The downstream region contains two long (52 and 51 bp), nearly identical (3 differences) direct repeats (DR3, DR4) separated by an 87-bp spacer (Figure  1). It is noteworthy that the four 5′-terminal residues of DR3 are located

within the RepA coding sequence. Moreover, a shorter sequence was identified 91 bp upstream of DR4 (DR5; 5′-GTCCGTCCGTATTACTTG-3′), that perfectly matches the core region of the DR3 and DR4 repeats (Figure  1). Such repeated sequences, placed downstream and upstream of the repA gene, were also identified within the REP region of the related plasmid RA3. It was demonstrated that the downstream repeats are crucial for the initiation of RA3 replication [45]. EPZ5676 price Based on the overall similarities of the REP regions, we assume that the origin of replication of pZM3H1 (oriV) is placed analogously to that of RA3, and contains the DR3, DR4 and DR5 repeats (Figure  1). The putative PAR module of pZM3H1 is composed of two non-overlapping ORFs (orf34 and orf35; 31-bp spacer) and a centromere-like site. The orf34 encodes a putative 214-aa protein, showing significant similarity to ATPases involved in chromosome

partitioning, assigned to COG1192 (cluster of orthologous group). This similarity includes the sequence Alpelisib manufacturer KGGVGKS (residues 11–17), which matches the highly conserved canonical deviant Walker A motif KGG(T/N/V)GKT of ParA-type proteins [47]. This predicted ParA also contains an N-terminally located putative HTH motif (YIIGVVSQKGGVGKSTISRAVAT; residues 3–24). The orf35 encodes an 80-aa polypeptide with sequence similarity to several hypothetical proteins, whose genes are usually located downstream from predicted parA genes (i.e. orf34 homologs). This strongly suggests that orf35 encodes a ParB-type protein: another important component of plasmid partitioning systems. Careful inspection of the nucleotide

sequence revealed the presence of several 7-bp imperfect inverted repeats, located close to the promoter region of the predicted par operon, which may constitute a plasmid centromere-like site (parS) (Figure  1). TA stabilization modules usually Glutathione peroxidase encode two components: a toxin which recognizes a specific cellular target and an antitoxin, which counteracts the toxin. The predicted TA module of pZM3H1 fits with this scheme, since it is composed of two short non-overlapping ORFs (orf29 and orf28) separated by a 9-bp spacer. One of the ORFs (orf29) encodes a putative protein with significant sequence homology to a large family of proteins assigned to COG4679 (DUF891). These proteins, referred to as phage-related (some are encoded by bacteriophages, e.g. gp49 of phage N15), were shown to be the toxic components (RelE/ParE toxin family) of a number of TA systems [48]. The downstream gene (orf28) encodes a putative protein with substantial similarity to antitoxins classified to COG5606 and COG1396. The predicted antitoxin contains a HTH domain typical for members of the Xre/Cro protein family.