7

March 9, 1948.

Dr. Michael. Doudoroff,
Department of Bacteriology,
University of California,
Dear Dr. Doudoroff,

Firat, I want to thank you for having sent so many of your reprints
when I requested them. They have been very helpful.

Since my arrival here, I have been working on the genetic control of
the fermentation enzymes of Escherichia coli, S-12. The approach has been
to irradiate dense suspensions on EMB plates containing a given sugar,
and to pick up fermentation mutants among the survivors by the failure of
the colony §or sestor thereof) to turn purple, in contrast to the fermenting,
non-mautants. With appropriate technique, as many as 1:k000 colonies will
be lactose- or maltose- negative mutants, which has made it very easy to
accumulate a large collection of diverse mutants. By the use of recombination
techniques it has been possible to classify the mtants obtained genetically,
and for example, mutations at any one of seven or sight loci will produce a
lactose negative (& glucose-or galactose—positive) mutant. This observation
suggests either that there is a very complex enzymatic scheme indeed for
the diddimilation of lactose, or that there is a more complex genetic control
of enzyme formation than has been thought. Although I am inclined to the latter,
the point is not settled, and I mst look forward to a lot of chemical work
to clear it up. I have a few observations on the specificities of some mutants
which may, on the one hand, interest you; on the other, I should like to hear
whether you have encountered anything similar in your experience.

The wild type (K-12) ferments gixkem hexoses, hexitols, lactose, maltose,

meliblose and trehalose, butbnot cellobiose, gentioblose or sucrose. Intensive
attempts to select out sucrose‘ mutants have failed.
«i Douderoff-—2-

Although melibiose is attacked, no utilization of raffinose can be detected,
nor have I been succesSful in shlecting out raffinose/ mutants. Is this differential
between melibiose and raffinose a frequent observation?

2, After irradiation a mutant (W-108) unable to ferment (or utilize for growth
under aerobic conditions) glucose was isolated,On subsequent testing it was found
not to ferment maltose or lactose, while it attacked galactose, pentoses, and
gluconic acid. In the 108 stock, after heavy inoculation into glucose medium, a
strain was developed which fermented glucoeg, but still not lactose or maltose or
trehalose. Genetic study showed that this adapted stock stiil carried the mtant
gens of W-108 (4ac_-) but that another gene (Sl.) had mutated so as partially to
suppress the effects pf the Las, mutation. By a similar procedure, the mutation
sl, which leads to Glu~Lacyiial—“pattern, was selected for in W-108. So far, while
géfietically interestaédg, none of this is very startling. However, 1 wus surprised
to find, from Maltose selections, still a third mutation, Slo/f which suppressed
part of the effects pf Lac.-, to give a strain which is glucose—nsgative, maltose-
positive! I suppese that these xkiannucka "suppressor" mutations are merely opening
up alternate pathways, possibly a maltose-phosphorylass, but this remains to be
shown. Have you found any clearcut indication of a phosphorylation of maltose or
of lactose in any material?

3. Another mitation has recurred (W-145), the locus labelled usc, which has
a particularly interesting pattern? Lac-~dul-Glu-Giuconic-. The me tabolisa eof gluconic
acid in £. coli is something we know very little about. as such, it is probably
not un intermeditae of glucoee utilization becuase glucose-adapted cells mst be
adapted to gluaonic before they will ferment(sic) it. This mutation has recurred
three or four times, and it is very unlikely that the pattern is merely a coincidence.
Maltobionic and laectobionic acids are not utilized even by wild type coli, so it
is not a yuestion of the first sten being oxidation of disacchurides to the
bionie acid. Unlike W-10G, however, I have not been abie to seiect for specific
suppressors of the tutation; i.e., whenever an adeptatlon to one of the sugzss
is found, it constitutes a reversion to the wild type. Have you uny suggestions
ag to a possible simple meaning of such a pattern?

4. Have you any data on trehakose preukdown: Some maltose ~ mutants are
trehaboss/, others are trekalose-. The SljALac -, italtogex strain mentioned above is
trehalose~, 3

5. I am very much interested in the behavior on analogues of the sugars.
For example, most of the Lac- mutants (Ly > 2, 4, 6, & 7) are specific, and still
ferment multose, giucose, ete. However, Luc,— utilizes p-nethyi galactoside, Lac>~
does not but an allela&c mutation can be selected for which is ie.gul-, lac/,}
nor do the others. If you should happen to have access to any other analogues of
lactose (synthetic galagtosides, allo-lactose, a-l-arabinosides } I would appreciate
very mich mont the fuvor of sufficient samples (downwards of 1 g.) to determine
the usefulness of having them synthesixed in larger amount).

Your discussion of any of these items would be greatly appreciated.
Yours sincer ely,

Joshua Lederberg _
Assistant Professor of Genetics.