Natural transformation of Thermotoga sp. strain RQ7

Abstract

Background: Thermotoga species are organisms of enormous interest from a biotechnological as well as evolutionary point of view. Genetic modifications of Thermotoga spp. are often desired in order to fully release their multifarious potentials. Effective transformation of recombinant DNA into these bacteria constitutes a critical step of such efforts. This study aims to establish natural competency in Thermotoga spp. and to provide a convenient method to transform these organisms.

Results: Foreign DNA was found to be relatively stable in the supernatant of a Thermotoga culture for up to 6 hours. Adding donor DNA to T. sp. strain RQ7 at its early exponential growth phase (OD600 0.18 ~ 0.20) resulted in direct acquisition of the DNA by the cells. Both T. neapolitana chromosomal DNA and Thermotoga-E. coli shuttle vectors effectively transformed T. sp. strain RQ7, rendering the cells resistance to kanamycin. The kan gene carried by the shuttle vector pDH10 was detected by PCR from the plasmid extract of the transformants, and the amplicons were verified by restriction digestions. A procedure for natural transformation of Thermotoga spp. was established and optimized. With the optimized method, T. sp. strain RQ7 sustained a transformation frequency in the order of 10⁻⁷ with both genomic and plasmid DNA.

Conclusions: T. sp. strain RQ7 cells are naturally transformable during their early exponential phase. They acquire DNA from both closely and distantly related species. Both chromosomal DNA and plasmid DNA serve as suitable substrates for transformation. Our findings lend a convenient technical tool for the genetic engineering of Thermotoga spp.

Figure 1

The genetic and physical map of pDH12. The bolded region in grey represents the sequence from pRQ7. The black bars denote the duplicated fragments. Unique enzyme sites are shown.

Figure 2

Degradation of C. saccharolyticus genomic DNA in SVO medium (A) and in the supernatant of an overnight culture of T. sp. strain RQ7 (B).

Figure 3

Transformability of T. sp. strain RQ7 at various time points of the early exponential phase. The number above each column represents the number of Kan^r colonies.

Figure 4

Four more independent experiments of transforming T. sp. strain RQ7 at OD₆₀₀ of 0.18 ~ 0.20. The number above each column represents the number of Kan^r colonies.

Figure 5

Plasmid extract of a T. sp. strain RQ7/pDH10 transformant (denoted as RQ7/pDH10). The host T. sp. strain RQ7 was used as the positive control (denoted as RQ7), and the recombinant strain T. maritima/pDH10, constructed in the previous study [3], served as the negative control (denoted as Tm/pDH10). The arrows point to the characteristic bands of pRQ7.

Figure 6

Amplification of the kan gene from the genomic DNA (G) and the plasmid DNA (P) prepared from four RQ7/pDH10 transformants. The plasmid extracts from DH5α/pDH10 (first lane after the marker) and T. sp. strain RQ7 (second lane after the marker) were used as positive and negative controls.

Figure 7

Restriction digestions of the kan gene amplicon of a RQ7/pDH10 transformant. Lanes 1, 3, and 5, positive controls prepared from DH5α/pDH10. Lanes 2, 4, and 6, RQ7/pDH10 transformant. Partial digestion is noticed in Lane 6.