RM Systems Research Articles

Genomic characterization of the antiviral arsenal of Actinobacteria.

Phages are ubiquitous in nature, and bacteria with very different genomics, metabolisms, and lifestyles are subjected to their predation. Yet, the defence systems that allow bacteria to resist their phages have rarely been explored experimentally outside a very limited number of model organisms. Actinobacteria (Actinomycetota) are a phylum of GC-rich Gram-positive bacteria, which often produce an important diversity of secondary metabolites. Despite being ubiquitous in a wide range of environments, from soil to fresh and sea water but also the gut microbiome, relatively little is known about the anti-phage arsenal of Actinobacteria. In this work, we used DefenseFinder to systematically detect 131 anti-phage defence systems in 22803 fully sequenced prokaryotic genomes, among which are 2253 Actinobacteria of more than 700 species. We show that, like other bacteria, Actinobacteria encode many diverse anti-phage systems that are often encoded on mobile genetic elements. We further demonstrate that most detected defence systems are absent or rarer in Actinobacteria than in other bacteria, while a few rare systems are enriched (notably gp29-gp30 and Wadjet). We characterize the spatial distribution of anti-phage systems on Streptomyces chromosomes and show that some defence systems (e.g. RM systems) tend to be encoded in the core region, while others (e.g. Lamassu and Wadjet) are enriched towards the extremities. Overall, our results suggest that Actinobacteria might be a source of novel anti-phage systems and provide clues to characterize mechanistic aspects of known anti-phage systems.

Delineation of a lactococcal conjugation system reveals a restriction-modification evasion system.

Plasmid pUC11B is a 49.3-kb plasmid harboured by the fermented meat isolate Lactococcus lactis subsp. lactis UC11. Among other features, pUC11B encodes a pMRC01-like conjugation system and tetracycline-resistance. In this study, we demonstrate that this plasmid can be conjugated at high frequencies to recipient strains. Mutational analysis of the 22 genes encompassing the presumed pUC11B conjugation cluster revealed the presence of several genes with essential conjugation functions, as well as a gene, trsR, encoding a putative transcriptional repressor of this conjugation cluster. Furthermore, plasmid pUC11B encodes an anti-restriction protein, TrsAR, which facilitates higher conjugation frequencies when pUC11B is transferred into recipient strains containing Type II or Type III RM systems. These findings demonstrate how RM mechanisms can be circumvented when they act as a biological barrier for conjugation events.

Efficiency of the new reciprocating and rotary systems with or without ultrasonics in removing root-canals filling with calcium silicate-based sealer (MTA)

BackgroundTo compare the efficiency of endodontic rotary and reciprocating systems in removing calcium silicate-sealer based fillings and to investigate the impact of passive ultrasonic irrigation (PUI) on their efficiency.Materials and methods160 root-canals were instrumented, filled with gutta-percha and calcium silicate based-sealer and divided into 10 equal groups. Five groups in which the reciprocating systems (WaveOne-Gold, Reciproc-Blue and R-Motion) and rotary systems (Fanta-AF-One and Tango-Endo) were used to remove root-canals’ fillings. In the other five groups the fillings were removed by the same systems then additionally with PUI. The times to complete retreatments procedures were recorded. Micro-computed tomography’s analysis of the root-canals fillings’ volume before and after retreatments was used to determine the remaining filling materials (RFMs) volumes.ResultsThe RFMs after using rotary systems (10.1%) was greater than after using reciprocating systems (3.8%) (P < 0.001). The RFMs after using WOG (2%) and RB systems (2.6%) were less than those in the RM (6.8%), TE (9.5%) and FAFO (10.7%) systems [P < 0.05]. The times required to remove the filling materials using the TE (3.7 min), FAFO (4.1 min) and RM (4.1 min) systems were shorter than those required by the RB (5.4 min) and WOG (4.9 min) systems [P < 0.05]. Using PUI resulted in less RFMs (1.44%) when compared to using only rotary or reciprocating systems (6.27%) [P < 0.001].ConclusionsEndodontic reciprocation systems were more effective, but needed longer times than rotary systems in removing calcium silicate based- sealers fillings. The PUI significantly improved removal of the root-canals’ filling materials.Clinical relevance: Reciprocating systems and PUI are recommended whenever root-canals retreatment is considered regardless of using calcium silicate-based sealers.

Complete Genome Sequencing Analysis of Deinococcus wulumuqiensis R12, an Extremely Radiation-Resistant Strain.

Genome sequencing was performed by the PacBio RS II platform and Illumina HiSeq 4000 platform to discover the metabolic profile of the Deinococcus wulumuqiensis R12, which was isolated from radiation-contaminated soils in Xinjiang Uygur Autonomous Region of northwest China. The genome of 3.5 Mbp comprises one circular chromosome and four circular plasmids with 3679 genes and a GC content of 66.97%. A total of 41 new transcriptional factors were identified using the DeepTFactor tool. Genomic analysis revealed the presence of genes for homologous recombination repair, which suggested high recombination efficiency in R12. Three Type I and one Type II RM systems, two CRISPR arrays, and one Cas-Type IC protein were found, allowing the development of endogenous CRISPR-Cas gene-editing tools. Additionally, we found that R12 has a broad spectrum of substrate utilization, which was validated by physiological experiments. Genes involved in the carotenoid biosynthesis pathway and the antioxidative system were also identified. Overall, the comprehensive description of the genome of R12 will facilitate the additional exploitation of this strain as a versatile cell factory for biotechnological applications.

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

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.

Nutrient Loading and Viral Memory Drive Accumulation of Restriction Modification Systems in Bloom-Forming Cyanobacteria.

ABSTRACTThe mechanisms driving cyanobacterial harmful algal blooms (HABs) like those caused by Microcystis aeruginosa remain elusive, but improved defense against viral predation has been implicated for success in eutrophic environments. Our genus-level analyses of 139,023 genomes revealed that HAB-forming cyanobacteria carry vastly more restriction modification systems per genome (RMPG) than nearly all other prokaryotic genera, suggesting that viral defense is a cornerstone of their ecological success. In contrast, picocyanobacteria that numerically dominate nutrient-poor systems have the fewest RMPG within the phylum Cyanobacteria. We used classic resource competition models to explore the hypothesis that nutrient enrichments drive ecological selection for high RMPG due to increased host-phage contact rate. These classic models, agnostic to the mechanism of defense, explain how nutrient loading can select for increased RMPG but, importantly, fail to explain the extreme accumulation of these defense systems. However, extreme accumulation of RMPG can be achieved in a novel “memory” model that accounts for a unique activity of restriction modification systems: the accidental methylation of viral DNA by the methyltransferase. The methylated virus “remembers” the RM defenses of its former host and can evade these defenses if they are present in the next host. This viral memory leads to continual RM system devaluation; RMs accumulate extensively because the benefit of each addition is diminished. Our modeling leads to the hypothesis that nutrient loading and virion methylation drive the extreme accumulation of RMPG in HAB-forming cyanobacteria. Finally, our models suggest that hosts with different RMPG values can coexist when hosts have unique sets of RM systems.

Last before-death transmission in patients with heart failure and implantable cardioverter-defibrillator followed by remote monitoring - insights from the COMMIT-HF registry

Abstract Funding Acknowledgements Type of funding sources: None. Background Remote monitoring (RM) of cardiac implantable electronic devices (CIED) in patients with heart failure allows to regularly analyze the devices" and patients" conditions. Purpose The purpose of this study was evaluation of the ultimate transmissions sent before death in patients monitored remotely. Methods The last transmissions delivered by the devices in patients enrolled into COMMIT-HF Registry (NCT02536443) who died when monitored remotely have been retrospectively analysed. The characteristics and contents of the transmissions and clinical reactions undertaken have been obtained from the RM systems of four major RM providers. Results Of 1,306 patients with CIEDs who were enrolled at the RM programme in our centre, 267 died and their last transmission occurred less than 90 days before death, of which 133 (49.8%) were scheduled and 134 (50.2%) alert-triggered. The median period between transmission and death was 31 days for scheduled and 8 days for alert-triggered transmissions. The most frequent alert-triggered transmissions were atrial fibrillation/flutter (35.8%) and ventricular tachyarrhythmias (24.6%). A clinical reaction has been undertaken after 9.8% of planned and 67.1% of alert-triggered transmissions and consisted mainly of telephone consultations and referrals for hospital admissions. Conclusions This is the first analysis of the ultimate transmissions delivered by CIEDs before death. In approximately 50% of patients, the last transmission has been alert-triggered. Hence, an appropriate organization of the RM facility, which should immediately analyse and react to the transmission, seems mandatory to obtain clinical benefit in patients with HF and RM. Causes of alerts and clinical reactionsCause of alertAll alert-triggered transmissions (N = 134)AF/AFL episode, n (%)48 (35.8%)Ventricular tachycardia, n (%)18 (13.4%)Ventricular fibrillation, n (%)15 (11.2%)Biventricular pacing percentage reduction, n (%)15 (11.2%)Others38 (28.3%)Congestion monitor indications, n (%)14 (10.4%)Clinical reactionPlanned transmission (N = 133)Alert-triggered transmission (N = 134)Telephone consultation10 (7.5%)58 (43.2%)Referral to the GP or outpatient specialist clinic visit2 (1.5%)12 (8.9%)Referral for hospital admission1 (0.7%)18 (13.4%)Pharmacotherapy modificationN/A2 (1.5%)Abstract Figure.

Designing Risk Management: Applying Value Stream Mapping to Risk Management

AbstractRisk management (RM) in new product development (NPD) is often implemented as a standardized framework and ends up being carried out as a tick the box, non-value adding activity. To avoid this problem, RM needs to be tailored to the organization and NPD project. This paper identifies a gap in both understanding and facilitating tailoring, i.e. design of RM systems in NPD. To understand how to design RM systems, we must better understand how RM adds value to NPD activities. We applied Product Development Value Stream Mapping (PDVSM) to RM and conceptualized a Risk Value Stream Mapping (RVSM) framework to support design. Through a state-of-the-art literature review, we identified typical categories of value and waste in RM as well as approaches to model the RM in NPD. We developed and tested components of the RVSM framework based on PDVSM in three case companies. In this paper, results are presented regarding waste, value and potential ways to model the value stream in RM. The framework enables a diagnosis of the current state of RM in companies and supports future design activities pertaining to RM systems. This paper is positioned at the intersection of design, lean thinking and RM

Conservation agriculture based sustainable intensification: Increasing yields and water productivity for smallholders of the Eastern Gangetic Plains

In the Eastern Gangetic Plains (EGP) region of South Asia cropping systems are predominantly rice-based and comprise rice and non-rice crops (e.g. maize, wheat, lentil, mungbean, jute) in rotation. There are large gaps between potential crop yields and those achieved in farmers’ fields, primarily due to poor crop and soil management practices. Increasing climate variability and injudicious use of natural resources, particularly water and soil, mean that cropping systems have little resilience and are showing signs of being unsustainable. We hypothesized that replacing traditional crop rotations, grown under conventional tillage (CT) and with full residue removal, with crops grown under zero tillage (ZT) and with partial or full residue retention would improve both crop and cropping system yields, and reduce crop water demand, thus increasing the water productivity of cropping systems across the EGP. We evaluated four tillage-and-crop-establishment scenarios in three major cropping systems (rice-wheat, RW; rice-maize, RM; rice-lentil, RL) across eight districts of the EGP in 433 farmers’ fields between 2015 and 2017. We compared farmers’ traditional crop establishment practices (T1) against three scenarios based on the principles of conservation agriculture based sustainable intensification (CASI) and comprising a “partial CASI” option (T2: only non-rice crops grown under CASI management) and two “full CASI” options in which both crops were grown under CASI management; in T3 rice was direct seeded rice (DSR) and in T4 rice was transplanted without prior land preparation such as puddling (UPTR).Rice yields under all systems were not significantly different between CT (PTR) and CASI (ZTDSR and UTPR) but there was a significant yield gain of around 5% in both full and partial CASI on the yield of wheat and maize in the RW and RM systems. There were significant spatial trends in rice yields across all cropping systems. Total in-crop water (irrigation plus rainfall) usage was similar for the rainfed PTR, DSR and UTPR in all cropping systems as rice was primarily a rainfed crop grown during the wet season, however CASI practices reduced the required irrigation water, and thus the total in-crop water, to the dry season wheat or maize (but not to lentil) crops, compared to CT. This resulted in increased irrigation water productivity (greater than 27%) and total water productivity (greater than 26%) in both the wheat and maize crops. In the RW and RM systems irrigation water productivity was increased by over 25% and total water productivity by around 10%. These results indicate that the uptake of full or partial CASI practices, particularly in RM and RW systems, can lead to improvements in both cropping system and water productivity for smallholder farmers in the EGP.

Assessment of Risk Management Maturity of Construction Organisations in Joint Venture Projects

Abstract For organizations to be successful, they should be committed in managing risk proactively and consistently throughout the project and equally important is the determination of the Risk Management Maturity (RMM) of organizations because it is the commencing point in the review of the current RM systems, practices, and culture of construction organisations. It was reported that construction organisations lack the knowledge of their capability to manage risk and as a result, JV projects continue to fail. Studies have identified, categorised and assessed risk associated with JV projects in their local and international context but no study has assessed the RMM of construction organisations in JV projects. Therefore, this study assessed the RMM of construction organisations in JV projects. The attributes and dimensions used to assess the RMM of construction organisations were identified from literature and used in the survey to collect data from respondents and subsequently analysed using the Fuzzy synthetic evaluation technique. Findings revealed that construction organisations undertaking JV projects are at the “defined maturity level” which means that these organisations only practice informal risk management and uses only qualitative risk assessment technique. The study recommends that further studies should focus on how to improve and move from the current RMM level of construction organisations to the next maturity level (managed and optimised).

Novel m4C modification in type I restriction-modification systems.

We identify a new subgroup of Type I Restriction-Modification enzymes that modify cytosine in one DNA strand and adenine in the opposite strand for host protection. Recognition specificity has been determined for ten systems using SMRT sequencing and each recognizes a novel DNA sequence motif. Previously characterized Type I systems use two identical copies of a single methyltransferase (MTase) subunit, with one bound at each half site of the specificity (S) subunit to form the MTase. The new m4C-producing Type I systems we describe have two separate yet highly similar MTase subunits that form a heterodimeric M1M2S MTase. The MTase subunits from these systems group into two families, one of which has NPPF in the highly conserved catalytic motif IV and modifies adenine to m6A, and one having an NPPY catalytic motif IV and modifying cytosine to m4C. The high degree of similarity among their cytosine-recognizing components (MTase and S) suggest they have recently evolved, most likely from the far more common m6A Type I systems. Type I enzymes that modify cytosine exclusively were formed by replacing the adenine target recognition domain (TRD) with a cytosine-recognizing TRD. These are the first examples of m4C modification in Type I RM systems.

Effect of climate variability on pasture-based dairy feeding systems in south-east Australia

The Australian dairy industry relies primarily on pasture for its feed supply. However, the variability in climate affects plant growth, leading to uncertainty in dryland pasture supply. This paper models the impact of climate variability on pasture production and examines the potential of two pasture-based dairy feeding systems: (1) to experience winter deficits; (2) to carry forward the conserved pasture surpluses as silage for future use; and (3) to conserve pasture surpluses as hay. The two dairy feeding systems examined were a traditional perennial ryegrass-based feeding system (ryegrass max. – RM) and a system that incorporated double cropping into the perennial ryegrass pasture base (complementary forage – CF). The conditional probability of the RM and CF systems to generate pasture deficits in winter were 94% and 96%, respectively. Both systems could carry forward the surplus silage into the following lactation almost once in every 4–5 years with the RM system performing slightly better than the CF system. The proportions of the grain-based concentrates fed in the two systems were 25% and 27% for the RM and CF systems, respectively. This study suggests that double-cropping systems have the potential to provide high-quality feed to support the feed gaps when pasture is not available due to increased variability in climatic conditions.

Development of an in vivo methylation system for the solventogen Clostridium saccharobutylicum NCP 262 and analysis of two endonuclease mutants

The genome of the biotechnologically important solventogenic Clostridium saccharobutylicum NCP 262 contains two operons coding for genes of presumed type I RM systems belonging to the families A and C. They represent a limiting factor for the development of transformation and conjugation protocols. We established an efficient triparental mating system to transfer DNA to C. saccharobutylicum by conjugation, which includes an in vivo methylation of the donor DNA. Furthermore we describe increased rates of conjugation in knock-out mutants of the restrictase subunits of both RM systems.

PsasM2I, a type II restriction-modification system in Pseudomonas savastanoi pv. savastanoi: differential distribution of carrier strains in the environment and the evolutionary history of homologous RM systems in the Pseudomonas syringae complex.

A type II restriction-modification system was found in a native plasmid of Pseudomonas savastanoi pv. savastanoi MLLI2. Functional analysis of the methyltransferase showed that the enzyme acts by protecting the DNA sequence CTGCAG from cleavage. Restriction endonuclease expression in recombinant Escherichia coli cells resulted in mutations in the REase sequence or transposition of insertion sequence 1A in the coding sequence, preventing lethal gene expression. Population screening detected homologous RM systems in other P. savastanoi strains and in the Pseudomonas syringae complex. An epidemiological survey carried out by sampling olive and oleander knots in two Italian regions showed an uneven diffusion of carrier strains, whose presence could be related to a selective advantage in maintaining the RM system in particular environments or subpopulations. Moreover, carrier strains can coexist in the same orchards, plants, and knot tissues with non-carriers, revealing unexpected genetic variability on a very small spatial scale. Phylogenetic analysis of the RM system and housekeeping gene sequences in the P. syringae complex demonstrated the ancient acquisition of the RM systems. However, the evolutionary history of the gene complex also showed the involvement of horizontal gene transfer between related strains and recombination events.

Increasing home-grown forage consumption and profit in non-irrigated dairy systems. 3. Intake, milk production and composition, bodyweight and body condition score

This paper reports the performance [intake, milk production, bodyweight and body condition score (BCS)] of cows managed under two feeding systems over 4 years (June 2005–May 2009) in south-west Victoria, Australia. The feeding systems were ‘Ryegrass Max’ (RM) a well managed perennial ryegrass pasture-based system stocked at 2.2 cows/ha, and a ‘Complementary Forage’ (CF) system based on perennial ryegrass, tall fescue and a double crop of winter-sown cereal and summer-sown brassica crops stocked at 2.82 cows/ha. There were no significant differences in milk production per lactation between feeding systems, with an average of 543 and 553 kg milk solids per cow (1246 and 1642 kg milk solids per ha) produced in the RM and CF systems, respectively. These production levels were 20.9 and 10.8% above pre-experimental model targets, respectively. The bodyweight and BCS of multiparous cows were not significantly different between the two feeding systems; however, bodyweight of heifers in early lactation declined more rapidly in the CF compared with RM feeding system. Over the 4-year study in the RM system pasture comprised 60% of total DM consumed (grazed + conserved), with 26% of the ration being concentrate and the balance being purchased hay supplements. In the CF system, home-grown forage contributed 57.9% of total DM consumed of which 46.6% was pasture (grazed + conserved) and 11.3% was from the double cropping system. These levels of home-grown forage consumption were lower than those predicted by the pre-experimental modelling, leading to higher than predicted levels of purchased concentrates and conserved forages being fed. The lower than expected levels of consumption of the forages produced in the double cropping system reflected both the low intake potential and moderate nutritive value of cereal silage, and the failure of establishment and subsequent low DM yields from the summer brassica crop. Further work is required to determine the risk of implementing CF in rain-fed dairy systems reflecting uncertainties in forage management planning and nutrient delivery to support high levels of milk production.

Increasing home-grown forage consumption and profit in non-irrigated dairy systems. 1. Rationale, systems design and management

The profitability of dairy businesses in southern Australia is closely related to the amount of feed consumed from perennial ryegrass-dominant pasture. Historically, the dairy industry has relied on improvements in pasture productivity and utilisation to support profitable increases in stocking rate and milk production per hectare. However, doubts surround the extent to which the industry can continue to rely on perennial ryegrass technology to provide the necessary productivity improvements required into the future. This paper describes the design and management of a dairy systems experiment at Terang in south-west Victoria (780-mm average annual rainfall) conducted over four lactations (June 2005–March 2009) to compare the production and profitability of two forage base options for non-irrigated dairy farms. These options were represented by two self-contained farmlets each milking 36 mixed-age, autumn-calving Holstein-Friesian cows at peak: (1) well managed perennial ryegrass pasture (‘Ryegrass Max’, or ‘RM’); and (2) perennial ryegrass plus complementary forages (‘CF’) including 15% of farmlet area under double cropping with annual species (winter cereal grown for silage followed by summer brassica for grazing on the same land) and an average of 25% of farmlet area in perennial pasture based on tall fescue for improved late spring–early summer feed supply. The design of these systems was informed by farming systems models (DairyMod, UDDER and Redsky), which were used to estimate the effects of introducing different forage options on farm profitability. The design of the CF system was selected based on modelled profitability increases assuming that all forage components could be managed to optimise forage production and be effectively integrated to optimise milk production per cow. Using the historical ‘average’ pasture growth curve for the Terang district and a mean milk price of $3.71 per kg milk solids, the models estimated that the return on assets of the RM and CF systems would be 9.4 and 15.0%, respectively. The objectives of the experiment described here were to test whether or not such differences in profitability could be achieved in practice, and to determine the risks associated with including complementary forages on a substantial proportion of the effective farm area. Key results of the experiment are presented in subsequent papers.

To be or not to be: regulation of restriction–modification systems and other toxin–antitoxin systems

One of the simplest classes of genes involved in programmed death is that containing the toxin–antitoxin (TA) systems of prokaryotes. These systems are composed of an intracellular toxin and an antitoxin that neutralizes its effect. These systems, now classified into five types, were initially discovered because some of them allow the stable maintenance of mobile genetic elements in a microbial population through postsegregational killing or the death of cells that have lost these systems. Here, we demonstrate parallels between some TA systems and restriction–modification systems (RM systems). RM systems are composed of a restriction enzyme (toxin) and a modification enzyme (antitoxin) and limit the genetic flux between lineages with different epigenetic identities, as defined by sequence-specific DNA methylation. The similarities between these systems include their postsegregational killing and their effects on global gene expression. Both require the finely regulated expression of a toxin and antitoxin. The antitoxin (modification enzyme) or linked protein may act as a transcriptional regulator. A regulatory antisense RNA recently identified in an RM system can be compared with those RNAs in TA systems. This review is intended to generalize the concept of TA systems in studies of stress responses, programmed death, genetic conflict and epigenetics.

Comprehensive analysis of the HEPN superfamily: identification of novel roles in intra-genomic conflicts, defense, pathogenesis and RNA processing

BackgroundThe major role of enzymatic toxins that target nucleic acids in biological conflicts at all levels has become increasingly apparent thanks in large part to the advances of comparative genomics. Typically, toxins evolve rapidly hampering the identification of these proteins by sequence analysis. Here we analyze an unexpectedly widespread superfamily of toxin domains most of which possess RNase activity.ResultsThe HEPN superfamily is comprised of all α-helical domains that were first identified as being associated with DNA polymerase β-type nucleotidyltransferases in prokaryotes and animal Sacsin proteins. Using sensitive sequence and structure comparison methods, we vastly extend the HEPN superfamily by identifying numerous novel families and by detecting diverged HEPN domains in several known protein families. The new HEPN families include the RNase LS and LsoA catalytic domains, KEN domains (e.g. RNaseL and Ire1) and the RNase domains of RloC and PrrC. The majority of HEPN domains contain conserved motifs that constitute a metal-independent endoRNase active site. Some HEPN domains lacking this motif probably function as non-catalytic RNA-binding domains, such as in the case of the mannitol repressor MtlR. Our analysis shows that HEPN domains function as toxins that are shared by numerous systems implicated in intra-genomic, inter-genomic and intra-organismal conflicts across the three domains of cellular life. In prokaryotes HEPN domains are essential components of numerous toxin-antitoxin (TA) and abortive infection (Abi) systems and in addition are tightly associated with many restriction-modification (R-M) and CRISPR-Cas systems, and occasionally with other defense systems such as Pgl and Ter. We present evidence of multiple modes of action of HEPN domains in these systems, which include direct attack on viral RNAs (e.g. LsoA and RNase LS) in conjunction with other RNase domains (e.g. a novel RNase H fold domain, NamA), suicidal or dormancy-inducing attack on self RNAs (RM systems and possibly CRISPR-Cas systems), and suicidal attack coupled with direct interaction with phage components (Abi systems). These findings are compatible with the hypothesis on coupling of pathogen-targeting (immunity) and self-directed (programmed cell death and dormancy induction) responses in the evolution of robust antiviral strategies. We propose that altruistic cell suicide mediated by HEPN domains and other functionally similar RNases was essential for the evolution of kin and group selection and cell cooperation. HEPN domains were repeatedly acquired by eukaryotes and incorporated into several core functions such as endonucleolytic processing of the 5.8S-25S/28S rRNA precursor (Las1), a novel ER membrane-associated RNA degradation system (C6orf70), sensing of unprocessed transcripts at the nuclear periphery (Swt1). Multiple lines of evidence suggest that, similar to prokaryotes, HEPN proteins were recruited to antiviral, antitransposon, apoptotic systems or RNA-level response to unfolded proteins (Sacsin and KEN domains) in several groups of eukaryotes.ConclusionsExtensive sequence and structure comparisons reveal unexpectedly broad presence of the HEPN domain in an enormous variety of defense and stress response systems across the tree of life. In addition, HEPN domains have been recruited to perform essential functions, in particular in eukaryotic rRNA processing. These findings are expected to stimulate experiments that could shed light on diverse cellular processes across the three domains of life.ReviewersThis article was reviewed by Martijn Huynen, Igor Zhulin and Nick Grishin

Insight into the specific virulence related genes and toxin-antitoxin virulent pathogenicity islands in swine streptococcosis pathogen Streptococcus equi ssp. zooepidemicus strain ATCC35246

BackgroundStreptococcus equi ssp. zooepidemicus (S. zooepidemicus) is an important pathogen causing swine streptococcosis in China. Pathogenicity islands (PAIs) of S. zooepidemicus have been transferred among bacteria through horizontal gene transfer (HGT) and play important roles in the adaptation and increased virulence of S. zooepidemicus. The present study used comparative genomics to examine the different pathogenicities of S. zooepidemicus.ResultsGenome of S. zooepidemicus ATCC35246 (Sz35246) comprises 2,167,264-bp of a single circular chromosome, with a GC content of 41.65%. Comparative genome analysis of Sz35246, S. zooepidemicus MGCS10565 (Sz10565), Streptococcus equi. ssp. equi. 4047 (Se4047) and S. zooepidemicus H70 (Sz70) identified 320 Sz35246-specific genes, clustered into three toxin-antitoxin (TA) systems PAIs and one restriction modification system (RM system) PAI. These four acquired PAIs encode proteins that may contribute to the overall pathogenic capacity and fitness of this bacterium to adapt to different hosts. Analysis of the in vivo and in vitro transcriptomes of this bacterium revealed differentially expressed PAI genes and non-PAI genes, suggesting that Sz35246 possess mechanisms for infecting animals and adapting to a wide range of host environments. Analysis of the genome identified potential Sz35246 virulence genes. Genes of the Fim III operon were presumed to be involved in breaking the host-restriction of Sz35246.ConclusionGenome wide comparisons of Sz35246 with three other strains and transcriptome analysis revealed novel genes related to bacterial virulence and breaking the host-restriction. Four specific PAIs, which were judged to have been transferred into Sz35246 genome through HGT, were identified for the first time. Further analysis of the TA and RM systems in the PAIs will improve our understanding of the pathogenicity of this bacterium and could lead to the development of diagnostics and vaccines.

Genome-wide mapping of methylated adenine residues in pathogenic Escherichia coli using single-molecule real-time sequencing

Single-molecule real-time (SMRT) DNA sequencing allows the systematic detection of chemical modifications such as methylation but has not previously been applied on a genome-wide scale. We used this approach to detect 49,311 putative 6-methyladenine (m6A) residues and 1,407 putative 5-methylcytosine (m5C) residues in the genome of a pathogenic Escherichia coli strain. We obtained strand-specific information for methylation sites and a quantitative assessment of the frequency of methylation at each modified position. We deduced the sequence motifs recognized by the methyltransferase enzymes present in this strain without prior knowledge of their specificity. Furthermore, we found that deletion of a phage-encoded methyltransferase-endonuclease (restriction-modification; RM) system induced global transcriptional changes and led to gene amplification, suggesting that the role of RM systems extends beyond protecting host genomes from foreign DNA.