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1.
Fish Shellfish Immunol ; 91: 306-314, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31121291

RESUMO

The flagellum is a complex surface structure necessary for a number of activities including motility, chemotaxis, biofilm formation and host attachment. Flagellin, the primary structural protein making up the flagellum, is an abundant and potent activator of innate and adaptive immunity and therefore expression of flagellin during infection could be deleterious to the infection process due to flagellin-mediated host recognition. Here, we use quantitative RT-PCR to demonstrate that expression of the flagellin locus fliC is repressed during the course of infection and subsequently up-regulated upon host mortality in a motile strain of Yersinia ruckeri. The kinetics of fliC repression during the infection process is relatively slow as full repression occurs 7-days after the initiation of infection and after approximately 3-logs of bacterial growth in vivo. These results suggests that Y. ruckeri possesses a regulatory system capable of sensing host and modulating the expression of motility in response. Examination of the master flagellar operon (flhDC) promoter region for evidence of transcriptional regulation and regulatory binding sites revealed potential interaction with the Rcs pathway through an Rcs(A)B Box. Deletion of rcsB (ΔrcsB) by marker-exchange mutagenesis resulted in overproduction of flagellin and unregulated motility, showing that the Rcs pathway negatively regulates biosynthesis of the flagellar apparatus. Experimental challenge with ΔrcsB and ΔrcsBΔfliC1ΔfliC2 mutants revealed that mutation of the Rcs pathway results in virulence attenuation which is dependent on presence of the flagellin gene. These results suggest that the inappropriate expression of flagellin during infection triggers host recognition and thus immune stimulation resulting in attenuation of virulence. In addition, RNAseq analyses of the ΔrcsB mutant strain verified the role of this gene as a negative regulator of the flagellar motility system and identified several additional genes regulated by the Rcs pathway.


Assuntos
Proteínas de Bactérias/genética , Flagelos/fisiologia , Yersinia ruckeri/fisiologia , Yersinia ruckeri/patogenicidade , Proteínas de Bactérias/metabolismo , Flagelina/genética , Flagelina/metabolismo , Virulência/genética , Yersinia ruckeri/genética
2.
BMC Genomics ; 20(1): 327, 2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31039790

RESUMO

BACKGROUND: Bacillus pumilus is a Gram-positive and endospore-forming bacterium broadly existing in a variety of environmental niches. Because it produces and secrets many industrially useful enzymes, a lot of studies have been done to understand the underlying mechanisms. Among them, scoC was originally identified as a pleiotropic transcription factor negatively regulating protease production and sporulation in B. subtilis. Nevertheless, its role in B. pumilus largely remains unknown. RESULTS: In this study we successfully disrupted scoC gene in B. pumilus BA06 and found increased total extracellular protease activity in scoC mutant strain. Surprisingly, we also found that scoC disruption reduced cell motility possibly by affecting flagella formation. To better understand the underlying mechanism, we performed transcriptome analysis with RNA sequencing. The result showed that more than one thousand genes were alternated at transcriptional level across multiple growth phases, and among them the largest number of differentially expressed genes (DEGs) were identified at the transition time point (12 h) between the exponential growth and the stationary growth phases. In accordance with the altered phenotype, many protease genes especially the aprE gene encoding alkaline protease were transcriptionally regulated. In contrast to the finding in B. subtilis, the aprN gene encoding neutral protease was transcriptionally downregulated in B. pumilus, implicating that scoC plays strain-specific roles. CONCLUSIONS: The pleiotropic transcription factor ScoC plays multiple roles in various cellular processes in B. pumilus, some of which were previously reported in B. subtilis. The supervising finding is the identification of ScoC as a positive regulator for flagella formation and bacterial motility. Our transcriptome data may provide hints to understand the underlying mechanism.


Assuntos
Bacillus pumilus/genética , Proteínas de Bactérias/antagonistas & inibidores , Regulação Bacteriana da Expressão Gênica , Pleiotropia Genética , Transcriptoma , Bacillus pumilus/citologia , Bacillus pumilus/crescimento & desenvolvimento , Bacillus pumilus/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Movimento Celular , Endopeptidases/metabolismo , Flagelos/fisiologia , Fenótipo
3.
MBio ; 10(3)2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31088927

RESUMO

Bordetella bronchiseptica encodes and expresses a flagellar apparatus. In contrast, Bordetella pertussis, the causative agent of whooping cough, has historically been described as a nonmotile and nonflagellated organism. The previous statements that B. pertussis was a nonmotile organism were consistent with a stop codon located in the flagellar biosynthesis gene, flhA, discovered when the B. pertussis Tohama I genome was sequenced and analyzed by Parkhill et al. in 2003 (J. Parkhill, M. Sebaihia, A. Preston, L. D. Murphy, et al., Nat Genet, 35:32-40, 2003, https://doi.org/10.1038/ng1227). The stop codon has subsequently been found in all annotated genomes. Parkhill et al. also showed, however, that B. pertussis contains all genetic material required for flagellar synthesis and function. We and others have determined by various transcriptomic analyses that these flagellar genes are differentially regulated under a variety of B. pertussis growth conditions. In light of these data, we tested for B. pertussis motility and found that both laboratory-adapted strains and clinical isolates can be motile. Upon isolation of motile B. pertussis, we discovered flagellum-like structures on the surface of the bacteria. B. pertussis motility appears to occur primarily in the Bvg(-) phase, consistent with regulation present in B. bronchiseptica Motility can also be induced by the presence of fetal bovine serum. These observations demonstrate that B. pertussis can express flagellum-like structures, and although it remains to be determined if B. pertussis expresses flagella during infection or if motility and/or flagella play roles during the cycle of infection and transmission, it is clear that these data warrant further investigation.IMPORTANCE This report provides evidence for motility and expression of flagella by B. pertussis, a bacterium that has been reported as nonmotile since it was first isolated and studied. As with B. bronchiseptica, B. pertussis cells can express and assemble a flagellum-like structure on their surface, which in other organisms has been implicated in several important processes that occur in vivo The discovery that B. pertussis is motile raises many questions, including those regarding the mechanisms of regulation for flagellar gene and protein expression and, importantly, the role of flagella during infection. This novel observation provides a foundation for further study of Bordetella flagella and motility in the contexts of infection and transmission.


Assuntos
Bordetella pertussis/fisiologia , Flagelos/fisiologia , Regulação Bacteriana da Expressão Gênica , Bordetella bronchiseptica/genética , Bordetella pertussis/genética , Flagelina/genética , Flagelina/isolamento & purificação , Movimento
4.
MBio ; 10(3)2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-31064826

RESUMO

Bacteria and archaea exhibit tactical behavior and can move up and down chemical gradients. This tactical behavior relies on a motility structure, which is guided by a chemosensory system. Environmental signals are sensed by membrane-inserted chemosensory receptors that are organized in large ordered arrays. While the cellular positioning of the chemotaxis machinery and that of the flagellum have been studied in detail in bacteria, we have little knowledge about the localization of such macromolecular assemblies in archaea. Although the archaeal motility structure, the archaellum, is fundamentally different from the flagellum, archaea have received the chemosensory machinery from bacteria and have connected this system with the archaellum. Here, we applied a combination of time-lapse imaging and fluorescence and electron microscopy using the model euryarchaeon Haloferax volcanii and found that archaella were specifically present at the cell poles of actively dividing rod-shaped cells. The chemosensory arrays also had a polar preference, but in addition, several smaller arrays moved freely in the lateral membranes. In the stationary phase, rod-shaped cells became round and chemosensory arrays were disassembled. The positioning of archaella and that of chemosensory arrays are not interdependent and likely require an independent form of positioning machinery. This work showed that, in the rod-shaped haloarchaeal cells, the positioning of the archaellum and of the chemosensory arrays is regulated in time and in space. These insights into the cellular organization of H. volcanii suggest the presence of an active mechanism responsible for the positioning of macromolecular protein complexes in archaea.IMPORTANCE Archaea are ubiquitous single cellular microorganisms that play important ecological roles in nature. The intracellular organization of archaeal cells is among the unresolved mysteries of archaeal biology. With this work, we show that cells of haloarchaea are polarized. The cellular positioning of proteins involved in chemotaxis and motility is spatially and temporally organized in these cells. This suggests the presence of a specific mechanism responsible for the positioning of macromolecular protein complexes in archaea.


Assuntos
Proteínas Arqueais/química , Polaridade Celular , Quimiotaxia , Haloferax volcanii/fisiologia , Citoplasma/química , Flagelos/fisiologia , Haloferax volcanii/ultraestrutura , Microscopia Eletrônica , Imagem com Lapso de Tempo
5.
MBio ; 10(2)2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30940700

RESUMO

The flagellar motor can spin in both counterclockwise (CCW) and clockwise (CW) directions. The flagellar motor consists of a rotor and multiple stator units, which act as a proton channel. The rotor is composed of the transmembrane MS ring made of FliF and the cytoplasmic C ring consisting of FliG, FliM, and FliN. The C ring is directly involved in rotation and directional switching. The Salmonella FliF-FliG deletion fusion motor missing 56 residues from the C terminus of FliF and 94 residues from the N terminus of FliG keeps a domain responsible for the interaction with the stator intact, but its motor function is reduced significantly. Here, we report the structure and function of the FliF-FliG deletion fusion motor. The FliF-FliG deletion fusion not only resulted in a strong CW switch bias but also affected rotor-stator interactions coupled with proton translocation through the proton channel of the stator unit. The energy coupling efficiency of the deletion fusion motor was the same as that of the wild-type motor. Extragenic suppressor mutations in FliG, FliM, or FliN not only relieved the strong CW switch bias but also increased the motor speed at low load. The FliF-FliG deletion fusion made intersubunit interactions between C ring proteins tighter compared to the wild-type motor, whereas the suppressor mutations affect such tighter intersubunit interactions. We propose that a change of intersubunit interactions between the C ring proteins may be required for high-speed motor rotation as well as direction switching.IMPORTANCE The bacterial flagellar motor is a bidirectional rotary motor for motility and chemotaxis, which often plays an important role in infection. The motor is a large transmembrane protein complex composed of a rotor and multiple stator units, which also act as a proton channel. Motor torque is generated through their cyclic association and dissociation coupled with proton translocation through the proton channel. A large cytoplasmic ring of the motor, called C ring, is responsible for rotation and switching by interacting with the stator, but the mechanism remains unknown. By analyzing the structure and function of the wild-type motor and a mutant motor missing part of the C ring connecting itself with the transmembrane rotor ring while keeping a stator-interacting domain for bidirectional torque generation intact, we found interesting clues to the change in the C ring conformation for the switching and rotation involving loose and tight intersubunit interactions.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Flagelos/fisiologia , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Salmonella typhimurium/fisiologia , Movimento (Física) , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Supressão Genética
6.
MBio ; 10(2)2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30992355

RESUMO

Microbially produced electrically conductive protein filaments are of interest because they can function as conduits for long-range biological electron transfer. They also show promise as sustainably produced electronic materials. Until now, microbially produced conductive protein filaments have been reported only for bacteria. We report here that the archaellum of Methanospirillum hungatei is electrically conductive. This is the first demonstration that electrically conductive protein filaments have evolved in Archaea Furthermore, the structure of the M. hungatei archaellum was previously determined (N. Poweleit, P. Ge, H. N. Nguyen, R. R. O. Loo, et al., Nat Microbiol 2:16222, 2016, https://doi.org/10.1038/nmicrobiol.2016.222). Thus, the archaellum of M. hungatei is the first microbially produced electrically conductive protein filament for which a structure is known. We analyzed the previously published structure and identified a core of tightly packed phenylalanines that is one likely route for electron conductance. The availability of the M. hungatei archaellum structure is expected to substantially advance mechanistic evaluation of long-range electron transport in microbially produced electrically conductive filaments and to aid in the design of "green" electronic materials that can be microbially produced with renewable feedstocks.IMPORTANCE Microbially produced electrically conductive protein filaments are a revolutionary, sustainably produced, electronic material with broad potential applications. The design of new protein nanowires based on the known M. hungatei archaellum structure could be a major advance over the current empirical design of synthetic protein nanowires from electrically conductive bacterial pili. An understanding of the diversity of outer-surface protein structures capable of electron transfer is important for developing models for microbial electrical communication with other cells and minerals in natural anaerobic environments. Extracellular electron exchange is also essential in engineered environments such as bioelectrochemical devices and anaerobic digesters converting wastes to methane. The finding that the archaellum of M. hungatei is electrically conductive suggests that some archaea might be able to make long-range electrical connections with their external environment.


Assuntos
Condutividade Elétrica , Flagelos/fisiologia , Methanospirillum/fisiologia , Eletricidade , Transporte de Elétrons , Fenilalanina/química
7.
Genes Cells ; 24(6): 408-421, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30963674

RESUMO

The flagellar protein export apparatus switches its substrate specificity when hook length has reached approximately 55 nm in Salmonella. The C-terminal cytoplasmic domain of FlhB (FlhBC ) is involved in this switching process. FlhBC consists of FlhBCN and FlhBCC polypeptides. FlhBCC has a flexible C-terminal tail (FlhBCCT ). FlhBCC is involved in substrate recognition, and conformational rearrangements of FlhBCN -FlhBCC boundary are postulated to be required for the export switching. However, it remains unknown how it occurs. To clarify this question, we carried out mutational analysis of highly conserved residues in FlhBC . The flhB(E230A) mutation reduced the FlhB function. The flhB(E11S) mutation restored the protein transport activity of the flhB(E230A) mutant to the wild-type level, suggesting that the interaction of FlhBCN with the extreme N-terminal region of FlhB is required for flagellar protein export. The flhB(R320A) mutation affected hydrophobic interaction networks in FlhBCC , thereby increasing insolubility of FlhBC . The R320A mutation also affected the export switching, thereby producing longer hooks with the filament attached. C-terminal truncations of FlhBCCT induced a conformational change of FlhBCN -FlhBCC boundary, resulting in a loose hook length control. We propose that FlhBCCT may control conformational arrangements of FlhBCN -FlhBCC boundary through the hydrophobic interaction networks of FlhBCC .


Assuntos
Proteínas de Bactérias/genética , Proteínas de Membrana/genética , Salmonella typhi/genética , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Transporte Biológico/genética , Análise Mutacional de DNA/métodos , Flagelos/genética , Flagelos/fisiologia , Proteínas de Membrana/metabolismo , Mutação , Domínios Proteicos , Transporte Proteico/genética , Salmonella/genética , Salmonella/metabolismo , Salmonella typhi/metabolismo , Especificidade por Substrato
8.
Nat Commun ; 10(1): 1792, 2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30996269

RESUMO

Motile subpopulations in microbial communities are believed to be important for dispersal, quest for food, and material transport. Here, we show that motile cells in sessile colonies of peritrichously flagellated bacteria can self-organize into two adjacent, centimeter-scale motile rings surrounding the entire colony. The motile rings arise from spontaneous segregation of a homogeneous swimmer suspension that mimics a phase separation; the process is mediated by intercellular interactions and shear-induced depletion. As a result of this self-organization, cells drive fluid flows that circulate around the colony at a constant peak speed of ~30 µm s-1, providing a stable and high-speed avenue for directed material transport at the macroscopic scale. Our findings present a unique form of bacterial self-organization that influences population structure and material distribution in colonies.


Assuntos
Bacillus subtilis/fisiologia , Escherichia coli/fisiologia , Flagelos/fisiologia , Microbiota/fisiologia , Proteus mirabilis/fisiologia , Hidrodinâmica
9.
Parasitol Res ; 118(4): 1205-1214, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30847613

RESUMO

Spermiogenesis in progenetic and adult stages of Archigetes sieboldi Leuckart, 1878, a tapeworm parasitic in oligochaetes and fish respectively, has been examined using transmission electron microscopy and cytochemical staining for glycogen. General pattern of spermiogenesis is essentially like that of other caryophyllideans, i.e., apical dense material in the zone of differentiation in the early stages of spermiogenesis, rotation of free flagellum and a flagellar bud, and proximo-distal fusion. Interestingly, rotation of a free flagellum and flagellar bud to the median cytoplasmic process (MCP) has been observed unconventionally at > 90° only in progenetic stages. Typical striated roots associated with the centrioles occur rarely in A. sieboldi, and only in form of faint structures in advanced stages of spermiogenesis. In contrast to most caryophyllideans studied to date, penetration of the nucleus into the spermatid body has started before the fusion of the free flagellum with the MCP. This feature has been reported rarely but exclusively in the family Caryophyllaeidae. The unipartite mature spermatozoon of A. sieboldi is composed of one axoneme of the 9 + '1' trepaxonematan pattern with its centriole, parallel nucleus, and parallel cortical microtubules which are situated in a moderately electron-dense cytoplasm with glycogen particles. An unusual arrangement of cortical microtubules in the two parallel rows in region I of the spermatozoon is described here for the first time in the Caryophyllidea. Ultrastructural data on spermiogenesis and the spermatozoon in A. sieboldi from tubuficids and carp are compared and discussed with those in other caryophyllideans and/or Neodermata.


Assuntos
Carpas/parasitologia , Cestoides/ultraestrutura , Infecções por Cestoides/veterinária , Doenças dos Peixes/parasitologia , Espermátides/citologia , Espermátides/ultraestrutura , Espermatogênese/fisiologia , Animais , Axonema/ultraestrutura , Núcleo Celular/fisiologia , Flagelos/fisiologia , Glicogênio/análise , Masculino , Microscopia Eletrônica de Transmissão , Coloração e Rotulagem
10.
MBio ; 10(2)2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30890608

RESUMO

Many motile bacteria swim or swarm using a filamentous rotating organelle, the flagellum. FliL, a component protein of the flagellar motor, is known to enhance the motor performance under high-load conditions in some bacteria. Here we determined the structure of the periplasmic region of FliL (FliLPeri) of the polar flagellum of Vibrio alginolyticus FliLPeri shows a remarkable structural similarity to the stomatin/prohibitin/flotillin/HflK/C (SPFH) domain of stomatin family proteins, some of which are involved in modulation of ion channel activities in various organisms. FliLPeri forms a ring assembly in the crystal with an inner diameter of around 8 nm, which is comparable to the size of the stator unit. Mutational analyses suggest that the FliL ring forms a complex with the stator unit and that the length of the periplasmic linkers of FliL and the stator B-subunit is essential for the complex formation. We propose a model of the FliL-stator complex to discuss how Vibrio FliL modulates stator function in the bacterial flagellar motor under conditions of high viscosity.IMPORTANCE Some flagellated bacteria regulate motor torque in response to the external load change. This behavior is critical for survival, but the mechanism has remained unknown. Here, we focused on a key protein, FliL of Vibrio alginolyticus, and solved the crystal structure of its periplasmic region (FliLPeri). FliLPeri reveals striking structural similarity to a conserved domain of stomatin, which is involved in ion channel regulation in some organisms, including mammals. FliLPeri forms a ring with an inner diameter that is comparable in size to the stator unit. The mutational analyses suggested that the presence of the ring-like assembly of FliL around the stator unit enhances the surface swarming of Vibrio cells. Our study data also imply that the structural element for the ion channel regulation is conserved from bacteria to mammals.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Flagelos/enzimologia , Flagelos/fisiologia , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Movimento (Física) , Vibrio alginolyticus/enzimologia , Vibrio alginolyticus/fisiologia , Proteínas de Bactérias/genética , Cristalografia por Raios X , Análise Mutacional de DNA , Proteínas de Membrana/genética , Conformação Proteica , Multimerização Proteica
11.
Proc Natl Acad Sci U S A ; 116(13): 6351-6360, 2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30850532

RESUMO

Leishmania kinetoplastid parasites infect millions of people worldwide and have a distinct cellular architecture depending on location in the host or vector and specific pathogenicity functions. An invagination of the cell body membrane at the base of the flagellum, the flagellar pocket (FP), is an iconic kinetoplastid feature, and is central to processes that are critical for Leishmania pathogenicity. The Leishmania FP has a bulbous region posterior to the FP collar and a distal neck region where the FP membrane surrounds the flagellum more closely. The flagellum is attached to one side of the FP neck by the short flagellum attachment zone (FAZ). We addressed whether targeting the FAZ affects FP shape and its function as a platform for host-parasite interactions. Deletion of the FAZ protein, FAZ5, clearly altered FP architecture and had a modest effect in endocytosis but did not compromise cell proliferation in culture. However, FAZ5 deletion had a dramatic impact in vivo: Mutants were unable to develop late-stage infections in sand flies, and parasite burdens in mice were reduced by >97%. Our work demonstrates the importance of the FAZ for FP function and architecture. Moreover, we show that deletion of a single FAZ protein can have a large impact on parasite development and pathogenicity.


Assuntos
Cílios/fisiologia , Flagelos/fisiologia , Leishmania/fisiologia , Leishmania/patogenicidade , Psychodidae/parasitologia , Animais , Membrana Celular/metabolismo , Cílios/genética , Cílios/ultraestrutura , Endocitose , Flagelos/genética , Flagelos/ultraestrutura , Deleção de Genes , Interações Hospedeiro-Parasita , Junções Intercelulares , Leishmania/genética , Leishmania/ultraestrutura , Camundongos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Virulência/genética
12.
Soft Matter ; 15(9): 2032-2042, 2019 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-30724307

RESUMO

It is known that some flagellated bacteria like Serratia marcescens, when deposited and affixed onto a surface to form a "bacterial carpet", self-organize in a collective motion of the flagella that is capable of pumping fluid through microfluidic channels. We set up a continuum model comprising two macroscopic variables that is capable of describing this self-organization mechanism as well as quantifying it to the extent that an agreement with the experimentally observed channel width dependence of the pumping is reached. The activity is introduced through a collective angular velocity of the helical flagella rotation, which is an example of a dynamic macroscopic preferred direction. Our model supports and quantifies the view that the self-coordination is due to a positive feedback loop between the bacterial flagella and the local flow generated by their rotation. Moreover, our results indicate that this biological active system is operating close to the self-organization threshold.


Assuntos
Microfluídica , Modelos Biológicos , Serratia marcescens/fisiologia , Elasticidade , Flagelos/fisiologia , Rotação
13.
J Biochem ; 166(1): 77-88, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30778544

RESUMO

YcgR, a cyclic diguanylate (c-di-GMP)-binding protein expressed in Escherichia coli, brakes flagellar rotation by binding to the motor in a c-di-GMP dependent manner and has been implicated in triggering biofilm formation. Vibrio alginolyticus has a single polar flagellum and encodes YcgR homologue, PlzD. When PlzD or PlzD-GFP was highly over-produced in nutrient-poor condition, the polar flagellar motility of V. alginolyticus was reduced. This inhibitory effect is c-di-GMP independent as mutants substituting putative c-di-GMP-binding residues retain the effect. Moderate over-expression of PlzD-GFP allowed its localization at the flagellated cell pole. Truncation of the N-terminal 12 or 35 residues of PlzD abolished the inhibitory effect and polar localization, and no inhibitory effect was observed by deleting plzD or expressing an endogenous level of PlzD-GFP. Subcellular fractionation showed that PlzD, but not its N-terminally truncated variants, was precipitated when over-produced. Moreover, immunoblotting and N-terminal sequencing revealed that endogenous PlzD is synthesized from Met33. These results suggest that an N-terminal extension allows PlzD to localize at the cell pole but causes aggregation and leads to inhibition of motility. In V. alginolyticus, PlzD has a potential property to associate with the polar flagellar motor but this interaction is too weak to inhibit rotation.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , GMP Cíclico/análogos & derivados , Flagelos/fisiologia , Movimento , Vibrio alginolyticus/química , GMP Cíclico/metabolismo
14.
J Bacteriol ; 201(6)2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30642987

RESUMO

The bacterial flagellar motor is composed of a rotor and a dozen stators and converts the ion flux through the stator into torque. Each stator unit alternates in its attachment to and detachment from the rotor even during rotation. In some species, stator assembly depends on the input energy, but it remains unclear how an electrochemical potential across the membrane (e.g., proton motive force [PMF]) or ion flux is involved in stator assembly dynamics. Here, we focused on pH dependence of a slow motile MotA(M206I) mutant of Salmonella The MotA(M206I) motor produces torque comparable to that of the wild-type motor near stall, but its rotation rate is considerably decreased as the external load is reduced. Rotation assays of flagella labeled with 1-µm beads showed that the rotation rate of the MotA(M206I) motor is increased by lowering the external pH whereas that of the wild-type motor is not. Measurements of the speed produced by a single stator unit using 1-µm beads showed that the unit speed of the MotA(M206I) is about 60% of that of the wild-type and that a decrease in external pH did not affect the MotA(M206I) unit speed. Analysis of the subcellular stator localization revealed that the number of functional stators is restored by lowering the external pH. The pH-dependent improvement of stator assembly was observed even when the PMF was collapsed and proton transfer was inhibited. These results suggest that MotA-Met206 is responsible for not only load-dependent energy coupling between the proton influx and rotation but also pH-dependent stator assembly.IMPORTANCE The bacterial flagellar motor is a rotary nanomachine driven by the electrochemical transmembrane potential (ion motive force). About 10 stators (MotA/MotB complexes) are docked around a rotor, and the stator recruitment depends on the load, ion motive force, and coupling ion flux. The MotA(M206I) mutation slows motor rotation and decreases the number of docked stators in Salmonella We show that lowering the external pH improves the assembly of the mutant stators. Neither the collapse of the ion motive force nor a mutation mimicking the proton-binding state inhibited stator localization to the motor. These results suggest that MotA-Met206 is involved in torque generation and proton translocation and that stator assembly is stabilized by protonation of the stator.


Assuntos
Proteínas de Bactérias/metabolismo , Flagelos/fisiologia , Proteínas Motores Moleculares/metabolismo , Proteínas Mutantes/metabolismo , Multimerização Proteica , ATPases Translocadoras de Prótons/metabolismo , Salmonella typhimurium/fisiologia , Concentração de Íons de Hidrogênio , Locomoção , Proteínas Motores Moleculares/genética , Proteínas Mutantes/genética , Mutação de Sentido Incorreto , ATPases Translocadoras de Prótons/genética , Torque
15.
Curr Microbiol ; 76(4): 393-397, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30600359

RESUMO

Salmonella Typhimurium is the causative agent of non-typhoidal, foodborne salmonellosis. Contamination of hen eggs by the bacterium is a common source of S. Typhimurium infection. S. Typhimurium is peritrichous, and flagellum-dependent motility and chemotaxis are believed to facilitate egg contamination despite the presence of many antimicrobial egg components. We performed motility and chemotaxis assays to demonstrate that S. Typhimurium cells are attracted to egg yolks and are repelled by albumen. The bacterial flagellar motor shows bidirectional rotation, and counterclockwise-biased rotation allows cells to swim smoothly. A rotation assay for a single flagellum showed that, in comparison with thin albumen, the thick albumen more strongly affected the directional bias of the flagellar rotation, resulting in a remarkable suppression of the migration distance. Nevertheless, the S. Typhimurium cells retained positive chemotaxis toward the yolk in the presence of the albumens, suggesting that motility facilitates the growth of S. Typhimurium and survival in eggs.


Assuntos
Clara de Ovo/microbiologia , Gema de Ovo/microbiologia , Microbiologia de Alimentos , Salmonella typhimurium/fisiologia , Animais , Quimiotaxia , Galinhas/microbiologia , Contagem de Colônia Microbiana , Gema de Ovo/metabolismo , Flagelos/fisiologia , Locomoção , Rotação
16.
J Bacteriol ; 201(5)2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30559113

RESUMO

The flagellar lipoprotein FlgP has been identified in several species of bacteria, and its absence provokes different phenotypes. In this study, we show that in the alphaproteobacterium Rhodobacter sphaeroides, a ΔflgP mutant is unable to assemble the hook and the filament. In contrast, the membrane/supramembrane (MS) ring and the flagellar rod appear to be assembled. In the absence of FlgP a severe defect in the transition from rod to hook polymerization occurs. In agreement with this idea, we noticed a reduction in the amount of intracellular flagellin and the chemotactic protein CheY4, both encoded by genes dependent on σ28 This suggests that in the absence of flgP the switch to export the anti-sigma factor, FlgM, does not occur. The presence of FlgP was detected by Western blot in samples of isolated wild-type filament basal bodies, indicating that FlgP is an integral part of the flagellar structure. In this regard, we show that FlgP interacts with FlgH and FlgT, indicating that FlgP should be localized closely to the L and H rings. We propose that FlgP could affect the architecture of the L ring, which has been recently identified to be responsible for the rod-hook transition.IMPORTANCE Flagellar based motility confers a selective advantage on bacteria by allowing migration to favorable environments or in pathogenic species to reach the optimal niche for colonization. The flagellar structure has been well established in Salmonella However, other accessory components have been identified in other species. Many of these have been implied in adapting the flagellar function to enable faster rotation, or higher torque. FlgP has been proposed to be the main component of the basal disk located underlying the outer membrane in Campylobacter jejuni and Vibrio fischeri Its role is still unclear, and its absence impacts motility differently in different species. The study of these new components will bring a better understanding of the evolution of this complex organelle.


Assuntos
Flagelos/metabolismo , Flagelina/metabolismo , Lipoproteínas/metabolismo , Rhodobacter sphaeroides/fisiologia , Western Blotting , Flagelos/fisiologia , Flagelina/genética , Deleção de Genes , Lipoproteínas/deficiência , Mapeamento de Interação de Proteínas , Rhodobacter sphaeroides/genética
17.
Virulence ; 9(1): 1163-1175, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30070169

RESUMO

Pseudomonas aeruginosa, an opportunistic pathogen involved in skin and lung diseases, possesses numerous virulence factors, including type 2 and 3 secretion systems (T2SS and T3SS) and its flagellum, whose functions remain poorly known during cutaneous infection. Using isogenic mutants deleted from genes encoding each or all of these three virulence factors, we investigated their role in induction of inflammatory response and in tissue invasiveness in human primary keratinocytes and reconstructed epidermis. Our results showed that flagellum, but not T2SS and T3SS, is involved in induction of a large panel of cytokine, chemokine, and antimicrobial peptide (AMP) mRNA in the infected keratinocytes. Chemokine secretion and AMP tissular production were also dependent on the presence of the bacterial flagellum. This pro-inflammatory effect was significantly reduced in keratinocytes infected in presence of anti-toll-like receptor 5 (TLR5) neutralizing antibody. Bacterial invasion of human epidermis and persistence in a mouse model of sub-cutaneous infection were dependent on the P. aeruginosa flagellum. We demonstrated that flagellum constitutes the main virulence factor of P. aeruginosa involved not only in early induction of the epidermis inflammatory response but also in bacterial invasion and cutaneous persistence. P. aeruginosa is mainly sensed by TLR5 during the early innate immune response of human primary keratinocytes.


Assuntos
Epiderme/microbiologia , Flagelos/fisiologia , Inflamação/microbiologia , Queratinócitos/imunologia , Pseudomonas aeruginosa/patogenicidade , Animais , Anticorpos Neutralizantes/farmacologia , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/imunologia , Células Cultivadas , Quimiocinas/genética , Quimiocinas/imunologia , Citocinas/genética , Citocinas/imunologia , Modelos Animais de Doenças , Humanos , Imunidade Inata , Queratinócitos/efeitos dos fármacos , Queratinócitos/microbiologia , Masculino , Camundongos , Mutação , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/imunologia , Pseudomonas aeruginosa/ultraestrutura , Receptor 5 Toll-Like/imunologia , Fatores de Virulência/deficiência , Fatores de Virulência/genética
18.
Phys Rev Lett ; 121(5): 058103, 2018 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-30118294

RESUMO

Active living organisms exhibit behavioral variability, partitioning between fast and slow dynamics. Such variability may be key to generating rapid responses in a heterogeneous, unpredictable environment wherein cellular activity effects continual exchanges of energy fluxes. We demonstrate a novel, noninvasive strategy for revealing nonequilibrium control of swimming-specifically, in an octoflagellate microalga. These organisms exhibit surprising features of flagellar excitability and mechanosensitivity, which characterize a novel, time-irreversible "run-stop-shock" motility comprising forward runs, knee-jerk shocks with dramatic beat reversal, and long stops during which cells are quiescent yet continue to exhibit submicron flagellar vibrations. Entropy production, associated with flux cycles arising in a reaction graph representation of the gait-switching dynamics, provides a direct measure of detailed balance violation in this primitive alga.


Assuntos
Clorófitas/fisiologia , Flagelos/fisiologia , Modelos Biológicos , Movimento
19.
Phys Rev E ; 97(6-1): 062604, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30011513

RESUMO

We studied the swimming of Escherichia coli bacteria in the vicinity of the critical point in a solution of the nonionic surfactant C_{12}E_{5} in buffer solution. In phase-contrast microscopy, each swimming cell produces a transient trail behind itself lasting several seconds. Comparing quantitative image analysis with simulations show that these trails are due to local phase reorganization triggered by differential adsorption. This contrasts with similar trails seen in bacteria swimming in liquid crystals, which are due to shear effects. We show how our trails are controlled, and use them to probe the structure and dynamics of critical fluctuations in the fluid medium.


Assuntos
Escherichia coli/fisiologia , Movimento , Tensoativos , Água , Simulação por Computador , Escherichia coli/genética , Éteres , Flagelos/fisiologia , Microscopia , Modelos Biológicos , Mutação , Polietilenoglicóis , Temperatura Ambiente
20.
Proc Natl Acad Sci U S A ; 115(31): E7341-E7350, 2018 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-30030284

RESUMO

The 9 + 2 axoneme structure of the motile flagellum/cilium is an iconic, apparently symmetrical cellular structure. Recently, asymmetries along the length of motile flagella have been identified in a number of organisms, typically in the inner and outer dynein arms. Flagellum-beat waveforms are adapted for different functions. They may start either near the flagellar tip or near its base and may be symmetrical or asymmetrical. We hypothesized that proximal/distal asymmetry in the molecular composition of the axoneme may control the site of waveform initiation and the direction of waveform propagation. The unicellular eukaryotic pathogens Trypanosoma brucei and Leishmania mexicana often switch between tip-to-base and base-to-tip waveforms, making them ideal for analysis of this phenomenon. We show here that the proximal and distal portions of the flagellum contain distinct outer dynein arm docking-complex heterodimers. This proximal/distal asymmetry is produced and maintained through growth by a concentration gradient of the proximal docking complex, generated by intraflagellar transport. Furthermore, this asymmetry is involved in regulating whether a tip-to-base or base-to-tip beat occurs, which is linked to a calcium-dependent switch. Our data show that the mechanism for generating proximal/distal flagellar asymmetry can control waveform initiation and propagation direction.


Assuntos
Dineínas/química , Flagelos/fisiologia , Axonema/química , Flagelos/química , Multimerização Proteica
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