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J Bacteriol. 2009 November; 191(22): 6960–6967.
Published online 2009 September 4. doi:  10.1128/JB.00594-09
PMCID: PMC2772473

Catabolism of Raffinose, Sucrose, and Melibiose in Erwinia chrysanthemi 3937 [down-pointing small open triangle]

Abstract

Erwinia chrysanthemi (Dickeya dadantii) is a plant pathogenic bacterium that has a large capacity to degrade the plant cell wall polysaccharides. The present study reports the metabolic pathways used by E. chrysanthemi to assimilate the oligosaccharides sucrose and raffinose, which are particularly abundant plant sugars. E. chrysanthemi is able to use sucrose, raffinose, or melibiose as a sole carbon source for growth. The two gene clusters scrKYABR and rafRBA are necessary for their catabolism. The phenotypic analysis of scr and raf mutants revealed cross-links between the assimilation pathways of these oligosaccharides. Sucrose catabolism is mediated by the genes scrKYAB. While the raf cluster is sufficient to catabolize melibiose, it is incomplete for raffinose catabolism, which needs two additional steps that are provided by scrY and scrB. The scr and raf clusters are controlled by specific repressors, ScrR and RafR, respectively. Both clusters are controlled by the global activator of carbohydrate catabolism, the cyclic AMP receptor protein (CRP). E. chrysanthemi growth with lactose is possible only for mutants with a derepressed nonspecific lactose transport system, which was identified as RafB. RafR inactivation allows the bacteria to the assimilate the novel substrates lactose, lactulose, stachyose, and melibionic acid. The raf genes also are involved in the assimilation of α- and β-methyl-d-galactosides. Mutations in the raf or scr genes did not significantly affect E. chrysanthemi virulence. This could be explained by the large variety of carbon sources available in the plant tissue macerated by E. chrysanthemi.

Pectinolytic erwiniae are enterobacteria that cause disease in a wide range of plants, including many crops of economic importance (23). The soft-rot symptom produced by Erwinia chrysanthemi (syn. Dickeya dadantii) results from the degradation of polysaccharides involved in the cohesion of the plant cell wall. The plant tissue maceration is concomitant with a large increase in the bacterial population (13). To ensure this multiplication, the bacteria assimilate various oligosaccharides released in the macerated tissue, which provide carbon and energy sources.

E. chrysanthemi is known to use several carbon sources for growth, including sugars ranging from monosaccharides to polysaccharides. The completion of the E. chrysanthemi strain 3937 genome provides a genome-scale view into its potential catabolic capacities. A substantial part of the E. chrysanthemi genome is dedicated to genes involved in carbohydrate catabolism. In plant tissues, the most abundant soluble carbohydrates are the two oligosaccharides sucrose and raffinose (32). The trisaccharide raffinose [α-d-Galp-(1→6)-α-d-Glcp-(1[left arrow over right arrow]2)β-d-Fruf] and the related disaccharides sucrose [α-d-Glcp-(1[left arrow over right arrow]2)β-d-Fruf] and melibiose [α-d-Galp-(1→6)-d-Glcp] are used as carbon sources for E. chrysanthemi growth. Previous studies suggested links between the transport of lactose and that of raffinose and melibiose (15). The E. chrysanthemi wild-type strain 3937 does not use lactose [β-d-Galp-(1→4)-d-Glcp] as a carbon source for growth. This is due to the lack of a specific lactose transport system. However, spontaneous mutants able to assimilate lactose (designated Lac+) are easily obtained; they show a deregulation of the transport system LmrT, which is able to mediate lactose, melibiose, and raffinose transport (15). Despite our current knowledge of the strain 3937 genome sequence, no open reading frame (ORF) could be assigned to the lmrT gene, the identity of which remains unknown. We analyzed the E. chrysanthemi genome for the presence of potential genes involved in the catabolism of α-galactosides or α-glucosides. It contains a complete scrKYABR gene cluster that is involved in sucrose catabolism in various enterobacteria and a truncated rafRBA locus that is involved in raffinose catabolism. The growth with raffinose, despite the presence of an incomplete raf cluster, suggests that the missing functions are provided by other genes. Moreover, while E. chrysanthemi can catabolize melibiose, its genome does not contain homologues of the Escherichia coli melABR genes (30). Thus, to assimilate melibiose, E. chrysanthemi exploits other genes, which have yet to be identified. The present study mainly reports the role of the E. chrysanthemi gene clusters scr and raf in the catabolism of the oligosaccharides sucrose, raffinose, melibiose, and lactose. The importance of such catabolic pathways for bacterial multiplication in the plant tissues also was assessed during the infection process.

MATERIALS AND METHODS

Strains, media, and growth conditions.

The E. chrysanthemi strains and the plasmids used in this study are listed in Table Table1.1. The [var phi]EC2 generalized transducing phage was used for transduction (25). E. chrysanthemi cells were grown at 30°C in M63 medium (20). Carbon sources were added at 2 g·liter−1. Escherichia coli cells were grown at 37°C in Luria-Bertani (LB) medium (20). The media were solidified with agar (15 g·liter−1). When required, antibiotics were added at the following concentrations: kanamycin (Km), 20 μg·ml−1; ampicillin (Ap), 50 μg·ml−1; and chloramphenicol (Cm), 20 μg·ml−1.

TABLE 1.
Bacterial strains, plasmids and primers used in this study

Strains were tested on Biolog Phenotype Microarray plates PM1 and PM2 using standard protocols (Biolog Corporation, Hayward CA). This system reports the bacterial utilization of various carbon sources in 96-well plates by detecting a color change due to the reduction of a tetrazolium dye in response to cellular respiration (4).

Recombinant DNA techniques.

The preparation of plasmid or chromosomal DNA, restriction digestions, ligations, DNA electrophoresis, and transformations were carried out as previously described (28).

Sequence data were obtained from the genome-sequencing project for E. chrysanthemi strain 3937 (http://asap.ahabs.wisc.edu/asap/ASAP1.htm).

PCR primers (22 to 26 mers) were designed to amplify 0.95 to 2.2 kb of E. chrysanthemi chromosomal DNA (Table (Table1).1). Restriction sites were added to each primer to facilitate the determination of the DNA orientation in the vector (usually BamHI at the 5′ end and XbaI at the 3′ end). The PCR products were purified (QIAquick PCR purification kit; Qiagen) and ligated to the pGEM-T vector (Promega), which has a protruding T nucleotide at each 3′ end.

Genetic fusions were constructed on the cloned genes by the insertion of the nonpolar uidA-Km cassettes (3) into a restriction site situated inside the corresponding ORF, namely the EcoRI site of rafA, the SmaI site of rafB, the EcoRI site of scrB, and the EcoRI site of scrY. The orientation of the uidA-Km cassette was determined by restriction analysis. Only plasmids in which uidA and the mutated gene have the same transcriptional direction were retained. The regulatory genes were inactivated by the insertion of the nonpolar CKC15 Cmr cassette (18) into the SalI and Eco47III sites situated in rafR and scrR, respectively. Plasmids bearing the uidA-Kmr or Cmr insertions then were introduced into E. chrysanthemi cells by electroporation. The insertions were integrated into the E. chrysanthemi chromosome by marker exchange recombination after successive cultures in low-phosphate medium in the presence of the appropriate antibiotic (27). The verification of the correct recombination of the insertions was performed by PCR.

Enzyme and uptake assays.

For α-galactosidase and ß-glucuronidase assays, strains were grown in minimal medium at 30°C to late exponential growth phase. Toluene was added to partially disrupt the cell membranes. To measure the α-galactosidase activity, the formation of p-nitrophenol, after the cleavage of p-nitrophenyl-α-d-galactoside (pNPαGal), was monitored at 405 nm. The reactions were performed with 1 mM substrate, and potential inhibitors were used at a final concentration of 10 mM. To estimate the expression of the gene fusions, ß-glucuronidase activity was measured by the degradation of p-nitrophenyl-ß-d-glucuronide (3.2 mM) into p-nitrophenol at 405 nm. The specific activities of α-galactosidase and ß-glucuronidase are expressed as nanomoles of product liberated per minute per milligram of bacterial dry weight.

To assay the lactose transport, the bacterial cultures were grown to mid-log phase. The uptake of [14C]lactose (1 mM) by whole cells was measured at room temperature by the Millipore filtration technique (15). Inhibitions were tested in the presence of a 10-fold excess of each unlabeled competitor.

Pathogenicity tests and competition assays.

Chicory leaves were inoculated as previously described (12, 34). Leaves were slightly wounded with pipette tips prior to inoculation. Ten leaves were infected for each strain, using 106 bacteria per inoculation site. After incubation in a dew chamber for 24 h, the length of rotted tissue was measured to estimate disease severity. The decayed tissue was recovered. β-Glucuronidase assays and bacterial cell numeration were performed on the macerated tissues to estimate the expression of gene fusions during plant infection (19).

Mutants resistant to kanamycin (Kmr) were used to analyze their competitive capacity when mixed with the wild-type strain during infection. Chicory leaves were inoculated with a mixture of the two strains at a ratio of 50/50. Macerated tissues were recovered 24 h after infection. After the serial dilution of the macerated tissue, the replica plating of the appropriate dilutions on medium containing kanamycin allowed us to determine the ratio between mutant (Kmr) and wild-type clones. The expected ratio after infection, if no competition occurred, was 50%.

RESULTS AND DISCUSSION

Genome analysis reveals two potential clusters for catabolism of sucrose and raffinose.

The strain 3937 genome was analyzed for the presence of genes potentially involved in the catabolism of sucrose, melibiose, and raffinose. It contains a group of genes homologous to those involved in sucrose assimilation in other enterobacteria. This cluster includes five adjacent genes transcribed in the same direction, scrKYABR (Fig. (Fig.1).1). Its products are homologous (55 to 81% identity) to proteins encoded by the chromosome of Erwinia amylovora and Klebsiella pneumoniae but also by enterobacterial mobile genetic elements, the plasmid pUR400 of E. coli or Salmonella spp., and the transposon Tn2555 of E. coli (2, 6, 9, 11, 17, 31). The genetic organization of these sucrose utilization loci is well conserved (24), having the same gene order as that observed in E. chrysanthemi (Fig. (Fig.1).1). In E. amylovora, these genes are organized in three transcriptional units, scrK, scrYAB, and scrR (6). This cluster encodes a fructose kinase (ScrK), an outer membrane porin (ScrY), the sucrose-specific enzyme II of a phosphoenolpyruvate-dependent phosphotransferase system (ScrA), a sucrose hydrolase (ScrB), and a regulator of the LacI family (ScrR).

FIG. 1.
Genetic organization of the scr and raf clusters in E. chrysanthemi strain 3937. E. chrysanthemi contains a cluster of scr genes (ID 16549, 16548, 16547, 16546, and 16544) probably organized in three contiguous operons, scrK-scrYAB-scrR, and coding for ...

The E. chrysanthemi genome also contains a group of three contiguous genes (Fig. (Fig.1)1) whose products share 55 to 73% identity with proteins involved in raffinose catabolism and are encoded by the plasmid pSRD2, which is found in a few E. coli strains (1). These plasmidic genes are organized in two adjacent operons, rafR and rafABDY. The gene rafR encodes a regulator of the LacI family, rafA encodes an α-galactosidase that cleaves raffinose into galactose and sucrose, rafB encodes a transport system of the LacY family, rafD encodes a sucrose hydrolase, and rafY encodes a porin (1, 33). The E. chrysanthemi cluster raf includes only the first three genes: rafR, rafA, and rafB (Fig. (Fig.1).1). Thus, in E. chrysanthemi, RafD and RafY have to be functionally replaced by another hydrolase and porin, respectively.

Assimilation of sucrose, melibiose, and raffinose by E. chrysanthemi.

The growth of the E. chrysanthemi wild-type strain 3937 was tested in minimal medium supplemented with various sugars as the sole carbon and energy source. Strain 3937 is able to use sucrose, melibiose, and raffinose as carbon sources for growth. In contrast, lactose was unable to support the growth of the wild-type strain. The growth rate was determined in the presence of these oligosaccharides and also with their constitutive monosaccharides, namely glucose, galactose, and fructose (Table (Table2).2). Doubling times shorter than 2 h were observed with fructose, glucose, and sucrose, which thus are readily utilizable carbon sources. Melibiose, galactose, and raffinose gave doubling times ranging between 2 and 3 h (Table (Table22).

TABLE 2.
Growth phenotype of E. chrysanthemi wild-type and mutant strains

Effect of raf and scr inactivation on oligosaccharide assimilation.

The structural genes rafA, rafB, scrB, and scrY were inactivated by nonpolar insertions (Fig. (Fig.1).1). The growth phenotypes of the mutants were compared to that of the wild-type strain (Table (Table2).2). The RafA and RafB mutants became unable to grow with either raffinose or melibiose. The ScrB mutant was incapable of growth with either sucrose or raffinose. The ScrY mutant showed reduced growth with either sucrose or raffinose. The doubling time of the ScrY mutant is about threefold longer than that of the wild-type strain in the presence of either sucrose or raffinose (Table (Table2).2). These growth data confirmed that the E. chrysanthemi genes raf and scr are involved in the catabolism of raffinose and sucrose, respectively. In addition, they reveal novel roles of these genes. First, the rafA and rafB genes are responsible for the catabolism of melibiose. Second, the genes scrB and scrY are involved in raffinose catabolism.

Even if their name does not reflect their double function, the raf genes appear to contribute equally to the catabolism of melibiose and raffinose in E. chrysanthemi (Fig. (Fig.2).2). The transporter and the α-galactosidase encoded by rafBA are sufficient for melibiose catabolism. Raffinose catabolism needs rafA, rafB, and two additional steps provided by scrY and scrB. The porin ScrY facilitates the diffusion of both sucrose and raffinose across the outer membrane. The enzyme ScrB hydrolyzes both sucrose and sucrose-6-phosphate (Fig. (Fig.2).2). Thus, ScrY and ScrB functionally replace the RafY and RafD proteins encoded by the plasmidic raffinose clusters. Previous studies reported functional homologies between Scr and Raf proteins. The substrate range of the plasmid-encoded porins ScrY and RafY were shown to be similar, and since they are not strictly specific, both are able to facilitate the diffusion of sucrose and raffinose (33). Similarly, it was noticed that the hydrolase ScrB resembles RafD in terms of substrate specificity (29). Thus, the genes rafY and rafD are dispensable when the genome contains the genes scrY and scrB. This explains the functionality of the incomplete raf cluster in E. chrysanthemi.

FIG. 2.
Role of the Raf and Scr proteins in the assimilation of sucrose, raffinose, melibiose, and lactose in E. chrysanthemi. The structure of the oligosaccharides is given. Scr, sucrose; Raf, raffinose; Mel, melibiose; Lac, lactose; Glc, glucose; Gal, galactose; ...

To test a large range of carbon sources, the mutants were compared to the wild-type strain on Biolog Phenotype Microarray plates for carbon catabolism (4). The RafB and ScrB mutants were affected only for the phenotypes reported in Table Table2.2. The ScrY mutant had no visible phenotype. The RafA mutant appeared unable to assimilate α-methyl-d-galactoside in addition to raffinose and melibiose. Thus, the α-galactosidase RafA also is necessary for the cleavage of α-methyl-d-galactoside.

Effect of regulator mutations on oligosaccharide assimilation.

Since the cyclic AMP receptor protein (CRP) is involved in the global control of carbohydrate catabolism, the assimilation of several sugars is abolished in a CRP mutant. An E. chrysanthemi CRP mutant was shown to be able to use glucose but was unable to grow with either glycerol, gluconate, galacturonate, or polygalacturonate as the sole carbon source (26). We tested its growth phenotype with other sugars (Table (Table2).2). The CRP mutant is unable to use raffinose, melibiose, and galactose. However, it remains able to assimilate fructose, showing normal growth, or sucrose, with decreased growth. Thus, the global regulator CRP is necessary for the activation of the catabolism of both raffinose and melibiose. CRP also is involved in sucrose catabolism, but with a weaker effect.

The potential repressor genes associated with each cluster, scrR and rafR, were inactivated by the insertion of a nonpolar Cmr cassette (Fig. (Fig.1).1). The RafR and ScrR mutants showed normal growth with sucrose, melibiose, raffinose, fructose, glucose, or galactose (Table (Table2).2). In contrast, the RafR mutant has acquired the capacity to use lactose as the sole carbon source for growth. This observation suggested a direct role of the raf cluster in lactose assimilation.

Using the Biolog Phenotype Microarray plates for carbon catabolism (4), the ScrR mutant had no visible modified phenotype. The RafR mutant became able to assimilate lactose and also lactulose [β-d-Galp-(1→4)-d-Fruf], melibionic acid [α-d-Galp-(1→6)-d-GlcA], and stachyose [α-d-Galp-(1→6)-α-d-Galp-(1→6)-α-d-Glcp-(1[left arrow over right arrow]2)β-d-Fruf]. The RafR mutant also showed increased growth with β-methyl-d-galactoside. Thus, the derepression of the raf genes could trigger the assimilation of various oligosaccharides related to their substrates, melibiose and raffinose. The raf cluster facilitates either the uptake of these compounds (lactose, lactulose, and β-methyl-d-galactoside), their cleavage (α-methyl-d-galactoside), or both activities (melibionic acid and stachyose).

Role of RafB in lactose assimilation.

Lac+ derivatives of strain 3937 results from the deregulation of a nonspecific transport system previously named LmrT (15). The ability of the RafR mutant to grow with lactose raises two hypotheses: (i) the Lac+ derivatives arise from RafR inactivation, and (ii) LmrT corresponds to RafB.

To verify the first hypothesis, 12 spontaneous Lac+ derivatives of the strain 3937 were analyzed. The strain A350 (also named L2), which often has been used to construct lacZ gene fusions, was included in this analysis. This strain is a spontaneous Lac+ mutant (lmrTc) that then was submitted to mutagenesis to inactivate the main β-galactosidase activity (16). Considering their genetic organization, the genes rafA and rafB probably are cotranscribed from a RafR-regulated promoter situated upstream of rafA (Fig. (Fig.1).1). The rafA expression was tested by a direct α-galactosidase assay. In the RafA mutant, no α-galactosidase activity could be detected, confirming that this activity is due entirely to RafA. In the strain 3937, the α-galactosidase activity was very low in the presence of glycerol as the carbon source, but it increased in the presence of raffinose, melibiose, or galactose (Table (Table3).3). In the RafR mutant, α-galactosidase was produced at a high level in all growth conditions used (Table (Table3).3). In the 12 Lac+ derivatives tested, the α-galactosidase activity also was highly derepressed (Table (Table3).3). Thus, the Lac+ phenotype is correlated with the derepression of rafA. Most of the spontaneous Lac+ mutants are RafR mutants.

TABLE 3.
α-Galactosidase activity in E. chrysanthemi cells grown in the presence of different carbon sources

The RafB mutant was unable to provide Lac+ derivatives, while they were obtained at the frequency of 10−7 with the wild-type strain. While the RafR mutant is able to assimilate lactose, the RafR RafB double mutant has lost this capacity. Thus, an intact rafB gene is required for lactose uptake. The transport system previously named LmrT is, in fact, RafB. This also is consistent with biochemical studies showing that LmrT is more specific to melibiose and raffinose than to lactose (10, 15). Lactose cannot induce the raf genes but will be taken up through RafB when expressed, as in the RafR mutant. In a previous report, it was shown that the β-galactosidase activity of E. chrysanthemi is due mostly to GanB, an enzyme involved in galactane degradation (8). Thus, in this enterobacterium adapted to a plant host, lactose assimilation results from the secondary activities of proteins involved in the catabolism of plant oligosaccharides. The truncated lactose operon found in the E. chrysanthemi genome (lacI-lacZ) has minor functional significance.

Specificity of RafB and RafA for raffinose and melibiose.

Since RafB is responsible for lactose transport in E. chrysanthemi, its activity was measured by the assay of [14C]lactose uptake in whole cells of a RafR mutant grown in glycerol minimal medium. To assess the RafB specificity, [14C]lactose uptake was assayed in the presence of a 10-fold excess of unlabeled oligosaccharides used as competitors (Table (Table4).4). Melibiose was a strong inhibitor of lactose uptake. Raffinose inhibited lactose uptake to a lesser extent. Sucrose did not exhibit any effect. This inhibition pattern suggests that RafB has a higher affinity for the disaccharide melibiose than for the trisaccharide raffinose.

TABLE 4.
Inhibition of RafB and RafA activities by different sugars

To analyze the affinity of RafA for melibiose and raffinose, inhibition experiments were performed using the α-galactosidase assay based on the cleavage of a chromogenic substrate (Table (Table4).4). This reaction was strongly inhibited by melibiose, and it was weakly inhibited by raffinose, glucose, and galactose. Sucrose had no effect. RafA thus is inhibited by its substrates and by its reaction products, glucose and galactose. The comparison of melibiose and raffinose effects suggests that RafA has more affinity for the disaccharide than for the trisaccharide.

These biochemical data indicate that, despite their names, the rafA and rafB genes of E. chrysanthemi are better adapted to melibiose catabolism than to raffinose catabolism. This also could be true in other bacteria in which the raf genes have been identified previously and in which melibiose catabolism was not analyzed simultaneously. In Enterobacter cloacae, the RafB homologue (77% identity) was named MelY (22), since it was identified as being a melibiose transporter, but its role in raffinose assimilation has not been tested.

Expression of the rafA and rafB genes.

The insertion of uidA-Km cassettes in the rafA and rafB genes allowed us to inactivate these genes and also to obtain transcriptional fusions (3). The expression of these fusions was monitored in various conditions (Table (Table5).5). The transcription of the rafA fusion strongly increased in the presence of raffinose, melibiose, and galactose (Table (Table5).5). The direct assay of α-galactosidase also indicated that RafA synthesis is induced in the presence of these sugars (Table (Table3).3). Since the RafA mutant is unable to metabolize raffinose and melibiose (Table (Table2),2), this induction indicates that the oligosaccharides are inducers without further cleavage by RafA (Fig. (Fig.2).2). The low induction of the rafB fusion in the presence of the rafA inducers (Table (Table5)5) could be explained by the absence of the specific uptake of the sugars when the RafB transporter is inactivated. Sucrose, lactose, or fructose did not induce the rafA or rafB fusions (data not shown).

TABLE 5.
Expression of raf transcriptional fusions in Erwinia chrysanthemi

To analyze the potential effect of the regulators, the rafA and rafB fusions were transferred to RafR and CRP mutants using transduction with the [var phi]EC2 phage. When the RafR repressor was inactivated, the expression of the rafA and rafB fusions highly increased in the absence of inducers (Table (Table5).5). The decreased rafA and rafB expression in the presence of glucose and in the CRP mutant (Table (Table5)5) indicated that these genes are submitted to catabolite repression and that CRP is necessary for their activation. This is consistent with the fact that a CRP mutant is unable to use raffinose or melibiose as its sole carbon and energy source (Table (Table2).2). The examination of the raf cluster for conserved regulatory sequences revealed the presence of potential CRP and RafR binding sites in the 5′ noncoding end of rafA (Fig. (Fig.1).1). The nucleotide sequence upstream of rafA contains two repeated 14-bp sequences (CCGAAACGTTTCGG) included in the imperfect palindrome identified as the RafR binding site (1). This suggests that regulation by RafR in E. chrysanthemi results from a mechanism similar to that previously characterized in E. coli (21). The absence of a potential CRP or RafR binding site in the 5′ noncoding end of rafB, despite its in vivo regulation by CRP and RafR (Table (Table5),5), is a strong indication that rafB is cotranscribed with rafA and that these regulation events occur at the level of the rafA promoter.

Expression of the scrB and scrY genes.

Transcriptional fusions in the scrB and scrY genes also were obtained by the insertion of uidA-Km cassettes. The transcription of the scrY fusion was highly stimulated in the presence of sucrose and raffinose (Table (Table6).6). Since the inactivation of the porin ScrY reduces the growth rate with each of these oligosaccharides (Table (Table1),1), it decreases their uptake without preventing induction. The transcription of the scrB fusion was not significantly affected by the addition of sucrose or raffinose (Table (Table6).6). This absence of induction suggests that ScrB is necessary for the formation of the intracellular inducer, with such formation being prevented in the ScrB mutant. The sucrose hydrolase ScrB cleaves sucrose-6-phosphate to give glucose-6-phosphate and fructose (Fig. (Fig.2).2). In E. amylovora, the inducer of the scr cluster is supposed to be fructose (6). In E. chrysanthemi, the addition of fructose to the culture medium provoked only a low induction of the scrY fusion (Table (Table6).6). Extracellular fructose is transported into the cells by a specific phosphoenolpyruvate-dependent phosphotransferase system; this transport is concomitant with substrate phosphorylation, resulting in the intracellular formation of fructose-1-phosphate (Fig. (Fig.2).2). Thus, fructose-1-phosphate is not a good inducer of the scrY gene. In E. chrysanthemi, the true scr inducers are formed after the ScrB step; they are either fructose, fructose-6-phosphate (formed by ScrK), or both compounds (Fig. (Fig.2).2). The addition of glucose decreased both scrB and scrY expression (Table (Table6),6), indicating that these genes are submitted to catabolite repression.

TABLE 6.
Expression of scr transcriptional fusions in Erwinia chrysanthemi

To verify the functionality of the ScrR repressor, the fusions were transferred by transduction into the ScrR mutant. The expression of the scrB and scrY fusions strongly increased in the ScrR mutant, surpassing the levels observed in the wild-type background (Table (Table6).6). The expression of scrB or scrY decreased in the CRP mutant (Table (Table6),6), indicating that CRP is involved in their activation. The examination of the scr cluster revealed a sequence homologous to the consensus of the E. coli CRP binding site (7) in the 5′ noncoding end of scrY and revealed a sequence homologous to the palindrome involved in ScrR binding (6, 17) upstream of scrK and scrY (Fig. (Fig.1).1). The absence of this palindrome upstream of scrB or scrA suggests that these two genes are cotranscribed with scrY.

The analysis of the scrB and scrY fusions in the RafR mutant indicated that RafR is not involved in the control of these genes. Similarly, the analysis of the rafA and rafB fusions in the ScrR mutant indicated that ScrR is not involved in the control of these genes either (data not shown). Despite common steps in the catabolism of raffinose and sucrose, scr and raf genes are independently controlled by ScrR and RafR, respectively.

Virulence of the Raf and Scr mutants and expression of the fusions during plant infection.

Chicory leaves were infected with the Raf and Scr mutants to analyze the effect of the mutations on virulence. We observed no significant difference in the degree of maceration caused by the RafA, RafB, ScrB, or ScrY mutant and the parental strain 3937 (Fig. (Fig.3A).3A). To determine whether the genes are expressed during infection, the expression of rafA, rafB, scrB, and scrY transcriptional fusions was assayed in the macerated tissues. A fusion in the highly inducible pectate lyase gene pelD (14) was used as the internal control. While no rafB expression was detected, the rafA, scrB, and scrY fusions all were expressed in the macerated tissue (Fig. (Fig.3B).3B). As shown previously, the absence of the uptake of the substrates in the RafB mutant prevents the induction of the fusion. The induction of rafA during infection shows that at least one of the substrates, raffinose or melibiose, is present in the macerated tissue. Similarly, the induction of the scrY fusion indicates that sucrose and/or raffinose is available in the macerated tissue. These oligosaccharides may provide nutritional resources for bacterial growth in planta. Bacterial numerations were performed 24 h after infection. No significant difference was observed between the cell densities of the mutants and the wild-type strain; the bacterial populations reached a level of about 4 × 109 bacteria per g of macerated tissue with each strain. The absence of an effect of the Raf or Scr mutation on bacterial multiplication is quite surprising, considering that sucrose and raffinose are available in the plant tissues. To highlight any fine differences between the mutants and the wild-type strain, we analyzed the ability of each mutant to compete with the wild-type strain during infection. The KdgM mutant, which is affected in the uptake of pectic oligomers (5), was used as the control. The RafA, RafB, or PelD mutant was able to multiply as well as the wild-type strain during infection (Fig. (Fig.3C).3C). In contrast, the KdgM and ScrY mutants were significantly impaired in their ability to compete with the wild-type strain. The decrease in the competitive ability of the KdgM mutant reflects the fact that oligogalacturonides are used by E. chrysanthemi for its multiplication in planta. Similarly, the decreased competitive capacity of the ScrY mutant suggests that the entry of sucrose and/or raffinose favors E. chrysanthemi growth in plant tissues. The inactivation of a catabolic pathway becomes apparent when the mutant has to compete with the wild-type strain. The variety of carbon sources available in the macerated tissues conceals the effect of a single mutation on the bacterial population.

FIG. 3.
Infection of chicory leaves with the Raf and Scr mutants. (A) After a 24-h infection with each mutant and the wild-type strain (WT), the length of rotted tissue was measured to estimate the disease severity. The mean values and the standard ...

Acknowledgments

We acknowledge members of the International Erwinia Consortium for the exchange of unpublished data concerning the E. chrysanthemi strain 3937 genome sequence. We thank Geraldine Effantin for her assistance with some experiments. We thank the ParMic Platform of UMR 5557 for the use of the Biolog equipment.

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

Footnotes

[down-pointing small open triangle]Published ahead of print on 4 September 2009.

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