SPONTANEOUS BACTERIAL MUTATIONS TO
RESISTANCE TO ANTIBACTERIAL AGENTS
                     S. E. LURIA
Bacterial genetics differs from the genetics of
higher organisms chiefly because of lack of informa-
tion about the mechanism of orderly transmission
of characters from generation to generation, owing
to the absence of well-established processes of con-
jugation and to the infant state of bacterial cytol-
0,~. Our only way of approach is the study of bac-
terial mutability, for which the field of bacterial
variation offers a wide range of material. One of the
first tasks we face is that of establishing how closely
mutations in bacteria are comparable to the so-
called gene mutations in higher organisms. Geneti-
cists as well as bacteriologists have often thought
that the genetic system of bacteria may be less
orderly; in particular, that properties may be trans-
mitted at fission by more or less random distribu-
tion of substances present in the protoplasm, with-
out special mechanisms for equational division.
If a case could be made, however, for similarity
of the processes of mutation in bacteria and in
higher organisms-that is, for the existence of
discrete, gene-like hereditary units in bacteria-
then these organisms might prove to be invaluable
material not only for the study of physiological
genetics, but also for an attack on the problems of
gene structure and mutability (4, 5).
The case would necessarily rest on analogies,
some of which may be listed as follows: (1) per-
manence of the mutated character; (2) "spontane-
ous" occurrence, independent of specific environ-
mental stimuli; (3) definite rate of occurrence; (4)
independence of different mutations in the same
organism; (5) inducibility by agents known to
produce mutations in higher organisms; (6) rever-
sibility, showing that the mutation is not neces-
sarily a loss, caused by unequal division or some
similar process; (7) physiological effects, fumish-
ing proof that bacterial mutations can bring about
the same type of metabolic changes known to be
produced by gene mutations in higher organisms.
One difficulty of bacterial genetics is that of
recognizing a given mutational step, since the same
character may be affected by a number of different
mutations, which we cannot attribute to different
genetic loci by classical genetic tests. Hence the
necessity of identifying each mutation by as many
of its phenotypic effects as possible.
Another peculiarity of bacterial genetics is that
we generally do not deal with the properties of
single individuals, but only with those of large
"clones" (colonies, single-cell cultures), each de-
rived from one individual whose characters must
be retraced from those of the clone. We must there.
fore take into account intraclonal variability. Cases
of high mutation frequency are generally unsuitable
for the study of the mutation process, since they
are likely to be balanced by reverse mutations; our
visible clones will then contain mutant and re
verted types in genetic equilibrium. These con-
siderations have been discussed in greater detail by
Delbriick (8).
More convenient are cases with low mutation
frequency, since these need not necessarily be
balanced by either reversion or adverse selection. In
these cases the mutants can be detected, however,
only if we can select them out in a suitable environ-
ment. Mutants capable of growth in media "in-
complete" for the wild type (22, 19) are in this
category. Other such mutations are those producing
increased resistance to some destructive agent.
We shall discuss in this paper the latter group of
mutations, on the basis of work on bacterial re-
sistance to bacteriophages, antibiotics, drugs, and
radiation (18, II, 1, 2, 9, 24, 21).
ORIGIN OF RESISTANT VARIANTS
The resistant variants are generally isolated as
colonies which appear after the parent strain has
been plated in the presence of a destructive agent.
The question then arises: were the cells that gave
these colonies resistant before the plating, or have
they acquired hereditary resistance as a response to
the action of the antibacterial agent? Unappealing
as the second alternative may be to classical genetic
thought, the question is by no means idle. Con-
siderable evidence has been interpreted as support-
ing the idea that environmental conditions, in
particular nutritional or inhibitory stimuli, may
alter the metabolic machinery of the bacterial cell,
not only temporarily, but in a permanent hereditary
way (14, 13). It may be conceivable that a small
fraction of the cells exposed to an antibacterial
agent respond to its action with a specific change
in properties, and that this change is somehow
transmitted to the offspring.
Mutational origin of the resistant variants, in-
dependent of the action of the destructive agent,
can only be proved either by direct isolation of
resistant substrains before the treatment (7)) or by
establishing the presence within sensitive cultures of
"clones" of resistant bacteria, each clone stemming
from a mutation that occurred during the growth
of the cultures prior to the test for resistance (IS).
If we define mutation rate as the probability that a
E 130 1

BACTERIAL
bacterium will mutate in a given interval of time,
the frequency of mutations will be proportional to
the number of bacteria present. Each mutation gives
Origin to a "clone" of mutants; the earlier the mu-
tation OCCUTS, the larger the clone derived from it
will be at the moment of the test. If the. mutants
sow at the same rate as the normal bacterra, clones
of a certain "size" will be twice as frequent as those
of double that size. The expectation should be,
therefore, that, of the mutants present in a culture
at any one time, equal numbers stem from muta-
tions that occurred in each of the previous genera-
tions. This situation, however, is never realized in
actual cases. In any actual culture, the likelihood
MUTATIONS                131
same culture. A detailed analysis of these considera-
tions has been given by Luria and Delbriick (18).
These expectations can best be tested in the
case of resistance to bacteriophages. The phage-
resistant variants are easily detected and counted in
a mixture with sensitive cells; they neither grow
the phage nor adsorb it, and their resistance is a
permanent new character of the strain.
As an example, Table 1 (from 18) shows the
results of the enumeration of bacteria resistant to
phage Tl in platings from similar cultures of
Escherkhia co.%, strain B. The presence and type
of fluctuations in the frequency of mutants provide
strong confirmation of the mutational origin of re-
TABLE 1. DISTRIBUTION OF THE NUMBERS OF BACTERIA RESISTANT TO PHAGE Tl IN SERIES OF SIMILAR CULTURES
                        OF SENSITIVE BACTE~UA
   Experiment No.
Number of cultures
Volume of cultures, cc.
Volume of samples, cc.
22                       23
100                     87
2:          1; *
Resistant
Bacteria
  0
:
i
Al
Resistant     Number of'
Bacteria       Cultures
  0           29
  1
3     17 4
610 5     :
      3
11-20
21-30     i          11-20        6
                       21-50
51-100     :         51-100     :
101-200                    101-200
201-500          0         201-500     i
501-1000          1         5014000          0
Average per sample
Variance (corrected for sampling)
Average per culture
Bacteria per culture
%t                    28.6
                       6431
40.48                    28.6
2.8 x 10"                 2.4 X 10'
* Cultures in synthetic medium.
of occurrence of early mutations is small, because
not enough individuals are present. When the popu-
lation reaches a size where mutations begin to occur,
the frequency of their occurrence will be subject to
tremendous fluctuations owing to their small aver-
age number. In a large number of trials (series of
similar cultures), therefore, we must expect to find
COrresponding large fluctuations of the frequency
of mutants; the presence of these fluctuations is a
direct consequence of the clonal distribution of the
mutants. When the proportion of variants in a
series of similar cultures shows such large fluctua-
tions! we may interpret them as proof of a clonal
distnbution, hence of a mutational origin of the
wiants.
Such fluctuations among similar cultures would
not be expected if resistance were actively produced
hy the antibacterial agent. The fluctuations should
not be greater than those among samples from the
sistance. A refinement of this type of analysis would
be afforded by a comparison of the actual distribu-
tion of the number of variant individuals with a
distribution calculated from the assumptions of the
mutation hypothesis. Mathematical difficulties have
been encountered, however, in attempts to calculate
the theoretical distribution.
It is important to notice in Table 1 (Exp. 23, in
which the whole contents of the cultures were
tested) the presence of large numbers of cultures
containing one mutant only. This suggests that the
spontaneous mutations may be phenotypically ex-
pressed without a lag longer than one cell genera-
tion.
The "fluctuation" test has been' applied to a
variety of cases-resistance of EC Goli B to several
phages (II) and to radiation (24) ;` resistance of
Staphylococcus 3 13 to penicillii (9)) and to sulfa-
thiazole (II)-with results of the same type, fndicat-

132
S. E. ZURIA
hg that in all these cases resistance arises as the're-
sult of mutations occurring before the test. The data
of Lewis (16) on Escherkhk cold mutabile show
that similar fluctuations between cultures were pres-
ent in the numbers of lactose-fermenting cells. Large
fluctuations in the time of appearance of fermenta-
tion variants were also present in experiments of
Kristensen (15). The mutational origin of bacterial
variants is well supported by all these observations.
The type of test described above shows only that
the variants were already a genetically modified type
before the test. It is possible that in some cases the
specific environment, besides selecting out the mu-
tants, may also act by rendering phenotypical a
new character otherwise masked by some cytoplas-
mic effect. In a sense, this may be considered to be
true of mutations affecting the production of adap-
tive enzymes. In the case of mutations to resistance
to antibacterial agents, similar instances might be
difficult to detect.
MUTATION RATES
In work on bacteriophage resistance, Luria and
Delbriick (18) have chosen as a definition of the
mutation rate the probability of mutation per bac-
terium per physiological time unit (generation).
This detition is satisfactory as long as it can be
proved that mutations occur only during growth
of the bacteria, and that their frequency is propor-
tional to the physiological rather than to the as-
tronomical time. These conditions appeared to be
fulfilled in our experiments on phage resistance
(mutation rate independent of generation time in
different media, no increase in mutants after maxi-
mum growth).
Several methods have been proposed for estimat-
ing the mutation rate. Since mutations occur in-
dependently of each other, when there are only a
few they will be distributed among different cultures
in a Poisson distribution, with the probability of any
given number of mutattons occurring in one culture
depending only on the average number of muta-
tions per culture.
The average number of mutations per culture,
and hence the mutation rate, can be calculated from
the number of cultures having no mutants and from
the number of bacteria per culture. If. in a series
of C simiir cultures, each containing N bacteria,
a fraction C& proves to contain no mutant (the
whole contents of the cultures must obviously be
tested), the mutation rate, a, can be obtained:
(1)     In 2 x In (G/C)
   a=-       N
This method, although it requires careful plan-
ning of the experiments, has the advantage of not
introducing the actual numbers of mutants and
thus avoiding complications due to possiile selec-
tion.
Another method that allows direct measurement
of mutation rates without making use of the num.
hers of mutants consists in applying the antibac.
terial agent (phage) to bacteria growing on solid
medium without disturbing the formation of micro.
colonies (20). One colony appears for each mute-
tion that has occurred. The difficulty here lies b
precise estimation of the number of bacteria pres-
ent at the moment of the test.
Mutation rate can also be calculated from the
actual number of mutants, estimating from this the
number of clones present. One must take into so
count that in cultures of limited size mutations are
likely to occur only during the last part of their
growth. If, for example, the mutation rate is 10-T,
mutations are not likely to occur, in actual Cultures,
until the population reaches a size of the order of
10' cells. Introducing the necessary corrections, the
mutation rate can be estimated from the average
number, r, of mutants from C similar cultures:
            aNC
(2) r=EhrF
A diagram for the use of this formula has been
given by Luria and Delbriick (18). Mutation rates
calculated by formula (2) are likely to be in-
accurate because of the fluctuations in frequency
of occurrence of the early mutations; in particular,
they will be too high whenever one or more of the
unlikely early mutations happens to occur. Impor-
tant corrections should be introduced if the growth
rates of normal and mutant types are different.
Altogether, our present methods of estimating mu-
tation rates are far from satisfactory.
Some values of mutation rates calculated with
formulas (1) and (2) are given as examples in
Table 2. These values are subject to the further
limitation that all mutations giving the same pheno-
type for the character investigated are lumped to-
gether, and that in some cases it is likely that not
all mutants present in a sample are detected. The
orders of magnitude, however, are probably correct.
Even for the same wild-type strains, mutation
rates for different mutations to resistance vary over
a wide range. That they are generally very small
is to be expected; in searching for stable resistance
to destructive agents, we particularly select for rare
mutations, since if stable mutants occurred very
frequently they would generally have displaced the
parent type, unless checked by strong adverse
selection. Much higher mutation rates may he
found for mutants that also show high rates of re-
version.
INDEPENDENT MUTATIONS
It has already been mentioned that the same by
terium can undergo different mutations. This 1s
clearly seen in the case of resistance to bacteria-
phages. Working with Escherichia co& B and bsc-
teriophages Tl-T7, one finds a series of bacterial
mutations, each producing resistance to one, two,

BACTERIAL MUTATIONS                     133
0r more phages. Indicating a mutant from B that is
,esistant to phage n by the symbol B/n, we have
ior instance the mutants: B/l; B/l, 5; B/6; B/3,
) 7. From each of these mutants one can obtain
&cr mutants with additional resistance to other
@ages: B/l/6; B/6/3, 4, 7, etc.
The successive mutations are clearly independent.
4 strain that has undergone a certain mutation will
&rally give the same mutations to resistance to
zther phages as the parent wild-type strain. Within
the limits of precision of the available methods of
measurement, the rates for the same mutation in
the parent strain and in any of its mutants appear
could decide by what mechanism resistance to un-
related phages is produced in one mutational step.
The answer to this question might be of the greatest
importance, also, in relation to the mechanism of
bacteriophage action.
A possible interpretation has been suggested by
E. H. Anderson's work on the nutritional require-
ments of some phage-resistant mutants (I, 2).
Anderson found that a number of B/l mutant
strains from Escherichia coli B differed from the
wild type also in being unable to grow in a mini-
mal synthetic medium. The metabolic disturbance
was identified as inability to synthesize Ltrypto-
TABLE 2. MUTATION RATES
Organism
Escherichia coli B
Staphylococcus aureus 313
=
--
Mutation            Reference
Resistance to phage Tl
Resistance to phage Tl
Resistance to phage T3
Resistance to phage T6
Resistance to radiation
Resistance to penicillin, one-step
Resistance to sulfathiazole, one-step
to be equal. For example, the mutations B + B/l,
and B/6 -+ B/6/1 occur with the same frequency
(11).
The same holds true for other types of mutation
as well. The mutant B/r resistant to radiation (24)
has the same pattern of mutability to phage re-
sistance as the wild-type B.
These results provide evidence of the independ-
ence of different mutations in bacteria, and agree
with the hypothesis of the existence of discrete,
mutable, gene-like units.
MULTIPLE EFFECTS OF MUTATIONS
In cases of resistance to drugs and antibiotics,
the resistance is often very specific; cross-resistance
is limited to related substances. In the case of phage
resistance, however, multiple resistance to several
phages may appear as one mutational step. The
phages to which resistance can be produced by one
mutation are often unrelated as judged by their
size, structure, serological specificity, and growth
characteristics (8). It is easy to prove, on the basis
of the mutation rates, that the multiple resistance
does not result from chance occurrence of two or
more independent mutations in the same bacterial
line. Moreover, resistance to the same group of
phages can be reached in one or two mutational
steps. It is clear, therefore, that tests of resistance
to a limited number of phages are not sufficient to
characterize the genotypes of a group of bacterial
mutants.
It would be an important step forward if we
Mutation Rate per Bacterium
   per Generation
         40-7
-10-g
phane, which therefore became a required nutrilite
for these strains. Moreover, these strains exhibited
another disturbance in their nitrogen metabolism,
reflected by their inability to utilize inorganic nitro-
gen in the absence of at least one of a series of
amino acids.
Additional inability to synthesize the amino acid
proline was found by Anderson in a strain B/1/3,
4, 7. Other phage-resistant strains that fail to
grow in a minimal medium have also been en-
countered.
Anderson (1) interpreted his findings as indicat-
ing the existence of common steps in the chains of
reactions leading to the synthesis of an essential
metabolite and of substances needed for phage
sensitivity, the mutation blocking one of the en-
zymatic reactions. As an extension of this inter-
pretation, it was suggested that multiple resistance
to various phages acquired in one mutational step
results from the blocking of a reaction common to
the syntheses of substances needed for growth or
adsorption of the phages. Common steps could oc-
cur in the chains of reactions leading to sensitivity
to unrelated phages. Different mutations producing
resistance to more phages would probably produce
blocks at different levels of the series of reactions.
Since bacteria resistant to a certain phage are
often sensitive to some mutants from that phage,
the "blocks" in the chains of synthetic reactions
are likely to alter, rather than suppress completely,
the synthesis of substances necessary for phage
sensitivity.

134                        S. E. LURIA
It was thought (17) that the mutational changes
might simply involve slight alterations in the con-
figuration of the bacterial receptors for phage ad-
sorption, which were supposed to react with the
phage by simple coming together of complementary
surface structures. Mutational alteration of the
surface receptors could be brought about either by
a primary change in the receptor itself or by an
alteration in the structure of a gene-like center act-
ing as template for the receptor, without involve-
ment of any long reaction chain. The association
of specific metabolic deficiencies with phage resist-
ance seems to contradict this idea. Moreover, the
fmding of T. F. Anderson (3) that certain amino
phage-resistant mutant of the usual type. Unfor-
tunately, the relation between the two types of
resistance was not thoroughly analyzed. The in-
dications were, however, that phage resistance in
the acridme-resistant strain was caused by a change
in an acridine-sensitive mechanism involved in
phage growth. Interestingly enough, both antiphage
action of the acridines and phage resistance of the
acridine-resistant strain could be antagonized by
the addition of ribose nucleic acid.
The idea that the usual type of phage resistance
is due to a block in a chain of synthetic reactions
would be convincingly proved if it were possible to
restore sensitivity to a resistant mutant by supply-
TABLE 3. ONE-STEP MUTANTS
Phenotype
Wild type
Frequent
mutants
   a
  b
Ii
;
Complex
mutants
f
f
Fl    ;i;s     T2 T6 T4
                   *
T3 T7   T7h   T7h'
rrypto-
phane
lequire
ment
-
+
-
-
+
i
kneration
Time,
Minutes,
in Broth
at 37" C.
19-20
19-20
19-20
19-20
25-26
25-26
29-30
29-30
28-29
28-29
40
* The braces indicate serological relationship between phages.
acids may act as necessary cofactors for phage
adsorption indicates that the latter process is likely
to involve some rather complex enzymatic activity
We found recently that some of the B/3, 4, 7
of the phage particle on the bacterial surface.
mutants, besides not adsorbing phages T3, T4, and
T7, also show a partial inability to grow phages
T2 and T6. They adsorb these phages like normal
wild-type bacteria, but 80-90s of the infected
bacteria fail to liberate any phage at all. Similar
observations were also made by Hershey (personal
communication). This phenomenon, which needs
further investigation, indicates a relation between
adsorption capacity and ability to grow phages,
and supports the idea that both phenomena in-
volve chains of reactions variously linked.
Additional evidence for relating phage resistance
to specific changes in enzymatic reactions is offered
by recent results of Fitzgerald and his collaborators
(22). It was found that some acridines exerted a
specific inhibition on phage growth in de bacterial
cell, pnd that a variant resistant to one of the
acrldmes was almost as resistant to phage as a
ing its medium with the intermediate metabolite
whose synthesis is supposedly blocked, and whose
Some additional information on multiple resist-
absence is assumed to be responsible for the phage
ance has been derived from a category of rare mu-
tants with unusual combinations of resistance to
resistance. Such cases have not yet been found.
various phages (11). We have recently isolated a
number of these "complex" mutants, and attempted
a further characterization of their phenotypes, com-
paring them with those of the more common my-
tams. This was done by taking into account sensl-
tivity to phages Tl-T7 and to some of their host-
range mutants (active on some of the resistant
bacterial mutants), nutritional requirements, and
growth rates, which will be considered later. The
complex mutants were isolated by plating large
numbers of sensitive bacteria in the presence of
suitable mixtures of phages. Most of them occur
with mutation rates of the order of lo-lo. The re-
sults of some of these tests are summarized in
Table 3.
We see that certain basic groupings of characters

BACTERIAL MUTATIONS                    135
awear in the phenotypes of both simple and com-
plex mutants: resistance to phages Tl and TS; to
ohages T3, T4, and T7; resistance to phage Tl and
tryptophane requirement. All of the complex mu-
tants incapable of growing in minimal medium
were found to have the same tryptophane require-
ment as the B/l mutants, although some of them
mro\v more poorly in the presence of the same
imount of the amino acid.
The coupling of resistance to phage T2 and to
@age T6, as present in the complex mutants, is
not shown by any of our simple mutants. This seems
due to the fact that all the frequent mutants isolated
from our B strain in the presence of phage T2 prove
sensitive to this phage after growth in phage-free
media, owing to some as yet unclear mechanism of
FIG. 1. Chains of synthetic reactions needed to account for
the coupling of resistance in the mutants of Table 3. The
symbol X in each case indicates the block corresponding to
the mutant phenotype indicated by the adjacent letter. The
broken lines indicate the coupling of resistance to T3, T4,
T7 with failure to grow T2 and Td.
partial reversion to sensitivity. Thus most of the
B/Z, 6 strains show an apparent B/6 phentoype
(phenotype c, Table 3).
The groups of characters shown by the "simple"
mutants appear in the complex mutants, associated
in a number of ways but not in all possible ways.
For example, resistance to phages T3, T4, T7 was
found associated either with resistance to Tl and
TS (phenotype h) or with resistance to Tl and
tryptophane requirement (phenotype g), or with
resistance to T2 and T6 (phenotype i). Resistance
to phages Tl and TS was not found associated with
Qptophane requirement.
Some of the complex phenotypes are clearly a
superposition of two of the simpler ones: phenotype
g is like phenotypes a + f; phenotype h, like
b + f. Other complex mutants differ from the sum
of two simple ones in some character, particularly
in sensitivity to various mutants from T7.
It may be possible to account for these various
combinations of characters, according to Ander-
son's scheme, on the basis of blocks or changes in
common steps of different chains of synthetic re-
actions, assuming that each phenotype corresponds
to a different synthetic block; the situation, as
shown in the diagram (Fig. l), becomes quite com-
plicated. One must assume that there are a number
of different reactions common to the chains leading
to sensitivity to certain groups of phages, and that
some of these reactions are shared by different
chains of reactions leading to sensitivity to other
phages. Further complications are revealed by dif-
ferences in sensitivity to various mutants of the
T7h group, indicating either a greater multiplicity
of pathways, or different alterations of the same
reaction. Each of the numerous reactions assumed
in order to explain multiple resistance to such a
small number of phages must be capable of being
altered by a nonlethal mutation. We shouuld also
assume that the rare frequency of occurrence of all
the more complex mutations is purely coincidental.
One way out of some of these difhculties would
be to suppose that some or all of the very complex
mutations are actually combinations of two or more
simple mutations occurring simultaneously. Such
simultaneous occurrence might be brought about by
some type of mass rearrangement of the genetic
material of the bacterial cell, possibly comparable
to that responsible for chromosomal rearrangements
in higher organisms.
Altogether, we feel that the problem of multiple
effects of a bacterial mutation cannot yet be in-
terpreted satisfactorily by any single hypothesis.
Recognition of a larger number of complex mutants,
their characterization by as many traits as possible,
study of the physiology of the changes involved,
and work on reverse mutations should help clarify
the still obscure situation. Cases of simultaneous
acquisition of several growth requirements may
offer good material for this study.
QU.~NTITATIVE RESISTANCE
In studying the acquisition of resistance to anti-
biotics (penicillin, 9) and drugs (sulfathiazole, 21),
it has been found that resistance to increasing con-
centrations of an antibacterial agent is built up
stepwise by accumulation of successive mutations,
each contributing further resistance, until strains
are obtained that for all practical purposes can be
considered as completely resistant. Mutants isolated
by the selective action of a certain concentration
of an agent are often resistant to much higher con-
centrations. Supposed correspondence of the re-
sistance level with the concentration used in the
"training" to an antibacterial agent has been con-
sidered (13) as one proof of the active induction of
resistance. The actual lack of such correspondence

S. E. LURIA
is corroborating evidence of its spontaneous, muta-
tional origin. The successive mutations to resistance
might represent either similar changes in a series
of gene-lihe units, or successive changes in the same
unit, or independent genetic changes each affecting
one of the metabolic processes on which the anti-
bacterial agent may act.
A study of sulfonamide resistance in Staphylo;
coccus (21) indicates that the last alternative is
probably correct. A number of different mutations
are involved in acquisition of thii resistance, most
of the recognized ones occurring at comparable
rates (between 1CF' and N-lo). These mutations
csn be differentiated first of all by the degree of
resistance produced, expressed by the drug con-
centration withstood by the mutant. Even more
important is the fact that different mutants with
increased sulfonamide resistance differ in other cor-
related characters, particularly in the production
of an extraceMar sulfonamide antagonist appar-
ently identical with paminobenzoic acid. Some of
the resistant mutants release during growth up to
30 times more p.a.b. than the parent strain, while
others do not produce, any increased amount of it.
The same mutation affecting p.a.b. production may
occur at the same rate either in the parent strain
or in mutants with already increased resistance but
without increased p.a.b. production.
The simplest explanation is that sulfonamides act
as inhibitors of a number of metabolic reactions
(whether all involving p.a.b. or not is immaterial
for our problem). Successive mutations to resistance
produce changes in different enzymatic mechanisms,
the residual level of sensitivity being determined
by the most sensitive mechanism left.
In the case of penicillin resistance (9)) the oc-
currence of mutations producing different degrees
of resistance indicates a similar situation. That
penicillin affects a number of different enzyme sys-
tems of the bacterial cell is indirectly indicated by
data of Treffers (23) on growth inhibition brought
about by associations of penicillm with each of a
number of metabolic poisons in noninhibitory
EUIlOUIItS.
The acquisition of resistance to drugs and anti-
biotics by mutation in bacteria seems, therefore, to
fall in line with the idea of genetic determination
of single enzyme reactions by individual genes. It
is interesting to note that, in the case of resistance
to radiation in E. co23 B, Within (24) found only
one mutational step increasing resistance. All at-
tempts to select mutants having higher resistance
failed. It is likely that only one of the synthetic
mechanisms responsible for sensitivity to radiation
can be modified by a nonlethal genetic change.
The increased production of p.a.b. by sulfona-
mide-resistant mutants of Staphylococcus exempli-
fies a metabolic change increasing the extracellular
production of a metabolite. P.a.b. is not a required
metabolite, and is normally produced by the organ-
km. It is possible, on the one hand, that p.ab. ac-
cumulates because of a block in a sulfonamide-sensi-
tive reaction that utiliies p.a.b. Provided an altema-
tive metabolic pathway for growth were available,
such a block would be expected to produce sulfona-
mide resistance. It is possible, on the other hand, that
an actual increase in p.a.b. synthesis is caused by the
mutation. That positive biochemical mutations may
be responsible for resistance to antibacterial drugs
seems indicated by the increased formation of
pantothenate by C. diptctheriae in the course of its
becoming pantoyltaurine-resistant and independent
of pantothenate for growth (20).
   GROWTH OF T= RESISTANT MUTANTS
The resistant mutants often show definite dif-
ferences in growth characteristics from the sensitive
wild-type parents, which can be used.ss additional
criteria for identification of a given mutational step.
In the case of phage-resistant mutants, some mu-
tations are correlated with definite changes in
growth rate (generation time). Some of the data
for the growth of one-step mutants in nutrient
broth at 37O C. are included in Table 3. The growth
rates of the mutants range from the same value as
for wild-type to values more than twice as great.
These differences again indicate that the mutations
produce profound changes in the metabolic pro-
cesses of the bacterial cell.
It was found that when successive mutations are
accumulated in the same bacterium the growth rate
is always determined by the mutation that by itself
gives the slowest growth.
We can see in Table 3 that those complex mu-
tants that appear to be the superposition of two
simple mutant types also have the same generation
time as the slower simple mutant type included.
This agrees with the suggestion that they actually
originate by combined mutations. Others of the
most complex mutants have a much slower division
time, probably indicating a more profound altera-
tion of their synthetic abilities.
The effects on growth of mutations to resistance
are not liited to changes of generation time in the
logarithmic phase. All phases of growth may be
affected Among mutants resistant to phages or to
penicillin, we found some having longer or shorter
lag phases, others with higher or lower maximum
titers than the wild-type strain.
Of a large number of mutants whose growth was
measured under comparable conditions, a majority
grew less well than their wild-type parents. Only s
few seemed to grow as well or better. This seems to
agree with the general principle that most muts-
tions arising in well-established wild-types produce
less favorable phenotypes.
Complications may arise, however, when one at-
tempts to predict the growth of parent and mutant
strains in mixtwes on the basis of their growth in
isolated cultures. Production of diffusible growth

BACTERIAL MUTATIOiVS                     137
$-&itOrS 01 StimUhtS, Or diffeRIltid Kh!S Of
utilixstion of some nutrilite, may greatly alter the
picture. For instance, we found that the viable
counts of a penicillm-resistant mutant in mixed
cuiture with the parent strain began to fall long
before the end of the growth phase for separate
cultures of either strain. Other instances of interac-
tions of this type have also been encountered. In
different phases of the life of a bacterial culture
either the parent type or its mutants may be
favored, and only detailed study of individual cases
can clarify the most complex situations. The indica-
tions are, however, that many cases of bacterial
variation, including some of the most complex types
of so-called life cycles, can be explained in terms
of the simplest hypothesis-mutation followed by
selection within the resulting mixtures of pheno-
types (6).
SUMMARY
Bacterial mutations to resistance have provided
favorable material for the quantitative study of
bacterial mutability. The mutations discussed in
this paper present suggestive analogies with gene
mutations in higher organisms, although the. similar-
ity of the genetic systems involved can only be
considered, for the time being, as a useful working
hypothesis. The problems of determination of mu-
tation rates, of identification of single mutation
steps in strains with different genotypes, and of
multiple effects of mutations have been discussed.
1.
2.
3.
4.
5.
6,
7.
         REFERENCES
ANDERSON, E. H.  Incidence of metabolic changes among
virus resistant mutants of a bacterial strain. Proc. Nat.
Acad. Sci. 30: 397-403. 1944.
ANDERSON, E. H.  Growth requirements of virus resistant
mutants of Escherichiu coli strain B. Proc. Nat. Acad.
Sci. 32: 120-128. 1946.
ANDERSON, T. F. The role of tryptophane in the adsorp-
tion of two bacterial viruses on their host, Esckerichiu
coli. J. Ceil. Comp. Physiol. 25: 14-26. 1945.
AVERY, 0. T., MACLEOD, C. M. and MCCARTY, M.
Studies on the chemical nature of the substance inducing
transformation of pneumococcai types. Induction of
transformation by a desoxyribonudeic acid fraction iso-
lated from Pneumococcus Type HI. J. Exp. Med. 79:
137-1.58. 1944.
BOMN, A., DELAUNAY, A., VENDRELY, R., and LEHOULT,
Y. L'acide thymonu&ique polymer%, principe parais-
sant susceptible de d6terminer la speciiicit6 skologiqua
et l'equipment enxymatique des bact6ries. Signification
pour la biochimie de i'h6r6diti. Experientia 1: 334-335.
194s.
BRAUN, WERNZR. Dissociation in B*uce&z abortw: a
demonstration of the role of inherent and environmental
factors in bacterial variation. J. Bact. 51: 327-349. 1946.
BURNET, F. M. Smooth-rough variation in bacteria in
its relation to bacteriophage. J. Path. Bact. 32: 15-42.
1929.
8. DFJ,BR%X, M.  Spontaneous mutations of bacteria. Ann.
Missouri Bot. Garden 32: 223-233. 194%
9. Dxacxaxc, M. Production of Staphylococcus strains
10.
11.
12.
13.
14.
1.5.
16.
17.
18.
19.
20.
21.
22.
23.
24.
resistant to various concentrations of peniciiiin. Proc.
Nat. Acad. Sci. 31: 16-24. 194s.
DEMEREC, M. Induced mutations and possible mecha-
nisms of the transmission of heredity in Eschet-ichM coli.
Proc. Nat. Acad. Sci. 32: 36-46. 1946.
D-EC, M., and FANO, U. Bacteriophageksistant
mutants in Ercherichia coli. Genetics 30: 119-136. 1945.
FITZGERALD, R. J., and LEE, M. E. Studies on bacterial
viruses. II. Observations on the mode of action of
acridines in inhibiting lysis of virus-infected bacteria.
J. Immunol. 52: 127-135. 1946.
HINSEIELWOOD, C. N. Bacterial growth. Biol. Rev. 19:
150-163. 1944.
KNIGHT, B. C. J. G. Bacterial nutrition. Material for a
comparative physiology of bacteria. Spec. Rep. Ser.
Med. Res. Coun. No. 210. 1936.
KRISTENSEN, M. Recherches sur la fermentation muta-
tive des bactkes. Acta path. microbial. stand. 17: 193-
231. 1940.
LEWIS, I. M. Bacterial variation with special reference
to behavior of some mutable strains of colon bacteria in
synthetic medium. J. Bact. 28: 619-639. 1934.
LURIA, S. E.  Genetics of bacterium-bacterial virus reia-
tionship. Ann. Missouri Bot. Garden 32: 235-242. 194.5.
Luam, S. E., and DELBR~~CK, M. Mutations of bacteria
from virus sensitivity to virus resistance. Genetics 28:
491-511. 1943.
LWOFF, A., and AUDUKZAU, A. Sur une mutation de
Morazella kuofi apte a se dCvelopper dams les milieux a
I'acide succinique. Ann. Inst. Past. 67: 94-111. 1941.
MCILWAIN, d. Nutritional studies of bacterial varia-
tion. IL The derivation of drug resistant strains in the
absence of any inhibitor. Brit. J. Exp. Path. 24: 212-217.
1943.
OAKBERC, E. F., and LURIA, S. E.  Mutations to sulfona-
mide resistance in Staphylococcus uureus. Genetics, in
Press.
PARR, L. W. A new `mutation" in the coiiform group
of bacteria. J. Hered. 29: 380384. 1938.
Txx~xxas, H. P. The potentiation of penicillin or
streptomycin action by certain enzyme inhibitors. J.
Bact. 52: 502-.X& 1946.
WITKIN, E. M. Inherited differences in sensitivity to
radiation in Eschetichia coli. Proc. Nat. Acad. Sci. 32:
59-68. 1946.
DISCUSSION
KIDD: I wonder if Dr. Luria has seen a resem-
blance between the complex races of phage-resistant
bacteria with which he has been working and the
strains of drug-fast trypanosomes which have inter-
ested parasitologists since the time of Ehrlich. While
the genesis of the drug-fast strains of trypanosomes
has not perhaps been particularly well studied as
judged by the criteria of critical quantitative genetics,
the fact has long been known that they represent
more or less stable heritable variations in microor-
ganisms. Furthermore, the work of Warrington Yorke
and his collaborators and that of Jan& and others
has made it abundantly plain that the lethal effects
of drugs on trypanosomes depend upon their fina-
tion by the parasites: arsenical and acridine com-
pounds, for example, are absorbed by strains of
normal trypanosomes, which then succumb to their

138                        S. E. LURIA
effects; but the compounds are not absorbed by the
. drug-fast strains, which in this way resist their ac-
tion. From a study of the phenomena of drug re-
sistance, moreover, Frank Hawking has distin-
guished four different kinds of receptors in tryp-
anosomes-those for arsenicals and acriflavine, for
parafuchsin, for diamidine compounds, and for
Bayer ZOS-and he has studied the relationships
among them.
It seems to me that the parasitologists have
managed to acquire a noteworthy comprehension of
the phenomena with which they were confronted-
the practical implications for chemotherapy in-
cluded-and it may become manifest that the prin-
ciples involved are much the same as those with
which Dr. Luria is dealing.
SONNEBORN: The finding of a single mutant
colony when an entire culture is plated out need
not .necessarily indicate that the mutation was
brought to phenotypic expression at once in the
absence of further cell reproduction. Suppose the
mutation had occurred several cell generations
earlier, with a lag of some cell generations between
mutation and phenotypic expression and with varia-
tion among the progeny of the mutated cell in the
number of cell generations preceding phenotypic ex-
pression. In Paramecium, for example, precisely
such variation is observed. Under such conditions
a single descendant cell might resist phage and the
others be destroyed. It would then be diicult to
distinguish thii from cases of immediate phenotypic
expression.