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Sample records for flagellar genes activates

  1. Deduction of upstream sequences of Xanthomonas campestris flagellar genes responding to transcription activation by FleQ

    SciTech Connect

    Hu, R.-M.; Yang, T.-C.; Yang, S.-H.; Tseng, Y.-H. . E-mail: yhtseng@chtai.ctc.edu.tw

    2005-10-07

    Xanthomonas campestris pv. campestris (Xcc), a close relative to Pseudomonas aeruginosa, is the pathogen causing black rot in cruciferous plants. In P. aeruginosa, FleQ serves as a cognate activator of {sigma}{sup 54} in transcription from several {sigma}{sup 54}-dependent promoters of flagellar genes. These P. aeruginosa promoters have been analyzed for FleQ-binding sequences; however, no consensus was deduced. Xcc, although lacks fleSR, has a fleQ homologue residing among over 40 contiguously clustered flagellar genes. A fleQ mutant, Xc17fleQ, constructed by insertional mutation is deficient in FleQ protein, non-flagellated, and immobile. Transcriptional fusion assays on six putative {sigma}{sup 54}-dependent promoters of the flagellar genes, fliE, fliQ, fliL, flgG, flgB, and flhF, indicated that each of them is also FleQ dependent. Each of these promoters has a sequence with weak consensus to 5'-gaaacCCgccgCcgctTt-3', immediately upstream of the predicted {sigma}{sup 54}-binding site, with an imperfect inverted repeat containing a GC-rich center flanked by several A and T at 5'- and 3'-ends, respectively. Replacing this region in fliE promoter with a HindIII recognition sequence abolished the transcription, indicating that this region responds to transcription activation by FleQ.

  2. Regulation of Flagellar Gene Expression in Bacteria.

    PubMed

    Osterman, I A; Dikhtyar, Yu Yu; Bogdanov, A A; Dontsova, O A; Sergiev, P V

    2015-11-01

    The flagellum of a bacterium is a supramolecular structure of extreme complexity comprising simultaneously both a unique system of protein transport and a molecular machine that enables the bacterial cell movement. The cascade of expression of genes encoding flagellar components is closely coordinated with the steps of molecular machine assembly, constituting an amazing regulatory system. Data on structure, assembly, and regulation of flagellar gene expression are summarized in this review. The regulatory mechanisms and correlation of the process of regulation of gene expression and flagellum assembly known from the literature are described.

  3. Aeromonas hydrophila Lateral Flagellar Gene Transcriptional Hierarchy

    PubMed Central

    Wilhelms, Markus; Gonzalez, Victor; Merino, Susana

    2013-01-01

    Aeromonas hydrophila AH-3 lateral flagella are not assembled when bacteria grow in liquid media; however, lateral flagellar genes are transcribed. Our results indicate that A. hydrophila lateral flagellar genes are transcribed at three levels (class I to III genes) and share some similarities with, but have many important differences from, genes of Vibrio parahaemolyticus. A. hydrophila lateral flagellum class I gene transcription is σ70 dependent, which is consistent with the fact that lateral flagellum is constitutively transcribed, in contrast to the characteristics of V. parahaemolyticus. The fact that multiple genes are included in class I highlights that lateral flagellar genes are less hierarchically transcribed than polar flagellum genes. The A. hydrophila lafK-fliEJL gene cluster (where the subscript L distinguishes genes for lateral flagella from those for polar flagella) is exclusively from class I and is in V. parahaemolyticus class I and II. Furthermore, the A. hydrophila flgAMNL cluster is not transcribed from the σ54/LafK-dependent promoter and does not contain class II genes. Here, we propose a gene transcriptional hierarchy for the A. hydrophila lateral flagella. PMID:23335410

  4. Genetic and Transcriptional Analyses of the Flagellar Gene Cluster in Actinoplanes missouriensis.

    PubMed

    Jang, Moon-Sun; Mouri, Yoshihiro; Uchida, Kaoru; Aizawa, Shin-Ichi; Hayakawa, Masayuki; Fujita, Nobuyuki; Tezuka, Takeaki; Ohnishi, Yasuo

    2016-08-15

    Actinoplanes missouriensis, a Gram-positive and soil-inhabiting bacterium, is a member of the rare actinomycetes. The filamentous cells produce sporangia, which contain hundreds of flagellated spores that can swim rapidly for a short period of time until they find niches for germination. These swimming cells are called zoospores, and the mechanism of this unique temporal flagellation has not been elucidated. Here, we report all of the flagellar genes in the bacterial genome and their expected function and contribution for flagellar morphogenesis. We identified a large flagellar gene cluster composed of 33 genes that encode the majority of proteins essential for assembling the functional flagella of Gram-positive bacteria. One noted exception to the cluster was the location of the fliQ gene, which was separated from the cluster. We examined the involvement of four genes in flagellar biosynthesis by gene disruption, fliQ, fliC, fliK, and lytA Furthermore, we performed a transcriptional analysis of the flagellar genes using RNA samples prepared from A. missouriensis grown on a sporangium-producing agar medium for 1, 3, 6, and 40 days. We demonstrated that the transcription of the flagellar genes was activated in conjunction with sporangium formation. Eleven transcriptional start points of the flagellar genes were determined using the rapid amplification of cDNA 5' ends (RACE) procedure, which revealed the highly conserved promoter sequence CTCA(N15-17)GCCGAA. This result suggests that a sigma factor is responsible for the transcription of all flagellar genes and that the flagellar structure assembles simultaneously. The biology of a zoospore is very interesting from the viewpoint of morphogenesis, survival strategy, and evolution. Here, we analyzed flagellar genes in A. missouriensis, which produces sporangia containing hundreds of flagellated spores each. Zoospores released from the sporangia swim for a short time before germination occurs. We identified a large

  5. Genetic and Transcriptional Analyses of the Flagellar Gene Cluster in Actinoplanes missouriensis

    PubMed Central

    Jang, Moon-Sun; Mouri, Yoshihiro; Uchida, Kaoru; Aizawa, Shin-Ichi; Hayakawa, Masayuki; Fujita, Nobuyuki; Tezuka, Takeaki

    2016-01-01

    ABSTRACT Actinoplanes missouriensis, a Gram-positive and soil-inhabiting bacterium, is a member of the rare actinomycetes. The filamentous cells produce sporangia, which contain hundreds of flagellated spores that can swim rapidly for a short period of time until they find niches for germination. These swimming cells are called zoospores, and the mechanism of this unique temporal flagellation has not been elucidated. Here, we report all of the flagellar genes in the bacterial genome and their expected function and contribution for flagellar morphogenesis. We identified a large flagellar gene cluster composed of 33 genes that encode the majority of proteins essential for assembling the functional flagella of Gram-positive bacteria. One noted exception to the cluster was the location of the fliQ gene, which was separated from the cluster. We examined the involvement of four genes in flagellar biosynthesis by gene disruption, fliQ, fliC, fliK, and lytA. Furthermore, we performed a transcriptional analysis of the flagellar genes using RNA samples prepared from A. missouriensis grown on a sporangium-producing agar medium for 1, 3, 6, and 40 days. We demonstrated that the transcription of the flagellar genes was activated in conjunction with sporangium formation. Eleven transcriptional start points of the flagellar genes were determined using the rapid amplification of cDNA 5′ ends (RACE) procedure, which revealed the highly conserved promoter sequence CTCA(N15–17)GCCGAA. This result suggests that a sigma factor is responsible for the transcription of all flagellar genes and that the flagellar structure assembles simultaneously. IMPORTANCE The biology of a zoospore is very interesting from the viewpoint of morphogenesis, survival strategy, and evolution. Here, we analyzed flagellar genes in A. missouriensis, which produces sporangia containing hundreds of flagellated spores each. Zoospores released from the sporangia swim for a short time before germination occurs

  6. Gene Expression Profiling of Flagellar Disassembly in Chlamydomonas reinhardtii

    PubMed Central

    Chamberlain, Kara L.; Miller, Steven H.; Keller, Laura R.

    2008-01-01

    Flagella are sensory organelles that interact with the environment through signal transduction and gene expression networks. We used microarray profiling to examine gene regulation associated with flagellar length change in the green alga Chlamydomonas reinhardtii. Microarrays were probed with fluorescently labeled cDNAs synthesized from RNA extracted from cells before and during flagellar assembly or disassembly. Evaluation of the gene expression profiles identified >100 clones showing at least a twofold change in expression during flagellar length changes. Products of these genes are associated not only with flagellar structure and motility but also with other cellular responses, including signal transduction and metabolism. Expression of specific genes from each category was further characterized at higher resolution by using quantitative real-time PCR (qRT–PCR). Analysis and comparison of the gene expression profiles coupled to flagellar assembly and disassembly revealed that each process involves a new and uncharacterized whole-cell response to flagellar length changes. This analysis lays the groundwork for a more comprehensive understanding of the cellular and molecular networks regulating flagellar length changes. PMID:18493036

  7. A Complete Set of Flagellar Genes Acquired by Horizontal Transfer Coexists with the Endogenous Flagellar System in Rhodobacter sphaeroides▿ †

    PubMed Central

    Poggio, Sebastian; Abreu-Goodger, Cei; Fabela, Salvador; Osorio, Aurora; Dreyfus, Georges; Vinuesa, Pablo; Camarena, Laura

    2007-01-01

    Bacteria swim in liquid environments by means of a complex rotating structure known as the flagellum. Approximately 40 proteins are required for the assembly and functionality of this structure. Rhodobacter sphaeroides has two flagellar systems. One of these systems has been shown to be functional and is required for the synthesis of the well-characterized single subpolar flagellum, while the other was found only after the genome sequence of this bacterium was completed. In this work we found that the second flagellar system of R. sphaeroides can be expressed and produces a functional flagellum. In many bacteria with two flagellar systems, one is required for swimming, while the other allows movement in denser environments by producing a large number of flagella over the entire cell surface. In contrast, the second flagellar system of R. sphaeroides produces polar flagella that are required for swimming. Expression of the second set of flagellar genes seems to be positively regulated under anaerobic growth conditions. Phylogenic analysis suggests that the flagellar system that was initially characterized was in fact acquired by horizontal transfer from a γ-proteobacterium, while the second flagellar system contains the native genes. Interestingly, other α-proteobacteria closely related to R. sphaeroides have also acquired a set of flagellar genes similar to the set found in R. sphaeroides, suggesting that a common ancestor received this gene cluster. PMID:17293429

  8. Interactions between chemotaxis genes and flagellar genes in Escherichia coli.

    PubMed Central

    Parkinson, J S; Parker, S R; Talbert, P B; Houts, S E

    1983-01-01

    Escherichia coli mutants defective in cheY and cheZ function are motile but generally nonchemotactic; cheY mutants have an extreme counterclockwise bias in flagellar rotation, whereas cheZ mutants have a clockwise rotational bias. Chemotactic pseudorevertants of cheY and cheZ mutants were isolated on semisolid agar and examined for second-site suppressors in other chemotaxis-related loci. Approximately 15% of the cheZ revertants and over 95% of the cheY revertants contained compensatory mutations in the flaA or flaB locus. When transferred to an otherwise wild-type background, most of these suppressor mutations resulted in a generally nonchemotactic phenotype: suppressors of cheY caused a clockwise rotational bias; suppressors of cheZ produced a counterclockwise rotational bias. Chemotactic double mutants containing a che and a fla mutation invariably exhibited flagellar rotation patterns in between the opposing extremes characteristic of the component mutations. This additive effect on flagellar rotation resulted in essentially wild-type swimming behavior and is probably the major basis of suppressor action. However, suppression effects were also allele specific, suggesting that the cheY and cheZ gene products interact directly with the flaA and flaB products. These interactions may be instrumental in establishing the unstimulated swimming pattern of E. coli. Images PMID:6305913

  9. Flagellar apparatus gene sequences of Aeromonas hydrophila AL09-73 isolate

    USDA-ARS?s Scientific Manuscript database

    Flagellar apparatus genes of recent outbreak Aeromonas hydrophila AL09-73 isolate were sequenced and characterized. Total 28 flagellar genes were identified. The sizes of the genes range from 318 to 2001 nucleotides, which potentially encode different complex flagellar proteins. At nucleotide and...

  10. A Protein Thermometer Controls Temperature-Dependent Transcription of Flagellar Motility Genes in Listeria monocytogenes

    PubMed Central

    Kamp, Heather D.; Higgins, Darren E.

    2011-01-01

    Facultative bacterial pathogens must adapt to multiple stimuli to persist in the environment or establish infection within a host. Temperature is often utilized as a signal to control expression of virulence genes necessary for infection or genes required for persistence in the environment. However, very little is known about the molecular mechanisms that allow bacteria to adapt and respond to temperature fluctuations. Listeria monocytogenes (Lm) is a food-borne, facultative intracellular pathogen that uses flagellar motility to survive in the extracellular environment and to enhance initial invasion of host cells during infection. Upon entering the host, Lm represses transcription of flagellar motility genes in response to mammalian physiological temperature (37°C) with a concomitant temperature-dependent up-regulation of virulence genes. We previously determined that down-regulation of flagellar motility is required for virulence and is governed by the reciprocal activities of the MogR transcriptional repressor and the bifunctional flagellar anti-repressor/glycosyltransferase, GmaR. In this study, we determined that GmaR is also a protein thermometer that controls temperature-dependent transcription of flagellar motility genes. Two-hybrid and gel mobility shift analyses indicated that the interaction between MogR and GmaR is temperature sensitive. Using circular dichroism and limited proteolysis, we determined that GmaR undergoes a temperature-dependent conformational change as temperature is elevated. Quantitative analysis of GmaR in Lm revealed that GmaR is degraded in the absence of MogR and at 37°C (when the MogR:GmaR complex is less stable). Since MogR represses transcription of all flagellar motility genes, including transcription of gmaR, changes in the stability of the MogR:GmaR anti-repression complex, due to conformational changes in GmaR, mediates repression or de-repression of flagellar motility genes in Lm. Thus, GmaR functions as a thermo

  11. Identification and characterization of flagellar biosynthetic genes in Yersinia ruckeri

    USDA-ARS?s Scientific Manuscript database

    Using transposon mutagenesis we have identified a Yersinia ruckeri serovar I mutant defective in both motility and production of secreted lipase activity. Sequence analysis of this mutant revealed a single transposon insertion in an open reading frame (ORF) with homology to flhA, a flagellar biosynt...

  12. Flagellar tip activation stimulated by membrane adhesions in Chlamydomonas gametes

    PubMed Central

    1980-01-01

    Membrane adhesions between the flagella of mating-type "plus" and "minus" gametes of Chlamydomonas reinhardi are shown to stimulate a rapid change in the ultrastructure of the flagellar tips, designated as flagellar tip activation (FTA). A dense substance, termed fibrous tip material (FTM), accumulates between the flagellar membrane and the nine single A microtubules of the tip. The A microtubules then elongate, growing into the distal region of the tip, increasing tip length by 30%. This study describes FTA kinetics during normal and mutant matings, presents experiments designed to probe its role in the mating reaction, and offers the following conclusions: (a) FTA is elicited by agents that cross-link flagellar membrane components (including natural sexual agglutinins, antiflagellar antisera, and concanavalin A) but not by flagellar adherence to polylysine-coated films. (b) FTA is reversed by flagellar disadhesion. (c) Gametes can undergo repeated cycles of FTA during successive rounds of adhesion/disadhesion. (d) FTA, flagellar tipping, and sexual signaling are simultaneously blocked by colchicine and by vinblastine, suggesting that tubulinlike molecules, perhaps exposed at the membrane surface, are involved in all three responses. (e) FTA is not blocked by short exposure to chymotrypsin, by cytochalasins B and D, nor by concanavalin A, even though all block cell fusion; the response is therefore autonomous and experimentally dissociable from later stages in the mating reaction. (f) Under no experimental conditions is mating-structure activation observed to occur unless FTA also occurs. This study concludes that FTA is a necessary event in the sexual signaling sequence, and presents a testable working model for its mechanism. PMID:7358792

  13. Active Phase and Amplitude Fluctuations of Flagellar Beating

    NASA Astrophysics Data System (ADS)

    Ma, Rui; Klindt, Gary S.; Riedel-Kruse, Ingmar H.; Jülicher, Frank; Friedrich, Benjamin M.

    2014-07-01

    The eukaryotic flagellum beats periodically, driven by the oscillatory dynamics of molecular motors, to propel cells and pump fluids. Small but perceivable fluctuations in the beat of individual flagella have physiological implications for synchronization in collections of flagella as well as for hydrodynamic interactions between flagellated swimmers. Here, we characterize phase and amplitude fluctuations of flagellar bending waves using shape mode analysis and limit-cycle reconstruction. We report a quality factor of flagellar oscillations Q =38.0±16.7 (mean±s.e.). Our analysis shows that flagellar fluctuations are dominantly of active origin. Using a minimal model of collective motor oscillations, we demonstrate how the stochastic dynamics of individual motors can give rise to active small-number fluctuations in motor-cytoskeleton systems.

  14. Interaction of the atypical prokaryotic transcription activator FlhD2C2 with early promoters of the flagellar gene hierarchy.

    PubMed

    Claret, Laurent; Hughes, Colin

    2002-08-09

    The transcriptional activator FlhD2C2 is the master regulator of bacterial flagellum biogenesis and swarming migration, activating the "early" class II promoters of the large flagellar gene hierarchy. Using primer extensions, band-shift assays, and enzymatic and chemical footprinting, we describe the binding of the FlhD2C2 heterotetramer to the promoter regions of four class II flagella operons, fliAZ, flhBA and the divergent flgAMN and flgBCD(EFGHIJ). Each of the promoter regions was bound by a single heterotetramer, i.e. the flgAMN and flgBCD operons are characterised by a single FlhD2C2 binding site. Binding affinity differed, and correlated with previously reported promoter strength and order of activation. Methylation protection and interference, and depurination and depyrimidation interference provided a detailed map of critical bases within a common 46-59bp DNaseI footprint overlapping the promoter -35 sequences. These data and compilation of the 12 known class II promoter sequences of Escherichia coli, Proteus mirabilis and Salmonella typhimurium allowed determination of a FlhD2C2 binding site with pseudo symmetry, comprising two 17-18bp inverted repeats, each a consensus FlhD2C2 box, separated by a 10-11bp spacer. DNaseI hypersensitivity indicated that binding may cause a conformational change in the promoter regions. Only the FlhC subunit can bind DNA independently, but the specificity and stability of the interaction is strengthened by FlhD. Here, photo-crosslinking established that both FlhC and the stabilising FlhD contact the DNA within the FlhD2C2 tetramer. Our data suggest that specificity of recognition and stability of the FlhD2C2/DNA complex require protein-protein interaction and interaction of both FlhC and FlhD subunits with DNA. These characteristics of the FlhD and FlhC subunits in the FlhD2C2/DNA complex are strikingly atypical of prokaryotic regulators.

  15. Functional Activation of the Flagellar Type III Secretion Export Apparatus

    PubMed Central

    Phillips, Andrew M.; Calvo, Rebecca A.; Kearns, Daniel B.

    2015-01-01

    Flagella are assembled sequentially from the inside-out with morphogenetic checkpoints that enforce the temporal order of subunit addition. Here we show that flagellar basal bodies fail to proceed to hook assembly at high frequency in the absence of the monotopic protein SwrB of Bacillus subtilis. Genetic suppressor analysis indicates that SwrB activates the flagellar type III secretion export apparatus by the membrane protein FliP. Furthermore, mutants defective in the flagellar C-ring phenocopy the absence of SwrB for reduced hook frequency and C-ring defects may be bypassed either by SwrB overexpression or by a gain-of-function allele in the polymerization domain of FliG. We conclude that SwrB enhances the probability that the flagellar basal body adopts a conformation proficient for secretion to ensure that rod and hook subunits are not secreted in the absence of a suitable platform on which to polymerize. PMID:26244495

  16. FlhG employs diverse intrinsic domains and influences FlhF GTPase activity to numerically regulate polar flagellar biogenesis in Campylobacter jejuni.

    PubMed

    Gulbronson, Connor J; Ribardo, Deborah A; Balaban, Murat; Knauer, Carina; Bange, Gert; Hendrixson, David R

    2016-01-01

    Flagellation in polar flagellates is one of the rare biosynthetic processes known to be numerically regulated in bacteria. Polar flagellates must spatially and numerically regulate flagellar biogenesis to create flagellation patterns for each species that are ideal for motility. FlhG ATPases numerically regulate polar flagellar biogenesis, yet FlhG orthologs are diverse in motif composition. We discovered that Campylobacter jejuni FlhG is at the center of a multipartite mechanism that likely influences a flagellar biosynthetic step to control flagellar number for amphitrichous flagellation, rather than suppressing activators of flagellar gene transcription as in Vibrio and Pseudomonas species. Unlike other FlhG orthologs, the FlhG ATPase domain was not required to regulate flagellar number in C. jejuni. Instead, two regions of C. jejuni FlhG that are absent or significantly altered in FlhG orthologs are involved in numerical regulation of flagellar biogenesis. Additionally, we found that C. jejuni FlhG influences FlhF GTPase activity, which may mechanistically contribute to flagellar number regulation. Our work suggests that FlhG ATPases divergently evolved in each polarly flagellated species to employ different intrinsic domains and extrinsic effectors to ultimately mediate a common output - precise numerical control of polar flagellar biogenesis required to create species-specific flagellation patterns optimal for motility.

  17. Genome-wide transcriptional analysis of flagellar regeneration in Chlamydomonas reinhardtii identifies orthologs of ciliary disease genes

    PubMed Central

    Stolc, Viktor; Samanta, Manoj Pratim; Tongprasit, Waraporn; Marshall, Wallace F.

    2005-01-01

    The important role that cilia and flagella play in human disease creates an urgent need to identify genes involved in ciliary assembly and function. The strong and specific induction of flagellar-coding genes during flagellar regeneration in Chlamydomonas reinhardtii suggests that transcriptional profiling of such cells would reveal new flagella-related genes. We have conducted a genome-wide analysis of RNA transcript levels during flagellar regeneration in Chlamydomonas by using maskless photolithography method-produced DNA oligonucleotide microarrays with unique probe sequences for all exons of the 19,803 predicted genes. This analysis represents previously uncharacterized whole-genome transcriptional activity profiling study in this important model organism. Analysis of strongly induced genes reveals a large set of known flagellar components and also identifies a number of important disease-related proteins as being involved with cilia and flagella, including the zebrafish polycystic kidney genes Qilin, Reptin, and Pontin, as well as the testis-expressed tubby-like protein TULP2. PMID:15738400

  18. Genome-wide transcriptional analysis of flagellar regeneration in Chlamydomonas reinhardtii identifies orthologs of ciliary disease genes

    NASA Technical Reports Server (NTRS)

    Stolc, Viktor; Samanta, Manoj Pratim; Tongprasit, Waraporn; Marshall, Wallace F.

    2005-01-01

    The important role that cilia and flagella play in human disease creates an urgent need to identify genes involved in ciliary assembly and function. The strong and specific induction of flagellar-coding genes during flagellar regeneration in Chlamydomonas reinhardtii suggests that transcriptional profiling of such cells would reveal new flagella-related genes. We have conducted a genome-wide analysis of RNA transcript levels during flagellar regeneration in Chlamydomonas by using maskless photolithography method-produced DNA oligonucleotide microarrays with unique probe sequences for all exons of the 19,803 predicted genes. This analysis represents previously uncharacterized whole-genome transcriptional activity profiling study in this important model organism. Analysis of strongly induced genes reveals a large set of known flagellar components and also identifies a number of important disease-related proteins as being involved with cilia and flagella, including the zebrafish polycystic kidney genes Qilin, Reptin, and Pontin, as well as the testis-expressed tubby-like protein TULP2.

  19. Genome-wide transcriptional analysis of flagellar regeneration in Chlamydomonas reinhardtii identifies orthologs of ciliary disease genes

    NASA Technical Reports Server (NTRS)

    Stolc, Viktor; Samanta, Manoj Pratim; Tongprasit, Waraporn; Marshall, Wallace F.

    2005-01-01

    The important role that cilia and flagella play in human disease creates an urgent need to identify genes involved in ciliary assembly and function. The strong and specific induction of flagellar-coding genes during flagellar regeneration in Chlamydomonas reinhardtii suggests that transcriptional profiling of such cells would reveal new flagella-related genes. We have conducted a genome-wide analysis of RNA transcript levels during flagellar regeneration in Chlamydomonas by using maskless photolithography method-produced DNA oligonucleotide microarrays with unique probe sequences for all exons of the 19,803 predicted genes. This analysis represents previously uncharacterized whole-genome transcriptional activity profiling study in this important model organism. Analysis of strongly induced genes reveals a large set of known flagellar components and also identifies a number of important disease-related proteins as being involved with cilia and flagella, including the zebrafish polycystic kidney genes Qilin, Reptin, and Pontin, as well as the testis-expressed tubby-like protein TULP2.

  20. The Salmonella Spi1 virulence regulatory protein HilD directly activates transcription of the flagellar master operon flhDC.

    PubMed

    Singer, Hanna M; Kühne, Caroline; Deditius, Julia A; Hughes, Kelly T; Erhardt, Marc

    2014-04-01

    Infection of intestinal epithelial cells is dependent on the Salmonella enterica serovar Typhimurium pathogenicity island 1 (Spi1)-encoded type III injectisome system and flagellar motility. Thus, the expression of virulence and flagellar genes is subject to tight regulatory control mechanisms in order to ensure the correct spatiotemporal production of the respective gene products. In this work, we reveal a new level of cross-regulation between the Spi1 and flagellar regulatory systems. Transposon mutagenesis identified a class of mutants that prevented flhDC autorepression by overexpressing HilD. HilD, HilC, RtsA, and HilA comprise a positive regulatory circuit for the expression of the Spi1 genes. Here, we report a novel transcriptional cross talk between the Spi1 and flagellar regulons where HilD transcriptionally activates flhDC gene expression by binding to nucleotides -68 to -24 upstream from the P5 transcriptional start site. We additionally show that, in contrast to the results of a previous report, HilA does not affect flagellar gene expression. Finally, we discuss a model of the cross-regulation network between Spi1 and the flagellar system and propose a regulatory mechanism via the Spi1 master regulator HilD that would prime flagellar genes for rapid reactivation during host infection.

  1. Opsonic activity of anti-flagellar serum against Clostridium chauvoei by mouse polymorphonuclear leucocytes.

    PubMed

    Tamura, Y; Tanaka, M

    1987-05-01

    The role of anti-flagellar serum against Clostridium chauvoei in phagocytosis by mouse polymorphonuclear leucocytes was examined. Anti-flagellar serum markedly increased phagocytic rate against the flagellated strain Okinawa but not against a non-flagellated mutant (NFM) derived from the same strain, while anti-NFM serum increased the phagocytic rate against both strains. These results indicate that anti-flagellar serum exerts its protective effect by opsonic activity.

  2. Brucella melitensis cyclic di-GMP phosphodiesterase BpdA controls expression of flagellar genes.

    PubMed

    Petersen, Erik; Chaudhuri, Pallab; Gourley, Chris; Harms, Jerome; Splitter, Gary

    2011-10-01

    Brucella melitensis encounters a variety of conditions and stimuli during its life cycle--including environmental growth, intracellular infection, and extracellular dissemination--which necessitates flexibility of bacterial signaling to promote virulence. Cyclic-di-GMP is a bacterial secondary signaling molecule that plays an important role in adaptation to changing environments and altering virulence in a number of bacteria. To investigate the role of cyclic-di-GMP in B. melitensis, all 11 predicted cyclic-di-GMP-metabolizing proteins were separately deleted and the effect on virulence was determined. Three of these cyclic-di-GMP-metabolizing proteins were found to alter virulence. Deletion of the bpdA and bpdB genes resulted in attenuation of virulence of the bacterium, while deletion of the cgsB gene produced a hypervirulent strain. In a Vibrio reporter system to monitor apparent alteration in levels of cyclic-di-GMP, both BpdA and BpdB displayed a phenotype consistent with cyclic-di-GMP-specific phosphodiesterases, while CgsB displayed a cyclic-di-GMP synthase phenotype. Further analysis found that deletion of bpdA resulted in a dramatic decrease in flagellar promoter activities, and a flagellar mutant showed similar phenotypes to the bpdA and bpdB mutant strains in mouse models of infection. These data indicate a potential role for regulation of flagella in Brucella melitensis via cyclic-di-GMP.

  3. T-POP array identifies EcnR and PefI-SrgD as novel regulators of flagellar gene expression.

    PubMed

    Wozniak, Christopher E; Lee, Changhan; Hughes, Kelly T

    2009-03-01

    The T-POP transposon was employed in a general screen for tetracycline (Tet)-induced chromosomal loci that exhibited Tet-activated or Tet-repressed expression of a fliC-lac transcriptional fusion. Insertions that activated flagellar transcription were located in flagellar genes. T-POP insertions that exhibited Tet-dependent fliC-lac inhibition were isolated upstream of the ecnR, fimZ, pefI-srgD, rcsB, and ydiV genes and in the flagellar gene flgA, which is located upstream of the anti-sigma(28) factor gene flgM. When expressed from the chromosomal P(araBAD) promoter, EcnR, FimZ, PefI-SrgD, and RcsB inhibited the transcription of the flagellar class 1 flhDC operon. YdiV, which is weakly homologous to EAL domain proteins involved in cyclic-di-GMP regulation, appears to act at a step after class 1 transcription. By using a series of deletions of the regulatory genes to try to disrupt each pathway, these regulators were found to act largely independently of one another. These results identify EcnR and PefI-SrgD as additional components of the complex regulatory network controlling flagellar expression.

  4. Transcriptional Control of the Lateral-Flagellar Genes of Bradyrhizobium diazoefficiens.

    PubMed

    Mongiardini, Elías J; Quelas, J Ignacio; Dardis, Carolina; Althabegoiti, M Julia; Lodeiro, Aníbal R

    2017-08-01

    Bradyrhizobium diazoefficiens, a soybean N2-fixing symbiont, possesses a dual flagellar system comprising a constitutive subpolar flagellum and inducible lateral flagella. Here, we analyzed the genomic organization and biosynthetic regulation of the lateral-flagellar genes. We found that these genes are located in a single genomic cluster, organized in two monocistronic transcriptional units and three operons, one possibly containing an internal transcription start site. Among the monocistronic units is blr6846, homologous to the class IB master regulators of flagellum synthesis in Brucella melitensis and Ensifer meliloti and required for the expression of all the lateral-flagellar genes except lafA2, whose locus encodes a single lateral flagellin. We therefore named blr6846 lafR (lateral-flagellar regulator). Despite its similarity to two-component response regulators and its possession of a phosphorylatable Asp residue, lafR behaved as an orphan response regulator by not requiring phosphorylation at this site. Among the genes induced by lafR is flbTL , a class III regulator. We observed different requirements for FlbTL in the synthesis of each flagellin subunit. Although the accumulation of lafA1, but not lafA2, transcripts required FlbTL, the production of both flagellin polypeptides required FlbTL Moreover, the regulation cascade of this lateral-flagellar regulon appeared to be not as strictly ordered as those found in other bacterial species.IMPORTANCE Bacterial motility seems essential for the free-living style in the environment, and therefore these microorganisms allocate a great deal of their energetic resources to the biosynthesis and functioning of flagella. Despite energetic costs, some bacterial species possess dual flagellar systems, one of which is a primary system normally polar or subpolar, and the other is a secondary, lateral system that is produced only under special circumstances. Bradyrhizobium diazoefficiens, an N2-fixing symbiont of soybean

  5. Codon-based phylogenetics introduces novel flagellar gene markers to oomycete systematics.

    PubMed

    Robideau, Gregg P; Rodrigue, Nicolas; André Lévesque, C

    2014-10-01

    Oomycete systematics has traditionally been reliant on ribosomal RNA and mitochondrial cytochrome oxidase sequences. Here we report the use of two single-copy protein-coding flagellar genes, PF16 and OCM1, in oomycete systematics, showing their utility in phylogenetic reconstruction and species identification. Applying a recently proposed mutation-selection model of codon substitution, the phylogenetic relationships inferred by flagellar genes are largely in agreement with the current views of oomycete evolution, whereas nucleotide- and amino acid-level models produce biologically implausible reconstructions. Interesting parallels exist between the phylogeny inferred from the flagellar genes and zoospore ontology, providing external support for the tree obtained using the codon model. The resolution achieved for species identification is ample using PF16, and quite robust using OCM1, and the described PCR primers are able to amplify both genes for a range of oomycete genera. Altogether, when analyzed with a rich codon substitution model, these flagellar genes provide useful markers for the oomycete molecular toolbox.

  6. Temporal regulation of genes encoding the flagellar proximal rod in Caulobacter crescentus.

    PubMed

    Boyd, C H; Gober, J W

    2001-01-01

    The gram-negative bacterium Caulobacter crescentus has a life cycle that includes two distinct and separable developmental stages, a motile swarmer phase and a sessile stalked phase. The cell cycle-controlled biogenesis of the single polar flagellum of the swarmer cell is the best-studied aspect of this developmental program. The flagellar regulon is arranged into a rigid trans-acting hierarchy of gene expression in which successful expression of early genes is required for the expression of genes that are later in the hierarchy and in which the order of gene expression mirrors the order of assembly of gene products into the completed flagellum. The flgBC-fliE genes were identified as a result of the C. crescentus genome sequencing project and encode the homologues of two flagellar proximal rod proteins, FlgB and FlgC, and one conserved protein, FliE, that is of unknown function. Footprint assays on a DNA fragment containing the operon promoter as well as in vivo mutant suppressor analysis of promoter mutations indicate that this operon is controlled by the cell cycle response regulator CtrA, which with sigma(70) is responsible for regulating transcription of other early flagellar genes in C. crescentus. Promoter analysis, timing of expression, and epistasis experiments place these genes outside of the flagellar regulatory hierarchy; they are expressed in class II mutants, and flgB deletions do not prevent class III gene expression. This operon is also unusual in that it is expressed from a promoter that is divergent from the class II operon containing fliP, which encodes a member of the flagellum-specific protein export apparatus.

  7. FlbD has a DNA-binding activity near its carboxy terminus that recognizes ftr sequences involved in positive and negative regulation of flagellar gene transcription in Caulobacter crescentus.

    PubMed Central

    Mullin, D A; Van Way, S M; Blankenship, C A; Mullin, A H

    1994-01-01

    G. Our results demonstrate that FlbD contains a sequence-specific DNA-binding activity within the 87 amino acids at its carboxy terminus, and the results suggest that FlbD exerts its effect as a positive and negative regulator of C. crescentus flagellar genes by binding to ftr sequences. Images PMID:7928958

  8. Loss of the WaaL O-Antigen Ligase Prevents Surface Activation of the Flagellar Gene Cascade in Proteus mirabilis▿

    PubMed Central

    Morgenstein, Randy M.; Clemmer, Katy M.; Rather, Philip N.

    2010-01-01

    Proteus mirabilis is a Gram-negative bacterium that undergoes a physical and biochemical change from a vegetative swimmer cell (a typical Gram-negative rod) to an elongated swarmer cell when grown on a solid surface. In this study, we report that a transposon insertion in the waaL gene, encoding O-antigen ligase, blocked swarming motility on solid surfaces but had little effect on swimming motility in soft agar. The waaL mutant was unable to differentiate into a swarmer cell. Differentiation was also prevented by a mutation in wzz, encoding a chain length determinant for O antigen, but not by a mutation in wzyE, encoding an enzyme that polymerizes enterobacterial common antigen, a surface polysaccharide different from the lipid A::core. In wild-type P. mirabilis, increased expression of the flhDC operon occurs after growth on solid surfaces and is required for the high-level expression of flagellin that is characteristic of swarmer cells. However, in both the waaL and the wzz mutants, the flhDC operon was not activated during growth on agar. A loss-of-function mutation in the rcsB response regulator or overexpression of flhDC restored swarming to the waaL mutant, despite the absence of O antigen. Therefore, although O antigen may serve a role in swarming by promoting wettability, the loss of O antigen blocks a regulatory pathway that links surface contact with the upregulation of flhDC expression. PMID:20382766

  9. Digital image analysis of flagellar beating and microtubule sliding of activated and hyperactivated sperm flagella.

    PubMed

    Ishijima, Sumio

    2007-01-01

    Flagellar beatings of Suncus, golden hamster, and monkey spermatozoa before and after hyperactivation were analysed using high-speed video microscopy and digital image processing in order to examine the sliding mechanism of the flagellar beating and the function of accessory fibres of the mammalian spermatozoa. Although these spermatozoa have different morphology and movement characteristics, the flagellar beatings of hyperactivated spermatozoa had a few common features; i.e., sharp bends at the base of the flagellum and a low beat frequency. While nonhyperactivated (activated) spermatozoa exhibited nearly constant-curvature beating, the hyperactivated spermatozoa displayed a constant-frequency beating. A detailed analysis of the microtubule sliding of the activated and hyperactivated sperm flagella revealed that the sharp bends at the base of the flagella were induced by an increase in the total length of the microtubule sliding at the base of the flagella and that the sliding velocity of the activated and hyperactivated sperm flagella was consistent within each species. A comparison of the sliding velocity of the flagellar beating of Suncus, golden hamster, and monkey spermatozoa with the moment of inertia of the cross section of the flagellar base suggests that the sliding velocity is involved in the hardness of a sperm flagellum.

  10. Identification of Multicomponent Histidine-Aspartate Phosphorelay System Controlling Flagellar and Motility Gene Expression in Geobacter Species*

    PubMed Central

    Ueki, Toshiyuki; Leang, Ching; Inoue, Kengo; Lovley, Derek R.

    2012-01-01

    Geobacter species play an important role in the natural biogeochemical cycles of aquatic sediments and subsurface environments as well as in subsurface bioremediation by oxidizing organic compounds with the reduction of insoluble Fe(III) oxides. Flagellum-based motility is considered to be critical for Geobacter species to locate fresh sources of Fe(III) oxides. Functional and comparative genomic approaches, coupled with genetic and biochemical methods, identified key regulators for flagellar gene expression in Geobacter species. A master transcriptional regulator, designated FgrM, is a member of the enhancer-binding protein family. The fgrM gene in the most studied strain of Geobacter species, Geobacter sulfurreducens strain DL-1, is truncated by a transposase gene, preventing flagellar biosynthesis. Integrating a functional FgrM homolog restored flagellar biosynthesis and motility in G. sulfurreducens DL-1 and enhanced the ability to reduce insoluble Fe(III) oxide. Interrupting the fgrM gene in G. sulfurreducens strain KN400, which is motile, removed the capacity for flagellar production and inhibited Fe(III) oxide reduction. FgrM, which is also a response regulator of the two-component His-Asp phosphorelay system, was phosphorylated by histidine kinase GHK4, which was essential for flagellar production and motility. GHK4, which is a hybrid kinase with a receiver domain at the N terminus, was phosphorylated by another histidine kinase, GHK3. Therefore, the multicomponent His-Asp phosphorelay system appears to control flagellar gene expression in Geobacter species. PMID:22362768

  11. Identification of multicomponent histidine-aspartate phosphorelay system controlling flagellar and motility gene expression in Geobacter species.

    PubMed

    Ueki, Toshiyuki; Leang, Ching; Inoue, Kengo; Lovley, Derek R

    2012-03-30

    Geobacter species play an important role in the natural biogeochemical cycles of aquatic sediments and subsurface environments as well as in subsurface bioremediation by oxidizing organic compounds with the reduction of insoluble Fe(III) oxides. Flagellum-based motility is considered to be critical for Geobacter species to locate fresh sources of Fe(III) oxides. Functional and comparative genomic approaches, coupled with genetic and biochemical methods, identified key regulators for flagellar gene expression in Geobacter species. A master transcriptional regulator, designated FgrM, is a member of the enhancer-binding protein family. The fgrM gene in the most studied strain of Geobacter species, Geobacter sulfurreducens strain DL-1, is truncated by a transposase gene, preventing flagellar biosynthesis. Integrating a functional FgrM homolog restored flagellar biosynthesis and motility in G. sulfurreducens DL-1 and enhanced the ability to reduce insoluble Fe(III) oxide. Interrupting the fgrM gene in G. sulfurreducens strain KN400, which is motile, removed the capacity for flagellar production and inhibited Fe(III) oxide reduction. FgrM, which is also a response regulator of the two-component His-Asp phosphorelay system, was phosphorylated by histidine kinase GHK4, which was essential for flagellar production and motility. GHK4, which is a hybrid kinase with a receiver domain at the N terminus, was phosphorylated by another histidine kinase, GHK3. Therefore, the multicomponent His-Asp phosphorelay system appears to control flagellar gene expression in Geobacter species.

  12. Flagellar structure and hyperthermophily: analysis of a single flagellin gene and its product in Aquifex pyrophilus.

    PubMed Central

    Behammer, W; Shao, Z; Mages, W; Rachel, R; Stetter, K O; Schmitt, R

    1995-01-01

    The polytrichously inserted flagella of Aquifex pyrophilus, a marine hyperthermophilic bacterium growing at 85 degrees C, were isolated and purified. Electron micrographs of the 19-nm-diameter flagellar filaments show prominent helical arrays of subunits. The primary structure of these 54-kDa flagellin monomers determining the helical shape and heat stability of filaments was of particular interest. The genomic region encoding the flagellin subunit (flaA gene) and an upstream open reading frame (orf1) were cloned and sequenced. The 1,503-bp flaA and 696-bp orf1 are preceded by separate sigma 28-like promoters and ribosome-binding motifs and succeeded by palindromic transcription terminators. Both genes are actively transcribed, but the nature and function of the orf1-encoded 231-residue polypeptide remain unknown. The deduced primary structure of the 501-amino-acid flagellin encoded by flaA consists of conserved N- and C-terminal regions and a variable 246-residue central domain. In comparison to mesophilic flagellins, the thermostable A. pyrophilus flagellin is characterized by increases in aromatic residues and prolines as well as by a 7.9% +/- 3.2% increase in all hydrophobic residues that is balanced by a respective decrease in hydrophilic residues. This composition is thought to form more compact flagellin monomers and stable interface contacts between neighboring subunits in the polymer. PMID:7592443

  13. Regulation of flagellar, motility and chemotaxis genes in Rhizobium leguminosarum by the VisN/R-Rem cascade.

    PubMed

    Tambalo, Dinah D; Del Bel, Kate L; Bustard, Denise E; Greenwood, Paige R; Steedman, Audrey E; Hynes, Michael F

    2010-06-01

    In this paper, we describe the regulatory roles of VisN, VisR and Rem in the expression of flagellar, motility and chemotaxis genes in Rhizobium leguminosarum biovar viciae strains VF39SM and 3841. Individual mutations in the genes encoding these proteins resulted in a loss of motility and an absence of flagella, indicating that these regulatory genes are essential for flagellar synthesis and function. Transcriptional experiments involving gusA-gene fusions in wild-type and mutant backgrounds were performed to identify the genes under VisN/R and Rem regulation. Results showed that the chemotaxis and motility genes of R. leguminosarum could be separated into two groups: one group under VisN/R-Rem regulation and another group that is independent of this regulation. VisN and VisR regulate the expression of rem, while Rem positively regulates the expression of flaA, flaB, flaC, flaD, motA, motB, che1 and mcpD. All of these genes except mcpD are located within the main motility and chemotaxis gene cluster of R. leguminosarum. Other chemotaxis and motility genes, which are found outside of the main motility gene cluster (che2 operon, flaH for VF39SM, and flaG) or are plasmid-borne (flaE and mcpC), are not part of the VisN/R-Rem regulatory cascade. In addition, all genes exhibited the same regulation pattern in 3841 and in VF39SM, except flaE and flaH. flaE is not regulated by VisN/R-Rem in 3841 but it is repressed by Rem in VF39SM. flaH is under VisN/R-Rem regulation in 3841, but not in VF39SM. A kinetics experiment demonstrated that a subset of the flagellar genes is continuously expressed in all growth phases, indicating the importance of continuous motility for R. leguminosarum under free-living conditions. On the other hand, motility is repressed under symbiotic conditions. Nodulation experiments showed that the transcriptional activators VisN and Rem are dramatically downregulated in the nodules, suggesting that the symbiotic downregulation of motility-related genes

  14. Activation loop phosphorylation of a protein kinase is a molecular marker of organelle size that dynamically reports flagellar length

    PubMed Central

    Cao, Muqing; Meng, Dan; Wang, Liang; Bei, Shuqing; Snell, William J.; Pan, Junmin

    2013-01-01

    Specification of organelle size is crucial for cell function, yet we know little about the molecular mechanisms that report and regulate organelle growth and steady-state dimensions. The biflagellated green alga Chlamydomonas requires continuous-length feedback to integrate the multiple events that support flagellar assembly and disassembly and at the same time maintain the sensory and motility functions of the organelle. Although several length mutants have been characterized, the requisite molecular reporter of length has not been identified. Previously, we showed that depletion of Chlamydomonas aurora-like protein kinase CALK inhibited flagellar disassembly and that a gel-shift–associated phosphorylation of CALK marked half-length flagella during flagellar assembly. Here, we show that phosphorylation of CALK on T193, a consensus phosphorylation site on the activation loop required for kinase activity, is distinct from the gel-shift–associated phosphorylation and is triggered when flagellar shortening is induced, thereby implicating CALK protein kinase activity in the shortening arm of length control. Moreover, CALK phosphorylation on T193 is dynamically related to flagellar length. It is reduced in cells with short flagella, elevated in the long flagella mutant, lf4, and dynamically tracks length during both flagellar assembly and flagellar disassembly in WT, but not in lf4. Thus, phosphorylation of CALK in its activation loop is implicated in the disassembly arm of a length feedback mechanism and is a continuous and dynamic molecular marker of flagellar length during both assembly and disassembly. PMID:23836633

  15. Expression of flagellin and key regulatory flagellar genes in the non-motile bacterium Piscirickettsia salmonis.

    PubMed

    Carril, Gabriela P; Gómez, Fernando A; Marshall, Sergio H

    2017-02-08

    The Piscirickettsia salmonis genome was screened to evaluate potential flagella-related open reading frames, as well as their genomic organization and eventual expression. A complete and organized set of flagellar genes was found for P. salmonis, although no structural flagellum has ever been reported for this bacterium. To gain further understanding, the hierarchical flagellar cascade described for Legionella pneumophila was used as a reference model for putative analysis in P. salmonis. Specifically, 5 of the most relevant genes from this cascade were chosen, including 3 regulatory genes (fleQ, triggers the cascade; fliA, regulates the σ28-coding gene; and rpoN, an RNA polymerase-dependent gene) and 2 terminal structural genes (flaA and flaB, flagellin and a flagellin-like protein, respectively). Kinetic experiments evaluated gene expressions over time, with P. salmonis assessed in 2 liquid, cell-free media and during infection of the SHK-1 fish cell line. Under all conditions, the 5 target genes were primarily expressed during early growth/infection and were differentially expressed when bacteria encountered environmental stress (i.e. a high-salt concentration). Intriguingly, the flagellin monomer was fully expressed under all growth conditions and was located near the bacterial membrane. While no structural flagellum was detected under any condition, the recombinant flagellin monomer induced a proinflammatory response in SHK-1 cells, suggesting a possible immunomodulatory function. The potential implications of these observations are discussed in the context of P. salmonis biology and pathogenic potential.

  16. Rapid Turnover of FlhD and FlhC, the Flagellar Regulon Transcriptional Activator Proteins, during Proteus Swarming

    PubMed Central

    Claret, Laurent; Hughes, Colin

    2000-01-01

    The enterobacterial flhDC master operon activates expression of the flagellar biogenesis gene hierarchy and also represses cell division. During Proteus mirabilis differentiation into elongated hyperflagellated swarm cells, flhDC transcription is strongly but transiently increased. We show that concentration of the FlhD and FlhC proteins is also tightly controlled at the posttranslational level. This is achieved by protein degradation, which is most severe after differentiation when the half-life of both proteins is ca. 2 min. Degradation is energy dependent and putatively involves the Lon protease. PMID:10633123

  17. Cloning of Flagellar Genes in Chlamydomonas Reinhardtii by DNA Insertional Mutagenesis

    PubMed Central

    Tam, L. W.; Lefebvre, P. A.

    1993-01-01

    Chlamydomonas is a popular genetic model system for studying many cellular processes. In this report, we describe a new approach to isolate Chlamydomonas genes using the cloned nitrate reductase gene (NIT1) as an insertional mutagen. A linearized plasmid containing the NIT1 gene was introduced into nit1 mutant cells by glass-bead transformation. Of 3000 Nit(+) transformants examined, 74 showed motility defects of a wide range of phenotypes, suggesting that DNA transformation is an effective method for mutagenizing cells. For 13 of 15 such motility mutants backcrossed to nit(-) mutant strains, the motility phenotype cosegregated with the Nit(+) phenotype, indicating that the motility defects of these 13 mutants may be caused by integration of the plasmid. Further genetic analysis indicated that three of these mutants contained alleles of previously identified loci: mbo2 (move backward only), pf13 (paralyzed flagella) and vfl1 (variable flagellar number). Three other abnormal-flagellar-number mutants did not map to any previously described loci at which mutations produce similar phenotypes. Genomic sequences flanking the integrated plasmid in the mbo2 and vfl1 mutants were isolated and used as probes to obtain wild-type genomic clones, which complemented the motility defects upon transformation into cells. Our results demonstrate the potential of this new approach for cloning genes identified by mutation in Chlamydomonas. PMID:8244002

  18. Quorum sensing controls flagellar morphogenesis in Burkholderia glumae.

    PubMed

    Jang, Moon Sun; Goo, Eunhye; An, Jae Hyung; Kim, Jinwoo; Hwang, Ingyu

    2014-01-01

    Burkholderia glumae is a motile plant pathogenic bacterium that has multiple polar flagella and one LuxR/LuxI-type quorum sensing (QS) system, TofR/TofI. A QS-dependent transcriptional regulator, QsmR, activates flagellar master regulator flhDC genes. FlhDC subsequently activates flagellar gene expression in B. glumae at 37°C. Here, we confirm that the interplay between QS and temperature is critical for normal polar flagellar morphogenesis in B. glumae. In the wild-type bacterium, flagellar gene expression and flagellar number were greater at 28°C compared to 37°C. The QS-dependent flhC gene was significantly expressed at 28°C in two QS-defective (tofI::Ω and qsmR::Ω) mutants. Thus, flagella were present in both tofI::Ω and qsmR::Ω mutants at 28°C, but were absent at 37°C. Most tofI::Ω and qsmR::Ω mutant cells possessed polar or nonpolar flagella at 28°C. Nonpolarly flagellated cells processing flagella around cell surface of both tofI::Ω and qsmR::Ω mutants exhibited tumbling and spinning movements. The flhF gene encoding GTPase involved in regulating the correct placement of flagella in other bacteria was expressed in QS mutants in a FlhDC-dependent manner at 28°C. However, FlhF was mislocalized in QS mutants, and was associated with nonpolar flagellar formation in QS mutants at 28°C. These results indicate that QS-independent expression of flagellar genes at 28°C allows flagellar biogenesis, but is not sufficient for normal polar flagellar morphogenesis in B. glumae. Our findings demonstrate that QS functions together with temperature to control flagellar morphogenesis in B. glumae.

  19. Quorum Sensing Controls Flagellar Morphogenesis in Burkholderia glumae

    PubMed Central

    Jang, Moon Sun; Goo, Eunhye; An, Jae Hyung; Kim, Jinwoo; Hwang, Ingyu

    2014-01-01

    Burkholderia glumae is a motile plant pathogenic bacterium that has multiple polar flagella and one LuxR/LuxI-type quorum sensing (QS) system, TofR/TofI. A QS-dependent transcriptional regulator, QsmR, activates flagellar master regulator flhDC genes. FlhDC subsequently activates flagellar gene expression in B. glumae at 37°C. Here, we confirm that the interplay between QS and temperature is critical for normal polar flagellar morphogenesis in B. glumae. In the wild-type bacterium, flagellar gene expression and flagellar number were greater at 28°C compared to 37°C. The QS-dependent flhC gene was significantly expressed at 28°C in two QS-defective (tofI::Ω and qsmR::Ω) mutants. Thus, flagella were present in both tofI::Ω and qsmR::Ω mutants at 28°C, but were absent at 37°C. Most tofI::Ω and qsmR::Ω mutant cells possessed polar or nonpolar flagella at 28°C. Nonpolarly flagellated cells processing flagella around cell surface of both tofI::Ω and qsmR::Ω mutants exhibited tumbling and spinning movements. The flhF gene encoding GTPase involved in regulating the correct placement of flagella in other bacteria was expressed in QS mutants in a FlhDC-dependent manner at 28°C. However, FlhF was mislocalized in QS mutants, and was associated with nonpolar flagellar formation in QS mutants at 28°C. These results indicate that QS-independent expression of flagellar genes at 28°C allows flagellar biogenesis, but is not sufficient for normal polar flagellar morphogenesis in B. glumae. Our findings demonstrate that QS functions together with temperature to control flagellar morphogenesis in B. glumae. PMID:24416296

  20. Nucleotide sequence and characterization of a Bacillus subtilis gene encoding a flagellar switch protein.

    PubMed Central

    Zuberi, A R; Bischoff, D S; Ordal, G W

    1991-01-01

    The nucleotide sequence of the Bacillus subtilis fliM gene has been determined. This gene encodes a 38-kDa protein that is homologous to the FliM flagellar switch proteins of Escherichia coli and Salmonella typhimurium. Expression of this gene in Che+ cells of E. coli and B. subtilis interferes with normal chemotaxis. The nature of the chemotaxis defect is dependent upon the host used. In B. subtilis, overproduction of FliM generates mostly nonmotile cells. Those cells that are motile switch less frequently. Expression of B. subtilis FliM in E. coli also generates nonmotile cells. However, those cells that are motile have a tumble bias. The B. subtilis fliM gene cannot complement an E. coli fliM mutant. A frameshift mutation was constructed in the fliM gene, and the mutation was transferred onto the B. subtilis chromosome. The mutant has a Fla- phenotype. This phenotype is consistent with the hypothesis that the FliM protein encodes a component of the flagellar switch in B. subtilis. Additional characterization of the fliM mutant suggests that the hag and mot loci are not expressed. These loci are regulated by the SigD form of RNA polymerase. We also did not observe any methyl-accepting chemotaxis proteins in an in vivo methylation experiment. The expression of these proteins is also dependent upon SigD. It is possible that a functional basal body-hook complex may be required for the expression of SigD-regulated chemotaxis and motility genes. Images PMID:1898932

  1. Identification of a gene cluster involved in flagellar basal body biogenesis in Caulobacter crescentus.

    PubMed

    Hahnenberger, K M; Shapiro, L

    1987-03-05

    The bacterial flagellum is a complex structure composed of a transmembrane basal body, a hook, and a filament. In Caulobacter crescentus the biosynthesis and assembly of this structure is under temporal and spatial control. To help to define the order of assembly of the flagellar components and to identify the genes involved in the early steps of basal body construction, mutants defective in basal body formation have been analyzed. Mutants in the flaD flaB flaC gene cluster were found to be unable to assemble a complete basal body. The flaD BC motC region was cloned and the genes were localized by subcloning and complementation analysis. A series of Tn5 insertion mutations in the flaD BC region were mapped. Complementation analysis of the Tn5 insertion mutants indicated the existence of at least four transcriptional units in the region and identified the presence of two new genes designated flbN and flbO. Mutants in flbN, flaB, flaC and flbO were unable to assemble any basal body structure and are likely to be involved in the early steps of basal body formation. The flaD mutant, however, was found to contain a partially assembled basal body consisting of the rod and three hook-distal rings. All of the mutants in this cluster exhibited pleiotropic effects on the expression of other flagellar and chemotaxis functions, including the level of synthesis of flagellins, the hook protein and hook protein precursor, and the level of chemotaxis methylation.

  2. Molecular Cloning and Characterization of the Helicobacter pylori fliD Gene, an Essential Factor in Flagellar Structure and Motility

    PubMed Central

    Seong Kim, Jang; Hoon Chang, Ji; Il Chung, Soo; Sun Yum, Jung

    1999-01-01

    Helicobacter pylori colonizes the human stomach and can cause gastroduodenal disease. Flagellar motility is regarded as a major factor in the colonizing ability of H. pylori. The functional roles of flagellar structural proteins other than FlaA, FlaB, and FlgE are not well understood. The fliD operon of H. pylori consists of flaG, fliD, and fliS genes, in the order stated, under the control of a ς28-dependent promoter. In an effort to elucidate the function of the FliD protein, a hook-associated protein 2 homologue, in flagellar morphogenesis and motility, the fliD gene (2,058 bp) was cloned and isogenic mutants were constructed by disruption of the fliD gene with a kanamycin resistance cassette and electroporation-mediated allelic-exchange mutagenesis. In the fliD mutant, morphologically abnormal flagellar appendages in which very little filament elongation was apparent were observed. The fliD mutant strain was completely nonmotile, indicating that these abnormal flagella were functionally defective. Furthermore, the isogenic fliD mutant of H. pylori SS1, a mouse-adapted strain, was not able to colonize the gastric mucosae of host mice. These results suggest that H. pylori FliD is an essential element in the assembly of the functional flagella that are required for colonization of the gastric mucosa. PMID:10559162

  3. The Type VI Secretion System Modulates Flagellar Gene Expression and Secretion in Citrobacter freundii and Contributes to Adhesion and Cytotoxicity to Host Cells.

    PubMed

    Liu, Liyun; Hao, Shuai; Lan, Ruiting; Wang, Guangxia; Xiao, Di; Sun, Hui; Xu, Jianguo

    2015-07-01

    The type VI secretion system (T6SS) as a virulence factor-releasing system contributes to virulence development of various pathogens and is often activated upon contact with target cells. Citrobacter freundii strain CF74 has a complete T6SS genomic island (GI) that contains clpV, hcp-2, and vgr T6SS genes. We constructed clpV, hcp-2, vgr, and T6SS GI deletion mutants in CF74 and analyzed their effects on the transcriptome overall and, specifically, on the flagellar system at the levels of transcription and translation. Deletion of the T6SS GI affected the transcription of 84 genes, with 15 and 69 genes exhibiting higher and lower levels of transcription, respectively. Members of the cell motility class of downregulated genes of the CF74ΔT6SS mutant were mainly flagellar genes, including effector proteins, chaperones, and regulators. Moreover, the production and secretion of FliC were also decreased in clpV, hcp-2, vgr, or T6SS GI deletion mutants in CF74 and were restored upon complementation. In swimming motility assays, the mutant strains were found to be less motile than the wild type, and motility was restored by complementation. The mutant strains were defective in adhesion to HEp-2 cells and were restored partially upon complementation. Further, the CF74ΔT6SS, CF74ΔclpV, and CF74Δhcp-2 mutants induced lower cytotoxicity to HEp-2 cells than the wild type. These results suggested that the T6SS GI in CF74 regulates the flagellar system, enhances motility, is involved in adherence to host cells, and induces cytotoxicity to host cells. Thus, the T6SS plays a wide-ranging role in C. freundii.

  4. Comprehensive Mapping of the Escherichia coli Flagellar Regulatory Network

    PubMed Central

    Fitzgerald, Devon M.; Bonocora, Richard P.; Wade, Joseph T.

    2014-01-01

    Flagellar synthesis is a highly regulated process in all motile bacteria. In Escherichia coli and related species, the transcription factor FlhDC is the master regulator of a multi-tiered transcription network. FlhDC activates transcription of a number of genes, including some flagellar genes and the gene encoding the alternative Sigma factor FliA. Genes whose expression is required late in flagellar assembly are primarily transcribed by FliA, imparting temporal regulation of transcription and coupling expression to flagellar assembly. In this study, we use ChIP-seq and RNA-seq to comprehensively map the E. coli FlhDC and FliA regulons. We define a surprisingly restricted FlhDC regulon, including two novel regulated targets and two binding sites not associated with detectable regulation of surrounding genes. In contrast, we greatly expand the known FliA regulon. Surprisingly, 30 of the 52 FliA binding sites are located inside genes. Two of these intragenic promoters are associated with detectable noncoding RNAs, while the others either produce highly unstable RNAs or are inactive under these conditions. Together, our data redefine the E. coli flagellar regulatory network, and provide new insight into the temporal orchestration of gene expression that coordinates the flagellar assembly process. PMID:25275371

  5. Select Acetophenones Modulate Flagellar Motility in Chlamydomonas

    PubMed Central

    Evans, Shakila K.; Pearce, Austin A.; Ibezim, Prudence K.; Primm, Todd P.; Gaillard, Anne R.

    2009-01-01

    Acetophenones were screened for activity against positive phototaxis of Chlamydomonas cells, a process that requires coordinated flagellar motility. The structure-activity relationships of a series of acetophenones are reported, including acetophenones that affect flagellar motility and cell viability. Notably, 4-methoxyacetophenone, 3,4-dimethoxyacetophenone, and 4-hydroxyacetophenone induced negative phototaxis in Chlamydomonas, suggesting interference with activity of flagellar proteins and control of flagellar dominance. PMID:20659114

  6. Regulation of sperm flagellar motility activation and chemotaxis caused by egg-derived substance(s) in sea cucumber.

    PubMed

    Morita, Masaya; Kitamura, Makoto; Nakajima, Ayako; Sri Susilo, Endang; Takemura, Akihiro; Okuno, Makoto

    2009-04-01

    The sea cucumber Holothuria atra is a broadcast spawner. Among broadcast spawners, fertilization occurs by means of an egg-derived substance(s) that induces sperm flagellar motility activation and chemotaxis. Holothuria atra sperm were quiescent in seawater, but exhibited flagellar motility activation near eggs with chorion (intact eggs). In addition, they moved in a helical motion toward intact eggs as well as a capillary filled with the water layer of the egg extracts, suggesting that an egg-derived compound(s) causes motility activation and chemotaxis. Furthermore, demembranated sperm flagella were reactivated in high pH (> 7.8) solution without cAMP, and a phosphorylation assay using (gamma-32P)ATP showed that axonemal protein phosphorylation and dephosphorylation also occurred in a pH-dependent manner. These results suggest that the activation of sperm motility in holothurians is controlled by pH-sensitive changes in axonemal protein phosphorylation. Ca2+ concentration affected the swimming trajectory of demembranated sperm, indicating that Ca2+-binding proteins present at the flagella may be associated with regulation of flagellar waveform. Moreover, the phosphorylation states of several axonemal proteins were Ca2+-sensitive, indicating that Ca2+ impacts both kinase and phosphatase activities. In addition, in vivo sperm protein phosphorylation occurred after treatment with a water-soluble egg extract. Our results suggest that one or more egg-derived compounds activate motility and subsequent chemotactic behavior via Ca2+-sensitive flagellar protein phosphorylation.

  7. Mutations in the Borrelia burgdorferi Flagellar Type III Secretion System Genes fliH and fliI Profoundly Affect Spirochete Flagellar Assembly, Morphology, Motility, Structure, and Cell Division

    PubMed Central

    Gao, Lihui; Zhao, Xiaowei; Liu, Jun; Norris, Steven J.

    2015-01-01

    ABSTRACT The Lyme disease spirochete Borrelia burgdorferi migrates to distant sites in the tick vectors and mammalian hosts through robust motility and chemotaxis activities. FliH and FliI are two cytoplasmic proteins that play important roles in the type III secretion system (T3SS)-mediated export and assembly of flagellar structural proteins. However, detailed analyses of the roles of FliH and FliI in B. burgdorferi have not been reported. In this study, fliH and fliI transposon mutants were utilized to dissect the mechanism of the Borrelia type III secretion system. The fliH and fliI mutants exhibited rod-shaped or string-like morphology, greatly reduced motility, division defects (resulting in elongated organisms with incomplete division points), and noninfectivity in mice by needle inoculation. Mutants in fliH and fliI were incapable of translational motion in 1% methylcellulose or soft agar. Inactivation of either fliH or fliI resulted in the loss of the FliH-FliI complex from otherwise intact flagellar motors, as determined by cryo-electron tomography (cryo-ET). Flagellar assemblies were still present in the mutant cells, albeit in lower numbers than in wild-type cells and with truncated flagella. Genetic complementation of fliH and fliI mutants in trans restored their wild-type morphology, motility, and flagellar motor structure; however, full-length flagella and infectivity were not recovered in these complemented mutants. Based on these results, disruption of either fliH or fliI in B. burgdorferi results in a severe defect in flagellar structure and function and cell division but does not completely block the export and assembly of flagellar hook and filament proteins. PMID:25968649

  8. Characterization of the ATP-phosphohydrolase activity of bovine spermatozoa flagellar extracts.

    PubMed

    Young, L G; Smithwick, E B

    1975-02-01

    The ATP-phosphohydrolase activity of extracts prepared from bovine spermatozoa flagella (BSFE), was characterized with respect to enzyme, substrate, activator ion and salt concentration, temperature dependence and time stability. BSFE required the presence of a divalent cation for activity: Mg++ or Ca++ could function as activator; Mn++, Zn++ and Cd++ could not. EDTA, but not EGTA, was inhibitory to enzymatic activity. Ca++ inhibited the Mg++ stimulated activity. ATP was dephosphorylated more rapidly than GTP greater than CTP greater than ITP, and ADP was dephosphorylated at 40% of the rate of ATP. The magnesium activated ATPase was stimulated by potassium and inhibited by sodium ions. Activation of BSFE ATP-phosphohydrolase was maximal in the presence of Mg++ and ATP in equimolar concentrations and K+ (0.05-0.3 M) at 30 degrees C. Although the enzymatic activity of the extract was found to decrease rapidly with time, it could be maintained for up to three days by the addition of 2-beta-mercaptoethanol to the bovine spermatozoa flagellar extracts.

  9. Characterization of the Alternative Sigma Factor σ54 and the Transcriptional Regulator FleQ of Legionella pneumophila, Which Are Both Involved in the Regulation Cascade of Flagellar Gene Expression

    PubMed Central

    Jacobi, Sebastian; Schade, Rüdiger; Heuner, Klaus

    2004-01-01

    We cloned and analyzed Legionella pneumophila Corby homologs of rpoN (encoding σ54) and fleQ (encoding σ54 activator protein). Two other genes (fleR and pilR) whose products have a σ54 interaction domain were identified in the genome sequence of L. pneumophila. An rpoN mutant strain was nonflagellated and expressed very small amounts of the FlaA (flagellin) protein. Like the rpoN mutant, the fleQ mutant strain of L. pneumophila was also nonflagellated and expressed only small amounts of FlaA protein compared to the amounts expressed by the wild type. In this paper we show that the σ54 factor and the FleQ protein are involved in regulation of flagellar gene operons in L. pneumophila. RpoN and FleQ positively regulate the transcription of FliM and FleN, both of which have a σ54-dependent promoter consensus sequence. However, they seemed to be dispensable for transcription of flaA, fliA, or icmR. Our results confirmed a recently described model of the flagellar gene regulation cascade in L. pneumophila (K. Heuner and M. Steinert, Int. J. Med. Microbiol. 293:133-145, 2003). Flagellar gene regulation was found to be different from that of Enterobacteriaceae but seems to be comparable to that described for Pseudomonas or Vibrio spp. PMID:15090493

  10. A conserved CaM- and radial spoke associated complex mediates regulation of flagellar dynein activity.

    PubMed

    Dymek, Erin E; Smith, Elizabeth F

    2007-11-05

    For virtually all cilia and eukaryotic flagella, the second messengers calcium and cyclic adenosine monophosphate are implicated in modulating dynein- driven microtubule sliding to regulate beating. Calmodulin (CaM) localizes to the axoneme and is a key calcium sensor involved in regulating motility. Using immunoprecipitation and mass spectrometry, we identify members of a CaM-containing complex that are involved in regulating dynein activity. This complex includes flagellar-associated protein 91 (FAP91), which shares considerable sequence similarity to AAT-1, a protein originally identified in testis as an A-kinase anchor protein (AKAP)- binding protein. FAP91 directly interacts with radial spoke protein 3 (an AKAP), which is located at the base of the spoke. In a microtubule sliding assay, the addition of antibodies generated against FAP91 to mutant axonemes with reduced dynein activity restores dynein activity to wild-type levels. These combined results indicate that the CaM- and spoke-associated complex mediates regulatory signals between the radial spokes and dynein arms.

  11. Antiprotozoal glutathione derivatives with flagellar membrane binding activity against T. brucei rhodesiense.

    PubMed

    Daunes, Sylvie; Yardley, Vanessa; Croft, Simon L; D'Silva, Claudius

    2017-02-15

    A new series of N-substituted S-(2,4-dinitrophenyl)glutathione dibutyl diesters were synthesized to improve in vitro anti-protozoal activity against the pathogenic parasites Trypanosoma brucei rhodesiense, Trypanosoma cruzi and Leishmania donovani. The results obtained indicate that N-substituents enhance the inhibitory properties of glutathione diesters whilst showing reduced toxicity against KB cells as in the cases of compounds 5, 9, 10, 16, 18 and 19. We suggest that the interaction of N-substituted S-(2,4-dinitrophenyl) glutathione dibutyl diesters with T. b. brucei occurs mainly by weak hydrophobic interactions such as London and van der Waals forces. A QSAR study indicated that the inhibitory activity of the peptide is associated negatively with the average number of C atoms, NC and positively to SZX, the ZX shadow a geometric descriptor related to molecular size and orientation of the compound. HPLC-UV studies in conjunction with optical microscopy indicate that the observed selectivity of inhibition of these compounds against bloodstream form T. b. brucei parasites in comparison to L. donovani under the same conditions is due to intracellular uptake via endocytosis in the flagellar pocket. Copyright © 2016. Published by Elsevier Ltd.

  12. Mutations in the Borrelia burgdorferi Flagellar Type III Secretion System Genes fliH and fliI Profoundly Affect Spirochete Flagellar Assembly, Morphology, Motility, Structure, and Cell Division.

    PubMed

    Lin, Tao; Gao, Lihui; Zhao, Xiaowei; Liu, Jun; Norris, Steven J

    2015-05-12

    The Lyme disease spirochete Borrelia burgdorferi migrates to distant sites in the tick vectors and mammalian hosts through robust motility and chemotaxis activities. FliH and FliI are two cytoplasmic proteins that play important roles in the type III secretion system (T3SS)-mediated export and assembly of flagellar structural proteins. However, detailed analyses of the roles of FliH and FliI in B. burgdorferi have not been reported. In this study, fliH and fliI transposon mutants were utilized to dissect the mechanism of the Borrelia type III secretion system. The fliH and fliI mutants exhibited rod-shaped or string-like morphology, greatly reduced motility, division defects (resulting in elongated organisms with incomplete division points), and noninfectivity in mice by needle inoculation. Mutants in fliH and fliI were incapable of translational motion in 1% methylcellulose or soft agar. Inactivation of either fliH or fliI resulted in the loss of the FliH-FliI complex from otherwise intact flagellar motors, as determined by cryo-electron tomography (cryo-ET). Flagellar assemblies were still present in the mutant cells, albeit in lower numbers than in wild-type cells and with truncated flagella. Genetic complementation of fliH and fliI mutants in trans restored their wild-type morphology, motility, and flagellar motor structure; however, full-length flagella and infectivity were not recovered in these complemented mutants. Based on these results, disruption of either fliH or fliI in B. burgdorferi results in a severe defect in flagellar structure and function and cell division but does not completely block the export and assembly of flagellar hook and filament proteins. Many bacteria are able to rapidly transport themselves through their surroundings using specialized organelles called flagella. In spiral-shaped organisms called spirochetes, flagella act like inboard motors and give the bacteria the ability to bore their way through dense materials (such as human

  13. Characterization of the relationship between polar and lateral flagellar structural genes in the deep-sea bacterium Shewanella piezotolerans WP3.

    PubMed

    Jian, Huahua; Wang, Han; Zeng, Xianping; Xiong, Lei; Wang, Fengping; Xiao, Xiang

    2016-12-22

    Bacteria with a dual flagellar system, which consists of a polar flagellum (PF) and several lateral flagella (LF), have been identified in diverse environments. Nevertheless, whether and how these two flagellar systems interact with each other is largely unknown. In the present study, the relationship between the structural genes for the PF and LF of the deep-sea bacterium Shewanella piezotolerans WP3 was investigated by genetic, phenotypic and phylogenetic analyses. The mutation of PF genes induced the expression of LF genes and the production of LF in liquid medium, while the defective LF genes led to a decrease in PF gene transcription. However, the level of PF flagellin remained unchanged in LF gene mutants. Further investigation showed that the flgH2 gene (encoding LF L-ring protein) can compensate for mutations of the flgH1 gene (encoding PF L-ring protein), but this compensation does not occur between the flagellar hook-filament junction proteins (FlgL1, FlgL2). Swarming motility was shown to specifically require LF genes, and PF genes cannot substitute for the LF genes in the lateral flagella synthesis. Considering the importance of flagella-dependent motility for bacterial survival in the abyssal sediment, our study thus provided a better understanding of the adaptation strategy of benthic bacteria.

  14. Characterization of the relationship between polar and lateral flagellar structural genes in the deep-sea bacterium Shewanella piezotolerans WP3

    PubMed Central

    Jian, Huahua; Wang, Han; Zeng, Xianping; Xiong, Lei; Wang, Fengping; Xiao, Xiang

    2016-01-01

    Bacteria with a dual flagellar system, which consists of a polar flagellum (PF) and several lateral flagella (LF), have been identified in diverse environments. Nevertheless, whether and how these two flagellar systems interact with each other is largely unknown. In the present study, the relationship between the structural genes for the PF and LF of the deep-sea bacterium Shewanella piezotolerans WP3 was investigated by genetic, phenotypic and phylogenetic analyses. The mutation of PF genes induced the expression of LF genes and the production of LF in liquid medium, while the defective LF genes led to a decrease in PF gene transcription. However, the level of PF flagellin remained unchanged in LF gene mutants. Further investigation showed that the flgH2 gene (encoding LF L-ring protein) can compensate for mutations of the flgH1 gene (encoding PF L-ring protein), but this compensation does not occur between the flagellar hook-filament junction proteins (FlgL1, FlgL2). Swarming motility was shown to specifically require LF genes, and PF genes cannot substitute for the LF genes in the lateral flagella synthesis. Considering the importance of flagella-dependent motility for bacterial survival in the abyssal sediment, our study thus provided a better understanding of the adaptation strategy of benthic bacteria. PMID:28004809

  15. Early Caulobacter crescentus genes fliL and fliM are required for flagellar gene expression and normal cell division.

    PubMed Central

    Yu, J; Shapiro, L

    1992-01-01

    The biogenesis of the Caulobacter crescentus polar flagellum requires the expression of more than 48 genes, which are organized in a regulatory hierarchy. The flbO locus is near the top of the hierarchy, and consequently strains with mutations in this locus are nonmotile and lack the flagellar basal body complex. In addition to the motility phenotype, mutations in this locus also cause abnormal cell division. Complementing clones restore both motility and normal cell division. Sequence analysis of a complementing subclone revealed that this locus encodes at least two proteins that are homologs of the Salmonella typhimurium and Escherichia coli flagellar proteins FliL and FliM. FliM is thought to be a switch protein and to interface with the flagellum motor. The C. crescentus fliL and fliM genes form an operon that is expressed early in the cell cycle. Tn5 insertions in the fliM gene prevent the transcription of class II and class III flagellar genes, which are lower in the regulatory hierarchy. The start site of the fliLM operon lies 166 bp from the divergently transcribed flaCBD operon that encodes several basal body genes. Sequence comparison of the fliL transcription start site with those of other class I genes, flaS and flaO, revealed a highly conserved 29-bp sequence in a potential promoter region that differs from sigma 70, sigma 54, sigma 32, and sigma 28 promoter sequences, suggesting that at least three class I genes share a unique 5' regulatory region. Images PMID:1315735

  16. Escherichia coli flagellar genes as target sites for integration and expression of genetic circuits.

    PubMed

    Juhas, Mario; Evans, Lewis D B; Frost, Joe; Davenport, Peter W; Yarkoni, Orr; Fraser, Gillian M; Ajioka, James W

    2014-01-01

    E. coli is a model platform for engineering microbes, so genetic circuit design and analysis will be greatly facilitated by simple and effective approaches to introduce genetic constructs into the E. coli chromosome at well-characterised loci. We combined the Red recombinase system of bacteriophage λ and Isothermal Gibson Assembly for rapid integration of novel DNA constructs into the E. coli chromosome. We identified the flagellar region as a promising region for integration and expression of genetic circuits. We characterised integration and expression at four candidate loci, fliD, fliS, fliT, and fliY, of the E. coli flagellar region 3a. The integration efficiency and expression from the four integrations varied considerably. Integration into fliD and fliS significantly decreased motility, while integration into fliT and fliY had only a minor effect on the motility. None of the integrations had negative effects on the growth of the bacteria. Overall, we found that fliT was the most suitable integration site.

  17. Escherichia coli Flagellar Genes as Target Sites for Integration and Expression of Genetic Circuits

    PubMed Central

    Juhas, Mario; Evans, Lewis D. B.; Frost, Joe; Davenport, Peter W.; Yarkoni, Orr; Fraser, Gillian M.; Ajioka, James W.

    2014-01-01

    E. coli is a model platform for engineering microbes, so genetic circuit design and analysis will be greatly facilitated by simple and effective approaches to introduce genetic constructs into the E. coli chromosome at well-characterised loci. We combined the Red recombinase system of bacteriophage λ and Isothermal Gibson Assembly for rapid integration of novel DNA constructs into the E. coli chromosome. We identified the flagellar region as a promising region for integration and expression of genetic circuits. We characterised integration and expression at four candidate loci, fliD, fliS, fliT, and fliY, of the E. coli flagellar region 3a. The integration efficiency and expression from the four integrations varied considerably. Integration into fliD and fliS significantly decreased motility, while integration into fliT and fliY had only a minor effect on the motility. None of the integrations had negative effects on the growth of the bacteria. Overall, we found that fliT was the most suitable integration site. PMID:25350000

  18. The Ability of Proteus mirabilis To Sense Surfaces and Regulate Virulence Gene Expression Involves FliL, a Flagellar Basal Body Protein

    PubMed Central

    Belas, Robert; Suvanasuthi, Rooge

    2005-01-01

    Proteus mirabilis is a urinary tract pathogen that differentiates from a short swimmer cell to an elongated, highly flagellated swarmer cell. Swarmer cell differentiation parallels an increased expression of several virulence factors, suggesting that both processes are controlled by the same signal. The molecular nature of this signal is not known but is hypothesized to involve the inhibition of flagellar rotation. In this study, data are presented supporting the idea that conditions inhibiting flagellar rotation induce swarmer cell differentiation and implicating a rotating flagellar filament as critical to the sensing mechanism. Mutations in three genes, fliL, fliF, and fliG, encoding components of the flagellar basal body, result in the inappropriate development of swarmer cells in noninducing liquid media or hyperelongated swarmer cells on agar media. The fliL mutation was studied in detail. FliL− mutants are nonmotile and fail to synthesize flagellin, while complementation of fliL restores wild-type cell elongation but not motility. Overexpression of fliL+ in wild-type cells prevents swarmer cell differentiation and motility, a result also observed when P. mirabilis fliL+ was expressed in Escherichia coli. These results suggest that FliL plays a role in swarmer cell differentiation and implicate FliL as critical to transduction of the signal inducing swarmer cell differentiation and virulence gene expression. In concert with this idea, defects in fliL up-regulate the expression of two virulence genes, zapA and hpmB. These results support the hypothesis that P. mirabilis ascertains its location in the environment or host by assessing the status of its flagellar motors, which in turn control swarmer cell gene expression. PMID:16166542

  19. Synthetic Cystic Fibrosis Sputum Medium Regulates Flagellar Biosynthesis through the flhF Gene in Burkholderia cenocepacia

    PubMed Central

    Kumar, Brijesh; Cardona, Silvia T.

    2016-01-01

    Burkholderia cenocepacia belongs to the Burkholderia cepacia complex (Bcc), a group of at least 18 distinct species that establish chronic infections in the lung of people with the genetic disease cystic fibrosis (CF). The sputum of CF patients is rich in amino acids and was previously shown to increase flagellar gene expression in B. cenocepacia. We examined flagellin expression and flagellar morphology of B. cenocepacia grown in synthetic cystic fibrosis sputum medium (SCFM) compared to minimal medium. We found that CF nutritional conditions induce increased motility and flagellin expression. Individual amino acids added at the same concentrations as found in SCFM also increased motility but not flagellin expression, suggesting a chemotactic effect of amino acids. Electron microscopy and flagella staining demonstrated that the increase in flagellin corresponds to a change in the number of flagella per cell. In minimal medium, the ratio of multiple: single: aflagellated cells was 2:3.5:4.5; while under SCFM conditions, the ratio was 7:2:1. We created a deletion mutant, ΔflhF, to study whether this putative GTPase regulates the flagellation pattern of B. cenocepacia K56-2 during growth in CF conditions. The ΔflhF mutant exhibited 80% aflagellated, 14% single and 6% multiple flagellated bacterial subpopulations. Moreover, the ratio of multiple to single flagella in WT and ΔflhF was 3.5 and 0.43, respectively in CF conditions. The observed differences suggest that FlhF positively regulates flagellin expression and the flagellation pattern in B. cenocepacia K56-2 during CF nutritional conditions. PMID:27379216

  20. Regulatory network controlling extracellular proteins in Erwinia carotovora subsp. carotovora: FlhDC, the master regulator of flagellar genes, activates rsmB regulatory RNA production by affecting gacA and hexA (lrhA) expression.

    PubMed

    Cui, Yaya; Chatterjee, Asita; Yang, Hailian; Chatterjee, Arun K

    2008-07-01

    Erwinia carotovora subsp. carotovora produces an array of extracellular proteins (i.e., exoproteins), including plant cell wall-degrading enzymes and Harpin, an effector responsible for eliciting hypersensitive reaction. Exoprotein genes are coregulated by the quorum-sensing signal, N-acyl homoserine lactone, plant signals, an assortment of transcriptional factors/regulators (GacS/A, ExpR1, ExpR2, KdgR, RpoS, HexA, and RsmC) and posttranscriptional regulators (RsmA, rsmB RNA). rsmB RNA production is positively regulated by GacS/A, a two-component system, and negatively regulated by HexA (PecT in Erwinia chrysanthemi; LrhA [LysR homolog A] in Escherichia coli) and RsmC, a putative transcriptional adaptor. While free RsmA, an RNA-binding protein, promotes decay of mRNAs of exoprotein genes, binding of RsmA with rsmB RNA neutralizes the RsmA effect. In the course of studies of GacA regulation, we discovered that a locus bearing strong homology to the flhDC operon of E. coli also controls extracellular enzyme production. A transposon insertion FlhDC(-) mutant produces very low levels of pectate lyase, polygalacturonase, cellulase, protease, and E. carotovora subsp. carotovora Harpin (Harpin(Ecc)) and is severely attenuated in its plant virulence. The production of these exoproteins is restored in the mutant carrying an FlhDC(+) plasmid. Sequence analysis and transcript assays disclosed that the flhD operon of E. carotovora subsp. carotovora, like those of other enterobacteria, consists of flhD and flhC. Complementation analysis revealed that the regulatory effect requires functions of both flhD and flhC products. The data presented here show that FlhDC positively regulates gacA, rsmC, and fliA and negatively regulates hexA (lrhA). Evidence shows that FlhDC controls extracellular protein production through cumulative effects on hexA and gacA. Reduced levels of GacA and elevated levels of HexA in the FlhDC(-) mutant are responsible for the inhibition of rsmB RNA

  1. Borrelia burgdorferi uniquely regulates its motility genes and has an intricate flagellar hook-basal body structure.

    PubMed

    Sal, Melanie S; Li, Chunhao; Motalab, M A; Shibata, Satoshi; Aizawa, Shin-ichi; Charon, Nyles W

    2008-03-01

    Borrelia burgdorferi is a flat-wave, motile spirochete that causes Lyme disease. Motility is provided by periplasmic flagella (PFs) located between the cell cylinder and an outer membrane sheath. The structure of these PFs, which are composed of a basal body, a hook, and a filament, is similar to the structure of flagella of other bacteria. To determine if hook formation influences flagellin gene transcription in B. burgdorferi, we inactivated the hook structural gene flgE by targeted mutagenesis. In many bacteria, completion of the hook structure serves as a checkpoint for transcriptional control of flagellum synthesis and other chemotaxis and motility genes. Specifically, the hook allows secretion of the anti-sigma factor FlgM and concomitant late gene transcription promoted by sigma28. However, the control of B. burgdorferi PF synthesis differs from the control of flagellum synthesis in other bacteria; the gene encoding sigma28 is not present in the genome of B. burgdorferi, nor are any sigma28 promoter recognition sequences associated with the motility genes. We found that B. burgdorferi flgE mutants lacked PFs, were rod shaped, and were nonmotile, which substantiates previous evidence that PFs are involved in both cell morphology and motility. Although most motility and chemotaxis gene products accumulated at wild-type levels in the absence of FlgE, mutant cells had markedly decreased levels of the flagellar filament proteins FlaA and FlaB. Further analyses showed that the reduction in the levels of flagellin proteins in the spirochetes lacking FlgE was mediated at the posttranscriptional level. Taken together, our results indicate that in B. burgdorferi, the completion of the hook does not serve as a checkpoint for transcriptional regulation of flagellum synthesis. In addition, we also present evidence that the hook protein in B. burgdorferi forms a high-molecular-weight complex and that formation of this complex occurs in the periplasmic space.

  2. A Complex LuxR-LuxI Type Quorum Sensing Network in a Roseobacterial Marine Sponge Symbiont Activates Flagellar Motility and Inhibits Biofilm Formation

    PubMed Central

    Zan, Jindong; Cicirelli, Elisha M.; Mohamed, Naglaa M.; Sibhatu, Hiruy; Kroll, Stephanie; Choi, Ohkee; Uhlson, Charis L.; Wysoczinski, Christina L.; Murphy, Robert C.; Churchill, Mair E.A.; Hill, Russell T.; Fuqua, Clay

    2012-01-01

    Summary Bacteria isolated from marine sponges, including the Silicibacter-Ruegeria (SR) subgroup of the Roseobacter clade, produce N-acylhomoserine lactone (AHL) quorum sensing signal molecules. This study is the first detailed analysis of AHL quorum sensing in sponge-associated bacteria, specifically Ruegeria sp. KLH11, from the sponge Mycale laxissima. Two pairs of luxR and luxI homologues and one solo luxI homologue were identified and designated ssaRI, ssbRI, and sscI (sponge-associated symbiont locus A, B, and C, luxRI or luxI homologue). SsaI produced predominantly long-chain 3-oxo-AHLs and both SsbI and SscI specified 3-OH-AHLs. Addition of exogenous AHLs to KLH11 increased the expression of ssaI but not ssaR, ssbI or ssbR, and genetic analyses revealed a complex interconnected arrangement between SsaRI and SsbRI systems. Interestingly, flagellar motility was abolished in the ssaI and ssaR mutants, with the flagellar biosynthesis genes under strict SsaRI control, and active motility only at high culture density. Conversely, ssaI and ssaR mutants formed more robust biofilms than wild type KLH11. AHLs and transcript of the ssaI gene were detected in M. laxissima extracts suggesting that AHL signaling contributes to the decision between motility and sessility and that it also may facilitate acclimation to different environments including the sponge host. PMID:22742196

  3. EscO, a Functional and Structural Analog of the Flagellar FliJ Protein, Is a Positive Regulator of EscN ATPase Activity of the Enteropathogenic Escherichia coli Injectisome

    PubMed Central

    Romo-Castillo, Mariana; Andrade, Angel; Espinosa, Norma; Monjarás Feria, Julia; Soto, Eduardo; Díaz-Guerrero, Miguel

    2014-01-01

    Type III secretion systems (T3SSs) are multiprotein molecular devices used by many Gram-negative bacterial pathogens to translocate effector proteins into eukaryotic cells. A T3SS is also used for protein export in flagellar assembly, which promotes bacterial motility. The two systems are evolutionarily related, possessing highly conserved components in their export apparatuses. Enteropathogenic Escherichia coli (EPEC) employs a T3SS, encoded by genes in the locus of enterocyte effacement (LEE) pathogenicity island, to colonize the human intestine and cause diarrheal disease. In the present work, we investigated the role of the LEE-encoded EscO protein (previously Orf15 or EscA) in T3SS biogenesis. We show that EscO shares similar properties with the flagellar FliJ and the Yersinia YscO protein families. Our findings demonstrate that EscO is essential for secretion of all categories of T3SS substrates. Consistent with its central role in protein secretion, it was found to interact with the ATPase EscN and its negative regulator, EscL, of the export apparatus. Moreover, we show that EscO stimulates EscN enzymatic activity; however, it is unable to upregulate ATP hydrolysis in the presence of EscL. Remarkably, EscO partially restored the swimming defect of a Salmonella flagellar fliJ mutant and was able to stimulate the ATPase activity of FliI. Overall, our data indicate that EscO is the virulence counterpart of the flagellar FliJ protein. PMID:24706741

  4. EscO, a functional and structural analog of the flagellar FliJ protein, is a positive regulator of EscN ATPase activity of the enteropathogenic Escherichia coli injectisome.

    PubMed

    Romo-Castillo, Mariana; Andrade, Angel; Espinosa, Norma; Monjarás Feria, Julia; Soto, Eduardo; Díaz-Guerrero, Miguel; González-Pedrajo, Bertha

    2014-06-01

    Type III secretion systems (T3SSs) are multiprotein molecular devices used by many Gram-negative bacterial pathogens to translocate effector proteins into eukaryotic cells. A T3SS is also used for protein export in flagellar assembly, which promotes bacterial motility. The two systems are evolutionarily related, possessing highly conserved components in their export apparatuses. Enteropathogenic Escherichia coli (EPEC) employs a T3SS, encoded by genes in the locus of enterocyte effacement (LEE) pathogenicity island, to colonize the human intestine and cause diarrheal disease. In the present work, we investigated the role of the LEE-encoded EscO protein (previously Orf15 or EscA) in T3SS biogenesis. We show that EscO shares similar properties with the flagellar FliJ and the Yersinia YscO protein families. Our findings demonstrate that EscO is essential for secretion of all categories of T3SS substrates. Consistent with its central role in protein secretion, it was found to interact with the ATPase EscN and its negative regulator, EscL, of the export apparatus. Moreover, we show that EscO stimulates EscN enzymatic activity; however, it is unable to upregulate ATP hydrolysis in the presence of EscL. Remarkably, EscO partially restored the swimming defect of a Salmonella flagellar fliJ mutant and was able to stimulate the ATPase activity of FliI. Overall, our data indicate that EscO is the virulence counterpart of the flagellar FliJ protein. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  5. Identification of flgZ as a flagellar gene encoding a PilZ domain protein that regulates swimming motility and biofilm formation in Pseudomonas.

    PubMed

    Martínez-Granero, Francisco; Navazo, Ana; Barahona, Emma; Redondo-Nieto, Miguel; González de Heredia, Elena; Baena, Irene; Martín-Martín, Irene; Rivilla, Rafael; Martín, Marta

    2014-01-01

    Diguanylate cyclase and phosphodiesterase enzymatic activities control c-di-GMP levels modulating planktonic versus sessile lifestyle behavior in bacteria. The PilZ domain is described as a sensor of c-di-GMP intracellular levels and the proteins containing a PilZ domain represent the best studied class of c-di-GMP receptors forming part of the c-di-GMP signaling cascade. In P. fluorescens F113 we have found two diguanylate cyclases (WspR, SadC) and one phosphodiesterase (BifA) implicated in regulation of swimming motility and biofilm formation. Here we identify a flgZ gene located in a flagellar operon encoding a protein that contains a PilZ domain. Moreover, we show that FlgZ subcellular localization depends on the c-di-GMP intracellular levels. The overexpression analysis of flgZ in P. fluorescens F113 and P. putida KT2440 backgrounds reveal a participation of FlgZ in Pseudomonas swimming motility regulation. Besides, the epistasis of flgZ over wspR and bifA clearly shows that c-di-GMP intracellular levels produced by the enzymatic activity of the diguanylate cyclase WspR and the phosphodiesterase BifA regulates biofilm formation through FlgZ.

  6. Identification of flgZ as a Flagellar Gene Encoding a PilZ Domain Protein That Regulates Swimming Motility and Biofilm Formation in Pseudomonas

    PubMed Central

    Redondo-Nieto, Miguel; González de Heredia, Elena; Baena, Irene; Martín-Martín, Irene; Rivilla, Rafael; Martín, Marta

    2014-01-01

    Diguanylate cyclase and phosphodiesterase enzymatic activities control c-di-GMP levels modulating planktonic versus sessile lifestyle behavior in bacteria. The PilZ domain is described as a sensor of c-di-GMP intracellular levels and the proteins containing a PilZ domain represent the best studied class of c-di-GMP receptors forming part of the c-di-GMP signaling cascade. In P. fluorescens F113 we have found two diguanylate cyclases (WspR, SadC) and one phosphodiesterase (BifA) implicated in regulation of swimming motility and biofilm formation. Here we identify a flgZ gene located in a flagellar operon encoding a protein that contains a PilZ domain. Moreover, we show that FlgZ subcellular localization depends on the c-di-GMP intracellular levels. The overexpression analysis of flgZ in P. fluorescens F113 and P. putida KT2440 backgrounds reveal a participation of FlgZ in Pseudomonas swimming motility regulation. Besides, the epistasis of flgZ over wspR and bifA clearly shows that c-di-GMP intracellular levels produced by the enzymatic activity of the diguanylate cyclase WspR and the phosphodiesterase BifA regulates biofilm formation through FlgZ. PMID:24504373

  7. A complex LuxR-LuxI type quorum sensing network in a roseobacterial marine sponge symbiont activates flagellar motility and inhibits biofilm formation.

    PubMed

    Zan, Jindong; Cicirelli, Elisha M; Mohamed, Naglaa M; Sibhatu, Hiruy; Kroll, Stephanie; Choi, Okhee; Choi, Ohkee; Uhlson, Charis L; Wysoczynski, Christina L; Wysoczinski, Christina L; Murphy, Robert C; Churchill, Mair E A; Hill, Russell T; Fuqua, Clay

    2012-09-01

    Bacteria isolated from marine sponges, including the Silicibacter-Ruegeria (SR) subgroup of the Roseobacter clade, produce N-acylhomoserine lactone (AHL) quorum sensing signal molecules. This study is the first detailed analysis of AHL quorum sensing in sponge-associated bacteria, specifically Ruegeria sp. KLH11, from the sponge Mycale laxissima. Two pairs of luxR and luxI homologues and one solo luxI homologue were identified and designated ssaRI, ssbRI and sscI (sponge-associated symbiont locus A, B and C, luxR or luxI homologue). SsaI produced predominantly long-chain 3-oxo-AHLs and both SsbI and SscI specified 3-OH-AHLs. Addition of exogenous AHLs to KLH11 increased the expression of ssaI but not ssaR, ssbI or ssbR, and genetic analyses revealed a complex interconnected arrangement between SsaRI and SsbRI systems. Interestingly, flagellar motility was abolished in the ssaI and ssaR mutants, with the flagellar biosynthesis genes under strict SsaRI control, and active motility only at high culture density. Conversely, ssaI and ssaR mutants formed more robust biofilms than wild-type KLH11. AHLs and the ssaI transcript were detected in M. laxissima extracts, suggesting that AHL signalling contributes to the decision between motility and sessility and that it may also facilitate acclimation to different environments that include the sponge host. © 2012 Blackwell Publishing Ltd.

  8. CRISPR/Cas9-Induced Disruption of Paraflagellar Rod Protein 1 and 2 Genes in Trypanosoma cruzi Reveals Their Role in Flagellar Attachment

    PubMed Central

    Lander, Noelia; Li, Zhu-Hong; Niyogi, Sayantanee

    2015-01-01

    ABSTRACT Trypanosoma cruzi is the etiologic agent of Chagas disease, and current methods for its genetic manipulation have been highly inefficient. We report here the use of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated gene 9) system for disrupting genes in the parasite by three different strategies. The utility of the method was established by silencing genes encoding the GP72 protein, which is required for flagellar attachment, and paraflagellar rod proteins 1 and 2 (PFR1, PFR2), key components of the parasite flagellum. We used either vectors containing single guide RNA (sgRNA) and Cas9, separately or together, or one vector containing sgRNA and Cas9 plus donor DNA for homologous recombination to rapidly generate mutant cell lines in which the PFR1, PFR2, and GP72 genes have been disrupted. We demonstrate that genome editing of these endogenous genes in T. cruzi is successful without detectable toxicity of Cas9. Our results indicate that PFR1, PFR2, and GP72 contribute to flagellar attachment to the cell body and motility of the parasites. Therefore, CRISPR/Cas9 allows efficient gene disruption in an almost genetically intractable parasite and suggest that this method will improve the functional analyses of its genome. PMID:26199333

  9. FlgM Is Secreted by the Flagellar Export Apparatus in Bacillus subtilis

    PubMed Central

    Calvo, Rebecca A.

    2014-01-01

    The bacterial flagellum is assembled from over 20 structural components, and flagellar gene regulation is morphogenetically coupled to the assembly state by control of the anti-sigma factor FlgM. In the Gram-negative bacterium Salmonella enterica, FlgM inhibits late-class flagellar gene expression until the hook-basal body structural intermediate is completed and FlgM is inhibited by secretion from the cytoplasm. Here we demonstrate that FlgM is also secreted in the Gram-positive bacterium Bacillus subtilis and is degraded extracellularly by the proteases Epr and WprA. We further demonstrate that, like in S. enterica, the structural genes required for the flagellar hook-basal body are required for robust activation of σD-dependent gene expression and efficient secretion of FlgM. Finally, we determine that FlgM secretion is strongly enhanced by, but does not strictly require, hook-basal body completion and instead demands a minimal subset of flagellar proteins that includes the FliF/FliG basal body proteins, the flagellar type III export apparatus components FliO, FliP, FliQ, FliR, FlhA, and FlhB, and the substrate specificity switch regulator FliK. PMID:25313396

  10. Listeria monocytogenes DNA Glycosylase AdlP Affects Flagellar Motility, Biofilm Formation, Virulence, and Stress Responses

    PubMed Central

    Zhang, Ting; Bae, Dongryeoul

    2016-01-01

    ABSTRACT The temperature-dependent alteration of flagellar motility gene expression is critical for the foodborne pathogen Listeria monocytogenes to respond to a changing environment. In this study, a genetic determinant, L. monocytogenes f2365_0220 (lmof2365_0220), encoding a putative protein that is structurally similar to the Bacillus cereus alkyl base DNA glycosylase (AlkD), was identified. This determinant was involved in the transcriptional repression of flagellar motility genes and was named adlP (encoding an AlkD-like protein [AdlP]). Deletion of adlP activated the expression of flagellar motility genes at 37°C and disrupted the temperature-dependent inhibition of L. monocytogenes motility. The adlP null strains demonstrated decreased survival in murine macrophage-like RAW264.7 cells and less virulence in mice. Furthermore, the deletion of adlP significantly decreased biofilm formation and impaired the survival of bacteria under several stress conditions, including the presence of a DNA alkylation compound (methyl methanesulfonate), an oxidative agent (H2O2), and aminoglycoside antibiotics. Our findings strongly suggest that adlP may encode a bifunctional protein that transcriptionally represses the expression of flagellar motility genes and influences stress responses through its DNA glycosylase activity. IMPORTANCE We discovered a novel protein that we named AlkD-like protein (AdlP). This protein affected flagellar motility, biofilm formation, and virulence. Our data suggest that AdlP may be a bifunctional protein that represses flagellar motility genes and influences stress responses through its DNA glycosylase activity. PMID:27316964

  11. The Global Regulatory hns Gene Negatively Affects Adhesion to Solid Surfaces by Anaerobically Grown Escherichia coli by Modulating Expression of Flagellar Genes and Lipopolysaccharide Production

    PubMed Central

    Landini, Paolo; Zehnder, Alexander J. B.

    2002-01-01

    The initial binding of bacterial cells to a solid surface is a critical and essential step in biofilm formation. In this report we show that stationary-phase cultures of Escherichia coli W3100 (a K-12 strain) can efficiently attach to sand columns when they are grown in Luria broth medium at 28°C in fully aerobic conditions. In contrast, growth in oxygen-limited conditions results in a sharp decrease in adhesion to hydrophilic substrates. We show that the production of lipopolysaccharide (LPS) and of flagella, as well as the transcription of the fliC gene, encoding the major flagellar subunit, increases under oxygen-limited conditions. Inactivation of the global regulatory hns gene counteracts increased production of LPS and flagella in response to anoxia and allows E. coli W3100 to attach to sand columns even when it is grown under oxygen-limited conditions. We propose that increased production of the FliC protein and of LPS in response to oxygen limitation results in the loss of the ability of E. coli W3100 to adhere to hydrophilic surfaces. Indeed, overexpression of the fliC gene results in a decreased adhesion to sand even when W3100 is grown in fully aerobic conditions. Our observations strongly suggest that anoxia is a negative environmental signal for adhesion in E. coli. PMID:11872702

  12. Vaccination with recombinant flagellar proteins FlgJ and FliN induce protection against Brucella abortus 544 infection in BALB/c mice.

    PubMed

    Li, Xianbo; Xu, Jie; Xie, Yongfei; Qiu, Yefeng; Fu, Simei; Yuan, Xitong; Ke, Yuehua; Yu, Shuang; Du, Xinying; Cui, Mingquan; Chen, Yanfen; Wang, Tongkun; Wang, Zhoujia; Yu, Yaqing; Huang, Kehe; Huang, Liuyu; Peng, Guangneng; Chen, Zeliang; Wang, Yufei

    2012-12-28

    Brucella has been considered as a non-motile, facultative intracellular pathogenic bacterium. However, the genome sequences of different Brucella species reveal the presence of the flagellar genes needed for the construction of a functional flagellum. Due to its roles in the interaction between pathogen and host, we hypothesized that some of the flagellar proteins might induce protective immune responses and these proteins will be good subunit vaccine candidates. This study was conducted to screening of protective antigens among these flagellar proteins. Firstly, according to the putative functional roles, a total of 30 flagellar genes of Brucella abortus were selected for in vitro expression. 15 of these flagellar genes were successfully expressed as his-tagged recombinant proteins in Escherichia coli ER2566. Then, these proteins were purified and used to analyze their T cell immunity induction activity by an in vitro gamma interferon (IFN-γ) assay. Five of the flagellar proteins could stimulate significantly higher levels of IFN-γ secretion in splenocytes from S19 immunized mice, indicating their T cell induction activity. Finally, immunogenicity and protection activity of these 5 flagellar proteins were evaluated in BALB/c mice. Results showed that immunization with FlgJ (BAB1_0260) or FliN (BAB2_0122) plus adjuvant could provide protection against B. abortus 544 infection. Furthermore, mice immunized with FlgJ and FliN developed a vigorous immunoglobulin G response, and in vitro stimulation of their splenocytes with immunizing proteins induced the secretion of IFN-γ. Altogether, these data suggest that flagellar proteins FlgJ and FliN are protective antigens that could produce humoral and cell-mediated responses in mice and candidates for use in future studies of vaccination against brucellosis.

  13. Genetic Analysis and Detection of fliCH1 and fliCH12 Genes Coding for Serologically Closely Related Flagellar Antigens in Human and Animal Pathogenic Escherichia coli

    PubMed Central

    Beutin, Lothar; Delannoy, Sabine; Fach, Patrick

    2016-01-01

    The E. coli flagellar types H1 and H12 show a high serological cross-reactivity and molecular serotyping appears an advantageous method to establish a clear discrimination between these flagellar types. Analysis of fliCH1 and fliCH12 gene sequences showed that they were 97.5% identical at the nucleotide level. Because of this high degree of homology we developed a two-step real-time PCR detection procedure for reliable discrimination of H1 and H12 flagellar types in E. coli. In the first step, a real-time PCR assay for common detection of both fliCH1 and fliCH12 genes is used, followed in a second step by real-time PCR assays for specific detection of fliCH1 and fliCH12, respectively. The real-time PCR for common detection of fliCH1 and fliCH12 demonstrated 100% sensitivity and specificity as it reacted with all tested E. coli H1 and H12 strains and not with any of the reference strains encoding all the other 51 flagellar antigens. The fliCH1 and fliCH12 gene specific assays detected all E. coli H1 and all E. coli H12 strains, respectively (100% sensitivity). However, both assays showed cross-reactions with some flagellar type reference strains different from H1 and H12. The real-time PCR assays developed in this study can be used in combination for the detection and identification of E. coli H1 and H12 strains isolated from different sources. PMID:26913025

  14. Mechanism of Flagellar Vaccine Protection Related to Pseudomonas Pathogenesis in Trauma Burns

    DTIC Science & Technology

    1989-01-19

    0 MECHANISM OF FLAGELLAR VACCINE PROTECTION RELATED6TO PSEUDOMONAS PATHOGENESIS IN TRAUMA BURNS Annual and Final Report Thomas C. Montie, Ph.D...Classificaun) Mechanism of Flagellar Vaccine Protection Related to Pseudomonas Pathogenesis itr Trauma ( Burns ) 12. PERSONAL AUTHOR(S) Thomas C. Montie, Ph.D. 13a...virulence. Isolated flagellar preparations have provided active protection in a burned mouse model. Passive protection with anti-flagellar sera (anti-LPS-free

  15. Polar flagellar motility of the Vibrionaceae.

    PubMed

    McCarter, L L

    2001-09-01

    Polar flagella of Vibrio species can rotate at speeds as high as 100,000 rpm and effectively propel the bacteria in liquid as fast as 60 microm/s. The sodium motive force powers rotation of the filament, which acts as a propeller. The filament is complex, composed of multiple subunits, and sheathed by an extension of the cell outer membrane. The regulatory circuitry controlling expression of the polar flagellar genes of members of the Vibrionaceae is different from the peritrichous system of enteric bacteria or the polar system of Caulobacter crescentus. The scheme of gene control is also pertinent to other members of the gamma purple bacteria, in particular to Pseudomonas species. This review uses the framework of the polar flagellar system of Vibrio parahaemolyticus to provide a synthesis of what is known about polar motility systems of the Vibrionaceae. In addition to its propulsive role, the single polar flagellum of V. parahaemolyticus is believed to act as a tactile sensor controlling surface-induced gene expression. Under conditions that impede rotation of the polar flagellum, an alternate, lateral flagellar motility system is induced that enables movement through viscous environments and over surfaces. Although the dual flagellar systems possess no shared structural components and although distinct type III secretion systems direct the simultaneous placement and assembly of polar and lateral organelles, movement is coordinated by shared chemotaxis machinery.

  16. Gene sequence and predicted amino acid sequence of the motA protein, a membrane-associated protein required for flagellar rotation in Escherichia coli.

    PubMed Central

    Dean, G E; Macnab, R M; Stader, J; Matsumura, P; Burks, C

    1984-01-01

    The motA and motB gene products of Escherichia coli are integral membrane proteins necessary for flagellar rotation. We determined the DNA sequence of the region containing the motA gene and its promoter. Within this sequence, there is an open reading frame of 885 nucleotides, which with high probability (98% confidence level) meets criteria for a coding sequence. The 295-residue amino acid translation product had a molecular weight of 31,974, in good agreement with the value determined experimentally by gel electrophoresis. The amino acid sequence, which was quite hydrophobic, was subjected to a theoretical analysis designed to predict membrane-spanning alpha-helical segments of integral membrane proteins; four such hydrophobic helices were predicted by this treatment. Additional amphipathic helices may also be present. A remarkable feature of the sequence is the existence of two segments of high uncompensated charge density, one positive and the other negative. Possible organization of the protein in the membrane is discussed. Asymmetry in the amino acid composition of translated DNA sequences was used to distinguish between two possible initiation codons. The use of this method as a criterion for authentication of coding regions is described briefly in an Appendix. PMID:6090403

  17. The endocytic activity of the flagellar pocket in Trypanosoma brucei is regulated by an adjacent phosphatidylinositol phosphate kinase

    PubMed Central

    Demmel, Lars; Schmidt, Katy; Lucast, Louise; Havlicek, Katharina; Zankel, Armin; Koestler, Tina; Reithofer, Viktoria; de Camilli, Pietro; Warren, Graham

    2014-01-01

    ABSTRACT Phosphoinositides are spatially restricted membrane signaling molecules. Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] – a phosphoinositide that is highly enriched in, and present throughout, the plasma membrane – has been implicated in endocytosis. Trypanosoma brucei has one of the highest known rates of endocytosis, a process it uses to evade the immune system. To determine whether phosphoinositides play a role in endocytosis in this organism, we have identified and characterized one of the enzymes that is responsible for generating PI(4,5)P2. Surprisingly, this phosphoinositide was found to be highly concentrated in the flagellar pocket, the only site of endocytosis and exocytosis in this organism. The enzyme (designated TbPIPKA, annotated as Tb927.10.1620) was present at the neck of the pocket, towards the anterior-end of the parasite. Depletion of TbPIPKA led to depletion of PI(4,5)P2 and enlargement of the pocket, the result of impaired endocytosis. Taken together, these data suggest that TbPIPKA and its product PI(4,5)P2 are important for endocytosis and, consequently, for homeostasis of the flagellar pocket. PMID:24639465

  18. Properties of flagellar "rigor waves" formed by abrupt removal of adenosine triphosphate from actively swimming sea urchin sperm.

    PubMed

    Gibbons, B H; Gibbons, I R

    1974-12-01

    Sea urchin sperm were demembranated and reactivated with a solution containing 0.04% Triton X-100 and 0.03 mM ATP. The ATP concentration was then lowered abruptly by diluting the sperm suspension 50-fold into reactivating solution containing no ATP. The flagella of the sperm in the diluted suspension were not motile, but they were bent into a variety of stationary rigor wave forms closely resembling the wave forms occurring at different stages of the flagellar bending cycle during normal movement. The form of these rigor waves was unchanged upon storage for several hours in the presence of dithiothreitol and EDTA. Addition of 1 microM ATP induced slow relaxation of the waves, with most of the sperm becoming partially straightened over a period of about 30 min; somewhat higher concentrations gave a more rapid and complete relaxation. Concentrations of ATP above 10 microM induced resumption of normal beating movements. Addition of ITP, GTP, or GDP (up to 1 mM) produced no relaxation of the rigor waves. Digestion with trypsin to an extent sufficient to disrupt the radial spokes and the nexin links caused no change in the rigor wave forms, suggesting that these wave forms could be maintained by the dynein cross-bridges between the outer doublet tubules of the flagellar axoneme. Study of the effects of viscous shear on the rigor wave axonemes has shown that they are resistant to distortion by bending, although they can be twisted relatively easily.

  19. Quorum sensing positively regulates flagellar motility in pathogenic Vibrio harveyi.

    PubMed

    Yang, Qian; Defoirdt, Tom

    2015-04-01

    Vibrios belonging to the Harveyi clade are among the major pathogens of aquatic organisms. Quorum sensing (QS) is essential for virulence of V. harveyi towards different hosts. However, most virulence factors reported to be controlled by QS to date are negatively regulated by QS, therefore suggesting that their impact on virulence is limited. In this study, we report that QS positively regulates flagellar motility. We found that autoinducer synthase mutants showed significantly lower swimming motility than the wild type, and the swimming motility could be restored by adding synthetic signal molecules. Further, motility of a luxO mutant with inactive QS (LuxO D47E) was significantly lower than that of the wild type and of a luxO mutant with constitutively maximal QS activity (LuxO D47A). Furthermore, we found that the expression of flagellar genes (both early, middle and late genes) was significantly lower in the luxO mutant with inactive QS when compared with wild type and the luxO mutant with maximal QS activity. Motility assays and gene expression also revealed the involvement of the quorum-sensing master regulator LuxR in the QS regulation of motility. Finally, the motility inhibitor phenamil significantly decreased the virulence of V. harveyi towards gnotobiotic brine shrimp larvae.

  20. Regulation of Eukaryotic Flagellar Motility

    NASA Astrophysics Data System (ADS)

    Mitchell, David R.

    2005-03-01

    The central apparatus is essential for normal eukaryotic flagellar bend propagation as evidenced by the paralysis associated with mutations that prevent central pair (CP) assembly. Interactions between doublet-associated radial spokes and CP projections are thought to modulate spoke-regulated protein kinases and phosphatases on outer doublets, and these enzymes in turn modulate dynein activity. To better understand CP control mechanisms, we determined the three-dimensional structure of the Chlamydomonas reinhardtii CP complex and analyzed CP orientation during formation and propagation of flagellar bending waves. We show that a single CP microtubule, C1, is near the outermost doublet in curved regions of the flagellum, and this orientation is maintained by twists between successive principal and reverse bends. The Chlamydomonas CP is inherently twisted; twists are not induced by bend formation, and do not depend on forces or signals transmitted through spoke-central pair interactions. We hypothesize that CP orientation passively responds to bend formation, and that bend propagation drives rotation of the CP and maintains a constant CP orientation in bends, which in turn permits signal transduction between specific CP projections and specific doublet-associated dyneins through radial spokes. The central pair kinesin, Klp1, although essential for normal motility, is therefore not the motor that drives CP rotation. The CP also acts as a scaffold for enzymes that maintain normal intraflagellar ATP concentration.

  1. Assembly mechanism of Trypanosoma brucei BILBO1 at the flagellar pocket collar.

    PubMed

    Vidilaseris, Keni; Lesigang, Johannes; Morriswood, Brooke; Dong, Gang

    2015-01-01

    The flagellar pocket is a bulb-like invagination of the plasma membrane that encloses the base of the single flagellum in trypanosomes. It is the site of all endo- and exocytic activity in the parasite and has thus been proposed to be a therapeutic target. At the neck of the flagellar pocket is an electron-dense cytoskeletal structure named the flagellar pocket collar. The protein BILBO1 was the first characterized and remains the only known component of the flagellar pocket collar, with essential functions in the biogenesis of both the flagellar pocket and flagellar pocket collar. We recently reported that the filamentous assembly of Trypanosoma brucei BILBO1 (TbBILBO1) is mediated by its central coiled coil domain and C-terminal leucine zipper. Here, we discuss how TbBILBO1 might assemble at the flagellar pocket collar in T. brucei.

  2. RflM functions as a transcriptional repressor in the autogenous control of the Salmonella Flagellar master operon flhDC.

    PubMed

    Singer, Hanna M; Erhardt, Marc; Hughes, Kelly T

    2013-09-01

    Motility of bacteria like Salmonella enterica is a highly regulated process that responds to a variety of internal and external stimuli. A hierarchy of three promoter classes characterizes the Salmonella flagellar system, and the onset of flagellar gene expression depends on the oligomeric regulatory complex and class 1 gene product FlhD(4)C(2). The flhDC promoter is a target for a broad range of transcriptional regulators that bind within the flhDC promoter region and either negatively or positively regulate flhDC operon transcription. In this work, we demonstrate that the RflM protein is a key component of flhDC regulation. Transposon mutagenesis was performed to investigate a previously described autoinhibitory effect of the flagellar master regulatory complex FlhD(4)C(2). RflM is a LuxR homolog that functions as a flagellar class 1 transcriptional repressor. RflM was found to be the negative regulator of flhDC expression that is responsible for the formerly described autoinhibitory effect of the FlhD(4)C(2) complex on flhDC operon transcription (K. Kutsukake, Mol. Gen. Genet. 254:440-448, 1997). We conclude that upon commencement of flagellar gene expression, the FlhD(4)C(2) complex initiates a regulatory feedback loop by activating rflM gene expression. rflM encodes a transcriptional repressor, RflM, which fine-tunes flhDC expression levels.

  3. Flagellar regeneration in the scaly green flagellate Tetraselmis striata (Prasinophyceae): regeneration kinetics and effect of inhibitors

    NASA Astrophysics Data System (ADS)

    Reize, I. B.; Melkonian, M.

    1987-06-01

    Flagellar regeneration after experimental amputation was studied in synchronized axenic cultures of the scaly green flagellate Tetraselmis striata (Prasinophyceae). After removal of flagella by mechanical shearing, 95% of the cells regrow all four flagella (incl. the scaly covering) to nearly full length with a linear velocity of 50 nm/min under standard conditions. Flagellar regeneration is independent of photosynthesis (no effect of DCMU; the same regeneration rate in the light or in the dark), but depends on de novo protein synthesis: cycloheximide at a low concentration (0.35 μM) blocks flagellar regeneration reversibly. No pool of flagellar precursors appears to be present throughout the flagellated phase of the cell cycle. A transient pool of flagellar precursors, sufficient to generate 2.5 μm of flagellar length, however, develops during flagellar regeneration. Tunicamycin (2 μg/ml) inhibits flagellar regeneration only after a second flagellar amputation, when flagella reach only one third the length of the control. Flagellar regeneration in T. striata differs considerably from that of Chlamydomonas reinhardtii and represents an excellent model system for the study of synchronous Golgi apparatus (GA) activation, and transport and exocytosis of GA-derived macromolecules (scales).

  4. The bacterial flagellar protein export apparatus processively transports flagellar proteins even with extremely infrequent ATP hydrolysis.

    PubMed

    Minamino, Tohru; Morimoto, Yusuke V; Kinoshita, Miki; Aldridge, Phillip D; Namba, Keiichi

    2014-12-22

    For self-assembly of the bacterial flagellum, a specific protein export apparatus utilizes ATP and proton motive force (PMF) as the energy source to transport component proteins to the distal growing end. The export apparatus consists of a transmembrane PMF-driven export gate and a cytoplasmic ATPase complex composed of FliH, FliI and FliJ. The FliI(6)FliJ complex is structurally similar to the α(3)β(3)γ complex of F(O)F(1)-ATPase. FliJ allows the gate to efficiently utilize PMF to drive flagellar protein export but it remains unknown how. Here, we report the role of ATP hydrolysis by the FliI(6)FliJ complex. The export apparatus processively transported flagellar proteins to grow flagella even with extremely infrequent or no ATP hydrolysis by FliI mutation (E211D and E211Q, respectively). This indicates that the rate of ATP hydrolysis is not at all coupled with the export rate. Deletion of FliI residues 401 to 410 resulted in no flagellar formation although this FliI deletion mutant retained 40% of the ATPase activity, suggesting uncoupling between ATP hydrolysis and activation of the gate. We propose that infrequent ATP hydrolysis by the FliI6FliJ ring is sufficient for gate activation, allowing processive translocation of export substrates for efficient flagellar assembly.

  5. Regulation of flagellar motility during biofilm formation

    PubMed Central

    Guttenplan, Sarah B.; Kearns, Daniel B.

    2013-01-01

    Many bacteria swim in liquid or swarm over solid surfaces by synthesizing rotary flagella. The same bacteria that are motile also commonly form non-motile multicellular aggregates held together by an extracellular matrix called biofilms. Biofilms are an important part of the lifestyle of pathogenic bacteria and it is assumed that there is a motility-to-biofilm transition wherein the inhibition of motility promotes biofilm formation. The transition is largely inferred from regulatory mutants that reveal the opposite regulation of the two phenotypes. Here we review the regulation of motility during biofilm formation in Bacillus, Pseudomonas, Vibrio, and Escherichia, and we conclude that the motility-to-biofilm transition, if necessary, likely involves two steps. In the short term, flagella are functionally regulated to either inhibit rotation or modulate the basal flagellar reversal frequency. Over the long term, flagellar gene transcription is inhibited and in the absence of de novo synthesis, flagella are likely diluted to extinction through growth. Both short term and long term control is likely important to the motility-to-biofilm transition to stabilize aggregates and optimize resource investment. We emphasize the newly discovered classes of flagellar functional regulators and speculate that others await discovery in the context of biofilm formation. PMID:23480406

  6. Genetic and Molecular Characterization of Flagellar Assembly in Shewanella oneidensis

    PubMed Central

    Wu, Lin; Wang, Jixuan; Tang, Peng; Chen, Haijiang; Gao, Haichun

    2011-01-01

    Shewanella oneidensis is a highly motile organism by virtue of a polar flagellum. Unlike most flagellated bacteria, it contains only one major chromosome segment encoding the components of the flagellum with the exception of the motor proteins. In this region, three genes encode flagellinsaccording to the original genome annotation. However, we find that only flaA and flaB encode functional filament subunits. Although these two genesare under the control of different promoters, they are actively transcribed and subsequently translated, producing a considerable number of flagellin proteins. Additionally, both flagellins are able to interact with their chaperon FliS and are subjected to feedback regulation. Furthermore, FlaA and FlaB are glycosylated by a pathwayinvolving a major glycosylating enzyme,PseB, in spite of the lack of the majority of theconsensus glycosylation sites. In conclusion, flagellar assembly in S. oneidensis has novel features despite the conservation of homologous genes across taxa. PMID:21731763

  7. Hydrodynamic synchronization of flagellar oscillators

    NASA Astrophysics Data System (ADS)

    Friedrich, Benjamin

    2016-11-01

    In this review, we highlight the physics of synchronization in collections of beating cilia and flagella. We survey the nonlinear dynamics of synchronization in collections of noisy oscillators. This framework is applied to flagellar synchronization by hydrodynamic interactions. The time-reversibility of hydrodynamics at low Reynolds numbers requires swimming strokes that break time-reversal symmetry to facilitate hydrodynamic synchronization. We discuss different physical mechanisms for flagellar synchronization, which break this symmetry in different ways.

  8. Inhibitio of Flagellar Coordination in Spirillum volutans

    PubMed Central

    Krieg, Noel R.; Tomelty, Joseph P.; Wells, J. Scott

    1967-01-01

    The motility of Spirillum volutans is caused by the rotation of each polar flagellar fascicle in a direction opposite to that of the more slowly rotating cell. Both flagella form cones of revolution oriented in the same direction. When the cell reverses its motion, both fascicles simultaneously reverse their rotation and also the orientation of their cones of revolution, with the tail fascicle becoming the head and vice versa. Chloral hydrate and phenol were found to cause uncoordination, with both fascicles becoming the head type; MgSO4, Mg(NO3)2, NiSO4, NiCl2, CuSO4, and CuCl2 also caused uncoordination, with both fascicles becoming the tail type. In all cases, the flagellar fascicles remained highly active but the cells were motionless because of the opposing propulsion; the rotation of the fascicles was in a constant direction without reversal. Uncoordinated states could be maintained for 30 to 60 min. Neutralization of the dual-tail flagellation caused by NiSO4 could be accomplished with chloral hydrate. At the null point, the flagellar orientation was intermediate between head and tail; the fascicles continually reversed direction of rotation, and, now coordinated, caused the cells to move back and forth. Higher concentrations of chloral hydrate completely overcame the effect of NiSO4 and caused dual-head flagellation. Optimal concentrations of test compounds were determined with the use of pure cultures and a reproducible growth medium. Images PMID:6057800

  9. Molecular Architecture of the Bacterial Flagellar Motor in Cells

    PubMed Central

    2015-01-01

    The flagellum is one of the most sophisticated self-assembling molecular machines in bacteria. Powered by the proton-motive force, the flagellum rapidly rotates in either a clockwise or counterclockwise direction, which ultimately controls bacterial motility and behavior. Escherichia coli and Salmonella enterica have served as important model systems for extensive genetic, biochemical, and structural analysis of the flagellum, providing unparalleled insights into its structure, function, and gene regulation. Despite these advances, our understanding of flagellar assembly and rotational mechanisms remains incomplete, in part because of the limited structural information available regarding the intact rotor–stator complex and secretion apparatus. Cryo-electron tomography (cryo-ET) has become a valuable imaging technique capable of visualizing the intact flagellar motor in cells at molecular resolution. Because the resolution that can be achieved by cryo-ET with large bacteria (such as E. coli and S. enterica) is limited, analysis of small-diameter bacteria (including Borrelia burgdorferi and Campylobacter jejuni) can provide additional insights into the in situ structure of the flagellar motor and other cellular components. This review is focused on the application of cryo-ET, in combination with genetic and biophysical approaches, to the study of flagellar structures and its potential for improving the understanding of rotor–stator interactions, the rotational switching mechanism, and the secretion and assembly of flagellar components. PMID:24697492

  10. In situ ellipsometric study of surface immobilization of flagellar filaments

    NASA Astrophysics Data System (ADS)

    Kurunczi, S.; Németh, A.; Hülber, T.; Kozma, P.; Petrik, P.; Jankovics, H.; Sebestyén, A.; Vonderviszt, F.; Fried, M.; Bársony, I.

    2010-10-01

    Protein filaments composed of thousands of subunits are promising candidates as sensing elements in biosensors. In this work in situ spectroscopic ellipsometry is applied to monitor the surface immobilization of flagellar filaments. This study is the first step towards the development of layers of filamentous receptors for sensor applications. Surface activation is performed using silanization and a subsequent glutaraldehyde crosslinking. Structure of the flagellar filament layers immobilized on activated and non-activated Si wafer substrates is determined using a two-layer effective medium model that accounted for the vertical density distribution of flagellar filaments with lengths of 300-1500 nm bound to the surface. The formation of the first interface layer can be explained by the multipoint covalent attachment of the filaments, while the second layer is mainly composed of tail pinned filaments floating upwards with the free parts. As confirmed by atomic force microscopy, covalent immobilization resulted in an increased surface density compared to absorption.

  11. FliH and FliI ensure efficient energy coupling of flagellar type III protein export in Salmonella.

    PubMed

    Minamino, Tohru; Kinoshita, Miki; Inoue, Yumi; Morimoto, Yusuke V; Ihara, Kunio; Koya, Satomi; Hara, Noritaka; Nishioka, Noriko; Kojima, Seiji; Homma, Michio; Namba, Keiichi

    2016-06-01

    For construction of the bacterial flagellum, flagellar proteins are exported via its specific export apparatus from the cytoplasm to the distal end of the growing flagellar structure. The flagellar export apparatus consists of a transmembrane (TM) export gate complex and a cytoplasmic ATPase complex consisting of FliH, FliI, and FliJ. FlhA is a TM export gate protein and plays important roles in energy coupling of protein translocation. However, the energy coupling mechanism remains unknown. Here, we performed a cross-complementation assay to measure robustness of the energy transduction system of the export apparatus against genetic perturbations. Vibrio FlhA restored motility of a Salmonella ΔflhA mutant but not that of a ΔfliH-fliI flhB(P28T) ΔflhA mutant. The flgM mutations significantly increased flagellar gene expression levels, allowing Vibrio FlhA to exert its export activity in the ΔfliH-fliI flhB(P28T) ΔflhA mutant. Pull-down assays revealed that the binding affinities of Vibrio FlhA for FliJ and the FlgN-FlgK chaperone-substrate complex were much lower than those of Salmonella FlhA. These suggest that Vibrio FlhA requires the support of FliH and FliI to efficiently and properly interact with FliJ and the FlgN-FlgK complex. We propose that FliH and FliI ensure robust and efficient energy coupling of protein export during flagellar assembly. © 2016 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  12. Load- and polysaccharide-dependent activation of the Na+-type MotPS stator in the Bacillus subtilis flagellar motor

    PubMed Central

    Terahara, Naoya; Noguchi, Yukina; Nakamura, Shuichi; Kami-ike, Nobunori; Ito, Masahiro; Namba, Keiichi; Minamino, Tohru

    2017-01-01

    The flagellar motor of Bacillus subtilis possesses two distinct H+-type MotAB and Na+-type MotPS stators. In contrast to the MotAB motor, the MotPS motor functions efficiently at elevated viscosity in the presence of 200 mM NaCl. Here, we analyzed the torque-speed relationship of the Bacillus MotAB and MotPS motors over a wide range of external loads. The stall torque of the MotAB and MotPS motors at high load was about 2,200 pN nm and 220 pN nm, respectively. The number of active stators in the MotAB and MotPS motors was estimated to be about ten and one, respectively. However, the number of functional stators in the MotPS motor was increased up to ten with an increase in the concentration of a polysaccharide, Ficoll 400, as well as in the load. The maximum speeds of the MotAB and MotPS motors at low load were about 200 Hz and 50 Hz, respectively, indicating that the rate of the torque-generation cycle of the MotPS motor is 4-fold slower than that of the MotAB motor. Domain exchange experiments showed that the C-terminal periplasmic domain of MotS directly controls the assembly and disassembly dynamics of the MotPS stator in a load- and polysaccharide-dependent manner. PMID:28378843

  13. Modulation of the Lytic Activity of the Dedicated Autolysin for Flagellum Formation SltF by Flagellar Rod Proteins FlgB and FlgF

    PubMed Central

    Herlihey, Francesca A.; Osorio-Valeriano, Manuel; Dreyfus, Georges

    2016-01-01

    ABSTRACT SltF was identified previously as an autolysin required for the assembly of flagella in the alphaproteobacteria, but the nature of its peptidoglycan lytic activity remained unknown. Sequence alignment analyses suggest that it could function as either a muramidase, lytic transglycosylase, or β-N-acetylglucosaminidase. Recombinant SltF from Rhodobacter sphaeroides was purified to apparent homogeneity, and it was demonstrated to function as a lytic transglycosylase based on enzymatic assays involving mass spectrometric analyses. Circular dichroism (CD) analysis determined that it is composed of 83.4% α-structure and 1.48% β-structure and thus is similar to family 1A lytic transglycosylases. However, alignment of apparent SltF homologs identified in the genome database defined a new subfamily of the family 1 lytic transglycosylases. SltF was demonstrated to be endo-acting, cleaving within chains of peptidoglycan, with optimal activity at pH 7.0. Its activity is modulated by two flagellar rod proteins, FlgB and FlgF: FlgB both stabilizes and stimulates SltF activity, while FlgF inhibits it. Invariant Glu57 was confirmed as the sole catalytic acid/base residue of SltF. IMPORTANCE The bacterial flagellum is comprised of a basal body, hook, and helical filament, which are connected by a rod structure. With a diameter of approximately 4 nm, the rod is larger than the estimated pore size within the peptidoglycan sacculus, and hence its insertion requires the localized and controlled lysis of this essential cell wall component. In many beta- and gammaproteobacteria, this lysis is catalyzed by the β-N-acetylglucosaminidase domain of FlgJ. However, FlgJ of the alphaproteobacteria lacks this activity and instead it recruits a separate enzyme, SltF, for this purpose. In this study, we demonstrate that SltF functions as a newly identified class of lytic transglycosylases and that its autolytic activity is uniquely modulated by two rod proteins, FlgB and FlgF. PMID

  14. Magnetic Propulsion of Microswimmers with DNA-Based Flagellar Bundles

    PubMed Central

    2016-01-01

    We show that DNA-based self-assembly can serve as a general and flexible tool to construct artificial flagella of several micrometers in length and only tens of nanometers in diameter. By attaching the DNA flagella to biocompatible magnetic microparticles, we provide a proof of concept demonstration of hybrid structures that, when rotated in an external magnetic field, propel by means of a flagellar bundle, similar to self-propelling peritrichous bacteria. Our theoretical analysis predicts that flagellar bundles that possess a length-dependent bending stiffness should exhibit a superior swimming speed compared to swimmers with a single appendage. The DNA self-assembly method permits the realization of these improved flagellar bundles in good agreement with our quantitative model. DNA flagella with well-controlled shape could fundamentally increase the functionality of fully biocompatible nanorobots and extend the scope and complexity of active materials. PMID:26821214

  15. Magnetic Propulsion of Microswimmers with DNA-Based Flagellar Bundles.

    PubMed

    Maier, Alexander M; Weig, Cornelius; Oswald, Peter; Frey, Erwin; Fischer, Peer; Liedl, Tim

    2016-02-10

    We show that DNA-based self-assembly can serve as a general and flexible tool to construct artificial flagella of several micrometers in length and only tens of nanometers in diameter. By attaching the DNA flagella to biocompatible magnetic microparticles, we provide a proof of concept demonstration of hybrid structures that, when rotated in an external magnetic field, propel by means of a flagellar bundle, similar to self-propelling peritrichous bacteria. Our theoretical analysis predicts that flagellar bundles that possess a length-dependent bending stiffness should exhibit a superior swimming speed compared to swimmers with a single appendage. The DNA self-assembly method permits the realization of these improved flagellar bundles in good agreement with our quantitative model. DNA flagella with well-controlled shape could fundamentally increase the functionality of fully biocompatible nanorobots and extend the scope and complexity of active materials.

  16. Studies on the mechanism of bacterial flagellar rotation and the flagellar number regulation.

    PubMed

    Kojima, Seiji

    2016-01-01

    Many motile bacteria have the motility organ, the flagellum. It rotates by the rotary motor driven by the ion-motive force and is embedded in the cell surface at the base of each flagellar filament. Many researchers have been studying its rotary mechanism for years, but most of the energy conversion processes have been remained in mystery. We focused on the flagellar stator, which works at the core process of energy conversion, and found that the periplasmic region of the stator changes its conformation to be activated only when the stator units are incorporated into the motor and anchored at the cell wall. Meanwhile, the physiologically important supramolecular complex is localized in the cell at the right place and the right time with a proper amount. How the cell achieves such a proper localization is the fundamental question for life science, and we undertake this problem by analyzing the mechanism for biogenesis of a single polar flagellum of Vibrio alginolyticus. Here I describe the molecular mechanism of how the flagellum is generated at the specific place with a proper number, and also how the flagellar stator is incorporated into the motor to complete the functional motor assembly, based on our studies.

  17. Structural diversity of bacterial flagellar motors

    PubMed Central

    Chen, Songye; Beeby, Morgan; Murphy, Gavin E; Leadbetter, Jared R; Hendrixson, David R; Briegel, Ariane; Li, Zhuo; Shi, Jian; Tocheva, Elitza I; Müller, Axel; Dobro, Megan J; Jensen, Grant J

    2011-01-01

    The bacterial flagellum is one of nature's most amazing and well-studied nanomachines. Its cell-wall-anchored motor uses chemical energy to rotate a microns-long filament and propel the bacterium towards nutrients and away from toxins. While much is known about flagellar motors from certain model organisms, their diversity across the bacterial kingdom is less well characterized, allowing the occasional misrepresentation of the motor as an invariant, ideal machine. Here, we present an electron cryotomographical survey of flagellar motor architectures throughout the Bacteria. While a conserved structural core was observed in all 11 bacteria imaged, surprisingly novel and divergent structures as well as different symmetries were observed surrounding the core. Correlating the motor structures with the presence and absence of particular motor genes in each organism suggested the locations of five proteins involved in the export apparatus including FliI, whose position below the C-ring was confirmed by imaging a deletion strain. The combination of conserved and specially-adapted structures seen here sheds light on how this complex protein nanomachine has evolved to meet the needs of different species. PMID:21673657

  18. The glycosylphosphatidylinositol-PLC in Trypanosoma brucei forms a linear array on the exterior of the flagellar membrane before and after activation.

    PubMed

    Hanrahan, Orla; Webb, Helena; O'Byrne, Robert; Brabazon, Elaine; Treumann, Achim; Sunter, Jack D; Carrington, Mark; Voorheis, H Paul

    2009-06-01

    Bloodstream forms of Trypanosoma brucei contain a glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) that cleaves the GPI-anchor of the variable surface glycoprotein (VSG). Its location in trypanosomes has been controversial. Here, using confocal microscopy and surface labelling techniques, we show that the GPI-PLC is located exclusively in a linear array on the outside of the flagellar membrane, close to the flagellar attachment zone, but does not co-localize with the flagellar attachment zone protein, FAZ1. Consequently, the GPI-PLC and the VSG occupy the same plasma membrane leaflet, which resolves the topological problem associated with the cleavage reaction if the VSG and the GPI-PLC were on opposite sides of the membrane. The exterior location requires the enzyme to be tightly regulated to prevent VSG release under basal conditions. During stimulated VSG release in intact cells, the GPI-PLC did not change location, suggesting that the release mechanism involves lateral diffusion of the VSG in the plane of the membrane to the fixed position of the GPI-PLC.

  19. Biochemical, immunological, metabolic, and molecular studies on flagellar development in Euglena gracilis

    SciTech Connect

    Levasseur, P.J.

    1989-01-01

    The emergent flagellum of Euglena gracilis arises from an anterior invagination of the organism and possesses, along with the typical eukaryotic axoneme, a glycoprotein surface layer, a complement of structurally complex mastigonemes and a paraxial rod. Nonionic detergent extraction of isolated flagella yielded a fraction containing 21% of the flagellar protein. This fraction contained at least 25 components. In vivo radiolabeling experiments indicated that Euglena possessed a pool of flagellar precursors. This was evidence by the observation that flagellar proteins radiolabeled during an initial regeneration could be mobilized to flagella of a subsequent regeneration. At least one component in the pool was present in sufficient quantity to support an entire regeneration. This protein was tentatively identified as a mastigonemal protein of M{sub r} {approximately} 220,000. A cDNA library was constructed to investigate flagellar gene expression in Euglena.

  20. H{sup +} and Na{sup +} are involved in flagellar rotation of the spirochete Leptospira

    SciTech Connect

    Islam, Md. Shafiqul; Morimoto, Yusuke V.; Kudo, Seishi; Nakamura, Shuichi

    2015-10-16

    Leptospira is a spirochete possessing intracellular flagella. Each Leptospira flagellar filament is linked with a flagellar motor composed of a rotor and a dozen stators. For many bacterial species, it is known that the stator functions as an ion channel and that the ion flux through the stator is coupled with flagellar rotation. The coupling ion varies depending on the species; for example, H{sup +} is used in Escherichia coli, and Na{sup +} is used in Vibrio spp. to drive a polar flagellum. Although genetic and structural studies illustrated that the Leptospira flagellar motor also contains a stator, the coupling ion for flagellar rotation remains unknown. In the present study, we analyzed the motility of Leptospira under various pH values and salt concentrations. Leptospira cells displayed motility in acidic to alkaline pH. In the presence of a protonophore, the cells completely lost motility in acidic to neutral pH but displayed extremely slow movement under alkaline conditions. This result suggests that H{sup +} is a major coupling ion for flagellar rotation over a wide pH range; however, we also observed that the motility of Leptospira was significantly enhanced by the addition of Na{sup +}, though it vigorously moved even under Na{sup +}-free conditions. These results suggest that H{sup +} is preferentially used and that Na{sup +} is secondarily involved in flagellar rotation in Leptospira. The flexible ion selectivity in the flagellar system could be advantageous for Leptospira to survive in a wide range of environment. - Highlights: • This is a study on input energy for motility in the spirochete Leptospira. • Leptospira biflexa exhibited active motility in acidic to alkaline pH. • Both H{sup +} and Na{sup +} are involved in flagellar rotation in Leptospira. • H{sup +} is a primary energy source, but Na{sup +} can secondarily enhance motility.

  1. Transcriptional analysis of the MrpJ network: modulation of diverse virulence-associated genes and direct regulation of mrp fimbrial and flhDC flagellar operons in Proteus mirabilis.

    PubMed

    Bode, Nadine J; Debnath, Irina; Kuan, Lisa; Schulfer, Anjelique; Ty, Maureen; Pearson, Melanie M

    2015-06-01

    The enteric bacterium Proteus mirabilis is associated with a significant number of catheter-associated urinary tract infections (UTIs). Strict regulation of the antagonistic processes of adhesion and motility, mediated by fimbriae and flagella, respectively, is essential for disease progression. Previously, the transcriptional regulator MrpJ, which is encoded by the mrp fimbrial operon, has been shown to repress both swimming and swarming motility. Here we show that MrpJ affects an array of cellular processes beyond adherence and motility. Microarray analysis found that expression of mrpJ mimicking levels observed during UTIs leads to differential expression of 217 genes related to, among other functions, bacterial virulence, type VI secretion, and metabolism. We probed the molecular mechanism of transcriptional regulation by MrpJ using transcriptional reporters and chromatin immunoprecipitation (ChIP). Binding of MrpJ to two virulence-associated target gene promoters, the promoters of the flagellar master regulator flhDC and mrp itself, appears to be affected by the condensation state of the native chromosome, although both targets share a direct MrpJ binding site proximal to the transcriptional start. Furthermore, an mrpJ deletion mutant colonized the bladders of mice at significantly lower levels in a transurethral model of infection. Additionally, we observed that mrpJ is widely conserved in a collection of recent clinical isolates. Altogether, these findings support a role of MrpJ as a global regulator of P. mirabilis virulence.

  2. Transcriptional Analysis of the MrpJ Network: Modulation of Diverse Virulence-Associated Genes and Direct Regulation of mrp Fimbrial and flhDC Flagellar Operons in Proteus mirabilis

    PubMed Central

    Bode, Nadine J.; Debnath, Irina; Kuan, Lisa; Schulfer, Anjelique; Ty, Maureen

    2015-01-01

    The enteric bacterium Proteus mirabilis is associated with a significant number of catheter-associated urinary tract infections (UTIs). Strict regulation of the antagonistic processes of adhesion and motility, mediated by fimbriae and flagella, respectively, is essential for disease progression. Previously, the transcriptional regulator MrpJ, which is encoded by the mrp fimbrial operon, has been shown to repress both swimming and swarming motility. Here we show that MrpJ affects an array of cellular processes beyond adherence and motility. Microarray analysis found that expression of mrpJ mimicking levels observed during UTIs leads to differential expression of 217 genes related to, among other functions, bacterial virulence, type VI secretion, and metabolism. We probed the molecular mechanism of transcriptional regulation by MrpJ using transcriptional reporters and chromatin immunoprecipitation (ChIP). Binding of MrpJ to two virulence-associated target gene promoters, the promoters of the flagellar master regulator flhDC and mrp itself, appears to be affected by the condensation state of the native chromosome, although both targets share a direct MrpJ binding site proximal to the transcriptional start. Furthermore, an mrpJ deletion mutant colonized the bladders of mice at significantly lower levels in a transurethral model of infection. Additionally, we observed that mrpJ is widely conserved in a collection of recent clinical isolates. Altogether, these findings support a role of MrpJ as a global regulator of P. mirabilis virulence. PMID:25847961

  3. Salmonella Enteritidis flagellar mutants have a colonization benefit in the chicken oviduct.

    PubMed

    Kilroy, Sofie; Raspoet, Ruth; Martel, An; Bosseler, Leslie; Appia-Ayme, Corinne; Thompson, Arthur; Haesebrouck, Freddy; Ducatelle, Richard; Van Immerseel, Filip

    2017-02-01

    Egg borne Salmonella Enteritidis is still a major cause of human food poisoning. Eggs can become internally contaminated following colonization of the hen's oviduct. In this paper we aimed to analyze the role of flagella of Salmonella Enteritidis in colonization of the hen's oviduct. Using a transposon library screen we showed that mutants lacking functional flagella are significantly more efficient in colonizing the hen's oviduct in vivo. A micro-array analysis proved that transcription of a number of flagellar genes is down-regulated inside chicken oviduct cells. Flagella contain flagellin, a pathogen associated molecular pattern known to bind to Toll-like receptor 5, activating a pro-inflammatory cascade. In vitro tests using primary oviduct cells showed that flagellin is not involved in invasion. Using a ligated loop model, a diminished inflammatory reaction was seen in the oviduct resulting from injection of an aflagellated mutant compared to the wild-type. It is hypothesized that Salmonella Enteritidis downregulates flagellar gene expression in the oviduct and consequently prevents a flagellin-induced inflammatory response, thereby increasing its oviduct colonization efficiency.

  4. Flagellar Cap Protein FliD Mediates Adherence of Atypical Enteropathogenic Escherichia coli to Enterocyte Microvilli

    PubMed Central

    Sampaio, Suely C. F.; Luiz, Wilson B.; Vieira, Mônica A. M.; Ferreira, Rita C. C.; Garcia, Bruna G.; Sinigaglia-Coimbra, Rita; Sampaio, Jorge L. M.; Ferreira, Luís C. S.

    2016-01-01

    The expression of flagella correlates with different aspects of bacterial pathogenicity, ranging from adherence to host cells to activation of inflammatory responses by the innate immune system. In the present study, we investigated the role of flagella in the adherence of an atypical enteropathogenic Escherichia coli (aEPEC) strain (serotype O51:H40) to human enterocytes. Accordingly, isogenic mutants deficient in flagellin (FliC), the flagellar structural subunit; the flagellar cap protein (FliD); or the MotAB proteins, involved in the control of flagellar motion, were generated and tested for binding to differentiated Caco-2 cells. Binding of the aEPEC strain to enterocytes was significantly impaired in strains with the fliC and fliD genes deleted, both of which could not form flagella on the bacterial surface. A nonmotile but flagellated MotAB mutant also showed impaired adhesion to Caco-2 cells. In accordance with these observations, adhesion of aEPEC strain 1711-4 to Caco-2 cells was drastically reduced after the treatment of Caco-2 cells with purified FliD. In addition, incubation of aEPEC bacteria with specific anti-FliD serum impaired binding to Caco-2 cells. Finally, incubation of Caco-2 cells with purified FliD, followed by immunolabeling, showed that the protein was specifically bound to the microvillus tips of differentiated Caco-2 cells. The aEPEC FliD or anti-FliD serum also reduced the adherence of prototype typical enteropathogenic, enterohemorrhagic, and enterotoxigenic E. coli strains to Caco-2 cells. In conclusion, our findings further strengthened the role of flagella in the adherence of aEPEC to human enterocytes and disclosed the relevant structural and functional involvement of FliD in the adhesion process. PMID:26831466

  5. Flagellar flows around bacterial swarms

    NASA Astrophysics Data System (ADS)

    Dauparas, Justas; Lauga, Eric

    2016-08-01

    Flagellated bacteria on nutrient-rich substrates can differentiate into a swarming state and move in dense swarms across surfaces. A recent experiment measured the flow in the fluid around an Escherichia coli swarm [Wu, Hosu, and Berg, Proc. Natl. Acad. Sci. USA 108, 4147 (2011)], 10.1073/pnas.1016693108. A systematic chiral flow was observed in the clockwise direction (when viewed from above) ahead of the swarm with flow speeds of about 10 μ m /s , about 3 times greater than the radial velocity at the edge of the swarm. The working hypothesis is that this flow is due to the action of cells stalled at the edge of a colony that extend their flagellar filaments outward, moving fluid over the virgin agar. In this work we quantitatively test this hypothesis. We first build an analytical model of the flow induced by a single flagellum in a thin film and then use the model, and its extension to multiple flagella, to compare with experimental measurements. The results we obtain are in agreement with the flagellar hypothesis. The model provides further quantitative insight into the flagella orientations and their spatial distributions as well as the tangential speed profile. In particular, the model suggests that flagella are on average pointing radially out of the swarm and are not wrapped tangentially.

  6. Flagellar oscillation: a commentary on proposed mechanisms.

    PubMed

    Woolley, David M

    2010-08-01

    Eukaryotic flagella and cilia have a remarkably uniform internal 'engine' known as the '9+2' axoneme. With few exceptions, the function of cilia and flagella is to beat rhythmically and set up relative motion between themselves and the liquid that surrounds them. The molecular basis of axonemal movement is understood in considerable detail, with the exception of the mechanism that provides its rhythmical or oscillatory quality. Some kind of repetitive 'switching' event is assumed to occur; there are several proposals regarding the nature of the 'switch' and how it might operate. Herein I first summarise all the factors known to influence the rate of the oscillation (the beating frequency). Many of these factors exert their effect through modulating the mean sliding velocity between the nine doublet microtubules of the axoneme, this velocity being the determinant of bend growth rate and bend propagation rate. Then I explain six proposed mechanisms for flagellar oscillation and review the evidence on which they are based. Finally, I attempt to derive an economical synthesis, drawing for preference on experimental research that has been minimally disruptive of the intricate structure of the axoneme. The 'provisional synthesis' is that flagellar oscillation emerges from an effect of passive sliding direction on the dynein arms. Sliding in one direction facilitates force-generating cycles and dynein-to-dynein synchronisation along a doublet; sliding in the other direction is inhibitory. The direction of the initial passive sliding normally oscillates because it is controlled hydrodynamically through the alternating direction of the propulsive thrust. However, in the absence of such regulation, there can be a perpetual, mechanical self-triggering through a reversal of sliding direction due to the recoil of elastic structures that deform as a response to the prior active sliding. This provisional synthesis may be a useful basis for further examination of the problem.

  7. Structure of the Flagellar Type III Export Chaperone FliT, and Its Regulatory Mechanism of Flagellar Assembly

    NASA Astrophysics Data System (ADS)

    Imada, Katsumi; Minamino, Tohru; Namba, Keiichi

    Flagellar export chaperones are multifunctional small proteins required for efficient construction of the bacterial flagellum. FliT is a flagellar export chaperone for the filament-capping protein FliD. We have determined the structure of FliT at 3.2 Å resolution. FliT adopts an anti-parallel α-helical bundle structure with a unique C-terminal helical segment of flexible orientation. The structure and following genetic and biochemical studies indicated that a conformational change of the C-terminal segment is responsible for switching the binding partners to regulate gene expression and protein export. This finding reveals how the complex pattern of interactions with various binding partners is regulated by a conformational change of such a small protein molecule.

  8. Rhythmicity, Recurrence, and Recovery of Flagellar Beating

    NASA Astrophysics Data System (ADS)

    Wan, Kirsty Y.; Goldstein, Raymond E.

    2014-12-01

    The eukaryotic flagellum beats with apparently unfailing periodicity, yet responds rapidly to stimuli. Like the human heartbeat, flagellar oscillations are now known to be noisy. Using the alga C. reinhardtii, we explore three aspects of nonuniform flagellar beating. We report the existence of rhythmicity, waveform noise peaking at transitions between power and recovery strokes, and fluctuations of interbeat intervals that are correlated and even recurrent, with memory extending to hundreds of beats. These features are altered qualitatively by physiological perturbations. Further, we quantify the recovery of periodic breaststroke beating from transient hydrodynamic forcing. These results will help constrain microscopic theories on the origins and regulation of flagellar beating.

  9. Genetic Diversity of the fliC Genes Encoding the Flagellar Antigen H19 of Escherichia coli and Application to the Specific Identification of Enterohemorrhagic E. coli O121:H19

    PubMed Central

    Beutin, Lothar; Delannoy, Sabine

    2015-01-01

    Enterohemorrhagic Escherichia coli (EHEC) O121:H19 belong to a specific clonal type distinct from other classical EHEC and major enteropathogenic E. coli groups and is regarded as one of the major EHEC serogroups involved in severe infections in humans. Sequencing of the fliC genes associated with the flagellar antigen H19 (fliCH19) revealed the genetic diversity of the fliCH19 gene sequences in E. coli. A cluster analysis of 12 fliCH19 sequences, 4 from O121 and 8 from non-O121 E. coli strains, revealed five different genotypes. All O121:H19 strains fell into one cluster, whereas a second cluster was formed by five non-O121:H19 strains. Cluster 1 and cluster 2 strains differ by 27 single nucleotide exchanges in their fliCH19 genes (98.5% homology). Based on allele discrimination of the fliCH19 genes, a real-time PCR test was designed for specific identification of EHEC O121:H19. The O121 fliCH19 PCR tested negative in 73 E. coli H19 strains that belonged to serogroups other than O121, including 28 different O groups, O-nontypeable H19, and O-rough:H19 strains. The O121 fliCH19 PCR reacted with all 16 tested O121:H19 strains and 1 O-rough:H19 strain which was positive for the O121 wzx gene. A cross-reaction was observed only with E. coli H32 strains which share sequence similarities in the target region of the O121 fliCH19 PCR. The combined use of O-antigen genotyping (O121 wzx) and the detection of O121 fliCH19 allele type contributes to improving the identification and molecular serotyping of EHEC O121:H19 motile and nonmotile strains and variants of these strains lacking stx genes. PMID:25862232

  10. Genetic Diversity of the fliC Genes Encoding the Flagellar Antigen H19 of Escherichia coli and Application to the Specific Identification of Enterohemorrhagic E. coli O121:H19.

    PubMed

    Beutin, Lothar; Delannoy, Sabine; Fach, Patrick

    2015-06-15

    Enterohemorrhagic Escherichia coli (EHEC) O121:H19 belong to a specific clonal type distinct from other classical EHEC and major enteropathogenic E. coli groups and is regarded as one of the major EHEC serogroups involved in severe infections in humans. Sequencing of the fliC genes associated with the flagellar antigen H19 (fliCH19) revealed the genetic diversity of the fliCH19 gene sequences in E. coli. A cluster analysis of 12 fliCH19 sequences, 4 from O121 and 8 from non-O121 E. coli strains, revealed five different genotypes. All O121:H19 strains fell into one cluster, whereas a second cluster was formed by five non-O121:H19 strains. Cluster 1 and cluster 2 strains differ by 27 single nucleotide exchanges in their fliCH19 genes (98.5% homology). Based on allele discrimination of the fliCH19 genes, a real-time PCR test was designed for specific identification of EHEC O121:H19. The O121 fliCH19 PCR tested negative in 73 E. coli H19 strains that belonged to serogroups other than O121, including 28 different O groups, O-nontypeable H19, and O-rough:H19 strains. The O121 fliCH19 PCR reacted with all 16 tested O121:H19 strains and 1 O-rough:H19 strain which was positive for the O121 wzx gene. A cross-reaction was observed only with E. coli H32 strains which share sequence similarities in the target region of the O121 fliCH19 PCR. The combined use of O-antigen genotyping (O121 wzx) and the detection of O121 fliCH19 allele type contributes to improving the identification and molecular serotyping of EHEC O121:H19 motile and nonmotile strains and variants of these strains lacking stx genes. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  11. Flagellar Synchronization Is a Simple Alternative to Cell Cycle Synchronization for Ciliary and Flagellar Studies.

    PubMed

    Dutta, Soumita; Avasthi, Prachee

    2017-01-01

    The unicellular green alga Chlamydomonas reinhardtii is an ideal model organism for studies of ciliary function and assembly. In assays for biological and biochemical effects of various factors on flagellar structure and function, synchronous culture is advantageous for minimizing variability. Here, we have characterized a method in which 100% synchronization is achieved with respect to flagellar length but not with respect to the cell cycle. The method requires inducing flagellar regeneration by amputation of the entire cell population and limiting regeneration time. This results in a maximally homogeneous distribution of flagellar lengths at 3 h postamputation. We found that time-limiting new protein synthesis during flagellar synchronization limits variability in the unassembled pool of limiting flagellar protein and variability in flagellar length without affecting the range of cell volumes. We also found that long- and short-flagella mutants that regenerate normally require longer and shorter synchronization times, respectively. By minimizing flagellar length variability using a simple method requiring only hours and no changes in media, flagellar synchronization facilitates the detection of small changes in flagellar length resulting from both chemical and genetic perturbations in Chlamydomonas. This method increases our ability to probe the basic biology of ciliary size regulation and related disease etiologies. IMPORTANCE Cilia and flagella are highly conserved antenna-like organelles that found in nearly all mammalian cell types. They perform sensory and motile functions contributing to numerous physiological and developmental processes. Defects in their assembly and function are implicated in a wide range of human diseases ranging from retinal degeneration to cancer. Chlamydomonas reinhardtii is an algal model system for studying mammalian cilium formation and function. Here, we report a simple synchronization method that allows detection of small

  12. Sequence Variations in the Flagellar Antigen Genes fliCH25 and fliCH28 of Escherichia coli and Their Use in Identification and Characterization of Enterohemorrhagic E. coli (EHEC) O145:H25 and O145:H28.

    PubMed

    Beutin, Lothar; Delannoy, Sabine; Fach, Patrick

    2015-01-01

    Enterohemorrhagic E. coli (EHEC) serogroup O145 is regarded as one of the major EHEC serogroups involved in severe infections in humans. EHEC O145 encompasses motile and non-motile strains of serotypes O145:H25 and O145:H28. Sequencing the fliC-genes associated with the flagellar antigens H25 and H28 revealed the genetic diversity of the fliCH25 and fliCH28 gene sequences in E. coli. Based on allele discrimination of these fliC-genes real-time PCR tests were designed for identification of EHEC O145:H25 and O145:H28. The fliCH25 genes present in O145:H25 were found to be very similar to those present in E. coli serogroups O2, O100, O165, O172 and O177 pointing to their common evolution but were different from fliCH25 genes of a multiple number of other E. coli serotypes. In a similar way, EHEC O145:H28 harbor a characteristic fliCH28 allele which, apart from EHEC O145:H28, was only found in enteropathogenic (EPEC) O28:H28 strains that shared some common traits with EHEC O145:H28. The real time PCR-assays targeting these fliCH25[O145] and fliCH28[O145] alleles allow better characterization of EHEC O145:H25 and EHEC O145:H28. Evaluation of these PCR assays in spiked ready-to eat salad samples resulted in specific detection of both types of EHEC O145 strains even when low spiking levels of 1-10 cfu/g were used. Furthermore these PCR assays allowed identification of non-motile E. coli strains which are serologically not typable for their H-antigens. The combined use of O-antigen genotyping (O145wzy) and detection of the respective fliCH25[O145] and fliCH28[O145] allele types contributes to improve identification and molecular serotyping of E. coli O145 isolates.

  13. Sequence Variations in the Flagellar Antigen Genes fliCH25 and fliCH28 of Escherichia coli and Their Use in Identification and Characterization of Enterohemorrhagic E. coli (EHEC) O145:H25 and O145:H28

    PubMed Central

    Beutin, Lothar; Delannoy, Sabine; Fach, Patrick

    2015-01-01

    Enterohemorrhagic E. coli (EHEC) serogroup O145 is regarded as one of the major EHEC serogroups involved in severe infections in humans. EHEC O145 encompasses motile and non-motile strains of serotypes O145:H25 and O145:H28. Sequencing the fliC-genes associated with the flagellar antigens H25 and H28 revealed the genetic diversity of the fliCH25 and fliCH28 gene sequences in E. coli. Based on allele discrimination of these fliC-genes real-time PCR tests were designed for identification of EHEC O145:H25 and O145:H28. The fliCH25 genes present in O145:H25 were found to be very similar to those present in E. coli serogroups O2, O100, O165, O172 and O177 pointing to their common evolution but were different from fliCH25 genes of a multiple number of other E. coli serotypes. In a similar way, EHEC O145:H28 harbor a characteristic fliCH28 allele which, apart from EHEC O145:H28, was only found in enteropathogenic (EPEC) O28:H28 strains that shared some common traits with EHEC O145:H28. The real time PCR-assays targeting these fliCH25[O145] and fliCH28[O145] alleles allow better characterization of EHEC O145:H25 and EHEC O145:H28. Evaluation of these PCR assays in spiked ready-to eat salad samples resulted in specific detection of both types of EHEC O145 strains even when low spiking levels of 1–10 cfu/g were used. Furthermore these PCR assays allowed identification of non-motile E. coli strains which are serologically not typable for their H-antigens. The combined use of O-antigen genotyping (O145wzy) and detection of the respective fliCH25[O145] and fliCH28[O145] allele types contributes to improve identification and molecular serotyping of E. coli O145 isolates. PMID:26000885

  14. Quantitative analysis and modeling of katanin function in flagellar length control

    PubMed Central

    Kannegaard, Elisa; Rego, E. Hesper; Schuck, Sebastian; Feldman, Jessica L.; Marshall, Wallace F.

    2014-01-01

    Flagellar length control in Chlamydomonas reinhardtii provides a simple model system in which to investigate the general question of how cells regulate organelle size. Previous work demonstrated that Chlamydomonas cytoplasm contains a pool of flagellar precursor proteins sufficient to assemble a half-length flagellum and that assembly of full-length flagella requires synthesis of additional precursors to augment the preexisting pool. The regulatory systems that control the synthesis and regeneration of this pool are not known, although transcriptional regulation clearly plays a role. We used quantitative analysis of length distributions to identify candidate genes controlling pool regeneration and found that a mutation in the p80 regulatory subunit of katanin, encoded by the PF15 gene in Chlamydomonas, alters flagellar length by changing the kinetics of precursor pool utilization. This finding suggests a model in which flagella compete with cytoplasmic microtubules for a fixed pool of tubulin, with katanin-mediated severing allowing easier access to this pool during flagellar assembly. We tested this model using a stochastic simulation that confirms that cytoplasmic microtubules can compete with flagella for a limited tubulin pool, showing that alteration of cytoplasmic microtubule severing could be sufficient to explain the effect of the pf15 mutations on flagellar length. PMID:25143397

  15. Transcriptional regulation of coordinate changes in flagellar mRNAs during differentiation of Naegleria gruberi amoebae into flagellates

    SciTech Connect

    Lee, J.H.; Walsh, C.J.

    1988-06-01

    The nuclear run-on technique was used to measure the rate of transcription of flagellar genes during the differentiation of Naegleria gruberi amebae into flagellates. Synthesis of mRNAs for the axonemal proteins ..cap alpha..- and BETA-tubulin and flagellar calmodulin, as well as a coordinately regulated poly(A)/sup +/ RNA that codes for an unidentified protein, showed transient increases averaging 22-fold. The rate of synthesis of two poly(A)/sup +/ RNAs common to ameobae and flagellates was low until the transcription of the flagellar genes began to decline, at which time synthesis of the RNAs found in ameobae increased 3- to 10-fold. The observed changes in the rate of transcription can account quantitatively for the 20-fold increase in flagellar mRNA concentration during the differentiation. The data for the flagellar calmodulin gene demonstrate transcriptional regulation for a nontubulin axonemal protein. The data also demonstrate at least two programs of transcriptional regulation during the differentiation and raise the intriguing possibility that some significant fraction of the nearly 200 different proteins of the flagellar axoneme is transcriptionally regulated during the 1 h it takes N. gruberi amebae to form visible flagella.

  16. Nonlinear amplitude dynamics in flagellar beating

    NASA Astrophysics Data System (ADS)

    Oriola, David; Gadêlha, Hermes; Casademunt, Jaume

    2017-03-01

    The physical basis of flagellar and ciliary beating is a major problem in biology which is still far from completely understood. The fundamental cytoskeleton structure of cilia and flagella is the axoneme, a cylindrical array of microtubule doublets connected by passive cross-linkers and dynein motor proteins. The complex interplay of these elements leads to the generation of self-organized bending waves. Although many mathematical models have been proposed to understand this process, few attempts have been made to assess the role of dyneins on the nonlinear nature of the axoneme. Here, we investigate the nonlinear dynamics of flagella by considering an axonemal sliding control mechanism for dynein activity. This approach unveils the nonlinear selection of the oscillation amplitudes, which are typically either missed or prescribed in mathematical models. The explicit set of nonlinear equations are derived and solved numerically. Our analysis reveals the spatio-temporal dynamics of dynein populations and flagellum shape for different regimes of motor activity, medium viscosity and flagellum elasticity. Unstable modes saturate via the coupling of dynein kinetics and flagellum shape without the need of invoking a nonlinear axonemal response. Hence, our work reveals a novel mechanism for the saturation of unstable modes in axonemal beating.

  17. Nonlinear amplitude dynamics in flagellar beating

    PubMed Central

    Casademunt, Jaume

    2017-01-01

    The physical basis of flagellar and ciliary beating is a major problem in biology which is still far from completely understood. The fundamental cytoskeleton structure of cilia and flagella is the axoneme, a cylindrical array of microtubule doublets connected by passive cross-linkers and dynein motor proteins. The complex interplay of these elements leads to the generation of self-organized bending waves. Although many mathematical models have been proposed to understand this process, few attempts have been made to assess the role of dyneins on the nonlinear nature of the axoneme. Here, we investigate the nonlinear dynamics of flagella by considering an axonemal sliding control mechanism for dynein activity. This approach unveils the nonlinear selection of the oscillation amplitudes, which are typically either missed or prescribed in mathematical models. The explicit set of nonlinear equations are derived and solved numerically. Our analysis reveals the spatio-temporal dynamics of dynein populations and flagellum shape for different regimes of motor activity, medium viscosity and flagellum elasticity. Unstable modes saturate via the coupling of dynein kinetics and flagellum shape without the need of invoking a nonlinear axonemal response. Hence, our work reveals a novel mechanism for the saturation of unstable modes in axonemal beating. PMID:28405357

  18. Limiting Speed of the Bacterial Flagellar Motor

    NASA Astrophysics Data System (ADS)

    Nirody, Jasmine; Berry, Richard; Oster, George

    The bacterial flagellar motor (BFM) drives swimming in a wide variety of bacterial species, making it crucial for several fundamental biological processes including chemotaxis and community formation. Recent experiments have shown that the structure of this nanomachine is more dynamic than previously believed. Specifically, the number of active torque-generating units (stators) was shown to vary across applied loads. This finding invalidates the experimental evidence reporting that limiting (zero-torque) speed is independent of the number of active stators. Here, we put forward a model for the torque generation mechanism of this motor and propose that the maximum speed of the motor increases as additional torque-generators are recruited. This is contrary to the current widely-held belief that there is a universal upper limit to the speed of the BFM. Our result arises from the assumption that stators disengage from the motor for a significant portion of their mechanochemical cycles at low loads. We show that this assumption is consistent with current experimental evidence and consolidate our predictions with arguments that a processive motor must have a high duty ratio at high loads.

  19. How molecular motors shape the flagellar beat

    PubMed Central

    Riedel-Kruse, Ingmar H.; Hilfinger, Andreas; Howard, Jonathon; Jülicher, Frank

    2007-01-01

    Cilia and eukaryotic flagella are slender cellular appendages whose regular beating propels cells and microorganisms through aqueous media. The beat is an oscillating pattern of propagating bends generated by dynein motor proteins. A key open question is how the activity of the motors is coordinated in space and time. To elucidate the nature of this coordination we inferred the mechanical properties of the motors by analyzing the shape of beating sperm: Steadily beating bull sperm were imaged and their shapes were measured with high precision using a Fourier averaging technique. Comparing our experimental data with wave forms calculated for different scenarios of motor coordination we found that only the scenario of interdoublet sliding regulating motor activity gives rise to satisfactory fits. We propose that the microscopic origin of such “sliding control” is the load dependent detachment rate of motors. Agreement between observed and calculated wave forms was obtained only if significant sliding between microtubules occurred at the base. This suggests a novel mechanism by which changes in basal compliance could reverse the direction of beat propagation. We conclude that the flagellar beat patterns are determined by an interplay of the basal properties of the axoneme and the mechanical feedback of dynein motors. PMID:19404446

  20. The Flagellar Protein FliL Is Essential for Swimming in Rhodobacter sphaeroides▿ †

    PubMed Central

    Suaste-Olmos, Fernando; Domenzain, Clelia; Mireles-Rodríguez, José Cruz; Poggio, Sebastian; Osorio, Aurora; Dreyfus, Georges; Camarena, Laura

    2010-01-01

    In this work we characterize the function of the flagellar protein FliL in Rhodobacter sphaeroides. Our results show that FliL is essential for motility in this bacterium and that in its absence flagellar rotation is highly impaired. A green fluorescent protein (GFP)-FliL fusion forms polar and lateral fluorescent foci that show different spatial dynamics. The presence of these foci is dependent on the expression of the flagellar genes controlled by the master regulator FleQ, suggesting that additional components of the flagellar regulon are required for the proper localization of GFP-FliL. Eight independent pseudorevertants were isolated from the fliL mutant strain. In each of these strains a single nucleotide change in motB was identified. The eight mutations affected only three residues located on the periplasmic side of MotB. Swimming of the suppressor mutants was not affected by the presence of the wild-type fliL allele. Pulldown and yeast two-hybrid assays showed that that the periplasmic domain of FliL is able to interact with itself but not with the periplasmic domain of MotB. From these results we propose that FliL could participate in the coupling of MotB with the flagellar rotor in an indirect fashion. PMID:20889747

  1. MotD of Sinorhizobium meliloti and Related α-Proteobacteria Is the Flagellar-Hook-Length Regulator and Therefore Reassigned as FliK

    PubMed Central

    Eggenhofer, Elke; Rachel, Reinhard; Haslbeck, Martin; Scharf, Birgit

    2006-01-01

    The flagella of the soil bacterium Sinorhizobium meliloti differ from the enterobacterial paradigm in the complex filament structure and modulation of the flagellar rotary speed. The mode of motility control in S. meliloti has a molecular corollary in two novel periplasmic motility proteins, MotC and MotE, that are present in addition to the ubiquitous MotA/MotB energizing proton channel. A fifth motility gene is located in the mot operon downstream of the motB and motC genes. Its gene product was originally designated MotD, a cytoplasmic motility protein having an unknown function. We report here reassignment of MotD as FliK, the regulator of flagellar hook length. The FliK gene is one of the few flagellar genes not annotated in the contiguous flagellar regulon of S. meliloti. Characteristic for its class, the 475-residue FliK protein contains a conserved, compactly folded Flg hook domain in its carboxy-terminal region. Deletion of fliK leads to formation of prolonged flagellar hooks (polyhooks) with missing filament structures. Extragenic suppressor mutations all mapped in the cytoplasmic region of the transmembrane export protein FlhB and restored assembly of a flagellar filament, and thus motility, in the presence of polyhooks. The structural properties of FliK are consistent with its function as a substrate specificity switch of the flagellar export apparatus for switching from rod/hook-type substrates to filament-type substrates. PMID:16513744

  2. Modulation of Toxin Production by the Flagellar Regulon in Clostridium difficile

    PubMed Central

    Aubry, Annie; Hussack, Greg; Chen, Wangxue; KuoLee, Rhonda; Twine, Susan M.; Fulton, Kelly M.; Foote, Simon; Carrillo, Catherine D.; Tanha, Jamshid

    2012-01-01

    We show in this study that toxin production in Clostridium difficile is altered in cells which can no longer form flagellar filaments. The impact of inactivation of fliC, CD0240, fliF, fliG, fliM, and flhB-fliR flagellar genes upon toxin levels in culture supernatants was assessed using cell-based cytotoxicity assay, proteomics, immunoassay, and immunoblotting approaches. Each of these showed that toxin levels in supernatants were significantly increased in a fliC mutant compared to that in the C. difficile 630 parent strain. In contrast, the toxin levels in supernatants secreted from other flagellar mutants were significantly reduced compared with that in the parental C. difficile 630 strain. Transcriptional analysis of the pathogenicity locus genes (tcdR, tcdB, tcdE, and tcdA) revealed a significant increase of all four genes in the fliC mutant strain, while transcription of all four genes was significantly reduced in fliM, fliF, fliG, and flhB-fliR mutants. These results demonstrate that toxin transcription in C. difficile is modulated by the flagellar regulon. More significantly, mutant strains showed a corresponding change in virulence compared to the 630 parent strain when tested in a hamster model of C. difficile infection. This is the first demonstration of differential flagellum-related transcriptional regulation of toxin production in C. difficile and provides evidence for elaborate regulatory networks for virulence genes in C. difficile. PMID:22851750

  3. Flagellar proteins of motile spores of Actinomycetes.

    PubMed

    Vesselinova, N I; Ensign, J C

    1996-06-01

    Flagella of some of the actinoplanete genera were purified and the molecular sizes of their flagellin subunits compared by SDS-PAGE analysis to flagellins of cells of other bacteria. Several species of Actinoplanes have a major flagellar protein of subunit sizes of 42-43 kDa and a lesser amount of a second protein, possibly a minor flagellin subunit, of 60 kDa. The flagellar protein sizes of other actinoplanetes ranged from 32-43 kDa (major) and 48-58 kDa (minor). Antibodies formed against the 42-kDa protein of A. rectilineatus showed cross-reactivity in Western blots against flagellar proteins of spores of other Actinoplanes species, two species of Dactylosporangium and an Ampullariella species. Cross-reactivity was also observed with motile cells of two other actinomycetes, Arthrobacter atrocyaneus and a Geodermatophilus species, and with Bacillus subtilis. No cross-reactivity was observed with Escherichia coli or Planomonospora parontospora flagellar proteins. The amino acid composition and partial N-terminal sequence of the 42-kDa flagellar protein of A. rectilineatus was compared to literature data for other bacterial flagellins and found to be most similar to B. subtilis 168.

  4. Na+-driven bacterial flagellar motors.

    PubMed

    Imae, Y; Atsumi, T

    1989-12-01

    Bacterial flagellar motors are the reversible rotary engine which propels the cell by rotating a helical flagellar filament as a screw propeller. The motors are embedded in the cytoplasmic membrane, and the energy for rotation is supplied by the electrochemical potential of specific ions across the membrane. Thus, the analysis of motor rotation at the molecular level is linked to an understanding of how the living system converts chemical energy into mechanical work. Based on the coupling ions, the motors are divided into two types; one is the H+-driven type found in neutrophiles such as Bacillus subtilis and Escherichia coli and the other is the Na+-driven type found in alkalophilic Bacillus and marine Vibrio. In this review, we summarize the current status of research on the rotation mechanism of the Na+-driven flagellar motors, which introduces several new aspects in the analysis.

  5. Hydrodynamic interaction of bacterial flagella - flagellar bundling

    NASA Astrophysics Data System (ADS)

    Lim, Sookkyung

    2013-11-01

    Flagellar bundling is an important aspect of locomotion in bacteria such as Escherichia coli. To study the hydrodynamic behavior of helical flagella, we present a computational model that is based on the geometry of the bacterial flagellar filament at the micrometer scale. We consider two model flagella, each of which has a rotary motor at its base with the rotation rate of the motor set at 100 Hz. Bundling occurs when both flagella are left-handed helices turning counterclockwise (when viewed from the nonmotor end of the flagellum looking back toward the motor) or when both flagella are right-handed helices turning clockwise. Helical flagella of the other combinations of handedness and rotation direction do not bundle. In this work we use the generalized immersed boundary method combined with the unconstrained Kirchhoff rod theory, which allows us to study the complicated hydrodynamics of flagellar behavior. This is a joint work with Charlie Peskin at NYU. NSF

  6. Rhythmicity, recurrence, and recovery of flagellar beating

    NASA Astrophysics Data System (ADS)

    Wan, Kirsty; Goldstein, Raymond

    2015-03-01

    The eukaryotic flagellum beats with apparently unfailing periodicity, yet responds rapidly to stimuli. Like the human heartbeat, flagellar oscillations are now known to be noisy. Using the unicellular alga Chlamydomonas reinhardtii, we explore three aspects of nonuniform flagellar beating. We report the existence of rhythmicity, waveform noise peaking at transitions between power and recovery strokes, and fluctuations of interbeat intervals that are correlated and even recurrent, with memory extending to hundreds of beats. These features are altered qualitatively by physiological perturbations. Further, we quantify the recovery of periodic breaststroke beating from transient hydrodynamic forcing. These results will help constrain microscopic theories on the origins and regulation of flagellar beating. Financial support is acknowledged from the EPSRC, ERC Advanced Investigator Grant No. 247333, and a Senior Investigator Award from the Wellcome Trust.

  7. Flagellar waveform analysis of swimming algal cells

    NASA Astrophysics Data System (ADS)

    Kurtuldu, Huseyin; Johnson, Karl; Gollub, Jerry

    2011-11-01

    The twin flagella of the green alga Chlamydomas reinhardtii are driven by dynein molecular motors to oscillate at about 50-60 Hz in a breaststroke motion. For decades, Chlamydomas has been used as a model organism for studies of flagellar motility, and of genetic disorders of ciliary motion. However, little is known experimentally about the flagellar waveforms, and the resulting time-dependent force distribution along the 250 nm diameter flagella. Here, we study flagellar dynamics experimentally by confining cells in quasi-2D liquid films. From simultaneous measurements of the cell body velocity and the time-dependent velocities along the center lines of the two flagella, we determine the drag coefficients, and estimate the power expended by the body and the flagella, comparing our findings with measurements based on the induced fluid flow field. We contrast the results for the quite different beating patterns of synchronous and asynchronous flagella, respectively. Supported by NSF Grant DMR-0803153.

  8. Listeria monocytogenes DNA glycosylase AdiP affects flagellar motility, biofilm formation, virulence, and stress responses

    USDA-ARS?s Scientific Manuscript database

    The temperature-dependent alteration of flagellar motility gene expression is critical for the foodborne pathogen Listeria monocytogenes to respond to a changing environment. In this study, a genetic determinant, L. monocytogenes f2365_0220 (lmof2365_0220), encoding a putative protein that is struct...

  9. Load Response of the Flagellar Beat

    NASA Astrophysics Data System (ADS)

    Klindt, Gary S.; Ruloff, Christian; Wagner, Christian; Friedrich, Benjamin M.

    2016-12-01

    Cilia and flagella exhibit regular bending waves that perform mechanical work on the surrounding fluid, to propel cellular swimmers and pump fluids inside organisms. Here, we quantify a force-velocity relationship of the beating flagellum, by exposing flagellated Chlamydomonas cells to controlled microfluidic flows. A simple theory of flagellar limit-cycle oscillations, calibrated by measurements in the absence of flow, reproduces this relationship quantitatively. We derive a link between the energy efficiency of the flagellar beat and its ability to synchronize to oscillatory flows.

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

    PubMed

    Reddy, G Srinivas; Mukhopadhyay, Aakash Gautam; Dey, Chinmoy Sankar

    2017-09-27

    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. Copyright © 2017. Published by Elsevier Inc.

  11. Brucella melitensis Cyclic di-GMP Phosphodiesterase BpdA Controls Expression of Flagellar Genes▿†

    PubMed Central

    Petersen, Erik; Chaudhuri, Pallab; Gourley, Chris; Harms, Jerome; Splitter, Gary

    2011-01-01

    Brucella melitensis encounters a variety of conditions and stimuli during its life cycle—including environmental growth, intracellular infection, and extracellular dissemination—which necessitates flexibility of bacterial signaling to promote virulence. Cyclic-di-GMP is a bacterial secondary signaling molecule that plays an important role in adaptation to changing environments and altering virulence in a number of bacteria. To investigate the role of cyclic-di-GMP in B. melitensis, all 11 predicted cyclic-di-GMP-metabolizing proteins were separately deleted and the effect on virulence was determined. Three of these cyclic-di-GMP-metabolizing proteins were found to alter virulence. Deletion of the bpdA and bpdB genes resulted in attenuation of virulence of the bacterium, while deletion of the cgsB gene produced a hypervirulent strain. In a Vibrio reporter system to monitor apparent alteration in levels of cyclic-di-GMP, both BpdA and BpdB displayed a phenotype consistent with cyclic-di-GMP-specific phosphodiesterases, while CgsB displayed a cyclic-di-GMP synthase phenotype. Further analysis found that deletion of bpdA resulted in a dramatic decrease in flagellar promoter activities, and a flagellar mutant showed similar phenotypes to the bpdA and bpdB mutant strains in mouse models of infection. These data indicate a potential role for regulation of flagella in Brucella melitensis via cyclic-di-GMP. PMID:21856843

  12. Flagellar region 3b supports strong expression of integrated DNA and the highest chromosomal integration efficiency of the Escherichia coli flagellar regions

    PubMed Central

    Juhas, Mario; Ajioka, James W

    2015-01-01

    The Gram-negative bacterium Escherichia coli is routinely used as the chassis for a variety of biotechnology and synthetic biology applications. Identification and analysis of reliable chromosomal integration and expression target loci is crucial for E. coli engineering. Chromosomal loci differ significantly in their ability to support integration and expression of the integrated genetic circuits. In this study, we investigate E. coli K12 MG1655 flagellar regions 2 and 3b. Integration of the genetic circuit into seven and nine highly conserved genes of the flagellar regions 2 (motA, motB, flhD, flhE, cheW, cheY and cheZ) and 3b (fliE, F, G, J, K, L, M, P, R), respectively, showed significant variation in their ability to support chromosomal integration and expression of the integrated genetic circuit. While not reducing the growth of the engineered strains, the integrations into all 16 target sites led to the loss of motility. In addition to high expression, the flagellar region 3b supports the highest efficiency of integration of all E. coli K12 MG1655 flagellar regions and is therefore potentially the most suitable for the integration of synthetic genetic circuits. PMID:26074421

  13. Protein Arginine Methyltransferases Interact with IFT Particles and Change Location During Flagellar Growth and Resorption.

    PubMed

    Mizuno, Katsutoshi; Sloboda, Roger D

    2017-03-15

    Changes in protein activity driven by post translational modifications comprise an important mechanism for the control of many cellular processes. Several flagellar proteins are methylated on arginine residues during flagellar resorption; however, the function is not understood. To learn more about the role of protein methylation during flagellar dynamics, we have focused on protein arginine methyltransferases (PRMTs) 1, 3, 5, and 10. These PRMTs localize to the tip of flagella and in a punctate pattern along the length, very similar, but not identical, to that of intraflagellar transport (IFT) components. In addition, we found that PRMTs 1 and 3 are also highly enriched at the base of the flagella, and the basal localization of these PRMTs changes during flagellar regeneration and resorption. Proteins with methyl arginine residues are also enriched at the tip and base of flagella, and their localization also changes during flagellar assembly and disassembly. PRMTs are lost from the flagella of fla10-1 cells, which carry a temperature sensitive mutation in the anterograde motor for IFT. The data define the distribution of specific PRMTs and their target proteins in flagella, and demonstrate that PRMTs are cargo for translocation within flagella by the process of IFT.

  14. Flagellar Synchronization Is a Simple Alternative to Cell Cycle Synchronization for Ciliary and Flagellar Studies

    PubMed Central

    Dutta, Soumita

    2017-01-01

    ABSTRACT The unicellular green alga Chlamydomonas reinhardtii is an ideal model organism for studies of ciliary function and assembly. In assays for biological and biochemical effects of various factors on flagellar structure and function, synchronous culture is advantageous for minimizing variability. Here, we have characterized a method in which 100% synchronization is achieved with respect to flagellar length but not with respect to the cell cycle. The method requires inducing flagellar regeneration by amputation of the entire cell population and limiting regeneration time. This results in a maximally homogeneous distribution of flagellar lengths at 3 h postamputation. We found that time-limiting new protein synthesis during flagellar synchronization limits variability in the unassembled pool of limiting flagellar protein and variability in flagellar length without affecting the range of cell volumes. We also found that long- and short-flagella mutants that regenerate normally require longer and shorter synchronization times, respectively. By minimizing flagellar length variability using a simple method requiring only hours and no changes in media, flagellar synchronization facilitates the detection of small changes in flagellar length resulting from both chemical and genetic perturbations in Chlamydomonas. This method increases our ability to probe the basic biology of ciliary size regulation and related disease etiologies. IMPORTANCE Cilia and flagella are highly conserved antenna-like organelles that found in nearly all mammalian cell types. They perform sensory and motile functions contributing to numerous physiological and developmental processes. Defects in their assembly and function are implicated in a wide range of human diseases ranging from retinal degeneration to cancer. Chlamydomonas reinhardtii is an algal model system for studying mammalian cilium formation and function. Here, we report a simple synchronization method that allows detection of

  15. Role of calmodulin and calcineurin in regulating flagellar motility and wave polarity in Leishmania.

    PubMed

    Mukhopadhyay, Aakash Gautam; Dey, Chinmoy Sankar

    2017-09-07

    We have previously reported the involvement of cyclic AMP in regulating flagellar waveforms in Leishmania. Here, we investigated the roles of calcium, calmodulin, and calcineurin in flagellar motility regulation in L. donovani. Using high-speed videomicroscopy, we show that calcium-independent calmodulin and calcineurin activity is necessary for motility in Leishmania. Inhibition of calmodulin and calcineurin induced ciliary beats interrupting flagellar beating in both live (in vivo) and ATP-reactivated (in vitro) parasites. Our results indicate that signaling mediated by calmodulin and calcineurin operates antagonistically to cAMP signaling in regulating the waveforms of Leishmania flagellum. These two pathways are possibly involved in maintaining the balance between the two waveforms, essential for responding to environmental cues, survival, and infectivity.

  16. bop5 mutations reveal new roles for the IC138 phosphoprotein in the regulation of flagellar motility and asymmetric waveforms

    PubMed Central

    VanderWaal, Kristyn E.; Yamamoto, Ryosuke; Wakabayashi, Ken-ichi; Fox, Laura; Kamiya, Ritsu; Dutcher, Susan K.; Bayly, Phillip V.; Sale, Winfield S.; Porter, Mary E.

    2011-01-01

    I1 dynein, or dynein f, is a highly conserved inner arm isoform that plays a key role in the regulation of flagellar motility. To understand how the IC138 IC/LC subcomplex modulates I1 activity, we characterized the molecular lesions and motility phenotypes of several bop5 alleles. bop5-3, bop5-4, and bop5-5 are null alleles, whereas bop5-6 is an intron mutation that reduces IC138 expression. I1 dynein assembles into the axoneme, but the IC138 IC/LC subcomplex is missing. bop5 strains, like other I1 mutants, swim forward with reduced swimming velocities and display an impaired reversal response during photoshock. Unlike mutants lacking the entire I1 dynein, however, bop5 strains exhibit normal phototaxis. bop5 defects are rescued by transformation with the wild-type IC138 gene. Analysis of flagellar waveforms reveals that loss of the IC138 subcomplex reduces shear amplitude, sliding velocities, and the speed of bend propagation in vivo, consistent with the reduction in microtubule sliding velocities observed in vitro. The results indicate that the IC138 IC/LC subcomplex is necessary to generate an efficient waveform for optimal motility, but it is not essential for phototaxis. These findings have significant implications for the mechanisms by which IC/LC complexes regulate dynein motor activity independent of effects on cargo binding or complex stability. PMID:21697502

  17. Taking control of the flagellar motor

    NASA Astrophysics Data System (ADS)

    Gauthier, Mathieu; Truchon, Dany; Rainville, Simon

    2008-06-01

    Numerous types of bacteria swim in their environment by rotating long helical filaments. At the base of each filament is a tiny rotary motor called the bacterial flagellar motor. A lot is already known about the structure, assembly and function of this splendid molecular machine of nanoscopic dimensions. Nevertheless many fundamental questions remain open and the study of the flagellar motor is a very exciting area of current research. We are developing an in vitro assay to enable studies of the bacterial flagellar motor in precisely controlled conditions and to gain direct access to the inner components of the motor. We partly squeeze a filamentous E. coli bacterium inside a micropipette, leaving a working flagellar motor outside. We then punch a hole through the cell wall at the end of the bacterium located inside the micropipette using a brief train of ultrashort (~60 fs) laser pulses. This enables us to control the rotation of the motor with an external voltage (for at least 15 minutes). In parallel, new methods to monitor the speed of rotation of the motor in the low load (high speed) regime are being developed using various nanoparticules.

  18. RNA-mediated gene activation

    PubMed Central

    Jiao, Alan L; Slack, Frank J

    2014-01-01

    The regulation of gene expression by non-coding RNAs (ncRNAs) has become a new paradigm in biology. RNA-mediated gene silencing pathways have been studied extensively, revealing diverse epigenetic and posttranscriptional mechanisms. In contrast, the roles of ncRNAs in activating gene expression remains poorly understood. In this review, we summarize the current knowledge of gene activation by small RNAs, long non-coding RNAs, and enhancer-derived RNAs, with an emphasis on epigenetic mechanisms. PMID:24185374

  19. Analysis of unstable modes distinguishes mathematical models of flagellar motion

    PubMed Central

    Bayly, P. V.; Wilson, K. S.

    2015-01-01

    The mechanisms underlying the coordinated beating of cilia and flagella remain incompletely understood despite the fundamental importance of these organelles. The axoneme (the cytoskeletal structure of cilia and flagella) consists of microtubule doublets connected by passive and active elements. The motor protein dynein is known to drive active bending, but dynein activity must be regulated to generate oscillatory, propulsive waveforms. Mathematical models of flagellar motion generate quantitative predictions that can be analysed to test hypotheses concerning dynein regulation. One approach has been to seek periodic solutions to the linearized equations of motion. However, models may simultaneously exhibit both periodic and unstable modes. Here, we investigate the emergence and coexistence of unstable and periodic modes in three mathematical models of flagellar motion, each based on a different dynein regulation hypothesis: (i) sliding control; (ii) curvature control and (iii) control by interdoublet separation (the ‘geometric clutch’ (GC)). The unstable modes predicted by each model are used to critically evaluate the underlying hypothesis. In particular, models of flagella with ‘sliding-controlled’ dynein activity admit unstable modes with non-propulsive, retrograde (tip-to-base) propagation, sometimes at the same parameter values that lead to periodic, propulsive modes. In the presence of these retrograde unstable modes, stable or periodic modes have little influence. In contrast, unstable modes of the GC model exhibit switching at the base and propulsive base-to-tip propagation. PMID:25833248

  20. Pseudomonas fluorescens F113 Can Produce a Second Flagellar Apparatus, Which Is Important for Plant Root Colonization

    PubMed Central

    Barahona, Emma; Navazo, Ana; Garrido-Sanz, Daniel; Muriel, Candela; Martínez-Granero, Francisco; Redondo-Nieto, Miguel; Martín, Marta; Rivilla, Rafael

    2016-01-01

    The genomic sequence of Pseudomonas fluorescens F113 has shown the presence of a 41 kb cluster of genes that encode the production of a second flagellar apparatus. Among 2,535 pseudomonads strains with sequenced genomes, these genes are only present in the genomes of F113 and other six strains, all but one belonging to the P. fluorescens cluster of species, in the form of a genetic island. The genes are homologous to the flagellar genes of the soil bacterium Azotobacter vinelandii. Regulation of these genes is mediated by the flhDC master operon, instead of the typical regulation in pseudomonads, which is through fleQ. Under laboratory conditions, F113 does not produce this flagellum and the flhDC operon is not expressed. However, ectopic expression of the flhDC operon is enough for its production, resulting in a hypermotile strain. This flagellum is also produced under laboratory conditions by the kinB and algU mutants. Genetic analysis has shown that kinB strongly represses the expression of the flhDC operon. This operon is activated by the Vfr protein probably in a c-AMP dependent way. The strains producing this second flagellum are all hypermotile and present a tuft of polar flagella instead of the single polar flagellum produced by the wild-type strain. Phenotypic variants isolated from the rhizosphere produce this flagellum and mutation of the genes encoding it, results in a defect in competitive colonization, showing its importance for root colonization. PMID:27713729

  1. Identification of Archaea-specific chemotaxis proteins which interact with the flagellar apparatus

    PubMed Central

    2009-01-01

    Background Archaea share with bacteria the ability to bias their movement towards more favorable locations, a process known as taxis. Two molecular systems drive this process: the motility apparatus and the chemotaxis signal transduction system. The first consists of the flagellum, the flagellar motor, and its switch, which allows cells to reverse the rotation of flagella. The second targets the flagellar motor switch in order to modulate the switching frequency in response to external stimuli. While the signal transduction system is conserved throughout archaea and bacteria, the archaeal flagellar apparatus is different from the bacterial one. The proteins constituting the flagellar motor and its switch in archaea have not yet been identified, and the connection between the bacterial-like chemotaxis signal transduction system and the archaeal motility apparatus is unknown. Results Using protein-protein interaction analysis, we have identified three proteins in Halobacterium salinarum that interact with the chemotaxis (Che) proteins CheY, CheD, and CheC2, as well as the flagella accessory (Fla) proteins FlaCE and FlaD. Two of the proteins belong to the protein family DUF439, the third is a HEAT_PBS family protein. In-frame deletion strains for all three proteins were generated and analyzed as follows: a) photophobic responses were measured by a computer-based cell tracking system b) flagellar rotational bias was determined by dark-field microscopy, and c) chemotactic behavior was analyzed by a swarm plate assay. Strains deleted for the HEAT_PBS protein or one of the DUF439 proteins proved unable to switch the direction of flagellar rotation. In these mutants, flagella rotate only clockwise, resulting in exclusively forward swimming cells that are unable to respond to tactic signals. Deletion of the second DUF439 protein had only minimal effects. HEAT_PBS proteins could be identified in the chemotaxis gene regions of all motile haloarchaea sequenced so far, but not

  2. Modulation of Chlamydomonas reinhardtii flagellar motility by redox poise

    PubMed Central

    Wakabayashi, Ken-ichi; King, Stephen M.

    2006-01-01

    Redox-based regulatory systems are essential for many cellular activities. Chlamydomonas reinhardtii exhibits alterations in motile behavior in response to different light conditions (photokinesis). We hypothesized that photokinesis is signaled by variations in cytoplasmic redox poise resulting from changes in chloroplast activity. We found that this effect requires photosystem I, which generates reduced NADPH. We also observed that photokinetic changes in beat frequency and duration of the photophobic response could be obtained by altering oxidative/reductive stress. Analysis of reactivated cell models revealed that this redox poise effect is mediated through the outer dynein arms (ODAs). Although the global redox state of the thioredoxin-related ODA light chains LC3 and LC5 and the redox-sensitive Ca2+-binding subunit of the docking complex DC3 did not change upon light/dark transitions, we did observe significant alterations in their interactions with other flagellar components via mixed disulfides. These data indicate that redox poise directly affects ODAs and suggest that it may act in the control of flagellar motility. PMID:16754958

  3. Silencing of a putative inner arm dynein heavy chain results in flagellar immotility in Trypanosoma brucei

    PubMed Central

    Springer, Amy L.; Bruhn, David F.; Kinzel, Kathryn W.; Rosenthal, Noël F.; Zukas, Randi; Klingbeil, Michele M.

    2010-01-01

    The Trypanosoma brucei flagellum controls motility and is crucial for cell polarity and division. Unique features of trypanosome motility suggest that flagellar beat regulation in this organism is unusual and worthy of study. The flagellar axoneme, required for motility, has a structure that is highly conserved among eukaryotes. Of the several dyneins in the axonemal inner arm complex, dynein f is thought to control flagellar waveform shape. A T. brucei gene predicted to encode the dynein f alpha heavy chain, TbDNAH10, was silenced using RNA interference in procyclic T. brucei cells. This resulted in immotile flagella, showing no movement except for occasional slight twitches at the tips. Cell growth slowed dramatically and cells were found in large clusters. Microscopic analysis of silenced cultures showed many cells with detached flagella, sometimes entangled between multiple cells. DAPI staining showed an increased frequency of mis-positioned kinetoplasts and multinucleate cells, suggesting that these cells experience disruption at an early cell cycle stage, probably secondary to the motility defect. TEM images showed apparently normal axonemes and no discernable defects in inner arm structure. This study demonstrates use of RNAi as an effective method to study very large genes such as dynein heavy chains (HCs), and the immotility phenotype of these dynein knockdowns suggests that an intact inner arm is necessary for flagellar beating in T. brucei. Since analogous mutants in Chlamydomonas reinhardtii retain motility, this phenotype likely reflects differences in requirements for motility and/or dynein assembly between the two organisms and these comparative studies will help elucidate the mechanisms of flagellar beat regulation. PMID:20888370

  4. Purification, crystallization and preliminary X-ray analysis of FliT, a bacterial flagellar substrate-specific export chaperone

    PubMed Central

    Kinoshita, Miki; Yamane, Midori; Matsunami, Hideyuki; Minamino, Tohru; Namba, Keiichi; Imada, Katsumi

    2009-01-01

    The assembly process of the bacterial flagellum is coupled to flagellar gene expression. FliT acts not only as a flagellar type III substrate-specific export chaperone for the filament-capping protein FliD but also as a negative regulator that suppresses flagellar gene expression through its specific interaction with the master regulator FlhD4C2 complex. In this study, FliT of Salmonella enterica serovar Typhimurium was expressed, purified and crystallized. Crystals of SeMet FliT were obtained by the sitting-drop vapour-diffusion technique with potassium/sodium tartrate as the precipitant. The crystals grew in the trigonal space group P3121 or P3221 and diffracted to 3.2 Å resolution. The anomalous difference Patterson map of the SeMet FliT crystal showed significant peaks in its Harker sections, indicating the usefulness of the derivative data for structure determination. PMID:19652350

  5. Proposed model for the flagellar rotary motor.

    PubMed

    Mitsui, Toshio; Ohshima, Hiroyuki

    2005-11-25

    Flagellated bacteria swim by rotating helical filaments driven by motors embedded in the cell wall and cytoplasmic membrane. A model is proposed to explain the mechanism of the motor. The protons passing through the channels induce a strong electric field in Mot molecules. This field originates an impulse force to cause the flagellar rotation if the following conditions are fulfilled: (a) Mot molecules have a spontaneous electric polarization. (b) The lipid bilayers are viscoelastic. (c) There is a delay of deformation in response to stress in Mot molecules. The conclusions driven from the model are in agreement with the following experimental observations, denoting the flagellar rotation velocity as omega. (1) The torque is practically constant independent of omega from 0 to a critical value omega(cr) and then decreases sharply. (2) When omega is smaller than omega(cr), the torque varies little with temperature. (3) The critical velocity omega(cr) shifts to lower speed at lower temperatures. (4) Where omega is larger than omega(cr), declining of the torque steepens at lower temperatures. (5) When omega is smaller than omega(cr), one revolution of the flagellar rotation consists of a constant number of steps. (6) When omega is smaller than omega(cr), omega is proportional to the transmembrane potential difference. (7) The stator produces constant torque even when the stator is rotated relative to the rotor by external forces. (8) How the flagellar rotation velocity changes when the direction of the proton passage is reversed. (9) The motor has a switch that reverses the sense of the flagelllar rotation with the same absolute value of torque.

  6. Steps in the Bacterial Flagellar Motor

    PubMed Central

    Mora, Thierry; Yu, Howard; Sowa, Yoshiyuki; Wingreen, Ned S.

    2009-01-01

    The bacterial flagellar motor is a highly efficient rotary machine used by many bacteria to propel themselves. It has recently been shown that at low speeds its rotation proceeds in steps. Here we propose a simple physical model, based on the storage of energy in protein springs, that accounts for this stepping behavior as a random walk in a tilted corrugated potential that combines torque and contact forces. We argue that the absolute angular position of the rotor is crucial for understanding step properties and show this hypothesis to be consistent with the available data, in particular the observation that backward steps are smaller on average than forward steps. We also predict a sublinear speed versus torque relationship for fixed load at low torque, and a peak in rotor diffusion as a function of torque. Our model provides a comprehensive framework for understanding and analyzing stepping behavior in the bacterial flagellar motor and proposes novel, testable predictions. More broadly, the storage of energy in protein springs by the flagellar motor may provide useful general insights into the design of highly efficient molecular machines. PMID:19851449

  7. Studies on flagellar shortening in Chlamydomonas reinhardtii

    SciTech Connect

    Cherniack, J.

    1985-01-01

    Flagellar shortening of Chlamydomonas reinhardtii was promoted by sodium chloride, pyrophosphate (sodium, potassium and ammonium salts), EDTA and EGTA, succinate, citrate and oxalate (sodium salts), caffeine and aminophylline. Removal of calcium from the medium potentiated the effects of these agents in inducing shortening. Investigations of the release of phosphorylated compounds to the medium during pyrophosphate-induced flagellar shortening of cells pre-labelled with /sup 32/P, revealed an as yet unidentified /sup 32/P-labelled compound with distinct chromatographic properties. Chromatography and electrophoresis indicates that it is a small, highly polar molecule with a high charge to mass ratio, containing thermo- and acid-labile phosphate linkages. Investigations showed of the release of /sup 35/S-labelled protein to the medium from cells pre-labelled with /sup 35/S-sulfate showed that flagellated cells released two prominent polypeptides which comigrated with ..cap alpha..- and ..beta..-flagellar tubulin on SDS polyacrylamide gel electrophoresis, while deflagellated cells did not.

  8. Shear stress transmission model for the flagellar rotary motor.

    PubMed

    Mitsui, Toshio; Ohshima, Hiroyuki

    2008-09-01

    Most bacteria that swim are propelled by flagellar filaments, which are driven by a rotary motor powered by proton flux. The mechanism of the flagellar motor is discussed by reforming the model proposed by the present authors in 2005. It is shown that the mean strength of Coulomb field produced by a proton passing the channel is very strong in the Mot assembly so that the Mot assembly can be a shear force generator and induce the flagellar rotation. The model gives clear calculation results in agreement with experimental observations, e g., for the characteristic torque-velocity relationship of the flagellar rotation.

  9. Monitoring bacterial chemotaxis by using bioluminescence resonance energy transfer: Absence of feedback from the flagellar motors

    PubMed Central

    Shimizu, Thomas S.; Delalez, Nicolas; Pichler, Klemens; Berg, Howard C.

    2006-01-01

    We looked for a feedback system in Escherichia coli that might sense the rotational bias of flagellar motors and regulate the activity of the chemotaxis receptor kinase. Our search was based on the assumption that any machinery that senses rotational bias will be perturbed if flagellar rotation stops. We monitored the activity of the kinase in swimming cells by bioluminescence resonance energy transfer (BRET) between Renilla luciferase fused to the phosphatase, CheZ, and yellow fluorescent protein fused to the response regulator, CheY. Then we jammed the flagellar motors by adding an antifilament antibody that crosslinks adjacent filaments in flagellar bundles. At steady state, the rate of phosphorylation of CheY is equal to the rate of dephosphorylation of CheY-P, which is proportional to the degree of association between CheZ and CheY-P, the quantity sensed by BRET. No changes were observed, even upon addition of an amount of antibody that stopped the swimming of >95% of cells within a few seconds. So, the kinase does not appear to be sensitive to motor output. The BRET technique is complementary to one based on FRET, described previously. Its reliability was confirmed by measurements of the response of cells to the addition of attractants. PMID:16452163

  10. A quantitative model of the switch cycle of an archaeal flagellar motor and its sensory control.

    PubMed

    Nutsch, Torsten; Oesterhelt, Dieter; Gilles, Ernst Dieter; Marwan, Wolfgang

    2005-10-01

    By reverse-engineering we have detected eight kinetic phases of the symmetric switch cycle of the Halobacterium salinarum flagellar motor assembly and identified those steps in the switch cycle that are controlled by sensory rhodopsins during phototaxis. Upon switching the rotational sense, the flagellar motor assembly passes through a stop state from which all subunits synchronously resume rotation in the reverse direction. The assembly then synchronously proceeds through three subsequent functional states of the switch: Refractory, Competent, and Active, from which the rotational sense is switched again. Sensory control of the symmetric switch cycle occurs at two steps in each rotational sense by inversely regulating the probabilities for a change from the Refractory to the Competent and from Competent to the Active rotational mode. We provide a mathematical model for flagellar motor switching and its sensory control, which is able to explain all tested experimental results on spontaneous and light-controlled motor switching, and give a mechanistic explanation based on synchronous conformational transitions of the subunits of the switch complex after reversible dissociation and binding of a response regulator (CheYP). We conclude that the kinetic mechanism of flagellar motor switching and its sensory control is fundamentally different in the archaeon H. salinarum and the bacterium Escherichia coli.

  11. Complex Interplay between FleQ, Cyclic Diguanylate and Multiple σ Factors Coordinately Regulates Flagellar Motility and Biofilm Development in Pseudomonas putida

    PubMed Central

    Jiménez-Fernández, Alicia; López-Sánchez, Aroa; Jiménez-Díaz, Lorena; Navarrete, Blanca; Calero, Patricia; Platero, Ana Isabel

    2016-01-01

    Most bacteria alternate between a free living planktonic lifestyle and the formation of structured surface-associated communities named biofilms. The transition between these two lifestyles requires a precise and timely regulation of the factors involved in each of the stages that has been likened to a developmental process. Here we characterize the involvement of the transcriptional regulator FleQ and the second messenger cyclic diguanylate in the coordinate regulation of multiple functions related to motility and surface colonization in Pseudomonas putida. Disruption of fleQ caused strong defects in flagellar motility, biofilm formation and surface attachment, and the ability of this mutation to suppress multiple biofilm-related phenotypes associated to cyclic diguanylate overproduction suggests that FleQ mediates cyclic diguanylate signaling critical to biofilm growth. We have constructed a library containing 94 promoters potentially involved in motility and biofilm development fused to gfp and lacZ, screened this library for FleQ and cyclic diguanylate regulation, and assessed the involvement of alternative σ factors σN and FliA in the transcription of FleQ-regulated promoters. Our results suggest a dual mode of action for FleQ. Low cyclic diguanylate levels favor FleQ interaction with σN-dependent promoters to activate the flagellar cascade, encompassing the flagellar cluster and additional genes involved in cyclic diguanylate metabolism, signal transduction and gene regulation. On the other hand, characterization of the FleQ-regulated σN- and FliA-independent PlapA and PbcsD promoters revealed two disparate regulatory mechanisms leading to a similar outcome: the synthesis of biofilm matrix components in response to increased cyclic diguanylate levels. PMID:27636892

  12. Structural Changes of the Paraflagellar Rod during Flagellar Beating in Trypanosoma cruzi

    PubMed Central

    Rocha, Gustavo Miranda; Teixeira, Dirceu Esdras; Miranda, Kildare; Weissmüller, Gilberto; Bisch, Paulo Mascarello; de Souza, Wanderley

    2010-01-01

    Background Trypanosoma cruzi, the agent of Chagas disease, is a protozoan member of the Kinetoplastidae family characterized for the presence of specific and unique structures that are involved in different cell activities. One of them is the paraflagellar rod (PFR), a complex array of filaments connected to the flagellar axoneme. Although the function played by the PFR is not well established, it has been shown that silencing of the synthesis of its major proteins by either knockout of RNAi impairs and/or modifies the flagellar motility. Methodology/Principal Findings Here, we present results obtained by atomic force microscopy (AFM) and transmission electron microscopy (TEM) of replicas of quick-frozen, freeze-fractured, deep-etched and rotary-replicated cells to obtain detailed information of the PFR structures in regions of the flagellum in straight and in bent state. The images obtained show that the PFR is not a fixed and static structure. The pattern of organization of the PFR filament network differs between regions of the flagellum in a straight state and those in a bent state. Measurements of the distances between the PFR filaments and the filaments that connect the PFR to the axoneme as well as of the angles between the intercrossed filaments supported this idea. Conclusions/Significance Graphic computation based on the information obtained allowed the proposal of an animated model for the PFR structure during flagellar beating and provided a new way of observing PFR filaments during flagellar beating. PMID:20613980

  13. The role of the dynein light intermediate chain in retrograde IFT and flagellar function in Chlamydomonas.

    PubMed

    Reck, Jaimee; Schauer, Alexandria M; VanderWaal Mills, Kristyn; Bower, Raqual; Tritschler, Douglas; Perrone, Catherine A; Porter, Mary E

    2016-08-01

    The assembly of cilia and flagella depends on the activity of two microtubule motor complexes, kinesin-2 and dynein-2/1b, but the specific functions of the different subunits are poorly defined. Here we analyze Chlamydomonas strains expressing different amounts of the dynein 1b light intermediate chain (D1bLIC). Disruption of D1bLIC alters the stability of the dynein 1b complex and reduces both the frequency and velocity of retrograde intraflagellar transport (IFT), but it does not eliminate retrograde IFT. Flagellar assembly, motility, gliding, and mating are altered in a dose-dependent manner. iTRAQ-based proteomics identifies a small subset of proteins that are significantly reduced or elevated in d1blic flagella. Transformation with D1bLIC-GFP rescues the mutant phenotypes, and D1bLIC-GFP assembles into the dynein 1b complex at wild-type levels. D1bLIC-GFP is transported with anterograde IFT particles to the flagellar tip, dissociates into smaller particles, and begins processive retrograde IFT in <2 s. These studies demonstrate the role of D1bLIC in facilitating the recycling of IFT subunits and other proteins, identify new components potentially involved in the regulation of IFT, flagellar assembly, and flagellar signaling, and provide insight into the role of D1bLIC and retrograde IFT in other organisms.

  14. The role of the dynein light intermediate chain in retrograde IFT and flagellar function in Chlamydomonas

    PubMed Central

    Reck, Jaimee; Schauer, Alexandria M.; VanderWaal Mills, Kristyn; Bower, Raqual; Tritschler, Douglas; Perrone, Catherine A.; Porter, Mary E.

    2016-01-01

    The assembly of cilia and flagella depends on the activity of two microtubule motor complexes, kinesin-2 and dynein-2/1b, but the specific functions of the different subunits are poorly defined. Here we analyze Chlamydomonas strains expressing different amounts of the dynein 1b light intermediate chain (D1bLIC). Disruption of D1bLIC alters the stability of the dynein 1b complex and reduces both the frequency and velocity of retrograde intraflagellar transport (IFT), but it does not eliminate retrograde IFT. Flagellar assembly, motility, gliding, and mating are altered in a dose-dependent manner. iTRAQ-based proteomics identifies a small subset of proteins that are significantly reduced or elevated in d1blic flagella. Transformation with D1bLIC-GFP rescues the mutant phenotypes, and D1bLIC-GFP assembles into the dynein 1b complex at wild-type levels. D1bLIC-GFP is transported with anterograde IFT particles to the flagellar tip, dissociates into smaller particles, and begins processive retrograde IFT in <2 s. These studies demonstrate the role of D1bLIC in facilitating the recycling of IFT subunits and other proteins, identify new components potentially involved in the regulation of IFT, flagellar assembly, and flagellar signaling, and provide insight into the role of D1bLIC and retrograde IFT in other organisms. PMID:27251063

  15. Probing the role of IFT particle complex A and B in flagellar entry and exit of IFT-dynein in Chlamydomonas.

    PubMed

    Williamson, Shana M; Silva, David A; Richey, Elizabeth; Qin, Hongmin

    2012-07-01

    Mediating the transport of flagellar precursors and removal of turnover products, intraflagellar transport (IFT) is required for flagella assembly and maintenance. The IFT apparatus is composed of the anterograde IFT motor kinesin II, the retrograde IFT motor IFT-dynein, and IFT particles containing two complexes, A and B. In order to have a balanced two-way transportation, IFT-dynein has to be carried into flagella and transported to the flagellar tip by kinesin II, where it is activated to drive the retrograde IFT back to the flagellar base. In this study, we investigated the role of complex A and complex B in the flagellar entry and exit of IFT-dynein. We showed that regardless of the amount of complex A, IFT-dynein accumulated proportionally to the amount of complex B in the flagella of fla15/ift144 and fla17-1/ift139, two complex A temperature-sensitive mutants. Complex A was depleted from both cellular and flagellar compartments in fla15/ift144 mutant. However, in fla17-1/ift139 mutant, the flagellar level of complex A was at the wild-type level, which was in radical contrast to the significantly reduced cellular amount of complex A. These results support that complex A is not required for the flagellar entry of IFT-dynein, but might be essential for the lagellar exit of IFT-dynein. Additionally, we confirmed the essential role of IFT172, a complex B subunit, in the flagellar entry of IFT-dynein. These results indicate that complexes A and B play complementary but distinct roles for IFT-dynein, with complex B carrying IFT-dynein into the flagella while complex A mediates the flagellar exit of IFT-dynein.

  16. A high-throughput screening assay for inhibitors of bacterial motility identifies a novel inhibitor of the Na+-driven flagellar motor and virulence gene expression in Vibrio cholerae.

    PubMed

    Rasmussen, Lynn; White, E Lucile; Pathak, Ashish; Ayala, Julio C; Wang, Hongxia; Wu, Jian-He; Benitez, Jorge A; Silva, Anisia J

    2011-09-01

    Numerous bacterial pathogens, particularly those that colonize fast-flow areas in the bladder and gastrointestinal tract, require motility to establish infection and spread beyond the initially colonized tissue. Vibrio cholerae strains of serogroups O1 and O139, the causative agents of the diarrheal illness cholera, express a single polar flagellum powered by sodium motive force and require motility to colonize and spread along the small intestine. Therefore, motility may be an attractive target for small molecules that can prevent and/or block the infective process. In this study, we describe a high-throughput screening (HTS) assay to identify small molecules that selectively inhibit bacterial motility. The HTS assay was used to screen an ∼8,000-compound structurally diverse chemical library for inhibitors of V. cholerae motility. The screen identified a group of quinazoline-2,4-diamino analogs that completely suppressed motility without affecting the growth rate in broth. A further study on the effects of one analog, designated Q24DA, showed that it induces a flagellated but nonmotile (Mot(-)) phenotype and is specific for the Na(+)-driven flagellar motor of pathogenic Vibrio species. A mutation conferring phenamil-resistant motility did not eliminate inhibition of motility by Q24DA. Q24DA diminished the expression of cholera toxin and toxin-coregulated pilus as well as biofilm formation and fluid secretion in the rabbit ileal loop model. Furthermore, treatment of V. cholerae with Q24DA impacted additional phenotypes linked to Na(+) bioenergetics, such as the function of the primary Na(+) pump, Nqr, and susceptibility to fluoroquinolones. The above results clearly show that the described HTS assay is capable of identifying small molecules that specifically block bacterial motility. New inhibitors such as Q24DA may be instrumental in probing the molecular architecture of the Na(+)-driven polar flagellar motor and in studying the role of motility in the

  17. A High-Throughput Screening Assay for Inhibitors of Bacterial Motility Identifies a Novel Inhibitor of the Na+-Driven Flagellar Motor and Virulence Gene Expression in Vibrio cholerae▿†

    PubMed Central

    Rasmussen, Lynn; White, E. Lucile; Pathak, Ashish; Ayala, Julio C.; Wang, Hongxia; Wu, Jian-He; Benitez, Jorge A.; Silva, Anisia J.

    2011-01-01

    Numerous bacterial pathogens, particularly those that colonize fast-flow areas in the bladder and gastrointestinal tract, require motility to establish infection and spread beyond the initially colonized tissue. Vibrio cholerae strains of serogroups O1 and O139, the causative agents of the diarrheal illness cholera, express a single polar flagellum powered by sodium motive force and require motility to colonize and spread along the small intestine. Therefore, motility may be an attractive target for small molecules that can prevent and/or block the infective process. In this study, we describe a high-throughput screening (HTS) assay to identify small molecules that selectively inhibit bacterial motility. The HTS assay was used to screen an ∼8,000-compound structurally diverse chemical library for inhibitors of V. cholerae motility. The screen identified a group of quinazoline-2,4-diamino analogs that completely suppressed motility without affecting the growth rate in broth. A further study on the effects of one analog, designated Q24DA, showed that it induces a flagellated but nonmotile (Mot−) phenotype and is specific for the Na+-driven flagellar motor of pathogenic Vibrio species. A mutation conferring phenamil-resistant motility did not eliminate inhibition of motility by Q24DA. Q24DA diminished the expression of cholera toxin and toxin-coregulated pilus as well as biofilm formation and fluid secretion in the rabbit ileal loop model. Furthermore, treatment of V. cholerae with Q24DA impacted additional phenotypes linked to Na+ bioenergetics, such as the function of the primary Na+ pump, Nqr, and susceptibility to fluoroquinolones. The above results clearly show that the described HTS assay is capable of identifying small molecules that specifically block bacterial motility. New inhibitors such as Q24DA may be instrumental in probing the molecular architecture of the Na+-driven polar flagellar motor and in studying the role of motility in the expression of

  18. Flagellar rotation in the archaeon Halobacterium salinarum depends on ATP.

    PubMed

    Streif, Stefan; Staudinger, Wilfried Franz; Marwan, Wolfgang; Oesterhelt, Dieter

    2008-12-05

    Halobacterium salinarum swims with the help of a polarly inserted flagellar bundle. In energized cells, the flagellar motors rotate continuously, occasionally switching the rotational sense. Starving cells become immotile as the energy level drops. Presumably, there is a threshold of energy required for flagellar rotation. When starved, immotile cells are energized by exposure to light, the speed of flagellar rotation increases gradually to its steady state over several minutes. Since the light-driven proton pump bacteriorhodopsin energizes the cell membrane to the maximal level within a fraction of a second, the delay in reaching the maximal swimming speed suggests that the halobacterial flagellar motor may not be driven directly by proton motive force. Swimming cells, which obtain their energy exclusively through light-driven proton pumping, become immotile within 20 min when treated with N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of the proton translocating ATP synthase. However, flagellar motility in DCCD-treated cells can be restored by the addition of L-arginine, which serves as a fermentative energy source and restores the cytoplasmic ATP level in the presence of DCCD. This suggests that flagellar motor rotation depends on ATP, and this is confirmed by the observation that motility is increased strongly by L-arginine at zero proton motive force levels. The flagellar motor may be driven either by ATP directly or by an ATP-generated ion gradient that is not coupled directly to the proton gradient or the proton motive force of the cell.

  19. Simultaneous measurement of bacterial flagellar rotation rate and swimming speed.

    PubMed Central

    Magariyama, Y; Sugiyama, S; Muramoto, K; Kawagishi, I; Imae, Y; Kudo, S

    1995-01-01

    Swimming speeds and flagellar rotation rates of individual free-swimming Vibrio alginolyticus cells were measured simultaneously by laser dark-field microscopy at 25, 30, and 35 degrees C. A roughly linear relation between swimming speed and flagellar rotation rate was observed. The ratio of swimming speed to flagellar rotation rate was 0.113 microns, which indicated that a cell progressed by 7% of pitch of flagellar helix during one flagellar rotation. At each temperature, however, swimming speed had a tendency to saturate at high flagellar rotation rate. That is, the cell with a faster-rotating flagellum did not always swim faster. To analyze the bacterial motion, we proposed a model in which the torque characteristics of the flagellar motor were considered. The model could be analytically solved, and it qualitatively explained the experimental results. The discrepancy between the experimental and the calculated ratios of swimming speed to flagellar rotation rate was about 20%. The apparent saturation in swimming speed was considered to be caused by shorter flagella that rotated faster but produced less propelling force. Images FIGURE 1 FIGURE 4 PMID:8580359

  20. The Bacterial Flagellar Type III Export Gate Complex Is a Dual Fuel Engine That Can Use Both H+ and Na+ for Flagellar Protein Export

    PubMed Central

    Minamino, Tohru; Morimoto, Yusuke V.; Hara, Noritaka; Aldridge, Phillip D.; Namba, Keiichi

    2016-01-01

    The bacterial flagellar type III export apparatus utilizes ATP and proton motive force (PMF) to transport flagellar proteins to the distal end of the growing flagellar structure for self-assembly. The transmembrane export gate complex is a H+–protein antiporter, of which activity is greatly augmented by an associated cytoplasmic ATPase complex. Here, we report that the export gate complex can use sodium motive force (SMF) in addition to PMF across the cytoplasmic membrane to drive protein export. Protein export was considerably reduced in the absence of the ATPase complex and a pH gradient across the membrane, but Na+ increased it dramatically. Phenamil, a blocker of Na+ translocation, inhibited protein export. Overexpression of FlhA increased the intracellular Na+ concentration in the presence of 100 mM NaCl but not in its absence, suggesting that FlhA acts as a Na+ channel. In wild-type cells, however, neither Na+ nor phenamil affected protein export, indicating that the Na+ channel activity of FlhA is suppressed by the ATPase complex. We propose that the export gate by itself is a dual fuel engine that uses both PMF and SMF for protein export and that the ATPase complex switches this dual fuel engine into a PMF-driven export machinery to become much more robust against environmental changes in external pH and Na+ concentration. PMID:26943926

  1. The Bacterial Flagellar Type III Export Gate Complex Is a Dual Fuel Engine That Can Use Both H+ and Na+ for Flagellar Protein Export.

    PubMed

    Minamino, Tohru; Morimoto, Yusuke V; Hara, Noritaka; Aldridge, Phillip D; Namba, Keiichi

    2016-03-01

    The bacterial flagellar type III export apparatus utilizes ATP and proton motive force (PMF) to transport flagellar proteins to the distal end of the growing flagellar structure for self-assembly. The transmembrane export gate complex is a H+-protein antiporter, of which activity is greatly augmented by an associated cytoplasmic ATPase complex. Here, we report that the export gate complex can use sodium motive force (SMF) in addition to PMF across the cytoplasmic membrane to drive protein export. Protein export was considerably reduced in the absence of the ATPase complex and a pH gradient across the membrane, but Na+ increased it dramatically. Phenamil, a blocker of Na+ translocation, inhibited protein export. Overexpression of FlhA increased the intracellular Na+ concentration in the presence of 100 mM NaCl but not in its absence, suggesting that FlhA acts as a Na+ channel. In wild-type cells, however, neither Na+ nor phenamil affected protein export, indicating that the Na+ channel activity of FlhA is suppressed by the ATPase complex. We propose that the export gate by itself is a dual fuel engine that uses both PMF and SMF for protein export and that the ATPase complex switches this dual fuel engine into a PMF-driven export machinery to become much more robust against environmental changes in external pH and Na+ concentration.

  2. Genome sequence of Xanthomonas fuscans subsp. fuscans strain 4834-R reveals that flagellar motility is not a general feature of xanthomonads

    PubMed Central

    2013-01-01

    Background Xanthomonads are plant-associated bacteria responsible for diseases on economically important crops. Xanthomonas fuscans subsp. fuscans (Xff) is one of the causal agents of common bacterial blight of bean. In this study, the complete genome sequence of strain Xff 4834-R was determined and compared to other Xanthomonas genome sequences. Results Comparative genomics analyses revealed core characteristics shared between Xff 4834-R and other xanthomonads including chemotaxis elements, two-component systems, TonB-dependent transporters, secretion systems (from T1SS to T6SS) and multiple effectors. For instance a repertoire of 29 Type 3 Effectors (T3Es) with two Transcription Activator-Like Effectors was predicted. Mobile elements were associated with major modifications in the genome structure and gene content in comparison to other Xanthomonas genomes. Notably, a deletion of 33 kbp affects flagellum biosynthesis in Xff 4834-R. The presence of a complete flagellar cluster was assessed in a collection of more than 300 strains representing different species and pathovars of Xanthomonas. Five percent of the tested strains presented a deletion in the flagellar cluster and were non-motile. Moreover, half of the Xff strains isolated from the same epidemic than 4834-R was non-motile and this ratio was conserved in the strains colonizing the next bean seed generations. Conclusions This work describes the first genome of a Xanthomonas strain pathogenic on bean and reports the existence of non-motile xanthomonads belonging to different species and pathovars. Isolation of such Xff variants from a natural epidemic may suggest that flagellar motility is not a key function for in planta fitness. PMID:24195767

  3. Instability of hooks during bacterial flagellar swimming

    NASA Astrophysics Data System (ADS)

    Jabbarzadeh, Mehdi; Fu, Henry C.; Henry Fu Team

    2016-11-01

    In bacteria, a flexible hook transmits torque from the rotary motor at the cell body to the flagellum. Previously, the hook has been modeled as a Kirchhoff rod between the cell body and rotating flagellum. To study effects of the hook's flexibility on the bacteria's swimming speed and trajectory for wide range hook stiffnesses and flagellum configurations, we develop an efficient simplified spring model for the hook by linearizing the Kirchhoff rod. We treat the hydrodynamics of the cell body and helical flagellum using resistance matrices calculated by the method of regularized Stokeslets. We investigate flagellar and swimming dynamics for a range of hook flexibilities and flagellar orientations relative to the cell body and compare the results to models without hook flexibility. We investigate in detail parameters corresponding to E. coli and Vibrio alginolyticus. Generally, the flagellum changes orientation relative to the cell body, undergoing an orbit with the period of the motor rotation. We find that as the hook stiffness decreases, steady-state orbits of the flagellum first become unstable before the hook buckles, which may suggest a new mechanism of flick initiation in run-reverse-flick motility. We also find that for some parameter ranges, there are multiple stable steady state orbits, which may have implications for the tumbling and turning of bacteria.

  4. On Flagellar Structure in Certain Flagellates

    PubMed Central

    Gibbons, I. R.; Grimstone, A. V.

    1960-01-01

    This paper describes the structure of the flagella, basal bodies, and some of the associated fibre systems in three genera of complex flagellates, Trichonympha, Pseudotrichonympha, and Holomastigotoides. Three groups of longitudinal fibres occur in a flagellum: two central and nine outer fibres such as have been repeatedly described in other material, and an additional set of nine smaller secondary fibres not previously identified as such. Each central fibre shows a helical substructure; the pair of them are enveloped in a common sheath. Each outer fibre is a doublet with one subfibre bearing projections—called arms—that extend toward the adjacent outer fibre. The basal body is formed by a cylinder of nine triplet outer fibres. Two subfibres of each triplet continue into the flagellum and constitute the doublets. The third subfibre terminates at the transition of basal body to flagellum, possibly giving rise to the nine radial transitional fibres that seem to attach the end of the basal body to the surface of the organism. The central and secondary flagellar fibres are not present in the lumen of the basal body, but other complex structures occur there. The form of these intraluminal structures differs from genus to genus. The flagellar unit is highly asymmetrical. All the flagella examined have possessed the same one of the two possible enantiomorphic forms. At least two systems of fibres are associated with the basal bodies of all three genera. PMID:13827900

  5. SEROLOGICAL SIMILARITY OF FLAGELLAR AND MITOTIC MICROTUBULES

    PubMed Central

    Fulton, Chandler; Kane, R. E.; Stephens, R. E.

    1971-01-01

    An antiserum to flagellar axonemes from sperm of Arbacia punctulata contains antibodies which react both with intact flagellar outer fibers and with purified tubulin from the outer fibers. Immunodiffusion tests indicate the presence of similar antigenic determinants on outer-fiber tubulins from sperm flagella of five species of sea urchins and a sand dollar, but not a starfish. The antibodies also react with extracts containing tubulins from different classes of microtubules, including central-pair fibers and both A- and B-subfibers from outer fibers of sperm flagella, an extract from unfertilized eggs, mitotic apparatuses from first cleavage embryos, and cilia from later embryos. Though most tubulins tested share similar antigenic determinants, some clear differences have been detected, even, in Pseudoboletia indiana, between the outer-fiber tubulins of sperm flagella and blastular cilia. Though tubulins are "actin-like" proteins, antitubulin serum does not react with actin from sea urchin lantern muscle. On the basis of these observations, we suggest that various echinoid microtubules are built of similar, but not identical, tubulins. PMID:4106543

  6. A study of bacterial flagellar bundling.

    PubMed

    Flores, Heather; Lobaton, Edgar; Méndez-Diez, Stefan; Tlupova, Svetlana; Cortez, Ricardo

    2005-01-01

    Certain bacteria, such as Escherichia coli (E. coli) and Salmonella typhimurium (S. typhimurium), use multiple flagella often concentrated at one end of their bodies to induce locomotion. Each flagellum is formed in a left-handed helix and has a motor at the base that rotates the flagellum in a corkscrew motion. We present a computational model of the flagellar motion and their hydrodynamic interaction. The model is based on the equations of Stokes flow to describe the fluid motion. The elasticity of the flagella is modeled with a network of elastic springs while the motor is represented by a torque at the base of each flagellum. The fluid velocity due to the forces is described by regularized Stokeslets and the velocity due to the torques by the associated regularized rotlets. Their expressions are derived. The model is used to analyze the swimming motion of a single flagellum and of a group of three flagella in close proximity to one another. When all flagellar motors rotate counterclockwise, the hydrodynamic interaction can lead to bundling. We present an analysis of the flow surrounding the flagella. When at least one of the motors changes its direction of rotation, the same initial conditions lead to a tumbling behavior characterized by the separation of the flagella, changes in their orientation, and no net swimming motion. The analysis of the flow provides some intuition for these processes.

  7. Analysis of flagellar phosphoproteins from Chlamydomonas reinhardtii.

    PubMed

    Boesger, Jens; Wagner, Volker; Weisheit, Wolfram; Mittag, Maria

    2009-07-01

    Cilia and flagella are cell organelles that are highly conserved throughout evolution. For many years, the green biflagellate alga Chlamydomonas reinhardtii has served as a model for examination of the structure and function of its flagella, which are similar to certain mammalian cilia. Proteome analysis revealed the presence of several kinases and protein phosphatases in these organelles. Reversible protein phosphorylation can control ciliary beating, motility, signaling, length, and assembly. Despite the importance of this posttranslational modification, the identities of many ciliary phosphoproteins and knowledge about their in vivo phosphorylation sites are still missing. Here we used immobilized metal affinity chromatography to enrich phosphopeptides from purified flagella and analyzed them by mass spectrometry. One hundred forty-one phosphorylated peptides were identified, belonging to 32 flagellar proteins. Thereby, 126 in vivo phosphorylation sites were determined. The flagellar phosphoproteome includes different structural and motor proteins, kinases, proteins with protein interaction domains, and many proteins whose functions are still unknown. In several cases, a dynamic phosphorylation pattern and clustering of phosphorylation sites were found, indicating a complex physiological status and specific control by reversible protein phosphorylation in the flagellum.

  8. Flagellar synchronization through direct hydrodynamic interactions.

    PubMed

    Brumley, Douglas R; Wan, Kirsty Y; Polin, Marco; Goldstein, Raymond E

    2014-07-29

    Flows generated by ensembles of flagella are crucial to development, motility and sensing, but the mechanisms behind this striking coordination remain unclear. We present novel experiments in which two micropipette-held somatic cells of Volvox carteri, with distinct intrinsic beating frequencies, are studied by high-speed imaging as a function of their separation and orientation. Analysis of time series shows that the interflagellar coupling, constrained by lack of connections between cells to be hydrodynamical, exhibits a spatial dependence consistent with theory. At close spacings it produces robust synchrony for thousands of beats, while at increasing separations synchrony is degraded by stochastic processes. Manipulation of the relative flagellar orientation reveals in-phase and antiphase states, consistent with dynamical theories. Flagellar tracking with exquisite precision reveals waveform changes that result from hydrodynamic coupling. This study proves unequivocally that flagella coupled solely through a fluid can achieve robust synchrony despite differences in their intrinsic properties.DOI: http://dx.doi.org/10.7554/eLife.02750.001.

  9. Synthesis, transport, and utilization of specific flagellar proteins during flagellar regeneration in Chlamydomonas

    PubMed Central

    1982-01-01

    We labeled gametes of Chlamydomonas with 10-min pulses of 35SO4(-2) before and at various times after deflagellation, and isolated whole cells and flagella immediately after the pulse. The labeled proteins were separated by one- or two-dimensional gel electrophoresis, and the amount of isotope incorporated into specific proteins was determined. Individual proteins were identified with particular structures by correlating missing axonemal polypeptides with ultrastructural defects in paralyzed mutants, or by polypeptide analysis of flagellar fractions. Synthesis of most flagellar proteins appeared to be coordinately induced after flagellar amputation. The rate of synthesis for most quantified proteins increased at least 4- to 10-fold after deflagellation. The kinetics of synthesis of proteins contained together within a structure (e.g., the radial spoke proteins [RSP] ) were frequently similar; however, the kinetics of synthesis of proteins contained in different structures (e.g., RSP vs. alpha- and beta- tubulins) were different. Most newly synthesized flagellar proteins were rapidly transported into the flagellum with kinetics reflecting the rate of growth of the organelle; exceptions included a central tubule complex protein (CT1) and an actinlike component, both of which appeared to be supplied almost entirely from pre-existing, unlabeled pools. Isotope dilution experiments showed that, for most quantified axonemal proteins, a minimum of 35-40% of the polypeptide chains used in assembling a new axoneme was synthesized during regeneration; these proteins appeared to have predeflagellation pools of approximately the same size relative to their stoichiometries in the axoneme. In contrast, CT1 and the actinlike protein had comparatively large pools. PMID:7118994

  10. Flagellar central pair assembly in Chlamydomonas reinhardtii

    PubMed Central

    2013-01-01

    Background Most motile cilia and flagella have nine outer doublet and two central pair (CP) microtubules. Outer doublet microtubules are continuous with the triplet microtubules of the basal body, are templated by the basal body microtubules, and grow by addition of new subunits to their distal (“plus”) ends. In contrast, CP microtubules are not continuous with basal body microtubules, raising the question of how these microtubules are assembled and how their polarity is established. Methods CP assembly in Chlamydomonas reinhardtii was analyzed by electron microscopy and wide-field and super-resolution immunofluorescence microscopy. To analyze CP assembly independently from flagellar assembly, the CP-deficient katanin mutants pf15 or pf19 were mated to wild-type cells. HA-tagged tubulin and the CP-specific protein hydin were used as markers to analyze de novo CP assembly inside the formerly mutant flagella. Results In regenerating flagella, the CP and its projections assemble near the transition zone soon after the onset of outer doublet elongation. During de novo CP assembly in full-length flagella, the nascent CP was first apparent in a subdistal region of the flagellum. The developing CP replaces a fibrous core that fills the axonemal lumen of CP-deficient flagella. The fibrous core contains proteins normally associated with the C1 CP microtubule and proteins involved in intraflagellar transport (IFT). In flagella of the radial spoke-deficient mutant pf14, two pairs of CPs are frequently present with identical correct polarities. Conclusions The temporal separation of flagellar and CP assembly in dikaryons formed by mating CP-deficient gametes to wild-type gametes revealed that the formation of the CP does not require proximity to the basal body or transition zone, or to the flagellar tip. The observations on pf14 provide further support that the CP self-assembles without a template and eliminate the possibility that CP polarity is established by interaction

  11. Regulation of flagellar motility by the conserved flagellar protein CG34110/Ccdc135/FAP50

    PubMed Central

    Yang, Yong; Cochran, Deborah A.; Gargano, Mary D.; King, Iryna; Samhat, Nayef K.; Burger, Benjain P.; Sabourin, Katherine R.; Hou, Yuqing; Awata, Junya; Parry, David A.D.; Marshall, Wallace F.; Witman, George B.; Lu, Xiangyi

    2011-01-01

    Eukaryotic cilia and flagella are vital sensory and motile organelles. The calcium channel PKD2 mediates sensory perception on cilia and flagella, and defects in this can contribute to ciliopathic diseases. Signaling from Pkd2-dependent Ca2+ rise in the cilium to downstream effectors may require intermediary proteins that are largely unknown. To identify these proteins, we carried out genetic screens for mutations affecting Drosophila melanogaster sperm storage, a process mediated by Drosophila Pkd2. Here we show that a new mutation lost boys (lobo) encodes a conserved flagellar protein CG34110, which corresponds to vertebrate Ccdc135 (E = 6e-78) highly expressed in ciliated respiratory epithelia and sperm, and to FAP50 (E = 1e-28) in the Chlamydomonas reinhardtii flagellar proteome. CG34110 localizes along the fly sperm flagellum. FAP50 is tightly associated with the outer doublet microtubules of the axoneme and appears not to be a component of the central pair, radial spokes, dynein arms, or structures defined by the mbo waveform mutants. Phenotypic analyses indicate that both Pkd2 and lobo specifically affect sperm movement into the female storage receptacle. We hypothesize that the CG34110/Ccdc135/FAP50 family of conserved flagellar proteins functions within the axoneme to mediate Pkd2-dependent processes in the sperm flagellum and other motile cilia. PMID:21289096

  12. Ovarian fluid impacts flagellar beating and biomechanical metrics of sperm between alternative reproductive tactics.

    PubMed

    Butts, Ian A E; Prokopchuk, Galina; Kašpar, Vojtěch; Cosson, Jacky; Pitcher, Trevor E

    2017-06-15

    Alternative reproductive tactics (ARTs) are prevalent in nature, where smaller parasitic males typically have better sperm quality than larger territorial guard males. At present, it is unclear what is causing this phenomenon. Our objective was to gain insights into sperm form and function by examining flagellar beating patterns (beat frequency, wave amplitude, bend length, bend angle, wave velocity) and biomechanical sperm metrics (velocity, hydrodynamic power output, propulsive efficiency) of wild spawning Chinook salmon ARTs. Ovarian fluid and milt were collected to form a series of eight experimental blocks, each composed of ovarian fluid from a unique female and sperm from a unique pair of parasitic jack and guard hooknose males. Sperm from each ART were activated in river water and ovarian fluid. Flagellar parameters were evaluated from recordings using high-speed video microscopy and biomechanical metrics were quantified. We show that ART has an impact on flagellar beating, where jacks had a higher bend length and bend angle than hooknoses. Activation media also impacted the pattern of flagellar parameters, such that beat frequency, wave velocity and bend angle declined, while wave amplitude of flagella increased when ovarian fluid was incorporated into activation media. Furthermore, we found that sperm from jacks swam faster than those from hooknoses and required less hydrodynamic power output to propel themselves in river water and ovarian fluid. Jack sperm were also more efficient at swimming than hooknose sperm, and propulsive efficiency increased when cells were activated in ovarian fluid. The results demonstrate that sperm biomechanics may be driving divergence in competitive reproductive success between ARTs. © 2017. Published by The Company of Biologists Ltd.

  13. Complex spatial organization and flagellin composition of flagellar propeller from marine magnetotactic ovoid strain MO-1.

    PubMed

    Zhang, Wei-Jia; Santini, Claire-Lise; Bernadac, Alain; Ruan, Juanfang; Zhang, Sheng-Da; Kato, Takayuki; Li, Ying; Namba, Keiichi; Wu, Long-Fei

    2012-03-02

    Marine magnetotactic ovoid bacterium MO-1 is capable of swimming along the geomagnetic field lines by means of its two sheathed flagellar bundles at a speed up to 300 μm/s. In this study, by using electron microscopy, we showed that, in each bundle, six individual flagella were organized in hexagon with a seventh in the middle. We identified 12 flagellin paralogs and 2 putative flagellins in the genome of MO-1. Among them, 13 were tandemly located on an ~ 17-kb segment while the 14th was on a separated locus. Using reverse transcription PCR and quantitative PCR, we found that all the 14 flagellin or putative flagellin genes were transcribed and that 2 of them were more abundantly expressed than others. A nLC (nanoliquid chromatography)-ESI (electrospray ionization)-MS/MS (mass spectrometry/mass spectrometry) mass spectrometry analysis identified all the 12 flagellin proteins in three glycosylated polypeptide bands resolved by one-dimensional denaturing polyacrylamide gel electrophoresis and 10 of them in 21 spots obtained by means of two-dimensional electrophoresis of flagellar extracts. Most spots contained more than one flagellin, and eight of the ten identified flagellins existed in multiple isoforms. Taken together, these results show unprecedented complexity in the spatial organization and flagellin composition of the flagellar propeller. Such architecture is observed only for ovoid-coccoid, bilophotrichously flagellated magnetotactic bacteria living in marine sediments, suggesting a species and environmental specificity.

  14. Two flagellar BAR domain proteins in Trypanosoma brucei with stage-specific regulation

    PubMed Central

    Cicova, Zdenka; Dejung, Mario; Skalicky, Tomas; Eisenhuth, Nicole; Hanselmann, Steffen; Morriswood, Brooke; Figueiredo, Luisa M.; Butter, Falk; Janzen, Christian J.

    2016-01-01

    Trypanosomes are masters of adaptation to different host environments during their complex life cycle. Large-scale proteomic approaches provide information on changes at the cellular level, and in a systematic way. However, detailed work on single components is necessary to understand the adaptation mechanisms on a molecular level. Here, we have performed a detailed characterization of a bloodstream form (BSF) stage-specific putative flagellar host adaptation factor Tb927.11.2400, identified previously in a SILAC-based comparative proteome study. Tb927.11.2400 shares 38% amino acid identity with TbFlabarin (Tb927.11.2410), a procyclic form (PCF) stage-specific flagellar BAR domain protein. We named Tb927.11.2400 TbFlabarin-like (TbFlabarinL), and demonstrate that it originates from a gene duplication event, which occurred in the African trypanosomes. TbFlabarinL is not essential for the growth of the parasites under cell culture conditions and it is dispensable for developmental differentiation from BSF to the PCF in vitro. We generated TbFlabarinL-specific antibodies, and showed that it localizes in the flagellum. Co-immunoprecipitation experiments together with a biochemical cell fractionation suggest a dual association of TbFlabarinL with the flagellar membrane and the components of the paraflagellar rod. PMID:27779220

  15. Variable Symmetry in Salmonella typhimurium Flagellar Motors

    PubMed Central

    Young, Howard S.; Dang, Hongyue; Lai, Yimin; DeRosier, David J.; Khan, Shahid

    2003-01-01

    Electron cryomicroscopy of rotor complexes of the Salmonella typhimurium flagellar motor, overproduced in a nonmotile Escherichia coli host, has revealed a variation in subunit symmetry of the cytoplasmic ring (C ring) module. C rings with subunit symmetries ranging from 31 to 38 were found. They formed a Gaussian distribution around a mean between 34 and 35, a similar number to that determined for native C rings. C-ring diameter scaled with the number of subunits, indicating that the elliptical-shaped subunits maintained constant intersubunit spacing. Taken together with evidence that the M ring does not correspondingly increase in size, this finding indicates that rotor assembly does not require strict stoichiometric interactions between the M- and C-ring subunits. Implications for motor function are discussed. PMID:12524310

  16. Flagellar hook protein from Salmonella SJ25.

    PubMed

    Kagawa, H; Owaribe, K; Asakura, S; Takahashi, N

    1976-01-01

    From acid-disintegrated flagellar hooks of Salmonella SJ25 an immunochemically pure preparation of hook protein was obtained by column chromatography. The molecular weight of the protein determined by sodium dodecyl sulfate-gel electrophoresis was 43,000, whereas that of SJ25 flagellin was 56,000. The amino-terminal residue of the hook protein was determined to be seryl. The amino acid composition of the protein was determined, the results being very similar to that for an Escheria coli hook protein reported by Silverman and Simon (1972). Within a wavelength range of 200 to 250 nm, our purified preparation of hook protein gave a circular dichroism spectrum with unusually small amplitudes, suggesting that the alpha-helix content of the protein was very low.

  17. Flagellar hook protein from Salmonella SJ25.

    PubMed Central

    Kagawa, H; Owaribe, K; Asakura, S; Takahashi, N

    1976-01-01

    From acid-disintegrated flagellar hooks of Salmonella SJ25 an immunochemically pure preparation of hook protein was obtained by column chromatography. The molecular weight of the protein determined by sodium dodecyl sulfate-gel electrophoresis was 43,000, whereas that of SJ25 flagellin was 56,000. The amino-terminal residue of the hook protein was determined to be seryl. The amino acid composition of the protein was determined, the results being very similar to that for an Escheria coli hook protein reported by Silverman and Simon (1972). Within a wavelength range of 200 to 250 nm, our purified preparation of hook protein gave a circular dichroism spectrum with unusually small amplitudes, suggesting that the alpha-helix content of the protein was very low. Images PMID:54355

  18. Chlamydomonas IFT172 is encoded by FLA11, interacts with CrEB1, and regulates IFT at the flagellar tip.

    PubMed

    Pedersen, Lotte B; Miller, Mark S; Geimer, Stefan; Leitch, Jeffery M; Rosenbaum, Joel L; Cole, Douglas G

    2005-02-08

    The transport of flagellar precursors and removal of turnover products from the flagellar tip is mediated by intraflagellar transport (IFT) , which is essential for both flagellar assembly and maintenance . Large groups of IFT particles are moved from the flagellar base to the tip by kinesin-2, and smaller groups are returned to the base by cytoplasmic dynein 1b. The IFT particles are composed of two protein complexes, A and B, comprising approximately 16-18 polypeptides. How cargo is unloaded from IFT particles, turnover products loaded, and active IFT motors exchanged at the tip is unknown. We previously showed that the Chlamydomonas microtubule end binding protein 1 (CrEB1) localizes to the flagellar tip and is depleted from the tips of the temperature-sensitive (ts) mutant fla11ts . We demonstrate here that FLA11 encodes IFT protein 172, a component of IFT complex B, and show that IFT172 interacts with CrEB1. Because fla11ts cells are defective in IFT particle turnaround at the tip, our results indicate that IFT172 is involved in regulating the transition between anterograde and retrograde IFT at the tip, perhaps by a mechanism involving CrEB1. Therefore, IFT172 is involved in the control of flagellar assembly/disassembly at the tip.

  19. A flagellar sheath protein of Helicobacter pylori is identical to HpaA, a putative N-acetylneuraminyllactose-binding hemagglutinin, but is not an adhesin for AGS cells.

    PubMed Central

    Jones, A C; Logan, R P; Foynes, S; Cockayne, A; Wren, B W; Penn, C W

    1997-01-01

    The gene encoding a 29-kDa flagellar sheath protein was cloned and found to be similar to hpaA, reported to encode an N-acetylneuraminyllactose-binding fibrillar hemagglutinin (D. G. Evans, T. K. Karjalainen, D. J. Evans, Jr., D. Y. Graham, and C. H. Lee, J. Bacteriol. 175:674-683, 1993). The transcriptional start was mapped by primer extension from Helicobacter pylori mRNA, indicating an active consensus promoter at a location different from that suggested by Evans et al. Immunogold labelling of the flagellar sheath with a monoclonal antibody to HpaA was demonstrated in four strains, contrary to previous reports of a surface (D. G. Evans, T. K. Karjalainen, D. J. Evans, Jr., D. Y. Graham, and C. H. Lee, J. Bacteriol. 175:674-683, 1993) or a cytoplasmic (P. W. O'Toole, L. Janzon, P. Doig, J. Huang, M. Kostrzynska, and T. J. Trust, J. Bacteriol. 177:6049-6057, 1995) locale. Agglutination of erythrocytes and adherence to AGS cells by a delta hpaA mutant were no different from those of the parent strain, confirming a recent finding of O'Toole et al. PMID:9287032

  20. A flagellar sheath protein of Helicobacter pylori is identical to HpaA, a putative N-acetylneuraminyllactose-binding hemagglutinin, but is not an adhesin for AGS cells.

    PubMed

    Jones, A C; Logan, R P; Foynes, S; Cockayne, A; Wren, B W; Penn, C W

    1997-09-01

    The gene encoding a 29-kDa flagellar sheath protein was cloned and found to be similar to hpaA, reported to encode an N-acetylneuraminyllactose-binding fibrillar hemagglutinin (D. G. Evans, T. K. Karjalainen, D. J. Evans, Jr., D. Y. Graham, and C. H. Lee, J. Bacteriol. 175:674-683, 1993). The transcriptional start was mapped by primer extension from Helicobacter pylori mRNA, indicating an active consensus promoter at a location different from that suggested by Evans et al. Immunogold labelling of the flagellar sheath with a monoclonal antibody to HpaA was demonstrated in four strains, contrary to previous reports of a surface (D. G. Evans, T. K. Karjalainen, D. J. Evans, Jr., D. Y. Graham, and C. H. Lee, J. Bacteriol. 175:674-683, 1993) or a cytoplasmic (P. W. O'Toole, L. Janzon, P. Doig, J. Huang, M. Kostrzynska, and T. J. Trust, J. Bacteriol. 177:6049-6057, 1995) locale. Agglutination of erythrocytes and adherence to AGS cells by a delta hpaA mutant were no different from those of the parent strain, confirming a recent finding of O'Toole et al.

  1. THE FLAGELLAR PHOTORESPONSE IN VOLVOX SPECIES (VOLVOCACEAE, CHLOROPHYCEAE)(1).

    PubMed

    Solari, Cristian A; Drescher, Knut; Goldstein, Raymond E

    2011-06-01

    Steering their swimming direction toward the light is crucial for the viability of Volvox colonies, the larger members of the volvocine algae. While it is known that this phototactic steering is achieved by a difference in behavior of the flagella on the illuminated and shaded sides, conflicting reports suggest that this asymmetry arises either from a change in beating direction or a change in beating frequency. Here, we report direct observations of the flagellar behavior of various Volvox species with different phyletic origin in response to light intensity changes and thereby resolve this controversy: Volvox barberi W. Shaw from the section Volvox sensu Nozaki (2003) changes the direction of the flagellar beating plane, while species encompassed in the group Eudorina (Volvox carteri F. Stein, Volvox aureus Ehrenb., and Volvox tertius Art. Mey.) decrease the flagellar beating frequency, sometimes down to flagellar arrest. © 2011 Phycological Society of America.

  2. Load-Dependent Assembly of the Bacterial Flagellar Motor

    PubMed Central

    Tipping, Murray J.; Delalez, Nicolas J.; Lim, Ren; Berry, Richard M.; Armitage, Judith P.

    2013-01-01

    ABSTRACT It is becoming clear that the bacterial flagellar motor output is important not only for bacterial locomotion but also for mediating the transition from liquid to surface living. The output of the flagellar motor changes with the mechanical load placed on it by the external environment: at a higher load, the motor runs more slowly and produces higher torque. Here we show that the number of torque-generating units bound to the flagellar motor also depends on the external mechanical load, with fewer stators at lower loads. Stalled motors contained at least as many stators as rotating motors at high load, indicating that rotation is unnecessary for stator binding. Mutant stators incapable of generating torque could not be detected around the motor. We speculate that a component of the bacterial flagellar motor senses external load and mediates the strength of stator binding to the rest of the motor. PMID:23963182

  3. Signal processing and flagellar motor switching during phototaxis of Halobacterium salinarum.

    PubMed

    Nutsch, Torsten; Marwan, Wolfgang; Oesterhelt, Dieter; Gilles, Ernst Dieter

    2003-11-01

    Prokaryotic taxis, the active search of motile cells for the best environmental conditions, is one of the paradigms for signal transduction. The search algorithm implemented by the cellular biochemistry modulates the probability of switching the rotational direction of the flagellar motor, a nanomachine that propels prokaryotic cells. On the basis of the well-known biochemical mechanisms of chemotaxis in Escherichia coli, kinetic modeling of the events leading from chemoreceptor activation by ligand binding to the motility response has been performed with great success. In contrast to Escherichia coli, Halobacterium salinarum, in addition, responds to visible light, which is sensed through specific photoreceptors of different wavelength sensitivity (phototaxis). Light stimuli of defined intensity and time course can be controlled precisely, which facilitates input-output measurements used for system analysis of the molecular network connecting the sensory receptors to the flagellar motor switch. Here, we analyze the response of halobacterial cells to single and double-pulse light stimuli and present the first kinetic model for prokaryotic cells that couples the signal-transduction pathway with the flagellar motor switch. Modeling based on experimental data supports the current biochemical model of halobacterial phototaxis. Moreover, the simulations demonstrate that motor switching occurs through subsequent rate-limiting steps, which are both under sensory control, suggesting that two signals may be involved in halobacterial phototaxis.

  4. Fuel of the Bacterial Flagellar Type III Protein Export Apparatus.

    PubMed

    Minamino, Tohru; Kinoshita, Miki; Namba, Keiichi

    2017-01-01

    The flagellar type III export apparatus utilizes ATP and proton motive force (PMF) across the cytoplasmic membrane as the energy sources and transports flagellar component proteins from the cytoplasm to the distal growing end of the growing structure to construct the bacterial flagellum beyond the cellular membranes. The flagellar type III export apparatus coordinates flagellar protein export with assembly by ordered export of substrates to parallel with their order of the assembly. The export apparatus is composed of a PMF-driven transmembrane export gate complex and a cytoplasmic ATPase complex. Since the ATPase complex is dispensable for flagellar protein export, PMF is the primary fuel for protein unfolding and translocation. Interestingly, the export gate complex can also use sodium motive force across the cytoplasmic membrane in addition to PMF when the ATPase complex does not work properly. Here, we describe experimental protocols, which have allowed us to identify the export substrate class and the primary fuel of the flagellar type III protein export apparatus in Salmonella enterica serovar Typhimurium.

  5. Genetic Interactions at the Fla10 Locus: Suppressors and Synthetic Phenotypes That Affect the Cell Cycle and Flagellar Function in Chlamydomonas Reinhardtii

    PubMed Central

    Lux-III, F. G.; Dutcher, S. K.

    1991-01-01

    Through the isolation of suppressors of temperature-sensitive flagellar assembly mutations at the FLA10 locus of Chlamydomonas reinhardtii, we have identified six other genes involved in flagellar assembly. Mutations at these suppressor loci, termed SUF1-SUF6, display allele specificity with respect to which fla10(-) mutant alleles they suppress. An additional mutation, apm1-122, which confers resistance to the plant herbicides amiprophos-methyl and oryzalin, was also found to interact with mutations at the FLA10 locus. The apm1-122 mutation in combination with three fla10(-) mutant alleles results in synthetic cold-sensitive cell division defects, and in combination with an additional pseudo-wild-type fla10(-) allele yields a synthetic temperature-sensitive flagellar motility phenotype. Based upon the genetic interactions of these loci, we propose that the FLA10 gene product interacts with multiple components of the flagellar apparatus and plays a role both in flagellar assembly and in the cell cycle. PMID:1874415

  6. Flagellar mutants of Chlamydomonas: Studies of radial spoke-defective strains by dikaryon and revertant analysis

    PubMed Central

    Luck, David; Piperno, Gianni; Ramanis, Zenta; Huang, B.

    1977-01-01

    The motility mutant of Chlamydomonas reinhardtii pf14 lacks radial spoke structures in its flagellar axonemes, and 12 proteins present in wild type are missing from a two-dimensional map (isoelectrofocusing/sodium dodecyl sulfate electrophoresis) of its 35S-labeled flagellar proteins. Six of these same proteins are missing in pf1, which lacks spoke-heads. To determine whether any of the missing proteins represent the mutant gene product two experimental approaches have been applied. The first makes use of the fact that gametes of either mutant strain when fused with wild-type gametes to form quadriflagellate dikaryons undergo recovery of flagellar function. Recovery at the molecular level was monitored by prelabeling the mutant proteins with 35S and allowing recovery to occur in the absence of protein synthesis. It is to be expected that the mutant gene product would not be restored as a radioactive protein and that recovery would depend on the assembly of the wild-type counterpart that is not labeled. The second technique makes use of revertants induced by UV irradiation. Dikaryon rescue in the case of pf14 leads to restoration of 11 radioactive components; only protein 3 fails to appear as a radioactive spot. For pf1 only two radioactive proteins are restored; proteins 4, 6, 9, and 10 were not radioactive. Analysis of revertants of pf1 gave evidence (altered map positions) that protein 4 is the mutant gene product. In the case of pf14, analysis of 22 revertants has not provided similar positive evidence that protein 3 is the gene product. Images PMID:269405

  7. Perturbation of FliL interferes with Proteus mirabilis swarmer cell gene expression and differentiation.

    PubMed

    Cusick, Kathleen; Lee, Yi-Ying; Youchak, Brian; Belas, Robert

    2012-01-01

    Proteus mirabilis is a dimorphic, motile bacterium often associated with urinary tract infections. Colonization of urinary tract surfaces is aided by swarmer cell differentiation, which is initiated by inhibition of flagellar rotation when the bacteria first contact a surface. Mutations in fliL, encoding a flagellar structural protein with an enigmatic function, result in the inappropriate production of differentiated swarmer cells, called pseudoswarmer cells, under noninducing conditions, indicating involvement of FliL in the surface sensing pathway. In the present study, we compared the fliL transcriptome with that of wild-type swarmer cells and showed that nearly all genes associated with motility (flagellar class II and III genes) and chemotaxis are repressed. In contrast, spontaneous motile revertants of fliL cells that regained motility yet produced differentiated swarmer cells under noninducing conditions transcribed flagellar class II promoters at consistent levels. Expression of umoA (a known regulator of swarmer cells), flgF, and flgI increased significantly in both swarmer and pseudoswarmer cells, as did genes in a degenerate prophage region situated immediately adjacent to the Rcs phosphorelay system. Unlike swarmer cells, pseudoswarmers displayed increased activity, rather than transcription, of the flagellar master regulatory protein, FlhD(4)C(2), and analyses of the fliL parent strain and its motile revertants showed that they result from mutations altering the C-terminal 14 amino acids of FliL. Collectively, the data suggest a functional role for the C terminus of FliL in surface sensing and implicate UmoA as part of the signal relay leading to the master flagellar regulator FlhD(4)C(2), which ultimately controls swarmer cell differentiation.

  8. Perturbation of FliL Interferes with Proteus mirabilis Swarmer Cell Gene Expression and Differentiation

    PubMed Central

    Cusick, Kathleen; Lee, Yi-Ying; Youchak, Brian

    2012-01-01

    Proteus mirabilis is a dimorphic, motile bacterium often associated with urinary tract infections. Colonization of urinary tract surfaces is aided by swarmer cell differentiation, which is initiated by inhibition of flagellar rotation when the bacteria first contact a surface. Mutations in fliL, encoding a flagellar structural protein with an enigmatic function, result in the inappropriate production of differentiated swarmer cells, called pseudoswarmer cells, under noninducing conditions, indicating involvement of FliL in the surface sensing pathway. In the present study, we compared the fliL transcriptome with that of wild-type swarmer cells and showed that nearly all genes associated with motility (flagellar class II and III genes) and chemotaxis are repressed. In contrast, spontaneous motile revertants of fliL cells that regained motility yet produced differentiated swarmer cells under noninducing conditions transcribed flagellar class II promoters at consistent levels. Expression of umoA (a known regulator of swarmer cells), flgF, and flgI increased significantly in both swarmer and pseudoswarmer cells, as did genes in a degenerate prophage region situated immediately adjacent to the Rcs phosphorelay system. Unlike swarmer cells, pseudoswarmers displayed increased activity, rather than transcription, of the flagellar master regulatory protein, FlhD4C2, and analyses of the fliL parent strain and its motile revertants showed that they result from mutations altering the C-terminal 14 amino acids of FliL. Collectively, the data suggest a functional role for the C terminus of FliL in surface sensing and implicate UmoA as part of the signal relay leading to the master flagellar regulator FlhD4C2, which ultimately controls swarmer cell differentiation. PMID:22081397

  9. Coordinated Reversal of Flagellar Motors on a Single Escherichia coli Cell

    PubMed Central

    Terasawa, Shun; Fukuoka, Hajime; Inoue, Yuichi; Sagawa, Takashi; Takahashi, Hiroto; Ishijima, Akihiko

    2011-01-01

    An Escherichia coli cell transduces extracellular stimuli sensed by chemoreceptors to the state of an intracellular signal molecule, which regulates the switching of the rotational direction of the flagellar motors from counterclockwise (CCW) to clockwise (CW) and from CW back to CCW. Here, we performed high-speed imaging of flagellar motor rotation and show that the switching of two different motors on a cell is controlled coordinatedly by an intracellular signal protein, phosphorylated CheY (CheY-P). The switching is highly coordinated with a subsecond delay between motors in clear correlation with the distance of each motor from the chemoreceptor patch localized at a cell pole, which would be explained by the diffusive motion of CheY-P molecules in the cell. The coordinated switching becomes disordered by the expression of a constitutively active CheY mutant that mimics the CW-rotation stimulating function. The coordinated switching requires CheZ, which is the phosphatase for CheY-P. Our results suggest that a transient increase and decrease in the concentration of CheY-P caused by a spontaneous burst of its production by the chemoreceptor patch followed by its dephosphorylation by CheZ, which is probably a wavelike propagation in a subsecond timescale, triggers and regulates the coordinated switching of flagellar motors. PMID:21539787

  10. Signal-dependent turnover of the bacterial flagellar switch protein FliM

    PubMed Central

    Delalez, Nicolas J.; Wadhams, George H.; Rosser, Gabriel; Xue, Quan; Brown, Mostyn T.; Dobbie, Ian M.; Berry, Richard M.; Leake, Mark C.; Armitage, Judith P.

    2010-01-01

    Most biological processes are performed by multiprotein complexes. Traditionally described as static entities, evidence is now emerging that their components can be highly dynamic, exchanging constantly with cellular pools. The bacterial flagellar motor contains ∼13 different proteins and provides an ideal system to study functional molecular complexes. It is powered by transmembrane ion flux through a ring of stator complexes that push on a central rotor. The Escherichia coli motor switches direction stochastically in response to binding of the response regulator CheY to the rotor switch component FliM. Much is known of the static motor structure, but we are just beginning to understand the dynamics of its individual components. Here we measure the stoichiometry and turnover of FliM in functioning flagellar motors, by using high-resolution fluorescence microscopy of E. coli expressing genomically encoded YPet derivatives of FliM at physiological levels. We show that the ∼30 FliM molecules per motor exist in two discrete populations, one tightly associated with the motor and the other undergoing stochastic turnover. This turnover of FliM molecules depends on the presence of active CheY, suggesting a potential role in the process of motor switching. In many ways the bacterial flagellar motor is as an archetype macromolecular assembly, and our results may have further implications for the functional relevance of protein turnover in other large molecular complexes. PMID:20498085

  11. Structural insights into bacterial flagellar hooks similarities and specificities

    PubMed Central

    Yoon, Young-Ho; Barker, Clive S.; Bulieris, Paula V.; Matsunami, Hideyuki; Samatey, Fadel A.

    2016-01-01

    Across bacteria, the protein that makes the flagellar hook, FlgE, has a high variability in amino acid residue composition and sequence length. We hereby present the structure of two fragments of FlgE protein from Campylobacter jejuni and from Caulobacter crescentus, which were obtained by X-ray crystallography, and a high-resolution model of the hook from Caulobacter. By comparing these new structures of FlgE proteins, we show that bacterial hook can be divided in two distinct parts. The first part comprises domains that are found in all FlgE proteins and that will make the basic structure of the hook that is common to all flagellated bacteria. The second part, hyper-variable both in size and structure, will be bacteria dependent. To have a better understanding of the C. jejuni hook, we show that a special strain of Salmonella enterica, which was designed to encode a gene of flgE that has the extra domains found in FlgE from C. jejuni, is fully motile. It seems that no matter the size of the hook protein, the hook will always have a structure made of 11 protofilaments. PMID:27759043

  12. The CckA-ChpT-CtrA Phosphorelay System Is Regulated by Quorum Sensing and Controls Flagellar Motility in the Marine Sponge Symbiont Ruegeria sp. KLH11

    PubMed Central

    Zan, Jindong; Heindl, Jason E.; Liu, Yue; Fuqua, Clay; Hill, Russell T.

    2013-01-01

    Bacteria respond to their environment via signal transduction pathways, often two-component type systems that function through phosphotransfer to control expression of specific genes. Phosphorelays are derived from two-component systems but are comprised of additional components. The essential cckA-chpT-ctrA phosphorelay in Caulobacter crescentus has been well studied and is important in orchestrating the cell cycle, polar development and flagellar biogenesis. Although cckA, chpT and ctrA homologues are widespread among the Alphaproteobacteria, relatively few is known about their function in the large and ecologically significant Roseobacter clade of the Rhodobacterales. In this study the cckA-chpT-ctrA system of the marine sponge symbiont Ruegeria sp. KLH11 was investigated. Our results reveal that the cckA, chpT and ctrA genes positively control flagellar biosynthesis. In contrast to C. crescentus, the cckA, chpT and ctrA genes in Ruegeria sp. KLH11 are non-essential and do not affect bacterial growth. Gene fusion and transcript analyses provide evidence for ctrA autoregulation and the control of motility-related genes. In KLH11, flagellar motility is controlled by the SsaRI system and acylhomoserine lactone (AHL) quorum sensing. SsaR and long chain AHLs are required for cckA, chpT and ctrA gene expression, providing a regulatory link between flagellar locomotion and population density in KLH11. PMID:23825536

  13. Hybrid flagellar motor/MEMS based TNT detection system

    NASA Astrophysics Data System (ADS)

    Kim, Jin-Woo; Tung, Steve

    2006-05-01

    Effective and rapid detection of nitroaromatic explosive compounds, especially trinitrotoluene (TNT), is very important to homeland security as well as to environmental monitoring of contaminants in soil and water, and landmine detection. In this research, we explore a novel nanoscale flagellar motor based TNT detection system (nFMTNT). The nFMTNT is a bio-hybrid MEMS system which combines genetically engineered flagellar motors and MEMS devices. The system consists of three major components: (1) a non-pathogenic, genetically modified Escherichia coli strain KAF95 with a rotating flagellar filament, (2) a microchannel with tethered cells, and (3) a sub-micron bead attached to a rotating flagellar filament. The operational principle of nFMTNT is based on detecting the change in the rotational behavior of the nanoscale flagellar filament in the presence of TNT. The rotational behavior of flagellar filaments of E. coli KAF95 was shown to be extremely sensitive to the presence of nitrate or nitrite. Normally, the flagellar filaments were locked in to rotate in the counterclockwise direction. However, when a nitrate or nitrite was present in the immediate environment, the filaments cease to rotate. Our results indicate that the threshold concentrations required for this response were 10 -4 M for nitrate and 10 -3 M for nitrite. This is equivalent to around 10 pg of nitrate and 100 pg of nitrite, based on the dimension of the MEMS-based reaction system used for the experiment (400 μm × 100 μm × 40 μm). These detection limits can be even lower when the size of the system is reduced.

  14. Functional characterization of FlgM in the regulation of flagellar synthesis and motility in Yersinia pseudotuberculosis.

    PubMed

    Ding, Lisha; Wang, Yao; Hu, Yangbo; Atkinson, Steve; Williams, Paul; Chen, Shiyun

    2009-06-01

    We describe here the functional characterization of the flgM gene in Yersinia pseudotuberculosis. Direct interaction of FlgM with the alternative sigma factor sigma(28) (FliA) was first confirmed. A conserved region in the C-terminus of FlgM was found which included the sigma(28) binding domain. By site-directed mutagenesis, bacterial two-hybrid analysis and Western blotting, the primary FlgM binding sites with sigma(28) were shown to be Ile85, Ala86 and Leu89. A role for FlgM in swimming motility was demonstrated by inactivation of flgM and subsequent complementation in trans. Transcriptional fusion analyses showed differential gene expression of flhDC, fliA, flgM and fliC in the fliA and flgM mutants compared with the wild-type. flhDC expression was not influenced by sigma(28) or FlgM while fliA expression was abolished in the fliA mutant and considerably reduced in the flgM mutant when compared to the wild-type, indicating that both FliA and FlgM can activate fliA transcription. Conversely, flgM transcription was higher in the fliA mutant when compared to the wild-type, suggesting that flgM transcription was repressed by sigma(28). Interestingly, fliC expression was markedly increased in the flgM mutant, suggesting a negative regulatory role for FlgM in fliC expression. The transcription of other sigma-dependent genes (cheW, flgD, flaA, csrA and fliZ) was also examined in fliA and flgM mutant backgrounds and this revealed that other sigma-factors apart from sigma(28) may be involved in flagellar biogenesis in Y. pseudotuberculosis. Taking together the motility phenotypes and effects of flgM mutation on the regulation of these key motility genes, we propose that the mechanisms regulating flagellar biogenesis in Y. pseudotuberculosis may differ from those described for other bacteria.

  15. The flagellar adenylate kinases of Trypanosoma cruzi.

    PubMed

    Camara, María de los Milagros; Bouvier, León A; Miranda, Mariana R; Pereira, Claudio A

    2015-01-01

    Adenylate kinases (ADK) are key enzymes involved in cell energy management. Trypanosomatids present the highest number of variants in a single cell in comparison with the rest of the living organisms. In this work, we characterized two flagellar ADKs from Trypanosoma cruzi, called TcADK1 and TcADK4, which are also located in the cell cytosol. Interestingly, TcADK1 presents a stage-specific expression. This variant was detected in epimastigotes cells, and was completely absent in trypomastigotes and amastigotes, while TcADK4 is present in the major life cycle stages of T. cruzi. Both variants are also regulated, in opposite ways, along the parasite growth curve suggesting that their expression depends on the intra- and extracellular conditions. Both, TcADK1 and TcADK4 present N-terminal extension that could be responsible for their subcellular localization. The presence of ADK variants in the flagellum would be critical for the provision of energy in a process of high ATP consumption such as cell motility. © The Author 2014. Published by Oxford University Press on behalf of FEMS. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  16. Loss of the lac operon contributes to Salmonella invasion of epithelial cells through derepression of flagellar synthesis.

    PubMed

    Jiang, Lingyan; Ni, Zhiwei; Wang, Lei; Feng, Lu; Liu, Bin

    2015-03-01

    Salmonella, a genus that is closely related to Escherichia coli, includes many pathogens of humans and other animals. A notable feature that distinguishes Salmonella from E. coli is lactose negativity, because the lac operon is lost in most Salmonella genomes. Here, we expressed the lac operon in Salmonella enterica serovar Typhimurium and compared the virulence of the Lac(+) strain to that of the wild-type strain in a murine model, invasion assays, and macrophage replication assays. We showed that the Lac(+) strain is attenuated in vivo and the attenuation of virulence is caused by its defect in epithelial cell invasion. However, the invasion-defective phenotype is unrelated to lactose utilization. Through sequencing and the comparison of the transcriptome profile between the Lac(+) and wild-type strains during invasion, we found that most flagellar genes were markedly downregulated in the Lac(+) strain, while other genes associated with invasion, such as the majority of genes encoded in Salmonella pathogenicity island 1, were not differentially expressed. Moreover, we discovered that lacA is the major repressor of flagellar gene expression in the lac operon. In conclusion, these data demonstrate that the lac operon decreases Salmonella invasion of epithelial cells through repression of flagellar biosynthesis. As the ability to invade epithelial cells is a critical virulence determinant of Salmonella, our results provide important evidence that the loss of the lac operon contributes to the evolution of Salmonella pathogenicity.

  17. The Helicobacter pylori Anti-Sigma Factor FlgM Is Predominantly Cytoplasmic and Cooperates with the Flagellar Basal Body Protein FlhA ▿ †

    PubMed Central

    Rust, Melanie; Borchert, Sophie; Niehus, Eike; Kuehne, Sarah A.; Gripp, Eugenia; Bajceta, Afrodita; McMurry, Jonathan L.; Suerbaum, Sebastian; Hughes, Kelly T.; Josenhans, Christine

    2009-01-01

    Helicobacter pylori requires flagellar motility and orientation to persist actively in its habitat. A particular feature of flagella in most Helicobacter species including H. pylori is a membraneous flagellar sheath. The anti-sigma factor FlgM of H. pylori is unusual, since it lacks an N-terminal domain present in other FlgM homologs, e.g., FlgM of Salmonella spp., whose regulatory function is intimately coupled to its secretion through the flagellar type III secretion system. The aim of the present study was to characterize the localization and secretion of the short H. pylori FlgM in the presence of a flagellar sheath and to elucidate its interaction with other flagellar proteins, such as the basal body protein FlhA, which was previously shown to cooperate with FlgM for regulation. H. pylori FlgM was only released into the medium in minor amounts in wild-type bacteria, where the bulk amount of the protein was retained in the cytoplasm. Some FlgM was detected in the flagellar fraction. FlgM was expressed in flhA mutants and was less soluble and differentially localized in bacterial fractions of the flhA mutant in comparison to wild-type bacteria. FlgM-green fluorescent protein and FlgM-V5 translational fusions were generated and expressed in H. pylori. FlgM displayed a predominantly polar distribution and interacted with the C-terminal domain of FlhA (FlhAC). We suggest that, in H. pylori, FlgM secretion may not be paramount for its regulatory function and that protein interactions at the flagellar basal body may determine the turnover and localization of functional FlgM. PMID:19465658

  18. A 12-base-pair deletion in the flagellar master control gene flhC causes nonmotility of the pathogenic German sorbitol-fermenting Escherichia coli O157:H- strains.

    PubMed

    Monday, Steven R; Minnich, Scott A; Feng, Peter C H

    2004-04-01

    An atypical, Stx2-producing, pathogenic Escherichia coli O157:H(-) strain has been isolated with increasing frequency from hemolytic uremic syndrome patients in Germany. The lack of the H7 antigen coupled with the strain's ability to ferment sorbitol and express beta-glucuronidase have complicated its detection and identification. In this study, we have determined that the loss of motility in these German sorbitol-fermenting (SF) O157 strains is due to a 12-bp in-frame deletion in flhC that is required for transcriptional activation of genes involved in flagellum biosynthesis. Either complementation with a functional flhC or repair of this mutation restored H7 antigen expression and motility. PCR analysis of several nonmotile E. coli O157 strains from various geographical sources confirmed that the 12-bp flhC deletion is found only in the cluster of German SF O157 strains, providing a potentially useful marker by which these atypical strains can be identified. The loss of motility via mutations in the flhDC operon that we observed in the German SF O157 strains is consistent with a similar phenomenon currently observed in a significant subset of other important gram-negative pathogens.

  19. Reactivation of flagellar motility in demembranated Leishmania reveals role of cAMP in flagellar wave reversal to ciliary waveform

    PubMed Central

    Mukhopadhyay, Aakash Gautam; Dey, Chinmoy Sankar

    2016-01-01

    The flagellum of parasitic trypanosomes is a multifunctional appendage essential for its viability and infectivity. However, the biological mechanisms that make the flagellum so dynamic remains unexplored. No method is available to access and induce axonemal motility at will to decipher motility regulation in trypanosomes. For the first time we report the development of a detergent-extracted/demembranated ATP-reactivated model for studying flagellar motility in Leishmania. Flagellar beat parameters of reactivated parasites were similar to live ones. Using this model we discovered that cAMP (both exogenous and endogenous) induced flagellar wave reversal to a ciliary waveform in reactivated parasites via cAMP-dependent protein kinase A. The effect was reversible and highly specific. Such an effect of cAMP on the flagellar waveform has never been observed before in any organism. Flagellar wave reversal allows parasites to change direction of swimming. Our findings suggest a possible cAMP-dependent mechanism by which Leishmania responds to its surrounding microenvironment, necessary for its survival. Our demembranated-reactivated model not only serves as an important tool for functional studies of flagellated eukaryotic parasites but has the potential to understand ciliary motility regulation with possible implication on human ciliopathies. PMID:27849021

  20. Flagellar membranes are rich in raft-forming phospholipids

    PubMed Central

    Serricchio, Mauro; Schmid, Adrien W.; Steinmann, Michael E.; Sigel, Erwin; Rauch, Monika; Julkowska, Daria; Bonnefoy, Serge; Fort, Cécile; Bastin, Philippe; Bütikofer, Peter

    2015-01-01

    ABSTRACT The observation that the membranes of flagella are enriched in sterols and sphingolipids has led to the hypothesis that flagella might be enriched in raft-forming lipids. However, a detailed lipidomic analysis of flagellar membranes is not available. Novel protocols to detach and isolate intact flagella from Trypanosoma brucei procyclic forms in combination with reverse-phase liquid chromatography high-resolution tandem mass spectrometry allowed us to determine the phospholipid composition of flagellar membranes relative to whole cells. Our analyses revealed that phosphatidylethanolamine, phosphatidylserine, ceramide and the sphingolipids inositol phosphorylceramide and sphingomyelin are enriched in flagella relative to whole cells. In contrast, phosphatidylcholine and phosphatidylinositol are strongly depleted in flagella. Within individual glycerophospholipid classes, we observed a preference for ether-type over diacyl-type molecular species in membranes of flagella. Our study provides direct evidence for a preferential presence of raft-forming phospholipids in flagellar membranes of T. brucei. PMID:26276100

  1. Loose coupling in the bacterial flagellar motor

    PubMed Central

    Boschert, Ryan; Adler, Frederick R.; Blair, David F.

    2015-01-01

    Physiological properties of the flagellar rotary motor have been taken to indicate a tightly coupled mechanism in which each revolution is driven by a fixed number of energizing ions. Measurements that would directly test the tight-coupling hypothesis have not been made. Energizing ions flow through membrane-bound complexes formed from the proteins MotA and MotB, which are anchored to the cell wall and constitute the stator. Genetic and biochemical evidence points to a “power stroke” mechanism in which the ions interact with an aspartate residue of MotB to drive conformational changes in MotA that are transmitted to the rotor protein FliG. Each stator complex contains two separate ion-binding sites, raising the question of whether the power stroke is driven by one, two, or either number of ions. Here, we describe simulations of a model in which the conformational change can be driven by either one or two ions. This loosely coupled model can account for the observed physiological properties of the motor, including those that have been taken to indicate tight coupling; it also accords with recent measurements of motor torque at high load that are harder to explain in tight-coupling models. Under loads relevant to a swimming cell, the loosely coupled motor would perform about as well as a two-proton motor and significantly better than a one-proton motor. The loosely coupled motor is predicted to be especially advantageous under conditions of diminished energy supply, or of reduced temperature, turning faster than an obligatorily two-proton motor while using fewer ions. PMID:25825730

  2. Flagellar duplication and migration during the Trichomonas vaginalis cell cycle.

    PubMed

    Zuo, Y; Riley, D E; Krieger, J N

    1999-04-01

    Trichomonas vaginalis is a flagellated protozoan, a representative of 1 of the earliest known eukaryotic lineages. Trichomonas vaginalis lacks centrioles but possesses basal bodies. We report here the cell cycle-dependent flagellar dynamics of T. vaginalis. By immunofluorescence, we found that T. vaginalis flagella duplicated during S-phase, segregated toward the nuclear poles, and then emanated from the spindle poles at mitosis. This behavior strongly parallels that of centrioles and other spindle pole-associated structures variously termed centrosomes, spindle pole bodies, or microtubule organizing centers. These observations support the hypothesis that flagellar forces may have provided motile forces for spindle pole alignment in an ancestral eukaryote.

  3. Crystallization and preliminary X-ray analysis of Salmonella FliI, the ATPase component of the type III flagellar protein-export apparatus

    SciTech Connect

    Minamino, Tohru; Imada, Katsumi; Tahara, Aiko; Kihara, May; Macnab, Robert M.; Namba, Keiichi

    2006-10-01

    Crystals of an N-terminally truncated variant of the Salmonella flagellar ATPase FliI, which exports substrate proteins into the central channel of the growing flagellar structure by utilizing the energy of ATP hydrolysis, have been obtained and characterized by X-ray diffraction. Most of the structural components making up the bacterial flagellum are translocated through the central channel of the growing flagellar structure by the type III flagellar protein-export apparatus in an ATPase-driven manner and are assembled at the growing end. FliI is the ATPase that drives flagellar protein export using the energy of ATP hydrolysis. FliI forms an oligomeric ring structure in order to attain maximum ATPase activity. In this study, FliI(Δ1–18), an N-terminally truncated variant of FliI lacking the first 18 residues, was purified and crystallized. Crystals were obtained using the hanging-drop vapour-diffusion technique with PEG 8000 as a precipitant. FliI(Δ1–18) crystals grew in the monoclinic space group P2{sub 1}, with unit-cell parameters a = 48, b = 73, c = 126 Å, β = 94°, and diffracted to 2.4 Å resolution. Anomalous difference Patterson maps of Os-derivative and Pt-derivative crystals showed significant peaks in their Harker sections, indicating that both derivatives are suitable for structure determination.

  4. Refining the Binding of the Escherichia coli Flagellar Master Regulator, FlhD4C2, on a Base-Specific Level ▿†

    PubMed Central

    Lee, Yi-Ying; Barker, Clive S.; Matsumura, Philip; Belas, Robert

    2011-01-01

    The Escherichia coli flagellar master regulator, FlhD4C2, binds to the promoter regions of flagellar class II genes, yet, despite extensive analysis of the FlhD4C2-regulated promoter region, a detailed consensus sequence has not emerged. We used in vitro and in vivo experimental approaches to determine the nucleotides in the class II promoter, fliAp, required for the binding and function of FlhD4C2. FlhD4C2 protects 48 bp (positions −76 to −29 relative to the σ70-dependent transcriptional start site) in the fliA promoter. We divided the 48-bp footprint region into 5 sections to determine the requirement of each DNA segment for the binding and function of FlhD4C2. Results from an in vitro binding competition assay between the wild-type FlhD4C2-protected fragment and DNA fragments possessing mutations in one section of the 48-bp protected region showed that only one-third of the 48 bp protected by FlhD4C2 is required for FlhD4C2 binding and fliA promoter activity. This in vitro binding result was also seen in vivo with fliA promoter-lacZ fusions carrying the same mutations. Only seven bases (A12, A15, T34, A36, T37, A44, and T45) are absolutely required for the promoter activity. Moreover, A12, A15, T34, T37, and T45 within the 7 bases are highly specific to fliA promoter activity, and those bases form an asymmetric recognition site for FlhD4C2. The implications of the asymmetry of the FlhD4C2 binding site and its potential impact on FlhD4C2 are discussed. PMID:21685294

  5. Computer simulation of flagellar movement. VI. Simple curvature-controlled models are incompletely specified.

    PubMed Central

    Brokaw, C J

    1985-01-01

    Computer simulation is used to examine a simple flagellar model that will initiate and propagate bending waves in the absence of viscous resistances. The model contains only an elastic bending resistance and an active sliding mechanism that generates reduced active shear moment with increasing sliding velocity. Oscillation results from a distributed control mechanism that reverses the direction of operation of the active sliding mechanism when the curvature reaches critical magnitudes in either direction. Bend propagation by curvature-controlled flagellar models therefore does not require interaction with the viscous resistance of an external fluid. An analytical examination of moment balance during bend propagation by this model yields a solution curve giving values of frequency and wavelength that satisfy the moment balance equation and give uniform bend propagation, suggesting that the model is underdetermined. At 0 viscosity, the boundary condition of 0 shear rate at the basal end of the flagellum during the development of new bends selects the particular solution that is obtained by computer simulations. Therefore, the details of the pattern of bend initiation at the basal end of a flagellum can be of major significance in determining the properties of propagated bending waves in the distal portion of a flagellum. At high values of external viscosity, the model oscillates at frequencies and wavelengths that give approximately integral numbers of waves on the flagellum. These operating points are selected because they facilitate the balance of bending moments at the ends of the model, where the external viscous moment approaches 0. These mode preferences can be overridden by forcing the model to operate at a predetermined frequency. The strong mode preferences shown by curvature-controlled flagellar models, in contrast to the weak or absent mode preferences shown by real flagella, therefore do not demonstrate the inapplicability of the moment-balance approach

  6. Two Distinct Ca2+ Signaling Pathways Modulate Sperm Flagellar Beating Patterns in Mice1

    PubMed Central

    Chang, Haixin; Suarez, Susan S.

    2011-01-01

    Hyperactivation, a swimming pattern of mammalian sperm in the oviduct, is essential for fertilization. It is characterized by asymmetrical flagellar beating and an increase of cytoplasmic Ca2+. We observed that some mouse sperm swimming in the oviduct produce high-amplitude pro-hook bends (bends in the direction of the hook on the head), whereas other sperm produce high-amplitude anti-hook bends. Switching direction of the major bends could serve to redirect sperm toward oocytes. We hypothesized that different Ca2+ signaling pathways produce high-amplitude pro-hook and anti-hook bends. In vitro, sperm that hyperactivated during capacitation (because of activation of CATSPER plasma membrane Ca2+ channels) developed high-amplitude pro-hook bends. The CATSPER activators procaine and 4-aminopyridine (4-AP) also induced high-amplitude pro-hook bends. Thimerosal, which triggers a Ca2+ release from internal stores, induced high-amplitude anti-hook bends. Activation of CATSPER channels is facilitated by a pH rise, so both Ca2+ and pH responses to treatments with 4-AP and thimerosal were monitored. Thimerosal triggered a Ca2+ increase that initiated at the base of the flagellum, whereas 4-AP initiated a rise in the proximal principal piece. Only 4-AP triggered a flagellar pH rise. Proteins were extracted from sperm for examination of phosphorylation patterns induced by Ca2+ signaling. Procaine and 4-AP induced phosphorylation of proteins on threonine and serine, whereas thimerosal primarily induced dephosphorylation of proteins. Tyrosine phosphorylation was unaffected. We concluded that hyperactivation, which is associated with capacitation, can be modulated by release of Ca2+ from intracellular stores to reverse the direction of the dominant flagellar bend and, thus, redirect sperm. PMID:21389347

  7. From conformational spread to allosteric and cooperative models of E. coli flagellar motor

    NASA Astrophysics Data System (ADS)

    Pezzotta, A.; Adorisio, M.; Celani, A.

    2017-02-01

    Escherichia coli swims using flagella activated by rotary motors. The direction of rotation of the motors is indirectly regulated by the binding of a single messenger protein. The conformational spread model has been shown to accurately describe the equilibrium properties as well as the dynamics of the flagellar motor. In this paper we study this model from an analytic point of view. By exploiting the separation of timescales observed in experiments, we show how to reduce the conformational spread model to a coarse-grained, cooperative binding model. We show that this simplified model reproduces very well the dynamics of the motor switch.

  8. DNA elements regulating alpha1-tubulin gene induction during regeneration of eukaryotic flagella.

    PubMed

    Periz, G; Keller, L R

    1997-07-01

    Eukaryotic flagella are complex organelles composed of more than 200 polypeptides. Little is known about the regulatory mechanisms governing synthesis of the flagellar protein subunits and their assembly into this complex organelle. The unicellular green alga Chlamydomonas reinhardtii is the premier experimental model system for studying such cellular processes. When acid shocked, C. reinhardtii excises its flagella, rapidly and coordinately activates transcription of a set of flagellar genes, and ultimately regenerates a new flagellar pair. To define functionally the regulatory sequences that govern induction of the set of genes after acid shock, we analyzed the alpha1-tubulin gene promoter. To simplify transcriptional analysis in vivo, we inserted the selectable marker gene ARG7 on the same plasmid with a tagged alpha1-tubulin gene and stably introduced it into C. reinhardtii cells. By deletion of various sequences, two promoter regions (-176 to -122 and -85 to -16) were identified as important for induction of the tagged alpha1-tubulin gene. Deleting the region between -176 and -122 from the transcription start site resulted in an induction level which was only 45 to 70% of that of the resident gene. Deleting the region upstream of -56 resulted in a complete loss of inducibility without affecting basal expression. The alpha1-tubulin promoter region from -85 to -16 conferred partial acid shock inducibility to an arylsulfatase (ARS) reporter gene. These results show that induction of the alpha1-tubulin gene after acid shock is a complex response that requires diverse sequence elements.

  9. Divalent Cation Control of Flagellar Motility in African Trypanosomes

    NASA Astrophysics Data System (ADS)

    Westergard, Anna M.; Hutchings, Nathan R.

    2005-03-01

    Changes in calcium concentration have been shown to dynamically affect flagellar motility in several eukaryotic systems. The African trypanosome is a monoflagellated protozoan parasite and the etiological agent of sleeping sickness. Although cell motility has been implicated in disease progression, very little is currently known about biochemical control of the trypanosome flagellum. In this study, we assess the effects of extracellular changes in calcium and nickel concentration on trypanosome flagellar movement. Using a flow through chamber, we determine the relative changes in motility in individual trypanosomes in response to various concentrations of calcium and nickel, respectively. Extracellular concentrations of calcium and nickel (as low as 100 micromolar) significantly inhibit trypanosome cell motility. The effects are reversible, as indicated by the recovery of motion after removal of the calcium or nickel from the chamber. We are currently investigating the specific changes in flagellar oscillation and coordination that result from calcium and nickel, respectively. These results verify the presence of a calcium-responsive signaling mechanism(s) that regulates flagellar beat in trypanosomes.

  10. Flagellar expression in clinical isolates of non-typeable Haemophilus influenzae.

    PubMed

    Carabarin-Lima, Alejandro; Lozano-Zarain, Patricia; Castañeda-Lucio, Miguel; Martínez de la Peña, Claudia Fabiola; Martinez-Garcia, Julieta; Flores, Norarizbeth Lara; Cruz, Elías Campos de la; González-Posos, Sirenia; Rocha-Gracia, Rosa Del Carmen

    2017-05-01

    Haemophilus influenzae is a commensal organism found in the upper respiratory tract of humans. When H. influenzae becomes a pathogen, these bacteria can move out of their commensal niche and cause multiple respiratory tract diseases such as otitis media, sinusitis, conjunctivitis and bronchitis in children, and chronic obstructive pulmonary disease in adults. However, H. influenzae is currently considered a non-flagellate bacterium. In this study, 90 clinical isolates of H. influenzae strains (typeable and non-typeable) showed different degrees of the swarm-motility phenotype in vitro.Keys findings. One of these strains, NTHi BUAP96, showed the highest motility rate and its flagella were revealed using transmission electron microscopy and Ryu staining. Moreover, the flagellar genes fliC and flgH exhibited high homology with those of Actinobacillus pleuropneumoniae, Escherichia coli and Shigella flexneri. Furthermore, Western blot analysis, using anti-flagellin heterologous antibodies from E. coli, demonstrated cross-reaction with a protein present in NTHi BUAP96. This study provides, for the first time, information on flagellar expression in H. influenzae, representing an important finding related to its evolution and pathogenic potential.

  11. Role of flgA for Flagellar Biosynthesis and Biofilm Formation of Campylobacter jejuni NCTC11168.

    PubMed

    Kim, Joo-Sung; Park, Changwon; Kim, Yun-Ji

    2015-11-01

    The complex roles of flagella in the pathogenesis of Campylobacter jejuni, a major cause of worldwide foodborne diarrheal disease, are important. Compared with the wild-type, an insertional mutation of the flgA gene (cj0769c) demonstrated significant decrease in the biofilm formation of C. jejuni NCTC11168 on major food contact surfaces, such as polystyrene, stainless steel, and borosilicate glass. The flgA mutant was completely devoid of flagella and non-motile whereas the wild-type displayed the full-length flagella and motility. In addition, the biofilm formation of the wild-type was inversely dependent on the viscosity of the media. These results support that flagellar-mediated motility plays a significant role in the biofilm formation of C. jejuni NCTC11168. Moreover, our adhesion assay suggests that it plays an important role during biofilm maturation after initial attachment. Furthermore, C. jejuni NCTC11168 wild-type formed biofilm with a net-like structure of extracellular fiber-like material, but such a structure was significantly reduced in the biofilm of the flgA mutant. It supports that the extracellular fiber-like material may play a significant role in the biofilm formation of C. jejuni. This study demonstrated that flgA is essential for flagellar biosynthesis and motility, and plays a significant role in the biofilm formation of C. jejuni NCTC11168.

  12. Defects in the Flagellar Motor Increase Synthesis of Poly-γ-Glutamate in Bacillus subtilis

    PubMed Central

    Chan, Jia Mun; Guttenplan, Sarah B.

    2014-01-01

    Bacillus subtilis swims in liquid media and swarms over solid surfaces, and it encodes two sets of flagellar stator homologs. Here, we show that B. subtilis requires only the MotA/MotB stator during swarming motility and that the residues required for stator force generation are highly conserved from the Proteobacteria to the Firmicutes. We further find that mutants that abolish stator function also result in an overproduction of the extracellular polymer poly-γ-glutamate (PGA) to confer a mucoid colony phenotype. PGA overproduction appeared to be the result of an increase in the expression of the pgs operon that encodes genes for PGA synthesis. Transposon mutagenesis was conducted to identify insertions that abolished colony mucoidy and disruptions in known transcriptional regulators of PGA synthesis (Com and Deg two-component systems) as well as mutants defective in transcription-coupled DNA repair (Mfd)-reduced expression of the pgs operon. A final class of insertions disrupted proteins involved in the assembly of the flagellar filament (FliD, FliT, and FlgL), and these mutants did not reduce expression of the pgs operon, suggesting a second mechanism of PGA control. PMID:24296669

  13. Structure and assembly of the flagellar hook-basal body complex of Salmonella typhimurium

    SciTech Connect

    Jones, C.J.

    1989-01-01

    The hook-basal body (HBB) complex is a multi-component structure which comprises a significant part of the bacterial flagellar motor. Electrophoretic mobility shifts of HBB complex component proteins from four non-flagellate mutants have enabled the author to assign each protein as being the product of the gene defective in each of the respective strains. The author has purified and characterized HBB complexes lacking either the L ring or both the P and L rings, and concluded that the 27-kDa basal-body protein is the major component of the L ring, and that the 38-kDa basal-body protein is the major component of the P ring. He has sequenced the genes encoding the subunit proteins of the M, P, and L rings of the basal body, and have examined both the gene and deduced amino acid sequences for clues regarding the regulation of these genes and the structure of their products. By quantitating the amount of {sup 35}S incorporated into the component protein vivo and correcting for the amount of contained in each protein (as deduced from gene sequencing data), he has determined the relative stoichiometries of most of the known component proteins of the HBB complex. He has developed a protocol for differential {sup 35}S-radiolabeling of HBB complexes in vivo and used it to examine the HBB complex assembly process. He has identified proteins required for M-ring assembly or stabilization and for the possible initiation of rod assembly. The rod is not stable until the P ring is assembled onto it. The monomers of the P and L rings are exported independent of flagellar assembly. These radiolabeling experiments have also enabled me to identify several new component proteins of the HBB complex.

  14. Mutational Analysis of the Flagellar Rotor Protein FliN: Identification of Surfaces Important for Flagellar Assembly and Switching†

    PubMed Central

    Paul, Koushik; Harmon, Jacob G.; Blair, David F.

    2006-01-01

    FliN is a component of the flagellar switch complex in many bacterial species. The crystal structure is known for most of FliN, and a targeted cross-linking study (K. Paul and D. F. Blair, J. Bacteriol. 188:2502-2511, 2006) showed that it is organized in ring-shaped tetramers at the bottom of the basal body C ring. FliN is essential for flagellar assembly and direction switching, but its precise functions have not been defined. Here, we identify functionally important regions on FliN by systematic mutagenesis. Nonconservative mutations were made at positions sampling the surface of the protein, and the effects on flagellar assembly and function were measured. Flagellar assembly was disrupted by mutations in a conserved hydrophobic patch centered on the dimer twofold axis or by mutations on the surface that forms the dimer-dimer interface in the tetramer. The assembly defect in hydrophobic-patch mutants was partially rescued by overexpression of the flagellar export proteins FliH and FliI, and coprecipitation assays demonstrated a binding interaction between FliN and FliH that was weakened by mutations in the hydrophobic patch. Thus, FliN might contribute to export by providing binding sites for FliH or FliH-containing complexes. The region around the hydrophobic patch is also important for switching; certain mutations in or near the patch caused a smooth-swimming chemotaxis defect that in most cases could be partially rescued by overexpression of the clockwise-signaling protein CheY. The results indicate that FliN is more closely involved in switching than has been supposed, possibly contributing to the binding site for CheY on the switch. PMID:16816196

  15. Preparation and preliminary X-ray diffraction analysis of crystals of bacterial flagellar sigma factor σ{sup 28} in complex with the σ{sup 28}-binding region of its antisigma factor, FlgM

    SciTech Connect

    Okada, Kengo; Ichihara, Hisako; Takahashi, Hiroyuki; Fujita, Nobuyuki; Ishihama, Akira; Hakoshima, Toshio

    2007-03-01

    A complex of E. coli flagellar and chemotaxis-specific sigma factor σ{sup 28} bound to the σ{sup 28}-binding region of its antisigma factor FlgM was crystallized. Diffraction data were collected to a resolution of 2.7 Å. The sigma 28 kDa (σ{sup 28}) factor is a transcription factor specific for the expression of bacterial flagellar and chemotaxis genes. Its antisigma factor, FlgM, binds σ{sup 28} factor and inhibits its activity as a transcription factor. In this study, crystals of the complex between Escherichia coli σ{sup 28} and the C-terminal σ{sup 28}-binding region of FlgM were obtained. The crystals belong to space group P3{sub 1}21 or P3{sub 2}21, with unit-cell parameters a = b = 106.7 (2), c = 51.74 (3) Å, containing one complex in the crystallographic asymmetric unit. An X-ray intensity data set was collected to a resolution of 2.7 Å.

  16. Evolution. Evolutionary resurrection of flagellar motility via rewiring of the nitrogen regulation system.

    PubMed

    Taylor, Tiffany B; Mulley, Geraldine; Dills, Alexander H; Alsohim, Abdullah S; McGuffin, Liam J; Studholme, David J; Silby, Mark W; Brockhurst, Michael A; Johnson, Louise J; Jackson, Robert W

    2015-02-27

    A central process in evolution is the recruitment of genes to regulatory networks. We engineered immotile strains of the bacterium Pseudomonas fluorescens that lack flagella due to deletion of the regulatory gene fleQ. Under strong selection for motility, these bacteria consistently regained flagella within 96 hours via a two-step evolutionary pathway. Step 1 mutations increase intracellular levels of phosphorylated NtrC, a distant homolog of FleQ, which begins to commandeer control of the fleQ regulon at the cost of disrupting nitrogen uptake and assimilation. Step 2 is a switch-of-function mutation that redirects NtrC away from nitrogen uptake and toward its novel function as a flagellar regulator. Our results demonstrate that natural selection can rapidly rewire regulatory networks in very few, repeatable mutational steps.

  17. CRIS—A Novel cAMP-Binding Protein Controlling Spermiogenesis and the Development of Flagellar Bending

    PubMed Central

    Krähling, Anke Miriam; Alvarez, Luis; Debowski, Katharina; Van, Qui; Gunkel, Monika; Irsen, Stephan; Al-Amoudi, Ashraf; Strünker, Timo; Kremmer, Elisabeth; Krause, Eberhard; Voigt, Ingo; Wörtge, Simone; Waisman, Ari; Weyand, Ingo; Seifert, Reinhard; Kaupp, Ulrich Benjamin; Wachten, Dagmar

    2013-01-01

    The second messengers cAMP and cGMP activate their target proteins by binding to a conserved cyclic nucleotide-binding domain (CNBD). Here, we identify and characterize an entirely novel CNBD-containing protein called CRIS (cyclic nucleotide receptor involved in sperm function) that is unrelated to any of the other members of this protein family. CRIS is exclusively expressed in sperm precursor cells. Cris-deficient male mice are either infertile due to a lack of sperm resulting from spermatogenic arrest, or subfertile due to impaired sperm motility. The motility defect is caused by altered Ca2+ regulation of flagellar beat asymmetry, leading to a beating pattern that is reminiscent of sperm hyperactivation. Our results suggest that CRIS interacts during spermiogenesis with Ca2+-regulated proteins that—in mature sperm—are involved in flagellar bending. PMID:24339785

  18. Applying torque to the Escherichia coli flagellar motor using magnetic tweezers.

    PubMed

    van Oene, Maarten M; Dickinson, Laura E; Cross, Bronwen; Pedaci, Francesco; Lipfert, Jan; Dekker, Nynke H

    2017-03-07

    The bacterial flagellar motor of Escherichia coli is a nanoscale rotary engine essential for bacterial propulsion. Studies on the power output of single motors rely on the measurement of motor torque and rotation under external load. Here, we investigate the use of magnetic tweezers, which in principle allow the application and active control of a calibrated load torque, to study single flagellar motors in Escherichia coli. We manipulate the external load on the motor by adjusting the magnetic field experienced by a magnetic bead linked to the motor, and we probe the motor's response. A simple model describes the average motor speed over the entire range of applied fields. We extract the motor torque at stall and find it to be similar to the motor torque at drag-limited speed. In addition, use of the magnetic tweezers allows us to force motor rotation in both forward and backward directions. We monitor the motor's performance before and after periods of forced rotation and observe no destructive effects on the motor. Our experiments show how magnetic tweezers can provide active and fast control of the external load while also exposing remaining challenges in calibration. Through their non-invasive character and straightforward parallelization, magnetic tweezers provide an attractive platform to study nanoscale rotary motors at the single-motor level.

  19. Applying torque to the Escherichia coli flagellar motor using magnetic tweezers

    PubMed Central

    van Oene, Maarten M.; Dickinson, Laura E.; Cross, Bronwen; Pedaci, Francesco; Lipfert, Jan; Dekker, Nynke H.

    2017-01-01

    The bacterial flagellar motor of Escherichia coli is a nanoscale rotary engine essential for bacterial propulsion. Studies on the power output of single motors rely on the measurement of motor torque and rotation under external load. Here, we investigate the use of magnetic tweezers, which in principle allow the application and active control of a calibrated load torque, to study single flagellar motors in Escherichia coli. We manipulate the external load on the motor by adjusting the magnetic field experienced by a magnetic bead linked to the motor, and we probe the motor’s response. A simple model describes the average motor speed over the entire range of applied fields. We extract the motor torque at stall and find it to be similar to the motor torque at drag-limited speed. In addition, use of the magnetic tweezers allows us to force motor rotation in both forward and backward directions. We monitor the motor’s performance before and after periods of forced rotation and observe no destructive effects on the motor. Our experiments show how magnetic tweezers can provide active and fast control of the external load while also exposing remaining challenges in calibration. Through their non-invasive character and straightforward parallelization, magnetic tweezers provide an attractive platform to study nanoscale rotary motors at the single-motor level. PMID:28266562

  20. Torque-speed relationship of the bacterial flagellar motor.

    PubMed

    Xing, Jianhua; Bai, Fan; Berry, Richard; Oster, George

    2006-01-31

    Many swimming bacteria are propelled by flagellar filaments driven by a rotary motor. Each of these tiny motors can generate an impressive torque. The motor torque vs. speed relationship is considered one of the most important measurable characteristics of the motor and therefore is a major criterion for judging models proposed for the working mechanism. Here we give an explicit explanation for this torque-speed curve. The same physics also can explain certain puzzling properties of other motors.

  1. Torque–speed relationship of the bacterial flagellar motor

    PubMed Central

    Xing, Jianhua; Bai, Fan; Berry, Richard; Oster, George

    2006-01-01

    Many swimming bacteria are propelled by flagellar filaments driven by a rotary motor. Each of these tiny motors can generate an impressive torque. The motor torque vs. speed relationship is considered one of the most important measurable characteristics of the motor and therefore is a major criterion for judging models proposed for the working mechanism. Here we give an explicit explanation for this torque–speed curve. The same physics also can explain certain puzzling properties of other motors. PMID:16432218

  2. Association of Lis1 with outer arm dynein is modulated in response to alterations in flagellar motility

    PubMed Central

    Rompolas, Panteleimon; Patel-King, Ramila S.; King, Stephen M.

    2012-01-01

    The cytoplasmic dynein regulatory factor Lis1, which induces a persistent tight binding to microtubules and allows for transport of cargoes under high-load conditions, is also present in motile cilia/flagella. We observed that Lis1 levels in flagella of Chlamydomonas strains that exhibit defective motility due to mutation of various axonemal substructures were greatly enhanced compared with wild type; this increase was absolutely dependent on the presence within the flagellum of the outer arm dynein α heavy chain/light chain 5 thioredoxin unit. To assess whether cells might interpret defective motility as a “high-load environment,” we reduced the flagellar beat frequency of wild-type cells through enhanced viscous load and by reductive stress; both treatments resulted in increased levels of flagellar Lis1, which altered the intrinsic beat frequency of the trans flagellum. Differential extraction of Lis1 from wild-type and mutant axonemes suggests that the affinity of outer arm dynein for Lis1 is directly modulated. In cytoplasm, Lis1 localized to two punctate structures, one of which was located near the base of the flagella. These data reveal that the cell actively monitors motility and dynamically modulates flagellar levels of the dynein regulatory factor Lis1 in response to imposed alterations in beat parameters. PMID:22855525

  3. Escherichia coli swimming is robust against variations in flagellar number

    PubMed Central

    Mears, Patrick J; Koirala, Santosh; Rao, Chris V; Golding, Ido; Chemla, Yann R

    2014-01-01

    Bacterial chemotaxis is a paradigm for how environmental signals modulate cellular behavior. Although the network underlying this process has been studied extensively, we do not yet have an end-to-end understanding of chemotaxis. Specifically, how the rotational states of a cell’s flagella cooperatively determine whether the cell ‘runs’ or ‘tumbles’ remains poorly characterized. Here, we measure the swimming behavior of individual E. coli cells while simultaneously detecting the rotational states of each flagellum. We find that a simple mathematical expression relates the cell’s run/tumble bias to the number and average rotational state of its flagella. However, due to inter-flagellar correlations, an ‘effective number’ of flagella—smaller than the actual number—enters into this relation. Data from a chemotaxis mutant and stochastic modeling suggest that fluctuations of the regulator CheY-P are the source of flagellar correlations. A consequence of inter-flagellar correlations is that run/tumble behavior is only weakly dependent on number of flagella. DOI: http://dx.doi.org/10.7554/eLife.01916.001 PMID:24520165

  4. Direct evidence of flagellar synchronization through hydrodynamic interactions

    NASA Astrophysics Data System (ADS)

    Brumley, Douglas; Polin, Marco; Wan, Kirsty; Goldstein, Raymond

    2013-11-01

    Eukaryotic cilia and flagella exhibit striking coordination, from the synchronous beating of two flagella in Chlamydomonas to the metachronal waves and large-scale flows displayed by carpets of cilia. However, the precise mechanisms responsible for flagellar synchronization remain unclear. We perform a series of experiments involving two individual flagella in a quiescent fluid. Cells are isolated from the colonial alga Volvox carteri, held in place at a fixed distance d, and oriented so that their flagellar beating planes coincide. In this fashion, we are able to explicitly assess the role of hydrodynamics in achieving synchronization. For closely separated cells, the flagella are capable of exhibiting a phase-locked state for thousands of beats at a time, despite significant differences in their intrinsic frequencies. For intermediate values of d, synchronous periods are interrupted by brief phase slips, while for d >> 1 the flagellar phase difference drifts almost linearly with time. The coupling strength extracted through analysis of the synchronization statistics exhibits excellent agreement with hydrodynamic predictions. This study unambiguously reveals that flagella coupled only through hydrodynamics are capable of exhibiting robust synchrony.

  5. Acid extrusion from human spermatozoa is mediated by flagellar voltage-gated proton channel.

    PubMed

    Lishko, Polina V; Botchkina, Inna L; Fedorenko, Andriy; Kirichok, Yuriy

    2010-02-05

    Human spermatozoa are quiescent in the male reproductive system and must undergo activation once introduced into the female reproductive tract. This process is known to require alkalinization of sperm cytoplasm, but the mechanism responsible for transmembrane proton extrusion has remained unknown because of the inability to measure membrane conductance in human sperm. Here, by successfully patch clamping human spermatozoa, we show that proton channel Hv1 is their dominant proton conductance. Hv1 is confined to the principal piece of the sperm flagellum, where it is expressed at unusually high density. Robust flagellar Hv1-dependent proton conductance is activated by membrane depolarization, an alkaline extracellular environment, endocannabinoid anandamide, and removal of extracellular zinc, a potent Hv1 blocker. Hv1 allows only outward transport of protons and is therefore dedicated to inducing intracellular alkalinization and activating spermatozoa. The importance of Hv1 for sperm activation makes it an attractive target for controlling male fertility.

  6. Helicobacter pylori CheZ(HP) and ChePep form a novel chemotaxis-regulatory complex distinct from the core chemotaxis signaling proteins and the flagellar motor.

    PubMed

    Lertsethtakarn, Paphavee; Howitt, Michael R; Castellon, Juan; Amieva, Manuel R; Ottemann, Karen M

    2015-09-01

    Chemotaxis is important for Helicobacter pylori to colonize the stomach. Like other bacteria, H. pylori uses chemoreceptors and conserved chemotaxis proteins to phosphorylate the flagellar rotational response regulator, CheY, and modulate the flagellar rotational direction. Phosphorylated CheY is returned to its non-phosphorylated state by phosphatases such as CheZ. In previously studied cases, chemotaxis phosphatases localize to the cellular poles by interactions with either the CheA chemotaxis kinase or flagellar motor proteins. We report here that the H. pylori CheZ, CheZ(HP), localizes to the poles independently of the flagellar motor, CheA, and all typical chemotaxis proteins. Instead, CheZ(HP) localization depends on the chemotaxis regulatory protein ChePep, and reciprocally, ChePep requires CheZ(HP) for its polar localization. We furthermore show that these proteins interact directly. Functional domain mapping of CheZ(HP) determined the polar localization motif lies within the central domain of the protein and that the protein has regions outside of the active site that participate in chemotaxis. Our results suggest that CheZ(HP) and ChePep form a distinct complex. These results therefore suggest the intriguing idea that some phosphatases localize independently of the other chemotaxis and motility proteins, possibly to confer unique regulation on these proteins' activities.

  7. Anatomical and Molecular Design of the Drosophila Antenna as a Flagellar Auditory Organ

    PubMed Central

    TODI, SOKOL V.; SHARMA, YASHODA; EBERL, DANIEL F.

    2007-01-01

    The molecular basis of hearing is less well understood than many other senses. However, recent studies in Drosophila have provided some important steps towards a molecular understanding of hearing. In this report, we summarize these findings and their implications on the relationship between hearing and touch. In Drosophila, hearing is accomplished by Johnston’s Organ, a chordotonal organ containing over 150 scolopidia within the second antennal segment. We will discuss anatomical features of the antenna and how they contribute to the function of this flagellar auditory receptor. The effects of several mutants, identified through mutagenesis screens or as homologues of vertebrate auditory genes, will be summarized. Based on evidence gathered from these studies, we propose a speculative model for how the chordotonal organ might function. PMID:15252880

  8. Function of the Histone-Like Protein H-NS in Motility of Escherichia coli: Multiple Regulatory Roles Rather than Direct Action at the Flagellar Motor

    PubMed Central

    Kim, Eun A

    2015-01-01

    ABSTRACT A number of investigations of Escherichia coli have suggested that the DNA-binding protein H-NS, in addition to its well-known functions in chromosome organization and gene regulation, interacts directly with the flagellar motor to modulate its function. Here, in a study initially aimed at characterizing the H-NS/motor interaction further, we identify problems and limitations in the previous work that substantially weaken the case for a direct H-NS/motor interaction. Null hns mutants are immotile, largely owing to the downregulation of the flagellar master regulators FlhD and FlhC. We, and others, previously reported that an hns mutant remains poorly motile even when FlhDC are expressed constitutively. In the present work, we use better-engineered strains to show that the motility defect in a Δhns, FlhDC-constitutive strain is milder than that reported previously and does not point to a direct action of H-NS at the motor. H-NS regulates numerous genes and might influence motility via a number of regulatory molecules besides FlhDC. To examine the sources of the motility defect that persists in an FlhDC-constitutive Δhns mutant, we measured transcript levels and overexpression effects of a number of genes in candidate regulatory pathways. The results indicate that H-NS influences motility via multiple regulatory linkages that include, minimally, the messenger molecule cyclic di-GMP, the biofilm regulatory protein CsgD, and the sigma factors σS and σF. The results are in accordance with the more standard view of H-NS as a regulator of gene expression rather than a direct modulator of flagellar motor performance. IMPORTANCE Data from a number of previous studies have been taken to indicate that the nucleoid-organizing protein H-NS influences motility not only by its well-known DNA-based mechanisms but also by binding directly to the flagellar motor to alter function. In this study, H-NS is shown to influence motility through diverse regulatory pathways

  9. Two flagellar stators and their roles in motility and virulence in Pseudomonas syringae pv. tabaci 6605.

    PubMed

    Kanda, Eiko; Tatsuta, Takafumi; Suzuki, Tomoko; Taguchi, Fumiko; Naito, Kana; Inagaki, Yoshishige; Toyoda, Kazuhiro; Shiraishi, Tomonori; Ichinose, Yuki

    2011-02-01

    The motor proteins around the flagellar basal body consist of two cytoplasmic membrane proteins, MotA and MotB, and function as a complex that acts as the stator to generate the torque that drives rotation. Genome analysis of several Pseudomonas syringae pathovars revealed that there are two sets of genes encoding motor proteins: motAB and motCD. Deduced amino acid sequences for MotA/B and MotC/D showed homologies to the H(+)-driven stator from Escherichia coli and Na(+)-driven stator from Vibrio alginolyticus, respectively. However, the swimming motility of P. syringae pv. tabaci (Pta) 6605 was inhibited by the protonophore carbonyl cyanide m-chlorophenylhydrazone but not by the sodium stator-specific inhibitor phenamil. To identify a gene encoding the stator protein required for motility, ∆motAB, ∆motCD, and ∆motABCD mutants were generated. The ∆motCD mutant had remarkably reduced and the ∆motABCD mutant completely abolished swimming motilities, whereas the ∆motAB mutant retained some degree of these abilities. The ∆motCD and ∆motABCD mutants did not produce N-acyl-homoserine lactones (AHLs), quorum-sensing molecules in this pathogen, and remarkably reduced the ability to cause disease in host tobacco leaves, as we previously observed in the ∆fliC mutant strain. These results strongly indicate that both stator pairs in Pta 6605 are proton-dependent and that MotCD is important for not only flagellar motility but also for production of AHLs and the ability to cause disease in host plants.

  10. Bone Regeneration Using Gene-Activated Matrices.

    PubMed

    D'Mello, Sheetal; Atluri, Keerthi; Geary, Sean M; Hong, Liu; Elangovan, Satheesh; Salem, Aliasger K

    2017-01-01

    Gene delivery to bone is a potential therapeutic strategy for directed, sustained, and regulated protein expression. Tissue engineering strategies for bone regeneration include delivery of proteins, genes (viral and non-viral-mediated delivery), and/or cells to the bone defect site. In addition, biomimetic scaffolds and scaffolds incorporating bone anabolic agents greatly enhance the bone repair process. Regional gene therapy has the potential of enhancing bone defect healing and bone regeneration by delivering osteogenic genes locally to the osseous lesions, thereby reducing systemic toxicity and the need for using supraphysiological dosages of therapeutic proteins. By implanting gene-activated matrices (GAMs), sustained gene expression and continuous osteogenic protein production in situ can be achieved in a way that stimulates osteogenesis and bone repair within osseous defects. Critical parameters substantially affecting the therapeutic efficacy of gene therapy include the choice of osteogenic transgene(s), selection of non-viral or viral vectors, the wound environment, and the selection of ex vivo and in vivo gene delivery strategies, such as GAMs. It is critical for gene therapy applications that clinically beneficial amounts of proteins are synthesized endogenously within and around the lesion in a sustained manner. It is therefore necessary that reliable and reproducible methods of gene delivery be developed and tested for their efficacy and safety before translating into clinical practice. Practical considerations such as the age, gender, and systemic health of patients and the nature of the disease process also need to be taken into account in order to personalize the treatments and progress towards developing a clinically applicable gene therapy for healing bone defects. This review discusses tissue engineering strategies to regenerate bone with specific focus on non-viral gene delivery systems.

  11. Bacterial flagellar microhydrodynamics: Laminar flow over complex flagellar filaments, analog archimedean screws and cylinders, and its perturbations.

    PubMed

    Trachtenberg, Shlomo; Fishelov, Dalia; Ben-Artzi, Matania

    2003-09-01

    The flagellar filament, the bacterial organelle of motility, is the smallest rotary propeller known. It consists of 1), a basal body (part of which is the proton driven rotary motor), 2), a hook (universal joint-allowing for off-axial transmission of rotary motion), and 3), a filament (propeller-a long, rigid, supercoiled helical assembly allowing for the conversion of rotary motion into linear thrust). Helically perturbed (so-called "complex") filaments have a coarse surface composed of deep grooves and ridges following the three-start helical lines. These surface structures, reminiscent of a turbine or Archimedean screw, originate from symmetry reduction along the six-start helical lines due to dimerization of the flagellin monomers from which the filament self assembles. Using high-resolution electron microscopy and helical image reconstruction methods, we calculated three-dimensional density maps of the complex filament of Rhizobium lupini H13-3 and determined its surface pattern and boundaries. The helical symmetry of the filament allows viewing it as a stack of identical slices spaced axially and rotated by constant increments. Here we use the closed outlines of these slices to explore, in two dimensions, the hydrodynamic effect of the turbine-like boundaries of the flagellar filament. In particular, we try to determine if, and under what conditions, transitions from laminar to turbulent flow (or perturbations of the laminar flow) may occur on or near the surface of the bacterial propeller. To address these questions, we apply the boundary element method in a manner allowing the handling of convoluted boundaries. We tested the method on several simple, well-characterized cylindrical structures before applying it to real, highly convoluted biological surfaces and to simplified mechanical analogs. Our results indicate that under extreme structural and functional conditions, and at low Reynolds numbers, a deviation from laminar flow might occur on the flagellar

  12. Individual Flagellar Waveform Affects Collective Behavior of Chlamydomonas reinhardtii.

    PubMed

    Kage, Azusa; Mogami, Yoshihiro

    2015-08-01

    Bioconvection is a form of collective motion that occurs spontaneously in the suspension of swimming microorganisms. In a previous study, we quantitatively described the "pattern transition," a phase transition phenomenon that so far has exclusively been observed in bioconvection of the unicellular green alga Chlamydomonas. We suggested that the transition could be induced by changes in the balance between the gravitational and shear-induced torques, both of which act to determine the orientation of the organism in the shear flow. As both of the torques should be affected by the geometry of the Chlamydomonas cell, alteration in the flagellar waveform might change the extent of torque generation by altering overall geometry of the cell. Based on this working hypothesis, we examined bioconvection behavior of two flagellar mutants of Chlamydomonas reinhardtii, ida1 and oda2, making reference to the wild type. Flagella of ida1 beat with an abnormal waveform, while flagella of oda2 show a normal waveform but lower beat frequency. As a result, both mutants had swimming speed of less than 50% of the wild type. ida1 formed bioconvection patterns with smaller spacing than those of wild type and oda2. Two-axis view revealed the periodic movement of the settling blobs of ida1, while oda2 showed qualitatively similar behavior to that of wild type. Unexpectedly, ida1 showed stronger negative gravitaxis than did wild type, while oda2 showed relatively weak gravitaxis. These findings suggest that flagellar waveform, not swimming speed or beat frequency, strongly affect bioconvection behavior in C. reinhardtii.

  13. Second-Chance Signal Transduction Explains Cooperative Flagellar Switching

    PubMed Central

    Zot, Henry G.; Hasbun, Javier E.; Van Minh, Nguyen

    2012-01-01

    The reversal of flagellar motion (switching) results from the interaction between a switch complex of the flagellar rotor and a torque-generating stationary unit, or stator (motor unit). To explain the steeply cooperative ligand-induced switching, present models propose allosteric interactions between subunits of the rotor, but do not address the possibility of a reaction that stimulates a bidirectional motor unit to reverse direction of torque. During flagellar motion, the binding of a ligand-bound switch complex at the dwell site could excite a motor unit. The probability that another switch complex of the rotor, moving according to steady-state rotation, will reach the same dwell site before that motor unit returns to ground state will be determined by the independent decay rate of the excited-state motor unit. Here, we derive an analytical expression for the energy coupling between a switch complex and a motor unit of the stator complex of a flagellum, and demonstrate that this model accounts for the cooperative switching response without the need for allosteric interactions. The analytical result can be reproduced by simulation when (1) the motion of the rotor delivers a subsequent ligand-bound switch to the excited motor unit, thereby providing the excited motor unit with a second chance to remain excited, and (2) the outputs from multiple independent motor units are constrained to a single all-or-none event. In this proposed model, a motor unit and switch complex represent the components of a mathematically defined signal transduction mechanism in which energy coupling is driven by steady-state and is regulated by stochastic ligand binding. Mathematical derivation of the model shows the analytical function to be a general form of the Hill equation (Hill AV (1910) The possible effects of the aggregation of the molecules of haemoglobin on its dissociation curves. J Physiol 40: iv–vii). PMID:22844429

  14. [Structure and function of the bacterial flagellar type III protein export system in Salmonella
].

    PubMed

    Minamino, Tohru

    2015-01-01

    The bacterial flagellum is a filamentous organelle that propels the bacterial cell body in liquid media. For construction of the bacterial flagellum beyond the cytoplasmic membrane, flagellar component proteins are transported by its specific protein export apparatus from the cytoplasm to the distal end of the growing flagellar structure. The flagellar export apparatus consists of a transmembrane export gate complex and a cytoplasmic ATPase ring complex. Flagellar substrate-specific chaperones bind to their cognate substrates in the cytoplasm and escort the substrates to the docking platform of the export gate. The export apparatus utilizes ATP and proton motive force across the cytoplasmic membrane as the energy sources to drive protein export and coordinates protein export with assembly by ordered export of substrates to parallel with their order of assembly. In this review, we summarize our current understanding of the structure and function of the flagellar protein export system in Salmonella enterica serovar Typhimurium.

  15. Growth rate control of flagellar assembly in Escherichia coli strain RP437

    PubMed Central

    Sim, Martin; Koirala, Santosh; Picton, David; Strahl, Henrik; Hoskisson, Paul A.; Rao, Christopher V.; Gillespie, Colin S.; Aldridge, Phillip D.

    2017-01-01

    The flagellum is a rotary motor that enables bacteria to swim in liquids and swarm over surfaces. Numerous global regulators control flagellar assembly in response to cellular and environmental factors. Previous studies have also shown that flagellar assembly is affected by the growth-rate of the cell. However, a systematic study has not yet been described under controlled growth conditions. Here, we investigated the effect of growth rate on flagellar assembly in Escherichia coli using steady-state chemostat cultures where we could precisely control the cell growth-rate. Our results demonstrate that flagellar abundance correlates with growth rate, where faster growing cells produce more flagella. They also demonstrate that this growth-rate dependent control occurs through the expression of the flagellar master regulator, FlhD4C2. Collectively, our results demonstrate that motility is intimately coupled to the growth-rate of the cell. PMID:28117390

  16. Involvement of the flagellar assembly pathway in Vibrio alginolyticus adhesion under environmental stresses

    PubMed Central

    Wang, Lu; Huang, Lixing; Su, Yongquan; Qin, Yingxue; Kong, Wendi; Ma, Ying; Xu, Xiaojin; Lin, Mao; Zheng, Jiang; Yan, Qingpi

    2015-01-01

    Adhesion is an important virulence factor of Vibrio alginolyticus. This factor may be affected by environmental conditions; however, its molecular mechanism remains unclear. In our previous research, adhesion deficient strains were obtained by culturing V. alginolyticus under stresses including Cu, Pb, Hg, and low pH. With RNA-seq and bioinformatics analysis, we found that all of these stress treatments significantly affected the flagellar assembly pathway, which may play an important role in V. alginolyticus adhesion. Therefore, we hypothesized that the environmental stresses of the flagellar assembly pathway may be one way in which environmental conditions affect adhesion. To verify our hypothesis, a bioinformatics analysis, QPCR, RNAi, in vitro adhesion assay and motility assay were performed. Our results indicated that (1) the flagellar assembly pathway was sensitive to environmental stresses, (2) the flagellar assembly pathway played an important role in V. alginolyticus adhesion, and (3) motility is not the only way in which the flagellar assembly pathway affects adhesion. PMID:26322276

  17. An ultrasonic motor model for bacterial flagellar motors.

    PubMed

    Atsumi, T

    2001-11-07

    A model for the transduction of energy occurring in bacterial flagellar motors is presented. In this model, the influx of ions across the channel causes the cyclic conformational change of the channel itself, which in turn produces travelling waves in one of the subcomponents of the motor, the C ring. This wave stabilizes the cyclical movement of the channel which generates the rotating force. The estimated frequency of cyclic conformational change is between 36 kHz and 6.3 MHz, i.e. in the ultrasonic range. This phenomenon is therefore referred to as the ultrasonic micromotor of microorganisms. Copyright 2001 Academic Press.

  18. The Deep-Sea Bacterium Photobacterium profundum SS9 Utilizes Separate Flagellar Systems for Swimming and Swarming under High-Pressure Conditions ▿ †

    PubMed Central

    Eloe, Emiley A.; Lauro, Federico M.; Vogel, Rudi F.; Bartlett, Douglas H.

    2008-01-01

    Motility is a critical function needed for nutrient acquisition, biofilm formation, and the avoidance of harmful chemicals and predators. Flagellar motility is one of the most pressure-sensitive cellular processes in mesophilic bacteria; therefore, it is ecologically relevant to determine how deep-sea microbes have adapted their motility systems for functionality at depth. In this study, the motility of the deep-sea piezophilic bacterium Photobacterium profundum SS9 was investigated and compared with that of the related shallow-water piezosensitive strain Photobacterium profundum 3TCK, as well as that of the well-studied piezosensitive bacterium Escherichia coli. The SS9 genome contains two flagellar gene clusters: a polar flagellum gene cluster (PF) and a putative lateral flagellum gene cluster (LF). In-frame deletions were constructed in the two flagellin genes located within the PF cluster (flaA and flaC), the one flagellin gene located within the LF cluster (flaB), a component of a putative sodium-driven flagellar motor (motA2), and a component of a putative proton-driven flagellar motor (motA1). SS9 PF flaA, flaC, and motA2 mutants were defective in motility under all conditions tested. In contrast, the flaB and motA1 mutants were defective only under conditions of high pressure and high viscosity. flaB and motA1 gene expression was strongly induced by elevated pressure plus increased viscosity. Direct swimming velocity measurements were obtained using a high-pressure microscopic chamber, where increases in pressure resulted in a striking decrease in swimming velocity for E. coli and a gradual reduction for 3TCK which proceeded up to 120 MPa, while SS9 increased swimming velocity at 30 MPa and maintained motility up to a maximum pressure of 150 MPa. Our results indicate that P. profundum SS9 possesses two distinct flagellar systems, both of which have acquired dramatic adaptations for optimal functionality under high-pressure conditions. PMID:18723648

  19. CDKL5 regulates flagellar length and localizes to the base of the flagella in Chlamydomonas.

    PubMed

    Tam, Lai-Wa; Ranum, Paul T; Lefebvre, Paul A

    2013-03-01

    The length of Chlamydomonas flagella is tightly regulated. Mutations in four genes-LF1, LF2, LF3, and LF4-cause cells to assemble flagella up to three times wild-type length. LF2 and LF4 encode protein kinases. Here we describe a new gene, LF5, in which null mutations cause cells to assemble flagella of excess length. The LF5 gene encodes a protein kinase very similar in sequence to the protein kinase CDKL5. In humans, mutations in this kinase cause a severe form of juvenile epilepsy. The LF5 protein localizes to a unique location: the proximal 1 μm of the flagella. The proximal localization of the LF5 protein is lost when genes that make up the proteins in the cytoplasmic length regulatory complex (LRC)-LF1, LF2, and LF3-are mutated. In these mutants LF5p becomes localized either at the distal tip of the flagella or along the flagellar length, indicating that length regulation involves, at least in part, control of LF5p localization by the LRC.

  20. Evidence for Loss of a Partial Flagellar Glycolytic Pathway during Trypanosomatid Evolution

    PubMed Central

    Brown, Robert W. B.; Collingridge, Peter W.; Gull, Keith; Rigden, Daniel J.; Ginger, Michael L.

    2014-01-01

    Classically viewed as a cytosolic pathway, glycolysis is increasingly recognized as a metabolic pathway exhibiting surprisingly wide-ranging variations in compartmentalization within eukaryotic cells. Trypanosomatid parasites provide an extreme view of glycolytic enzyme compartmentalization as several glycolytic enzymes are found exclusively in peroxisomes. Here, we characterize Trypanosoma brucei flagellar proteins resembling glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK): we show the latter associates with the axoneme and the former is a novel paraflagellar rod component. The paraflagellar rod is an essential extra-axonemal structure in trypanosomes and related protists, providing a platform into which metabolic activities can be built. Yet, bioinformatics interrogation and structural modelling indicate neither the trypanosome PGK-like nor the GAPDH-like protein is catalytically active. Orthologs are present in a free-living ancestor of the trypanosomatids, Bodo saltans: the PGK-like protein from B. saltans also lacks key catalytic residues, but its GAPDH-like protein is predicted to be catalytically competent. We discuss the likelihood that the trypanosome GAPDH-like and PGK-like proteins constitute molecular evidence for evolutionary loss of a flagellar glycolytic pathway, either as a consequence of niche adaptation or the re-localization of glycolytic enzymes to peroxisomes and the extensive changes to glycolytic flux regulation that accompanied this re-localization. Evidence indicating loss of localized ATP provision via glycolytic enzymes therefore provides a novel contribution to an emerging theme of hidden diversity with respect to compartmentalization of the ubiquitous glycolytic pathway in eukaryotes. A possibility that trypanosome GAPDH-like protein additionally represents a degenerate example of a moonlighting protein is also discussed. PMID:25050549

  1. The de novo synthesis of GDP-fucose is essential for flagellar adhesion and cell growth in Trypanosoma brucei.

    PubMed

    Turnock, Daniel C; Izquierdo, Luis; Ferguson, Michael A J

    2007-09-28

    The protozoan parasite Trypanosoma brucei causes human African sleeping sickness in sub-Saharan Africa. The parasite makes several essential glycoproteins, which has led to the investigation of the sugar nucleotides and glycosyltransferases required to synthesize these structures. Fucose is a common sugar in glycoconjugates from many organisms; however, the sugar nucleotide donor GDP-fucose was only recently detected in T. brucei, and the importance of fucose metabolism in this organism is not known. In this paper, we identified the genes encoding functional GDP-fucose biosynthesis enzymes in T. brucei and created conditional null mutants of TbGMD, the gene encoding the first enzyme in the pathway from GDP-mannose to GDP-fucose, in both bloodstream form and procyclic form parasites. Under nonpermissive conditions, both life cycle forms of the parasite became depleted in GDP-fucose and suffered growth arrest, demonstrating that fucose metabolism is essential to both life cycle stages. In procyclic form parasites, flagellar detachment from the cell body was also observed under nonpermissive conditions, suggesting that fucose plays a significant role in flagellar adhesion. Fluorescence microscopy of epitope-tagged TbGMD revealed that this enzyme is localized in glycosomes, despite the absence of PTS-1 or PTS-2 target sequences.

  2. Gains of Bacterial Flagellar Motility in a Fungal World

    PubMed Central

    Pion, Martin; Bshary, Redouan; Bindschedler, Saskia; Filippidou, Sevasti; Wick, Lukas Y.; Job, Daniel

    2013-01-01

    The maintenance of energetically costly flagella by bacteria in non-water-saturated media, such as soil, still presents an evolutionary conundrum. Potential explanations have focused on rare flooding events allowing dispersal. Such scenarios, however, overlook bacterial dispersal along mycelia as a possible transport mechanism in soils. The hypothesis tested in this study is that dispersal along fungal hyphae may lead to an increase in the fitness of flagellated bacteria and thus offer an alternative explanation for the maintenance of flagella even in unsaturated soils. Dispersal along fungal hyphae was shown for a diverse array of motile bacteria. To measure the fitness effect of dispersal, additional experiments were conducted in a model system mimicking limited dispersal, using Pseudomonas putida KT2440 and its nonflagellated (ΔfliM) isogenic mutant in the absence or presence of Morchella crassipes mycelia. In the absence of the fungus, flagellar motility was beneficial solely under conditions of water saturation allowing dispersal, while under conditions limiting dispersal, the nonflagellated mutant exhibited a higher level of fitness than the wild-type strain. In contrast, in the presence of a mycelial network under conditions limiting dispersal, the flagellated strain was able to disperse using the mycelial network and had a higher level of fitness than the mutant. On the basis of these results, we propose that the benefit of mycelium-associated dispersal helps explain the persistence of flagellar motility in non-water-saturated environments. PMID:23995942

  3. Gains of bacterial flagellar motility in a fungal world.

    PubMed

    Pion, Martin; Bshary, Redouan; Bindschedler, Saskia; Filippidou, Sevasti; Wick, Lukas Y; Job, Daniel; Junier, Pilar

    2013-11-01

    The maintenance of energetically costly flagella by bacteria in non-water-saturated media, such as soil, still presents an evolutionary conundrum. Potential explanations have focused on rare flooding events allowing dispersal. Such scenarios, however, overlook bacterial dispersal along mycelia as a possible transport mechanism in soils. The hypothesis tested in this study is that dispersal along fungal hyphae may lead to an increase in the fitness of flagellated bacteria and thus offer an alternative explanation for the maintenance of flagella even in unsaturated soils. Dispersal along fungal hyphae was shown for a diverse array of motile bacteria. To measure the fitness effect of dispersal, additional experiments were conducted in a model system mimicking limited dispersal, using Pseudomonas putida KT2440 and its nonflagellated (ΔfliM) isogenic mutant in the absence or presence of Morchella crassipes mycelia. In the absence of the fungus, flagellar motility was beneficial solely under conditions of water saturation allowing dispersal, while under conditions limiting dispersal, the nonflagellated mutant exhibited a higher level of fitness than the wild-type strain. In contrast, in the presence of a mycelial network under conditions limiting dispersal, the flagellated strain was able to disperse using the mycelial network and had a higher level of fitness than the mutant. On the basis of these results, we propose that the benefit of mycelium-associated dispersal helps explain the persistence of flagellar motility in non-water-saturated environments.

  4. Structure of the microtubule-binding domain of flagellar dynein.

    PubMed

    Kato, Yusuke S; Yagi, Toshiki; Harris, Sarah A; Ohki, Shin-ya; Yura, Kei; Shimizu, Youské; Honda, Shinya; Kamiya, Ritsu; Burgess, Stan A; Tanokura, Masaru

    2014-11-04

    Flagellar dyneins are essential microtubule motors in eukaryotes, as they drive the beating motions of cilia and flagella. Unlike myosin and kinesin motors, the track binding mechanism of dyneins and the regulation between the strong and weak binding states remain obscure. Here we report the solution structure of the microtubule-binding domain of flagellar dynein-c/DHC9 (dynein-c MTBD). The structure reveals a similar overall helix-rich fold to that of the MTBD of cytoplasmic dynein (cytoplasmic MTBD), but dynein-c MTBD has an additional flap, consisting of an antiparallel b sheet. The flap is positively charged and highly flexible. Despite the structural similarity to cytoplasmic MTBD, dynein-c MTBD shows only a small change in the microtubule- binding affinity depending on the registry change of coiled coil-sliding, whereby lacks the apparent strong binding state. The surface charge distribution of dynein-c MTBD also differs from that of cytoplasmic MTBD, which suggests a difference in the microtubule-binding mechanism.

  5. Surface organization and composition of Euglena. II. Flagellar mastigonemes

    PubMed Central

    1978-01-01

    The surface of the Euglena flagellum is coated with about 30,000 fine filaments of two distinct types. The longer of these nontubular mastigonemes (about 3 micron) appear to be attached to the paraflagellar rod whereas the shorter nontubular mastigonemes (about 1.5 micron) are the centrifugally arranged portions of a larger complex, which consists of an attached unit parallel to and outside of the flagellar membrane. Units are arranged laternally in near registration and longitudinally overlap by one-half of a unit length. Rows of mastigoneme units are firmly attached to the axoneme microtubules or to the paraflagellar rod as evidenced by their persistence after removal of the flagellar membrane with neutral detergents. SDS-acrylamide gels of whole flagella revealed about 30 polypeptides, of which two gave strong positive staining with the periodic acid-Schiff (PAS) procedure. At least one of these two bands (glycoproteins) has been equated with the surface mastigonemes by parallel analysis of isolated and purified mastigonemes, particularly after phenol extraction. The faster moving glycoprotein has been selectively removed from whole flagella and from the mastigoneme fraction with low concentrations of neutral detergents at neutral or high pH. The larger glycoprotein was found to be polydisperse when electrophoresed through 1% agarose/SDS gels. Thin-layer chromatography of hydrolysates of whole flagella or of isolated mastigonemes has indicated that the major carbohydrate moiety is the pentose sugar, xylose, with possibly a small amount of glucose and an unknown minor component. PMID:98532

  6. An Element of Determinism in a Stochastic Flagellar Motor Switch

    PubMed Central

    Xie, Li; Altindal, Tuba; Wu, Xiao-Lun

    2015-01-01

    Marine bacterium Vibrio alginolyticus uses a single polar flagellum to navigate in an aqueous environment. Similar to Escherichia coli cells, the polar flagellar motor has two states; when the motor is counter-clockwise, the cell swims forward and when the motor is clockwise, the cell swims backward. V. alginolyticus also incorporates a direction randomization step at the start of the forward swimming interval by flicking its flagellum. To gain an understanding on how the polar flagellar motor switch is regulated, distributions of the forward Δf and backward Δb intervals are investigated herein. We found that the steady-state probability density functions, P(Δf) and P(Δb), of freely swimming bacteria are strongly peaked at a finite time, suggesting that the motor switch is not Poissonian. The short-time inhibition is sufficiently strong and long lasting, i.e., several hundred milliseconds for both intervals, which is readily observed and characterized. Treating motor reversal dynamics as a first-passage problem, which results from conformation fluctuations of the motor switch, we calculated P(Δf) and P(Δb) and found good agreement with the measurements. PMID:26554590

  7. Bacterial flagellar capping proteins adopt diverse oligomeric states

    PubMed Central

    Postel, Sandra; Deredge, Daniel; Bonsor, Daniel A; Yu, Xiong; Diederichs, Kay; Helmsing, Saskia; Vromen, Aviv; Friedler, Assaf; Hust, Michael; Egelman, Edward H; Beckett, Dorothy; Wintrode, Patrick L; Sundberg, Eric J

    2016-01-01

    Flagella are crucial for bacterial motility and pathogenesis. The flagellar capping protein (FliD) regulates filament assembly by chaperoning and sorting flagellin (FliC) proteins after they traverse the hollow filament and exit the growing flagellum tip. In the absence of FliD, flagella are not formed, resulting in impaired motility and infectivity. Here, we report the 2.2 Å resolution X-ray crystal structure of FliD from Pseudomonas aeruginosa, the first high-resolution structure of any FliD protein from any bacterium. Using this evidence in combination with a multitude of biophysical and functional analyses, we find that Pseudomonas FliD exhibits unexpected structural similarity to other flagellar proteins at the domain level, adopts a unique hexameric oligomeric state, and depends on flexible determinants for oligomerization. Considering that the flagellin filaments on which FliD oligomers are affixed vary in protofilament number between bacteria, our results suggest that FliD oligomer stoichiometries vary across bacteria to complement their filament assemblies. DOI: http://dx.doi.org/10.7554/eLife.18857.001 PMID:27664419

  8. Bacterial flagellar capping proteins adopt diverse oligomeric states.

    PubMed

    Postel, Sandra; Deredge, Daniel; Bonsor, Daniel A; Yu, Xiong; Diederichs, Kay; Helmsing, Saskia; Vromen, Aviv; Friedler, Assaf; Hust, Michael; Egelman, Edward H; Beckett, Dorothy; Wintrode, Patrick L; Sundberg, Eric J

    2016-09-24

    Flagella are crucial for bacterial motility and pathogenesis. The flagellar capping protein (FliD) regulates filament assembly by chaperoning and sorting flagellin (FliC) proteins after they traverse the hollow filament and exit the growing flagellum tip. In the absence of FliD, flagella are not formed, resulting in impaired motility and infectivity. Here, we report the 2.2 Å resolution X-ray crystal structure of FliD from Pseudomonas aeruginosa, the first high-resolution structure of any FliD protein from any bacterium. Using this evidence in combination with a multitude of biophysical and functional analyses, we find that Pseudomonas FliD exhibits unexpected structural similarity to other flagellar proteins at the domain level, adopts a unique hexameric oligomeric state, and depends on flexible determinants for oligomerization. Considering that the flagellin filaments on which FliD oligomers are affixed vary in protofilament number between bacteria, our results suggest that FliD oligomer stoichiometries vary across bacteria to complement their filament assemblies.

  9. Modes of flagellar assembly in Chlamydomonas reinhardtii and Trypanosoma brucei

    PubMed Central

    Höög, Johanna L; Lacomble, Sylvain; O’Toole, Eileen T; Hoenger, Andreas; McIntosh, J Richard; Gull, Keith

    2014-01-01

    Defects in flagella growth are related to a number of human diseases. Central to flagellar growth is the organization of microtubules that polymerize from basal bodies to form the axoneme, which consists of hundreds of proteins. Flagella exist in all eukaryotic phyla, but neither the mechanism by which flagella grow nor the conservation of this process in evolution are known. Here, we study how protein complexes assemble onto the growing axoneme tip using (cryo) electron tomography. In Chlamydomonas reinhardtii microtubules and associated proteins are added simultaneously. However, in Trypanosoma brucei, disorganized arrays of microtubules are arranged into the axoneme structure by the later addition of preformed protein complexes. Post assembly, the T. brucei transition zone alters structure and its association with the central pair loosens. We conclude that there are multiple ways to form a flagellum and that species-specific structural knowledge is critical before evaluating flagellar defects. DOI: http://dx.doi.org/10.7554/eLife.01479.001 PMID:24448408

  10. Mechanics of torque generation in the bacterial flagellar motor

    PubMed Central

    Mandadapu, Kranthi K.; Nirody, Jasmine A.; Berry, Richard M.; Oster, George

    2015-01-01

    The bacterial flagellar motor (BFM) is responsible for driving bacterial locomotion and chemotaxis, fundamental processes in pathogenesis and biofilm formation. In the BFM, torque is generated at the interface between transmembrane proteins (stators) and a rotor. It is well established that the passage of ions down a transmembrane gradient through the stator complex provides the energy for torque generation. However, the physics involved in this energy conversion remain poorly understood. Here we propose a mechanically specific model for torque generation in the BFM. In particular, we identify roles for two fundamental forces involved in torque generation: electrostatic and steric. We propose that electrostatic forces serve to position the stator, whereas steric forces comprise the actual “power stroke.” Specifically, we propose that ion-induced conformational changes about a proline “hinge” residue in a stator α-helix are directly responsible for generating the power stroke. Our model predictions fit well with recent experiments on a single-stator motor. The proposed model provides a mechanical explanation for several fundamental properties of the flagellar motor, including torque–speed and speed–ion motive force relationships, backstepping, variation in step sizes, and the effects of key mutations in the stator. PMID:26216959

  11. Mechanism for adaptive remodeling of the bacterial flagellar switch

    PubMed Central

    Lele, Pushkar P.; Branch, Richard W.; Nathan, Vedhavalli S. J.; Berg, Howard C.

    2012-01-01

    The bacterial flagellar motor has been shown in previous work to adapt to changes in the steady-state concentration of the chemotaxis signaling molecule, CheY-P, by changing the FliM content. We show here that the number of FliM molecules in the motor and the fraction of FliM molecules that exchange depend on the direction of flagellar rotation, not on CheY-P binding per se. Our results are consistent with a model in which the structural differences associated with the direction of rotation modulate the strength of FliM binding. When the motor spins counterclockwise, FliM binding strengthens, the fraction of FliM molecules that exchanges decreases, and the ring content increases. The larger number of CheY-P binding sites enhances the motor’s sensitivity, i.e., the motor adapts. An interesting unresolved question is how additional copies of FliM might be accommodated. PMID:23169659

  12. Bacterial flagellar capping proteins adopt diverse oligomeric states

    SciTech Connect

    Postel, Sandra; Deredge, Daniel; Bonsor, Daniel A.; Yu, Xiong; Diederichs, Kay; Helmsing, Saskia; Vromen, Aviv; Friedler, Assaf; Hust, Michael; Egelman, Edward H.; Beckett, Dorothy; Wintrode, Patrick L.; Sundberg, Eric J.

    2016-09-24

    Flagella are crucial for bacterial motility and pathogenesis. The flagellar capping protein (FliD) regulates filament assembly by chaperoning and sorting flagellin (FliC) proteins after they traverse the hollow filament and exit the growing flagellum tip. In the absence of FliD, flagella are not formed, resulting in impaired motility and infectivity. Here, we report the 2.2 Å resolution X-ray crystal structure of FliD fromPseudomonas aeruginosa, the first high-resolution structure of any FliD protein from any bacterium. Using this evidence in combination with a multitude of biophysical and functional analyses, we find thatPseudomonasFliD exhibits unexpected structural similarity to other flagellar proteins at the domain level, adopts a unique hexameric oligomeric state, and depends on flexible determinants for oligomerization. Considering that the flagellin filaments on which FliD oligomers are affixed vary in protofilament number between bacteria, our results suggest that FliD oligomer stoichiometries vary across bacteria to complement their filament assemblies.

  13. Mechanics of torque generation in the bacterial flagellar motor.

    PubMed

    Mandadapu, Kranthi K; Nirody, Jasmine A; Berry, Richard M; Oster, George

    2015-08-11

    The bacterial flagellar motor (BFM) is responsible for driving bacterial locomotion and chemotaxis, fundamental processes in pathogenesis and biofilm formation. In the BFM, torque is generated at the interface between transmembrane proteins (stators) and a rotor. It is well established that the passage of ions down a transmembrane gradient through the stator complex provides the energy for torque generation. However, the physics involved in this energy conversion remain poorly understood. Here we propose a mechanically specific model for torque generation in the BFM. In particular, we identify roles for two fundamental forces involved in torque generation: electrostatic and steric. We propose that electrostatic forces serve to position the stator, whereas steric forces comprise the actual "power stroke." Specifically, we propose that ion-induced conformational changes about a proline "hinge" residue in a stator α-helix are directly responsible for generating the power stroke. Our model predictions fit well with recent experiments on a single-stator motor. The proposed model provides a mechanical explanation for several fundamental properties of the flagellar motor, including torque-speed and speed-ion motive force relationships, backstepping, variation in step sizes, and the effects of key mutations in the stator.

  14. Constraints on models for the flagellar rotary motor.

    PubMed Central

    Berg, H C

    2000-01-01

    Most bacteria that swim are propelled by flagellar filaments, each driven at its base by a rotary motor embedded in the cell wall and cytoplasmic membrane. A motor is about 45 nm in diameter and made up of about 20 different kinds of parts. It is assembled from the inside out. It is powered by a proton (or in some species, a sodium-ion) flux. It steps at least 400 times per revolution. At low speeds and high torques, about 1000 protons are required per revolution, speed is proportional to protonmotive force, and torque varies little with temperature or hydrogen isotope. At high speeds and low torques, torque increases with temperature and is sensitive to hydrogen isotope. At room temperature, torque varies remarkably little with speed from about -100 Hz (the present limit of measurement) to about 200 Hz, and then it declines rapidly reaching zero at about 300 Hz. These are facts that motor models should explain. None of the existing models for the flagellar rotary motor completely do so. PMID:10836502

  15. Analysis of Flagellar Phosphoproteins from Chlamydomonas reinhardtii▿ †

    PubMed Central

    Boesger, Jens; Wagner, Volker; Weisheit, Wolfram; Mittag, Maria

    2009-01-01

    Cilia and flagella are cell organelles that are highly conserved throughout evolution. For many years, the green biflagellate alga Chlamydomonas reinhardtii has served as a model for examination of the structure and function of its flagella, which are similar to certain mammalian cilia. Proteome analysis revealed the presence of several kinases and protein phosphatases in these organelles. Reversible protein phosphorylation can control ciliary beating, motility, signaling, length, and assembly. Despite the importance of this posttranslational modification, the identities of many ciliary phosphoproteins and knowledge about their in vivo phosphorylation sites are still missing. Here we used immobilized metal affinity chromatography to enrich phosphopeptides from purified flagella and analyzed them by mass spectrometry. One hundred forty-one phosphorylated peptides were identified, belonging to 32 flagellar proteins. Thereby, 126 in vivo phosphorylation sites were determined. The flagellar phosphoproteome includes different structural and motor proteins, kinases, proteins with protein interaction domains, and many proteins whose functions are still unknown. In several cases, a dynamic phosphorylation pattern and clustering of phosphorylation sites were found, indicating a complex physiological status and specific control by reversible protein phosphorylation in the flagellum. PMID:19429781

  16. Resurrection of the flagellar rotary motor near zero load

    PubMed Central

    Yuan, Junhua; Berg, Howard C.

    2008-01-01

    Flagellated bacteria, such as Escherichia coli, are propelled by helical flagellar filaments, each driven at its base by a reversible rotary motor, powered by a transmembrane proton flux. Torque is generated by the interaction of stator proteins, MotA and MotB, with a rotor protein FliG. The physiology of the motor has been studied extensively in the regime of relatively high load and low speed, where it appears to operate close to thermodynamic equilibrium. Here, we describe an assay that allows systematic study of the motor near zero load, where proton translocation and movement of mechanical components are rate limiting. Sixty-nanometer-diameter gold spheres were attached to hooks of cells lacking flagellar filaments, and light scattered from a sphere was monitored at the image plane of a microscope through a small pinhole. Paralyzed motors of cells carrying a motA point mutation were resurrected at 23°C by expression of wild-type MotA, and speeds jumped from zero to a maximum value (≈300 Hz) in one step. Thus, near zero load, the speed of the motor is independent of the number of torque-generating units. Evidently, the units act independently (they do not interfere with one another), and there are no intervals during which a second unit can add to the speed generated by the first (the duty ratio is close to 1). PMID:18202173

  17. Sperm chemotaxis and regulation of flagellar movement by Ca2+.

    PubMed

    Yoshida, Manabu; Yoshida, Kaoru

    2011-08-01

    The chemotaxis of sperm towards eggs is a widespread phenomenon that occurs in most forms of life from lower plants to mammals and plays important roles in ensuring fertilization. In spermatozoa, the attractants act as beacons, indicating the path leading to the eggs from the same species. The existence of species-specific sperm chemotaxis has been demonstrated in marine invertebrates; thus, sperm chemotaxis may be involved in preventing crossbreeding, especially in marine invertebrates with external fertilization. However, the mechanisms of sperm chemotaxis in mammalian species differ from those of marine invertebrates. In mammals, the attractant source is not the egg, but follicular fluids or cumulus cells and chemotactic behaviour is shown only in small populations of sperm. Nevertheless, the fundamental mechanisms underlying sperm chemotaxis are likely to be common among all species. Among these mechanisms, intracellular Ca(2+) concentration ([Ca(2+)](i)) is an important factor for the regulation of chemotactic behaviour in spermatozoa. Sperm attractants induce the entry of extracellular Ca(2+), resulting in [Ca(2+)](i) increase in the sperm cells. Furthermore, [Ca(2+)](i) modulates sperm flagellar movement. However, the relationship between [Ca(2+)](i) and the chemotactic response of a sperm flagellum is not well known. Investigation of the dynamic responses of sperm cells to their attractants is important for our understanding of the regulation of fertilization. Here, we reviewed sperm chemotaxis focusing on the mechanisms that regulate sperm flagellar beating during the chemotactic response.

  18. Effects of osmolality on sperm morphology, motility and flagellar wave parameters in Northern pike (Esox lucius L.).

    PubMed

    Alavi, S M Hadi; Rodina, Marek; Viveiros, Ana T M; Cosson, Jacky; Gela, David; Boryshpolets, Sergei; Linhart, Otomar

    2009-07-01

    Northern pike (Esox lucius L.) spermatozoa are uniflagellated cells differentiated into a head without acrosome, a midpiece and a flagellar tail region flanked by a fin structure. Total, flagellar, head and midpiece lengths of spermatozoa were measured and show mean values of 34.5, 32.0, 1.32, 1.17 microm, respectively, with anterior and posterior widths of the midpiece measuring 0.8 and 0.6 microm, respectively. The osmolality of seminal plasma ranged from 228 to 350 mOsmol kg(-1) (average: 283.88+/-33.05). After triggering of sperm motility in very low osmolality medium (distilled water), blebs appeared along the flagellum. At later periods in the motility phase, the tip of the flagellum became curled into a loop shape which resulted in a shortening of the flagellum and a restriction of wave development to the proximal part (close to head). Spermatozoa velocity and percentage of motile spermatozoa decreased rapidly as a function of time postactivation and depended on the osmolality of activation media (P<0.05). In general, the greatest percentage of motile spermatozoa and highest spermatozoa velocity were observed between 125 and 235 mOsmol kg(-1). Osmolality above 375 mOsmol kg(-1) inhibited the motility of spermatozoa. After triggering of sperm motility in activation media, beating waves propagated along the full length of flagella, while waves appeared dampened during later periods in the motility phase, and were absent at the end of the motility phase. By increasing osmolality, the velocity of spermatozoa reached the highest value while wave length, amplitude, number of waves and curvatures also were at their highest values. This study showed that sperm morphology can be used for fish classification. Sperm morphology, in particular, the flagellar part showed several changes during activation in distilled water. Sperm motility of pike is inhibited due to high osmolality in the seminal plasma. Osmolality of activation medium affects the percentage of motile

  19. Adenosine 3',5'-cyclic monophosphate in Chlamydomonas reinhardtii. Influence on flagellar function and regeneration.

    PubMed

    Rubin, R W; Filner, P

    1973-03-01

    Adenosine 3',5'-cyclic monophosphate (cAMP) influences both flagellar function and flagellar regeneration in Chlamydomonas reinhardtii. The methylxanthine, aminophylline, which can cause a tenfold increase in cAMP level in C. reinhardtii, inhibits flagellar movement and flagellar regeneration by wild-type cells, without inhibiting cell multiplication. Caffeine, a closely related inhibitor, also inhibits flagellar movement and regeneration, but it inhibits cell multiplication too. Regeneration by a mutant lacking the central pair of flagellar microtubules was found to be more sensitive than wild type to inhibition by caffeine and to be subject to synergistic inhibition by aminophylline plus dibutyryl cAMP. Regeneration by three out of seven mutants with different flagellar abnormalities was more sensitive than wild type to these inhibitors. We interpret these results to mean that cAMP affects a component of the flagellum directly or indirectly, and that the responsiveness of that component to cAMP is enhanced by mutations which affect the integrity of the flagellum. The component in question could be microtubule protein.

  20. A Novel Trypanosoma cruzi Protein Associated to the Flagellar Pocket of Replicative Stages and Involved in Parasite Growth

    PubMed Central

    Durante, Ignacio M.; Cámara, María de los Milagros; Buscaglia, Carlos A.

    2015-01-01

    The flagellar pocket constitutes an active and strategic site in the body of trypanosomatids (i.e. parasitic protozoa that cause important human and/or livestock diseases), which participates in several important processes such as cell polarity, morphogenesis and replication. Most importantly, the flagellar pocket is the unique site of surface protein export and nutrient uptake in trypanosomatids, and thus constitutes a key portal for the interaction with the host. In this work, we identified and characterized a novel Trypanosoma cruzi protein, termed TCLP 1, that accumulates at the flagellar pocket area of parasite replicative forms, as revealed by biochemical, immuno-cytochemistry and electron microscopy techniques. Different in silico analyses revealed that TCLP 1 is the founding member of a family of chimeric molecules restricted to trypanosomatids bearing, in addition to eukaryotic ubiquitin-like and protein-protein interacting domains, a motif displaying significant structural homology to bacterial multi-cargo chaperones involved in the secretion of virulence factors. Using the fidelity of an homologous expression system we confirmed TCLP 1 sub-cellular distribution and showed that TCLP 1-over-expressing parasites display impaired survival and accelerated progression to late stationary phase under starvation conditions. The reduced endocytic capacity of TCLP 1-over-expressors likely underlies (at least in part) this growth phenotype. TCLP 1 is involved in the uptake of extracellular macromolecules required for nutrition and hence in T. cruzi growth. Due to the bacterial origin, sub-cellular distribution and putative function(s), we propose TCLP 1 and related orthologs in trypanosomatids as appealing therapeutic targets for intervention against these health-threatening parasites. PMID:26086767

  1. Proteomics on the rims; insights into the biology of the nuclear envelope and flagellar pocket of trypanosomes

    PubMed Central

    Field, Mark C.; Adung’a, Vincent; Obado, Samson; Chait, Brian T.; Rout, Michael P.

    2014-01-01

    SUMMERY Trypanosomatids represent the causative agents of major diseases in humans, livestock and plants, with inevitable suffering and economic hardship as a result. They are also evolutionarily highly divergent organisms, and the many unique aspects of trypanosome biology provide opportunities in terms of identification of drug targets, the challenge of exploiting these putative targets, and at the same time significant scope for exploration of novel and divergent cell biology. We can estimate from genome sequences that the degree of divergence of trypanosomes from animals and fungi is extreme, with perhaps one third to one half of predicted trypanosome proteins having no known function based on homology or recognizable protein domains/architecture. Two highly important aspects of trypanosome biology are the flagellar pocket and the nuclear envelope, where in silico analysis clearly suggests great potential divergence in the proteome. The flagellar pocket is the sole site of endo- and exocytosis in trypanosomes and plays important roles in immune evasion via variant surface glycoprotein (VSG) trafficking and providing a location for sequestration of various invariant receptors. The trypanosome nuclear envelope has been largely unexplored, but by analogy with higher eukaryotes, roles in the regulation of chromatin and most significantly, in controlling VSG gene expression are expected. Here we discuss recent successful proteomics-based approaches towards characterization of the nuclear envelope and the endocytic apparatus, the identification of conserved and novel trypanosomatid-specific features, and the implications of these findings. PMID:22309600

  2. Proteomics on the rims: insights into the biology of the nuclear envelope and flagellar pocket of trypanosomes.

    PubMed

    Field, Mark C; Adung'a, Vincent; Obado, Samson; Chait, Brian T; Rout, Michael P

    2012-08-01

    Trypanosomatids represent the causative agents of major diseases in humans, livestock and plants, with inevitable suffering and economic hardship as a result. They are also evolutionarily highly divergent organisms, and the many unique aspects of trypanosome biology provide opportunities in terms of identification of drug targets, the challenge of exploiting these putative targets and, at the same time, significant scope for exploration of novel and divergent cell biology. We can estimate from genome sequences that the degree of divergence of trypanosomes from animals and fungi is extreme, with perhaps one third to one half of predicted trypanosome proteins having no known function based on homology or recognizable protein domains/architecture. Two highly important aspects of trypanosome biology are the flagellar pocket and the nuclear envelope, where in silico analysis clearly suggests great potential divergence in the proteome. The flagellar pocket is the sole site of endo- and exocytosis in trypanosomes and plays important roles in immune evasion via variant surface glycoprotein (VSG) trafficking and providing a location for sequestration of various invariant receptors. The trypanosome nuclear envelope has been largely unexplored but, by analogy with higher eukaryotes, roles in the regulation of chromatin and most significantly, in controlling VSG gene expression are expected. Here we discuss recent successful proteomics-based approaches towards characterization of the nuclear envelope and the endocytic apparatus, the identification of conserved and novel trypanosomatid-specific features, and the implications of these findings.

  3. Profilin is associated with transcriptionally active genes

    PubMed Central

    Söderberg, Emilia; Hessle, Viktoria; von Euler, Anne; Visa, Neus

    2012-01-01

    We have raised antibodies against the profilin of Chironomus tentans to study the location of profilin relative to chromatin and to active genes in salivary gland polytene chromosomes. We show that a fraction of profilin is located in the nucleus, where profilin is highly concentrated in the nucleoplasm and at the nuclear periphery. Moreover, profilin is associated with multiple bands in the polytene chromosomes. By staining salivary glands with propidium iodide, we show that profilin does not co-localize with dense chromatin. Profilin associates instead with protein-coding genes that are transcriptionally active, as revealed by co-localization with hnRNP and snRNP proteins. We have performed experiments of transcription inhibition with actinomycin D and we show that the association of profilin with the chromosomes requires ongoing transcription. However, the interaction of profilin with the gene loci does not depend on RNA. Our results are compatible with profilin regulating actin polymerization in the cell nucleus. However, the association of actin with the polytene chromosomes of C. tentans is sensitive to RNase, whereas the association of profilin is not, and we propose therefore that the chromosomal location of profilin is independent of actin. PMID:22572953

  4. Torque and rotation rate of the bacterial flagellar motor.

    PubMed Central

    Läuger, P

    1988-01-01

    This paper describes an analysis of microscopic models for the coupling between ion flow and rotation of bacterial flagella. In model I it is assumed that intersecting half-channels exist on the rotor and the stator and that the driving ion is constrained to move together with the intersection site. Model II is based on the assumption that ion flow drives a cycle of conformational transitions in a channel-like stator subunit that are coupled to the motion of the rotor. Analysis of both mechanisms yields closed expressions relating the torque M generated by the flagellar motor to the rotation rate v. Model I (and also, under certain assumptions, model II) accounts for the experimentally observed linear relationship between M and v. The theoretical equations lead to predictions on the relationship between rotation rate and driving force which can be tested experimentally. PMID:3342270

  5. Flagellar swimmers oscillate between pusher- and puller-type swimming

    NASA Astrophysics Data System (ADS)

    Klindt, Gary S.; Friedrich, Benjamin M.

    2015-12-01

    Self-propulsion of cellular microswimmers generates flow signatures, commonly classified as pusher and puller type, which characterize hydrodynamic interactions with other cells or boundaries. Using experimentally measured beat patterns, we compute that the flagellated green alga Chlamydomonas oscillates between pusher and puller, rendering it an approximately neutral swimmer, when averaging over its full beat cycle. Beyond a typical distance of 100 μ m from the cell, inertia attenuates oscillatory microflows. We show that hydrodynamic interactions between cells oscillate in time and are of similar magnitude as stochastic swimming fluctuations. From our analysis, we also find that the rate of hydrodynamic dissipation varies in time, which implies that flagellar beat patterns are not optimized with respect to this measure.

  6. Flagellar generated flow mediates attachment of Giardia lamblia

    NASA Astrophysics Data System (ADS)

    Urbach, Jeffrey; Luo, Haibei; Picou, Theodore; McAllister, Ryan; Elmendorf, Heidi

    2011-03-01

    Giardia lamblia is a protozoan parasite responsible for widespread diarrheal disease in humans and animals worldwide. Attachment to the host intestinal mucosa and resistance to peristalsis is necessary for establishing infection, but the physical basis for this attachment is poorly understood. We report results from TIRF and confocal fluorescence microscopy that demonstrate that the regular beating of the posterior flagella generate a flow through the ventral disk, a suction-cup shaped structure that is against the substrate during attachment. Finite element simulations are used to compare the negative pressure generated by the flow to the measured attachment force and the expected performance of the flagellar pump. NIH grant 1R21AI062934-0.

  7. Antiphase synchronization in a flagellar-dominance mutant of Chlamydomonas.

    PubMed

    Leptos, Kyriacos C; Wan, Kirsty Y; Polin, Marco; Tuval, Idan; Pesci, Adriana I; Goldstein, Raymond E

    2013-10-11

    Groups of beating flagella or cilia often synchronize so that neighboring filaments have identical frequencies and phases. A prime example is provided by the unicellular biflagellate Chlamydomonas reinhardtii, which typically displays synchronous in-phase beating in a low-Reynolds number version of breaststroke swimming. We report the discovery that ptx1, a flagellar-dominance mutant of C. reinhardtii, can exhibit synchronization in precise antiphase, as in the freestyle swimming stroke. High-speed imaging shows that ptx1 flagella switch stochastically between in-phase and antiphase states, and that the latter has a distinct waveform and significantly higher frequency, both of which are strikingly similar to those found during phase slips that stochastically interrupt in-phase beating of the wild-type. Possible mechanisms underlying these observations are discussed.

  8. Flagellar Kinematics and Swimming of Algal Cells in Viscoelastic Fluids

    PubMed Central

    Qin, B.; Gopinath, A.; Yang, J.; Gollub, J. P.; Arratia, P. E.

    2015-01-01

    The motility of microorganisms is influenced greatly by their hydrodynamic interactions with the fluidic environment they inhabit. We show by direct experimental observation of the bi-flagellated alga Chlamydomonas reinhardtii that fluid elasticity and viscosity strongly influence the beating pattern - the gait - and thereby control the propulsion speed. The beating frequency and the wave speed characterizing the cyclical bending are both enhanced by fluid elasticity. Despite these enhancements, the net swimming speed of the alga is hindered for fluids that are sufficiently elastic. The origin of this complex response lies in the interplay between the elasticity-induced changes in the spatial and temporal aspects of the flagellar cycle and the buildup and subsequent relaxation of elastic stresses during the power and recovery strokes. PMID:25778677

  9. Flagellar kinematics and swimming of algal cells in viscoelastic fluids.

    PubMed

    Qin, B; Gopinath, A; Yang, J; Gollub, J P; Arratia, P E

    2015-03-17

    The motility of microorganisms is influenced greatly by their hydrodynamic interactions with the fluidic environment they inhabit. We show by direct experimental observation of the bi-flagellated alga Chlamydomonas reinhardtii that fluid elasticity and viscosity strongly influence the beating pattern - the gait - and thereby control the propulsion speed. The beating frequency and the wave speed characterizing the cyclical bending are both enhanced by fluid elasticity. Despite these enhancements, the net swimming speed of the alga is hindered for fluids that are sufficiently elastic. The origin of this complex response lies in the interplay between the elasticity-induced changes in the spatial and temporal aspects of the flagellar cycle and the buildup and subsequent relaxation of elastic stresses during the power and recovery strokes.

  10. Hybrid-fuel bacterial flagellar motors in Escherichia coli.

    PubMed

    Sowa, Yoshiyuki; Homma, Michio; Ishijima, Akihiko; Berry, Richard M

    2014-03-04

    The bacterial flagellar motor rotates driven by an electrochemical ion gradient across the cytoplasmic membrane, either H(+) or Na(+) ions. The motor consists of a rotor ∼50 nm in diameter surrounded by multiple torque-generating ion-conducting stator units. Stator units exchange spontaneously between the motor and a pool in the cytoplasmic membrane on a timescale of minutes, and their stability in the motor is dependent upon the ion gradient. We report a genetically engineered hybrid-fuel flagellar motor in Escherichia coli that contains both H(+)- and Na(+)-driven stator components and runs on both types of ion gradient. We controlled the number of each type of stator unit in the motor by protein expression levels and Na(+) concentration ([Na(+)]), using speed changes of single motors driving 1-μm polystyrene beads to determine stator unit numbers. De-energized motors changed from locked to freely rotating on a timescale similar to that of spontaneous stator unit exchange. Hybrid motor speed is simply the sum of speeds attributable to individual stator units of each type. With Na(+) and H(+) stator components expressed at high and medium levels, respectively, Na(+) stator units dominate at high [Na(+)] and are replaced by H(+) units when Na(+) is removed. Thus, competition between stator units for spaces in a motor and sensitivity of each type to its own ion gradient combine to allow hybrid motors to adapt to the prevailing ion gradient. We speculate that a similar process may occur in species that naturally express both H(+) and Na(+) stator components sharing a common rotor.

  11. Protein export through the bacterial flagellar type III export pathway.

    PubMed

    Minamino, Tohru

    2014-08-01

    For construction of the bacterial flagellum, which is responsible for bacterial motility, the flagellar type III export apparatus utilizes both ATP and proton motive force across the cytoplasmic membrane and exports flagellar proteins from the cytoplasm to the distal end of the nascent structure. The export apparatus consists of a membrane-embedded export gate made of FlhA, FlhB, FliO, FliP, FliQ, and FliR and a water-soluble ATPase ring complex consisting of FliH, FliI, and FliJ. FlgN, FliS, and FliT act as substrate-specific chaperones that do not only protect their cognate substrates from degradation and aggregation in the cytoplasm but also efficiently transfer the substrates to the export apparatus. The ATPase ring complex facilitates the initial entry of the substrates into the narrow pore of the export gate. The export gate by itself is a proton-protein antiporter that uses the two components of proton motive force, the electric potential difference and the proton concentration difference, for different steps of the export process. A specific interaction of FlhA with FliJ located in the center of the ATPase ring complex allows the export gate to efficiently use proton motive force to drive protein export. The ATPase ring complex couples ATP binding and hydrolysis to its assembly-disassembly cycle for rapid and efficient protein export cycle. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.

  12. Structural Analysis of the Flagellar Component Proteins in Solution by Small Angle X-Ray Scattering.

    PubMed

    Lee, Lawrence K

    2017-01-01

    Small angle X-ray scattering is an increasingly utilized method for characterizing the shape and structural properties of proteins in solution. The technique is amenable to very large protein complexes and to dynamic particles with different conformational states. It is therefore ideally suited to the analysis of some flagellar motor components. Indeed, we recently used the method to analyze the solution structure of the flagellar motor protein FliG, which when combined with high-resolution snapshots of conformational states from crystal structures, led to insights into conformational transitions that are important in mediating the self-assembly of the bacterial flagellar motor. Here, we describe procedures for X-ray scattering data collection of flagellar motor components, data analysis, and interpretation.

  13. CsrA-FliW interaction governs flagellin homeostasis and a checkpoint on flagellar morphogenesis in Bacillus subtilis

    PubMed Central

    Mukherjee, Sampriti; Yakhnin, Helen; Kysela, Dave; Sokoloski, Josh; Babitzke, Paul; Kearns, Daniel B.

    2011-01-01

    CsrA is a widely distributed RNA binding protein that regulates translation initiation and/or mRNA stability of target transcripts. CsrA activity is antagonized by sRNA(s) containing multiple CsrA binding sites in several Gram-negative bacterial species. Here we discover FliW, the first protein antagonist of CsrA activity that constitutes a partner switching mechanism to control flagellin synthesis in the Gram-positive organism Bacillus subtilis. Following the flagellar assembly checkpoint of hook completion, secretion of flagellin (Hag) releases FliW protein from a FliW-Hag complex. FliW then binds to CsrA and relieves CsrA-mediated translational repression of hag for flagellin synthesis concurrent with filament assembly. Thus, flagellin homeostatically restricts its own translation. Homeostatic autoregulation may be a general mechanism to precisely control structural subunits required at specific times and in finite amounts such as those involved in the assembly of flagella, type III secretion machines, and pili. Finally, phylogenetic analysis suggests that CsrA, a highly pleiotropic virulence regulator in many bacterial pathogens, had an ancestral role in flagellar assembly and evolved to co-regulate various cellular processes with motility. PMID:21895793

  14. CsrA-FliW interaction governs flagellin homeostasis and a checkpoint on flagellar morphogenesis in Bacillus subtilis.

    PubMed

    Mukherjee, Sampriti; Yakhnin, Helen; Kysela, Dave; Sokoloski, Josh; Babitzke, Paul; Kearns, Daniel B

    2011-10-01

    CsrA is a widely distributed RNA binding protein that regulates translation initiation and/or mRNA stability of target transcripts. CsrA activity is antagonized by sRNA(s) containing multiple CsrA binding sites in several Gram-negative bacterial species. Here we discover FliW, the first protein antagonist of CsrA activity that constitutes a partner switching mechanism to control flagellin synthesis in the Gram-positive organism Bacillus subtilis. Following the flagellar assembly checkpoint of hook completion, secretion of flagellin (Hag) releases FliW protein from a FliW-Hag complex. FliW then binds to CsrA and relieves CsrA-mediated translational repression of hag for flagellin synthesis concurrent with filament assembly. Thus, flagellin homeostatically restricts its own translation. Homeostatic autoregulation may be a general mechanism to precisely control structural subunits required at specific times and in finite amounts such as those involved in the assembly of flagella, type III secretion machines and pili. Finally, phylogenetic analysis suggests that CsrA, a highly pleiotropic virulence regulator in many bacterial pathogens, had an ancestral role in flagellar assembly and evolved to co-regulate various cellular processes with motility. © 2011 Blackwell Publishing Ltd.

  15. Curcumin Reduces the Motility of Salmonella enterica Serovar Typhimurium by Binding to the Flagella, Thereby Leading to Flagellar Fragility and Shedding

    PubMed Central

    Balakrishnan, Arjun; Negi, Vidya Devi; Sakorey, Deepika; Chandra, Nagasuma

    2016-01-01

    ABSTRACT One of the important virulence properties of the pathogen is its ability to travel to a favorable environment, cross the viscous mucus barrier (intestinal barrier for enteric pathogens), and reach the epithelia to initiate pathogenesis with the help of an appendage, like flagella. Nonetheless, flagella can act as an “Achilles heel,” revealing the pathogen's presence to the host through the stimulation of innate and adaptive immune responses. We assessed whether curcumin, a dietary polyphenol, could alter the motility of Salmonella, a foodborne pathogen. It reduced the motility of Salmonella enterica serovar Typhimurium by shortening the length of the flagellar filament (from ∼8 μm to ∼5 μm) and decreasing its density (4 or 5 flagella/bacterium instead of 8 or 9 flagella/bacterium). Upon curcumin treatment, the percentage of flagellated bacteria declined from ∼84% to 59%. However, no change was detected in the expression of the flagellin gene and protein. A fluorescence binding assay demonstrated binding of curcumin to the flagellar filament. This might make the filament fragile, breaking it into smaller fragments. Computational analysis predicted the binding of curcumin, its analogues, and its degraded products to a flagellin molecule at an interface between domains D1 and D2. Site-directed mutagenesis and a fluorescence binding assay confirmed the binding of curcumin to flagellin at residues ASN120, ASP123, ASN163, SER164, ASN173, and GLN175. IMPORTANCE This work, to our knowledge the first report of its kind, examines how curcumin targets flagellar density and affects the pathogenesis of bacteria. We found that curcumin does not affect any of the flagellar synthesis genes. Instead, it binds to the flagellum and makes it fragile. It increases the torsional stress on the flagellar filament that then breaks, leaving fewer flagella around the bacteria. Flagella, which are crucial ligands for Toll-like receptor 5, are some of the most important

  16. Specificity of motor components in the dual flagellar system of Shewanella putrefaciens CN-32.

    PubMed

    Bubendorfer, Sebastian; Held, Susanne; Windel, Natalie; Paulick, Anja; Klingl, Andreas; Thormann, Kai M

    2012-01-01

    Bacterial flagellar motors are intricate nanomachines in which the stator units and rotor component FliM may be dynamically exchanged during function. Similar to other bacterial species, the gammaproteobacterium Shewanella putrefaciens CN-32 possesses a complete secondary flagellar system along with a corresponding stator unit. Expression of the secondary system occurs during planktonic growth in complex media and leads to the formation of a subpopulation with one or more additional flagella at random positions in addition to the primary polar system. We used physiological and phenotypic characterizations of defined mutants in concert with fluorescent microscopy on labelled components of the two different systems, the stator proteins PomB and MotB, the rotor components FliM(1) and FliM(2), and the auxiliary motor components MotX and MotY, to determine localization, function and dynamics of the proteins in the flagellar motors. The results demonstrate that the polar flagellum is driven by a Na(+)-dependent FliM(1)/PomAB/MotX/MotY flagellar motor while the secondary system is rotated by a H(+)-dependent FliM(2)/MotAB motor. The components were highly specific for their corresponding motor and are unlikely to be extensively swapped or shared between the two flagellar systems under planktonic conditions. The results have implications for both specificity and dynamics of flagellar motor components. © 2011 Blackwell Publishing Ltd.

  17. The Histone-Like Nucleoid Structuring Protein (H-NS) Is a Negative Regulator of the Lateral Flagellar System in the Deep-Sea Bacterium Shewanella piezotolerans WP3

    PubMed Central

    Jian, Huahua; Xu, Guanpeng; Gai, Yingbao; Xu, Jun

    2016-01-01

    Although the histone-like nucleoid structuring protein (H-NS) is well known for its involvement in the adaptation of mesophilic bacteria, such as Escherichia coli, to cold environments and high-pressure stress, an understanding of the role of H-NS in the cold-adapted benthic microorganisms that live in the deep-sea ecosystem, which covers approximately 60% of the earth's surface, is still lacking. In this study, we characterized the function of H-NS in Shewanella piezotolerans WP3, which was isolated from West Pacific sediment at a depth of 1,914 m. An hns gene deletion mutant (WP3Δhns) was constructed, and comparative whole-genome microarray analysis was performed. H-NS had a significant influence (fold change, >2) on the expression of a variety of WP3 genes (274 and 280 genes were upregulated and downregulated, respectively), particularly genes related to energy production and conversion. Notably, WP3Δhns exhibited higher expression levels of lateral flagellar genes than WP3 and showed enhanced swarming motility and lateral flagellar production compared to those of WP3. The DNA gel mobility shift experiment showed that H-NS bound specifically to the promoter of lateral flagellar genes. Moreover, the high-affinity binding sequences of H-NS were identified by DNase I protection footprinting, and the results support the “binding and spreading” model for H-NS functioning. To our knowledge, this is the first attempt to characterize the function of the universal regulator H-NS in a deep-sea bacterium. Our data revealed that H-NS has a novel function as a repressor of the expression of genes related to the energy-consuming secondary flagellar system and to swarming motility. PMID:26873312

  18. The Histone-Like Nucleoid Structuring Protein (H-NS) Is a Negative Regulator of the Lateral Flagellar System in the Deep-Sea Bacterium Shewanella piezotolerans WP3.

    PubMed

    Jian, Huahua; Xu, Guanpeng; Gai, Yingbao; Xu, Jun; Xiao, Xiang

    2016-04-01

    Although the histone-like nucleoid structuring protein (H-NS) is well known for its involvement in the adaptation of mesophilic bacteria, such as Escherichia coli, to cold environments and high-pressure stress, an understanding of the role of H-NS in the cold-adapted benthic microorganisms that live in the deep-sea ecosystem, which covers approximately 60% of the earth's surface, is still lacking. In this study, we characterized the function of H-NS in Shewanella piezotolerans WP3, which was isolated from West Pacific sediment at a depth of 1,914 m. Anhns gene deletion mutant (WP3Δhns) was constructed, and comparative whole-genome microarray analysis was performed. H-NS had a significant influence (fold change, >2) on the expression of a variety of WP3 genes (274 and 280 genes were upregulated and downregulated, respectively), particularly genes related to energy production and conversion. Notably, WP3Δhnsexhibited higher expression levels of lateral flagellar genes than WP3 and showed enhanced swarming motility and lateral flagellar production compared to those of WP3. The DNA gel mobility shift experiment showed that H-NS bound specifically to the promoter of lateral flagellar genes. Moreover, the high-affinity binding sequences of H-NS were identified by DNase I protection footprinting, and the results support the "binding and spreading" model for H-NS functioning. To our knowledge, this is the first attempt to characterize the function of the universal regulator H-NS in a deep-sea bacterium. Our data revealed that H-NS has a novel function as a repressor of the expression of genes related to the energy-consuming secondary flagellar system and to swarming motility.

  19. Nuclear actin activates human transcription factor genes including the OCT4 gene.

    PubMed

    Yamazaki, Shota; Yamamoto, Koji; Tokunaga, Makio; Sakata-Sogawa, Kumiko; Harata, Masahiko

    2015-01-01

    RNA microarray analyses revealed that nuclear actin activated many human transcription factor genes including OCT4, which is required for gene reprogramming. Oct4 is known to be activated by nuclear actin in Xenopus oocytes. Our findings imply that this process of OCT4 activation is conserved in vertebrates and among cell types and could be used for gene reprogramming of human cells.

  20. Independent evolution of neurotoxin and flagellar genetic loci in proteolytic Clostridium botulinum

    PubMed Central

    Carter, Andrew T; Paul, Catherine J; Mason, David R; Twine, Susan M; Alston, Mark J; Logan, Susan M; Austin, John W; Peck, Michael W

    2009-01-01

    Background Proteolytic Clostridium botulinum is the causative agent of botulism, a severe neuroparalytic illness. Given the severity of botulism, surprisingly little is known of the population structure, biology, phylogeny or evolution of C. botulinum. The recent determination of the genome sequence of C. botulinum has allowed comparative genomic indexing using a DNA microarray. Results Whole genome microarray analysis revealed that 63% of the coding sequences (CDSs) present in reference strain ATCC 3502 were common to all 61 widely-representative strains of proteolytic C. botulinum and the closely related C. sporogenes tested. This indicates a relatively stable genome. There was, however, evidence for recombination and genetic exchange, in particular within the neurotoxin gene and cluster (including transfer of neurotoxin genes to C. sporogenes), and the flagellar glycosylation island (FGI). These two loci appear to have evolved independently from each other, and from the remainder of the genetic complement. A number of strains were atypical; for example, while 10 out of 14 strains that formed type A1 toxin gave almost identical profiles in whole genome, neurotoxin cluster and FGI analyses, the other four strains showed divergent properties. Furthermore, a new neurotoxin sub-type (A5) has been discovered in strains from heroin-associated wound botulism cases. For the first time, differences in glycosylation profiles of the flagella could be linked to differences in the gene content of the FGI. Conclusion Proteolytic C. botulinum has a stable genome backbone containing specific regions of genetic heterogeneity. These include the neurotoxin gene cluster and the FGI, each having evolved independently of each other and the remainder of the genetic complement. Analysis of these genetic components provides a high degree of discrimination of strains of proteolytic C. botulinum, and is suitable for clinical and forensic investigations of botulism outbreaks. PMID:19298644

  1. Cloning, expression and purification flagellar sheath adhesion of Helicobacter pylori in Escherichia coli host as a vaccination target

    PubMed Central

    2016-01-01

    Purpose Helicobacter pylori is a widely distributed gram-negative bacterium that infects the human stomach and duodenum. HpaA is a H. pylori–specific lipoprotein that has been shown to be an effective protective antigen against H. pylori infection. HpaA of H. pylori as a vaccine antigen is fully competent for stimulation of immune responses. The aim of this project is cloning, expression, and purification flagellar sheath adhesion of H. pylori in Escherichia coli host by fast protein liquid chromatography (FPLC) as a vaccination target. Materials and Methods The hpaA gene was inserted into pET28a (+) as cloning and expression vectors respectively. The recombinant plasmid (pET-hpaA) was subjected to sequencing other than polymerase chain reaction (PCR) and digestion analysis. Protein expression was induced by adding 1 mM isopropyl-β-D-thiogalactoside to cultures of E. coli strain BL21 transformed with pET-hpaA. Protein expression assessed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Protein purification of flagellar sheath adhesion was by FPLC. Results The restriction endonuclease digestion, PCR amplification analysis showed that the hpaA gene of 730 bp was amplified from H. pylori DNA and sequencing analysis of the pET-hpaA confirmed the cloning accuracy and in frame insertion of hpaA fragment. SDS-PAGE analysis showed the expression of an approximately 29,000 Da protein. Conclusion Sequencing results along with SDS-PAGE analysis confirms the expression of recombinant hpaA in the heterologous E. coli BL21. Conclusion A prokaryotic expression system for hpaA gene was successfully constructed. These results indicate that production of a specific recombinant protein is an alternative and potentially more expeditious strategy for development of H. pylori vaccine. PMID:26866020

  2. Cloning, expression and purification flagellar sheath adhesion of Helicobacter pylori in Escherichia coli host as a vaccination target.

    PubMed

    Soleimani, Neda; Mohabati Mobarez, Ashraf; Farhangi, Baharak

    2016-01-01

    Helicobacter pylori is a widely distributed gram-negative bacterium that infects the human stomach and duodenum. HpaA is a H. pylori-specific lipoprotein that has been shown to be an effective protective antigen against H. pylori infection. HpaA of H. pylori as a vaccine antigen is fully competent for stimulation of immune responses. The aim of this project is cloning, expression, and purification flagellar sheath adhesion of H. pylori in Escherichia coli host by fast protein liquid chromatography (FPLC) as a vaccination target. The hpaA gene was inserted into pET28a (+) as cloning and expression vectors respectively. The recombinant plasmid (pET-hpaA) was subjected to sequencing other than polymerase chain reaction (PCR) and digestion analysis. Protein expression was induced by adding 1 mM isopropyl-β-D-thiogalactoside to cultures of E. coli strain BL21 transformed with pET-hpaA. Protein expression assessed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Protein purification of flagellar sheath adhesion was by FPLC. The restriction endonuclease digestion, PCR amplification analysis showed that the hpaA gene of 730 bp was amplified from H. pylori DNA and sequencing analysis of the pET-hpaA confirmed the cloning accuracy and in frame insertion of hpaA fragment. SDS-PAGE analysis showed the expression of an approximately 29,000 Da protein. Sequencing results along with SDS-PAGE analysis confirms the expression of recombinant hpaA in the heterologous E. coli BL21. Conclusion A prokaryotic expression system for hpaA gene was successfully constructed. These results indicate that production of a specific recombinant protein is an alternative and potentially more expeditious strategy for development of H. pylori vaccine.

  3. Following the Viterbi Path to Deduce Flagellar Actin-Interacting Proteins of Leishmania spp.: Report on Cofilins and Twinfilins

    NASA Astrophysics Data System (ADS)

    Pacheco, Ana Carolina L.; Araújo, Fabiana F.; Kamimura, Michel T.; Medeiros, Sarah R.; Viana, Daniel A.; Oliveira, Fátima de Cássia E.; Filho, Raimundo Araújo; Costa, Marcília P.; Oliveira, Diana M.

    2007-11-01

    For performing vital cellular processes, such as motility, eukaryotic cells rely on the actin cytoskeleton, whose structure and dynamics are tightly controlled by a large number of actin-interacting (AIP) or actin-related/regulating (ARP) proteins. Trypanosomatid protozoa, such as Leishmania, rely on their flagellum for motility and sensory reception, which are believed to allow parasite migration, adhesion, invasion and even persistence on mammalian host tissues to cause disease. Actin can determine cell stiffness and transmit force during mechanotransduction, cytokinesis, cell motility and other cellular shape changes, while the identification and analyses of AIPs can help to improve understanding of their mechanical properties on physiological architectures, such as the present case regarding Leishmania flagellar apparatus. This work conveniently apply bioinformatics tools in some refined pattern recognition techniques (such as hidden Markov models (HMMs) through the Viterbi algorithm/path) in order to improve the recognition of actin-binding/interacting activity through identification of AIPs in genomes, transcriptomes and proteomes of Leishmania species. We here report cofilin and twinfilin as putative components of the flagellar apparatus, a direct bioinformatics contribution in the secondary annotation of Leishmania and trypanosomatid genomes.

  4. Flagellar mitochondrial association of the male-specific Don Juan protein in Drosophila spermatozoa.

    PubMed

    Santel, A; Blümer, N; Kämpfer, M; Renkawitz-Pohl, R

    1998-11-01

    The Drosophila don juan gene encodes a basic protein (Don Juan protein), which is solely expressed postmeiotically during spermiogenesis in elongated spermatids and in mature sperm. Transgenic expression of a GFP-tagged Don Juan protein (DJ-GFP) in the male germ line showed an association of the fusion protein with the sperm tail. Detailed examination of DJ-GFP localization revealed novel insights into its distinct temporal and spatial distribution along the sperm tail during the last phase of spermatid maturation. Co-localization of DJ-GFP with actin-labeled cysts demonstrated its emergence in elongated spermatids during individualization. Additionally, the endogenous Don Juan protein was detected with epitope-specific antibodies in finally elongated nuclei of spermatids. After completion of nuclear shaping Don Juan is no longer detectable in the sperm heads with the onset of individualization. Mislocalization of the DJ-GFP protein in flagella of a mutant with defective mitochondrial differentiation provides evidence of mitochondrial association of the fusion protein with flagellar mitochondrial arrays. Ectopically expressed DJ-GFP in premeiotic germ cells as well as salivary gland cells confirmed the capability of the fusion protein to associate with mitochondria. Therefore we suppose that Don Juan is a nuclear-encoded, germ-cell specifically expressed mitochondrial protein, which might be involved in the final steps of mitochondrial differentiation within the flagellum.

  5. Cancer genes: rare recombinants instead of activated oncogenes (a review).

    PubMed Central

    Duesberg, P H

    1987-01-01

    The 20 known transforming (onc) genes of retroviruses are defined by sequences that are transduced from cellular genes termed protooncogenes or cellular oncogenes. Based on these sequences, viral onc genes have been postulated to be transduced cellular cancer genes, and proto-onc genes have been postulated to be latent cancer genes that can be activated from within the cell to cause virus-negative tumors. The hypothesis is popular because it promises direct access to cellular cancer genes. However, the existence of latent cancer genes presents a paradox, since such genes are clearly undesirable. The hypothesis predicts that viral onc genes and proto-onc genes are isogenic; that expression of proto-onc genes induces tumors; that activated proto-onc genes transform diploid cells upon transfection, like viral onc genes; and that diploid tumors exist. As yet, none of these predictions is confirmed. Instead: Structural comparisons between viral onc genes, essential retroviral genes, and proto-onc genes show that all viral onc genes are indeed new genes, rather than transduced cellular cancer genes. They are recombinants put together from truncated viral and truncated proto-onc genes. Proto-onc genes are frequently expressed in normal cells. To date, not one activated proto-onc gene has been isolated that transforms diploid cells. Above all, no diploid tumors with activated proto-onc genes have been found. Moreover, the probability of spontaneous transformation in vivo is at least 10(9) times lower than predicted from the mechanisms thought to activate proto-onc genes. Therefore, the hypothesis that proto-onc genes are latent cellular oncogenes appears to be an overinterpretation of sequence homology to structural and functional homology with viral onc genes. Here it is proposed that only rare truncations and illegitimate recombinations that alter the germ-line configuration of cellular genes generate viral and possibly cellular cancer genes. The clonal chromosome

  6. Activities of Human Gene Nomenclature Committee

    SciTech Connect

    2002-07-16

    The objective of this project, shared between NIH and DOE, has been and remains to enable the medical genetics communities to use common names for genes that are discovered by different gene hunting groups, in different species. This effort provides consistent gene nomenclature and approved gene symbols to the community at large. This contributes to a uniform and consistent understanding of genomes, particularly the human as well as functional genomics based on comparisons between homologous genes in related species (human and mice).

  7. Acute physical activity effects on cardiac gene expression.

    PubMed

    Simonsen, Michelle L; Alessio, Helaine M; White, Peter; Newsom, David L; Hagerman, Ann E

    2010-11-01

    Regular bouts of physical activity may cause changes in gene expression that accumulate over time and ultimately affect phenotypes, such as body weight, blood lipid profile and tumour development. Furthermore, acute activity may affect gene expression and phenotypes differently depending on whether the individual is regularly inactive or active. One-month-old male Sprague-Dawley rats (n = 72) were equally divided into SED (standard laboratory cage, n = 24), PA (large activity box, n = 24) and EX groups (exercise wheel inside standard cage, n = 24). At 3 months of age, half the animals from each group were killed at rest and the other half following 30 min of physical activity. The RNA was extracted from cardiac tissue, and microarray analysis was performed on 27,000 genes. Select gene results were validated using quantitative PCR. No gene expression differences occurred when comparing all 3-month-old groups at rest. A relatively small percentage of genes (1.9%) were differentially expressed (P < 0.05) following acute swimming activity in all groups, but only 37 unique and identifiable genes reached or exceeded twofold differences in expression. The genes Atf3, Fos, Apold1 and Pxdn were expressed differently among SED, PA and EX following acute activity, with a clear separation of the magnitude in gene expression with SED > PA > EX. Differences in gene expression levels in young physically inactive and active animals following acute activity have different regulatory roles in gene networks that affect health-related phenotypes.

  8. Helix Rotation Model of the Flagellar Rotary Motor

    PubMed Central

    Schmitt, Rüdiger

    2003-01-01

    A new model of the flagellar motor is proposed that is based on established dynamics of the KcsA potassium ion channel and on known genetic, biochemical, and biophysical facts, which accounts for the mechanics of torque generation, force transmission, and reversals of motor rotation. It predicts that proton (or in some species sodium ion) flow generates short, reversible helix rotations of the MotA-MotB channel complex (the stator) that are transmitted by Coulomb forces to the FliG segments at the rotor surface. Channels are arranged as symmetric pairs, S and T, that swing back and forth in synchrony. S and T alternate in attaching to the rotor, so that force transmission proceeds in steps. The sense of motor rotation can be readily reversed by conformationally switching the position of charged groups on the rotor so that they interact with the stator during the reverse rather than forward strokes. An elastic device accounts for the observed smoothness of rotation and a prolonged attachment of the torque generators to the rotor, i.e., a high duty ratio of each torque-generating unit. PMID:12885632

  9. Model Studies of the Dynamics of Bacterial Flagellar Motors

    SciTech Connect

    Bai, F; Lo, C; Berry, R; Xing, J

    2009-03-19

    The Bacterial Flagellar Motor is a rotary molecular machine that rotates the helical filaments which propel swimming bacteria. Extensive experimental and theoretical studies exist on the structure, assembly, energy input, power generation and switching mechanism of the motor. In our previous paper, we explained the general physics underneath the observed torque-speed curves with a simple two-state Fokker-Planck model. Here we further analyze this model. In this paper we show (1) the model predicts that the two components of the ion motive force can affect the motor dynamics differently, in agreement with the latest experiment by Lo et al.; (2) with explicit consideration of the stator spring, the model also explains the lack of dependence of the zero-load speed on stator number in the proton motor, recently observed by Yuan and Berg; (3) the model reproduces the stepping behavior of the motor even with the existence of the stator springs and predicts the dwelling time distribution. Predicted stepping behavior of motors with two stators is discussed, and we suggest future experimental verification.

  10. Transient pauses of the bacterial flagellar motor at low load

    NASA Astrophysics Data System (ADS)

    Nord, A. L.; Pedaci, F.; Berry, R. M.

    2016-11-01

    The bacterial flagellar motor (BFM) is the molecular machine responsible for the swimming and chemotaxis of many species of motile bacteria. The BFM is bidirectional, and changes in the rotation direction of the motor are essential for chemotaxis. It has previously been observed that many species of bacteria also demonstrate brief pauses in rotation, though the underlying cause of such events remains poorly understood. We examine the rotation of Escherichia coli under low mechanical load with high spatial and temporal resolution. We observe and characterize transient pauses in rotation in a strain which lacks a functional chemosensory network, showing that such events are a phenomenon separate from a change in rotational direction. Rotating at low load, the BFM of E. coli exhibits about 10 pauses s-1, lasting on average 5 ms, during which time the rotor diffuses with net forwards rotation. Replacing the wild type stators with Na+ chimera stators has no substantial effect on the pausing. We discuss possible causes of such events, which are likely a product of a transient change in either the stator complex or the rotor.

  11. Model studies of the dynamics of bacterial flagellar motors.

    PubMed

    Bai, Fan; Lo, Chien-Jung; Berry, Richard M; Xing, Jianhua

    2009-04-22

    The bacterial flagellar motor is a rotary molecular machine that rotates the helical filaments that propel swimming bacteria. Extensive experimental and theoretical studies exist on the structure, assembly, energy input, power generation, and switching mechanism of the motor. In a previous article, we explained the general physics underneath the observed torque-speed curves with a simple two-state Fokker-Planck model. Here, we further analyze that model, showing that 1), the model predicts that the two components of the ion motive force can affect the motor dynamics differently, in agreement with latest experiments; 2), with explicit consideration of the stator spring, the model also explains the lack of dependence of the zero-load speed on stator number in the proton motor, as recently observed; and 3), the model reproduces the stepping behavior of the motor even with the existence of the stator springs and predicts the dwell-time distribution. The predicted stepping behavior of motors with two stators is discussed, and we suggest future experimental procedures for verification.

  12. Polar features in the flagellar propulsion of E. coli bacteria.

    PubMed

    Bianchi, S; Saglimbeni, F; Lepore, A; Di Leonardo, R

    2015-06-01

    E. coli bacteria swim following a run and tumble pattern. In the run state all flagella join in a single helical bundle that propels the cell body along approximately straight paths. When one or more flagellar motors reverse direction the bundle unwinds and the cell randomizes its orientation. This basic picture represents an idealization of a much more complex dynamical problem. Although it has been shown that bundle formation can occur at either pole of the cell, it is still unclear whether these two run states correspond to asymmetric propulsion features. Using holographic microscopy we record the 3D motions of individual bacteria swimming in optical traps. We find that most cells possess two run states characterized by different propulsion forces, total torque, and bundle conformations. We analyze the statistical properties of bundle reversal and compare the hydrodynamic features of forward and backward running states. Our method is naturally multi-particle and opens up the way towards controlled hydrodynamic studies of interacting swimming cells.

  13. [A new type of flagellar structure. Type 9+n

    PubMed Central

    1977-01-01

    The ultrastructural study of the Eoacanthocephala sperm cell shows a variation from 0 to 5 in the number of the axial fibers in the axoneme. All the species of the order Eoacanthocephala available to us show this variation; moreover, every individual possesses simultaneously several different structural types. So, we are dealing with a new flagellar organization: 9+n, with 0 less than or equal to n less than or equal to 5. In the Quadrigyridae and the Tenuisentidae families, n varies from 0 to 4, with a maximum of 2 for most individuals, exceptionally at 1 for some individuals. In the Neoechinorhynchidae family, n varies from 0 to 5 with a conspicuous prevalence of 3 (from 84 to 99%, according to the individual). These results prompted us to reexamine the two other orders of Acanthocephala in which the structural types 9+2 or 9+0 have been considered as fixed. Indeed, we have found a few flagella the structure of which is different from the prevalent one. It seems, therefore, that the number of the central fibers of the axoneme in the Acanthocephala sperm cell is never absolutely fixed. PMID:557042

  14. Real-Time Imaging of Fluorescent Flagellar Filaments

    PubMed Central

    Turner, Linda; Ryu, William S.; Berg, Howard C.

    2000-01-01

    Bacteria swim by rotating flagellar filaments that are several micrometers long, but only about 20 nm in diameter. The filaments can exist in different polymorphic forms, having distinct values of curvature and twist. Rotation rates are on the order of 100 Hz. In the past, the motion of individual filaments has been visualized by dark-field or differential-interference-contrast microscopy, methods hampered by intense scattering from the cell body or shallow depth of field, respectively. We have found a simple procedure for fluorescently labeling cells and filaments that allows recording their motion in real time with an inexpensive video camera and an ordinary fluorescence microscope with mercury-arc or strobed laser illumination. We report our initial findings with cells of Escherichia coli. Tumbles (events that enable swimming cells to alter course) are remarkably varied. Not every filament on a cell needs to change its direction of rotation: different filaments can change directions at different times, and a tumble can result from the change in direction of only one. Polymorphic transformations tend to occur in the sequence normal, semicoiled, curly 1, with changes in the direction of movement of the cell body correlated with transformations to the semicoiled form. PMID:10781548

  15. Bacteria can exploit a flagellar buckling instability to change direction

    NASA Astrophysics Data System (ADS)

    Son, Kwangmin; Guasto, Jeffrey S.; Stocker, Roman

    2013-08-01

    Bacteria swim by rotating rigid helical flagella and periodically reorienting to follow environmental cues. Despite the crucial role of reorientations, their underlying mechanism has remained unknown for most uni-flagellated bacteria. Here, we report that uni-flagellated bacteria turn by exploiting a finely tuned buckling instability of their hook, the 100-nm-long structure at the base of their flagellar filament. Combining high-speed video microscopy and mechanical stability theory, we demonstrate that reorientations occur 10ms after the onset of forward swimming, when the hook undergoes compression, and that the associated hydrodynamic load triggers the buckling of the hook. Reducing the load on the hook below the buckling threshold by decreasing the swimming speed results in the suppression of reorientations, consistent with the critical nature of buckling. The mechanism of turning by buckling represents one of the smallest examples in nature of a biological function stemming from controlled mechanical failure and reveals a new role for flexibility in biological materials, which may inspire new microrobotic solutions in medicine and engineering.

  16. Real-Time Imaging of Fluorescent Flagellar Filaments

    NASA Astrophysics Data System (ADS)

    Ryu, William

    2003-03-01

    Bacteria swim by rotating flagellar filaments that are several micrometers long, but only about 18 nm in diameter. The filaments can exist in different polymorphic forms, having distinct values of curvature and twist. Rotation rates are on the order of 100 Hz. In the past, the motion of individual filaments has been visualized by dark-field or differential-interference-contrast microscopy, methods hampered by intense scattering from the cell body or shallow depth of field, respectively. We have found a simple procedure for fluorescently labeling cells and filaments that allows recording their motion in real time with an inexpensive video camera and an ordinary fluorescence microscope with mercury-arc or strobed laser illumination. We report our initial findings with cells of Escherichia coli. Tumbles (events that enable swimming cells to alter course) are remarkably varied. Not every filament on a cell needs to change its direction of rotation: different filaments can change directions at different times, and a tumble can result from the change in direction of only one. Polymorphic transformations tend to occur in the sequence normal, semicoiled, curly 1, with changes in the direction of movement of the cell body correlated with transformations to the semicoiled form.

  17. Polar features in the flagellar propulsion of E. coli bacteria

    NASA Astrophysics Data System (ADS)

    Bianchi, S.; Saglimbeni, F.; Lepore, A.; Di Leonardo, R.

    2015-06-01

    E. coli bacteria swim following a run and tumble pattern. In the run state all flagella join in a single helical bundle that propels the cell body along approximately straight paths. When one or more flagellar motors reverse direction the bundle unwinds and the cell randomizes its orientation. This basic picture represents an idealization of a much more complex dynamical problem. Although it has been shown that bundle formation can occur at either pole of the cell, it is still unclear whether these two run states correspond to asymmetric propulsion features. Using holographic microscopy we record the 3D motions of individual bacteria swimming in optical traps. We find that most cells possess two run states characterized by different propulsion forces, total torque, and bundle conformations. We analyze the statistical properties of bundle reversal and compare the hydrodynamic features of forward and backward running states. Our method is naturally multi-particle and opens up the way towards controlled hydrodynamic studies of interacting swimming cells.

  18. Novel Membrane-Bound eIF2α Kinase in the Flagellar Pocket of Trypanosoma brucei▿

    PubMed Central

    Moraes, Maria Carolina S.; Jesus, Teresa C. L.; Hashimoto, Nilce N.; Dey, Madhusudan; Schwartz, Kevin J.; Alves, Viviane S.; Avila, Carla C.; Bangs, James D.; Dever, Thomas E.; Schenkman, Sergio; Castilho, Beatriz A.

    2007-01-01

    Translational control mediated by phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF2α) is central to stress-induced programs of gene expression. Trypanosomatids, important human pathogens, display differentiation processes elicited by contact with the distinct physiological milieu found in their insect vectors and mammalian hosts, likely representing stress situations. Trypanosoma brucei, the agent of African trypanosomiasis, encodes three potential eIF2α kinases (TbeIF2K1 to -K3). We show here that TbeIF2K2 is a transmembrane glycoprotein expressed both in procyclic and in bloodstream forms. The catalytic domain of TbeIF2K2 phosphorylates yeast and mammalian eIF2α at Ser51. It also phosphorylates the highly unusual form of eIF2α found in trypanosomatids specifically at residue Thr169 that corresponds to Ser51 in other eukaryotes. T. brucei eIF2α, however, is not a substrate for GCN2 or PKR in vitro. The putative regulatory domain of TbeIF2K2 does not share any sequence similarity with known eIF2α kinases. In both procyclic and bloodstream forms TbeIF2K2 is mainly localized in the membrane of the flagellar pocket, an organelle that is the exclusive site of exo- and endocytosis in these parasites. It can also be detected in endocytic compartments but not in lysosomes, suggesting that it is recycled between endosomes and the flagellar pocket. TbeIF2K2 location suggests a relevance in sensing protein or nutrient transport in T. brucei, an organism that relies heavily on posttranscriptional regulatory mechanisms to control gene expression in different environmental conditions. This is the first membrane-associated eIF2α kinase described in unicellular eukaryotes. PMID:17873083

  19. Giardia Flagellar Motility Is Not Directly Required to Maintain Attachment to Surfaces

    PubMed Central

    House, Susan A.; Richter, David J.; Pham, Jonathan K.; Dawson, Scott C.

    2011-01-01

    Giardia trophozoites attach to the intestinal microvilli (or inert surfaces) using an undefined “suction-based” mechanism, and remain attached during cell division to avoid peristalsis. Flagellar motility is a key factor in Giardia's pathogenesis and colonization of the host small intestine. Specifically, the beating of the ventral flagella, one of four pairs of motile flagella, has been proposed to generate a hydrodynamic force that results in suction-based attachment via the adjacent ventral disc. We aimed to test this prevailing “hydrodynamic model” of attachment mediated by flagellar motility. We defined four distinct stages of attachment by assessing surface contacts of the trophozoite with the substrate during attachment using TIRF microscopy (TIRFM). The lateral crest of the ventral disc forms a continuous perimeter seal with the substrate, a cytological indication that trophozoites are fully attached. Using trophozoites with two types of molecularly engineered defects in flagellar beating, we determined that neither ventral flagellar beating, nor any flagellar beating, is necessary for the maintenance of attachment. Following a morpholino-based knockdown of PF16, a central pair protein, both the beating and morphology of flagella were defective, but trophozoites could still initiate proper surface contacts as seen using TIRFM and could maintain attachment in several biophysical assays. Trophozoites with impaired motility were able to attach as well as motile cells. We also generated a strain with defects in the ventral flagellar waveform by overexpressing a dominant negative form of alpha2-annexin::GFP (D122A, D275A). This dominant negative alpha2-annexin strain could initiate attachment and had only a slight decrease in the ability to withstand normal and shear forces. The time needed for attachment did increase in trophozoites with overall defective flagellar beating, however. Thus while not directly required for attachment, flagellar motility is

  20. High-speed holographic microscopy of malaria parasites reveals ambidextrous flagellar waveforms

    PubMed Central

    Wilson, Laurence G.; Carter, Lucy M.; Reece, Sarah E.

    2013-01-01

    Axonemes form the core of eukaryotic flagella and cilia, performing tasks ranging from transporting fluid in developing embryos to the propulsion of sperm. Despite their abundance across the eukaryotic domain, the mechanisms that regulate the beating action of axonemes remain unknown. The flagellar waveforms are 3D in general, but current understanding of how axoneme components interact stems from 2D data; comprehensive measurements of flagellar shape are beyond conventional microscopy. Moreover, current flagellar model systems (e.g., sea urchin, human sperm) contain accessory structures that impose mechanical constraints on movement, obscuring the “native” axoneme behavior. We address both problems by developing a high-speed holographic imaging scheme and applying it to the (male) microgametes of malaria (Plasmodium) parasites. These isolated flagella are a unique, mathematically tractable model system for the physics of microswimmers. We reveal the 3D flagellar waveforms of these microorganisms and map the differential shear between microtubules in their axonemes. Furthermore, we overturn claims that chirality in the structure of the axoneme governs the beat pattern [Hirokawa N, et al. (2009) Ann Rev Fluid Mech 41:53–72], because microgametes display a left- or right-handed character on alternate beats. This breaks the link between structural chirality in the axoneme and larger scale symmetry breaking (e.g., in developing embryos), leading us to conclude that accessory structures play a critical role in shaping the flagellar beat. PMID:24194551

  1. Coordinated regulation of multiple flagellar motors by the Escherichia coli chemotaxis system.

    PubMed

    Fukuoka, Hajime; Inoue, Yuichi; Ishijima, Akihiko

    2012-01-01

    Escherichia coli cells swim toward a favorable environment by chemotaxis. The chemotaxis system regulates the swimming behavior of the bacteria by controlling the rotational direction of their flagellar motors. Extracellular stimuli sensed by chemoreceptors are transduced to an intracellular signal molecule, phosphorylated CheY (CheY-P), that switches the rotational direction of the flagellar motors from counterclockwise (CCW) to clockwise (CW) or from CW to CCW. Many studies have focused on identifying the proteins involved in the chemotaxis system, and findings on the structures and intracellular localizations of these proteins have largely elucidated the molecular pathway. On the other hand, quantitative evaluations of the chemotaxis system, including the process of intracellular signaling by the propagation of CheY-P and the rotational switching of flagellar motor by binding of CheY-P molecules, are still uncertain. For instance, scientific consensus has held that the flagellar motors of an E. coli cell switch rotational direction asynchronously. However, recent work shows that the rotational switching of any two different motors on a single E. coli cell is highly coordinated; a sub-second switching delay between motors is clearly correlated with the relative distance of each motor from the chemoreceptor patch located at one pole of the cell. In this review of previous studies and our recent findings, we discuss the regulatory mechanism of the multiple flagellar motors on an individual E. coli cell and the intracellular signaling process that can be inferred from this coordinated switching.

  2. Coordinated regulation of multiple flagellar motors by the Escherichia coli chemotaxis system

    PubMed Central

    Fukuoka, Hajime; Inoue, Yuichi; Ishijima, Akihiko

    2012-01-01

    Escherichia coli cells swim toward a favorable environment by chemotaxis. The chemotaxis system regulates the swimming behavior of the bacteria by controlling the rotational direction of their flagellar motors. Extracellular stimuli sensed by chemoreceptors are transduced to an intracellular signal molecule, phosphorylated CheY (CheY-P), that switches the rotational direction of the flagellar motors from counterclockwise (CCW) to clockwise (CW) or from CW to CCW. Many studies have focused on identifying the proteins involved in the chemotaxis system, and findings on the structures and intracellular localizations of these proteins have largely elucidated the molecular pathway. On the other hand, quantitative evaluations of the chemotaxis system, including the process of intracellular signaling by the propagation of CheY-P and the rotational switching of flagellar motor by binding of CheY-P molecules, are still uncertain. For instance, scientific consensus has held that the flagellar motors of an E. coli cell switch rotational direction asynchronously. However, recent work shows that the rotational switching of any two different motors on a single E. coli cell is highly coordinated; a sub-second switching delay between motors is clearly correlated with the relative distance of each motor from the chemoreceptor patch located at one pole of the cell. In this review of previous studies and our recent findings, we discuss the regulatory mechanism of the multiple flagellar motors on an individual E. coli cell and the intracellular signaling process that can be inferred from this coordinated switching. PMID:27857608

  3. Nonconventional cation-coupled flagellar motors derived from the alkaliphilic Bacillus and Paenibacillus species.

    PubMed

    Ito, Masahiro; Takahashi, Yuka

    2017-01-01

    Prior to 2008, all previously studied conventional bacterial flagellar motors appeared to utilize either H(+) or Na(+) as coupling ions. Membrane-embedded stator complexes support conversion of energy using transmembrane electrochemical ion gradients. The main H(+)-coupled stators, known as MotAB, differ from Na(+)-coupled stators, PomAB of marine bacteria, and MotPS of alkaliphilic Bacillus. However, in 2008, a MotAB-type flagellar motor of alkaliphilic Bacillus clausii KSM-K16 was revealed as an exception with the first dual-function motor. This bacterium was identified as the first bacterium with a single stator-rotor that can utilize both H(+) and Na(+) for ion-coupling at different pH ranges. Subsequently, another exception, a MotPS-type flagellar motor of alkaliphilic Bacillus alcalophilus AV1934, was reported to utilize Na(+) plus K(+) and Rb(+) as coupling ions for flagellar rotation. In addition, the alkaline-tolerant bacterium Paenibacillus sp. TCA20, which can utilize divalent cations such as Ca(2+), Mg(2+), and Sr(2+), was recently isolated from a hot spring in Japan, which contains a high Ca(2+) concentration. These findings show that bacterial flagellar motors isolated from unique environments utilize unexpected coupling ions. This suggests that bacteria that grow in different extreme environments adapt to local conditions and evolve their motility machinery.

  4. Entosiphon sulcatum (Euglenophyceae): flagellar roots of the basal body complex and reservoir region

    SciTech Connect

    Solomon, J.A.; Walne, P.L.; Kivic, P.A.

    1987-03-01

    The flagellar root system of Entosiphon sulcatum (Dujardin) Stein (Euglenophyceae) is described and compared with kinetoplastid and other euglenoid systems. An asymmetric pattern of three microtubular roots, one between the two flagellar basal bodies and one on either side (here called the intermediate, dorsal, and ventral roots), is consistent within the euglenoid flagellates studied thus far. The dorsal root is associated with the basal body of the anterior flagellum (F1) and lies on the left dorsal side of the basal body complex. Originating between the two flagellar basal bodies, and associated with the basal body of the trailing flagellum (F2), the intermediate root is morphologically distinguished by fibrils interconnecting the individual microtubules to one another and to the overlying reservoir membrane. The intermediate root is often borne on a ridge projecting into the reservoir. The ventral root originates near the F2 basal body and lies on the right ventral side of the cell. Fibrillar connections link the membrane of F2 with the reservoir membrane at the reservoir-canal transition level. A large cross-banded fiber joins the two flagellar basal bodies, and a series of smaller striated fibers links the anterior accessory and flagellar basal bodies. Large nonstriated fibers extend from the basal body complex posteriorly into the cytoplasm.

  5. Building predictive gene signatures through simultaneous assessment of transcription factor activation and gene expression.

    EPA Science Inventory

    Building predictive gene signatures through simultaneous assessment of transcription factor activation and gene expression Exposure to many drugs and environmentally-relevant chemicals can cause adverse outcomes. These adverse outcomes, such as cancer, have been linked to mol...

  6. Building predictive gene signatures through simultaneous assessment of transcription factor activation and gene expression.

    EPA Science Inventory

    Building predictive gene signatures through simultaneous assessment of transcription factor activation and gene expression Exposure to many drugs and environmentally-relevant chemicals can cause adverse outcomes. These adverse outcomes, such as cancer, have been linked to mol...

  7. Replication timing-related and gene body-specific methylation of active human genes.

    PubMed

    Aran, Dvir; Toperoff, Gidon; Rosenberg, Michael; Hellman, Asaf

    2011-02-15

    Understanding how the epigenetic blueprint of the genome shapes human phenotypes requires systematic evaluation of the complex interplay between gene activity and the different layers of the epigenome. Utilizing microarray-based techniques, we explored the relationships between DNA methylation, DNA replication timing and gene expression levels across a variety of human tissues and cell lines. The analyses revealed unequal methylation levels among early- and late-replicating fractions of the genome: late-replicating DNA was hypomethylated compared with early-replicating DNA. Moreover, late-replicating regions were gradually demethylated with cell divisions, whereas the methylation of early-replicating regions was better maintained. As active genes concentrate at early-replicating regions, they are overall hypermethylated relative to inactive genes. Accordingly, we show that the previously reported positive correlation between gene-body methylation (methylation of the transcribed portion of genes) and gene expression is restricted to proliferative tissues and cell lines, whereas in tissues containing few proliferating cells, active and inactive genes have similar methylation levels. We further show that active gene bodies are hypermethylated not only compared with inactive gene bodies, but also compared with their flanking sequences. This specific hypermethylation of the active gene bodies is severely disrupted in cells of an immunodeficiency, centromeric region instability, facial anomalies (ICF) syndrome patient bearing mutated DNA methyltransferase 3B (DNMT3B). Our data show that a high methylation level is preferentially maintained in active gene bodies through independent cellular processes. Rather than serving as a distinctive mark between active and inactive genes, gene-body methylation appears to serve a vital, currently unknown function in active genes.

  8. Zipping and entanglement in flagellar bundle of E. coli: Role of motile cell body

    NASA Astrophysics Data System (ADS)

    Adhyapak, Tapan Chandra; Stark, Holger

    2015-11-01

    The course of a peritrichous bacterium, such as E. coli, crucially depends on the level of synchronization and self-organization of several rotating flagella. However, the rotation of each flagellum generates countermovements of the body which in turn affect the flagellar dynamics. Using a detailed numerical model of an E. coli, we demonstrate that flagellar entanglement, besides fluid flow relative to the moving body, dramatically changes the dynamics of flagella from that compared to anchored flagella. In particular, bundle formation occurs through a zipping motion in a remarkably rapid time, affected little by initial flagellar orientation. A simplified analytical model supports our observations. Finally, we illustrate how entanglement, hydrodynamic interactions, and body movement contribute to zipping and bundling.

  9. Preparing well-oriented sols of straight bacterial flagellar filaments for X-ray fiber diffraction.

    PubMed

    Yamashita, I; Vonderviszt, F; Noguchi, T; Namba, K

    1991-01-20

    Well-oriented sols of straight bacterial flagellar filaments have been obtained by preparing reconstituted flagellar filaments with an appropriate length distribution and choosing appropriate solvent conditions. An average filament length of 300 to 500 nm and the use of solvents with very low concentrations of salt has allowed us to prepare highly fluid sols that make flow orientation possible. X-ray fiber diffraction from these sols has shown distinct layer-line reflections to 3.5 A resolution in the meridional direction. Layer-line intensities have been collected by the angular deconvolution method up to 5 A resolution. The possibility of using a magnetic field to further improve the orientation has been explored and a solvent condition that makes flagellar sols sensitive to the magnetic field has been found. General applicability of the method to other systems is also discussed.

  10. Biochemical characterization of tektins from sperm flagellar doublet microtubules

    PubMed Central

    1987-01-01

    Tektins, protein components of stable protofilaments from sea urchin sperm flagellar outer doublet microtubules (Linck, R. W., and G. L. Langevin, 1982, J. Cell Sci., 58:1-22), are separable by preparative SDS PAGE into 47-, 51-, and 55-kD equimolar components. High resolution two-dimensional tryptic peptide mapping reveals 63-67% coincidence among peptides of the 51-kD tektin chain and its 47- and 55-kD counterparts, greater than 70% coincidence between the 47- and 55-kD tektins, but little obvious similarity to either alpha- or beta- tubulin. With reverse-phase HPLC on a C18 column, using 6 M guanidine- HCl solubilization and a 0.1% trifluoroacetic acid/CH3CN gradient system (Stephens, R. E., 1984, J. Cell Biol. 90:37a [Abstr.]), the relatively less hydrophobic 51-kD tektin elutes at greater than 45% CH3CN, immediately followed by the 55-kD chain. The 47-kD tektin is substantially more hydrophobic, eluting between the two tubulins. The amino acid compositions of the tektins are very similar to each other but totally distinct from tubulin chains, being characterized by a greater than 50% higher arginine plus lysine content (in good agreement with the number of tryptic peptides) and about half the content of glycine, histidine, proline, and tyrosine. The proline content correlates well with the fact that tektin filaments have twice as much alpha-helical content as tubulin. Total hydrophobic amino acid content correlates with HPLC elution times for the tektins but not tubulins. The average amino acid composition of the tektins indicates that they resemble intermediate filament proteins, as originally postulated from structural, solubility, and electrophoretic properties. Tektins have higher cysteine and tryptophan contents than desmin and vimentin, which characteristically have only one residue of each, more closely resembling certain keratins in these amino acids. PMID:3558479

  11. Flagellar coordination in Chlamydomonas cells held on micropipettes.

    PubMed

    Rüffer, U; Nultsch, W

    1998-01-01

    The two flagella of Chlamydomonas are known to beat synchronously: During breaststroke beating they are generally coordinated in a bilateral way while in shock responses during undulatory beating coordination is mostly parallel [Rüffer and Nultsch, 1995: Botanica Acta 108:169-276]. Analysis of a great number of shock responses revealed that in undulatory beats also periods of bilateral coordination are found and that the coordination type may change several times during a shock response, without concomitant changes of the beat envelope and the beat period. In normal wt cells no coordination changes are found during breaststroke beating, but only short temporary asynchronies: During 2 or 3 normal beats of the cis flagellum, the trans flagellum performs 3 or 4 flat beats with a reduced beat envelope and a smaller beat period, resulting in one additional trans beat. Long periods with flat beats of the same shape and beat period are found in both flagella of the non-phototactic mutant ptx1 and in defective wt 622E cells. During these periods, the coordination is parallel, the two flagella beat alternately. A correlation between normal asynchronous trans beats and the parallel-coordinated beats in the presumably cis defective cells and also the undulatory beats is discussed. In the cis defective cells, a perpetual spontaneous change between parallel beats with small beat periods (higher beat frequency) and bilateral beats with greater beat periods (lower beat frequency) are observed and render questionable the existence of two different intrinsic beat frequencies of the two flagella cis and trans. Asynchronies occur spontaneously but may also be induced by light changes, either step-up or step-down, but not by both stimuli in turn as breaststroke flagellar photoresponses (BFPRs). Asynchronies are not involved in phototaxis. They are independent of the BFPRs, which are supposed to be the basis of phototaxis. Both types of coordination must be assumed to be regulated

  12. Biochemical characterization of tektins from sperm flagellar doublet microtubules.

    PubMed

    Linck, R W; Stephens, R E

    1987-04-01

    Tektins, protein components of stable protofilaments from sea urchin sperm flagellar outer doublet microtubules (Linck, R. W., and G. L. Langevin, 1982, J. Cell Sci., 58:1-22), are separable by preparative SDS PAGE into 47-, 51-, and 55-kD equimolar components. High resolution two-dimensional tryptic peptide mapping reveals 63-67% coincidence among peptides of the 51-kD tektin chain and its 47- and 55-kD counterparts, greater than 70% coincidence between the 47- and 55-kD tektins, but little obvious similarity to either alpha- or beta-tubulin. With reverse-phase HPLC on a C18 column, using 6 M guanidine-HCl solubilization and a 0.1% trifluoroacetic acid/CH3CN gradient system (Stephens, R. E., 1984, J. Cell Biol. 90:37a [Abstr.]), the relatively less hydrophobic 51-kD tektin elutes at greater than 45% CH3CN, immediately followed by the 55-kD chain. The 47-kD tektin is substantially more hydrophobic, eluting between the two tubulins. The amino acid compositions of the tektins are very similar to each other but totally distinct from tubulin chains, being characterized by a greater than 50% higher arginine plus lysine content (in good agreement with the number of tryptic peptides) and about half the content of glycine, histidine, proline, and tyrosine. The proline content correlates well with the fact that tektin filaments have twice as much alpha-helical content as tubulin. Total hydrophobic amino acid content correlates with HPLC elution times for the tektins but not tubulins. The average amino acid composition of the tektins indicates that they resemble intermediate filament proteins, as originally postulated from structural, solubility, and electrophoretic properties. Tektins have higher cysteine and tryptophan contents than desmin and vimentin, which characteristically have only one residue of each, more closely resembling certain keratins in these amino acids.

  13. Intraflagellar transport (IFT) cargo: IFT transports flagellar precursors to the tip and turnover products to the cell body.

    PubMed

    Qin, Hongmin; Diener, Dennis R; Geimer, Stefan; Cole, Douglas G; Rosenbaum, Joel L

    2004-01-19

    Intraflagellar transport (IFT) is the bidirectional movement of multisubunit protein particles along axonemal microtubules and is required for assembly and maintenance of eukaryotic flagella and cilia. One posited role of IFT is to transport flagellar precursors to the flagellar tip for assembly. Here, we examine radial spokes, axonemal subunits consisting of 22 polypeptides, as potential cargo for IFT. Radial spokes were found to be partially assembled in the cell body, before being transported to the flagellar tip by anterograde IFT. Fully assembled radial spokes, detached from axonemal microtubules during flagellar breakdown or turnover, are removed from flagella by retrograde IFT. Interactions between IFT particles, motors, radial spokes, and other axonemal proteins were verified by coimmunoprecipitation of these proteins from the soluble fraction of Chlamydomonas flagella. These studies indicate that one of the main roles of IFT in flagellar assembly and maintenance is to transport axonemal proteins in and out of the flagellum.

  14. Cloning, sequencing, and disruption of the Bacillus subtilis sigma 28 gene.

    PubMed Central

    Helmann, J D; Márquez, L M; Chamberlin, M J

    1988-01-01

    Bacillus subtilis contains multiple forms of RNA polymerase holoenzyme, distinguished by the presence of different specificity determinants known as sigma factors. The sigma 28 factor was initially purified as a unique transcriptional activity in vegetatively growing B. subtilis cells. Purification of the sigma 28 protein has allowed tryptic peptides to be prepared and sequenced. The sequence of one tryptic peptide fragment was used to prepare an oligonucleotide probe specific for the sigma 28 structural gene, and the gene was isolated from a B. subtilis subgenomic library. The complete nucleotide sequence of the sigma 28 gene was determined, and the cloned sigma 28 gene was used to construct a mutant strain which does not express the sigma 28 protein. This strain also failed to synthesize flagellin protein and grew as long filaments. The predicted sigma 28 gene product is a 254-amino-acid polypeptide with a calculated molecular weight of 29,500. The sigma 28 protein sequence was similar to that of other sequenced sigma factors and to the flbB gene product of Escherichia coli. Since the flbB gene product is a positive regulator of flagellar synthesis in E. coli, it is likely that sigma 28 functions to regulate flagellar synthesis in B. subtilis. Images PMID:2832368

  15. Correspondence between resting state activity and brain gene expression

    PubMed Central

    Wang, Guang-Zhong; Belgard, T. Grant; Mao, Deng; Chen, Leslie; Berto, Stefano; Preuss, Todd M.; Lu, Hanzhang; Geschwind, Daniel H.; Konopka, Genevieve

    2015-01-01

    SUMMARY The relationship between functional brain activity and gene expression has not been fully explored in the human brain. Here, we identify significant correlations between gene expression in the brain and functional activity by comparing fractional Amplitude of Low Frequency Fluctuations (fALFF) from two independent human fMRI resting state datasets to regional cortical gene expression from a newly generated RNA-seq dataset and two additional gene expression datasets to obtain robust and reproducible correlations. We find significantly more genes correlated with fALFF than expected by chance, and identify specific genes correlated with the imaging signals in multiple expression datasets in the default mode network. Together, these data support a population-level relationship between regional steady state brain gene expression and resting state brain activity. PMID:26590343

  16. Evolution of Brain Active Gene Promoters in Human Lineage Towards the Increased Plasticity of Gene Regulation.

    PubMed

    Gunbin, Konstantin V; Ponomarenko, Mikhail P; Suslov, Valentin V; Gusev, Fedor; Fedonin, Gennady G; Rogaev, Evgeny I

    2017-02-24

    Adaptability to a variety of environmental conditions is a prominent feature of Homo sapiens. We hypothesize that this feature can be explained by evolutionary changes in gene promoters active in the brain prefrontal cortex leading to a more flexible gene regulation network. The genotype-dependent range of gene expression can be broader in humans than in other higher primates. Thus, we searched for specific signatures of evolutionary changes in promoter architectures of multiple hominid genes, including the genes active in human cortical neurons that may indicate an increase of variability of gene expression rather than just changes in the level of expression, such as downregulation or upregulation of the genes. We performed a whole-genome search for genetic-based alterations that may impact gene regulation "flexibility" in a process of hominids evolution, such as (i) CpG dinucleotide content, (ii) predicted nucleosome-DNA dissociation constant, and (iii) predicted affinities for TATA-binding protein (TBP) in gene promoters. We tested all putative promoter regions across the human genome and especially gene promoters in active chromatin state in neurons of prefrontal cortex, the brain region critical for abstract thinking and social and behavioral adaptation. Our data imply that the origin of modern man has been associated with an increase of flexibility of promoter-driven gene regulation in brain. In contrast, after splitting from the ancestral lineages of H. sapiens, the evolution of ape species is characterized by reduced flexibility of gene promoter functioning, underlying reduced variability of the gene expression.

  17. Genetic dissection of the Leishmania paraflagellar rod, a unique flagellar cytoskeleton structure.

    PubMed

    Maga, J A; Sherwin, T; Francis, S; Gull, K; LeBowitz, J H

    1999-08-01

    The paraflagellar rod (PFR) is a unique network of cytoskeletal filaments that lies alongside the axoneme in the flagella of most trypanosomatids. While little is known about how two major Leishmania mexicana PFR protein components, PFR1 and PFR2, assemble into this complex structure, previous analysis of PFR2 null mutants demonstrated that the PFR is essential for proper cell motility. The structural roles of PFR1 and PFR2 are now examined through comparison of PFR2 null mutants with new PFR1 null mutant and PFR1/PFR2 double null mutant parasites. Both PFR1 and PFR2 were essential for PFR formation and cell motility. When elimination of one PFR gene prevented assembly of a native PFR structure, the other PFR protein accumulated at the distal flagellar tip. Comparison of PFR substructures remaining in each mutant revealed that: (1) fibers that attach the PFR to the axoneme did not contain PFR1 or PFR2, and assemble in the absence of a PFR. (2) PFR1 was synthesized and transported to the flagella in the absence of PFR2, where it formed a stable association with the axoneme attachment fibers. (3) PFR2 was synthesized and transported to the flagella in the absence of PFR1, though it was not found associated with the axoneme attachment fibers. (4) PFR1 and PFR2 were located throughout the subdomains of the PFR. These data suggest that while PFR filaments contain both PFR1 and PFR2, the PFR is attached to the axoneme by interaction of PFR1 with the axoneme attachment fibers.

  18. Pausing of flagellar rotation is a component of bacterial motility and chemotaxis.

    PubMed Central

    Lapidus, I R; Welch, M; Eisenbach, M

    1988-01-01

    When bacterial cells are tethered to glass by their flagella, many of them spin. On the basis of experiments with tethered cells it has generally been thought that the motor which drives the flagellum is a two-state device, existing in either a counterclockwise or a clockwise state. Here we show that a third state of the motor is that of pausing, the duration and frequency of which are affected by chemotactic stimuli. We have recorded on video tape the rotation of tethered Escherichia coli and Salmonella typhimurium cells and analyzed the recordings frame by frame and in slow motion. Most wild-type cells paused intermittently. The addition of repellents caused an increase in the frequency and duration of the pauses. The addition of attractants sharply reduced the number of pauses. A chemotaxis mutant which lacks a large part of the chemotaxis machinery owing to a deletion of the genes from cheA to cheZ did not pause at all and did not respond to repellents by pausing. A tumbly mutant of S. typhimurium responded to repellents by smooth swimming and to attractants by tumbling. When tethered, these cells exhibited a normal rotational response but an inverse pausing response to chemotactic stimuli: the frequency of pauses decreased in response to repellents and increased in response to attractants. It is suggested that (i) pausing is an integral part of bacterial motility and chemotaxis, (ii) pausing is independent of the direction of flagellar rotation, and (iii) pausing may be one of the causes of tumbling. PMID:3042756

  19. Orthogonal gene knock out and activation with a catalytically active Cas9 nuclease

    PubMed Central

    Dahlman, James E.; Abudayyeh, Omar O.; Joung, Julia; Gootenberg, Jonathan S.; Zhang, Feng; Konermann, Silvana

    2015-01-01

    We have developed a CRISPR-based method that uses catalytically active Cas9 and distinct sgRNA constructs to knock out and activate different genes in the same cell. These sgRNAs, with 14 15 bp target sequences and MS2 binding loops, can activate gene expression using an active Cas9 nuclease, without inducing DSBs. We use these ‘dead RNAs’ to perform orthogonal gene knockout and transcriptional activation in human cells. PMID:26436575

  20. FlhF, a signal recognition particle-like GTPase, is involved in the regulation of flagellar arrangement, motility behaviour and protein secretion in Bacillus cereus.

    PubMed

    Salvetti, Sara; Ghelardi, Emilia; Celandroni, Francesco; Ceragioli, Mara; Giannessi, Francesco; Senesi, Sonia

    2007-08-01

    Flagellar arrangement is a highly conserved feature within bacterial species. However, only a few genes regulating cell flagellation have been described in polar flagellate bacteria. This report demonstrates that the arrangement of flagella in the peritrichous flagellate Bacillus cereus is controlled by flhF. Disruption of flhF in B. cereus led to a reduction in the number of flagella from 10-12 to 1-3 filaments per cell in the insertion mutant MP06. Moreover, compared to the parental strain, MP06 exhibited: (i) shorter smooth swimming phases, causing reduced swimming motility but not affecting chemotaxis; (ii) complete inhibition of swarming motility, as differentiated swarm cells were never detected; (iii) an increased amount of extracellular proteins; and (iv) differential export of virulence determinants, such as haemolysin BL (HBL), phosphatidylcholine-preferring phospholipase C (PC-PLC) and non-haemolytic enterotoxin (NHE). Introduction of a plasmid harbouring flhF (pDGflhF) into MP06 completely restored the wild-type phenotype in the trans-complemented strain MP07. B. cereus flhF was found to constitute a monocistronic transcriptional unit and its overexpression did not produce abnormal features in the wild-type background. Characterization of a B. cereus mutant (MP05) carrying a partial flhF deletion indicated that the last C-terminal domain of FlhF is involved in protein export while not required for flagellar arrangement and motility behaviour. Taken together, these data suggest that B. cereus FlhF is a promising candidate for connecting diverse cellular functions, such as flagellar arrangement, motility behaviour, pattern of protein secretion and virulence phenotype.

  1. The murine Sry gene encodes a nuclear transcriptional activator

    SciTech Connect

    Dubin, R.A.; Ostrer, H.

    1994-09-01

    The Sry gene functions as a genetic switch in gonadal ridge initiating testis determination. The murine Sry and human SRY open reading frames (ORF) share a conserved 79 amino acid motif, the HMG-box, that binds DNA. Outside this region the two genes share no additional homology. These studies were undertaken to determine whether the Sry/SRY genes encode nuclear transcriptional regulators. As judged by the accumulation of lacZ-SRY hybrid proteins in the nucleus, both the human and murine SRY ORFs contain a nuclear localization signal. The murine Sry HMG-box selectively binds the sequence NACAAT in vitro when presented with a random pool of oligonucleotides and binds AACAAT with the highest affinity. The murine Sry ORF, when expressed in HeLa cells, activates transcription of a reporter gene containing multiple copies of the AACAAT binding site. Activation was observed for a GAL4-responsive gene when the murine Sry ORF was linked to the DNA-binding domain of GAL4. Using this system, the activation function was mapped to a C-terminal glutamine/histidine-rich domain. In addition, LexA-Sry fusion genes activated a LexA-responsive gene in yeast. In contrast, a GAL4-human SRY fusion gene did not cause transcriptional activation. These studies suggest that both the human and mouse SRY ORFs encode nuclear, DNA-binding proteins, and that the mouse Sry ORF can function as a transcriptional activator with separable DNA-binding and activator domains.

  2. Identification of the flagellar chaperone FlgN in the phytopathogen Xanthomonas axonopodis pathovar citri by its interaction with hook-associated FlgK.

    PubMed

    Khater, Letícia; Alegria, Marcos C; Borin, Paula F L; Santos, Túlio M; Docena, Cássia; Tasic, Ljubica; Farah, Chuck S; Ramos, Carlos H I

    2007-09-01

    Genome annotation of the plant pathogen Xanthomonas axonopodis pv. citri (Xac), identified flagellar genes in a 15.7 kb gene cluster. However, FlgN, a secretion chaperone for hook-associated proteins FlgK and FlgL, was not identified. We performed extensive screening of the X. axonopodis pv. citri genome with the yeast two-hybrid system to identify a protein with the characteristics of the flagellar chaperone FlgN. We found a candidate (XAC1990) encoded by an operon for components of the flagellum apparatus that interacted with FlgK. In order to further support this finding, Xac FlgK and XAC1990 were cloned, expressed, and purified. The recombinant proteins were characterized by spectroscopic methods and their interaction in vitro confirmed by pull-down assays. We, therefore, conclude that XAC1990 and its homologs in other Xanthomonas species are, in fact, FlgN proteins. These observations extend the sequence diversity covered by this family of proteins.

  3. Computer simulation of flagellar movement. III. Models incorporating cross-bridge kinetics.

    PubMed

    Brokaw, C J; Rintala, D R

    1975-01-01

    A computer simulation procedure is used to analyze the generation of propagated bending waves by flagellar models in which active sliding is generated by a cycle of cross-bridge activity. Two types of cross-bridge cycle have been examined in detail. In both cycles, cross-bridge attachment is followed immediately by a configurational change in the cross-bridge, which transfers energy to a stretched elastic element and generates a shearing force between the filaments. In the first model, which has cross-bridge behavior close to current ideas about cross-bridge behavior in muscle, cross-bridge attachment is proportional to curvature of the flagellum and detachment is an exponential decay process. The configurational change is equivalent to an angular deviation of pi/5 radians. In the second type of cross-bridge cycle, cross-bridge attachment occurs rapidly when a critical curvature is reached, and detachment occurs when a critical curvature in the opposite direction is reached. With this cycle, an unrealistically large angular deviation of the cross-bridges, equivalent to 3.0 radians, is required to obtain bending waves of normal amplitude. Both models generate bending wave patterns similar to those obtained in earlier work. However, the behavior of the second type of cross-bridge model more closely matches the actual behavior of flagella under experimental conditions: the chemical turnover rate per beat cycle remains constant as the viscosity is increased, and reduction in the number of active cross-bridges can cause a reduction in beat frequency, with little change in amplitude or wavelength.

  4. Central-pair-linked regulation of microtubule sliding by calcium in flagellar axonemes.

    PubMed

    Nakano, Izumi; Kobayashi, Takeshi; Yoshimura, Misako; Shingyoji, Chikako

    2003-04-15

    The movement of eukaryotic flagella and cilia is regulated by intracellular calcium. We have tested a model in which the central pair of microtubules mediate the effect of Ca(2+) to modify the dynein activity. We used a novel microtubule sliding assay that allowed us to test the effect of Ca(2+) in the presence or absence of the central-pair microtubules. When flagellar axonemes of sea-urchin sperm were exposed to ATP in the presence of elastase, they showed different types of sliding disintegration depending on the ATP concentration: at low concentrations of ATP (/=100 micro M), a large proportion of the axonemes showed limited sliding and split lengthwise into a pair of two microtubule bundles, one of which was thicker than the other. The sliding behaviour of the axonemes was also influenced by Ca(2+). Thus, at 1 mM ATP, the proportion of axonemes that split into two bundles increased from 25% at <10(-9) M Ca(2+) to 60% at 10(-4) M Ca(2+), whereas the sliding velocity of doublets during the splitting did not change. Electron microscopy of split bundles showed that the thicker bundles contained five or six doublets and the central pair, whereas the thinner bundles contained three or four doublets but not the central pair. Closer examinations revealed that the thicker bundles were dominated by four patterns of doublet combinations: doublets 8-9-1-2-3-4, 8-9-1-2-3, 4-5-6-7-8 and 3-4-5-6-7-8. This indicates that the sliding occurred preferentially at one or two fixed interdoublet sites on either side of the central-pair microtubules, whereas the sliding at the remaining interdoublet sites was inhibited under these conditions. Ca(2+) reduced the appearance of the 4-5-6-7-8 and 3-4-5-6-7-8 patterns and increased the 8-9-1-2-3-4 and 8-9-1-2-3 patterns. The splitting patterns are possibly related to the switching mechanism of the

  5. FlrA, a sigma54-dependent transcriptional activator in Vibrio fischeri, is required for motility and symbiotic light-organ colonization.

    PubMed

    Millikan, Deborah S; Ruby, Edward G

    2003-06-01

    Flagellum-mediated motility of Vibrio fischeri is an essential factor in the bacterium's ability to colonize its host, the Hawaiian squid Euprymna scolopes. To begin characterizing the nature of the flagellar regulon, we have cloned a gene, designated flrA, from V. fischeri that encodes a putative sigma(54)-dependent transcriptional activator. Genetic arrangement of the flrA locus in V. fischeri is similar to motility master-regulator operons of Vibrio cholerae and Vibrio parahaemolyticus. In addition, examination of regulatory regions of a number of flagellar operons in V. fischeri revealed apparent sigma(54) recognition motifs, suggesting that the flagellar regulatory hierarchy is controlled by a similar mechanism to that described in V. cholerae. However, in contrast to its closest known relatives, flrA mutant strains of V. fischeri ES114 were completely abolished in swimming capability. Although flrA provided in trans restored motility to the flrA mutant, the complemented strain was unable to reach wild-type levels of symbiotic colonization in juvenile squid, suggesting a possible role for the proper expression of FlrA in regulating symbiotic colonization factors in addition to those required for motility. Comparative RNA arbitrarily primed PCR analysis of the flrA mutant and its wild-type parent revealed several differentially expressed transcripts. These results define a regulon that includes both flagellar structural genes and other genes apparently not involved in flagellum elaboration or function. Thus, the transcriptional activator FlrA plays an essential role in regulating motility, and apparently in modulating other symbiotic functions, in V. fischeri.

  6. Chromatin remodeling inactivates activity genes and regulates neural coding.

    PubMed

    Yang, Yue; Yamada, Tomoko; Hill, Kelly K; Hemberg, Martin; Reddy, Naveen C; Cho, Ha Y; Guthrie, Arden N; Oldenborg, Anna; Heiney, Shane A; Ohmae, Shogo; Medina, Javier F; Holy, Timothy E; Bonni, Azad

    2016-07-15

    Activity-dependent transcription influences neuronal connectivity, but the roles and mechanisms of inactivation of activity-dependent genes have remained poorly understood. Genome-wide analyses in the mouse cerebellum revealed that the nucleosome remodeling and deacetylase (NuRD) complex deposits the histone variant H2A.z at promoters of activity-dependent genes, thereby triggering their inactivation. Purification of translating messenger RNAs from synchronously developing granule neurons (Sync-TRAP) showed that conditional knockout of the core NuRD subunit Chd4 impairs inactivation of activity-dependent genes when neurons undergo dendrite pruning. Chd4 knockout or expression of NuRD-regulated activity genes impairs dendrite pruning. Imaging of behaving mice revealed hyperresponsivity of granule neurons to sensorimotor stimuli upon Chd4 knockout. Our findings define an epigenetic mechanism that inactivates activity-dependent transcription and regulates dendrite patterning and sensorimotor encoding in the brain. Copyright © 2016, American Association for the Advancement of Science.

  7. Cationic Amino Acid Uptake Constitutes a Metabolic Regulation Mechanism and Occurs in the Flagellar Pocket of Trypanosoma cruzi

    PubMed Central

    Bouvier, León A.; Cámara, María de los Milagros; Montserrat, Javier; Pereira, Claudio A.

    2012-01-01

    Trypanosomatids' amino acid permeases are key proteins in parasite metabolism since they participate in the adaptation of parasites to different environments. Here, we report that TcAAP3, a member of a Trypanosoma cruzi multigene family of permeases, is a bona fide arginine transporter. Most higher eukaryotic cells incorporate cationic amino acids through a single transporter. In contrast, T. cruzi can recognize and transport cationic amino acids by mono-specific permeases since a 100-fold molar excess of lysine could not affect the arginine transport in parasites that over-express the arginine permease (TcAAP3 epimastigotes). In order to test if the permease activity regulates downstream processes of the arginine metabolism, the expression of the single T. cruzi enzyme that uses arginine as substrate, arginine kinase, was evaluated in TcAAP3 epimastigotes. In this parasite model, intracellular arginine concentration increases 4-folds and ATP level remains constant until cultures reach the stationary phase of growth, with decreases of about 6-folds in respect to the controls. Interestingly, Western Blot analysis demonstrated that arginine kinase is significantly down-regulated during the stationary phase of growth in TcAAP3 epimastigotes. This decrease could represent a compensatory mechanism for the increase in ATP consumption as a consequence of the displacement of the reaction equilibrium of arginine kinase, when the intracellular arginine concentration augments and the glucose from the medium is exhausted. Using immunofluorescence techniques we also determined that TcAAP3 and the specific lysine transporter TcAAP7 co-localize in a specialized region of the plasma membrane named flagellar pocket, staining a single locus close to the flagellar pocket collar. Taken together these data suggest that arginine transport is closely related to arginine metabolism and cell energy balance. The clinical relevance of studying trypanosomatids' permeases relies on the

  8. Kinetically resolved states of the Halobacterium halobium flagellar motor switch and modulation of the switch by sensory rhodopsin I.

    PubMed Central

    McCain, D A; Amici, L A; Spudich, J L

    1987-01-01

    Spontaneous switching of the rotation sense of the flagellar motor of the archaebacterium Halobacterium halobium and modulation of the switch by attractant and repellent photostimuli were analyzed by using a computerized cell-tracking system with 67-ms resolution coupled to electronic shutters. The data fit a three-state model of the switch, in which a Poisson process governs the transition from state N (nonreversing) to state R (reversing). After a reversal, the switch returns to state N, passing through an intermediate state I (inactive), which produces a ca. 2-s period of low reversal frequency before the state N Poisson rate is restored. The stochastic nature of the H. halobium switch reveals a close similarity to Escherichia coli flagellar motor properties as elucidated previously. Sensory modulation of the switch by both photoattractant and photorepellent signals can be interpreted in terms of modulation of the single forward rate constant of the N to R transition. Insight into the mechanism of modulation by the phototaxis receptor sensory rhodopsin I (SR-I) was gained by increasing the lifetime of the principal photointermediate of the SR-I photochemical reaction cycle, S373, by replacing the native chromophore, all-trans-retinal, with the acyclic analog, 3,7,11-trimethyl-2,4,6,8-dodecapentaenal. Flash photolysis of analog-containing cells revealed an eightfold decrease in the rate of thermal decay of S373, and behavioral analysis showed longer periods of reversal suppression than that of cells with the native chromophore over similar ranges of illumination intensities. This indicates that attractant signaling is governed by the lifetime of the S373 intermediate rather than by the frequency of photocycling. In this sense, SR-I is similar to rhodopsin, whose function depends on an active photoproduct (Meta-II). PMID:3654583

  9. A Comparative Overview of the Flagellar Apparatus of Dinoflagellate, Perkinsids and Colpodellids

    PubMed Central

    Okamoto, Noriko; Keeling, Patrick J.

    2014-01-01

    Dinoflagellates are a member of the Alveolata, and elucidation of the early evolution of alveolates is important for our understanding of dinoflagellates, and vice versa. The ultrastructure of the flagellar apparatus has been described from several dinoflagellates in the last few decades, and the basic components appear to be well conserved. The typical dinoflagellate apparatus is composed of two basal bodies surrounded by striated collars attached to a connective fiber. The longitudinal basal body is connected to a longitudinal microtubular root (LMR; equivalent of R1) and single microtubular root (R2), whereas the transverse basal body is connected to a transverse microtubular root (TMR; R3) and transverse striated root (TSR) with a microtubule (R4). Some of these components, especially the connective fibers and collars, are dinoflagellate specific characteristics that make their flagellar apparatus relatively complex. We also compare these structures with the flagellar apparatus from a number of close relatives of dinoflagellates and their sister, the apicomplexans, including colpodellids, perkinsids, and Psammosa. Though the ultrastructural knowledge of these lineages is still relatively modest, it provides us with an interesting viewpoint of the character evolution of the flagellar apparatus among those lineages. PMID:27694777

  10. Non-equilibrium effect in the allosteric regulation of the bacterial flagellar switch

    NASA Astrophysics Data System (ADS)

    Wang, Fangbin; Shi, Hui; He, Rui; Wang, Renjie; Zhang, Rongjing; Yuan, Junhua

    2017-07-01

    The switching mechanism of the flagellar motor provides the basis for the motile behaviour of flagellated bacteria. Its highly sensitive response has previously been understood in terms of equilibrium models, either the classical two-state concerted allosteric model, or more generally, the Ising-type conformation spread model. Here, we systematically study motor switching under various load conditions from high to zero load, under different proton motive force (pmf) conditions and varying the number of torque-generating units (stators). In doing so, we reveal the signature of a non-equilibrium effect. To consistently account for the motor-switching dependence on each those conditions, a previously neglected non-equilibrium effect--the energy input from the motor torque--has to be incorporated into models of the flagellar switch. We further show that this effect increases the sensitivity of the flagellar switch. Exploiting a very small fraction of the energy expense of the flagellar motor for functional regulation increases its sensitivity greatly. Similar mechanisms are expected to be found in other protein complexes.

  11. DRC3 connects the N-DRC to dynein g to regulate flagellar waveform

    PubMed Central

    Awata, Junya; Song, Kangkang; Lin, Jianfeng; King, Stephen M.; Sanderson, Michael J.; Nicastro, Daniela; Witman, George B.

    2015-01-01

    The nexin-dynein regulatory complex (N-DRC), which is a major hub for the control of flagellar motility, contains at least 11 different subunits. A major challenge is to determine the location and function of each of these subunits within the N-DRC. We characterized a Chlamydomonas mutant defective in the N-DRC subunit DRC3. Of the known N-DRC subunits, the drc3 mutant is missing only DRC3. Like other N-DRC mutants, the drc3 mutant has a defect in flagellar motility. However, in contrast to other mutations affecting the N-DRC, drc3 does not suppress flagellar paralysis caused by loss of radial spokes. Cryo–electron tomography revealed that the drc3 mutant lacks a portion of the N-DRC linker domain, including the L1 protrusion, part of the distal lobe, and the connection between these two structures, thus localizing DRC3 to this part of the N-DRC. This and additional considerations enable us to assign DRC3 to the L1 protrusion. Because the L1 protrusion is the only non-dynein structure in contact with the dynein g motor domain in wild-type axonemes and this is the only N-DRC–dynein connection missing in the drc3 mutant, we conclude that DRC3 interacts with dynein g to regulate flagellar waveform. PMID:26063732

  12. Dynamics in the Dual Fuel Flagellar Motor of Shewanella oneidensis MR-1.

    PubMed

    Brenzinger, Susanne; Thormann, Kai M

    2017-01-01

    The stator is an eminent component of the flagellar motor and determines a number of the motor's properties, such as the rotation-energizing coupling ion (H(+) or Na(+)) or the torque that can be generated. The stator consists of several units located in the cytoplasmic membrane surrounding the flagellar drive shaft. Studies on flagellar motors of several bacterial species have provided evidence that the number as well as the retention time of stators coupled to the motor is highly dynamic and depends on the environmental conditions. Notably, numerous species possess more than a single distinct set of stators. It is likely that the presence of different stator units enables these bacteria to adjust the flagellar motor properties and function to meet the environmental requirements. One of these species is Shewanella oneidensis MR-1 that is equipped with a single polar flagellum and two stator units, the Na(+)-dependent PomAB and the H(+)-dependent MotAB. Here, we describe a method to determine stator dynamics by fluorescence microscopy, demonstrating how bacteria can change the composition of an intricate molecular machine according to environmental conditions.

  13. Swimming performance of Bradyrhizobium diazoefficiens is an emergent property of its two flagellar systems.

    PubMed

    Quelas, J Ignacio; Althabegoiti, M Julia; Jimenez-Sanchez, Celia; Melgarejo, Augusto A; Marconi, Verónica I; Mongiardini, Elías J; Trejo, Sebastián A; Mengucci, Florencia; Ortega-Calvo, José-Julio; Lodeiro, Aníbal R

    2016-04-07

    Many bacterial species use flagella for self-propulsion in aqueous media. In the soil, which is a complex and structured environment, water is found in microscopic channels where viscosity and water potential depend on the composition of the soil solution and the degree of soil water saturation. Therefore, the motility of soil bacteria might have special requirements. An important soil bacterial genus is Bradyrhizobium, with species that possess one flagellar system and others with two different flagellar systems. Among the latter is B. diazoefficiens, which may express its subpolar and lateral flagella simultaneously in liquid medium, although its swimming behaviour was not described yet. These two flagellar systems were observed here as functionally integrated in a swimming performance that emerged as an epistatic interaction between those appendages. In addition, each flagellum seemed engaged in a particular task that might be required for swimming oriented toward chemoattractants near the soil inner surfaces at viscosities that may occur after the loss of soil gravitational water. Because the possession of two flagellar systems is not general in Bradyrhizobium or in related genera that coexist in the same environment, there may be an adaptive tradeoff between energetic costs and ecological benefits among these different species.

  14. Swimming performance of Bradyrhizobium diazoefficiens is an emergent property of its two flagellar systems

    PubMed Central

    Quelas, J. Ignacio; Althabegoiti, M. Julia; Jimenez-Sanchez, Celia; Melgarejo, Augusto A.; Marconi, Verónica I.; Mongiardini, Elías J.; Trejo, Sebastián A.; Mengucci, Florencia; Ortega-Calvo, José-Julio; Lodeiro, Aníbal R.

    2016-01-01

    Many bacterial species use flagella for self-propulsion in aqueous media. In the soil, which is a complex and structured environment, water is found in microscopic channels where viscosity and water potential depend on the composition of the soil solution and the degree of soil water saturation. Therefore, the motility of soil bacteria might have special requirements. An important soil bacterial genus is Bradyrhizobium, with species that possess one flagellar system and others with two different flagellar systems. Among the latter is B. diazoefficiens, which may express its subpolar and lateral flagella simultaneously in liquid medium, although its swimming behaviour was not described yet. These two flagellar systems were observed here as functionally integrated in a swimming performance that emerged as an epistatic interaction between those appendages. In addition, each flagellum seemed engaged in a particular task that might be required for swimming oriented toward chemoattractants near the soil inner surfaces at viscosities that may occur after the loss of soil gravitational water. Because the possession of two flagellar systems is not general in Bradyrhizobium or in related genera that coexist in the same environment, there may be an adaptive tradeoff between energetic costs and ecological benefits among these different species. PMID:27053439

  15. Bacteria exploit a polymorphic instability of the flagellar filament to escape from traps.

    PubMed

    Kühn, Marco J; Schmidt, Felix K; Eckhardt, Bruno; Thormann, Kai M

    2017-06-13

    Many bacterial species swim by rotating single polar helical flagella. Depending on the direction of rotation, they can swim forward or backward and change directions to move along chemical gradients but also to navigate their obstructed natural environment in soils, sediments, or mucus. When they get stuck, they naturally try to back out, but they can also resort to a radically different flagellar mode, which we discovered here. Using high-speed microscopy, we monitored the swimming behavior of the monopolarly flagellated species Shewanella putrefaciens with fluorescently labeled flagellar filaments at an agarose-glass interface. We show that, when a cell gets stuck, the polar flagellar filament executes a polymorphic change into a spiral-like form that wraps around the cell body in a spiral-like fashion and enables the cell to escape by a screw-like backward motion. Microscopy and modeling suggest that this propagation mode is triggered by an instability of the flagellum under reversal of the rotation and the applied torque. The switch is reversible and bacteria that have escaped the trap can return to their normal swimming mode by another reversal of motor direction. The screw-type flagellar arrangement enables a unique mode of propagation and, given the large number of polarly flagellated bacteria, we expect it to be a common and widespread escape or motility mode in complex and structured environments.

  16. A novel type bacterial flagellar motor that can use divalent cations as a coupling ion

    PubMed Central

    Imazawa, Riku; Takahashi, Yuka; Aoki, Wataru; Sano, Motohiko; Ito, Masahiro

    2016-01-01

    The bacterial flagellar motor is a sophisticated nanomachine embedded in the cell envelope and powered by an electrochemical gradient of H+, Na+, or K+across the cytoplasmic membrane. Here we describe a new member of the bacterial flagellar stator channel family (MotAB1 of Paenibacillus sp. TCA20 (TCA-MotAB1)) that is coupled to divalent cations (Ca2+and Mg2+). In the absence of divalent cations of alkaline earth metals, no swimming was observed in Paenibacillus sp. TCA20, which grows optimally in Ca2+-rich environments. This pattern was confirmed by swimming assays of a stator-free Bacillus subtilis mutant expressing TCA-MotAB1. Both a stator-free and major Mg2+uptake system-deleted B. subtilis mutant expressing TCA-MotAB1 complemented both growth and motility deficiency under low Mg2+conditions and exhibited [Mg2+]in identical to that of the wild-type. This is the first report of a flagellar motor that can use Ca2+and Mg2+as coupling ions. These findings will promote the understanding of the operating principles of flagellar motors and molecular mechanisms of ion selectivity. PMID:26794857

  17. Quantification of flagellar motor stator dynamics through in vivo proton-motive force control.

    PubMed

    Tipping, Murray J; Steel, Bradley C; Delalez, Nicolas J; Berry, Richard M; Armitage, Judith P

    2013-01-01

    The bacterial flagellar motor, one of the few rotary motors in nature, produces torque to drive the flagellar filament by ion translocation through membrane-bound stator complexes. We used the light-driven proton pump proteorhodopsin (pR) to control the proton-motive force (PMF) in vivo by illumination. pR excitation was shown to be sufficient to replace native PMF generation, and when excited in cells with intact native PMF generation systems increased motor speed beyond the physiological norm. We characterized the effects of rapid in vivo PMF changes on the flagellar motor. Transient PMF disruption events from loss of illumination caused motors to stop, with rapid recovery of their previous rotation rate after return of illumination. However, extended periods of PMF loss led to stepwise increases in rotation rate upon PMF return as stators returned to the motor. The rate constant for stator binding to a putative single binding site on the motor was calculated to be 0.06 s(-1). Using GFP-tagged MotB stator proteins, we found that transient PMF disruption leads to reversible stator diffusion away from the flagellar motor, showing that PMF presence is necessary for continued motor integrity, and calculated a stator dissociation rate of 0.038 s(-1).

  18. Constitutive androstane receptor activation evokes the expression of glycolytic genes.

    PubMed

    Yarushkin, Andrei A; Kazantseva, Yuliya A; Prokopyeva, Elena A; Markova, Diana N; Pustylnyak, Yuliya A; Pustylnyak, Vladimir O

    2016-09-23

    It is well-known that constitutive androstane receptor (CAR) activation by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) increases the liver-to-body weight ratio. CAR-mediated liver growth is correlated with increased expression of the pleiotropic transcription factor cMyc, which stimulates cell cycle regulatory genes and drives proliferating cells into S phase. Because glycolysis supports cell proliferation and cMyc is essential for the activation of glycolytic genes, we hypothesized that CAR-mediated up-regulation of cMyc in mouse livers might play a role in inducing the expression of glycolytic genes. The aim of the present study was to examine the effect of long-term CAR activation on glycolytic genes in a mouse model not subjected to metabolic stress. We demonstrated that long-term CAR activation by TCPOBOP increases expression of cMyc, which was correlated with reduced expression of gluconeogenic genes and up-regulation of glucose transporter, glycolytic and mitochondrial pyruvate metabolising genes. These changes in gene expression after TCPOBOP treatment were strongly correlated with changes in levels of glycolytic intermediates in mouse livers. Moreover, we demonstrated a significant positive regulatory effect of TCPOBOP-activated CAR on both mRNA and protein levels of Pkm2, a master regulator of glucose metabolism and cell proliferation. Thus, our findings provide evidence to support the conclusion that CAR activation initiates a transcriptional program that facilitates the coordinated metabolic activities required for cell proliferation.

  19. Modeling the Activity of Single Genes

    NASA Technical Reports Server (NTRS)

    Mjolsness, Eric; Gibson, Michael

    1999-01-01

    The central dogma of molecular biology states that information is stored in DNA, transcribed to messenger RNA (mRNA) and then translated into proteins. This picture is significantly augmentated when we consider the action of certain proteins in regulating transcription. These transcription factors provide a feedback pathway by which genes can regulate one another's expression as mRNA and then as protein. To review: DNA, RNA and proteins have different functions. DNA is the molecular storehouse of genetic information. When cells divide, the DNA is replicated, so that each daughter cell maintains the same genetic information as the mother cell. RNA acts as a go-between from DNA to proteins. Only a single copy of DNA is present, but multiple copies of the same piece of RNA may be present, allowing cells to make huge amounts of protein. In eukaryotes (organisms with a nucleus), DNA is found in the nucleus only. RNA is copied in the nucleus then translocates(moves) outside the nucleus, where it is transcribed into proteins. Along the way, the RNA may be spliced, i.e., may have pieces cut out. RNA then attaches to ribosomes and is translated to proteins. Proteins are the machinery of the cell other than DNA and RNA, all the complex molecules of the cell are proteins. Proteins are specialized machines, each of which fulfills its own task, which may be transporting oxygen, catalyzing reactions, or responding to extracellular signals, just to name a few. One of the more interesting functions a protein may have is binding directly or indirectly to DNA to perform transcriptional regulation, thus forming a closed feedback loop of gene regulation. The structure of DNA and the central dogma were understood in the 50s; in the early 80s it became possible to make arbitrary modifications to DNA and use cellular machinery to transcribe and translate the resulting genes; more recently, genomes (i.e., the complete DNA sequence) of many organisms have been sequenced. This large

  20. Carcinogen-induced trans activation of gene expression

    SciTech Connect

    Kleinberger, T.; Flint, Y.B.; Blank, M.; Etkin, S.; Lavi, S.

    1988-03-01

    The authors report a new mechanism of carcinogen action by which the expression of several genes was concomitantly enhanced. This mechanism involved the altered activity of cellular factors which modulate the expression of genes under their control. The increased expression was regulated at least in part on the transcriptional level and did not require amplification of the overexpressed genes. This phenomenon was transient; it was apparent as early as 24 h after carcinogen treatment and declined a few days later.

  1. Carcinogen-induced trans activation of gene expression.

    PubMed Central

    Kleinberger, T; Flint, Y B; Blank, M; Etkin, S; Lavi, S

    1988-01-01

    We report a new mechanism of carcinogen action by which the expression of several genes was concomitantly enhanced. This mechanism involved the altered activity of cellular factors which modulate the expression of genes under their control. The increased expression was regulated at least in part on the transcriptional level and did not require amplification of the overexpressed genes. This phenomenon was transient; it was apparent as early as 24 h after carcinogen treatment and declined a few days later. Images PMID:2835673

  2. A species-specific periplasmic flagellar protein of Serpulina (Treponema) hyodysenteriae.

    PubMed Central

    Li, Z; Dumas, F; Dubreuil, D; Jacques, M

    1993-01-01

    We have previously reported that a 46-kDa protein present in an outer membrane protein preparation seemed to be a species-specific antigen of Serpulina hyodysenteriae (Z. S. Li, N. S. Jensen, M. Bélanger, M.-C. L'Espérance, and M. Jacques, J. Clin. Microbiol. 30:2941-2947, 1992). The objective of this study was to further characterize this antigen. A Western blot (immunoblot) analysis and immunogold labeling with a monospecific antiserum against this protein confirmed that the protein was present in all S. hyodysenteriae reference strains but not in the nonpathogenic organism Serpulina innocens. The immunogold labeling results also indicated that the protein was associated with the periplasmic flagella of S. hyodysenteriae. N-terminal amino acid sequencing confirmed that the protein was in fact a periplasmic flagellar sheath protein. The molecular mass of this protein, first estimated to be 46 kDa by Western blotting, was determined to be 44 kDa when the protein was evaluated more precisely by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the protein was glycosylated, as determined by glycoprotein staining and also by N-glycosidase F treatment. Five other periplasmic flagellar proteins of S. hyodysenteriae, which may have been the core proteins and had molecular masses of 39, 35, 32, 30, and 29 kDa, were antigenically related and cross-reacted with the periplasmic flagellar proteins of S. innocens. Finally, serum from a pig experimentally infected with S. hyodysenteriae recognized the 44-kDa periplasmic flagellar sheath protein. Our results suggest that the 44-kDa periplasmic flagellar sheath protein of S. hyodysenteriae is a species-specific glycoprotein antigen. Images PMID:8253687

  3. Sequential development of flagellar defects in spermatids and epididymal spermatozoa of selenium-deficient rats.

    PubMed

    Olson, Gary E; Winfrey, Virginia P; Hill, Kristina E; Burk, Raymond F

    2004-03-01

    In this study cauda epididymal spermatozoa of rats maintained on a selenium-deficient diet for 5 and 7 months exhibited an array of flagellar defects. Spermatids and spermatozoa were analyzed by light and electron microscopy to define the appearance of flagellar abnormalities during spermiogenesis and post-testicular sperm development. Late spermatids of selenium-deficient rats displayed normal structural organization of the flagellar plasma membrane, axoneme, outer dense fibers, fibrous sheath and annulus, but they exhibited a premature termination of the mitochondrial sheath. A comparison of late spermatids and caput epididymal spermatozoa revealed that a late step in flagellar differentiation was the structural remodeling of the annulus and its accompanying fusion with both the fibrous sheath and the mitochondrial sheath. In selenium-deficient animals, however, the annulus failed to fuse with the mitochondrial sheath, generating an apparent weak point in the flagellum. After epididymal passage, cauda epididymal spermatozoa of selenium-deficient animals also exhibited extensive flagellar disorganization resulting from the apparent sliding and extrusion of specific outer dense fiber-doublet microtubule complexes from the proximal and the distal ends of the mitochondrial sheath and the accompanying loss of the midpiece plasma membrane. Only fiber complex number 4 was extruded proximally, whereas fibers 4, 5, 6 and 7 were extruded from the mitochondrial sheath-deficient posterior midpiece. Axonemal fibers 8, 9, 1, 2 and 3 retained their normal geometric relationships. These data suggest that the known loss of male fertility in selenium deficiency results from the sequential development of sperm defects expressed during both spermiogenesis and maturation in the epididymis.

  4. Correlated gene expression supports synchronous activity in brain networks

    PubMed Central

    Richiardi, Jonas; Altmann, Andre; Milazzo, Anna-Clare; Chang, Catie; Chakravarty, M. Mallar; Banaschewski, Tobias; Barker, Gareth J.; Bokde, Arun L.W.; Bromberg, Uli; Büchel, Christian; Conrod, Patricia; Fauth-Bühler, Mira; Flor, Herta; Frouin, Vincent; Gallinat, Jürgen; Garavan, Hugh; Gowland, Penny; Heinz, Andreas; Lemaître, Hervé; Mann, Karl F.; Martinot, Jean-Luc; Nees, Frauke; Paus, Tomáš; Pausova, Zdenka; Rietschel, Marcella; Robbins, Trevor W.; Smolka, Michael N.; Spanagel, Rainer; Ströhle, Andreas; Schumann, Gunter; Hawrylycz, Mike; Poline, Jean-Baptiste; Greicius, Michael D.

    2016-01-01

    During rest, brain activity is synchronized between different regions widely distributed throughout the brain, forming functional networks. However, the molecular mechanisms supporting functional connectivity remain undefined. We show that functional brain networks defined with resting-state functional magnetic resonance imaging can be recapitulated by using measures of correlated gene expression in a post mortem brain tissue data set. The set of 136 genes we identify is significantly enriched for ion channels. Polymorphisms in this set of genes significantly affect resting-state functional connectivity in a large sample of healthy adolescents. Expression levels of these genes are also significantly associated with axonal connectivity in the mouse. The results provide convergent, multimodal evidence that resting-state functional networks correlate with the orchestrated activity of dozens of genes linked to ion channel activity and synaptic function. PMID:26068849

  5. Dietary Methanol Regulates Human Gene Activity

    PubMed Central

    Komarova, Tatiana V.; Sheshukova, Ekaterina V.; Kosorukov, Vyacheslav S.; Kiryanov, Gleb I.; Dorokhov, Yuri L.

    2014-01-01

    Methanol (MeOH) is considered to be a poison in humans because of the alcohol dehydrogenase (ADH)-mediated conversion of MeOH to formaldehyde (FA), which is toxic. Our recent genome-wide analysis of the mouse brain demonstrated that an increase in endogenous MeOH after ADH inhibition led to a significant increase in the plasma MeOH concentration and a modification of mRNA synthesis. These findings suggest endogenous MeOH involvement in homeostasis regulation by controlling mRNA levels. Here, we demonstrate directly that study volunteers displayed increasing concentrations of MeOH and FA in their blood plasma when consuming citrus pectin, ethanol and red wine. A microarray analysis of white blood cells (WBC) from volunteers after pectin intake showed various responses for 30 significantly differentially regulated mRNAs, most of which were somehow involved in the pathogenesis of Alzheimer's disease (AD). There was also a decreased synthesis of hemoglobin mRNA, HBA and HBB, the presence of which in WBC RNA was not a result of red blood cells contamination because erythrocyte-specific marker genes were not significantly expressed. A qRT-PCR analysis of volunteer WBCs after pectin and red wine intake confirmed the complicated relationship between the plasma MeOH content and the mRNA accumulation of both genes that were previously identified, namely, GAPDH and SNX27, and genes revealed in this study, including MME, SORL1, DDIT4, HBA and HBB. We hypothesized that human plasma MeOH has an impact on the WBC mRNA levels of genes involved in cell signaling. PMID:25033451

  6. Entropy and information in flagellar axoneme cybernetics: a radial spokes integrative function.

    PubMed

    Cibert, Christian

    2003-04-01

    Radial spokes and the consequences of their relationships with the central apparatus seem to play a very important role in the regulation of axonemal activity. We modeled their behavior and observed that it appears to differ in the cilium and the flagellum with respect to the development of bending as a function of time. Specifically, our calculation raises the question of the real function of the radial spokes in the regulation of the axoneme, because a given curvature of the flagellar axoneme may correspond to two opposite of their tilts. The stable nil/low amplitude shear points that we had characterized along the flagellum allowed us to describe their axoneme as a series of modules [Cibert, 2002: Cell Motil. Cytoskeleton 51:89-111]. We observed that a nil/low shearing point moves along each module during beating when a new bend is created at the base of the flagellum [Cibert, 2001: Cell Motil. Cytoskeleton 49:161-175]. We propose that the structural gradients of isoforms of tubulin could be basic verniers that act as structural references for the axonemal machinery during the beating. This allowed us to interpret the axonemal organization as a segmented structure, that could be analyzed according to the complexion(1) theory and Shannon's information theory, which associate entropy and probability in the creation of information. The important consequence of this interpretation is that regulation of the axonemal machinery appears to be due to the upstream and downstream cross-talk between the axonemal segments that do not involve any dedicated integrative structure but depend on the energy level of the entire length of each module.

  7. Cloning, overexpression, purification, crystallization and preliminary X-ray analysis of CheY3, a response regulator that directly interacts with the flagellar 'switch complex' in Vibrio cholerae.

    PubMed

    Khamrui, Susmita; Biswas, Maitree; Sen, Udayaditya; Dasgupta, Jhimli

    2010-08-01

    Vibrio cholerae is the aetiological agent of the severe diarrhoeal disease cholera. This highly motile organism uses the processes of motility and chemotaxis to travel and colonize the intestinal epithelium. Chemotaxis in V. cholerae is far more complex than that in Escherichia coli or Salmonella typhimurium, with multiple paralogues of various chemotaxis genes. In contrast to the single copy of the chemotaxis response-regulator protein CheY in E. coli, V. cholerae contains four CheYs (CheY1-CheY4), of which CheY3 is primarily responsible for interacting with the flagellar motor protein FliM, which is one of the major constituents of the ;switch complex' in the flagellar motor. This interaction is the key step that controls flagellar rotation in response to environmental stimuli. CheY3 has been cloned, overexpressed and purified by Ni-NTA affinity chromatography followed by gel filtration. Crystals of CheY3 were grown in space group R3, with a calculated Matthews coefficient of 2.33 A3 Da(-1) (47% solvent content) assuming the presence of one molecule per asymmetric unit.

  8. appR gene product activates transcription of microcin C7 plasmid genes.

    PubMed Central

    Díaz-Guerra, L; Moreno, F; San Millán, J L

    1989-01-01

    Microcin C7 (MccC7) is encoded by Escherichia coli plasmid pMccC7. However, some strains of E. coli K-12 carrying this plasmid do not produce this antibiotic. Here we show that these strains differ in the gene locus appR. This chromosomal gene product controls MccC7 production by activating the transcription of some, but not all, MccC7 plasmid genes. PMID:2651423

  9. Endogenous Methanol Regulates Mammalian Gene Activity

    PubMed Central

    Komarova, Tatiana V.; Petrunia, Igor V.; Shindyapina, Anastasia V.; Silachev, Denis N.; Sheshukova, Ekaterina V.; Kiryanov, Gleb I.; Dorokhov, Yuri L.

    2014-01-01

    We recently showed that methanol emitted by wounded plants might function as a signaling molecule for plant-to-plant and plant-to-animal communications. In mammals, methanol is considered a poison because the enzyme alcohol dehydrogenase (ADH) converts methanol into toxic formaldehyde. However, the detection of methanol in the blood and exhaled air of healthy volunteers suggests that methanol may be a chemical with specific functions rather than a metabolic waste product. Using a genome-wide analysis of the mouse brain, we demonstrated that an increase in blood methanol concentration led to a change in the accumulation of mRNAs from genes primarily involved in detoxification processes and regulation of the alcohol/aldehyde dehydrogenases gene cluster. To test the role of ADH in the maintenance of low methanol concentration in the plasma, we used the specific ADH inhibitor 4-methylpyrazole (4-MP) and showed that intraperitoneal administration of 4-MP resulted in a significant increase in the plasma methanol, ethanol and formaldehyde concentrations. Removal of the intestine significantly decreased the rate of methanol addition to the plasma and suggested that the gut flora may be involved in the endogenous production of methanol. ADH in the liver was identified as the main enzyme for metabolizing methanol because an increase in the methanol and ethanol contents in the liver homogenate was observed after 4-MP administration into the portal vein. Liver mRNA quantification showed changes in the accumulation of mRNAs from genes involved in cell signalling and detoxification processes. We hypothesized that endogenous methanol acts as a regulator of homeostasis by controlling the mRNA synthesis. PMID:24587296

  10. Physical activity in adulthood: genes and mortality

    PubMed Central

    Karvinen, Sira; Waller, Katja; Silvennoinen, Mika; Koch, Lauren G.; Britton, Steven L.; Kaprio, Jaakko; Kainulainen, Heikki; Kujala, Urho M.

    2015-01-01

    Observational studies report a strong inverse relationship between leisure-time physical activity and all-cause mortality. Despite suggestive evidence from population-based associations, scientists have not been able to show a beneficial effect of physical activity on the risk of death in controlled intervention studies among individuals who have been healthy at baseline. On the other hand, high cardiorespiratory fitness is known to be a strong predictor of reduced mortality, even more robust than physical activity level itself. Here, in both animals and/or human twins, we show that the same genetic factors influence physical activity levels, cardiorespiratory fitness, and risk of death. Previous observational follow-up studies in humans suggest that increasing fitness through physical activity levels could prolong life; however, our controlled interventional study with laboratory rats bred for low and high intrinsic fitness contrast with these findings. Also, we find no evidence for the suggested association using pairwise analysis among monozygotic twin pairs who are discordant in their physical activity levels. Based on both our animal and human findings, we propose that genetic pleiotropy might partly explain the frequently observed associations between high baseline physical activity and later reduced mortality in humans. PMID:26666586

  11. Peroxisome proliferator-activated receptor alpha target genes.

    PubMed

    Rakhshandehroo, Maryam; Knoch, Bianca; Müller, Michael; Kersten, Sander

    2010-01-01

    The peroxisome proliferator-activated receptor alpha (PPARα) is a ligand-activated transcription factor involved in the regulation of a variety of processes, ranging from inflammation and immunity to nutrient metabolism and energy homeostasis. PPARα serves as a molecular target for hypolipidemic fibrates drugs which bind the receptor with high affinity. Furthermore, PPARα binds and is activated by numerous fatty acids and fatty acid-derived compounds. PPARα governs biological processes by altering the expression of a large number of target genes. Accordingly, the specific role of PPARα is directly related to the biological function of its target genes. Here, we present an overview of the involvement of PPARα in lipid metabolism and other pathways through a detailed analysis of the different known or putative PPARα target genes. The emphasis is on gene regulation by PPARα in liver although many of the results likely apply to other organs and tissues as well.

  12. Peroxisome Proliferator-Activated Receptor Alpha Target Genes

    PubMed Central

    Rakhshandehroo, Maryam; Knoch, Bianca; Müller, Michael; Kersten, Sander

    2010-01-01

    The peroxisome proliferator-activated receptor alpha (PPARα) is a ligand-activated transcription factor involved in the regulation of a variety of processes, ranging from inflammation and immunity to nutrient metabolism and energy homeostasis. PPARα serves as a molecular target for hypolipidemic fibrates drugs which bind the receptor with high affinity. Furthermore, PPARα binds and is activated by numerous fatty acids and fatty acid-derived compounds. PPARα governs biological processes by altering the expression of a large number of target genes. Accordingly, the specific role of PPARα is directly related to the biological function of its target genes. Here, we present an overview of the involvement of PPARα in lipid metabolism and other pathways through a detailed analysis of the different known or putative PPARα target genes. The emphasis is on gene regulation by PPARα in liver although many of the results likely apply to other organs and tissues as well. PMID:20936127

  13. Mechanisms of specificity in neuronal activity-regulated gene transcription

    PubMed Central

    Lyons, Michelle R.; West, Anne E.

    2011-01-01

    The brain is a highly adaptable organ that is capable of converting sensory information into changes in neuronal function. This plasticity allows behavior to be accommodated to the environment, providing an important evolutionary advantage. Neurons convert environmental stimuli into long-lasting changes in their physiology in part through the synaptic activity-regulated transcription of new gene products. Since the neurotransmitter-dependent regulation of Fos transcription was first discovered nearly 25 years ago, a wealth of studies have enriched our understanding of the molecular pathways that mediate activity-regulated changes in gene transcription. These findings show that a broad range of signaling pathways and transcriptional regulators can be engaged by neuronal activity to sculpt complex programs of stimulus-regulated gene transcription. However, the shear scope of the transcriptional pathways engaged by neuronal activity raises the question of how specificity in the nature of the transcriptional response is achieved in order to encode physiologically relevant responses to divergent stimuli. Here we summarize the general paradigms by which neuronal activity regulates transcription while focusing on the molecular mechanisms that confer differential stimulus-, cell-type-, and developmental-specificity upon activity-regulated programs of neuronal gene transcription. In addition, we preview some of the new technologies that will advance our future understanding of the mechanisms and consequences of activity-regulated gene transcription in the brain. PMID:21620929

  14. Inhibition of Stat1-mediated gene activation by PIAS1

    PubMed Central

    Liu, Bin; Liao, Jiayu; Rao, Xiaoping; Kushner, Steven A.; Chung, Chan D.; Chang, David D.; Shuai, Ke

    1998-01-01

    STAT (signal transducer and activator of transcription) proteins are latent cytoplasmic transcription factors that become activated by tyrosine phosphorylation in response to cytokine stimulation. Tyrosine phosphorylated STATs dimerize and translocate into the nucleus to activate specific genes. Different members of the STAT protein family have distinct functions in cytokine signaling. Biochemical and genetic analysis has demonstrated that Stat1 is essential for gene activation in response to interferon stimulation. Although progress has been made toward understanding STAT activation, little is known about how STAT signals are down-regulated. We report here the isolation of a family of PIAS (protein inhibitor of activated STAT) proteins. PIAS1, but not other PIAS proteins, blocked the DNA binding activity of Stat1 and inhibited Stat1-mediated gene activation in response to interferon. Coimmunoprecipitation analysis showed that PIAS1 was associated with Stat1 but not Stat2 or Stat3 after ligand stimulation. The in vivo PIAS1–Stat1 interaction requires phosphorylation of Stat1 on Tyr-701. These results identify PIAS1 as a specific inhibitor of Stat1-mediated gene activation and suggest that there may exist a specific PIAS inhibitor in every STAT signaling pathway. PMID:9724754

  15. CsrA impacts survival of Yersinia enterocolitica by affecting a myriad of physiological activities.

    PubMed

    LeGrand, Karen; Petersen, Shane; Zheng, Yan; Liu, Kang K; Ozturk, Gulustan; Chen, Jing-Yu; Young, Glenn M

    2015-02-14

    A previous study identified a Yersinia enterocolitica transposon mutant, GY448, that was unable to export the flagellar type three secretion system (T3SS)-dependent phospholipase, YplA. This strain was also deficient for motility and unable to form colonies on Lauria-Bertani agar medium. Preliminary analysis suggested it carried a mutation in csrA. CsrA in Escherichia coli is an RNA-binding protein that is involved in specific post-transcriptional regulation of a myriad of physiological activities. This study investigated how CsrA affects expression of the flagellar regulatory cascade that controls YplA export and motility. It also explored the effect of csrA mutation on Y. enterocolitica in response to conditions that cue physiological changes important for growth in environments found both in nature and the laboratory. The precise location of the transposon insertion in GMY448 was mapped within csrA. Genetic complementation restored disruptions in motility and the YplA export phenotype (Yex), which confirmed this mutation disrupted CsrA function. Mutation of csrA affected expression of yplA and flagellar genes involved in flagellar T3SS dependent export and motility by altering expression of the master regulators flhDC. Mutation of csrA also resulted in increased sensitivity of Y. enterocolitica to various osmolytes, temperatures and antibiotics. The results of this study reveal unique aspects of how CsrA functions in Y. enterocolitica to control its physiology. This provides perspective on how the Csr system is susceptible to adaptation to particular environments and bacterial lifestyles.

  16. Absence of canonical active chromatin marks in developmentally regulated genes

    PubMed Central

    Ruiz-Romero, Marina; Corominas, Montserrat; Guigó, Roderic

    2015-01-01

    The interplay of active and repressive histone modifications is assumed to play a key role in the regulation of gene expression. In contrast to this generally accepted view, we show that transcription of genes temporally regulated during fly and worm development occurs in the absence of canonically active histone modifications. Conversely, strong chromatin marking is related to transcriptional and post-transcriptional stability, an association that we also observe in mammals. Our results support a model in which chromatin marking is associated to stable production of RNA, while unmarked chromatin would permit rapid gene activation and de-activation during development. In this case, regulation by transcription factors would play a comparatively more important regulatory role. PMID:26280901

  17. Purification and characterization of the flagellar hook-basal body complex of Bacillus subtilis.

    PubMed

    Kubori, T; Okumura, M; Kobayashi, N; Nakamura, D; Iwakura, M; Aizawa, S I

    1997-04-01

    The flagellar hook-basal body (HBB) complex of the Gram-positive bacterium Bacillus subtilis was purified and analysed by electron microscopy, gel electrophoresis, and amino acid sequencing of the major component proteins. The purified HBB complex consisted of the inner (M and S) rings, a rod and a hook. There were no outer (P and L) rings that are found in Gram-negative bacteria. The hook was 15 nm in thickness and 70 nm in length, which is thinner and longer than the hook of Salmonella typhimurium. The hook protein had an apparent molecular mass of 29 kDa, and its N-terminal sequence was identical to that of B. subtilis FIgG, which was previously reported as a rod protein. The sequence of the reported FIgG protein of B. subtilis is more closely related to that of FIgE (the hook protein) rather than FIgG (the rod protein) of S. typhimurium, in spite of the difference of the apparent molecular masses between the two hook proteins (29 kDa versus 42 kDa). The hook-basal body contained six major proteins (with apparent molecular masses of 82, 59, 35, 32, 29 and 20 kDa) and two minor proteins (23 kDa and 13 kDa), which consistently appeared from preparation to preparation. The N-terminus of each of these proteins was sequenced. Comparison with protein databases revealed the following polypeptide-gene correspondences: 82 kDa, fIiF; 59 kDa, fIgK; 35 kDa, orfF; 32 kDa, yqhF; 23 kDa, orf3 of the fIaA locus; 20 kDa, fIgB and fIgC; 13 kDa, not determined. The band at 20 kDa was a mixture of FIgB and FIgC, as revealed by two-dimensional gel analysis. Characteristic features of B. subtilis HBB are discussed in comparison with those of S. typhimiurium.

  18. Flagellar filament bio-templated inorganic oxide materials - towards an efficient lithium battery anode

    NASA Astrophysics Data System (ADS)

    Beznosov, Sergei N.; Veluri, Pavan S.; Pyatibratov, Mikhail G.; Chatterjee, Abhijit; Macfarlane, Douglas R.; Fedorov, Oleg V.; Mitra, Sagar

    2015-01-01

    Designing a new generation of energy-intensive and sustainable electrode materials for batteries to power a variety of applications is an imperative task. The use of biomaterials as a nanosized structural template for these materials has the potential to produce hitherto unachievable structures. In this report, we have used genetically modified flagellar filaments of the extremely halophilic archaea species Halobacterium salinarum to synthesize nanostructured iron oxide composites for use as a lithium-ion battery anode. The electrode demonstrated a superior electrochemical performance compared to existing literature results, with good capacity retention of 1032 mAh g-1 after 50 cycles and with high rate capability, delivering 770 mAh g-1 at 5 A g-1 (~5 C) discharge rate. This unique flagellar filament based template has the potential to provide access to other highly structured advanced energy materials in the future.

  19. The phylogeny of swimming kinematics: The environment controls flagellar waveforms in sperm motility

    NASA Astrophysics Data System (ADS)

    Guasto, Jeffrey; Burton, Lisa; Zimmer, Richard; Hosoi, Anette; Stocker, Roman

    2013-11-01

    In recent years, phylogenetic and molecular analyses have dominated the study of ecology and evolution. However, physical interactions between organisms and their environment, a fundamental determinant of organism ecology and evolution, are mediated by organism form and function, highlighting the need to understand the mechanics of basic survival strategies, including locomotion. Focusing on spermatozoa, we combined high-speed video microscopy and singular value decomposition analysis to quantitatively compare the flagellar waveforms of eight species, ranging from marine invertebrates to humans. We found striking similarities in sperm swimming kinematics between genetically dissimilar organisms, which could not be uncovered by phylogenetic analysis. The emergence of dominant waveform patterns across species are suggestive of biological optimization for flagellar locomotion and point toward environmental cues as drivers of this convergence. These results reinforce the power of quantitative kinematic analysis to understand the physical drivers of evolution and as an approach to uncover new solutions for engineering applications, such as micro-robotics.

  20. Low flagellar motor torque and high swimming efficiency of Caulobacter crescentus swarmer cells.

    PubMed

    Li, Guanglai; Tang, Jay X

    2006-10-01

    We determined the torque of the flagellar motor of Caulobacter crescentus for different motor rotation rates by measuring the rotation rate and swimming speed of the cell body and found it to be remarkably different from that of other bacteria, such as Escherichia coli and Vibrio alginolyticus. The average stall torque of the Caulobacter flagellar motor was approximately 350 pN nm, much smaller than the values of the other bacteria measured. Furthermore, the torque of the motor remained constant in the range of rotation rates up to those of freely swimming cells. In contrast, the torque of a freely swimming cell for V. alginolyticus is typically approximately 20% of the stall torque. We derive from these results that the C. crescentus swarmer cells swim more efficiently than both E. coli and V. alginolyticus. Our findings suggest that C. crescentus is optimally adapted to low nutrient aquatic environments.

  1. Cell-body rocking is a dominant mechanism for flagellar synchronization in a swimming alga.

    PubMed

    Geyer, Veikko F; Jülicher, Frank; Howard, Jonathon; Friedrich, Benjamin M

    2013-11-05

    The unicellular green alga Chlamydomonas swims with two flagella that can synchronize their beat. Synchronized beating is required to swim both fast and straight. A long-standing hypothesis proposes that synchronization of flagella results from hydrodynamic coupling, but the details are not understood. Here, we present realistic hydrodynamic computations and high-speed tracking experiments of swimming cells that show how a perturbation from the synchronized state causes rotational motion of the cell body. This rotation feeds back on the flagellar dynamics via hydrodynamic friction forces and rapidly restores the synchronized state in our theory. We calculate that this "cell-body rocking" provides the dominant contribution to synchronization in swimming cells, whereas direct hydrodynamic interactions between the flagella contribute negligibly. We experimentally confirmed the two-way coupling between flagellar beating and cell-body rocking predicted by our theory.

  2. Flagellar filament bio-templated inorganic oxide materials - towards an efficient lithium battery anode.

    PubMed

    Beznosov, Sergei N; Veluri, Pavan S; Pyatibratov, Mikhail G; Chatterjee, Abhijit; MacFarlane, Douglas R; Fedorov, Oleg V; Mitra, Sagar

    2015-01-13

    Designing a new generation of energy-intensive and sustainable electrode materials for batteries to power a variety of applications is an imperative task. The use of biomaterials as a nanosized structural template for these materials has the potential to produce hitherto unachievable structures. In this report, we have used genetically modified flagellar filaments of the extremely halophilic archaea species Halobacterium salinarum to synthesize nanostructured iron oxide composites for use as a lithium-ion battery anode. The electrode demonstrated a superior electrochemical performance compared to existing literature results, with good capacity retention of 1032 mAh g(-1) after 50 cycles and with high rate capability, delivering 770 mAh g(-1) at 5 A g(-1) (~5 C) discharge rate. This unique flagellar filament based template has the potential to provide access to other highly structured advanced energy materials in the future.

  3. Amphipathic helical ordering of the flagellar secretion signal of Salmonella flagellin.

    PubMed

    Tőke, Orsolya; Vonderviszt, Ferenc

    2016-08-05

    Export of external flagellar proteins requires a signal located within their N-terminal disordered part, however, these regions do not share any significant sequence similarity suggesting that the secondary/tertiary structure might be important for recognition by the export gate. NMR experiments were performed to reveal the conformational properties of the flagellin signal sequence in vitro. It assumed a largely disordered fluctuating structure in aqueous environment, but acquired a folded structure containing an amphipathic helical portion in 50% MeOH or upon addition of SDS micelles which are known to promote hydrophobic interactions. Our observations raise the possibility that the signal sequence may partially undergo amphipathic helical ordering upon interaction with the recognition unit of the flagellar export machinery in a similar way as revealed for protein import into intracellular eukaryotic organelles mediated by targeting signals of high diversity. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Cell-body rocking is a dominant mechanism for flagellar synchronization in a swimming alga

    PubMed Central

    Geyer, Veikko F.; Jülicher, Frank; Howard, Jonathon; Friedrich, Benjamin M.

    2013-01-01

    The unicellular green alga Chlamydomonas swims with two flagella that can synchronize their beat. Synchronized beating is required to swim both fast and straight. A long-standing hypothesis proposes that synchronization of flagella results from hydrodynamic coupling, but the details are not understood. Here, we present realistic hydrodynamic computations and high-speed tracking experiments of swimming cells that show how a perturbation from the synchronized state causes rotational motion of the cell body. This rotation feeds back on the flagellar dynamics via hydrodynamic friction forces and rapidly restores the synchronized state in our theory. We calculate that this “cell-body rocking” provides the dominant contribution to synchronization in swimming cells, whereas direct hydrodynamic interactions between the flagella contribute negligibly. We experimentally confirmed the two-way coupling between flagellar beating and cell-body rocking predicted by our theory. PMID:24145440

  5. Flagellar filament bio-templated inorganic oxide materials – towards an efficient lithium battery anode

    PubMed Central

    Beznosov, Sergei N.; Veluri, Pavan S.; Pyatibratov, Mikhail G.; Chatterjee, Abhijit; MacFarlane, Douglas R.; Fedorov, Oleg V.; Mitra, Sagar

    2015-01-01

    Designing a new generation of energy-intensive and sustainable electrode materials for batteries to power a variety of applications is an imperative task. The use of biomaterials as a nanosized structural template for these materials has the potential to produce hitherto unachievable structures. In this report, we have used genetically modified flagellar filaments of the extremely halophilic archaea species Halobacterium salinarum to synthesize nanostructured iron oxide composites for use as a lithium-ion battery anode. The electrode demonstrated a superior electrochemical performance compared to existing literature results, with good capacity retention of 1032 mAh g−1 after 50 cycles and with high rate capability, delivering 770 mAh g−1 at 5 A g−1 (~5 C) discharge rate. This unique flagellar filament based template has the potential to provide access to other highly structured advanced energy materials in the future. PMID:25583370

  6. Tbx16 regulates hox gene activation in mesodermal progenitor cells

    PubMed Central

    Payumo, Alexander Y.; McQuade, Lindsey E.; Walker, Whitney J.; Yamazoe, Sayumi; Chen, James K.

    2016-01-01

    The transcription factor T-box 16 (Tbx16/Spadetail) is an essential regulator of paraxial mesoderm development in zebrafish (Danio rerio). Mesodermal progenitor cells (MPCs) fail to differentiate into trunk somites in tbx16 mutants and instead accumulate within the tailbud in an immature state. The mechanisms by which Tbx16 controls mesoderm patterning have remained enigmatic, and we describe here the application of photoactivatable morpholino oligonucleotides to determine the Tbx16 transcriptome in MPCs. We identify 124 Tbx16-regulated genes that are expressed in zebrafish gastrulae, including several developmental signaling proteins and regulators of gastrulation, myogenesis, and somitogenesis. Unexpectedly, we observe that loss of Tbx16 function precociously activates posterior hox genes in MPCs, and overexpression of a single posterior hox gene is sufficient to disrupt MPC migration. Our studies support a model in which Tbx16 regulates the timing of collinear hox gene activation to coordinate the anterior-posterior fates and positions of paraxial MPCs. PMID:27376691

  7. Evolutionary relationships between miRNA genes and their activity.

    PubMed

    Zhu, Yan; Skogerbø, Geir; Ning, Qianqian; Wang, Zhen; Li, Biqing; Yang, Shuang; Sun, Hong; Li, Yixue

    2012-12-22

    The emergence of vertebrates is characterized by a strong increase in miRNA families. MicroRNAs interact broadly with many transcripts, and the evolution of such a system is intriguing. However, evolutionary questions concerning the origin of miRNA genes and their subsequent evolution remain unexplained. In order to systematically understand the evolutionary relationship between miRNAs gene and their function, we classified human known miRNAs into eight groups based on their evolutionary ages estimated by maximum parsimony method. New miRNA genes with new functional sequences accumulated more dynamically in vertebrates than that observed in Drosophila. Different levels of evolutionary selection were observed over miRNA gene sequences with different time of origin. Most genic miRNAs differ from their host genes in time of origin, there is no particular relationship between the age of a miRNA and the age of its host genes, genic miRNAs are mostly younger than the corresponding host genes. MicroRNAs originated over different time-scales are often predicted/verified to target the same or overlapping sets of genes, opening the possibility of substantial functional redundancy among miRNAs of different ages. Higher degree of tissue specificity and lower expression level was found in young miRNAs. Our data showed that compared with protein coding genes, miRNA genes are more dynamic in terms of emergence and decay. Evolution patterns are quite different between miRNAs of different ages. MicroRNAs activity is under tight control with well-regulated expression increased and targeting decreased over time. Our work calls attention to the study of miRNA activity with a consideration of their origin time.

  8. Human DJ-1-specific Transcriptional Activation of Tyrosine Hydroxylase Gene*

    PubMed Central

    Ishikawa, Shizuma; Taira, Takahiro; Takahashi-Niki, Kazuko; Niki, Takeshi; Ariga, Hiroyoshi; Iguchi-Ariga, Sanae M. M.

    2010-01-01

    Loss-of-function mutation in the DJ-1 gene causes a subset of familial Parkinson disease. The mechanism underlying DJ-1-related selective vulnerability in the dopaminergic pathway is, however, not known. DJ-1 has multiple functions, including transcriptional regulation, and one of transcriptional target genes for DJ-1 is the tyrosine hydroxylase (TH) gene, the product of which is a key enzyme for dopamine biosynthesis. It has been reported that DJ-1 is a neuroprotective transcriptional co-activator that sequesters a transcriptional co-repressor polypyrimidine tract-binding protein-associated splicing factor (PSF) from the TH gene promoter. In this study, we found that knockdown of human DJ-1 by small interference RNA in human dopaminergic cell lines attenuated TH gene expression and 4-dihydroxy-l-phenylalanine production but that knockdown or knock-out of mouse DJ-1 in mouse cell lines or in mice did not affect such expression and TH activity. In reporter assays using the human TH gene promoter linked to the luciferase gene, stimulation of TH promoter activity was observed in human cells, but not mouse cells, that had been transfected with DJ-1. Although human DJ-1 and mouse DJ-1 were associated either with human or with mouse PSF, TH promoter activity inhibited by PSF was restored by human DJ-1 but not by mouse DJ-1. Chromatin immunoprecipitation assays revealed that the complex of PSF with DJ-1 bound to the human but not the mouse TH gene promoter. These results suggest a novel species-specific transcriptional regulation of the TH promoter by DJ-1 and one of the mechanisms for no reduction of TH in DJ-1-knock-out mice. PMID:20938049

  9. Human DJ-1-specific transcriptional activation of tyrosine hydroxylase gene.

    PubMed

    Ishikawa, Shizuma; Taira, Takahiro; Takahashi-Niki, Kazuko; Niki, Takeshi; Ariga, Hiroyoshi; Iguchi-Ariga, Sanae M M

    2010-12-17

    Loss-of-function mutation in the DJ-1 gene causes a subset of familial Parkinson disease. The mechanism underlying DJ-1-related selective vulnerability in the dopaminergic pathway is, however, not known. DJ-1 has multiple functions, including transcriptional regulation, and one of transcriptional target genes for DJ-1 is the tyrosine hydroxylase (TH) gene, the product of which is a key enzyme for dopamine biosynthesis. It has been reported that DJ-1 is a neuroprotective transcriptional co-activator that sequesters a transcriptional co-repressor polypyrimidine tract-binding protein-associated splicing factor (PSF) from the TH gene promoter. In this study, we found that knockdown of human DJ-1 by small interference RNA in human dopaminergic cell lines attenuated TH gene expression and 4-dihydroxy-L-phenylalanine production but that knockdown or knock-out of mouse DJ-1 in mouse cell lines or in mice did not affect such expression and TH activity. In reporter assays using the human TH gene promoter linked to the luciferase gene, stimulation of TH promoter activity was observed in human cells, but not mouse cells, that had been transfected with DJ-1. Although human DJ-1 and mouse DJ-1 were associated either with human or with mouse PSF, TH promoter activity inhibited by PSF was restored by human DJ-1 but not by mouse DJ-1. Chromatin immunoprecipitation assays revealed that the complex of PSF with DJ-1 bound to the human but not the mouse TH gene promoter. These results suggest a novel species-specific transcriptional regulation of the TH promoter by DJ-1 and one of the mechanisms for no reduction of TH in DJ-1-knock-out mice.

  10. Analysis of flagellar bending in hamster spermatozoa: characterization of an effective stroke.

    PubMed

    Kinukawa, Masashi; Ohmuro, Junko; Baba, Shoji A; Murashige, Sunao; Okuno, Makoto; Nagata, Masao; Aoki, Fugaku

    2005-12-01

    The mechanism by which flagella generate the propulsive force for movement of hamster spermatozoa was analyzed quantitatively. Tracing points positioned 30, 60, 90, and 120 microm from the head-midpiece junction on the flagellum revealed that they all had zigzag trajectories. These points departed from and returned to the line that crossed the direction of progression. They moved along the concave side (but not the convex side) of the flagellar envelope that was drawn by tracing the trajectory of the entire flagellum. To clarify this asymmetry, the bending rate was analyzed by measuring the curvatures of points 30, 60, 90, and 120 microm from the head-midpiece junction. The bending rate was not constant through the cycle of flagellar bending. The rate was higher when bending was in the direction described by the curve of the hook-shaped head (defined as a principal bend [P-bend]) to the opposite side (R-bend). We measured a lower bending rate in the principal direction (R-bend to P-bend). To identify the point at which the propulsive force is generated efficiently within the cycle of flagellar bending, we calculated the propulsive force generated at each point on the flagellum. The value of the propulsive force was positive whenever the flagellum bent from an R-bend to a P-bend (when the bending rate was lowest). By contrast, the propulsive force value was zero or negative when the flagellum bent in the other direction (when the bending rate was higher). These results indicate that flagellar bending in hamster spermatozoa produces alternate effective and ineffective strokes during propulsion.

  11. Bio-Hybrid Micro/Nanodevices Powered by Flagellar Motor: Challenges and Strategies

    PubMed Central

    Kim, Jin-Woo; Tung, Steve

    2015-01-01

    Molecular motors, which are precision engineered by nature, offer exciting possibilities for bio-hybrid engineered systems. They could enable real applications ranging from micro/nano fluidics, to biosensing, to medical diagnoses. This review describes the fundamental biological insights and fascinating potentials of these remarkable sensing and actuation machines, in particular, bacterial flagellar motors, as well as their engineering perspectives with regard to applications in bio-engineered hybrid systems. PMID:26284237

  12. A solid-state control system for dynein-based ciliary/flagellar motility

    PubMed Central

    2013-01-01

    Ciliary and flagellar beating requires the coordinated action of multiple dyneins with different enzymatic and motor properties. In this issue, Yamamoto et al. (2013. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201211048) identify the MIA (modifier of inner arms) complex within the Chlamydomonas reinhardtii axoneme that physically links to a known regulatory structure and provides a signaling conduit from the radial spokes to an inner arm dynein essential for waveform determination. PMID:23569213

  13. A Xanthomonas citri subsp citri hypothetical protein related to virulence contains a non-functional HD domain and is implicated in flagellar motility.

    PubMed

    Vieira, F C F; Gonçalves, A M; Mendoza, E F R; Ferreira, R M; Costa, M L M; Balbuena, T S; Sebinelli, H G; Ciancaglini, P; Pizauro Junior, J M; Ferro, J A

    2017-08-31

    Citrus canker, caused by the Gram-negative bacterium Xanthomonas citri subsp citri (Xac), severely affects most economically important citrus varieties worldwide. A previous study showed that disruption of the ORF XAC1201 from the Xac 306 strain by transposon Tn5 decreased bacterium virulence in the Rangpur lime host (Citrus limonia L. Osbeck). However, little is known regarding the possible function of the hypothetical protein XAC1201 and how it affects the virulence of Xac 306. Here, we confirmed that disruption of ORF XAC1201 reduces Xac 306 virulence in two different hosts, delaying the onset of typical symptoms. In silico analysis suggested that XAC1201 interacts with the flagellar proteins FliM and FliL, known to be an important factor for virulence. In fact, motility assays revealed that the XAC1201 mutant has a significant difference in motility compared to the wild-type Xac 306. Also, a 3-D structure model revealed modified cofactor binding sites and suggested that XAC1201 has a non-functional HD domain. This hypothesis was confirmed by enzymatic assays performed in purified, XAC1201 recombinant protein expressed in Escherichia coli, which revealed no significant activities previously associated with HD domains for the tested substrates. Thus, the role of the XAC1201 protein in Xac 306 virulence seems to be related to flagellar motility, although a non-classic role for the HD domain cannot be dismissed.

  14. A monoclonal antibody against the dynein IC1 peptide of sea urchin spermatozoa inhibits the motility of sea urchin, dinoflagellate, and human flagellar axonemes.

    PubMed Central

    Gagnon, C; White, D; Huitorel, P; Cosson, J

    1994-01-01

    To investigate the role of axonemal components in the mechanics and regulation of flagellar movement, we have generated a series of monoclonal antibodies (mAb) against sea urchin (Lytechinus pictus) sperm axonemal proteins, selected for their ability to inhibit the motility of demembranated sperm models. One of these antibodies, mAb D1, recognizes an antigen of 142 kDa on blots of sea urchin axonemal proteins and of purified outer arm dynein, suggesting that it acts by binding to the heaviest intermediate chain (IC1) of the dynein arm. mAb D1 blocks the motility of demembranated sea urchin spermatozoa by modifying the beating amplitude and shear angle without affecting the ATPase activity of purified dynein or of demembranated immotile spermatozoa. Furthermore, mAb D1 had only a marginal effect on the velocity of sliding microtubules in trypsin-treated axonemes. This antibody was also capable of inhibiting the motility of flagella of Oxyrrhis marina, a primitive dinoflagellate, and those of demembranated human spermatozoa. Localization of the antigen recognized by mAb D1 by immunofluorescence reveals its presence on the axonemes of flagella from sea urchin spermatozoa and O. marina but not on the cortical microtubule network of the dinoflagellate. These results are consistent with a dynamic role for the dynein intermediate chain IC1 in the bending and/or wave propagation of flagellar axonemes. Images PMID:7841521

  15. The RABL5 homolog IFT22 regulates the cellular pool size and the amount of IFT particles partitioned to the flagellar compartment in Chlamydomonas reinhardtii.

    PubMed

    Silva, David A; Huang, Xiaomeng; Behal, Robert H; Cole, Douglas G; Qin, Hongmin

    2012-01-01

    Cilia and flagella, sensory and motile structures protruding from the cell body, rely on the continuous bidirectional traffic of intraflagellar transport (IFT) particles to ferry flagellar precursors into flagella for assembly. Cells synthesize a large pool of IFT particle proteins in the cell body, but only a small portion engages in active transport within the flagella at any given time. The atypical small G protein Rab-like 5 (RABL5) has been shown to move in an IFT-like manner in the flagella, but its function in ciliogenesis is controversial. In this report, we demonstrate that IFT22, the Chlamydomonas reinhardtii homolog of RABL5, is a bona fide IFT particle complex B subunit. Although the amount of IFT22 remains unaffected by depletion of either complex A or B, depletion of IFT22 leads to a smaller pool of both complex A and B. Strikingly, the smaller cellular pool of IFT particles does not lead to a reduced distribution of IFT particles to flagella. Instead, the amount of IFT particle proteins, including IFT22 itself, increase in the flagella. Moreover, cells over-expressing IFT22 also accumulate IFT particles in their flagella. Taken together, these data indicate that, in C. reinhardtii, IFT22 controls the cellular levels of both complex A and B, thus plays a critical role in determining the cellular availability of IFT particles. In addition, although IFT22 may not directly carry any precursors for flagellar assembly, it controls how many IFT particles participate in ferrying precursors into flagella.

  16. Flagellar phenotypic plasticity in volvocalean algae correlates with Péclet number

    PubMed Central

    Solari, Cristian A.; Drescher, Knut; Ganguly, Sujoy; Kessler, John O.; Michod, Richard E.; Goldstein, Raymond E.

    2011-01-01

    Flagella-generated fluid stirring has been suggested to enhance nutrient uptake for sufficiently large micro-organisms, and to have played a role in evolutionary transitions to multicellularity. A corollary to this predicted size-dependent benefit is a propensity for phenotypic plasticity in the flow-generating mechanism to appear in large species under nutrient deprivation. We examined four species of volvocalean algae whose radii and flow speeds differ greatly, with Péclet numbers (Pe) separated by several orders of magnitude. Populations of unicellular Chlamydomonas reinhardtii and one- to eight-celled Gonium pectorale (Pe ∼ 0.1–1) and multicellular Volvox carteri and Volvox barberi (Pe ∼ 100) were grown in diluted and undiluted media. For C. reinhardtii and G. pectorale, decreasing the nutrient concentration resulted in smaller cells, but had no effect on flagellar length and propulsion force. In contrast, these conditions induced Volvox colonies to grow larger and increase their flagellar length, separating the somatic cells further. Detailed studies on V. carteri found that the opposing effects of increasing beating force and flagellar spacing balance, so the fluid speed across the colony surface remains unchanged between nutrient conditions. These results lend further support to the hypothesized link between the Péclet number, nutrient uptake and the evolution of biological complexity in the Volvocales. PMID:21367778

  17. Flagellar phenotypic plasticity in volvocalean algae correlates with Péclet number.

    PubMed

    Solari, Cristian A; Drescher, Knut; Ganguly, Sujoy; Kessler, John O; Michod, Richard E; Goldstein, Raymond E

    2011-10-07

    Flagella-generated fluid stirring has been suggested to enhance nutrient uptake for sufficiently large micro-organisms, and to have played a role in evolutionary transitions to multicellularity. A corollary to this predicted size-dependent benefit is a propensity for phenotypic plasticity in the flow-generating mechanism to appear in large species under nutrient deprivation. We examined four species of volvocalean algae whose radii and flow speeds differ greatly, with Péclet numbers (Pe) separated by several orders of magnitude. Populations of unicellular Chlamydomonas reinhardtii and one- to eight-celled Gonium pectorale (Pe ∼ 0.1-1) and multicellular Volvox carteri and Volvox barberi (Pe ∼ 100) were grown in diluted and undiluted media. For C. reinhardtii and G. pectorale, decreasing the nutrient concentration resulted in smaller cells, but had no effect on flagellar length and propulsion force. In contrast, these conditions induced Volvox colonies to grow larger and increase their flagellar length, separating the somatic cells further. Detailed studies on V. carteri found that the opposing effects of increasing beating force and flagellar spacing balance, so the fluid speed across the colony surface remains unchanged between nutrient conditions. These results lend further support to the hypothesized link between the Péclet number, nutrient uptake and the evolution of biological complexity in the Volvocales.

  18. Structure of Salmonella FlhE, conserved member of a flagellar Type III secretion operon

    DOE PAGES

    Lee, Jaemin; Monzingo, Arthur F.; Keatinge-Clay, Adrian T.; ...

    2014-12-26

    In this paper, the bacterial flagellum is assembled by a multicomponent transport apparatus categorized as a type III secretion system. The secretion of proteins that assemble into the flagellum is driven by the proton motive force. The periplasmic protein FlhE is a member of the flhBAE operon in the majority of bacteria where FlhE is found. FlhA and FlhB are established components of the flagellar type III secretion system. The absence of FlhE results in a proton leak through the flagellar system, inappropriate secretion patterns, and cell death, indicating that FlhE regulates an important aspect of proper flagellar biosynthesis. Wemore » isolated FlhE from the periplasm of Salmonella and solved its structure to 1.5 Å resolution. The structure reveals a β-sandwich fold, with no close structural homologs. Finally, possible roles of FlhE, including that of a chaperone, are discussed.« less

  19. Emergence of flagellar beating from the collective behavior of individual ATP-powered dyneins

    NASA Astrophysics Data System (ADS)

    Namdeo, S.; Onck, P. R.

    2016-10-01

    Flagella are hair-like projections from the surface of eukaryotic cells, and they play an important role in many cellular functions, such as cell-motility. The beating of flagella is enabled by their internal architecture, the axoneme, and is powered by a dense distribution of motor proteins, dyneins. The dyneins deliver the required mechanical work through the hydrolysis of ATP. Although the dynein-ATP cycle, the axoneme microstructure, and the flagellar-beating kinematics are well studied, their integration into a coherent picture of ATP-powered flagellar beating is still lacking. Here we show that a time-delayed negative-work-based switching mechanism is able to convert the individual sliding action of hundreds of dyneins into a regular overall beating pattern leading to propulsion. We developed a computational model based on a minimal representation of the axoneme consisting of two representative doublet microtubules connected by nexin links. The relative sliding of the microtubules is incorporated by modeling two groups of ATP-powered dyneins, each responsible for sliding in opposite directions. A time-delayed switching mechanism is postulated, which is key in converting the local individual sliding action of multiple dyneins into global beating. Our results demonstrate that an overall nonreciprocal beating pattern can emerge with time due to the spatial and temporal coordination of the individual dyneins. These findings provide insights in the fundamental working mechanism of axonemal dyneins and could possibly open new research directions in the field of flagellar motility.

  20. Diverse high-torque bacterial flagellar motors assemble wider stator rings using a conserved protein scaffold

    PubMed Central

    Ribardo, Deborah A.; Brennan, Caitlin A.; Ruby, Edward G.; Jensen, Grant J.; Hendrixson, David R.

    2016-01-01

    Although it is known that diverse bacterial flagellar motors produce different torques, the mechanism underlying torque variation is unknown. To understand this difference better, we combined genetic analyses with electron cryo-tomography subtomogram averaging to determine in situ structures of flagellar motors that produce different torques, from Campylobacter and Vibrio species. For the first time, to our knowledge, our results unambiguously locate the torque-generating stator complexes and show that diverse high-torque motors use variants of an ancestrally related family of structures to scaffold incorporation of additional stator complexes at wider radii from the axial driveshaft than in the model enteric motor. We identify the protein components of these additional scaffold structures and elucidate their sequential assembly, demonstrating that they are required for stator-complex incorporation. These proteins are widespread, suggesting that different bacteria have tailored torques to specific environments by scaffolding alternative stator placement and number. Our results quantitatively account for different motor torques, complete the assignment of the locations of the major flagellar components, and provide crucial constraints for understanding mechanisms of torque generation and the evolution of multiprotein complexes. PMID:26976588

  1. Diverse high-torque bacterial flagellar motors assemble wider stator rings using a conserved protein scaffold.

    PubMed

    Beeby, Morgan; Ribardo, Deborah A; Brennan, Caitlin A; Ruby, Edward G; Jensen, Grant J; Hendrixson, David R

    2016-03-29

    Although it is known that diverse bacterial flagellar motors produce different torques, the mechanism underlying torque variation is unknown. To understand this difference better, we combined genetic analyses with electron cryo-tomography subtomogram averaging to determine in situ structures of flagellar motors that produce different torques, from Campylobacter and Vibrio species. For the first time, to our knowledge, our results unambiguously locate the torque-generating stator complexes and show that diverse high-torque motors use variants of an ancestrally related family of structures to scaffold incorporation of additional stator complexes at wider radii from the axial driveshaft than in the model enteric motor. We identify the protein components of these additional scaffold structures and elucidate their sequential assembly, demonstrating that they are required for stator-complex incorporation. These proteins are widespread, suggesting that different bacteria have tailored torques to specific environments by scaffolding alternative stator placement and number. Our results quantitatively account for different motor torques, complete the assignment of the locations of the major flagellar components, and provide crucial constraints for understanding mechanisms of torque generation and the evolution of multiprotein complexes.

  2. Structure and function of the bi-directional bacterial flagellar motor.

    PubMed

    Morimoto, Yusuke V; Minamino, Tohru

    2014-02-18

    The bacterial flagellum is a locomotive organelle that propels the bacterial cell body in liquid environments. The flagellum is a supramolecular complex composed of about 30 different proteins and consists of at least three parts: a rotary motor, a universal joint, and a helical filament. The flagellar motor of Escherichia coli and Salmonella enterica is powered by an inward-directed electrochemical potential difference of protons across the cytoplasmic membrane. The flagellar motor consists of a rotor made of FliF, FliG, FliM and FliN and a dozen stators consisting of MotA and MotB. FliG, FliM and FliN also act as a molecular switch, enabling the motor to spin in both counterclockwise and clockwise directions. Each stator is anchored to the peptidoglycan layer through the C-terminal periplasmic domain of MotB and acts as a proton channel to couple the proton flow through the channel with torque generation. Highly conserved charged residues at the rotor-stator interface are required not only for torque generation but also for stator assembly around the rotor. In this review, we will summarize our current understanding of the structure and function of the proton-driven bacterial flagellar motor.

  3. Structure and Function of the Bi-Directional Bacterial Flagellar Motor

    PubMed Central

    Morimoto, Yusuke V.; Minamino, Tohru

    2014-01-01

    The bacterial flagellum is a locomotive organelle that propels the bacterial cell body in liquid environments. The flagellum is a supramolecular complex composed of about 30 different proteins and consists of at least three parts: a rotary motor, a universal joint, and a helical filament. The flagellar motor of Escherichia coli and Salmonella enterica is powered by an inward-directed electrochemical potential difference of protons across the cytoplasmic membrane. The flagellar motor consists of a rotor made of FliF, FliG, FliM and FliN and a dozen stators consisting of MotA and MotB. FliG, FliM and FliN also act as a molecular switch, enabling the motor to spin in both counterclockwise and clockwise directions. Each stator is anchored to the peptidoglycan layer through the C-terminal periplasmic domain of MotB and acts as a proton channel to couple the proton flow through the channel with torque generation. Highly conserved charged residues at the rotor–stator interface are required not only for torque generation but also for stator assembly around the rotor. In this review, we will summarize our current understanding of the structure and function of the proton-driven bacterial flagellar motor. PMID:24970213

  4. Secondary bacterial flagellar system improves bacterial spreading by increasing the directional persistence of swimming

    PubMed Central

    Bubendorfer, Sebastian; Koltai, Mihaly; Rossmann, Florian; Sourjik, Victor; Thormann, Kai M.

    2014-01-01

    As numerous bacterial species, Shewanella putrefaciens CN-32 possesses a complete secondary flagellar system. A significant subpopulation of CN-32 cells induces expression of the secondary system under planktonic conditions, resulting in formation of one, sometimes two, filaments at lateral positions in addition to the primary polar flagellum. Mutant analysis revealed that the single chemotaxis system primarily or even exclusively addresses the main polar flagellar system. Cells with secondary filaments outperformed their monopolarly flagellated counterparts in spreading on soft-agar plates and through medium-filled channels despite having lower swimming speed. While mutant cells with only polar flagella navigate by a “run-reverse-flick” mechanism resulting in effective cell realignments of about 90°, wild-type cells with secondary filaments exhibited a range of realignment angles with an average value of smaller than 90°. Mathematical modeling and computer simulations demonstrated that the smaller realignment angle of wild-type cells results in the higher directional persistence, increasing spreading efficiency both with and without a chemical gradient. Taken together, we propose that in S. putrefaciens CN-32, cell propulsion and directional switches are mainly mediated by the polar flagellar system, while the secondary filament increases the directional persistence of swimming and thus of spreading in the environment. PMID:25049414

  5. Bimodal rheotactic behavior reflects flagellar beat asymmetry in human sperm cells

    PubMed Central

    Bukatin, Anton; Kukhtevich, Igor; Stoop, Norbert; Dunkel, Jörn; Kantsler, Vasily

    2015-01-01

    Rheotaxis, the directed response to fluid velocity gradients, has been shown to facilitate stable upstream swimming of mammalian sperm cells along solid surfaces, suggesting a robust physical mechanism for long-distance navigation during fertilization. However, the dynamics by which a human sperm orients itself relative to an ambient flow is poorly understood. Here, we combine microfluidic experiments with mathematical modeling and 3D flagellar beat reconstruction to quantify the response of individual sperm cells in time-varying flow fields. Single-cell tracking reveals two kinematically distinct swimming states that entail opposite turning behaviors under flow reversal. We constrain an effective 2D model for the turning dynamics through systematic large-scale parameter scans, and find good quantitative agreement with experiments at different shear rates and viscosities. Using a 3D reconstruction algorithm to identify the flagellar beat patterns causing left or right turning, we present comprehensive 3D data demonstrating the rolling dynamics of freely swimming sperm cells around their longitudinal axis. Contrary to current beliefs, this 3D analysis uncovers ambidextrous flagellar waveforms and shows that the cell’s turning direction is not defined by the rolling direction. Instead, the different rheotactic turning behaviors are linked to a broken mirror symmetry in the midpiece section, likely arising from a buckling instability. These results challenge current theoretical models of sperm locomotion. PMID:26655343

  6. Flagellar biosynthesis exerts temporal regulation of secretion of specific Campylobacter jejuni colonization and virulence determinants.

    PubMed

    Barrero-Tobon, Angelica M; Hendrixson, David R

    2014-09-01

    The Campylobacter jejuni flagellum exports both proteins that form the flagellar organelle for swimming motility and colonization and virulence factors that promote commensal colonization of the avian intestinal tract or invasion of human intestinal cells respectively. We explored how the C. jejuni flagellum is a versatile secretory organelle by examining molecular determinants that allow colonization and virulence factors to exploit the flagellum for their own secretion. Flagellar biogenesis was observed to exert temporal control of secretion of these proteins, indicating that a bolus of secretion of colonization and virulence factors occurs during hook biogenesis with filament polymerization itself reducing secretion of these factors. Furthermore, we found that intramolecular and intermolecular requirements for flagellar-dependent secretion of these proteins were most reminiscent to those for flagellin secretion. Importantly, we discovered that secretion of one colonization and virulence factor, CiaI, was not required for invasion of human colonic cells, which counters previous hypotheses for how this protein functions during invasion. Instead, secretion of CiaI was essential for C. jejuni to facilitate commensal colonization of the natural avian host. Our work provides insight into the versatility of the bacterial flagellum as a secretory machine that can export proteins promoting diverse biological processes.

  7. Identification and Validation of Novel Chromosomal Integration and Expression Loci in Escherichia coli Flagellar Region 1

    PubMed Central

    Juhas, Mario; Ajioka, James W.

    2015-01-01

    Escherichia coli is used as a chassis for a number of Synthetic Biology applications. The lack of suitable chromosomal integration and expression loci is among the main hurdles of the E. coli engineering efforts. We identified and validated chromosomal integration and expression target sites within E. coli K12 MG1655 flagellar region 1. We analyzed five open reading frames of the flagellar region 1, flgA, flgF, flgG, flgI, and flgJ, that are well-conserved among commonly-used E. coli strains, such as MG1655, W3110, DH10B and BL21-DE3. The efficiency of the integration into the E. coli chromosome and the expression of the introduced genetic circuit at the investigated loci varied significantly. The integrations did not have a negative impact on growth; however, they completely abolished motility. From the investigated E. coli K12 MG1655 flagellar region 1, flgA and flgG are the most suitable chromosomal integration and expression loci. PMID:25816013

  8. Flagellar cells and ciliary cells in the renal tubule of elasmobranchs.

    PubMed

    Lacy, E R; Luciano, L; Reale, E

    1989-01-01

    Flagella or cilia are present on most epithelial cells in the renal tubule of elasmobranch fishes (little skate, spiny dogfish, smooth dogfish, Atlantic sharpnose, scalloped hammerhead, cow-nosed ray). Flagellar cells, those with numerous flagella ordered in one, two, or more rows on the luminal surface, are shown here for the first time in a vertebrate. The flagellar cells are intercalated among other epithelial cells, each bearing a single cilium, from Bowman's capsule to the third subdivision of the intermediate segment of the nephron. The flagella form undulated ribbons up to 55 microns long. In every ribbon the axis of the central pair of microtubules in the axoneme is oriented parallel to the long axis of the flagellar row. This suggests a beat perpendicular to these two axes. The arrangement of the flagella in ribbons most likely promotes movement of glomerular filtrate down the renal tubule. Cells bearing numerous cilia occur in the large collecting ducts of spiny dogfish but without apparent preferential orientation of the cilia.

  9. Correlation between supercoiling and conformational motions of the bacterial flagellar filament.

    PubMed

    Stadler, Andreas M; Unruh, Tobias; Namba, Keiichi; Samatey, Fadel; Zaccai, Giuseppe

    2013-11-05

    The bacterial flagellar filament is a very large macromolecular assembly of a single protein, flagellin. Various supercoiled states of the filament exist, which are formed by two structurally different conformations of flagellin in different ratios. We investigated the correlation between supercoiling of the protofilaments and molecular dynamics in the flagellar filament using quasielastic and elastic incoherent neutron scattering on the picosecond and nanosecond timescales. Thermal fluctuations in the straight L- and R-type filaments were measured and compared to the resting state of the wild-type filament. Amplitudes of motion on the picosecond timescale were found to be similar in the different conformational states. Mean-square displacements and protein resilience on the 0.1 ns timescale demonstrate that the L-type state is more flexible and less resilient than the R-type, whereas the wild-type state lies in between. Our results provide strong support that supercoiling of the protofilaments in the flagellar filament is determined by the strength of molecular forces in and between the flagellin subunits.

  10. Structure of the torque ring of the flagellar motor and the molecular basis for rotational switching

    SciTech Connect

    Lee, Lawrence K.; Ginsburg, Michael A.; Crovace, Claudia; Donohoe, Mhairi; Stock, Daniela

    2010-09-13

    The flagellar motor drives the rotation of flagellar filaments at hundreds of revolutions per second, efficiently propelling bacteria through viscous media. The motor uses the potential energy from an electrochemical gradient of cations across the cytoplasmic membrane to generate torque. A rapid switch from anticlockwise to clockwise rotation determines whether a bacterium runs smoothly forward or tumbles to change its trajectory. A protein called FliG forms a ring in the rotor of the flagellar motor that is involved in the generation of torque through an interaction with the cation-channel-forming stator subunit MotA. FliG has been suggested to adopt distinct conformations that induce switching but these structural changes and the molecular mechanism of switching are unknown. Here we report the molecular structure of the full-length FliG protein, identify conformational changes that are involved in rotational switching and uncover the structural basis for the formation of the FliG torque ring. This allows us to propose a model of the complete ring and switching mechanism in which conformational changes in FliG reverse the electrostatic charges involved in torque generation.

  11. Characterization of ciliobrevin A mediated dynein ATPase inhibition on flagellar motility of Leishmania donovani.

    PubMed

    Reddy, G Srinivas; Mukhopadhyay, Aakash Gautam; Dey, Chinmoy Sankar

    2017-04-05

    Axonemal dyneins are members of AAA+ proteins involved in force generation and are responsible for flagellar motility in eukaryotes. In this study, we characterized the effects of ciliobrevin A (CbA), a dynein ATPase inhibitor, on flagella driven motility of the protozoan parasite Leishmania donovani. Using fast-capture video microscopy, we observed that CbA decreased flagellar beat frequency of swimming parasites in a concentration-dependent manner. Beat frequency of live and reactivated L. donovani decreased by approximately 89% and 41% respectively in the presence of 250μM CbA. This inhibition was lost when CbA was removed, suggesting its effects were reversible. CbA also altered wavelength and amplitude of the flagellum of live parasites. Waveform analysis of live and reactivated L. donovani revealed that CbA significantly affected flagellar waveform by inducing non-uniform bends with the flagellum beating away from the cell axis. These results suggest that CbA sensitive dynein ATPases possibly are responsible for power generation and waveform maintenance of the flagellum of L. donovani. This ability to inhibit axonemal dyneins also emphasizes the use of dynein inhibitors as valuable tools in studying functional roles of axonemal dyneins of flagellated eukaryotes.

  12. Evidence for symmetry in the elementary process of bidirectional torque generation by the bacterial flagellar motor

    PubMed Central

    Nakamura, Shuichi; Kami-ike, Nobunori; Yokota, Jun-ichi P.; Minamino, Tohru; Namba, Keiichi

    2010-01-01

    The bacterial flagellar motor can rotate in both counterclockwise (CCW) and clockwise (CW) directions. It has been shown that the sodium ion-driven chimeric flagellar motor rotates with 26 steps per revolution, which corresponds to the number of FliG subunits that form part of the rotor ring, but the size of the backward step is smaller than the forward one. Here we report that the proton-driven flagellar motor of Salmonella also rotates with 26 steps per revolution but symmetrical in both CCW and CW directions with occasional smaller backward steps in both directions. Occasional shift in the stepping positions is also observed, suggesting the frequent exchange of stators in one of the 11–12 possible anchoring positions around the rotor. These observations indicate that the elementary process of torque generation by the cyclic association/dissociation of the stator with every FliG subunit along the circumference of the rotor is symmetric in CCW and CW rotation even though the structure of FliG is highly asymmetric and suggests a 180° rotation of a FliG domain for the rotor-stator interaction to reverse the direction of rotation. PMID:20876126

  13. Selective Purification of Recombinant Neuroactive Peptides Using the Flagellar Type III Secretion System

    PubMed Central

    Singer, Hanna M.; Erhardt, Marc; Steiner, Andrew M.; Zhang, Min-Min; Yoshikami, Doju; Bulaj, Grzegorz; Olivera, Baldomero M.; Hughes, Kelly T.

    2012-01-01

    ABSTRACT The structure, assembly, and function of the bacterial flagellum involves about 60 different proteins, many of which are selectively secreted via a specific type III secretion system (T3SS) (J. Frye et al., J. Bacteriol. 188:2233–2243, 2006). The T3SS is reported to secrete proteins at rates of up to 10,000 amino acid residues per second. In this work, we showed that the flagellar T3SS of Salmonella enterica serovar Typhimurium could be manipulated to export recombinant nonflagellar proteins through the flagellum and into the surrounding medium. We translationally fused various neuroactive peptides and proteins from snails, spiders, snakes, sea anemone, and bacteria to the flagellar secretion substrate FlgM. We found that all tested peptides of various sizes were secreted via the bacterial flagellar T3SS. We subsequently purified the recombinant μ-conotoxin SIIIA (rSIIIA) from Conus striatus by affinity chromatography and confirmed that T3SS-derived rSIIIA inhibited mammalian voltage-gated sodium channel NaV1.2 comparably to chemically synthesized SIIIA. PMID:22647788

  14. Function of the conserved FHIPEP domain of the flagellar type III export apparatus, protein FlhA.

    PubMed

    Barker, Clive S; Inoue, Tomoharu; Meshcheryakova, Irina V; Kitanobo, Seiya; Samatey, Fadel A

    2016-04-01

    The Type III flagellar protein export apparatus of bacteria consists of five or six membrane proteins, notably FlhA, which controls the export of other proteins and is homologous to the large family of FHIPEP export proteins. FHIPEP proteins contain a highly-conserved cytoplasmic domain. We mutagenized the cloned Salmonella flhA gene for the 692 amino acid FlhA, changing a single, conserved amino acid in the 68-amino acid FHIPEP region. Fifty-two mutations at 30 positions mostly led to loss of motility and total disappearance of microscopically visible flagella, also Western blot protein/protein hybridization showed no detectable export of hook protein and flagellin. There were two exceptions: a D199A mutant strain, which produced short-stubby flagella; and a V151L mutant strain, which did not produce flagella and excreted mainly un-polymerized hook protein. The V151L mutant strain also exported a reduced amount of hook-cap protein FlgD, but when grown with exogenous FlgD it produced polyhooks and polyhook-filaments. A suppressor mutant in the cytoplasmic domain of the export apparatus membrane protein FlhB rescued export of hook-length control protein FliK and facilitated growth of full-length flagella. These results suggested that the FHIPEP region is part of the gate regulating substrate entry into the export apparatus pore.

  15. Aberrant rel/nfkb genes and activity in human cancer.

    PubMed

    Rayet, B; Gélinas, C

    1999-11-22

    Rel/NF-kappaB transcription factors are key regulators of immune, inflammatory and acute phase responses and are also implicated in the control of cell proliferation and apoptosis. Remarkable progress has been made in understanding the signal transduction pathways that lead to the activation of Rel/NF-kappaB factors and the consequent induction of gene expression. Evidence linking deregulated Rel/NF-kappaB activity to oncogenesis in mammalian systems has emerged in recent years, consistent with the acute oncogenicity of the viral oncoprotein v-Rel in animal models. Chromosomal amplification, overexpression and rearrangement of genes coding for Rel/NF-kappaB factors have been noted in many human hematopoietic and solid tumors. Persistent nuclear NF-kappaB activity was also described in several human cancer cell types, as a result of constitutive activation of upstream signaling kinases or mutations inactivating inhibitory IkappaB subunits. Studies point to a correlation between the activation of cellular gene expression by Rel/NF-kappaB factors and their participation in the malignant process. Experiments implicating NF-kappaB in the control of the apoptotic response also support a role in oncogenesis and in the resistance of tumor cells to chemotherapy. This review focuses on the status of the rel, nfkb and ikb genes and their activity in human tumors and their association with the onset or progression of malignancies.

  16. Computer simulation of flagellar movement VIII: coordination of dynein by local curvature control can generate helical bending waves.

    PubMed

    Brokaw, Charles J

    2002-10-01

    Computer simulations have been carried out with a model flagellum that can bend in three dimensions. A pattern of dynein activation in which regions of dynein activity propagate along each doublet, with a phase shift of approximately 1/9 wavelength between adjacent doublets, will produce a helical bending wave. This pattern can be termed "doublet metachronism." The simulations show that doublet metachronism can arise spontaneously in a model axoneme in which activation of dyneins is controlled locally by the curvature of each outer doublet microtubule. In this model, dyneins operate both as sensors of curvature and as motors. Doublet metachronism and the chirality of the resulting helical bending pattern are regulated by the angular difference between the direction of the moment and sliding produced by dyneins on a doublet and the direction of the controlling curvature for that doublet. A flagellum that is generating a helical bending wave experiences twisting moments when it moves against external viscous resistance. At high viscosities, helical bending will be significantly modified by twist unless the twist resistance is greater than previously estimated. Spontaneous doublet metachronism must be modified or overridden in order for a flagellum to generate the planar bending waves that are required for efficient propulsion of spermatozoa. Planar bending can be achieved with the three-dimensional flagellar model by appropriate specification of the direction of the controlling curvature for each doublet. However, experimental observations indicate that this "hard-wired" solution is not appropriate for real flagella. Copyright 2002 Wiley-Liss, Inc.

  17. Recombination events that activate, diversify, and delete immunoglobulin genes.

    PubMed

    Leder, P; Max, E E; Seidman, J G; Kwan, S P; Scharff, M; Nau, M; Norman, B

    1981-01-01

    Immunoglobulin kappa light-chain diversity arises, in large part, from an array of germ-line V-region genes that undergo somatic recombination with one of four active J-region segments. The diversity provided by this combinational system is increased by a recombination mechanism that allows variation of crossover points so as to generate additional diversity at a critical region of the light chain. The elaborate mechanism for generating diversity is accompanied not only by considerable waste, in terms of unused V and J regions in a given cell, but also by a range of aberrant recombinants that fail to produce active immunoglobulin genes.

  18. Methylation of an alpha-foetoprotein gene intragenic site modulates gene activity.

    PubMed Central

    Opdecamp, K; Rivière, M; Molné, M; Szpirer, J; Szpirer, C

    1992-01-01

    By comparing the methylation pattern of Mspl/Hpall sites in the 5' region of the mouse alpha-foetoprotein (AFP) gene of different cells (hepatoma cells, foetal and adult liver, fibroblasts), we found a correlation between gene expression and unmethylation of a site located in the first intron of the gene. Other sites did not show this correlation. In transfection experiments of unmethylated and methylated AFP-CAT chimeric constructions, we then showed that methylation of the intronic site negatively modulates expression of CAT activity. We also found that a DNA segment centered on this site binds nuclear proteins; however methylation did not affect protein binding. Images PMID:1371343

  19. [Cascade of gene activation in Landouzy Dejerine muscular dystrophy].

    PubMed

    Belayew, A

    2010-01-01

    Our laboratory studies the Landouzy Dejerine muscular dystrophy or FSHD, a genetic disease which affects 7 in 100,000 individuals. The genetic defect is a deletion on chromosome 4 that decreases the copy number of a repeated DNA element, disturbs chromatin structure and activates the expression of neighbouring genes. The originality of our team has been to identify a gene within the repeated element itself and to show its activation in FSHD muscle cells. This gene expresses DUX4, a transcription factor that targets tens of genes, some of which express other transcription factors which target other genes, leading to a general deregulation. This DUX4-mediated cascade recapitulates by itself the major pathological features of FSHD: muscle atrophy, differentiation defect, oxidative stress... The homologous DUX4c gene located 42 kb from the repeat array expresses a protein that triggers myoblast proliferation. Its high expression level in severe cases of FSHD most probably contributes to the pathology by interfering with myoblast fusion with the muscle fibers at the last steps of muscle regeneration. We are performing global analyses of proteins and metabolites in healthy and FSHD myotubes (collaboration R Wattiez and JM Colet, UMONS) to identify abnormalities and their links with DUX4 or DUX4C.

  20. The neurotensin gene is a downstream target for Ras activation.

    PubMed Central

    Evers, B M; Zhou, Z; Celano, P; Li, J

    1995-01-01

    Ras regulates novel patterns of gene expression and the differentiation of various eukaryotic cell types. Stable transfection of Ha-ras into the human colon cancer line CaCo2 results in the morphologic differentiation to a small bowel phenotype. The purpose of our study was to determine whether the Ras regulatory pathway plays a role in the expression of the neurotensin gene (NT/N), a terminally differentiated endocrine product specifically localized in the gastrointestinal tract to the adult small bowel. We found that CaCo2-ras cells, but not parental CaCo2, express high levels of the human NT/N gene and, moreover, that this increase in gene expression is regulated at the level of transcription. Transfection experiments using NT/N-CAT mutation constructs identify the proximal 200 bp of NT/N flanking sequence as sufficient for maximal Ras-mediated NT/N reporter gene induction. Furthermore, a proximal AP-1/CRE motif is crucial for this Ras-mediated NT/N activation. Wild-type Ha-ras induces NT/N gene expression, albeit at lower levels than activated Ras; a dominant-negative Raf blocks this NT/N induction, suggesting that Raf lies down-stream of Ras in this pathway. In addition, postconfluent cultures of CaCo2 cells, which are differentiated to a small bowel phenotype, express the NT/N gene by 6 d after reaching confluency; this increase of NT/N expression is associated with concomitant increases of cellular p21ras protein. We conclude that Ras (both wild-type and activated) enhances expression of the NT/N gene in the gut-derived CaCo2 cell line, suggesting an important role for the Ras signaling pathway in NT/N gene transcription. Our results underscore the possibility that tissue-specific genes (such as NT/N) expressed in distinct subpopulations of the gut may be subject to Ras regulation. Finally, we speculate that the NT/N gene and the CaCo2 and CaCo2-ras cell systems will provide unique models to further define the cellular mechanisms leading to mammalian

  1. Transcriptional activation of virulence genes of Rhizobium etli.

    PubMed

    Wang, Luyao; Lacroix, Benoît; Guo, Jianhua; Citovsky, Vitaly

    2017-01-09

    Recently, Rhizobium etli has emerged, in addition to Agrobacterium spp., as a prokaryotic species that encodes a functional machinery for DNA transfer to plant cells. To understand this R. etli-mediated genetic transformation, it would be useful to define how its vir genes respond to the host plants. Here, we explored the transcriptional activation of the vir genes contained on the R. etli p42a plasmid. Using a reporter construct harboring lacZ under the control of the R. etli virE promoter, we showed that the signal phenolic molecule acetosyringone (AS) induced R. etli vir gene expression both in R. etli and in A. tumefaciens background. Furthermore, in both bacterial backgrounds, the p42a plasmid also promoted plant genetic transformation with a reporter T-DNA. Importantly, the R. etli vir genes were transcriptionally activated by AS in a bacterial species-specific fashion in regard to the VirA/VirG signal sensor system, and this activation was induced by signals from the natural host species of this bacterium, but not from non-host plants. Early kinetics of transcriptional activation of the major vir genes of R. etli also revealed several features distinct from those known for A. tumefaciens: the expression of the virG gene reached saturation relatively quickly, and virB2, which in R. etli is located outside of the virB operon, was expressed only at low levels and did not respond to AS. These differences in vir gene transcription may contribute to the lower efficiency of T-DNA transfer of R. etli p42a versus pTiC58 of A. tumefaciens IMPORTANCE: The region encoding homologs of Agrobacterium tumefaciens virulence genes in the Rhizobium etli CE3 p42a plasmid was the first endogenous virulence system encoded by a non-Agrobacterium species demonstrated to be functional in DNA transfer and stable integration into plant cell genome. In this study, we explore the transcriptional regulation and induction of virulence genes in R. etli and show similarities and differences

  2. RNA activation of haploinsufficient Foxg1 gene in murine neocortex

    PubMed Central

    Fimiani, Cristina; Goina, Elisa; Su, Qin; Gao, Guangping; Mallamaci, Antonello

    2016-01-01

    More than one hundred distinct gene hemizygosities are specifically linked to epilepsy, mental retardation, autism, schizophrenia and neuro-degeneration. Radical repair of these gene deficits via genome engineering is hardly feasible. The same applies to therapeutic stimulation of the spared allele by artificial transactivators. Small activating RNAs (saRNAs) offer an alternative, appealing approach. As a proof-of-principle, here we tested this approach on the Rett syndrome-linked, haploinsufficient, Foxg1 brain patterning gene. We selected a set of artificial small activating RNAs (saRNAs) upregulating it in neocortical precursors and their derivatives. Expression of these effectors achieved a robust biological outcome. saRNA-driven activation (RNAa) was limited to neural cells which normally express Foxg1 and did not hide endogenous gene tuning. saRNAs recognized target chromatin through a ncRNA stemming from it. Gene upregulation required Ago1 and was associated to RNApolII enrichment throughout the Foxg1 locus. Finally, saRNA delivery to murine neonatal brain replicated Foxg1-RNAa in vivo. PMID:27995975

  3. Linear topology confers in vivo gene transfer activity to polyethylenimines.

    PubMed

    Brissault, B; Leborgne, C; Guis, C; Danos, O; Cheradame, H; Kichler, A

    2006-01-01

    Although polyethylenimines (PEIs) are frequently used transfection agents, it is still unclear which of their properties are required for efficient gene delivery. This is even more striking when working in vivo since some PEIs are able to generate significant gene expression, whereas others are not. To facilitate a rational development of compounds with improved transfection activities, studies aimed at identifying the properties involved in the transfection process seem indispensable. In the present work, we investigated how transfection with linear PEI of 22 kDa allows for high reporter gene expression in lungs after intravenous injection, whereas the branched PEI of 25 kDa does not. To this end, we synthesized L-PEI derivatives that are intermediates between linear and branched PEIs. Our results show that the topology plays a crucial role in obtaining in vivo reporter gene expression, whereas the content of primary, secondary, and tertiary amines is only of minor importance.

  4. Periplasmic flagellar export apparatus protein, FliH, is involved in post-transcriptional regulation of FlaB, motility and virulence of the relapsing fever spirochete Borrelia hermsii.

    PubMed

    Guyard, Cyril; Raffel, Sandra J; Schrumpf, Merry E; Dahlstrom, Eric; Sturdevant, Daniel; Ricklefs, Stacy M; Martens, Craig; Hayes, Stanley F; Fischer, Elizabeth R; Hansen, Bryan T; Porcella, Stephen F; Schwan, Tom G

    2013-01-01

    Spirochetes are bacteria characterized in part by rotating periplasmic flagella that impart their helical or flat-wave morphology and motility. While most other bacteria rely on a transcriptional cascade to regulate the expression of motility genes, spirochetes employ post-transcriptional mechanism(s) that are only partially known. In the present study, we characterize a spontaneous non-motile mutant of the relapsing fever spirochete Borrelia hermsii that was straight, non-motile and deficient in periplasmic flagella. We used next generation DNA sequencing of the mutant's genome, which when compared to the wild-type genome identified a 142 bp deletion in the chromosomal gene encoding the flagellar export apparatus protein FliH. Immunoblot and transcription analyses showed that the mutant phenotype was linked to the posttranscriptional deficiency in the synthesis of the major periplasmic flagellar filament core protein FlaB. Despite the lack of FlaB, the amount of FlaA produced by the fliH mutant was similar to the wild-type level. The turnover of the residual pool of FlaB produced by the fliH mutant was comparable to the wild-type spirochete. The non-motile mutant was not infectious in mice and its inoculation did not induce an antibody response. Trans-complementation of the mutant with an intact fliH gene restored the synthesis of FlaB, a normal morphology, motility and infectivity in mice. Therefore, we propose that the flagellar export apparatus protein regulates motility of B. hermsii at the post-transcriptional level by influencing the synthesis of FlaB.

  5. DNA Adenine Methylation Regulates Virulence Gene Expression in Salmonella enterica Serovar Typhimurium▿

    PubMed Central

    Balbontín, Roberto; Rowley, Gary; Pucciarelli, M. Graciela; López-Garrido, Javier; Wormstone, Yvette; Lucchini, Sacha; García-del Portillo, Francisco; Hinton, Jay C. D.; Casadesús, Josep

    2006-01-01

    Transcriptomic analyses during growth in Luria-Bertani medium were performed in strain SL1344 of Salmonella enterica serovar Typhimurium and in two isogenic derivatives lacking Dam methylase. More genes were repressed than were activated by Dam methylation (139 versus 37). Key genes that were differentially regulated by Dam methylation were verified independently. The largest classes of Dam-repressed genes included genes belonging to the SOS regulon, as previously described in Escherichia coli, and genes of the SOS-inducible Salmonella prophages ST64B, Gifsy-1, and Fels-2. Dam-dependent virulence-related genes were also identified. Invasion genes in pathogenicity island SPI-1 were activated by Dam methylation, while the fimbrial operon std was repressed by Dam methylation. Certain flagellar genes were repressed by Dam methylation, and Dam− mutants of S. enterica showed reduced motility. Altered expression patterns in the absence of Dam methylation were also found for the chemotaxis genes cheR (repressed by Dam) and STM3216 (activated by Dam) and for the Braun lipoprotein gene, lppB (activated by Dam). The requirement for DNA adenine methylation in the regulation of specific virulence genes suggests that certain defects of Salmonella Dam− mutants in the mouse model may be caused by altered patterns of gene expression. PMID:16997949

  6. Interplay between the localization and kinetics of phosphorylation in flagellar pole development of the bacterium Caulobacter crescentus.

    PubMed

    Tropini, Carolina; Huang, Kerwyn Casey

    2012-01-01

    Bacterial cells maintain sophisticated levels of intracellular organization that allow for signal amplification, response to stimuli, cell division, and many other critical processes. The mechanisms underlying localization and their contribution to fitness have been difficult to uncover, due to the often challenging task of creating mutants with systematically perturbed localization but normal enzymatic activity, and the lack of quantitative models through which to interpret subtle phenotypic changes. Focusing on the model bacterium Caulobacter crescentus, which generates two different types of daughter cells from an underlying asymmetric distribution of protein phosphorylation, we use mathematical modeling to investigate the contribution of the localization of histidine kinases to the establishment of cellular asymmetry and subsequent developmental outcomes. We use existing mutant phenotypes and fluorescence data to parameterize a reaction-diffusion model of the kinases PleC and DivJ and their cognate response regulator DivK. We then present a systematic computational analysis of the effects of changes in protein localization and abundance to determine whether PleC localization is required for correct developmental timing in Caulobacter. Our model predicts the developmental phenotypes of several localization mutants, and suggests that a novel strain with co-localization of PleC and DivJ could provide quantitative insight into the signaling threshold required for flagellar pole development. Our analysis indicates that normal development can be maintained through a wide range of localization phenotypes, and that developmental defects due to changes in PleC localization can be rescued by increased PleC expression. We also show that the system is remarkably robust to perturbation of the kinetic parameters, and while the localization of either PleC or DivJ is required for asymmetric development, the delocalization of one of these two components does not prevent

  7. Human monoamine oxidase A gene determines levels of enzyme activity.

    PubMed Central

    Hotamisligil, G S; Breakefield, X O

    1991-01-01

    Monoamine oxidase (MAO) is a critical enzyme in the degradative deamination of biogenic amines throughout the body. Two biochemically distinct forms of the enzyme, A and B, are encoded in separate genes on the human X chromosome. In these studies we investigated the role of the structural gene for MAO-A in determining levels of activity in humans, as measured in cultured skin fibroblasts. The coding sequence of the mRNA for MAO-A was determined by first-strand cDNA synthesis, PCR amplification, and direct dideoxy sequencing. Two single-basepair substitutions were observed in cDNAs from cells with a 30-fold difference in activity levels. These two substitutions were in the third base of a triplet codon and hence did not affect the deduced amino acid sequence but did affect the presence or absence of restriction-enzyme sites for EcoRV and Fnu4HI, which could be elucidated on PCR fragments derived from genomic DNA or cDNAs. A third polymorphism for MspI in the noncoding region of the MAOA gene was also evaluated by Southern blot analysis using genomic DNA. Statistically significant associations were observed between the alleles for MAOA and levels of MAO activity in human male fibroblast lines. This association indicates that the MAOA gene itself is a major determinant of activity levels, apparently, in part, through noncoding, regulatory elements. Images Figure 3 Figure 4 Figure 5 PMID:1678250

  8. Activity-Regulated Genes as Mediators of Neural Circuit Plasticity

    PubMed Central

    Leslie, Jennifer H.; Nedivi, Elly

    2011-01-01

    Modifications of neuronal circuits allow the brain to adapt and change with experience. This plasticity manifests during development and throughout life, and can be remarkably long lasting. Many electrophysiological and molecular mechanisms are common to the seemingly diverse types of activity-dependent functional adaptation that take place during developmental critical periods, learning and memory, and alterations to sensory map representations in the adult. Experience-dependent plasticity is triggered when neuronal excitation activates cellular signaling pathways from the synapse to the nucleus that initiate new programs of gene expression. The protein products of activity-regulated genes then work via a diverse array of cellular mechanisms to modify neuronal functional properties. They fine-tune brain circuits by strengthening or weakening synaptic connections or by altering synapse numbers. Their effects are further modulated by posttranscriptional regulatory mechanisms, often also dependent on activity, that control activity-regulated gene transcript and protein function. Thus, the cellular response to neuronal activity integrates multiple tightly coordinated mechanisms to precisely orchestrate long-lasting, functional and structural changes in brain circuits. PMID:21601615

  9. Redistribution and shedding of flagellar membrane glycoproteins visualized using an anti-carbohydrate monoclonal antibody and concanavalin A

    PubMed Central

    1986-01-01

    Two carbohydrate-binding probes, the lectin concanavalin A and an anti- carbohydrate monoclonal antibody designated FMG-1, have been used to study the distribution of their respective epitopes on the surface of Chlamydomonas reinhardtii, strain pf-18. Both of these ligands bind uniformly to the external surface of the flagellar membrane and the general cell body plasma membrane, although the labeling is more intense on the flagellar membrane. In addition, both ligands cross- react with cell wall glycoproteins. With respect to the flagellar membrane, both concanavalin A and the FMG-1 monoclonal antibody bind preferentially to the principal high molecular weight glycoproteins migrating with an apparent molecular weight of 350,000 although there is, in addition, cross-reactivity with a number of minor glycoproteins. Western blots of V-8 protease digests of the high molecular weight flagellar glycoproteins indicate that the epitopes recognized by the lectin and the antibody are both repeated multiple times within the glycoproteins and occur together, although the lectin and the antibody do not compete for the same binding sites. Incubation of live cells with the monoclonal antibody or lectin at 4 degrees C results in a uniform labeling of the flagellar surface; upon warming of the cells, these ligands are redistributed along the flagellar surface in a characteristic manner. All of the flagellar surface-bound antibody or lectin collects into a single aggregate at the tip of each flagellum; this aggregate subsequently migrates to the base of the flagellum, where it is shed into the medium. The rate of redistribution is temperature dependent and the glycoproteins recognized by these ligands co-redistribute with the lectin or monoclonal antibody. This dynamic flagellar surface phenomenon bears a striking resemblance to the capping phenomenon that has been described in numerous mammalian cell types. However, it occurs on a structure (the flagellum) that lacks most of the

  10. Transcriptional activation of cloned human beta-globin genes by viral immediate-early gene products.

    PubMed

    Green, M R; Treisman, R; Maniatis, T

    1983-11-01

    When the human beta-globin gene is transfected into Hela cells, no beta-globin RNA is detected unless the gene is linked to a viral transcription enhancer. In this paper we show that trans-acting adenovirus and herpesvirus (pseudorabies) transcriptional regulatory proteins can circumvent this enhancer requirement for detectable beta-globin transcription in transient expression assays. The viral gene products can be provided by constitutively expressed, integrated viral genes in established cell lines, by viral infection of permissive cells, or by transfection of cells with bacterial plasmids carrying the viral immediate-early genes. These results demonstrate the utility of transient expression assays for studying regulatory mechanisms involving trans-acting factors. Analysis of beta-globin promoter mutants indicates that between 75 and 128 bp of sequence 5' to the mRNA cap site is required for enhancer-dependent transcription in Hela cells. In contrast, beta-globin transcription in the presence of viral immediate-early gene products requires only 36 bp of 5'-flanking sequence, which includes the TATA box. Thus both cis and trans-acting viral factors activate beta-globin gene transcription in transient expression experiments, but the mechanisms by which they act appear to be fundamentally different.

  11. FleQ DNA Binding Consensus Sequence Revealed by Studies of FleQ-Dependent Regulation of Biofilm Gene Expression in Pseudomonas aeruginosa.

    PubMed

    Baraquet, Claudine; Harwood, Caroline S

    2015-10-19

    The transcription factor FleQ from Pseudomonas aeruginosa derepresses expression of genes involved in biofilm formation when intracellular levels of the second messenger cyclic diguanosine monophosphate (c-di-GMP) are high. FleQ also activates transcription of flagellar genes, and the expression of these genes is highest at low intracellular c-di-GMP. FleQ thus plays a central role in mediating the transition between planktonic and biofilm lifestyles of P. aeruginosa. Previous work showed that FleQ controls expression of the pel operon for Pel exopolysaccharide biosynthesis by converting from a repressor to an activator upon binding c-di-GMP. To explore the activity of FleQ further, we carried out DNase I footprinting at three additional biofilm gene promoters, those of psl, cdrAB, and PA2440. The expression of cdrAB, encoding a cell surface adhesin, was sufficiently responsive to FleQ to allow us to carry out in vivo promoter assays. The results showed that, similarly to our observations with the pel operon, FleQ switches from a repressor to an activator of cdrAB gene expression in response to c-di-GMP. From the footprinting data, we identified a FleQ DNA binding consensus sequence. A search for this conserved sequence in bacterial genome sequences led to the identification of FleQ binding sites in the promoters of the siaABCD operon, important for cell aggregation, and the bdlA gene, important for biofilm dispersal, in P. aeruginosa. We also identified FleQ binding sites upstream of lapA-like adhesin genes in other Pseudomonas species. The transcription factor FleQ is widely distributed in Pseudomonas species. In all species examined, it is a master regulator of flagellar gene expression. It also regulates diverse genes involved in biofilm formation in P. aeruginosa when intracellular levels of the second messenger c-di-GMP are high. Unlike flagellar genes, biofilm-associated genes are not always easy to recognize in genome sequences. Here, we identified a

  12. Synapsins are late activity-induced genes regulated by birdsong

    PubMed Central

    Velho, Tarciso A. F.; Mello, Claudio V.

    2008-01-01

    The consolidation of long-lasting sensory memories requires the activation of gene expression programs in the brain. In spite of considerable knowledge about the early components of this response, little is known about late components (i.e. genes regulated 2-6 hr after stimulation) and the relationship between early and late genes. Birdsong represents one of the best natural behaviors to study sensory-induced gene expression in awake, freely behaving animals. Here we show that the expression of several isoforms of synapsins, a group of phosphoproteins thought to regulate the dynamics of synaptic vesicle storage and release, is induced by auditory stimulation with birdsong in the caudomedial nidopallium (NCM) of the zebra finch (Taeniopygia guttata) brain. This induction occurs mainly in excitatory (non-GABAergic) neurons and is modulated (suppressed) by early song-inducible proteins. We also show that ZENK, an early song-inducible transcription factor, interacts with the syn3 promoter in vivo, consistent with a direct regulatory effect and an emerging novel view of ZENK action. These results demonstrate that synapsins are a late component of the genomic response to neuronal activation and that their expression depends on a complex set of regulatory interactions between early and late regulated genes. PMID:19005052

  13. KHARON Is an Essential Cytoskeletal Protein Involved in the Trafficking of Flagellar Membrane Proteins and Cell Division in African Trypanosomes*

    PubMed Central

    Sanchez, Marco A.; Tran, Khoa D.; Valli, Jessica; Hobbs, Sam; Johnson, Errin; Gluenz, Eva; Landfear, Scott M.

    2016-01-01

    African trypanosomes and related kinetoplastid parasites selectively traffic specific membrane proteins to the flagellar membrane, but the mechanisms for this trafficking are poorly understood. We show here that KHARON, a protein originally identified in Leishmania parasites, interacts with a putative trypanosome calcium channel and is required for its targeting to the flagellar membrane. KHARON is located at the base of the flagellar axoneme, where it likely mediates targeting of flagellar membrane proteins, but is also on the subpellicular microtubules and the mitotic spindle. Hence, KHARON is probably a multifunctional protein that associates with several components of the trypanosome cytoskeleton. RNA interference-mediated knockdown of KHARON mRNA results in failure of the calcium channel to enter the flagellar membrane, detachment of the flagellum from the cell body, and disruption of mitotic spindles. Furthermore, knockdown of KHARON mRNA induces a lethal failure of cytokinesis in both bloodstream (mammalian host) and procyclic (insect vector) life cycle stages, and KHARON is thus critical for parasite viability. PMID:27489106

  14. Gene Transfer in Eukaryotic Cells Using Activated Dendrimers

    NASA Astrophysics Data System (ADS)

    Dennig, Jörg

    Gene transfer into eukaryotic cells plays an important role in cell biology. Over the last 30 years a number of transfection methods have been developed to mediate gene transfer into eukaryotic cells. Classical methods include co-precipitation of DNA with calcium phosphate, charge-dependent precipitation of DNA with DEAE-dextran, electroporation of nucleic acids, and formation of transfection complexes between DNA and cationic liposomes. Gene transfer technologies based on activated PAMAM-dendrimers provide another class of transfection reagents. PAMAM-dendrimers are highly branched, spherical molecules. Activation of newly synthesized dendrimers involves hydrolytic removal of some of the branches, and results in a molecule with a higher degree of flexibility. Activated dendrimers assemble DNA into compact structures via charge interactions. Activated dendrimer - DNA complexes bind to the cell membrane of eukaryotic cells, and are transported into the cell by non-specific endocytosis. A structural model of the activated dendrimer - DNA complex and a potential mechanism for its uptake into cells will be discussed.

  15. Metallothionein gene activation in the earthworm (Lumbricus rubellus).

    PubMed

    Höckner, M; Dallinger, R; Stürzenbaum, S R

    2015-05-08

    In order to cope with changing environmental conditions, organisms require highly responsive stress mechanisms. Heavy metal stress is handled by metallothioneins (MTs), the regulation of which is evolutionary conserved in insects and vertebrates and involves the binding of metal transcription factor 1 (MTF-1) to metal responsive elements (MREs) positioned in the promoter of MT genes. However, in most invertebrate phyla, the transcriptional activation of MTs is different and the exact mechanism is still unknown. Interestingly, although MREs are typically present also in invertebrate MT gene promoters, MTF-1 is notably absent. Here we use Lumbricus rubellus, the red earthworm, to study the elusive mechanism of wMT-2 activation in control and Cd-exposed conditions. EMSA and DNase I footprinting approaches were used to pinpoint functional binding sites within the wMT-2 promoter region, which revealed that the cAMP responsive element (CRE) is a promising candidate which may act as a transcriptional activator of invertebrate MTs.

  16. "Active" cancer immunotherapy by anti-Met antibody gene transfer.

    PubMed

    Vigna, Elisa; Pacchiana, Giovanni; Mazzone, Massimiliano; Chiriaco, Cristina; Fontani, Lara; Basilico, Cristina; Pennacchietti, Selma; Comoglio, Paolo M

    2008-11-15

    Gene therapy provides a still poorly explored opportunity to treat cancer by "active" immunotherapy as it enables the transfer of genes encoding antibodies directed against specific oncogenic proteins. By a bidirectional lentiviral vector, we transferred the cDNA encoding the heavy and light chains of a monoclonal anti-Met antibody (DN-30) to epithelial cancer cells. In vitro, the transduced cells synthesized and secreted correctly assembled antibodies with the expected high affinity, inducing down-regulation of the Met receptor and strong inhibition of the invasive growth response. The inhibitory activity resulted (a) from the interference of the antibody with the Met receptor intracellular processing ("cell autonomous activity," in cis) and (b) from the antibody-induced cleavage of Met expressed at the cell surface ("bystander effect," in trans). The monoclonal antibody gene transferred into live animals by systemic administration or by local intratumor delivery resulted in substantial inhibition of tumor growth. These data provide proof of concept both for targeting the Met receptor and for a gene transfer-based immunotherapy strategy.

  17. CRISPR RNA-guided activation of endogenous human genes.

    PubMed

    Maeder, Morgan L; Linder, Samantha J; Cascio, Vincent M; Fu, Yanfang; Ho, Quan H; Joung, J Keith

    2013-10-01

    Short guide RNAs (gRNAs) can direct catalytically inactive CRISPR-associated 9 nuclease (dCas9) to repress endogenous genes in bacteria and human cells. Here we show that single or multiple gRNAs can direct dCas9 fused to a VP64 transcriptional activation domain to increase expression of endogenous human genes. This proof-of-principle work shows that clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems can target heterologous effector domains to endogenous sites in human cells.

  18. Dynamics of the bacterial flagellar motor: the effects of stator compliance, back steps, temperature, and rotational asymmetry.

    PubMed

    Meacci, Giovanni; Lan, Ganhui; Tu, Yuhai

    2011-04-20

    The rotation of a bacterial flagellar motor (BFM) is driven by multiple stators tethered to the cell wall. Here, we extend a recently proposed power-stroke model to study the BFM dynamics under different biophysical conditions. Our model explains several key experimental observations and reveals their underlying mechanisms. 1), The observed independence of the speed at low load on the number of stators is explained by a force-dependent stepping mechanism that is independent of the strength of the stator tethering spring. Conversely, without force-dependent stepping, an unrealistically weak stator spring is required. 2), Our model with back-stepping naturally explains the observed absence of a barrier to backward rotation. Using the same set of parameters, it also explains BFM behaviors in the high-speed negative-torque regime. 3), From the measured temperature dependence of the maximum speed, our model shows that stator-stepping is a thermally activated process with an energy barrier. 4), The recently observed asymmetry in the torque-speed curve between counterclockwise- and clockwise-rotating BFMs can be quantitatively explained by the asymmetry in the stator-rotor interaction potentials, i.e., a quasilinear form for the counterclockwise motor and a quadratic form for the clockwise motor.

  19. Dynamics of the Bacterial Flagellar Motor: The Effects of Stator Compliance, Back Steps, Temperature, and Rotational Asymmetry

    PubMed Central

    Meacci, Giovanni; Lan, Ganhui; Tu, Yuhai

    2011-01-01

    The rotation of a bacterial flagellar motor (BFM) is driven by multiple stators tethered to the cell wall. Here, we extend a recently proposed power-stroke model to study the BFM dynamics under different biophysical conditions. Our model explains several key experimental observations and reveals their underlying mechanisms. 1), The observed independence of the speed at low load on the number of stators is explained by a force-dependent stepping mechanism that is independent of the strength of the stator tethering spring. Conversely, without force-dependent stepping, an unrealistically weak stator spring is required. 2), Our model with back-stepping naturally explains the observed absence of a barrier to backward rotation. Using the same set of parameters, it also explains BFM behaviors in the high-speed negative-torque regime. 3), From the measured temperature dependence of the maximum speed, our model shows that stator-stepping is a thermally activated process with an energy barrier. 4), The recently observed asymmetry in the torque-speed curve between counterclockwise- and clockwise-rotating BFMs can be quantitatively explained by the asymmetry in the stator-rotor interaction potentials, i.e., a quasilinear form for the counterclockwise motor and a quadratic form for the clockwise motor. PMID:21504735

  20. Flagellar Movement in Two Bacteria of the Family Rickettsiaceae: A Re-Evaluation of Motility in an Evolutionary Perspective

    PubMed Central

    Vannini, Claudia; Boscaro, Vittorio; Ferrantini, Filippo; Benken, Konstantin A.; Mironov, Timofei I.; Schweikert, Michael; Görtz, Hans-Dieter; Fokin, Sergei I.; Sabaneyeva, Elena V.; Petroni, Giulio

    2014-01-01

    Bacteria of the family Rickettsiaceae have always been largely studied not only for their importance in the medical field, but also as model systems in evolutionary biology. In fact, they share a recent common ancestor with mitochondria. The most studied species, belonging to genera Rickettsia and Orientia, are hosted by terrestrial arthropods and include many human pathogens. Nevertheless, recent findings show that a large part of Rickettsiaceae biodiversity actually resides outside the group of well-known pathogenic bacteria. Collecting data on these recently described non-conventional members of the family is crucial in order to gain information on ancestral features of the whole group. Although bacteria of the family Rickettsiaceae, and of the whole order Rickettsiales, are formally described as non-flagellated prokaryotes, some recent findings renewed the debate about this feature. In this paper we report the first finding of members of the family displaying numerous flagella and active movement inside their host cells. These two new taxa are hosted in aquatic environments by protist ciliates and are described here by means of ultrastructural and molecular characterization. Data here reported suggest that the ancestor of Rickettsiales displayed flagellar movement and re-evaluate the hypothesis that motility played a key-role in the origin of mitochondria. Moreover, our study highlights that the aquatic environment represents a well exploited habitat for bacteria of the family Rickettsiaceae. Our results encourage a deep re-consideration of ecological and morphological traits of the family and of the whole order. PMID:24505307

  1. Activating GNAS1 gene mutations in patients with premature thelarche.

    PubMed

    Román, Rossana; Johnson, Mara Cecilia; Codner, Ethel; Boric, Mara Angélica; áVila, Alejandra; Cassorla, Fernando

    2004-08-01

    To identify GNAS1 gene mutations in girls with exaggerated and/or chronic fluctuating thelarche for at least 1-year duration with no other signs of precocious puberty, skeletal dysplasia, or typical skin lesions of McCune-Albright syndrome. We studied the GNAS1 gene mutation by allele-specific polymerase chain reaction and enzymatic digestion in leukocyte DNA in 23 girls previously described. Fluctuating thelarche was present in 14 girls and exaggerated thelarche was observed in 9. Molecular study revealed that 6 girls had a substitution of arginine by histidine in codon 201 (R201H [+]). Three R201H (+) girls reached their menarche at a mean chronologic age of 10.8 years and 9 of the R201H (-) girls at a mean age of 11 years. Activating mutations of GNAS1 gene may be observed in some girls with chronic fluctuating and/or exaggerated thelarche, without other classic signs of McCune-Albright syndrome.

  2. Tumor suppressor genes are larger than apoptosis-effector genes and have more regions of active chromatin: Connection to a stochastic paradigm for sequential gene expression programs.

    PubMed

    Garcia, Marlene; Mauro, James A; Ramsamooj, Michael; Blanck, George

    2015-08-03

    Apoptosis- and proliferation-effector genes are substantially regulated by the same transactivators, with E2F-1 and Oct-1 being notable examples. The larger proliferation-effector genes have more binding sites for the transactivators that regulate both sets of genes, and proliferation-effector genes have more regions of active chromatin, i.e, DNase I hypersensitive and histone 3, lysine-4 trimethylation sites. Thus, the size differences between the 2 classes of genes suggest a transcriptional regulation paradigm whereby the accumulation of transcription factors that regulate both sets of genes, merely as an aspect of stochastic behavior, accumulate first on the larger proliferation-effector gene "traps," and then accumulate on the apoptosis effector genes, thereby effecting sequential activation of the 2 different gene sets. As IRF-1 and p53 levels increase, tumor suppressor proteins are first activated, followed by the activation of apoptosis-effector genes, for example during S-phase pausing for DNA repair. Tumor suppressor genes are larger than apoptosis-effector genes and have more IRF-1 and p53 binding sites, thereby likewise suggesting a paradigm for transcription sequencing based on stochastic interactions of transcription factors with different gene classes. In this report, using the ENCODE database, we determined that tumor suppressor genes have a greater number of open chromatin regions and histone 3 lysine-4 trimethylation sites, consistent with the idea that a larger gene size can facilitate earlier transcriptional activation via the inclusion of more transactivator binding sites.

  3. Flagellar apparatus of south-seeking many-celled magnetotactic prokaryotes.

    PubMed

    Silva, Karen Tavares; Abreu, Fernanda; Almeida, Fernando P; Keim, Carolina Neumann; Farina, Marcos; Lins, Ulysses

    2007-01-01

    Magnetotactic bacteria orient and migrate along geomagnetic field lines. Each cell contains membrane-enclosed, nano-scale, iron-mineral particles called magnetosomes that cause alignment of the cell in the geomagnetic field as the bacteria swim propelled by flagella. In this work we studied the ultrastructure of the flagellar apparatus in many-celled magnetotactic prokaryotes (MMP) that consist of several Gram-negative cells arranged radially around an acellular compartment. Flagella covered the organism surface, and were observed exclusively at the portion of each cell that faced the environment. The flagella were helical tubes never as long as a complete turn of the helix. Flagellar filaments varied in length from 0.9 to 3.8 micro m (average 2.4 +/- 0.5 micro m, n = 150) and in width from 12.0 to 19.5 nm (average 15.9 +/- 1.4 nm, n = 52), which is different from previous reports for similar microorganisms. At the base of the flagella, a curved hook structure slightly thicker than the flagellar filaments was observed. In freeze-fractured samples, macromolecular complexes about 50 nm in diameter, which possibly corresponded to part of the flagella basal body, were observed in both the P-face of the cytoplasmic membrane and the E-face of the outer membrane. Transmission electron microscopy showed that magnetosomes occurred in planar groups in the cytoplasm close and parallel to the organism surface. A striated structure, which could be involved in maintaining magnetosomes fixed in the cell, was usually observed running along magnetosome chains. The coordinated movement of the MMP depends on the interaction between the flagella of each cell with the flagella of adjacent cells of the microorganism.

  4. Novel Cationic Lipids with Enhanced Gene Delivery and Antimicrobial Activity

    PubMed Central

    Fein, David E.; Bucki, Robert; Byfield, Fitzroy; Leszczynska, Katarzyna; Janmey, Paul A.

    2010-01-01

    Cationic lipids facilitate plasmid delivery, and some cationic sterol-based compounds have antimicrobial activity because of their amphiphilic character. These dual functions are relevant in the context of local ongoing infection during intrapulmonary gene transfer for cystic fibrosis. The transfection activities of two cationic lipids, dexamethasone spermine (DS) and disubstituted spermine (D2S), were tested as individual components and mixtures in bovine aortic endothelial cells and A549 cells. The results showed a 3- to 7-fold improvement in transgene expression for mixtures of DS with 20 to 40 mol% D2S. D2S and coformulations with DS, dioleoyl phosphatidylethanolamine, and DNA exhibited potent bactericidal activity against Escherichia coli MG1655, Bacillus subtilis, and Pseudomonas aeruginosa PAO1, which was maintained in bronchoalveolar lavage fluid. Complete bacterial killing was demonstrated at ∼5 μM, including gene delivery formulations, with 2 orders of magnitude higher tolerance before eukaryotic membrane disruption (erythrocyte hemolysis). D2S also exhibited lipopolysaccharide (LPS) scavenging activity resulting in significant inhibition of LPS-mediated activation of human neutrophils with 85 and 65% lower interleukin-8 released at 12 and 24 h, respectively. Mixtures of DS and D2S can improve transfection activity over common lipofection reagents, and D2S has strong antimicrobial action suited for the suppression of bacterial-mediated inflammation. PMID:20573781

  5. Transcriptional Activators of Human Genes with Programmable DNA-Specificity

    PubMed Central

    Hahn, Simone; Streubel, Jana; Bonas, Ulla; Behrens, Sven-Erik; Boch, Jens

    2011-01-01

    TAL (transcription activator-like) effectors are translocated by Xanthomonas bacteria into plant cells where they activate transcription of target genes. DNA target sequence recognition occurs in a unique mode involving a central domain of tandem repeats. Each repeat recognizes a single base pair in a contiguous DNA sequence and a pair of adjacent hypervariable amino acid residues per repeat specifies which base is bound. Rearranging the repeats allows the design of novel TAL proteins with predictable DNA-recognition specificities. TAL protein-based transcriptional activation in plant cells is mediated by a C-terminal activation domain (AD). Here, we created synthetic TAL proteins with designed repeat compositions using a novel modular cloning strategy termed “Golden TAL Technology”. Newly programmed TAL proteins were not only functional in plant cells, but also in human cells and activated targeted expression of exogenous as well as endogenous genes. Transcriptional activation in different human cell lines was markedly improved by replacing the TAL-AD with the VP16-AD of herpes simplex virus. The creation of TAL proteins with potentially any desired DNA-recognition specificity allows their versatile use in biotechnology. PMID:21625585

  6. Rapid activation of the bivalent gene Sox21 requires displacement of multiple layers of gene-silencing machinery

    PubMed Central

    Chakravarthy, Harini; Ormsbee, Briana D.; Mallanna, Sunil K.; Rizzino, Angie

    2011-01-01

    The rapid formation of numerous tissues during development is highly dependent on the swift activation of key developmental regulators. Recent studies indicate that many key regulatory genes are repressed in embryonic stem cells (ESCs), yet poised for rapid activation due to the presence of both activating (H3K4 trimethylation) and repressive (H3K27 trimethylation) histone modifications (bivalent genes). However, little is known about bivalent gene regulation. In this study, we investigated the regulation of the bivalent gene Sox21, which is activated rapidly when ESCs differentiate in response to increases in Sox2. Chromatin immunoprecipitation demonstrated that prior to differentiation, the Sox21 gene is bound by a complex array of repressive and activating transcriptional machinery. Upon activation, all identified repressive machinery and histone modifications associated with the gene are lost, but the activating modifications and transcriptional machinery are retained. Notably, these changes do not occur when ESCs differentiate in response to retinoic acid. Moreover, ESCs lacking a functional PRC2 complex fail to activate this gene, apparently due to its association with other repressive complexes. Together, these findings suggest that bivalent genes, such as Sox21, are silenced by a complex set of redundant repressive machinery, which exit rapidly in response to appropriate differentiation signals.—Chakravarthy, H., Ormsbee, B. D., Mallanna, S. K., Rizzino, A. Rapid activation of the bivalent gene Sox21 requires displacement of multiple layers of gene-silencing machinery. PMID:20876214

  7. The effect of flagellar motor-rotor complexes on twitching motility in P. aeruginosa

    NASA Astrophysics Data System (ADS)

    Zhao, Kun; Utada, Andrew; Gibiansky, Maxsim; Xian, Wujing; Wong, Gerard

    2013-03-01

    P. aeruginosa is an opportunistic bacterium responsible for a broad range of biofilm infections. In order for biofilms to form, P. aeruginosa uses different types of surface motility. In the current understanding, flagella are used for swarming motility and type IV pili are used for twitching motility. The flagellum also plays important roles in initial surface attachment and in shaping the architectures of mature biofilms. Here we examine how flagella and pili interact during surface motility, by using cell tracking techniques. We show that the pili driven twitching motility of P. aeruginosa can be affected by the motor-rotor complexes of the flagellar system.

  8. How Biophysics May Help Us Understand the Flagellar Motor of Bacteria Which Cause Infections.

    PubMed

    Baker, Matthew A B

    2016-01-01

    Motor proteins are molecules which convert chemical energy to mechanical work and are responsible for motility across all levels: for transport within a cell, for the motion of an individual cell in its surroundings, and for movement in multicellular aggregates, such as muscles. The bacterial flagellar motor is one of the canonical examples of a molecular complex made from several motor proteins, which self-assembles on demand and provides the locomotive force for bacteria. This locomotion provides a key aspect of bacteria's prevalence. Here, we outline the biophysics behind the assembly, the energetics, the switching and the rotation of this remarkable nanoscale electric motor that is Nature's first wheel.

  9. A Delicate Nanoscale Motor Made by Nature—The Bacterial Flagellar Motor

    PubMed Central

    Xue, Ruidong; Ma, Qi

    2015-01-01

    The bacterial flagellar motor (BFM) is a molecular complex ca. 45 nm in diameter that rotates the propeller that makes nearly all bacteria swim. The motor self‐assembles out of ca. 20 different proteins and can not only rotate at up to 50 000 rpm, but can also switch rotational direction in milliseconds and navigate its environment to maneuver, on average, towards regions of greater benefit. The BFM is a pinnacle of evolution that informs and inspires the design of novel nanotechnology in the new era of synthetic biology. PMID:27980978

  10. Modeling torque versus speed, shot noise, and rotational diffusion of the bacterial flagellar motor.

    PubMed

    Mora, Thierry; Yu, Howard; Wingreen, Ned S

    2009-12-11

    We present a minimal physical model for the flagellar motor that enables bacteria to swim. Our model explains the experimentally measured torque-speed relationship of the proton-driven E. coli motor at various pH and temperature conditions. In particular, the dramatic drop of torque at high rotation speeds (the "knee") is shown to arise from saturation of the proton flux. Moreover, we show that shot noise in the proton current dominates the diffusion of motor rotation at low loads. This suggests a new way to probe the discreteness of the energy source, analogous to measurements of charge quantization in superconducting tunnel junctions.

  11. Modeling Torque Versus Speed, Shot Noise, and Rotational Diffusion of the Bacterial Flagellar Motor

    NASA Astrophysics Data System (ADS)

    Mora, Thierry; Yu, Howard; Wingreen, Ned S.

    2009-12-01

    We present a minimal physical model for the flagellar motor that enables bacteria to swim. Our model explains the experimentally measured torque-speed relationship of the proton-driven E. coli motor at various pH and temperature conditions. In particular, the dramatic drop of torque at high rotation speeds (the “knee”) is shown to arise from saturation of the proton flux. Moreover, we show that shot noise in the proton current dominates the diffusion of motor rotation at low loads. This suggests a new way to probe the discreteness of the energy source, analogous to measurements of charge quantization in superconducting tunnel junctions.

  12. Adaptation of muscle gene expression to changes in contractile activity

    NASA Technical Reports Server (NTRS)

    Booth, F. W.; Babij, P.; Thomason, D. B.; Wong, T. S.; Morrison, P. R.

    1987-01-01

    A review of the existing literature regarding the effects of different types of physical activities on the gene expression of adult skeletal muscles leads us to conclude that each type of exercise training program has, as a result, a different phenotype, which means that there are multiple mechanisms, each producing a unique phenotype. A portion of the facts which support this position is presented and interpreted here. [Abstract translated from the original French by NASA].

  13. Adaptation of muscle gene expression to changes in contractile activity

    NASA Technical Reports Server (NTRS)

    Booth, F. W.; Babij, P.; Thomason, D. B.; Wong, T. S.; Morrison, P. R.

    1987-01-01

    A review of the existing literature regarding the effects of different types of physical activities on the gene expression of adult skeletal muscles leads us to conclude that each type of exercise training program has, as a result, a different phenotype, which means that there are multiple mechanisms, each producing a unique phenotype. A portion of the facts which support this position is presented and interpreted here. [Abstract translated from the original French by NASA].

  14. A numerical study of the effects of fluid rheology and stroke kinematics on flagellar swimming in complex fluids

    NASA Astrophysics Data System (ADS)

    Li, Chuanbin; Guy, Robert; Thomases, Becca

    2016-11-01

    It is observed in experiments that as the fluid rheology is changed, Chlamydomonas reinhardtii exhibits changes in both flagellar kinematics and the swimming speed. To understand this phenomenon, we develop a computational model of the swimmer, using flagellar strokes fit from experimental data. We conduct numerical simulations by changing strokes and fluid rheology independently to dissect the effects of these two factors. We discover that stroke patterns extracted from viscoelastic fluids generate much lower stress and have higher efficiency at the cost of lower swimming speed. We also discover that higher fluid elasticity hinders swimming for a fixed stroke pattern.

  15. Metronidazole activation and isolation of Clostridium acetobutylicum electron transport genes.

    PubMed Central

    Santangelo, J D; Jones, D T; Woods, D R

    1991-01-01

    An Escherichia coli F19 recA, nitrate reductase-deficient mutant was constructed by transposon mutagenesis and shown to be resistant to metronidazole. This mutant was a most suitable host for the isolation of Clostridium acetobutylicum genes on recombinant plasmids, which activated metronidazole and rendered the E. coli F19 strain sensitive to metronidazole. Twenty-five E. coli F19 clones containing different recombinant plasmids were isolated and classified into five groups on the basis of their sensitivity to metronidazole. The clones were tested for nitrate reductase, pyruvate-ferredoxin oxidoreductase, and hydrogenase activities. DNA hybridization and restriction endonuclease mapping revealed that four of the C. acetobutylicum insert DNA fragments on recombinant plasmids were linked in an 11.1-kb chromosomal fragment. DNA sequencing and amino acid homology studies indicated that this DNA fragment contained a flavodoxin gene which encoded a protein of 160 amino acids that activated metronidazole and made the E. coli F19 mutant very sensitive to metronidazole. The flavodoxin and hydrogenase genes which are involved in electron transfer systems were linked on the 11.1-kb DNA fragment from C. acetobutylicum. Images PMID:1991710

  16. Protein inhibitor of activated STAT3 inhibits adipogenic gene expression

    SciTech Connect

    Deng Jianbei; Hua Kunjie; Caveney, Erica J.; Takahashi, Nobuyuki; Harp, Joyce B. . E-mail: jharp@unc.edu

    2006-01-20

    Protein inhibitor of activated STAT3 (PIAS3), a cytokine-induced repressor of signal transducer and activator of transcription 3 (STAT3) and a modulator of a broad array of nuclear proteins, is expressed in white adipose tissue, but its role in adipogenesis is not known. Here, we determined that PIAS3 was constitutively expressed in 3T3-L1 cells at all stages of adipogenesis. However, it translocated from the nucleus to the cytoplasm 4 days after induction of differentiation by isobutylmethylxanthine, dexamethasone, and insulin (MDI). In ob/ob mice, PIAS3 expression was increased in white adipose tissue depots compared to lean mice and was found in the cytoplasm of adipocytes. Overexpression of PIAS3 in differentiating preadipocytes, which localized primarily to the nucleus, inhibited mRNA level gene expression of adipogenic transcription factors C/EBP{alpha} and PPAR{gamma}, as well as their downstream target genes aP2 and adiponectin. PIAS3 also inhibited C/EBP{alpha} promoter activation mediated specifically by insulin, but not dexamethasone or isobutylmethylxanthine. Taken together, these data suggest that PIAS3 may play an inhibitory role in adipogenesis by modulating insulin-activated transcriptional activation events. Increased PIAS3 expression in adipose tissue may play a role in the metabolic disturbances of obesity.

  17. Recurrent epimutations activate gene body promoters in primary glioblastoma.

    PubMed

    Nagarajan, Raman P; Zhang, Bo; Bell, Robert J A; Johnson, Brett E; Olshen, Adam B; Sundaram, Vasavi; Li, Daofeng; Graham, Ashley E; Diaz, Aaron; Fouse, Shaun D; Smirnov, Ivan; Song, Jun; Paris, Pamela L; Wang, Ting; Costello, Joseph F

    2014-05-01

    Aberrant DNA hypomethylation may play an important role in the growth rate of glioblastoma (GBM), but the functional impact on transcription remains poorly understood. We assayed the GBM methylome with MeDIP-seq and MRE-seq, adjusting for copy number differences, in a small set of non-glioma CpG island methylator phenotype (non-G-CIMP) primary tumors. Recurrent hypomethylated loci were enriched within a region of chromosome 5p15 that is specified as a cancer amplicon and also encompasses TERT, encoding telomerase reverse transcriptase, which plays a critical role in tumorigenesis. Overall, 76 gene body promoters were recurrently hypomethylated, including TERT and the oncogenes GLI3 and TP73. Recurring hypomethylation also affected previously unannotated alternative promoters, and luciferase reporter assays for three of four of these promoters confirmed strong promoter activity in GBM cells. Histone H3 lysine 4 trimethylation (H3K4me3) ChIP-seq on tissue from the GBMs uncovered peaks that coincide precisely with tumor-specific decrease of DNA methylation at 200 loci, 133 of which are in gene bodies. Detailed investigation of TP73 and TERT gene body hypomethylation demonstrated increased expression of corresponding alternate transcripts, which in TP73 encodes a truncated p73 protein with oncogenic function and in TERT encodes a putative reverse transcriptase-null protein. Our findings suggest that recurring gene body promoter hypomethylation events, along with histone H3K4 trimethylation, alter the transcriptional landscape of GBM through the activation of a limited number of normally silenced promoters within gene bodies, in at least one case leading to expression of an oncogenic protein.

  18. Sensation-seeking genes and physical activity in youth.

    PubMed

    Wilkinson, A V; Gabriel, K P; Wang, J; Bondy, M L; Dong, Q; Wu, X; Shete, S; Spitz, M R

    2013-03-01

    Many studies examining genetic influences on physical activity (PA) have evaluated the impact of single nucleotide polymorphisms (SNPs) related to the development of lifestyle-related chronic diseases, under the hypothesis that they would be associated with PA. However, PA is a multidetermined behavior and associated with a multitude of health consequences. Thus, examining a broader range of candidate genes associated with a broader range of PA correlates may provide new insights into the genetic underpinnings of PA. In this study, we focus on one such correlate - sensation-seeking behavior. Participants (N = 1130 Mexican origin youth) provided a saliva sample and data on PA and sensation-seeking tendencies in 2008-2009. Participants were genotyped for 630 functional and tagging variants in the dopamine, serotonin and cannabinoid pathways. Overall 30% of participants (males - 37.6% and females - 22.0%) reported ≥60 min of PA on 5 of 7 days. After adjusting for gender, age and population stratification, and applying the Bayesian False Discovery Probability approach for assessing noteworthiness, four gene variants were significantly associated with PA. In a multivariable model, being male, having higher sensation-seeking tendencies and at least one copy of the minor allele for SNPs in angiotensin I-converting enzyme gene [ACE; rs8066276 odds ratio (OR) = 1.44; P = 0.012] and tryptophan hydroxylase 2 gene (TPH2; rs11615016 OR = 1.73; P = 0.021) were associated with increased likelihood of meeting PA recommendations. Participants with at least one copy of the minor allele for SNPs in synaptosomal-associated protein 25 gene (SNAP25; rs363035 OR = 0.53; P = 0.005) and cannabinoid receptor 1 gene (CNR1; rs6454672 OR = 0.62; P = 0.022) have decreased likelihood of meeting PA recommendations. Our findings extend current knowledge of the complex relationship between PA and possible genetic underpinnings.

  19. A mathematical model for synergistic eukaryotic gene activation.

    PubMed

    Wang, J; Ellwood, K; Lehman, A; Carey, M F; She, Z S

    1999-02-19

    The precise biochemical mechanism underlying the synergistic action of gene activators on eukaryotic transcription has eluded a solution, largely because of the technical difficulties inherent in analyzing the mechanics of a 2.5 MDa complex comprising greater than 50 polypeptide components. To complement the biochemical approach we have employed mathematical modeling as a means to understand the mechanism of synergy. Parameters relevant to activated transcription were varied in a simple biochemical system and the data were compared to the transcriptional response predicted by a multi-component statistical model. We found that the model achieved a consistent, semi-quantitative description of the measured transcriptional response, and enabled the characterization and measurement of thermodynamic parameters in the in vitro system. The results provide evidence for the existence of cooperativity in the activation process beyond what would be predicted from one current model suggesting that activators function solely by simple recruitment of the general transcription machinery to the promoter. Copyright 1999 Academic Press.

  20. Analysis of the swimming activity of Pseudomonas aeruginosa by using photonic force microscope

    NASA Astrophysics Data System (ADS)

    Chan, Chia-Han; Chang, Bo-Jui; Huang, Ying-Jung; Fan, Chia-Chieh; Peng, Hwei-Ling; Chi, Sien; Hsu, Long

    2005-08-01

    Swimming activity of flagella is a main factor of the motility of bacteria. Flagella expressed on the surface of bacterial species serve as a primary means of motility including swimming. We propose to use optical tweezers to analyze the swimming activity of bacteria. The sample bacteria in the work is Pseudomonas aeruginosa, and it is a gram-negative bacterium and often causes leading to burn wound infections, urinary-tract infections, and pneumonia. The single polar flagellum of P. aeruginosa has been demonstrated to be important virulence and colonization factor of this opportunistic pathogen. We demonstrate a gene to regulate the bacterial swimming activity in P. aeruginosa PAO1 by biological method. However, the change of flagellar morphology was not observed by electron microscopy analysis, suggesting that the gene regulates the flagellar rotation that could not be detected by biological method. PFM exhibits a spatial resolution of a few nanometers to detect the relative position of the probe at an acquisition rate over 1 MHz. By binding a probe such as a bead or a quantum dot on the flagella, we expect the rotation of the probe due to the flagella could be detected. It is expected that the study of the swimming activity of P. aeruginosa provide potent method for the pathogenic role of the flagella in P. aeruginosa.

  1. Direct optical monitoring of flow generated by bacterial flagellar rotation

    SciTech Connect

    Kirchner, Silke R.; Nedev, Spas; Carretero-Palacios, Sol; Lohmüller, Theobald E-mail: feldmann@lmu.de; Feldmann, Jochen E-mail: feldmann@lmu.de; Mader, Andreas; Opitz, Madeleine

    2014-03-03

    We report on a highly sensitive approach to measure and quantify the time dependent changes of the flow generated by the flagella bundle rotation of single bacterial cells. This is achieved by observing the interactions between a silica particle and a bacterium, which are both trapped next to each other in a dual beam optical tweezer. In this configuration, the particle serves as a sensitive detector where the fast-Fourier analysis of the particle trajectory renders, it possible to access information about changes of bacterial activity.

  2. Titanium nanotubes activate genes related to bone formation in vitro

    PubMed Central

    Pozio, Alfonso; Palmieri, Annalisa; Girardi, Ambra; Cura, Francesca; Carinci, Francesco

    2012-01-01

    Background: Titanium is used worldwide to make osseointegrable devices, thanks to its favorable characteristics as mechanical proprieties and biocompatibility, demonstrated by in vivo studies with animal models and clinical trials over a forty-year period. However, the exact genetic effect of the titanium layer on cells is still not well characterized. Materials and Methods: To investigate how titanium nanotubes stimulate osteoblasts differentiation and proliferation, some osteoblast genes (SP7, RUNX2, COL3A1, COL1A1, ALPL, SPP1 and FOSL1) were analyzed by quantitative Real Time RT- PCR. Results: After 15 days, osteoblasts cultivated on titanium naotube showed the up-regulation of bone related genes SP7, ENG, FOSL1 and SPP1 and the down-regulation of RUNX2, COL3A1, COL1A1, and ALPL. After 30 days of treatment, the bone related genes SP7, ENG, FOSL1 and RUNX2 were up-regulated while COL3A1, COL1A1, ALPL and SPP1 were down-regulated. Conclusions: Our results, demonstrates that titanium nanotubes can lead to osteoblast differentiation and extracellular matrix deposition and mineralization in dental pulp stem cells by the activation of osteoblast related genes SPP1, FOSL1 and RUNX2. PMID:23814577

  3. The Trypanosome Flagellar Pocket Collar and Its Ring Forming Protein—TbBILBO1

    PubMed Central

    Perdomo, Doranda; Bonhivers, Mélanie; Robinson, Derrick R.

    2016-01-01

    Sub-species of Trypanosoma brucei are the causal agents of human African sleeping sickness and Nagana in domesticated livestock. These pathogens have developed an organelle-like compartment called the flagellar pocket (FP). The FP carries out endo- and exocytosis and is the only structure this parasite has evolved to do so. The FP is essential for parasite viability, making it an interesting structure to evaluate as a drug target, especially since it has an indispensible cytoskeleton component called the flagellar pocket collar (FPC). The FPC is located at the neck of the FP where the flagellum exits the cell. The FPC has a complex architecture and division cycle, but little is known concerning its organization. Recent work has focused on understanding how the FP and the FPC are formed and as a result of these studies an important calcium-binding, polymer-forming protein named TbBILBO1 was identified. Cellular biology analysis of TbBILBO1 has demonstrated its uniqueness as a FPC component and until recently, it was unknown what structural role it played in forming the FPC. This review summarizes the recent data on the polymer forming properties of TbBILBO1 and how these are correlated to the FP cytoskeleton. PMID:26950156

  4. A molecular mechanism of direction switching in the flagellar motor of Escherichia coli

    PubMed Central

    Paul, Koushik; Brunstetter, Duncan; Titen, Sienna; Blair, David F.

    2011-01-01

    The direction of flagellar rotation is regulated by a rotor-mounted protein assembly, termed the “switch complex,” formed from multiple copies of the proteins FliG, FliM, and FliN. The structures of major parts of these proteins are known, and the overall organization of proteins in the complex has been elucidated previously using a combination of protein-binding, mutational, and cross-linking approaches. In Escherichia coli, the switch from counterclockwise to clockwise rotation is triggered by the signaling protein phospho-CheY, which binds to the lower part of the switch complex and induces small movements of FliM and FliN subunits relative to each other. Direction switching also must produce movements in the upper part of the complex, particularly in the C-terminal domain of FliG (FliGC), which interacts with the stator to generate the torque for flagellar rotation. In the present study, protein movements in the middle and upper parts of the switch complex have been probed by means of targeted cross-linking and mutational analysis. Switching induces a tilting movement of the FliM domains that form the middle part of the switch and a consequent rotation of the affixed FliGC domains that reorients the stator interaction sites by about 90°. In a recently proposed hypothesis for the motor mechanism, such a reorientation of FliGC would reverse the direction of motor rotation. PMID:21969567

  5. Complete structure of the bacterial flagellar hook reveals extensive set of stabilizing interactions

    PubMed Central

    Matsunami, Hideyuki; Barker, Clive S.; Yoon, Young-Ho; Wolf, Matthias; Samatey, Fadel A.

    2016-01-01

    The bacterial flagellar hook is a tubular helical structure made by the polymerization of multiple copies of a protein, FlgE. Here we report the structure of the hook from Campylobacter jejuni by cryo-electron microscopy at a resolution of 3.5 Å. On the basis of this structure, we show that the hook is stabilized by intricate inter-molecular interactions between FlgE molecules. Extra domains in FlgE, found only in Campylobacter and in related bacteria, bring more stability and robustness to the hook. Functional experiments suggest that Campylobacter requires an unusually strong hook to swim without its flagella being torn off. This structure reveals details of the quaternary organization of the hook that consists of 11 protofilaments. Previous study of the flagellar filament of Campylobacter by electron microscopy showed its quaternary structure made of seven protofilaments. Therefore, this study puts in evidence the difference between the quaternary structures of a bacterial filament and its hook. PMID:27811912

  6. Architecture of a flagellar apparatus in the fast-swimming magnetotactic bacterium MO-1.

    PubMed

    Ruan, Juanfang; Kato, Takayuki; Santini, Claire-Lise; Miyata, Tomoko; Kawamoto, Akihiro; Zhang, Wei-Jia; Bernadac, Alain; Wu, Long-Fei; Namba, Keiichi

    2012-12-11

    The bacterial flagellum is a motility organelle that consists of a rotary motor and a helical propeller. The flagella usually work individually or by forming a loose bundle to produce thrust. However, the flagellar apparatus of marine bacterium MO-1 is a tight bundle of seven flagellar filaments enveloped in a sheath, and it has been a mystery as to how the flagella rotate smoothly in coordination. Here we have used electron cryotomography to visualize the 3D architecture of the sheathed flagella. The seven filaments are enveloped with 24 fibrils in the sheath, and their basal bodies are arranged in an intertwined hexagonal array similar to the thick and thin filaments of vertebrate skeletal muscles. This complex and exquisite architecture strongly suggests that the fibrils counter-rotate between flagella in direct contact to minimize the friction of high-speed rotation of individual flagella in the tight bundle within the sheath to enable MO-1 cells to swim at about 300 µm/s.

  7. Autonomously responsive pumping by a bacterial flagellar forest: A mean-field approach

    NASA Astrophysics Data System (ADS)

    Martindale, James D.; Fu, Henry C.

    2017-09-01

    This study is motivated by a microfluidic device that imparts a magnetic torque on an array of bacterial flagella. Bacterial flagella can transform their helical geometry autonomously in response to properties of the background fluid, which provides an intriguing mechanism allowing their use as an engineered element for the regulation or transport of chemicals in microscale applications. The synchronization of flagellar phase has been widely studied in biological contexts, but here we examine the synchronization of flagellar tilt, which is necessary for effective pumping. We first examine the effects of helical geometry and tilt on the pumping flows generated by a single rotating flagellum. Next, we explore a mean-field model for an array of helical flagella to understand how collective tilt arises and influences pumping. The mean-field methodology allows us to take into account possible phase differences through a time-averaging procedure and to model an infinite array of flagella. We find array separation distances, magnetic field strengths, and rotation frequencies that produce nontrivial self-consistent pumping solutions. For individual flagella, pumping is reversed when helicity or rotation is reversed; in contrast, when collective effects are included, self-consistent tilted pumping solutions become untilted nonpumping solutions when helicity or rotation is reversed.

  8. Metachronal waves in the flagellar beating of Volvox and their hydrodynamic origin

    PubMed Central

    Brumley, Douglas R.; Polin, Marco; Pedley, Timothy J.; Goldstein, Raymond E.

    2015-01-01

    Groups of eukaryotic cilia and flagella are capable of coordinating their beating over large scales, routinely exhibiting collective dynamics in the form of metachronal waves. The origin of this behaviour—possibly influenced by both mechanical interactions and direct biological regulation—is poorly understood, in large part due to a lack of quantitative experimental studies. Here we characterize in detail flagellar coordination on the surface of the multicellular alga Volvox carteri, an emerging model organism for flagellar dynamics. Our studies reveal for the first time that the average metachronal coordination observed is punctuated by periodic phase defects during which synchrony is partial and limited to specific groups of cells. A minimal model of hydrodynamically coupled oscillators can reproduce semi-quantitatively the characteristics of the average metachronal dynamics, and the emergence of defects. We systematically study the model's behaviour by assessing the effect of changing intrinsic rotor characteristics, including oscillator stiffness and the nature of their internal driving force, as well as their geometric properties and spatial arrangement. Our results suggest that metachronal coordination follows from deformations in the oscillators' limit cycles induced by hydrodynamic stresses, and that defects result from sufficiently steep local biases in the oscillators' intrinsic frequencies. Additionally, we find that random variations in the intrinsic rotor frequencies increase the robustness of the average properties of the emergent metachronal waves. PMID:26040592

  9. The N Terminus of FliM Is Essential To Promote Flagellar Rotation in Rhodobacter sphaeroides

    PubMed Central

    Poggio, Sebastian; Osorio, Aurora; Corkidi, Gabriel; Dreyfus, Georges; Camarena, Laura

    2001-01-01

    FliM is part of the flagellar switch complex. Interaction of this protein with phospho-CheY (CheY-P) through its N terminus constitutes the main information relay point between the chemotactic system and the flagellum. In this work, we evaluated the role of the N terminus of FliM in the swimming behavior of Rhodobacter sphaeroides. Strains expressing the FliM protein with substitutions in residues previously reported in Escherichia coli as being important for interaction with CheY showed an increased stop frequency compared with wild-type cells. In accordance, we observed that R. sphaeroides cells expressing FliM lacking either the first 13 or 20 amino acids from the N terminus showed a stopped phenotype. We show evidence that FliMΔ13 and FliMΔ20 are stable proteins and that cells expressing them allow flagellin export at levels indistinguishable from those detected for the wild-type strain. These results suggest that the N-terminal region of FliM is required to promote swimming in this bacterium. The role of CheY in controlling flagellar rotation in this organism is discussed. PMID:11325943

  10. Metachronal waves in the flagellar beating of Volvox and their hydrodynamic origin.

    PubMed

    Brumley, Douglas R; Polin, Marco; Pedley, Timothy J; Goldstein, Raymond E

    2015-07-06

    Groups of eukaryotic cilia and flagella are capable of coordinating their beating over large scales, routinely exhibiting collective dynamics in the form of metachronal waves. The origin of this behavior--possibly influenced by both mechanical interactions and direct biological regulation--is poorly understood, in large part due to a lack of quantitative experimental studies. Here we characterize in detail flagellar coordination on the surface of the multicellular alga Volvox carteri, an emerging model organism for flagellar dynamics. Our studies reveal for the first time that the average metachronal coordination observed is punctuated by periodic phase defects during which synchrony is partial and limited to specific groups of cells. A minimal model of hydrodynamically coupled oscillators can reproduce semi-quantitatively the characteristics of the average metachronal dynamics, and the emergence of defects. We systematically study the model's behaviour by assessing the effect of changing intrinsic rotor characteristics, including oscillator stiffness and the nature of their internal driving force, as well as their geometric properties and spatial arrangement. Our results suggest that metachronal coordination follows from deformations in the oscillators' limit cycles induced by hydrodynamic stresses, and that defects result from sufficiently steep local biases in the oscillators' intrinsic frequencies. Additionally, we find that random variations in the intrinsic rotor frequencies increase the robustness of the average properties of the emergent metachronal waves.

  11. A “Mechanistic” Explanation of the Multiple Helical Forms Adopted by Bacterial Flagellar Filaments

    PubMed Central

    Calladine, C.R.; Luisi, B.F.; Pratap, J.V.

    2013-01-01

    The corkscrew-like flagellar filaments emerging from the surface of bacteria such as Salmonella typhimurium propel the cells toward nutrient and away from repellents. This kind of motility depends upon the ability of the flagellar filaments to adopt a range of distinct helical forms. A filament is typically constructed from ~ 30,000 identical flagellin molecules, which self-assemble into a tubular structure containing 11 near-longitudinal protofilaments. A “mechanical” model, in which the flagellin building block has the capacity to switch between two principal interfacial states, predicts that the filament can assemble into a “canonical” family of 12 distinct helical forms, each having unique curvature and twist: these include two “extreme” straight forms having left- and right-handed twists, respectively, and 10 intermediate helical forms. Measured shapes of the filaments correspond well with predictions of the model. This report is concerned with two unanswered questions. First, what properties of the flagellin determine which of the 12 discrete forms is preferred? Second, how does the interfacial “switch” work, at a molecular level? Our proposed solution of these problems is based mainly on a detailed examination of differences between the available electron cryo-microscopy structures of the straight L and R filaments, respectively. PMID:23274110

  12. Constant torque in flagellar bacterial motors optimizes space exploration

    NASA Astrophysics Data System (ADS)

    Condat, Carlos A.; di Salvo, Mario E.

    2012-02-01

    Experiments indicate that the torque provided by the bacterial rotary motor is approximately constant over a large range of angular speeds. Constant torque implies that the power spent in active motion is proportional to the instantaneous bacterial speed, if the relation between angular speed and swimming speed is linear. Here we show that a constant torque maximizes the volume of the region explored by a bacterium in a resource-depleted medium. Given that nutrients in the ocean are often concentrated in separate, ephemeral patches, we propose that the observed constancy of the torque may be a trait evolved to maximize bacterial survival in the ocean. We also discuss the dependence of the explored volume with the particular features of the bacterial propulsion mechanism.

  13. [Effects related to gene-gene interactions of peroxisome proliferator-activated receptor on essential hypertension].

    PubMed

    Yu, Hao; Chen, Qiu; Yang, Jie; Hu, Xiao-shu; Zhou, Zheng-yuan; Guo, Zhi-rong; Wu, Ming

    2013-04-01

    To explore the impact of the gene-gene interaction among the single nucleotide polymorphisms (SNPs) of peroxisome proliferator-activated receptor α/δ/γ on essential hypertension (EH). Participants were recruited based on the previous work of the PMMJS (Prevention of Multiple Metabolic Disorders and Metabolic Syndrome in Jiangsu Province) cohort study in Jiangsu province of China. A total number of 820 subjects were randomly selected from the cohort and received gene polymorphism detection covered ten SNPs:PPARα/δ/γ (PPARα: rs135539, rs1800206 and rs4253778; PPARδ: rs2016520 and rs9794; PPARγ: rs10865710, rs1805192, rs4684847, rs709158 and rs3856806). Generalized Multifactor Dimensionality Reduction (GMDR) model was used to evaluate the association between gene-gene interaction among the ten SNPs and EH. After adjusting factors as gender, age, BMI, FPG, TG, HDL-C, high fat diet, low fiber diet and physical activity, results from the GMDR analysis showed that the best qualitative trait models were 7/9-dimensional model (EH: cross-validation consistency were 9/10 and 10/10, prediction accuracy were 0.5862 and 0.5885), 5/9-dimensional model (SBP:cross-validation consistency were 10/10 and 8/10, prediction accuracy were 0.6055 and 0.6011), and 8/9-dimensional model (DBP: cross-validation consistency both were 10/10, prediction accuracy were 0.5926 and 0.5972), while the best quantitative trait models were 4/5-dimensional model (SBP: cross-validation consistency were 10/10 and 8/10, prediction accuracy were 0.6111 and 0.6072), and 5-dimensional model (DBP: cross-validation consistency were 9/10, prediction accuracy were 0.5753). Interactions among ten SNPs of PPARs seemed to have existed and with significant impact on the levels of blood pressure.

  14. Three faces of recombination activating gene 1 (RAG1) mutations.

    PubMed

    Patiroglu, Turkan; Akar, Himmet Haluk; Van Der Burg, Mirjam

    2015-12-01

    Severe combined immune deficiency (SCID) is a group of genetic disorder associated with development of T- and/or B-lymphocytes. Recombination-activating genes (RAG1/2) play a critical role on VDJ recombination process that leads to the production of a broad T-cell receptor (TCR) and B-cell receptor (BCR) repertoire in the development of T and B cells. RAG1/2 genes mutations result in various forms of primary immunodeficiency, ranging from classic SCID to Omenn syndrome (OS) to atypical SCID with such as granuloma formation and autoimmunity. Herein, we reported 4 patients with RAG1 deficiency: classic SCID was seen in two patients who presented with recurrent pneumonia and chronic diarrhoea, and failure to thrive. OS was observed in one patient who presented with chronic diarrhoea, skin rash, recurrent lower respiratory infections, and atypical SCID was seen in one patient who presented with Pyoderma gangrenosum (PG) and had novel RAG1 mutation.

  15. Transcriptional activation of ribosomal RNA genes during compensatory renal hypertrophy

    SciTech Connect

    Ouellette, A.J.; Moonka, R.; Zelenetz, A.; Malt, R.A.

    1986-05-01

    The overall rate of rDNA transcription increases by 50% during the first 24 hours of compensatory renal hypertrophy in the mouse. To study mechanisms of ribosome accumulation after uninephrectomy, transcription rates were measured in isolated kidneys by transcriptional runoff. /sup 32/P-labeled nascent transcripts were hybridized to blots containing linearized, denatured cloned rDNA, and hybridization was quantitated autoradiographically and by direct counting. Overall transcriptional activity of rDNA was increased by 30% above control levels at 6 hrs after nephrectomy and by 50% at 12, 18, and 24 hrs after operation. Hybridizing RNA was insensitive to inhibiby alpha-amanitin, and no hybridization was detected to vector DNA. Thus, accelerated rDNA transcription is one regulatory element in the accretion of ribosomes in renal growth, and the regulatory event is an early event. Mechanisms of activation may include enhanced transcription of active genes or induction of inactive DNA.

  16. Regulator of complement activation (RCA) gene cluster in Xenopus tropicalis.

    PubMed

    Oshiumi, Hiroyuki; Suzuki, Yuzuru; Matsumoto, Misako; Seya, Tsukasa

    2009-05-01

    Genome and expressed sequence tag information of Xenopus tropicalis suggested that short-consensus repeat (SCR)-containing proteins are encoded by three genes that are mapped within a 300-kb downstream of PFKFB2, which is a marker gene for the regulator of complement activation (RCA) loci in human and chicken. Based on this observation, we cloned the three cDNAs of these proteins using 3'- or 5'-RACE technique. Since their primary structures and locations of the proximity to the PFKFB2 locus, we named them amphibian RCA protein (ARC) 1, 2, and 3. Expression in human HEK293 or CHO cells suggested that ARC1 is a soluble protein of Mr approximately 67 kDa, ARC2 is a membrane protein with Mr 44 kDa, and ARC3 a secretary protein with a putative transmembrane region. They were N-glycosylated during maturation. In human and chicken RCA clusters, the order in which genes for soluble, GPI-anchored, and membrane forms of SCR proteins are arranged is from the distant to proximity to the PFKFB2 gene. However, the amphibian ARC1, 2, and 3 resembled one another and did not reflect the same order found in human and chicken RCA genes. This may be due to self-duplication of ARCs to form a family, and it evolved after the amphibia separated from the ancestor of the amniotes, which possessed soluble, GPI-anchored, and membrane forms of SCR protein members. Taken together, frog possesses a RCA locus, but the constitution of the ARC proteins differs from that of the amniotes with a unique self-resemblance.

  17. Behavioral meaningful opioidergic stimulation activates kappa receptor gene expression

    PubMed Central

    Teodorov, E.; Ferrari, M.F.R.; Fior-Chadi, D.R.; Camarini, R.; Felício, L.F.

    2012-01-01

    The periaqueductal gray (PAG) has been reported to be a location for opioid regulation of pain and a potential site for behavioral selection in females. Opioid-mediated behavioral and physiological responses differ according to the activity of opioid receptor subtypes. The present study investigated the effects of the peripheral injection of the kappa-opioid receptor agonist U69593 into the dorsal subcutaneous region of animals on maternal behavior and on Oprk1 gene activity in the PAG of female rats. Female Wistar rats weighing 200-250 g at the beginning of the study were randomly divided into 2 groups for maternal behavior and gene expression experiments. On day 5, pups were removed at 7:00 am and placed in another home cage that was distant from their mother. Thirty minutes after removing the pups, the dams were treated with U69593 (0.15 mg/kg, sc) or 0.9% saline (up to 1 mL/kg) and after 30 min were evaluated in the maternal behavior test. Latencies in seconds for pup retrieval, grouping, crouching, and full maternal behavior were scored. The results showed that U69593 administration inhibited maternal behavior (P < 0.05) because a lower percentage of U69593 group dams showed retrieval of first pup, retrieving all pups, grouping, crouching and displaying full maternal behavior compared to the saline group. Opioid gene expression was evaluated using real-time reverse-transcription polymerase chain reaction (RT-PCR). A single injection of U69593 increased Oprk1 PAG expression in both virgin (P < 0.05) and lactating female rats (P < 0.01), with no significant effect on Oprm1 or Oprd1 gene activity. Thus, the expression of kappa-opioid receptors in the PAG may be modulated by single opioid receptor stimulation and behavioral meaningful opioidergic transmission in the adult female might occur simultaneously to specific changes in gene expression of kappa-opioid receptor subtype. This is yet another alert for the complex role of the opioid system in female

  18. Stimulation of human spermatozoa with progesterone gradients to simulate approach to the oocyte. Induction of [Ca(2+)](i) oscillations and cyclical transitions in flagellar beating.

    PubMed

    Harper, Claire V; Barratt, Christopher L R; Publicover, Stephen J

    2004-10-29

    Progesterone is present at micromolar concentrations in the cumulus matrix, which surrounds mammalian oocytes. Exposure of human spermatozoa to a concentration gradient of progesterone (0-3 microM) to simulate approach to the oocyte induced a slowly developing increase in [Ca(2+)](i) upon which, in many cells, slow oscillations were superimposed. [Ca(2+)](i) oscillations often started at very low progesterone (<10 nm), and their frequency did not change during the subsequent rise in concentration. Oscillations also occurred, but in a much smaller proportion of cells, in response to stepped application of progesterone (3 microM). When progesterone was removed, [Ca(2+)](i) oscillations often persisted or quickly resumed. Superfusion with low-Ca(2+) bathing medium (no added Ca(2+)) did not prevent [Ca(2+)](i) oscillations, but they could be abolished by addition of EGTA or La(3+). Inhibitors of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPases or inositol trisphosphate signaling had no effect on [Ca(2+)](i) oscillations, but pharmacological manipulation of ryanodine receptors affected both their frequency and amplitude. Staining of live spermatozoa with BODIPY FL-X ryanodine showed localization of ryanodine binding primarily to the caudal part of the head and mid-piece. [Ca(2+)](i) oscillations did not induce acrosome reaction, but in cells generating oscillations, the flagellar beat mode alternated in synchrony with the oscillation cycle. Flagellar bending and lateral movement of the sperm head during [Ca(2+)](i) peaks were markedly increased compared with during [Ca(2+)](i) troughs. This alternating pattern of activity is likely to facilitate zona penetration. These observations show that progesterone initiates unusual and complex store-mediated [Ca(2+)](i) signaling in human spermatozoa and identify a previously unrecognized effect of progesterone in regulating sperm "behavior" during fertilization.

  19. Mechanisms guiding Polycomb activities during gene silencing in Arabidopsis thaliana

    PubMed Central

    He, Chongsheng; Huang, Hai; Xu, Lin

    2013-01-01

    Polycomb group (PcG) proteins act in an evolutionarily conserved epigenetic pathway that regulates chromatin structures in plants and animals, repressing many developmentally important genes by modifying histones. PcG proteins can form at least two multiprotein complexes: Polycomb Repressive Complexes 1 and 2 (PRC1 and PRC2, respectively). The functions of Arabidopsis thaliana PRCs have been characterized in multiple stages of development and have diverse roles in response to environmental stimuli. Recently, the mechanism that precisely regulates Arabidopsis PcG activity was extensively studied. In this review, we summarize recent discoveries in the regulations of PcG at the three different layers: the recruitment of PRCs to specific target loci, the polyubiquitination and degradation of PRC2, and the antagonism of PRC2 activity by the Trithorax group proteins. Current knowledge indicates that the powerful activity of the PcG pathway is strictly controlled for specific silencing of target genes during plant development and in response to environmental stimuli. PMID:24312106

  20. Recovering glycoside hydrolase genes from active tundra cellulolytic bacteria.

    PubMed

    Pinnell, Lee J; Dunford, Eric; Ronan, Patrick; Hausner, Martina; Neufeld, Josh D

    2014-07-01

    Bacteria responsible for cellulose hydrolysis in situ are poorly understood, largely because of the relatively recent development of cultivation-independent methods for their detection and characterization. This study combined DNA stable-isotope probing (DNA-SIP) and metagenomics for identifying active bacterial communities that assimilated carbon from glucose and cellulose in Arctic tundra microcosms. Following DNA-SIP, bacterial fingerprint analysis of gradient fractions confirmed isotopic enrichment. Sequenced fingerprint bands and clone library analysis of 16S rRNA genes identified active bacterial taxa associated with cellulose-associated labelled DNA, including Bacteroidetes (Sphingobacteriales), Betaproteobacteria (Burkholderiales), Alphaproteobacteria (Caulobacteraceae), and Chloroflexi (Anaerolineaceae). We also compared glycoside hydrolase metagenomic profiles from bulk soil and heavy DNA recovered from DNA-SIP incubations. Active populations consuming [(13)C]glucose and [(13)C]cellulose were distinct, based on ordinations of light and heavy DNA. Metagenomic analysis demonstrated a ∼3-fold increase in the relative abundance of glycoside hydrolases in DNA-SIP libraries over bulk-soil libraries. The data also indicate that multiple displacement amplification introduced bias into the resulting metagenomic analysis. This research identified DNA-SIP incubation conditions for glucose and cellulose that were suitable for Arctic tundra soil and confirmed that DNA-SIP enrichment can increase target gene frequencies in metagenomic libraries.