Flagella-driven Motility Research Articles

The p38 MAP kinase inhibitor, PD 169316, inhibits flagellar motility in Leishmania donovani

Mitogen-activated protein kinases (MAPKs) have been demonstrated to regulate flagellar/ciliary motility of spermatozoa and miracidia of Schistosoma mansoni. However, the role of MAPKs in mediating flagella-driven motility of Leishmania donovani is unexplored. We investigated the function of MAPKs in motility regulation of L. donovani using pharmacological inhibitors and activators of various MAPKs and fast-capture videomicroscopy. Our studies have revealed that the inhibitor of p38 MAPK, PD 169316, significantly affected various motility parameters such as flagellar beat frequency, parasite swimming speed, waveform of the flagellum and resulted in reduced parasite motility. Together, our results suggest that a MAPK, similar to human p38 MAPK, is implicated in flagellar motility regulation of L. donovani.

Importance of Campylobacter jejuni FliS and FliW in Flagella Biogenesis and Flagellin Secretion.

Flagella-driven motility enables bacteria to reach their favorable niche within the host. The human foodborne pathogen Campylobacter jejuni produces two heavily glycosylated structural flagellins (FlaA and FlaB) that form the flagellar filament. It also encodes the non-structural FlaC flagellin which is secreted through the flagellum and has been implicated in host cell invasion. The mechanisms that regulate C. jejuni flagellin biogenesis and guide the proteins to the export apparatus are different from those in most other enteropathogens and are not fully understood. This work demonstrates the importance of the putative flagellar protein FliS in C. jejuni flagella assembly. A constructed fliS knockout strain was non-motile, displayed reduced levels of FlaA/B and FlaC flagellin, and carried severely truncated flagella. Pull-down and Far Western blot assays showed direct interaction of FliS with all three C. jejuni flagellins (FlaA, FlaB, and FlaC). This is in contrast to, the sensor and regulator of intracellular flagellin levels, FliW, which bound to FlaA and FlaB but not to FlaC. The FliS protein but not FliW preferred binding to glycosylated C. jejuni flagellins rather than to their non-glycosylated recombinant counterparts. Mapping of the binding region of FliS and FliW using a set of flagellin fragments showed that the C-terminal subdomain of the flagellin was required for FliS binding, whereas the N-terminal subdomain was essential for FliW binding. The separate binding subdomains required for FliS and FliW, the different substrate specificity, and the differential preference for binding of glycosylated flagellins ensure optimal processing and assembly of the C. jejuni flagellins.

Imaging the motility and chemotaxis machineries in Helicobacter pylori by cryo-electron tomography.

Helicobacter pylori is a highly motile bacterial pathogen that colonizes approximately 50% of the world's population. H. pylori can move readily within the viscous mucosal layer of the stomach. It has become increasingly clear that its unique flagella-driven motility is essential for successful gastric colonization and pathogenesis. Here we use advanced imaging techniques to visualize novel in situ structures with unprecedented detail in intact H. pylori cells. Remarkably, H. pylori possesses multiple unipolar flagella, which are driven by one of the largest flagellar motors found in bacteria. These large motors presumably provide higher torque needed by the bacterial pathogens to navigate in viscous environment of the human stomach.

Oxylipins produced by Pseudomonas aeruginosa promote biofilm formation and virulence.

The oxygenation of unsaturated fatty acids by dioxygenases occurs in all kingdoms of life and produces physiologically important lipids called oxylipins. The biological roles of oxylipins have been extensively studied in animals, plants, algae and fungi, but remain largely unidentified in prokaryotes. The bacterium Pseudomonas aeruginosa displays a diol synthase activity that transforms several monounsaturated fatty acids into mono- and di-hydroxylated derivatives. Here we show that oxylipins derived from this activity inhibit flagellum-driven motility and upregulate type IV pilus-dependent twitching motility of P. aeruginosa. Consequently, these oxylipins promote bacterial organization in microcolonies, increasing the ability of P. aeruginosa to form biofilms in vitro and in vivo (in Drosophila flies). We also demonstrate that oxylipins produced by P. aeruginosa promote virulence in Drosophila flies and lettuce. Our study thus uncovers a role for prokaryotic oxylipins in the physiology and pathogenicity of bacteria.

Identification of the nitrogen-fixing Pseudomonas stutzeri major flagellar gene regulator FleQ and its role in biofilm formation and root colonization

AbstractFlagellar biosynthesis and motility are subject to a four-tiered transcriptional regulatory circuit in Pseudomonas, and the master regulator FleQ appears to be the highest-level regulator in this hierarchical regulatory cascade. Pseudomonas stutzeri A1501 is motile by a polar flagellum; however, the motility and regulatory mechanisms involved in this process are unknown. Here, we searched the A1501 genome for flagella and motility genes and found that approximately 50 genes, which were distributed in three non-contiguous chromosomal regions, contribute to the formation, regulation and function of the flagella. The non-polar mutation of fleQ impaired flagellar biosynthesis, motility and root colonization but enhanced biofilm formation. FleQ positively regulates the expression of flagellar class II–IV genes, suggesting a regulatory cascade that is coordinated similar to that of the well-known P. aeruginosa. Based on our results, we propose that flagellar genes in P. stutzeri A1501 are regulated in a cascade regulated by FleQ and that flagellum-driven motility properties may be necessary for competitive rhizosphere colonization.

The Pseudomonas aeruginosa diguanylate cyclase GcbA, a homolog of P. fluorescens GcbA, promotes initial attachment to surfaces, but not biofilm formation, via regulation of motility.

Cyclic di-GMP is a conserved signaling molecule regulating the transitions between motile and sessile modes of growth in a variety of bacterial species. Recent evidence suggests that Pseudomonas species harbor separate intracellular pools of c-di-GMP to control different phenotypic outputs associated with motility, attachment, and biofilm formation, with multiple diguanylate cyclases (DGCs) playing distinct roles in these processes, yet little is known about the potential conservation of functional DGCs across Pseudomonas species. In the present study, we demonstrate that the P. aeruginosa homolog of the P. fluorescens DGC GcbA involved in promoting biofilm formation via regulation of swimming motility likewise synthesizes c-di-GMP to regulate surface attachment via modulation of motility, however, without affecting subsequent biofilm formation. P. aeruginosa GcbA was found to regulate flagellum-driven motility by suppressing flagellar reversal rates in a manner independent of viscosity, surface hardness, and polysaccharide production. P. fluorescens GcbA was found to be functional in P. aeruginosa and was capable of restoring phenotypes associated with inactivation of gcbA in P. aeruginosa to wild-type levels. Motility and attachment of a gcbA mutant strain could be restored to wild-type levels via overexpression of the small regulatory RNA RsmZ. Furthermore, epistasis analysis revealed that while both contribute to the regulation of initial surface attachment and flagellum-driven motility, GcbA and the phosphodiesterase DipA act within different signaling networks to regulate these processes. Our findings expand the complexity of c-di-GMP signaling in the regulation of the motile-sessile switch by providing yet another potential link to the Gac/Rsm network and suggesting that distinct c-di-GMP-modulating signaling pathways can regulate a single phenotypic output.

Crowning: a novel Escherichia coli colonizing behaviour generating a self-organized corona

BackgroundEncased in a matrix of extracellular polymeric substances (EPS) composed of flagella, adhesins, amyloid fibers (curli), and exopolysaccharides (cellulose, β-1,6-N-acetyl-D-glucosamine polymer-PGA-, colanic acid), the bacteria Escherichia coli is able to attach to and colonize different types of biotic and abiotic surfaces forming biofilms and colonies of intricate morphological architectures. Many of the biological aspects that underlie the generation and development of these E. coli’s formations are largely poorly understood.ResultsHere, we report the characterization of a novel E. coli sessile behaviour termed "crowning" due to the bacterial generation of a new 3-D architectural pattern: a corona. This bacterial pattern is formed by joining bush-like multilayered "coronal flares or spikes" arranged in a ring, which self-organize through the growth, self-clumping and massive self-aggregation of cells tightly interacting inside semisolid agar on plastic surfaces. Remarkably, the corona’s formation is developed independently of the adhesiveness of the major components of E. coli’s EPS matrix, the function of chemotaxis sensory system, type 1 pili and the biofilm master regulator CsgD, but its formation is suppressed by flagella-driven motility and glucose. Intriguingly, this glucose effect on the corona development is not mediated by the classical catabolic repression system, the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex. Thus, corona formation departs from the canonical regulatory transcriptional core that controls biofilm formation in E. coli.ConclusionsWith this novel "crowning" activity, E. coli expands its repertoire of colonizing collective behaviours to explore, invade and exploit environments whose critical viscosities impede flagella driven-motility.

Changes in the proteome of the cadmium-tolerant bacteria Cupriavidus taiwanensis KKU2500-3 in response to cadmium toxicity

Cupriavidus taiwanensis KKU2500-3 is a cadmium (Cd)-tolerant bacterial strain that was previously isolated from rice fields contaminated with high levels of Cd. In 500 μmol/L CdCl2, the KKU2500-3 strain grew slower and with a more prolonged lag-phase than when grown in the absence of Cd. A proteomic approach was used to characterize the protein expression in the Cd-tolerant bacteria C. taiwanensis KKU2500-3 during growth under Cd stress. When compared with the untreated cells, a total of 982 differentially expressed protein spots were observed in the CdCl2-treated cells, and 59 and 10 spots exhibited >2- and >4-fold changes, respectively. The level of up- and downregulation varied from 2.01- to 11.26-fold and from 2.01- to 5.34-fold, respectively. Of the 33 differentially expressed protein spots analyzed by MALDI TOF MS/MS, 19 spots were successfully identified, many of which were involved in stress responses. The most highly upregulated protein (+7.95-fold) identified was the chaperone GroEL, which indicated that this factor likely contributed to the bacterial survival and growth in response to Cd toxicity. Detection of the downregulated protein flagellin (-3.52-fold) was consistent with the less effective ATP-mediated and flagella-driven motility. The flagella-losing cells were also observed in the Cd-treated bacteria when analyzed by scanning electron microscopy. Thus, the Cd-stressed cells may downregulate pathways involving ATP utilization in favor of other mechanisms in response to Cd toxicity. When the KKU2500-3 strain was grown in the presence of Cd, H2S was not detected, suggesting a possible role of the sulfur in precipitation with Cd. Apart from a general response, no specific process could be determined using the present proteomic approach. However, the potential role of protein folding-mediated GroEL, flagella-mediated motility and CdS biotransformation in Cd toxicity response observed in this study as well as the extent of Cd-tolerant mechanisms using other methods could facilitate the future application of this strain in addressing Cd environmental contamination.

Expression of nipP.w of Pectobacterium wasabiae is dependent on functional flgKL flagellar genes

While flagellum-driven motility is hypothesized to play a role in the virulence of Pectobacterium species, there is no direct evidence that genes involved in flagellum assembly regulate the synthesis of virulence factors. The purpose of this study was to identify genes that affect the production or secretion of necrosis-inducing protein (Nip) in the strain SCC3193. Transposon mutagenesis of an RpoS strain overexpressing NipP.w was performed, and a mutant associated with decreased necrosis of tobacco leaves was detected. The mutant contained a transposon in the regulatory region upstream of the flagellar genes flgK and flgL. Additional mutants were generated related to the flagellar genes fliC and fliA. The mutation in flgKL, but not those in fliC and fliA, inhibited nipP.w transcription. Moreover, the regulatory effect of the flgKL mutation on nipP.w transcription was partially dependent on the Rcs phosphorelay. Secretion of NipP.w was also dependent on a type II secretion mechanism. Overall, the results of this study indicate that the flgKL mutation is responsible for reduced motility and lower levels of nipP.w expression.

Defining Protein Complexes that Mediate Bacterial Chemotaxis by Pulsed Dipolar ESR Spectroscopy

Bacterial chemotaxis is the process whereby cells modulate their flagella-driven motility in response to environmental cues. This widespread behavior relies upon a complex sensory apparatus composed of transmembrane receptors, histidine kinases and coupling proteins to achieve great sensitivity, gain and dynamic range in signal processing. We have applied pulsed-dipolar ESR spectroscopy (PDS) combined with site-directed spin labeling to probe the structures and mechanisms of proteins that compose these regulatory circuits. Inherent symmetries within the protein complexes require interpretation of signals from systems that contain more than two spins. Tikhonov regularization and maximum entropy refinement when combined with model simulation reproduce accurate inter-spin distance distributions from such multiply labeled species. Disulfide crosslinking and disruptive mutagenesis verify component interfaces predicted by PDS. Weak but specific interactions that produce low population aggregates relevant to complex assembly are detected by an approach that relies on magnetic dilution and baseline analysis of dipolar spectra. The resulting PDS-based models capture key architectural features of the receptor kinase arrays and the flagellar motor. Moreover, distance distributions derived from the multi- spin sites reveal changes in conformation and dynamics that accompany kinase activation and motor switching. Application to the chemotaxis system demonstrates how PDS effectively reports on key structural features of transient protein complexes and in doing so fills the resolution gap between other well-established biophysical techniques such as electron microscopy and x-ray crystallography.

Spaceflight Promotes Biofilm Formation by Pseudomonas aeruginosa

Understanding the effects of spaceflight on microbial communities is crucial for the success of long-term, manned space missions. Surface-associated bacterial communities, known as biofilms, were abundant on the Mir space station and continue to be a challenge on the International Space Station. The health and safety hazards linked to the development of biofilms are of particular concern due to the suppression of immune function observed during spaceflight. While planktonic cultures of microbes have indicated that spaceflight can lead to increases in growth and virulence, the effects of spaceflight on biofilm development and physiology remain unclear. To address this issue, Pseudomonas aeruginosa was cultured during two Space Shuttle Atlantis missions: STS-132 and STS-135, and the biofilms formed during spaceflight were characterized. Spaceflight was observed to increase the number of viable cells, biofilm biomass, and thickness relative to normal gravity controls. Moreover, the biofilms formed during spaceflight exhibited a column-and-canopy structure that has not been observed on Earth. The increase in the amount of biofilms and the formation of the novel architecture during spaceflight were observed to be independent of carbon source and phosphate concentrations in the media. However, flagella-driven motility was shown to be essential for the formation of this biofilm architecture during spaceflight. These findings represent the first evidence that spaceflight affects community-level behaviors of bacteria and highlight the importance of understanding how both harmful and beneficial human-microbe interactions may be altered during spaceflight.

Adaptation of Salmonella enterica Hadar under static magnetic field: effects on outer membrane protein pattern

BackgroundSalmonella enterica serovar Hadar (S. Hadar) is a highly prevalent foodborne pathogen and therefore a major cause of human gastroenteritis worldwide. Outer membrane proteins whose production is often regulated by environmental conditions also play important roles in the adaptability of bacterial pathogens to various environments.ResultsThe present study investigated the adaptation of S. Hadar under the effect of acute static magnetic field exposure (200 mT, 9 h) and the impact on the outer membrane protein pattern. Via two-dimensional electrophoresis (2-DE) and LC-MS/MS spectrometry, we compared the proteome of enriched-outer membrane fraction before and after exposure to a magnetic field. A total of 11 proteins, displaying more than a two-fold change, were differentially expressed in exposed cells, among which 7 were up-regulated and 4 down-regulated. These proteins were involved in the integrity of cell envelope (TolB, Pal), in the response to oxidative stress (OmpW, dihydrolipoamide dehydrogenase, UspF), in the oxidative stress status (bacterioferritin), in virulence (OmpX, Yfgl) or in motility (FlgE and UspF). Complementary experiments associated the down-regulation of FlgE and UspF with an alteration of swarming, a flagella-driven motility, under SMF. Furthermore, the antibiotic disc diffusion method confirmed a decrease of gentamicin susceptibility in exposed cells. This decrease could be partly associated with the up-regulation of TolC, outer membrane component of an efflux pump. OmpA, a multifunctional protein, was up-regulated.ConclusionsSMF (200 mT) seems to maintain the cell envelope integrity and to submit the exposed cells to an oxidative stress. Some alterations suggest an increase of the ability of exposed cells to form biofilms.

Regulatory Linkages between Flagella and Surfactant during Swarming Behavior: Lubricating the Flagellar Propeller?

Many bacteria explore their immediate environment using flagellar locomotion, and on semi-solid surfaces this type of flagella-driven motility is known as swarming (22).…

Involvement of Flagella-Driven Motility and Pili in <i>Pseudomonas aeruginosa</i> Colonization at the Air-Liquid Interface

Many aerobic microorganisms can colonize at the air-liquid interface and form a multicellular structure, known as a pellicle. In this study, the involvement of motility and attachment traits in the Pseudomonas aeruginosa pellicle formation process was investigated. Flagella- and flagellar-motor-deficient mutants exhibited delayed pellicle formation and unusual pellicle morphology, indicating the large contribution of flagella-driven motility to structural development of the pellicle. A pili-deficient mutant showed normal pellicle formation properties, while the disruption of the pilus gene in the flagella-deficient mutant restored normal pellicle morphology. These results indicate that flagella and pili play key roles in P. aeruginosa pellicle development.

PapX, a P Fimbrial Operon-Encoded Inhibitor of Motility in Uropathogenic Escherichia coli

Motility and adherence are two integral aspects of bacterial pathogenesis. Adherence, often mediated by fimbriae, permits bacteria to attach to host cells and establish infection, whereas flagellum-driven motility allows bacteria to disseminate to sites more advantageous for colonization. Both fimbriae and flagella have been proven important for virulence of uropathogenic Escherichia coli (UPEC). Reciprocal regulation is one mechanism by which bacteria may reconcile the contradictory actions of adherence and motility. PapX, a P fimbrial gene product of UPEC strain CFT073, is a functional homolog of MrpJ of Proteus mirabilis; ectopic expression of papX in P. mirabilis reduces motility. To define the connection between P fimbria expression and motility in UPEC, the role of papX in the regulation of motility of strain CFT073 was examined. Overexpression of papX decreased motility of CFT073, which correlated with both a significant reduction in flagellin protein synthesized and flagella assembled on the cell surface. Conversely, an increase in motility and flagellin production was seen in an isogenic papX deletion mutant of CFT073. Microarray and quantitative reverse transcription-PCR analysis indicated that repression of motility of CFT073 by PapX appears to occur at the transcriptional level; expression of many motility-associated genes, including flhDC, the master regulator of motility, is decreased when papX is overexpressed. Transcription of motility genes is increased in the papX mutant compared to wild type. Electrophoretic mobility gel shift analysis revealed that PapX binds to the flhD promoter. We conclude that synthesis of P fimbriae regulates flagellum synthesis to repress motility via PapX.

A novel role for RecA under non-stress: promotion of swarming motility in Escherichia coli K-12

BackgroundBacterial motility is a crucial factor in the colonization of natural environments. Escherichia coli has two flagella-driven motility types: swimming and swarming. Swimming motility consists of individual cell movement in liquid medium or soft semisolid agar, whereas swarming is a coordinated cellular behaviour leading to a collective movement on semisolid surfaces. It is known that swimming motility can be influenced by several types of environmental stress. In nature, environmentally induced DNA damage (e.g. UV irradiation) is one of the most common types of stress. One of the key proteins involved in the response to DNA damage is RecA, a multifunctional protein required for maintaining genome integrity and the generation of genetic variation.ResultsThe ability of E. coli cells to develop swarming migration on semisolid surfaces was suppressed in the absence of RecA. However, swimming motility was not affected. The swarming defect of a ΔrecA strain was fully complemented by a plasmid-borne recA gene. Although the ΔrecA cells grown on semisolidsurfaces exhibited flagellar production, they also presented impaired individual movement as well as a fully inactive collective swarming migration. Both the comparative analysis of gene expression profiles in wild-type and ΔrecA cells grown on a semisolid surface and the motility of lexA1 [Ind-] mutant cells demonstrated that the RecA effect on swarming does not require induction of the SOS response. By using a RecA-GFP fusion protein we were able to segregate the effect of RecA on swarming from its other functions. This protein fusion failed to regulate the induction of the SOS response, the recombinational DNA repair of UV-treated cells and the genetic recombination, however, it was efficient in rescuing the swarming motility defect of the ΔrecA mutant. The RecA-GFP protein retains a residual ssDNA-dependent ATPase activity but does not perform DNA strand exchange.ConclusionThe experimental evidence presented in this work supports a novel role for RecA: the promotion of swarming motility. The defective swarming migration of ΔrecA cells does not appear to be associated with defective flagellar production; rather, it seems to be associated with an abnormal flagellar propulsion function. Our results strongly suggest that the RecA effect on swarming motility does not require an extensive canonical RecA nucleofilament formation. RecA is the first reported cellular factor specifically affecting swarming but not swimming motility in E. coli. The integration of two apparently disconnected biologically important processes, such as the maintenance of genome integrity and motility in a unique protein, may have important evolutive consequences.

Requirement of flhA for swarming differentiation, flagellin export, and secretion of virulence-associated proteins in Bacillus thuringiensis.

Bacillus thuringiensis is being used worldwide as a biopesticide, although increasing evidence suggests that it is emerging as an opportunistic human pathogen. While phospholipases, hemolysins, and enterotoxins are claimed to be responsible for B. thuringiensis virulence, there is no direct evidence to indicate that the flagellum-driven motility plays a role in parasite-host interactions. This report describes the characterization of a mini-Tn10 mutant of B. thuringiensis that is defective in flagellum filament assembly and in swimming and swarming motility as well as in the production of hemolysin BL and phosphatidylcholine-preferring phospholipase C. The mutant strain was determined to carry the transposon insertion in flhA, a flagellar class II gene encoding a protein of the flagellar type III export apparatus. Interestingly, the flhA mutant of B. thuringiensis synthesized flagellin but was impaired in flagellin export. Moreover, a protein similar to the anti-sigma factor FlgM that acts in regulating flagellar class III gene transcription was not detectable in B. thuringiensis, thus suggesting that the flagellar gene expression hierarchy of B. thuringiensis differs from that described for Bacillus subtilis. The flhA mutant of B. thuringiensis was also defective in the secretion of hemolysin BL and phosphatidylcholine-preferring phospholipase C, although both of these virulence factors were synthesized by the mutant. Since complementation of the mutant with a plasmid harboring the flhA gene restored swimming and swarming motility as well as secretion of toxins, the overall results indicate that motility and virulence in B. thuringiensis may be coordinately regulated by flhA, which appears to play a crucial role in the export of flagellar as well as nonflagellar proteins.