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Applied and Environmental Microbiology, July 2009, p. 4362-4373, Vol. 75, No. 13
0099-2240/09/$08.00+0     doi:10.1128/AEM.02521-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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

Bomba Dam,1,{ddagger} Wriddhiman Ghosh,1,2 and Sujoy K. Das Gupta1*

Department of Microbiology,1 Department of Biophysics, Bose Institute, P-1/12, C. I. T. Scheme VII-M, Kolkata 700054, India2

Received 4 November 2008/ Accepted 27 April 2009

Tetrathiobacter spp. and other members of the Alcaligenaceae are metabolically versatile and environmentally significant. A novel, ~60-kb conjugative plasmid, pBTK445, from the sulfur chemolithoautotroph Tetrathiobacter 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 a repA gene encoding a protein belonging to the RPA (replication protein A) superfamily and an upstream, iteron-based oriV. 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 the tagB (transfer-associated gene homologous to virB) locus. The core and peripheral TagB components show different phylogenetic affinities, suggesting that this system has evolved by assembling components from evolutionarily divergent T4SSs. A virD4 homolog, putatively involved in nucleoprotein transfer, is also present downstream of the tagB locus. Although pBTK445 resembles IncP plasmids in terms of its genomic organization and the presence of an IncP-specific trbM homolog, it also shows several unique features. Unlike that of IncP, the oriT of pBTK445 is located in close proximity to the oriV, and a traL homolog, which is generally present in the TraI locus of IncP, is present in pBTK445 in isolation, upstream of the tagB locus. A significant outcome of this study is the construction of conjugative shuttle vectors for Tetrathiobacter and related members of the Alkaligenaceae.

* Corresponding author. Mailing address: Department of Microbiology, Bose Institute, P-1/12, C. I. T. Scheme VII-M, Kolkata 700 054, India. Phone: 91-033-2355-9416. Fax: 91-33-2355-3886. E-mail: sujoy{at}boseinst.ernet.in

{triangledown} Published ahead of print on 1 May 2009.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

{ddagger} Present address: Microbiology Laboratory, Department of Botany, Institute of Sciences, Visva-Bharati Central University, Santiniketan, West-Bengal 731 235, India.

Applied and Environmental Microbiology, July 2009, p. 4362-4373, Vol. 75, No. 13
0099-2240/09/$08.00+0     doi:10.1128/AEM.02521-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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