Characterization of cleavage intermediate and star sites of RM.Tth111II

Zhu, Zhenyu; Guan, Shengxi; Robinson, Derek; Fezzazi, Hanna El; Quimby, Aine; Xu, Shuang-yong

doi:10.1038/srep03838

Cited by 9 publications

(8 citation statements)

References 34 publications

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“…Interestingly, the TdtA protein is only present in a few of the sequenced strains of T. thermophilus, encoded by a mobile genetic element (ICETh1) reminiscent of the integrative and conjugative elements (ICE) found in many conjugative bacteria (Blesa et al, 2017). This 14 kb element encodes a typical phage-like recombinase of the XerC family and a four-gene operon encoding: a type IIG restriction endonuclease identical in sequence to Tth111II (Zhu et al, 2014); a putative nuclease of the NurA family; TdtA; and a putative DNA methylase. Additional genes in the ICETh1 include a transposase for which two more copies exist in the genome, and a putative hydrolase of unknown function.…”

Section: ! 28mentioning

confidence: 99%

Horizontal Gene Transfer in Thermus spp.

Blesa¹,

Averhoff²,

Berenguer³

2018

Current Issues in Molecular Biology

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The small amount of genetic content in thermophiles generally limits their adaptability to environmental changes. In Thermus spp., very active horizontal gene transfer (HGT) mechanisms allow the rapid spread of strain-specific adaptive gene modules among the entire population. Constitutive expression of a rather particular and highly efficient DNA transport apparatus (DTA) is at the center of this HGT-mediated enhanced adaptability. The function of the DTA is dependent on the integrity and longevity of the extracellular DNA (eDNA) being transformed, which can be improved by the production of extracellular vesicles (EV) through lysis of a fraction of the population. The DTA must also contend with the recipient cell's defensive barriers, namely restriction enzymes, a panoply of CRISPR-Cas systems, and the argonaute-like protein TtAgo, which may be bypassed by transjugation, a new class of bidirectional transformation-dependent conjugation. Efficient transjugation depends on the presence of the ICETh1, an integrative and conjugative element which promotes simultaneous, generalized DNA transfer from several points in the genome. Transjugation shows preference for genes located within a megaplasmid replicon, where the main strain-specific adaptive modules are located. Contribution of transformation, vesicle-mediated eDNAs, and transjugation to HGT in this genus is discussed.

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Section: ! 28mentioning

confidence: 99%

Horizontal Gene Transfer in Thermus spp.

Blesa¹,

Averhoff²,

Berenguer³

2018

Current Issues in Molecular Biology

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“…It is known that some Type II REases can be stimulated by duplex oligos with cognate DNA recognition sequences (Senesac and Romanin 1997 ; Zhu et al 2014 ). Thus, the activity of Tso I REase was investigated in the presence of different concentrations of the following oligo duplexes: A (no Tso I site), B (one TAACCA site), C (one TAGCCA), D (one SCS site TAGCtc) and E (one TAACCA site; cleavage-like product) (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The activity of Tso I REase on supercoiled pUC19 with additional duplex oligos at different concentrations was investigated similarly as described by Zhu et al 2014 . The following concentrations of duplex oligo were used: 4.1, 2.05, 1.025, 0.51, 0.26, 0.13, 0.06, 0.03; 0.016, 0.008, 0.004, 0.002 μM.…”

Section: Methodsmentioning

confidence: 99%

Novel parameter describing restriction endonucleases: Secondary-Cognate-Specificity and chemical stimulation of TsoI leading to substrate specificity change

Żebrowska

Jeżewska-Frąckowiak

Wieczerzak

et al. 2019

Appl Microbiol Biotechnol

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Over 470 prototype Type II restriction endonucleases (REases) are currently known. Most recognise specific DNA sequences 4–8 bp long, with very few exceptions cleaving DNA more frequently. Tso I is a thermostable Type IIC enzyme that recognises the DNA sequence TARCCA (R = A or G) and cleaves downstream at N11/N9. The enzyme exhibits extensive top-strand nicking of the supercoiled single-site DNA substrate. The second DNA strand of such substrate is specifically cleaved only in the presence of duplex oligonucleotides containing a cognate site. We have previously shown that some Type IIC/IIG/IIS enzymes from the Thermus -family exhibit ‘affinity star’ activity, which can be induced by the S-adenosyl-L-methionine (SAM) cofactor analogue—sinefungin (SIN). Here, we define a novel type of inherently built-in ‘star’ activity, exemplified by Tso I. The Tso I ‘star’ activity cannot be described under the definition of the classic ‘star’ activity as it is independent of the reaction conditions used and cannot be separated from the cognate specificity. Therefore, we define this phenomenon as Secondary-Cognate-Specificity (SCS). The Tso I SCS comprises several degenerated variants of the cognate site. Although the efficiency of Tso I SCS cleavage is lower in comparison to the cognate Tso I recognition sequence, it can be stimulated by S-adenosyl-L-cysteine (SAC). We present a new route for the chemical synthesis of SAC. The Tso I/SAC REase may serve as a novel tool for DNA manipulation. Electronic supplementary material The online version of this article (10.1007/s00253-019-09731-0) contains supplementary material, which is available to authorized users.

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“…Further upstream, TTC1877 encodes a large protein (1106 aa) that is identical to Tth111II, a type IIG restriction endonuclease previously characterized from another strain of T . thermophilus [21]. Downstream from tdtA , TTC1880 encodes a hypothetical 410 aa protein of the N4-N6 methylase family (pfam 01555).…”

Section: Resultsmentioning

confidence: 99%

“…Compared to TraB, TdtA is smaller with no putative DNA binding domains detected by sequence analysis, thus the recognition of any hypothetical oriT would likely depend on other proteins. The best candidate for this role is the endonuclease Tth111II [21], whose deletion produced a 1,000-fold decrease in transjugation efficiency. This type IIG restriction enzyme has a rather low specific activity and preferentially nicks the top strand at position N11 downstream from its CAARCA recognition sequence.…”

Section: Discussionmentioning

confidence: 99%

The transjugation machinery of Thermus thermophilus: Identification of TdtA, an ATPase involved in DNA donation

et al. 2017

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In addition to natural competence, some Thermus thermophilus strains show a high rate of DNA transfer via direct cell-to-cell contact. The process is bidirectional and follows a two-step model where the donor cell actively pushes out DNA and the recipient cell employs the natural competence system to take up the DNA, in a hybrid transformation-dependent conjugation process (transjugation). While the DNA uptake machinery is well known as in other bacterial species that undergo transformation, the pushing step of transjugation remains to be characterized. Here we have searched for hypothetical DNA translocases putatively involved in the pushing step of transjugation. Among candidates encoded by T. thermophilus HB27, the TdtA protein was found to be required for DNA pushing but not for DNA pulling during transjugation, without affecting other cellular processes. Purified TdtA shows ATPase activity and oligomerizes as hexamers with a central opening that can accommodate double-stranded DNA. The tdtA gene was found to belong to a mobile 14 kbp-long DNA element inserted within the 3′ end of a tRNA gene, flanked by 47 bp direct repeats. The insertion also encoded a homolog of bacteriophage site-specific recombinases and actively self-excised from the chromosome at high frequency to form an apparently non-replicative circular form. The insertion also encoded a type II restriction endonuclease and a NurA-like nuclease, whose activities were required for efficient transjugation. All these data support that TdtA belongs to a new type of Integrative and Conjugative Element which promotes the generalized and efficient transfer of genetic traits that could facilitate its co-selection among bacterial populations.

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Characterization of cleavage intermediate and star sites of RM.Tth111II

Cited by 9 publications

References 34 publications

Horizontal Gene Transfer in Thermus spp.

Horizontal Gene Transfer in Thermus spp.

Novel parameter describing restriction endonucleases: Secondary-Cognate-Specificity and chemical stimulation of TsoI leading to substrate specificity change

The transjugation machinery of Thermus thermophilus: Identification of TdtA, an ATPase involved in DNA donation

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