Sulfoquinovose degraded by pure cultures of bacteria with release of Crorganosulfonates: complete degradation in two-member communities
Karin Denger', Thomas Huhn2, Klaus Hollemeyer3, David Schleheck' & Alasdair M. Cook'
'Department of Biology, University of Konstanz, Konstanz, Germany; 2Department of Chemistry, University of Konstanz, Konstanz, Germany; and 'Institute of Biochemical Engineering, University of the Saarland, Saarbrücken, Germany
Abstract
Correspondence: Alasdair M. Cook, Department of Biology, University of Konstanz, D-78457 Konstanz, Germany. Tel.:
+49 753 1884247; fax: +49 753 188 2966;
e-mail: alasdair.cook@uni-konstanz.de
DOI: 10.1111/j.1574-6968.2011.02477.x
Editor: Christiane Dahl
Keywords
Cupriavidus pinatubonensis;
dihydroxypropanesulfonate; Klebsiella oxytoca; Paracoccus pantotrophus;
Pseudomonas putida; 3-sulfolactate.
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-l-sulfonate (DHPS), was developed. A set of 10 genome-sequenced, sulfonate-utilizing bacteria did not utilize SQ, but an iso- late, Pseudomonas putida SQl, 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) -I 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 TauNI, could utilize SQ, forming 1 mol DHPS (mol SQ)-I; the molar growth yield with SQ was half of that with glucose. This OHPS could be degraded by Cupriavidus pinatubonensis JMPI34, with quantitative recovery of the sulfonate sulfur as sulfate. We presume that SQ can be degraded by communities in the environment.
Introduction
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 sul- foacetate (Martelli & Benson, 1964; Martelli & Souza, 1970), but these organisms were lost (Cook & Denger, 2002). All five SQ-degrading bacteria from Europe, including astrain of Pseudomonas putida, released sub- stoichiometric amounts of sulfate from SQ (Roy et a/., 2000, 2003). Two organisms (e.g. Pseudomonas sp. and Klebsiella sp. strain ABRI!) excreted organosulfonates (and, e.g. acetate), which were identified in the medium by C 13 -NMR as 3-sulfolactate and 2,3-dihydroxypropane- I-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 ABRll also expressed an NAO+ -dependent SQ- dehydrogenase activity (Roy et a/., 2003).
More recently, organisms able to utilize sulfolactate and/or OHPS have been discovered, and corresponding degradative pathways elucidated (e.g. Oenger & Cook, 2010; Mayer et a/., 2010). Further, sulfonate excretion systems in degradative pathways have been proposed (e.g. Weinitschke et al., 2007; Mayer & Cook, 2009;
Krejcik et
ai.,
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 astrain of P. putida, as found earlier by Roy et a/. (2000), so we followed their lead to Klebsiella sp. and found that our sulfonate-utilizing
Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-176904
HO~ S03-
0HO OH
Growth of Pseudomonas putida S01
--
OH Growth of
Sulfoquinovose (SO)
~ Klebsiella oxytoca TauN1
~ --
Growth of
Paracoccus pantotrophus NKNCYSA
l ) Y S O , -
~ --
OH
3-sulfolactate (Slac)
Growth of Cupriavidus pinatubonensis JMP134
HO~SO,- ~
OH rv~
--
sOI-
sOI-
2.3-dihydroxypropane-1-sulfonate (DHPS)
Fig. 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.
Klebsiella oxytoca TauNI (Styp von Rekowski et al., 2005) also utilized SQ. Each organism excreted a Crsulfonate, which could be completely degraded by a second bacte- rium.
Materials and methods
Chemicalsyntheses
Synthesis of SQ was achieved following in part the proto- cols of Miyano & Benson (1962) and of Roy & Hewlins (1997) without the need to form .its barium saIt for puri- fication. The starting material for the preparation of SQ, 1,2-0-isopropylidene-6-0-tosyl-D-glucofuranose was pre- pa red from 1,2-0-isopropylidene-D-glucofuranose by tosylation (Valverde et al., 1987) and isolated chromato- graphically pure. The tosylate (2.0 g) dissolved in ethanol (20 mL) was refluxed with an aqueous solution of NazS03 (1.21 gin 20 mL) under an inert gas atmosphere.
Complete consumption of the starting tosyl compound (Re: 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 c1eaved 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-Iike.
This gum was triturated with methanol upon wh ich it partly solidified. Decanting off the methanol and repeat- ing the procedure with fresh methanol led finally to a
complete solidification. The IH-NMR spectrum, analo- gous to that of Roy & Hewlins (1997), showed an enrich- ment 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, wh ich is consistent with SQ (M = 444).
The syntheses of DHPS and racemic sulfolactate were described e1sewhere (Roy et al., 2003; Mayer et al., 2010).
Other chemicals were available commercially from Sigma- A1drich, Fluka, Merck or Biomol.
Bacteria and growth conditions
Burkholderia phymatum STM815 (DSM 17167) (e.g. Elli- ott 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 TauNI (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 Fl (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 saIts 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 (Krejcik et al., 2008). Strain Och 149 was grown at 25°C and strain 217 required the addition of vitamins (Pfennig, 1978). Roseovarius nubinhi- bens ISM (Gonzalez et al., 2003) was grown in modified
Silicibacter basal medium (Oenger et al., 2006) and needed a supplement of 0.05% yeast extract (Oenger 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 incu- bated in a roller. For growth experiments, 12-mL cultures were grown in a beaker on a shaker, and 0.8 mL sampIes 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, subcliltures 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, cuItures were considered pure.
Analytical methods
Growth was measured as turbidity at 580 nm and corre- lated with protein that was quantified in a Lowry-type reaction (Cook & Hütter, 1981). Sulfate was quantified turbidimetrically as a suspension of BaS04 (Sörbo, 1987).
3-Sulfolactate was quantified by ion chromatography (IC) with the conditions described for sulfoacetate (Denger et al., 2004). OHPS was assayed qualitatively by the reac- tion of OHPS dehydrogenase [HpsN (EC 1.1.1.308) cata- Iyzes the NAO+-dependent oxidation of OHPS to sulfolactatel from the soluble fraction of C. pinatubonen- sis JMP134 (Mayer et al., 2010). The reaction mixture contained in 50 mM Tris/HCl, pH 9.0, 2 mM NAO+, sol- uble fraction (about 0.3 mg protein mL .... I) and outgrown medium of K. oxytoca TauN1 after growth with sulfoqui- novose. Standard methods were used for the Gram reac- tion 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 Nucleo- dur HILlC (hydrophylic-interaction liquid chromatogra- phy) column (125 x 3 mm) (Macherey-Nagel, Oüren, Germany) and evaporative Iight-scattering detection (ELSO). The isocratic e1uent was 0.1 M ammonium acetate in 80 % acetonitrile with a flow rate of 0.5 mL min -1.
SampIes were dissolved in the eluent. Und er those conditions, OHPS, taurine (2-aminoethanesulfonate), and glucose could also be analyzed directly in cuIture medium, which did not interfere with the analyses (Fig. 2); sulfolac-
1.0
Chloride
~
~ '(ji
c: 0.5
.E 2 OHPS
o
2 4 6 8 10Retention time (min)
Fig. 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.
tate could also be quantified, but it interfered with the peak of sulfoquinovose.
Results
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-tolu- enesulfonate, 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 organ- isms, 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 concentra- tions 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-sul- folactate (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, OHPS, which we could not detect by IC.
What was needed was a detector which was sensitive for nonchromophores and a column which could sepa- rate highly polar compounds. The ELSD detector and the HILlC column met our demands (Fig. 2). We optimized
(a) (b)
0,6
" "
4 00 0 tE c 0,5
"
000 ~ 0
•
co 3 0
Ln tJ.
.s
1ii 0,4 00 c 0
•
C;- 0 o
~ 0
'~ 0,3
"
0 'E 2•
0Q)
'0 0 Q)
~ 0,2 0 () c
•
"
0 0a
0 0 0 ü 00 0,1
"
0••
Fig, 3. Growth of Pseudomonas putida SQ1:~ " 0
• • ....
B (a) growth with 4 mM glucose (6.) or 4 mM0.0 0
•
sulfoquinovose (0) and (b) concentrations of0 3 6 9 12 0.0 0,1 0,2 0,3 0,4 sulfoquinovose ([l) and of sulfolactate ("') as
Time (h) Optical density at 580 nm a function of growth,
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 Cr or Crsulfonates within 1 'week as sole carbon and energy sources, for the ability to degrade SQ. No candidate was detected. The organisms were the Alpha- proteobacteria 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 Betaproteo- bacteria B. phymatum STM815, B. xenovoral1s LB400, C. necator H16, and C. pinatubonel1sis 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 &om Lake Con- stance yielded at least one positive culture per inoculum.
One representative, rapidly growing, pure culture, strain SQ 1 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. p,utida SQ1
Pseudomonas putida SQ 1 grew in glucose salts medium with a molar growth yield of 5.0 g protein (mol C)-I (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). Analy- sis of the spent growth medium showed that the SQ had disappeared completely, measured as reducing sugar, and that a product was visible by Ie. This product co-eluted with authentic 3-sulfolactate and 1 mol sulfolactate (mol SQ)-I 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
r-
I 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. pal1totrophus NKNCYSA, a freshwater bacterium from our culture collection known to degrade sulfolactate (Rein et al., 2005) and which did not utilize SQ. Strain NKN- CYSA 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 ABRll, was found to uti- Iize SQ and to excrete DHPS (Roy et al., 2003). So, we tried a sulfonate-utilizing organism from our strain collection, K. oxytoca TauNI, whose genome is not sequenced (Styp von Rekowski et al., 2005) but which represents the genus of Klebsiella sp. strain ABR11.
Klebsiella oxytoca TauNl grew overnight with SQ as sole SOl\rce of carbon and energy, du ring wh ich SQ dis- appeared (Fig. 4) and a compound was formed which could be oxidized with soluble fraction of C. pinatubol1- ensis JMP134 plus NAD+ by the reaction of DHPS dehy- drogen ase, HpsN. The growth yield with SQ was half of that with glucose (not shown), consistent with excretion
Strain TauN1 + strain JMP134
5 0
0 0
~ 4
.s
c:3
0
~ -E
'"
2() c 0 U
o
0.0 0.2 0.4 0.4 0.6 0.8
Growth (00580 nm)
Fig. 4. Degradation of 4 mM sulfoquinovose (0) and formation of dihydroxypropanesulfonate (T) by Klebsiel/a oxytoca TauNl. After growth of strain TauNl, Cupriavidus pinatubonensis JMP.134 was added, which degraded DHPS to sulfate (.) and cell material. Further explanation is given in the Results section.
of 1 mol DHPS (mol SQ) '-1, wh ich 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, wh ich developed during growth, m/z = 155 = [M- lj-I, matched the Mcalcd = 156 for DHPS. Addition of the DHPS utilizer, C. pinatubonensis JMPI34, to outgrown K. oxytoca TauNI medium allowed growth (Fig. 4), and the DHPS disappeared while equimolar sulfate was released into the medium. As with P. putida SQ 1 and P. pal1totrophus NKNCYSA, there was mass balance for the conversion of SQ to bacterial bio- mass and sulfate.
Discussion
The ease with wh ich Martelli' (in North and South Amer- ica) (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 main- land. 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 quan- titatively (Fig. 3), and DHPS, which was also recovered quantitatively (Fig, 4) (cf. Roy et al., 2003). These com- pounds 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 environ- ment to take place in communities (Fig. 4) that presum- ably indude organisms of the type examined by Martelli (Martelli & Benson, 1964; Martelli & Souza, 1970).
Our data make dear that the advances made by Roy et al. (2003) are one key to understanding sulfoglycolysis at the molecular basis. They anticipate sulfoglycolysis (c1eavage of 6-deoxy-6-sulfofructose-l-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
The project was supported by the University of Konstanz and by the German Research Foundation (DFG) (SCHL
1936/1-1 to DS).
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