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Characterization of pra, a gene for replication control in pSAM2, the integrating element of Streptomyces ambofaciens
G Sezonov1, J Hagège, J L Pernodet
1Laboratoire de Biologie et Génétique Moléculaire, Institut de Génétique et Microbiologie, URA CNRS 1354, Orsay, France.
Abstract:
pSAM2 is a genetic element found integrated in Streptomyces ambofaciens (B2) and additionally in a replicating form in two mutants B3 and B4. The presence of the pSAM2 replicating form in these mutants was the result of mutations located on pSAM2 in the pra locus, named pra3 and pra4, respectively. The pra gene is not directly involved in replication, but its inactivation led to the disappearance of the pSAM2 free form; therefore, it was considered as a replication regulator. The pra3 and pra4 mutations were located in the pra promoter and were shown to be point substitutions that increase the promoter strength. The replication regulator role of pra was demonstrated by the fact that its constitutive expression in cells harbouring pSAM2B2, which is normally only integrated, led to the appearance of the pSAM2 replicating form. Northern analysis showed that the pra gene transcript can be detected only for the replicating mutants B3 and B4 and that the three adjacent genes korSA, pra and traSA were transcribed separately. As replication of pSAM2 is not needed for its maintenance but is an indispensable stage of its transfer, the pra gene, described formally as an activator of pSAM2 replication, is patently involved in pSAM2 transfer.
Insights
Mutations in the pra gene promoter of the pSAM2 genetic element increase its strength, activating pSAM2 replication and transfer in Streptomyces ambofaciens. This regulatory role is crucial for the mobile genetic element
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- pSAM2 is a genetic element present in Streptomyces ambofaciens.
- pSAM2 exists in an integrated form in wild-type strains and a replicating form in specific mutants (B3 and B4).
Purpose of the Study:
- To investigate the role of the pra gene and its mutations (pra3 and pra4) in the replication and transfer of the pSAM2 genetic element.
- To understand the regulatory mechanism controlling pSAM2 replication.
Main Methods:
- Genetic analysis of pSAM2 mutants (B2, B3, B4).
- Identification and characterization of mutations in the pra locus and its promoter.
- Northern blot analysis to study gene transcription.
- Assessing the effect of constitutive pra gene expression on pSAM2 replication.
Main Results:
- Mutations pra3 and pra4 in the pra promoter increase its strength, leading to the replicating form of pSAM2.
- Inactivation of the pra gene results in the loss of the pSAM2 free form, indicating its regulatory role.
- The pra gene transcript is detectable only in replicating mutants, and adjacent genes (korSA, pra, traSA) are transcribed separately.
- Constitutive expression of pra activates pSAM2 replication in strains where it is normally integrated.
Conclusions:
- The pra gene acts as a replication regulator for pSAM2, with specific promoter mutations enhancing its activity.
- Enhanced pra promoter strength is directly linked to the activation of pSAM2 replication.
- The pra gene is fundamentally involved in the transfer of pSAM2, as replication is a prerequisite for this process.