EFFECT OF SODIUM SULFATE AND MAGNESIUM SULFATE ONHETEROPOLYSACCHARIDE SYNTHESIS IN GRAM-NEGATIVE

SOIL BACTERIA

ALVIN MARKOVITZ AND SUSAN SYLVANThe 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 ofsodium sulfate and magnesium sulfate on hetero-polysaccharide synthesis in gram-negative soilbacteria. J. Bacteriol. 83:483-489. 1962.-Theeffect of Na2SO4 and MgSO4 on heteropolysac-charide biosynthesis has been investigated ingram-negative bacteria isolated from soil. Thesebacteria may be divided into three arbitrarygroups on the basis of the effect of Na2SO4 andMgSO4 on heteropolysaccharide synthesis: group1, synthesis of polysaccharides containing uronicacid is inhibited by increasing the concentrationof sulfate ion; group 2, synthesis of polysac-charides containing uronic acid is stimulated bysulfate ions; group 3, synthesis of polysaccharidenot containing uronic acid is stimulated mini-mally by Na2SO4.

High concentrations of sulfate ions in liquidenrichment cultures resulted in the selection ofbacteria that synthesized polysaccharides con-taining uronic acid (Markovitz, 1961a). To studythis phenomenon further, two classes of bacteriawere obtained. Class I strains were isolated bythe liquid enrichment cuilture technique in mediacontaining high concentrations of sulfate ions;class II strains were isolated directly on agarplates of the same medium without high concen-trations of sulfate ions.

Representatives of both classes have beengrown on varying concentrations of MgSO4 andNa2SO4. The effects of these salts on the compo-sition and synthesis of polysaccharide are thesubject of this communication. Preliminary re-ports of this work have been presented (Marko-vitz, 1960; Markovitz, 1961b).

MATERIALS AND METHODS

Media. The inorganic medium described byPalleroni and Doudoroff (1956) was used. It con-

tained 0.033 M KH2PO4-Na2HPO4 (pH 6.8),0.05% MgSO4 (0.0042 M), 0.1% NH4Cl, 0.01%ferric ammonium citrate, and 0.001% CaCl2.Isolates obtained from basal agar plates contain-ing sucrose (strains designated, SE, SR, SW, andST in Table 1) were grown in liquid media con-taining 0.5% sucrose. Other strains of bacteriawere isolated and grown in liquid media contain-ing 0.5% glucose. The pH of culture media con-taining salts, after sterilization in the autoclave,was as follows: 0.042 M MgSO4, pH 6.0; 0.083 MMgSO4, pH 5.8; 0.25 M MgSO4, pH 5.6; 0.42 MMgSO4, pH 5.5; 0.035 to 0.91 M Na2SO4, pH 6.6to 6.8. It was noted that the addition of MgSO4caused an increase in the amount of precipitatein the medium over that usually present. Agar(1.5%, Difco) was used for preparing solid media.

Growth. Bacterial growth in liquid media wasestimated turbidimetrically at 600 m,u. Quanti-ties (30 ml) of medium in 125-ml Erlenmeyerflasks or 500-ml quantities of medium in 2-literErlenmeyer flasks were aerated on a reciprocalshaker at room temperature. The cultures wereaerated until optical density measurements indi-cated that growth had terminated (usuallv 24 to48 hr). When poor growth was apparent afterthis interval, shaking was continued for an addi-tional 24 to 48 hr.

Bacteria were harvested by centrifugation forfrom 10 to 30 min at 30,000 X g at 4 C. In certaincases, one-half of the culture was placed in aboiling bath for 5 min before the bacteria wereremoved by centrifugation, in an attempt to re-lease loosely bound capsular material. Thethoroughly dialyzed supernatant liquids wereanalyzed as indicated. Similar ratios of compo-nents were found when analyses were performedon material precipitated with alcohol from con-centrated supernatant liquids. Little protein ornucleic acid was found in the dialyzed superna-tant liquids from unheated cultures.

Fractionation of polysaccharides with cetylpyri-483

MARKOVITZ AND SYLVAN

dinium chloride (CPC). Uronic acid-containingpolysaccharides were precipitated by addition ofa 17% solution of CPC to concentrated crudepolysaccharides in 0.04 N NaCl or Na2SO4. Neu-tral polysaccharides that remained in the super-natant 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-bu-tanone saturated with water (Krauss et al.,

1960); 3) 2 ,6-lutidine-water (65:35; Dent, 1948);4) pyridine-n-butanol-water (4:6:3); 5) n-bu-tanol saturated with water (Krauss et al., 1960);6) n-butanol-ethanol (4:1) saturated with pH8.9 borate buffer (Krauss et al., 1960); 7) n-bu-tanol-2-butanone (1:1) saturated with pH 8.9borate buffer (Krauss et al., 1960). Anilineoxalate was used to develop spots of hexoses,pentoses, and 6-deoxyhexoses; ninhydrin was

used to develop spots of hexosamines.

TABLE 1. Composition of polysaccharides and optimal .alt concentrations for polysaccharidesynthesis in gram-negative soil isolates

Hexoa "Total" GrowthGroup Number Organisma Mediumb Uronic acid c Methylpentose bh drate in basalmne bohyrt mediumd

mm mm msu mm %

1 1 0.5-F Basal 0.59 (1.5)- 0.03 0.71 (1.7)h 1.6 1002 0.5-R 0.070 M Na2SO4 0.30 0.06f 0.04h 1.3 1103 1-M 0.083 M MgSO4 0.23 0.04 1.6 1004 1-T 0.042 M MgSO4 0. 10 0. 12f, g 0. 24h,i 0.56 1105 3-M 0.042 M MgSO4 0.48 0.03 1.9 1206 5-Z 0.070 M Na2SO4 1.0 0.05 - 2.3 1107 BT Basal 0.08 0. 07f 0.11 0.37 1008 GJ Basal 0.15 0.119 0.06 0.31 1009 TGB Basal 0.06 0. 13f a 0.03 0.33 10010 SR Basal 1.5 (2.4) 0.07 3.8 10011 SW Basal 0.26 (0.24) 2.6 10012 STk Basal 0.05 _ 0.33 100

2a 13 GL 0.56 M Na2SO4 1.2 0.13 2.4 5.5 8914 GS 0.35 M Na2SO4 0.76 (0.77) - 0.01 (0.07)i 3.5 (3.2) 8915 GC 0.21 M Na2SO4 0.33 (0.34) 1.1 15016 GI 0 .35 M Na2SO4 0.09 - 0.04 0.27 76

2b 17 GM 0.63 M Na2SO4 0.13 - 0.04 0.39 3003a 18 SE 0.070 M Na2SO4 0.06 0.09 0.80 923b 19 GG 0.21 M Na2SO4 _ - 0.03 0.83 77

20 GK 0.070 M Na2SO4 0. 39f 0.09 0.23 9721 GP 0.21 M Na2SO4 0.03 0.85 140

a The liquid enrichment media from which the organisms were isolated were as follows: 0.5-F and0.5-R, 0.042 M MgSO4; 1-M and 1-T, 0.083 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-mediumagar 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 bacteriawere removed by centrifugation and the thoroughly dialyzed culture supernatants were analyzed asindicated.

c Figures are given for organisms liberating hexosamine into the medium at a concentration of 0.03mm 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.

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TABLE 2. Effect of salts on polysaccharide synthesis in organism O.5-Ra

Medium Uronic acid Glucosamineb L-Rhamnoseb "Total" Growth incarbohydrate basal medium"

mM mm mM mM %

Basal 0.27 (0.91)d 0.06 0.07 1.2 1000.083 M MgSO4 0.10 0.10 0.08 0.43 1300.25 M MgSO4 0.08 (0.28) 0.09 0.08 0.59 1000.42 M MgSO4 0.03 0.02 0.02 0.10 880.035 M Na2SO4 0.23 0.06 0.05 0.97 1200.070 M Na2SO4 0.30 0.05 0.04 1.3 1100. 21 M Na2SO4 0.03 (0.27) 0.02 0.01 0.19 1100.35 M Na2SO4 0.02 0.01 0.00 0.10 87

a The bacteria were removed by centrifugation, and the thoroughly dialyzed supernatant liquidswere analyzed as indicated.

b Quantities were determined by colorimetric procedures (see Materials and Methods). The identitiesof 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-Rhamnosewas detected as described by Englesberg andBaron (1959), using L-rhamnose isomerase fromSalmonella typhosa; a crude extract of S. typhosagrown in the presence of L-rhamnose was a giftfrom E. Englesberg.

Chemical analyses. Uronic acid was determinedby the carbazole method (Dische, 1947) withglucuronolactone as a standard. When uronic acidvalues were 10% or less of the "total" carbohy-drate values or showed abnormal color, the ab-sorption spectrum of the color complex was ex-amined to confirm the presence of uronic acid.Methylpentose was determined by the cvsteine-sulfuric acid method at 100 C for 10 min (Discheand Shettles, 1948), with L-rhamnose as a stand-ard; "total" carbohydrate was determined bythe phenol-sulfuric acid method of Dubois et al.(1951), with glucose as a standard. The lattermethod does not include hexosamines. Hexos-amine was determined by the Elson-Morgan re-action as modified by Boas (1953), afterhydrolysis for 14 hr with 4 N HCI at 100 C, withglucosamine as a standard. Since hydrolyzateswere not treated with Dowex-50 ion-exchangeresins to remove compounds that interfered withthe hexosamine analyses, less than 0.03 jumole ofhexosamine per ml was not considered significant.Analyses for the presence of 3-substituted hexos-amines (muramic acid) were performed by themethod of Cifonelli and Dorfman (1958).N-Acetylhexosamines were measured by themethod of Reissig, Strominger, and Leloir (1955),

with N-acetylglucosamine as standard. Ketosugars were measured by the method of Discheand Borenfreund (1951), with fructose andD-ribulose-o-nitrophenyl hydrazone (CaliforniaCorp. for Biochemical Research, Los Angeles) asstandards. Protein and nucleic acid were esti-mated on the basis of ultraviolet absorption ac-cording to the method of Warburg and Christianas described by Layne (1957).

Isolation of hexosamines from polysaccharidesfor paper chromatography. Polysaccharide washydrolyzed for 14 hr in 4 N HCl at 100 C insealed tubes (hexosamine concentration of 1 mg/ml). Acid and water were removed in vacuo atroom temperature. The hexosamines were thenadsorbed to Dowex 50 H+ resins (8 X, 200 to 400mesh, 0.8 by 2.5 cm) and the column was washedwith water. Elution was carried out with 0.3 NHCI. Fractions reacting with Nessler reagentKoch (Scientific Supply Co., Chicago, Ill.) werepooled, dried in vacuo, and spotted for paperchromatography.

Isolation of monosaccharides from polysaccha-rides for paper chromatography. Polysaccharideswere hydrolyzed in 1 N H2SO4 for 3 hr at 100 C,neutralized with BaCO3, centrifuged, the super-natant solution deionized by passage through aDowex 50 H+ column, and the effluent concen-trated to dryness.

RESULTS

The 21 strains of bacteria isolated from soilwere grown on the basal medium supplemented

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with varying concentrations of MgSO4 andNa2SO4. The strains of bacteria have been di-vided into three groups on the basis of the effectsof salts on polysaccharide composition or synthe-sis or both. Table 1 lists the strains studied, theconcentration of salt found necessary for maxi-mal polysaccharide synthesis, and chemicalanalyses of the polysaccharides synthesized. Thearbitrary basis of the division into three groupsis described below. At least one detailed exampleof the response of each group to salt is presentedin Tables 2 through 6.Group 1. Polysaccharide synthesis, including

synthesis of polysaccharides containing uronicacid, may be inhibited by increasing the concen-tration of MgSO4 or Na2SO4, with little effect on

TABLE 3. Effect of salts on polysaccharidesynthesis in organism GLa

Uroi Methyl- "Total" Growth inMedium aronic pento carbo- basalbacidpenosehydrate mediumb

mM mM mM %

Basal 0.07 0.21 0.36 1000.083 M MgSO4 0.05 0.13 0.20 1100.42 M MgSO4 0.06 0.14 0.25 950.21 M Na2SO4 0.08 0.18 0.37 1000.35 M Na2SO4 0.78 1.6 3.4 1100.42 M Na2SO4 1.1 1.9 3.8 970.49 M Na2SO4 1.4 2.2 5.4 850.56 M Na2SO4 1.2 2.4 5.5 890.77 M Na2SO4 0.86 1.6 3.4 720.91 M Na2SO4 0.07 0.29 0.65 24

a As in footnote a of Table 2.b As in footnote c of Table 2.

growth (Table 1, no. 1 through 12). This includesthe six strains isolated from the liquid enrich-ment cultures that contained varying concentra-tions of MgSO4, as well as six strains isolated fromplates streaked with soil. Although Table 1 indi-cates that optimal polysaccharide synthesis re-

quires some Na2SO4 or MgSO4 in five of the sixorganisms isolated from MgSO4 enrichment cul-tures, the difference in polysaccharide synthesisbetween the optimal salt concentration and thatproduced on the basal medium is not great; inhi-bition of polysaccharide synthesis, but notgrowth, becomes apparent as the salt concentra-tion is raised.Organism 0.5-R is a typical example of group 1;

Table 2 shows the depression in synthesis of poly-saccharide containing uronic acid with increasingNa2SO4 or MgSO4 concentrations. However, a

comparison of the analyses of the polysaccharidesynthesized in the basal medium and in 0.25 MMgSO4 medium indicates that the quantities ofpolysaccharide containing glucosamine andL-rhamnose are not reduced by growth in thelatter, although they are reduced during growthin media containing 0.42 M MgSO4 or 0.21 M

Na2SO4. Fractionation of polysaccharide fromthis organism with CPC permitted the separationof a polysaccharide containing uronic acid fromone containing glucosamine and L-rhamnose. (Inthe CPC-precipitable fraction, the molar ratioswere: uronic acid, 1.00; glucosamine, 0.13;L-rhamnose, 0.07; "total" carbohydrate, 3.8. Inthe fraction not precipitable by CPC, the molarratios 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 inMedium 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) 1000.042 MMgSO4 0.21 0.13 0.89 1000.083 MMgSO4 0.17 0.10 0.69 930.25 M MgSO4 0.28 0.07 1.7 (3.2) 980.42 M MgSO4 0.61 (0.75) 0.04 (0.18) 2.8 1000.035 M Na2SO4 0.43 0.12 1.6 1100.070 M Na2SO4 0.59 0.09 2.1 970.21 M Na2SO4 0.89 0.02 3.1 960.35 M Na2SO4 0.76 (0-77) 0.01 (0.07) 3.6 (3.2) 89

a As in footnote a of Table 2.As in footnote c of Table 2.

c As in footnote d of Table 2.

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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) 1000.25 M MgSO4 0.00 (0.00) 0.03 (0.03) 0.10 (0.11) 2300.42 M MgSO4 0.00 (0.02) 0.00 (0.01) 0.07 (0.08) 310.070 M Na2SO4 0.00 (0.02) - (0.03) 0.08 (0.15) 1200.21 M Na2SO4 0.03 (0.04) 0.04 (0.07) 0.16 (0.31) 4400.35 M Na2SO4 0.07 (0.07) 0.02 (0.06) 0.22 (0.39) 4100.49 M Na2SO4 0.11 0.02 0.24 4000.63 M Na2SO4 0.13 (0.13) 0.04 0.39 (0.47) 3000.70M Na2SO4 0.04 (0-04) 0.01 0.12 (0.13) 23

a As in footnote a of Table 2.a As in footnote c of Table 2.b As in footnote d of Table 2.

TABLE 6. Effect of salts on polysaccharidesynthesis in organism SEa

Uronic Hexosa- "Total" Growth inMedium acid He carbohy- basalmie drate mediumb

mM mM mM SI'.isal 0.06 0.07 0.39 1000.042 M MgSO4 0.08 0.06 0.35 910.083 M MgSO4 0.04 0.07 0.33 930.25 M MgSO4 0.05 0.10 0.40 480.42 M MgSO4 0.06 0.11 0.32 220.035M Na2SO4 0.03 0.06 0.44 829.070 M Na2SO4 0.06 0.09 0.80 920.21 M Na2SO4 0.07 0.11 0.74 840.35M Na2SO4 0.09 0.08 0.45 54

a As in footnote a of Table 2.b As in footnote c of Table 2.

Group 2a. Synthesis of polysaccharide contain-ing uronic acid is stimulated by Na2SO4 or MgSO4or both with little effect on growth (Table 1,

13 through 16). The response of organism GL isshown in Table 3. It is apparent that a 20-foldincrease in synthesis of polysaccharide containinguronic acid occurred when this organism was

grown in media containing 0.49 M Na2SO4. Re-gardless of the composition of the growth medium,the centrifuged cells retained little polysaccharidecontaining uronic acid. In contrast, 0.42 M

MgSO4 had little effect on polysaccharide syn-thesis by this organism.

In group 2a, organism GS is also of particularinterest. Synthesis of a polysaccharide containinguronic acid was stimulated by either MgSO4 or

Na2SO4 but synthesis of a methylpentose com-

ponent 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 fromstrain GS with CPC permitted the separation ofa polysaccharide fraction containing uronic acidfrom one containing the methylpentoses, D-rhamnose and D-talomethylose (Table 1) (Marko-vitz, 1961b). A comparison of the data on boiledvs. untreated culture fluids (Table 4) indicatedthat the uronic acid component was completelyin the medium but the methylpentose componentwas loosely bound to the cells and could be re-leased by boiling the suspension.Group 2b. Synthesis of a uronic acid-containing

polysaccharide is initiated only in the presenceof Na2SO4. The only isolate responding in thisfashion was GM (Table 1, no. 17 and Table 5).Enhancement of growth was coincident with theinitiation of synthesis of the uronic acid-contain-ing component in Na2SO4-containing media(Table 5, last column). However, growth in thebasal medium was of a granular nature and ad-hered to the growth flask; in media containingNa2SO4 or MgSO4 the growth was evenly sus-pended. It should be noted that 0.25 M MgSO4appeared to increase growth, but did not initiatesynthesis of uronic acid-containing polysac-charide.

Concentration and subsequent analysis ofsupernatant liquids from strain GM grown in thebasal medium failed to reveal evidence of uronicacid. Bacteria from the medium containing 0.35 MNa2SO4 were subcultured in the same mediumseveral times and then either inoculated into thebasal medium or plated on the basal agar. Iso-

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lated clones from the latter were also grown inthe basal medium. Such attempts to obtainmutants that synthesized polysaccharide con-taining uronic acid in the absence of Na2SO4 wereunsuccessful. Therefore, it appears likely that thephenotypic expression of the ability to synthesizepolysaccharide containing uronic acid requiresthe presence of Na2SO4 in strain GM.Group 3a. Polysaccharide synthesis, but not

the component containing uronic acid, is stimu-lated 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 ofNa2SO4, although no polysaccharide containinguronic acid is synthesized (Table 1, no. 19, 20,and 21).

DISCUSSION

Previous results demonstrated that bacteriacapable of synthesizing polysaccharides contain-ing uronic acid could be selected from soil inliquid enrichment cultures containing high con-centrations of sulfate ions (Markovitz, 1961a).Six of these isolates (Table 1, no. 1 through 6)reacted to increased quantities of sulfate ions byinhibiting synthesis of polysaccharides containinguronic acid. Other isolates, obtained by streakingsoil samples directly on basal agar, also producedless polysaccharide containing uronic acid whengrown in media with increased quantities ofsulfate ions (Table 1, no. 7 through 12).These results lead to the conclusion that the

effect of the concentration of sulfate ions on thesynthesis of polysaccharides in other bacterialstrains warrants investigation, since this appearsto be an important parameter affecting poly-saccharide synthesis that is not mandatorillylinked to growth. The remarkable stimulation ofsynthesis of polysaccharide containing uronicacid observed in strains GL (Table 3) and GS(Table 4) by Na2SO4 suggests that the potentialto synthesize these polysaccharides may requireNa2SO4 or MgSO4 for full expression in some bac-teria. GM (Table 5) may be an extreme case ofthis type, since it appeared that Na2SO4 wasnecessary for initiation of synthesis of polysac-charide containing uronic acid.

Stimulation of polysaccharide synthesis byNa2SO4 or MgSO4 was not limited to bacteriathat synthesize polysaccharides containing uronicacid; one group in which no uronic acid-contain-

ing polysaccharide was synthesized was stimu-lated (Table 1, group 3b). In addition, oneorganism (strain SE, Tables 1 and 6) respondedto increased concentrations of Na2SO4 by synthe-sizing more total polysaccharide, without affect-ing synthesis of the polysaccharide containinguronic acid.The effect of sulfate ion concentration on

changes in the ratio of certain constituents ofpolysaccharide fractions may become a usefultool in indicating whether one or more poly-saccharides are synthesized by a particular or-ganism. Changes in the ratio of glucosamine orL-rhamnose to uronic acid were apparent in0.5-R (Table 2), and fractionation with CPCproved that two polysaccharides were present. Asimilar situation was even more apparent in GS(Table 4), and again two polysaccharides wereseparated by fractionation with CPC. On theother hand, 0.5-F revealed no changes in the ratioof uronic acid to L-rhamnose with increasing saltconcentration, and fractionation with CPC didnot separate the uronic acid component from theL-rhamnose component. Historically, bacteriol o-gists have believed that bacteria ordinarily syn-thesize very complex heteropolysaccharides, i.e.,single heteropolysaccharides containing five ormore different monosaccharide units. However,few attempts have been made to separate thecomponents, and, as a result, studies on the bio-synthesis of such polysaccharide fractions havenot been pursued because of their presumed com-plexity. The complexity of certain plant gumsmay also be more apparent than real (Smith andMontgomery, 1959).Whether growth of bacteria in media of high

salt concentration affects the synthesis of enzymesnecessary for polysaccharide synthesis, or theactivity of the enzymes, remains to be deter-mined. In this connection, work on feedback in-hibition of enzyme activity and repression ofenzyme synthesis may be relevant (Yates andPardee, 1956; Umbarger, 1956; Vogel, 1957;Ames and Garry, 1959).

ACKNOWLEDGMENTS

The authors wish to acknowledge the technicalassistance of Sarah M. Moxham and MinoruMayeda. The advice and encouragement of A.Dorfman and J. A. Cifonelli during these experi-ments is gratefully acknowledged.

This work was aided by grants from The

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National Foundation and the National HeartInstitute of the U. S. Public Health Service(H-311).

LITERATURE CITED

AMES, B. N., AND B. GARRY. 1959. Coordinaterepression of the synthesis of four histidinebiosynthetic enzymes by histidine. Proc.Natl. Acad. Sci. U. S. 45:1453-1461.

BOAS, N. F. 1953. Method for the determination ofhexosamines in tissues. J. Biol. Chem. 204:553-563.

CIFONELLI, J. A., AND A. DORFMAN. 1958. Acolorimetric method for determination of link-age in hexosamine-containing compounds. J.Biol. Chem. 231:11-18.

DENT, C. E. 1948. A study of the behavior of somesixty amino acids and other ninhydrin-react-ing substances on phenol-"collidine" filterpaper chromatograms, with notes as to theoccurrence of some of them in biologicalfluids. Biochem. J. 43:169-180.

DISCHE, Z. 1947. A new specific color reaction ofhexuronic acids. J. Biol. Chem. 167:189-198.

DISCHE, Z., AND E. BORENFREUND. 1951. A new

spectrophotometric method for the detectionand determination of keto sugars and trioses.J. Biol. Chem. 192:583-587.

DISCHE, Z., AND L. B. SHETTLES. 1948. A specificcolor reaction of methylpentoses and a spec-

trophotometric micromethod for their deter-mination. J. Biol. Chem. 175:595-603.

DuBoIs, M., K. GILLES, J. K. HAMILTON, P. A.REBERS, AND F. SMITH. 1951. A colorimetricmethod for the determination of sugars.Nature 168:167.

ENGLESBERG, E., AND L. S. BARON. 1959. Mutationto L-rhamnose resistance and transductionto L-rhamnose utilization in Salmonellatyphosa. J. Bacteriol. 78:675-686.

KRAUSS, M. T., H. JAGER, 0. SCHINDLER, AND T.

REICHSTEIN. 1960. Desoxyzucker. 33. Mittei-lung. Papierchromatographische Differen-zierung der Hexamethylosen und ihrer 3-o-methylderivate. J. Chromatography 3:63-74.

LAYNE, E. 1957. In S. P. Colowick and N. 0. Kap-lan [ed.], Methods in enzymology, vol. 3, p.

447-454, Academic Press, Inc., New York.MARKOVITZ, A. 1960. Polysaccharide composition

and synthesis in bacteria isolated from soil.Bacteriol. Proc., p. 31.

MARKOVITZ, A. 1961a. A method for the selectionof bacteria that synthesize uronic acid-con-taining polysaccharides. J. Bacteriol. 82:436-441.

MARKOVITZ, A. 1961b. Studies on a bacterium thatsynthesizes a polysaccharide containing D-talomethylose and D-rhamnose. Bacteriol.Proc., p. 193.

PALLERONI, N. J., AND M. DOUDOROFF. 1956.Mannose isomerase of Pseudomonas saccharo-phila. J. Biol. Chem. 218:535-548.

REISSIG, J. L., J. L. STROMINGER, AND L. F.LELOIR. 1955. A modified colorimetric methodfor the estimation of N-acetylamino sugars.

J. Biol. Chem. 217:959-966.SMITH, F., AND R. MONTGOMERY. 1959. The chem-

istry of plant gums and mucilages. ReinholdPublishing Corp., New York.

UMBARGER, H. F. 1956. Evidence for a negative-feedback mechanism in the biosynthesis ofisoleucine. Science 123:848.

VOGEL, H. J. 1957. Repression and induction as

control mechanisms of enzyme biogenesis: theadaptive formation of acetylornithinase. InW. D. McElroy and B. Glass [ed.], The chem-ical basis of heredity, p. 276-289. Johns Hop-kins Press, Baltimore.

YATES, R. A., AND A. B. PARDEE. 1956. Control ofpyrimidine biosynthesis in Escherichia coliby a feed-back mechanism. J. Biol. Chem.221:757-770.

1962] 489