GENERALIZED IRANSDUCTION OF A
GALACTQSE MARKER IN SALMONELLA
GALUNARUM-PUtLORUM

Theslsx ‘09 the Dogma of M. 5.

MECHIGAN STATE UNWERSETY

Richard Willis Vaughan
1962

THE-SIS

 

LIBRARY

Michigan State
University

 

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I

 

ABSTRACT

GENERALIZED TRANSDUCTION OF A GALACTOSE MARKER
IN SALMONELLA GALLINARUM—PULLORUM

by Richard Willis Vaughan

Phage mediated transfer of the ga1+ character in
Salmonella gallinarum-pullorum was demonstrated and partially
characterized. Transductions were performed with phage
lysates produced by ultraviolet induction of strain 38 and
with lysates of phage P38, propagated on strain 35 or 53.
Galactose fermenting clones were distinguished from a back—
ground lawn of galactose nonfermenting cells by their appear—
ance when overlaid with agar containing 0.05% 2,3,5-triphenyl-
2H-tetrazolium chloride. Fermenting clones appeared red on
a white nonfermenting background. Inter- and intrastrain
transductions were obtained. Little difference was observed
between the transducing efficiency of UV-induced P38 lysates
and lysates of P38 propagated on strain 53. At a multiplicity
of input of 1, 90-99% of the P38 phage particles were adsorbed
within 25 min.

GENERALIZED TRANSDUCTION OF A GALACTOSE MARKER

IN SALMONELLA GALLINARUM-PULLORUM

By

Richard Willis Vaughan

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Microbiology and Public Health

1962

Dedication

To my wife for her understanding and

moral support.

ii

ACKNOWLEDGMENTS

The author wishes to express his appreciation to
Dr. Delbert E. Schoenhard for his encouragement and
guidance in this work.

The author is also grateful to Miss Mary J. Robinson
for her gift of mutant cultures and to Mr. Richard B. Dardas
for his help in producing the phage antiserum.

Financial assistance from the Agricultural Experiment
Station for the period 1961-1962 is also gratefully appre-
ciated.

iii

TABLE OF CONTENTS

INTRODUCTION . .
HISTORICAL. . .
MATERIALS AND METHODS

Bacterial strains

Phage . . . . . . . . .

Media .

Incubation . . . . . .

Ultraviolet source. . . .

UV survival curve . . .

UV induction of mutants

Preparation of transducing lysates
Spontaneous . . . . . . .
UV induction of P38

Method for phage assays

Preparation of P38 antiserum

Phage adsorption determination.

Transduction procedure

Test for lysogeny of transductants

RESULTS. . .

Production of mutants.
Adsorption of P38 to strain 53.
Assay of P38 antiserum

iv

21
23
23

Page

Detection and specificity of gal+ clones . . . 26
Transduction . . . . . . i. . . . . . 28
DISCUSSION . . . . . . . . . . . . . . 35
Production of mutants . . . . . . . . . 35
Adsorption of phage P38. . . . . . . . . 36
Detection and specificity of gal+ clones . . . 37
Transduction . . . . . . . . . . . . 38
SUMMARY . . . . . . . . . . . . . . . 42
BIBLIOGRAPHY. . . . . . . . . . . . . . 44
APPENDIX . . . . . . . . . . . . . . . 49

Table
l.
2.
3.

LIST OF TABLES

Bacterial mutants . . . .
Specificity of red papillae.

Transduction of the gal+ character to
gal” recipients. . . . . . . . .

Infection of 53 gal'2R with transducing
lysates . . . . . . . . . .

Efficiency of transduction as a function
of MI . . . . .

vi

Page

28

29

32

34

LIST OF FIGURES

 

Figure Page
1. UV inactivation of strain 53 . . . . . . 22
2. Adsorption of P38 to strain 53 . . . . . 24
3. Neutralization of P38 with a 1:1000

dilution of anti-P38 serum. . . . . . . 25
r
4’ £38§3S DNAse transductants . . . . . . 31
53 gal 2
5. 53 8&1'2 control . . . . . . . . . . 31
6. E3§E3§-- transductants. . . . . . . . 31
35 gal 6
7. 35 3&1'6 control . . . . . . . . . . 31
8. Transductant papillae . . . . . . . . 31

\

vii

APPENDIX TABLE

Table Page

6. Typical reactions of Salmonella
allinarum~pullorum strains on
erential tube media . . . . . . . . 5O

 

 

viii

INTRODUCTION

Transduction, a form of infectious heredity, was first
observed by Zinder and Lederberg (1952). Transduction
mediated with bacteriophages involves: (1) infection of
donor cells with phage particles, (2) multiplication of
phage particles within donor bacteria (1ytic cycle), (3)
acquisition of a fragment of donor bacterial genetic comple-
ment, presumably at random, (4) infection of recipient
bacteria with the matured donor phage, and (5) expression
of the acquired genetic fragment in recipient bacteria
(transductants). Direct evidence that the genetic comple-
ment involved in transduction is deoxyribonucleic acid (DNA)
has been provided by Kaiser and Hogness (1960), who have
transformed recipient bacteria with DNA isolated from a
transducing phage preparation.

The experiments studied in this research were prompted
by the report of transduction of galactose (gal) characters

in Salmonella gallinarum—pullorum (Snyder and Schoenhard,

 

1960, 1961; Snyder, 1961). It seemed desirable to clarify
and partially characterize the general transducing system of

S, gallinarum-pullorum using the gal marker.

 

Galactose negative mutants were obtained from two

strains of S, gallinarum-pullorum, and successful transduction

 

l

2
was performed. Transduction was demonstrated with lysates
of phage 38 (P38) prepared by UV irradiation of strain 38
or with lysates of infected wild type strain 53 or 35.
The results reported are expected to serve a twofold
purpose: (1) a foundation for further experiments to map
the gal chromosomal region and (2) a model for elucidation

of the arginine segment.

HISTORICAL

Since the original discovery of transduction, three
processes have been shown toLbe possible: general, special,
and abortive transduction.

Generalized transduction has been observed with phage

PLT22 in Salmonella typhimurium (Zinder and Lederberg, 1952),

 

phage P1 in Escherichia coli (Lennox, 1955; Jacob, 1955).

staphylococcal typing phage 53 in Staphylococcus aureus

 

(Morse, 1959), and bacteriophage SP—lO in Bacillus subtilis

 

(Thorne, 1961). The phenomenon has also been shown to occur

 

in Vibrio cholerae (Bhaskaran, 1958), Pseudomonas aeruginosa
(Loutit, 1958; Holloway and Monk, 1960) and Proteus mirabilis
(Coetzee and Sacks, 1960a). Any of the genetic characters of
a donor bacterium can be transferred by phage particles to a
recipient bacterium, singly or in pairs (Lennox, 1955) and
the transductants may or may not be lysogenic for the trans-
ducing phage (Adams and Luria, 1958; Coetzee and Sacks, 1960b).
The frequency of genetic transfer in generalized transduction
is quite low, 1 x 105 - l x 108 per infecting phage particle,
for any one trait (Adams and Luria, 1958).

Specialized transduction involving phage I3 of E,
coli (Morse et.gl,, 1956a,b) results when A. is reduced to a

defective state. Luria, Adams, and Ting (1960) reported an

3

LL
analogous mechanism utilizing phage P1 to transduce from g,
ggli_lactose fermenting (1ac+) donors to Shigella dysen-
teriae Sh lac“ recipients.

Phage 2. becomes defective when the bacterial genome
galactose (gal) region substitutes for a segment of the A.
genome (Arber et_gl,, 1957). Defective lambda ( A.dg) is
genetically blocked in its ability to enter the 1ytic cycle,
however, ultraviolet (UV) irradiation of bacteria carrying a_
or Adg induces 7x or Adg to enter the lytic cycle (Weigel
and Delbruck, 1951; Morse et_a1,, 1956a). The induced lambda
lysate will transduce only characters of the gal region, 70%
of which are unstable. Upon repeated isolation unstable
transductants (heterogenotes) segregate gal' progeny. UV
induction of heterogenotes produces lysates in which trans-
ducing particles approximate the number of 7Lpartic1es
(Morse 333;” 1956a, b).

Abortive transduction was discovered by Stocker,
Zinder, and Lederberg (1953). In abortive transduction there
is only one bacterium present in the transductant clone, at
any given time, which contains the donor genetic fragment
(Stacker, 1956; Lederberg, 1956). General transduction
involves integration into the replicating genome, resulting
in true clonal inheritance of the transduced trait. Abortive
transduction occurs when the genetic fragment is not inte-
grated and does not replicate, being passed unilinearly from

the recipient cell to only one of the daughter cells at each

5

cellular division (Hartman and Goodgal, smear).

One maJor problem in genetic research is the develop-
ment of a suitable technique to study a desired trait.
Since mutants deficient in their capacity to ferment galac-
tose were the object of this investigation, a suitable
indicator medium which would permit colony classification
was required. Eosinemethylene blue (EMB) agar (Lederberg,
1950) is an extensively used indicator medium for this
purpose. Although EMB is satisfactory for transduction
analysis of fermentation mutants of S, gg}l_(Morse g§_§l,,

1956a) and s. typhimurium (Zinder and Lederberg, 1952), it

 

does not work well with S, gallinarum-pullorum. Lederberg

 

(1948) reports the use of triphenyltetrazolium chloride
where fermenting clones remain white and nonfermenting
clones stain red with formazan. Ogur §£_§;, (1957) report
the use of a triphenyltetrazolium chloride agar overlay
technique to detect respiration-deficient mutants in yeast,
the deficient mutants remaining white and the respiring
colonies turning red. Jordan 32 El- (1962) report the use
of triphenyltetrazolium chloride incorporated into the
plate agar to detect fermentation clones, the nonfermenters

remaining white and the fermenters staining red.

MATERIALS AND METHODS

Bacterial Strains

Four strains of S, gallinarum—pullorum were employed:

 

strain SP25 (Massachusetts 1), strain SP35 (South Carolina
C), strain SP38 (South Carolina F), and strain SP53 (Kentucky
CD03522). Mutants of SP35 and SP53 are listed in Table 1.

TABLE l.--Bacteria1 mutants

 

Designation Production

 

39-35 gal—i UV-induced
SP-35 gal-6 . HNO2 induced*
SP—35 gal—l HNo2 induced*
SP-35 galfié HNo2 induced*
SP-53 galwi UV-induced**
SP-53 galIé Spontaneous
SP-53 gal—'2R Spontaneous
SP—53 gal—é UV-induced
SP-53 Sr Spontaneous***

 

*Obtained from D. E. Schoenhard.
**0btained from J. J. Robinson.
***Obtained from R. w. Snyder

7
Strain 38 carries the bacteriophage P38. Strains 25,
35, and 53 are sensitive to and lysogenized by P38. All
bacterial stocks were maintained in duplicate 0.7% Brain
Heart Infusion (BHI) agar stab cultures kept at A C and at
room temperature. Working stocks were maintained at room

temperature on BHI agar slants in screw cap vials.

Phage
Phage P38 is a temperate phage isolated from mixtures

of bacterial strains 38-35 or 38-53. Initial isolation was
done on BHI agar plates according to the standard soft agar
overlay method of Adams (1959). The phage particles were

suspended in T2 buffer (Hershey and Chase, 1952) and stored

at h C in screw cap vials over chloroform.

£23.13.

Difco BHI agar, BHI broth, nutrient agar, and nutrient
broth were used where indicated.

BHI soft agar contained 7 g Bacto-Difco agar, 37 g
dehydrated BHI broth, and 1000 ml distilled water. The
sterilized agar was dispensed 2.5 ml per sterile 13 mm x 100
mm test tube.

Soft agar (7 g Bacto-Difco agar/1000 ml distilled
water) was dispensed 2.5 ml per 13 mm x 100 mm test tube.

M-9 broth (Dr. Myron Levine, personal communication)
was employed for production of phage lysates.

KHQPOA 1.67 S

NaeHPoh 3-33 8

NH401 0.56 g
Casamino acids (Difco) 7.50 g

NaCl 0.50 g

Glucose 2.00 g 7
M3804 1M 2.50 ml
Distilled water 1000.00 m1

A casein hydrolysate broth was used for producing

galactose negative mutants.

NH4N03 1.0 g
Naesou 2.0 g
KQHP04 7.0 g
KHQPOQ 3.0 g
NHQCl 2.5 g
MgSOu.7H20 0.1 g
Casein hydrolysate 3.0 g
(Nutritional Biochemical Co.)
Glycerol 15.0 ml
Distilled water 1000.0 ml

Casein hydrolysate and glycerol were added to 925 m1
of distilled water and autoclaved. Separate 10X solutions
of MgSOu.7H2O and NH401 were prepared and autoclaved. One
ml of 10X MgSOh.7H20 and 10 m1 of 10X NHhCl were added to
the casein hydrolysate-glycerol solution. A solution con-
taining 10X by weight of the above amounts of NH4N03,
NaQSOA, K2HP04, and KH2P04 in 500 ml of distilled water was

prepared. The solution was autoclaved and 50 m1 added to

9
the casein hydrolysate-glycerol solution.

Casein hydrolysate agar was prepared by addition of
15 g agar to the casein hydrolysate-glycerol solution before
autoclaving.

Casein hydrolysate—galactose broth was made by supple-
menting casein hydrolysate broth with 10 m1 of a 20% galac-
tose solution.

Eosin—methylene blue agar (Lederberg, 1950) supple-
mented with 1.0% galactose (Difco) (EMBGal) was used for
isolation of gal- mutants and purification of gal+ transduc-
tants.

Casein hydrolysate (acid 8.0 g

hydrolyzed, Nutritional
Biochemical Co.)

Yeast Extract (Difco) 1.0 g
NaCl 5.0 g
K2HP04 2.0 g
Eosin Y 0.4 g
Methylene Blue 0.065 g
Galactose (Difco) 10.0 g
Agar 15.0 g
Distilled water 1000.0 ml

A11 transduétions were performed on a modification
(LGal agar) of Lederberg's (1950) EMB agar.
Casein hydrolysate (acid 8.0 g
hydrolyzed, Nutritional
Biochemical Co.)

Yeast Extract (Difco) 1.0 g

10

NaCl 5-0 g
K2HP04 (anhydrous) 2.0 g
Bacto agar (Difco) 15.0 g
Galactose (Pfanstiehl) 10.0 g
Distilled water 1000.0 ml

Five hundred m1 of LGal agar were prepared by the
following method. Seven and one-half g of agar were added
to 360 m1 of distilled water in a 500 ml flask, heated to
boiling, and autoclaved. Twenty g of casein hydrolysate
were dissolved in 500 ml of distilled water, dispensed 100
ml per 6 oz prescription bottle, and autoclaved. One
hundred ml were poured into melted agar kept at 50 C.

One hundred m1 solutions of the following were dispensed
into 6 oz prescription bottles and autoclaved: 25% NaCl,
10%1yeast extract, 20% K2HP04, and 25% galactose. Five m1
of NaCl, 2.5 m1 of yeast extract, 2.5 m1 of K2HP04, and 10
m1 galactose were added to the melted agar. The final
solution was shaken and 20-25 m1 poured into 100 mm x 15 mm
plastic Petri dishes. All agar plates were dried overnight:
at 37 C before use.

After suitable incubation of the transduction plates,
15 ml of 2, 3, 5-tripheny1—2H-tetrazolium chloride agar
(Ogur gt §;,, 1957; Nagai _£_al,, 1961) were overlayed on
them.

Agar (Bacto Difco) 15.0 g

Galactose (Pfanstiehl) 10.0 g

ll
2,3,5-triphenyl-2H- 0.5 g
tetrazolium chloride
(TTC, Eastman)
KHQPOA 0.067 M
Distilled water 1000.0 ml
Solutions of 0.402 M KH2P04 buffer adjusted to pH
7.0 with lON NaOH, 0.15% TTC, and 6.0% galactose were
separately dispensed in the amount of 250 ml per 16 oz
bottle. Five ml of 4.5% agar were placed in 20 mm x 150
mm test tubes. Before use, the tubes of agar were melted
and allowed to cool to 50 C. Two and one-half ml of buffer,
2.5 ml of galactose and 5.0 m1 of TTC were added. The TTC
agar tubes were allowed to stand at 50 C for 30 minutes
before overlaying.
All dilutions were made in 0.5% saline or T2 buffer
(Hershey and Chase, 1952).

Gelatin 0.01 g (1 m1 of a 1%
solution)

Na2HP04.12 H20 7.4 g (or NaQHPou, 3g)
KHQPOu 1.5 8
NaCl 4.0 g
K2804 5.0 g
MgSOu 0.1 M 10.0 ml
CaCl2 0.01 M 10.0 ml
Distilled water 1000.0 m1

Solutions of gelatin, MgSOu, and CaCl2 were prepared
separately and added after autoclaving. The final buffer
had a pH of 7.0.

12
Saline contained 5 g NaCl per 1000 ml distilled water.
All autoclaving was done at 15 pounds pressure for

20 min.

Incubation

Incubation was carried out at 37 C unless otherwise

stated.

Ultraviolethource

A 30 watt, 35 inch long General Electric Germicidal
Lamp was employed for inducing mutation and determination
of a UV survival curve. Irradiation was done at a distance
of 41 cm using a Superior Electric Company type 116-11056
powerstat set at 80 to control line voltage. The lamp was
calibrated by measuring the impinging radiation with a
General Electric light meter recording in foot candles.
Foot candles were converted to ergs/mm2 by the following
method:

x foot candles
10,000

2
2252?... = ems/mm?
10

UV Survival Curve

x 1320 watts/m2 x 107 = ergs/m2

Strain 53 was inoculated into 10 m1 of nutrient broth
and incubated 12 hours. One ml of culture was centrifuged,
washed twice with 0.5% saline and resuspended in 10 m1 of

saline. One-tenth m1 samples of suitable dilutions were

13
spread on duplicate nutrient agar plates to determine the
number of viable organisms. Seven m1 of saline culture were
irradiated in a plastic Petri dish. One-tenth m1 samples
were withdrawn at 10 sec intervals. Suitable dilutions were

spread on duplicate nutrient agar plates.

UV Induction 9f Mutants

Strain 35 or 53 was inoculated from a BHI slant culture
into 10 m1 of casein hydrolysate—galactose broth. The
culture was incubated with aeration for 20 hours (stationary
phase cell concentration a 1-2 x 109/ml). Seven m1 of a
1:500 dilution were irradiated 15 seconds (approximately 10%
survival) in a plastic Petri dish. The irradiated sample
was centrifuged (6000 x G) for 15 minutes. The pellet was
resuspended in 7 ml of casein hydrolysate broth and incubated
2-3.hours. One-tenth ml samples of 1:10 and 1:100 dilutions
were spread on casein hydrolysate agar plates or EMBGal.
After 48 hours incubation, EMBGal plates were observed for
white gal- colonies. After 24 hours incubation, casein
hydrolysate agar plates were replicated (Lederberg, 1952)
with velveteen to EMBGal agar. Replicated EMBGal plates
were incubated 48 additional hours before observation. All
mutants were carried through a minimum of 3 subcultures
before tests were made to classify them. Gram stains,
fermentation patterns, morphological comparisons, and P38

phage sensitivity were used to confirm that the mutants were

strains of S, gallinarum-pullorum.

14

Preparation of Transducing Lysates

Spontaneous.--Salmonella gallinarum-pullorum bacterio-
phage P38 was prepared according to a modified method of Z.
Hartman (1956). One-tenth m1 of an overnight M-9 broth
culture of bacteria was transferred into 10 m1 of M-9 broth.
The culture was aerated for 3 hours. Phage particles were

added to a final concentration of 1-2 x 106

per m1, and the
culture aerated 6-8 hours. The culture was centrifuged at
6000 x G for 10 minutes followed by filtration of the super-
natant fluid. Filtration was done through a glass micro-
millipore filter using a 0.45‘n HA membrane without a
supporting pad. Phage suspensions were assayed and stored

over 0.5 ml chloroform at 4 C.

UV induction of P38 (UVIWP38).—-0ne ml of an over-
night strain 38 M-9 broth culture was transferred to 10 ml
of M-9 broth. The culture was incubated 11 hours with
aeration (1-2 x 109 bacteria/m1), centrifuged and resuspended
in 10 ml of saline. The saline suspension was irradiated,
centrifuged and resuspended in 10 m1 of M-9 broth. Further
incubation (2-3 hours) with aeration was done in the dark.
The culture was centrifuged and the supernatant fluid
filtered through a millipore filter. Lysates were assayed
and stored over chloroform. UV induced lysates usually con-

tained 1-4 x 108 plaque forming units per ml (pfu/ml).

15

Method for Phage Assays (after Adams, 19592.

Two-tenths ml or an overnight BHI broth culture of
indicator strain 25 were transferred into melted 0.7% soft
agar tubes kept at 45 C. The phage to be assayed were
serially diluted in T2 buffer. One-tenth ml portions of a
suitable phage dilution were transferred into duplicate
bacteria-soft agar tubes. The tubes were poured onto BHI
agar plates. Poured plates were rocked gently with a
circular motion to spread the soft agar over the entire
surface of the plates. The assay plates were incubated
face-up for 10-12 hours. Plaques were counted on plates
showing 30-300 plaques per plate. Phage titers (pfu/ml)
were calculated from the average number of plaques on the
duplicate plates multiplied by the dilution factor for the

assay.

Preparation of P38 Antisera

Bacteriophage suspensions to be injected were prepared
as follows: (1) Strain 53 was grown overnight in 10 ml
BHI broth, (2) one ml was inoculated into 100 ml of BHI
broth contained in a fluted 500 ml shaker flask, (3) the
culture was shaken on a rotary shaker for 3 hours, (4)
phage P38 was added to give 1-2 x 106 phage particles per
m1 final concentration, (5) the culture was shaken an addi-
tional 8 hours, heated at 60 C for 30 minutes and placed in

a refrigerator overnight, (6) the culture was centrifuged

16

at 6000 x G for 15 minutes (25 ml per 50 ml centrifuge tube),
(7) the supernatant fluid was centrifuged at 25,000 x c for
2 hours, (8) resuspension of the pellet was done in 5 ml of
a solution of 0.001% gelatin and 0.5% NaCl by letting it
stand in the cold for 2 hours with occasional gentle agita-
tion, (9) phage particle suspensions were combined and centri-
fuged at 6000 x G for 15 minutes, (10) the supernatant fluid
was decanted and Deoxyribonuclease (DNAse) (Nutritional Bio-
chemical Co.)added to a final concentration of 5‘u g/ml, (11)
the solution was incubated 1 hour at 37 C, filtered through
a millipore filter and centrifuged at 25,000 x G for 2 hours,
(12) the phage pellet was resuspended as per step 8, and (13)
the phage suspension was sterilized by filtration through a
millipore filter. All centrifuge procedures were carried out
in a Serval SS-l Angle head centrifuge in a 4 C cold room.
Phage titers usually were between 5 x 1010 and 5 x lollpfu/ml.
Five ml of phage suspension diluted to l x 1010 pfu/ml
in saline were emulsified with 5 ml of incomplete Freund's
Adjuvant (Difco). Once a week, for 3 weeks, 2.5 ml were
injected intramuscularly (IM) into both rear legs of 2 two-
month-old rabbits. The rabbits were rested for 2 weeks and
injected (booster injection) with 2.5 ml per rear leg with
l x 1010 pfu/ml in 0.85% saline. After two weeks the rabbits
were bled via cardiac puncture. The booster injection and
bleeding cycle were repeated twice at lO-day intervals.

The serum of each bleeding was ooled, centrifuged,

decanted, sterilized by filtration through a millipore

l7
membrane, and frozen. All serum was heat-inactivated at
56 C before use. Assay of phage neutralizing titer was per—
formed according to Adams (1959) with the exception that the

initial phage dilution was 1 x 108 instead of 1 x 107 pfu/ml.

Phgge Adsorption Determination (Adams, 1959)

In order to perform a satisfactory transduction, it
was desirable to obtain a knowledge of the approximate time
required for adsorption of 90-99% of input phage. Strain 53
was inoculated from a stock BHI slant into 10 ml of LC broth
(Luria g£_§;,, 1960) and incubated with aeration. A direct
estimation of cell concentration was made with a Petroff-
Hausser counter. The culture was diluted to 2 x 107 bacteria/
ml in LC broth. Phage from a stock of P38 propagated on

8 and

strain 53 (P38/53) was diluted in LG broth to 2 x 10
2 x 107 pfu/ml. Bacteria and diluted phage were equili-
brated1x>and kept at 37 C before further use. Phage P38/53
(2 x 108 pfu/ml) was diluted in LC broth and assayed to
serve as a stock titer control. One ml of 2 x 107 bacteria
was transferred to each of two 13 mm x 100 mm test tubes.
One ml of 2 x 107 pfu/ml was added to one tube to give a

8 pfu/ml

multiplicity of input (MI) of 1. One ml of 2 x 10
was added 90 sec later to the second tube to give a MI of 10.
Immediately after addition of phage 0.1 m1 samples were with-
drawn from each tube (0 time) and diluted 1:100 in T2 buffer

containing chloroform to stop phage adsorption. One-tenth m1

samples were withdrawn and diluted at 3 minute intervals for

18
a period of 21 minutes. Diluted phage suspensions (free
phage) were freed from chloroform by 15-30 minutes of
aeration at 37 C. Free phage particles were diluted
appropriately and assayed using LC soft agar and LC agar
(Luria g£_§;,, 1960).

Transductign Procedure

Overnight 10 ml BHI broth cultures (recipient cells)
were centrifuged, supernatant fluid decanted, the pellet
rinsed with 2 m1 of T2 buffer and resuspended in an equal
volume of T2 buffer. They were recentrifuged, rinsed, and
resuspended in 3 ml of the same buffer. The organisms of
the suspension were then counted in a Petroff-Hausser
counter and diluted with T2 buffer to 2 x 109 or 2 x 108
bacteria/m1. Five-tenths ml of suitably diluted phage
particles, depending upon the desired MI, were added to
0.5 ml of culture. The mixture was allowed to adsorb for
25 minutes at 37 C in a water bath. One-tenth m1 samples
of the mixture were spread on 5 LGal agar plates. Incuba—
tion was at room temperature for 2 hours (Luria §§_§;,, 1960)
to increase lysogenization before incubating an additional
70 hours at 37 C. Bertani (1958) has shown that decrease
‘in temperature of incubation increases the frequency of
lysogenization. Each plate was carefully overlaid with 15
ml of TTC agar. Overlaid plates were incubated 24 hours

before scoring.

l9

Recipient cells were diluted to l x 108 or 1 x 109
and 0.1 m1 samples were spread on 5 LGal plates to serve as
a control to detect back mutants. Treatment of these plates
was identical to that of the plates for transduction. One-
tenth ml of T2 buffer and phage stocks were spotted on BHI
agar plates to serve as a control to determine the purity
of the phage stocks.

Frequency of transduction was calculated by subtracting
the average number of gal+ colonies appearing on reversion
plates from the average number of gal+ colonies on the trans-
duction plates, multiplying by the plating factor and dividing
by the total pfu.

Transductants were checked for galactose fermentation
by streaking with a needle on EMBGal agar plates. Plates
were scored for fermentation after 48 hours incubation.

In some experiments, 0.1 ml of transduction mixture
was diluted 1:100, 2 ml centrifuged and the supernatant fluid
assayed for free phage content to determine the per cent of

phage adsorption.

Test for Lysogeny of Transductants_(Levine;1957)
Transductants were carried through 5 subcultures on
EMBGal agar. An isolated colony from subculture 5 was
suspended in 0.5 m1 of P38 antisera diluted 1:100 with 0.85%
saline. The mixture was incubated at 37 C for 30 minutes in
a water bath. A loopful was streaked on EMBGal plates con-

taining P38 antisera at a dilution of 1:250. After 48 hours

20
incubation an isolated colony was suspended in 0.5 m1 of
saline. This procedure frees bacteria from passively
carried phage. A BHI plate was overlaid with soft agar con-
taining indicator strain 25 to form a bacterial lawn and
spotted with a loopful of cell suspension. The seeded BHI
plates were observed for plaque formation after lO-l2 hours
incubation. If plaques appeared in the area spotted with

the test organism, then it was classified as lysogenic.

RESULTS

Production of Mutants

 

Induction of mutation with ultraviolet light has been
reported to be a complex process affected by many factors
(Doudney and Haas, 1958, 1959; Haas and Doudney, 1957a,
1957b, 1959; and Witkin, 1956). One factor was the amount
of irradiation employed. An ultraviolet survival curve
(Figure l) was determined to serve as a basis for obtaining
a mutagenic dose.

Strain 53 was subjected to the following dosage: 15,
25, 40, 43, 45, 55, and 60 seconds. The number of plates
employed per dose varied from 30-100 while the number of
colonies per plate ranged from 1-150. In some cases induc-
tion was repeated 2-3 times for a single UV dose. One gal"
mutant (53 gal-3) was isolated from strain 53. Four gal—
mutants (35 gal-l, 35 gal-2, 35 gal-3, and 35 gal-4) were
isolated from strain 35 of which 35 gal“2 and 35 gal'u are
sensitive to galactose. All mutants isolated were induced
with 15 sec (10% survival) of irradiation. It is of inter-
est that the one induction experiment performed with strain

35 utilized 20 plates and resulted in 4 gal- mutants.

21

LOG. SURVIVING FRACTION

22

ENERGY (sacs/mmz)

 

 

 

0 46 I38 230 322 4M 506
I TA r r A I i
L
l I I I I I
O S '5 25 35 45 SS
TIMI: (sec )
FIG.| UV INACTIVATION OF STRAIN $3

23
Adsorption ofP38 to Strain 53
In studying the principle of transduction it was
desirable to know the percentage of phage adsorbed to
recipient cells as a function of time. The results of such
an experiment were recorded in Figure 2. The velocity con-

stants were calculated from the formula

K = 23%. x log_%2.(Adams, 1959)

where, po a phage assay at zero time, p = phage not adsorbed
at time t min, B = bacteria/m1, and K a velocity constant
ml/min. The P38-strain 53 system had a K = 1.59 x 10'8
ml/min for a MI of 1 and a K = 1.2 x 10"8 ml/min for a MI
of 10.

Since 90-99% phage adsorption was assumed to be ade-
quate for transducing procedures, the adsorption time was
calculated by solving the above equation for t. At a MI of
1, 99% adsorption required 28.6 minutes. Hence, 25 minutes

seemed sufficient to obtain 90-99% phage adsorption.

Assay of P38 Antiserum

Phage antiserum was essential for the proof of lysogeny
in transductants. The results recorded in Figure 3 demon-
strate the ability of the serum to inactivate P38. The
velocity constant was calculated from the formula

K = 2.3 %.x log $9. (Adams, 1959)

where, p0 = phage assay at zero time, p = phage not

LOG SURVIVING FRACTION

24

 

 

 

'0-2 I I I - I I I I
O 3 6 9 l2 l5 l8 2|
TIME(mim)

FIG. 2 . ADSORPTION OF P38 TO STRAIN 53

LOG SURVIVING FRACTION

25

 

 

IO":5 ‘ I I I I I I I
O 3 6 9 I2 l5 l8 2|
TIME (min)

FIG.3. NEUTRALIZATION OF P38 WITH A
IIIOOO DILUTION OF ANTI-P38
SERUM.

26
inactivated at time t min, D = final dilution of serum in
the phage serum mixture, and K = velocity constant. Figure 3
indicated first order kinetics were followed over the
limited range 93-99.3% inactivation. A velocity constant

K a 217.2 min“1

was determined for this region. Adams
(1959) stated the equation describes the course of the re-
action only over a limited range of inactivation, usually
between 90-99%. Figure 3 indicated a serum diluted 1:1000
should inactivate 99% within 13 min. Hence, a 1:100 and a

1:250 serum dilution was considered sufficient to validate

the test for lysogeny.

Detection and Specificipy of Ga1+ Clones

 

Early attempts to transduce the gal character met
with little success. One of the problems was a suitable
medium to detect the transductants. It was known that
transduction occurred in a liquid medium, therefore, the
solid medium being used probably was not satisfactory.

Reconstruction experiments were performed by mixing
approximately 103 gal+ cells with 2 x 108 53 gal‘l or
53 gal-2 cells and plating 0.1 m1 on EMBGal agar. Upon
incubation, papillae developed on the white gal_ bacterial
lawn but failed to give the characteristic color of fer-
menting clones. Mutant 53 gal-l is leaky and results were

not decisive after 48 hours. When 53 gal“ cells and gal+

2
cells of strain 53 (W53) were streaked next to each other

on EMBGal agar, it was observed that a substance diffused

27
from the gal- cells. This material obscured the character-
istic color of fermenting clones. Neutral red agar (Morse
and Alaire, 1958), another acid indicating medium, was tried
but abandoned.

A medium (LGal agar) employing TTC, an oxidation-
reduction indicator, as an agar overlay was successful.

One hundred and sixteen dark red papillae were observed on

a lawn of 53 gal'2 cells as compared to an expected 108.
Thirty papillae were picked of which 1 was gal', 20 were
gal+, and 9 failed to grow. A 48 hour incubation period
was determined to be insufficient as papillae were too small
to be picked with consistency. It was noted that isolated
53 gal.2 clones gave a pink-light red color when overlaid
with TTC agar, making it difficult to distinguish between
fermenting and nonfermenting clones. However, a lawn of
gal- cells remained white.

Donor phage UV-induced P38 were mixed with 53 ga1'2
recipient cells and plated on LGal agar with and without
galactose. This was done to decide whether galactose was
an essential ingredient for papillae development. No
papillae developed on the medium without galactose while an
average of 21 papillae per plate was observed on the galac-
tose containing medium. Recipient cells were plated on the
same medium which allowed a transduction frequency of 1.11 x
10'6 to be calculated.

Further evidence that the red papillae are gal+ clones

was obtained by picking the papillae from 53 gal-2 transduc-
tion plates (Table 2).

28

TABLE 2.--Specificity of red papillae

 

 

========================== in===================I
Lysate of Total Number Picked Gal+ Gal-
UV-induced P38* 48 48 O
UV-induced P38* 27 23 4
P38/WS3* 16 15 1
P38/W53* 12 12 0
P38/W53* 42 41 ' 0

 

*Different experiments.

Transduction

 

The preliminary evidence for transduction reported in
Table 2 was further substantiated by more complete experi-
ments (Table 3).

Transduction frequencies of the homologous systems
(recipient cells infected with phage propagated on the
recipient) were considered to be insignificant. The average
difference between the phenotypic reversions and trans-
ductants varied from -1.0 to 0.93. Results of the homologous
infection experiments eliminate the phenomenon of phage
conversion.

The transformation of gal‘ to gal+ by P38 is illustrated
in Figures 4, 6, and 8. Control plates of mutants without
phage are shown in Figures 5 and 7. Each dark red papilla

is a clone of glactose fermenting cells.

29

 

 

m-6fiwm6.fi mm.m 6:46 66.6H
66.6 66.H mm.m 66.6H
66.6 6:.6 6:.H 66.H
6.mm m-6Hs66.m 6e.H 6m.H 66.H
m.mm 6-6Hamm.a 6m.6 6m.6 66.H m-Hsm mm mIst
cmm\mm3mmm
m.sm AI6wa6-e 6.m: 6m.H 66.H
m.mm s-6HsH6.a 6.6m ms.6 66.H
6-6mem.a e.aa m.a mm.o
s-6aaee.m 6.6 e.m mam.6 m-asm mm mmz\mma
6.3m 66.6 6.6 ms.6 66.6H m-asw mm m-st
mm\wmmH>a
s.mm 6-6erm.m m.e 6m.H 66e.6
:.mm 6-6Hwes.s m.:H ms.6 66:.6
6-6mem.H m.Hm 6m.6 :mm.6
6-6HwHH.H 6.6m 6w.6 smm.6 m-Hsm mm mmmH>6
coauanomp< mosoduonm mpdmpoznmcmne tachomha ocv Hz onzuaso opmmmq
owned R Goaposomcmne Homecoo psoadfioom

 

opmam\cmaaadmm .Hmw hoxnonssz owmno>d

mpmoadfioon Iamw on nopomnmno +Hmw on» no coauozpmsmneII.m mqm¢a

30.

.onmzm seas possess macs message smmm\mmd one mm3\wma chess

.NIHsm mm 66 noosmsoosd mm3\mma n muasm mm\mm3mma+

 

 

 

.cofimno>on msoocmBCOQm one mopmHQ Honpcoo one do omaafldmd +Hmm 058*

s-6aa66.6 6.36 66.: 66.H m-st mm **omszo
nmmm\mma

n-6Hamm.m m.m6 6s.m 66.H 6-Hsm mm swcmszm
mm3\mma

m-6aa66.6 66.m 6:.H 66.H mIst
m \mmzwmm
AI6ere.H 6m.ma 6m.6 66.H w-asm mm mmzxwma
p-6Ham.e ms.me 6m.6 66.H m-asm mm mm3\mma

66.6 66.H 6m.m 66.H WIst
m \mmzmma
w-6HA6m.s 62.6 6m.H 66.H A-st mm mm3\mma
sIofiamwzc. 6e.mm 6m.H 66.H s-~sm mm mm3\mmm

66.6 66.m 6m.m 66.H 6-st
mm\mm3mmm
A-6Hawfi.fi 6m.mH 66.m 66.H 6IHsm mm mm3\mmm
e.6Hxi.m 6:.mm 66.m 66.H muasm mm mm3\mma

moapdnomp< zocosvonm monopozomcmne *Aow%W%%ownv Hz endpaso opmmmq
owmnm R coapooomcmse oumam\omHaadmwm . peoadfioom
. +Hmw mo nonesz o no>¢

 

 

UmSGHuCOUIIm mgmdfi

 

Figure 4. Transductants

 

 

 

 

__ .—_—_ |—_._.—_-

Figure 5.

 

 

Figure 6. Transductants

 

Control

 

 

Figure 7.

 

 

Figure 8.

Transductant papillae

Control

32

The carbohydrate fermentation pattern (Appendix, Table
6) was determined for strain 35, strain 53 and their derived
mutants. It was noted that 53 gal“2 differed from W53 in its
ability to ferment rhamnose and galactose. One transductant
derived from 53 gal’g infected with P38/W53 was also tested.
The transductant had regained its ability to ferment galac-
tose but not rhamnose. . :

During the course of this research, a spontaneous rough
form of 53 gal"2 (53 gal—2R) was isolated. Experiments were

performed to determine its susceptibility to transduction

 

 

 

(Table 4).
TABLE 4.—-Infection of 53 gal"2R with
transducing lysates
I Average Number of
Gal apillae/Plate
Control Transduce
Recipient (no Transduc- tion % Phage
Lysate Culture PHI lysate) tants Frequency Adsorption
UVIP38 53 gal;R 0.4 1.0 2.0 1.18x10-6 57.5
0.4 0.8 1.4 3.64x10‘7 87.9
P38/ 53 gal” 1.0 1.5 1.0 0.00 26.0
'W53 2R 8
1.0 1.4 2.2 1.14X10_ 0.0

 

 

 

 

 

 

 

The transduction frequencies reported in Table 4 were
not considered to be significant. There was an average dif-
ference of l gal+ clone between the transduction and control

plates. Concurrent transduction experiments employed the

33

same lysates with 53 gal-2 as the recipients. These results,
including the per cent phage adsorption, were recorded in

Table 3. The inability of 53 gal" to be transduced was

2R
believed to be caused by insufficient phage adsorption.

Transductions were performed with DNAse treated lysates
to determine whether a transforming principle was operative.
The results recorded in Table 3 indicated transformation did
not occur. In fact, DNAse treatment of lysates seemed to
enhance the transduction frequencies. .

Ten P38/W53 to 53 gal“2 and 15 UV-induced P38 to 53
gal-2 transductants were tested for lysogeny and segregation
of gal“ clones. All were found to be stable gal+ and lysogenic.

Transduction frequencies were determined as a function
of the multiplicity of input (Table 5). Although the most
efficient MI appeared to be 0.1, there was a difference of
approximately 3 gal+ clones between the control and trans-
duction plates. A ratio of 1 phage per bacterium was chosen
for most transduction procedures as it was felt the results
would be more significant.

Galactose negative mutants 35 gal-1 and 53 gal-3 were
not amenable to transduction. Mutant 35 gal'l adsorbed and
propagated phage but could not be transduced. A lawn of 53
gal'3 cells turned red when overlaid with TTC agar. Conse-
quently, gal+ transductants were not distinguishable from

the background of mutants.

34

TABLE 5.--Efficiency of transduction as a function of MI

Average Number

 

 

of Ga} Trans-

Recipient P3911138 Plate Transduc— duction
Lysate Culture MI Control tants Frequency
P38/W53 53 ga1“2 45.98 1.75 94.2 1.24x10"7
10.45 1.75 10.2 7.91x10‘8
1.045 1.75 9.4 4.06x10'7

0.1045 1.75 4.6 1.562(10‘6

0.0145 1.75 1.2 0.00

UV-induced -6

P38 53 gal’2 1.04 2.6 7.6 3.80x10
0.52 1.4 17.2 3.04x10“6
0.052 1.4 4.2 5.40x10"6

 

Mutant 53 gal-1 formed papillae but when overlaid with
TTC agar, the papillae,appeared as star shaped craters.
Sixteen craters were picked and streaked on EMBGal agar.

No ga1+ colonies were isolated.

DISCUSSION

Production of Mutants

Transduction studies have as their first prerequisite,
an adequate supply of mutants. Hence, much time was
'allotted to this problem.

Haas and Doudney (l957a,b) found in S, g9;;_that pre-
irradiation incubation in a minimal medium supplemented with
yeast extract or ribonucleic acid precursors increased
mutation frequencies. Witkin (1956) observed post-irradiation
incubation in aimedium containing an adequate supply of amino
acids increased the frequency of UV-induced prototrophs with
§;_92;;_B/r and Salmonella typhimurium LT2. Therefore, S,
gallinarum-pullorum was incubated in a medium containing
casein hydrolysate as a source of amino acids and yeast
extract.

Mutation induction is at a maximum when a synchronously
dividing culture is irradiated 10-15 min before cellular
division (Haas and Doudney, 1957b). The procedure of Haas
and Doudney (1957b) was used with strain 53. However, no
gal" mutants were obtained. Employing a method of Abbo and
Pardee (1960), 70% synchrony was obtained with strain 53.

No mutants were isolated. Consequently, further use of

synchronous cultures was abandoned.

35

36

A range of UV dosage was tried sufficient to induce
mutation in §_. typhimurium (Witkin, 1956) and g. _c_9_1__i_ (Haas
and Doudney, 1957b). Induction with nitrous acid resulted
in the isolation of 5 gal‘ mutants from strain 35 but none
from strain 53 (J. M. Robinson, personal communication).
Although the nitrous acid system was not thoroughly investi-
gated, the results combined with data obtained from UV
experiments, suggested strain 53 was refractory to mutation
at the gal loci.

Mutants of S, pgli_have been described as being
genetically blocked and unable to use galactose as a carbon
source (Lederberg and Lederberg, 1953). Biochemical studies
have shown these mutants to be deficient in the following
enzymes of the Leloire pathway (Kurahaski, 1957; Kalckar
g£_§l,, 1959): (1) mutants which will grow in the presence
of but will not metabolize galoctose lack galactokinase,

(2) sensitive mutants are blocked in galactose-l-phosphate
uridyl transferase or uridine diphosphogalactose-4-epimerase.
Two galactose sensitive mutants have been isolated from
strain 35 which may be of the type described for S, £913,
Six gal' mutants of strain 35 and 3 gal" mutants of strain

53 may lack galactokinase.

Adsorption of Phage P38

 

Adams (1959) concluded that adsorption—rate constants
in the range 10"8—10‘9 per min could be expected under

optimal conditions. Thus, LC broth provided excellent

37
conditions for adsorption of P38 to strain 53.

Since transduction experiments employed different con-
ditions (e.g., T2 buffer, strain 35, strain 35 gal" mutants
and strain 53 gal' mutants) the per cent adsorption was
determined after the 25 min incubation period. Adsorption

was found to be in the 90-99% range.

Detection and Specificity of Gal+ Clones

A sufficient number of mutants were obtained to allow
partial characterization of the transducing system. However,
a method was required to detect gal+ clones among a majority
of gal- cells.

Classical media used to indicate fermentation are de-
pendent upon acid production with consequent change in color
of a pH indicator. It was apparent from the reconstruction
experiments that such media (EMBGal and Neutral Red) were
not acceptable.

Application of a technique developed to detect respir-
ation-deficient mutants in yeast (Ogur 22.2l3: 1957; Nagai
§£.§l:: 1961) was successful. Mudd §£_§S, (1951) have used
TTC as an indicator of sites of dehydrogenases in bacteria
and also gave reference to a concurrent oxidation-reduction
interaction between diphosphropyridine nucleotide and flavo-
protein, causing reduction of the colorless TTC to red formazan..
Nagai 22.2l: (1961) stated the exact nature of the enzymatic
locus affecting the reduction of TTC into formazan is not

yet identified, although succinic dehydrogenase, which

38
catalyzes the succinate-to-fumarate conversion at a redox
potential near that of TTC, and which is absent in the
respiration deficient mutant, is assumed to be responsible.

The difference between TTC overlaid gal- and gal+
clones grown in the presence of galactose as a carbon source
is probably a differential in the amount of dehydrogenases
present.

The following information indicated the observed red
papillae were gal+ clones: (1) reconstruction experiments
showed excellent agreement between the expected number of
gal+ papillae and the observed number of red clones, (2)
twenty of the 30 subcultured red clones from the reconstruc-
tion experiment were ga1+, l was gal“ and 9 clones failed
to grow, suggesting the one gal” clone isolated could be
attributed to a failure to pick the complete clone, (3)
one hundred and thirty-four of the 139 subcultured red
clones assumed to be transductants were gal+ and 5 were gal”,
and (4) galactose was required for the development of the

red papillae.

Transduction

 

Transformation, conjugation, and phage mediated trans-
fer were three general phenomena of genetic exchange in
bacteria which had to be considered.

Transformation (Avery §£_§S,, 1944) involves a trans-
forming principle which is inactivated by DNAse. Experi-

ments with DNAse treated phage preparations precluded the

39
occurrence of transformation.

Conjugation (Lederberg and Tatum, 1946), which requires
physical contadt between two mating types, was considered
negligible as bacteria-free phage lysates were used and the
gal+ clones on the control plates with no lysates could be
attributed to phenotypic reversion.

Active plaque forming phage lysates were found to be
essential for transfer of genetic information. The number
of gal+ clones which appeared on the LGal agar plates seemed
to be dependent upon whether the mutant culture had been
mixed with phage lysate. Mutant 53 gal‘g adsorbed P38/W53
(greater than 93%),and the gal+ character was transferred.
Mutant 53 gal-QR, spontaneously derived from 53 gal-2,
adsorbed P38/W53 poorly (less than 27%), and the gal+
character was not transferred suggesting phage were the
vectors of genetic exchange.

Therefore, the genetic system was assumed to be a
type of phage mediated exchange. Phage conversion, special-
ized and generalized transduction were considered as possi—
bilities.

Phage conversion (Freeman and Morse, 1952; Groman,
1953) has been shown to be a property of the phage irrespec-
tive of the bacterial genome. Phage conversion did not
occur in the P38 system since infection of gal" mutants
with homologous phage lysates resulted in none to an occa-

sional gal+ clone.

40

Specialized transduction (Morse _§__S,, 1956a,b; Luria
g£_§S,, 1960) is characterized by high frequencies (greater
than 1 x 10'“) and unstable heterogenotes. UV-induced P38
infected 53 gal'2 cells transduced at a frequency of 1-8 x
10'"6 and were found to be stable. Strain 38 carries phage
P38, but under the conditions employed, specialized transduc-
ing lysates were not produced. The P38/W53—53 gal”2 system
transduced at a frequency of 1 x 10"6 - 4 x 10-7. Ten trans-
ductants from this system were stable and lysogenic. A
specialized transducing system may be present in the P38/W53-
53 gal'2 system, but the gal+ transductants would have to be
irradiated and the resultant lysates used for transduction.
This was not done. Lysogeny of the 25 transductants tested
suggested that transfer of the gal marker is intimately
associated with P38.

The transduction frequency for the galactose marker was
comparable to that found for general transduction in S, 991;,

l x 10'6 (Morse t 1., 1956a) and S, Syphimurium, 1 x 10'"6

 

(Hartman, 1957). Lysis, evident on the transduction plates,
may have lowered the transduction frequency somewhat from a
more optimal rate.

Intrastrain transduction with both strain 35 and 53
was demonstrated. Interstrain transduction from strain
53 to strain 35 suggested a close genetic homology between
the two.

Comparison of the P38/W53 and UV—induced P38 trans-

ducing systems indicated little, if any, difference.

41
Behavior of the transductants was identical although they
occurred at a higher frequency. It was felt, due to the low
phage titer obtained, that further work with UV-induced
lysates was not warranted.
Mutant 35 gal“l was not transducible,and other similar

situations have been reported (Hartman and Goodgal, 1959).

SUMMARY

Spontaneous, nitrous acid-, and UV—induced nonfer-
menting galactose mutants were obtained on two strains (35

and 53) of S, gallinarum-pullorum.

 

A method for detecting respiration—deficient mutants
of yeast was successfully adapted to distinguish gal+ clones
on a background lawn of gal‘ cells. Reconstruction experi-
ments demonstrated almost quantitative recovery of the
introduced gal+ cells.

The gal- mutants, suspended in T2 buffer, were observed
to adsorb 90-99% of the P38 phage particles within 25 min.
Wild type 53, using LC broth as a suspending medium, adsorbed
90% of the phage within 18 minutes.

Inter- and intrastrain generalized transduction was
demonstrated using P38 as a vector. At a MI of 1 the ga1+
character was transduced to 53 gal—2 at a frequency of 1-8 x
10‘6 by UV-induced P38 or at a frequency of 1 x 10‘6 to
4 x 10‘7 by P38 propagated on W53. A spontaneous rough
form of 53 gal-2 was found not to be transducible and to
adsorb considerably less phage than 53 gal’g. The gal+
character was transferred to mutants 35 gal—6, 35 gal-7,
and 35 ga1'8 but not to 35 gal-l by P38/35 phage particles.

Interstrain transduction was accomplished by infecting 35

42

43
ga1'6, 35 ga1'7, and 35 gal-8 with lysates of P38/53. Mutant
35 gal‘l was also refractory to interstrain transduction.
Twenty-five gal+ transductants were tested and found to be
actively lysogenic. The efficiency of transduction and type
of transductants produced appear to be independent of method

of preparing phage lysates.

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Adams, M. H. 1959. Bacteriophages. Interscience Pub-
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47

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48

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APPENDIX

49

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