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.

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.

BackgroundRapidly 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.ResultsWe 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.ConclusionsSimple 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.

Xylella fastidiosa is a phytopathogenic bacterium that causes serious diseases in a wide range of economically important crops. Despite extensive comparative analyses of genome sequences of Xylella pathogenic strains from different plant hosts, nonpathogenic strains have not been studied. In this report, we show that X. fastidiosa strain J1a12, associated with citrus variegated chlorosis (CVC), is nonpathogenic when injected into citrus and tobacco plants. Furthermore, a DNA microarray-based comparison of J1a12 with 9a5c, a CVC strain that is highly pathogenic and had its genome completely sequenced, revealed that 14 coding sequences of strain 9a5c are absent or highly divergent in strain J1a12. Among them, we found an arginase and a fimbrial adhesin precursor of type III pilus, which were confirmed to be absent in the nonpathogenic strain by PCR and DNA sequencing. The absence of arginase can be correlated to the inability of J1a12 to multiply in host plants. This enzyme has been recently shown to act as a bacterial survival mechanism by down-regulating host nitric oxide production. The lack of the adhesin precursor gene is in accordance with the less aggregated phenotype observed for J1a12 cells growing in vitro. Thus, the absence of both genes can be associated with the failure of the J1a12 strain to establish and spread in citrus and tobacco plants. These results provide the first detailed comparison between a nonpathogenic strain and a pathogenic strain of X. fastidiosa, constituting an important step towards understanding the molecular basis of the disease.

Xylella fastidiosa is a phytopathogenic bacterium that is responsible for diseases in many economically important crops. Although different strains have been studied, little is known about X. fastidiosa stress responses. One of the better characterized stress responses in bacteria is the heat shock response, which induces the expression of specific genes to prevent protein misfolding and aggregation and to promote degradation of the irreversibly denatured polypeptides. To investigate X. fastidiosa genes involved in the heat shock response, we performed a whole-genome microarray analysis in a time course experiment. Globally, 261 genes were induced (9.7%) and 222 genes were repressed (8.3%). The expression profiles of the differentially expressed genes were grouped, and their expression patterns were validated by quantitative reverse transcription-PCR experiments. We determined the transcription start sites of six heat shock-inducible genes and analyzed their promoter regions, which allowed us to propose a putative consensus for 32 promoters in Xylella and to suggest additional genes as putative members of this regulon. Besides the induction of classical heat shock protein genes, we observed the up-regulation of virulence-associated genes such as vapD and of genes for hemagglutinins, hemolysin, and xylan-degrading enzymes, which may indicate the importance of heat stress to bacterial pathogenesis. In addition, we observed the repression of genes related to fimbriae, aerobic respiration, and protein biosynthesis and the induction of genes related to the extracytoplasmic stress response and some phage-related genes, revealing the complex network of genes that work together in response to heat shock.

Fbxo7 is a clinically relevant F-box protein, associated with both cancer and Parkinson's disease (PD). Additionally, SNPs within FBXO7 are correlated with alterations in red blood cell parameters. Point mutations within FBXO7 map within specific functional domains, including near its F-box domain and its substrate recruiting domains, suggesting that deficiencies in SCFFbxo7/PARK15 ubiquitin ligase activity are mechanistically linked to early-onset PD. To date, relatively few substrates of the ligase have been identified. These include HURP (hepatoma up-regulated protein), whose ubiquitination results in proteasome-mediated degradation, and c-IAP1 (inhibitor of apoptosis protein 1), TNF receptor-associated factor 2 (TRAF2), and NRAGE, which are not destabilized as a result of ubiquitination. None of these substrates have been linked directly to PD, nor has it been determined whether they would directly engage neuronal cell death pathways. To discover ubiquitinated substrates of SCFFbxo7 implicated more directly in PD aetiology, we conducted a high-throughput screen using protein arrays to identify new candidates. A total of 338 new targets were identified and from these we validated glycogen synthase kinase 3β (Gsk3β), which can phosphorylate α-synuclein, and translocase of outer mitochondrial membrane 20 (Tomm20), a mitochondrial translocase that, when ubiquitinated, promotes mitophagy, as SCFFbxo7 substrates both in vitro and in vivo. Ubiquitin chain restriction analyses revealed that Fbxo7 modified Gsk3β using K63 linkages. Our results indicate that Fbxo7 negatively regulates Gsk3β activity, rather than its levels or localization. In addition, Fbxo7 ubiquitinated Tomm20, and its levels correlated with Fbxo7 expression, indicating a stabilizing effect. None of the PD-associated mutations in Fbxo7 impaired Tomm20 ubiquitination. Our findings demonstrate that SCFFbxo7 has an impact directly on two proteins implicated in pathological processes leading to PD.

The relatively small numbers of proteins and fewer possible posttranslational modifications in microbes provides a unique opportunity to comprehensively characterize their dynamic proteomes. We have constructed a Peptide Atlas (PA) for 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 helped highlight 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.

Background: High-density tiling arrays and new sequencing technologies are generating rapidly increasing volumes of transcriptome and protein-DNA interaction data. Visualization and exploration of this data is critical to understanding the regulatory logic encoded in the genome by which the cell dynamically affects its physiology and interacts with its environment. Results:The Gaggle Genome Browser is a cross-platform desktop program for interactively visualizing highthroughput data in the context of the genome. Important features include dynamic panning and zooming, keyword search and open interoperability through the Gaggle framework. Users may bookmark locations on the genome with descriptive annotations and share these bookmarks with other users. The program handles large sets of usergenerated data using an in-process database and leverages the facilities of SQL and the R environment for importing and manipulating data.