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GENETEC TRANSDUCTION IN ESCHERICHIA COLI
By’

MELVIN LAURANCE MORSE

A Thesis Submitted in Partial Fulfillment
of the Requirements for the Degree of
DO6TOR OF PHILOSOPHY

UNIVERSITY OF WISCONSIN

1955
f

TABLE OF CONTENTS
log

Introduction - \

Experimental results -\,
\,7 General observations on tasnsduction”
Observation on galactose negative cultures- | 4
BAe de of lysates of wild type awhile Cultue:
ior of lysates of galactose negative Halle cuties - 1 %
br of lysates of reverted gAhactose negative delle cul hues
Considerations of the method of assay of transducing activity ~ e
The necessity of lambda adsorption for transduction - ]
YHELABHERKEIX AF AL SHA ELE XT EXSE AR GABELID EXEIO BEEN SEAR
The activity of lytic lambda—- 4
fhe trensfemmed—-oebts franvducthon clenes—4
i dncidence | of lysogenicity in the transduction clones derived
from Lp® recipient cells-\o
Existence of transductions stable for gatactose fermentation-|?~
The segregants from the unstable transductions -14
Galactose negative cultures giving lysates with HFT property-/ 5S
Experiments with lysates giving a high frguency of transduction- 7
The relationship of lysogenization to transduction -! %
. The interaction of Gal, and Gal, (Position effect) -\4
The action of HFT lysates on Lanbda~2 resistant cultures -2©
Crossing behavior of the transduction clones - J}
z Galactose negative cultures that are not transformed by lysates -2 a

~ Disenssion ~ ol.

}-
Sa Materials and methods ” 4
e-

  

yWwrvevyan dam

cya.

Summary - 32
~ Bibliography -3 |
Figures

~ Tables
OO @

INTRODUCTION
Exchanges of genetic matérial between bacterial cells can be
classified into two main categories ( Lederberg, J., 1954). The first
category is exemplified by the recombinational process found in
Escherichia coli K-12 by Tatum and Lederberg (1947). This form of gentic change
includes a syngamic process, that is, the conjunction of large blocts of
genetic material, and there is evidence of linkage groups, linearity of
genes, and requirement for intact cells ( Lederberg, J., et al, 1951,
| Lederberg, Je, 1954).

| Under the second max main category are found the exchanges

where one of the participating cells is not found in intact form, but

whose genetic material is presented as a solution or suspension of

 

J
particles much smaller tha the cell, —— This category

has been given the general ‘title of transduction (Zinder and Lederberg, 1952,

4.
Lederberg, 1954) J and is readily subdivided into two classes on the basis of

sub of thansduchor-
the vector of recombination. The first clase is exemplified by the pneumococcs

transformation sy stem, (Austrian, 1952), where. the genetic changes are , prought
; {D -

about by means of purified preparations of desoxyribonucleic nein In the

second subclass the genetic changes are mediated by Bacterse® viruges or

bacteriophages ¢ Zinder and Lederberg, 1952, Waianae TORRES iq Pome,

syn aw,
“WORD. In contrast watt Ll lee ater cng : genetic transduction usually

results in monofactorial genic changes, although dual changee have been
noted ( Stocker, Zinder and hederbere, 1953. Hotchkise, 1954).

The frequency of occurrence of these exchange processes among the

uw

|
|
‘various genera of bacteria is not known, Genetic recombination of the E. coli *
2

+

‘K-12 type has been observed in about 50 additional strains of E. coli of over
[Ons Sim low te
2000 Santas (kederberg and Tatum, 1953). fransductim rinse atta — ©

pneumococcal teecmetersetst=a— ime have been observed in flomophilus infl infl nensag
A?
~@

( Alexander and Leidy, 1951), Weisseria menigitidis ( Alexander and

Redman, 1952), and Escherichia coli ( Boivin, 1947). While strains

of BE. coli are reported to show syngamy and traneduction, ESUELIEK

Boivin's culture has been lost and farther studies with it are

impossible. Att8mpts to transfer genetic material via desoxyrivonmerst:
acid preparations in E. coli K-12 have been unsucessful. ( patchly, 1951).

In Salmonella, Zinder and Lederberg (1952) demonstk&ted phage mediated
transductions but failed to show the occurrence of syngamic recombinatéon.
Thus, of the three forms of recombination considered, no one culture has
previously been observed to exhibit more than one of the exchange processes.
It is the purpose of this thesis to describe a limited system of transduction
in BE. coli mediated by the lysogenic phage of strain K-12, lambda. The

occurrence within the same sgrain of syngamic recombination and of phage

mediated transduction promises to improve our underatanding of both

processes.
(3)
@

The principal cultures used are listed in table 1. In summary

MATBRIALS A.D METHODS

they represent mutations at three distinct loci which lead to the 1688
of ability to ferment galactose. Such matations have been obtained by
irradiating galactose positive cultures on an indicator medium, EMB
galactose agar. The different loci have been atstinentenothy intercroasing
the various stocks and. finding galactose positive recombinants in
certain crosses (Lederberg, E, 1950). *he Gal,- and
Gal, stocks are the result of a single mutation’ to (- ) in each case,
while Gal. , stocks ‘represent two independent motations to (-) whose
identity is vased. upon the observation that no galactose positive
recombinants have been observed in more than 11,000 prototrophic -
recombinants from crosses between them and upon the synonymous behavior
of the stocks in transduction experiments. These three loci are closely
linked to one ‘another as indicated by the data in table 2, but the order
of the loci is not specified.

In addition, each of these loci is known ( Lederberg, 3 and
Lederberg, a 1983 ) to be closely Linked to Aa Le, latent phage )
Locus of 3 col K-12. Tiree alleles are known to exist at the Lp locusg ¢
(2) Lp’, ¢ overtly lysogenic am (showing evidence of free phage in cross

rashes with Lps forms} and resistant to lysis by free lambda phage,

 

(2 2) Lp* ‘f hot overtly lysogenic

Ss Ee seer eee, wot! (Peststant to lyste by free lambda

i>
rhage, (3) Lp 8, not lysogenic, and — lysed or lysogenized by free

hage.
\

At least two other loci affect the interaction of lambda with
E. coli E-12. and are scored by resistance to lambda-2, the lytic

mutant of lambda, One of these shows a coincidence change in maltose

4h
fermentation. Both mtations result in a loss by the cell of ability
either
to adit lambas or lambda~2 regardless of the state at the Lp locus.

Methods and media were as detailed in Lederberg, J.(1950).
Liquid cultures were in penassay broth, with or without aeration; solid
media were of EMB base, either with or without added sugar, or Dikco
nutrient agar with 0.5 percent saCl. Vor crosses, a synthetic form of
EMB, EMS)was used. | .

High titeréd lambda phage lysates were prepared by two methods.
The first ank most commanly used was that of Weigle and Delbriick(195/)
in which induction by ultraviolet radiation (UV) is used. The UV was
administered to penassay grown cells resuspended in saline at a density
of about 10? per ml, After irradiation the cells were diluted with double
strength penassay broth and incubated at 37C with aeration until maximal
clearing was obtained. " Lytic " lambda was prepared by infacting lambda
sensitive cells with UV-induced lambda; the infected cells were resuspended
in nutrient saline broth. These suspensions were then incubated at 37C

with aeration until maximal clearing was obtained. Lysates prepared by UV

10 per ml, whereas the lysates prepared

induction had titers in excess of 10
by the other method had slightly lower titers. Unless otherwise specified,
the lambda used in the following experiments was obtained by UV induction
of lysogenic bacteria.

Crosses were performed by mixing % salinre suspensions of penassay
grown cells either before plating on the EMS synthetic medium (usually with

added galactose) or directly upon the plates
“@

in which the culture is atreaked screee either phage or phage sensitive

Teats of sultyres for phage reaction vere by the cross brush method

cells to ascertain whether or not 4t ‘earrying phage or sensitive te

phege ( Lederberg, M% ani Lederberg, %% 1953).
. lysates qving 4
" Pransduotion aseaya were made in the case of <2 normal, hus
frequency of transduction Snes by adding 0.1 ml of lysate to the
sppropriate cells on EMB galactose agar and incubating the’ mate for a
48 | hours, A separate plate with 0 lysate bddod. served as an cttinete
in ether cases
of the amount of spontaneous reversion occurring, ox, the lysate was
spread only apen one-half of the plate, With tn lysates elving 5... Oa

(Her)
high frequency © of frenaGngstonsthe lysate was eross brushed cerns oe

 

on the cells, ry pa lis He tosts phage sensitivity.
x

©

EXPERIMENTAL RESULTS

General @deervations on transduction

of a number of loci selected at yvaudom

 
 

t for ability to -

 

Tests
he eave negative

 

be transduced #4

results (table 3). ‘The tests for thansduction of the auxotrophic markers
were performed by adding lysate to cells on minimal medium, the tests
on fermentation markers on EMB medium with the appropriate sugar, Agdis

was performed
“The teat for transduction of streptomycin resistance ey growling the

“G- addition = © | : ne roe
oe Glactose negative cultures unable to fermert an additional
‘weer carbohydrate such as lactose, xylose, and arabinose ( Ee Lederberg,

unpublished) will give apparent transductions wheh plated with phage |

on media containine these substances. Such apparent tnpfpauctions are

, “hot ‘for the fermentation of the carbohydrate in the medium, but for

wih £4 veh

* palactoge: fermentation,’ ainse: after purification: “the” ‘traneductions | ,

  

” Slonee’a are ound only galactose positive. Media containing these substances

. . Veet ae
a ke ney tee Oh Lotaeg

have some selective: action’ on galactose fermenting clones. ;

st dheghee. ek ote ce whew oe, bese d feces
Bo SI Stk Ee ALS ee wee bh ADV anee

in the mumber of galactose fermenting papillae are observed (table 4),

 

The number of galactose fermenting clones is proportional to the

amount of lysate added (figure 1). Since eack of these mmtations to
inability to ferment galactose ia capable of reverse mtation the data
mast be corrected, {n-each—case. This haa been done for the data in
figure 1 by subtracting the mmber of spontaneous reversions as
determined from control platings with no added lysate. In addition to
indicating proportionality, the data in figure 1 indicate that the cells
show the effect irrespective of the Lp genotype of the cell, and that
O.

rk

dambda sonsitive om are more capable of showing the effect of added
lysate than lysogehia chkltures,
See AY: ot reJ
ae of _galactose negative Sakis. |
When lysates of galactose negative cultures are mixed vith

the various galactose negative cells results similar to those shown in
table 4 are obtained. With the possible exception of the interactions
of Se and Galys seach of the lysates is capable of evoking galactose
fernenting popilise upon plates apread with nemhonologous negative

- celle. with the umal lysates Gals Cady, interactions are erratic, sone-
tines giving stentficant differences detween control and Lysate added
plates, sougtines not. This lateraction will be dealt with in more
detail in a later section, it will be eufficient to state here that

pos drvl

such interaction does not produce elenes that are phenotypically SB.

ot Mese loci
The differentiation aby lysate interaction corresponds to the

differentiation Apppest-b6ek by recombinational analysis.

Actiuit
- 3, Babotte of Lysates of reverted Balactose negative cultfires.
: Reverse mation restores the ability of lysates of a galactose

Md 2s hoe _-_ a FP oo daw

Rte Seyi St wes boat
4 OR PRESS . PAR Ye on . ‘

: Ae .

, ay

wee 3
FP oseae Be eat eas aS as an Let ha rn ee
; 1 4 ve ‘ oR Ra eR ne ES geegtoos.

Minte: reversals should be able to evoke: pepttiee | fron. cells of a a
original matant type only in the improbable: event, that they | are located

| _ in the restricted genetic. sognent, thet appears to be capable ¢ of genetic

ee t4 ay oy Tee SR EE aT 2 ee at

ee RE Te ee ee

. e eet oo Mae wag ek ae Kg dpe atin nae hha Shee

Aue wees a
ne My ME GALA Sha ELEE

eae 6! ef the transducing activity of a lysate by the method
sakithpcles
of mixing lysate and cells on the plates appears to be gam in the case
of lysogenic cultures, the variation being lesa than two-fold over a

thousand~fold change in the number of cells plated. Cell concentrations
©)

oPTIMAL

Detween 5 X 107 and 5 X 108 appear to give Ee cscctson of lysat:
activity. When the assay cells are lambda sensitive the variation is
two to three fold greater over the thousand-fold range of cell values
from 106 to 107, with increasing assay values as the number of cekls
increases. Since the ratiog of phage particles to transducing particles
in a lysate is very large the interaction between lysate and sensitive
cells is compl ex, axa xan with. the great probability that the inactive

phage particles Rion influence the expression of the transducing particles. |

a ee ee ee

 

The ratio of transductions to phage content of the lysates varies,
-6

approximating 107? for lysogenic assay cells, about 10 ~ for sensitive

cells, that is, about a ten-fold difference in efficiency.

The necessity of lambda adsorption for transduction
The necessity for lambda adsorption for transduction is illustrated

by the results given in table 9, When the various galactose negative cultures
are lambda-2 resistant, a combination which is incapable of adsorbing

either lambda or lambda-2, transductions are not obtained. The ability to
transform a galactose negative locus found coupled with lambda~2 resistance
is demonstrable when a suitable out cross is made and the galactose negative
lambda-2 sensitive recombinant obtained. Lambda-2 resistance does not

effect the ability of a lysogenic culture to give rise to phage and

transducing particles after UV induction,
g
oot vty q)

he palfterot lytic lambda. * ~
tHe transductions described thus far have been effected by

 
  
  

eans of yeates Prepared, by the ultraviolet intuotion technique.
Lyeates prepared by igtic grovth of the phage ona sensitive calture
pparently havé no transducing activity end have lpst the transducing

  
  

activity included in the starting Sam phage {nooulun (table ®): a

JN

The pasihe chm gfgnes

With the exception of the Lp locus in the case of lambda

 

sensitive cells, no changes have been observed in any of the other genetic
characteristics of the transformed cells. Many of the galactose fermenting
clones produced vy transduction are different from the spontaneous reversions
in their instabilfty for galactose fermentation and in some cases for
lambda reaction. That is, they continue to segregate galactose negative
clones in the course of many serial {solations. In addition, in the case
of the transductions with Lp” reaction there is seer dgétion for lambda
sensitivity with segregation for galactose fermentation. Lysates from unstable
transduction clones also differ from lysates of galactose reversions: in
the former the ratio of transductions to plaques 4s much closer to unityy
(table 8).

Lysates of the cultures unstable for galacbose fermentation

when prepared in the manner of the other cultures
© @

have lower phage titers. The reason for this is not known but the

production of phage in these lysates is being studied further, With
the exceRtionyl of transductions formed with wild type lysates, the
transduction titer of these lysates is dependent on the genotype of

the assay culture.

When portions of these lysates are cross brushed on galactose
negative cultures the intersection of the streaks is converted principally -
to galactose positive growth because of the high frequency of traneduction

(HFT), The problem of the HFT lysates will be dealt with in more detail

in a later section.

 

Incidence Sfclysogentcity in the transduction clones derived from Lp®

recipient calls,
When MIT lysates are used in transductions to Lp® recipient cells,

about 90 percent of the resultant transduction clones are lysogenic (Ip’)
or Lp’. There is some slight evidence for lambda sensitive transductions,
but these putative transductions have been found stable for galactose
fermenattion and it has not been possible to distinguish them from

spontaneous reVersions except by their frequency of occurrence,
@

When Lp” cultures'are treated with lysates a small fraction
(3-5 percent) of the segregants from the resultant transductions are
lysogenic whereas it had not. been possible to lysogenize Lp’ cultures
with previous methods (Lederberg and Ledenberg, 1953).

The high incidence of lysogehicity in the transduction clones
may be misleading owing to the excess of phage, and it cannot be ascertained
whether lysogenization took place before, concomitant with, or after
transduction, by the NFT phage. In the section on HFT lysates the rdefftonentp
between transduction and lysogenization will be shown more clearly.

The segregants from the transductions with Lp" reaction are
Lp’, while the segregants from the Lp” transductions are Lp” and Lp’.

In speaking of the Lp* reaction it shpnld be noted that the
classification of Lp” is more subject to quantitative considerations than
the other alleles of Lp. The two cultures (W1924,W1027) derived from sources
other than transduction that showed no plaque forming phage in cross brushes
with sensitive cultures gave plaque forking phage after induction with
ultraviolet radiation, The amount of phage was greatly reduced over that
obtained from Lp’ cultures under similar conditions. These two cultures
were obtained after separate procedures, one from an ultraviolet
irradiated Lp’ GW1tti@, the other from an Lp* culture treated with lambda
(KE. Lederberg, unpublished), Both were stable as regards their lambda
reactions, The Lp” clones observed after transductiion have not given plaque

they |
forming phage after U.V. exposure, but differ from those which have given

phage, by instability at the Lp locus

Whether the bransductions with Lyp* reaction are the results of

heterogeneity among the phage particles, the cells, or as the results of a

"defective" >
act of lysogenization is not known, but presumably the problem could
be investigated by statistical or |
Existence of transductions stable for galactose fermentation.

Te evidence for the occurrence of stable transductions is the
increased number of stable galactose positive clones a on lysate
platen gamer “than eqpucrtet Geom combust piakines{ tabséa 4). Although the
increase could also be explained on the assumption of a change in

oe fayorin Spmrancout reversions ©: tinding that molt oo Chen ave alts ;
selective condi tions 6 fac eated, lysates ( 56C for 30 minutes y : “yr peme
tn Phe Coan |

hh or filtrates of galactose positive, lambda sensitive cultures gave 7 et
no increase in number of papillac, suggests. that change in selective Gud

conditions is ‘not the case.
THE S&6¢téGanre From THe UNL TABLE TRANS OUCH NS ae
— The non-fermenting segregants from the unstable treneincttiy

   

et

negative cultures against tyeates of the segregants,(3) by cresses wih
known galactose negative types. In Classifying the segregants it will

be convenient to fefer to the BSERSKARERXSEXERS parental source of the
negative allele or alleles by generalized designations. By idiotype is
meant the genotype of the recipient cell parent, by allotype the genotype
of the donor source of the transducing lysate, Amphitypic will designate
cultures whtch at some joci are. idiotypke and at others are allotypic. le
Unstable. or. segregaine stocks,.as will appear, are heterogenotes and

the underlying state is described as heterogenic to distinguish it from [|
euploid heterosygoeis for satire gence, oo aa . wo |
= For further analysis tt will ultimately be desirably * rsp
ee rut single cell pedigrees. The following observations on wy }

     
 

cues Age MADE
Yaolations, with due regard to the complexities of colonkey fo
three
methods, and ‘some cates (table 10) by all mothods., Tables 1% and 19.
set nt Behe esos oo Boga Bay coe: ‘ a
Present wmitmarios of the analysis ag transduction zeeipients and ag
#. felgee PRAM 8 apo nay cE Eee May eee Cae RET

: for this purpose
stock is ts aratighg

fe ON SS a Shen etal ot tant See eeegts. brag
is, a culture Slassified: by the. first method mas: = aly was alec classified
as: this wits by. the other tire teste, : pe no
mo | ree segregint . re classifi hi ty
against Iyeaton of known cultures, 3 ¥ re a 3 - Gat,-, and one was
Gal,- Gal,~. “The former Were Prototrophi and it was not possible to

 

   

exanine their behavior in crosses, the al, Gad, culture is Croseable

but has not veon tested Rereteceiidl a0 yet. ce

Because ot the Gal;- ean, interaction it is not Feasible to test
loc

any of the anph typie segregants weing only. the threes 80 far considered,

. Attempts were made te analyse the amphitypes. further oy the action of.
their lysates on an m addi tional locus, Gale-. Lysates of the two Gal, ~Gal,-
were plated with cells of a alge culture, Both lysates had little action
in producing papillae . (This perhaps might have been expected since ated, ae 8
@al,- fauna on, Galg-). Several unstable galactose fernenting
clones wétte ‘obtained from each Interaction, Aoverer, and a amunber of:

segr egants were tested. or 16 segrogante trom the ‘transtuotions by the
lysate of one aephitypic culture, 15 were dal, - ® and one was ‘classified as
Gal)- Gal,-. From the action of the lysate ef the second amphitypic culture
five Gal,- and two Gal,- segregants vere obtained. Although both lysades
@)

wag ale ofiels
transmitted Gal,- and Galo-, confirming the existence of these Gm in-
the: parental. enltures,: the failare to recover the idiotypis Galg-~ locas

 

among the negregants is disturbing.
Beyond the fact that Gal, is @ locus transduced by lysates nothing is

known of. its behavior. MEE a teat)

Al th th ic cultures ar % tranef ed to wild.
ough o amphitypic, ef are no orm we bind

, 4
type. by, the. action of a Miisiaahe: pure. ven

   
    
 

. cE. the. _stattatios, of, the interaction of cols and, Lysate hate not OS ik det
_ deen investigated, wat the greatly reduced pander: of transinetions produced gril eed ¢
y the mixed lysate is expected on the. assumption of indepenient interaction a

 

‘Dehween the cells and each of the. transducing activities,
. The tranesductions produced by the action of. nixed lysates on
. amphitypie segregants appear to be less atable than traneiuetions of caltpres ~~ oe
| Saenecative at a single galactose locus. In addition they give rise to
_ Sintermediate" segregants in whieh only one of the éwo transducing activities
has Been los} from the WMA clone. grese "intermediate" segregants in turn
give rise to Teregante from which both transducing activities have heen

lost, t ‘
Under the section on transformed cella itk was noted that in lysates

of the unstable galactose positive clones the ratio of transduction titer
oe eck PE MARKABLY

0 Kew plague titer was gubye, high ny, Aenea herent apenarre—

Ye te sn ceenenaTE clamped these cultures were not the

first ‘to cive HY? lysates. In the course of eine ergata fron

A lysates
traneduotion, by means of lysates of then, several excepite waitress were

 
 
 
  

of transduction. aq

 
     

ee for the

ee =a bn meee eet
. ilar
these exceptional culture no aifrerent: fron -. other secregants.

 

vere: ‘unstable -

guachy

Regarding the Latter inatabtlity, HFT : cultures which were negative ‘at a.

 

for this property ond uastanle, on, rare. oncastons ‘for ng

single locus segregated MFT segregants. that were negative at this lecus |

and pip Miah ‘wane negative at sh addi tional locus as well. In most...

instances , hovever, the BFE segregants were of > ome nesative Sea Kr THe SAME
os the parent galactose negative HF? culture.

the galactose positive yeversions of the ‘ae cal tures that have

been studied are still capable of giving HY? lysates, Tat are unstable
for galactose fermentation, , The galactose negative sogregante fron the

reverted ne ‘cultures are at, are elther negative at the same locus as.

the original negative HY? segregant, or negative at this locus and negative

which proved tole th thd ovig (nad

- at an additional locus, one, was th tadotyst locus in the fornation
Zz