Substrate Gradients Research Articles

A general method for determining secondary active transporter substrate stoichiometry.

The number of ions required to drive substrate transport through a secondary active transporter determines the protein's ability to create a substrate gradient, a feature essential to its physiological function, and places fundamental constraints on the transporter's mechanism. Stoichiometry is known for a wide array of mammalian transporters, but, due to a lack of readily available tools, not for most of the prokaryotic transporters for which high-resolution structures are available. Here, we describe a general method for using radiolabeled substrate flux assays to determine coupling stoichiometries of electrogenic secondary active transporters reconstituted in proteoliposomes by measuring transporter equilibrium potentials. We demonstrate the utility of this method by determining the coupling stoichiometry of VcINDY, a bacterial Na+-coupled succinate transporter, and further validate it by confirming the coupling stoichiometry of vSGLT, a bacterial sugar transporter. This robust thermodynamic method should be especially useful in probing the mechanisms of transporters with available structures.

Directional Migration of MDA-MB-231 Cells Under O2/pH Gradients.

We hypothesized that cancer cells actively migrate toward intratumor microvessels, guided by tissue gradients of metabolic substrates (such as O2) and/or metabolites (such as CO2/H+). To test this hypothesis, we developed an in vitro model in which cellular energy metabolism establishes gradients of O2/nutrient/metabolite in monolayer cells cultured in a conventional culture dish. When gradients of O2 ranging from 3% to ~0% were produced, MDA-MB-231 cells located at 300, 500 and 1500 μm downstream in the gradient demonstrated significant directional migrations (Rayleigh z test). We also found a similar directionality in cell migration at the same location even when the initial O2 level in the O2 gradient was raised from 3% to 21%. Interestingly, such directionalities were no longer demonstrated when the cell density was lowered from 1.8 × 106 to 0.9 × 106 cells/ml. In the former, the magnitude of the extracellular pH gradient in regions 300 and 500 μm downstream in the gradient was significantly larger. Thus, the direction of cell migrations appeared to depend on the gradient of extracellular pH rather than on O2.

Predicting N2O emissions from nitrifying and denitrifying biofilms: a modeling study.

Wastewater treatment plants can be significant sources of nitrous oxide (N2O), a potent greenhouse gas. While our understanding of N2O emissions from suspended-growth processes has advanced significantly, less is known about emissions from biofilm processes. Biofilms may behave differently due to their substrate gradients and microbial stratification. In this study, we used mathematical modeling to explore the mechanisms of N2O emissions from nitrifying and denitrifying biofilms. Our ammonia-oxidizing bacteria biofilm model suggests that N2O emissions from biofilm can be significantly greater than from suspended-growth systems. The driving factor is the diffusion of hydroxylamine, a nitrification intermediate, from the aerobic to the anoxic regions of the biofilm. The presence of nitrite-oxidizing bacteria further increased emissions. For denitrifying biofilms, our results suggest that emissions are generally greater than for suspended-growth systems. However, the magnitude of the difference depends on the bulk dissolved oxygen, chemical oxygen demand, and nitrate concentrations, as well as the biofilm thickness. Overall, the accumulation and diffusion of key intermediates, i.e. hydroxylamine and nitrite, distinguish biofilms from suspended-growth systems. Our research suggests that the mechanisms of N2O emissions from biofilms are much more complex than suspended-growth systems, and that emissions may be higher in many cases.

Reaction-diffusion theory explains hypoxia and heterogeneous growth within microbial biofilms associated with chronic infections.

Reaction–diffusion models were applied to gain insight into the aspects of biofilm infection and persistence by comparing mathematical simulations with the experimental data from varied bacterial biofilms. These comparisons, including three in vitro systems and two clinical investigations of specimens examined ex vivo, underscored the central importance of concentration gradients of metabolic substrates and the resulting physiological heterogeneity of the microorganisms. Relatively simple one-dimensional and two-dimensional (2D) models captured the: (1) experimentally determined distribution of specific growth rates measured in Pseudomonas aeruginosa cells within sputum from cystic fibrosis patients; (2) pattern of relative growth rate within aggregates of streptococcal biofilm harboured in an endocarditis vegetation; (3) incomplete penetration of oxygen into a Pseudomonas aeruginosa biofilm under conditions of exposure to ambient air and also pure oxygen; (4) localisation of anabolic activity around the periphery of P. aeruginosa cell clusters formed in a flow cell and attribution of this pattern to iron limitation; (5) very low specific growth rates, as small as 0.025 h−1, in the interior of cell clusters within a Klebsiella pneumoniae biofilm in a complex 2D domain of variable cell density.

Performance loss of Corynebacterium glutamicum cultivations under scale‐down conditions using complex media

Substrate and oxygen gradients appear in industrial‐scale fed‐batch processes due to limitations in the achievable power input and concomitantly increased mixing times. In order to mimic these gradients at lab scale, scale‐down reactors are applied. Previous studies in such reactor systems suggest that Corynebacterium glutamicum is robust against oscillatory oxygen and substrate availability in relation to growth and side product accumulation. Usually, defined mineral salt media are applied contrary to the industrial case, in which complex media containing different carbon sources are used. Therefore, this study investigated the cultivation performance using complex medium based on sucrose, molasses, and corn steep liquor in a three‐compartment scale‐down reactor. The reactor consisted of a stirred tank and two plug flow reactor modules. This approach was applied based on assumptions of gradient distributions in bottom‐fed bioreactors. A drastic reduction of growth and volumetric product yield of a cadaverine producing strain was observed while several short chain fatty acids accumulated, among them l‐lactate and acetate. Growth was depleted after several hours of cultivation, while the substrate uptake rate was reduced by 20%. Hence, the main carbon source sucrose accumulated after 10 h of fed‐batch cultivation. Despite growth cessation, neither reduction of cell vitality nor increased cell lysis were observed.

Shoreface storm morphodynamics and mega-rip evolution at an embayed beach: Bondi Beach, NSW, Australia

Embayed beach dynamics differ from open beaches due to the nature of headland control. Their resultant morphologies and morphodynamic behaviour are poorly understood due in part to a critical lack of surfzone and nearshore bathymetry observations. This study describes the morphodynamic storm response of a high-energy intermediate, 850m long embayed beach over a three week period spanning a cluster of storms. A headland and subaqueous ridge protects the northern end of the beach, resulting in an alongshore wave height gradient. Contrary to existing beach state conceptual models, under energetic forcing the beach did not ‘reset’ or enter a ‘cellular mega-rip’ beach state. The protected northern end persisted in a low energy state, while the wave exposed southern section transitioned from transverse-bar-and-rip to a complex double-bar system, a process previously undescribed in the literature. Bar-rip morphology at the exposed end of the beach migrated offshore to greater depths, leaving an inner-reflective beach and longshore trough, while a mega-rip channel with 3m relief developed at the exposed headland. The number of rip channels remained near constant over multiple storm events. Offshore sediment flux was 350m3/m at the exposed headland and 20m3/m at the protected end. Alongshore bathymetric non-uniformity decreased over the sub-aerial beach and inner surfzone, but increased in the outer surfzone and beyond. Suggested mechanisms for the persistence of 3D morphology during the cluster of storms include: (i) wave refraction to shore normal within the embayment; (ii) alongshore energy gradients; and (iii) pre-existing bar-rip morphology. Formation of the complex multi-bar state may be related to antecedent morphology, headland geometry, substrate gradient and localised hydrodynamic interactions near the headland. A new conceptual embayed beach state model is proposed for asymmetric, transitional embayed beaches. The model describes a pre-storm embayment where beach state changes gradually alongshore, while the post-storm embayment exhibits an extreme alongshore morphological gradient, from low-energy intermediate to a “complex multi-bar and mega-rip” state at the exposed end of the beach. Further observations are required to determine the prevalence of this high-energy state and to provide inputs to future numerical models designed to examine the dominant forcing controls involved in its formation.

Micromotors Powered by Enzyme Catalysis.

Active biocompatible systems are of great current interest for their possible applications in drug or antidote delivery at specific locations. Herein, we report the synthesis and study of self-propelled microparticles powered by enzymatic reactions and their directed movement in substrate concentration gradient. Polystyrene microparticles were functionalized with the enzymes urease and catalase using a biotin-streptavidin linkage procedure. The motion of the enzyme-coated particles was studied in the presence of the respective substrates, using optical microscopy and dynamic light scattering analysis. The diffusion of the particles was found to increase in a substrate concentration dependent manner. The directed chemotactic movement of these enzyme-powered motors up the substrate gradient was studied using three-inlet microfluidic channel architecture.

Scale-down of continuous protein producing Saccharomyces cerevisiae cultivations using a two-compartment system.

In the biotechnological industry, economic decisions in investment are typically based on laboratory-scale experiments. Scale-down as a tool is therefore of high industrial importance to transfer the processes into larger production scale without loss in performance. In this study, large-scale prolonged continuous cultivations with a heterologous protein producing Saccharomyces cerevisiae strain have been scaled-down to a two-compartment scale-down reactor system. The effects of glucose, pH, and oxygen concentration gradients have been investigated by comparison with corresponding 300 mL standard continuous cultivations. It was found that substrate gradients within a limited range result in increased productivity of the heterologous protein under regulation of glycolytic TPI promoter and delay the decrease of protein and trehalose production during continuous cultivation. Based on these results, it is argued that introduction of variations in substrate concentration can be beneficial for industrial continuous cultivations.

A new approach to simultaneous ammonium and dissolved methane removal from anaerobic digestion liquor: A model-based investigation of feasibility

The presence of a high level of dissolved methane (e.g., 20–26 g m−3) in the anaerobic sludge digestion liquor represents a major challenge to the treatment of this stream, as its emission to the atmosphere contributes significantly to the carbon footprint of wastewater treatment. Here we propose a new approach to simultaneous ammonium and dissolved methane removal from the anaerobic digestion liquor through integrating partial nitritation-Anammox and denitrifying anaerobic methane oxidation (DAMO) processes in a single-stage membrane biofilm reactor (MBfR). In such an MBfR, the anaerobic digestion liquor is provided in the bulk liquid, while oxygen is supplied through gas-permeable membranes to avoid dissolved methane stripping. A previously developed model with appropriate extensions was applied to assess the system performance under different operational conditions and the corresponding microbial interactions. Both influent surface loading (or hydraulic retention time) and oxygen surface loading are found to significantly influence the total nitrogen (TN) and dissolved methane removal, which jointly determine the overall system performance. The counter diffusion and concentration gradients of substrates cause microbial stratification in the biofilm, where ammonia-oxidizing bacteria (AOB) attach close to the membrane surface (biofilm base) where oxygen and ammonium are available, while Anammox and DAMO microorganisms jointly grow in the biofilm layer close to the bulk liquid where methane, ammonium, and nitrite are available with the latter produced by AOB. These results provide first insights and useful information for the design and operation of this new technology for simultaneous ammonium and dissolved methane removal in its potential future applications.

Anatomy of Nanoscale Propulsion.

Nature supports multifaceted forms of life. Despite the variety and complexity of these forms, motility remains the epicenter of life. The applicable laws of physics change upon going from macroscales to microscales and nanoscales, which are characterized by low Reynolds number (Re). We discuss motion at low Re in natural and synthetic systems, along with various propulsion mechanisms, including electrophoresis, electrolyte diffusiophoresis, and nonelectrolyte diffusiophoresis. We also describe the newly uncovered phenomena of motility in non-ATP-driven self-powered enzymes and the directional movement of these enzymes in response to substrate gradients. These enzymes can also be immobilized to function as fluid pumps in response to the presence of their substrates. Finally, we review emergent collective behavior arising from interacting motile species, and we discuss the possible biomedical applications of the synthetic nanobots and microbots.

Random forests to evaluate biotic interactions in fish distribution models

Previous research indicated that high predictive performance in species distribution modelling can be obtained by combining both biotic and abiotic habitat variables. However, models developed for fish often only address physical habitat characteristics, thus omitting potentially important biotic factors. Therefore, we assessed the impact of biotic variables on fish habitat preferences in four selected stretches of the upper Cabriel River (E Spain). The occurrence of Squalius pyrenaicus and Luciobarbus guiraonis was related to environmental variables describing biotic interactions (inferred by relationships among fish abundances) and channel hydro-morphological characteristics. Random Forests (RF) models were trained and then validated using independent datasets. To build RF models, the conditional variable importance was used together with the model improvement ratio technique. The procedure showed effectiveness in identifying a parsimonious set of not correlated variables, which minimize noise and improve model performance in both training and validation phases. Water depth, channel width, fine substrate and water-surface gradient were selected as most important habitat variables for both fish. Results showed clear habitat overlapping between fish species and suggest that competition is not a strong factor in the study area.

Carbonate build-ups in lacustrine, hydrothermal and fluvial settings: comparing depositional geometry, fabric types and geochemical signature

Abstract Carbonate build-ups in lakes, hydrothermal and fluvial settings are characterized by distinctive geometry, spatial distribution, fabrics and geochemical signature but also by some comparable features. Lake margin bioherms form continuous belts for hundreds of metres to kilometres, subparallel to shorelines. Sublacustrine spring mounds are spaced at hundreds of metres to kilometres and aligned along faults. Hydrothermal travertine mounds and aprons with planar clinoforms or terraced slopes are controlled by faults, thermal water discharge and substrate topography. Fluvial tufa barrages, cascades and terraced slopes are controlled by climate, vegetation and substrate gradient. The wide spectrum of carbonate microfabrics ranges from clotted peloidal micrite and laminated boundstone to crystalline dendrite cementstone. Non-marine carbonate microfabrics cannot be linked to specific depositional environments, and are not deterministic proxies for the interpretation of build-up architecture. Microfabric associations can be indicative, but not exclusive, of specific depositional environments and geometry. Stable isotope geochemistry is a useful tool to distinguish between hydrothermal, karstic freshwater and evaporative lake carbonates. Carbonate precipitation results from a continuum of abiotic and biologically influenced/induced processes in settings where carbonate supersaturation is largely driven by physico-chemical mechanisms and microbial biofilms, even if acting as passive low-energy surface sites for nucleation, are widely present.

Effects of hydroperiod and substrate properties on tree alpha diversity and composition in Amazonian floodplain forests

It is well established that tree assemblage diversity and composition in Amazonian floodplain forest are influenced by substrate properties and hydroperiod. However, no study to date has tried to disentangle the individual effects of these variables on floodplain tree assemblages. In this study, we investigated the influence of several edaphic variables and hydroperiod on alpha diversity and individual tree species distribution. We carried out floristic inventories in 12 ha of floodplain forests across central-western Amazonia and sampled associated data on substrate properties and hydroperiod. Generalized linear mixed models were used to evaluate the importance of each predictor on the response variables. Flooding was the only variable significantly influencing alpha diversity and also significantly influenced the distribution of the highest number of tree species. Edaphic variables had a lesser effect, and a large number of tree species were not significantly influenced by any of the predictors tested. However, iron had a strong negative influence on several species. We conclude that floristic diversity and composition are more influenced by the hydroperiod than by substrate conditions, and substrate conditions become decreasingly important for increasingly flood-tolerant species. Many floodplain species are generalists occurring across the entire substrate and hydroperiod gradient.

CO₂ /HCO₃⁻ perturbations of simulated large scale gradients in a scale-down device cause fast transcriptional responses in Corynebacterium glutamicum.

The exploration of scale-down models to imitate the influence of large scale bioreactor inhomogeneities on cellular metabolism is a topic with increasing relevance. While gradients of substrates, pH, or dissolved oxygen are often investigated, oscillating CO2/HCO3 (-) levels, a typical scenario in large industrial bioreactors, is rarely addressed. Hereby, we investigate the metabolic and transcriptional response in Corynebacterium glutamicum wild type as well as the impact on L-lysine production in a model strain exposed to pCO2 gradients of (75-315) mbar. A three-compartment cascade bioreactor system was developed and characterized that offers high flexibility for installing gradients and residence times to mimic industrial-relevant conditions and provides the potential of accurate carbon balancing. The phenomenological analysis of cascade fermentations imposed to the pCO2 gradients at industry-relevant residence times of about 3.6 min did not significantly impair the process performance, with growth and product formation being similar to control conditions. However, transcriptional analysis disclosed up to 66 differentially expressed genes already after 3.6 min under stimulus exposure, with the overall change in gene expression directly correlateable to the pCO2 gradient intensity and the residence time of the cells.

Mathematical modeling of competition and coexistence of sulfate-reducing bacteria, acetogens, and methanogens in multispecies biofilms

This work presents an integrated mathematical model able to simulate the physical, chemical, and biological processes prevailing in a sulfate-reducing biofilm under dynamic conditions. The model includes sulfate reduction by complete and incomplete sulfate-reducing bacteria (SRB); lactate removal by sulfate reduction and by acetogenic bacteria and acetate consumption via methanogenesis. Numerical integration based on the method of characteristics has been developed. The major problem of sulfate-reducing fixed-growth reactors is the formation of undesired bacterial species, which compete for space and substrate within the biofilm with SRB. The effect of COD/SO42-ratio on the reactor performances in terms of bacterial species distribution and substrate diffusion trends in the biofilm has been assessed. The simulation results reveal a stratification of microbial activities in biofilm reflecting the different ecological niches created by substrate gradients.

Water and substrate control of cliff communities: patterns among species and phyla

AbstractAimsCliff communities tend to be compositionally simple but taxonomically diverse at the phylum/division level. As a result, they provide a setting for examining the responses of distinct phyla (e.g. Anthophyta, Bryophyta and lichens), species within these phyla and communities comprised of these phyla, to the same environmental gradients. Our aims were both descriptive and hypothesis‐driven. We sought to identify major community shifts across water–substrate gradients in a poorly studied system (inland North American cliffs), while considering extant hypotheses concerning the relationships of organisms to environments at different taxonomic scales.LocationSub‐alpine cliffs in northeastern Yellowstone National Park.MethodsCliff faces on two mountains were blocked at nine waterfalls. At each block, community composition was recorded from random samples obtained for three levels of water availability (xeric, mesic and hygric) on two substrates (limestone and andesite). Patterns of cover and richness were compared using Friedman's Rank F‐tests. Community patterns were discerned using permutation MANOVAs, ordinations, and cluster analyses.ResultsPhyletic composition varied in response to water availability. In general, lichens dominated dry sites, while vascular plants dominated wet sites. Anthophyta was the most narrowly specialized phylum, and Bryophyta was the most generalized. Beta diversity was higher at drier sites, due to the dominance of the diverse lichen group. As a result, communities on lime and andesitic substrates were indistinguishable in hygric environments, but significantly different at drier sites. This may have occurred because, while water is limiting for neither substrate in hygric environments, andesites are more likely to absorb and store atmospheric water in drier environments. Indicator species analyses identified 27 distinct species distinguishing water and substrate conditions, or their interactions.ConclusionsOur experimental design uniquely accounted for extraneous variables in its examination of water and substrate effects on cliff communities. Water strongly controlled community composition both among and within phyla through both direct effects and interactions with substrate.

Effects of feeding pattern and dissolved oxygen concentration on microbial morphology and community structure: The competition between floc-forming bacteria and filamentous bacteria

Understanding of the competition between floc-formers and filaments is critical to prevent filamentous bulking in practice. This study aimed to investigate the effects of feeding pattern and dissolved oxygen (DO) concentration on their competition in four sequencing batch reactors (SBRs). Short feeding under anoxic condition (fill time<10min) resulted in a well-settling sludge (sludge volume index (SVI)<100mL/g), in spite of DO concentrations. Sludge settleability deteriorated (SVI>200mL/g) and filamentous bulking was observed when the substrate was added in a limiting rate by prolonging the anoxic fill time up to 90min. In contrast, sludge settleability in fully aerobic systems was quite poor (SVI>500mL/g) in spite of the feeding length. Compared to the systems with an anoxic fill phase, more types and abundant filamentous bacteria were identified in fully aerobic systems. Microscopic observation, staining reactions and fluorescence in situ hybridization analysis indicated that the extensive filaments, including Thiothrix nivea, Type 021N, Type 1851 and Microthrix parvicella, proliferated in fully aerobic systems. The results of this study indicated that substrate gradients played an important role on the competition between filaments and floc-formers. It is recommend the adoption of plug-flow selector configurations, with anoxic conditions in order to maintain good and robust sludge settleability.

Functional Coupling of a Urate-Anion Exchanger URAT1 and Sodium-Dependent Anion Transporter SMCT2 on a PDZK1 Scaffold; Proposal of “Transportsome” for Urate-Transport

Transporters utilize electrochemical gradients across membranes to translocate their substrates through the membranes. Many eukaryotic plasma membrane symporters transport substrates in a Na+-dependent manner by using an electrochemical Na+ gradient across plasma membranes, while exchangers couple the movement of a substrate against its electrochemical gradient with the movement of another substrate down its electrochemical gradient in the opposite direction. Although the Na+ gradient is ubiquitous and stable, the electrochemical gradients of substrates for exchangers usually depend on cells and are affected by cellular conditions. The concentrations of intracellular substrates limit the accumulation of extracellular substrates thorough exchangers. To overcome the constraint, exchangers need to couple with other proteins that help accumulation of substrates around exchangers. We have been proposing that such functional coupling of transporters is general and constitutes the functional unit of transport that we call “transportsome”. Here, we show one of the examples. Proteomics analysis following co-immunoprecipitation revealed that urate-anion exchanger URAT1 and Na+-dependent anion transporter SMCT2 formed a complex through scaffold protein PDZK1 in the apical membrane of renal proximal tubules. FRET experiment clearly indicated that PDZK1 assembled URAT1 and SMCT2 in living cells. To elucidate functional coupling of the transporters, URAT1 and SMCT2 were overexpressed in insect cells, purified and reconstituted into proteoliposomes in the presence or absence of PDZK1. The initial rate of urate-uptake was increased in the presence of Na+ when co-reconstituted with PDZK1 while Na+ did not alter the rate without PDZK1. These results suggest that SMCT2 generate an electrochemical gradient of the substrate which drives the urate-uptake via URAT1 located in close proximity to SMCT2 with PDZK1 scaffold. Taken together, the transporters and the scaffold protein formed Transportsome for urate-transport.

Modeling of biofilm systems: a review.

The modeling of biochemical processes in biofilms is more complex compared to those in suspended biomass due to the existence of substrate gradients. The diffusion and reaction of substrates within the biofilms were simulated in 1D models in the 1970s. The quality of these simulation results was later improved by consideration of mass transfer at the bulk/biofilm interface and detachment of biomass from the surface. Furthermore, modeling of species distribution along the axis perpendicular to the substratum helped to simulate productivity and long-term behavior in multispecies biofilms. Multidimensional models that were able to give a realistic prediction of the surface structure of biofilms were published in the 1990s. The 2D or 3D representation of the distribution of the species in a matrix of extracellular polymeric substances (EPS) helped predict the behavior of multispecies biofilm systems. The influence of shear forces on such 2D or 3D biofilm structures was included by solving the Navier-Stokes equation for the liquid phase above the biofilm. More recently, the interaction between the fluid and biofilm structures was addressed more seriously by no longer considering the biofilm structures as being rigid. The latter approach opened a new door, enabling one to describe biofilms as viscoelastic systems that are not only able to grow and produce but also be deformed or even dislodged if external forces are applied.

Flux response of glycolysis and storage metabolism during rapid feast/famine conditions in Penicillium chrysogenum using dynamic 13C labeling

The scale-up of fermentation processes frequently leads to a reduced productivity compared to small-scale screening experiments. Large-scale mixing limitations that lead to gradients in substrate and oxygen availability could influence the microorganism performance. Here, the impact of substrate gradients on a penicillin G producing Penicillium chrysogenum cultivation was analyzed using an intermittent glucose feeding regime. The intermittent feeding led to fluctuations in the extracellular glucose concentration between 400 μM down to 6.5 μM at the end of the cycle. The intracellular metabolite concentrations responded strongly and showed up to 100-fold changes. The intracellular flux changes were estimated on the basis of dynamic (13) C mass isotopomer measurements during three cycles of feast and famine using a novel hybrid modeling approach. The flux estimations indicated a high turnover of internal and external storage metabolites in P. chrysogenum under feast/famine conditions. The synthesis and degradation of storage requires cellular energy (ATP and UTP) in competition with other cellular functions including product formation. Especially, 38% of the incoming glucose was recycled once in storage metabolism. This result indicated that storage turnover is increased under dynamic cultivation conditions and contributes to the observed decrease in productivity compared to reference steady-state conditions.