Stationary-phase variation due to transposition of novel insertion elements in Xanthomonas oryzae pv. oryzae

Abstract

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, a serious disease of rice. Spontaneous mutants which are deficient for virulence and extracellular polysaccharide (Eps) production accumulate in large numbers in stationary-phase cultures of this bacterium, a phenomenon which we have called stationary-phase variation. A clone (pSD1) carrying the Eps biosynthetic gene (gum) cluster of X. oryzae pv. oryzae restored Eps production and virulence to several spv (for stationary-phase variation) mutants. Data from localized recombination analysis, Southern hybridization, PCR amplification, and sequence analysis showed that the mutations are due to insertion of either one of two novel endogenous insertion sequence (IS) elements, namely, ISXo1 and ISXo2, into gumM, the last gene of the gum gene cluster. The results of Southern analysis indicate the presence of multiple copies of both IS elements in the genome of X. oryzae pv. oryzae. These results demonstrate the role of IS elements in stationary-phase variation in X. oryzae pv. oryzae.

FIG. 1

Virulence of X. oryzae pv. oryzae strains on rice. Inoculations were performed on 40 one-day-old plants of rice cultivar TN-1 as described in Materials and Methods. The data are the averages of measurements from 10 leaves that had been inoculated with particular strains: BXO1 (wild type), BXO125 (spv mutant), BXO152 (BXO125/pSD1), and BXO150 (an Eps⁺ Vir⁺ recombinant obtained in a BXO125/pSD2 background). Error bars indicate standard deviations. Similar results were obtained in independent experiments.

FIG. 2

DNA rearrangement at the gum locus in several spv strains. Southern analyses of EcoRI-digested genomic DNA were performed using α-³²P-labeled probes as described in Materials and Methods. pSD2 (A) and pSD1 (B) were used as probes. Lanes: M, ladder marker DNA from Stratagene; 1, BXO1 (wild type); 2, BXO125 (spv mutant); 3 and 4, BXO113 and BXO114 (both are spv mutants which cannot be complemented by pSD1). The 7.8-kb fragment is present as a 9-kb fragment in BXO125.

FIG. 3

(A) The gumI-to-gumM region of the gum locus, included in the pRR7 clone. The locations where PCR primers were designed are indicated. F1 to F6 are forward primers, whereas R1 to R6 are reverse primers. (B) The gumM region of the gum locus. Sites of insertions of the IS elements are shown. ▿, sites of ISXo1 insertion in BXO121, -123, and -125. ISXo1 is inserted in the same site in BXO121 and -125. ▾, site of ISXo2 insertion in BXO119. Note that the putative transposase genes of ISXo1 and ISXo2 insertions are of a transcriptional orientation (←) opposite to that of the gumM gene (→). (C) Schematic representation of the organization of ISXo1 and ISXo2 elements. ▧, the ORF encoding putative transposase; ░⃞, transposon-specific flanking sequences; ■, terminal inverted repeats. ATG is the start codon; TAA and TGA are the stop codons.

FIG. 4

PCR amplification of mutant alleles from spv strains. Agarose gel electrophoresis of PCR products was performed by using the F6-R6 (lanes 1 to 5) and F5-R5 (lanes 6 to 10) sets of gum-specific primers with genomic DNA from X. oryzae pv. oryzae strains. Lanes: 1 and 6, BXO112; 2 and 7, BXO119; 3 and 8, BXO123; 4 and 9, BXO121; 5 and 10, BXO125. The molecular size marker (lane M) is a BstEII digest of lambda DNA.

FIG. 5

Distribution of ISXo1 and ISXo2 in the X. oryzae pv. oryzae genome. Southern analyses of BamHI-digested genomic DNA were performed using α-³²P-labeled probes ISXo1 (A) and ISXo2 (B). Lanes: 1, HindIII-digested lambda DNA marker; 2, BXO1; 3, BXO125; 4, BXO119. An additional 5.7-kb band (indicated by arrows) was detected in BXO125 with the ISXo1 probe and in BXO119 with the ISXo2 probe.