UNIVERSITY of WASHINGTON
SCHOOL OF MEDICINE
DEPARTMENT OF MICROBIOLOGY
SEATTLE 5

March 10, 1954

Dear Dr, Lederberg:

It amazed me to check back on your last letter and find that the date on it
was February, 1953, It is possible to report a certain amount of progrees with
the C. diphtheriae system, I have just finished a manuscript of some of the
work and I feel that the best way I can convey the information is to send you
a copy. Unfortunately I can't vouch for the speed of the secretarial service
so I'll convey the basic conclusions in this letter and forward the paper as
soon as possible, I would certainly appreciate your comments,

In essence the hypothesis that lysogenization and toxigenicity conversion,
in the particular system under study, are intimately connected seems to have
held up. In agreement with your original suspicion the evidence I have ac=
cumulated does not conform to transduction. Although Barksdale and Pappenheimer
have indicated a similar conclusion I felt a certain mental reservation since
in their work and my awn the non-toxigenic strain used to propagate the phage
was itself susceptible to toxigenicity conversion. Ideally a strain capable
of being lysogenized, without being converted to toxigenicity, should have been
used, but at present there is no strain which fits this description, Never~
theless it has been possible to show that in a series of 5 consecutive single
plaque transfers on the C4 strain that every plaque forming particle, through-
out the whole transfer series, still retains the pgqtentiality of conversion
to toxigenicity. While the argument is somewhat lengthy it is possible to
reason that the dilution effect on a transducing particle would have precluded
such results,

The second significant point is that it has been possible to isolate a
non-lysogenic revertant from a culture of ca(P). Strain C4(f) is toxigenic
and lysogenic; in the reverted strain loss of lysogenicity i$ accompanied
by a loss of toxigenicity. Furthermore these reverted cells when grown and
re-exposed to the converting (? phage yield toxigenic-lyosgenic cells once
more, This concommitant gain and loss of toxigenicity and lysogehicity
strongly supports the hypothesis entertained above, - Reversion has been ac-
complished in three distinct, widely separated experiments. In one experi-
ment 4 such revertants were obtained and in two others there was 1] per
experiment, The screening technique is laborious accounting in part for
the low numbers recovered, The technique of obtaining these reverted cells
involves exposing the ca cells to a virulent phage mutant and screening
the resistant clones for non-toxigenic and/or non-lysogenic cells, As yet
we have not isolated a cell which has lost either characteristic indepencently
although I do not consider this situation impossible subsequent to the ini-
tial conversion process,

The third point and one which has opened up the probiém is the isolation
of a phage which lysogenized the C4 dells but does not convert them to toxigeni-
city. We are now exploring the ramifications of this observation, It is ob-
vious at once that conversion to toxigenicity is a phage specific phenomenon
that lysogenization with phage does not necessarily mean toxigenicity conversions
The other question as to whether toxigenicity is always related to jysagenization
with a particular type or types of phage is really not answered yet if one ex—
pands this phenomenon to include all toxigenic C. diphtheriae. The difficulties
which can arise in demonstrating the presence of a prophage in a given cell may
—2—

make an answer to this question well nigh impossible to obtain. For example
"mutation" at a prophage locus, assuming now that the locus is chrumosonal,
could result in the retention of toxigenicity but loss of inductive properties
on the part of théfphage. Or it is possible that a non-toxigenic C. diphtheriae
cell may mutate to a condition of toxin production independent of phage action.
To my mind a method of discriminating between these two mechanisms with the

same demonstrable end result is not yet at hand,

Beyond the point of having isolated this new phage there is some interesiing
data accumulating but it is by no means at the quotable stage. We have found
that a cell lysogenized by the_new phage (lvsogmic but non-toxigenic) is still
susceptible to action by the (converting phage, At present we are involved in
attempting to isolate the doubly lysogenic celis and any possible variants there-
from, It does seem certain that a cell carrying the new phage can be converted
to toxigenicity by exposure to ),. We have not critically determined the sero-
logical relationship of these two phages, but using a P antiserum which had a
rather low neutrmalizing titer it appeared that the rate of neutralization of
these two phages was identical, We are in the process of trying to get better
antisera, but low titers and rapid inactivahion of the diphtheria phages makes
this somewhat difficult. One other interesting point which we are striving to
nail down is whether conversion to toxigenicity is a function of the entire
prophage or whether it is a function of a Bubunit, At the moment there are
some indigations that .conversion to toxigenicity is a sub-unit function and
I hope we will have more information on the point very soon.

Having first spewed forth what I know, due credit goes to Barksdale for
the simultaneous isolation of a phage which lysogenized but which does not con-
vert to toxigenicity. Furthermore he has written about some interference ef-
fects of another phage on the ability of § to convert to toxigenicity when
cells are doubly lysogenized. Of course he should speak for himself, but
I simply waated to indicate that there is considerable ferment at the moment
and I would suggest that you write directly to him for the information. Our
correspondence has been regular and to me very rewarding. His present address
is, Institut Pasteur, 2& Rue du Dr. Roux, Paris, France c/@ Service Iwoff,
There are a few other workers who have corresponded with me, but of these the
only suggestion that progeess was being made came from Dr. L. F. Hewitt.

He did not relay any specific information, A note from a Dr, Hatano in Japan
suggests that he, too, has observed reversion from the toxigenic-—lysogenic
state to one of non-toxigenicity and non-—lysogenicity.

I shall try to get the manuscript to you soon and would be grateful for
any comments and suggestions you might have in pursuit of the work.

Sincerely yours,

Nedl B, Groman
Assistant Professor