Alexey Fomenkov

In prokaryotes, host-dependent restriction-modification (HDRM) systems comprise a major category of defense against DNA invasion. Such systems monitor the pattern of DNA modification conferred by site-specific DNA methyltransferases (MTase), attacking with nuclease when specific sites do not carry the host modification pattern. NTPase activity is often used to regulate activation of nuclease activity. Bioinformatic analysis yielded provisional identifications of nuclease, MTase and NTPase domains in a new configuration of component domains with novel connectivity. Confirmation of domain activity employed in vitro transcription-translation of epitope-tagged proteins yielding assignment of catalytic activity to protein segments. In vivo heterologous expression of the corresponding protein segments followed by affinity purification allowed us to characterize DNA site-requirements for cleavage, ATPase and MTase activities. Three representatives from different phyla employ methylating MTase components divided into two parts, with the parts connected to regulatory NTPase and defensive nuclease domains in novel organizations. The DrdVI system from Deinococcus wulumuqiensis NEB479 (formerly Deinococcus radiodurans ) recognizes 5’-GCAGCC-3’, modifying only one strand. Two systems, PenI ( Porphyromonas endodontalis ATCC 35406) and SmoLII ( Streptobacillus moniliformis DSM12112), each recognize 5’-GCAGT-3’, again modifying the A of that strand. Organizationally, the genes and proteins of these MTase activities have separated catalytic motifs from DNA-binding components in distinct ways. All have a component subunit that connects an endonuclease to an NTP-hydrolyzing translocase domain. The endonucleases of DrdVI and PenI require (d)ATP and divalent cations (Mg ²⁺ or Mn ²⁺ ) as cofactors. Cleavage occurs when head-to-head inverted recognition sites are present. DNA cleavage does not occur at a fixed site. The one-strand modification and inverted cleavage site configuration resemble typical Type III enzymes, but the connectivity of domain components sets them apart. Bioinformatic analysis reveals numerous similar putative HDRM systems. This suggests that they may represent a new subtype containing features of both Type I and Type III systems.

Bacteriophage Exclusion (BREX) systems are a diverse family of bacterial and archaeal defense mechanisms defined by two conserved genes encoding a putative alkaline phosphatase BrxZ (PglZ) and an ATPase BrxC (PglY). Here, we characterize a Type V BREX system from the archaeon Haloarcula hispanica . Similar to bacterial Type I BREX systems, host–virus discrimination relies on methylation of specific non-palindromic DNA motifs. Notably, the H. hispanica BREX locus encodes two methyltransferases, BrxX1 and BrxX2, which independently target distinct motifs (GTAYCCG and GACCCC). The system protects H. hispanica against three of seven tested archaeal viruses. However, two related viruses, SH1 and HHIV-2, despite encoding multiple BREX target sites, escape BREX-mediated defense. We demonstrate that a large virion protein, VP1, encoded by these viruses inhibits the BREX system, as mutant viruses carrying partial deletions of VP1 lose resistance to the host defense. Together, these findings provide the first characterization of a Type V BREX system and demonstrate that archaeal viruses can counteract BREX through virion-associated anti-defense proteins.

Salinibacter ruber strain M31 ^T , an extremely halophilic bacterium, was isolated from a saltern crystallizer pond in Spain. Single-molecule real-time sequencing revealed a 3.6-Mbp genome with a single 3.55-Mbp circular chromosome and a 35.5-kbp plasmid. The highly acidic proteome includes a total of 2,962 proteins, some of which are archaeal-like.

The genomes of 43 distinct lactococcal strains were reconstructed by a combination of long- and short-read sequencing, resolving the plasmid complement and methylome of these strains. The genomes comprised 43 chromosomes of approximately 2.5 Mb each and 269 plasmids ranging from 2 to 211 kb (at an average occurrence of 6 per strain). A total of 953 antiphage genes representing 538 phage defence systems were identified in the 43 strains and were catalogued and cross-correlated with co-occurrent mobile elements, which indicated that almost 60% of these systems are predicted to be mobile. Detailed analysis established that restriction-modification (R-M) systems form a significant portion of this mobile phage defensome. As such, all detected Type I, II, and III-associated methylated motifs (46 of which were unique to this study) were matched to their corresponding methylating enzymes by homology detection or molecular cloning. The cumulative antiphage activity of selected systems and the ability of truncated R-M genes to contribute to methylation were demonstrated. This study reveals, for the first time, the dairy lactococcal plasmidome to be a rich reservoir of orphan HsdS-encoding genes, in a comprehensive survey of (mobile) phage defence systems in lactic acid bacteria.

We report the 3.8-Mbp genome sequence of an extreme Haloarchaeon, Natrinema pallidum strain NMX15-1, isolated from 250-million-year-old halite samples of the Permian Period. PacBio’s long-read sequencing revealed a genome comprising a chromosome (3.5-Mbp) and three plasmids (203, 71, and 56-Kbp). The proteome is highly acidic and includes 3,784 proteins.

The availability of large databases of biological sequences presents an opportunity for in-depth exploration of gene diversity and function. Bacterial defense systems are a rich source of diverse but difficult to annotate genes with biotechnological applications. In this work, we present Domainator, a flexible and modular software suite for domain-based gene neighborhood and protein search, extraction and clustering. We demonstrate the utility of Domainator through three examples related to bacterial defense systems. First, we cluster CRISPR-associated Rossman fold (CARF) containing proteins with difficult to annotate effector domains, classifying most of them as likely transcriptional regulators and a subset as likely RNases. Second, we extract and cluster P4-like phage satellite defense hotspots, identify an abundant variant of Lamassu defense systems and demonstrate its in vivo activity against several T-even phages. Third, we integrate a protein language model into Domainator and use it to identify restriction endonucleases with low similarity to known reference sequences, validating the activity of one example in vitro. Domainator is made available as an open-source package with detailed documentation and usage examples.

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 scepticism. Recent ONT advancements, such as new Flow cells (R10.4.1), chemistry (V14) and duplex mode, improve data quality. Our study compares ONT with PacBio and Illumina, including hybrid data. We used Propionibacterium freudenreichii, a bacterium with a genome known for being difficult to reconstruct. By combining data from ONT’s Native Barcoding and a custom-developed BARSEQ method, we achieved high-quality, near-perfect genome assemblies. Our findings demonstrate, for the first time, that the combination of nanopore-only long-native with shorter PCR DNA reads (~3 kb) results in high-quality genome reconstruction, comparable to hybrid data assembly from two sequencing platforms. This endorses ONT as a cost-effective, stand-alone strategy for bacterial genome reconstruction. Additionally, we compared methylated motif detection between PacBio and ONT R10.4.1 data, showing that results comparable to PacBio are achievable using ONT, especially when utilizing the advanced Nanomotif tool.

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-resistance. Here, we describe the systematic identification and functional analysis of the phage resistome of S. thermophilus using a collection of 27 strains as representatives of the species. In addition to CRISPR-Cas and R/M systems, we uncover nine distinct phage-resistance systems including homologues of Kiwa, Gabija, Dodola, defence-associated sirtuins and classical lactococcal/streptococcal abortive infection systems. The genes encoding several of these newly identified S. thermophilus antiphage systems are located in proximity to the genetic determinants of CRISPR-Cas systems thus constituting apparent Phage Defence Islands. Other phage-resistance systems whose encoding genes are not co-located with genes specifying CRISPR-Cas systems may represent anchors to identify additional Defence Islands harbouring, as yet, uncharacterised phage defence systems. We estimate that up to 2.5% of the genetic material of the analysed strains is dedicated to phage defence, highlighting that phage-host antagonism plays an important role in driving the evolution and shaping the composition of dairy streptococcal genomes.

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-quality complete genomes, yet remains one of the most expensive sequencing strategies. Oxford Nanopore Technology (ONT) offers the advantage of producing the longest reads, being at the same time the most cost-effective option in terms of platform costs, as well as library preparation, and sequencing. However, ONTs error rate, although significantly reduced lately, still holds a certain level of distrust in the scientific community. In recent years, hybrid assembly of Nanopore and Illumina data has been used to solve ONTs issue with error rate and has yielded the best results in terms of genome completeness, quality, and price. However, the latest advancements in Nanopore technology, including new flow cells (R10.4.1), new library preparation chemistry (V14) and duplex-mode, updated basecallers (Dorado v0.4.1), and the realization that sequencing in dark mode results in significantly increased throughput, have had a significant impact on the quality of generated data and, thus, the recovery of complete genomes by ONT sequencing alone. In this study, we compared the data generated by ONT using three sequencing strategies (Native barcoding, RAPID barcoding, and custom-developed: BARSEQ) against PacBio and Illumina (NextSeq) as well as Illumina-ONT hybrid data. For this purpose, we employed three strains of the actinobacteria Propionibacterium freudenreichii , whose genomes have been proven difficult to reconstruct due to high GC content, regions of repeated sequences and massive genome rearrangements. Our data indicate that DNA libraries prepared with the native barcoding kit, sequenced with V14 chemistry on R10.4.1 flow cell, and assembled with Flye resulted in the reconstruction of complete genomes of overall quality highly similar to that of genomes reconstructed with PacBio. The highest level of quality can be achieved by hybrid assembly of data from the Native barcoding kit complemented with data from custom-developed BARSEQ, both sequenced on R10.4.1 flow cell. In conclusion, our results demonstrate that ONT can be used as a cost-effective sequencing strategy, without the need for complementing with other sequencing technologies, for the reconstruction of complete genomes of the highest quality.