Nanocalorimetry Reveals the Growth Dynamics of Escherichia coli Cells Undergoing Adaptive Evolution during Long-Term Stationary Phase.

Bacterial populations in long-term stationary-phase (LTSP) laboratory cultures can provide insights into physiological and genetic adaptations to low-energy conditions and population dynamics in natural environments. While overall population density remains stable, these communities are very dynamic and are characterized by the rapid emergence and succession of distinct mutants expressing the growth advantage in stationary phase (GASP) phenotype, which can reflect an increased capacity to withstand energy limitations and environmental stress. Here, we characterize the metabolic heat signatures and growth dynamics of GASP mutants within an evolving population using isothermal calorimetry. We aged Escherichia coli in anaerobic batch cultures over 20 days inside an isothermal nanocalorimeter and observed distinct heat events related to the emergence of three mutant populations expressing the GASP phenotype after 1.5, 3, and 7 days. Given the heat produced by each population, the maximum number of GASP mutant cells was calculated, revealing abundances of ∼2.5 × 107, ∼7.5 × 106, and ∼9.9 × 106 cells in the populations, respectively. These data indicate that mutants capable of expressing the GASP phenotype can be acquired during the exponential growth phase and subsequently expressed in LTSP culture.IMPORTANCE The present study is innovative in that we have identified previously unknown growth dynamics related to the temporal expression of the growth advantage in stationary phase (GASP) phenotype that allow mutants in long-term stationary-phase cultures to capitalize on the decrease of energy over prolonged incubation periods. By remaining in an active, but growth-limited, metabolic state similar to that observed in GASP cells grown in vitro, natural microbial communities might be able to prevail over much longer time scales. We believe this report to be a remarkable methodological and conceptual breakthrough in the study of the long-term survival and evolution of bacteria.

Changes in Microbial Energy Metabolism Measured by Nanocalorimetry during Growth Phase Transitions.

Calorimetric measurements of the change in heat due to microbial metabolic activity convey information about the kinetics, as well as the thermodynamics, of all chemical reactions taking place in a cell. Calorimetric measurements of heat production made on bacterial cultures have recorded the energy yields of all co-occurring microbial metabolic reactions, but this is a complex, composite signal that is difficult to interpret. Here we show that nanocalorimetry can be used in combination with enumeration of viable cell counts, oxygen consumption rates, cellular protein content, and thermodynamic calculations to assess catabolic rates of an isolate of Shewanella oneidensis MR-1 and infer what fraction of the chemical energy is assimilated by the culture into biomass and what fraction is dissipated in the form of heat under different limiting conditions. In particular, our results demonstrate that catabolic rates are not necessarily coupled to rates of cell division, but rather, to physiological rearrangements of S. oneidensis MR-1 upon growth phase transitions. In addition, we conclude that the heat released by growing microorganisms can be measured in order to understand the physiochemical nature of the energy transformation and dissipation associated with microbial metabolic activity in conditions approaching those found in natural systems.

Nanocalorimetric Characterization of Microbial Activity in Deep Subsurface Oceanic Crustal Fluids.

Although fluids within the upper oceanic basaltic crust harbor a substantial fraction of the total prokaryotic cells on Earth, the energy needs of this microbial population are unknown. In this study, a nanocalorimeter (sensitivity down to 1.2 nW ml(-1)) was used to measure the enthalpy of microbially catalyzed reactions as a function of temperature in samples from two distinct crustal fluid aquifers. Microorganisms in unamended, warm (63°C) and geochemically altered anoxic fluids taken from 292 meters sub-basement (msb) near the Juan de Fuca Ridge produced 267.3 mJ of heat over the course of 97 h during a step-wise isothermal scan from 35.5 to 85.0°C. Most of this heat signal likely stems from the germination of thermophilic endospores (6.66 × 10(4) cells ml(-1) FLUID) and their subsequent metabolic activity at temperatures greater than 50°C. The average cellular energy consumption (5.68 pW cell(-1)) reveals the high metabolic potential of a dormant community transported by fluids circulating through the ocean crust. By contrast, samples taken from 293 msb from cooler (3.8°C), relatively unaltered oxic fluids, produced 12.8 mJ of heat over the course of 14 h as temperature ramped from 34.8 to 43.0°C. Corresponding cell-specific energy turnover rates (0.18 pW cell(-1)) were converted to oxygen uptake rates of 24.5 nmol O2 ml(-1) FLUID d(-1), validating previous model predictions of microbial activity in this environment. Given that the investigated fluids are characteristic of expansive areas of the upper oceanic crust, the measured metabolic heat rates can be used to constrain boundaries of habitability and microbial activity in the oceanic crust.

Activity and community structures of sulfate-reducing microorganisms in polar, temperate and tropical marine sediments.

Temperature has a fundamental impact on the metabolic rates of microorganisms and strongly influences microbial ecology and biogeochemical cycling in the environment. In this study, we examined the catabolic temperature response of natural communities of sulfate-reducing microorganisms (SRM) in polar, temperate and tropical marine sediments. In short-term sediment incubation experiments with (35)S-sulfate, we demonstrated how the cardinal temperatures for sulfate reduction correlate with mean annual sediment temperatures, indicating specific thermal adaptations of the dominant SRM in each of the investigated ecosystems. The community structure of putative SRM in the sediments, as revealed by pyrosequencing of bacterial 16S rRNA gene amplicons and phylogenetic assignment to known SRM taxa, consistently correlated with in situ temperatures, but not with sediment organic carbon concentrations or C:N ratios of organic matter. Additionally, several species-level SRM phylotypes of the class Deltaproteobacteria tended to co-occur at sites with similar mean annual temperatures, regardless of geographic distance. The observed temperature adaptations of SRM imply that environmental temperature is a major controlling variable for physiological selection and ecological and evolutionary differentiation of microbial communities.

Activity and phylogenetic diversity of sulfate-reducing microorganisms in low-temperature subsurface fluids within the upper oceanic crust.

The basaltic ocean crust is the largest aquifer system on Earth, yet the rates of biological activity in this environment are unknown. Low-temperature (<100°C) fluid samples were investigated from two borehole observatories in the Juan de Fuca Ridge (JFR) flank, representing a range of upper oceanic basement thermal and geochemical properties. Microbial sulfate reduction rates (SRR) were measured in laboratory incubations with (35)S-sulfate over a range of temperatures and the identity of the corresponding sulfate-reducing microorganisms (SRM) was studied by analyzing the sequence diversity of the functional marker dissimilatory (bi)sulfite reductase (dsrAB) gene. We found that microbial sulfate reduction was limited by the decreasing availability of organic electron donors in higher temperature, more altered fluids. Thermodynamic calculations indicate energetic constraints for metabolism, which together with relatively higher cell-specific SRR reveal increased maintenance requirements, consistent with novel species-level dsrAB phylotypes of thermophilic SRM. Our estimates suggest that microbially-mediated sulfate reduction may account for the removal of organic matter in fluids within the upper oceanic crust and underscore the potential quantitative impact of microbial processes in deep subsurface marine crustal fluids on marine and global biogeochemical carbon cycling.

Effects of freeze-thaw cycles on anaerobic microbial processes in an Arctic intertidal mud flat.

Insight into the effects of repeated freezing and thawing on microbial processes in sediments and soils is important for understanding sediment carbon cycling at high latitudes acutely affected by global warming. Microbial responses to repeated freeze-thaw conditions were studied in three complementary experiments using arctic sediment collected from an intertidal flat that is exposed to seasonal freeze-thaw conditions (Ymerbukta, Svalbard, Arctic Ocean). The sediment was subjected to oscillating freeze-thaw incubations, either gradual, from -5 to 4 degrees C, or abrupt, from -20 to 10 degrees C. Concentrations of low-molecular weight carboxylic acids (volatile fatty acids) were measured and sulfate reduction was assessed by measuring (35)S sulfate reduction rates (SRRs). Gradual freeze-thaw incubation decreased microbial activity in the frozen state to 0.25 % of initial levels at 4 degrees C, but activity resumed rapidly reaching >60 % of initial activity in the thawed state. Exposure of sediments to successive large temperature changes (-20 versus 10 degrees C) decreased SRR by 80% of the initial activity, suggesting that a fraction of the bacterial community recovered rapidly from extreme temperature fluctuations. This is supported by 16S rRNA gene-based denaturing gradient gel electrophoresis profiles that revealed persistence of the dominant microbial taxa under repeated freeze-thaw cycles. The fast recovery of the SRRs suggests that carbon mineralization in thawing arctic sediment can resume without delay or substantial growth of microbial populations.

The impact of temperature change on the activity and community composition of sulfate-reducing bacteria in arctic versus temperate marine sediments.

Arctic regions may be particularly sensitive to climate warming and, consequently, rates of carbon mineralization in warming marine sediment may also be affected. Using long-term (24 months) incubation experiments at 0°C, 10°C and 20°C, the temperature response of metabolic activity and community composition of sulfate-reducing bacteria were studied in the permanently cold sediment of north-western Svalbard (Arctic Ocean) and compared with a temperate habitat with seasonally varying temperature (German Bight, North Sea). Short-term (35)S-sulfate tracer incubations in a temperature-gradient block (between -3.5°C and +40°C) were used to assess variations in sulfate reduction rates during the course of the experiment. Warming of arctic sediment resulted in a gradual increase of the temperature optima (T(opt)) for sulfate reduction suggesting a positive selection of psychrotolerant/mesophilic sulfate-reducing bacteria (SRB). However, high rates at in situ temperatures compared with maximum rates showed the predominance of psychrophilic SRB even at high incubation temperatures. Changing apparent activation energies (E(a)) showed that increasing temperatures had an initial negative impact on sulfate reduction that was weaker after prolonged incubations, which could imply an acclimatization response rather than a selection process of the SRB community. The microbial community composition was analysed by targeting the 16S ribosomal RNA using catalysed reporter deposition fluorescence in situ hybridization (CARD-FISH). The results showed the decline of specific groups of SRB and confirmed a strong impact of increasing temperatures on the microbial community composition of arctic sediment. Conversely, in seasonally changing sediment sulfate reduction rates and sulfate-reducing bacterial abundance changed little in response to changing temperature.

Endosymbioses between bacteria and deep-sea siboglinid tubeworms from an Arctic Cold Seep (Haakon Mosby Mud Volcano, Barents Sea).

Siboglinid tubeworms do not have a mouth or gut and live in obligate associations with bacterial endosymbionts. Little is currently known about the phylogeny of frenulate and moniliferan siboglinids and their symbionts. In this study, we investigated the symbioses of two co-occurring siboglinid species from a methane emitting mud volcano in the Arctic Ocean (Haakon Mosby Mud Volcano, HMMV): Oligobrachia haakonmosbiensis (Frenulata) and Sclerolinum contortum (Monilifera). Comparative sequence analysis of the host-specific 18S and the symbiont-specific 16S rRNA genes of S. contortum showed that the close phylogenetic relationship of this host to vestimentiferan siboglinids was mirrored in the close relationship of its symbionts to the sulfur-oxidizing gammaproteobacterial symbionts of vestimentiferans. A similar congruence between host and symbiont phylogeny was observed in O. haakonmosbiensis: both this host and its symbionts were most closely related to the frenulate siboglinid O. mashikoi and its gammaproteobacterial symbiont. The symbiont sequences from O. haakonmosbiensis and O. mashikoi formed a clade unaffiliated with known methane- or sulfur-oxidizing bacteria. Fluorescence in situ hybridization indicated that the dominant bacterial phylotypes originated from endosymbionts residing inside the host trophosome. In both S. contortum and O. haakonmosbiensis, characteristic genes for autotrophy (cbbLM) and sulfur oxidation (aprA) were present, while genes diagnostic for methanotrophy were not detected. The molecular data suggest that both HMMV tubeworm species harbour chemoautotrophic sulfur-oxidizing symbionts. In S. contortum, average stable carbon isotope values of fatty acids and cholesterol of -43 per thousand were highly negative for a sulfur oxidizing symbiosis, but can be explained by a (13)C-depleted CO(2) source at HMMV. In O. haakonmosbiensis, stable carbon isotope values of fatty acids and cholesterol of -70 per thousand are difficult to reconcile with our current knowledge of isotope signatures for chemoautotrophic processes.