1949 Annual Progress Report
Wational Institute of Health, Research Grant 1445 (Bact.)
THE GENETICS OF SALMONELLA

Joshua Lederberg, Ass't Professor of Genetics, University of Wisconsin

Our immediate objective is to determine the role that genetic recombination
may play in the formation of new types in the Salmonella group. If, as in
Escherichia coli, recombination should be found in Salmonella, it can then be
used as a tool for the analysis of specific genetic problems in this group, in
particular, the relationships of somatic antigens, phase variation in flagellar
antigens, and determination of virulence.

It is hoped to apply to Salmonella the procedures which worked with EB. coli
as detailed in 1, Complementary biochemical mtants would be mixed and plated
into minimal agar. The development of prototroph colonies with no nutritional
deficiencies would be a sign that recombination of factors had occurred.

The progress which may be reported for the period July 1, 1948, to March l,
1949, consists primarily of (A) the development of an improved technique for the
isolation of biochemical mutants, and (B) preliminary recombination experiments
on three strains of 5. typhimurium.

In the exploration of a new bacterial group for gene recombination, most of
the labor would be spent on the isolation of the multiple biochemical mutants
needed to look for the formation of prototroph recombinants. For this reason,
considerable attention was given to the possibility of improving methods for
selecting biochemical mutants. Penicillin might be a useful reagant for this
purpose since it has been established that this antibiotic has a bactericidal
action specific for growing cells. After a number of preliminary reconstruc—
tion experiments, a procedure was worked out with which the mutant cells present
in small numbers in ultra-violet treated susnensions could be concentrated so as
to facilitate their isolation.

The suspensions are inoculated into synthetic medium containing penicillin.
The mitants are protected from bacteriolysis by their inability to grow in this
medium, while the non-mtant cells are rapidly killed. Details have been pub
lished<. Just as we submitted this manuscript for publication, we learned that
Bernard Davis, Senior Surgeon, U.S.P.H.S., at the Tuberculosis Research Laboratory
in New York, had independently developed the same technique. We arranged to
publish our findings as adjoining notes in the same journal, and found it
economical to reprint the notes on the same sheets.

This method has been applied with considerable success to three salmonella
typhimurtfum strains, with our designations: SY-20, SY-21, and SY-23. SY-20 and
SY=-21 are monophasic variants, in the first and second flagellar phase respectively,
originally received from Dr. P. R. Edwards, while SY¥-23 is a typical diphasic
culture. The strains also differ in their fermentative reactions, and in their
phage resistance patterns,

 

1. lederberg, J. Gene recombination and linked segregations in Escherichia coli.
Genetics 32:505-525 (1947).

2, Lederberg, J., and Zinder, N. Concentration of biochemical mutants of
bacteria with penicillin. J, Am. Chem, Soc,, 70, 4267 (1948).
-~2-

A number of mutants, and then, double mutants, were produced in strains
5-20 and 5-21, Using the principle described atove, no indication of recon-
bination was found between mutants either of the same or of the two different
strains.

Somewhat more encouraging results were obtained with SY~23. However, we
have been disappointed to discover that it has not been possible to secure
unequivocal evidence for or against the occurrence of recombination in Salmonella
with the same ease as in E, coli.

Combinations of mutants within strain SY-23 have erratically led to the
production of prototrophs, particularly in a "cross" of a proline-histidineless
with a leucine-methionineless, rough, mutant. In this case, it is unlikely that
prototrophs could have originated by reversion of either of the parents, although
a syntrophic effect is not excluded.

More consistent results have teen ottained in an interstrain cross of an
isoleucine-valineless mutant (SW-13, from SY-21) with a histidine-threonineless
(SW-25, from SY-23). In about every other experiment, a few prototroohs have
been found from this combination. These cultures also differ in the fermentation
of maltose, xylose, galactose, and arabinose. The prototrophs which could be
tested usually showed the fermentation pattern of the SW-13 parent, although
a very few have teen like SW-25. However, it was noted during these fermentation
tests on indicator agar that the prototrophs isolated from these experiments are
frankly lysogenic, i.e., when spread out on nutrient agar, they are ridden with
plaques of bacteriophage. In spite of repeated serial single colony isolation,
it has not been possible to free the bacterial cultures from this phage. This
contamination probably also accounts for the relatively poor growth of these
prototrophs in minimal medium, as compared with either of the original wild
type parents,

Further study showed that each of the two strains carries, in lysogenic
condition, a phage active against the other. SY-21 also carries a second phage
which is active on S. gallinarum, but not on other typhimurium isolates, It
has not been possible to obtain these phages in high titer as yet. Our attempts,
so far, to obtain resistant mutants which would be uneffected by either of the
phages have also been unsuccessful.

The suicidal behavior of the prototrophs obtained from this experiment
makes it very difficult to study them, and therefore to draw any definite con-
clusions as to the validity of the prototrophs which have appeared in these experi-
ments. Hspecially perplexing is the fact that prototrophs isolated from the
intrastrain cross in SY-23 are also suicidal, although both parents were shown
to carry, and to be resistant to lysis by, the same phage.

By coincidence, lysogenicity has also become a vital issue in other genetic
work béing done in this laboratory on the genetics of BE. coli, with the finding
that E, coli K~12 is an "immune carrier" of a phage that remained undiscovered
until certain cultures were disinfected (and rendered susceptible) by ultra-
violet light,

Although lysogenicity probably has no direct relationship to genetic
recombination, we are imposed with the obligation to it, (a) as a factor which
interferes with "interbreeding", i,e., as an important isolation mechanism, and
(b) as a type of intereaction between cultures comparable to type transformation,
’ ~ 3 ~
For the present, our interest in lysogenicity in Salmonella is confined to its
intrusion in our recombination experiments, Attempts to disinfect Salmonella
cultures with ultra-violet, along the lines which were successful with E. coli,
have not been fruitful so far, However, it has been noted that the action of
the lysogenic phages is much less marked at room temperature, and concordantly
that the yield of prototrophs is higher at lower temperatures.

The last line of approach is to proceed with the development of other strains,
in hopes of escaping interference from phages. However, lysogenicity in this
group appears to be exceedingly common émeatikemmep, and it is doubtful whether
one could safely assert that a given culture was not contaminated with a
symbiotic phage until it had been exhaustively tested with a very large number
of suitable indicator strains,

Incidentally to the genetic studies, a mutant of SY-23 was isolated which
has interesting properties from a biochemical point of view. The mutant, SW-38,
responds either to phenylalanine or to tyrosine, and preferably to the combination,
This pattern suggested that the mutant was deficient in the synthesis of the aromat-—
ic ring, so a number of related phenols were tested. Many of these proved to be
active, especially in combination with serine, including phenol, hydroquinone,
catechol and cyclohexanol, On the other hand, resorcinol and naphthol were in-
active. Unfortunately, the mutant is highly unstable, and reversions are se-
lected for, and lead to growth in unsupplemented medium within a relatively
short time. However, the response to cyclohexanol plus serine has been verified
by plating experiments in which small numbers of cells were added to synthetic
agar with various supplements: This finding suggests that the aromatic ring may
be synthesized from a saturated cyclic intermediate such as cyclohexanol, and
that it is subsequently condensed with an "alanyl" residue to form the aromatic
amino acids. However, this study is net being pursued further, as it is too far
from our primary genetic objectives,