In addition, ICEVpaChn3 SB273005 price shows a 5′-region truncated version of the HS2 of ICEVchMex1 [36], and contains a homologous gene to previously described mex02 (98% amino acid identity) (GenBank: KF411062). Finally, amplification of the HS2 yielded no PCR product from ICEVchChn2, ICEVpaChn2 and ICEVnaChn1, which may resulted from large DNA insertions, e.g. a 29.2-kb insertion in the ICESpuPO1 HS2 carrying heavy metal efflux gene clusters [28]. Hotspot3. Transposon-like structures carrying genes involved in trimethoprim resistance or DNA modification, recombination

or repair in diverse putative restriction-modification systems were found within the hotspot3 [23]. As illustrated in Figure 1, about 5.4-kb DNA insertion was identified in five ICEs

including ICEVchChn1, ICEVchChn3, ICEVchChn4, ICEVchChn5 and ICEVchChn6, respectively. BLAST BKM120 molecular weight analysis revealed the same gene content as that in the HS3 of SXTLAOS[38], encoding an exonuclease and a helicase (99% amino acid identity) (GenBank: KF411063). In addition, a large DNA fragment was amplified from the HS3 (GenBank: KF411064) of ICEValChn1. It is 9.7 kb in length and shows no significant similarity in gene content with any known ICEs that have been characterized to date. Database searches revealed that besides the LEE011 boundary genes, the DNA insertion contains at least three more genes, encoding a putative glucose-1-phosphate adenylyltransferase and a RNA-directed DNA polymerase, displaying

high sequence identities (60-100%) at the amino acid level to corresponding homologs in the genomes of Vibrios and closely related species in the public databases. It also contains a novel gene with 76% amino acid sequence identity to a transposase of the Vibrio metschnikovi CIP 69.14 (GenBank: eex38460.1). Moreover, BLAST search yielded no significant similarity in its 3′-region sequence of the insertion, almost half of its full length, indicating completely novel genes carried by this ICE. Finally, ICEVpaChn1 harbored no DNA insertion in the HS3, from Glutamate dehydrogenase which only the boundary gene sequences were amplified, while four ICEs including ICEVchChn2, ICEVpaChn2, ICEVpaChn3 and ICEVnaChn1 failed to yield any PCR products in their respective HS3 locus. Hotspot4. Extensive differences in molecular profiles of hotspot4 were reported in the SXT/R391 ICEs [23]. Amplification and sequencing of the HS4 yielded about 5.6-kb inserted sequence from five ICEs (Figure 1). Database searches showed the SXT-specific molecular profile in their respective HS4 site (GenBank: KF411068). These elements contain three homologous genes (94-100% amino acid identity) to previously described s060 to s062 in the SXT HS4, encoding a putative nuclease and two conserved hypothetical proteins of unknown functions in the current literature. Similarly, ICEVpaChn3 has R391-specific genes orf64 in the HS4 (2.