Intracellular excision and reintegration dynamics of the ICEclc genomic island of Pseudomonas knackmussii sp. strain B13

Abstract

SummaryGenomic islands are DNA elements acquired by horizontal gene transfer that are common to a large number of bacterial genomes, which can contribute specific adaptive functions, e.g. virulence, metabolic capacities or antibiotic resistances. Some genomic islands are still self-transferable and display an intricate life-style, reminiscent of both bacteriophages and conjugative plasmids. Here we studied the dynamical process of genomic island excision and intracellular reintegration using the integrative and conjugative element ICEclc from Pseudomonas knackmussii B13 as model. By using self-transfer of ICEclc from strain B13 to Pseudomonas putida and Cupriavidus necator as recipients, we show that ICEclc can target a number of different tRNA Gly genes in a bacterial genome, but only those which carry the GCC anticodon. Two conditional traps were designed for ICEclc based on the attR sequence, and we could show that ICEclc will insert with different frequencies in such traps producing brightly fluorescent cells. Starting from clonal primary transconjugants we demonstrate that ICEclc is excising and reintegrating at detectable frequencies, even in the absence of recipient. Recombination site analysis provided evidence to explain the characteristics of a larger number of genomic island insertions observed in a variety of strains, including Bordetella petri, Pseudomonas aeruginosa and Burkholderia.

Highly variable individual donor cell fates characterize robust horizontal gene transfer of an integrative and conjugative element

“…A 752-bp promoterless egfp gene was amplified from a derivative of plasmid pJAMA23 (47) and inserted 27 bp downstream of the stop codon of intB13 (Fig. S1) in P. putida carrying WT ICEclc (strain 2737) or ICEclc-ΔmfsR [strain 4322 (27)], with the ICE inserted in the gene for tRNA-gly5 (40). The resulting strains (P. putida 4611 and 4612, respectively) were further used to create an 18-bp deletion in attL of ICEclc by using the same double-recombination techniques (producing P. putida strains 4754 and 4755, respectively).…”

“…Cells were allowed to grow for 4 h and then collected by centrifuging the 20-mL cultures (early exponential phase). DNA was extracted using the Wizard Genomic DNA purification kit (Promega) and used to quantify by real-time PCR the relative abundances of the clcB gene of ICEclc and of attP (formed exclusively when ICEclc excises) (40). The primer set for clcB amplified a 177-bp fragment (5′-GGTTCAGAGAGCGTGCCTTC + 5′-GCGCTGAAACCAT-CAAGGTC), whereas the attP primer set (5′-CTTCGCTGGCCACCTCGG + 5′-GTGGCGCTCGCTGGAATGA) amplified a 166-bp fragment.…”

“…P. putida UWC1 strains containing an attR-mcherry conditional trap for ICEclc insertion (Fig. 1B) were constructed by cloning the attR fragment [formerly named ΔP int -fragment (40)] in front of a promoterless mcherry on plasmid pJAMA39 (17). The NotI attR-mcherry fragment was then recovered and cloned into pCK218 (48), and the resulting plasmid was electroporated into P. putida UWC1 to deliver the mini-Tn5 cassette, as previously described (40), to produce strains 5001-5003.…”

“…1C). As recipients, we used derivatives of P. putida UWC1 constitutively expressing mcherry (29) to distinguish them from donor cells or equipped with a conditionally fluorescent "trap" (lighting up after ICEclc insertion) (40). Recipient cells producing EGFP were observed exclusively when they had been in direct contact with tc starters or their daughters (57 transfers among 328 observed donorrecipient contacts with P. putida ICEclc-intB13-egfp,ΔmfsR in triplicate independent experiments; Table 1), but never with non-tc cells (n = 202 observed contacts with recipient cells).…”

Fatores envolvidos com a mobilização de PAPI-1

“…Finally, in order to verify interactions involving the conserved proteins in PAPI-1 and XAC, two hybrid assays were carried out, but only false positives results were obtained. (Koonin, et al 2001;Mohd-Zain, et al 2004;Juhas, et al 2007;Mathee, et al 2008;Battle, et al 2009;Sentchilo, et al 2009 …”