DNA-binding directs the localization of a membrane-integrated receptor of the ToxR family

Brameyer, Sophie; Rösch, Thomas C.; Andari, Jihad El; Hoyer, Elisabeth; Schwarz, J.; Graumann, Peter L.; Jung, Kirsten

doi:10.1038/s42003-018-0248-7

Cited by 21 publications

(38 citation statements)

References 51 publications

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“…One of the observations arguing against proteolytic cleavage was that external signals can rapidly deactivate the CadC response after the original stimulus, whereas cleavage would irreversibly separate the DNA-binding domain from the signaling input [26]. In contrast, the diffusion and capture mechanism appeared consistent with experiments that imaged fluorophore-labeled CadC in vivo [28]. These experiments showed that localized CadC spots in fluorescent microscopic images form after cells were shifted to a medium that simultaneously provides acid stress and a lysine-rich environment, the two input signals required to stimulate the CadC response [26].…”

Section: Introductionmentioning

confidence: 78%

“…A drop in external pH induces dimerization of the periplasmic sensory domain of CadC followed by structural rearrangement of its cytoplasmic linker, permitting the DNA-binding domain of CadC to homodimerize [21,32,35]. The CadC protein number is extremely low (on average 1-3 molecules per cell [28]), mainly due to a low translation rate caused by polyproline stalling, which is only partially relieved by elongation factor P [36].…”

Section: Choice Of Cadc As Experimental Model Systemmentioning

confidence: 99%

“…To probe the kinetics of this search process in individual cells, it is crucial to have a well-defined initial state and clear 'start' and 'stop' events. When cells are initially grown in a medium with neutral pH, CadC is inactive and homogeneously distributed in the membrane [28]. A sudden medium shift to low pH and rich lysine conditions then serves as a suitable 'start' trigger, which activates CadC via the signal transduction mechanism described above and starts the target search process.…”

Section: Choice Of Cadc As Experimental Model Systemmentioning

confidence: 99%

“…For the case of E. coli CadC, a well characterized model system [25], indirect evidence [26,27] paired with direct observation [28] argues against the proteolytic cleavage and transertion scenarios, and instead supports the diffusion and capture mechanism. For instance, the transertion mechanism should be sensitive to relocating the cadC gene to a locus far from its native position, which is close to the CadC target site at the cadBA promoter.…”

Section: Introductionmentioning

confidence: 96%

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Dynamics of chromosomal target search by a membrane-integrated one-component receptor

Martini

Brameyer

Hoyer

et al. 2020

Preprint

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AbstractWhile the classical DNA target search problem of cytoplasmic transcription factors is well studied, it remains unclear how a membrane-integrated protein can kinetically establish contact with its specific binding site on the genome. Here, we address this question for the case of CadC, the membrane-integrated pH-receptor of the acid stress response Cad system in E. coli. CadC is a prime example of a one-component signaling protein that directly binds to its cognate target site on the chromosome to regulate transcription. We combined fluorescence microscopy experiments, mathematical analysis, and kinetic Monte Carlo simulations to probe this target search process. Using fluorescently labeled CadC, we measured the time from activation of the receptor until successful binding to the DNA in single cells, exploiting that stable receptor-DNA complexes are visible as fluorescent foci. Our experimental data indicate that CadC is highly mobile in the membrane and finds its target by 2D diffusion. DNA mobility is constrained due to the overall chromosome organization, but a labeled DNA locus in the vicinity of the target site appears sufficiently mobile to randomly come close to the membrane. Relocation of the DNA target site to a distant position on the chromosome had almost no effect on the mean search time, which was between four and five minutes in either case. However, a mutant strain with two binding sites displayed a mean search time that was reduced by about a factor of two. This behavior is consistent with simulations of a coarse-grained lattice model for the coupled dynamics of DNA within a cell volume and proteins on its surface. The model also rationalizes the experimentally determined distribution of search times. Overall our findings reveal that DNA target search does not present a much bigger kinetic challenge for membrane-integrated proteins than for cytoplasmic proteins.Author summaryAdaptation to changing environments is vital to bacteria and is enabled by sophisticated signal transduction systems. While classical two-component signaling is well studied, the dynamics of one-component signaling, where one protein performs both sensing and response regulation, are insufficiently understood. Specifically, how can a membrane-integrated protein bind to specific sites on the genome to regulate transcription? Here, we study the kinetics of this process, which involves both protein diffusion within the membrane and conformational fluctuations of the genomic DNA. A well-suited model system for this question is CadC, the signaling protein of the E. coli Cad system involved in pH stress response. Fluorescently labeled CadC forms visible foci in single cells upon stable DNA-binding, marking the end of the protein-DNA search process. Moreover, the start of the search is triggered by a medium shift exposing cells to pH stress. We probe the underlying mechanism by varying the number and position of DNA target sites. We combine these experiments with mathematical analysis and kinetic Monte Carlo simulations of lattice models for the search process. Our results suggest that CadC diffusion in the membrane is pivotal for this search, while the DNA target site is just mobile enough to reach the membrane.

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Section: Introductionmentioning

confidence: 78%

Section: Choice Of Cadc As Experimental Model Systemmentioning

confidence: 99%

Section: Choice Of Cadc As Experimental Model Systemmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 96%

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Dynamics of chromosomal target search by a membrane-integrated one-component receptor

Martini

Brameyer

Hoyer

et al. 2020

Preprint

Self Cite

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“…Such co-regulatory mechanisms may not only be widespread for carbohydrate transporters, but also for amino acid transport proteins such as CadC, which is part of an E. coli onecomponent system that receives information on lysine availability from LysP, a lysine-specific membrane-associated amino acid permease ( Figure 2D) (Tetsch and Jung, 2009;Haneburger et al, 2011;Rauschmeier et al, 2014;Brameyer et al, 2019). When lysine is available and the cell is under stress due to low pH, the sensor protein CadC activates the expression of the cadBA genes.…”

Section: Regulatory Influence Of Membrane Transport Proteins and Intrmentioning

confidence: 99%

Protein Activity Sensing in Bacteria in Regulating Metabolism and Motility

2020

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Bacteria have evolved complex sensing and signaling systems to react to their changing environments, most of which are present in all domains of life. Canonical bacterial sensing and signaling modules, such as membrane-bound ligand-binding receptors and kinases, are very well described. However, there are distinct sensing mechanisms in bacteria that are less studied. For instance, the sensing of internal or external cues can also be mediated by changes in protein conformation, which can either be implicated in enzymatic reactions, transport channel formation or other important cellular functions. These activities can then feed into pathways of characterized kinases, which translocate the information to the DNA or other response units. This type of bacterial sensory activity has previously been termed protein activity sensing. In this review, we highlight the recent findings about this non-canonical sensory mechanism, as well as its involvement in metabolic functions and bacterial motility. Additionally, we explore some of the specific proteins and protein-protein interactions that mediate protein activity sensing and their downstream effects. The complex sensory activities covered in this review are important for bacterial navigation and gene regulation in their dynamic environment, be it host-associated, in microbial communities or free-living.

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Structural and DNA-binding properties of the cytoplasmic domain of Vibrio cholerae transcription factor ToxR

Gubensäk

Schrank

Hartlmüller

et al. 2021

Journal of Biological Chemistry

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This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

show abstract

DNA-binding directs the localization of a membrane-integrated receptor of the ToxR family

Cited by 21 publications

References 51 publications

Dynamics of chromosomal target search by a membrane-integrated one-component receptor

Dynamics of chromosomal target search by a membrane-integrated one-component receptor

Protein Activity Sensing in Bacteria in Regulating Metabolism and Motility

Structural and DNA-binding properties of the cytoplasmic domain of Vibrio cholerae transcription factor ToxR

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