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PB264, a small, mobilizable, temperature sensitive plasmid from Rhodococcus.

ABSTRACT: 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. B264-1 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 degrees 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 cis-acting 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.

SUBMITTER: Lessard PA

PROVIDER: S-EPMC419973 | biostudies-literature | 2004 Apr

REPOSITORIES: biostudies-literature

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pB264, a small, mobilizable, temperature sensitive plasmid from Rhodococcus.

Lessard Philip A PA O'Brien Xian M XM Currie Devin H DH Sinskey Anthony J AJ

BMC microbiology 20040414

<h4>Background</h4>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.<h4>Results</h4>We isolated and sequenced pB264, a 4,970 bp cryptic plasmid from Rhodococcus sp. B2 ...[more]

PMID: 15084226

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Project description:Plasmids are the key element in horizontal gene transfer in the microbial community. Recently, a large number of experimental and computational methods have been developed to obtain the plasmidomes of microbial communities. Distinguishing transmissible plasmid sequences, which are derived from conjugative or at least mobilizable plasmids, from non-transmissible plasmid sequences in the plasmidome is essential for understanding the diversity of plasmids and how they regulate the microbial community. Unfortunately, due to the highly fragmented characteristics of DNA sequences in the plasmidome, effective identification methods are lacking. In this work, we used information entropy from information theory to assess the randomness of synonymous codon usage over 4424 plasmid genomes. The results showed that for all amino acids, the choice of a synonymous codon in conjugative and mobilizable plasmids is more random than that in non-transmissible plasmids, indicating that transmissible plasmids have different sequence signatures from non-transmissible plasmids. Inspired by this phenomenon, we further developed a novel algorithm named PlasTrans. PlasTrans takes the triplet code sequences and base sequences of plasmid DNA fragments as input and uses the convolutional neural network of the deep learning technique to further extract the more complex signatures of the plasmid sequences and identify the conjugative and mobilizable DNA fragments. Tests showed that PlasTrans could achieve an AUC of as high as 84-91%, even though the fragments only contained hundreds of base pairs. To the best of our knowledge, this is the first quantitative analysis of the difference in sequence signatures between transmissible and non-transmissible plasmids, and we developed the first tool to perform transferability annotation for DNA fragments in the plasmidome. We expect that PlasTrans will be a useful tool for researchers who analyse the properties of novel plasmids in the microbial community and horizontal gene transfer, especially the spread of resistance genes and virulence factors associated with plasmids. PlasTrans is freely available via https://github.com/zhenchengfang/PlasTrans.

Dataset Information

PB264, a small, mobilizable, temperature sensitive plasmid from Rhodococcus.

Publications

pB264, a small, mobilizable, temperature sensitive plasmid from Rhodococcus.

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