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Yu Qiu,
Harish Nagarajan,
Mallory Embree,
Wendy Shieu,
Elisa Abate,
Katy Juárez, Byung-Kwan Cho,
James G Elkins,
Kelly P Nevin,
Christian L Barrett,
Derek R Lovley,
Bernhard O Palsson,
Karsten Zengler
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ABSTRACT: Bacteria contain multiple sigma factors, each targeting diverse, but often overlapping sets of promoters, thereby forming a complex network. The layout and deployment of such a sigma factor network directly impacts global transcriptional regulation and ultimately dictates the phenotype. Here we integrate multi-omic data sets to determine the topology, the operational, and functional states of the sigma factor network in Geobacter sulfurreducens, revealing a unique network topology of interacting sigma factors. Analysis of the operational state of the sigma factor network shows a highly modular structure with σ(N) being the major regulator of energy metabolism. Surprisingly, the functional state of the network during the two most divergent growth conditions is nearly static, with sigma factor binding profiles almost invariant to environmental stimuli. This first comprehensive elucidation of the interplay between different levels of the sigma factor network organization is fundamental to characterize transcriptional regulatory mechanisms in bacteria.
Nature Communications 04/2013; 4:1755. · 7.40 Impact Factor
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ABSTRACT: BACKGROUND: The increasing number of infections caused by strains of Klebsiella pneumoniae that are resistant to multiple antibiotics has developed into a major medical problem worldwide. The development of next-generation sequencing technologies now permits rapid sequencing of many K. pneumoniae isolates, but sequence information alone does not provide important structural and operational information for its genome. RESULTS: Here we take a systems biology approach to annotate the K. pneumoniae MGH 78578 genome at the structural and operational levels. Through the acquisition and simultaneous analysis of multiple sample-matched --omics data sets from two growth conditions, we detected 2677, 1227, and 1066 binding sites for RNA polymerase, RpoD, and RpoS, respectively, 3660 RNA polymerase-guided transcript segments, and 3585 transcription start sites throughout the genome. Moreover, analysis of the transcription start site data identified 83 probable leaderless mRNAs, while analysis of unannotated transcripts suggested the presence of 119 putative open reading frames, 15 small RNAs, and 185 antisense transcripts that are not currently annotated. CONCLUSIONS: These findings highlight the strengths of systems biology approaches to the refinement of sequence-based annotations, and to provide new insight into fundamental genome-level biology for this important human pathogen.
BMC Genomics 11/2012; 13(1):679. · 4.07 Impact Factor
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ABSTRACT: Genome-wide transcription start site (TSS) profiles of the enterobacteria Escherichia coli and Klebsiella pneumoniae were experimentally determined through modified 5' RACE followed by deep sequencing of intact primary mRNA. This identified 3,746 and 3,143 TSSs for E. coli and K. pneumoniae, respectively. Experimentally determined TSSs were then used to define promoter regions and 5' UTRs upstream of coding genes. Comparative analysis of these regulatory elements revealed the use of multiple TSSs, identical sequence motifs of promoter and Shine-Dalgarno sequence, reflecting conserved gene expression apparatuses between the two species. In both species, over 70% of primary transcripts were expressed from operons having orthologous genes during exponential growth. However, expressed orthologous genes in E. coli and K. pneumoniae showed a strikingly different organization of upstream regulatory regions with only 20% identical promoters with TSSs in both species. Over 40% of promoters had TSSs identified in only one species, despite conserved promoter sequences existing in the other species. 662 conserved promoters having TSSs in both species resulted in the same number of comparable 5' UTR pairs, and that regulatory element was found to be the most variant region in sequence among promoter, 5' UTR, and ORF. In K. pneumoniae, 48 sRNAs were predicted and 36 of them were expressed during exponential growth. Among them, 34 orthologous sRNAs between two species were analyzed in depth, and the analysis showed that many sRNAs of K. pneumoniae, including pleiotropic sRNAs such as rprA, arcZ, and sgrS, may work in the same way as in E. coli. These results reveal a new dimension of comparative genomics such that a comparison of two genomes needs to be comprehensive over all levels of genome organization.
PLoS Genetics 08/2012; 8(8):e1002867. · 8.69 Impact Factor
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ABSTRACT: Although metabolic networks have been reconstructed on a genome scale, the corresponding reconstruction and integration of governing transcriptional regulatory networks has not been fully achieved. Here we reconstruct such an integrated network for amino acid metabolism in Escherichia coli. Analysis of ChIP-chip and gene expression data for the transcription factors ArgR, Lrp and TrpR showed that 19 out of 20 amino acid biosynthetic pathways are either directly or indirectly controlled by these regulators. Classifying the regulated genes into three functional categories of transport, biosynthesis and metabolism leads to the elucidation of regulatory motifs that constitute the integrated network's basic building blocks. The regulatory logic of these motifs was determined on the basis of relationships between transcription factor binding and changes in the amount of transcript in response to exogenous amino acids. Remarkably, the resulting logic shows how amino acids are differentiated as signaling and nutrient molecules, revealing the overarching regulatory principles of the amino acid stimulon.
Nature Chemical Biology 11/2011; 8(1):65-71. · 14.69 Impact Factor
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ABSTRACT: Interactions between cis-regulatory elements and trans-acting factors are fundamental for cellular functions such as transcription. With the revolution in microarrays and sequencing technologies, genome-wide binding locations of trans-acting factors are being determined in large numbers. The richness of the genome-scale information has revealed that the nature of the bacterial transcriptome and regulome are considerably more complex than previously expected. In addition, the emerging view of the bacterial transcriptome is revising the concept of the operon organization of the genome. This review describes current advances in the genome-scale analysis of the interaction between cis-regulatory elements and trans-acting factors in microorganisms.
Biotechnology Journal 08/2011; 6(9):1052-63.
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ABSTRACT: The PurR transcription factor plays a critical role in transcriptional regulation of purine metabolism in enterobacteria. Here, we elucidate the role of PurR under exogenous adenine stimulation at the genome-scale using high-resolution chromatin immunoprecipitation (ChIP)-chip and gene expression data obtained under in vivo conditions. Analysis of microarray data revealed that adenine stimulation led to changes in transcript level of about 10% of Escherichia coli genes, including the purine biosynthesis pathway. The E. coli strain lacking the purR gene showed that a total of 56 genes are affected by the deletion. From the ChIP-chip analysis, we determined that over 73% of genes directly regulated by PurR were enriched in the biosynthesis, utilization and transport of purine and pyrimidine nucleotides, and 20% of them were functionally unknown. Compared to the functional diversity of the regulon of the other general transcription factors in E. coli, the functions and size of the PurR regulon are limited.
Nucleic Acids Research 05/2011; 39(15):6456-64. · 8.03 Impact Factor
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ABSTRACT: Immuno-precipitation of protein-DNA complexes followed by microarray hybridization is a powerful and cost-effective technology for discovering protein-DNA binding events at the genome scale. It is still an unresolved challenge to comprehensively, accurately and sensitively extract binding event information from the produced data. We have developed a novel strategy composed of an information-preserving signal-smoothing procedure, higher order derivative analysis and application of the principle of maximum entropy to address this challenge. Importantly, our method does not require any input parameters to be specified by the user. Using genome-scale binding data of two Escherichia coli global transcription regulators for which a relatively large number of experimentally supported sites are known, we show that ∼90% of known sites were resolved to within four probes, or ∼88 bp. Over half of the sites were resolved to within two probes, or ∼38 bp. Furthermore, we demonstrate that our strategy delivers significant quantitative and qualitative performance gains over available methods. Such accurate and sensitive binding site resolution has important consequences for accurately reconstructing transcriptional regulatory networks, for motif discovery, for furthering our understanding of local and non-local factors in protein-DNA interactions and for extending the usefulness horizon of the ChIP-chip platform.
Nucleic Acids Research 11/2010; 39(5):1656-65. · 8.03 Impact Factor
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ABSTRACT: Specific small deletions within the rpoC gene encoding the β'-subunit of RNA polymerase (RNAP) are found repeatedly after adaptation of Escherichia coli K-12 MG1655 to growth in minimal media. Here we present a multiscale analysis of these mutations. At the physiological level, the mutants grow 60% faster than the parent strain and convert the carbon source 15-35% more efficiently to biomass, but grow about 30% slower than the parent strain in rich medium. At the molecular level, the kinetic parameters of the mutated RNAP were found to be altered, resulting in a 4- to 30-fold decrease in open complex longevity at an rRNA promoter and a ∼10-fold decrease in transcriptional pausing, with consequent increase in transcript elongation rate. At a genome-scale, systems biology level, gene expression changes between the parent strain and adapted RNAP mutants reveal large-scale systematic transcriptional changes that influence specific cellular processes, including strong down-regulation of motility, acid resistance, fimbria, and curlin genes. RNAP genome-binding maps reveal redistribution of RNAP that may facilitate relief of a metabolic bottleneck to growth. These findings suggest that reprogramming the kinetic parameters of RNAP through specific mutations allows regulatory adaptation for optimal growth in new environments.
Proceedings of the National Academy of Sciences 11/2010; 107(47):20500-5. · 9.68 Impact Factor
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ABSTRACT: Prokaryotic genomes can be annotated based on their structural, operational, and functional properties. These annotations provide the pivotal scaffold for understanding cellular functions on a genome-scale, such as metabolism and transcriptional regulation. Here, we describe a systems approach to simultaneously determine the structural and operational annotation of the Geobacter sulfurreducens genome. Integration of proteomics, transcriptomics, RNA polymerase, and sigma factor-binding information with deep-sequencing-based analysis of primary 5'-end transcripts allowed for a most precise annotation. The structural annotation is comprised of numerous previously undetected genes, noncoding RNAs, prevalent leaderless mRNA transcripts, and antisense transcripts. When compared with other prokaryotes, we found that the number of antisense transcripts reversely correlated with genome size. The operational annotation consists of 1453 operons, 22% of which have multiple transcription start sites that use different RNA polymerase holoenzymes. Several operons with multiple transcription start sites encoded genes with essential functions, giving insight into the regulatory complexity of the genome. The experimentally determined structural and operational annotations can be combined with functional annotation, yielding a new three-level annotation that greatly expands our understanding of prokaryotic genomes.
Genome Research 09/2010; 20(9):1304-11. · 13.61 Impact Factor
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ABSTRACT: Bacterial genomes are organized by structural and functional elements, including promoters, transcription start and termination sites, open reading frames, regulatory noncoding regions, untranslated regions and transcription units. Here, we iteratively integrate high-throughput, genome-wide measurements of RNA polymerase binding locations and mRNA transcript abundance, 5' sequences and translation into proteins to determine the organizational structure of the Escherichia coli K-12 MG1655 genome. Integration of the organizational elements provides an experimentally annotated transcription unit architecture, including alternative transcription start sites, 5' untranslated region, boundaries and open reading frames of each transcription unit. A total of 4,661 transcription units were identified, representing an increase of >530% over current knowledge. This comprehensive transcription unit architecture allows for the elucidation of condition-specific uses of alternative sigma factors at the genome scale. Furthermore, the transcription unit architecture provides a foundation on which to construct genome-scale transcriptional and translational regulatory networks.
Nature Biotechnology 11/2009; 27(11):1043-9. · 29.50 Impact Factor
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ABSTRACT: In a recent issue of Molecular Cell, Vora et al. (2009) introduce an in vivo protein occupancy display (IPOD) technology and propose that the domains extensively occupied by proteins with inactive transcription in bacteria are mainly topologically isolated chromosomal domains.
Molecular cell 09/2009; 35(3):255-6. · 14.61 Impact Factor
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ABSTRACT: Transposon mutagenesis coupled with microarray analysis helps to rapidly generate information about changing genotype-phenotype relationships in laboratory-evolved bacteria.
Nature Methods 09/2009; 6(8):565-6. · 19.28 Impact Factor
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Journal of bacteriology 05/2009; 191(11):3437-44. · 3.94 Impact Factor
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ABSTRACT: Broad-acting transcription factors (TFs) in bacteria form regulons. Here, we present a 4-step method to fully reconstruct the leucine-responsive protein (Lrp) regulon in Escherichia coli K-12 MG 1655 that regulates nitrogen metabolism. Step 1 is composed of obtaining high-resolution ChIP-chip data for Lrp, the RNA polymerase and expression profiles under multiple environmental conditions. We identified 138 unique and reproducible Lrp-binding regions and classified their binding state under different conditions. In the second step, the analysis of these data revealed 6 distinct regulatory modes for individual ORFs. In the third step, we used the functional assignment of the regulated ORFs to reconstruct 4 types of regulatory network motifs around the metabolites that are affected by the corresponding gene products. In the fourth step, we determined how leucine, as a signaling molecule, shifts the regulatory motifs for particular metabolites. The physiological structure that emerges shows the regulatory motifs for different amino acid fall into the traditional classification of amino acid families, thus elucidating the structure and physiological functions of the Lrp-regulon. The same procedure can be applied to other broad-acting TFs, opening the way to full bottom-up reconstruction of the transcriptional regulatory network in bacterial cells.
Proceedings of the National Academy of Sciences 01/2009; 105(49):19462-7. · 9.68 Impact Factor
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ABSTRACT: We determined the genome-wide distribution of the nucleoid-associated protein Fis in Escherichia coli using chromatin immunoprecipitation coupled with high-resolution whole genome-tiling microarrays. We identified 894 Fis-associated regions across the E. coli genome. A significant number of these binding sites were found within open reading frames (33%) and between divergently transcribed transcripts (5%). Analysis indicates that A-tracts and AT-tracts are an important signal for preferred Fis-binding sites, and that A(6)-tracts in particular constitute a high-affinity signal that dictates Fis phasing in stretches of DNA containing multiple and variably spaced A-tracts and AT-tracts. Furthermore, we find evidence for an average of two Fis-binding regions per supercoiling domain in the chromosome of exponentially growing cells. Transcriptome analysis shows that approximately 21% of genes are affected by the deletion of fis; however, the changes in magnitude are small. To address the differential Fis bindings under growth environment perturbation, ChIP-chip analysis was performed using cells grown under aerobic and anaerobic growth conditions. Interestingly, the Fis-binding regions are almost identical in aerobic and anaerobic growth conditions-indicating that the E. coli genome topology mediated by Fis is superficially identical in the two conditions. These novel results provide new insight into how Fis modulates DNA topology at a genome scale and thus advance our understanding of the architectural bases of the E. coli nucleoid.
Genome Research 07/2008; 18(6):900-10. · 13.61 Impact Factor
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ABSTRACT: Interactions between cis-acting elements and proteins play a key role in transcriptional regulation of all known organisms. To better understand these interactions, researchers developed a method that couples chromatin immunoprecipitation with microarrays (also known as ChIP-chip), which is capable of providing a whole-genome map of protein-DNA interactions. This versatile and high-throughput strategy is initiated by formaldehyde-mediated cross-linking of DNA and proteins, followed by cell lysis, DNA fragmentation, and immunopurification. The immunoprecipitated DNA fragments are then purified from the proteins by reverse-cross-linking followed by amplification, labeling, and hybridization to a whole-genome tiling microarray against a reference sample. The enriched signals obtained from the microarray then are normalized by the reference sample and used to generate the whole-genome map of protein-DNA interactions. The protocol described here has been used for discovering the genomewide distribution of RNA polymerase and several transcription factors of Escherichia coli.
Methods in molecular biology (Clifton, N.J.) 02/2008; 439:131-45.
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ABSTRACT: Reconstruction of transcriptional regulatory and metabolic networks is the foundation of large-scale microbial systems and synthetic biology. An enormous amount of information including the annotated genomic sequences and the genomic locations of DNA-binding regulatory proteins can be used to define metabolic and regulatory networks in cells. In particular, advances in experimental methods to map regulatory networks in microbial cells have allowed reliable data-driven reconstruction of these networks. Recent work on metabolic engineering and experimental evolution of microbes highlights the key role of global regulatory networks in controlling specific metabolic processes and the need to consider the integrated function of multiple types of networks for both scientific and engineering purposes.
Current Opinion in Biotechnology 09/2007; 18(4):360-4. · 7.71 Impact Factor
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ABSTRACT: ArcA is a global transcription factor required for optimal growth of Escherichia coli during anaerobic growth. In this study, the role of ArcA on the transcriptional regulatory subnetwork of the fad regulon was investigated. Gene expression profiles of deletion mutants (Delta arcA, Delta fadR and Delta arcA/Delta fadR) indicated that (i) ArcA is a major transcription factor for the transcriptional regulation of fatty acid metabolism in the absence of oxygen, and (ii) ArcA and FadR cooperatively regulate the fad regulon under anaerobic conditions. To determine the direct interaction between ArcA and the promoters of the fad regulon genes, chromatin immunoprecipitation (ChIP) analysis was performed. ChIP analysis suggested that ArcA directly binds to the promoter regions of the fad regulon genes in vivo. An ArcA-binding motif was identified from known binding sequences and predicted putative binding sites in the promoter regions of the fad regulon genes. These results indicate that ArcA directly represses the expression of fad regulon genes during anaerobic growth.
Microbiology 09/2006; 152(Pt 8):2207-19. · 3.06 Impact Factor
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ABSTRACT: Biological discovery in the postgenomic era requires a systematic and high-throughput experimental approach. To this end, a versatile PCR-based tandem epitope tagging system is described, which inserts a tandem epitope coding sequence into any desired position of the Escherichia coli chromosome. Template plasmids were constructed that carry tandem copies of the epitope encoding sequence, Flp recombinase target (FRT) sites, and antibiotic resistance genes. The linear DNA fragment, amplified from the template plasmid with extensions homologous to the end of the target gene and part of its downstream region, was transformed into E. coli K-12 MG1655 harboring the bacteriophage gamma Red recombination system. The antibiotic resistance gene was then removed from the inserted heterologous PCR fragment using Flp recombinase. This epitope tagging system was applied to global transcription factors of E. coli to obtain proteins fused with tandem c-myc epitope tags. The tandem myc epitope-fused transcription factors were successfully detected by Western blot analysis and chromatin immunoprecipitation with increased detection sensitivity and higher yield. Higher copy numbers of the epitope molecule allowed the use of more stringent experimental conditions to increase the signal-to-noise ratio in subsequent experimental applications. Furthermore, judging from the measurement of gene expression using reverse transcription PCR (RT-PCR), the epitope-fused transcription factors retained their normal function for gene regulation in vivo.
BioTechniques 02/2006; 40(1):67-72. · 2.67 Impact Factor
