Role of Genetic Recombination in DNA Replication of Bacteriophage Lambda I. Genetic Characterization of the Delta Gene

Copyright i 1974 American Society for Microbiology Printed in U.S.A.

Role

of

Genetic Recombination

in DNA

Replication

of

Bacteriophage Lambda

I.

Genetic Characterization

of

the

Delta

Gene

K. BARTA, P. TAVERNIER, AND J. ZISSLER

Department ofMicrobiology, University of Minnesota Medical School,Minneapolis, Minnesota55455

Received forpublication 12February 1974

We describe the isolation and genetic characterization of point mutations in

genedelta, includingatemperature-sensitive mutation

(del206,).

Genetic methods

enable the extraction ofadelta mutation from the triplemutant (del,red,gam)

and the construction of new genotypes, including del,red and del,gam double

mutants. Tests of plating efficiency indicate gene delta is essential for normal

phase growth on the polA host. The possible association of delta in a system

involving alpha, beta, andgamma is considered.

Bacteriophage lambda encodes proteins

which act in genetic recombination (the Red

system).Theredalpha gene codes for X

exonu-clease (2, 7-9, 11, 12), and the red beta gene

codes for beta protein (2, 8, 11, 12). The

phage gamma gene codes for gamma protein (15), which interacts with the host

recombina-tion protein, recBC nuclease (10); gamma also

mayparticipate in the A recombination process.

Although theseproteins function in

recombi-nation, it is clear that they also affect DNA

replication (3). Phagesmutant inAexonuclease

orbeta protein make somewhat lower levels of

DNAthanX+ (approximately50% ofXA in rec+

hosts), and concatemers are somewhat shorter

than thoseobserved forX+.

Phages mutant in gamma also make lower levels of DNAthan X+ (30%). Gamma mutants

makesignificantly fewer concatemers than

X+,

andtheycontinue to make throughout infection

early replication intermediates consisting of

nicked circles and supercoils. These results

suggest recombination functions might act

di-rectly in DNAreplication, possibly topromote formation of concatemers (3).

In aseries of experiments (1), we are

investi-gating the role of the delta gene in DNA replication. In this paper are reported the isola-tion and characterization ofpoint mutations in gene delta, and the observation that delta

mutants plate with somewhat reduced

effi-ciencyonhostsdeficientinDNApolymerase I. Together with biochemical studies on DNA

replication reportedelsewhere (1), theseresults

suggest that gene delta may be an additional

component in a system

involving

genes for X

exonuclease, beta protein, andgamma

protein.

MATERIALS AND METHODS

Media, procedures, and phages have been

de-scribed previously (12). The bacterial strains used

here aredescribedinTable 1.

RESULTS

Isolation of delta mutants. Phage lambda

failstoplateon

lysogens

ofthe unrelated

phage

P2; thisphenotypeiscalled

Spi+ (sensitive

toP2 interference) (5). Lambda phages able to plate

on P2

lysogens

(Spi-)

were

originally

detected by Lindahl et al.

(5)

as

biotin-transducing

phages deleted for lambda genes between the

attsiteand the CIII gene (Fig. 1). Zissleret al.

(15)

subsequently

demonstrated that mutations

in three genes are requiredforthe

Spi-

pheno-type. Theseare mutations ina redgene

(alpha

or beta), in the gamma gene, and in the delta

gene.

In previous

studies,

the delta gene was de-fined

by

studies using the bio deletion

phage,

bio7-20,

which removes genes between the att

site and the gene for A exonuclease

(Fig. 1).

Thus,aseries of

Spi- phages

wasconstructedof

the general family: bio7-20,

red,gam.

These

phages are notsuitableforgeneticor biochemi-cal studies to elucidate the function of gene

delta because the bio7-20

phage

is deleted for

several genes, and because Xbio

phage

behave

anomalously in other respects,

perhaps

due to

the bio insertion (see [6]).

Therefore,

we setout

(i) to isolate a series ofpointmutations in

delta,

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TABLE 1. Bacterial strainsa

Strain Characteristics Source

W3550 (X) K-12(A)

W3110 thy- DeLuciaandCairns

P3478 W3110polA-thy- DeLucia andCairns

QR161 AB1157(P2)sup-37 Luria

WA5022 W3110 (P2) E. R.Signer

Q5175 thr-leuilac-P2) supE C600 (P2)

MR42 F-gal-spcRrecA+ E. R. Signer

MR43 F-gal2-spcRrecA- Isogenic to MR42

KRO F-lac-trpamSRrecA E. R.Signer

aGenetic symbols used: K-12, E. coli K-12;

(X),

lysogenic for phage A; thy-, nutritional requirement

for thymine; polI, deficiency in DNA polymerase

I; (P), lysogenic for phage P2; sup, carries amber

suppressor; thr-, nutritional requirement for

threo-nine; leu-, nutritional requirement for leucine; lac-,

unable to ferment lactose; gal-, unable to ferment

galactose; F-, female strainmissing Fsexfactor;spcR,

spectinomycin resistance; recA-, deficient in

recom-bination; trpam, suppressible nutritional

require-ment for tryptophan; and SI', streptomycin resist-ance.

and (ii) to construct a new series of phages

carrying delta mutations in combination with other mutations.

Ethyl methane sulfonate-mutagenized stocks

ofgam phage were plated on P2 lysogens (see

Table 1 for strains). Small plaques arise from

mutagenizedgam mutants at afrequencyof2x

10-'. Thisisapproximately a 100-fold

stimula-tion in mutastimula-tion ascompared with

unmutagen-ized stocks. Analysis of these Spi- mutants

from Agamphage shows that these phages carry

three mutations: the original gam mutation, a

mutation indel, and athirdmutation ineither

alphaorbeta.

Spi- phage derived from Fec-

(red,gam)

phage are obtainedwith difficulty because the leaky growth ofthered,gam phage reduces the

background

against whichaSpi- plaquecanbe visualized. However, when ideal plating

condi-tions are achieved small Spi- plaques are

de-tected.

Tem perature-sensitive del mutations were

obtainedby a different technique. We

mutagen-ized with ethyl methane sulfonate

beta270gaM210, which has an amber mutation in

bothbeta and gamma. In Su- P2 lysogens, this

phage is already partially Spi-. In Escherichia

coli strain QR161, which is a P2 lysogen of

AB1157 carrying the weak amber suppressor

sup-37,

beta270gaMi210

is unable to form a

plaque. Since sup-37 incompletely suppresses

the beta and gam mutations, the phage is still

partially beta,gam. Nevertheless, partial

sup-pression of beta,gam eliminates the leaky

growth of this phage on Su- P2 lysogens. This

enables us to detect more easily Spi- phage

which contain new mutations in delta. Among

these phage tested, approximately 1% are spi

at40C and spi+ at32C.

All the delta mutationsdescribed hereappear

tobe of thesamegeneralclass.They constitute

a Spi- phenotypein combinationwith redand

gam mutations, and they allextractand

back-cross as described below. At a much lower

frequency (106 to 10-8), Spi- phages arise

which are also Fec+. These may be A reverse,

which are also fec+ because they contain an

insertion of new recombination genes ([16]);

Zissler, unpublished data). Another subclass

ap-pears to have a chi mutation such as that described originally by D. Henderson (personal communication; [4] and [8]). By genetic and

physiological tests, chi mutations differ from

the del mutations described here (Barta and

Zissler, unpublished data).

Extraction of the del mutation from the

del,beta, gam triplemutant. To have the del

mutation by itself, we extracted it from the

triple mutant del,beta,gam which have been

isolated previouslyas aSpi-phage (see above).

The extraction involves a cross between the

del,beta,gam phage and Xbio72 (Fig. 2). Each

parentfails toplateonthe polA host (deficient

in DNA polymerase I). The del,beta+,gam+

recombinant canbe isolatedas aplaque onthe

polA host (even though the efficiency of plating

(EDP) of the isolated delta mutant onpolA is

0.03 [see below]). The deltamutantextracted in

thiswaywasthen confirmedbyabackcross; the

cross between del and beta,gam produces Spi-phage. The delta mutations which have been

extracted aregiven in Table 2.

Construction of the del,gam double

mutant. We constructed new genotypes with

the delta mutation in combination with other

mutations because we hoped that additional

del b2 Ott imt alpha betagum Cm NCI

bio

17.2

bio

bio 11

b2 I

I b13191

FIG. 1. Map oftheAchromosomeshowingtheorder

ofrelevant genes.

-4

del beta gam

FIG. 2. Diagram illustrating the extraction of del.

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TABLE 2. Genotype and growth properties of 6 mutantsa

Phenotype Growth

Phage

6(delta) Exo(exo) | (beta) -y(gamma) polA recA (P,)lysogen Single

62 NC + + + + ++

_-61601 NC + + + + ++

_-6206 ts + + + + ++

_-Double

62Y5

NC + + NC - ++

-627210 NC + + AM11 - + +

-62.113 NC + NC + - ++

-640175 NC + + NC - ++

-64017201 NC + + AM11 - + +

-620.75 ts + + NC _ ++

-620,7210 ts + + AM11 - ++

-Triple

6201137210 NC + NC AMW - __ +

6206 2707210 NC NC + AM11 - _- +

61575 NC ts + NC -

_-

+

62red329Y210

NC NC +

AM,1

- -- +

6401#11375 NC + NC NC - __ +

6401161137210

NC + NC

AM,1

- _ +

6401redlY210

NC NC +

AM,1

-

_-

+

6206011837

ts + NC NC - __ +

620&82?0Y210

ts + AM,, AM,, -

I

+

aGenetic symbols used: NC, non-conditional mutation; AM1, suppressible by supE (carries amber

suppressor); ts, temperature-sensitive mutation; +, forms small- to medium-size plaque; ++, formslarge

plaque;and -, forms no plaque.

cluestothefunction of delta could beobtained fromdouble mutants.

Toconstructthe

del,gam

doublemutants, we

crossed the triple mutant

del,beta,gam

and

Xbio72,

gam. Each of the parents fails to

plate

on the recA

host;

the

del,gam

recombinant,

however,

isFec+ and

plates

onthis host(Fig. 3). The genotype of the

del,gam

mutant, once

isolated, was confirmed

by

additional tests;

crosses between the

del,gam

mutantand a red

single

mutant

produce

the

triple

mutant

del,red,gam

at the expected frequency. The

del,gam

double mutant checked as having the

gam mutation because it plated on the

polA

hostwith an

extremely

lowefficiencyof

plating

EOP

(10-8).

Construction of the

del,beta

double

mutant. We constructed

del,beta

double

mu-tants

by

crossing the triple mutant

del,beta,-gam with Xbioll (Fig. 4). Each parent fails to

plate on the recA host, i.e., is Fec-. Since the gaM210 mutation maps to the right ofthe bio end point, the cross produces Fec+

recombi-nants which are

del,beta.

The genotype ofthe

Fec+ mutants selectedinthis way isconfirmed

because crossesbetween

del,beta

and gam pro-duce

Spi-

phage. The presence of the beta

bio72 (aam

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del beta 22m!

FIG. 3. Diagram illustrating the construction of

the

del,gam

doublemutant.

bio~~~~~~

del beta gaOi

FIG. 4. Diagram illustrating the construction of

thedel,betadouble mutant.

mutationin the double mutant is indicated

by

thelow EOPofthedouble mutantonthepolA

host (Table3).

Summary

of delta mutants constructed.

The mutations in deltanowavailablearelisted

inTable 2. Deltamutations del2 and

del40,

are

nonconditional, whereas del20, is conditional.

(Mutation

del20,,

is

temperature-sensitive

in

delta.)

Genotypes

involving

delta point

muta-tions in combination with other mutations are

also listed. Fordouble mutants, different delta

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BARTA,TAVERNIER, AND ZISSLER

TABLE 3. EOPof delta mutants

Phage W3110 polAa (P2)8 recAc

62 1.0 0.033 2x 10-6 1.0

6401 1.0 0.030 1.5x 10-6 1.0

6102 1.0 0.010 <1x 10-6 0.9

6275 1.0 1.3x 10-7 1.5 x10-6 0.80

62Y210 1.0 <5x 10-8 2.3x10-5 1.0

62 113 1.0 1.3 x 10-8 1.4 x 10-5 1.0

62redl'Y210 1.0 <1 x 10-8 0.6 <1 x

10-6401011375 1.0 <1 X 10-8 0.2 <1x 10-1

6201137210 1.0 <1X 10-8 0.6 <1 x 10-8

A+ 1.0 0.16 4x 10-6 1.00

red,

1.0 <1X 10-7 3x 10-6 0.86

7210 1.0 7x 10-f 1.3x 10-1 0.60

redl.Y210

1.0 <1X 10-8 <1 X 10-6 <1 X 10-7

bio7-20 1.0 0.11 5 x 10-5 1.10

aNumber of plaques at 37C on strain P3478

dividedby the numberonstrain W3110.

bNumber of plaques on strain WA5022 dividedby

the number on strain W3110.

cNumber ofplaques on strain MR43dividedbythe

number on strain MR42.

point mutations exist in combination with a

beta mutation (i.e.,

del,beta)

orwitha gamma

mutation (i.e.,

del,gam),

wheregamma iseither nonconditional (gaM5) or conditional (gaM210,

an amber mutation suppressed by SuII). Triple

mutants are listed, which mutants have

differ-ent delta mutations reconstructed with various

other

red,gam

combinations. The type of the point mutation in any gene (conditional, or

nonconditional) is given for each mutant.

Role ofgene delta in

phage growth.

The delta mutants constructed in crosses (see above) are listed in Table 2,

together

with the phenotype for growth on various hosts. The

EOP valuesare presented inTable 3.

Whereas the red or gam mutants have an

extremely low EOP on the

polA

host (10-7), delta mutants have a higher EOP (3 x 10-2, Table 3). The delta mutants, however, have a

lowerEOPonthe

polA

host than A+. The EOP

ofdelta mutants on

polA

is 0.03, whereas the

EOP of X+ under these conditions is 0.16. In

addition, delta mutantshave alower burstsize

(8) than A+ (62) in the

polA

host.

Table 4 gives data for the burst size ofthe del,gam double mutant on the recA host. These data show that the del mutation in

combinationwith agam mutation (either gaM5

orgaM210) significantly increases the burst size

inthe recA hostfrom the low level observed for

the gam control. This indicates that the

in-creased growth observed for del,gam does not

depend on an intact host rec system. This

contrasts with the gam,chi mutant, which

others (D. Henderson and J. Weil, unpublished

data)have shown doesrequireanintact hostrec

system for increased growth.

Mapposition of delta. The delta

point

muta-tionsdescribed hereappear to maptothe left of

alpha. We conclude this form crosses between

del,red,gam and Xbio72 (Fig. 2).Ten

independ-entrecombinantsselected from these crosseson

a polA indicator were del, as tested in

subse-quentbackcrosses withred,gam phageto create

Spi-

phage. This indicates the del mutations

are to the left of the beta113 mutation and are

possibly to the left of the deletion endpointin

Xbio72.

We obtained furthermappinginformation in

the crossbetween del mutants(del2 and

del206)

and the deletion phage, A b1319,CI857 (Fig. 5). Mutant A b1319,CI857, which produces a min-uteplaqueon aP2lysogen,isdeletedfor A genes

from the att site to gene CIII. Del mutations

successfully backcross with A b1319,CI857 to

producealarge Spi- plaqueon aP2 lysogenat a

frequency of 1%. This maps del mutations outside the regionfrom attto CIII.

The del2 and

del201

mutations can be

reex-tracted from the corresponding

del,b1319

re-combinant with a crossbetween

del,b1319

and

XsusJ,6.

Neitherparent grows ontherecA strain

KRO. Recombinants (dashed line in Fig. 6) arise on this host at a frequency of 1 to 2%. Backcrossing ofthese recombinants with either b1319 or

red,gam

again

yields Spi-

phage,

indicating that del is to the leftand outsideof

the b1319 deletion.In thecase of

del206

(a

tem-perature-sensitive del mutation), only crosses

plated at 41 C yield Spi- recombinants. Thus, thetemperature-sensitive marker ofthe

original

isolate maps to the left and outside the b1319 deletion. Crosses between

red,gam

and A b2, a

TABLE 4. Burst-sizeafter infection ofarecAhosta

Phage (5.0 input phage Infective Yield Burst

per cell) centers" size'

del2gam5 0.75 8.8 117

gami 0.91 5.3 58

del2gam210CI857

1.30 6.3 49

gam210CIs57

1.50 3.0 20

aStrain KRO(recA

Su-)

wasgrown to108/mland

infected. After 15 min of adsorption at 37 C, the

mixture was washed in warm broth, diluted

1,000-fold, grown for 90 min at 37 C, and treated with chloroform.

'Number of cells releasing at least one phage,

measured at 15 min after infection. Number equals

valuetimes 10-7.

'Values to bemultiplied by

10-6.

dCalculatedastheratioofyield to infective centers

x 1,000(dilution factor).

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[ b 1319 Ott

FIG. 5. Diagram illustratingthe back

mutation tothe deletionmutant b1319.

d.cJ

Ott

b 1319

SUsJ

FIG. 6. Diagram illustrating the extrc

from

the

del,b1319

recombinant.

phage deleted for 13% of thegenome

ofatt, fail toyield Spi- phage,sugg

del mutations maptothe left of this

DISCUSSION In this paper, we describe the i

point mutations ingene delta. Ate(

extracting the delta mutation from

mutant (del,red,gam) permits isola

del mutation itself. Other techni(

construction of thedelmutation inc

with red mutations (del,exo; del,i

gamma mutations (del,gam) and wi gamma mutations (del,red,gam). Si genes may act with delta insome pi

helpful to study the delta gene in

where theproducts of othergenes (a

and gamma) also can be controlled

tion.The series ofpointmutantsdes

make this possible.

The Spi- phenotype of the

trip

(del,red,gam) implicates delta in so

involving the function of the red s

gamma. First, however, consider t gamma. Gamma protein has beer

inhibit directly the host recBC nu

Sironi et al. (13) discovered that r(

tants are killed by prophage P2 ift]

old+ but not if the phage is old

thesefactssuggest that infection of

by Xgam+ converts the host to

phenocopy (13) leadingtothe death

and perhaps also the infecting A.

Since a gamma mutation is requi

Spi- phenotype, DNA replication b

P2 lysogen should be restricted to

modeproposed byEnquist and Skall

model could provide a clue to the

delta inthe Spi phenomenon. In the

gamma, delta inhibits phage growth. Thus,

when concatemer synthesis is prevented by a

gam mutation, delta might in some way block

theDNA replication occurring in theabnormal

cm

mode.

cm'

The low burst size ofdel mutants on polA

cross

of

adel strains issignificantinterms of the possible role

of delta in DNA replication. Other mutants

such as red orgam fail to grow on polA hosts

c}m

(15) and are defective in DNA replication in

these hosts (L. W. Enquist and A. Skalka, personal communication).

Inthe recA host, delta also has a phenotype

and inhibits phage growth when gamma is

action of del

missing.

Perhaps

deltaaffectsDNA

replication

inthis case, since X replication isabnormal in

the recA host whengamma is missing (3).

DNAcanbecut intomonomersduring

matu-tothe left ration

only

if it contains at least two cos

resting

that sequences, andthustheminimum

packageable

deletion. DNA size must be longer than a monomer; when concatemer synthesis in a recA host is

blocked by a gamma mutation, circular DNA

Lsolation

of

might

be recombined to a DNAform thatcan

chnique

for be

packaged,

and this could occur

by phage

then

triple

recombination functions alpha andbeta (3). If

ti

of

the

delmutations increase gam phage production in

ques

allow a recA host

through

an effect on

replication

)mbination

(possibly through

an effect on circle

produc-beta)

with tion),we therefore mightexpectredgenes tobe

ith

red

an

essential, perhaps for making packageable

tnce

severa

DNA. Infact, weobservethatredgenes arestill

rocess, it is essential for

phage growth,

because the

del,red,-situations gam

triple

mutant fails to grow on the recA

tlph,

bta,host.

i by muta- A paradox arises in the Spi phenomenon,

cribed

here because the phage recombination proteins

alpha

and beta apparently do not promote

le mutant phage growth. If

circles

predominate in the

ime process replication of

Xspi,

it is not clear whythe Red

mesprocess

system (which "rescues" the DNA in a recA

the

role of

host)

islethal in the

P2

lysogen.

a found to ACKNOWLEDGMENTS

clease (10). We thank PhillyPengforexcellent technical assistance.

ecBC- mu- Thisinvestigationconstitutespartof the thesis submittedby

he

phage is K. Barta in partial fulfillment ofthe requirementsforthe

Together Ph.D.degreefromtheUniversityofMinnesota,Minneapolis, Minn.

P2lysogens This investigation was supported by research grant a recBC- GB20677A1 from the National Science Foundation and a

ofthe host grantfrom the Graduate SchooloftheUniversityof

Min-nesota.K. Barta and P. Tavernier were supportedbyU.S. Public HealthService traininggrant No.AI-00090fromthe ired for the National Instituteof Allergyand Infectious Diseases.

oy Xspiin a

the circle LITERATURE CITED

ka(3). This 1. Barta, K., and J.Zissler. 1974. Role ofgenetic

recombina-tion in DNA replicarecombina-tion of bacteriophagelambda.II.

function of Effect in DNA replication by gene delta. J. Virol.

> absenceof 14:1451-1457.

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BARTA, TAVERNIER, AND

2. Echols, H., and R. Gingery. 1968.Mutants of bacterio-phage defective in vegetative geneticrecombination.J. Mol. Biol.34:251.

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37:177.

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14. Stahl, F. W., K. D. McMilin, M. M. Stahl, J. M.

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The gamma gene,p.455-469. In A. D. Hershey (ed.),

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York.

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