Near-zero growth kinetics of Pseudomonas putida deduced from proteomic analysis.

Intensive microbial growth typically observed in laboratory rarely occurs in nature. Because of severe nutrient deficiency, natural populations exhibit near-zero growth (NZG). There is a long-standing controversy about sustained NZG, specifically whether there is a minimum growth rate below which cells die or whether cells enter a non-growing maintenance state. Using chemostat with cell retention (CCR) of Pseudomonas putida, we resolve this controversy and show that under NZG conditions, bacteria differentiate into growing and VBNC (viable but not non-culturable) forms, the latter preserving measurable catabolic activity. The proliferating cells attained a steady state, their slow growth balanced by VBNC production. Proteomic analysis revealed upregulated (transporters, stress response, self-degrading enzymes and extracellular polymers) and downregulated (ribosomal, chemotactic and primary biosynthetic enzymes) proteins in the CCR versus batch culture. Based on these profiles, we identified intracellular processes associated with NZG and generated a mathematical model that simulated the observations. We conclude that NZG requires controlled partial self-digestion and deep reconfiguration of the metabolic machinery that results in the biosynthesis of new products and development of broad stress resistance. CCR allows efficient on-line control of NZG including VBNC production. A well-nuanced understanding of NZG is important to understand microbial processes in situ and for optimal design of environmental technologies.

Oribacterium parvum sp. nov. and Oribacterium asaccharolyticum sp. nov., obligately anaerobic bacteria from the human oral cavity, and emended description of the genus Oribacterium.

Three strictly anaerobic, Gram-positive, non-spore-forming, rod-shaped, motile bacteria, designated strains ACB1(T), ACB7(T) and ACB8, were isolated from human subgingival dental plaque. All strains required yeast extract for growth. Strains ACB1(T) and ACB8 were able to grow on glucose, lactose, maltose, maltodextrin and raffinose; strain ACB7(T) grew weakly on sucrose only. The growth temperature range was 30-42 °C with optimum growth at 37 °C. Major metabolic fermentation end products of strain ACB1(T) were acetate and lactate; the only product of strains ACB7(T) and ACB8 was acetate. Major fatty acids of strain ACB1(T) were C(14 : 0), C(16 : 0), C(16 : 1)ω7c dimethyl aldehyde (DMA) and C(18 : 1)ω7c DMA. Major fatty acids of strain ACB7(T) were C(12 : 0), C(14 : 0), C(16 : 0), C(16 : 1)ω7c and C(16 : 1)ω7c DMA. The hydrolysate of the peptidoglycan contained meso-diaminopimelic acid, indicating peptidoglycan type A1γ. Genomic DNA G+C content varied from 42 to 43.3% between strains. According to 16S rRNA gene sequence phylogeny, strains ACB1(T), ACB8 and ACB7(T) formed two separate branches within the genus Oribacterium, with 98.1-98.6% sequence similarity to the type strain of the type species, Oribacterium sinus. Predicted DNA-DNA hybridization values between strains ACB1(T), ACB8, ACB7(T) and O. sinus F0268 were <70%. Based on distinct genotypic and phenotypic characteristics, strains ACB1(T) and ACB8, and strain ACB7(T) are considered to represent two distinct species of the genus Oribacterium, for which the names Oribacterium parvum sp. nov. and Oribacterium asaccharolyticum sp. nov. are proposed. The type strains are ACB1(T) ( = DSM 24637(T) = HM-481(T) = ATCC BAA-2638(T)) and ACB7(T) ( = DSM 24638(T) = HM-482(T) = ATCC BAA-2639(T)), respectively.

Stomatobaculum longum gen. nov., sp. nov., an obligately anaerobic bacterium from the human oral cavity.

A strictly anaerobic Gram-stain-variable but positive by structure, non-spore-forming bacterium designated Lachnospiraceae bacterium ACC2 strain DSM 24645(T) was isolated from human subgingival dental plaque. Bacterial cells were 4-40 µm long non-motile rods, often swollen and forming curved filaments up to 200 µm. Cells contained intracellular, poorly crystalline, nanometre-sized iron- and sulfur-rich particles. The micro-organism was able to grow on yeast extract, trypticase peptone, milk, some sugars and organic acids. The major metabolic end-products of glucose fermentation were butyrate, lactate, isovalerate and acetate. The growth temperature and pH ranges were 30-42 °C and 4.9-7.5, respectively. Major fatty acids were C14 : 0, C14 : 0 DMA (dimethyl aldehyde), C16 : 0, C16 : 1ω7c DMA. The whole-cell hydrolysate contained meso-diaminopimelic acid, indicating peptidoglycan type A1γ. The DNA G+C content was calculated to be 55.05 mol% from the whole-genome sequence and 55.3 mol% as determined by HPLC. There were no predicted genes responsible for biosynthesis of respiratory lipoquinones, mycolic acids and lipopolysaccharides. Genes associated with synthesis of teichoic and lipoteichoic acids, diaminopimelic acid, polar lipids and polyamines were present. According to the 16S rRNA gene sequence phylogeny, strain DSM 24645(T) formed, together with several uncultured oral clones, a separate branch within the family Lachnospiraceae, with the highest sequence similarity to the type strain of Moryella indoligenes at 94.2 %. Based on distinct phenotypic and genotypic characteristics, we suggest that strain DSM 24645(T) represents a novel species in a new genus, for which the name Stomatobaculum longum gen. nov., sp. nov. is proposed. The type strain of Stomatobaculum longum is DSM 24645(T) ( = HM-480(T); deposited in BEI Resources, an NIH collection managed by the ATCC).

Draft genome sequence for Microbacterium laevaniformans strain OR221, a bacterium tolerant to metals, nitrate, and low pH.

Microbacterium laevaniformans strain OR221 was isolated from subsurface sediments obtained from the Field Research Center (FRC) in Oak Ridge, TN. It was characterized as a bacterium tolerant to heavy metals, such as uranium, nickel, cobalt, and cadmium, as well as nitrate and low pH. We present its draft genome sequence.

Genome-Scale Reconstruction of Microbial Dynamic Phenotype: Successes and Challenges.

This review is a part of the SI 'Genome-Scale Modeling of Microorganisms in the Real World'. The goal of GEM is the accurate prediction of the phenotype from its respective genotype under specified environmental conditions. This review focuses on the dynamic phenotype; prediction of the real-life behaviors of microorganisms, such as cell proliferation, dormancy, and mortality; balanced and unbalanced growth; steady-state and transient processes; primary and secondary metabolism; stress responses; etc. Constraint-based metabolic reconstructions were successfully started two decades ago as FBA, followed by more advanced models, but this review starts from the earlier nongenomic predecessors to show that some GEMs inherited the outdated biokinetic frameworks compromising their performances. The most essential deficiencies are: (i) an inadequate account of environmental conditions, such as various degrees of nutrients limitation and other factors shaping phenotypes; (ii) a failure to simulate the adaptive changes of MMCC (MacroMolecular Cell Composition) in response to the fluctuating environment; (iii) the misinterpretation of the SGR (Specific Growth Rate) as either a fixed constant parameter of the model or independent factor affecting the conditional expression of macromolecules; (iv) neglecting stress resistance as an important objective function; and (v) inefficient experimental verification of GEM against simple growth (constant MMCC and SGR) data. Finally, we propose several ways to improve GEMs, such as replacing the outdated Monod equation with the SCM (Synthetic Chemostat Model) that establishes the quantitative relationships between primary and secondary metabolism, growth rate and stress resistance, process kinetics, and cell composition.

True and Illusory Benefits of Modeling: Comment on "Genome-Scale Metabolic Network Reconstruction and In Silico Analysis of Hexanoic Acid Producing Megasphaera elsdenii. Microorganisms 2020, 8, 539".

Lee et al. [1] recently published a paper that is part of the special issue "Genome-Scale Modeling of Microorganisms in the Real World"[...].

A simple cleanup method for the removal of humic substances from soil protein extracts using aluminum coagulation.

Soil proteomics, the large-scale characterization of the entire protein complement in soils, provides a promising approach for deciphering the role of microbial functioning in terrestrial ecosystems. However, the extraction of soil proteins in sufficient quantities and of adequate purity remains a challenging task mainly due to the co-extraction of interfering humic substances. Up to now, the treatment of soil extracts with liquid phenol has been the "gold standard" for reducing humics, while the NoviPure cleanup kit was recently launched as a non-toxic approach. The present study describes an alternative method for delivering high-purity proteins based on humic coagulation with trivalent aluminum ions (Al3+). Various experimental parameters were optimized individually in order to maximize protein yield and diminish co-extracted humics. The optimized method was applied on a set of soil samples with diverse physicochemical characteristics and a comparison with the other two techniques was conducted. The amount of residual humics resulting from Al3+-based method was 26 and 35% higher than that from phenol treatment and NoviPure Kit, respectively, but these differences were of marginal statistical significance. With regard to extracted proteins, the average yields of the three methods were comparable, without showing any statistically significant differences. Overall, humic coagulation with Al3+ offers comparable cleanup performance in terms of protein yield and purity, but it is less toxic and less complex than the phenol-partitioning method, whereas it is far less expensive than the NoviPure Kit. The new technique is expected to facilitate the implementation of proteomic studies in soils.

Growth kinetics of microorganisms isolated from Alaskan soil and permafrost in solid media frozen down to -35 degrees C.

We developed a procedure to culture microorganisms below freezing point on solid media (cellulose powder or plastic film) with ethanol as the sole carbon source without using artificial antifreezes. Enrichment from soil and permafrost obtained on such frozen solid media contained mainly fungi, and further purification resulted in isolation of basidiomycetous yeasts of the genera Mrakia and Leucosporidium as well as ascomycetous fungi of the genus Geomyces. Contrary to solid frozen media, the enrichment of liquid nutrient solutions at 0 degrees C or supercooled solutions stabilized by glycerol at -1 to -5 degrees C led to the isolation of bacteria representing the genera Polaromonas, Pseudomonas and Arthrobacter. The growth of fungi on ethanol-microcrystalline cellulose media at -8 degrees C was exponential with generation times of 4.6-34 days, while bacteria displayed a linear or progressively declining curvilinear dynamic. At -17 to -0 degrees C the growth of isolates and entire soil community on 14C-ethanol was continuous and characterized by yields of 0.27-0.52 g cell C (g of C-substrate)(-1), similar to growth above the freezing point. The 'state of maintenance,' implying measurable catabolic activity of non-growing cells, was not confirmed. Below -18 to -35 degrees C, the isolated organisms were able to grow only transiently for 3 weeks after cooling with measurable respiratory and biosynthetic (14CO2 uptake) activity. Then metabolic activity declined to zero, and microorganisms entered a state of reversible dormancy.

Novel facultative anaerobic acidotolerant Telmatospirillum siberiense gen. nov. sp. nov. isolated from mesotrophic fen.

Three facultative anaerobic acidotolerant Gram-negative motile spirilla strains designated 26-4b1, 26-2 and K-1 were isolated from mesotrophic Siberian fen as a component of methanogenic consortia. The isolates were found to grow chemoorganotrophically on several organic acids and glucose under anoxic and low oxygen pressure in the dark, tolerant up to 5kPa of oxygen. At low oxygen supply, faint autotrophic growth on the H(2):CO(2) mixture was also observed. All three isolates were able to fix N(2). Major cellular fatty acids were 18:1 omega7c, 17:0 cyclopropane and 16:0. Phylogenetic analyses of the 16S rRNA gene sequences revealed that they formed a deep branch within the family Rhodospirillaceae of the Alphaproteobacteria with the highest similarity of 90.9-92.5% with members of genera Phaeospirillum and Magnetospirillum. Phylogenetic study of nifH (nitrogenase) and cbbL (RuBisCO) amino acid sequence identities confirmed that the new isolates represent a novel group. Based on the phylogenetic analyses and distinct phenotypic characteristics, we are of the opinion that strains 26-4b1, 26-2 and K-1 represent a new species of a novel genus for which the name Telmatospirillum siberiense gen. nov. sp. nov. is proposed.

Respiration of Pseudomonas fluorescens as a function of intracellular substrate concentration.

A kinetic method to measure the intracellular concentration of respiratory substrates in short-term starvation-enrichment experiments is proposed. Samples of bacterial suspension from steady-state chemostat cultures were subjected to 25 min starvation, followed by pulse addition of [14C]glucose. Residual substrate utilization rates and respiration rates (uptake of dissolved O2) before and after amendment were recorded. Increases in pool sizes (δL) during transients were calculated on the basis of C balance. The dependence of respiration rate qresp on δL was found to obey modified Michaelis-Menten kinetics: q resp = Q resp (LC+δL)/(K L+LC+δL) [Q resp is maximal respiration rate (29.1 mmol O2 h-1per g biomass C), K L = 12.14 mg C per g biomass C], where LC is the absolute value of the pool size before amendment. Direct chemical determination of LC in cold TCA extracts revealed two fractions. The first fraction was mobile and showed a close correlation with both respiration and L. The second, 'stable', fraction did not correlate with respiration dynamics and was interpreted as material formed artifactually by acid degradation of polymeric cell components.

Past changes in Arctic terrestrial ecosystems, climate and UV radiation.

At the last glacial maximum, vast ice sheets covered many continental areas. The beds of some shallow seas were exposed thereby connecting previously separated landmasses. Although some areas were ice-free and supported a flora and fauna, mean annual temperatures were 10-13 degrees C colder than during the Holocene. Within a few millennia of the glacial maximum, deglaciation started, characterized by a series of climatic fluctuations between about 18,000 and 11,400 years ago. Following the general thermal maximum in the Holocene, there has been a modest overall cooling trend, superimposed upon which have been a series of millennial and centennial fluctuations in climate such as the "Little Ice Age spanning approximately the late 13th to early 19th centuries. Throughout the climatic fluctuations of the last 150,000 years, Arctic ecosystems and biota have been close to their minimum extent within the most recent 10,000 years. They suffered loss of diversity as a result of extinctions during the most recent large-magnitude rapid global warming at the end of the last glacial stage. Consequently, Arctic ecosystems and biota such as large vertebrates are already under pressure and are particularly vulnerable to current and projected future global warming. Evidence from the past indicates that the treeline will very probably advance, perhaps rapidly, into tundra areas, as it did during the early Holocene, reducing the extent of tundra and increasing the risk of species extinction. Species will very probably extend their ranges northwards, displacing Arctic species as in the past. However, unlike the early Holocene, when lower relative sea level allowed a belt of tundra to persist around at least some parts of the Arctic basin when treelines advanced to the present coast, sea level is very likely to rise in future, further restricting the area of tundra and other treeless Arctic ecosystems. The negative response of current Arctic ecosystems to global climatic conditions that are apparently without precedent during the Pleistocene is likely to be considerable, particularly as their exposure to co-occurring environmental changes (such as enhanced levels of UV-B, deposition of nitrogen compounds from the atmosphere, heavy metal and acidic pollution, radioactive contamination, increased habitat fragmentation) is also without precedent.

Effects on the function of Arctic ecosystems in the short- and long-term perspectives.

Historically, the function of Arctic ecosystems in terms of cycles of nutrients and carbon has led to low levels of primary production and exchanges of energy, water and greenhouse gases have led to low local and regional cooling. Sequestration of carbon from atmospheric CO2, in extensive, cold organic soils and the high albedo from low, snow-covered vegetation have had impacts on regional climate. However, many aspects of the functioning of Arctic ecosystems are sensitive to changes in climate and its impacts on biodiversity. The current Arctic climate results in slow rates of organic matter decomposition. Arctic ecosystems therefore tend to accumulate organic matter and elements despite low inputs. As a result, soil-available elements like nitrogen and phosphorus are key limitations to increases in carbon fixation and further biomass and organic matter accumulation. Climate warming is expected to increase carbon and element turnover, particularly in soils, which may lead to initial losses of elements but eventual, slow recovery. Individual species and species diversity have clear impacts on element inputs and retention in Arctic ecosystems. Effects of increased CO2 and UV-B on whole ecosystems, on the other hand, are likely to be small although effects on plant tissue chemisty, decomposition and nitrogen fixation may become important in the long-term. Cycling of carbon in trace gas form is mainly as CO2 and CH4. Most carbon loss is in the form of CO2, produced by both plants and soil biota. Carbon emissions as methane from wet and moist tundra ecosystems are about 5% of emissions as CO2 and are responsive to warming in the absence of any other changes. Winter processes and vegetation type also affect CH4 emissions as well as exchanges of energy between biosphere and atmosphere. Arctic ecosystems exhibit the largest seasonal changes in energy exchange of any terrestrial ecosystem because of the large changes in albedo from late winter, when snow reflects most incoming radiation, to summer when the ecosystem absorbs most incoming radiation. Vegetation profoundly influences the water and energy exchange of Arctic ecosystems. Albedo during the period of snow cover declines from tundra to forest tundra to deciduous forest to evergreen forest. Shrubs and trees increase snow depth which in turn increases winter soil temperatures. Future changes in vegetation driven by climate change are therefore, very likely to profoundly alter regional climate.

Uncertainties and recommendations.

An assessment of the impacts of changes in climate and UV-B radiation on Arctic terrestrial ecosystems, made within the Arctic Climate Impacts Assessment (ACIA), highlighted the profound implications of projected warming in particular for future ecosystem services, biodiversity and feedbacks to climate. However, although our current understanding of ecological processes and changes driven by climate and UV-B is strong in some geographical areas and in some disciplines, it is weak in others. Even though recently the strength of our predictions has increased dramatically with increased research effort in the Arctic and the introduction of new technologies, our current understanding is still constrained by various uncertainties. The assessment is based on a range of approaches that each have uncertainties, and on data sets that are often far from complete. Uncertainties arise from methodologies and conceptual frameworks, from unpredictable surprises, from lack of validation of models, and from the use of particular scenarios, rather than predictions, of future greenhouse gas emissions and climates. Recommendations to reduce the uncertainties are wide-ranging and relate to all disciplines within the assessment. However, a repeated theme is the critical importance of achieving an adequate spatial and long-term coverage of experiments, observations and monitoring of environmental changes and their impacts throughout the sparsely populated and remote region that is the Arctic.

Biodiversity, distributions and adaptations of Arctic species in the context of environmental change.

The individual of a species is the basic unit which responds to climate and UV-B changes, and it responds over a wide range of time scales. The diversity of animal, plant and microbial species appears to be low in the Arctic, and decreases from the boreal forests to the polar deserts of the extreme North but primitive species are particularly abundant. This latitudinal decline is associated with an increase in super-dominant species that occupy a wide range of habitats. Climate warming is expected to reduce the abundance and restrict the ranges of such species and to affect species at their northern range boundaries more than in the South: some Arctic animal and plant specialists could face extinction. Species most likely to expand into tundra are boreal species that currently exist as outlier populations in the Arctic. Many plant species have characteristics that allow them to survive short snow-free growing seasons, low solar angles, permafrost and low soil temperatures, low nutrient availability and physical disturbance. Many of these characteristics are likely to limit species' responses to climate warming, but mainly because of poor competitive ability compared with potential immigrant species. Terrestrial Arctic animals possess many adaptations that enable them to persist under a wide range of temperatures in the Arctic. Many escape unfavorable weather and resource shortage by winter dormancy or by migration. The biotic environment of Arctic animal species is relatively simple with few enemies, competitors, diseases, parasites and available food resources. Terrestrial Arctic animals are likely to be most vulnerable to warmer and drier summers, climatic changes that interfere with migration routes and staging areas, altered snow conditions and freeze-thaw cycles in winter, climate-induced disruption of the seasonal timing of reproduction and development, and influx of new competitors, predators, parasites and diseases. Arctic microorganisms are also well adapted to the Arctic's climate: some can metabolize at temperatures down to -39 degrees C. Cyanobacteria and algae have a wide range of adaptive strategies that allow them to avoid, or at least minimize UV injury. Microorganisms can tolerate most environmental conditions and they have short generation times which can facilitate rapid adaptation to new environments. In contrast, Arctic plant and animal species are very likely to change their distributions rather than evolve significantly in response to warming.

Effects on the structure of Arctic ecosystems in the short- and long-term perspectives.

Species individualistic responses to warming and increased UV-B radiation are moderated by the responses of neighbors within communities, and trophic interactions within ecosystems. All of these responses lead to changes in ecosystem structure. Experimental manipulation of environmental factors expected to change at high latitudes showed that summer warming of tundra vegetation has generally led to smaller changes than fertilizer addition. Some of the factors manipulated have strong effects on the structure of Arctic ecosystems but the effects vary regionally, with the greatest response of plant and invertebrate communities being observed at the coldest locations. Arctic invertebrate communities are very likely to respond rapidly to warming whereas microbial biomass and nutrient stocks are more stable. Experimentally enhanced UV-B radiation altered the community composition of gram-negative bacteria and fungi, but not that of plants. Increased plant productivity due to warmer summers may dominate food-web dynamics. Trophic interactions of tundra and sub-Arctic forest plant-based food webs are centered on a few dominant animal species which often have cyclic population fluctuations that lead to extremely high peak abundances in some years. Population cycles of small rodents and insect defoliators such as the autumn moth affect the structure and diversity of tundra and forest-tundra vegetation and the viability of a number of specialist predators and parasites. Ice crusting in warmer winters is likely to reduce the accessibility of plant food to lemmings, while deep snow may protect them from snow-surface predators. In Fennoscandia, there is evidence already for a pronounced shift in small rodent community structure and dynamics that have resulted in a decline of predators that specialize in feeding on small rodents. Climate is also likely to alter the role of insect pests in the birch forest system: warmer winters may increase survival of eggs and expand the range of the insects. Insects that harass reindeer in the summer are also likely to become more widespread, abundant and active during warmer summers while refuges for reindeer/caribou on glaciers and late snow patches will probably disappear.

Synthesis of effects in four Arctic subregions.

An assessment of impacts on Arctic terrestrial ecosystems has emphasized geographical variability in responses of species and ecosystems to environmental change. This variability is usually associated with north-south gradients in climate, biodiversity, vegetation zones, and ecosystem structure and function. It is clear, however, that significant east-west variability in environment, ecosystem structure and function, environmental history, and recent climate variability is also important. Some areas have cooled while others have become warmer. Also, east-west differences between geographical barriers of oceans, archipelagos and mountains have contributed significantly in the past to the ability of species and vegetation zones to relocate in response to climate changes, and they have created the isolation necessary for genetic differentiation of populations and biodiversity hot-spots to occur. These barriers will also affect the ability of species to relocate during projected future warming. To include this east-west variability and also to strike a balance between overgeneralization and overspecialization, the ACIA identified four major sub regions based on large-scale differences in weather and climate-shaping factors. Drawing on information, mostly model output that can be related to the four ACIA subregions, it is evident that geographical barriers to species re-location, particularly the distribution of landmasses and separation by seas, will affect the northwards shift in vegetation zones. The geographical constraints--or facilitation--of northward movement of vegetation zones will affect the future storage and release of carbon, and the exchange of energy and water between biosphere and atmosphere. In addition, differences in the ability of vegetation zones to re-locate will affect the biodiversity associated with each zone while the number of species threatened by climate change varies greatly between subregions with a significant hot-spot in Beringia. Overall, the subregional synthesis demonstrates the difficulty of generalizing projections of responses of ecosystem structure and function, species loss, and biospheric feedbacks to the climate system for the whole Arctic region and implies a need for a far greater understanding of the spatial variability in the responses of terrestrial arctic ecosystems to climate change.

Effects of changes in climate on landscape and regional processes, and feedbacks to the climate system.

Biological and physical processes in the Arctic system operate at various temporal and spatial scales to impact large-scale feedbacks and interactions with the earth system. There are four main potential feedback mechanisms between the impacts of climate change on the Arctic and the global climate system: albedo, greenhouse gas emissions or uptake by ecosystems, greenhouse gas emissions from methane hydrates, and increased freshwater fluxes that could affect the thermohaline circulation. All these feedbacks are controlled to some extent by changes in ecosystem distribution and character and particularly by large-scale movement of vegetation zones. Indications from a few, full annual measurements of CO2 fluxes are that currently the source areas exceed sink areas in geographical distribution. The little available information on CH4 sources indicates that emissions at the landscape level are of great importance for the total greenhouse balance of the circumpolar North. Energy and water balances of Arctic landscapes are also important feedback mechanisms in a changing climate. Increasing density and spatial expansion of vegetation will cause a lowering of the albedo and more energy to be absorbed on the ground. This effect is likely to exceed the negative feedback of increased C sequestration in greater primary productivity resulting from the displacements of areas of polar desert by tundra, and areas of tundra by forest. The degradation of permafrost has complex consequences for trace gas dynamics. In areas of discontinuous permafrost, warming, will lead to a complete loss of the permafrost. Depending on local hydrological conditions this may in turn lead to a wetting or drying of the environment with subsequent implications for greenhouse gas fluxes. Overall, the complex interactions between processes contributing to feedbacks, variability over time and space in these processes, and insufficient data have generated considerable uncertainties in estimating the net effects of climate change on terrestrial feedbacks to the climate system. This uncertainty applies to magnitude, and even direction of some of the feedbacks.

Responses to projected changes in climate and UV-B at the species level.

Environmental manipulation experiments showed that species respond individualistically to each environmental-change variable. The greatest responses of plants were generally to nutrient, particularly nitrogen, addition. Summer warming experiments showed that woody plant responses were dominant and that mosses and lichens became less abundant. Responses to warming were controlled by moisture availability and snow cover. Many invertebrates increased population growth in response to summer warming, as long as desiccation was not induced. CO2 and UV-B enrichment experiments showed that plant and animal responses were small. However, some microorganisms and species of fungi were sensitive to increased UV-B and some intensive mutagenic actions could, perhaps, lead to unexpected epidemic outbreaks. Tundra soil heating, CO2 enrichment and amendment with mineral nutrients generally accelerated microbial activity. Algae are likely to dominate cyanobacteria in milder climates. Expected increases in winter freeze-thaw cycles leading to ice-crust formation are likely to severely reduce winter survival rate and disrupt the population dynamics of many terrestrial animals. A deeper snow cover is likely to restrict access to winter pastures by reindeer/caribou and their ability to flee from predators while any earlier onset of the snow-free period is likely to stimulate increased plant growth. Initial species responses to climate change might occur at the sub-species level: an Arctic plant or animal species with high genetic/racial diversity has proved an ability to adapt to different environmental conditions in the past and is likely to do so also in the future. Indigenous knowledge, air photographs, satellite images and monitoring show that changes in the distributions of some species are already occurring: Arctic vegetation is becoming more shrubby and more productive, there have been recent changes in the ranges of caribou, and "new" species of insects and birds previously associated with areas south of the treeline have been recorded. In contrast, almost all Arctic breeding bird species are declining and models predict further quite dramatic reductions of the populations of tundra birds due to warming. Species-climate response surface models predict potential future ranges of current Arctic species that are often markedly reduced and displaced northwards in response to warming. In contrast, invertebrates and microorganisms are very likely to quickly expand their ranges northwards into the Arctic.

Bacterial genome replication at subzero temperatures in permafrost.

Microbial metabolic activity occurs at subzero temperatures in permafrost, an environment representing ∼25% of the global soil organic matter. Although much of the observed subzero microbial activity may be due to basal metabolism or macromolecular repair, there is also ample evidence for cellular growth. Unfortunately, most metabolic measurements or culture-based laboratory experiments cannot elucidate the specific microorganisms responsible for metabolic activities in native permafrost, nor, can bulk approaches determine whether different members of the microbial community modulate their responses as a function of changing subzero temperatures. Here, we report on the use of stable isotope probing with (13)C-acetate to demonstrate bacterial genome replication in Alaskan permafrost at temperatures of 0 to -20 °C. We found that the majority (80%) of operational taxonomic units detected in permafrost microcosms were active and could synthesize (13)C-labeled DNA when supplemented with (13)C-acetate at temperatures of 0 to -20 °C during a 6-month incubation. The data indicated that some members of the bacterial community were active across all of the experimental temperatures, whereas many others only synthesized DNA within a narrow subzero temperature range. Phylogenetic analysis of (13)C-labeled 16S rRNA genes revealed that the subzero active bacteria were members of the Acidobacteria, Actinobacteria, Chloroflexi, Gemmatimonadetes and Proteobacteria phyla and were distantly related to currently cultivated psychrophiles. These results imply that small subzero temperature changes may lead to changes in the active microbial community, which could have consequences for biogeochemical cycling in permanently frozen systems.

Comparative proteomic analysis reveals mechanistic insights into Pseudomonas putida F1 growth on benzoate and citrate.

Pseudomonas species are capable to proliferate under diverse environmental conditions and thus have a significant bioremediation potential. To enhance our understanding of their metabolic versatility, this study explores the changes in the proteome and physiology of Pseudomonas putida F1 resulting from its growth on benzoate, a moderate toxic compound that can be catabolized, and citrate, a carbon source that is assimilated through central metabolic pathways. A series of repetitive batch cultivations were performed to ensure a complete adaptation of the bacteria to each of these contrasting carbon sources. After several growth cycles, cell growth stabilized at the maximum level and exhibited a reproducible growth profile. The specific growth rates measured for benzoate (1.01 ± 0.11 h-1) and citrate (1.11 ± 0.12 h-1) were similar, while a higher yield was observed for benzoate (0.6 and 0.3 g cell mass per g of benzoate and citrate, respectively), reflecting the different degrees of carbon reduction in the two substrates. Comparative proteomic analysis revealed an enrichment of several oxygenases/dehydrogenases in benzoate-grown cells, indicative of the higher carbon reduction of benzoate. Moreover, the upregulation of all 14 proteins implicated in benzoate degradation via the catechol ortho-cleavage pathway was observed, while several stress-response proteins were increased to aid cells to cope with benzoate toxicity. Unexpectedly, citrate posed more challenges than benzoate in the maintenance of pH homeostasis, as indicated by the enhancement of the Na+/H+ antiporter and carbonic anhydrase. The study provides important mechanistic insights into Pseudomonas adaptation to varying carbon sources that are of great relevance to bioremediation efforts.