View publications by year:
-
Raman imaging of the phycosphere reveals sharp gradients of organic matter exuded by single phytoplankton cells
Abstract
Phytoplankton cells exude a wide array of chemicals in the water column, generating a localized microenvironment known as the phycosphere. Although it is now well accepted that the phycosphere mediates interactions between phytoplankton and bacteria, the chemical gradients around individual phytoplankton cells have never been explicitly measured, and their shape has been classically assumed to be set by ideal diffusion. Here we used Raman microspectroscopy to obtain micrometer-scale measurements of the concentration profile of a phytoplankton metabolite (fucoxanthin) around individual phytoplankton cells of different species, having radii between and 60 μm. We found that fucoxanthin concentration decreases more rapidly with distance from the cell than predicted by ideal diffusion, showing that the phycosphere includes compounds whose diffusion is characterized by nonideal effects. We explain this observation using a space-dependent diffusivity model where nonideality arises from viscosity and solubility gradients in the extracellular environment. Our results suggest an onion-structured model of the phycosphere, in which small hydrophilic solutes that obey ideal diffusion generate broad but weak gradients, whereas insoluble compounds are retained within to μ from the phytoplankton cell surface and yield steep gradients of organic matter. These observations, supported by evidence that fucoxanthin can act as an effective chemoattractant for marine bacteria, show the existence of strong and highly localized chemical cues with potentially far-reaching impacts on microbial interactions in aquatic environments. These findings highlight the importance of directly measuring the microscale chemical landscape experienced by marine microbes.
-
A Continuous Culture Millifluidic Device for the Study of Escherichia coli under Low and Fluctuating Nutrient Conditions
Abstract
Microbial habitats in nature are often characterized by low concentrations of mixed nutrients, spatial heterogeneity, and temporal fluctuations. However, traditional laboratory culturing methods fail to replicate these conditions. Batch cultures cannot sustain growth in low-nutrient environments, while chemostats maintain steady-state growth with a single limiting nutrient but are challenging to implement when the goal is to maintain defined low concentrations of nutrient mixtures or to introduce rapid fluctuations. Microfluidic systems generate dynamic environments but yield insufficient biomass for population-level omic analyses. To address these limitations, we introduce the millifluidic continuous culture device (MCCD), a versatile platform for studying microbial responses to stable and fluctuating nutrient conditions. The MCCD houses bacterial populations inside a Sterivex filter (0.45 µm polyvinylidene fluoride [PVDF] porous filtering membrane), where a continuous flow of media sustains stable culture conditions while preventing nutrient depletion. A three-way solenoid valve system, controlled via custom Matlab software, enables precise, minute-scale nutrient fluctuations. This protocol provides a step-by-step guide to operating the MCCD in two modes: (1) constant low-nutrient conditions and (2) fluctuating-nutrient conditions. Using this system, Escherichia coli grew exponentially in a mixture of amino acids and nucleobases present at tens to hundreds of nanomolar concentrations, reaching cell concentrations on the order of 109 cells/mL. By recreating key features of natural microbial habitats, the MCCD enables the study of bacterial growth and physiology under controlled yet ecologically relevant conditions in E. coli and other microbial species.
-
The coccosphere of the heavy calcifying coccolithophore Coccolithus braarudii provides defense against bacteria
Abstract
Coccolithophores produce 40–60% of marine calcium carbonate, largely through biomineralization of plates which encase cells in a ‘coccosphere’. Despite the importance of coccolithophore calcification in ocean biogeochemistry, its function remains unresolved. A hypothesis suggesting it acts as a physical deterrent has been investigated in grazers and viruses, but not in bacteria. Here we show bacterial pathogenicity in heavily-calcified C. braarudii treated with Gephyrocapsa huxleyi bacterial-pathogen, Phaeobacter inhibens, is only observed with decalcified algae, leading to algal-cell death within as little as 15 hours. Decalcified algal cell mortality is P. inhibens-specific and likely requires close proximity, since treatment with bacterial supernatant or growth-inhibiting concentrations of indole-3-acetic acid shows no detrimental effect. Additionally, scanning electron microscopy shows visible bacterial attachment only on decalcified C. braarudii. These findings provide the first experimental evidence that the coccosphere can act as a barrier against specific bacteria, highlighting its defensive role in coccolithophores.
-
Deep-sea anaerobic microbial communities couple degradation of insoluble chitin to extracellular electron transfer
Abstract
Chitin, a major structural component of arthropod exoskeletons, is an abundant carbon and nitrogen source in marine ecosystems. While its degradation is well studied in oxic waters, the microbial processes and interactions that mediate its anaerobic breakdown in deep-sea sediments remain poorly understood. Iron oxides are predicted to be energetically favorable electron acceptors for anaerobic chitin degradation, yet the spatial separation of insoluble substrates and the required microbial partnerships in sediments are not well defined. Here, we used potentiostatically controlled bioelectrochemical reactors poised at +0.22 V vs. SHE, mimicking iron-reducing conditions, to enrich and characterize a chitin-degrading, metal-reducing microbial community from an anoxic deep-sea whale-fall sediment. Amendment with crystalline chitin generated stable anodic currents, which increased upon addition of chitin-associated metabolites (N-acetylglucosamine, glucose, acetate). 16S rRNA gene sequencing revealed a deep-sea affiliated assemblage dominated by Firmicutes (Vallitalea), Spirochaetota, Gammaproteobacteria, and Desulfobacterota (Trichloromonas). Exoenzyme assays, metabolite profiling, and current measurements confirmed that active chitin degradation provided substrate(s) for extracellular electron transfer (EET). Single-cell analyses using FISH-BONCAT and nanoSIMS showed that Vallitalea (primary degrader) and electrode-respiring Desulfobacterota exhibited highest activity within the electrode biofilm, particularly within ca.10 μm of the surface. We isolated a chitin-degrading Vallitalea sp. and an iron-reducing, electrogenic Trichloromonas sp., and demonstrated that, when reconstituted in co-culture, they cooperatively degrade chitin via acetate cross-feeding coupled to EET. This integrated electrochemical and ecophysiological study reveals microbial interactions linking chitin degradation with iron-oxide respiration in deep-sea sediments and provides a defined electrogenic model community for future syntrophy research.
-
Metabolic feedbacks drive population dynamics and can lead to oscillations among leaf bacteria
Abstract
Metabolic interactions are fundamental to the assembly and function of microbiomes. Yet, our understanding of how specific interaction mechanisms can drive broader ecological outcomes and population dynamics remains limited. Here, we monitor interactions resulting from plant oligosaccharide degradation by leaf-associated bacteria using a microfluidic device that enables direct cell observation and quantitative metabolite detection. This approach enables the identification of key metabolic mediators, revealing recipient-specific patterns of carbon substrate and cofactor complementation. By linking these patterns to emergent dynamics observed between pairs of bacteria, we identify metabolically driven feedbacks that could lead to a variety of ecological outcomes – from outcompetition to coexistence characterized by oscillating population abundances. Investigating these observations with metabolic modeling allows us to systematically assess the impact of specific molecular mediators on population dynamics, yielding predictions of interaction outcomes that we validate experimentally. Our results provide a detailed mapping of metabolic mechanisms to emergent population trajectories among environmental microbes and help inform strategies for designing microbiomes with desired steady states.
-
Precise 3D Tracking of Highly Non-Planar Eukaryotic Flagellar Beating Patterns Using Digital Holographic Microscopy
Abstract
Precise tracking of the rapid and complex 3D movement of eukaryotic flagella is important for understanding their roles in cellular motility, sensory functions, and resource acquisition. Yet, achieving accurate 3D kinematic reconstruction of flagellar beating patterns, particularly highly non-planar ones, remains challenging. Here we present holoV3C, a method based on Digital Holographic Microscopy (DHM) that allows precise, label-free 3D tracking of highly non-planar eukaryotic flagella with high temporal resolution. This algorithm leverages phase anomaly detection to provide a combination of high temporal and axial resolution, with 0.25 µm for beating mouse sperm flagella and down to 53 nm for polystyrene particles, across large sampling volumes in a computationally efficient manner. Algorithmic validation is performed by tracking mouse sperm flagella over time, capturing approximately 600 points along a single flagellum to achieve high axial resolution. Furthermore, we apply holoV3C to reconstruct the highly non-planar beating dynamics of the 200-nm-diameter flagellum of the protist Reclinomonas americana with a temporal resolution of 200 frames per second. By enabling 3D tracking of non-planar eukaryotic flagella, holoV3C can yield important insights to advance our understanding of flagellar dynamics, opening new avenues in the study of microorganism motility and its ecological roles.
-
The ecology of bacterial attachment to phytoplankton
Abstract
Phytoplankton are responsible for approximately half of Earth’s net primary production and, together with heterotrophic bacteria—the main consumers of organic matter—play a pivotal role in biogeochemical cycles. Their key ecological importance has led to growing interest in the interactions between these two groups. Yet, our understanding of the microscale mechanisms driving these interactions remains limited. Recent work highlighted the contribution of bacterial motility and chemotaxis to promoting encounters and nutrient exchange between bacteria and phytoplankton. In contrast, the ecological role of bacterial attachment—an important adaptation enabling bacteria to establish the closest contact with their phytoplankton host and retain it over extended periods of time—remains less explored. Here we describe the current evidence and understanding of bacterial attachment to phytoplankton and highlight recent insights from single-cell studies. Motivated by the implications for large-scale ecosystem processes, we discuss promising research avenues to further unveil the ecological relevance of bacterial attachment to phytoplankton.
-
Defining metabolic niches for marine microbial heterotrophs
Abstract
Ocean microbial communities are made up of thousands of diverse taxa whose metabolic demands set the rates of both biomass production and degradation. Thus, these microscopic organisms play a critical role in ecosystem dynamics, global carbon cycling, and climate. While we have frameworks for relating phytoplankton diversity to rates of carbon fixation, our knowledge of how variations in heterotrophic microbial populations drive changes in carbon cycling is in its infancy. Here, we leverage global metagenomic datasets and metabolic models to identify a set of metabolic niches with distinct growth strategies. These groupings provide a simplifying framework for describing microbial communities in different oceanographic regions and for understanding how heterotrophic microbial populations function. This framework, predicated directly on metabolic capability rather than taxonomy, will enable us to tractably link heterotrophic diversity directly to biogeochemical rates in large scale ecosystem models.
-
A universal surface functionalization technique to chemically enhance live microbial cells
Abstract
Microbial surface functionalization is a powerful strategy for endowing microbes with novel, non-genetic functions. However, existing methods are often species-specific, limited in scope, and compromise cell viability. Here, we present a universal and modular platform for high-density, reproducible surface functionalization across diverse microbial species-including Gram-positive, Gram-negative, aerobic, and anaerobic bacteria-using multiple molecular classes such as fluorophores, enzymes, and nucleic acids. Our method preserves cell viability and achieves 50× higher functionalization efficiency than previous methods with a standardized protocol applicable to any azide-containing molecule. Applications of the method show reproducible and tunable phenotypic outcomes at the single-cell level: fluorophore labeling yielded adjustable fluorescence, β-lactamase conferred scalable antibiotic resistance, and DNA coatings modulated adhesion and aggregation. This platform provides quantitative, non-genetic control over microbial phenotypes and complements genetic engineering approaches. It enables new possibilities for microbial design in biotechnology, medicine, and environmental applications where genetic modification is impractical or undesirable.
-
Stochastic resilience enables particle foraging in oligotrophic marine environments
Abstract
Heterotrophic bacteria play a central role in attenuating the sequestration of carbon to the deep ocean by degrading sinking marine particles. The role of certain copiotrophic adaptations such as surface attachment and motility in particle degradation has remained unclear outside of coastal regions, where the sparsity of particles would appear to preclude a foraging lifestyle based on particle hopping. We show here instead that many oligotrophic marine environments are much more amenable to copiotrophic particle foraging than would be inferred from average-based estimates, because the foraging process samples a broad distribution of particle–bacteria interactions, with large variation in encounter times, particle sizes, and associated survival outcomes, and due to the disproportionate benefit of a particle encounter. We develop a generalized branching process model for particle foraging to assess environment viability and population growth rates based on encounters with particles, for different oceanographic particle size spectra. The results indicate that even bathypelagic environments can support particle foraging bacteria without requiring long-term starvation tolerance or multiyear feast–famine cycles, because stochastic encounters generate sufficient short-interval, high-reward events to sustain population growth despite long mean encounter times. More generally, stochasticity can confer resilience to microbial populations in resource-scarce marine environments.
-
Illuminating the newly produced viruses within the virosphere with bioorthogonal noncanonical amino acid tagging and single-virus genomic sequencing technologies
Abstract
Marine viruses impact biogeochemical cycles through cell lysis, releasing organic matter and nutrients that fuel ocean productivity. Identifying and quantifying the specific viruses active in these processes remain a priority in the field. Here, we introduce a click-chemistry method to fluorescently label, sort, and sequence the genomes of newly produced viral particles (viral progeny) released from transcriptionally active host microbial cells, alongside the analysis of co-occurring inactive cells and pre-existing viruses in environmental samples. This approach, called viral bioorthogonal noncanonical amino acid tagging (BONCAT)-fluorescence-activated cell sorting (FACS), combines BONCAT with environmental sample incubation, followed by single-virus and single-cell sorting by flow cytometry (FACS). Genomic analysis of translationally active cells and new viral progeny in coastal seawater incubations confirmed BONCAT labeling and successful sorting of diverse marine bacteria, microeukaryotic cells, and virioplankton, with stark differences in the predicted turnover of specific groups of infecting viruses, including pelagiphages, methylophages, a Flavobacteriales-associated novel “Far-T4” clade, noncanonical DNA viruses of Naomiviridae using dU instead of dT, algae-infecting giant NCLDV viruses, and parasitic virophages. Sequenced BONCAT-active cells showed a strong enrichment in viral contigs relative to the inactive cell fraction, suggestive of a large proportion of translationally active virocells. This study illustrates the effectiveness of viral BONCAT-FACS for uncovering genome-resolved virus–host dynamics. By providing a direct approach for tracking active viral infections in natural environments, this method enhances our ability to investigate behavior and interactions of these nanoscale predators, expanding our understanding of their role in ecosystem dynamics.
-
Bacterial iron acquisition by Escherichia coli is facilitated by amino acid complexation in a rapid-renewal environment
Abstract
In natural environments, bacteria often encounter low concentrations of nutrient mixtures that are continuously replenished by physical processes such as fluid flow. Studying bacterial physiology under such conditions is experimentally challenging because it is difficult to maintain steady, low nutrient concentrations with rapid renewal. Most studies on nutrient limitation have used approaches such as the chemostat, which rely on long renewal times to sustain low concentrations. We developed a Millifluidic Continuous Culture Device (MCCD), inspired by microfluidics, that enables bacterial cultivation in nutrient mixtures at low micromolar concentrations with rapid renewal driven by fluid flow. Unlike microfluidic systems, the MCCD retains sufficient culture volume to support batch-scale ‘omic analyses. Using the MCCD, we cultured Escherichia coli in a mixture of amino acids and nucleobases at three concentration ranges spanning a fivefold difference in growth rates. Surprisingly, at the lowest concentration range, cells exhibited proteomic signatures of iron limitation despite equal total ferrous iron across conditions. Uptake experiments with labeled iron–histidine and iron–cysteine complexes confirmed that amino acids facilitated ferrous iron acquisition. Under continuous flow, siderophores were washed out, rendering this pathway ineffective and revealing a previously unrecognized mechanism of iron acquisition via soluble ferrous iron–amino acid complexes. These findings highlight the importance of studying bacterial physiology at low nutrient concentrations and also suggest a broader role for other organic substrates capable of complexing iron as potential iron sources in environments with rapid renewal.
-
Phosphate deprivation restricts bacterial degradation of the marine polysaccharide fucoidan
Abstract
Brown algae and diatoms convert carbon dioxide into the polysaccharide fucoidan, which sequesters carbon in the ocean despite the prevalence of marine bacterial fucoidanase genes. Bacteria with fucoidanase genes also have high-affinity phosphate transporters, suggesting that phosphate could impact fucoidan degradation and subsequent carbon sequestration. Here, to test this hypothesis, we assembled a system consisting of a microalga that produces and a bacterium that degrades fucoidan. The fixation of carbon dioxide into fucoidan by the microalga Glossomastix sp. PLY432 occurred independent of the phosphate concentration. In contrast, the fucoidan-degrading Verrucomicrobiaceae bacterium 227 was inhibited by a lack of phosphate. Degradation of the structurally simpler polysaccharide laminarin was less affected by the phosphate concentration. Phosphate deprivation enabled the fixation of carbon dioxide in fucoidan and disabled its degradation. These conclusions suggest that phosphate deprivation could be a potential strategy to promote the fixation and sequestration of carbon dioxide as fucoidan.
-
Phytoplankton community composition in the oligotrophic Argo Basin of the eastern Indian Ocean
Abstract
Phytoplankton, the foundational organisms in ocean food webs, have been little studied in the Indonesian Throughflow region of the eastern Indian Ocean, the spawning area of Southern Bluefin Tuna. Here, we assess phytoplankton abundance, biomass, size structure, pigment composition, taxonomic diversity and percent functional mixotrophs of that region based on complementary approaches of flow cytometry, microscopy, taxon-specific pigments and rRNA gene sequencing. During summer (January–February) 2022, the region was characterized by warm (up to 30.5 °C), stratified, oligotrophic (nitrogen-limited) waters, with integrated euphotic zone (EZ) chlorophyll a (CHLa) of 13 mg m−2. EZ mean CHLa was low in the upper layer (85 ng L−1) and 3.8 times higher (320 ng L−1) at the pronounced deep CHLa maximum. EZ-integrated phytoplankton carbon averaged 1229 mg C m−2. Prochlorococcus dominated throughout the EZ, but eukaryotic carbon biomass was ∼4-times greater in the lower than upper EZ, along with a distinct community. In the upper EZ, haptophytes, dinoflagellates and prasinophycean taxa without prasinoxanthin contributed most to monovinyl chlorophyll a (MV-CHLa). In the more diverse lower EZ, haptophytes, dinoflagellates, prasinophycean taxa with prasinoxanthin, pelagophytes, and cryptophytes were the main contributors to MV-CHLa. Diatoms were a minor part of the community. A higher percentage of the upper EZ community showed mixotrophy (35–84%) relative to the lower EZ (30–51%). Nitrogen-fixing organisms (as symbionts of diatoms and free-living cyanobacteria taxa) were ubiquitous, but low in abundance. Overall, community characteristics were similar to those at the Hawaii Ocean Time-series site and the central Gulf of Mexico.
-
Dynamic reworking of marine diatom endometabolomes in response to temperature and a model bacterium
Abstract
A large annual carbon flux occurs through the surface ocean’s labile dissolved organic carbon (DOC) pool, with influx dominated by phytoplankton-derived metabolites and outflux by heterotrophic bacterioplankton uptake. We addressed the dynamics of this carbon flow between microbial primary and secondary producers through analysis of the Thalassiosira pseudonana CCMP1335 endometabolome, a proxy for the labile DOC released upon phytoplankton lysis, as temperature and bacterial presence were altered. Diatom strains acclimated at one of three different temperatures (14°C, 20°C, or 28°C) were cultured either axenically or with the bacterium Ruegeria pomeroyi DSS-3, and their endometabolites analyzed by NMR. Median concentration variation between conditions was ~1.5-fold across all identified endometabolites. Those with roles as osmolytes varied most, exhibiting concentration differences up to 170-fold across conditions with the largest variations triggered by the presence/absence of the heterotrophic bacterium. Differential expression observed for diatom metabolite synthesis pathways suggested changes in synthesis rates as a mechanism for endome tabolome remodeling. Consistent with expectations of high turnover by heterotrophic bacteria, endometabolite mean lifetimes in a DOC pool were <2 h to 12 h.