TRANSDUCTION IN ESCHERICHIA COLI K-12

M. L. MORSE, ESTHER M. LEDERBERG, AND JOSHUA LEDERBERG
De$artment of Genetics, Uwiversity of Wisconsin, Madison, Wisconsin

Reprinted from GENETICS
Vol. 41, No. 1, January, 1956
  Printed in U.S.A.


TRANSDUCTIOJJ IN ES(`HkXIC'HI:\ C`OLI K-1,'

A S\iSTE?\I of genetic Iransduction has been discovered in the sexually fertile
  K-12 strain of Escherichia di. This transduction is mediated hy lambda, a
temperate phage for which K-12 is normally Iysogenic.

The distinctive features of the lambda-K-12 system include the follolving: (1)
The transductions are limited to a cluster of genes for galactose fcrmcnlation. The
Gal loci are closely linked to each other and to Lp, the locus for latnbda-mainte-
nance. (2) The transducing competence of lambda depends on how it is prepared.
Competent lambda is produced by induct ion of lysogenic bacteria; lambda har-
vested from infected, sensitive hosts is incompetent. (3) The transduction clones
are often heterogenotic, that is, heterozygous for the Gul genes which they con-
tinue to segregate. Technical advantages of the lambda systetn include recombina-
tional analysis by the sexual cycle and the availability of lysates in which nearly
every lambda particle is competent.

The origin and history of the Escherichia cnli K-12 cultures studied hct\ye alread)
been described (E. TxDt3izmxc; 1950, 19.52; IA:IW:II~~IIK(; and L~CDKKHI;KG 105.1).
The emphasis will be placed here on the Gal loci i+ = fermenting galac.tose; - =
nonfermenting and on the locus which controls the maintenance 0i lamlxla iLpt).
The phenotypes of cultures with different alleles of l.pt are as follmvs:

                               I,, SC<1 11s. h,i,,l`i     t.pr> Lp," r"ltllrc
     Lp," culture (semilive)              )-c's            110
     I-p,' cultttrc (I>wgenic)              no            JW
     J,plr culture (immune)               110            no

Regardless of their Lpt genotyl)e, cultures have been fountl to aclsorb Iamlxla.
Thus /,pt+ and 1,~~~ are resistant to lysis by lambda in spite of their ability to atl-
sorb the phage. In contrast with this, mutants resistant to lambdn-2. a virulent
mutant of lambda, are resistant because they do not adsorb either lambda or
lambda-2 under Ihe experimental conditions used here.


hf. 1.. MORSE, E. M. I.EDERBEKG Ah-D J. I,EDERBEKG         143

sugar; minimal agar, U(0); and minimal indicator agar, EMS (J. LEDERBERG 1950).
Special supplements were added where indicated. ,411 dilutions of phage lysates
were made in either penassay or nutrient saline broth, and cell suspensions were
diluted in either 0.5 percent saline or penassay broth.

General methods

Plates and tubes were incubated at 37oC. When high cell densities were desired,
broth cultures were aerated by bubbling filtered air through them. Propylene glycol
monolaurate (Glyco Products Co., Inc.) at a final concentration of 0.01 percent
was added to bubbled cultures to lessen foaming. Phage assays were made either
in agar layer or by spreading a portion of dilute lysate with Gal- cells on EnIB
galactose agar.

Lysates containing lambda in high titer were prepared by two methods: (1)
"Induced lambda" was liberated from lysogenic bacteria after treatment with
ultraviolet (CV) (WEIGLE and DEI.BR~~CK 1951); (2) "Lytic lambda" was har-
vested from sensitive bacteria infected with free lambda. The induced lambda was
prepared as follows: aerated, penassay grown cultures of an Lpf strain (ca. 10g
cells per ml) were sedimented in the centrifuge, the broth discarded, and the cells
resuspended in 0.5 percent saline. The cell suspensions (10 ml) were exposed to the
radiation from a GE Sterilamp (45 seconds at 50 cm) in open petri dishes on a plat-
form shaker. .4fter irradiation the suspensions were diluted with an equivalent
volume of double strength penassay broth and aerated at 37oC until maximal clear-
ing was obtained. This usually required from 2 to 3 hours. To produce lytic lambda,
an inoculum of induced lambda was adsorbed on to penassay grown sensitive cells.
After the adsorption period the cells were sedimented to separate them from the
penassay broth and resuspended in nutrient saline broth. The suspension was then
aerated until maximal clearing was obtained (-1-S hours). Induced lysates have
phage plaque titers of about 3 X lOlo particles per ml, while lytic lysates have
about lOlo.

Induced lambda was used in all experiments unless otherwise stated.

Melhods for lesfilzg for lramductiorl

In order to detect infrequent genetic changes, selective agar media were used:
Ei\IB agar for fermentation markers; EMB agar plus 100 micrograms per ml strep-
tomycin for streptomycin resistance; minimal agar for nutritional markers. About
1Os mutant cells in 0.1 ml broth or saline, and 0.14.2 ml of lysate were added to
the surface of each agar plate and then spread with a bent glass rod. The plates
were incubated 2-3 days before being scored.

Transduction clones selected by these methods develop in a heavy background of
unchanged celis. On ERIB medium, negative cells grow at the expense of the pep-
tone; by using sugar as well, positive clones form papillate outgrowths from the
negative background. EhIB agar serves as an indicator as well as a selective me-
dium; isolated positive colonies are deeply colored, while negative colonies remain
translucent (ilmstrated in fig. 3).

The transduction clones were purified by the following procedure. Papillae were
picked with a needle and suspended in 1 ml of sterile water. A loopful (ca. 0.001


144                    TRANSDUCTION IN E. COLI

ml) of this suspension was then streaked upon a portion of another plate of the
EMB agar. These primary dispersals of the transduction clones were nearly always
mixed. Direct picking and streaking, or spotting without any purification cannot
be trusted. From the primary streaks a single colony that looked pure was picked
to water and streaked as before. This operation was repeated once again, and a
single colony from the last streaking was taken to represent the transduction clone.
In addition to freeing the transduction clone from unchanged background cells,
this method of purification may also act selectively within an unstable clone. Pick-
ing apparently pure colonies leads to an overestimate of the fraction of non-segregat-
ing clones.

RESULTS

Although a number of different loci affecting diverse portions of the genotype
were tested, only genes of a cluster of loci for galactose fermentation were trans-
duced by lambda lysates (MORSE 1954). The Gal loci, of which about seven have
been investigated thus far, are closely linked to one another (less than one percent
recombination) and to Lpi, the locus for lambda maintenance (LEDERHEKG and
LEDERBERG 1953, and unpublished).

The transformation of Gal- cells to Gal+ by induced lambda is illustrated in figure
1. Each papilla is a clone of galactose fermenting cells; on the area of the plate to
which lysate was added, most of the Gal+ papillae are transduction clones. The

FIGURE l.-The protluction of Gal+ papillae from a GO- background of ceils 1,~ a lambda lysate.
I,&, the control, no lysate atldetl. Right. 0.1 ml of lysate iron1 a G(rl+ culture. Some ol the papillae
have I)een picked xvith a needle.


>I. L. MORSE, E. hf. LEDERBERG AND J. LEDERBERG        145

           TABLE 1
Transformalio~t of Gal- cultures by lysales of Gal+

Recipient culture

GalrLp+

GakLp+

Gd-Lp+

Ga&Lpa

Gall-Lpq

Gd-L~~
Gal,-Lpr ~
Galr-Lp'$     I
Gd-LP'$

Lysste of:

Gal+
Gal,-
Gal+
Gd-
Gal+
Gold-
Gulf
Gald-
Gdf
Gala--
Gal+
Gal+
Gal+
Gal+

1  Number of Cal+ papillae

Lambda titer in 1
I"." .

l"L" per ml   Control (no (
          lysate)'

I.1 ml lysate

  ~ 2
1.4
2.4          7

1.4         20
4.9         20
1.4        47
1.7        47
1.4          4
1.7        163
2.3         10
1.7         10
2.3         5
2.3         40
1.4         29
1.6         28

Gal+ papillae pvr
10' lambda

405       2.9
  2        -

3.56       2.4
10        -

394       2.5
SO        -

2112      15.1
86        -

3020       13.1
18        -

1296       5 6
161       0.5
129       0.7
92       0.4

* The Gal' papillae on the control are spontaneous reversals of phenotype
t Different stocks.

1 Different experiments.

quantitative relationships are illustrated in figure 2. The data can be summarized:
(1) Regardless of the LPI genotype of the recipient, transductions were obtained;
(2) with each genotype the number of transductions was proportional to the amount
of lysate plated; (3) Lp8 recipient cultures gave 5 to IO-fold more papillae per unit
of lysate than either Lpif or LPI'. Further, the transducing activity of lysates
(which contain 1Oro lambda per ml) varies according to the number of cells plated:
(1) with LPI+ Gal- cultures there is a two-fold increase between 106-lo9 cells per
plate, with a plateau of maximal yield around lo* cells per plate; (2) Lp Gal- re-
cipient cultures show about a six-fold increase over a similar range of cell platings,
with the highest yield at the highest cell density.

The transducing activity of lysates is specific; that is, a lysate of a Gal, culture
will not transform Gal,- cultures (table 1) but Gal+ papillae were found with a
Gal, culture. The specificity is extended further in that some galactose positive
phenotypic reversals of a Gal- culture give lysates with transducing activity on the
original Gal, indicator (table 2). The different types of phenotypic reversals may be
understood under the following hypothesis: (1) reverse mutations (Gal, to Gal,+)
yield cultures that give active lysates, and (2) suppressor mutations (Gul, Sup-
pressor- to Gal,- Suppressor+) will yield incompetent cultures when the suppressor
lies outside the region transduced.

From the data in table 1 and figure 2 the ratio of the transducing particles to the
lambda particles in a lysate may be obtained. Lp;* recipient cultures give about one
transduction per lo6 lambda; Lp+ recipients, one per 10'. One per 106-10' lambda
will be referred to as LFT (low frequency of transduction).


146                    TRANSDUCTION Ih- E. COT,1

TABLE 2

L.i- recipient culture       Lysate of reversion

Gall-
G&-

Gd-

Gall+ N 1
Gdz f# 1
Gal?' #2
Gal?+ $4.5
Gala+ % 8

Control (no lysate)     0.1 ml lysate

    0            648
    10             96
     6            552
    39            204
    2.5             291

O-






O-

Gal,- Lp+

. /

/

Lambda PARTICLES

FIGURE 2.-Proportionality between amount of lambda lysate (LFT) plated and number of
papillae formed from Lp*`, Lp+ and Ly Gal- cultures. The ratio of papillae to lamhda particles is
tOP for an Lp* culture, lo-' for Lp+ and LF cultures.

      Examimdion of the Gal+ clo?zes formed by frartsductiorz
After purification the transduction clones were examined for changes at loci other
than the Gal series. A number of markers were examined, including fermentative,
nutritional, and phage and drug resistance mutations. The only changes at other
loci were Lp" to Lp+ in lambda sensitive recipients, and occasionally Lpr to Lpi in
lambda immune cultures. Transduction clones from Lp+ recipients were invari-
ably Lp+.

To determine whether lysogeny was causally related to transduction, a recon-
struction experiment was done. To a mixture of lysate and Gal- Lp8 cells, Gal+ Lps
cells labelled with a mutant character were added to estimate the frequency of
chance lysogenization in the untransformed cells in a transduction mixture. After
papillae had formed, they were picked, purified, and on the basis of the differential
label, divided into: (1) the inserted Gal+, and (2) the transductions. The frequency


M. L. MORSE, E. M. LEDEKBEKG AND J. LEDERBERG        l-ii

Types recovered from mixture' of Lp" bacteria and       Number of clones
          LFT lysate                    examined

Percent of CloncS
lysopenized

Inserted GaL+Lac+ `P
             L                        16            68.5
Recipient Gal-La-9 (,non-transformed)           `IQ            i2.5
Transduction Gnl+Lac-9              I      103            100.
                                                      -. ~~
* 10" G&La-S', 100 Gal+Lac+S, 10" lamlxla particles.

of lysogeny was determined in the two classes, and in the Gal- background. Whereas
unchanged Gal- cells and the inserted Gal+ were each only 70 percent lysogenized,
the transduction clones were 100 percent lysogenized (table 3).

When Lp' cultures were used as recipients, 14/112 (12 percent) of transduction
clones formed were Lp+. Although the fraction is small, all previous attempts to
lysogenize these cultures have been unsuccessful. The isolation of transduction
clones evidently selects for these cells that have been infected with lambda par-
ticles from the input lysate.

The original GuZ+ strain and spontaneous reversions of the Gal- mutants have
all been stable in ordinary culture. However, the Gal+ clones formed by transduc-
tion are unstable for galactose fermentation as shown by the recurrence of negative
and mosaic colonies (fig. 3). Despite many serial single colony isolations the galac-
tose-positives continue to segregate galsctose negative progeny. They behave as if

I~IGUKE 3.--EMB galactose ngar plate spread with cells form aculture of a heterogynote, showing
Gal+, Gal- and sectoring colonies.


14x

TKAh-SDUCTION IN E. COLI

                    TABLE 4
Frequency oj instability for galactosefernlel2tation among the transdrtctioiz. clones

Recipient cells         Unstable clones/total examined         Perrent unstable

Gall- Lja             9/22                   41
Gall- Lpi            40/48                  83
Gait- Lp+            22/24                   92
Gal,- Lp            13/24                  54
   Lp'            20/24                   83
   -W            29/48                  60
Gale- L,b            W                  7.5
G& Lp             28/48                  58
  LPC            16/24                 67

heterozygous for a single gene (or short chromosome segment) and may be desig-
nated as heterogenotes. Instability among the transduction clones is quite frequent;
484 of 600 clones (70 percent) were found unstable (representative data are given
in table 4). This estimate is probably low because the purification procedure acts
selectively against unstable clones.

The frequency of segregation has been estimated from the incidence of Gai- in
small clones of heterogenotes. The probability of segregation per bacterial division
is about 2 X 1W3 (table 5). By repeated reisolation, however, heterogenotic lines
can be maintained indefinitely.

The segregants from the heterogenotic clones were examined with regard to their
GUI and Lp character. Lysates of the segregants have no transducing activity on
the Gal- culture that was used as the recipient in forming the transduction clone
and are therefore allelic to it. The same lysates continue to give one transduction
per 1P107 lambda (LET ratio) on non-allelic GnZ- cultures. With different recipient
cultures the Lp alleles of the segregants were (1) Lp+ recipient, all segregants Lp+;
(2) Lp,` recipients, all segregants Lp +; (3) Lp' cells, the segregants were usually
Lp'. In one instance, a heterogenote segregated both Lp+/Lpr and Gal+/Gal-.

Lysates prepared from the heterogenotes have two outstanding features: (1)
instead of containing 1W lambda particles per ml, they seldom have titers higher
than 5 X 108, particularly if they originate from cultures containing few Gal- segre-
gants; (2) the number of transducing particles in these lysates is often nearly equal
to the number of lambda particles in the lysate (table 6). These lysates mill be re-
ferred to as HFT (giving a high frequency of transduction).

         Tram-duclions wi/lt lysafes oj heterogemtes
l'latings of highly diluted HFT lysate with LpS and Lp+ bacteria give a number
of papillae. The number of papillae obtained with Lp" cells is, however, less than
that obtained with Lpf. The lower yield with Lp recipients may result at least in
part from the loss of potential transductions through lysis of the recipient cell or of
some of its early progeny.

With HI:T lysates it is possible to transform a large fraction of a cell population,
and to observe transduction without strong selection. By adsorbing HFT lambda
onto cells, diluting and plating on EMB galactose to obtain well isolated colonies


M. I.. MORSE, E. M. LEDERBERG AND J. L)IDICRBERG        119


               TABLE 5

Heterogenote   ~ :

Vumber of cells in
  inoculum

2.1$

I-

Test clones*

Number of Gal- cells

6
3
4
23
9

19
103

319
22
0

1.5s           11
              2
              8
             3
            52
              0

36
3
6

,     I      5.5
I

Total cells

- :
I

Probability of
qregation per 10"
xxterial ilivisianst

1169
595
251
1252
1113
897
2750
1622
1966
237

~~~-. -

1
1
4
3.6
2
1.3
6.6
36.8
2.0
0

323          8.1
176          3
1669          0.9
317          2
1236          8.2
10          0
1055          6.7
299          2
386          4
1965          .5 1

* .A fully grown culture in penassay broth was diluted to give about 10 cells per ml. Twenty
samples of 0.1 ml mere taken up in 0.2 ml serological pipettes which were supported in a horizontal
position on a tray. The pipettes were incubated at 37oC for 4.5 hours. Each pipette was then blown
out on to an EMB galactose agar plate, and the inside of the pipette washed with 0.1 ml of Ixoth.
The washing was added to the plate, and the inoculum spread with a glass rod. .\fter 18 hours
incubation at 37oC the number of Gal+ and Gal- colonies on the plates was determined.
t Using the equation u = 0.602r/N log N, ( modified for the indicated units from Lr~ra and
DELBK~CK 1943) where I = the number oi Gal- segregants and ,V = the clone size. The probability
of segregation is also estimated by the fraction of cultures containing no segregants.
   n = 2; log&P, = fraction of cultures with no segregants.)

In the first experiment, using N = 21o

   2.3
0 =
   1024 log l/1/19 = 2.8 x 10-a

In the second exl)eriment, using N = 21o
                     2.3
                   a = ~~
                      1024 log l/1/11 = 2.6 X 10m3

1 The assay plates showed this culture to have Gal': Gulk in the ratio 106:4. Of the txventy sam-
ples in this experiment, one contained only Gal+, one contained only Gulk, ant1 18, both Gal F and
Gal-. Only the plates that were counted are given. Nine plates were too crowded to be counted
accurately.

$ The ratio Df Gd+:G& in the parent culture was 128: 19. The twenty cultures were distributed
as follows: failed to grow, 9; contained only Gd +, 1; contained both Gal+ and Gal-, 10. One plate
had approximately equal numbers of Cd+ and Gal- and was assumctl to have come from a mixed
inoculum.


150

TRANSDUCTION IN E. COLI

.
Lp+ Gal-

   . /
/

     :
/       Lp' Gol-
/'         .,:
     `/.

  .I ;/'
I'
     0.05     0.10
ML. of HFT LYSATE

I'JGUKE ~.-I'rol")rtionalit~. Idween amount of HFT Iysste and nurnl~er of papillae formed
from Lp' and L/I+ GulF cultures. For the assays, a Iysnte containing 1.6 X 108 phage/ml was tli-
lutetl a thousandfold.

it is possible to study individual transduction clones derived from single particle
infections of isolated bacteria. At the optimal ratio of about 10 lambda particles
per cell, the fraction of cells transformed ranged from 5 to 15 percent.

       Edenc-e that lambda is the elector (f tramductiorz
That lambda is the vector of Gal transduction is suggested by previous experi-
ments: (1) the 100 percent lysogenization of I-p' recipients by LIT lysate trans-
ductions; (2) the incidence of lysogenicity in transduction to f,pr recipients. In


M. I,. NORSE, E. M. IXDXRBERG AND J. IXDERBERG        151

                                        Number of fermenting clones
Keripient cells (Lp+!        Lambda-2 reactwn' I--
                                    No lysate added
              ---:p. Im~zG.;sate

    Gd-              sensitive              1         126 (LFT)
                     resistant              1             2
    G&              sensitive             20           356
                     resistant             14
    Gd-              sensitive            xc)  ~ l4
                                                  206
                     resistant             SO            .i7
    Gal,-             sensitive              2          10' (HI'T)
                     resistant             3             4

* Lamlxla-2 resistant mutants do not atlsorb lambda or lamlda-2.

addition, lambda and the transducing agent are adsorbed to about the same degree
by Lp8 cells, and both are inactivated by crude anti-lambda serum. Fore definite
evidence was the failure of lambda-2 resistant cells to adsorb either lambda or trans-
ducing activity or to be transformed even by HFT lysates (table 7).

Conclusive evidence that lambda is the vector of transduction is found from the
behavior of single transduction clones: (1) Heterogenotes formed from HE'1 lysate
and I@ cells at low lambda multiplicity are always either overtly lysogenic (Lp+)
or carry a defective prophage (Lp') (table 8). (2) Proportionality between number
of transductions and amount of lysate at high dilution (fig. 4). For a tmo-factor-
system to be invoked at these dilutions, the accessory factor would have to exceed
the lambda by at least lOto, which would imply a concentration of this fancied ele-
ment in undiluted HFT lysate of 10'8 per ml, which should be compared with Avo-
gadro's number.

Early segregatiw of` Lp and Gal irz transduclion clorles

HFT Gal+ lambda was mixed with a culture of Gal, bacteria to give 2.6 X 10'
lambda and 7 X lo8 cells per ml, a multiplicity ratio of 0.04. The suspension was
then diluted and plated on ERIB Gal to give about 100 cells per plate. After 24
hours incubation, on 7 plates, a total of 8 colonies with Gal+ sectors was noted.
Each of these colonies was sectored, with a large Gal- component. Each colony was
restreaked, and 20 to 30 Gal- reisolated from each line. Of the 8 lines, the Gal-
from 3 gave only Lp+, from 5 gave mainly Lp" with a few Lp+. Ten Gal+ (heter-
ogenote) colonies were also picked from each line. All of them were ~!,p+ and of a
total of 297 Gal- segregants subsequently reisolated from these 60 heterogenotic
colonies, all were Lpf also. The frequent segregation of Lp+;`Lp" subclones from
lambda-infected Lp" cells has been noted previously (LEDEKBEKG and LEDEKUEKG
19.53; LIEB 1953). The correlation of Lp+ and Gal+ evidently extended, in the j/8
clones that segregated both markers, to the early intraclonal progeny. Since the
heterogenotes do not continue to segregate Lp", these results are economically in-
terpreted on the basis of the multinucleate character of the bacterial cells. The early
segregation would represent the separation of unaltered Lp" Gal- nuclei from the


152

TRANSDUCTION IN E. COLI

TABLE 8

Incidence of lysogenicity in isolated helerogenoles
--

        1. The fmnsduclions

Gd- cells cxposrd to:



Broth
HFT Iysate*

Unaltered Gal-


  3280
  2801

Kumber of colonies observed
  With Cal+ -    car partially lywl


       0              0
      31              54

Colony type

Number of colonies
  examined

LP"

Number of colonies


I     LP'    I     LP'

*One ml of cell suspension (4.1 X lo9 cells) was added to one ml of HFT lysate (1.2 X 109
plaques per ml, 3.0 X 108 transducing particles per ml) and the mixture incubated at 37oC for 10
minutes. The cells were then centrifugetl down, the supernatant discarded, and the cells resus-
pended in one ml of broth. The suspension was then diluted and plated on EMB galactose agar.
The tube contained 3.5 X log cells after HFT lysate exposure. 1.1 percent of exposed cells vere
transformed, and 1.8 X 10' transductions per ml were accomplished.

nucleus with which the prophage-Gal+ complex has associated. The segregation of
Gal and stability of Lp in the heterogenotic subclones will be taken in later com-
munications.

       The failure to observe trawdltctio~r with lytic lamb&
The experiments described above employed U\' induced lysates. That lytic
lambda, prepared by the growth of lambda on sensitive cells is incompetent in
transduction is evident from the following: (I) lytic lambda failed to augment the
number of papillae when added to Gal- cells on EMB galactose agar; (2) the oc-
casional Gal+ clones that were found on plates to which lytic lambda was added
were all stable and were presumably spontaneous reversions. The lysates used in
these experiments were made by growing induced lambda from a Gal, culture on
a Gal+ culture, and the initial tests of competence of the lysates were made on Gall
cultures. In this way, confusion by "carry-over" of the inoculum phage was avoided.
The experiments were executed on a scale that should have detected as little as 3 %
of the activity per phage of LFT induced lysates.

Pailwe to observe fransdztcfiolc at loci other than Gal

Attempts with LFT, HFT, or lytic lysates to transduce genes at other loci were
unsuccessful.

The unsuccessful tests for transductions of prototrophy to auxotrophic cultures
involved: histidine; leucine (two loci); methionine; proline; glycine or serine; tryp-
tophane.


M. L. NORSE, E. M. LEDERBERG Ah-D J. LEDERBERG        153

The fermentation markers that were not transduced included: lactose (Lacr);
maltose (two loci); arabinose (two loci); xylose; glucose.

The attempt to transduce streptomycin resistance to sensitive cells was unsuc-
cessful

In the E. coli compatibility system, failure to transduce the following was noted:
(1) by lysates of Hfr cultures, F+ and F- recipients to Hfr; and F- recipients to
F+; (2) by lysates of I;+, I;- recipients to F+.

The most extensive tests were made on genes at loci known to be linked to the
Gal series (Hfr, histidine; CAVALLI-SFORZA personal communication, and proline),
or mutations other than Gal (W435, Lac:,-, LEDERBERG and LEDERBERG 1953 and
some auxotrophs) which had occurred coincidently with changes of Lp+ cultures
to LP".

In considering the transduction of specific loci, interactive effects should be kept
in mind. For example, papillae were observed on EfiIR lactose, arabinose, and
xylose, respectively, in tests with multiple marker stocks. m'hen purified, however,
these papillae were negative for the indicated sugar, but gave galactose-positive
colonies. Historically, transduction papillae were first observed in platings of a
treated Gal- Lac- culture on E3\IB lactose. The papillae proved to be Gal+ LUG-
rather than Gal- Iszc+. Evidently, all these sugars have slight selective potentials
for Gal+ clones.

@her obserralioris

hlost lambda lysates are viscous when first obtained. The viscosity is destroyed:
(1) by DNAase, an indication that DNA is the cause of viscosity; (2) spontaneously
at a slow rate. Exposure of lambda lysates to DNAase has not affected either trans-
duction or plaque titers.

Transduction of the Gal gene is not restricted when either the donor or the re-
cipient culture is (1) a prototroph or any of a variety of auxotrophs; (2) f1jr, F+
or I;-, in any combination. Transduction is controlled (1) by the method of lysate
production, and (2) the ability of the recipient cells to adsorb lambda. The only
genes transduced are the Gal loci.

Gal- mutants in E. coli strains other than K-12 that adsorb lambda can be trans-
formed. As in strain K-12 the transformation does not require that the recipient
be sensitive; among the susceptible strains are lambda sensitives, lambda immunes,
and host modifiers of K-12 lambda (E. LEDEKBERG 1954). However, lambda was
incompetent when tested on galactose negative mutants of Salmonella, and trans-
ducing Salmonella phage @INDEX and LEDERBERG 19.52) failed to transform E.
cdi.

DISCUSSION

Galactose-negative cultures of E. coli are transformed to galactose-positive by
certain lysates containing the phage lambda. That this process is genetic transduc-
tion by lambda particles is established by the following: (1) Gal, cells are trans-
formed to Gal+ by lysates of Gal,+ cultures but not by Gal,-. (2) However, Gal,+
obtained by reversion regains its ability to transform the Gal,-, which emphasizes


151                    TKASSDUCTION IN E. COT.1

the role of the donor genotype in effective transformation. (3) The transformed
positives are unstable, and segregate Gal, and not other galactose types. The
various "Gall" used for these experiments include Gull, Gal?, Ga&, Gala, Gul,, Gali
and Gals. (4) All transduction clones obtained from Lp" recipients become lysogenic
for lambda (either f,p+ or Lp'). (3) I ' `ransduction is not obtained with cells unable
to adsorb lambda.

The contrasting features of the E. toll-lambda and the Salmonella systems of
transduction are summarized as follows:

                                          I
Range of penes transduced         R. m/i K-12 phage lambda       Salmonella l,hage I'LTZ?
              I          only Gal             any selertabl~~ marker

Localizaztion of prophage
Competence of lytic phage
Transduction clones
IXficienc~- of transduction, Ixr
phage
Sexual fertility of the host

Lp locus linked to Gd
No
unslal~le heterogenotes
LFT 10-G
HFT lo-'

Fertile, subject to I: compatildity
system

The two systems are alike in the following respects: (1) Genetic factors are carried
by phage particles; (2) The specificity of the transducing particles is determined
by the genetic content of the donor bacteria in contrast to lysogenic conversions
(UE:T.AKE, ET AL. 1955); (3) The genetic material is inaccessible to DNAase and
other enzymes; (4) Transduction occurs without regard (except for quantitative
changes in yield) to the lysogenic or sensitive status of the recipient cells. In both
systems UV induced phage is competent, but lytic phage is competent only in
Salmonella.

However, the two systems evidently do not cross-react; lambda does not trans-
form Salmonella and conversely, probably because of the specificity of phage ad-
sorption.

Several of these features may be related in origin. For example, the limitation
both on the mode of inclusion in the phage (i.e., only after induction of a lysogenic
bacterium), and on the genetic material that can be transduced suggest that the
physical proximity of the Gal loci to the prophage site determines transduction
competence of lambda. This is supported by the linkage observed in crosses of Lp
to Gal. Presumably the linked Gal genes may sometimes accompany the prophage
into the maturing lambda particle when lysogenic bacteria are irradiated. The
failure to obtain lambda particles with transducing activity when the phage is grown
lytically on sensitive cells would be explained on this hypothesis, since the lambda
may have no speciiic association with the Lp-Gal chromosomal segment during
lytic growth.

The heterogenotic clones which result from transduction are isolated through the
effectiveness of the Gal genes that accompany the prophage. In LFT transductions,
this is a rare event; the HFT quality of lysates from heterogenotes may result in
part from the prior selection of an effective fragment and its reproduction as such
in the growth of the clone.


M. 1.. MORSE, E. M. LEDERBERG AKD J. LEDERBERG        155

The persistence of the fragment in transduction clones requires an ad hoc explana-
tion, possibly related to the presence of an LJ!J region in the fragment. For example,
I-p might be closely linked to a centromere; it may function as a centromere itself;
it may be adapted to synapse with the homologous site of an intact chromosome.

At any rate, the Lp region is singular in at least two respects: it is close to a regu-
lar point of breakage in crosses determined by F polarity (LEDEKBEKG and LEDER-
BERG 1953; NELSON and LEDERBERG 1954; CAV.AT.LI-SFORZ.~ and JINKS 1956) and
the I,p segment (considered as prophage) is capable of independent replication as a
phage. If comparable singular regions exist in Salmonella, they have not yet been
revealed in the occurrence of heterogenotes.

The occurrence of sexual recombination and transduction in the same organism
raises the technical question of their experimental confusion. Since sexual recom-
bination requires intact cells, and transduction is accomplished with a cell-free
lysate, sexual recombination can have no direct bearing on transduction experi-
ments. Furthermore, although crossing is completely blocked between F- cultures,
the compatibility status has 110 effect on transduction. On the other hand, the
rarity of LFT transduction makes it a priori unlikely that transduction will signifi-
cantly interfere with segregation ratios in crosses.

Crosses of the various combinations of cultures carrying different Lp alleles will
be presented in detail in further reports. However, they have indicated that com-
binations involving Lp+ (where transduction could occur) do not give appreciably
different frequencies of Gal+ than /-pr X I,p8 crosses (where lambda transduction is
not possible). In addition, the Gul' prototrophic recombinants obtained are stable
for galactose fermentation. Even crosses of known heterogenotes (capable of HFT
lambda) have not given increased frequencies of GaZ+. These observations suggest
that lambda transduction has IIO~ significantly affected results obtained by crossing.

The mosaic colonies of heterogenotic cultures (tig. 3) are reminiscent of those
formed by segregating diploids of B. cnli K-12. The latter, of course, are segregating
blocks of many linked markers, not merely the Gal genes. Diploids are, however,
more diihcult to maintain without the benefit of balanced selective markers. They
segregate twenty times as frequently as heterogenotes, as can be judged from the
appearance of the colonies and from rates calculated from cell pedigrees (7,~t.t.~
and LKDEKH~G 1952 and unpublished).

Further studies involving the use of two or more Gal markers, and relating trans-
duction to sexual recombination analysis will be presented shortly, together with
further consideration of the genetics of the prophage.

Transduction of several Gal+ genes from galactose positive (Gal+) to galactose
negative cells (Gal-) by the bacteriophage lambda has been demonstrated. The
resultant galactose positive clones have been found to be heterozygous for the Gal
region and have been designated as heterogenotes (Gal-i/Gal+). Segregation and
the reappearance of GaZ- from the heterogenotes occurs about once per lo3 bacterial
divisions. The low frequency of lambda particles with Gal genes (1/106) from haploid
cultures resembles other transduction systems, However, heterogenotic cultures


156                    TK.4KSDUCTION Ih: E. COLI

produce lysates in which nearly every lambda particle carries Gal genes. No other
markers have been transduced by lambda, and the competence of lambda in trans-
duction depends upon its production from lysogenic cells, rather than by lytic
growth on sensitive bacteria.

LITERATUKE CITED

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LIEB, M., 1953 The establishment of lysogenicity in Esd~erichia coli. J. Bacterial. 65: 642-651.
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