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.T hermus spp. are extremely thermophilic bacteria that can grow at high temperatures from 50 to 82°C. Their thermophilic enzymes, e.g., DNA polymerase for PCR, have been used in industrial applications, and their potential as cell factories to produce enzymes from other thermophiles has been demonstrated (1). The bacteria themselves have also been extensively investigated as model organisms for systems biology including structural genomics (1, 2), and their genomic DNAs, with GϩC contents as high as about 70%, are an interesting target for synthetic biology (3). Thermus-related studies have been encouraged by the existence of an established genetic engineering system based on natural competence (4-6), with thermostabilized drug resistance markers (7-10) and other thermostable genetic tools (1,(11)(12)(13)(14)(15)(16).Complete genomic sequences of T. scotoductus SA-01, T. oshimai JL-2, Thermus sp. strain CCB_US3_UF1, T. aquaticus Y51MC23, T. thermophilus strains HB8, HB27, SG0.5JP17-16, and JL-18, and others are available online. Thermus spp. often harbor a megaplasmid larger than 100 kbp, whereas in some strains, the megaplasmid genes seem to be transferred to the chromosome (17, 18). Similar megaplasmids have been observed in other bacteria of the Thermus-Deinococcus group showing resistance to extreme stresses such as high temperature, rad...

Random Chromosome Partitioning in the Polyploid BacteriumThermus thermophilusHB27

“…To this end, initially, two homozygous strains (Δ pyrE :: kat and Δ pyrE :: blm ) were created by replacing the pyrE gene with a kanamycin and a bleomycin resistant cassette, respectively (Figure 2A). Numerous examples have shown that homozygous gene deletion mutant could be obtained in T. thermophilus in spite of its polyploid genomic background (Angelov et al 2013; Leis et al 2014; Li et al 2015; Ohtani et al 2015; Wang et al 2016). In the experience, when a non-essential gene was targeted by a vector containing an antibiotic resistant marker sandwiched by two homology arms of that target region, near 90% of the resultant transformants were homozygous deletion mutant of that gene.…”

A high-efficiency scar-free genome editing toolkit for Acinetobacter baumannii

“…Curing native plasmids, usually of unknown function, often involves tedious counterselection screenings. [37][38][39] Else, spontaneous plasmid-cured strains can be found serendipitously. 36,40 Given the high frequency of A. baumannii strains bearing multiple native plasmids and the difficulties entailed by mutating and manipulating them, this methodology shows a remarkable potential for their study.…”

Efficient genome editing of an extreme thermophile, Thermus thermophilus, using a thermostable Cas9 variant

“…The genome of T. thermophilus strain HB27 40 is composed of a 1.9 Mb chromosome and a 0.2 Mb megaplasmid with a GC content of 69% 37 . The bacterium is polyploid, containing an estimated 4-5 copies of the chromosome and megaplasmid each 41,42 . Genes encoding enzymes of a carotenoid biosynthesis pathway are located on the megaplasmid, making the cells yellow-pigmented.…”

“…The crtI gene, which encodes a phytoene desaturase, converts colorless phytoene to lycopene early in the carotenoid biosynthesis pathway, and crtI knock-out mutants therefore appear white 43 . Cells are viable without the megaplasmid, but considerably growth retarded 41 . The purA gene, which encodes adenylosuccinate synthase, is located on the chromosome and is essential in adenine biosynthesis.…”