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Histidine kinases (HKs) are sensor proteins found ubiquitously in prokaryotes. They are the first protein in two-component systems (TCSs), signaling pathways that respond to a myriad of environmental stimuli. TCSs are typically comprised of a HK and its cognate response regulator (RR) which often acts as a transcription factor. RRs will bind DNA and ultimately lead to a cellular response. These cellular outputs vary widely, but HKs are particularly interesting as they are tied to antibiotic resistance and virulence pathways in pathogenic bacteria, making them promising drug targets. We anticipate that HK inhibitors could serve as either standalone antibiotics or antivirulence therapies. Additionally, while the cellular response mediated by the HKs is often well-characterized, very little is known about which stimuli trigger the sensor kinase to begin the phosphorylation cascade. Studying HK activity and enrichment of active HKs through activity-based protein profiling will enable these stimuli to be elucidated, filling this fundamental gap in knowledge. Here, we describe methods to evaluate the potency of putative HK inhibitors in addition to methods to calculate kinetic parameters of various activity-based probes designed for the HKs.
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ELIHKSIR Web Server: Evolutionary Links Inferred for Histidine Kinase Sensors Interacting with Response Regulators
Two-component systems (TCS) are signaling machinery that consist of a histidine kinases (HK) and response regulator (RR). When an environmental change is detected, the HK phosphorylates its cognate response regulator (RR). While cognate interactions were considered orthogonal, experimental evidence shows the prevalence of crosstalk interactions between non-cognate HK–RR pairs. Currently, crosstalk interactions have been demonstrated for TCS proteins in a limited number of organisms. By providing specificity predictions across entire TCS networks for a large variety of organisms, the ELIHKSIR web server assists users in identifying interactions for TCS proteins and their mutants. To generate specificity scores, a global probabilistic model was used to identify interfacial couplings and local fields from sequence information. These couplings and local fields were then used to construct Hamiltonian scores for positions with encoded specificity, resulting in the specificity score. These methods were applied to 6676 organisms available on the ELIHKSIR web server. Due to the ability to mutate proteins and display the resulting network changes, there are nearly endless combinations of TCS networks to analyze using ELIHKSIR. The functionality of ELIHKSIR allows users to perform a variety of TCS network analyses and visualizations to support TCS research efforts.
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- Award ID(s):
- 1943442
- PAR ID:
- 10300761
- Date Published:
- Journal Name:
- Entropy
- Volume:
- 23
- Issue:
- 2
- ISSN:
- 1099-4300
- Page Range / eLocation ID:
- 170
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Spiers, Andrew (Ed.)Introduction:Two-component response systems (TCRS) are the main mechanism by which prokaryotes acclimate to changing environments. These systems are composed of a membrane bound histidine kinase (HK) that senses external signals and a response regulator (RR) that activates transcription of response genes. Despite their known role in acclimation, little is known about the role TCRS play in environmental adaptation. Several experimental evolution studies have shown the acquisition of mutations in TCRS during adaptation, therefore here we set out to characterize the adaptive mechanism resulting from these mutations and evaluate whether single nucleotide changes in one gene could induce variable genotype-by-environment (GxE) interactions. Methods:To do this, we assessed fitness changes and differential gene expression for four adaptive mutations incusS, the gene that encodes the HK CusS,acquired byEscherichia coliduring silver adaptation. Results:Fitness assays showed that as the environment changed, each mutant displayed a unique fitness profile with greatest fitness in the original selection environment. RNAseq then indicated that, in ± silver nitrate, each mutant induces a primary response that upregulatescusS,its RRcusR, and constitutively expresses the target response genescusCFBA. This then induces a secondary response via differential expression of genes regulated by the CusR through TCRS crosstalk. Finally, each mutant undergoes fitness tuning through unique tertiary responses that result in gene expression patterns specific for the genotype, the environment and optimized for the original selection conditions. Discussion:This three-step response shows that different mutations in a single gene leads to individualized phenotypes governed by unique GxE interactions that not only contribute to transcriptional divergence but also to phenotypic plasticity.more » « less
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/e23020170
