COHEN, GARY H. (University of Vermont, Burlington), AND DONALD B. JOHNSTONE. Capsular polysaccharide of Azotobacter agilis. J. Bacteriol. 88:1695-1699. 1964.-Capsular polysaccharide from Azotobacter agilis strain 132 was recovered from washed cells by alkaline digestion. The polysaccharide was purified by centrifugation, repeated alcohol precipitation, Sevag deproteinization, and treatment with ribonuclease and charcoal-cellulose. Methods of isolation and purification appeared to provide a polymer showing no evidence of heterogeneity when examined by chemical and physical methods. Colorimetric, paper chromatographic, and enzymatic analyses on both intact and acid-hydrolyzed polysaccharide indicated that the polymer contained galactose and rhamnose at a molar ratio of approximately 1.0:0.7. A sialic acid-like component was also present in the polysaccharide. The study shows significant differences in the chemical composition of the extracellular polysaceharide of A. agilis and that of A. vinelandii. This adds further biochemical evidence for the right of these species to independent status. Recently, we have been interested in the extracellular polysaccharides synthesized by Azotobacter vinelandii (Cohen and Johnstone, 1964). It was of interest, therefore, to extend the study to the capsular material of A. agilis and compare it with that of A. vinelandii. Such information may provide additional help in the differentiation of these frequently confused species, the distinguishing characteristics of which have been reviewed (Johnstone, 1962a). Although the extracellular polysaccharides of A. vinelandii are found as both cell-free slime and capsular material, the extracellular polysaccharide of A. agilis ' From a dissertation submitted by the senior author in partial fulfillment of the requirements for the Ph.D. degree. Contribution from the University of Vermont Agricultural Experiment Station, Journal Article No. 142. 2 Present address: Department of Veterinary Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia. is confined to well-defined capsules. To our knowledge, no reports have appeared in the literature concerning the chemistry of the extracellular polysaccharide of A. agilis. MATERIALS AND METHODS Growth of the organisms. A. agilis (ATCC 12838) used throughout this study was originally isolated in this laboratory from water (Johnstone, 1957) and designated in subsequent reports as strain 132 (Johnstone, Pfeffer, and Blanchard, 1959; Johnstone, 1962b). Burk's nitrogen-free broth (Wilson and Knight, 1952) at pH 7.0 supplemented with 2% sucrose was inoculated with cells growing in the logarithmic phase. Cultures were incubated at 31 C in 7.5-liter New Brunswick fermentors with sterile moist air supplied at the rate of 4 liters per min and an impeller rotation of 130 rev/min. Incubation was discontinued after 72 hr. Isolation and purification of polysaccharide. The cells were harvested by centrifugation in a Servall continuous-flow centrifuge at 10,000 X g, washed in 0.05 M phosphate buffer (pH 7.3), treated with 1% (v/v) formaldehyde (Dudman and Wilkinson, 1956) for 15 min, and suspended in 0.1 N NaOH for 1 hr on a rotary shaker at room temperature to remove the capsules. The digest was then centrifuged at 10,000 X g for 1 hr and neutralized with HCl; the sedimented cell mass was discarded, and the supernatant fluid was added to 4 volumes of cold ethanol. The presence of electrolyte (NaCl) was required for precipitation of A. agilis capsular polysaccharide. The polysaccharide was purified by three additional alcohol precipitations followed by 17 Sevag deproteinization cycles, as outlined previously (Cohen and Johnstone, 1964). Ribonucleic acid (RNA) was indicated in the polysaceharide preparation by strong absorption at 260 m,u and the presence of ribose, as shown by paper chromatography of acid hydrolysates. RNA was removed by incubating the preparation with ribonuclease (Worthington Biochemical 1695 on A uust 0, 2017 by gest http/jb.asm .rg/ D ow nladed fom COHEN AND) JOHNSTONE Corp., Freehold, N.J.) in 0.1 M acetate buffer at pH 5.0 for 1 hr at 37 C. The mixture was dialyzed against 0.1 M acetate buffer, until no ultraviolet absorption at 260 m,u was observed. The polysaccharide was passed through a charcoal-cellulose pad (Cifonelli and MIayeda, 1957) to remove remaining ultraviolet-absorbing material, dialyzed against distilled water, and precipitated with ethanol and NaCl. The precipitate was washed successively in 80% ethanol, absolute ethanol, and acetone, and dried in vacuo at room temperature. Homogeneity of A. agilis polysaccharide was determined in a Spinco model E ultracentrifuge at 52,640 rev/min. Spectrophotometric measurements were performed in a Beckman model DU spectrophotometer. Hexuronic acid was determined by the carbazole test (Dische, 1947), methylpentose by the L-cysteine-sulfuric acid test (Dische and Shettles, 1948), hexose by the primary L-cysteine-sulfuric acid method (Dische, Shettles, and Osnos, 1949), and sialic acid by the thiobarbituric acid method (Warren, 1959) and the modified Ehrlich test (Barry, Abbott, and Tsai, 1962). The standards were L-rhamnose, D-galactose, and N-acetylneuraminic acid, respectively. A purified sample of N-acetylneuraminic acid isolated from bovine submaxillary gland was the gift of R. C. Woodworth, University of Vermont. Quantitative estimation of the sugar moieties of the polysaceharide, as well as methods for detection of protein, were as previously described (Cohen and Johnstone, 1964). Chromatography. Paper chromatography was carried out by the descending technique on Whatman 3 MM paper. The following solvent systems were used: (i) n-butanol-pyridine-water (9:5:8, v/v); (ii) ethyl acetate-pyridine-water (12:5:4, v/v); (iii) isopropanol-butanol-water (14:2:4, v/v); (iv) ethyl acetate-pyridine-acetic acid-water (5:5:1:3, v/v). The sugars were located with aniline hydrogen phthalate (Partridge, 1949), aniline diphenylamine (Smith, 1958), glucose oxidase, and galactose oxidase reagents (Worthington Biochemical Corp., Freehold, N.J.) (Salton, 1960), and 0.2% (w/v) ninhydrin in acetone for amino sugars. Acid hydrolysis. Acid hydrolyses were carried out in Teflon-lined screw-capped tubes with 1 N HCl at 100 C for 1 hr. A time course was run to obtain maximal reducing values, as determined by the Nelson modification (1944) of Somogyi (1952). The hydrolysate was evaporated to dryness under reduced pressure and redissolved; the procedure was repeated four times to remove HCl.