, 2009). However, tblastn returned two putative regions within plasmids pLJ42 and pLB925A03, the latter isolated from Lactobacillus brevis (Wada et al., 2009), that could code for proteins with high identity to Orf2. Because orf2 was not included in the original annotation of either of the latter plasmids, we re-annotated them using the same bioinformatics tools as with pREN. orf2 was indeed predicted in the afore-mentioned plasmids (positions 5170–5499 nt for pLB925A03 and 2415–2744 nt for pLJ42). The deduced orf2 products exhibited a high degree of conservation (Fig. 2a). It should be mentioned that a terminator sequence Nutlin-3a chemical structure within the orf2

locus (position 2515–2579 nt) was initially deposited for pLJ42; however, our analysis with findterm did not support the existence of this terminator. In fact, orf2 was located downstream of repA, followed by a terminator in both pLJ42 and pLB925A03, resulting in a conserved operon structure as shown for pREN. The four remaining orfs were all found to encode different types of mobilization proteins. The orf3 product (112 amino acids) displayed the highest identity to MobC of pLJ42 (100% query coverage, 100% identity, e-value 9e−58). Orf4 (195 amino acids) and Orf5 (208

amino acids) proteins were identified as MobA (MobA1 and MobA2, respectively), both receiving top scores for the MobA of pLB925A03 (68% query coverage, 100% identity with e-value 2e−76 and 100% query coverage, 98% PI3K signaling pathway identity with e-value 5e−114, respectively). Initial analysis clearly excluded the possibility of a gene duplication event. interproscan indicated that while MobA1 carried a significant proportion of the N-terminal pfam03432 signal of the family of relaxases, MobA2 carried the remaining distal sequence of the signal’s C-terminus. The alignment of these proteins with

the MobA of pLB925A03, carrying Alectinib datasheet a full pfam03432 signal, demonstrated that MobA1 and MobA2 were originally a single full-length peptide (Fig. 2b). Inspection of the mobA1 gene revealed that it was disrupted by a frameshift mutation at position 2339 nt, causing premature termination at position 2526 nt. Furthermore, orf6 was predicted as a mobB gene. Interestingly, in contrast to the other pREN Mob proteins, this MobB molecule was detected only in a very limited number of bacteria, all of which were LAB. Sequence comparison among the MobB proteins showed a considerable degree of conservation that was more pronounced at the C-terminus (Fig. 2c). This annotation transfer through sequence identity was based on a previous observation that the protein product of an orf in plasmid pNZ4000 of Lactococcus lactis (van Kranenburg et al., 2000) shared a moderate homology to MobB of Staphylococcus aureus plasmid pC223 (Smith & Thomas, 2004).