Impact of modeled microgravity stress on innate immunity in a beneficial animal-microbe symbiosis.

The innate immune response is the first line of defense for all animals to not only detect invading microbes and toxins but also sense and interface with the environment. One such environment that can significantly affect innate immunity is spaceflight. In this study, we explored the impact of microgravity stress on key elements of the NFκB innate immune pathway. The symbiosis between the bobtail squid Euprymna scolopes and its beneficial symbiont Vibrio fischeri was used as a model system under a simulated microgravity environment. The expression of genes associated with the NFκB pathway was monitored over time as the symbiosis progressed. Results revealed that although the onset of the symbiosis was the major driver in the differential expression of NFκB signaling, the stress of simulated low-shear microgravity also caused a dysregulation of expression. Several genes were expressed at earlier time points suggesting that elements of the E. scolopes NFκB pathway are stress-inducible, whereas expression of other pathway components was delayed. The results provide new insights into the role of NFκB signaling in the squid-vibrio symbiosis, and how the stress of microgravity negatively impacts the host immune response. Together, these results provide a foundation to develop mitigation strategies to maintain host-microbe homeostasis during spaceflight.

Modelled microgravity impacts Vibrio fischeri population structure in a mutualistic association with an animal host.

Perturbations to host-microbe interactions, such as environmental stress, can alter and disrupt homeostasis. In this study, we examined the effects of a stressor, simulated microgravity, on beneficial bacteria behaviours when colonising their host. We studied the bacterium Vibrio fischeri, which establishes a mutualistic association in a symbiosis-specific organ within the bobtail squid, Euprymna scolopes. To elucidate how animal-microbe interactions are affected by the stress of microgravity, squid were inoculated with different bacterial strains exhibiting either a dominant- or sharing-colonisation behaviour in High Aspect Ratio Vessels, which simulate the low-shear environment of microgravity. The colonisation behaviours of the sharing and dominant strains under modelled microgravity conditions were determined by counting light-organ homogenate of squids as well as confocal microscopy to assess the partitioning of different strains within the light organ. The results indicated that although the colonisation behaviours of the strains did not change, the population levels of the sharing strains were at lower relative abundance in single-colonised animals exposed to modelled microgravity compared to unit gravity; in addition, there were shifts in the relative abundance of strains in co-colonised squids. Together these results suggest that the initiation of beneficial interactions between microbes and animals can be altered by environmental stress, such as simulated microgravity.

Integration of multi-omics data to elucidate keystone unknown taxa within microbialite-forming ecosystems.

Microbes continually shape Earth's biochemical and physical landscapes by inhabiting diverse metabolic niches. Despite the important role microbes play in ecosystem functioning, most microbial species remain unknown highlighting a gap in our understanding of structured complex ecosystems. To elucidate the relevance of these unknown taxa, often referred to as "microbial dark matter," the integration of multiple high throughput sequencing technologies was used to evaluate the co-occurrence and connectivity of all microbes within the community. Since there are no standard methodologies for multi-omics integration of microbiome data, we evaluated the abundance of "microbial dark matter" in microbialite-forming communities using different types meta-omic datasets: amplicon, metagenomic, and metatranscriptomic sequencing previously generated for this ecosystem. Our goal was to compare the community structure and abundances of unknown taxa within the different data types rather than to perform a functional characterization of the data. Metagenomic and metatranscriptomic data were input into SortMeRNA to extract 16S rRNA gene reads. The output, as well as amplicon sequences, were processed through QIIME2 for taxonomy analysis. The R package mdmnets was utilized to build co-occurrence networks. Most hubs presented unknown classifications, even at the phyla level. Comparisons of the highest scoring hubs of each data type using sequence similarity networks allowed the identification of the most relevant hubs within the microbialite-forming communities. This work highlights the importance of unknown taxa in community structure and proposes that ecosystem network construction can be used on several types of data to identify keystone taxa and their potential function within microbial ecosystems.

Forages and pastures symposium: an update on in vitro and in situ experimental techniques for approximation of ruminal fiber degradation.

Static quantification measures of chemical components are commonly used to make certain assumptions about forage or feed nutritive value and quality. In order for modern nutrient requirement models to estimate intake and digestibility more accurately, kinetic measures of ruminal fiber degradation are necessary. Compared to in vivo experiments, in vitro (IV) and in situ (IS) experimental techniques are relatively simple and inexpensive methods to determine the extent and rate of ruminal fiber degradation. This paper summarizes limitations of these techniques and statistical analyses of the resulting data, highlights key updates to these techniques in the last 30 yr, and presents opportunities for further improvements to these techniques regarding ruminal fiber degradation. The principle biological component of these techniques, ruminal fluid, is still highly variable because it is influenced by ruminally fistulated animal diet type and timing of feeding, and in the case of the IV technique by collection and transport procedures. Commercialization has contributed to the standardization, mechanization, and automation of the IV true digestibility technique, for example, the well-known DaisyII Incubator. There has been limited commercialization of supplies for the IS technique and several review papers focused on standardization in the last 30 yr; however, the IS experimental technique is not standardized and there remains variation within and among laboratories. Regardless of improved precision resulting from enhancements of these techniques, the accuracy and precision of determining the indigestible fraction are fundamental to modeling digestion kinetics and the use of these estimates in more complex dynamic nutritional modeling. Opportunities for focused research and development are additional commercialization and standardization, methods to improve the precision and accuracy of indigestible fiber fraction, data science applications, and statistical analyses of results, especially for IS data. In situ data is typically fitted to one of a few first-order kinetic models, and parameters are estimated without determining if the selected model has the best fit. Animal experimentation will be fundamental to the future of ruminant nutrition and IV and IS techniques will remain vital to bring together nutritive value with forage quality. It is feasible and important to focus efforts on improving the precision and accuracy of IV and IS results.

In vitro and in situ techniques are important to studying ruminant nutrition because these procedures go beyond measures of components of a feedstuff in a laboratory by fermenting a sample in ruminal fluid. The in situ procedure was first described regarding ruminant nutrition in 1938 and in vitro in 1966 and both techniques have been refined over time to improve the reliability of results. This review focused on the state of knowledge 30 yr ago and significant discoveries that have impacted these techniques in the last 30 yr and shared a vision for future opportunities to refine these methods further. Commercialization of equipment and supplies has resulted in increased standardization of these methods; however, efforts should be made to continue to improve the standardization, and reliability of the results, of these procedures. Statistical analyses and data science applications are opportunities to expand these techniques to modern nutritional models and/or forecasting animal performance. The amount and kinetics of ruminal degradation estimate that in vitro and in situ techniques provide continue to be crucial to advance the efficiency and sustainability of ruminant animal production.

Multiscale stiffness characterisation of both healthy and osteoporotic bone tissue using subject-specific data.

Severe bone fractures are often treated by appending internal fixations. In unhealthy or osteoporotic patients, post-implantation bone fractures can occur due to external impact (e.g. from a fall), day-to-day activities in highly-osteoporotic cases and mismatches in the stiffness of bone and the implant's biomaterial, since this causes stress concentrations. One approach to alleviating this problem is to use biomaterials that closely mimic the effective stiffness of real bone, thereby more seamlessly integrating the fixation. This requires to know the properties target (bone properties) and therefore, it highlights the relevance of the evaluation of the bone's mechanical properties which is impractical via direct measurement. This work presents a methodology (multistage homogenisation) for predicting the anisotropic stiffness of bone given the porosity and mineral fraction, both of which are more readily obtained than the mechanical properties themselves. Unlike previous work we: (i) account for finger-like morphology of the mineral phase at the nanoscale; (ii) use microscopy data to model the osteon geometry and its curvilinear anisotropy at the microscale, and (iii) use data to define the trabecular (microCT) and cortical (microscopy) bone geometries at the mesoscale. The predicts have been shown to agree favourably with experimental data in the literature as well as previous modelling works. The results are summarised in a database containing anisotropic stiffness tensors applicable to a broad range of degrees of bone health (e.g. mineral fractions and mesoscale porosities); thus, this work is a contribution towards being able to design more robust patient-specific bone implants in practice.

Data-driven optimal closures for mean-cluster models: Beyond the classical pair approximation.

This study concerns the mean-clustering approach to modeling the evolution of lattice dynamics. Instead of tracking the state of individual lattice sites, this approach describes the time evolution of the concentrations of different cluster types. It leads to an infinite hierarchy of ordinary differential equations which must be closed by truncation using a so-called closure condition. This condition approximates the concentrations of higher-order clusters in terms of the concentrations of lower-order ones. The pair approximation is the most common form of closure. Here, we consider its generalization, termed the "optimal approximation," which we calibrate using a robust data-driven strategy. To fix attention, we focus on a recently proposed structured lattice model for a nickel-based oxide, similar to that used as cathode material in modern commercial Li-ion batteries. The form of the obtained optimal approximation allows us to deduce a simple sparse closure model. In addition to being more accurate than the classical pair approximation, this "sparse approximation" is also physically interpretable which allows us to a posteriori refine the hypotheses underlying construction of this class of closure models. Moreover, the mean-cluster model closed with this sparse approximation is linear and hence analytically solvable such that its parametrization is straightforward, although it offers a good approximation of the actual time evolution of the cluster concentrations on short timescales only. On the other hand, parametrization of the mean-cluster model closed with the pair approximation is shown to lead to an ill-posed inverse problem.

Modeled microgravity alters apoptotic gene expression and caspase activity in the squid-vibrio symbiosis.

BACKGROUND: Spaceflight is a novel and profoundly stressful environment for life. One aspect of spaceflight, microgravity, has been shown to perturb animal physiology thereby posing numerous health risks, including dysregulation of normal developmental pathways. Microgravity can also negatively impact the interactions between animals and their microbiomes. However, the effects of microgravity on developmental processes influenced by beneficial microbes, such as apoptosis, remains poorly understood. Here, the binary mutualism between the bobtail squid, Euprymna scolopes, and the gram-negative bacterium, Vibrio fischeri, was studied under modeled microgravity conditions to elucidate how this unique stressor alters apoptotic cell death induced by beneficial microbes. RESULTS: Analysis of the host genome and transcriptome revealed a complex network of apoptosis genes affiliated with extrinsic/receptor-mediated and intrinsic/stress-induced apoptosis. Expression of apoptosis genes under modeled microgravity conditions occurred earlier and at high levels compared to gravity controls, in particular the expression of genes encoding initiator and executioner caspases. Functional assays of these apoptotic proteases revealed heightened activity under modeled microgravity; however, these increases could be mitigated using caspase inhibitors. CONCLUSIONS: The outcomes of this study indicated that modeled microgravity alters the expression of both extrinsic and intrinsic apoptosis gene expression and that this process is mediated in part by caspases. Modeled microgravity-associated increases of caspase activity can be pharmacologically inhibited suggesting that perturbations to the normal apoptosis signaling cascade can be mitigated, which may have broader implications for maintaining animal-microbial homeostasis in spaceflight.

Emergence of novel cephalopod gene regulation and expression through large-scale genome reorganization.

Coleoid cephalopods (squid, cuttlefish, octopus) have the largest nervous system among invertebrates that together with many lineage-specific morphological traits enables complex behaviors. The genomic basis underlying these innovations remains unknown. Using comparative and functional genomics in the model squid Euprymna scolopes, we reveal the unique genomic, topological, and regulatory organization of cephalopod genomes. We show that coleoid cephalopod genomes have been extensively restructured compared to other animals, leading to the emergence of hundreds of tightly linked and evolutionary unique gene clusters (microsyntenies). Such novel microsyntenies correspond to topological compartments with a distinct regulatory structure and contribute to complex expression patterns. In particular, we identify a set of microsyntenies associated with cephalopod innovations (MACIs) broadly enriched in cephalopod nervous system expression. We posit that the emergence of MACIs was instrumental to cephalopod nervous system evolution and propose that microsyntenic profiling will be central to understanding cephalopod innovations.

Lithifying and Non-Lithifying Microbial Ecosystems in the Wetlands and Salt Flats of the Central Andes.

The wetlands and salt flats of the Central Andes region are unique extreme environments as they are located in high-altitude saline deserts, largely influenced by volcanic activity. Environmental factors, such as ultraviolet (UV) radiation, arsenic content, high salinity, low dissolved oxygen content, extreme daily temperature fluctuation, and oligotrophic conditions, resemble the early Earth and potentially extraterrestrial conditions. The discovery of modern microbialites and microbial mats in the Central Andes during the past decade has increased the interest in this area as an early Earth analog. In this work, we review the current state of knowledge of Central Andes region environments found within lakes, small ponds or puquios, and salt flats of Argentina, Chile, and Bolivia, many of them harboring a diverse range of microbial communities that we have termed Andean Microbial Ecosystems (AMEs). We have integrated the data recovered from all the known AMEs and compared their biogeochemistry and microbial diversity to achieve a better understanding of them and, consequently, facilitate their protection.

Effects of roughage type on particle separation, rumination, fiber mat characteristics, in situ degradation, and ruminal fermentation parameters in beef steers.

Six ruminally cannulated steers (average BW = 791 ± 71 kg) were used in a replicated 3 × 3 Latin square experiment to determine the effects of roughage type on rumination, fiber mat characteristics, and rumen fermentation variables. Three roughages were included at 7% (DM basis) in a steam flaked corn-based diet: cotton burrs (CB), wheat silage (WS), or corn stalks (CS). Steers were fitted with a sensory collar to record rumination behaviors in 2-h intervals at the beginning of the experiment. Each 30-d period consisted of 7 d of recovery, 14 d of diet adaptation, 7 d of rumination data collection (daily and bi-hourly average rumination), 1 d of rumen fluid collection, and 1 d of rumen evacuations. In situ degradation of individual roughages was determined for 4 d after period 3 evacuations. During rumen evacuations, ruminal contents were removed; the rumen fiber mat (RF) was separated from the liquid portion with a 2-mm sieve, weighed, and a subsample was dried. Data were analyzed using the MIXED procedure of SAS with steer as the experimental unit and roughage (CB, WS, and CS) as the main effect. Dry matter intake (DMI) was not different for CB and WS (P = 0.25) and greatest for steers consuming CS diet (P ≤ 0.01). Roughage type did not influence the weight of the RF dry matter (%; DM; P = 0.92), RF weight (P = 0.69), or RF:DMI ratio (P = 0.29). Daily rumination (min/d) did not differ among roughages (P = 0.40), but min of rumination/kg of DMI was greatest for CS (18.0 min), min/kg of NDF was greatest for WS (89.8 min; P = 0.02), and min/kg of peNDF was greatest for CS (132.4 min; P ≤ 0.01). Wheat silage had the greatest percentage of soluble and degradable DM. Rumen fiber mat did not differ for roughages, although rumination min/kg of DMI and peNDF was greatest for steers consuming CS and WS. In situ degradation determined that CB-R and CS-R had the greatest percentage of ruminal undegraded DM. Based on the objective of the experiment, roughage type did not influence daily rumination or fiber mat characteristics.

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.

Modeled microgravity alters lipopolysaccharide and outer membrane vesicle production of the beneficial symbiont Vibrio fischeri.

Reduced gravity, or microgravity, can have a pronounced impact on the physiology of animals, but the effects on their associated microbiomes are not well understood. Here, the impact of modeled microgravity on the shedding of Gram-negative lipopolysaccharides (LPS) by the symbiotic bacterium Vibrio fischeri was examined using high-aspect ratio vessels. LPS from V. fischeri is known to induce developmental apoptosis within its symbiotic tissues, which is accelerated under modeled microgravity conditions. In this study, we provide evidence that exposure to modeled microgravity increases the amount of LPS released by the bacterial symbiont in vitro. The higher rates of shedding under modeled microgravity conditions are associated with increased production of outer-membrane vesicles (OMV), which has been previously correlated to flagellar motility. Mutants of V. fischeri defective in the production and rotation of their flagella show significant decreases in LPS shedding in all treatments, but levels of LPS are higher under modeled microgravity despite loss of motility. Modeled microgravity also appears to affect the outer-membrane integrity of V. fischeri, as cells incubated under modeled microgravity conditions are more susceptible to cell-membrane-disrupting agents. These results suggest that, like their animal hosts, the physiology of symbiotic microbes can be altered under microgravity-like conditions, which may have important implications for host health during spaceflight.

A network approach to elucidate and prioritize microbial dark matter in microbial communities.

Microbes compose most of the biomass on the planet, yet the majority of taxa remain uncharacterized. These unknown microbes, often referred to as "microbial dark matter," represent a major challenge for biology. To understand the ecological contributions of these Unknown taxa, it is essential to first understand the relationship between unknown species, neighboring microbes, and their respective environment. Here, we establish a method to study the ecological significance of "microbial dark matter" by building microbial co-occurrence networks from publicly available 16S rRNA gene sequencing data of four extreme aquatic habitats. For each environment, we constructed networks including and excluding unknown organisms at multiple taxonomic levels and used network centrality measures to quantitatively compare networks. When the Unknown taxa were excluded from the networks, a significant reduction in degree and betweenness was observed for all environments. Strikingly, Unknown taxa occurred as top hubs in all environments, suggesting that "microbial dark matter" play necessary ecological roles within their respective communities. In addition, novel adaptation-related genes were detected after using 16S rRNA gene sequences from top-scoring hub taxa as probes to blast metagenome databases. This work demonstrates the broad applicability of network metrics to identify and prioritize key Unknown taxa and improve understanding of ecosystem structure across diverse habitats.

Digital Proxy of a Bio-Reactor (DIYBOT) combines sensor data and data analytics to improve greywater treatment and wastewater management systems.

Technologies to treat wastewater in decentralized systems are critical for sustainable development. Bioreactors are suitable for low-energy removal of inorganic and organic compounds, particularly for non-potable applications where a small footprint is required. One of the main problems associated with bioreactor use is sporadic spikes of chemical toxins, including nanoparticles. Here, we describe the development of DIYBOT (Digital Proxy of a Bio-Reactor), which enables remote monitoring of bioreactors and uses the data to inform decisions related to systems management. To test DIYBOT, a household-scale membrane aerated bioreactor with real-time water quality sensors was used to treat household greywater simulant. After reaching steady-state, silver nanoparticles (AgNP) representative of the mixture found in laundry wastewater were injected into the system to represent a chemical contamination. Measurements of carbon metabolism, effluent water quality, biofilm sloughing rate, and microbial diversity were characterized after nanoparticle exposure. Real-time sensor data were analyzed to reconstruct phase-space dynamics and extrapolate a phenomenological digital proxy to evaluate system performance. The management implication of the stable-focus dynamics, reconstructed from observed data, is that the bioreactor self-corrects in response to contamination spikes at AgNP levels below 2.0 mg/L. DIYBOT may help reduce the frequency of human-in-the-loop corrective management actions for wastewater processing.

Inclusion of quebracho tannin extract in a high-roughage cattle diet alters digestibility, nitrogen balance, and energy partitioning.

Condensed tannins (CT) might improve animal and system-level efficiency due to enhanced protein efficiency and reduced CH4. This study evaluated the impact of quebracho tannin (QT) extract fed at 0%, 1.5%, 3%, and 4.5% of dry matter (DM), within a roughage-based diet on apparent digestibility of DM, organic matter (OM), fibrous fractions, and N retention and energy partitioning of growing steers (236 ± 16 kg BW). A Latin rectangle design with eight animals and four periods was used to determine the whole-animal exchange of CO2, O2, and CH4 as well as the collection of total feces and urine over a 48-h period, using two open-circuit, indirect calorimetry respiration chambers. Following the removal of steers from respiration chambers, rumen inoculum was collected to determine ruminal parameter, including volatile fatty acids (VFA) and ammonia. Animals were fed a 56.5% roughage diet at 1.7% BW (dry matter basis). Dry matter and gross energy intakes were influenced by the level of QT inclusion (P ≤ 0.036). Digestibility of DM, OM, and N was reduced with QT inclusion (P < 0.001), and fiber digestibility was slightly impacted (P > 0.123). QTs altered the N excretion route, average fecal N-to-total N ratio excreted increased 14%, and fecal N-to-urinary N ratio increased 38% (P < 0.001) without altering the retained N. Increased fecal energy with QT provision resulted in reduced dietary digestible energy (DE) concentration (Mcal/kg DM; P = 0.024). There were no differences in urinary energy (P = 0.491), but CH4 energy decreased drastically (P = 0.007) as QT inclusion increased. Total ruminal VFA concentration did not differ across treatments, but VFA concentration increased linearly with QT inclusion (P = 0.049). Metabolizable energy (ME) was not affected by the QT rate, and the conversion efficiency of DE-to-ME did not differ. Heat energy decreased (P = 0.013) with increased QT provision likely due to changes in the DE intake, but there was no difference in retained energy. There were no differences for retained energy or N per CO2 equivalent emission produced (P = 0.774 and 0.962, respectively), but improved efficiency for energy retention occurred for 3% QT. We concluded that QT provided up to 4.5% of dry matter intake (about 3.51% of CT, dry matter basis) does not affect N and energy retention within the current setting. Feeding QT reduced energy losses in the form of CH4 and heat, but the route of energy loss appears to be influenced by the rate of QT inclusion.

Symbiotic organs shaped by distinct modes of genome evolution in cephalopods.

Microbes have been critical drivers of evolutionary innovation in animals. To understand the processes that influence the origin of specialized symbiotic organs, we report the sequencing and analysis of the genome of Euprymna scolopes, a model cephalopod with richly characterized host-microbe interactions. We identified large-scale genomic reorganization shared between E. scolopes and Octopus bimaculoides and posit that this reorganization has contributed to the evolution of cephalopod complexity. To reveal genomic signatures of host-symbiont interactions, we focused on two specialized organs of E. scolopes: the light organ, which harbors a monoculture of Vibrio fischeri, and the accessory nidamental gland (ANG), a reproductive organ containing a bacterial consortium. Our findings suggest that the two symbiotic organs within E. scolopes originated by different evolutionary mechanisms. Transcripts expressed in these microbe-associated tissues displayed their own unique signatures in both coding sequences and the surrounding regulatory regions. Compared with other tissues, the light organ showed an abundance of genes associated with immunity and mediating light, whereas the ANG was enriched in orphan genes known only from E. scolopes Together, these analyses provide evidence for different patterns of genomic evolution of symbiotic organs within a single host.

Transcriptional profiling of the mutualistic bacterium Vibrio fischeri and an hfq mutant under modeled microgravity.

For long-duration space missions, it is critical to maintain health-associated homeostasis between astronauts and their microbiome. To achieve this goal it is important to more fully understand the host-symbiont relationship under the physiological stress conditions of spaceflight. To address this issue we examined the impact of a spaceflight analog, low-shear-modeled microgravity (LSMMG), on the transcriptome of the mutualistic bacterium Vibrio fischeri. Cultures of V. fischeri and a mutant defective in the global regulator Hfq (∆hfq) were exposed to either LSMMG or gravity conditions for 12 h (exponential growth) and 24 h (stationary phase growth). Comparative transcriptomic analysis revealed few to no significant differentially expressed genes between gravity and the LSMMG conditions in the wild type or mutant V. fischeri at exponential or stationary phase. There was, however, a pronounced change in transcriptomic profiles during the transition between exponential and stationary phase growth in both V. fischeri cultures including an overall decrease in gene expression associated with translational activity and an increase in stress response. There were also several upregulated stress genes specific to the LSMMG condition during the transition to stationary phase growth. The ∆hfq mutants exhibited a distinctive transcriptome profile with a significant increase in transcripts associated with flagellar synthesis and transcriptional regulators under LSMMG conditions compared to gravity controls. These results indicate the loss of Hfq significantly influences gene expression under LSMMG conditions in a bacterial symbiont. Together, these results improve our understanding of the mechanisms by which microgravity alters the physiology of beneficial host-associated microbes.

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.