Anaerobic degradation of resorcinol
(1,3-dihydroxybenzene) by Azoarcus anaerobius:
Biochemical aspects of the degradation pathway and identification of involved genes
Dissertation
zur Erlangung des Grades eines Doktors der Naturwissenschaften im Fachbereich Biologie der naturwissenschaftlichen Sektion der Universität Konstanz
vorgelegt von: Jutta Anette Hellstern Konstanz 2005
Mündliche Prüfung: 23. September 2005
Referenten: Prof. Dr. Bernard Schink Prof. Dr. Alasdair Cook Prof. Dr. Juan Luis Ramos
Success consists of going from failure to failure without loss of enthusiasm.
Winston Churchill (1874-1965)
Die vorliegende Arbeit wurde im Zeitraum von März 2001 bis Juni 2005 am Lehrstuhl für Mikrobielle Ökologie im Fachbereich Biologie der naturwissenschaftlichen Sektion der Universität Konstanz unter Anleitung von Prof. Dr. Bernhard Schink angefertigt.
Dabei gilt mein besonderer Dank:
Herrn Professor Dr. B. Schink für die Überlassung des Themas und für sein Interesse an der Arbeit sowie für die Möglichkeit, diese Arbeit sowohl an seinem Lehrstuhl als auch im Institut CSIC, Granada (Spanien),durchzuführen.
Herrn Prof. Dr. J.L. Ramos Martín, Leiter der Arbeitsgruppe degradacíon de tóxicos organicos im Institut CSIC in Granada, für seine Unterstützung während meiner Arbeitsaufenthalte in seinem Labor.
Herrn Prof. A. Cook als meinem Koreferenten.
Herrn Dr. B. Philipp für seine fruchtbaren Ideen und für seine Unterstützung während meiner gesamten Doktorarbeit.
Frau Dr. S. Marqués Martin für ihr Interesse und ihre Energie.
Allen derzeitigen und ehemaligen Mitarbeitern des Lehrstuhls von Prof. Dr. B. Schink und der Arbeitsgruppe von Prof. Dr. J.L. Ramos Martín für konstruktive Gespräche und für die angenehme Arbeitsatmosphäre. Stellvertretend seien genannt: Abraham, Antje, Antonio, Carlos, Janosch, Javier, Lissy, Melanie, Olga, Oli, Patricia, Rike, Sascha und Uli.
Meinen Eltern und Geschwistern als verlässlichem Ruhepol in meinem Leben und steter Quelle neuer Kraft.
Allen lieben Personen, denen ich im Laufe meiner Doktorarbeit begegnet bin. Ganz besonders Stefan.
Table of contents
1 Summary in German/ deutsche Zusammenfassung 1
2 Summary in English 4
3 Introduction 6
3.1 Importance of aromatic compounds 6
3.2 Degradation of aromatic compounds 6
3.3 General information on resorcinol 8
3.4 Degradation of resorcinol 9
3.5 Description of Azoarcus anaerobius and Thauera aromatica strain AR-1 10
3.6 Aim of dissertation 10
4 Degradation of 2-hydroxy-1,4-benzoquinone (HBQ), the first non-aromatic intermediate in anaerobic resorcinol (1,3-dihydroxybenzene) degradation, by cell-free
extracts of Azoarcus anaerobius 12
4.1 Abstract 12
4.2 Introduction 12
4.3 Materials and methods 13
4.3.1 Medium and growth conditions 13
4.3.2 Preparation of cell suspensions and cell-free extracts 13
4.3.3 Measurements of enzyme activities 14
4.3.4 Analytical methods 14
4.3.5 Dialysis 15
4.3.6 Derivatisation and analysis of ketones and aldehydes 15
4.3.7 Chemicals 15
4.4 Results 16
4.4.1 HBQ degradation by cell suspensions 16
4.4.2 HBQ degradation in cell-free extracts 16
4.4.3 Identification of end products 16
4.4.4 Influence of NADH 17
4.4.5 Induction of HBQ degradation 19
4.4.6 Identification of ring fission products 19
4.5 Discussion 20
4.5.1 Pathway of resorcinol degradation. 21
4.5.2 Function of NADH 23
4.6 References 24
5 Heterologous expression of the genes of Azoarcus anaerobius involved in anaerobic degradation of resorcinol (1,3-dihydroxybenzene) in Thauera aromatica
strains AR-1 and K 172 27
5.1 Abstract 27
5.2 Introduction 28
5.3 Materials and Methods 29
5.3.1 Medium and growth conditions 29
5.3.2 Molecular biology techniques 29
5.3.3 Transconjugation of Thauera aromatica AR-1 30
5.3.4 Biochemical methods 31
5.4 Results 31
5.4.1 Heterologous expression of genes involved in resorcinol degradation. 31
5.4.2 Enzyme activity measurements in cell-free extracts 33
5.4.3 Sequence analysis 35
5.5 Discussion 39
5.6 References 43
6 Characterisation of resorcinol (1,3-dihydroxybenzene) hydroxylase and detection of hydroxyhydroquinone (1,2,4-trihydroxybenzene, HHQ) dehydrogenase, the first two enzymes in anaerobic resorcinol degradation by Azoarcus anaerobius. 49
6.1 Abstract 49
6.2 Introduction 50
6.3 Materials and methods 51
6.3.1 Medium and growth conditions 51
6.3.2 Preparation of cell-free extracts 51
6.3.3 Measurements of enzyme activity 52
6.3.4 Protein determination 52
6.3.5 Molecular biology methods 52
6.3.6 Analytical methods 53
6.4 Results 53
6.4.1 Molybdenum dependency in growth experiments 53
6.4.2 Resorcinol hydroxylase in the presence of inhibitors 54
6.4.3 Detection of HHQ dehydrogenase 55
6.4.4 Proteins involved in resorcinol degradation 58
6.5 Discussion 59
6.6 References 62
7 Discussion 65
8 References 73
9 Appendix 85
9.1 Record of achievement 85
9.2 Publications 85
1 Summary in German/ deutsche Zusammenfassung
Diese Arbeit beschäftigt sich mit der Untersuchung eines bislang unbekannten Weges des Resorcin (1,3-Dihydroxybenzol) Abbaus durch das Nitrat- reduzierende Bakterium Azoarcus anaerobius.
Resorcin wird durch membrangebundene Enzyme über Hydroxyhydrochinon (HHQ, 1,2,4-Trihydroxybenzol) zu Hydroxybenzochinon (HBQ, 2-Hydroxy-1,4- Benzochinon) oxidiert. Der Schwerpunkt der Dissertation liegt auf der Aufklärung des weiteren Abbaus von HBQ und in der Identifizierung der am Resorcinabbau beteiligten Gene.
Der Abbau von HBQ wurde, ausgehend von Resorcin, im zellfreien Extrakt von A. anaerobius untersucht. Resorcin wurde mit Nitrat und NADH stöchiometrisch zu Acetat, Malat und Succinat abgebaut. Während des Resorcinabbaus konnten instabile Aldehyde und Ketone nach Derivatisierung mit 2,4-Dinitrophenylhydrazin als Zwischenprodukte detektiert werden. Der weitere Abbau von HBQ zu Acetat, Malat und Succinat wurde nur in zellfreiem Extrakt von Resorcin-gewachsenen Zellen katalysiert.
Durch heterologe Expression einer Genbibliothek, die Fragmente vom A. anaerobius-Genom enthielt, wurde ein Fragment R+ identifiziert, welches zwei Thauera aromatica Stämme, AR-1 and K 172, befähigte, mit Resorcin als einziger Kohlenstoff- und Energiequelle plus Nitrat als Elektronenakzeptor zu wachsen. Das Genomfragment R+ hatte eine Größe von 29.88 Kilobasen (kb).
Durch physiologische und biochemische Experimente konnte gezeigt werden, dass R+ die Gene für die Resorcinhydroxylase, die HHQ-Dehydrogenase und die Gene für die Umwandlung von HBQ zu Acetat, Malat und Succinat enthalten muss. Die am Resorcinstoffwechsel beteiligten Gene sowie die dazugehörigen regulatorischen Einheiten wurden in vier benachbarte Gengruppen zusammengefasst.
Physiologische Experimente und Enzymaktivitätsmessungen in Gegenwart von Hemmstoffen (Arsenit, Selenit, Wolframat) für molybdänhaltige Enzyme legten nahe, daß ein molybdänabhängiges Enzym für die Hydroxylierung von
Resorcin zu HHQ verantwortlich war. Typische Motive für molybdänhaltige Enzyme (Molybdän- und Eisen-Schwefel-Bindestelle) wurden auf dem Genomfragment R+ gefunden. Die entsprechenden offenen Leseraster (ORFs) zeigten eine Identität von ca. 50% zu den α- und β- Untereinheiten der Pyrogallol-Phloroglucin-Transhydroxylase von Pelobacter acidigallici und wurden den Untereinheiten der potentielle Resorcinhydroxylase zugewiesen.
Die Oligopeptidsequenzen von einem membrangebundenen Protein mit einer Größe von 50 kDa von A. anaerobius waren identisch mit Sequenzen der potentiellen HHQ-Dehydrogenase auf dem Genomfragment R+. Das 50 kDa große Protein war unabhängig vom verwendeten Wachstumssubstrat in der Membranfraktion von A. anaerobius, Wildtyp T. aromatica AR-1 (wt), und in den Membranfraktionen der beiden Transkonjuganten T. aromatica AR-1 R+
and K 172 R+ vorhanden. Die Dicke dieser Proteinbande korrelierte mit den spezifischen Aktivitäten der HHQ-Dehydrogenase in den entsprechenden Extrakten. Die Tatsache, dass der Gehalt des Proteins mit der Größe von 50 kDa quantitativ größer und die spezifische Aktivität der HHQ- Dehydrogenase um einen Faktor 2 höher war in der Transkonjuganten T. aromatica AR-1 R+ als in T. aromatica AR-1 wt während des Wachstums auf Resorcin oder α-Resorcylat (3,5-Dihydroxybenzoat) legt nahe, dass die Transkonjugante T. aromatica AR-1 R+ über zwei Genkopien für die HHQ- Dehydrogenase verfügt.
Die Gene für die HBQ-Ringspaltung wurden den ORFs des Multienzymkomplexes einer Pyruvat-Dehydrogenase auf dem Genomfragment R+ zugeordnet. Typische Bindemotive für Coenzyme und -substrate der Pyruvat-Dehydrogenase (CoA, FAD, NAD+, TPP) wurden lokalisiert. Jedoch unterstützten die zugegebenen Coenzyme und/ oder -substrate in Experimenten mit zellfreiem Extrakt nicht den Abbau von Resorcin. HBQ wird wahrscheinlich in einer nicht-oxidativen hydrolytischen Reaktion zu einer β-Ketosäure gespalten, die durch eine weitere Spaltung in eine C2 und eine C4 Einheit zerlegt wird.
Die Gene für die den weiteren Abbau zu Acetat, Malat and schließlich zu Succinat wurden potentiellen NADH-abhängigen, löslichen Dehydrogenasen
zugeordnet. Diese Enzyme können die C2-Einheit zu Acetat und die C4-Einheit über Malat zu Succinat umwandeln.
2 Summary in English
The present thesis deals with investigations on an hitherto unknown pathway of resorcinol (1,3-dihydroxybenzene) degradation by the nitrate-reducing bacterium Azoarcus anaerobius.
Resorcinol is oxidized by membrane-bound enzymes via hydroxyhydroquinone (1,2,4-trihydroxybenzene, HHQ) to 2-hydroxy-1, 4-benzoquinone (hydroxybenzoquinone, HBQ). In the focus of this thesis is the further degradation of HBQ and the identification of genes involved in resorcinol degradation.
Degradation of HBQ was investigated in cell-free extracts of A. anaerobius.
Resorcinol was converted into stoichiometric amounts of acetate, malate, and succinate in the presence of nitrate and NADH. During resorcinol degradation, unstable aldehydes and ketones were trapped as intermediates by derivatization with 2,4-dinitrophenylhydrazine. The conversion of HBQ to acetate, malate, and succinate was catalyzed only by cell-free extracts of resorcinol-grown cells.
By heterologous expression of a gene library containing fragments of the A. anaerobius genome, a genome fragment R+ was obtained which enabled two Thauera aromatica strains, AR-1 and K 172, to grow with resorcinol as sole carbon and energy source plus nitrate as electron acceptor. The genome fragment R+ was 29.88 kb in size. Physiological and biochemical experiments showed that R+ must harbour genes for resorcinol hydroxylase and HHQ dehydrogenase, and genes for the conversion of HBQ to acetate, malate, and succinate. Genes involved in resorcinol metabolism including the specific regulatory units were assigned to four adjacent gene clusters.
Growth experiments and enzyme activity tests with inhibitors for molybdenum- containing enzymes (arsenite, tungstate, selenite) suggested that a molybdenum-containing enzyme catalysed the hydroxylation of resorcinol to HHQ. Specific motifs of molybdenum-containing enzymes (molybdenum and iron-sulfur binding sites) were found in the genome fragment R+. The
respective ORFs, assigned as putative resorcinol hydroxylase, showed identities of ∼50% to the α- and β-subunits of the pyrogallol-phloroglucinol transhydroxylase of Pelobacter acidigallici.
Sequences of oligopeptides of a 50 kDa protein from membrane fractions of A. anaerobius grown with resorcinol aligned to the predicted HHQ dehydrogenase in the genome fragment R+. The 50 kDa protein was present in membrane fractions of A. anaerobius, wild-type T. aromatica AR-1 (wt), and the two transconjugantes T. aromatica AR-1 R+ and K 172 R+, independent of the used growth substrate. The intensity of this protein band correlated with the specific activity of HHQ dehydrogenase in the respective cell-free extracts.
The observation that the intensity of the 50 kDa protein band and the specific activity of HHQ dehydrogenase both were substantially higher in trans- conjugate T. aromatica AR-1 R+ than in wild-type T. aromatica AR-1 during growth on resorcinol or α-resorcylate suggested that trans-conjugate T. aromatica AR-1 R+ possesses two gene copies for the HHQ dehydrogenase.
The genes for HBQ ring cleavage were assigned to the ORFs that resembled the multi-enzyme complex of pyruvate dehydrogenase on the genome fragment R+. Typical binding-motifs for the co-enzymes and -substrates of pyruvate dehydrogenase (CoA, FAD, NAD+, TPP) were localized. However, addition of these co-enzymes and/or -substrates in cell-free extract experiments did not support resorcinol degradation. HBQ was cleaved most likely in a non-oxidative hydrolytic reaction to a β-ketoacid, which was subsequently cleaved to a C2 and a C4 moiety.
The genes for the subsequent reactions to form acetate, malate, and finally succinate were assigned to putative NADH-dependent, soluble dehydrogenases. These enzymes transformed the C2 moiety to acetate and the C4 moiety via malate to succinate.
3 Introduction
3.1 Importance of aromatic compounds
The benzene ring is the most widely distributed structure in biosphere after the glucosyl residue (Dagley, 1981). Naturally occurring aromatic compounds are produced mainly by plants: The polymer lignin comprises about 25 % of the land-based biomass on earth. Other aromatic compounds act as protectants which are either toxic or polymerize through peroxidase activity to act as a wound seal (Sitte et al., 2002). However, aromatic compounds are ubiquitous in the living world. They are found in aromatic amino acids (tryptophan, phenylalanine, and tyrosine), in certain steroids, and in components of electron transfer systems (quinones, flavonoids). Since the industrialization aromatic compounds are produced in great amounts and are released into the environment by human activities. They are components of crude oil and fuels, pharmaceuticals, agrochemicals, polymers, explosives and many other products (Smith, 1990). Benzene, toluene, ethylbenzene, styrene, and xylenes belong to the 50 largest-volume industrial aromatic products, with production of millions of tonnes per year (Smith, 1990). Xenobiotic aromatic compounds exhibit a hazardous potential for living organisms, not only because of their cytotoxicity (Shen, 1998), but also because their combustion increases significantly CO2 atmospheric level (Díaz, 2004).
Microorganisms shoulder the major part for mineralization, transformation, and immobilization of pollutants.
3.2 Degradation of aromatic compounds
Microorganisms, particularly bacteria, use aromatic compounds for diverse purposes. Chlorinated aromatic compounds are used as electron acceptor (Holliger et al. 1999), humic substances are used as electron shuttle to make inorganic electron acceptors available (e.g. insoluble iron oxides) (Lovely et al.
1996), substituents that are attached to aromatic rings, as the methyl group of phenylmethyl ethers, are used as growth substrate (Kreft and Schink, 1993;
Bache and Pfennig, 1981), or aromatic compounds are degraded completely to serve as carbon and energy source (Schink et al. 2000, Heider and Fuchs, 1997).
Environmental conditions have a great influence on the degradation of aromatic compounds. The presence or absence of oxygen and the availability of other electron acceptors such as nitrate or sulfate determine the rate and pathway of degradation of aromatic compounds. Under aerobic conditions molecular oxygen is required not only as terminal electron acceptor but also for activation by incorporation into the compound during hydroxylation and ring cleavage (Harwood and Parales, 1996). In the absence of oxygen, bacteria use different mechanisms for activation of aromatic compounds and ring cleavage. To enter the benzoyl-CoA pathway, the most common degradation pathway under anoxic conditions, the aromatic compounds must carry a carboxyl group or must be carboxylated to form an aromatic acid in one of the initial steps (Heider et al., 1999). Thioesterification at the carboxyl group leads to benzoyl-CoA formation, which is prone to ring reduction and fission (Harwood et al., 1999). The aromatic character of a benzene ring with two or more hydroxyl groups is diminished and the compound can be degraded more easily by further hydroxylations or rearrangements, followed by reductive ring fission (Schink et al., 2000).
Common to aerobic and anaerobic metabolism is the separation into peripheral and central degradation pathways (Heider and Fuchs, 1997). In the peripheral pathways the large variety of aromatic compounds is converted into few central intermediates. Under oxic conditions these are catechol, protocatechuate, or gentisate (Harwood and Parales, 1996), under anoxic conditions phloroglucinol, resorcinol, benzoyl-CoA, or hydroxyhydroquinone (Schink et al., 2000; Boll et al., 2002). In the central pathway these intermediates are ready to be de-aromatized and cleaved to common metabolites such as acetyl-CoA or succinate.
In the following the aromatic compound resorcinol, 1,3-dihydroxybenzene, will be in the focus of investigations.
3.3 General information on resorcinol
Resorcinol is a monoaromatic compound with two hydroxyl group in meta position to each other. It occurs naturally in fossil fuels, in heartwood of Artocarpus and Morus (Moraceae) species (Hegnauer, 1990), and in exudates of Nuphar lutea (Sütfeld et al. 1996). Alkylated resorcinol compounds occur in tissues of several Ononis (Leguminosae) species (Barrero et al., 1994) and in the peel or flesh of mango fruits (Droby et al. 1987). Accumulation of resorcinol derivates is considered as defensive response of plants against bacterial infection (Droby et al. 1987). During encystment alkylresorcinols are synthesized and replace other membrane lipids in Azotobacter and Pseudomonas strains (Kozubek et al., 1996; Bitkov et al., 1992). The biological role of these alkylresorcinols in bacterial membranes is not known yet.
Resorcinol is used primarily in the production of special adhesives and/ or improvers of tires and wood products, because of its resistance to high temperature and durability under mechanical stress. Other uses include UV stabilizers, the manufacture of dyes, pharmaceuticals, flame retardants, agricultural chemicals (herbicides, pesticides, fertilizers), fungicidal creams and lotions, explosive primers, antioxidants, and the use in paper industry to improve mechanical and chemical resistance of paper fabrics (www.indespec- chem.com, INSPEC chemical corporation, 2004).
In the year 2000 the global production of resorcinol reached 46,000 metric tonnes, with the United States as the largest producer and consumer. The world consumption of resorcinol is growing at an average annual rate of 0.6 % (www.sriconsulting.com/CEH/Public/Reports/691.7000, CEH report, 2005).
Even though resorcinol was found to be less toxic than phenol, it exhibits similar toxicity (Windholz, 1983). Resorcinol is a skin, eye, and mucous membrane irritant (Windholz, 1983). Exposure to resorcinol causes, with increasing exposure, skin burns, cyanosis, methemoglobinemia, convulsions, and death.
3.4 Degradation of resorcinol
Photochemical transformation of aromatic compounds occurs if the compound absorbs solar light. However, the chemical changes after the absorption of a photon are in general slow processes (Vialaton and Richard, 2002) and do not produce CO2. Therefore, mainly biochemical degradation leads resorcinol back to the global carbon cycle and simultaneously detoxifies the pollutant.
Several microorganisms have the capacity to use resorcinol as growth substrate. Fungi degrade resorcinol by enzymatic hydroxylation forming pyrogallol (1,2,3-trihydroxybenzene) or hydroxyhydroquinone (1,2,4 trihydroxybenzene, HHQ) as intermediates (Shailubhai et al., 1983). Also aerobic pseudomonads (Pseudomonas putida ORC and O1) have been documented to generate HHQ, which is the substrate for ortho or meta ring cleavage (Chapman and Ribbons, 1976) to form either maleylacetate or 2,4,6- trioxohexanoate. Azotobacter vinelandii transforms resorcinol to pyrogallol, which is cleaved to form oxalocrotonate (Groseclose and Ribbons, 1981).
Under anoxic conditions resorcinol is degraded almost quantitatively to methane in undefined cultures from a sludge digester (Chou et al., 1978). A defined co-culture of a Clostridium sp. with Campylobacter sp. ferments resorcinol over 1,3-cyclohexanedione to stoichiometric amounts of acetate and butyrate (Tschech and Schink, 1985). The function of the second organism is not known yet. Strain RE10, a sulfate-reducing bacterium, oxidizes resorcinol completely to CO2 through a pathway similar to that described for the co-culture Clostridium sp. with Campylobacter sp. (Schnell et al., 1989).
Although not all reactions involved in anaerobic degradation of resorcinol are understood in detail yet, most of them involve a reductive attack on the ring system. However, the obligately denitrifying bacterium Azoarcus anaerobius degrades resorcinol by hydroxylation to the intermediate HHQ followed by dehydrogenation to the non-aromatic compound 2-hydroxy-1, 4-benzoquinone (HBQ) (Philipp and Schink, 1998). Both oxidation reactions are performed by membrane-bound enzymes.
So far, the pathway of HHQ degradation has been confirmed only for two nitrate-reducing bacteria, namely A. anaerobius and Thauera aromatica strain
AR-1 (Schink et al., 2000). While A. anaerobius hydroxylates resorcinol to the central intermediate HHQ, Thauera aromatica strain AR-1 degrades α- resorcylate (3,5-dihydroxybenzoate) via hydroxylation and subsequent decarboxylation to HHQ thus entering the HHQ-pathway (Philipp and Schink, 2000).
3.5 Description of Azoarcus anaerobius and Thauera aromatica strain AR-1 A. anaerobius (DSM 12081) was isolated with resorcinol (Springer et al., 1998) and T. aromatica AR-1 (DSM 11528) with α-resorcylate (Gallus et al., 1997) as sole carbon and energy source plus nitrate as electron acceptor, from sewage sludge in Tübingen (Germany). Both bacteria are Gram-negative and belong to the β-subclass of proteobacteria.
A. anaerobius (anaerobius; not living in air) is a rod-shaped bacterium, 2.7- 3.3 µm x 1.5 µm in size, and motile. Its metabolism is strictly oxidative with nitrate as the only electron acceptor. Nitrate is quantitatively reduced to N2, while nitrite accumulates intermediately. Resorcinol is oxidized completely to CO2. A. anaerobius contains no nitrogenase, no catalase, but dismutase activity.
T. aromatica AR-1 is rod-shaped, 1.5-3.0 µm x 0.5 µm in size, and motile. Its metabolism is strictly oxidative with either oxygen or nitrate as electron acceptor. Nitrate is quantitatively reduced to N2, while nitrite accumulates intermediately. α-resorcylate is oxidized to CO2 only with nitrate as electron acceptor. Resorcinol is not degraded.
3.6 Aim of dissertation
The aim of this study was to investigate the resorcinol degradation by A. anaerobius. In the first part, the biochemical investigations on the degradation of HBQ, the first non-aromatic intermediate in resorcinol degradation by A. anaerobius is presented. In the second part, the genes involved in resorcinol metabolism including their regulatory units are identified by heterologous expression in two Thauera aromatica strains. In the third part, the study focuses on resorcinol hydroxylase, the first enzyme in resorcinol
degradation, and on HHQ dehydrogenase, the first enzyme in the central pathway of HHQ degradation.
4 Degradation of 2-hydroxy-1,4-benzoquinone (HBQ), the first non- aromatic intermediate in anaerobic resorcinol (1,3-dihydroxybenzene) degradation, by cell-free extracts of Azoarcus anaerobius
Jutta A. Hellstern, Bodo Philipp and Bernhard Schink
4.1 Abstract
Anaerobic degradation of resorcinol (1,3-dihydroxybenzene) by the denitrifying bacterium Azoarcus anaerobius is initiated by two oxidative reaction steps catalysed by membrane-bound enzymes leading to 2-hydroxy-1, 4-benzoquinone (HBQ). We investigated the further fate of HBQ in cell-free extracts. The degradation of resorcinol further than HBQ depended on cytosolic enzymes, nitrate, and substoichiometric amounts of NADH.
Acetate, malate, and succinate were identified as products by HPLC analysis.
Derivatization with 2,4-dinitrophenylhydrazine indicated that unstable aldehydes or ketones were formed as intermediates. On the basis of these results we propose pathways for cleavage of the HBQ ring.
Keywords: Anaerobic degradation; Aromatic compound; Azoarcus anaerobius;
resorcinol; ring fission.
4.2 Introduction
Many anaerobic bacteria degrade aromatic compounds completely to CO2. The degradation proceeds through the central intermediates benzoyl-CoA, phloroglucinol (1,3,5-trihydroxybenzene), and resorcinol (1,3- dihydroxybenzene) (Boll et al., 2002; Schink et al., 2000; Heider and Fuchs, 1997). These central intermediates are subject to a reductive reaction to overcome the high resonance stability of the aromatic ring (> 100 kJ/mol).
Denitrifying bacteria can destabilize aromatic compounds also by oxidative reactions. Thauera aromatica strain AR-1 transforms α-resorcylate (3,5-diydroxybenzoate) by hydroxylation and subsequent decarboxylation to the fourth central intermediate under anaerobic conditions,
hydroxyhydroquinone (HHQ,1,2,4-trihydroxybenzene) (Schink et al., 2000, Philipp and Schink, 2000). Azoarcus anaerobius hydroxylates resorcinol directly to HHQ (Philipp and Schink, 1998). In both strains, the central intermediate HHQ is not reduced but oxidized to form 2-hydroxy-1,4-benzoquinone (HBQ) as the first non-aromatic degradation intermediate. The further fate of HBQ is unknown so far.
The hydroxylation of the respective substrates and the dehydrogenation of HHQ are catalysed by membrane-bound enzymes and can be measured in vitro. Production of HBQ is indicated by formation of a red-coloured compound in the assay. HHQ and HBQ are rapidly oxidized chemically by an artificial electron acceptor with a positive redox potential, but they do not react chemically with nitrate (Philipp and Schink, 1998). Therefore, nitrate can be used as an electron acceptor in these in vitro assays. Furthermore, HBQ tends to form dimers or easily undergoes nucleophilic attack by e.g. thiol or imidazol groups, leading to addition products (Philipp and Schink, 1998). This reactivity rendered it impossible to investigate the further degradation of HBQ in cell- free extracts by applying this compound directly. Thus, we had to find ways to investigate the subsequent reactions by avoiding undesired side reactions. We selected two strategies, that is, the degradation of chemically prepared HBQ by cell suspensions and the establishment of a cell-free reaction system for degradation of resorcinol further than HBQ.
4.3 Materials and methods 4.3.1 Medium and growth conditions
A. anaerobius (DSM 12081) was grown as described before (Philipp and Schink, 1998).
4.3.2 Preparation of cell suspensions and cell-free extracts
Cell suspensions and cell-free extracts were prepared as described elsewhere (Philipp and Schink, 1998). Cells were permeabilized by addition of 10-15 µg cetyltrimethylammonium bromide (CTAB) per mg dry cell mass. Cells were incubated for 5-10 min with CTAB before the reaction was started.
Protein was quantified by the BCA protein assay (Pierce, USA) (Smith et al., 1985), with bovine serum albumin as standard.
4.3.3 Measurements of enzyme activities
All measurements were performed under strictly anoxic conditions at 30°C in 5-ml Hungate tubes, which were closed with butyl rubber septa and made anoxic by evacuation and subsequent flushing with N2 five times. All buffers and solutions were anoxic. All additions and samplings were done with gas- tight microliter syringes (Macherey-Nagel, Germany). Resorcinol or HHQ degradation assays were performed in Tris HCl (50 mM, pH 7.0) or potassium phosphate buffer (50 mM, pH 7.0). A standard reaction mixture contained cell- free extract (0.5 to 2 mg protein), NaNO3 (4 to 8 mM), and the reaction was started by addition of resorcinol (1 or 2 mM). Degradation of the substrate was measured discontinuously by high performance liquid chromatography (HPLC) analysis. For reversed phase HPLC analysis, 25 µl samples were taken at certain time intervals and mixed immediately with 25 µl ice-cold potassium phosphate buffer. These samples had to be analysed immediately to avoid auto-oxidation of HHQ to HBQ. For ion-exchange HPLC analysis, 150 µl samples were taken at certain time intervals, mixed immediately with 50 µl 1 M H2SO4, and centrifuged at 14,000 x g for 10 min at room temperature. These samples were stable and could be stored at -20°C before analysis.
4.3.4 Analytical methods
Resorcinol, HHQ, and HBQ were analysed with an HPLC system consisting of a C18 reversed phase column (Grom, Herrenberg, Germany), a diode array detector (SPD M10A, Shimadzu, Duisburg, Germany), two pumping units (LC- 10ATvp, Shimadzu) and an auto sampler (Gilson 234, Ohio, USA). The eluents were ammonium phosphate buffer (100 mM, pH 2.6) plus methanol for analysis of resorcinol, or ammonium acetate (10 mM, pH 4,76, by addition of acetic acid) plus methanol for analysis of resorcinol, HHQ, and HBQ. The separation program was started with 5% methanol. After 1 min, the methanol concentration was gradually increased over 6 min to 45% and lowered to 5%
within 0.5 min, followed by 7.5 min equilibration. The concentrations of the compounds were calculated via external standards. Nitrate and nitrite were determined by an anion exchange HPLC system (Sykam A06 column,
Fürstenfeldbruck, Germany) through an isocratic elution with 40 mM NaCl.
Aliphatic intermediates were analysed by an HPLC system equipped with an Aminex column (HPX-87H, Bio-Rad, München, Germany) at 40°C, a refraction detector (Sykam 7512), one pump (Sykam S1000) and an auto sampler (Gilson 234). The eluent was 5 mM H2SO4.
4.3.5 Dialysis
Cell-free extracts, membrane, or cytosolic fractions were dialysed in a cellophane dialysis tubes (cut-off 12-14 kDa, Serva, Germany) against 500 ml potassium phosphate buffer (50 mM, pH 7.0) for 24 hours. The buffer was changed twice. Dialysis was performed anoxically at 4°C. Dialysed cell-free extract, membrane, and cytosolic fraction were transferred anoxically into tubes and were kept at -20°C until use.
4.3.6 Derivatisation and analysis of ketones and aldehydes
At certain time intervals after starting a standard reaction mixture, 1.5 ml reaction mixture was withdrawn, mixed with 40 µl 20% TCA, and centrifuged at 14,000 x g for 10 min at room temperature. All further steps were done at 25°C. After addition of 1 ml 0.1% hydrazine reagent (0.5 g 2,4-dinitrophenylhydrazine in 500 ml 2 N HCl) (Friedemann and Haugen, 1943), the derivatization reaction took place for exactly 5 min. Addition of ethyl acetate and bubbling with N2 for 2 min separated the hydrazine from the hydrazones. Hydrazones passed into the ester phase and could be easily removed. Addition of 3 ml 10% Na2CO3 (filtered) to the ester phase followed by bubbling with N2 resulted in two phases, upon which the hydrazones passed into the Na2CO3 solution. The lower phase was removed, mixed with 5 ml 1,5 N NaOH, and incubated for another 5 min. Subsequently, the absorption spectra were measured in a quartz cuvette with a photometer (Uvikon 930, Switzerland) (Friedemann and Haugen, 1943).
4.3.7 Chemicals
All chemicals used were of analytical grade and highest purity available. HBQ was prepared through auto-oxidation of HHQ with atmospheric oxygen, followed by evacuation and subsequent flushing with N2 to make the solution anoxic.
4.4 Results
4.4.1 HBQ degradation by cell suspensions
HBQ, prepared by auto-oxidation of HHQ, was added to suspensions of intact or permeabilized cells of A. anaerobius in the presence of nitrate. HPLC analysis revealed that HBQ was not degraded and no products were formed.
4.4.2 HBQ degradation in cell-free extracts
A standard reaction mixture consisted of membrane fraction plus cytosolic fraction, and nitrate in excess to allow further oxidation. Resorcinol was degraded faster (5.8 mU (mg protein)-1) than by the membrane fraction alone (2.1 mU (mg protein)-1). Formation of a red colour was still observable, even though not so intense as with the membrane fraction only.
Addition of ascorbic acid, ATP, biotin, coenzyme A, cystein, dithiothreitol, glutathione, lipoic acid, NAD(P)+, naphthoquinone, or thiamine pyrophosphate had no effect on resorcinol degradation by cell-free extracts. However, addition of NAD(P)H (1 mM) prevented the formation of red colour in the reaction assay while the resorcinol degradation rate was not influenced.
4.4.3 Identification of end products
HPLC analysis revealed no accumulation of HBQ in NAD(P)H containing assays. In ion exchange HPLC analysis, three peaks were detected while resorcinol disappeared. These peaks were identified by co-chromatography with authentic compounds as malate (10.5 min retention time), succinate (13.0 min), and acetate (15.7 min).
A typical process of degradation in a standard reaction mixture with cell-free extract (0.5 mg ml-1) is shown in Fig. 1. Within the first 6 hours, 1 mM resorcinol was degraded nearly completely. Then, the degradation rate decelerated before resorcinol was metabolized completely after 24 hours.
Acetate was measured from the start of the reaction and the concentration increased linearly over time until all resorcinol was metabolized. Formation of malate began simultaneously with resorcinol degradation and ceased as resorcinol degradation decelerated. Furthermore, malate was degraded and succinate was formed. The final concentration of these products (acetate
1.56 mM, malate 0.33 mM, succinate 0.63 mM) was nearly stoichiometric to the amount of resorcinol degraded (1 mM).
0.00 0.20 0.40 0.60 0.80 1.00 1.20
0 1 2 3 4 5 6 7
time [h]
conc. [mM]
Fig. 1 Nitrate- and NADH-dependent resorcinol (?) degradation and formation of acetate (♦), malate (?) and succinate (¦) by cell free extract of A. anaerobius grown with resorcinol.
4.4.4 Influence of NADH
The effect of NADH was studied with the standard reaction mixture with or without NADH and a control mixture with NADH, but without resorcinol (Fig. 2).
During resorcinol degradation, nitrate was reduced to nitrite. The formation of nitrite depended on the presence of NADH. In reaction mixtures with NADH, more nitrate than required for the first two oxidative reactions was reduced to nitrite. Only little nitrate reduction was detected in a control assay without resorcinol. Therefore, the higher nitrate reduction by the reaction mixture in the presence of NADH plus resorcinol could not be caused by reduction of nitrate with NADH. Additional control experiments showed no NADH- dependent back reaction to resorcinol.
(a)
0.00 1.00 2.00
0 2 22
time [h]
resorcinol concentration [mM]
(b)
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00
0 2 22
time [h]
nitrate concentration [mM]
(c)
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00
0 2 22
time [h]
nitrite concentration [mM]
Fig. 2 Effect of NADH on nitrate-dependent degradation of resorcinol by cell-free extract of A. anaerobius grown on resorcinol. Shown are resorcinol (a), nitrate (b), and nitrite (c) concentrations determined after 0, 2 and 22 hours in a standard reaction mixture with NADH (¦ ), without NADH (¦ ), and with NADH, but without resorcinol (¦ ).
The clearest product formation pattern without formation of a red-coloured side-product was achieved with 1 mM resorcinol, 8 mM nitrate and 1 mM NADH.
Dialysis of the cell-free extract had no influence on the rate of resorcinol degradation or on product formation. Furthermore, no effect could be observed by addition of Mg2+ or K+ ions (2.5- 5 mM) in standard reaction mixtures buffered with Tris HCl.
4.4.5 Induction of HBQ degradation
The induction of HBQ degradation was investigated in a standard reaction mixture with NADH and with the cytosolic fraction of A. anaerobius cells grown with acetate. The resorcinol degradation rate was 2.0 mU (mg protein)-1. The assay mixture turned red and no formation of acetate, malate, or succinate was detected. These results were equivalent to the data obtained in the experiment omitting the cytosolic fraction (see above).
4.4.6 Identification of ring fission products
Addition of inhibitors of presumptive enzymes involved in HBQ degradation, namely, chloramphenicol for NADH-dehydrogenase, glyoxylic acid for succinate-semialdehyde dehydrogenase, and malonic acid for succinate dehydrogenase (Zollner, 1990), did not affect resorcinol degradation.
Derivatization experiments with 2,4-dinitrophenylhydrazine were performed to trap presumptive aldehydes or ketones. Sampling of a standard reaction mixture after certain time intervals and subsequent derivatization with 2,4- dinitrophenylhydrazine led to 2,4-dinitrophenylhydrazones (DNPHs). The formation of DNPHs was time dependent. At the reaction start, UV/ VIS spectra showed no absorption at the characteristic wavelength for DNPHs between 400 and 500 nm (Friedemann and Haugen, 1943). Within the first four hours after reaction start, an increase of absorption at four different wavelengths was observed (340, 425, 450, 550 nm). The absorption at every wavelength decreased over time, but the absorptions at 450 and 550 nm were still visible after 24 hours.
Fig. 3 shows the absorption of a standard reaction mixture with NADH, and two controls consisting of a standard reaction mixture with NADH, but without addition of resorcinol, and a HBQ solution, prepared by auto-oxidation of HHQ (1 mM). All reaction assays were incubated for 24 h before derivatization was performed. The control assay without resorcinol showed no significant
increase in absorption after derivatization, whereas the HBQ solution exhibited absorption at 340 nm.
Fig. 3 Absorption spectra of standard resorcinol degradation assays with NADH (-), without resorcinol (-), and a HBQ solution (-) after derivatization with 2,4-dinitrophenylhydrazine.
Analysis of reference solutions of derivatized aldehydes and ketones (acetone, acetaldehyde, and succinic semialdehyde) showed the characteristic absorption for DNPHs between 400 and 500 nm. Thus, the experiment demonstrated that aldehydes and/or ketones were formed and were further degraded. Quantification of the formed aldehydes or ketones was not possible because absorption wavelength, and absorption maxima differed for each reference compound.
4.5 Discussion
This study presents investigations on the degradation of HBQ which is the first non-aromatic compound in anaerobic degradation of resorcinol by A. anaerobius. HBQ was not degraded by cell suspensions. As a polar compound, HBQ probably cannot pass the cytosolic membrane efficiently by simple diffusion. Obviously, A. anaerobius lacks a transport system for HBQ.
However, permeabilized cells did not degrade HBQ either. Since HBQ reacts with components of the cell-free extracts (Philipp and Schink, 1998), it could possibly damage enzymes involved in its further degradation. In resorcinol degradation experiments with cell-free extract, HBQ always accumulated unless NAD(P)H was added to the assay. Only in the presence of NAD(P)H,
resorcinol was degraded further beyond HBQ, and aliphatic compounds (acetate, malate, and succinate) accumulated.
4.5.1 Pathway of resorcinol degradation.
Acetate and malate were formed simultaneously during resorcinol degradation. Malate decreased later while succinate was formed, indicating that malate was a primary degradation product.
The pathway of HBQ degradation to acetate and malate has not yet been elucidated. However, our experiments suggest that HBQ was cleaved in such a manner that aldehydes and/or ketones were formed and later degraded.
These compounds had only a short lifetime because detection of these intermediates was possible only through stabilization by an irreversible derivatization reaction. On the basis of these investigations we postulate alternative degradation pathways (Fig. 4). HBQ cleavage might occur by a reaction analogous to the oxidative-thiolytic cleavage of acetoin by the strict anaerobe bacterium Pelobacter carbinolicus (Oppermann et al., 1989).
Acetoin is cleaved into acetyl-CoA and acetaldehyde by a complex of three enzymes which depend on coenzyme A (CoA), thiamine pyrophosphate (TPP), and NAD+ (Oppermann et al., 1991). Assignment of this type of reaction to HBQ cleavage would propose a cleavage between the hydroxyl and the oxo group to produce a maleylacetatsemialdehyde. However, TPP and/ or CoA addition did not support formation of acetate, malate, and succinate. We therefore suggest a hydrolytic rather than a thiolytic cleavage of HBQ, to form a C2 and a C4 moiety. The C4 moiety could be maleinate.
However, in standard reaction mixture with NADH, no maleinate was detected, and no maleinate reductase nor hydratase activity was found in cell-free extract of A. anaerobius. Alternative degradation routes start from a tautomeric form of HBQ, 1,2,4-trioxo-hex-5,6-ene. The cleavage could proceed between the 1,2 or 1,3 diol. Carbon-carbon bound cleavage between a 1,3 diol has been described for several fermenting (Tschech and Schink, 1985; Krumholz et al., 1987; Brune and Schink, 1992), sulfate-reducing (Schnell et al., 1989) and nitrate-reducing (Dangel et al., 1989) bacteria. The two oxo groups in meta position destabilize the ring and make hydrolytic cleavage possible.
OH
OH
O
OH OH
OH
OH O
O
O O
O
O O
O
O O
O
O O O-
O-
O-
O- O
O O-
O
O OH Acetate
Malate
Malate C CH
O
Acetate C CH
O O
O
O OH
O- O
O
O O
O
O OH O O
O
O O
O-
O
O O O
OH
O O O
OH
O O
O- O
C CH O
Acetate Malate
Fig. 4 Degradation of resorcinol over HBQ and possible ring cleavage reactions leading to the formation of acetate, malate and succinate.
Depending on the cleavage site, HBQ could be hydrolized to 4,5-dioxohex- 2,3-enoic acid or to 2,5-dioxohex-3,4-enoic acid. The further degradation of 4,5-dioxohex-2,3-enoic acid could proceed in a similar manner as described above. However, 2,5-dioxohex-3,4-enoic acid could be lactonized and, after
isomerization, be hydrolysed to 2-hydroxy-4,5-dioxohexanoic acid. Similar degradation reactions have been described for aerobic degradation of aromatic compounds. Pseudomonas putida cleaves catechol by a dioxygenase to muconate, which is subsequently transformed to muconolactone (Ornston, 1966). A hydrolase forms β-ketoadipate, which is then further degraded to an acetyl and succinyl residue. Carbon-carbon bound cleavage between a 1,2 diol has been described in anaerobic cyclohexane- 1,2-diol degradation by Azoarcus strain 22Lin (Harder, 1997). Cyclohexane- 1,2-diol is oxidized to cyclohexane-1,2-dione which is cleaved by hydrolysis.
The resulting 6-oxohexanoate is oxidized to adipate. Assignment of this intradiol cleavage to HBQ degradation would result in 3,6-dioxohex-4,5-enoic acid. After subsequent reactions of lactonizing enzyme, isomerase, and hydrolase, 3-hydroxy-4,6-dioxohexanoic acid is cleaved to acetaldehyde and malate.
Independent of the cleavage reaction resorcinol would be degraded stoichiometrically to acetate, and malate. This correlates with our observations in standard reaction mixtures with membrane and cytosolic fractions obtained from resorcinol-grown cells of A. anaerobius. However, with cytosolic fractions from cells grown with acetate no product formation was detected. Therefore, we conclude that the enzymes for the peripheral and for the ring fission reactions are induced during growth with resorcinol. This correlates with observations in Thauera aromatica, in which enzymes for the peripheral and ring fission reactions are present only after growth with the respective substrate (Heider et al., 1998).
4.5.2 Function of NADH
Aromatic compounds with hydroxyl groups in meta-position can be reduced with common physiological reductants (Boll, 2005). NAD(P)H acts as electron donor in HHQ reductase in the sulfate-reducing bacterium Desulfovibrio inopinatus (Reichenbecher et al., 2000) and in reductases in fermenting bacteria during trihydroxybenzene degradation (Schink et al., 2000). In A. anaerobius, no such reduction of HHQ was observed (Philipp and Schink, 1998). NAD(P)H could not be replaced by other electron donors. However, NADH could act as an electron carrier involved in redox reactions and thus,
oscillating between the reduced and the oxidized form. However, NADH could not be replaced by NAD+ indicating that NADH acts initially as a reducing agent, while subsequent reactions could involve a NAD+/ NADH cycling.
However, NADH could also act as an allosteric effector for activation of enzymes. This has been shown for proline dehydrogenase, where NADH could replace proline as a promoter for membrane association of proline dehydrogenase under anaerobic reaction conditions (Wood, 1987).
Imaginable would be also a combination of both functions, an activator in initial reactions and an electron mediator in further redox reactions, thus explaining the substoichiometric consumption of NADH.
At present further investigations concerning the involved enzymes are in progress in our lab. Genetic approaches will reveal new insights into this novel pathway of resorcinol degradation.
4.6 References
Boll, M. (2005) Key enzymes in the anaerobic aromatic metabolism catalysing Birch-like reductions. Biochim Biophys Acta 1707: 34-50.
Boll, M., G. Fuchs, and J. Heider (2002) Anaerobic oxidation of aromatic compounds and hydrocarbons. Curr Opin Chem Biol 6: 604-611.
Brune, A., and B. Schink (1992) Phloroglucinol pathway in the strictly anaerobic Pelobacter acidigallici: Fermentation of trihydroxybenzenes to acetate via triacetic acid. Arch Microbiol 157: 417-424.
Dangel, W., A. Tschech, and G. Fuchs (1989) Enzyme reactions involved in anaerobic cyclohexanol metabolism by a denitrifying Pseudomonas species. Arch Microbiol 152: 273-279.
Friedemann, T.E., and G.E. Haugen (1943) The determination of the keto acids in blood and urine. J Biol Chem 147: 415-442.
Harder, J. (1997) Anaerobic degradation of cyclohexane-1,2-diol by a new Azoarcus species. Arch Microbiol 168: 199-204.
Heider, J., and G. Fuchs (1997) Anaerobic metabolism of aromatic compounds. Eur J Biochem 243: 577-596.
Heider, J., M. Boll, K. Breese, S. Breinig, C. Ebenau-Jehle, U. Feil, N. Gad’on, D. Laempe, B. Leuther, M. El-Said Mohamed, S. Schneider, G.
Burchhardt, and G. Fuchs (1998) Differential induction of enzymes involved in anaerobic metabolism of aromatic compounds in the denitrifying bacterium Thauera aromatica. Arch Microbiol 170: 120-131.
Krumholz, L.R., R.L. Crawford, M.E. Hemling, and M.P. Bryant (1987) Metabolism of gallate and phloroglucinol in Eubacterium oxidoreducens via 3-hydroxy-5-oxohexanoate. J Bacteriol 169: 1886-1890.
Oppermann, F. B., B. Schmidt, and A. Steinbüchel (1991) Purification and characterization of acetoin: 2,6-dichlorophenolindophenol oxidoreductase, dihydrolipoamide dehydrogenase, and dihydrolipoamide acetyltransferase of the Pelobacter carbinolicus acetoin dehydrogenase enzyme system. J Bacteriol 173: 757-767.
Oppermann, F.B., A. Steinbüchel, and H.G: Schlegel (1989) Evidence for oxidative thiolytic cleavage of acetoin in Pelobacter carbinolicus analogous to aerobic oxidative decarboxylation of pyruvate. FEMS Microbiol Lett 60: 113-118.
Ornston, L.N. (1966) The conversion of catechol and protocatechuate to β- ketoadipate by Pseudomonas putida. IV. Regulation. J Biol Chem 241:
3800-3810.
Philipp, B., and B. Schink (1998) Evidence of two oxidative reaction steps initiating anaerobic degradation of resorcinol (1,2-dihydroxybenzene) by the denitrifying bacterium Azoarcus anaerobius. J Bacteriol 180: 3644- 3649.
Philipp, B., and B. Schink (2000) Two distinct pathways for anaerobic degradation of aromatic compounds in the denitrifying bacterium Thauera aromatica AR-1. Arch Microbiol 173: 91-96.
Reichenbecher, W., B. Philipp, M.J.-F. Suter, and B. Schink (2000) Hydroxyhydroquinone reductase, the initial enzyme involved in the degradation of hydroxyhydroquinone (1,2,4-Trihydroxybenzene) by Desulfovibrio inopinatus. Arch Mircobiol 173: 206-212.
Schink, B., B. Philipp, and J. Mueller (2000) Anaerobic degradation of phenolic compounds. Naturwissenschaften 87, 12-23.
Schnell, S., F. Bak, and N. Pfennig (1989) Anaerobic degradation of aniline and dihydroxybenzenes by newly isolated sulfate-reducing bacteria and description of Desulfobacterium anilini. Arch Microbiol 152: 556-563.
Smith, P.K., R.I. Krohn, G.T. Hermanson, A.K. Mallia, F.H. Gartner, M.D.
Provenzano, E.K. Fujimoto, N.M. Goeke., B.J. Olson, and D.C. Klenk (1985) Measurements of protein using bicinchoninic acid. Analy Biochem 150: 76-85.
Tschech, A., and B. Schink (1985) Fermentative degradation of resorcinol and resorcylic acids. Arch Microbiol 143: 52-59.
Wood, J.M. (1987) Membrane association of proline dehydrogenase in Escherichia coli is redox dependent. Proc Natl Acad Sci USA 84: 373- 377.
Zollner, H. (1990) Handbook of enzyme inhibitors. VCH Weinheim.
5 Heterologous expression of the genes of Azoarcus anaerobius involved in anaerobic degradation of resorcinol (1,3-dihydroxybenzene) in Thauera aromatica strains AR-1 and K 172
Jutta A. Hellstern, Bodo Philipp, Silvia Marqués Martin, Javier Medina Bellver, and Bernhard Schink
5.1 Abstract
The obligately denitrifying bacterium, Azoarcus anaerobius, grows with resorcinol (1,3- dihydroxybenzene) as sole carbon and energy source.
Degradation proceeds via hydroxylation of resorcinol to hydroxyhydroquinone (1,2,4-trihydroxybenzene, HHQ) which is dehydrogenated to 2-hydroxy-1,4- benzoquinone (hydroxybenzoquinone, HBQ). The further degradation leads to the formation of acetate, malate, and finally succinate.
To identify the genes involved in this novel degradation pathway, a genome library of A. anaerobius was constructed and randomly transformed in the closely related bacterium Thauera aromatica strain AR-1. The genome fragment R+ of A. anaerobius enabled T. aromatica AR-1 to use resorcinol as growth substrate. T. aromatica strain K 172 received this genome fragment by transconjugation, too. In contrast to T. aromatica AR-1 harbouring the genes for the HHQ-degradation pathway, T. aromatica K 172 was not known to degrade any aromatic compound over this pathway. However, heterologous expression of genome fragment R+ enabled T. aromatica K 172 to use resorcinol as sole carbon and energy source.
The genome fragment R+ was 29 880 base pairs long. Physiological and biochemical experiments demonstrated that this fragment contained the genes for resorcinol hydroxylase and for HHQ dehydrogenase, and possibly the genes for the further degradation to intermediates of the central metabolism.
Sequence analysis of this genome fragment R+ revealed 22 open reading frames concentrated in 5 gene clusters with different orientation.
Keywords: Anaerobic degradation; Aromatic compound; resorcinol;
Azoarcus anaerobius; Thauera aromatica; heterologous expression.
5.2 Introduction
Bacteria degrade aromatic compounds always after the same principle:
various aromatic compounds are channelled into few central intermediates, which are degraded in few central degradation pathways. In the absence of oxygen, bacteria channel aromatic compounds into either one of the central intermediates benzoyl- CoA, hydroxyhydroquinone, phloroglucinol, or resorcinol (Boll et al. 2002; Schink et al., 2000). In most cases the aromatic compound is destabilized by a reductive reaction followed by a hydrolytic cleavage of the ring. Due to the use of an electron acceptor of comparably high redox potential (NO3-
/ NO2-
E°’= +430 mV), nitrate-reducing bacteria hold an exceptional position and can also use oxidative steps for destabilization (Schink et al., 2000). The facultatively nitrate-reducing bacterium Thauera aromatica strain AR-1 harbours two distinct degradation pathways for aromatic compounds: benzoate is degraded via the benzoyl-CoA pathway, and α-resorcylate (3,5-dihydroxybenzoate) via the hydroxyhydroquinone pathway (1,2,4-trihydroxybenzene, HHQ) (Philipp and Schink, 2000). α- Resorcylate is hydroxylated to 2,3,5-trihydroxybenzoate before it can be decarboxylated to the central intermediate HHQ (Gallus and Schink, 1998).
The obligately nitrate-reducing bacterium Azoarcus anaerobius initiates degradation of resorcinol by hydroxylation to HHQ (Philipp and Schink, 1998).
Both organisms destabilize the aromatic nucleus of the central intermediate HHQ by an oxidative reaction to 2-hydroxy-1,4-benzoquinone (hydroxybenzoquinone, HBQ) (Philipp and Schink, 2000; Philipp and Schink, 1998). The further degradation is catalysed by soluble enzymes. It depends on NADH and nitrate as co-substrates and leads over unidentified aldehydes and ketones to acetate, malate, and succinate (Hellstern et al., unpublished).
The present article deals with the identification of genes involved in resorcinol degradation by A. anaerobius. T. aromatica strain AR-1 and K 172 which are close relatives of A. anaerobius were used for heterologous expression of genes of A. anaerobius. Both strains are unable to degrade resorcinol.
T. aromatica AR-1 harbouring the HHQ-pathway lacks only the first enzyme which converts resorcinol to HHQ (Gallus and Schink, 1998). T. aromatica
K 172 is not known to degrade resorcinol or α-resorcylate, the two aromatic compounds known to be degraded through the HHQ-pathway (Anders et al., 1995; Mechichi et al., 2002).
5.3 Materials and Methods 5.3.1 Medium and growth conditions
A. anaerobius (DSM 12081), T. aromatica strain AR-1 (DSM 11528) and strain K 172 (DSM 6984) were grown in mineral medium as described before (Philipp and Schink, 1998), except that 3-[N-morpholino] propane sulfonic acid (30 mM, pH 7.2) was used as buffering system. Pseudomonas strain JLR 11 (CECT 4460) and Klebsiella oxytoca (PSC 44) grown in M9 minimal medium (Sambrook et al., 1989) supplemented with 1 mM MgSO4, 50 µM iron citrate, and trace metals, with glucose (20 mM) as carbon source. Escherichia coli strain HB 101 and strain HB 101 RK 600 (cmR, tra function) were grown in Luria-Bertani broth (LB) (Sambrook et al., 1989). Where appropriate, tetracycline or chloramphenicol were added at 10 µg/ ml.
Agar shake dilutions were performed as described by Pfennig (Pfennig, 1978).
5.3.2 Molecular biology techniques
For construction of a gene library of A. anaerobius, genomic DNA was purified and partially digested with restriction enzyme Pst I. Fragments of 20 to 30 kb were ligated into a Pst I digested pLAFR 3 vector (tetR, cos-site) (Staskawicz et al., 1987). The gene library was packed in lambda phage heads (Gigapack III packaging extract, Stratagene, USA) and incubated with E. coli HB 101 to allow the phage to attach to the cells (Kretz et al., 1991). Screening was carried out in the presence of tetracycline.
16S rDNA was amplified by PCR with primer 27F (5'-AGA GTT TGA TCC TGG CTC AG-3') and 1492R (5'-TAC GGY TAC CTT GTT ACG ACT T-3') with 30 cycles as described before (Henckel et al., 1999), except that the annealing temperature was 52°C. PCR products were purified with the E.Z.N.A cycle-pure kit (Peqlab, Erlangen, Germany). Phylogenetic analysis of the sequence was done using the automated tools of the ARB software package (http://www.arb-home.de).
Cosmids were purified using Qiagen Large-Construct Kit (Hilden, Germany).
The nucleotide sequence was determined by GATC (Konstanz, Germany).
Final sequence quality of > 99.9 % was based on sequencing both strands.
Evaluation of the sequence was done using DNAStar software (http://www.dnastar.com). The program was adjusted to bacterial genomes.
Nucleotide and protein sequence similarity searches were carried out by BLAST programs at the BLAST server of NCBI (http://www.ncbi.nlm.nih.gov/blast/) (Altschul et al., 1997) and hydrophilicity analysis of the protein sequences was done using the TMHMM server 2.0 (http://www.cbs.dtu.dk/servicesTMHMM) (Krogh et al., 2001).
5.3.3 Transconjugation of Thauera aromatica AR-1
In order to receive a strain of T. aromatica AR-1 able to use resorcinol as growth substrate, tri-parental mating was performed with E. coli HB 101 containing the gene library of A. anaerobius, and E. coli HB 101 RK 600 as a helper strain. In the middle of the exponential growth phase 500 µl cell suspension of E. coli HB 101 containing the gene library of A. anaerobius, 500 µl cell suspension of E. coli HB 101 RK 600, and 1 000 µl cell suspension of T. aromatica strain AR-1 were separately centrifuged at 14 000 x g for 10 min. Pellets were washed with LB and centrifuged again. All cells were combined in a volume < 40 µl and transferred onto a sterile filter (Schleicher and Schell, Dassel, Germany) on solid LB medium. The mixture was incubated overnight at 28 °C. The filter was transferred into a new sterile tube with minimal medium and completely re-suspended by vigorous shaking.
100 ml infusion bottle (Müller-Krempel) filled with 50 ml anoxic minimal medium, containing 2 mM resorcinol plus 8 mM nitrate was inoculated with the re-suspended cells, and incubated anoxically at 28 °C for two weeks. E. coli HB 101, HB 101 RK 600, and T. aromatica AR-1 were incubated separately as controls.
The same procedure was applied with T. aromatica strain K 172 R+ after isolating a cosmid with genome fragment R+, containing genes for resorcinol metabolism.
5.3.4 Biochemical methods
Cell-free extract, cytosolic, and membrane fractions were prepared as described before (Philipp and Schink, 1998).
Protein was quantified by the BCA protein assay (Pierce, USA) (Smith et al., 1985), with bovine serum albumin as the standard.
All enzyme activity measurements were performed under strictly anoxic conditions at 30 °C in 1.5 ml cuvettes (Hellma, Germany) or 5 ml Hungate tubes, which were closed with butyl rubber septa and made anoxic by evacuation and subsequent flushing with N2 (five times). Tris HCl buffer (50 mM, pH 7.0), potassium phosphate buffer (50 mM, pH 7.0), and all other solutions were anoxic. All additions and samplings were done with gas-tight microliter syringes (Macherey-Nagel, Germany). Resorcinol hydroxylase was measured with potassium hexacyanoferrate (1 mM) as electron acceptor.
Degradation of resorcinol was continuously observed via reduction of potassium hexacyanoferrate by a spectrophotometer (Hitachi 100-40, Japan) at 420 nm wavelength (ε420= 0.9 mM-1 cm -1). HHQ dehydrogenase was measured discontinuously by high performance liquid chromatography (HPLC) analysis with nitrate as electron acceptor as described before (Hellstern et al., unpublished). Formation of acetate, malate, and succinate was measured as described before (Hellstern et al., unpublished).
5.4 Results
5.4.1 Heterologous expression of genes involved in resorcinol degradation.
Numerous attempts were made to express genes of A. anaerobius in various organisms. E. coli HB 101 containing the gene library of A. anaerobius was grown under anoxic conditions in liquid LB medium with additional 1 mM of resorcinol and 4 mM of nitrate. After preparation of cell-free extracts, no resorcinol hydroxylase activity was detected. The gene library was transferred into Pseudomonas strain JLR 11 and into Klebsiella oxytoca. No transconjugante which could use resorcinol as growth substrate was received from these hosts.
p-Toluidine reacts with phenolic compounds containing two hydroxyl groups in ortho position as HHQ to form a coloured compound (Parke, 1992). No coloured colony was observed after addition of resorcinol and p-toluidine (1.5 mM p-toluidine in N,N-dimethylformamide) to growing P. putida JLR 11 and K. oxytoca cultures, indicating that resorcinol was not hydroxylated to HHQ. T. aromatica AR-1, a bacterium closely related to A. anaerobius, but so far never used as a recipient strain for genetic work, was chosen for heterologous expression of genes of A. anaerobius. Due to the high cell density in the transconjugation assay, no increase in optical density of the transconjugation assay could be observed. After two weeks of incubation, 2 % of the inoculum was transferred into fresh anoxic minimal medium with resorcinol as growth substrate plus nitrate. After two days of incubation an increase in optical density at 578 nm (OD578) was observed. This culture was diluted in agar shakes to receive a single clone. This clone was named T. aromatica AR-1 R+. R+ referred to the gene fragment of A. anaerobius harbouring genes for resorcinol metabolism. Cells were rod shaped and motile as described for wildtype T. aromatica AR-1 (Gallus and Schink, 1997).
16S rDNA analysis proved the transconjugante as T. aromatica. Growth experiments showed that the increase in OD578 was coupled to degradation of resorcinol with simultaneous reduction of nitrate to nitrite (Fig. 1(a)).
After purification of the cosmid containing genome fragment R+ and its transformation into E. coli HB 101, this cosmid was used for a second tri- parental mating with T. aromatica K 172 as acceptor. After selection for growth with resorcinol and isolation of a single colony, a second transconjugante was obtained (T. aromatica K 172 R+). This strain was confirmed by 16S rDNA analysis as T. aromatica. The increase of OD578 was coupled to degradation of resorcinol and to formation of nitrite (Fig. 1(b)).
