pickettii 12J Position Accession no. Z-DEVD-FMK concentration         Start Stop   CirIm ~220 RE1 GCATGGAAGACTTGACAG LE1 GAGCTTGAGTTTTGCCACG 54 N\A N\A FM244490 int 1035 intFor1 TTTCATTTCACCATGACTCCAG intRev1 GAGAGCAGTCGATAGGCTTCC 61.7 2715201 2716235 FM244486 RepA, ParA ParB 1657 RepAF GAGACTACCAGCGCCTCAAG

RepAR ACGTGTTCATGAGGACTTCTCC 55 2734598 2736255 FM244487 traG 1483 traGF GTTCGAGTGGTGGTTCTTCTTC traGR GAAATTGCTGTCCGCGTAGTAG 61 2757179 2758661 FM244488 trbI 1597 trbIF AACTGACCATGAGCCAGGAC trbIR AAAGCTCCTCAAAAGCGAAAG 62 2767516 2769113 FM244489 The attL and attR region of Tn4371 ICEs Analysis of hosts harbouring Tn4371-like elements indicated that integration occurred at an 8-bp attB site generating attL and attR element chromosomal junctions [[11], Fig. 7a]. An alignment of the first and last 200 bp of the elements analysed in this study Selleckchem Temsirolimus with Tn4371-like element from previous studies showed the attL site had a sequence of TTTTC/TA/GT and attR had a sequence of TTTTC/TA/GT for some bacteria, while others had no direct repeats. These alignments can be seen in Additional file 4. The exact sequence of the direct repeat for each element is presented in Table 4. The absence of direct repeats in some of these elements may mean that they are no longer mobile. Tn4371 has been shown to excise from the RP4 plasmid in Ralstonia eutropha forming a Selleckchem mTOR inhibitor circular extrachromosomal intermediate [[10], Fig. 7a] as a transfer

intermediate. The strains in which we detected Tn4371-like elements were examined to see if they also excised forming extrachromosomal intermediates [CirIm] using a PCR assay that allowed amplification across the circular junction but which would not amplify if the element were integrated. Primer LE1 is specific to integrated Tn4371-like ICE DNA at the attL left-end where as primer RE1 is specific to integrated Tn4371-like ICE at the attR right-end [Fig. 7a, Table 3]. Both primers are oriented towards the Tn4371- like ICE junctions, and PCR product

will be generated only if the respective left and right ends [attL and attR sites] excise from the chromosome and circularise [CirIm], reconstituting attP [attachment locus on the element]. Exoribonuclease A model of integration and excision of the ICE can be seen in Fig. 7a. PCR products of ~220-bp were obtained from ICETn4371 6043 [ULM001] and ICETn4371 6044 [ULM003] [Fig. 7b.], indicating that a circular extrachromosomal form of the element is present in these cells, while no PCR product was obtained from ULM006 [Fig. 7b]. The sequencing of the attP region of ICETn4371 6043 gave an attL region of TTTTTCAT and an attR region of TACTTTTT. This rapid amplification across the circular attP junction can also be utilised for the rapid identification of Tn4371-like elements. It is possible that the PCR may have picked up tandems of the element if those happened to be intermediates in “”transposition”".