Swarmer Cells Research Articles

Switching and Torque Generation in Swarming E. coli.

Escherichia coli swarm on semi-solid surfaces with the aid of flagella. It has been hypothesized that swarmer cells overcome the increased viscous drag near surfaces by developing higher flagellar thrust and by promoting surface wetness with the aid of a flagellar switch. The switch enables reversals between clockwise (CW) and counterclockwise (CCW) directions of rotation of the flagellar motor. Here, we measured the behavior of flagellar motors in swarmer cells. Results indicated that although the torque was similar to that in planktonic cells, the tendency to rotate CCW was higher in swarmer cells. This suggested that swarmers likely have a smaller pool of phosphorylated CheY. Results further indicated that the upregulation of the flagellin gene was not critical for flagellar thrust or swarming. Consistent with earlier reports, moisture added to the swarm surface restored swarming in a CCW-only mutant, but not in a FliG mutant that rotated motors CW-only (FliGCW). Fluorescence assays revealed that FliGCW cells grown on agar surfaces carried fewer flagella than planktonic FliGCW cells. The surface-dependent reduction in flagella correlated with a reduction in the number of putative flagellar preassemblies. These results hint toward a possibility that the conformational dynamics of switch proteins play a role in the proper assembly of flagellar complexes and flagellar export, thereby aiding bacterial swarming.

A cell length-dependent transition in MinD-dynamics promotes a switch in division-site placement and preservation of proliferating elongated Vibrio parahaemolyticus swarmer cells.

Vibrio parahaemolyticus exists as swimmer and swarmer cells, specialized for growth in liquid and on solid environments respectively. Swarmer cells are characteristically highly elongated due to an inhibition of cell division, but still need to divide in order to proliferate and expand the colony. It is unknown how long swarmer cells divide without diminishing the population of long cells required for swarming behavior. Here we show that swarmer cells divide but the placement of the division site is cell length-dependent; short swarmers divide at mid-cell, while long swarmers switch to a specific non-mid-cell placement of the division site. Transition to non-mid-cell positioning of the Z-ring is promoted by a cell length-dependent switch in the localization-dynamics of the division regulator MinD from a pole-to-pole oscillation in short swarmers to a multi-node standing-wave oscillation in long swarmers. Regulation of FtsZ levels restricts the number of divisions to one and SlmA ensures sufficient free FtsZ to sustain Z-ring formation by preventing sequestration of FtsZ into division deficient clusters. By limiting the number of division-events to one per cell at a specific non-mid-cell position, V. parahaemolyticus promotes the preservation of long swarmer cells and permits swarmer cell division without the need for dedifferentiation.

In long bacterial cells, the Min system can act off-center.

In many rod-shaped bacteria, the Min system is well-known for generating a cell-pole to cell-pole standing wave oscillation with a single node at mid-cell to align cell division. In filamentous E. coli cells, the single-node standing wave transitions into a multi-nodal oscillation. These multi-nodal dynamics have largely been treated simply as an interesting byproduct of artificially elongated cells. However, a recent in vivo study by Muraleedharan et al. shows how multi-nodal Min dynamics are used to align non-mid-cell divisions in the elongated swarmer cells of Vibrio parahaemolyticus. The authors propose a model where the combined actions of cell-length dependent Min dynamics, in concert with nucleoid occlusion along the cell length and regulation of FtsZ levels ensures Z ring formation and complete chromosome segregation at a single off-center position. By limiting the number of cell division events to one per cell at an off-center position, long swarmer cells are preserved within a multiplying population. The findings unveil an elegant mechanism of cell-division regulation by the Min system that allows long swarmer cells to divide without the need to 'dedifferentiate'.

Positive autoregulation of the flhDC operon in Proteus mirabilis

Using a variety of techniques, we demonstrate the Class I regulator of the flagellar cascade, FlhD4C2, can activate its own expression in Proteus mirabilis. This activation was direct, as the FlhD4C2 protein specifically bound to its promoter region. The expression of bacterial genes under a positive feedback control can exhibit varying levels between cells due to stochastic fluctuations that activate the feedback loop and result in some cells in an “ON” state. Cells containing a chromosomal flhDC::gfp transcriptional fusion exhibited a heterogeneous pattern of expression within the population during growth on agar surfaces and the percentage of cells expressing GFP increased as cells approached swarmer cell differentiation. Disrupting the FlhD4C2 –mediated positive feedback loop significantly reduced the frequency of cells exhibiting GFP expression.

Swarming Migration of Co-attracting Mesenchymal Cells into Fractal-Like Epithelial Clusters

Cell-secreted factors can function as biochemical “signal relays” to coordinate collective migration over long distances. For instance, cells “co-attract” by releasing chemokines that they also chemotax towards, driving aggregation and cohesion during neural crest development, mesenchymal condensation, and neutrophil homing. Here, we show that mammary epithelial cells cultured in reduced serum conditions display a mutual attraction that results in self-organizing migration patterns. Ordinarily, the migration of spatially dispersed cells in EGF-supplemented media can be understood as a persistent random walk. Instead, cells cultured in EGF-depleted media display directed migration towards each other through co-attraction, resulting in multicellular aggregation. Subsequently, aggregated cells establish cell-cell junctions with E-cadherin, reminiscent of a mesenchymal-to-epithelial transition (MET). Furthermore, cells exhibit substantial morphological changes, extending outward protrusions towards other cells in the proximity. As a consequence, these aggregates display a highly branched, fractal-like architecture. This biological behavior has physical analogies with diffusion-limited aggregation of colloidal particles, which results in a gelation or jamming transition even at very subconfluent cell densities. We implement a computational model based on dissipative particle dynamics that recapitulates clustering and aggregation. These results may reveal new quantitative insights into swarming cell migration during neural crest development, mesenchymal condensation, and tumor metastasis.

Multi-objective structural-acoustic optimization of beams made of functionally graded materials

This paper presents a study of multi-objective structural-acoustic optimization of elastic beams made of functionally graded materials. The goal of this research is to discover the potentials to design multi-objective optimal volume fraction for better acoustic performance. The transfer matrix method is applied to obtain the structural-acoustic response of functionally graded beams. The hybrid of particle swarm optimization and cell mapping method is used to search for the Pareto optimal solutions. Two well-known distribution laws of material distribution are considered and compared with the spline interpolation of material distribution when spatially sampled material properties are used as design variables. Several cases are studied to demonstrate the multi-objective optimal design of functionally graded beams.

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells.

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ability to obtain single cells for fluorescence microscopy, which can be particularly challenging when imaging cells that exist within bacterial communities. For example, the human pathogen Vibrio parahaemolyticus exists as short rod-shaped swimmer cells in liquid conditions that upon surface contact differentiate into a subpopulation of highly elongated swarmer cells specialized for growth on solid surfaces. This paper presents a method to perform single cell fluorescence microscopy analysis of V. parahaemolyticus in its two differential states. This protocol very reproducibly induces differentiation of V. parahaemolyticus into a swarmer cell life-cycle and facilitates their proliferation over solid surfaces. The method produces flares of differentiated swarmer cells extending from the edge of the swarm-colony. Notably, at the very tip of the swarm-flares, swarmer cells exist in a single layer of cells, which allows for their easy transfer to a microscope slide and subsequent fluorescence microscopy imaging of single cells. Additionally, the workflow of image analysis for demographic representation of bacterial societies is presented. As a proof of principle, the analysis of the intracellular localization of chemotaxis signaling arrays in swimmer and swarmer cells of V. parahaemolyticus is described.

Bacillus subtilis Swarmer Cells Lead the Swarm, Multiply, and Generate a Trail of Quiescent Descendants.

ABSTRACTBacteria adopt social behavior to expand into new territory, led by specialized swarmers, before forming a biofilm. Such mass migration of Bacillus subtilis on a synthetic medium produces hyperbranching dendrites that transiently (equivalent to 4 to 5 generations of growth) maintain a cellular monolayer over long distances, greatly facilitating single-cell gene expression analysis. Paradoxically, while cells in the dendrites (nonswarmers) might be expected to grow exponentially, the rate of swarm expansion is constant, suggesting that some cells are not multiplying. Little attention has been paid to which cells in a swarm are actually multiplying and contributing to the overall biomass. Here, we show in situ that DNA replication, protein translation and peptidoglycan synthesis are primarily restricted to the swarmer cells at dendrite tips. Thus, these specialized cells not only lead the population forward but are apparently the source of all cells in the stems of early dendrites. We developed a simple mathematical model that supports this conclusion.

Differential Localization of Chemotactic Signaling Arrays during the Lifecycle of Vibrio parahaemolyticus.

When encountering new environments or changes to their external milieu, bacteria use elaborate mechanisms to respond accordingly. Here, we describe how Vibrio parahaemolyticus coordinates two such mechanisms – differentiation and chemotaxis. V. parahaemolyticus differentiates between two distinct cell types: short rod-shaped swimmer cells and highly elongated swarmer cells. We show that the intracellular organization of chemotactic signaling arrays changes according to the differentiation state. In swimmer cells chemotaxis arrays are strictly polarly localized, but in swarmer cells arrays form both at the cell poles and at irregular intervals along the entire cell length. Furthermore, the formation of lateral arrays increases with cell length of swarmer cells. Occurrence of lateral signaling arrays is not simply a consequence of the elongated state of swarmer cells, but is instead differentiation state-specific. Moreover, our data suggest that swarmer cells employ two distinct mechanisms for localization of polar and lateral signaling arrays, respectively. Furthermore, cells show a distinct differentiation and localization pattern of chemosensory arrays, depending on their location within swarm colonies, which likely allows for the organism to simultaneously swarm across surfaces while sustaining a pool of swimmers immediately capable of exploring new liquid surroundings.

FlhF Is Required for Swarming Motility and Full Pathogenicity of Bacillus cereus.

Besides sporulation, Bacillus cereus can undergo a differentiation process in which short swimmer cells become elongated and hyperflagellated swarmer cells that favor migration of the bacterial community on a surface. The functionally enigmatic flagellar protein FlhF, which is the third paralog of the signal recognition particle (SRP) GTPases Ffh and FtsY, is required for swarming in many bacteria. Previous data showed that FlhF is involved in the control of the number and positioning of flagella in B. cereus. In this study, in silico analysis of B. cereus FlhF revealed that this protein presents conserved domains that are typical of SRPs in many organisms and a peculiar N-terminal basic domain. By proteomic analysis, a significant effect of FlhF depletion on the amount of secreted proteins was found with some proteins increased (e.g., B component of the non-hemolytic enterotoxin, cereolysin O, enolase) and others reduced (e.g., flagellin, L2 component of hemolysin BL, bacillolysin, sphingomyelinase, PC-PLC, PI-PLC, cytotoxin K) in the extracellular proteome of a ΔflhF mutant. Deprivation of FlhF also resulted in significant attenuation in the pathogenicity of this strain in an experimental model of infection in Galleria mellonella larvae. Our work highlights the multifunctional role of FlhF in B. cereus, being this protein involved in bacterial flagellation, swarming, protein secretion, and pathogenicity.

Defining the daughter swarmer cell

A DNA-binding protein may organize DNA to enable Caulobacter to divide into morphologically different daughter cells.

Cell cycle progression in Caulobacter requires a nucleoid-associated protein with high AT sequence recognition

Faithful cell cycle progression in the dimorphic bacterium Caulobacter crescentus requires spatiotemporal regulation of gene expression and cell pole differentiation. We discovered an essential DNA-associated protein, GapR, that is required for Caulobacter growth and asymmetric division. GapR interacts with adenine and thymine (AT)-rich chromosomal loci, associates with the promoter regions of cell cycle-regulated genes, and shares hundreds of recognition sites in common with known master regulators of cell cycle-dependent gene expression. GapR target loci are especially enriched in binding sites for the transcription factors GcrA and CtrA and overlap with nearly all of the binding sites for MucR1, a regulator that controls the establishment of swarmer cell fate. Despite constitutive synthesis, GapR accumulates preferentially in the swarmer compartment of the predivisional cell. Homologs of GapR, which are ubiquitous among the α-proteobacteria and are encoded on multiple bacteriophage genomes, also accumulate in the predivisional cell swarmer compartment when expressed in Caulobacter The Escherichia coli nucleoid-associated protein H-NS, like GapR, selectively associates with AT-rich DNA, yet it does not localize preferentially to the swarmer compartment when expressed exogenously in Caulobacter, suggesting that recognition of AT-rich DNA is not sufficient for the asymmetric accumulation of GapR. Further, GapR does not silence the expression of H-NS target genes when expressed in E. coli, suggesting that GapR and H-NS have distinct functions. We propose that Caulobacter has co-opted a nucleoid-associated protein with high AT recognition to serve as a mediator of cell cycle progression.

Visualizing Flagella while Tracking Bacteria

A complete description of the swimming behavior of a bacterium requires measurement of the displacement and orientation of the cell body together with a description of the movement of the flagella. We rebuilt a tracking microscope so that we could visualize flagellar filaments of tracked cells by fluorescence. We studied Escherichia coli (cells of various lengths, including swarm cells), Bacillus subtilis (wild-type and a mutant with fewer flagella), and a motile Streptococcus (now Enterococcus). The run-and-tumble statistics were nearly the same regardless of cell shape, length, and flagellation; however, swarm cells rarely tumbled, and cells of Enterococcus tended to swim in loops when moving slowly. There were events in which filaments underwent polymorphic transformations but remained in bundles, leading to small deflections in direction of travel. Tumble speeds were ∼2/3 as large as run speeds, and the rates of change of swimming direction while running or tumbling were smaller when cells swam more rapidly. If a smaller fraction of filaments were involved in tumbles, the tumble intervals were shorter and the angles between runs were smaller.

Caulobacter crescentus intrinsic dimorphism provides a prompt bimodal response to copper stress.

Stress response to fluctuating environments often implies a time-consuming reprogramming of gene expression. In bacteria, the so-called bet hedging strategy, which promotes phenotypic stochasticity within a cell population, is the only fast stress response described so far(1). Here, we show that Caulobacter crescentus asymmetrical cell division allows an immediate bimodal response to a toxic metals-rich environment by allocating specific defence strategies to morphologically and functionally distinct siblings. In this context, a motile swarmer cell favours negative chemotaxis to flee from a copper source, whereas a sessile stalked sibling engages a ready-to-use PcoAB copper homeostasis system, providing evidence of a prompt stress response through intrinsic bacterial dimorphism.

A Bacterial Pathogen Displaying Temperature-Enhanced Virulence of the Microalga Emiliania huxleyi.

Emiliania huxleyi is a globally abundant microalga that plays a significant role in biogeochemical cycles. Over the next century, sea surface temperatures are predicted to increase drastically, which will likely have significant effects on the survival and ecology of E. huxleyi. In a warming ocean, this microalga may become increasingly vulnerable to pathogens, particularly those with temperature-dependent virulence. Ruegeria is a genus of Rhodobacteraceae whose population size tracks that of E. huxleyi throughout the alga’s bloom–bust lifecycle. A representative of this genus, Ruegeria sp. R11, is known to cause bleaching disease in a red macroalga at elevated temperatures. To investigate if the pathogenicity of R11 extends to microalgae, it was co-cultured with several cell types of E. huxleyi near the alga’s optimum (18°C), and at an elevated temperature (25°C) known to induce virulence in R11. The algal populations were monitored using flow cytometry and pulse-amplitude modulated fluorometry. Cultures of algae without bacteria remained healthy at 18°C, but lower cell counts in control cultures at 25°C indicated some stress at the elevated temperature. Both the C (coccolith-bearing) and S (scale-bearing swarming) cell types of E. huxleyi experienced a rapid decline resulting in apparent death when co-cultured with R11 at 25°C, but had no effect on N (naked) cell type at either temperature. R11 had no initial negative impact on C and S type E. huxleyi population size or health at 18°C, but caused death in older co-cultures. This differential effect of R11 on its host at 18 and 25°C suggest it is a temperature-enhanced opportunistic pathogen of E. huxleyi. We also detected caspase-like activity in dying C type cells co-cultured with R11, which suggests that programmed cell death plays a role in the death of E. huxleyi triggered by R11 – a mechanism induced by viruses (EhVs) and implicated in E. huxleyi bloom collapse. Given that E. huxleyi has recently been shown to have acquired resistance against EhVs at elevated temperature, bacterial pathogens with temperature-dependent virulence, such as R11, may become much more important in the ecology of E. huxleyi in a warming climate.

Light and electron microscopic observations of the reproductive swarmer cells of nassellarian and spumellarian polycystines (Radiolaria)

We observed reproductive swarmer cells of the nassellarian and spumellarian polycystine radiolarians Didymocyrtis ceratospyris, Pterocanium praetextum, Tetrapyle sp., and Triastrum aurivillii using light, scanning and transmission electron microscopy. The swarmer cells had subspherical to ovoid or spindle shapes with two unequal flagella tapered to whip-like ends. The cell size was approximately 2.5–5.5μm long and 1.6–2.2μm wide, which is significantly smaller than that of the collodarian (colonial or naked) polycystine radiolarians. Transmission electron microscopy revealed that the swarmer cells possessed a nucleus, mitochondria with tubular cristae, Golgi body, and small lipid droplets in the cytoplasm; they also had a large vacuole in which a single crystalline inclusion (approx. 1.0–1.5μm) that was probably celestite (SrSO4) was enclosed. The swarmer cells were released directly from the parent cells. At that time, morphological change such as encystment was not observed in the parent cells, and the axopodia remained extended in a period of swarmer reproduction for floating existence. This may have prevented the polycystine swarmers from rapidly sinking down to great depths. Thus, we concluded that the polycystine radiolarians release the swarmer cells into the photic layer in the same way as the symbiotic acantharians.

The Rcs regulon in Proteus mirabilis: implications for motility, biofilm formation, and virulence.

The overall role of the Rcs phosphorelay in Proteus mirabilis is largely unknown. Previous work had demonstrated that the Rcs phosphorelay represses the flhDC operon and activates the minCDE cell division inhibition system. To identify additional cellular functions regulated by the Rcs phosphorelay, an analysis of RNA-seq data was undertaken. In this report, the results of the RNA-sequencing are discussed with an emphasis on the predicted roles of the Rcs phosphorelay in swarmer cell differentiation, motility, biofilm formation, and virulence. RcsB is shown to activate genes important for differentiation and fimbriae formation, while repressing the expression of genes important for motility and virulence. Additionally, to follow up on the RNA-Seq data, we demonstrate that an rcsB mutant is deficient in its ability to form biofilm and exhibits enhanced virulence in a Galleria mellonella waxworm model. Overall, these results indicate the Rcs regulon in P. mirabilis extends beyond flagellar genes to include those involved in biofilm formation and virulence. Furthermore, the information presented in this study may provide clues to additional roles of the Rcs phosphorelay in other members of the Enterobacteriaceae.

Dynamics of Snake-like Swarming Behavior of Vibrio alginolyticus

Swarming represents a special case of bacterial behavior where motile bacteria migrate rapidly and collectively on surfaces. Swarming and swimming motility of bacteria has been studied well for rigid, self-propelled rods. In this study we report a strain of Vibrio alginolyticus, a species that exhibits similar collective motility but a fundamentally different cell morphology with highly flexible snake-like swarming cells. Investigating swarming dynamics requires high-resolution imaging of single cells with coverage over a large area: thousands of square microns. Researchers previously have employed various methods of motion analysis but largely for rod-like bacteria. We employ temporal variance analysis of a short time-lapse microscopic image series to capture the motion dynamics of swarming Vibrio alginolyticus at cellular resolution over hundreds of microns. Temporal variance is a simple and broadly applicable method for analyzing bacterial swarming behavior in two and three dimensions with both high-resolution and wide-spatial coverage. This study provides detailed insights into the swarming architecture and dynamics of Vibrio alginolyticus isolate B522 on carrageenan agar that may lay the foundation for swarming studies of snake-like, nonrod-shaped motile cell types.

Bacterial swarmer cells in confinement: a mesoscale hydrodynamic simulation study.

A wide spectrum of Peritrichous bacteria undergo considerable physiological changes when they are inoculated onto nutrition-rich surfaces and exhibit a rapid and collective migration denoted as swarming. Thereby, the length of such swarmer cells and their number of flagella increases substantially. In this article, we investigated the properties of individual E. coli-type swarmer cells confined between two parallel walls via mesoscale hydrodynamic simulations, combining molecular dynamics simulations of the swarmer cell with the multiparticle particle collision dynamics approach for the embedding fluid. E. coli-type swarmer cells are three-times longer than their planktonic counter parts, but their flagella density is comparable. By varying the wall separation, we analyze the confinement effect on the flagella arrangement, on the distribution of cells in the gap between the walls, and on the cell dynamics. We find only a weak dependence of confinement on the bundle structure and dynamics. The distribution of cells in the gap changes from a geometry-dominated behavior for very narrow to fluid-dominated behavior for wider gaps, where cells are preferentially located in the gap center for narrower gaps and stay preferentially next to one of the walls for wider gaps. Dynamically, the cells exhibit a wide spectrum of migration behaviors, depending on their flagella bundle arrangement, and ranges from straight swimming to wall rolling.

Mycosphere Essays 3. Myxomycete spore and amoeboflagellate biology: a review

The spore and amoeboflagellate stages of the heterothallic myxomycete life cycle are generally a uninucleate haploid alternative to the multinucleate diploid plasmodial stage. The spore is produced in a sporangium which develops from the plasmodium, and is a dispersal and resistant stage which has ornamentations that can be used in taxonomy. These small spores have thick pigmented walls and are generally wind dispersed and require water and other biological and physical conditions in order to germinate; this dispersal can occur over long distances and the germination conditions apparently vary between and within a species. The amoeboflagellate stage has three alternative phases: myxamoeba, swarm cell and cyst. The myxamoeba is a typical pleomorphic amoeboid cell that is the major phase, since it is the vegetative phase that ingests bacteria and yeasts, and divides mitotically to form a clonal population of cells. The myxamoeba of heterothallic strains can also, after reaching a certain cell density, become sexually competent and fuse to produce the plasmodial stage. The swarm cell phase develops from the myxamoebal stage when free water is present; it also feeds on bacteria but does not divide unless it reconverts to the myxamoebal phase. The cyst phase develops from the myxamoebal stage, when growth conditions become adverse, by the condensing of the cell and the formation of a resistant wall.