Cloning and characterization of the TneDI restriction–modification system of Thermotoga neapolitana

Xu, Zhaohui; Han, Dongmei; Cao, Jingjing; Saini, Uksha

doi:10.1007/s00792-011-0397-9

Cited by 8 publications

(8 citation statements)

References 30 publications

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“…Performing the experiment in an anaerobic chamber should help to improve the transformation efficiencies, albeit the operations would be cumbersome. In addition, restriction-modification systems have been discovered in Thermotoga , for instance, we have recently characterized a Thermotoga -specific Type II restriction-modification system [23]. Unmethylated foreign DNA will be restricted by host-specific restriction nucleases as soon as they enter Thermotoga cells.…”

Section: Discussionmentioning

confidence: 99%

Construction and transformation of a Thermotoga-E. colishuttle vector

Han

Norris

2012

BMC Biotechnol

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BackgroundThermotoga spp. are attractive candidates for producing biohydrogen, green chemicals, and thermostable enzymes. They may also serve as model systems for understanding life sustainability under hyperthermophilic conditions. A lack of genetic tools has hampered the investigation and application of these organisms. This study aims to develop a genetic transfer system for Thermotoga spp.ResultsMethods for preparing and handling Thermotoga solid cultures under aerobic conditions were optimized. A plating efficiency of ~50% was achieved when the bacterial cells were embedded in 0.3% Gelrite. A Thermotoga-E. coli shuttle vector pDH10 was constructed using pRQ7, a cryptic mini-plasmid found in T. sp. RQ7. Plasmid pDH10 was introduced to T. maritima and T. sp. RQ7 by electroporation and liposome-mediated transformation. Transformants were isolated, and the transformed kanamycin resistance gene (kan) was detected from the plasmid DNA extracts of the recombinant strains by PCR and was confirmed by restriction digestions. The transformed DNA was stably maintained in both Thermotoga and E. coli even without the selective pressure.ConclusionsThermotoga are transformable by multiple means. Recombinant Thermotoga strains have been isolated for the first time. A heterologous kan gene is functionally expressed and stably maintained in Thermotoga.

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Section: Discussionmentioning

confidence: 99%

Construction and transformation of a Thermotoga-E. colishuttle vector

Han

Norris

2012

BMC Biotechnol

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“…A Type II RM system has been characterized in T. neapolitana and has also been found in T. maritima , T. petrophila , T. sp. strain RQ2, and T. naphthophila [33]. In addition, REBASE has identified a few putative methylases that might be involved in restriction-modification mechanisms in Thermotoga , such as M.TspRQ2ORF1808P in T. sp.…”

Section: Discussionmentioning

confidence: 99%

Natural transformation of Thermotoga sp. strain RQ7

Han

Puranik

et al. 2014

BMC Biotechnol

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BackgroundThermotoga 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.ResultsForeign 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.ConclusionsT. 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.

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“…The Type II R-M system TneDI has been characterized in 10.1601/nm.465 10.1601/strainfinder?urlappend=%3Fid%3DDSM+4359 and overexpressed in 10.1601/nm.3093 [32, 33]. The nuclease R.TneDI cleaves at the center of the recognition site (CG↓CG), and the methylase M.TneDI modifies one of the cytosines.…”

Section: Insights From the Genome Sequencementioning

confidence: 99%

“…M.TneDI has been predicted to be a m 4 C methylase based on sequence analysis [32]. It has also been noticed that m 4 C methylation is more common than m 5 C in thermophiles, probably due to a reduced risk of deamination [34].…”

Section: Insights From the Genome Sequencementioning

confidence: 99%

Complete genome sequence of Thermotoga sp. strain RQ7

Puranik

et al. 2017

Stand in Genomic Sci

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Thermotoga sp. strain RQ7 is a member of the family Thermotogaceae in the order Thermotogales. It is a Gram negative, hyperthermophilic, and strictly anaerobic bacterium. It grows on diverse simple and complex carbohydrates and can use protons as the final electron acceptor. Its complete genome is composed of a chromosome of 1,851,618 bp and a plasmid of 846 bp. The chromosome contains 1906 putative genes, including 1853 protein coding genes and 53 RNA genes. The genetic features pertaining to various lateral gene transfer mechanisms are analyzed. The genome carries a complete set of putative competence genes, 8 loci of CRISPRs, and a deletion of a well-conserved Type II R-M system.

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Cloning and characterization of the TneDI restriction–modification system of Thermotoga neapolitana

Cited by 8 publications

References 30 publications

Construction and transformation of a Thermotoga-E. colishuttle vector

Construction and transformation of a Thermotoga-E. colishuttle vector

Natural transformation of Thermotoga sp. strain RQ7

Complete genome sequence of Thermotoga sp. strain RQ7

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