Аннотация:Primary objective of this investigation is the characterization of two new AT-rich plasmids isolated from thermophilic bacterium Thermoanaerobacterium saccharolyticum ( clostridia ) as a result of a search of extrachromosomal elements among thermophilic bacteria. We suggest that studies of functions encoded by this plasmid DNA and other new plasmids, isolated from different groups thermophilic bacteria having quite a low melting point might provide the additional information that can help us to understand how thermophilic bacteria can grow at extremely high temperatures.
The streaking feature of thermophilic clostridia is low guanine-plus-cytosine content of their DNA (38%) that does not correlate with their high temperature tolerance. Nucleotide sequence analysis of one of two clostridial plasmids (pNB2, 1882 bp in length) revealed that its G+C composition is very low (about 27%), suggesting that this plasmid may serves as a model for studying modes of maintenance and replication of AT-rich DNA under conditions of thermophilic bacteria growth.
We have found that the cryptic plasmid pNB2 replicates in their host, thermophilic bacterium Thermoanaerobacterium saccharolyticum via a rolling circle (RC) mode and identified the plasmid gene encoding the RC replication initiator protein (RepN) having all motifs that are well conserved among numerous related Rep proteins. We found that in contrast to all known RC Rep proteins that initiate the plasmid RC replication, RepN protein is very toxic for E. coli cells in spite of it is extremely insoluble in bacterial cells. Mutation of tyrosine residue (Y211F) that seems to have the catalytic function and be indispensable for Rep proteins did not affect the lethality associated with the activity of RepN, suggesting that this protein may have the additional activity that differs from a common relaxase function. Our mutational analysis of RepN and the studies on its in vivo activities led us to the proposal that the RepN protein may have two mode of action and function (i) as a common relaxase and (ii) as a site-specific recombinase (the Int family). To verify this suggestion the mutational analysis of RepN will be continued to identify the amino acid residues that are essential for unusual recombinase/lethality activity of RepN protein.