PN PACE
interactions during the growth of mutating PUY \

poputations of bacteria. \ |

Adaptation in its most general sense means a satisfactory
adjustment to the environment. It is a behavior frequently found
among micreorganisme. In the red bread mold, Neurospora, it has
been possible to show that adaptation can involve genic mtation
(1), A diochemical mutant of Neurospora, requiring a supply of
leucine for growth, can back-mutate to dispense with that require-
ment and grow in the absence of leucine, Such adaptation is com
plicated by the fact that, in the heterocaryon formed by baeke
mutation, the deficient leucine-dependent nuclel may, under certain
conditions have a selective advantage over the leucine-independ-
ent back-mutated nuclei, Attempts to discover the mechanism of
this selection or competition of nuclei in Neurospora have not met
with success,

Since there were clues in the literature that a similar sel-
ection in favor of biechemically deficient cells occurred among
bacteria, it was thought that a mechanism which operated through
culture medium might be more easily studied than one which acted
through the cytoplasm of a heterocaryon, Therefore, an investige
ation of adaptation in biochemical mutants of bacterla was begun.
The organiam chosen was a mutent strain of Becherichia coli which
requires « supply of histidine for growth. It was secured after
X-radistion (2) and will be referred to as he. After purification
by repeated isolation from single colonies, a stock culture was
established on agar medium containing histidine. The growth of

this organiem was studied in stationary test tubes each containing
(2)

10 ml. of a synthetic medium. Unless otherwise mentioned inocula
consisted of from 10° to 10°
tical ddnsity.

cells and growth was measured as op~

On different concentrations of histidine the results shown in
Pig. 1 were obtained. After 10 hours there is no growth in the
absence of histidine but increasing amounts of growth occur with
increasing concentrations of histidine. The upper curve is for an
optimum concentration. After about 17 hours adaptation occurs.
Cultures without any histidine show complete growth but with im
ereasing concentrationa of histidine adaptation ie progrdasively
less complete. At intermediate concentrations adaptation does not
occur even after 7 days. When an adapted culture is washed and
plated into agar gnd synthetic medium devoid of histidine, colonies
were formed. These colonies, when isolated and transferred to
liquid medium without histidine, grow like wild type Ee coli, i.e.
without the long initial period of the h- strain. These new hist-
idine-~independent cultures, which are called he, also differ from
he cultures inaemich as they will form colonies on agar devoid of
histidine while h- cells, even after 7 days, will not form colonies
visible to the naked eye. Partially adapted cultures can be shown
in a similar way to contain smaller numbers of he cells, All of
these he cells have arisen from the he cells by “nmtation", The
adapted growth may be due to he cella already present in the rather
large ineculum but it is also posaible to have the same type of
adsp tation after the inoculation of a single he cell into medium
without histidine. Adaptation then 1s due to a change in the syn-~
thetic capacities of the cell or to mitation,
(3)

When the amounts of growth achieved after 11 hours and after
29 hours are plotted against histidine concentration the curves
shown in Fig. 2 are obtained. The decreasing amounts of adaptation
that can occur with increasing concentrations of histidine are
clearly shown. This is the same type of relationship that was
found in leucineless Neurospora. Since it can be shown by plating
out, that there are he cells in the incompletely adapted cultures
the problem is to discover what prevents these celle from contin-
uing growth, We might assume, as in the case of leucineless Reur-
ospora, that the he organisms are prevented from growing by the
presence of too many h- cells. We know that the proportion of h-
cells in these cultures which never adapt is greater than 9S per-
cent. There are progressively smaller numbers and percentages of
h- cells as adaptation is more complete.

It is possible to eliminate the depression of growth which
occw's on intermediate concentrations of histidine by decreasing
the percentage of he cells in the inoculum. ‘When the inoculum cone
esiste almost entirely of h¢ cells there ts nq depression of crowth.
Consequently we conclude thet this depression is brought about by
the h- cells. The proportion of h- and he celle remains about the
seme ae in the inoculum when growth is allowed to take place in the
presence of an optimum amount of histidine. Under these conditions,
then, the growth characteristics of the h~ and the he bacteria
are the same. It is only on limiting concentrations of histidine
that selection of the he cells takes place during adaptation and

the ratio introduced in the inoculum changes. But even
(4)

though selection is in favor of the he cells we know that they are
eventually inhibited by the he bacteria,

We have been able to reveal some of the factors involved in
this inhibition. Under the conditions of our experiments we know
that acid production is proportional to growth. This resulte in
varying reductions in the pH of the culture medium After adapte
ation is complete hydrogen ion concentration is limiting, for when
the pH is brought back to 7 erowth resumes. The addition of hist~
idine, or other components of the medium, will not tring sbout a
reinitiation of growth. But the limiting pH is different for
different histidine coneentrations. It is highest at the intermed-
late concentrations where growth is most depressed. One hypothesis
which explains this assumes that an inhibitor is formed to varying
extents by the he cells with maximm production at intermediate
histidine concentrations, Where the least amount of growth has
eceurred after 2 hours (Fig, 2) and where the pH is decreased during
growth only to 6.54, there would be the maximum amount of inhibitor.
Thies situation would parallel the accwmlation of precursers by
some biochemical mutants of Neurospora which can occur only in ine
termediate concentrations of the required growth factor (3).

Although the inhibitor (8), has not b-en characterized there
are several lines of evidence indicating that it existe. Cultures
of he bacteria, allowed to adapt on different histidine concentrat-
ions were sterile filtered, brought to om tn the presence of an

optimum amount of histidine and inoculated with either he or he
(5)

bacteria, The amount of growth secured was a function of the hist~
idine concentration on which the bacteria had been allowed to ad~
apt. Culture filtrates from those concentrations which supported
the leas® growth, although their pH was brought to 7 and they were
supplemented with an optimum amount of histidine, allowed the least
growth of the he and h- cells with which they were reinowulated.
fhe amount of new growth supported was proportional toe the amount
of growth which had been allowed on the original histidine concen-
trations, Once again where the least new growth occurred the pH
was brought to pH 6.5. The limitation of this new growth was not
due to the presence of an inhibitor (#) which was in greatest con~
centration in those filtrates which had originally contained inter~
mediate histidine concentrations, This inhibitory effect is not
destroyed by heat. In another series of experiments the phosphate
concentration and buffer capacity of the medium was increased. WNever-
theless, at intermediate histidine concentrations the same amounts
of growth were obtained on the different media — and thie despite the
fact that that the pH was decreased by growth to different extents.
This inhibition is a function of the inability of h- bacteria
to synthesise histidine. One of the main physiological differences
between he and h- cells is the probable accumlation of precurser
in the latter, especially at intermediate histidine concentrations.
Perhaps the inhibitor is in some way related to a histidine pre~
curser, To speculate further would involve an unwise extrapolation

of the data, It would also be inappropriate to discuss notions with
(6)

regerd to the mutation from be te he or of the mutation which also
occurs from he te he. Oar knowledge is not yet extensive enouch
mat a worl of conution may be stated, These mutations may not be
independent of histidine ox the genes or factors controlling hist-
idine synthesis are se unstable ae to raise questions -bout eslling
changes that ocour in them mutations in the ordinary genatie sense.
We should be prepared in these new investigations on the cenetice
of microorganiems te encounter new phenomens and new concepts.

In conclusion, the growth of a bacterial culture 1¢ 2 popul-
ation event. It may involve mutation in growth abilities and when
competitions and interactione occur they may bring ebout rapid
changes in the populstione These studies on histidineles« Regher=
ichts coli confirm, in a sense, the work on leucinelees Neurozpor:.
They enable one to postulate that celle with biochemical deficien-
cles may be able tc compete favorably with eynthesiaing celle in a
way that does not involve differencea in growth rate,
Tig. 1. fhe growth of histidineless Eacherichia coli on.
different concentrations of histidine expressed as ¥ per ml.

Fig. 2. The effect of histidine concentration on the smount

of growth produced by histidineless Escherichia coli before and
after adaptation.
i.

Ze

3e

Ryan, Fe Je 1946 Back-mutation and adaptation of nutritional
matents, Cold Spring Herbor Symp. Quant. Biol, 112215=227.

Roepke, RoR., Libby, Rel. and Small, MoH. 1944 Mutations or

veriation of Escherichia coli with respect to growth require-~
mentee. de Bact. LS2ho1-l12.

Mitchell, H.K. and Houlahen, M.B. 1945 Adenine«requiring mut-

ante of Neurospora crassa. Fed. Proce §:370=375.