Abstract
Functional transposable elements (TEs) of several Pseudomonas spp. strains isolated from black shale ore of Lubin mine and from post-flotation tailings of Zelazny Most in Poland, were identified using a positive selection trap plasmid strategy. This approach led to the capture and characterization of (i) 13 insertion sequences from 5 IS families (IS3, IS5, ISL3, IS30 and IS1380), (ii) isoforms of two Tn3-family transposons – Tn5563a and Tn4662a (the latter contains a toxin-antitoxin system), as well as (iii) non-autonomous TEs of diverse structure, ranging in size from 262 to 3892 bp. The non-autonomous elements transposed into AT-rich DNA regions and generated 5- or 6-bp sequence duplications at the target site of transposition. Although these TEs lack a transposase gene, they contain homologous 38-bp-long terminal inverted repeat sequences (IRs), highly conserved in Tn5563a and many other Tn3-family transposons. The simplest elements of this type, designated TIMEs (Tn3 family-derived Inverted-repeat Miniature Elements) (262 bp), were identified within two natural plasmids (pZM1P1 and pLM8P2) of Pseudomonas spp. It was demonstrated that TIMEs are able to mobilize segments of plasmid DNA for transposition, which results in the generation of more complex non-autonomous elements, resembling IS-driven composite transposons in structure. Such transposon-like elements may contain different functional genetic modules in their core regions, including plasmid replication systems. Another non-autonomous element “captured” with a trap plasmid was a TIME derivative containing a predicted resolvase gene and a res site typical for many Tn3-family transposons. The identification of a portable site-specific recombination system is another intriguing example confirming the important role of non-autonomous TEs of the TIME family in shuffling genetic information in bacterial genomes. Transposition of such mosaic elements may have a significant impact on diversity and evolution, not only of transposons and plasmids, but also of other types of mobile genetic elements.
Highlights
Transposable elements (TEs) play a very important role in bacterial evolution
Plasmids and culture conditions The bacterial strains analyzed in this study were isolated from black shale ore collected in Lubin copper mine (LM) (51u249N– 16u129E) and postflotation tailings sampled from Zelazny Most (ZM) (51u309N–16u129E) in Poland, during the Bioshale FP6 European project [15]
Isolation and identification of Pseudomonas spp. strains The bacterial strains analyzed in this study were isolated from black shale ore from the active Lubin copper mine and from postflotation tailings from Zelazny Most reservoir (Lower Silesia province, Poland) [26]
Summary
Transposable elements (TEs) play a very important role in bacterial evolution They can shape the structure of bacterial genomes and influence the expression of many genes by the generation of various DNA rearrangements. TEs are highly diverse with respect to their structure, specific properties and the regulation of the transposition process. Due to their genetic organization they may be divided into two transposon classes. TEs as well as the distribution of homologous sequences in the Pseudomonas spp. genomes were analyzed by DNA-DNA hybridization. Molecular probes specific for TIME elements were prepared by (i) PCR amplification of selected DNA fragments (specific oligonucleotide primer pairs are listed in Table S2) and (ii) labelling of gel-purified amplified DNA fragments with digoxigenin (DIG, Roche). The number of DNA bands hybridizing with each probe was equivalent to the minimum number of copies of a given element within the genome
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