multimedia: Multimodal Mediation Analysis of Microbiome Data.

Mediation analysis has emerged as a versatile tool for answering mechanistic questions in microbiome research because it provides a statistical framework for attributing treatment effects to alternative causal pathways. Using a series of linked regression models, this analysis quantifies how complementary data modalities relate to one another and respond to treatments. Despite these advances, the rigid modeling assumptions of existing software often results in users viewing mediation analysis as a black box, not something that can be inspected, critiqued, and refined. We designed the multimedia R package to make advanced mediation analysis techniques accessible to a wide audience, ensuring that all statistical components are easily interpretable and adaptable to specific problem contexts. The package provides a uniform interface to direct and indirect effect estimation, synthetic null hypothesis testing, and bootstrap confidence interval construction. We illustrate the package through two case studies. The first re-analyzes a study of the microbiome and metabolome of Inflammatory Bowel Disease patients, uncovering potential mechanistic interactions between the microbiome and disease-associated metabolites, not found in the original study. The second analyzes new data about the influence of mindfulness practice on the microbiome. The mediation analysis identifies a direct effect between a randomized mindfulness intervention and microbiome composition, highlighting shifts in taxa previously associated with depression that cannot be explained by diet or sleep behaviors alone. A gallery of examples and further documentation can be found at https://go.wisc.edu/830110.

Massively parallel mutant selection identifies genetic determinants of Pseudomonas aeruginosa colonization of Drosophila melanogaster.

Pseudomonas aeruginosa is recognized for its ability to colonize diverse habitats and cause disease in a variety of hosts, including plants, invertebrates, and mammals. Understanding how this bacterium is able to occupy wide-ranging niches is important for deciphering its ecology. We used transposon sequencing [Tn-Seq, also known as insertion sequencing (INSeq)] to identify genes in P. aeruginosa that contribute to fitness during the colonization of Drosophila melanogaster. Our results reveal a suite of critical factors, including those that contribute to polysaccharide production, DNA repair, metabolism, and respiration. Comparison of candidate genes with fitness determinants discovered in previous studies on P. aeruginosa identified several genes required for colonization and virulence determinants that are conserved across hosts and tissues. This analysis provides evidence for both the conservation of function of several genes across systems, as well as host-specific functions. These findings, which represent the first use of transposon sequencing of a gut pathogen in Drosophila, demonstrate the power of Tn-Seq in the fly model system and advance the existing knowledge of intestinal pathogenesis by D. melanogaster, revealing bacterial colonization determinants that contribute to a comprehensive portrait of P. aeruginosa lifestyles across habitats.IMPORTANCEDrosophila melanogaster is a powerful model for understanding host-pathogen interactions. Research with this system has yielded notable insights into mechanisms of host immunity and defense, many of which emerged from the analysis of bacterial mutants defective for well-characterized virulence factors. These foundational studies-and advances in high-throughput sequencing of transposon mutants-support unbiased screens of bacterial mutants in the fly. To investigate mechanisms of host-pathogen interplay and exploit the tractability of this model host, we used a high-throughput, genome-wide mutant analysis to find genes that enable the pathogen P. aeruginosa to colonize the fly. Our analysis reveals critical mediators of P. aeruginosa establishment in its host, some of which are required across fly and mouse systems. These findings demonstrate the utility of massively parallel mutant analysis and provide a platform for aligning the fly toolkit with comprehensive bacterial genomics.

Surface colonization by Flavobacterium johnsoniae promotes its survival in a model microbial community.

Flavobacterium johnsoniae is a ubiquitous soil and rhizosphere bacterium, but despite its abundance, the factors contributing to its success in communities are poorly understood. Using a model microbial community, The Hitchhikers of the Rhizosphere (THOR), we determined the effects of colonization on the fitness of F. johnsoniae in the community. Insertion sequencing, a massively parallel transposon mutant screen, on sterile sand identified 25 genes likely to be important for surface colonization. We constructed in-frame deletions of candidate genes predicted to be involved in cell membrane biogenesis, motility, signal transduction, and transport of amino acids and lipids. All mutants poorly colonized sand, glass, and polystyrene and produced less biofilm than the wild type, indicating the importance of the targeted genes in surface colonization. Eight of the nine colonization-defective mutants were also unable to form motile biofilms or zorbs, thereby suggesting that the affected genes play a role in group movement and linking stationary and motile biofilm formation genetically. Furthermore, we showed that the deletion of colonization genes in F. johnsoniae affected its behavior and survival in THOR on surfaces, suggesting that the same traits are required for success in a multispecies microbial community. Our results provide insight into the mechanisms of surface colonization by F. johnsoniae and form the basis for further understanding its ecology in the rhizosphere. IMPORTANCE: Microbial communities direct key environmental processes through multispecies interactions. Understanding these interactions is vital for manipulating microbiomes to promote health in human, environmental, and agricultural systems. However, microbiome complexity can hinder our understanding of the underlying mechanisms in microbial community interactions. As a first step toward unraveling these interactions, we explored the role of surface colonization in microbial community interactions using The Hitchhikers Of the Rhizosphere (THOR), a genetically tractable model community of three bacterial species, Flavobacterium johnsoniae, Pseudomonas koreensis, and Bacillus cereus. We identified F. johnsoniae genes important for surface colonization in solitary conditions and in the THOR community. Understanding the mechanisms that promote the success of bacteria in microbial communities brings us closer to targeted manipulations to achieve outcomes that benefit agriculture, the environment, and human health.

Massively parallel mutant selection identifies genetic determinants of Pseudomonas aeruginosa colonization of Drosophila melanogaster.

Pseudomonas aeruginosa is recognized for its ability to colonize diverse habitats and cause disease in a variety of hosts, including plants, invertebrates, and mammals. Understanding how this bacterium is able to occupy wide-ranging niches is important for deciphering its ecology. We used transposon sequencing (Tn-Seq, also known as INSeq) to identify genes in P. aeruginosa that contribute to fitness during colonization of Drosophila melanogaster. Our results reveal a suite of critical factors, including those that contribute to polysaccharide production, DNA repair, metabolism, and respiration. Comparison of candidate genes with fitness determinants discovered in previous studies of P. aeruginosa identified several genes required for colonization and virulence determinants that are conserved across hosts and tissues. This analysis provides evidence for both the conservation of function of several genes across systems, as well as host-specific functions. These findings, which represent the first use of transposon sequencing of a gut pathogen in Drosophila, demonstrate the power of Tn-Seq in the fly model system and advance existing knowledge of intestinal pathogenesis by D. melanogaster, revealing bacterial colonization determinants that contribute to a comprehensive portrait of P. aeruginosa lifestyles across habitats.

A triangulated approach for understanding scientists' perceptions of public engagement with science.

Scientists are expected to engage with the public, especially when society faces challenges like the COVID-19 pandemic or climate change, but what public engagement means to scientists is not clear. We use a triangulated, mixed-methods approach combining survey and focus group data to gain insight into how pre-tenure and tenured scientists personally conceptualize public engagement. Our findings indicate that scientists' understanding of public engagement is similarly complex and diverse as the scholarly literature. While definitions and examples of one-way forms of engagement are the most salient for scientists, regardless of tenure status, scientists also believe public engagement with science includes two-way forms of engagement, such as citizen and community involvement in research. These findings suggest that clear definitions of public engagement are not necessarily required for its application but may be useful to guide scientists in their engagement efforts, so they align with what is expected of them.

Microbiome composition modulates secondary metabolism in a multispecies bacterial community.

Bacterial secondary metabolites are a major source of antibiotics and other bioactive compounds. In microbial communities, these molecules can mediate interspecies interactions and responses to environmental change. Despite the importance of secondary metabolites in human health and microbial ecology, little is known about their roles and regulation in the context of multispecies communities. In a simplified model of the rhizosphere composed of Bacillus cereus, Flavobacterium johnsoniae, and Pseudomonas koreensis, we show that the dynamics of secondary metabolism depend on community species composition and interspecies interactions. Comparative metatranscriptomics and metametabolomics reveal that the abundance of transcripts of biosynthetic gene clusters (BGCs) and metabolomic molecular features differ between monocultures or dual cultures and a tripartite community. In both two- and three-member cocultures, P. koreensis modified expression of BGCs for zwittermicin, petrobactin, and other secondary metabolites in B. cereus and F. johnsoniae, whereas the BGC transcriptional response to the community in P. koreensis itself was minimal. Pairwise and tripartite cocultures with P. koreensis displayed unique molecular features that appear to be derivatives of lokisin, suggesting metabolic handoffs between species. Deleting the BGC for koreenceine, another P. koreensis metabolite, altered transcript and metabolite profiles across the community, including substantial up-regulation of the petrobactin and bacillibactin BGCs in B. cereus, suggesting that koreenceine represses siderophore production. Results from this model community show that bacterial BGC expression and chemical output depend on the identity and biosynthetic capacity of coculture partners, suggesting community composition and microbiome interactions may shape the regulation of secondary metabolism in nature.

Achieving STEM diversity: Fix the classrooms.

Outdated teaching methods amount to discrimination.

Public engagement: Faculty lived experiences and perspectives underscore barriers and a changing culture in academia.

The idea of faculty engaging in meaningful dialogue with different publics instead of simply communicating their research to interested audiences has gradually morphed from a novel concept to a mainstay within most parts of the academy. Given the wide variety of public engagement modalities, it may be unsurprising that we still lack a comprehensive and granular understanding of factors that influence faculty willingness to engage with public audiences. Those nuances are not always captured by quantitative surveys that rely on pre-determined categories to assess scholars' willingness to engage. While closed-ended categories are useful to examine which factors influence the willingness to engage more than others, it is unlikely that pre-determined categories comprehensively represent the range of factors that undermine or encourage engagement, including perceptual influences, institutional barriers, and scholars' lived experiences. To gain insight into these individual perspectives and lived experiences, we conducted focus group discussions with faculty members at a large midwestern land-grant university in the United States. Our findings provide context to previous studies of public engagement and suggest four themes for future research. These themes affirm the persistence of institutional barriers to engaging with the public, particularly the expectations in the promotion process for tenure-track faculty. However, we also find a perception that junior faculty and graduate students are challenging the status quo by introducing a new wave of attention to public engagement. This finding suggests a "trickle-up" effect through junior faculty and graduate students expecting institutional support for public engagement. Our findings highlight the need to consider how both top-down factors such as institutional expectations and bottom-up factors such as graduate student interest shape faculty members' decisions to participate in public engagement activities.

AJEDI in Science: Leveraging Instructor Communities to Create Antiracist Curricula.

Gateway college science courses continue to exclude students from science, disproportionately discriminating against students of color. As the higher education system strives to reduce discrimination, we need a deliberate, iterative process to modify, supplement, or replace current modalities. By incorporating antiracist, just, equitable, diverse, and inclusive (AJEDI) principles throughout course design, instructors create learning environments that provide an antidote to historically oppressive systems. In this paper, we describe how a community of microbiology instructors who all teach Tiny Earth, a course-based undergraduate research experience, created and rapidly integrated antiracist content and pivoted to an online format in response to the social unrest and pandemic of 2020. The effort strengthened an existing teaching community of practice and produced collective change in classrooms across the nation. We provide a perspective on how instructor communities of practice can be leveraged to design and disseminate AJEDI curriculum.

Evolutionary Genome Mining for the Discovery and Engineering of Natural Product Biosynthesis.

Genome mining has become an invaluable tool in natural products research to quickly identify and characterize the biosynthetic pathways that assemble secondary or specialized metabolites. Recently, evolutionary principles have been incorporated into genome mining strategies in an effort to better assess and prioritize novelty and understand their chemical diversification for engineering purposes. Here, we provide an introduction to the principles underlying evolutionary genome mining, including bioinformatic strategies and natural product biosynthetic databases. We introduce workflows for traditional genome mining, focusing on the popular pipeline antiSMASH, and methods to predict enzyme substrate specificity from genomic information. We then provide an in-depth discussion of evolutionary genome mining workflows, including EvoMining, CORASON, ARTS, and others, as adopted by our group for the discovery and prioritization of natural products biosynthetic gene clusters and their products.

THOR's Hammer: the Antibiotic Koreenceine Drives Gene Expression in a Model Microbial Community.

Microbial interactions dictate the structure and function of microbiomes, but the complexity of natural communities can obscure the individual interactions. Model microbial communities constructed with genetically tractable strains known to interact in natural settings can untangle these networks and reveal underpinning mechanisms. Our model system, The Hitchhikers of the Rhizosphere (THOR), is composed of three species-Bacillus cereus, Flavobacterium johnsoniae, and Pseudomonas koreensis-that co-isolate from field-grown soybean roots. Comparative metatranscriptomics on THOR revealed global patterns of interspecies transcriptional regulation. When grown in pairs, each member of THOR exhibits unique signaling behavior. In the community setting, gene expression is dominated by pairwise interactions with Pseudomonas koreensis mediated either directly or indirectly by its production of the antibiotic koreenceine-the apparent "hammer" of THOR. In pairwise interactions, the koreenceine biosynthetic cluster is responsible for 85 and 22% of differentially regulated genes in F. johnsoniae and B. cereus, respectively. Although both deletion of the koreenceine locus and reduction of P. koreensis inoculum size increase F. johnsoniae populations, the transcriptional response of P. koreensis is only activated when it is a relative minority member at the beginning of coculture. The largest group of upregulated P. koreensis genes in response to F. johnsoniae are those without functional annotation, indicating that focusing on genes important for community interactions may offer a path toward functional assignments for unannotated genes. This study illustrates the power of comparative metatranscriptomics of microorganisms encountering increasing microbial complexity for understanding community signal integration, antibiotic responses, and interspecies communication. IMPORTANCE The diversity, ubiquity, and significance of microbial communities is clear. However, the predictable and reliable manipulation of microbiomes to impact human, environmental, and agricultural health remains a challenge. Effective remodeling of microbiomes will be enabled by understanding the interspecies interactions that govern community processes. The extreme complexity of most microbiomes has impeded characterization of the relevant interactions. Investigating the genetics and biochemistry of simplified, model microbiomes could unearth specific interactions and generate predictions about community-governing principles. Here, we use one such model community to quantify changes in gene expression of individual species as they encounter stimuli from one or more species, directly mapping combinatorial interspecies interactions. A surprising amount of gene expression is regulated by a single molecule, the antibiotic koreenceine, which appears to impact gene regulation across community networks.

Needles in haystacks: reevaluating old paradigms for the discovery of bacterial secondary metabolites.

Covering: up to 2021Natural products research is in the midst of a renaissance ushered in by a modern understanding of microbiology and the technological explosions of genomics and metabolomics. As the exploration of uncharted chemical space expands into high-throughput discovery campaigns, it has become increasingly clear how design elements influence success: (bio)geography, habitat, community dynamics, culturing/induction methods, screening methods, dereplication, and more. We explore critical considerations and assumptions in natural products discovery. We revisit previous estimates of chemical rediscovery and discuss their relatedness to study design and producer taxonomy. Through frequency analyses of biosynthetic gene clusters in publicly available genomic data, we highlight phylogenetic biases that influence rediscovery rates. Through selected examples of how study design at each level determines discovery outcomes, we discuss the challenges and opportunities for the future of high-throughput natural product discovery.

Social motility of biofilm-like microcolonies in a gliding bacterium.

Bacterial biofilms are aggregates of surface-associated cells embedded in an extracellular polysaccharide (EPS) matrix, and are typically stationary. Studies of bacterial collective movement have largely focused on swarming motility mediated by flagella or pili, in the absence of a biofilm. Here, we describe a unique mode of collective movement by a self-propelled, surface-associated biofilm-like multicellular structure. Flavobacterium johnsoniae cells, which move by gliding motility, self-assemble into spherical microcolonies with EPS cores when observed by an under-oil open microfluidic system. Small microcolonies merge, creating larger ones. Microscopic analysis and computer simulation indicate that microcolonies move by cells at the base of the structure, attached to the surface by one pole of the cell. Biochemical and mutant analyses show that an active process drives microcolony self-assembly and motility, which depend on the bacterial gliding apparatus. We hypothesize that this mode of collective bacterial movement on solid surfaces may play potential roles in biofilm dynamics, bacterial cargo transport, or microbial adaptation. However, whether this collective motility occurs on plant roots or soil particles, the native environment for F. johnsoniae, is unknown.

Tiny Earth: A Big Idea for STEM Education and Antibiotic Discovery.

The world faces two seemingly unrelated challenges-a shortfall in the STEM workforce and increasing antibiotic resistance among bacterial pathogens. We address these two challenges with Tiny Earth, an undergraduate research course that excites students about science and creates a pipeline for antibiotic discovery.

Gender Can Influence Student Experiences in MD-PhD Training.

Background: Female physician-scientists have led major advances in medicine broadly and particularly in women's health. Women remain underrepresented in dual MD-PhD degree programs that train many physician-scientists despite gender parity among medical and biomedical research students. Materials and Methods: To explore how the training environment might be experienced differently for male and female students in one MD-PhD program, the authors analyzed gender differences in annual symposium speakers with exact binomial tests, student participation as question-askers at a weekly seminar with logistic regression, and number of publications with quasi-Poisson generalized linear models. They compared male and female students' perceptions of gender-based discrimination using a survey, including qualitative analysis of free text responses. The program consisted of 71 total students in the 2017-2018 and 2018-2019 academic years. Female students comprised 42.0% (81/191) of program matriculants from 1997 to 2019. Results: Male and female students were equally likely to present at the annual program symposium, but faculty (p = 0.001) and keynote (p = 0.012) presenters were more likely to be male. Compared with male counterparts, female students asked fewer seminar questions (p < 0.005) and female speakers received more questions (p = 0.03). Female students perceived less support and differed from men in reasons for asking or not asking seminar questions. Free text responses described repeated small acts of discrimination toward women with cumulative impact. Positive program changes followed presentation of findings to program leaders and students. Conclusions: The authors identified several aspects of one MD-PhD program that could discourage career or training persistence of female students. Increasing awareness of these issues was temporally related to positive programmatic changes.

Chemically informed analyses of metabolomics mass spectrometry data with Qemistree.

Untargeted mass spectrometry is employed to detect small molecules in complex biospecimens, generating data that are difficult to interpret. We developed Qemistree, a data exploration strategy based on the hierarchical organization of molecular fingerprints predicted from fragmentation spectra. Qemistree allows mass spectrometry data to be represented in the context of sample metadata and chemical ontologies. By expressing molecular relationships as a tree, we can apply ecological tools that are designed to analyze and visualize the relatedness of DNA sequences to metabolomics data. Here we demonstrate the use of tree-guided data exploration tools to compare metabolomics samples across different experimental conditions such as chromatographic shifts. Additionally, we leverage a tree representation to visualize chemical diversity in a heterogeneous collection of samples. The Qemistree software pipeline is freely available to the microbiome and metabolomics communities in the form of a QIIME2 plugin, and a global natural products social molecular networking workflow.

Broadening Participation in Scientific Conferences during the Era of Social Distancing.

Virtual conferences can offer significant benefits but require considerable planning and creativity to be successful. Here we describe the successes and failures of a hybrid in-person/virtual conference model. The COVID-19 epidemic presents the scientific community with an opportunity to pioneer novel models that effectively engage virtual participants to advance conference goals.

A Chemical Counterpunch: Chromobacterium violaceum ATCC 31532 Produces Violacein in Response to Translation-Inhibiting Antibiotics.

Antibiotics produced by bacteria play important roles in microbial interactions and competition Antibiosis can induce resistance mechanisms in target organisms, and at sublethal doses, antibiotics have been shown to globally alter gene expression patterns. Here, we show that hygromycin A from Streptomyces sp. strain 2AW. induces Chromobacterium violaceum ATCC 31532 to produce the purple antibiotic violacein. Sublethal doses of other antibiotics that similarly target the polypeptide elongation step of translation likewise induced violacein production, unlike antibiotics with different targets. C. violaceum biofilm formation and virulence against Drosophila melanogaster were also induced by translation-inhibiting antibiotics, and we identified an antibiotic-induced response (air) two-component regulatory system that is required for these responses. Genetic analyses indicated a connection between the Air system, quorum-dependent signaling, and the negative regulator VioS, leading us to propose a model for induction of violacein production. This work suggests a novel mechanism of interspecies interaction in which a bacterium produces an antibiotic in response to inhibition by another bacterium and supports the role of antibiotics as signal molecules.IMPORTANCE Secondary metabolites play important roles in microbial communities, but their natural functions are often unknown and may be more complex than appreciated. While compounds with antibiotic activity are often assumed to underlie microbial competition, they may alternatively act as signal molecules. In either scenario, microorganisms might evolve responses to sublethal concentrations of these metabolites, either to protect themselves from inhibition or to change certain behaviors in response to the local abundance of another species. Here, we report that violacein production by C. violaceum ATCC 31532 is induced in response to hygromycin A from Streptomyces sp. 2AW, and we show that this response is dependent on inhibition of translational polypeptide elongation and a previously uncharacterized two-component regulatory system. The breadth of the transcriptional response beyond violacein induction suggests a surprisingly complex metabolite-mediated microbe-microbe interaction and supports the hypothesis that antibiotics evolved as signal molecules. These novel insights will inform predictive models of soil community dynamics and the unintended effects of clinical antibiotic administration.