The Biokinetic Spectrum for Temperature and optimal Darwinian fitness.

Darwinian fitness is maximised at a temperature below Topt, but what this temperature is remains unclear. By linking our previous work on the Biokinetic Spectrum for Temperature with a model for temperature-dependent biological growth rate we obtain a plausible value for such a temperature. We find this approach reveals considerable commonalities in how life responds to temperature with implications that follow in evolution, physiology and ecology. We described a data set consisting of 17,021 observations of temperature-dependent population growth rates from 2411 bacterial, archaeal and eukaryal strains. We fitted a thermodynamic model to describe the strains' temperature-dependent growth rate curves that assumed growth was limited by a single rate-limiting enzyme. We defined Umes as an empirical measure of the temperature at which strains grew as fast and also as efficiently as possible. We propose that Darwinian fitness is optimised at Umes by trading-off growth rate and physiological efficiency. Using the full data set we calculated the Biokinetic Spectrum for Temperature (BKST): the distribution of temperature-dependent growth rates for each temperature. We used quantile regression to fit alternative models to the BKST to obtain quantile curves. A quantile is a value that contains a particular proportion of the data. The quantile curves suggested commonalities in temperature-dependencies spanning taxa and ecotype, consistent with the single rate-limiting enzyme concept. We showed that on the log scale, the slopes of the quantile curves were the same as the slopes of the thermodynamic model growth curves at Umes. This was true for Bacteria, Archaea, and Eukarya, and across other conditions (pH, water activity, metabolic type and trophic type). We showed that the quantile curves were the loci of the temperatures and growth rates that optimised Darwinian fitness for each strain at a given temperature-dependence and independently of other conditions. The quantile curves for Archaea and Bacteria shared a number of similarities attributable to the influence of the properties of water on protein folding. Other implications have impact on evolutionary biology, ecology, and physiology. The model predicts the existence of eurythermic strains that grow with about equal efficiency over a broad temperature range. These strains will have higher evolutionary rates with lower mutational costs that are independent of environmental conditions, a factor likely to have been significant during the Precambrian if the early Earth was warmer than today. The model predicts that random mutations are likely to result in shifts along the quantile curves and not across them. It predicts that some psychrophiles will be capable of performing well under climate change, and that selection will favour faster growth rates as the temperature increases. Last, it predicts trade-offs between growth rate and soma production, so that temperature-dependence, and possibly Darwinian fitness, remain constant over a broad temperature range and growth rates.

Correction: The Biokinetic Spectrum for Temperature.

[This corrects the article DOI: 10.1371/journal.pone.0153343.].

The Biokinetic Spectrum for Temperature.

We identify and describe the distribution of temperature-dependent specific growth rates for life on Earth, which we term the biokinetic spectrum for temperature. The spectrum has the potential to provide for more robust modeling in thermal ecology since any conclusions derived from it will be based on observed data rather than using theoretical assumptions. It may also provide constraints for systems biology model predictions and provide insights in physiology. The spectrum has a Δ-shape with a sharp peak at around 42°C. At higher temperatures up to 60°C there was a gap of attenuated growth rates. We found another peak at 67°C and a steady decline in maximum rates thereafter. By using Bayesian quantile regression to summarise and explore the data we were able to conclude that the gap represented an actual biological transition between mesophiles and thermophiles that we term the Mesophile-Thermophile Gap (MTG). We have not identified any organism that grows above the maximum rate of the spectrum. We used a thermodynamic model to recover the Δ-shape, suggesting that the growth rate limits arise from a trade-off between activity and stability of proteins. The spectrum provides underpinning principles that will find utility in models concerned with the thermal responses of biological processes.

Protein thermodynamics can be predicted directly from biological growth rates.

Life on Earth is capable of growing from temperatures well below freezing to above the boiling point of water, with some organisms preferring cooler and others hotter conditions. The growth rate of each organism ultimately depends on its intracellular chemical reactions. Here we show that a thermodynamic model based on a single, rate-limiting, enzyme-catalysed reaction accurately describes population growth rates in 230 diverse strains of unicellular and multicellular organisms. Collectively these represent all three domains of life, ranging from psychrophilic to hyperthermophilic, and including the highest temperature so far observed for growth (122 °C). The results provide credible estimates of thermodynamic properties of proteins and obtain, purely from organism intrinsic growth rate data, relationships between parameters previously identified experimentally, thus bridging a gap between biochemistry and whole organism biology. We find that growth rates of both unicellular and multicellular life forms can be described by the same temperature dependence model. The model results provide strong support for a single highly-conserved reaction present in the last universal common ancestor (LUCA). This is remarkable in that it means that the growth rate dependence on temperature of unicellular and multicellular life forms that evolved over geological time spans can be explained by the same model.

Thraustochytrids can be grown in low-salt media without affecting PUFA production.

Marine microheterotrophs thraustochytrids are emerging as a potential source for commercial production of polyunsaturated fatty acids (PUFA) that have nutritional and pharmacological values. With prospective demand for PUFAs increasing, biotechnological companies are looking for potential increases in those valuable products. However, high levels of NaCl in the culture media required for optimal thraustochytrid growth and PUFA production poses a significant problem to the biotechnological industry due to corrosion of fermenters calling for a need to reduce the amount of NaCl in the culture media, without imposing penalties on growth and yield of cultured organisms. Earlier, as reported by Shabala et al. (Environ Microbiol 11:1835-1843, 2009), we have shown that thraustochytrids use sodium predominantly for osmotic adjustment purposes and, as such, can be grown in low-salt environment without growth penalties, providing the media osmolality is adjusted. In this study, we verify if that conclusion, made for one specific strain and osmolyte only, is applicable to the larger number of strains and organic osmotica, as well as address the issue of yield quality (e.g., PUFA production in low-saline media). Using mannitol and sucrose for osmotic adjustment of the growth media enabled us to reduce NaCl concentration down to 1 mM; this is 15-100-fold lower than any method proposed so far. At the same time, the yield of essential PUFAs was increased by 15 to 20 %. Taken together, these results suggest that the proposed method can be used in industrial fermenters for commercial PUFA production.

Universality of thermodynamic constants governing biological growth rates.

BACKGROUND: Mathematical models exist that quantify the effect of temperature on poikilotherm growth rate. One family of such models assumes a single rate-limiting 'master reaction' using terms describing the temperature-dependent denaturation of the reaction's enzyme. We consider whether such a model can describe growth in each domain of life. METHODOLOGY/PRINCIPAL FINDINGS: A new model based on this assumption and using a hierarchical Bayesian approach fits simultaneously 95 data sets for temperature-related growth rates of diverse microorganisms from all three domains of life, Bacteria, Archaea and Eukarya. Remarkably, the model produces credible estimates of fundamental thermodynamic parameters describing protein thermal stability predicted over 20 years ago. CONCLUSIONS/SIGNIFICANCE: The analysis lends support to the concept of universal thermodynamic limits to microbial growth rate dictated by protein thermal stability that in turn govern biological rates. This suggests that the thermal stability of proteins is a unifying property in the evolution and adaptation of life on earth. The fundamental nature of this conclusion has importance for many fields of study including microbiology, protein chemistry, thermal biology, and ecological theory including, for example, the influence of the vast microbial biomass and activity in the biosphere that is poorly described in current climate models.

Ion transport and osmotic adjustment in Escherichia coli in response to ionic and non-ionic osmotica.

Bacteria respond to osmotic stress by a substantial increase in the intracellular osmolality, adjusting their cell turgor for altered growth conditions. Using Escherichia coli as a model organism we demonstrate here that bacterial responses to hyperosmotic stress specifically depend on the nature of osmoticum used. We show that increasing acute hyperosmotic NaCl stress above approximately 1.0 Os kg(-1) causes a dose-dependent K(+) leak from the cell, resulting in a substantial decrease in cytosolic K(+) content and a concurrent accumulation of Na(+) in the cell. At the same time, isotonic sucrose or mannitol treatment (non-ionic osmotica) results in a gradual increase of the net K(+) uptake. Ion flux data are consistent with growth experiments showing that bacterial growth is impaired by NaCl at the concentration resulting in a switch from net K(+) uptake to efflux. Microarray experiments reveal that about 40% of upregulated genes shared no similarity in their responses to NaCl and sucrose treatment, further suggesting specificity of osmotic adjustment in E. coli to ionic and non-ionic osmotica. The observed differences are explained by the specificity of the stress-induced changes in the membrane potential of bacterial cells highlighting the importance of voltage-gated K(+) transporters for bacterial adaptation to hyperosmotic stress.

Osmotic adjustment and requirement for sodium in marine protist thraustochytrid.

A non-invasive ion-selective microelectrode technique was used to elucidate the ionic mechanisms of osmotic adjustment in a marine protist thraustochytrid. Hypoosmotic stress caused significant efflux of Na(+), Cl(-) and K(+) from thraustochytrid cells. Model calculations showed that almost complete osmotic adjustment was achieved within the first 30 min after stress onset. Of these, sodium was the major contributor (more than half of the total osmotic adjustment), with chloride being the second major contributor. The role of K(+) in the process of osmotic adjustment was relatively small. Changes in Ca(2+) and H(+) flux were attributed to intracellular signalling. Ion flux data were confirmed by growth experiments. Thraustochytrium cells showed normal growth patterns even when grown in a sodium-free solution provided the medium osmolality was adjusted by mannitol to one of the seawater. That suggests that the requirement of sodium for thraustochytrid growth cycle is due to its role in cell osmotic adjustment rather than because of the direct Na(+) involvement in cell metabolism. Altogether, these data demonstrate the evidence for turgor regulation in thraustochytrids and suggest that these cells may be grown in the absence of sodium providing that cell turgor is adjusted by some other means.

The future of predictive microbiology: strategic research, innovative applications and great expectations.

This paper considers the future of predictive microbiology by exploring the balance that exists between science, applications and expectations. Attention is drawn to the development of predictive microbiology as a sub-discipline of food microbiology and of technologies that are required for its applications, including a recently developed biological indicator. As we move into the era of systems biology, in which physiological and molecular information will be increasingly available for incorporation into models, predictive microbiologists will be faced with new experimental and data handling challenges. Overcoming these hurdles may be assisted by interacting with microbiologists and mathematicians developing models to describe the microbial role in ecosystems other than food. Coupled with a commitment to maintain strategic research, as well as to develop innovative technologies, the future of predictive microbiology looks set to fulfil "great expectations".

Listeria innocua and Lactobacillus delbrueckii subsp. bulgaricus employ different strategies to cope with acid stress.

Responses of Listeria innocua and Lactobacillus delbrueckii subsp. bulgaricus to a rapid change in extracellular pH (pHex) from pHex 6 to a range of concentrations down to pHex 3.0 were examined, using HCl and lactic acid (LA) as acidulants. A new fluorescent probe 5-(and-6)-carboxy-2', 7'-dichlorofluorescein diacetate succinimidyl ester (CDCFDA-SE) was employed that enabled reliable measurements of intracellular pH (pHi) to a minimum pHi of 4.0. Changes in pHi and H+ fluxes from immobilised bacteria were measured using fluorescence ratio imaging microscopy (FRIM) and a non-invasive ion flux measuring technique (MIFE), respectively. L. innocua maintained a relatively constant pHi of 5.5-6.1 at pHex 4 and 5 via H+ extrusion. In contrast, L. delbrueckii subsp. bulgaricus progressively lowered pHi towards pHex over the entire pHex range examined. The type of acidulant used influenced pH regulation with both pHi and H+ -fluxes being more severely affected by LA compared to HCl. Overall, our data demonstrated different adaptive strategies in these two bacteria. While L. innocua expels protons to maintain a constant pHi, L. delbrueckii subsp. bulgaricus allows proton entry after acidic treatment so that pHi follows pHex.

Non-invasive microelectrode ion flux measurements to study adaptive responses of microorganisms to the environment.

The regulation of membrane-transport activity is crucial for intracellular pH homeostasis, maintenance of cell osmotic potential, nutrient acquisition, signalling, and adaptation of bacterial cells. The non-invasive microelectrode ion flux estimation (MIFE) technique is a powerful tool for kinetic studies of membrane-transport processes across cellular membranes. Since 2001, when this technique was first applied to the study of membrane-transport processes in bacterial cells (J Microbiol Methods 46, 119-129), a large amount of information has been accumulated. This review summarizes some of these findings and discusses the advantages and applicability of this technique in studying bacterial adaptive responses to adverse environmental conditions. First, various methodological aspects of the application of this novel technique in microbiology are discussed. Then, several practical examples ('case studies') are described. The latter include changes in membrane-transport activity in response to various stresses (acidic, osmotic, and temperature stresses) as well as flux changes as a function of bacterial growth stage and nutrient availability. It is shown that non-invasive ion flux measurements may provide a significant conceptual advance in our understanding of adaptive responses in bacteria, fungi and biofilms to a variety of environmental conditions. The technique can also be used for the rapid assessment of food-processing treatments aimed at reducing bacterial contamination of food and for the development of strategies to assess the resistance of organisms to antimicrobial agents.

Variation of branched-chain fatty acids marks the normal physiological range for growth in Listeria monocytogenes.

The fatty acid composition of Listeria monocytogenes Scott A was determined by close-interval sampling over the entire biokinetic temperature range. There was a high degree of variation in the percentage of branched-chain fatty acids at any given temperature. The percentage of branched C17 components increased with growth temperature in a linear manner. However, the percentages of iso-C15:0 (i15:0) and anteiso-C15:0 (a15:0) were well described by third-order and second-order polynomial curves, respectively. There were specific temperature regions where the proportion of branched-chain fatty acids deviated significantly from the trend established over the entire growth range. In the region from 12 to 13 degrees C there were significant deviations in the percentages of both i15:0 and a15:0 together with a suggested deviation in a17:0, resulting in a significant change in the total branched-chain fatty acids. In the 31 to 33 degrees C region the percentage of total branched-chain components exhibited a significant deviation. The observed perturbations in fatty acid composition occurred near the estimated boundaries of the normal physiological range for growth.

Responses of Listeria monocytogenes to acid stress and glucose availability monitored by measurements of intracellular pH and viable counts.

Physiological aspects of the response of Listeria monocytogenes to acidic conditions and effect of glucose availability were studied by fluorescence ratio-imaging microscopy (FRIM) as compared with traditional viable counts. Three types of experiments were conducted: (i) static with measurements of intracellular pH (pHi) at extracellular pH (pHo) values ranging from pH 3.0 to 6.0 at 0.5 pH unit intervals; (ii) kinetic with monitoring of bacterial responses to changes in the pHo from the value of 6.0 to 4.0 or 3.0; (iii) survival experiments studying bacterial recovery in response to a shift to favourable conditions after a treatment at low pH. All the experiments were performed at three levels of glucose in the medium (0, 1, and 10 mM). Both survival and pHi were greatly affected by pHo and glucose availability with the highest values for CFU and pHi at highest glucose concentration and pHo values in the medium in all trials. A high correlation (R2 = 0.995) between pHi and CFU counts was observed. The pH gradient started to collapse at pHo 4 and below for trials with glucose in the medium and at pHo 5.5 and below without glucose. A recovery step was proposed after the apparently lethal treatment to assess cell viability by FRIM.

Responses of Listeria monocytogenes to acid stress and glucose availability revealed by a novel combination of fluorescence microscopy and microelectrode ion-selective techniques.

Fluorescence ratio imaging microscopy and microelectrode ion flux estimation techniques were combined to study mechanisms of pH homeostasis in Listeria monocytogenes subjected to acid stress at different levels of glucose availability. This novel combination provided a unique opportunity to measure changes in H(+) at either side of the bacterial membrane in real time and therefore to evaluate the rate of H(+) flux across the bacterial plasma membrane and its contribution to bacterial pH homeostasis. Responses were assessed at external pHs (pH(o)) between 3.0 and 6.0 for three levels of glucose (0, 1, and 10 mM) in the medium. Both the intracellular pH (pH(i)) and net H(+) fluxes were affected by the glucose concentration in the medium, with the highest absolute values corresponding to the highest glucose concentration. In the presence of glucose, the pH(i) remained above 7.0 within a pH(o) range of 4 to 6 and decreased below pH(o) 4. Above pH(o) 4, H(+) extrusion increased correspondingly, with the maximum value at pH(o) 5.5, and below pH(o) 4, a net H(+) influx was observed. Without glucose in the medium, the pH(i) decreased, and a net H(+) influx was observed below pH(o) 5.5. A high correlation (R = 0.75 to 0.92) between the pH(i) and net H(+) flux changes is reported, indicating that the two processes are complementary. The results obtained support other reports indicating that membrane transport processes are the main contributors to the process of pH(i) homeostasis in L. monocytogenes subjected to acid stress.

Biomarker techniques to screen for bacteria that produce polyunsaturated fatty acids.

The production of polyunsaturated fatty acids (PUFA) by bacteria has been firmly established for over two decades although it is still commonly ignored. Investigations of Antarctic sea ice have revealed a high diversity of novel bacterial taxa with the ability to produce PUFA. The majority are psychrophilic (requiring low temperatures for growth) and halophilic (requiring the presence of salts for growth), in contrast to the bacterial community present in the underlying water column. Specific fatty acids may be used as indicators of PUFA-producing bacteria in environmental samples. Structural studies of bacterial phospholipids have been particularly revealing in suggesting biomarkers specific for prokaryotic PUFA input. The use of negative ion fast atom bombardment tandem mass spectrometry for the analysis of bacterial phospholipids has identified species specific for certain groups of bacterial PUFA producers. The phylogeny of PUFA production in the gamma-Proteobacteria also suggests the future use of PUFA genes for the assessment of marine bacterial biodiversity.

Psychroflexus torquis gen. nov., sp. nov., a psychrophilic species from Antarctic sea ice, and reclassification of Flavobacterium gondwanense (Dobson et al. 1993) as Psychroflexus gondwanense gen. nov., comb. nov.

A group of sea-ice-derived psychrophilic bacterial strains possessing the unusual ability to synthesize the polyunsaturated fatty acids eicosapentaenoic acid (20:5 omega 3) and arachidonic acid (20:4 omega 6) belong to the Family Flavobacteriaceae (Flexibacter-Bacteroides-Flavobacterium phylum), according to 16S rRNA sequence analysis. Surprisingly, the isolates were also found to cluster closely to the moderately halophilic and psychrotrophic species [Flavobacterium] gondwanense (sequence similarity 97.8-98.1%). The whole-cell fatty acid profiles of this group and [Flavobacterium] gondwanense were very similar and distinct from other related flavobacteria. The sea ice strains and [Flavobacterium] gondwanense differed substantially in terms of ecophysiology, possibly representing divergent adaptations to sympagic and planktonic marine habitats, respectively. Evidence based on phylogeny and fatty acid profiles supports the conclusion that the taxa are close relatives distinct from other bacterial groups. It is thus proposed that the sea ice strains represent a novel taxon designated Psychroflexus torquis gen. nov., sp. nov. (type strain ACAM 623T) while [Flavobacterium] gondwanense becomes Psychroflexus gondwanense gen. nov., comb. nov.