Differences in the regulatory strategies of marine oligotrophs and copiotrophs reflect differences in motility.

Aquatic bacteria frequently are divided into lifestyle categories oligotroph or copiotroph. Oligotrophs have proportionately fewer transcriptional regulatory genes than copiotrophs and are generally non-motile/chemotactic. We hypothesized that the absence of chemotaxis/motility in oligotrophs prevents them from occupying nutrient patches long enough to benefit from transcriptional regulation. We first confirmed that marine oligotrophs are generally reduced in genes for transcriptional regulation and motility/chemotaxis. Next, using a non-motile oligotroph (Ca. Pelagibacter st. HTCC7211), a motile copiotroph (Alteromonas macleodii st. HOT1A3), and [14 C]l-alanine, we confirmed that l-alanine catabolism is not transcriptionally regulated in HTCC7211 but is in HOT1A3. We then found that HOT1A3 took 2.5-4 min to initiate l-alanine oxidation at patch l-alanine concentrations, compared to <30 s for HTCC7211. By modelling cell trajectories, we predicted that, in most scenarios, non-motile cells spend <2 min in patches, compared to >4 min for chemotactic/motile cells. Thus, the time necessary for transcriptional regulation to initiate prevents transcriptional regulation from being beneficial for non-motile oligotrophs. This is supported by a mechanistic model we developed, which predicted that HTCC7211 cells with transcriptional regulation of l-alanine metabolism would produce 12% of their standing ATP stock upon encountering an l-alanine patch, compared to 880% in HTCC7211 cells without transcriptional regulation.

Fresh Ideas Bloom in Gut Healthcare to Cross-Fertilize Lake Management.

Harmful bacteria may be the most significant threat to human gut and lake ecosystem health, and they are often managed using similar tools, like poisoning with antibiotics or algicides. Out-of-the-box thinking in human microbiome engineering is leading to novel methods, like engineering bacteria to kill pathogens, "persuade" them not to produce toxins, or "mop up" their toxins. The bacterial agent can be given a competitive edge via an exclusive nutrient, and they can be engineered to commit suicide once their work is done. Viruses can kill pathogens with specific DNA sequences or knock out their antibiotic resistance genes using CRISPR technology. Some of these ideas may work for lakes. We critically review novel methods for managing harmful bacteria in the gut from the perspective of managing toxic cyanobacteria in lakes, and discuss practical aspects such as modifying bacteria using genetic engineering or directed evolution, mass culturing and controlling the agents. A key knowledge gap is in the ecology of strains, like toxigenic vs nontoxigenic Microcystis, including allelopathic and Black Queen interactions. Some of the "gut methods" may have future potential for lakes, but there presently is no substitute for established management approaches, including reducing N and P nutrient inputs, and mitigating climate change.

Heterotrophic substrate specificity in the aquatic environment: The role of microscale patchiness investigated using modelling.

Recent observations of natural bacterial communities show high genetic diversity in organic carbon uptake systems (microdiversity) and specificity in substrate species taken up. This seemingly contradicts a large body of literature from laboratory experiments under nutrient-limiting conditions, where mixed substrate use is the rule. This apparent discrepancy can be resolved by realizing that bacteria in the natural aquatic environment encounter nutrients as high-concentration patches. They may live in an ecologically nutrient-limiting environment, but they are rarely in a biologically nutrient-limited state. Rather they switch between non-growing and nutrient-replete states. During nutrient-replete growth the metabolism is saturated and assimilating additional substrates does not increase the growth rate, but carrying the assimilation system constitutes a cost. Consequently, the specialist strategy is beneficial, which is consistent with observations from laboratory experiments. When the bacteria are not growing, the added cost also reduces the fitness of the generalist species. A simple mathematical model encompassing the relevant mechanisms is developed and parameterized realistically based on the literature. The model predicts that, under pulsed conditions, specialization is beneficial when the metabolic cost of an additional uptake system is more than ~0.5% of the total metabolic cost, which is a reasonable estimate and illustrates that this is a plausible hypothesis.

Neutral Evolution and Dispersal Limitation Produce Biogeographic Patterns in Microcystis aeruginosa Populations of Lake Systems.

Molecular observations reveal substantial biogeographic patterns of cyanobacteria within systems of connected lakes. An important question is the relative role of environmental selection and neutral processes in the biogeography of these systems. Here, we quantify the effect of genetic drift and dispersal limitation by simulating individual cyanobacteria cells using an agent-based model (ABM). In the model, cells grow (divide), die, and migrate between lakes. Each cell has a full genome that is subject to neutral mutation (i.e., the growth rate is independent of the genome). The model is verified by simulating simplified lake systems, for which theoretical solutions are available. Then, it is used to simulate the biogeography of the cyanobacterium Microcystis aeruginosa in a number of real systems, including the Great Lakes, Klamath River, Yahara River, and Chattahoochee River. Model output is analyzed using standard bioinformatics tools (BLAST, MAFFT). The emergent patterns of nucleotide divergence between lakes are dynamic, including gradual increases due to accumulation of mutations and abrupt changes due to population takeovers by migrant cells (coalescence events). The model predicted nucleotide divergence is heterogeneous within systems, and for weakly connected lakes, it can be substantial. For example, Lakes Superior and Michigan are predicted to have an average genomic nucleotide divergence of 8200 bp or 0.14%. The divergence between more strongly connected lakes is much lower. Our results provide a quantitative baseline for future biogeography studies. They show that dispersal limitation can be an important factor in microbe biogeography, which is contrary to the common belief, and could affect how a system responds to environmental change.

Community Biological Ammonium Demand: A Conceptual Model for Cyanobacteria Blooms in Eutrophic Lakes.

Cyanobacterial harmful algal blooms (CyanoHABs) are enhanced by anthropogenic pressures, including excessive nutrient (nitrogen, N, and phosphorus, P) inputs and a warming climate. Severe eutrophication in aquatic systems is often manifested as non-N2-fixing CyanoHABs (e.g., Microcystis spp.), but the biogeochemical relationship between N inputs/dynamics and CyanoHABs needs definition. Community biological ammonium (NH4+) demand (CBAD) relates N dynamics to total microbial productivity and NH4+ deprivation in aquatic systems. A mechanistic conceptual model was constructed by combining nutrient cycling and CBAD observations from a spectrum of lakes to assess N cycling interactions with CyanoHABs. Model predictions were supported with CBAD data from a Microcystis bloom in Maumee Bay, Lake Erie, during summer 2015. Nitrogen compounds are transformed to reduced, more bioavailable forms (e.g., NH4+ and urea) favored by CyanoHABs. During blooms, algal biomass increases faster than internal NH4+ regeneration rates, causing high CBAD values. High turnover rates from cell death and remineralization of labile organic matter consume oxygen and enhance denitrification. These processes drive eutrophic systems to NH4+ limitation or colimitation under warm, shallow conditions and support the need for dual nutrient (N and P) control.

Dynamic, mechanistic, molecular-level modelling of cyanobacteria: Anabaena and nitrogen interaction.

Phytoplankton (eutrophication, biogeochemical) models are important tools for ecosystem research and management, but they generally have not been updated to include modern biology. Here, we present a dynamic, mechanistic, molecular-level (i.e. gene, transcript, protein, metabolite) model of Anabaena - nitrogen interaction. The model was developed using the pattern-oriented approach to model definition and parameterization of complex agent-based models. It simulates individual filaments, each with individual cells, each with genes that are expressed to yield transcripts and proteins. Cells metabolize various forms of N, grow and divide, and differentiate heterocysts when fixed N is depleted. The model is informed by observations from 269 laboratory experiments from 55 papers published from 1942 to 2014. Within this database, we identified 331 emerging patterns, and, excluding inconsistencies in observations, the model reproduces 94% of them. To explore a practical application, we used the model to simulate nutrient reduction scenarios for a hypothetical lake. For a 50% N only loading reduction, the model predicts that N fixation increases, but this fixed N does not compensate for the loading reduction, and the chlorophyll a concentration decreases substantially (by 33%). When N is reduced along with P, the model predicts an additional 8% reduction (compared to P only).

Accounting for nitrogen fixation in simple models of lake nitrogen loading/export.

Coastal eutrophication, an important global environmental problem, is primarily caused by excess nitrogen and management efforts consequently focus on lowering watershed N export (e.g., by reducing fertilizer use). Simple quantitative models are needed to evaluate alternative scenarios at the watershed scale. Existing models generally assume that, for a specific lake/reservoir, a constant fraction of N loading is exported downstream. However, N fixation by cyanobacteria may increase when the N loading is reduced, which may change the (effective) fraction of N exported. Here we present a model that incorporates this process. The model (Fixation and Export of Nitrogen from Lakes, FENL) is based on a steady-state mass balance with loading, output, loss/retention, and N fixation, where the amount fixed is a function of the N/P ratio of the loading (i.e., when N/P is less than a threshold value, N is fixed). Three approaches are used to parametrize and evaluate the model, including microcosm lab experiments, lake field observations/budgets and lake ecosystem model applications. Our results suggest that N export will not be reduced proportionally with N loading, which needs to be considered when evaluating management scenarios.

Internal carbon recycling by heterotrophic prokaryotes compensates for mismatches between phytoplankton production and heterotrophic consumption.

Molecular observational tools are useful for characterizing the composition and genetic endowment of microbial communities but cannot measure fluxes, which are critical for the understanding of ecosystems. To overcome these limitations, we used a mechanistic inference approach to estimate dissolved organic carbon (DOC) production and consumption by phytoplankton operational taxonomic units and heterotrophic prokaryotic amplicon sequence variants and inferred carbon fluxes between members of this microbial community from Western English Channel time-series data. Our analyses focused on phytoplankton spring and summer blooms, as well as bacteria summer blooms. In spring blooms, phytoplankton DOC production exceeds heterotrophic prokaryotic consumption, but in bacterial summer blooms heterotrophic prokaryotes consume three times more DOC than produced by the phytoplankton. This mismatch is compensated by heterotrophic prokaryotic DOC release by death, presumably from viral lysis. In both types of summer blooms, large amounts of the DOC liberated by heterotrophic prokaryotes are reused through internal recycling, with fluxes between different heterotrophic prokaryotes being at the same level as those between phytoplankton and heterotrophic prokaryotes. In context, internal recycling accounts for approximately 75% and 30% of the estimated net primary production (0.16 vs 0.22 and 0.08 vs 0.29 µmol l-1 d-1) in bacteria and phytoplankton summer blooms, respectively, and thus represents a major component of the Western English Channel carbon cycle. We have concluded that internal recycling compensates for mismatches between phytoplankton DOC production and heterotrophic prokaryotic consumption, and we encourage future analyses on aquatic carbon cycles to investigate fluxes between heterotrophic prokaryotes, specifically internal recycling.

Genome reduction occurred in early Prochlorococcus with an unusually low effective population size.

In the oligotrophic sunlit ocean, the most abundant free-living planktonic bacterial lineages evolve convergently through genome reduction. The cyanobacterium Prochlorococcus responsible for 10% global oxygen production is a prominent example. The dominant theory known as "genome streamlining" posits that they have extremely large effective population sizes (Ne) such that selection for metabolic efficiency acts to drive genome reduction. Because genome reduction largely took place anciently, this theory builds on the assumption that their ancestors' Ne was similarly large. Constraining Ne for ancient ancestors is challenging because experimental measurements of extinct organisms are impossible and alternatively reconstructing ancestral Ne with phylogenetic models gives large uncertainties. Here, we develop a new strategy that leverages agent-based modeling to simulate the changes in the genome-wide ratio of radical to conservative nonsynonymous nucleotide substitution rate (dR/dC) in a possible range of Ne in ancestral populations. This proxy shows expected increases with decreases of Ne only when Ne falls to about 10 k - 100 k or lower, magnitudes characteristic of Ne of obligate endosymbiont species where drift drives genome reduction. Our simulations therefore strongly support a scenario where the primary force of Prochlorococcus genome reduction is drift rather than selection.

Use of agent-based modeling to explore the mechanisms of intracellular phosphorus heterogeneity in cultured phytoplankton.

There can be significant intraspecific individual-level heterogeneity in the intracellular P of phytoplankton, which can affect the population-level growth rate. Several mechanisms can create this heterogeneity, including phenotypic variability in various physiological functions (e.g., nutrient uptake rate). Here, we use modeling to explore the contribution of various mechanisms to the heterogeneity in phytoplankton grown in a laboratory culture. An agent-based model simulates individual cells and their intracellular P. Heterogeneity is introduced by randomizing parameters (e.g., maximum uptake rate) of daughter cells at division. The model was calibrated to observations of the P quota of individual cells of the centric diatom Thalassiosira pseudonana, which were obtained using synchrotron X-ray fluorescence (SXRF). A number of simulations, with individual mechanisms of heterogeneity turned off, then were performed. Comparison of the coefficient of variation (CV) of these and the baseline simulation (i.e., all mechanisms turned on) provides an estimate of the relative contribution of these mechanisms. The results show that the mechanism with the largest contribution to variability is the parameter characterizing the maximum intracellular P, which, when removed, results in a CV of 0.21 compared to a CV of 0.37 with all mechanisms turned on. This suggests that nutrient/element storage capabilities/mechanisms are important determinants of intrapopulation heterogeneity.

A Reduction of Transcriptional Regulation in Aquatic Oligotrophic Microorganisms Enhances Fitness in Nutrient-Poor Environments.

In this review, we consider the regulatory strategies of aquatic oligotrophs, microbial cells that are adapted to thrive under low-nutrient concentrations in oceans, lakes, and other aquatic ecosystems. Many reports have concluded that oligotrophs use less transcriptional regulation than copiotrophic cells, which are adapted to high nutrient concentrations and are far more common subjects for laboratory investigations of regulation. It is theorized that oligotrophs have retained alternate mechanisms of regulation, such as riboswitches, that provide shorter response times and smaller amplitude responses and require fewer cellular resources. We examine the accumulated evidence for distinctive regulatory strategies in oligotrophs. We explore differences in the selective pressures copiotrophs and oligotrophs encounter and ask why, although evolutionary history gives copiotrophs and oligotrophs access to the same regulatory mechanisms, they might exhibit distinctly different patterns in how these mechanisms are used. We discuss the implications of these findings for understanding broad patterns in the evolution of microbial regulatory networks and their relationships to environmental niche and life history strategy. We ask whether these observations, which have emerged from a decade of increased investigation of the cell biology of oligotrophs, might be relevant to recent discoveries of many microbial cell lineages in nature that share with oligotrophs the property of reduced genome size.

Microcystin aids in cold temperature acclimation: Differences between a toxic Microcystis wildtype and non-toxic mutant.

For Microcystis aeruginosa PCC 7806, temperature decreases from 26 °C to 19 °C double the microcystin quota per cell during growth in continuous culture. Here we tested whether this increase in microcystin provided M. aeruginosa PCC 7806 with a fitness advantage during colder-temperature growth by comparing cell concentration, cellular physiology, reactive oxygen species damage, and the transcriptomics-inferred metabolism to a non-toxigenic mutant strain M. aeruginosa PCC 7806 ΔmcyB. Photo-physiological data combined with transcriptomic data revealed metabolic changes in the mutant strain during growth at 19 °C, which included increased electron sinks and non-photochemical quenching. Increased gene expression was observed for a glutathione-dependent peroxiredoxin during cold treatment, suggesting compensatory mechanisms to defend against reactive oxygen species are employed in the absence of microcystin in the mutant. Our observations highlight the potential selective advantages of a longer-term defensive strategy in management of oxidative stress (i.e., making microcystin) vs the shorter-term proactive strategy of producing cellular components to actively dissipate or degrade oxidative stress agents.

Cool temperature acclimation in toxigenic Microcystis aeruginosa PCC 7806 and its non-toxigenic mutant.

For Microcystis aeruginosa PCC 7806, temperature decreases from 26° C to 19° C double the microcystin quota per cell during growth in continuous culture. Here we tested whether this increase in microcystin provided M. aeruginosa PCC 7806 with a fitness advantage during colder-temperature growth by comparing cell concentration, cellular physiology, and the transcriptomics-inferred metabolism to a non-toxigenic mutant strain M. aeruginosa PCC 7806 ΔmcyB. Photo-physiological data combined with transcriptomic data revealed metabolic changes in the mutant strain during growth at 19° C, which included increased electron sinks and non-photochemical quenching. Increased gene expression was observed for a glutathione-dependent peroxiredoxin during cold treatment, suggesting compensatory mechanisms to defend against reactive oxygen species are employed in the absence of microcystin in the mutant. Our observations highlight the potential selective advantages of a longer-term defensive strategy in management of oxidative stress (i.e., making microcystin) vs the shorter-term proactive strategy of producing cellular components to actively dissipate or degrade oxidative stress agents.

Heterogeneity of intracellular polymer storage states in enhanced biological phosphorus removal (EBPR)--observation and modeling.

A number of agent-based models (ABMs) for biological wastewater treatment processes have been developed, but their skill in predicting heterogeneity of intracellular storage states has not been tested against observations due to the lack of analytical methods for measuring single-cell intracellular properties. Further, several mechanisms can produce and maintain heterogeneity (e.g., different histories, uneven division) and their relative importance has not been explored. This article presents an ABM for the enhanced biological phosphorus removal (EBPR) treatment process that resolves heterogeneity in three intracellular polymer storage compounds (i.e., polyphosphate, polyhydroxybutyrate, and glycogen) in three functional microbial populations (i.e., polyphosphate-accumulating, glycogen-accumulating, and ordinary heterotrophic organisms). Model predicted distributions were compared to those based on single-cell estimates obtained using a Raman microscopy method for a laboratory-scale sequencing batch reactor (SBR) system. The model can reproduce many features of the observed heterogeneity. Two methods for introducing heterogeneity were evaluated. First, biological variability in individual cell behavior was simulated by randomizing model parameters (e.g., maximum acetate uptake rate) at division. This method produced the best fit to the data. An optimization algorithm was used to determine the best variability (i.e., coefficient of variance) for each parameter, which suggests large variability in acetate uptake. Second, biological variability in individual cell states was simulated by randomizing state variables (e.g., internal nutrient) at division, which was not able to maintain heterogeneity because the memory in the internal states is too short. These results demonstrate the ability of ABM to predict heterogeneity and provide insights into the factors that contribute to it. Comparison of the ABM with an equivalent population-level model illustrates the effect of accounting for the heterogeneity in models.

Models predict planned phosphorus load reduction will make Lake Erie more toxic.

Harmful cyanobacteria are a global environmental problem, yet we lack actionable understanding of toxigenic versus nontoxigenic strain ecology and toxin production. We performed a large-scale meta-analysis including 103 papers and used it to develop a mechanistic, agent-based model of Microcystis growth and microcystin production. Simulations for Lake Erie suggest that the observed toxigenic-to-nontoxigenic strain succession during the 2014 Toledo drinking water crisis was controlled by different cellular oxidative stress mitigation strategies (protection by microcystin versus degradation by enzymes) and the different susceptibility of those mechanisms to nitrogen limitation. This model, as well as a simpler empirical one, predicts that the planned phosphorus load reduction will lower biomass but make nitrogen and light more available, which will increase toxin production, favor toxigenic cells, and increase toxin concentrations.

Response to Comment on "Models predict planned phosphorus load reduction will make Lake Erie more toxic".

Huisman et al. claim that our model is poorly supported or contradicted by other studies and the predictions are "seriously flawed." We show their criticism is based on an incomplete selection of evidence, misinterpretation of data, or does not actually refute the model. Like all ecosystem models, our model has simplifications and uncertainties, but it is better than existing approaches hat ignore biology and do not predict toxin concentration.

Dynamic carbon flux network of a diverse marine microbial community.

The functioning of microbial ecosystems has important consequences from global climate to human health, but quantitative mechanistic understanding remains elusive. The components of microbial ecosystems can now be observed at high resolution, but interactions still have to be inferred e.g., a time-series may show a bloom of bacteria X followed by virus Y suggesting they interact. Existing inference approaches are mostly empirical, like correlation networks, which are not mechanistically constrained and do not provide quantitative mass fluxes, and thus have limited utility. We developed an inference method, where a mechanistic model with hundreds of species and thousands of parameters is calibrated to time series data. The large scale, nonlinearity and feedbacks pose a challenging optimization problem, which is overcome using a novel procedure that mimics natural speciation or diversification e.g., stepwise increase of bacteria species. The method allows for curation using species-level information from e.g., physiological experiments or genome sequences. The product is a mass-balancing, mechanistically-constrained, quantitative representation of the ecosystem. We apply the method to characterize phytoplankton-heterotrophic bacteria interactions via dissolved organic matter in a marine system. The resulting model predicts quantitative fluxes for each interaction and time point (e.g., 0.16 µmolC/L/d of chrysolaminarin to Polaribacter on April 16, 2009). At the system level, the flux network shows a strong correlation between the abundance of bacteria species and their carbon flux during blooms, with copiotrophs being relatively more important than oligotrophs. However, oligotrophs, like SAR11, are unexpectedly high carbon processors for weeks into blooms, due to their higher biomass. The fraction of exudates (vs. grazing/death products) in the DOM pool decreases during blooms, and they are preferentially consumed by oligotrophs. In addition, functional similarity of phytoplankton i.e., what they produce, decouples their association with heterotrophs. The methodology is applicable to other microbial ecosystems, like human microbiome or wastewater treatment plants.

SAR11 Cells Rely on Enzyme Multifunctionality To Metabolize a Range of Polyamine Compounds.

In the ocean surface layer and cell culture, the polyamine transport protein PotD of SAR11 bacteria is often one of the most abundant proteins detected. Polyamines are organic cations at seawater pH produced by all living organisms and are thought to be an important component of dissolved organic matter (DOM) produced in planktonic ecosystems. We hypothesized that SAR11 cells uptake and metabolize multiple polyamines and use them as sources of carbon and nitrogen. Metabolic footprinting and fingerprinting were used to measure the uptake of five polyamine compounds (putrescine, cadaverine, agmatine, norspermidine, and spermidine) in two SAR11 strains that represent the majority of SAR11 cells in the surface ocean environment, "Candidatus Pelagibacter" strain HTCC7211 and "Candidatus Pelagibacter ubique" strain HTCC1062. Both strains took up all five polyamines and concentrated them to micromolar or millimolar intracellular concentrations. Both strains could use most of the polyamines to meet their nitrogen requirements, but polyamines did not fully substitute for their requirements of glycine (or related compounds) or pyruvate (or related compounds). Our data suggest that potABCD transports all five polyamines and that spermidine synthase, speE, is reversible, catalyzing the breakdown of spermidine and norspermidine, in addition to its usual biosynthetic role. These findings provide support for the hypothesis that enzyme multifunctionality enables streamlined cells in planktonic ecosystems to increase the range of DOM compounds they metabolize. IMPORTANCE Genome streamlining in SAR11 bacterioplankton has resulted in a small repertoire of genes, yet paradoxically, they consume a substantial fraction of primary production in the oceans. Enzyme multifunctionality, referring to enzymes that are adapted to have broader substrate and catalytic range than canonically defined, is hypothesized to be an adaptation that increases the range of organic compounds metabolized by cells in environments where selection favors genome minimization. We provide experimental support for this hypothesis by demonstrating that SAR11 cells take up and metabolize multiple polyamine compounds and propose that a small set of multifunctional enzymes catalyze this metabolism. We report that polyamine uptake rates can exceed metabolic rates, resulting in both high intracellular concentrations of these nitrogen-rich compounds (in comparison to native polyamine levels) and an increase in cell size.