Cloning of genes encoding pectolytic enzymes from a genomic library of the phytopathogenic bacterium, Erwinia chrysanthemi

Gene, 35 (1985) 121-130 Elsevier

121

GENE 1273

Cloning of genes encoding pectolytic enzymes from a genomic library of the phytopathogenic bacterium, Enviniu chysunthemi (Recombinant DNA; broad-host-range cleavage; physical map)

cosmid; Escherichiu coli host; pBR322 plasmid; polygalacturonate

S. Reverchon, N. Hugouvieux-Cot&Pat-tat and J. Robert-Baudouy Laboratoirede Microbiologic, Blit. 406,Institit Nationaldes Sciences Appliqukes, 20 Avenue Albert Einstein, 69621 Villeurbanne (France) Tel. (7) 894.80.88 (Received September 26th, 1984) (Revision received October 31st, 1984) (Accepted November 27th, 1984)

SUMMARY

Erwinia chrysanthemi are phytopathogenic enterobacteria causing soft-rot disease due to pectolytic enzymes degrading plant cell walls. We constructed a genomic library from Sau3Adigested E. chrpanthemi B374 DNA cloned in the BamHI site of the broad-host-range cosmid pMMB33 grown in Escherichia coli. Out of 1500 kanamycin-resistant (KmR) transductants of E. coli, nine pectolytic-enzyme-positive clones were identified. One of these contained the PEW325 cosmid with a 35-kb insert of Enviniu DNA. Cell extracts of E. coli harboring the cosmid pEW325 were fractionated on a polyacrylamide electrofocusing gel; bands with pectolytic activity were found to co-focus with pectolytic enzymes of E. chrpanthemi B374 strain. Cosmid pEW325 encodes three pectolytic enzymes PLlO, PL20 and PL130 with isoelectric points of about 9.3,9.2 and 4.6, respectively. These enzymes are lyases that cleave polygalacturonate by transelimination, and give rise to unsaturated products. A 15-kb Hind111 fragment coding for polygalacturonate lyases was subcloned in pBR322, and a physical map of the resulting plasmid pPLO1 was constructed. Starting from the pPLO1, various endonuclease-generated fragments were subcloned into pBR322. Genes encoding pectate lyases were localized within an 8-kb fragment (pPLO4) and then in a 2.7-kb fragment (pPLO3). Polygalacturonate lyases are expressed at various levels; they accumulated in the periplasmic space of E. coli host, whereas E. chrysanthemi secreted these enzymes into the culture medium.

INTRODUCI’ION

E. chrysanthemi are phytopathogenic bacteria, responsible for soft-rot disease of many plant species.

Abbreviations: Ap, ampicillin; bp, base pairs; kb, 1000 bp; Km, kanamycin; PL, pectate lyases; R, resistant; Sm, streptomycin; Tc, tetracycline; [I, indicates plasmid-carrier state; 0, specifies prophage; ::, novel joint. 0378-l 119/85/$03.30 0 1985 Elsevier Science Publishers

Their phytopathogenicity is related to their ability to produce and to secrete pectolytic enzymes which degrade plant cell-wall pectins. This action leads to tissue breakdown and cell lysis by osmotic shock (Garibaldi and Bateman, 1970; 1971; Mount et al., 1970; Tseng and Mount, 1974). Pectic acid is a homopolysaccharide made up of linear chains of a-( 1,4)-linked/D-galacturonic acid residues; pectin itself is pectic acid with varying

PECTINS 1 POLY-METHYLGALACTURONATE

GALACTURONATE

DIG AL AC TURONATE

-

TAGATURONATE

5-KETO-C-DEOXY-URONATE

I

F (kdul)

III luxaB)

I

Z-5-DIKETO-3-DEOXY-GLUCONATE I

ALTRONATE liYluxdA~

1

_

lslldrU,

Z-KETO-3-DEOXY-GLUCONATE Y lkdgKI I 6-PHOSPHO-Z-KETO-3-DEOXY-GLUCONATE YI lkdgAl

I PYRUVATE + 3- PHOSPHOGLYCERALDEHYDE Fig. 1. Degradative pathway for pectin and poiyg~acturonate in E. c~~~u#~~ernj.The different steps are catalysed by the following enzymes: A, pectin-methyles~rase (EC 3.1.1.11); B, polyg~acturonase (EC 3.2.1.15); C and E, poIyg~~t~onate and pectin lyases (EC 4.2.2.2.); D, oligog~a~uronide lyase (EC 4.2.2.6); F, 5-keto~~eox~ronate isomerase (EC 5.3.1.17); G, 2-keto-3~eoxy~uconate oxidoreductase (EC 1.1.1.127); H, hexuronate transport system; If, uronate isomerase (EC 5.3.1.12); III, m~nonate oxidoreductase (EC 1.1.1.57); IV, mannonate hydrolyase (EC 4.2.1.8); V, Zketo-3-deoxygiuconate kinase (EC 2.7.1.45); VI, 2-keto-3-deoxy-6-phosphogluconate aldolase (EC 4.2.1.14). For each step, the symbols in parentheses designate the structural genes of the corresponding enzymes. The galacturonate pathway is similar to that described in E. coii (Kilgore and Starr, 1959; Ashwell, 1962). The 5-keto-4-deoxyuronate pathway has been first described in Pseudomonas (Preiss and Ashwell, 1963) and confirmed in E. chrysunthemi (Condemine et al., 1984).

extents of methyl esteritication. Erwinia phytopathogenie species (chrysanthemi or carotowra) produce a large variety of intra- and extracellular pectolytic enzymes (see Fig. 1). Pad-me~ylesterase (Fig. 1, A) demethoxylates pectin to form polyg~act~onic acid (Goto and Okabe, 1962). A whole array of PL, including random- and end-splitting types, specific for pectin or polygalacturonic acid has been found (Fig. 1, C and E). These enzymes act by transelimination and generate unsaturated products

(Moran et al., 1968; Garibaldi and Bateman, 1970; 1971; Stack et al., 1980). Polygalacturonases (Fig. 1, B) split the glycosidic bonds in a hydrolytic manner resulting in formation of saturated products (Nasuno and Starr, 1966; Collmer et al., 1982). An oligogalacturonide lyase was isolated from E. carotovora (Moran et al., 1968; Stack et al., 1980). At pH 8.5, this enzyme converts unsaturated digalacturonic acid to two molecules of 5-keto-4-deoxyuronate. At pH 6.0, it can convert

123

unsaturated and saturated digalacturonic acid into monomeric D-galacturonic acid and Sketo&deoxyuronate (Fig. 1). E. chrysanthemi excretes pectolytic enzymes, cellulases (Andro et al., 1984), and proteases (C. Wandersman, pers. commun.) into the culture medium. To permit a genetic study of genes involved in pectolysis, cellulolysis, proteolysis or in excretory mechanisms of E. chrysanthemi, we decided to construct a genomic library from the E. chrysanthemi B374 strain (Hamon and Peron, 1961).

MATERIALS AND METHODS

(a) Bacterial strains and plasmids The bacterial strains and plasmids used in this study are given in Table I. (b) Media and culture conditions E. chrysanthemi and E. coli were grown at 30°C and 37’ C, respectively, with aeration. LB rich medi-

and M63 minimal medium were both prepared according to Miller (1972), and the media were solidified by addition of 15 g/l agar. Glycerol was added in minimal medium as sole carbon source at 0.2%. Antibiotics were used in selective media at the following concentrations: 20 pg Km/ml, 50 pg Ap/ml and 5 pg Tc/ml.

um

(c) Extraction and manipulation of DNA Chromosomal DNA from E. chrysunthemi B374 was purified by phenol extraction. The cells were lysed by incubation in 50 mM Tris * HCl pH 8, 50 mM EDTA, 1 mg/ml lysozyme (Boehringer) at 0°C for 30 min followed by the addition of 1 mg/ml sarkosyl, 0.1 M EDTA, 0.2 mg/ml proteinase K (Sigma) and incubation at 50” C for 1 h. The lysate was then extracted three times with phenol, twice with phenol-chloroform (1: 1) and once with chloroform-isoamyloalcohol (24 : 1). After precipitation with 2.5 vol. of ethanol, the DNA was dissolved in TE buffer (10 mM Tris * HCl pH 8, 1 mM EDTA) and treated with 100 pg/ml BNase for 1 h at 37°C. DNA was extracted again with phenol and phenolchloroform, precipitated with 2.5 ~01s. of ethanol

TABLE I Bacterial strains and plasmids Strains E. chrysanthemi B374 E. coli JD75 HBlOl BHB2688 BHB2690 LE392

Genotype or phenotype

Source or reference

wild type

Hamon and Peron (1961)

F-,

recA1, endAl, gyrA96, thi-1, hsdR17 (r-m+), supE44, Iz[R64.11] F-, hsdS20 (r-m-), recAl3, am-14, proA2, leu, lucY1, guZK2, 1psL20, ~1-5, m&l, supE44, IN205 recA(1 Eam4, b2, red3, imm434, cIr.r, Sam7)/1 N205 recA(r2Dam4, b2, red3, imm434, cI&r, Sam7)/Iz

Frey et al. (1983)

F~,hsdR5l4(r~m~),supE44,supF58,lacYl,galK2,galT22,metBl,

Boyer and Roulland-Dussoix (1969) Hohn (1979) Hohn (1979) Maniatis et al. (1982)

hpR55,1PhWllllds” pMMB33 R64.11 pBR322

Rmn, cosl SmR, Ten, tra + Apn, TcR

Frey et al. (1983) Meynell and Cooke (1969) Bolivar et al. (1977)

*The broad-host-range cosmid pMMB33 is a 13.75-kb derivative of RSF 1010 and pHC79. It contains a single BamHI restriction enzyme site and confers resistance to Km. It is not self-transmissible but can be mobilized in the presence of a helper plasmid such as R64.11 which carries the conjugal transfer genes and confers resistance to Tc and Sm. R64.11 is a narrow-host-range plasmid which replicates in E. cob but not in E. chrysanrhemi, as tested in our laboratory.

124

were performed as described elsewhere (Maniatis et al., 1982).

and finally dissolved in TE buffer. Plasmid was purified by the method of Birnboim and Doly (1979). Digestion of DNA with restriction endonucleases, separation of DNA fragments by gel electrophoresis, and ligation of DNA fragments with T4 DNA ligase

(d) Construction of a genomic library The procedure used is summarized in Fig. 2.

Bamtil

digestion 1

Hpal

OFI

with Sau3.d

Sau 3A

t;;;;

wmh3”

Km

-

BamHl SmaI

I

I

-m

ligation I -ios

on

hU3A

SadA

Km

z

38-51 Kb in

vitro

vr, I

4P

packaging

cosmid lysate

V

infection

of

Escherichia

coli

Fig. 2. Cloning of Sau3A fragments of E. chrysanthemi DNA in the pMMB33 cosmid vector. Following digestion of pMMB33 DNA with HpaI or SmaI that both generate blunt ends, the linear vector DNAs are cleaved with BamHI. 25-40-kb fragments of E’. chrysunthemi chromosomal DNA generated by partial digestion with Sau3A are ligated to the cos-carrying fragments. The ligation conditions (5 mM ATP, 5 mM MgCl,) prevent the blunt end ligation (Ferretti and Sgaramella, 1981). The concatemers thus constructed contain two cos sites and are suitable substrate for in vitro 1 packaging. Following transfection into E. coli, the cosmid DNA recircularizes and replicates in the form of a large plasmid. The plasmid contains a gene that confers resistance to Km on the host bacterium. In vitro packaging was performed as described by Mania& et al. (1982). Freeze-thaw lysate (FTL) was prepared from the strain BHB 2688 and sonicated extract (SE) from the strain BHB 2690 using the procedure of Ish-Horowitz and Burke (1981). Packaging efficiency was tested using U&m17 DNA and the E. co& LE392 strain, and found to be 4 x lo* plaque-fo~g units per gg of 1 DNA.

125

LKB ampholine ultroPAG plates in the 3.5-9.5 pH range were used to focus polygalacturonate lyases. The electrofocusing was performed at 30 W for 3 h with 0.1 M NaOH and 0.1 M H,SO,, respectively, as catholyte and anolyte. After protein separation pectolytic activities were detected as described by Bertheau et al. (1984).

RESULTS

(a) Construction of the gene bank of E. clrrysanthemi A gene bank of E. chrysanthemi strain B374 total DNA, partially digested with Sau3A, was constructed using the broad host range cosmid pMMB33 and was rn~t~~ in E. coli JD75. KmR ~~sduct~ts were obtained at a frequency of 5 x 103/,ug of E. ch~~unt~mi DNA. The resulting gene bank contained about 5000 independent clones. 1500 clones were stored individually, and the remaining clones were stored as mixture. 20 clones were chosen at random for analysis of their plasmid

DNA. Gel el~~ophoresis of BarnHI digests of these plasmids showed that all contained inserted DNA fragments ranging between 25 and 40 kb and averaging 30 kb in length. If the size of the E. chrysanthemi genome is assumed to be about the same as that of the E. coli genome, i.e., 4200 kb (Cairns, 1963), then only about 650 members of the gene bank are needed to guarantee a 99% chance that a given sequence of DNA will be represented (Clarke and Carbon, 1976). To verify that any sequence could be found in the bank, we examined our 1500 ~~~du~ transductams for the presence of specific genes of E. chrysanthemi encoding cellulases, proteases, pectolytic enzymes and for genes conferring on E. coli the ability to grow on saccharose, cellobiose or raffinose as a sole carbon sonrce (E. coli strains are unable to degrade these sugars). All these genes were found in several independent clones in our library (Table II), suggesting that the library is a good representation of the entire genome. (b) Detection of peetolytic activities in E. cdi Out of 1500 transductants, nine pectolytic enzymepositive clones were isolated (Table II). One of these clones was studied in detail. The cosmid it harbors

TABLE II Specific E. chrysanrhemimarkers found in the cosmid library Marker screened”

Cosmids bearing this marker Number

Pectolytic enzyme synthesis

9

Cellulase synthesis

8

Protease synthesis

11

Growth of E. coli on saccharose

14

Growth of E. co&on cellobiose Growtb of E. co&on raflinose

7 8

Name pEW168, pEW325, pEW508, pEW673, pEW892, pEW971, pEW1460, pEW1466, pEWI495. pEW362, pEW457, pEW498, pEW1159, pEW1302, pEW1332, pEW1348, pEW1487. pEW1, pEW273, pEW294, pEW393, pEW495, pEW587, pEW733, pEW738, pEW778, pEW1196, pEW1441. pEW122, pEW140, pEW340, pEW352, pEW358, pEW394, pEW420, pEW608, pEW682, pEW819, pEW1305, pEW1389, pEW1444, pEW1491. pEW365, pEW485, pEW797, pEW1096, pEW1345, pEW1365, pEW1401. pEW164, pEW398, pEW473, pEW774, pEW785, pEW891, pEWY41, pEW1147.

BThe 1500 individual E. co&clones constituting the E. chrysunrhemi gene bank were screened to find markers present in E. chrysanthemi but &sent in E. co& The tests for detection of pectolytic enzymes, cellulases or proteases were based on the degradation of an enxyme substrate surrounclmg positive clones in solid medium. When necessary, an appropriate dye which forms complex with the polymer, per&s to see clear haloes around these clones. Cellulases were detected on carboxymethylcellulose (0.4%), trypan blue (0.05 %) medium (Wood, 1981). Pectinases were screened on plates containing polygalacturonate (0.4x), stained aher incubation with 10% copper acetate. Proteases were detected on medium containing 1% skim milk powder.

126

was designated pEW325 and contains an insert of about 35 kb. The plasmid DNA was extracted from this clone and used to transform HBlOl. All the KmR colonies isolated &er ~~sfo~a~on displayed pectolytic activities. This result confmed the presence of pectolytic enzyme genes in the cosmid pEW325.

(c) alley

of the phmid

pEW325 and expres-

sion in B. co6

In an attempt to locate the coding sequences for the pectolytic enzymes on the large insert of 35 kb, we frosttried to subclone PstI, BarnHI, and Hind111 fr~ents into pBR322. pEW325 Cas totally digested with one of these enzymes and ligated with

lkb

BarnWI1

BarnHII

pPLos

Sam @

,EcdU3

pPCo4

lkb Fig. 3. ~est~~tion map of the 15.kb cbromosom~ DNA fra~ent inserted into the ~~~d1~~site of pBR322 and ~nt~g genes codixq for pectolytic enzymes of E. chrysan&emi. The pBR322 portion is represented by the heavy line. Five fragments suboloned in pBR322 are shown below. Only E. coli transformants bearing pPLO3 and pPLO4 showed pectolytic activities. A more precise restriction map of the piasmid pPLO4 is given in the tower part of the figure. The restriction sites are numbered (l-3) from left to right.

121

pBR322 linearized with the same enzyme. The ligation mixture was used to transform E. coli strain HB 101 and transformants were screened for pectolytic activity. Plasmids were extracted from all positive clones. One, containing a 15-kb Hind111 fragment was selected for further characterization and called pPLO1. A restriction map of this plasmid is shown in Fig. 3. Based on this restriction map, and to localize more precisely the regions encoding the pectolytic activities, we subcloned the different endonuclease-generated fragments of pPLO1 into pBR322. The resulting pPL plasmids are shown on Fig. 3. E. coli transformants carrying the different pPL plasmids were screened for pectolytic activity. Only E. coli transformants bearing pPLO3 and pPLO4 showed a positive test. When the 15-kb Hind111 fragment of pPLO1 was inserted in the opposite direction, in the resulting plasmid pectolytic activity was also detected. Cell extracts of E. cob harboring the initial cosmid pEW325 were fractionated on a polyacrylamide electrofocusing gel and stained for pectolytic activity. These extracts contained three bands of pectolytic activity with isoelectric points of 9.3, 9.2 and 4.6; these bands cofocus with PL recovered from supernatant of E. chrysanthemicultures. From cultures of B374, five major PL isoenzymes are detected PLlO, PL20, PL50, PL70 and PL130 which have isoelectric points of 9.3, 9.2, 8.5, 8.2 and 4.6, respectively (Bertheau et al., 1984). We conclude that the cosmid pEW325 contains the genes pelE, pelD and pel4 encoding pectolytic enzymes PLlO, PL20 and PL130, respectively. Some or all of these genes are located within the 15-kb fragment of pPLO1, then in the 8-kb fragment of pPLO4 and in the 2.7-kb fragment of pPLO3 (Fig. 3). A more precise restriction map of the plasmid pPLO4 was constructed (Fig. 3). The fragments BgflIl-BglII2 and EcoRI2-BamHIl showed no pectolytic activity. E. coli strains harboring plasmids pEW325, pPLO1, pPLO3 and pPLO4 could degrade polygalacturonate by transelimination, giving rise to unsaturated products whose appearance was followed at 235 nm, thus indicating that the genes present in our plasmids encode PL (enzyme C, Fig. 1). The level of expression of these enzymes in E. coli transformed by the four different plasmids was subsequently analyzed (Table III). A large difference in specific activity was observed when E. coli cells were transformed either by the

pEW325 cosmid or by the derivative plasmids pPLO1, pPLO3 and pPLO4. Strains carrying plasmid pPLO3 produce high levels of PL, about threefold the rate of the wild-type E. chrysanthemi B374 strain. TABLE HI Polygalacturonate lyase assays in E. coli strains bearing E. chrysanthemi-cloned genes. Polygalacturonate lyase activity was assayed on extracts of exponentially growing cells in glycerol minimal medium. The unsaturated compounds released from polygalacturonate by the enzyme were followed spectrophotometrically at 235 nm. The reaction mixture consisted of 0.1 M Tris . HCl buffer at pH 8.5, 0.125% polygalacturonic acid, 1 mM CaCl, and the enzyme at the correct concentration. The increase of 0.52 A units in the absorbance at 235 nm in a l-ml reaction mixture under the above conditions is equivalent to the release of 0.1 pmol of aldehyde groups (Moran et al., 1968). Specific activity is expressed as pmol of aldehyde groups liberated/min/mg of bacterial dry weight. Strain [plasmid]

Specific activitya

JD75[pEW325] HBlOl[pPLOl] HBlOl[pPL03] HBlOl[pPL04] HBlOl[pBR322] B374

0.005 0.07 1 0.336 0.060 ND 0.102

a ND, not detectable.

(d) Localization of polygalacturonate lyase activity in E. coli transformants The localization of polygalacturonate lyase activity in relation to marker enzymes was analyzed in an exponential phase culture of E. coli bearing the plasmid pPLO1 (Table IV). More than 85% of the total activity of the marker enzymes was found in the expected fractions. Polygalacturonate lyases, which are extracellular enzymes in E. chrysanthemi, were found in the periplasmic region in E. coli transformants.

DISCUSSION

In this report, we have described the construction of a genomic library of E. chrysanthemi strain B374 using the cosmid pMMB33, which is transmissible in

128 TABLE IV Localization of polygalacturonate lyase activity in E. coii [pPLOl] After centrifugation, the cell pellet ofE. coli harboring recombinant plasmids with pectolytic enzyme genes was washed twice with 10 mM Tris *HCI buffer (pH 8.0). Washed cells were treated with 2 mg/ml lysozyme (Boehringer) at 37°C for 25 min in 10 mM Tris - HCI buffer (pH 8.0) containing 20% sucrose and 10 mM EDTA. The periplasmic enzymes were recovered in the supernatant fraction after centrifugation at 10000 x g, for 10 min. The pellet containing spheroplasts was suspended in 10 mM Tris . HCI to cause the cells to burst. Cytoplasmic and membrane fractions were separated by centrifirgation at 27000 x g for 20 min. A marker enzyme in the cytoplasm, fi-galactosidase, was measured by using o-nitrophenyl-@.)-galactopyranoside as substrate (Miller, 1972); the released o-nitrophenol was followed at 420nm. One unit was defined as the amount of enzyme producing 1 nmol o-nitrophenol in 1 min. Cultures were grown in presence of 5 mM isopropyl-~-D-thiog~actopyranoside to induce @-gdactosidase. p-lactamase, a typical periplasmic enzyme, was measured by microi~omet~ as described by Novick (1962). One unit was defined as the amount of enzyme which catalyzes the hydrolysis of 1 pmol benxyl penicillin in 1 min. NADH oxidase, a membr~e-bond activity in E. co&was measured by monitoring s~c~ophotomet~c~y the decrease in absorbance at 340 nm in presence of NADH (Osborn et al., $972). Enzyme activities were expressed as % of total activities. Distribution of enzymes in fractions

Polygalacturonate lyase /J-Galactosidase &Lactamase NADH-oxyd~e

Medium

Periplasm

Membrane

Cytoplasm

(%I

(%I

(%)

(%)

15 0.3 2 0

82 0.6 86 5

0.5 6.3 0 88

2.5 92.8 12 7

the presence of the conjugative functions of helper plasmid R64.11. We chose the pMMB33 cloning system, because conjugation is a useful method for introducing cloned genes in E. chrysanthemi. This gene bank could be mated en masse into recipient strains that contained various mutations. Among the merodiploid strains thus constructed, clones carrying the wild-type allele could be obtained by screening for complementation of the mutant phenotype, this is particularly attractive for genes that could not be selected in E. coli. Some genomic libraries from other Erwinia strains have been constructed using different vectors: one from the E. chrysanthemi strain Ec16 in the vector pHC79 (Keen et al., 1984), one from E. chrysanthemi 3937j in the phage IL47.1 (A. Kotoujansky and A. Diolez, pers. commun.), one from E. chrysanthemi 1237 in pBR322 (A. Collmer, pers. co~un.), one from E. carotovora Ecc71 in the cosmid pHC79 (AK. Chatterjee, pers. commun.) and one from the E. c~~santhemi strain 3665 in a cosmid vector (F. Barras et al., pers. commun.). In all these cases, it is diicult to introduce the cloned genes in the parental Erwinia strains. In vivo cloning of some genes of the E. chrysanthemi B374 has been performed using RP4 : : mini-Mu plasmids (Van Gij-

segem et al., 1983 and pers. commun.). Our genomic library was constructed to identify genes governing pectin degradation. Hydrolysis of pectin to monomer sugars requires at least three types of enzymes: polygalacturonate lyases, polygalacturonases and oligogalacturonate lyase. We have determined that E. co& extracts carrying pEW325 possess three proteins with pectolytic activities with isoelectric points of 9.3, 9.2 and 4.6. These proteins are polygalacturonate lyases because these E. coli extracts can give rise to unsaturated products by polygalacturonate degradation. Polygalacturonate lyase activity was mainly located in the periplasmic space of E. coli cells, suggesting that the signal sequence of E. chrysanthemi polygalacturonate lyase is correctly processed in E. coli cells. In E. chlysanthemi, these enzymes are mainly excreted into the medium (Andro et al., 1984). The cosmid pEW325 contains an insert of about 35 kb bearing the pel genes encoding PLIO, PL20 and PL130. Some pef genes were located within a 15-kb Hind111 fragment (pPLOl), then in an 8-kb Hind111 1-BamHIl fragment (pPLO4) and in the 2.7-kb HindIIIl-Sal11 fragment (pPLO3) (Fig. 3). The 15-kb HlndIII fragment was inserted in the two opposite directions; PL activity was detected in both

129

cases. This result suggests that the pel genes are expressed under their own promoter and not under the TcR gene promoter. Since the Hind111 site of pBR322 cuts in the “ -10” region of the TcR gene, leaving the “-35” region intact, reconstitution of the promoter might occur. The fact that BgflIl-BgZII2 and EcoRI2-Bum HI 1 fragments showed no pectolytic activity suggests that thepel genes located on the HindIIIl-BumHI fragment could constitute an operon which transcription direction could be from Hind1111 to Sal1 1 and with a regulatory region located between Hind1111 and BgflIl. Subcloning new fragments of the plasmids pPLO3 and pPLO4 will allow the determination of the number and the relative order of the pel genes. Keen et al. (1984) have also cloned PL genes from another E. chrysanthemi strain EC16. This strain possesses only two pectinases with isoelectric points of 9.8 and 8.8 and the genes coding for these two pectinases are separated by at least 40 kb. In contrast, our strain B374 possesses five pectinases with isoelectricpointsof4.6,8.2,8.5,9.2and9.3,andthe three genes cloned in our work are gathered on a 35-kb fragment and could constitute an operon. In conclusion the PL genes of the strains Ec16 and B374 are different in their product, organization and regulation. These results underline the complexity of the pectin degradation by Erwinia. A large difference in the level of expression of the PL genes was observed in E. coli cells transformed either by the pEW325 cosmid or by the plasmids pPLO1, pPLO3 and pPLO4. This fact was probably due to a greater number of copies of pBR322 derivatives (pPLO1, pPLO3 and pPLO4) than the pMMB33 derivative (pEW325). E. coli strain carrying plasmid pPLO3, produce high levels of pectate lyases, about threefold the rate of the wild-type E. chrysanthemi B374 strain. This could be a first stage in overproduction of pectinases for industrial applications. In conclusion, the isolation of three structural genes encoding pectolytic enzymes of E. chrysanthemi will allow us to further characterize their expression and to determine the genetic structures governing their regulation. This is an important preliminary result for future investigations into the genetic basis of the pectinolysis of Erwinia and for the comprehension of factors regulating its phytopathogenicity.

ACKNOWLEDGEMENTS

This work was supported by grants from the Centre National de La Recherche Scientifique (Laboratoire Propre du C.N.R.S. No. 05421 and Action Thematique programmee Microbiologic 198 l), from the Minis&e de La Recherche et de L’Industrie (Aide a La Recherche Biologie Moleculaire) and from the Commission of the European Communities (Biomolecular Engineering Program). We thank J. Frey for supplying us with strains and for advice about handling pMMB33, A. Kotoujansky and Y. Bertheau for the separation of the PL of E. chrysanthemi by electrofocusing. We thank A. Hinnebusch, J. Hughes and M. Cashel for critical reading the manuscript, and F. Stoeber, in whose laboratory this work was carried out.

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