JOURNAL OF BACTERIOLOGY Vol. 88, No. 6, p. 1695-1699 Decemnber, 1964 Copyright © 1964 American Society for Microbiology Printed in U.S.A. CAPSULAR OF AZOTOBACTER AGILIS' GARY H. COHEN2 AND DONALD B. JOHNSTONE Department of Agricultural Biochemistry, University of Vermont, Burlington, Vermont Received for publication 19 June 1964

ABSTRACT is confined to well-defined capsules. To our COHEN, GARY H. (University of Vermont, Bur- knowledge, no reports have appeared in the lington), AND DONALD B. JOHNSTONE. Capsular literature concerning the chemistry of the extra- polysaccharide of Azotobacter agilis. J. Bacteriol. cellular polysaccharide of A. agilis. 88:1695-1699. 1964.-Capsular polysaccharide from Azotobacter agilis strain 132 was recovered from MATERIALS AND METHODS washed cells by alkaline digestion. The polysac- Growth of the organisms. A. agilis (ATCC charide was purified by centrifugation, repeated 12838) used alcohol precipitation, Sevag deproteinization, and throughout this study was originally treatment with ribonuclease and charcoal-cellu- isolated in this laboratory from water (Johnstone, lose. Methods of isolation and purification ap- 1957) and designated in subsequent reports as peared to provide a polymer showing no evidence strain 132 (Johnstone, Pfeffer, and Blanchard, of heterogeneity when examined by chemical and 1959; Johnstone, 1962b). Burk's nitrogen-free physical methods. Colorimetric, paper chromato- broth (Wilson and Knight, 1952) at pH 7.0 graphic, and enzymatic analyses on both intact supplemented with 2% was inoculated and acid-hydrolyzed polysaccharide indicated with cells growing in the logarithmic phase. that the polymer contained and rham- Cultures were incubated at 31 C in 7.5-liter New nose at a molar ratio of approximately 1.0:0.7. A Brunswick fermentors with sterile moist air sialic acid-like component was also present in the polysaccharide. The study shows significant dif- supplied at the rate of 4 liters per min and an ferences in the chemical composition of the extra- impeller rotation of 130 rev/min. Incubation cellular polysaceharide of A. agilis and that of A. was discontinued after 72 hr. vinelandii. This adds further biochemical evidence Isolation and purification of polysaccharide. for the right of these species to independent status. The cells were harvested by centrifugation in a Servall continuous-flow centrifuge at 10,000 X g, Recently, we have been interested in the extra- washed in 0.05 M phosphate buffer (pH 7.3), cellular synthesized by Azoto- treated with 1% (v/v) formaldehyde (Dudman bacter vinelandii (Cohen and Johnstone, 1964). and Wilkinson, 1956) for 15 min, and suspended It was of interest, therefore, to extend the study in 0.1 N NaOH for 1 hr on a rotary shaker at to the capsular material of A. agilis and compare room temperature to remove the capsules. The it with that of A. vinelandii. Such information digest was then centrifuged at 10,000 X g for may provide additional help in the differentiation 1 hr and neutralized with HCl; the sedimented of these frequently confused species, the distin- cell mass was discarded, and the supernatant guishing characteristics of which have been fluid was added to 4 volumes of cold ethanol. reviewed (Johnstone, 1962a). Although the The presence of electrolyte (NaCl) was required extracellular polysaccharides of A. vinelandii are for precipitation of A. agilis capsular polysac- found as both cell-free slime and capsular mate- charide. The polysaccharide was purified by rial, the extracellular polysaccharide of A. agilis three additional alcohol precipitations followed by 17 Sevag deproteinization cycles, as outlined ' From a dissertation submitted by the senior previously (Cohen and Johnstone, 1964). author in partial fulfillment of the requirements Ribonucleic acid (RNA) was indicated in the for the Ph.D. degree. Contribution from the Uni- versity of Vermont Agricultural Experiment Sta- polysaceharide preparation by strong absorption tion, Journal Article No. 142. at 260 m,u and the presence of , as shown 2 Present address: Department of Veterinary by paper chromatography of acid hydrolysates. Biology, School of Veterinary Medicine, Univer- RNA was removed by incubating the preparation sity of Pennsylvania, Philadelphia. with ribonuclease (Worthington Biochemical 1695 1696 COHEN AND) JOHNSTONE J. BACTERIOI,.

Corp., Freehold, N.J.) in 0.1 M acetate buffer at by the Nelson modification (1944) of Somogyi pH 5.0 for 1 hr at 37 C. The mixture was dialyzed (1952). The hydrolysate was evaporated to against 0.1 M acetate buffer, until no ultraviolet dryness under reduced pressure and redissolved; absorption at 260 m,u was observed. The poly- the procedure was repeated four times to remove saccharide was passed through a charcoal-cellu- HCl. lose pad (Cifonelli and MIayeda, 1957) to remove remaining ultraviolet-absorbing material, dia- RESULTS lyzed against distilled water, and precipitated The cells of A. agilis 132 were surrounded by with ethanol and NaCl. The precipitate was a well-defined capsule, but cell-free slime, as washed successively in 80% ethanol, absolute found with A. vinelandii, was not produced. ethanol, and acetone, and dried in vacuo at room Because the slime-synthesizing capacity of some temperature. bacteria depends much upon cultural conditions Homogeneity of A. agilis polysaccharide was (Anderson and Rogers, 1963), several attempts determined in a Spinco model E ultracentrifuge were made to induce slime production in A. at 52,640 rev/min. Spectrophotometric measure- agilis. Cells were grown in Burk's broth supple- ments were performed in a Beckman model DU mented with high concentrations (1 to 20%, w/v) spectrophotometer. of , , or sucrose. Slime was not Hexuronic acid was determined by the carba- detected in the culture supernatant fluid by zole test (Dische, 1947), methylpentose by the acid or alcohol precipitation, nor was free slime L-cysteine-sulfuric acid test (Dische and Shettles, observed microscopically by India ink negative 1948), by the primary L-cysteine-sulfuric staining (Duguid, 1951). acid method (Dische, Shettles, and Osnos, 1949), The capsular polysaccharide was isolated as a and sialic acid by the thiobarbituric acid method flocculent light-brown material. Purified poly- (Warren, 1959) and the modified Ehrlich test saccharide was white, and, although readily (Barry, Abbott, and Tsai, 1962). The standards soluble in water at high concentrations (10 mg/ were L-rhamnose, D-galactose, and N-acetyl- ml), it produced solutions of low viscosity. , respectively. A purified sample The homogeneity of the highly purified poly- of N-acetylneuraminic acid isolated from bo- saccharide was examined by ultracentrifugation vine submaxillary gland was the gift of R. (Fig. 1). Although this method strongly indicated C. Woodworth, University of Vermont. the polysaccharide to be homogeneous in com- Quantitative estimation of the moieties position, further studies are required for confirma- of the polysaceharide, as well as methods for tion. The sharpness of the sedimentation pattern detection of protein, were as previously described suggested a relatively narrow distribution of (Cohen and Johnstone, 1964). molecular weight species for the polymer. The Chromatography. Paper chromatography was ultraviolet spectrum of a 0.1 % (w/v) aqueous carried out by the descending technique on solution of polysaceharide revealed no maxima Whatman 3 MM paper. The following solvent in the region of 230 to 300 m,u, indicating removal systems were used: (i) n-butanol-pyridine-water of the RNA, and protein was not found by the (9:5:8, v/v); (ii) ethyl acetate-pyridine-water method of Lowry et al. (1951). (12:5:4, v/v); (iii) isopropanol-butanol-water Paper chromatography of acid hydrolysates (14:2:4, v/v); (iv) ethyl acetate-pyridine-acetic (Table 1) revealed two major components which acid-water (5:5:1:3, v/v). The were were identified by the following methods as located with aniline hydrogen phthalate (Part- galactose and rhamnose. Rhamnose was indicated ridge, 1949), aniline diphenylamine (Smith, as one component of the polysaccharide on the 1958), glucose oxidase, and galactose oxidase basis of chromatographic evidence, identical reagents (Worthington Biochemical Corp., Free- color reactions with developing sprays, and a hold, N.J.) (Salton, 1960), and 0.2% (w/v) positive L-cysteine-sulfuric acid test of Dische ninhydrin in acetone for amino sugars. and Shettles (1948). The identical absorption Acid hydrolysis. Acid hydrolyses were carried spectra for unhydrolyzed polysaccharide, a out in Teflon-lined screw-capped tubes with 1 N fraction isolated from an acid hydrolysate, and an HCl at 100 C for 1 hr. A time course was run to authentic sample of L-rhamnose are illustrated obtain maximal reducing values, as determined in Fig. 2. Galactose was indicated on the basis l'OL. 88, 1964 CAPSULAR POLYSACCHARIDE OF A. AGILIS 1697 of chiromatographic evidence, identical color reactions with developing sprays, and a positive 0.5 - primary L-eysteine-sulfuric acid test. Figure 3 illustrates the identical absorption spectra for A unhydrolyzed polysaceharide, a fraction isolated from an acid hydrolysate, and an authentic H04 z 0 0.3-

O.-Jm 0- 0 0.1 .

350 375 400 425 450

FIG. 2. Absorption spectrafor intact extracellular polysaccharide of Azotobacter agilis (A), rhamnose (B), and a fraction isolated front a polysaccharide hydrolysate (C), in the L-cysteine-sulfuric acid test for methylpentose.

sample of galactose. Paper chromatograms of hydrolysates of the polymer and authentic FIG. 1. Ultracentrifuge pattern of purified capsu- galactose were sprayed with Galactostat reagent lar polysaccharide of Azotobacter agilis. The picture (Worthington Biochemical Corp.). A green color was taken 192 min after the rotor reached a speed of developed with both the hexose spot and the 52,640 rev/min. Polysaccharide concentration was authentic galactose. Results from Galactostat 10 nmg/nitl in 0.85% NVaCl. S20 = 1.6S. Sedimentation was toward the left. spray treatment were read when the paper was wet, because the color faded upon drying. TABLE 1. Paper chromnatography* of sugar residues When a duplicate chromatogram was sprayed fronm a hydrolysate of extracellular polysaccharide with Glucostat reagent, only the glucose stand- of Azotobactet agilis 132 and known sugars ard was observed. Thus, galactose appeared to be the hexose in this polymer. Quantitative Solvent system colorimetric analysis of the p)olysaccharide indi- Ethyl cated a molar ratio for galactose-rhamnose of Sugar n-Buta- Ethyl Isopro- acetate- 1.0:0.7, based on moles of galactose. No uronic nol-pyri- acetate- panol- pyridine- dine- pyridine- butanol- acetic acid was detected in the polysaccharide of A. water water water acid- water agilis, nor were hexosamines. A positive thiobarbituric acid (TBA) test Glucose ...... 10Ot 100 100 100 (Warren, 1959) was observed with hydrolyzed Galactose ..... 96 94 89 91 (0.1 N H2SO4 at 80 C for 1 hr) or unhydrolyzed Hexose spot. 96 94 89 91 polysaccharide. The absorption spectrum of the Ilhaninose ... 129 136 175 171 color the in the Methyl pen- produced by polymer TBA test tose spot 129 136 175 171 was identical to that produced with an N-acetyl- neuraminic acid standard. Sialic acid was not * Whatman 3MM paper was used. detected in purified polysaceharide by the modi- t lResults are expressed as Rglucote values. fied Ehrlich reaction (Barry et al., 1962); how- 1698 COHEN AND JOHNSTONE J. BACTERI OL.

contained no uronic acid or glucose. Rhamnose, 0.7 however, was a major component in both species. A TBA-positive component, similar to that found in polysaccharide from one strain of A. 0.6 vinelandii, was present in the capsular polymer of A. agilis. ACKNOWLEDGMENTS 0.5 C/) We express our appreciation to Jane Wark z and T. B. Tomasi, Division of Experimental w 0.4 Medicine, University of Vermont Medical School, for conducting the ultracentrifugal -J study. 0 0.3 LITERATURE CITED ANDERSON, E. S., AND A. H. ROGERS. 1963. Slime 0 polysaccharides of the Enterobacteriaceae. 0.2 Nature 198:714-715. BARRY, G. T., V. ABBOTT, AND T. TSAI. 1962. Re- lationship of colominic neuraminic acid. J. Gen. Microbiol. 29:335-352. 0.11. CIFONELLI, J. A., AND M. MAYEDA. 1957. The purification of hyaluronic acid by the use of charcoal. Biochim. Biophys. Acta 24:397-400. L I I I I COHEN, G. H., AND D. B. JOHNSTONE. 1964. Extra- 350 370 390 410 430 450 cellular polysaccharides of Azotobacter vine- landii. J. Bacteriol. 88:329-338. m,u DISCHE, Z. 1947. A new specific color reaction of FIG. 3. Absorption spectra for galactose (A), the hexuronic acids. J. Biol. Chem. 167:189-198. intact polysaccharide of Azotobacter agilis (B), and DISCHE, Z., AND L. B. SHETTLES. 1948. A specific afraction isolatedfrom a polysaccharide hydrolysate color reaction of methylpentoses and a spec- (C), in the primary L-cysteine-sulfuric acid test for trophotometric micromethod for their deter- hexose. mination. J. Biol. Chem. 175:595-603. DISCHE, Z., L. B. SHETTLES, AND M. OSNOS. 1949. ever, dried cell material of A. agilis did yield posi- New specific color reactions of and tive results with this method. Significance of this spectrophotometric micromethods for their moiety was previously discussed (Cohen and determination. Arch. Biochem. Biophvs. 22: 169-184. Johnstone, 1964). Further studies are now in D)UDMAN, W. F., AND J. F. WILKINSON. 1956. The progress to determine the conditions for hydroly- composition of the extracellular polysac- sis of the TBA-positive component from the charides of A erobacter-Klebsiella strains. polymer, so that methods other than colorimetric Biochem. J. 62:389-395. may be used for identification. DUGUID, J. P. 1951. The demonstration of bacterial DISCUSSION capsules and slime. J. Pathol. Bacteriol. 63:673-685. The evidence reported here indicates that the JOHNSTONE, 1). B. 1957. Isolation of Azotobacter capsular polysaccharide of A. agilis 132 consists agile from strawboard waste water. Ecology of galactose, rihamnose, and a thiobarbituric 38:156. acid positive moiety. In comparison, extracellular JOHNSTONE, D. B. 1962a. Azotobacter agilis or A. slime and capsular polysaccharide synthesized vinelandii? Soil Microbiol. Newsletter 1:6-8. JOHNSTONE, 1). B. 1962b. Growth of Azotobacter- in by A. vinelandii in common, contained, galac- deuterium oxide. J. Bacteriol. 83:867-870. turonic acid, a-D-glucose, rhamnose, and a JOHNSTONE, D. B., M. PFEFFER, AND G. C. BLAN- hexuronic acid lactone, probably mannuronolac- CHARD. 1959. Fluorescence of AIzotobacter. Can. tone (Cohen and Johnstone, 1964). A. agilis J. Microbiol. 5:299-304. capsular polysaccharide, in shari) contrast, LOWRY, 0. H., N. J. 11OSEBRo7(;1I , A. L. FARR, VOL. 88, 1964 CAPSULAR POLYSACCHARIDE OF A. AGILIS 1699

AND R. J. RANDALL. 1951. Protein measure- SMITH, I. 1958. Sugars, p. 164-177. In I. Smith ment with the Folin phenol reagent. J. Biol. [ed.], Chromatographic techniques-clinical Chem. 193:265-275. and biochemical applications. Interscience NELSON, N. 1944. A photometric adaption of the Publishers, Inc., New York. Somogyi method for the determination of SOMOGYI, M. 1952. Notes on sugar determination. glucose. J. Biol. Chem. 153:375-380. J. Biol. Chem. 195:19-23. PARTRIDGE, S. M. 1949. Aniline hydrogen phthal- WARREN, L. 1959. The thiobarbituric acid assay of ate as a spraying reagent for chromatography sialic acids. J. Biol. Chem. 234:1971-1975. of sugars. Nature 164:443. WILSON, P. W., AND S. G. KNIGHT. 1952. Experi- SALTON, M. R. J. 1960. Specific detection of glucose ments in bacterial physiology. Burgess Pub- on paper chromatograms. Nature 186:966-967. lishing Co., Minneapolis.