Genetically Alterable Transport ofAmethopterin Diplococcus ...

JOURNAL OF BACTERIOLOGY, Jan., 1967, p. 315-319 Vol. 93, No. I
Copyright ( 1967 American Society for Microbiology Printed in U.S.A.

Genetically Alterable Transport of Amethopterin Downloaded from http://jb.asm.org/ on August 11, 2016 by guest

in Diplococcus pneumoniae

II. Impairment of the System Associated with Various Mutant Genotypes

FRANCIS M. SIROTNAK, M. GAY SARGENT, AND DORRIS J. HUTCHISON

Division of Experimental Chemotherapy, Sloan-Kettering Institute for Cancer Research, and Sloan-Kettering
Division, Graduate School of Medical Sciences, Cornell University Medical College, New York

Received for publication 27 July 1966

ABsTRAc-r

Six mutations determining resistance to amethopterin were examined for their
effects on the active transport of the drug. In strains bearing each of the mutations
and exhibiting resistance levels varying from 10- to 100-fold, transport at limiting
concentrations of H3-amethopterin was reduced from 2.5 to 10 times the rate char-
acteristic of the wild type. Kinetic analysis of transport showed an increase in the
value for Km of the system in all of the mutants. Values for the wild-type system
were 0.9 X 10-6 M and for the mutants varied between 2.5 x 10-6 M and 9.0 X 10-6
M. Values for Vmax were approximately the same for each system. The mutant trans-
port systems also exhibited a shift in pH optimum from near 6.0 (wild-type) to below
5.0. The results were interpreted as an alteration in the binding properties of the
permease in the mutant strains.

Resistance to antifolates involving a decrease in of the transport capacity in the mutant strains was
the ability to transport the drug has been ob- always made by a direct comparison with transport
served in Bacillus subtilis (8), and in murine leu- in the wild-type strain in the same experiment. Fur-
kemic cell lines both in vitro (4) and in vivo (5, ther experimental details are given in the text or the
16). An understanding of this physiological legends accompanying each figure. Resistance levels
mechanism of resistance to these antimetabolites of the various strains were determined as described
should be of some importance to future cancer previously (14).
chemotherapy, particularly of leukemia in man.
During studies of resistance to the antifolate, The amethopterin-resistant mutants used during
amethopterin, in Diplococcus pneumoniae, one these studies were all prepared by genetic transforma-
class of mutation was identified which was unre- tion of the drug-sensitive parent strain (wild-type R6,
lated to the well-documented effects on the "tar- obtained from R. D. Hotchkiss, Rockefeller Univer-
get enzyme," dihydrofolate reductase (11, 12). sity, New York, N.Y.) with DNA isolated from ap-
This finding has led to an examination of strains propriate donor strains. It can, therefore, be assumed
bearing these mutations for a possible impairment that the genotype of each strain, both mutant and
in the transport of amethopterin. The properties wild-type, is identical, with the exception of the re-
of the transport system in the wild-type organism sistance determinant which was transferred. Each of
have been described in a companion report (15). the mutant strains bears one of six mutations (Table
Presented here are results relating to both quanti- 1) which was either spontaneously derived (ame-r) or
tative and qualitative mutationally determined induced with nitrous acid (AR-16). The latter was
effects observed in the resistant strains. obtained from A. M. Sicard, Laboratorie de Genetique
Physiologique du C.N.R.S., Gif-sur Yvette, France.
MATERiALS AND METHODS Each mutation determines a characteristic level of re-
sistance and a uniformly depressed rate of growth.
Cultural conditions, a description of the H3-ame- Mutations in the ame-r group were also distinguish-
thopterin uptake system, and the procedure for the able by their individual efficiencies of transformation
determination of radioactivity have been given (15). and chromosomal location (14).
In general, uptake was measured by determining the
radioactivity remaining after the washing of cells RESULTS

(7.7 X 109) which had been incubated in growth Uptake of H3-amethopterin in mutant and wild-
medium containing Hs-amethopterin. An evaluation type strains. The intracellular accumulation of
H3-amethopterin by wild-type D. pneumoniae has
been shown to occur primarily by an active trans-

315

316 SIROTNAK, SARGENT, AND HUTCHISON J. BAcTmRioL.

TABLE 1. Some characteristics of the various
mutant strains of Diplococcus pneumoniae

Resi-s-

StrSatirnain DDerrmiuvtaattiioonn of Reglraotwitvhe" atmaetneterhbionpt-o

rate (X 10-61 )

R6 ame-r-l Spontaneous 0.60 0.44 (A
R6 ame-r-2
R6 ame-r-7 Spontaneous 0.68 4.4 -i
R6 ame-r-9 Spontaneous 0.60 1.3
R6 ame-r-10 Spontaneous 0.51 4.4 0.
R6 AR-16 Spontaneous 0.46 4.4
Nitrous acid 0.51 4.4 CD)

induced Downloaded from http://jb.asm.org/ on August 11, 2016 by guest

a As related to the growth rate for the wild
type in a semisynthetic enzymatic casein hydroly-
sate medium (13).

b The wild type is resistant to 0.044 X 10-6 M

drug.

port process (15). The effects of mutation on this T (MIN)

process were estimated by comparing the rates of FIG. 1. Uptake of HI-amethopterin by wild-type
and various mutant strains ofDiplococcus pneumoniae.
H3-amethopterin uptake at 37 C (corrected for Samples (1-mi) of suspension (7.7 X 109 cells per
milliliter) were incubated simultaneously at 37 and 0 C
uptake at 0 C) at a concentration of 0.2 ,ug/ml by with 0.2 ,ug/ml of HI-amethopterin. Solid lines repre-
similarly prepared cell suspensions from both sent uptake at 37 C correctedfor uptake at 0 C, whdch
is shown by the broken line. Strains are represented as
wild-type and mutant cultures. The results are follows: 0, wild type; 0, ame-r-1; X, ame-r-2; A
ame-r-7; A, ame-r-9; U, ame-r-10; E, AR-16.
shown in Fig. 1. Uptake of drug at 0 C (presumed
to be due, in part at least, to diffusion) occurred TABLE 2. Comparison of resistance and
in an identical fashion in all of the strains ex- H3-amethopterin transport properties of
various mutant and wild-type strains of
amined. Maximal accumulation occurred ap-
Diplococcus pneumoniae
proximately 4 min after the addition of drug. For
reasons of clarity, this finding has been repre- Rela- H'-amethopter in transport
sented by a single set of data. Active transport of
drug (uptake at 37 C) was markedly affected in all tiveG
of the mutant strains examined. In a manner Strain resis- R(Xa) a1-xKVVCmmdd~ pH

similar to that previously observed with the wild- tance (X (X 16(-1X3) opti-
type strain, transport of IP-amethopterin in all level 10-6) lo-,)
of the strains did not assume a constant rate until mum

after 4 min of incubation. This was the point at R6 (wild type). 1 5.10 0.9 1.90 5.9
which zero-degree uptake no longer contributed R6 ame-r-. 10 1.70 <5.0
to total uptake. At limiting drug concentration 2.04 2.5 2.42 <5.0
(0.2 ,ug/ml), the rate of uptake in the ame-r-2, R6 ame-r-2. 100
ame-r-9, ame-r-10, and AR-16 strains was reduced R6 ame-r-7. 30 0.59 9.1 1.43 <5.0
1.82 <5.0
by a factor of 10, but it was lowered only by a R6 ame-r-9. 100 1.02 3.6
R6 ame-r-10 100 1.82 <5.0
factor of 2.5 and 5 in the ame-r-l and ame-r-7 R6 AR-16. 100 0.51 7.1 2.04 <5.0
strains, respectively. Quantitative values for the
0.46 7.1
rate of uptake in the individual strains are given 0.46 9.0
in Table 2.
a Mutant/wild type.
Kinetic analyses ofHI-amethopterin transport in
b IP-amethopterin uptake at a concentration of
the mutant and wild-type strains. As an approach
toward experimentally defining the nature of the 0.2 ,ug/ml. Rate = moles per minute per milligram
impairment observed in each of the mutant (dry weight).

strains, analyses of the individual transport ca- * Moles per liter.
pacities according to the method of Lineweaver d Moles per minute per milligram (dry weight).
and Burk (7) were carried out. Uptake of HA-
amethopterin by the wild-type system has already equilibration at 0 C, to allow maximal zero-de-
been shown to conform to typical saturation gree uptake to occur, the rates of transport (up-
(Michaelis-Menten) kinetics (15). After a 5-min take at 37 C) were determined over a concentra-
tion range of 0.3 ,g/ml (0.66 x 10-6 M) to 2

VOL. 93, 1967 TRANSPORT OF AMETHOPTERIN IN MUTANT D. PNEUMONIAE 317

Ag/ml (4.4 x 10-6 M). The results, represented as corresponding differences (among the same Downloaded from http://jb.asm.org/ on August 11, 2016 by guest

double reciprocal plots in Fig. 2, reveal a simi- strains) in the rates of transport at limiting con-
larity in the kinetics of botih mutant and wild-type
systems but with definite q[uantitative differences. centration. On the other hand, values for maximal
The calculated value for tihe Km of the transport transport velocity (Vmax) for the mutant systems
system in each of the mutaLnts (Table 2) was con- were approximately the same as that for the
sistently greater (2.5 x 10-6 to 9.0 x 10-6 M) wild type (Table 2).
than that obtained for the vvild-type system (0.9 X
10-6 M). These differences in the value for Km Transport of H3-amethopterin in the various mu-
could almost be quantitatively compared to
tant and wild-type strains as a function ofpH. The
20 FA results of the kinetic experiments seem best in-
terpreted as a qualitative alteration of the trans-
15 F
port system in the mutant strains. Additional
cli evidence was obtained from a comparison of the
00 pH optimum for transport in the various strains.
Samples of the original culture (10 ml containing
_px> 10
7.7 X 108 cells per milliliter) were adjusted to
5 I0 I5 varying increments of pH and incubated with 0.2

,ug/ml of H3-amethopterin for 10 min at 37 C.
Controls with the same amount of drug were held
at 0 C for the same time. The results are shown in
Fig. 3. As observed previously (15), and shown
here as well, the pH optimum for the wild-type
system was near 6.0. In sharp contrast to this, the
optimum for each of the mutant systems was
shifted to a pH below 5.0. An estimation of trans-
port rates at a pH lower than this was not at-
tempted because of adverse physiological effects
on the cells.

0 5 10 15 DIscUSSION

[AMETH] X105 Functional similarity among the ame-r mu-

FIG. 2. Double reciprocal plot of H3-amethopterin tations examined here was suggested earlier
transport at varying concentrations by wild-type and
mutant strains of Diplococcus pneumoniae. After a by data on cross-resistance and collateral sen-
5-min equilibration at 0 C, 1-ml samples of suspension sitivity to a number of antimetabolites (14).
(7.7 X 109 cells per milliliter) were incubated for 10 The findings derived from the present studies
min with H3-amethopterin at the indicated concentra-
tions. Data shown represents uptake at 37 C, corrected specifically relate these mutations to an impair-
for uptake at 0 C. Strain designation: 0, wild type; ment of the same physiological property, namely,
0, ame-r-J; X, ame-r-2; A, ame-r-7; A, ame-r-9; transport capacity for H3-amethopterin. Indi-
U, ame-r-10; O, AR-16. vidual effects on transport determined by each
mutation reveal a definite correlation with the

corresponding resistance properties (Table 1).
Transport capacity, however, was only reduced by
2.5- to 10-fold by mutations determining a 10- to
100-fold increase in resistance. This lack of pro-
portionality is understandable when one considers
that the total uptake of drug in the wild-type was
a net result of two independent processes, one
physiological with defined kinetic limits and the
other a passive phenomenon of unlimited ca-
pacity, but far less efficient (15). Genetic effects,
as would be expected, were only related to the
first process. The level of resistance determined
by each mutation appears to be a direct reflection
of the additional amount of drug which must be
externally applied to reach intracellular inhibi-
tory levels no longer attainable by active trans-
port alone. Resistance of this type, consequently,
would have theoretical limits, becoming maximal
as the intracellular level maintained by transport

318 SIROTNAK, SARGENT, AND HUTCHISON J. BACTERIOL.

10

CD)

2 Downloaded from http://jb.asm.org/ on August 11, 2016 by guest

4.5 5.0 5.5 6.0 6.5 7.0 7.5

pH

FIG. 3. Effect of the variation in pH on the transport of I-awnethopterin by wild-type and mutant strains of
Diplococcus pneumoniae. Samples (10-mO of suspension (7.7 X 108 cells per milliliter) were incubated with 0.2
,ug/ml of Ha-amethopterin for 10 min at the pH levels indicated. The data represent uptake at 37 C less uptake
at 0 C. Strain designations: 0, wild type; 0, ame-r-1; X, ame-r-2; A, ame-r-7; L\, ame-r-9; U, ame-r-10;

O, AR-16.

is diminished to that level maintained solely by probably at sites within the same cistron speci-
fying the structure of the permease for the trans-
diffusion. port system. Available genetic data from studies
with the ame-r mutations (13) are not incongruous
Comparative kinetic analysis of H3-amethop- with this idea, even though only amer-r-2 and
ame-r-7 have been conclusively mapped in re-
terin uptake in the various mutant strains showing spect to one another within the same region.

an increase in the Michaelis constant (Km) for the The exact role of the permease in active trans-
transport system, but no significant change in port has not been fully defined [see review by
Cirillo (1)]. Cohen and Monod (2) suggested that
Vmax, is taken as evidence for the probable altera- this specific component is probably the active
membrane carrier. Recent findings by Koch (6),
tion in binding properties of a specific compo- on the other hand, support the idea that the
nent, probably a permease, in the transport system. permease is only a specific catalytic component
Other findings related to a concomitant shift in mediating attachment of the transported com-
pH optimum for transport in the same mutants pound to the carrier. In either case, the explana-
tion offered here for the mutational effects ob-
are compatible with this conclusion. Evidence served would appear to be acceptable.
presented elsewhere (15) reveals a remarkable
affinity of this transport component in the wild- The AR-16 mutation is one of many determin-
type system for H3-amethopterin when com- ing resistance to aminopterin mapped by recom-
pared with normal analogues. The mutations binational analysis (3, 9) at the amiA locus in D.
examined here all appear to determine a decrease pneumoniae. The similarity in the effects of the
in this affinity. Any alternate explanation which AR-16 mutation and the five ame-r mutations on
proposes an alteration at some other physiological the properties of a specific transport component
site seems improbable, since an identical effect relates them, functionally, to the same gene locus.
on both transport and diffusion should be ex-
pected. More specifically, to propose an effect on ACKNOWLEDGMENTS
the intracellular receptor, in this case, the "target
This investigation was supported by Public Health
enzyme," dihydrofolate reductase, would seem Service grant CA 08748 from the National Cancer
unacceptable for other reasons as well. Bio- Institute, and by grant T-107 from the American
chemical studies (10, Sirotnak and Hutchison, Cancer Society.

unpublished data) have already revealed the ab-
sence of any effect of these mutations on the

properties of this enzyme. In addition, their
chromosomal location is quite remote (11, 14)
from a region identified earlier as the cistron for
this enzyme. The six mutations, therefore, are

VOL. 93, 1967 TRANSPORT OF AMETHOPTERIN IN MUTANT D. PNEUMONIAE 319

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genotypically distinguishable amethopterin-
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2. COHEN, G. M., AND J. MONOD. 1957. Bacterial
permeases. Bacteriol. Rev. 21:169-194. 11. SIROTNAK, F. M., G. J. DONATI, AND D. J.
HUTCHISON. 1964. Genetic modification of the
3. EPHRUSSI-TAYLOR, H., A. M. SICARD, AND R. structure and amount of dihydrofolate reduc-
KAMEN. 1965. Genetic recombination in DNA- tase in amethopterin-resistant Diplococcus
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15. SIROTNAK, F. M., M. G. SARGENT, AND D. J.
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