Alexey Fomenkov

New England Biolabs

The reconstruction of complete bacterial genomes is essential for microbial research, offering insights into genetic content, ontology and regulation. While Pacific Biosciences (PacBio) provides high-quality genomes, its cost remains a limitation. Oxford Nanopore Technologies (ONT) offers long reads at a lower cost, yet its error rate raises scepti...

Bacteria possess (bacterio)phage defence systems to ensure their survival. The thermophilic lactic acid bacterium, Streptococcus thermophilus, which is used in dairy fermentations, harbours multiple CRISPR-Cas and restriction and modification (R/M) systems to protect itself against phage attack, with limited reports on other types of phage-resistan...

Reconstruction of complete bacterial genomes is a vital aspect of microbial research, as it provides complex information about genetic content, gene ontology, and regulation. It has become a domain of 3rd generation, long-read sequencing platforms, as short-read technologies can deliver mainly fragmented genomes. PacBio platform can provide high-qu...

Although restriction-modification systems are found in both Eubacterial and Archaeal kingdoms, comparatively less is known about patterns of DNA methylation and genome defense systems in archaea. Here we report the complete closed genome sequence and methylome analysis of Methanococcus aeolicus PL15/Hp, a strain of the CO2-reducing methanogenic arc...

Phosphorothioation (PT), in which a non-bridging oxygen is replaced by a sulfur, is one of the rare modifications discovered in bacteria and archaea that occurs on the sugar-phosphate backbone as opposed to the nucleobase moiety of DNA. While PT modification is widespread in the prokaryotic kingdom, how PT modifications are distributed in the genom...

Phosphorothioation (PT), in which a non-bridging oxygen is replaced by a sulfur, is one of the rare modifications discovered in bacteria and archaea that occurs on the sugar-phosphate backbone as opposed to the nucleobase moiety of DNA. While PT modification is widespread in the prokaryotic kingdom, how PT modifications are distributed in the genom...

Understanding mechanisms that shape horizontal exchange in prokaryotes is a key problem in biology. A major limit on DNA entry is imposed by restriction-modification (RM) processes that depend on the pattern of DNA modification at host-specified sites. In classical RM, endonucleolytic DNA cleavage follows detection of unprotected sites on entering...

Understanding mechanisms that shape horizontal exchange in prokaryotes is a key problem in biology. A major limit on DNA entry is imposed by restriction-modification (RM) processes that depend on the pattern of DNA modification at host-specified sites. In classical RM, endonucleolytic DNA cleavage follows detection of unprotected sites on entering...

Two strains of filamentous, colorless sulfur bacteria were isolated from bacterial fouling in the outflow of hydrogen sulfide-containing waters from a coal mine (Thiothrix sp. Ku-5) and on the seashore of the White Sea (Thiothrix sp. AS). Metagenome-assembled genome (MAG) A52 was obtained from a sulfidic spring in the Volgograd region, Russia. Phyl...

The genome of halophilic archaeon Haloarcula sinaiiensis ATCC 33800 was sequenced and assembled and comprises seven replicons. Four m6A and one m4C modified motifs and their responsible methyltransferase genes have been identified in the genome by single-molecule real-time (SMRT) sequencing and bioinformatic analysis.

DNA methylation is widespread amongst eukaryotes and prokaryotes to modulate gene expression and confer viral resistance. 5-Methylcytosine (m5C) methylation has been described in genomes of a large fraction of bacterial species as part of restriction-modification systems, each composed of a methyltransferase and cognate restriction enzyme. Methylas...

The Salmonella research community has used strains and bacteriophages over decades, exchanging useful new isolates among laboratories for study of cell surface antigens, metabolic pathways and restriction-modification studies. Here we present the sequences of two laboratory Salmonella strains (STK005, an isolate of LB5000; and its descendant ER3625...

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...

Paenibacillus spp. exopolysaccharides (EPSs) have become a growing interest recently as a source of biomaterials. In this study, we characterized Paenibacillus polymyxa 2020 strain, which produces a large quantity of EPS (up to 68 g/L),and was isolated from wasp honeycombs. Here we report its complete genome sequence and full methylome analysis det...

BisI is a sequence-specific and 5-methylcytosine (m5C)-dependent restriction endonuclease (REase), that cleaves the modified DNA sequence Gm5CNGC (G indicates that the cytosine opposite to G is modified). We expressed and purified a number of BisI homologs from sequenced bacterial genomes and used Illumina sequencing to determine the Pam7902I (Esp6...

The SARS-CoV-2 viral genome contains a positive-strand single-stranded RNA of ∼30 kb. Human ACE2 protein is the receptor for SARS-CoV-2 virus attachment and infection. We propose to use ribonucleases (RNases) as antiviral agents to destroy the viral genome in vitro. In the virions, the RNA is protected by viral capsid proteins, membrane proteins, a...

Single-molecule Real-Time (SMRT) sequencing can easily identify sites of N6-methyladenine and N4-methylcytosine within DNA sequences, but similar identification of 5-methylcytosine sites is not as straightforward. In prokaryotic DNA, methylation typically occurs within specific sequence contexts, or motifs, that are a property of the methyltransfer...

The Salmonella research community has used strains and bacteriophages over decades, exchanging useful new isolates among laboratories for study of cell surface antigens, metabolic pathways and restriction-modification studies. Here we present the sequences of two laboratory Salmonella strains (STK005, an isolate of LB5000; and its descendant ER3625...

A rising incidence of meningococcal serogroup W disease has been evident in many countries worldwide. Serogroup W isolates belonging to the sequence type (ST)-11 clonal complex have been associated with atypical symptoms and increased case fatality rates. The continued expansion of this clonal complex in the later part of the 2010s has been largely...

The genome of Salmonella enterica serovar Typhimurium LT7 comprises a chromosome and two plasmids. One plasmid is very close to pSLT of Salmonella Typhimurium LT2; the second harbors a shufflon region. Prophage content is distinct: LT7 lacks Fels-1, while Gifsy-1 and Fels-2 show island-like divergence and likely programmed inversion, respectively.

DNA methylation is widespread amongst eukaryotes and prokaryotes to modulate gene expression and confer viral resistance. 5-methylcytosine (m5C) methylation has been described in genomes of a large fraction of bacterial species as part of restriction-modification systems, each composed of a methyltransferase and cognate restriction enzyme. Methylas...

Single-molecule Real-Time (SMRT) sequencing can easily identify sites of N6-methyladenine and N4-methylcytosine within DNA sequences, but similar identification of 5-methylcytosine sites is not as straightforward. In prokaryotic DNA, methylation typically occurs within specific sequence contexts, or motifs, that are a property of the methyltransfer...

The SARS-CoV-2 viral genome contains a positive-strand single-stranded RNA of ~30 kb. Human ACE2 protein is the receptor for SARS-CoV-2 virus attachment and initiation of infection. We propose to use ribonucleases (RNases) as antiviral agents to destroy the viral genome in vitro. In the virions the RNA is protected by viral capsid proteins, membran...

Analysis of genomic DNA from pathogenic strains of Burkholderia cenocepacia J2315 and Escherichia coli O104:H4 revealed the presence of two unusual MTase genes. Both are plasmid-borne ORFs, carried by pBCA072 for B. cenocepacia J2315 and pESBL for E. coli O104:H4. Pacific Biosciences SMRT sequencing was used to investigate DNA methyltransferases M....

The genomes of gut Bacteroidales contain numerous invertible regions, many of which contain promoters that dictate phase-variable synthesis of surface molecules such as polysaccharides, fimbriae, and outer surface proteins. Here, we characterize a different type of phase-variable system of Bacteroides fragilis, a Type I restriction modification sys...

Modification dependent restriction endonucleases (MDREs) restrict modified DNA, typically with limited sequence specificity (∼2–4 bp). Here, we focus on MDREs that have an SRA and/or SBD (sulfur binding domain) fused to an HNH endonuclease domain, cleaving cytosine modified or phosphorothioated (PT) DNA. We independently characterized the SBD-SRA-H...

Here, we report the finished closed genomes of two environmental bacteria, Oceanispirochaeta crateria K2 and Thiospirochaeta perfilievii P (formally known as Spirochaeta perfilievii P). In addition, we provide methylation data and the associated enzymes predicted and confirmed to be responsible for each modified motif.

Invasive meningococcal disease (IMD) due to serogroup Y Neisseria meningitidis emerged in Europe during the 2000s. Draft genomes of serogroup Y isolates in Sweden revealed that although the population structure of these isolates was similar to other serogroup Y isolates internationally, a distinct strain (YI) and more specifically a sublineage (1)...

HhaI, a Type II restriction endonuclease, recognizes the symmetric sequence 5'-GCG↓C-3' in duplex DNA and cleaves ('↓') to produce fragments with 2-base, 3'-overhangs. We determined the structure of HhaI in complex with cognate DNA at an ultra-high atomic resolution of 1.0 Å. Most restriction enzymes act as dimers with two catalytic sites, and clea...

Sphaerotilus natans subsp. sulfidivorans D-507 is an environmental isolate from a sulfate spring in the northern Caucasus region of Russia. This heterotrophic bacterium is involved in the oxidation of reduced sulfur derivatives. This report includes the finished genome of this strain. In addition, we provide methylation data and the associated enzy...

Modification dependent restriction endonucleases (MDREs) often have separate catalytic and modification dependent domains. We systematically looked for previously uncharacterized fusion proteins featuring a PUA or DUF3427 domain and HNH or PD-(D/E)XK catalytic domain. The enzymes were clustered by similarity of their putative modification sensing d...

Here, we report the complete genome sequence and full methylome analysis of a newly isolated, aerobic, thermophilic, Gram-positive actinomycete, a strain of Thermoactinomyces vulgaris designated strain 2H.

The genomes of two extremely halophilic Archaea species, Haloarcula marismortui and Haloferax mediterranei , were sequenced using single-molecule real-time sequencing. The ∼4-Mbp genomes are GC rich with multiple large plasmids and two 4-methyl-cytosine patterns. Methyl transferases were incorporated into the Restriction Enzymes Database (REBASE),...

Deinococcus wulumuqiensis 479 (formerly known as Deinococcus radiodurans 479) is the original source strain for the restriction enzyme DrdI. Its complete sequence and full methylome were determined using Pacific Biosciences single-molecule real-time (SMRT) sequencing.

We report the complete genome sequences of two strains of the Alphaproteobacteria genus Rhodobacter , Rhodobacter blasticus 28/5, the source of the commercially available enzyme RsaI, and a new isolate of Rhodobacter sphaeroides 2.4.1. Both strains contain multiple restriction-modification systems, and their DNA methylation motifs are included in t...

Here, we report the complete closed genome sequence and methylome analysis of Beggiatoa leptomitoformis strain D-401 (DSM 14945, UNIQEMU 779), which is quite different from the previously described Beggiatoa leptomitoformis neotype strain D-402 T (DSM 14946, UNIQEM U 779) with regard to morphology and lithotrophic growth in the presence of thiosulf...

Bacillus caldolyticus NEB414 is the original source strain for the restriction enzyme BclI. Its complete sequence and full methylome were determined using single-molecule real-time sequencing.

We describe the cloning, expression and characterization of the first truly non-specific adenine DNA methyltransferase, M.EcoGII. It is encoded in the genome of the pathogenic strain Escherichia coli O104:H4 C227-11, where it appears to reside on a cryptic prophage, but is not expressed. However, when the gene encoding M.EcoGII is expressed in vivo...

Prevotella intermedia, a major periodontal pathogen, is increasingly implicated in human respiratory tract and cystic fibrosis lung infections. Nevertheless, the specific mechanisms employed by this pathogen remain only partially characterized and poorly understood, largely due to its total lack of genetic accessibility. Here, using Single Molecule...

Additional restriction enzyme assays to determine methylation status of 5’-GATC-3’ motifs on P. intermedia genomic DNA. A) Extended 12-hour incubation of P. intermedia ATCC25611F gDNA with excessive concentrations (5U/ug DNA) of restriction enzymes recognizing GATC, each with different methylation sensitivity, Lane 1, undigested control DNA; lane 2...

Restriction enzyme assay to determine methylation status of 5’-GATC-3’ motifs on plasmid DNA isolated from E.coli ER2796: M2.Pin17FORF7650P+. The methyltransferase gene was cloned to plasmid pRRS (primers listed in S1 Table) and expressed in E. coli ER2796, a strain deficient in methyltransferase activity. Plasmid DNA (1 μg) isolated from recombina...

Multiple sequence alignment of CRISPR direct repeats (DR) and anti-repeats in P. intermedia strains. A) Comparison of ATCC25611F and 17F CRISPR-I system DR sequences. B) Sequence alignment of CRISPR-I system DR with putative tracRNA sites anti-repeat_1 and anti-repeat_2, identified by CRISPROne (http://omics.informatics.indiana.edu/CRISPRone). C) S...

Site directed bisulfite sequencing of P. intermedia ATCC-25611F demonstrates 5-methylcytosine modifications within 5’-GCWGC-3’ motifs. Multiple sequence alignment of native and bisulfite treated ATCC25611F genomic DNA. Two regions, region 1(A) and region 2 (B), were selected based on the density of GCWGC motifs present and PCR amplified using prime...

Analysis of P. intermedia CRISPR-I leader sequences. A) Multiple sequence alignment of putative leader sequences and the first repeats in P. intermedia. The putative leader sequence of P. intermedia (120 bp) and the first repeat (47 bp) of the CRISPR-I locus is shown. The conserved palindromic sequence is highlight in red, the first DR of the array...

Methylated motifs identified in Prevotella intermedia ATCC-25611F and 17F. a The modified base within each motif is bolded while the modified base in the complementary strand is italicized. b Novel recognition sequences. c Detected only after tet-assisted SMRT library preparation. d The total number includes motifs occurring on the “+” and “–” stra...

Prevotella intermedia genome annotations. Subsystem distribution based on RAST annotation of individual chromosomes I and II of Prevotella intermedia ATCC-25611F (A and B) and Strain 17F (C and D). (TIF)

Site directed bisulfite sequencing of P. intermedia ATCC-25611F indicates the absence of 5-methylcytosine modifications within 5’-GATC-3’ motifs. Multiple sequence alignment of native and bisulfite treated ATCC25611F genomic DNA. Two regions, region 1(A) and region 2 (B), were selected based on the density of GATC motifs present and PCR amplified u...

Prediction of PAM consensus sequence. Putative protospacers flanking sequence (10 bp on each side, 5’: -1 to-10 and 3’:+1 to +10) were extracted and aligned. The alignment of these regions was used to create the sequence logo by WebLogo (http://weblogo.berkeley.edu/logo.cgi) for CRISPR-I and CRISPR-II of P. intermedia strains. The height of the let...

PacBio SMRT sequence basemod motif summary of recombinant E. coli ER2796 clones expressing P. intermedia R-M system methyltranserase genes. motifString: Detected motif sequence. centerPos: Position in motif of modification. fraction: Fraction of instances of this motif with modification Quality Value (QV) above the QV threshold. nDetected: Number o...

CRISPR systems spacers of P. intermedia ATCC-25611F. (XLSX)

CRISPR systems protospacers of P. intermedia ATCC25611F and 17F. (XLSX)

Oligonucleotides used in this study. Primers are shown in 5’ to 3’ orientation. (XLSX)

CRISPR systems spacers of P. intermedia 17F. (XLSX)

Two restriction–modification systems have been previously discovered in Thermus aquaticus YT-1. TaqI is a 263-amino acid (aa) Type IIP restriction enzyme that recognizes and cleaves within the symmetric sequence 5′-TCGA-3′. TaqII, in contrast, is a 1105-aa Type IIC restriction-and-modification enzyme, one of a family of Thermus homologs. TaqII was...

In this report, we announce the availability of a whole-genome sequence and methylome analysis of Thioflexothrix psekupsii strain D3.

The bacterial strains, plasmids and phages used in this study are listed in Tables 1-2. Defective Tn10 transposons Tn10dTet (element 104) or Tn10dCm (element 105) [1] are also known as miniTn10 elements, and appear as mTn10 TcR or mTn10 CmR in Figure A. ER2683 (Table 2) was used for plasmid constructions. Media and reagents Media for constructions...