Bacterial chemotaxis to saccharides is governed by a trade-off between sensing and uptake.

To swim up gradients of nutrients, E. coli senses nutrient concentrations within its periplasm. For small nutrient molecules, periplasmic concentrations typically match extracellular concentrations. However, this is not necessarily the case for saccharides, such as maltose, which are transported into the periplasm via a specific porin. Previous observations have shown that, under various conditions, E. coli limits maltoporin abundance so that, for extracellular micromolar concentrations of maltose, there are predicted to be only nanomolar concentrations of free maltose in the periplasm. Thus, in the micromolar regime, the total uptake of maltose from the external environment into the cytoplasm is limited not by the abundance of cytoplasmic transport proteins but by the abundance of maltoporins. Here, we present results from experiments and modeling suggesting that this porin-limited transport enables E. coli to sense micromolar gradients of maltose despite having a high-affinity ABC transport system that is saturated at these micromolar levels. We used microfluidic assays to study chemotaxis of E. coli in various gradients of maltose and methyl-aspartate and leveraged our experimental observations to develop a mechanistic transport-and-sensing chemotaxis model. Incorporating this model into agent-based simulations, we discover a trade-off between uptake and sensing: although high-affinity transport enables higher uptake rates at low nutrient concentrations, it severely limits the range of dynamic sensing. We thus propose that E. coli may limit periplasmic uptake to increase its chemotactic sensitivity, enabling it to use maltose as an environmental cue.

Chemotaxis shapes the microscale organization of the ocean's microbiome.

The capacity of planktonic marine microorganisms to actively seek out and exploit microscale chemical hotspots has been widely theorized to affect ocean-basin scale biogeochemistry1-3, but has never been examined comprehensively in situ among natural microbial communities. Here, using a field-based microfluidic platform to quantify the behavioural responses of marine bacteria and archaea, we observed significant levels of chemotaxis towards microscale hotspots of phytoplankton-derived dissolved organic matter (DOM) at a coastal field site across multiple deployments, spanning several months. Microscale metagenomics revealed that a wide diversity of marine prokaryotes, spanning 27 bacterial and 2 archaeal phyla, displayed chemotaxis towards microscale patches of DOM derived from ten globally distributed phytoplankton species. The distinct DOM composition of each phytoplankton species attracted phylogenetically and functionally discrete populations of bacteria and archaea, with 54% of chemotactic prokaryotes displaying highly specific responses to the DOM derived from only one or two phytoplankton species. Prokaryotes exhibiting chemotaxis towards phytoplankton-derived compounds were significantly enriched in the capacity to transport and metabolize specific phytoplankton-derived chemicals, and displayed enrichment in functions conducive to symbiotic relationships, including genes involved in the production of siderophores, B vitamins and growth-promoting hormones. Our findings demonstrate that the swimming behaviour of natural prokaryotic assemblages is governed by specific chemical cues, which dictate important biogeochemical transformation processes and the establishment of ecological interactions that structure the base of the marine food web.

A tradeoff between physical encounters and consumption determines an optimal droplet size for microbial degradation of dispersed oil.

Immiscible hydrocarbons occur in the ocean water column as droplets of varying diameters. Although microbial oil degradation is a central process in the remediation of hydrocarbon pollution in marine environments, the relationship between droplet size distribution and oil degradation rates by bacteria remains unclear, with a conflicting history of laboratory studies. Despite this knowledge gap, the use of chemical dispersants in oil spill response and mitigation is based on the rationale that increasing the surface-area-to-volume ratio of droplets will enhance net bacterial biodegradation rates. We demonstrate that this intuitive argument does not apply to most natural marine environments, where the abundance of oil droplets is much lower than in laboratory experiments and droplet-bacteria encounters are the limiting factor. We present a mechanistic encounter-consumption model to predict the characteristic time for oil degradation by marine bacteria as a function of the initial oil concentration, the distribution of droplet sizes, and the initial abundance of oil-degrading bacteria. We find that the tradeoff between the encounter time and the consumption time leads to an optimal droplet size larger than the average size generated by the application of dispersants. Reducing droplet size below this optimum can increase the persistence of oil droplets in the environment from weeks to years. The new perspective granted by this biophysical model of biodegradation that explicitly accounts for oil-microbe encounters changes our understanding of biodegradation particularly in the deep ocean, where droplets are often small and oil concentrations low, and explains degradation rate discrepancies between laboratory and field studies.

Coral mucus rapidly induces chemokinesis and genome-wide transcriptional shifts toward early pathogenesis in a bacterial coral pathogen.

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.

Mechanistic model of nutrient uptake explains dichotomy between marine oligotrophic and copiotrophic bacteria.

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.

PhenoChip: A single-cell phenomic platform for high-throughput photophysiological analyses of microalgae.

Photosynthetic microorganisms are key players in aquatic ecosystems with strong potential for bioenergy production, yet their systematic selection at the single-cell level for improved productivity or stress resilience ("phenotyping") has remained largely inaccessible. To facilitate the phenotyping of microalgae and cyanobacteria, we developed "PhenoChip," a platform for the multiparametric photophysiological characterization and selection of unicellular phenotypes under user-controlled physicochemical conditions. We used PhenoChip to expose single cells of the coral symbiont Symbiodinium to thermal and chemical treatments and monitor single-cell photophysiology via chlorophyll fluorometry. This revealed strain-specific thermal sensitivity thresholds and distinct pH optima for photosynthetic performance, and permitted the identification of single cells with elevated resilience toward rising temperature. Optical expulsion technology was used to collect single cells from PhenoChip, and their propagation revealed indications of transgenerational preservation of photosynthetic phenotypes. PhenoChip represents a versatile platform for the phenotyping of photosynthetic unicells relevant to biotechnology, ecotoxicology, and assisted evolution.

Aging a little: On the optimality of limited senescence in Escherichia coli.

Recent studies have shown that even in the absence of extrinsic stress, the morphologically symmetrically dividing model bacteria Escherichia coli do not generate offspring with equal reproductive fitness. Instead, daughter cells exhibit asymmetric division times that converge to two distinct growth states. This represents a limited senescence/rejuvenation process derived from asymmetric division that is stable for hundreds of generations. It remains unclear why the bacteria do not continue the senescence beyond this asymptote. Although there are inherent fitness benefits for heterogeneity in population growth rates, the two growth equilibria are surprisingly similar, differing by a few percent. In this work we derive an explicit model for the growth of a bacterial population with two growth equilibria, based on a generalized Fibonacci recurrence, in order to quantify the fitness benefit of a limited senescence process and examine costs associated with asymmetry that could generate the observed behavior. We find that a simple saturating effect of asymmetric partitioning of subcellular components is sufficient to explain why two distinct but similar growth states may be optimal while providing evolutionarily significant growth advantages.

Single-cell bacterial transcription measurements reveal the importance of dimethylsulfoniopropionate (DMSP) hotspots in ocean sulfur cycling.

Dimethylsulfoniopropionate (DMSP) is a pivotal compound in marine biogeochemical cycles and a key chemical currency in microbial interactions. Marine bacteria transform DMSP via two competing pathways with considerably different biogeochemical implications: demethylation channels sulfur into the microbial food web, whereas cleavage releases sulfur into the atmosphere. Here, we present single-cell measurements of the expression of these two pathways using engineered fluorescent reporter strains of Ruegeria pomeroyi DSS-3, and find that external DMSP concentration dictates the relative expression of the two pathways. DMSP induces an upregulation of both pathways, but only at high concentrations (>1 µM for demethylation; >35 nM for cleavage), characteristic of microscale hotspots such as the vicinity of phytoplankton cells. Co-incubations between DMSP-producing microalgae and bacteria revealed an increase in cleavage pathway expression close to the microalgae's surface. These results indicate that bacterial utilization of microscale DMSP hotspots is an important determinant of the fate of sulfur in the ocean.

Publisher Correction: An automated Raman-based platform for the sorting of live cells by functional properties.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

An automated Raman-based platform for the sorting of live cells by functional properties.

Stable-isotope probing is widely used to study the function of microbial taxa in their natural environment, but sorting of isotopically labelled microbial cells from complex samples for subsequent genomic analysis or cultivation is still in its early infancy. Here, we introduce an optofluidic platform for automated sorting of stable-isotope-probing-labelled microbial cells, combining microfluidics, optical tweezing and Raman microspectroscopy, which yields live cells suitable for subsequent single-cell genomics, mini-metagenomics or cultivation. We describe the design and optimization of this Raman-activated cell-sorting approach, illustrate its operation with four model bacteria (two intestinal, one soil and one marine) and demonstrate its high sorting accuracy (98.3 ± 1.7%), throughput (200-500 cells h-1; 3.3-8.3 cells min-1) and compatibility with cultivation. Application of this sorting approach for the metagenomic characterization of bacteria involved in mucin degradation in the mouse colon revealed a diverse consortium of bacteria, including several members of the underexplored family Muribaculaceae, highlighting both the complexity of this niche and the potential of Raman-activated cell sorting for identifying key players in targeted processes.

A Foraging Mandala for Aquatic Microorganisms.

Aquatic environments harbor a great diversity of microorganisms, which interact with the same patchy, particulate, or diffuse resources by means of a broad array of physiological and behavioral adaptations, resulting in substantially different life histories and ecological success. To date, efforts to uncover and understand this diversity have not been matched by equivalent efforts to identify unifying frameworks that can provide a degree of generality and thus serve as a stepping stone to scale up microscale dynamics to predict their ecosystem-level consequences. In particular, evaluating the ecological consequences of different resource landscapes and of different microbial adaptations has remained a major challenge in aquatic microbial ecology. Here, inspired by Ramon Margalef's mandala for phytoplankton, we propose a foraging mandala for microorganisms in aquatic environments, which accounts for both the local environment and individual adaptations. This biophysical framework distills resource acquisition into two fundamental parameters: the search time for a new resource and the growth return obtained from encounter with a resource. We illustrate the foraging mandala by considering a broad range of microbial adaptations and environmental characteristics. The broad applicability of the foraging mandala suggests that it could be a useful framework to compare disparate microbial strategies in aquatic environments and to reduce the vast complexity of microbe-environment interactions into a minimal number of fundamental parameters.

Synthesis and degradation of FtsZ quantitatively predict the first cell division in starved bacteria.

In natural environments, microbes are typically non-dividing and gauge when nutrients permit division. Current models are phenomenological and specific to nutrient-rich, exponentially growing cells, thus cannot predict the first division under limiting nutrient availability. To assess this regime, we supplied starving Escherichia coli with glucose pulses at increasing frequencies. Real-time metabolomics and microfluidic single-cell microscopy revealed unexpected, rapid protein, and nucleic acid synthesis already from minuscule glucose pulses in non-dividing cells. Additionally, the lag time to first division shortened as pulsing frequency increased. We pinpointed division timing and dependence on nutrient frequency to the changing abundance of the division protein FtsZ. A dynamic, mechanistic model quantitatively relates lag time to FtsZ synthesis from nutrient pulses and FtsZ protease-dependent degradation. Lag time changed in model-congruent manners, when we experimentally modulated the synthesis or degradation of FtsZ. Thus, limiting abundance of FtsZ can quantitatively predict timing of the first cell division.

Segmentation and the Entropic Elasticity of Modular Proteins.

Single-molecule force spectroscopy utilizes polyproteins, which are composed of tandem modular domains, to study their mechanical and structural properties. Under the application of external load, the polyproteins respond by unfolding and refolding domains to acquire the most favored extensibility. However, unlike single-domain proteins, the sequential unfolding of the each domain modifies the free energy landscape (FEL) of the polyprotein nonlinearly. Here we use force-clamp (FC) spectroscopy to measure unfolding and collapse-refolding dynamics of polyubiquitin and poly(I91). Their reconstructed unfolding FEL involves hundreds of kB T in accumulating work performed against conformational entropy, which dwarfs the ∼30 kB T that is typically required to overcome the free energy difference of unfolding. We speculate that the additional entropic energy caused by segmentation of the polyprotein to individual proteins plays a crucial role in defining the "shock absorber" properties of elastic proteins such as the giant muscle protein titin.

A microfluidics-based in situ chemotaxis assay to study the behaviour of aquatic microbial communities.

Microbial interactions influence the productivity and biogeochemistry of the ocean, yet they occur in miniscule volumes that cannot be sampled by traditional oceanographic techniques. To investigate the behaviours of marine microorganisms at spatially relevant scales, we engineered an in situ chemotaxis assay (ISCA) based on microfluidic technology. Here, we describe the fabrication, testing and first field results of the ISCA, demonstrating its value in accessing the microbial behaviours that shape marine ecosystems.

Logarithmic sensing in Bacillus subtilis aerotaxis.

Aerotaxis, the directed migration along oxygen gradients, allows many microorganisms to locate favorable oxygen concentrations. Despite oxygen's fundamental role for life, even key aspects of aerotaxis remain poorly understood. In Bacillus subtilis, for example, there is conflicting evidence of whether migration occurs to the maximal oxygen concentration available or to an optimal intermediate one, and how aerotaxis can be maintained over a broad range of conditions. Using precisely controlled oxygen gradients in a microfluidic device, spanning the full spectrum of conditions from quasi-anoxic to oxic (60 n mol/l-1 m mol/l), we resolved B. subtilis' 'oxygen preference conundrum' by demonstrating consistent migration towards maximum oxygen concentrations ('monotonic aerotaxis'). Surprisingly, the strength of aerotaxis was largely unchanged over three decades in oxygen concentration (131 n mol/l-196 µ mol/l). We discovered that in this range B. subtilis responds to the logarithm of the oxygen concentration gradient, a rescaling strategy called 'log-sensing' that affords organisms high sensitivity over a wide range of conditions. In these experiments, high-throughput single-cell imaging yielded the best signal-to-noise ratio of any microbial taxis study to date, enabling the robust identification of the first mathematical model for aerotaxis among a broad class of alternative models. The model passed the stringent test of predicting the transient aerotactic response despite being developed on steady-state data, and quantitatively captures both monotonic aerotaxis and log-sensing. Taken together, these results shed new light on the oxygen-seeking capabilities of B. subtilis and provide a blueprint for the quantitative investigation of the many other forms of microbial taxis.

Microbial Morphology and Motility as Biosignatures for Outer Planet Missions.

Meaningful motion is an unambiguous biosignature, but because life in the Solar System is most likely to be microbial, the question is whether such motion may be detected effectively on the micrometer scale. Recent results on microbial motility in various Earth environments have provided insight into the physics and biology that determine whether and how microorganisms as small as bacteria and archaea swim, under which conditions, and at which speeds. These discoveries have not yet been reviewed in an astrobiological context. This paper discusses these findings in the context of Earth analog environments and environments expected to be encountered in the outer Solar System, particularly the jovian and saturnian moons. We also review the imaging technologies capable of recording motility of submicrometer-sized organisms and discuss how an instrument would interface with several types of sample-collection strategies. Key Words: In situ measurement-Biosignatures-Microbiology-Europa-Ice. Astrobiology 16, 755-774.

Chemotaxis toward phytoplankton drives organic matter partitioning among marine bacteria.

The microenvironment surrounding individual phytoplankton cells is often rich in dissolved organic matter (DOM), which can attract bacteria by chemotaxis. These "phycospheres" may be prominent sources of resource heterogeneity in the ocean, affecting the growth of bacterial populations and the fate of DOM. However, these effects remain poorly quantified due to a lack of quantitative ecological frameworks. Here, we used video microscopy to dissect with unprecedented resolution the chemotactic accumulation of marine bacteria around individual Chaetoceros affinis diatoms undergoing lysis. The observed spatiotemporal distribution of bacteria was used in a resource utilization model to map the conditions under which competition between different bacterial groups favors chemotaxis. The model predicts that chemotactic, copiotrophic populations outcompete nonmotile, oligotrophic populations during diatom blooms and bloom collapse conditions, resulting in an increase in the ratio of motile to nonmotile cells and in the succession of populations. Partitioning of DOM between the two populations is strongly dependent on the overall concentration of bacteria and the diffusivity of different DOM substances, and within each population, the growth benefit from phycospheres is experienced by only a small fraction of cells. By informing a DOM utilization model with highly resolved behavioral data, the hybrid approach used here represents a new path toward the elusive goal of predicting the consequences of microscale interactions in the ocean.

Vortical ciliary flows actively enhance mass transport in reef corals.

The exchange of nutrients and dissolved gasses between corals and their environment is a critical determinant of the growth of coral colonies and the productivity of coral reefs. To date, this exchange has been assumed to be limited by molecular diffusion through an unstirred boundary layer extending 1-2 mm from the coral surface, with corals relying solely on external flow to overcome this limitation. Here, we present direct microscopic evidence that, instead, corals can actively enhance mass transport through strong vortical flows driven by motile epidermal cilia covering their entire surface. Ciliary beating produces quasi-steady arrays of counterrotating vortices that vigorously stir a layer of water extending up to 2 mm from the coral surface. We show that, under low ambient flow velocities, these vortices, rather than molecular diffusion, control the exchange of nutrients and oxygen between the coral and its environment, enhancing mass transfer rates by up to 400%. This ability of corals to stir their boundary layer changes the way that we perceive the microenvironment of coral surfaces, revealing an active mechanism complementing the passive enhancement of transport by ambient flow. These findings extend our understanding of mass transport processes in reef corals and may shed new light on the evolutionary success of corals and coral reefs.

Extended Kalman filter estimates the contour length of a protein in single molecule atomic force microscopy experiments.

Atomic force microscopy force spectroscopy has become a powerful biophysical technique for probing the dynamics of proteins at the single molecule level. Extending a polyprotein at constant velocity produces the now familiar sawtooth pattern force-length relationship. Customarily, manual fits of the wormlike chain (WLC) model of polymer elasticity to sawtooth pattern data have been used to measure the contour length L(c) of the protein as it unfolds one module at a time. The change in the value of L(c) measures the number of amino acids released by an unfolding protein and can be used as a precise locator of the unfolding transition state. However, manual WLC fits are slow and introduce inevitable operator-driven errors which reduce the accuracy of the L(c) estimates. Here we demonstrate an extended Kalman filter that provides operator-free real time estimates of L(c) from sawtooth pattern data. The filter design is based on a cantilever-protein arrangement modeled by a simple linear time-invariant cantilever model and by a nonlinear force-length relationship function for the protein. The resulting Kalman filter applied to sawtooth pattern data demonstrates its real time, operator-free ability to accurately measure L(c). These results are a marked improvement over the earlier techniques and the procedure is easily extended or modified to accommodate further quantities of interest in force spectroscopy.