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.

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.

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.

Structure of a bacterial homologue of vitamin K epoxide reductase.

Vitamin K epoxide reductase (VKOR) generates vitamin K hydroquinone to sustain gamma-carboxylation of many blood coagulation factors. Here, we report the 3.6 A crystal structure of a bacterial homologue of VKOR from Synechococcus sp. The structure shows VKOR in complex with its naturally fused redox partner, a thioredoxin-like domain, and corresponds to an arrested state of electron transfer. The catalytic core of VKOR is a four transmembrane helix bundle that surrounds a quinone, connected through an additional transmembrane segment with the periplasmic thioredoxin-like domain. We propose a pathway for how VKOR uses electrons from cysteines of newly synthesized proteins to reduce a quinone, a mechanism confirmed by in vitro reconstitution of vitamin K-dependent disulphide bridge formation. Our results have implications for the mechanism of the mammalian VKOR and explain how mutations can cause resistance to the VKOR inhibitor warfarin, the most commonly used oral anticoagulant.

The significance of cooking for early hominin scavenging.

Meat scavenged by early Homo could have contributed importantly to a higher-quality diet. However, it has been suggested that because carrion would normally have been contaminated by bacteria it would have been dangerous and therefore eaten rarely prior to the advent of cooking. In this study, we quantified bacterial loads on two tissues apparently eaten by hominins, meat and bone marrow. We tested the following three hypotheses: (1) the bacterial loads on exposed surfaces of raw meat increase within 24 h to potentially dangerous levels, (2) simple roasting of meat on hot coals kills most bacteria, and (3) fewer bacteria grow on marrow than on meat, making marrow a relatively safe food. Our results supported all three hypotheses. Our experimental data imply that early hominins would have found it difficult to scavenge safely without focusing on marrow, employing strategies of carrion selection to minimize pathogen load, or cooking.

Overexpression of the rhodanese PspE, a single cysteine-containing protein, restores disulphide bond formation to an Escherichia coli strain lacking DsbA.

Escherichia coli uses the DsbA/DsbB system for introducing disulphide bonds into proteins in the cell envelope. Deleting either dsbA or dsbB or both reduces disulphide bond formation but does not entirely eliminate it. Whether such background disulphide bond forming activity is enzyme-catalysed is not known. To identify possible cellular factors that might contribute to the background activity, we studied the effects of overexpressing endogenous proteins on disulphide bond formation in the periplasm. We find that overexpressing PspE, a periplasmic rhodanese, partially restores substantial disulphide bond formation to a dsbA strain. This activity depends on DsbC, the bacterial disulphide bond isomerase, but not on DsbB. We show that overexpressed PspE is oxidized to the sulphenic acid form and reacts with substrate proteins to form mixed disulphide adducts. DsbC either prevents the formation of these mixed disulphides or resolves these adducts subsequently. In the process, DsbC itself gets oxidized and proceeds to catalyse disulphide bond formation. Although this PspE/DsbC system is not responsible for the background disulphide bond forming activity, we suggest that it might be utilized in other organisms lacking the DsbA/DsbB system.

Real-time metabolomics on living microorganisms using ambient electrospray ionization flow-probe.

Microorganisms such as bacteria and fungi produce a variety of specialized metabolites that are invaluable for agriculture, biological research, and drug discovery. However, the screening of microbial metabolic output is usually a time-intensive task. Here, we utilize a liquid microjunction surface sampling probe for electrospray ionization-mass spectrometry to extract and ionize metabolite mixtures directly from living microbial colonies grown on soft nutrient agar in Petri-dishes without any sample pretreatment. To demonstrate the robustness of the method, this technique was applied to observe the metabolic output of more than 30 microorganisms, including yeast, filamentous fungi, pathogens, and marine-derived bacteria, that were collected worldwide. Diverse natural products produced from different microbes, including Streptomyces coelicolor , Bacillus subtilis , and Pseudomonas aeruginosa are further characterized.

Inhibition of bacterial disulfide bond formation by the anticoagulant warfarin.

Blood coagulation in humans requires the activity of vitamin K epoxide reductase (VKOR), the target of the anticoagulant warfarin (Coumadin). Bacterial homologs of VKOR were recently found to participate in a pathway leading to disulfide bond formation in secreted proteins of many bacteria. Here we show that the VKOR homolog from the bacterium Mycobacterium tuberculosis, the causative agent of human tuberculosis, is inhibited by warfarin and that warfarin-resistant mutations of mycobacterial VKOR appear in similar locations to mutations found in human patients who require higher doses of warfarin. Deletion of VKOR results in a severe growth defect in mycobacteria, and the growth of M. tuberculosis is inhibited by warfarin. The bacterial VKOR homolog may represent a target for antibiotics and a model for genetic studies of human VKOR. We present a simple assay in Escherichia coli, based on a disulfide-sensitive beta-galactosidase, which can be used to screen for stronger inhibitors of the M. tuberculosis VKOR homolog.

Longitudinal, Multi-Platform Metagenomics Yields a High-Quality Genomic Catalog and Guides an In Vitro Model for Cheese Communities.

Microbiomes are intricately intertwined with human health, geochemical cycles, and food production. While many microbiomes of interest are highly complex and experimentally intractable, cheese rind microbiomes have proven to be powerful model systems for the study of microbial interactions. To provide a more comprehensive view of the genomic potential and temporal dynamics of cheese rind communities, we combined longitudinal, multi-platform metagenomics of three ripening washed-rind cheeses with whole-genome sequencing of community isolates. Sequencing-based approaches revealed a highly reproducible microbial succession in each cheese and the coexistence of closely related Psychrobacter species and enabled the prediction of plasmid and phage diversity and their host associations. In combination with culture-based approaches, we established a genomic catalog and a paired 16-member in vitro washed-rind cheese system. The combination of multi-platform metagenomic time-series data and an in vitro model provides a rich resource for further investigation of cheese rind microbiomes both computationally and experimentally. IMPORTANCE Metagenome sequencing can provide great insights into microbiome composition and function and help researchers develop testable hypotheses. Model microbiomes, such as those composed of cheese rind bacteria and fungi, allow the testing of these hypotheses in a controlled manner. Here, we first generated an extensive longitudinal metagenomic data set. This data set reveals successional dynamics, yields a phyla-spanning bacterial genomic catalog, associates mobile genetic elements with their hosts, and provides insights into functional enrichment of Psychrobacter in the cheese environment. Next, we show that members of the washed-rind cheese microbiome lend themselves to in vitro community reconstruction. This paired metagenomic data and in vitro system can thus be used as a platform for generating and testing hypotheses related to the dynamics within, and the functions associated with, cheese rind microbiomes.

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 molecule mediated 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 spp. 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 their role in complex communities should continue to be a priority.

Taking a metagenomic view of human nutrition.

PURPOSE OF REVIEW: Humans harbor microbial communities throughout the gastrointestinal tract that both respond to and modify orally ingested macronutrients, bioactive compounds, and xenobiotics; for example, the metabolism of polyphenols, heterocyclic amines, and phosphatidylcholine. However, the composition and physiological impact of our diet is also linked to the methods of food production, preparation, and consumption, which are altered by environmental and food-borne microbial communities. Metagenomic analyses spanning these various steps in human nutrition will be critical for a more comprehensive view. RECENT FINDINGS: Studies in humans and animal models have highlighted the key role that diet plays in shaping gut microbial ecology, and how the trillions of microbes in the gut (microbiota) enable the digestion of substrates inaccessible to our own human enzymes. These transformations have been implicated in a variety of diseases and disorders, ranging from obesity, inflammatory bowel disease, heart disease, to cancer. SUMMARY: In order to move towards personalized nutrition and medicine, it is important to take into account both our host and microbial genomes. The resulting metagenomic view of human nutrition, ranging from the initial biotransformations of food to digestion and the end result on human physiology, could have wide-ranging implications for food science, human evolutionary biology, and microbial ecology.

Putting microbial interactions back into community contexts.

Microbial interactions are key aspects of the biology of microbiomes. Recently, there has been a shift in the field towards studying interactions in more representative contexts, whether using multispecies model microbial communities or by looking at interactions in situ. Across diverse microbial systems, these studies have begun to identify common interaction mechanisms. These mechanisms include interactions related to toxic molecules, nutrient competition and cross-feeding, access to metals, signaling pathways, pH changes, and interactions within biofilms. Leveraging technological innovations, many of these studies have used an interdisciplinary approach combining genetic, metabolomic, imaging, and/or microfluidic techniques to gain insight into mechanisms of microbial interactions and into the impact of these interactions on microbiomes.

Higher-order interactions shape microbial interactions as microbial community complexity increases.

Non-pairwise interactions, or higher-order interactions (HOIs), in microbial communities have been described as significant drivers of emergent features in microbiomes. Yet, the re-organization of microbial interactions between pairwise cultures and larger communities remains largely unexplored from a molecular perspective but is central to our understanding and further manipulation of microbial communities. Here, we used a bottom-up approach to investigate microbial interaction mechanisms from pairwise cultures up to 4-species communities from a simple microbiome (Hafnia alvei, Geotrichum candidum, Pencillium camemberti and Escherichia coli). Specifically, we characterized the interaction landscape for each species combination involving E. coli by identifying E. coli's interaction-associated mutants using an RB-TnSeq-based interaction assay. We observed a deep reorganization of the interaction-associated mutants, with very few 2-species interactions conserved all the way up to a 4-species community and the emergence of multiple HOIs. We further used a quantitative genetics strategy to decipher how 2-species interactions were quantitatively conserved in higher community compositions. Epistasis-based analysis revealed that, of the interactions that are conserved at all levels of complexity, 82% follow an additive pattern. Altogether, we demonstrate the complex architecture of microbial interactions even within a simple microbiome, and provide a mechanistic and molecular explanation of HOIs.

Native mass spectrometry-based metabolomics identifies metal-binding compounds.

Although metals are essential for the molecular machineries of life, systematic methods for discovering metal-small molecule complexes from biological samples are limited. Here, we describe a two-step native electrospray ionization-mass spectrometry method, in which post-column pH adjustment and metal infusion are combined with ion identity molecular networking, a rule-based data analysis workflow. This method enabled the identification of metal-binding compounds in complex samples based on defined mass (m/z) offsets of ion species with the same chromatographic profiles. As this native electrospray metabolomics approach is suited to the use of any liquid chromatography-mass spectrometry system to explore the binding of any metal, this method has the potential to become an essential strategy for elucidating metal-binding molecules in biology.

Direct interaction of FliX and FlbD is required for their regulatory activity in Caulobacter crescentus.

BACKGROUND: The temporal and spatial expression of late flagellar genes in Caulobacter crescentus is activated by the transcription factor FlbD and its partner trans-acting factor FliX. The physical interaction of these two proteins represents an alternative mechanism for regulating the activity of σ54 transcription factors. This study is to characterize the interaction of the two proteins and the consequences of the interaction on their regulatory activity. RESULTS: FliX and FlbD form stable complexes, which can stand the interference of 2.65 M NaCl. The stability of FliX and FlbD was affected by the co-existence of each other. Five FliX mutants (R71A, L85K, Δ117-118, T130L, and L136K) were created by site-directed mutagenesis in conserved regions of the protein. All mutants were successfully expressed in both wild-type and ΔfliX Caulobacter strains. All but FliXL85K could rescue the motility and cell division defects of a ΔfliX mutant strain. The ability of FliX to regulate the transcription of class II and class III/IV flagellar promoters was fully diminished due to the L85K mutation. Co-immunoprecipitation experiment revealed that FliXL85K was unable to physically interact with FlbD. CONCLUSIONS: FliX interacts with FlbD and thereby directly regulates the activity of FlbD in response to flagellar assembly. Mutations in highly conserved regions of FliX could severely affect the recognition between FliX and FlbD and hence interrupt the normal progression of flagellar synthesis and other developmental events in Caulobacter.

Bacterial species exhibit diversity in their mechanisms and capacity for protein disulfide bond formation.

Protein disulfide bond formation contributes to the folding and activity of many exported proteins in bacteria. However, information about disulfide bond formation is limited to only a few bacterial species. We used a multifaceted bioinformatic approach to assess the capacity for disulfide bond formation across this biologically diverse group of organisms. We combined data from a cysteine counting method, in which a significant bias for even numbers of cysteine in proteins is taken as an indicator of disulfide bond formation, with data on the presence of homologs of known disulfide bond formation enzymes. These combined data enabled us to make predictions about disulfide bond formation in the cell envelope across bacterial species. Our bioinformatic and experimental results suggest that many bacteria may not generally oxidatively fold proteins, and implicate the bacterial homolog of the enzyme vitamin K epoxide reductase, a protein required for blood clotting in humans, as part of a disulfide bond formation pathway present in several major bacterial phyla.

Bacterial-fungal interactions revealed by genome-wide analysis of bacterial mutant fitness.

Microbial interactions are expected to be major determinants of microbiome structure and function. Although fungi are found in diverse microbiomes, their interactions with bacteria remain largely uncharacterized. In this work, we characterize interactions in 16 different bacterial-fungal pairs, examining the impacts of 8 different fungi isolated from cheese rind microbiomes on 2 bacteria (Escherichia coli and a cheese-isolated Pseudomonas psychrophila). Using random barcode transposon-site sequencing with an analysis pipeline that allows statistical comparisons between different conditions, we observed that fungal partners caused widespread changes in the fitness of bacterial mutants compared to growth alone. We found that all fungal species modulated the availability of iron and biotin to bacterial species, which suggests that these may be conserved drivers of bacterial-fungal interactions. Species-specific interactions were also uncovered, a subset of which suggested fungal antibiotic production. Changes in both conserved and species-specific interactions resulted from the deletion of a global regulator of fungal specialized metabolite production. This work highlights the potential for broad impacts of fungi on bacterial species within microbiomes.

Experimental approaches to tracking mobile genetic elements in microbial communities.

Horizontal gene transfer is an important mechanism of microbial evolution and is often driven by the movement of mobile genetic elements between cells. Due to the fact that microbes live within communities, various mechanisms of horizontal gene transfer and types of mobile elements can co-occur. However, the ways in which horizontal gene transfer impacts and is impacted by communities containing diverse mobile elements has been challenging to address. Thus, the field would benefit from incorporating community-level information and novel approaches alongside existing methods. Emerging technologies for tracking mobile elements and assigning them to host organisms provide promise for understanding the web of potential DNA transfers in diverse microbial communities more comprehensively. Compared to existing experimental approaches, chromosome conformation capture and methylome analyses have the potential to simultaneously study various types of mobile elements and their associated hosts. We also briefly discuss how fermented food microbiomes, given their experimental tractability and moderate species complexity, make ideal models to which to apply the techniques discussed herein and how they can be used to address outstanding questions in the field of horizontal gene transfer in microbial communities.