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Optofluidic Raman-activated cell sorting for targeted genome retrieval or cultivation of microbial cells with specific functions
Abstract
Stable isotope labeling of microbial taxa of interest and their sorting provide an efficient and direct way to answer the question “who does what?” in complex microbial communities when coupled with fluorescence in situ hybridization or downstream ‘omics’ analyses. We have developed a platform for automated Raman-based sorting in which optical tweezers and microfluidics are used to sort individual cells of interest from microbial communities on the basis of their Raman spectra. This sorting of cells and their downstream DNA analysis, such as by mini-metagenomics or single-cell genomics, or cultivation permits a direct link to be made between the metabolic roles and the genomes of microbial cells within complex microbial communities, as well as targeted isolation of novel microbes with a specific physiology of interest. We describe a protocol from sample preparation through Raman-activated live cell sorting. Subsequent cultivation of sorted cells is described, whereas downstream DNA analysis involves well-established approaches with abundant methods available in the literature. Compared with manual sorting, this technique provides a substantially higher throughput (up to 500 cells per h). Furthermore, the platform has very high sorting accuracy (98.3 ± 1.7%) and is fully automated, thus avoiding user biases that might accompany manual sorting. We anticipate that this protocol will empower in particular environmental and host-associated microbiome research with a versatile tool to elucidate the metabolic contributions of microbial taxa within their complex communities. After a 1-d preparation of cells, sorting takes on the order of 4 h, depending on the number of cells required.
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Raman microspectroscopy for microbiology
Abstract
Raman microspectroscopy offers microbiologists a rapid and non-destructive technique to assess the chemical composition of individual live microorganisms in near real time. In this Primer, we outline the methodology and potential for its application to microbiology. We describe the technical aspects of Raman analyses and practical approaches to apply this method to microbiological questions. We discuss recent and potential future applications to determine the composition and distribution of microbial metabolites down to subcellular scale; to investigate the host–microorganism, cell–cell and cell–environment molecular exchanges that underlie the structure of microbial ecosystems from the ocean to the human gut microbiomes; and to interrogate the microbial diversity of functional roles in environmental and industrial processes — key themes in modern microbiology. We describe the current technical limitations of Raman microspectroscopy for investigation of microorganisms and approaches to minimize or address them. Recent technological innovations in Raman microspectroscopy will further reinforce the power and capacity of this method for broader adoptions in microbiology, allowing microbiologists to deepen their understanding of the microbial ecology of complex communities at nearly any scale of interest.
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A traveling-wave solution for bacterial chemotaxis with growth
Abstract
Bacterial cells navigate their environment by directing their movement along chemical gradients. This process, known as chemotaxis, can promote the rapid expansion of bacterial populations into previously unoccupied territories. However, despite numerous experimental and theoretical studies on this classical topic, chemotaxis-driven population expansion is not understood in quantitative terms. Building on recent experimental progress, we here present a detailed analytical study that provides a quantitative understanding of how chemotaxis and cell growth lead to rapid and stable expansion of bacterial populations. We provide analytical relations that accurately describe the dependence of the expansion speed and density profile of the expanding population on important molecular, cellular, and environmental parameters. In particular, expansion speeds can be boosted by orders of magnitude when the environmental availability of chemicals relative to the cellular limits of chemical sensing is high. Analytical understanding of such complex spatiotemporal dynamic processes is rare. Our analytical results and the methods employed to attain them provide a mathematical framework for investigations of the roles of taxis in diverse ecological contexts across broad parameter regimes.
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Relationships between community composition, productivity and invasion resistance in semi-natural bacterial microcosms
Abstract
Common garden experiments that inoculate a standardised growth medium with synthetic microbial communities (i.e. constructed from individual isolates or using dilution cultures) suggest that the ability of the community to resist invasions by additional microbial taxa can be predicted by the overall community productivity (broadly defined as cumulative cell density and/or growth rate). However, to the best of our knowledge, no common garden study has yet investigated the relationship between microbial community composition and invasion resistance in microcosms whose compositional differences reflect natural, rather than laboratory-designed, variation. We conducted experimental invasions of two bacterial strains (Pseudomonas fluorescens and Pseudomonas putida) into laboratory microcosms inoculated with 680 different mixtures of bacteria derived from naturally occurring microbial communities collected in the field. Using 16S rRNA gene amplicon sequencing to characterise microcosm starting composition, and high-throughput assays of community phenotypes including productivity and invader survival, we determined that productivity is a key predictor of invasion resistance in natural microbial communities, substantially mediating the effect of composition on invasion resistance. The results suggest that similar general principles govern invasion in artificial and natural communities, and that factors affecting resident community productivity should be a focal point for future microbial invasion experiments.
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Polysaccharide-Bacteria Interactions From the Lens of Evolutionary Ecology
Abstract
Microbes have the unique ability to break down the complex polysaccharides that make up the bulk of organic matter, initiating a cascade of events that leads to their recycling. Traditionally, the rate of organic matter degradation is perceived to be limited by the chemical and physical structure of polymers. Recent advances in microbial ecology, however, suggest that polysaccharide persistence can result from non-linear growth dynamics created by the coexistence of alternate degradation strategies, metabolic roles as well as by ecological interactions between microbes. This complex “landscape” of degradation strategies and interspecific interactions present in natural microbial communities appears to be far from evolutionarily stable, as frequent gene gain and loss reshape enzymatic repertoires and metabolic roles. In this perspective, we discuss six challenges at the heart of this problem, ranging from the evolution of genetic repertoires, phenotypic heterogeneity in clonal populations, the development of a trait-based ecology, and the impact of metabolic interactions and microbial cooperation on degradation rates. We aim to reframe some of the key questions in the study of polysaccharide-bacteria interactions in the context of eco-evolutionary dynamics, highlighting possible research directions that, if pursued, would advance our understanding of polysaccharide degraders at the interface between biochemistry, ecology and evolution.
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Sinking enhances the degradation of organic particles by marine bacteria
Abstract
The sinking of organic particles in the ocean and their degradation by marine microorganisms is one of the main drivers of the biological pump. Yet, the mechanisms determining the magnitude of the pump remain poorly understood, limiting our ability to predict this carbon flux in future ocean scenarios. Current ocean models assume that the biological pump is governed by the competition between sinking speed and degradation rate, with the two processes independent from one another. Contrary to this paradigm, we show that sinking itself is a primary determinant of the rate at which bacteria degrade particles. Heterotrophic bacterial degradation rates were obtained from a laboratory study on model surface-colonized particles at atmospheric pressure under a range of flow speeds to mimic different sinking velocities. We find that even modest sinking speeds of 8 m day−1 enhance degradation rates more than 10-fold compared with degradation rates of non-sinking particles. We discovered that the molecular mechanism underlying this sinking-enhanced degradation is the flow-induced removal from the particles of the oligomeric breakdown products, which otherwise compete for enzymatic activity. This mechanism applies across several substrates and bacterial strains, suggesting its potentially broad occurrence under natural marine conditions. Integrating our findings into a mathematical model of particulate carbon flux, we propose that the coupling of sinking and degradation may contribute, in conjunction with other processes, to determining the magnitude of the vertical carbon flux in the ocean.
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Resource–diversity relationships in bacterial communities reflect the network structure of microbial metabolism
Abstract
The relationship between the number of available nutrients and community diversity is a central question in ecological research that remains unanswered. Here we studied the assembly of hundreds of soil-derived microbial communities on a wide range of well-defined resource environments, from single carbon sources to combinations of up to 16. We found that, while single resources supported multispecies communities varying from 8 to 40 taxa, mean community richness increased only one-by-one with additional resources. Cross-feeding could reconcile these seemingly contrasting observations, with the metabolic network seeded by the supplied resources explaining the changes in richness due to both the identity and the number of resources, as well as the distribution of taxa across different communities. By using a consumer–resource model incorporating the inferred cross-feeding network, we provide further theoretical support to our observations and a framework to link the type and number of environmental resources to microbial community diversity.
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Nationwide genomic atlas of soil-dwelling Listeria reveals effects of selection and population ecology on pangenome evolution
Abstract
Natural bacterial populations can display enormous genomic diversity, primarily in the form of gene content variation caused by the frequent exchange of DNA with the local environment. However, the ecological drivers of genomic variability and the role of selection remain controversial. Here, we address this gap by developing a nationwide atlas of 1,854 Listeria isolates, collected systematically from soils across the contiguous United States. We found that Listeria was present across a wide range of environmental parameters, being mainly controlled by soil moisture, molybdenum and salinity concentrations. Whole-genome data from 594 representative strains allowed us to decompose Listeria diversity into 12 phylogroups, each with large differences in habitat breadth and endemism. ‘Cosmopolitan’ phylogroups, prevalent across many different habitats, had more open pangenomes and displayed weaker linkage disequilibrium, reflecting higher rates of gene gain and loss, and allele exchange than phylogroups with narrow habitat ranges. Cosmopolitan phylogroups also had a large fraction of genes affected by positive selection. The effect of positive selection was more pronounced in the phylogroup-specific core genome, suggesting that lineage-specific core genes are important drivers of adaptation. These results indicate that genome flexibility and recombination are the consequence of selection to survive in variable environments.
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Empowering the crowd: feasible strategies for epidemic management in high-density informal settlements. The case of COVID-19 in Northwest Syria
Abstract
More than 1 billion people live in informal settlements worldwide, where precarious living conditions pose unique challenges to managing a COVID-19 outbreak. Taking Northwest Syria as a case study, we simulated an outbreak in high-density informal Internally Displaced Persons (IDP) camps using a stochastic Susceptible-Exposed-Infectious-Recovered model. Expanding on previous studies, taking social conditions and population health/structure into account, we modelled several interventions feasible in these settings: moderate self-distancing, self-isolation of symptomatic cases and protection of the most vulnerable in ‘safety zones’. We considered complementary measures to these interventions that can be implemented autonomously by these communities, such as buffer zones, health checks and carers for isolated individuals, quantifying their impact on the micro-dynamics of disease transmission. All interventions significantly reduce outbreak probability and some of them reduce mortality when an outbreak does occur. Self-distancing reduces mortality by up to 35% if contacts are reduced by 50%. A reduction in mortality by up to 18% can be achieved by providing one self-isolation tent per eight people. Protecting the most vulnerable in a safety zone reduces the outbreak probability in the vulnerable population and has synergistic effects with the other interventions. Our model predicts that a combination of all simulated interventions may reduce mortality by more than 90% and delay an outbreak’s peak by almost 2 months. Our results highlight the potential for non-medical interventions to mitigate the effects of the pandemic. Similar measures may be applicable to controlling COVID-19 in other informal settlements, particularly IDP camps in conflict regions, around the world.
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Public good exploitation in natural bacterioplankton communities
Abstract
Bacteria often interact with their environment through extracellular molecules that increase access to limiting resources. These secretions can act as public goods, creating incentives for exploiters to invade and “steal” public goods away from producers. This phenomenon has been studied extensively in vitro, but little is known about the occurrence and impact of public good exploiters in the environment. Here, we develop a genomic approach to systematically identify bacteria that can exploit public goods produced during the degradation of polysaccharides. Focusing on chitin, a highly abundant marine biopolymer, we show that public good exploiters are active in natural chitin degrading microbial communities, invading early during colonization, and potentially hindering degradation. In contrast to in vitro studies, we find that exploiters and degraders belong to distant lineages, facilitating their coexistence. Our approach opens novel avenues to use the wealth of genomic data available to infer ecological roles and interactions among microbes.
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Environmental fluctuations and their effects on microbial communities, populations and individuals
Abstract
From the homeostasis of human health to the cycling of Earth’s elements, microbial activities underlie environmental, medical and industrial processes. These activities occur in chemical and physical landscapes that are highly dynamic and experienced by bacteria as fluctuations. In this review, we first discuss how bacteria can experience both spatial and temporal heterogeneity in their environments as temporal fluctuations of various timescales (seconds to seasons) and types (nutrient, sunlight, fluid flow, etc.). We then focus primarily on nutrient fluctuations to discuss how bacterial communities, populations and single cells respond to environmental fluctuations. Overall, we find that environmental fluctuations are ubiquitous and diverse, and strongly shape microbial behavior, ecology and evolution when compared with environments in which conditions remain constant over time. We hope this review may serve as a guide toward understanding the significance of environmental fluctuations in microbial life, such that their contributions and implications can be better assessed and exploited.
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Coral mucus rapidly induces chemokinesis and genome-wide transcriptional shifts toward early pathogenesis in a bacterial coral pathogen
Abstract
Elevated seawater temperatures have contributed to the rise of coral disease mediated by bacterial pathogens, such as the globally distributed Vibrio coralliilyticus, which utilizes coral mucus as a chemical cue to locate stressed corals. However, the physiological events in the pathogens that follow their entry into the coral host environment remain unknown. Here, we present simultaneous measurements of the behavioral and transcriptional responses of V. coralliilyticus BAA-450 incubated in coral mucus. Video microscopy revealed a strong and rapid chemokinetic behavioral response by the pathogen, characterized by a two-fold increase in average swimming speed within 6 min of coral mucus exposure. RNA sequencing showed that this bacterial behavior was accompanied by an equally rapid differential expression of 53% of the genes in the V. coralliilyticus genome. Specifically, transcript abundance 10 min after mucus exposure showed upregulation of genes involved in quorum sensing, biofilm formation, and nutrient metabolism, and downregulation of flagella synthesis and chemotaxis genes. After 60 min, we observed upregulation of genes associated with virulence, including zinc metalloproteases responsible for causing coral tissue damage and algal symbiont photoinactivation, and secretion systems that may export toxins. Together, our results suggest that V. coralliilyticus employs a suite of behavioral and transcriptional responses to rapidly shift into a distinct infection mode within minutes of exposure to the coral microenvironment.
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A distinct growth physiology enhances bacterial growth under rapid nutrient fluctuations
Abstract
It has long been known that bacteria coordinate their physiology with their nutrient environment, yet our current understanding offers little intuition for how bacteria respond to the second-to-minute scale fluctuations in nutrient concentration characteristic of many microbial habitats. To investigate the effects of rapid nutrient fluctuations on bacterial growth, we couple custom microfluidics with single-cell microscopy to quantify the growth rate of E. coli experiencing 30 s to 60 min nutrient fluctuations. Compared to steady environments of equal average concentration, fluctuating environments reduce growth rate by up to 50%. However, measured reductions in growth rate are only 38% of the growth loss predicted from single nutrient shifts. This enhancement derives from the distinct growth response of cells grown in environments that fluctuate rather than shift once. We report an unexpected physiology adapted for growth in nutrient fluctuations and implicate nutrient timescale as a critical environmental parameter beyond nutrient identity and concentration.
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Mechanistic model of nutrient uptake explains dichotomy between marine oligotrophic and copiotrophic bacteria
Abstract
Marine bacterial diversity is immense and believed to be driven in part by trade-offs in metabolic strategies. Here we consider heterotrophs that rely on organic carbon as an energy source and present a molecular-level model of cell metabolism that explains the dichotomy between copiotrophs—which dominate in carbon-rich environments—and oligotrophs—which dominate in carbon-poor environments—as the consequence of trade-offs between nutrient transport systems. While prototypical copiotrophs, like Vibrios, possess numerous phosphotransferase systems (PTS), prototypical oligotrophs, such as SAR11, lack PTS and rely on ATP-binding cassette (ABC) transporters, which use binding proteins. We develop models of both transport systems and use them in proteome allocation problems to predict the optimal nutrient uptake and metabolic strategy as a function of carbon availability. We derive a Michaelis–Menten approximation of ABC transport, analytically demonstrating how the half-saturation concentration is a function of binding protein abundance. We predict that oligotrophs can attain nanomolar half-saturation concentrations using binding proteins with only micromolar dissociation constants and while closely matching transport and metabolic capacities. However, our model predicts that this requires large periplasms and that the slow diffusion of the binding proteins limits uptake. Thus, binding proteins are critical for oligotrophic survival yet severely constrain growth rates. We propose that this trade-off fundamentally shaped the divergent evolution of oligotrophs and copiotrophs.
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Experimentally-validated correlation analysis reveals new anaerobic methane oxidation partnerships with consortium-level heterogeneity in diazotrophy
Abstract
Archaeal anaerobic methanotrophs (“ANME”) and sulfate-reducing Deltaproteobacteria (“SRB”) form symbiotic multicellular consortia capable of anaerobic methane oxidation (AOM), and in so doing modulate methane flux from marine sediments. The specificity with which ANME associate with particular SRB partners in situ, however, is poorly understood. To characterize partnership specificity in ANME-SRB consortia, we applied the correlation inference technique SparCC to 310 16S rRNA amplicon libraries prepared from Costa Rica seep sediment samples, uncovering a strong positive correlation between ANME-2b and members of a clade of Deltaproteobacteria we termed SEEP-SRB1g. We confirmed this association by examining 16S rRNA diversity in individual ANME-SRB consortia sorted using flow cytometry and by imaging ANME-SRB consortia with fluorescence in situ hybridization (FISH) microscopy using newly-designed probes targeting the SEEP-SRB1g clade. Analysis of genome bins belonging to SEEP-SRB1g revealed the presence of a complete nifHDK operon required for diazotrophy, unusual in published genomes of ANME-associated SRB. Active expression of nifH in SEEP-SRB1g within ANME-2b—SEEP-SRB1g consortia was then demonstrated by microscopy using hybridization chain reaction (HCR-) FISH targeting nifHtranscripts and diazotrophic activity was documented by FISH-nanoSIMS experiments. NanoSIMS analysis of ANME-2b—SEEP-SRB1g consortia incubated with a headspace containing CH4 and 15N2 revealed differences in cellular 15N-enrichment between the two partners that varied between individual consortia, with SEEP-SRB1g cells enriched in 15N relative to ANME-2b in one consortium and the opposite pattern observed in others, indicating both ANME-2b and SEEP-SRB1g are capable of nitrogen fixation, but with consortium-specific variation in whether the archaea or bacterial partner is the dominant diazotroph.
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A unified theory for organic matter accumulation
Abstract
Organic matter constitutes a key reservoir in global elemental cycles. However, our understanding of the dynamics of organic matter and its accumulation remains incomplete. Seemingly disparate hypotheses have been proposed to explain organic matter accumulation: the slow degradation of intrinsically recalcitrant substrates, the depletion to concentrations that inhibit microbial consumption, and a dependency on the consumption capabilities of nearby microbial populations. Here, using a mechanistic model, we develop a theoretical framework that explains how organic matter predictably accumulates in natural environments due to biochemical, ecological, and environmental factors. Our framework subsumes the previous hypotheses. Changes in the microbial community or the environment can move a class of organic matter from a state of functional recalcitrance to a state of depletion by microbial consumers. The model explains the vertical profile of dissolved organic carbon in the ocean and connects microbial activity at subannual timescales to organic matter turnover at millennial timescales. The threshold behavior of the model implies that organic matter accumulation may respond nonlinearly to changes in temperature and other factors, providing hypotheses for the observed correlations between organic carbon reservoirs and temperature in past earth climates.
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Nutrient complexity triggers transitions between solitary and colonial growth in bacterial populations
Abstract
Microbial populations often experience fluctuations in nutrient complexity in their natural environment such as between high molecular weight polysaccharides and simple monosaccharides. However, it is unclear if cells can adopt growth behaviors that allow individuals to optimally respond to differences in nutrient complexity. Here, we directly control nutrient complexity and use quantitative single-cell analysis to study the growth dynamics of individuals within populations of the aquatic bacterium Caulobacter crescentus. We show that cells form clonal microcolonies when growing on the polysaccharide xylan, which is abundant in nature and degraded using extracellular cell-linked enzymes; and disperse to solitary growth modes when the corresponding monosaccharide xylose becomes available or nutrients are exhausted. We find that the cellular density required to achieve maximal growth rates is four-fold higher on xylan than on xylose, indicating that aggregating is advantageous on polysaccharides. When collectives on xylan are transitioned to xylose, cells start dispersing, indicating that colony formation is no longer beneficial and solitary behaviors might serve to reduce intercellular competition. Our study demonstrates that cells can dynamically tune their behaviors when nutrient complexity fluctuates, elucidates the quantitative advantages of distinct growth behaviors for individual cells and indicates why collective growth modes are prevalent in microbial populations.
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Niche dimensions of a marine bacterium are identified using invasion studies in coastal seawater
Abstract
Niche theory is a foundational ecological concept that explains the distribution of species in natural environments. Identifying the dimensions of any organism’s niche is challenging because numerous environmental factors can affect organism viability. We used serial invasion experiments to introduce Ruegeria pomeroyi DSS-3, a heterotrophic marine bacterium, into a coastal phytoplankton bloom on 14 dates. RNA-sequencing analysis of R. pomeroyi was conducted after 90 min to assess its niche dimensions in this dynamic ecosystem. We identified ~100 external conditions eliciting transcriptional responses, which included substrates, nutrients, metals and biotic interactions such as antagonism, resistance and cofactor synthesis. The peak bloom was characterized by favourable states for most of the substrate dimensions, but low inferred growth rates of R. pomeroyi at this stage indicated that its niche was narrowed by factors other than substrate availability, most probably negative biotic interactions with the bloom dinoflagellate. Our findings indicate chemical and biological features of the ocean environment that can constrain where heterotrophic bacteria survive.
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Salt-Tolerant Metabolomics for Exometabolomic Measurements of Marine Bacterial Isolates
Abstract
Identifying and quantifying metabolites secreted by microbial isolates can aid in understanding the physiological traits of diverse species and their interaction with the environment. Mass spectrometry-based metabolomics has potential to provide a holistic view of the exometabolism of marine isolates, but the high salt content of such samples interferes with chromatography and ionization during the measurement of polar exometabolites. The most common desalting methods are faced with major limitations, including limited separation of small polar metabolites from salts, the use of organic solvents that cannot accommodate large salt quantities, and sample throughput. Here, we utilize a cyano stationary phase to develop a high-throughput, isocratic liquid chromatography-mass spectrometry (LC-MS) desalting method that mitigates these shortcomings. We demonstrate that counterions present in a common marine growth medium experience distinct elution times, which prevents their coelution with 73 physiologically relevant polar metabolites, effectively minimizing the effects of salt content on ion suppression. We determined optimal salt concentrations for quadrupole time-of-flight (QTOF) MS measurements and limits of quantification in the low micromolar range in the salty matrix. The efficacy of this method was demonstrated through the measurement of exometabolites secreted by three marine bacterial isolates originating from a carrageenan degrading microbial community. This method provides a simple, versatile desalting method for measuring exometabolites of environmental isolates and other biological matrices.
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Phylogenetic Core Groups: a promising concept in search of a consistent methodological framework
Abstract
In this comment, we analyse the conceptual framework proposed by Aguirre de Cárcer (Microbiome 7:142, 2019), introducing the novel concept of Phylogenetic Core Groups (PCGs). This notion aims to complement the traditional classification in operational taxonomic units (OTUs), widely used in microbial ecology, to provide a more intrinsic taxonomical classification which avoids the use of pre-determined thresholds. However, to introduce this concept, the author frames his proposal in a wider theoretical framework based on a conceptualization of selection that we argue is a tautology. This blurs the subsequent formulation of an assembly principle for microbial communities, favouring that some contradictory examples introduced to support the framework appear aligned in their conclusions. And more importantly, under this framework and its derived methodology, it is not possible to infer PCGs from data in a consistent way. We reanalyse the proposal to identify its logical and methodological flaws and, through the analysis of synthetic scenarios, we propose a number of methodological refinements to contribute towards the determination of PCGs in a consistent way. We hope our analysis will promote the exploration of PCGs as a potentially valuable tool, helping to bridge the gap between environmental conditions and community composition in microbial ecology.
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Importance of environmental factors over habitat connectivity in shaping bacterial communities in microbial mats and bacterioplankton in an Antarctic freshwater system
Abstract
Freshwater ecosystems are considered hotspots of biodiversity in Antarctic polar deserts. Anticipated warming is expected to change the hydrology of these systems due to increased meltwater and reduction of ice cover, with implications for environmental conditions and physical connectivity between habitats. Using 16S rRNA gene sequencing, we evaluated microbial mat and planktonic communities within a connected freshwater system in the McMurdo Wright Valley, Antarctica, to determine the roles of connectivity and habitat conditions in controlling microbial assemblage composition. We examined communities from glacial Lake Brownworth, the perennially ice-covered Lake Vanda and the Onyx River, which connects the two. In Lake Vanda, we found distinct microbial assemblages occupying sub-habitats at different lake depths, while the communities from Lake Brownworth and Onyx River were structurally similar. Despite the higher physical connectivity and dispersal opportunities between bacterial communities in the shallow parts of the system, environmental abiotic conditions dominated over dispersal in driving community structure. Functional metabolic pathway predictions suggested differences in the functional gene potential between the microbial mat communities located in shallower and deeper water depths. The findings suggest that increasing temperatures and meltwater due to future climate change will affect bacterial diversity and functioning in Antarctic freshwater ecosystems.
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Resource partitioning of phytoplankton metabolites that support bacterial heterotrophy
Abstract
The communities of bacteria that assemble around marine microphytoplankton are predictably dominated by Rhodobacterales, Flavobacteriales, and families within the Gammaproteobacteria. Yet whether this consistent ecological pattern reflects the result of resource-based niche partitioning or resource competition requires better knowledge of the metabolites linking microbial autotrophs and heterotrophs in the surface ocean. We characterized molecules targeted for uptake by three heterotrophic bacteria individually co-cultured with a marine diatom using two strategies that vetted the exometabolite pool for biological relevance by means of bacterial activity assays: expression of diagnostic genes and net drawdown of exometabolites, the latter detected with mass spectrometry and nuclear magnetic resonance using novel sample preparation approaches. Of the more than 36 organic molecules with evidence of bacterial uptake, 53% contained nitrogen (including nucleosides and amino acids), 11% were organic sulfur compounds (including dihydroxypropanesulfonate and dimethysulfoniopropionate), and 28% were components of polysaccharides (including chrysolaminarin, chitin, and alginate). Overlap in phytoplankton-derived metabolite use by bacteria in the absence of competition was low, and only guanosine, proline, and N-acetyl-d-glucosamine were predicted to be used by all three. Exometabolite uptake pattern points to a key role for ecological resource partitioning in the assembly marine bacterial communities transforming recent photosynthate.
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Experimental Evolution of Interference Competition
Abstract
The importance of interference competition, where individuals compete through antagonistic traits such as the production of toxins, has long been recognized by ecologists, yet understanding how these types of interactions evolve remains limited. Toxin production is thought to be beneficial when competing with a competitor. Here, we explore if antagonism can evolve by long-term selection of the toxin (pyocin) producing strain Pseudomonas aeruginosa PAO1 in the presence (or absence) of one of three clinical isolates of the same species (Recipient) over ten serial transfers. We find that inhibition decreases in the absence of a recipient. In the presence of a recipient, antagonism evolved to be different depending on the recipient used. Our study shows that the evolution of interference competition by toxins can decrease or increase, experimentally demonstrating the importance of this type of interaction for the evolution of species interactions.
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Bistability in oxidative stress response determines the migration behavior of phytoplankton in turbulence
Abstract
Turbulence is an important determinant of phytoplankton physiology, often leading to cell stress and damage. Turbulence affects phytoplankton migration both by transporting cells and by triggering switches in migratory behavior, whereby vertically migrating cells can actively invert their direction of migration upon exposure to turbulent cues. However, a mechanistic link between single-cell physiology and vertical migration of phytoplankton in turbulence is currently missing. Here, by combining physiological and behavioral experiments with a mathematical model of stress accumulation and dissipation, we show that the mechanism responsible for the switch in the direction of migration in the marine raphidophyte Heterosigma akashiwo is the integration of reactive oxygen species (ROS) signaling generated by turbulent cues. Within timescales as short as tens of seconds, the emergent downward-migrating subpopulation exhibited a twofold increase in ROS, an indicator of stress, 15% lower photosynthetic efficiency, and 35% lower growth rate over multiple generations compared to the upward-migrating subpopulation. The origin of the behavioral split as a result of a bistable oxidative stress response is corroborated by the observation that exposure of cells to exogenous stressors (H2O2, UV-A radiation, or high irradiance), in lieu of turbulence, caused comparable ROS accumulation and an equivalent split into the two subpopulations. By providing a mechanistic link between the single-cell mechanics of swimming and physiology on the one side and the emergent population-scale migratory response and impact on fitness on the other, the ROS-mediated early warning response we discovered contributes to our understanding of phytoplankton community composition in future ocean conditions.
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Evidence of streamlined extracellular electron transfer pathway from biofilm structure, metabolic stratification, and long-range electron transfer parameters
Abstract
A strain of Geobacter sulfurreducens, an organism capable of respiring solid extracellular substrates, lacking four of five outer membrane cytochrome complexes (extABCD+ strain) grows faster and produces greater current density than the wild type grown under identical conditions. To understand cellular and biofilm modifications in the extABCD+ strain responsible for this increased performance, biofilms grown using electrodes as terminal electron acceptors were sectioned and imaged using electron microscopy to determine changes in thickness and cell density, while parallel biofilms incubated in the presence of nitrogen and carbon isotopes were analyzed using NanoSIMS (nanoscale secondary ion mass spectrometry) to quantify and localize anabolic activity. Long-distance electron transfer parameters were measured for wild-type and extABCD+ biofilms spanning 5-μm gaps. Our results reveal that extABCD+ biofilms achieved higher current densities through the additive effects of denser cell packing close to the electrode (based on electron microscopy), combined with higher metabolic rates per cell compared to the wild type (based on increased rates of 15N incorporation). We also observed an increased rate of electron transfer through extABCD+ versus wild-type biofilms, suggesting that denser biofilms resulting from the deletion of unnecessary multiheme cytochromes streamline electron transfer to electrodes. The combination of imaging, physiological, and electrochemical data confirms that engineered electrogenic bacteria are capable of producing more current per cell and, in combination with higher biofilm density and electron diffusion rates, can produce a higher final current density than the wild type.
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Controls on Interspecies Electron Transport and Size Limitation of Anaerobically Methane-Oxidizing Microbial Consortia
Abstract
About 382 Tg yr−1 of methane rising through the seafloor is oxidized anaerobically (W. S. Reeburgh, Chem Rev 107:486–513, 2007, https://doi.org/10.1021/cr050362v), preventing it from reaching the atmosphere, where it acts as a strong greenhouse gas. Microbial consortia composed of anaerobic methanotrophic archaea and sulfate-reducing bacteria couple the oxidation of methane to the reduction of sulfate under anaerobic conditions via a syntrophic process. Recent experimental studies and modeling efforts indicate that direct interspecies electron transfer (DIET) is involved in this syntrophy. Here, we explore a fluorescent in situ hybridization-nanoscale secondary ion mass spectrometry data set of large, segregated anaerobic oxidation of methane (AOM) consortia that reveal a decline in metabolic activity away from the archaeal-bacterial interface and use a process-based model to identify the physiological controls on rates of AOM. Simulations reproducing the observational data reveal that ohmic resistance and activation loss are the two main factors causing the declining metabolic activity, where activation loss dominated at a distance of <8 μm. These voltage losses limit the maximum spatial distance between syntrophic partners with model simulations, indicating that sulfate-reducing bacterial cells can remain metabolically active up to ∼30 μm away from the archaeal-bacterial interface. Model simulations further predict that a hybrid metabolism that combines DIET with a small contribution of diffusive exchange of electron donors can offer energetic advantages for syntrophic consortia.
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Spatially Resolved Electron Transport through Anode-Respiring Geobacter sulfurreducens Biofilms: Controls and Constraints
Abstract
Microbial fuel cells (MFCs) with Geobacter sulfurreducens have been shown to produce high current densities; however, electron transport in G. sulfurreducens biofilms is not fully understood. Here, we utilize a spatially resolved numerical model describing this electron transfer to constrain mechanisms and controls on metabolic activity. Our model reproduces the metabolic activity profile obtained using nanoSIMS under positive (+0.24 V SHE) and negative (−0.1 V SHE) anode potentials. The simulations indicate that the distribution of the electric potential and pH both control cellular metabolism. Model simulations reproducing the experimentally determined activity patterns also support the presence of two activity modes in G. sulfurreducens biofilms, with a shift from a redox mid-potential of −0.07 V SHE to −0.15 V SHE. Our model provides valuable insights into the fundamental mechanisms of electron transfer at Micron-scale in conductive biofilms which can inform MFCs designs that maximize current production by minimizing the impact of inhibitory factors.