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DNA phosphorothioate (PT) modifications, with the nonbridging phosphate oxygen replaced by sulfur, governed by DndABCDE or SspABCD, are widely distributed in prokaryotes and have a highly unusual feature of occupying only a small portion of available consensus sequences in a genome. Despite the presence of plentiful non-PT-protected consensuses, DNA PT modification is still employed as a recognition tag by the restriction cognate, for example, DndFGH or SspE, to discriminate and destroy PT-lacking foreign DNA. This raises a fundamental question about how PT modifications are distributed along DNA molecules to keep the restriction components in check. Here, we present two single-molecule strategies that take advantage of the nucleophilicity of PT in combination with fluorescent markers for optical mapping of both single- and double-stranded PT modifications across individual DNA molecules. Surprisingly, PT profiles vary markedly from molecule to molecule, with different PT locations and spacing distances between PT pairs, even in the presence of DndFGH or SspE. The results revealed unprecedented PT modification features previously obscured by ensemble averaging, providing novel insights into the riddles regarding unusual target selection by PT modification and restriction components.

Phosphorothioate (PT) DNA modifications—in which a nonbonding phosphate oxygen is replaced with sulfur—represent a widespread, horizontally transferred epigenetic system in prokaryotes and have a highly unusual property of occupying only a small fraction of available consensus sequences in a genome. Using Salmonella enterica as a model, we asked a question of fundamental importance: How do the PT-modifying DndA-E proteins select their G PS AAC/G PS TTC targets? Here, we applied innovative analytical, sequencing, and computational tools to discover a novel behavior for DNA-binding proteins: The Dnd proteins are “parked” at the G 6m ATC Dam methyltransferase consensus sequence instead of the expected GAAC/GTTC motif, with removal of the 6m A permitting extensive PT modification of GATC sites. This shift in modification sites further revealed a surprising constancy in the density of PT modifications across the genome. Computational analysis showed that GAAC, GTTC, and GATC share common features of DNA shape, which suggests that PT epigenetics are regulated in a density-dependent manner partly by DNA shape-driven target selection in the genome.

DNA damage and epigenetic marks are well established to have profound influences on genome stability and cell phenotype, yet there are few technologies to obtain high-resolution genomic maps of the many types of chemical modifications of DNA. Here we present Nick-seq for quantitative, sensitive, and accurate mapping of DNA modifications at single-nucleotide resolution across genomes. Pre-existing breaks are first blocked and DNA modifications are then converted enzymatically or chemically to strand-breaks for both 3′-extension by nick-translation to produce nuclease-resistant oligonucleotides and 3′-terminal transferase tailing. Following library preparation and next generation sequencing, the complementary datasets are mined with a custom workflow to increase sensitivity, specificity and accuracy of the map. The utility of Nick-seq is demonstrated with genomic maps of site-specific endonuclease strand-breaks in purified DNA from Eschericia coli, phosphorothioate epigenetics in Salmonella enterica Cerro 87, and oxidation-induced abasic sites in DNA from E. coli treated with a sublethal dose of hydrogen peroxide. Nick-seq applicability is demonstrated with strategies for >25 types of DNA modification and damage.

Phages are regarded as powerful antagonists of bacteria, especially in industrial fermentation processes involving bacteria. While bacteria have developed various defense mechanisms, most of which are effective against a narrow range of phages and consequently exert limited protection from phage infection. Here, we report a strategy for developing phage-resistant Escherichia coli strains through the simultaneous genomic integration of a DNA phosphorothioation-based Ssp defense module and mutations of components essential for the phage life cycle. The engineered E. coli strains show strong resistance against diverse phages tested without affecting cell growth. Additionally, the resultant engineered phage-resistant strains maintain the capabilities of producing example recombinant proteins, D-amino acid oxidase and coronavirus-encoded nonstructural protein nsp8, even under high levels of phage cocktail challenge. The strategy reported here will be useful for developing engineered E. coli strains with improved phage resistance for various industrial fermentation processes for producing recombinant proteins and chemicals of interest.

Sepsis, a widely recognized disease, is characterized by multiple pathogen infections. Therefore, it is imperative to develop methods that can efficiently identify and neutralize pathogen species. Phage cocktail therapy utilizes the host specificity of phages to adapt to infect resistant bacteria. However, its low sterilization stability efficiency and lack of imaging units seriously restrict its application. Here, a novel strategy combining the aggregation-induced emission photosensitizer (AIE-PS) TBTCP-PMB with phages through a nucleophilic substitution reaction between benzyl bromide and sulfhydryl groups to remove pathogenic bacteria for sepsis treatment is proposed. This strategy retains the phage's host specificity while possessing AIE-PS characteristics with a fluorescence imaging function and reactive oxygen species (ROS) for detecting and sterilizing bacteria. This synergetic strategy combining phage cocktail therapy and photodynamic therapy (PDT) shows a strong "1 + 1 2" bactericidal efficacy and superior performance in sepsis mouse models with good biocompatibility. Furthermore, the strategy can quickly diagnose blood infections of clinical blood samples. This simple and accurate strategy provides a promising therapeutic platform for rapid pathogen detection and point-of-care diagnosis. Moreover, it presents a new method for expanding the library of antibacterial drugs to develop new strain identification and improve infectious disease treatment, thereby demonstrating strong translational potential.

We recently found that SspABCD, catalyzing single-stranded (ss) DNA phosphorothioate (PT) modification, coupled with SspE provides protection against phage infection. SspE performs both PT-simulated NTPase and DNA-nicking nuclease activities to damage phage DNA, rendering SspA-E a PT-sensing defense system., Unlike nucleobase modifications in canonical restriction-modification systems, DNA phosphorothioate (PT) epigenetic modification occurs in the DNA sugar-phosphate backbone when the nonbridging oxygen is replaced by sulfur in a double-stranded (ds) or single-stranded (ss) manner governed by DndABCDE or SspABCD, respectively. SspABCD coupled with SspE constitutes a defense barrier in which SspE depends on sequence-specific PT modifications to exert its antiphage activity. Here, we identified a new type of ssDNA PT-based SspABCD-SspFGH defense system capable of providing protection against phages through a mode of action different from that of SspABCD-SspE. We provide further evidence that SspFGH damages non-PT-modified DNA and exerts antiphage activity by suppressing phage DNA replication. Despite their different defense mechanisms, SspFGH and SspE are compatible and pair simultaneously with one SspABCD module, greatly enhancing the protection against phages. Together with the observation that the sspBCD-sspFGH cassette is widely distributed in bacterial genomes, this study highlights the diversity of PT-based defense barriers and expands our knowledge of the arsenal of phage defense mechanisms.

COVID‐19 pandemic, caused by severe acute respiratory syndrome coronavirus 2, has resulted in global social and economic disruption, putting the world economy to the largest global recession since the Great Depression. To control the spread of COVID‐19, cutting off the transmission route is a critical step. In this work, the efficient inactivation of human coronavirus with photodynamic therapy (PDT) by employing photosensitizers with aggregation‐induced emission characteristics (DTTPB) is reported. DTTPB is designed to bear a hydrophilic head and two hydrophobic tails, mimicking the structure of phospholipids on biological membranes. DTTPB demonstrates a broad absorption band covering the whole visible light range and high molar absorptivity, as well as excellent reactive oxygen species sensitizing ability, making it an excellent candidate for PDT. Besides, DTTPB can target membrane structure, and bind to the envelope of human coronaviruses. Upon light irradiation, DTTPB demonstrates highly effective antiviral behavior: human coronavirus treated with DTTPB and white‐light irradiation can be efficiently inactivated with complete loss of infectivity, as revealed by the significant decrease of virus RNA and proteins in host cells. Thus, DTTPB sensitized PDT can efficiently prevent the infection and the spread of human coronavirus, which provides a new avenue for photodynamic combating of COVID‐19., In this work, we report the application of a novel photosensitizer, DTTPB, with aggregation‐induced emission characteristics for photodynamic inactivation of human coronaviruses. With high molar absorbance coefficient, broad absorption band covering the whole white light region, and high membrane targeting and ROS sensitizing efficiency, DTTPB could effectively inactivate human coronaviruses and may contribute to preventing the spread of COVID‐19 pandemic.

Ongoing studies have determined that the gut microbiota is a major factor influencing both health and disease. Host genetic factors and environmental factors contribute to differences in gut microbiota composition and function. Intestinal dysbiosis is a cause or a contributory cause for diseases in multiple body systems, ranging from the digestive system to the immune, cardiovascular, respiratory, and even nervous system. Investigation of pathogenesis has identified specific species or strains, bacterial genes, and metabolites that play roles in certain diseases and represent potential drug targets. As research progresses, gut microbiome-based diagnosis and therapy are proposed and applied, which might lead to considerable progress in precision medicine. We further discuss the limitations of current studies and potential solutions.

Archaea and Bacteria have evolved different defence strategies that target virtually all steps of the viral life cycle. The diversified virion morphotypes and genome contents of archaeal viruses result in a highly complex array of archaea-virus interactions. However, our understanding of archaeal antiviral activities lags far behind our knowledges of those in bacteria. Here we report a new archaeal defence system that involves DndCDEA-specific DNA phosphorothioate (PT) modification and the PbeABCD-mediated halt of virus propagation via inhibition of DNA replication. In contrast to the breakage of invasive DNA by DndFGH in bacteria, DndCDEA-PbeABCD does not degrade or cleave viral DNA. The PbeABCD-mediated PT defence system is widespread and exhibits extensive interdomain and intradomain gene transfer events. Our results suggest that DndCDEA-PbeABCD is a new type of PT-based virus resistance system, expanding the known arsenal of defence systems as well as our understanding of host-virus interactions., The prokaryote defence system Dnd relies on phosphorothioation (PT) of DNA backbone to distinguish between self and non-self DNA. Here, Xiong et al. describe a previously uncharacterized PT-based antiviral system that is independent of the canonical Dnd and is widespread in Archaea and Bacteria.

Engineering and modifying synthetic microbial chassis is one of the best ways not only to unravel the fundamental principles of life but also to enhance applications in the health, medicine, agricultural, veterinary, and food industries. The two primary strategies for constructing a microbial chassis are the top-down approach (genome reduction) and the bottom-up approach (genome synthesis). Research programs on this topic have been funded in several countries. The ‘Minimum genome factory’ (MGF) project was launched in 2001 in Japan with the goal of constructing microorganisms with smaller genomes for industrial use. One of the best examples of the results of this project is E. coli MGF-01, which has a reduced-genome size and exhibits better growth and higher threonine production characteristics than the parental strain [1]. The ‘cell factory’ project was carried out from 1998 to 2002 in the Fifth Framework Program of the EU (European Union), which tried to comprehensively understand microorganisms used in the application field. One of the outstanding results of this project was the elucidation of proteins secreted by Bacillus subtilis, which was summarized as the ‘secretome’ [2]. The GTL (Genomes to Life) program began in 2002 in the United States. In this program, researchers aimed to create artificial cells both in silico and in vitro, such as the successful design and synthesis of a minimal bacterial genome by John Craig Venter's group [3]. This review provides an update on recent advances in engineering, modification and application of synthetic microbial chassis, with particular emphasis on the value of learning about chassis as a way to better understand life and improve applications. Keywords: Microbial chassis, Systems biology, Synthetic biology

Arsenic, distributed pervasively in the natural environment, is an extremely toxic substance which can severely impair the normal functions of living cells. Research on the genetic mechanisms of arsenic metabolism is of great importance for remediating arsenic-contaminated environments. Many organisms, including bacteria, have developed various strategies to tolerate arsenic, by either detoxifying this harmful element or utilizing it for energy generation. This review summarizes arsenic detoxification as well as arsenic respiratory metabolic pathways in bacteria and discusses novel arsenic resistance pathways in various bacterial strains. This knowledge provides insights into the mechanisms of arsenic biotransformation in bacteria. Multiple detoxification strategies among bacteria imply possible functional relationships among different arsenic detoxification/metabolism pathways. In addition, this review sheds light on the bioremediation of arsenic-contaminated environments and prevention of antibiotic resistance.

Synthetic phosphorothioate (PT) internucleotide linkages, in which a nonbridging oxygen is replaced by a sulphur atom, share similar physical and chemical properties with phosphodiesters but confer enhanced nuclease tolerance on DNA/RNA, making PTs a valuable biochemical and pharmacological tool. Interestingly, PT modification was recently found to occur naturally in bacteria in a sequence-selective and RP configuration-specific manner. This oxygen–sulphur swap is catalysed by the gene products of dndABCDE, which constitute a defence barrier with DndFGH in some bacterial strains that can distinguish and attack non-PT-modified foreign DNA, resembling DNA methylation-based restriction-modification (R-M) systems. Despite their similar defensive mechanisms, PT- and methylation-based R-M systems have evolved to target different consensus contexts in the host cell because when they share the same recognition sequences, the protective function of each can be impeded. The redox and nucleophilic properties of PT sulphur render PT modification a versatile player in the maintenance of cellular redox homeostasis, epigenetic regulation and environmental fitness. The widespread presence of dnd systems is considered a consequence of extensive horizontal gene transfer, whereas the lability of PT during oxidative stress and the susceptibility of PT to PT-dependent endonucleases provide possible explanations for the ubiquitous but sporadic distribution of PT modification in the bacterial world., DNA phosphorothioation is a sulphur-containing physiological modification on the DNA backbone with multiple functions.

Apart from its roles in Fe-S cluster assembly, tRNA thiolation, and sulfur-containing cofactor biosynthesis, cysteine desulfurase serves as a sulfur donor in the DNA PT modification, in which a sulfur atom substitutes a nonbridging oxygen in the DNA phosphodiester backbone. The initial sulfur mobilization from l-cysteine is catalyzed by the SspA cysteine desulfurase in the SspABCD-mediated DNA PT modification system. By determining the crystal structure of SspA, the study presents the molecular mechanism that SspA employs to recognize its cysteine substrate and PLP cofactor. To overcome the long distance (8.9 Å) between the catalytic Cys314 and the cysteine substrate, a conformational change occurs to bring Cys314 to the vicinity of the substrate, allowing for nucleophilic attack., DNA phosphorothioate (PT) modification, in which the nonbridging oxygen in the sugar-phosphate backbone is substituted by sulfur, is catalyzed by DndABCDE or SspABCD in a double-stranded or single-stranded manner, respectively. In Dnd and Ssp systems, mobilization of sulfur in PT formation starts with the activation of the sulfur atom of cysteine catalyzed by the DndA and SspA cysteine desulfurases, respectively. Despite playing the same biochemical role, SspA cannot be functionally replaced by DndA, indicating its unique physiological properties. In this study, we solved the crystal structure of Vibrio cyclitrophicus SspA in complex with its natural substrate, cysteine, and cofactor, pyridoxal phosphate (PLP), at a resolution of 1.80 Å. Our solved structure revealed the molecular mechanism that SspA employs to recognize its cysteine substrate and PLP cofactor, suggesting a common binding mode shared by cysteine desulfurases. In addition, although the distance between the catalytic Cys314 and the substrate cysteine is 8.9 Å, which is too far for direct interaction, our structural modeling and biochemical analysis revealed a conformational change in the active site region toward the cysteine substrate to move them close to each other to facilitate the nucleophilic attack. Finally, the pulldown analysis showed that SspA could form a complex with SspD, an ATP pyrophosphatase, suggesting that SspD might potentially accept the activated sulfur atom directly from SspA, providing further insights into the biochemical pathway of Ssp-mediated PT modification.

Glioblastoma is the most aggressive primary brain tumor in adults. The prognosis of patients with primary glioblastoma treated with the current standard of care, tumor resection followed by radiation therapy and auxiliary temozolomide, remains poor. Integrative genomic analyses have identified essential core signaling pathways and frequent genetic aberrations, which provide potential drug targets for glioblastoma treatment. Drugs against these therapeutic targets have been developed rapidly in recent years. Although some have shown promising effects on models in preclinical studies, many have shown only modest efficacy in clinical trials. New therapeutic strategies and potent drugs are urgently needed to improve the prognosis of patients with glioblastoma. The goal of this review is to summarize the current advances in drug development for targeted glioblastoma therapies and to reveal the major challenges encountered in clinical trials or treatment. This study will provide new perspectives for future studies of targeted therapeutic drug development and provide insights into the clinical treatment of glioblastoma.

Circulating tumor cells (CTCs) are cancer cells that shed from a primary tumor and circulate in the bloodstream. As a form of "tumor liquid biopsy", CTCs provide important information for the mechanistic investigation of cancer metastasis and the measurement of tumor genotype evolution during treatment and disease progression. However, the extremely low abundance of CTCs in the peripheral blood and the heterogeneity of CTCs make their isolation and characterization major technological challenges. Recently, nanotechnologies have been developed for sensitive CTC detection; such technologies will enable better cell and molecular characterization and open up a wide range of clinical applications, including early disease detection and evaluation of treatment response and disease progression. In this review, we summarize the nanotechnology-based strategies for CTC isolation, including representative nanomaterials (such as magnetic nanoparticles, gold nanoparticles, silicon nanopillars, nanowires, nanopillars, carbon nanotubes, dendrimers, quantum dots, and graphene oxide) and microfluidic chip technologies that incorporate nanoroughened surfaces and discuss their key challenges and perspectives in CTC downstream analyses, such as protein expression and genetic mutations that may reflect tumor aggressiveness and patient outcome.

Explosive growth in the study of microbial epigenetics has revealed a diversity of chemical structures and biological functions of DNA modifications in restriction-modification (R-M) and basic genetic processes. Here, we describe the discovery of shared consensus sequences for two seemingly unrelated DNA modification systems, 6mA methylation and phosphorothioation (PT), in which sulfur replaces a nonbridging oxygen in the DNA backbone. Mass spectrometric analysis of DNA from Escherichia coli B7A and Salmonella enterica serovar Cerro 87, strains possessing PT-based R-M genes, revealed d(GPS6mA) dinucleotides in the GPS6mAAC consensus representing ∼5% of the 1,100 to 1,300 PT-modified d(GPSA) motifs per genome, with 6mA arising from a yet-to-be-identified methyltransferase. To further explore PT and 6mA in another consensus sequence, GPS6mATC, we engineered a strain of E. coli HST04 to express Dnd genes from Hahella chejuensis KCTC2396 (PT in GPSATC) and Dam methyltransferase from E. coli DH10B (6mA in G6mATC). Based on this model, in vitro studies revealed reduced Dam activity in GPSATC-containing oligonucleotides whereas single-molecule real-time sequencing of HST04 DNA revealed 6mA in all 2,058 GPSATC sites (5% of 37,698 total GATC sites). This model system also revealed temperature-sensitive restriction by DndFGH in KCTC2396 and B7A, which was exploited to discover that 6mA can substitute for PT to confer resistance to restriction by the DndFGH system. These results point to complex but unappreciated interactions between DNA modification systems and raise the possibility of coevolution of interacting systems to facilitate the function of each.

Antibiotic production is often governed by large gene clusters composed of genes related to antibiotic scaffold synthesis, tailoring, regulation, and resistance. With the expansion of genome sequencing, a considerable number of antibiotic gene clusters has been isolated and characterized. The emerging genome engineering techniques make it possible towards more efficient engineering of antibiotics. In addition to genomic editing, multiple synthetic biology approaches have been developed for the exploration and improvement of antibiotic natural products. Here, we review the progress in the development of these genome editing techniques used to engineer new antibiotics, focusing on three aspects of genome engineering: direct cloning of large genomic fragments, genome engineering of gene clusters, and regulation of gene cluster expression. This review will not only summarize the current uses of genomic engineering techniques for cloning and assembly of antibiotic gene clusters or for altering antibiotic synthetic pathways but will also provide perspectives on the future directions of rebuilding biological systems for the design of novel antibiotics.

Fluorescence imaging, as a commonly used scientific tool, is widely applied in various biomedical and material structures through visualization technology. Highly selective and sensitive luminescent biological probes, as well as those with good water solubility, are urgently needed for biomedical research. In contrast to the traditional aggregation-caused quenching of fluorescence, in the unique phenomenon of aggregation-induced emission (AIE), the individual luminogens have extremely weak or no emissivity because they each have free intramolecular motion; however, when they form aggregates, these components immediately "light up". Since the discovery of "turn-on" mechanism, researchers have been studying and applying AIE in a variety of fields to develop more sensitive, selective, and efficient strategies for the AIE dyes. There are numerous advantages to the use of AIE-based methods, including low background interference, strong contrast, high performance in intracellular imaging, and the ability for long-term monitoring in vivo. In this review, two typical examples of AIEgens, TPE-Cy and TPE-Ph-In, are described, including their structure properties and applications. Recent progress in the biological applications is mainly focused on. Undoubtedly, in the near future, an increasing number of encouraging and practical ideas will promote the development of more AIEgens for broad use in biomedical applications.

Bacteria have evolved diverse mechanisms to fend off predation by bacteriophages. We previously identified the Dnd system, which uses DndABCDE to insert sulfur into the DNA backbone as a double-stranded phosphorothioate (PT) modification, and DndFGH, a restriction component. Here, we describe an unusual SspABCD-SspE PT system in Vibrio cyclitrophicus, Escherichia coli and Streptomyces yokosukanensis, which has distinct genetic organization, biochemical functions and phenotypic behaviour. SspABCD confers single-stranded and high-frequency PTs with SspB acting as a nickase and possibly introducing nicks to facilitate sulfur incorporation. Strikingly, SspABCD coupled with SspE provides protection against phages in unusual ways: (1) SspE senses sequence-specific PTs by virtue of its PT-stimulated NTPase activity to exert its anti-phage activity, and (2) SspE inhibits phage propagation by introducing nicking damage to impair phage DNA replication. These results not only expand our knowledge about the diversity and functions of DNA PT modification but also enhance our understanding of the known arsenal of defence systems.

Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems, especially type II (Cas9) systems, have been widely used in gene/genome targeting. Modifications of Cas9 enable these systems to become platforms for precise DNA manipulations. However, the utilization of CRISPR-Cas systems in RNA targeting remains preliminary. The discovery of type VI CRISPR-Cas systems (Cas13) shed light on RNA-guided RNA targeting. Cas13d, the smallest Cas13 protein, with a length of only ~930 amino acids, is a promising platform for RNA targeting compatible with viral delivery systems. Much effort has also been made to develop Cas9, Cas13a and Cas13b applications for RNA-guided RNA targeting. The discovery of new RNA-targeting CRISPR-Cas systems as well as the development of RNA-targeting platforms with Cas9 and Cas13 will promote RNA-targeting technology substantially. Here, we review new advances in RNA-targeting CRISPR-Cas systems as well as advances in applications of these systems in RNA targeting, tracking and editing. We also compare these Cas protein-based technologies with traditional technologies for RNA targeting, tracking and editing. Finally, we discuss remaining questions and prospects for the future.

DNA phosphorothioate (PT) modification was recently identified to occur naturally in diverse bacteria and to be governed by DndABCDE proteins. The nuclease resistance as well as the redox and nucleophilic properties of PT sulfur make PT modification a versatile player in restriction-modification (R-M) defense, epigenetic regulation, environmental fitness and the maintenance of cellular redox homeostasis. In this study, we discovered that tight control of PT levels is mediated by the ATPase activity of DndB. The ATP-binding activity of DndB stimulates the dissociation of the DndB-DNA complex, allowing transcriptional initiation, whereas its ATP hydrolysis activity promotes the conversion of DndB-ATP to free DndB that is capable of rebinding to promoter DNA for transcriptional inhibition. Since sulfur incorporation is an ATP-consuming process, these activities provide an economical way to fine-tune PT modification in an ATP-sensing manner. To our knowledge, this ATP-mediated regulation is a rare example among DNA epigenetic modification systems; the features of autoregulation and the repeated usage of DndB allow the dedicated regulation of PT levels in response to cellular ATP concentrations, providing insight into PT function and its role in physiology.

Phosphorothioate (PT) modification by the dnd gene cluster is the first identified DNA backbone modification and constitute an epigenetic system with multiple functions, including antioxidant ability, restriction modification, and virus resistance. Despite these advantages for hosting dnd systems, they are surprisingly distributed sporadically among contemporary prokaryotic genomes. To address this ecological paradox, we systematically investigate the occurrence and phylogeny of dnd systems, and they are suggested to have originated in ancient Cyanobacteria after the Great Oxygenation Event. Interestingly, the occurrence of dnd systems and prophages is significantly negatively correlated. Further, we experimentally confirm that PT modification activates the filamentous phage SW1 by altering the binding affinity of repressor and the transcription level of its encoding gene. Competition assays, concurrent epigenomic and transcriptomic sequencing subsequently show that PT modification affects the expression of a variety of metabolic genes, which reduces the competitive fitness of the marine bacterium Shewanella piezotolerans WP3. Our findings strongly suggest that a series of negative effects on microorganisms caused by dnd systems limit horizontal gene transfer, thus leading to their sporadic distribution. Overall, our study reveals putative evolutionary scenario of the dnd system and provides novel insights into the physiological and ecological influences of PT modification., Phosphorothioate (PT) modification by the dnd gene cluster is the first identified DNA backbone modification and has been shown to constitute a multifunctional epigenetic system. Despite a variety of advantages for hosting dnd systems, these systems are surprisingly distributed sporadically among contemporary microbial genomes. To address this ecological paradox, Jian et al. systematically investigated the occurrence and phylogeny of dnd systems in prokaryotes, and provided evidence to suggest that dnd systems have originated in ancient Cyanobacteria (probably Nostocales) after the Great Oxygenation Event.

Since the original report that Halomonas sp. strain GFAJ-1 was capable of using arsenic instead of phosphorus to sustain growth, additional studies have been conducted, and GFAJ-1 is now considered a highly arsenic-resistant but phosphorus-dependent bacterium. However, the mechanisms supporting the extreme arsenic resistance of the GFAJ-1 strain remain unknown. In this study, we show that GFAJ-1 has multiple distinct arsenic resistance mechanisms. It lacks the genes to reduce arsenate, which is the essential step in the well-characterized resistance mechanism of arsenate reduction coupled to arsenite extrusion. Instead, GFAJ-1 has two arsenic resistance operons, arsH1 - acr3 - 2 - arsH2 and mfs1 - mfs2 - gapdh , enabling tolerance to high levels of arsenate. mfs2 and gapdh encode proteins homologous to Pseudomonas aeruginosa ArsJ and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively, which constitute the equivalent of an As(V) efflux system to catalyze the transformation of inorganic arsenate to pentavalent organoarsenical 1-arseno-3-phosphoglycerate and its subsequent extrusion. Surprisingly, the arsH1 - acr3 - 2 - arsH2 operon seems to consist of typical arsenite resistance genes, but this operon is sufficient to confer both arsenite and arsenate resistance on Escherichia coli AW3110 even in the absence of arsenate reductase, suggesting a novel pathway of arsenic detoxification. The simultaneous occurrence of these two unusual detoxification mechanisms enables the adaptation of strain GFAJ-1 to the particularly arsenic-rich environment of Mono Lake. IMPORTANCE Halomonas sp. strain GFAJ-1 was previously reported to use arsenic as a substitute for phosphorus to sustain life under phosphate-limited conditions. Although this claim was later undermined by several groups, how GFAJ-1 can thrive in environments with high arsenic concentrations remains unclear. Here, we determined that this ability can be attributed to the possession of two arsenic detoxification operons, arsH1 - acr3 - 2 - arsH2 and mfs1 - mfs2 - gapdh . mfs2 and gapdh encode proteins homologous to ArsJ and GAPDH in Pseudomonas aeruginosa ; these proteins create an arsenate efflux pathway to reduce cellular arsenate accumulation. Interestingly, the combination of acr3 - 2 with either arsH gene was sufficient to confer resistance to both arsenite and arsenate in E. coli AW3110, even in the absence of arsenate reductase, suggesting a new strategy for bacterial arsenic detoxification. This study concludes that the survival of GFAJ-1 in high arsenic concentrations is attributable to the cooccurrence of these two unusual arsenic detoxification mechanisms.

Bacterial epigenetic modifications play key roles in cellular processes such as stress responses, DNA replication, segregation, antimicrobial resistance, etc. In recent years, emerging new sequencing technologies, including single-molecule real-time (SMRT) sequencing, and nanopore sequencing, have enabled the directed reading of epigenetic modifications without pre-treatment of DNA or DNA amplification. The applications of SMRT and nanopore sequencing open the door for the identification of more diverse epigenetic modifications of DNA bases and backbones and potentially facilitate the understanding of the novel functions of these epigenetic markers in cell physiology. With ongoing improvements in throughput and accuracy, new-generation sequencing has become a contender as an alternative to second-generation sequencing technologies. Here, the authors review recent advances in bacterial epigenetic analysis using SMRT sequencing and nanopore sequencing to provide insights regarding the detection and analysis of DNA epigenetic modifications in bacterial genomes.

The chemical diversity of physiological DNA modifications has expanded with the identification of phosphorothioate (PT) modification in which the nonbridging oxygen in the sugar-phosphate backbone of DNA is replaced by sulfur. Together with DndFGH as cognate restriction enzymes, DNA PT modification, which is catalyzed by the DndABCDE proteins, functions as a bacterial restriction-modification (R-M) system that protects cells against invading foreign DNA. However, the occurrence of dnd systems across a large number of bacterial genomes and their functions other than R-M are poorly understood. Here, a genomic survey revealed the prevalence of bacterial dnd systems: 1,349 bacterial dnd systems were observed to occur sporadically across diverse phylogenetic groups, and nearly half of these occur in the form of a solitary dndBCDE gene cluster that lacks the dndFGH restriction counterparts. A phylogenetic analysis of 734 complete PT R-M pairs revealed the coevolution of M and R components, despite the observation that several PT R-M pairs appeared to be assembled from M and R parts acquired from distantly related organisms. Concurrent epigenomic analysis, transcriptome analysis, and metabolome characterization showed that a solitary PT modification contributed to the overall cellular redox state, the loss of which perturbed the cellular redox balance and induced Pseudomonas fluorescens to reconfigure its metabolism to fend off oxidative stress. An in vitro transcriptional assay revealed altered transcriptional efficiency in the presence of PT DNA modification, implicating its function in epigenetic regulation. These data suggest the versatility of PT in addition to its involvement in R-M protection.

In this study, we investigated the formation mechanism and chemical structure of melanin that results from the self-assembly of L-3,4-dihydroxyphenylalanine (L-DOPA). Using a combination of "top-down" and "bottom-up" approaches, and on the basis of state-of-the-art electrospray ionization mass spectrometry (ESI-MS) results, we propose a new formation mechanism and an alternative structure for melanin. Specifically, our study of the self-aggregation of L-DOPA based on L-DOPA clusters revealed that melanin is comprised partially of noncovalent supramolecular aggregate that is formed by self-aggregation of L-DOPA and with the individual monomers linked together by a combination of hydrogen bonds, π-π stacking, and ionic bonds. Furthermore, our study showed that unmodified L-DOPA may be part of the building block for melanin in addition to the previously proposed indole derivative based on L-DOPA cyclization. A similar self-aggregation phenomenon was also observed in other structurally related catecholamines, for example, adrenaline.