A Rationally Designed c-di-AMP Förster Resonance Energy Transfer Biosensor To Monitor Nucleotide Dynamics.

3'3'-Cyclic di-AMP (c-di-AMP) is an important nucleotide second messenger found throughout the bacterial domain of life. c-di-AMP is essential in many bacteria and regulates a diverse array of effector proteins controlling pathogenesis, cell wall homeostasis, osmoregulation, and central metabolism. Despite the ubiquity and importance of c-di-AMP, methods to detect this signaling molecule are limited, particularly at single-cell resolution. In this work, crystallization of the Listeria monocytogenes c-di-AMP effector protein Lmo0553 enabled structure-guided design of a Förster resonance energy transfer (FRET)-based biosensor, which we have named CDA5. CDA5 is a fully genetically encodable, specific, and reversible biosensor which allows the detection of c-di-AMP dynamics both in vitro and within live cells in a nondestructive manner. Our initial studies identified a distribution of c-di-AMP in Bacillus subtilis populations first grown in Luria broth and then resuspended in diluted Luria broth compatible with fluorescence analysis. Furthermore, we found that B. subtilis mutants lacking either a c-di-AMP phosphodiesterase and cyclase have higher and lower FRET responses, respectively. These findings provide novel insight into the c-di-AMP distribution within bacterial populations and establish CDA5 as a powerful platform for characterizing new aspects of c-di-AMP regulation. IMPORTANCE c-di-AMP is an important nucleotide second messenger for which detection methods are severely limited. In this work we engineered and implemented a c-di-AMP-specific FRET biosensor to remedy this dearth. We present this biosensor, CDA5, as a versatile tool to investigate previously intractable facets of c-di-AMP biology.

In Vitro Methods for the Investigation of sRNA-mRNA Interactions in Bacillus subtilis.

So far, in Bacillus subtilis, only four trans-encoded and 11 cis-encoded sRNAs and their targets have been investigated in detail, the majority of them in our group (rev. in 1, 2). Here, we describe in vitro methods for the analysis of sRNA/mRNA interactions. All these methods have been either elaborated or significantly improved in our group and successfully applied to characterize a number of sRNA/target mRNA systems in Bacillus subtilis for which we provide examples from our own work. The in vitro methods comprise the synthesis and purification of labeled and unlabeled RNA, the analysis of sRNA/mRNA interactions in electrophoretic mobility shift assays (EMSAs) including the calculation of their apparent binding rate constants (kapp) and equilibrium dissociation constants (Kd), the localization of minimal regulatory regions of an sRNA, the determination of the secondary structures of both interacting RNAs and their complex as well as the analysis of RNA chaperones that may promote the sRNA/mRNA interaction.

A mass spectrometry-based non-radioactive differential radial capillary action of ligand assay (DRaCALA) to assess ligand binding to proteins.

Binding of ligands to macromolecules changes their physicochemical and enzymatic characteristics. Cyclic di-GMP is a second messenger involved in motility/sessility and acute/chronic infection life style transition. Although the GGDEF domain, predominantly a diguanylate cyclase, represents one of the most abundant bacterial domain superfamilies, the number of cyclic di-GMP receptors falls short. To facilitate screening for cyclic di-nucleotide binding proteins, we describe a non-radioactive, matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF)-based modification of the widely applied differential radial capillary action of ligand assay (DRaCALA). The results of this assay suggest that the diguanylate cyclase/phosphodiesterase variant YciRFec101 , but not selected catalytic mutants, bind cyclic di-GMP. HIGHLIGHTS: Cyclic di-nucleotides are ubiquitous second messengers in bacteria. However, few receptors have been identified. Previous screening of cell lysates by differential radial capillary action of ligand assay (DRaCALA) using radioactive ligand identified cyclic di-nucleotide binding proteins. A MALDI-TOF-based DRaCALA was developed to detect cyclic di-nucleotide binding as a non-radioactive alternative. Known cyclic di-GMP binding proteins were verified and potential cyclic di-GMP binding proteins were identified.

Novel (p)ppGpp Binding and Metabolizing Proteins of Escherichia coli.

The alarmone (p)ppGpp plays pivotal roles in basic bacterial stress responses by increasing tolerance of various nutritional limitations and chemical insults, including antibiotics. Despite intensive studies since (p)ppGpp was discovered over 4 decades ago, (p)ppGpp binding proteins have not been systematically identified in Escherichia coli We applied DRaCALA (differential radial capillary action of ligand assay) to identify (p)ppGpp-protein interactions. We discovered 12 new (p)ppGpp targets in E. coli that, based on their physiological functions, could be classified into four major groups, involved in (i) purine nucleotide homeostasis (YgdH), (ii) ribosome biogenesis and translation (RsgA, Era, HflX, and LepA), (iii) maturation of dehydrogenases (HypB), and (iv) metabolism of (p)ppGpp (MutT, NudG, TrmE, NadR, PhoA, and UshA). We present a comprehensive and comparative biochemical and physiological characterization of these novel (p)ppGpp targets together with a comparative analysis of relevant, known (p)ppGpp binding proteins. Via this, primary targets of (p)ppGpp in E. coli are identified. The GTP salvage biosynthesis pathway and ribosome biogenesis and translation are confirmed as targets of (p)ppGpp that are highly conserved between E. coli and Firmicutes In addition, an alternative (p)ppGpp degradative pathway, involving NudG and MutT, was uncovered. This report thus significantly expands the known cohort of (p)ppGpp targets in E. coliIMPORTANCE Antibiotic resistance and tolerance exhibited by pathogenic bacteria have resulted in a global public health crisis. Remarkably, almost all bacterial pathogens require the alarmone (p)ppGpp to be virulent. Thus, (p)ppGpp not only induces tolerance of nutritional limitations and chemical insults, including antibiotics, but is also often required for induction of virulence genes. However, understanding of the molecular targets of (p)ppGpp and the mechanisms by which (p)ppGpp influences bacterial physiology is incomplete. In this study, a systematic approach was used to uncover novel targets of (p)ppGpp in E. coli, the best-studied model bacterium. Comprehensive comparative studies of the targets revealed conserved target pathways of (p)ppGpp in both Gram-positive and -negative bacteria and novel targets of (p)ppGpp, including an alternative degradative pathway of (p)ppGpp. Thus, our discoveries may help in understanding of how (p)ppGpp increases the stress resilience and multidrug tolerance not only of the model organism E. coli but also of the pathogenic organisms in which these targets are conserved.

Differential Radial Capillary Action of Ligand Assay (DRaCALA) for High-Throughput Detection of Protein-Metabolite Interactions in Bacteria.

Bacteria rely on numerous nucleotide second messengers for signal transduction such as cyclic AMP, cyclic-di-GMP, and cyclic-di-AMP. Although a number of receptors responsible for known regulated phenotypes have been established, the completeness of protein receptors in any given organism remains elusive. We have developed a method called differential radial capillary action of ligand assay (DRaCALA) that allows for an unbiased, systematic high-throughput screen for the detection of ligand binding proteins encoded by a genome. DRaCALA permits interrogation of ligand binding directly to an overexpressed protein in a cell lysate and bypasses the need of protein purification. Gateway-cloning-compatible open reading frame libraries are available for a diverse range of bacterial species and permits generation of the lysates overexpressing each open reading frame. These lysates can be assessed by DRaCALA in a 96-well format to allow rapid identification of protein-ligand interactions, including previously unknown proteins. Here, we present the protocols for generating the expression library, conducting the DRaCALA screen, data analysis, and hit validation.

Assessing RNA interactions with proteins by DRaCALA.

Discovery of RNA elements, including riboswitches and regulatory RNAs, has revealed additional regulatory mechanisms for transcript stability, transcript termination, and translational initiation. These regulatory RNA molecules act through direct binding to cellular targets including other RNA molecules, proteins, and low molecular weight metabolites. RNA-RNA interactions based on complementarity can be identified through bioinformatic analysis. However, identification of novel interactions between these regulatory RNA molecules and their partners other than complementary sequences is more challenging. We have developed a technique called Differential Radial Capillary Action of Ligand Assay (DRaCALA) to facilitate the detection of direct binding between RNA elements to proteins or low molecular weight ligands. Previously, we have described the adaptation of this technique to detect the binding interaction between Vc2 riboswitch to a signaling cyclic dinucleotide called cyclic-di-GMP. Here, we describe the adaptation of DRaCALA for identifying sequence-specific RNA-binding proteins directly from E. coli cell lysates expressing the recombinant binding protein. DRaCALA can be used to qualitatively and quantitatively assess RNA-protein interaction in whole cell lysate, determine the kinetics of the binding, and test for competitors. Using DRaCALA in a high-throughput format has the potential to rapidly identify sequence-specific RNA-binding proteins.