The coordination of cell cycle progression and flagellar synthesis is a complex process in motile bacteria. In γ-proteobacteria, the localization of the flagellum to the cell pole is mediated by the SRP-type GTPase FlhF. However, the mechanism of action of FlhF, and its relationship with the cell pole landmark protein HubP remain unclear. In this study, we discovered a novel protein called FipA that is required for normal FlhF activity and function in polar flagellar synthesis. We demonstrated that membrane-localized FipA interacts with FlhF and is required for normal flagellar synthesis in Vibrio parahaemolyticus , Pseudomonas putida , and Shewanella putrefaciens , and it does so independently of the polar localization mediated by HubP. FipA exhibits a dynamic localization pattern and is present at the designated pole before flagellar synthesis begins, suggesting its role in licensing flagellar formation. This discovery provides insight into a new pathway for regulating flagellum synthesis and coordinating cellular organization in bacteria that rely on polar flagellation and FlhF-dependent localization.

Failure of the cell to properly regulate the number and intracellular positioning of their flagella, has detrimental effects on the cells' swimming ability. The flagellation pattern of numerous bacteria is regulated by the NTPases FlhF and FlhG. In general, FlhG controls the number of flagella produced, whereas FlhF coordinates the position of the flagella. In the human pathogen Vibrio parahaemolyticus, its single flagellum is positioned and formed at the old cell pole. Here, we describe the spatiotemporal localization of FlhF and FlhG in V. parahaemolyticus and their effect on swimming motility. Absence of either FlhF or FlhG caused a significant defect in swimming ability, resulting in absence of flagella in a ΔflhF mutant and an aberrant flagellated phenotype in ΔflhG. Both proteins localized to the cell pole in a cell cycle-dependent manner, but displayed different patterns of localization throughout the cell cycle. FlhF transitioned from a uni- to bi-polar localization, as observed in other polarly flagellated bacteria. Localization of FlhG was strictly dependent on the cell pole-determinant HubP, while polar localization of FlhF was HubP independent. Furthermore, localization of FlhF and FlhG was interdependent and required for each other's proper intracellular localization and recruitment to the cell pole. In the absence of HubP or FlhF, FlhG forms non-polar foci in the cytoplasm of the cell, suggesting the possibility of a secondary localization site within the cell besides its recruitment to the cell poles.

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.

A major form of transcriptional regulation in bacteria occurs through the exchange of the primary σ factor of RNA polymerase (RNAP) with an alternative extracytoplasmic function (ECF) σ factor1. ECF σ factors are generally intrinsically active and are retained in an inactive state via the sequestration into σ factor-anti-σ factor complexes until their action is warranted2-20. Here, we report a previously uncharacterized mechanism of transcriptional regulation that relies on intrinsically inactive ECF σ factors, the activation of which and interaction with the β'-subunit of RNAP depends on σ factor phosphorylation. In Vibrio parahaemolyticus, the threonine kinase PknT phosphorylates the σ factor EcfP, which results in EcfP activation and expression of an essential polymyxin-resistant regulon. EcfP phosphorylation occurs at a highly conserved threonine residue, Thr63, positioned within a divergent region in the σ2.2 helix. Our data indicate that EcfP is intrinsically inactive and unable to bind the β'-subunit of RNAP due to the absence of a negatively charged DAED motif in this region. Furthermore, our results indicate that phosphorylation at residue Thr63 mimics this negative charge and licenses EcfP to interact with the β'-subunit in the formation of the RNAP holoenzyme, which in turn results in target gene expression. This regulatory mechanism is a previously unrecognized paradigm in bacterial signal transduction and transcriptional regulation, and our data suggest that it is widespread in bacteria.

How complex, multi-component macromolecular machines evolved remains poorly understood. Here we reveal the evolutionary origins of the chemosensory machinery that controls flagellar motility in Escherichia coli . We first identify ancestral forms still present in Vibrio cholerae , Pseudomonas aeruginosa , Shewanella oneidensis and Methylomicrobium alcaliphilum , characterizing their structures by electron cryotomography and finding evidence that they function in a stress response pathway. Using bioinformatics, we trace the evolution of the system through γ-Proteobacteria, pinpointing key evolutionary events that led to the machine now seen in E. coli . Our results suggest that two ancient chemosensory systems with different inputs and outputs (F6 and F7) existed contemporaneously, with one (F7) ultimately taking over the inputs and outputs of the other (F6), which was subsequently lost.

How complex, multi-component macromolecular machines evolved remains poorly understood. Here we reveal the evolutionary origins of the chemosensory machinery that controls flagellar motility in Escherichia coli . We first identified ancestral forms still present in Vibrio cholerae, Pseudomonas aeruginosa, Shewanella oneidensis and Methylomicrobium alcaliphilum , characterizing their structures by electron cryotomography and finding evidence that they function in a stress response pathway. Using bioinformatics, we then traced the evolution of the system through γ-Proteobacteria, pinpointing key evolutionary events that led to the machine now seen in E. coli. Our results suggest that two ancient chemosensory systems with different inputs and outputs (F6 and F7) existed contemporaneously, with one (F7) ultimately taking over the inputs and outputs of the other (F6), which was subsequently lost.

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.

Most motile bacteria are able to bias their movement toward more favorable environments or to escape from obnoxious substances by a process called chemotaxis. Chemotaxis depends on a chemosensory system that is able to sense specific environmental signals and generate a behavioral response.

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.

The chemoreceptor array, a remarkably ordered supramolecular complex, is composed of hexagonally packed trimers of receptor dimers networked by a histidine kinase and one or more coupling proteins. Even though the receptor packing is universal among chemotactic bacteria and archaea, the array architecture has been extensively studied only in selected model organisms. Here, we show that even in the complete absence of the kinase, the cluster II arrays in Vibrio cholerae retain their native spatial localization and the iconic hexagonal packing of the receptors with 12-nm spacing. Our results demonstrate that the chemotaxis array is versatile in composition, a property that allows auxiliary chemotaxis proteins such as ParP and CheV to integrate directly into the assembly. Along with its compositional variability, cluster II arrays exhibit a low degree of structural stability compared with the ultrastable arrays in Escherichia coli. We propose that the variability in chemoreceptor arrays is an important mechanism that enables the incorporation of chemotaxis proteins based on their availability.

The ability of most bacterial flagellar motors to reverse the direction of rotation is crucial for efficient chemotaxis. In Escherichia coli, motor reversals are mediated by binding of phosphorylated chemotaxis protein CheY to components of the flagellar rotor, FliM and FliN, which induces a conformational switch of the flagellar C-ring. Here, we show that for Shewanella putrefaciens, Vibrio parahaemolyticus and likely a number of other species an additional transmembrane protein, ZomB, is critically required for motor reversals as mutants lacking ZomB exclusively exhibit straightforward swimming also upon full phosphorylation or overproduction of CheY. ZomB is recruited to the cell poles by and is destabilized in the absence of the polar landmark protein HubP. ZomB also co-localizes to and may thus interact with the flagellar motor. The ΔzomB phenotype was suppressed by mutations in the very C-terminal region of FliM. We propose that the flagellar motors of Shewanella, Vibrio and numerous other species harboring orthologs to ZomB are locked in counterclockwise rotation and may require interaction with ZomB to enable the conformational switch required for motor reversals. Regulation of ZomB activity or abundance may provide these species with an additional means to modulate chemotaxis efficiency.

The ability to quickly sense and interpret the environmental signals is crucial for microbes to survive. Most motile bacteria have evolved delicate protein networks to detect the gradient changes of surrounding conditions and switch the direction of flagellar rotation in response [1]. Within this chemotaxis pathway, the signal transduction and regulation is carried out by the large, highly ordered supramolecular complexes termed chemoreceptor arrays. The Chemoreceptor arrays have been most intensively studied in Escherichia.coli. In this organism, receptors, the histadine kinase CheA and coupling protein CheW form extended arrays with strict stoichiometry [2]. These clusters form highly ordered, hexagonally packed arrays with a strict 12-nm spacing at cell pole, which is proposed to be the structural basis for signal amplification, cooperativity and sensitivity adaptation [3].

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.

Chemotaxis proteins organize into large, highly ordered, chemotactic signaling arrays, which in Vibrio species are found at the cell pole. Proper localization of signaling arrays is mediated by ParP, which tethers arrays to a cell pole anchor, ParC. Here we show that ParP’s C-terminus integrates into the core-unit of signaling arrays through interactions with MCP-proteins and CheA. Its intercalation within core-units stimulates array formation, whereas its N-terminal interaction domain enables polar recruitment of arrays and facilitates its own polar localization. Linkage of these domains within ParP couples array formation and localization and results in controlled array positioning at the cell pole. Notably, ParP’s integration into arrays modifies its own and ParC’s subcellular localization dynamics, promoting their polar retention. ParP serves as a critical nexus that regulates the localization dynamics of its network constituents and drives the localized assembly and stability of the chemotactic machinery, resulting in proper cell pole development.

Nearly all motile bacterial cells use a highly sensitive and adaptable sensory system to detect changes in nutrient concentrations in the environment and guide their movements toward attractants and away from repellents. The best-studied bacterial chemoreceptor arrays are membrane-bound. Many motile bacteria contain one or more additional, sometimes purely cytoplasmic, chemoreceptor systems. Vibrio cholerae contains three chemotaxis clusters (I, II, and III). Here, using electron cryotomography, we explore V. cholerae's cytoplasmic chemoreceptor array and establish that it is formed by proteins from cluster I. We further identify a chemoreceptor with an unusual domain architecture, DosM, which is essential for formation of the cytoplasmic arrays. DosM contains two signaling domains and spans the two-layered cytoplasmic arrays. Finally, we present evidence suggesting that this type of receptor is important for the structural stability of the cytoplasmic array.

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.

The diarrheal pathogen Vibrio cholerae contains three gene clusters that encode chemotaxis-related proteins, but only cluster II appears to be required for chemotaxis. Here, we present the first characterization of V.cholerae's cluster III' chemotaxis system. We found that cluster III proteins assemble into foci at bacterial poles, like those formed by cluster II proteins, but the two systems assemble independently and do not colocalize. Cluster III proteins are expressed in vitro during stationary phase and in conjunction with growth arrest linked to carbon starvation. This expression, as well as expression in vivo in suckling rabbits, is dependent upon RpoS. V.cholerae's CAI-1 quorum sensing (QS) system is also required for cluster III expression in stationary phase and modulates its expression in vivo, but is not required for cluster III expression in response to carbon starvation. Surprisingly, even though the CAI-1 and AI-2 QS systems are thought to feed into the same signaling pathway, the AI-2 system inhibited cluster III gene expression, revealing that the outputs of the two QS systems are not always the same. The distinctions between genetic determinants of cluster III expression in vitro and in vivo highlight the distinctive nature of the in vivo environment.

Vibrio cholerae, the agent of cholera, is a motile non-invasive pathogen that colonizes the small intestine (SI). Most of our knowledge of the processes required for V. cholerae intestinal colonization is derived from enumeration of wt and mutant V. cholerae recovered from orogastrically infected infant mice. There is limited knowledge of the distribution of V. cholerae within the SI, particularly its localization along the villous axis, or of the bacterial and host factors that account for this distribution. Here, using confocal and intravital two-photon microscopy to monitor the localization of fluorescently tagged V. cholerae strains, we uncovered unexpected and previously unrecognized features of V. cholerae intestinal colonization. Direct visualization of the pathogen within the intestine revealed that the majority of V. cholerae microcolonies attached to the intestinal epithelium arise from single cells, and that there are notable regiospecific aspects to V. cholerae localization and factors required for colonization. In the proximal SI, V. cholerae reside exclusively within the developing intestinal crypts, but they are not restricted to the crypts in the more distal SI. Unexpectedly, V. cholerae motility proved to be a regiospecific colonization factor that is critical for colonization of the proximal, but not the distal, SI. Furthermore, neither motility nor chemotaxis were required for proper V. cholerae distribution along the villous axis or in crypts, suggesting that yet undefined processes enable the pathogen to find its niches outside the intestinal lumen. Finally, our observations suggest that host mucins are a key factor limiting V. cholerae intestinal colonization, particularly in the proximal SI where there appears to be a more abundant mucus layer. Collectively, our findings demonstrate the potent capacity of direct pathogen visualization during infection to deepen our understanding of host pathogen interactions.

Bacterial chemotaxis proteins are organized into ordered arrays. In peritrichous organisms, such as Escherichia coli, stochastic assembly processes are thought to account for the placement of chemotaxis arrays, which are nonuniformly distributed. In contrast, we previously found that chemotactic signaling arrays in polarly flagellated vibrios are uniformly polar and that array localization is dependent on the ParA-like ATPase ParC. However, the processes that enable ParC to facilitate array localization have not been described. Here, we show that a previously uncharacterized protein, ParP, interacts with ParC and that ParP is integral to array localization in Vibrio parahaemolyticus. ParC's principal contribution to chemotaxis appears to be via positioning of ParP. Once recruited to the pole by ParC, ParP sequesters arrays at this site by capturing and preventing the dissociation of chemotactic signaling protein (CheA). Notably, ParP also stabilizes chemotactic protein complexes in the absence of ParC, indicating that some of its activity is independent of this interaction partner. ParP recruits CheA via CheA's localization and inheritance domain, a region found only in polarly flagellated organisms that encode ParP, ParC, and CheA. Thus, a tripartite (ParC-ParP-CheA) interaction network enables the polar localization and sequestration of chemotaxis arrays in polarly flagellated organisms. Localization and sequestration of chemotaxis clusters adjacent to the flagella-to which the chemotactic signal is transmitted-facilitates proper chemotaxis as well as accurate inheritance of these macromolecular machines.

PprA is known to contribute to Deinococcus radiodurans' remarkable capacity to survive a variety of genotoxic assaults. The molecular bases for PprA's role(s) in the maintenance of the damaged D. radiodurans genome are incompletely understood, but PprA is thought to promote D. radiodurans's capacity for DSB repair. PprA is found in a multiprotein DNA processing complex along with an ATP type DNA ligase, and the D. radiodurans toposiomerase IB (DraTopoIB) as well as other proteins. Here, we show that PprA is a key contributor to D. radiodurans resistance to nalidixic acid (Nal), an inhibitor of topoisomerase II. Growth of wild type D. radiodurans and a pprA mutant were similar in the absence of exogenous genotoxic insults; however, the pprA mutant exhibited marked growth delay and a higher frequency of anucleate cells following treatment with DNA-damaging agents. We show that PprA interacts with both DraTopoIB and the Gyrase A subunit (DraGyrA) in vivo and that purified PprA enhances DraTopoIB catalysed relaxation of supercoiled DNA. Thus, besides promoting DNA repair, our findings suggest that PprA also contributes to preserving the integrity of the D. radiodurans genome following DNA damage by interacting with DNA topoisomerases and by facilitating the actions of DraTopoIB.