- Email: [email protected]
[77] UDP-glucose: Polyglycerolteichoic acid glucosyltransferase from Bacillus subtilis
436
ENZYMES OF COMPLEX SACCHARIDE SYNTHESIS
[77]
[77] UDP-Glucose: PolyglycerolteichoicAcid Glucosylt r a n s f c r a s c f r o m Bacillus subtilis 1
By Lcls GLASERand M. M. BURGER
CHz--CH--CHz--O--.P--O-vCH~--.CH--CI~--O--.P--O--,
I OH
I OH
J O_
| (
I OH
+ n UDP-glucose ~
c ~ - - cI-I- c ~ - o - P - o ~ c ~ I
J
O_
1
]n
/
n UDP
CH-- CH,-- O--P--O~, I
I
|
OH ? O_ ( O-glucose O_ )n glucose FIG. 1. Glucosylationof polyglycerolphosphate. Assay Method
Principle. The assay is based on the incorporation of 1~C from UDPD-glucose-14C into polymeric form, as measured by radioactivity remaining at the origin after paper chromatography. Reagents Tris-C1, 0.05 M, pH 8.0-0.01 M MgCl~-0.001 M EDTA UDP-D-glucose-~4C, 0.001 M (100,000 cpm/micromole). This can be prepared enzymatically from UTP and glucose-l-P-~4C or glucose-6-P. 2 It can also be prepared from ~4C-glucose-l-P by the morpholidate procedure? Polyglycerolphosphate, 5 micromoles/ml (expressed in micromoles of glucose free glycerol in polyglycerolphosphate). This material is prepared as described below. Pyridine 1L. Glaser a n d M. M. Burger, J. Biol. Chem. 239, 3187 (1964). T h e strains of B. subtilis ( A T C C 6051, N C T C 3610) used in this work contain a fully glucosylated polyglycerolphosphate in the cell wall, and a mixture of polyglycerolphosphate and fully glucosylated polyglycerolphosphate in the "intracellular" fraction. 2 L. Glaser, J. Biol. Chem. 232, 627 (1958). 3 M. Smith a n d H. G. Khorana, Vol. ¥ I [94].
[77]
UDP-GLUCOSE: POLYGLYCEROLTEICHOICTRANSFERASE
437
Procedure. The reaction mixture contains 0.2 ml of Tris-Mg-EDTA buffer pH 8.0, 0.05 ml of polyglycerolphosphate, 0.04 ml of UDP-glucose-14C and 0.01-0.05 ml of enzyme (see below) and is incubated at 37 ° for 30 minutes. The reaction is stopped by the addition of an equal volume of pyridine, streaked on strips of Whatman No. 1 paper (4 X 50 cm), and chromatographed by descending paper chromatography in ethanol 1 M NH4-acetate pH 3.8 (7.5:3.0). 4 The solvent is allowed to reach the end of the paper (approximately 18 hours). The strips are scanned for radioactivity in a paper strip counter and the radioactivity remaining at the origin determined. It is found convenient to determine the ratio of the counts per minute at the origin, to the total counts on the paper, automatically correcting for errors in spotting and for variations in the efficiency of the paper strip counter. When the required accuracy is not large, it is necessary only to count the origin. Although they have not been used by the author, other methods for counting 14C on paper may be equally satisfactory. However, only part of the radioactivity can be eluted from the paper, and counting paper eluates does not provide a satisfactory assay. A control stopped at 0 time is always included. Pyridine is used to stop the reaction because it gives a homogeneous suspension that can be spotted on paper. Attempts to deproteinize the solution with acid or heat prior to chromatography lead to partial losses of glucosylated polyglycerolphosphate by adsorption on the protein. The assay described should be used with considerable caution. Other strains of B. subtilis than the ones used may contain different teichoic acids and the enzymatic products be completely different.5 In related organisms such as B. licheniformis ATCC 9945 a polymer of the same overall composition has been isolated from the cell wall and synthesized enzymatically.6 In this new polymer the hexose is a part of the polymer backbone.
Preparation of B. subtilis membranes These are prepared from B. subtilis NCTC 3610 or ATCC 6051 by lysozyme digestion as described for the enzymes that synthesize polyglycerolphosphate2 The enzyme preparation contains considerable glucosyl acceptor. In order to have a preparation dependent on the addition of 4D. C. Paladini and L. F. Leloir, Biochem. J. 51, 426 (1952). 5In B. subtilis W23 a similar enzyme preparation will catalyze glucosyl transfer to polyribitol phosphate (T. Chin and L. Glaser, unpublished observations). M. M. Burger and L. Glaser, J. Biol. Chem. in press. 7This volume [76].
438
ENZYMES OF COMPLEX SACCHARIDE SYNTHESIS
[77]
exogenous glucosyl acceptor the enzyme is preincubated with cold UDPD-glucose as follows: 1 ml of membrane suspension is incubated at 37 ° with 0.5 ml of 0.01 M UDP-glucose and 2 ml of 0.05 M Tris-C1-0.01 M MgC12-0.001 M EDTA pH 8.0 at 37 ° for 2 hours. At the end of the incubation the reaction medium is diluted to l0 ml with cold 0.05M Tris-C1-0.01 M MgC12-0.001 M EDTA pH 8.0 centrifuged and the membranes twice suspended in 10 ml of the same buffer with a loose fitting TenBroeck homogenizer and collected by centrifugation. Finally they are suspended in 1 ml of the same buffer and stored frozen. The preparation can be stored frozen for at least one month without significant loss of activity. Under optimal conditions the activity of this enzyme is about 40 millimicromoles of glucose transferred per milligram membrane, dry weight, per hour. Properties of the Enzyme In Tris buffer the pH optimum is 8. The enzyme has an absolute requirement for divalent cation. The optimal concentration of MgC12 is 3 X 10-2 M. At 1 X 10-2 M MgCI2 the activity is 80% of maximal and at 5 X 10-3 M, 50%. The Km for UDP-glucose is 4 X 10-5 M at pH 8.0, and the Km for polyglycerolphosphate (expressed as glycerolphosphate units) is 2 X 10-3 M. Preparation of Polyglycerolphosphate This material can be prepared enzymaticallyT; it is most easily obtained as a partially glucosylated "intracellular" glycerol teichoic acid from B. subtilis NCTC 3610 or ATCC 6051. This organism is grown and harvested as described in this volume [76]. The cells are disrupted in a Nossal shaker (8 g of 0.2 mm glass beads, 8 ml of cell suspension, 1 drop capryl alcohol, are shaken for six 30-second periods with intermittent cooling in an ice bath). Intact cells and cell walls are removed by centrifugation at 10,000 g for 10 minutes. The supernatant fluid is centrifuged at 105,000 g for 60 minutes. The sediment is suspended in water and stirred with an equal volume of 80% phenol at 0 ° for 30 minutes. The suspension is centrifuged at 15,000 g for 10 minutes; the upper phase is removed and the phenol layer is washed with an equal volume of water. The pooled aqueous phase is extracted with an equal volume of chloroform and then dialyzed overnight against distilled water. This material can be further purified by digestion with 10 #g/ml each of RNase and DNase in 0.05 M Tris-C1 buffer pH 8.0 for 3 hours at 37 ° followed by a second phenol extraction. The dialyzed polyglycerolphosphate is concentrated by lyophilization and then chromatographed on Sephadex G-200 equilibrated with 0.1 M triethylammonium acetate pH
[77]
UDP-GLUCOSE: POLYGLYCEROLTEICHOICTRANSFERASE
439
6.8. The teichoic acid is eluted with the void volume and can be freed of buffer by lyophilization. Alternatively teichoic acid may be prepared by extraction of an acetone powder of whole cells with 107~ trichloroacetic acid, as described by Kelemen and Baddiley.s Material prepared by this method appears to be of lower molecular weight than material prepared by phenol extraction, but is equally satisfactory as a glucosyl acceptor. The teichoic acid obtained by either method is analyzed by hydrolyzing an aliquot in 1 N HC1 for 3 hours at 100 °, and, after evaporating the HC1 in vacuo, digesting the sample with E. coli alkaline phosphatase. The inorganic phosphate in the digest is determined by the method of Chen, Toribara and Warner, 9 glucose with hexokinase and glucose 6-phosphate dehydrogenase11 and glycerol with glycerol dehydrogenase.1° The ratio of glycerol to phosphate should be close to unity. The difference between the glycerol concentration and the glucose concentration is a measure of the concentration of unglueosylated polyglycerolphosphate. Alternatively since glucosylpolyglycerolphosphate is alkali stable the sample can be hydrolyzed in 1 N KOH for 2 hours, KOH precipitated by neutralization with HCI04 and the sample digested with E. coli alkaline phosphatase. Essentially all the inorganic phosphate is liberated from free (not glucosylated) glycerolphosphate residues. Isolation of 14C-Glucosyl Polyglycerolphosphate Since it is difficult to elute glucosylpolyglycerolphosphate quantitatively from paper chromatograms, the product is best isolated by phenol treatment of the whole reaction mixture, as described for the preparation of polyglycerolphosphate (see above). The final chromatography on Sephadex G-200 will remove any remaining traces of UDP-glucose. Identification of 14C-Glucosylpolyglycerolphosphate The identification of glucosylated polyglycerolphosphate is best carried out by degradation to glucosylglycerol or glucosylglycerolphosphate. Fully glucosylated polyglycerolphosphate is alkali stable, and the only successful method of degradation is fluorolysis in 60% HF at 0%1,11 This method is generally applicable to all fully substituted polyglycerolphosphate. s M. V. Kelemen and J. Baddiley, Bichem. J. 80, 246 (1961). 9p. S. Chert, Jr., T. Y. Toribara, and H. Warner, Anal. Chem. 28, 1756 (1956). See also this volume [10]. 1oSee Vol. I [59]. This enzyme is not absolutely specific and should be used with caution. Glycerol can also be determined with glycerol kinase and glycerolphosphate dehydrogenase (Vol. V [46], [65a]). ,1Unpublished procedure kindly made available by Dr. D. Lipkin and Mr. J. AbreU. See also J. Abrell, Ph.D. Thesis, Washington University, 1965.
440
ENZYMES OF COMPLEX SACCHARIDE SYNTHESIS
[77]
To 1 mg of dry polymer (up to 5 mg) in a 12-ml conical polyethylene centrifuge tube is added 0.! ml of 60% H F at 0°; this is best done with a drawn out polyethylene tube attached to a plastic syringe. The sample is incubated at 0 ° for 5 hours and cooled in a dry ice-Cellosolve bath; 0.7-0.75 ml of saturated LiOH is added. The tube is allowed to warm to 0 ° and neutralization completed by the addition of saturated Li2C03. After 1 hour at 0 ° the insoluble LiF is removed by centrifugation and washed twice with 1 ml of water. The pooled supernatant fluids are deionized by passage through a 1 X 5-cm column of Amberlite MB-3 and concentrated in a flash evaporator. Glucosylglycerol can be isolated by putting the sample on a charcoal Celite column (1 g of Darco-G-60 and 1 g of Celite for 1 mg or less of glucosylpolyglycerolphosphate) and eluting free glycerol and glucose with H20 and glucosylglycerol with 10% ethanol. TM The structure of the glucosylglycerol obtained from the cell wall teichoic acid has been determined as 2- (a-D-glucopyranosyl) glycerol, since it is cleaved completely with ~-glucosidase18 to glucose and glycerol and since no formaldehyde is formed on periodate oxidation. For the identification of the enzymatically synthesized polyglycerolphosphate, the synthesis is best carried out using ~4C-polyglycerolphosphate as an aeceptor. (This acceptor can be prepared enzymatically.~) The degradation of the polymer after addition of carrier glucosylpolyglycerolphosphate is carried out as described above. The ~4Cglucosyl-~4C-glycerol is identified by its elution pattern from a charcoal Celite column, by cleavage to ~4C-glucose and ~4C-glycerol with a-glucosidase, and by the absence of l~C-formaldehyde formation after periodate oxidation. ~ Since no satisfactory paper chromatography system is available for separating glucosylglycerol from glucose, the products of a-glucosidase action after deionization on an Amberlite MB-3 column, are first fractionated on a charcoal Celite column. This separates glucose and glycerol from residual glucosylglycerol. The glycerol can be identified by chromatography in butanol-pyridine-H_~O (6:4: 3) ,~4 and by conversion to L-a-glycerolphosphate with glycerol-kinase.~5 The L-a-glycerol-P is identified by chromatography in ethanol-1 M ammonium acetate, pH 7.8.4 The glucose eluted from the charcoal Celite column is most easily 1,R. L. Whistler and J. N. BeMiller, in "Methods in Carbohydrate Chemistry" (R. L. Whistler and M. L. Wolfrom, eds.), Vol. I, p. 42. Academic Press, New York, 1962. 1~I. R. Lehman and E. A. Pratt, J. Biol. Chem. ~.35, 3254 (1960). 14A. Jeanes, C. S. Wise, and R. J. Dimler, Anal. Chem. 23, 415 (1961). E. P. Kennedy, Vol. V [65a]. G. Bublitz and O. Wieland, Vol. V [46].
[78]
FORMATION OF RHAMNOLIPIDS OF P. aeruginosa
441
identified by its chromatographic behavior, and by oxidation to gluconic acid with glucose oxidase followed by c h r o m a t o g r a p h y in b u t a n o l p y r i d i n e - H 2 0 (6: 4: 3) .14 Although in the original identification of the enzymatically synthesized glucosylpolyglycerolphosphate, the identification of 2-(a-D-glucopyranosyl)glycerol was based on the degradation scheme described above, 1 the same compound has been prepared chemically and could be conveniently used as a reference compound or as carrier for crystallization of the radioactive product. 16 '~A. J. Charlson and A. S. Perlin, Can. J. Chem. 34, 1200 (1956). See also A. J. Charlson, P. A. J. Gorin, and A. S. Perlin, Can. J. Chem. 35, 365 (1957).
[78] Formation
of Rhamnolipids
of Pseudornonas
aeruginosa 1
B y M. M. BURGER, L. GLASER, and R. M. BURTON 2 flOH-decanoyl-CoA --~ --~ ~OH-decanoyl-~OH-decanoic acid (1) ~OH-decanoyl-flOtt-decanoic acid ~- dTDP-L-rhamnose -~ dTDP -b a-L-rhamnospyranosyl-~OH-decanoyl-~OH-decanoic acid (2) L-RhamnosyI-~OH-decanoyl-BOH-decanoic acid q- dTDP-L-rhamnose dTDP q- a-L-rhamnopyranosyl-1 --~ 2-a-L-rhamnopyranosyl-/~OH-decanoyl-flOH-decanoic acid~ (3) Assay M e t h o d Principle. The products of the reactions are extracted into ether after acidifying the incubation mixture. The radioactivity contained in the products is measured and used as the estimate of enzymatic synthesis. Reactions 2 and 3 are assayed by employing dTDP-L-rhamnose-14C as the isotope donor and either flOH-dec-flOH-decanoic acid or R h - f i O H dec-fiOH-decanoic acid as the acceptor, as appropriate. In crude extracts, when flOH-dec-flOH-decanoic acid is used as an acceptor the activity measured is the sum of Reactions 2 and 3, since the product of ~M. M. Burger, L. Glaser, and R. M. Burton, J. Biol. Chem. 238, 2595 (1963). 2For simplicity the following abbreviations will be used: Rh-rhamnopyranosyl residues in the rhamnolipid, flOH-dec ~-~flOH-decanoyl residues in the rhamnolipid. The structure of the rhamnolipid was originally determined by 17. J. Jarvis and M. J. Johnson [J. Am. Chem. Soc. 71, 4124 (1949)]. Recent observations by J. A. Hayashi and J. R. Edwards [Arch. Biochem. Biophys. 111, 415 (1965)] indicate a 1 ---)2 rhamnosyl rhamnose instead of the suggested 1 --->3 linkage ; both glycosidic linkages are a, and the sugars exist in pyranose form. Figure 1 illustrates this latter structure.
ENZYMES OF COMPLEX SACCHARIDE SYNTHESIS
[77]
[77] UDP-Glucose: PolyglycerolteichoicAcid Glucosylt r a n s f c r a s c f r o m Bacillus subtilis 1
By Lcls GLASERand M. M. BURGER
CHz--CH--CHz--O--.P--O-vCH~--.CH--CI~--O--.P--O--,
I OH
I OH
J O_
| (
I OH
+ n UDP-glucose ~
c ~ - - cI-I- c ~ - o - P - o ~ c ~ I
J
O_
1
]n
/
n UDP
CH-- CH,-- O--P--O~, I
I
|
OH ? O_ ( O-glucose O_ )n glucose FIG. 1. Glucosylationof polyglycerolphosphate. Assay Method
Principle. The assay is based on the incorporation of 1~C from UDPD-glucose-14C into polymeric form, as measured by radioactivity remaining at the origin after paper chromatography. Reagents Tris-C1, 0.05 M, pH 8.0-0.01 M MgCl~-0.001 M EDTA UDP-D-glucose-~4C, 0.001 M (100,000 cpm/micromole). This can be prepared enzymatically from UTP and glucose-l-P-~4C or glucose-6-P. 2 It can also be prepared from ~4C-glucose-l-P by the morpholidate procedure? Polyglycerolphosphate, 5 micromoles/ml (expressed in micromoles of glucose free glycerol in polyglycerolphosphate). This material is prepared as described below. Pyridine 1L. Glaser a n d M. M. Burger, J. Biol. Chem. 239, 3187 (1964). T h e strains of B. subtilis ( A T C C 6051, N C T C 3610) used in this work contain a fully glucosylated polyglycerolphosphate in the cell wall, and a mixture of polyglycerolphosphate and fully glucosylated polyglycerolphosphate in the "intracellular" fraction. 2 L. Glaser, J. Biol. Chem. 232, 627 (1958). 3 M. Smith a n d H. G. Khorana, Vol. ¥ I [94].
[77]
UDP-GLUCOSE: POLYGLYCEROLTEICHOICTRANSFERASE
437
Procedure. The reaction mixture contains 0.2 ml of Tris-Mg-EDTA buffer pH 8.0, 0.05 ml of polyglycerolphosphate, 0.04 ml of UDP-glucose-14C and 0.01-0.05 ml of enzyme (see below) and is incubated at 37 ° for 30 minutes. The reaction is stopped by the addition of an equal volume of pyridine, streaked on strips of Whatman No. 1 paper (4 X 50 cm), and chromatographed by descending paper chromatography in ethanol 1 M NH4-acetate pH 3.8 (7.5:3.0). 4 The solvent is allowed to reach the end of the paper (approximately 18 hours). The strips are scanned for radioactivity in a paper strip counter and the radioactivity remaining at the origin determined. It is found convenient to determine the ratio of the counts per minute at the origin, to the total counts on the paper, automatically correcting for errors in spotting and for variations in the efficiency of the paper strip counter. When the required accuracy is not large, it is necessary only to count the origin. Although they have not been used by the author, other methods for counting 14C on paper may be equally satisfactory. However, only part of the radioactivity can be eluted from the paper, and counting paper eluates does not provide a satisfactory assay. A control stopped at 0 time is always included. Pyridine is used to stop the reaction because it gives a homogeneous suspension that can be spotted on paper. Attempts to deproteinize the solution with acid or heat prior to chromatography lead to partial losses of glucosylated polyglycerolphosphate by adsorption on the protein. The assay described should be used with considerable caution. Other strains of B. subtilis than the ones used may contain different teichoic acids and the enzymatic products be completely different.5 In related organisms such as B. licheniformis ATCC 9945 a polymer of the same overall composition has been isolated from the cell wall and synthesized enzymatically.6 In this new polymer the hexose is a part of the polymer backbone.
Preparation of B. subtilis membranes These are prepared from B. subtilis NCTC 3610 or ATCC 6051 by lysozyme digestion as described for the enzymes that synthesize polyglycerolphosphate2 The enzyme preparation contains considerable glucosyl acceptor. In order to have a preparation dependent on the addition of 4D. C. Paladini and L. F. Leloir, Biochem. J. 51, 426 (1952). 5In B. subtilis W23 a similar enzyme preparation will catalyze glucosyl transfer to polyribitol phosphate (T. Chin and L. Glaser, unpublished observations). M. M. Burger and L. Glaser, J. Biol. Chem. in press. 7This volume [76].
438
ENZYMES OF COMPLEX SACCHARIDE SYNTHESIS
[77]
exogenous glucosyl acceptor the enzyme is preincubated with cold UDPD-glucose as follows: 1 ml of membrane suspension is incubated at 37 ° with 0.5 ml of 0.01 M UDP-glucose and 2 ml of 0.05 M Tris-C1-0.01 M MgC12-0.001 M EDTA pH 8.0 at 37 ° for 2 hours. At the end of the incubation the reaction medium is diluted to l0 ml with cold 0.05M Tris-C1-0.01 M MgC12-0.001 M EDTA pH 8.0 centrifuged and the membranes twice suspended in 10 ml of the same buffer with a loose fitting TenBroeck homogenizer and collected by centrifugation. Finally they are suspended in 1 ml of the same buffer and stored frozen. The preparation can be stored frozen for at least one month without significant loss of activity. Under optimal conditions the activity of this enzyme is about 40 millimicromoles of glucose transferred per milligram membrane, dry weight, per hour. Properties of the Enzyme In Tris buffer the pH optimum is 8. The enzyme has an absolute requirement for divalent cation. The optimal concentration of MgC12 is 3 X 10-2 M. At 1 X 10-2 M MgCI2 the activity is 80% of maximal and at 5 X 10-3 M, 50%. The Km for UDP-glucose is 4 X 10-5 M at pH 8.0, and the Km for polyglycerolphosphate (expressed as glycerolphosphate units) is 2 X 10-3 M. Preparation of Polyglycerolphosphate This material can be prepared enzymaticallyT; it is most easily obtained as a partially glucosylated "intracellular" glycerol teichoic acid from B. subtilis NCTC 3610 or ATCC 6051. This organism is grown and harvested as described in this volume [76]. The cells are disrupted in a Nossal shaker (8 g of 0.2 mm glass beads, 8 ml of cell suspension, 1 drop capryl alcohol, are shaken for six 30-second periods with intermittent cooling in an ice bath). Intact cells and cell walls are removed by centrifugation at 10,000 g for 10 minutes. The supernatant fluid is centrifuged at 105,000 g for 60 minutes. The sediment is suspended in water and stirred with an equal volume of 80% phenol at 0 ° for 30 minutes. The suspension is centrifuged at 15,000 g for 10 minutes; the upper phase is removed and the phenol layer is washed with an equal volume of water. The pooled aqueous phase is extracted with an equal volume of chloroform and then dialyzed overnight against distilled water. This material can be further purified by digestion with 10 #g/ml each of RNase and DNase in 0.05 M Tris-C1 buffer pH 8.0 for 3 hours at 37 ° followed by a second phenol extraction. The dialyzed polyglycerolphosphate is concentrated by lyophilization and then chromatographed on Sephadex G-200 equilibrated with 0.1 M triethylammonium acetate pH
[77]
UDP-GLUCOSE: POLYGLYCEROLTEICHOICTRANSFERASE
439
6.8. The teichoic acid is eluted with the void volume and can be freed of buffer by lyophilization. Alternatively teichoic acid may be prepared by extraction of an acetone powder of whole cells with 107~ trichloroacetic acid, as described by Kelemen and Baddiley.s Material prepared by this method appears to be of lower molecular weight than material prepared by phenol extraction, but is equally satisfactory as a glucosyl acceptor. The teichoic acid obtained by either method is analyzed by hydrolyzing an aliquot in 1 N HC1 for 3 hours at 100 °, and, after evaporating the HC1 in vacuo, digesting the sample with E. coli alkaline phosphatase. The inorganic phosphate in the digest is determined by the method of Chen, Toribara and Warner, 9 glucose with hexokinase and glucose 6-phosphate dehydrogenase11 and glycerol with glycerol dehydrogenase.1° The ratio of glycerol to phosphate should be close to unity. The difference between the glycerol concentration and the glucose concentration is a measure of the concentration of unglueosylated polyglycerolphosphate. Alternatively since glucosylpolyglycerolphosphate is alkali stable the sample can be hydrolyzed in 1 N KOH for 2 hours, KOH precipitated by neutralization with HCI04 and the sample digested with E. coli alkaline phosphatase. Essentially all the inorganic phosphate is liberated from free (not glucosylated) glycerolphosphate residues. Isolation of 14C-Glucosyl Polyglycerolphosphate Since it is difficult to elute glucosylpolyglycerolphosphate quantitatively from paper chromatograms, the product is best isolated by phenol treatment of the whole reaction mixture, as described for the preparation of polyglycerolphosphate (see above). The final chromatography on Sephadex G-200 will remove any remaining traces of UDP-glucose. Identification of 14C-Glucosylpolyglycerolphosphate The identification of glucosylated polyglycerolphosphate is best carried out by degradation to glucosylglycerol or glucosylglycerolphosphate. Fully glucosylated polyglycerolphosphate is alkali stable, and the only successful method of degradation is fluorolysis in 60% HF at 0%1,11 This method is generally applicable to all fully substituted polyglycerolphosphate. s M. V. Kelemen and J. Baddiley, Bichem. J. 80, 246 (1961). 9p. S. Chert, Jr., T. Y. Toribara, and H. Warner, Anal. Chem. 28, 1756 (1956). See also this volume [10]. 1oSee Vol. I [59]. This enzyme is not absolutely specific and should be used with caution. Glycerol can also be determined with glycerol kinase and glycerolphosphate dehydrogenase (Vol. V [46], [65a]). ,1Unpublished procedure kindly made available by Dr. D. Lipkin and Mr. J. AbreU. See also J. Abrell, Ph.D. Thesis, Washington University, 1965.
440
ENZYMES OF COMPLEX SACCHARIDE SYNTHESIS
[77]
To 1 mg of dry polymer (up to 5 mg) in a 12-ml conical polyethylene centrifuge tube is added 0.! ml of 60% H F at 0°; this is best done with a drawn out polyethylene tube attached to a plastic syringe. The sample is incubated at 0 ° for 5 hours and cooled in a dry ice-Cellosolve bath; 0.7-0.75 ml of saturated LiOH is added. The tube is allowed to warm to 0 ° and neutralization completed by the addition of saturated Li2C03. After 1 hour at 0 ° the insoluble LiF is removed by centrifugation and washed twice with 1 ml of water. The pooled supernatant fluids are deionized by passage through a 1 X 5-cm column of Amberlite MB-3 and concentrated in a flash evaporator. Glucosylglycerol can be isolated by putting the sample on a charcoal Celite column (1 g of Darco-G-60 and 1 g of Celite for 1 mg or less of glucosylpolyglycerolphosphate) and eluting free glycerol and glucose with H20 and glucosylglycerol with 10% ethanol. TM The structure of the glucosylglycerol obtained from the cell wall teichoic acid has been determined as 2- (a-D-glucopyranosyl) glycerol, since it is cleaved completely with ~-glucosidase18 to glucose and glycerol and since no formaldehyde is formed on periodate oxidation. For the identification of the enzymatically synthesized polyglycerolphosphate, the synthesis is best carried out using ~4C-polyglycerolphosphate as an aeceptor. (This acceptor can be prepared enzymatically.~) The degradation of the polymer after addition of carrier glucosylpolyglycerolphosphate is carried out as described above. The ~4Cglucosyl-~4C-glycerol is identified by its elution pattern from a charcoal Celite column, by cleavage to ~4C-glucose and ~4C-glycerol with a-glucosidase, and by the absence of l~C-formaldehyde formation after periodate oxidation. ~ Since no satisfactory paper chromatography system is available for separating glucosylglycerol from glucose, the products of a-glucosidase action after deionization on an Amberlite MB-3 column, are first fractionated on a charcoal Celite column. This separates glucose and glycerol from residual glucosylglycerol. The glycerol can be identified by chromatography in butanol-pyridine-H_~O (6:4: 3) ,~4 and by conversion to L-a-glycerolphosphate with glycerol-kinase.~5 The L-a-glycerol-P is identified by chromatography in ethanol-1 M ammonium acetate, pH 7.8.4 The glucose eluted from the charcoal Celite column is most easily 1,R. L. Whistler and J. N. BeMiller, in "Methods in Carbohydrate Chemistry" (R. L. Whistler and M. L. Wolfrom, eds.), Vol. I, p. 42. Academic Press, New York, 1962. 1~I. R. Lehman and E. A. Pratt, J. Biol. Chem. ~.35, 3254 (1960). 14A. Jeanes, C. S. Wise, and R. J. Dimler, Anal. Chem. 23, 415 (1961). E. P. Kennedy, Vol. V [65a]. G. Bublitz and O. Wieland, Vol. V [46].
[78]
FORMATION OF RHAMNOLIPIDS OF P. aeruginosa
441
identified by its chromatographic behavior, and by oxidation to gluconic acid with glucose oxidase followed by c h r o m a t o g r a p h y in b u t a n o l p y r i d i n e - H 2 0 (6: 4: 3) .14 Although in the original identification of the enzymatically synthesized glucosylpolyglycerolphosphate, the identification of 2-(a-D-glucopyranosyl)glycerol was based on the degradation scheme described above, 1 the same compound has been prepared chemically and could be conveniently used as a reference compound or as carrier for crystallization of the radioactive product. 16 '~A. J. Charlson and A. S. Perlin, Can. J. Chem. 34, 1200 (1956). See also A. J. Charlson, P. A. J. Gorin, and A. S. Perlin, Can. J. Chem. 35, 365 (1957).
[78] Formation
of Rhamnolipids
of Pseudornonas
aeruginosa 1
B y M. M. BURGER, L. GLASER, and R. M. BURTON 2 flOH-decanoyl-CoA --~ --~ ~OH-decanoyl-~OH-decanoic acid (1) ~OH-decanoyl-flOtt-decanoic acid ~- dTDP-L-rhamnose -~ dTDP -b a-L-rhamnospyranosyl-~OH-decanoyl-~OH-decanoic acid (2) L-RhamnosyI-~OH-decanoyl-BOH-decanoic acid q- dTDP-L-rhamnose dTDP q- a-L-rhamnopyranosyl-1 --~ 2-a-L-rhamnopyranosyl-/~OH-decanoyl-flOH-decanoic acid~ (3) Assay M e t h o d Principle. The products of the reactions are extracted into ether after acidifying the incubation mixture. The radioactivity contained in the products is measured and used as the estimate of enzymatic synthesis. Reactions 2 and 3 are assayed by employing dTDP-L-rhamnose-14C as the isotope donor and either flOH-dec-flOH-decanoic acid or R h - f i O H dec-fiOH-decanoic acid as the acceptor, as appropriate. In crude extracts, when flOH-dec-flOH-decanoic acid is used as an acceptor the activity measured is the sum of Reactions 2 and 3, since the product of ~M. M. Burger, L. Glaser, and R. M. Burton, J. Biol. Chem. 238, 2595 (1963). 2For simplicity the following abbreviations will be used: Rh-rhamnopyranosyl residues in the rhamnolipid, flOH-dec ~-~flOH-decanoyl residues in the rhamnolipid. The structure of the rhamnolipid was originally determined by 17. J. Jarvis and M. J. Johnson [J. Am. Chem. Soc. 71, 4124 (1949)]. Recent observations by J. A. Hayashi and J. R. Edwards [Arch. Biochem. Biophys. 111, 415 (1965)] indicate a 1 ---)2 rhamnosyl rhamnose instead of the suggested 1 --->3 linkage ; both glycosidic linkages are a, and the sugars exist in pyranose form. Figure 1 illustrates this latter structure.