Molecular Microbiology (1989) 3(12). 1785-1795

Extracellular and periplasmic isoenzymes of pectatelyase from Erwinia carotovora subspecies carotovorabelong to different gene families

J. C. D. Hinton,* J. M. Sidebotham, D. R. Gill andG. P. C. SalmondDepartment of Biological Sciences. University ofWarwick. Coventry CV4 7AL, UK.

Summary

Pectate lyase (Pel) plays a crucial role in the mac-eration of vegetables by soft rot Erwinia spp. We havecharacterized the four Pel isoenzymes of Erwiniacarotovora subspecies carotovora strain SCRI193. Inthis paper we concentrate on two isoenzymes whichhave different locations in SCRI193: PLb is periplasmicand PLc is extracellular. Comparison of the geneproducts and nucleotide sequences of pelB and peICallowed us to assign them to different gene families. Inaddition, we have identified a number of conservedamino acid residues that are common to all extra-cellular Pel isoenzymes.

Introduction

Pectate lyase (Pet) has been identified as a major virulencefactor produced by members of the soft rot Erwinia group(Collmer and Keen. 1986). This bacterial group, whichincludes Erwinia carotovora subspecies carotovora {Ecc),E. carotovora ssp. atroseptica (Eca) and £ chrysanthemi(Echr), causes the soft rot of a number of vegetable cropsduring storage. Annual economic losses due to soft rotexceed $100 million worldwide (Perombelon and Kelman,1980). and crops including potato, carrot, green pepper,chicory, celery and cucumber are particularly susceptible.

Biochemical and genetical techniques have been usedto study the role of Pel in the development of soft rotdisease {Collmer and Keen, 1986). Pel genes have beencloned from a variety of Erwinia species (Kotoujansky.1987), including our laboratory Ecc strain SCRI193 (Plas-tow etai.. 1986). tsoelectric focusing (lEF) techniques havebeen used to characterize the multiple isoenzyme profilesof Pel produced by different Erwinia species (Bertheau eta/., 1984; Ried and Collmer, 1985; 1986). Recently the role

Received 16 June, 1989. 'For correspondence. Tel. (0203) 523523, ext.2572.

of Specific Pel isoenzymes in causing plant tissue mac-eration has been studied (Barras ef ai., 1987; Boccara etai. 1988; Tamaki etai., 1988).

The majority of Pel isoenzymes are secreted fromEn/vinia into the extracellular environment, but there areexamples of intracellular Pel isoenzymes (Chatterjee et al.,1979; Stack ef a/.. 1980; Starred a/., 1977; Trollinger eta/..1989). Willis ef ai. (1987) showed that as well as producingthree basic extracellular isoenzymes. Ecc strain DB71 alsoproduced two neutral periplasmic isoenzymes.

In this paper we describe the characterization of extra-cellular and periplasmic Pel isoenzymes from Ecc strainSCRI193. We present evidence that the pet genes thatencode periplasmic isoenzymes are completely unrelatedto those encoding extracellular isoenzymes.

Results

Analysis of the Pel isoenzyme profile of SCRI193

Several pel genes have been cloned from SCRI193 byPtastow et ai (1986). These workers reported thatSCRI193 produced two major Pel isoenzymes with pis of9,1 and 9.3. In repeating this work, we obtained moredetailed data, as shown in Fig. 1. SCRI193 appears tomake three extracellular Pel isoenzymes with pis ofapproximately 10.3, 10.5 and 11.0. In addition. SCRI193produces two periplasmic Pel isoenzymes with pis of 7.2and 8.2. This profile resembles that of Ecc DB71 (Willis efa/.. 1987).

To determine which Pel isoenzymes were carried bywhich clones, samples were prepared from various E. coliderivatives, analysed by lEF. and Pel isoenzymes revealedby activity stain overlays. Preliminary sequence data fromthe Sp^l-Sacl fragment of pJS6161 has suggested thatthe extracellular Pel activity at pi 10.5 is not encoded by adistinct structural gene. We suspect that this activityrepresents a breakdown product of PLd, and haveobserved that its relative intensity can vary a great dealbetween different samples {data not shown). Clone pBIproduced the isoenzymes PLc and d {pis 10.3 and 11.respectively); clone pH5 encoded the PLa isoenzyme (pi7.2; data not shown) and clone pB6 produced the PLbisoenzyme {pi 8.2; Fig. 1). Subclones were constructed

1786 J. C. D. Hinton. J. M. Sidebotham. D. R. Gill and G. P. C. Salmond

2 3 4 Pl

PeiD

PeiC

PelB

Pel A

10.6

8.3

7.3

Fig. 1. Activity stain overlay from an isoelectrte focusing gel (pH range7-11). The pl values determined from markers are shown. Lane 1:SCRI193 supernatant. Lane 2: SCRI193 periplasm. Lane 3: sonicate otTG1(pJS6197). Lane 4: sonicate of TG1(pB6).

from pBI to locate the two Pel genes (Fig. 2). One clone,PJS6161, produced PLd (data not shown), while pJS6197encoded PLc alone {Fig. 1).

We are interested in the mechanism of secretion of thePel isoenzymes, and therefore we decided to characterizeone extracellular and one periplasmic isoenzyme in detail.We chose the extracellular isoenzyme PLc and the peri-plasmic isoenzyme PLb, encoded by pJS6197 and p86.respectively (Fig. 1).

Localization of Pel in E. coli

Pel isoenzymes from Erwinia have been shown to beperiplasmic when expressed in E. co/( (Collmer ef a/., 1985;Keen etal.. 1984; Lei etal.. 1985; Reverchon etai. 1985;Willis ef ai. 1987). We wanted to know whether the four Pelisoenzymes of SCRI193 had similar localizations in thishost.

Preliminary experiments involved the use of the pUC-based Pel clones carried by TG2, and the osmotic shockmethod of Neu and Heppel {1965) to prepare periplasmicfractions. However, much cell lysis was observed, and themajority of the Pel activity remained in the cell-associatedfraction (data not shown).

Since this result disagreed with published data tromother workers, we repeated the isolation of periplasmicfractions by a different method. Since it was possible thatthe cell lysis we observed was related to the use of highcopy-number. pUC-based vectors, we used derivativesbased on the pBR322 replicon. Spheroplasts were pre-pared from DH1 derivatives carrying pJH6161, pJH6197,pJHB6 and pH5. (J-lactamase (Bla) and p-galactosidase((i-gal.) were used as control enzymes for the periplasmand cytoplasm, respectively.

The majority (76-90%) of PLa, B, C and D activities waslocated in the periplasmic fraction, as was 90-97% of theBla activity (Table 1). However, 32-57% of the (i-gal.activity was found in the periplasm, suggesting that someleakage between the cytoplasmic and periplasmiccompartments had occurred. It is not clear whethercontamination with cytoplasmic proteins is commonbecause, apart from Reverchon et ai (1985), previous

BL.

pai

PJS6I6

Bg

So

PBg\

I I

PJS6I6I

So

Bt

Bg

P \ ^ Bg1

o

So

Bg\LJ

Bg B

Sa

/ rp HP

I Bg

PJS6I9

So

B

I kbPJS6I97

Fig. 2. Subcloning of pBI . Restriction sites are:B. SsmHI; Bg. BglW: C. Ctel: H, Wndlll: Hp,Hpa\\ P. Rsfl: S. SphV. Sa, Sac\. PLc is encodedby pJS6197. and PLd iB encoded by pJS6161.The location and direction of transcription of PLcare shown by an arrow.

PLbc sequences 1787

Table 1.

Enzyme

PelBlap-Gal

Pectate lyase localization in E.coli DHl.

05

54

P

769038

pH5 (PU)

S»

2458

Total"

0.20.79.5

C

132

54

P

779732

pJHB6 (PLb)

S

101

14

Total

0.0227.0

2.6

C

43

45

P

909647

pJH6197(PLc)

S

618

Total

0.16.31.9

C

61

37

PJH6161

P

819557

S

1346

(PLd)

Total

0.00516.4

110

The localization experiments were performed three times; the values shown are from a single representative experiment. Cultures were grown in LB, and theunits of activity have been defined previously (Hinton and Salmond, 1987).a. Percentage of the total activity present in cell lysate (C). periplasmic fraction (P) and culture supematant (S).b. Total enzyme activity in C + P + S.

reports on Pel location do not quote cytoplasmic controlenzyme data (Collmer ef ai, 1985; Keen et ai, 1984; Lei etai, 1985; Willis ef ai. 1987; Zink and Chatterjee, 1985). Wesuggest that the leakage of p-gal. activity into the peri-plasmic fraction reflects a degree of cell lysis duringspheroplast preparation. It is possible that the fragility ofthe £ coti cells resulted from a deleterious effect of theperiptasmic accumulation of the Pel proteins. We do notunderstand the reason for the wide range of Bla and (5-gal.total activities shown in Table 1. However, this range oftotal activities was observed in three separate exper-iments.

Temperature stability of Pel isoenzymes

In an attempt to differentiate between the various Pelisoenzymes, their temperature stabilities were investi-gated. The PLa, PLb and the SCR1193 periplasmic activi-ties (i.e. PLa plus PLb) were relatively unstable, withhalf-lives of 0.42,0.56 and 0.75 min, respectively, at 55°C.However the SCRI193 supernatant (containing PLc andPLd), and the individual isoenzymes PLc and PLd weremore stable, with half-lives of 4.1, 3.4 and 1.3 min,respectively. Such differences between the thermal stabi-lity of three Eca Pel isoenzymes have been reportedpreviously (Quantick et ai, 1983).

Identification of the PLb and PLc gene products

The PLb and PLc gene products were identified bylabelling the proteins with [^^S]-methionine in maxicellexperiments, and in vitro using the Zubay DNA-directedtranscription translation system. The use of the Zubaysystem allowed us to determine the size of theunprocessed form of each protein, because this systemdoes not possess leader peptidase activity (Pratt et ai,1981). The maxicell system allowed us to visualize pro-cessed forms of the proteins (Sancar et ai, 1979).

PLc was synthesized as a pre- and a processed proteinof molecular weights 44 000 and 42 000, respectively (Fig.3, lanes 1 and 2). In contrast, PLb was synthesized as aprotein of molecular weight 59500 in both the Zubay andmaxicell systems (Fig. 3, lanes 3 and 4).

Nucleotide sequence of pe\B

The complete sequence of the 2201 bp chromosomalBamHI fragment of pB6 was determined (Fig. 4). A singleopen reading frame (ORF) was identified, starting at base49 and ending at base 1752, and coding for a product witha calculated molecular weight of 63532. This 568-residueprotein includes a putative 19-amino-acid signalsequence which resembles a typical E. coli signal peptide(Oliver, 1985); it contains two positively charged aminoacids at positions 2 and 3, and 12 hydrophobic residues.However, the predicted cleavage site would produce amature protein of molecular weight 61589. which corres-ponds well with the protein of molecular weight 59500identified in maxicell experiments. Thus the 1704bp ORFis the pelB gene. The identification of products with similar

2 3 4 MWst-andards

' — 92.5 kD

— 69 kD

PelB —

— 46 kD

pre PeiCpro PeIC

pre Bla —

pro Bio —— 30 kD

Fig. 3. Identification of pelB and peIC gene products. Sizes determinedfrom '"C-labelled protein standards are shown. Lane 1: pJH6197 Zubay.Lane 2: pJH6197 maxicell. Lane 3: pJHB6 Zubay. Lane 4: pJHB6maxicell.

1788 J. C. D. Hinton, J. M. Sidebotham, D. R. Gill and G. P. C. Salmond

i>0 . . . 80 . . . 1 2 0GATCCTTTTATTACAGCTCrGGATTAATCATCGTTACiSGlAGATAAAATGAAAAGATTTCCGCTGTCGCTCCTTGCGGGTCTGGTCGCTTTACAGGCCAGCGCCGCTACCCCTGACCCC

H K R F A L S L L A G L V A t f Q A S A A T P D R

160 . . . 200 . . . 240

L T I V N Q Y V D N V L T K A G D H Y H G Q S P T P L L A D G V D P R T G K Q M

280 . . . 320 . . . 360

E W I f P D G R H A V L S N F S A Q Q N L M R V L V G L S N L S G H P S Y K Q R

^ 0 0 . . . 440 , . , 4B0GGCGAAGCGATTGTGAAATACCATTTTCAACATTATCAGGATGAGAGCGGCCTGGTGATTTGGGGCCGCCACGGTTTTGTGGATCTAAAAACGCTGCAACCGGAAGGCCCCAGCGAAAAA

A E A I V K Y H F Q H Y Q D E S G L L I U G G H R F V D L K T L Q P E G P S E K

520 . , . 560 . . . 600GAGATGGTGGATGAGCTGAAAAATGCCTATGGGTACTAGGATTTGATGTTGAGCGTCGATAAAGACGGAACCGCAGGGTTTATTCGCGGTTTCTGGAATGGGCAGGTTTATGACTGGAAGE M V H E L K N A Y P Y Y D L M F S V D K D A T A R F I R G F W N A H V Y D W K

640 . . . 680 . . . 720ATTATGGAAAGGAGTCGTGAGGGTAAATAGGGGGAAAAAATAGGTGCGCTGTGGCAAAGTGGGTTTGAGCAACAGGGGGCCTTCTTGGCCACGAAAGGGGTGAGGTTGCTGAATGCGGGGI M E T S R H G K Y G Q K I G A L W Q S P F E Q Q P P F F A T K G L S F L N A G

760 . . . 800 . . 840AAGGATCTGATCTATTCCGGATCAGTACTGTAGAAATACAATAAAGAAGAGGGGGCGGTGGTCTGGGGAAAACGTCTGGCACAGCAGTACGTGCTGGCAGGTGATAAAGCGAGCGGGGTC' ^ ' ^ ' - ^ ^ S A S L L Y K Y N K E D G A L V W A K R L A Q Q Y V L P R D K A T G L

880 , . . 920 . . . 960GGCGTATATCAGTTTAGGCAGGGGGTGAAGGGGGATGAAAGTACCGACGATGCCGATACGCATTGAAAATACGGCGATCGCGCCCAGGGTGAGTTTGGGCGGGAATTCGGCCGGAGTGGG

G V Y Q F T Q A L K R D E T T D D A D T H S K Y G D R A Q R Q F G P E F G P T A

1000 . . . 1040 . . 1080CTGGAAGGCAATATGATGGTGAAAGGAGGCACCAGCACAATGTATTCCGAAAATGGGGTGATGGAGCTCCAGTTGGGCAAAGATTTAGGTGGGGAAGGCAAAGAACTTGTGAGGTGGAGA

L E G N M M L K G R T S T I Y S E N A L M Q L Q L G K D L G A E G K E L L T W T

. . . 1160 , . 1200AGGGATCGGCTGAAAGCCTTTGCCAAATATGCCTATAACGAGTCAGATAACAGGTTGCGCCCGATGCTGGGTAACGGGAAAGATCTGTGCAACTACGTTGTGGGGCGTGAGGGTTAGTAGT D G L K A F A K Y A Y N E S D N T F R P K L A N G K D L S N Y V L P R D G Y Y

1240 . . , 1280 . . . 1320

GGTAAAAAAGGGACCGTGATCAAGCGTTATGGTGCGGATAATTCATTCCTGCTTTGCTATGGTCGCGGGTATACCGTCTTACCGGACGCGGAGGTGTGGCGTGTGGGAGGGGGGATGGCTG K K G T V I K P Y P A D N S F L L S Y A R A Y T V L P D A E L W R V A R G I A

1360 . . . 1400 . , 1440GGGGCACAGGGGTTAGGTGAGTTAGGTTCAGCACCGGGTAAAGACGTTAAAGTGGATCTGGGGACGAAGAACAAGGATCCTTACGCGCTGTTGGCGTTGCTGGATGTGTATCAGGCTAGGR A Q G L G E L G S A P G K D V K V D L A T K N N D P Y A L F A L L D L Y Q A S

. . . 1520 . , . 1560AAAGTGAAAGACTATGTGTCGCTGGCGGAAAAAGTGGCCGATAAGATTATGAGGACAGGTTATGAAAACGGCTTGTTTATGGCCGAGGGCAACAGACAATATGCCGATGTCGATAGGATGK V K D Y L S L A E K V G D N I I S T R Y Q N G F F M A E P N R Q Y A D V D T I

. . . 1640 . . . 1680GAGGGTTATGGGGTGTTAGGGTTGGAAGGTGGGGTGGGCAATGAGGCACAGTCGGTTGCACGGTTCGTGAATGGTGCCGGGTTGACCGAAGGTGGGTACCGTATGGAAGATGGCTCAAGAE P Y A L L A L E A A V R N Q P Q S V A P F L N G A G F T E G G y R M E D C S T

. , . 1760 . 1800CGTGTGTGGAGTCGGGATAACGAGATCTTCCTGTTGAAGGTTGGCGAAAGGTTGAAAGGGAACAACAAGAAATAAGGGTTAATCCTCAAGAGGGCAATGGTGCTTGGGTGGGATTGGCAAR V S T R D N E l F L L N V G E T L K P K N K K

18'iO , , , 1880 1920TGGGGTCTTGGTTACTCTACTCAAGGGTGAAGGGAACAAGAGGGACATTATGGGGTGTGATAGTTAATCGTGTGGGGGTTAATGATGTGCTTTTAGAGGATGCAATGCCGGGGACGAGTA

, . . 2000 2040AGGTGACCAGGATGAAGTAAGGCAATAATTTTAAGGGTTAGTTAACCAGAGGCACGTCTGGGTTGACACCAAGGGAGCCCGGCGTGGGTTGGGGTTTCGCTTGAGCGAGATAGAGGCTGA

. , . 2120 2160TCTGTGAGGGGAAGGGCTCGAGCGGAGGGGGATCGTAGATGAAATGAGGAAATGGCTGGGGACGAGTGTTAGGGTATTCCCAGATATGTTTGTAACCTACTGGCCGCTGAAGGTGGGTGG

2200GGACCGAGCACATAGGGGAGAGGTGAGTGTGGGTTTGGATC

BapiHI

Fig. 4. Nucleotide sequence of the 2201 bp SamHI fragment of pB6. containing the pelB gene. The sequence is that of the non-coding strand. Theputative ribosome-binding site is underlined. The predicted amino acid sequence of PLb is shown, and the putative signal sequence is underlined. Thesesequence data will appear in the EMBLyGenBanWDDBJ Nucleotide Sequence Database under the accession number X16397 pelB.

PLbo sequences 1789

molecular weights in both the Zubay and maxicell systemsmay suggest that the signal sequence is not oleaved inmaxioells. Further experiments will be required to clarifythis observation, because maxicells have been shownpreviously to process plasmid-encoded gene products(Sancar ef a/., 1979).

A Shine-Dalgarno sequence, AGGTAG, lies at - 6relative to ATG, with AAAA located at +3 (Stormo, 1986).No r/7o-in dependent terminator sequences were identifiedin the 3' region of the pelB gene (von Heijne, 1987). Theshort stretoh of DNA 5' to pelB does not contain anyobvious putative promoter sequences, suggesting thattranscription occurs by readthrough from a plasmid pro-moter. Restriction mapping of the pB6 plasmid has shownthat the 2201 bp fragment is inserted in anti-orientation tothe p/ac promoter of pUC8. This could account for the lowlevel of Pel activity encoded by pB6 {Plastow etal., 1986)and the faint band observed in gene product identificationexperiments.

Nucleotide sequence ofpe\C

The sequence of the 1812bp Sac\-Hind\l\ fragment ofpJS6197 was determined (Fig. 5). A single ORF waslocated running from bases 605 to 1726, coding for aproduct of 374 amino acids with a deduced molecularweight of 40571. The first 17 amino acids of the proteinconstitute an E. coli-lype signal sequence, with a posi-tively charged lysine residue at position 2, and contains 12hydrophobic amino acids (Oliver, 1985).

The predicted cleavage site would produce a protein ofmolecular weight 38872, slightly smaller than the pro-cessed product of molecular weight 42000 that wasobserved in maxiceils. Such anomalies in migration havebeen observed previously for Pel proteins (Tamaki ef a/.,1988). The difference in mobility between the pre- and theprocessed forms of PLc corresponded to a reduction ofmolecular weight of 2000, as shown in Fig. 3, which agreeswell with the sequence prediction.

A Shine-Dalgarno sequence of GGAGAG lies at - 8relative to the ATG initiation codon, which is followed byAAA, thus constituting a strong ribosome-binding site(Stormo, 1986). Although there is an in-frame ATG at base566, we consider the ATG at base 605 to be the mostprobable translational start because of the strong Shine-Dalgarno sequence, and the typical signal sequence of thepredicted product. Thus the 1122 bp ORF starting at base605 probably corresponds to pelC.

A putative E. coli Sigma 70-type promoter was found atbases 513 (TTCATT), 537 (TAATAT) and 552 fTATl whichshares 9 out of 15 base identities, and appropriatespacing, with the consensus sequence (von Heijne, 1987).A putative KdgR-binding site is located at bases 500(AATAAA) and 512 (TTTCAT) which shares 10 out of 12

bases of the consensus sequence (Reverchon et ai,1989). The KdgR-binding site is thought to allow negativeregulation of Pel gene expression by the KdgR repressor(Reverchon et ai. 1989). Interestingly, the second domainof the putative KdgR-binding site of peIC lies at thepredicted ' -35 ' region of the peIC promoter. Thus, KdgRbinding could physically occlude RNA polymerase, and soprevent transcription of pelC.

A putative catabolite activator protein (CAP) binding sitelies at base 234 (CAA TGA GAA ATT TTT ACT TAT TTT)which matches 11 out of 14 bases of the E co//consensussequence (de Crombrugghe et ai, 1984). However, thisputative site does lie at -317 relative to the prediotedtranscriptional start, which is much further than has beenobserved previously in E. coli (de Crombrugghe et al.,1984).

Discussion

The sequencing of several pel genes has allowed theidentification of three distinct gene families. The PLbcfamily comprises genes which encode extracellular Pelsproduced by Ecc, Eca and Echr (Fig. 6). These Pets share70% to 85% overall amino acid identity (Ito etai, 1988;Keen and Tamaki, 1986; Lei etai. 1987; 1988; Tamaki etai, 1988). The PLade family Is made up of genes whichencode extracellular Pels from Echr alone (Fig. 6), andshare 58% to 90% overall amino acid identity (Keen andTamaki, 1986; Tamaki etai, 1988; Reverohon etai, 1989;van Gijsegem, 1989). The third family only includes twointracellular Pels, from Yersinia pseudotuberculosisICPB3821 (PLY) and Ecc EC153 (PL153), which share80% overall amino acid identity (ManuNs et ai, 1988;Trollingerefa/., 1989).

The peIC gene from SCR1193 belongs to the PLbc familyof extracellular Pels. PLc shares 74-82% overall aminoacid identity, and 71-79% overall DNA identity with othermembers of the family.

The pelB gene of SCRI193 belongs to the third genefamily of intracellutar Pels. PLb shares 80% overall aminoaoid identity with PLY and 98% with PL153.

There is a greater homology between PLb of SCRI193and PL153 than has been observed between any pelgenes from the same or from different Erwinia strains,implying that the strains SCRI193 and EC153 are veryclosely related. However the DNA homology over thecoding regions of PL153 and PLc of SCRI193 is only 91 %.This suggests that some genetic drift has occurredbetween the pel genes of the two strains, while theprotein sequences have remained fairly homologous.

It has been shown previously that 99% of the Pel activityof Y. pseudotuberculosis ICPB3821 was intracellular(Starr etai, 1977). Similarly, Troliinger etai (1989) couldnot detect any PL153 activity outside the oell, and further

1790 J. C. D. Hinton, J. M. Sidebotham. D. R. Gill and G. P. C Salmond

sad . . . 40 . . . 80 . . . 120

GAGCTCGCTGCGTCTTTTTATGGAATCTACGCyvTGAGAGTGGATAGGCATrCCTAAAMCTATGCATACATTAMTACCAAGCTAATTATTTTCATGGTAAATTAATGGCTTGATTTATT

A--T Ca-ATT-

. Clal 160

280

400

200

320

240CGAG

CA-TGtG

360

480

:TTTTATTTTTATGGT

520 560 600SGAOAGTAC

AtgAAA TTTCAT

PBtl . 6 4 0 . . . 6 8 0 . . . 720

M K Y L L P S A A A G L L L L A A Q P T M A A N T G G V A T T D G G O V A G A

760 . . . 800 . . . 8iO

V K K T A R S M Q D I I D I I E A A K L D S N G K K V K G C A Y P L V I T Y N G

880 . . . 920 . . , 960

N E D A L I K A A E N D I C G Q W K K D A R G V E I K E F T K G I T I I G T N G

1 0 0 0 . . . lO^tO . . . 1 0 8 0

S S A N F G I U L T K S S D I V I R N M R F G Y K P G G A Q D G D A I R l D N T

1 1 2 0 . . . 1 1 6 0 . . . 1 2 0 0GCCGAACGTCTGGATCGAGCACAACGAAATCTTCGCCAAAAACTTTGAATGGGCAGGTACAAAAGACGGTGACACGACATTCGAATGGGGGATTGATATCAAGAAAGCGTCAAGCAACGT

P N V U I D H N E I F A K N F E C A G T K D G D T T F E S A I D I K K A S T N V

1 2 4 0 . . . 1 2 8 0 . . .fipal 1320

T V S Y N Y I H G I K K V G L S G F S S S D T G R D L T Y H H N I Y D D V N A R

1360 . . . 1400 . . . l't'iO

L P L Q R G G Q V H A Y N N L Y T G I T S S G L N V R Q K G I A L I E R N W F E

U 8 0 . . . 1520 . . . 1560

K A K N P V T S R Y D G S N F G T W E L R N N N V M S P A D F A K Y N I T W D K

1600 . . . 1640 , . . 1680

D T K P Y V N A E D W K S T G T F A S V P Y S Y S P V S A Q C V K D K L A N Y A

1720 . ' . . 1760 . . . 1800

G V N K f J L A V L T A A N C N

HlndlllGCAAACAAGCTT

Fig. 5. Nucleotide sequence of the 1812bp Sacl-H/ndlll fragment of pJS6197 containing the pe'C gene. Ttie sequence is thai of the non-coding strand.The ribosome-binding site and the potential -10 and -35 regions of the promoter are underlined, and discussed in the text. The KdgR-binding andCAP-binding consensus sequences are shown at bases 500 and 234. respectively. Upper-case letters are used to show Identity. The predicted aminoacid sequence of PLc is shown, and the putative signal sequence is underlined. These sequence data will appear in the EMBUGenBank/DDBJNucleotide Sequence Databases under the accession number X16398 pelC.

workby Chatterjee ef a/. (1979) showed that a significant sequence, we think it likely that PLY and PL153 are

proportion of the total Pel activity produced by strains periplasmic in their parent strains. Hence the third gene

iCPB3821 and EC153 was periplasmic (33% and 11%. family should be known as the famiiy of periplasmic Pel

respectively), in this paper we have shown that the PLb isoenzymes.

isoenzyme is localized within the peripiasm of SCRI193. it is interesting to speculate on the role that the Pels of

Given the high degree of homology between PLb, PLY and the periplasmic family have in the virulence of Ecc. It has

PL153, and the fact that each possesses a good signal been reported that members of this family are not required

PLbc sequences 1791

for efficient tissue maceration: the PL153 gene, and itsanaiogue from the Ecc strain DB71, have been inactivatedv 'ithout a significant effect upon maceration ability (J.Engwail, personal communication; Trollinger etai., 1989).In contrast, similar experiments with the extracellularisoenzyme PLe of Echr demonstrated a crucial role intissue maceration (Boccara et ai., 1988; Payne et ai.,1987).

It is intriguing that the periplasmic gene family has beenfound in Ecc but not in any of the Echr strains analysed todate. This may reflect the aetiological differences betweenfee and Echr in terms of their host range and worldwidedistribution (Perombelon, 1987).

We considered whether the function of the periplasmicPel family could be to degrade galacturonate oligomerswhich are produced by extracellular Pel isoenzymes andtransported into the periplasm. This remains a possibility,although PLY, PL153 and PLb have no homology with theoligogalacturonate Iyase protein (Reverchon etal., 1989).Another possibility is that the secretion of the periplasmicPel family could be induced in planta, and this hypothesisis under investigation.

The PLbc and PLade families have been shown to sharetwo domains of homology of 20 residues each (Tamaki efai., 1988). We have carried out computer searches whichhave shown that the family of periplasmic pels do notcontain any regions homologous to the PLbc and PLadefamilies. It may be significant that all members of thePLade and PLbc families contain between two and fourcysteine residues, whereas the three members of theperiplasmic family do not contain cysteine. Nishikawa efai. (1983) observed that intracellular proteins rarely havedisulphide bonds, and this would support the hypothesisthat the periplasmic Pels are true intracellular proteins.Furthermore, PLb of the periplasmic famly has bio-chemical properties different from those of PLc of thePLbc family, as judged by the temperature-stability data.Such differences may reflect the fact that, unlike theperiplasmic Pels, isoenzymes belonging to the PLbc orPLade families are secreted across the outer membrane.

We have been investigating whether members of thePLbc and PLade families have certain conserved residuesthat could be important in constituting a simple Pelstructure. Conserved residues may be required to allowpassage across the outer membrane by interaction withsecretion or Out proteins, and/or may be involved informing the active site of the enzyme.

As shown in Fig. 6, we have been able to align all 12extracellular Pels that have been sequenced, with minimalpadding, to reveal conserved residues running throughoutthe proteins. The rudimentary consensus sequence ismade up of 49 exact matches between the 12 proteins,and 29 matches which are conserved in at least 10 out of12 Pels. The figure is arranged with six members of the

PLade family positioned above six members of the PLbcfamily.

The homology between positions 235 and 310 has beenobserved previously (Tamaki ef a/., 1988). We haveextended this homology, and demonstrated that similari-ties exist throughout the whole length of the Pel proteins,although the signal sequence region of the 12 Pelsappeared to be distinct. This multiple alignment shouldfacilitate more accurate prediction of the secondarystructure of the Pel proteins as outlined by Zvelebi! e( ai.(1987).

It is intriguing to speculate on the significance of theconsensus sequence to our understanding of the activityand secretion of pectate Iyase isoenzymes. We considerthat the production of extracellular Pel and cellulase (Cei)by EnAfinia spp. represents a fascinating system for thestudy of protein secretion (i.e. the translocation of proteinsacross the bacterial outer membrane). A specializedmechanism must be involved in the selective secretion ofPel and Cel, without allowing the leakage of other peri-plasmic proteins. Several Erwinia out mutants have beenisolated which are unable to secrete Pei and Cel, accumu-lating the enzymes in the periplasm (Andro et al., 1984;Chatterjeeefa/., 1985; Gibson e(a/., 1988; Ji efa/., 1987;Thurn and Chatterjee, 1985), and thus suggesting acommon mechanism for the secretion of Pel and Cel. Theconsensus sequence could allow us to identify 'func-tionally important residues' (Zvelebil et al., 1987) thatmake up a fundamental 'backbone' structure required forcatalysis and/or secretion of Pel.

We have attempted to align the consensus sequencewith other proteins secreted from Gram-negative bacteria.These included CelZ (Guiseppi ef a/., 1988) and pectinmethylesterase (Plastow, 1988), which are also secretedby the out pathway from Echr.. as well as pullulanase fromKlebsieila pneumoniae (Katsuragi ef ai., 1987), chitinasefrom Serratia marcescens (Jones ef ai., 1986), and HlyAfrom E coii (Felmlee ef ai, 1985). Even when screening forconservative changes as well as amino acid identities, wewere unable to find any significant match with the consen-sus sequence, suggesting that these proteins do notcontain common residues that are involved in secretion.

However, it is possible that out mutants are affected in ascaffolding function for another class of proteins thatinteract directly with Pel, or other secreted proteins. Thusit would be possible that each secreted enzyme wouldinteract with a different protein, but still share some part ofthe translocation process in common. Consequently,conservation of particular residues would not be expec-ted. We are left with two possibilities: either the consensussequence simply reflects the conservation of particularresidues required for pectate Iyase activity, or the consen-sus sequence does contain information required forsecretion, but we are not able to recognize it. Thus, the

1792 J. C. D. Hinton. J. M. Sidebotham. D. R. Gill and G. P. C. Salmond

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Table 2, Plasmids.Plasmid

pBI

pB6

pH5

pJS616

PJS6161

pJS619

PJS6197

pJHB6

PJH6161

PJH6197

Description

8.7kb BamHi fragment cloned intopUC8 and corrtaining the peIC and Dgenes

2201 bp SamHI fragment cloned intopUC8 and carrying the pelB gene

7.0kb H/ndlll fragment cloned intopBR322. and containing the pelA gene

4.1 kb SamHI-SacI fragment from pBIcloned into pUC19, carrying the pelDgene

2.4kb Sph\-Sac\ fragment from pJS616cloned into pUC19, carrying the pe/Dgene

3.6kb Sacl-SamHI fragment from pBIcloned into pUC19, canying the pe/C gene

1813bp Sacl-HmdIM fragment frompJS619 cloned into pUC19, carrying thepeIC gene

2201 bp 8amHI fragment from pB6cloned Into pBR322

2.4kb SpM'Sac\ fragment from pJS6161cloned into PJRD184

1813bp Sacl-Hi/idlll fragment fromPJS6197 cloned into pJRD184

Source

Plastow eta/. (1966)

Plastow eta/. (1986)

Plastow eta/. (1986)

This paper

This paper

This paper

This paper

This paper

This paper

This paper

biological significance of these conserved residues mustbe determined by an experimental approach, which is nowin progress in our laboratory.

Experimental procedures

Bacterial strains and plasmids

The wild-type Ecc strain SCR1193 was described by Hinton andSalmond (1987). Three E. co//strains were used in this study: DHl(Hanahan. 1983); TG1, an ecoK'derivative (T. Gibson, personalcommunication)of JM101 {Yanisch-Perron etai, 1985); andTG2.a recA derivative of TGI (T. Gibson, personal communication).Plasmid constructions are described in Table 2 and Fig. 2. The pBand pJS plasmids were based on pUC8 (Vieira and Messing,1982) or pUC19 (Yanisch-Perron et al., 1985) respectively. ThepH5 and pJH plasmids were based on pBR322 (Bolivar ef ai.1977) or pJRD184 (Heusterspreute ef ai. 1985). pJRD184 is a3793bp derivative of pBR322 which contains 43 unique cloningsites.

Media and enzyme assays

Nutrient media. Pel-detection media and antibiotic concentra-tions, as well as Pel, Bta and p-gal. assays have been describedpreviously (Hinton and Salmond. 1987).

Cell localizations

Ecc localizations were performed by the method of Neu andHeppel (1965), as outlined by Hinton and Salmond (1987).

Isolation of periplasmic proteins from E. coli was initially per-formed using the method of Neu and Heppel (1965). Sub-sequently, spheroplasts were prepared using the method ofOsborn and Munson (1974).

Analysis of Pel isoenzymes

The temperature stability of Pel isoenzymes was determined byholding samples at 55°C for increasing periods of time, storingthem on ice, and assaying for Pel activity. Sonicated whole-celllysates were prepared from Tg1(pB6), TG1(pH5), TGl(pJS6197)and TG1(pJS6161). Preparation of Ecc samples has been des-cribed above.

Thin-layer lEF was carried out exactly as described by Willis efai (1987) on a water-cooled LKB Multiphor II rig, using LKBampholytes. Approximately 0.05 A253 min ' m l " ' Units of Pelactivity were loaded per gel track. We realize that it is not passibleto assign accurate values above a pl of 10, but we show theapproximate pis of the three extracellular isoenzymes to rep-resent their relative positions on the lEF gel.

Putative gene designations were made according to theProposals for Uniform Genetic Nomenclature (EMBO Workshopon Soft Rot Erwiniae, Marseille. July 1984). Genes A to E refer toPel isoenzymes with acidic to basic pis.

Gene cloning and DNA-sequencing techniques

All DNA manipulations, subcloning. etc. were performed accord-ing to standard methods (Maniatis et ai, 1982). Random DNAsequencing was performed using the same strategy as that usedby Gill et al. (1986). We used the ohain-termination sequencingprocedure described in Focus (Bethesda Research Laboratories.1987). The dITP analogue was used to resolve 'compressions' in

1794 J. C. D. Hinton, J. M. Sidebotham, D. R. Gill and G. P. C. Saimond

GC-rich regions (Gough and Murray, 1983). For sequence compi-lation, the fragments were sequenced at least twice on eachstrand, and up to seven times in some regions. Sequencecompilation and analysis of DNA and proteins was performedusing the Beckman MicroGenie programs (Queen and Korn,1984).

Gene-product identification

For maxicells, the strain CSH26AF6 was used (Hinton et al.,1987). For coupled in vitro transcription-translation ('Zubays'),the Amersham Kit was used according to the manufacturer'sinstructions. Plasmids with the same copy number as pBR322(pJH series) were used for gene-product identification. Suchplasmids gave a cleaner background than pUC-based clones.

Acknowledgements

We would like to thank Noel Keen, Sylvie Reverchon andFrederique van Gijsegem for sending us unpublished data, PhilReeves for help with spheropiast preparation, and DaveWhitcombe for his assistance with Fig. 6. This work wassupported by CEC/BAP contract No. BAP-0191-UK(H1), and byproject grants GR/D/47110 and GR/E/93503 from the Scienceand Engineering Research Council to G.P.C.S. The work wasdone under MAFF licence number PHF 248/37(27), Plant Pests(Great Britain) order 1980. We are grateful to Carol Howes fortyping the manuscript.

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