A type Ib plasmid segregation machinery of the Advenella kashmirensis plasmid pBTK445

pBTK445 is a newly described large (∼60Kb), low-copy number, conjugative plasmid indigenous to the sulfur-chemolithoautotroph Advenella kashmirensis. Based on its minimal replication region, a shuttle vector, pBTKS was constructed which can be used for diverse Alcaligenaceae members. The construct was found to be stably maintained both in the native host as well as in Escherichia coli in the absence of selective pressure which indicated that pBTKS harbors the stabilizing system of pBTK445, that are commonly coded by low-copy-number plasmids. Deletion analyzes of pBTKS confirmed the essentiality of parA (encoding a Walker-type ATPase of 214 amino acids) and the downstream located small parB (encoding an 85 amino acid protein having no sequence homolog in the database) in the faithful partitioning of pBTK445. A 1075bp PCR product, containing parA, parB and an upstream sequence having nine 11bp direct repeats (parS site) was found to comprise the partition functions of pBTK445, stabilizing both low-copy or high-copy number homologous and heterologous replicons in diverse hosts. The incompatibility determinant and the par promoter, P(par) were both found to be present within a 191bp iterated sequence present upstream of parA. ParB was found to regulate the expression of the Par proteins from P(par). The presence of a typical Walker-type ATPase motif in ParA, a short phylogenetically unrelated ParB, that acts as a repressor of P(par), and location of the iterated parS site upstream of parA, confirm that the active partition system of pBTK445 belongs to the type Ib.