Regulation of Pectinolysis in Erwinia Chrysanthemi

August 2, 1996 19:58 Annual Reviews HUGOTEXT.TXT AR15-07

Annu. Rev. Microbiol. 1996. 50:213–57Copyright c© 1996 by Annual Reviews Inc. All rights reserved

REGULATION OF PECTINOLYSIS INERWINIA CHRYSANTHEMI

Nicole Hugouvieux-Cotte-Pattat, Guy Condemine, WilliamNasser, and Sylvie ReverchonLaboratoire de Génetique Moleculaire des Microorganismes, UMR-CNRS 5577,INSA Batiment 406, 20 Avenue Albert Einstein, 69621 Villeurbanne Cedex, France

KEY WORDS: plant pathogens, virulence factors, pectinases, transcriptional regulation, DNA-protein interactions

ABSTRACT

Erwinia chrysanthemiis an enterobacterium that causes various plant diseases.Its pathogenicity results from the secretion of pectinolytic enzymes responsiblefor the disorganization of the plant cell wall. TheE. chrysanthemistrain 3937produces two pectin methylesterases, at least seven pectate lyases, a polygalac-turonase, and a pectin lyase. The extracellular degradation of the pectin leads tothe formation of oligogalacturonides that are catabolized through an intracellularpathway. The pectinase genes are expressed from independent cistrons, and theirtranscription is favored by environmental conditions such as presence of pectinand plant extracts, stationary growth phase, low temperature, oxygen or iron lim-itation, and so on. Moreover, transcription of the pectin lyase gene respondsto DNA-damaging agents. The differential expressions of individual pectinasegenes presumably reflect their role during plant infection. The regulation ofpelgenes requires several regulatory systems, including the KdgR repressor, whichmediates the induction of all the pectinolysis genes in the presence of pectincatabolites. KdgR also controls the genes necessary for pectinase secretion andother pectin-inducible genes not yet characterized. PecS, a cytoplasmic proteinhomologous to other transcriptional regulators, can bind in vitro to the regulatoryregions of pectinase and cellulase genes. The PecT protein, a member of the LysRfamily of transcriptional regulators, represses the expression of some pectinasegenes and also affects other metabolic pathways of the bacteria. Other proteinsinvolved in global regulations, such as CRP or HNS, can bind to the regulatoryregions of the pectinase genes and affect their transcription.

2130066-4227/96/1001-0213$08.00

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214 HUGOUVIEUX-COTTE-PATTAT ET AL

CONTENTS

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214

PECTIN DEGRADATION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215The Pectate Lyases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217The Other Pectinases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218Maturation and Secretion of Pectinases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220Contribution of Individual Pectinases to Plant Maceration and Bacterial Virulence. . . . 220Catabolism of Monomers Derived from Pectin Cleavage. . . . . . . . . . . . . . . . . . . . . . . . . . 222The Steps of Galacturonate and Glucuronate Catabolism. . . . . . . . . . . . . . . . . . . . . . . . . 225

STIMULI AFFECTING PECTINASE PRODUCTION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225Expression of Pectinase Genes in Planta. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226Induction by Pectin-Degradation Intermediates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227Other Inducers from Plant Extracts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228Catabolite Repression. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229Stationary-Phase Induction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229Osmoregulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230Thermoregulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232Oxygen and Nitrogen Availability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232Iron Deprivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233Regulation ofpnl by DNA-Damaging Agents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233

THE REGULATORY ELEMENTS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234Regulation of theexu-uxa-uxuGenes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234The KdgR Regulon. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236The PecS-PecM Regulatory Couple. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241The PecT Regulator. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243Additional Regulatory Systems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244Regulatory Sites. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246

CONCLUSION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248

INTRODUCTION

The enterobacteriaErwinia chrysanthemiandErwinia carotovorabelong tothe soft-rot group ofErwiniae. These bacteria are commonly found in soil orassociated with plants. They are responsible for important economic lossessince they cause soft-rotting, wilting, and dwarfing, among other dysfunctions,in a wide range of plants (108). These symptoms result from the disorganizationof the plant cell wall caused by a set of enzymes secreted from the bacteria.Disease initiation and extent of rotting depend on both plant sensitivity andthe occurrence of favorable environmental conditions (temperature, oxygenavailability, etc). A response to these environmental parameters requires thecoordinated expression of the bacterial virulence genes. Therefore, the switchbetween the saprophytic and the pathogenic way of life is determined by aregulated response of the virulence factors. The main characteristic of soft-rotErwiniae is their ability to produce large quantities of extracellular plant cellwall–degrading enzymes. E. chrysanthemisynthesizes pectin methylesterases,pectate lyases, a pectin lyase, a polygalacturonase, cellulases, proteases, and aphospholipase (25). Most of the pectinases and cellulases are secreted in theexternal medium via a common secretion system, the Out machinery. Among

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PECTINOLYSIS REGULATION INE. CHRYSANTHEMI 215

all these degrading enzymes, pectate lyases have a predominant role in plant-tissue maceration (25).E. chrysanthemidifferentially regulates the synthesis ofseveral isoenzymes of pectate lyases. This bacterium’s ability to modulate itspectinolytic machinery depends on complex regulatory pathways, which allowfine-tuning of gene expression.

Besides the cell wall–degrading enzymes, other factors are important forEr-winia pathogenicity.E. chrysanthemicells synthesize siderophores that enablethem to overcome the low iron availability in plant intercellular fluids (102).Screening for reduced virulence mutants inE. carotovoraled to the isolationof mutants defective in motility, exoenzyme synthesis, or exoenzyme secre-tion (96, 111). A second approach to identify the pathogenicity genes ofE.chrysanthemiinvolved the selection of plant-inducible gene fusions (8). Manyof these mutants showed a reduced virulence. Some are defective in iron trans-port, the synthesis of exoenzymes, or pectin catabolism; others define novelvirulence loci (8). These two approaches illustrate the fact that pathogenicityis a multifunctional process.

The roles of extracellular enzymes in pathogenicity and the secretory mech-anisms were recently reviewed in depth (5, 128). In this paper, we focus on thepectinolytic pathway and its regulation. After the presentation of the structuralgenes of pectinolysis, the main part of this review addresses recent findingson factors modulating gene expression and on the regulatory mechanisms thathave evolved inErwinia species.

PECTIN DEGRADATION

The maceration process involves the depolymerization of the pectin in plantcell walls. Pectin is a heteropolysaccharide with a backbone consisting ofα-1,4-linkedD-galacturonate residues and some rhamnose molecules. Naturalpectins present a high percentage of methylesterification on the carboxyl groupof galacturonate residues (from 40 to 65%). The variety of enzymes produced bypectinolytic bacteria reflects the complexity of pectin. Pectinases can be classi-fied according to their preferential substrate, pectin or polygalacturonate (PGA),and their reaction mechanism (β-elimination or hydrolysis). Endo-pectinasescut the polymer at random sites within the chain to give a mixture of oligomers,while exo-pectinases attack the reducing end of the polymer and produce onlydimers. The pectinase equipment of theE. chrysanthemistrain 3937 is par-ticularly well characterized. This strain produces five types of pectinases andmultiple isoenzymes (Figure 1): at least six endo-pectate lyases (PelA to PelEand PelL), an exo-pectate lyase (PelX), two pectin methylesterases (PemA andPemB), a pectin lyase (PnlA), and an exo-polygalacturonase (PehX). In contrastto these pectinases, which degrade long polymeric chains, oligogalacturonate

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Figure 1 Pectin catabolism inErwinia chrysanthemi.The upper part of the figure shows thedetailed structure of pectin. Beneath the structure, a diagram outlines the action of the differentpectinases outside the bacterial cell (but note that PelX and PemB are a periplasmic and a membraneprotein, respectively). The Out proteins (large arrow) are involved in the secretion of most ofthe pectinases and of the cellulase CelZ.Double arrowsindicate sugar transport systems. Thecatabolism of oligogalacturonides takes place in the cytoplasm.

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lyase (Ogl) recognizes pectic oligomers of two to four residues. While PelX,PemB, and Ogl are intracellular enzymes, the other pectinases are secreted intothe extracellular medium byE. chrysanthemicells.

The Pectate LyasesPectate lyases appear to be the major pectolytic enzymes produced byE.chrysanthemi,and they play a main role in the soft-rot symptom. Pectate lyasescleave internal glycosidic bonds in PGA viaβ-elimination to yield oligomersthat are 4,5-unsaturated at the nonreducing end. They exhibit reduced activityon pectin.

Separation ofE. chrysanthemipectate lyases by means of electrofocusing inthin polyacrylamide gels, followed by an activity detection (9, 123), revealedin most strains five major isoenzymes with endo-pectate lyase activity: onewith an acidic isoelectric point (PelA, pI 4.5), two slightly alkaline (PelBand PelC, pI 7.5–8.5), and two very alkaline (PelD and PelE, pI 9.5–10.5).The molecular biology approach confirmed that the pectate lyase activity inE. chrysanthemilargely results from the cumulative action of these five majorendo-enzymes encoded by the five genespelA, pelB, pelC, pelD,andpelE(74).Thepelgenes have been cloned from variousE. chrysanthemistrains. They areorganized in two clusters—pelB, pelCandpelA, pelE, pelD—which are widelyseparated on the bacterial chromosome (Figure 2) (63). Despite clustering,eachpel gene is an individual transcriptional unit (74). Genes belonging to agiven cluster are homologous, which suggests that genes of the same clusterhave appeared because of duplication of an ancestral gene (71, 140, 148). Simi-larly, the three secreted isoenzymes ofE. carotovoraencoded by three adjacentgenes are homologous (57, 78). The reason for such gene duplications is stillunclear.

An exo-pectate lyase activity was first reported inE. chrysanthemiCUCP-B1237 (24). Exo-pectate lyase generates a sole product identified as unsatu-rated digalacturonate. ThepelX gene was cloned from twoE. chrysanthemistrains, EC16 (16, 24) and 3937 (V Shevchik, personal communication). PelXcan utilize PGA and also methylated pectins as substrates (16, 24). The cel-lular location of the enzyme has not been determined. InE. chrysanthemiCUCPB1237, the exo-pectate lyase activity was cell bound (24). We wouldexpect this type of enzyme to be present in the bacterial periplasm because itacts better on oligomers produced by endo-pectate lyases than on polymericsubstrates (16).

The deletion ofpelXand the five majorpelgenes ofE. chrysanthemifailed tototally eliminate the bacteria’s capacity for tissue maceration (122). Analysisof the macerated tissue by means of electrofocusing followed by an activitydetection revealed the presence of a new set of pectate lyases (up to 5 forms)

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Figure 2 Genetic map of theE. chrysanthemi3937 chromosome. Positions of the markers weredescribed in Hugouvieux-Cotte-Pattat et al (63). The genes involved in pectin catabolism are inbold type and the transcriptional regulators of pectinolysis are in larger type.

(7, 72). Because of their low activity in synthetic medium, they are describedas secondary pectinases. The gene for one secondary endo-pectate lyase, PelL,has been characterized in twoE. chrysanthemistrains. InE. chrysanthemi3937,pelLwas isolated from a genomic bank of a strain lacking the five majorpel genes (84). Insertion mutagenesis of the1pelA,E,B,C,X, pehXderivativeof E. chrysanthemiEC16 with a transposon containing an inducible promoterallowed hyperexpression of thepelLgene (1). ThepelLgene is adjacent to themajor cellulase genecelZ (Figure 2). The PelL protein shows some sequencesimilarity, restricted to the C-terminal region, with the exo-pectate lyase PelXof E. chrysanthemi(16). PelL and PelX define a new class of pectic enzymessince they display no homology with other pectinases.

The Other PectinasesPECTIN METHYLESTERASES Pectin methylesterases facilitate pectate lyase ac-tion by removing the methoxyl groups of pectin to yield PGA and methanol.Despite its pectin methylesterase activity,E. chrysanthemicannot grow onhighly (98%) methylesterified pectin as carbon source (135). The genepemA,which encodes an extracellular pectin methylesterase, is linked to thepelA,pelE, pelDlocus encoding three major pectate lyases (Figure 2) (74). This

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gene was cloned and characterized from twoE. chrysanthemistrains, B374 and3937 (77, 112). The genepemB,which encodes a novel pectin methylesterase,was also recently cloned (135). PemB is an outer-membrane lipoprotein whoseactivity is approximately 100-fold higher on pectic oligomers than on naturalpectins. The action of extracellular pectinases on pectin probably liberatessmall methylated oligogalacturonides that can enter the periplasm by diffusion,and the role of PemB might be to degrade such oligomers. Sequence similaritybetween PemA and PemB is quite low (135). Thus the presence inE. chrysan-themiof two pemgenes probably does not result from a recent duplication ofan ancestral gene, as supposed forpelgenes.

PECTIN LYASE Pectin lyase can cleave natural pectin and highly (98%) methy-lesterified PGA throughβ-elimination, but it otherwise has no activity towardPGA. Most soft-rotErwiniaeproduce an endo-pectin lyase activity in responseto DNA-damaging agents (147). Pectin lyase activity is generally higher inE. carotovorathan inE. chrysanthemiand has been more closely analyzed inthe former species. TheE. carotovoragene encoding pectin lyase has beencharacterized (20, 104), but theE. chrysanthemigene has not.

POLYGALACTURONASE Polygalacturonases are distinguishable from the lyasesby their hydrolytic reaction mechanism. WhereasE. carotovorapresents endo-polygalacturonase activity (74), the only hydrolase activity found inE. chrysan-themiis an exo-cleaving polygalacturonase. Exo-polygalacturonases hydrolyzeα-1,4-glycosidic bonds and attack the nonreducing end of PGA to release di-galacturonate. The enzyme isolated fromE. chrysanthemiCUCPB1237 has apreference for chains bearing a 4,5-unsaturated residue at the nonreducing end(such as those generated by pectate lyases). ThepehXgene was cloned fromE. chrysanthemiEC16 (55) and 3937 (V Shevchik, personal communication).PehX, which has a molecular weight of 67,000, is the largest of the pectinasesfound in the supernatant ofE. chrysanthemicultures (55).

OLIGOGALACTURONATE LYASE Oligogalacturonate lyase, encoded by theoglgene, converts the oligogalacturonides that result from the extracellular ac-tion of pectinases into monomeric sugars. This cytoplasmic enzyme cleavesthe α-1,4-glycosidic bond by transelimination. Unsaturated digalacturonate,the best substrate, is cleaved into two molecules of 5-keto-4-deoxyuronate [4-deoxy-L-threo-5-hexosulose uronic acid (DKI)]. Digalacturonate is degradedto yield equimolar concentrations of galacturonate and DKI. The rate of degra-dation decreases strongly when the length of the substrate increases. Tetra-galacturonides are attacked at a very slow rate. Mutants ofE. chrysanthemideficient in oligogalacturonate lyase production (22, 24, 120) cannot use PGA

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or digalacturonate for growth. Theogl gene ofE. chrysanthemi3937 wascloned via complementation of anogl mutation (117, 120), and theogl geneof E. carotovorawas characterized recently; the two derived protein sequencesshare 88% of identity (151).

Maturation and Secretion of PectinasesPectin-degrading enzymes must be secreted outside the bacteria to reach theirsubstrate, the plant cell wall pectins. MostE. chrysanthemipectinases areextracellular enzymes: the five major pectate lyases PelA to PelE, the sec-ondary pectate lyases including PelL, the polygalacturonase PehX, and thepectin methylesterase PemA. In contrast, PelX and PemB are periplasmic andmembrane proteins, respectively. The secretion of PnlA probably results fromsemispecific leakage from the bacterial envelope owing to the presence of DNA-damaging agents, as suggested for the secretion of colicins inEscherichia coli(20). The other secreted pectinases reach the outer medium in a common two-step process (128). They are synthesized with a signal sequence that is cleavedduring transfer across the inner membrane by the general Sec machinery. Theyare then exported across the outer membrane by the specific Out system. Isola-tion of mutants incapable of pectate lyase secretion led to the identification oftheout mutants in which the pectinases PelA to PelE, PelL, PehX, and PemAand the cellulase CelZ accumulate in the periplasm. Theout locus consistsof 15 genes organized in 5 transcriptional units:outS, outB, outT, outCDE-FGHIJKLM, andoutO (Figure 2) (128). The products of these genes exhibithomology with secretory proteins of other Gram-negative bacteria and form atype II secretion machinery. The exact function of most of the Out proteins insecretion is not yet known (128). Although expression ofoutSandoutBseemsconstitutive, expression ofoutTand theoutCoperon is coregulated with that ofpectinases (26).

Once exported to the periplasm, secreted proteins can adopt their final con-formation. Protein folding can be influenced by periplasmic chaperones orenzymes involved in structural modifications. For instance, all the secretedE.chrysanthemipectinases present intramolecular disulfide bonds, and mutationsin genes involved in disulfide bond formation prevent the secretion of the ma-jor pectate lyases (136). Thus the modifications taking place in the periplasmappear to be important for protein secretion.

Contribution of Individual Pectinases to Plant Macerationand Bacterial VirulenceEarly investigations showed that pectate lyases are able to macerate plant tissueand are responsible for the symptoms of soft rot (5, 25). Although all the pectatelyases showed similar biochemical properties when assayed on purified pectic

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polymers, their abilities to macerate plant tissues varied (4, 48). The basicisoenzymes, PelD and PelE, most effectively macerate plant tissue, causingelectrolyte loss and plant cell death. PelB and PelC can also macerate planttissues, but the acidic isoenzyme PelA cannot. Analysis of purified proteinsindicated that the specific activity of PelA is at least 10-fold lower than that ofthe other major pectate lyases (42). However, the secondary isoenzyme, PelL,which exhibits a specific activity approximately 100-fold lower than that ofthe major pectate lyases, appeared able to macerate plant tissue quite efficiently(84). Therefore, the maceration capacity is not linked to the specific activity. Noevidence was found for a role of the exo-enzymes PelX or PehX in maceration(16, 55).

Analysis of mutants in which onepelgene is inactivated indicated that noneof these genes are essential for the maceration of potato tubers or other planttissues (7, 122). Use of the natural host plant African violet (Saintpauliaionantha) of the 3937 strain to assess the virulence ofpel mutants gave newinsights into the role of each isoenzyme (11). Inactivation ofpelA, pelD,orpelEsignificantly reduces the systemic-invasion capacity of the bacteria. PelA,which as discussed above is incapable of causing tissue maceration, is crucialfor symptom development. In contrast, inactivation ofpelBorpelChas no effecton the outcome of the disease. Therefore, in the interaction ofS. ionanthaandstrain 3937, enzymes encoded by thepelA, pelE, pelDcluster appear to be moreimportant for virulence than those of thepelB, pelCcluster. ThepelL mutantdisplayed a reduced virulence onS. ionantha,which suggests an important roleof secondary isoenzymes in soft-rot disease (84). Analysis ofpemAmutantsdemonstrated that the secreted pectin methylesterase also has a crucial role invirulence in African violets and potato tubers (10, 39).

The contribution to the pathogenicity of each pectate lyase isoenzyme variesaccording to the plant infected (7). For instance, on pea plantlets, onlypelAappears necessary for soft-rot disease, whereas on chicory leaf, all thepelgenes,exceptpelE, contribute to maceration. In addition, electrofocusing analysisof the isoenzymes indicated that synthesis of both the major and secondaryisoenzymes can vary qualitatively and quantitatively within the different plants(7). Moreover, analysis of the isoenzyme profiles of natural strains isolatedfrom different hosts suggested a correlation between the host range and thepectate lyase profiles (13, 123).

Pectin is a common component of the middle lamella and the primary cellwall of plants. However, the degree of methylesterification or acetylation andthe presence of other chemical groups vary according to the plant, tissue, andcell wall compartment. Gold-labeling and microscope analysis demonstratedthat PelB and PelC are preferentially located in some domains of the middle

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lamella and cell junctions (142), whereas PelD and PelE are found along theplasmalemma of the cell wall (Y Bertheau & B Vian, personal communication),confirming a possible specialization of each isoenzyme toward different pecticpolymers. The capacity to attack a wide range of plants may necessitate thepresence of several isoenzymes that exhibit minor differences in their abilityto recognize and cleave pectin. All these enzymes can act cooperatively in thebreakdown of various pectic polymers. Indeed, a synergism in pectate lyaseaction toward pectin was recently observed inE. carotovora(6).

Catabolism of Monomers Derived from Pectin CleavageE. chrysantheminot only is able to cleave the pectic polymers, but also can usethem as carbon and energy sources for growth. Breakdown of pectic polymersresults in the formation of two kinds of monomers,D-galacturonate and DKI,which are catabolized by two independent pathways leading to a common inter-mediate, namely 2-keto-3-deoxygluconate (KDG) (Figure 1). The biochemicalcharacterization of the galacturonate catabolic pathway was first reported forE. carotovora(73) and a few years later forE. coli (3). The intracellularsteps involved in DKI catabolism were first characterized in a phytopathogenicPseudomonasspecies (115). The more recent work done inE. chrysanthemiconfirmed the presence of similar pathways in this bacterium. DKI, galactur-onate, and glucuronate are converted to KDG, which is then phosphorylatedand cleaved to give intermediates of general cellular metabolism (Figure 1).

The main monomer produced after the action of pectate lyases and oligogalac-turonate lyase on pectin is DKI. Moreover, polygalacturonase activity is verylow in E. chrysanthemi,and only small amounts of galacturonate are generatedfrom pectin degradation. The galacturonate catabolism is thus of secondaryimportance for the growth ofE. chrysanthemion pectic polymers. Indeed,mutations in DKI catabolism genes (kduD, kduI) prevented growth on PGA asthe sole carbon source, whereas mutants with altered galacturonate catabolismgenes (uxaC, uxaB, uxaA) are capable of normal growth on pectin or PGA (28).

Because the first steps of pectin degradation are extracellular, the intracel-lular catabolism depends on the permeability of the membrane to the externalsubstrates. Oligogalacturonides are known to enter the cells (24), and a digalac-turonate transport system has been recently described (131a). Two transportsystems mediate entry of monomers: ExuT, for galacturonate and glucuronateuptake, and KdgT, for KDG uptake (Figure 1) (25, 29, 61, 131).

Cloning of large chromosomal fragments on R-prime plasmids and muta-tional analysis established that the genes involved in these pathways are orga-nized in five clusters on theE. chrysanthemichromosome (Figure 2) (63). Onelocus contains the genesogl, kduI,andkduD(31, 118). A second locus containsthe genesexuT, uxaC, uxaB,anduxaA(65, 149, 150), andkdgK, kdgA,andkdgT

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are located in three different loci. These genes constitute independent transcrip-tional units, except for the threeuxagenes that form theuxaCBAoperon.

THE exuTTRANSPORT SYSTEM E. chrysanthemicontains an active transportsystem encoded by theexuTgene that can mediate uptake of galacturonateand glucuronate (46, 61, 131, 150). This gene has been cloned from twoE.chrysanthemistrains, B374 and EC16 (46, 61, 131, 150). AnE. chrysanthemiexuTmutant was unable to grow on galacturonate, and thus ExuT may be the solesystem by which galacturonate enters the cells. TheexuTexpression is inducedby galacturonate but not by glucuronate. In consequence,E. chrysanthemiwild-type strains cannot use glucuronate as a carbon source for growth becauseof this lack of induction. However, mutants capable of using glucuronate as acarbon source have been isolated (65). This ability is linked to the constitutivityof exuTexpression (see below).

THE kdgT TRANSPORT SYSTEM In E. chrysanthemi,the kdgT gene encodesan active transport system responsible for the uptake of DKI, 2,5-diketo-3-deoxygluconate [or 3-deoxy-D-glycero-2,5-hexodiulosonate (DKII)], and KDG.It can also mediate the entry of glucuronate, although with low affinity (29).DKII uptake was also observed in strain EC16 (24).kdgTwas cloned fromstrains B374 and 3937 through complementation of anE. coli kdgTmutationusing a RP4 derivative plasmid (2, 29, 150).

The 3937 strain can grow on DKI or DKII as the sole carbon source butnot on KDG. In mutants able to grow on KDG, transcription of thekdgTgeneis derepressed.E. chrysanthemi kdgTmutants cannot grow on any of thesesubstrates, which indicates that KdgT is the only system for DKI, DKII, orKDG to enter the cells (29). Failure to grow on KDG can be attributed totwo factors: The basal level ofkdgTtranscription is very low, and KdgT has alow affinity for KDG. ThuskdgTtranscription is not induced in the presenceof extracellular KDG. The high affinity of KdgT for DKI and DKII probablyallows accumulation of inducing quantities of substrates, derepression of thekdgTtranscription, and hence growth on these compounds.

DEOXYURONATE ISOMERASE The isomerization reaction is the least well-characterized step of pectin catabolism. Preiss & Ashwell (115) purified from aPseudomonasspecies a fraction with isomerase activity that converted DKI toDKII. A similar catabolic step was generally believed to occur inE. carotovoraandE. chrysanthemi.However, amongE. chrysanthemimutants unable to growon PGA, no mutation affecting this step has been found. Genetic analysis of theDNA region near theogl gene revealed that it contains other genes involved inpectin catabolism (31, 118). One of these genes was identified as the putative

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gene of desoxyuronate isomerase,kduI,since the corresponding mutant couldnot grow on PGA or digalacturonate but could grow on DKII.

KDG OXIDOREDUCTASE KDG oxidoreductase converts DKII into KDG with aconcomitant oxidation of NADH. This enzyme, first discovered in a pectinolyticPseudomonasspecies (115), was also found inE. chrysanthemi(27). Mutants ofE. chrysanthemialtered in KDG oxidoreductase production cannot use PGA asa carbon source for growth (22, 28). ThekduDmutation also prevented growthon digalacturonides but did not affect growth on galacturonate. ThekduDgeneis located approximately 30 kilobases (kb) from theogl gene, and some of theR-prime plasmids selected for the complementation of anogl mutation alsocontain thekduDgene (31, 118).kduD is located downstream fromkduI andis transcribed in the same direction.

KDG KINASE The KDG kinase catalyzes the phosphorylation of KDG into6-phospho-KDG (KDGP) (Figure 1).E. chrysanthemi kdgKmutants cannotuse galacturonate, glucuronate, or PGA for growth. Furthermore,kdgK mu-tants are unable to metabolize KDG, which accumulates in these cells (64). TheE. chrysanthemi kdgKgene was cloned by complementing anE. coli kdgKmu-tation using a RP4 derivative (60).kdgKwas localized within 80 kb of theuxuAanduxuBgenes involved in glucuronate catabolism and resides approximately4 kb from thecelYgene coding for anE. chrysanthemicellulase.

KDGP-ALDOLASE The KDGP-aldolase catalyzes an aldol cleavage of KDGPto form pyruvate and glyceraldehyde-3-phosphate (Figure 1). This enzyme isin fact multifunctional and can use various substrates, such as oxaloacetate or2-keto-4-hydroxyglutarate (KHG) (33). The action of KDGP-aldolase sub-strates affects cellular functions in that KDGP is a toxic metabolic intermediateand glyoxalate (a product of KHG cleavage) and KHG inhibit enzymes of thecitric acid cycle. InE. coli, the activity of KDGP-aldolase occurs at a branchpoint where the catabolism of gluconate and hexuronic acids converges, and itis one of the key enzymes of the Entner-Doudoroff pathway (33). Because ofits role in the Entner-Doudoroff pathway, theE. coli gene of KDGP-aldolasewas namededa.

E. chrysanthemi kdgAmutants cannot use galacturonate, glucuronate, orpectin as a carbon source for growth (64). Moreover, the addition of one ofthese substrates to a culture containing another carbon source inhibits growthbecause toxic KDGP accumulates in the cell. InE. chrysanthemi, kdgAis notinvolved in gluconate catabolism, an observation suggesting the absence ofthe Entner-Doudoroff pathway in this bacterium (68). ThekdgAgene of theE. chrysanthemistrain 3937 was cloned by complementing anE. coli kdgAmutation (68). ThekdgA structural gene begins 153 bases downstream of

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an open reading frame (ORF) highly homologous to theE. coli zwfgene en-coding glucose-6-phosphate dehydrogenase. In Gram-negative bacteria, genesinvolved in glucose metabolism, such asedaor zwf,are often clustered (33),but organization of these genes varies greatly depending on the bacteria.

The Steps of Galacturonate and Glucuronate CatabolismIn E. chrysanthemi,galacturonate is catabolized in a pathway similar to thatfirst described inE. coli (113, 124). After its transport into the cell via the ExuTtransport system, KDG is formed through the successive action of the uronateisomerase, encoded byuxaC;the altronate oxydoreductase, encoded byuxaB;and the altronate hydrolyase, encoded byuxaA(Figure 1).E. chrysanthemicanuse galacturonate as a carbon source for growth, but it cannot grow on the otherhexuronate, glucuronate, except in mutants with derepressed expression of theExuT transport system. Glucuronate is catabolized via a pathway consisting ofthree steps, as is galacturonate catabolism. After action of the UxaC isomerase,the pathway involves two specific enzymes, the mannonate oxydoreductase andthe mannonate hydrolyase, which are encoded byuxuBanduxuA,respectively.

E. chrysanthemi uxaCmutants can use neither galacturonate nor glucuronatefor growth. Only galacturonate is unusable byuxaAanduxaBmutants, whereasonly glucuronate is unusable byuxuAanduxuBmutants (Figure 1). Growth ofany of these mutants on pectin or polygalacturonate is normal. The organizationand localization of these genes inE. chrysanthemihave both similarities anddifferences with these features inE. coli (65, 113, 124, 149, 150). InE. coli,exuTis located nearuxaCAat 67 min on the chromosome;uxaBis not linked tothis cluster and lies at 52 min. InE. chrysanthemi, uxaBis linked to theexu-uxacluster, which is organized in two operons,exuTanduxaCBA(Figure 2). InE.chrysanthemi, uxuAanduxuBconstitute two independent transcriptional unitslocated near thekdgK gene (Figure 2), whereas inE. coli they constitute anuxuABoperon located at 98 min.

STIMULI AFFECTING PECTINASE PRODUCTION

Studies comparing pathogenic and nonpathogenic pectinolytic bacteria havesuggested that differences in the rates of synthesis and secretion of pectinasesmight be crucial in the elicitation of tissue-macerating diseases (21, 154). For in-stance, in nonphytopathogenic bacteria such asKlebsiella pneumoniae, Yersiniapseudotuberculosis,andYersinia enterocolitica,levels of pectate lyases are lowcompared with those ofErwinia species, and their synthesis is not induced inthe presence of pectin (21). Thus to understand the expression of virulencein soft-rot bacteria, we must consider the physiological factors controlling thesynthesis of pectinases. Such an analysis conducted in bothE. chrysanthemiandE. carotovoraled to similar conclusions. Ecological factors favoring the

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development of the soft-rot diseases very often stimulate pectinase production(108). These factors also affect bacterial growth but usually in opposite ways.Pectinase synthesis is stimulated by stress conditions rather than by favorablegrowth conditions (59, 107).

In E. chrysanthemi,the presence of pectin is the main signal required forinduction of the expression of all the genes of pectinolysis. In addition, the tran-scription of pectinase genes is subject to other physiological controls, includinggrowth phase–dependent induction; catabolite repression; and variations in en-vironmental conditions, such as the presence of plant extracts, temperature,anaerobiosis, iron limitation, osmolarity, or nitrogen starvation (15, 59, 132).These conditions do not act in synergy, and response to them probably requiresseveral independent regulatory mechanisms. Some environmental stimuli af-fect not only pectinase production but also that of other extracellular enzymes,namely cellulases and proteases, which permits a coordinate synthesis of allthe plant cell wall–degrading enzymes. Pectin lyase synthesis responds to to-tally different stimuli;pnl expression is induced in response to DNA-damagingagents (147).

Regulation analysis was performed by using transcriptional fusions in eachgene of the pectinolytic pathway (28, 31, 59, 60, 68, 120). Such fusions areparticularly important in thepel genes since the total pectate lyase activityresults from the cumulative action of at least seven enzymes. When followedindividually, the distinctpelgenes each respond differently to the same signalsand sometimes exhibit opposite behaviors. Comparison of the basal level ofeachpel fusion (59) revealed the major role played bypelE in the productionof pectate lyase in the absence of pectin. ThepelAgene is weakly transcribedin synthetic medium whatever the conditions tested, whereas thepelBandpelCgenes are expressed at intermediate levels. ThepelDexpression has a low basallevel but is strongly inducible. This high inducibility allows a greater degreeof pelD expression than that of the otherpel genes. Generally,pelD andpelEgenes are more sensitive to variations in the environment than otherpel genes(59). The predominant role of basic isoenzymes during infection could beexplained by (a) the high basal level ofpelEexpression, which allows the rapiddegradation of plant pectin before the induction of plant defense mechanisms,and (b) the strongly induced expression ofpelD, which results in the highpectinase activity necessary for the generalization of infection.

Expression of Pectinase Genes in PlantaTranscriptional fusions with the six genes of major pectinases (pelA–EandpemA) were used to study the genes’ expression after plant infection (39, 83,87). In these experiments, the reporter geneuidA (or GUS) was used since itsproduct is totally absent in plant tissues and is thus a useful tool for analysis

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of gene expression during plant-microbe interactions. The first plant testedwas potato (Solanum tuberosum), which has distinct types of tissues (tubers,leaves, etc) and diverse varieties with different levels of sensitivity to soft-rotdisease. During infection of susceptible potato tubers (83), pectinase genescould be separated into two groups on the basis of their expression level: (a)the moderately expressed genes,pelA, pelB, pelC,andpemA,and (b) the highlyexpressed genes,pelD and pelE. Expression of all pectinase genes, exceptpelA, in potato tubers was similar to that obtained in synthetic medium underinducing conditions. Expression of thepelAfusion was 23-fold higher in tubersthan in a synthetic medium, suggesting that at least one plant factor modulatesthe expression of this gene. In contrast, inoculation of potato plantlets didnot stimulatepelAexpression (83). These results indicate that stimulation ofpelAexpression is tissue specific. In a resistant line of potato tubers, nopelAinduction was observed, whereaspelEexpression partially decreased andpemAexpression was stimulated (39). Thus expression of each pectinase gene couldbe modulated as a function of the composition of the infected tissue.

In the natural host plant of the 3937 strain,S. ionantha,only thepelDgene washighly expressed. The expression ofpelE, pelB,andpelCwas moderate, and thepelAexpression was weak (87). The kinetics ofpel expression were analyzedduring the early phase of the disease. All thepel genes were induced 9 h afterinfection, exceptpelD,which was turned on earlier, after 6 h. Comparison oftheSaintpauliaand potato systems clearly demonstrates that thepel genes aredifferentially regulated depending on the host plant. This differential regulationcould result either from biochemical signals, such as the presence of inducingmolecules, or from biophysical conditions linked to the microenvironment inplanta, such as osmolarity or pH.

Induction by Pectin-Degradation IntermediatesIn E. chrysanthemi,the synthesis of pectate lyases, polygalacturonase, andpectin methylesterases is induced approximately 40-fold in the presence of PGA(24, 59). The inducer is not PGA itself but its breakdown products, presumablyinitially generated by the basal level of pectate lyases (145). Indeed, pectinasesynthesis is induced in the presence of galacturonate, saturated digalacturonate,or unsaturated digalacturonate (24). The analysis of mutants that are deficientin each step of the pectinolytic pathway allowed for the identification of thetrue intracellular inducers, namely KDG, DKI, and DKII (Table 1). Inoglmutants, digalacturonates no longer induce pectinase synthesis, demonstratingthat the formation of DKI or DKII is necessary for induction in the presence ofoligomers (22, 24, 120). Similarly, in anuxaAmutant, pectinase synthesis isno longer inducible in the presence of galacturonate, demonstrating that KDGformation is necessary for induction in the presence of galacturonate (65). In

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kdgKmutants, which accumulate KDG intracellularly, pectinase production isstrongly induced in the presence of either PGA or galacturonate, confirmingthat KDG is a true inducer (65). InkduDandkduImutants, which accumulateDKII and DKI, respectively, pectinase synthesis is also strongly induced in thepresence of PGA, so these compounds are also true inducers (22, 28).

Individual investigation of the regulation of each gene of the pectinolyticpathway (pemA,B; pelA–E,L; pehX; ogl; kduD; kduI; kdgT; kdgK; kdgA)demonstrated that they are all induced in the presence of PGA and that they areall sensitive to the intracellular inducers DKI, DKII, or KDG. The isolation ofregulatory mutants overproducing pectate lyases and constitutively expressingall genes of the catabolic pathway led to the identification of thekdgRregulatorygene, which mediates induction by KDG, DKI, and DKII. The characteristicsof this regulation, which ensures coordinate control of pectin catabolism, aredetailed below. However, the residual induction of pectate lyase synthesis ob-served in a doublekdgR-kdgKmutant in the presence of PGA or galacturonatedemonstrated that other regulatory proteins respond to KDG (65). Althoughits expression is inducible by PGA, the pectate lyase genepelL is not regulatedby kdgR(84), confirming that KdgR is not the only regulator triggering PGAinduction.

Other Inducers from Plant ExtractsPart of the induction observed in planta could result from signaling moleculesnot originating from pectin catabolism but present in the plant tissue. Carrot rootextracts induce pectate lyase synthesis synergistically with PGA (15). Prelimi-nary characterization of the inducing factor present in plant extract revealed thatit is a low-molecular-weight (350–1000 Daltons), thermoresistant compoundthat displays no affinity for hydrophobic-interaction columns. Such propertiessuggested that the inducing factor could be a glucidic molecule composed oftwo to six residues (15). A similar induction by carrot root extracts was ob-

Table 1 Effect of mutations in pectinolysis on pectate lyaseinduction

Accumulated Induction in the presence ofGenotype intermediate galacturonatea PGAa

Wild-type + +uxaA Altronate − +ogl Digalacturonate + −kduI DKI + +++kduD DKII + +++kdgK KDG +++ +++

a−, no induction;+, normal induction;+++, super-induction.

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served for polygalacturonase synthesis inAgrobacterium tumefaciens(125).The inducer is a complex pectic polysaccharide that contains both arabinoseand galacturonic acid and is approximately 100-fold more active than is PGA.Such a complex pectic oligosaccharide could be an inducer of pectate lyasesynthesis inE. chrysanthemi.

Catabolite RepressionPectate lyase production inErwiniaspecies is subjected to cyclic AMP (cAMP)-controlled catabolite repression (58). Catabolite repression can be observedduring growth in the presence of glucose and also in the presence of pectincatabolic products. For instance, high concentrations of unsaturated digalac-turonate exert cAMP–reversible self-catabolite repression on pectate lyase pro-duction (146). Similarly, galacturonate mediates both induction and cataboliterepression of pectinase synthesis. Moreover, the spectrum of pectinases pro-duced byErwinia strongly depends on the nature of the carbon source presentin the growth medium, which suggests that individual genes are differentiallysensitive to catabolite repression (137). Catabolite repression is conservedwhen pectate lyases are expressed inE. coli,so the catabolite activator proteins(CRP) are probably interchangeable between the two bacterial genera (68a,70). Molecular cloning of theE. chrysanthemi crpgene, encoding CRP, wasrecently performed by means of complementation of anE. coli crp mutation(W Nasser, unpublished data). TheE. chrysanthemiCRP, which is 210 aminoacids long with a molecular mass of 24 kDa, displays 97% identity with theE.coli CRP. An E. chrysanthemi crpmutant still catabolizes glucose but loses itsability to grow on several carbohydrates, including PGA, galacturonate, glyc-erol, mannitol, and galactose. Thecrp mutant is also deficient in pectate lyaseproduction. Similarly, acyamutant ofE. carotovorathat cannot produce cAMPis defective in pectate lyase synthesis (95). Mutants ofE. chrysanthemiB374,in which extracellular enzyme production became insensitive to the cataboliterepression, were designated ascri (62). InE. coli, mutations allowing expres-sion of catabolite-sensitive operons in the absence of CRP and cAMP werelocalized in therpoD gene, which encodes the sigma-70 factor (144). Thecrimutations ofE. chrysanthemimay also affect the transcription apparatus.

Stationary-Phase InductionAnalysis of genetic fusions demonstrated that in the late exponential growthphase, when the bacterial population has reached its maximum, the genespemA,pemB, pelA, pelB, pelC, pelD, pelE, pelLandoutare expressed in levels 5- to 60-fold higher than during early exponential growth phase (26, 59, 84, 135). Thistiming correlates with the observation of soft-rot symptoms only after extensivebacterial multiplication. Expression ofE. chrysanthemi pelgenes cloned intoE. coli is still induced in the late exponential growth phase, indicating that the

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mechanism responsible for this regulation is conserved inE. coli (67, 68a). InE. coli, the expression of genes induced in the stationary-growth phase requiresa specific sigma factor, the product of therpoSgene (75). However, pectatelyase expression remains growth phase dependent inrpoSmutants, so therpoSproduct is probably not necessary for pectate lyase expression inE. coli (67).In several Gram-negative bacteria, a regulation similar to that mediated by theLuxI-LuxR system ofVibrio fischeri(89) is responsible for the growth-phaseinducibility. The LuxR family comprises regulators that respond to small dif-fusible molecules related to homoserine lactone (HSL) with a variableN-linkedacyl side chain. InE. carotovora,production of all extracellular enzymes isinduced byN-(3-oxohexanoyl)-HSL, which is synthesized by the action of theExpI protein (a LuxI homologue) (69, 110). In binding to the ExpR regulatoryprotein (a LuxR homologue), acyl-HSL can activate the expression of both theexoenzyme genes and theexpIgene. Because acyl-HSL induces the productionof ExpI, the system is auto-induced. At high cell density, acyl-HSL concen-tration reaches a sufficient level to fully activate ExpR. Acyl-HSL productionpeaks during the transition between the late log phase and the stationary phaseof growth, a period corresponding to the maximal induction of exoenzymesynthesis.

Acyl-HSL is a widespread regulatory signal; it controls various mechanismsin species ofVibrio, Pseudomonas, Agrobacterium, Rhizobium, Yersinia,andErwinia (129). Pseudomonas aeruginosaemploys distinct acyl-HSL and mul-tiple LuxI-LuxR homologues to coordinate the synthesis of virulence factors(76). Thelux box, identified as the LuxR binding site, also appears to repre-sent a conserved regulatory element of genes regulated by cell density (52).No sequence displaying homology with thelux box was found upstream oftheE. chrysanthemi pelgenes. Homologues of theexpIandexpRgenes wererecently isolated fromE. chrysanthemi3937 (W Nasser, unpublished data).Whether acyl-HSL may also mediates the growth-phase dependence of pectatelyase synthesis in this species remains to be tested. Mutants ofE. chrysanthemiB374, in which extracellular enzyme production became independent of thegrowth phase, were designatedgpi (62). According to the ExpI-ExpR model,gpi mutants may constitutively produce the acyl-HSL signal.

OsmoregulationMedium osmolarity is an environmental factor that acts on most living organ-isms. The inhibitory effect of osmolarity can be reversed by molecules designedas osmoprotectants, such as glycine betaine and proline (34). In bacteria, os-molarity and osmoprotectants influence cellular growth and various metabolicprocesses as well as the expression of virulence determinants. For instance,osmolarity affects the expression of the cholera toxin ofVibrio choleraeand

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of the invasion geneinvA of Salmonella typhimurium(47, 94). Plants are animportant source of osmoprotectants in nature (121), and osmoprotectants alsooccur in soil and sediments (49). For phytopathogenic bacteria, osmoprotectioninfluences both bacterial survival in soil and the plant infection process.

Early reports demonstrated that growth and pectinase production ofE. chrys-anthemiare inhibited when water activity is reduced (92, 93), and recent studiesconfirm that elevated osmolarity inhibits the growth ofE. chrysanthemi(51,116). Exogenously provided osmoprotectants, such as glycine betaine, pro-line, ectoine, pipecolate, carnitine, or dimethyl glycine, permitted growth toresume at an inhibitory osmolarity (51, 116). These osmoprotectants accumu-late inE. chrysanthemicells via specific transport systems (51). Osmolarity ofthe medium also influences pectate lyase production. An increase in mediumosmolarity inhibits the secretion of pectate lyases, decreasing the extracellularpool with a concomitant periplasmic accumulation (91, 92). InE. chrysanthemistrain 3937, the total pectate lyase production increased at moderate osmolar-ity, then shifted to a basal level at higher osmolyte concentrations (51). Thisincrease of pectate lyase activity was attributed to the transcriptional inductionof the major pectate lyase gene,pelE.Expression ofpelA, pelB,andpelCwasnot affected by elevated osmolarity, and that ofpelD andpelL was weakly re-pressed (59). The presence of osmoprotectants in the medium prevented theinduction ofpelEat high osmolarity (51). In conclusion, osmolarity influencesbacterial growth and PelE production in opposite ways (51). The osmolarityof the bacterial environment probably increases after degradation of plant cellwall constituents. The induction ofpelEat high osmolarity could result froman adaptive mechanism compensating for the inhibition of growth. The releaseof osmoprotectants from the macerated plant tissue may allow the restorationof the bacterial growth.

In V. cholerae,the osmoregulation of different virulence determinants ap-peared to be mediated through two specific regulatory genes,toxRand toxT(38). In S. typhimurium,various factors influencing supercoiling affect theosmoregulation of theinvAgene (47). InE. coli,mutations affecting osmoreg-ulation were isolated in theosmZlocus–an allele of thehnsgene, which encodesthe histone-like protein HNS (56). HNS is involved in the expression of manyvirulence genes but is probably not directly involved in a specific type of regu-lation, such as osmoregulation. Instead, it plays a role in a general adaptationof the bacterial metabolism to stress conditions. Analysis ofpel expression inE. coli hnsmutants suggested that the HNS protein modulates the expression oftheE. chrysanthemigenespelA–E,but this effect is not connected to osmoreg-ulation (V James, personal communication). A more specific regulation mustexist to explain the strong effect of osmolarity onpelEexpression.

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ThermoregulationTemperature is a primary environmental factor affecting the virulence of pecti-nolytic Erwiniae (107). It differentially influences the bacterial growth andpectinase production: Pectinase production decreases when temperature risesabove the optimal growth temperature and vice versa. InE. chrysanthemi,pec-tate lyase synthesis increased approximately sevenfold at 25◦C and decreasedthree- to sevenfold at 37◦C, compared with synthesis levels at 30◦C (59). Ther-moregulation is more pronounced on thepelA, pelE, pelDlocus than on genesof thepelB, pelC. As expression of clonedpelgenes inE. coli is not temperaturedependent (68a), thermoregulation is not conserved inE. coli. In Bordetellapertusis,thermoregulation of a set of virulence genes is coordinated by theproducts ofbvgAandbvgS(90), which encode a two-component regulatorysystem that responds to modulators such as temperature, SO4, and nicotinate(126). Thermoregulation of virulence determinants in other bacteria dependson thehnsgene. A mutatedhnsgene affected thermoregulation of invasiongenes inShigella flexneriinfluenced Pap pilus production inE. coli (54, 88).Whether thermoregulation of pectate lyase synthesis inErwinia species is de-pendent on HNS or is mediated by a specific sensing system remains to betested.

Oxygen and Nitrogen AvailabilityThe incidence and severity ofErwinia soft rot increases under low oxygen con-centration (36, 108). Anaerobic conditions inhibit plant resistance and increasethe production ofErwinia pectinases but disfavor growth of the facultativeanaerobeErwinia. The switch between aerobic and anaerobic respiration de-pends on the presence of an appropriate alternative electron acceptor. In planttissue, nitrate is present in sufficient amounts to play this role. The proposedmodel supposes that anaerobic respiration on nitrate facilitates the proliferationof invading bacteria in the plant tissue (138). Indeed, augmentation of nitro-gen fertilization in fields favors bacterial soft rot (18). In addition, nitrogenstarvation inhibits expression of thepelgenes (59).

In E. chrysanthemi,pectate lyase synthesis increases about sixfold undersemiaerobic conditions (59), although the individualpel genes responddifferently. Expression ofpelA, pelD,and pelE is stimulated in anaerobio-sis;pelBandpelCtranscription is not affected; andpelLexpression is reduced.The increase ofpelexpression in semiaerobic conditions is conserved whenpelgenes are cloned inE. coli (68a). In this bacterium, anaerobiosis regulation ismediated mainly by thefnr gene product (53). However, in anE. coli fnr mu-tant, pectate lyase synthesis remained at an increased level under semiaerobicconditions, indicating that the FNR protein is not necessary for the induction

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of pel expression (68a). Mutants affecting theE. chrysanthemi hmpXgene(previously namedpecX), which is adjacent to thepelA, pelE, pelDregion,synthesize reduced amounts of pectate lyases under low oxygen tension (43).ThehmpXgene codes for a flavohemoglobin that has two distinct regions, anN-terminal hemoglobin domain and a C-terminal flavin-containing reductaseregion. This protein could provide a new electron pathway to the bacteriumin the plant’s oxygen-limiting environment (43). The regulatory effects of thehmpXmutations could be explained by a defect in oxygen status sensing. EitherHmpX has a direct sensing role, or a secondary effect results from mutationsin hmpXthat disturb the anaerobic metabolism.

Iron DeprivationIron has a central role in bacterial infections of animals, not only in iron trans-port functions but also in the synthesis of virulence factors such as toxins andextracellular enzymes (106). A parallel can be drawn for the phytopathogenicE.chrysanthemi.During infection,Erwiniacells multiply in the intercellular fluidof plants, which is very poor in available iron. In response to iron-deprivation,E. chrysanthemisynthesize two siderophores, chrysobactin and achromobactin(85, 86, 109). Chrysobactin is a stronger iron ligand than achromobactin. Thechrysobactin-dependent iron transport pathway entails about 25 functions en-coded from a single region of the chromosome and involved in the biosynthesisand excretion of the siderophore and in the uptake of the siderophore ferric com-plex (45). If any of these functions are impaired by insertional mutation, themutant fails to incite a systemic disease inS. ionantha(41). A low iron level alsoinduces pectate lyase synthesis independently of the presence of PGA (132).ThepelB, pelC, pelE,andpelLgenes are induced under limited iron deprivation,whereaspelD is only induced under severe iron starvation (87). A mutation inferri-achromobactin uptake (cbr) that provokes limited iron deprivation resultsin chrysobactin synthesis derepression, thus permitting iron uptake (85). Thecbrmutant is then less susceptible to iron deprivation than the wild-type cells inplanta. InS. ionanthainfected withcbrmutants, pectate lyase production by thebacteria and symptom development in the plant are delayed (132). This resultsuggests that iron level is probably one of the first signals to induce pectate lyasesynthesis recorded byE. chrysanthemi(87). Iron sensing inE. chrysanthemiseems to be partially mediated by a Fur-like protein; in afur mutant ofE. coli,the chrysobactin operon is no longer iron regulated (133). However, the Furrepressor is probably not the only regulator involved in iron sensing.

Regulation ofpnl by DNA-Damaging AgentsIn E. chrysanthemiandE. carotovora,pectin lyase synthesis is subject to a formof control completely different from that of other pectinases. Expression ofpnl

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is induced by DNA-damaging agents such as mitomycin C, nalidixic acid, or UVlight, but it is not sensitive to PGA induction (147). InE. carotovora,RecA is re-quired for transcriptional activation of thepnlAgene (153). ThisrecA-mediatedregulation was initially thought to be dependent on the SOS regulon ofE. coli.However, subsequent studies revealed that the transcription ofpnlA was notcontrolled directly by LexA, the SOS repressor, but rather depended upon twogenes,rdgA andrdgB (81). In the proposed model, RdgB is a direct activatorof pnlA transcription, and its production depends on RdgA. Under noninduc-ing conditions, RdgA represses RdgB synthesis and preventspnlAexpression.In the presence of DNA-damaging agents, the activated proteolytic form ofRecA processes RdgA in a form that activatesrdgB expression, which in turnactivatespnlA expression (81). This regulation may be important inErwiniapathogenicity since the plant defense response includes the production of mu-tagenic compounds. The bacteria answer by inducing the SOS system to repairthe DNA damage and by producing an additional virulence factor, pectin lyase.

THE REGULATORY ELEMENTS

The response to the various stimuli received by the bacteria requires severalintervening elements. Changes in the environment must be transduced intoan understandable signal, frequently a small molecule that can be rapidly syn-thesized and degraded. Then this signal has to interact with a transcriptionalregulator, either directly or through sensor and transducer proteins. Such aninteraction will modify the affinity of the regulator for its DNA-binding sites,permitting an effect on the transcription of the controlled genes. The isola-tion of regulatory mutations is crucial for the identification of the elementsinvolved in the regulation mechanisms. This approach was used to investigatethe regulation of pectinolytic genes inE. chrysanthemi.Selection of up- ordown-regulated mutants covered the possibilities of both positive and negativecontrol. The genes belonging to the same regulon were detected by their abnor-mal expression in the regulatory mutant. Often the main difficulty was to findthe stimulus and the signal triggering the regulation altered by the mutation.Several loci involved in the regulation ofE. chrysanthemipectinolytic geneswere characterized by the mutant analysis (Table 2).

Regulation of theexu-uxa-uxuGenesThe regulatory genes acting on galacturonate and glucuronate catabolism inE. chrysanthemiwere studied to identify possible connections between theregulatory mechanisms governing these pathways and pectin catabolism. Genesinvolved in galacturonate utilization are under the control of theexuRgeneproduct, which probably acts as a negative regulator since inactivation ofexuRleads to constitutive expression of the genesexuT, uxaC, uxaB,anduxaA(65).

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Tabl

e2

Prot

eins

invo

lved

inth

ere

gula

tion

ofpe

ctin

olys

isin

Erw

inia

spp.

Md

rFa

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orN

ame

Spec

iesa

Invi

vosp

ectr

umSi

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c(k

Da)

hom

olog

ues

Reg

ulat

ory

func

tione

Ref

eren

ces

Aep

HE

cape

l,pe

h,ce

l,pr

t?

0.5

Acc

esso

ryfa

ctor

?98

,130

CR

PE

ca,E

chpe

l,su

gar

cata

bolis

mcA

MP

24T

rans

crip

tiona

lact

ivat

orW

Nas

ser,

unpu

blis

hed

data

Cya

Eca

,Ech

pel,

suga

rca

tabo

lism

cAM

PN

Df

Sign

albi

osyn

thes

is58

,95

Exp

IE

ca

pe

l,pe

h,pn

l,ce

l,pr

i,ex

pI...

acyl

-HSL

25L

uxIf

amily

Sign

albi

osyn

thes

is69

,110

Exp

RE

ca

pe

l,pe

h,pn

l,ce

l,pr

i,ex

pI...

acyl

-HSL

28L

uxR

fam

ilyT

rans

crip

tiona

lact

ivat

or?

TPa

lva

Exu

RE

chex

uT,u

xaC

BA

gala

ctur

onat

eN

DT

rans

crip

tiona

lrep

ress

or?

65H

NS

Eca

,Ech

pel..

.?

ND

Glo

balr

egul

ator

WN

asse

r,un

publ

ishe

dda

taIH

FE

chpe

l...

?N

DG

loba

lreg

ulat

or40

Kdg

RE

chpe

lA,B

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, E;

pem

A,B

;ou

tC;

KD

G,D

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35G

ylR

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R,

Tra

nscr

iptio

nalr

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ssor

29,3

0,62

, 100

,10

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8og

l;kd

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kdgK

;kd

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DK

IIPo

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Out

TE

chou

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ator

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Z,o

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Mem

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4,11

9Pe

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chpe

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rR,S

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crip

tiona

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4,11

9M

arR

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cTE

chpe

lC,D

,E,L

,cel

l?

35Ly

sRfa

mily

Tra

nscr

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ssor

?13

9asu

rfac

epr

oper

ties

PecY

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pem

A?

6810

;NC

otte

-Pat

tat,

unpu

blis

hed

data

PehR

Eca

pehA

?N

DT

rans

crip

tiona

lact

ivat

or?

127

Rec

AE

ca,E

chpn

lAD

NA

dam

age

ND

Rdg

Aac

tivat

ion

81R

dgA

Eca

pnlA

DN

Ada

mag

e27

880

cI,4

34cI

,R

egul

ator

�of

rdgB

?81

λcI

,Lex

A,P

rtR

Rdg

BE

capn

lAD

NA

dam

age

14M

uM

or,M

uC

Act

ivat

orof

pnlA

?81

Rsm

AE

ca,E

chpe

l,pe

h,ce

l,pr

t,ex

pI,c

ells

urfa

cepr

oper

ties

?6.

8C

srA

Dec

reas

eR

NA

stab

ility

?19

,35

Uxu

RE

chux

uAFr

uctu

rona

te?

ND

Tra

nscr

iptio

nalr

epre

ssor

?65

a Eca

,Erw

inia

caro

tovo

ra;

Ech

,Erw

inia

chry

sant

hem

i.

c?

indi

cate

sun

dete

rmin

edsi

gnal

.dM

rar

ede

duce

dfr

omse

quen

cing

data

for

the

mon

omer

icpr

otei

n.T

hena

tura

lfor

mof

Kdg

Ran

dPe

cSis

adi

mer.

.e?

deno

tes

prop

osed

butn

otde

mon

stra

ted

regu

lato

ryac

tivity

.f N

D,n

otde

term

ined

.

?

b

cel a

nd p

rt: g

enes

enc

odin

g ce

llula

ses

and

prot

ease

s, r

espe

ctiv

ely.

b

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Intermediates of the pathway (galacturonate, tagaturonate, or altronate) arethe direct inducers of the ExuR regulon. TheexuRgene does not affect theexpression of the genes involved in the catabolism of glucuronate, KDG, orpectin.

The two genes specifically involved in glucuronate catabolism,uxuA anduxuB,are independent transcriptional units under different controls. TheuxuBgene appeared to be constitutively expressed. In contrast, the presence of glu-curonate, fructuronate, or mannonate inducesuxuA,an observation suggestingthat a regulatory gene,uxuR,controls theuxuAexpression (65). The inducers ofuxuAexpression completely differ from those of pectinolysis or galacturonatecatabolism, indicating independent regulatory circuits. The various interme-diates of the galacturonate or glucuronate catabolism do not inducepel geneexpression by themselves. They can act as inducers only after their transfor-mation into KDG.

The KdgR RegulonInduction of all the genes involved in pectinolysis and in pectate lyase secretionin the presence of PGA and galacturonate suggests a coordinated regulation ofthese genes. The genekdgRis responsible for this common regulation. Threedifferent selection procedures yieldedkdgRmutants: (a) Mutants able to growon KDG as the sole carbon source showed a derepressed expression ofkdgT(29). (b) A kduD-lacZfusion was used to obtain mutants with a derepressedexpression of the fusion (30). (c) An in situ detection test allowed selectionof mutants that synthesized increased levels of pectate lyases in the absenceof inducer (62). In fact,kdgRinactivation results in a derepressed expressionof all the steps of pectin catabolism (30). Genes regulated bykdgRcould beseparated into two groups according to their expression in akdgRmutant. Theexpression ofpemB, ogl, kduI, kduD, kdgT, kdgK, kdgA,andoutTis constitutiveand no longer inducible, whereas expression ofpemA, pelA, pelB, pelC, pelD,pelE,andoutC is derepressed but remains inducible in the presence of PGA.Comparison of the regulatory regions of the KdgR-controlled genes revealedthe existence of a conserved motif proposed as the KdgR-binding site (KdgRbox) (Figure 3) (31, 67, 117).

The kdgRgene resides in a region encoding several steps of pectinolysis,just downstream ofogl. These two genes form independent transcriptionalunits, although thekdgRpromoter overlaps the end of theogl coding sequence(118). KdgR is a 306–amino acid protein with a molecular mass of 35,029. It ishomologous with other regulatory proteins controlling catabolic pathways, suchas GylR, a regulator of the glycerol operon inStreptomyces coelicolor(139);IclR, a repressor of the acetate operon inS. typhimuriumandE. coli (103); andPobR, which controls the metabolism ofp-hydroxybenzoate inAcinetobacter

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Figure 3 Consensus of the KdgR box and alignment of the KdgR operators. Abbreviations: R,A or G; Y, C or T; N, A, C, G, or T. Underlined bases are in contact with KdgR, as deduced frommethylation interference experiments (100). In the sequence alignment, nucleotides that fit withthe consensus are shown in bold letters. Two distinct operators, O1 and O2, are present in frontof theogl gene. The divergently transcribedkduI andkdgFgenes share a common KdgR box. Invitro functionality of operators tested by means of gel retardation is indicated by+ or− signs (ND,not determined). For many genes, footprinting experiments confirm that KdgR binding protectsthe corresponding operator (100). The dissociation constants of the KdgR-operator complexes aregiven (note that forogl, the constant corresponds to both the O1 and O2 sites) (100).

calcoaceticus(37). The N-terminal part of all these regulatory proteins containsa region that could fold into a helix-turn-helix motif, a feature characteristicof DNA-binding proteins. However, these proteins share little homology withthe known transcriptional regulators that have a helix-turn-helix motif in theirN-terminal domain and consequently may constitute a new family of regulatoryproteins.

The native KdgR repressor was purified in the fraction corresponding to amolecular mass of 68 kDa, indicating that the natural form of KdgR is a dimer.Gel retardation assays showed that the purified repressor binds to a syntheticKdgR box (101), and physiological studies suggested that KDG, DKI, andDKII are the inducing molecules interacting with KdgR. In vitro, KDG can

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dissociate the KdgR-operator complex (100, 101). TheKm of KdgR for KDGvaried from 0.3 to 0.6 mM, depending on the operator (100). Analysis ofthe inducing properties of KDG analogues allowed for the determination of thestructure of the inducer recognized by KdgR: All the inducing molecules containthe motif COOH-CO-CH2-CHOH-C-C in a pyranic cycle (99). This motif isalso found in DKI and DKII, but a direct interaction of these two compoundswith KdgR has not been verified. Modifying the substituent on carbon 5 ofKDG has no influence on the inducing properties but renders the moleculesnonmetabolizable because the KDG-kinase is unable to phosphorylate them.In consequence, C5-KDG derivatives are gratuitous inducers of the expressionof the KdgR regulon’s genes (99).

Gel retardation and filter binding assays permitted in vitro analysis of theKdgR interaction with the operator regions of 11 genes involved in pectinolysis(pemA, pelA, pelB, pelC, pelE, ogl, kduI, kduD, kdgT, kdgK,andkdgA) and of2 involved in enzyme secretion (outTandoutC) (60, 100). In vitro, KdgR canbind to only nine of these operators (pelA, pelB, pelC, pelE, ogl, kduI, kdgT,kdgK,andoutT). DNaseI experiments revealed that binding of KdgR to its op-erators allowed for the protection of the KdgR box(es) present in the regulatoryregion of the controlled genes. These footprint experiments showed that theKdgR-protected regions usually overlap or reside close to the sequences corre-sponding to the−35 or−10 regions of the putative promoters. Thus the KdgRprotein and the RNA polymerase probably compete for adjacent binding siteson DNA, explaining that KdgR binding prevents gene expression. Alignmentof the different KdgR boxes showed that they correspond to imperfect invertedrepeats of about nine nucleotides with two strictly conserved regions, RAAA-TTTY, separated by five nucleotides. The moderate variations in the sequenceof individual sites allow some modulation in the strength of repressor bind-ing. Indeed, KdgR binds to its operators with different affinities. In general,the affinity of KdgR for an operator and the derepression of the correspondinggene in akdgRmutant are not strictly correlated. However, the KdgR repressordisplays the strongest affinity, 1.1×10−10 M, for thekdgTgene, which exhibitsthe strongest derepression, 500-fold. Similarly, the weakest affinity, 1.1×10−8

M, was observed for thepelE gene, which shows the smallest derepression,three-fold (100). The duplication of the KdgR box upstream of several genes(ogl, pelE, kdgT,andpelC) and the relative position of this operator within thedifferent regulatory regions (Figure 4) could affect how often KdgR occupiesthese operators and modulate the efficiency of repression. Duplication of oper-ators allows stronger repressor binding through cooperative interaction and canprovide for a more flexible response to changing environmental conditions (23).

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Although gene fusion studies proved that the expression ofkduD, pemA,kdgA,andoutC is controlled by KdgR, no direct interaction was detected invitro between the KdgR protein and the regulatory regions of these genes (100).Missing-contact experiments performed onpelEandogl operators allowed forthe identification of the precise nucleotides that make close contact with theKdgR protein. The refined KdgR box (Figure 3) is well conserved in theoperators that interact in vitro with the KdgR repressor. In contrast, this motifis degenerate in the operators that cannot interact in vitro with KdgR. Theseresults suggest that the KdgR protein acts through different mechanisms: KdgRdirectly controls the genes whose operators interact in vitro with the repressorand a more complex mechanism governs expression of the genes that cannotinteract in vitro with the KdgR protein. Two hypotheses explain the regulation

Figure 4 Simplified view of regulatory regions of some pectinolysis genes fromE. chrysanthemi3937. The main structural elements involved in the transcription ofpelA, pelB, pelC, pelD, pelE,andogl are indicated. Promoters ofpelB, pelC,andpelD (i.e. pelEof strain EC16) were identifiedby primer extension (50, 67). KdgR boxes ofpelA, pelB, pelC, pelE,andogl and CRP sites ofpelB, pelC, pelE,andoglwere confirmed by footprinting experiments (100; W Nasser, unpublisheddata). The other sites are proposed based on their homology with the consensus sequences. Theregion involved in transcription ofpelD (i.e. pelE of strain EC16) (dotted line) and the putativeactivator site were defined by deletion mutagenesis (50).

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of genes in this second group: (a) KdgR binding may require either a cofactor(s)or an additional regulatory protein(s). (b) the KdgR effect may be mediated viaa cascade of regulatory proteins. For example, the in vivo regulation of theoutCoperon by KdgR requires a functionaloutT gene product (32), whereasoutTexpression is directly controlled by KdgR (100). The proposed model is thatthe outC operon is activated by the OutT protein whose production is KdgRdependent (32). This apparent complexity may contribute to some specificcharacteristics concerning the regulation of the secretion operon.

Potential KdgR boxes found in the regulatory regions of genes involved inpectin degradation inE. carotovorasuggest that this species contains akdgRhomologue. TheE. chrysanthemiKdgR protein can interact in vitro with theE. carotovoraKdgR boxes (W Nasser, unpublished data). Moreover, the use ofantibodies directed against theE. chrysanthemiKdgR confirms the existencein E. carotovoraof a immunologically cross-reacting protein (W Nasser, un-published data). Recently, a protein involved in the regulation of exoenzymeproduction that had 26% identity with theE. chrysanthemiKdgR was found intheE. carotovorastrain GS101 (130). This protein does not cross-react withthe KdgR antibodies and, unlike KdgR, acts as a positive regulator to controlpectinase, protease, and cellulase production (W Nasser & G Salmond, unpub-lished data). The molecular mechanism of this additional regulator of the KdgRfamily remains to be determined.

The KdgR regulon includes at least 13 operons involved in pectinolysis orsecretion but also several genes of unknown function. Isolation oflac fusionswhose expression is induced in the presence of PGA allowed for the charac-terization of a set ofpgi (PGA inducible) genes (66). Some of these insertionmutants showed a reduced virulence in plants. Mutations inpgi sometimesaffect the pectinolysis genes. However, severalpgi genes do not seem to playa role in PGA metabolism and their functions remain to be determined. Nev-ertheless, most of these genes are controlled by KdgR and thus belong to theKdgR regulon.

Two additional genes of the KdgR regulon,kdgCandkdgF,were identifiedon the basis of the presence of a KdgR box in their regulatory region (31).kdgCresides downstream ofkduDand encodes a 47,059-Dalton protein that displayshomology with pectate lyases of the periplasmic family.kdgF is transcribeddivergently fromkduI,with which it shares a common KdgR box. The productof kdgFis a 12,423-Dalton protein that has no homology with proteins of knownfunction. A mutation inkdgFresults in reduced induction of pectate lyases inthe presence of PGA, suggesting that KdgF plays a role in pectin degradation(31). However, no pectinolytic activity could be associated with KdgF. Theseobservations indicate that KdgR has numerous targets and that its role may not

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be restricted to pectinolysis. Instead, it may be a more general regulator ofvirulence factors.

The PecS-PecM Regulatory CoupleIsolation of mutations altering pectinase synthesis led to the characterization ofthepecSlocus (119). Inactivation ofpecSresults in derepressed synthesis ofpectinases, cellulases, and secretion-machinery proteins. Moreover, thepecSmutant produces an extracellular, insoluble blue pigment not synthesized in thewild-type strain. This blue pigment is thought to be indigoidine, based on itsbiochemical characteristics. In contrast tokdgR,the inactivation ofpecSdoesnot affect expression of the intracellular steps of pectinolysis.

ThepecSgene was cloned by selection of an R-prime plasmid carrying theadjacent genesxyl andargG. Complementation of thepecSmutation by thisplasmid demonstrated that thepecSproduct actsin trans to modulate geneexpression. Insertion mutagenesis of this locus revealed two divergently tran-scribed genes,pecSandpecM,which have the same spectrum of action. How-ever, the level of derepression of the controlled genes is higher in thepecSmutant (15-fold) than in thepecMmutant (4-fold). Pectate lyase production inpecSandpecMmutants was still inducible in the presence of PGA, and the syn-ergistic effect of plant extracts was also conserved. Similarly, no modificationof the pectate lyase regulation in response to growth phase, temperature fluctu-ation, anaerobiosis, nitrogen starvation, or catabolite repression was observedin pecSandpecMmutants, which suggests that these two genes participate inthe pectinase and cellulase regulation in response to another unidentified signal.

Boccara et al (12) recently reported that incompatible infection of tobaccocells by the wild-typeE. chrysanthemistrain 3937 led to a hypersensitive re-sponse of the plant cells and to the secretion of a blue pigment by the bacteria.SincepecSandpecMmutants constitutively synthesize a blue pigment, thisfinding suggests that the PecM/PecS proteins may be involved in a reaction toa signal generated during the plant hypersensitive response, such as productionof phytoalexins, oxygen radicals, or pH variation.

ThepecSgene encodes a protein of 166 amino acids with a calculated molec-ular mass of 19,287 (119). This protein displays homology with a family ofsmall (20-kDa) regulatory proteins, including EmrR, SlyA, MarR, and HpcR(114). Although all these proteins are involved in the transcriptional regulationof various genes, none of them display obvious motifs, such as helix-turn-helix,leucine zipper, or zinc finger, known to be involved in protein-DNA interactions(105). The alignment of the five protein sequences reveals the conservation ofthe motif GX9DRRX5LT in the central part of the proteins. This short regioncould correspond to the DNA-binding domain of this new class of transcrip-tional regulators.

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ThepecMgene encodes a protein of 297 amino acids with a calculated molec-ular mass of 31,913. The predicted PecM protein displays the characteristics ofan integral membrane protein: It is largely hydrophobic with 10 potential trans-membrane domains. Subcellular fractionation confirmed that PecM is anchoredin the bacterial inner membrane, whereas PecS is located in the cytoplasm. Inthe proposed model, PecM is involved in the sensing and transduction of theexternal stimulus (119). Despite its putative role, PecM displays no similaritiesto any signal-transducing receptors and, more particularly, no similarities to thewell-defined sensor proteins present in the bacterial two-component regulatorysystems (14).

The native PecS repressor was purified in the fraction corresponding to amolecular mass of 40 kDa, indicating that the natural form of PecS is a dimer(114). Gel retardation assays demonstrated that the PecS protein can inter-act with the regulatory regions of several controlled genes (pelA, pelE, pelL,celZ,and theoutCoperon) with relatively low affinities (Kd−app close to 10−8

M). In all cases, three types of PecS-DNA complexes were observed. The C1complexes, characterized by the least-retarded migration and obtained with lowPecS concentrations, correspond to the binding of one PecS dimer. The C2 com-plexes result from the binding of two PecS dimers. The C3 complexes, whichexhibit a highly retarded migration, appear for saturating PecS concentrationsand correspond to the binding of many PecS dimers. Competition experimentsdemonstrated that C1 and C2 complexes result from specific binding to DNA,whereas the C3 complexes are the result of less specific interactions. Analysisof the PecS-binding sites in DNaseI protection experiments on specific com-plexes revealed no well-conserved sequences. The PecS-protected sequenceshave a high AT content, which suggests that the PecS repressor may not rec-ognize a precise motif but rather some of the structural features of the DNAtargets. The PecS-binding sites are not bent DNA regions, nor are they bent byPecS binding (114). They either overlap the promoters (pelE) or are locateddownstream from the promoters of the controlled genes (pelA, celZ). The tran-scriptional control exerted by PecS could be explained either by competitionwith RNA polymerase for binding to the regulatory regions or by interferencewith RNA elongation. However, the relatively low affinity of PecS for its op-erators, as observed in vitro with protein purified fromE. coli, suggests thateither cofactors are required or a posttranslational modification increases thePecS affinity and specificity.

The PecM protein would be a good candidate for PecS modification. Ourmodel implies that, in the absence of an inducing signal, PecM modifies PecS sothat its affinity for DNA increases. When sensing the external signal, the PecMprotein becomes inactive, resulting in an unmodified PecS form incapable of

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strong repression. The phenotypes ofpecSandpecMmutants are consistentwith this model. In apecSmutant, the absence of PecS repressor results in a highlevel of derepression, whereas in apecMmutant, the presence of the unmodifiedPecS form, with low DNA affinity, results in a weaker derepression.

The PecT RegulatorA third type of mutation, resulting in elevated synthesis of pectate lyases in theabsence of inducer, was identified after Tn5 mutagenesis. The correspondingregulatory gene is calledpecT (139a). ThepecTmutation derepresses theexpression of a restricted set of pectinase genes:pelC, pelD, pelE, pelL,andkdgC.This mutation has no effect on the expression of the intracellular stepsof pectinolysis or on genes of other extracellular enzymes. However, the rangeof pecTaction is larger, andpecTmutants look quite different from cells of thewild-type strain (139a). They appear as mucoid clones when grown on solidminimal medium, and the cells floculate when grown in liquid minimal medium.Mucoidy often results from an elevated synthesis of exopolysaccharides (EPSs).Synthesis of EPS is a virulence factor for bacteria such asErwinia stewartiiandPseudomonas solanacearum.In the latter bacterium, synthesis of EPS iscoregulated with other virulence factors (79). Such a coordinate regulation,mediated bypecT,could exist inE. chrysanthemi.The floculation ofpecTmutants is relieved when leucine is added to the medium. InE. coli, leucine isa regulatory signal that plays a key role in many metabolic processes. Leucinemodifies the affinity of the general regulator Lrp for the regulatory regions ofthe controlled genes (17). The leucine effect observed in theE. chrysanthemipecTmutant could result from an interaction between PecT and Lrp for thecontrol of some genes.

ThepecTgene encodes a 34,761-Dalton protein belonging to the LysR fam-ily of transcriptional regulators. This family contains more than 50 identifiedmembers, which are usually activators and which have well-conserved features:They encode proteins with sizes ranging from 276 to 324 residues; their N ter-minus contains the helix-turn-helix DNA-binding motif; they usually bind tolong DNA regions that lack a conserved motif; they negatively autoregulate;and they are transcribed divergently from a gene that they regulate (134). PecThas 316 residues and a potential helix-turn-helix DNA-binding motif. In con-trast, as in a few other exceptions,pecTlacks any divergently controlled geneand is positively autoregulated. Alignment of the regulatory regions of thecontrolled genes revealed no motif that could correspond to PecT binding sites.However, PecT most probably acts as a negative transcriptional regulator onpelexpression, in response to an as yet unidentified signal.

Double or triple mutants were constructed (139a) to test whether the threeregulators KdgR, PecS, and PecT have a joint action or operate via independent

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regulatory pathways. All the double mutants exhibited an increased derepres-sion of pectate lyase synthesis compared with that in each single mutant. More-over, the triplekdgR-pecS-pecTmutant synthesizes more pectate lyase in unin-duced conditions than any of the double mutants. The additive effects of thethree mutations suggest that KdgR, PecS, and PecT regulatepelexpression viaindependent regulatory networks.

Additional Regulatory SystemsChemical or insertion mutagenesis ofE. chrysanthemiled to the isolation ofmany mutants with derepressed pectate lyase synthesis. Some of these mu-tants, which have not yet been characterized, differ from thekdgR, pecS,andpecTmutants with regard to their phenotypes and chromosomal location. Incontrast, very few mutants with decreased pectate lyase production could beobtained, despite an active search. Thus positive regulation seems less im-portant than negative regulation for the control ofpel gene expression inE.chrysanthemi.One mutant with decreased pectate lyase production,pecG,wasrecently obtained in strain 3937, but the corresponding insertion does not residein a transcribed region and more probably affects the transcription of a negativeregulatory gene (A Castillo, personal communication). In contrast, manyE.carotovoramutants with decreased pectinase production have been obtained,suggesting the prevalence of positive regulation in this bacteria. However,both Erwinia species probably have some homologous regulatory networks,for instance the KdgR and ExpR regulators.

One type ofE. carotovoramutation resulting in a decreased production ofextracellular enzymes was designatedaepor rex(98, 130). DNA fragments thatrestored exoenzyme production were cloned by direct complementation. Theaep locus contains two potential coding regions: theaepAgene coding for a465–amino acid protein (82) and a small ORF,aepH,encoding a 47–amino acidprotein (97). The smaller fragment required for the activator function is 508base pairs long. It contains theaepHputative ORF and, in its 5′ region, threeKdgR boxes and a series of imperfect direct repeats of 16 nucleotides (97). TheaepAgene itself does not appear to be necessary for the activator function. Achromosomal insertion inaepHresults in a deficiency in exoenzyme production.The smaller fragment of therex locus able to complement therexmutations is250 base pairs long and shows extensive sequence identity with theaepHregion(130). When these smallaepHor rex DNA fragments are transferred into thewild-type E. carotovorastrains, they cause exoenzyme hyperproduction. Therex fragment can also restore exoenzyme production in other down-regulatedmutants, such asexpI.In addition, the activation ofpelorcelexpression dependsupon the dosage ofaepH,a circumstance observed inE. coli as well. Theseobservations demonstrate thataepHor rex regions are not responsible for a

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simple allelic complementation of the initialaepor rexmutations but rather fora trans-acting activation.

Two models are proposed to explain the regulatory effect of these regions.Murata et al (97) conclude that the small AepH protein may act as an acces-sory factor to activate the production of extracellular enzymes in conjunctionwith other transcriptional regulators. In contrast, Salmond et al (130) infer thatthe regulatory effect results from a multicopy suppression. The 250–base pairrex fragment encodes no protein but would titrate out a putative repressor. Inconclusion, the role of theaepor rex region remains to be clarified. In anycase, care must be taken in the interpretation of the complementation of reg-ulatory mutations. The complexity of the exoenzyme control necessitates anintensive characterization of the genes and their products to deduce their role inregulation.

A second type of regulatory gene,rsmA,controlling extracellular enzymeproduction, was described inE. carotovora. The rsmAmutants overproducepectinases, cellulases, and proteases (19). ThersmAgene encodes a 6.8-kDaprotein with extensive homology with theE. coliCsrA protein, which is a globalnegative regulator affecting carbon storage and cell-surface properties (35). InE. carotovora, rsmAmay also function as a global regulator, as multiple copiesof this gene affect diverse phenotypes, such as the production of polysaccha-rides, antibiotics, and pigments; flagellum formation; and motility (19). Thecontrol exerted by RsmA may result from a modulation of the acyl-HSL levelsin the cells, as evidenced by reduction ofexpItranscription (which is responsiblefor acyl-HSL production) by thersmAmutation. However, in anexpImutant,the rsmAmutation provoked derepressed exoenzyme production. This resultsuggests that RsmA also acts onpel expression through a second mechanismindependent of acyl-HSL. ThersmAgene is widespread amongEnterobacte-riaceaeand also occurs inE. chrysanthemi(35). TheE. coli CsrA proteinpresents a RNA-binding motif and acts by decreasing mRNA stability (80).Whether this type of posttranscriptional regulation is involved in exoenzymeproduction inErwinia is an interesting problem for future study.

The integration host factor (IHF) affectspel transcription, as shown by thedecrease in pectate lyase production in ahimAmutant ofE. chrysanthemi3937(40). IHF usually acts as a cofactor to enhance the efficiency of transcriptionalactivators or helps the regulatory mechanisms by bending the DNA. Amongthe majorpelgenes, onlypelEpresents a putative IHF-binding site (Figure 4).If IHF affects the expression of otherpelgenes, the action could be indirect.

In addition to the global regulators, genetic evidence demonstrates the ex-istence of gene-specific regulators. For instance, theE. carotovora pehRgeneencodes a specific regulator of polygalacturonase production (127). ThepehR

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mutation was obtained through an original selection procedure: restoration ofnormal growth for bacteria overexpressing apehA::blatranslational fusion thatis toxic for the cells. ThepehRmutation, which is extragenic topehA,decreasespehAtranscription but does not affect the production of other extracellular en-zymes. ThepehRgene may encode atrans-acting positive regulator ofpehA.PehA production is repressed at a high calcium concentration (44). Such re-pression is specific for PehA and is not observed in the production of otherexoenzymes. Calcium sensing may thus be one of the signals triggering PehRcontrol.

In E. chrysanthemi,two gene-specific regulators,pecYandpecZ,were local-ized in the vicinity of the controlled genes.pecZ(or ptlR), the gene adjacentto pelC,had been supposed to be involved in regulation on the basis of its ef-fectsin transon the expression ofpel genes cloned inE. coli (67, 152). Morecareful analysis proved that this effect results from the titration of a regulatoryprotein (C Pissavin, personal communication). SomepecYmutations enhancepemAexpression (10), but other insertional mutations inpecYdecreasepemAexpression.pecYcodes for a 68-kDa protein that displays no similarities to anyfamilies of transcriptional regulators (N Hugouvieux-Cotte-Pattat, unpublisheddata). The specific-gene regulators could explain the differential expression ofpectinase genes and could allow for subtle variations, according to plant hostsor microenvironments.

Regulatory SitesOther elements essential in the regulatory mechanisms are the DNA sites in-volved in transcription initiation. Thepelgenes are subject to control by globalsystems as well as by specific regulators. How do their regulatory regionsaccommodate this variety of controls?

Despite their common transcriptional direction and their vicinity within thetwo clusterspelB-pelCandpelA-pelE-pelD,thepelgenes are independent tran-scriptional units. Each of the cloned genes can be transcribed independently,and insertions in the first genes of each cluster do not inactivate transcriptionof the other genes (74). All the sequenced pectate lyase genes ofE. chrysan-themistrains have a TAA stop codon followed by a sequence similar to Rho-independent terminators. In the case of thepelBgene of strain 3937, S1 mappingconfirmed that this sequence really functions as a transcriptional terminator (67).The transcripts initiated from thepelB-pelCregion were identified using North-ern blot analysis, and the results indicated the presence of only one transcriptper gene, the length of which matched that of the corresponding gene (67). Thisindependent organization for genes of the same regulon is preferable to a largeoperon arrangement because it allows individual flexibility of expression.

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Primer extension experiments were performed to locate precisely the pro-moter elements ofpelB, pelC,andpelE(50, 67). In each case, a single definedstart site was observed (Figure 4). Inspection of the DNA sequence revealedreasonable transcriptional start sites. Because of sequence similarities, the lo-cation of the 3937pelD or pelE promoters is supposed to be similar to thatof the EC16pelE promoter. Comparison of the differentpel promoters withthe classic sigma-70 promoter consensus shows that homology with the−35region is higher (4/6 to 5/6) than homology with the−10 region (4/6 to 3/6).Spacing between the−35 and the−10 regions could be either 17 or 16 nu-cleotides. Weakly conserved−10 regions are often found for promoters whoserecognition is favored by a specific sigma factor different from the sigma-70factor. Thepelpromoters must respond to multiple signals but lack homologywith promoters characteristic of the other known sigma factors, i.e.σ 28, σ 32,σ 60, or σS.

The AT content in the noncoding regions of thepel genes is clearly higherthan that of the coding regions (67). The base composition of theE. chrysan-themichromosome is approximately 57% GC. Thepel 5′ noncoding regionsare significantly different, with only 30–42% GC. This distinction is not ageneral property ofE. chrysanthemiregulatory regions, as the GC content of5′ noncoding regions of other genes is not so low. AT-rich regions inducebending of DNA, and the presence of curved regions downstream from pro-moters may increase their transcription-initiation rate (143). AT-rich regionsmay also correspond to the binding sites of histone-like proteins, such as theglobal transcriptional regulator HNS (141). Therefore, these AT-rich regionsmay modulate the transcription ofpelgenes inE. chrysanthemi.

Analysis of the promoters of equivalent genes in several bacterial strains canhelp to detect the important DNA sites that are better conserved than the non-significant regions. Comparison of the nucleotide sequences of the regulatoryregions of theE. chrysanthemi pelgenes allows the distinction of three classes:pelA, pelBC,andpelDE(67). Regulatory regions of genes inside thepelBCorpelDEclasses have conserved sequences, mainly in the promoter region. ThepelAgene exhibits very restricted homology with the otherpel genes in its 5′

noncoding region. Deletion analysis and site-directed mutagenesis showed thatsequences involved inpelEexpression inE. chrysanthemiEC16 are situated be-tween positions−112 to+50, relative to the transcriptional start (50). Deletionof a few bases from the putative CRP-binding site eliminatedpelEtranscription.Mutations in two imperfect palindromic sequences, showing homology with theKdgR box, result in constitutive promoter activity, as expected for repressor-binding sites. In addition, a region of approximately 40 base pairs appears to

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be necessary forpelE expression and could contain an activator-binding site.Partial conservation of this site in thepelA, pelE,andpelD regulatory regions(Figure 4) suggests that it is involved in a type of regulation that they have incommon.

The regulatory regions of thepelgenes contain various sequences exhibitingthe typical symmetry of operator sites. However, palindromes differ among thepelgenes, except for the one overlapping thepelBandpelCpromoters (Figure4) (67). This sequence could be important for specific regulation of the genesencoding the neutral pectate lyases. In addition, the 5′-untranslated ends ofeachpelgene contain sequences homologous with theE. coliCRP-binding site(Figure 4) (50, 117). Purification of theE. chrysanthemiCRP demonstratedthat it specifically interacts with the regulatory regions ofpel genes. DNaseIfootprinting experiments confirm that theE. chrysanthemiCRP-binding sitesare closely related to the consensus of theE. coli CRP-binding site (W Nasser,unpublished data).

CONCLUSION

The studies presented in this review underline the exceptional complexity andsubtlety of the pectinolysis regulation that evolved inErwinia species. The dif-ferent characterized regulators define distinct regulons that sometimes overlapbut are never identical. Some regulators control a set of pectinase genes or evenan individual gene—e.g. PehR forpehAexpression and RdgA-RdgB forpnlexpression. Action of ExuR is restricted to the galacturonate catabolic pathway.KdgR controls expression of the pectin catabolism and related functions, suchas pectinase secretion. The ExpI-ExpR system coordinates the production ofall extracellular degradative enzymes. Other regulators affect, in addition topectinase production, cell-surface properties (PecT, RsmA) or pigment biosyn-thesis (PecS). Whether the other genes belonging to these regulons are requiredfor Erwinia pathogenicity remains a subject for future study.

Other systems correspond to classical global regulators, such as CRP, whichis involved in carbon utilization. These mechanisms probably also containregulatory cascades, in which one regulator controls the synthesis of a secondregulator; an example is the OutT KdgR–dependent regulation of the secretionoperonoutC.

The nature of the signal inducing the cell response has been established inonly a few cases. The catabolic intermediates DKI, DKII, and KDG indicatepectin presence and directly modify the DNA-binding properties of the KdgRrepressor. The small diffusible molecule acyl-HSL is the signal through whichErwinia species perceive and respond to high cell populations. For other reg-ulons (PecS, PecT), the nature of the corresponding environmental stimulus

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and the means by which cells sense this stimulus are not understood. More-over, many environmental conditions affecting pectinase production are not yetassociated with any regulatory protein.

Additional black boxes in regulation mechanisms concern the DNA targetsof the regulators. Motifs conserved between promoters belonging to the sameregulon are useful in identifying the regulator-binding site. This approachsuccessfully identified KdgR binding site that was fully confirmed by in vitroexperiments. However, sequence homologies are not always manifest and thisapproach was deceptive in the hunt for PecS- and PecT-binding sites. Morelikely, this type of regulator does not recognize a precise motif. Since virulencegenes are subject to a variety of controls, their promoter region must harmonizethese multiple regulations. The organization of several binding sites must allowthe cohabitation of RNA polymerase, repressor(s), and activator(s). Moreover,the strength of the various controls could lead to the differential expression ofeach gene. The genetic organization of thepelgenes appears to be appropriatefor the personalized responses of individual genes.

Multiple regulations allow different signals to activate the virulence factorsindependently. They probably play an essential role during the first steps of theplant-pathogen interaction. Various conditions, such as iron deprivation, oxy-gen limitation, and nitrate presence may be the first signals recorded byErwiniato induce expression of the genes necessary for bacterial dissemination in theplant tissues, such as those genes encoding pectinases. Intracellular formationof KDG would then enhance pectin catabolism, leading to an increase in thebacterial population. Subsequently, high cell density would allow maximalproduction of all the extracellular degradative enzymes and other pathogenicfactors. The bacterial virulence is thus completely expressed, and generaliza-tion of the disease can progress rapidly. This scenario will undoubtedly bemodified when more data have been obtained concerning the expression ofvirulence genes in planta. Still, although the actual regulation scheme takingplace during the plant-pathogen battle may vary from the model proposed, itmost probably involves a sequential action of regulatory mechanisms.

In recent years, a lot of data have been gathered in the field of virulence-factor regulation. However, as we have pointed out forE. chrysanthemi,manyquestions remain unanswered. The future promises to be productive, providingnew information to complete the proposed model of the regulation of pecti-nolytic gene expression.

ACKNOWLEDGMENTS

We gratefully acknowledge the many colleagues who sent us published andunpublished material to help in the writing of this review. We thank V James

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for her careful reading of the text. Work on various aspects of pectinolysisregulation has been supported in our laboratory by grants from the CNRS andfrom the Ministere de l’Education Nationale, de l’Enseignement Supérieur, dela Recherche et de l’Insertion Professionnelle.

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Regulation of Pectinolysis in Erwinia Chrysanthemi

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