October 10, 1956

Dear Dr. Dienes:

Thank you for your letter on Proteus Depely brief examination of
it was rather puzzling. Having learned a great deal from studying
your Proteus strains, we are now entirely prececupled whkth E. coli.

Some K-12 strains (including Y-10) give primary L—colonies quite
readily when plated into the following medium: Agar, 1.0-1.2%; sucrose,
10%; MgSO, 0.2%; penassay broth/ Difco; maat extract Difco 0.5%. Unlike
Proteus , the coli L-form requires a rather high agar concentration, a fact
which mictum eluded and embarrassed us for some time. About 10% of the
inectlated bacteria form L-colonies, which seem to be of the B type.

We have not been very lucky so far in getting serial passages, al-
though we have carried some four or five transfers. (We usually cut out a
block of agar and mince it in some added broth (as above) with a Virtis
tissue grinier.) There is negligible L-colony deve lopment on the surface
of the agar. The progressive decrease in viability on passage suggests
that there 1s a special growth factor, memo presumbly synthesized by
normal bacteria: we are locking along these and other lines to improve
the medium. Serum is no help.

I am sending you Y-10 and an Hfr strain W-1695, which are an extremely
fertile combination. The L-cycle plays no part in the usual conjugation
process. + know that protoplasts of W-1895 are quite fertile. I have not
been able to obtain, so far, a aatisfactory l—colony development from W-
1895, but there are a number of other Hfr strains which may be more satis-
factory and are being tested. The experiment we are trying at the moment
to to look for recombination between two F- strains, which cultures do not
undergo conjugation in ¢he ba terial form, as an index of new processes
of genetic interaction. We have had one clean experimental trial so far,
and the results were quite negative. We have to do more with varying con-
ditions.

My own observations and conclusions agree with yours almost wherever
they coinmide. I hope the final paragraph of the (fa merly missing) enclosure
is a clearer statement of the protoplast—L colony relationship. However, I
have not (with E. colj)seen the segnentation of a large body to form bac-

tgria; instead, there is an elongation, and a bacterium is cut off, then
proliferates. I have no fixed opinton about the mode of proliferation of
the large body itself: your observations will certainly be an indd&pensable
guide to my own. However, I am seeking to establikh conditions where the
entire cycle (in familiar material, E. coli) can be observed on the sane
specimens in living condition.

I have also noticed the tendency of nearly divided cells to swell from
the point of dividion. Kideneberger's fancies of the role of fusion in the
Ireycle mst be based on the appearance of two-legged balloons (Mellon's

 
de. uiitely
eparated

"‘zygospores"). But there are other better authenticated instances of fusion
oftprotoplasts or large bodies, though not associated with direct observations
on further development. The question is whether this can have any genetic con-
sequences, and whether it can oceur between bacteria that would be unable to
conjugate ahyhow,

As I hope I have emphasized sufficiently, it is obvious that many pieces
to this puzzle have been lying around already, and probably there are mang
more not yet recdgnized. The proposition (or definition if you like) that L-
forms are protoplasts will at least have the virtue of atbracting the attention
of another group of investigators into this atea.

You my be intetested that Dr. B. Davis has found that bacterial mtants
deprived of diaminopimelic acdéd will form near-protoplasts when deprived of
DAP in a protective medium, ctherwise lyse. DAP is of special interest as being
uniquely found in bacterdal cell walls. I have also communicated whth Dr.

Park and find he is in full agreement as to the significance of the uridine-
diphosphate derivatives.

Yours sincerely,

Joshua Lederberg