Nov 27, 1974 - mutants of Penicillium charlesii incapable of growing on galactose were investigated. ..... Uridine 5'-diphosphate (UDP)-D-galactose is.
JOURNAL OF BACTERIOLOGY, Feb. 1975, p. 675-681 Copyright 0 1975 American Society for Microbiology
Vol. 121, No. 2 Printed in U.S.A.
Exocellular Glycopeptide from a Penicillium charlesii Mutant Incapable of Growth on D-Galactosel L. R. DREWES' AND J. E. GANDER* Department of Biochemistry, College of Biological Sciences, University of Minnesota, St. Paul, Minnesota 55101 Received for publication 27 November 1974
The compositions of exocellular saccharide-containing polymers from six mutants of Penicillium charlesii incapable of growing on galactose were investigated. The polymers from the mutants contain a much smaller percentage
of galactose than that reported for the peptidophosphogalactomannan (PPGM) from the wild-type organism (Gander et al. 1974). A polymer containing only one galactosyl residue per 49 mannosyl residues was investigated in detail. This polymer is a glycopeptide (peptidomannan) with an amino acid composition similar to that of peptidophospogalactomannan and a mass of about 23,000 daltons. Treatment of peptidomannan with 0.4 N NaOH releases mannan, mannopentaose, mannotetraose, mannotriose, mannobiose, and mannose residues, which are attached to the peptide by 0-glycosidic linkage to seryl and threonyl groups. The quantity of glycerol and threitol, derived from mannosyl and internal galactofuranosyl residues, respectively, following Smith degradation, showed that peptidomannan contains 2 mol of internal galactofuranosyl residues per mol of polymer. The polymer contains only 3 mol of (1-5)-linked galactofuranosyl residues per mol of polymer, as described by analysis of the methylation products. Methylation analysis also indicates that the polysaccharide contains primarily (1-2)-linked (67.5%) and (1,6)-linked (20.2%) mannopyranosyl residues. However, acetolysis of the polymer suggests that 37% of the residues are (1->6)-linked. Mannopentaose, mannotetraose, mannotriose, mannobiose, and mannose in a molar ratio of 0.30:0.11:0.15:0.39:0.06, respectively, are released by acetolysis. This result is similar to that obtained with peptidophosphogalactomannan. We conclude that the occurrence of large numbers of galactofuranosyl residues in the major extracellular glycopeptide is not an obligatory requirement for glycopeptide formation.
Aerated Penicillium charlesii cultures produce an exocellular glycopeptide (peptidophosphogalactomannan; PPGM), which contains a polysaccharide, (phosphogalactomannan; PGM), mannosyl-containing oligosaccharides, and mannosyl residues (7), attached to seryl and threonyl residues of the polypeptide (16). The polypeptide contains 30 aminoacyl residues (16). The phosphogalactomannan is comprised of a mannan backbone to which 8 to 10 chains, each chain containing a variable number of 5-0-,8-D-galactofuranosyl residues, are attached (7). The number of galactofuranosyl residues per chain appears to be a function of the pH of the culture and the quantity of extracellular exo-,/-D-galactofuranosidase in the culture medium (Fed. Proc. 32:528). PGM also contains 10
phosphodiester residues and ethanolamine attached to the mannan (16). We reported recently (6) that 2-deoxy-Dglucose blocks the synthesis of exo-f3-D-galactofuranosidase but has little effect on the synthesis of PPGM in the same culture. In Saccharomyces species 2-deoxy-D-glucose blocks the biosynthesis of exocellular glyco-enzymes (11), and also is effective in blocking cell wall glycan biosynthesis (5). This suggests that PPGM is not derived from an exocellular glycoenzyme or a cell wall glycan. PPGM biosynthesis occurs primarily from substrates remaining in the growth medium after the culture attains one-half its maximal dry weight and the medium is depleted of ammonium ion and 50% of the glucose. Currently, the metabolic role of PPGM in ' Scientific journal series no. 8829, University of Minnesota the fungus is unknown. Further, it is not known Agricultural Experiment Station. 'Present address: Department of Physiology and Surgery, if the occurrence of galactofuranosyl residues in the glycopeptide is obligatory. University of Wisconsin, Madison, Wis. 53706. 675
676
DREWES AND GANDER
This paper reports the results of investigations to determine if the occurrence of galactofuranosyl-containing chains is obligatory in the formation of exocellular glycopeptide. Mutants incapable of growth on D-galactose were isolated and the composition of each exocellular glycopeptide was examined. One mutant produced an exocellular glycopeptide containing as few as two to five galactofuranosyl residues per mole. MATERIALS AND METHODS Growth of P. charlesii and mutants of P. charlesii. Stocks cultures of P. charlesii G. Smith ATCC 1887 were maintained as described (14). Routinely, 125 ml of modified Raulin-Thom medium (10) containing 5% D-glucose and 20.2 mM ammonium phosphate in 500-ml notched Erlenmeyer flask was inocultated with a suspension of 8 x 107 spores in 2 ml of 0.9% NaCl-1% Tween 20. Cultures were incubated at 20 C on a gyratory shaker (model 10, New Brunswick Scientific Corp.) at a setting of 8. Modified Raulin-Thom medium containing 5% galactose substituted for glucose, and Raulin-Thom yeast extract medium containing 1% yeast extract (Difco), 4% D-glucose, and salts of the Raulin-Thom medium were used to assist in isolation of mutants. Czapek-Dox (CD-Glc), Czapek-Dox galactose (CDGal), and nutrient-yeast extract agars were also used in the isolation of mutants of P. charlesii. D-galactose was substituted for D-glucose in the CD-Gal agar medium. The nutrient-yeast extract agar medium was composed of 4.5 g of yeast extract, 4.5 g of nutrient broth (Difco), 20 g of D-glucose, 10 g of galactose, 20 g of agar, and the Raulin-Thom salts in a liter of water. Isolation of P. charlesii mutants. A stirred suspension P. charlesii spores (4 x 10' spores per ml) was irradiated with ultraviolet light for 2 min. This treatment resulted in a logarithmic decrease in the number of viable spores and a survival of about 2% of the spores. The suspension was then maintained in the dark at 20 C for 2 h. One-milliliter portions were transferred to nutrient-yeast extract agar and the culture was incubated for 5 to 7 days at 20 C. The resulting spores were suspended in 0.9% NaCl-1% Tween 20 and approximately 8 x 107 spores were inoculated into 125 ml of modified Raulin-Thom medium containing 5% galactose and incubated at 20 C on a gyratory shaker. After 36 h the medium was filtered under aseptic conditions through four layers of cheese cloth to remove any mycelial growth. Filtration was continued every 6 to 8 h for an additional 72 h. The remaining culture, which contained viable spores that were unable to use galactose as a carbon source, was diluted 100-fold and 1-ml portions were plated on nutrient-yeast extract agar. After 3 days at 20 C small colonies developed, and after an addtional 2 days at 4 C mature conidia developed. The plates were then replica plated, first on CDGal agar, then on to CD-Glc agar, and the plates were incubated at 20 C. Colonies which appeared within 4
J. BACTERIOL.
days on CD-Glc agar but not on CD-Gal agar were selected and single spore isolates were obtained. Stock cultures of the mutant strains were maintained in vials of sterilized soil at -15 C in the dark. These cultures were tested from time to time for viablility on CD-Glc agar and their inability to grow on CD-Gal agar. Isolation of heteropolysaccharides. Cultures of P. charlesii mutants were filtered and the heteropolysaccharides in these filtrates were precipitated as a borate-cetyltrimethylammonium bromide complex as described previously (7). The precipitate was dissolved in distilled water and protein, and lipids were removed by at least three extractions with CHClI and 1-pentanol (9:1, vol/vol). The aqueous phase was made 0.05 M with K2B407 and chromatographed on diethylaminoethyl-cellulose. Usually at least 95% of the carbohydrate applied to the column eluted as a discrete peak with 0.01 N HCl-0.06 M LiCl. The fractions containing carbohydrate were combined, neutralized, concentrated, and chromatographed on Sephadex G-50 with 0.1 M NaCl as the eluant. The carbohydrate appeared in the void volume from the Sephadex G-50 column and the fractions containing carbohydrate were dialyzed to remove NaCl, and concentrated or lyophilized. Analytical procedures. Total carbohydrate was determined by the phenol-sulfuric acid method (4). Values obtained were compared to a reference solution of mannose. Total phosphate was determined after ashing the sample (2), by either the Fiske-SubbaRow (1) or the more sensitive Parvin and Smith (13) method. Smith degradation of the glycopeptide was carried out as described (16a). Glucose, galactose, mannose, glycerol, and threitol were determined by gas-liquid chromatography of their trimethylsilyl ether derivatives according to Sweeley et al. (18). Gas chromatography was performed with an F and M model 810 dual column gas chromatograph equipped with flame ionization detector and a Honeywell model Electronik 16 chart recorder with disk integrator. Separations were carried out
on
8-foot (ca. 2.4 m) copper columns ( 36
inch [ca. 0.45 cm] outer diameter) packed with 3% SE-52 on 80- to 100-mesh Gas-Chrom Q. The columns were conditioned at 225 C for 24 hr before use. The various permethylated alditol acetates which were obtained following methylation of glycopeptide (3), hydrolysis of the polymer (7), reduction acetylation, were quantitated by gas-liquid chromatography. The permethylated alditol acetates were separated on a 7-foot (ca. 2.1 m) column (1/4 inch [ca. 0.6 cm] outer diameter) of 3% polyphenyl ether (6 rings) at 180 C. The retention times for the various species were compared to those obtained from the wild-type organism and authentic mannopyranosyl and galactofuranosyl derivatives. Acetolysis was carried out as described by Stewart et al. (17) and the products were separated on Bio-Gel P-2 (minus 400 mesh) as described previously (7). A sample of glycopeptide (6 Amol of amino acids) was prepared for amino acid analysis by treatment with 6 N HCl at 110 C for 20 h. HCl was removed by repeated evaporation to dryness at 35 C. The sample
VOL. 121, 1975
677
FUNGAL MUTANT GLYCOPEPTIDE
dissolved in sodium citrate buffer (0.1 M Na+), agar and normal or only slightly retarded pH 2.2, and volumes were analyzed on the long and growth on CD-Glc agar were obtained by the short columns of a Beckman-Spinco amino acid procedure described in Materials and Methods. analyzer. Tryptophan and tyrosine were determined in the intact glycopeptide by the spectrophotometric Each mutant was cultured in modified RaulinThom media and the resultant heteropolysacmethod of Goodwin and Morton (8). Alkaline degradation of the glycopeptide. A charide formed during an 8-day culture period sample of peptidomannan (PM) containing approxi- was isolated and examined for carbohydrate. mately 2 ,mol of hexosyl residues was treated with 0.4 The polymers produced by six mutants were N NaOH. The change in absorbance of the solution at examined in more detail because these poly241 nm was monitored for approximately 4 h in a mers contained an abnormally small quantity of Kintrac spectrophotometer. The number of hydroxyl- galactose. The percentage of mannose, galacsubstituted seryl and threonyl residues undergoing tose, and glucose in each mutant polymer was B-elimination was calculated by using an extinction coefficient of 4200 for the dehydro derivatives of both determined as a function of total carbohydrate serine (a-amino acrylic acid) and threonine (a-amino and compared to the polymer composition of crotonic acid) (15). Another sample of PM (approxi- the wild-type organism (Table 1) (7). The molar mately 100 umol of hexosyl residues) was treated ratios of galactose to mannose, phosphate to similarly with 0.4 N NaOH and the released saccha- galactose, and phosphate to mannose are also rides were fractionated on a Bio-Gel P-2 column (92 shown. All heteropolysaccharides contained a by 2.5 cm) as described previously (16). measurable quantity of galactose. Although the Determination of ethanolamine. Amino acid phosphate to galactose ratio is relatively conanalysis of hydrolyzed PM indicated the presence of stant in all of the polymers, the galactose to ninhydrin-positive material with the same elution time as ethanolamine. PM (30 ,gmol of hexosyl resi- mannose and phosphate to mannose ratios for dues) was dansylated (9), treated with 6 N HCI for 6 h the mutant polymers are consistently less than at 110 C, and chromatographed on polyamide layer that of the wild type. The polymer from mutant sheets with (i) 1.5% formic acid followed by (ii) 17D contained the smallest percentage of galacbenzene-acetic acid (9:1 vol/vol) in the first and tose and phosphate and the smallest percentage second directions, respectively. The dansyl deriva- of galactose and phosphate and the smallest tives were visualized without ultraviolet light. The phosphate to mannose ratio. This polymer was occurrence of ethanolamine in PM was also demonexamined in more detail. The remainder of the strated by two-dimensional thin-layer chromatogra- paper reports the results of experiments that phy of the dansyl ethanolamine derivative. Sedimentation velocity and molecular weight compare the composition of the polymer from estimation. Four concentrations (15, 30, 45, and 60 the mutant 17D to the glycopeptide obtained umol of hexosyl residues per ml of 0.1 M NaCl) of from the wild-type organism. Amino acid composition of polymer. Hypolymer were centrifuged at 59,780 rpm and 20 C in a Beckman model E ultracentrifuge for 184 min. Pic- drolysis of the polymer in 6 N HCl followed by tures were taken at 16-min intervals. The diffusion amino acid analysis (Table 2) shows that PM coefficient, partial specific volume, and molecular contains an amino acid composition similar to weight were calculated as described previously (7). that obtained for the glycopeptide from the wild-type organism (16). The amino acids serine and theronine are the most abundant and RESULTS account for nearly half of the total residues. A Composition of heteropolysaccharides significant quantity of ethanolamine is also from mutants. Sixteen mutants of P. charlesii present in the mutant polymer. Molecular weight of glycopeptides. The that showed abnormal or no growth on CD-Gal was
TABLE 1. Composition of saccharide-containing Source of
polymer
Wild type 3B 12B 17A 17B 17C 17D
polymers from Penicillium charlesii strains
Monosaccharides relative (%) Mannose Galactose Glucose
84.0 81.0 83.7 90.1 95.5 84.6 98
{
15.5 12.0 14.3 9.4 4.5 9.3 2.0
