High-level expression of the cloned ada gene of Escherichia coli by deletion of its regulatory sequence

Gfw.

64 ( 1988) m-3

305

1I

Elsevier GEN 02345,

High-level expression of the cloned ada gene of ~sc~e$ic~iu coli by deletion of its regulatory sequence (Recombinant

DNA;

site-directed

DNA methyltransferase;

mutagenesis;

adaptive

response;

regulon;

terminator;

Oh-methylguanine-

DNA repair)

Keizo Tano h, Robert S. Foote b and Sankar Mitra a

Received

18 August

Revised

30 November

1987

Accepted

1X Deccmbcr

Received

by publisher

lYX7 1987 21 January

1988

SUMMARY

The Ada protein, a nlethyltransferase for repair of several alkyl adducts in DNA, was expressed in its native form at a high level in Escherichia coli from a pUC9 recombinant plasmid carrying ada gene from which the sequence controlling the Ada induction was deleted. The regulatory sequence appears to act as a terminator of transcription initiated from the lac promoter of the vector. However, deletion of the regulatory sequence resulted in elimination of ada induction by alkylating agents, providing confirmation of its role in activation of ada expression.

INTRODUCTION

Exposure of E. coli cells to a submutagenic concentration of a simple alkylating agent such as MNNG causes an increase in resistance to both mutagenic and killing effects of the same or other alkylating agents (Samson and Cairns, 1977; Jeggo Correspondenceto: Dr. S. Mitra, Oak Ridge National Biology

Division,

Laboratory,

P.O. Box Y, Oak Ridge, TN 37831 (U.S.A.)

et al., 1977). This phenomenon, called the adaptive response, was shown to be due to the induction of several genes involved in repair of alkyl adducts in DNA (Jeggo, 1979; Evensen and Seeberg, 1982; Mitra et al., 1982; Karran et al., 1982; Volkert and Nguyen, 1984). The gene controlling the coordinate expression chromatography; base(s)

or

soguauidine~

Tel. (615)574-0963.

E. c&DNA Abbreviations:

ada, gene coding for Ada protein

adaptive

response;

stranded;

D7T,

Ap, ampicillin;

dithiothreitol;

and controlling

bp, base pair(s);

HPLC,

ds, double

high-performance

of these activities

cative Na,

liquid

0378-l 1l~i88/~03.50 0 1988 Elsevier Science Publishers B.V. (Biomedical Division)

form; citrate,

IPTG, 1000

bp;

isopropyl

[I-thiogalactoslde;

MNNG.

N-methyl-iV’-nitro-N-nitro-

nt, nucleotide(s); polymerasc

kb. kilo-

Polik, Klenow (large) fragme~lt of

I; PTH, phenylthiohydant[~in;

ss, single stranded: pH 7.0;

is called adu (Lemotte

[ 1, designates

RF, reph-

SSC, 0.15 M NaCI-0.015 plasmid-carrier

state

M

306

and Walker, 1984; Teo et al., 1984; Nakabeppu et al., 1985). Ada protein, the 39-kDa product of this

region,

gene, is a methyltransferase.

a strong signal for transcription

fragment, guanine

first characterized methyltransferase,

Its 19-kDa C-terminal as DNA-@-methylrepairs

ine in DNA by in situ demethylation

(Foote

1980) in a stoichiometric

reaction

methyl group is transferred

to a cysteinyl

the protein

(Olsson

and

Lindahl,

and

Oh-methylguanin which

with its inverted

potential

for forming

Gottesman,

possibility

repeat

sequence

with

a stem-loop

structure,

acts as

termination

1978). Experiments

are described

a

(Adhya

to test this

in this paper.

et al., the

residue of

1980;

Lindahl

et al., 1982). Recently, Teo et al. (1986) showed that the 39-kDa Ada protein also accepts methyl groups from Q4-methylthymine and methylphosphotriesters in alkylated DNA and that the methylated Ada protein is the activator for expression of the udu regulon. The E. coli adu gene was first cloned by Sedgwick (1983) and subsequently by several other groups (Lemotte and Walker, 198.5; Margison et al., 1985; Nakabeppu et al., 1985). The regulatory system has been characterized in vitro (Teo et al., 1986; Nakabeppu and Sekiguchi, 1986) and the ada gene completely sequenced (Demple et al., 1985; Nakabeppu et al., 1985). Thus, the wild-type Ada protein can be produced in a large quantity from multicopy plasmids containing the adu gene by induction with MNNG or methyl methanesulfonate. The sequence upstream from the initiator codon contains, in addition to promoter and ribosome-binding sequences, a set of inverted and direct repeats, which appears to be the regulatory sequence for activation. In the 39-kDa Ada protein, Cys-69 accepts methyl groups from phosphotriesters and Cys-321 is the methyl acceptor for Oh-methylguanine. The Ada protein methylated at Cys-69 binds to the regulatory sequence and stimulates transcription of the udu gene (and alI& gene) (Teo et al., 1986; Nakabeppu and Sekiguchi, 1986). Induction of udu by alkylating agents is not always desirable because the recovered Ada protein would not be homogeneous as a result of its partial methyIation. Furthermore, the induction is not possible for many mutant udu genes (Mitra et al., 1982; unpublished observation). Nakabeppu et al. (1985) cloned the ada gene in pUC9 from which Ada protein was produced in significant amounts by transcription driven from the upstream luc promoter of the vector. In our const~ction of a similar plasmid, we observed that the luc promoter-initiated transcription is far less efficient than that induced by MNNG. It appeared possible that the udu regulatory

(a) Bacterial

strains and plasmids

The standard

E. cdi strains

for carrying

lac and

i [>I. pronloter-~ont~~ining plasmids were JMl07 (dlrc-pm et&A 1 gt:,‘vA96 thi-1 hsdR 17 s11pE44 relA 1 [F’truD36 pmA ’ I.3’ lacP luc.ZAM 151) and WPSlX (ltr(,.1Ul69 proC’: :TnlO le1r thi h.rdR, h.sdM,

(/,dBam

N +p,i cIts857dHl)) (Sisk et al., 19X6), respectively. For site-directed mutagenesis, a metll~l~~tictn-deficierlt strain. GM373 (F-4’[F’luc]/dam-3 dew-6 hsdR 17 tonA lucYI t.s.x7# .su~)E44 gulK2 galT33 thi- 1) and a mismatch repairdeficient strain, GM374 (F-42[F’ktc] mufL25 thr-1 leuB6 thi- I urgE3 hixG4 proA IacYI p/K2 mtl- 1 .~$-5 urn-13 rpsL3 1 tsx-33 &r/44 tflD1 E;&K41) (Marinus, 1987) were used. Bacteria were routinely grown in L broth medium, supplemented with 100 118 Ap,iml where indicated. Plasmid pCS70 (Teo et al., 1984) was provided by B. Sedgwick. and pTK23 (Nakamur~~ et al., 1983) was a gift of 11. Court. Plasmid DNA was prepared by the alkaline lysis method (Birnboim and Doly. 1979). DNA reslriction fragments were analyzed by agarose gel electrophorcsis. and, when needed, electroeluted from the gel by the DEAE-paper method (S~~koyanla et al., 198.5). (b) Construction

of ah-containing

plasmids

Oligodeoxynucleotide-directed mutagenesis (Zollcr and Smith, 1983) was used to create a unique ilplrl site in the 5’-proximal noncoding sequence of the r&gene as follows. The HindIII-XmaI fragment containing the udu gene in pCS70 was ligated at the corresponding cohesive sites in the polylinker region of ~113nlpl9 RF DNA digested with Hind111 + litnul; the recombinant DNA was used for transfcction of E. coli JM 107. M 13mpl9-adu ss DNA was isolated from purified progeny phage

307

DNA (0.8 pmol) was annealed with the ol~godeoxynu~Ieotide primer 5 ’ -d(~TGGTTAA~G,4TAGCCT), synthesized by the phospl~oramjdite method (Sinha et al.. 1984) and 5’-phosphorylated (Niyogi et al., 1986) before use. The primer was complementary to the ( + > strand of the a&gene at nt 74-90 (numbered from the Hind111 site; Demple et al., 1985), except for the substitution of C in place of G opposite nt 82, to create a unique &@I site. The primed-template was converted into RF DNA with 12 units of PolIk and 2 units of T4 DNA ligase at 30°C for 2 h in two stages according to Neisbet and Beilharz (I 985). The RF I DNrZ was purified by band sedimentation in alkaline sucrose gradient (Niyogi et aI., 1986) and used to transfect E. coli strain GM374. Mini-preparations of phage DNA obtained from single plaques were screened for the desired mutation by dot hybridization on nitrocelIulose with the 5’-[ 32P]primer oligodeox~u~leotide. The nitrocellulose membrane was then washed at 48’ C in 6 x SSC and autoradiographed. The mutant sequence was further checked by &a1 sensitivity of the RF DNA. Finally the mutated phage ss DNA was sequenced by the dideo~ynucIeotide ch~n~termination method in which a 5’-d(~CAGCG~GAT~GTC) primer (complementary to nt 139-125) was used. The sequence of the entire regulatory region including the segment, was determined and found to be in complete agreement with that pubIished by Dempie et al. (1985) and Nakabeppu et al. (1985). The mutant adu gene (HindHI-XmaI fragment) was transferred to pUC9 and the resulting recombinant plasmid (pSM4~~, after testing for HpuI sensitivity, was used as the starting material for deleting the a& regulatory sequence. pSM40 plasmid DNA was digested with HindHI and the cohesive ends were filled in with PolIk followed by digestion with E&f. The large fragment was purified by agarose gel electrophoresis and recircularized by blunt-end ligation with T4 DNA ligase. The resulting plasmid (pSM41) was purified after tr~sformation of E. co& strain JM107. The t&r structural gene fragment (f!/~~I-&zc~I) was also isolated from pSM40 and inserted in i I’,_-based plasmid pTK23 via blunt-end ligation at the Qtll site. The resulting plasmid, pKT-1, was propagated in E. coli WPS18.

grown

in E. coli GM373.

(c) Analysis

of Ada protein

The presence of Ada protein in E. co& extracts, prepared according to Nakabeppu et at. (1985), was determined by electrophoresis in 0.1 P/; SDS-12.50,;; polyacrylamide gels (Laemmli, 1970). The relative amount of Ada protein in each sample was estimated from a densitometric scan using an LKB 2202 Ultroscan Laser Densitometer. For N-teeing sequencing, 800 pg of the Ada protein, purified to 80% homogeneity (not shown), was further purified by electrophoresis on a 3-mm 0.1% SDS-Q% polyacrylamide slab gel and extracted according to Hager and Burgess (1980). The recovered protein (approx. 30 pg) was then subjected to automated edman degradation by use of a gasphase sequencer (Applied Biosystems Model 470A) and PTH-amino acids were identified by HPLC (Porter et al., 1986). (d) Expression

of ada gene

We compared the expression of a& gene in three recombinant plasmids pSM31, pSM41 and pKT-1 (Fig. 1A). pSM31 carries the entire uda gene which is under the control of the iuc promoter of the vector as well as its own promoter. The creation of an HpaI site, 5’ proximal to the ribosome-binding site of the a& gene, allowed us to delete the regulatory and promoter sequences of the crdu gene to create pSM4 L. The Hpal-.%QI fragment containing the coding sequence of the gene was placed under the control of i, 11,.promoter in pKT-1. E. co/i bearing these plasmids were subjected to induction treatment with IPTG or MNNG, and the Ada protein levels were measured by electrophoresis in SDS-polyacrylamide gels (Fig. 2). It is evident that the Ada protein was overproduced in E. coli carrying pSM31 when treated with MNNG but was not induced to a large extent with IPTG. On the other hand, a high level of Ada protein was observed in E, co&harboring pSM41 when the cells were pretreated with IPTG but no induction was observed with MNNG. The biological activity of the Ada protein in various E. 0% extracts is in quaIitative agreement with the gel data (Table I). Based on the size and specific activity, the Ada protein constituted about 1.8”, of the total soluble protein of E. coli carrying

308

B ,C -

A, A

G G

-\C

/ * G,A

‘A

.

T’

lot Of T,

T I A I A

/acOP

* . .

I A I A I

Sm

HP

* *

I G *

T--G-G

J

I

c * I

T

5’-G Fig. 1. Schematic plasmid

representation

DNA. The structural

the ribosome-binding is the same as pSM31 created (positive

of the plasmids sequence

by open boxes, the regulatory

sites by black bars. pSM3 1 contains

the wild-type

except for the HpaI site (HP). pSM41

by ligation of HpaI-SmaI(Sm) strand)

ada DNA. (A) The ada genes are shown

containing

is indicated

indicated

of the ada gene (Demple

fragment

by facing arrows

by deletion stem-loop

A

I

* C-3’

as linear segments

without

(not drawn to scale) by hatched

ada under the control

was created

to pKT23. (B) A potential

in the hatched

sequence

.

I

(la&P) of pUC9; pSM40

of lac promoter

of HindIII(H)-HpaI(Hp) structure

fragment.

of the regulatory

box in panel A. The 5’ end corresponds

the

boxes and

sequence

pKT-1 was of ada DNA

to nt 10, and the 3’ end to nt 61

et al., 1985).

11 12

pSM41

and

induced

with

IPTG.

Densitometric

analysis of protein bands (Fig. 2) yielded a value of about 7U;. This discrepancy may not be due to synthesis of inactive Ada protein because there was Fig. 2. Plasmid-coded were analyzed phoresis

expression

of Ada protein. E. coli extracts

by 0.1% SDS-12.50/,

as described

in section

polyacrylamide

c. Electrophoresis

out in a 1.5-mm thick slab gel for 3 h at constant mA. The protein brilliant

bands

were

blue R250. Lanes:

B, bovine albumin, inhibitor

stained

1, protein

ovalbumin,

of 20

(phosphorylase

anhydrase,

and trypsin

of 92.5, 66, 45, 31, and 21.5 kDa, respectively);

JMl07[pUC9];

5-7, JM107[pSM31];

5 and 8, untreated

bacteria;

MNNG/ml

for 1 h at 37°C;

3, 6 and 9, bacteria

The arrow

indicates

treated

the position

2,

with 0.5

treated with 5 pg

11 and 12, WPSl8

and with prior incubation

2-4,

8-10, JMl07[pSM41];

mM IPTG for 4 h at 37°C; 4.7, and 10, bacteria without

current

with 0.1 Y, Coomassie

standards

carbonic

gel electrowas carried

carrying

pKT-1

at 42°C for 1 h, respectively. of Ada protein.

309

TABLE

I

DNA-Oh-methylguanine

methyltransferase

E. coli strain a

Treatment

of Escherichiu coli extracts

activity

b

[plasmid]

Protein

Specific

concentration

of methyltransferase

activity

in extract

(pmol/mg

of protein)d

(mgiml) ’

JM 107[pSM3 l]

JM107[pSM41]

WPS18[pKT-l]

,’ The strains

same fresh medium

at 42’C

5.7

None

7.5

29.9

IPTG

8.5

51.5

MNNG

2.7

288.5

None

7.4

20.4

IPTG

9.3

449.2

MNNG

1.5

18.5

42°C

8.6

155.3

32°C

8.1

4.8

are described

in section

JM 107 was grown overnight and incubated

0.5 mM and the cultures were treated

containing

0.26

4.5

and the plasmids

h The plasmid-carrying

cultures

11.3

MNNG

IPTG

JM107[pUC9]

at 37°C until A,,,, reached

were incubated

for

a.

at 37°C in L broth containing

for another

4 h. Alternatively,

for adaptive

IPTG was added

treatment,

MNNG

was diluted

1: 50 with

at a final concentration

of

was added at 5 pg/ml and the

I h. In the case of WPS18 harboring I pL based expression plasmids, overnight culture grown in L broth 1 : 50 with fresh medium and incubated at 32°C until A,,, reached about 0.5. Induction was by heating

Ap at 32’C was diluted for 1 h.

’ Cells were harvested M NaCl (Nakabeppu

and resuspended

as the standard ‘i The extracts

with 20 mM Tris’ HCI (pH 8.5) containing

et al.. 1985) and disrupted

min at 4°C and its protein (Smith

concentration

in an ultrasonic

was assayed

and alcohol

10s; glycerol,

The extract

reagent

(Pierce

1 mM EDTA,

was centrifuged

Chemical

1 mM DTT. and 0.4

at 40000

rev.jmin

for 20

Co.) using bovine serum albumin

et al.. 1985).

were incubated

precipitation

disintegrator.

with BCA protein

at 37’C for

I h, with poly(dC,dG,[&‘H]m’dG)

(pH 7.9). 2 mM DTT, 2 mM EDTA and 0.1 mM spermidine

and redissolved

at 7O’C for 30 min and were separated

O”-methylguanine

demcthylated

was calculated

containing

1 pmol(2900

cpm) ofm6dG,

in a total volume of 0.1 ml. Nucleic acids were isolated

in water (90 ~1). The purines

mM and heating (Lindahl

100 pg/ml Ap. The culture

about 0.3. The lac inducer

by HPLC

from the radioactivity

were released on Aminex in guanine

from the aqueous A-6 (Foote

solution

in 50 mM Hepes

by phenol extraction by adding

et al., 1983). The molar

and is equal to the amount

HCI to 20 amount

of

of methyltransferase

et al.. 1982).

no evidence of heterogeneity in the protein during its purification (data not shown). It is likely that this difference resulted from uncertainties in the quantitation of protein and variability of induction in different experiments, We confirmed that the pSM41-encoded Ada protein was identical to the native form, by determining its N-terminal sequence through nine amino acids: Met-Lys-Lys-Ala-Thr-Cys-Leu-Thr-Asp (Demple et al., 1985; Nakabeppu et al., 1985). The contribution of the protein by the host cells is negligible (Fig. 2 and Table I).

(e) Conclusions

and discussion

The regulatory sequence of the a& gene is identical in two E. coli strains, B and K-12 (Demple et al., 1985; Nakabeppu et al., 1985) and the sequence upstream from the promoter contains a dyad symmetry. The molecular basis of activation of the adu gene (and alkA) was shown to be the binding of the methylated Ada protein to this sequence (Nakabeppu and Sekiguchi, 1986; Teo et al., 1986). We observed a poor induction of the Ada protein when the entire ada gene was placed under the control of the luc promoter in pUC9, and we suspected that the potential stem-loop structure of the regulato-

310

ry sequence 5’ upstream from the coding (Fig. IB) acts as an effective transcription tor (Adhya and Gottesman, consistent

1978). This possibility

with the high level of protein

when the regulatory The

presence

sequence

of nonsense

starting

ATG codons

pSM31

and pSM41

sequence termina-

was deleted codons

is

synthesized (Fig. 2).

between

the

of lac and ada genes in both predicts

synthesis

of the Ada

protein in the native form and this was confirmed by N-terminal sequencing of the protein expressed from pSM41. The present experiments induction

School,

Worcester,

of the ada gene by MNNG

requires

the

upstream sequences which were deleted in our highexpression plasmid. This is an in vivo confirmation of the earlier in vitro studies which indicated the regulatory role of this sequence in the activation of the ada gene (Demple et al., 1985; Nakabeppu et al., 1985; Nakabeppu and Sekiguchi, 1986). Thus, the regulatory sequence of the ada gene may have two opposing roles in vivo, namely, a negative (termination) role in transcription initiated upstream from the gene and a positive role in transcription initiated at the natural promoter when interacting with methylated Ada protein. Finally, our objective was to develop an expression system for both wild-type and mutant Ada proteins which does not require activation by an alkylating agent. To achieve this, we have expressed the wild-type ada gene in both pUC-based plasmid and a 2 p,,-expression vector. However, the i. p, construction, unlike the pUC recombinant plasmid, failed to produce several mutant Ada proteins (not shown). It is possible that the rather high temperature (42-C) required for induction of the i 11~ promoter resulted in degradation of these labile proteins. In any case, the pUC-expression system should be more useful for the production of temperature-sensitive Ada mutants.

sion for suggestions

and

in the construction

of plasmids

appreciate the generous gift of Dr. D. Court, NCIFrederick Cancer Research Facility, Frederick, MD of i 11~ expression kindly

vector

and its host as well as

Dr. F.C. Hartman

provided

the facilities

of this Division

for the N-terminal

sequencing of the Ada protein C.D. Stringer.

carried

out by Mr.

The submitted manuscript has been authored by a contractor of the U.S. Government under contract No. DE-AC05840R21400. Accordingly, the U.S. Government retains a nonexclusive, royalty-free license to publish or reproduce the published form of this contribution, or allow others to do so, for U.S. Government purposes. This research was jointly sponsored by the Office of Health and Environmental Research, U.S. Department of Energy, under contract DE-ACOS840R21400 with the Martin Marietta Energy Systems, Inc., and by a National Cancer Institute Grant CA 31721.

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mutaMethods