Complex two-component signaling regulates the general stress response in Alphaproteobacteria

Kaczmarczyk, Andreas; Hochstrasser, Ramon; Vorholt, Julia A.; Francez‐Charlot, Anne

doi:10.1073/pnas.1410095111

Cited by 47 publications

(126 citation statements)

References 52 publications

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“…Although this integrated computational and experimental approach proved to be a useful tool to disentangle molecular interactions in the circadian signaling system of S. elongatus, its applicability is much broader. The ability to infer function partners in a complex signaling network can be of use for understanding the stress response of Alphaproteobacteria, where the role of HRXXN kinases and response regulators has not been fully characterized (38,39). Also, our framework might inform the study of organisms like myxobacteria, which have the largest number of TCS in any organism and a remarkably large number of unpaired TCS proteins (40).…”

Section: Resultsmentioning

confidence: 99%

A Combined Computational and Genetic Approach Uncovers Network Interactions of the Cyanobacterial Circadian Clock

et al. 2016

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Two-component systems (TCS) that employ histidine kinases (HK) and response regulators (RR) are critical mediators of cellular signaling in bacteria. In the model cyanobacterium Synechococcus elongatus PCC 7942, TCSs control global rhythms of transcription that reflect an integration of time information from the circadian clock with a variety of cellular and environmental inputs. The HK CikA and the SasA/RpaA TCS transduce time information from the circadian oscillator to modulate downstream cellular processes. Despite immense progress in understanding of the circadian clock itself, many of the connections between the clock and other cellular signaling systems have remained enigmatic. To narrow the search for additional TCS components that connect to the clock, we utilized direct-coupling analysis (DCA), a statistical analysis of covariant residues among related amino acid sequences, to infer coevolution of new and known clock TCS components. DCA revealed a high degree of interaction specificity between SasA and CikA with RpaA, as expected, but also with the phosphate-responsive response regulator SphR. Coevolutionary analysis also predicted strong specificity between RpaA and a previously undescribed kinase, HK0480 (herein CikB). A knockout of the gene for CikB (cikB) in a sasA cikA null background eliminated the RpaA phosphorylation and RpaA-controlled transcription that is otherwise present in that background and suppressed cell elongation, supporting the notion that CikB is an interactor with RpaA and the clock network. This study demonstrates the power of DCA to identify subnetworks and key interactions in signaling pathways and of combinatorial mutagenesis to explore the phenotypic consequences. Such a combined strategy is broadly applicable to other prokaryotic systems. IMPORTANCESignaling networks are complex and extensive, comprising multiple integrated pathways that respond to cellular and environmental cues. A TCS interaction model, based on DCA, independently confirmed known interactions and revealed a core set of subnetworks within the larger HK-RR set. We validated high-scoring candidate proteins via combinatorial genetics, demonstrating that DCA can be utilized to reduce the search space of complex protein networks and to infer undiscovered specific interactions for signaling proteins in vivo. Significantly, new interactions that link circadian response to cell division and fitness in a light/dark cycle were uncovered. The combined analysis also uncovered a more basic core clock, illustrating the synergy and applicability of a combined computational and genetic approach for investigating prokaryotic signaling networks. Bacterial two-component systems (TCS) are ubiquitous signaling modules consisting of at least one histidine kinase (HK) and one response regulator (RR) (1), which transduce signals via transfer of a phosphate group from the HK to a receiver domain on the RR. Over the years, studies have expanded the common paradigm of TCS signaling via phosphotransfer between single HK-RR pa...

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Section: Resultsmentioning

confidence: 99%

A Combined Computational and Genetic Approach Uncovers Network Interactions of the Cyanobacterial Circadian Clock

et al. 2016

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“…1A and B). PkrF was previously shown to be a negative regulator of the GSR, an effect that depends on PakF, and we proposed that PkrF might be a phosphate sink, competing with PhyR and SdrG for phosphorylation by PakF (7). In this study, we tested the idea that three genes-pakF, pkrF, and kipF-work in the same pathway and, in particular, that KipF is the actual sensor kinase of stresses, activating the GSR through PakF by phosphorylation of its REC domain.…”

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confidence: 92%

“…PakB and PakC seem to be largely redundant in TBHP sensing, i.e., either kinase alone is sufficient to almost fully activate the GSR. In contrast, PakF and PakC seem to have nonredundant roles in GSR activation by HS, since both pakF and pakC single mutants show reduced GSR activation, and this effect is additive in the corresponding double mutant (7). Although the actual signals sensed are currently unknown, PakB and PakC have classical sensory input domains (i.e., Per-Arnt-Sim [PAS] and cyclic GMP phosphodiesterase, adenylyl cyclase, FhlA [GAF] domains), suggesting that they are genuine sensor kinases.…”

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confidence: 94%

“…PhyR is a response regulator, and as such, its anti-anti-sigma factor activity depends on phosphorylation of a conserved Asp residue in its receiver (REC) domain. Histidine kinases belonging to the HWE (3) or HisKA_2 (4) family are emerging as stress sensors, triggering the GSR through phosphorylation of PhyR (5)(6)(7)(8)(9)(10). Because HWE and HisKA_2 kinases share an HRXXN amino acid motif in their H boxes, they are collectively referred to as HRXXN kinases (7).…”

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confidence: 99%

“…Paks also phosphorylate the single-domain response regulator (SDRR) SdrG, which acts genetically upstream of phyR and plays an important role in the GSR, through a currently unknown mechanism (7). The S. melonis Fr1 GSR is involved in resistance to several stresses, and a number of conditions that induce the GSR in a PhyR-dependent manner have been identified (6,7). Three of the seven Paks have been associated with particular inducing conditions, namely, PakB with t-butyl hydroperoxide (TBHP) sensing, PakC with sensing of TBHP and heat shock (HS), and PakF with HS and NaCl sensing.…”

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confidence: 99%

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Two-Tiered Histidine Kinase Pathway Involved in Heat Shock and Salt Sensing in the General Stress Response of Sphingomonas melonis Fr1

et al. 2015

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The general stress response (GSR) allows bacteria to monitor and defend against a broad set of unrelated, adverse environmental conditions. In Alphaproteobacteria, the key step in GSR activation is phosphorylation of the response regulator PhyR. In Sphingomonas melonis Fr1, seven PhyR-activating kinases (Paks), PakA to PakG, are thought to directly phosphorylate PhyR under different stress conditions, but the nature of the activating signals remains obscure. PakF, a major sensor of NaCl and heat shock, lacks a putative sensor domain but instead harbors a single receiver (REC) domain (PakF REC ) N-terminal to its kinase catalytic core. Such kinases are called "hybrid response regulators" (HRRs). How HRRs are able to perceive signals in the absence of a true sensor domain has remained largely unexplored. In the present work, we show that stresses are actually sensed by another kinase, KipF (kinase of PakF), which phosphorylates PakF REC and thereby activates PakF. KipF is a predicted transmembrane kinase, harboring a periplasmic CHASE3 domain flanked by two transmembrane helices in addition to its cytoplasmic kinase catalytic core. We demonstrate that KipF senses different salts through its CHASE3 domain but is not a sensor of general osmotic stress. While salt sensing depends on the CHASE3 domain, heat shock sensing does not, suggesting that these stresses are perceived by different mechanisms. In summary, our results establish a two-tiered histidine kinase pathway involved in activation of the GSR in S. melonis Fr1 and provide the first experimental evidence for the so far uncharacterized CHASE3 domain as a salt sensor. IMPORTANCEHybrid response regulators (HRRs) represent a particular class of histidine kinases harboring an N-terminal receiver (REC) domain instead of a true sensor domain. This suggests that the actual input for HRRs may be phosphorylation of the REC domain. In the present study, we addressed this question by using the HRR PakF. Our results suggest that PakF is activated through phosphorylation of its REC domain and that this is achieved by another kinase, KipF. KipF senses heat shock and salt stress, with the latter requiring the periplasmic CHASE3 domain. This work not only suggests that HRRs work in two-tiered histidine kinase pathways but also provides the first experimental evidence for a role of the so far uncharacterized CHASE3 domain in salt sensing. Many bacteria possess a so-called general stress response (GSR) that allows them to monitor a broad range of stressful conditions and to launch an appropriate response to adapt to adverse environments. In Alphaproteobacteria, the GSR is controlled by an alternative sigma factor, EcfG , whose activity is regulated via a partner-switching mechanism (1, 2). Under nonstress conditions, EcfG is inactivated by binding to the anti-sigma factor NepR. Upon stress, NepR is sequestered by the anti-anti-sigma factor PhyR, thereby releasing EcfG and allowing it to activate transcription of stress-related genes. PhyR is a response regulator, and a...

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The Extracytoplasmic Function Sigma Factor–mediated Response to Heavy Metal Stress in Caulobacter Crescentus

Lourenço

Gomes

2016

Stress and Environmental Regulation of Gene Expression and Adaptation in Bacteria

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Complex two-component signaling regulates the general stress response in Alphaproteobacteria

Cited by 47 publications

References 52 publications

A Combined Computational and Genetic Approach Uncovers Network Interactions of the Cyanobacterial Circadian Clock

A Combined Computational and Genetic Approach Uncovers Network Interactions of the Cyanobacterial Circadian Clock

Two-Tiered Histidine Kinase Pathway Involved in Heat Shock and Salt Sensing in the General Stress Response of Sphingomonas melonis Fr1

The Extracytoplasmic Function Sigma Factor–mediated Response to Heavy Metal Stress in Caulobacter Crescentus

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