Vol. 168, No. 2, 1990 April 30, 1990

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 801-808

BIOCHEMICAL

Cloning

and Expression

Erwinia

of

carotovora

Pectin

Lyase

Gene from

in Escherichia

coli

Toshihiko Suzuki, Kazutoshi Tamotsu Nishida, Yoshiyuki Kamio and Kazuo Izaki Department Agriculture,

of

Ito,

Agricultural Chemistry, Faculty of Tohoku University, Sendai 981, Japan

Received March 12, 1990 SUMMARY: A pectin lyase (PNL; EC 4.2.2.10) gene of Erwinia carotovora Er was cloned and expressed in Escherichia coli. The analysisof the nucleotide sequenceofthe 0.6 kb StuI-EcoRI fragment, which was hybridized with the mixed oligonucleotideprobe for PNL the presence of an open reading frame (ORF)andcorgene, revealed related exactly with the known N-terminal18 amino acid sequence of PNL. When a plasmid pTN2159, which has a BamHI-EcoRI fragment -containing this ORF, was introduced into -E.coli JM109, PNL was not expressed. When a tat-promoter was inserted in front of the ORF, PNL was efficiently expressed in -E.coli. Synthesis of PNL by E.coli was also confirmed by immunoblot analysis. 01990 Academic Press,

Inc.

Pectolytic organisms

enzymes

which

Among pectinolytic

(pectate

lyase,

tion

with

as well

strains

PL;

carotovora

studied

and Erwinia

respect

fungi

vora

Er

rarely

to nalidixic

Tsuyumu and Chatterjee atroseptica examined amount

acid, surveyed

and E.chrysanthemi produced of the

PNL

(3).

lyase,

which

to

PL, some

is

found

in

The PNL from -~ E.caroto-

mitomycin

when bacterial

cultures

C, or WV light

(6,7,8).

a number of and found

of produc-

In addition

only

upon mitomycin

enzyme produced

has been extensively

(4,5,6).

abundantly

lyase

Erwinias,i.e.,

and regulation

a pectin

in bacteria

in plants acid

soft-rot-causing

chrysanthemi,

micro-

diseases

polygalacturonic

properties produce

was synthesized

were exposed

of

causing

to pathogenicity

as enzymatic

but

in phytopathogenic

for

enzymes,

EC 4.2.2.2)

of E.carotovora

some

found

are responsible

(1,2).

Erwinia

are often

that

E.carotovora all

of

C treatment,

was different

from

the

subsp. strains

although one strain

the to

0006-291X/90$1.50 801

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

168, No. 2, 1990

another

(9).

is required tovora

(10).

-ra Er and its

BIOCHEMICAL

Zink for

the

In this

et --

al

also

production report,

expression

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

demonstrated of

the

PNL in E.carotovora

cloning

in E.coli --

that

of

PNL gene from

recA

product

subsp.caroE.carotovo-

are described.

MATERIALS AND METHODS Bacterial strains and plasmids. E.coli JM109 [( Alac-proAB), -7 F :traD36, recA1, endAl, gyrA96, c, hsdR17, supE44, &Al, proAB, lacIqZ@fl51 was used asahost strain and a plasmid pUC19 was used as a vector for cloning. Plasmid pUC118, pUC119, were used as vector and -E.coli MVI 184 [e, A(lac-pro), -A, m, ($80, lacZaM15),(srl-recA)306::Tn10(tetr), F':traD36, proAB, 1acIq zAM151, and M13K07 were used as a host, andahelper phage respectively, for preparation of single stranded DNA. Erwinia carotovora Er was used as a source of chromosomal DNA. Plasmid pKK223-3 was used as anexpression vector. Preparation of mixed oligonucleotide probe for cloning of PNL gene. The NH2-terminal sequence of PNL was determined by Edman degradation. Oligonucleotides were synthesized by Applied Biosystems model 381A DNA synthesizer. Synthetic oligonucleotides were labeled with Y-32P-ATP (>11OTBq/mmol) as described by Maniatis et -al (11). Genomic Southern blot hybridization. About 10 pg of purified DNA of E.carotovora Er was completely digested with a series of restriction enzymes and electrophoresed in 0.7 % agarose gel. Fragment of DNA were transferred from the gel to nylon membrane filand hybridized ters with the labeled probe. Hybridization was done at 55°C for 18 hr in a solution containing 6 x SSC (1 x SSC: 0.15 M NaCl, 0.015 M sodium citrate), 5 x Denhardt's reagent (1 x Denhardt's reagent is 0.02 % each of Ficoll, polyvinylpyrrolidone and bovine serum albumin), 0.5 % SDS and 200 ug/ml denatured salmon sperm DNA. Filters were washed twice (15 min. per wash) with 2 x SSC at room temperature and once with 2 x SSC at 55'C for 5 min. DNA preparation and cloning. Genomic DNA from E.carotovora Er was purified by the method of Rodriguez digested com--et al (12), pletely with EcoRI and BamHI, and was fractionated in size on a 0.7% agarose gel. DNAragments of desired size range were extracted by the procedure of Maniatis et al (11). Plasmid pUC19 DNA was double digested with EcoRI and BamHI. The digested plasmid DNA was then treated with bacterial alkaline phosphatase. EcoRI-BamHI fragments of the chromosomal DNA were ligated to the phosphatase-treated pUC19 DNA. The ligated DNA was transformed into -E.coli JM109 and the transformants were selected by ampicillin resistance and absence of 8-galactosidase activity (white colonies) on LB plates containing 75 ug/ml of ampicillin and 40 ug/ml of 5-bromo-4-chloro-3-indolyl 8-D-galactopyranogide (K-gal). White colonies on the agar plate were screened by the colony hybridization technique using the Y-32P-labeled probe. The plasmid, which was isolated from the positive clone,was designated as ~TN2159.

DNA sequencing. DNA of pTN2159 was subcloned into the plasmids pUC119 and pUC118 and the resultinq nlasmids were introduced into E.coli MV1184 to isolate ssDNA using bacteriophage M13KO7 as de-scribed previously(l3). DNA sequencing was performed by the method of Sangar et using Ml3 sequencing kit 141, (Takara Shuzo -- al co., Kyoto) and a-3 1 P-dCTP (>lSTBq/mmol). 802

Vol.

BIOCHEMICAL

168, No. 2, 1990

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Construction of expression plasmids. A StuI-EcoRI fragment (2.1 from pTN2159 was isolated, and thefraqment was trimmed to kb) about 200 bp each from bothendswith Bal31 exonuclease and filled up with the aid of T4 DNA polymerase and ligated to SmaI-digested and phosphatase-treated expression vector pKK223-3 (Pharmacia, Inc. Uppsala) according to the strategy illustrated in Fig. 3. The ligated DNA was transformed into -E.coli JM109. The plasmids containing the fragments, which were trimmed from both the ends of the -StuI-EcoRI fragment at various lengths, were obtained from the transformed cells (pDSE74, pDSE85, pDSE147). Preparation of cell extracts and assay of PNL. The cells of the plasmid-containing E.coli JM109 were inoculated into LB medium -containing ampicillin (75 pg/ml) and were grown at 37'C to an early log phase (3 x IO8 cells/ml). At this stage, IPTG (Isopropyl-p-D-thiogalactopyranoside) was added to the medium to a final concentration of 2 mM, and the cells were allowed to grow for an additional 2 hr. The cells were harvested by centrifugation at 10,000 x g for 10 min. at 4'C and washed once with 10 mM Tris-HCl buffer (pH 7.5). The cells were suspended in 10 mM Tris-HCl buffsonicated and then centrifuged at 15,000 x g er (pH7.5), for 10 min. (4" C). The supernatant was used directly for PNL assay. PNL activity was determined by the method as described by Itoh -et al (15). Protein content was determined by the method of Lowry et al -(16).

Polyacrylamide qel electrophoresis and Western immunoblot analya. SDS-polyacrylamide gel electrophoresis was done by the method of Laemmli (17). Proteins in the gel were electrophoretically transferred to a nitrocellulose filter. The blotted filter was treated with an anti-PNL polyclonal antibody and the protein reacting with this antibody was detected with alkaline phosphataselabeled anti-rabbit immunoglobulin antibody (Promega).

RESULTS AND DISCUSSION Analysis

of N-terminal

Er was purified minal

by the

amino acids

18

sequence method

of

of

PNL.

as described

of

synthesis

N-terminal

of

previously

PNL were determined

AsnLeuThrGlyLeuIleGlyPheAlaLysAlaAlaLys sequence

PNL from E.carotovora N-ter-

to be AlaTyrProThrThr

by Edman degradation.

residues

53 mer mixed

(7).

1 to

18 allowed

oligonucleotide

probe:

us to design

The the

5'-GCITATCCIACIACIAATTTIACIGGITTIATIGGITTTGCIAAAGCIGCIAA-3'. C cc C C G Cloning probe

of

was labeled

was carried tovora

beled mately

PNL gene in E.coli

out

digested probe.

with with with

After

8 kb position

JM109.

y- 32P-ATP. the

fragments

several

Southern of

restriction

radioautography, was detected

Synthetic

hybridization

chromosomal enzymes a discrete

inthecaseof 803

oligonucleotide

DNA

analysis of -E.caro-

using band at

BamHIand

this

la-

approxiEcoRI di-

Vol.

BIOCHEMICAL

168, No. 2, 1990

Fig.

B

S

I

I

1.

Restriction

AND BIOPHYSICAL

R5

RESEARCH COMMUNICATIONS

R5 R.5 T22

I

I

endonuclease

map

of

Rl

I I I 1 0.5 kb 1

8.2

kb

insert

in

plasmid pTN2159. 0.6 kb StuI-EcoRV fragment hybridized probe is underlined. Abbreviations: E,QRI; B,mHI; R5,gRV ; T22,ET22 sites.

gested

chromosomal DNA

somal

in

approximately

screened

9 kb in

colonies

with

the

gel

library

the

mixed

restriction

Four

insert

oligonucleotide the

colonies were all

130

140

150

GGTAWGAATTATTOXXAGGTTG 200

190

160

were colonies

strongly not all

the

120

180

170

TlTTWl'AlTAAACTCGAlTAATAAGCGTAATAAGCGTAAn;AAA 210

220

230

240

290

300

TCC'R"l'CTATACAATl"l"lTAA~TCGGA~~A~A~A~TCAATT~T~TAC 260

250

270

280

~n;GMGACATTATTRTTCA~A~T~~~C~A~~A~CMCMC m . 310 AAATCTTA(X'GGGCT

320

&tM~TyrPKlThShK

.

330 340 350 360 AAAACJXGCAAAAGTTACCGGAGGAACGGGCGG

AsnLeuThrGlyLeuIleGlyPheRlaLysAlaAlaLy~alThrGl~l~rGl~ly 370

380

390

400

410

420

TAAAGTCGTTACGGTAAA 'lXTWXCCGAl'Tl.TAAATCAGCGGTGACffiTTCCGCAAAA LysValValThrValAsnSerLeuAlaAspPheLysSerAlaVal~rValPr~ln

Fig. 2. Partial nucleotide ment of plasmid pTN2159. 78

amino

site is numbered

acids

labeled beginning

of

PNL

as

S.D. from

is

and the

sequence

of

0.6

kb

aI--RI

frag-

Corresponding sequence of N-terminal boxed. The presumed ribosome-binding underlined. StuI site.

The

base

positions

hy-

shown).

60

100 110 70 80 90 AGGCTGGTGATGATAATCGTAGCGC'ECCA'TTlTACTAAAAGATGGCGGCGTAlW,ATTG

and

containing

(data

50

of

Approxi-

colonies)

showed that

40

stu1 10 20 30 C~TATCAGTCTGATGAAGTACAGGCTGCGAACCGTATA

by

gel,

positive

probe

plasmids

the

JM109.

(white

probe.

chromo-

fragments

from

DNAs from positive

map of

DNA

and

inserts

the

was fractionated

in E.coli --

8.2 kb -BamHI-EcoRI

to

Accordingly,

were extracted

containing

Plasmid

an approximately

Partial

size

oligonucleotide

were identified.

bridized

% agarose

a subgenomic

1,000

shown).

BamHI and EcoRI,

a 0.7

6 to

used to prepare

not

with

digested

electrophoresis

mately

DNA (data

the

with the S,GI;

are

plas-

Vol.

168,

No.

mids

were

I).

To confirm

the

of

quence strongly

This

the 0.6

kb

an open

exactly

to

that

resuit

fragment

of

Expression

fragment

mixed

the

as

pTN2159, of

pTN2159

oligonucleotide

that (ORF)

in

COMMUNICATIONS

the

N-terminal

18

the

presence

of

lyase

activity

in

of

JM109

amino

acids at

(Fig. DNA se-

which

was

was

fragment

sequence

PNL gene

the

probe

S&I-EcoRV

which

of

pTN2159

of 2.lkb

decon-

corresponds PNL (Fig.2). --StuI-EcoRV

pTN2159.

of in

StuI-EcoRV the

RESEARCH

designated

PNL gene

frame

suggests of

was

indicates

reading

BIOPHYSICAL

of

with

The result

AND

plasmid

presence

hybridized

tains

tected

same.

the

termined.

This

BIOCHEMICAL

2, 1990

pectin

the

crude

extract

B

-Y

E.coli. harboring

As PNL was pTN2159,

not

we at-

pTN2159 10.9 kb

B +$$

L-l

&I StuI-EcoRI 2.1 kb fragment Alkaline'p~sphatase / Ba131 nudase

& T4 DNi polymerase \

T4 DNA ligase H B

pDSE147 pDSEs5 pDSE74

@k-

8 Construction of Fig. 3. The position and direction solid arrow. Abbreviations: st,cu1.

the

plasmid pDSE74, pDSE85, pDSE147. by the of the PNL gene is indicated B,wHI; E,mRI; H,=dIII; S,aI;

805

de-

Vol. 168~, No. 2, 1990 Table

BIOCHEMICAL

1.

Activity of containing

Plasmid puc19 ;TN2159 pKK223-3 pDSE147 pDSE85 pDSE74

the

plasmids

PNL gene in Fig.

each of

those

ities

of

ted

of

level

E coli -*-

JM109

protein)

plasmids

(pDSE147,

50-fold the

higher

was inserted

upstream

the

in -E.coli

than

plasmid with

of a protein

A

12

JM109,

illus-

1).

Er. the

were solubilized brilliant

Coomassie

12

B

Activ-

pDSE85 or

in E.carotovora

PNL in E.coli,

containing

In

sonicawith blue.

10%

A

pDSE74

3

Fig. 4. The expression of PNL in E.coli. (A)10 % SDS polyacrylamide gel electrophoresis pattern of proteins from the cell containing pDSE74 and pKK223-3. Cells containing were grown and collected as described in MATERIALS AND METHODS, and equivalent amounts of proteins were analyzed by SDS-PAGE. (B) Immunoblot of total cell lysates from the cells containin'g pDSE74 or pKK223-3 is shown. Equivalent amounts of proteins from total cell lysates were separated by SDS-PAGE, transferred electrophoretically to nitrocellulose filter, and cross reacted with anti-PNL serum. Lanes: 1, purified PNL from E.carotovora Er; 2, pDSE74; 3, pKK223-3; MW, molecular weight standards. The arrows indicate the position of PNL. 806

of

containing

pDSE147,

in E.coli

3

strategy

pDSE74)(Table

that of

and stained

kD!'iw

tat-promoter.

JM109 containing

harboring

expression

of

to

pDSE85,

overexpression

E.coli --

control

according

3. PNL was expressed

SDS, electrophoresed high

the

tat-promoter

PNL shown by -E.coli

to verify cells

gene under

in which

pDSE74 wereabout order

this

were constructed

trated

of

(U/mq

ND ND ND 4680 4790 3200

in

detected.

to express

Several

PNL synthesized the plasmids

PNL activity

ND: Not

tempted

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol.

166,

No.

2, 1990

BIOCHEMICAL

which

was

detected

from

E.carotovora

the

same

samples

the

overexpressed

PNL (Fig.

of

promoter

reason into

why

of

From

Er was

cloned

in

E.coli.

Preliminary

two

palindromes

ing

site.

region immediately

The presence

failure

of

the

expression

the

We have

not

of

--E.coli

the

of

PNL gene

indeed

evident

that

explanation pTN2159

was

of

revealed

palindromes in

was

a tac-

PNL was

effi-

for

the

introduced

nucleotide

the

of the putative

of

that

and when

analysis

PNL gene

upstream

was

PNL gene,

on the

PNL of

confirmed

it

when

of

analysis

pDSE74

no clear

expressed data

weight

antibodies containing

of

COMMUNICATIONS

Immunoblot

result,

in

promoter

2).

E.coli --

front

RESEARCH

molecular

above

in

was

the

the

inserted

PNL gene

as

anti-PNL in

E.carotovora

-E.coli ---

quence

rabbit

2).

expressed

same

BIOPHYSICAL

4A,Lane

protein

was

ciently

(Fig.

with

4B,Lane

PNL gene

the

was

Er

AND

se-

presence

of

ribosome-bind-

might

be related

to

E.coli.

ACKNOWLEDGMENT We thank manuscript.

Mr.

A.Rahman

for

his

critical

reading

of

this

REFERENCES

3) 4)

Chatterjee,A.K. and Starr,M.P. (1980) Ann. Rev. Microbial. 645-6'76. Collmer,A., Berman,P. and Mount,M.S. (1982) In Phytopathogenic Prokaryotes,ed. M.S.Mount and G.H.Lacy, 1, 395-422. Academic Press, New York. Kotoujansky,A. (1987) Ann. Rev. Phytopathol. 25, 405-430. Erdstorm,R.D. and Phaff,H.J. (1964) J. Biol. Chem. 239,

5)

Ishii,S.

1)

2)

2403-2415.

and

Yokotsuka,T.

(1975)

Agric.

Biol.

Chem.

39,

313-321. 6)

Tomizawa,H.

and

Takahashi,H.

(1971)

Agric.

Biol.

Chem.

35,

191-200.

7)

Kamimiya,S., Nishiya,T., Izaki,K. and Takahashi,H. Agric. Biol. Chem. 38, 1071-1078. 8) Itoh,Y. ,Sugiura,J., Izaki,K. and Takahashi,H. (1982) Agric. Biol. Chem. 46, 199-205. and Chatterjee,A.K. (1984) Physiol. Plant 9) Tsuyumu,S.

10)

4,

11)

Pathol.

291-302.

Zink,R.T.,

Engwall,J.K., McEvoy,J.L. and Bacterial. 164, 390-396. Maniatis,T., Fritsch,E.F. and Sambrook,J. Cloning: A Laboratory Manual, Cold Spring Cold Spring Harbor, N.Y. (1985)

(1974)

Chatterjee,A.K.

J.

807

(1982)

Harbor

Molecular Laboratory,

34,

Vol. 168, No. 2, 1990

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Rodriguez,R.L. (1983) Recombinant DNA Techniques: An Introduction, Addison-Wesley Publ. Co. 13) Vieira,J. and Messing,J. (1987) Methods in Enzymology

12)

153,

3-11. 14) 15)

Sanger,F., Nicklen,S. and Coulson,A.R. Acad. Sci. USA 74, 5463-5467. and Takahashi,H.(1980) Itoh,Y., Izaki,K. 44,

16) 17)

(1977)

Agric.

Proc.

Natl.

Biol.

1135-1140.

Lowry,O.H., Rosebrough,N.J., (1951) J. Biol. Chem. 193, Laemmli,U.K. (1970) Nature

Farr,A.L. and Randal1,R.J. 265-275. 227, 680-685.

808

Chem.