Alimchandani, H. R.

Sreenivasan, A.

PubMed

Publications of the IAS Fellows

INACTIVITY OF PTEROYLGLUTAMIC ACID AND LEUCOVORIN
IN OVERCOMING SULFONAMIDE GROWTH INHIBITION
OF ESCHERICHIA COLI
H. R. ALIMCHANDANI AND A. SREENIVASAN
Department of Chemical Technology, University of Bombay, Bombay, India
Received for publication January 28, 1957
It was reported earlier (Alimchandani and
Sreenivasan, 1957a) that in Escherichia coli (Mac-
Leod strain) pteroylglutamic acid (PGA) andleu-
covorin (5-formyl,5-6-7-8,tetrahydrofolic acid;
LV) are inactive in overcoming growth inhibition
by sulfonamides or in substituting for any of the
other metabolites (methionine purines, serine,
thymine, valine, and glycine) which are active in
reversing the same in spite of the vitamin being
known to be intimately connected with the syn-
thesis of most of these metabolites. It was also
observed (Alimchandani and Sreenivasan, 1957b)
that PGA and LV are unable to decrease the
accumulation of arylamine by E. coli during sul-
fonamide bacteriostasis, though in the presence of
aminopterin the biosynthesis of purines is known
to be blocked at this step (Woolley and Pringle,
1950).
The following three points must be examined
before definite conclusions can be drawn: (1)
Folic acid (FA) is not involved in these systems
in E. coli, (2) the functionally active form of FA
for E. coli is different from PGA and LV and
these can not be utilized as intermediates in its
synthesis by the organism, and (3) PGA and LV
are not utilized by the cell.
The experiments reported here are an attempt
to find an explanation for the observed inactivity
of PGA derivatives in replacing p-aminobenzoic
acid (PABA) in overcoming inhibition of E. coli
by sulfonamides.
METHODS AND RESULTS
Characterization of folic acid active factor elabo-
rated by E. coli (MacLeod strain). Growth studies:
-The response of Streptococcus faecalis to graded
doses of E. coli culture filtrate was compared
with that of PGA and LV.
E. coli was grown in 100 ml quantities in the
medium of Green and Sevag (1946) dispensed
in 250 ml Erlenmeyer flasks. Incubation was for
24 hr. Following this the contents were adjusted
to pH 7, steamed for 5 min and the cells spun
down in a centrifuge. Aliquots of the supernatant
were used for the assay. Assays were done with
S. faecalis using the method of Luckey et al.
(1944) as modified by Mitbander and Sreenivasan
(1954).
Growth curves with PGA, LV, and a suitably
diluted culture filtrate concentrate of E. coli are
shown in figure 1. The FA synthesised by the
organism gave a response curve which differed
from that for PGA and LV in that heavier growth
was produced.
Since E. coli culture filtrate could support
growth of both S. faecalis strain ATCC 8043 and
Leuconostoc citrovorum strain ATCC 8081 (now
identified as Pediococcuk cerevisiae by Felton and
Niven, 1953), a differential assay was carried out.
The total S. faecalis activity was measured in
terms of PGA using S. faecalis as assay organism.
The citrovorum factor activity, expressed in
terms of LV, was assayed by the method of
Sauberlich and Baumann (1948) using Leuco-
nostoc citrovorum.
In a typical experiment, S. faecalis activity
was 1.2 mAg PGA/ml and CF activity was 0.97
m,ug LV/ml. As LV is only half as active as
natural CF (Keresztesy and Silverman, 1951;
Cosulich et al., 1952) the CF activity is in fact
only 0.48 m,ug/ml.
Inhibition analysis:-The activity of E. coli
culture filtrate was compared with that of PGA
and LV in overcoming growth inhibition of S.
faecalis by the PGA analogue antagonists methyl
folic acid (1 ,ug/ml) and aminopterin (0.03 ,ug/
ml). Other details of the method were as above.
Using a series of concentrations of the three sub-
stances, the inhibition indices were determined
from the concentration of the compounds required
to effect reversal. The inhibition index represents
the ratio of the concentration of the inhibitor to
that of the metabolite (McIlwain, 1942; Shive
and Macow, 1946).
171
ALIMCHANDANI AND SREENIVASAN
ml. E.coli culture filtrate
0 0.1 0.2 0.3
100
80
60
40
Culture filtrate-X-x_
20
______________
1 2 3 4 5
mY FGA
2 4 6 8 10
mY LV
Figure 1. Growth response of Streptococcus faecalis strain R to culture filtrate of Escherichia coli
TABLE 1
Activity of Escherichia coli culture filtrate in over-
coming growth inhibiton of Streptococcus faecalis
by pteroylglutamic acid (PGA) antagonists
1
14p
Inhibition
Index With:
mi,/On3° 7-ymethy
0.03
DI/M g/Mi
It was found that the factor elaborated by E.
coli was more active than PGA in overcoming
growth inhibition by both the antagonists. The
activity of the factor approaches that of LV
though it is slightly less with aminopterin and a
little higher with 7-methyl folic acid (table 1).
Bioautography:-The technique followed was
based on that of Winsten and Eigen (1948). A
100 fold concentrate of E. coli culture filtrate was
prepared after neutralization by freeze-drying
and was spotted on paper strips along with PGA
and LV. Chromatograms were developed for 4 to
5 hr at room temperature in 0.1 M phosphate
172 [VOL. 74
0.4 0.5
Pteroylglutamic acid (PGA).. . 0.2 120
Leucovorin (LV) ................. 3.1 400
E. coli culture filtrate............ 2.7 430
g;/m.
BACTERIOSTASIS OF E. COLI
TABLE 2
Bioautography of folic acid (FA) active factors in
Escherichia coli culture filtrate
Sample Rf Value
1. Pteroylglutamic acid (PGA) ..... 0.51
2. Leucovorin (LV) ................ 0.81
3. Escherichia coli culture filtrate
concentrate
(1).......................... 0.59
(2).......................... 0.74
The E. coli culture filtrate concentrate gave
two spots with Rf values between those for PGA
and LV. Thus it would seem that E. coli elabo-
rates two FA active factors different from PGA
and LV.
buffer of pH 7 (Zakrzewski and Nichol, 1953) by
descending chromatography. Strips were allowed
to dry and laid on agar basal medium seeded with
S. faecalis. The use of the dye triphenyl-tetrazo-
lium bromide in the medium was of value because
of its reduction of red color at the region of
growth, thus making spotting easy. The results
(Rf values) of the bioautographic analysis are
given in table 2.
Permeability of cells of E. coli to PGA and LV:
-Cells of E. coli were harvested after 24 hr
growth in the medium of Green and Sevag (1946)
and washed twice with sterile saline. The cells
were incubated aseptically with PGA and LV in
presence of phosphate buffer of pH 7 in a final
concentration of 0.1 M at 30 C. At the end of 2
hr, the cells were spun down in a centrifuge and
suitable aliquots of the supernatant were used for
assay. It was found that the cells excrete an ac-
tive compound of S. faecalis into the medium; how-
ever, the difference between the experimental and
TABLE 3
Permeability of Escherichia coli cells to pteroyl-
glutamic acid (PGA) and leucovorin (LV)
Streptococcus
System Total Volume 5 ml Activity/mi in
Terms of PGA
1. Cells*........................... 8
2. Cells + PGA 10 mjAg/ml......... 17
3. Cells + LV 20 m,sg/ml. ....................18.5
4. PGA 10 m,ug/ml ................. 9.5
5. LV 20 m,g/ml................... 10.8
* Dry weight 3.2 mg in a final volume of 5 ml.
control values was equivalent to the PGA or LV
added as the case may be.
DISCUSSION
The FA active compound elaborated by E. coli
gives higher growth response than PGA or LV
with S. faecalis, indicatingperhaps thatthis factor
is different and more active than PGA or LV for
this organism. The possibility exists that the cul-
ture filtrate contains activators. In a study of
synthesis of FA activity by Lactobacillus arabino-
sus, Mitbander and Sreenivasan (1951) also re-
ported on the differential response to growth of
S. faecalis to the L. arabinosus culture factor.
While the present work was in progress, Lascelles
and Woods (1952) reported on the FA activity of
E. coli culture filtrate with similar conclusions.
The observations reported here show that the
compound(s) elaborated by E. coli possesses citro-
vorum factor activity. However, the CF activity
can not account for all the S. faecalis activity. This
might be due to the fact that the compound has
a low CF activity or that it is a mixture of two
compounds, one lacking in CF activity.
The activity of the culture filtrate in reversing
growth inhibition due to PGA antagonists is
comparable to that of LV. Considering however
that natural CF is twice as active as LV (Ke-
resztesy and Silverman, 1951; Cosulich et al.,
1952) the factor can not be identical to the nat-
ural L-form of LV.
Bioautographic studies reveal the existence of
two active factors distinct from PGA and LV
though it may be possible that one is derived
from the other.
The S. faecalis activity of these factors rules
out the possibility that the factors are glutamic
acid conjugates, as all known conjugates of PGA
are inactive for this organism (Hutchings and
Mowat, 1948).
Recent work (Blakley, 1954; Kisliuk and Sa-
kami, 1955; Alexander and Greenberg, 1955) has
indicated that tetrahydrofolic acid and its hy-
droxymethyl derivative are more closely related
to the functionally active form of FA.
In the permeability experiment it was observed
that there was an excretion of FA active com-
pound into the medium. Nevertheless, the whole
of the added PGA or LV is recoverable quantita-
tively from the medium indicating no diffusion of
added compounds into the cell.
The FA active factors synthesized by E. coli
1957] 173
ALIMCHANDANI AND SREENIVASAN
are different from PGA and LV as indicated by
S. faecalis growth response curve, inhibition in-
dices and bioautography. The possibility that
PGA or LV may be interiimediates in, or are con-
cerned with, the biosynthesis of these factors can
not be excluded. The inability of PGA and LV to
diffuse into the cells may account for their inac-
tivity in overcoming sulfonamide growth in-
hibition.
SUMMARY
Culture filtrate of Escherichia coli shows folic
acid and citrovorum factor activities.
With Streptococcus faecalis strain R a dose re-
sponse curve diffeIent from those for pteroylglu-
tamic acid and leucovorin (5-formyl,5-6-7-8, tet-
rahydrofolic acid) is obtained.
Using methyl folic aci(d and aminopterin as
growth inhibitors of S. faecalis it is found that
the inhibition index of the culture filtrate is dif-
ferent from those of pteroylglutamic acid and Leu-
covorin (5-formyl,5-6-7-8, tetrahydr ofolic acid).
Bioautography of the cultuie filtrate shows the
existence of at least two factors distinct fiom both
pteroylglutamic acid and leucovorin (5-formyl,-
5-6-7-8, tetrahydrofolic acid).
Evidence is presented which indicates that
cells of E. coli are impermeable to PGA or LV.
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[VOL. 74174


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Inactivity of pteroylglutamic acid and leucovorin in overcoming sulfonamide growth inhibition of Escherichia coli

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INACTIVITY OF PTEROYLGLUTAMIC ACID AND LEUCOVORIN IN OVERCOMING SULFONAMIDE GROWTH INHIBITION OF ESCHERICHIA COLI

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