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.

Functional and Compositional Changes in the Fecal Microbiome of a Shorebird during Migratory Stopover.

Shorebirds migrate long distances twice annually, which requires intense physiological and morphological adaptations, including the ability to rapidly gain weight via fat deposition at stopover locations. The role of the microbiome in weight gain in avian hosts is unresolved, but there is substantial evidence to support the hypothesis that the microbiome is involved with host weight from mammalian microbiome literature. Here, we collected 100 fecal samples of Ruddy Turnstones to investigate microbiome composition and function during stopover weight gain in Delaware Bay, USA. Using 16S rRNA sequencing on 90 of these samples and metatranscriptomic sequencing on 22, we show that taxonomic composition of the microbiome shifts during weight gain, as do functional aspects of the metatranscriptome. We identified 10 genes that are associated with weight class, and polyunsaturated fatty acid biosynthesis in the microbiota is significantly increasing as birds gain weight. Our results support that the microbiome is a dynamic feature of host biology that interacts with both the host and the environment and may be involved in the rapid weight gain of shorebirds. IMPORTANCE Many animals migrate long distances annually, and these journeys require intense physiological and morphological adaptations. One such adaptation in shorebirds is the ability to rapidly gain weight at stopover locations in the middle of their migrations. The role of the microbiome in weight gain in birds is unresolved but is likely to play a role. Here, we collected 100 fecal samples from Ruddy Turnstones to investigate microbiome composition (who is there) and function (what they are doing) during stopover weight gain in Delaware Bay, USA. Using multiple molecular methods, we show that both taxonomic composition and function of the microbiome shifts during weight gain. We identified 10 genes that are associated with weight class, and polyunsaturated fatty acid biosynthesis in the microbiota is significantly increasing as birds gain weight. Our results support that the microbiome is a dynamic feature of host biology that interacts with both the host and the environment and may be involved in the rapid weight gain of shorebirds.

Effect of a Stannous Fluoride Dentifrice on Biofilm Composition, Gene Expression and Biomechanical Properties.

An in situ study was conducted to examine the mode of action of a 0.454% stannous fluoride (SnF2)-containing dentifrice in controlling the composition and properties of oral biofilm. Thirteen generally healthy individuals participated in the study. Each participant wore an intra-oral appliance over a 48-h period to measure differences in the resulting biofilm's architecture, mechanical properties, and bacterial composition after using two different toothpaste products. In addition, metatranscriptomics analysis of supragingival plaque was conducted to identify the gene pathways influenced. The thickness and volume of the microcolonies formed when brushing with the SnF2 dentifrice were dramatically reduced compared to the control 0.76% sodium monofluorophosphate (MFP)-containing toothpaste. Similarly, the biophysical and nanomechanical properties measured by atomic force microscopy (AFM) demonstrated a significant reduction in biofilm adhesive properties. Metatranscriptomic analysis identified pathways associated with biofilm formation, cell adhesion, quorum sensing, and N-glycosylation that are significantly downregulated with SnF2. This study provides a clinically relevant snapshot of how the use of a stabilized, SnF2 toothpaste formulation can change the spatial organization, nanomechanical, and gene expression properties of bacterial communities.

The Evolutionary Origins of Extreme Halophilic Archaeal Lineages.

Interest and controversy surrounding the evolutionary origins of extremely halophilic Archaea has increased in recent years, due to the discovery and characterization of the Nanohaloarchaea and the Methanonatronarchaeia. Initial attempts in explaining the evolutionary placement of the two new lineages in relation to the classical Halobacteria (also referred to as Haloarchaea) resulted in hypotheses that imply the new groups share a common ancestor with the Haloarchaea. However, more recent analyses have led to a shift: the Nanohaloarchaea have been largely accepted as being a member of the DPANN superphylum, outside of the euryarchaeota; whereas the Methanonatronarchaeia have been placed near the base of the Methanotecta (composed of the class II methanogens, the Halobacteriales, and Archaeoglobales). These opposing hypotheses have far-reaching implications on the concepts of convergent evolution (distantly related groups evolve similar strategies for survival), genome reduction, and gene transfer. In this work, we attempt to resolve these conflicts with phylogenetic and phylogenomic data. We provide a robust taxonomic sampling of Archaeal genomes that spans the Asgardarchaea, TACK Group, euryarchaeota, and the DPANN superphylum. In addition, we assembled draft genomes from seven new representatives of the Nanohaloarchaea from distinct geographic locations. Phylogenies derived from these data imply that the highly conserved ATP synthase catalytic/noncatalytic subunits of Nanohaloarchaea share a sisterhood relationship with the Haloarchaea. We also employ a novel gene family distance clustering strategy which shows this sisterhood relationship is not likely the result of a recent gene transfer. In addition, we present and evaluate data that argue for and against the monophyly of the DPANN superphylum, in particular, the inclusion of the Nanohaloarchaea in DPANN.

Evaluating the lettuce metatranscriptome with MinION sequencing for future spaceflight food production applications.

Healthy plants are vital for successful, long-duration missions in space, as they provide the crew with life support, food production, and psychological benefits. The microorganisms that associate with plant tissues play a critical role in improving plant health and production. To that end, we developed a methodology to investigate the transcriptional activities of the microbiome of red romaine lettuce, a key salad crop that was grown under International Space Station (ISS)-like conditions. Microbial transcripts enriched from host-microbe total RNA were sequenced using the Oxford Nanopore MinION sequencing platform. Results show that this enrichment approach was highly reproducible and could be an effective approach for the on-site detection of microbial transcriptional activity. Our results demonstrate the feasibility of using metatranscriptomics of enriched microbial RNA as a potential method for on-site monitoring of the transcriptional activity of crop microbiomes, thereby helping to facilitate and maintain plant health for on-orbit space food production.

Draft Genome Sequence of the Free-Living, Iridescent Bacterium Tenacibaculum mesophilum Strain ECR.

Here, we report the genome sequence of Tenacibaculum mesophilum strain ECR, which was isolated from the river/ocean interface at Trunk River in Falmouth, Massachusetts. The isolation and sequencing were performed as part of the 2016 and 2018 Microbial Diversity courses at the Marine Biological Laboratory in Woods Hole, Massachusetts.

Insights into gene expression changes under conditions that facilitate horizontal gene transfer (mating) of a model archaeon.

Horizontal gene transfer is a means by which bacteria, archaea, and eukaryotes are able to trade DNA within and between species. While there are a variety of mechanisms through which this genetic exchange can take place, one means prevalent in the archaeon Haloferax volcanii involves the transient formation of cytoplasmic bridges between cells and is referred to as mating. This process can result in the exchange of very large fragments of DNA between the participating cells. Genes governing the process of mating, including triggers to initiate mating, mechanisms of cell fusion, and DNA exchange, have yet to be characterized. We used a transcriptomic approach to gain a more detailed knowledge of how mating might transpire. By examining the differential expression of genes expressed in cells harvested from mating conditions on a filter over time and comparing them to those expressed in a shaking culture, we were able to identify genes and pathways potentially associated with mating. These analyses provide new insights into both the mechanisms and barriers of mating in Hfx. volcanii.

Complete Genome Sequence of Halorubrum ezzemoulense Strain Fb21.

Isolated from Aran-Bidgol Lake in Iran, and reported here, Halorubrum ezzemoulense strain Fb21 represents the first complete genome from this archaeal species. Local recombination in this genome is in stark contrast to equidistant recombination events in bacteria. The genome's GC bias, however, points to a genome architecture and origin that resemble those of a bacterium. Its availability, genome signatures, and frequent intragenomic recombination mean that Fb21 presents an attractive model organism for this species.

The Patchy Distribution of Restriction⁻Modification System Genes and the Conservation of Orphan Methyltransferases in Halobacteria.

Restriction⁻modification (RM) systems in bacteria are implicated in multiple biological roles ranging from defense against parasitic genetic elements, to selfish addiction cassettes, and barriers to gene transfer and lineage homogenization. In bacteria, DNA-methylation without cognate restriction also plays important roles in DNA replication, mismatch repair, protein expression, and in biasing DNA uptake. Little is known about archaeal RM systems and DNA methylation. To elucidate further understanding for the role of RM systems and DNA methylation in Archaea, we undertook a survey of the presence of RM system genes and related genes, including orphan DNA methylases, in the halophilic archaeal class Halobacteria. Our results reveal that some orphan DNA methyltransferase genes were highly conserved among lineages indicating an important functional constraint, whereas RM systems demonstrated patchy patterns of presence and absence. This irregular distribution is due to frequent horizontal gene transfer and gene loss, a finding suggesting that the evolution and life cycle of RM systems may be best described as that of a selfish genetic element. A putative target motif (CTAG) of one of the orphan methylases was underrepresented in all of the analyzed genomes, whereas another motif (GATC) was overrepresented in most of the haloarchaeal genomes, particularly in those that encoded the cognate orphan methylase.

Comparative Metagenomics Provides Insight Into the Ecosystem Functioning of the Shark Bay Stromatolites, Western Australia.

Stromatolites are organosedimentary build-ups that have formed as a result of the sediment trapping, binding and precipitating activities of microbes. Today, extant systems provide an ideal platform for understanding the structure, composition, and interactions between stromatolite-forming microbial communities and their respective environments. In this study, we compared the metagenomes of three prevalent stromatolite-forming microbial mat types in the Spaven Province of Hamelin Pool, Shark Bay located in Western Australia. These stromatolite-forming mat types included an intertidal pustular mat as well as a smooth and colloform mat types located in the subtidal zone. Additionally, the metagenomes of an adjacent, non-lithifying mat located in the upper intertidal zone were also sequenced for comparative purposes. Taxonomic and functional gene analyses revealed distinctive differences between the lithifying and non-lithifying mat types, which strongly correlated with water depth. Three distinct populations emerged including the upper intertidal non-lithifying mats, the intertidal pustular mats associated with unlaminated carbonate build-ups, and the subtidal colloform and smooth mat types associated with laminated structures. Functional analysis of metagenomes revealed that amongst stromatolite-forming mats there was an enrichment of photosynthesis pathways in the pustular stromatolite-forming mats. In the colloform and smooth stromatolite-forming mats, however, there was an increase in the abundance of genes associated with those heterotrophic metabolisms typically associated with carbonate mineralization, such as sulfate reduction. The comparative metagenomic analyses suggest that stromatolites of Hamelin Pool may form by two distinctive processes that are highly dependent on water depth. These results provide key insight into the potential adaptive strategies and synergistic interactions between microbes and their environments that may lead to stromatolite formation and accretion.

A year in the life of a thrombolite: comparative metatranscriptomics reveals dynamic metabolic changes over diel and seasonal cycles.

Microbialites are one of the oldest known ecosystems on Earth and the coordinated metabolisms and activities of these mineral-depositing communities have had a profound impact on the habitability of the planet. Despite efforts to understand the diversity and metabolic potential of these systems, there has not been a systematic molecular analysis of the transcriptional changes that occur within a living microbialite over time. In this study, we generated metatranscriptomic libraries from actively growing thrombolites, a type of microbialite, throughout diel and seasonal cycles and observed dynamic shifts in the population and metabolic transcriptional activity. The most transcribed genes in all seasons were associated with photosynthesis, but only transcripts associated with photosystem II exhibited diel cycling. Photosystem I transcripts were constitutively expressed at all time points including midnight and sunrise. Transcripts associated with nitrogen fixation, methanogenesis and dissimilatory sulfate reduction exhibited diel cycling, and variability between seasons. Networking analysis of the metatranscriptomes showed correlated expression patterns helping to elucidate how metabolic interactions are coordinated within the thrombolite community. These findings have identified distinctive temporal patterns within the thrombolites and will serve an important foundation to understand the mechanisms by which these communities form and respond to changes in their environment.

A Study of the Microbial Spatial Heterogeneity of Bahamian Thrombolites Using Molecular, Biochemical, and Stable Isotope Analyses.

Thrombolites are buildups of carbonate that exhibit a clotted internal structure formed through the interactions of microbial mats and their environment. Despite recent advances, we are only beginning to understand the microbial and molecular processes associated with their formation. In this study, a spatial profile of the microbial and metabolic diversity of thrombolite-forming mats of Highborne Cay, The Bahamas, was generated by using 16S rRNA gene sequencing and predictive metagenomic analyses. These molecular-based approaches were complemented with microelectrode profiling and in situ stable isotope analysis to examine the dominant taxa and metabolic activities within the thrombolite-forming communities. Analyses revealed three distinctive zones within the thrombolite-forming mats that exhibited stratified populations of bacteria and archaea. Predictive metagenomics also revealed vertical profiles of metabolic capabilities, such as photosynthesis and carboxylic and fatty acid synthesis within the mats that had not been previously observed. The carbonate precipitates within the thrombolite-forming mats exhibited isotopic geochemical signatures suggesting that the precipitation within the Bahamian thrombolites is photosynthetically induced. Together, this study provides the first look at the spatial organization of the microbial populations within Bahamian thrombolites and enables the distribution of microbes to be correlated with their activities within modern thrombolite systems. Key Words: Thrombolites-Microbial diversity-Metagenome-Stable isotopes-Microbialites. Astrobiology 17, 413-430.

Genetic structure, admixture and invasion success in a Holarctic defoliator, the gypsy moth (Lymantria dispar, Lepidoptera: Erebidae).

Characterizing the current population structure of potentially invasive species provides a critical context for identifying source populations and for understanding why invasions are successful. Non-native populations inevitably lose genetic diversity during initial colonization events, but subsequent admixture among independently introduced lineages may increase both genetic variation and adaptive potential. Here we characterize the population structure of the gypsy moth (Lymantria dispar Linnaeus), one of the world's most destructive forest pests. Native to Eurasia and recently introduced to North America, the current distribution of gypsy moth includes forests throughout the temperate region of the northern hemisphere. Analyses of microsatellite loci and mitochondrial DNA sequences for 1738 individuals identified four genetic clusters within L. dispar. Three of these clusters correspond to the three named subspecies; North American populations represent a distinct fourth cluster, presumably a consequence of the population bottleneck and allele frequency change that accompanied introduction. We find no evidence that admixture has been an important catalyst of the successful invasion and range expansion in North America. However, we do find evidence of ongoing hybridization between subspecies and increased genetic variation in gypsy moth populations from Eastern Asia, populations that now pose a threat of further human-mediated introductions. Finally, we show that current patterns of variation can be explained in terms of climate and habitat changes during the Pleistocene, a time when temperate forests expanded and contracted. Deeply diverged matrilines in Europe imply that gypsy moths have been there for a long time and are not recent arrivals from Asia.