The two-component system GacS/A and the posttranscriptional control system Rsm constitute a genetic regulation pathway in Gammaproteobacteria; in some species of Pseudomonas, this pathway is part of a multikinase network (MKN) that regulates the activity of the Rsm system. In this network, the activity of GacS is controlled by other kinases. One of the most studied MKNs is the MKN-GacS of Pseudomonas aeruginosa, where GacS is controlled by the kinases RetS and LadS; RetS decreases the kinase activity of GacS, whereas LadS stimulates the activity of the central kinase GacS. Outside of the Pseudomonas genus, the network has been studied only in Azotobacter vinelandii. In this work, we report the study of the RetS kinase of A. vinelandii; as expected, the phenotypes affected in gacS mutants, such as production of alginates, polyhydroxybutyrate, and alkylresorcinols and swimming motility, were also affected in retS mutants. Interestingly, our data indicated that RetS in A. vinelandii acts as a positive regulator of GacA activity. Consistent with this finding, mutation in retS also negatively affected the expression of small regulatory RNAs belonging to the Rsm family. We also confirmed the interaction of RetS with GacS, as well as with the phosphotransfer protein HptB.

Phytochromes (Phys) are a divergent cohort of bili-proteins that detect light through reversible interconversion between dark-adapted Pr and photoactivated Pfr states. While our understandings of downstream events are emerging, it remains unclear how Phys translate light into an interpretable conformational signal. Here, we present models of both states for a dimeric Phy with histidine kinase (HK) activity from the proteobacterium Pseudomonas syringae, which were built from high-resolution cryo-EM maps (2.8-3.4-Å) of the photosensory module (PSM) and its following signaling (S) helix together with lower resolution maps for the downstream output region augmented by RoseTTAFold and AlphaFold structural predictions. The head-to-head models reveal the PSM and its photointerconversion mechanism with strong clarity, while the HK region is interpretable but relatively mobile. Pr/Pfr comparisons show that bilin phototransformation alters PSM architecture culminating in a scissoring motion of the paired S-helices linking the PSMs to the HK bidomains that ends in reorientation of the paired catalytic ATPase modules relative to the phosphoacceptor histidines. This action apparently primes autophosphorylation enroute to phosphotransfer to the cognate DNA-binding response regulator AlgB which drives quorum-sensing behavior through transient association with the photoreceptor. Collectively, these models illustrate how light absorption conformationally translates into accelerated signaling by Phy-type kinases.

Phytochromes are red-light photoreceptors discovered in plants with homologs in bacteria and fungi that regulate a variety of physiological responses. They display a reversible photocycle between two distinct states: a red-light-absorbing Pr state and a far-red light-absorbing Pfr state. The photoconversion regulates the activity of an enzymatic domain, usually a histidine kinase (HK). The molecular mechanism that explains how light controls the HK activity is not understood because structures of unmodified bacterial phytochromes with HK activity are missing. Here, we report three cryo-electron microscopy structures of a wild-type bacterial phytochrome with HK activity determined as Pr and Pfr homodimers and as a Pr/Pfr heterodimer with individual subunits in distinct states. We propose that the Pr/Pfr heterodimer is a physiologically relevant signal transduction intermediate. Our results offer insight into the molecular mechanism that controls the enzymatic activity of the HK as part of a bacterial two-component system that perceives and transduces light signals.

Bacteria rely on two-component systems to sense environmental cues and regulate gene expression for adaptation. The PhoQ/PhoP system exemplifies this crucial role, playing a key part in sensing magnesium (Mg2+) levels, antimicrobial peptides, mild acidic pH, osmotic upshift, and long-chain unsaturated fatty acids, promoting virulence in certain bacterial species. However, the precise details of PhoQ activation remain elusive. To elucidate PhoQ\'s signaling mechanism at atomic resolution, we combined AlphaFold2 predictions with molecular modeling and carried out extensive Molecular Dynamics (MD) simulations. Our MD simulations revealed three distinct PhoQ conformations that were validated by experimental data. Notably, one conformation was characterized by Mg2+ bridging the acidic patch in the sensor domain to the membrane, potentially representing a repressed state. Furthermore, the high hydration observed in a putative intermediate state lends support to the hypothesis of water-mediated conformational changes during PhoQ signaling. Our findings not only revealed specific conformations within the PhoQ signaling pathway, but also hold significant promise for understanding the broader histidine kinase family due to their shared structural features. Our approach paves the way for a more comprehensive understanding of histidine kinase signaling mechanisms across various bacterial species and opens the door for developing novel therapeutics that target PhoQ modulation.

The two-component regulatory system CenK-CenR has recently emerged as a regulator of cell envelope and cell division processes in the alpha-proteobacteria. In Sinorhizobium meliloti, CenK-CenR regulates the expression of SrlA, a thioredoxin-domain protein of unknown function. Deletion of srlA causes sensitivity to salt and oxidizing agents on solid growth medium. In this work, we report that the response regulator CenR, but not the histidine kinase CenK, is essential for cell viability in S. meliloti. We also demonstrate that phosphorylation of the target residue D55 is not required for viability, suggesting that the unphosphorylated transcription factor sufficiently regulates expression of one or more essential genes in the genome. Using transcription assays and phenotype testing we examine CenK-CenR-dependent activation of the srlA promoter and demonstrate its absolute dependence on phosphoryl-CenR for activity and that the CenR substitution D55E acts as a phosphomimetic that partially restores activity at the srlA promoter in the absence of phosphorylation by CenK. Finally, we report a mutational analysis of the CenR binding site in the srlA promoter required for transcriptional activation.

Phosphohistidine (pHis) is a reversible protein post-translational modification (PTM) that is currently poorly understood. The P-N bond in pHis is heat and acid-sensitive, making it more challenging to study than the canonical phosphoamino acids pSer, pThr, and pTyr. As advancements in the development of tools to study pHis have been made, the roles of pHis in cells are slowly being revealed. To date, a handful of enzymes responsible for controlling this modification have been identified, including the histidine kinases NME1 and NME2, as well as the phosphohistidine phosphatases PHPT1, LHPP, and PGAM5. These tools have also identified the substrates of these enzymes, granting new insights into previously unknown regulatory mechanisms. Here, we discuss the cellular function of pHis and how it is regulated on known pHis-containing proteins, as well as cellular mechanisms that regulate the activity of the pHis kinases and phosphatases themselves. We further discuss the role of the pHis kinases and phosphatases as potential tumor promoters or suppressors. Finally, we give an overview of various tools and methods currently used to study pHis biology. Given their breadth of functions, unraveling the role of pHis in mammalian systems promises radical new insights into existing and unexplored areas of cell biology.

Protein phosphorylation by kinases regulates mammalian cell functions, such as growth, division, and signal transduction. Among human kinases, NME1 and NME2 are associated with metastatic tumor suppression but remain understudied due to the lack of tools to monitor their cellular substrates. In particular, NME1 and NME2 are multispecificity kinases phosphorylating serine, threonine, histidine, and aspartic acid residues of substrate proteins, and the heat and acid sensitivity of phosphohistidine and phosphoaspartate complicates substrate discovery and validation. To provide new substrate monitoring tools, we established the γ-phosphate-modified ATP analog, ATP-biotin, as a cosubstrate for phosphorylbiotinylation of NME1 and NME2 cellular substrates. Building upon this ATP-biotin compatibility, the Kinase-catalyzed Biotinylation with Inactivated Lysates for Discovery of Substrates method enabled validation of a known substrate and the discovery of seven NME1 and three NME2 substrates. Given the paucity of methods to study kinase substrates, ATP-biotin and the Kinase-catalyzed Biotinylation with Inactivated Lysates for Discovery of Substrates method are valuable tools to characterize the roles of NME1 and NME2 in human cell biology.

Two-component systems (TCSs) are prevalent signaling pathways in bacteria. These systems mediate phosphotransfer between histidine kinase and a response regulator, facilitating responses to diverse physical, chemical, and biological stimuli. Advancements in synthetic and structural biology have repurposed TCSs for applications in monitoring heavy metals, disease-associated biomarkers, and the production of bioproducts. However, the utility of many TCS biosensors is hindered by undesired performance due to the lack of effective engineering methods. Here, we briefly discuss the architectures and regulatory mechanisms of TCSs. We also summarize the recent advancements in TCS engineering by experimental or computational-based methods to fine-tune the biosensor functional parameters, such as response curve and specificity. Engineered TCSs have great potential in the medical, environmental, and biorefinery fields, demonstrating a crucial role in a wide area of biotechnology.

Two-component signal transduction systems (TCSs) are regulatory systems widely distributed in eubacteria, archaea, and a few eukaryotic organisms, but not in mammalian cells. A typical TCS consists of a histidine kinase and a response regulator protein. Functional and mechanistic studies on different TCSs have greatly advanced the understanding of cellular phosphotransfer signal transduction mechanisms. In this concept paper, we focus on the His-Asp phosphotransfer mechanism, the ATP synthesis function, antimicrobial drug design, cellular biosensors design, and protein allostery mechanisms based on recent TCS investigations to inspire new applications and future research perspectives.

In human pathogenic fungi, receiver domains from hybrid histidine kinases (hHK) have to recognize one HPt. To understand the recognition mechanism, we have assessed phosphorelay from receiver domains of five hHKs of group III, IV, V, VI, and XI to HPt from Chaetomium thermophilum and obtained the structures of Ct_HPt alone and in complex with the receiver domain of hHK group VI. Our data indicate that receiver domains phosphotransfer to Ct_HPt, show a low affinity for complex formation, and prevent a Leu-Thr switch to stabilize phosphoryl groups, also derived from the structures of the receiver domains of hHK group III and Candida albicans Sln1. Moreover, we have elucidated the envelope structure of C. albicans Ypd1 using small-angle X-ray scattering which reveals an extended flexible conformation of the long loop αD-αE which is not involved in phosphotransfer. Finally, we have analyzed the role of salt bridges in the structure of Ct_HPt alone.