The succinate:menaquinone reductase of Bacillus cereus — characterization of the membrane-bound and purified enzyme

The succinate:menaquinone reductase of Bacillus cereus — characterization of the membrane- bound and purified enzyme L.M. Garcı́a, M.L. Contreras-Zentella, R. Jaramillo, M.C. Benito-Mercadé, G. Mendoza-Hernández, I.P. del Arenal, J. Membrillo-Hernández, and J.E. Escamilla Abstract: Utilization of external succinate by Bacillus cereus and the properties of the purified succinate:menaquinone-7 reductase (SQR) were studied. Bacillus cereus cells showed a poor ability for the uptake of and respiratory utilization of exogenous succinate, thus suggesting that B. cereus lacks a specific succinate uptake system. Indeed, the genes coding for a succinate–fumarate transport system were missing from the genome database of B. cereus. Kinetic studies of membranes indicated that the reduction of menaquinone-7 is the rate-limiting step in succinate respiration. In accordance with its mo- lecular characteristics, the purified SQR of B. cereus belongs to the type-B group of SQR enzymes, consisting of a 65- kDa flavoprotein (SdhA), a 29-kDa iron–sulphur protein (SdhB), and a 19-kDa subunit containing 2 b-type cytochromes (SdhC). In agreement with this, we could identify the 4 conserved histidines in the SdhC subunit predicted by the B. cereus genome database. Succinate reduced half of the cytochrome b content. Redox titrations of SQR-cytochrome b-557 detected 2 components with apparent midpoint potential values at pH 7.6 of 79 and –68 mV, respectively; the com- ponents were not spectrally distinguishable by their maximal absorption bands as those of Bacillus subtilis. The physiolog- ical properties and genome database analyses of B. cereus are consistent with the cereus group ancestor being an opportunistic pathogen. Key words: succinate dehydrogenase, C4-dicarboxylate transport, Bacillus cereus. Résumé : L’utilisation de succinate exogène par Bacillus cereus et les propriétés de la succinate:menaquinone-7 (MK7) ré- ductace (SQR) purifiée ont été étudiées. Les cellules de B. cereus démontrent une faible capacité de captation et d’utili- sation respiratoire de succinate exogène, ce qui suggère qu’un système de captation spécifique de succinate soit absent chez B. cereus. En effet, les gènes codant le système de transport succinate:fumarate sont absents des bases de données gé- nomiques de B. cereus. Des études cinétiques faites à partir de membranes ont indiqué que la réduction de MK7 constituait l’étape limitante de la respiration à partir du succinate. Conformément à ses caractéristiques moléculaires, la SQR purifiée de B. cereus appartient au groupe des enzymes SQR de type B, consistant en une flavoprotéine de 65 kDa (SdhA), une protéine à fer–soufre de 29 kDa (SdhB) et une sous-unité de 19 kDa contenant 2 cytochromes de type-b (SdhC). Conformément à ce modèle, nous avons pu identifier les 4 histidines conservées la sous-unité SdhC prédites par les bases de données génomiques de B. cereus. Le succinate réduisait la moitié de contenu en cytochrome b. La titration redox de la SQR-cytochrome b-557 a détecté 2 composantes ayant des valeurs apparentes de potentiel central (Em,7.6) de 79 et –68 mV respectivement qui ne pouvaient se distinguer de façon spectrale par leurs bandes d’absorption maximales, comparative- ment à leurs contreparties chez B.acillus subtilis. Les analyses des propriétés physiologiques et des bases de données géno- miques de B. cereus concordent avec le fait que l’ancêtre du groupe cereus soit un pathogène opportuniste. Mots-clés : succinate déshydrogénase, transport des C4-dicarboxylates, Bacillus cereus. [Traduit par la Rédaction] Received 14 December 2007. Revision received 18 April 2008. Accepted 21 April 2008. Published on the NRC Research Press Web site at cjm.nrc.ca on 5 June 2008. L.M. Garcı́a,1 M.L. Contreras-Zentella, R. Jaramillo,2 M.C. Benito-Mercadé,3 and J.E. Escamilla.4 Departamento de Bioquı́mica, Instituto de Fisiologı́a Celular, Apdo. Postal 70-242, Universidad Nacional Autónoma de México, México 04510, D.F. México. G. Mendoza-Hernández and I.P. del Arenal. Departamento de Bioquı́mica, Facultad de Medicina, Apdo. Postal 70-159, Universidad Nacional Autónoma de México, México 04510, D.F. México. J. Membrillo-Hernández. Avenida Copilco 162, Coyoacán, México 04319, D.F. México. 1Present address: Escuela de Biologı́a, Benemérita Universidad Autónoma de Puebla, Edificio 76, Ciudad Universitaria, Puebla, México. 2Present address: Programa de Biologı́a, Universidad de Sucre No. 5-267, Sincelejo, Sucre, Colombia. 3Present address: Laboratorios Roche, Avenida Santa Fe No. 485, Col. Cruz Manca Santa Fe, Cuajimalpa, México 05349, D.F. México. 4Corresponding author (e-mail: [email protected]). 456 Can. J. Microbiol. 54: 456–466 (2008) doi:10.1139/W08-037 # 2008 NRC Canada Introduction Succinate:quinone oxidoreductase (EC 1.3.5.1) comprises succinate:quinone reductase (SQR, SDH, or Complex II) and quinol:fumarate reductase (QFR). The former is found in aerobic organisms and catalyses succinate oxidation in the citric acid cycle, transferring electrons to the respiratory quinones in the membrane. QFRs, on the other hand, can be found in organisms growing anaerobically, using fumarate as the terminal electron acceptor (Hägerhäll 1997). SQRs and QFRs have a hydrophilic domain composed of 2 sub- units (i) a flavoprotein (SdhA) bearing the substrate-binding site and (ii) an iron–sulphur protein (SdhB) that contains 3 different [Fe-S] centres involved in intramolecular electron transfer. In addition, there is a hydrophobic domain consti- tuted by 1 (SdhC) or 2 (SdhC, SdhD) polypeptides contain- ing 0, 1, or 2 associated b-type cytochromes; this is the site for quinone reduction (Hägerhäll 1997; Ackrell 2000). On the basis of their hydrophobic domain and heme content, these enzymes have been classified into 5 groups (Hägerhäll and Hederstedt 1996; Hederstedt 1999; Lancaster 2002): (i) type A, enzymes possessing 2 subunits and 2 cytochromes b, e.g., SQR from archaea; (ii) type B, enzymes with 1 sub- unit and 2 cytochromes b, e.g., SQR from gram-positive and 3-Proteobacteria; (iii) type C, enzymes with 2 hydrophobic subunits and 1 cytochrome b, e.g., SQR from eucaryotic mitochondria, Escherichia coli, and Paracoccus denitrifi- cans; (iv) type D, enzymes containing 2 subunits and no heme group, e.g., QFR of E. coli; and (v) type E, enzymes, such as the ‘‘non-classical’’ archaeal SQRs, that have no associated heme but have 2 hydrophobic subunits (SdhE and SdhF). There are 2 functional classes of SQR enzymes (Hägerhäll 1997; Matsson et al. 2000), those using a high- potential quinone as the physiological electron acceptor (e.g., ubiquinone) and those that reduce a low-potential qui- none (e.g., menaquinone). The electron transfer from succi- nate (Em,7 = +30 mV) to ubiquinone (Em,7 = +112 mV) is an exergonic reaction (DG8’ = –15 kJ�mol–1), whereas the electron transfer from succinate to menaquinone (Em,7 = –74) is endergonic (DG8’ = 21 kJ�mol–1). As a rule, succinate:ubiquinone reductases contain only one heme b; whereas the succinate:menaquinone reductases contain 2. Here we report on the properties of cells and membrane particles of Bacillus cereus in relation to succinate utiliza- tion. In addition, we purified and determined the molecular and kinetic properties of the succinate:menaquinone reduc- tase. Our results indicated that B. cereus cells have limited capacity for the uptake of succinate and an even lower ca- pacity for its utilization as exogenous substrate for respira- tion. According to its molecular characteristics, the purified SQR of B. cereus belongs to the type B group of enzymes having 1 SdhC subunit and 2 b-type cytochromes; 1 of them is reduced by succinate. SQR cytochromes b of B. cereus had redox midpoint potential values close to those previously reported for Bacillus subtilis; however, they were not spectrally distinguishable by their maximal absorption bands as those of B. subtilis (Hägerhäll et al. 1992). Materials and methods Bacteria, growth, and cell disruption Bacillus cereus ATCC 9373 and B. subtilis 168 (Marburg strain requiring tryptophan, kindly donated by Dr. Jorge Vázquez, Facultad de Quı́mica, Universidad Nacional Autónoma de México) were used in the present study. Cells were grown at 30 8C in nutrient sporulation medium with phosphate (NSMP) containing 8.0 g�L–1 nutrient broth (Difco, Detroit, Michigan, USA), 0.1 mol�L–1 potassium phosphate (pH 6.5), and metal salts (Sigma-Aldrich Corpor- ation, St. Louis, Missouri, USA), as described by Fortnagel and Freese (1968), in a Bioflo 5000 New Brunswick fermen- tor (New Brunswick Scientific, New Jersey, USA) contain- ing 60 L of medium and were stirred at 150 r�min–1 and sparged with 55 L air�min–1. Cells were harvested during sporulation at stage II (2 h into stationary phase). For ex- periments with intact cells of B. cereus and B. subtilis, cul- tures were grown in 1.0 L of NSMP supplemented with 20 mmol�L–1 succinate (Sigma-Aldrich) to early log phase (OD600 = 0.5) at 30 8C in 2.8 L Fernbach flasks. The utiliza- tion of C4-dicarboxylates as carbon sources by B. cereus was monitored as described previously (Asai et al. 2000). Succinate, fumarate, or malate (Sigma-Aldrich) were added at the final concentration of 0.5% (m/v), when cells growing in the minimal salt medium containing 0.5% yeast extract Sigma-Aldrich) (Willecke and Lange 1974) reached an OD600 = 0.1. For disruption, cells (750 g, wet mass) were washed twice and suspended in 1 L of cold 50 mmol�L–1 potassium phos- phate buffer, pH 7.4. One tablet of ‘‘Complete’’ protease in- hibitor cocktail (Roche Diagnostics, Mannheim, Germany) plus 0.1 mmol�L–1 phenylmethylsulfonyl fluoride (PMSF; Sigma-Aldrich) was added. Disruption was carried out by 10 cycles of 30 s (resting 5 min between cycles) at 4 8C in a bead beater cell fractionator (Biospec Products, Bartles- ville, Oklahoma, USA). Glass beads of 150–200 mm dia- meter were used. Unbroken cells and debris were eliminated by centrifugation at 8600g for 10 min. Mem- branes pelleted at 125 000g were washed twice with the same buffer without protease inhibitors. Succinate uptake by intact cells Vegetative cells of B. cereus and B. subtilis grown to an OD600 = 0.5 in NSMP supplemented with 20 mmol�L–1 suc- cinate were harvested and washed twice with 0.1 mol�L–1 potassium phosphate or sodium phosphate, 1.0 mmol�L–1 MgCl2, and 100 mg�mL–1 chloramphenicol, pH 7.4 (Sigma- Aldrich) and were resuspended in the same buffer to an OD600 = 1.0. The bacteria were energized by incubation for 10 min in 20 mmol�L–1 glucose (Sigma-Aldrich), as described by Schirawski and Unden (1998), and then 2,3- [14C]succinate (100 mmol�L–1; 40 MBq�mmol–1 succinate; New England Nuclear, Massachusetts, USA) was added. At the times noted in Fig. 1a, samples (100 mL) were withdrawn, filtered (Millipore HA 0.45 mm filters), and washed with 2.0 mL of cold 250 mmol�L–1 succinate. Dry filters were counted for radioactivity in the scintillation liquid. UV photoinactivation of membranes Ultraviolet irradiation (360 nm) was used to destroy endo- genous menaquinone-7 (MK7) of membrane particles, ac- cording to the method described earlier (Escamilla et al. 1988). Samples withdrawn at different times of irradiation Garcı́a et al. 457 # 2008 NRC Canada (10–60 min) were used to determine the remaining oxidase activities with NADH (Sigma-Aldrich) or succinate as sub- strates. Enzymatic assays Succinate and NADH oxidoreductase activities were measured at 30 8C, according to methods reported elsewhere (Escamilla et al. 1988), with 1.1 mmol�L–1 phenazine metho- sulfate (PMS) and 75 mmol�L–1 dichlorophenolindophenol (DCPIP) (Sigma-Aldrich) for succinate dehydrogenase and with 75 mmol�L–1 DCPIP for NADH dehydrogenase, as elec- tron acceptors. In the case of succinate:quinone DCPIP re- ductase activity, PMS was replaced by 50 mmol�L–1 decylubiquinone (Sigma-Aldrich). Oxidase activities with NADH or succinate were measured polarographically with a Clark oxygen electrode at 30 8C, according to methods previously described by Escamilla et al. (1988), in 2.0 mL of 100 mmol�L–1 potas- sium phosphate buffer (pH 7.6) and 1 mg of membrane protein. The reaction was started by the addition of 0.5 mmol�L–1 NADH or 20 mmol�L–1 succinate (final con- centrations). Oxidase activities in the presence of 50 mmol�L–1 ubiquinone analogues (Sigma-Aldrich), as elec- tron mediators, were measured at pH 6.6 to avoid significant spontaneous oxidation of the tested quinones (Escamilla et al. 1988). For all succinate-dependent activities, samples were preincubated (5 min at 30 8C) in 20 mmol�L–1 succi- nate for activation of SDH. Purification of the SQR Membranes (6 g protein; 10 mg�mL–1) in 30 mmol�L–1 Tris–H2SO4 (pH 8.0) containing 2% sodium cholate, 1% so- dium deoxycholate, 0.2 mol�L–1 sodium sulphate, 0.5 mmol�L–1 dithiotreitol (Sigma-Aldrich), and one tablet of ‘‘complete’’ protease inhibitor cocktail plus 0.1 mmol�L–1 PMSF were sonicated on an ice bath at 4 8C for 10 min and centrifuged at 125 000g for 40 min. The obtained pellet was washed twice and homogenized (5 mg protein�mL–1) in 50 mmol�L–1 Tris–HCl and 1 mmol�L–1 MgSO4 (pH 8.0) (Sigma-Aldrich), and the protease inhibitors were supple- mented as described above. Then, Triton X-100 (4% final concentration; Sigma-Aldrich) was added and stirred for 30 min. The mixture was centrifuged at 125 000g for 40 min. The reddish-brown cytochrome supernatant was re- covered and applied to a DEAE-Sepharose CL-6B column (7 cm � 9 cm; SUPELCO-Sigma-Aldrich), equilibrated with 50 mmol�L–1 Tris–HCl (pH 8), 1.0 mmol�L–1 MgSO4, and 0.1% Triton X-100. After the column was washed with the equilibration buffer, the retained protein was eluted with a 0–200 mmol�L–1 NaCl (Sigma-Aldrich) gradient in equili- bration buffer containing 5% glycerol (Sigma-Aldrich). Fractions containing SDH activity were pooled, diluted 1:4 with equilibration buffer, and applied to a QAE-Toyopearl column (2.5 cm � 17 cm; SUPELCO-Sigma-Aldrich), equilibrated as for the previous column and eluted with a 100–300 mmol�L–1 NaCl gradient containing 5% glycerol. Fractions containing enzyme activity were pooled, aliquoted, and stored at 4 8C in the presence of 10 mg�mL–1 of soybean asolectin (Sigma-Aldrich). All purification procedures were carried out at 4 8C. Spectroscopic analysis of cytochromes Membranes suspended in 80 mmol�L–1 potassium phos- phate buffer (pH 7.6) containing 40% glycerol were ana- lysed in an OLIS-SLM DW 2000 spectrophotometer (Olis, Inc., Bogart, Georgia, USA) at room temperature or at 77 K, in cuvettes of 10 and 2 mm light path, respectively. The concentration of cytochromes was calculated from re- duced (dithionite or substrate) minus persulfate-oxidized spectra at room temperature using the reported molar extinc- tion coefficients (Escamilla et al. 1988). Purified SQR was reduced by dithionite or succinate in buffer previously satu- Fig. 1. Utilization of exogenous succinate by intact cells of Bacillus cereus. (a) Comparative uptake of [14C]succinate by (*) B. subtilis and (&) B. cereus cells. (b) Growth of B. cereus in minimal salt medium containing 0.5% yeast extract without (&) and with the added C4- dicarboxylates 0.5% succinate (~), fumarate (*), or malate (!). Time 0 indicates the time when the C4-dicarboxylate was added to the growing cultures. 458 Can. J. Microbiol. Vol. 54, 2008 # 2008 NRC Canada rated with dinitrogen gas (AGA Gas S.A. de C.V., Tlane- pantla, Estado de México, México). SDS–PAGE and N-terminal sequence analysis The purified enzyme was analysed by the SDS–PAGE method of Laemmli (1970), performed in 16 cm � 14 cm plates with a 10%–16% polyacrylamide (Sigma-Aldrich) gradient, and electroblotted on a PVDF membrane Immobilon-P (Millipore Corporation, Bedford, Massachu- setts, USA) as described by Towbin et al. (1979). The 3 re- solved polypeptides were recovered, and their N-terminal sequences were determined by an automated Edman degra- dation on a gas-phase protein sequencer (LF 3000, Beckman instruments, Fullerton, California, USA) equipped with an on-line Beckman Gold high performance liquid chroma- tography system using a model 126 pump and a model 168 diode array detector setting at 268 and 293 nm for signal and reference, respectively. A Beckman Spherogel Micro PTH column (2 mm � 150 mm) and the standard Beckman sequencing reagents were used for analysis. Redox titration of the di-heme cytochrome b of SQR Anaerobic redox titrations of di-heme cytochrome b in the purified SQR (0.8 mg protein) were done essentially as described by Hägerhäll et al. (1992). The 7 mL glass cuvette (1.0 cm light path) in which the titrations were performed was fitted with an Ag/AgCl platinum combination electrode. Oxygen was excluded by flushing continuously with dinitrogen gas. Titrations were carried out in 50 mmol�L–1 Tris–HCl (pH 7.6) buffer in the presence or absence of 40 mmol�L–1 2-n-heptyl-4-hydroxyquinoline N-oxide (HOQNO; Sigma-Aldrich). The electrode was previously calibrated by measuring the potential (Em,7 = +285 mV) with a saturated solution of quinhydrone (Sigma-Aldrich). A mixture of redox mediators was used as follows (Em,7): 2-hydroxy-1,4-naphthoquinone (–152 mV), duroquinone (+7 mV), 1,4-naphthoquinone, (+50 mV), phenazine metho- sulphate (+70 mV), 1,2 naphthoquinone (–24 mV), anthra- quinone-2-sulfonate (–225 mV), menadione (–1 mV), and 1,4-benzoquinone (+280 mV) (Sigma-Aldrich). Each media- tor was dissolved in dimethyl sulfoxide (Sigma-Aldrich); PMS was dissolved in water. Mediators were added to a fi- nal concentration of 50 mmol�L–1. Titration was performed by stepwise addition of small volumes of anaerobic 50 mmol�L–1 sodium dithionite (Sigma-Aldrich) or 50 mmol�L–1 potassium ferricyanide (Sigma-Aldrich) to the Tris–HCl buffer. The reduction of cytochrome b was re- corded as the difference in absorbance between the a-band maximum at 557 nm and the isosbestic point at 570 nm, us- ing dual-wavelength spectroscopy. Each heme component contributed approximately 50% to the total absorption at 557 nm of the fully reduced cytochrome. The redox mid- point value for the high potential was calculated from data in the interval 0%–50% of total cytochrome b-557 reduc- tion, using the Nernst equation; for the low-potential heme it was calculated in the same way from data in the 50%– 100% reduction interval. Other methods Protein was determined by the method of Markwell et al. (1978). Acid nonextractable flavin was determined fluoro- metrically in a model DMX-1000 OLIS-Aminco spectro- fluorometer (Olis, Inc., Bogart, Georgia, USA), as described by Hederstedt (1980). Cytochrome b in the purified SQR complex was quantified as the reduced hemochromogen by the method described by Lin and Beattie (1978). The puri- fied complex was treated with an equal volume of a 4.4 mol�L–1 pyridine in a 0.2 mol�L–1 sodium hydroxide sol- ution. The difference spectrum of pyridine hemochromogen was obtained by subtracting the ferricyanide-oxidized spec- trum from the dithionite-reduced spectrum. The cytochrome b concentration in the sample was calculated with an index of DA556–575 = 30 mmol�L–1�cm–1. Results Utilization of external succinate by whole cells Endogenous respiration of starved vegetative cells of B. cereus grown in NSMP was poorly stimulated by the ad- dition of succinate or fumarate; albeit, the addition of ma- late, glutamate, but mainly alanine stimulated respiration by severalfold (Table 1). By contrast, endogenous respiration in B. subtilis cells was 4.0-fold higher when succinate or fuma- rate was added (Table 1). A comparison of the uptake of [14C]-succinate into the bacteria (Fig. 1a) revealed that the uptake rate in B. cereus (0.5 nmol�min–1�(mg dry cells)–1) was lower by a factor of 3.5 than that in B. subtilis. The time-dependent kinetics and transport rates obtained for B. subtilis were consistent with those reported by Schirawski and Unden (1998). The ability of B. cereus to utilize C4-dicarboxylates as the sole carbon source was monitored as described in B. subtilis (Asai et al. 2000). The growth of B. cereus in minimal salts medium containing 0.05% yeast extract as sole carbon source was Table 1. Comparative stimulation by several substrates of the endogenous respiration of intact cells of Bacillus cereus and Bacillus subtilis.a Relative respirationb Substrate B. cereus B. subtilis Stimulation-fold of B. subtilis / B. cereus Endogenous 1.0 1.0 1.0 Succinate 1.2 4.0 3.3 Fumarate 1.2 4.0 3.3 Malate 3.5 6.0 1.7 Pyruvate 1.4 1.4 1.0 Citrate 1.5 2.0 1.3 a-Ketoglutarate 1.0 1.0 1.0 Aspartate 1.4 2.0 1.4 Glutamate 4.0 3.0 0.75 Alanine 9.0 6.0 0.7 aBacillus cereus and B. subtilis grown in nutrient sporulation medium with phosphate to early exponential phase (OD600 = 0.5) were washed twice with 50 mmol�L–1 potassium phosphate buffer (pH 7.6) and starved for 1 h in 100 mL of the same buffer (stirred at 200 r�min–1, 30 8C). bRespiration (nmol O2�min–1�(mg dry cells)–1) was measured with a Clark O2 electrode in the above buffer. Endogenous substrate respiration activities in starved cells were 24 and 20 nmol O2�min–1�(mg dry cells)–1 for B. cereus and B. subtilis, respectively. The substrates listed were used at 20 mmol�L–1. Garcı́a et al. 459 # 2008 NRC Canada readily stimulate by malate but not in the cases where the medium was supplemented with succinate or fumarate (Fig. 1b). Thus, the low uptake transport rates for succinate registered in B. cereus cells could explain, at least in part, the poor utilization of exogenous succinate for respiration and as a carbon source. Genomic database analyses In B. subtilis, the ydbEFGH operon codes for the C4- dicarboxylate transport and regulatory systems. Indeed, in- frame deletion of each of the individual genes causes an impaired utilization of succinate and fumarate (Asai et al. 2000). Since the genome sequences of several strains of B. cereus were released (B. cereus ATCC 14579, GenBank entry AE016877; B. cereus ATCC 10987, GenBank entry AE017194; B. cereus E33L, GenBank entry CP000001), we explored if B. cereus had a ydbEFGH orthologue, using the Blast facility of the NCBI (http://www.ncbi.nlm.nih.gov/ blast/Blast.cgi) and The Institute for Genomic Research (http://www.tigr.org/). Sequences with significant similarity to the ydbE, ydbF, or ydbG genes in any of the B. cereus databases where not detected. Interestingly, a gene highly similar to ydbH (coding for the transporter protein of C4- dicarboxylate) was identified as a separate operon in B. cereus ATCC 10987 (dctA, 83% similarity; GenBank en- try AE017194) and B. cereus E33L (dctA, 71% similarity; GenPept YP 084880) but was absent in B. cereus ATCC 14579 databases. These results strongly suggest that B. cereus lacks the C4-dicarboxylate transport system de- scribed for B. subtilis (Asai et al. 2000). This may explain the poor ability of exogenous succinate and fumarate utiliza- tion (Fig. 1 and Table 1). Reduction of MK7 by succinate and NADH in membranes Membrane particles of B. cereus showed poor respiratory activity with succinate when compared with NADH- supported respiration (10 versus 75 nmol O2�min–1�mg1, re- spectively). However, succinate oxidation with the artificial electron acceptors PMS plus DCPIP was 25-fold higher. This result suggests that the reduction of endogenous MK7 by SQR was a rate-limiting step for succinate respiration in membranes. Therefore, the time courses of cytochrome re- duction induced by NADH and succinate were compared (Fig. 2). In the presence of NADH, membrane preparations rapidly reached anaerobiosis, causing the full reduction of b-, c-, and a-type cytochromes (Figs. 2a and 2c). On the other hand, in the presence of succinate (Figs. 2b and 2c), only a b-type (558 nm) cytochrome was significantly re- duced within the first minute of incubation, and no further spectral changes were observed after 12 min. Photoinactiva- tion (UV360) of endogenous MK7 caused a severe impair- ment of the respiratory electron transport capacity with NADH, as judged by the abolition of NADH oxidase activ- ity (not shown) and by the lack of reduction of cytochromes by NADH (Figs. 2a and 2c). However, the reduction pattern evoked by succinate (Figs. 2b and 2c) on intact and photo- inactivated membranes was similar, suggesting that cyto- chrome b-558 is located before the MK7 reaction step and is very likely a functional part of the SQR complex. Respiration and reduction of cytochromes with NADH and succinate in photoinactivated membranes could be re- stored by the addition of the hydrophilic naphthoquinones (50 mmol�L–1 final concentrations) plumbagine, menadione, and juglone. Interestingly, in the presence of the hydrophilic naphthoquinones, succinate caused the full reduction of Fig. 2. Photoinactivation (UV360) of membrane menaquinone-7 and its effect on the reduction kinetics of cytochromes by (a) 2 mmol�L–1 NADH and (b) 30 mmol�L–1 succinate. Trace sets show the time course for the reduction of cytochromes. Solid-line traces are for intact membranes while broken-line traces are for membranes previously photoirratiated for 60 min. (c) Reduction (calculated from recorded traces) of cytochrome b (DA558–575) induced by (*, *) NADH and (~, ~) succinate of intact (open symbols) and photoinactivated (filled symbols) membranes. 460 Can. J. Microbiol. Vol. 54, 2008 # 2008 NRC Canada cytochromes and a 2- to 5-fold increase in succinate oxidase activity as compared with the activity registered in native membranes (not shown). Restored activities were inhibited by KCN or HOQNO (not shown). Purification and characterization of SQR SQR has been purified from several sources and it is shown to contain 2, 1, or 0 b-type cytochromes (Pennoyer et al. 1988; Kita et al. 1989). According to the data in Fig. 2, SQR of B. cereus contains a b-type cytochrome that could be readily reduced by succinate, even after photo- inactivation of the endogenous MK7. To get insight into the molecular and kinetic characteristics of SQR of B. cereus, we decided to purify the enzyme. Membranes (6.1 g of pro- tein) prepared from stage II sporulating cells were used as starting material. The final preparation analysed by SDS– PAGE (Fig. 3a), showed the presence of 2 well-defined bands with Mrs of 65 and 29 kDa. A third, poorly stained band of 19 kDa was also observed. The N-terminal se- quence for the first 20 residues of the 65-kDa subunit was MKGKLIVVGGGLAGLMATIK. After a BLAST analysis of this sequence, a high degree of identity with the FAD subunit of B. subtilis 168 (78%; GenBank entry AL009126) was found. Moreover, it was identical to the predicted flavoprotein of SQR in B. cereus ATCC 14579 (GenBank entry AE016877). In the case of the 29-kDa polypeptide, the first 13 terminal residues of the N-termi- nal end were SEKTIRLIITRQD. The BLAST analysis showed a high degree of identity with the iron–sulfur sub- unit of B. subtilis 168 (85%; GenBank AL009126), and this sequence was identical to the predicted SQR Fe-S protein in the B. cereus ATCC 14579 database (GenBank entry AE016877). After several attempts, we could not obtain the sequence at the N-terminal end of the 19-kDa subunit (cytochrome b-558), probably because it was blocked. The succinate-reduced minus ammonium persulfate- oxidized spectrum at room temperature (Fig. 3b) of the purified Complex II, showed the typical pattern of a cytochrome b with symmetrical peaks at 425, 526, and 557 nm. A comparison of the specific activities of initial and final stages of purification (0.45 and 7.6 mmol�min–1�mg–1, re- spectively) of B. cereus Complex II (Table 2) yielded an enrichment of 17-fold and a recovery of 16%. Considering that this enzyme was purified to homogeneity, its specific activity was 3-fold lower than that of the purified enzyme of B. subtilis (22.5 mmol�min–1�mg–1) (Hägerhäll et al. 1992). The SQR purified here was activated by about 1.7-fold upon incubation with substrate (not shown). The enzyme showed an optimal pH of 7.8 with either PMS–DCPIP or decylubiquinone–DCPIP as electron acceptors. Dose re- sponse to succinate, PMS, and decylubiquinone showed hyperbolic kinetics (Figs. 4a–4c) with Km values of 640, 330, and 11 mmol�L–1, respectively, and Vmax values of 8.1, 8.5, and 3.8 mmol�min–1�mg–1, respectively. Considering that Complex II of B. cereus has 1 FAD�(mol of enzyme)–1 (Table 3), a turnover number of 77 s–1 was calculated (PMS–DCPIP acceptor). The oxidation of succinate by purified SQR was inhibited in a competitive manner by malonate (not shown). From the Dixon plot, a Ki for malonate of 88 mmol�L–1 was obtained (Table 3). Thenoyltrifluoroacetone (TTFA) or HOQNO did not affect PMS–DCPIP acceptor activity. On the other hand, decylubiquinone acceptor activity was fully inhibited by 25 mmol�L–1 HOQNO (Fig. 4d). Partial inhibition with HOQNO on quinone reductase activity of purified SQR of B. subtilis was reported earlier (Hägerhäll et al. 1992; Smirnova et al. 1995). Fig. 3. (a) SDS–PAGE depicting the molecular and spectral prop- erties of succinate:quinone reductase (SQR) purified from Bacillus cereus. Lanes: 1, Mr markers (Mark VII Sigma-Aldrich); 2, purified succinate dehydrogenase (20 mg). (b) Light absorption dif- ference spectrum (succinate reduced minus ammonium persulfate oxidized) registered at room temperature. The SQR protein concen- tration was 0.3 mg�mL–1. Garcı́a et al. 461 # 2008 NRC Canada Low-temperature absorption spectroscopy In comparison with the room-temperature spectrum (Fig. 3b), the spectra registered at 77 K showed sharp reduc- tion peaks that were blue shifted by 1 nm (424, 525, and 556 nm; Fig. 5). The a peaks at 556 nm induced by dith- ionite or succinate were symmetrical (Figs. 5a and 5b, respectively). It is noteworthy that when the succinate- reduced spectrum was subtracted from that reduced by dith- ionite, a single symmetrical peak at 556 nm was obtained (Fig. 5c). Reduction levels calculated from spectra at 77 K (DA556–575) showed that succinate reduced close to half (44%) of the total cytochrome b reduced by dithionite. Thus, despite the fact that succinate induced partial reduc- tion of cytochrome b, the subtraction of the succinate- reduced enzyme from the dithionite-reduced enzyme did not result in the spectral resolution of 2 types of cytochrome b (i.e., 553 and 558 nm), as it has been described for the SQR of B. subtilis (Hägerhäll et al. 1992). Interestingly, the spectral properties of the purified SQR of B. cereus re- sembled those of the purified SQR of Bacillus firmus QF4 (Gilmour and Krulwich 1996) and Bacillus sp. strain YN2000 (Qureshi et al. 1996). Potentiometric analysis of SQR cytochrome b Redox titration of cytochrome b in the purified SQR of B. cereus (Fig. 6) revealed the presence of 2 single-electron components with apparent midpoint potentials of –68 and +79 mV (hemes bL (low-potential heme component) and bH (high-potential heme component), respectively). In the presence of 40 mmol�L–1 HOQNO, the apparent Em,7.6 of heme bL was shifted to –87 mV, without affecting the Em,7.6 of the high potential heme, which is in agreement with reported data in B. subtilis (Smirnova et al. 1995). The Em,7.6 values determined for the purified SQR of B. cereus were close to those reported for the purified SQR of B. subtilis (–96 and +65 mV) (Hägerhäll et al. 1992; Smirnova et al. 1995). Chemical composition and kinetic properties of SQR The chemical composition and kinetic constants of puri- fied SQR of B. cereus are summarized in Table 3. The en- zyme contained about 2.2 nmol covalently bound FAD�(mg protein)–1. Protoheme was present in a nearly 2:1 stoichiom- etry with FAD covalently bound, indicating the presence of 2 hemes per SQR complex. These ratios are in good agree- ment with those reported for the SQR complex of B. subtilis (Hägerhäll et al. 1992). Discussion Utilization of external succinate Bacillus cereus is an opportunistic pathogen and is closely related to the well-recognized pathogens Bacillus anthracis and Bacillus thuringiensis. Indeed, the comparison of the Table 2. Purification data of the succinate dehydrogenase of Bacillus cereus. Fraction Total protein (mg) Specific activitya Purification fold Yield (%) Membranes 6125 0.45 1.0 100 Bile salts 3101 1.06 2.4 119 Triton X-100 3051 0.56 1.2 62 DEAE Sepharose 300 2.20 5.0 24 QAE Toyopearl 57 7.63 17.0 16 aSpecific activities were expressed as reduced mmol dichlorophenolindophenol�(min�mg protein)–1. Fig. 4. Kinetic properties of the succinate:quinone reductase (SQR) purified from Bacillus cereus. Dose response to (a) succinate, (b) phe- nazine methosulfate (PMS), and (c) decylubiquinone. (d) Inhibition of the succinate:decylubiquinone reductase activity by HOQNO (2-n- heptyl-4-hydroxyquinoline N-oxide). Enzyme activity was determined by the PMS–DCPIP (dichlorophenolindophenol) reductase assay (a and b) or the quinone–DCPIP reductase assay (c and d) described in Materials and methods. 462 Can. J. Microbiol. Vol. 54, 2008 # 2008 NRC Canada Table 3. Comparison of succinate:quinone reductase (SQR) from Bacillus subtilis, Paracoccus denitrifi- cans, and Bacillus cereus. Characteristic B. subtilis P. denitrificans B. cereus Polypeptide (No. of residues) SdhA (584)a SdhA (600)a SdhA (586)b SdhB (251)a SdhB (259)a SdhB (253)b SdhC (201)a SdhC (130)a SdhC (208)b SdhD (129)a Prostetic group FAD, heme bp, and heme bd FAD, heme bpa FAD, heme bp, and heme bdb Heme b / FAD 2 1 2 Electron acceptor Menaquinonea Ubiquinonea Menaquinoned Enzyme turnoverh (s–1) 116a 140e 77b Inhibitori HOQNO Ki = 0.2 mmol�L–1f No Ki = 0.6 mmol�L–1b Malonate — No Ki = 88 mmol�L–1b TTFA Noc Ki = 5.52 mmol�L–1g Nob Carboxin Noc Ki = 13.2 mmol�L–1g — Note: FAD, flavin adenine dinucleotide; heme bp, proximal heme b; heme bd, distal heme b; HOQNO, 2-n-heptyl-4- hydroxyquinoline N-oxide; TTFA, 2-thenoyltrifluoroacetone; DCPIP, dichlorophenolindophenol; PMS, phenazine meth- osulfate. aData from Hederstedt (2002). bData from this work. cData from Hägerhäll et al. (1992). dData from Collins and Jones (1981). eData from Matsson et al. (1998). fData from Smirnova et al. (1995). gData from Pennoyer et al. (1988). hActivity was measured by DCPIP reduction using PMS as primary electron acceptor (succinate–PMS–DCPIP). iActivity was measured by DCPIP reduction using quinone as primary electron acceptor (succinate–quinone–DCPIP). Fig. 5. Light absorption spectra (at 77 K) of Bacillus cereus succinate:quinone reductase (SQR) reduced by (a) dithionite and (b) succinate. (c) Difference spectrum (a minus b). The 4th derivatives of the spectra are shown in the upper part of the panels. The protein concentration of purified SQR was 0.3 mg�mL–1. Garcı́a et al. 463 # 2008 NRC Canada metabolic potential encoded by the large core set of genes conserved between B. cereus and B. anthracis contradicts the hypothesis proposing that the common ancestor for the cereus group was a soil bacterium (Ivanova et al. 2003). The main feature of soil bacteria, such as B. subtilis, is the multiplicity of carbohydrate catabolic pathways. In contrast, a limited number of genes for degradation of carbohydrates are present in B. cereus and B. anthracis (Ivanova et al. 2003). The lack of a succinate–fumarate transport system in B. cereus seems to reflect a limited ability for carbohydrate degradation. Interestingly, genomic database analyses of B. anthracis and B. thuringiensis (NCBI, http//ncbi.nlm.nih. gob/Blast; The Institute for Genomic Research, http://www. tigr.org) suggested that as known for B. cereus, a C4- dicarboxylate transport system is also absent in these spe- cies. Instead, B. cereus and B. anthracis possess a large number of genes coding for proteolytic enzymes and a mul- tiplicity of peptide and amino acid transporters (Ivanova et al. 2003). This strongly suggests that proteins and their deri- vatives are the preferred nutrient source for B. cereus and B. anthracis. Accordingly, we found that alanine is the best exogenous substrate for respiration in B. cereus, while gluta- mate is also used. Besides the common potential metabolic properties, B. cereus and B. anthracis also share genes cod- ing numerous factors for invasion, establishment, and propa- gation of bacteria within the host. The presence of these genes in B. cereus was rather unexpected (Ivanova et al. 2003), but it is consistent with the cereus group ancestor being an opportunistic pathogen in insects (Margulis et al. 1998). SQR and MK7 The comparison of the kinetics of the reduction of membrane cytochromes by NADH and succinate (Fig. 2) indicated that reduction of membrane MK7 was the rate- limiting step in the succinate-supported respiration. Accord- ingly, NADH induced a rapid reduction of all cytochromes; this was abolished by photoinactivation (UV360) of membrane-bound MK7. On the other hand, succinate caused a fast reduction of only cytochrome b-558 associated with the SQR complex, and this was not altered by photoinactiva- tion of the MK7. Reconstitution with several quinone ana- logues restored the respiratory activity to even higher levels than those in non reated membranes (not shown). In the case of succinate, the quinone anologues tested significantly in- creased the rate of reduction of all cytochromes (not shown), suggesting that the quinone analogues tested were better electron acceptors for the SQR of B. cereus than its endoge- nous MK7. The SQR complex of B. cereus The molecular and kinetic properties of the purified SQR of B. cereus (Table 3) were similar to those of SQR of B. subtilis (Hägerhäll et al. 1992) and somewhat different from the SQR of P. denitrificans (Pennoyer et al. 1988; Matsson et al. 1998). Notably, Bacillus spp. SQRs consist Fig. 6. Potentiometric analysis of cytochrome b-558 in the purified succinate:quinone reductase (SQR) of Bacillus cereus. Redox titrations of 0.8 mg�mL–1 SQR were performed under anoxic conditions in the absence (*) and in the presence (*) of 40 mmol�L–1 HOQNO. Ex- perimental points were fitted by Nernst curves for 2 noninteracting single-electron components with unknown midpoint potentials (Em). Inset shows the semilog plot of the experimental data, yielding the best fit for the following parameters. Without HOQNO: for heme bH, Em = +79 mV ,and for heme bL, Em = –68 mV. In the presence of HOQNO: for heme bH, Em = +77 mV,and for heme bL, Em = –87 mV. 464 Can. J. Microbiol. Vol. 54, 2008 # 2008 NRC Canada of 3 subunits containing 2 cytochromes b and they use me- naquinone as an electron acceptor. They are highly sensitive to HOQNO, whereas TTFA and carboxin are not inhibitory. On the other hand, the SQR of P. denitrificans is formed by 4 subunits, containing one cytochrome b and uses ubiqui- none as an electron acceptor. TTFA and carboxin are strong inhibitors but HOQNO is not. The presence of 2 cytochromes b in SQRs that use mena- quinone as sole electron acceptor seems to be instrumental (Schirawski and Unden 1998). However, the SQR of the al- kaliphilic Bacillus sp. strain YN-2000 contains only one cytochrome b (Qureshi et al. 1996). We found that the SQR of B. cereus contained 2 cytochromes b per FAD and that succinate reduced about 50% of the total dithionite-reducible cytochrome b. Our chemical quantification of cytochrome b is in line with the presence of the 4 invariant histidines in positions 27, 69, 112, and 154 of the SdhC subunit, as pre- dicted by the genome sequence of B. cereus ATCC 14579 (GenBank entry AE016877). In the SQR of B. subtilis, these 2 cytochromes b were spectrally resolved by subtracting the succinate-reduced spectrum from the dithionite-reduced spectrum. The difference spectrum showed peaks at 553 and 558 nm (Hägerhäll et al. 1992). In contrast to the SQR from B. subtilis, and following the same experimental proto- col, we were not able to resolve the 2 cytochromes b of the B. cereus SQR by means of absorption bands (Fig. 5). How- ever, the redox titrations of our purified SQR unequivocally showed the presence of a high-potential heme component (cytochrome bH) and a low-potential heme component (cyto- chrome bL) with apparent midpoint potential (Em,7.6) values of +79 and –68 mV, respectively (Fig. 6). These values were not far from those reported by Hägerhäll et al. (1992) for the SQR of B. subtilis (+65 and –96 mV at pH 7.4, re- spectively). A detailed comparison of the proteins predicted from the sdh genes of the B. cereus ATCC 10987 genomes databases (GenBank entry AE017194) with the corresponding SQR proteins of B. subtilis 168 (GenBank entry AL009126), Pae- nibacillus macerans DSM24 (GenBank entryY08563), B. anthracis Ames (GenBank entry AE016879), and B. halodurans C-125 (GenBank entry BA000004), showed a high sequence similarity. For instance, the SdhA protein of B. cereus was 100%, 93%, 82%, and 74% identical to that of B. anthracis, B. halodurans, B. subtilis, and P. macerans, respectively. The predicted amino acid sequence of the SdhB subunit of B. cereus was 100% identical to the same protein of B. anthracis Ames, 83% to B. subtilis 168, 82% to B. halodurans C-125, and 74% to P. macerans DSM24 homologues. The predicted amino acid sequence of the membrane anchor protein (SdhC) of B. cereus was 99% identical to the same protein of B. anthracis, 72% to B. subtilis, 72% of B. halodurans, and 46% of P. macerans. Figure 7 shows the position of B. cereus SQR in a phylo- genetic tree based on the protein sequences of the flavo- protein subunit (SdhA) from several Bacillales and Corynebacteriae species (EMBL/GenPet/GenBank data- bases) reported to contain SQRs type B. As expected, B. cereus and B. anthracis are closely related, forming a separate clade from other Bacillales. A more complete tree including SdhA sequences of SQRs types A–C and QFRs has been already documented (Kurokawa and Sakamoto 2005). 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The succinate:menaquinone reductase of Bacillus cereus — characterization of the membrane-bound and purified enzyme

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