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Keywords:

  • Tn5;
  • Extracellular polysaccharide;
  • gumG;
  • Xanthomonas campestris pv. campestris;
  • Xanthomonas oryzae pv. oryzae

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

Xanthomonas oryzae pv. oryzae causes a serious disease of rice called bacterial leaf blight. It produces copious amounts of extracellular polysaccharide (EPS). An EPS- and virulence-deficient mutant of X. oryzae pv. oryzae was isolated by Tn5 mutagenesis. The mutant allele in this strain was cloned by transposon tagging in the Escherichia coli vector pBluescript and the DNA sequences flanking the transposon insertion site were determined. Computer-based similarity searches in the DNA database using the BLAST algorithm showed these sequences to be 78% identical at the nucleotide level to a gene, gumG, in the gum cluster, which is required for EPS biosynthesis in Xanthomonas campestris pv. campestris. A 36-kb X. oryzae pv. oryzae genomic clone containing the putative EPS biosynthetic gene cluster of X. oryzae pv. oryzae restored both EPS production and virulence proficiency to the gumGXo::Tn5 mutant. The results suggest that EPS is an important virulence factor of X. oryzae pv. oryzae.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

The genus Xanthomonas comprises bacteria that are specialized for causing plant disease. More than 250 plant diseases worldwide are caused by members of this genus. All xanthomonads produce copious amounts of an extracellular polysaccharide (EPS). Loss of EPS production has been correlated with loss of virulence in many plant pathogens [1]. However, the genes affected in such mutants have been often found to be regulatory in function affecting other putative virulence factors in addition to EPS production [2–4]. Therefore, it has been suggested that specific mutations in EPS biosynthetic genes should be obtained (preferably by using transposons) to assess the role of EPS in virulence of a plant pathogenic bacterium [5].

The EPS produced by Xanthomonas campestris pv. campestris is an industrially important product called xanthan gum. The structure of the EPS made by X. campestris pv. campestris has been shown to be composed of a repeating pentamer composed of two subunits of glucose, two subunits of mannose and one of glucuronic acid along with certain modifications like acetylation [1]. A cluster of genes spanning 16 kb has been shown to encode enzymes involved in EPS synthesis [6].

The role of EPS in the virulence of X. campestris pv. campestris is not very clear. A mutation in the gumD gene drastically reduces virulence on broccoli seedlings [7]. However, the gumD mutant begins to show symptoms 12 days after inoculation (as compared to 3 days for the wild-type strain of X. campestris pv. campestris). In another study, defined non-polar transposon insertions in specific genes of the EPS biosynthetic pathway show no more than 25–50% reduction in virulence when inoculated on cabbage leaves [6]. Therefore, it was concluded that EPS is involved in but not essential for virulence of X. campestris pv. campestris.

Spontaneous mutants which accumulate in stationary phase cultures and affect EPS production and virulence have been reported in X. oryzae pv. oryzae[8]. The nature of the mutations in these strains has not yet been defined. We report the isolation of a Tn5 insertion in the gumG homologue (henceforth referred to as gumGXo) of X. oryzae pv. oryzae. The X. campestris pv. campestris homologue of this gene has been shown to be involved in the acetylation of xanthan gum [6]. The gumGXo::Tn5 mutant is severely deficient in EPS production and virulence. Introduction of a clone containing the putative EPS biosynthetic gene cluster of X. oryzae pv. oryzae restored EPS production and virulence to the gumGXo::Tn5 mutant suggesting that EPS may play a significant role in the virulence of X. oryzae pv. oryzae.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

2.1Bacterial strains, media, EPS estimation etc.

All bacterial strains used are listed in Table 1. X. oryzae pv. oryzae strains were grown in peptone sucrose (PS) medium [9] at 28°C. Escherichia coli strains were grown in Luria-Bertani medium [10] at 37°C. Where appropriate, antibiotics used were rifampicin (Rf; 50 μg ml−1), spectinomycin (Sp; 50 μg ml−1), ampicillin (Ap; 50 μg ml−1), kanamycin (Kn; 50 μg ml−1), chloramphenicol (Cm; 20 μg ml−1), cycloheximide (Cy; 75 μg ml−1) and cephalexin (Cp; 20 μg ml−1). X. oryzae pv. oryzae is naturally resistant to cephalexin.

Table 1.  Strain list
  1. aIsolated by plating a saturated culture of BXO1 on PS agar with rifampicin (100 μg ml−1).

  2. bIsolated following random Tn5gusA40 mutagenesis of BXO151.

StrainRelevant characteristicsReference/source
Plasmids  
pBluescript (KS)AprStratagene, La Jolla, CA, USA
pRK600pRK2013 npt::Tn9, CmrLab collection
pEPS2Apr, Spr; Tn5 insertion from BXO1002 cloned in KpnI site of pBS(KS)This study
pEPS5Apr, Spr; Tn5 insertion from BXO1002 cloned in SalI site of pBS(KS)This study
pSD1, pSD3pUFR034+36 kb of X. oryzae pv. oryzae DNA that encodes genes required for EPS biosynthesisThis study
pSD2pUFR034+14.2 kb of X. oryzae pv. oryzae DNA that contains a part of the EPS biosynthetic regionThis study
E. coli strains
DH5αF′, endA1 hsdR17 (rk mk+) supE44 thi− 1 recA1 gyrA relA1 φ80dlacZΔM15 Δ(lacZYA-argF) U169Lab collection
S17-1RP4-2-Tc::Mu-Km::Tn7 pro hsdR recALab collection
X. oryzae pv. oryzae strains
BXO1Natural isolate; Chinsuria, West Bengal, IndiaLab collection
BXO151arif-10; derivative of BXO1This study
BXO1002bgumGXo::Tn5gusA40 rif-10 (derived from BXO151)This study
BXO1009gumGXo::Tn5gusA40/pSD1This study
BXO1010gumGXo::Tn5gusA40/pSD2This study
BXO1011gumGXo::Tn5gusA40/pSD3This study
eps indicates a mutation that confers extracellular polysaccharide deficiency. rif indicates a mutation that confers resistance to rifampicin. Apr and Spr confer resistance to ampicillin and spectinomycin, respectively.

Transposon mutagenesis of X. oryzae pv. oryzae was performed in biparental matings [11] between E. coli strain S17-1 carrying the Tn5gusA40 derivative on a suicide plasmid [12] and BXO151. Four EPS-deficient mutants were identified by visual screening of approximately 5000 exconjugants. EPS was isolated by precipitation with acetone as previously described [13] and quantitated by the colorimetric method for estimation of pentoses and hexoses [14]. The analysis of one particular Tn5-induced mutant, BXO1002 is described in this paper.

2.2Virulence assays on rice plants and re-isolation of bacteria from infected leaves

Forty-day-old greenhouse-grown rice plants of the susceptible rice cultivar Taichung Native-1 (TN-1) were inoculated by clipping leaf tips with sterile scissors dipped in saturated cultures of X. oryzae pv. oryzae[15]. Lesion lengths were measured at regular intervals (see Section 3).

2.3Marker exchange and GUS assay

Genomic DNA was isolated from BXO1002 [16] and electroporated into competent cells of BXO151 using a BTX electroporator (BTX, CA, USA) and a 2-mm gap cuvette according to the manufacturer's instructions. The transformants were plated on PS agar plus spectinomycin and incubated at 28°C.

2.4Cloning procedure

Genomic DNA was isolated from BXO1002 [16] and digested to completion with either KpnI or SalI (New England Biolabs, MA, USA). pBluescript was isolated by the alkaline lysis method [17]. pEPS2 was obtained after ligation of BXO1002 genomic DNA with pBluescript (both digested with KpnI). Tn5gusA40 does not have a KpnI site whereas SalI cuts once between the spec and gus genes of this Tn5 derivative [12]. pEPS5 was obtained after ligation of BXO1002 genomic DNA with pBluescript (both digested with SalI). pEPS5, therefore, contained a fragment that had the spectinomycin marker of the transposon and its adjacent flanking sequence from genomic DNA but does not contain the gus gene present in Tn5gusA40. Ligation mixtures were electroporated into E. coli DH5α and transformants were selected on LB plus ampicillin and spectinomycin.

2.5Sequencing of Tn5 insertion and analysis

Sequencing of the region flanking the Tn5 insertion was performed by using primers to the spec (5′-TCTAGCGAGGGCTTTACTA-3′) and gus (5′-CGCGATCCAGACTGAATGCC-3′) genes on the transposon [12]. The primers for pBluescript (KS), M13 forward primer and M13 reverse primer (Perkin Elmer, USA) were also used. Sequencing reactions, polyacrylamide gel electrophoresis and sequence output processing were carried out on an automated sequencing unit (ABI Prism 377, Perkin Elmer, USA) according to the manufacturer's instructions. Computer based sequence homology searches were performed using the BLAST algorithm [18] available on the World Wide Web.

2.6Screening of a genomic library and functional complementation

Five hundred and seventy-six individual clones of a genomic library of X. oryzae pv. oryzae[19] were screened in colony hybridization with pEPS5 as a probe as described [20]. The positive clones identified were first mobilized from E. coli DH5α to S17-1 (using pRK600 as the helper plasmid) and hence to BXO1002.

3Results

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

3.1The Tn5-induced mutant (BXO1002) produces very low amounts of EPS and is virulence-deficient

EPS was isolated and estimated from BXO1002 and BXO151 as described in Section 2. Fig. 1 shows that BXO1002 is severely deficient in EPS production. The wild-type typically produces 1950–2000 μg of glucose/109 cells whereas the mutant produces only 10–20 μg for the same number of cells.

image

Figure 1. Quantitation of EPS levels in wild-type and EPS-deficient strain of X. oryzae pv. oryzae. EPS was isolated and estimated as described in Section 2. The Y-axis shows the amount of glucose in μg, 10−9 cells of each strain. Each data point represents the average and S.D. obtained from three independent experiments. BXO151 is the wild-type strain, BXO1002 is the EPS-deficient mutant and BXO1009 is the mutant with a clone (pSD1) that restores EPS production.

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Inoculation of leaves of 40-day-old plants of the TN-1 rice cultivar indicates that BXO1002 is severely virulence-deficient (Fig. 2). Also, 14 days after inoculation, inoculated rice leaves were cut into 1 cm long pieces (after surface sterilization) and placed in serial order on PS agar plates with cycloheximide (to control fungal contamination). Bacteria oozed out of cut ends of the leaves infected with BXO151 and formed colonies on the plate all along the leaf indicating bacterial migration throughout the length of the leaf blade. In case of BXO1002, no bacteria oozed out of the cut ends and no colonies were formed on the plates except at the leaf tips (1.5 cm from the point of inoculation) indicating minimal bacterial migration.

image

Figure 2. Virulence phenotypes of BXO151, BXO1002 and BXO1009 on rice plants. Inoculation of 40-day-old rice plants of the TN-1 cultivar was performed as described in Section 2. The data at each time point represent the average and standard deviation of lesion lengths obtained from 12 inoculated leaves. BXO151 is the wild-type strain, BXO1002 is the EPS-deficient mutant and BXO1009 is the mutant with the clone that restores EPS production.

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3.2Sequence analysis of Tn5 insertion in pEPS2

Using the spec primer, we obtained (see Section 2) 574 bp of X. oryzae pv. oryzae sequence flanking the Tn5 insertion on pEPS2. In computer based similarity searches by the BLAST algorithm [18], this sequence showed 78% (574/733 bases) identity to the gumG and gumH genes of X. campestris pv. campestris at the nucleotide sequence level. The gum cluster is an operon with twelve open reading frames (ORFs) from gumB–gumM that encode the enzymes required for xanthan gum (EPS) biosynthesis [21] in X. campestris pv. campestris (GenBank accession number XCU22511). The high degree of homology indicates that the X. oryzae pv. oryzae sequence is a homologue of the gum sequence. The initial 117 bp of the sequence obtained from the spec primer was homologous to the gumGXc gene of the gum cluster indicating that the transposon has inserted in the gumGXo gene. The subsequent 457 bases were homologous to the gumHXc gene. The gumGXc and gumHXc genes are colinear on the gumXc cluster and the homology of the X. oryzae pv. oryzae sequence to these two genes suggests that their homologues are present in the same order on the genome of X. oryzae pv. oryzae.

Using the gus primer, the sequence of 159 bp of X. oryzae pv. oryzae DNA was obtained from the pEPS2 clone. The flanking region on this side was very short as the transposon had fortuitously inserted very close to the KpnI site. This sequence is also homologous to gumGXc gene confirming that the Tn5 has inserted into the X. oryzae pv. oryzae homologue of gumG (Fig. 3).

image

Figure 3. The Tn5 insertion in BXO1002 is located in the gumGXo gene. DNA sequence of the region flanking the transposon insertion in BXO1002 was obtained by using spec and gus primers with pEPS2 as template (as described in Section 2). The stop codon of the gumGXo gene (indicated with an asterisk) overlaps with the start codon of the gumHXo gene. The transposon (shown as a filled triangle) is inserted in the gumG homologue of X. oryzae pv. oryzae. The deduced amino acid sequence of the gumGXo and gumHXo genes is shown below the DNA sequence.

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A sequence of 630 bp was obtained by using the M13 reverse primer which is complementary to a region upstream of the KpnI site in pBluescript(KS). It showed 82% (518/630 bases) identity to the gumI gene of the gum cluster of X. campestris pv. campestris (data not shown). We also found that the sequence obtained with the M13 forward primer is identical to that obtained with the gus primer. The partial sequences of the X. oryzae pv. oryzae homologues of gumG, gumH and gumI of X. campestris pv. campestris have been deposited in GenBank under the accession numbers AF147035, AF147036 and AF147037, respectively. The gumGXc gene encodes an acetyl transferase; the gumHXc and the gumIXc encode glycosyl transferases.

3.3Marker exchange of Tn5 insertion in BXO1002

Genomic DNA was isolated from the EPS-deficient strain, BXO1002, and electroporated into competent cells of the wild-type strain, BXO151 (as described in Section 2). After 4 days of incubation at 28°C, all 10 spectinomycin colonies that appeared were EPS-deficient as determined by visual inspection. Three individual colonies were inoculated on rice plants (as described in Section 2) and found to be virulence-deficient (data not shown). This suggested that the single Tn5 insertion in BXO1002 (now marker exchanged on to the wild-type chromosome of BXO151) resulted in the concomitant loss of EPS production and virulence.

3.4Functional complementation

A genomic library of X. oryzae pv. oryzae in the E. coli strain DH5α[15] was screened by colony hybridization using the plasmid pEPS5 as a probe (see Section 2). pEPS5 was chosen for colony hybridization instead of pEPS2 in order to exclude homology of the Tn5gusA40 gus gene with its chromosomal copy in DH5α.

Three clones (designated pSD1, pSD2 and pSD3) were identified as positively hybridizing clones. The respective plasmids were digested with EcoRI and run on an agarose gel (data not shown). The clones pSD1 and pSD3 were identical and had six fragments of 1.2, 5.2, 5.5, 6.5, 7.8 and 10 kb for a total insert size of 36 kb. pSD2 had three fragments of sizes 1.2, 5.2 and 7.8 kb.

The pSD1, pSD2 and pSD3 plasmids were mobilized to BXO1002. pSD1 and pSD3 restored EPS production to BXO1002 as observed by visual inspection. EPS was also isolated from BXO1009 (BXO1002/pSD1) and quantitated as described in Section 2 (Fig. 1). The pSD1 clone restored wild-type levels of EPS production to the BXO1002 mutant. Neither pSD2 nor pUFRO34 (the vector in which the genomic library was constructed) restored EPS production to BXO1002 (data not shown). Southern hybridization on EcoRI digested genomic DNA of BXO1002 using pSD1 as a probe indicated that the Tn5 insertion is in the 7.8-kb fragment contained in this clone (data not shown). The lesion lengths caused by BXO1009 were comparable with the wild-type derivative, BX0151 (Fig. 2) showing that the clone pSD1 restored both EPS production and virulence proficiency to BXO1002.

4Discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

X. oryzae pv. oryzae causes a serious disease of rice called bacterial leaf blight. This bacterium, like other members of the genus Xanthomonas produces large amounts of EPS. In this paper, we report the isolation of a Tn5 insertion in the gumG homologue of X. oryzae pv. oryzae. The gumGXo::Tn5 insertion is also likely to have a polar effect on the expression of downstream genes in the gum operon [6]. This strain is severely deficient in EPS production and virulence. Introduction of a clone (pSD1) that restores EPS production also restores virulence of BXO1002. Since the gumG gene of X. campestris pv. campestris is required for EPS biosynthesis it is reasonable to expect that the gumGXo gene is also involved in EPS biosynthesis. Therefore, the virulence deficiency of the gumGXo::Tn5 mutant suggests that EPS is an important virulence factor of X. oryzae pv. oryzae.

In X. campestris pv. campestris, the EPS biosynthetic gene cluster has been shown to contain 12 genes designated gumB–gumM[6]. Sequence analysis presented here indicates that the gumHXo and gumIXo genes are located adjacent to the gumGXo gene and in that order. Additional sequencing data (R. Rajeshwari and R.V. Sonti, unpublished data) indicates that the gumFXo–gumMXo genes are encoded in that order on the 7.8-kb EcoRI fragment that is present in pSD1. This suggests that the organization of the gum cluster homologue of X. oryzae pv. oryzae is similar to that of X. campestris pv. campestris. Additional sequencing data are needed to determine if this similarity extends to the entire gum cluster.

In an earlier study, a Tn5 insertion in the rpfC homologue of X. oryzae pv. oryzae has been shown to affect EPS production as well as lesion formation on rice leaves [22]. The rpfC gene of X. campestris pv. campestris is a regulatory gene that affects EPS production as well as secretion of extracellular enzymes [23]. Therefore, loss of other factors besides EPS might have contributed to the lesion formation defect of the rpfCXo::Tn5 mutant. It has also been shown that although the rpfCXo::Tn5 insertion carries a defect in lesion formation, it does not affect bacterial growth and migration within the rice leaf [22]. The inability of the gumGXo::Tn5 mutant to migrate within the rice leaf and cause lesions might be a reflection of the complexity of the regulatory pathways that control EPS production and virulence in xanthomonads.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

We thank R. Rajeshwari for the marker exchange experiments. The Tn5gusA40 construct was provided by R.A. Jefferson and K. Wilson. S.D. is supported by a senior research fellowship from the Council for Scientific and Industrial Research (CSIR), India. This work was supported by a grant to R.V.S. from the Department of Biotechnology, Government of India.

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  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References
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