title: Curing the Megaplasmid pTT27 from Thermus thermophilus HB27 and Maintaining Exogenous Plasmids in the Plasmid-Free Strain.
abstract: Stepwise deletions in the only plasmid in Thermus thermophilus HB27, megaplasmid pTT27, showed that two distantly located loci were important for maintenance of the plasmid. One is a minimum replicon including one gene, repT, coding a replication initiator, and the other encodes subunits of class I ribonucleotide reductase (RNR) for deoxynucleoside triphosphate (dNTP) synthesis. Since the initiator protein, RepT, bound to direct repeats downstream from its own gene, it was speculated that a more-downstream A+T-rich region, which was critical for replication ability, could be unwound for replication initiation. On the other hand, the class I RNR is not necessarily essential for cell growth, as evidenced by the generation of the plasmid-free strain by the loss of pTT27. However, the plasmid-free strain culture has fewer viable cells than the wild-type culture, probably due to a dNTP pool imbalance in the cell. This is because of the introduction of the class I RNR genes or the supplementation of 5'-deoxyadenosylcobalamin, which stimulated class II RNR encoded in the chromosome, resolved the decrease in the number of viable cells in the plasmid-free strain. Likewise, these treatments dramatically enhanced the efficiency of transformation by exogenous plasmids and the stability of the plasmids in the strain. Therefore, the class I RNR would enable the stable maintenance of plasmids, including pTT27, as a result of genome replication normalized by reversing the dNTP pool imbalance. The generation of this plasmid-free strain with great natural competence and its analysis in regard to exogenous plasmid maintenance will expand the availability of HB27 for thermophilic cell factories.
authors.affiliation: Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, JapanMcMaster University ohtani@ttck.keio.ac.jp.
authors.forename: Naoto
authors.initials: N
authors.lastname: Ohtani
authors.affiliation: Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, JapanMcMaster University.
authors.forename: Masaru
authors.initials: M
authors.lastname: Tomita
authors.affiliation: Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, JapanMcMaster University.
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Itaya
mesh_terms: D004265:DNA Helicases / Q000235:genetics; D004261:DNA Replication; D017353:Gene Deletion; D005798:Genes, Bacterial; D042822:Genomic Instability; D010957:Plasmids / Q000096:biosynthesis*; D012093:Replicon; D012264:Ribonucleotide Reductases / Q000235:genetics; D016963:Thermus thermophilus / Q000235:genetics*; D015534:Trans-Activators / Q000235:genetics; D014169:Transformation, Bacterial
chemical_list: D015534:Trans-Activators; C045264:replication initiator protein; D012264:Ribonucleotide Reductases; D004265:DNA Helicases