scholarly journals Surface Sensing Stimulates Cellular Differentiation in Caulobacter crescentus

2019 ◽  
Author(s):  
Rhett A. Snyder ◽  
Courtney K. Ellison ◽  
Geoffrey B. Severin ◽  
Christopher M. Waters ◽  
Yves V. Brun
Keyword(s):  
Cell Cycle ◽  
Cell Differentiation ◽  
Cellular Differentiation ◽  
Cell Cycle Progression ◽  
Caulobacter Crescentus ◽  
Chromosome Replication ◽  
Fundamental Aspect ◽  
Cycle Progression ◽  
Surface Sensing ◽  
Surface Contact

AbstractCellular differentiation is a fundamental strategy used by cells to generate specialized functions at specific stages of development. The bacterium C. crescentus employs a specialized dimorphic life cycle consisting of two differentiated cell types. How environmental cues, including mechanical inputs such as contact with a surface, regulate this cell cycle remain unclear. Here, we find that surface sensing by the physical perturbation of retracting extracellular pilus filaments accelerates cell cycle progression and cellular differentiation. We show that physical obstruction of dynamic pilus activity by chemical perturbation or by a mutation in the outer membrane pilus pore protein, CpaC, stimulates early initiation of chromosome replication. In addition, we find that surface contact stimulates cell cycle progression by demonstrating that surface-stimulated cells initiate early chromosome replication to the same extent as planktonic cells with obstructed pilus activity. Finally, we show that obstruction of pilus retraction stimulates the synthesis of the cell cycle regulator, cyclic diguanylate monophosphate (c-di-GMP) through changes in the activity and localization of two key regulatory histidine kinases that control cell fate and differentiation. Together, these results demonstrate that surface contact and mechanosensing by alterations in pilus activity stimulate C. crescentus to bypass its developmentally programmed temporal delay in cell differentiation to more quickly adapt to a surface-associated lifestyle.SignificanceCells from all domains of life sense and respond to mechanical cues [1–3]. In eukaryotes, mechanical signals such as adhesion and surface stiffness are important for regulating fundamental processes including cell differentiation during embryonic development [4]. While mechanobiology is abundantly studied in eukaryotes, the role of mechanical influences on prokaryotic biology remains under-investigated. Here, we demonstrate that mechanosensing mediated through obstruction of the dynamic extension and retraction of tight adherence (tad) pili stimulates cell differentiation and cell cycle progression in the dimorphic α-proteobacterium Caulobacter crescentus. Our results demonstrate an important intersection between mechanical stimuli and the regulation of a fundamental aspect of cell biology.

scholarly journals Surface sensing stimulates cellular differentiation inCaulobacter crescentus

2020 ◽  
Vol 117 (30) ◽  
pp. 17984-17991 ◽  
Author(s):  
Rhett A. Snyder ◽  
Courtney K. Ellison ◽  
Geoffrey B. Severin ◽  
Gregory B. Whitfield ◽  
Christopher M. Waters ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Fate ◽  
Cellular Differentiation ◽  
Cell Cycle Progression ◽  
Caulobacter Crescentus ◽  
Control Cell ◽  
Chromosome Replication ◽  
Cycle Progression ◽  
Surface Sensing ◽  
Surface Contact

Cellular differentiation is a fundamental strategy used by cells to generate specialized functions at specific stages of development. The bacteriumCaulobacter crescentusemploys a specialized dimorphic life cycle consisting of two differentiated cell types. How environmental cues, including mechanical inputs such as contact with a surface, regulate this cell cycle remain unclear. Here, we find that surface sensing by the physical perturbation of retracting extracellular pilus filaments accelerates cell-cycle progression and cellular differentiation. We show that physical obstruction of dynamic pilus activity by chemical perturbation or by a mutation in the outer-membrane pilus secretin CpaC stimulates early initiation of chromosome replication. In addition, we find that surface contact stimulates cell-cycle progression by demonstrating that surface-stimulated cells initiate early chromosome replication to the same extent as planktonic cells with obstructed pilus activity. Finally, we show that obstruction of pilus retraction stimulates the synthesis of the cell-cycle regulator cyclic diguanylate monophosphate (c-di-GMP) through changes in the activity and localization of two key regulatory histidine kinases that control cell fate and differentiation. Together, these results demonstrate that surface contact and sensing by alterations in pilus activity stimulateC. crescentusto bypass its developmentally programmed temporal delay in cell differentiation to more quickly adapt to a surface-associated lifestyle.

scholarly journals The type IV pilin PilA couples surface attachment and cell cycle initiation in Caulobacter crescentus

2019 ◽  
Author(s):  
Luca Del Medico ◽  
Dario Cerletti ◽  
Matthias Christen ◽  
Beat Christen
Keyword(s):  
Cell Cycle ◽  
Biofilm Formation ◽  
Cell Cycle Progression ◽  
Caulobacter Crescentus ◽  
Peptide Mimetics ◽  
Cycle Progression ◽  
Surface Attachment ◽  
Type Iv ◽  
Surface Sensing ◽  
Type Iv Pilin

Understanding how bacteria colonize surfaces and regulate cell cycle progression in response to cellular adhesion is of fundamental importance. Here, we used transposon sequencing in conjunction with FRET microscopy to uncover the molecular mechanism how surface sensing drives cell cycle initiation in Caulobacter crescentus. We identified the type IV pilin protein PilA as the primary signaling input that couples surface contact to cell cycle initiation via the second messenger c-di-GMP. Upon retraction of pili filaments, the monomeric pilin reservoir in the inner membrane is sensed by the 17 amino-acid transmembrane helix of PilA to activate the PleC-PleD two component signaling system, increase cellular c-di-GMP levels and signal the onset of the cell cycle. We termed the PilA signaling sequence CIP for cell cycle initiating pilin peptide. Addition of the chemically synthesized CIP peptide initiates cell cycle progression and simultaneously inhibits surface attachment. The broad conservation of the type IV pili and their importance in pathogens for host colonization suggests that CIP peptide mimetics offer new strategies to inhibit surface-sensing, prevent biofilm formation and control persistent infections.Significance StatementPili are hair-like appendages found on the surface of many bacteria to promote adhesion. Here, we provide systems-level findings on a molecular signal transduction pathway that interlinks surface sensing with cell cycle initiation. We propose that surface attachment induces depolymerization of pili filaments. The concomitant increase in pilin sub-units within the inner membrane function as a stimulus to activate the second messenger c-di-GMP and trigger cell cycle initiation. Further-more, we show that the provision of a 17 amino acid synthetic peptide corresponding to the membrane portion of the pilin sub-unit mimics surface sensing, activates cell cycle initiation and inhibits surface attachment. Thus, synthetic peptide mimetics of pilin may represent new chemotypes to control biofilm formation and treat bacterial infections.

scholarly journals The type IV pilin PilA couples surface attachment and cell-cycle initiation in Caulobacter crescentus

2020 ◽  
Vol 117 (17) ◽  
pp. 9546-9553 ◽  
Author(s):  
Luca Del Medico ◽  
Dario Cerletti ◽  
Philipp Schächle ◽  
Matthias Christen ◽  
Beat Christen
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Caulobacter Crescentus ◽  
Resonance Energy ◽  
Cellular Adhesion ◽  
Cycle Progression ◽  
Surface Attachment ◽  
Type Iv ◽  
Surface Sensing ◽  
Type Iv Pilin

Understanding how bacteria colonize surfaces and regulate cell-cycle progression in response to cellular adhesion is of fundamental importance. Here, we use transposon sequencing in conjunction with fluorescence resonance energy transfer (FRET) microscopy to uncover the molecular mechanism for how surface sensing drives cell-cycle initiation in Caulobacter crescentus. We identify the type IV pilin protein PilA as the primary signaling input that couples surface contact to cell-cycle initiation via the second messenger cyclic di-GMP (c-di-GMP). Upon retraction of pili filaments, the monomeric pilin reservoir in the inner membrane is sensed by the 17-amino acid transmembrane helix of PilA to activate the PleC-PleD two-component signaling system, increase cellular c-di-GMP levels, and signal the onset of the cell cycle. We termed the PilA signaling sequence CIP for “cell-cycle initiating pilin” peptide. Addition of the chemically synthesized CIP peptide initiates cell-cycle progression and simultaneously inhibits surface attachment. The broad conservation of the type IV pili and their importance in pathogens for host colonization suggests that CIP peptide mimetics offer strategies to inhibit surface sensing, prevent biofilm formation and control persistent infections.

scholarly journals Polar Remodeling and Histidine Kinase Activation, Which Is Essential for Caulobacter Cell Cycle Progression, Are Dependent on DNA Replication Initiation

2010 ◽  
Vol 192 (15) ◽  
pp. 3893-3902 ◽  
Author(s):  
Antonio A. Iniesta ◽  
Nathan J. Hillson ◽  
Lucy Shapiro
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Histidine Kinase ◽  
Cell Cycle Progression ◽  
Caulobacter Crescentus ◽  
Replication Initiation ◽  
Cycle Progression ◽  
Dna Replication Initiation ◽  
Flagellar Biosynthesis ◽  
Initiation Of Dna Replication

ABSTRACT Caulobacter crescentus initiates a single round of DNA replication during each cell cycle. Following the initiation of DNA replication, the essential CckA histidine kinase is activated by phosphorylation, which (via the ChpT phosphotransferase) enables the phosphorylation and activation of the CtrA global regulator. CtrA∼P then blocks the reinitiation of replication while regulating the transcription of a large number of cell cycle-controlled genes. It has been shown that DNA replication serves as a checkpoint for flagellar biosynthesis and cell division and that this checkpoint is mediated by the availability of active CtrA. Because CckA∼P promotes the activation of CtrA, we addressed the question of what controls the temporal activation of CckA. We found that the initiation of DNA replication is a prerequisite for remodeling the new cell pole, which includes the localization of the DivL protein kinase to that pole and, consequently, the localization, autophosphorylation, and activation of CckA at that pole. Thus, CckA activation is dependent on polar remodeling and a DNA replication initiation checkpoint that is tightly integrated with the polar phospho-signaling cascade governing cell cycle progression.

scholarly journals Regulation of Bacterial Cell Cycle Progression by Redundant Phosphatases

2020 ◽  
Vol 202 (17) ◽  
Author(s):  
Jérôme Coppine ◽  
Andreas Kaczmarczyk ◽  
Kenny Petit ◽  
Thomas Brochier ◽  
Urs Jenal ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Environmental Changes ◽  
Caulobacter Crescentus ◽  
Response Regulator ◽  
Model Organism ◽  
Replication Initiation ◽  
Cycle Progression ◽  
Content Type ◽  
Two Component

ABSTRACT In the model organism Caulobacter crescentus, a network of two-component systems involving the response regulators CtrA, DivK, and PleD coordinates cell cycle progression with differentiation. Active phosphorylated CtrA prevents chromosome replication in G1 cells while simultaneously regulating expression of genes required for morphogenesis and development. At the G1-S transition, phosphorylated DivK (DivK∼P) and PleD (PleD∼P) accumulate to indirectly inactivate CtrA, which triggers DNA replication initiation and concomitant cellular differentiation. The phosphatase PleC plays a pivotal role in this developmental program by keeping DivK and PleD phosphorylation levels low during G1, thereby preventing premature CtrA inactivation. Here, we describe CckN as a second phosphatase akin to PleC that dephosphorylates DivK∼P and PleD∼P in G1 cells. However, in contrast to PleC, no kinase activity was detected with CckN. The effects of CckN inactivation are largely masked by PleC but become evident when PleC and DivJ, the major kinase for DivK and PleD, are absent. Accordingly, mild overexpression of cckN restores most phenotypic defects of a pleC null mutant. We also show that CckN and PleC are proteolytically degraded in a ClpXP-dependent way before the onset of the S phase. Surprisingly, known ClpX adaptors are dispensable for PleC and CckN proteolysis, raising the possibility that as yet unidentified proteolytic adaptors are required for the degradation of both phosphatases. Since cckN expression is induced in stationary phase, depending on the stress alarmone (p)ppGpp, we propose that CckN acts as an auxiliary factor responding to environmental stimuli to modulate CtrA activity under suboptimal conditions. IMPORTANCE Two-component signal transduction systems are widely used by bacteria to adequately respond to environmental changes by adjusting cellular parameters, including the cell cycle. In Caulobacter crescentus, PleC acts as a phosphatase that indirectly protects the response regulator CtrA from premature inactivation during the G1 phase of the cell cycle. Here, we provide genetic and biochemical evidence that PleC is seconded by another phosphatase, CckN. The activity of PleC and CckN phosphatases is restricted to the G1 phase since both proteins are degraded by ClpXP protease before the G1-S transition. Degradation is independent of any known proteolytic adaptors and relies, in the case of CckN, on an unsuspected N-terminal degron. Our work illustrates a typical example of redundant functions between two-component proteins.

scholarly journals A Localized Complex of Two Protein Oligomers Controls the Orientation of Cell Polarity

mBio ◽  
2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Adam M. Perez ◽  
Thomas H. Mann ◽  
Keren Lasker ◽  
Daniel G. Ahrens ◽  
Michael R. Eckart ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Polarity ◽  
Histidine Kinase ◽  
Bacterial Cell ◽  
Cell Cycle Progression ◽  
Caulobacter Crescentus ◽  
Cycle Progression ◽  
Self Assembling ◽  
Membrane Bound ◽  
Ordered Assembly

ABSTRACT Signaling hubs at bacterial cell poles establish cell polarity in the absence of membrane-bound compartments. In the asymmetrically dividing bacterium Caulobacter crescentus, cell polarity stems from the cell cycle-regulated localization and turnover of signaling protein complexes in these hubs, and yet the mechanisms that establish the identity of the two cell poles have not been established. Here, we recapitulate the tripartite assembly of a cell fate signaling complex that forms during the G1-S transition. Using in vivo and in vitro analyses of dynamic polar protein complex formation, we show that a polymeric cell polarity protein, SpmX, serves as a direct bridge between the PopZ polymeric network and the cell fate-directing DivJ histidine kinase. We demonstrate the direct binding between these three proteins and show that a polar microdomain spontaneously assembles when the three proteins are coexpressed heterologously in an Escherichia coli test system. The relative copy numbers of these proteins are essential for complex formation, as overexpression of SpmX in Caulobacter reorganizes the polarity of the cell, generating ectopic cell poles containing PopZ and DivJ. Hierarchical formation of higher-order SpmX oligomers nucleates new PopZ microdomain assemblies at the incipient lateral cell poles, driving localized outgrowth. By comparison to self-assembling protein networks and polar cell growth mechanisms in other bacterial species, we suggest that the cooligomeric PopZ-SpmX protein complex in Caulobacter illustrates a paradigm for coupling cell cycle progression to the controlled geometry of cell pole establishment. IMPORTANCE Lacking internal membrane-bound compartments, bacteria achieve subcellular organization by establishing self-assembling protein-based microdomains. The asymmetrically dividing bacterium Caulobacter crescentus uses one such microdomain to link cell cycle progression to morphogenesis, but the mechanism for the generation of this microdomain has remained unclear. Here, we demonstrate that the ordered assembly of this microdomain occurs via the polymeric network protein PopZ directly recruiting the polarity factor SpmX, which then recruits the histidine kinase DivJ to the developing cell pole. Further, we find that overexpression of the bridge protein SpmX in Caulobacter disrupts this ordered assembly, generating ectopic cell poles containing both PopZ and DivJ. Together, PopZ and SpmX assemble into a cooligomeric network that forms the basis for a polar microdomain that coordinates bacterial cell polarity. IMPORTANCE Lacking internal membrane-bound compartments, bacteria achieve subcellular organization by establishing self-assembling protein-based microdomains. The asymmetrically dividing bacterium Caulobacter crescentus uses one such microdomain to link cell cycle progression to morphogenesis, but the mechanism for the generation of this microdomain has remained unclear. Here, we demonstrate that the ordered assembly of this microdomain occurs via the polymeric network protein PopZ directly recruiting the polarity factor SpmX, which then recruits the histidine kinase DivJ to the developing cell pole. Further, we find that overexpression of the bridge protein SpmX in Caulobacter disrupts this ordered assembly, generating ectopic cell poles containing both PopZ and DivJ. Together, PopZ and SpmX assemble into a cooligomeric network that forms the basis for a polar microdomain that coordinates bacterial cell polarity.

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