Fermented foods as experimentally tractable microbial ecosystems.

Microbial communities of fermented foods have provided humans with tools for preservation and flavor development for thousands of years. These simple, reproducible, accessible, culturable, and easy-to-manipulate systems also provide opportunities for dissecting the mechanisms of microbial community formation. Fermented foods can be valuable models for processes in less tractable microbiota.

Cheese rind communities provide tractable systems for in situ and in vitro studies of microbial diversity.

Tractable microbial communities are needed to bridge the gap between observations of patterns of microbial diversity and mechanisms that can explain these patterns. We developed cheese rinds as model microbial communities by characterizing in situ patterns of diversity and by developing an in vitro system for community reconstruction. Sequencing of 137 different rind communities across 10 countries revealed 24 widely distributed and culturable genera of bacteria and fungi as dominant community members. Reproducible community types formed independent of geographic location of production. Intensive temporal sampling demonstrated that assembly of these communities is highly reproducible. Patterns of community composition and succession observed in situ can be recapitulated in a simple in vitro system. Widespread positive and negative interactions were identified between bacterial and fungal community members. Cheese rind microbial communities represent an experimentally tractable system for defining mechanisms that influence microbial community assembly and function.

Microbiome Assembly in Fermented Foods.

For thousands of years, humans have enjoyed the novel flavors, increased shelf-life, and nutritional benefits that microbes provide in fermented foods and beverages. Recent sequencing surveys of ferments have mapped patterns of microbial diversity across space, time, and production practices. But a mechanistic understanding of how fermented food microbiomes assemble has only recently begun to emerge. Using three foods as case studies (surface-ripened cheese, sourdough starters, and fermented vegetables), we use an ecological and evolutionary framework to identify how microbial communities assemble in ferments. By combining in situ sequencing surveys with in vitro models, we are beginning to understand how dispersal, selection, diversification, and drift generate the diversity of fermented food communities. Most food producers are unaware of the ecological processes occurring in their production environments, but the theory and models of ecology and evolution can provide new approaches for managing fermented food microbiomes, from farm to ferment.

A phylogenomic perspective on interspecific competition.

Evolutionary processes may have substantial impacts on community assembly, but evidence for phylogenetic relatedness as a determinant of interspecific interaction strength remains mixed. In this perspective, we consider a possible role for discordance between gene trees and species trees in the interpretation of phylogenetic signal in studies of community ecology. Modern genomic data show that the evolutionary histories of many taxa are better described by a patchwork of histories that vary along the genome rather than a single species tree. If a subset of genomic loci harbour trait-related genetic variation, then the phylogeny at these loci may be more informative of interspecific trait differences than the genome background. We develop a simple method to detect loci harbouring phylogenetic signal and demonstrate its application through a proof-of-principle analysis of Penicillium genomes and pairwise interaction strength. Our results show that phylogenetic signal that may be masked genome-wide could be detectable using phylogenomic techniques and may provide a window into the genetic basis for interspecific interactions.

Genomic, transcriptomic, and ecological diversity of Penicillium species in cheese rind microbiomes.

Although Penicillium molds can have significant impacts on agricultural, industrial, and biomedical systems, the ecological roles of Penicillium species in many microbiomes are not well characterized. Here we utilized a collection of 35 Penicillium strains isolated from cheese rinds to broadly investigate the genomic potential for secondary metabolism in cheese-associated Penicillium species, the impact of Penicillium on bacterial community assembly, and mechanisms of Penicillium-bacteria interactions. Using antiSMASH, we identified 1558 biosynthetic gene clusters, 406 of which were mapped to known pathways, including several mycotoxins and antimicrobial compounds. By measuring bacterial abundance and fungal mRNA expression when culturing representative Penicillium strains with a cheese rind bacterial community, we observed divergent impacts of different Penicillium strains, from strong inhibitors of bacterial growth to those with no impact on bacterial growth or community composition. Through differential mRNA expression analyses, Penicillium strains demonstrated limited differential gene expression in response to the bacterial community. We identified a few shared responses between the eight tested Penicillium strains, primarily upregulation of nutrient metabolic pathways, but we did not identify a conserved fungal response to growth in a multispecies community. These results in tandem suggest high variation among cheese-associated Penicillium species in their ability to shape bacterial community development and highlight important ecological diversity within this iconic genus.

Death caps (Amanita phalloides) frequently establish from sexual spores, but individuals can grow large and live for more than a decade in invaded forests.

Global change is reshaping Earth's biodiversity, but the changing distributions of nonpathogenic fungi remain largely undocumented, as do mechanisms enabling invasions. The ectomycorrhizal Amanita phalloides is native to Europe and invasive in North America. Using population genetics and genomics, we sought to describe the life history traits of this successfully invading symbiotic fungus. To test whether death caps spread underground using hyphae, or aboveground using sexual spores, we mapped and genotyped mushrooms from European and US sites. Larger genetic individuals (genets) would suggest spread mediated by vegetative growth, while many small genets would suggest dispersal mediated by spores. To test whether genets are ephemeral or persistent, we also sampled from populations over time. At nearly every site and across all time points, mushrooms resolve into small genets. Individuals frequently establish from sexual spores. But at one Californian site, a single individual measuring nearly 10 m across dominated. At two Californian sites, the same genetic individuals were discovered in 2004, 2014, and 2015, suggesting single individuals (both large and small) can reproduce repeatedly over relatively long timescales. A flexible life history strategy combining both mycelial growth and spore dispersal appears to underpin the invasion of this deadly perennial ectomycorrhizal fungus.

Metabolomics of bacterial-fungal pairwise interactions reveal conserved molecular mechanisms.

Bacterial-fungal interactions (BFIs) can shape the structure of microbial communities, but the small molecules mediating these BFIs are often understudied. We explored various optimization steps for our microbial culture and chemical extraction protocols for bacterial-fungal co-cultures, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed that metabolomic profiles are mainly comprised of fungi derived features, indicating that fungi are the key contributors to small molecules in BFIs. LC-inductively coupled plasma MS (LC-ICP-MS) and MS/MS based dereplication using database searching revealed the presence of several known fungal specialized metabolites and structurally related analogues in these extracts, including siderophores such as desferrichrome, desferricoprogen, and palmitoylcoprogen. Among these analogues, a novel putative coprogen analogue possessing a terminal carboxylic acid motif was identified from Scopulariopsis sp. JB370, a common cheese rind fungus, and its structure was elucidated via MS/MS fragmentation. Based on these findings, filamentous fungal species appear to be capable of producing multiple siderophores with potentially different biological roles (i.e. various affinities for different forms of iron). These findings highlight that fungal species are important contributors to microbiomes via their production of abundant specialized metabolites and that elucidating their role in complex communities should continue to be a priority.

Spatial variability in localization biases predicts crowding performance.

Visual processing varies substantially across individuals, and prior work has shown significant individual differences in fundamental processes such as spatial localization. For example, when asked to report the location of a briefly flashed target in the periphery, different observers systematically misperceive its location in an idiosyncratic manner, showing different patterns of reproduction error across visual field locations. In this study, we tested whether these individual differences may propagate to other stages of visual processing, affecting the strength of visual crowding, which depends on the spacing between objects in the periphery. We, therefore, investigated the relationship between observers' idiosyncratic biases in localization and the strength of crowding to determine whether these spatial biases limit peripheral object recognition. To examine this relationship, we measured the strength of crowding at 12 locations at 8° eccentricity, in addition to the perceived spacing between pairs of Gaussian patches at these same locations. These measurements show an association between variability in crowding strength and perceived spacing at the same visual field locations: at locations where a participant experienced stronger crowding, their perceived spacing was smaller, and vice versa. We demonstrate that spatial heterogeneity in perceived spacing affects observers' ability to recognize objects in the periphery. Our results support the idea that variability in both spatial sensitivity and bias contribute to variability in the strength of crowding and bolster the account that variability in spatial coding may propagate across multiple stages of visual processing.

Road Hazard Stimuli: Annotated naturalistic road videos for studying hazard detection and scene perception.

Driving requires vision, yet there is little empirical data about how vision and cognition support safe driving. It is difficult to study perception during natural driving because the experimental rigor required would be dangerous and unethical to implement on the road. The driving environment is complex, dynamic, and immensely variable, making it extremely challenging to accurately replicate in simulation. Our proposed solution is to study vision using stimuli which reflect this inherent complexity by using footage of real driving situations. To this end, we curated a set of 750 crowd-sourced video clips (434 hazard and 316 no-hazard clips), which have been spatially, temporally, and categorically annotated. These annotations describe where the hazard appears, what it is, and when it occurs. In addition, perceived dangerousness changes from moment to moment and is not a simple binary detection judgement. To capture this more granular aspect of our stimuli, we asked 48 observers to rate the perceived hazardousness of 1356 brief video clips taken from these 750 source clips on a continuous scale. These ratings span the entire scale, have high interrater agreement, and are robust to driving history. This novel stimulus set is not only useful for understanding drivers' ability to detect hazards, but is also a tool for studying dynamic scene perception and other aspects of visual function. While this stimulus set was originally designed for behavioral studies, researchers interested in other areas such as traffic safety or computer vision may also find this dataset a useful resource.

Chronic stress and captivity alter the cloacal microbiome of a wild songbird.

There are complex interactions between an organism's microbiome and its response to stressors, often referred to as the 'gut-brain axis'; however, the ecological relevance of this axis in wild animals remains poorly understood. Here, we used a chronic mild stress protocol to induce stress in wild-caught house sparrows (Passer domesticus), and compared microbial communities among stressed animals, those recovering from stress, captive controls (unstressed) and a group not brought into captivity. We assessed changes in microbial communities and abundance of shed microbes by culturing cloacal samples on multiple media to select for aerobic and anaerobic bacteria and fungi. We complemented this with cultivation-independent 16S and ITS rRNA gene amplification and sequencing, pairing these results with host physiological and immune metrics, including body mass change, relative spleen mass and plasma corticosterone concentrations. We found significant effects of stress and captivity on the house sparrow microbiomes, with stress leading to an increased relative abundance of endotoxin-producing bacteria - a possible mechanism for the hyperinflammatory response observed in captive avians. While we found evidence that the microbiome community partially recovers after stress cessation, animals may lose key taxa, and the abundance of endotoxin-producing bacteria persists. Our results suggest an overall link between chronic stress, host immune system and the microbiome, with the loss of potentially beneficial taxa (e.g. lactic acid bacteria), and an increase in endotoxin-producing bacteria due to stress and captivity. Ultimately, consideration of the host's microbiome may be useful when evaluating the impact of stressors on individual and population health.

Toward a Theory of Visual Information Acquisition in Driving.

OBJECTIVE: The aim of this study is to describe information acquisition theory, explaining how drivers acquire and represent the information they need. BACKGROUND: While questions of what drivers are aware of underlie many questions in driver behavior, existing theories do not directly address how drivers in particular and observers in general acquire visual information. Understanding the mechanisms of information acquisition is necessary to build predictive models of drivers' representation of the world and can be applied beyond driving to a wide variety of visual tasks. METHOD: We describe our theory of information acquisition, looking to questions in driver behavior and results from vision science research that speak to its constituent elements. We focus on the intersection of peripheral vision, visual attention, and eye movement planning and identify how an understanding of these visual mechanisms and processes in the context of information acquisition can inform more complete models of driver knowledge and state. RESULTS: We set forth our theory of information acquisition, describing the gap in understanding that it fills and how existing questions in this space can be better understood using it. CONCLUSION: Information acquisition theory provides a new and powerful way to study, model, and predict what drivers know about the world, reflecting our current understanding of visual mechanisms and enabling new theories, models, and applications. APPLICATION: Using information acquisition theory to understand how drivers acquire, lose, and update their representation of the environment will aid development of driver assistance systems, semiautonomous vehicles, and road safety overall.

American artisan cheese quality and spoilage: A survey of cheesemakers' concerns and needs.

Production of artisan cheeses, including surface-ripened cheeses, has increased in the United States over the past 2 decades. Although many of these cheesemakers report unique quality and spoilage problems during production, a systematic assessment of the quality concerns facing this sector of specialty cheese production has not been conducted. Here we report the effects of microbial spoilage and quality issues on US artisan cheese production. In a survey of 61 cheesemakers, the most common issues reported were undesirable surface molds (71%) and incorrect or unexpected colors or pigments on rinds (54%). When asked, 18% of participants indicated that they were extremely concerned about quality and spoilage problems, and they indicated that their quality standards are frequently not met, either annually (39%) or monthly (33%). Although most of the respondents (62%) said that just 0 to 5% of their cheese was lost or rendered less valuable due to quality issues annually, a small number (7% combined) reported large losses of 20 to 30% or >30% of their product lost or rendered less valuable. Almost all respondents (95%) agreed that improved quality would reduce waste, increase profits, and improve production. The survey respondents indicated in open response questions that they want access to more online resources related to quality issues and digital forums to discuss issues with experts and peers when problems arise. These findings represent the first attempt to document and estimate the effect of quality and spoilage on the American artisan cheese industry. Future work should investigate what technologies, interventions, or information could reduce losses from these problems.

Fungal volatiles mediate cheese rind microbiome assembly.

In vitro studies in plant, soil, and human systems have shown that microbial volatiles can mediate microbe-microbe or microbe-host interactions. These previous studies have often used artificially high concentrations of volatiles compared to in situ systems and have not demonstrated the roles volatiles play in mediating community-level dynamics. We used the notoriously volatile cheese rind microbiome to identify bacteria responsive to volatiles produced by five widespread cheese fungi. Vibrio casei had the strongest growth stimulation when exposed to all fungi. In multispecies community experiments, fungal volatiles caused a shift to a Vibrio-dominated community, potentially explaining the widespread occurrence of Vibrio in surface-ripened cheeses. RNA sequencing identified activation of the glyoxylate shunt as a possible mechanism underlying volatile-mediated growth promotion and community assembly. Our study demonstrates how airborne chemicals could be used to control the composition of microbiomes and illustrates how volatiles may impact the development of cheese rinds.

Diet rapidly and reproducibly alters the human gut microbiome.

Long-term dietary intake influences the structure and activity of the trillions of microorganisms residing in the human gut, but it remains unclear how rapidly and reproducibly the human gut microbiome responds to short-term macronutrient change. Here we show that the short-term consumption of diets composed entirely of animal or plant products alters microbial community structure and overwhelms inter-individual differences in microbial gene expression. The animal-based diet increased the abundance of bile-tolerant microorganisms (Alistipes, Bilophila and Bacteroides) and decreased the levels of Firmicutes that metabolize dietary plant polysaccharides (Roseburia, Eubacterium rectale and Ruminococcus bromii). Microbial activity mirrored differences between herbivorous and carnivorous mammals, reflecting trade-offs between carbohydrate and protein fermentation. Foodborne microbes from both diets transiently colonized the gut, including bacteria, fungi and even viruses. Finally, increases in the abundance and activity of Bilophila wadsworthia on the animal-based diet support a link between dietary fat, bile acids and the outgrowth of microorganisms capable of triggering inflammatory bowel disease. In concert, these results demonstrate that the gut microbiome can rapidly respond to altered diet, potentially facilitating the diversity of human dietary lifestyles.

Glanceable, legible typography over complex backgrounds.

Modern digital interfaces display typeface in ways new to the 500 year old art of typography, driving a shift in reading from primarily long-form to increasingly short-form. In safety-critical settings, such at-a-glance reading competes with the need to understand the environment. To keep both type and the environment legible, a variety of 'middle layer' approaches are employed. But what is the best approach to presenting type over complex backgrounds so as to preserve legibility? This work tests and ranks middle layers in three studies. In the first study, Gaussian blur and semi-transparent 'scrim' middle layer techniques best maximise legibility. In the second, an optimal combination of the two is identified. In the third, letter-localised middle layers are tested, with results favouring drop-shadows. These results, discussed in mixed reality (MR) including overlays, virtual reality (VR), and augmented reality (AR), considers a future in which glanceable reading amidst complex backgrounds is common. Practitioner summary: Typography over complex backgrounds, meant to be read and understood at a glance, was once niche but today is a growing design challenge for graphical user interface HCI. We provide a technique, evidence-based strategies, and illuminating results for maximising legibility of glanceable typography over complex backgrounds. Abbreviations: AR: augmented reality; VR: virtual reality; HUD: head-up display; OLED: organic light-emitting diode; UX: user experience; MS: millisecond; CM: centimeter.

Establishment Limitation Constrains the Abundance of Lactic Acid Bacteria in the Napa Cabbage Phyllosphere.

Patterns of phyllosphere diversity have become increasingly clear with high-throughput sequencing surveys, but the processes that control phyllosphere diversity are still emerging. Through a combination of lab and field experiments using Napa cabbage and lactic acid bacteria (LAB), we examined how dispersal and establishment processes shape the ecological distributions of phyllosphere bacteria. We first determined the abundance and diversity of LAB on Napa cabbage grown at three sites using both culture-based approaches and 16S rRNA gene amplicon sequencing. Across all sites, LAB made up less than 0.9% of the total bacterial community abundance. To assess whether LAB were low in abundance in the Napa cabbage phyllosphere due to a limited abundance in local species pools (source limitation), we quantified LAB in leaf and soil samples across 51 vegetable farms and gardens throughout the northeastern United States. Across all sites, LAB comprised less than 3.2% of the soil bacterial communities and less than 1.6% of phyllosphere bacterial communities. To assess whether LAB are unable to grow in the phyllosphere even if they dispersed at high rates (establishment limitation), we used a gnotobiotic Napa cabbage system in the lab with experimental communities mimicking various dispersal rates of LAB. Even at high dispersal rates, LAB became rare or completely undetectable in experimental communities, suggesting that they are also establishment limited. Collectively, our data demonstrate that the low abundance of LAB in phyllosphere communities may be explained by establishment limitation.IMPORTANCE The quality and safety of vegetable fermentations are dependent on the activities of LAB naturally present in the phyllosphere. Despite their critical role in determining the success of fermentation, the processes that determine the abundance and diversity of LAB in vegetables used for fermentation are poorly characterized. Our work demonstrates that the limited ability of LAB to grow in the cabbage phyllosphere environment may constrain their abundance on cabbage leaves. These results suggest that commercial fermentation of Napa cabbage proceeds despite low and variable abundances of LAB across different growing regions. Propagule limitation may also explain ecological distributions of other rare members of phyllosphere microbes.

Predicting road scenes from brief views of driving video.

If a vehicle is driving itself and asks the driver to take over, how much time does the driver need to comprehend the scene and respond appropriately? Previous work on natural-scene perception suggests that observers quickly acquire the gist, but gist-level understanding may not be sufficient to enable action. The moving road environment cannot be studied with static images alone, and safe driving requires anticipating future events. We performed two experiments to examine how quickly subjects could perceive the road scenes they viewed and make predictions based on their mental representations of the scenes. In both experiments, subjects performed a temporal-order prediction task, in which they viewed brief segments of road video and indicated which of two still frames would come next after the end of the video. By varying the duration of the previewed video clip, we determined the viewing duration required for accurate prediction of recorded road scenes. We performed an initial experiment on Mechanical Turk to explore the space, and a follow-up experiment in the lab to address questions of road type and stimulus discriminability. Our results suggest that representations which enable prediction can be developed from brief views of a road scene, and that different road environments (e.g., city versus highway driving) have a significant impact on the viewing durations drivers require to make accurate predictions of upcoming scenes.

Unifying Visual Space Across the Left and Right Hemifields.

Visual space is perceived as continuous and stable even though visual inputs from the left and right visual fields are initially processed separately within the two cortical hemispheres. In the research reported here, we examined whether the visual system utilizes a dynamic recalibration mechanism to integrate these representations and to maintain alignment across the visual fields. Subjects adapted to randomly oriented moving lines that straddled the vertical meridian; these lines were vertically offset between the left and right hemifields. Subsequent vernier alignment judgments revealed a negative aftereffect: An offset in the same direction as the adaptation was required to correct the perceived misalignment. This aftereffect was specific to adaptation to vertical, but not horizontal, misalignments and also occurred following adaptation to movie clips and patterns without coherent motion. Our results demonstrate that the visual system unifies the left and right halves of visual space by continuously recalibrating the alignment of elements across the visual fields.

Indigo- and indirubin-producing strains of Proteus and Psychrobacter are associated with purple rind defect in a surface-ripened cheese.

The rinds of surface-ripened cheeses have expected aesthetic properties, including distinct colors, that contribute to overall quality and consumer acceptance. Atypical rind pigments are frequently reported in small-scale cheese production, but the causes of these color defects are largely unknown. We provide a potential microbial explanation for a striking purple rind defect in a surface-ripened cheese. A cheese producer in the United States reported to us several batches of a raw-milk washed-rind cheese with a distinctly purple rind. We isolated a Proteus species from samples with purple rind defect, but not from samples with typical rind pigments, suggesting that this strain of Proteus could be causing the defect. When provided tryptophan, a precursor in the indigo and indirubin biosynthesis pathway, the isolated strain of Proteus secreted purple-red pigments. A Psychrobacter species isolated from both purple and normal rinds also secreted purple-red pigments. Using thin-layer chromatography and liquid chromatography-mass spectrometry, we confirmed that these bacteria produced indigo and indirubin from tryptophan just as closely related bacteria make these compounds in purple urine bag syndrome in medical settings. Experimental cheese communities with or without Proteus and Psychrobacter confirmed that these Proteobacteria cause purple pigmentation of cheese rinds. Reports of purple rinds in two other cheeses from Europe and the observation of pigment production by Proteus and Psychrobacter strains isolated from other cheese rinds suggest that purple rind defect has the potential to be widespread in surface-ripened cheeses.

Single cells (put a ring on it).

Proper spatial and temporal regulation of the small GTPase RhoA at the equatorial cortex represents a critical step in the specification of the division plane in eukaryotes. Despite increased understanding of the mechanisms whereby RhoA becomes active following chromosome segregation, far less is known about how RhoA is spatially regulated so that it concentrates precisely at the division site. In the April 1, 2009, issue of Genes & Development, Yoshida and colleagues (pp. 810-823) uncovered two genetically separable mechanisms whereby Rho1 is recruited to the bud neck in the budding yeast Saccharomyces cerevisiae to facilitate cytokinesis.