Systematic genome-wide discovery of host factors governing bacteriophage infectivity.

Bacterial host factors regulate the infection cycle of bacteriophages. Except for some well-studied host factors (e.g., receptors or restriction-modification systems), the contribution of the rest of the host genome on phage infection remains poorly understood. We developed PHAGEPACK, a pooled assay that systematically and comprehensively measures each host-gene impact on phage fitness. PHAGEPACK combines CRISPR interference with phage packaging to link host perturbation to phage fitness during active infection. Using PHAGEPACK, we constructed a genome-wide map of genes impacting T7 phage fitness in permissive E. coli, revealing pathways previously unknown to affect phage packaging. When applied to the non-permissive E. coli O121, PHAGEPACK identified pathways leading to host resistance; their removal increased phage susceptibility up to a billion-fold. Bioinformatic analysis indicates phage genomes carry homologs or truncations of key host factors, potentially for fitness advantage. In summary, PHAGEPACK offers valuable insights into phage-host interactions, phage evolution, and bacterial resistance.

Massively parallel single-cell sequencing of diverse microbial populations.

Single-cell genetic heterogeneity is ubiquitous in microbial populations and an important aspect of microbial biology; however, we lack a broadly applicable and accessible method to study this heterogeneity in microbial populations. Here, we show a simple, robust and generalizable method for high-throughput single-cell sequencing of target genetic loci in diverse microbes using simple droplet microfluidics devices (droplet targeted amplicon sequencing; DoTA-seq). DoTA-seq serves as a platform to perform diverse assays for single-cell genetic analysis of microbial populations. Using DoTA-seq, we demonstrate the ability to simultaneously track the prevalence and taxonomic associations of >10 antibiotic-resistance genes and plasmids within human and mouse gut microbial communities. This workflow is a powerful and accessible platform for high-throughput single-cell sequencing of diverse microbial populations.

Shaping human gut community assembly and butyrate production by controlling the arginine dihydrolase pathway.

The arginine dihydrolase pathway (arc operon) present in a subset of diverse human gut species enables arginine catabolism. This specialized metabolic pathway can alter environmental pH and nitrogen availability, which in turn could shape gut microbiota inter-species interactions. By exploiting synthetic control of gene expression, we investigated the role of the arc operon in probiotic Escherichia coli Nissle 1917 on human gut community assembly and health-relevant metabolite profiles in vitro and in the murine gut. By stabilizing environmental pH, the arc operon reduced variability in community composition across different initial pH perturbations. The abundance of butyrate producing bacteria were altered in response to arc operon activity and butyrate production was enhanced in a physiologically relevant pH range. While the presence of the arc operon altered community dynamics, it did not impact production of short chain fatty acids. Dynamic computational modeling of pH-mediated interactions reveals the quantitative contribution of this mechanism to community assembly. In sum, our framework to quantify the contribution of molecular pathways and mechanism modalities on microbial community dynamics and functions could be applied more broadly.

ViWrap: A modular pipeline to identify, bin, classify, and predict viral-host relationships for viruses from metagenomes.

Viruses are increasingly being recognized as important components of human and environmental microbiomes. However, viruses in microbiomes remain difficult to study because of the difficulty in culturing them and the lack of sufficient model systems. As a result, computational methods for identifying and analyzing uncultivated viral genomes from metagenomes have attracted significant attention. Such bioinformatics approaches facilitate the screening of viruses from enormous sequencing datasets originating from various environments. Though many tools and databases have been developed for advancing the study of viruses from metagenomes, there is a lack of integrated tools enabling a comprehensive workflow and analyses platform encompassing all the diverse segments of virus studies. Here, we developed ViWrap, a modular pipeline written in Python. ViWrap combines the power of multiple tools into a single platform to enable various steps of virus analysis, including identification, annotation, genome binning, species- and genus-level clustering, assignment of taxonomy, prediction of hosts, characterization of genome quality, comprehensive summaries, and intuitive visualization of results. Overall, ViWrap enables a standardized and reproducible pipeline for both extensive and stringent characterization of viruses from metagenomes, viromes, and microbial genomes. Our approach has flexibility in using various options for diverse applications and scenarios, and its modular structure can be easily amended with additional functions as necessary. ViWrap is designed to be easily and widely used to study viruses in human and environmental systems. ViWrap is publicly available via GitHub (https://github.com/AnantharamanLab/ViWrap). A detailed description of the software, its usage, and interpretation of results can be found on the website.

Genomic and transcriptomic insights into complex virus-prokaryote interactions in marine biofilms.

Marine biofilms are complex communities of microorganisms that play a crucial ecological role in oceans. Although prokaryotes are the dominant members of these biofilms, little is known about their interactions with viruses. By analysing publicly available and newly sequenced metagenomic data, we identified 2446 virus-prokaryote connections in 84 marine biofilms. Most of these connections were between the bacteriophages in the Uroviricota phylum and the bacteria of Proteobacteria, Cyanobacteria and Bacteroidota. The network of virus-host pairs is complex; a single virus can infect multiple prokaryotic populations or a single prokaryote is susceptible to several viral populations. Analysis of genomes of paired prokaryotes and viruses revealed the presence of 425 putative auxiliary metabolic genes (AMGs), 239 viral genes related to restriction-modification (RM) systems and 38,538 prokaryotic anti-viral defence-related genes involved in 15 defence systems. Transcriptomic evidence from newly established biofilms revealed the expression of viral genes, including AMGs and RM, and prokaryotic defence systems, indicating the active interplay between viruses and prokaryotes. A comparison between biofilms and seawater showed that biofilm prokaryotes have more abundant defence genes than seawater prokaryotes, and the defence gene composition differs between biofilms and the surrounding seawater. Overall, our study unveiled active viruses in natural biofilms and their complex interplay with prokaryotes, which may result in the blooming of defence strategists in biofilms. The detachment of bloomed defence strategists may reduce the infectivity of viruses in seawater and result in the emergence of a novel role of marine biofilms.

Viromes vs. mixed community metagenomes: choice of method dictates interpretation of viral community ecology.

Background: Viruses, the majority of which are uncultivated, are among the most abundant biological entities on Earth. From altering microbial physiology to driving community dynamics, viruses are fundamental members of microbiomes. While the number of studies leveraging viral metagenomics (viromics) for studying uncultivated viruses is growing, standards for viromics research are lacking. Viromics can utilize computational discovery of viruses from total metagenomes of all community members (hereafter metagenomes) or use physical separation of virus-specific fractions (hereafter viromes). However, differences in the recovery and interpretation of viruses from metagenomes and viromes obtained from the same samples remain understudied. Results: Here, we compare viral communities from paired viromes and metagenomes obtained from 60 diverse samples across human gut, soil, freshwater, and marine ecosystems. Overall, viral communities obtained from viromes were more abundant and species rich than those obtained from metagenomes, although there were some exceptions. Despite this, metagenomes still contained many viral genomes not detected in viromes. We also found notable differences in the predicted lytic state of viruses detected in viromes vs metagenomes at the time of sequencing. Other forms of variation observed include genome presence/absence, genome quality, and encoded protein content between viromes and metagenomes, but the magnitude of these differences varied by environment. Conclusions: Overall, our results show that the choice of method can lead to differing interpretations of viral community ecology. We suggest that the choice of whether to target a metagenome or virome to study viral communities should be dependent on the environmental context and ecological questions being asked. However, our overall recommendation to researchers investigating viral ecology and evolution is to pair both approaches to maximize their respective benefits.

Global diversity and inferred ecophysiology of microorganisms with the potential for dissimilatory sulfate/sulfite reduction.

Sulfate/sulfite-reducing microorganisms (SRM) are ubiquitous in nature, driving the global sulfur cycle. A hallmark of SRM is the dissimilatory sulfite reductase encoded by the genes dsrAB. Based on analysis of 950 mainly metagenome-derived dsrAB-carrying genomes, we redefine the global diversity of microorganisms with the potential for dissimilatory sulfate/sulfite reduction and uncover genetic repertoires that challenge earlier generalizations regarding their mode of energy metabolism. We show: (i) 19 out of 23 bacterial and 2 out of 4 archaeal phyla harbor uncharacterized SRM, (ii) four phyla including the Desulfobacterota harbor microorganisms with the genetic potential to switch between sulfate/sulfite reduction and sulfur oxidation, and (iii) the combination as well as presence/absence of different dsrAB-types, dsrL-types and dsrD provides guidance on the inferred direction of dissimilatory sulfur metabolism. We further provide an updated dsrAB database including > 60% taxonomically resolved, uncultured family-level lineages and recommendations on existing dsrAB-targeted primers for environmental surveys. Our work summarizes insights into the inferred ecophysiology of newly discovered SRM, puts SRM diversity into context of the major recent changes in bacterial and archaeal taxonomy, and provides an up-to-date framework to study SRM in a global context.

Genetic risk score in multiple sclerosis is associated with unique gut microbiome.

Multiple sclerosis (MS) is a complex autoimmune disease in which both the roles of genetic susceptibility and environmental/microbial factors have been investigated. More than 200 genetic susceptibility variants have been identified along with the dysbiosis of gut microbiota, both independently have been shown to be associated with MS. We hypothesize that MS patients harboring genetic susceptibility variants along with gut microbiome dysbiosis are at a greater risk of exhibiting the disease. We investigated the genetic risk score for MS in conjunction with gut microbiota in the same cohort of 117 relapsing remitting MS (RRMS) and 26 healthy controls. DNA samples were genotyped using Illumina's Infinium Immuno array-24 v2 chip followed by calculating genetic risk score and the microbiota was determined by sequencing the V4 hypervariable region of the 16S rRNA gene. We identified two clusters of MS patients, Cluster A and B, both having a higher genetic risk score than the control group. However, the MS cases in cluster B not only had a higher genetic risk score but also showed a distinct gut microbiome than that of cluster A. Interestingly, cluster A which included both healthy control and MS cases had similar gut microbiome composition. This could be due to (i) the non-active state of the disease in that group of MS patients at the time of fecal sample collection and/or (ii) the restoration of the gut microbiome post disease modifying therapy to treat the MS. Our study showed that there seems to be an association between genetic risk score and gut microbiome dysbiosis in triggering the disease in a small cohort of MS patients. The MS Cluster A who have a higher genetic risk score but microbiome profile similar to that of healthy controls could be due to the remitting phase of the disease or due to the effect of disease modifying therapies.

Ecogenomics and cultivation reveal distinctive viral-bacterial communities in the surface microlayer of a Baltic Sea slick.

Visible surface films, termed slicks, can extensively cover freshwater and marine ecosystems, with coastal regions being particularly susceptible to their presence. The sea-surface microlayer (SML), the upper 1-mm at the air-water interface in slicks (herein slick SML) harbors a distinctive bacterial community, but generally little is known about SML viruses. Using flow cytometry, metagenomics, and cultivation, we characterized viruses and bacteria in a brackish slick SML in comparison to non-slick SML as well as seawater below slick and non-slick areas (subsurface water = SSW). Size-fractionated filtration of all samples distinguished viral attachment to hosts and particles. The slick SML contained higher abundances of virus-like particles, prokaryotic cells, and dissolved organic carbon compared to non-slick SML and SSW. The community of 428 viral operational taxonomic units (vOTUs), 426 predicted as lytic, distinctly differed across all size fractions in the slick SML compared to non-slick SML and SSW. Specific metabolic profiles of bacterial metagenome-assembled genomes and isolates in the slick SML included a prevalence of genes encoding motility and carbohydrate-active enzymes (CAZymes). Several vOTUs were enriched in slick SML, and many virus variants were associated with particles. Nine vOTUs were only found in slick SML, six of them being targeted by slick SML-specific clustered-regularly interspaced short palindromic repeats (CRISPR) spacers likely originating from Gammaproteobacteria. Moreover, isolation of three previously unknown lytic phages for Alishewanella sp. and Pseudoalteromonas tunicata, abundant and actively replicating slick SML bacteria, suggests that viral activity in slicks contributes to biogeochemical cycling in coastal ecosystems.

Methionine restriction-induced sulfur deficiency impairs antitumour immunity partially through gut microbiota.

Restriction of methionine (MR), a sulfur-containing essential amino acid, has been reported to repress cancer growth and improve therapeutic responses in several preclinical settings. However, how MR impacts cancer progression in the context of the intact immune system is unknown. Here we report that while inhibiting cancer growth in immunocompromised mice, MR reduces T cell abundance, exacerbates tumour growth and impairs tumour response to immunotherapy in immunocompetent male and female mice. Mechanistically, MR reduces microbial production of hydrogen sulfide, which is critical for immune cell survival/activation. Dietary supplementation of a hydrogen sulfide donor or a precursor, or methionine, stimulates antitumour immunity and suppresses tumour progression. Our findings reveal an unexpected negative interaction between MR, sulfur deficiency and antitumour immunity and further uncover a vital role of gut microbiota in mediating this interaction. Our study suggests that any possible anticancer benefits of MR require careful consideration of both the microbiota and the immune system.

Determinants of Gastrointestinal Group B Streptococcus Carriage in Adults.

Background: Streptococcus agalactiae (Group B Streptococcus, GBS) is a commensal Gram-positive bacterium found in the human gastrointestinal and urogenital tracts. Much of what is known about GBS relates to the diseases it causes in pregnant people and neonates. However, GBS is a common cause of disease in the general population with 90% of GBS mortality occurring in non-pregnant people. There are limited data about the predisposing factors for GBS and the reservoirs in the body. To gain an understanding of the determinants of gastrointestinal GBS carriage, we used stool samples and associated metadata to determine the prevalence and abundance of GBS in the gut microbiome of adults and find risk factors for GBS status. Methods: We used 754 stool samples collected from adults in Wisconsin from 2016-2017 to test for the prevalence and abundance of GBS using a Taqman probe-based qPCR assay targeting two GBS-specific genes: cfp and sip. We compared the microbiome compositions of the stool samples by GBS status using 16S rRNA analysis. We compared associations with GBS status and 557 survey variables collected during sample acquisition (demographics, diet, overall health, and reproductive health) using univariate and multivariate analyses. Results: We found 137/754 (18%) of participants had detectable GBS in their stool samples with a median abundance of 104 copies per nanogram of starting DNA. There was no difference in GBS status or abundance based on gender. Beta-diversity, Bray-Curtis and Unweighted UniFrac, was significantly different based on carrier status of the participant. Prior to p-value correction, 59/557 (10.6%) survey variables were significantly associated with GBS carrier status and 11/547 (2.0%) variables were significantly associated with abundance (p-value<0.05). After p-value correction, 2/547 (0.4%) variables were associated with GBS abundance: an increased abundance of GBS was associated with a decreased frequency since last dental checkup (p<0.001) and last dental cleaning (p<0.001). Increased GBS abundance was significantly associated with increased frequency of iron consumption (p=0.007) after p-value correction in multivariate models. Conclusions: GBS is found in stool samples from adults in Wisconsin at similar frequencies as pregnant individuals screened with rectovaginal swabs. We did not find associations between risk factors historically associated with GBS in pregnant people, suggesting that risk factors for GBS carriage in pregnancy may differ from those in the general population. We found that frequency of iron consumption and dental hygiene are risk factors for GBS carriage in Wisconsin adults. Given that these variables were not assayed in previous GBS surveys, it is possible they also influence carriage in pregnant people. Taken together, this work serves as a foundation for future work in developing approaches to decrease GBS abundance in carriers.

Physiological and genomic evidence of cysteine degradation and aerobic hydrogen sulfide production in freshwater bacteria.

The sulfur-containing amino acid cysteine is abundant in the environment, including in freshwater lakes. Biological cysteine degradation can result in hydrogen sulfide (H2S), a toxic and ecologically relevant compound that is a central player in biogeochemical cycling in aquatic environments. Here, we investigated the ecological significance of cysteine in oxic freshwater, using isolated cultures, controlled experiments, and multiomics. We screened bacterial isolates enriched from natural lake water for their ability to produce H2S when provided cysteine. We identified 29 isolates (Bacteroidota, Proteobacteria, and Actinobacteria) that produced H2S. To understand the genomic and genetic basis for cysteine degradation and H2S production, we further characterized three isolates using whole-genome sequencing (using a combination of short-read and long-read sequencing) and tracked cysteine and H2S levels over their growth ranges: Stenotrophomonas maltophilia (Gammaproteobacteria), S. bentonitica (Gammaproteobacteria), and Chryseobacterium piscium (Bacteroidota). Cysteine decreased and H2S increased, and all three genomes had genes involved in cysteine degradation. Finally, to assess the presence of these organisms and genes in the environment, we surveyed a 5-year time series of metagenomic data from the same isolation source (Lake Mendota, Madison, WI, USA) and identified their presence throughout the time series. Overall, our study shows that diverse isolated bacterial strains can use cysteine and produce H2S under oxic conditions, and we show evidence using metagenomic data that this process may occur more broadly in natural freshwater lakes. Future considerations of sulfur cycling and biogeochemistry in oxic environments should account for H2S production from the degradation of organosulfur compounds. IMPORTANCE Hydrogen sulfide (H2S), a naturally occurring gas with both biological and abiotic origins, can be toxic to living organisms. In aquatic environments, H2S production typically originates from anoxic (lacking oxygen) environments, such as sediments, or the bottom layers of thermally stratified lakes. However, the degradation of sulfur-containing amino acids such as cysteine, which all cells and life forms rely on, can be a source of ammonia and H2S in the environment. Unlike other approaches for biological H2S production such as dissimilatory sulfate reduction, cysteine degradation can occur in the presence of oxygen. Yet, little is known about how cysteine degradation influences sulfur availability and cycling in freshwater lakes. In our study, we identified diverse bacteria from a freshwater lake that can produce H2S in the presence of O2. Our study highlights the ecological importance of oxic H2S production in natural ecosystems and necessitates a change in our outlook on sulfur biogeochemistry.

zol & fai: large-scale targeted detection and evolutionary investigation of gene clusters.

Many universally and conditionally important genes are genomically aggregated within clusters. Here, we introduce fai and zol, which together enable large-scale comparative analysis of different types of gene clusters and mobile-genetic elements (MGEs), such as biosynthetic gene clusters (BGCs) or viruses. Fundamentally, they overcome a current bottleneck to reliably perform comprehensive orthology inference at large scale across broad taxonomic contexts and thousands of genomes. First, fai allows the identification of orthologous or homologous instances of a query gene cluster of interest amongst a database of target genomes. Subsequently, zol enables reliable, context-specific inference of protein-encoding ortholog groups for individual genes across gene cluster instances. In addition, zol performs functional annotation and computes a variety of statistics for each inferred ortholog group. These programs are showcased through application to: (i) longitudinal tracking of a virus in metagenomes, (ii) discovering novel population-genetic insights of two common BGCs in a fungal species, and (iii) uncovering large-scale evolutionary trends of a virulence-associated gene cluster across thousands of genomes from a diverse bacterial genus.

Assembly and Annotation of Viral Metagenomes from Short-Read Sequencing Data.

Viral metagenomics enables the detection, characterization, and quantification of viral sequences present in shotgun-sequenced datasets of purified virus-like particles and whole metagenomes. Next generation sequencing (Illumina) derived short single or paired-end read runs are a principal platform for metagenomics, and assembly of short reads allows for the identification of distinguishing viral signatures and complex genomic features for taxonomy and functional annotation. Here we describe the identification and characterization of viral genome sequences, bacteriophages, and eukaryotic viruses, from a cohort of human stool samples, using multiple methods. Following the purification of virus-like particles, sequencing, quality refinement, and genome assembly, we begin the protocol with raw short reads deposited in an open-source nucleotide archive. We highlight the use of VIBRANT, an automated computational tool for the characterization of microbial viruses and their viral community function. Finally, we also describe an alternative assembly-free option of mapping reads to established databases of reference genomes and previously characterized metagenome-assembled viral genomes.

Sulfur cycling connects microbiomes and biogeochemistry in deep-sea hydrothermal plumes.

In globally distributed deep-sea hydrothermal vent plumes, microbiomes are shaped by the redox energy landscapes created by reduced hydrothermal vent fluids mixing with oxidized seawater. Plumes can disperse over thousands of kilometers and their characteristics are determined by geochemical sources from vents, e.g., hydrothermal inputs, nutrients, and trace metals. However, the impacts of plume biogeochemistry on the oceans are poorly constrained due to a lack of integrated understanding of microbiomes, population genetics, and geochemistry. Here, we use microbial genomes to understand links between biogeography, evolution, and metabolic connectivity, and elucidate their impacts on biogeochemical cycling in the deep sea. Using data from 36 diverse plume samples from seven ocean basins, we show that sulfur metabolism defines the core microbiome of plumes and drives metabolic connectivity in the microbial community. Sulfur-dominated geochemistry influences energy landscapes and promotes microbial growth, while other energy sources influence local energy landscapes. We further demonstrated the consistency of links among geochemistry, function, and taxonomy. Amongst all microbial metabolisms, sulfur transformations had the highest MW-score, a measure of metabolic connectivity in microbial communities. Additionally, plume microbial populations have low diversity, short migration history, and gene-specific sweep patterns after migrating from background seawater. Selected functions include nutrient uptake, aerobic oxidation, sulfur oxidation for higher energy yields, and stress responses for adaptation. Our findings provide the ecological and evolutionary bases of change in sulfur-driven microbial communities and their population genetics in adaptation to changing geochemical gradients in the oceans.

Metagenome-based metabolic modelling predicts unique microbial interactions in deep-sea hydrothermal plume microbiomes.

Deep-sea hydrothermal vents are abundant on the ocean floor and play important roles in ocean biogeochemistry. In vent ecosystems such as hydrothermal plumes, microorganisms rely on reduced chemicals and gases in hydrothermal fluids to fuel primary production and form diverse and complex microbial communities. However, microbial interactions that drive these complex microbiomes remain poorly understood. Here, we use microbiomes from the Guaymas Basin hydrothermal system in the Pacific Ocean to shed more light on the key species in these communities and their interactions. We built metabolic models from metagenomically assembled genomes (MAGs) and infer possible metabolic exchanges and horizontal gene transfer (HGT) events within the community. We highlight possible archaea-archaea and archaea-bacteria interactions and their contributions to the robustness of the community. Cellobiose, D-Mannose 1-phosphate, O2, CO2, and H2S were among the most exchanged metabolites. These interactions enhanced the metabolic capabilities of the community by exchange of metabolites that cannot be produced by any other community member. Archaea from the DPANN group stood out as key microbes, benefiting significantly as acceptors in the community. Overall, our study provides key insights into the microbial interactions that drive community structure and organisation in complex hydrothermal plume microbiomes.

ViWrap: A modular pipeline to identify, bin, classify, and predict viral-host relationships for viruses from metagenomes.

Viruses are increasingly being recognized as important components of human and environmental microbiomes. However, viruses in microbiomes remain difficult to study because of difficulty in culturing them and the lack of sufficient model systems. As a result, computational methods for identifying and analyzing uncultivated viral genomes from metagenomes have attracted significant attention. Such bioinformatics approaches facilitate screening of viruses from enormous sequencing datasets originating from various environments. Though many tools and databases have been developed for advancing the study of viruses from metagenomes, there is a lack of integrated tools enabling a comprehensive workflow and analyses platform encompassing all the diverse segments of virus studies. Here, we developed ViWrap, a modular pipeline written in Python. ViWrap combines the power of multiple tools into a single platform to enable various steps of virus analysis including identification, annotation, genome binning, species- and genus-level clustering, assignment of taxonomy, prediction of hosts, characterization of genome quality, comprehensive summaries, and intuitive visualization of results. Overall, ViWrap enables a standardized and reproducible pipeline for both extensive and stringent characterization of viruses from metagenomes, viromes, and microbial genomes. Our approach has flexibility in using various options for diverse applications and scenarios, and its modular structure can be easily amended with additional functions as necessary. ViWrap is designed to be easily and widely used to study viruses in human and environmental systems. ViWrap is publicly available via GitHub (https://github.com/AnantharamanLab/ViWrap). A detailed description of the software, its usage, and interpretation of results can be found on the website.

Deep metagenomic mining reveals bacteriophage sequence motifs driving host specificity.

Bacteriophages can adapt to new hosts by altering sequence motifs through recombination or convergent evolution. Where such motifs exist and what fitness advantage they confer remains largely unknown. We report a new method, Bacteriophage Library Informed Sequence Scoring (BLISS), to discover sequence motifs in metagenomic datasets governing phage activity. BLISS uses experimental deep mutational scanning data to create sequence profiles to enable deep mining of metagenomes for functional motifs which are otherwise invisible to searches. We experimentally tested 10,073 BLISS-derived sequence motifs for the receptor-binding protein of the T7 phage. The screen revealed hundreds of T7 variants with novel host specificity with functional motifs sourced from distant families besides other major phyla. Position, substitution and location preferences on T7 dictated different specificities. To demonstrate therapeutic utility, we engineered highly active T7 variants against urinary tract pathogens. BLISS is a powerful tool to unlock the functional potential encoded in phage metagenomes.