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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, T he H itchhikers o f the R hizosphere (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 T he H itchhikers O f the R hizosphere (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., Competing Interests: David J. Beebe holds equity in Bellbrook Labs LLC, Tasso Inc., Salus Discovery LLC, Lynx Biosciences Inc., Stacks to the Future LLC, Flambeau Diagnostics LLC, and Onexio Biosystems LLC. Jo Handelsman holds equity in Wacasa Inc. and Ascribe, Inc.

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, T he H itchhikers o f the R hizosphere (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.

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., (Copyright © 2021 Hurley et al.)

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 a ntibiotic- i nduced r esponse ( 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., (Copyright © 2020 Lozano et al.)

The quest to manipulate microbiomes has intensified, but many microbial communities have proven to be recalcitrant to sustained change. Developing model communities amenable to genetic dissection will underpin successful strategies for shaping microbiomes by advancing an understanding of community interactions. We developed a model community with representatives from three dominant rhizosphere taxa, the Firmicutes , Proteobacteria , and Bacteroidetes We chose Bacillus cereus as a model rhizosphere firmicute and characterized 20 other candidates, including "hitchhikers" that coisolated with B. cereus from the rhizosphere. Pairwise analysis produced a hierarchical interstrain-competition network. We chose two hitchhikers, Pseudomonas koreensis from the top tier of the competition network and Flavobacterium johnsoniae from the bottom of the network, to represent the Proteobacteria and Bacteroidetes , respectively. The model community has several emergent properties, induction of dendritic expansion of B. cereus colonies by either of the other members, and production of more robust biofilms by the three members together than individually. Moreover, P. koreensis produces a novel family of alkaloid antibiotics that inhibit growth of F. johnsoniae , and production is inhibited by B. cereus We designate this community THOR, because the members are t he h itchhikers o f the r hizosphere. The genetic, genomic, and biochemical tools available for dissection of THOR provide the means to achieve a new level of understanding of microbial community behavior. IMPORTANCE The manipulation and engineering of microbiomes could lead to improved human health, environmental sustainability, and agricultural productivity. However, microbiomes have proven difficult to alter in predictable ways, and their emergent properties are poorly understood. The history of biology has demonstrated the power of model systems to understand complex problems such as gene expression or development. Therefore, a defined and genetically tractable model community would be useful to dissect microbiome assembly, maintenance, and processes. We have developed a tractable model rhizosphere microbiome, designated THOR, containing Pseudomonas koreensis , Flavobacterium johnsoniae , and Bacillus cereus , which represent three dominant phyla in the rhizosphere, as well as in soil and the mammalian gut. The model community demonstrates emergent properties, and the members are amenable to genetic dissection. We propose that THOR will be a useful model for investigations of community-level interactions., (Copyright © 2019 Lozano et al.)

Microbial communities typically contain many rare taxa that make up the majority of the observed membership, yet the contribution of this microbial "rare biosphere" to community dynamics is unclear. Using 16S rRNA amplicon sequencing of 3,237 samples from 42 time series of microbial communities from nine different ecosystems (air; marine; lake; stream; adult human skin, tongue, and gut; infant gut; and brewery wastewater treatment), we introduce a new method to detect typically rare microbial taxa that occasionally become very abundant (conditionally rare taxa [CRT]) and then quantify their contributions to temporal shifts in community structure. We discovered that CRT made up 1.5 to 28% of the community membership, represented a broad diversity of bacterial and archaeal lineages, and explained large amounts of temporal community dissimilarity (i.e., up to 97% of Bray-Curtis dissimilarity). Most of the CRT were detected at multiple time points, though we also identified "one-hit wonder" CRT that were observed at only one time point. Using a case study from a temperate lake, we gained additional insights into the ecology of CRT by comparing routine community time series to large disturbance events. Our results reveal that many rare taxa contribute a greater amount to microbial community dynamics than is apparent from their low proportional abundances. This observation was true across a wide range of ecosystems, indicating that these rare taxa are essential for understanding community changes over time. Importance: Microbial communities and their processes are the foundations of ecosystems. The ecological roles of rare microorganisms are largely unknown, but it is thought that they contribute to community stability by acting as a reservoir that can rapidly respond to environmental changes. We investigated the occurrence of typically rare taxa that very occasionally become more prominent in their communities ("conditionally rare"). We quantified conditionally rare taxa in time series from a wide variety of ecosystems and discovered that not only were conditionally rare taxa present in all of the examples, but they also contributed disproportionately to temporal changes in diversity when they were most abundant. This result indicates an important and general role for rare microbial taxa within their communities., (Copyright © 2014 Shade et al.)

Application of manure from antibiotic-treated animals to crops facilitates the dissemination of antibiotic resistance determinants into the environment. However, our knowledge of the identity, diversity, and patterns of distribution of these antibiotic resistance determinants remains limited. We used a new combination of methods to examine the resistome of dairy cow manure, a common soil amendment. Metagenomic libraries constructed with DNA extracted from manure were screened for resistance to beta-lactams, phenicols, aminoglycosides, and tetracyclines. Functional screening of fosmid and small-insert libraries identified 80 different antibiotic resistance genes whose deduced protein sequences were on average 50 to 60% identical to sequences deposited in GenBank. The resistance genes were frequently found in clusters and originated from a taxonomically diverse set of species, suggesting that some microorganisms in manure harbor multiple resistance genes. Furthermore, amid the great genetic diversity in manure, we discovered a novel clade of chloramphenicol acetyltransferases. Our study combined functional metagenomics with third-generation PacBio sequencing to significantly extend the roster of functional antibiotic resistance genes found in animal gut bacteria, providing a particularly broad resource for understanding the origins and dispersal of antibiotic resistance genes in agriculture and clinical settings. IMPORTANCE The increasing prevalence of antibiotic resistance among bacteria is one of the most intractable challenges in 21st-century public health. The origins of resistance are complex, and a better understanding of the impacts of antibiotics used on farms would produce a more robust platform for public policy. Microbiomes of farm animals are reservoirs of antibiotic resistance genes, which may affect distribution of antibiotic resistance genes in human pathogens. Previous studies have focused on antibiotic resistance genes in manures of animals subjected to intensive antibiotic use, such as pigs and chickens. Cow manure has received less attention, although it is commonly used in crop production. Here, we report the discovery of novel and diverse antibiotic resistance genes in the cow microbiome, demonstrating that it is a significant reservoir of antibiotic resistance genes. The genomic resource presented here lays the groundwork for understanding the dispersal of antibiotic resistance from the agroecosystem to other settings.

Unlabelled: We investigated the hypothesis that the gender of conveners at scientific meetings influenced the gender distribution of invited speakers. Analysis of 460 symposia involving 1,845 speakers in two large meetings sponsored by the American Society for Microbiology revealed that having at least one woman member of the convening team correlated with a significantly higher proportion of invited female speakers and reduced the likelihood of an all-male symposium roster. Our results suggest that inclusion of more women as conveners may increase the proportion of women among invited speakers at scientific meetings., Importance: The proportion of women entering scientific careers has increased substantially, but women remain underrepresented in academic ranks. Participation in meetings as a speaker is a factor of great importance for academic advancement. We found that having a woman as a convener greatly increased women's participation in symposia, suggesting that one mechanism for achieving gender balance at scientific meetings is to involve more women as conveners.

Unlabelled: We report that establishment and maintenance of the Drosophila melanogaster microbiome depend on ingestion of bacteria. Frequent transfer of flies to sterile food prevented establishment of the microbiome in newly emerged flies and reduced the predominant members, Acetobacter and Lactobacillus spp., by 10- to 1,000-fold in older flies. Flies with a normal microbiome were less susceptible than germfree flies to infection by Serratia marcescens and Pseudomonas aeruginosa. Augmentation of the normal microbiome with higher populations of Lactobacillus plantarum, a Drosophila commensal and probiotic used in humans, further protected the fly from infection. Replenishment represents an unexplored strategy by which animals can sustain a gut microbial community. Moreover, the population behavior and health benefits of L. plantarum resemble features of certain probiotic bacteria administered to humans. As such, L. plantarum in the fly gut may serve as a simple model for dissecting the population dynamics and mode of action of probiotics in animal hosts., Importance: Previous studies have defined the composition of the Drosophila melanogaster microbiome in laboratory and wild-caught flies. Our study advances current knowledge in this field by demonstrating that Drosophila must consume bacteria to establish and maintain its microbiome. This finding suggests that the dominant Drosophila symbionts remain associated with their host because of repeated reintroduction rather than internal growth. Furthermore, our study shows that one member of the microbiome, Lactobacillus plantarum, protects the fly from intestinal pathogens. These results suggest that, although not always present, the microbiota can promote salubrious effects for the host. In sum, our work provides a previously unexplored mechanism of microbiome maintenance and an in vivo model system for investigating the mechanisms of action of probiotic bacteria.

Unlabelled: Despite its importance to the host, the flower microbiome is poorly understood. We report a culture-independent, community-level assessment of apple flower microbial diversity and dynamics. We collected flowers from six apple trees at five time points, starting before flowers opened and ending at petal fall. We applied streptomycin to half of the trees when flowers opened. Assessment of microbial diversity using tag pyrosequencing of 16S rRNA genes revealed that the apple flower communities were rich and diverse and dominated by members of TM7 and Deinococcus-Thermus, phyla about which relatively little is known. From thousands of taxa, we identified six successional groups with coherent dynamics whose abundances peaked at different times before and after bud opening. We designated the groups Pioneer, Early, Mid, Late, Climax, and Generalist communities. The successional pattern was attributed to a set of prevalent taxa that were persistent and gradually changing in abundance. These taxa had significant associations with other community members, as demonstrated with a cooccurrence network based on local similarity analysis. We also detected a set of less-abundant, transient taxa that contributed to general tree-to-tree variability but not to the successional pattern. Communities on trees sprayed with streptomycin had slightly lower phylogenetic diversity than those on unsprayed trees but did not differ in structure or succession. Our results suggest that changes in apple flower microbial community structure are predictable over the life of the flower, providing a basis for ecological understanding and disease management., Importance: Flowering plants (angiosperms) represent a diverse group of an estimated 400,000 species, and their successful cultivation is essential to agriculture. Yet fundamental knowledge of flower-associated microbiotas remains largely unknown. Even less well understood are the changes that flower microbial communities experience through time. Flowers are particularly conducive to comprehensive temporal studies because they are, by nature, ephemeral organs. Here, we present the first culture-independent time series of bacterial and archaeal communities associated with the flowers of apple, an economically important crop. We found unexpected diversity on apple flowers, including a preponderance of taxa affiliated with Deinococcus-Thermus and TM7, phyla that are understudied but thought to be tolerant to an array of environmental stresses. Our results also suggest that changes in microbial community structure on the apple flower may be predictable over the life of the flower, providing the basis for ecological understanding and disease management.

Unlabelled: A dynamic homeostasis is maintained between the host and native bacteria of the gastrointestinal tract in animals, but migration of bacteria from the gut to other organs can lead to disease or death. Enterococcus faecalis is a commensal of the gastrointestinal tract; however, Enterococcus spp. are increasingly frequent causes of nosocomial infections with a high mortality rate. We investigated the commensal-to-pathogen switch undergone by E. faecalis OG1RF in the lepidopteran model host Manduca sexta associated with its location in the host. E. faecalis persists in the harsh midgut environment of M. sexta larvae without causing apparent illness, but injection of E. faecalis directly into the larval hemocoel is followed by rapid death. Additionally, oral ingestion of E. faecalis in the presence of Bacillus thuringiensis insecticidal toxin, a pore-forming toxin that targets the midgut epithelium, induces an elevated mortality rate. We show that the loss of gut integrity due to B. thuringiensis toxin correlates with the translocation of E. faecalis from the gastrointestinal tract into the hemolymph. Upon gaining access to the hemolymph, E. faecalis induces an innate immune response, illustrated by hemocyte aggregation, in larvae prior to death. The degree of hemocyte aggregation is dependent upon the route of E. faecalis entry. Our data demonstrate the efficacy of the M. sexta larval model system in investigating E. faecalis-induced sepsis and clarifies controversies in the field regarding the events leading to larval death following B. thuringiensis toxin exposure., Importance: This study advances our knowledge of Enterococcus faecalis-induced sepsis following translocation from the gut and provides a model for mammalian diseases in which the spatial distribution of bacteria determines disease outcomes. We demonstrate that E. faecalis is a commensal in the gut of Manduca sexta and a pathogen in the hemocoel, resulting in a robust immune response and rapid death, a process we refer to as the "commensal-to-pathogen" switch. While controversy remains regarding Bacillus thuringiensis toxin-induced killing, our laboratory previously found that under some conditions, the midgut microbiota is essential for B. thuringiensis toxin killing of Lymantria dispar (N. A. Broderick, K. F. Raffa, and J. Handelsman, Proc. Natl. Acad. Sci. U. S. A. 103:15196-15199, 2006; B. Raymond, et al., Environ. Microbiol. 11:2556-2563, 2009; P. R. Johnston, and N. Crickmore, Appl. Environ. Microbiol. 75:5094-5099, 2009). We and others have demonstrated that the role of the midgut microbiota in B. thuringiensis toxin killing is dependent upon the lepidopteran species and formulation of B. thuringiensis toxin (N. A. Broderick, K. F. Raffa, and J. Handelsman, Proc. Natl. Acad. Sci. U. S. A. 103:15196-15199, 2006; N. A. Broderick, et al., BMC Biol. 7:11, 2009). This work reconciles much of the apparently contradictory previous data and reveals that the M. sexta-E. faecalis system provides a model for mammalian sepsis.