequent conversion to intracellular nitrite took some time, delayed the reaction about 14% conversion rate. Based on these observations, the strain C27 can be applied to a nitrate-containing or a nitrite-containing medium for denitrification, and since nitrite is a better nitrogen source for C27 than is nitrate, this FAB strain has a niche in competition among various nitrite processes, including partial nitrification, comammox and anammox processes. Further metabolic flux analyses can be performed to verify these claims. The reported data herein can be used as a basis to synthesize biological processes combined with other cycles such as P or Fe cycles.
Pseudomonas aeruginosa. Mol. Microbiol. 25, 1141–1148. Chen, C., Ren, N.Q., Wang, A.J., Yu, Z.G., Lee, D.J., 2008. Simultaneous biological removal of sulfur, nitrogen and carbon using EGSB reactor. Appl. Microbiol. Biotechnol. 78, 1057–1063. Chen, C., Ho, K.L., Liu, F.C., Ho, M., Wang, A.J., Ren, N.Q., Lee, D.J., 2013. Autotrophic and heterotrophic denitrification by a newly isolated strain Pseudomonas sp. C27. Bioresour. Technol. 145, 351–356. Chen, C., Shao, B., Zhang, R.C., Xu, X.J., Zhou, X., Yuan, Y., Ren, N.Q., Lee, D.J., 2018. Mitigating adverse impacts of varying sulfide/nitrate ratios on denitrifying sulfide removal process performance. Bioresour. Technol. 267, 782–788. Edwards, E.S., Rondeau, S.S., Demoss, J.A., 1983. chlC (nar) operon of Escherichia coli includes structural genes for alpha and beta subunits of nitrate reductase. J. Bacteriol. 153, 1513–1520. Foglino, M., Borne, F., Bally, M., Ball, M., Patte, J.C., 1995. A direct sulfhydrylation pathway is used for methionine biosynthesis in Pseudomonas aeruginosa. Microbiolgy 141, 431–439. Gruez, A., Pignol, D., Zeghouf, M., Coves, J., Fontecave, M., Ferrer, J.L., FontecillaCamps, J.C., 2000. Four crystal structures of the 60 kDa flavoprotein monomer of the sulfite reductase indicate a disordered flavodoxin-like module. J. Mol. Biol. 299, 199–212. Guo, H.L., Chen, C., Lee, D.J., Wang, A.J., Ren, N.Q., 2014. Coupled carbon, sulfur and nitrogen cycles of mixotrophic growth of Pseudomonas sp. C27 under denitrifying sulfide removal conditions. Bioresour. Technol. 171, 120–126. Guo, H.L., Chang, Y.J., Lee, D.J., 2018a. Enzymatic saccharification of lignocellulosic biorefinery: research focuses. Bioresour. Technol. 252, 198–215. Guo, H.L., Wang, X.D., Lee, D.J., 2018b. Proteomic researches for lignocellulose-degrading enzymes: a mini-review. Bioresour. Technol. 265, 532–541. Jia, W., Cole, J.A., 2005. Nitrate and nitrite transport in Escherichia coli. Biochem. Soc. Trans. 33, 159–161. Lee, D.J., Pan, X.L., Wang, A.J., Ho, K.L., 2013. Facultative autotrophic denitrifiers in denitrifying sulfide removal granules. Bioresour. Technol. 132, 356–360. Nakahara, K., Tanimoto, T., Hatano, K., Usuda, K., Shoun, H., 1993. Cytochrome P-450 55A1 (P-450dNIR) acts as nitric oxide reductase employing NADH as the direct electron donor. J. Biol. Chem. 268, 8350–8355. Noriega, C.E., Lin, H.Y., Chen, L.L., Williams, S.B., Stewart, V., 2010. Asymmetric crossregulation between the nitrate-responsive NarX-NarL and NarQ-NarP two-component regulatory systems from Escherichia coli K-12. Mol. Microbiol. 75, 394–412. Pauleta, S.R., Dell’Acqua, S., Moura, I., 2013. Nitrous oxide reductase. Coord. Chem. Rev. 257, 332–349. Sorokin, D.Y., 2003. Oxidation of inorganic sulfur compounds by obligatively organotrophic bacteria. Microbiology 72, 641–653. Show, K.Y., Lee, D.J., Pan, X.L., 2013. Simultaneous biological removal of nitrogen-sulfurcarbon: recent advances and challenges. Biotechnol. Adv. 31, 409–420. Sugio, T., Ako, A., Takeuchi, F., 2010. Sulfite oxidation catalyzed by aa3-Type cytochrome c oxidase in Acidithiobacillus ferrooxidans. Biosci. Biotechnol. Biochem. 74, 2242–2247. Sun, C., Yuan, J., Xu, H., Huang, S., Wen, X., Tong, N., Zhang, Y., 2019. Simultaneous removal of nitric oxide and sulfur dioxide in a biofilter under micro-oxygen thermophilic conditions; Removal performance, competitive relationship and bacterial community structure. Bioresour. Technol. 290, 121768. Tran, T.H., Krishnamoorthy, K., Begley, T.P., Ealick, S.E., 2011. A novel mechanism of sulfur transfer catalyzed by O-acetylhomoserine sulfhydrylase in the methioninebiosynthetic pathway of Wolinella succinogenes. Biol. Crystallogr. 67, 831–838. Wanner, C., Soppa, J., 1999. Genetic identification of three ABC transporters as essential elements for nitrate respiration in Haloferax volcanii. Genetics 152, 1417–1428. Wakamatsu, T., Higashi, C., Ohmori, T., Doi, K., Ohshima, T., 2013. Biochemical characterization of two glutamate dehydrogenases with different cofactor specificities from a hyperthermophilic archaeon Pyrobaculum calidifontis. Extremophiles 17, 379─389. Yan, H.C., Huang, W.Y., Yan, C.Y., 2013. Structure and mechanism of a nitrate transporter. Cell Rep. 3, 716–723. Zhang, R.C., Xu, X.J., Chen, C., Xing, D.F., Shao, B., Liu, W.Z., Wang, A.J., Lee, D.J., Ren, N.Q., 2018. Interactions of functional bacteria and their contributions to the performance in integrated autotrophic and heterotrophic denitrification. Water Res. 143, 355–366. Zumft, W.G., Braun, C., Cuypers, H., 2010. Nitric-oxide reductase from PseudomonasStutzeri – primary structure and gene organization of a novel bacterial cytochrome bc complex. FEBS J. 219, 481–490.
4. Conclusion By a quantitative proteomic analysis, 47 proteins that are involved in the nitrogen metabolism and seven proteins that are involved in the sulfur metabolism of C27 in mixotrophic growth with nitrate or nitrite as a sole nitrogen source were identified using iTRAQ and LC-MS/MS. The proteins, including transport proteins, denitrifying proteins, denitrifying protein regulators, ammonia cycle proteins, phosphate-converting proteins and others, were identified for the first time. The nitrogen metabolism pathway from external nitrate to nitrogen gas and phosphate with coupled ammonia cycles and the sulfur metabolism pathway of strain C27 were proposed based on the identified proteins. Acknowledgments The authors gratefully acknowledge funding supported by University Nursing Program for Young Scholars with Creative Talents in Heilongjiang Province (UNPYSCT-2018137) and Scientific Research Project of Harbin University of Commerce (No. 18XN044), Open Project of State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology (No. HC201710), and Harbin Applied Technology Research and Development Project (2017RAXXJ025). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biortech.2019.122169. References Arai, H., Kodama, T., Igarashi, Y., 1997. Cascade regulation of the two CRP/FNR-related transcriptional regulators (ANR and DNR) and the denitrification enzymes in
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