CHARACTERIZATION OF AN INSERTION SEQUENCE ELEMENT ASSOCIATED WITH GENETICALLY DIVERSE PLANT PATHOGENIC STREPTOMYCES SPP.

Insertion sequence (IS) elements constitute the simplest class of prokaryotic transposable genetic elements and are commonly found within prokaryotic genomes, plasmids, and phages. While they vary in size, typical IS elements generally share several properties, including (i) the presence of terminal inverted repeats, (ii) the absence of detectable IS-encoded activities other than those required for transposition or its regulation, and (iii) the generation of direct repeats of target nucleotide sequences upon insertion (5, 10). They have been found to effect a variety of genetic changes, vis-a-vis genetic organization (e.g., through deletion or duplication-inversion events) and gene expression (e.g., through insertional mutagenesis or the introduction of new cis-acting regulatory elements). Gram-positive filamentous streptomycetes are cosmopolitan members of soil microbial communities, and close to 500 species have been described. They share a complex developmental program of growth and differentiation characterized by the elaboration of substrate mycelia followed by a phase of filamentous aerial growth, which culminates in the transformation of aerial filaments into chains of spores. Their soil-dwelling nature would suggest that many have evolved in close association with plants and derive nutrients from decaying organic matter. Four species have been described, however, which have developed the means to incite disease on subterranean parts of economically important tuber crops, e.g., the potato. These species—Streptomyces scabies, S. acidiscabies, S. turgidiscabies, and S. ipomoeae—are quite unrelated to one another based on several criteria, including 16S ribosomal DNA sequence and DNA-DNA relatedness data, as well as morphological and biochemical attributes (8, 11, 12, 17, 30). Despite this diversity, however, all of these species share the production of one or more of a family of phytotoxins, thaxtomins, which mimic disease symptomatology on host plants (1, 13). Since the production of this unusual phytotoxin is common to unrelated species, it has been suggested that pathogenicity factor(s) have been horizontally transferred among these diverse pathogens (8, 13). Previous efforts aimed at elucidating other pathogenicity determinants in S. scabies 84.34 (ATCC 49173) identified a putative pathogenicity or virulence gene, nec1, which was sufficient to confer a necrogenic phenotype upon the nonpathogen S. lividans 66 TK24 (2). It was also found that nec1 is conserved in plant pathogens and is absent from nonpathogens. Subsequently, a strong correlation was shown to exist between thaxtomin production and the presence of nec1 in S. scabies, S. acidiscabies, and S. turgidiscabies strains (1). Evidence for the transfer of pathogenicity genes among diverse pathogenic species has recently been presented. The codon bias and GC content (54%) of nec1 are atypical relative to high-GC coding regions characteristic of Streptomyces strains (32), suggesting that it was acquired from another taxon. Sequence analysis of the 5′ end of nec1 identified an IS256 transposase homolog, designated ORFtnp, which is apparently nonfunctional due to a frameshift mutation (2). It was subsequently found that the nucleotide sequence of the ORFtnp-nec1 region was identical among strains of S. scabies (three strains examined), S. acidiscabies (two strains examined), and S. turgidiscabies (two strains examined) (1). Taken together, these data strongly suggest that this region has recently been mobilized among these unrelated Streptomyces pathogens and may be involved in the evolution of plant pathogenicity within the genus. It was therefore of interest to explore the genetic organization of regions adjacent to the 3′ end of nec1 in S. scabies 84.34, reasoning that other novel pathogenicity factors or elements that may have played a role in the mobilization and horizontal transfer of pathogenicity genes could be identified in this region. We report here the identification and characterization of a functional pathogenicity-associated IS element, designated IS1629, from our analysis of the 3′ flanking region of nec1. We present evidence for the unidirectional transfer of an element that contains IS1629, from IS1629-containing (type II) S. scabies isolates to S. acidiscabies and S. turgidiscabies. This region may constitute a portion of an as-yet-uncharacterized pathogenicity island in filamentous gram-positive plant pathogens.