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The Use of Flagella and Motility for Plant Colonization and Fitness by Different Strains of the Foodborne Pathogen Listeria monocytogenes  [PDF]
Lisa Gorski, Jessica M. Duhé, Denise Flaherty
PLOS ONE , 2009, DOI: 10.1371/journal.pone.0005142
Abstract: The role of flagella and motility in the attachment of the foodborne pathogen Listeria monocytogenes to various surfaces is mixed with some systems requiring flagella for an interaction and others needing only motility for cells to get to the surface. In nature this bacterium is a saprophyte and contaminated produce is an avenue for infection. Previous studies have documented the ability of this organism to attach to and colonize plant tissue. Motility mutants were generated in three wild type strains of L. monocytogenes by deleting either flaA, the gene encoding flagellin, or motAB, genes encoding part of the flagellar motor, and tested for both the ability to colonize sprouts and for the fitness of that colonization. The motAB mutants were not affected in the colonization of alfalfa, radish, and broccoli sprouts; however, some of the flaA mutants showed reduced colonization ability. The best colonizing wild type strain was reduced in colonization on all three sprout types as a result of a flaA deletion. A mutant in another background was only affected on alfalfa. The third, a poor alfalfa colonizer was not affected in colonization ability by any of the deletions. Fitness of colonization was measured in experiments of competition between mixtures of mutant and parent strains on sprouts. Here the flaA and motAB mutants of the three strain backgrounds were impaired in fitness of colonization of alfalfa and radish sprouts, and one strain background showed reduced fitness of both mutant types on broccoli sprouts. Together these data indicate a role for flagella for some strains to physically colonize some plants, while the fitness of that colonization is positively affected by motility in almost all cases.
A defined medium to investigate sliding motility in a Bacillus subtilis flagella-less mutant
Ray Fall, Daniel B Kearns, Tam Nguyen
BMC Microbiology , 2006, DOI: 10.1186/1471-2180-6-31
Abstract: Using a defined semi-solid medium we determined that a B. subtilis hag mutant colonized the surface in two stages, first as tendril-like clusters of cells followed by a profuse pellicle-like film. We determined the levels of macro- and micro-nutrients required for the tendril-to-film transition. Sufficient levels of each of the macronutrients, glycerol, Na-glutamate, and Na-phosphate, and inorganic nutrients, K+, Mg2+, Fe2+ and Mn2+, were required for robust film formation. The K+ requirement was quantified in more detail, and the thresholds for complete tendril coverage (50 μM KCl) or film coverage (2–3 mM KCl) were determined. In addition, disruption of the genes for the higher affinity K+ transporter (KtrAB), but not the lower affinity K+ transporter (KtrCD), strongly inhibited the formation of both tendrils and films, and could be partially overcome by high levels of KCl. Examination of hag tendrils by confocal scanning laser microscopy revealed that tendrils are multicellular structures, but that the cells are not as highly organized as cells in wild-type B. subtilis pellicles.These results suggest that B. subtilis can use sliding motility to colonize surfaces, using a tendril-like growth mode when various macronutrients or micronutrients are limiting. If nutrients are balanced and sufficient, the surfaces between tendrils can be colonized by robust surface films. Sliding motility may represent a strategy for nutrient-deprived cells to colonize surfaces in natural environments, such as plant roots, and the media described here may be useful in investigations of this growth phenotype.Bacteria use a variety of motility mechanisms to colonize environments, including flagella-dependent swimming and swarming, and flagella-independent, twitching, gliding, and sliding (reviewed in [1]). Of these motility mechanisms, the least investigated is sliding motility, which Henrichsen [2] defined as surface translocation produced by expansive forces in the growing colony combi
Isolation of Salmonella Mutants Resistant to the Inhibitory Effect of Salicylidene acylhydrazides on Flagella-Mediated Motility  [PDF]
Isabel Martinez-Argudo, Andreas K. J. Veenendaal, Xia Liu, A. Dorothea Roehrich, Maria C. Ronessen, Giulia Franzoni, Katerine N. van Rietschoten, Yusuke V. Morimoto, Yumiko Saijo-Hamano, Matthew B. Avison, David J. Studholme, Keiichi Namba, Tohru Minamino, Ariel J. Blocker
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0052179
Abstract: Salicylidene acylhydrazides identified as inhibitors of virulence-mediating type III secretion systems (T3SSs) potentially target their inner membrane export apparatus. They also lead to inhibition of flagellar T3SS-mediated swimming motility in Salmonella enterica serovar. Typhimurium. We show that INP0404 and INP0405 act by reducing the number of flagella/cell. These molecules still inhibit motility of a Salmonella ΔfliH-fliI-fliJ/flhB(P28T) strain, which lacks three soluble components of the flagellar T3S apparatus, suggesting that they are not the target of this drug family. We implemented a genetic screen to search for the inhibitors' molecular target(s) using motility assays in the ΔfliH-fliI/flhB(P28T) background. Both mutants identified were more motile than the background strain in the absence of the drugs, although HM18 was considerably more so. HM18 was more motile than its parent strain in the presence of both drugs while DI15 was only insensitive to INP0405. HM18 was hypermotile due to hyperflagellation, whereas DI15 was not hyperflagellated. HM18 was also resistant to a growth defect induced by high concentrations of the drugs. Whole-genome resequencing of HM18 indicated two alterations within protein coding regions, including one within atpB, which encodes the inner membrane a-subunit of the FOF1-ATP synthase. Reverse genetics indicated that the alteration in atpB was responsible for all of HM18's phenotypes. Genome sequencing of DI15 uncovered a single A562P mutation within a gene encoding the flagellar inner membrane protein FlhA, the direct role of which in mediating drug insensitivity could not be confirmed. We discuss the implications of these findings in terms of T3SS export apparatus function and drug target identification.
Nickel and low CO2-controlled motility in Chlamydomonas through complementation of a paralyzed flagella mutant with chemically regulated promoters
Paola Ferrante, Dennis R Diener, Joel L Rosenbaum, Giovanni Giuliano
BMC Plant Biology , 2011, DOI: 10.1186/1471-2229-11-22
Abstract: We fused the CYC6 and CAH1 promoters to an HA-tagged RSP3 gene, encoding a protein of the flagellar radial spoke complex. The constructs were used for chemically regulated complementation of the pf14 mutant, carrying an ochre mutation in the RSP3 gene. 7 to 8% of the transformants showed cells with restored motility after induction with nickel or transfer to low CO2 conditions, but not in non-inducing conditions. Maximum complementation (5% motile cells) was reached with very different kinetics (5-6 hours for CAH1, 48 hours for CYC6). The two inducible promoters drive much lower levels of RSP3 protein expression than the constitutive PSAD promoter, which shows almost complete rescue of motility.To our knowledge, this is the first example of the use of the CYC6 or CAH1 promoters to perform a chemically regulated complementation of a Chlamydomonas mutant. Based on our data, the CYC6 and CAH1 promoters should be capable of fully complementing mutants in genes whose products exert their biological activity at low concentrations.Chlamydomonas reinhardtii is a unicellular green alga, capable of both photosynthetic and fermentative growth. A plethora of mutants in relevant biological processes are available, and nuclear and chloroplast transformation are easy to perform [1]. Its 120-megabase genome has been completely sequenced [2]. Chlamydomonas combines functions typical of higher plants, such as the presence of a chloroplast endowed with two photosystems [3], of protozoa, such as the presence of motile flagella for swimming [4], and of archaea, such as the presence of sensory rhodopsins mediating phototaxis [5].Flagellar motility in Chlamydomonas is dependent on dynein motors, which drive microtubule sliding, and a multitude of accessory proteins that control dynein activity, including radial spokes and the central pair complex. Immotile mutants missing individual subunits of these components have been identified and, in many cases, rescued by introducing the correspond
Flagella Overexpression Attenuates Salmonella Pathogenesis  [PDF]
Xinghong Yang, Theresa Thornburg, Zhiyong Suo, SangMu Jun, Amanda Robison, Jinquan Li, Timothy Lim, Ling Cao, Teri Hoyt, Recep Avci, David W. Pascual
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0046828
Abstract: Flagella are cell surface appendages involved in a number of bacterial behaviors, such as motility, biofilm formation, and chemotaxis. Despite these important functions, flagella can pose a liability to a bacterium when serving as potent immunogens resulting in the stimulation of the innate and adaptive immune systems. Previous work showing appendage overexpression, referred to as attenuating gene expression (AGE), was found to enfeeble wild-type Salmonella. Thus, this approach was adapted to discern whether flagella overexpression could induce similar attenuation. To test its feasibility, flagellar filament subunit FliC and flagellar regulon master regulator FlhDC were overexpressed in Salmonella enterica serovar Typhimurium wild-type strain H71. The results show that the expression of either FliC or FlhDC alone, and co-expression of the two, significantly attenuates Salmonella. The flagellated bacilli were unable to replicate within macrophages and thus were not lethal to mice. In-depth investigation suggests that flagellum-mediated AGE was due to the disruptive effects of flagella on the bacterial membrane, resulting in heightened susceptibilities to hydrogen peroxide and bile. Furthermore, flagellum-attenuated Salmonella elicited elevated immune responses to Salmonella presumably via FliC’s adjuvant effect and conferred robust protection against wild-type Salmonella challenge.
Transcriptome Profiling Reveals Stage-Specific Production and Requirement of Flagella during Biofilm Development in Bordetella bronchiseptica  [PDF]
Tracy L. Nicholson, Matt S. Conover, Rajendar Deora
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0049166
Abstract: We have used microarray analysis to study the transcriptome of the bacterial pathogen Bordetella bronchiseptica over the course of five time points representing distinct stages of biofilm development. The results suggest that B. bronchiseptica undergoes a coordinately regulated gene expression program similar to a bacterial developmental process. Expression and subsequent production of the genes encoding flagella, a classical Bvg? phase phenotype, occurs and is under tight regulatory control during B. bronchiseptica biofilm development. Using mutational analysis, we demonstrate that flagella production at the appropriate stage of biofilm development, i.e. production early subsequently followed by repression, is required for robust biofilm formation and maturation. We also demonstrate that flagella are necessary and enhance the initial cell-surface interactions, thereby providing mechanistic information on the initial stages of biofilm development for B. bronchiseptica. Biofilm formation by B. bronchiseptica involves the production of both Bvg-activated and Bvg-repressed factors followed by the repression of factors that inhibit formation of mature biofilms.
Shape of optimal active flagella  [PDF]
Christophe Eloy,Eric Lauga
Physics , 2013, DOI: 10.1017/jfm.2013.370
Abstract: Many eukaryotic cells use the active waving motion of flexible flagella to self-propel in viscous fluids. However, the criteria governing the selection of particular flagellar waveforms among all possible shapes has proved elusive so far. To address this question, we derive computationally the optimal shape of an internally-forced periodic planar flagellum deforming as a travelling wave. The optimum is here defined as the shape leading to a given swimming speed with minimum energetic cost. To calculate the energetic cost though, we consider the irreversible internal power expanded by the molecular motors forcing the flagellum, only a portion of which ending up dissipated in the fluid. This optimisation approach allows us to derive a family of shapes depending on a single dimensionless number quantifying the relative importance of elastic to viscous effects: the Sperm number. The computed optimal shapes are found to agree with the waveforms observed on spermatozoon of marine organisms, thus suggesting that these eukaryotic flagella might have evolved to be mechanically optimal.
Nonlinear Dynamics of Cilia and Flagella  [PDF]
Andreas Hilfinger,Amit K Chattopadhyay,Frank Julicher
Quantitative Biology , 2009, DOI: 10.1103/PhysRevE.79.051918
Abstract: Cilia and flagella are hair-like extensions of eukaryotic cells which generate oscillatory beat patterns that can propel micro-organisms and create fluid flows near cellular surfaces. The evolutionary highly conserved core of cilia and flagella consists of a cylindrical arrangement of nine microtubule doublets, called the axoneme. The axoneme is an actively bending structure whose motility results from the action of dynein motor proteins cross-linking microtubule doublets and generating stresses that induce bending deformations. The periodic beat patterns are the result of a mechanical feedback that leads to self-organized bending waves along the axoneme. Using a theoretical framework to describe planar beating motion, we derive a nonlinear wave equation that describes the fundamental Fourier mode of the axonemal beat. We study the role of nonlinearities and investigate how the amplitude of oscillations increases in the vicinity of an oscillatory instability. We furthermore present numerical solutions of the nonlinear wave equation for different boundary conditions. We find that the nonlinear waves are well approximated by the linearly unstable modes for amplitudes of beat patterns similar to those observed experimentally.
Flagella from Five Cronobacter Species Induce Pro-Inflammatory Cytokines in Macrophage Derivatives from Human Monocytes  [PDF]
Ariadnna Cruz-Córdova, Luz M. Rocha-Ramírez, Sara A. Ochoa, Bertha Gónzalez-Pedrajo, Norma Espinosa, Carlos Eslava, Ulises Hernández-Chi?as, Guillermo Mendoza-Hernández, Alejandra Rodríguez-Leviz, Pedro Valencia-Mayoral, Stanislaw Sadowinski-Pine, Rigoberto Hernández-Castro, Iris Estrada-García, Onofre Mu?oz-Hernández, Irma Rosas, Juan Xicohtencatl-Cortes
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0052091
Abstract: Cronobacter spp. are opportunistic pathogens linked to lie-threatening infections in neonates and contaminated powdered infant formula that has been epidemiologically associated with these cases. Clinical symptoms of Cronobacter include necrotizing enterocolitis, bacteremia, and meningitis. Flagella from C. sakazakii are involved in biofilm formation and its adhesion to epithelial cells. We investigated the role of flagella from C. sakazakii ST1 and ST4, C. malonaticus, C. muytjensii, C. turicensis and C. dublinensis during the activation of cytokines (IL-8, TNF-α, and IL-10) in macrophage derivatives from human monocytes, which has not been extensively studied. The production and identity of flagella from the five Cronobacter species were visualized and recognized with anti-flagella antibodies by immunogold labeling through transmission electron microscopy. Purified flagella were dissociated into monomers in 12% SDS-PAGE Coomassie blue-stained gels showing a band of ~28 kDa and, in addition, mass spectrometry revealed the presence of several peptides that correspond to flagellin. Flagella (100 ng) induced the release of IL-8 (3314–6025 pg/ml), TNF-α (39–359 pg/ml), and IL-10 (2–96 pg/ml), in macrophage isolates from human monocytes and similar results were obtained when flagella were dissociated into monomers. Inhibition assays using three dilutions of anti-flagella antibodies (1:10, 1:100, and 1:200) suppressed the secretion of IL-8, TNF-α, and IL-10 between 95–100% using 100 ng of protein. A transfection assay using 293-hTLR5 cells showed IL-8 release of 197 pg/ml and suppression in the secretion of IL-8 when anti-hTLR5-IgA antibodies were used at different concentrations. These observations suggest that flagella and flagellin are involved in an inflammatory response dependent on TLR5 recognition, which could contribute to the pathogenesis of the bacteria.
The flhDC gene affects motility and biofilm formation in Yersinia pseudotuberculosis
Yao Wang,LiSha Ding,YangBo Hu,Yong Zhang,BaoYu Yang,ShiYun Chen
Science China Life Sciences , 2007, DOI: 10.1007/s11427-007-0101-6
Abstract: The flagella master regulatory gene flhDC of Yersinia pseudotuberculosis serotype III (YPIII) was mutated by deleting the middle region and replaced by a tetracycline resistant gene, and the subsequent mutant strain named YPIIIΔflhDC was obtained. Swimming assay showed that the swimming motility of the mutant strain was completely abolished. The promoter region of the flagella second-class regulatory gene fliA was fused with the lux box, and was conjugated with the mutant and the parent strains respectively for the first cross. LUCY assay result demonstrated that flhDC regulated the expression of fliA in YPIII as reported in E. coli. Biofilm formation of the mutant strain on abiotic and biotic surfaces was observed and quantified. The results showed that mutation of flhDC decreased biofilm formation on both abiotic and biotic surfaces, and abated the infection on Caenorhabdtis elegans. Our results suggest that mutation of the flagella master regulatory gene flhDC not only abolished the swimming motility, but also affected biofilm formation of YPIII on different surfaces. The new function of flhDC identified in this study provides a novel viewpoint for the control of bacterial biofilm formation.
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