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Journal of Bacteriology, August 2007, p. 5955-5962, Vol. 189, No. 16
0021-9193/07/$08.00+0     doi:10.1128/JB.00218-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Differential Regulation of Two Oligogalacturonate Outer Membrane Channels, KdgN and KdgM, of Dickeya dadantii (Erwinia chrysanthemi){triangledown}

Guy Condemine1* and Alexandre Ghazi2

Université de Lyon, F-69003 Lyon, Université Lyon 1, F-69622 Lyon, INSA-Lyon, F-69621 Villeurbanne, and CNRS UMR5240, Unité Microbiologie Adaptation et Pathogénie, F-69622 Lyon, France,1 Groupe Canaux Ioniques, CNRS UMR8619, Université Paris-Sud, F-91405 Orsay, France2

Received 9 February 2007/ Accepted 6 June 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The entry of oligogalacturonates into Dickeya dadantii occurs through the specific channel KdgM. The genome of the bacterium encodes a second member of this family of outer membrane proteins, KdgN. We showed that this protein is also involved in the uptake of oligogalacturonates. When KdgN was reconstituted in proteoliposomes, it formed channels with a conductance of about 450 pS at a positive potential. These channels had weak anionic selectivity. The regulation of kdgN is complex, and five genes controlling the expression of kdgN have been identified: kdgR, pecS, ompR, hns, and crp. Moreover, kdgN was regulated by growth phase but only when bacteria were grown in rich medium. Most of these regulators of kdgN also control kdgM expression, but some of them regulate kdgM in the opposite manner: while PecS and OmpR are repressors of kdgM, they are activators of kdgN. This pattern resembles the regulation of the Escherichia coli general porins OmpF and OmpC, but such opposite regulation of two specific outer membrane channels has never been described before. KdgN may allow the bacteria to collect oligogalacturonates under saprophytic conditions, when virulence genes, including kdgM, are not expressed.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The plant pathogenic bacterium Dickeya dadantii (Erwinia chrysanthemi) causes the soft rot disease on many plants by the massive production of plant cell wall-degrading enzymes. The main part of these enzymes consists of pectinases. D. dadantii synthesizes more than 25 enzymes involved in the degradation and the catabolism of pectin. However, the functions of many of these enzymes are redundant: nine pectate lyases (PelA, PelB, PelC, PelD, PelE, PelI, PelL, PelW, and PelX), two pectin methylesterases (PemA and PemB), two pectin acetylesterases (PaeX and PaeY), and four polygalacturonases (PehV, PehW, PehX, and PehN) have been identified (9, 24). All these enzymes cleave pectin into small oligogalacturonates. Two transport systems allow the entry of these oligogalacturonates into the cytoplasm: the ABC transporter TogMNAB and the symporter TogT (10). The multiplicity of these enzymes does not mean that they are equivalent. Enzymes with similar activities may have different localization patterns. PemA is a secreted enzyme, while PemB is outer membrane anchored (30). PelW is cytoplasmic, PelX is periplasmic, and the other Pels are secreted (31). Another difference may lie in the regulation of their synthesis. All the pel genes except pelL are regulated by kdgR (11). They may also have different catalytic properties (33). This strategy of multiplication of enzymes with slightly different characteristics allows D. dadantii to degrade very efficiently and completely the plant cell wall pectin.

The entry of nutriments into the periplasm occurs through general porins or, in a few cases, through specific outer membrane channels that allow the diffusion of molecules that would be too large to enter through general porins (17). In D. dadantii, the uptake of long oligogalacturonides resulting from the degradation of pectin by pectate lyases requires the presence of a specific channel, KdgM (2). The gene encoding this channel is within the pelW-togMNAB-kdgM-paeX operon. All these genes are involved in pectin degradation, and their expression is controlled by KdgR, a protein which regulates the expression of most of the genes of pectinolysis, and by the cyclic AMP receptor protein (CRP) activator. However, a strong internal promoter located in front of kdgM allows for additional control of the expression of the last two genes of the operon: kdgM is repressed by PecS, a regulator of several functions involved in the virulence of the bacterium (pectate lyase synthesis, motility, and resistance to oxidative stress) (2, 23, 27). KdgM was the first characterized member of a new family of outer membrane channels, the Transport Classification Database family 1.B.35 (http://www.tcdb.org). These channels consist of small proteins (216 amino acids for KdgM) and do not seem to form oligomers. A structural model suggests that KdgM may form a 14-strand ß-barrel (18). Another member of this family has been characterized: the outer membrane sialic acid-inducible channel NanC is required for the growth of Escherichia coli with this compound as the sole carbon source in the absence of the general porins (4).

The genome of D. dadantii contains a gene encoding another member of the KdgM outer membrane channel family, named KdgN. We show here that this protein is also involved in the transport of oligogalacturonides. The regulation of kdgN is complex and, in some aspects, opposite to that of kdgM. This differential regulation may explain this new example of redundancy of genes involved in pectin metabolism in D. dadantii.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial strains, plasmids, and culture conditions. Bacterial strains and plasmids used in this study are described in Table 1. D. dadantii and E. coli cells were grown at 30 and 37°C, respectively, in Luria broth (LB) medium or M63 minimal medium (13) supplemented with a carbon source (0.2%) and, when required, with antibiotics at the following concentrations: ampicillin, 100 µg/ml; kanamycin, 50 µg/ml; chloramphenicol, 20 µg/ml; and tetracycline, 2 µg/ml. To prepare conditioned medium, an overnight LB medium culture was centrifuged, the supernatant was sterilized by filtration, and 1 volume was added to 3 volumes of fresh LB medium. Primers kdgN5 (5'-CCGCGGTATGAAAGCCGCCACCCGC-3') and kdgN6 (5'-TTACGTCTGGTGATACACGGAACGG-3') were used to amplify a 1,368-bp DNA fragment containing the kdgN coding sequence. The amplified fragment was cloned into plasmid pGEM-T (Promega, Madison, WI) to give plasmid pTN1. The kdgN::uidA transcriptional fusions were constructed by the insertion of a uidA-Km or a uidA-Cm cassette into the first BsrGI site of kdgN. To construct the ompR mutant, a DNA fragment containing 1,000 bp upstream and downstream of ompR was amplified with the primers OmpR+ (5'-GCCCTCCGTAATCATTGAGGCCACGG-3') and OmpR– (5'-ACGGGCTGACTTTCATTGGCAACTC-3') and cloned into plasmid pGEM-T. A Tc cassette (5) was inserted between the two NruI sites of ompR, and the construct was recombined into the D. dadantii chromosome. Marker exchange recombinations were obtained after growth in low-phosphate-concentration medium, as described by Roeder and Collmer (26). Transduction with phage {phi}EC2 was carried out as described by Résibois et al. (21).


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  		TABLE 1. Bacterial strains used in this study

 
Recombinant-DNA techniques. Preparations of chromosomal and plasmid DNA, restriction digestions, ligations, DNA electrophoresis, transformations, and electroporations were carried out as described by Sambrook et al. (28).

RNA isolation and primer extension analysis. RNA was extracted from a culture of D. dadantii strain A576 grown in LB medium supplemented with galacturonate (2 mg/ml). RNA extraction and primer extension experiments were performed essentially as described previously (22). The primers used for the specific detection of mRNA were 5'-end-labeled kdgN12 (5'-CGGACGCTACCATCATCGTCAG-3') and kdgN13 (5'-ATTGTTATCACCCTCATCAGAG-3'), which anneal to kdgN mRNA molecules at positions +16 to +37 and –20 to +2 (relative to the translation initiation codon ATG). The extension products were resolved on a 6% sequencing gel and visualized by autoradiography. The lengths of the transcripts were identified by using the corresponding dideoxy sequencing reactions for a reference.

Production and purification of KdgN. An NcoI-SacI fragment from plasmid pTN1 was introduced into the same sites of the overexpression plasmid pKSM717 (12) to give plasmid p17N1. This plasmid was introduced into the porin-negative overexpression strain BL21(DE3)omp8/pLys. The cells were grown in LB with ampicillin and chloramphenicol to an optical density at 600 nm of 0.6. IPTG (isopropyl-ß-D-thiogalactopyranoside) was added to a final concentration of 1 mM, and the culture was grown for an additional 2 h. Cells were harvested and disrupted in a French press in 50 mM Tris-HCl (pH 8.0)-5 mM EDTA. Unbroken cells were removed by centrifugation at 10,000 x g for 20 min, and the crude cell membrane fraction was sedimented by centrifugation at 100,000 x g for 2 h. The membranes were resuspended in 50 mM Tris-HCl (pH 8.0)-5 mM EDTA-0.5% octylglucoside, incubated for 30 min with gentle agitation at room temperature, and centrifuged at 100,000 x g for 2 h. The supernatant was loaded onto a 12% preparative Tris-glycine-sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gel. After electrophoresis, the band corresponding to KdgN was cut and the protein was electroeluted. The protein was further purified on a preparative Tris-tricine-SDS-PAGE gel and electroeluted.

Membrane purification. D. dadantii cells were grown overnight in minimal medium supplemented with glycerol, pelleted by centrifugation, and resuspended in a 1/20 volume of 10 mM Tris-HCl (pH 8.0)-1 mM EDTA. Cells were broken by disruption in a French press. Unbroken cells were removed by centrifugation at 10,000 x g for 20 min, and the crude cell membrane fraction was sedimented by centrifugation at 100,000 x g for 2 h.

Enzyme assays. ß-Glucuronidase assays were performed with toluenized cells grown to exponential phase, with p-nitrophenyl-ß-D-glucuronate as the substrate (29). Pectate lyase activity was determined by monitoring the appearance of unsaturated oligomers at 230 nm, according to the method of Shevchik et al. (29).

Reconstitution of KdgN in liposomes. The purified protein KdgN (500 ng) was added to 2 ml of a buffer of 100 mM KCl, 10 mM HEPES-KOH (pH 7.4), and 33 mM octylglucoside containing 1 mg of sonicated lipids (asolectin from soybean, type IV-S). After incubation for 15 min at room temperature, 160 mg (wet weight) of SM-2 BIOBEADs (Bio-Rad) was added to the suspension to remove the detergent. Incubation was carried out for 4 h at room temperature. The BIOBEADs were discarded, and the suspension was centrifuged for 30 min at 344,000 x g at 4°C. The proteoliposomes were resuspended in 100 µl of 100 mM KCl-10 mM HEPES-KOH (pH 7.4).

Electrophysiological recording. Bilayers from a solution of asolectin lipids dissolved in n-decane (30 mg/ml) were formed across a 250-µm-diameter hole. Proteoliposomes (3 to 5 ng of protein/ml, final concentration) were added to the cis compartment. Fusion was induced by imposing a salt gradient between the two chambers: 800 mM KCl-10 mM HEPES-KOH (pH 7.4) in the cis compartment versus 100 mM KCl-10 mM HEPES-KOH (pH 7.4) in the trans compartment. The bilayer setup was connected to the external circuit through salt bridges with Ag/AgCl electrodes. Unitary currents were recorded using an Axon 200B patch clamp amplifier and an Axon Digidata 1322A interface. Records were filtered at 1 kHz through a four-pole Bessel filter and digitized offline at 2 kHz. The membrane potential is the potential of the cis side minus the potential of the trans side.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
A second gene of the kdgM family is present in the D. dadantii genome. An analysis of the D. dadantii genome showed the presence of a gene, ABF-0015532, coding for a protein with 60% identity to the oligogalacturonate-specific channel KdgM (J. D. Glasner, C.-H. Yang, S. Reverchon, N. Hugouvieux-Cotte-Pattat, G. Condemine, J.-P. Bohin, F. van Gijsegem, S. Yang, T. Franza, D. Expert, G. Plunkett, M. San Francisco, A. Charkowski, B. Py, L. Grandemange, K. Bell, L. Rauscher, P. Rodriguez-Palenzuela, A. Toussaint, M. Holeva, S.-Y. He, V. Douet, M. Boccara, C. Blanco, I. Toth, A. D. Anderson, B. Biehl, B. Mau, S. M. Flynn, F. Barras, M. Lindeberg, P. Birch, S. Tsuyumu, X. Shi, M. Hibbing, M.-N. Yap, U. Masahiro, J. F. Kim, P. Soni, G. F. Mayhew, D. Fouts, S. Gill, F. R. Blattner, N. T. Keen, and N. T. Perna, submitted for publication). As observed for kdgM and other members of the kdgM family, the G+C content of ABF-0015532 (45%) was much lower than the average G+C content of the corresponding organism (57% for D. dadantii). The size of the mature protein (26,020 Da) was a little larger than that of KdgM (24,688 Da) (Fig. 1). In contrast to kdgM, which is included in a cluster of genes involved in pectin degradation (2), ABF-0015532 is located downstream of the tryptophan synthesis genes trpEDCBA and upstream of the gene of an outer membrane protein encoded by the other strand, and thus, no information about its function could be deduced from its localization. However, an in silico search for potential KdgR-binding sites in the regulatory region of D. dadantii genes detected one site in front of ABF-0015532, and in consequence, the gene was renamed kdgN (25).


Figure 1
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  		FIG. 1. Protein compositions of different D. dadantii strain membranes. Membranes from several M63-glycerol-grown D. dadantii strains were prepared, and proteins were separated by SDS-PAGE and stained with Coomassie blue. Lane 1, wild-type strain; lane 2, kdgR mutant; lane 3, kdgR kdgM mutant; lane 4, kdgR kdgN mutant; lane 5, pecS mutant; lane 6, ompR mutant; lane 7, ompF mutant. Positions of KdgN, KdgM, and OmpF are indicated. MW, molecular weight.

 
Characterization of KdgN. Purified KdgN was reconstituted in liposomes. The addition of proteoliposomes to the cis compartment of a bilayer chamber under asymmetrical conditions (800 mM KCl against 100 mM KCl) resulted in the insertion into the bilayer of large conductance channels which were open at 0 mV and at low positive or negative voltages. The application of a high positive membrane potential induced the closure of the channels. Such closures were observed only at +140 mV or above (data not shown). As shown in Fig. 2, when the voltage was stepped up to +160 mV, the channels started to block up until they reached a closed state, from which they could not reopen. However, a return to 0 mV generally resulted in the complete opening of the channels. Both slow and fast kinetics upon closure were observed, and several subconductances could frequently be observed (Fig. 2). Negative membrane potentials were less effective. Partial closures at high negative potentials could be observed (Fig. 2), but complete closure could never be observed. The most frequent conductance was in the range from 450 to 500 pS in 800 mM symmetrical media. Under asymmetrical conditions (800 mM KCl against 100 mM KCl), the reversal potential was 21 ± 3 mV (n = 7). This result corresponds to a 2.9-fold preference for chloride over potassium, and KdgN is thus, as KdgM, weakly selective for anions. In contrast to KdgM, KdgN was insensitive to trigalacturonate, up to 60 mM added in both chambers, whatever the applied potential (data not shown).


Figure 2
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  		FIG. 2. Channel activity of KdgN. The recordings were performed under asymmetric conditions (100 mM KCl versus 800 mM KCl). The bilayer was subjected to various positive and negative potentials as indicated. Between pulses, the bilayer was held at 0 mV for several seconds. The beginning of the trace corresponds to the onset of the pulse. (A) KdgN channels closed at a high positive membrane potential. Three seconds’ worth of the recording on an expanded time scale, showing the existence of different subconductances, is displayed. (B) Only partial closure at a high negative potential was observed. O, open level; C, closed level.

 
Analysis of a kdgN mutant. The presence of a binding site for KdgR, the regulator of pectinolysis genes in D. dadantii, in the regulatory region of kdgN let us suppose that the product of kdgN might be involved in the uptake of oligogalacturonides and that its function and that of KdgM may be redundant. A kdgN mutant (A4205) was constructed by the insertion of a Km cassette into the gene. The synthesis of pectate lyases requires the release of KdgR repression by 2-keto-3-deoxygluconate, a polygalacturonate degradation product formed intracellularly. Thus, the level of production of pectate lyases is a function of that of oligogalacturonates entering the bacteria. Pectate lyase synthesis in a kdgN mutant in the presence of polygalacturonate was reduced compared to that in the wild-type strain (Fig. 3), almost to the level observed in the kdgM mutant. In the double kdgM kdgN mutant, induction was only slightly reduced. Galacturonate also induces pectate lyase synthesis but does not require specific outer membrane channels to enter the cells. The similar levels of induction of pectate lyase synthesis by galacturonate observed in the four strains indicates that the kdgN and kdgM mutations do not modify the inducibility of pectate lyases but the amounts of inducing compounds that can enter the bacteria to induce their synthesis (Fig. 3). Thus, both KdgN and KdgM appear to be required for the transport of oligogalacturonides into the bacteria.


Figure 3
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  		FIG. 3. Induction of pectate lyase synthesis in a kdgN, a kdgM, or a kdgM kdgN mutant. Strains containing the indicated mutations were grown in M63 medium with glycerol as the carbon source (black bars) supplemented with galacturonate (white bars) or PGA (gray bars), and the pectate lyase specific activity in the supernatant was measured and expressed as micromoles of unsaturated products formed per minute per milligram of bacteria (dry weight). The bars indicate the means of results from four experiments, and the error bars indicate the standard deviations. WT, wild type.

 
The role of these channels in the transport of trigalacturonate has been estimated by measuring the growth rate of D. dadantii with a limiting (10 µM) concentration of a substrate in the absence of KdgM or KdgN. Nonspecific transport through the general porin (OmpF) has been prevented by the use of an ompR mutant, since a characteristic of the ompR mutant is the absence of the general porin OmpF (Fig. 1). A preculture in the presence of galacturonate was performed to induce the complete catabolic pathway. An increased doubling time for the kdgN ompR strain compared to the ompR strain (3 h and 10 min versus 2 h and 24 min) or for the ompR kdgM kdgN strain compared to the ompR kdgM strain (13 h and 50 min versus 9 h and 20 min) confirmed that KdgN is involved in trigalacturonate uptake.

Regulation of kdgN expression. A kdgN-uidA gene fusion was constructed to assay kdgN expression under various conditions. In the presence of galacturonate, the expression of the fusion was induced nearly twofold (Table 2). In a kdgK mutant, which accumulates 2-keto-3-deoxygluconate, a galacturonate catabolism compound that relieves KdgR-mediated repression, the induction level rose sixfold. In a kdgR background, the level of fusion expression was fivefold higher than that in the wild-type strain. These results are consistent with the functionality of the KdgR-binding site found in front of the gene. Since kdgN may be involved in the uptake of rhamnogalacturonides or other oligosaccharides resulting from the degradation of various pectic polymers, we tested its induction by sugars present in these polysaccharides (rhamnose, galactose, arabinose, and xylose). None of them were inducers of the kdgN fusion (data not shown). PecS is a regulator of pectate lyase genes and other virulence functions. It is a repressor of kdgM (2). The expression of the kdgN fusion decreased in a pecS background, showing that PecS is an activator of this gene (Table 2; Fig. 1). The addition of a kdgR mutation in this pecS mutant elevated the expression of the fusion, indicating that the two regulators act independently. kdgM is repressed by ExuR, the regulator of the galacturonate degradation pathway (2). The introduction of an exuR mutation did not modify kdgN expression (data not shown). Growth with glucose as the sole carbon source lowered the level of expression of the fusion, which let us suppose activation by the catabolite repression protein CRP (Table 2). The level of expression of kdgN in a crp mutant was indeed reduced to 5% of that in the wild-type strain. It was further lowered by the introduction of a pecS mutation (Table 2).


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  		TABLE 2. Effect of various carbon sources and regulatory mutations on the expression of the kdgN-uidA fusiona

 
Osmoregulation of kdgN. Since OmpR is required for the regulation of several porin genes (4, 6, 19), we tested whether it controlled kdgM and kdgN expression. While in an ompR background, kdgN expression decreased fivefold (Table 2), that of kdgM increased fourfold (data not shown). The effect of additional mutations in kdgR or pecS in the ompR mutant was analyzed. The kdgR mutation elevated kdgN expression, indicating that OmpR and KdgR regulate this gene by two independent pathways. In contrast, the level of kdgN expression in the double ompR pecS mutant was equivalent to that in each single mutant. Thus, ompR and pecS may be involved in the same regulation pathway. To verify that the OmpR regulation observed at the transcriptional level results in a variation at the protein level, we examined the protein content in the cell envelope. As measured by fusion assays, the amount of KdgM increased greatly in the ompR mutant while that of KdgN decreased (Fig. 1). The EnvZ-OmpR regulatory system has been reported to be involved in the osmoregulation of porin expression (19). We therefore tested kdgN transcription in media of increasing osmolarity (Fig. 4). In the range tested, kdgN expression was modified only moderately (1.5-fold) by the osmolarity of the medium and was always higher than that in an ompR mutant. hns is a global regulator of D. dadantii pectate lyase genes (15), but it is also, in other bacteria, a regulator of some porin gene expression (6). The level of expression of kdgN decreased in an hns mutant (Table 2). Since hns is often involved in regulation in response to variations in environmental conditions, we also tested the regulation of kdgN by osmolarity in this background. The expression of kdgN was strongly modified. The optimum observed at medium osmolarity was no longer present, and the activity decreased greatly when the osmolarity increased (Fig. 4).


Figure 4
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  		FIG. 4. Osmoregulation of kdgN. Strain A4205 (kdgN uidA; gray bars) and an hns (white bars) or an ompR (black bars) derivative were grown in M63 medium diluted twofold and containing glycerol as the carbon source and increasing concentrations of NaCl. The ß-glucuronidase specific activity of the fusion was measured and expressed as nanomoles of o-nitrophenol formed per minute per milligram of bacteria (dry weight). The bars indicate the means of results from four experiments, and the error bars indicate the standard deviations.

 
Growth phase regulation of kdgN. When the expression of kdgN during the growth of bacteria in LB-rich medium was monitored, a strong increase in the activity of the gene was observed, beginning at optical densities of 0.5 to 0.7 (Fig. 5). This effect was not observed when M63 minimal medium was used for growth. We tried to identify the gene responsible for this effect. Neither the kdgN regulators identified in this study (kdgR, pecS, ompR, hns, and crp) nor the regulator known to be involved in quorum sensing in D. dadantii (expR) (14) was found to be involved in this growth phase-dependent regulation. Growth in conditioned LB medium did not change the induction profile (data not shown). Thus, a diffusible compound is not responsible for this effect.


Figure 5
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  		FIG. 5. Growth phase dependence of kdgN expression. Strain A4205 (kdgN uidA) was grown in LB medium (diamonds) or M63 minimal medium plus mannitol (squares). Aliquots were taken, and ß-glucuronidase activity was measured. The specific activity of the culture is expressed in nanomoles of o-nitrophenol formed per minute per milligram of bacteria (dry weight) as a function of the optical density (OD) at 600 nm.

 
Characterization of the kdgN promoter. Primer extension analysis with RNA extracted from the kdgK strain A576 induced with galacturonate allowed us to determine a single kdgN transcription start site positioned 64 bases upstream of the translation start site (Fig. 6A). A good –10 sequence (TAGAAT) is separated by 18 nucleotides from a poor –35 region (CACACA). Regulatory proteins controlling kdgN expression, whose binding consensus is known, may be positioned relative to the promoter. A potential CRP-binding site is present, centered at –40.5, a position optimal for a CRP-activated gene. A good KdgR-binding site (ATGAAAN5TTTCAT) is centered at position –68. This position corresponds to the UP site of the promoter, and the binding of KdgR there may explain the observed repressor role. A genome-wide search for PecS-binding sites detected a potential site in front of kdgN at position –84 (27). At this position, PecS may interact with the C-terminal part of the {alpha} subunit of the RNA polymerase, which would explain its activator role. An OmpR-binding consensus has been defined using the OmpR sites located in front of the E. coli ompF and ompC genes (8). No sequence presenting homology to this consensus in the regulatory region of kdgN could be detected.


Figure 6
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  		FIG. 6. (A) Sequence of the kdgN promoter region. The translation initiation codon is shown in bold letters. The determined promoter is indicated by an arrow. The –10 and –35 regions are highlighted in gray. The position of a potential CRP-binding site is indicated by a light gray box, that of a KdgR-binding site is indicated by a white box, and that of a PecS-binding site is highlighted in black. The numbering is from the transcription start site. (B) PCR fragments 1 (from –50 to +100), 2 (from –142 to +8), 3 (from –222 to –72), and 4 (from –302 to –152) separated on 7.5% PAGE at +4°C. The numbering is from the transcription start site. The size of the DNA standard (bp) is indicated on the right.

 
H-NS has been shown to bind preferentially to curved DNA (32). To test the possibility that the kdgN promoter may be curved, we tested the migration of four overlapping 150-bp DNA fragments covering the kdgN promoter by PAGE at 4°C. At this temperature, curved DNA migrates more slowly than straight DNA, and the more centered the curvature is, the slower the migration is. Fragment 3 (–222 to –72) was the most retarded, and fragment 2 (–142 to +8) had an intermediate rate of migration (Fig. 6B). Thus, a curvature is present near the middle of fragment 3 and close to the end of fragment 2, probably around position –130 relative to the kdgN transcription start site. Thus, H-NS may interact with this region.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
KdgN was detected in D. dadantii as a homologue of KdgM, a monomeric outer membrane channel involved in the transport of oligogalacturonates. The main biochemical and electrophysiological characteristics of KdgM and KdgN are similar. Both proteins behave as monomers in SDS-PAGE and form voltage-dependent pores of similar conductances under identical conditions. KdgM channels were observed to close at high membrane potentials, while negative potentials had no effect, a feature which is rather unusual for porin channels. Such an asymmetry, but less marked, was also observed for KdgN channels. However, in contrast to KdgM, KdgN displayed various subconductances. KdgM was found to be weakly selective for anions, and in keeping with its role in oligogalacturonate transport, it was observed to be blocked by trigalacturonate (2). Although KdgN shows the same anion selectivity as KdgM, we could not detect any obvious block of this channel by trigalacturonate. It is worth noting that NanC, another anionic channel of the KdgM family found in E. coli, was also not blocked by its substrate, sialic acid (4). Here, our growth experiments clearly showed that KdgN is able to provide a pathway for trigalacturonate through the outer membrane. Therefore, the present studies have not revealed a major functional difference between the two D. dadantii proteins. As discussed below, this redundancy of outer membrane channels involved in the same metabolic pathway can probably be accounted for by a difference in regulation.

Indeed, the regulation of kdgN expression is very complex. We have identified five regulators controlling its expression: KdgR, PecS, CRP, OmpR, and H-NS. Other regulators may be involved in this regulation since we could not identify the mechanism responsible for strong regulation by growth phase in rich medium. This feature has not been described for other growth phase-regulated D. dadantii genes and may rely on a yet-unidentified regulation mechanism. The positions of KdgR- and PecS-binding sites on the kdgN promoter predicted by bioinformatic analysis can explain the results obtained with assays of the kdgN transcriptional fusion: the KdgR-binding site is centered at position –68 from the transcription start site, which corresponds to the UP site of the promoter, and KdgR may prevent RNA polymerase binding. The PecS-binding site is predicted to be located at position –84, where PecS may interact with the C-terminal part of the {alpha} subunit of the RNA polymerase and serve as an activator. PecS is usually a repressor, but an activator role of PecS for the polygalacturonase gene pehX has been described previously (16). However, in that case, PecS was acting as an antirepressor by preventing KdgR binding. This type of regulation does not apply to kdgN since fusion assays show that the two regulators act independently. Thus, a direct activation of kdgN by PecS would be a new mode of action of this versatile regulatory protein (27). OmpR is an activator of kdgN. In E. coli, OmpR is partly responsible for the osmoregulation of ompF and ompC (7). The effect of osmolarity on kdgN expression is much weaker than that of an ompR mutation, and the strong regulation of kdgN by osmolarity only in an hns mutant was observed. Thus, the effects of osmolarity and ompR on kdgN regulation seem complex and may involve other partners such as regulatory RNAs, like the E. coli MicF and MicC (3).

kdgM and kdgN are both repressed by KdgR and activated by CRP. ExuR controls only kdgM, and kdgM and kdgN are regulated in opposite ways by PecS and OmpR. The opposite regulation of a couple of porins has already been described. The E. coli general porins OmpF and OmpC are differentially regulated by OmpR and CpxR (1). A possible explanation for the regulation by OmpR implicated a difference in the pore size of the proteins. The OmpF pore seems larger than that of OmpC (17), and compounds such as antibiotics, toxins, and bile salts diffuse better through the OmpF channel. In environments such as freshwater, a medium of low osmolarity, OmpF synthesis is favored, allowing easier entry of diluted nutrients. When the bacteria are in intestinal tracts, a medium of high osmolarity, OmpF synthesis is decreased and that of OmpC is increased, thus preventing the uptake of toxic bile salts. Regulation by CpxR of porin synthesis may respond to the same rationale: the induction of the Cpx pathway by toxins reduces OmpF synthesis, limiting the entry of the toxins into the bacteria (1). A pattern of opposite regulation of a couple of specific outer membrane channels, such as those of kdgM and kdgN by PecS and OmpR, has never been described before. KdgM and KdgN seem to have the same substrates, oligogalacturonates. There may be differences in their affinity for a type of substrate (involving the length of the galacturonate chain or the presence of a double bond on the sugar at the nonreducing end), but the nonavailability of these potential substrates prevented such studies. kdgM is located in an operon of genes involved in pectin degradation. It is probably expressed, with all the pectinolysis genes, when the bacteria infect a plant. Under this condition, the bacteria do not need two oligogalacturonate-specific channels. This may explain the repression of KdgN by PecS, a regulator of virulence factor synthesis. However, it may be advantageous for D. dadantii, which is also able to live as a saprophytic bacterium, to be able to get pectin or pectin breakdown products, a good carbon source, when it is in the soil. Under this condition, KdgM expression would be at a basal level and KdgN expression would be induced. The higher level of KdgN expression than of KdgM expression observed in bacteria grown in glycerol (Fig. 1), an example of noninducing conditions, may be an illustration of the more important role of KdgN when the bacteria are outside a plant. Interestingly, Yersinia pestis and Y. enterocolitica also possess two KdgM-type proteins, and Pectobacterium atrosepticum possesses four (25). In these bacteria, the gene of the KdgM orthologue is clustered with genes required for oligogalacturonate transport, while the genes of other homologues are either clustered with a pectate lyase gene (Y. pestis, Y. enterocolitica, and two P. atrosepticum genes) or found alone (one P. atrosepticum gene). Although their regulation has not been studied, the presence of several copies of these genes may also reflect the need for the differential expression of the corresponding porins.


   ACKNOWLEDGMENTS
 
We thank William Nasser for strains and advice, Sylvie Reverchon, Vladimir Shevchik, and Nicole Cotte-Pattat for reading the manuscript, and Samia Habbas for experimental help.

This work was supported by grants from the Centre National de la Recherche Scientifique and from the Ministère de l'Enseignement Supérieur et de la Recherche.


   FOOTNOTES
 
* Corresponding author. Mailing address: Unité Microbiologie Adaptation et Pathogénie, UMR 5240 CNRS-UCB-INSA-Bayer CropScience, Bat Lwoff, 10 rue Raphaël Dubois, Université Lyon 1, 69622 Villeurbanne, France. Phone: (33) 472 44 58 27. Fax: (33) 472 43 15 84. E-mail: guy.condemine{at}insa-lyon.fr Back

{triangledown} Published ahead of print on 15 June 2007. Back


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Journal of Bacteriology, August 2007, p. 5955-5962, Vol. 189, No. 16
0021-9193/07/$08.00+0     doi:10.1128/JB.00218-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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