Integrative FourD omics approach profiles the target network of the carbon storage regulatory system.

Multi-target regulators represent a largely untapped area for metabolic engineering and anti-bacterial development. These regulators are complex to characterize because they often act at multiple levels, affecting proteins, transcripts and metabolites. Therefore, single omics experiments cannot profile their underlying targets and mechanisms. In this work, we used an Integrative FourD omics approach (INFO) that consists of collecting and analyzing systems data throughout multiple time points, using multiple genetic backgrounds, and multiple omics approaches (transcriptomics, proteomics and high throughput sequencing crosslinking immunoprecipitation) to evaluate simultaneous changes in gene expression after imposing an environmental stress that accentuates the regulatory features of a network. Using this approach, we profiled the targets and potential regulatory mechanisms of a global regulatory system, the well-studied carbon storage regulatory (Csr) system of Escherichia coli, which is widespread among bacteria. Using 126 sets of proteomics and transcriptomics data, we identified 136 potential direct CsrA targets, including 50 novel ones, categorized their behaviors into distinct regulatory patterns, and performed in vivo fluorescence-based follow up experiments. The results of this work validate 17 novel mRNAs as authentic direct CsrA targets and demonstrate a generalizable strategy to integrate multiple lines of omics data to identify a core pool of regulator targets.

Advances in synthetic dynamic circuits design: using novel synthetic parts to engineer new generations of gene oscillations.

As bioengineering applications expand, the need to design and implement circuits that exhibit dynamic properties increases. In particular, schemes that control precise patterns of gene expression as a function of time are essential for balancing multiple metabolic objectives in natural and synthetic systems. Given that modularity has been an important component of dynamic circuits, recent efforts to improve dynamic circuits have focused on replacing old parts for new components that increase the robustness, stability, and tunability. In this review, we show that incorporation of novel components such as regulatory noncoding RNAs (ncRNAs), promoter-transcription factor pairs, and metabolite sensors have allowed traditional dynamic circuits to obtain more robust functionality and improved dynamic properties.

Adaptation of Tri-molecular fluorescence complementation allows assaying of regulatory Csr RNA-protein interactions in bacteria.

sRNAs play a significant role in controlling and regulating cellular metabolism. One of the more interesting aspects of certain sRNAs is their ability to make global changes in the cell by interacting with regulatory proteins. In this work, we demonstrate the use of an in vivo Tri-molecular Fluorescence Complementation assay to detect and visualize the central regulatory sRNA-protein interaction of the Carbon Storage Regulatory system in E. coli. The Carbon Storage Regulator consists primarily of an RNA binding protein, CsrA, that alters the activity of mRNA targets and of an sRNA, CsrB, that modulates the activity of CsrA. We describe the construction of a fluorescence complementation system that detects the interactions between CsrB and CsrA. Additionally, we demonstrate that the intensity of the fluorescence of this system is able to detect changes in the affinity of the CsrB-CsrA interaction, as caused by mutations in the protein sequence of CsrA. While previous methods have adopted this technique to study mRNA or RNA localization, this is the first attempt to use this technique to study the sRNA-protein interaction directly in bacteria. This method presents a potentially powerful tool to study complex bacterial RNA protein interactions in vivo.

Group II intron-ribosome association protects intron RNA from degradation.

The influence of the cellular environment on the structures and properties of catalytic RNAs is not well understood, despite great interest in ribozyme function. Here we report on ribosome association of group II introns, which are ribozymes that are important because of their putative ancestry to spliceosomal introns and retrotransposons, their retromobility via an RNA intermediate, and their application as gene delivery agents. We show that group II intron RNA, in complex with the intron-encoded protein from the native Lactoccocus lactis host, associates strongly with ribosomes in vivo. Ribosomes have little effect on intron ribozyme activities; rather, the association with host ribosomes protects the intron RNA against degradation by RNase E, an enzyme previously shown to be a silencer of retromobility in Escherichia coli. The ribosome interacts strongly with the intron, exerting protective effects in vivo and in vitro, as demonstrated by genetic and biochemical experiments. These results are consistent with the ribosome influencing the integrity of catalytic RNAs in bacteria in the face of degradative nucleases that regulate intron mobility.

Discovery of posttranscriptional regulatory RNAs using next generation sequencing technologies.

Next generation sequencing (NGS) has revolutionized the way by which we engineer metabolism by radically altering the path to genome-wide inquiries. This is due to the fact that NGS approaches offer several powerful advantages over traditional methods that include the ability to fully sequence hundreds to thousands of genes in a single experiment and simultaneously detect homozygous and heterozygous deletions, alterations in gene copy number, insertions, translocations, and exome-wide substitutions that include "hot-spot mutations." This chapter describes the use of these technologies as a sequencing technique for transcriptome analysis and discovery of regulatory RNA elements in the context of three main platforms: Illumina HiSeq, 454 pyrosequencing, and SOLiD sequencing. Specifically, this chapter focuses on the use of Illumina HiSeq, since it is the most widely used platform for RNA discovery and transcriptome analysis. Regulatory RNAs have now been found in all branches of life. In bacteria, noncoding small RNAs (sRNAs) are involved in highly sophisticated regulatory circuits that include quorum sensing, carbon metabolism, stress responses, and virulence (Gorke and Vogel, Gene Dev 22:2914-2925, 2008; Gottesman, Trends Genet 21:399-404, 2005; Romby et al., Curr Opin Microbiol 9:229-236, 2006). Further characterization of the underlying regulation of gene expression remains poorly understood given that it is estimated that over 60% of all predicted genes remain hypothetical and the 5' and 3' untranslated regions are unknown for more than 90% of the genes (Siegel et al., Trends Parasitol 27:434-441, 2011). Importantly, manipulation of the posttranscriptional regulation that occurs at the level of RNA stability and export, trans-splicing, polyadenylation, protein translation, and protein stability via untranslated regions (Clayton, EMBO J 21:1881-1888, 2002; Haile and Papadopoulou, Curr Opin Microbiol 10:569-577, 2007) could be highly beneficial to metabolic engineering.