Cloning and nucleotide sequence of the genes coding for the<i>Sau</i>961 restriction and modification enzymes

Szilák, László; Venetianer, Pál; Kiss, Antal

doi:10.1093/nar/18.16.4659

Cited by 39 publications

(19 citation statements)

References 42 publications

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“…Moreover, chromosomal DNA prepared from strain CD6 was shown to be resistant to digestion with this enzyme, whereas the genomic DNA of strain CD3 was readily cleaved. To confirm the hypothesis that strain CD6 contains a restriction system with equivalent specificity to Sau 96I, we obtained a pACYC184 plasmid clone carrying the Sau 96I methylase (M. Sau 96I) gene (Szilak et al ., 1990). When this plasmid was introduced into an E. coli host carrying pJIR418, both plasmid DNAs were found to be resistant to digestion by either CD6 lysate or Sau 96I.…”

Section: Resultsmentioning

confidence: 99%

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Conjugative transfer of clostridial shuttle vectors from Escherichia coli to Clostridium difficile through circumvention of the restriction barrier

Purdy¹,

O'Keeffe²,

Elmore³

et al. 2002

Molecular Microbiology

241

210

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SummaryProgress towards understanding the molecular basis of virulence in Clostridium difficile has been hindered by the lack of effective gene transfer systems. We have now, for the first time, developed procedures that may be used to introduce autonomously replicating vectors into this organism through their conjugative, oriT -based mobilization from Escherichia coli donors. Successful transfer was achieved through the use of a plasmid replicon isolated from an indigenous C. difficile plasmid, pCD6, and through the characterization and subsequent circumvention of host restriction/modification (RM) systems. The characterized replicon is the first C. difficile plasmid replicon to be sequenced and encodes a large replication protein (RepA) and a repetitive region composed of a 35 bp iteron sequence repeated seven times. Strain CD6 has two RM systems, Cdi CD6I/M .Cdi CD6I and Cdi CD6II/M. Cdi CD6II, with equivalent specificities to Sau 96I/M.

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Section: Resultsmentioning

confidence: 99%

“…Plasmid pACYC184::Sau5 was made by isolating a 2.4 kb Nru I– Eco RV fragment encoding the M. Sau 96I gene from plasmid pSau5 (Szilak et al ., 1990) and inserting it between the Nru I and Eco RV sites of plasmid pACYC184.…”

Section: Methodsmentioning

confidence: 99%

Conjugative transfer of clostridial shuttle vectors from Escherichia coli to Clostridium difficile through circumvention of the restriction barrier

Purdy¹,

O'Keeffe²,

Elmore³

et al. 2002

Molecular Microbiology

241

210

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“…RM systems are categorized into four major types based on their homology and associated functions. Types I, II, and IV have been observed in both S. aureus (8)(9)(10)(11) and S. epidermidis (4,12,13); the type III system appears infrequently in both species. The type I RM system is the most diverse among different clonal types and consists of restriction, modification, and specificity units, designated HsdR, HsdM, and HsdS, respectively, assembled into multisubunit complexes (14,15).…”

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confidence: 99%

Bypassing the Restriction System To Improve Transformation of Staphylococcus epidermidis

et al. 2017

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Staphylococcus epidermidis is the leading cause of infections on indwelling medical devices worldwide. Intrinsic antibiotic resistance and vigorous biofilm production have rendered these infections difficult to treat and, in some cases, require the removal of the offending medical prosthesis. With the exception of two widely passaged isolates, RP62A and 1457, the pathogenesis of infections caused by clinical S. epidermidis strains is poorly understood due to the strong genetic barrier that precludes the efficient transformation of foreign DNA into clinical isolates. The difficulty in transforming clinical S. epidermidis isolates is primarily due to the type I and IV restriction-modification systems, which act as genetic barriers. Here, we show that efficient plasmid transformation of clinical S. epidermidis isolates from clonal complexes 2, 10, and 89 can be realized by employing a plasmid artificial modification (PAM) in Escherichia coli DC10B containing a Δdcm mutation. This transformative technique should facilitate our ability to genetically modify clinical isolates of S. epidermidis and hence improve our understanding of their pathogenesis in human infections.IMPORTANCE Staphylococcus epidermidis is a source of considerable morbidity worldwide. The underlying mechanisms contributing to the commensal and pathogenic lifestyles of S. epidermidis are poorly understood. Genetic manipulations of clinically relevant strains of S. epidermidis are largely prohibited due to the presence of a strong restriction barrier. With the introductions of the tools presented here, genetic manipulation of clinically relevant S. epidermidis isolates has now become possible, thus improving our understanding of S. epidermidis as a pathogen.KEYWORDS Staphylococcus epidermidis, transformation, restriction system, DC10B, clonal complexes, HsdS, efficiency, methylation, restriction barrier S taphylococcus epidermidis is a ubiquitous human commensal that normally colonizes the human skin and mucous membranes (1). S. epidermidis can also assume an invasive lifestyle by colonizing indwelling medical devices, which in some cases can lead to invasive bacteremia. Indeed, due to the common use of implanted medical devices in modern medicine, S. epidermidis is now the number one cause of nosocomial bacteremia in the United States (2). Although these device-related infections are less severe than those due to their Staphylococcus aureus counterparts, these diseases tend to be more persistent, with most being described as subacute or chronic in nature. In many cases, infection cannot be eliminated unless the offending medical devices, such as catheters, prostheses, or pacemakers, are removed, thus leading to high morbidity rates (3). Treatment of S. epidermidis infections is also complicated by the high level of innate antibiotic resistance as well as robust biofilm formation, which can render host defenses and antibiotics ineffective (1).

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“…A regulatory mechanism based on the different efficiencies of translation signals was suggested to operate also in some other R-M systems (18,49). Such a mechanism could be an alternative to transcriptional control coordinating REase and MTase expression (51).…”

Section: Discussionmentioning

confidence: 99%

BspRI restriction endonuclease: cloning, expression in Escherichia coli and sequential cleavage mechanism

Raskó

Dér

Klement

et al. 2010

Nucleic Acids Research

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The GGCC-specific restriction endonuclease BspRI is one of the few Type IIP restriction endonucleases, which were suggested to be a monomer. Amino acid sequence information obtained by Edman sequencing and mass spectrometry analysis was used to clone the gene encoding BspRI. The bspRIR gene is located adjacently to the gene of the cognate modification methyltransferase and encodes a 304 aa protein. Expression of the bspRIR gene in Escherichia coli was dependent on the replacement of the native TTG initiation codon with an ATG codon, explaining previous failures in cloning the gene using functional selection. A plasmid containing a single BspRI recognition site was used to analyze kinetically nicking and second-strand cleavage under steady-state conditions. Cleavage of the supercoiled plasmid went through a relaxed intermediate indicating sequential hydrolysis of the two strands. Results of the kinetic analysis of the first- and second-strand cleavage are consistent with cutting the double-stranded substrate site in two independent binding events. A database search identified eight putative restriction-modification systems in which the predicted endonucleases as well as the methyltransferases share high sequence similarity with the corresponding protein of the BspRI system. BspRI and the related putative restriction endonucleases belong to the PD-(D/E)XK nuclease superfamily.

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Cloning and nucleotide sequence of the genes coding for theSau961 restriction and modification enzymes

Cited by 39 publications

References 42 publications

Conjugative transfer of clostridial shuttle vectors from Escherichia coli to Clostridium difficile through circumvention of the restriction barrier

Conjugative transfer of clostridial shuttle vectors from Escherichia coli to Clostridium difficile through circumvention of the restriction barrier

Bypassing the Restriction System To Improve Transformation of Staphylococcus epidermidis

BspRI restriction endonuclease: cloning, expression in Escherichia coli and sequential cleavage mechanism

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