Molecular Microbiology (1994) 14(5), 999-1010

organization and expression ofrdgA and rdgB genes that regulate pectin lyaseproduction in the plant pathogenic bacterium Erwiniacarotovora subsp^ carotovora in response toDNA-damaging agentsYang Liu, Asita Chatterjee and Arun K. Chatterjee*Department of Plant Pathoiogy, University of Missouri,Columbia, Missouri 65211, USA.

Summary

in most soft-rotting Erwinia spp., including E. caroto-i^ ora subsp. carofovora strain 71 (Ecc71), productionof the plant cell wall degrading enzyme pectin lyase(Pnl) is activated by DNA-damaging agents such asmitomycin C (MC). Induction of Pnl production inEcc71 requires a functional recA gene and the rdglocus. DNA sequencing and RNA analyses revealedthat the rdg locus contains two regulatory genes,rdgA and rdgB, in separate transcriptional units.There is high homology between RdgA and repres-sers of iambdoid phages, specially ii)80. RdgB, how-ever, has significant homology with transcriptionalactivators of Mu phage. Both RdgA and RdgB arealso predicted to possess heiix-turn-helix motifs. Byreplacing the rdgB promoter with the IPTG-inducibletac promoter, we have determined that rdgB by itselfcan activate Pnl production in Escherichia coli. How-ever, deletion analysis of rdg' DNA indicated that,when driven by their native promoters, functions ofboth rdgA and rdgB are required for the induction ofpnIA expression by MC treatment. While rdgB tran-scription occurs only after MC treatment, a substan-tial level of rdgA mRNA is detected in the absence ofMC treatment. Moreover, upon induction with MC, anew rdgA mRNA species, initiated from a differentstart site, is produced at a high level. Thus, the twoclosely linked rdgA and rdgB genes, required for theregulation of Pnl production, are expressed dif-ferently in Ecc71.

Introduction

Erwinia carotovora subsp. carotovora (Ecc) and most other

Received 21 June, 1994; revised 16 August, 1994: accepted 23August, 1994. *For correspondence- Tel. (314) 882 2940; Fax (314)882 0588,

soft-rotting Enmnia produce extracellular enzymes thatcause degradation of plant cell wall components (Barrasef ai.. 1994; Chatterjee and Vidaver, 1986; Collmer andKeen, 1986). Noteworthy among these are pectinasessuch as pectate lyase (Pel), poiygalacturonase (Peh) andpectin lyase (Pnl). These enzymes cause plant tissuemaceration, i.e. ceil separation and cell death. These find-ings and genetic data demonstrated that pectinases areimportant determinants of pathogenicity in soft-rottingErwinia (Barras et ai., 1994).

Previous research in our laboratory (Chatterjee et ai.,1991; McEvoy etai, 1987; 1990; Tsuyumu and Chatter-jee, 1984; Zink et ai. 1985) and elsewhere (Itoh et ai.1980; Kamimiya etai, 1972; 1974; Tomizawa and Taka-hashi, 1971; Tsuyumu et ai, 1985) have revealed thatPnl production is regulated differently fTom Pel and Pehproduction, In Ecc strain 71 (Ecc71), production of thelatter enzymes, along with protease and cellulase. isco-ordinately activated by plant components and severalregulator genes such as aepA. aepB and aepH (Chatter-jee et ai. 1992; Liu et ai, 1993; Murata ef ai, 1991;1994). Pnl production in Ecc71 is not affected by tiieAEP regulatory system (Barras et ai, 1994); instead, it isactivated together with carotovoricin (Ctv) synthesis andeel! lysis (Lss) by DNA-damaging agents, such as miio-mycin C (MC), nalidixic acid or u.v. ligiit (McEvoy et ai,1990; Tsuyumu and Ghatterjee, 1984; Zink et ai. 1985).Activation of Pnl production by DNA-damaging agentscommonly occurs in other strains of Ecc as well as inmost soft-rotting Erwinia spp., including Erwinia ciiry-santiiemi, EnA/inia carotovora subsp. atroseptica. andErwinia rhapontici (McEvoy el ai, 1990; Tsuyumu andChatterjee, 1984).

Our work with Ecc71 has revealed that the induction ofPnl production in plant tissue or in culture requires a func-tional recA (McEvoy etai, 1987; 1992; Zink etai, 1985).However, several lines of evidence indicated that tran-scription of pnIA, the structural gene for Pnl, was notdirectly controlled by LexA, as would be expected if Pnlwas a component of the SOS regulon (Walker, 1985).Multiple copies of iexA or the lexA(def) mutation had nosignificant effect on the induction of pnIA expression by

1000 y. Liu, A. Chatterjee and A. K. Chatterjee

o oH Hu uB UB G

uGGB

G

oCDUOOEi^ 1

uuG

1 ^UE-.^BHUGGGfTi;' ^

CJoCJ

G

H

a

G

tJ ^

^

Bi t ^

UUBBBCJCD

G[ ^B

.-1

1 ^

w

U >B

CJo

oGOouBOCJCl

AA

A

C3G

CTG

C/H

G

O

GGGCDUBi

M

H

UBG

'iGOUG 5uCJuG

uGGU

AA

A

uGOBOB/^ciE-ICJOOGUBBf - iGO

B

U l

t i l

^

O

W

G

13

,^

1 - ^

S

[ l ]

G-

CS

;>

Bi

E-IBUG

UBBOCJU ^

UGGGCD^

GCJ

P3rfjooGUU

u

(J(J

o

u

uuEHoGCJBC)

IS

(^

U l

^

B

G

3:

r j

Ul

Pi

P.

H

U l

p.

W

O

w

a;

GEHE-I

BCJG

B

aHCJ

w

M

Q

O A.BO

GGBClO

GO

oGGCJB[1u

oCJ

>

w

Q

W

Ul

h:i

BBOOOBOO^BBBaGGUr.

i aa j

ro

C O S

F -S

i l "3

E ^O

MC (McEvcy, 1991; McEvoy at ai. 1992), The nucleoiidesequence of pnIA did not reveal the presence of a LexAbox upstream of the coding region (Chatterjee st at..1991). Moreover, pnIA expression was no\ activated byDNA-damaging agents in Escherichia coti MC4100, aRecA"' and LexA* strain, prompting the notion that apositive reguiator was required for the activation of pntAtranscription. Subsequent studies with regulatoiy mutantsand a cloned rdg locus confirmed this prediction (McEvoyet at., 1992), The rdg locus, when present in low copy,activated pnIA expression in RecA' E. coti or RecA"Ecc, but not in RecA" derivatives ot these bacteria.However, Pnl production remained non-inducibie whenthe rdg locus was pi'esent in high copy, i.e. cioned intopBluescript-ll vector SK-H, This suppressive effect wasnot Ihe result of plasmid instability or high-copy toxicity.These observations prompted the hypothesis that the rdglocus contains both positive and negative regulatorygenes controlling pnIA induction by DNA-damagingagents (McEvoy et a!.. 1992), We report here the structureand organization of the regulator genes rdgA and rdgB andprovide evidence for the activation of pnIA transcription invivo by rdgB. Our data also show that rdgA and rdgB areexpressed differently in Ecc71,

Results and Discussion

Nudeotide sequence determination of rdg"^ DNA

We had previously localized the rdg locus to a 2.6 kb DNAsegment of Eoc71 (McEvoy el ai. 1992). To analyse the

Regulation of pectin lyase production 1001

regulatory genes present within this DNA segment, wedetermined Ihe complete nucleotide sequence of bothstrands. The first open reading frame (ORF), Irom nucleo-tide (nt) 78 to 810, and the second ORF, between 1267 andI618nt, were designated rdgA and rdgB. respectively(Fig, 1), rdgA is predicted to encode a polypeptide of244 amino acid residues with a moiecuiar mass of26 785 Da and an isoelectric point of 5,09, The rdgBgene could encode a polypeptide ot 117 amino acidresidues with a molecular mass of 13551 Da and an Jso-etectric point of 9.56.

The deduced amino acid sequence ol RdgA hassimilarities with several bacteriophage and bacterialrepressor proteins. The alignment of sequences of E. coliphage cj>80 cl repressor (Ogawa el ai, 1988), 434 clrepressor (Sauer ef al.. 1982) and RdgA is presented inFig, 2. RdgA is 64.8% similar to c|>80 cl repressor. The/V-terminal region of RdgA (residues 1 to 72) is 47,2%similar to the DNA-binding domain (residues 1 to 69) of434 repressor (Neri ef at.. 1992), aithough homologydecreases to 29.5% when sequences of the entire pro-teins are compared. RdgA also is 33.2% similar to X clrepressor (Sauer and Anderegs, 1978), 29.1% similar toE. carotovora LexA repressor (Garriga et al.. 1992),31,1% similar to E. coii LexA repressor (Horii ef at.,1981) and 55,7% simiiar to PrtR, a repressor that wasrecently found to control pyocin production in Pseudo-monoas aeruginosa in response to DNA-damagingagents (Matsui etat.. 1993).

A heiix-turn-helix (HTH) motif is found in the W-!erminalHclil Turn Helix

C l SSISERIKFLLAREGLKQFJ^LASALSTSPQTVI-iNWIKRDALSREAAQQ A^

RdgA HKTTLAERLKTAJ^TAQGLSQiCALGDMIGVSQAAIQKIEVGKASQTTKIVE 50

'134 c l SISSRVKSKRIQLGLMQAELAQKVGTTQQSIEQLENGKTKRPRFLPE 4 7

3NIPPESEWGTVDAWDKNTPiJiaO c l I5EKFGYSLDWLLNGEG3PKKDL

RdgA LSNNLR^/RPEWLATJGEGPMRSSEVTRSLQEPSIPPKSEWGTVSAWDSTTE

^34 c r LASALGVSVDWLLNGTSDSNVRFVGHVEPKGKYPLISMVPJIGSWCEACEP

(|)6G cl LPDDEVSVPFLKDIEFACGDGRVHDEPHNGFKLRFSKATLEIRVGANSDGSRdgA LSEDEVEVPFLKDIEFACGDGRIQSEDYNGFKLRFSKATLPJ

1002 Y. Liu, A. Chatterjee and A. K. Chatterjee

Mor 26 SRFPALLAELNDLLRGELSRLGVDPAHSLEIVVAICKHL GGGQVRdgB 3 SKGPELLVELSQHVADTVKTVTELDPQTAELVGNARFAKHMMTVWGGQMV

c

Mor

RdgE

C

29 SRWPRSWDLrDVLENELKRQNVSNPRELARKQAVALSCFL GGRQFHeliK Tum HaLix

YIPRGQALDSLIRDLRIWNDFNGRNVSELTTRYGVTFNTVYKAIRRiMRR 118

YFPMGISWRASQRDLQIYEEFDGRNHSALAKKYNVSLQWIYKIVKTMRK 106

YrPCGDTILTALRDDLLYCQFNGRNMEELRRQYRLSQPQIYQIIARQRK 123

Fig. 3. Alignment ol deduced amino acidsequenc;e of idgB product (residues 8 to 106)and Mor (residues 26 to 118) and C (residues29 to 123) proteins oi temperate pliage Mu.The HTH motifs found, in RdgB, Mor and Care indicated by arrows. Identical amino acidsare marked by asterisks and chemicallysimilar amino acids with dots. The i^imiiaramino acids are grouped as in Fig. 2.Numbers indicate amino acid positions ineach protein.

region of the postulated RdgA protein. The occurrence ofGly in position 5, Gly in position 9 and lie in position 15within the HTH motif of RdgA (Fig. 2) demonstratesthat these three highly conserved amino acid residues(Brennan and Matthews, 1989) are properly positioned.Statistical analysis by Dodd and Egan (1987) yielded ascore of 1913 for the stretch of 20 amino acid residuescomprising the predicted HTH motif. All proteins scoringmore than 1700 have been found to be Cro-like DNA-binding proteins (Dodd and Egan, 1987). The C-terminalregion (residues 155 to 244) of RdgA shows 55.8% simi-larity to the C-terminal domain (residues 147 to 244) ofthe (])80 cl repressor (Fig. 2), which is known to be impor-tant for protein dimerization. These observations suggestthat the C-terminal domain of RdgA could vei7 well beinvolved in dimerization and that RdgA could bind DNAas a homodimer. a feature that is common to most pro-karyotJc HTH proteins.

Another area of strong homology between RdgA andcjiBO cl repressor is centred around the RecA'^ coproteasecleavage site of (|i80 cl repressor (Eguchi et ai, 1988)(Fig. 2). Therefore, we postulate that RecA* cleavagecould take place between the Cys-118-Gly-119 residuesin RdgA as in the tjiBO cl repressor (Eguchi et al., 1988).The presence of a putative RecA-processing site inRdgA is consistent with our previous finding that RecA,activated by DNA-damaging agents, is required for theinduction of pnIA (McEvoy ef ai, 1987; 1992; Zink ef ai,1985).

The derived amino acid sequence of RdgB has signifi-cant homology with Mor and C proteins of the temperatephage Mu within their C-terminal regions (Fig. 3). The

Mor and C proteins are the transcriptiona! activators ofMu middie and late operons, respectively (Bolker et aL,1989; Margolin and Howe, 1986; Mathee and Howe,1990). The putative DNA-binding domains of Mor and Cproteins contain HTH motifs in their C-terminal regions.Within the C-terminal region (i.e. the region spanningfrom residues 53 to 106), RdgB is 43% identical to theC-terminal region (residues 65 to 118) of Mor and 35%identical to the corresponding domain (residues 70 to123) of the C protein. Similarities increase to 65% and48% when chemically similar amino acids are included(Fig. 3). An HTH motif aiso is present near the C-terminalend ol RdgB. Statistical analysis according to Dodd andEgan (1987) yielded a score of 1820 for this HTH motif,although one of the three most highly conserved aminoacids within RdgB (i.e. the N residue at position 9;Fig. 3) does not match with the consensus (Brennan andMatthews, 1989). Based upon these observations, wepredicted that RdgB is a transcriptional activator of pnlA.

Since the closely linked rdgA and rdgB genes comprisethe rdg iocus, it was of interest to ascertain if each genecontained its own regulatory sequences. Analysis of the5' non-coding regions revealed the presence of sequencestypical of the E. coli a'' promoter, i.e. - 1 0 and 35boxes upstream of rdgA and rdgB genes (Fig. 1). Thesequence (5-GGTGAT-3') iocated about 2bp precedingihe rdgB start codon is complementary to the sequence(3'-CCAGUA-5') within the 3' end of 16s rRNA (3'-AUUC-CUCCACUA-5', Schererefa/., 1980). Thus, m^B appearsto have a respectable ribosome-binding site. However, wecould not detect a typical ribosome-binding site upstreamof the rdgr/^ -coding region.

4 3 4 Operator Box

rdaA

rdqB

SOB Box

5 ACAAnnnnnnTTGT 3'I ! ; 4 ^ ^ ^ M :! 1_ *p *p^

5' TTGATAAATGACAACCTAGGTTGTACATTGAACGGA 3- ^ 5 ' i , - 1 0 *

i' TTGACACAAAAACAACTATAGTTTTAAATAGACACfi 3'1 I I I I I I I1 I I I I I I I

5 ' GAACnnnnGTTC 3 '

Fig. 4. Comparison of the palindromic sequences within rdgA and rdgB promoters with the operator sequence tor phage 434 c! represser(Harrison and AgganA/al. 1990) and SOB consensus sequence of B. subtilis DNA-ciamage inducible genes (Cheo el aL. 1991). Stars representthe transcriptional start sites o\ Ihe rdg genes. The - 10 and the -35 boxes are underlined. The palindromic sequences are indicated byarrows.

Regulation of pectin lyase production 1003

rdoA

+MC -MC +MC -MC

aso-700' m

Fig. 5. Northern analyses of rdg mRNA produced by E carotovorasiibsp, carotovora strain 71. Cultures were grown and induced withmitomycin C (MC) as described in tha Experiiventat procedures.Total RNA was isolated according to the procedure of Aiba etal.(1981) and hybridized with rdgA and rdgB probes. The arrowsindicate the size (bases) of rdgA and rdgB mRNAs, Lanes indicatedby +MC and - M C were loaded with RNA samples from MC-treatedand non-MC-treafed bacteria, respectively.

The two palindromic structures within the 10 and 35boxes of rdgA and rdgB promoters (Fig. 1) are goodcandidates for operators. The 14-mer (5'-ACAACCTAG-GTTGT-3') palindromic structure of rdgA matches wellthe operator sequence (5'-ACAAnnnnnnTTGT-3') of bac-teriophage 434 cl lepressor (Fig. 4: Harrison and Aggar-wal, 1990). The corresponding region of rdgB (5'-ACAACTATAGi I I 1-3'). containing a smaller palindromicstructure {5'-AACTATAGTT-3'), also is similar to theoperator sequence of bacteriophage 434 ci repressor(Fig. 4), although a guanine residue is replaced by anadenine residue within the 3' core sequence. These rdgpalindromes have some homology with the SOB box(Fig. 4), a consensus element found upstream of Bacitiussubtilis damage-inducible genes (Gheo et at.. 1991). Thehigh homologies between the W-terminal region of RdgAand the DNA-binding domain of 434 repressor (Fig. 2)and between the operator sequence of 434 ol and thepalindromes in rdgA and rdgB (Fig, 4) strongly suggestthat RdgA is a repressor which regulates its own expres-sion and possibly also the expression of rdgB by bindingto the palindromic 'operators' upstream of both genes.

MC, and rdg RNAs were analysed on Northern blots.Induction with MG allows detection of transcription ofboth genes (Fig. 5). The rdgA mRNA is about 850 bpand the rdgB mRNA is about 700 bp. These sizes,together with the failure to detect the ITIRNAS of largersize, lead to the conclusion that rdgA and rdgB are tran-scribed independently.

In the absence of MC (i,e. non-inducing conditions),rdgA mRNA is found though at lower levels than in thepresence of MC (Fig. 5). By contrast, rdgB mRNA isdetected only after induction with MG, The molecularbasis for the presence ol a low basal level ol rdgA tran-scripts and high induced levels following MC treatment isnot yet known. Assuming that RdgA, like the 434 repres-sor (Ptashne, 1992), is autogenousiy regulated, the lowbasal level may be attributed to the binding ot RdgA tothe operator-like palindrome thereby restricting transcrip-tion. Upon MC treatment, the RdgA pool may be depletedbecause of RecA processing, resulting in the activation ofrdgA transcription. The absence of a basal level of rdgBtranscription may also be caused by the binding of RdgAto the putative operator-like palindrome present withinthe promoter region of rdgB (Fig. 4),

Primer extension analysis of rdgB reveaied a single 5'endpoint, at the adenine residue located 77 nt upstream

G A

Characterization of tbe rdgA and rdgB transcripts

if RdgA regulates expression of both itself and rdgB, thenwe would expect the two genes to be transcribed indepen-dentiy of one another. To test this idea, RNAs were iso-lated from ceils grown in the absence and presence of

Fig. 6. Primer extension analyses of the 5' end ot the rdgB mRNA,Lane - M C represents RNA sampie from the cullurc withoulmitomycin C treatment. Lane +MC contains an RNA sampie fromliie mitomycin C-treated culture. The nucleotides on the left refer tothe nucleotide sequence beyond the transcripiional start site.Asterisk denotes the nucleotide at which transcription was initiafed.

1004 y. Liu. A. Chatterjee and A. K- Chatterjee

(J OC G A

i 21 2

Fig. 7. Analysis of 5' end of rdgA mRNA.U U Lanes -MC and +MC represent RNA^ ^ T C G A samples tram Ihe cultufes in !lie absence and

- presence o! mitomycin C (MC), respectively.: ^ ^ -^^ " A. Primer extension anaiyses of rdgA mRNA. 9 1 The porlion of the sequence pertinent fo ihe_. 2^1.. ~'' transcriptional start site is shown. Asterisks

.J::L ^^^ indicate the in vitro initiation points of_^ transcription,

: ^ ^ ^ .,__ B. RNase protection assay ol rdgA niFlNA.- "^z: The numbers at Ihe lefl indicate the size of

, "^ ^^ protected RNA products determined by aligninen! with a sequencing ladder.

iof the ATG start codon (Fig. 6). This endpoint was onlydetected in RNA isolated from celJs induced with MC,consistent with the results obtained by Northern blots.Analysis ot rdgA mRNA by both primer extension(Fig. 7A) and RNase protection (Fig, 7B) showed two 5'endpoints. We infer that these endpoints represent dif-ferent transcription initiation sites: PM is the initiation site

used in the absence of MC and P| is the initiation siteused foltowing induction with MC. The weak band (Prj) inthe non-MC-induced RNA samples represents a basallevel of rdgA mRNA that was actually transcribed fromthe adenine residue of the initiation (ATG) codon. Itshould be noted that there are several instances wheremRNA species without leader sequences, like the PN

100 bp

DR CD A B ADRN

rdgA rdgB Pnl Specific Rctivlty

pAKC74O-1

pAKC?'-10-19

PAKC740-I4

pAKG740-16

PAKC7.0-35

pAKC740~33

-MC

0 .85

0. 90

0 . 7 5

0 .73

0 . 8 6

0.6B

+MC

41.6

4 2 . 6

4 7 . 8

D.74

0 . 9 1

0 . 8 2

Fig. 8. InciucUon ol pectin iyase (Pnl) production by mitomycin G (MC) in ttie . coii recA' strain MC41QQ carrving pn/M' and deleted rdggeness. The arrows inc!ica!e the location of rdgA and rdgB coding regions and Ihe direction of franscription. A, AvaW. B, BsmHI: C, Hinc.W. D,Oral; F, Fspl; N, SnaBI; R, Rsa\-. and S, Slui. Specific activity is defined as units of activity per mg protein.

Regulation of pectin iyase production 1005

Table 1. Bacterial slrain.s and plasmids.

Slrain/Piasmid Relevant characlerislics Source/Reference

StrainE, coliDH5(x

MC4100

A{tacZYA~argF).0169. recAI. tlv-1

arsD139A{laclP0ZYA}U169 recA^ Ihi, Slr'^

E. carotovora subsp. carotovoraEcc71 Wild-type serogroup

PlasmidpBluescript-ll Ap"

vector KS-i-and vectorSK+

pCL1920 Stf" Spc'^

pAKC278 pnIA". Ap"pDK7 Cm''PAKC700 rdgA\^ rdgB\ Amp"PAKC730 rdgA'. rdgB ', '^

pAKC740

pAKC740-1

rdgA*. rdgB\ Str",Spc"

, rdgB'\

pAKC740-14 rdgA', rdgB*. Str",Spc"

PAKC740-16 rdgA-. rdgB^. Str'^,Spc"

PAKC740-19 rdgA-, rdgB\ Str".Spc"

pAKC740-32 rdgA^\ rdgB~. Str",Spc"

PAKC740-35 rdgA\Spc"

pAKC762 Plac-rdgB, Cm"

pAKC775

8RL

Laboratory collection

Laboratory collection

Slratagene

Lerner and Inouye(1990)

McEvoy (1991)Kleiner et al. (1988)McEvoy (1991)2.1 kb Fsp\-SnaB\

tragment ot pAKG700cloned into EcoRVsite pBluescript-ltvector SK-I-; this work

2.1kb Fspl-SnaBIfragment of pAKC730cloned into Smal siteof pCL1920 orientedopposite to p/ac; thiswork

Exo III nuclease deletionplasmid of pAKC7

1006 Y. LIU, A. Chatterjee and A. K. Chatterjee

ReoA

Acllvollon o(SOS f&omon

Fig. 9. A teiitetjve modef depicting theregulation of pectin lyase (Pri!) production andother damage-inrJucible traits in E. carotovorasubsp. carotovora strain 71. C\, native FtdgA;n , RecA"' processed RdgA; and fl, RcigB,See text for the details.

pnlA

c t v

PnIA

Ctv

Lysislas

Since rdgA and rdgB are organized as separatetranscriptional units, these results indicated that both rdggenes must be expressed co-ordinatGly for the activationof PnlA production. It is rather puzzling that a function ofthe rdgA gene, which is presumed to encode a repres-sor, is required for the activation of pntA expression inthe presence of MC- This apparent paradox may be recon-ciled by invoking the hypothesis that RdgA^\ derived byRecA-processing of RdgA, is involved in the activation ofrc/gS transcription or RdgB function. Support for this viewcomes from the precedent that UmuD protein in E. coti ispost-translationally modified via RecA hydrolytic cleavageto a form (UmuD*) that is active in SOS mutagenesis(Nohmi etat., 1988; Shinagawa etai, 1988).

rdgB aotivates pnIA expression

Based upon nucieotide sequence data (see above), rdgBwas predicted to encode a transcriptional factor activatingpnM transcription, In order to test this idea, the 636 bp Dralfragment containing the coding region of rdgB was clonedbehind the tao promoter in pDK7 (Kleiner et ai. 1988) toproduce pAKC762. In this construct, rdgB expressionshould be induced by the addition of IPTG. E. coti DH5ycarrying the pnIA piasmid pAKC278 was transformedwith either pAKC762 or the vector pDK7 (Table 1). Thedata (Table 2) show that Pnl was induced in bacteria carry-ing pAKC278 and pAKC762, but not in bacteria carryingpAKC278 and vector pDK7. While there was no detect-able expression of pntA in DH5a{pAKC278) carryingpDK7, there was a basal level of Pnl activity in DH5'/-(pAKC278) carrying pAKC762 wherein rdgB expressionwas driven by Ptac. We attribute this basal expression,in spite of the presence of tacl'^ in the vector, to the resi-dual activity of the strong tac promoter. However, theievel of Pnl was substantially higher in the presence ofIPTG, which was expected to activate the expression ofrdgB. Pnl specific activities at 2, 4 and 18 h incubationafter IPTG addition were twofold, threefold and sevenfold

higher, respectively, in the induced culture than in thenon-induced culture (Tabie 2). These data cleariy demon-strated that rdgB by itself activates pnlA transcription in theabsence of a functional recA.

In conclusion, our pubiished obseivations and the datapresented in this report show that the regulation of Pnl pro-duction in Ecc71 is mediated by rdgA. rdgB and recA geneproducts. In Fig. 9. we outline the possible mechanisms bywhich they control Pnl production, Ctv synthesis and induc-tion of cellular lysis. DNA damage generates a signal(s)that converts RecA to the activated form RecA^. Then,RecA^ '^ is believed to activate at least two regulatoiy sys-tems: the SOS regulon, principally directed at repairingDNA damage, and the RDG regulon controiiing Pnl pro-duction, Ctv synthiesis and cell lysis. RecA* most likelyactivates the SOS regulon in Ecc71 by a mechanismsimilar to that in E. coti, namely by participating in thecleavage of LexA. (he repressor of the SOS genes (Little.1991). The characteristics of the cloned Ecc texA(Garriga ef a/., 1992) as well as the effect of E. coli texAin Ecc71 (McEvoy et at., 1987) clearly support this hypo-thesis. However, the RDG regulon is proposed to beactivated by RecA^'-mediated cleavage of a repressorencoded by rdgA. !n its native state, RdgA may repressitself and the idgB gene, whereas the RecA'^-pracessedRdgA, i.e. RdgA*, presumably becomes an 'activator' of/'c/giS transcription. RdgB, in turn, aotivates the expressionof pntA, ctv and Iss genes, These characteristics of RdgAmay allow the bacterium to respond quickly and moreefficiently than if it had to rely solely upon de novo syn-thesis of a new activator. Although we have not formallyeliminated the possibility that an interaction of RdgA^ ^and RdgB leads to the activation of pnlA transcription,we consider it less iikely since, as demonstrated above,rdgB by itself can stimulate Pnl production in E. coti.

The RDG regulon of Ecc71 has some similarity with tliepyocin regulatory system of P. aerugiriosa (fviatsui et ai,1993). Pyocin pi'oduction in response to DNA-damagingagents involves positive and negative reguiator genes.

Regulation of pectin Iyase production 1007

prtN and prfR. respectively. However. PrtR functionssolely as a repressor in contrast to the requirement forfunctional RdgA in fhe activation ot pnIA transcription inEcc71. Another difference is that PrtN has no homoiogywith other regLilatory proteins, whereas RdgB has signifi-cant homology with transcriptional activators such asMor and C (see above). These differences notwithstand-ing, the two systems demonstrate that certain damage-inducible genes require transcriptional activators, whichcontrasts with the SOS regulon wherein LexA mediatesnegative control of d/n and SOS genes of coti and manyother bacteria (Garriga ef a/., 1992; Lewis et ai, 1992;Raymond-Denise and Guiilen, 1991; Walker, 1985).Therefore, some regulatoi7 pathways may have evolvedsuch that the RecA-LexA global system is used butother regulatory components are superimposed thatpermit fine tuning of seemingly unrelated physiologicalprocesses responding to common signals.

The ecological significance of the unusual induction ofpn/awaits elucidation. However, RecA-mediated expres-sion of pnl occurs in planta and in response to plantmetabolites produced either constitutiveiy or induced byany of a number of stresses (Ames. 1983: Tsuyumuet ai, 1985). In view of the tissue-macerating ability of Pniand a partial attenuation of virulence in a Pnl mutant(Pirhonen et ai. 1991), it is possible that this unusualand rather elaborate regulatory system has a role inthe host-pathogen interactions (see Barras etai, 1994;Chatterjee and Vidaver, 1986; for reviews).

The activation of Pnl in soft-rot Erwinia probably alsooccurs in environments oufside the host, such as insoil or plant debris, when the bacteria encounter DNA-damaging agents. In these situations, Pnl may serve to (i)release products that can be assimilated as carbonsources and (ii) yield substances that activate the produc-tion of other degradalive enzymes such as pectate lyases,cellulases or proteases. The induction of extracellularnuclease. chitinase and lipase by MC in RecA"'" Serratiamarcescens (Ball et ai, 1990) not only lends credenceto that hypothesis but also raises the possibility thatdamage-inducible production of degradative enzymesmay be more common in bacteria than has been recog-nized thus far.

Experimental procedures

Bacterial sfrains, construction of plasmids and growthconditions

The bacterial strains and plasmids used in this work are listedin Table 1. Basic genetic manipulations were carried out bystandard procedures (Sambrook e! a!.. 1989). For DNA andRNA manipulations, E. coli and E. carotovora ceils weregrown in Luria-Berlani (LB) broth at 37 C and 28 C. respec-tively, with shaking at 150r,p.m. Antibiotics were added as

needed: ampicillin (SOfigml" '), spectinomycin (50|igi"nl '},streptomycin (SOjigml ') and chlaramphenicol (20),igml ^).

DNA manipulationsDNA isoiation, exonuclease III deletion, DNA fragment purifi-cation and gel electrophoresis were performed as describedin Sambrook ef ai (1989). DNA sequences were determinedby the dideoxy method (Sanger et ai. 1977) using the Seque-nase Version 2 DNA Sequencing Kit (US Biochemical Corp,).Restriction enzymes, T4 DNA iigase, T3 and T7 RNA poly-merases and Klenow tragment DNA polymerase were pur-chased trom Promega: dNTPs and rNTPs from BoehringerMannheim, [7.--'''^ P]-dCTP, [Y-^^PI -ATP and [r/-^^''P]-UTP tromDu Pont,

Computer methodsRecognition of protein-coding region, search of protein homo-logy, analysis of protein secondary structure and alignmenl ofprotein sequences were performed using the POOENIE programpackage (Release 6.8, IntelliGenetics, Inc.).

HNA preparationTotal RNA was obtained from Ecc71. The bacterium wasgrown in 20 ml LB at 28 C to an optical density at 600 nm(Aeoo) of 1.0. The culture was equally divided into two flasks.Mitomycin C (f^C, M-0503, Sigma Chemical Co.) was addedto one flask at a final concentration of 500ngml ', and theother haif of the culture was used as the conliol. These cul-tures were incubated at 28 C for an additional 8 h. TotalRNA was then extracted by the 'hot phenol' method asdescribed by Aiba etai (1981).

Northern hybridization experiments

RNA samples (20 ).ig RNA) dissolved in 13 (.il denaturing buffer(50% formamide, 6% formaldehyde, (10 mM) morpholinopro-pane sulphate (MOPS) buffer) were incubated for 5 min at65 C and then immediately placed on ice. Each sample wastreated with 4^1 loading dye (40% giycerol, 0.1 M EDTA,0.03% bromophenol blue) and then loaded on a 6% formalde-hyde. 1% agarose gel and run in MOPS buffer. Followingtransfer to Biotrans nylon membranes (ICN), the membraneswere briefly washed in 2\ SSC and the RNA was fixed bybaking for 2h at 80 C in a vacuum oven.

The 1053bp Fsp\~Stu\ DNA fragment from pAKC740containing a part of rdgA (377 bp upstream sequence and675 bp of rdgA coding region; Fig. 8) and the 500 bpSamHI-SnaBI DNA fragment from pAKC740 containing apart of rdgB (259 bp /-cygS-coding region and 241 bp down-stream sequence: Fig, B) were labelled with [r;:--'''P]-dCTPby random-priming according to the manufacturer's instruc-tions (US Biochemical Corp.). Prehybridization (4h at 42 C)and hybridization (18h at 42 C) were performed in the pre-hybridization buffer (5x SSC, 10^ Denhardfs, 0 .1% (w/v)SDS. 0.1 M KH3PO4 pH6.8, 100Mgml ' rJenatured salmonsperm DNA) and the hybridization buffer (5 ' SSC. 10xDenhardfs, 0.1 M KH^PO,! pH6.8, 100 ng ml ^ denatured

1008 y. Liu, A. Chatterjee and A. K. Chatterjee

salmon sperm DNA, 10% (w/v) dextran sulphate, 30% (v/v)deionized tormamide). After hybridization, filters werewashed twice for 20n-iin at 50 C in 2x SSC, 0.5% (w/v)SDS. and then for 30 min at 65 C in 0.5x SSC, 0.5% (w/v)SDS. and the filters were then examined by autoradiographywith X-ray film (Kodak).

Primer extension analysis

Primer extension was performed according to Ihefacturer's instructions (Promega Corp.). Two oligonucleotideprimers were used: primer 5-CTTGGGCAGTTCTGGCGG-TC-3'. corresponding to base positions 126 to 107 for rdgAmRNA, and primer 5-GCGAAA1TGTGGCTCAG-3', corre-sponding to base positions 1287 to 1271 for rdgB mRNA(Fig. 1). Ten picamoles ot each primer was end-labelledwith T4 polynucieotide kinase and [y-'^^Pj-ATP and lOOfmolof this primer was incubated with 20jig RNA in 11 jil primerextension buffer al 58 C for 20min and cooled for 10min atroom temperature for annealing. Reverse transcriptionreaction was carried out by AMV reverse transcriptase af4 2 X for 30 min.

HNase ptotection assay

RNase protection assay was carried ouf as described by Liuetai (1993). Briefly, 0.5^lg SamHl-digested pAKC775 DNAwas mixed with 4MI 5^ transcription buffer (0.2M Tris-HCIpH7.5, 30mM MgCl2, 5mM spermidine), 2MI 0.1 M DTT, 1 piRNAsin (35Unr^ ) , 4}il rNTPs (2.5mM each of ATP. CTP.GTP and 125nM UTP), 5MI b,-^^Pl-UTP and 50U T7 poly-merase in a total volume of 20 pi. Reaction was carried outfor 50 min at 37 C for the synthesis of the RNA probe whichcorresponds to base positions 1 to 492 shown in Fig. 1.DNA template was then removed from the RNA probe byDNasG treatment. A 20 MQ RNA sample and lO^c.p.m. of theRNA probe were incubated in 30fii hybridization buffer (80%(v/v) formamide, 40 mM Pipes pH6.7, 400 mM f^aCI, 1 mMEDTA) overnight at 45 C. then reacted with 3001.1! RNasesolution (10mM Tiis pH7.5, 5mM EDTA, 300 mM NaCL4 0 p g m r ' RNase A. 2 ug ml ' RNase T1) at 30 C for 1 h.

Pectin iyase assay

E. CO//strains containing the pnIA gene and either deleted rdggenes or the Ptac-rdgB fusion were grown in LB supple-mented with appropriate anfibiotics at 28 C with shaking at150r.p.m, The cultures were treated with MC (2.5ngml ')or IPTG (1 niM) when they reached an ^QQQ value of 1.0. Por-tions of the cultures not treated with MC or IPTG were used ascontrols. Cells were collected after the desired incubationperiod, extracts were prepared and pectin lyase was assayedas described by IVlcEvoy et ai. (1990).

Acknowledgements

This investigation was supported by National ScienceFoundation (Grant DMB-9018733) and the Food for the 21stCentury Program of the University of Missouri. We thank

Karen Cone, Judy Wall and Jim Schoeiz for critical commentsand D. L, Pinkerton for photography. This manuscript is journalseries 12 117 of the Missouri Agriculture Experiment Station.

References

Aiba, H., Adhya, S., and de Crombrugghe, B. (1981)Evidence for two functional gal promoters in intactEscherichia coii J Biol Chem 256: 11905-11910-

Ames, B.N. (1983) Dietary carcinogens and anticarcinogens.Oxygen radicals and degenerative diseases. Science 221:1256^1264-

August, P.R.. Flickinger, M.C, and Sherman, D.H. (1994)Cloning and analysis of a iocus {mar) involved in mitomycinC resistance in Streptomyces lavendulae. J Bacterioi 176;4448-4454.

Ball, T.K., Wasmuth, C.R., Braunagel, S.C., and Benedik,M.J. (1990) Expression of Serratia marcescens extracel-lular proteins requires recA. J Bacterioi 112.: 342-349.

Barras. F,, van Gijsegem. F,, and Chatterjee, A.K. (1994)Extracellular enzymes and pathogenesis of soft-rot Erwinia.Annu Rev Piiytopath 32: 201-234.

Bolker. M,, Wulczyn, F.G.. and Kahmann, R. (1989) Role ofbacteriophage Mu C protein in activation of the mom genepromoter. J Bacterioi 171: 2019-2027.

Brennan, R,G-. and iVlatthews. B.W, (1989) The helix-turn-helix DNA binding motif. J Bioi Chem 264: 1903-1906.

Chatterjee, A.K., and Vidaver. A.K. (1986) Genetics otpathogenicity factors: application to phytopathogenicbacteria. In Advances in Plant Pathoiogy, Vol. 4. Ingram,D.S., and Williams, P.H. (eds). London: Academic Press,pp. 1-224.

Chatterjee, A.. McEvoy, J.L., Chambost. J.P.. Blasco, F.. andChatterjee, A.K. (1991) Nucleotide sequence and molecu-!ar characterization of pnIA, the structural gene for damage-inducible pectin lyase of Erwitiia carotovora subsp.catotovora 71. J Bacterioi 173: 1765-1769.

Chatterjee, A.K., Murata, H., Liu. Y., and Chatterjee, A. (1992)Regulation of the production of plant cell wall degradingenzyme in the bacterial plant pathogen, Erwinia carotovorasubsp. carotovora. In Agricuitural Biotechnology. You,C.B., and Chen, Z.L. (eds). Beijing: China Science andTechnology Press, pp. 690-693.

Cheo, D.L, Bayles. K.W., and Yasbin, R.E. (1991) Cloningand characterization of DNA damage-inducible promoterregions from Bacillus subtilis. J Bacterioi 173: 1696-1703.

Collmer, A., and Keen, N.T. (1986) The role of pectic;enzymes in plant pathogenesis. Annu Rev Phytopathol24: 383-409,

Dodd, I.B.. and Egan, J.B, (1987) Systematic method for thedetection of potential X Cro-like DNA-binding regions inproteins. J Moi Biol 194: 557-564.

Eguchi, Y., Ogawa, T., and Ogawa, H. (1988) Cleavage ofbacteriophage

Regulation of pectin Iyase production 1009

Harrison, S,C,. and Aggaiwal, A.K. (1990) DNA recognitionby proteins with the helix-turn-helix motif. Annu RevBiochem 59: 933-969.

Horii, T., Ogawa, T., and Ogama, H, (1981) Nucleotidesequence of the texA gene of E. coli. Cell 23: 689-697.

itoh, Y., Izaki, K., and Takahashi, H. (1980) Simultaneoussynthesis of pectin Iyase and carotovoricin induced bymitomycin C. nalidixic acid or ultraviolet light irradiation inEminia carotovora. Agric Biol Chem 44: 1135-T1 AO.

Kamimiya, S., izaki, K., and Takahashi. H. (1972) A newpectolytic enzyme in Erwinia aroideae formed in thepresence of nalidixic acid. AgrIc Biol Chem 36: 2367-2372.

Kamimiya. S.. Nishiya, T.. Izaki. K., and Takahashi, H. (1974)Purification and properties of a pectin ^rans-eiiminase inEminia aroideae formed in the presence of nalidixic acid.Agric Bioi Chem 38: 1071-1078.

Kleiner, D., Paul, W.. and Merrick, M.J. (1988) Construction ofmulticopy expression vectors for regulated overproductionof proteins in Klebsielia pneumoniae and other entericbacteria. J Gen M/croWo/134: 1779-1784.

Lerner, C.G,, and Inouye, M. (1990) Low copy numberplasmids for regulated low-level expression of clonedgenes in Esdierichia coii with blue/white insert screeningcapability. Nud Acids Hes 18: 4631.

Lewis, L.K., Jenkins, M.E.. and Mount, D. (1992) Isolation ofDNA damage-inducibie promoters in Escherichia coti:regulation of polB {dinA), dinG, and dinH by LexArepressor, J Bacterioi 17A: 3377-3385.

Litile, J.W. (1991) Mechanism of specific LexA cleavage:autodigestion and the role of RecA coprotease. Biochimie73; 411-422.

Liu, Y.. Murata, H., Chatterjee, A., and Chatterjee, A.K. (1993)Characterization of a novel regulatory gene aepA thatcontrols extracellular enzyme production tn the phytopatho-genic bacterium Erwinia carotovora subsp. carotovora. MotPlant-Microbe interact 6: 299-308.

Margolin, W., and Howe, N.M. (1986) Localization and DNAsequence analysis of C gene of bacteriophage Mu, thepositive regulator, Nud Adds Res 14: 4881-4897.

Mathee, K., and Howe, M.M, (1990) Identification of a positiveregulator of the Mu middle operon. J Bactoi'iol 172: 6641 -6650.

Matsui, H., Sano, Y., Ishihara, H., and Shinomiya, T. (1993)Regulation of pyocin genes in Pseudomonas aeruginosaby positive (prtN) and negative [prtR) regulatory genes, JBacteriol 175: 1257--!263.

McEvoy, J.L- (1991) Regulation of PNL production in Eiwiniacarotovora. PhD Thesis, University ot Missouri, Columbia,MO, USA.

McEvoy. J.L., Thurn. K.K., and Chatterjee, A.K. (1987)Expression of the E.coli IexA'' gene in Eiwinia carotovorasubsp, carotovora and its effect on production of pectiniyase and carotovoricin. FEMS Microbioi Lett42: 205-208-

McEvoy^ J.L., Murata, H.. and Chatterjee. A.K. (1990) Moiecu-iar cioning and characterization of an Erwinia carotovorasubsp. carotovora pectin Iyase gene that responds to DNA-damaging agents. J Bacterioi 172: 3284-3289.

McEvoy, J.L. Murata, H-, and Chatterjee, A.K. (1992) Geneticevidence for an activator required for induction of pectinIyase in Erwinia carotovora subsp. carotovora by DNA-damage agents, J Bacteriot 174: 5471-5474.

Murata, H., McEvoy. J.L. Chatterjee, A., Collmer, A., andChatterjee, A.K. (1991) Molecular cloning of an aepA genethat activates production of extracellular pectolytio, cellulo-lytic, and proteolytic enzymes In Erwinia carotovoia subsp.carotovora. Moi Plant-Microbe Interact 1: 239-246.

Murata, H., Chatterjee, A., Liu, Y., and Chatterjee, A.K. (1994)Regulation of the production of extracellular pectinase,cellulase and protease in the soft-rotting bacterium Erwiniacarotovora subsp. carotovora [Ecc]; evidence that aepH ofEcc strain 71 activates gene expression in Ecc, Eminiacarotovora subsp. atroseptica and Escherichia coii. AppiEnviron Microbtot 60: 3150-3159.

Neri, D.. Billeter, M-, and Wuthrich, K. (1992) Deteiminationof the nuclear magnetic resonance solution structure of theDNA-binding domain (residues 1 to 69) of the 434repressor and comparison with the X-ray crystal struc-ture. J Mot Biol 223: 743-767,

Nohmi, T., Batfista, J.R., Dodson, L.A.. and Walker, G.C.(1988) RecA-mediated cleavage activates UmuD formutagenesis: mechanistic relationship between transcrip-tional derepression and posttranslaiional activation. ProcNati Acad Sd USA 85: 1816-1820,

Ogawa, T., Ogawa, H., and Tomizawa, J. (1988) Organizationof the early region of bacteriophage ij)80, genes andproteins. J Mot Bioi 202: 537-550.

Pedro, G., Liu, Y., Orbovi, V,, Verkamp, E., Poff, K.L,, andGreen, P.J. (1994) Characterization of the auxin-inducibleSAUP-AC1 gene for use as a molecular genetic tool inArabidopsis. Plant Physionm: 777-784.

Ptashne, M. (1992) A Genetic Switch. Cambridge, MA:Blackwell Scientific Publications, Inc.

Pirhonen, M., Saarilahti, H., Karlsson, M-B., and Palva, E.T.(1991) Identification of pathogenicity determinants otEiwinia carotovora subsp. carotovora by transposonmutagenesis, Mol Plant-Microbe interact 4: 276-283.

Raymond-Denise. A., and Guillen, N. (1991) Idenlitication ofdinR, a DNA damage-inducibie regulator gene of Bacillussubtilis. J Bacteriol 173: 7084-7091,

Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) InMotecutar Ctoning. A Laboratory Manuai 2nd edn. ColdSpring Harbor, New York: Cold Spring Harbor LaboratoryPress.

Sangor, F., Nicklen, S., and Coulson, A.R. (1977) DNAsequencing with chain-terminating inhibitors. Proc NattAcad Sd USA 74: 5463-5467.

Sauer, R.T., and Anderegs, R, (1978) X Cl repressorsequence. Biochemistry M: 1092-1100.

Sauer, R.T., Yocum, R.R., Dooliltie, R.F., Lewis, M., andPabo, CO. (1982) Homology among DNA-binding proteinssuggests use of a conserved super-secondary structure.Nature 298: 447-451.

Scheren G.F.E., Walkinshaw, M,D., Arnott, S-, and Morre.D.J, (1980) The ribosome binding sites recognized by E.coll ribosomes have regions with signal character in boththe leader and protein coding segments. Nud Acids Res 8:3895-3907.

Shinagawa, H.. Iwasaki, H-, Kato, T., and Nakata, A. (1988)RecA protein-dependent cleavage of UmuD protein andSOS mutagenesis, Proc Nati Acad Sd USA 85: 1808-1810.

Tomizawa, H., and Takahashi, H. (1971) Stimulation ot

1010 Y. Liu, A. Chatterjee and A. K. Chatterjee

pectolytic enzyme formation of Erwinia aroideae by the possible inducers of pectin lyases of soft-rot Erwinia;nalidixic acid, mitomycin C and bleomycin. Agric Biot Ann Phytopath Soc Japan 51: 294-302,Chem 35: 191-200. Walker. G.C. (1985) Inducible DNA repair systems. Annu Rev

Tsuyumu, S., and Chatterjee, A.K. (1984) Pectin lyase Biochem 54: 425-457.production in Erwinia chrysanthemi and other soft-rot Zink, R.T., Engwall, J.K., McEvoy, J.L, and Chatterjee, A.K,Erwinia species. Physlot PtanI Pathot24: 291-302. (1985) recA is required in the induction of pectin lyase and

Tsuyumu, S,, Funakubo, T., Hori. K., Takikawa, Y., and Goto, carotovoricin in Erwinia carotovora subsp. carotovora- JU. (1985) Presence of DNA damaging agents in plants as Bacterioi 164: 390-396.