Objective
Characterization of processes which underlie segregational and structural genetic instability in Bacillus subtilis and decreasing the instability to industrially acceptable levels.
Stable maintenance of foreign genetic information is essential for amelioration of industrially important microorganisms. Small plasmids which replicate as rolling circles are widely used for deoxyribonucleic acid (DNA) cloning in Gram positive bacteria, which is an important class of industrial microorganisms, and their biology is relatively well known. In contrast, large theta-replicating plasmids have only been used recently for cloning in these bacteria and are less well characterised. The well-known small plasmids seem to be intrinsically less stable than the large less-known plasmids. Part of the research during the past year was orientated towards understanding the instability and improving the stability of the small plasmids and the part other towards isolating new and better characterising the known larger plasmids. Bacillus subtilis was used as a model host and processes known to affect stability of genetic information, such as DNA replication of plasmids and phage were studied. Better understanding of the genetic stability is expected to have impact on a number of biotechnology applications.
Significant highlights of the study to date include unexpected activity of the Rep protein of a rolling circle replication (RCR) plasmid was revealed; CHI sequence was shown to be a critical factor in the formation of high molecular weight (HMW) plasmid multimers, which affect the stability of RCR plasmids; phage PBSX lysis gene was identified; replication region of a representative large plasmid was characterised; minus origin of a RCR plasmid was characterised.
The project consists of five main tasks.
Characterization of DNA replication. Three representative replication systems will be studied:
small single stranded DNA plasmids which replicate as rolling circles;
large plasmids which replicate as tereta-forms;
bacteriophage phi29 which replicates as a linear molecule.
Characterization of the role of topoisomerases. Roles of topoisomerase I and DNA gyrase in structural and segregational stability will be determined.
Characterization of the partition systems. Active partition systems will be sought, identified and used to stabilise heterologous replicons.
Construction of post segregational killing systems. These will be based on the repressor and lysis functions of the phage PBSX. Lysis gene will be integrated in the chromosome and the repressor gene will be carried on plasmids, such that the loss of plasmids provokes lysis of the host cell.
Characterization of the stability of genes inserted in the chromosome. The effects of neighbouring structures and functions as well as the chromosomal location will be determined. Segregationally and structurally stable cloning systems should result from these studies.
Fields of science (EuroSciVoc)
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
- natural sciences biological sciences microbiology bacteriology
- natural sciences biological sciences microbiology virology
- natural sciences biological sciences genetics DNA
- natural sciences biological sciences biochemistry biomolecules proteins
- natural sciences biological sciences genetics chromosomes
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Coordinator
78352 Jouy-en-Josas
France
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