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Keywords:

  • Cupriavidus pinatubonensis;
  • dihydroxypropanesulfonate;
  • Klebsiella oxytoca;
  • Paracoccus pantotrophus;
  • Pseudomonas putida;
  • 3-sulfolactate

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Sulfoquinovose (SQ, 6-deoxy-6-sulfoglucose) was synthesized chemically. An HPLC-ELSD method to separate SQ and other chromophore-free sulfonates, e.g. 2,3-dihydroxypropane-1-sulfonate (DHPS), was developed. A set of 10 genome-sequenced, sulfonate-utilizing bacteria did not utilize SQ, but an isolate, Pseudomonas putida SQ1, from an enrichment culture did so. The molar growth yield with SQ was half of that with glucose, and 1 mol 3-sulfolactate (mol SQ)−1 was formed during growth. The 3-sulfolactate was degraded by the addition of Paracoccus pantotrophus NKNCYSA, and the sulfonate sulfur was recovered quantitatively as sulfate. Another isolate, Klebsiella oxytoca TauN1, could utilize SQ, forming 1 mol DHPS (mol SQ)−1; the molar growth yield with SQ was half of that with glucose. This DHPS could be degraded by Cupriavidus pinatubonensis JMP134, with quantitative recovery of the sulfonate sulfur as sulfate. We presume that SQ can be degraded by communities in the environment.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Sulfoquinovose (SQ; 6-deoxy-6-sulfoglucose) (Fig. 1) is the polar head group of the plant sulfolipid (Benson, 1963), the annual production of SQ by phototrophs is about 10 000 000 000 tonnes (Harwood & Nicholls, 1979), and very little is known about its biodegradation. Bacteria from the Americas degrade SQ quantitatively to sulfate and cell material via intracellular cysteate and sulfoacetate (Martelli & Benson, 1964; Martelli & Souza, 1970), but these organisms were lost (Cook & Denger, 2002). All five SQ-degrading bacteria from Europe, including a strain of Pseudomonas putida, released sub-stoichiometric amounts of sulfate from SQ (Roy et al., 2000, 2003). Two organisms (e.g. Pseudomonas sp. and Klebsiella sp. strain ABR11) excreted organosulfonates (and, e.g. acetate), which were identified in the medium by C13-NMR as 3-sulfolactate and 2,3-dihydroxypropane-1-sulfonate (DHPS, sulfopropanediol) (Roy et al., 2003) (chemical structures in Fig. 1). Two organisms expressed phosphofructokinase, consistent with the operation of a glycolytic-type degradative pathway for SQ. Klebsiella sp. strain ABR11 also expressed an NAD+-dependent SQ-dehydrogenase activity (Roy et al., 2003).

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Figure 1. Sulfoquinovose degraded by two pure cultures to 3-sulfolactate or DHPS, and the degradation of these two compounds by two SQ-negative pure cultures to yield sulfate stoichiometrically.

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More recently, organisms able to utilize sulfolactate and/or DHPS have been discovered, and corresponding degradative pathways elucidated (e.g. Denger & Cook, 2010; Mayer et al., 2010). Further, sulfonate excretion systems in degradative pathways have been proposed (e.g. Weinitschke et al., 2007; Mayer & Cook, 2009; Krejčík et al., 2010).

We wanted to use genome-sequenced organisms to expand on the work of Roy et al. (2000, 2003), but had little success with this approach, so we isolated an organism able to utilize SQ as a sole source of carbon and energy for growth. It was identified as a strain of P. putida, as found earlier by Roy et al. (2000), so we followed their lead to Klebsiella sp. and found that our sulfonate-utilizing Klebsiella oxytoca TauN1 (Styp von Rekowski et al., 2005) also utilized SQ. Each organism excreted a C3-sulfonate, which could be completely degraded by a second bacterium.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Chemical syntheses

Synthesis of SQ was achieved following in part the protocols of Miyano & Benson (1962) and of Roy & Hewlins (1997) without the need to form its barium salt for purification. The starting material for the preparation of SQ, 1,2-O-isopropylidene-6-O-tosyl-d-glucofuranose was prepared from 1,2-O-isopropylidene-d-glucofuranose by tosylation (Valverde et al., 1987) and isolated chromatographically pure. The tosylate (2.0 g) dissolved in ethanol (20 mL) was refluxed with an aqueous solution of Na2SO3 (1.21 g in 20 mL) under an inert gas atmosphere. Complete consumption of the starting tosyl compound (Rf: 0.62) was detected after 24 h by TLC in ethyl acetate on silica gel. Excess sodium sulfite was dissolved by the addition of water (50 mL) and the ethanol removed in vacuo. The aqueous solution was freed from sodium ions by passing it through a strongly acidic Amberlite IR 120 ion exchange column (45 g). Concentration of the acidic eluate under reduced pressure removed sulfur dioxide and cleaved the isopropylidene protecting group, leaving behind a syrup that consisted of equimolar amounts of p–toluenesulfonic acid and 6-sulfo-d-quinovose. Drying was continued at the lower pressure of an oil rotary vane pump upon which the syrup became gum-like. This gum was triturated with methanol upon which it partly solidified. Decanting off the methanol and repeating the procedure with fresh methanol led finally to a complete solidification. The 1H-NMR spectrum, analogous to that of Roy & Hewlins (1997), showed an enrichment of SQ as a mixture of its anomers over p-toluenesulfonic acid (≤ 10%) and no other organic impurities. Data from MALDI-TOF-MS in the negative ion mode gave m/z = 443 = [M−1]−1, which is consistent with SQ (M = 444).

The syntheses of DHPS and racemic sulfolactate were described elsewhere (Roy et al., 2003; Mayer et al., 2010). Other chemicals were available commercially from Sigma-Aldrich, Fluka, Merck or Biomol.

Bacteria and growth conditions

Burkholderia phymatum STM815 (DSM 17167) (e.g. Elliott et al., 2007), Burkholderia xenovorans LB400 (e.g. Chain et al., 2006), Cupriavidus necator H16 (DSM 428) (e.g. Pohlmann et al., 2006), Cupriavidus pinatubonensis JMP134 (DSM 4058) (Sato et al., 2006), K. oxytoca TauN1 (DSM 16963) (Styp von Rekowski et al., 2005), Paracoccus pantotrophus NKNCYSA (DSM 12449) (e.g. Rein et al., 2005), Sinorhizobium meliloti Rm1021 (e.g. Finan et al., 2001), Rhodopseudomonas palustris CGA009 (e.g. Larimer et al., 2004), Rhodobacter sphaeroides 2.4.1 (e.g. Mackenzie et al., 2001), P. putida F1 (e.g. Zylstra & Gibson, 1989), and P. putida KT2440 (e.g. Nelson et al., 2002) were grown aerobically at 30 °C in a phosphate-buffered mineral salts medium, pH 7.2 (Thurnheer et al., 1986). Roseobacter litoralis Och 149 (DSM 6996) (e.g. Kalhoefer et al., 2011) and Roseovarius sp. strain 217 (Schäfer et al., 2005) were cultured in a Tris-buffered artificial seawater medium (Krejčík et al., 2008). Strain Och 149 was grown at 25 °C and strain 217 required the addition of vitamins (Pfennig, 1978). Roseovarius nubinhibens ISM (González et al., 2003) was grown in modified Silicibacter basal medium (Denger et al., 2006) and needed a supplement of 0.05% yeast extract (Denger et al., 2009).

The sole carbon source was 5 mM sulfoquinovose or as a control 20 mM acetate or taurine or 10 mM succinate or 5 mM 4-toluenesulfonate or 5 mM glucose. Cultures on the 3-mL scale in 30-mL screw-cap tubes were incubated in a roller. For growth experiments, 12-mL cultures were grown in a beaker on a shaker, and 0.8 mL samples were taken at intervals to measure the optical density at 580 nm and to analyze concentrations of substrate and product.

Enrichment cultures were set up in a 3-mL scale in the freshwater mineral salts medium with 5 mM SQ as sole added carbon source. If turbidity developed and bacteria could be seen under the microscope, subcultures in fresh selective medium were inoculated. After four or five transfers, cultures were streaked on LB-agar plates and colonies were picked into fresh selective medium. After three rounds of plating and picking from homogeneous plates, cultures were considered pure.

Analytical methods

Growth was measured as turbidity at 580 nm and correlated with protein that was quantified in a Lowry-type reaction (Cook & Hütter, 1981). Sulfate was quantified turbidimetrically as a suspension of BaSO4 (Sörbo, 1987). 3-Sulfolactate was quantified by ion chromatography (IC) with the conditions described for sulfoacetate (Denger et al., 2004). DHPS was assayed qualitatively by the reaction of DHPS dehydrogenase [HpsN (EC 1.1.1.308) catalyzes the NAD+-dependent oxidation of DHPS to sulfolactate] from the soluble fraction of C. pinatubonensis JMP134 (Mayer et al., 2010). The reaction mixture contained in 50 mM Tris/HCl, pH 9.0, 2 mM NAD+, soluble fraction (about 0.3 mg protein mL−1) and outgrown medium of K. oxytoca TauN1 after growth with sulfoquinovose. Standard methods were used for the Gram reaction and to assay catalase or cytochrome c-oxidase activity (Gerhardt et al., 1994).

SQ was assayed with a colorimetric assay for reducing sugars (2,3-dinitrosalicylic acid method; Sturgeon, 1990). SQ was quantified by HPLC after separation on a Nucleodur HILIC (hydrophylic-interaction liquid chromatography) column (125 × 3 mm) (Macherey-Nagel, Düren, Germany) and evaporative light-scattering detection (ELSD). The isocratic eluent was 0.1 M ammonium acetate in 80 % acetonitrile with a flow rate of 0.5 mL min−1. Samples were dissolved in the eluent. Under those conditions, DHPS, taurine (2-aminoethanesulfonate), and glucose could also be analyzed directly in culture medium, which did not interfere with the analyses (Fig. 2); sulfolactate could also be quantified, but it interfered with the peak of sulfoquinovose.

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Figure 2. HPLC-chromatogram showing separation of three sulfonates and glucose when using a HILIC column and an ELSD detector. The chloride is from the culture medium.

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Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Problems with the syntheses of SQ

The chemical synthesis of SQ is simple: two hydroxyl groups of glucose are protected, and the hydroxyl group at C-6 tosylated and the tosyl group are displaced by sulfite. This yields two organic products, SQ and 4-toluenesulfonate, and, finally, sodium sulfate. The problem is to separate the two organic products, in which we were not fully successful. The consequence was that all organisms, with which we worked, had to be checked for growth with 4-toluenesulfonate. No organism used in the work utilized (or was inhibited by) 4-toluenesulfonate.

Separation and determination of SQ and its metabolites

We initially assayed SQ, a reducing sugar, with a standard method (Sturgeon, 1990) (e.g. Fig. 3). At low concentrations of sugar, the standard curve is, indeed, a curve and the interpolation had to be made manually. We required a different method, IC, for the metabolic product, 3-sulfolactate (Fig. 3), which eluted on the tail of the peak for sulfate (not shown). These methods were just adequate (Fig. 3), but inadequate for the next product, DHPS, which we could not detect by IC.

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Figure 3. Growth of Pseudomonas putida SQ1: (a) growth with 4 mM glucose (△) or 4 mM sulfoquinovose (○) and (b) concentrations of sulfoquinovose (□) and of sulfolactate (▲) as a function of growth.

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What was needed was a detector which was sensitive for nonchromophores and a column which could separate highly polar compounds. The ELSD detector and the HILIC column met our demands (Fig. 2). We optimized the system for our purposes and had linear standard curves between 0 and 5 pmol per injection (R2 > 0.99); a fresh standard curve was needed with each set of experiments.

Genome-sequenced organisms

We tested ten genome-sequenced organisms, which could utilize C2- or C3-sulfonates within 1 week as sole carbon and energy sources, for the ability to degrade SQ. No candidate was detected. The organisms were the Alphaproteobacteria R. sphaeroides 2.4.1, R. palustris CGA009, R. litoralis Och 149, R. nubinhibens ISM, Roseovarius sp. strain 217, and S. meliloti Rm1021, and the Betaproteobacteria B. phymatum STM815, B. xenovorans LB400, C. necator H16, and C. pinatubonensis JMP134.

Enrichment cultures

A set of aerobic enrichment cultures in SQ-mineral salts medium with an inoculum from forest soil, sediment from a forest pond or littoral sediment from Lake Constance yielded at least one positive culture per inoculum. One representative, rapidly growing, pure culture, strain SQ1 from the littoral sediment, was chosen for further work because it grew homogeneously in suspended culture. Its molar growth yield with SQ was half of that with glucose (Fig. 3a). The organism was identified as P. putida SQ1 by its 16S rRNA gene sequence and by its physiology (Holt et al., 1994): a rod-shaped, motile, nonspore-forming, Gram negative, catalase- and oxidase-positive aerobic bacterium.

Growth physiology of P. putida SQ1

Pseudomonas putida SQ1 grew in glucose salts medium with a molar growth yield of 5.0 g protein (mol C)−1 (Fig. 3a), a value which indicated complete utilization of the carbon source (Cook, 1987); glucose, measured as reducing sugar, disappeared. The organism grew only half as much in equimolar SQ-salts medium (Fig. 3a). Analysis of the spent growth medium showed that the SQ had disappeared completely, measured as reducing sugar, and that a product was visible by IC. This product co-eluted with authentic 3-sulfolactate and 1 mol sulfolactate (mol SQ)−1 was formed (Fig. 3b). The identity of this tentative 3-sulfolactate was confirmed by MALDI-TOF-MS in the negative ion mode. A novel signal at m/z = 169 = [M−1]−1 was found after growth, which corresponded to the Mcalcd = 170 for 3-sulfolactate. After growth of P. putida SQ1, we inoculated the outgrown medium with P. pantotrophus NKNCYSA, a freshwater bacterium from our culture collection known to degrade sulfolactate (Rein et al., 2005) and which did not utilize SQ. Strain NKNCYSA grew, sulfolactate was degraded, and stoichiometric amounts of sulfate were excreted into the medium (not shown). There was mass balance for the conversion of SQ to bacterial biomass and sulfate.

We had two genome-sequenced strains (F1 and KT2440) of P. putida in our strain collection, but neither organism utilized SQ, so we altered our strategy and used nonsequenced organism(s).

Organisms found in recent work

An isolate of Klebsiella sp., strain ABR11, was found to utilize SQ and to excrete DHPS (Roy et al., 2003). So, we tried a sulfonate-utilizing organism from our strain collection, K. oxytoca TauN1, whose genome is not sequenced (Styp von Rekowski et al., 2005) but which represents the genus of Klebsiella sp. strain ABR11.

Klebsiella oxytoca TauN1 grew overnight with SQ as sole source of carbon and energy, during which SQ disappeared (Fig. 4) and a compound was formed which could be oxidized with soluble fraction of C. pinatubonensis JMP134 plus NAD+ by the reaction of DHPS dehydrogenase, HpsN. The growth yield with SQ was half of that with glucose (not shown), consistent with excretion of 1 mol DHPS (mol SQ)−1, which was supported by HPLC (Fig. 4). These tentative identifications of DHPS were confirmed by MALDI-TOF-MS in the negative ion mode: A novel signal, which developed during growth, m/z = 155 = [M−1]−1, matched the Mcalcd = 156 for DHPS. Addition of the DHPS utilizer, C. pinatubonensis JMP134, to outgrown K. oxytoca TauN1 medium allowed growth (Fig. 4), and the DHPS disappeared while equimolar sulfate was released into the medium. As with P. putida SQ1 and P. pantotrophus NKNCYSA, there was mass balance for the conversion of SQ to bacterial biomass and sulfate.

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Figure 4. Degradation of 4 mM sulfoquinovose (□) and formation of dihydroxypropanesulfonate (▼) by Klebsiella oxytoca TauN1. After growth of strain TauN1, Cupriavidus pinatubonensis JMP134 was added, which degraded DHPS to sulfate (•) and cell material. Further explanation is given in the Results section.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The ease with which Martelli (in North and South America) (Martelli & Benson, 1964; Martelli, 1967; Martelli & Souza, 1970) and Roy et al. (2000) (on a European island) obtained bacteria able to utilize SQ was expanded on by our positive enrichment cultures on the European mainland. The American isolates, where studied (Martelli & Benson, 1964; Martelli & Souza, 1970), did not involve an excreted intermediate, whereas all of the seven European isolates (this paper and Roy et al., 2000, 2003) did so. The excreted intermediates were 3-sulfolactate, recovered quantitatively (Fig. 3), and DHPS, which was also recovered quantitatively (Fig. 4) (cf. Roy et al., 2003). These compounds are widespread, as are degradative organisms (see 'Introduction') which can degrade them in co-culture (e.g. Fig. 4). So, we presume SQ degradation in the environment to take place in communities (Fig. 4) that presumably include organisms of the type examined by Martelli (Martelli & Benson, 1964; Martelli & Souza, 1970).

Our data make clear that the advances made by Roy et al. (2003) are one key to understanding sulfoglycolysis at the molecular basis. They anticipate sulfoglycolysis (cleavage of 6-deoxy-6-sulfofructose-1-phosphate by an aldolase) on the one hand and an Entner-Doudoroff-type (or pentose-phosphate-type) pathway (oxidation of SQ to the lactone) on the other.

We anticipated rapid access to genome-sequenced SQ degraders, to allow rapid identification of genes, e.g. via peptide-mass fingerprint, and then pathways (e.g. Mayer et al., 2010). But neither our screen of genome-sequenced sulfonate utilizers nor our change from wild-type P. putida SQ to genome-sequenced P. putida spp. brought success, though we still believe in this approach.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The project was supported by the University of Konstanz and by the German Research Foundation (DFG) (SCHL 1936/1-1 to DS).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References