EFFECT OF SODIUM SULFATE AND MAGNESIUM SULFATE ON

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1 EFFECT OF SODIUM SULFATE AND MAGNESIUM SULFATE ON HETEROPOLYSACCHARIDE SYNTHESIS IN GRAM-NEGATIVE SOIL BACTERIA ALVIN MARKOVITZ AND SUSAN SYLVAN The LaRabida-University of Chicago Institute and Department of Microbiology, University of Chicago, Chicago, Illinois Received for publication August 22, 1961 ABSTRACT MARKOVITZ, ALVIN (University of Chicago, Chicago, Ill.) AND SUSAN SYLVAN. Effect of sodium sulfate and magnesium sulfate on heteropolysaccharide synthesis in gram-negative soil bacteria. J. Bacteriol. 83: The effect of Na2SO4 and MgSO4 on heteropolysaccharide biosynthesis has been investigated in gram-negative bacteria isolated from soil. These bacteria may be divided into three arbitrary groups on the basis of the effect of Na2SO4 and MgSO4 on heteropolysaccharide synthesis: group 1, synthesis of polysaccharides containing uronic acid is inhibited by increasing the concentration of sulfate ion; group 2, synthesis of polysaccharides containing uronic acid is stimulated by sulfate ions; group 3, synthesis of polysaccharide not containing uronic acid is stimulated minimally by Na2SO4. High concentrations of sulfate ions in liquid enrichment cultures resulted in the selection of bacteria that synthesized polysaccharides containing uronic acid (Markovitz, 1961a). To study this phenomenon further, two classes of bacteria were obtained. Class I strains were isolated by the liquid enrichment cuilture technique in media containing high concentrations of sulfate ions; class II strains were isolated directly on agar plates of the same medium without high concentrations of sulfate ions. Representatives of both classes have been grown on varying concentrations of MgSO4 and Na2SO4. The effects of these salts on the composition and synthesis of polysaccharide are the subject of this communication. Preliminary reports of this work have been presented (Markovitz, 1960; Markovitz, 1961b). 483 MATERIALS AND METHODS Media. The inorganic medium described by Palleroni and Doudoroff (1956) was used. It contained M KH2PO4-Na2HPO4 (ph 6.8), 0.05% MgSO4 ( M), 0.1% NH4Cl, 0.01% ferric ammonium citrate, and 0.001% CaCl2. Isolates obtained from basal agar plates containing sucrose (strains designated, SE, SR, SW, and ST in Table 1) were grown in liquid media containing 0.5% sucrose. Other strains of bacteria were isolated and grown in liquid media containing 0.5% glucose. The ph of culture media containing salts, after sterilization in the autoclave, was as follows: M MgSO4, ph 6.0; M MgSO4, ph 5.8; 0.25 M MgSO4, ph 5.6; 0.42 M MgSO4, ph 5.5; to 0.91 M Na2SO4, ph 6.6 to 6.8. It was noted that the addition of MgSO4 caused an increase in the amount of precipitate in the medium over that usually present. Agar (1.5%, Difco) was used for preparing solid media. Growth. Bacterial growth in liquid media was estimated turbidimetrically at 600 m,u. Quantities (30 ml) of medium in 125-ml Erlenmeyer flasks or 500-ml quantities of medium in 2-liter Erlenmeyer flasks were aerated on a reciprocal shaker at room temperature. The cultures were aerated until optical density measurements indicated that growth had terminated (usuallv 24 to 48 hr). When poor growth was apparent after this interval, shaking was continued for an additional 24 to 48 hr. Bacteria were harvested by centrifugation for from 10 to 30 min at 30,000 X g at 4 C. In certain cases, one-half of the culture was placed in a boiling bath for 5 min before the bacteria were removed by centrifugation, in an attempt to release loosely bound capsular material. The thoroughly dialyzed supernatant liquids were analyzed as indicated. Similar ratios of components were found when analyses were performed on material precipitated with alcohol from concentrated supernatant liquids. Little protein or nucleic acid was found in the dialyzed supernatant liquids from unheated cultures. Fractionation of polysaccharides with cetylpyri-

2 484 MARKOVITZ AND SYLVAN [VOL. 83 dinium chloride (CPC). Uronic acid-containing polysaccharides were precipitated by addition of a 17% solution of CPC to concentrated crude polysaccharides in 0.04 N NaCl or Na2SO4. Neutral polysaccharides that remained in the supernatant liquid were precipitated with ethanol. Paper chromatography. The following solvents were used for identification of monosaccharides: 1) acetic acid-n-butanol-water (1:4:1); 2) 2-butanone saturated with water (Krauss et al., TABLE ); 3) 2,6-lutidine-water (65:35; Dent, 1948); 4) pyridine-n-butanol-water (4:6:3); 5) n-butanol saturated with water (Krauss et al., 1960); 6) n-butanol-ethanol (4:1) saturated with ph 8.9 borate buffer (Krauss et al., 1960); 7) n-butanol-2-butanone (1:1) saturated with ph 8.9 borate buffer (Krauss et al., 1960). Aniline oxalate was used to develop spots of hexoses, pentoses, and 6-deoxyhexoses; ninhydrin was used to develop spots of hexosamines. Composition of polysaccharides and optimal.alt concentrations for polysaccharide synthesis in gram-negative soil isolates Hexoa "Total" Growth Group Number Organisma Mediumb Uronic acid c Methylpentose bh drate in basal mne bohyrt mediumd mm mm msu mm % F Basal 0.59 (1.5) (1.7)h R M Na2SO f 0.04h M M MgSO T M MgSO f, g 0. 24h,i M M MgSO Z M Na2SO BT Basal f GJ Basal TGB Basal f a SR Basal 1.5 (2.4) SW Basal 0.26 (0.24) STk Basal 0.05 _ a 13 GL 0.56 M Na2SO GS 0.35 M Na2SO (0.77) (0.07)i 3.5 (3.2) GC 0.21 M Na2SO (0.34) GI 0.35 M Na2SO b 17 GM 0.63 M Na2SO a 18 SE M Na2SO b 19 GG 0.21 M Na2SO4 _ GK M Na2SO f GP 0.21 M Na2SO a The liquid enrichment media from which the organisms were isolated were as follows: 0.5-F and 0.5-R, M MgSO4; 1-M and 1-T, M MgSO4; 3-M, 0.25 M MgSO4; 5-Z, 0.42 M MgSO4 (Markovitz, 1961a, method II). The rest of the organisms were isolated by selecting large colonies from basal-medium agar plates that were streaked directly with soil samples (Markovitz, 1961a, method I). b For maximal polysaccharide synthesis and for which analyses in the table are given. The bacteria were removed by centrifugation and the thoroughly dialyzed culture supernatants were analyzed as indicated. c Figures are given for organisms liberating hexosamine into the medium at a concentration of 0.03 mm or greater. d Growth in the basal medium without added salts was taken as 100%. e Figures in parentheses were obtained on samples that were boiled before bacteria were removed. f Glucosamine, solvent 3. g Galactosamine, solvent 3. h L-Rhamnose, solvents 1 and 2, and L-rhamnose isomerase. D-Rhamnose and D-talomethylose (Markovitz, 1961b) and solvents 1, 2, 4, 5, 6, and 7. i Fucose, solvents 1 and 2. k ST was the only gram-positive organism studied.

3 1962] EFFECTS OF SALTS ON POLYSACCHARIDE SYNTHESIS 485 TABLE 2. Effect of salts on polysaccharide synthesis in organism O.5-Ra Medium Uronic acid Glucosamineb L-Rhamnoseb "Total" Growth in carbohydrate basal medium" mm mm mm mm % Basal 0.27 (0.91)d M MgSO M MgSO (0.28) M MgSO M Na2SO M Na2SO M Na2SO (0.27) M Na2SO a The bacteria were removed by centrifugation, and the thoroughly dialyzed supernatant liquids were analyzed as indicated. b Quantities were determined by colorimetric procedures (see Materials and Methods). The identities of the components were ascertained as indicated in Table 1. c Growth in the basal medium without added salts was taken as 100%. d Figures in parentheses represent analyses of bacterial cultures that were boiled before bacteria were removed. Enzymatic detection of L-rhamnose. L-Rhamnose was detected as described by Englesberg and Baron (1959), using L-rhamnose isomerase from Salmonella typhosa; a crude extract of S. typhosa grown in the presence of L-rhamnose was a gift from E. Englesberg. Chemical analyses. Uronic acid was determined by the carbazole method (Dische, 1947) with glucuronolactone as a standard. When uronic acid values were 10% or less of the "total" carbohydrate values or showed abnormal color, the absorption spectrum of the color complex was examined to confirm the presence of uronic acid. Methylpentose was determined by the cvsteinesulfuric acid method at 100 C for 10 min (Dische and Shettles, 1948), with L-rhamnose as a standard; "total" carbohydrate was determined by the phenol-sulfuric acid method of Dubois et al. (1951), with glucose as a standard. The latter method does not include hexosamines. Hexosamine was determined by the Elson-Morgan reaction as modified by Boas (1953), after hydrolysis for 14 hr with 4 N HCI at 100 C, with glucosamine as a standard. Since hydrolyzates were not treated with Dowex-50 ion-exchange resins to remove compounds that interfered with the hexosamine analyses, less than 0.03 jumole of hexosamine per ml was not considered significant. Analyses for the presence of 3-substituted hexosamines (muramic acid) were performed by the method of Cifonelli and Dorfman (1958). N-Acetylhexosamines were measured by the method of Reissig, Strominger, and Leloir (1955), with N-acetylglucosamine as standard. Keto sugars were measured by the method of Dische and Borenfreund (1951), with fructose and D-ribulose-o-nitrophenyl hydrazone (California Corp. for Biochemical Research, Los Angeles) as standards. Protein and nucleic acid were estimated on the basis of ultraviolet absorption according to the method of Warburg and Christian as described by Layne (1957). Isolation of hexosamines from polysaccharides for paper chromatography. Polysaccharide was hydrolyzed for 14 hr in 4 N HCl at 100 C in sealed tubes (hexosamine concentration of 1 mg/ ml). Acid and water were removed in vacuo at room temperature. The hexosamines were then adsorbed to Dowex 50 H+ resins (8 X, 200 to 400 mesh, 0.8 by 2.5 cm) and the column was washed with water. Elution was carried out with 0.3 N HCI. Fractions reacting with Nessler reagent Koch (Scientific Supply Co., Chicago, Ill.) were pooled, dried in vacuo, and spotted for paper chromatography. Isolation of monosaccharides from polysaccharides for paper chromatography. Polysaccharides were hydrolyzed in 1 N H2SO4 for 3 hr at 100 C, neutralized with BaCO3, centrifuged, the supernatant solution deionized by passage through a Dowex 50 H+ column, and the effluent concentrated to dryness. RESULTS The 21 strains of bacteria isolated from soil were grown on the basal medium supplemented

4 486 (VOL. 83 MARKOVITZ AND SYLVAN with varying concentrations of MgSO4 and Na2SO4. The strains of bacteria have been divided into three groups on the basis of the effects of salts on polysaccharide composition or synthesis or both. Table 1 lists the strains studied, the concentration of salt found necessary for maximal polysaccharide synthesis, and chemical analyses of the polysaccharides synthesized. The arbitrary basis of the division into three groups is described below. At least one detailed example of the response of each group to salt is presented in Tables 2 through 6. Group 1. Polysaccharide synthesis, including synthesis of polysaccharides containing uronic acid, may be inhibited by increasing the concentration of MgSO4 or Na2SO4, with little effect on TABLE 3. Effect of salts on polysaccharide synthesis in organism GLa Uroi Methyl- "Total" Growth in Medium aronic pento carboacidpenosehydrate mediumb basalb mm mm mm % Basal M MgSO M MgSO M Na2SO M Na2SO M Na2SO M Na2SO M Na2SO M Na2SO M Na2SO b As in footnote c of Table 2. growth (Table 1, no. 1 through 12). This includes the six strains isolated from the liquid enrichment cultures that contained varying concentrations of MgSO4, as well as six strains isolated from plates streaked with soil. Although Table 1 indicates that optimal polysaccharide synthesis requires some Na2SO4 or MgSO4 in five of the six organisms isolated from MgSO4 enrichment cultures, the difference in polysaccharide synthesis between the optimal salt concentration and that produced on the basal medium is not great; inhibition of polysaccharide synthesis, but not growth, becomes apparent as the salt concentration is raised. Organism 0.5-R is a typical example of group 1; Table 2 shows the depression in synthesis of polysaccharide containing uronic acid with increasing Na2SO4 or MgSO4 concentrations. However, a comparison of the analyses of the polysaccharide synthesized in the basal medium and in 0.25 M MgSO4 medium indicates that the quantities of polysaccharide containing glucosamine and L-rhamnose are not reduced by growth in the latter, although they are reduced during growth in media containing 0.42 M MgSO4 or 0.21 M Na2SO4. Fractionation of polysaccharide from this organism with CPC permitted the separation of a polysaccharide containing uronic acid from one containing glucosamine and L-rhamnose. (In the CPC-precipitable fraction, the molar ratios were: uronic acid, 1.00; glucosamine, 0.13; L-rhamnose, 0.07; "total" carbohydrate, 3.8. In the fraction not precipitable by CPC, the molar ratios were: uronic acid, 0.18; glucosamine, 1.25; L-rhamnose, 1.00; total carbohydrate, 2.0.) TABLE 4. Effect of salts on polysaccharide synthesis in organism GS- Growth in Medium Uronic acid Methylpentose "Total" carbohydrate basal mediumb mm mm mm S Basal 0.17 (0.17)e 0.37 (0.73) 0.83 (1.4) MMgSO MMgSO M MgSO (3.2) M MgSO (0.75) 0.04 (0.18) M Na2SO M Na2SO M Na2SO M Na2SO (0-77) 0.01 (0.07) 3.6 (3.2) 89 As in footnote c of Table 2. c As in footnote d of Table 2.

5 1962] EFFECTS OF SALTS ON POLYSACCHARIDE SYNTHESIS 487 TABLE 5. Effect of salts on polysaccharide synthesis in organism GMa Medium Uronic acid Methylpentose "Total" carbohydrate mediumb mm mm mm % Basal 0.00 (0.00)c 0.02 (0.02) 0.04 (0.05) M MgSO (0.00) 0.03 (0.03) 0.10 (0.11) M MgSO (0.02) 0.00 (0.01) 0.07 (0.08) M Na2SO (0.02) - (0.03) 0.08 (0.15) M Na2SO (0.04) 0.04 (0.07) 0.16 (0.31) M Na2SO (0.07) 0.02 (0.06) 0.22 (0.39) M Na2SO M Na2SO (0.13) (0.47) M Na2SO (0-04) (0.13) 23 a As in footnote c of Table 2. b As in footnote d of Table 2. TABLE 6. Effect of salts on polysaccharide synthesis in organism SEa Uronic Hexosa- "Total" Growth in Medium acid He carbohy- basal mie drate mediumb mm mm mm S I'.isal M MgSO M MgSO M MgSO M MgSO M Na2SO M Na2SO M Na2SO M Na2SO b As in footnote c of Table 2. Group 2a. Synthesis of polysaccharide containing uronic acid is stimulated by Na2SO4 or MgSO4 or both with little effect on growth (Table 1, 13 through 16). The response of organism GL is shown in Table 3. It is apparent that a 20-fold increase in synthesis of polysaccharide containing uronic acid occurred when this organism was grown in media containing 0.49 M Na2SO4. Regardless of the composition of the growth medium, the centrifuged cells retained little polysaccharide containing uronic acid. In contrast, 0.42 M MgSO4 had little effect on polysaccharide synthesis by this organism. In group 2a, organism GS is also of particular interest. Synthesis of a polysaccharide containing uronic acid was stimulated by either MgSO4 or Na2SO4 but synthesis of a methylpentose component was inhibited by these salts (Table 4). Similar results were obtained with NaCl, LiCl, and (NH4)2SO4 at a concentration of 0.35 M. Fractionation of crude polysaccharide from strain GS with CPC permitted the separation of a polysaccharide fraction containing uronic acid from one containing the methylpentoses, D- rhamnose and D-talomethylose (Table 1) (Markovitz, 1961b). A comparison of the data on boiled vs. untreated culture fluids (Table 4) indicated that the uronic acid component was completely in the medium but the methylpentose component was loosely bound to the cells and could be released by boiling the suspension. Group 2b. Synthesis of a uronic acid-containing polysaccharide is initiated only in the presence of Na2SO4. The only isolate responding in this fashion was GM (Table 1, no. 17 and Table 5). Enhancement of growth was coincident with the initiation of synthesis of the uronic acid-containing component in Na2SO4-containing media (Table 5, last column). However, growth in the basal medium was of a granular nature and adhered to the growth flask; in media containing Na2SO4 or MgSO4 the growth was evenly suspended. It should be noted that 0.25 M MgSO4 appeared to increase growth, but did not initiate synthesis of uronic acid-containing polysaccharide. Concentration and subsequent analysis of supernatant liquids from strain GM grown in the basal medium failed to reveal evidence of uronic acid. Bacteria from the medium containing 0.35 M Na2SO4 were subcultured in the same medium several times and then either inoculated into the basal medium or plated on the basal agar. Iso-

6 488 MARKOVITZ AND SYLVAN [VOL. 83 lated clones from the latter were also grown in the basal medium. Such attempts to obtain mutants that synthesized polysaccharide containing uronic acid in the absence of Na2SO4 were unsuccessful. Therefore, it appears likely that the phenotypic expression of the ability to synthesize polysaccharide containing uronic acid requires the presence of Na2SO4 in strain GM. Group 3a. Polysaccharide synthesis, but not the component containing uronic acid, is stimulated minimally by Na2SO4 and not by MgSO4 (Table 1, no. 18 and Table 6). Group 3b. Polysaccharide synthesis is increased up to twofold with increasing concentrations of Na2SO4, although no polysaccharide containing uronic acid is synthesized (Table 1, no. 19, 20, and 21). DISCUSSION Previous results demonstrated that bacteria capable of synthesizing polysaccharides containing uronic acid could be selected from soil in liquid enrichment cultures containing high concentrations of sulfate ions (Markovitz, 1961a). Six of these isolates (Table 1, no. 1 through 6) reacted to increased quantities of sulfate ions by inhibiting synthesis of polysaccharides containing uronic acid. Other isolates, obtained by streaking soil samples directly on basal agar, also produced less polysaccharide containing uronic acid when grown in media with increased quantities of sulfate ions (Table 1, no. 7 through 12). These results lead to the conclusion that the effect of the concentration of sulfate ions on the synthesis of polysaccharides in other bacterial strains warrants investigation, since this appears to be an important parameter affecting polysaccharide synthesis that is not mandatorilly linked to growth. The remarkable stimulation of synthesis of polysaccharide containing uronic acid observed in strains GL (Table 3) and GS (Table 4) by Na2SO4 suggests that the potential to synthesize these polysaccharides may require Na2SO4 or MgSO4 for full expression in some bacteria. GM (Table 5) may be an extreme case of this type, since it appeared that Na2SO4 was necessary for initiation of synthesis of polysaccharide containing uronic acid. Stimulation of polysaccharide synthesis by Na2SO4 or MgSO4 was not limited to bacteria that synthesize polysaccharides containing uronic acid; one group in which no uronic acid-containing polysaccharide was synthesized was stimulated (Table 1, group 3b). In addition, one organism (strain SE, Tables 1 and 6) responded to increased concentrations of Na2SO4 by synthesizing more total polysaccharide, without affecting synthesis of the polysaccharide containing uronic acid. The effect of sulfate ion concentration on changes in the ratio of certain constituents of polysaccharide fractions may become a useful tool in indicating whether one or more polysaccharides are synthesized by a particular organism. Changes in the ratio of glucosamine or L-rhamnose to uronic acid were apparent in 0.5-R (Table 2), and fractionation with CPC proved that two polysaccharides were present. A similar situation was even more apparent in GS (Table 4), and again two polysaccharides were separated by fractionation with CPC. On the other hand, 0.5-F revealed no changes in the ratio of uronic acid to L-rhamnose with increasing salt concentration, and fractionation with CPC did not separate the uronic acid component from the L-rhamnose component. Historically, bacteriol o- gists have believed that bacteria ordinarily synthesize very complex heteropolysaccharides, i.e., single heteropolysaccharides containing five or more different monosaccharide units. However, few attempts have been made to separate the components, and, as a result, studies on the biosynthesis of such polysaccharide fractions have not been pursued because of their presumed complexity. The complexity of certain plant gums may also be more apparent than real (Smith and Montgomery, 1959). Whether growth of bacteria in media of high salt concentration affects the synthesis of enzymes necessary for polysaccharide synthesis, or the activity of the enzymes, remains to be determined. In this connection, work on feedback inhibition of enzyme activity and repression of enzyme synthesis may be relevant (Yates and Pardee, 1956; Umbarger, 1956; Vogel, 1957; Ames and Garry, 1959). ACKNOWLEDGMENTS The authors wish to acknowledge the technical assistance of Sarah M. Moxham and Minoru Mayeda. The advice and encouragement of A. Dorfman and J. A. Cifonelli during these experiments is gratefully acknowledged. This work was aided by grants from The

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