Functional analysis and expressional regulation of wxoE and wxoF in lipopolysaccharide (lps) biosynthesis gene cluster I of Xanthomonas oryzae pv. oryzae

Physiological and Molecular Plant Pathology 75 (2011) 129e136

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Functional analysis and expressional regulation of wxoE and wxoF in lipopolysaccharide (lps) biosynthesis gene cluster I of Xanthomonas oryzae pv. oryzae Ji-Chun Wang a, Ulambayar Temuujin a, Jong-Gun Kim a, c, Young-Jin Park b, Byoung-Moo Lee b, Chang-Sun Choi a, Laura Ann Silo-Suh d, Hee-Wan Kang a, c, * a

Graduate School of Biotechnology and Information Technology, Hankyong National University, Ansung 456-749, Republic of Korea National Academy of Agricultural Science, Suwon 441-707, Republic of Korea Institute of Genetic Engineering, Hankyong National University, Ansung 456-749, Republic of Korea d Biological Sciences, 101 Rouse Life Sciences Bldg, Auburn University, AL 36849, USA b c

a r t i c l e i n f o

a b s t r a c t

Article history: Accepted 29 September 2010

wxoE and wxoF, two genes in the lipopolysaccharide (LPS) biosynthesis cluster I of Xanthomonas oryzae pv. oryzae (Xoo) that have not been characterized, were mutated by transposon insertion. Transposon mutants of wxoE and wxoF were nonpathogenic to rice. In LPS analysis on SDS-PAGE, Low mobility bands regarded as LPS O-antigen complex were observed in wild-type strain KACC10859 and mutant wxoD, but not in LPS profiles of wxoA, wxoB, wxoC, wxoE and wxoF mutants. In addition, exopolysaccharide (EPS) production from wxoE and wxoF mutant strains were dramatically reduced. WxoE protein showed enzymatic activity resembling that of cystathionine g-lyase and specificity to cystathionine substrates. WxoF showed significant homology with methyltransferases that may function in the methylation of sugars in LPS biochemical modifications. Western blot analysis demonstrated WxoF is located in membrane and the lps genes involving wxoE and wxoF in cluster I are cotranslated in an operon that is dependent on a promoter with a polar fashion. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Xanthomonas oryzae pv. oryzae LPS genes wxoE wxoF Functions

1. Introduction Xanthomonas oryzae pv. oryzae (Xoo), a causal agent of bacterial blight (BB), is economically the most important bacterial disease in rice plants (Oryza sativa L.). Currently, pathogenecity related genes of Xoo related to hypersensitive response and pathogenicity (hrp), productions of extracellular polysaccharide (EPS), extracellular enzymes and lipopolysaccharides (LPS) have been isolated and characterized [23,26]. LPS of gram negative bacterial pathogens including X. oryzae pv. oryzae (Xoo) have long been recognized as critical virulence determinants [4,6,7,36]. LPSs from certain plant pathogens, such as Xanthomonas campestris, Pseudomonas syringae, and Ralstonia solanacearum, have been shown to activate a number of defense-related responses in plants [3,13,19,30]. It has also been

* Corresponding author. Graduate School of Biotechnology and Information Technology, Hankyong National University, Ansung 456-749, Republic of Korea. Tel.: þ82 31 670 5420; fax: þ82 31 676 2602. E-mail address: [email protected] (H.-W. Kang). 0885-5765/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.pmpp.2010.09.003

suggested that LPS is likely to be involved in the association of bacteria with plant cell walls during the infection process [2]. Recently, the complete sequences of the LPS gene clusters present in Xoo strains were compared with the LPS gene clusters of other bacterial species that belong to Xanthomonacea, including X. axonopodis pv. citri and X. oryzae pv. oryzicola [21]. Interestingly, the study found that the lps locus was absent in two Xoo strains (Indian pathotype BOX8 and Nepal 642) and in X. oryzae pv. oryzicola strains, but different sets of lps genes were present at this locus in other species such as X. campestris pv. campestris [21,22]. Therefore, it was concluded that multiple horizontal gene transfer (HGT) events of lps genes occurred in xanthomonads. An LPS is an amphipathic molecule divided into the following three distinct components: a hydrophobic membrane anchor (lipid A), a short chain of sugar residues with multiple phosphoryl constituents (core oligosaccharide), and a structurally diverse, serospecific polymer composed of oligosaccharide repeats (O-antigen) [38]. The gene clusters containing the 15 genes related to LPS biosynthesis were identified in the X. campestris pv. campestris genome [34]. Based on results from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), mutations in

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these genes have been held responsible for deficiencies in the LPS core and LPS O-antigen. A previous study reported the complete 12.2-kb sequence and genomic organization of a locus containing 5 genes (from metB to wxoD), whose products are involved in LPS biosynthesis [21]. On the basis of full genomic sequence of X. oryzae pv. oryzae KACC10331 [16], an additional gene, smtA have been annotated as a gene between metB and wxoA. It was considerable that the cysB gene upstream an metB is also involved in the locus. These genes have been divided into two major clusters [20]. Cluster I includes cysB, metB, smtA, and wxoABCD, which encode methionine synthesis-related proteins, a predicted epimerase, glycosyl transferases, and an O-antigen acetylase, respectively. Among the cluster I genes, it was reported that wxoABC genes were associated with pathogenicity as well as EPS and LPS biosynthesis in X. oryzae pv. oryzae [4]. However, the functional roles including pathogenecity of, cysB, metB and smtA genes in cluster I (Fig. 1A) and further their expressional relationships to other lps genes in the locus are remained to elucidate. We conducted functional analyses of metB and smtA genes in an LPS gene cluster I of Xoo. metB and smtA genes were renamed as the wxoE and wxoF based on the nomenclature of lps genes in the cluster region of Xoo. Our results are the first to provide evidence that wxoE and wxoF genes are essential for pathogenicity, LPS biosynthesis, and EPS production of Xoo. Furthermore, Western blot analyses demonstrated the translational gene expression in lps gene cluster I i.e., wxoE, wxoF, and wxoABCD is corelated.

Korean Agricultural Culture Collection (KACC) at the National Academy of Agricultural Science (NAAS), Suwon, Korea. Other wildtype X. oryzae pv. oryzae and X. oryzae pv. oryzicola strains were a gift from Dr. Sonti. Wild-type and mutant strains of Xoo were cultured on peptone sucrose (PS) [20] or NB medium (Duchefa) at 28  C for 3 d. Escherichia coli was grown in LB medium (Duchefa) at 37  C for 18 h. Antibiotics were added to final concentrations (mg/liter) as follows: for E. coli, ampicillin, 80; gentamycin, 50; and kanamycin, 50; for X. oryzae pv. oryzae, ampicillin, 50; gentamycin, 20; and kanamycin, 20.

2. Materials & methods

The genes in lps cluster I (cysB, wxoE, wxoF, wxoA, wxoB, wxoC, and wxoD) were PCR-amplified from plasmid pBACF6 using the primer sets given in Table 2. In addition, the primer set (50 -CATC GACCTGCTCAACTACC-30 /50 -GTCATGGCTATGAGTGGTGC-30 ) was used for PCR amplification of a 250-bp amplicon that contained the noncoding region (91 bp) between cysB and wxoE. Each PCR product was

2.1. Bacterial strains, plasmids, and culture conditions The strains and plasmids used in this study are listed in Table 1. The X. oryzae pv. oryzae KACC10859 strain was obtained from the

2.2. Genomic DNA and Southern blot hybridization Genomic DNA of X. oryzae pv. oryzae strains were extracted from bacterial cells grown in 5 ml PS broth for 3 d at 28  C using a previously described method [20]. For Southern blot hybridization, about 5 mg of each genomic DNA was digested with the restriction enzyme EcoRI, fractionated on 0.9% agarose gel in TAE buffer (10 mM Tris-acetate, 1 mM EDTA, pH 8.0) and transferred to a HybondÔ Nþ nylon membrane (Amersham Biosciences, UK). The DNA fragments on the membrane were hybridized with a DNA probe labeled with AlkPhos Direct Labeling Reagents (GE Healthcare, UK). Hybridization signals were detected on X-ray film using detection systems according to the protocol provided by the manufacturer (Amersham Biosciences). 2.3. Disruption and marker exchange of target genes

Fig. 1. Genetic organization of LPS biosynthesis-related genes in X. oryzae pv. oryzae (A) and pathogenicity assay of transposon inserted mutant strains in cluster I (B). The genetic structure of the genes was drawn from sequencing data (NCBI BLAST database) of X. oryzae pv. oryzae KACC 10331 [13]. Black triangle bars on each gene indicate transposon insertion sequence sites and IS indicates insertional sequence. Pathogenicity values are represented as the mean  standard deviation from three independent experiments.

J.-C. Wang et al. / Physiological and Molecular Plant Pathology 75 (2011) 129e136 Table 1 Bacterial strains and plasmid used in this study. Strain/plasmid E. coli strains DH5a

BL21(DE3)

Relevant characteristics

reference/source

f80dlacZ6M156(lacAYA-argF)U169 deoR recAl endAl hsdR17(rk- mk-) supE44I- thi-l gyrA96 reL41 F- dcm ompT hsdS(rB- mB-) gal l(DE3)

Lab. collection

Novagen

Plasmids pBACF6

pGEM-T-Easy vector pXOPR

pET15b pETwxoC pETwxoF pETwxoE pML122 pMLOT

pTP27 pTP84

This This This This This This This This This This

Kanamycin;

Gen:

Gentamycin;

study study study study study study study study study study

2.5. LPS and EPS analyses Wild-type and lps mutant strains of Xoo were grown on PSplate [20]. The Cell density of 5  108 cells were pelleted by centrifugation at 6000 rpm for 10 min. LPS was extracted from the Xoo cell pellet by using method of Hitchcock and Brown [13], subjected to 15% SDS-PAGE, and visualized bya silver staining [30]. The amount of EPS produced by each mutant and wild-type strain was determined by a modified version of the phenol-sulfuric acid method [8]. Xoo strains were cultured in 5 ml of PS broth until a density of 2  108 (cfu) and then harvested by centrifugation at 10,000 rpm for 20 min. The supernatant was filtered through a 0.45-mm filter (Whatman) to completely remove the Xoo cells. EPS was precipitated from the 1-ml filtrate solution with 3 volumes of absolute ethanol and then dried. The EPS was dissolved in 200 ml of distilled water and then suspended in 10 ml of 80% phenol. Then, 1 ml of 100% sulfuric acid was added to the phenol mixture, suspended, and placed at room temperature for 30 min. The concentration of EPS was spectrophotometrically determined by measuring the absorbance at 420 nm. Commercial xanthan gum (2 g/liter) was subjected to quantitative analysis as a standard.

Lab. collection

Promega

pGEM vector harboring 250 bp containing non-coding region between cysB and wxoE Overexpression vectot, laczAþ, Ampr pET15b harboring wxoC pET15b harboring wxoF pET15b harboring wxoE Complementation Vector, Genr, Kanr pML122 containing 7.5 kb DNA fragment with cysB-wxoE-wxoF-wxoABC genes derived from pBACF6, Genr Tn5 insertion in 27 bp upstream wxoE of pXOPR Tn5 insertion in 84 bp upstream wxoE of pXOPR

Abrebreations Km: chroramphenicol.

Inoculums (w1 106 cells) prepared from wild-type and mutant strains of Xoo were grown as PSA cultures for 3 d. Virulence assays were tested on 60-day-old leaves of a susceptible rice cultivar (variety, Milyang 23) by leaf-clipping and punching methods [20]. Pathogenicity was observed after 14 d of inoculation.

This study

A BAC plasmid including LPS biosynthesis related gene clusters I and II, Chr. vector, Apmr , lacZaþ

a target gene with the transposon insertion was transformed into X. oryzae pv. oryzae KACC10859 by electroporation and then plated on Nutrient Agar media containing kanamycin (20 mg/liter). The kanamycin-resistant colonies were selected as marker-exchanged clones; this was confirmed by PCR amplification and Southern blot hybridization. 2.4. Virulence assays

X. oryzae pv. oryzae (Wild type strains) KACC 10859 X. oryzae pv. oryzae (mutant strains) McysB KACC10859 cysB:: Tn5 Kmr MwxoE KACC10859 wxoE:: Tn5 Kmr MwxoF KACC10859 wxoF:: Tn5 Kmr MwxoA KACC10859 wxoA :: Tn5 Kmr MwxoB KACC10859 wxoB:: Tn5 Kmr MwxoC KACC10859 WxoC:: Tn5 Kmr MwxoD KACC10859 WxoD:: Tn5 Kmr CwxoE MwxoE harboring pMLOT, Kmr, Gmr CwxoF MwxoF harboring pMLOT, Kmr, Gmr XoPT27 Marker exchanged KACC10859 with pTP47 XoPT84 Marker exchanged KACC10859 with pTP47

131

This study

Novagen This study This study This study Lab. collection This study

This study

2.6. Complementation test

This study

Am:

Ampicillin,

Chr:

ligated into pGEM-T Easy vector (Promega) and transformed into E. coli DH5a using the MicroPulserÔ Electroporation apparatus (BioRad). Plasmids carrying target genes were disrupted by transposon insertion using the EZ::TN Insertion kit (Epicentre Technologies) in accordance with the manufacturer’s instructions. Transposon insertion sites in the target genes were confirmed by both PCR amplification and sequencing analyses. Each plasmid containing

BACF6 plasmids carrying LPS loci of Xoo were partially digested with Sau3A, and the DNA fragments ranging from 6 kb to 10 kb were ligated into the pML122 vector, which was cut with BamHI and transformed into E. coli DH5a by electroporation (MicropulserÔ; Bio-Rad). A recombinant clone, pMLOT, containing a DNA fragment with the cysB, wxoE, wxoF, wxoA, wxoB, and wxoC genes was obtained. pMLOT was transformed into MwxoE- and MwxoFmutant strains by electroporation, and the strains were plated on PSA medium containing gentamycin and kanamycin. After 4 d, the colonies formed on the medium were isolated as the complementation clones CwxoE and CwxoF. 2.7. Overexpression of lps genes and protein purification

Table 2 Enzyme activity of WxoE protein to different substrates. Substrate

Final conc.(mM)

Thiol determination PLP ()

PLP (þ)

L-Cystathionine

2.5 2.5 2.5 25 25 25

80.5 0.0 ND ND 0.0 0.0

100.0 0.0 ND ND 0.0 0.0

O-Succinylhomoserine DL-Homocysteine L-Cysteine L-Serine L-Methionine

Enzyme activity was measured in the presence (þ) or the absence () of PLP. The relative activity of L-cystathionine was set as 100%. Values are the mean of three determinations. ND, not determined.

The wxoF, wxoE, and wxoC genes were amplified from BAC plasmid (pBACF6) carrying the lps gene cluster I of Xoo by using a forward primer (wxoE-F: 50 -CAGAATCTCATATGTCCAACCGCACCAC30 ) containing a NdeI restriction site (underlined) at the start codon of the ORF and a reverse primer (wxoEeR: 50 -CTGCTCGAGTCAATTTT GATTCACCAAC-30 ) containing an XhoI restriction site after the stop codon. The wxoF gene was amplified with a forward primer containing an NheI restriction site (underlined) at the start codon of the ORF (wxoF-F: 50 -CAGAAGATCTCATATGCTAAAGAATTTTTCAAA-30 ) and a reverse primer (wxoF-R: 50 -CAGTCTCTCGAGTCAAGCACGTAG CAGCAAC-30 ) containing a XhoI restriction site at the stop codon. For PCR amplification of the wxoC gene, a forward primer containing an

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engineered NheI restriction site (underlined) at the start codon (wxoC-F: 50 -CAGAAGATCTCATATGTCTGTAATGGACCGTC-30 ) and a reverse primer containing a XhoI restriction site (underlined) at the stop codon (wxoC-R: 50 -AGTCTCTCGAGCTATACTCGCTCAGGATAA TTAC-30 ) were used. The PCR amplicons were double-digested with NdeI and XhoI, ligated into a pET15b expression vector (Novagen) containing a 6xHis tag upstream of a thrombin cleavage site and the multiple cloning site, and transformed into E. coli BL21 (DE3) pLysS, yielding the recombinant clones pET-WxoE, pETWxoF, and pETWxoC. To purify the overexpressed proteins, pET-WxoE, pETWxoF, and pETWxoC were grown in 1 liter of LB medium containing ampicillin to OD600 ¼ 0.5 at 37  C. Overexpression was induced by adding 0.5 mM IPTG for 3 h. The bacterial cells were pelleted and suspended in 200 ml buffer (20 mM Tris; 5 mM imidazole; pH 8.0), sonicated, and centrifuged at 104 rpm for 15 min. The supernatant was loaded on a column packed with nickel nitrilotriacetic acid (Ni-NTA) equilibrated to buffer solution (20 mM TriseHCl; 0.5 M NaCl; 5 mM imidazole; 8.0 M urea; pH 8.0). The column was first washed with 50-mM imidazole buffer, and the fusion protein was then eluted using a 250-mM imidazole buffer. The eluted fractions were analyzed on SDS-PAGE. 2.8. Western blot analysis The purified WxoF and WxoC proteins were used for raising rabbit polyclonal antibodies following a standard protocol [25]. Cellular fractions of Xoo and purified WxoF and WxoC proteins were separated by SDS-PAGE (12%) and electroblotted onto a polyvinylidene fluoride (PVDF) membrane at 60 mA for 1 h in a transfer buffer (25 mM TriseHCl, 190 mM glycine, and 10% methanol). The blotted membrane was incubated at room temperature for 2 h in blocking solution and then hybridized with primary antibodies at a dilution of 1:2000 for 1 h. After washing 3 times (15 min each time) with washing solution (Tris 6 g, NaCl 8 g, KCl 0.2 g, 0.1% Tween 20 in 1 L, pH 8), the PVDF membrane was hybridized with the secondary antibody (anti-rabbit antibody; GE Healthcare) for 1 h. After washing the membrane 3 times with TTBS solution, the membrane was humidified with a detection reagent based on the protocol provided with the kit (GE Healthcare). The hybridization signals on the membrane were detected by exposure to X-ray film (Fuji). 2.9. Cellular fractionation Cellular fractionation from wild-type Xoo cells was performed according to the reported method with slight modifications [(Ray et al., 2000). The cells were grown in 40 ml of PS medium for 3 d at 28  C and pelleted by centrifugation. The pellet was suspended in 5 ml of 50 mM TriseHCl (pH 8.0), containing sucrose (20%), 2 mM EDTA, and 200 mg/ml lysozyme (from chicken egg white, Sigma, USA), and incubated at 28  C for 30 min with gentle shaking. The cell suspension was centrifuged and the supernatant was collected as the periplasmic fraction. The cell pellet was resuspended in 5 ml of 50 mM TriseHCl pH 8.0, sonicated briefly to break the cells, and then centrifuged to remove the undisrupted cells. The resulting supernatant was ultracentrifuged at 2.5  104 g for 2 h. The supernatant and precipitant were collected as cytoplasmic and membrane fractions, respectively. Periplasmic and cytoplasmic fractions were precipitated by adding 50% ammonium sulfate (wt/vol). The membrane fraction was dissolved in 50 mM TriseHCl (pH 8.0) containing 2% SDS. 2.10. Enzymatic activity of WxoE Cystathionine lyase (cystathioninase; EC 4.4.1.8) activity was measured by the determination of free-thiol group formation with

5,50 -dithio-bis 2-nitrobenzonic acid (DTNB) as described by Uren [33]. Pyridoxal-50 -phosphate (PLP) was added to the reaction mixture at a final concentration of 20 mM. In order to investigate the specificity of the purified WxoE, sulfur-containing amino acids or amino acid derivatives were used as substrates. For WxoE, a protein concentration of 1 mg in 1 ml of final volume was used for enzymatic reactions. 3. Results 3.1. wxoE and wxoF are organized in lps gene cluster I and necessary for pathogenecity As shown in Fig. 1(A), we noted the presence of 2 lps biosynthesis gene clusters organized with 17 genes between three transposon elements. In homology search, wxoE (previously named as metB) and wxoF is found restricted in X. oryze pv. oryzae genomes as lps genes. Especially, we newly annotated wxoE as a gene member in lps cluster I. Therefore, our primary interest in this study was focused on wxoE and wxoF in gene cluster I since their functional roles remained be demonstrated. wxoE and wxoF, as well as cysB, wxoA, wxoB, wxoC, and wxoD in the cluster I were PCRamplified, cloned, and disrupted by transposon insertion and marker-exchanged with wild-type strain KACC10859. Verification of the transposon insertions in the target genes and single transposon insertion in each gene was further confirmed by PCR and Southern blot analysis (data not shown). The transposon insertion sequence in each gene of the LPS locus is shown in Fig. 1A. The growth rate of the mutants on PSA medium was similar to that of the wild-type strain. Pathogenicities of the mutant strains were assayed on leaves of a susceptible rice variety, Milyang 23, by using leaf-clipping and punching methods. Virulence assays revealed that wxoE and wxoF, were nonpathogenic to rice leaves, similar to wxoA, wxoB, and wxoC mutants, while the cysB and wxoD mutants maintained their virulent phenotypes akin to the wild-type strain (Fig. 1B). On the other hand, complementation using CwxoE and CwxoF restored the pathogenicity of the respective mutants. In an earlier study, the wxoA, wxoB, and wxoC genes in lps gene cluster I of Xoo were reported to be required for virulence [4]. Our data presented here demonstrate that wxoE and wxoF are also novel virulence genes. 3.2. wxoF and wxoE is likely associated with biosynthesis of LPS O antigen The wxoE and wxoF genes had not previously been implicated in LPS biosyntheses. Therefore, we focused on these two genes in this study to demonstrate their role in LPS biosynthesis of X. oryzae pv. oryzae. In addition, we investigated if LPS structure of the bacterium was affected by mutations in each gene in of the lps gene cluster I including wxoE and wxoF. LPS extracts from the wild-type strain and the mutant strains were prepared from proteinase K-treated whole-cell lysates and analyzed by SDS-PAGE. The silverstained LPS gel is shown in Fig. 2. A ladder-like structure was observed for E. coli LPS that was used as a comparative sample. Upper LPS bands with low mobility were observed in wild-type strain KACC10859 and mutant MwxoD, but not in the other mutant LPS profiles. The low mobility bands corresponding to high molecular weight molecules on the SDS-PAGE are complete LPS consisting of a long chain of repeating O-antigens, core oligosaccharides and lipid A [9,12,13,34]. The high mobility bands corresponding to low molecular weight molecules likely represent lipooligosaccharides (LOS) consisting of the core oligosaccharides and lipid A prior to O-antigen attachment. The intermediate mobility bands likely represent the presence of different lengths of O-antigen subunits attached to LOS.

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3.3. wxoE and wxoF effect EPS production Total EPS fractions were extracted from the wild-type strain KACC10859, MwxoE, MwxoF, MwxoA, MwxoB, MwxoC, and MwxoD. Under the tested conditions, EPS production by MwxoE, MwxoF, MwxoA, MwxoB, and MwxoC was dramatically reduced in comparison to that by the wild-type strain (Fig. 3). In contrast, MwxoD produced EPS comparable to the wild-type strain KACC10859. Generally, a wild-type Xoo colony is yellow and has a smooth and mucoid morphology on PSA medium due to EPS production. However, colonies of the MwxoE and MwxoF, similar to MwxoA, B, and C colonies, were rough and showed a reduced mucoid morphology as compared to the wild-type strain KACC10859, indicating a defect in EPS production. The EPS analytical result suggests that wxoE and wxoF also effect EPS production likely to wxoABC.

Fig. 2. SDS-PAGE profile of LPS extracts isolated from wild-type (KACC10859) and lps mutant strains of X. oryzae pv. oryzae. The names of the analyzed strains are given above the lanes. The LPS samples were run on 15% polyacrylamide gel and detected with silver staining. The arrowheads indicate LPS bands present on wild type and mutant MwxoD strains.

These result strongly suggested that the genes affected in these mutants were necessary for synthesis of the full length of O-antigen chains of X.oryzae pv. oryzae LPS. In homology search (Fig. 4A), WxoF showed significant sequence identity of about 23e31% similarity to different methyltransferases derived from various bacterial species [18,35]. However, WxoF was not identified in genome of other Xanthomonas species, showing unique gene of X. oryzae pv. oryzae. WxoF protein was similar to the Salmonella enterica rfbT gene, which is thought to encode a component of the ligase that links O-antigen to core-lipid A [33,35]. Therefore, it was presumed that WxoF played an important role in the modification of O-antigen LPS. The wxoE, wxoF, and wxoC genes were overexpressed in the E. coli system, and the fusion proteins of WxoF and WxoE along with WxoC with the (His)6 tag were purified. The expected molecular sizes 29 kDa, 44 kDa, and 45 kDa of WxoF, WxoE, and WxoC proteins were separated on SDS-PAGE. Polyclonal antibodies were raised against WxoF and WxoC. In the hydropathy profile, 54 amino acids in the N-terminus of the WxoF protein were hydrophilic, but the WxoF protein was largely hydrophobic, with a mean hydrophobicity of 0.26 (Fig. 4B), indicating features of being a membrane protein. To confirm this nature of WxoF, the cytosplasmic, periplasmic, and membrane proteins were fractionated from the whole cells of X. oryzae pv. oryzae strain KACC10859 and subjected to SDS-PAGE, immunoblotted, and probed with antiWxoF. An intense band of 29 kDa was detected in the membrane fraction but not in other fractions (Fig. 4B). It was therefore concluded that WxoF is a membrane protein. On the other hand, WxoE demonstrated 54% sequence identity with human cystathione g-lyase, 48% with yeast cystathione g-lyase, and 46% with P. putida methionine g-lyase, strongly suggesting that WxoE is a cystathione g-lyase. In order to demonstrate such enzymatic activity of WxoE, the enzymatic activity of the purified WxoE using different substrates including cystathione was tested. Table 2 shows the results of this test; purified WxoE possessed enzymatic activity with substrate specificity to cystathione as measured by thio group determination. Thus, it was reasonably assumed that that wxoE and wxoF is associated with methylation in O-antigen synthesis of LPS together with other genes in lps gene cluster I.

3.4. Translational expressions of wxoE and wxoF are correlated to lps genes in cluster I It was revealed that wxoE, wxoF wxoA, wxoB and wxoC exept wxoD was correlated to Pathogenecity, LPS biosynthesis and EPS production, suggesting expression of the genes is regulated under an operon. Therefore, the protein expression pattern of the lps genes in cluster I was studied by western blot hybridization. Polyclonal antibodies were raised against the purified WxoF and WxoC and then were used for western blot analysis. As shown in Fig. 5, WxoF antiserum produced positive signals in the wild-type strain KACC10859 and some mutant strains (McysB, MwxoA, MwxoB, and MwxoC) but not in MwxoF. On the other hand, WxoC antiserum detected a band of 49 kDa in total protein samples in the wild-type strain KACC10859, McysB, and XoMTP84, but not in XoMTP27 and other mutant strains. The results of western blot analysis suggested that the translational expression of the lps genes was polar and dependent on the putative promoter region located approximately 27 bp upstream of the wxoE gene. In addition, XoMTP84 was pathogenic to rice similar to the wild-type strain, while XoMTP27 showed attenuated pathogenicity (data not shown), indicating that the 27-bp upstream wxoE gene is an important sequence region for inducing pathogenicity. Consequently, we considered that the reduction in pathogenicity was due to disruption of the promoter region.

Fig. 3. EPS production in X. oryzae pv. oryzae strains. Total EPS was extracted from culture filtrates of the Xoo strains and measured by the phenol-sulfuric acid method. Commercial xanthan gum was used as a standard sample to compare the amounts of EPS produced by Xoo strains. Values are represented as the mean  standard deviation from three independent experiments.

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Fig. 4. Alignment of the amino acid sequences between GeneBank accessions and WxoF protein (A) and hydropathy profile of the deduced protein from wxoF (B). CAA42150: RfbT of Vibrio cholerae; EDN54060: methyltransferase FkbM family of Methylobacterium extorquens PA1; YP_113626: methyltransferase, FkbM family Methylococcus capsulatus str. Bath; YP_468309: lipopolysaccharide biosynthesis protein (SAM-dependent methyltransferase protein). Hydropathy profile of WxoF was analyzed by Kyte and Doolitte algorithm. The abscissa is the amino acid reside number and ordinate is the hydrophobic index. The western blot data in hydropathy profile show the cellular localization of WxoF; C: cytoplasmic fraction, P: periplasmic fraction, and M: membrane fraction of Xoo strain KACC10859.

Fig. 5. Western blot analysis of lps gene cluster I using antibodies raised against WxoF and WxoC. The predicted promoter sequences (e10 and e35 regions) and ribosomal binding sites are marked by underlining and with boxes, respectively (Panel A). P27 and P84 indicate the sequence positions of transposon insertion. Total proteins from wild-type and LPS mutant strains of X. oryzae pv. oryzae, subjected to SDS-PAGE and blotted onto PVDF membrane and then hybridized with anti-WxoF (a) and anti-WxoC (b).

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4. Discussion Despite the fact that LPS is a very important factor for virulence in X. oryzae pv. oryzae [4], the functional roles of LPS biosynthesisrelated genes still remain poorly understood. In this study, we focused on the biological functions of the previously uncharacterized wxoE and wxoF genes in lps gene cluster I. Mutant strains of wxoE and wxoF were generated by transposon mutagenesis. Pathogenicity test using the mutant strains showed that wxoE and wxoF are novel virulence genes comparable to wxoA, wxoB, and wxoC reported previously [4]. We conducted LPS analysis of the lps cluster I genes containing wxoE and wxoF. Interestingly, the diffused band of low- mobility was only present in the wild-type strain and wxoD, but not absent in wxoE, wxoF, wxoA, wxoB, and wxoC mutants. In a previous report, low- and high-mobility bands of LPS in X. campestris pv. campestris were respectively defined as O-antigen and core oligosaccharide [34]. The LPS pattern showed a good agreement to that of our result. Generally, the high molecular LPS portions of O-antigen was formed as the repeated unit with ladder type on SDS-PAGE in bacteria [5,9,12]. Therefore, it was concluded that the lps genes in cluster I may participate in LPS O-antigen biosynthesis in Xoo. Vicariously, LPS bands with the intermediate mobility were mostly formed in the mutant strains unlikely to wild-type strain. In E. coli [12], intermediate LPS portions were added to lipid A/core, when was in the absence of the complete O antigene The biosynthesis of LPS O-antigen is organized in two successive steps. First, nucleotide sugars are synthesized, which serve as precursors for the second step. Then, the repeating units of the polysaccharides are assembled on the cytosolic face of the inner membrane. The repeating units are then polymerized and transferred through the cell membrane [37,38]. The wxoB and wxoC genes encode predicted glycosyl transferases, thereby assuming functional roles in LPS biosynthesis. The glycosyl transferase proteins sequentially transfer the various precursor sugars (UDP-, ADP-, GDP-, or CMP-linked) to form an oligosaccharide on a carrier lipid, undecaprenyl phosphate (UndP), which is situated in the inner membrane facing the cytoplasmic side, or transfer them to a variety of substrates such as LPS, glycogen, and fructose-6phosphate [24]. wxoA encodes a predicted epimerase and resembles the rmd gene of X. campestris pv. campestris that converts GDP-4keto-6-deoxy-D-mannose to GDP-D-rhamnose in O-antigen LPS biosynthesis [15,37]. The wxoD gene encodes a putative O-antigen acetylase involved in LPS modification, although the gene affected neither the pathogenicity nor LPS biosynthesis. Interestingly, CysB and WxoE resemble key enzymes in the biosynthesis of methionine from cysteine. CysB was reported to share significant sequence identity with cystathionine synthase [11], but was not associated with pathogenicity of Xoo. It was reported that mutation in metC of animal pathogen, Salmonella enterica serotype Gallinarum attenuated virulence in mice and chickens [27], supporting the association with pathogenecity of wxoE. WxoE resembles cystathione g-lyase that converts cystathione to homocysteine in the biochemical pathway for methionine biosynthesis [10,11]. In the next step of this biochemical cycle, homocysteine is altered along with S-adenosylmethionine, which is the methyl donor in a number of essential biochemical reactions. The methylation of sugars is a typical feature in chemical structures of O-antigens of different Xanthomonas species [28]. Actually, it was revealed that the purified WxoE possessed enzymatic activity of cystathione g-lyase by specifically using cystathionine as a substrate. Also, it has been known that sulfur-containing amino acids such as cysteine are major components in inducing Xanthomonas pathogenicity locus [26]. However, we do not know the manner in which WxoE participates in pathogenecity and LPS biosynthesis as a member of lps gene cluster I.

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On the other hand, WxoF showed significant sequence identity with proteins associated with methylation as well as the RfbT protein related to O-antigen conversion in Vibrio cholerae [29]. The O-antigen is the most variable portion in LPS and is heavily substituted with moieties that can confer the hydrophobic characteristic, such as O-methylation, acetylation, and esterification [37,38]. In chemical structure analysis of the lipopolysaccharides of X. campestris strains, it was revealed the O-polysaccharide chains include methylated sugars with 2,3,5-tri-O-methylfucose, 3,4-di-O-methylrhamnose, 2,4-di-Omethylrhamnose, and 2-O-methylrhamnose, respectively. In addition, in Rhizobium etli, a plant signal such as anthocyanin causes a change in the sugar composition of LPS O-antigen, leading to di- and tri-O-methyl on fructose terminals [9]. In western blot analysis using anti-WxoF, it was revealed that WxoF is localized in the membrane. Previously, it was reported that the RfbT protein, which is homologous to WxoF, is also a membrane protein [29], supporting the results obtained in this study. From the predicted functions of WxoE and WxoF, we hypothesized that they played a functional role in generating the methyl donor and transferring the methyl on to sugar residues for the modification of O-LPS. Xanthomonas species produce copious amounts of exopolysaccharides, mainly xanthan gum, as a virulence factor on media complemented with glucose [1,3,4,14,17]. In this study, the mutation of wxoE and wxoF caused a dramatic reduction in EPS production, leading to rough mucoid colony morphologies. The mutant strains of wxoA, wxoB, and wxoC also showed EPS deficiency, which was in good agreement with a previous report [4]. Reports from gram negative bacteria including X. campestris pv. campestris have shown that defects in LPS biosynthesis are correlated with other mutant phenotypes, such as rough colony morphology, autoagglutiation in liquid medium, and loss of motility [15,31]. Correspondence among wxoE, wxoF, wxoA, wxoB, and wxoC in LPS and EPS patterns speculated that the genes might be correlated in gene expression. Western blot analysis showed that lps genes containing wxoE and wxoF in cluster I are cotranslated in an operon with a polar fashion. However, molecular technology such as the primer extension will further be performed for finding real molecular region of promoter, although we demonstrated their translational expressions are dependent on the putative promoter region by mutation experiment. LPSs from certain plant pathogens, such as X. campestris, P. syringae, and R. solanacearum, have been shown to activate a number of defense-related responses in plants [3,14,19,32]. It has also been suggested that LPS is likely to be involved in the association of bacteria with plant cell walls during the infection process [2]. This study provides additional insights into the functional roles of wxoE and wxoF genes in LPS biosynthesis of X. oryzae pv. oryzae. Subsequently, it was postulated that LPS O-antigen components of X. oryzae pv. oryzae are critical factors in interaction between pathogen and host for pathogenesis. However, additional research should further be performed on the biochemical modifications on LPS chemical structure to elucidate each functional role of LPS biosynthesis-related genes in X. oryzae pv. oryzae Acknowledgments This research was supported by a grant from the Agenda research program (Code #200901FHT020710285) in the Rural Development Administration of Korea. References [1] Barrere GC, Barber CE, Daniels MJ. Molecular cloning of genes involved in the production of the extracellular polysaccharide xanthan by Xanthomonas campestris pv. campestris. Int J Biol Macromol 1986;8:372e4.

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