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1.
Mol Biol Cell ; 35(7): mr4, 2024 Jul 01.
Article in English | MEDLINE | ID: mdl-38717434

ABSTRACT

Cryogenic electron tomography (cryo-ET) is the highest resolution imaging technique applicable to the life sciences, enabling subnanometer visualization of specimens preserved in their near native states. The rapid plunge freezing process used to prepare samples lends itself to time-resolved studies, which researchers have pursued for in vitro samples for decades. Here, we focus on developing a freezing apparatus for time-resolved studies in situ. The device mixes cellular samples with solution-phase stimulants before spraying them directly onto an electron microscopy grid that is transiting into cryogenic liquid ethane. By varying the flow rates of cell and stimulant solutions within the device, we can control the reaction time from tens of milliseconds to over a second before freezing. In a proof-of-principle demonstration, the freezing method is applied to a model bacterium, Caulobacter crescentus, mixed with an acidic buffer. Through cryo-ET we resolved structural changes throughout the cell, including surface-layer protein dissolution, outer membrane deformation, and cytosolic rearrangement, all within 1.5 s of reaction time. This new approach, Time-Resolved cryo-ET (TR-cryo-ET), enhances the capabilities of cryo-ET by incorporating a subsecond temporal axis and enables the visualization of induced structural changes at the molecular, organelle, or cellular level.


Subject(s)
Caulobacter crescentus , Cryoelectron Microscopy , Electron Microscope Tomography , Electron Microscope Tomography/methods , Cryoelectron Microscopy/methods , Caulobacter crescentus/ultrastructure , Caulobacter crescentus/metabolism , Caulobacter crescentus/physiology , Freezing
2.
Life Sci Alliance ; 7(8)2024 Aug.
Article in English | MEDLINE | ID: mdl-38806218

ABSTRACT

Cell size regulation has been extensively studied in symmetrically dividing cells, but the mechanisms underlying the control of size asymmetry in asymmetrically dividing bacteria remain elusive. Here, we examine the control of asymmetric division in Caulobacter crescentus, a bacterium that produces daughter cells with distinct fates and morphologies upon division. Through comprehensive analysis of multi-generational growth and shape data, we uncover a tightly regulated cell size partitioning mechanism. We find that errors in division site positioning are promptly corrected early in the division cycle through differential growth. Our analysis reveals a negative feedback between the size of daughter cell compartments and their growth rates, wherein the larger compartment grows slower to achieve a homeostatic size partitioning ratio at division. To explain these observations, we propose a mechanistic model of differential growth, in which equal amounts of growth regulators are partitioned into daughter cell compartments of unequal sizes and maintained over time via size-independent synthesis.


Subject(s)
Caulobacter crescentus , Cell Division , Caulobacter crescentus/metabolism , Caulobacter crescentus/cytology , Caulobacter crescentus/growth & development , Caulobacter crescentus/physiology , Asymmetric Cell Division , Bacterial Proteins/metabolism , Models, Biological
3.
Mol Biol Cell ; 35(6): ar78, 2024 Jun 01.
Article in English | MEDLINE | ID: mdl-38598301

ABSTRACT

Microfluidic platforms enable long-term quantification of stochastic behaviors of individual bacterial cells under precisely controlled growth conditions. Yet, quantitative comparisons of physiological parameters and cell behaviors of different microorganisms in different experimental and device modalities is not available due to experiment-specific details affecting cell physiology. To rigorously assess the effects of mechanical confinement, we designed, engineered, and performed side-by-side experiments under otherwise identical conditions in the Mother Machine (with confinement) and the SChemostat (without confinement), using the latter as the ideal comparator. We established a protocol to cultivate a suitably engineered rod-shaped mutant of Caulobacter crescentus in the Mother Machine and benchmarked the differences in stochastic growth and division dynamics with respect to the SChemostat. While the single-cell growth rate distributions are remarkably similar, the mechanically confined cells in the Mother Machine experience a substantial increase in interdivision times. However, we find that the division ratio distribution precisely compensates for this increase, which in turn reflects identical emergent simplicities governing stochastic intergenerational homeostasis of cell sizes across device and experimental configurations, provided the cell sizes are appropriately mean-rescaled in each condition. Our results provide insights into the nature of the robustness of the bacterial growth and division machinery.


Subject(s)
Caulobacter crescentus , Cell Division , Stochastic Processes , Caulobacter crescentus/physiology , Caulobacter crescentus/metabolism , Caulobacter crescentus/cytology , Microfluidics/methods
4.
Curr Opin Microbiol ; 79: 102468, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38579360

ABSTRACT

Bacteria utilize type IV pili (T4P) to interact with their environment, where they facilitate processes including motility, adherence, and DNA uptake. T4P require multisubunit, membrane-spanning nanomachines for assembly. The tight adherence (Tad) pili are an Archaea-derived T4P subgroup whose machinery exhibits significant mechanistic and architectural differences from bacterial type IVa and IVb pili. Most Tad biosynthetic genes are encoded in a single locus that is widespread in bacteria due to facile acquisition via horizontal gene transfer. These loci experience extensive structural rearrangements, including the acquisition of novel regulatory or biosynthetic genes, which fine-tune their function. This has permitted their integration into many different bacterial lifestyles, including the Caulobacter crescentus cell cycle, Myxococcus xanthus predation, and numerous plant and mammalian pathogens and symbionts.


Subject(s)
Fimbriae, Bacterial , Fimbriae, Bacterial/metabolism , Fimbriae, Bacterial/genetics , Fimbriae, Bacterial/physiology , Caulobacter crescentus/genetics , Caulobacter crescentus/metabolism , Caulobacter crescentus/physiology , Bacteria/genetics , Bacteria/metabolism , Bacterial Adhesion/genetics , Gene Transfer, Horizontal , Fimbriae Proteins/genetics , Fimbriae Proteins/metabolism , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Myxococcus xanthus/genetics , Myxococcus xanthus/physiology , Myxococcus xanthus/metabolism
5.
Nat Commun ; 15(1): 3590, 2024 Apr 27.
Article in English | MEDLINE | ID: mdl-38678033

ABSTRACT

Predatory bacteria feed upon other bacteria in various environments. Bdellovibrio exovorus is an obligate epibiotic predator that attaches on the prey cell surface, where it grows and proliferates. Although the mechanisms allowing feeding through the prey cell envelope are unknown, it has been proposed that the prey's proteinaceous S-layer may act as a defensive structure against predation. Here, we use time-lapse and cryo-electron microscopy to image the lifecycle of B. exovorus feeding on Caulobacter crescentus. We show that B. exovorus proliferates by non-binary division, primarily generating three daughter cells. Moreover, the predator feeds on C. crescentus regardless of the presence of an S-layer, challenging its assumed protective role against predators. Finally, we show that apparently secure junctions are established between prey and predator outer membranes.


Subject(s)
Bdellovibrio , Caulobacter crescentus , Cell Membrane , Cryoelectron Microscopy , Caulobacter crescentus/physiology , Caulobacter crescentus/ultrastructure , Bdellovibrio/physiology , Cell Membrane/ultrastructure , Cell Membrane/metabolism , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Membrane Glycoproteins/metabolism , Time-Lapse Imaging
6.
Biophys J ; 122(7): 1254-1267, 2023 04 04.
Article in English | MEDLINE | ID: mdl-36814380

ABSTRACT

Proliferating bacterial cells exhibit stochastic growth and size dynamics, but the regulation of noise in bacterial growth and morphogenesis remains poorly understood. A quantitative understanding of morphogenetic noise control, and how it changes under different growth conditions, would provide better insights into cell-to-cell variability and intergenerational fluctuations in cell physiology. Using multigenerational growth and width data of single Escherichia coli and Caulobacter crescentus cells, we deduce the equations governing growth and size dynamics of rod-like bacterial cells. Interestingly, we find that both E. coli and C. crescentus cells deviate from exponential growth within the cell cycle. In particular, the exponential growth rate increases during the cell cycle irrespective of nutrient or temperature conditions. We propose a mechanistic model that explains the emergence of super-exponential growth from autocatalytic production of ribosomes coupled to the rate of cell elongation and surface area synthesis. Using this new model and statistical inference on large datasets, we construct the Langevin equations governing cell growth and size dynamics of E. coli cells in different nutrient conditions. The single-cell level model predicts how noise in intragenerational and intergenerational processes regulate variability in cell morphology and generation times, revealing quantitative strategies for cellular resource allocation and morphogenetic noise control in different growth conditions.


Subject(s)
Caulobacter crescentus , Escherichia coli , Models, Biological , Cell Division , Cell Cycle , Caulobacter crescentus/physiology
7.
Elife ; 122023 01 19.
Article in English | MEDLINE | ID: mdl-36475544

ABSTRACT

In their natural environment, most bacteria preferentially live as complex surface-attached multicellular colonies called biofilms. Biofilms begin with a few cells adhering to a surface, where they multiply to form a mature colony. When conditions deteriorate, cells can leave the biofilm. This dispersion is thought to be an important process that modifies the overall biofilm architecture and that promotes colonization of new environments. In Caulobacter crescentus biofilms, extracellular DNA (eDNA) is released upon cell death and prevents newborn cells from joining the established biofilm. Thus, eDNA promotes the dispersal of newborn cells and the subsequent colonization of new environments. These observations suggest that eDNA is a cue for sensing detrimental environmental conditions in the biofilm. Here, we show that the toxin-antitoxin system (TAS) ParDE4 stimulates cell death in areas of a biofilm with decreased O2 availability. In conditions where O2 availability is low, eDNA concentration is correlated with cell death. Cell dispersal away from biofilms is decreased when parDE4 is deleted, probably due to the lower local eDNA concentration. Expression of parDE4 is positively regulated by O2 and the expression of this operon is decreased in biofilms where O2 availability is low. Thus, a programmed cell death mechanism using an O2-regulated TAS stimulates dispersal away from areas of a biofilm with decreased O2 availability and favors colonization of a new, more hospitable environment.


Bacteria are more social than what had long been expected. While they can swim around on their own, most of them in fact settle down as part of a surface-bound community. The plaque on our teeth and the gooey deposit in our bathroom pipes are the visible results of this communal lifestyle. Inside this slimy 'biofilm', cells share resources and are protected from toxic substances such as antibiotics. However, being tied to one spot is not always a good thing: it may be advantageous for a cell in a biofilm to strike out on its own and resume 'single life' if local conditions deteriorate. Caulobacter crescentus bacteria do not always have this choice, as adult cells in this species become permanently glued into place upon joining a biofilm. When these divide, however, their daughters have a choice: swim away, or stick with the group. Previous research has shown that this decision is influenced by the health of the community. Dying cells release DNA fragments which disable the structures allowing newborn cells to adhere to the environment, and a high mortality rate in the biofilm therefore forces unattached cells to leave the colony. Berne et al. wanted to build on these results and examine how exactly cells die in the biofilm. In particular, the deaths could be sudden and random, with the bacteria succumbing to injury; or they could result from cells activating one of their built-in self-destruct programs. To investigate this question, genetically modified C. crescentus bacteria were grown in the laboratory and exposed to different environments. Combining genetic and microscopic approaches revealed that as a biofilm becomes too crowded, certain individuals self-destruct via a cell death program known as the toxin-antitoxin system. Further experiments showed that low oxygen availability was the signal that triggered self-destruction. Drops in oxygen levels can happen when the environment becomes hostile or when a colony is overpopulated. The results by Berne et al. therefore suggest that by triggering self-destruction in certain members of the community, reduced oxygen access leads to newborn cells swimming away, which in turn prevents further overcrowding and allows new, more hospitable locations to be colonized. Biofilms are of growing interest in a wide range of human industries, but they also present formidable challenges. This is particularly the case in healthcare, as they tend to infest medical devices and help disease-causing species to resist treatments. Understanding how bacteria are encouraged to join or leave their colony is necessary to better control biofilms to our advantage.


Subject(s)
Caulobacter crescentus , Toxin-Antitoxin Systems , Humans , Infant, Newborn , Caulobacter crescentus/physiology , Biofilms , DNA/metabolism , DNA, Bacterial/metabolism
8.
Elife ; 102021 10 25.
Article in English | MEDLINE | ID: mdl-34693909

ABSTRACT

The highly conserved protease Lon has important regulatory and protein quality control functions in cells from the three domains of life. Despite many years of research on Lon, only a few specific protein substrates are known in most organisms. Here, we used a quantitative proteomics approach to identify novel substrates of Lon in the dimorphic bacterium Caulobacter crescentus. We focused our study on proteins involved in polar cell differentiation and investigated the developmental regulator StaR and the flagella hook length regulator FliK as specific Lon substrates in detail. We show that Lon recognizes these proteins at their C-termini, and that Lon-dependent degradation ensures their temporally restricted accumulation in the cell cycle phase when their function is needed. Disruption of this precise temporal regulation of StaR and FliK levels in a Δlon mutant contributes to defects in stalk biogenesis and motility, respectively, revealing a critical role of Lon in coordinating developmental processes with cell cycle progression. Our work underscores the importance of Lon in the regulation of complex temporally controlled processes by adjusting the concentrations of critical regulatory proteins. Furthermore, this study includes the first characterization of FliK in C. crescentus and uncovers a dual role of the C-terminal amino acids of FliK in protein function and degradation.


Subject(s)
Bacterial Proteins/genetics , Caulobacter crescentus/physiology , Cell Differentiation/genetics , Polar Bodies/physiology , Protease La/genetics , Bacterial Proteins/metabolism , Caulobacter crescentus/genetics , Protease La/metabolism
9.
Commun Biol ; 4(1): 669, 2021 06 03.
Article in English | MEDLINE | ID: mdl-34083715

ABSTRACT

How motile bacteria navigate environmental chemical gradients has implications ranging from health to climate science, but the underlying behavioral mechanisms are unknown for most species. The well-studied navigation strategy of Escherichia coli forms a powerful paradigm that is widely assumed to translate to other bacterial species. This assumption is rarely tested because of a lack of techniques capable of bridging scales from individual navigation behavior to the resulting population-level chemotactic performance. Here, we present such a multiscale 3D chemotaxis assay by combining high-throughput 3D bacterial tracking with microfluidically created chemical gradients. Large datasets of 3D trajectories yield the statistical power required to assess chemotactic performance at the population level, while simultaneously resolving the underlying 3D navigation behavior for every individual. We demonstrate that surface effects confound typical 2D chemotaxis assays, and reveal that, contrary to previous reports, Caulobacter crescentus breaks with the E. coli paradigm.


Subject(s)
Algorithms , Chemotaxis/physiology , Escherichia coli/physiology , High-Throughput Screening Assays/methods , Microfluidic Analytical Techniques/methods , Models, Biological , Caulobacter crescentus/physiology , Species Specificity
10.
J Mol Biol ; 433(13): 167004, 2021 06 25.
Article in English | MEDLINE | ID: mdl-33891903

ABSTRACT

The bacterial flagellum consists of a long extracellular filament that is rotated by a motor embedded in the cell envelope. While flagellar assembly has been extensively studied,1 the disassembly process remains less well understood. In addition to the programmed flagellar ejection that occurs during the life cycle of Caulobacter crescentus, we and others have recently shown that many bacterial species lose their flagella under starvation conditions, leaving relic structures in the outer membrane.2-7 However, it remains unknown whether the programmed flagellar ejection of C. crescentus leaves similar relics or not. Here, we imaged the various stages of the C. crescentus life cycle using electron cryo-tomography (cryo-ET) and found that flagellar relic subcomplexes, akin to those produced in the starvation-induced process, remain as a result of flagellar ejection during cell development. This similarity suggests that the programmed flagellar ejection of C. crescentus might share a common evolutionary path with the more general, and likely more ancient,3 starvation-related flagellar loss.


Subject(s)
Caulobacter crescentus/physiology , Cell Wall/metabolism , Flagella/physiology , Lipopolysaccharides/metabolism , Peptidoglycan/metabolism , Basal Bodies/physiology , Basal Bodies/ultrastructure , Caulobacter crescentus/metabolism , Caulobacter crescentus/ultrastructure , Cell Wall/ultrastructure , Electron Microscope Tomography/methods , Fimbriae, Bacterial/physiology , Fimbriae, Bacterial/ultrastructure , Flagella/ultrastructure
11.
Proc Natl Acad Sci U S A ; 118(13)2021 03 30.
Article in English | MEDLINE | ID: mdl-33753507

ABSTRACT

Asymmetric cell division generates two daughter cells with distinct characteristics and fates. Positioning different regulatory and signaling proteins at the opposing ends of the predivisional cell produces molecularly distinct daughter cells. Here, we report a strategy deployed by the asymmetrically dividing bacterium Caulobacter crescentus where a regulatory protein is programmed to perform distinct functions at the opposing cell poles. We find that the CtrA proteolysis adaptor protein PopA assumes distinct oligomeric states at the two cell poles through asymmetrically distributed c-di-GMP: dimeric at the stalked pole and monomeric at the swarmer pole. Different polar organizing proteins at each cell pole recruit PopA where it interacts with and mediates the function of two molecular machines: the ClpXP degradation machinery at the stalked pole and the flagellar basal body at the swarmer pole. We discovered a binding partner of PopA at the swarmer cell pole that together with PopA regulates the length of the flagella filament. Our work demonstrates how a second messenger provides spatiotemporal cues to change the physical behavior of an effector protein, thereby facilitating asymmetry.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Asymmetric Cell Division , Bacterial Proteins/metabolism , Caulobacter crescentus/physiology , Caulobacter crescentus/cytology , Caulobacter crescentus/metabolism , Cyclic GMP/analogs & derivatives , Cyclic GMP/metabolism , Endopeptidase Clp/metabolism , Protein Multimerization , Proteolysis
12.
mBio ; 12(1)2021 02 09.
Article in English | MEDLINE | ID: mdl-33563824

ABSTRACT

Bacteria carry out sophisticated developmental programs to colonize exogenous surfaces. The rotary flagellum, a dynamic machine that drives motility, is a key regulator of surface colonization. The specific signals recognized by flagella and the pathways by which those signals are transduced to coordinate adhesion remain subjects of debate. Mutations that disrupt flagellar assembly in the dimorphic bacterium Caulobacter crescentus stimulate the production of a polysaccharide adhesin called the holdfast. Using a genomewide phenotyping approach, we compared surface adhesion profiles in wild-type and flagellar mutant backgrounds of C. crescentus We identified a diverse set of flagellar mutations that enhance adhesion by inducing a hyperholdfast phenotype and discovered a second set of mutations that suppress this phenotype. Epistasis analysis of the flagellar signaling suppressor (fss) mutations demonstrated that the flagellum stimulates holdfast production via two genetically distinct pathways. The developmental regulator PleD contributes to holdfast induction in mutants disrupted at both early and late stages of flagellar assembly. Mutants disrupted at late stages of flagellar assembly, which assemble an intact rotor complex, induce holdfast production through an additional process that requires the MotAB stator and its associated diguanylate cyclase, DgcB. We have assigned a subset of the fss genes to either the stator- or pleD-dependent networks and characterized two previously unidentified motility genes that regulate holdfast production via the stator complex. We propose a model through which the flagellum integrates mechanical stimuli into the C. crescentus developmental program to coordinate adhesion.IMPORTANCE Understanding how bacteria colonize solid surfaces is of significant clinical, industrial and ecological importance. In this study, we identified genes that are required for Caulobacter crescentus to activate surface attachment in response to signals from a macromolecular machine called the flagellum. Genes involved in transmitting information from the flagellum can be grouped into separate pathways, those that control the C. crescentus morphogenic program and those that are required for flagellar motility. Our results support a model in which a developmental and a mechanical signaling pathway operate in parallel downstream of the flagellum and converge to regulate adhesion. We conclude that the flagellum serves as a signaling hub by integrating internal and external cues to coordinate surface colonization and emphasize the role of signal integration in linking complex sets of environmental stimuli to individual behaviors.


Subject(s)
Adhesins, Bacterial/genetics , Adhesins, Bacterial/metabolism , Bacterial Adhesion , Caulobacter crescentus/physiology , Flagella/genetics , Flagella/metabolism , Polysaccharides, Bacterial/genetics , Polysaccharides, Bacterial/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Caulobacter crescentus/genetics , Gene Expression Regulation, Bacterial , Mutation , Signal Transduction
13.
BMC Bioinformatics ; 21(Suppl 14): 408, 2020 Sep 30.
Article in English | MEDLINE | ID: mdl-32998723

ABSTRACT

BACKGROUND: Second messengers, c-di-GMP and (p)ppGpp, are vital regulatory molecules in bacteria, influencing cellular processes such as biofilm formation, transcription, virulence, quorum sensing, and proliferation. While c-di-GMP and (p)ppGpp are both synthesized from GTP molecules, they play antagonistic roles in regulating the cell cycle. In C. crescentus, c-di-GMP works as a major regulator of pole morphogenesis and cell development. It inhibits cell motility and promotes S-phase entry by inhibiting the activity of the master regulator, CtrA. Intracellular (p)ppGpp accumulates under starvation, which helps bacteria to survive under stressful conditions through regulating nucleotide levels and halting proliferation. (p)ppGpp responds to nitrogen levels through RelA-SpoT homolog enzymes, detecting glutamine concentration using a nitrogen phosphotransferase system (PTS Ntr). This work relates the guanine nucleotide-based second messenger regulatory network with the bacterial PTS Ntr system and investigates how bacteria respond to nutrient availability. RESULTS: We propose a mathematical model for the dynamics of c-di-GMP and (p)ppGpp in C. crescentus and analyze how the guanine nucleotide-based second messenger system responds to certain environmental changes communicated through the PTS Ntr system. Our mathematical model consists of seven ODEs describing the dynamics of nucleotides and PTS Ntr enzymes. Our simulations are consistent with experimental observations and suggest, among other predictions, that SpoT can effectively decrease c-di-GMP levels in response to nitrogen starvation just as well as it increases (p)ppGpp levels. Thus, the activity of SpoT (or its homologues in other bacterial species) can likely influence the cell cycle by influencing both c-di-GMP and (p)ppGpp. CONCLUSIONS: In this work, we integrate current knowledge and experimental observations from the literature to formulate a novel mathematical model. We analyze the model and demonstrate how the PTS Ntr system influences (p)ppGpp, c-di-GMP, GMP and GTP concentrations. While this model does not consider all aspects of PTS Ntr signaling, such as cross-talk with the carbon PTS system, here we present our first effort to develop a model of nutrient signaling in C. crescentus.


Subject(s)
Caulobacter crescentus/physiology , Models, Theoretical , Second Messenger Systems , Cell Cycle Checkpoints , Cyclic GMP/analogs & derivatives , Cyclic GMP/metabolism , Nitrogen/metabolism , Phosphotransferases/metabolism , Second Messenger Systems/physiology
14.
Commun Biol ; 3(1): 587, 2020 10 16.
Article in English | MEDLINE | ID: mdl-33067555

ABSTRACT

Active dispersal of microorganisms is often attributed to the cells' motile organelles. However, much less is known about whether sessile cells can access such motility through aggregation with motile counterparts. Here, we show that the rosette aggregates of the bacterium Caulobacter crescentus, although predominantly sessile, can actively disperse through the flagellar motors of motile members. Comparisons in kinematics between the motile rosettes and solitary swimming cells indicate that the rosettes can be powered by as few as a single motor. We further reconstructed the 3D movements of the rosettes to reveal that their proximity to a solid-liquid interface promotes a wheel-like rolling, as powered by the flagellar torque. This rolling movement also features a sequence of sharp turns, a reorientation mechanism distinct from that of swimming cells. Overall, our study elucidates an unexplored regime of aggregation-based motility that can be widely applied to sessile-motile composites.


Subject(s)
Bacterial Physiological Phenomena , Caulobacter crescentus/physiology , Bacterial Adhesion , Biophysical Phenomena , Caulobacter crescentus/cytology , Caulobacter crescentus/ultrastructure , Models, Theoretical , Movement
15.
Biochemistry ; 59(37): 3508-3516, 2020 09 22.
Article in English | MEDLINE | ID: mdl-32844640

ABSTRACT

The bacterium Caulobacter crescentus is known to attach irreversibly to underwater surfaces by utilizing an adhesive structure called the holdfast, which exhibits the greatest known adhesive strength of any organism. The very small size of the holdfast (∼400 nm wide and ∼40 nm high) has made direct chemical analysis difficult, and its structure remains poorly understood. In this study, we employ spectroscopic techniques, including attenuated total reflection infrared spectroscopy (ATR-IR) and X-ray photoelectron spectroscopy, to probe holdfast chemistry. The data indicate the presence of a peptide signal within the holdfast polymer. By comparing the ATR-IR spectrum of the holdfast to peptidoglycan spectra from other bacterial species, we demonstrate the similarity of the holdfast chemistry to that of peptidoglycan, suggesting peptide cross-linking may play a role in holdfast architecture. To probe the molecular groups at the interface, surface-sensitive sum frequency generation spectroscopy was used to show that aromatic and hydroxyl groups related to this protein content at the adhesive interface could be playing a crucial role in adhesion. On the basis of these results, we propose a model of the holdfast architecture with similarities to the peptide cross-linking observed in the peptidoglycan polymer of the bacterial cell wall. These results not only provide information about the development of adhesives that could be based on holdfast chemical architecture but also reveal a potentially yet unexplored biosynthetic pathway in holdfast synthesis that has not yet been revealed by genetic approaches, thereby opening up a potentially new avenue of research in holdfast synthesis.


Subject(s)
Adhesins, Bacterial/metabolism , Bacterial Adhesion , Bacterial Proteins/chemistry , Carrier Proteins/chemistry , Caulobacter crescentus/physiology , Peptide Fragments/chemistry , Peptidoglycan/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cross-Linking Reagents/chemistry , Spectrophotometry, Infrared
16.
Proc Natl Acad Sci U S A ; 117(30): 17984-17991, 2020 07 28.
Article in English | MEDLINE | ID: mdl-32661164

ABSTRACT

Cellular differentiation is a fundamental strategy used by cells to generate specialized functions at specific stages of development. The bacterium Caulobacter 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 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 stimulate C. crescentus to bypass its developmentally programmed temporal delay in cell differentiation to more quickly adapt to a surface-associated lifestyle.


Subject(s)
Bacterial Physiological Phenomena , Caulobacter crescentus/physiology , Gram-Negative Bacterial Infections/microbiology , Cell Cycle , Cyclic GMP/analogs & derivatives , Cyclic GMP/metabolism , DNA Replication , Fimbriae, Bacterial/physiology , Models, Biological , Mutation
17.
Nat Rev Microbiol ; 18(12): 677-689, 2020 12.
Article in English | MEDLINE | ID: mdl-32710089

ABSTRACT

Advances in imaging technologies have revealed that many bacteria possess organelles with a proteomically defined lumen and a macromolecular boundary. Some are bound by a lipid bilayer (such as thylakoids, magnetosomes and anammoxosomes), whereas others are defined by a lipid monolayer (such as lipid bodies), a proteinaceous coat (such as carboxysomes) or have a phase-defined boundary (such as nucleolus-like compartments). These diverse organelles have various metabolic and physiological functions, facilitating adaptation to different environments and driving the evolution of cellular complexity. This Review highlights that, despite the diversity of reported organelles, some unifying concepts underlie their formation, structure and function. Bacteria have fundamental mechanisms of organelle formation, through which conserved processes can form distinct organelles in different species depending on the proteins recruited to the luminal space and the boundary of the organelle. These complex subcellular compartments provide evolutionary advantages as well as enabling metabolic specialization, biogeochemical processes and biotechnological advances. Growing evidence suggests that the presence of organelles is the rule, rather than the exception, in bacterial cells.


Subject(s)
Bacterial Proteins/chemistry , Macromolecular Substances/chemistry , Magnetosomes/ultrastructure , Organelle Biogenesis , Organelles/ultrastructure , Bacterial Proteins/ultrastructure , Caulobacter crescentus/physiology , Caulobacter crescentus/ultrastructure , Cell Compartmentation/physiology , Cell Engineering/methods , Desulfovibrio/physiology , Desulfovibrio/ultrastructure , Escherichia coli/physiology , Escherichia coli/ultrastructure , Macromolecular Substances/ultrastructure , Magnetosomes/physiology , Magnetospirillum/physiology , Magnetospirillum/ultrastructure , Organelles/classification , Organelles/physiology , Shewanella putrefaciens/physiology , Shewanella putrefaciens/ultrastructure , Species Specificity
18.
ACS Synth Biol ; 9(7): 1705-1713, 2020 07 17.
Article in English | MEDLINE | ID: mdl-32559383

ABSTRACT

The signal processing capabilities of bacterial signaling networks offer immense potential for advanced phospho-signaling systems for synthetic biology. Emerging models suggest that complex development may require interconnections between what were once thought to be isolated signaling arrays. For example, Caulobacter crescentus achieves the feat of asymmetric division by utilizing a novel pseudokinase DivL, which senses the output of one signaling pathway to modulate a second pathway. It has been proposed that DivL reverses signal flow by exploiting conserved kinase conformational changes and protein-protein interactions. We engineered a series of DivL-based modulators to synthetically stimulate reverse signaling of the network in vivo. Stimulation of conformational changes through the DivL signal transmission helix resulted in changes to hallmark features of the network: C. crescentus motility and DivL accumulation at the cell poles. Additionally, mutations to a conserved PAS sensor transmission motif disrupted reverse signaling flow in vivo. We propose that synthetic stimulation and sensor disruption provide strategies to define signaling circuit organization principles for the rational design and validation of synthetic pathways.


Subject(s)
Bacterial Proteins/metabolism , Caulobacter crescentus/metabolism , Histidine Kinase/metabolism , Signal Transduction/genetics , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Caulobacter crescentus/physiology , Histidine Kinase/chemistry , Histidine Kinase/genetics , Leucine Zippers/genetics , Microscopy, Fluorescence , Protein Conformation
19.
J Bacteriol ; 202(17)2020 08 10.
Article in English | MEDLINE | ID: mdl-32571969

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.


Subject(s)
Bacterial Proteins/metabolism , Caulobacter crescentus/physiology , Gene Expression Regulation, Bacterial/physiology , Gene Expression Regulation, Enzymologic/physiology , Phosphoric Monoester Hydrolases/metabolism , Bacterial Proteins/genetics , Cell Cycle , Phosphoric Monoester Hydrolases/genetics
20.
Proc Natl Acad Sci U S A ; 117(17): 9546-9553, 2020 04 28.
Article in English | MEDLINE | ID: mdl-32295877

ABSTRACT

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.


Subject(s)
Bacterial Adhesion/physiology , Caulobacter crescentus/physiology , Cell Cycle/physiology , Fimbriae Proteins/metabolism , Gene Expression Regulation, Bacterial/physiology , Fimbriae Proteins/genetics
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