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RESEARCH LETTER Acetate formation in the photoheterotrophic bacterium Chloroflexus aurantiacus involves an archaeal type ADP-forming acetyl-CoA synthetase isoenzyme I € nheit Marcel Schmidt & Peter Scho €r Allgemeine Mikrobiologie, Christian-Albrechts-Universit€ Institut fu at Kiel, Kiel, Germany €nheit, Institut Correspondence: Peter Scho €r Allgemeine Mikrobiologie, Christianfu Albrechts-Universit€ at Kiel, Am Botanischen Garten 1-9, D-24118 Kiel, Germany. Tel.: +49 431 880 4328/4330; fax: +49 431 880 2194; e-mail: [email protected] Received 20 September 2013; revised 21 October 2013; accepted 21 October 2013. Final version published online 13 November 2013. DOI: 10.1111/1574-6968.12312 MICROBIOLOGY LETTERS Editor: Dieter Jahn Keywords Chloroflexus aurantiacus; acetate formation; adenosine diphosphate-forming acetyl-CoA synthetase; acetate kinase; phosphotransacetylase; Bacteria. Abstract The bacterium Chloroflexus aurantiacus excreted significant amounts of acetate during photohetero trophic growth on glucose and in resting cell suspensions. Up to 1.5 mol acetate per mol glucose were formed. In acetate-forming cells, the activities of phosphotransacetylase and acetate kinase, usually involved in acetate formation in Bacteria, could not be detected; instead, the cells contained an acetyl-CoA synthetase (ADP-forming) (ACD) (acetyl-CoA + ADP + Pi ? acetate + ATP + CoA), an enzyme so far reported in prokaryotes to be specific for acetate-forming Archaea. ACD, which was induced 10-fold during growth on glucose, was purified and the encoding gene was identified as Caur_3920. The recombinant enzyme, a homotetrameric 300-kDa protein composed of 75-kDa subunits, was characterized as functional ACD. Substrate specificities and kinetic constants for acetyl-CoA/acetate and other acyl-CoA esters/ acids were determined, showing similarity of the C. aurantiacus ACD to archaeal ACD I isoenzymes, which are involved in acetate formation from sugars. This is the first report of a functional ACD involved in acetate formation in the domain of Bacteria. Introduction Acetate is an important product of fermentation processes in many anaerobic and facultative prokaryotes of both the bacterial and the archaeal domains. Analyses of the mechanism of acetate formation from acetyl-CoA indicate differences in Bacteria and Archaea. All acetate-forming Bacteria analyzed so far, catalyze acetate formation from acetyl-CoA by the classical mechanism involving the two enzymes phosphotransacetylase (PTA) and acetate kinase (AK). In contrast, in all acetate-forming Archaea, including anaerobic hyperthermophiles and aerobic halophiles, the conversion of acetyl-CoA to acetate is catalyzed by one enzyme, an acetyl-CoA synthetase (ADP-forming) (ACD; Sch€afer et al., 1993). ACD catalyzes the conversion of acetyl-CoA to acetate and couples this reaction with the synthesis of ATP from ADP and Pi via substrate level phosphorylation (acetyl-CoA + ADP + Pi ? acetate + ATP + HSCoA). Thus, the mechanism of acetate formaFEMS Microbiol Lett 349 (2013) 171–179 tion from acetyl-CoA in prokaryotes appears to be domain-specific (Sch€afer et al., 1993; Br€asen et al., 2008). ACD is also present in eukaryotic protists, e.g. Entamoeba histolytica and Giardia lamblia (Reeves et al., 1977; Sanchez & M€ uller, 1996), and ACD homologs have been found in many bacterial genomes (Sanchez et al., 2000). The metabolic functions of these putative ACDs have not been studied so far. Several Bacteria, including the phototroph Chloroflexus aurantiacus and the syntrophs Pelotomaculum thermopropionicum and Syntrophus aciditrophicus, have been reported to generate acetate in their metabolism, but lack genes for AK and PTA. However, these Bacteria contain one or more homologs of ACD genes (Kondratieva et al., 1992; McInerney et al., 2007; Kosaka et al., 2008; Tang et al., 2011), suggesting that these putative ACDs might have a functional role in acetate formation. In this work we studied the formation of acetate and the enzyme involved in the photoheterotrophic bacterium C. aurantiacus. It is shown that C. aurantiacus ferments ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved €nheit M. Schmidt & P. Scho 172 glucose to high amounts of acetate and that acetate formation is catalyzed by an ACD. The C. aurantiacus ACD was purified and the encoding gene identified. The ACD showed similar substrate specificity as ACD isoenzymes I from Archaea. This is the first report of a functional ACD involved in acetate formation in the domain of Bacteria. It is proposed that C. aurantiacus acquired its ACD via lateral gene transfer from Archaea. Materials and methods Growth conditions Chloroflexus aurantiacus strain J-10-fl (DSM 635; Pierson & Castenholz, 1974) was obtained from the Deutsche Sammlung f€ ur Mikroorganismen und Zellkulturen (DSMZ). Chloroflexus aurantiacus was grown under anaerobic conditions at pH 8.3 and at 55 °C in 1-L bottles filled with 1000 mL medium with N2 as gas phase. The bottles were shaken at 35 r.p.m. and illuminated with two 40-W reflector lamps at 600–800 lux. Medium for growth of C. aurantiacus was prepared according to Herter et al. (2001) with a few modifications. The medium contained (per liter): 1 g yeast extract, 2.5 g casamino acids, 3.33 g glycylglycine, 100 mL solution A, 0.5 mL solution B, pH 8.3, 1.25 mL solution C, 1 mL solution D and 20 mM glucose. Solution A contained (per liter): 1 g EDTA, 0.6 g CaSO4H2O, 1 g MgSO47H2O, 0.08 g NaCl, 1.11 g Na2HPO4. Solution B contained (per liter): 2.28 g MnSO47H2O, 0.5 g ZnSO47H2O, 0.5 g H3BO3, 0.025 g CuSO45H2O, 0.025 g Na2MoO42H2O, 0.045 g CoCl26H2O. Solution C contained (per liter): 0.08 g cobalamin, 0.024 g thiamine hydrochloride, 0.4 g niacin, 0.04 g 4-aminobenzoic acid, 0.08 g biotin, 0.04 g calciumD-pantothenic acid, 0.008 g pyridoxal hydrochloride, 0.4 g myoinosite, 0.008 g folic acid, and the pH was adjusted to pH 7 with NaOH. Solution D consisted of a filter-sterilized solution of 2.9 g L 1 FeCl36H2O. The medium was autoclaved and then filter-sterilized solutions C and D, and glucose were added. Growth was monitored by measuring the optical density at 600 nm (DOD600). An DOD600 of 1 corresponded to a protein content of 0.72 mg mL 1 as determined by the Biuret method. Acetate and glucose were determined enzymatically (Kunst et al., 1986; Sch€afer et al., 1993). Preparation of cell extracts For determination of enzyme activity, cells of the stationary phase of growth were harvested by centrifugation (1100 g, 8 °C, 20 min) and suspended in 100 mM Hepes-KOH pH 7.5 and 5 mM MgCl2. Cells were disrupted by passing four times through a French pressure ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved cell at 14 000 psi. Cell debris was removed by centrifugation for 120 min at 4 °C with 100 000 g. Protein was determined by the Bradford method. Cell-suspension experiments Cell suspensions of C. aurantiacus were prepared after growth on media (see above) containing glucose but omitting yeast extract. Cells of the exponential phase of growth were harvested by centrifugation and washed three times in the same volume of suspension buffer (100 mM Hepes-KOH pH 8.5 with 10% Solution A). Suspended cells were incubated at 55 °C for 45 min to degrade potentially accumulated reserve substances, washed once and then suspended in the same buffer at a protein concentration of 13 mg mL 1. Cell-suspension experiments were performed in 100-mL bottles filled with a 20-mL cell suspension at 55 °C, under anaerobic conditions (N2 gas phase) in the dark. Purification of ACD from C. aurantiacus (Caur-ACD) Cell extracts was prepared from 96 g wet weight cells obtained after photoheterotrophic growth on glucose and was incubated at 70 °C for 10 min and centrifuged for 45 min at 100 000 g and 4 °C. The supernatant was adjusted to 0.5 M (NH4)2SO4 followed by an additional centrifugation step, and was applied to a Phenyl Sepharose 26/10 HiLoad column equilibrated with 100 mM Hepes-KOH pH 7.5 containing 5 mM MgCl2, 500 mM (NH4)2SO4 and 10% (v/v) glycerol. Protein was desorbed by linear gradient to 200 mM (NH4)2SO4. Fractions containing the highest ACD activity eluting at 280–230 mM (NH4)2SO4 were pooled, diluted 50-fold in buffer A (50 mM Hepes-KOH, pH 8.0, containing 5 mM MgCl2 and 10% glycerol) and applied to a Q-Sepharose 16/10column equilibrated with buffer A. The column was washed with buffer A and protein was eluted by increasing NaCl concentrations up to 1 M. The highest ACD activity eluted at 140–180 mM NaCl. The fractions with the highest activity were concentrated by Amicon ultrafiltration (cutoff 30 kDa) to a final volume of 1 mL and applied to a Superdex 200 HiLoad column (1.6 9 60 cm) equilibrated with 100 mM Tris–HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2 and 10% glycerol (buffer B). Isocratic elution was performed with 120 mL buffer B. Fractions with the highest activity were diluted 50-fold in buffer A and applied to Mono-Q-Sepharose column (1 mL) equilibrated with buffer A. The column was washed and protein was eluted with increasing NaCl concentrations up to 170 mM. The highest ACD activity eluted at 115 mM NaCl. At this step, ACD was almost pure as analyzed by FEMS Microbiol Lett 349 (2013) 171–179 173 ADP-forming acetyl-CoA synthetase in Chloroflexus SDS PAGE. The gene encoding ACD was identified by matrix-assisted laser desorption-time of flight (MALDITOF) mass spectrometry (Schaffer et al., 2001). Cloning and expression of Caur-ACD and purification of the recombinant enzyme Caur_3920 was amplified from genomic DNA of C. aurantiacus by PCR and cloned into pET19b by two restricition sites (NdeI and XhoI) created with the primers: 5′ CCCATCATATGCTAGAAGC and 5′CTCGAGCATTAG TTGAGGA (restriction sites are underlined). For expression of Caur_3920 the vector pET19bCaur_ACD was transformed into Escherichia coli BL21(DE3) codon plus RIL (Stratagene). Growth was performed in Luria–Bertani medium with chloramphenicol (34 lg mL 1) and carbenicillin (100 lg mL 1) at 37 °C. Expression was induced by the addition of 1 mM isopropyl-1-thio-b-D-galactopyranoside at an OD600 of 0.8. After 4 h of further growth, the cells were harvested by centrifugation (20 min at 8 °C and 4500 g) and resuspended in 100 mM Tris–HCl, pH 8.2, containing 300 mM NaCl and 10 mM imidazole (buffer C). Cells were disrupted by sonication followed by centrifugation at 100 000 g for 90 min. The supernatant was applied to a Nickel-nitrilotriacetic acid (Ni-NTA) column equilibrated with buffer C. Protein was eluted with increasing concentrations of imidazole. ACD-containing fractions eluted at 250 mM imidazole were concentrated by ultrafiltration to a total volume of 1.2 mL (30-kDa cutoff) and applied to a Superdex 200 HiLoad 16/60 column equilibrated with 100 mM Tris–HCl pH 7.5 and 150 mM NaCl. Protein was eluted by an isocratic flow, yielding pure protein. Enzyme assays ACD activity was measured at 55 °C in both reaction directions. In the direction of acetate formation, ACD activity was followed as ADP- and Pi-dependent release of HSCoA from acetyl-CoA with Ellman’s thiol reagent, 5,5dithiobis (2-nitrobenzoic acid) (DTNB), by measuring the formation of thiophenolate anion at 412 nm (e412 = 13.6 mM 1 cm 1) (Srere et al., 1963). The assay mixture (1 mL) contained 100 mM Hepes-KOH pH 7.5, 0.1 mM DTNB, 5 mM MgCl2, 2 mM ADP, 0.1 mM acetyl-CoA, 20 mM KH2PO4. In the direction of acetate activation, ACD activity was measured as HSCoA and ATP-dependent formation of ADP by coupling the reaction with the oxidation of NADH via pyruvate kinase (PK) and lactate dehydrogenase (LDH). The assay contained 100 mM Hepes-KOH, 5 mM MgCl2, 2.5 mM PEP, 0.3 mM NADH, 4 U PK, 6 U LDH, 10 mM acetate, 2 mM ATP, 1 mM HSCoA. The pH- and temperature-dependence of FEMS Microbiol Lett 349 (2013) 171–179 recombinant enzyme was monitored with Ellman’s thiol reagent. The temperature-dependence was determined between 20 and 85 °C adjusting the pH of the buffer to 7.5 at the specific temperature. The pH-dependence was determined between pH 5.0 and 8.5 using piperazine (pH 5.0–6.0), MES (pH 6.0–7.0), HEPES (pH 7.0–8.0) or glycylglycine (pH 8.0–8.5). Phosphotransacetylase activity was measured as Pi-dependent release of HSCoA from acetyl-CoA with DTNB using the same assay as described above for ACD but omitting ADP. Activity of AK was determined as described in Sch€afer et al. (1993). Results Acetate formation from glucose by C. aurantiacus Chloroflexus aurantiacus was grown photoheterotrophically on glucose (20 mM) in the presence of casamino acids and yeast extract under a 100% N2 gas phase. The cells grew with a doubling time of about 19 h to a cell density DOD600 of 7.5. During growth, glucose (12 mM) was consumed and, in parallel, acetate (about 8 mM) was formed (Fig. 1a). In the absence of glucose, cells grew to an DOD600 of 4 with a doubling time of 22 h and about 2.5 mM acetate was formed from complex constituents (Fig. 1b). To prove acetate formation from glucose, resting cells of glucose-grown C. aurantiacus were incubated in the dark in the absence of complex constituents. Under these conditions the cells consumed glucose and formed acetate at a ratio of 1.5 mol acetate per mol glucose (Fig. 2), indicating that acetate is the main product of C. aurantiacus fermentation of glucose. Acetate formation in C. aurantiacus is catalyzed by an ACD Extracts of glucose-grown cells were analyzed for enzyme activities of acetate formation from acetyl CoA. Activities of PTA and AK could not be detected. Instead, cell extracts contained an ADP-forming ACD activity of 0.2 U mg 1. The ACD activity in cells grown in the absence of glucose was 0.022 U mg 1, indicating an c. 10-fold induction of ACD upon growth on glucose. Purification of ACD from C. aurantiacus and identification of the encoding gene ACD was purified from glucose-grown cells involving heat precipitation followed by four chromatographic steps (Supporting Information, Table S1). In this procedure, ACD was purified 507-fold to a specific activity of 45.6 U mg 1. SDS-PAGE revealed one subunit with an apparent ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved €nheit M. Schmidt & P. Scho 174 (a) (b) Fig. 1. (a, b) Growth of Chloroflexus aurantiacus on 20 mM glucose, 0.1% yeast extract and 0.25% casamino acids (a) or on yeast extract/ casamino acids (b). The cultures were incubated under anaerobic conditions in the light. Growth (■), glucose consumption (●) and acetate formation (▲) were followed over time. molecular mass of 75 kDa (Fig. 3). The molecular mass of the native ACD, determined by gel filtration on Superdex 200, was 300 kDa, indicating a homotetrameric structure. The gene encoding ACD was determined by peptide mass fingerprinting of the 75-kDa subunit, resulting in the identification of a single ORF, Caur_3920, in the genome of C. aurantiacus. The matched peptides covered 42% of the protein. Thus, Caur_3920 represents the ACD encoding gene, acd, in C. aurantiacus. This was proved by functional overexpression of Caur_3920 in E. coli. Expression of the acd gene and purification of recombinant ACD Caur_3920 consisted of 2091-bp coding for a polypeptide of 696 amino acids with a calculated molecular mass of 75.1 kDa. Caur_3920 was cloned into pET19b and transformed into E. coli BL21 (DE3)-codon plus RIL and expressed. A 77-kDa His-tagged protein was produced in accordance with the calculated molecular mass. The recombinant ACD was purified to homogeneity by NiNTA affinity chromatography and gel filtration on Superdex 200 (Fig. 3b). The apparent molecular mass of recombinant His-tagged ACD determined by gel filtration was ca. 317 kDa. SDS-PAGE revealed one subunit of 77 kDa, indicating a homotetrameric structure of the native enzyme. ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved Catalytic properties and substrate specificity of recombinant ACD Kinetic properties of ACD catalyzing the reversible conversion of acetyl-CoA/acetate (acetyl-CoA + ADP + Pi ↔ acetate + ATP + CoA) were determined at 55 °C, pH 7.5. In both directions of the reaction, the rate dependence on the substrate concentrations followed Michaelis–Menten kinetics. The apparent Vmax and Km values in the direction of acetate formation were 51 U mg 1 and 0.037 mM (acetyl-CoA), 0.091 mM (ADP), and 1.0 mM (phosphate), respectively. In the direction of acetate activation the apparent Vmax and Km values were 49 U mg 1, and 0.9 mM (acetate), 0.57 mM (ATP), and 0.024 mM (HSCoA), respectively. The pH optimum was at pH 7.5, with remaining activities of about 40% at pH 7.3 and 59% at pH 7.8. ACD activity was dependent on MgCl2, showing the highest activity at about 5 mM. The temperature optimum was at 55 °C. From the linear part of the Arrhenius plot between 20 and 55 °C, an activation energy of 49 kJ mol 1 was calculated. Different acyl- and aryl-acids were tested as substrates (10 mM) for ACD. Besides acetate (100%), the ACD effectively utilized propionate (100%) and butyrate (98%), thioglycolate (110%), and branched chain fatty acids isovalerate (65%), isobutyrate (46%) and 2-methylbutyrate (75%). With lower activities, ACD accepted the FEMS Microbiol Lett 349 (2013) 171–179 175 ADP-forming acetyl-CoA synthetase in Chloroflexus This is the first report of a functional ACD involved in acetate formation in the domain of Bacteria. Acetate formation from glucose in C. aurantiacus involves an ACD Fig. 2. Glucose conversion to acetate in cell suspensions from Chloroflexus aurantiacus. Glucose-grown cells were incubated under anaerobic conditions in the dark in the presence of 10 mM glucose. Glucose consumption (▲) and acetate formation (■) were followed over time. As a control, acetate formation (●) was followed in the absence of glucose. aromatic acids imidazole-4-acetate (17%) and phenylacetate (10%). 4-Hydroxyphenylacetate, indol-3-acetate and succinate were not utilized by ACD. Culture and cell suspension experiments showed that C. aurantiacus converted glucose to acetate as the major fermentation product. Up to 1.5 mol acetate per mol glucose were formed. The formation of acetate by C. aurantiacus in glucose-containing media has been reported earlier (Krasil’nikova & Kondrat’eva, 1987); however, acetate/glucose stoichiometries have not been determined. Glucose degradation to pyruvate has been proposed to involve the classical EM pathway, as concluded from enzyme measurements and genome information (Kondratieva et al., 1992; Tang et al., 2011). However, the enzymes involved in acetate formation have not been analyzed. Here we report that in cell extracts of glucosegrown C. aurantiacus, activities of PTA and AK, that is the enzymes typical for acetate-forming Bacteria, could not be detected. This is in accordance with the absence of the respective genes in the genome of the organism (Tang et al., 2011). Instead, the cells contained an ADP-forming ACD activity that was induced 10-fold in glucose-grown cells, indicating a functional involvement in acetate formation from glucose. So far, ACDs have been reported in prokaryotes to be typical for acetate-forming Archaea, and this is the first report of the presence of ACD in a bacterium. Thus, glucose degradation to acetate in the bacterium C. aurantiacus appears to be a chimeric pathway consisting of a bacterial-like Embden–Meyerhof pathway, and an archaeal-like acetate-forming enzyme, ACD. Discussion The present study shows that the photoheterotrophic bacterium C. aurantiacus converted glucose to acetate as the main product and that acetate formation from acetylCoA is catalyzed by an archael type ADP-forming ACD. (a) Caur-ACD is related to archaeal ACD I isoenzymes ACD from C. aurantiacus was purified and the encoding gene acd was identified as Caur_3920, which was overex(b) Fig. 3. (a, b) Purified ADP-forming ACD from Chloroflexus aurantiacus (a) and recombinant His-tagged enzyme isolated from transformed Escherichia coli (b) as analyzed by SDS-PAGE. (a) Lane 1, molecular mass markers, lane 2 purified enzyme after Mono-Q-Sepharose; (b) Lane 1, molecular mass markers; lane 2, purified recombinant enzyme. FEMS Microbiol Lett 349 (2013) 171–179 ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved €nheit M. Schmidt & P. Scho 176 Fig. 4. Comparison of subunit composition and oligomeric structures of Chloroflexus aurantiacus ACD and characterized archaeal ACD I isoenzymes. Arrows indicate the ORFs encoding the subunits and the subunit sizes. Homologous regions of a-subunit are shaded in dark gray and homologous regions of b-subunit in light gray. Amino acid sequence identities in comparison with the homologous region of C. aurantiacus ACD are given as percent. Substrate C. aurantiacus* Direction of CoA-ester formation (%) Acetate 100 Isobutyrate 91 Isovalerate 65 Indol-3-acetate NM Phenylacetate 10 Succinate NM Km-value (mM) Acetate 0.90 ATP 0.57 HSCoA 0.02 Direction of acid formation (%) Acetyl-CoA 100 Phenylacetyl-CoA 13 Isobutyryl-CoA 32 Km-value (mM) Acetyl-CoA 0.037 ADP 0.09 1.0 KH2PO4 A. fulgidus† H. marismortui‡ P. furiosus§ 100 56 10 4 8 9 100 38 25 2 3 NM 100 85 ND NM NM NM 0.34 0.133 0.027 100 NM ND 0.01 0.01 0.11 1.7 0.38 0.06 100 1 ND 0.41 0.02 1.3 Table 1. Comparative analysis of substrate specificities of recombinant Chloroflexus aurantiacus ACD to characterized archaeal ACD I isoenzymes 1.1 0.48 0.018 100 NM 32 0.025 0.15 0.396 Specific activities (U mg 1) are shown as percent activity in the direction of CoA-ester formation and acid formation, respectively. One hundred percent correspond to either 49 U mg 1 (acetate) or 51 U mg 1 (acetyl-CoA) (C. aurantiacus), 70 or 60 U mg 1 (Archaeoglobus fulgidus), 40 or 48 U mg 1 (Haloarcula marismortui) and 27 or 65 U mg 1 (Pyrococcus furiosus). *Kinetic constants were determined at 55 °C as described in Materials and methods. Assay mixture contained 10 mM of acid and 0.1 mM of CoA-ester. † €nheit (2002). Musfeldt & Scho ‡ €nheit (2004b). Br€ asen & Scho § Mai & Adams (1996). NM, not measurable; ND, not determined. pressed in E. coli to yield a recombinant ACD with similar properties as the native enzyme. Chloroflexus aurantiacus ACD, designated as Caur-ACD, was compared with characterized ACDs from hyperthermophilic and halophilic Archaea with respect to molecular properties and ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved substrate specificities. ACD from the hyperthermophiles Pyrococcus furiosus and Thermococcus kodakaraensis comprise 145-kDa heterotetramers composed of two a-subunits (47 kDa) and two b-subunits (25 kDa) (Fig. 4; Mai & Adams, 1996; Glasemacher et al., 1997; Shikata et al., FEMS Microbiol Lett 349 (2013) 171–179 177 ADP-forming acetyl-CoA synthetase in Chloroflexus 2007). The ACDs from the halophilic archaeon Haloarcula marismortui, and the hyperthermophilic Archaea Archaeoglobus fulgidus and Methanococcus jannaschii are 145-kDa homodimeric proteins composed of subunits representing fusions of homologous a- and b-subunits of the P. furiosus ACD (Musfeldt & Sch€ onheit, 2002; Br€asen & Sch€ onheit, 2004b). ACD from the eukaryotic protist G. lamblia is composed of 78-kDa subunits (Sanchez et al., 1999). With respect to their substrate specificities, several ACD isoenzymes have been identified in Archaea; for example, in P. furiosus two ACD isoenzymes, I and II, have been characterized. ACD I preferentially utilizes acetyl-CoA over aryl-CoA esters such as phenylacetyl-CoA and is primarily involved in sugar fermentation, whereas ACD II showed a preference for aryl-CoA esters over acetyl-CoA and is primarily implicated in the degradation of (aromatic) amino acids (Mai & Adams, 1996; Glasemacher et al., 1997; Adams et al., 2001). ACD I and II enzymes have also been characterized in A. fulgidus, an ACD II in T. kodakaraensis and an ACD I in H. marismortui (Musfeldt & Sch€ onheit, 2002; Br€asen & Sch€ onheit, 2004b; Shikata et al., 2007). Besides ACD I and II, other ACD isoenzymes with different substrate specificities have been reported, for example, for Pyrobaculum aerophilum and T. kodakaraensis and for the eukaryotic protist G. lamblia (Sanchez & M€ uller, 1996; Br€asen & Sch€ onheit, 2004b; Shikata et al., 2007). Caur-ACD is composed of 75-kDa subunits which are fusions of the a- and b-subunit homologs of P. furiosus ACD. With a molecular mass of 300 kDa for the native enzyme, Caur-ACD represents the largest of all ACDs characterized so far. Based on substrate specificities for CoA ester and the corresponding acids, Caur-ACD can be attributed to ACD I isoenzymes from Archaea. A comparison of substrate specificities and kinetic constants of the Caur-ACD and characterized archaeal ACD I isoenzymes is given in Table 1. Caur-ACD and archaeal ACD I isoenzymes showed a strong preference for acetyl-CoA/acetate over phenylacetyl-CoA/phenylacetate and accepted the branched chain fatty acids isobutyrate and isovalerate, but did not utilize succinate. These properties are characteristic of ACD I isoenzymes. Also, kinetic constants for selected CoA esters and acids were similar in all ACD I isoenzymes. ACD I of the anaerobic P. furiosus constitutes a major energy-conserving site in sugar degradation (Mai & Adams, 1996; Glasemacher et al., 1997; Adams et al., 2001). In the aerobic H. marismortui, ACD I is proposed to be involved in acetate formation from glucose as part of an overflow mechanism (Br€asen & Sch€ onheit, 2004a). 41 Fig. 5. Phylogenetic relationships of Chloroflexus aurantiacus ACD to selected ACD of Archaea, Bacteria and Eukarya. The numbers at the nodes are bootstrapping values (%) according to maximum parsimony (500 replicates). Only bootstrapping values above 40% are shown. Alignment was performed using Pyrococcus furiosus ACD I a-subunit homologous region of the selected ACDs. Analyses were conducted in MEGA 5.2. Accession no.: Archaeoglobus fulgidus 1211 ACD I NP_070039.1; Chloroflexus aggregans ZP_01517440.1; C. aurantiacus NC_010175; Entamoeba histolytica XP_656290.1; Giardia lamblia XP_001705744.1; Haloarcula marismortui YP_135572.1; Haloferax volcanii YP_003535057.1; Methanocaldococcus jannaschii NP_247570.1; Methanosarcina €1 NP_632382.1 Pelotomaculum mazei Go thermopropionicum YP_001212057.1; Pyrobaculum aerophilum NP_560604; P. furiosus ACD I NP_579269.1; P. furiosus ACD II NP_578261.1; Roseiflexus castenholzii YP_001430231.1; Syntrophus aciditrophicus YP_460839.1; Thermococcus kodakaraensis YP_183078.1. FEMS Microbiol Lett 349 (2013) 171–179 G. lamblia P. thermopropionicum 60 R. castenholzii 65 C. aggregans 99 100 C. aurantiacus A. fulgidus 1211 ACDI H. marismortui ACDI 46 100 H. volcanii E. histolytica M. jannaschii S. aciditrophicus 74 P. aerophilum M. mazei P. furiosus ACDII P. furiosus ACDI 67 100 T. kodakaraensis ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved €nheit M. Schmidt & P. Scho 178 The similarity of Caur-ACD with ACD I isoenzymes of Archaea is in accordance with its role in acetate formation from glucose in C. aurantiacus, reported in this paper. In summary, Caur-ACD represents the first functional ACD within the domain of Bacteria. It is involved in the formation of acetate from acetyl-CoA and thus likely substitutes for the classical bacterial acetate-forming enzymes AK/PTA, which are missing in C. aurantiacus. It is proposed that Caur-ACD originates from archaeal ACD I type isoenzymes, which are taken up by C. aurantiacus via lateral gene transfer. To identify the archaeal ACD I most closely related to Caur-ACD, the degree of sequence identity, the subunit size and the phylogenetic relationship of Caur-ACD were analyzed in comparison with characterized archaeal ACD I isoenzymes (Fig. 4). Caur-ACD showed similar high sequence identity, about 45–50%, to all archaeal ACD I isoenzymes. However, based on subunit size, Caur-ACD is more closely related to ACD I isoenzymes from A. fulgidus and H. marismortui, which are all composed of ‘fused’ 70–85 kDa subunits. In contrast, P. furiosus ACD I consists of separate 25 kDa a- and 47 kDa b-subunits (Fig. 4). In accordance with these findings, a phylogenetic tree of Caur-ACD and selected bacterial homologs, characterized archaeal ACD I enzymes and other ACDs mentioned in text (Fig. 5), indicates that Caur-ACD is more closely related to the archaeal ACD I enzymes from H. marismortui and A. fulgidus, rather than to ACD I from P. furiosus. Thus, one might speculate that C. aurantiacus acquired its ACD by lateral gene transfer of an ACD I isoenzyme from Archaeoglobus/Haloarcula species. ACD homologs with high sequence identity, about 50%, of Caur-ACD were found in all Chloroflexaceae analyzed, suggesting a similar function in acetate formation as in C. aurantiacus. Further, several syntrophic Bacteria such as S. aciditrophicus and P. thermopropionicum, which form acetate in their metabolism, do not contain AK/PTA genes (McInerney et al., 2007; Kosaka et al., 2008) but do contain ACD homologs. 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