Functional Annotation of Bacterial Signal Transduction Systems: Progress and Challenges

Abstract

Bacteria possess a large number of signal transduction systems that sense and respond to different environmental cues. Most frequently these are transcriptional regulators, two-component systems and chemosensory pathways. A major bottleneck in the field of signal transduction is the lack of information on signal molecules that modulate the activity of the large majority of these systems. We review here the progress made in the functional annotation of sensor proteins using high-throughput ligand screening approaches of purified sensor proteins or individual ligand binding domains. In these assays, the alteration in protein thermal stability following ligand binding is monitored using Differential Scanning Fluorimetry. We illustrate on several examples how the identification of the sensor protein ligand has facilitated the elucidation of the molecular mechanism of the regulatory process. We will also discuss the use of virtual ligand screening approaches to identify sensor protein ligands. Both approaches have been successfully applied to functionally annotate a significant number of bacterial sensor proteins but can also be used to study proteins from other kingdoms. The major challenge consists in the study of sensor proteins that do not recognize signal molecules directly, but that are activated by signal molecule-loaded binding proteins.

Hybrid Two-Component Sensors for Identification of Bacterial Chemoreceptor Function

“…Genetic approaches, such as the generation of chemoreceptor mutants, can be difficult depending on the bacterial species and whether the strain of interest is amenable to genetic manipulation. In many cases, MCPs are functionally redundant, which can mask the phenotype of single mutants (16), which was the case with propionate detection by P. putida F1 as shown here. Recently, a fluorescence-based thermal shift assay was developed to rapidly link the LBDs of MCPs to their ligands (31).…”

“…To date, relatively few MCPs and MCP-like proteins from soil bacteria have been characterized, but ligands for several MCPs from various pseudomonads and rhizobia have been characterized in recent years (14,16). Although there are numerous known attractants and repellents for various soil bacteria, the relevant chemoreceptors often remain unidentified due to various technical constraints in current methods (16). Previous studies have utilized the construction of hybrid receptors to help elucidate the protein function of E. coli MCPs (17,18), E. coli Nar proteins (3), and Nar-MCP chimeras (19,20).…”

The use of isothermal titration calorimetry to unravel chemotactic signalling mechanisms

“…Identification of novel chemoreceptors by the combined use of thermal shift assays and ITC. A powerful approach that has accelerated the identification of novel chemoreceptors consists in the combined use of thermal shift assays (TSA) and isothermal titration calorimetry (McKellar et al, 2015;Fernández et al, 2016;Martin-Mora et al, 2018b). Ligand binding to proteins does typically increase the T m value or the midpoint of protein unfolding transition (Chiu and Prenner, 2011).…”

Unraveling the role of the CbrA histidine kinase in the signal transduction of the CbrAB two-component system in Pseudomonas putida

“…Two-component systems (TCS) are one of the main mechanisms of signal transduction for bacteria to sense physicochemical and biological constraints to adjust their gene expression program and to respond to changing conditions properly. TCS regulate chemotaxis, sporulation, nutrient uptake and utilization among other important cellular processes 1,2 . The prototypical TCS consists of a membrane-bound sensor histidine kinase (HK) and a cytosolic response regulator (RR), which interacts with promoter regions to modulate DNA transcription.…”

“…Signal transduction processes are typically initiated by the interaction of signal molecules with the sensor domains of the histidine kinases, which leads to an alteration of their autokinase activity and subsequently a change in the phosphorylation state of their RR partners, although a variety of alternative modes have evolved 3,4 . The enormous diversity of environmental signals and the fact that its perception has evolved through diverse mechanisms results in relatively few signals being identified in TCS systems 2,5 . Amongst the wide variety of sensor domains (more than 14 different types have been described in HKs), PAS domains are most frequently found.…”