August 27, 1956

Professor H. Kikkawa
Department of Gene tics
Faculty of Medicine
@axa Osaka, Japan

Dear Professor Kikkawa:

Thank you for your letter of May 3, and for Mr. Hirota's ms. We
Have also seen the publication in NATURE. My K Ada

We are still interested in trying to reprb S result here, and
I am sorry to say still unsuccessfully. He has, for example, recently done

the following experiment:

A 2&4 hr. nutrient broth culture of WF+ was spun down, washed with saline
and resuspended in saline. The culture was then starved 1 hr. at 37°. Co**
was then added to give .O2M. After 3 hrs., at 37°, the culture was plated
on nutrient agar containing .04H citrate. The survival was 50%. Of 40 colonies
teste: all were F+ still. Ina repbtition with various Co conc., the
following results were obtained:

CoM Survival F teste

002 003 18/18 F+
04 1074 18/18 F+
206 107? 18/18 F+

It seems very likely that our strain(s) have deviated from yours. For
that reason, we would greatly appreciate getting back the 58-161 or Y-40
strain that Mr. Hirota has currently and successfully bem using for these
experiments. If you can offer ang other suggests that wouldhelp us to
confirm your results we would apprechate that very much too. It might alss
be helpful if we could have one of the derived F- strains (if reinfected with
F+, this derivative might be more readily amenable tc the exptl. effect).

We are also greatly concerned about the extent of killing which occurs in
Mr, Hirota's experiments, which is not specifically mentioned in the paper.
Can you get the formation of F- under conditions of high survivorship?

In discussing the mtter further with Mr. Richter, we notice that he
had added citrate to the nutrient agar in the 3~hour treatment—expts. just
summarized, since this was described for the earlier 24-hour treatments.
However, your letter does not mention the addition of citrate here. Rich-
ter found that plating, after 3 hours, on medium whthout gitrate, led to
quite low survivals. The question of extent of survival. may be the key
to our problem. ge a

I am very sorry. ‘that. t will not be “able ‘after wall | ‘to discuss these
matters with you in person at the Tokyo symposium. My good friend and
colleague, Professor Crow will, however, be there and also. intends to
visit Osaka afterwards. May I name him as intermediary, both to
facilitate the exchange of the cultures requested, and to discuss t’_
questions I have raised here. It would prohably be helpful af you |
show him this letter. —
Some years agec, Dr. Skaar discowered = method for obtaining F= fron F-.
namely by passage of motile cultures through "motility agar” (See Nelson &
Lederberg,1954, Proc. Nat. Acad, Sei. U.S. 40:419, fuctnote 10.) We have
not published this in detail because we were waiting for definite eeidence
whether ‘the treatment selected or induced the F~ variants. We were especially
hopeful of the latter, and were thinking in terms of the dilution of a cyto-
plasmic factor (like kappa in Paramedium) during prolonged rapid fission of the
_ bacteria. Recent experiments by Richter MAMIMMMMnow indicate, however, that
‘it is a selective effect after all, the F+ parents being slightly more sluggish
than F- variants from them. The problem was compliwated because the rate of
motility is also conditioned by a polygenic background.

Yours sincerely,

Joshvwa Lederberg
Professor of Genetics

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Wh US