a-gnotobiotic-system-for-studying-microbiome-assembly-phyllosphere

A Gnotobiotic System for Studying Microbiome Assembly in the Phyllosphere and in Vegetable Fermentation

The phyllosphere, the above ground portion of the plant that can be colonized by microbes, is a useful model system to identify processes of microbial ...

Tufts University

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. 

Fermented foods as experimentally tractable microbial ecosystems.

Emerging evidence supports novel roles for vitamin K in cardiometabolic health, some of which may be unique to the bacterially synthesized vitamin K forms known as menaquinones. However, factors influencing menaquinone biosynthesis by the gut microbiota and associations with cardiometabolic health have not been examined.

Fecal menaquinone profiles of overweight adults are associated with gut microbiota composition during a gut microbiota-targeted dietary intervention.

Many metagenomic sequencing studies have observed the presence of closely related bacterial species or genotypes in the same microbiome. Previous attempts to explain these patterns of microdiversity have focused on the abiotic environment, but few have considered how biotic interactions could drive patterns of microbiome diversity. We dissected the patterns, processes, and mechanisms shaping the ecological distributions of three closely related Staphylococcus species in cheese rind biofilms. Paradoxically, the most abundant species (S. equorum) is the slowest colonizer and weakest competitor based on growth and competition assays in the laboratory. Through in vitro community reconstructions, we determined that biotic interactions with neighboring fungi help resolve this paradox. Species-specific stimulation of the poor competitor by fungi of the genus Scopulariopsis allows S. equorum to dominate communities in vitro as it does in situ Results of comparative genomic and transcriptomic experiments indicate that iron utilization pathways, including a homolog of the S. aureus staphyloferrin B siderophore operon pathway, are potential molecular mechanisms underlying Staphylococcus-Scopulariopsis interactions. Our integrated approach demonstrates that fungi can structure the ecological distributions of closely related bacterial species, and the data highlight the importance of bacterium-fungus interactions in attempts to design and manipulate microbiomes.

Biotic Interactions Shape the Ecological Distributions of Staphylococcus Species.

Erratum for Kastman et al., "Biotic Interactions Shape the Ecological Distributions of  Species".

Regeneration is regulated not only by chemical signals but also by physical processes, such as bioelectric gradients. How these may change in the absence of the normal gravitational and geomagnetic fields is largely unknown. Planarian flatworms were moved to the International Space Station for 5 weeks, immediately after removing their heads and tails. A control group in spring water remained on Earth. No manipulation of the planaria occurred while they were in orbit, and space-exposed worms were returned to our laboratory for analysis. One animal out of 15 regenerated into a double-headed phenotype-normally an extremely rare event. Remarkably, amputating this double-headed worm again, in plain water, resulted again in the double-headed phenotype. Moreover, even when tested 20 months after return to Earth, the space-exposed worms displayed significant quantitative differences in behavior and microbiome composition. These observations may have implications for human and animal space travelers, but could also elucidate how microgravity and hypomagnetic environments could be used to trigger desired morphological, neurological, physiological, and bacteriomic changes for various regenerative and bioengineering applications.

Planarian regeneration in space: Persistent anatomical, behavioral, and bacteriological changes induced by space travel.

Acquisition of genes through horizontal gene transfer (HGT) allows microbes to rapidly gain new capabilities and adapt to new or changing environments. Identifying widespread HGT regions within multispecies microbiomes can pinpoint the molecular mechanisms that play key roles in microbiome assembly. We sought to identify horizontally transferred genes within a model microbiome, the cheese rind. Comparing 31 newly sequenced and 134 previously sequenced bacterial isolates from cheese rinds, we identified over 200 putative horizontally transferred genomic regions containing 4733 protein coding genes. The largest of these regions are enriched for genes involved in siderophore acquisition, and are widely distributed in cheese rinds in both Europe and the US. These results suggest that HGT is prevalent in cheese rind microbiomes, and that identification of genes that are frequently transferred in a particular environment may provide insight into the selective forces shaping microbial communities.

Extensive horizontal gene transfer in cheese-associated bacteria.

Most studies of bacterial motility have examined small-scale (micrometer-centimeter) cell dispersal in monocultures. However, bacteria live in multispecies communities, where interactions with other microbes may inhibit or facilitate dispersal. Here, we demonstrate that motile bacteria in cheese rind microbiomes use physical networks created by filamentous fungi for dispersal, and that these interactions can shape microbial community structure. Serratia proteamaculans and other motile cheese rind bacteria disperse on fungal networks by swimming in the liquid layers formed on fungal hyphae. RNA-sequencing, transposon mutagenesis, and comparative genomics identify potential genetic mechanisms, including flagella-mediated motility, that control bacterial dispersal on hyphae. By manipulating fungal networks in experimental communities, we demonstrate that fungal-mediated bacterial dispersal can shift cheese rind microbiome composition by promoting the growth of motile over non-motile community members. Our single-cell to whole-community systems approach highlights the interactive dynamics of bacterial motility in multispecies microbiomes.

Fungal networks shape dynamics of bacterial dispersal and community assembly in cheese rind microbiomes.

As troves of microbiome sequencing data provide improved resolution of patterns of microbial diversity, new approaches are needed to understand what controls these patterns. Many microbial ecologists are using cultivated model microbial communities to address this challenge. These systems provide opportunities to identify drivers of microbiome assembly, but key challenges and limitations need to be carefully considered in their development, implementation, and interpretation. How well do model microbial communities mimic  communities in terms of taxonomic diversity, trophic levels, intraspecific diversity, and the abiotic environment? What are the best ways to manipulate and measure inputs and outputs in model community experiments? In this perspective, I briefly address some of these challenges on the basis of our experience developing fermented food model communities. Future work integrating genetic and molecular approaches with cultivated model microbial communities will allow microbial ecology to develop a more mechanistic understanding of microbiome diversity.

Using Cultivated Microbial Communities To Dissect Microbiome Assembly: Challenges, Limitations, and the Path Ahead.

 The plant-based form of vitamin K (phylloquinone, vitamin K-1) has been well quantified in the US diet. Menaquinones (vitamin K-2) are another class of vitamin K compounds that differ from phylloquinone in the length and saturation of their side chain, but they have not been well characterized in foods.  The objectives of this study were to ) quantify phylloquinone and the different forms of menaquinones [menaquinone (MK) 4-MK13] in milk, yogurt, Greek yogurt, creams, and cheeses and ) compare the menaquinone contents of full-fat, reduced-fat, and nonfat dairy products.  All dairy samples were either obtained from the USDA National Food and Nutrient Analysis Program or purchased from retail outlets. Phylloquinone and menaquinone concentrations in these dairy products were quantified by mass spectrometry technology.  Full-fat dairy products contained appreciable amounts of menaquinones, primarily in the forms of MK9, MK10, and MK11. We also measured modest amounts of phylloquinone, MK4, MK8, and MK12 in these products. In contrast, there was little MK5-7 or MK13 detected in the majority of dairy products. The total vitamin K contents of soft cheese, blue cheese, semi-soft cheese, and hard cheese were (means ± SEMs): 506 ± 63, 440 ± 41, 289 ± 38, and 282 ± 5.0 µg/100 g, respectively. Nonfermented cheeses, such as processed cheese, contained lower amounts of vitamin K (98 ± 11 µg/100 g). Reduced-fat or fat-free dairy products contained ∼5-22% of the vitamin K found in full-fat equivalents. For example, total vitamin K contents of full-fat milk (4% fat), 2%-fat milk, 1%-fat milk, and nonfat milk were 38.1 ± 8.6, 19.4 ± 7.7, 12.9 ± 2.0, and 7.7 ± 2.9 µg/100 g, respectively.  To the best of our knowledge, this is the first report of menaquinone contents of US dairy products. Findings indicate that the amount of vitamin K contents in dairy products is high and proportional to the fat content of the product.

Multiple Vitamin K Forms Exist in Dairy Foods.

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.

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

Microbial communities of fermented food microbiomes typically exhibit predictable patterns of microbial succession. However, the biochemical mechanisms that control the diversity and dynamics of these communities are not well described. Interactions between bacteria and fungi may be one mechanism controlling the development of cheese rind microbiomes. This study characterizes a specific bacterium-fungus interaction previously discovered on cheese rinds between the bacterium Glutamicibacter arilaitensis (formerly Arthrobacter arilaitensis) and fungi of the genus  and identifies the specialized metabolites produced during cocultures. G. arilaitensis was previously shown to produce an unknown pink pigment in response to the presence of . Using a combination of mass spectrometry, nuclear magnetic resonance (NMR), and transcriptome sequencing (RNA-seq), we determined that this pigment production is associated with production of coproporphyrin III. The discovery that coproporphyrin III preferentially bound zinc over other trace metals found in cheese curds highlights the value of using analytical chemistry to confirm identity of predicted chemical species.  Bacterium-fungus interactions play key roles in the assembly of cheese rind microbial communities, but the molecular mechanisms underlying these interactions are poorly characterized. Moreover, millions of people around the world enjoy eating cheeses and cheese rinds, but our understanding of the diversity of microbial metabolites ingested during cheese consumption is limited. The discovery of zinc coproporphyrin III as the cause of pink pigment production by Glutamicibacter arilaitensis suggests that secretion of this molecule is important for microbial acquisition of trace metals.

Coproporphyrin III Produced by the Bacterium  Binds Zinc and Is Upregulated by Fungi in Cheese Rinds.

Planarian flatworms have been used for over a century as models for regeneration. Planarians live in aquatic environments with constant exposure to microbes, but the mechanisms by which bacteria may mediate planarian regeneration are largely unknown. We characterized the microbiome of laboratory populations of the planarian Dugesia japonica and determined how individual bacteria impact D. japonica regeneration. Eight to ten taxa in the phyla Bacteroidetes and Proteobacteria consistently occur across planarian colonies housed in different research laboratories. Individual members of the D. japonica microbiome can delay regeneration including the development of eye spots and blastema formation. The microbial metabolite indole is produced in significant quantities by two bacteria that are consistently found in the D. japonica microbiome and contributes to delays in regeneration. Collectively, these results provide a baseline understanding of the bacteria associated with the planarian D. japonica and demonstrate how metabolite production by host-associated microbes can affect regeneration.

The Bacterial Metabolite Indole Inhibits Regeneration of the Planarian Flatworm Dugesia japonica.

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. 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.

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

Fermented foods provide novel ecological opportunities for natural populations of microbes to evolve through successive recolonization of resource-rich substrates. Comparative genomic data have reconstructed the evolutionary histories of microbes adapted to food environments, but experimental studies directly demonstrating the process of domestication are lacking for most fermented food microbes. Here, we show that during adaptation to cheese, phenotypic and metabolomic traits of wild  molds rapidly change to produce domesticated phenotypes with properties similar to those of the industrial cultures used to make Camembert and other bloomy rind cheeses. Over a period of just a few weeks, populations of wild  strains serially passaged on cheese had reduced pigment, spore, and mycotoxin production. Domesticated strains also had a striking change in volatile metabolite production, shifting from production of earthy or musty volatile compounds (e.g., geosmin) to fatty and cheesy volatiles (e.g., 2-nonanone, 2-undecanone). RNA sequencing demonstrated a significant decrease in expression of 356 genes in domesticated strains, with an enrichment of many secondary metabolite production pathways in these downregulated genes. By manipulating the presence of neighboring microbial species and overall resource availability, we demonstrate that the limited competition and high nutrient availability of the cheese environment promote rapid trait evolution of  molds. Industrial cultures of filamentous fungi are used to add unique aesthetics and flavors to cheeses and other microbial foods. How these microbes adapted to live in food environments is generally unknown as most microbial domestication is unintentional. Our work demonstrates that wild molds closely related to the starter culture  can readily lose traits and quickly shift toward producing desirable aroma compounds. In addition to experimentally demonstrating a putative domestication pathway for , our work suggests that wild  isolates could be rapidly domesticated to produce new flavors and aesthetics in fermented foods.

Rapid Phenotypic and Metabolomic Domestication of Wild  Molds on Cheese.

An integrative pattern-process-mechanism approach is revealing the roles of biotic interactions in microbiome assembly. Patterns of microbiome diversity observed in metagenomic studies can be partly explained by interaction processes (e.g. competition, facilitation) and underlying molecular or genetic mechanisms (e.g. antibiotic production, nutrient cross-feeding). Exciting opportunities remain to fully understand the significance and generalizability of biotic interactions within microbiomes. Many microbial interactions have been studied by chasing easily quantifiable phenotypes including changes in growth or pigmentation, but it is likely that diverse cryptic interactions occur without obvious growth changes or macroscopic phenotypes. A narrow phylogenetic breadth of well-studied microbes limits our understanding of whether there are conserved genetic or molecular mechanisms of microbial interactions. Biotic interactions can impose strong selective pressures that could shape rates and modes of microbial evolution, but few studies have examined the evolutionary consequences of interactions within microbiomes. Continued exploration of the chemical and genetic mechanisms underlying biotic interactions may provide novel tools to manipulate and manage microbiomes.

Causes and consequences of biotic interactions within microbiomes.

Cheese rind microbiomes are useful model systems for identifying the mechanisms that control microbiome diversity. Here, we describe the methods we have optimized to first deconstruct in situ cheese rind microbiome diversity and then reconstruct that diversity in laboratory environments to conduct controlled microbiome manipulations. Most cheese rind microbial species, including bacteria, yeasts, and filamentous fungi, can be easily cultured using standard lab media. Colony morphologies of taxa are diverse and can often be used to distinguish taxa at the phylum and sometimes even genus level. Through the use of cheese curd agar medium, thousands of unique community combinations or microbial interactions can be assessed. Transcriptomic experiments and transposon mutagenesis screens can pinpoint mechanisms of interactions between microbial species. Our general approach of creating a tractable synthetic microbial community from cheese can be easily applied to other fermented foods to develop other model microbiomes. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Isolation of cheese rind microbial communities Support Protocol 1: Preparation of plate count agar with milk and salt Basic Protocol 2: Identification of cheese rind bacterial and fungal isolates using 16S and ITS sequences Basic Protocol 3: Preparation of experimental glycerol stocks of yeasts and bacteria Basic Protocol 4: Preparation of experimental glycerol stocks of filamentous fungi Basic Protocol 5: Reconstruction of cheese rind microbial communities in vitro Support Protocol 2: Preparation of lyophilized and powdered cheese curd Support Protocol 3: Preparation of 10% cheese curd agar plates and tubes Basic Protocol 6: Interaction screens using responding lawns Support Protocol 4: Preparation of liquid 2% cheese curd Basic Protocol 7: Experimental evolution Basic Protocol 8: Measuring community function: pH/acidification Basic Protocol 9: Measuring community function: Pigment production Basic Protocol 10: RNA sequencing of cheese rind biofilms.

Article	Year
Fermented foods as experimentally tractable microbial ecosystems. Cell\| PubMed ID: 25815984	2015
Fecal menaquinone profiles of overweight adults are associated with gut microbiota composition during a gut microbiota-targeted dietary intervention. The American journal of clinical nutrition\| PubMed ID: 26016865	2015
Biotic Interactions Shape the Ecological Distributions of Staphylococcus Species. mBio\| PubMed ID: 27795388	2016
Erratum for Kastman et al., "Biotic Interactions Shape the Ecological Distributions of Species". mBio\| PubMed ID: 28325768	2017
Planarian regeneration in space: Persistent anatomical, behavioral, and bacteriological changes induced by space travel. Regeneration (Oxford, England)\| PubMed ID: 28616247	2017
Extensive horizontal gene transfer in cheese-associated bacteria. eLife\| PubMed ID: 28644126	2017
Fungal networks shape dynamics of bacterial dispersal and community assembly in cheese rind microbiomes. Nature communications\| PubMed ID: 29362365	2018
Using Cultivated Microbial Communities To Dissect Microbiome Assembly: Challenges, Limitations, and the Path Ahead. mSystems\| PubMed ID: 29629414	2018
Multiple Vitamin K Forms Exist in Dairy Foods. Current developments in nutrition\| PubMed ID: 29955705	2017
Indigo- and indirubin-producing strains of Proteus and Psychrobacter are associated with purple rind defect in a surface-ripened cheese. Food microbiology\| PubMed ID: 30166186	2018
Coproporphyrin III Produced by the Bacterium Binds Zinc and Is Upregulated by Fungi in Cheese Rinds. mSystems\| PubMed ID: 30175236	2018
The Bacterial Metabolite Indole Inhibits Regeneration of the Planarian Flatworm Dugesia japonica. iScience\| PubMed ID: 30521984	2018
Establishment Limitation Constrains the Abundance of Lactic Acid Bacteria in the Napa Cabbage Phyllosphere. Applied and environmental microbiology\| PubMed ID: 31003989	2019
Rapid Phenotypic and Metabolomic Domestication of Wild Molds on Cheese. mBio\| PubMed ID: 31615965	2019
Causes and consequences of biotic interactions within microbiomes. Current opinion in microbiology\| PubMed ID: 31627129	2019
Deconstructing and Reconstructing Cheese Rind Microbiomes for Experiments in Microbial Ecology and Evolution. Current protocols in microbiology\| PubMed ID: 31891451	2020

Benjamin E. Wolfe

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