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Reconstruction of rod self-aggregates of natural bacteriochlorophylls-c from Chloroflexus aurantiacus
Chemical Physics Letters 578 (2013) 102–105
Contents lists available at SciVerse ScienceDirect
Chemical Physics Letters journal homepage: www.elsevier.com/locate/cplett
Reconstruction of rod self-aggregates of natural bacteriochlorophylls-c from Chloroflexus aurantiacus Sunao Shoji, Tadashi Mizoguchi, Hitoshi Tamiaki ⇑ Graduate School of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan
a r t i c l e
i n f o
Article history: Received 30 April 2013 In final form 10 June 2013 Available online 17 June 2013
a b s t r a c t Bacteriochlorophyll(BChl)s-c were extracted, isolated and purified from cultured cells of a green photosynthetic bacterium, Chloroflexus (Cfl.) aurantiacus. Their self-aggregates were prepared from a hydrophobic hexane-based solution and the obtained self-aggregate solids were examined by electronic absorption and circular dichroism (CD) spectroscopy as well as atomic force microscopy (AFM). Visible/near-infrared absorption and CD spectra of the BChl-c self-aggregates were very similar to those in cells of Cfl. aurantiacus. AFM analysis indicated that some self-aggregates had rods with a 5-nm diameter and a 3.5-lm length at longest. The rod diameter was identical to that reported for natural chlorosomal rods of Cfl. aurantiacus. Rod self-aggregates of naturally occurring BChls-c with a 5-nm diameter were first reconstructed here in vitro. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Chlorosomes are major light-harvesting antennas in green photosynthetic bacteria and have attracted much attention from the viewpoint of their supramolecular structures [1–6]. Chlorosomes contain bacteriochlorophyll(BChl)-c, d and e molecules [7] (further, BChl-f was recently found in the mutants lacking bchU of Chlorobaculum (Cba.) limnaeum [8–10]), and a large number of these pigments self-aggregate without any assistance from the peptides in a hydrophobic environment composed of a lipid monolayer. Supramolecular nanostructures of such BChls have been investigated by microscopic [11–17] and spectroscopic techniques [18–20] as well as molecular modelling [21–24], and identified as rod (5-nm or 10-nm diameter) and/or lamellar nanostructures. For instance, self-aggregates of natural BChl-c in Chloroflexus (Cfl.) aurantiacus Ok-70-fl were observed by freeze fracture transmission electron microscopy and identified as rods with a 5-nm diameter [14]. Chlorosomal BChls are magnesium complexes of 31-hydroxy131-oxo-chlorins (left drawing of Figure 1). Their central magnesium as well as peripheral 31-hydroxy and 131-oxo substituents are requisite for their self-aggregation in a chlorosome [25–29]. BChl-c is the most extensively researched chlorosomal pigment and several molecular variants are found in chlorosomes of Cfl. aurantiacus. These chlorosomes consist of 8-ethyl-12-methyl-
⇑ Corresponding author. Fax: +81 77 561 2659. E-mail address: [email protected] (H. Tamiaki). 0009-2614/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cplett.2013.06.012
BChls-c which possess various hydrocarbon chains at the 17-propionate residue: R17 = cetyl (C16), stearyl (C18), oleyl (C18), phytyl (C20) and geranylgeranyl groups (C20) as shown in Figure 1 [30– 32]. These BChls-c are an epimeric mixture at the chiral 31-position, and the ratio of (31R)/(31S) was 2/1 in Cfl. aurantiacus [32]. Therefore, BChls-c from Cfl. aurantiacus are also depicted as R/ S[E,M]BChl-c; the first R/S indicates the 31-stereochemistry and the terms in brackets show the 8-ethyl (E) and 12-methyl (M) groups. Self-aggregates of natural chlorosomal BChls have been prepared in a non-polar organic solvent as well as in an aqueous solution [33–46]. Their self-aggregates were investigated by spectroscopic techniques [electronic/vibrational absorption, linear/circular dichroism (CD) and NMR spectra] in the solution state. In the solid state, only the self-aggregates of R[E,E]BChlc, which was a major BChl pigment in Cba. tepidum, were measured on a highly oriented pyrolytic graphite (HOPG) by scanning tunneling microscopy [47]; the images showed chains of 2.2-nm circular spots. Recently, we reported that synthetic BChl-d analogs (Zn-1–n) possessing an oligomethylene chain at the 17-propionate residue (right drawing of Figure 1) selfaggregated in the solid state to give rods with a 5-nm diameter [48]. Here we report self-aggregates of naturally occurring BChls-c from Cfl. aurantiacus that were reconstructed in a non-polar organic solution to give precipitates that on a substrate showed as rod supramolecular nanostructures with a 5-nm or 10-nm diameter. Visible/near-infrared (NIR) absorption and CD spectra of such in vitro BChl-c self-aggregates were very similar to those in green photosynthetic bacterial cells.
S. Shoji et al. / Chemical Physics Letters 578 (2013) 102–105
Figure 1. Molecular structures of BChls-c from Cfl. aurantiacus and synthetic BChl-d analogs (Zn-1–n; n = 1–18, 24): stereochemistry at the 31-position = R or S; R17 = cetyl, stearyl, oleyl, phytyl and geranylgeranyl.
2. Experimental 2.1. Isolation, purification and determination of BChls-c Cfl. aurantiacus Ok-70-fl cells were cultured according to the reported method [49]. Pigments of Cfl. aurantiacus were extracted as follows. A mixture of acetone and MeOH (9/1, v/v) was added to the harvested cells, filtrated and evaporated. A mixture of Et2O and petroleum ether (1/1, v/v) was added and washed with distilled water. The ether phase was dried over sodium sulfate and evaporated. The residue was washed with hexane, purified by FCC (3–10% Et2O–CH2Cl2) and isolated BChls-c were further purified by HPLC (a Shimadzu SCL-10Avp system controller, LC-20AT pump and SPD-M10Avp photodiode-array detector). Reversephase HPLC was performed under the following conditions: column, Cosmosil 5C18-AR-II (10/ 250 mm, Nacalai Tesque); eluent, MeOH; flow rate 3.0 mL/min. BChls-c possessing several 17-propionates were determined by LCMS (a Shimadzu LCMS2010EV system) comprised of the above HPLC system and a quadrupole mass spectrometer equipped with an atmospheric pressure chemical ionization (APCI) probe. APCI-MS spectra were measured as follows: resolution, ±0.15 Da; capillary temperature, 250 °C; APCI vaporizer temperature, 400 °C; ionization voltage, 4.5 kV; sheath gas flow, 2.5 L/min; drying gas pressure, 0.02 MPa. Figure 2 shows isolated BChls-c possessing geranylgeranyl (27.0%), cetyl and oleyl (14.0%), phytyl (6.3%) and stearyl (50.6%) at the 17-propionate residue. The assignment of each BChls-c was consistent with the literature [32].
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Figure 3. Photographs of self-aggregates of BChls-c (100 lM) in 1% (v/v) THF– hexane.
precipitated within a few minutes (Figure 3). After allowing this suspended solution to stand at room temperature for 1 week, the precipitates were ultrasonicated for dispersal, and the resulting suspensions were drop-cast on quartz and freshly cleaved HOPG substrates. 2.3. Analysis of self-aggregates of BChls-c Visible/NIR absorption and CD spectra of self-aggregates were measured on a quartz substrate at room temperature using a Hita-chi U-3500 spectrophotometer and a Jasco J-720W spectropolarimeter, respectively. Self-aggregates of BChls-c on an HOPG substrate were measured by atomic force microscopy (AFM) with a Multimode 8 system (Bruker AXS) in tapping mode at room temperature in air, and a silicon cantilever (MPP-11100-10, Bruker AXS) was used. 3. Results and discussion Absorption spectra of self-aggregates of BChls-c on a quartz substrate gave Soret and Qy bands at 462 and 743 nm, respectively (Figure 4a). Cells of Cfl. aurantiacus were measured in an aqueous Tris–HCl buffer, and gave 464- and 742-nm maxima. The visible/
2.2. Preparation of self-aggregates of BChls-c Self-aggregates of BChls-c were prepared according to the reported procedure [48]. When the solution of BChls-c in THF (10 mM) was added to 99-fold hexane, almost all the samples were
Figure 2. Reverse-phase HPLC profile of isolated BChls-c. HPLC conditions: column, Cosmosil 5C18-AR-II (4.6/ 150 mm), eluent, H2O/MeOH = 5/95, flow rate, 1.0 mL/ min.
Figure 4. Visible/NIR absorption (a) and CD spectra (b) of self-aggregates of BChls-c on a quartz substrate (red lines) and cells of Cfl. aurantiacus in 50 mM Tris–HCl buffer (pH 8.0) (blue lines). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Figure 5. AFM analysis of self-aggregates of BChls-c on an HOPG substrate: (a–c) height images, (d–g) cross-section analysis along the white (d and f) and black arrows (e and g). Height images (d) and (e) are obtained from (b), and (f) and (g) from (c).
S. Shoji et al. / Chemical Physics Letters 578 (2013) 102–105
NIR spectra in vivo and in vitro were similar to each other. Additionally, strongly exciton-coupled CD signals were observed in the Qy region and both the CD signals were very similar (Figure 4b). These spectroscopic evidences indicated that in vitro supramolecular self-aggregates of BChls-c from Cfl. aurantiacus were reconstructed in the solid state, similarly as in natural chlorosomes. It is noted that the cells contain carotenoids and BChl-a with absorbances at around 500 and 800 nm, respectively. AFM images of in vitro self-aggregates of naturally occurring BChls-c are shown in Figure 5a–c. Two long rods as well as amorphous structures are visible in Figure 5a (shown by white arrows). These rods were about 5-nm (Figure 5b, d and e) and 10-nm heights (Figure 5c, f and g). The lengths of 5-nm and 10-nm rods in Figure 5a were 3.5 lm and 13.7 lm corresponding to aspect ratios of 700 and 1370, respectively. The modelling gave inverted micelle and bilayer cylinder structures for 5- and 10-nm rods, respectively [21–24]. Accordingly, the rods in Figure 5 probably represent respective structures. The in vitro self-aggregates of BChls-c from Cfl. aurantiacus gave a rod nanostructure with a 5nm diameter, which was the same as in natural chlorosomal rods from this organism. This is the first confirmation that such rod architecture can be reconstructed from naturally occurring BChlsc. In vitro self-aggregates of BChls-c also formed a rod with a 10nm diameter, while such a diameter was not observed in Cfl. aurantiacus. In our previous work, self-aggregates of synthetic BChl-d analogs possessing cetyl (Zn-1–16) and stearyl (Zn-1–18) did not form rods with a 10-nm diameter [48]. BChls-c from Cfl. aurantiacus possess geranylgeranyl, phytyl and oleyl at the 17-propionate residue other than cetyl and stearyl, so the former hydrocarbon chains would affect in vitro formation of 10-nm rods; the effect of the 31-methyl and central magnesium could not be ruled out. It is noted that self-aggregates of BChl-c in chlorosomes of the BchQ BchR mutant of Cba. tepidum, which consisted of only R[E,M]BChl-c possessing a farnesyl group at the 17-propionate residue, were observed as tubular supramolecular nanostructures [20]. 4. Conclusion BChls-c were isolated from a natural green photosynthetic bacterium, Cfl. aurantiacus, and their self-aggregates were prepared from a hexane-based solution. Visible/NIR absorption and CD spectra of the prepared self-aggregate solids were very similar to those in cells of Cfl. aurantiacus. Thus, the local (short-range) structure of BChl-c self-aggregates in the solid state was almost identical to that in natural chlorosomal systems. AFM images indicated BChls-c from Cfl. aurantiacus could reconstruct a rod nanostructure with a 5-nm diameter and this value was similar to that in natural chlorosomal rods. Rod architectures were first visible in the reconstructed solid state using naturally occurring BChl-c composites. Their supramolecular nanostructures would be slightly affected by the environments for their self-aggregation. Natural chlorosomes contain carotenoids, quinones and waxes, and the selfaggregates were surrounded by lipids. Thus these components would help the rod self-aggregation in a natural system. Acknowledgement This work was partially supported by Grants-in-Aid for Scientific Research (A) (No. 22245030) and on Innovative Areas ‘Artificial photosynthesis (AnApple)’ (No. 24107002) from the Japan Society for the Promotion of Science (JSPS).
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Contents lists available at SciVerse ScienceDirect
Chemical Physics Letters journal homepage: www.elsevier.com/locate/cplett
Reconstruction of rod self-aggregates of natural bacteriochlorophylls-c from Chloroflexus aurantiacus Sunao Shoji, Tadashi Mizoguchi, Hitoshi Tamiaki ⇑ Graduate School of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan
a r t i c l e
i n f o
Article history: Received 30 April 2013 In final form 10 June 2013 Available online 17 June 2013
a b s t r a c t Bacteriochlorophyll(BChl)s-c were extracted, isolated and purified from cultured cells of a green photosynthetic bacterium, Chloroflexus (Cfl.) aurantiacus. Their self-aggregates were prepared from a hydrophobic hexane-based solution and the obtained self-aggregate solids were examined by electronic absorption and circular dichroism (CD) spectroscopy as well as atomic force microscopy (AFM). Visible/near-infrared absorption and CD spectra of the BChl-c self-aggregates were very similar to those in cells of Cfl. aurantiacus. AFM analysis indicated that some self-aggregates had rods with a 5-nm diameter and a 3.5-lm length at longest. The rod diameter was identical to that reported for natural chlorosomal rods of Cfl. aurantiacus. Rod self-aggregates of naturally occurring BChls-c with a 5-nm diameter were first reconstructed here in vitro. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Chlorosomes are major light-harvesting antennas in green photosynthetic bacteria and have attracted much attention from the viewpoint of their supramolecular structures [1–6]. Chlorosomes contain bacteriochlorophyll(BChl)-c, d and e molecules [7] (further, BChl-f was recently found in the mutants lacking bchU of Chlorobaculum (Cba.) limnaeum [8–10]), and a large number of these pigments self-aggregate without any assistance from the peptides in a hydrophobic environment composed of a lipid monolayer. Supramolecular nanostructures of such BChls have been investigated by microscopic [11–17] and spectroscopic techniques [18–20] as well as molecular modelling [21–24], and identified as rod (5-nm or 10-nm diameter) and/or lamellar nanostructures. For instance, self-aggregates of natural BChl-c in Chloroflexus (Cfl.) aurantiacus Ok-70-fl were observed by freeze fracture transmission electron microscopy and identified as rods with a 5-nm diameter [14]. Chlorosomal BChls are magnesium complexes of 31-hydroxy131-oxo-chlorins (left drawing of Figure 1). Their central magnesium as well as peripheral 31-hydroxy and 131-oxo substituents are requisite for their self-aggregation in a chlorosome [25–29]. BChl-c is the most extensively researched chlorosomal pigment and several molecular variants are found in chlorosomes of Cfl. aurantiacus. These chlorosomes consist of 8-ethyl-12-methyl-
⇑ Corresponding author. Fax: +81 77 561 2659. E-mail address: [email protected] (H. Tamiaki). 0009-2614/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cplett.2013.06.012
BChls-c which possess various hydrocarbon chains at the 17-propionate residue: R17 = cetyl (C16), stearyl (C18), oleyl (C18), phytyl (C20) and geranylgeranyl groups (C20) as shown in Figure 1 [30– 32]. These BChls-c are an epimeric mixture at the chiral 31-position, and the ratio of (31R)/(31S) was 2/1 in Cfl. aurantiacus [32]. Therefore, BChls-c from Cfl. aurantiacus are also depicted as R/ S[E,M]BChl-c; the first R/S indicates the 31-stereochemistry and the terms in brackets show the 8-ethyl (E) and 12-methyl (M) groups. Self-aggregates of natural chlorosomal BChls have been prepared in a non-polar organic solvent as well as in an aqueous solution [33–46]. Their self-aggregates were investigated by spectroscopic techniques [electronic/vibrational absorption, linear/circular dichroism (CD) and NMR spectra] in the solution state. In the solid state, only the self-aggregates of R[E,E]BChlc, which was a major BChl pigment in Cba. tepidum, were measured on a highly oriented pyrolytic graphite (HOPG) by scanning tunneling microscopy [47]; the images showed chains of 2.2-nm circular spots. Recently, we reported that synthetic BChl-d analogs (Zn-1–n) possessing an oligomethylene chain at the 17-propionate residue (right drawing of Figure 1) selfaggregated in the solid state to give rods with a 5-nm diameter [48]. Here we report self-aggregates of naturally occurring BChls-c from Cfl. aurantiacus that were reconstructed in a non-polar organic solution to give precipitates that on a substrate showed as rod supramolecular nanostructures with a 5-nm or 10-nm diameter. Visible/near-infrared (NIR) absorption and CD spectra of such in vitro BChl-c self-aggregates were very similar to those in green photosynthetic bacterial cells.
S. Shoji et al. / Chemical Physics Letters 578 (2013) 102–105
Figure 1. Molecular structures of BChls-c from Cfl. aurantiacus and synthetic BChl-d analogs (Zn-1–n; n = 1–18, 24): stereochemistry at the 31-position = R or S; R17 = cetyl, stearyl, oleyl, phytyl and geranylgeranyl.
2. Experimental 2.1. Isolation, purification and determination of BChls-c Cfl. aurantiacus Ok-70-fl cells were cultured according to the reported method [49]. Pigments of Cfl. aurantiacus were extracted as follows. A mixture of acetone and MeOH (9/1, v/v) was added to the harvested cells, filtrated and evaporated. A mixture of Et2O and petroleum ether (1/1, v/v) was added and washed with distilled water. The ether phase was dried over sodium sulfate and evaporated. The residue was washed with hexane, purified by FCC (3–10% Et2O–CH2Cl2) and isolated BChls-c were further purified by HPLC (a Shimadzu SCL-10Avp system controller, LC-20AT pump and SPD-M10Avp photodiode-array detector). Reversephase HPLC was performed under the following conditions: column, Cosmosil 5C18-AR-II (10/ 250 mm, Nacalai Tesque); eluent, MeOH; flow rate 3.0 mL/min. BChls-c possessing several 17-propionates were determined by LCMS (a Shimadzu LCMS2010EV system) comprised of the above HPLC system and a quadrupole mass spectrometer equipped with an atmospheric pressure chemical ionization (APCI) probe. APCI-MS spectra were measured as follows: resolution, ±0.15 Da; capillary temperature, 250 °C; APCI vaporizer temperature, 400 °C; ionization voltage, 4.5 kV; sheath gas flow, 2.5 L/min; drying gas pressure, 0.02 MPa. Figure 2 shows isolated BChls-c possessing geranylgeranyl (27.0%), cetyl and oleyl (14.0%), phytyl (6.3%) and stearyl (50.6%) at the 17-propionate residue. The assignment of each BChls-c was consistent with the literature [32].
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Figure 3. Photographs of self-aggregates of BChls-c (100 lM) in 1% (v/v) THF– hexane.
precipitated within a few minutes (Figure 3). After allowing this suspended solution to stand at room temperature for 1 week, the precipitates were ultrasonicated for dispersal, and the resulting suspensions were drop-cast on quartz and freshly cleaved HOPG substrates. 2.3. Analysis of self-aggregates of BChls-c Visible/NIR absorption and CD spectra of self-aggregates were measured on a quartz substrate at room temperature using a Hita-chi U-3500 spectrophotometer and a Jasco J-720W spectropolarimeter, respectively. Self-aggregates of BChls-c on an HOPG substrate were measured by atomic force microscopy (AFM) with a Multimode 8 system (Bruker AXS) in tapping mode at room temperature in air, and a silicon cantilever (MPP-11100-10, Bruker AXS) was used. 3. Results and discussion Absorption spectra of self-aggregates of BChls-c on a quartz substrate gave Soret and Qy bands at 462 and 743 nm, respectively (Figure 4a). Cells of Cfl. aurantiacus were measured in an aqueous Tris–HCl buffer, and gave 464- and 742-nm maxima. The visible/
2.2. Preparation of self-aggregates of BChls-c Self-aggregates of BChls-c were prepared according to the reported procedure [48]. When the solution of BChls-c in THF (10 mM) was added to 99-fold hexane, almost all the samples were
Figure 2. Reverse-phase HPLC profile of isolated BChls-c. HPLC conditions: column, Cosmosil 5C18-AR-II (4.6/ 150 mm), eluent, H2O/MeOH = 5/95, flow rate, 1.0 mL/ min.
Figure 4. Visible/NIR absorption (a) and CD spectra (b) of self-aggregates of BChls-c on a quartz substrate (red lines) and cells of Cfl. aurantiacus in 50 mM Tris–HCl buffer (pH 8.0) (blue lines). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Figure 5. AFM analysis of self-aggregates of BChls-c on an HOPG substrate: (a–c) height images, (d–g) cross-section analysis along the white (d and f) and black arrows (e and g). Height images (d) and (e) are obtained from (b), and (f) and (g) from (c).
S. Shoji et al. / Chemical Physics Letters 578 (2013) 102–105
NIR spectra in vivo and in vitro were similar to each other. Additionally, strongly exciton-coupled CD signals were observed in the Qy region and both the CD signals were very similar (Figure 4b). These spectroscopic evidences indicated that in vitro supramolecular self-aggregates of BChls-c from Cfl. aurantiacus were reconstructed in the solid state, similarly as in natural chlorosomes. It is noted that the cells contain carotenoids and BChl-a with absorbances at around 500 and 800 nm, respectively. AFM images of in vitro self-aggregates of naturally occurring BChls-c are shown in Figure 5a–c. Two long rods as well as amorphous structures are visible in Figure 5a (shown by white arrows). These rods were about 5-nm (Figure 5b, d and e) and 10-nm heights (Figure 5c, f and g). The lengths of 5-nm and 10-nm rods in Figure 5a were 3.5 lm and 13.7 lm corresponding to aspect ratios of 700 and 1370, respectively. The modelling gave inverted micelle and bilayer cylinder structures for 5- and 10-nm rods, respectively [21–24]. Accordingly, the rods in Figure 5 probably represent respective structures. The in vitro self-aggregates of BChls-c from Cfl. aurantiacus gave a rod nanostructure with a 5nm diameter, which was the same as in natural chlorosomal rods from this organism. This is the first confirmation that such rod architecture can be reconstructed from naturally occurring BChlsc. In vitro self-aggregates of BChls-c also formed a rod with a 10nm diameter, while such a diameter was not observed in Cfl. aurantiacus. In our previous work, self-aggregates of synthetic BChl-d analogs possessing cetyl (Zn-1–16) and stearyl (Zn-1–18) did not form rods with a 10-nm diameter [48]. BChls-c from Cfl. aurantiacus possess geranylgeranyl, phytyl and oleyl at the 17-propionate residue other than cetyl and stearyl, so the former hydrocarbon chains would affect in vitro formation of 10-nm rods; the effect of the 31-methyl and central magnesium could not be ruled out. It is noted that self-aggregates of BChl-c in chlorosomes of the BchQ BchR mutant of Cba. tepidum, which consisted of only R[E,M]BChl-c possessing a farnesyl group at the 17-propionate residue, were observed as tubular supramolecular nanostructures [20]. 4. Conclusion BChls-c were isolated from a natural green photosynthetic bacterium, Cfl. aurantiacus, and their self-aggregates were prepared from a hexane-based solution. Visible/NIR absorption and CD spectra of the prepared self-aggregate solids were very similar to those in cells of Cfl. aurantiacus. Thus, the local (short-range) structure of BChl-c self-aggregates in the solid state was almost identical to that in natural chlorosomal systems. AFM images indicated BChls-c from Cfl. aurantiacus could reconstruct a rod nanostructure with a 5-nm diameter and this value was similar to that in natural chlorosomal rods. Rod architectures were first visible in the reconstructed solid state using naturally occurring BChl-c composites. Their supramolecular nanostructures would be slightly affected by the environments for their self-aggregation. Natural chlorosomes contain carotenoids, quinones and waxes, and the selfaggregates were surrounded by lipids. Thus these components would help the rod self-aggregation in a natural system. Acknowledgement This work was partially supported by Grants-in-Aid for Scientific Research (A) (No. 22245030) and on Innovative Areas ‘Artificial photosynthesis (AnApple)’ (No. 24107002) from the Japan Society for the Promotion of Science (JSPS).
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