EFFECT OF SODIUM SULFATE AND MAGNESIUM SULFATE ON HETEROPOLYSACCHARIDE SYNTHESIS IN GRAM-NEGATIVE SOIL BACTERIA
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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
tained 0.033 M KH2PO4-Na2HPO4 (pH 6.8),
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ABSTRACT
MARKOVITZ, ALVIN (University of Chicago, 0.05% MgSO4 (0.0042 M), 0.1% NH4Cl, 0.01%
Chicago, Ill.) AND SUSAN SYLVAN. Effect of ferric ammonium citrate, and 0.001% CaCl2.
sodium sulfate and magnesium sulfate on hetero- Isolates obtained from basal agar plates contain-
polysaccharide synthesis in gram-negative soil ing sucrose (strains designated, SE, SR, SW, and
bacteria. J. Bacteriol. 83:483-489. 1962.-The ST in Table 1) were grown in liquid media con-
effect of Na2SO4 and MgSO4 on heteropolysac- taining 0.5% sucrose. Other strains of bacteria
charide biosynthesis has been investigated in were isolated and grown in liquid media contain-
gram-negative bacteria isolated from soil. These ing 0.5% glucose. The pH of culture media con-
bacteria may be divided into three arbitrary taining salts, after sterilization in the autoclave,
groups on the basis of the effect of Na2SO4 and was as follows: 0.042 M MgSO4, pH 6.0; 0.083 M
MgSO4 on heteropolysaccharide synthesis: group MgSO4, pH 5.8; 0.25 M MgSO4, pH 5.6; 0.42 M
1, synthesis of polysaccharides containing uronic MgSO4, pH 5.5; 0.035 to 0.91 M Na2SO4, pH 6.6
acid is inhibited by increasing the concentration to 6.8. It was noted that the addition of MgSO4
of sulfate ion; group 2, synthesis of polysac- caused an increase in the amount of precipitate
charides containing uronic acid is stimulated by in the medium over that usually present. Agar
sulfate ions; group 3, synthesis of polysaccharide (1.5%, Difco) was used for preparing solid media.
not containing uronic acid is stimulated mini- Growth. Bacterial growth in liquid media was
mally by Na2SO4. estimated turbidimetrically at 600 m,u. Quanti-
ties (30 ml) of medium in 125-ml Erlenmeyer
flasks or 500-ml quantities of medium in 2-liter
High concentrations of sulfate ions in liquid Erlenmeyer flasks were aerated on a reciprocal
enrichment cultures resulted in the selection of shaker at room temperature. The cultures were
bacteria that synthesized polysaccharides con- aerated until optical density measurements indi-
taining uronic acid (Markovitz, 1961a). To study cated that growth had terminated (usuallv 24 to
this phenomenon further, two classes of bacteria 48 hr). When poor growth was apparent after
were obtained. Class I strains were isolated by this interval, shaking was continued for an addi-
the liquid enrichment cuilture technique in media tional 24 to 48 hr.
containing high concentrations of sulfate ions; Bacteria were harvested by centrifugation for
class II strains were isolated directly on agar from 10 to 30 min at 30,000 X g at 4 C. In certain
plates of the same medium without high concen- cases, one-half of the culture was placed in a
trations of sulfate ions. boiling bath for 5 min before the bacteria were
Representatives of both classes have been removed by centrifugation, in an attempt to re-
grown on varying concentrations of MgSO4 and lease loosely bound capsular material. The
Na2SO4. The effects of these salts on the compo- thoroughly dialyzed supernatant liquids were
sition and synthesis of polysaccharide are the analyzed as indicated. Similar ratios of compo-
subject of this communication. Preliminary re- nents were found when analyses were performed
ports of this work have been presented (Marko- on material precipitated with alcohol from con-
vitz, 1960; Markovitz, 1961b). centrated supernatant liquids. Little protein or
MATERIALS AND METHODS nucleic acid was found in the dialyzed superna-
Media. The inorganic medium described by tant liquids from unheated cultures.
Palleroni and Doudoroff (1956) was used. It con- Fractionation of polysaccharides with cetylpyri-
483484 MARKOVITZ AND SYLVAN [VOL. 83
dinium chloride (CPC). Uronic acid-containing 1960); 3) 2 ,6-lutidine-water (65:35; Dent, 1948);
polysaccharides were precipitated by addition of 4) pyridine-n-butanol-water (4:6:3); 5) n-bu-
a 17% solution of CPC to concentrated crude tanol saturated with water (Krauss et al., 1960);
polysaccharides in 0.04 N NaCl or Na2SO4. Neu- 6) n-butanol-ethanol (4:1) saturated with pH
tral polysaccharides that remained in the super- 8.9 borate buffer (Krauss et al., 1960); 7) n-bu-
natant liquid were precipitated with ethanol. tanol-2-butanone (1:1) saturated with pH 8.9
Paper chromatography. The following solvents borate buffer (Krauss et al., 1960). Aniline
were used for identification of monosaccharides: oxalate was used to develop spots of hexoses,
1) acetic acid-n-butanol-water (1:4:1); 2) 2-bu- pentoses, and 6-deoxyhexoses; ninhydrin was
tanone saturated with water (Krauss et al., used to develop spots of hexosamines.
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TABLE 1. Composition of polysaccharides and optimal .alt concentrations for polysaccharide
synthesis in gram-negative soil isolates
Hexoa "Total" Growth
Group Number Organisma Mediumb Uronic acid mne c Methylpentose bh drate
bohyrt in basal
mediumd
mm mm msu mm %
1 1 0.5-F Basal 0.59 (1.5)- 0.03 0.71 (1.7)h 1.6 100
2 0.5-R 0.070 M Na2SO4 0.30 0.06f 0.04h 1.3 110
3 1-M 0.083 M MgSO4 0.23 0.04 1.6 100
4 1-T 0.042 M MgSO4 0. 10 0. 12f, g 0. 24h,i 0.56 110
5 3-M 0.042 M MgSO4 0.48 0.03 1.9 120
6 5-Z 0.070 M Na2SO4 1.0 0.05 - 2.3 110
7 BT Basal 0.08 0. 07f 0.11 0.37 100
8 GJ Basal 0.15 0.119 0.06 0.31 100
9 TGB Basal 0.06 0. 13f a 0.03 0.33 100
10 SR Basal 1.5 (2.4) 0.07 3.8 100
11 SW Basal 0.26 (0.24) 2.6 100
12 STk Basal 0.05 _ 0.33 100
2a 13 GL 0.56 M Na2SO4 1.2 0.13 2.4 5.5 89
14 GS 0.35 M Na2SO4 0.76 (0.77) - 0.01 (0.07)i 3.5 (3.2) 89
15 GC 0.21 M Na2SO4 0.33 (0.34) 1.1 150
16 GI 0 .35 M Na2SO4 0.09 - 0.04 0.27 76
2b 17 GM 0.63 M Na2SO4 0.13 - 0.04 0.39 300
3a 18 SE 0.070 M Na2SO4 0.06 0.09 0.80 92
3b 19 GG 0.21 M Na2SO4 _ - 0.03 0.83 77
20 GK 0.070 M Na2SO4 0. 39f 0.09 0.23 97
21 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 and
0.5-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-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.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 0.06 0.07 1.2 100
0.083 M MgSO4 0.10 0.10 0.08 0.43 130
0.25 M MgSO4 0.08 (0.28) 0.09 0.08 0.59 100
0.42 M MgSO4 0.03 0.02 0.02 0.10 88
0.035 M Na2SO4 0.23 0.06 0.05 0.97 120
0.070 M Na2SO4 0.30 0.05 0.04 1.3 110
0. 21 M Na2SO4 0.03 (0.27) 0.02 0.01 0.19 110
0.35 M Na2SO4 0.02 0.01 0.00 0.10 87
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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 with N-acetylglucosamine as standard. Keto
was detected as described by Englesberg and sugars were measured by the method of Dische
Baron (1959), using L-rhamnose isomerase from and Borenfreund (1951), with fructose and
Salmonella typhosa; a crude extract of S. typhosa D-ribulose-o-nitrophenyl hydrazone (California
grown in the presence of L-rhamnose was a gift Corp. for Biochemical Research, Los Angeles) as
from E. Englesberg. standards. Protein and nucleic acid were esti-
Chemical analyses. Uronic acid was determined mated on the basis of ultraviolet absorption ac-
by the carbazole method (Dische, 1947) with cording to the method of Warburg and Christian
glucuronolactone as a standard. When uronic acid as described by Layne (1957).
values were 10% or less of the "total" carbohy- Isolation of hexosamines from polysaccharides
drate values or showed abnormal color, the ab- for paper chromatography. Polysaccharide was
sorption spectrum of the color complex was ex- hydrolyzed for 14 hr in 4 N HCl at 100 C in
amined to confirm the presence of uronic acid. sealed tubes (hexosamine concentration of 1 mg/
Methylpentose was determined by the cvsteine- ml). Acid and water were removed in vacuo at
sulfuric acid method at 100 C for 10 min (Dische room temperature. The hexosamines were then
and Shettles, 1948), with L-rhamnose as a stand- adsorbed to Dowex 50 H+ resins (8 X, 200 to 400
ard; "total" carbohydrate was determined by mesh, 0.8 by 2.5 cm) and the column was washed
the phenol-sulfuric acid method of Dubois et al. with water. Elution was carried out with 0.3 N
(1951), with glucose as a standard. The latter HCI. Fractions reacting with Nessler reagent
method does not include hexosamines. Hexos- Koch (Scientific Supply Co., Chicago, Ill.) were
amine was determined by the Elson-Morgan re- pooled, dried in vacuo, and spotted for paper
action as modified by Boas (1953), after chromatography.
hydrolysis for 14 hr with 4 N HCI at 100 C, with Isolation of monosaccharides from polysaccha-
glucosamine as a standard. Since hydrolyzates rides for paper chromatography. Polysaccharides
were not treated with Dowex-50 ion-exchange were hydrolyzed in 1 N H2SO4 for 3 hr at 100 C,
resins to remove compounds that interfered with neutralized with BaCO3, centrifuged, the super-
the hexosamine analyses, less than 0.03 jumole of natant solution deionized by passage through a
hexosamine per ml was not considered significant. Dowex 50 H+ column, and the effluent concen-
Analyses for the presence of 3-substituted hexos- trated to dryness.
amines (muramic acid) were performed by the
RESULTS
method of Cifonelli and Dorfman (1958).
N-Acetylhexosamines were measured by the The 21 strains bacteria isolated from soil
of
method of Reissig, Strominger, and Leloir (1955), were grown on the basal medium supplemented486 MARKOVITZ AND SYLVAN (VOL. 83
with varying concentrations of MgSO4 and growth (Table 1, no. 1 through 12). This includes
Na2SO4. The strains of bacteria have been di- the six strains isolated from the liquid enrich-
vided into three groups on the basis of the effects ment cultures that contained varying concentra-
of salts on polysaccharide composition or synthe- tions of MgSO4, as well as six strains isolated from
sis or both. Table 1 lists the strains studied, the plates streaked with soil. Although Table 1 indi-
concentration of salt found necessary for maxi- cates that optimal polysaccharide synthesis re-
mal polysaccharide synthesis, and chemical quires some Na2SO4 or MgSO4 in five of the six
analyses of the polysaccharides synthesized. The organisms isolated from MgSO4 enrichment cul-
arbitrary basis of the division into three groups tures, the difference in polysaccharide synthesis
is described below. At least one detailed example between the optimal salt concentration and that
of the response of each group to salt is presented produced on the basal medium is not great; inhi-
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in Tables 2 through 6. bition of polysaccharide synthesis, but not
Group 1. Polysaccharide synthesis, including growth, becomes apparent as the salt concentra-
synthesis of polysaccharides containing uronic tion is raised.
acid, may be inhibited by increasing the concen- Organism 0.5-R is a typical example of group 1;
tration of MgSO4 or Na2SO4, with little effect on Table 2 shows the depression in synthesis of poly-
saccharide containing uronic acid with increasing
TABLE 3. Effect of salts on polysaccharide Na2SO4 or MgSO4 concentrations. However, a
synthesis in organism GLa comparison of the analyses of the polysaccharide
synthesized in the basal medium and in 0.25 M
Uroi Methyl- "Total" Growth in
MgSO4 medium indicates that the quantities of
Medium aronic pento carbo- basalb
acidpenosehydrate mediumb
polysaccharide containing glucosamine and
mM mM mM % L-rhamnose are not reduced by growth in the
Basal 0.07 0.21 0.36 100 latter, although they are reduced during growth
0.083 M MgSO4 0.05 0.13 0.20 110 in media containing 0.42 M MgSO4 or 0.21 M
0.42 M MgSO4 0.06 0.14 0.25 95 Na2SO4. Fractionation of polysaccharide from
0.21 M Na2SO4 0.08 0.18 0.37 100 this organism with CPC permitted the separation
0.35 M Na2SO4 0.78 1.6 3.4 110 of a polysaccharide containing uronic acid from
0.42 M Na2SO4 1.1 1.9 3.8 97 one containing glucosamine and L-rhamnose. (In
0.49 M Na2SO4 1.4 2.2 5.4 85 the CPC-precipitable fraction, the molar ratios
0.56 M Na2SO4 1.2 2.4 5.5 89 were: uronic acid, 1.00; glucosamine, 0.13;
0.77 M Na2SO4 0.86 1.6 3.4 72
0.91 M Na2SO4 0.07 0.29 0.65 24 L-rhamnose, 0.07; "total" carbohydrate, 3.8. In
the fraction not precipitable by CPC, the molar
a As in footnote a of Table 2. ratios were: uronic acid, 0.18; glucosamine, 1.25;
b As in footnote c of Table 2. L-rhamnose, 1.00; total carbohydrate, 2.0.)
TABLE 4. Effect of salts on polysaccharide synthesis in organism GS-
Medium Uronic acid Methylpentose "Total" carbohydrate Growth in
basal mediumb
mM mM mM S
Basal 0.17 (0.17)e 0.37 (0.73) 0.83 (1.4) 100
0.042 MMgSO4 0.21 0.13 0.89 100
0.083 MMgSO4 0.17 0.10 0.69 93
0.25 M MgSO4 0.28 0.07 1.7 (3.2) 98
0.42 M MgSO4 0.61 (0.75) 0.04 (0.18) 2.8 100
0.035 M Na2SO4 0.43 0.12 1.6 110
0.070 M Na2SO4 0.59 0.09 2.1 97
0.21 M Na2SO4 0.89 0.02 3.1 96
0.35 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 2.1962] 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) 100
0.25 M MgSO4 0.00 (0.00) 0.03 (0.03) 0.10 (0.11) 230
0.42 M MgSO4 0.00 (0.02) 0.00 (0.01) 0.07 (0.08) 31
0.070 M Na2SO4 0.00 (0.02) - (0.03) 0.08 (0.15) 120
0.21 M Na2SO4 0.03 (0.04) 0.04 (0.07) 0.16 (0.31) 440
0.35 M Na2SO4 0.07 (0.07) 0.02 (0.06) 0.22 (0.39) 410
0.49 M Na2SO4 0.11 0.02 0.24 400
0.63 M Na2SO4 0.13 (0.13) 0.04 0.39 (0.47) 300
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0.70 M 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 polysaccharide Similar results were obtained with NaCl, LiCl,
synthesis in organism SEa and (NH4)2SO4 at a concentration of 0.35 M.
"Total" Growth in
Fractionation of crude polysaccharide from
Uronic Hexosa- carbohy-
Medium acid He mie drate basal
mediumb strain GS with CPC permitted the separation of
a polysaccharide fraction containing uronic acid
mM mM mM S from one containing the methylpentoses, D-
I'.isal 0.06 0.07 0.39 100 rhamnose and D-talomethylose (Table 1) (Marko-
0.042 M MgSO4 0.08 0.06 0.35 91 vitz, 1961b). A comparison of the data on boiled
0.083 M MgSO4 0.04 0.07 0.33 93 vs. untreated culture fluids (Table 4) indicated
0.25 M MgSO4 0.05 0.10 0.40 48 that the uronic acid component was completely
0.42 M MgSO4 0.06 0.11 0.32 22 in the medium but the methylpentose component
0.035 M Na2SO4 0.03 0.06 0.44 82 was loosely bound to the cells and could be re-
9.070 M Na2SO4 0.06 0.09 0.80 92
0.21 M Na2SO4 0.07 0.11 0.74 84 leased by boiling the suspension.
0.35 M Na2SO4 0.09 0.08 0.45 54 Group 2b. Synthesis of a uronic acid-containing
polysaccharide is initiated only in the presence
a As in footnote a of Table 2. of Na2SO4. The only isolate responding in this
b As in footnote c of Table 2. fashion was GM (Table 1, no. 17 and Table 5).
Enhancement of growth was coincident with the
Group 2a. Synthesis of polysaccharide contain- initiation of synthesis of the uronic acid-contain-
ing uronic acid is stimulated by Na2SO4 or MgSO4 ing component in Na2SO4-containing media
or both with little effect on growth (Table 1,
(Table 5, last column). However, growth in the
13 through 16). The response of organism GL is basal medium was of a granular nature and ad-
shown in Table 3. It is apparent that a 20-fold hered to the growth flask; in media containing
increase in synthesis of polysaccharide containing Na2SO4 or MgSO4 the growth was evenly sus-
uronic acid occurred when this organism was pended. It should be noted that 0.25 M MgSO4
grown in media containing 0.49 M Na2SO4. Re-
gardless of the composition of the growth medium, appeared to increase growth, but did not initiate
the centrifuged cells retained little polysaccharide synthesis of uronic acid-containing polysac-
containing uronic acid. In contrast, 0.42 M charide.
MgSO4 had little effect on polysaccharide syn- Concentration and subsequent analysis of
thesis by this organism. supernatant liquids from strain GM grown in the
In group 2a, organism GS is also of particular basal medium failed to reveal evidence of uronic
interest. Synthesis of a polysaccharide containing acid. Bacteria from the medium containing 0.35 M
uronic acid was stimulated by either MgSO4 or Na2SO4 were subcultured in the same medium
Na2SO4 but synthesis of a methylpentose com- several times and then either inoculated into the
ponent was inhibited by these salts (Table 4). basal medium or plated on the basal agar. Iso-488 MARKOVITZ AND SYLVAN [VOL. 83
lated clones from the latter were also grown in ing polysaccharide was synthesized was stimu-
the basal medium. Such attempts to obtain lated (Table 1, group 3b). In addition, one
mutants that synthesized polysaccharide con- organism (strain SE, Tables 1 and 6) responded
taining uronic acid in the absence of Na2SO4 were to increased concentrations of Na2SO4 by synthe-
unsuccessful. Therefore, it appears likely that the sizing more total polysaccharide, without affect-
phenotypic expression of the ability to synthesize ing synthesis of the polysaccharide containing
polysaccharide containing uronic acid requires uronic acid.
the presence of Na2SO4 in strain GM. The effect of sulfate ion concentration on
Group 3a. Polysaccharide synthesis, but not changes in the ratio of certain constituents of
the component containing uronic acid, is stimu- polysaccharide fractions may become a useful
lated minimally by Na2SO4 and not by MgSO4 tool in indicating whether one or more poly-
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(Table 1, no. 18 and Table 6). saccharides are synthesized by a particular or-
Group 3b. Polysaccharide synthesis is increased ganism. Changes in the ratio of glucosamine or
up to twofold with increasing concentrations of L-rhamnose to uronic acid were apparent in
Na2SO4, although no polysaccharide containing 0.5-R (Table 2), and fractionation with CPC
uronic acid is synthesized (Table 1, no. 19, 20, proved that two polysaccharides were present. A
and 21). similar situation was even more apparent in GS
(Table 4), and again two polysaccharides were
DISCUSSION separated by fractionation with CPC. On the
Previous results demonstrated that bacteria other hand, 0.5-F revealed no changes in the ratio
capable of synthesizing polysaccharides contain- of uronic acid to L-rhamnose with increasing salt
ing uronic acid could be selected from soil in concentration, and fractionation with CPC did
liquid enrichment cultures containing high con- not separate the uronic acid component from the
centrations of sulfate ions (Markovitz, 1961a). L-rhamnose component. Historically, bacteriol o-
Six of these isolates (Table 1, no. 1 through 6) gists have believed that bacteria ordinarily syn-
reacted to increased quantities of sulfate ions by thesize very complex heteropolysaccharides, i.e.,
inhibiting synthesis of polysaccharides containing single heteropolysaccharides containing five or
uronic acid. Other isolates, obtained by streaking more different monosaccharide units. However,
soil samples directly on basal agar, also produced few attempts have been made to separate the
less polysaccharide containing uronic acid when components, and, as a result, studies on the bio-
grown in media with increased quantities of synthesis of such polysaccharide fractions have
sulfate ions (Table 1, no. 7 through 12). not been pursued because of their presumed com-
These results lead to the conclusion that the plexity. The complexity of certain plant gums
effect of the concentration of sulfate ions on the may also be more apparent than real (Smith and
synthesis of polysaccharides in other bacterial Montgomery, 1959).
strains warrants investigation, since this appears Whether growth of bacteria in media of high
to be an important parameter affecting poly- salt concentration affects the synthesis of enzymes
saccharide synthesis that is not mandatorilly necessary for polysaccharide synthesis, or the
linked to growth. The remarkable stimulation of activity of the enzymes, remains to be deter-
synthesis of polysaccharide containing uronic mined. In this connection, work on feedback in-
acid observed in strains GL (Table 3) and GS hibition of enzyme activity and repression of
(Table 4) by Na2SO4 suggests that the potential enzyme synthesis may be relevant (Yates and
to synthesize these polysaccharides may require Pardee, 1956; Umbarger, 1956; Vogel, 1957;
Na2SO4 or MgSO4 for full expression in some bac- Ames and Garry, 1959).
teria. GM (Table 5) may be an extreme case of
this type, since it appeared that Na2SO4 was ACKNOWLEDGMENTS
necessary for initiation of synthesis of polysac- The authors wish to acknowledge the technical
charide containing uronic acid. assistance of Sarah M. Moxham and Minoru
Stimulation of polysaccharide synthesis by Mayeda. The advice and encouragement of A.
Na2SO4 or MgSO4 was not limited to bacteria Dorfman and J. A. Cifonelli during these experi-
that synthesize polysaccharides containing uronic ments is gratefully acknowledged.
acid; one group in which no uronic acid-contain- This work was aided by grants from The1962] EFFECTS OF SALTS ON POLYSACCHARIDE SYNTHESIS 489
National Foundation and the National Heart REICHSTEIN. 1960. Desoxyzucker. 33. Mittei-
Institute of the U. S. Public Health Service lung. Papierchromatographische Differen-
(H-311). zierung der Hexamethylosen und ihrer 3-o-
methylderivate. J. Chromatography 3:63-74.
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