Phenotypic impact of regulatory noise in cellular stress-response pathways

Recent studies indicate that intrinsic promoter-mediated gene expression noise can confer a selective advantage under acute environmental stress by providing beneficial phenotypic diversity within cell populations. To investigate how extrinsic gene expression noise impacts the fitness of cell populations under stress, we engineered two nearly isogenic budding yeast strains; one carrying a two-step regulatory cascade that allows for precise control of the noise transmitted from a transcriptional regulator to a downstream stress-inducing gene, and one carrying a network with low constant upstream noise. The fitness and gene expression of these strains were compared under acute and prolonged stress exposure. Using a phenomenological modeling approach, we predicted that increased noise should confer a fitness advantage under high stress conditions, but reciprocally reduce the resistance of the population to low stress. The model also predicted that extrinsic noise might serve as a basis for phenotypic plasticity whereby gene expression distributions are modulated in response to prolonged stress. Experimentally, we confirmed the predicted differential fitness advantage of extrinsic noise under acute stress, as well as the predicted modulation of gene expression under prolonged stress. However, contrary to model predictions, strains with low and high extrinsic noise showed very similar adaptive responses to prolonged stress. This suggests that while phenotypic heterogeneity generated by noise in regulatory signals can confer increased robustness to acute stress, it is not a requirement for the observed long-term phenotypic plasticity

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

Prevalence of transcription promoters within archaeal operons and coding sequences

Despite the knowledge of complex prokaryotic-transcription mechanisms, generalized rules, such as the simplified organization of genes into operons with well-defined promoters and terminators, have had a significant role in systems analysis of regulatory logic in both bacteria and archaea. Here, we have investigated the prevalence of alternate regulatory mechanisms through genome-wide characterization of transcript structures of ∼64% of all genes, including putative non-coding RNAs in Halobacterium salinarum NRC-1. Our integrative analysis of transcriptome dynamics and protein–DNA interaction data sets showed widespread environment-dependent modulation of operon architectures, transcription initiation and termination inside coding sequences, and extensive overlap in 3′ ends of transcripts for many convergently transcribed genes. A significant fraction of these alternate transcriptional events correlate to binding locations of 11 transcription factors and regulators (TFs) inside operons and annotated genes—events usually considered spurious or non-functional. Using experimental validation, we illustrate the prevalence of overlapping genomic signals in archaeal transcription, casting doubt on the general perception of rigid boundaries between coding sequences and regulatory elements

Phenotypic impact of regulatory noise in cellular stress-response pathways

Recent studies indicate that intrinsic promoter-mediated gene expression noise can confer a selective advantage under acute environmental stress by providing beneficial phenotypic diversity within cell populations. To investigate how extrinsic gene expression noise impacts the fitness of cell populations under stress, we engineered two nearly isogenic budding yeast strains; one carrying a two-step regulatory cascade that allows for precise control of the noise transmitted from a transcriptional regulator to a downstream stress-inducing gene, and one carrying a network with low constant upstream noise. The fitness and gene expression of these strains were compared under acute and prolonged stress exposure. Using a phenomenological modeling approach, we predicted that increased noise should confer a fitness advantage under high stress conditions, but reciprocally reduce the resistance of the population to low stress. The model also predicted that extrinsic noise might serve as a basis for phenotypic plasticity whereby gene expression distributions are modulated in response to prolonged stress. Experimentally, we confirmed the predicted differential fitness advantage of extrinsic noise under acute stress, as well as the predicted modulation of gene expression under prolonged stress. However, contrary to model predictions, strains with low and high extrinsic noise showed very similar adaptive responses to prolonged stress. This suggests that while phenotypic heterogeneity generated by noise in regulatory signals can confer increased robustness to acute stress, it is not a requirement for the observed long-term phenotypic plasticity

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

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

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

Cell death upon exposure to darkness or <i>H.salinarum</i> cells triggers the release of glycerol by <i>D. salina</i>.

<p>(A) <i>D. salina</i> accumulates and utilizes glycerol as an osmoprotectant in hypersaline growth conditions. Accumulation of glycerol in <i>D. salina</i> cultures is correlated to increasing salinity in the growth medium peaking at 150 PSU (inset). (B) <i>D. salina</i> releases glycerol by cell death. Illustrated and merged phase contrast/fluorescence photomicrographs of a <i>D. salina</i> cell undergoing cell death. <i>D. salina</i> stores glycerol and other byproducts of photosynthesis inside secretory vesicles that are localized to the apical flagellar pole (top). The green color of the vesicles is due to quinacrine staining of glycerol and the red fluorescence corresponds to chloroplasts. The image show dramatic disruption of the cell membrane and complete loss of internal glycerol in a cell that has undergone cell death (bottom). (C) A shift of live light acclimated cultures (100–150 µmol-photon m⁻²sec⁻¹) to complete darkness (0 µmol-photon m⁻² sec⁻¹) results in release of glycerol by <i>D. salina</i>. The intracellular glycerol is measured by flow cytometry analysis of quinacrine stained vesicles <i>D. salina</i> cells. (D) Representative confocal laser micrograph of <i>D. salina</i> cells stained with FITC-Annexin V to highlight the externalization of PS (green fluorescence), and red corresponds to red chlorophyll autofluorescence. (E) Representative confocal laser micrographs of dead <i>D. salina</i> stained with FITC-Annexin V and SYTOX® blue highlighting PS completely externalized and the ejection of the nucleus indicating cell death.</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