Amy K. Schmid

A Predictive Model for Transcriptional Control of Physiology in a Free Living Cell

The environment significantly influences the dynamic expression and assembly of all components encoded in the genome of an organism into functional biological networks. We have constructed a model for this process in Halobacterium salinarum NRC-1 through the data-driven discovery of regulatory and functional interrelationships among approximately 80% of its genes and key abiotic factors in its hypersaline environment. Using relative changes in 72 transcription factors and 9 environmental factors (EFs) this model accurately predicts dynamic transcriptional responses of all these genes in 147 newly collected experiments representing completely novel genetic backgrounds and environments-suggesting a remarkable degree of network completeness. Using this model we have constructed and tested hypotheses critical to this organisms interaction with its changing hypersaline environment. This study supports the claim that the high degree of connectivity within biological and EF networks will enable the construction of similar models for any organism from relatively modest numbers of experiments.

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.

A single transcription factor regulates evolutionarily diverse but functionally linked metabolic pathways in response to nutrient availability

During evolution, enzyme‐coding genes are acquired and/or replaced through lateral gene transfer and compiled into metabolic pathways. Gene regulatory networks evolve to fine tune biochemical fluxes through such metabolic pathways, enabling organisms to acclimate to nutrient fluctuations in a competitive environment. Here, we demonstrate that a single TrmB family transcription factor in Halobacterium salinarum NRC‐1 globally coordinates functionally linked enzymes of diverse phylogeny in response to changes in carbon source availability. Specifically, during nutritional limitation, TrmB binds a cis‐regulatory element to activate or repress 113 promoters of genes encoding enzymes in diverse metabolic pathways. By this mechanism, TrmB coordinates the expression of glycolysis, TCA cycle, and amino‐acid biosynthesis pathways with the biosynthesis of their cognate cofactors (e.g. purine and thiamine). Notably, the TrmB‐regulated metabolic network includes enzyme‐coding genes that are uniquely archaeal as well as those that are conserved across all three domains of life. Simultaneous analysis of metabolic and gene regulatory network architectures suggests an ongoing process of co‐evolution in which TrmB integrates the expression of metabolic enzyme‐coding genes of diverse origins.

The Firegoose: two-way integration of diverse data from different bioinformatics web resources with desktop applications

BackgroundInformation resources on the World Wide Web play an indispensable role in modern biology. But integrating data from multiple sources is often encumbered by the need to reformat data files, convert between naming systems, or perform ongoing maintenance of local copies of public databases. Opportunities for new ways of combining and re-using data are arising as a result of the increasing use of web protocols to transmit structured data.ResultsThe Firegoose, an extension to the Mozilla Firefox web browser, enables data transfer between web sites and desktop tools. As a component of the Gaggle integration framework, Firegoose can also exchange data with Cytoscape, the R statistical package, Multiexperiment Viewer (MeV), and several other popular desktop software tools. Firegoose adds the capability to easily use local data to query KEGG, EMBL STRING, DAVID, and other widely-used bioinformatics web sites. Query results from these web sites can be transferred to desktop tools for further analysis with a few clicks.Firegoose acquires data from the web by screen scraping, microformats, embedded XML, or web services. We define a microformat, which allows structured information compatible with the Gaggle to be embedded in HTML documents.We demonstrate the capabilities of this software by performing an analysis of the genes activated in the microbe Halobacterium salinarum NRC-1 in response to anaerobic environments. Starting with microarray data, we explore functions of differentially expressed genes by combining data from several public web resources and construct an integrated view of the cellular processes involved.ConclusionThe Firegoose incorporates Mozilla Firefox into the Gaggle environment and enables interactive sharing of data between diverse web resources and desktop software tools without maintaining local copies. Additional web sites can be incorporated easily into the framework using the scripting platform of the Firefox browser. Performing data integration in the browser allows the excellent search and navigation capabilities of the browser to be used in combination with powerful desktop tools.

Two transcription factors are necessary for iron homeostasis in a salt-dwelling archaeon

Because iron toxicity and deficiency are equally life threatening, maintaining intracellular iron levels within a narrow optimal range is critical for nearly all known organisms. However, regulatory mechanisms that establish homeostasis are not well understood in organisms that dwell in environments at the extremes of pH, temperature, and salinity. Under conditions of limited iron, the extremophile Halobacterium salinarum, a salt-loving archaeon, mounts a specific response to scavenge iron for growth. We have identified and characterized the role of two transcription factors (TFs), Idr1 and Idr2, in regulating this important response. An integrated systems analysis of TF knockout gene expression profiles and genome-wide binding locations in the presence and absence of iron has revealed that these TFs operate collaboratively to maintain iron homeostasis. In the presence of iron, Idr1 and Idr2 bind near each other at 24 loci in the genome, where they are both required to repress some genes. By contrast, Idr1 and Idr2 are both necessary to activate other genes in a putative a feed forward loop. Even at loci bound independently, the two TFs target different genes with similar functions in iron homeostasis. We discuss conserved and unique features of the Idr1–Idr2 system in the context of similar systems in organisms from other domains of life.

Halobacterium salinarum NRC-1 PeptideAtlas: Toward Strategies for Targeted Proteomics and Improved Proteome Coverage

The relatively small numbers of proteins and fewer possible post-translational modifications in microbes provide a unique opportunity to comprehensively characterize their dynamic proteomes. We have constructed a PeptideAtlas (PA) covering 62.7% of the predicted proteome of the extremely halophilic archaeon Halobacterium salinarum NRC-1 by compiling approximately 636 000 tandem mass spectra from 497 mass spectrometry runs in 88 experiments. Analysis of the PA with respect to biophysical properties of constituent peptides, functional properties of parent proteins of detected peptides, and performance of different mass spectrometry approaches has highlighted plausible strategies for improving proteome coverage and selecting signature peptides for targeted proteomics. Notably, discovery of a significant correlation between absolute abundances of mRNAs and proteins has helped identify low abundance of proteins as the major limitation in peptide detection. Furthermore, we have discovered that iTRAQ labeling for quantitative proteomic analysis introduces a significant bias in peptide detection by mass spectrometry. Therefore, despite identifying at least one proteotypic peptide for almost all proteins in the PA, a context-dependent selection of proteotypic peptides appears to be the most effective approach for targeted proteomics.

Genome-Wide Assessment of Outer Membrane Vesicle Production in Escherichia coli

The production of outer membrane vesicles by Gram-negative bacteria has been well documented; however, the mechanism behind the biogenesis of these vesicles remains unclear. Here a high-throughput experimental method and systems-scale analysis was conducted to determine vesiculation values for the whole genome knockout library of Escherichia coli mutant strains (Keio collection). The resultant dataset quantitatively recapitulates previously observed phenotypes and implicates nearly 150 new genes in the process of vesiculation. Gene functional and biochemical pathway analyses suggest that mutations that truncate outer membrane structures such as lipopolysaccharide and enterobacterial common antigen lead to hypervesiculation, whereas mutants in oxidative stress response pathways result in lower levels. This study expands and refines the current knowledge regarding the cellular pathways required for outer membrane vesiculation in E. coli.

HspR is a global negative regulator of heat shock gene expression in Deinococcus radiodurans

The HspR protein functions as a negative regulator of chaperone and protease gene expression in a diversity of bacteria. Here we have identified, cloned and deleted the Deinococcus radiodurans HspR homologue, DR0934. ΔhspR mutants exhibit moderate growth defects when shifted to mild heat shock temperatures, but are severely impaired for survival at 48°C. Using quantitative reverse transcription polymerase chain reaction and global transcriptional analysis, we have identified 14 genes that are derepressed in the absence of stress in the ΔhspR background, 11 of which encode predicted chaperones and proteases, including dnaKJgrpE, ftsH, lonB, hsp20 and clpB. Promoter mapping indicated that the transcription of these genes initiates from a promoter bearing a σ70‐type consensus, and that putative HspR binding sites (HAIR) were present in the 5′‐untranslated regions. Electrophoretic mobility shift assays indicated that HspR binds to these promoters at the HAIR site in vitro. These results strongly suggest that DR0934 encodes the HspR‐like global negative regulator of D. radiodurans that directly represses chaperone and protease gene expression by binding to the HAIR site in close proximity to promoter regions.

Halobacterium salinarum NRC-1 PeptideAtlas: strategies for targeted proteomics

The relatively small numbers of proteins and fewer possible post-translational modifications in microbes provide a unique opportunity to comprehensively characterize their dynamic proteomes. We have constructed a PeptideAtlas (PA) covering 62.7% of the predicted proteome of the extremely halophilic archaeon Halobacterium salinarum NRC-1 by compiling approximately 636 000 tandem mass spectra from 497 mass spectrometry runs in 88 experiments. Analysis of the PA with respect to biophysical properties of constituent peptides, functional properties of parent proteins of detected peptides, and performance of different mass spectrometry approaches has highlighted plausible strategies for improving proteome coverage and selecting signature peptides for targeted proteomics. Notably, discovery of a significant correlation between absolute abundances of mRNAs and proteins has helped identify low abundance of proteins as the major limitation in peptide detection. Furthermore, we have discovered that iTRAQ labeling for quantitative proteomic analysis introduces a significant bias in peptide detection by mass spectrometry. Therefore, despite identifying at least one proteotypic peptide for almost all proteins in the PA, a context-dependent selection of proteotypic peptides appears to be the most effective approach for targeted proteomics.

Global Transcriptional and Proteomic Analysis of the Sig1 Heat Shock Regulon of Deinococcus radiodurans

The sig1 gene, predicted to encode an extracytoplasmic function-type heat shock sigma factor of Deinococcus radiodurans, has been shown to play a central role in the positive regulation of the heat shock operons groESL and dnaKJ. To determine if Sig1 is required for the regulation of additional heat shock genes, we monitored the global transcriptional and proteomic profiles of a D. radiodurans R1 sig1 mutant and wild-type cells in response to elevated temperature stress. Thirty-one gene products were identified that showed heat shock induction in the wild type but not in the sig1 mutant. Quantitative real-time PCR experiments verified the transcriptional requirement of Sig1 for the heat shock induction of the mRNA of five of these genes-dnaK, groES, DR1314, pspA, and hsp20. hsp20 appears to encode a new member of the small heat shock protein superfamily, DR1314 is predicted to encode a hypothetical protein with no recognizable orthologs, and pspA is predicted to encode a protein involved in maintenance of membrane integrity. Deletion mutation analysis demonstrated the importance in heat shock protection of hsp20 and DR1314. The promoters of dnaKJE, groESL, DR1314, pspA, and hsp20 were mapped and, combined with computer-based pattern searches of the upstream regions of the 26 other Sig1 regulon members, these results suggested that Sig1 might recognize both sigma70-type and sigma(W)-type promoter consensus sequences. These results expand the D. radiodurans Sig1 heat shock regulon to include 31 potential new members, including not only factors with cytoplasmic functions, such as groES and dnaK, but also those with extracytoplasmic functions, like pspA.