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Guangxi Key Laboratory of Subtropical Bioresources Conservation and Utilization, The Key Laboratory of Ministry of Education for Microbial and Plant Genetic Engineering, and College of Life Science and Technology, Guangxi University, 100 Daxue Road, Nanning, Guangxi 530004, China
Correspondence
Ji-Liang Tang
jltang{at}gxu.edu.cn
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Swings & Civerolo, 1993). This bacterium produces extracellular polysaccharide (EPS) and extracellular enzymes (including amylase, endoglucanase, polygalacturonate lyase and protease), which are collectively essential for pathogenesis (Dow & Daniels, 1994). EPS, also called xanthan gum, is an important industrial biopolymer which is used in a great variety of food and industrial applications as a viscosifying, thickening, stabilizing and suspending agent (Kennedy & Bradshaw, 1984).
Carbohydrate metabolism in Xcc has been investigated in some detail, as it is central to both EPS production and the mechanism of bacterial colonization. Xcc metabolizes sugars through a central metabolic network similar to that described for Pseudomonas aeruginosa (Temple et al., 1998). Glucose and sucrose have been shown to be the best carbon sources for EPS production (García-Ochoa et al., 2000). Two distinct pathways are employed to mobilize extracellular glucose for EPS production and energy generation. In the first pathway, glucose is transported directly into the cell by a permease and undergoes an intracellular phosphorylation catalysed by glucose kinase (EC 2 . 7 . 1 . 2, hereafter Glk) to yield glucose 6-phosphate (Lessie & Phibbs, 1984; Letisse et al., 2001). Glucose 6-phosphate is then converted to gluconate by glucose-6-phosphate 1-dehydrogenase. Extracellular glucose can also be catabolized by an oxidative periplasmic pathway in which glucose is oxidized to gluconate via a periplasmic NAD(P)+-independent glucose dehydrogenase (Whitfield et al., 1982). This enzyme uses pyrroloquinoline quinone (PQQ) as cofactor (Duine & Jongejan, 1989). Gluconate is transported into the cell by an active transport system involving gluconate permease. Gluconate from the two pathways is converted to 6-phosphogluconate and may be further metabolized via the Entner–Doudoroff pathway to yield glyceraldehyde 3-phosphate and pyruvate (Zagallo & Wang, 1967). Some of these products may be recycled via gluconeogenesis for glucose phosphate production (Banerjee et al., 1987), while the remainder is oxidized via the tricarboxylic acid (TCA) cycle.
Glk has been predicted to be important for EPS production in Xcc (Letisse et al., 2001, 2002). The enzyme is responsible for phosphorylation of glucose using ATP as a donor to give glucose 6-phosphate and ADP. Although the eukaryotic Glks, such as yeast hexokinase B, Arabidopsis thaliana hexokinases and human hexokinase IV (HK4 or GCK, EC 2 . 7 . 1 . 1), are well characterized (Catanzano et al., 1997; Moore et al., 2003; Wilson, 2003), little is known about their bacterial and archaeal counterparts. A survey of the genome sequence data of two Xcc strains has revealed three genes predicted to encode Glk enzymes: XCC2137, XCC2886 and XCC2943 in strain ATCC 33913 (da Silva et al., 2002), and XC1166, XC1223 and XC1976 in strain 8004 (Qian et al., 2005). However, these genes and their protein products have not been experimentally investigated. Here we have examined the biochemical characteristics and physiological roles (particularly in EPS production) of the multiple Glk enzymes in Xcc strain 8004.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Escherichia coli strains were grown in LB medium (Miller, 1972) at 37 °C. Xcc strains were grown at 28 °C in NYG medium (per litre: 5 g peptone, 3 g yeast extract and 20 g glycerol; Daniels et al., 1984b) or the modified minimal medium MMX [per litre: 2.0 g (NH4)2SO4, 4.0 g K2HPO4, 6.0 g KH2PO4, 0.2 g MgSO4 . 7H2O, 1.0 g citric acid, 5.0 g sugar; Daniels et al., 1984a]. Antibiotics were added at the following concentrations as required: kanamycin (Kan), 25 µg ml–1; rifampicin (Rif), 50 µg ml–1; ampicillin (Amp), 100 µg ml–1; spectinomycin (Spc), 50 µg ml–1 and tetracycline (Tet), 5 µg ml–1 for Xcc and 15 µg ml–1 for E. coli.
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were used for preparation of the plasmid and chromosomal DNAs, restriction digestion, DNA ligation, agarose gel electrophoresis and DNA transformation of E. coli. The conjugation between Xcc and E. coli strains was performed as described by Turner et al. (1984). The restriction endonucleases, T4 DNA ligase and Pfu polymerase were provided by Promega (Shanghai).
Construction of insertional mutants.
Mutants of the ORFs XC1166, XC1223 and XC1976 were constructed using the method previously described by Lu et al. (2007). A 300–500 bp internal fragment of the target ORF was amplified by PCR using the corresponding primers (Table 2). The amplified DNA fragments were cloned into suicide plasmid pK18mob (Schäfer et al., 1994; Windgassen et al., 2000) in the same orientation as the lacZ promoter to guarantee the construction of non-polar mutants. The resulting recombinant plasmid was introduced from E. coli strain JM109 (Yanisch-Perron et al., 1985) into the Xcc wild-type strain 8004 by triparental conjugation using pRK2073 (Leong et al., 1982) as the helper plasmid. Mutants were confirmed by PCR using oligonucleotide P18conF, and the corresponding oligonucleotide located downstream of the target ORF, as primers (Table 2).
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), and the amplified DNA fragment was cloned into the plasmid pLAFR3 (Staskawicz et al., 1987) to generate the recombinant plasmid pLATC1976 (Table 1). The recombinant plasmid was transferred into the XC1976 mutant by triparental conjugation, resulting in strain C1976nk (Table 1).
RT-PCR analysis.
To analyse transcriptional expression levels, total RNA was isolated using Trizol reagent (Promega) and treated with RNase-free DNase (Promega). RT-PCR analysis was carried out with total RNA and specific primers for XC1166 (O1166F/R), XC1223 (O1223F/R) and XC1976 (O1976F/R) (Table 2). One microgram of total RNA was used for each reaction in a 20 µl volume, and 4 µl of the reaction mixture was subjected to PCR amplification. The PCR products were analysed by electrophoresis on 1.5 % (w/v) agarose gels, and verified by DNA sequencing.
Overproduction and purification of protein.
For overproduction of XC1166, XC1223 and XC1976, their corresponding ORFs were amplified using the primers O1166F/R, O1223F/R and O1976F/R (Table 2), respectively. After being confirmed by sequencing, the amplified DNA fragments were cloned into the expression vector pQE-30 (Qiagen) (Table 1) to generate recombinant plasmids pQE-30-1166, pQE-30-1223 and pQE-30-1976 (Table 1), respectively. In these plasmids, XC1166, XC1223 or XC1976 is fused N-terminally in-frame to the His6-tag coding region of the plasmid pQE-30. The recombinant plasmids obtained were transformed into E. coli strain JM109, resulting in strains JM109/pQE-30-1166, JM109/pQE-30-1223 and JM109/pQE-30-1976 (Table 1), respectively. For fused proteins XC1166-His6, XC1223-His6 and XC1976-His6 overproduction and purification, strains were grown to OD600 0.6, and then induced by addition of 1.0 mM IPTG. The cultures were grown for a further 4 h. The fused proteins were purified by Ni-NTA resin (Qiagen). The proteins were checked by 12 % SDS-PAGE and used for enzymic assay.
Cell-free extract preparation.
Xcc strains were grown in NYG medium supplemented with 2 % glucose or fructose for 20 h. Cells were harvested and washed twice in sterile water by centrifugation. The cells were then resuspended in 2 ml 20 mM potassium phosphate (pH 7.5) containing 1 mM dithiothreitol and 0.1 mM EDTA and disrupted by sonication. The sonicates were centrifuged and the supernatant fractions were used in the enzyme assay.
Glk assay.
Glk activity from purified proteins or cell-free extracts was measured by an enzyme-linked assay based on the NADP+/NADPH ratio (Gonzali et al., 2001). Ten microlitres of the protein sample was transferred to a 5 ml tube containing 2 ml reaction buffer [100 mM Tris/HCl (pH 7.4), 100 mM KCl, 7.5 mM MgSO4, 5 mM ATP, 0.5 mM NADP+, 5 mM β-mercaptoethanol, 100 mM glucose and 0.2 U glucose-6-phosphate dehydrogenase ml–1]. After incubation for 10 min at 37 °C, A340 was measured. A unit of enzyme activity was taken as 1 nmol NADPH formed min–1 (mg protein)–1; the protein concentration in the samples was determined by the BCA protein assay kit (Pierce Biotechnology). For characterizing kinetic parameters of the purified proteins, the reaction mixture including 1.0 µg purified Glk, and a range of D-glucose or ATP concentrations (0.05–20 mM), was used under standard assay conditions. Michaelis–Menten kinetic parameters were evaluated by the double reciprocal-plot method.
The phosphorylation of different sugars by the purified proteins was monitored by TLC, as described by Meyer et al. (1997). Purified proteins were incubated with 50 mM sugar in 50 mM Tris/HCl (pH 7.4) containing 10 mM MgCl2 and 50 mM ATP. The assay volume was 100 µl containing 10 µg protein. After 2 h incubation, an 8 µl sample was spotted onto the silica-coated TLC plate (Qingdao Haiyang Chemical) and the plate was then developed with butanol-ethanol-water (5 : 3 : 2, by vol.) for 3 h. After drying the TLC plate, the sugar-containing spots were visualized by dipping the plate in methanol containing 2 % concentrated H2SO4, followed by drying and charring for 8 min at 150 °C.
EPS assay.
To estimate EPS production, strains were cultured in 100 ml NYG liquid medium containing 2 % (w/v) various sugars at 28 °C with shaking at 200 r.p.m. for 3 days. EPS was precipitated from the culture supernatant with ethanol, dried and weighed as described by Tang et al. (1991).
Virulence assay.
The virulence of Xcc to Chinese radish (Raphanus sativus) was tested by the leaf-clipping method (Dow et al., 2003). Leaves were cut with scissors dipped in the bacterial suspensions of an OD600 of 0.1. Lesion length was measured 10 days after inoculation, and data were analysed by t test. The growth of bacteria in radish leaf tissue was determined as previously described (Lu et al., 2007).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). This suggests that the three proteins have distinct functions. The three predicted Glk proteins in Xcc possess the same Pfam domain (PF02685) and they are the only proteins with such a domain in the Xcc proteome. Wider similarity searches revealed that these proteins also share limited identical residues with characterized Glks from other microbial sources: XC1976 has 38.4 and 19.3 % identity, respectively, to the E. coli enzyme (accession no. AAC75447) and the Streptomyces coelicolor A3(2) enzyme (accession no. CAA46727). XC1166 and XC1223 share 31 and 27.9 % identity, respectively, with the E. coli enzyme, and 16.3 and 14.5 % identity with the S. coelicolor enzyme.
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). Fusion proteins with a His6-tag were purified using a Ni-NTA column, to obtain preparations apparently free from contaminating proteins (Fig. 2).
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), was used to test enzyme properties and kinetic constants of the fusion proteins (see Methods). All three fusion proteins exhibited Glk activity in the presence of ATP by this method; however, no significant enzymic activity was observed without ATP (data not shown). The specific activity values were determined to be 1.16x106 U (mg protein)–1 for XC1166-His6, 4.36x107 U mg–1 for XC1223-His6 and 2.63x108 U mg–1 for XC1976-His6. The apparent Km values for glucose were 4.02 mM for XC1166-His6, 4.20 mM for XC1223-His6 and 2.52 mM for XC1976-His6, while the apparent Km values for ATP were 2.50 mM (XC1166-His6), 1.85 mM (XC1223-His6) and 1.25 mM (XC1976-His6).
The enzymic activities of the purified proteins were further assessed by examination of glucose phosphorylation by TLC (see Methods). Purified proteins were incubated with 50 mM glucose and 50 mM ATP for 2 h at 37 °C before TLC separation of substrates and products. Although phosphorylation of glucose by XC1976-His6 could be observed by this method (Fig. 3), no phosphorylation of glucose by XC1166-His6 and XC1223-His6 was detected. To examine the sugar substrate specificity of XC1166, XC1223 and XC1976, these assays were repeated under the same conditions using a range of sugars, including arabinose, xylose, sorbitol (glucitol), mannose, mannitol, sorbose, fructose, galactose, rhamnose, sucrose and maltose. No phosphorylation of any of these sugars by XC1166-His6, XC1223-His6 or XC1976-His6 could be detected (data not shown). Taken together, these results indicate that XC1976 is a Glk and not a hexokinase.
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), suggesting a role for XC1976 in this pathway of glucose metabolism.
XC1976 makes the major contribution to Glk activity in Xcc
To demonstrate the physiological role of the three genes, non-polar mutants carrying insertional disruption within the ORF XC1166, XC1223 or XC1976 were constructed and named 1166nk, 1223nk and 1976nk, respectively (Table 1; see Methods). RT-PCR analysis showed that full-length transcripts for each gene are present in the wild-type strain but not in the corresponding mutant (Fig. 4).
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); this might reflect the glucose dehydrogenase activity of Xcc. As summarized in Table 3, the Glk activities in the XC1166 and XC1223 mutants were not different from that in the wild-type strain 8004. However, the XC1976 mutant showed a substantial reduction in enzymic activity, whereby the level of ATP-dependent activity was only 6 % of the wild-type level. XC1976 was cloned as a 1222 bp DNA fragment in plasmid pLAFR3 to give pLATC1976 (see Methods). Introduction of pLATC1976 into the XC1976 mutant resulted in a strain that expressed the XC1976 transcript (Fig. 4) and that had a level of Glk activity that was considerably elevated over that of the wild-type (Table 3). XC1976 is the central gene in the XC1977-XC1975 operon (see above) and is unlikely to have its own promoter. The lac promoter of the vector pLAFR3, which is active in Xcc (Soby & Daniels, 1996), probably drives expression of XC1976 in pLATC1976. Consistent with this interpretation, when the 1222 bp fragment was cloned into the promoterless vector pLAFR6 (Huynh et al., 1989), the resulting construct could not restore the Glk activity of the XC1976 mutant (data not shown). Overall, these results indicate that XC1976 makes the most significant contribution to the total Glk activity in Xcc, when the bacterium grows in NYG medium supplemented with glucose or fructose.
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XC1976 influences EPS production
To evaluate the EPS production of these mutants, strains were grown on NYG agar plates supplied with a range of sugars. The XC1166 and XC1223 mutants showed the same colony size as the wild-type strain 8004 on all agar plates tested, indicating equivalent production of EPS. However, the XC1976 mutant displayed colonies that were smaller than the wild-type on plates containing arabinose, glucose, sorbitol, mannitol, galactose, sucrose or maltose, larger than the wild-type on plates containing fructose or sorbose, and the same as the wild-type on plates containing xylose, mannose or rhamnose. These findings suggest that the XC1976 gene might have complex effects on EPS production, promoting it in arabinose-, glucose-, sorbitol-, mannitol-, galactose-, sucrose- or maltose-containing medium, but inhibiting it in fructose- or sorbose-containing medium.
To quantitatively estimate the EPS production of the mutants, strains were grown in NYG liquid medium supplemented with 2 % of various carbohydrates for 3 days, and EPS was extracted from the cultures (see Methods). As summarized in Table 4, the XC1976 mutant produced about 25–60 % less EPS than the wild-type strain 8004 when cultured in arabinose-, glucose-, galactose-, sucrose- or maltose-containing medium. In addition, the EPS yield of the complemented mutant strain showed no significant difference from that of the wild-type. Conversely, the XC1976 mutant produced more EPS than the wild-type when cultured in fructose-containing medium. The EPS yield of the mutant was increased about 30 % compared to that of the wild-type strain, and reached 8.5 g l–1, which is approaching the level of the wild-type strain cultured in glucose- or sucrose-containing medium. Surprisingly, complementation did not restore EPS production on fructose to wild-type level.
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). Ten days after inoculation, the XC1976 mutant showed a mean lesion length of 5.48 mm on the leaves, while the wild-type strain, as well as the mutants of XC1166 and XC1223, showed a mean lesion length of about 12 mm. As analysed by t test, the mean lesion length caused by the XC1976 mutant was significantly shorter than that caused by the wild-type strain (P=0.01). In contrast, when the XC1976 mutant was transformed with plasmid pLATC1976, the virulence of the mutant could be restored to wild-type; the lesion lengths caused by the complemented strain and the wild-type strain were not significantly different (P=0.05) (Fig. 5b). These results show that Glk activity is required for the full virulence of Xcc.
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). The growth of the mutant in planta could be completely restored by pLATC1976. These results show that Glk activity affects the growth of Xcc as well as the production of symptoms in planta. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The insertional mutants of XC1166 and XC1223 still had Glk activity similar to that of the wild-type (Table 3), and no phosphorylation of glucose by either XC1166-His6 or XC1223-His6 could be observed on silica-coated TLC plates. We thus suppose that XC1166 and XC1223 do not serve as a Glk in Xcc, despite the close similarity of their amino acid sequences to those of other microbial Glks (Fig. 1) and the Glk activity of the purified proteins as measured in the linked assay (Table 3). These proteins could not phosphorylate any of a range of other sugars tested. Analysis of the deduced protein sequence using the SMART algorithm (http://smart.embl-heidelberg.de) showed differences in domain structure among XC1166, XC1223 and XC1976. All three proteins contain a Glk domain (PF02685); however, XC1166 and XC1223 have in addition a Rok domain [residues 24–218 of XC1166 (8.70e–3) and residues 20–215 of XC1223 (1.10e–2)]. The family of proteins with a Rok domain consists of repressors, ORFs with unidentified functions, and sugar kinases (Titgemeyer et al., 1994). This difference in domain composition suggests that XC1166 and XC1223 fulfil other cellular functions, which remain obscure and require more work before they can be understood.
In Xcc, glucose uptake relies on two discrete systems: the intracellular phosphorylation pathway and the periplasmic oxidative pathway (Whitfield et al., 1982; Lessie & Phibbs, 1984). Extracellular glucose entering cellular metabolism via the intracellular phosphorylation pathway will lead to the production of glucose 6-phosphate. Some of the glucose 6-phosphate will enter the pathways for polysaccharide production via phosphoglucomutase, and the remainder will enter the central metabolic network via glucose-6-phosphate dehydrogenase. Metabolic flux modelling predicts that if all external glucose enters the cytosol via the periplasmic pathway, no EPS will be synthesized. Conversely, if most glucose enters metabolism via intracellular phosphorylation, less EPS will be produced because the availability of biochemical energy is limited (Letisse et al., 2002). Here we have shown that inactivation of XC1976 greatly reduced the Glk activity in Xcc. One consequence of this may be to impede glucose mobilization via the permease/phosphorylation pathway and lead to a decrease in the cellular level of precursors for EPS synthesis, with a resulting reduction in EPS yield.
The XC1976 mutant also produced less EPS than the wild-type when cultured in medium supplemented with arabinose, galactose, sucrose or maltose as carbon source. The sucrose-utilization system in Xcc has recently been described in some detail and is clearly different from that of other bacteria (Blanvillain et al., 2007; Kim et al., 2004; Reid & Abratt, 2005). In Xcc, extracellular sucrose is transported through the outer membrane via a TonB-dependent receptor (SuxA), and through the inner membrane via a sugar transporter (SuxC). The intracellular sucrose is then hydrolysed by sucrose hydrolase (SUH) to yield glucose and fructose. The impeding of glucose phosphorylation through inactivation of XC1976 may thus lead to less sucrose utilization via EPS synthesis. Little is known about the pathways for utilization of arabinose, galactose or maltose in Xcc, although conversion of maltose (a disaccharide of glucose) to glucose seems likely.
Interestingly, the XC1976 mutant produced more EPS than the wild-type in fructose-containing medium. When plasmid pLATC1976, which resulted from the cloning of XC1976 in plasmid pLAFR3, was introduced into the XC1976 mutant, the resulting complemented strain displayed about 50 % higher Glk activity than the wild-type strain when cultured in NYG medium containing 2 % fructose, but still produced more EPS than the wild-type (Table 3). To confirm the effect of XC1976 mutation, three other isolates of the XC1976 mutant were selected at random to test in fructose-containing medium; all produced more EPS than the wild-type (data not shown).
A similar phenomenon has been observed in S. coelicolor, in which both inactivation and overexpression of Glk result in a loss of carbon catabolite repression (CCR) of glycerol kinase and agarase (Kwakman & Postma, 1994). Glk has been implicated in the mechanism of CCR in several bacteria from the genera Streptomyces, Staphylococcus and Bacillus (Angell et al., 1992, 1994; Kwakman & Postma, 1994; Wagner et al., 1995; Spath et al., 1997). CCR ensures that micro-organisms adjust their metabolic activities to utilize specific carbon sources in their environment (Brückner & Titgemeyer, 2002). A phosphoenolpyruvate-dependent phosphotransferase system (PTS) identified to be responsible for fructose transportation and phosphorylation in other bacteria (Prior & Kornberg, 1988; Geerse et al., 1989; Wu et al., 1990) has been demonstrated in Xcc, and a pathway for fructose catabolism has also been proposed (de Crécy-Lagard et al., 1991a, b, 1995). However, although some details of fructose metabolism are understood, little is known about either the regulation of fructose utilization or CCR in Xcc. Further investigations are needed to fully understand the contribution of XC1976 to such regulatory processes.
Mutation of XC1976 also attenuates the virulence of Xcc to Chinese radish and leads to a reduction of growth in this host plant. It is possible that these effects result from an impairment of EPS synthesis by bacteria in planta. EPS plays an important role during bacterial infection. It can enhance the susceptibility of host plants by suppressing defence responses such as callose formation (Yun et al., 2006), and contribute to biofilm formation (Dow et al., 2003) and bacterial resistance against host defences. It may also serve to mask the bacterium to prevent recognition by the host and to enable colonization of host tissues (Alvarez, 2000). An alternative, but not mutually exclusive, view is simply that the reduction in ability to utilize available carbohydrates in the plant reduces bacterial growth with consequences for the aggressiveness of the pathogen. Consistent with this, it has recently been demonstrated that sucrose utilization is required for full pathogenicity in Xcc (Blanvillain et al., 2007). Previously, we have shown that an Xcc ppsA mutant, which is unable to utilize C4-dicarboxylates as carbon source, has limited growth in plant hosts and reduced virulence compared to the wild-type (Tang et al., 2005). Collectively, our findings suggest that the utilization of plant carbohydrates as well as C4-dicarboxylates is required for the full virulence of Xcc. These carbon sources may be available in different amounts at different stages of infection, as bacteria initially utilize components of the xylem fluid before mobilizing carbon sources by enzymic degradation of host polymers as black rot disease progresses.
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ACKNOWLEDGEMENTS |
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Edited by: M. F. Hynes
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Received 11 June 2007;
revised 29 August 2007;
accepted 31 August 2007.
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