Columbia Gniversity
inthe Citpof Hew Pork

[NEW YorK 27.N, Yo]

DEPARTMENT OF ZOOLOGY

June 6, 1949
Dr. Joshua Lederberg
Department of Genetics
University of Wisconsin
Madison, Wisconsin

Dear Dr. Lederberg:

Unfortunately, your recent visit to our laboratory was of such short dura-
tion and our conversation was so inadequate, that I have decided to take

the liberty of wariting to you. I should like to tell you of my experi-
ences with the citrate-utilizing (C/) mutant of E.coli (dtrain K12) up to
the present time, and of what experiments I intend to do in the near future.

As I told you, my initial attempts to obtain a C/ mtant of E. coli Kl2
were fruitless, whether I tried heavy inocula into standing citrate media,
or irradiation followed by plating on citrate agar, or inoculating into
citrate plus limiting glucose medium (the latter method to encourage enough
C- growth to make probable a C/ mtation, which might then overgrow the
culture). It was not until I inoculated C- cells from standing glucose
plus G&T salts medium into rolled-aerated citrate medium that I obtained
adaptation to citrate-utilization. ‘The adaptation time depends upon the
medium employed: adaptation occurs between 24 and 48 hours with Koser's
citrate; it takes somewhat longer (about 12 hours more) and growth reaches
a lower final level in citrate plus G&T salts medium. The presence of
glucose may prevent adaptation (in 2 of 15 cases).

The CH mutants are small, thin, Gram-negative rods, morphologically similar
to E. coli and A. aerogenes, They give a non-lactose~fermenting reaction
on Endo's agar, however, and growth is very feeble on this medium, moreover,
A complete testing of the C/ mtants for coliform characteristics is in
progress. I have found that 0.25% citrate plus G&T salts medium gives
maximal growth under aerated conditions, but that growth is much slower

in this medium than in Koser's citrate, I am presently investigating the
possible influence of the trace elements used in G&T Salts in depressing
the C/ growth rate, (I already know that buffering action in Koser's
citrate is no better than in G&T salts-citrate medium.)

In preliminary experiments, I have learned that Cf growth in citrate is
characterized by (1) increase in pH proportional to amount of growth, (2)
autolysis immediately following end of logarithmic phase of growth; the
decrease in optical density of the culture is correlated with the maximal
growth level: the greater the maximal growth, the greater the decrease in
optical density, (3) a more or less stationary phase following the abrupt
autolytic period, (4) production of gummy material in the late stationary
Phase. If viable Cf cells are desired from liquid culture, they must be
taken from the logarithmic phase of growth. The longer the culture remains

in the stationary phase, the fewer viable C/ cells will be obtained in
Leo

samples taken from it. Furthermore, inocula from stationary phase- ;
adapted cultures into citrate media will result in either no growth or
delayed growth, The possibility of C- mutants predominating in the
stationary phase and which inhibit C/ cells will be investigated,

The C?# mutants obtained thus far can utilize citrate but cannot utilize
glucose as a source of carbon. Howver, a recent case of a "C$ Ge" ;
mitant adapting to glucose-utilization has been noted, and the biochemical
characteristics of the mutant parent end the Nadapted" cells will be
studied, Wild-type EZ. coli K12 is, of course, citrate-negative and
Blucose-positive. Furthermore, despite the fact that C/ mutants are
obtained only from aerated citrate cultures of C-, C/ can utilize citrate
in standing cultures and in aerated cultures. ‘Final level and rate of
growth of CH in citrate are higher under aerated conditions, however,

An interesting case of what I call "plate selection" has been observed in
mixing C- and C# cells on glucose agar: C- colonies prevent C/ colonies
from appearing after layering with citrate ager. If mixed on citrate agar,

colonies appear first; then after layering with glucose agar, complete
C- recovery is obtained.

I intend to work this "citrate-utilization" locus for all that it is worth,
and I hope that it will merit being the major part of a Ph. B. dissertation.
My planned expcriments include:

(1) doing a variance analysis on C- cultures plated on citrate,
in the expeetation that citrate does not induce the C- to Cf mutation but
acts only to select those cf mutants regularly present in C+ cultures:

(2) Mapping the "Iq" locus, to demonstrate the genic basis of
citrate-utilization;:

(3) checking the utilizability of all the obtainable substrates
involved in ths carbohydrate metabolism in C- and in Cf, with subsequent
experiments to determine tha nature of the action of the "c" locus. (If a
trace element is found to be responsible for the decreased growth rate of
Cf in citrate, it may provide a clue as to the "sempitive" reactions in
the citrate metabolism of C4);

(4) investigating all the possible engles éf C~ and Cf intern
action and selection in standing and aerated, in glucose, citrate and
glucose-citrate cultures.

Planned also are experimehts to obtain C- mutants of A. aerogenes by the
penicillin method.

At first a sub-project in the investigation of the mutability of the bio-
chemical characteristics which distinguish E. coli from A, aerogenes, the
investigation of citrate-utilization is bedoming a fulltime project in
itself. I would appreciate hearing from you concerning sugee stions or
questions about my work. I would especially like to hear if your non~
glucose-fermenters are citrate-utilizers, and what some of the character-
istics of your non-£lucose fermenters are, and how obtained.

TI hope that the "C4 G-" culture I gave you will be useful to you. I should
have remembered to remark on the "gumminess" or "stickiness" of the ce
growth on either citrate agar or nutrient ager slants. This "gumminess!
increases with prolonged incubation of freshly-prepared slants, and makes
transfers very difficult, I suggest making stock transfers at least once
@ week, even if stocks are refrigerated,

I expect soon to publish a note on the preliminary experiments and early
findings, and I shall be glad to send you a reprint of it. In connection
with reprints, may I remind you of my desire to possess reprints of the
publications of your extremely interesting and significant work?

Sincerely yours,

wuld Keo

Arnold Y. Ravin