M, 1 kb DNA ladder (Fermentas);

M2, 1 kb DNA ladder (Roche). Figure 3 Schematic representation of new IS 711 loci found in B. abortus field isolates. B12 (upper panel) and B16 and its check details related isolates (lower panel). The full-length 842 bp IS711 elements and their overlapping ORFs appear in grey. The Bru-RS1 element is shown as hatched box. The duplicated TA at the consensus YTAR site is shown below. Small black arrows represent the positions of site-specific primers. Numbers between primers indicate the molecular size of PCR products. The coordinates are based on the B. abortus 9-941 annotation. ORFs BruAb1_0734, BruAb1_0735 and BruAb1_0736 encode hypothetical proteins; lldP, L-lactate permease (BruAb1_0737); BruAb2_462 encodes a putative

D-amino acid oxidase family protein; asnC, transcriptional regulator AsnC family (BruAb2_0459). The x-B12 and x-B16 IS711 sequences were BIIB057 in vivo nearly identical to that of IS711_1a and depicted only changes in a few nucleotides (Figure 4A). On the basis of the high IS711 sequence similarity across sequenced B. abortus strains, we performed Bcl-2 inhibitor a cluster analysis between the IS711 copies of B. abortus 9-941 and those additional ones found in 2308, RB51, B12 and B16 strains to get insight about their origin (Figure 4B). Although as expected, the analysis disclosed only low sequence dissimilarity, it suggested that the new copies might derive from IS711_1a. Since a previous work has shown Sclareol that the IS711_xa in the B. abortus alkB locus and the IS711_x-08 in strain 2308 are identical to IS711_1a [3], the inclusion of IS711_x-B12 and IS711_x-B16 in the same cluster supports the hypothesis that IS711_1a is more active than other copies in the B. abortus genome and can transpose into new sites or even into sites shared with related species. Figure 4 Sequence analysis of IS 711 copies found in B. abortus strains. (A), Sequence alignment (IS711_1a is from

B. abortus 9-941). Single nucleotide polymorphisms are shadowed and numbered according to IS ORFs coordinates. (B), Clustering of full-length B. abortus IS711 copies found in B. abortus 9-941 (note that truncated 5a copy was excluded), additional IS711 copy carried by B. abortus 2308 (x-08) and B. abortus RB51 (x-RB51, accession no M94960), and the additional copies found in field isolates (x-B12, x-B16). IS transposition can disrupt genes and produce negative polar effects, but also cause beneficial changes by remodeling genomes through long range recombination [15]. In the case of strain B12, it is uncertain whether the intergenic position of IS711 disturbs the expression of nearby genes. Most IS711 studied in detail (1a, 2a, 3a, 5a, 6a, xa and x-08) are also located within intergenic regions showing that transposition is mostly viable when occurring into neutral sites.