Using the pan-genomic framework for the discovery of genomic islands in the haloarchaeon Halorubrum ezzemoulense.

In this study, we use pan-genomics to characterize the genomic variability of the widely dispersed halophilic archaeal species Halorubrum ezzemoulense (Hez). We include a multi-regional sampling of newly sequenced, high-quality draft genomes. The pan-genome graph of the species reveals 50 genomic islands that represent rare accessory genetic capabilities available to members. Most notably, we observe rearrangements that have led to the insertion/recombination/replacement of mutually exclusive genomic islands in equivalent genome positions ("homeocassettes"). These conflicting islands encode for similar functions, but homologs from islands located between the same core genes exhibit high divergence on the amino acid level, while the neighboring core genes are nearly identical. Both islands of a homeocassette often coexist in the same geographic location, suggesting that either island may be beyond the reach of selective sweeps and that these loci of divergence between Hez members are maintained and persist long term. This implies that subsections of the population have different niche preferences and rare metabolic capabilities. After an evaluation of the gene content in the homeocassettes, we speculate that these islands may play a role in the speciation, niche adaptability, and group selection dynamics in Hez. Though homeocassettes are first described in this study, similar replacements and divergence of genes on genomic islands have been previously reported in other Haloarchaea and distantly related Archaea, suggesting that homeocassettes may be a feature in a wide range of organisms outside of Hez.IMPORTANCEThis study catalogs the rare genes discovered in strains of the species Halorubrum ezzemoulense (Hez), an obligate halophilic archaeon, through the perspective of its pan-genome. These rare genes are often found to be arranged on islands that confer metabolic and transport functions and contain genes that have eluded previous studies. The discovery of divergent, but homologous islands occupying equivalent genome positions ("homeocassettes") in different genomes, reveals significant new information on genome evolution in Hez. Homeocassette pairs encode for similar functions, but their dissimilarity and distribution imply high rates of recombination, different specializations, and niche preferences in Hez. The coexistence of both islands of a homeocassette pair in multiple environments demonstrates that both islands are beyond the reach of selective sweeps and that these genome content differences between strains persist long term. The switch between islands through recombination under different environmental conditions may lead to a greater range of niche adaptability in Hez.

Host-Microbiome Interactions in a Changing Sea: The Gill Microbiome of an Invasive Oyster under Drastic Temperature Changes.

The gill tissue of bivalve mollusks hosts rich symbiotic microbial communities that may contribute to host health. Spondylus spinosus is an invasive Lessepsian oyster in the Eastern Mediterranean Sea that has become highly abundant while constantly expanding its range northwestward. Using 16S rRNA gene amplicon sequencing, we examined how temperature affects S. spinosus oysters and their gill microbiota in a series of experiments: exposing them to the current annual seawater temperature range, to the colder temperature of the Western Mediterranean Sea, and to the elevated temperature as predicted under global warming scenarios. The bacterial genus Endozoicomonas dominated the communities of the S. spinosus, mainly upon exposure to winter-like (16 °C) temperatures. Exposure to the elevated seawater temperature resulted in a significant change in the bacterial communities, while the oysters maintained normal functioning, suggesting that the oyster may survive a seawater warming scenario. Exposure to 11 °C led to the health deterioration of the oysters, the emergence of opportunistic pathogens, such as Arcobacter, Vibrio, Colwelliaceae, and Pseudoalteromonas, and a decline in the relative abundance of Endozoicomonas, suggesting that S. spinosus might not survive Western Mediterranean Sea winters. Both the host and its gill bacteria are thus greatly affected by temperature, which could consequently restrict the range of expansion of this and other invasive oysters.

Neighboring inteins interfere with one another's homing capacity.

Inteins are mobile genetic elements that invade conserved genes across all domains of life and viruses. In some instances, a single gene will have several intein insertion sites. In Haloarchaea, the minichromosome maintenance (MCM) protein at the core of replicative DNA helicase contains four intein insertion sites within close proximity, where two of these sites (MCM-a and MCM-d) are more likely to be invaded. A haloarchaeon that harbors both MCM-a and MCM-d inteins, Haloferax mediterranei, was studied in vivo to determine intein invasion dynamics and the interactions between neighboring inteins. Additionally, invasion frequencies and the conservation of insertion site sequences in 129 Haloferacales mcm homologs were analyzed to assess intein distribution across the order. We show that the inteins at MCM-a and MCM-d recognize and cleave their respective target sites and, in the event that only one empty intein invasion site is present, readily initiate homing (i.e. single homing). However, when two inteins are present co-homing into an intein-free target sequence is much less effective. The two inteins are more effective when invading alleles that already contain an intein at one of the two sites. Our in vivo and computational studies also support that having a proline in place of a serine as the first C-terminal extein residue of the MCM-d insertion site prevents successful intein splicing, but does not stop recognition of the insertion site by the intein's homing endonuclease.

Skin microbiome bacteria enriched following long sun exposure can reduce oxidative damage.

Sun exposure is harmful to the skin and increases the risk of skin aging and skin cancer. Here we examined the effects of daily exposure to sun radiation on the skin microbiome in order to determine whether skim microbiome bacteria can contribute to protection from solar damage. Skin swabs were collected from ten lifeguards before and after the summer to analyse the skin microbiome. The results indicate that specific skin microbiome bacteria were enriched following the seasonal sun exposure. Especially interesting were two bacterial families - Sphingomonas and Erythrobacteraceae - which may have the ability to protect against UV radiation as they produce potentially protective compounds. We concentrated on a Sphingomonas strain and could show that it was highly resistant to UV irradiation and was able to reduce reactive oxygen species levels in human keratinocytes. These results provide a proof-of-concept for the role of the skin microbiome in protection from solar radiation.

Relationships of the gut microbiome with cognitive development among healthy school-age children.

Background: The gut microbiome might play a role in neurodevelopment, however, evidence remains elusive. We aimed to examine the relationship between the intestinal microbiome and cognitive development of school-age children. Methods: This cross-sectional study included healthy Israeli Arab children from different socioeconomic status (SES). The microbiome was characterized in fecal samples by implementing 16S rRNA gene sequencing. Cognitive function was measured using Stanford-Binet test, yielding full-scale Intelligence Quotient (FSIQ) score. Sociodemographics and anthropometric and hemoglobin measurements were obtained. Multivariate models were implemented to assess adjusted associations between the gut microbiome and FSIQ score, while controlling for age, sex, SES, physical growth, and hemoglobin levels. Results: Overall, 165 children (41.2% females) aged 6-9 years were enrolled. SES score was strongly related to both FSIQ score and the gut microbiome. Measures of α-diversity were significantly associated with FSIQ score, demonstrating a more diverse, even, and rich microbiome with increased FSIQ score. Significant differences in fecal bacterial composition were found; FSIQ score explained the highest variance in bacterial ß-diversity, followed by SES score. Several taxonomic differences were significantly associated with FSIQ score, including Prevotella, Dialister, Sutterella, Ruminococcus callidus, and Bacteroides uniformis. Conclusions: We demonstrated significant independent associations between the gut microbiome and cognitive development in school-age children.

An archaeal Cas3 protein facilitates rapid recovery from DNA damage.

CRISPR-Cas systems provide heritable acquired immunity against viruses to archaea and bacteria. Cas3 is a CRISPR-associated protein that is common to all Type I systems, possesses both nuclease and helicase activities, and is responsible for degradation of invading DNA. Involvement of Cas3 in DNA repair had been suggested in the past, but then set aside when the role of CRISPR-Cas as an adaptive immune system was realized. Here we show that in the model archaeon Haloferax volcanii a cas3 deletion mutant exhibits increased resistance to DNA damaging agents compared with the wild-type strain, but its ability to recover quickly from such damage is reduced. Analysis of cas3 point mutants revealed that the helicase domain of the protein is responsible for the DNA damage sensitivity phenotype. Epistasis analysis indicated that cas3 operates with mre11 and rad50 in restraining the homologous recombination pathway of DNA repair. Mutants deleted for Cas3 or deficient in its helicase activity showed higher rates of homologous recombination, as measured in pop-in assays using non-replicating plasmids. These results demonstrate that Cas proteins act in DNA repair, in addition to their role in defense against selfish elements and are an integral part of the cellular response to DNA damage.

Skin microbiome of people living at the Dead Sea area - The lowest place on earth.

The Dead Sea is a salt lake with surface water at about 430 m below sea level and considered the lowest place on Earth. The Dead Sea basin is characterized by relatively high temperatures, attenuated UV radiation and the air above it has a relatively high-salt aerosol content. When we compared the skin microbiome of individuals from the hot, salty and arid Dead Sea area with that of individuals from the humid Mediterranean regions we observed a significantly lower bacterial diversity in the Dead Sea group as well as distinct differences in the composition of bacterial species. Our results suggest that these factors have a profound effect on the skin microbiome. Further study is required to understand how the local environment influences the skin microbiome, as well as the functional implications of these effects.

Mining metatranscriptomes reveals a vast world of viroid-like circular RNAs.

Viroids and viroid-like covalently closed circular (ccc) RNAs are minimal replicators that typically encode no proteins and hijack cellular enzymes for replication. The extent and diversity of viroid-like agents are poorly understood. We developed a computational pipeline to identify viroid-like cccRNAs and applied it to 5,131 metatranscriptomes and 1,344 plant transcriptomes. The search yielded 11,378 viroid-like cccRNAs spanning 4,409 species-level clusters, a 5-fold increase compared to the previously identified viroid-like elements. Within this diverse collection, we discovered numerous putative viroids, satellite RNAs, retrozymes, and ribozy-like viruses. Diverse ribozyme combinations and unusual ribozymes within the cccRNAs were identified. Self-cleaving ribozymes were identified in ambiviruses, some mito-like viruses and capsid-encoding satellite virus-like cccRNAs. The broad presence of viroid-like cccRNAs in diverse transcriptomes and ecosystems implies that their host range is far broader than currently known, and matches to CRISPR spacers suggest that some cccRNAs replicate in prokaryotes.

Strains Colonizing Different Intestinal Sites within an Individual Are Derived from a Single Founder Population.

Metagenomics has improved our understanding of commensal bacteria that colonize human intestines yet relies almost exclusively on fecal samples. Thus, spatial information about the niche range of these gut microbes and the level of specialized adaptation that they undergo has been inaccessible to fecal metagenomic studies. Here, we leveraged metagenomic data obtained through colonoscopy aspirates from three intestinal sites of healthy adults, and reconstructed metagenome-assembled genomes of several common gut bacteria to address intestinal site-specific evolution. We show that the genomes of bacterial strains at specific intestinal sites are clearly distinct yet are interrelated and are derived from a single founder strain colonizing multiple sites. We also reveal that within those intestinal sites, purifying selection is the dominant evolutionary force acting on Escherichia coli genomes within human hosts. Importantly, no site-specific adaptations at the level of accessory genes were detected, implying that these commensals are well-adapted to several host microniches and can therefore colonize multiple intestinal sites with high efficiency. Nevertheless, bacterial in situ growth rates differ markedly across different sections of the intestine. Metagenomics of aspirate samples can reveal unique strain- and intestinal tissue-specific genomic information. Such information may be critical for understanding bacterial contribution to gastrointestinal diseases, which involve only a part of the intestine, as is often the case in inflammatory bowel disease. IMPORTANCE By reconstructing bacterial genomes from samples taken from specific sites within the human intestines, via aspiration, we show that strains at specific intestinal sites are genetically distinct yet interrelated and are derived from a single founder population. Organ-specific metagenomic information represents a powerful tool to generate insights into gastrointestinal diseases, which involve only a part of the intestine, such as inflammatory bowel disease.

Differences in homologous recombination and maintenance of heteropolyploidy between Haloferax volcanii and Haloferax mediterranei.

Polyploidy, the phenomenon of having more than one copy of the genome in an organism, is common among haloarchaea. While providing short-term benefits for DNA repair, polyploidy is generally regarded as an "evolutionary trap" that by the notion of the Muller's ratchet will inevitably conclude in the species' decline or even extinction due to a gradual reduction in fitness. In most reported cases of polyploidy in archaea, the genetic state of the organism is considered as homoploidy i.e. all copies of the genome are identical. Here we demonstrate that while this is indeed the prevalent genetic status in the halophilic archaeon Haloferax volcanii, its close relative H. mediterranei maintains a prolonged heteroploidy state in a nonselective environment once a second allele is introduced. Moreover, a strong genetic linkage was observed between two distant loci in H. mediterranei indicating a low rate of homologous recombination while almost no such linkage was shown in H. volcanii indicating a high rate of recombination in the latter species. We suggest that H. volcanii escapes Muller's ratchet by means of an effective chromosome-equalizing gene-conversion mechanism facilitated by highly active homologous recombination, whereas H. mediterranei must elude the ratchet via a different, yet to be elucidated mechanism.

Et tu, Vibrio cholerae? Kin-cannibalism and a bacterial secretion system.

Type VI secretion systems are molecular syringes used by Gram-negative bacteria to kill heterospecific (non-kin) niche competitors. In this issue of Cell, Mashruwala et al. show that colonies of the pathogen Vibrio cholera can also exhibit T6SS-mediated cell killing of kin cells and that this process benefits emerging resistant mutants, thereby increasing genetic diversity.

Expansion of the global RNA virome reveals diverse clades of bacteriophages.

High-throughput RNA sequencing offers broad opportunities to explore the Earth RNA virome. Mining 5,150 diverse metatranscriptomes uncovered >2.5 million RNA virus contigs. Analysis of >330,000 RNA-dependent RNA polymerases (RdRPs) shows that this expansion corresponds to a 5-fold increase of the known RNA virus diversity. Gene content analysis revealed multiple protein domains previously not found in RNA viruses and implicated in virus-host interactions. Extended RdRP phylogeny supports the monophyly of the five established phyla and reveals two putative additional bacteriophage phyla and numerous putative additional classes and orders. The dramatically expanded phylum Lenarviricota, consisting of bacterial and related eukaryotic viruses, now accounts for a third of the RNA virome. Identification of CRISPR spacer matches and bacteriolytic proteins suggests that subsets of picobirnaviruses and partitiviruses, previously associated with eukaryotes, infect prokaryotic hosts.

Effect of Solar Radiation on Skin Microbiome: Study of Two Populations.

Here, we examined the skin microbiome of two groups of healthy volunteers living on the Mediterranean coast with different exposures to sun radiation. One group, exposed to the sun in the summer, was compared with a group covered with clothing throughout the year. The seasonal effects on the skin microbiome of three body sites were determined before and after summer. Surprisingly, at the phyla level, there were no significant differences in microbiome diversity between the groups. Furthermore, within each group, there were no significant seasonal differences in high-abundance species at any of the sampling sites. These results suggest that the skin microbiome, developed over years, remains stable even after several months of exposure to summer weather, direct sunlight and humidity. However, in the group exposed to the sun during the summer months, there were significant differences in low-abundance species in sun-exposed areas of the skin (the inner and outer arm). These subtle changes in low-abundance species are interesting, and their effect on skin physiology should be studied further.

Isolation of a virus causing a chronic infection in the archaeal model organism Haloferax volcanii reveals antiviral activities of a provirus.

Viruses are important ecological, biogeochemical, and evolutionary drivers in every environment. Upon infection, they often cause the lysis of the host cell. However, some viruses exhibit alternative life cycles, such as chronic infections without cell lysis. The nature and the impact of chronic infections in prokaryotic host organisms remains largely unknown. Here, we characterize a novel haloarchaeal virus, Haloferax volcanii pleomorphic virus 1 (HFPV-1), which is currently the only virus infecting the model haloarchaeon Haloferax volcanii DS2, and demonstrate that HFPV-1 and H. volcanii are a great model system to study virus-host interactions in archaea. HFPV-1 is a pleomorphic virus that causes a chronic infection with continuous release of virus particles, but host and virus coexist without cell lysis or the appearance of resistant cells. Despite an only minor impact of the infection on host growth, we uncovered an extensive remodeling of the transcriptional program of the host (up to 1,049 differentially expressed genes). These changes are highlighted by a down-regulation of two endogenous provirus regions in the host genome, and we show that HFPV-1 infection is strongly influenced by a cross-talk between HFPV-1 and one of the proviruses mediated by a superinfection-like exclusion mechanism. Furthermore, HFPV-1 has a surprisingly wide host range among haloarchaea, and purified virus DNA can cause an infection after transformation into the host, making HFPV-1 a candidate for being developed into a genetic tool for a range of so far inaccessible haloarchaea.

Horizontal Gene Transfer in Archaea-From Mechanisms to Genome Evolution.

Archaea remains the least-studied and least-characterized domain of life despite its significance not just to the ecology of our planet but also to the evolution of eukaryotes. It is therefore unsurprising that research into horizontal gene transfer (HGT) in archaea has lagged behind that of bacteria. Indeed, several archaeal lineages may owe their very existence to large-scale HGT events, and thus understanding both the molecular mechanisms and the evolutionary impact of HGT in archaea is highly important. Furthermore, some mechanisms of gene exchange, such as plasmids that transmit themselves via membrane vesicles and the formation of cytoplasmic bridges that allows transfer of both chromosomal and plasmid DNA, may be archaea-specific. This review summarizes what we know about HGT in archaea, and the barriers that restrict it, highlighting exciting recent discoveries and pointing out opportunities for future research.

Escherichia coli Strains from Patients with Inflammatory Bowel Diseases have Disease-specific Genomic Adaptations.

BACKGROUND AND AIMS: Escherichia coli is over-abundant in the gut microbiome of patients with inflammatory bowel disease [IBD]. Here, we aimed to identify IBD-specific genomic functions of diverse E. coli lineages. METHODS: We investigated E. coli genomes from patients with ulcerative colitis [UC], Crohn's disease [CD] or a pouch, and healthy subjects. The majority of genomes were reconstructed from metagenomic samples, including newly sequenced faecal metagenomes. Clinical metadata were collected. Functional analysis at the gene and mutation level were performed and integrated with IBD phenotypes and biomarkers. RESULTS: Overall, 530 E. coli genomes were analysed. The E. coli B2 lineage was more prevalent in UC compared with other IBD phenotypes. Genomic metabolic capacities varied across E. coli lineages and IBD phenotypes. Host mucin utilisation enzymes were present in a single lineage and depleted in patients with a pouch, whereas those involved in inulin hydrolysis were enriched in patients with a pouch. E. coli strains from patients with UC were twice as likely to encode the genotoxic molecule colibactin than strains from patients with CD or a pouch. Strikingly, patients with a pouch showed the highest inferred E. coli growth rates, even in the presence of antibiotics. Faecal calprotectin did not correlate with the relative abundance of E. coli. Finally, we identified multiple IBD-specific non-synonymous mutations in E. coli genes encoding for bacterial cell envelope components. CONCLUSIONS: Comparative genomics indicates that E. coli is a commensal species adapted to the overactive mucosal immune milieu in IBD, rather than causing it. Our results reveal mutations that may lead to attenuated antigenicity in some E. coli strains.

Socioeconomic disparities and household crowding in association with the fecal microbiome of school-age children.

The development of the gut microbiome occurs mainly during the first years of life; however, little is known on the role of environmental and socioeconomic exposures, particularly within the household, in shaping the microbial ecology through childhood. We characterized differences in the gut microbiome of school-age healthy children, in association with socioeconomic disparities and household crowding. Stool samples were analyzed from 176 Israeli Arab children aged six to nine years from three villages of different socioeconomic status (SES). Sociodemographic data were collected through interviews with the mothers. We used 16 S rRNA gene sequencing to characterize the gut microbiome, including an inferred analysis of metabolic pathways. Differential analysis was performed using the analysis of the composition of microbiomes (ANCOM), with adjustment for covariates. An analysis of inferred metagenome functions was performed implementing PICRUSt2. Gut microbiome composition differed across the villages, with the largest difference attributed to socioeconomic disparities, with household crowding index being a significant explanatory variable. Living in a low SES village and high household crowding were associated with increased bacterial richness and compositional differences, including an over-representation of Prevotella copri and depleted Bifidobacterium. Secondary bile acid synthesis, d-glutamine and d-glutamate metabolism and Biotin metabolism were decreased in the lower SES village. In summary, residential SES is a strong determinant of the gut microbiome in healthy school-age children, mediated by household crowding and characterized by increased bacterial richness and substantial taxonomic and metabolic differences. Further research is necessary to explore possible implications of SES-related microbiome differences on children's health and development.

Microbiome-related aspects of locust density-dependent phase transition.

Locust plagues are a notorious, ancient phenomenon. These swarming pests tend to aggregate and perform long migrations, decimating cultivated fields along their path. When population density is low, however, the locusts will express a cryptic, solitary, non-aggregating phenotype that is not considered a pest. Although the transition from the solitary to the gregarious phase has been well studied, associated shifts in the locust's microbiome have yet to be addressed. Here, using 16S rRNA amplicon sequencing, we compared the bacterial composition of solitary desert locusts before and after a phase transition. Our findings revealed that the microbiome is altered during the phase transition, and that a major aspect of this change is the acquisition of Weissella (Firmicutes). Our findings led us to hypothesize that the locust microbiome plays a role in inducing aggregation behaviour, contributing to the formation and maintenance of a swarm. Employing a mathematical model, we demonstrate the potential evolutionary advantage of inducing aggregation under different conditions; specifically, when the aggregation-inducing microbe exhibits a relatively high horizontal transmission rate. This is the first report of a previously unknown and important aspect of locust phase transition, demonstrating that the phase shift includes a shift in the gut and integument bacterial composition.

Long-term Antibiotic Treatment in Pouchitis-Patterns of Use and Safety.

INTRODUCTION: Pouchitis, often developing after colectomy and ileal pouch-anal anastomosis for ulcerative colitis, is highly responsive to antibiotics. Ciprofloxacin and/or metronidazole are commonly used, often for prolonged periods. We report patterns of antibiotic use, adverse events, and resistant infections in patients with pouchitis with long-term antibiotic treatment. METHODS: In a cohort of patients following pouch surgery, a retrospective nested case-control analysis was performed between 2010 and 2017. Ultra-long-term use, defined as the top 10% of users, was compared with the remaining users. Patterns of antibiotic use, adverse events, and resistant infections were analyzed. RESULTS: The cohort included 205 patients with UC, of whom 167 (81.5%) used antibiotics for pouchitis, predominantly ciprofloxacin. The long-term antibiotic use rate was 18% and 42% at 5 and 20 years postsurgery, respectively. Mean antibiotic use of at least 1, 3, and 6 months/year was noted in 54 (26.3%), 31 (15.1%), and 14 (6.8%) patients, respectively. Twenty-two (13.2%) and 4 (2.4%) patients reported mild and severe (transient) adverse events, respectively, without mortalities, tendinopathies or arrhythmias. Adverse event rates for ciprofloxacin and metronidazole were 1per 10,000 and 6 per 10,000 use-days, respectively. Longer, but not ultra-long antibiotic use, was associated with mild adverse events. There was no association between antibiotic use and resistant infections. Thirteen (6.3%) patients required ileostomy procedures-more commonly in the ultra-long-term antibiotic users. CONCLUSIONS: Patients with pouchitis may require prolonged antibiotic treatment, reflecting clinical benefit and favorable safety profile. Few adverse events and resistant infections were observed with long-term antibiotics use. However, resistant microbial strains selection, which are potentially transmittable, warrants consideration of different therapeutic alternatives.