Studies in Bacterial Genetics: an interim report bo the Director,
Jane Coffin Childs Fund, July 1, 1946.

Growth involves the integrated synthesis of a series of substances:
ayb,~.ee. The steps in the synthesis of these suhstances are controlled by
a set of genes, essentially homologous to the substances, which we shall designate
as A, Bese. or more specifically, A+, Bt, #ff// ...... We use the + terminology
because it is possible to modify the activity of specific genes so that the synthetic
step under their control is blocked. Such genes are designated A-, Be, .e.This is
demonstrable in Neurospora, where the occurrence of A~ rather than A+ is assoca&ated
with the inability to synthesize & and the consequent appearance of a as a nutritional
rquirement of the strain. Such strains are known as biochemical mutants, since the
modiéication of the homologous gene is a mutation, The mutation of a particular
'gene' has been achieved only in the pneumococcus transform tbbns. In Neurospora,
gene mutations are randomly induced by radiation, and detected by the alteration
in grwoth factor requirements in the resulting strain.

By similar treatments, it is possible to isolate strains of Bacteria (5.
colijin particular)with similar alterations in nutritional requirements. Because of
the eviient analogy with Neurospora, we believe these alterations to be based on
gene mutations, and it is the purpose of this investiga tion to use these characters
in investigating the gnetics of bacteria. On the basis of the Neurospora work we
can make partial deductions of the genotype on the kucem basis of the nutritional
behvaior of a strain, If a substance k is not required by a particular strain,
it is K+, However there may also be present K- ‘genes' masked by the activity of
K+, se proceed now to our experinenis,.

There are two modes of approach, in general: the study of the appearance
of K~ strains after various treatments, particular with respect to the time of
such appearance which affords evidence of the segreration of K- from K+; the other
the appearnce of new types in mixed cultures. While we are using both approaches,
it is developments in the akx latter which mve proven so exciting as Lo be the

basis of this re-uest for extension, The main result is that there appear,
under certain conditions in mixed cultures of[ A-,B-,C+, D+,...Jand [A+,B+,C~,D=, tesee]
cells of wild type or prototroph constitution, i.e. [A+,B+,C+, Dty cecels

The interpretation of this result depends on the results of certain
other experiments which occupied most of my first three months on this project.
Firstly it was necessary to use double mutants [KX-,B-, ++++] rather than only
single mutants |K-L+ +++] becaguie had previously been found (Ryan and Lederberg
unpubl.) that the change from K- to K+ occurs in generalZ with a measurable fre~

corncid

quency of the order of 10 8 However, the nutation of aii te genes K- and L-
to their + analogues is independent ard should occur. inth ‘an

cted (but un-
measurable frequency of the order of 10 ib It was necessary to deonnstrate
this independence, and in my experiments I have found no instance of the occurrence
of a wild type ( [K¢L+] ) in cultures inoculated with [K-L-].

Secondly, it was found during the last three months that different
mutanss can interact through the medium, so that a mixed population might grow
on minimal medbum : If K+l~ and K~L+ are inoculated together, the former manufactures
and releases into the medium sufficient k for the latter to grow, and conversely.
Analysis of the populations after such 'syntrophic' growth demonstrated this pheno~
menon independently of any genetic interaction that might have taken place.
A similar situation applies to the combined growth of a pair of Wouble mutants.
If k is proline(so that K- is proline—less) it was found thab K+ produces con-
siderable quantities of proline ami releases it into tle medium during early growth
but it is evidently reabsorbed in the adter p hases of the growth cycle, an indi-
cation that there exists a continual interchange between cells and medium.

In view of the occurrence of syntrophism, assurance would be required
that the prototrophs (A+B+C+....) mentioned above did not merely represent cells
temporarily stuck together, The stability of these protrophs wwas tested, and
confimmed, even when most of the cells had been killed by ultraviolet. “nder

these conditions one wouid expect many cell pairs to have been repres ented
by only a single mutant survivor; none were found under ideal testing conditions
covering thousands of cells, further evidence that these prototrophs are
geretically unique is provded b} the results of certain experiments stigll

in progress whprein bacteriophage susce:titili.y was used as a marker, We

found, to our surprise and fortune that the “ coli strain we have been using

is susceptible to virus T-l which has been the subject of investigation by the
Cold Spring Harbour Group. Designating resistance and susceptibility as Tlr and
Tis, the former obtained in various strains by mutation and selection, the
following 'crosses'were undertaken : [A~B-C+D4+Tlr] + [A+B+C-D-Tl1s] a wild type
susceptible strain could readily be isolated. [A+t5+C+D+T1ls]. If celis had merely
been stuck together one should have been left with « large proprta&qn of resiatant
mutant cells after applicet'on of the virus.

Sponteneous mutation has been eliminated as the source of these

monotonously + strains. “e have looked for these because they are patently
the easies& to find in large populations, and their rate of occurrence in

our best experiments has not exceeded 10° « To complete the proof that gene
recombination is the basis of these results it will be necessary to isolate
Kum gene recombinant types ( such as [AtB-C+D-] and other permtations of +,-)
from A~B- and C-D-..'crosses', There is a little evidence that they may ocgur.
The possibilities of the detailed genetic mechanims = occurrence of linkage groups,
haploidy or polyploidy, the life cycle, etc. are too mmnifold to be adecuately
investigated in one suw:mer.I could, I think, accomplish a great deal in another
year, and if these suppositions turn out correctly, open to investiggtion a broad

field obscured hitherto by the lack of basic know edge of micr@bial inheritance.