RESEARCH ARTICLE
Conversion of phenyl methyl ethers by Desulfitobacterium spp. and screening for the genes involved Felix S. Mingo, Sandra Studenik & Gabriele Diekert Department of Applied and Ecological Microbiology, Institute of Microbiology, Friedrich Schiller University Jena, Jena, Germany
Correspondence: Sandra Studenik, Department of Applied and Ecological Microbiology, Institute of Microbiology, Friedrich Schiller University Jena, Philosophenweg 12, 07743 Jena, Germany. Tel.: +49 3641 949300; fax: +49 3641 949302; e-mail: [email protected] Received 25 June 2014; revised 9 September 2014; accepted 30 September 2014. Final version published online 20 October 2014. DOI: 10.1111/1574-6941.12433
MICROBIOLOGY ECOLOGY
Editor: Alfons Stams Keywords metabolism; anaerobic bacteria; O-demethylase; Desulfitobacterium.
Abstract Microbial growth coupled to O-demethylation of phenyl methyl ethers, which are lignin decomposition products, was described for acetogenic bacteria and recently also for two species belonging to the nonacetogenic genus Desulfitobacterium. To elucidate the potential role of desulfitobacteria in the O-demethylation of phenyl methyl ethers in the environment, we cultivated Desulfitobacterium chlororespirans, D. dehalogenans, D. metallireducens, and different strains of D. hafniense with phenyl methyl ethers as sole electron donors. With the exception of D. metallireducens, all species and strains tested were able to demethylate at least three of the four phenyl methyl ethers applied with fumarate, nitrate, or thiosulfate as electron acceptor. Furthermore, a high number of operons putatively encoding demethylase systems were identified in the genomes of Desulfitobacterium spp., although discrimination between O-, S-, N- and, Cl-demethylases was not possible. These findings provide evidence for the importance of the methylotrophic metabolism for desulfitobacteria and point to their involvement in the O-demethylation of phenyl methyl ethers in the environment.
Introduction Desulfitobacteria, which are most commonly described as reductively dehalogenating bacteria, are able to utilize phenyl methyl ethers as growth substrates when an appropriate electron acceptor (fumarate or a chlorinated phenol) is provided (Neumann et al., 2004). Prior to this finding, utilization of these substrates had only been described for members of the acetogenic bacteria (Bache & Pfennig, 1981; Daniel et al., 1991; Traunecker et al., 1991; Stupperich & Konle, 1993; Liesack et al., 1994). Phenyl methyl ethers are the main decomposition products of lignin and therefore widespread in the environment, especially in soil. Abundant representatives of this class include, for example, syringate, vanillate, and isovanillate (Chen et al., 1982, 1983; K€ ogel, 1986). Phenyl methyl ethers typically result from the conversion of lignin by ligninolytic enzymes that are produced by white rot fungi under aerobic conditions (Higuchi, 1990). Once they are released from the lignin macrostructure, they become available to the soil microbial community. In acetogens and desulfitobacteria, the methyl groups of phenyl methyl ethers are cleaved off by O-demethylase FEMS Microbiol Ecol 90 (2014) 783–790
enzyme systems (Kaufmann et al., 1997; Naidu & Ragsdale, 2001; Studenik et al., 2012) and the further oxidation of the methyl moiety to CO2 plays a key role for energy conservation (for a review see Drake et al., 2006). O-demethylases, which are induced by their respective substrates (Engelmann et al., 2001; Peng et al., 2012), consist of two methyltransferases (MTs) (MT I and MT II), a corrinoid protein (CP), and an activating enzyme (AE) (Kaufmann et al., 1997; Schilhabel et al., 2009; Studenik et al., 2012). The reaction mechanism is depicted in Fig. 1. In the first step, MT I cleaves the ether bond of the methoxylated substrate and transfers the methyl group to the super-reduced corrinoid cofactor ([CoI]) of CP. Subsequently, MT II transfers the methyl group from the methylated corrinoid to tetrahydrofolate (FH4) yielding methyltetrahydrofolate (CH3-FH4). The last protein component of the system, AE, has a repair function after inadvertent oxidation of the super-reduced corrinoid cofactor ([CoI]) to inactive [CoII]-CP. It catalyzes the ATP-dependent electron transfer from an unknown electron donor to [CoII]-CP yielding physiologically active [CoI]-CP, allowing CP to participate in further O-demethylation reactions (Siebert et al., 2005; ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
784
F.S. Mingo et al.
fumarate, nitrate, and thiosulfate, was followed in growth experiments of D. chlororespirans, D. dehalogenans, D. metallireducens, and different strains of D. hafniense. Additionally, bioinformatic analyses of available genome sequences of various Desulfitobacterium species were carried out to uncover the O-demethylation potential of these organisms.
Materials and methods Bacterial strains
Fig. 1. Scheme of the O-demethylase reaction adapted from Schilhabel et al. (2009). AE, activating enzyme; [CoI-III], corrinoid protein (CP) with cobalt in the respective oxidation state; FH4, tetrahydrofolate; MT, methyltransferase.
Sperfeld et al., 2014). For acetogenic bacteria, it has been described that the phenyl methyl ether-derived methyl group in CH3-FH4 undergoes oxidation to CO2. The reducing equivalents generated by methyl group oxidation are transferred to CO2 that is reduced to enzyme-bound carbon monoxide. Acetyl-CoA is formed in the carbon monoxide dehydrogenase/acetyl-CoA synthase reaction from the enzyme-bound CO and methyl groups derived from phenyl methyl ether demethylation. Acetyl-CoA can then either be converted to acetate to conserve energy or it can be incorporated into cell carbon (Ragsdale & Pierce, 2008). In contrast to acetogens, desulfitobacteria appear to be unable to use CO2 as a terminal electron acceptor for O-demethylation (Neumann et al., 2004; Kreher et al., 2008), which renders their reductive acetylCoA pathway nonfunctional for energy conservation. Instead, they rely on alternate electron acceptors that might be provided by the microbial community or by the environment. It was previously shown that Desulfitobacterium hafniense DCB-2 and D. hafniense PCE-S use fumarate as terminal electron acceptor and that D. hafniense DCB-2 was also able to couple O-demethylation of vanillate to the reductive dechlorination of 3-chloro-4hydroxyphenylacetic acid to 4-hydroxyphenylacetic acid (Neumann et al., 2004). However, nothing is known about possible physiological electron acceptors that might be present in natural environments such as forest soil, where phenyl methyl ethers are available as substrates for the soil microbial community. The objective of this study was to shed light on the general importance of the methylotrophic metabolism for Desulfitobacterium spp. and to elucidate their potential role in the O-demethylation of phenyl methyl ethers in the environment. For this purpose, phenyl methyl ether consumption with different electron acceptors, namely ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
Desulfitobacterium hafniense strains DCB-2 (DSM-10664), DP7 (DSM-13498), G2 (DSM-16228), PCE-S (DSM14645), PCP-1 (DSM-12420), and TCP-A (DSM-13557) as well as D. chlororespirans (DSM-11544), D. dehalogenans (DSM-9161), and D. metallireducens (DSM-15288) were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany). Desulfitobacterium hafniense strain Y51 was taken from the strain collection of our laboratory and was kindly provided by Taiki Futagami (Kagoshima University, Kagoshima, Japan). Growth of bacteria
Desulfitobacterium spp. was grown anoxically in rubberstoppered serum bottles under 100% N2 atmosphere as described by Neumann et al. (2004). The electron donors (syringate, vanillate, isovanillate, or 4-hydroxyanisole) and acceptors (fumarate, nitrate, Fe(III), or thiosulfate) were supplied from separately autoclaved anoxic stock solutions. The concentrations applied were as follows: phenyl methyl ethers, 2 mM; fumarate, 20 mM; nitrate and thiosulfate, 5 mM; Fe(III) citrate, 12 mM. Media were inoculated with precultures grown with pyruvate and fumarate (each 40 mM) as electron donor and acceptor, respectively. For precultures of D. metallireducens, Fe (III) (c. 20 mM) was used as electron acceptor instead of fumarate. The culture volume for inoculation was 10% (v/v) of the final volume of the main culture. Cultures were incubated in a water bath shaker at 28 °C and 150 r.p.m. Samples were taken and analyzed for growth and metabolites. Analytical methods
The protein concentration was used as an indicator of bacterial growth and was determined according to the method of Bradford (1976) after cell lysis by alkaline treatment. The concentrations of phenyl methyl ethers and fumarate were determined by HPLC using a LiChrospher 100 FEMS Microbiol Ecol 90 (2014) 783–790
785
Phenyl methyl ether conversion by desulfitobacteria
RP-8 125 9 4 mm column (Merck KGaA, Darmstadt, Germany) and 25% methanol (v/v) plus 0.3% H3PO4 (v/v) in water as eluent. A flow rate of 0.4 mL min1 was applied. Signals were detected at 210 nm. Under these conditions, the retention times were as follows: fumarate, 6.3 min; vanillate, 8.3 min; 4-hydroxyanisole, 8.6 min; isovanillate, 8.8 min; syringate, 9.2 min. For separation of fumarate, vanillate, and its demethylated end product 3,4dihydroxybenzoate, a gradient with a flow rate of 0.3 mL min1 was applied: 0–11 min, 0.3% H3PO4; 11– 21 min, 0–25% methanol plus 0.3% H3PO4; 21–30 min, 25% methanol plus 0.3% H3PO4. Under these conditions, the retention times were as follows: fumarate, 7.1 min; 3,4-dihydroxybenzoate, 14.9 min; vanillate, 24.0 min. Nitrite was measured by a diazotization reaction described by Lunge (1904). The colorimetric reaction was started by the addition of 50 lL of 1% (w/v) sulphanilic acid and 50 lL of 0.3% (w/v) 1-naphthylamine to 50 lL of the diluted sample. After 5 min incubation at room temperature, absorption was measured at a wavelength of 525 nm on a VERSAmax tunable microplate reader (Molecular Devices, Biberach an der Riss, Germany). For nitrate determination, the method of Bosch Serrat (1998) was followed. Diluted samples were mixed in a 1 : 1 ratio with 200 lL of a chloride solution consisting of 0.28 M NaCl dissolved in 9.35% (v/v) H3PO4. Subsequently, 1 mL of 0.55% (w/v) resorcinol dissolved in 62.5% (v/v) H2SO4 was added to each sample. The mixtures were incubated at 95 °C for 8 min. They were allowed to cool down for 20 min and absorbance was measured at 505 nm. As in this assay both nitrite and nitrate were detected, nitrate concentration was calculated by subtracting the nitrite concentration from the value obtained. Thiosulfate was measured according to Quentin & Prachmayr (1964). Diluted samples were mixed in a 3 : 1 ratio with 0.006% (w/v) methylene blue in 6 M HCl. After 3 h of incubation at room temperature, the absorbance was measured at 660 nm. In silico mapping of putative O-demethylase operons
Mapping of putative O-demethylase operons of Desulfitobacterium spp. was carried out with the NCBI BLAST server (Altschul et al., 1990) and the Integrated Microbial Genomes (IMG) system (Markowitz et al., 2014). As template for BLASTP searches, the amino acid sequences of Dhaf_4610, Dhaf_4611, Dhaf_4612, and Dhaf_2573, which represent the protein components of an O-demethylase previously identified in D. hafniense DCB-2 (Studenik et al., 2012), were used. The putative O-demethylase operons identified in the genomes of D. hafniense DCB-2 FEMS Microbiol Ecol 90 (2014) 783–790
(Kim et al., 2012), DP7, PCE-S, PCP-1, TCE-1, TCP-A, and Y51 (Nonaka et al., 2006) as well as D. dehalogenans, D. dichloroeliminans, and D. metallireducens were compared in terms of organization and similarity of the encoded proteins.
Results Growth of Desulfitobacterium spp. with phenyl methyl ethers as electron donors
Desulfitobacterium hafniense (strains DCB-2, DP7, G2, PCE-S, PCP-1 TCP-A, Y51), D. chlororespirans, D. dehalogenans, and D. metallireducens were cultivated with 4hydroxyanisole, syringate, vanillate, or isovanillate in the presence of fumarate, nitrate, or thiosulfate as electron acceptors (Table 1). The results were obtained from at least duplicates of the experiments. All species with the exception of D. metallireducens were able to grow on at least three of the supplied phenyl methyl ethers by cleaving the ether bond. Vanillate and isovanillate were O-demethylated to 3,4-dihydroxybenzoate, 4-hydroxyanisole to hydroquinone, and syringate via 3,4-dihydroxy5-methoxybenzoate to 3,4,5-trihydroxybenzoate. The stoichiometry of demethylated end product formed per substrate consumed was about 1 : 1. Fumarate as well as nitrate or thiosulfate served as terminal electron acceptors for methyl group oxidation following O-demethylation. The substrate spectra and the demethylation rates of the Desulfitobacterium strains used in this study are summarized in Table 1. O-demethylation was independent of the type of electron acceptor used; however, the rates of phenyl methyl ether consumption were different. For most phenyl methyl ethers, the rates of demethylation and cell growth were highest in the presence of fumarate followed by nitrate and thiosulfate. As reported before for D. hafniense DCB-2 and PCE-S (Neumann et al., 2004; Kreher et al., 2008), no demethylation of the substrates occurred when CO2 was applied as sole terminal electron acceptor (data not shown). Figure 2 shows the consumption of vanillate by D. hafniense Y51 in the presence of fumarate, nitrate, or thiosulfate, respectively. The stoichiometries of fumarate/nitrate/thiosulfate reduced per mol methyl group consumed were usually higher than the values expected from the substrate conversion according to the following equations: 1 R-O-CH3 þ 3 fumarate þ 2 H2 O ! 1 R-OH þ 3 succinate þ CO2
(1)
þ 4 R-O-CH3 þ 3 NO 3 þ 6 H ! 4 R-OH þ 4 CO2 þ 3 NHþ 4 þ H2 O
(2)
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
786
F.S. Mingo et al.
Table 1. Maximal rate of phenyl methyl ether consumption by growing Desulfitobacterium spp. (lM h1) in the presence of fumarate (a; first row), nitrate (b; second row), or thiosulfate (c; third row) as electron acceptor. For further details, see ‘Materials and Methods’ Demethylation rates (lM h1)
Species and strain
4-Hydroxyanisole OH
Syringate
H3 CO
OCH3 Desulfitobacterium hafniense
DCB-2
DP7
G2
PCE-S
PCP-1
TCP-A
Y51
D. dehalogenans
D. chlororespirans
39 28 37 26 14 17 75 21 31 54 17 26 14
Conversion of phenyl methyl ethers by Desulfitobacterium spp. and screening for the genes involved Felix S. Mingo, Sandra Studenik & Gabriele Diekert Department of Applied and Ecological Microbiology, Institute of Microbiology, Friedrich Schiller University Jena, Jena, Germany
Correspondence: Sandra Studenik, Department of Applied and Ecological Microbiology, Institute of Microbiology, Friedrich Schiller University Jena, Philosophenweg 12, 07743 Jena, Germany. Tel.: +49 3641 949300; fax: +49 3641 949302; e-mail: [email protected] Received 25 June 2014; revised 9 September 2014; accepted 30 September 2014. Final version published online 20 October 2014. DOI: 10.1111/1574-6941.12433
MICROBIOLOGY ECOLOGY
Editor: Alfons Stams Keywords metabolism; anaerobic bacteria; O-demethylase; Desulfitobacterium.
Abstract Microbial growth coupled to O-demethylation of phenyl methyl ethers, which are lignin decomposition products, was described for acetogenic bacteria and recently also for two species belonging to the nonacetogenic genus Desulfitobacterium. To elucidate the potential role of desulfitobacteria in the O-demethylation of phenyl methyl ethers in the environment, we cultivated Desulfitobacterium chlororespirans, D. dehalogenans, D. metallireducens, and different strains of D. hafniense with phenyl methyl ethers as sole electron donors. With the exception of D. metallireducens, all species and strains tested were able to demethylate at least three of the four phenyl methyl ethers applied with fumarate, nitrate, or thiosulfate as electron acceptor. Furthermore, a high number of operons putatively encoding demethylase systems were identified in the genomes of Desulfitobacterium spp., although discrimination between O-, S-, N- and, Cl-demethylases was not possible. These findings provide evidence for the importance of the methylotrophic metabolism for desulfitobacteria and point to their involvement in the O-demethylation of phenyl methyl ethers in the environment.
Introduction Desulfitobacteria, which are most commonly described as reductively dehalogenating bacteria, are able to utilize phenyl methyl ethers as growth substrates when an appropriate electron acceptor (fumarate or a chlorinated phenol) is provided (Neumann et al., 2004). Prior to this finding, utilization of these substrates had only been described for members of the acetogenic bacteria (Bache & Pfennig, 1981; Daniel et al., 1991; Traunecker et al., 1991; Stupperich & Konle, 1993; Liesack et al., 1994). Phenyl methyl ethers are the main decomposition products of lignin and therefore widespread in the environment, especially in soil. Abundant representatives of this class include, for example, syringate, vanillate, and isovanillate (Chen et al., 1982, 1983; K€ ogel, 1986). Phenyl methyl ethers typically result from the conversion of lignin by ligninolytic enzymes that are produced by white rot fungi under aerobic conditions (Higuchi, 1990). Once they are released from the lignin macrostructure, they become available to the soil microbial community. In acetogens and desulfitobacteria, the methyl groups of phenyl methyl ethers are cleaved off by O-demethylase FEMS Microbiol Ecol 90 (2014) 783–790
enzyme systems (Kaufmann et al., 1997; Naidu & Ragsdale, 2001; Studenik et al., 2012) and the further oxidation of the methyl moiety to CO2 plays a key role for energy conservation (for a review see Drake et al., 2006). O-demethylases, which are induced by their respective substrates (Engelmann et al., 2001; Peng et al., 2012), consist of two methyltransferases (MTs) (MT I and MT II), a corrinoid protein (CP), and an activating enzyme (AE) (Kaufmann et al., 1997; Schilhabel et al., 2009; Studenik et al., 2012). The reaction mechanism is depicted in Fig. 1. In the first step, MT I cleaves the ether bond of the methoxylated substrate and transfers the methyl group to the super-reduced corrinoid cofactor ([CoI]) of CP. Subsequently, MT II transfers the methyl group from the methylated corrinoid to tetrahydrofolate (FH4) yielding methyltetrahydrofolate (CH3-FH4). The last protein component of the system, AE, has a repair function after inadvertent oxidation of the super-reduced corrinoid cofactor ([CoI]) to inactive [CoII]-CP. It catalyzes the ATP-dependent electron transfer from an unknown electron donor to [CoII]-CP yielding physiologically active [CoI]-CP, allowing CP to participate in further O-demethylation reactions (Siebert et al., 2005; ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
784
F.S. Mingo et al.
fumarate, nitrate, and thiosulfate, was followed in growth experiments of D. chlororespirans, D. dehalogenans, D. metallireducens, and different strains of D. hafniense. Additionally, bioinformatic analyses of available genome sequences of various Desulfitobacterium species were carried out to uncover the O-demethylation potential of these organisms.
Materials and methods Bacterial strains
Fig. 1. Scheme of the O-demethylase reaction adapted from Schilhabel et al. (2009). AE, activating enzyme; [CoI-III], corrinoid protein (CP) with cobalt in the respective oxidation state; FH4, tetrahydrofolate; MT, methyltransferase.
Sperfeld et al., 2014). For acetogenic bacteria, it has been described that the phenyl methyl ether-derived methyl group in CH3-FH4 undergoes oxidation to CO2. The reducing equivalents generated by methyl group oxidation are transferred to CO2 that is reduced to enzyme-bound carbon monoxide. Acetyl-CoA is formed in the carbon monoxide dehydrogenase/acetyl-CoA synthase reaction from the enzyme-bound CO and methyl groups derived from phenyl methyl ether demethylation. Acetyl-CoA can then either be converted to acetate to conserve energy or it can be incorporated into cell carbon (Ragsdale & Pierce, 2008). In contrast to acetogens, desulfitobacteria appear to be unable to use CO2 as a terminal electron acceptor for O-demethylation (Neumann et al., 2004; Kreher et al., 2008), which renders their reductive acetylCoA pathway nonfunctional for energy conservation. Instead, they rely on alternate electron acceptors that might be provided by the microbial community or by the environment. It was previously shown that Desulfitobacterium hafniense DCB-2 and D. hafniense PCE-S use fumarate as terminal electron acceptor and that D. hafniense DCB-2 was also able to couple O-demethylation of vanillate to the reductive dechlorination of 3-chloro-4hydroxyphenylacetic acid to 4-hydroxyphenylacetic acid (Neumann et al., 2004). However, nothing is known about possible physiological electron acceptors that might be present in natural environments such as forest soil, where phenyl methyl ethers are available as substrates for the soil microbial community. The objective of this study was to shed light on the general importance of the methylotrophic metabolism for Desulfitobacterium spp. and to elucidate their potential role in the O-demethylation of phenyl methyl ethers in the environment. For this purpose, phenyl methyl ether consumption with different electron acceptors, namely ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
Desulfitobacterium hafniense strains DCB-2 (DSM-10664), DP7 (DSM-13498), G2 (DSM-16228), PCE-S (DSM14645), PCP-1 (DSM-12420), and TCP-A (DSM-13557) as well as D. chlororespirans (DSM-11544), D. dehalogenans (DSM-9161), and D. metallireducens (DSM-15288) were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany). Desulfitobacterium hafniense strain Y51 was taken from the strain collection of our laboratory and was kindly provided by Taiki Futagami (Kagoshima University, Kagoshima, Japan). Growth of bacteria
Desulfitobacterium spp. was grown anoxically in rubberstoppered serum bottles under 100% N2 atmosphere as described by Neumann et al. (2004). The electron donors (syringate, vanillate, isovanillate, or 4-hydroxyanisole) and acceptors (fumarate, nitrate, Fe(III), or thiosulfate) were supplied from separately autoclaved anoxic stock solutions. The concentrations applied were as follows: phenyl methyl ethers, 2 mM; fumarate, 20 mM; nitrate and thiosulfate, 5 mM; Fe(III) citrate, 12 mM. Media were inoculated with precultures grown with pyruvate and fumarate (each 40 mM) as electron donor and acceptor, respectively. For precultures of D. metallireducens, Fe (III) (c. 20 mM) was used as electron acceptor instead of fumarate. The culture volume for inoculation was 10% (v/v) of the final volume of the main culture. Cultures were incubated in a water bath shaker at 28 °C and 150 r.p.m. Samples were taken and analyzed for growth and metabolites. Analytical methods
The protein concentration was used as an indicator of bacterial growth and was determined according to the method of Bradford (1976) after cell lysis by alkaline treatment. The concentrations of phenyl methyl ethers and fumarate were determined by HPLC using a LiChrospher 100 FEMS Microbiol Ecol 90 (2014) 783–790
785
Phenyl methyl ether conversion by desulfitobacteria
RP-8 125 9 4 mm column (Merck KGaA, Darmstadt, Germany) and 25% methanol (v/v) plus 0.3% H3PO4 (v/v) in water as eluent. A flow rate of 0.4 mL min1 was applied. Signals were detected at 210 nm. Under these conditions, the retention times were as follows: fumarate, 6.3 min; vanillate, 8.3 min; 4-hydroxyanisole, 8.6 min; isovanillate, 8.8 min; syringate, 9.2 min. For separation of fumarate, vanillate, and its demethylated end product 3,4dihydroxybenzoate, a gradient with a flow rate of 0.3 mL min1 was applied: 0–11 min, 0.3% H3PO4; 11– 21 min, 0–25% methanol plus 0.3% H3PO4; 21–30 min, 25% methanol plus 0.3% H3PO4. Under these conditions, the retention times were as follows: fumarate, 7.1 min; 3,4-dihydroxybenzoate, 14.9 min; vanillate, 24.0 min. Nitrite was measured by a diazotization reaction described by Lunge (1904). The colorimetric reaction was started by the addition of 50 lL of 1% (w/v) sulphanilic acid and 50 lL of 0.3% (w/v) 1-naphthylamine to 50 lL of the diluted sample. After 5 min incubation at room temperature, absorption was measured at a wavelength of 525 nm on a VERSAmax tunable microplate reader (Molecular Devices, Biberach an der Riss, Germany). For nitrate determination, the method of Bosch Serrat (1998) was followed. Diluted samples were mixed in a 1 : 1 ratio with 200 lL of a chloride solution consisting of 0.28 M NaCl dissolved in 9.35% (v/v) H3PO4. Subsequently, 1 mL of 0.55% (w/v) resorcinol dissolved in 62.5% (v/v) H2SO4 was added to each sample. The mixtures were incubated at 95 °C for 8 min. They were allowed to cool down for 20 min and absorbance was measured at 505 nm. As in this assay both nitrite and nitrate were detected, nitrate concentration was calculated by subtracting the nitrite concentration from the value obtained. Thiosulfate was measured according to Quentin & Prachmayr (1964). Diluted samples were mixed in a 3 : 1 ratio with 0.006% (w/v) methylene blue in 6 M HCl. After 3 h of incubation at room temperature, the absorbance was measured at 660 nm. In silico mapping of putative O-demethylase operons
Mapping of putative O-demethylase operons of Desulfitobacterium spp. was carried out with the NCBI BLAST server (Altschul et al., 1990) and the Integrated Microbial Genomes (IMG) system (Markowitz et al., 2014). As template for BLASTP searches, the amino acid sequences of Dhaf_4610, Dhaf_4611, Dhaf_4612, and Dhaf_2573, which represent the protein components of an O-demethylase previously identified in D. hafniense DCB-2 (Studenik et al., 2012), were used. The putative O-demethylase operons identified in the genomes of D. hafniense DCB-2 FEMS Microbiol Ecol 90 (2014) 783–790
(Kim et al., 2012), DP7, PCE-S, PCP-1, TCE-1, TCP-A, and Y51 (Nonaka et al., 2006) as well as D. dehalogenans, D. dichloroeliminans, and D. metallireducens were compared in terms of organization and similarity of the encoded proteins.
Results Growth of Desulfitobacterium spp. with phenyl methyl ethers as electron donors
Desulfitobacterium hafniense (strains DCB-2, DP7, G2, PCE-S, PCP-1 TCP-A, Y51), D. chlororespirans, D. dehalogenans, and D. metallireducens were cultivated with 4hydroxyanisole, syringate, vanillate, or isovanillate in the presence of fumarate, nitrate, or thiosulfate as electron acceptors (Table 1). The results were obtained from at least duplicates of the experiments. All species with the exception of D. metallireducens were able to grow on at least three of the supplied phenyl methyl ethers by cleaving the ether bond. Vanillate and isovanillate were O-demethylated to 3,4-dihydroxybenzoate, 4-hydroxyanisole to hydroquinone, and syringate via 3,4-dihydroxy5-methoxybenzoate to 3,4,5-trihydroxybenzoate. The stoichiometry of demethylated end product formed per substrate consumed was about 1 : 1. Fumarate as well as nitrate or thiosulfate served as terminal electron acceptors for methyl group oxidation following O-demethylation. The substrate spectra and the demethylation rates of the Desulfitobacterium strains used in this study are summarized in Table 1. O-demethylation was independent of the type of electron acceptor used; however, the rates of phenyl methyl ether consumption were different. For most phenyl methyl ethers, the rates of demethylation and cell growth were highest in the presence of fumarate followed by nitrate and thiosulfate. As reported before for D. hafniense DCB-2 and PCE-S (Neumann et al., 2004; Kreher et al., 2008), no demethylation of the substrates occurred when CO2 was applied as sole terminal electron acceptor (data not shown). Figure 2 shows the consumption of vanillate by D. hafniense Y51 in the presence of fumarate, nitrate, or thiosulfate, respectively. The stoichiometries of fumarate/nitrate/thiosulfate reduced per mol methyl group consumed were usually higher than the values expected from the substrate conversion according to the following equations: 1 R-O-CH3 þ 3 fumarate þ 2 H2 O ! 1 R-OH þ 3 succinate þ CO2
(1)
þ 4 R-O-CH3 þ 3 NO 3 þ 6 H ! 4 R-OH þ 4 CO2 þ 3 NHþ 4 þ H2 O
(2)
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
786
F.S. Mingo et al.
Table 1. Maximal rate of phenyl methyl ether consumption by growing Desulfitobacterium spp. (lM h1) in the presence of fumarate (a; first row), nitrate (b; second row), or thiosulfate (c; third row) as electron acceptor. For further details, see ‘Materials and Methods’ Demethylation rates (lM h1)
Species and strain
4-Hydroxyanisole OH
Syringate
H3 CO
OCH3 Desulfitobacterium hafniense
DCB-2
DP7
G2
PCE-S
PCP-1
TCP-A
Y51
D. dehalogenans
D. chlororespirans
39 28 37 26 14 17 75 21 31 54 17 26 14
