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A rolling-circle plasmid from Psychrobacter sp. TA144: evidence for a novel rep subfamily
M L Tutino1, A Duilio, M A Moretti
1Dipartimento di Chimica Organica e Biologica, University of Naples Federico II, via Mezzocannone, Naples, 16 80134, Italy. tutino@unina.it
Abstract:
In this paper we report the cloning and sequencing of two small plasmids, pTAUp and pTADw, from the Antarctic Gram-negative Psychrobacter sp strain TA144. The observation that pTAUp contains a putative Rep-coding gene (Psyrep) suggested that its duplication occurs via a rolling-circle replication mechanism. This hypothesis was confirmed by the identification of the pTAUp single-stranded DNA form. The putative pTAUp plus origin of replication was found at the 3' end of the Psyrep by using an in vivo complementation assay. Structural similarities at the level of (i) gene organization, (ii) protein sequence, and (iii) nick site sequences strongly suggest that the psychrophilic enzyme belongs to a new subfamily of replication enzymes.
Insights
Researchers cloned and sequenced two plasmids from Antarctic bacteria. They discovered a novel rolling-circle replication mechanism, identifying a new subfamily of replication enzymes.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Psychrobacter sp strain TA144 is an Antarctic Gram-negative bacterium.
- Plasmids are extrachromosomal DNA elements crucial for bacterial adaptation and evolution.
Purpose of the Study:
- To clone and sequence plasmids from Antarctic Psychrobacter sp strain TA144.
- To investigate the replication mechanism of the plasmid pTAUp.
- To characterize the putative Rep-coding gene (Psyrep) and its role in replication.
Main Methods:
- Plasmid DNA isolation and sequencing.
- Identification of single-stranded DNA forms.
- In vivo complementation assays to determine the origin of replication.
- Comparative analysis of gene organization, protein sequences, and nick site sequences.
Main Results:
- Two plasmids, pTAUp and pTADw, were successfully cloned and sequenced.
- Evidence confirmed a rolling-circle replication mechanism for pTAUp, including identification of its single-stranded DNA form.
- The origin of replication for pTAUp was localized to the 3' end of the Psyrep gene.
- Structural similarities indicated that the Psyrep enzyme belongs to a new subfamily of replication enzymes.
Conclusions:
- The study elucidates the replication mechanism of plasmids in Antarctic bacteria.
- A novel rolling-circle replication mechanism was identified in Psychrobacter sp.
- The findings suggest the existence of a new subfamily of replication enzymes adapted to psychrophilic environments.