pB 264 , a small , mobilizable , temperature sensitive

Background: Gram-positive bacteria of the genus Rhodococcus have shown an extraordinary capacity for metabolizing recalcitrant organic compounds. One hindrance to the full exploitation of Rhodococcus is the dearth of genetic tools available for strain manipulation. To address this issue, we sought to develop a plasmid-based system for genetic manipulation of a variety of Rhodococcus strains. Results: We isolated and sequenced pB264, a 4,970 bp cryptic plasmid from Rhodococcus sp. B2641 with features of a theta-type replication mechanism. pB264 was nearly identical to pKA22, a previously sequenced but uncharacterized cryptic plasmid. Derivatives of pB264 replicate in a diverse range of Rhodococcus species, showing that this plasmid does not bear the same host range restrictions that have been exhibited by other theta replicating plasmids. Replication or maintenance of pB264 is inhibited at 37°C, making pB264 useful as a suicide vector for genetic manipulation of Rhodococcus. A series of deletions revealed that ca. 1.3 kb from pB264 was sufficient to support replication and stable inheritance of the plasmid. This region includes two open reading frames that encode functions (RepAB) that can support replication of pB264 derivatives in trans. Rhodococcus sp. B264-1 will mobilize pB264 into other Rhodococcus species via conjugation, making it possible to genetically modify bacterial strains that are otherwise difficult to transform. The cisacting element (oriT) required for conjugal transfer of pB264 resides within a ca. 0.7 kb region that is distinct from the regions responsible for replication. Conclusion: Shuttle vectors derived from pB264 will be useful for genetic studies and strain improvement in Rhodococcus, and will also be useful for studying the processes of theta replication and conjugal transfer among actinomycetes. Background Gram-positive bacteria of the genus Rhodococcus have shown an extraordinary capacity for metabolizing recalcitrant organic compounds. Due to these diverse enzymatic activities and a demonstrated solvent tolerance, rhodococci have many practical applications for bioconversion and bioremediation (reviewed in [1,2]). One hindrance to the full exploitation of Rhodococcus is the dearth of genetic tools available for strain manipulation. Among the most basic of tools for genetic manipulation of bacteria are plasmids. Plasmids can be used to introduce new or extra copies of genes into bacterial cells and to complement mutations in bacteria. They can be used to manipulate expression levels of different gene products, impacting a Published: 14 April 2004 BMC Microbiology 2004, 4:15 Received: 16 January 2004 Accepted: 14 April 2004 This article is available from: http://www.biomedcentral.com/1471-2180/4/15 © 2004 Lessard et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.