Expression of the phage λ recombination genes exo and bet under lacPO control on a multi-copy plasmid

Gene, 23 (1983) 277-292

277

l%Wif%

GEN 00803

Expression of the phage X recombination genes exu and Iret under lhcP0 control on a multi-copy plasmid (Recombinant

DNA;

genetic complementation;

recA_ host; deletions;

recombinogenic

structures)

Robert J. Zagursky * and John B. Hays ** &partment

of Chemistry,

(Received

July lOth, 1982)

(Revision

received

Uniuersity of Maryhd

and accepted

March

Baltimore County, Cutonsville, MD 21228

CU. S.A.)

(301) 455 - 2560

Tel

25th, 1983)

SUMMARY

The bacteriophage phenotype) mediate

X genes exe and bet, whose products (X exonuclease and p protein, respectively; Red homologous recombination of X phages, have been cloned under la&PO lad4 control

on multi-copy plasmids. Induction of vecA3 cells harboring these plasmids with isopropylthiogalactoside (IPTG) resulted in X exonuclease levels (assayed in vitro) that were proportional to the time of induction (for at least 4 h); recombination of h Red- phages in vivo was similarly inducible. Only one out of 25 fret4 plasmids (constructed by a variety of in vitro techniques) expressed X exonuclease, a result consistent with the polarity of several known phage bef mutations. A general method for transferring phage exe and bef mutations to plasmids was devised and plasmids bearing polar (bet3) and nonpolar (bet1 13) mutations were constructed. Mutant derivatives of the plasmid showed the same complementation pattern as analogous phage red mutants. When hbet3 phages (Exe-Bet-) infected IPTG-induced recA3 bacteria containing em+bet+ plasmids, recombination frequencies were nc, more than twice those typical for infection of plasmid-free recA3 cells with exo’bet+ phages, even in the case of IPTG induction sufficient to elevate the production of X exonuclease about 100-fold. Even when pl.asmid induction was delayed till as late as 50 min after infection, recombination was significant. Preliminary experiments suggest that these plasmids encode a polypeptide with Gam activity that corresponds to the %-amino acid “shorter” open reading frame assigned to gam by Sanger et al. (J. Mol. Bid. 162 (1982) 729-773).

* Present address:

NCI-Frederick

Cancer

Research

INTRODUCTION

P.O. Box B, Frederick,

*yTo whom all correspondence

should be addressed.

Genetic analysis of recombination-deficient (red) mutants has established that for phage h, general (homology-dependent) recombination is absolutely dependent, in vecA bacteria, on the function of at least two X genes (Echols and

Abbreviations:

sensitivity;

0378-l I 19/83/$03.00

0 1983 Elsevier Science Publishers

3.V.

deletion; galactoside; ming

units;

ethidium

kb,

MATERIALS resistance;

ApS, ampicillin

EtBr,

kilobase

bromide; pairs;

AND METHODS, SDS,

Facility,

MD 21701 (U.S.A.) Tel. (301) 695-1279.

sodium

dodecyl

1 %X equals 485 bp.

IPTG,

bp, base pairs;

A,

isopropylthio-P-D-

LB, TBM,

TBY,

XG

(see

section e); PFU, plaque-forsulfate;

Tc’,

tetracycline

278

Gingery, 1968; Signer and Weil, 1968a; Schulman et al., 1970). These genes, distinct from those

phage

responsible

mutant

been

for X site-specific

designated

recombination,

exo and bet, respectively

man et al., 1970). The product exonuclease, and

an

a marked,

but

double-stranded product,

enzyme not

p protein,

was

preference

originally

and antigenicity was readily p protein

for

1967). The bet gene

enzymatic

al., 1971). Recently,

the

plasmid-encoded

and (iii) the complementation derivatives

pattern

of

of these plasmids.

(Schul-

5’ --) 3’ specificity

absolute

means of physical activity

functions,

by

of the exo gene is h

with

DNA (Little,

have

recombination

purified

by

MATERIALS

(Carter

et

METHODS

(a) Bacteria and bacteriophages

assays, since no

apparent

AND

The following N99 (Meselson

E. coli K-12 strains

were used:

28) galK2 strR; NlOO (Meselson

has been shown to

152), galK2 strR recA3; N2668, strR kg-7 (ts). N99

catalyze the renaturation of single-stranded DNA (Kmiec and Holloman, 1981). Tight coupling between the action of the h general recombination system and phage DNA replication has been inferred from two important observations. Concomitant replication of X DNA is obligatory for recombination mediated by the phage Red system, unlike recombination by the

and NlOO are described by Gottesman and Yarmolinsky (1968); N2268 was obtained from M.E. Gottesman (Gottesman et al., 1974). Strain RZ146, which is N99 (F’ZacIqlacP8), and strain RZ15.5, which is NlOO (F’laclqlacP8), were constructed for this work by conjugal transfer of the F’ episome from strain JL384, from J. Little (simi-

bacterial Ret versely, either

procedure was described by Miller (1972). The h bacteriophages from the collection at the National Institutes of Health (Max Gottesman) were W159, Xbio7-20 exoaml017 and Y2082, hb538 bet113 ~1857. The following phages were constructed in this laboratory, as part of these or previous studies: W1713, ha106-19 b538 bet3 ~126; W1717, Xa106-19 b538 ~126; W1728, Xa106-19 b538 betam ~126; W1741, ha106-19 b538 bet3 ci- (in&); W1807, Xa106-19 b538 exoam1017; Wl809, Xa106-19 b538 betl13; Y1750, Xb538 bet3 ~1857. The Abetam and hexoaml017 mutants

system (Stahl et al., 1972). Conan exo or bet mutation renders X

phages unable to plate on Escherichia coli polA or ligg- mutants (the Feb- phenotype; see Skalka, 1974). The mechanism of Red-mediated recombination remains to be determined, although Cassuto et al. (1971) have used model substrates to demonstrate that X exonuclease catalyzes at least one plausible recombination step, namely strand assimilation. On the basis of these and other observations, several models for replication-linked, Red-mediated recombination have been proposed (Skalka, 1974; Stahl et al., 1974; 1978; Radding, 1978). Study of the X recombination system would be facilitated by cloning the genes under lacP0 control, for several reasons. First, the phage recombination activities could be studied in the absence of other phage gene expression. Second, induction of exo-bet expression at different times during A growth should facilitate studies of Red action during DNA replication. Finally, exo-bet induction by a mechanism that does not depend on temperature shifts would permit studies in bacteria carrying temperature-sensitive mutations of interest, e.g., polA(ts). We describe here (i) the cloning of the h exo and bet genes under lacP0 control on a derivative of plasmid pBR322, (ii) catalysis of h

lar

to JL385;

Little

et al.,

1980).

The

mating

are recombination-proficient (Red+) in Su+ bacteria, but Redotherwise. The Xbet3 and Abet113 phages are Red- in all bacteria. The a10619 tandem duplication extends from 2.2 to 20.2 %h on the h map (Emmons and Thomas, 1975). The b538 deletion removes the region from 43.0 to 60.11 % h (Szybalski and Szybalski, 1979). (b) Plasmids A list of the plasmids constructed work is shown in Table I.

during

this

(c) Media, chemicals and buffers TBM routinely,

and TBY broth and LB plates, used are described elsewhere (Hays et al.,

219

TABLE

I

Plasmids

constructed

for this work Genotype

Plasmid

a

A Red

Size

Figure

Phenotype

(kb)

describing the structure

pz105

AXiS-CIII

none ’

10.9

1

pZ106

Axis - gam

none ’

9.4

1

pz107

hex0 - gam

none ’

5.7

1

pZlO8

IacPO

none

4.6

1

pz109

Xbet+exo+

none ’

5.8

1

pz114

IacPO

none

2.9

1

pz115

lacP0 bet + exe+

Exe+ Bet+

4.4

1

pZ116

lacP0 bet + exe’

Exe-

Bet+

3.9

3

pz117

lacIqlacP0

none

6.1

2

pZ118

lacP0 bet”exo+

Exe+ Bet-

4.0

3

pz120

lacI~lacP0

none

5.2

2

pz122

lad9 (lacPO)=bet

Exe+ Bet+

6.7

2

pZ124

lacIq(lacP0)2bet+exoA

Exe-

Bet+

6.3

(as pZ116)

pZ128

lacPObet3

Exe-

Bet-

4.4

5

pZ129

Abet”-exoA( HpaI-HpaI)

ExoABetA ’

5.4

5

pz130

Abet113 exe+

Exe+ Bet-

5.8

5

pz131

lacIq(lacP0)2bet3

6.1

(as pZ122)

~2132

lacZ~(lacP0)2betl13

Exe+ Bet-

6.1

(as pZ122)

pz133

lacP0 bet 113 exe+

Exe+ Bet-

4.4

(as pZ115)

pz134

la~I~(lacPO)~bet

Exe-

5.3

3

a All plasmids b (lacP0)’

+exo+ b

exe+

exe+

Exo

exe+ +exoATc’Aps

encode Ap’ only, except as indicated

represents

lacP0

Bet

Bet+

’

otherwise.

lacP0 duplication.

’ The X genes in these plasmids

are not adjacent

to any known promoter.

1980). When required, ampicillin and tetracycline were added to final concentrations of 50 pg/ml, and 10 pg/ml, respectively. XC plates, which contained 0.0025% 5-bromo-6chloro-3-indolyl-P-Dgalactoside (Aldrich), were prepared as described

vided by the supplier. Bacterial strains were transformed by the procedure of Cohen et al. (1972).

by Smith and Sadler (1971). IPTG and Dobtained from Sigma; cycloserine were [ 35Slmethionine and [ 14C]protein M, standards were purchased from New England Nuclear. TM buffer contained 10 mM Tris * HCl, pH 7.4, 10 mM MgSO,.

Plasmids were amplified by addition of chloramphenicol (Sigma), 150 pg/ml, to log-phase

(d) Restriction enzymes Restriction endonucleases AccI, AvaI, BarnHI, HindII, HindIII, SalI, PvuI, EcoRI, HpaI and PvuII were obtained from New England Biolabs. (e) DNA ligation and transformations

with plasmids

Phage T4 DNA ligase, from New England Biolabs, was used according to the instructions pro-

(f) Preparation of DNA

cells in TBY broth and subsequent overnight growth. Preparation of cleared lysates and EtBrCsCl purification of the plasmid DNA were according to the procedure of Clewell and Helinski DNA isolation was “ Rapid-plasmid” (1969). according to Holmes and Quigley (198 1). DNA fragments to be cloned were initially generated by restriction enzymes, resolved by electrophoresis in 1% low-melting agarose [Bethesda Research Laboratories (BRL)], and stained with EtBr. DNA bands were cut out of the gel and extracted according to a procedure supplied by BRL.

280

(g) Attachment of BumHI linkers

glycine,

pH 9.4 supplemented

albumin BarnHI Research, procedure

linkers,

purchased

were

(Superfine)

resolved

column,

were ligated of T4 DNA

digested with S-200 column,

on

a Sephadex

ethanol-precipitated

solved in water. About units

Collaborative

were phosphorylated according to the of Maxam and Gilbert (1977). The [5’-

32P]linkers

linkers

from

G-25

and dis-

5 pg of the phosphorylated

to blunt-end

DNA,

ligase. The ligated

BumHI, resolved ethanol-precipitated

using 300

mixture

was

on a Sephacryl and dissolved

in BTE buffer (50 mM Tris - HCl, pH 8.0-

Endonucleases from BRL and

with

single-strand-specific

(100 pg/ml),

15 min

essentially

and incubated

at 37°C.

as described

The

assay

serum

with DNA

procedure

was

by Little (1966) except that

t3H]DNA extracted from X phages was the substrate; use of sonicated h DNA increased apparent activities.

In all experiments,

made acid-soluble amount

of extract

than

15% of the radioactive Blank

values,

of DNA

proportional

used and corresponded

tion.

of DNA without

the amount

was directly

DNA

corresponding

added protein,

to the to less

in the incubato incubations

were usually 30-70

1 mM

cpm and were subtracted

endo-

ues. Assay mixtures contained 0,002 to 0.04 units of h exonuclease (one unit corresponds to 1 nmol of nucleotide made acid-soluble in 1 min at 37°C). Protein concentrations were determined by the method of Lowry et al. (1951).

EDTA). (h) Digestion n&eases

for

with bovine

Sl and BAL31 were obtained used according to the supplier’s

(k) Phage r~ombination

from experimental

val-

assays

procedures. (i) Analysis of plasmid encoded proteins Plas~d-encoded proteins, labeled with [ 35S]methionine by the “maxicell’” technique of Sancar et al. (1979), as modified by Little et al. (19X0), were electrophoresed in a 5% stacking, 12.5% polyacrylamide-SDS gel, as described by Laemmli (197(l), along with radioactive protein M,. standards. Labeled protein bands were visualized by fluorography using the “EN” HANCE” procedure of New England (j) X exonuclease

Nuclear

Recombination of h tandem duplication phages (conversion to EDTA-resistant ‘fsingle-copy” phages) was measured as described by Hays and Zagursky f 1978). The recombination frequency was equated with the fraction of EDTA-resistant PFU.

RESULTS

(a) Plasmid construction

and characterization

Corp.

assay

Bacteria were grown in TBM broth to exponential phase. IPTG was added to 1 mM, and incubation continued for up to 4 h. Broth plus IPTG was added where necessary to maintain exponential growth. Cells were harvested by ~ent~fugation, washed in one volume TM buffer, resuspended in 0.1 volume of BTE buffer, then mixed with lysosyme (Calbiochem) at 0.2 mg/ml, and subjected immediately to three freeze-thaw cycles in liquid nitrogen and centrifugation in a Beckman Ti5O rotor at 28 000 rev./mm (52 000 X g) for 30 min at 4°C. Supematants were stored at -7O’C. For assays, extracts were diluted in 10 mM

(1) pZl1.5, a pIasmid with the h exo and bet genes on pBR322 under 1acPO control Cloning the phage Xx&c111 region into pBR322, removal of all h genes except exo and bet, and replacement of the Sal1 site just to the right of gam by a BumHI site, yielded plasmid pZ109, as outlined in Fig. 1 (left side). Restriction analysis of pZIO9 by S&I + BamHI revealed that about 100 bp to the left of the SuII site had been inadvertently deleted during Step IV. Thus pZ109 would encode only the distal portion of the gum coding region, if the Sal1 site were actually in gam (Szybalski and Szybaiski, 1979), or were only 15 bp upstream of the translational start (Ineichen et al,, 1981). However, if the gam start were 132 bp downstream from the Sal1 site (Sanger et al.,

281

1982), then

pZ109

and

its derivatives

to express gam activity

expected

Construction (pZ108), (pZ114),

might

activity

be

This

(see DISCUSSION).

of the IacPO-containing

plasmid

deletion of DNA between PvuII sites and insertion of exo-bet X DNA in front

in the absence

presumably

of induction

reflects

genuine

IacPO expression,

plasmid

pZ116

was observed.

a constitutive since neither

(described

below),

pZ109, which has no lac promoter,

level

of

the exe’

nor

plasmid

show this back-

of lacP0, to yield pZ115, are outlined in Fig. 1 (right side). Expression of the exo gene encoded by

ground activity in extracts (Table IIA, lines 2, 3). The only enzymatic activity demonstrated for p

pZ115

protein, (Kmiec

was demonstrated

by in vitro exonuclease

assay (Table IIA, line 1). In the presence specific

TABLE

activity

increased

of IPTG,

renaturation and Holloman,

in crude extracts.

for at least 4 h. Some

of single-stranded 1981) is difficult

Since X phage recombination

activities

(A) Plasmid-encoded

exonuclease

Plasmid

Relative

X Red genotype

specific activity

at indicated

pz115

bet +exo+

pz109

bet+exo+

pZ116

bet + exe’

pz122

bet+exo+

units) (min)

60 8

a

(arbitrary

times after induction

0

180

120

28

131

73

I1
I1 6

66

(41)

104

bet + exe’

I1

pz131

bet3 exe+

11

pZ132

bet113 exe+

a The X genes are not adjacent

(B) Phage-encoded

84

to any known promoter.

and plasmid-encoded

exonucleases Time after

Source of X exonuclease

induction

of X exo+bet+cI857

Induction

of pZ122

X exonuclease

A Hl prophage

assays were performed

for Table II(B). The relative

obtained units/mg) obtained

activity

relative

was arbitrarily

induced

Phage-encoded lysogenic

Although

bacteria

indicated

times. Relative

substrate

sources:

activities

41 AND METHODS,

corresponding

section j, except that cells were grown at

of data from several different

5 X 10’ cpm,$mol)

(specific activity,

in each experiment,

was arbitrarily

to several different

thus providing

genes to the right are deleted, shutoff

(Castellazzi

shown as 5 1 (in Table II(A)) correspond

exceed the blank values by an amount

greater

than the experimental

variation

with a relative activity

(sonicated

an extract for estimating

+~I857 AH1 prophage

to determinations in the latter.

(approx.

10

of 41 (value

DNA) in Table II(B) (actually

reference

permitting

were

the level

X [ 3H]DNA

(on unsonicated

experiments,

et al., 1972).] Plasmids

experiments, to increase

with exe+ bet + phages activity

equated

an internal

X exo+bet i phage, W1717 (5 per cell); X exo+bet

cro-mediated

was sonicated,

to 30 min after infection

25 units/mg)

the data in Table II(A) correspond

without

28

of 1.0. In Table II(A), the exonuclease

pZ122 (approx.

in which cro and all prophage

(e.g., exe, bet) to continue

30 60

to the relative value of 41 for 60 min of pZ122 induction

for 60 min was assayed X exonuclease

(1.0) 8

values in this table, a composite

as relative activity

of plasmid

so as to correspond

400 units/mg).

activity

30

under MATERIALS

activity

to blank values. The activity

designated

by 60 min of induction

in parentheses), bacteria

as described specific

as follows, In Table II(B), the X [ 3H]DNA

of apparent

specific

(min)

30

with exe+ bet + phages

Induction

Relative or

infection Infection

240


~2124

about

in

II

X exonuclease

42°C

DNA to assay

of pZ122-containing the relative [heat-induced

transcription

of leftward

were induced

with IPTG (1 mM) for the

in which the apparent

prophage

values. (42°C)

activity

genes did not

r

(II 1

lacPO fraament1

t

(VI 1

[PZl@I

_

I

//cl-&&?%

c PZ114 1

t

[ PZ107 ]

( IV 1 Y (4.4Kb )

[ pBR322 ]

B.

i

int xis 28

30 1

I

exo, bet gam 32 1

Clll

34 I

{

Bamtil

BarnHI Hpa I

Fig. 1. Structure

of exe- bet region and construction

fragment

in multi-copy

selection

of ~2105

partial

among

Sal1 digestion,

has a single remaining site and

Step I: insertion

Tcs Ap* transformants

conversion

(conversion

BarnHI

site (in X

displaying

desired

(A) Isolation

digestion

of phage X exo- bet region and fusion to lucP0

(6534 bp) of phage X xis-~111 into BamHI insert.

Step II: removal

site of pBR322

and

of X genes right of bet (~111, etc.) by

and in h (near gam, but not at site in h bet), yielding

digest), 40 bp left of the exe transiational

to blunt ends by BAL31 treatment;

linkers;

plasmids. fragment

pZ106 (9.4 kb), which

int). Step III: removal of h genes left of exe (Ea9, etc.) by cleavage at the pBR322 Hind111

of Sal1 site at X-pBR322 junction

to BarnHI

of BarnHI

so as to cleave at sites in pBR322

at the Act1 site (partial

fragment; ligation

plasmid.

of exe-bet

to BarnHI

of this linker-attached

religation site): partiat product

stop (Sanger

et al., 1982): isolation

to yield pZ107 (5.7 kb lacking HindIII Sal1 digestion and pBR322

of pZlO7;

conversion

with Pm1 and BamHI

of desired

and BarnHI

5740-bp

sites). Step IV

to blunt ends (nuclease enzymes;

ligation

together

Sl), of

283

event assaye d, cam&m ofa X dupL&n &age to an EDTA-resistant “single-copy” phage (Hays and Zagursky, 1978), was mediated by IPTG-in-

Plasmid Time afier

duced (recA3) bacteria containing pZ115 (Table III). Recombination was not induced beyond a plateau level (about lo%), suggesting that at

pz115

3

10

pz115

1.5

10

pz115

0

12

pz115

no inducer

0.27

saturating levels of exonuclease and some other factor becomes rate-limiting.

none

0

0.16

p protein,

mation of bacteria lacking F’luclq suggested that “constitutive” synthesis of plasmid-encoded functions, presumably due to titration of lac repressor expressed by the chromosomal iacl gene, might be

frequency

induction

Growth

(2) pZ12.2, a clasped containing h exo bet under lacIq IacPO contr02 The small size of colonies arising from transfor-

~e~m~inati~n

IPTG

lacP0

of recA3[F’lacZq Ibacteria bet em plasmid

indicated (fraction “no

added

AND

inducer”)

(strain

RZ155 harboring

with h 0106-19 b538 red3 ~126 phage of recombination

frequency

PFU in lysate) were as described

METHODS,

section

mixture

in

k. In all cases (ex-

100 ~01s. of IPTG-containing

to the phage-cell

the

in broth plus IPTG (1 mM) for

determination

EDTA-resistant

MATERIALS cept

pZ115)

times, infection

(5 per cell) and

(X 10’)

(h)

broth

were

(in TM buffer).

somewhat deleterious. To circumvent the need for F’luclq in strains containing pZ115, a plasmid encoding

la0

as well as lacP0

bet exo was con-

structed. Transfer of the facIq lacP0 segment from pMC7 to the lacPO-containing pZI 14 is outlined in Fig. 2 (Steps I and II). The product plasmid, pZ120, contains the configuration IucIq lacP0 IacPO, followed by pBR322 BarnHI, Hind111 and EcoRI restriction sites downstream. Such a plasmid may be of general use, since la0 is present in as many copies as any gene placed under &PO control. In this case, h exo bet was inserted downstream, yielding pZ122 (Fig. 2, Step III). Surprisingly, h exonuclease was expressed at approximately the same low constitutive level (and induced to the same extent with IPTG) from pZ122 as from pZ115, despite the additional copies of

desired PouI-RumHI fhe

nrpridv

fragments

rnmnl~m~ntnrv

(2500 bp of pZ107 containing C-terminal

nortion

of

&lactamase.

1acIq encoded by pZ122 (Table IIA, line 4). Induced levels of plasmid-encoded X exonuclease were about four times as high as those resulting from expression of a single exe+ gene expressed from the h pL promoter in the ~I857 AH1 prophage, and 28 times as high as typical lytic-infection values (Table IIB). Recombination of X bet3 duplication phages in vivo was mediated by induced pZ122 and induced pZ 115 with the same efficiency (frequencies 11.7% and 12.5%, respectively). In another experiment, phage recombination mediated by ~2122 was found to be maximal when IPTG was added to 0.1 mh4 at the time of infection; neither higher IPTG concentration nor longer induction times significantly increased recombination.

N-terminal

portion

of /3-lactamase

as well as the olasmid

gene, 3359 bp of pBR322

renlication

oriein):

selection

containing

of oZlO9

from

284

[pBR322]

[PZ114]

I PZ1151 of lad4 (lacP0)’

Fig. 2. Construction partial

Hind11 restriction

plates

containing

(clockwise)

0.1 PM IPTG;

opposite

with nuclease

of pMC7)

bet+exo+

identification

to that of P-lactamase.

Sl (destruction

plasmid

pZl22.

into PvuII site of pBR322;

of PvuI

of desired

Step II: restriction

site, hence

Step I: insertion

screening

insert

of 1725bp

IacI‘JlacPO fragment

of Ap’ Tc’ transformants

in plasmid

pZ117,

in which

showing

lacl is transcribed

of pZ1 I7 with both PvuI and Am1 enzymes,

loss of ampicillin

resistance);

ligation

of blunt-end

(obtained

white colonies

in the direction

conversion

mixture

by

on XG

to blunt ends

to PouII-restricted

pZ116 (3.9Kb

[pBR322]

T

Fig. 3. Construction BumHI

enzymes;

of plasmids conversion

sites); and religation

to yield pZ118.

TcrApS):

restriction

fragment

(containing

screening

of ApS Tc* transformants

of pZ116

X bet, plasmid

complementation

deletions

Step II (exe’):

with PuuI

are in the same (counterclockwise)

plasmid

encoding

of X exo and bet. Step I (bet”):

to blunt ends with Sl nuclease enzyme,

ori) and ligation

restriction

or with BAL31 endonuclease

EcoRI

digestion,

conversion

to blunt

to 2067-bp

dilution,

and religation

ends (Sl), subsequent

EcoRI-PuuII

to obtain pZ134, in which transcription

fragment

of plasmid

pZ115

(so as to destroy

to yield pZ116. EcoRI

of pBR322

digestion,

(encoding

with Sal1 and

both Sal1 and BarnHI Step III (exe’, isolation

but

of 3350-bp

tetracycline

resistance);

from lacP0 and from the tetracycline-resistance

promoter

direction.

studies, it was necessary

of bet’ mutations,

and to provide

a wider range of

PhageX -(b538h

, *-.

_/* ,_c-, , exo

Cl

,betllY,

X(A)

X(B)

exo’

I iA)

Fig. 4,Transfer ret+ cells (strain “A”)

so as to insert ~2129

containing replaced screening “b”.

of phage excf or 6ez mutations N99) harboring

h::pZ129 of bacteria

Step III: growth

to plasmids

the Hpal-HpaI

into phage chromosome.

cointegrate;

by exe+ bet ‘); phenol

b538

-

internal

of rapid-plasmid

to detect ezo - bet 113 plasmids of h b538 bet3 ~1857 phage

by in viva recombination. (exe bet) deletion

Step II: tr~sduction

rapid-plasrkd-electrophoresis extraction

‘bet PSI v+ +

screening DNA

of strain

(e.g.. pZ130) among in ret+ cells (strain

_

(pZ129);

residual

those resolved RZl46)

+

of X b538 befl13

reciprocal

for plasmids, phages)

(crossover

at 38°C by lysate

in which (exe-bet)”

and transformation

from the cointegrate

h~bo~ng

cI857 phage in

recombination

N99 to ampicillin-resistance

of Ap’ colonies

(to remove

113 C f

bet

(b)

Step I: growth

plasmid

exo

Amp'

oti

the ~XO+ beti

has been

of N99: in vivo

by recombination IucPO plasmid

at site (pZ1 IS);

of the progeny

phage

other

4 of 7, presumably

Febi

rescue frequencies

An

analogous

but

pZ115,

HpaI deletion

bet+ plasmids, of about

more

was used to transfer plasmid

on lig bacteria.

plated

showed

selected on ampicillin

10e3.

laborious

trophoresis

procedure

plasmids

the exo bet lacP0

bet3 into

which

bet” exe+ plasmid pZ118 or the lacP0 bet1 13 exo’ plasmid pZ133, and double transform~ts

The

does not carry

the HpaI-

by analysis

of plasmid

was not possible,

only 1 of 23 plasmids

the marker-rescue

assay (pZ128)

length

of plasmid-encoded

plasmid

exo bet1 13 and

exo bet3 with iacig facP0 lacP0, the exo bet-encoding PuuI-BaPnHI fragments of pZl30 and pZ12X were joined to the FLUI BatnHI ~ac~~~aeP~ EacPO fragment of pZ120. The products, pZ131 and pZ132, are identical in structure to pZ122, except that pZ131 encodes exo beb3, and pZ132 encodes exe bet1 13. These genotypes were verified

of both of either

with phage

with exe+ bet + plasmids.

Thus

chromosomes.

However,

and Red-independent)

pZ134

during cannot

infection

recombination

hbet3 and a bet + exe+

between

to produce

be ruled out.

(2) Plasmid-phage complementation

Tltese studies were performed by infection of recA3 bacteria harboring various exo and bet mutant derivatives of the iadq &PO la@0 bet + exo’ plasmid pZ122 with four different Redphages (Table V). Wild-type phages showed more recombination in cells containing an exe+ bet + plasmid than in those harboring mutant plasmids (Table V, line 1). Thus phage-encoded levels of both h exonuclease and /? protein appear somewhat rate-limiting during ordinary lytic infections.

Neither the exe’ bet+ plasmid pZ124 nor pZ131 expressed exonuclease {Table IIA, lines 5, 6). Thus bet3 is polar on exe in plasmids as it is in phages (Signer et al., 1968). The IacPO bet1 13 exe+ plasmid pZ 133 was constructed from pZ 115 and pZ 130 in an analogous manner.

Recombination of XRed- phages was less than 0.1% in all cases where plasmids lacked the complementary activity, except in the case of h ~2106-19

for h Red-mediated recombi-

b538 exoamlOl7, where Red- levels were 0.4% (Table V, line 2). Thus, this mutation appears slightly leaky for recombination in vivo, even though exoaml017 phages show the Feb- phenotype. This mutation also appears quite leaky when h exonuclease is assayed 30 min after infection

For these experiments, recA3 [F’lac/ql bacteria were transformed with pZ 134 plus either the lacP0

TABLE

Infection

(IPTG-induced)

(RecAgenome

by in vitro exonuclease assays, IPTG induction of pZ132 and pZ122 (exe’ bet+) resulted in approximately equal X exonuclease levels (Table IIA, lines 4, 7).

(b) ~omplementation nation

plates. Gel elecpresence

efficient complementation was obtained even when the two Red functions were encoded on separate

the

bet3 mutation. For fusion

shown).

transformant

value was obtained

tested by

had acquired

not

the

expressing neither exo nor bet (Xbet3), resulted in about 10% recombination (Table IV); the same

(Fig. 4, Steps III, IV). In this case,

where prescreening

(data

double

tetracycline

demonstrated

IV

Plasmid-plasmid

complementation

Plasmids

for Red function h Red genotypes

Phage recombination

frequency

in vivo (X IO*) Uninduced pZ134+pZ118

exo’bez + + exe i

pz134+pz133

exa’bet+

Growth

of recA3 bacteria

IPTG induction

(strain

RZISS)

(I mM) concomitant

PFU in lysate) were as described

bera

+ em+ber113 containing

in MATERIALS

9.1

0.18

the indicated

with phage infection,

Induced

0.12

plasmids,

and determination

AND METHODS,

infection

11 with Xa106-19

of recombination

section k.

b538 red3 phage (5 per cell),

frequency

(fraction

EDTA-resistant

288

TABLE

V

Phage-plas~d Growth legend

complementation

of rec.43 bacteria

(strain

NlOO) containing

to Table III. All re~mbination

were done separately. exonucIease, two separate

For assay of h exonuclease

and measurement

enough

phage infections

ex~+ bet *_ W1717; tzo + bet +, ~2122;

(two extract

exoam1017beti, emAbet+,

~2124;

Recombination

Relative h

genotype

exonuclease

infecting

of h

activity of

was as described

was directly

W1807;

exo+betl13, pZ132;

exa+ bet +

exo t bet ’

(1) 0.42+0.17

11

exo+b&113

0.64+ 0.01

exe + betam

0.18~0.07

exo+bet3

a

NDb

’ bet3 phages are phenotypically exe+ bet3 phages

Exe-

frequency

with indicated

phages

9.3 13

infections,

cells were harvested

by

for the assay of h

section j.Extracts

AND METHODS,

in

which

Values correspond

were

to average

for

range of values is indicated).

X phages

used:

W1728;

Plasmids

used:

exo+bef3,

W1741.

pZ131.

Phage recombination plasmids

phages

exoaml017bet

exo+beiam270,

were as described

of crude cell-free extracts

concentration.

for each infection;

Wl809; exo’bet3,

to protein

phages

the X exe+ befam270

except that infected

Preparation

in MATERIALS

proportional

measured

with indicated

except

were similar,

and IPTG induction,

concentrations

wo+beil13,

and infection

simultaneously,

in vitro, phage infections

concentration

radioacti~ty

plasmids

were performed

phage injection

of protein

so that acid-soluble

the indicated

experiments

30 mill after simultaneous

centrifugation diluted

for recombination

(X IO*) for

genotype

exo’bet ’

exo+betlt3

exo * bet3 a

6.9

6.7

6.8

0.44

5.4

0.45

0.06

0.10

9.0

0.36

0.05

0.05

8.0

0.06

0.09

0.05

10

Bet- (Signer et al., 1968b). We have never detected

expression

of X exonuclease

activity

from

or plasmids.

b ND, not determined.

(Table V, column 2). In all cases but one, exo + bet- phages were complemented by exe- bet+ pIasmids (and conversely) so as to yield about the same recombination frequencies as red’ phages. The poor complementation of the Xa106-19 b538 bet~270 phage by the exo”bet plasmid (Table V, line 2) parallels the low yield of X exonuclease from the phage. Thus, this amber mutation ap-

-69 -46

-30

pears partially polar on exo despite the presence of h N function (Adhya et al., 1974).

-18

(c) Gel electrophoresis of piasmid-encoded proteins The proteins encoded by the lacfg lacP0 la&W-controlled plasmids, pZ 120 (vector), pZ 122 (exe’ bet +), pZ124 (exe’ bet’), and pZ131 (exebet -> were examined by the maxicell technique of Sancar et al. (1979), as modified by Little et al. (1980). The X exonuclease polypeptide encoded by pZ122 was identified as a band migrating just below a non-lambda-encoded peptide (Fig. 5, lanes c, d) and absent from the gel corresponding to the induced exe’ plasmid pZ124 (Fig. 5, lane f). The bet polypeptide was identified as a band migrating

Fig. 5. Gel electrophoresis of plasmid-encoded proteins. Bacteria (strain RZ155) encoding the indicated plasmids were to the maxicell

subjected ALS

AND

METHODS,

technique, section

as described i. Plasmids

in MATERItested:

~2122,

lanes b, c, d; pZ124, lanes e, f; pZ120, lanes g, h; pZ13 I, lanes i, j. Lanes Lane

c, d, f. h, j correspond

d is as lane c, except

to IPTG-induced

that IPTG-induction

time of labeling

with [s5Sfmethionine.

protein

(subunit

markers

ovalbumin

(46000),

upper

anhydrase

(30080);

c (12 300). Arrows

and bet lower polypeptide

bands.

at

Lanes a, k: 14C-labeled

iM,): bovine serum albumin

carbonic

A (18 367); cytochrome

bacteria. occurred

point

(69~~0);

Iactoglobuhn to the exn

289

above the exo band, produced by induction both pZ122

and pZ124.

The polypeptide

of

M,s,

estimated by comparison with radiolabeled protein standards (Fig. 5, lanes a, k) were 27000

for /3

protein and 25 000 for X exonuclease. The respective reported M,s are 28000 (by gel electrophoresis; Kmiec and Holloman,

1981) and 25 830 (by

analysis of DNA sequence determined by Sanger et al., 1982). Of the remaining bands, those showing M,s of 38 000, 28 000, and 31600 were identified as 1acI repressor, mature /3-lactamase and pre+lactamase, Sutcliffe, 1978).

respectively

(Bayreuther,

1978;

(d) Initiation of phage recombination by exe+ bet + plasmid induction at late times during lytic growth To investigate the times at which X DNA substrates for Red-mediated recombination are present during the lytic cycle, plasrnid-encoded Exo and Bet functions were induced with IPTG at various times after phage infection (Fig. 6). [Timing begins when the phage-cell mixture (5 phage/ cell) is diluted lOO-fold into warm broth, adsorption having taken place during the preceding 20 min at O’C.] Although frequencies were lower when Red function was induced later in the lytic cycle, recombination was significant even when IPTG was added 50 min after infection. A lo-fold increase in inducer concentration did not affect the recombination frequencies. In another experiment, the synchrony of the infection was analyzed by assaying at various times phage particles released (PFU in supernatant after centrifugation) and for total particles produced (PFU in chloroform-treated aliquot of infected cells). The initial level of PFU, corresponding to 10% unadsorbed phage, did not change for 20 min. Thus, there was no significant further adsorption and most cells should have begun the lytic cycle at the same time. Production and release of new phage particles was about 12% complete at 32 min and at least 90% complete at 48 min. Nevertheless, IPTG induction at 55 min resulted in significant recombination frequencies: 0.3% above (uninduced) control levels (0.15%) at 70 min, 0.7% above at 100 min.

I 0

I 20

10

TIME OF INDUCTION Fig. 6. Recombination

frequency

plasmid-encoded

Red function.

NlOO) containing

plasmid

MATERIALS

PFU

(0),

)

of recA3 bacteria with Xa106-19

at indicated

of recombination in lysate)

AND METHODS,

(0) 1 mM IPTG;

50

of X phages after induction Growth

~2122, infection

and determination

tion of EDTA-resistant

40

( min. after infection

red3 phage (5 per cell), IPTG induction infection,

0

I

I 30

of

(strain b538

times after

frequency

(frac-

were as described

in

section k. ( X ), 10 mM IPTG;

no IPTG.

(e) Effect of phage recombination functions on cell growth and viability The effect of Red gene expression on cell growth was tested by growing bacteria containing an exe’ bet + plasmid (RZ155[pZ115]), and control bacteria, with and without inducer for 7 h. Only a slight decrease in growth rate, as judged by turbidity measurements, was observed as a result of exe+ bet + induction (not shown). However, transformation of pZ115 into bacteria that did not contain F’Zaclq resulted in ampicillin-resistant colonies that were much smaller than the corresponding F’laclq-containing transformants, even without added lactose in the plates. Another long-term effect on viability, loss of about 75% of colony-forming ability after overnight growth in IPTG, was seen with Exe- or Bet- as well as Red+ plasmids, but not with the vector plasmid pZ120 (not shown).

290

and R. Zagursky, work in progress). Although this

DISCUSSION

seems likely to be the protein with M, of 11600 The X general recombination genes, exo and bet, have been cloned and placed under IacPO control on a multi-copy plasmid. Both X exonuclease activity in crude extracts, and recombination of X Red-

encoded by the more p,-distal

gum ATG,

the

possibility that the apparent Gam activity resides in a polypeptide in which 108 C-terminal amino acids of gam

are fused to 8 N-terminal

amino

phages in vivo, depend on prior induction with the lactose analog IPTG. In the absence of induction,

acids of /?-galactosidase cannot yet be excluded.

there is a significant

plasmids may be noted:

onuclease

activity,

constitutive

level of X ex-

even when the lac repressor

gene is also present in high copy number. Presumably

this

repressor-independent

expression

corresponds in part to the low-level constitutive lac expression that enables wild-type cells to transport and hydrolyze the lactose molecules that initiate the induction process (Miller et al., 1968; Jobe and Bourgeois, 1972), but we cannot explain the relatively high values seen here. It is surprising that constitutive expression of phage recombination frequencies in the presence of uninduced plasmids in vivo are not higher than the observed 0.2-0.5%. These plasmids, which can be induced to produce more than 100 times as much Red function as is produced during phage infections, should prove useful in studying the mechanism of Red-mediated recombination and its coupling to h lytic growth. Recent DNA sequence data leave the question of the translational start of the gum gene unresolved. There are two plausible gum translational start sequences promoter-proximal to bet. Ineichen et al. (1981) assigned the morep,-proximal one to gam, since the coding sequence corresponded to an M, of 16 500, the value reported by Karu et al. (1976). However, Sanger et al. (1982) designated the morep,-distal ATG as the gam start, since the adjacent ribosome-binding site appeared stronger. The corresponding M, would be 11600. On the basis of preliminary restriction analysis and DNA sequence determination, the exo bet plasmids described here appear to encode only the more p,-distal of the two possible gam translational start sequences, because about 100 bp of h sequence was inadvertently deleted during the construction of pZ109. Furthermore, recA3 bacteria harboring pZ131, a bet3 IacPO IacPO lac14 derivative of pZ109, support the growth of X bio256 (Red-Gam-) phages, as if a polypeptide with Gam activity were being expressed (S. Friedman

Three results of initial studies with these exo bet constructed

(i) most bet mutations

were polar on exo; (ii) levels of X

exonuclease and /I protein were nearly saturating in phage infections; (iii) these gene products could act very late in the lytic cycle. (i) The polarity of many bet mutations on exo Alexpression warrants further investigation. though bet deletion mutations were constructed by a variety of techniques, 21 of 25 resulted in loss of exo expression as well. The polarity of many known phage bet mutations on exo, taken with our results, suggests that another feature of h gene expression remains to be elucidated. It cannot be that both gene products must be made from the same chromosome, since some bet mutants do complement exo phages (Schulman et al., 1970), and we show here plasmid-phage and plasmid-plasmid complementation for bet113 and for bet* mutation. It may be that translation of the exo message is affected by the secondary structure and/or efficiency of translation of the bet message. Further complexity in exo-bet interaction is suggested by the lack of correlation between exonuclease production and recombination proficiency seen in Table V. Phages encoding exo+betll3 produce only 60% of wild-type exonuclease levels (presumably because of a mild polar effect), yet show full recombination activity when excess of p protein is supplied from a plasmid. However, both exoaml017 phages, which actually show 40% exonuclease activity, and exo + betam phages, in which exonuclease is reduced to 20% of normal level by a strong polar effect, show only l/25 as much recombination under similar circumstances. It seems likely that the role of X exonuclease in Red-mediated recombination involves more than exonuclease activity per se. (ii) The high level of exo bet plasmid gene expression after 3 h of IPTG induction (lOO-fold increase above phage-infection value, by X exonuclease assay) increased recombination less than

291

2-fold. tained gens

This using

observation, thermally

(R. Zagursky,

similar

to results

that which

In contrast,

recombination overproduce

data),

early X functions, in Red+

(but

not

from the difference

Franklin products

were elevated Red-

Ret+)

Work in this laboratory

suggests substrates, enzymes,

(1973) found

for cro-

frequencies red gene

ACKNOWLEDGEMENTS

Xc1857 cro’ lyso-

induced unpublished

that the level of recombinogenic DNA rather than the supply of recombination is rate-limiting.

ob-

phages,

and

other

GM22586

J.B.H. gratefully an

was supported

from the National

American

Award.

Institutes

acknowledges Cancer

We thank

salary support

Society

Connie

Holt for technical

by grant of Health.

Faculty

from

Research

Summers

and

Maria

assistance.

as much as 50-fold infections.

in events assayed

Aside

(recombina-

tion between closely spaced genetic markers vs. recombination between chromosomal duplications), two important experimental differences may be noted. First, we measured recombination in cro+ infections. Since DNA synthesis is delayed in crop infections, and the product is highly nicked (L. Enquist and A.-M. Skalka, cited by Franklin, 1973), structures that are recombinogenic for the phage Red system but not for the bacterial Ret system may have been produced. Second, our plasmids overproduce only A exonuclease and /3 protein, (and perhaps gum protein). Thus, the interesting possibility that another h early function is rate-limiting for recombination remains. (iii) Since the timing and degree of expression h red genes from the plasmids described here is independent of phage growth and gene expression, new approaches to study the nature of the recombinogenic substrates for Red-mediated recombination are possible. Wilkins and Mistry (1974) found that when Red+ phages infected recA bacteria, the first recombinant molecules appeared about 13 min later. They suggested that the transition from circle-circle to rolling-circle replication was involved in the recombination process. We find that recombination is significant when Red function is not induced until as late as 55 min after infection, a time when most cells have already lysed. One interpretation would be that the recombinants are produced by the few cells in which lysis is delayed, since high multiplicity of initial infection, and the subsequent lOO-fold dilution, make secondary infection highly unlikely.

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