Regulation of the expression of prtW::gusA fusions in Erwinia carotovora subsp. carotovora

Reet Marits1, Marina Tshuikina1, Minna Pirhonen2, Eve Laasik1 and Andres Mäe1

Department of Genetics Institute of Molecular and Cell Biology, Tartu University Estonian Biocentre, Riia 23, Tartu 51010, Estonia1
Department of Plant Biology, Swedish University of Agricultural Sciences (SLU), PO Box 7080, SE-75007 Uppsala, Sweden2

Author for correspondence: Andres Mäe. Tel: +372 7 375 014. Fax: +372 7 420 286. e-mail: amae{at}tamm.ebc.ee


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Erwinia carotovora subsp. carotovora, a Gram-negative phytopathogenic bacterium, secretes an extracellular metalloprotease, PrtW. Previous results demonstrated that protease activity is necessary for the normal progression of disease symptoms caused by this bacterium. The present study revealed that the prtW gene constitutes an independent transcriptional unit. It is demonstrated that introduction of the prtW+ plasmid in trans into the prtW- mutant restores the protease activity in this strain. Gene fusions to the gusA (ß-glucuronidase) reporter were employed to analyse the transcription of prtW. The transcription of prtW is dependent on many environmental signals. When the bacteria were grown in the presence of potato extract, the expression of the protease gene was markedly higher at the beginning of the exponential phase of growth than that observed when cells were grown in the presence of polygalacturonate (PGA). Analysis of the promoter revealed that an essential regulatory region resided between 371 and 245 bp 5' of the translational start site. As this sequence showed no homology to the KdgR box it may be involved in the binding of an unknown negative regulator protein in E. carotovora subsp. carotovora. The differential responses of prtW expression to potato extract and to PGA appeared to be dependent on the KdgR repressor and the response regulator ExpA. According to the results presented here, it is conceivable that the multiple regulatory network allows flexibility in the expression of the prtW gene during different stages of infection.

Keywords: phytopathogenic bacteria, protease, promoter fusions

Abbreviations: E. carotovora, Erwinia carotovora subsp. carotovora; PCWDE, plant-cell-wall-degrading enzyme; PGA, polygalacturonate; pNP, p-nitrophenol


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The phytopathogenicity of the soft-rot enterobacteria of the genus Erwinia is correlated with their ability to produce and secrete plant-cell-wall-degrading enzymes (PCWDEs) (Pérombelon & Kelman, 1980 ; Barras et al., 1994 ). Many of these PCWDEs have been shown to trigger plant defence responses (probably by releasing cell wall fragments as active elicitors). The mechanism by which extracellular enzymes of Erwinia carotovora subsp. carotovora (E. carotovora) induce defence responses in plants has been proposed to involve the release of oligogalacturonide elicitors from plant cell wall pectin (Palva et al., 1993 ; Vidal et al., 1997 ).

To be able to overcome a plant defence response and to survive in plant tissue, the invading pathogen produces toxins or substances called suppressors, which act as pathogenicity factors by suppressing the expression of the defence response(s) of the host plant (Basse et al., 1993 ; Kato et al., 1993 ; Bender et al., 1999 ). As E. carotovora lacks these systems it has to find other means to escape the plant defence response(s). The expression of virulence in the soft-rot erwinias seems to depend on a fine balance between avoiding the plant defence reaction and rapid killing of the plant cells. To ensure that the balance is on the side of the pathogen, when the right environmental conditions prevail, members of the genus Erwinia have evolved multiple strategies to sense their environment and to modulate their gene expression both by positive expA/expS, aepA, rdgA/rdgB, rpfA, rpoS and hor regulators, and by negative hexA and kdgR regulators (Liu et al., 1993 , 1996 ; Mukherjee et al., 1996 ; Frederick et al., 1997 ; Thompson et al., 1997 ; Eriksson et al., 1998 ; Harris et al., 1998 ; Liu et al., 1999 ). Some of these regulators respond to environmental stimuli by modulating the expression of the controlled genes (Eriksson et al., 1998 ; Liu et al., 1999 ).

Microbial resistance to plant defence(s) during infection can also be achieved by the direct degradation of defence proteins, or proteins involved in signal transduction, in the host plant (Heilbronn et al., 1995 ). Plant response(s) triggered by E. carotovora have shown that the transcriptional activation of several defence genes is already achieved 4–6 h after infection (Vidal et al., 1997 ). The expression of the protease gene prtW during the early exponential phase of growth and the observation that the prtW mutant exhibits reduced virulence on potato tubers suggest the possibility that protease might be necessary for the supression of plant defence responses (Marits et al., 1999 ).

Here we report the construction and analysis of promoter fusions between the E. carotovora protease gene prtW and the reporter gene gusA (ß-glucuronidase), to identify sequences 5' to the prtW coding region that might account for the expression of the gene. We have analysed the expression of each fusion in the wild-type strain (SCC3193) in response to the presence of potato extract or polygalacturonate (PGA), to verify the different effect(s) these inducers have on gene expression. We have also tested the expression of each of the fusions in the kdgR- mutant strain SCC510, in the expA- mutant strain SCC3060 and in the expA- kdgR- double mutant strain SCC500, to determine if these regulators modulate prtW expression in response to different physiological inducers.


   METHODS
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INTRODUCTION
METHODS
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DISCUSSION
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Bacterial strains, plasmids, media and culture conditions.
Bacterial strains and plasmids used or constructed in this study are described in Table 1 and Fig. 2(b). E. carotovora and Escherichia coli were grown at 28 or 37 °C, respectively, in L-broth (Miller, 1972 ) or in M9 minimal medium (Sambrook et al., 1982 ) supplemented with 0·4% glycerol and trace elements (Bauchop & Elsden, 1960 ). When required, media were supplemented with 0·4% PGA, 16% potato extract and antibiotics at the following concentrations: 150 µg ampicillin ml-1, 25 µg kanamycin ml-1 and 25 µg chloramphenicol ml-1. Crude potato extract was prepared according to Marits et al. (1999) .


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Table 1. Bacterial strains and plasmids used in this study

 
DNA manipulation, plasmid construction and determination of the nucleotide sequence.
Unless otherwise stated, DNA cloning and gel analysis of plasmid DNA were done by established procedures (Sambrook et al., 1982 ). Plasmid preparations and the isolation of restriction fragments from agarose gels were performed with the QIAprep Spin Miniprep Kit and the QIAquick Gel Extraction Kit (both from Qiagen). Escherichia coli cells were transformed by the CaCl2 method (Sambrook et al., 1982 ). Plasmids were introduced into E. carotovora cells by electroporation (Ausubel et al., 1987 ; Py et al., 1991 ).

DNA was sequenced by the dideoxy method of Sanger et al. (1977) . The sequencing kit used was obtained from USB.

For the PrtW-His6-tagged overexpression plasmid, the coding region of prtW was amplified by PCR with primers Qia1 (5'-GAGAAGGATCCATGGCTTTACGAGATGACG-3') and Qia2 (5'-TCCTCGTCGACTCACACGATAAAATCGGTT-3'). The PCR product was digested with BamHI and SalI, and cloned into the vector pQE30 to yield pRT1. All PCR amplifications were performed with the proof-reading DNA polymerase Pwo (Boehringer Mannheim).

RNA isolation and Northern blot analysis.
Total RNA was isolated with the RNeasy RNA isolation kit (Qiagen). Ten micrograms of total RNA was denaturated in formamide, separated by electrophoresis through formaldehyde/agarose gels and blotted onto nylon filters (Sambrook et al., 1982 ). To generate a prtW-specific probe, prtW was amplified from the wild-type strain (SCC3193) by PCR using primers Qia1 and Qia2. The probe was labelled by using the multiprime DecaLabel DNA labelling Kit (MBI Fermentas). Prehybridization (1 h at 65 °C) and hybridization (12 h at 65 °C) were performed in prehybridization buffer (6xSSC, 2xDenhardt’s, 0·1% SDS and 100 µg denatured salmon-sperm DNA ml-1). After hybridization, the nylon membranes were washed twice for 20 min at 65 °C in 2xSSC plus 0·5% SDS, followed by 30 min at 65 °C in 0·5xSSC plus 0·5% SDS. The membranes were then examined by autoradiography.

Primer extension.
The primer extension assay was performed according to the manufacturer’s instructions (MBI Fermentas) with primer PROM1reverse (5'-GTCTCTTGGCGGGATA-3') and 10 µg RNA. The plasmid pROT4, primed with PROM1reverse, was used as a size marker.

Enzyme assays.
ß-Glucuronidase activity was assayed by using p-nitrophenyl ß-D-glucuronide as substrate (Novel et al., 1974 ). The degradation product, p-nitrophenol (pNP), was detected at an absorbance of 405 nm; the specific activity of ß-glucuronidase was expressed as nmol pNP liberated min-1 (OD600 unit)-1. The activity of protease was detected on L-agar plates containing 5% skim milk.

Expression of the prtW gene product in E. coli.
E. coli M15(pRT1) was grown at 37 °C in L-broth containing kanamycin and ampicillin until the culture reached OD600 0·4. One hundred microlitre aliquots of cell suspensions were withdrawn from samples which had been incubated for 4 h with 1·0 mM IPTG or without IPTG. The cells were collected by centrifugation and solubilized in SDS sample buffer (20% glycerol, w/v; 10% ß-mercaptoethanol, w/v; 0·02% SDS, w/v; bromophenol blue in 0·25 M Tris/HCl, pH 6·8) to yield a preparation of total cellular proteins. The samples were analysed by SDS-PAGE according to Laemmli (1970) and visualized by Coomassie-blue staining.


   RESULTS
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METHODS
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DISCUSSION
REFERENCES
 
Identification of the PrtW product
The small DNA fragment containing prtW was amplified by PCR (using primers Qia1 and Qia2) from strain SCC3193 and cloned into the BamHI–SalI site of the pBluescript(+) vector, yielding pROT3 and pROT3A, respectively. This produced both orientations of the gene with respect to the lac promoter. Only the orientation in pROT3, corresponding to the transcriptional direction of the predicted prtW ORF, allowed the complementation of the prtW mutant SCC6004 in trans (data not shown).

Cloning the BamHI–SalI restriction fragment containing the prtW gene into the compatible sites of a pQE30 vector allowed PrtW to be overproduced in E. coli M15. SDS-PAGE analysis of crude protein extracts of the E. coli M15(pRT1) strain showed, after IPTG induction, marked overproduction of a protein with an estimated molecular mass of 50 kDa (Fig. 1). This value is consistent with the 51 kDa protein predicted from the PrtW sequence (Marits et al., 1999 ).



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Fig. 1. PrtW protein expression in Escherichia coli. Crude extracts were analysed by SDS-PAGE in a 12% (w/v) polyacrylamide gel. Lanes: M, molecular mass marker; 1, lysate of E. coli M15(pRT1) after 4 h growth without IPTG; 2, lysate of E. coli M15(pRT1) after 4 h growth with IPTG.

 
Mapping the transcriptional site of the prtW gene
RNA was extracted from SCC3193 cells and used in primer extension experiments. Primer extension terminated abruptly at an A base located 186 bp upstream from the translational start site of prtW; no other significant start sites were detected. Inspection of the DNA sequence of this prtW region disclosed the presence of a possible {sigma}70 promoter, with a -10 box 10 bp and a -35 box 33 bp upstream of the transcriptional start site (Fig. 2a).



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Fig. 2. (a) Summary of the promoter region of the prtW gene in E. carotovora SCC3193. The translational initiation sequence (ATG) is shown in bold. The transcriptional start site, identified by primer extension analysis, is indicated by an asterisk and bold-type A. Potential -35 and -10 promoter elements are bold and underlined, the potential Shine–Dalgarno (SD) sequence is underlined. HindIII, ClaI and PaeI restriction sites utilized for the construction of gusA fusions are also shown (bold and italic). The beginning of pROT7 is indicated by an arrow. (b) Endonuclease restriction site maps for the inserts of prtW in plasmids pROT5, pROT6, pROT7 and pROT8. The transcriptional start site of prtW is indicated by an arrow. The construction of the plasmids is described in Results and in Table 1.

 
In Erwinia chrysanthemi the secretion genes belong to one gene cluster along with the protease, the structural and the inhibitor genes (Létoffé et al., 1990 ). The introduction of prtW (on pROT3) into the prtW- mutant strain SCC6004 restored its proteolytic activity. This suggests that the secretion genes must be functional in SCC6004 and that they are transcribed separately from prtW. As a further test of the fidelity of this hypothesis, Northern blots of total RNA isolated from SCC3193 were hybridized with the prtW probe. The results indicate that when prtW-specific DNA was used as a probe only a single transcript with a size of 1500 bp was visible, which corresponds to the calculated size of the protease gene transcript (1421 bp; data not shown).

Deletions in the prtW promoter region
To define the DNA regions necessary for prtW promoter activity, restriction-site deletions and PCR constructs were made from the prtW promoter regions (Fig. 2b; Table 1). Expression of prtW was examined during bacterial growth on minimal medium (M9+glycerol) in the presence and in the absence of PGA or potato extract using the prtW::gusA transcriptional fusions.

To determine the 5' extent of the prtW promoter, a 2038 bp KpnI–PaeI fragment, which had a 3' terminus at the 43 bp within the prtW coding region and a 5' terminus 1995 bp upstream from the first codon, was cloned from E. carotovora into the pBluescript(+) vector, yielding pROT4. The corresponding DNA fragment was then cloned into the low-copy vector pMW119::gusA, yielding pROT5.

In non-inducing conditions, pROT5 showed a low level of ß-glucuronidase activity which remained constant throughout the whole growth curve (data not shown). ß-Glucuronidase expression clearly increased when potato extract or PGA was added to the minimal medium. When the bacteria were grown in the presence of potato extract the level of ß-glucuronidase activity was markedly higher at the beginning of the exponential phase of growth compared to when the cells were grown in the presence of PGA. The maximum expression of ß-glucuronidase occurred in a short period during which the cells were still in the mid-exponential phase of growth. The maximum expression of gusA was transient and was followed by a decline (Fig. 3). In the presence of PGA the timing of ß-glucuronidase expression was somewhat delayed, relative to its expression in the presence of potato extract, and started to increase only after 6 h of growth. The plasmid pROT5 yielded ß-glucuronidase activity data that were indistinguishable from those of SCC6004 under all conditions tested (Fig. 3). These results indicate that no essential promoter elements reside 3' to the PaeI site or 5' to the KpnI site.



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Fig. 3. Expression of the prtW::gusA fusion in strain SCC6004 and in strain SCC3193 pROT5 under various growth conditions. Cultures were grown in M9 minimal medium containing 0·4% glycerol and supplemented as indicated. (a) ß-Glucuronidase activity of the bacterial cultures. (b) Growth of the bacterial cultures followed by measurement of OD600. For both graphs: {blacksquare}, SCC6004 in minimal medium plus 0·4% PGA; {blacktriangleup}, SCC3193(pROT5) in minimal medium plus 0·4% PGA; {square}, SCC6004 in minimal medium plus 16% potato extract; {triangleup}, SCC3193(pROT5) in minimal medium plus 16% potato extract. Each value represents the mean of three experiments; the SD is indicated by the error bars.

 
As the rate of induction of the prtW promoter with the potato extract or the PGA is maximal after 8 h of growth, the expression of different promoter constructs was observed at this time point. In the first construct, a HindIII site located 683 bp upstream of the first codon in the prtW promoter region was exploited to delete the region upstream of this site in pROT5. The new construct was designated pROT6. E. carotovora SCC3193 cells carrying pROT6 showed the same basal level of ß-glucuronidase activity when growing under non-inducing conditions as when carrying pROT5 (Fig. 4a). The induction rates of ß-glucuronidase activity in the presence of PGA or potato extract were the same as in pROT5; however, the values attained were lower (Fig. 4a).



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Fig. 4. Expression of pROT5, pROT6 and pROT8 in different strains of E. carotovora. ß-Glucuronidase activity was measured 8 h after inoculation. Cultures were grown in M9 minimal medium containing 0·4% glycerol and supplemented as indicated. (a) Wild-type SCC3193; (b) SCC510 kdgR-; (c) SCC3060 expA-; (d) SCC500 expA- kdgR-. Unsupplemented medium (open bars), medium plus 0·4% PGA (bricks) and medium plus 16% potato extract (solid bars). Each value represents the mean of three experiments; the SD is indicated by the error bars.

 
The promoter region of prtW from -371 to +43 nt (Fig. 2a) together with gusA was cloned from pROT5 by PCR, using primers PROM5 (5'-ATGTGAGCTCGTAGCGCGGTTT-3') and M13/pUC Reverse Primer (-24), into the low-copy vector pMW119; the resulting plasmid was designated pROT7. The basal level of ß-glucuronidase activity in pROT7 was low, but was comparable to that of pROT5. However, pROT7 was completely incapable of inducing ß-glucuronidase activity, even when cells were grown in the presence of potato extract or PGA (data not shown). The results of these experiments refer to a hypothetical negative regulatory sequence spanning the region from -371 to -245 nt (Fig. 2a).

The prtW promoter region was also restricted at a unique ClaI site located 245 bp upstream of the first codon. The 2700 bp ClaI fragment together with miniTn5CmR::gusA from pROT1 was cloned into the ClaI site of the low-copy vector pACYC184 to produce plasmid pROT8. The ß-glucuronidase activity was measured in the SCC3193(pROT8) cells under the same conditions as with pROT5 and pROT6. In the case of pROT8, ß-glucuronidase expression increased eightfold relative to pROT5 in the absence of the inducer (Fig. 4a). It was interesting that ß-glucuronidase activity was still stimulated about two- to threefold in the presence of potato extract or PGA (Fig. 4a).

Expression of prtW::gusA in various regulatory backgrounds
PCWDE synthesis is subjected to a wide range of plant signal molecules, including cell-wall fragments released by the action of PCWDEs and substances from the lysing plant cells. We compared prtW::gusA fusion activities in the wild-type strain and in expA- and kdgR- mutants under different induction conditions to see whether there were changes in regulation in response to different signals.

To analyse the effect of KdgR on protease expression, we analysed the expression of the transcriptional prtW::gusA fusions (pROT5, pROT6 and pROT8) in the kdgR- mutant. In non-inducing conditions, prtW::gusA transcriptional fusions showed an approximately two- to fourfold increase in the expression of ß-glucuronidase activity in comparison to the wild-type strain (Fig. 4a, b). The expression of pROT5, pROT6 and pROT8 clearly increased when the cells were grown in the presence of potato extract. The addition of PGA to the medium, however, did not result in further enhancement of promoter activity in comparison to the wild-type strain (Fig. 4a, b). In the case of pROT8 the induction rate of ß-glucuronidase activity was lower, probably due to the high non-induced level of ß-glucuronidase activity (Fig. 4b). To determine whether the promoter activity in pROT7 was affected by KdgR, pROT7 was introduced into SCC510; pROT7 was completely devoid of promoter activity on any medium tested (data not shown). These results indicate that expression of the prtW::gusA fusion in pROT7 appeared to be unaffected by KdgR.

We also studied the expression of the prtW::gusA transcriptional fusions (pROT5, pROT6 and PROT8) in the expA- mutant strain SCC3060. When pROT5 and pROT6 were introduced into SCC3060, ß-glucuronidase activity was almost undetectable on any medium tested (Fig. 4c). However, when pROT8 was introduced into SCC3060, ß-glucuronidase activities in all conditions tested were on the same level as the non-induced activity in the wild-type strain (Fig. 4c).

When pROT5 and pROT6 were introduced into the expA- kdgR- double mutant, SCC500, the ß-glucuronidase activities were higher than in the expA- mutant, although the activities did not reach the levels observed in the wild-type strain (Fig. 4a, d). When pROT8 was introduced into the expA- kdgR- double mutant, the ß-glucuronidase activities remained at the same levels in both the presence and the absence of PGA. However, when medium with potato extract was used, the relative upregulation of pROT5, pROT6 and pROT8 activity was even greater than when strains carrying these plasmids were grown in the presence of medium with PGA (Fig. 4d).

As in the case of the wild-type and the kdgR- mutant strains, pROT7 was completely devoid of promoter activity in the expA- and expA- kdgR- mutants on any medium tested (data not shown).


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
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In this study we have explored some aspects of the transcriptional regulation of the E. carotovora protease prtW gene. We analysed the effect of PGA and of potato extract on the different prtW::gusA fusions in the wild-type strain (SCC3193) and in different regulatory mutants.

Identification of the prtW transcriptional signal
Evidence from Northern analysis and from the complementation of the protease-negative phenotype in SCC6004 with the cloned prtW wild-type allele established that the protease gene is transcribed separately from the inhibitor and secretion genes. In spite of the extensive similarity of PrtW from E. carotovora with proteases of Erwinia chrysanthemi, the operon structure of the protease, the inhibitor and the secretions genes in these two species is different. These differences might result from the rearrangements that have occurred during the transfer of these genes between different Erwinia strains (Létoffé et al., 1990 ; Ghigo & Wandersman, 1992 ; Marits et al., 1999 ).

Effect of growth conditions on the expression of prtW::gusA fusions
Genes that encode the PCWDEs are often subject to coordinated regulation; these regulatory systems are able to respond to the various environmental signals that may be encountered during the cycle of infection (Liu et al., 1993 , 1999 ; Eriksson et al., 1998 ; Harris et al., 1998 ). The most important signals originate from the infected plant tissues, as this is the main environment for the pathogen during the plant–microbe interaction. To mimic in planta conditions low nutrient medium (M9+glycerol) was used to culture the E. carotovora strains.

The expression of protease already during the early stages of infection correlates with the early expression of pathogenesis-related genes in infected plants, as shown by Vidal et al. (1997) . Such a rapid response to the incoming signal, activating the expression of the protease, could facilitate the establishment of successful infection and makes the protease a possible candidate for the repression of plant defences. The signal to which prtW responds is, for the moment, unknown, but as the induction had already occurred by 4 h after infection this signal may be a plant protein(s), phenolic compounds or other factors that are released from the damaged plant cells. We also observed the activator effect of PGA on prtW expression; this function could result from the degradation products of PGA. These degradation products are the result of the enzymic activity of different pectinolytic enzymes whose expression is usually activated at the start of the stationary phase of growth (Pirhonen et al., 1991 ; Eriksson et al., 1998 ). This explains our results, which showed that expression of the prtW fusions reached its maximum in the presence of PGA only in the initial stages of the stationary phase of growth (Fig. 3).

Interestingly, although the induction rates were similar, we observed differences in the level of prtW expression in pROT5 and pROT6 in the wild-type (Fig. 4a). It is possible that these differences may be caused by the occurrence of regulatory elements in the upstream region of pROT5 that are necessary for the full expression of the prtW promoter. Huang et al. (1998) have described the occurrence of promoters in Ralstonia solanacearum that have distant cis-acting DNA sequences enhancing the expression of different virulence genes.

Role of the regulators KdgR and ExpA on the expression of prtW
The complex regulatory network controlling the production of virulence factors has been the subject of intensive studies in many Gram-negative pathogens (Liu et al., 1993 , 1996 ; Frederick et al., 1997 ; Thompson et al., 1997 ; Harris et al., 1998 ). The data presented by Hyytiäinen et al. (2001) show that the global regulators ExpA and KdgR modulate extracellular enzyme gene expression through the RsmA–rsmB system. We now provide additional evidence for the role of KdgR and ExpA in the differential responses of prtW to potato extract and PGA. The interesting fact is that although the KdgR mutant shows increased prtW promoter activity in the absence of PGA, we could still observe the inducing effect of PGA on the kdgR- mutant (Fig. 4b). This may indicate that KdgR on its own does not mediate the induction of protease upon the addition of PGA, but that further levels of control are required for protease induction. The effect of potato extract seems to be more pronounced when used as an inducer than the effect of PGA on the expression of protease in the kdgR- mutant SCC510 (Fig. 4a, b). These results might refer to the possibility that KdgR negatively regulates the expression of a regulator responding to the signal present in the potato extract.

The level of expression of the pROT5, pROT6 and pROT8 fusions in the expA- mutant remained in all conditions tested at the same level as that in the wild-type strain under non-inducing conditions (Fig. 4a, c). In the expA- kdgR- double mutant the promoter activities of pROT5, pROT6 and pROT8 were similar or slightly increased when compared to those in the expA- mutant (Fig. 4a, d). Similarly, Hyytiäinen et al. (2001) showed that under the non-induced conditions the production of protease was only partly restored in the expA- kdgR- mutant. Taken together, these data suggest that the presence of ExpA plays an important role in the expression of prtW under non-induced as well as under induced growth conditions.

Deletion analysis identified an essential regulatory element that resides between the nucleotides -371 and -245 relative to the prtW translational start site (Fig. 2a). The construct pROT7, which was lacking the region upstream from the -371 nt, was completely devoid of promoter activity in the wild-type strain as well as in the kdgR- mutant (not shown). Furthermore, we searched for potential KdgR sites between nucleotides -371 and -245 and were unable to find any convincing matches to the E. carotovora consensus (Liu et al., 1999 ). The deletion of this region restored the promoter activity of prtW, as was observed in the case of pROT8 (Fig. 4b). These results refer to the possibility that the corresponding DNA region may be involved in the binding of an unknown negative regulatory protein.

Differential responses of protease expression to physiological inducers, such as potato extract and PGA, probably allow flexibility in selectively expressing the prtW gene. Further studies of different regulatory systems, which must guarantee the coordinated expression of protease, may shed some light on the complex regulation of this virulence factor.


   ACKNOWLEDGEMENTS
 
We thank Heidi Hyytiäinen (Dept of Biosciences, Division of Genetics, Viikki Biocenter, University of Helsinki, Finland) for the E. carotovora strains SCC500 and SCC510. We also thank Sulev Kuuse for help with the photography. Our work was supported by the Estonian Science Foundation (Grant TBGMR510) and the European Community (Contract ERBIC15-CT96-0908).


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 6 August 2001; revised 17 October 2001; accepted 25 October 2001.



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