Fig. 4
ICEPae1161 identified in P. aeruginosa PAO1161 genome is a functional PAPI-1 family integrative and conjugative element conferring resistance to mercury. a Comparative genomics of ICEPae1161 and selected PAPI-1 family ICEs. Mauve alignment of ICEPae1161 and PAPI-1 [59], PAGI-5 [60], PFGI-1 [61], pKLC102 [62] and ICEPaePACS2–1 (position 896,693:1002644 of NZ_AAQW01000001) [35] is presented. Three blocks of sequences which are free of genome rearrangements, such as inversions and duplications, are marked with rectangles connected with lines. Green segments indicate sequences conserved in all ICEs (backbone). Regions conserved among subsets of analysed ICEs are color coded. White region is specific only to one analysed ICE. Arrows indicate the location and orientation of coding sequences. Boundary genes and the mercury resistance operon are shown on top. b Schematic model of linear and excised (circular) ICEPae1161. c PCR analysis of ICEPae1161 excision. PCR was performed with indicated primer pairs flanking att sequences using PAO1161 genomic DNA as a template and products were separated on a 1.2% agarose gel followed by DNA visualization using ethidium bromide staining. d Growth of P. putida KT2440 strain and P. putida KT2440 ICEPae1161::aadA (SmR). Strains were grown in L broth with or without 40 μM HgCl2. Data represent mean OD600nm ± SD for 6 clones analysed in 3 biological replicates. e Genomic context of three potential ICEPae1161 integration sites (attB) in P. putida KT2440 genome. The sites were identified based on the presence of 48 bp sequence flanking ICEPae1161 in PAO1161 genome. Blue arrows indicate the orientation of the sequences. Schematic model of ICEPae1161 integration at each attB site in orientation corresponding to the one observed in PAO1161 genome. f PCR analysis of ICEPae1161 presence in P. putida KT2440. Genomic DNA of the wild-type P. putida KT2440 (WT) and six independent transconjugants (1–6) was used as a template in PCR with the indicated primer pairs. Products were separated on 1% agarose gel followed by DNA visualization using ethidium bromide staining. Primer binding sites and names are indicated in red (Additional file 6: Table S6)