IMG/PR: a database of plasmids from genomes and metagenomes with rich annotations and metadata.

Plasmids are mobile genetic elements found in many clades of Archaea and Bacteria. They drive horizontal gene transfer, impacting ecological and evolutionary processes within microbial communities, and hold substantial importance in human health and biotechnology. To support plasmid research and provide scientists with data of an unprecedented diversity of plasmid sequences, we introduce the IMG/PR database, a new resource encompassing 699 973 plasmid sequences derived from genomes, metagenomes and metatranscriptomes. IMG/PR is the first database to provide data of plasmid that were systematically identified from diverse microbiome samples. IMG/PR plasmids are associated with rich metadata that includes geographical and ecosystem information, host taxonomy, similarity to other plasmids, functional annotation, presence of genes involved in conjugation and antibiotic resistance. The database offers diverse methods for exploring its extensive plasmid collection, enabling users to navigate plasmids through metadata-centric queries, plasmid comparisons and BLAST searches. The web interface for IMG/PR is accessible at https://img.jgi.doe.gov/pr. Plasmid metadata and sequences can be downloaded from https://genome.jgi.doe.gov/portal/IMG_PR.

Plasmid classifications.

Plasmids are universally present in bacteria and play key roles in the dissemination of genes such as antibiotic resistance determinants. Major concepts in Plasmid Biology derive from the efforts to classify plasmids. Here, we review the main plasmid classification systems, starting by phenotype-based methods, such as fertility inhibition and incompatibility, followed by schemes based on a single gene (replicon type and MOB class), and finishing with recently developed approaches that use genetic distances between whole plasmid sequences. A comparison of the latter highlights significant differences between them. We further discuss the need for an operational definition of plasmid species that reveals their biological features, akin to plasmid taxonomic units (PTUs).

Highlighting the potential of Synechococcus elongatus PCC 7942 as platform to produce α-linolenic acid through an updated genome-scale metabolic modeling.

Cyanobacteria are prokaryotic organisms that capture energy from sunlight using oxygenic photosynthesis and transform CO2 into products of interest such as fatty acids. Synechococcus elongatus PCC 7942 is a model cyanobacterium efficiently engineered to accumulate high levels of omega-3 fatty acids. However, its exploitation as a microbial cell factory requires a better knowledge of its metabolism, which can be approached by using systems biology tools. To fulfill this objective, we worked out an updated, more comprehensive, and functional genome-scale model of this freshwater cyanobacterium, which was termed iMS837. The model includes 837 genes, 887 reactions, and 801 metabolites. When compared with previous models of S. elongatus PCC 7942, iMS837 is more complete in key physiological and biotechnologically relevant metabolic hubs, such as fatty acid biosynthesis, oxidative phosphorylation, photosynthesis, and transport, among others. iMS837 shows high accuracy when predicting growth performance and gene essentiality. The validated model was further used as a test-bed for the assessment of suitable metabolic engineering strategies, yielding superior production of non-native omega-3 fatty acids such as α-linolenic acid (ALA). As previously reported, the computational analysis demonstrated that fabF overexpression is a feasible metabolic target to increase ALA production, whereas deletion and overexpression of fabH cannot be used for this purpose. Flux scanning based on enforced objective flux, a strain-design algorithm, allowed us to identify not only previously known gene overexpression targets that improve fatty acid synthesis, such as Acetyl-CoA carboxylase and ß-ketoacyl-ACP synthase I, but also novel potential targets that might lead to higher ALA yields. Systematic sampling of the metabolic space contained in iMS837 identified a set of ten additional knockout metabolic targets that resulted in higher ALA productions. In silico simulations under photomixotrophic conditions with acetate or glucose as a carbon source boosted ALA production levels, indicating that photomixotrophic nutritional regimens could be potentially exploited in vivo to improve fatty acid production in cyanobacteria. Overall, we show that iMS837 is a powerful computational platform that proposes new metabolic engineering strategies to produce biotechnologically relevant compounds, using S. elongatus PCC 7942 as non-conventional microbial cell factory.

Targeted bacterial conjugation mediated by synthetic cell-to-cell adhesions.

Genetic interventions on microbiomes, for clinical or biotechnological purposes, remain challenging. Conjugation-based delivery of genetic cargo is still unspecific and limited by low conjugation rates. Here we report an approach to overcome these problems, based on a synthetic bacterial adhesion system. Mating assemblers consist on a synthetic adhesion formed by the expression on the surface of donor and target cells of specific nanobodies (Nb) and their cognate antigen (Ag). The Nb-Ag bridge increased 1-3 logs transfer of a variety of plasmids, especially in liquid media, confirming that cell-cell docking is a main determinant limiting mating efficiency. Synthetic cell-to-cell adhesion allows efficient conjugation to targeted recipients, enhancing delivery of desired genes to a predefined subset of prey species, or even specific pathogenic strains such as enterohemorrhagic Escherichia coli (EHEC), within a bacterial community. The synthetic conjugation enhancer presented here optimizes plasmid delivery by selecting the target hosts with high selectivity.

Structural basis of direct and inverted DNA sequence repeat recognition by helix-turn-helix transcription factors.

Some transcription factors bind DNA motifs containing direct or inverted sequence repeats. Preference for each of these DNA topologies is dictated by structural constraints. Most prokaryotic regulators form symmetric oligomers, which require operators with a dyad structure. Binding to direct repeats requires breaking the internal symmetry, a property restricted to a few regulators, most of them from the AraC family. The KorA family of transcriptional repressors, involved in plasmid propagation and stability, includes members that form symmetric dimers and recognize inverted repeats. Our structural analyses show that ArdK, a member of this family, can form a symmetric dimer similar to that observed for KorA, yet it binds direct sequence repeats as a non-symmetric dimer. This is possible by the 180° rotation of one of the helix-turn-helix domains. We then probed and confirmed that ArdK shows affinity for an inverted repeat, which, surprisingly, is also recognized by a non-symmetrical dimer. Our results indicate that structural flexibility at different positions in the dimerization interface constrains transcription factors to bind DNA sequences with one of these two alternative DNA topologies.

Genome Sequences of 18 Salmonella enterica Serotype Hadar Strains Collected from Patients in the United States.

Despite being linked to a number of recent poultry-associated outbreaks in the United States, few reference genomes are available for Salmonella enterica serotype Hadar. Here, we address this need by reporting 18 Salmonella Hadar genomes from samples collected from patients in the United States between 2014 and 2020.

Characterization of the DNA Binding Domain of StbA, A Key Protein of A New Type of DNA Segregation System.

Low-copy-number plasmids require sophisticated genetic devices to achieve efficient segregation of plasmid copies during cell division. Plasmid R388 uses a unique segregation mechanism, based on StbA, a small multifunctional protein. StbA is the key protein in a segregation system not involving a plasmid-encoded NTPase partner, it regulates the expression of several plasmid operons, and it is the main regulator of plasmid conjugation. The mechanisms by which StbA, together with the centromere-like sequence stbS, achieves segregation, is largely uncharacterized. To better understand the molecular basis of R388 segregation, we determined the crystal structure of the conserved N-terminal domain of StbA to 1.9 Å resolution. It folds into an HTH DNA-binding domain, structurally related to that of the PadR subfamily II of transcriptional regulators. StbA is organized in two domains. Its N-terminal domain carries the specific stbS DNA binding activity. A truncated version of StbA, deleted of its C-terminal domain, displays only partial activities in vivo, indicating that the non-conserved C-terminal domain is required for efficient segregation and subcellular plasmid positioning. The structure of StbA DNA-binding domain also provides some insight into how StbA monomers cooperate to repress transcription by binding to the stbDR and to form the segregation complex with stbS.

Five Complete Salmonella enterica Serotype Reading Genomes Recovered from Patients in the United States.

Between 2018 and 2019, Salmonella enterica serotype Reading caused a large, multistate outbreak linked to contact with raw turkey products in the United States. Here, we provide five Salmonella Reading reference genomes collected from US patients between 2016 and 2018.

Synechococcus elongatus PCC 7942 as a Platform for Bioproduction of Omega-3 Fatty Acids.

Alpha-linolenic acid and stearidonic acid are precursors of omega-3 polyunsaturated fatty acids, essential nutrients in the human diet. The ability of cyanobacteria to directly convert atmospheric carbon dioxide into bio-based compounds makes them promising microbial chassis to sustainably produce omega-3 fatty acids. However, their potential in this area remains unexploited, mainly due to important gaps in our knowledge of fatty acid synthesis pathways. To gain insight into the cyanobacterial fatty acid biosynthesis pathways, we analyzed two enzymes involved in the elongation cycle, FabG and FabZ, in Synechococcus elongatus PCC 7942. Overexpression of these two enzymes led to an increase in C18 fatty acids, key intermediates in omega-3 fatty acid production. Nevertheless, coexpression of these enzymes with desaturases DesA and DesB from Synechococcus sp. PCC 7002 did not improve alpha-linolenic acid production, possibly due to their limited role in fatty acid synthesis. In any case, efficient production of stearidonic acid was not achieved by cloning DesD from Synechocystis sp. PCC 6803 in combination with the aforementioned DesA and DesB, reaching maximum production at 48 h post induction. According to current knowledge, this is the first report demonstrating that S. elongatus PCC 7942 can be used as an autotrophic chassis to produce stearidonic acid.

Evolution of Plasmid Mobility: Origin and Fate of Conjugative and Nonconjugative Plasmids.

Conjugation drives the horizontal transfer of adaptive traits across prokaryotes. One-fourth of the plasmids encode the functions necessary to conjugate autonomously, the others being eventually mobilizable by conjugation. To understand the evolution of plasmid mobility, we studied plasmid size, gene repertoires, and conjugation-related genes. Plasmid gene repertoires were found to vary rapidly in relation to the evolutionary rate of relaxases, for example, most pairs of plasmids with 95% identical relaxases have fewer than 50% of homologs. Among 249 recent transitions of mobility type, we observed a clear excess of plasmids losing the capacity to conjugate. These transitions are associated with even greater changes in gene repertoires, possibly mediated by transposable elements, including pseudogenization of the conjugation locus, exchange of replicases reducing the problem of incompatibility, and extensive loss of other genes. At the microevolutionary scale of plasmid taxonomy, transitions of mobility type sometimes result in the creation of novel taxonomic units. Interestingly, most transitions from conjugative to mobilizable plasmids seem to be lost in the long term. This suggests a source-sink dynamic, where conjugative plasmids generate nonconjugative plasmids that tend to be poorly adapted and are frequently lost. Still, in some cases, these relaxases seem to have evolved to become efficient at plasmid mobilization in trans, possibly by hijacking multiple conjugative systems. This resulted in specialized relaxases of mobilizable plasmids. In conclusion, the evolution of plasmid mobility is frequent, shapes the patterns of gene flow in bacteria, the dynamics of gene repertoires, and the ecology of plasmids.

Isolation and phenotypic and genomic characterization of Tetragenococcus spp. from two Spanish traditional blue-veined cheeses made of raw milk.

High throughput sequencing has recently revealed the presence of Tetragenococcus-related DNA sequences in dairy environments such as brine and cheeses. In the present work, a selective medium was developed to isolate Tetragenococcus spp. from two ripened, traditional, Spanish, blue-veined cheese varieties made from raw milk. The strains recovered belonged to either Tetragenococcus koreensis or Tetragenococcus halophilus species. Twenty of these isolates (15 of T. koreensis and 5 of T. halophilus) were then subjected to a battery of phenotypic and genetic tests, and six strains (4 T. koreensis and 2 T. halophilus) to genome sequencing. Wide genetic and phenotypic diversity was noted. All strains grew poorly in milk, producing small quantities of lactic and acetic acids. Most strains used lactose as a carbon source and ferment milk citrate. In agreement, genome analysis detected in the genome of the six strains analyzed gene clusters harboring several lactose/galactose-related genes and genes encoding citrate metabolic enzymes (permease, citrate lyase, and oxaloacetate decarboxylase). Most of the tested strains were resistant to erythromycin and clindamycin, and a few to other antimicrobial agents, but neither known mutations nor acquired genes conferring resistance to antibiotics were identified in their genomes. Neither were genes coding for pathogenicity or virulence factors detected. Decarboxylase-encoding genes involved in biogenic amine production were not identified, in keeping with the strains' negative biogenic amine-producer phenotype. Genome comparison revealed vast arrays of genes (similar in number to those described in other lactic acid bacteria) coding for components of proteolytic and lipolytic systems. Tetragenococcus strains showing desirable traits plus the absence of detrimental features might be exploitable in the form of secondary, adjunct or ripening cultures to ensure the typical bouquet of traditional blue-veined cheeses is obtained, or to diversify the final flavor in other varieties.

PLASmid TAXonomic PCR (PlasTax-PCR), a Multiplex Relaxase MOB Typing to Assort Plasmids into Taxonomic Units.

Plasmids transmissible by conjugation are responsible for disseminating antibiotic-resistance genes, making plasmid detection relevant for pathogen tracking. We describe the use of a multiplex PCR method for the experimental identification of specific plasmid taxonomic units (PTUs) of transmissible plasmids. The PCR primers were designed to target conserved segments of the relaxase MOB gene of PTUs encoding adaptive traits for enterobacteria (antimicrobial resistance, virulence, and metabolism). In this way, PlasTax-PCR detects the presence of these plasmids and allows their direct assignation to a PTU.

Conjugation Inhibitors Effectively Prevent Plasmid Transmission in Natural Environments.

Plasmid conjugation is a major route for the spread of antibiotic resistance genes. Inhibiting conjugation has been proposed as a feasible strategy to stop or delay the propagation of antibiotic resistance genes. Several compounds have been shown to be conjugation inhibitors in vitro, specifically targeting the plasmid horizontal transfer machinery. However, the in vivo efficiency and the applicability of these compounds to clinical and environmental settings remained untested. Here we show that the synthetic fatty acid 2-hexadecynoic acid (2-HDA), when used as a fish food supplement, lowers the conjugation frequency of model plasmids up to 10-fold in controlled water microcosms. When added to the food for mice, 2-HDA diminished the conjugation efficiency 50-fold in controlled plasmid transfer assays carried out in the mouse gut. These results demonstrate the in vivo efficiency of conjugation inhibitors, paving the way for their potential application in clinical and environmental settings. IMPORTANCE The spread of antibiotic resistance is considered one of the major threats for global health in the immediate future. A key reason for the speed at which antibiotic resistance spread is the ability of bacteria to share genes with each other. Antibiotic resistance genes harbored in plasmids can be easily transferred to commensal and pathogenic bacteria through a process known as bacterial conjugation. Blocking conjugation is thus a potentially useful strategy to curtail the propagation of antibiotic resistance. Conjugation inhibitors (COINS) are a series of compounds that block conjugation in vitro. Here we show that COINS efficiently block plasmid transmission in two controlled natural environments, water microcosms and the mouse gut. These observations indicate that COIN therapy can be used to prevent the spread of antibiotic resistance.

COPLA, a taxonomic classifier of plasmids.

BACKGROUND: Plasmids are mobile genetic elements, key in the dissemination of antibiotic resistance, virulence determinants and other adaptive traits in bacteria. Obtaining a robust method for plasmid classification is necessary to better understand the genetics and epidemiology of many pathogens. Until now, plasmid classification systems focused on specific traits, which limited their precision and universality. The definition of plasmid taxonomic units (PTUs), based on average nucleotide identity metrics, allows the generation of a universal plasmid classification scheme, applicable to all bacterial taxa. Here we present COPLA, a software able to assign plasmids to known and novel PTUs, based on their genomic sequence. RESULTS: We implemented an automated pipeline able to assign a given plasmid DNA sequence to its cognate PTU, and assessed its performance using a sample of 1000 unclassified plasmids. Overall, 41% of the samples could be assigned to a previously defined PTU, a number that reached 63% in well-known taxa such as the Enterobacterales order. The remaining plasmids represent novel PTUs, indicating that a large fraction of plasmid backbones is still uncharacterized. CONCLUSIONS: COPLA is a bioinformatic tool for universal, species-independent, plasmid classification. Offered both as an automatable pipeline and an open web service, COPLA will help bacterial geneticists and clinical microbiologists to quickly classify plasmids.

Genomic Insights into Drug Resistance and Virulence Platforms, CRISPR-Cas Systems and Phylogeny of Commensal E. coli from Wildlife.

Commensal bacteria act as important reservoirs of virulence and resistance genes. However, existing data are generally only focused on the analysis of human or human-related bacterial populations. There is a lack of genomic studies regarding commensal bacteria from hosts less exposed to antibiotics and other selective forces due to human activities, such as wildlife. In the present study, the genomes of thirty-eight E. coli strains from the gut of various wild animals were sequenced. The analysis of their accessory genome yielded a better understanding of the role of the mobilome on inter-bacterial dissemination of mosaic virulence and resistance plasmids. The study of the presence and composition of the CRISPR/Cas systems in E. coli from wild animals showed some viral and plasmid sequences among the spacers, as well as the relationship between CRISPR/Cas and E. coli phylogeny. Further, we constructed a single nucleotide polymorphisms-based core tree with E. coli strains from different sources (humans, livestock, food and extraintestinal environments). Bacteria from humans or highly human-influenced settings exhibit similar genetic patterns in CRISPR-Cas systems, plasmids or virulence/resistance genes-carrying modules. These observations, together with the absence of significant genetic changes in their core genome, suggest an ongoing flow of both mobile elements and E. coli lineages between human and natural ecosystems.

Population genomics and antimicrobial resistance dynamics of Escherichia coli in wastewater and river environments.

Aquatic environments are key niches for the emergence, evolution and dissemination of antimicrobial resistance. However, the population diversity and the genetic elements that drive the dynamics of resistant bacteria in different aquatic environments are still largely unknown. The aim of this study was to understand the population genomics and evolutionary events of Escherichia coli resistant to clinically important antibiotics including aminoglycosides, in anthropogenic and natural water ecosystems. Here we show that less different E. coli sequence types (STs) are identified in wastewater than in rivers, albeit more resistant to antibiotics, and with significantly more plasmids/cell (6.36 vs 3.72). However, the genomic diversity within E. coli STs in both aquatic environments is similar. Wastewater environments favor the selection of conserved chromosomal structures associated with diverse flexible plasmids, unraveling promiscuous interplasmidic resistance genes flux. On the contrary, the key driver for river E. coli adaptation is a mutable chromosome along with few plasmid types shared between diverse STs harboring a limited resistance gene content.

Pathways for horizontal gene transfer in bacteria revealed by a global map of their plasmids.

Plasmids can mediate horizontal gene transfer of antibiotic resistance, virulence genes, and other adaptive factors across bacterial populations. Here, we analyze genomic composition and pairwise sequence identity for over 10,000 reference plasmids to obtain a global map of the prokaryotic plasmidome. Plasmids in this map organize into discrete clusters, which we call plasmid taxonomic units (PTUs), with high average nucleotide identity between its members. We identify 83 PTUs in the order Enterobacterales, 28 of them corresponding to previously described archetypes. Furthermore, we develop an automated algorithm for PTU identification, and validate its performance using stochastic blockmodeling. The algorithm reveals a total of 276 PTUs in the bacterial domain. Each PTU exhibits a characteristic host distribution, organized into a six-grade scale (I-VI), ranging from plasmids restricted to a single host species (grade I) to plasmids able to colonize species from different phyla (grade VI). More than 60% of the plasmids in the global map are in groups with host ranges beyond the species barrier.

A Role for Gut Microbiome Fermentative Pathways in Fatty Liver Disease Progression.

Non-alcoholic fatty liver disease (NAFLD) is a multifactorial disease in which environmental and genetic factors are involved. Although the molecular mechanisms involved in NAFLD onset and progression are not completely understood, the gut microbiome (GM) is thought to play a key role in the process, influencing multiple physiological functions. GM alterations in diversity and composition directly impact disease states with an inflammatory course, such as non-alcoholic steatohepatitis (NASH). However, how the GM influences liver disease susceptibility is largely unknown. Similarly, the impact of strategies targeting the GM for the treatment of NASH remains to be evaluated. This review provides a broad insight into the role of gut microbiota in NASH pathogenesis, as a diagnostic tool, and as a therapeutic target in this liver disease. We highlight the idea that the balance in metabolic fermentations can be key in maintaining liver homeostasis. We propose that an overabundance of alcohol-fermentation pathways in the GM may outcompete healthier, acid-producing members of the microbiota. In this way, GM ecology may precipitate a self-sustaining vicious cycle, boosting liver disease progression.

ArdC, a ssDNA-binding protein with a metalloprotease domain, overpasses the recipient hsdRMS restriction system broadening conjugation host range.

Plasmids, when transferred by conjugation in natural environments, must overpass restriction-modification systems of the recipient cell. We demonstrate that protein ArdC, encoded by broad host range plasmid R388, was required for conjugation from Escherichia coli to Pseudomonas putida. Expression of ardC was required in the recipient cells, but not in the donor cells. Besides, ardC was not required for conjugation if the hsdRMS system was deleted in P. putida recipient cells. ardC was also required if the hsdRMS system was present in E. coli recipient cells. Thus, ArdC has antirestriction activity against the HsdRMS system and consequently broadens R388 plasmid host range. The crystal structure of ArdC was solved both in the absence and presence of Mn2+. ArdC is composed of a non-specific ssDNA binding N-terminal domain and a C-terminal metalloprotease domain, although the metalloprotease activity was not needed for the antirestriction function. We also observed by RNA-seq that ArdC-dependent conjugation triggered an SOS response in the P. putida recipient cells. Our findings give new insights, and open new questions, into the antirestriction strategies developed by plasmids to counteract bacterial restriction strategies and settle into new hosts.

Biochemical interactions between LPS and LPS-binding molecules.

Lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria, often pose a serious risk not only when delivered in the bloodstream but also in air, the environment and several industrial fields such as pharmaceutics or food. LPS is constituted of three regions; the O-specific chain, the core region and the lipid A, which is the responsible segment of the toxicity. Previous literature dealt with the study of lipid A, its potential ligands as well as the mechanisms of Lipid A interactions that, among other applications, establish the basis for detection methods such as Limulus Amebocyte Lysate (LAL) assays and emerging biosensoring techniques. However, quantifying LPS binding affinity is an urgent need that still requires thorough studies. In this context, this work reviews the molecules that bind LPS, highlighting quantitative affinity parameters. Moreover, state of the art methods to analyze the affinity and kinetics of lipid-ligand interactions are also reviewed and different techniques have been briefly described. Thus, first, we review existing information on LPS ligands, classifying them into three main groups and targeting the comparison of molecules in terms of their interaction affinities and, second, we establish the basis for further research aimed at the development of effective methods for LPS detection and removal.