THE UNIVERSITY OF TEXAS
AUSTIN 12

DEPARTMENT OF ZOOLOGY

January 29, 1952

Dr. Joshua Lederberg
Department of Genetics

The University of Wisconsin
College of Agriculture
Madison, Wisconsin

Dear Dr. Lederberg:

In answer to your letter of January 18, 1952 the details of the experiments
published in the American Naturalist, 84; 261-274 (1950) follow:

Thirty ml aliquots of nutrient broth (see Hershey, A.D. and Rotman, Re,
Genetics, 34, 89-106 (1948) for composition) were placed in sterile petri
dishes and irradiated with a Hanovia double U = SC~2537 ultraviolet mercury
vapor lamp operating at 100 milliamperes. The samples were 10 cms from the
lamp during the exposure. The lengths of exposure time were 0, 20, 0, and
60 minutes. Twenty ml aliquots were then pipetted to large sterile aeration
tubes. Each of the tubes was then inoculated with approximately 100 thousand
organisms per ml from , hour log phase cultures of E. coli B or E. coli 58=16l.
These cultures were then incubated with aeration in a 37°C water bath until
there was good turbidity (4 to 6 hours). After incubation each of these cultures
was treated as follows:

Twenty tubes gontaining 2 ml soft (0.7%) agar at 45°C and seeded with approxi-
mately 5 X 10” phage Tl particles were each inoculated with 6.1 ml of the
culture. The tubes were then plated via the soft agar technique and incubated
at 37°C overnight. At the same time one to one million and one to 10 million
dilutions of the cultures were prepared and plated in triplicate in nutrient
agar containing no phage to determine the number of bacteria subjected to the
action of the phage. All plates were examined and counted after 2); hours
incubation.

‘numberof the ¢olonies:wersuthen. picked: from the plates of each experiment
( one or two colonies from a plate) and checked for resistance. Each colony
tested was emulsified in broth then streaked on an agar plate seeded with Tl
phage and on one with no phage. At the same time an agar slant was inoculated
with the emulsion. WNinffy six percent of the colonies tested in this manner
proved to be resistant. The false positive slants were discarded and the others
stored in the ice box.

To determine if phage-resistant mutants also possessed nutritional
deficiencies a small inoculum from each agar slant was emulsified in liquid
minimal medium without washing and then streaked onto minimal agar plates
and nutFient agar plates. If the organism failed to grow on the minimal
medium or if it grew much more slowly than on the nutrient agar plate it
was considered to be a possible biochemical mutant. The agar slants
containing phage-resistant biochemical mutants were retained and the others
discarded. Many of the mutants were derived from strain 58-161 and whenever
such mutants were tested for deficiencies the minimal medium was supplemented
with methionine and biotin.

"Since both B/1 and B/1,5 mutants are obtained together on complete medium it
was suspected that many of the biochemical mutants would be B/1 and require
tryptophane, thus mutants not growing on minimal were tested for growth on
minimal supplemented with tryptophane (50 gamme/ml). No biochemical mutant
grew on this medium. Since this indicated that only B/1,5 mutants were being
obtained two more series of Tl resistant mutants were taken from plates after
lysis and tested for tryptophane requirement. None of these colonies
exhibited such a requirement. The cultures were then tested for their resistance
pattern with all 7 phages of the T series by the cross-streak method and all
proved to be B/1,5« It was also found that none of the 58-161 mutants
required tryptophane. These were not tested to determine if they were resistant
to T5 as well as Tl however. A control culture of B/l tested at the same time
as the previously mentioned mutants showed a definite requirement for tryptophane.

The resistant mutants of strain 58=161 which were suspected of having
different biochemical requirements foom the parent strain were tested on
minimal medium supplemented with a vitamin solution, purine and pyrimidine
solution, or one of two different “group™ amino acid solutions. Washed heavy
suspensions of each of the mutants were tested by streaking onto plates of
each of the following media;

nutrient agar
minimal agar
minimal agar # biotin # methionine

minimal agar " 7 f# amino acid solution A.
minimal agar £ nog " f amino acid solution B.
minimal agar nf " f purines and pyrimidines
minimal agar # mn 4 " 7 vitamines

minimal agar 7 all supplements.

Using these methods it was found that 19 of 72 virus resistant mutants
from untreated media, le of 72 from ultraviolet irradiated broth, and 26 of
72 from hydrogen peroxide treated broth exhibited some definite type of
stable nutritional deficiency. Of these mutants 11 were found on further
testing with single growth factors to have a definite requirement for proline.
The remainder show no good growth on any single amino acid, vitamin or purine
“9% pyrimidine but grow as well as the parent strains when yeast extract is
added to the medium.

A few of the resistant mutants appeared at first to be vitamin requiring,
however these were not stable enough to be certain and on further testing
exhibited more and more growth on unsupplemented minimal medium. Therefore
they are not included as biochemical mutants but as non-mutants in the above
figurese

Each of the positive mutants was checked by the auxanographic method.
The results of this test were the same as those of the streak method.
Tests were then carried out on the proline requiring mutants. Three of
these would grow only on proline supplemented medium or complete medium. Five
would grow on arginine supplemented medium but not nearly as well as on the
proline medium. Three of them grew on medium supplemented with either phenyl-
alanine, ornithine, arginine or glutamic acid but not as well as on medium
supplemented with proline. All except three of these mutants came from
different experiments and from different treatments (i.e. no treatment, treat-
ment with H,0o, or treatment with ultraviolet). Two of these three mutants
coming from the same experiment (but different plates) were the same (would
grow on arginine as well as proline) and one was different (would grow only
on proline).

In answer to the questions and suggestions in the last paragraph of
your letter I offer the following;

Only one resistant mutant was taken from each plate in the original
isolation unless there were distinct colony differences in which case the
different colony was taken also. In no case were more than two colonies
taken froma plate. It is possible that some of the mutants could come from
a single clone since a growth period of 4 to 6 hours was permitted before
a culture was tested. This seems unlikely however in view of the differences
in the proline mutants and since the mutants finally tested for biochemical
requirements came from a total of eleven seperate experiments employing
approximately 200 completely seperate cultures.

I have not carried out any genetic tests on any of these mutants. I have
intended doing this and also doing some more work on the biochemical aspects.
However, last year I was working with Neurospora at Cal Tech. and did not
have a chance to do anything with bacteria during this time. On my return
to Texas I found that due to stock culture technician difficulties many of
the stocks not in immediate use had been allowed to die, including most of

mines I have a couple of Drosophila problems going at present and I am
trying to finish up some Neurospora work. For these reasons I haven't

gotten back to the bacteria yet. I hope to be able to do this in a couple
of months.

I hope that this gives you the information that you need. If there are
any other questions about this or if you would like to have any of the cultures
please let me know and I will send them if they are still available. Please
let me know of any suggestions, criticism, etc. that you may have in regard
to these experiments. I can see no reason why our results should differ unless
there is a difference in the strains. This seems unlikely since the 58-161
Strein used for most of the biochemical mutant work was obtained from you.

Also will you please send me copies of your‘reprints which are available.

Sincerely yours

Felix L. Haas

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