An integrated systems approach for understanding cellular responses to gamma radiation

Cellular response to stress entails complex mRNA and protein abundance changes, which translate into physiological adjustments to maintain homeostasis as well as to repair and minimize damage to cellular components. We have characterized the response of the halophilic archaeon Halobacterium salinarum NRC-1 to (60)Co ionizing gamma radiation in an effort to understand the correlation between genetic information processing and physiological change. The physiological response model we have constructed is based on integrated analysis of temporal changes in global mRNA and protein abundance along with protein–DNA interactions and evolutionarily conserved functional associations. This systems view reveals cooperation among several cellular processes including DNA repair, increased protein turnover, apparent shifts in metabolism to favor nucleotide biosynthesis and an overall effort to repair oxidative damage. Further, we demonstrate the importance of time dimension while correlating mRNA and protein levels and suggest that steady-state comparisons may be misleading while assessing dynamics of genetic information processing across transcription and translation

Large scale physiological readjustment during growth enables rapid, comprehensive and inexpensive systems analysis

Abstract Background Rapidly characterizing the operational interrelationships among all genes in a given organism is a critical bottleneck to significantly advancing our understanding of thousands of newly sequenced microbial and eukaryotic species. While evolving technologies for global profiling of transcripts, proteins, and metabolites are making it possible to comprehensively survey cellular physiology in newly sequenced organisms, these experimental techniques have not kept pace with sequencing efforts. Compounding these technological challenges is the fact that individual experiments typically only stimulate relatively small-scale cellular responses, thus requiring numerous expensive experiments to survey the operational relationships among nearly all genetic elements. Therefore, a relatively quick and inexpensive strategy for observing changes in large fractions of the genetic elements is highly desirable. Results We have discovered in the model organism Halobacterium salinarum NRC-1 that batch culturing in complex medium stimulates meaningful changes in the expression of approximately two thirds of all genes. While the majority of these changes occur during transition from rapid exponential growth to the stationary phase, several transient physiological states were detected beyond what has been previously observed. In sum, integrated analysis of transcript and metabolite changes has helped uncover growth phase-associated physiologies, operational interrelationships among two thirds of all genes, specialized functions for gene family members, waves of transcription factor activities, and growth phase associated cell morphology control. Conclusions Simple laboratory culturing in complex medium can be enormously informative regarding the activities of and interrelationships among a large fraction of all genes in an organism. This also yields important baseline physiological context for designing specific perturbation experiments at different phases of growth. The integration of such growth and perturbation studies with measurements of associated environmental factor changes is a practical and economical route for the elucidation of comprehensive systems-level models of biological systems

Diurnally Entrained Anticipatory Behavior in Archaea

By sensing changes in one or few environmental factors biological systems can anticipate future changes in multiple factors over a wide range of time scales (daily to seasonal). This anticipatory behavior is important to the fitness of diverse species, and in context of the diurnal cycle it is overall typical of eukaryotes and some photoautotrophic bacteria but is yet to be observed in archaea. Here, we report the first observation of light-dark (LD)-entrained diurnal oscillatory transcription in up to 12% of all genes of a halophilic archaeon Halobacterium salinarum NRC-1. Significantly, the diurnally entrained transcription was observed under constant darkness after removal of the LD stimulus (free-running rhythms). The memory of diurnal entrainment was also associated with the synchronization of oxic and anoxic physiologies to the LD cycle. Our results suggest that under nutrient limited conditions halophilic archaea take advantage of the causal influence of sunlight (via temperature) on O2 diffusivity in a closed hypersaline environment to streamline their physiology and operate oxically during nighttime and anoxically during daytime

A Role for Programmed Cell Death in the Microbial Loop

<div><p>The microbial loop is the conventional model by which nutrients and minerals are recycled in aquatic eco-systems. Biochemical pathways in different organisms become metabolically inter-connected such that nutrients are utilized, processed, released and re-utilized by others. The result is that unrelated individuals end up impacting each others' fitness directly through their metabolic activities. This study focused on the impact of programmed cell death (PCD) on a population's growth as well as its role in the exchange of carbon between two naturally co-occurring halophilic organisms. Flow cytometric, biochemical, ¹⁴C radioisotope tracing assays, and global transcriptomic analyses show that organic algal photosynthate released by <i>Dunalliela salina</i> cells undergoing PCD complements the nutritional needs of other non-PCD <i>D. salina</i> cells. This occurs <i>in vitro</i> in a carbon limited environment and enhances the growth of the population. In addition, a co-occurring heterotroph <i>Halobacterium salinarum</i> re-mineralizes the carbon providing elemental nutrients for the mixoheterotrophic chlorophyte. The significance of this is uncertain and the archaeon can also subsist entirely on the lysate of apoptotic algae. PCD is now well established in unicellular organisms; however its ecological relevance has been difficult to decipher. In this study we found that PCD in <i>D. salina</i> causes the release of organic nutrients such as glycerol, which can be used by others in the population as well as a co-occurring halophilic archaeon. <i>H. salinarum</i> also re-mineralizes the dissolved material promoting algal growth. PCD in <i>D. salina</i> was the mechanism for the flow of dissolved photosynthate between unrelated organisms. Ironically, programmed death plays a central role in an organism's own population growth and in the exchange of nutrients in the microbial loop.</p></div

Initial and fitted parameter values for model of growth for pure <i>D. salina</i> and <i>D. salina</i> + <i>H</i>. <i>salinarum</i> co-cultures.

<p>“±CI_95%” are parameter 95% confidence intervals such that the lower and upper bound of estimated values are X-CI_95% and X+CI_95%, respectively.</p

Diurnally synchronized syntrophic interaction with <i>H.salinarum</i> increases productivity of <i>D. salina</i>.

<p>(A) Intra- and (B) extra-cellular glycerol concentrations in <i>D. salina</i> culture individually (blue) or with <i>H. salinarum</i> (red) over several day: night cycles, dash lines represent +/− standard deviation. (C) Radiolabel incorporation and tracing shows daytime uptake and nighttime release of ¹⁴C by <i>D. salina</i>. Uptake of ¹⁴C by <i>D. salina</i> at night is enhanced two-fold in co-cultures relative to pure cultures indicating nighttime assimilation of ¹⁴C in presence of <i>H. salinarum</i>. (D) Simultaneous tracing of C within <i>H. salinarum</i> cells demonstrates uptake and processing of ¹⁴C in sync with the diurnal cycle.</p

Dissolved organic material (DOM or photosynthate) released by <i>D.salina</i> fully complements nutritional requirements of <i>H. salinarum</i>.

<p>Supernatant of <i>D. salina</i> culture in artificial seawater (MM1) supported <i>H. salinarum</i> growth at a level that was comparable to its growth in MM1 supplemented with amino acids at naturally occurring concentrations.</p

Cell death is triggered at nighttime as part of the diurnal synchronized program of <i>D.salina</i>.

<p>(A) Cell numbers for <i>D. salina</i> in pure and co-cultures with <i>H. salinarum</i> over several diurnal cycles. Live cell concentration measured using flow cytometry are indicated with blue (pure culture) and red (co-culture) points while lines are fitted model simulations. Green boxed region indicates time frame reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062595#pone-0062595-g003" target="_blank"><b>Fig. 3D</b></a> over which caspase-3 activity was assayed. (B) <i>H. salinarum</i> induces cell death in <i>D. salina</i> under continuous light regime. Intracellular glycerol within <i>D. salina</i> was stained with quinacrine and quantified with flow cytometry. Decrease of intracellular glycerol proportionally with higher cell density of <i>H. salinarum</i>. Unstimulated (pure <i>D. salina</i> culture and dark shifted samples are shown as controls. (L/L>L, cultures grown on a 24 h constant light regime maintained in the light during the measurements, LL>D, cultures grown in constant light shifted to dark conditions (0 µmoles m²s⁻¹). (C) The decrease in <i>D. salina</i> cell number in the model (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062595#pone-0062595-g004" target="_blank"><b>Fig. 4A</b></a> (blue line) due to cell death is supported by the time course of annexin V labeled cells (blue line) indicating percentage of cells exhibiting externalization of PS and SYTOX® blue stained cells indicating the percent dead cells (red line). (D) The decrease in cell number in the model (blue dotted line) due to cell death is also supported by higher levels of caspase-3 during nighttime. Red line is Savitsky-Golay smoothed (span of 5) average of two replicate measurements for each time point.</p

Mechanisms of communication and interactions in the syntrophic interaction.

<p>Transcriptional response of <i>H. salinarum</i> NRC-1 to <i>D. salina</i> conditioned artificial seawater amended with nutrients (MM1).</p

Diurnally synchronized cell death drives C-flux in an algal-archaeal syntrophic interaction.

<p>At night a stochastic process determines the fate of each algal cell resulting in up to 74% of cells undergoing death to release DOM (byproducts of photosynthetic C assimilation) into the surrounding media. The DOM are further metabolized and remineralized by archaea into a form that is readily consumed by algae. With onset of the subsequent day cycle, the algal population rapidly regenerates with up to 3 doublings with a cell division rate of 1.4 hrs. This entire process iterates over the next diurnal cycle.</p