Conjugative Type 4 Secretion System of a Novel Large Plasmid from the Chemoautotroph Tetrathiobacter kashmirensis and Construction of Shuttle Vectors for Alcaligenaceae

Tetrathiobacter spp. and other members of theAlcaligenaceae are metabolically versatile and environmentally significant. A novel, ∼60-kb conjugative plasmid, pBTK445, from the sulfur chemolithoautotrophTetrathiobacter kashmirensis , was identified and characterized. This plasmid exists at a low copy number of 2 to 3 per host chromosome. The portion of pBTK445 sequenced so far (∼25 kb) harbors genes putatively involved in replication, transfer functions, partition, and UV damage repair. A 1,373-bp region was identified as the minimal replicon. This region contains arepA gene encoding a protein belonging to the RPA (replication protein A) superfamily and an upstream, iteron-basedoriV . A contiguous 11-gene cluster homologous to various type 4 secretion systems (T4SSs) was identified. Insertional inactivation demonstrated that this cluster is involved in the conjugative transfer functions of pBTK445, and thus, it was named thetagB (transfer- associated gene homologous tovirB ) locus. The core and peripheral TagB components show different phylogenetic affinities, suggesting that this system has evolved by assembling components from evolutionarily divergent T4SSs. AvirD4 homolog, putatively involved in nucleoprotein transfer, is also present downstream of thetagB locus. Although pBTK445 resembles IncP plasmids in terms of its genomic organization and the presence of an IncP-specifictrbM homolog, it also shows several unique features. Unlike that of IncP, theoriT of pBTK445 is located in close proximity to theoriV , and atraL homolog, which is generally present in the TraI locus of IncP, is present in pBTK445 in isolation, upstream of thetagB locus. A significant outcome of this study is the construction of conjugative shuttle vectors forTetrathiobacter and related members of theAlkaligenaceae .