Chapter 45
BACTERIAL GENETICS: GENETIC TRANSDUCTION

PRE-LECTURE ASSIGNMENT

1. Quickly review notes for the previous lec-
ture.
2. Suggested readings:
a. General genetics textbooks
Altenburg: Chap. 22, pp. 392-393, 396-
397, 398-399.
Sinnott, Dunn, and Dobzhansky: Chap.
23, pp. 318-319; Chap. 28, pp. 384-

386,

Snyder and David: Chap. 26, pp. 410-
413,

Winchester: Chap. 23, pp. 324-326,
322-324,

b. Additional references
Lederberg, E. M., and Lederberg, J.
1953. Genetic studies of lysogenicity in
Escherichia coli, Genetics, 38: 51-64.
Lederberg, J. 1956. Genetic transduc-
tion. Amer. Scient., 44: 264-280.
Lederberg, J. 1959. A view of genetics.
Stanford Med. Bull., 17: 120-132. This
Nobel Prize lecture is published also in
"Science",
Lederberg, J. 1959. Bacterial repro-
duction, Harvey Lect., 53: 69-82,
Zinder, N. D. 1958. "Transduction"
in bacteria. Scient. Amer., 199: 38-43.

D.

LECTURE NOTES

A.

256

Chap. 44 described a sexual mechanism in

Escherichia coli involving the mediation of F

which, like fertilization in higher forms,

1. involved intact cells as participants, and

2. had an entire genome as the unit of transfer.
Relatives of E. coli and some filamentous
bacteria also show sexual processes.

Genetic transductions refer to processes of

fragmentary genetic exchange. One type, dis-

cussed at length here, involves bacteriophage.

Salmonella typhimurium

E.

Lecturer—J. LEDERBERG

1. causes mouse typhoid and is an agent in
human food poisoning.

2. Like its close relative E. coli, itis cul-
tured on simple medium.

3. Zinder and Lederberg found genetic recom-
bination, of erratic pattern, between certain
strains.

a. Mixing an M* T™ strain with an M~ T*
strain produced a filterable, heat-resis-
tant agent with Mt activity.

b. This agent made prototrophs when added
to an M™ indicator strain,

ce. The agent, smaller than a bacterium,
could not be isolated from a pure Mt T~
culture although this must have donated
the M* factor when mixed with the M7 T+
strain.

d. Yet a drop of filtrate from the mixed cul-
ture added to fresh M*carrying cells
evoked more filterable M* factor.

e. Two activities were involved ~- evoking
and transferring Mt.

Bacteriophage as the evoking agent

 

1." The phage P22 is lysogenic in the M7 Tt
strain,

A stock suspension of P22 grown on the
Mt T™ strain yields particles with Mt acti-
vity.

The indicator strain M7 was derived from
the M~ Tt strain and is, therefore, lyso-
genic for P22,

Bacteriophage as the transferring agent

2.

 

1. P22 acquires fragments of genetic material
from the host on which it is grown.

2. Evidences for the association of bacterio-~
phage with the genetic transferring capacity
of the phage suspension -- the transductional
capacity -- include:

a. both show the same temperature inacti-
vation pattern;
b. both have the same susceptibility to an
3.

antiserum that blocks phage attachment
to cells;

c. both attach to susceptible cells simul-
taneously;

d. size and mass of both are the same, as
determined by filtration and sedimenta-
tion tests.

It is strongly suspected that phage that car-

ries part of a bacterial genome is defective

for virus genome -- that there is a replace-
ment of the latter by the former.

Example of transduction experiment in

 

Salmonella

1,

2.

Phage 22 is grown on bacteria genetically
Part of the crop of phage harvested is then
tested on suitable indicator strains (M7,
T, X, Y_, Z_) one at a time.

This is done to show that the phage filtrate
has the same range of activity as the bac-
teria on which it was grown.

. Another part of the crop is now grown on a

new bacterial strain, for example, M? T-

XT yt Ze.

a. The new crop of phage harvested has now
lost T+ and gained Yt.

b. The phage is passive with respect to the
content of the genes it transduces.

. To harvest the phage, the liquid culture is

centrifuged and the supernate heated at
60°C for 20 to 30 minutes (to kill any re-
maining bacteria).

. To detect transduction of Mt, phage is

grown on M* bacteria, harvested as de-

scribed, mixed with M™ bacteria and plated

on agar containing methionine-deficient
medium.

a. Phage attaches and injects its DNA into
the M7 bacterium (see Chap. 46).

b. If the bacterium survives this attack and
if it acquires the Mt fragment from the
phage a clone will be formed.

c. This transduction can by symbolized:

Mt —t@ x m- —>mt

G. Genetic scope of transduced material
1. Usually a single bacterial marker is trans-

duced.

a. Mt rt xt P28 Mot x

b. The latter bacteria are grown on differ-
ent media -- one which selects for Mt,
another for T*, and a third for Xt.

c. When the Mt clones are further typed

they are still T- X-. Similarly, T+

clones are still M7 X~, and Xt clones

are still M~ T7. _

2. In contrast, in sexual recombination, large
blocks of genes are transmitted together
from Hfr to F™ cells.

3. Several markers may be transduced to-
gether in linked transduction or co-trans-
duction.

a. Demerec has shown, using transduction,
that the genes for the biosynthetic se-
quence: anthranilic acid to indol to tryp-
tophan (see Chap. 40), are closely linked
to each other.

b. Different mutants involved in defecting a
particular enzyme are even more closely
linked.

c. Histidine biosynthesis is also controlled
by a cluster of genes.

d. This correspondence between biosynthe-
tic and genetic association, though it does
not apply to some higher organisms (e.g.
Neurospora) may be adaptive in provid-
ing a mechanism for turning on or off a
whole series of enzymes.

4. Any locus in Salmonella is transducable by
P22,

Co-transduction in E. coli

1. From bacterial crosses, Lp and Gal are
known to be closely linked (Chap. 44).

2. When lambda is harvested from Gal* Lp*
prophage cells, with the aid of ultraviolet
light (Chap. 44), it has Galt transducing ac-
tivity.

3. Gal is the only marker known to be trans-
duced by lambda, The typical rate is one
transduction per 100, 000 phage particles.

4, The transduced Gal7 strain is a hetero-
genote (partial heterozygote), having one
complete and unchanged chromosome of the
host (Gal~ Lp) and a fragment (perhaps at-
tached to the chromosome), carried over
with lambda, containing Galt Lpt.

5. Heterogenotes can undergo reduction during
which the Galt may exchange places with
Gal*.

6. Nearly every lambda obtained from a
heterogenote contains Gal.

This was demonstrated by obtaining
lambda from a Galt heterogenote and
cross~brushing it over a Gal™ clone
streaked on galactose-deficient nutrient
agar. The Galt-carrying lambda particles
can be counted by the number of Galt colo-
nies that grow at the zone of intersection.

257
I.

7.

About one hundredth of the bacterial DNA is
transduced at one time. ‘

Recombination mechanisms in bacteria may

 

1,

2.

involve whole nuclei (sexuality).

a. Heterokaryosis, in certain filamentous
fungi, involves concurrence of nuclei in
common cytoplasm and leads to

b. heterozygosis, asin E. coli K-12,

involve fragments of genomes (transduc-

tion) via

a. bacteriophages, like P22,

b. episomes (see Chap. 44), like F and
lambda, and

c. purified DNA, in bacterial transforma-
tion (see Chap. 40).

POST-LECTURE ASSIGNMENT

258

1.

Read the notes immediately after the lec-
ture or as soon thereafter as possible,
making additions to them as desired.

. Review the reading assignment.
. Be able to discuss or define orally or in

writing the items underlined in the lecture
notes.

. Complete any additional assignment.
QUESTIONS FOR DISCUSSION

45, 1. Is the infective F particle (Chap. 44) ap-
propriately placed under the heading of gene-
tic transduction? Explain.

45, 2. What evidence can you cite that the genetic
recombination observed in Salmonella is not
accomplished by a sexual process ?

45. 3. Describe how you would perform an ex-
periment to transduce the X” Z™ loci present
in a given strain of Salmonella.

45. 4. What evidence would you accept as proof
that phage P22 is passive with respect to the
genes it transduces ?

45. 5. How would the results in G of the lecture
notes be changed if co-transduction occurred

a. between M and T only?
b. between T and X only?

45. 6. Should lambda be called a virus or a seg-
ment of a bacterial chromosome? Explain.

45. 7. Compare P22 with lambda, as to similari-
ties and differences.

45. 8. How is it possible to estimate the propor-
tion of the total bacterial chromosome which
can be carried in a transducing phage ?

45. 9. Of all the types of transduction, what is
unique to bacterial transformation?

45,10. Compare the genetic behavior of E. coli
and S. typhimurium.

45.11. Learn what colicins are from the sug-
gested readings. Have they any bearing upon
genetic recombination in bacteria? Explain.

45.12. Do you suppose transduction occurs also
in higher organisms? Explain.

45.13, What are the possible advantages and dis-
advantages of transduction as compared with
sexuality ?

45.14. Do bacteria obey Mendel's laws of inheri-
tance? Justify your answer.

259