Characterization of an insertion sequence element associated with genetically diverse plant pathogenic Streptomyces spp

Abstract

Streptomycetes are common soil inhabitants, yet few described species are plant pathogens. While the pathogenicity mechanisms remain unclear, previous work identified a gene, nec1, which encodes a putative pathogenicity or virulence factor. nec1 and a neighboring transposase pseudogene, ORFtnp, are conserved among unrelated plant pathogens and absent from nonpathogens. The atypical GC content of nec1 suggests that it was acquired through horizontal transfer events. Our investigation of the genetic organization of regions adjacent to the 3' end of nec1 in Streptomyces scabies 84.34 identified a new insertion sequence (IS) element, IS1629, with homology to other IS elements from prokaryotic animal pathogens. IS1629 is 1,462 bp with 26-bp terminal inverted repeats and encodes a putative 431-amino-acid (aa) transposase. Transposition of IS1629 generates a 10-bp target site duplication. A 77-nucleotide (nt) sequence encompassing the start codon and upstream region of the transposase was identified which could function in the posttranscritpional regulation of transposase synthesis. A functional copy of IS1629 from S. turgidiscabies 94.09 (Hi-C-13) was selected in the transposon trap pCZA126, through its insertion into the lambda cI857 repressor. IS1629 is present in multiple copies in some S. scabies strains and is present in all S. acidiscabies and S. turgidiscabies strains examined. A second copy of IS1629 was identified between ORFtnp and nec1 in S. acidiscabies strains. The diversity of IS1629 hybridization profiles was greatest within S. scabies. IS1629 was absent from the 27 nonpathogenic Streptomyces strains tested. The genetic organization and nucleotide sequence of the nec1-IS1629 region was conserved and identical among representatives of S. acidiscabies and S. turgidiscabies. These findings support our current model for the unidirectional transfer of the ORFtnp-nec1-IS1629 locus from IS1629-containing S. scabies (type II) to S. acidiscabies and S. turgidiscabies.

FIG. 1

Genetic organization of ORFtnp-nec1-IS1629 region in S. scabies (type I and II), S. acidiscabies, and S. turgidiscabies strains.

FIG. 2

Nucleotide sequence of IS1629, with putative transposase sequence given below. Terminal inverted repeats are double underlined; the nucleotide sequence involved in the proposed RNA secondary structure is underlined once (also see Fig. 6). The probable Shine-Dalgarno sequence is boxed.

FIG. 3

Clustal alignment of putative IS1629 transposase with transposase homologs. M.u., putative transposase from Mycobacterium ulcerans; P.g. PGIS2, Porphyromonas gingivalis IS2; A.s. AsIS1, Aeromonas salmonicida IS1; V.c. IS1358, V. cholerae IS1358.

FIG. 4

Southern hybridization of 1.4-kb [α-³²P]dCTP-labeled ApaI fragment of IS1629 with KpnI-digested total DNA from the indicated Streptomyces pathogens (IS1629 does not have a KpnI site) (A) and dot blot analysis with the same fragment with the total DNA from nonpathogenic strains. (B) Panel B is arranged as follows: row A1 to A6, 83.40, 84.05, 84.29, 84.130, 84.222, and 86.31; row B1 to B6, 87.80, 88.24, 88.25, 89.05, 89.08, and 89.18; row C1 to C6, 92.01, 92.03, ATCC 10246, ATCC 12309, ATCC 14975, and ATCC 23920; row D1 to D6, ATCC 25435, ATCC 25497, ATCC 27449, IMRU 3018, IFO 13350, and NRRL 3585; row E1 to E5, TK24, M146, C581, 84.34, and calf thymus DNA.

FIG. 5

IS1629 selection in pCZA126 (adapted from Solenberg and Burgett [28]) (A) and nucleotide sequences of duplicated IS1629 target insertion sites from the following: the 3′ region of nec1 in S. scabies 84.34, S. turgidiscabies, and S. acidiscabies; λ cI857; and S. acidiscabies 84.104 and 84.110 (5′ of nec1) (B).

FIG. 6

Proposed RNA secondary structure adopted by IS1629 transposase transcribed from exogenous promoter sequences. Nucleotides are numbered from the start of the IS1629 sequence. The probable ribosome binding site is underlined, and the putative start codon is boxed.