Variation suggestive of horizontal gene transfer at a lipopolysaccharide (lps) biosynthetic locus in Xanthomonas oryzae pv. oryzae, the bacterial leaf blight pathogen of rice

Abstract

Background: In animal pathogenic bacteria, horizontal gene transfer events (HGT) have been frequently observed in genomic regions that encode functions involved in biosynthesis of the outer membrane located lipopolysaccharide (LPS). As a result, different strains of the same pathogen can have substantially different lps biosynthetic gene clusters. Since LPS is highly antigenic, the variation at lps loci is attributed to be of advantage in evading the host immune system. Although LPS has been suggested as a potentiator of plant defense responses, interstrain variation at lps biosynthetic gene clusters has not been reported for any plant pathogenic bacterium.

Results: We report here the complete sequence of a 12.2 kb virulence locus of Xanthomonas oryzae pv. oryzae (Xoo) encoding six genes whose products are homologous to functions involved in LPS biosynthesis and transport. All six open reading frames (ORFs) have atypical G+C content and altered codon usage, which are the hallmarks of genomic islands that are acquired by horizontal gene transfer. The lps locus is flanked by highly conserved genes, metB and etfA, respectively encoding cystathionine gamma lyase and electron transport flavoprotein. Interestingly, two different sets of lps genes are present at this locus in the plant pathogens, Xanthomonas campestris pv. campestris (Xcc) and Xanthomonas axonopodis pv. citri (Xac). The genomic island is present in a number of Xoo strains from India and other Asian countries but is not present in two strains, one from India (BXO8) and another from Nepal (Nepal624) as well as the closely related rice pathogen, Xanthomonas oryzae pv. oryzicola (Xoor). TAIL-PCR analysis indicates that sequences related to Xac are present at the lps locus in both BXO8 and Nepal624. The Xoor strain has a hybrid lps gene cluster, with sequences at the metB and etfA ends, being most closely related to sequences from Xac and the tomato pathogen, Pseudomonas syringae pv. tomato respectively.

Conclusion: This is the first report of hypervariation at an lps locus between different strains of a plant pathogenic bacterium. Our results indicate that multiple HGT events have occurred at this locus in the xanthomonad group of plant pathogens.

Figure 1

Genetic organization of a locus encoding LPS biosynthetic genes in Xoo strain BXO1. a. Overall G+C content of the locus and the flanking regions. The G+C content of the genomic island was calculated without including the sequences of IS elements. The overall G+C content of the genome is ~65%. b. Organization and G+C content of individual genes and transposases of IS elements. IS1114 encodes a truncated ORF. Arrows indicate transcriptional orientation. c. and d. Presence (+) and absence (-) of genes/PCR products in particular strains. ^§ Indicates PCR products obtained using primer pairs directed against either metB and wxoA or etfA and wzm. ^# similar results were obtained with all Xoo strains tested excepting BXO8 and Nepal 624. * similar results were obtained with the Nepal 624 strain.

Figure 2

Genes encoded in the lps locus exhibit altered Codon Usage Pattern (CUP). Eight amino acids, each of which has atleast four synonymous codons, are represented on the X-axis. The % of codons ending with G/C for each of these amino acids is represented on the Y-axis as mean ± SD. The lower line represents CUP for eight aminoacids of the six genes (excluding transposase ORFs) encoded in the lps locus. The upper line represents CUP of six Xoo genes from elsewhere in the genome (Please refer Methods).

Figure 3

The lps locus is absent from the genomes of Xoo strains BXO8, Nepal624 and Xoor strain BXORI. (A) PCR analysis using primers that are specific to wxoA gene. M is the λ HindIII Marker lane. An expected band of 1 kb (indicated by arrow) is present in the Xoo strains, BXO1 (lane 1), BXO5 (lane 3), BXO6 (lane 4) and BXO20 (lane 6) but absent in BXO8 (lane 5), Nepal624 (lane 7) and BXORI (lane 2). (B) Southern hybridization analysis of EcoRI digested genomic DNA using α-³²-P labeled wxoA specific probe (see Methods). A 4 kb band can be seen (indicated by arrow) in BXO1 (lane 1) but not in BXORI (lane 2), BXO8 (lane 3) and Nepal624 (lane 4). Similar results were obtained for wxoB, wxoC, wxoD, wzm and wzt genes. (C) The blot from (B) was deprobed and was hybridized with α-³²P labeled probe specific to the metB gene. A specific band can be seen in all the lanes. Note the sizes of the bands indicating that metB is present in different EcoRI fragments in BXO1, BXORI and BXO8/Nepal624.

Figure 4

Invert repeat sequences flanking the lps gene cluster in the BXO1 strain of Xoo. The horizontal arrows represent the ORFs of metB and etfA. I, II, III represent three different invert repeats in the 3' regions of metB and etfA genes. The vertical arrows represent the breakpoints of homology between BXO1 and BXO8. The distances of the break points from the stop codons of the metB and etfA genes are indicated. Dashed lines indicate the remainder of the metB and etfA genes. The sequence of the corresponding inverse repeats in Xac, Xcc and Xoor are also indicated along with the derived consensus sequence for the repeats. The numbers in brackets indicate distances in bp between individual repeats.

Figure 5

Restriction fragment length polymorphism analysis of Xoo strains. Southern analysis of EcoRI-digested genomic DNA was performed using α-³²P labeled IS1112 as a probe (see Methods). Lanes: 1; BXO1, 2; BXO5, 3; BXO6, 4; BXO8, 5; BXO20, 6; Nepal624, 7; Xoor strain BXORI. M; indicates the size of molecular weight markers in kb.

Figure 6

Cluster analysis of Xoo strains. The dendrogram was constructed using the UPGMA option of PHYLIP on the basis of restriction fragment length polymorphism data obtained with IS1112 probe. Numbers or symbols at the internal branches indicate bootstrap values for clusters. The BXORI (Xoor) strain constitutes the outgroup.

Figure 7

Variation in lps gene clusters within the xanthomonads. The genes that are adjacent to metB and etfA in different xanthomonads are indicated. Dashed lines represent the remainder of the lps cluster. Empty and filled boxes represent sequences specific to Xoo and Xcc respectively. Boxes with dots indicate that the sequences are either from or related to Xac genes. Box with stripes represents sequences that are related to Pseudomonas syringae pv. tomato. Arrows indicate transcriptional orientation. The wzm gene encodes a predicted ABC transporter permease protein, wxoA encodes a predicted epimerase, wxcA encodes a glycosyl transferase and wxcH encodes a hypothetical protein.