Browsing by Author "Schmid, Amy K"

Microbial population growth measurements are widespread in the study of microorganisms, providing insight into areas including genetics, physiology, and engineering. The most common models of microbial population growth data are parametric, and are derived from specific assumptions about the underlying growth process. While useful in cases where these assumptions are valid, these models are inadequate in many cases typically found in microbial growth studies, including presence of significant population death and the presence of multiple growth phases (e.g. diauxie). Here, we explore the use of the Bayesian non-parametric model Gaussian processes on microbial population growth. We first develop a general hypothesis-test using Gaussian process regression and false-discovery rate corrected Bayes factor scores. We then explore a fully Bayesian model with Gaussian process priors that can capture the latent growth processes of many population measurements under a single model. Finally, we develop hierarchical Bayesian model with GP priors in order to capture random effects in microbial population growth data.

Hypersaline-adapted archaea, or haloarchaea, inhabit extreme environments where changes in near-saturated salinity, oxygen, and nutrients require rapid regulation of essential cellular processes. Transcriptional regulatory networks govern the dynamical responses enabling these organisms to sense and respond to rapidly changing conditions. Additionally, timely regulation of carbon metabolic pathways is essential to respond to intermittent nutrient availability and prevent futile cycling of intracellular metabolites. In Halobacterium salinarum, a hypersaline-adapted archaeon that does not rely on carbohydrates for carbon or energy, over 1,500 transcriptome profiling experiments have yielded a genome-scale model of regulatory interactions and revealed a general transcriptional response to diverse stressors (Chapter 2). Among regulators in Hbt. salinarum, a sugar-sensing TrmB family protein has previously been shown to control gluconeogenesis and other biosynthetic pathways. This characterization expanded the set of regulatory functions known for TrmB-family proteins in archaea, which regulate carbohydrate metabolism in hyperthermophilic archaea. TrmB regulators are particularly interesting in Haloarchaea because an expansion of the protein family is presumed to have occurred alongside a diversification of carbohydrate catabolic pathways (Chapter 3).

To investigate whether the expanded set of TrmB functions is shared among haloarchaea with different metabolic capabilities and better understand how regulatory variation arises in extremophiles, we characterized the role of TrmB in two haloarchaeal model species that catabolize carbohydrates: Haloarcula hispanica and Haloferax volcanii (Chapters 4 and 5, respectively). We hypothesized that TrmB would maintain a role in the regulation of gluconeogenesis through homologous targets but would acquire targets involved in the concordant catabolic processes of these saccharolytic models. To characterize the role of TrmB homologs, we conducted high-throughput growth assays, microscopy and other microbiological phenotyping techniques, gene expression profiling via RNA-seq, promoter activity analysis, and protein-DNA binding assays of TrmB homologs in Har. hispanica and Hfx. volcanii. Our results show that TrmB homologs indeed activate gluconeogenesis through the recognition of conserved cis-regulatory motifs. However, contrary to its role in Hbt. salinarum, TrmB does not act as a global regulator in Har. hispanica or Hfx. volcanii: it does not directly repress the expression of peripheral pathways such as cofactor biosynthesis or catabolism of other carbon sources. A key bidirectional control point, activation of ppsA and repression of pyk, is lost in Har. hispanica.

Our results indicate substantial rewiring of the TrmB regulon in Hfx. volcanii. A novel transcriptional regulator, TbsP, is responsible for repressing gluconeogenic genes when glucose is available. TrmB and TbsP appear to compete for partially overlapping binding sites in the promoter of gapII, which encodes the gluconeogenic-specific glyceraldehyde-3-phosphate dehydrogenase. Loss-of-function mutations in tbsP are sufficient to recover partial gapII expression and gluconeogenic activity when trmB is deleted. In Hfx. volcanii, TrmB is predicted to activate ppsA and repress the gene encoding bacterial phosphoenopyruvate carboxylase, perhaps preserving the dynamical and functional behavior of TrmB in Hbt salinarum, but reflecting species-specific anaplerotic strategies. Moreover, TrmB is predicted to repress the expression of bacterial type I GAPDH: gapI and gapII may comprise an additional bidirectional control point in Hfx. volcanii, although this hypothesis requires additional testing.

Cumulatively, this dissertation outlines specific examples of TRN rewiring highlighting the incorporation of metabolic enzymes gained through inter-domain horizontal gene transfer, suggesting rewiring of the TrmB regulon via gain and loss of binding sites alongside metabolic network evolution in Haloarchaea.

The regulation of metabolism is one of the key challenges faced by organisms across all domains of life. Despite fluctuating environments, cells must produce the same metabolic outputs to thrive. Although much is known about the regulation of metabolism in the bacteria and the eukaryotes, relatively little is known about the regulation of metabolism in archaea. Previous work identified the winged helix-turn-helix transcription factor TrmB as a major regulator of metabolism in the model archaeon Halobacterium salinarum. TrmB was found to bind to the promoter of 113 genes in the absence of glucose. Many of these genes encode enzymes involved in metabolic processes, including central carbon metabolism, purine synthesis, and amino acid degradation. Although much is known about TrmB, it remains unclear how it dynamically regulates its ~100 metabolic enzyme-coding gene targets, what the effect of transcriptional regulation is on metabolite levels, and why TrmB regulates so many metabolic processes in response to glucose. Using dynamic gene expression and TrmB-DNA binding assays, we found that that TrmB functions alone to regulate central metabolic enzyme-coding genes, but cooperates with various regulators to control peripheral metabolic pathways. After determining the temporal pattern of gene expression changes and their dependence on TrmB, we used dynamic metabolite profiling to investigate the effects of transcriptional changes on metabolite levels and phenotypes. We found that TrmB-mediated transcriptional changes resulted in substantial changes in metabolite levels. Additionally, we showed that mis-regulation of genes encoding enzymes involved in gluconeogenesis in the ΔtrmB mutant strain in the absence of glucose results in low PRPP levels, which cause a metabolic block in de novo purine synthesis that is partially responsible for the growth defect of the ΔtrmB mutant strain. Finally, using a series of quantitative phenotyping experiments, we showed that TrmB regulates the gluconeogenic production of sugars incorporated into the cell surface S-layer glycoprotein. Because S-layer glycosylation is proportional to growth, we hypothesize that TrmB transduces a growth rate signal to co-regulated metabolic pathways including amino acid, purine, and cobalamin biosynthesis. Taken together, our results suggest that TrmB is a global regulator of archaeal metabolism that works in concert with other transcription factors to regulate diverse metabolic pathways in response to nutrients and growth rate.

Protein-based compaction of the genome is a feature found in species across the tree of life. In Archaea, the majority of species contain a histone fold domain-containing protein, and these have been shown to compact DNA through the formation of nucleosomes and extended structures called hypernucleosomes. However, the role of the histone-like proteins of halophilic archaea is unclear. Previous work in the model species Halobacterium salinarum indicated that its sole histone gene, hpyA, is dispensable for growth and is expressed at very low levels. I hypothesize that the unique high-salt environment of halophilic archaea has selected for an alternative histone function, and that they function instead as transcription factors.

This hypothesis was addressed with genetic approaches including the creation of knockout and complementation strains, traditional microbiology techniques including growth assays and microscopy, and high-throughput genomics approaches: ChIP-Seq to study genome-wide binding, and RNA-Seq to study differential expression in ΔhpyA strain. It was found that hpyA is required for optimal growth in hypo-osmotic conditions, and exhibits strong salt-dependent binding patterns and gene regulation. It directly regulates genes involved in iron uptake, and indirectly regulates genes in ion transport and nucleotide metabolism. These results validate the link between histone function and the high-salt environment of halophilic archaea.

Similar to hpyA, I found that the sole histone gene of another model halophile: hstA of Haloferax volcanii, could be deleted, and that knockout cells remained viable. The genome-wide binding of both halophilic histones was studied, and compared with publicly available data regarding the binding patterns from transcription factors (TFs), nucleoid-associated proteins (NAPs), and eukaryotic histones. Halophilic histones bind in narrow, discrete, and relatively rare peaks, just like TFs; however, this binding is not enriched at the promoter, and they instead bind evenly in both intergenic and coding regions (like some NAPs). Their occupancy profile across gene start sites do not resemble those of histones or TFs. In terms of sequence specificity, HpyA exhibits a histone-like preference for 10bp periodicity, while HstA exhibits a TF-like trait in preferentially binding a palindromic sequence motif. When considering all the data, I conclude that halophilic histones blur the line between TFs, NAPs, and histones.

A major technical challenge in generating this data was the removal of rRNA prior to carrying out RNA-Seq. Several approaches were tested across four model species of halophiles, and the reasons for differences in performance for these approaches were analyzed. Methods that deliver efficient rRNA removal targeted to a particular species, or to halophilic archaea in general, are highlighted.

Together these results shed light on the unusual function and binding modes of the histone-like proteins of halophilic archaea. In combination with other recent work, they suggest that histone function is linked with the physical environment of archaeal species.

Like most Archaea, the hypersaline-adapted organism Halobacterium salinarum exhibits characteristics from all three domains of life, including a eukaryotic histone protein, a universal propensity to genetic rearrangements, and homologs of bacterial cell division proteins. Here we investigate the ancestral function of histone protein in the Archaea. Transcriptomics, proteomics, and phenotypic assays of histone mutants determine that histone regulates gene expression and cell shape but not genome compaction in H. salinarum. We further explore the regulation of gene expression on a genome-wide scale through the study of genomic instability. Genomic deletions and duplications are detected through the meta-analysis of 1154 previously published gene expression arrays and 48 chromatin immunoprecipitation arrays. We discover that a 90 kb duplication event in the megaplasmid pNRC100 directly leads to increased gene expression, and find evidence that the chromosome is far more unstable than previously assumed. These events are all linked with the presence of mobile insertion elements. Finally, in response to questions generated by these experiments, we develop a novel time-lapse protocol for H. salinarum and ask basic questions about single cell dynamics during division. Fluorescent labeling of homologs to bacterial cell division proteins confirms their involvement in cell division but localization dynamics contradict the basic bacterial model. The discovery of unusual facets of morphology during cell division is consistent with these novel protein dynamics and opens up new avenues of inquiry into archaeal cell division.

Many important biological processes are temporally regulated. For periodic biological processes—such as the cell cycle, the circadian rhythm, and the malaria developmental cycle—temporal ordering of dynamic cellular events is controlled by large programs of periodic gene expression. A large portion of the genome oscillates in these dynamic biological processes (between 20 and 90 percent of the genome for the dynamic biological processes above). These coordinated programs of gene expression are controlled by gene regulatory networks (GRNs) consisting of transcription factors (TFs), kinases, and ubiquitin ligases. GRNs serve to generate and transmit a pulse of transcription, order temporal events, and maintain oscillations over multiple cycles. Historically, uncovering these regulatory mechanisms has taken coordinated effort over decades. An important challenge in biology today is accelerating the rate of discovery for these high-dimensional and complex biological phenomena. To this end, high-dimensional data and complex analytical tools are required. Time-series transcriptomic analyses have uncovered many important insights into dynamic processes, as they enable the characterization of gene-expression profiles for thousands of genes simultaneously. Furthermore, these time-series transcriptomic datasets can be used to infer GRN models from the data. However, these analyses can be complex. Many new computational tools have been developed to enable complex analyses. In Chapters 2, 3, and 4 of this dissertation, I describe methods for analysis of time-series transcriptomic data, network inference, and comparison across experiments. These computational tools were then applied to the Saccharomyces cerevisiae cell cycle to understand the regulation of the cell-cycle period in unfavorable growth conditions. The cell cycle is a vitally important dynamic biological process, which relies on multiple layers of regulation to ensure correct temporal ordering of cell-cycle events and the cell-cycle transcriptional program. Checkpoints monitor cell-cycle progression to ensure correct temporal ordering without catastrophic errors. This ordering is vital to guarantee faithful duplication of the cell. Cell-cycle progression is also affected by environmental conditions. In response to acute environmental stress, the S. cerevisiae cell cycle halts or pauses as a stress responsive and preparative mechanism. In response to chronic environmental stress, the cell-cycle period slows, providing mild stress-resistance. However, the mechanism underlying cell-cycle period control remains under debate. Early studies suggested that this regulation occurs in late G1 via a size or resource threshold at START. More recent studies show that cell-cycle period regulation can occur outside of G1 as well, indicating a need for other regulatory models. One such alternative model comes from the GRN models described above. Turning to other biological oscillators, the circadian period is controlled by the complex circadian GRN. The circadian period is tightly regulated to ensure a 24-hour cycle, matching the natural day-night cycle. However, the circadian period is altered upon experimental perturbation of GRN components, indicating that the circadian GRN controls the period of oscillation. Similarly, perturbation of the cell-cycle GRN alters the cell-cycle period, indicating that the cell-cycle period could similarly be controlled by its regulatory network. Using the computational tools outlined in Chapter 2, 3, and 4, Chapter 5 of this dissertation seeks to understand the mechanisms by which the cell-cycle period slows in response to unfavorable growth conditions. I propose a network model for cell-cycle period control, consisting of stress-regulatory interactions with the cell-cycle GRN. These regulatory models serve as experimental guidance, thus enabling more rapid identification of regulatory mechanisms for complex and high-dimensional biological processes.