Swarmer Cells Research Articles

Paired high‐throughput, in situ imaging and high‐throughput sequencing illuminate acantharian abundance and vertical distribution

AbstractAcantharians (supergroup Rhizaria) can be important contributors to surface primary production and to carbon flux to the deep sea, but are often underestimated because their delicate structures are destroyed by plankton nets or dissolved by chemical fixatives. As they are also uncultured, relatively little is known about acantharian biology, especially regarding their life cycles. Here, we take a paired approach, bringing together high‐throughput, in situ imaging and metabarcode sequencing, to investigate acantharian abundance, vertical distribution, and life history in the western North Pacific. Concentrations of imaged acantharian cells correlated well with relative abundances of 18S rRNA gene sequences from acantharians with known, recognizable morphologies, but not to sequences corresponding to acantharians with unknown morphology. These results suggest that morphologically undescribed clades may lack the characteristic star‐shaped acantharian skeleton or are much smaller than described acantharians. The smaller size of acantharians imaged at depth supports current hypotheses regarding nonsymbiotic acantharian life cycles: cysts or vegetative cells release reproductive swarmer cells in deep water and juvenile cells grow as they ascend toward the surface. Moreover, sequencing data present the possibility that some photosymbiotic acantharians may also reproduce at depth, like their nonsymbiotic, encysting relatives, which is counter to previous hypotheses. Finally, in situ imaging captured a new acantharian behavior that may be a previously undescribed predation strategy.

Swarming Motility Without Flagellar Motor Switching by Reversal of Swimming Direction in E. coli.

In a crowded environment such as a bacterial swarm, cells frequently got jammed and came to a stop, but were able to escape the traps by backing up in their moving course with a head-to-tail change (a reversal). Reversals are essential for the expansion of a bacterial swarm. Reversal for a wildtype cell usually involved polymorphic transformation of the flagellar filaments induced by directional switching of the flagellar motors. Here we discovered a new way of reversal in cells without motor switching and characterized its mechanisms. We further found that this type of reversal was not limited to swarmer cells, but also occurred for cells grown in a bulk solution. Therefore, reversal was a general way of escaping when cells got jammed in their natural complex habitats. The new way of reversal we discovered here offered a general strategy for cells to escape traps and explore their environment.

Specific proteomic adaptation to distinct environments in Vibrio parahaemolyticus includes significant fluctuations in expression of essential proteins.

Bacteria constantly experience changes to their external milieu and need to adapt accordingly to ensure their survival. Certain bacteria adapt by means of cellular differentiation, resulting in the development of a specific cell type that is specialized for life in a distinct environment. Furthermore, to understand how bacteria adapt, it is essential to appreciate the significant changes that occur at the proteomic level. By analysing the proteome of our model organism Vibrio parahaemolyticus from distinct environmental conditions and cellular differential states, we demonstrate that the proteomic expression profile is highly flexible, which likely allows it to adapt to life in different environmental conditions and habitats. We show that, even within the same swarm colony, there are specific zones of cells with distinct expression profiles. Furthermore, our data indicate that cell surface attachment and swarmer cell differentiation are distinct programmes that require specific proteomic expression profiles. This likely allows V. parahaemolyticus to adapt to life in different environmental conditions and habitats. Finally, our analyses reveal that the expression profile of the essential protein pool is highly fluid, with significant fluctuations that dependent on the specific life-style, environment and differentiation state of the bacterium.

Vibrio parahaemolyticus FcrX, a Fur-controlled regulator that inhibits repression by Fur.

Iron is an essential nutrient for most organisms, but its limited availability and inherent toxicity necessitate the strict regulation of iron homeostasis. In bacteria, iron starvation affects a broad range of phenotypes including virulence, motility and biofilm formation. For Vibrio parahaemolyticus, a marine bacterium and pathogen, iron limitation is a signal modulating swarmer cell differentiation. In this work, we show the iron regulation of swarming works through the ferric uptake regulator protein Fur. We identified a new Fur-controlled regulator that is upregulated upon iron starvation. FcrX is a 144-amino acid protein containing a domain of unknown function (DUF2753) with three tetratricopeptide repeats. We found that overexpressing fcrX+ was sufficient to induce swarming, luminescence and iron uptake gene expression in multiple Vibrio species; furthermore, ectopic expression increased the transcription of a Fur-controlled gene in Escherichia coli. FcrX production increased intracellular iron. Thus, the overexpression of fcrX+ phenocopied a fur mutant and may prove a generally useful tool to ectopically derepress the Fur regulon. Both V. parahaemolyticus and E. coli Fur interacted with FcrX, and this interaction was altered by iron availability. These data support a model in which this new regulator of iron homeostasis limits the repressive action of Fur.

Robust Modulation of a Bacterial Kinase by Protein Phase Separation

The diversity of molecular function and material properties in cytoplasmic biomolecular condensates contributes to the robustness of enzymatic interactions in a crowded cellular environment. However, molecular principles that organize and control chemical reactions in these condensates are poorly understood. We utilized a multi-scale approach combining biochemical reconstitution and single molecule imaging to gain insights into a kinase signaling reaction localized in a phase separated polar microdomain in the Gram-negative bacterium Caulobacter crescentus. Caulobacter divides asymmetrically to yield a sessile stalked cell and a flagellated swarmer cell. Much of this asymmetric division is regulated through different signaling pathways that reside in microdomains at the two cell poles prior to cell division. Specifically, at the stalked pole, two intrinsically disordered proteins, PopZ and SpmX, form a stable complex and sequester the histidine kinase DivJ whose autophosphorylation contributes to the regulation of cell cycle progression. Here, I will discuss the biophysical basis for phase separation of SpmX and PopZ and the implications of this phenomenon in the context of the Caulobacter cell cycle. Using biochemistry and state of the art 3-D single molecule imaging, we have identified molecular features that regulate DivJ diffusion and autophosphorylation in the polar microdomain. We further establish the role of protein topology, crowding, and the extracellular environment on DivJ regulation via phase separation. Our results underscore the relationship between enzyme activity and unstructured biomolecular condensates that govern the physical environment in signaling hubs for robust modulation of biochemical reactions a crowded cytoplasm.

A Transitory Signaling Center Controls Timing of Primordial Germ Cell Differentiation

Organogenesis requires exquisite spatio-temporal coordination of cell morphogenesis, migration, proliferation and differentiation of multiple cell types. For gonads, this involves complex interactions between somatic and germline tissues. During Drosophila ovary morphogenesis primordial germ cells (PGCs) are either sequestered in stem cell niches and maintained in an undifferentiated, germline stem cell state, or transition directly towards differentiation. Here, we identify a mechanism that links hormonal triggers of somatic tissue morphogenesis with PGC differentiation. An early ecdysone pulse initiates somatic swarm cell (SwC) migration, positioning them close to PGCs. A second hormone peak activates Torso-like signal in SwCs, which stimulates the Torso RTK signaling pathway in PGCs promoting their differentiation by de-repression of the differentiation gene bag of marbles. Thus, systemic temporal cues generate a transitory signaling center that coordinate ovarian morphogenesis with stem cell self-renewal and differentiation programs, a concept applicable broadly to the integration of stem cells and their niches.

Inhibitors of Bacterial Swarming Behavior.

Bacteria can migrate in groups of flagella‐driven cells over semisolid surfaces. This coordinated form of motility is called swarming behavior. Swarming is associated with enhanced virulence and antibiotic resistance of various human pathogens and may be considered as favorable adaptation to the diverse challenges that microbes face in rapidly changing environments. Consequently, the differentiation of motile swarmer cells is tightly regulated and involves multi‐layered signaling networks. Controlling swarming behavior is of major interest for the development of novel anti‐infective strategies. In addition, compounds that block swarming represent important tools for more detailed insights into the molecular mechanisms of the coordination of bacterial population behavior. Over the past decades, there has been major progress in the discovery of small‐molecule modulators and mechanisms that allow selective inhibition of swarming behavior. Herein, an overview of the achievements in the field and future directions and challenges will be presented.

The release of a distinct cell type from swarm colonies facilitates dissemination of Vibrio parahaemolyticus in the environment.

Bacteria experience changes in their environment and have developed various strategies to respond accordingly. To accommodate environmental changes, certain bacteria differentiate between specialized cell types. Vibrio parahaemolyticus is a marine bacterium, a worldwide human pathogen and the leading agent of seafood-borne gastroenteritis. It exists as swimmer or swarmer cells, specialized for life in liquid and on solid environments, respectively. Swarmer cells are characteristically highly elongated-a morphology important for swarming behavior. When attached to surfaces it forms swarm colonies, however, it is not known how cells within swarming populations respond to changes in the external milieu and how its distinct life cycle influences its ecological dissemination. The worldwide distribution of V. parahaemolyticus accentuates the need for understanding the factors contributing to its dissemination. Here we determine the stage-wise development of swarm colonies and show how the swarm colony architecture fluctuates with changing environmental conditions. Swarm colonies act as a continuous source of cells that are released from the swarm colony into the environment. Surprisingly, the cell length distribution of released cells was very homogenous and almost no long cells were detected, indicating that swarmer cells are not released into the liquid environment but stay surface attached during flooding. Released cells comprise a distinct cell type that is morphologically optimized for swimming behavior and is capable of spreading in the liquid environment and attach to new surfaces. Release of this distinct cell type facilitates the dissemination of V. parahaemolyticus in the environment and likely influences the ecology of this bacterium.

Bacterial Swarming Reduces Proteus mirabilis and Vibrio parahaemolyticus Cell Stiffness and Increases β-Lactam Susceptibility

Swarmer cells of the Gram-negative uropathogenic bacteria Proteus mirabilis and Vibrio parahaemolyticus become long (>10 to 100 μm) and multinucleate during their growth and motility on polymer surfaces. We demonstrated that the increasing cell length is accompanied by a large increase in flexibility. Using a microfluidic assay to measure single-cell mechanics, we identified large differences in the swarmer cell stiffness (bending rigidity) of P. mirabilis (5.5 × 10-22 N m2) and V. parahaemolyticus (1.0 × 10-22 N m2) compared to vegetative cells (1.4 × 10-20 N m2 and 2.2 × 10-22 N m2, respectively). The reduction in bending rigidity (∼2-fold to ∼26-fold) was accompanied by a decrease in the average polysaccharide strand length of the peptidoglycan layer of the cell wall from 28 to 30 disaccharides to 19 to 22 disaccharides. Atomic force microscopy revealed a reduction in P. mirabilis peptidoglycan thickness from 1.5 nm (vegetative cells) to 1.0 nm (swarmer cells), and electron cryotomography indicated changes in swarmer cell wall morphology. P. mirabilis and V. parahaemolyticus swarmer cells became increasingly sensitive to osmotic pressure and susceptible to cell wall-modifying antibiotics (compared to vegetative cells)-they were ∼30% more likely to die after 3 h of treatment with MICs of the β-lactams cephalexin and penicillin G. The adaptive cost of "swarming" was offset by the increase in cell susceptibility to physical and chemical changes in their environment, thereby suggesting the development of new chemotherapies for bacteria that leverage swarming for the colonization of hosts and for survival.IMPORTANCE Proteus mirabilis and Vibrio parahaemolyticus are bacteria that infect humans. To adapt to environmental changes, these bacteria alter their cell morphology and move collectively to access new sources of nutrients in a process referred to as "swarming." We found that changes in the composition and thickness of the peptidoglycan layer of the cell wall make swarmer cells of P. mirabilis and V. parahaemolyticus more flexible (i.e., reduce cell stiffness) and that they become more sensitive to osmotic pressure and cell wall-targeting antibiotics (e.g., β-lactams). These results highlight the importance of assessing the extracellular environment in determining antibiotic doses and the use of β-lactam antibiotics for treating infections caused by swarmer cells of P. mirabilis and V. parahaemolyticus.

Life Cycles of Myxogastria Stemonitopsis typhina and Stemonitis fusca on Agar Culture.

Myxogastria is a group of protozoa characterized by cellular uninucleate amoeboflagellates (myxamoebae and flagellated swarm cell), acellular multinucleate plasmodia, and stationary spore‐bearing sporocarps. The Stemonitales is a large order in the Myxogastria and contains approximately 230 species, but only 13 species have their completed life cycles observed so far. Here, we described the life cycles of two species in Stemonitales, Stemonitopsis typhina and Stemonitis fusca by culturing in water agar medium and observing the morphogenesis of their spore germination, plasmodium, and sporocarp development. The spore‐to‐spore life cycles of Ste. typhina and S. fusca were completed in approximately 67 and 12 d, respectively. Both species possessed an aphanoplasmodium. However, the spores of Ste. typhina and S. fusca germinated by the V‐shape split and pore methods, respectively. Unlike S. fusca with an evanescent peridium, Ste. typhina produced a shiny persistent peridium which was continuous with the membrane surrounding its stalk. The information will contribute to a better understanding of their taxonomy and phylogeny.

Sulfur Assimilation Alters Flagellar Function and Modulates the Gene Expression Landscape of Serratia marcescens.

Sulfur is an essential nutrient that contributes to cellular redox homeostasis, transcriptional regulation, and translation initiation when incorporated into different biomolecules. Transport and reduction of extracellular sulfate followed by cysteine biosynthesis is a major pathway of bacterial sulfur assimilation. For the opportunistic pathogen Serratia marcescens, function of the cysteine biosynthesis pathway is required for extracellular phospholipase activity and flagellum-mediated surface motility, but little else is known about the influence of sulfur assimilation on the physiology of this organism. In this work, it was determined that an S. marcescens cysteine auxotroph fails to differentiate into hyperflagellated and elongated swarmer cells and that cysteine, but not other organic sulfur molecules, restores swarming motility to these bacteria. The S. marcescens cysteine auxotroph further exhibits reduced transcription of phospholipase, hemolysin, and flagellin genes, each of which is subject to transcriptional control by the flagellar regulatory system. Based on these data and the central role of cysteine in sulfur assimilation, it was reasoned that environmental sulfur availability may contribute to the regulation of these functions in S. marcescens Indeed, bacteria that are starved for sulfate exhibit substantially reduced transcription of the genes for hemolysin, phospholipase, and the FlhD flagellar master regulator. A global transcriptomic analysis further defined a large set of S. marcescens genes that are responsive to extracellular sulfate availability, including genes that encode membrane transport, nutrient utilization, and metabolism functions. Finally, sulfate availability was demonstrated to alter S. marcescens cytolytic activity, suggesting that sulfate assimilation may impact the virulence of this organism.IMPORTANCE Serratia marcescens is a versatile bacterial species that inhabits diverse environmental niches and is capable of pathogenic interactions with host organisms ranging from insects to humans. This report demonstrates for the first time the extensive impacts that environmental sulfate availability and cysteine biosynthesis have on the transcriptome of S. marcescens The finding that greater than 1,000 S. marcescens genes are differentially expressed depending on sulfate availability suggests that sulfur abundance is a crucial factor that controls the physiology of this organism. Furthermore, the high relative expression levels for the putative virulence factors flagella, phospholipase, and hemolysin in the presence of sulfate suggests that a sulfur-rich host environment could contribute to the transcription of these genes during infection.

Asymmetric division yields progeny cells with distinct modes of regulating cell cycle-dependent chromosome methylation

The cell cycle-regulated methylation state of Caulobacter DNA mediates the temporal control of transcriptional activation of several key regulatory proteins. Temporally controlled synthesis of the CcrM DNA methyltransferase and Lon-mediated proteolysis restrict CcrM to a specific time in the cell cycle, thereby allowing the maintenance of the hemimethylated state of the chromosome during the progression of DNA replication. We determined that a chromosomal DNA-based platform stimulates CcrM degradation by Lon and that the CcrM C terminus both binds to its DNA substrate and is recognized by the Lon protease. Upon asymmetric cell division, swarmer and stalked progeny cells employ distinct mechanisms to control active CcrM. In progeny swarmer cells, CcrM is completely degraded by Lon before its differentiation into a replication-competent stalked cell later in the cell cycle. In progeny stalked cells, however, accumulated CcrM that has not been degraded before the immediate initiation of DNA replication is sequestered to the cell pole. Single-molecule imaging demonstrated physical anticorrelation between sequestered CcrM and chromosomal DNA, thus preventing DNA remethylation. The distinct control of available CcrM in progeny swarmer and stalked cells serves to protect the hemimethylated state of DNA during chromosome replication, enabling robustness of cell cycle progression.

The life cycles of two species of Myxomycetes in Physarales, Physarum rigidum and Didymium squamulosum

Myxomycetes are eukaryotic microorganisms containing characteristics akin to both fungi and amoebae. They can complete their whole life cycles while being cultured on agar media, and under-laboratory conditions, which favors taxonomic, phylogenetic, and cytological researches. Here, we describe the life cycles of two such species: Didymium squamulosum collected from the field and Physarum rigidum cultured from moist chamber both belonging to the Order Physarales. Three percent oat-agar media (OAM) was used to culture the plasmodia until they aggregated and were almost starved. Natural light was then applied to the plasmodia to induce fructification. Their life cycles share the same common stages, namely: spore, myxamoebae, swarm cell, plasmodia, and sporulation. In this study, we describe the morphogenesis from spore to spore of two species by differential interference contrast (DIC) and stereoscopic microscopies, as well as discuss the differences between the development of both species and interspecies. We found that the spore germination method of both species was the same. However, there were differences noted in time taken and fruiting body formation. Unlike P. rigidum, the species D. squamulosum did not require natural light stimulation. Moreover, the maturation process of both species had similar color transitions but exhibited distinct morphology in each developmental stage except during the swarm cell stage.

Microfluidic Synchronizer Using a Synthetic Nanoparticle-Capped Bacterium.

Conventional techniques to synchronize bacterial cells often require manual manipulations and lengthy incubation lacking precise temporal control. An automated microfluidic device was recently developed to overcome these limitations. However, it exploits the stalk property of Caulobacter crescentus that undergoes asymmetric stalked and swarmer cell cycle stages and is therefore restricted to this species. To address this shortcoming, we have engineered Escherichia coli cells to adhere to microchannel walls via a synthetic and inducible "stalk". The pole of E. coli is capped by magnetic fluorescent nanoparticles via a polar-localized outer membrane protein. A mass of cells is immobilized in a microfluidic chamber by an externally applied magnetic field. Daughter cells are formed without the induced stalk and hence are flushed out, yielding a synchronous population of "baby" cells. The stalks can be tracked by GFP and nanoparticle fluorescence; no fluorescence signal is detected in the eluted cell population, indicating that it consists solely of daughters. The collected daughter cells display superb synchrony. The results demonstrate a new on-chip method to synchronize the model bacterium E. coli and likely other bacterial species, and also foster the application of synthetic biology to the study of the bacterial cell cycle.

Pseudomonas aeruginosa Swarmer Cells Adaptation Toward UVc Radiations.

Swarming is the most rapid surface motility allowing Pseudomonas aeruginosa bacteria to rapidly colonize new surfaces. However, swarming behavior is affected by environmental factors like ultraviolet irradiation (UVc). UVc radiation is the most disinfection technology usually applied for wastewater and proven to be effective to inactivate microorganisms. However, efficiency against motile bacteria is not yet studied. This study aims to explain the mechanisms of resistance of swarmer P. aeruginosa cells toward UVc exposure. P. aeruginosa liquid cultures were allowed to swarm across a semisolid surface for 18 h and directly exposed to UVc radiations. Emergent swarmer colonies, revealed after re-incubation, were selected to study biofilm formation, fatty acid (FA) composition, and ultrastructure. Our results showed that membrane adaptation to UVc radiations was seen in Pseudomonas cells by an increase of cyclic fatty acid (CFA) content, confirming the role of cyclopropane in radio-resistance of swarmer cells. Furthermore, electron microscopic study confirmed that over production of S-layer is believed to be a protective form adopted by P. aeruginosa swarmer cells to resist after 5 min of UVc exposure. Moreover, membrane disintegration is the lethal effect observed after 15 min of UVc exposure. In the other hand, study of biofilm production showed an enhancement of biofilm formation, of swarmer cells mainly after 15 min of UVc exposure. There results confirmed that swarming process is highly correlated with particular FA composition of P. aeruginosa membrane and that radio-resistance of swarmer cells is highly supported by CFA biosynthesis and S-layer overproduction.

Knockdown of Dinoflagellate Cellulose Synthase CesA1 Resulted in Malformed Intracellular Cellulosic Thecal Plates and Severely Impeded Cyst-to-Swarmer Transition.

Cellulose synthesis (CS) is conducted by membrane-bound cellulose synthase complexes (CSCs), containing cellulose synthases (CesA), that are either arranged in hexagonal structures in higher plants or in linear arrays in most microbial organisms, including dinoflagellates. Dinoflagellates are a major phytoplankton group having linear-type CSCs and internal cellulosic thecal plates (CTPs) in large cortical vesicles. Immunological study suggested CesA1p were cortically localized to the periphery of CTPs. During cyst-to-swarmer transition (TC–S), synchronized peaks of CesA1 transcription, CesA1p expression, CS and CTP formation occurred in respective order, over 12–16 h, strategically allowing the study of CS regulation and CTP biogenesis. CesA1-knockdown resulted in 40% reduction in CesA1p level and time required for swarmer cells reappearance. CTPs were severely malformed with reduced cellulose content. As CTPs are deposited in internal organelle, the present study demonstrated dinoflagellate CesA1 ortholog was adapted for non-surface deposition; this is different to paradigm of other CesAps which require plasmamembrane for cellulose fiber deposition. This pioneer gene-knockdown study demonstrated the requirement of a gene for dinoflagellate cell wall remodeling and proper TC–S, which are prominent in dinoflagellate life-cycles.

Multilayered control of chromosome replication in Caulobacter crescentus.

The environmental Alphaproteobacterium Caulobacter crescentus is a classical model to study the regulation of the bacterial cell cycle. It divides asymmetrically, giving a stalked cell that immediately enters S phase and a swarmer cell that stays in the G1 phase until it differentiates into a stalked cell. Its genome consists in a single circular chromosome whose replication is tightly regulated so that it happens only in stalked cells and only once per cell cycle. Imbalances in chromosomal copy numbers are the most often highly deleterious, if not lethal. This review highlights recent discoveries on pathways that control chromosome replication when Caulobacter is exposed to optimal or less optimal growth conditions. Most of these pathways target two proteins that bind directly onto the chromosomal origin: the highly conserved DnaA initiator of DNA replication and the CtrA response regulator that is found in most Alphaproteobacteria. The concerted inactivation and proteolysis of CtrA during the swarmer-to-stalked cell transition license cells to enter S phase, while a replisome-associated Regulated Inactivation and proteolysis of DnaA (RIDA) process ensures that initiation starts only once per cell cycle. When Caulobacter is stressed, it turns on control systems that delay the G1-to-S phase transition or the elongation of DNA replication, most probably increasing its fitness and adaptation capacities.

Testing the Ability of Compounds to Induce Swarming.

One of the most distinctive features of Proteus mirabilis is its ability to undergo differentiation from short, rod-shaped vegetative cells with peritrichous flagella to massively elongated swarm cells that express hundreds to thousands of flagella. The unique bull's-eye pattern that forms from cycles of active swarming and consolidation back to the vegetative state has long been a distinguishing characteristic of this species. Many factors involved in regulation of flagellar synthesis and swarm cell differentiation have been characterized, but the exact conditions sensed by P. mirabilis that send a signal to initiate differentiation and motility have yet to be fully elucidated. Here we describe a method for using several types of media to investigate compounds that induce swarming motility under conditions that would not normally be permissive.

Pathogenesis of Proteus mirabilis Infection.

Proteus mirabilis, a Gram-negative rod-shaped bacterium most noted for its swarming motility and urease activity, frequently causes catheter-associated urinary tract infections (CAUTIs) that are often polymicrobial. These infections may be accompanied by urolithiasis, the development of bladder or kidney stones due to alkalinization of urine from urease-catalyzed urea hydrolysis. Adherence of the bacterium to epithelial and catheter surfaces is mediated by 17 different fimbriae, most notably MR/P fimbriae. Repressors of motility are often encoded by these fimbrial operons. Motility is mediated by flagella encoded on a single contiguous 54-kb chromosomal sequence. On agar plates, P. mirabilis undergoes a morphological conversion to a filamentous swarmer cell expressing hundreds of flagella. When swarms from different strains meet, a line of demarcation, a "Dienes line," develops due to the killing action of each strain's type VI secretion system. During infection, histological damage is caused by cytotoxins including hemolysin and a variety of proteases, some autotransported. The pathogenesis of infection, including assessment of individual genes or global screens for virulence or fitness factors has been assessed in murine models of ascending urinary tract infections or CAUTIs using both single-species and polymicrobial models. Global gene expression studies performed in culture and in the murine model have revealed the unique metabolism of this bacterium. Vaccines, using MR/P fimbria and its adhesin, MrpH, have been shown to be efficacious in the murine model. A comprehensive review of factors associated with urinary tract infection is presented, encompassing both historical perspectives and current advances.

Feedback regulation of Caulobacter crescentus holdfast synthesis by flagellum assembly via the holdfast inhibitor HfiA.

To permanently attach to surfaces, Caulobacter crescentusproduces a strong adhesive, the holdfast. The timing of holdfast synthesis is developmentally regulated by cell cycle cues. When C. crescentusis grown in a complex medium, holdfast synthesis can also be stimulated by surface sensing, in which swarmer cells rapidly synthesize holdfast in direct response to surface contact. In contrast to growth in complex medium, here we show that when cells are grown in a defined medium, surface contact does not trigger holdfast synthesis. Moreover, we show that in a defined medium, flagellum synthesis and regulation of holdfast production are linked. In these conditions, mutants lacking a flagellum attach to surfaces over time more efficiently than either wild-type strains or strains harboring a paralyzed flagellum. Enhanced adhesion in mutants lacking flagellar components is due to premature holdfast synthesis during the cell cycle and is regulated by the holdfast synthesis inhibitor HfiA. hfiA transcription is reduced in flagellar mutants and this reduction is modulated by the diguanylate cyclase developmental regulator PleD. We also show that, in contrast to previous predictions, flagella are not necessarily required for C. crescentus surface sensing in the absence of flow, and that arrest of flagellar rotation does not stimulate holdfast synthesis. Rather, our data support a model in which flagellum assembly feeds back to control holdfast synthesis via HfiA expression in a c-di-GMP-dependent manner under defined nutrient conditions.