Elucidation of insertion elements carried on plasmids and in vitro construction of shuttle vectors from the toxic cyanobacterium Planktothrix

Abstract

Several gene clusters that are responsible for toxin synthesis in bloom-forming cyanobacteria have been found to be associated with transposable elements (TEs). In particular, insertion sequence (IS) elements were shown to play a role in the inactivation or recombination of the genes responsible for cyanotoxin synthesis. Plasmids have been considered important vectors of IS element distribution to the host. In this study, we aimed to elucidate the IS elements propagated on the plasmids and the chromosome of the toxic cyanobacterium Planktothrix agardhii NIVA-CYA126/8 by means of high-throughput sequencing. In total, five plasmids (pPA5.5, pPA14, pPA50, pPA79, and pPA115, of 5, 6, 50, 79, and 120 kbp, respectively) were elucidated, and two plasmids (pPA5.5, pPA115) were found to propagate full IS element copies. Large stretches of shared DNA information between plasmids were constituted of TEs. Two plasmids (pPA5.5, pPA14) were used as candidates to engineer shuttle vectors (named pPA5.5SV and pPA14SV, respectively) in vitro by PCR amplification and the subsequent transposition of the Tn5 cat transposon containing the R6Kγ origin of replication of Escherichia coli. While pPA5.5SV was found to be fully segregated, pPA14SV consistently co-occurred with its wild-type plasmid even under the highest selective pressure. Interestingly, the Tn5 cat transposon became transferred by homologous recombination into another plasmid, pPA50. The availability of shuttle vectors is considered to be of relevance in investigating genome plasticity as a consequence of homologous recombination events. Combining the potential of high-throughput sequencing and in vitro production of shuttle vectors makes it simple to produce species-specific shuttle vectors for many cultivable prokaryotes.

FIG 1

Ethidium bromide-stained agarose gels showing PCR amplicons (10 kbp) obtained from Planktothrix agardhii NIVA-CYA126/8 with primer pairs amplifying the entire plasmid (pPA5.5, pPA14, pPA50, pPA79, pPA115) without interruption. For clarity, only the nucleotide positions of the forward primer according to the sequence with GenBank accession no. ASAK00000000 are indicated (see Table S1 in the supplemental material). Lanes M, DNA size markers (in kbp; range, 0.5 to 10 kbp). The expected PCR product sizes are marked with arrows. A few smaller PCR amplification by-products deviating from the expected size represent unspecific amplification.

FIG 2

Schematic representation of annotated plasmids occurring in Planktothrix agardhii NIVA-CYA126/8 and construction of the shuttle vectors pPA5.5SV and pPA14SV. ORFs in gray, ORFs for proteins putatively involved in polyketide synthesis; ORFs in black, transposable elements. Small ORFs (<650 bp) are marked as black bars (transposable elements) and blue bars (other proteins). MCS, multicloning site.

FIG 3

Amplification of cat transposon Tn5 from the Planktothrix agardhii NIVA-CYA126/8 transformant using pPA5.5 and pPA14 plasmid-specific primer pairs (shuttle vector and transformant). Lanes M, DNA size markers (in kbp; range, 0.5 to 3 kbp).

FIG 4

Absolute (A) and relative (B) quantification (mean ± SD) of the number of copies of the Planktothrix agardhii NIVA-CYA126/8 chromosome and the number of plasmids per individual cell. PC-IGS, phycocyanin-intergenic spacer region.

FIG 5

Amplification of cat transposon Tn5 from the Planktothrix agardhii NIVA-CYA126/8 transformant using pPA50 plasmid-specific primer pairs. 1 and 2, different harvests at 4 μg Cm ml⁻¹.