scholarly journals Structural and functional diversity among Type III restriction-modification systems that confer host DNA protection via methylation of the N4 atom of cytosine

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0253267
Author(s):  
Iain A. Murray ◽  
Yvette A. Luyten ◽  
Alexey Fomenkov ◽  
Nan Dai ◽  
Ivan R. Corrêa ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Methyltransferase ◽  
Protein A ◽  
Single Copy ◽  
Atpase Subunit ◽  
Type Iii ◽  
Host Protection ◽  
Rich Diversity ◽  
Restriction Modification ◽  
Restriction Modification Systems

We report a new subgroup of Type III Restriction-Modification systems that use m4C methylation for host protection. Recognition specificities for six such systems, each recognizing a novel motif, have been determined using single molecule real-time DNA sequencing. In contrast to all previously characterized Type III systems which modify adenine to m6A, protective methylation of the host genome in these new systems is achieved by the N4-methylation of a cytosine base in one strand of an asymmetric 4 to 6 base pair recognition motif. Type III systems are heterotrimeric enzyme complexes containing a single copy of an ATP-dependent restriction endonuclease-helicase (Res) and a dimeric DNA methyltransferase (Mod). The Type III Mods are beta-class amino-methyltransferases, examples of which form either N6-methyl adenine or N4-methyl cytosine in Type II RM systems. The Type III m4C Mod and Res proteins are diverged, suggesting ancient origin or that m4C modification has arisen from m6A MTases multiple times in diverged lineages. Two of the systems, from thermophilic organisms, required expression of both Mod and Res to efficiently methylate an E. coli host, unlike previous findings that Mod alone is proficient at modification, suggesting that the division of labor between protective methylation and restriction activities is atypical in these systems. Two of the characterized systems, and many homologous putative systems, appear to include a third protein; a conserved putative helicase/ATPase subunit of unknown function and located 5’ of the mod gene. The function of this additional ATPase is not yet known, but close homologs co-localize with the typical Mod and Res genes in hundreds of putative Type III systems. Our findings demonstrate a rich diversity within Type III RM systems.

scholarly journals Genetic variation regulates the activation and specificity of Restriction-Modification systems in Neisseria gonorrhoeae

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Leonor Sánchez-Busó ◽  
Daniel Golparian ◽  
Julian Parkhill ◽  
Magnus Unemo ◽  
Simon R. Harris
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Single Molecule ◽  
Regulation Of Gene Expression ◽  
Phase Variation ◽  
Type I ◽  
Dam Methylase ◽  
Different Strains ◽  
Methylation Patterns ◽  
Restriction Modification ◽  
Restriction Modification Systems

Abstract Restriction-Modification systems (RMS) are one of the main mechanisms of defence against foreign DNA invasion and can have an important role in the regulation of gene expression. The obligate human pathogen Neisseria gonorrhoeae carries one of the highest loads of RMS in its genome; between 13 to 15 of the three main types. Previous work has described their organization in the reference genome FA1090 and has inferred the associated methylated motifs. Here, we studied the structure of RMS and target methylated motifs in 25 gonococcal strains sequenced with Single Molecule Real-Time (SMRT) technology, which provides data on DNA modification. The results showed a variable picture of active RMS in different strains, with phase variation switching the activity of Type III RMS, and both the activity and specificity of a Type I RMS. Interestingly, the Dam methylase was found in place of the NgoAXI endonuclease in two of the strains, despite being previously thought to be absent in the gonococcus. We also identified the real methylation target of NgoAXII as 5′-GCAGA-3′, different from that previously described. Results from this work give further insights into the diversity and dynamics of RMS and methylation patterns in N. gonorrhoeae.

scholarly journals Genetic variation regulates the activation and specificity of Restriction-Modification systems in Neisseria gonorrhoeae

2019 ◽  
Author(s):  
Leonor Sánchez-Busó ◽  
Daniel Golparian ◽  
Julian Parkhill ◽  
Magnus Unemo ◽  
Simon R. Harris
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Single Molecule ◽  
Regulation Of Gene Expression ◽  
Phase Variation ◽  
Type I ◽  
Dam Methylase ◽  
Different Strains ◽  
Methylation Patterns ◽  
Restriction Modification ◽  
Restriction Modification Systems

ABSTRACTRestriction-Modification systems (RMS) are one of the main mechanisms of defence against foreign DNA invasion and can have an important role in the regulation of gene expression. The obligate human pathogen Neisseria gonorrhoeae carries one of the highest loads of RMS in its genome; between 13 to 15 of the three main types. Previous work has described their organization in the reference genome FA1090 and has experimentally inferred the associated methylated motifs. Here, we studied the structure of RMS and target methylated motifs in 25 gonococcal strains sequenced with Single Molecule Real-Time (SMRT) technology, which provides data on DNA modification. The results showed a variable picture of active RMS in different strains, with phase variation switching the activity of Type III RMS, and both the activity and specificity of a Type I RMS. Interestingly, the Dam methylase was found in place of the NgoAXI endonuclease in two of the strains, despite being previously thought to be absent in the gonococcus. We also identified the real methylation target of NgoAX as 5’-GCAGA-3’, different from that previously described. Results from this work give further insights into the diversity and dynamics of RMS and methylation patterns in N. gonorrhoeae.

scholarly journals Epigenomics, Genomics, Resistome, Mobilome, Virulome and Evolutionary Phylogenomics of Carbapenem-resistant Klebsiella pneumoniae clinical strains

2020 ◽  
Author(s):  
Katlego Kopotsa ◽  
Nontombi M Mbelle ◽  
Osei Sekyere John
Keyword(s):  
South Africa ◽  
Klebsiella Pneumoniae ◽  
Evolutionary Relationship ◽  
Multidrug Resistant ◽  
Type I ◽  
Bacteriophage Therapy ◽  
Carbapenem Resistant ◽  
Rich Diversity ◽  
Restriction Modification ◽  
Restriction Modification Systems

AbstractBackgroundCarbapenem-resistant Klebsiella pneumoniae (CRKP) remains a major clinical pathogen and public health threat with few therapeutic options. The mobilome, resistome, methylome, virulome and phylogeography of CRKP were characterised.MethodsCRKP collected in 2018 were subjected to antimicrobial susceptibility testing, screening by multiplex-PCR, genotyping by Repetitive Element Palindromic-Polymerase Chain Reaction (REP-PCR), plasmid size, number, incompatibility, and mobility analyses, and PacBio’s SMRT sequencing (n=6).Results & conclusionThere were 56 multidrug-resistant CRKP, having blaOXA-48-like and blaNDM-1/7 carbapenemases on self-transmissible IncF, A/C, IncL/M and IncX3 plasmids endowed with prophages, traT, resistance islands and type I and II restriction modification systems (RMS). These plasmids were of close evolutionary relationship to several plasmids globally whilst the strains also clustered with several global clades, evincing transboundary horizontal and vertical dissemination. Reduced susceptibility to colistin occurred in 23 strains. Common clones included ST307, ST607, ST17, ST39, and ST3559. IncFIIk virulent plasmid replicon was present in 56 strains. The six strains contained at least 41 virulence genes and four different K- and O-loci types: KL2, KL25, KL27, KL102, O1, O2, O4 and O5. Types I, II, and III RMS, conferring m6A (GATC, GATGNNNNNNTTG, CAANNNNNNCATC motifs) and m4C (CCWGG) modifications on chromosomes and plasmids, were found.There is plasmid-mediated, clonal, and multiclonal dissemination of blaOXA-48-like and blaNDM-1 in South Africa, mirroring international epidemiology of similar clones and plasmids. Plasmid-mediated transmission of RMS, virulome and prophages influence bacterial evolution, epidemiology, pathogenicity, and resistance, threatening infection treatment. RMS influence on antimicrobial and bacteriophage therapy needs urgent investigation.Highlights/ImportanceK. pneumoniae is a major pathogen implicated in numerous nosocomial infections. Worryingly, we show that K. pneumoniae isolates from South Africa, Africa and globally are endowed with rich resistomes and mobilomes that make them almost pandrug resistant. The isolates in this study contained rich virulomes and prophages on both chromosomes and plasmids, with close evolutionary kith or kin to other plasmids identified worldwide. There was a rich diversity of restriction modification systems that regulate virulence, transcription, and plasmid mobility in bacteria, facilitating the epidemiology, resistance, pathogenicity and genomic evolution of the strains, and threatening antimicrobial and bacteriophage therapy.

Cloning and sequence comparison ofAvaI andBsoBI restriction-modification systems

1996 ◽  
Vol 252 (6) ◽  
pp. 695-699 ◽  
Author(s):  
H. Ruan ◽  
K. D. Lunnen ◽  
M. E. Scott ◽  
L. S. Moran ◽  
B. E. Slatko ◽  
...  
Keyword(s):  
Sequence Comparison ◽  
Restriction Modification ◽  
Restriction Modification Systems

scholarly journals Single-copy detection of somatic variants from solid and liquid biopsy

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ana-Luisa Silva ◽  
Paulina Klaudyna Powalowska ◽  
Magdalena Stolarek ◽  
Eleanor Ruth Gray ◽  
Rebecca Natalie Palmer ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Real Time Pcr ◽  
Clinical Decision Making ◽  
High Sensitivity ◽  
Clinical Decision ◽  
Single Copy ◽  
Turnaround Time ◽  
Copy Detection ◽  
Allele Specific

AbstractAccurate detection of somatic variants, against a background of wild-type molecules, is essential for clinical decision making in oncology. Existing approaches, such as allele-specific real-time PCR, are typically limited to a single target gene and lack sensitivity. Alternatively, next-generation sequencing methods suffer from slow turnaround time, high costs, and are complex to implement, typically limiting them to single-site use. Here, we report a method, which we term Allele-Specific PYrophosphorolysis Reaction (ASPYRE), for high sensitivity detection of panels of somatic variants. ASPYRE has a simple workflow and is compatible with standard molecular biology reagents and real-time PCR instruments. We show that ASPYRE has single molecule sensitivity and is tolerant of DNA extracted from plasma and formalin fixed paraffin embedded (FFPE) samples. We also demonstrate two multiplex panels, including one for detection of 47 EGFR variants. ASPYRE presents an effective and accessible method that simplifies highly sensitive and multiplexed detection of somatic variants.

scholarly journals Live-cell imaging reveals the spatiotemporal organization of endogenous RNA polymerase II phosphorylation at a single gene

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Linda S. Forero-Quintero ◽  
William Raymond ◽  
Tetsuya Handa ◽  
Matthew N. Saxton ◽  
Tatsuya Morisaki ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Computational Models ◽  
Spatial Organization ◽  
Fluorescent Antibody ◽  
Single Gene ◽  
Single Copy ◽  
Living Cells ◽  
Live Cell

AbstractThe carboxyl-terminal domain of RNA polymerase II (RNAP2) is phosphorylated during transcription in eukaryotic cells. While residue-specific phosphorylation has been mapped with exquisite spatial resolution along the 1D genome in a population of fixed cells using immunoprecipitation-based assays, the timing, kinetics, and spatial organization of phosphorylation along a single-copy gene have not yet been measured in living cells. Here, we achieve this by combining multi-color, single-molecule microscopy with fluorescent antibody-based probes that specifically bind to different phosphorylated forms of endogenous RNAP2 in living cells. Applying this methodology to a single-copy HIV-1 reporter gene provides live-cell evidence for heterogeneity in the distribution of RNAP2 along the length of the gene as well as Serine 5 phosphorylated RNAP2 clusters that remain separated in both space and time from nascent mRNA synthesis. Computational models determine that 5 to 40 RNAP2 cluster around the promoter during a typical transcriptional burst, with most phosphorylated at Serine 5 within 6 seconds of arrival and roughly half escaping the promoter in ~1.5 minutes. Taken together, our data provide live-cell support for the notion of efficient transcription clusters that transiently form around promoters and contain high concentrations of RNAP2 phosphorylated at Serine 5.

scholarly journals Sfi1p has conserved centrin-binding sites and an essential function in budding yeast spindle pole body duplication

2003 ◽  
Vol 162 (7) ◽  
pp. 1211-1221 ◽  
Author(s):  
John V. Kilmartin
Keyword(s):  
Single Molecule ◽  
Protein A ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Binding Motif ◽  
Temperature Sensitive ◽  
Pole Body ◽  
Human Proteins ◽  
Z Domain ◽  
Essential Function

Centrins are calmodulin-like proteins present in microtubule-organizing centers. The Saccharomyces cerevisiae centrin, Cdc31p, was functionally tagged with a single Z domain of protein A, and used in pull-down experiments to isolate Cdc31p-binding proteins. One of these, Sfi1p, localizes to the half-bridge of the spindle pole body (SPB), where Cdc31p is also localized. Temperature-sensitive mutants in SFI1 show a defect in SPB duplication and genetic interactions with cdc31-1. Sfi1p contains multiple internal repeats that are also present in a Schizosaccharomyces pombe protein, which also localizes to the SPB, and in several human proteins, one of which localizes close to the centriole region. Cdc31p binds directly to individual Sfi1 repeats in a 1:1 ratio, so a single molecule of Sfi1p binds multiple molecules of Cdc31p. The centrosomal human protein containing Sfi1 repeats also binds centrin in the repeat region, showing that this centrin-binding motif is conserved.

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