Prophage Induction by High Temperature in Thermosensitive dna Mutants Lysogenic for Bacteriophage Lambda

Copyright 01973 AmericanSocietyforMicrobiology Printed inU.SA.

Prophage

Induction

by High Temperature

in

Thermosensitive dna

Mutants

Lysogenic

for

Bacteriophage Lambda

H. SCHUSTER, D. BEYERSMANN, M. MIKOLAJCZYK, AND M. SCHLICHT Max-Planck-Institut fiirMolekulare Genetik, 1Berlin 33(Dahlem), Germany

Received forpublication 14December 1972

High-temperature treatment of thermosensitive dna mutants lysogenic for

phage A leads to prophage induction and release of phage (at the permissive

temperature) in elongation-defective mutantsof thegenotypesdnaB,dnaE, and

dnaG. In initiation-defective mutants no prophage inductionoccurs at42 C in mutants ofthe genotype dnaA, whereas with a dnaC mutant as well as with strain HfrH 252 (map positionnotyetknown) phagesarereleased at 42C. DNA degradation atthe replication fork at 42 C is observed in all dnaB(A) mutants

tested, but not in mutants ofthe genotypes dnaE(X) and dnaG(A). Therefore,

degradation of replication fork DNA is not a prerequisite for prophage induction.

In

X-lysogenic bacteria, phage development

canbe induced

by

a variety oftreatments that

damage the bacterial chromosome and inhibit

DNAreplication. Since the bacterial recA

func-tionisrequired for induction (3), it issupposed

that induction results from recA-mediated

re-pair of

damaged

DNA. The mechanism of

induction isstill obscure (5).

In thermosensitive dna mutants, DNA

re-plication can be interrupted at high

tempera-ture (42 C and

above),

and the mode of

cessa-tion of DNA synthesis varies markedly in the

different classesofdnamutants.In most ofthe

mutant types known to be defective in chain

elongation

(dnaB, dnaE, dnaG),

DNAsynthesis

stops

immediately

after reaching the critical

temperature (42 C; reference 23). A residual

DNA synthesis at the elevated temperature is

observedin mutant typesdefective inthe

proc-essof initiation (dnaA, dnaC, strain HfrH 252;

1, 4, 12). In dnaBmutantsDNAatthe

replica-tionfork is

degraded

extensively at 42C (19).

It seemed worthwhileto us toexamine

X-lyso-genic dna mutants of different genotypes for

their ability to induce the prophage at high temperature inorder to find out whether or not

a damaged bacterial DNA is a necessary

pre-requisite for induction to occur. It hasalready

been reported that prophage A is not induced

when DNA synthesis ceases at 42 C in an

Escherichia colidnaA mutant. However,

induc-tionoccurredinthis mutant when the cellswere

UV-irradiated after

completion

of rounds of

replication at 42 C (20). On the other

hand,

it

has been found that

prophage

X is induced at

high temperature in bacterial mutants with

temperature-sensitive

but unknown

genetic

de-fects (15,

22).

Induction of

prophage

Xhas also

been inferred from the loss of

colony-forming

ability

of a dnaB mutant after temperature

shifts to 42C (21).

MATERIALS AND METHODS

Radioisotopes. Radioisotopesusedwere 3H-thymi-dine,22 to 27Ci/mmol, and3H-thymine,5Ci/mmol,

from The Radiochemical Centre, Amersham,

Eng-land.

Bacteria. The E. coli strains listed in Table1 were used. All strains were lysogenized by phage A (wild

type) in our laboratory. In addition, strain HfrH

165/70 was lysogenized by phage Aind-. Strain K-12 C600wasusedas indicator for A.

Media. Tryptone broth: 1% tryptone (Difco) and 0.5% NaCl. K medium: 1: 1 mixture of

double-strength M9 medium and 3% Difco Casamino Acids (27); A medium (7); M9 medium (27). All synthetic mediaweresupplementedwith20

Mig

ofthymine and thiamineperml.Inthe case of strain HfrH252, the media contained 20

Mlg

ofproline per ml in addition.

Measurement of induction of prophage X by temperature shift.Lambda-lysogenic bacteria grown

overnightintryptone broth at room temperature were usually diluted1:1,000 into K medium and grown at 30Ctoabout1 x 101cells per ml. The temperature of theculturewasthen raisedto 42C (or46Cinthe case of dnaE mutants) and incubation was continued. 879

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TABLE 1. Escherichia coli strainsa

E. coli strain dna Remarks Reference

locus

HfrH 252 ? 1,17

HfrH 252rev dna+ Selected for growth This paper at 42C, our

lab-oratory

PC5 A 4,23

E508 A 23

CRT46 A 13, 23

HfrH 165 dna+ Parental strain of 2, 19 HfrH165/70

HfrH165/70rev dna+ Selected for growth Thispaper at 42C, our

lab-oratory

HfrH165/70 B 2,19

Hfr 100 B 19

PC6 B 4, 11

PC8 B 4,11

PC2 C 4,23

PC7 D 4,23

BT1126 E 24

E486 E 23

E101 F 9,23

JW185 G dna allelenumber 11

308

PC3 G 4,23

aSource of dna mutants: HfrH 252, HfrH 165, HfrH

165/70, Hfr 100, BT1126 (H. Schaller and F. Bonhoeffer);

PC2, 3, 5, 6, 7, 8 (J. Zeuthen); E486, E101, JW185 (J.

Wechsler).

Since the time period for optimal induction at ele-vated temperatures might be different for different dna mutants,ananalysisasshowninFig.1and3 was carriedoutwithatleastone mutantofeach genotype. For this purposesampleswerewithdrawnatintervals and (i) assayed immediately for surviving bacteria (colony-forming ability at 30 C), induced prophage,

and free phage (after treatment with CHCl3); (ii) diluted 1: 100 into K medium and incubation

con-tinuedfor 2 to 3hat30C,after which theculturewas

againassayed for freephage.

In someexperiments, K medium was replaced by

tryptonebroth,M9or Amedium.Thenumber of cells containingan inducedprophageweremeasuredbya

spottestin ordernot tooverlook induction processes yielding only low phage bursts. A serial dilution of bacteria(in the rangeof 50to2,000 cells perspot)was

spottedonto alawnofK-12 C600 indicator bacteria. Plates were subsequently incubated at 27 to 30 C. Control cells(grownat30C)wereincludedinthespot test to correct for the spontaneously induced pro-phages.

Measurement of the stability of radioactively

labeled chromosomal DNA at elevated tempera-ture:(i)pulselabeling(replicationforkDNA).To1

volume oflog-phase grownbacteria in K medium at

30C,0.03volume of3H-thymidine(1mCiperml)was

addedfor50s,after which theculturewastransferred

to 42 C (or 46C in thecase ofdnaEmutants) with

simultaneous addition of 0.05 volume ofthymidine

(10 mg/ml).

(ii) Uniform labeling (preexisting DNA).

Bac-teriawere grownfor sixtosevengenerationsat30C in

K medium supplemented with 3H-thymine (10 uCi and 20mg perml). Ata titer of108perml, the cells

were harvested by filtration, washed, and resus-pended in the same volume of K medium

supple-mented with 200 ug ofunlabeled thymine. After an additional 15 minofincubationat30 C,the cultures

weredivided. Onepartwasshiftedto42C (or46C in

thecaseofdnaE mutants), and anotherpartwaskept at30C.

Samples (0.1 ml) wereremoved successively from

theradioactive cultures.Incorporationofradioactive DNA precursors wasterminated, and trichloroacetic acid-insoluble radioactivity was determined as de-scribed elsewhere (19).

RESULTS

Induction bytemperatureshift of prophage

X in lysogenic dna mutants: (i) induction of

X-lysogenic initiation-defective mutants. In initiation-defective mutants, X is able to repli-cate at temperatures at which host DNA syn-thesis hascome toahalt (13, 17). Here, induc-tion of prophage and release of phages can be followed directly at the elevated temperature.

The initiation-defective mutants dnaA E508(X) and dnaA CRT46(X) do not undergo

spontaneous prophage induction after cessation of DNA synthesis at 42 C (20). Another dnaA

mutant (PC5), isolated by Carl (4), also does

not induce X prophage efficiently. When this

strain is incubated for2to3hat42C, at most

4% ofthe cellsare induced(Table 2).

We would conclude from theseresults that in initiation-defective mutants cessation of host DNA synthesis perse is not a sufficient condi-tion for X induction to occur. However, this is

not true for an initiation-defective mutant of the genotype dnaC PC2 (4). With the nonlyso-genic strain, thecell numberstillincreasesbya factorof3.3reachingaplateau after2to3 hat 42C. Under thesameconditionstheX-lysogenic mutantinduces the prophageand the cells die

(Table 2).

Still another initiation-defective mutant,

strain HfrH 252(X) (whose map location is not yet known), behaves similarly to the strain

dnaC PC2. In Fig. 1 phage release at 42 C is

followedover aperiodof 4 h in A medium for the

mutantaswellasforarevertantof thismutant.

In both strains, the number of spontaneously induced phages increases as a consequence of

thetemperatureshiftto42C.Theeffect ismore pronounced with the mutant than with the revertant strain. However, no cell death is

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TABLE 2. Induction of prophage Xindna(X) mutants byhigh temperaturea dna(X)mutant dna locus Induction Fractionofcells induced Remarks

HfrH 252 ? + In A medium:seeFig.1

In K medium: phage bursts >2 after 3 h at 42 C (surviving cell fraction = 0.15)

PC5 A _ <0.04(2-3 h42C)

E508 _ } Measured by Monk and Gross (20)

CRT46

HfrH165/70 B + 0.4-0.7(2-3h42C)

Hfr 100 + Maximal phage release after 1-2 h

PC6 + at42C+3hat 30C (seeFig. 2)

PC8 +

PC2 C + Phage bursts >2after 3h at42C

(surviving cell fraction = 0.03)

PC7 D _ Slight increase of spontaneously

induced prophages at 42 C

BT1126 E + 0.1-0.4(1.5 h 46C) Phage bursts usually <1

E486 + 0.7-0.9(1.5-2h46C) Maximal phage release after 1.5 h at46C + 3hat 30C

Ell F _

JW185 G + 0.3(3 h42C) Maximalphage release after3h

at42C+3hat30C

PC3 0.005(2 h42C)

aInduction isregardedaspositive,whenatleast10%ofthemutantcellsyieldinfectivecenters.The fraction ofcellsinduced is difficult to determineexactlywith the strains dnaCPC2(X) and HfrH252(X),sinceinthese mutants A isable to replicate at42C. Here phage burstsareexpressedasthe number ofphagereleasedat42C perviable cell at time -zero, 42 C.

102 [

101I

1.

* 10

@ 10 2[

A

bacteria

/ phage

A

B

102

-1o1l bacteria _._

-,/ phage

0-3-~ -0_ _

_I I

0 1 2 3 4 0 1 2 3 4

hours at 42 °C

FIG. 1. Phage release at 42 C of the strains HfrH252(X) and HfrH252rev(X). Bacteria were grown in A medium at 30 C andincubated in the same medium at 42 C. Cell concentration at time zero, 42 C: A = HfrH

252(A),5 x 107per ml;B = HfrH252rev(A),1 x 108 perml. .1_

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observed with strain HfrH 252 afterDNA

syn-thesis and cell divisions have come to a halt,

indicating that only a minor fraction of cells

have been induced. Only the subsequent

ir-radiation with UV light or treatment with

mitomycin (both at 42 C) leads to induction

(data not shown). However, when K medium

wasused instead of Amedium, the cells diedat

42 C and a larger number of phages were

released (Table 2).

(ii) Induction of X-lysogenic

elongation-defectivemutants. In the

elongation-defective

mutants investigated, X DNA is not

replicated

atthe restrictivetemperature (6, 13, 17).

There-fore, induction of

prophage,

provoked

by

the

interruption of DNA

synthesis,

is

subsequently

measuredattemperatures

permissive

for DNA

synthesis. Phage bursts of

prophage-induced

bacteria are

determined

at 30 C after the

induction

period

at 42 C.

dnaBmutants. In

Fig.

2

phage

release at 30

C of strain HfrH

165/70(X)

after an induction

period

of 100 min at 42C is shown. Amaximal

burstisobtained after3to4hofincubation at

30 C. The

phage

yield (after

3 h at 30

C)

in

relationtothe

preceding

induction

period

at42

108

io7

._

106

105

0 1 2 3 4

[image:4.493.59.247.347.590.2]

hours at 30 °C

FIG. 2. Phage release at 30 C of strain HfrH 165/70(X) after 100 min of preincubation at 42 C. Bacteria were grown in K medium at 30 C and incubated in thesamemediumat42C. Cell concen-trationattimezero,42 C: 9x101perml.The culture

was diluted1:100 into K mediumafter100minand incubationwascontinuedat30 C.

C is shown in Fig. 3A. Largest bursts are

obtainedafter 1 to 2 h ofincubation at 42 C, and

induction is accompanied by a decrease in the

numberofsurviving bacteria. On the contrary,

intherevertant strain, growth at 42 C only leads

to aslight increase in the numberof

spontane-ously induced phages (Fig. 3B).

According to the experimental conditions

outlined in Fig. 3, all dnaB mutants examined

induce X prophage very efficiently following an

incubation periodat 42 C of 1 to 2 h (Table 2).

However,different dnaBmutants differ slightly

with respect to the length of time at 42 C

required for optimal induction. It is also

com-mon tothe (lysogenicaswell as the nonlysogen-ic) dnaB mutants tested that at 42 C they

degrade their DNAatthereplication fork

exten-sively (see below andreference 19).

Prophage induction studies were done in

moredetail withstrainHfrH165/70(X).Usually

40 to70% of thecells induced the prophageat 42

C. Induction and phage release in K medium

werealsoveryefficientathighconcentrations of

bacteria (omitting the 1: 100 dilution step, see

Materialsand Methods). By this means phage

titerslarger than1 x 1010permlwereobtained.

Prophage induction also occurred when K

me-diumwasreplaced bytryptonebroth. However,

forunknownreasonsphageyieldwasextremely

low if the 1:100 dilution step (into tryptone

broth)

was omitted. In M9 medium, phage

bursts lower than0.1 wereobtained after50 min

at 42C and2h at 30C. ProphageXind-, which

is not inducible by UV (14), also cannot be

induced by high temperature in

165/70(Xind-).

dnaE mutants. In mutants having a

ther-mosensitive lesion at the dnaE locus, DNA

polymerase III activity is thermosensitive (10).

Inthe strains E486 andBT1126, DNAsynthesis

stops at 42 C and 46 C, respectively. No DNA

degradation at the replication fork is observed

at these temperatures (see below). Both

lyso-genicstrains induce their prophage (Table 2).

dnaG mutants. In the strains JW185 and

PC3,

the synthesis of short deoxynucleotide

chains (Okazaki DNA) is inhibited at 42 C (18). At that temperature nodegradationofDNA at

thereplicationfork isobserved in strain JW185

(see belowandFig.4).Strain

JW185(X)

induces

the prophage at 42 C; strain PC3(X) does not

(Table2). UV irradiation of the bacteria at

30

C

leads to induction of

JW185(X),

but

PC3(X)

is

only poorly induced. As it was found later,

phageXisonly weakly reproducedinPC3at 30

C (in comparison to growth in C600). In this

mutant, either the mutation in dnaG affects

growthof X at 30C orthepoorgrowthisdue to

someotherproperty ofthe strain.

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102 1

10'

1.0 1

10o-lo03

0 1 2 3 4 0 1 2 3 4

hours at 42 C

FIG. 3. Phagereleaseat30 Caftervariouspreincubation periodsat42C. BacteriaweregrowninKmedium

at30C and incubatedin thesamemediumat42C. Cell concentrationattimezero,42C. A = HfrH 165/70(X),8

x 107per ml;B = HfrH 165/70rev(X), 7x 105per ml. Relative numberofbacteria (----0)andrespective

phage (A A) afterthe indicated timeat42C. Relative numberof phage(A A)afterthe indicated time

at42Cplusanadditional timeof3 hat30 C in eachcase.

Stabilityof chromosomal DNA and X

pro-phage induction in dna mutants: (i) fate of

30 C pulse-labeled DNA atelevated

temper-atures. With X-lysogenic as well as with some

nonlysogenic elongation-defective mutants, the

effectofhightemperatureon(i) thereplication

fork DNA(the mostrecently made DNA at 30

C) and (ii) the uniformly labeled chromosomal

DNAwas studied. In the formercase theDNA

was pulse-labeled with 3H-thymidine

immedi-ately before the temperature shift (see

Mate-rials and Methods). Aswas already mentioned

above, all the dnaB mutantstested (see Table

2) degrade replication fork DNA. As an

exam-ple, the kinetics of DNA degradation in strain

dnaB PC8(X) are shown in Fig. 4. However,

when mutants ofthe genotype dnaE [E486(X)

and BT1126] and dnaG [JW185(X) and PC3]

were tested in the same way, no significant

degradation was observed. The stability of

pulse-labeledDNAat42C is showninFig.4for

the strains E486(A) andJW185(A). Both these

mutants, aswellasstrainBT1126(X), do induce theirprophage by high temperature treatment

(Table 2).Obviously degradationof the DNAat

the replication fork is not a prerequisite for

inductiontooccur.

(ii) Fate of 30 Cuniformly labeledDNAat 42 C. Elongation-defective mutants with

3H-thymine uniformly labeled DNA were

in-cubated in K medium at30C and42C for 3h,

and trichloroacetic acid-insoluble radioactivity

was determined at 30-min time intervals (see 1.

I

.21.

0

cc

0 10 20 30

Minutes atelevated temperature

FIG. 4. Fate of 30 C pulse-labeled DNA in dna

mutants at elevated temperatures. Bacteria were

grownin Kmedium at 30 C and pulse labeled with 3H-thymidineat30C, and trichloroacetic acid-insolu-bleradioactivity wasfollowed aftertemperatureshift (see Materials andMethods).A,Strain dnaBPC8(A), 42 C;0, strain dnaEE486(X),46C; 0,strain dnaG

JW185(G),42 C.

also Materials and Methods). With the strains

dnaBHfrH165/70(X), dnaEE486(X), and dnaG

JW185(X), between 15 and20% of the 3H label

became acid-soluble after 3 h of incubation at

bothtemperatures. However, degradation at42

C wassomewhat faster thanat30C in all three

mutants. Under the same conditions in strain

4) .0

0

B

,

bacteria ,

_~~~~~~~~~.

0

~0

4

F ,phage,

742°C.3h30°C/'

A

hage

/I

.5

0

.0 0

.0 0~

~~~~~

0

o L

1.5

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dna+165(X) the acid-insoluble 3H label

in-creased by about5% at 30 C (probably due to a

depletion of the residual 3H-labeled precursor

pool).Theseresults indicate that in the mutant

strainsthednadefectalreadyleads to alimited

turnover of the chromosomal DNA at 30 C.

Surprisingly, in strain dna+165(X) 13% of the

DNA was also made trichloroaceticacidsoluble after 3 h at 42 C. Therefore, it is unlikely that

the DNA turnoverat 42C in thedna mutantsis

thereason forinduction.

DISCUSSION

Lambda-lysogenic elongation-defective

mu-tants of the genotype dnaB, dnaE, and dnaG

induce their prophage at high temperature.

Thus, the inactivationof afunctionresponsible

forthe elongationof areplicating DNA has the

same effect as the variety ofother treatments

whichall leadtoprophageinduction by

damag-ing the DNA and inhibiting DNA replication.

Consistent with this are the findings that dna

mutants with onlyareducedDNAsynthesisat

elevated temperature

(dnaD

PC7, residual

DNA synthesis withoutreaching a plateau [4];

dnaF E101, DNA

synthesis

with animmediate

reductioninrate [23] dueto adefective

ribonu-cleoside

diphosphate

reductase

[9])

do not

in-ducetheprophage (Table 2).

It is striking that all dnaB mutants which

degrade their DNAatthe

replication

forkat42

C also induce their

prophage

and

yield

large

phage bursts. (In

comparison

to the induction

by UV irradiation this "temperature induction"

iseasier tohandle, andwe areusingit

routinely

for the preparation ofphage

stocks.)

However,

these DNA

degradation

products

are

probably

not the reason for the release of

prophage

re-pression since other

elongation-defective

mu-tants

(dnaE,

dnaG)

which donot

degrade

their

replication fork DNA arealso inducible

by high

temperature. It is also

unlikely

that a more

general

degradation or abreakdown and

resyn-thesis ofpreexisting DNAat42C isthereason

forinduction.

Although

aturnoverofthis DNA

isobserved at 42 C in dna mutants, it also takes

place in a lysogenic wild type which is not

induced atthistemperature.

When DNA

synthesis

is

stopped by thymine

starvation in E. coli strains

auxotrophic.

for

thymine, an endonuclease activity appears

which introduces nicks into the DNA(8). Since

X prophage can also be induced

by thymine

starvation (16),nickingofthe DNAmaytrigger

in some way the induction process. Such an

eventcould also be thereasonfortheinduction of X in dna mutants. However,

preliminary

experiments with a (nonlysogenic) dnaB

mu-tantruled out an extensive nicking of chromoso-mal DNA (19). A more careful analysis of all

lysogenic dna mutants tested is in progress.

Thatinduction should result from the action

of therecA-mediatedrepair ofdamaged DNAis

difficult to explain in the case of such

elonga-tion-defective dnamutants which do not show a

noticeable

DNA breakdown but nevertheless

induce their prophage (dnaE E486, dnaG

JW185). However, the involvement ofthe recA

product in prophage induction may have noth-ing to do with DNA repair processes. In

(non-lysogenic) dnaB mutants asymmetric

chromo-some reinitiations can be triggered by

tempo-rary blocks in DNA chain elongation (25).

Double mutants of the genotype dnaB recA,

however, fail to reinitiate chromosome replica-tion after temperature shifts (A. Worcel, M.

Schwartz, personal communication). Such

ab-normal initiations may also trigger prophage

induction. They have in fact been observed

during inductionof aBacillus subtilisprophage

(26). It has to be shown whether abnormal

chromosomal initiationscanbe observed alsoin

all inducibleelongation-defective dnamutants.

In the initiation-defective dnaA mutant

PC5(X),

theprophage isnotinducedat 42C. As

wasobserved earlier, cessation of DNA

synthe-sis at 42 C in two other X-lysogenic dnaA

mutants also did not lead to induction (20).

Comparing the inducibilityof

elongation-defec-tive dna mutants with the non-inducibility of

initiation-defective dnaA mutantsby

tempera-tureshifts,onecouldassumethat the

induction.

process is onlytriggered in such cells in which

the chromosomal DNA is in the process of

replication. However,sincetwoother

initiation-defectivemutants,namely dnaC PC2 and HfrH

252

(unmapped),

can induce their

prophage

at

42C, the explanationcannotbeso

simple.

The

factthat thetwolatter strainsbehave

similarly

with regard to induction may indicate that

strain HfrH252isalso adnaC mutanttype.

ACKNOWLEDGMENTS

We aremuch indebtedto F.Bonhoeffer,H. Schaller, J.A.

Wechsler, and J. Zeuthen for providing us with the dna mutants. We would like to thank H. A. Rolton for her assistance inthe correctionofthemanuscript.

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