Small molecule in situ resin capture provides a compound first approach to natural product discovery.

Culture-based microbial natural product discovery strategies fail to realize the extraordinary biosynthetic potential detected across earth's microbiomes. Here we introduce Small Molecule In situ Resin Capture (SMIRC), a culture-independent method to obtain natural products directly from the environments in which they are produced. We use SMIRC to capture numerous compounds including two new carbon skeletons that were characterized using NMR and contain structural features that are, to the best of our knowledge, unprecedented among natural products. Applications across diverse marine habitats reveal biome-specific metabolomic signatures and levels of chemical diversity in concordance with sequence-based predictions. Expanded deployments, in situ cultivation, and metagenomics facilitate compound discovery, enhance yields, and link compounds to candidate producing organisms, although microbial community complexity creates challenges for the later. This compound-first approach to natural product discovery provides access to poorly explored chemical space and has implications for drug discovery and the detection of chemically mediated biotic interactions.

Microbial evolution-An under-appreciated driver of soil carbon cycling.

Although substantial advances in predicting the ecological impacts of global change have been made, predictions of the evolutionary impacts have lagged behind. In soil ecosystems, microbes act as the primary energetic drivers of carbon cycling; however, microbes are also capable of evolving on timescales comparable to rates of global change. Given the importance of soil ecosystems in global carbon cycling, we assess the potential impact of microbial evolution on carbon-climate feedbacks in this system. We begin by reviewing the current state of knowledge concerning microbial evolution in response to global change and its specific effect on soil carbon dynamics. Through this integration, we synthesize a roadmap detailing how to integrate microbial evolution into ecosystem biogeochemical models. Specifically, we highlight the importance of microscale mechanistic soil carbon models, including choosing an appropriate evolutionary model (e.g., adaptive dynamics, quantitative genetics), validating model predictions with 'omics' and experimental data, scaling microbial adaptations to ecosystem level processes, and validating with ecosystem-scale measurements. The proposed steps will require significant investment of scientific resources and might require 10-20 years to be fully implemented. However, through the application of multi-scale integrated approaches, we will advance the integration of microbial evolution into predictive understanding of ecosystems, providing clarity on its role and impact within the broader context of environmental change.

MAR4 Streptomyces: A Unique Resource for Natural Product Discovery.

Marine-derived Streptomyces have long been recognized as a source of novel, pharmaceutically relevant natural products. Among these bacteria, the MAR4 clade within the genus Streptomyces has been identified as metabolically rich, yielding over 93 different compounds to date. MAR4 strains are particularly noteworthy for the production of halogenated hybrid isoprenoid natural products, a relatively rare class of bacterial metabolites that possess a wide range of biological activities. MAR4 genomes are enriched in vanadium haloperoxidase and prenyltransferase genes, thus accounting for the production of these compounds. Functional characterization of the enzymes encoded in MAR4 genomes has advanced our understanding of halogenated, hybrid isoprenoid biosynthesis. Despite the exceptional biosynthetic capabilities of MAR4 bacteria, the large body of research they have stimulated has yet to be compiled. Here we review 35 years of natural product research on MAR4 strains and update the molecular diversity of this unique group of bacteria.

Small Molecule in situ Resin Capture - A Compound First Approach to Natural Product Discovery.

Microbial natural products remain an important resource for drug discovery. Yet, commonly employed discovery techniques are plagued by the rediscovery of known compounds, the relatively few microbes that can be cultured, and laboratory growth conditions that do not elicit biosynthetic gene expression among myriad other challenges. Here we introduce a culture independent approach to natural product discovery that we call the Small Molecule In situ Resin Capture (SMIRC) technique. SMIRC exploits in situ environmental conditions to elicit compound production and represents a new approach to access poorly explored chemical space by capturing natural products directly from the environments in which they are produced. In contrast to traditional methods, this compound-first approach can capture structurally complex small molecules across all domains of life in a single deployment while relying on Nature to provide the complex and poorly understood environmental cues needed to elicit biosynthetic gene expression. We illustrate the effectiveness of SMIRC in marine habitats with the discovery of numerous new compounds and demonstrate that sufficient compound yields can be obtained for NMR-based structure assignment. Two new compound classes are reported including one novel carbon skeleton that possesses a functional group not previously observed among natural products and a second that possesses potent biological activity. We introduce expanded deployments, in situ cultivation, and metagenomics as methods to facilitate compound discovery, enhance yields, and link compounds to producing organisms. This compound first approach can provide unprecedented access to new natural product chemotypes with broad implications for drug discovery.

Metagenomic data reveals type I polyketide synthase distributions across biomes.

Microbial polyketide synthase (PKS) genes encode the biosynthesis of many biomedically or otherwise commercially important natural products. Despite extensive discovery efforts, metagenomic analyses suggest that only a small fraction of nature's polyketide biosynthetic potential has been realized. Much of this potential originates from type I PKSs (T1PKSs), which can be further delineated based on their domain organization and the structural features of the compounds they encode. Notably, phylogenetic relationships among ketosynthase (KS) domains provide an effective method to classify the larger and more complex T1PKS genes in which they occur. Increased access to large metagenomic data sets from diverse habitats provides opportunities to assess T1PKS biosynthetic diversity and distributions through their smaller and more tractable KS domain sequences. Here, we used the web tool NaPDoS2 to detect and classify over 35,000 type I KS domains from 137 metagenomic data sets reported from eight diverse, globally distributed biomes. We found biome-specific separation with soils enriched in KSs from modular cis-acetyltransferase (AT) and hybrid cis-AT KSs relative to other biomes and marine sediments enriched in KSs associated with polyunsaturated fatty acid and enediyne biosynthesis. We linked the phylum Actinobacteria to soil-derived enediyne and cis-AT KSs while marine-derived KSs associated with enediyne and monomodular PKSs were linked to phyla from which the compounds produced by these biosynthetic enzymes have not been reported. These KSs were phylogenetically distinct from those associated with experimentally characterized PKSs suggesting they may be associated with novel structures or enzyme functions. Finally, we employed our metagenome-extracted KS domains to evaluate the PCR primers commonly used to amplify type I KSs and identified modifications that could increase the KS sequence diversity recovered from amplicon libraries. IMPORTANCE Polyketides are a crucial source of medicines, agrichemicals, and other commercial products. Advances in our understanding of polyketide biosynthesis, coupled with the increased availability of metagenomic sequence data, provide new opportunities to assess polyketide biosynthetic potential across biomes. Here, we used the web tool NaPDoS2 to assess type I polyketide synthase (PKS) diversity and distributions by detecting and classifying ketosynthase (KS) domains across 137 metagenomes. We show that biomes are differentially enriched in type I KS domains, providing a roadmap for future biodiscovery strategies. Furthermore, KS phylogenies reveal biome-specific clades that do not include biochemically characterized PKSs, highlighting the biosynthetic potential of poorly explored environments. The large metagenome-derived KS data set allowed us to identify regions of commonly used type I KS PCR primers that could be modified to capture a larger extent of environmental KS diversity. These results facilitate both the search for novel polyketides and our understanding of the biogeographical distribution of PKSs across Earth's major biomes.

Biogeographic patterns of biosynthetic potential and specialized metabolites in marine sediments.

While the field of microbial biogeography has largely focused on the contributions of abiotic factors to community patterns, the potential influence of biotic interactions in structuring microbial communities, such as those mediated by the production of specialized metabolites, remains largely unknown. Here, we examined the relationship between microbial community structure and specialized metabolism at local spatial scales in marine sediment samples collected from the Long-Term Ecological Research (LTER) site in Moorea, French Polynesia. By employing a multi-omic approach to characterize the taxonomic, functional, and specialized metabolite composition within sediment communities, we find that biogeographic patterns were driven by local scale processes (e.g., biotic interactions) and largely independent of dispersal limitation. Specifically, we observed high variation in biosynthetic potential (based on Bray-Curtis dissimilarity) between samples, even within 1 m2 plots, that reflected uncharacterized chemical space associated with site-specific metabolomes. Ultimately, connecting biosynthetic potential to community metabolomes facilitated the in situ detection of natural products and revealed new insights into the complex metabolic dynamics associated with sediment microbial communities. Our study demonstrates the potential to integrate biosynthetic genes and metabolite production into assessments of microbial community dynamics.

Investigating the eco-evolutionary response of microbiomes to environmental change.

Microorganisms are the primary engines of biogeochemical processes and foundational to the provisioning of ecosystem services to human society. Free-living microbial communities (microbiomes) and their functioning are now known to be highly sensitive to environmental change. Given microorganisms' capacity for rapid evolution, evolutionary processes could play a role in this response. Currently, however, few models of biogeochemical processes explicitly consider how microbial evolution will affect biogeochemical responses to environmental change. Here, we propose a conceptual framework for explicitly integrating evolution into microbiome-functioning relationships. We consider how microbiomes respond simultaneously to environmental change via four interrelated processes that affect overall microbiome functioning (physiological acclimation, demography, dispersal and evolution). Recent evidence in both the laboratory and the field suggests that ecological and evolutionary dynamics occur simultaneously within microbiomes; however, the implications for biogeochemistry under environmental change will depend on the timescales over which these processes contribute to a microbiome's response. Over the long term, evolution may play an increasingly important role for microbially driven biogeochemical responses to environmental change, particularly to conditions without recent historical precedent.

Metagenomic Data Reveal Type I Polyketide Synthase Distributions Across Biomes.

Microbial polyketide synthase (PKS) genes encode the biosynthesis of many biomedically important natural products, yet only a small fraction of nature's polyketide biosynthetic potential has been realized. Much of this potential originates from type I PKSs (T1PKSs), which can be delineated into different classes and subclasses based on domain organization and structural features of the compounds encoded. Notably, phylogenetic relationships among PKS ketosynthase (KS) domains provide a method to classify the larger and more complex genes in which they occur. Increased access to large metagenomic datasets from diverse habitats provides opportunities to assess T1PKS biosynthetic diversity and distributions through the analysis of KS domain sequences. Here, we used the webtool NaPDoS2 to detect and classify over 35,000 type I KS domains from 137 metagenomic data sets reported from eight diverse biomes. We found biome-specific separation with soils enriched in modular cis -AT and hybrid cis -AT KSs relative to other biomes and marine sediments enriched in KSs associated with PUFA and enediyne biosynthesis. By extracting full-length KS domains, we linked the phylum Actinobacteria to soil-specific enediyne and cis -AT clades and identified enediyne and monomodular KSs in phyla from which the associated compound classes have not been reported. These sequences were phylogenetically distinct from those associated with experimentally characterized PKSs suggesting novel structures or enzyme functions remain to be discovered. Lastly, we employed our metagenome-extracted KS domains to evaluate commonly used type I KS PCR primers and identified modifications that could increase the KS sequence diversity recovered from amplicon libraries. Importance: Polyketides are a crucial source of medicines, agrichemicals, and other commercial products. Advances in our understanding of polyketide biosynthesis coupled with the accumulation of metagenomic sequence data provide new opportunities to assess polyketide biosynthetic potential across biomes. Here, we used the webtool NaPDoS2 to assess type I PKS diversity and distributions by detecting and classifying KS domains across 137 metagenomes. We show that biomes are differentially enriched in KS domain classes, providing a roadmap for future biodiscovery strategies. Further, KS phylogenies reveal both biome-specific clades that do not include biochemically characterized PKSs, highlighting the biosynthetic potential of poorly explored environments. The large metagenome-derived KS dataset allowed us to identify regions of commonly used type I KS PCR primers that could be modified to capture a larger extent of KS diversity. These results facilitate both the search for novel polyketides and our understanding of the biogeographical distribution of PKSs across earth's major biomes.

Structure and Candidate Biosynthetic Gene Cluster of a Manumycin-Type Metabolite from Salinispora pacifica.

A new manumycin-type natural product named pacificamide (1) and its candidate biosynthetic gene cluster (pac) were discovered from the marine actinobacterium Salinispora pacifica CNT-855. The structure of the compound was determined using NMR, electronic circular dichroism, and bioinformatic predictions. The pac gene cluster is unique to S. pacifica and found in only two of the 119 Salinispora genomes analyzed across nine species. Comparative analyses of biosynthetic gene clusters encoding the production of related manumycin-type compounds revealed genetic differences in accordance with the unique pacificamide structure. Further queries of manumycin-type gene clusters from public databases revealed their limited distribution across the phylum Actinobacteria and orphan diversity that suggests additional products remain to be discovered in this compound class. Production of the known metabolite triacsin D is also reported for the first time from the genus Salinispora. This study adds two classes of compounds to the natural product collective isolated from the genus Salinispora, which has proven to be a useful model for natural product research.

Vertical Inheritance Facilitates Interspecies Diversification in Biosynthetic Gene Clusters and Specialized Metabolites.

While specialized metabolites are thought to mediate ecological interactions, the evolutionary processes driving chemical diversification, particularly among closely related lineages, remain poorly understood. Here, we examine the evolutionary dynamics governing the distribution of natural product biosynthetic gene clusters (BGCs) among 118 strains representing all nine currently named species of the marine actinobacterial genus Salinispora. While much attention has been given to the role of horizontal gene transfer (HGT) in structuring BGC distributions, we find that vertical descent facilitates interspecies BGC diversification over evolutionary timescales. Moreover, we identified a distinct phylogenetic signal among Salinispora species at both the BGC and metabolite level, indicating that specialized metabolism represents a conserved phylogenetic trait. Using a combination of genomic analyses and liquid chromatography-high-resolution tandem mass spectrometry (LC-MS/MS) targeting nine experimentally characterized BGCs and their small molecule products, we identified gene gain/loss events, constrained interspecies recombination, and other evolutionary processes associated with vertical inheritance as major contributors to BGC diversification. These evolutionary dynamics had direct consequences for the compounds produced, as exemplified by species-level differences in salinosporamide production. Together, our results support the concept that specialized metabolites, and their cognate BGCs, can represent phylogenetically conserved functional traits with chemical diversification proceeding in species-specific patterns over evolutionary time frames. IMPORTANCE Microbial natural products are traditionally exploited for their pharmaceutical potential, yet our understanding of the evolutionary processes driving BGC evolution and compound diversification remain poorly developed. While HGT is recognized as an integral driver of BGC distributions, we find that the effects of vertical inheritance on BGC diversification had direct implications for species-level specialized metabolite production. As such, understanding the degree of genetic variation that corresponds to species delineations can enhance natural product discovery efforts. Resolving the evolutionary relationships between closely related strains and specialized metabolism can also facilitate our understanding of the ecological roles of small molecules in structuring the environmental distribution of microbes.

Adaptive differentiation and rapid evolution of a soil bacterium along a climate gradient.

Microbial community responses to environmental change are largely associated with ecological processes; however, the potential for microbes to rapidly evolve and adapt remains relatively unexplored in natural environments. To assess how ecological and evolutionary processes simultaneously alter the genetic diversity of a microbiome, we conducted two concurrent experiments in the leaf litter layer of soil over 18 mo across a climate gradient in Southern California. In the first experiment, we reciprocally transplanted microbial communities from five sites to test whether ecological shifts in ecotypes of the abundant bacterium, Curtobacterium, corresponded to past adaptive differentiation. In the transplanted communities, ecotypes converged toward that of the native communities growing on a common litter substrate. Moreover, these shifts were correlated with community-weighted mean trait values of the Curtobacterium ecotypes, indicating that some of the trait variation among ecotypes could be explained by local adaptation to climate conditions. In the second experiment, we transplanted an isogenic Curtobacterium strain and tracked genomic mutations associated with the sites across the same climate gradient. Using a combination of genomic and metagenomic approaches, we identified a variety of nonrandom, parallel mutations associated with transplantation, including mutations in genes related to nutrient acquisition, stress response, and exopolysaccharide production. Together, the field experiments demonstrate how both demographic shifts of previously adapted ecotypes and contemporary evolution can alter the diversity of a soil microbiome on the same timescale.

Grincamycins P-T: Rearranged Angucyclines from the Marine Sediment-Derived Streptomyces sp. CNZ-748 Inhibit Cell Lines of the Rare Cancer Pseudomyxoma Peritonei.

While marine natural products have been investigated for anticancer drug discovery, they are barely screened against rare cancers. Thus, in our effort to discover potential drug leads against the rare cancer pseudomyxoma peritonei (PMP), which currently lacks effective drug treatments, we screened extracts of marine actinomycete bacteria against the PMP cell line ABX023-1. This effort led to the isolation of nine rearranged angucyclines from Streptomyces sp. CNZ-748, including five new analogues, namely, grincamycins P-T (1-5). The chemical structures of these compounds were unambiguously established based on spectroscopic and chemical analyses. Particularly, grincamycin R (3) possesses an S-containing α-l-methylthio-aculose residue, which was discovered in nature for the first time. All of the isolated compounds were evaluated against four PMP cell lines and some exhibited low micromolar inhibitory activities. To identify a candidate biosynthetic gene cluster (BGC) encoding the grincamycins, we sequenced the genome of the producing strain, Streptomyces sp. CNZ-748, and compared the BGCs detected with those linked to the production of angucyclines with different aglycon structures.

High-Fiber, Whole-Food Dietary Intervention Alters the Human Gut Microbiome but Not Fecal Short-Chain Fatty Acids.

Dietary shifts can have a direct impact on the gut microbiome by preferentially selecting for microbes capable of utilizing the various dietary nutrients. The intake of dietary fiber has decreased precipitously in the last century, while consumption of processed foods has increased. Fiber, or microbiota-accessible carbohydrates (MACs), persist in the digestive tract and can be metabolized by specific bacteria encoding fiber-degrading enzymes. The digestion of MACs results in the accumulation of short-chain fatty acids (SCFAs) and other metabolic by-products that are critical to human health. Here, we implemented a 2-week dietary fiber intervention aiming for 40 to 50 g of fiber per day within the context of a course-based undergraduate research experience (CURE) (n = 20). By coupling shotgun metagenomic sequencing and targeted gas chromatography-mass spectrometry (GC-MS), we found that the dietary intervention significantly altered the composition of individual gut microbiomes, accounting for 8.3% of the longitudinal variability within subjects. Notably, microbial taxa that increased in relative abundance as a result of the diet change included known MAC degraders (i.e., Bifidobacterium and Lactobacillus). We further assessed the genetic diversity within Bifidobacterium, assayed by amplification of the groEL gene. Concomitant with microbial composition changes, we show an increase in the abundance of genes involved in inositol degradation. Despite these changes in gut microbiome composition, we did not detect a consistent shift in SCFA abundance. Collectively, our results demonstrate that on a short-term timescale of 2 weeks, increased fiber intake can induce compositional changes of the gut microbiome, including an increase in MAC-degrading bacteria.IMPORTANCE A profound decrease in the consumption of dietary fiber in many parts of the world in the last century may be associated with the increasing prevalence of type II diabetes, colon cancer, and other health problems. A typical U.S. diet includes about ∼15 g of fiber per day, far less fiber than the daily recommended allowance. Changes in dietary fiber intake affect human health not only through the uptake of nutrients directly but also indirectly through changes in the microbial community and their associated metabolism. Here, we conducted a 2-week diet intervention in healthy young adults to investigate the impact of fiber consumption on the gut microbiome. Participants increased their average fiber consumption by 25 g/day on average for 2 weeks. The high-fiber diet intervention altered the gut microbiome of the study participants, including increases in known fiber-degrading microbes, such as Bifidobacterium and Lactobacillus.

Six novel species of the obligate marine actinobacterium Salinispora, Salinispora cortesiana sp. nov., Salinispora fenicalii sp. nov., Salinispora goodfellowii sp. nov., Salinispora mooreana sp. nov., Salinispora oceanensis sp. nov. and Salinispora vitiensis sp. nov., and emended description of the genus Salinispora.

Ten representative actinobacterial strains isolated from marine sediments collected worldwide were studied to determine their taxonomic status. The strains were previously identified as members of the genus Salinispora and shared >99 % 16S rRNA gene sequence similarity to the three currently recognized Salinispora species. Comparative genomic analyses resulted in the delineation of six new species based on average nucleotide identity and digital DNA-DNA hybridization values below 95 and 70 %, respectively. The species status of the six new groups was supported by a core-genome phylogeny reconstructed from 2106 orthologs detected in 118 publicly available Salinispora genomes. Chemotaxonomic and physiological studies were used to complete the phenotypic characterization of the strains. The fatty acid profiles contained the major components iso-C16 : 0, C15 : 0, iso-17 : 0 and anteiso C17 : 0. Galactose and xylose were common in all whole-sugar patterns but differences were found between the six groups of strains. Polar lipid compositions were also unique for each species. Distinguishable physiological and biochemical characteristics were also recorded. The names proposed are Salinispora cortesiana sp. nov., CNY-202T (=DSM 108615T=CECT 9739T); Salinispora fenicalii sp. nov., CNT-569T (=DSM 108614T=CECT 9740T); Salinispora goodfellowii sp. nov., CNY-666T (=DSM 108616T=CECT 9738T); Salinispora mooreana sp. nov., CNT-150T (=DSM 45549T=CECT 9741T); Salinispora oceanensis sp. nov., CNT-138T (=DSM 45547T=CECT 9742T); and Salinispora vitiensis sp. nov., CNT-148T (=DSM 45548T=CECT 9743T).

Fiber Force: A Fiber Diet Intervention in an Advanced Course-Based Undergraduate Research Experience (CURE) Course.

Course-based undergraduate research experiences (CUREs) are an effective way to introduce students to contemporary scientific research. Research experiences have been shown to promote critical thinking, improve understanding and proper use of the scientific method, and help students learn practical skills including writing and oral communication. We aimed to improve scientific training by engaging students enrolled in an upper division elective course in a human microbiome CURE. The "Fiber Force" course is aimed at studying the effect of a wholesome high-fiber diet (40 to 50 g/day for two weeks) on the students' gut microbiomes. Enrolled students participated in a noninvasive diet intervention, designed health surveys, tested hypotheses on the effect of a diet intervention on the gut microbiome, and analyzed their own samples (as anonymized aggregates). The course involved learning laboratory techniques (e.g., DNA extraction, PCR, and 16S sequencing) and the incorporation of computational techniques to analyze microbiome data with QIIME2 and within the R software environment. In addition, the learning objectives focused on effective student performance in writing, data analysis, and oral communication. Enrolled students showed high performance grades on writing, data analysis and oral communication assignments. Pre- and post-course surveys indicate that the students found the experience favorable, increased their interest in science, and heightened awareness of their diet habits. Fiber Force constitutes a validated case of a research experience on microbiology with the capacity to improve research training and promote healthy dietary habits.

Maintenance of Sympatric and Allopatric Populations in Free-Living Terrestrial Bacteria.

For free-living bacteria and archaea, the equivalent of the biological species concept does not exist, creating several obstacles to the study of the processes contributing to microbial diversification. These obstacles are particularly high in soil, where high bacterial diversity inhibits the study of closely related genotypes and therefore the factors structuring microbial populations. Here, we isolated strains within a single Curtobacterium ecotype from surface soil (leaf litter) across a regional climate gradient and investigated the phylogenetic structure, recombination, and flexible gene content of this genomic diversity to infer patterns of gene flow. Our results indicate that microbial populations are delineated by gene flow discontinuities, with distinct populations cooccurring at multiple sites. Bacterial population structure was further delineated by genomic features allowing for the identification of candidate genes possibly contributing to local adaptation. These results suggest that the genetic structure within this bacterium is maintained both by ecological specialization in localized microenvironments (isolation by environment) and by dispersal limitation between geographic locations (isolation by distance).IMPORTANCE Due to the promiscuous exchange of genetic material and asexual reproduction, delineating microbial species (and, by extension, populations) remains challenging. Because of this, the vast majority of microbial studies assessing population structure often compare divergent strains from disparate environments under varied selective pressures. Here, we investigated the population structure within a single bacterial ecotype, a unit equivalent to a eukaryotic species, defined as highly clustered genotypic and phenotypic strains with the same ecological niche. Using a combination of genomic and computational analyses, we assessed the phylogenetic structure, extent of recombination, and flexible gene content of this genomic diversity to infer patterns of gene flow. To our knowledge, this study is the first to do so for a dominant soil bacterium. Our results indicate that bacterial soil populations, similarly to those in other environments, are structured by gene flow discontinuities and exhibit distributional patterns consistent with both isolation by distance and isolation by environment. Thus, both dispersal limitation and local environments contribute to the divergence among closely related soil bacteria as observed in macroorganisms.

Experimental Evidence that Stochasticity Contributes to Bacterial Composition and Functioning in a Decomposer Community.

Stochasticity emerging from random differences in replication, death, mutation, and dispersal is thought to alter the composition of ecological communities. However, the importance of stochastic effects remains somewhat speculative because stochasticity is not directly measured but is instead inferred from unexplained variations in beta-diversity. Here, we performed a field experiment to more directly disentangle the role of stochastic processes, environmental selection, and dispersal in the composition and functioning of a natural bacterial decomposer community in the field. To increase our ability to detect these effects, we reduced initial biological and environmental heterogeneity using replicate nylon litterbags in the field. We then applied two treatments: ambient/added precipitation and bacterial and fungal dispersal using "open" litterbags (made from 18.0-µm-pore-size mesh) ("open bacterial dispersal") versus bacterial and fungal dispersal using "closed" litterbags (made from 22.0-µm-pore-size mesh) ("closed bacterial dispersal"). After 5 months, we assayed composition and functioning by the use of three subsamples from each litterbag to disentangle stochastic effects from residual variation. Our results indicate that stochasticity via ecological drift can contribute to beta-diversity in bacterial communities. However, residual variation, which had previously been included in stochasticity estimates, accounted for more than four times as much variability. At the same time, stochastic effects on beta-diversity were not attenuated at the functional level, as measured by genetic functional potential and extracellular enzyme activity. Finally, dispersal was found to interact with precipitation availability to influence the degree to which stochasticity contributed to functional variation. Together, our results demonstrate that the ability to quantify stochastic processes is key to understanding microbial diversity and its role in ecosystem functioning.IMPORTANCE Randomness can alter the diversity and composition of ecological communities. Such stochasticity may therefore obscure the relationship between the environment and community composition and hinder our ability to predict the relationship between biodiversity and ecosystem functioning. This study investigated the role of stochastic processes, environmental selection, and dispersal in microbial composition and its functioning on an intact field community. To do this, we used a controlled and replicated experiment that was similar to that used to study population genetics in the laboratory. Our study showed that, while the stochastic effects on taxonomic composition are smaller than expected, the degree to which stochasticity contributes to variability in ecosystem processes may be much higher than previously assumed.

Cystic Fibrosis-Associated Stenotrophomonas maltophilia Strain-Specific Adaptations and Responses to pH.

The airway fluids of cystic fibrosis (CF) patients contain local pH gradients and are more acidic than those of healthy individuals. pH is a critical factor that is often overlooked in studies seeking to recapitulate the infection microenvironment. We sought to determine the impact of pH on the physiology of a ubiqituous yet understudied microbe, Stenotrophomonas maltophilia Phylogenomics was first used to reconstruct evolutionary relationships between 74 strains of S. maltophilia (59 from CF patients). Neither the core genome (2,158 genes) nor the accessory genome (11,978 genes) distinguish the CF and non-CF isolates; however, strains from similar isolation sources grouped into the same subclades. We grew two human and six CF S. maltophilia isolates from different subclades at a range of pH values and observed impaired growth and altered antibiotic tolerances at pH 5. Transcriptomes revealed increased expression of both antibiotic resistance and DNA repair genes in acidic conditions. Although the gene expression profiles of S. maltophilia in lab cultures and CF sputum were distinct, we found that the same genes associated with low pH were also expressed during infection, and the higher pH cultures were more similar to sputum metatranscriptomes. Our findings suggest that S. maltophilia is not well adapted to acidity and may cope with low pH by expressing stress response genes and colonizing less acidic microenvironments. As a whole, our study underlines the impact of microenvironments on bacterial colonization and adaptation in CF infections.IMPORTANCE Understanding bacterial responses to physiological conditions is an important priority for combating opportunistic infections. The majority of CF patients succumb to inflammation and necrosis in the airways, arising from chronic infection due to ineffective mucociliary clearance. Steep pH gradients characterize the CF airways but are not often incorporated in standard microbiology culture conditions. Stenotrophomonas maltophilia is a prevalent CF opportunistic pathogen also found in many disparate environments, yet this bacterium's contribution to CF lung damage and its response to changing environmental factors remain largely understudied. Here, we show that pH impacts the physiology and antibiotic susceptibility of S. maltophilia, with implications for the development of relevant in vitro models and assessment of antibiotic sensitivity.