JouRNAL OF BACTERIOLOGY, Sept. 1967, p. 707-711 Vol. 94, No.

3
Copyright © 1967 American Society for Microbiology Printed in U.S.A.

Growth Inhibition by Hexoses of a Temperature-

Sensitive Thiazoleless Mutant of
Salmonella typhimurium
JOSE L. PARADA AmN MANUEL V. ORTEGA
Departamento de Bioquimica, Centro de Investigacio'n y de Estudios Avanzados del 1. P. N., Apartado Postal
14-740, Mexico 14, D.F., Mexico
Received for publication 12 June 1967

A Salmonella typhimurium mutant showing impairment in the utilization of

hexoses was isolated after treatment with N-methyl-N'-nitro-N-nitrosoguanidine.
At 30 C, it grew with hexoses (glucose, fructose, galactose, mannitol), glycerol,
succinate, or acid-hydrolyzed casein. At 37 C, it failed to grow with any of the hexo-
ses. Enzymatic determinations demonstrated, however, that the enzymes of
the glycolytic pathway (up to the formation of triose phosphates) were present
and active at 25 and 37 C. At 42 C, the mutant did not grow with any of the
carbon sources used. At both 37 and 42 C, the mutant grew perfectly well with hexo-
ses if yeast extract was present. The metabolite required for growth was thiamine
or, specifically, its thiazole moiety. If glucose was added to a culture growing in
glycerol, at 37 C, growth was inhibited. This inhibition was relieved by the addition
of thiamine or thiazole. Thus, at 37 C and only in the presence of hexoses, the mu-
tant manifests a requirement for thiazole. This auxotrophy is absolute at 42 C.
These data indicate that, in this mutant, some derivative of hexoses inhibits the syn-
thesis of thiazole, and that this inhibition is also dependent on the temperature of
incubation. The position in the bacterial chromosome of the genetic locus of this
lesion (thz-) was determined by conjugation and found to coincide with the only
thiamine (thi) locus so far reported.

Although the biochemical potential of an or- source used in growing the cells. At 37 C, in the
ganism is determined by its genetic composition, absence of hexoses, the mutant behaved as a
the final phenotypic expression of a given charac- prototroph. Hexoses inhibited growth; this in-
teristic can be modified by the existing environ- hibition was relieved, exclusively, by either thi-
mental conditions. amine or its thiazole moiety. At 42 C, the require-
This is the case, for instance, in the adenine- ment for thiazole was absolute.
sensitive mutants of Salmonella typhimurium MATERIALS AND METHODS
studied by Dalal et al. (2). These mutants are Bacterial strains. S. typhimurium LT-2 was kindly
prototrophic in their growth requirements, but, provided by the late M. Demerec. Mutant A-33 was
in the presence of adenine, there is inhibition of isolated after treatment of the parental strain with
growth. The adenine inhibition is relieved non- N-methyl-N'-nitro-N-nitrosoguanidine (K & K Lab-
competitively by the pyrimidine moiety of thia- oratories, Inc.), by the method of Adelberg et al. (1).
mine, by pantoyl lactone, and by methionine. At 30 C, it grew in minimal media with any of the
Kinetic analysis of this phenomenon demon- following carbon sources: glucose, fructose, galactose,
strated that adenine inhibits the synthesis of the mannitol, glycerol, succinate, or acid-hydrolyzed
casein. At 37 C, it failed to grow with any of the
reversing agents at the level of the biosynthesis of
folic acid coenzymes, common factors required aforementioned hexoses. It was maintained on nutrient
for the synthesis of the three mentioned metabo- broth-glycerol-agar slants. The Hfr strains used were:
lites. S. abony SW1444 (met- aro- srr; obtained from
E. Lederberg), S. abony SW1452 (trp-), and S. typhi-
We here report a temperature-sensitive mutant murium SR305 (his- gal-; obtained from K. E. San-
of S. typhimurium with a thiazoleless genotype. derson).
This genotype was made apparent both by the Culture media. The minimal medium used was that
temperature of incubation and by the carbon of Davis and Mingioli (3), citrate omitted. The carbon
707
708 PARADA AND ORTEGA J. BACTERIOL.

sources were added at a final concentration of 0.4%0 When the growth response of the mutant at
(w/v); yeast extract, at 0.04% (w/v). For solid media, 42 C was investigated, it was found that the mu-
agar (Difco) was added (1.5% w/v). Purine and tant did not grow at this temperature with any of
pyrimidine bases and vitamins were used at the con- the carbon sources used (hexoses, glycerol, suc-
centrations suggested by Guirard and Snell (5). For cinate, and acid-hydrolyzed casein), unless yeast
the final experiments in the determination of the extract was added to the medium. The same re-
growth requirement, thiamine, thiazole, and pyrimi-
dine were used at a concentration of 10 mgg/ml. sults were found when the mutant was grown at
(Both thiazole and pyrimidine were gifts of K. Pfister, 37 C in the presence of hexoses.
Merck Sharp & Dohme). 5-(2-Hydroxyethyl)-4- Growth in minimal-glucose liquid media, with
methylthiazole and 4-amino-5-hydroxymethyl-2-meth- added vitamins and purine and pyrimidine bases,
ylpyrimidine, the moieties of thiamine, are called, was studied. It was found that the mutant grew
throughout, thiazole and pyrimidine, respectively. only when thiamine was present; none of the
Growth in liquid media. Cells were grown overnight other substances tested had any effect (Fig. 1).
at 37 C, aerobically, in liquid glycerol-minimal me- When the pyrimidine and thiazole moieties of
dium. They were harvested by centrifugation, washed thiamine were tested, alone or in combination,
with sterile saline solution, resuspended in sterile
saline, and used to inoculate 50-ml portions of the growth occurred only when thiazole was present;
minimal medium, with the corresponding carbon it did not matter whether the experiment was
sources and additions, in 300-ml Bellco flasks equipped carried out at 37 or 42 C (Fig. 2). In liquid media,
with side arms (diameter, 19 mm). The cultures were at 30 C, there was no requirement whatsoever;
incubated aerobically, at the required temperature, in the mutant grew at the same rate with glucose or
a New Brunswick rotatory shaker. Growth was fol- glycerol, with or without thiazole.
lowed by reading, every hour, the optical density of
the cultures, at 660 mu, using a Coleman Junior
spectrophotometer.
Conjugation. Conjugation experiments were done
following the instructions of Sanderson and Demerec
(7). Appropriate dilutions of samples of the mating
mixture, taken at various times and vigorously shaken
for 1 min, were plated, in triplicate, in minimal-
glucose plates. The plates were incubated at 42 C, and
the recombinants were counted after 36 hr of incuba-
tion. It should be pointed out that, with strains SW-
1452 and SR305, the "dead time" necessary before
any chromosomal markers can be detected in the
recipient cell by the interrupted-mating technique was
5 to 6 min after time zero, as defined by Sanderson
and Demerec (7). We verified these results by inter-
rupted-conjugation experiments, using, as recipients,
different mutant strains with well-located chromoso-
mal lesions. There is a difference, especially in strain
SW1452, in the "dead time" values given by Sanderson
and Demerec and those found by us. We do not know
the reason for this; however, since our results were
reproducible, we used, for the mating experiments
done with any of those strains, a "dead time" of 5 to 6
min.
RESULTS
Characterization of the thiazoleless genotype of
the mutant. As stated before, mutant A-33 was
isolated as a temperature-sensitive mutant that,
at 37 C, was unable to utilize hexoses for growth.
A lesion in the early steps of the utilization of
glucose was suspected. It was found, however,
that cell-free extracts of glycerol-grown bacteria,
both of the parental and the mutant strains, HOURS OF INCUBATION
presented, at 25 and 37 C, similar enzymes: FIG. 1. Growth of mutant A-33 in minimal glucose
glucokinase, phosphoglucoisomerase, phospho- medium, at 37 C, in the presence of vitamins. (0) No
fructokinase, aldolase, and glucose-6-phosphate additions; (A) with all of the vitamins; (O) with all
dehydrogenase. vitamins except thiamine; (@) with only thiamine.
VOL. 94,1967 HEXOSES AND INHIBITION OF A SALMONELLA MUTANT 709
Li.0 was added to the culture at the time of the tem-
perature change, growth continued unarrested
(Fig. 4). The inhibition observed by a temperature
o. shift disappeared when the inhibited culture was
incubated again at 30 C (Fig. 5).
The same inhibition phenomenon was ob-
0..8 served when the mutant was growing in a gly-
cerol-minimal medium and the shift in tempera-
ture was from 30 to 42 C; when the change was
01.7 from 30 to 37 C, there was no delay in growth.
Location of the affected locus on the bacterial
E
0
chromosome. Table 1 shows the results of several
t0 interrupted-mating experiments, done to locate
the affected locus on the bacterial chromosome.
1-
U) From these results, it appears that the mutation
z
C]
i.5- is located at 129 min from the zero point of the
-J bacterial chromosome, according to the map of
Sanderson and Demerec (7). It is of interest that
I-.
a.oi.4- these investigators located a locus for thiamine
0
at this point, although they did not test the re-
sponse of their mutant to the isolated moieties of
0O1.3

0.1.2

O 2 3 4 5 6 7 8 9 10
HOURS OF INCUBATION

FIG. 2. Thiazole requirement of mutant A-33 growing

in minimal glucose medium at 37 C (0) No additions;
(A) with thiamine; (O) with thiazole; (0) with pyrimi-
dine.

Inhibition of growth by glucose at 37 C. When

glucose was added to a culture growing in gly-
cerol-liquid medium at 37 C, growth was in-
hibited. This inhibition was relieved by the addi-
tion of thiamine or thiazole, but not by pyrimi-
dine (Fig. 3).
The inhibition of growth was also observed
when glucose was present, at the beginning of the
experiment, in a glycerol medium. If thiamine or
thiazole were also present, no inhibition occurred.
When, after several hours of incubation, thiamine
or thiazole was added to a glycerol-grown culture
inhibited by glucose at zero-time, the inhibition
of growth disappeared (Fig. 3). HOURS OF INCUBATION
Inhibition and re-establishment of growth by FIG. 3. Inhibition of growth of mutant A-33 by glu-
temperature shifts. When the incubation tempera- cose. Cells growing at 3 7 C in minimal glycerol medium.
ture of the mutant, growing in a glucose-minimal (0) No additions; (A) glucose added at time A; (-)
medium, was changed from 30 to either 37 or glucose added at time A and thiamine added at time B;
42 C, there was a decrease in the rate of growth; (O) glucose added at zero-time; (U) glucose added at
after a few hours, growth ceased. When thiazole zero time and thiamine added at time C.
710 PARADA AND ORTEGA J. BAm- RioL.

inhibited, at 37 C, by a hexose metabolite. At

42 C, the enzyme ceases to function, and the
synthesis of thiazole stops completely; growth can
proceed only if the cells are provided with thiazole
or thiamine.
At present, nothing is known about the enzy-
matic steps leading to the biosynthesis of thiazole
in bacterial systems, and very little is known about
the precursors of this compound. D. J. Howells

!E
f-
0
u)
z
w
a
-
4
E'
tL o -11a
0

F- O."
0
7
z
0~

2
0.;
0
O
369
a.
[. . . .A
. . . . .

O 2 3 4 5 6 7 O 9 10

HOURS OF INCUBATION
FIG. 4. Effect of the addition of thiazole on the in- 1 2 4' 7' 90

hibition of growth produced by a temperature shift. HOURS OF INCUBATION

Cells growing at 30 C in minimal glucose medium. (0)
No addition, no temperature shift; (A) no addition, FIG. 5. Effect of temperature shifts on growth of
temperature shift (42 C) at time indicated by arrow; mutant A-33. Cells growing at 30 C in minimal glucose
(0) addition and temperature shift (42 C) at time indi- medium. (0) No temperature shifts; (A) shift to 37 C
cated by arrow. at time A; (-) shift to 37 C at time A, then shift to
30 C at time B; (0) shift to 42 C at time A; (U) shift
this vitamin (K. E. Sanderson, personal communi- to 42 C at time A, then shift to 30 C at time C.
cation).
TABLE 1. Map position of the thz locus
DIscussIoN determined by conjugation
The biochemical and genetic characteristics of
S. typhimurium mutant A-33 indicate that it is a
Donor Hfr strain
TmofApparent
Time of
Cross no. correction map
single-lesion, temperature-senstitive, thiazoleless position
mutant and that the manifestation of its genotype
o~f 1hz
depends on environmental conditions. At 30 C, min min
the mutant behaves as a prototroph; at higher Salmonella typhimu- 1 14 128
temperatures (37 and 42 C), its response to the rium SR305 2 15 129
environment is different. At 37 C, when succinate, 3 15 129
glycerol, or acid-hydrolyzed casein is used as a
carbon source, the behavior is still that of a S. abony SW1452 1 7 129
prototroph; if hexoses are used, the auxotrophy 2 8 128
for thiazole is made apparent. At 42 C, it does 3 7 129
not matter which carbon source is used; the S. abony SW1444 1 NRa
mutant behaves as a thiazole-exacting auxotroph. 2 NR
It thus appears that, in this mutant, some en- 3 NR
zyme in the biosynthetic pathway of thiazole is
temperature-sensitive and that its activity can be No recombinants.
VOL. 94,1967 HEXOSES AND INHIBITION OF A SALMONELLA MUTANT
[Ph.D. Thesis, University of Wales, 1962, as
quoted by Goodwin (4)] found that growing
cultures of Bacillus subtilis incorporate the methyl
carbon of methionine into the thiazole moiety of
thiamine.
More is known about the precursors of thiazole
in bakers' yeast. The recent communication of
Johnson et al. (6) indicates that, in this micro-
organism, at least the methyl carbon and the sul-
fur of methionine are incorporated, to the same
extent, in the thiazole structure. The two carbons
of acetate and two carbons of alanine (both com-
pounds probably as acetaldehyde) are also in-
corporated.
If bacterial systems use the same precursors for
thiazole synthesis as yeast, it could be postulated
that the five-carbon chain present in thiazole is
formed from acetaldehyde and a three-carbon
compound, by means of an aldolase- or trans-
aldolase-type reaction, giving a pentose-like
compound. The theoretical enzyme required for
this condensation could represent the lesion in
mutant A-33. At 37 C, its active center would be
deformed and could accept a molecule of some
derivative of hexose that would block it from
carrying out its function. In that case, both the
observed inhibition by hexose and the relieving
effect of thiazole could be easily explained.
In any case, a sound explanation of the hexose
inhibitory effect observed with this thiazoleless
mutant will have to wait until more knowledge
about the precursors of thiazole and the enzy-
matic steps of its synthesis, in bacterial systems,
is obtained.
The fact that the chromosomal lesion respon-
sible for the thiazole requirement of mutant A-33
is located at the same point at which Sanderson
and Demerec (7) reported a locus concerned with
thiamine biosynthesis can have two explanations.
As these investigators did not test the response of
their thiamine-requiring mutant to the thiazole
and pyrimidine moieties, it could be that the thi
locus is rather a thz locus. The other possibility
is that there are two loci, thi and thz, closely
711
linked, that map at the point indicated by Sander-
son and Demerec and by our experiments. Fur-
ther work with some other thiamineless and
thiazoleless mutants will clear up this point.
ADDENDUM IN PROOF
At the suggestion of K. E. Sanderson, and to
maintain a coherent nomenclature of S. typhimurium
mutants, mutant A-33 will in the future be designated
as SM33.
ACKNOWLEDGMENTS
Thanks are due to Jose Chavez for technical as-
sistance.
One of us (J.L.P.) was supported by a Predoctoral
Fellowship, Organization of the American States
(1965-1966) and a CIEA Latin-American Fellowship
(1967).
LITERATURE CITED
1. ADELBERG, E. A., M. MANDEL, AND G. C. CHING
CHEN. 1965. Optimal conditions for mutagene-
sis by N-methyl-N'-nitro-N-nitrosoguanidine in
Escherichia coli K12. Biochem. Biophys. Res.
Commun. 18:788-795.
2. DALAL, F. R., R. E. GOTS, AND J. S. Gos. 1966.
Mechanism of adenine inhibition in adenine-
sensitive mutants of Salmonella typhimurium. J.
Bacteriol. 91:507-513.
3. DAvis, B. D., AND E. S. MINGIOLI. 1950. Mutants
of Escherichia coli requiring methionine or
vitamin B12. J. Bacteriol. 60:17-28.
4. GOODWIN, T. W. 1963. The biosynthesis of vita-
mins and related compounds, p. 10. Academic
Press, Inc., New York.
5. GUIRARD, B. M., AND E. E. SNELL. 1962. Nutri-
tional requirements of microorganisms, p. 33-
94. In I. C. Gunsalus and R. Y. Stanier [ed.],
The bacteria, vol. 4. Academic Press, Inc., New
York.
6. JOHNSON, D. B., D. J. HOWELLS, AND T. W. GOOD-
WIN. 1966. Observations on the biosynthesis of
thiamine in yeast. Biochem. J. 98:30-37.
7. SANDERSON, K. E., AND M. DEMEREC. 1965. The
linkage map of Salmonella typhimurium.
Genetics 51:897-913.