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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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). The pathogen produces extracellular polysaccharide (EPS), also called xanthan gum, and a series of extracellular enzymes including amylase, endoglucanase, polygalacturonate lyase and protease that collectively contribute to pathogenesis (Dow & Daniels, 1994). EPS plays an important role during bacterial infection. It can enhance the susceptibility of host plants by suppressing defence responses such as callus formation (Yun et al., 2006). EPS may also mask the bacterium to prevent host recognition and to enable colonization of host tissues (Alvarez, 2000). In addition to a role in bacterial virulence, xanthan has a wide variety of applications in agriculture, petroleum production and the food industry as a stabilizing, viscosifying, emulsifying, thickening and suspending agent (Kennedy & Bradshaw, 1984).
The commercial value and role in pathogenesis of xanthan has prompted a number of studies on the genetics and biochemistry of biosynthesis of this EPS in recent decades. Two loci within the Xcc genome, the gum cluster and a 35.3 kb gene cluster, have been demonstrated to be involved in the biosynthesis of EPS (Capage et al., 1987; Hotte et al., 1990; Vanderslice et al., 1990). The gum cluster, which is composed of 12 genes (gumB to gumM), is responsible for the sequential assembly and polymerization of pentasaccharide repeating units, and also the release of polymers into the growth medium (Ielpi et al., 1993). Certain genes such as xanA and xanB, which are located within the 35.3 kb gene cluster, are responsible for the biosynthesis of the sugar nucleotide precursors, and are involved in both EPS and lipopolysaccharide (LPS) biosynthesis (Köplin et al., 1992). In addition, the products of rpf, clp and pigB were found to be implicated in either the positive or negative regulation of EPS production (Tang et al., 1990, 1991; De Crècy-Lagard et al., 1990; Poplawsky & Chun, 1998).
The recent rapid development of genomics has brought about a paradigm shift in gene function research. The genomes of two Xcc strains, ATCC 33913 (da Silva et al., 2002) and 8004 (Qian et al., 2005), have recently been sequenced and provide a profile of genetic information with which to explore the biological characteristics of Xcc. The functions of about one-third of the ORFs are, however, yet to be assigned, and a large repertoire of genes has not been experimentally defined. One aim of the work in our laboratory is to identify further genes that influence EPS biosynthesis in Xcc. In preliminary work we isolated a number of transposon insertion mutants in Xcc strain 8004 with defects in EPS synthesis. Here we investigate one of these mutated loci in more detail.
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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 in NYG medium (per litre: 5 g peptone, 3 g yeast extract and 20 g glycerol; Daniels et al., 1984b) or the 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 glucose; Daniels et al., 1984a) at 28 °C. Antibiotics were added at the following concentrations as required: kanamycin (Kan) 25 µg ml–1; rifampicin (Rif) 50 µg ml–1; gentamicin (Gm) 5 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 plasmid and chromosomal DNAs, restriction digestion, DNA ligation, agarose gel electrophoresis and DNA transformation of E. coli. 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 ORFs XC3811, XC3812, XC3813, XC3814, XC3815 and XC3816 were constructed using the suicide plasmid pK18mob (Schafer et al., 1994; Windgassen et al., 2000). A 300–500 bp internal fragment of the target ORF was amplified by PCR using Xcc chromosomal DNA as the template and the corresponding oligonucleotides as primers (Table 2). Primers were designed based on the ORF sequences in the genome of Xcc strain 8004 (accession number NC_007086). After being confirmed by sequencing, the amplified DNA fragments were cloned into pK18mob to create the recombinant plasmids. Primers were modified to give BamHI- or HindIII-compatible ends to ensure that the internal fragment was cloned in the same orientation as the lacZ promoter in pK18mob. The recombinant plasmid was introduced from E. coli JM109 (Yanisch-Perron et al., 1985) into Xcc strain 8004 by triparental conjugation using pRK2073 (Leong et al., 1982) as the helper plasmid. Mutants were selected on NYG agar plates containing rifampicin and kanamycin. Transconjugants with a mutation in the target ORF were confirmed by PCR. Confirmation PCR was performed using the total DNA of the transconjugants as the template, oligonucleotide P18conF, which is located in pK18mob, and the corresponding oligonucleotide, which is located downstream of the target ORF, as primers. The total DNA of Xcc strain 8004 was used as the negative control. The expected PCR products were further confirmed by sequencing analysis.
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), respectively, and the amplified DNA fragments were cloned into plasmid pLAFR3 (Staskawicz et al., 1987) or pLAFR6 (Huynh et al., 1989) to generate recombinant plasmids. The recombinant plasmids were transferred into the corresponding mutant by triparental conjugation. The transconjugants carrying the recombinant plasmid were screened on NYG agar plates with rifampicin, kanamycin and tetracycline. The resulting strains were named, respectively, C3813nk, C3814nk and C3815nk (Table 1).
Extracellular polysaccharide assay.
To estimate EPS production, strains were cultured in 100 ml NYG liquid medium containing 2 % (w/v) glucose 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). EPS structure analysis was performed with Fourier transform infrared (FT-IR) spectra. FT-IR spectra were recorded on a Nicolet 5DX spectrometer. The dry sample powder was mixed with KBr and pressed into pellets under reduced pressure. The FT-IR spectra were obtained by scanning between 4000 and 450 cm–1.
Lipopolysaccharide analysis.
LPS was prepared using an LPS extraction kit (iNtRON Biotechnology). LPS preparations were analysed using Tricine-SDS-PAGE (Lesse et al., 1990) and visualized by silver staining (Kittelberger & Hilbink, 1993).
-Glucuronidase (GUS) activity assay.
Xcc strains were cultured overnight and diluted to an OD600 of 0.5, and 1.0 ml of each was inoculated into 200 ml NYG medium held in 500 ml flasks. GUS activities were determined at 12 h intervals until 60 h by measurement of the A415 using p-nitrophenyl -D-glucuronide as the substrate, as described by Henderson et al. (1985).
Virulence assay.
The virulence of Xcc to Chinese radish (Raphanus sativus) was tested by the leaf-clipping method (Dow et al., 2003). Seedlings with four fully expanded leaves were used for inoculation. Bacteria grown overnight in NYG liquid medium were washed and resuspended in water to an OD600 of 0.1. Two or three fully expanded leaves per plant were cut with scissors dipped in the bacterial suspensions. Sixty leaves were inoculated in each independent experiment. Each treatment was repeated three times. Lesion length was measured 10 days after inoculation, and data were analysed by t-test.
The growth of bacteria in radish leaf tissue was measured by homogenizing a group of leaves (five leaves for each sample) in 9 ml sterile water. Diluted homogenates were plated on NYG agar plates supplemented with rifampicin (for wild-type) or rifampicin plus kanamycin (for mutants). Bacterial c.f.u. were counted after incubation at 28 °C for 3 days.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). In the reported genome sequence of strain 8004, the protein encoded by XC3814 (YP_244874) was assigned as a glycosyltransferase (Qian et al., 2005). The upstream ORFs XC3811, XC3812 and XC3813, and the downstream ORFs XC3815 and XC3816 are poorly characterized genes, and the proteins encoded by these ORFs (YP_244871, YP_244872, YP_244873, YP_244875, YP_244876) were annotated as ‘conserved hypothetical proteins’" (Qian et al., 2005). These six ORFs are located in the genome sequence between position 4505214 and position 4512185 (Fig. 1).
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; see Methods). To test the EPS production, these insertional mutants were grown on NYG agar plates supplemented with 2 % glucose for 5 days. Mutants 3813nk, 3814nk and 3815nk displayed smaller colonies than the wild-type strain (Fig. 2a), while the other mutants formed normal wild-type colonies. The growth rates of the mutants did not differ from that of the wild-type in NYG liquid medium (Fig. 2b) or in MMX minimal medium (data not shown). These findings suggested that mutants 3813nk, 3814nk and 3815nk might produce less EPS than the wild-type. To quantitatively estimate the EPS production of the mutants, strains were grown in NYG liquid medium containing 2 % glucose for 3 days and EPS was extracted from the cultures. The results showed that the mutants 3813nk, 3814nk and 3815nk produced significantly less EPS than the wild-type strain 8004 (Table 3). For complementation, plasmids pLATC3813, pLATC3814 and pLATC3815 (Table 1) were constructed by cloning a 1301 bp fragment, a 1096 bp fragment and a 1478 bp fragment, each of which included the corresponding ORF (Fig. 1), into the vector pLAFR3. These plasmids were introduced into mutants 3813nk, 3814nk and 3815nk to form the complemented mutants C3813nk, C3814nk and C3815nk (Table 1; see Methods for details). The EPS yield of the complemented mutants showed no significant difference from that of the wild-type strain (Table 3, Fig. 2a), indicating that the mutations in XC3813, XC3814 and XC3815 could be complemented by the corresponding ORF in trans. The promoterless plasmid pLAFR6 harbouring the 1301 bp fragment, the 1096 bp fragment or the 1478 bp fragment could also complement the corresponding mutant (data not shown), suggesting that XC3813, XC3814 and XC3815 have their own promoter.
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-glucuronic acid-1,2--mannose-1,3-
-cellobiose (Jansson et al., 1975). The assembly and polymerization of the repeated units have been attributed to the protein products of the gum genes (Ielpi et al., 1993). To estimate the effect of inactivation of XC3813, XC3814 or XC3815 on the EPS chemical structure, EPS from the mutant strains and the wild-type strain was analysed using FT-IR spectra. These spectra revealed that the EPS patterns of the mutants were similar to that of the wild-type (data not shown). The characteristic vibration peaks appeared in all the EPS samples and no obvious shift of these characteristic peaks was observed. This indicates that mutation in any of the three ORFs does not result in change in the pentasaccharide repeating units.
Characterization of XC3813, XC3814 and XC3815
Domain analysis with the SMART program (http://smart.embl-heidelberg.de/) showed that the 352 amino acid protein XC3813 contains a glycerophosphotransferase domain (PF04464, 4.7e–08) (residues 148–317). It has been reported that in Staphylococcus epidermidis, glycerophosphotransferase, encoded by the tagF gene, is responsible for polymerization of the main chain of teichoic acid (Fitzgerald & Foster, 2000). The deduced XC3814 protein comprises 274 amino acids and contains a domain characteristic of glycosyltransferase family 2 (PF00535, 2.80e–20) at its N-terminus (residues 14–177). Glycosyltransferase family 2 is a diverse family that transfers the sugar from UDP-glucose, UDP-N-acetylgalactosamine, GDP-mannose or CDP-abequose to a range of substrates including cellulose, dolichol phosphate and teichoic acids (Campbell et al., 1997). The deduced XC3815 protein, of 429 amino acids, harbours a Wzy conserved domain (PF04932, 1.7e–19), residues 270–341. The protein Wzy, an O-antigen polymerase encoded by the gene wzy (Reeves et al., 1996), is responsible for polymerization of the repeated O-antigen unit of LPS (Bengoechea et al., 2002). One of the features of the Wzy protein is that it has multiple putative membrane-spanning domains (Collins & Hackett, 1991; De Kievit et al., 1995; Bengoechea et al., 2002). The deduced protein XC3815 is predicted to have 11 transmembrane segments, suggesting an integral membrane location.
These bioinformatic analyses suggested that XC3814 and XC3815 might affect LPS synthesis. To address this issue, LPS from the mutant and wild-type strains was prepared and analysed using SDS-PAGE. Under these conditions, the banding patterns of LPS from the mutant strains 3814nk and 3815nk were indistinguishable from the wild-type strain 8004 (data not shown). This result indicated that the XC3814 and XC3815 may not be involved in LPS synthesis.
To determine whether the inactivation of XC3813, XC3814 or XC3815 reduces the expression level of the gum genes, the reporter plasmid pL6gumGUS, which carries the promoter region of the gum operon fused to the coding region for gusA (Vojnov et al., 2001), was transferred to the wild-type and mutant strains by triparental mating with the E. coli DH5 donor strain and helper strain HB101/pRK2073. In the wild-type background the GusA level, determined enzymically, mirrors gum gene expression and is correlated closely with the level of EPS production (Vojnov et al., 2001). The transconjugant strains 8004/pL6gumGUS, 3813nk/pL6gumGUS, 3814nk/pL6gumGUS and 3815nk/pL6gumGUS (Table 1) were cultured in NYG liquid medium containing 2 % (w/v) glucose with shaking at 200 r.p.m. GUS activities were assayed as described by Henderson et al. (1985) at 12 h intervals. The results showed that the GUS activities in the mutant backgrounds were not significantly different (P=0.05; t-test) from each other and from the wild-type background at each of the time points tested (Table 4). This indicates that inactivation of any of the ORFs XC3813, XC3814 or XC3815 has no effect on the transcription level of gum genes.
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). Xcc is a vascular pathogen and the leaf-clipping inoculation introduces the bacteria directly into the vascular system. Ten days after inoculation XC3813 and XC3815 mutant strains showed markedly reduced virulence compared with the wild-type strain: the mean lesion lengths caused by 3813nk and 3815nk were significantly shorter than that caused by the wild-type strain 8004 at P=0.01 (t-test). Furthermore the mutant strain 3814nk almost completely lost virulence and hardly induced any symptoms on the inoculated leaves (Fig. 3a,b). In contrast, lesion lengths caused by the complemented strains and the wild-type strain were not significantly different at P=0.05 (Fig. 3b), indicating that the virulence of the mutants 3813nk, 3814nk and 3815nk could be fully restored by the recombinant plasmids pLATC3813, pLATC3814 and pLATC3815, respectively.
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). For mutant 3814nk, the number of bacterial cells was approximately 100-fold lower than that of the wild-type strain 5 days post-inoculation and onward (Fig. 3c). The growth of 3813nk, 3814nk and 3815nk in planta could be completely restored by pLATC3813, pLATC3814 and pLATC3815, respectively. These results reveal that XC3813, XC3814 and XC3815 affect growth of Xcc as well as symptom production in planta. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The biosynthesis of EPS in Xcc occurs in at least two stages. Firstly, the repeating pentasaccharide unit is sequentially assembled while linked to a polyprenol through a diphosphate bridge. Secondly, the repeating units are polymerized and the polymer is exported from the cell. This second stage is still poorly understood. The protein products of the gum gene cluster direct assembly of pentasaccharide repeating units, polymerization and the export of EPS (Vanderslice et al., 1990; Ielpi et al., 1993). Mutations in certain gum genes that affect EPS structure also reduce the level of EPS production (Katzen et al., 1998). In this work, we have shown that mutation of XC3813, XC3814 or XC3815 causes reduced EPS production but neither the structure of the pentasaccharide repeating unit nor the transcription of the gum genes is altered. How then do XC3813, XC3814 and XC3815 influence EPS production?
Bioinformatic analysis suggested that XC3814 and XC3815 have a role in LPS synthesis. XC3814 is annotated as encoding an LPS core biosynthesis glycosyltransferase by reason of the shared limited similarity with the protein KdtX (39 % identity and 54 % similarity) from Serratia marcescens (accession no. Q54435; Guasch et al., 1996), the WaaE protein (39 % identity and 51 % similarity) from Klebsiella pneumoniae (accession no. Q9XC90; Regue et al., 2001), and the LpsC protein (33 % identity and 48 % similarity) from Sinorhizobium meliloti (accession no. Q9R9M9; Lagares et al., 2001). KdtX, WaaE and LpsC have been predicted to be LPS core biosynthesis glycosyltransferases. Consistent with this assertion, mutation of kdtX, waaE and lpsC led to changes in LPS pattern on SDS-polyacrylamide gels such that the core LPS from the mutant strains migrated faster than that of the wild-type strain (Guasch et al., 1996; Regue et al., 2001; Lagares et al., 2001). XC3815 was predicted bioinformatically to be a cytoplasmic membrane protein containing the Wzy domain. Wzy has been shown to be responsible for polymerization of the repeated O-antigen unit of LPS in some bacteria, where inactivation of the wzy gene led to the loss of the O-antigen ladder in the LPS electrophoresis pattern (Collins & Hackett, 1991; Bengoechea et al., 2002; Grozdanov et al., 2002; Tao et al., 2004). In this work, inactivation of XC3814 or XC3815 did not alter the LPS pattern, suggesting that XC3814 and XC3815 are not involved in LPS synthesis. Bioinformatic analysis suggested that XC3813 has a glycerol-phosphate transfer domain. However, structural analyses of LPS have not indicated the presence of glycerol-phosphate residues (Corsaro et al., 2001; Raetz & Whitfield, 2002; Molinaro et al., 2003), and our analysis of LPS failed to detect an effect of mutation of XC3813 on the electrophoretic mobility of LPS bands.
Although XC3813, 3814 and 3815 have no apparent role in LPS biosynthesis, it is possible that these proteins direct the synthesis of a different (as yet unidentified) surface polysaccharide, and that the loss of this polymer adversely affects EPS export or polymerization. A second possibility is that XC3813, 3814 and 3815 may act as accessory elements to Gum proteins involved in EPS export or polymerization. Further work is required to understand the molecular basis of the influence of this novel gene cluster on EPS production and this will be the subject of future investigations.
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ACKNOWLEDGEMENTS |
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Received 17 August 2006;
revised 7 November 2006;
accepted 9 November 2006.
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