150

Studies of a Yeast Exacting towards

-
p Aminobenzoic Acid

SUMMARY: Certain single-cell strains of brewers top fermentation yeasts require

p-aminobenzoic acid as an essential nutrient. Relatively large quantities of adenine,
together with certain amino-acids, of which methionine and histidine were of prime
importance, could substitute for the p-aminobenzoic acid.
In the presence of adenine, histidine, and suboptimal amounts of methionine, the
addition of any one of seven other amino-acids caused a marked stimulation of
growth. These amino-acids were leucine, isoleucine, norleucine, valine, laorvaline,
tyrosine and phenylalanine.
Rainbow (1948)and Cutts & Rainbow (1949)found that certain strains of
Saccharomyces cerevisiae isolated from British brewery top fermentation yeasts ,
required p-aminobenzoic acid (PABA) as an essential nutrient. The present
work is a study of the nutrition of one of Rainbows PABA-requiring strains
of yeast and of its possible use in the microbiological assay of PABA.

METHODS
Two techniques were mainly used.
Auzanographic tests. For the preliminary qualitative establishment of
growth requirements, Beijerincks auxanographic test, as described by Ponte-
corvo (1949),was used. Plates were made of a layer of basal synthetic medium
containing a heavy seeding of yeast (of the order of 500,000 cells per plate).
Minute amounts of substances under test were then touched on to the surface
of the agar at marked points. After incubation at 25 for 18-48 hr., the plates
were examined. A zone of growth at the point of application of a test substance
indicated a positive effect as a growth stimulant.
Tube tests. The information elicited by auxanographs was studied further
by growth tests in tubes, by the usual procedure for microbiological assays.
Tubes (6 x g in.) containing 6 ml. of test medium were sterilized for 10 min. at
10 lb. pressure. After inoculation, the tubes were incubated in a vertical
position a t 25 in a water bath and shaken every 24 hr. Growth was determined
turbidimetrically in the Spekker absorptiometer, using a 1 cm. cell, for which
6 ml. of cell suspension is suitable. Unless otherwise stated, each response
quoted in the Tables in this paper is the average Spekker reading of duplicate
tubes after 72 hr. incubation at 25O.
Test organim a d inocula. This work was carried out entirely with the single-
cell strain of an English top fermentation brewery yeast designated Yeast 47
by Rainbow (1948). The organism was maintained on malt agar slants, transfers
being made weekly, or more frequently when necessary. Inocula for the tube
tests were prepared by transferring a little of the surface growth from a 16 to

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 A yeast requiring p-aminobertxoic acid 151
24 hr. slope culture into sterile 0.85 yo saline, shaking to a uniform suspension
and inoculating each tube with one drop (about 0.03 ml.) of the suspension.
Such a suspension gave a reading between 0.08 and 0-20 on the Spekker,
corresponding to 20,000-50,000 cells per tube.
Medium. The composition of the basal defined medium used for all tube
tests was, per 100 ml. final volume, in g. : glucose, 4.0; (NH4)$PO4,O*4;
.
KKPO,, 0.2; MgSO,. 7H20, 0.1 ; CaCl, 6H,O, 0.025; in pg. : inositol, 1000;
&-biotin, 0.2; Ca d-pantothenate, 100; aneurin, 100; pyridoxin HC1, 100;
nicotinic acid, 100; riboflavin, 5; KI, 10; in ml., lactic acid (syrupy A.R.), 0.3;
potassium lactate (50 % ,vlv), 1.2; trace element solution (Emery, McLeod &
Robinson, 1946), 0.1. Adjustment to pH 5-0-5-2 was made, if necessary, with
lactic acid-or sodium hydroxide.
For auxanographic tests the same basal medium was used with two modi-
fications; the glucose concentration was decreased to 1% and the medium was
solidified with 2 % agar that had been repeatedly washed with distilled water.

RESULTS
p-Aminobenzoic acid as a growth factor for Yeast 47. The basal PABA-free
medium supported the growth of some twenty single-cell strains of yeast
isolated from British breweries, the essential growth factors for which were
biotin, pantothenic acid and, in some cases, inositol. Four strains, however,
failed to grow in this medium. Awanographic tests identified the missing
factor in each case as PABA. From these four strains, that designated Yeast 47
was selected for further study. In addition to PABA, this yeast required biotin
and pantothenic acid; inositol was strongly stimulatory.
The response of Yeast 47 in basal medium to different concentrations of
PABA was determined by tube tests. The response curve was sigmoid and
a half-maximum growth was produced by c. 0-007pg. PABA/ml. medium
(Table 2). In the absence of PABA, Yeast 47 failed to grow appreciably.
Replacement of PABA by prilzes and amino-acids. Snell & Mitchell (1942)
and Shive & Roberts (1946) found that certain purine bases and methionine
were effective in reversing sulphanilamideinhibition whilst Lampen, Roepke &
Jones (1946) found that an amino-acid medium containing adenine could
support the growth of a PABA-requiring mutant of Escherichia coli. Tests
were therefore made to see whether purine and pyrimidine bases and amino-
acids could replace PABA for Yeast 47.
The amnographic technique was used for the preliminary experiments.
To test the bases a medium was used in which the ammonium phosphate of
the basal medium was replaced by 0.4 g./100 ml. vitamin-free casein acid-
hydrolysate (Ashe Labs. Ltd.). The bases adenine, guanine, xanthine, uracil,
thymine and cytosine were tested singly and in pairs. PABA was included as
a test substance to ensure at least one positive result. Results were read after
24 hr. incubation. A positive result was observed at those places on the plate
only where adenine or PABA had been applied. The other bases had no effect
even when incubation was prolonged to 96 hr. Tube tests showed that about

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 152 N . S . Czctts and C . Rainbow
10,ug. adenine HCl/ml. was sufficient to induce a half-maximum growth in
casein hydrolysate medium. Adenine possessed no growth-promoting activity
in the absence of amino-acids.
Nature of the active amirzo-acids. A preliminary survey of the amino-acids
responsible for promoting the growth of Yeast 47 in the presence of adenine in
PABA-free media was made by the auxanographic test. For this purpose,
10,ug. adenine hydrochloride/ml. were incorporated into the basal medium and
twenty-two amino-acids tested. Of these only methionine had any action.
The auxanographs were then repeated, with adenine (lO,ug./ml.) and DL-
methionine (20pg.lml.) incorporated in the basal medium, to test whether
other amino-acids had supplementary effects. There was now a positive
response to histidine, visible within 24 hr., after which period general growth
of the plates took place.
Taking the auxanographs a stage further by including lOpg./ml. histidine as
well as adenine and methionine in the basal medium and testing the remaining
amino-acids, it was found that any one of seven amino-acids had a positive
effect after 18 hr. incubation. This group of amino-acids (referred to below as
the leucine group) consisted of :leucine, norleucine, isoleucine, valine, norvaline,
tyrosine and phenylalanine. The response to tryptophan was also positive, but
less strongly marked. DL-Alanine and DL-threonine had a positive effect on
one occasion only.

Table 1. The importance of ahnine, methimine, histidine and leuciw

in PABA-free medium for the growth of Yeast 47
Growth recorded as Spekker readings;half-maximumgrowth =0.75 ;uninoculated medium
reads 0.0. The supplements were as follows, in ,ug./ml. medium: A = 10 adenine HCl;
M =20 DL-methionbe; M =100 DL-methionhe ; H =10 L-histidine HCI; L = 10 L-leucine;
P =O-OOS PABA.
Basalmedium plus ... A M H AML AHL M H L A M H L AMiH AMHL P
Spekker readings
A
>
...
f
Relative growth 0.05 0.02 0.01 0.01 0-39 0.34 0.33 0.51

The results of these auxanographic tests were confirmed by tube tests.

Table 1 illustrates the importance of adenine, methionine, histidine and a
member of the leucine group in the nutrition of Yeast 47 when PABA was
omitted from the medium. Omission of any one of these substances severely
restricts growth, good growth being obtained when the three substances and
one of the leucine group are present together. In Table 1,L-leucine is quoted as
typical of the leucine group. The effect of the leucine was less evident, however,
when the methionine content of the medium was increased, until, a t 1OOpg.
DL-m ethionine/ml., no effect was observed.
Effect of admifie and amino-acids in diminishing the requirement of Yeast 47
for PABA. The growth-promoting effect of PABA for Yeast 47 could thus be
imitated in PABA-free media by adenine in the presence of methionine,
histidine, and, where the amount of methionine was small, by one of the leucine
group of amino-acids. However, whereas the quantity of PABA required to

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 A yeast requiring p-anaimbenzoic acid 153
promote a half-maximum growth was of the order of 0-007pg./mld,the corre-
sponding quantities of adenine and the amino-acids were relatively large, of
the order of lOpg./ml., i.e. about 1000times greater in weight. This suggests that
PABA functions in an enzyme system involved in the synthesis of these
substances.
Supporting evidence was sought by measuring the response of Yeast 47 to
different concentrations of PABA in the basal medium and in media supple-
mented with adenine, methionine, histidine and other amino-acids. A decrease
in the amount of PABA required to cause half-maximum growth (corresponding
to a Spekker reading of 0-75) in the basal medium supplemented with a
particular substance, as compared with the basal medium alone, is regarded as
evidence that PABA is involved in the synthesis of that substance (Shive &
R O W , 1946). Supplements to the basal medium were added as follows:
adenhe hydrochloride, lO,ug./ml. ; DL-methionhe, 20,ug./ml.; L-histidine
monohydrochloride, lO,ug./ml. ; amino-acids of the leucine group, 1Opg. of
the L-isomer/ml,
The results are set out in Table 2. The amount of PABA required to give
half-maximum growth has been interpolated from curves drawn from the data

Table 2.. Response of Yeast 47 to PABA in media supplemented with

adenine and certain amino-acids
Growth recorded as Spekker readings; half maximum growth reads 0.75; uninoculated
medium reads 0.0. The various supplements were as follows, in pg./ml. medium: M =20 DL-
methionine; H =10 L-histidine HCl; L = 10 L-leucine; C = 10 L-cystine ; A = 10 adenine HCl.
mpg. PABA/ml.
Basalmedium
A
' mpg. PABA/ml. Percentage
0-0 1-07 3-33 5-00 6-07 8.33 10.00 for half- relative
Spekker readings maximum PABA-sparing
r , growth effect
Experiment 1
No additions 0.0 - - 0.30 0.78 1-28 1.42 6.5 -
M 0.0 0.12 0.38 1-04 1-44) - - 4.3 34l
H 0.0 - - 0.51 0.93 1.38 1.45 5.9 9
1
0.0 - - 0.43 0.98 1.34 1-44! 6.0 7
0.01 0.19 0.58 1.15 1.42 - - 3.8 41
0.0 0.68 1.28 1.36 1-40 - - 1-8 72
0.0 - - 0.45 0.97 1.33 1.50 6.0 7
0.01 0.82 1-37' 1.43 1.48 - - 1.5 77

Experiment 2
No additions 0.01 - - 0.17 0.43 0.85 1.17 7-9 -
A 0.01 - - 0.59 0.90 1.13' 1-33 5.8 26
M 0-02 0.09 0.29 0.67 1.23 - - 5.2 34
A+M 0.02 0.29 0.82 1.18 1-30 - - 4.3 61
A+H 0.01 - - 0.55 0.83 1.04 1.30 6.2 22
M+H 0.02 0.15 0.40 0.78 1.25 - - 4.8 39

Experiment 3
No additions 0.01 - - 0.35 0.81 1.31 - 6-4 -
M+H 0.02 - 0.51 1.17 1.46 - - 3.9 39
M+H+C 0.02 - 0.56 1.23 1.48 -' - 3-8 41

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154 N . S . C U B and C . Rainbow
contained in the table. The relative PABA-sparing effects were calculated from
the formula :
percentage relative PABA-sparing effect Ez-yx 100,
X
where II: and y are the quantities of PABA required to give half-maximal growth
in unsupplemented aud supplemented media respectively.
The results may be summarized:
(a)Addition of methionine to the basal medium decreased the amount of
PABA required to induce a given growth response. The half-maximum growth
level was promoted by 6.5 mpg. PABA/ml. basal medium, the corresponding
figure in the presence of methionine being 4.3 mpg., i.e. methionine exerts
a relative PABA-sparing effect of 34 yo (Table 2, Exp. 1).
(b) The relative PABA-sparingeffect of histidine was relatively small, about
9 yo. In the presence of methionine the effectof histidine was roughly additive
(Table 2, Exp. 1).
( c ) Leucine alone exerted a small PABA-sparing effect (7yo). In presence
of methionine, however, a marked interaction occurred. From Table 2 the
relative PABA-sparing effect of leucine in presence of methionine, calculated
on an additive basis, is 41 yo. The experimentally determined relative PABA-
sparing effect was greater than this (72%), indicating an interaction effect
between methionine and leucine. Other members of the leucine group showed,
qualitatively, the same effect. Tryptophan, alanine and threonine also exerted
a PABA-sparing effect in presence of methionine, but to a much smaller degree.
( d ) Adenine exerted a relative PABA-sparing action of 26 Yo (Table 2,
Exp. 2). The sum of the single effects due to adenine and methionine is equal
to their effect together (61 %); i.e. no interaction was observed.
(e) The following amino-acids showed no growth-promoting effect and pos-
sessed little or no PABA-sparing effect, either alone or in the presence of
methionine : arginine, asparagine, aspartic acid, cystine, cysteine, glutamic
acid, glycine, lysine, proline, hydroxyproline, serine. In Table 2 cystine is
quoted as typical of these amino-acids,

DISCUSSION
The essential growth factors required by most brewery top fermentation yeasts
are biotin, pantothenic acid and sometimes inositol, but strains exist which
also require PABA. These strains, however, may not be suitable for the micro-
biological assay of PABA. In the case of the strain studied, the presence of
adenine and certain amino-acids in test samples would interfere with assays.
Shive & Roberts (1946),as a result of sulphanilamide inhibition experiments
on Escherichia COG,believe that PABA takes part in the synthesis of purines
and methionine; our results support this view. In the absence of PABA,
Yeast 47 responds to adenine and certain amino-acids, of which methionine
is the most important. The fact that the amounts of adenine and methionine
required to induce half-maximal growth are relatively large compared with the
amount of PABA required to give a similar response suggests that PABA

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 A yeast requiring p-arninoberaoic acid 155
functions in enzymic systems which synthesizeadenineand methionine. Further
support for this view is given by the PABA-sparing effect of methionine and
adenine. Histidine showed a small PABA-sparing effect, which was significant
but not so large as that of methionine. H a l (1947)found that histidine replaced
folic acid as a growth factor for Streptococms lactis R and thus has already
established an indirect connexion between histidine and PABA, since the
latter is part of the folic acid (pteroylglutamic acid) molecule. More evidence
is required before an interpretation of the connexion between methionine and
the members of the leucine group of amino-acids can be offered.

REFERENCES
Cmm, N. S. & RAINBOW, C. (19419). Nutrition of a strain of brewers yeast exacting
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HAIZ,D. A. (1947). Histidine and its relation to folic acid. Biochem. J . 41, 299.
LAMPEN,J. O., ROEPEE,R. R. & JONES, M.J. (1946). The replacement of p-amino-
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PONTECORVO, G. (194I9). Auxanographic techniques in biochemical genetics. J . gen.
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RAINBOW, C. (1948). Para-aminobenzoic acid a growth factor for certain brewers
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SEIVE,W. & ROBERTS, E. C. (1946). Biochemical transformations as determined by
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(Received 1 July 1949)

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