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Sample records for coli mg1655 flagellar

  1. Engineering Escherichia coli K12 MG1655 to use starch

    PubMed Central

    2014-01-01

    Background To attain a sustainable bioeconomy, fuel, or valuable product, production must use biomass as substrate. Starch is one of the most abundant biomass resources and is present as waste or as a food and agroindustry by-product. Unfortunately, Escherichia coli, one of the most widely used microorganisms in biotechnological processes, cannot use starch as a carbon source. Results We engineered an E. coli strain capable of using starch as a substrate. The genetic design employed the native capability of the bacterium to use maltodextrins as a carbon source plus expression and secretion of its endogenous α-amylase, AmyA, in an adapted background. Biomass production improved using 35% dissolved oxygen and pH 7.2 in a controlled bioreactor. Conclusion The engineered E. coli strain can use starch from the milieu and open the possibility of optimize the process to use agroindustrial wastes to produce biofuels and other valuable chemicals. PMID:24886307

  2. [The directed modification of Escherichia coli MG1655 to obtain histidine-producing mutants].

    PubMed

    Doroshenko, V G; Lobanov, A O; Fedorina, E A

    2013-01-01

    Strain MG 1655+hisGr hisL'-Delta, purR, which produces histidine with a weight yield of approximately 12% from glucose, was constructed through directed chromosomal modifications of the laboratory Escherichia coli strain MG 1655+, which has a known genome sequence. A feedback-resistant ATP-phosphoribosyl transferase encoded by the mutant hisGr (E271 K) was the main determinant of histidine production. A further increase in histidine production was achieved by the expression enhance of a mutant his operon containing hisGr through the deleting attenuator region (hisL'-Delta). An increase in the expression of the wildtype his operon did not result in histidine accumulation. Deletion of the transcriptional regulator gene purR increased the biomass produced and maintained the level of histidine production per cell under the fermentation conditions used.

  3. The Escherichia coli MG1655 in silico metabolic genotype: Its definition, characteristics, and capabilities

    NASA Astrophysics Data System (ADS)

    Edwards, J. S.; Palsson, B. O.

    2000-05-01

    The Escherichia coli MG1655 genome has been completely sequenced. The annotated sequence, biochemical information, and other information were used to reconstruct the E. coli metabolic map. The stoichiometric coefficients for each metabolic enzyme in the E. coli metabolic map were assembled to construct a genome-specific stoichiometric matrix. The E. coli stoichiometric matrix was used to define the system's characteristics and the capabilities of E. coli metabolism. The effects of gene deletions in the central metabolic pathways on the ability of the in silico metabolic network to support growth were assessed, and the in silico predictions were compared with experimental observations. It was shown that based on stoichiometric and capacity constraints the in silico analysis was able to qualitatively predict the growth potential of mutant strains in 86% of the cases examined. Herein, it is demonstrated that the synthesis of in silico metabolic genotypes based on genomic, biochemical, and strain-specific information is possible, and that systems analysis methods are available to analyze and interpret the metabolic phenotype.

  4. Identification of riboflavin: revealing different metabolic characteristics between Escherichia coli BL21(DE3) and MG1655.

    PubMed

    Wang, Xinran; Wang, Qian; Qi, Qingsheng

    2015-06-01

    There are many physiological differences between Escherichia coli B and K-12 strains, owing to their different origins. Deeper insight into the metabolic and regulative mechanisms of these strains will inform improved usage of these industrial workhorses. In the present study, we observed that BL21 fermentation broth gradually turned yellow during cultivation. By spectral analysis and liquid chromatography-mass spectrometry identification, we confirmed for the first time that the yellow substance accumulated in the fermentation broth is riboflavin. Comparing the enzyme sequences involved in riboflavin metabolism between BL21 and MG1655, we identified a site mutation on the 115 residue of bifunctional riboflavin kinase/FMN adenylyltransferase (RibF) in BL21. This His115Leu mutation was found to reduce enzyme activity to 55% of that of MG1655, which is probably one reason for riboflavin accumulation in BL21. Quantitative PCR analysis showed that genes of the entire branch of the riboflavin and FAD biosynthesis pathways in BL21 were up-regulated. Several physiological and metabolic characteristics of BL21 and MG1655 were found to be different, and may also be related to the riboflavin accumulation.

  5. Identification of riboflavin: revealing different metabolic characteristics between Escherichia coli BL21(DE3) and MG1655.

    PubMed

    Wang, Xinran; Wang, Qian; Qi, Qingsheng

    2015-06-01

    There are many physiological differences between Escherichia coli B and K-12 strains, owing to their different origins. Deeper insight into the metabolic and regulative mechanisms of these strains will inform improved usage of these industrial workhorses. In the present study, we observed that BL21 fermentation broth gradually turned yellow during cultivation. By spectral analysis and liquid chromatography-mass spectrometry identification, we confirmed for the first time that the yellow substance accumulated in the fermentation broth is riboflavin. Comparing the enzyme sequences involved in riboflavin metabolism between BL21 and MG1655, we identified a site mutation on the 115 residue of bifunctional riboflavin kinase/FMN adenylyltransferase (RibF) in BL21. This His115Leu mutation was found to reduce enzyme activity to 55% of that of MG1655, which is probably one reason for riboflavin accumulation in BL21. Quantitative PCR analysis showed that genes of the entire branch of the riboflavin and FAD biosynthesis pathways in BL21 were up-regulated. Several physiological and metabolic characteristics of BL21 and MG1655 were found to be different, and may also be related to the riboflavin accumulation. PMID:25926527

  6. Genome-derived minimal metabolic models for Escherichia coli MG1655 with estimated in vivo respiratory ATP stoichiometry.

    PubMed

    Taymaz-Nikerel, Hilal; Borujeni, Amin Espah; Verheijen, Peter J T; Heijnen, Joseph J; van Gulik, Walter M

    2010-10-01

    Metabolic network models describing growth of Escherichia coli on glucose, glycerol and acetate were derived from a genome scale model of E. coli. One of the uncertainties in the metabolic networks is the exact stoichiometry of energy generating and consuming processes. Accurate estimation of biomass and product yields requires correct information on the ATP stoichiometry. The unknown ATP stoichiometry parameters of the constructed E. coli network were estimated from experimental data of eight different aerobic chemostat experiments carried out with E. coli MG1655, grown at different dilution rates (0.025, 0.05, 0.1, and 0.3 h(-1)) and on different carbon substrates (glucose, glycerol, and acetate). Proper estimation of the ATP stoichiometry requires proper information on the biomass composition of the organism as well as accurate assessment of net conversion rates under well-defined conditions. For this purpose a growth rate dependent biomass composition was derived, based on measurements and literature data. After incorporation of the growth rate dependent biomass composition in a metabolic network model, an effective P/O ratio of 1.49 +/- 0.26 mol of ATP/mol of O, K(X) (growth dependent maintenance) of 0.46 +/- 0.27 mol of ATP/C-mol of biomass and m(ATP) (growth independent maintenance) of 0.075 +/- 0.015 mol of ATP/C-mol of biomass/h were estimated using a newly developed Comprehensive Data Reconciliation (CDR) method, assuming that the three energetic parameters were independent of the growth rate and the used substrate. The resulting metabolic network model only requires the specific rate of growth, micro, as an input in order to accurately predict all other fluxes and yields.

  7. The PurR regulon in Escherichia coli K-12 MG1655.

    PubMed

    Cho, Byung-Kwan; Federowicz, Stephen A; Embree, Mallory; Park, Young-Seoub; Kim, Donghyuk; Palsson, Bernhard Ø

    2011-08-01

    The PurR transcription factor plays a critical role in transcriptional regulation of purine metabolism in enterobacteria. Here, we elucidate the role of PurR under exogenous adenine stimulation at the genome-scale using high-resolution chromatin immunoprecipitation (ChIP)-chip and gene expression data obtained under in vivo conditions. Analysis of microarray data revealed that adenine stimulation led to changes in transcript level of about 10% of Escherichia coli genes, including the purine biosynthesis pathway. The E. coli strain lacking the purR gene showed that a total of 56 genes are affected by the deletion. From the ChIP-chip analysis, we determined that over 73% of genes directly regulated by PurR were enriched in the biosynthesis, utilization and transport of purine and pyrimidine nucleotides, and 20% of them were functionally unknown. Compared to the functional diversity of the regulon of the other general transcription factors in E. coli, the functions and size of the PurR regulon are limited.

  8. Adaptive laboratory evolution of Escherichia coli K-12 MG1655 for growth at high hydrostatic pressure

    PubMed Central

    Marietou, Angeliki; Nguyen, Alice T. T.; Allen, Eric E.; Bartlett, Douglas H.

    2015-01-01

    Much of microbial life on Earth grows and reproduces under the elevated hydrostatic pressure conditions that exist in deep-ocean and deep-subsurface environments. In this study adaptive laboratory evolution (ALE) experiments were conducted to investigate the possible modification of the piezosensitive Escherichia coli for improved growth at high pressure. After approximately 500 generations of selection, a strain was isolated that acquired the ability to grow at pressure non-permissive for the parental strain. Remarkably, this strain displayed growth properties and changes in the proportion and regulation of unsaturated fatty acids that indicated the acquisition of multiple piezotolerant properties. These changes developed concomitantly with a change in the gene encoding the acyl carrier protein, which is required for fatty acid synthesis. PMID:25610434

  9. Global Gene Expression Analysis of Long-Term Stationary Phase Effects in E. coli K12 MG1655

    PubMed Central

    Arunasri, Kotakonda; Adil, Mohammed; Khan, Pathan Akbar Ali; Shivaji, Sisinthy

    2014-01-01

    Global gene expression was monitored in long-term stationary phase (LSP) cells of E. coli K12 MG1655 and compared with stationary phase (SP) cells that were sub-cultured without prolonged delay to get an insight into the survival strategies of LSP cells. The experiments were carried out using both LB medium and LB supplemented with 10% of glycerol. In both the media the LSP cells showed decreased growth rate compared to SP cells. DNA microarray analysis of LSP cells in both the media resulted in the up- and down-regulation of several genes in LSP cells compared to their respective SP cells in the corresponding media. In LSP cells grown in LB 204 genes whereas cells grown in LB plus glycerol 321 genes were differentially regulated compared to the SP cells. Comparison of these differentially regulated genes indicated that irrespective of the medium used for growth in LSP cells expression of 95 genes (22 genes up-regulated and 73 down-regulated) were differentially regulated. These 95 genes could be associated with LSP status of the cells and are likely to influence survival and growth characteristics of LSP cells. This is indeed so since the up- and down-regulated genes include genes that protect E. coli LSP cells from stationary phase stress and genes that would help to recover from stress when transferred into fresh medium. The growth phenotype in LSP cells could be attributed to up-regulation of genes coding for insertion sequences that confer beneficial effects during starvation, genes coding for putative transposases and simultaneous down-regulation of genes coding for ribosomal protein synthesis, transport-related genes, non-coding RNA genes and metabolic genes. As yet we still do not know the role of several unknown genes and genes coding for hypothetical proteins which are either up- or down-regulated in LSP cells compared to SP cells. PMID:24858919

  10. Biochemical characterization of the class B acid phosphatase (AphA) of Escherichia coli MG1655.

    PubMed

    Passariello, Claudio; Forleo, Costantino; Micheli, Vanna; Schippa, Serena; Leone, Rosalida; Mangani, Stefano; Thaller, Maria Cristina; Rossolini, Gian Maria

    2006-01-01

    The AphA enzyme of Escherichia coli, a molecular class B periplasmic phosphatase that belongs to the DDDD superfamily of phosphohydrolases, was purified and subjected to biochemical characterization. Kinetic analysis with several substrates revealed that the enzyme essentially behaves as a broad-spectrum nucleotidase highly active on 3'- and 5'-mononucleotides and monodeoxynucleotides, but not active on cyclic nucleotides, or nucleotides di- and triphosphate. Mononucleotides are degraded to nucleosides, and AphA apparently does not exhibit any nucleotide phosphomutase activity. However, it can transphosphorylate nucleosides in the presence of phosphate donors. Kinetic properties of AphA are consistent with structural data, and suggest a role for the hydrophobic pocket present in the active site crevice, made by residues Phe 56, Leu71, Trp77 and Tyr193, in conferring preferential substrate specificity by accommodating compounds with aromatic rings. AphA was inhibited by several chelating agents, including EDTA, EGTA, 1,10-phenanthroline and dipicolinic acid, with EDTA being apparently the most powerful inhibitor.

  11. Stock culture heterogeneity rather than new mutational variation complicates short-term cell physiology studies of Escherichia coli K-12 MG1655 in continuous culture.

    PubMed

    Nahku, Ranno; Peebo, Karl; Valgepea, Kaspar; Barrick, Jeffrey E; Adamberg, Kaarel; Vilu, Raivo

    2011-09-01

    Nutrient-limited continuous cultures in chemostats have been used to study microbial cell physiology for over 60 years. Genome instability and genetic heterogeneity are possible uncontrolled factors in continuous cultivation experiments. We investigated these issues by using high-throughput (HT) DNA sequencing to characterize samples from different phases of a glucose-limited accelerostat (A-stat) experiment with Escherichia coli K-12 MG1655 and a duration regularly used in cell physiology studies (20 generations of continuous cultivation). Seven consensus mutations from the reference sequence and five subpopulations characterized by different mutations were detected in the HT-sequenced samples. This genetic heterogeneity was confirmed to result from the stock culture by Sanger sequencing. All the subpopulations in which allele frequencies increased (betA, cspG/cspH, glyA) during the experiment were also present at the end of replicate A-stats, indicating that no new subpopulations emerged during our experiments. The fact that ~31 % of the cells in our initial cultures obtained directly from a culture stock centre were mutants raises concerns that even if cultivations are started from single colonies, there is a significant chance of picking a mutant clone with an altered phenotype. Our results show that current HT DNA sequencing technology allows accurate subpopulation analysis and demonstrates that a glucose-limited E. coli K-12 MG1655 A-stat experiment with a duration of tens of generations is suitable for studying cell physiology and collecting quantitative data for metabolic modelling without interference from new mutations.

  12. An Escherichia coli MG1655 Lipopolysaccharide Deep-Rough Core Mutant Grows and Survives in Mouse Cecal Mucus but Fails To Colonize the Mouse Large Intestine

    PubMed Central

    Møller, Annette K.; Leatham, Mary P.; Conway, Tyrrell; Nuijten, Piet J. M.; de Haan, Louise A. M.; Krogfelt, Karen A.; Cohen, Paul S.

    2003-01-01

    The ability of E. coli strains to colonize the mouse large intestine has been correlated with their ability to grow in cecal and colonic mucus. In the present study, an E. coli MG1655 strain was mutagenized with a mini-Tn5 Km (kanamycin) transposon, and mutants were tested for the ability to grow on agar plates with mouse cecal mucus as the sole source of carbon and nitrogen. One mutant, designated MD42 (for mucus defective), grew poorly on cecal-mucus agar plates but grew well on Luria agar plates and on glucose minimal-agar plates. Sequencing revealed that the insertion in MD42 was in the waaQ gene, which is involved in lipopolysaccharide (LPS) core biosynthesis. Like “deep-rough” E. coli mutants, MD42 was hypersensitive to sodium dodecyl sulfate (SDS), bile salts, and the hydrophobic antibiotic novobiocin. Furthermore, its LPS core oligosaccharide was truncated, like that of a deep-rough mutant. MD42 initially grew in the large intestines of streptomycin-treated mice but then failed to colonize (<102 CFU per g of feces), whereas its parent colonized at levels between 107 and 108 CFU per g of feces. When mouse cecal mucosal sections were hybridized with an E. coli-specific rRNA probe, MD42 was observed in cecal mucus as clumps 24 h postfeeding, whereas its parent was present almost exclusively as single cells, suggesting that clumping may play a role in preventing MD42 colonization. Surprisingly, MD42 grew nearly as well as its parent during growth in undiluted, highly viscous cecal mucus isolated directly from the mouse cecum and, like its parent, survived well after reaching stationary phase, suggesting that there are no antimicrobials in mucus that prevent MD42 colonization. After mini-mariner transposon mutagenesis, an SDS-resistant suppressor mutant of MD42 was isolated. The mini-mariner insertion was shown to be in the bipA gene, a known regulator of E. coli surface components. When grown in Luria broth, the LPS core of the suppressor mutant remained

  13. 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

  14. Genome-wide Reconstruction of OxyR and SoxRS Transcriptional Regulatory Networks under Oxidative Stress in Escherichia coli K-12 MG1655.

    PubMed

    Seo, Sang Woo; Kim, Donghyuk; Szubin, Richard; Palsson, Bernhard O

    2015-08-25

    Three transcription factors (TFs), OxyR, SoxR, and SoxS, play a critical role in transcriptional regulation of the defense system for oxidative stress in bacteria. However, their full genome-wide regulatory potential is unknown. Here, we perform a genome-scale reconstruction of the OxyR, SoxR, and SoxS regulons in Escherichia coli K-12 MG1655. Integrative data analysis reveals that a total of 68 genes in 51 transcription units (TUs) belong to these regulons. Among them, 48 genes showed more than 2-fold changes in expression level under single-TF-knockout conditions. This reconstruction expands the genome-wide roles of these factors to include direct activation of genes related to amino acid biosynthesis (methionine and aromatic amino acids), cell wall synthesis (lipid A biosynthesis and peptidoglycan growth), and divalent metal ion transport (Mn(2+), Zn(2+), and Mg(2+)). Investigating the co-regulation of these genes with other stress-response TFs reveals that they are independently regulated by stress-specific TFs.

  15. Genome-wide Reconstruction of OxyR and SoxRS Transcriptional Regulatory Networks under Oxidative Stress in Escherichia coli K-12 MG1655.

    PubMed

    Seo, Sang Woo; Kim, Donghyuk; Szubin, Richard; Palsson, Bernhard O

    2015-08-25

    Three transcription factors (TFs), OxyR, SoxR, and SoxS, play a critical role in transcriptional regulation of the defense system for oxidative stress in bacteria. However, their full genome-wide regulatory potential is unknown. Here, we perform a genome-scale reconstruction of the OxyR, SoxR, and SoxS regulons in Escherichia coli K-12 MG1655. Integrative data analysis reveals that a total of 68 genes in 51 transcription units (TUs) belong to these regulons. Among them, 48 genes showed more than 2-fold changes in expression level under single-TF-knockout conditions. This reconstruction expands the genome-wide roles of these factors to include direct activation of genes related to amino acid biosynthesis (methionine and aromatic amino acids), cell wall synthesis (lipid A biosynthesis and peptidoglycan growth), and divalent metal ion transport (Mn(2+), Zn(2+), and Mg(2+)). Investigating the co-regulation of these genes with other stress-response TFs reveals that they are independently regulated by stress-specific TFs. PMID:26279566

  16. Use of adaptive laboratory evolution to discover key mutations enabling rapid growth of Escherichia coli K-12 MG1655 on glucose minimal medium.

    PubMed

    LaCroix, Ryan A; Sandberg, Troy E; O'Brien, Edward J; Utrilla, Jose; Ebrahim, Ali; Guzman, Gabriela I; Szubin, Richard; Palsson, Bernhard O; Feist, Adam M

    2015-01-01

    Adaptive laboratory evolution (ALE) has emerged as an effective tool for scientific discovery and addressing biotechnological needs. Much of ALE's utility is derived from reproducibly obtained fitness increases. Identifying causal genetic changes and their combinatorial effects is challenging and time-consuming. Understanding how these genetic changes enable increased fitness can be difficult. A series of approaches that address these challenges was developed and demonstrated using Escherichia coli K-12 MG1655 on glucose minimal media at 37°C. By keeping E. coli in constant substrate excess and exponential growth, fitness increases up to 1.6-fold were obtained compared to the wild type. These increases are comparable to previously reported maximum growth rates in similar conditions but were obtained over a shorter time frame. Across the eight replicate ALE experiments performed, causal mutations were identified using three approaches: identifying mutations in the same gene/region across replicate experiments, sequencing strains before and after computationally determined fitness jumps, and allelic replacement coupled with targeted ALE of reconstructed strains. Three genetic regions were most often mutated: the global transcription gene rpoB, an 82-bp deletion between the metabolic pyrE gene and rph, and an IS element between the DNA structural gene hns and tdk. Model-derived classification of gene expression revealed a number of processes important for increased growth that were missed using a gene classification system alone. The methods described here represent a powerful combination of technologies to increase the speed and efficiency of ALE studies. The identified mutations can be examined as genetic parts for increasing growth rate in a desired strain and for understanding rapid growth phenotypes.

  17. Deletion of genes encoding cytochrome oxidases and quinol monooxygenase blocks the aerobic-anaerobic shift in Escherichia coli K-12 MG1655.

    PubMed

    Portnoy, Vasiliy A; Scott, David A; Lewis, Nathan E; Tarasova, Yekaterina; Osterman, Andrei L; Palsson, Bernhard Ø

    2010-10-01

    The constitutive activation of the anoxic redox control transcriptional regulator (ArcA) in Escherichia coli during aerobic growth, with the consequent production of a strain that exhibits anaerobic physiology even in the presence of air, is reported in this work. Removal of three terminal cytochrome oxidase genes (cydAB, cyoABCD, and cbdAB) and a quinol monooxygenase gene (ygiN) from the E. coli K-12 MG1655 genome resulted in the activation of ArcA aerobically. These mutations resulted in reduction of the oxygen uptake rate by nearly 98% and production of d-lactate as a sole by-product under oxic and anoxic conditions. The knockout strain exhibited nearly identical physiological behaviors under both conditions, suggesting that the mutations resulted in significant metabolic and regulatory perturbations. In order to fully understand the physiology of this mutant and to identify underlying metabolic and regulatory reasons that prevent the transition from an aerobic to an anaerobic phenotype, we utilized whole-genome transcriptome analysis, (13)C tracing experiments, and physiological characterization. Our analysis showed that the deletions resulted in the activation of anaerobic respiration under oxic conditions and a consequential shift in the content of the quinone pool from ubiquinones to menaquinones. An increase in menaquinone concentration resulted in the activation of ArcA. The activation of the ArcB/ArcA regulatory system led to a major shift in the metabolic flux distribution through the central metabolism of the mutant strain. Flux analysis indicated that the mutant strain had undetectable fluxes around the tricarboxylic acid (TCA) cycle and elevated flux through glycolysis and anaplerotic input to oxaloacetate. Flux and transcriptomics data were highly correlated and showed similar patterns.

  18. Use of adaptive laboratory evolution to discover key mutations enabling rapid growth of Escherichia coli K-12 MG1655 on glucose minimal medium.

    PubMed

    LaCroix, Ryan A; Sandberg, Troy E; O'Brien, Edward J; Utrilla, Jose; Ebrahim, Ali; Guzman, Gabriela I; Szubin, Richard; Palsson, Bernhard O; Feist, Adam M

    2015-01-01

    Adaptive laboratory evolution (ALE) has emerged as an effective tool for scientific discovery and addressing biotechnological needs. Much of ALE's utility is derived from reproducibly obtained fitness increases. Identifying causal genetic changes and their combinatorial effects is challenging and time-consuming. Understanding how these genetic changes enable increased fitness can be difficult. A series of approaches that address these challenges was developed and demonstrated using Escherichia coli K-12 MG1655 on glucose minimal media at 37°C. By keeping E. coli in constant substrate excess and exponential growth, fitness increases up to 1.6-fold were obtained compared to the wild type. These increases are comparable to previously reported maximum growth rates in similar conditions but were obtained over a shorter time frame. Across the eight replicate ALE experiments performed, causal mutations were identified using three approaches: identifying mutations in the same gene/region across replicate experiments, sequencing strains before and after computationally determined fitness jumps, and allelic replacement coupled with targeted ALE of reconstructed strains. Three genetic regions were most often mutated: the global transcription gene rpoB, an 82-bp deletion between the metabolic pyrE gene and rph, and an IS element between the DNA structural gene hns and tdk. Model-derived classification of gene expression revealed a number of processes important for increased growth that were missed using a gene classification system alone. The methods described here represent a powerful combination of technologies to increase the speed and efficiency of ALE studies. The identified mutations can be examined as genetic parts for increasing growth rate in a desired strain and for understanding rapid growth phenotypes. PMID:25304508

  19. Emergence of Hyper-Resistant Escherichia coli MG1655 Derivative Strains after Applying Sub-Inhibitory Doses of Individual Constituents of Essential Oils

    PubMed Central

    Chueca, Beatriz; Berdejo, Daniel; Gomes-Neto, Nelson J.; Pagán, Rafael; García-Gonzalo, Diego

    2016-01-01

    The improvement of food preservation by using essential oils (EOs) and their individual constituents (ICs) is attracting enormous interest worldwide. Until now, researchers considered that treatments with such antimicrobial compounds did not induce bacterial resistance via a phenotypic (i.e., transient) response. Nevertheless, the emergence of genotypic (i.e., stable) resistance after treatment with these compounds had not been previously tested. Our results confirm that growth of Escherichia coli MG1655 in presence of sub-inhibitory concentrations of the ICs carvacrol, citral, and (+)-limonene oxide do not increase resistance to further treatments with either the same IC (direct resistance) or with other preservation treatments (cross-resistance) such as heat or pulsed electric fields (PEF). Bacterial mutation frequency was likewise lower when those IC's were applied; however, after 10 days of re-culturing cells in presence of sub-inhibitory concentrations of the ICs, we were able to isolate several derivative strains (i.e., mutants) displaying an increased minimum inhibitory concentration to those ICs. Furthermore, when compared to the wild type (WT) strain, they also displayed direct resistance and cross-resistance. Derivative strains selected with carvacrol and citral also displayed morphological changes involving filamentation along with cell counts at late-stationary growth phase that were lower than the WT strain. In addition, co-cultures of each derivative strain with the WT strain resulted in a predominance of the original strain in absence of ICs, indicating that mutants would not out-compete WT cells under optimal growth conditions. Nevertheless, growth in the presence of ICs facilitated the selection of these resistant mutants. Thus, as a result, subsequent food preservation treatments of these bacterial cultures might be less effective than expected for WT cultures. In conclusion, this study recommends that treatment with ICs at sub

  20. 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

  1. Transcriptional and Physiological Characterizations of Escherichia coli MG1655 that have been grown under Low Shear Stress Environment for 1000 Generations

    NASA Astrophysics Data System (ADS)

    Karouia, Fathi; Tirumalai, Madhan R.; Nelman-Gonzalez, Mayra A.; Sams, Clarence F.; Ott, Mark C.; Pierson, Duane L.; Fofanov, Yuriy; Willson, Richard C.; Fox, George E.

    Human space travelers experience a unique environment that affects homeostasis and physio-logic adaptation. One of the important regulatory biology interactions affected by space flight is the alteration of the immune response. As such, the impairment of the immune system may lead to higher risk of bacterial and/or viral infection during human space flight missions. Mi-crobiological contaminants have been a source of concern over the years for NASA and there is evidence to suggest that microbes in space do not behave like they do on Earth. Previ-ous studies have examined the physiological response of bacteria when exposed to short-term microgravity either during spaceflight or in a Low Shear Modeled Microgravity (LSMMG) en-vironment. Exposure to these environments has been found to induce increased resistance to stresses and antibiotics, and in one case increase of virulence. As NASA increases the duration of space flight missions and is starting to envision human presence on the lunar surface and Mars, it becomes legitimate to question the long-term effects of microgravity on bacteria. The effect of long-term exposure to LSMMG on microbial gene expression and physiology in Escherichia coli (E. coli) is being examined using functional genomics, and molecular tech-niques. In previous E. coli short term studies, reproducible changes in transcription were seen but no direct responses to changes in the gravity vector were identified. Instead, absence of shear and a randomized gravity vector appeared to cause local extra-cellular environmental changes, which elicited cellular responses. In order to evaluate the long-term effects of micro-gravity on bacteria, E. coli was grown under simulated microgravity for 1000 generations and gene expression patterns and cellular physiology were analyzed in comparison with short-term exposure. The analysis revealed that the long-term response differed significantly from the short-term exposure and 357 genes were expressed

  2. An individual-based modeling approach to simulate the effects of cellular nutrient competition on Escherichia coli K-12 MG1655 colony behavior and interactions in aerobic structured food systems.

    PubMed

    Tack, Ignace L M M; Logist, Filip; Noriega Fernández, Estefanía; Van Impe, Jan F M

    2015-02-01

    Traditional kinetic models in predictive microbiology reliably predict macroscopic dynamics of planktonically-growing cell cultures in homogeneous liquid food systems. However, most food products have a semi-solid structure, where microorganisms grow locally in colonies. Individual colony cells exhibit strongly different and non-normally distributed behavior due to local nutrient competition. As a result, traditional models considering average population behavior in a homogeneous system do not describe colony dynamics in full detail. To incorporate local resource competition and individual cell differences, an individual-based modeling approach has been applied to Escherichia coli K-12 MG1655 colonies, considering the microbial cell as modeling unit. The first contribution of this individual-based model is to describe single colony growth under nutrient-deprived conditions. More specifically, the linear and stationary phase in the evolution of the colony radius, the evolution from a disk-like to branching morphology, and the emergence of a starvation zone in the colony center are simulated and compared to available experimental data. These phenomena occur earlier at more severe nutrient depletion conditions, i.e., at lower nutrient diffusivity and initial nutrient concentration in the medium. Furthermore, intercolony interactions have been simulated. Higher inoculum densities lead to stronger intercolony interactions, such as colony merging and smaller colony sizes, due to nutrient competition. This individual-based model contributes to the elucidation of characteristic experimentally observed colony behavior from mechanistic information about cellular physiology and interactions.

  3. An individual-based modeling approach to simulate the effects of cellular nutrient competition on Escherichia coli K-12 MG1655 colony behavior and interactions in aerobic structured food systems.

    PubMed

    Tack, Ignace L M M; Logist, Filip; Noriega Fernández, Estefanía; Van Impe, Jan F M

    2015-02-01

    Traditional kinetic models in predictive microbiology reliably predict macroscopic dynamics of planktonically-growing cell cultures in homogeneous liquid food systems. However, most food products have a semi-solid structure, where microorganisms grow locally in colonies. Individual colony cells exhibit strongly different and non-normally distributed behavior due to local nutrient competition. As a result, traditional models considering average population behavior in a homogeneous system do not describe colony dynamics in full detail. To incorporate local resource competition and individual cell differences, an individual-based modeling approach has been applied to Escherichia coli K-12 MG1655 colonies, considering the microbial cell as modeling unit. The first contribution of this individual-based model is to describe single colony growth under nutrient-deprived conditions. More specifically, the linear and stationary phase in the evolution of the colony radius, the evolution from a disk-like to branching morphology, and the emergence of a starvation zone in the colony center are simulated and compared to available experimental data. These phenomena occur earlier at more severe nutrient depletion conditions, i.e., at lower nutrient diffusivity and initial nutrient concentration in the medium. Furthermore, intercolony interactions have been simulated. Higher inoculum densities lead to stronger intercolony interactions, such as colony merging and smaller colony sizes, due to nutrient competition. This individual-based model contributes to the elucidation of characteristic experimentally observed colony behavior from mechanistic information about cellular physiology and interactions. PMID:25500383

  4. A model-driven quantitative metabolomics analysis of aerobic and anaerobic metabolism in E. coli K-12 MG1655 that is biochemically and thermodynamically consistent.

    PubMed

    McCloskey, Douglas; Gangoiti, Jon A; King, Zachary A; Naviaux, Robert K; Barshop, Bruce A; Palsson, Bernhard O; Feist, Adam M

    2014-04-01

    The advent of model-enabled workflows in systems biology allows for the integration of experimental data types with genome-scale models to discover new features of biology. This work demonstrates such a workflow, aimed at establishing a metabolomics platform applied to study the differences in metabolomes between anaerobic and aerobic growth of Escherichia coli. Constraint-based modeling was utilized to deduce a target list of compounds for downstream method development. An analytical and experimental methodology was developed and tailored to the compound chemistry and growth conditions of interest. This included the construction of a rapid sampling apparatus for use with anaerobic cultures. The resulting genome-scale data sets for anaerobic and aerobic growth were validated by comparison to previous small-scale studies comparing growth of E. coli under the same conditions. The metabolomics data were then integrated with the E. coli genome-scale metabolic model (GEM) via a sensitivity analysis that utilized reaction thermodynamics to reconcile simulated growth rates and reaction directionalities. This analysis highlighted several optimal network usage inconsistencies, including the incorrect use of the beta-oxidation pathway for synthesis of fatty acids. This analysis also identified enzyme promiscuity for the pykA gene, that is critical for anaerobic growth, and which has not been previously incorporated into metabolic models of E coli.

  5. The plasticity of global proteome and genome expression analyzed in closely related W3110 and MG1655 strains of a well-studied model organism, Escherichia coli-K12.

    PubMed

    Vijayendran, Chandran; Polen, Tino; Wendisch, Volker F; Friehs, Karl; Niehaus, Karsten; Flaschel, Erwin

    2007-03-10

    The use of Escherichia coli as a model organism has provided a great deal of basic information in biomolecular sciences. Examining trait differences among closely related strains of the same species addresses a fundamental biological question: how much diversity is there at the single species level? The main aim of our research was to identify significant differences in the activities of groups of genes between two laboratory strains of an organism closely related in genome structure. We demonstrate that despite strict and controlled growth conditions, there is high plasticity in the global proteome and genome expression in two closely related E. coli K12 sub-strains (W3110 and MG1655), which differ insignificantly in genome structure. The growth patterns of these two sub-strains were very similar in a well-equipped bioreactor, and their genome structures were shown to be almost identical by DNA microarray. However, detailed profiling of protein and gene expression by 2-dimensional gel electrophoresis and microarray analysis showed many differentially expressed genes and proteins, combinations of which were highly correlated. The differentially regulated genes and proteins belonged to the following functional categories: genes regulated by sigma subunit of RNA polymerase (RpoS), enterobactin-related genes, and genes involved in central metabolism. Genes involved in central cell metabolism - the glycolysis pathway, the tricarboxylic acid cycle and the glyoxylate bypass - were differentially regulated at both the mRNA and proteome levels. The strains differ significantly in central metabolism and thus in the generation of precursor metabolites and energy. This high plasticity probably represents a universal feature of metabolic activities in closely related species, and has the potential to reveal differences in regulatory networks. We suggest that unless care is taken in the choice of strains for any validating experiment, the results might be misleading. PMID

  6. The plasticity of global proteome and genome expression analyzed in closely related W3110 and MG1655 strains of a well-studied model organism, Escherichia coli-K12.

    PubMed

    Vijayendran, Chandran; Polen, Tino; Wendisch, Volker F; Friehs, Karl; Niehaus, Karsten; Flaschel, Erwin

    2007-03-10

    The use of Escherichia coli as a model organism has provided a great deal of basic information in biomolecular sciences. Examining trait differences among closely related strains of the same species addresses a fundamental biological question: how much diversity is there at the single species level? The main aim of our research was to identify significant differences in the activities of groups of genes between two laboratory strains of an organism closely related in genome structure. We demonstrate that despite strict and controlled growth conditions, there is high plasticity in the global proteome and genome expression in two closely related E. coli K12 sub-strains (W3110 and MG1655), which differ insignificantly in genome structure. The growth patterns of these two sub-strains were very similar in a well-equipped bioreactor, and their genome structures were shown to be almost identical by DNA microarray. However, detailed profiling of protein and gene expression by 2-dimensional gel electrophoresis and microarray analysis showed many differentially expressed genes and proteins, combinations of which were highly correlated. The differentially regulated genes and proteins belonged to the following functional categories: genes regulated by sigma subunit of RNA polymerase (RpoS), enterobactin-related genes, and genes involved in central metabolism. Genes involved in central cell metabolism - the glycolysis pathway, the tricarboxylic acid cycle and the glyoxylate bypass - were differentially regulated at both the mRNA and proteome levels. The strains differ significantly in central metabolism and thus in the generation of precursor metabolites and energy. This high plasticity probably represents a universal feature of metabolic activities in closely related species, and has the potential to reveal differences in regulatory networks. We suggest that unless care is taken in the choice of strains for any validating experiment, the results might be misleading.

  7. 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

  8. 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

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

    PubMed

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

    2014-03-01

    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. PMID:24550452

  10. 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.

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

    PubMed

    Adhyapak, Tapan Chandra; Stark, Holger

    2015-01-01

    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. PMID:26651717

  12. 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.

  13. Populational Heterogeneity vs. Temporal Fluctuation in Escherichia coli Flagellar Motor Switching

    PubMed Central

    Bai, Fan; Che, Yong-Suk; Kami-ike, Nobunori; Ma, Qi; Minamino, Tohru; Sowa, Yoshiyuki; Namba, Keiichi

    2013-01-01

    The dynamic switching of the bacterial flagellar motor regulates cell motility in bacterial chemotaxis. It has been reported under physiological conditions that the switching bias of the flagellar motor undergoes large temporal fluctuations, which reflects noise propagating in the chemotactic signaling network. On the other hand, nongenetic heterogeneity is also observed in flagellar motor switching, as a large group of switching motors show different switching bias and frequency under the same physiological condition. In this work, we present simultaneous measurement of groups of Escherichia coli flagellar motor switching and compare them to long time recording of single switching motors. Consistent with previous studies, we observed temporal fluctuations in switching bias in long time recording experiments. However, the variability in switching bias at the populational level showed much higher volatility than its temporal fluctuation. These results suggested stable individuality in E. coli motor switching. We speculate that uneven expression of key regulatory proteins with amplification by the ultrasensitive response of the motor can account for the observed populational heterogeneity and temporal fluctuations. PMID:24209857

  14. 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

  15. Torque generated by the flagellar motor of Escherichia coli.

    PubMed Central

    Berg, H C; Turner, L

    1993-01-01

    Cells of the bacterium Escherichia coli were tethered and spun in a high-frequency rotating electric field at a series of discrete field strengths. This was done first at low field strengths, then at field strengths generating speeds high enough to disrupt motor function, and finally at low field strengths. Comparison of the initial and final speed versus applied-torque plots yielded relative motor torque. For backward rotation, motor torque rose steeply at speeds close to zero, peaking, on average, at about 2.2 times the stall torque. For forward rotation, motor torque remained approximately constant up to speeds of about 60% of the zero-torque speed. Then the torque dropped linearly with speed, crossed zero, and reached a minimum, on average, at about -1.7 times the stall torque. The zero-torque speed increased with temperature (about 90 Hz at 11 degrees C, 140 Hz at 16 degrees C, and 290 Hz at 23 degrees C), while other parameters remained approximately constant. Sometimes the motor slipped at either extreme (delivered constant torque over a range of speeds), but eventually it broke. Similar results were obtained whether motors broke catastrophically (suddenly and completely) or progressively or were de-energized by brief treatment with an uncoupler. These results are consistent with a tightly coupled ratchet mechanism, provided that elastic deformation of force-generating elements is limited by a stop and that mechanical components yield at high applied torques. PMID:8298044

  16. Characterization of Escherichia coli Flagellar Mutants That are Insensitive to Catabolite Repression

    PubMed Central

    Silverman, Michael; Simon, Melvin

    1974-01-01

    In Escherichia coli, the synthesis of the flagellar organelle is sensitive to catabolite repression. Synthesis requires the presence of the cyclic adenosine monophosphate receptor protein (Crp) and 3′,5′-cyclic adenosine monophosphate (cAMP); i.e., mutants that lack Crp or adenylcyclase (Cya) synthesize no flagella. We isolated and characterized a series of mutants (cfs) that restored flagella-forming ability in a Crp strain of E. coli. The mutations in these strains were transferred onto episomes and they were then introduced into a variety of other strains. The presence of the mutation resulted in flagella synthesis in Cya and Crp strains as well as in the wild type grown under conditions of catabolite repression. Deletion analysis and other genetic studies indicated that: (i) the cfs mutations had a dominant effect when they were in the transconfiguration in merodiploids: (ii) they occurred in or very close to the flaI gene: and (iii) their expression required the presence of an intact flaI gene adjacent to the cfs mutation. Biochemical studies showed that the synthesis of at least two flagellar polypeptides, the hook subunit and an amber fragment of flagellin, were absent in strains that carried a cya mutation. Their synthesis was depressed in strains grown under conditions of catabolite repression. The presence of the cfs mutation restored the specific synthesis of these two polypeptides. We suggest that the formation of the flaI gene product is the step in flagellar synthesis that is catabolite sensitive and requires cAMP. We propose a regulatory function for the product of the flaI gene. Images PMID:4373438

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

    PubMed

    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; Gomes, Tânia A T

    2016-04-01

    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 fliCa nd 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 a EPEC strain 1711-4 to Caco-2 cells was drastically reduced after the treatment of Caco-2 cells with purified FliD. In addition, incubation of a EPEC 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 a EPEC 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 a EPEC to human enterocytes and disclosed the relevant structural and functional involvement of FliD in the adhesion process. PMID:26831466

  18. 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

  19. DksA and ppGpp Directly Regulate Transcription of the Escherichia coli Flagellar Cascade

    PubMed Central

    Lemke, Justin J.; Durfee, Tim; Gourse, Richard L.

    2009-01-01

    The components of the Escherichia coli flagella apparatus are synthesized in a three-level transcriptional cascade activated by the master regulator FlhDC. The cascade coordinates the synthesis rates of a large number of gene products with each other and with nutritional conditions. Recent genome-wide studies have reported that flagellar transcription is altered in cells lacking the transcription regulators DksA or ppGpp, but some or all reported effects could be indirect, and some are contradictory. We report here that the activities of promoters at all three levels of the cascade are much higher in strains lacking dksA, resulting in overproduction of flagellin and hyperflagellated cells. In vitro, DksA/ppGpp inhibits the flhDC promoter and the σ70-dependent fliA promoter transcribing the gene for σ28. However, DksA and ppGpp do not affect the σ28-dependent fliA promoter or the σ28-dependent fliC promoter in vitro, suggesting that the dramatic effects on expression of those genes in vivo are mediated indirectly through direct effects of DksA/ppGpp on FlhDC and σ28 expression. We conclude that DksA/ppGpp inhibits expression of the flagellar cascade during stationary phase and following starvation, thereby coordinating flagella and ribosome assembly and preventing expenditure of scarce energy resources on synthesis of two of the cell’s largest macromolecular complexes. PMID:19889089

  20. Fluctuations in rotation rate of the flagellar motor of Escherichia coli.

    PubMed Central

    Kara-Ivanov, M; Eisenbach, M; Caplan, S R

    1995-01-01

    The purpose of this work was to study the changes in rotation rate of the bacterial motor and to try to discriminate between various sources of these changes with the aim of understanding the mechanism of force generation better. To this end Escherichia coli cells were tethered and videotaped with brief stroboscopic light flashes. The records were scanned by means of a computerized motion analysis system, yielding cell size, radius of rotation, and accumulated angle of rotation as functions of time for each cell selected. In conformity with previous studies, fluctuations in the rotation rate of the flagellar motor were invariably found. Employing an exclusively counterclockwise rotating mutant ("gutted" RP1091 strain) and using power spectral density, autocorrelation and residual mean square angle analysis, we found that a simple superposition of rotational diffusion on a steady rotary motion is insufficient to describe the observed rotation. We observed two additional rotational components, one fluctuating (0.04-0.6 s) and one oscillating (0.8-7 s). However, the effective rotational diffusion coefficient obtained after taking these two components into account generally exceeded that calculated from external friction by two orders of magnitude. This is consistent with a model incorporating association and dissociation of force-generating units. PMID:7669902

  1. Estimation of the adhesive force distribution for the flagellar adhesion of Escherichia coli on a glass surface.

    PubMed

    Yoshihara, Akinori; Nobuhira, Noritaka; Narahara, Hisaya; Toyoda, Syunsuke; Tokumoto, Hayato; Konishi, Yasuhiro; Nomura, Toshiyuki

    2015-07-01

    The effects of the presence or absence of microbial flagella and the microbial motility on the colloidal behaviors of microbial cells were quantitatively evaluated. The microbial cell attachment and detachment processes on a glass surface were observed directly using a parallel-plate flow chamber. Wild-type, flagellar paralyzed, and nonflagellated Escherichia coli strains were used as model microbial cells. In the cell attachment tests, the microbial adhesion rate in a 160mM NaCl solution was approximately 10 times higher than that in a 10mM solution, for all E. coli strains. The colloidal behavior of the microbial cells agreed well with the predictions of the DLVO theory. In addition, the microbial flagella and motility did not significantly affect the cell attachment, regardless of the existence of a potential barrier between the cell and the glass substratum. In the cell detachment tests, the cumulative number of microbial cells detached from the glass substratum with increasing flow rate was fit well with the Weibull distribution function. The list of strains arranged in order of increasing median drag force required to remove them was nonflagellated strain, flagellar paralyzed strain, and wild-type strain. These results indicated that the flagella and the flagellar motility inhibited the cell detachment from the glass substratum. Furthermore, a large external force would likely be required to inhibit the microbial adhesion in the early stage of the biofilm formation.

  2. Draft Genome Sequences of 14 Escherichia coli Phages Isolated from Cattle Slurry.

    PubMed

    Smith, R; O'Hara, M; Hobman, J L; Millard, A D

    2015-01-01

    The diversity of bacteriophages in slurry from dairy cows remains largely unknown. Here, we report the draft genome sequences of 14 bacteriophages isolated from dairy cow slurry using Escherichia coli K-12 MG1655 as a host. PMID:26722010

  3. Detection of flagellar antigen of Campylobacter jejuni and Campylobacter coli in canine faeces with an enzyme-linked immunosorbent assay (ELISA)--new prospects for diagnosis.

    PubMed

    Monfort, J D; Bech-Nielsen, S; Stills, H F

    1994-01-01

    A new diagnostic procedure was developed to detect the flagellar antigen of Campylobacter jejuni and Campylobacter coli in canine faecal specimens and was tested on faecal samples from random-source dogs obtained from the local dog pound. Extraction of acid-soluble proteins was performed on faecal specimens and the extracted material was evaluated using species-specific monoclonal antibodies in an enzyme-linked immunosorbent assay. The assay detected all C. jejuni or C. coli infected specimens compared with direct selective faecal culture. One of 18 faecal specimens culture-negative for C. jejuni was identified as positive by the assay, i.e. a false positive rate of 1 of 18 (5.6%) and a corresponding specificity of 94.4%. These results suggest that the screening procedure developed to detect flagellar antigens of C. jejuni and C. coli in canine faecal samples should be further investigated as a diagnostic alternative to culture.

  4. pH regulates genes for flagellar motility, catabolism, and oxidative stress in Escherichia coli K-12.

    PubMed

    Maurer, Lisa M; Yohannes, Elizabeth; Bondurant, Sandra S; Radmacher, Michael; Slonczewski, Joan L

    2005-01-01

    Gene expression profiles of Escherichia coli K-12 W3110 were compared as a function of steady-state external pH. Cultures were grown to an optical density at 600 nm of 0.3 in potassium-modified Luria-Bertani medium buffered at pH 5.0, 7.0, and 8.7. For each of the three pH conditions, cDNA from RNA of five independent cultures was hybridized to Affymetrix E. coli arrays. Analysis of variance with an alpha level of 0.001 resulted in 98% power to detect genes showing a twofold difference in expression. Normalized expression indices were calculated for each gene and intergenic region (IG). Differential expression among the three pH classes was observed for 763 genes and 353 IGs. Hierarchical clustering yielded six well-defined clusters of pH profiles, designated Acid High (highest expression at pH 5.0), Acid Low (lowest expression at pH 5.0), Base High (highest at pH 8.7), Base Low (lowest at pH 8.7), Neutral High (highest at pH 7.0, lower in acid or base), and Neutral Low (lowest at pH 7.0, higher at both pH extremes). Flagellar and chemotaxis genes were repressed at pH 8.7 (Base Low cluster), where the cell's transmembrane proton potential is diminished by the maintenance of an inverted pH gradient. High pH also repressed the proton pumps cytochrome o (cyo) and NADH dehydrogenases I and II. By contrast, the proton-importing ATP synthase F1Fo and the microaerophilic cytochrome d (cyd), which minimizes proton export, were induced at pH 8.7. These observations are consistent with a model in which high pH represses synthesis of flagella, which expend proton motive force, while stepping up electron transport and ATPase components that keep protons inside the cell. Acid-induced genes, on the other hand, were coinduced by conditions associated with increased metabolic rate, such as oxidative stress. All six pH-dependent clusters included envelope and periplasmic proteins, which directly experience external pH. Overall, this study showed that (i) low pH accelerates acid

  5. 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

  6. 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

  7. An Escherichia coli Nissle 1917 missense mutant colonizes the streptomycin-treated mouse intestine better than the wild type but is not a better probiotic.

    PubMed

    Adediran, Jimmy; Leatham-Jensen, Mary P; Mokszycki, Matthew E; Frimodt-Møller, Jakob; Krogfelt, Karen A; Kazmierczak, Krystyna; Kenney, Linda J; Conway, Tyrrell; Cohen, Paul S

    2014-02-01

    Previously we reported that the streptomycin-treated mouse intestine selected for two different Escherichia coli MG1655 mutants with improved colonizing ability: nonmotile E. coli MG1655 flhDC deletion mutants that grew 15% faster in vitro in mouse cecal mucus and motile E. coli MG1655 envZ missense mutants that grew slower in vitro in mouse cecal mucus yet were able to cocolonize with the faster-growing flhDC mutants. The E. coli MG1655 envZ gene encodes a histidine kinase that is a member of the envZ-ompR two-component signal transduction system, which regulates outer membrane protein profiles. In the present investigation, the envZP41L gene was transferred from the intestinally selected E. coli MG1655 mutant to E. coli Nissle 1917, a human probiotic strain used to treat gastrointestinal infections. Both the E. coli MG1655 and E. coli Nissle 1917 strains containing envZP41L produced more phosphorylated OmpR than their parents. The E. coli Nissle 1917 strain containing envZP41L also became more resistant to bile salts and colicin V and grew 50% slower in vitro in mucus and 15% to 30% slower on several sugars present in mucus, yet it was a 10-fold better colonizer than E. coli Nissle 1917. However, E. coli Nissle 1917 envZP41L was not better at preventing colonization by enterohemorrhagic E. coli EDL933. The data can be explained according to our "restaurant" hypothesis for commensal E. coli strains, i.e., that they colonize the intestine as sessile members of mixed biofilms, obtaining the sugars they need for growth locally, but compete for sugars with invading E. coli pathogens planktonically. PMID:24478082

  8. An Escherichia coli Nissle 1917 missense mutant colonizes the streptomycin-treated mouse intestine better than the wild type but is not a better probiotic.

    PubMed

    Adediran, Jimmy; Leatham-Jensen, Mary P; Mokszycki, Matthew E; Frimodt-Møller, Jakob; Krogfelt, Karen A; Kazmierczak, Krystyna; Kenney, Linda J; Conway, Tyrrell; Cohen, Paul S

    2014-02-01

    Previously we reported that the streptomycin-treated mouse intestine selected for two different Escherichia coli MG1655 mutants with improved colonizing ability: nonmotile E. coli MG1655 flhDC deletion mutants that grew 15% faster in vitro in mouse cecal mucus and motile E. coli MG1655 envZ missense mutants that grew slower in vitro in mouse cecal mucus yet were able to cocolonize with the faster-growing flhDC mutants. The E. coli MG1655 envZ gene encodes a histidine kinase that is a member of the envZ-ompR two-component signal transduction system, which regulates outer membrane protein profiles. In the present investigation, the envZP41L gene was transferred from the intestinally selected E. coli MG1655 mutant to E. coli Nissle 1917, a human probiotic strain used to treat gastrointestinal infections. Both the E. coli MG1655 and E. coli Nissle 1917 strains containing envZP41L produced more phosphorylated OmpR than their parents. The E. coli Nissle 1917 strain containing envZP41L also became more resistant to bile salts and colicin V and grew 50% slower in vitro in mucus and 15% to 30% slower on several sugars present in mucus, yet it was a 10-fold better colonizer than E. coli Nissle 1917. However, E. coli Nissle 1917 envZP41L was not better at preventing colonization by enterohemorrhagic E. coli EDL933. The data can be explained according to our "restaurant" hypothesis for commensal E. coli strains, i.e., that they colonize the intestine as sessile members of mixed biofilms, obtaining the sugars they need for growth locally, but compete for sugars with invading E. coli pathogens planktonically.

  9. Mutations upregulating the flhDC operon of Escherichia coli K-12.

    PubMed

    Lee, Changhan; Park, Chankyu

    2013-02-01

    Bacterial motility is governed by the flhDC master operon that is under the control of factors like OmpR, LrhA, HdfR, and H-NS. Previously, derivatives of the wild-type MG1655 strain of E. coli K-12 with enhanced motility were found to contain insertion sequences (ISs) in the regulatory region of the flhDC operon. Here, we report that not only integrations of IS insertion sequences into the regulatory region of the flhDC operon, but also a missense mutation in the lrhA gene enhances motility by relieving transcriptional repression of the flhDC operon. Two novel IS insertions were found upstream of flhDC. So far, the relationships between the trans- acting factors and the cis-acting regulatory sequences associated with the flhDC operon have not been clearly established. In this study, it was found that effects of the cis- and trans-acting mutations were acting in parallel, suggesting their apparently independent regulation of flagellar expression.

  10. 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.

  11. 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

  12. 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

  13. [Construction of the new Escherichia coli K-12 wild-type strain with improved growth characteristics for application in metabolic engineering].

    PubMed

    Biriukova, I V; Krylov, A A; Kiseleva, E M; Minaeva, N I; Mashko, S V

    2010-03-01

    MG1655 of Escherichia coli K-12 is frequently used in metabolic engineering as the wild-type strain. However, its two mutations, ilvG and rph-1 provide a negative effect on culture growth. The "polar effect" of rph-1 decreases the level of pyrE expression, causing partial auxotrophy for pyrimidines. Mutation ilvG leading to the appearance of Val(S) phenotype causes retardation of cell growth rate on media containing amino acids. In this work, the substitution of two loci in the genome of MG1655 with the recovery of the wild-type phenotype was accomplished. Gene rph(wt) from the chromosome of E. coli TG1 was marked via Red-dependent integration of DNA fragment carrying lambda attL-Cm(R)-lambda attR and transduced with phage P1 into MG1655; later, the Cm(R) marker was removed with the use of lambda Xis/Int recombinase. Parallel to this procedure, a spontaneous Val(R) mutant of E. coli MG1655 yielding colonies of maximal size on M9 medium with glucose in the presence of Val (50 microg/ml) was isolated. It was shown that a nucleotide deletion in the isolated Val(R) strain had been generated in the region of the identified E. coli K-12 ilvG mutation, which led to the recovery of the reading frame and active protein synthesis. This mutation named ilvG-15, which is the only reason for the Val(R) phenotype in the obtained strain, was transferred to MG1655-rph(wt) using cotransduction, by analogy to the transfer of rph(wt). Evaluation of rates of aerobically growing cells (microm, hour(-1)) on M9 medium with glucose produced the following values: 0.56, 0.69, and 0.73 for strains MG1655, MG1655-rph(wt), and MG1655-(rph(wt), ilvG-15), respectively.

  14. Influence of cyclopropane fatty acids on heat, high pressure, acid and oxidative resistance in Escherichia coli.

    PubMed

    Chen, Yuan Yao; Gänzle, Michael G

    2016-04-01

    Heat and high pressure resistant strains of Escherichia coli are a challenge to food safety. This study investigated effects of cyclopropane fatty acids (CFAs) on stress tolerance in the heat- and pressure-resistant strain E. coli AW1.7 and the sensitive strain E. coli MG1655. The role of CFAs was explored by disruption of cfa coding for CFA synthase with an in-frame, unmarked deletion method. Both wild-type strains consumed all the unsaturated fatty acids (C16:1 and C18:1) that were mostly converted to CFAs and a low proportion to saturated fatty acid (C16:0). Moreover, E. coli AW1.7 contained a higher proportion of membrane C19:0 cyclopropane fatty acid than E. coli MG1655 (P<0.05). The Δcfa mutant strains did not produce CFAs, and the corresponding substrates C16:1 and C18:1 accumulated in membrane lipids. The deletion of cfa did not alter resistance to H2O2 but increased the lethality of heat, high pressure and acid treatments in E. coli AW1.7, and E. coli MG1655. E. coli AW1.7 and its Δcfa mutant were more resistant to pressure and heat but less resistant to acid stress than E. coli MG1655. Heat resistance of wild-type strains and their Δcfa mutant was also assessed in beef patties grilled to an internal temperature of 71 °C. After treatment, cell counts of wild type strains were higher than those of the Δcfa mutant strains. In conclusion, CFA synthesis in E. coli increases heat, high pressure and acid resistance, and increases heat resistance in food. This knowledge on mechanisms of stress resistance will facilitate the design of intervention methods for improved pathogen control in food production.

  15. Protective role of E. coli outer membrane vesicles against antibiotics.

    PubMed

    Kulkarni, Heramb M; Nagaraj, R; Jagannadham, Medicharla V

    2015-12-01

    The outer membrane vesicles (OMVs) from bacteria are known to posses both defensive and protective functions and thus participate in community related functions. In the present study, outer membrane vesicles have been shown to protect the producer bacterium and two other bacterial species from the growth inhibitory effects of some antibiotics. The OMVs isolated from E. coli MG1655 protected the bacteria against membrane-active antibiotics colistin, melittin. The OMVs of E. coli MG1655 could also protect P. aeruginosa NCTC6751 and A. radiodioresistens MMC5 against these membrane-active antibiotics. However, OMVs could not protect any of these bacteria against the other antibiotics ciprofloxacin, streptomycin and trimethoprim. Hence, OMVs appears to protect the bacterial community against membrane-active antibiotics and not other antibiotics, which have different mechanism of actions. The OMVs of E. coli MG1655 sequester the antibiotic colistin, whereas their protein components degrade the antimicrobial peptide melittin. Proteomic analysis of OMVs revealed the presence of proteases and peptidases which appear to be involved in this process. Thus, the protection of bacteria by OMVs against antibiotics is situation dependent and the mechanism differs for different situations. These studies suggest that OMVs of bacteria form a common defense for the bacterial community against specific antibiotics. PMID:26640046

  16. Protective role of E. coli outer membrane vesicles against antibiotics.

    PubMed

    Kulkarni, Heramb M; Nagaraj, R; Jagannadham, Medicharla V

    2015-12-01

    The outer membrane vesicles (OMVs) from bacteria are known to posses both defensive and protective functions and thus participate in community related functions. In the present study, outer membrane vesicles have been shown to protect the producer bacterium and two other bacterial species from the growth inhibitory effects of some antibiotics. The OMVs isolated from E. coli MG1655 protected the bacteria against membrane-active antibiotics colistin, melittin. The OMVs of E. coli MG1655 could also protect P. aeruginosa NCTC6751 and A. radiodioresistens MMC5 against these membrane-active antibiotics. However, OMVs could not protect any of these bacteria against the other antibiotics ciprofloxacin, streptomycin and trimethoprim. Hence, OMVs appears to protect the bacterial community against membrane-active antibiotics and not other antibiotics, which have different mechanism of actions. The OMVs of E. coli MG1655 sequester the antibiotic colistin, whereas their protein components degrade the antimicrobial peptide melittin. Proteomic analysis of OMVs revealed the presence of proteases and peptidases which appear to be involved in this process. Thus, the protection of bacteria by OMVs against antibiotics is situation dependent and the mechanism differs for different situations. These studies suggest that OMVs of bacteria form a common defense for the bacterial community against specific antibiotics.

  17. 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.

  18. 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.

  19. Evolution of Escherichia coli to 42 °C and subsequent genetic engineering reveals adaptive mechanisms and novel mutations.

    PubMed

    Sandberg, Troy E; Pedersen, Margit; LaCroix, Ryan A; Ebrahim, Ali; Bonde, Mads; Herrgard, Markus J; Palsson, Bernhard O; Sommer, Morten; Feist, Adam M

    2014-10-01

    Adaptive laboratory evolution (ALE) has emerged as a valuable method by which to investigate microbial adaptation to a desired environment. Here, we performed ALE to 42 °C of ten parallel populations of Escherichia coli K-12 MG1655 grown in glucose minimal media. Tightly controlled experimental conditions allowed selection based on exponential-phase growth rate, yielding strains that uniformly converged toward a similar phenotype along distinct genetic paths. Adapted strains possessed as few as 6 and as many as 55 mutations, and of the 144 genes that mutated in total, 14 arose independently across two or more strains. This mutational recurrence pointed to the key genetic targets underlying the evolved fitness increase. Genome engineering was used to introduce the novel ALE-acquired alleles in random combinations into the ancestral strain, and competition between these engineered strains reaffirmed the impact of the key mutations on the growth rate at 42 °C. Interestingly, most of the identified key gene targets differed significantly from those found in similar temperature adaptation studies, highlighting the sensitivity of genetic evolution to experimental conditions and ancestral genotype. Additionally, transcriptomic analysis of the ancestral and evolved strains revealed a general trend for restoration of the global expression state back toward preheat stressed levels. This restorative effect was previously documented following evolution to metabolic perturbations, and thus may represent a general feature of ALE experiments. The widespread evolved expression shifts were enabled by a comparatively scant number of regulatory mutations, providing a net fitness benefit but causing suboptimal expression levels for certain genes, such as those governing flagellar formation, which then became targets for additional ameliorating mutations. Overall, the results of this study provide insight into the adaptation process and yield lessons important for the future

  20. Campylobacter jejuni Increases Flagellar Expression and Adhesion of Noninvasive Escherichia coli: Effects on Enterocytic Toll-Like Receptor 4 and CXCL-8 Expression

    PubMed Central

    Reti, Kristen L.; Tymensen, Lisa D.; Davis, Shevaun P.; Amrein, Matthias W.

    2015-01-01

    Campylobacter jejuni is the most common cause of bacterium-induced gastroenteritis, and while typically self-limiting, C. jejuni infections are associated with postinfectious intestinal disorders, including flares in patients with inflammatory bowel disease and postinfectious irritable bowel syndrome (PI-IBS), via mechanisms that remain obscure. Based on the hypothesis that acute campylobacteriosis may cause pathogenic microbiota dysbiosis, we investigated whether C. jejuni may activate dormant virulence genes in noninvasive Escherichia coli and examined the epithelial pathophysiological consequences of these alterations. Microarray and quantitative real-time PCR analyses revealed that E. coli adhesin, flagellum, and hemolysin gene expression were increased when E. coli was exposed to C. jejuni-conditioned medium. Increased development of bacterial flagella upon exposure to live C. jejuni or C. jejuni-conditioned medium was observed under transmission electron microscopy. Atomic force microscopy demonstrated that the forces of bacterial adhesion to colonic T84 enterocytes, and the work required to rupture this adhesion, were significantly increased in E. coli exposed to C. jejuni-conditioned media. Finally, C. jejuni-modified E. coli disrupted TLR4 gene expression and induced proinflammatory CXCL-8 gene expression in colonic enterocytes. Together, these data suggest that exposure to live C. jejuni, and/or to its secretory-excretory products, may activate latent virulence genes in noninvasive E. coli and that these alterations may directly trigger proinflammatory signaling in intestinal epithelia. These observations shed new light on mechanisms that may contribute, at least in part, to postcampylobacteriosis inflammatory disorders. PMID:26371123

  1. Optimisation of engineered Escherichia coli biofilms for enzymatic biosynthesis of l-halotryptophans

    PubMed Central

    2013-01-01

    Engineered biofilms comprising a single recombinant species have demonstrated remarkable activity as novel biocatalysts for a range of applications. In this work, we focused on the biotransformation of 5-haloindole into 5-halotryptophan, a pharmaceutical intermediate, using Escherichia coli expressing a recombinant tryptophan synthase enzyme encoded by plasmid pSTB7. To optimise the reaction we compared two E. coli K-12 strains (MC4100 and MG1655) and their ompR234 mutants, which overproduce the adhesin curli (PHL644 and PHL628). The ompR234 mutation increased the quantity of biofilm in both MG1655 and MC4100 backgrounds. In all cases, no conversion of 5-haloindoles was observed using cells without the pSTB7 plasmid. Engineered biofilms of strains PHL628 pSTB7 and PHL644 pSTB7 generated more 5-halotryptophan than their corresponding planktonic cells. Flow cytometry revealed that the vast majority of cells were alive after 24 hour biotransformation reactions, both in planktonic and biofilm forms, suggesting that cell viability was not a major factor in the greater performance of biofilm reactions. Monitoring 5-haloindole depletion, 5-halotryptophan synthesis and the percentage conversion of the biotransformation reaction suggested that there were inherent differences between strains MG1655 and MC4100, and between planktonic and biofilm cells, in terms of tryptophan and indole metabolism and transport. The study has reinforced the need to thoroughly investigate bacterial physiology and make informed strain selections when developing biotransformation reactions. PMID:24188712

  2. Comparative reaction engineering studies for succinic acid production from sucrose by metabolically engineered Escherichia coli in fed-batch-operated stirred tank bioreactors.

    PubMed

    Hoefel, Torben; Faust, Georg; Reinecke, Liv; Rudinger, Nicolas; Weuster-Botz, Dirk

    2012-10-01

    This study presents a comparative reaction engineering analysis of metabolically engineered sucrose-utilizing Escherichia coli derived from E. coli K12 MG1655 for the anaerobic production of succinic acid. Production capacities of 16 different recombinant strains were evaluated in 48 parallel fed-batch-operated milliliter-scale stirred tank bioreactors (10 mL) with continuous CO₂ sparging. The effects of recombinant sucrose-utilization systems (csc-operon or scr-operon), enhancements of anaplerotic reactions (pck, ppc, maeA, maeB or heterologous pyc) and gene deletions (ldhA, adhE, ack-pta and ptsG) were studied with respect to the overall process performances of the respective recombinant strains. Both sucrose-utilization systems enabled the production of succinic acid from sucrose in E. coli K12 MG1655. Maximum succinate production was observed by overexpressing the pyruvate carboxylase from Corynebacterium glutamicum resulting in a succinate concentration of 26.8 g L⁻¹ after 48 h and a cell-specific productivity of 0.14 g g⁻¹ h⁻¹. Further experiments in a fed-batch-operated laboratory-scale stirred tank bioreactor (2 L) showed that micro-aerobic conditions preceding the anaerobic phase enhance succinic acid production of E. coli K12 MG1655-derived strains. The work demonstrates the importance of parallel approaches within the scope of applied metabolic engineering studies.

  3. FLAGELLAR REGENERATION IN PROTOZOAN FLAGELLATES

    PubMed Central

    Rosenbaum, Joel L.; Child, F. M.

    1967-01-01

    The flagella of populations of three protozoan species (Ochromonas, Euglena, and Astasia) were amputated and allowed to regenerate. The kinetics of regeneration in all species were characterized by a lag phase during which there was no apparent flagellar elongation; this phase was followed by elongation at a rate which constantly decelerated as the original length was regained. Inhibition by cycloheximide applied at the time of flagellar amputation showed that flagellar regeneration was dependent upon de novo protein synthesis. This was supported by evidence showing that a greater amount of leucine was incorporated into the proteins of regenerating than nonregenerating flagella. The degree of inhibition of flagellar elongation observed with cycloheximide depended on how soon after flagellar amputation it was applied: when applied to cells immediately following amputation, elongation was almost completely inhibited, but its application at various times thereafter permitted considerable elongation to occur prior to complete inhibition of flagellar elongation. Hence, a sufficient number of precursors were synthesized and accumulated prior to addition of cycloheximide so that their assembly (elongation) could occur for a time under conditions in which protein synthesis had been inhibited. Evidence that the site of this assembly may be at the tip of the elongating flagellum was obtained from radioautographic studies in which the flagella of Ochromonas were permitted to regenerate part way in the absence of labeled leucine and to complete their regeneration in the presence of the isotope. Possible mechanisms which may be operating to control flagellar regeneration are discussed in light of these and other observations. PMID:6033540

  4. Determining the Relative Contribution and Hierarchy of hha and qseBC in the Regulation of Flagellar Motility of Escherichia coli O157:H7

    PubMed Central

    Sharma, Vijay K.; Casey, Thomas A.

    2014-01-01

    In recent studies, we demonstrated that a deletion of hha caused increased secretion of locus of enterocyte encoded adherence proteins and reduced motility of enterohemorrhagic Escherichia coli (EHEC) O157:H7. In addition to the importance of hha in positive regulation of motility, a two-component quorum sensing pathway encoded by the qseBC genes has been shown to activate bacterial motility in response to mammalian stress hormones epinephrine and norepinephrine as well as bacterially produced autoinducer-3. In this study, we compared regulatory contribution and hierarchy of hha, a member of the Hha/YmoA family of nucleoid-associated proteins, to that of qseBC in the expression of EHEC O157:H7 motility. Since norepinephrine affects motility of EHEC O157:H7 through a qseBC-encoded two-component quorum sensing signaling, we also determined whether the hha-mediated regulation of motility is affected by norepinephrine and whether this effect is qseBC dependent. We used single (Δhha or ΔqseC) and double (Δhha ΔqseC) deletion mutants to show that hha exerts a greater positive regulatory effect in comparison to qseBC on the expression of motility by EHEC O157:H7. We also show that Hha is hierarchically superior in transcriptional regulation of motility than QseBC because transcription of qseC was significantly reduced in the hha deletion mutant compared to that in the parental and the hha-complemented mutant strains. These results suggest that hha regulates motility of EHEC O157:H7 directly as well as indirectly by controlling the transcription of qseBC. PMID:24465756

  5. Escherichia coli EDL933 requires gluconeogenic nutrients to successfully colonize the intestines of streptomycin-treated mice precolonized with E. coli Nissle 1917.

    PubMed

    Schinner, Silvia A C; Mokszycki, Matthew E; Adediran, Jimmy; Leatham-Jensen, Mary; Conway, Tyrrell; Cohen, Paul S

    2015-05-01

    Escherichia coli MG1655, a K-12 strain, uses glycolytic nutrients exclusively to colonize the intestines of streptomycin-treated mice when it is the only E. coli strain present or when it is confronted with E. coli EDL933, an O157:H7 strain. In contrast, E. coli EDL933 uses glycolytic nutrients exclusively when it is the only E. coli strain in the intestine but switches in part to gluconeogenic nutrients when it colonizes mice precolonized with E. coli MG1655 (R. L. Miranda et al., Infect Immun 72:1666-1676, 2004, http://dx.doi.org/10.1128/IAI.72.3.1666-1676.2004). Recently, J. W. Njoroge et al. (mBio 3:e00280-12, 2012, http://dx.doi.org/10.1128/mBio.00280-12) reported that E. coli 86-24, an O157:H7 strain, activates the expression of virulence genes under gluconeogenic conditions, suggesting that colonization of the intestine with a probiotic E. coli strain that outcompetes O157:H7 strains for gluconeogenic nutrients could render them nonpathogenic. Here we report that E. coli Nissle 1917, a probiotic strain, uses both glycolytic and gluconeogenic nutrients to colonize the mouse intestine between 1 and 5 days postfeeding, appears to stop using gluconeogenic nutrients thereafter in a large, long-term colonization niche, but continues to use them in a smaller niche to compete with invading E. coli EDL933. Evidence is also presented suggesting that invading E. coli EDL933 uses both glycolytic and gluconeogenic nutrients and needs the ability to perform gluconeogenesis in order to colonize mice precolonized with E. coli Nissle 1917. The data presented here therefore rule out the possibility that E. coli Nissle 1917 can starve the O157:H7 E. coli strain EDL933 of gluconeogenic nutrients, even though E. coli Nissle 1917 uses such nutrients to compete with E. coli EDL933 in the mouse intestine. PMID:25733524

  6. Escherichia coli EDL933 requires gluconeogenic nutrients to successfully colonize the intestines of streptomycin-treated mice precolonized with E. coli Nissle 1917.

    PubMed

    Schinner, Silvia A C; Mokszycki, Matthew E; Adediran, Jimmy; Leatham-Jensen, Mary; Conway, Tyrrell; Cohen, Paul S

    2015-05-01

    Escherichia coli MG1655, a K-12 strain, uses glycolytic nutrients exclusively to colonize the intestines of streptomycin-treated mice when it is the only E. coli strain present or when it is confronted with E. coli EDL933, an O157:H7 strain. In contrast, E. coli EDL933 uses glycolytic nutrients exclusively when it is the only E. coli strain in the intestine but switches in part to gluconeogenic nutrients when it colonizes mice precolonized with E. coli MG1655 (R. L. Miranda et al., Infect Immun 72:1666-1676, 2004, http://dx.doi.org/10.1128/IAI.72.3.1666-1676.2004). Recently, J. W. Njoroge et al. (mBio 3:e00280-12, 2012, http://dx.doi.org/10.1128/mBio.00280-12) reported that E. coli 86-24, an O157:H7 strain, activates the expression of virulence genes under gluconeogenic conditions, suggesting that colonization of the intestine with a probiotic E. coli strain that outcompetes O157:H7 strains for gluconeogenic nutrients could render them nonpathogenic. Here we report that E. coli Nissle 1917, a probiotic strain, uses both glycolytic and gluconeogenic nutrients to colonize the mouse intestine between 1 and 5 days postfeeding, appears to stop using gluconeogenic nutrients thereafter in a large, long-term colonization niche, but continues to use them in a smaller niche to compete with invading E. coli EDL933. Evidence is also presented suggesting that invading E. coli EDL933 uses both glycolytic and gluconeogenic nutrients and needs the ability to perform gluconeogenesis in order to colonize mice precolonized with E. coli Nissle 1917. The data presented here therefore rule out the possibility that E. coli Nissle 1917 can starve the O157:H7 E. coli strain EDL933 of gluconeogenic nutrients, even though E. coli Nissle 1917 uses such nutrients to compete with E. coli EDL933 in the mouse intestine.

  7. Reconstitution of flagellar sliding.

    PubMed

    Alper, Joshua; Geyer, Veikko; Mukundan, Vikram; Howard, Jonathon

    2013-01-01

    The motile structure within eukaryotic cilia and flagella is the axoneme. This structure typically consists of nine doublet microtubules arranged around a pair of singlet microtubules. The axoneme contains more than 650 different proteins that have structural, force-generating, and regulatory functions. Early studies on sea urchin sperm identified the force-generating components, the dynein motors. It was shown that dynein can slide adjacent doublet microtubules in the presence of ATP. How this sliding gives rise to the beating of the axoneme is still unknown. Reconstitution assays provide a clean system, free from cellular effects, to elucidate the underlying beating mechanisms. These assays can be used to identify the components that are both necessary and sufficient for the generation of flagellar beating. PMID:23498749

  8. Reconstitution of flagellar sliding.

    PubMed

    Alper, Joshua; Geyer, Veikko; Mukundan, Vikram; Howard, Jonathon

    2013-01-01

    The motile structure within eukaryotic cilia and flagella is the axoneme. This structure typically consists of nine doublet microtubules arranged around a pair of singlet microtubules. The axoneme contains more than 650 different proteins that have structural, force-generating, and regulatory functions. Early studies on sea urchin sperm identified the force-generating components, the dynein motors. It was shown that dynein can slide adjacent doublet microtubules in the presence of ATP. How this sliding gives rise to the beating of the axoneme is still unknown. Reconstitution assays provide a clean system, free from cellular effects, to elucidate the underlying beating mechanisms. These assays can be used to identify the components that are both necessary and sufficient for the generation of flagellar beating.

  9. Reducing acetate excretion from E. coli K-12 by over-expressing the small RNA SgrS.

    PubMed

    Negrete, Alejandro; Majdalani, Nadim; Phue, Je-Nie; Shiloach, Joseph

    2013-01-25

    When exposed to the nonmetabolized glucose derivative alpha methyl glucoside (αMG), both Escherichia coli K-12 (JM109 and MG1655) and E. coli B (BL21) respond by reducing the concentration of the mRNA of the ptsG gene which is responsible for the biosynthesis of the glucose transporter EIICB(glu). This occurs through the over-expression of the noncoding small RNA SgrS, which interacts specifically with the mRNA of the ptsG gene and prevents its translation. However, when these bacteria are exposed to a glucose concentration of 40 g/L, over-expression of SgrS is observed only in E. coli B (BL21). Unlike E. coli K-12 (JM109 and MG1655), which are affected by high glucose concentration and produce higher levels of acetate, E. coli B (BL21) is not affected. Based on this information, it was assumed that over-expression of SgrS enables E. coli B (BL21) to reduce its acetate excretion by controlling the glucose transport. When SgrS was over-expressed in both E. coli K-12 strains from a multicopy plasmid, it was possible to reduce their acetate excretion levels to those seen in E. coli B. This observation opens a new approach towards controlling bacterial metabolism through the use of noncoding RNA.

  10. Variable symmetry in Salmonella typhimurium flagellar motors.

    PubMed

    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.

  11. Analysis of flagellar protein ubiquitination.

    PubMed

    Long, Huan; Huang, Kaiyao

    2013-01-01

    Flagella/cilia are conserved organelles existing in unicellular protists and multicellular animals, where they perform essential motile and sensory functions. Their assembly and disassembly are coordinated with the cell cycle, and recent evidence shows that posttranslational modifications such as phosphorylation, methylation, and ubiquitination are involved in these two processes, perhaps through interacting with intraflagellar transport (IFT), a specialized intracellular transport that is required for the assembly and maintenance of flagella/cilia. In this chapter, we summarize the components of the ubiquitination system published in proteomic databases of flagella/cilia. Furthermore, we describe procedures to analyze the ubiquitin-conjugating system in Chlamydomonas flagella and to analyze flagellar protein ubiquitination during flagellar shortening and the mating process. These results and tools will be valuable for the characterization of substrates of ubiquitination and their roles in flagellar disassembly and in regulating signal transduction pathways in flagella/cilia.

  12. YbcL of uropathogenic Escherichia coli suppresses transepithelial neutrophil migration.

    PubMed

    Lau, Megan E; Loughman, Jennifer A; Hunstad, David A

    2012-12-01

    Uropathogenic Escherichia coli (UPEC) strains suppress the acute inflammatory response in the urinary tract to ensure access to the intracellular uroepithelial niche that supports the propagation of infection. Our understanding of this initial cross talk between host and pathogen is incomplete. Here we report the identification of a previously uncharacterized periplasmic protein, YbcL, encoded by UPEC that contributes to immune modulation in the urinary tract by suppressing acute neutrophil migration. In contrast to wild-type UPEC, an isogenic strain lacking ybcL expression (UTI89 ΔybcL) failed to suppress transepithelial polymorphonuclear leukocyte (PMN) migration in vitro, a defect complemented by expressing ybcL episomally. YbcL homologs are present in many E. coli genomes; expression of the YbcL variant encoded by nonpathogenic E. coli K-12 strain MG1655 (YbcL(MG)) failed to complement the UTI89 ΔybcL defect, whereas expression of the UPEC YbcL variant (YbcL(UTI)) in MG1655 conferred the capacity for suppressing PMN migration. This phenotypic difference was due to a single amino acid difference (V78T) between the two YbcL homologs, and a majority of clinical UPEC strains examined were found to encode the suppressive YbcL variant. Purified YbcL(UTI) protein suppressed PMN migration in response to live or killed MG1655, and YbcL(UTI) was detected in the supernatant during UPEC infection of bladder epithelial cells or PMNs. Lastly, early PMN influx to murine bladder tissue was augmented upon in vivo infection with UTI89 ΔybcL compared with wild-type UPEC. Our findings demonstrate a role for UPEC YbcL in suppression of the innate immune response during urinary tract infection.

  13. Long-Term Evolution Studies of E. Coli under Combined Effects of Simulated Microgravity and Antibiotic.

    NASA Astrophysics Data System (ADS)

    Karouia, Fathi; Tirumalai, Madhan R.; Ott, Mark C.; Pierson, Duane L.; Fox, George E.; Tran, Quyen

    2016-07-01

    Multiple spaceflight and simulated microgravity experiments have shown changes in phenotypic microbial characteristics such as microbial growth, morphology, metabolism, genetic transfer, antibiotic and stress susceptibility, and an increase in virulence factors. However, while these studies have contributed to expand our understanding of the short-term effects of spaceflight or simulated microgravity on biological systems, it remains unclear the type of responses subsequent to long-term exposure to space environment and microgravity in particular. As such, organisms exposed to the space environment for extended periods of time may evolve in unanticipated ways thereby negatively impacting long duration space missions. We report here for the first time, an experimental study of microbial evolution in which the effect of long-term exposure to Low Shear Modeled MicroGravity (LSMMG) on microbial gene expression and physiology in Escherichia coli (E. coli) MG1655 was examined using functional genomics, and molecular techniques with and without simultaneous exposure to broad spectrum antibiotic chloramphenicol. E. coli cells were grown under simulated microgravity for 1000 generations in High Aspect Ratio Vessels (HARVs) that were either heat-sterilized (115 deg C, 15 min) or by using/rinsing the HARVs with a saturated solution of the broad-spectrum antibiotic chloramphenicol. In the case of the cells evolved using the antibiotic sterilized HARVs, the expression levels of 357 genes were significantly changed. In particular, fimbriae encoding genes were significantly up-regulated whereas genes encoding the flagellar motor complex were down-regulated. Re-sequencing of the genome revealed that a number of the flagellar genes were actually deleted. The antibiotic resistance levels of the evolved strains were analyzed using VITEK analyzer. The evolved strain was consistently resistant to the antibiotics used (viz., Ampicillin, Cefalotin, Cefurox-ime, Cefuroxime Axetil

  14. Flagellar apparatus structure of choanoflagellates.

    PubMed

    Karpov, Sergey A

    2016-01-01

    Phylum choanoflagellata is the nearest unicellular neighbor of metazoa at the phylogenetic tree. They are single celled or form the colonies, can be presented by naked cells or live in theca or lorica, but in all cases they have a flagellum surrounded by microvilli of the collar. They have rather uniform and peculiar flagellar apparatus structure with flagellar basal body (FB) producing a flagellum, and non-flagellar basal body (NFB) lying orthogonal to the FB. Long flagellar transition zone contains a unique structure among eukaryotes, the central filament, which connects central microtubules to the transversal plate. Both basal bodies are composed of triplets and interconnected with fibrillar bridge. They also contain the internal arc-shaped connectives between the triplets. The FB has prominent transitional fibers similar to those of chytrid zoospores and choanocytes of sponges, and a radial microtubular root system. The ring-shaped microtubule organizing center (MTOC) produces radial root microtubules, but in some species a MTOC is represented by separate foci. The NFB has a narrow fibrillar root directed towards the Golgi apparatus in association with membrane-bounded sac. Prior to cell division, the basal bodies replicate and migrate to poles of elongated nucleus. The basal bodies serve as MTOCs for the spindle microtubules during nuclear division by semiopen orthomitosis. PMID:27148446

  15. Biofilm modifies expression of ribonucleotide reductase genes in Escherichia coli.

    PubMed

    Cendra, Maria del Mar; Juárez, Antonio; Torrents, Eduard

    2012-01-01

    Ribonucleotide reductase (RNR) is an essential enzyme for all living organisms since is the responsible for the last step in the synthesis of the four deoxyribonucleotides (dNTPs) necessary for DNA replication and repair. In this work, we have investigated the expression of the three-RNR classes (Ia, Ib and III) during Escherichia coli biofilm formation. We show the temporal and spatial importance of class Ib and III RNRs during this process in two different E. coli wild-type strains, the commensal MG1655 and the enteropathogenic and virulent E2348/69, the prototype for the enteropathogenic E. coli (EPEC). We have established that class Ib RNR, so far considered cryptic, play and important role during biofilm formation. The implication of this RNR class under the specific growth conditions of biofilm formation is discussed. PMID:23050019

  16. Robust Parameter Identification to Perform the Modeling of pta and poxB Genes Deletion Effect on Escherichia Coli.

    PubMed

    Guerrero-Torres, V; Rios-Lozano, M; Badillo-Corona, J A; Chairez, I; Garibay-Orijel, C

    2016-08-01

    The aim of this study was to design a robust parameter identification algorithm to characterize the effect of gene deletion on Escherichia coli (E. coli) MG1655. Two genes (pta and poxB) in the competitive pathways were deleted from this microorganism to inhibit pyruvate consumption. This condition deviated the E. coli metabolism toward the Krebs cycle. As a consequence, the biomass, substrate (glucose), lactic, and acetate acids as well as ethanol concentrations were modified. A hybrid model was proposed to consider the effect of gene deletion on the metabolism of E. coli. The model parameters were estimated by the application of a least mean square method based on the instrument variable technique. To evaluate the parametric identifier method, a set of robust exact differentiators, based on the super-twisting algorithm, was implemented. The hybrid model was successfully characterized by the parameters obtained from experimental information of E. coli MG1655. The significant difference between parameters obtained with wild-type strain and the modified (with deleted genes) justifies the application of the parametric identification algorithm. This characterization can be used to optimize the production of different byproducts of commercial interest.

  17. Robust Parameter Identification to Perform the Modeling of pta and poxB Genes Deletion Effect on Escherichia Coli.

    PubMed

    Guerrero-Torres, V; Rios-Lozano, M; Badillo-Corona, J A; Chairez, I; Garibay-Orijel, C

    2016-08-01

    The aim of this study was to design a robust parameter identification algorithm to characterize the effect of gene deletion on Escherichia coli (E. coli) MG1655. Two genes (pta and poxB) in the competitive pathways were deleted from this microorganism to inhibit pyruvate consumption. This condition deviated the E. coli metabolism toward the Krebs cycle. As a consequence, the biomass, substrate (glucose), lactic, and acetate acids as well as ethanol concentrations were modified. A hybrid model was proposed to consider the effect of gene deletion on the metabolism of E. coli. The model parameters were estimated by the application of a least mean square method based on the instrument variable technique. To evaluate the parametric identifier method, a set of robust exact differentiators, based on the super-twisting algorithm, was implemented. The hybrid model was successfully characterized by the parameters obtained from experimental information of E. coli MG1655. The significant difference between parameters obtained with wild-type strain and the modified (with deleted genes) justifies the application of the parametric identification algorithm. This characterization can be used to optimize the production of different byproducts of commercial interest. PMID:27093969

  18. Escherichia coli mutants resistant to inactivation by high hydrostatic pressure.

    PubMed Central

    Hauben, K J; Bartlett, D H; Soontjens, C C; Cornelis, K; Wuytack, E Y; Michiels, C W

    1997-01-01

    Alternating cycles of exposure to high pressure and outgrowth of surviving populations were used to select for highly pressure-resistant mutants of Escherichia coli MG1655. Three barotolerant mutants (LMM1010, LMM1020, and LMM1030) were isolated independently by using outgrowth temperatures of 30, 37, and 42 degrees C, respectively. Survival of these mutants after pressure treatment for 15 min at ambient temperature was 40 to 85% at 220 MPa and 0.5 to 1.5% at 800 MPa, while survival of the parent strain, MG1655, decreased from 15% at 220 MPa to 2 x 10(-8)% at 700 MPa. Heat resistance of mutants LMM1020 and LMM1030 was also altered, as evident by higher D values at 58 and 60 degrees C and reduced z values compared to those for the parent strain. D and z values for mutant LMM1010 were not significantly different from those for the parent strain. Pressure sensitivity of the mutants increased from 10 to 50 degrees C, as opposed to the parent strain, which showed a minimum around 40 degrees C. The ability of the mutants to grow at moderately elevated pressure (50 MPa) was reduced at temperatures above 37 degrees C, indicating that resistance to pressure inactivation is unrelated to barotolerant growth. The development of high levels of barotolerance as demonstrated in this work should cause concern about the safety of high-pressure food processing. PMID:9055412

  19. Reduced Protein Synthesis Fidelity Inhibits Flagellar Biosynthesis and Motility.

    PubMed

    Fan, Yongqiang; Evans, Christopher R; Ling, Jiqiang

    2016-01-01

    Accurate translation of the genetic information from DNA to protein is maintained by multiple quality control steps from bacteria to mammals. Genetic and environmental alterations have been shown to compromise translational quality control and reduce fidelity during protein synthesis. The physiological impact of increased translational errors is not fully understood. While generally considered harmful, translational errors have recently been shown to benefit cells under certain stress conditions. In this work, we describe a novel regulatory pathway in which reduced translational fidelity downregulates expression of flagellar genes and suppresses bacterial motility. Electron microscopy imaging shows that the error-prone Escherichia coli strain lacks mature flagella. Further genetic analyses reveal that translational errors upregulate expression of a small RNA DsrA through enhancing its transcription, and deleting DsrA from the error-prone strain restores motility. DsrA regulates expression of H-NS and RpoS, both of which regulate flagellar genes. We demonstrate that an increased level of DsrA in the error-prone strain suppresses motility through the H-NS pathway. Our work suggests that bacteria are capable of switching on and off the flagellar system by altering translational fidelity, which may serve as a previously unknown mechanism to improve fitness in response to environmental cues. PMID:27468805

  20. Reduced Protein Synthesis Fidelity Inhibits Flagellar Biosynthesis and Motility

    PubMed Central

    Fan, Yongqiang; Evans, Christopher R.; Ling, Jiqiang

    2016-01-01

    Accurate translation of the genetic information from DNA to protein is maintained by multiple quality control steps from bacteria to mammals. Genetic and environmental alterations have been shown to compromise translational quality control and reduce fidelity during protein synthesis. The physiological impact of increased translational errors is not fully understood. While generally considered harmful, translational errors have recently been shown to benefit cells under certain stress conditions. In this work, we describe a novel regulatory pathway in which reduced translational fidelity downregulates expression of flagellar genes and suppresses bacterial motility. Electron microscopy imaging shows that the error-prone Escherichia coli strain lacks mature flagella. Further genetic analyses reveal that translational errors upregulate expression of a small RNA DsrA through enhancing its transcription, and deleting DsrA from the error-prone strain restores motility. DsrA regulates expression of H-NS and RpoS, both of which regulate flagellar genes. We demonstrate that an increased level of DsrA in the error-prone strain suppresses motility through the H-NS pathway. Our work suggests that bacteria are capable of switching on and off the flagellar system by altering translational fidelity, which may serve as a previously unknown mechanism to improve fitness in response to environmental cues. PMID:27468805

  1. Mechanobiology of Antimicrobial Resistant Escherichia coli and Listeria innocua.

    PubMed

    Tajkarimi, Mehrdad; Harrison, Scott H; Hung, Albert M; Graves, Joseph L

    2016-01-01

    A majority of antibiotic-resistant bacterial infections in the United States are associated with biofilms. Nanoscale biophysical measures are increasingly revealing that adhesive and viscoelastic properties of bacteria play essential roles across multiple stages of biofilm development. Atomic Force Microscopy (AFM) applied to strains with variation in antimicrobial resistance enables new opportunities for investigating the function of adhesive forces (stickiness) in biofilm formation. AFM force spectroscopy analysis of a field strain of Listeria innocua and the strain Escherichia coli K-12 MG1655 revealed differing adhesive forces between antimicrobial resistant and nonresistant strains. Significant increases in stickiness were found at the nanonewton level for strains of Listeria innocua and Escherichia coli in association with benzalkonium chloride and silver nanoparticle resistance respectively. This advancement in the usage of AFM provides for a fast and reliable avenue for analyzing antimicrobial resistant cells and the molecular dynamics of biofilm formation as a protective mechanism.

  2. Mechanobiology of Antimicrobial Resistant Escherichia coli and Listeria innocua.

    PubMed

    Tajkarimi, Mehrdad; Harrison, Scott H; Hung, Albert M; Graves, Joseph L

    2016-01-01

    A majority of antibiotic-resistant bacterial infections in the United States are associated with biofilms. Nanoscale biophysical measures are increasingly revealing that adhesive and viscoelastic properties of bacteria play essential roles across multiple stages of biofilm development. Atomic Force Microscopy (AFM) applied to strains with variation in antimicrobial resistance enables new opportunities for investigating the function of adhesive forces (stickiness) in biofilm formation. AFM force spectroscopy analysis of a field strain of Listeria innocua and the strain Escherichia coli K-12 MG1655 revealed differing adhesive forces between antimicrobial resistant and nonresistant strains. Significant increases in stickiness were found at the nanonewton level for strains of Listeria innocua and Escherichia coli in association with benzalkonium chloride and silver nanoparticle resistance respectively. This advancement in the usage of AFM provides for a fast and reliable avenue for analyzing antimicrobial resistant cells and the molecular dynamics of biofilm formation as a protective mechanism. PMID:26914334

  3. Mechanobiology of Antimicrobial Resistant Escherichia coli and Listeria innocua

    PubMed Central

    Tajkarimi, Mehrdad; Harrison, Scott H.; Hung, Albert M.; Graves, Joseph L.

    2016-01-01

    A majority of antibiotic-resistant bacterial infections in the United States are associated with biofilms. Nanoscale biophysical measures are increasingly revealing that adhesive and viscoelastic properties of bacteria play essential roles across multiple stages of biofilm development. Atomic Force Microscopy (AFM) applied to strains with variation in antimicrobial resistance enables new opportunities for investigating the function of adhesive forces (stickiness) in biofilm formation. AFM force spectroscopy analysis of a field strain of Listeria innocua and the strain Escherichia coli K-12 MG1655 revealed differing adhesive forces between antimicrobial resistant and nonresistant strains. Significant increases in stickiness were found at the nanonewton level for strains of Listeria innocua and Escherichia coli in association with benzalkonium chloride and silver nanoparticle resistance respectively. This advancement in the usage of AFM provides for a fast and reliable avenue for analyzing antimicrobial resistant cells and the molecular dynamics of biofilm formation as a protective mechanism. PMID:26914334

  4. CorA affects tolerance of Escherichia coli and Salmonella enterica serovar Typhimurium to the lactoperoxidase enzyme system but not to other forms of oxidative stress.

    PubMed

    Sermon, Jan; Wevers, Eva M-R P; Jansen, Leentje; De Spiegeleer, Philipp; Vanoirbeek, Kristof; Aertsen, Abram; Michiels, Chris W

    2005-11-01

    The enzyme lactoperoxidase is part of the innate immune system in vertebrates and owes its antimicrobial activity to the formation of oxidative reaction products from various substrates. In a previous study, we have reported that, with thiocyanate as a substrate, the lactoperoxidase system elicits a distinct stress response in Escherichia coli MG1655. This response is different from but partly overlapping with the stress responses to hydrogen peroxide and to superoxide. In the current work, we constructed knockouts in 10 lactoperoxidase system-inducible genes to investigate their role in the tolerance of E. coli MG1655 to this antimicrobial system. Five mutations resulted in a slightly increased sensitivity, but one mutation (corA) caused hypersensitivity to the lactoperoxidase system. This hypersensitive phenotype was specific to the lactoperoxidase system, since neither the sensitivity to hydrogen peroxide nor to the superoxide generator plumbagin was affected in the corA mutant. Salmonella enterica serovar Typhimurium corA had a similar phenotype. Although corA encodes an Mg2+ transporter and at least three other inducible open reading frames belonged to the Mg2+ regulon, repression of the Mg stimulon by Mg2+ did not change the lactoperoxidase sensitivity of either the wild-type or corA mutant. Prior exposure to 0.3 mM Ni2+, which is also transported by CorA, strongly sensitized MG1655 but not the corA mutant to the lactoperoxidase system. Furthermore, this Ni2+-dependent sensitization was suppressed by the CorA-specific inhibitor Co(III) hexaammine. These results indicate that CorA affects the lactoperoxidase sensitivity of E. coli by modulating the cytoplasmic concentrations of transition metals that enhance the toxicity of the lactoperoxidase system.

  5. Domain-swap polymerization drives the self-assembly of the bacterial flagellar motor.

    PubMed

    Baker, Matthew A B; Hynson, Robert M G; Ganuelas, Lorraine A; Mohammadi, Nasim Shah; Liew, Chu Wai; Rey, Anthony A; Duff, Anthony P; Whitten, Andrew E; Jeffries, Cy M; Delalez, Nicolas J; Morimoto, Yusuke V; Stock, Daniela; Armitage, Judith P; Turberfield, Andrew J; Namba, Keiichi; Berry, Richard M; Lee, Lawrence K

    2016-03-01

    Large protein complexes assemble spontaneously, yet their subunits do not prematurely form unwanted aggregates. This paradox is epitomized in the bacterial flagellar motor, a sophisticated rotary motor and sensory switch consisting of hundreds of subunits. Here we demonstrate that Escherichia coli FliG, one of the earliest-assembling flagellar motor proteins, forms ordered ring structures via domain-swap polymerization, which in other proteins has been associated with uncontrolled and deleterious protein aggregation. Solution structural data, in combination with in vivo biochemical cross-linking experiments and evolutionary covariance analysis, revealed that FliG exists predominantly as a monomer in solution but only as domain-swapped polymers in assembled flagellar motors. We propose a general structural and thermodynamic model for self-assembly, in which a structural template controls assembly and shapes polymer formation into rings. PMID:26854663

  6. The evolution of metabolic networks of E. coli

    PubMed Central

    2011-01-01

    Background Despite the availability of numerous complete genome sequences from E. coli strains, published genome-scale metabolic models exist only for two commensal E. coli strains. These models have proven useful for many applications, such as engineering strains for desired product formation, and we sought to explore how constructing and evaluating additional metabolic models for E. coli strains could enhance these efforts. Results We used the genomic information from 16 E. coli strains to generate an E. coli pangenome metabolic network by evaluating their collective 76,990 ORFs. Each of these ORFs was assigned to one of 17,647 ortholog groups including ORFs associated with reactions in the most recent metabolic model for E. coli K-12. For orthologous groups that contain an ORF already represented in the MG1655 model, the gene to protein to reaction associations represented in this model could then be easily propagated to other E. coli strain models. All remaining orthologous groups were evaluated to see if new metabolic reactions could be added to generate a pangenome-scale metabolic model (iEco1712_pan). The pangenome model included reactions from a metabolic model update for E. coli K-12 MG1655 (iEco1339_MG1655) and enabled development of five additional strain-specific genome-scale metabolic models. These additional models include a second K-12 strain (iEco1335_W3110) and four pathogenic strains (two enterohemorrhagic E. coli O157:H7 and two uropathogens). When compared to the E. coli K-12 models, the metabolic models for the enterohemorrhagic (iEco1344_EDL933 and iEco1345_Sakai) and uropathogenic strains (iEco1288_CFT073 and iEco1301_UTI89) contained numerous lineage-specific gene and reaction differences. All six E. coli models were evaluated by comparing model predictions to carbon source utilization measurements under aerobic and anaerobic conditions, and to batch growth profiles in minimal media with 0.2% (w/v) glucose. An ancestral genome

  7. Flagellar membrane proteins in kinetoplastid parasites.

    PubMed

    Landfear, Scott M; Tran, Khoa D; Sanchez, Marco A

    2015-09-01

    All kinetoplastid parasites, including protozoa such as Leishmania species, Trypanosoma brucei, and Trypanosoma cruzi that cause devastating diseases in humans and animals, are flagellated throughout their life cycles. Although flagella were originally thought of primarily as motility organelles, flagellar functions in other critical processes, especially in sensing and signal transduction, have become more fully appreciated in the recent past. The flagellar membrane is a highly specialized subdomain of the surface membrane, and flagellar membrane proteins are likely to be critical components for all the biologically important roles of flagella. In this review, we summarize recent discoveries relevant to flagellar membrane proteins in these parasites, including the identification of such proteins, investigation of their biological functions, and mechanisms of selective trafficking to the flagellar membrane. Prospects for future investigations and current unsolved problems are highlighted.

  8. Flagellar membrane proteins in kinetoplastid parasites.

    PubMed

    Landfear, Scott M; Tran, Khoa D; Sanchez, Marco A

    2015-09-01

    All kinetoplastid parasites, including protozoa such as Leishmania species, Trypanosoma brucei, and Trypanosoma cruzi that cause devastating diseases in humans and animals, are flagellated throughout their life cycles. Although flagella were originally thought of primarily as motility organelles, flagellar functions in other critical processes, especially in sensing and signal transduction, have become more fully appreciated in the recent past. The flagellar membrane is a highly specialized subdomain of the surface membrane, and flagellar membrane proteins are likely to be critical components for all the biologically important roles of flagella. In this review, we summarize recent discoveries relevant to flagellar membrane proteins in these parasites, including the identification of such proteins, investigation of their biological functions, and mechanisms of selective trafficking to the flagellar membrane. Prospects for future investigations and current unsolved problems are highlighted. PMID:26599841

  9. Flagellar Membrane Proteins in Kinetoplastid Parasites

    PubMed Central

    Landfear, Scott M.; Tran, Khoa D.; Sanchez, Marco A.

    2015-01-01

    All kinetoplastid parasites, including protozoa such as Leishmania species, Trypanosoma brucei, and Trypanosoma cruzi that cause devastating diseases in humans and animals, are flagellated throughout their life cycles. While flagella were originally thought of primarily as motility organelles, flagellar functions in other critical processes, especially in sensing and signal transduction, have become more fully appreciated in the recent past. The flagellar membrane is a highly specialized subdomain of the surface membrane, and flagellar membrane proteins are likely to be critical components for all the biologically important roles of flagella. In this review, we summarize recent discoveries relevant to flagellar membrane proteins in these parasites including the identification of such proteins, investigation of their biological functions, and mechanisms of selective trafficking to the flagellar membrane. Prospects for future investigations and current unsolved problems are highlighted. PMID:26599841

  10. Decoding genome-wide GadEWX-transcriptional regulatory networks reveals multifaceted cellular responses to acid stress in Escherichia coli.

    PubMed

    Seo, Sang Woo; Kim, Donghyuk; O'Brien, Edward J; Szubin, Richard; Palsson, Bernhard O

    2015-01-01

    The regulators GadE, GadW and GadX (which we refer to as GadEWX) play a critical role in the transcriptional regulation of the glutamate-dependent acid resistance (GDAR) system in Escherichia coli K-12 MG1655. However, the genome-wide regulatory role of GadEWX is still unknown. Here we comprehensively reconstruct the genome-wide GadEWX transcriptional regulatory network and RpoS involvement in E. coli K-12 MG1655 under acidic stress. Integrative data analysis reveals that GadEWX regulons consist of 45 genes in 31 transcription units and 28 of these genes were associated with RpoS-binding sites. We demonstrate that GadEWX directly and coherently regulate several proton-generating/consuming enzymes with pairs of negative-feedback loops for pH homeostasis. In addition, GadEWX regulate genes with assorted functions, including molecular chaperones, acid resistance, stress response and other regulatory activities. These results show how GadEWX simultaneously coordinate many cellular processes to produce the overall response of E. coli to acid stress. PMID:26258987

  11. Decoding genome-wide GadEWX-transcriptional regulatory networks reveals multifaceted cellular responses to acid stress in Escherichia coli

    PubMed Central

    Seo, Sang Woo; Kim, Donghyuk; O'Brien, Edward J.; Szubin, Richard; Palsson, Bernhard O.

    2015-01-01

    The regulators GadE, GadW and GadX (which we refer to as GadEWX) play a critical role in the transcriptional regulation of the glutamate-dependent acid resistance (GDAR) system in Escherichia coli K-12 MG1655. However, the genome-wide regulatory role of GadEWX is still unknown. Here we comprehensively reconstruct the genome-wide GadEWX transcriptional regulatory network and RpoS involvement in E. coli K-12 MG1655 under acidic stress. Integrative data analysis reveals that GadEWX regulons consist of 45 genes in 31 transcription units and 28 of these genes were associated with RpoS-binding sites. We demonstrate that GadEWX directly and coherently regulate several proton-generating/consuming enzymes with pairs of negative-feedback loops for pH homeostasis. In addition, GadEWX regulate genes with assorted functions, including molecular chaperones, acid resistance, stress response and other regulatory activities. These results show how GadEWX simultaneously coordinate many cellular processes to produce the overall response of E. coli to acid stress. PMID:26258987

  12. Flagellar root contraction and nuclear movement during flagellar regeneration in Chlamydomonas reinhardtii

    PubMed Central

    1987-01-01

    When Chlamydomonas cells are deflagellated by pH shock or mechanical shear the nucleus rapidly moves toward the flagellar basal apparatus at the anterior end of the cell. During flagellar regeneration the nucleus returns to a more central position within the cell. The nucleus is connected to the flagellar apparatus by a system of fibers, the flagellar roots (rhizoplasts), which undergo a dramatic contraction that coincides with anterior nuclear movement. A corresponding extension of the root system, back to its preshock configuration is observed as the nucleus retracts to a central position. Anterior displacement of the nucleus and flagellar root contraction require free calcium in the medium. Nuclear movement and flagellar root contraction and extension are not sensitive to inhibitors of protein synthesis (cycloheximide), or drugs that influence either microtubules (colchicine) or actin-based microfilaments (cytochalasin D). Detergent- extracted cell models contract and extend their flagellar roots and move their nuclei in response to alterations of free calcium levels in the medium. Cycles of nuclear movement in detergent-extracted models require ATP to potentiate the contractile mechanism for subsequent calcium-induced contraction. Flagellar root contraction and nuclear movement in Chlamydomonas may be causally related to signaling of induction of flagellar precursor genes or to the transport of flagellar precursors or their messages to sites of synthesis or assembly near the basal apparatus of the cell. PMID:3667698

  13. 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.

  14. Comparative analysis of envelope proteomes in Escherichia coli B and K-12 strains.

    PubMed

    Han, Mee-Jung; Lee, Sang Yup; Hong, Soon Ho

    2012-04-01

    Recent genome comparisons of E. coli B and K-12 strains have indicated that the makeup of the cell envelopes in these two strains is quite different. Therefore, we analyzed and compared the envelope proteomes of E. coli BL21(DE3) and MG1655. A total of 165 protein spots, including 62 nonredundant proteins, were unambiguously identified by two-dimensional gel electrophoresis and mass spectrometry. Of these, 43 proteins were conserved between the two strains, whereas 4 and 16 strain-specific proteins were identified only in E. coli BL21(DE3) and MG1655, respectively. Additionally, 24 proteins showed more than 2-fold differences in intensities between the B and K-12 strains. The reference envelope proteome maps showed that E. coli envelope mainly contained channel proteins and lipoproteins. Interesting proteomic observations between the two strains were as follows: (i) B produced more OmpF porin with a larger pore size than K-12, indicating an increase in the membrane permeability; (ii) B produced higher amounts of lipoproteins, which facilitates the assembly of outer membrane beta-barrel proteins; and (iii) motility- (FliC) and chemotaxis-related proteins (CheA and CheW) were detected only in K-12, which showed that E. coli B is restricted with regard to migration under unfavorable conditions. These differences may influence the permeability and integrity of the cell envelope, showing that E. coli B may be more susceptible than K-12 to certain stress conditions. Thus, these findings suggest that E. coli K-12 and its derivatives will be more favorable strains in certain biotechnological applications, such as cell surface display or membrane engineering studies.

  15. Metabolic engineering of the Stevia rebaudiana ent-kaurene biosynthetic pathway in recombinant Escherichia coli.

    PubMed

    Kong, Min Kyung; Kang, Hyun-Jun; Kim, Jin Ho; Oh, Soon Hwan; Lee, Pyung Cheon

    2015-11-20

    The ent-kaurene is a dedicated precursor pool and is responsible for synthesizing natural sweeteners such as steviol glycosides. In this study, to produce ent-kaurene in Escherichia coli, we modularly constructed and expressed two ent-kaurene genes encoding ent-copalyl diphosphate synthase (CPPS) and ent-kaurene synthase (KS) from Stevia rebaudiana known as a typical plant producing steviol glycoside. The CPPS and KS from S. rebaudiana were functionally expressed in a heterologous host E. coli. Furthermore, in order to enhance ent-kaurene production in E. coli, six geranylgeranyl diphosphate synthases (GGPPS) from various microorganisms and eight strains of E. coli as host were compared by measuring ent-kaurene production. The highest ent-kaurene production of approximately 41.1mg/L was demonstrated in E. coli strain MG1655 co-expressing synthetic CPPS-KS module and GGPPS from Rhodobacter sphaeroides. The ent-kaurene production was further increased up to 179.6 mg/L by overexpression of the three key enzymes for isoprenoid precursor, 1-deoxyxylulose-5-phosphate synthase (DXS), farnesyl diphosphate synthase (IspA) and isopentenyl diphosphate isomerase (IDI) from E. coli. Finally, the highest titer of ent-kaurene (578 mg/L) with a specific yield of ent-kaurene of 143.5mg/g dry cell weight was obtained by culturing E. coli strain MG1655 co-expressing the ent-kaurene module, DXS, IDI and IspA in 1L bioreactor containing 20 g/L glycerol. PMID:26392384

  16. Metabolic engineering of the Stevia rebaudiana ent-kaurene biosynthetic pathway in recombinant Escherichia coli.

    PubMed

    Kong, Min Kyung; Kang, Hyun-Jun; Kim, Jin Ho; Oh, Soon Hwan; Lee, Pyung Cheon

    2015-11-20

    The ent-kaurene is a dedicated precursor pool and is responsible for synthesizing natural sweeteners such as steviol glycosides. In this study, to produce ent-kaurene in Escherichia coli, we modularly constructed and expressed two ent-kaurene genes encoding ent-copalyl diphosphate synthase (CPPS) and ent-kaurene synthase (KS) from Stevia rebaudiana known as a typical plant producing steviol glycoside. The CPPS and KS from S. rebaudiana were functionally expressed in a heterologous host E. coli. Furthermore, in order to enhance ent-kaurene production in E. coli, six geranylgeranyl diphosphate synthases (GGPPS) from various microorganisms and eight strains of E. coli as host were compared by measuring ent-kaurene production. The highest ent-kaurene production of approximately 41.1mg/L was demonstrated in E. coli strain MG1655 co-expressing synthetic CPPS-KS module and GGPPS from Rhodobacter sphaeroides. The ent-kaurene production was further increased up to 179.6 mg/L by overexpression of the three key enzymes for isoprenoid precursor, 1-deoxyxylulose-5-phosphate synthase (DXS), farnesyl diphosphate synthase (IspA) and isopentenyl diphosphate isomerase (IDI) from E. coli. Finally, the highest titer of ent-kaurene (578 mg/L) with a specific yield of ent-kaurene of 143.5mg/g dry cell weight was obtained by culturing E. coli strain MG1655 co-expressing the ent-kaurene module, DXS, IDI and IspA in 1L bioreactor containing 20 g/L glycerol.

  17. Comparative analysis of envelope proteomes in Escherichia coli B and K-12 strains.

    PubMed

    Han, Mee-Jung; Lee, Sang Yup; Hong, Soon Ho

    2012-04-01

    Recent genome comparisons of E. coli B and K-12 strains have indicated that the makeup of the cell envelopes in these two strains is quite different. Therefore, we analyzed and compared the envelope proteomes of E. coli BL21(DE3) and MG1655. A total of 165 protein spots, including 62 nonredundant proteins, were unambiguously identified by two-dimensional gel electrophoresis and mass spectrometry. Of these, 43 proteins were conserved between the two strains, whereas 4 and 16 strain-specific proteins were identified only in E. coli BL21(DE3) and MG1655, respectively. Additionally, 24 proteins showed more than 2-fold differences in intensities between the B and K-12 strains. The reference envelope proteome maps showed that E. coli envelope mainly contained channel proteins and lipoproteins. Interesting proteomic observations between the two strains were as follows: (i) B produced more OmpF porin with a larger pore size than K-12, indicating an increase in the membrane permeability; (ii) B produced higher amounts of lipoproteins, which facilitates the assembly of outer membrane beta-barrel proteins; and (iii) motility- (FliC) and chemotaxis-related proteins (CheA and CheW) were detected only in K-12, which showed that E. coli B is restricted with regard to migration under unfavorable conditions. These differences may influence the permeability and integrity of the cell envelope, showing that E. coli B may be more susceptible than K-12 to certain stress conditions. Thus, these findings suggest that E. coli K-12 and its derivatives will be more favorable strains in certain biotechnological applications, such as cell surface display or membrane engineering studies. PMID:22534293

  18. 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.

  19. Rhythmicity, recurrence, and recovery of flagellar beating.

    PubMed

    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.

  20. The Complete Genome Sequence of Escherichia coli DH10B: Insights into the Biology of a Laboratory Workhorse▿ †

    PubMed Central

    Durfee, Tim; Nelson, Richard; Baldwin, Schuyler; Plunkett, Guy; Burland, Valerie; Mau, Bob; Petrosino, Joseph F.; Qin, Xiang; Muzny, Donna M.; Ayele, Mulu; Gibbs, Richard A.; Csörgő, Bálint; Pósfai, György; Weinstock, George M.; Blattner, Frederick R.

    2008-01-01

    Escherichia coli DH10B was designed for the propagation of large insert DNA library clones. It is used extensively, taking advantage of properties such as high DNA transformation efficiency and maintenance of large plasmids. The strain was constructed by serial genetic recombination steps, but the underlying sequence changes remained unverified. We report the complete genomic sequence of DH10B by using reads accumulated from the bovine sequencing project at Baylor College of Medicine and assembled with DNAStar's SeqMan genome assembler. The DH10B genome is largely colinear with that of the wild-type K-12 strain MG1655, although it is substantially more complex than previously appreciated, allowing DH10B biology to be further explored. The 226 mutated genes in DH10B relative to MG1655 are mostly attributable to the extensive genetic manipulations the strain has undergone. However, we demonstrate that DH10B has a 13.5-fold higher mutation rate than MG1655, resulting from a dramatic increase in insertion sequence (IS) transposition, especially IS150. IS elements appear to have remodeled genome architecture, providing homologous recombination sites for a 113,260-bp tandem duplication and an inversion. DH10B requires leucine for growth on minimal medium due to the deletion of leuLABCD and harbors both the relA1 and spoT1 alleles causing both sensitivity to nutritional downshifts and slightly lower growth rates relative to the wild type. Finally, while the sequence confirms most of the reported alleles, the sequence of deoR is wild type, necessitating reexamination of the assumed basis for the high transformability of DH10B. PMID:18245285

  1. Global gene expression analysis of glucose overflow metabolism in Escherichia coli and reduction of aerobic acetate formation.

    PubMed

    Veit, Andrea; Polen, Tino; Wendisch, Volker F

    2007-02-01

    During aerobic growth on glucose, Escherichia coli produces acetate in the so-called overflow metabolism. DNA microarray analysis was used to determine the global gene expression patterns of chemostat cultivations of E. coli MG1655 that were characterized by different acetate formation rates during aerobic growth on glucose. A correlation analysis identified that expression of ten genes (sdhCDAB, sucB, sucC, acnB, lpdA, fumC and mdh) encoding the TCA cycle enzymes succinate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinyl-CoA synthetase, aconitase, fumarase and malate dehydrogenase, respectively, and of the acs-yjcH-actP operon for acetate utilization correlated negatively with acetate formation. Relieving transcriptional control of the sdhCDAB-b0725-sucABCD operon by chromosomal promoter exchange mutagenesis yielded a strain with increased specific activities of the TCA cycle enzymes succinate dehydrogenase, alpha-ketoglutarate dehydrogenase and succinyl-CoA synthetase, which are encoded by this operon. The resulting strain produced less acetate and directed more carbon towards carbon dioxide formation than the parent strain MG1655 while maintaining high growth and glucose consumption rates. PMID:17273855

  2. 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

  3. 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

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

    PubMed

    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

  5. Deciphering Fur transcriptional regulatory network highlights its complex role beyond iron metabolism in Escherichia coli.

    PubMed

    Seo, Sang Woo; Kim, Donghyuk; Latif, Haythem; O'Brien, Edward J; Szubin, Richard; Palsson, Bernhard O

    2014-09-15

    The ferric uptake regulator (Fur) plays a critical role in the transcriptional regulation of iron metabolism. However, the full regulatory potential of Fur remains undefined. Here we comprehensively reconstruct the Fur transcriptional regulatory network in Escherichia coli K-12 MG1655 in response to iron availability using genome-wide measurements. Integrative data analysis reveals that a total of 81 genes in 42 transcription units are directly regulated by three different modes of Fur regulation, including apo- and holo-Fur activation and holo-Fur repression. We show that Fur connects iron transport and utilization enzymes with negative-feedback loop pairs for iron homeostasis. In addition, direct involvement of Fur in the regulation of DNA synthesis, energy metabolism and biofilm development is found. These results show how Fur exhibits a comprehensive regulatory role affecting many fundamental cellular processes linked to iron metabolism in order to coordinate the overall response of E. coli to iron availability.

  6. EchoBASE: an integrated post-genomic database for Escherichia coli.

    PubMed

    Misra, Raju V; Horler, Richard S P; Reindl, Wolfgang; Goryanin, Igor I; Thomas, Gavin H

    2005-01-01

    EchoBASE (http://www.ecoli-york.org) is a relational database designed to contain and manipulate information from post-genomic experiments using the model bacterium Escherichia coli K-12. Its aim is to collate information from a wide range of sources to provide clues to the functions of the approximately 1500 gene products that have no confirmed cellular function. The database is built on an enhanced annotation of the updated genome sequence of strain MG1655 and the association of experimental data with the E.coli genes and their products. Experiments that can be held within EchoBASE include proteomics studies, microarray data, protein-protein interaction data, structural data and bioinformatics studies. EchoBASE also contains annotated information on 'orphan' enzyme activities from this microbe to aid characterization of the proteins that catalyse these elusive biochemical reactions. PMID:15608209

  7. Deciphering Fur transcriptional regulatory network highlights its complex role beyond iron metabolism in Escherichia coli.

    PubMed

    Seo, Sang Woo; Kim, Donghyuk; Latif, Haythem; O'Brien, Edward J; Szubin, Richard; Palsson, Bernhard O

    2014-01-01

    The ferric uptake regulator (Fur) plays a critical role in the transcriptional regulation of iron metabolism. However, the full regulatory potential of Fur remains undefined. Here we comprehensively reconstruct the Fur transcriptional regulatory network in Escherichia coli K-12 MG1655 in response to iron availability using genome-wide measurements. Integrative data analysis reveals that a total of 81 genes in 42 transcription units are directly regulated by three different modes of Fur regulation, including apo- and holo-Fur activation and holo-Fur repression. We show that Fur connects iron transport and utilization enzymes with negative-feedback loop pairs for iron homeostasis. In addition, direct involvement of Fur in the regulation of DNA synthesis, energy metabolism and biofilm development is found. These results show how Fur exhibits a comprehensive regulatory role affecting many fundamental cellular processes linked to iron metabolism in order to coordinate the overall response of E. coli to iron availability. PMID:25222563

  8. Overexpression of the Lactobacillus plantarum peptidoglycan biosynthesis murA2 gene increases the tolerance of Escherichia coli to alcohols and enhances ethanol production.

    PubMed

    Yuan, Yongbo; Bi, Changhao; Nicolaou, Sergios A; Zingaro, Kyle A; Ralston, Matthew; Papoutsakis, Eleftherios T

    2014-10-01

    A major challenge in producing chemicals and biofuels is to increase the tolerance of the host organism to toxic products or byproducts. An Escherichia coli strain with superior ethanol and more generally alcohol tolerance was identified by screening a library constructed by randomly integrating Lactobacillus plantarum genomic DNA fragments into the E. coli chromosome via Cre-lox recombination. Sequencing identified the inserted DNA fragment as the murA2 gene and its upstream intergenic 973-bp sequence, both coded on the negative genomic DNA strand. Overexpression of this murA2 gene and its upstream 973-bp sequence significantly enhanced ethanol tolerance in both E. coli EC100 and wild type E. coli MG1655 strains by 4.1-fold and 2.0-fold compared to control strains, respectively. Tolerance to n-butanol and i-butanol in E. coli MG1655 was increased by 1.85-fold and 1.91-fold, respectively. We show that the intergenic 973-bp sequence contains a native promoter for the murA2 gene along with a long 5' UTR (286 nt) on the negative strand, while a noncoding, small RNA, named MurA2S, is expressed off the positive strand. MurA2S is expressed in E. coli and may interact with murA2, but it does not affect murA2's ability to enhance alcohol tolerance in E. coli. Overexpression of murA2 with its upstream region in the ethanologenic E. coli KO11 strain significantly improved ethanol production in cultures that simulate the industrial Melle-Boinot fermentation process.

  9. Escherichia coli Nissle 1917 enhances bioavailability of serotonin in gut tissues through modulation of synthesis and clearance.

    PubMed

    Nzakizwanayo, Jonathan; Dedi, Cinzia; Standen, Guy; Macfarlane, Wendy M; Patel, Bhavik A; Jones, Brian V

    2015-01-01

    Accumulating evidence shows indigenous gut microbes can interact with the human host through modulation of serotonin (5-HT) signaling. Here we investigate the impact of the probiotic Escherichia coli Nissle 1917 (EcN) on 5-HT signalling in gut tissues. Ex-vivo mouse ileal tissue sections were treated with either EcN or the human gut commensal MG1655, and effects on levels of 5-HT, precursors, and metabolites, were evaluated using amperometry and high performance liquid chromatography with electrochemical detection (HPLC-EC). Exposure of tissue to EcN cells, but not MG1655 cells, was found to increase levels of extra-cellular 5-HT. These effects were not observed when tissues were treated with cell-free supernatant from bacterial cultures. In contrast, when supernatant recovered from untreated ileal tissue was pre-incubated with EcN, the derivative cell-free supernatant was able to elevate 5-HT overflow when used to treat fresh ileal tissue. Measurement of 5-HT precursors and metabolites indicated EcN also increases intracellular 5-HTP and reduces 5-HIAA. The former pointed to modulation of tryptophan hydroxylase-1 to enhance 5-HT synthesis, while the latter indicates an impact on clearance into enterocytes through SERT. Taken together, these findings show EcN is able to enhance 5-HT bioavailability in ileal tissues through interaction with compounds secreted from host tissues. PMID:26616662

  10. Escherichia coli Nissle 1917 enhances bioavailability of serotonin in gut tissues through modulation of synthesis and clearance

    PubMed Central

    Nzakizwanayo, Jonathan; Dedi, Cinzia; Standen, Guy; Macfarlane, Wendy M.; Patel, Bhavik A.; Jones, Brian V.

    2015-01-01

    Accumulating evidence shows indigenous gut microbes can interact with the human host through modulation of serotonin (5-HT) signaling. Here we investigate the impact of the probiotic Escherichia coli Nissle 1917 (EcN) on 5-HT signalling in gut tissues. Ex-vivo mouse ileal tissue sections were treated with either EcN or the human gut commensal MG1655, and effects on levels of 5-HT, precursors, and metabolites, were evaluated using amperometry and high performance liquid chromatography with electrochemical detection (HPLC-EC). Exposure of tissue to EcN cells, but not MG1655 cells, was found to increase levels of extra-cellular 5-HT. These effects were not observed when tissues were treated with cell-free supernatant from bacterial cultures. In contrast, when supernatant recovered from untreated ileal tissue was pre-incubated with EcN, the derivative cell-free supernatant was able to elevate 5-HT overflow when used to treat fresh ileal tissue. Measurement of 5-HT precursors and metabolites indicated EcN also increases intracellular 5-HTP and reduces 5-HIAA. The former pointed to modulation of tryptophan hydroxylase-1 to enhance 5-HT synthesis, while the latter indicates an impact on clearance into enterocytes through SERT. Taken together, these findings show EcN is able to enhance 5-HT bioavailability in ileal tissues through interaction with compounds secreted from host tissues. PMID:26616662

  11. Escherichia coli Nissle 1917 enhances bioavailability of serotonin in gut tissues through modulation of synthesis and clearance.

    PubMed

    Nzakizwanayo, Jonathan; Dedi, Cinzia; Standen, Guy; Macfarlane, Wendy M; Patel, Bhavik A; Jones, Brian V

    2015-11-30

    Accumulating evidence shows indigenous gut microbes can interact with the human host through modulation of serotonin (5-HT) signaling. Here we investigate the impact of the probiotic Escherichia coli Nissle 1917 (EcN) on 5-HT signalling in gut tissues. Ex-vivo mouse ileal tissue sections were treated with either EcN or the human gut commensal MG1655, and effects on levels of 5-HT, precursors, and metabolites, were evaluated using amperometry and high performance liquid chromatography with electrochemical detection (HPLC-EC). Exposure of tissue to EcN cells, but not MG1655 cells, was found to increase levels of extra-cellular 5-HT. These effects were not observed when tissues were treated with cell-free supernatant from bacterial cultures. In contrast, when supernatant recovered from untreated ileal tissue was pre-incubated with EcN, the derivative cell-free supernatant was able to elevate 5-HT overflow when used to treat fresh ileal tissue. Measurement of 5-HT precursors and metabolites indicated EcN also increases intracellular 5-HTP and reduces 5-HIAA. The former pointed to modulation of tryptophan hydroxylase-1 to enhance 5-HT synthesis, while the latter indicates an impact on clearance into enterocytes through SERT. Taken together, these findings show EcN is able to enhance 5-HT bioavailability in ileal tissues through interaction with compounds secreted from host tissues.

  12. Simultaneous utilization of glucose and mannose from woody hydrolysate for free fatty acid production by metabolically engineered Escherichia coli.

    PubMed

    Wu, Hui; Lee, Jane; Karanjikar, Mukund; San, Ka-Yiu

    2015-06-01

    In this study, the Escherichia coli strain MG1655 with fadD mutant (named as ML103), and MG1655 with fadD and ptsG double mutant (named as ML190) carrying the plasmid with the acyl-ACP thioesterase (TE) from Ricinus communis (pXZ18) or the plasmid with the combination of the TE and the native (3R)-hydroxyacyl-ACP dehydrase (fabZ) (pXZ18Z), produced free fatty acids (FFAs) efficiently using mannose as the sole carbon source. Due to the carbon catabolite repression (CCR) regulation, ML103(pXZ18) utilized glucose and mannose sequentially in the mixed sugar culture, while ML190(pXZ18) and ML190(pXZ18Z), with ptsG mutation, used glucose and mannose simultaneously. The highest total FFA concentration from the mixed sugar culture reached 2.96g/L by ML190(pXZ18Z). Furthermore, the strain ML190(pXZ18Z) can produce 2.86g/L FFAs with a high yield of 0.23g/g using hydrolysate mainly contained glucose and mannose from a commercial plant.

  13. 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.

  14. 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.

  15. Mesoscopic modeling of bacterial flagellar microhydrodynamics.

    PubMed

    Gebremichael, Yeshitila; Ayton, Gary S; Voth, Gregory A

    2006-11-15

    A particle-based hybrid method of elastic network model and smooth-particle hydrodynamics has been employed to describe the propulsion of bacterial flagella in a viscous hydrodynamic environment. The method explicitly models the two aspects of bacterial propulsion that involve flagellar flexibility and long-range hydrodynamic interaction of low-Reynolds-number flow. The model further incorporates the molecular organization of the flagellar filament at a coarse-grained level in terms of the 11 protofilaments. Each of these protofilaments is represented by a collection of material points that represent the flagellin proteins. A computational model of a single flexible helical segment representing the filament of a bacterial flagellum is presented. The propulsive dynamics and the flow fields generated by the motion of the model filament are examined. The nature of flagellar deformation and the influence of hydrodynamics in determining the shape of deformations are examined based on the helical filament.

  16. Torque generation by the flagellar rotary motor.

    PubMed Central

    Berg, H C

    1995-01-01

    A review is given of the structure and dynamics of the flagellar rotary motor. Force-generating elements in a motor driving a tethered bacterium (a cell fixed to the substratum by a single flagellum) exert forces of order 20 pN while moving at speeds of order 1 micron/s. Force-generating elements in a motor driving a flagellar filament in a bundle exert forces some 10-fold lower but move at speeds more than 10-fold higher. The motor torque-speed relationship has been measured over a wide dynamic range. Motors strongly resist being driven backwards and are easily broken. PMID:7787060

  17. 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. PMID:26615435

  18. Investigation into the resistance of lactoperoxidase tolerant Escherichia coli mutants to different forms of oxidative stress.

    PubMed

    De Spiegeleer, Philipp; Vanoirbeek, Kristof; Lietaert, Annelies; Sermon, Jan; Aertsen, Abram; Michiels, Chris W

    2005-11-15

    Six lactoperoxidase tolerant Escherichia coli transposon mutants isolated and characterized in an earlier study, and some newly constructed double mutants, were subjected to peroxide, superoxide and hypochlorite stress, and their inactivation was compared to that of the wild type strain MG1655. Knock out mutants of waaQ and waaO, which owed their lactoperoxidase tolerance to an impaired outer membrane permeability due to a reduced porin content, also exhibited higher resistance to hypochlorite, as did a knock-out strain of lrp, encoding a regulatory protein affecting a wide range of cellular functions. Unlike the outer membrane mutants however, the lrp strain was also more resistant to t-butyl hydroperoxide, but more susceptible to the superoxide generating compound plumbagin. Finally, a lactoperoxidase tolerant knock-out strain of ulaA, involved in ascorbic acid uptake, did not show resistance to any of the other oxidants. The possible modes of action of these different oxidants are discussed.

  19. Unique stress response to the lactoperoxidase-thiocyanate enzyme system in Escherichia coli.

    PubMed

    Sermon, Jan; Vanoirbeek, Kristof; De Spiegeleer, Philipp; Van Houdt, Rob; Aertsen, Abram; Michiels, Chris W

    2005-03-01

    Using a differential fluorescence induction approach, we screened a promoter trap library constructed in a vector with a promoterless gfp gene for Escherichia coli MG1655 promoters that are induced upon challenge with the antimicrobial lactoperoxidase-thiocyanate enzyme system. None of the thirteen identified lactoperoxidase-inducible open reading frames was inducible by H(2)O(2) or by the superoxide generator plumbagin. However, analysis of specific promoters of known stress genes showed some of these, including recA, dnaK and sodA, to be inducible by the lactoperoxidase-thiocyanate enzyme system. The results show that the lactoperoxidase-thiocyanate enzyme system elicits a distinct stress response different from but partly overlapping other oxidative stress responses. Several of the induced genes or pathways may be involved in bacterial defense against the toxic effects of the lactoperoxidase-thiocyanate enzyme system.

  20. Automated Immobilized Metal Affinity Chromatography System for Enrichment of Escherichia coli Phosphoproteome

    SciTech Connect

    Qu, Yi; Wu, Si; Zhao, Rui; Zink, Erika M.; Orton, Daniel J.; Moore, Ronald J.; Meng, Da; Clauss, Therese RW; Aldrich, Joshua T.; Lipton, Mary S.; Pasa-Tolic, Ljiljana

    2013-06-05

    Enrichment of bacterial phosphopeptides is an essential step prior to bottom-up mass spectrometry-based analysis of the phosphoproteome, which is fundamental to understanding the role of phosphoproteins in cell signaling and regulation of protein activity. We developed an automated IMAC system to enrich strong cation exchange-fractionated phosphopeptides from the soluble proteome of Escherichia coli MG1655 grown on minimal medium. Initial demonstration of the system resulted in identification of 75 phosphopeptides covering 52 phosphoproteins. Consistent with previous studies, many of these phosphoproteins are involved in the carbohydrate portion of central metabolism. The automated system utilizes a large capacity IMAC column that can effectively enrich phosphopeptides from a bacterial sample by increasing peptide loading and reducing the wash time. An additional benefit of the automated IMAC system is reduced labor and associated costs.

  1. The flagellar cytoskeleton of the spirochetes.

    PubMed

    Wolgemuth, Charles W; Charon, Nyles W; Goldstein, Stuart F; Goldstein, Raymond E

    2006-01-01

    The recent discoveries of prokaryotic homologs of all three major eukaryotic cytoskeletal proteins (actin, tubulin, intermediate filaments) have spurred a resurgence of activity in the field of bacterial morphology. In spirochetes, however, it has long been known that the flagellar filaments act as a cytoskeletal protein structure, contributing to their shape and conferring motility on this unique phylum of bacteria. Therefore, revisiting the spirochete cytoskeleton may lead to new paradigms for exploring general features of prokaryotic morphology. This review discusses the role that the periplasmic flagella in spirochetes play in maintaining shape and producing motility. We focus on four species of spirochetes: Borrelia burgdorferi, Treponema denticola, Treponema phagedenis and Leptonema (formerly Leptospira) illini. In spirochetes, the flagella reside in the periplasmic space. Rotation of the flagella in the above species by a flagellar motor induces changes in the cell morphology that drives motility. Mutants that do not produce flagella have a markedly different shape than wild-type cells. PMID:16983197

  2. Flagellar Synchronization Independent of Hydrodynamic Interactions

    NASA Astrophysics Data System (ADS)

    Friedrich, Benjamin M.; Jülicher, Frank

    2012-09-01

    Inspired by the coordinated beating of the flagellar pair of the green algae Chlamydomonas, we study theoretically a simple, mirror-symmetric swimmer, which propels itself at low Reynolds number by a revolving motion of a pair of spheres. We show that perfect synchronization between these two driven spheres can occur due to the motion of the swimmer and local hydrodynamic friction forces. Hydrodynamic interactions, though crucial for net propulsion, contribute little to synchronization for this free-moving swimmer.

  3. Small Intestine Early Innate Immunity Response during Intestinal Colonization by Escherichia coli Depends on Its Extra-Intestinal Virulence Status.

    PubMed

    Tourret, Jérôme; Willing, Benjamin P; Croxen, Matthew A; Dufour, Nicolas; Dion, Sara; Wachtel, Sarah; Denamur, Erick; Finlay, B Brett

    2016-01-01

    Uropathogenic Escherichia coli (UPEC) strains live as commensals in the digestive tract of the host, but they can also initiate urinary tract infections. The aim of this work was to determine how a host detects the presence of a new UPEC strain in the digestive tract. Mice were orally challenged with UPEC strains 536 and CFT073, non-pathogenic strain K12 MG1655, and ΔPAI-536, an isogenic mutant of strain 536 lacking all 7 pathogenicity islands whose virulence is drastically attenuated. Intestinal colonization was measured, and cytokine expression was determined in various organs recovered from mice after oral challenge. UPEC strain 536 efficiently colonized the mouse digestive tract, and prior Enterobacteriaceae colonization was found to impact strain 536 colonization efficiency. An innate immune response, detected as the production of TNFα, IL-6 and IL-10 cytokines, was activated in the ileum 48 hours after oral challenge with strain 536, and returned to baseline within 8 days, without a drop in fecal pathogen load. Although inflammation was detected in the ileum, histology was normal at the time of cytokine peak. Comparison of cytokine secretion 48h after oral gavage with E. coli strain 536, CFT073, MG1655 or ΔPAI-536 showed that inflammation was more pronounced with UPECs than with non-pathogenic or attenuated strains. Pathogenicity islands also seemed to be involved in host detection, as IL-6 intestinal secretion was increased after administration of E. coli strain 536, but not after administration of ΔPAI-536. In conclusion, UPEC colonization of the mouse digestive tract activates acute phase inflammatory cytokine secretion but does not trigger any pathological changes, illustrating the opportunistic nature of UPECs. This digestive tract colonization model will be useful for studying the factors controlling the switch from commensalism to pathogenicity. PMID:27096607

  4. Small Intestine Early Innate Immunity Response during Intestinal Colonization by Escherichia coli Depends on Its Extra-Intestinal Virulence Status.

    PubMed

    Tourret, Jérôme; Willing, Benjamin P; Croxen, Matthew A; Dufour, Nicolas; Dion, Sara; Wachtel, Sarah; Denamur, Erick; Finlay, B Brett

    2016-01-01

    Uropathogenic Escherichia coli (UPEC) strains live as commensals in the digestive tract of the host, but they can also initiate urinary tract infections. The aim of this work was to determine how a host detects the presence of a new UPEC strain in the digestive tract. Mice were orally challenged with UPEC strains 536 and CFT073, non-pathogenic strain K12 MG1655, and ΔPAI-536, an isogenic mutant of strain 536 lacking all 7 pathogenicity islands whose virulence is drastically attenuated. Intestinal colonization was measured, and cytokine expression was determined in various organs recovered from mice after oral challenge. UPEC strain 536 efficiently colonized the mouse digestive tract, and prior Enterobacteriaceae colonization was found to impact strain 536 colonization efficiency. An innate immune response, detected as the production of TNFα, IL-6 and IL-10 cytokines, was activated in the ileum 48 hours after oral challenge with strain 536, and returned to baseline within 8 days, without a drop in fecal pathogen load. Although inflammation was detected in the ileum, histology was normal at the time of cytokine peak. Comparison of cytokine secretion 48h after oral gavage with E. coli strain 536, CFT073, MG1655 or ΔPAI-536 showed that inflammation was more pronounced with UPECs than with non-pathogenic or attenuated strains. Pathogenicity islands also seemed to be involved in host detection, as IL-6 intestinal secretion was increased after administration of E. coli strain 536, but not after administration of ΔPAI-536. In conclusion, UPEC colonization of the mouse digestive tract activates acute phase inflammatory cytokine secretion but does not trigger any pathological changes, illustrating the opportunistic nature of UPECs. This digestive tract colonization model will be useful for studying the factors controlling the switch from commensalism to pathogenicity.

  5. Small Intestine Early Innate Immunity Response during Intestinal Colonization by Escherichia coli Depends on Its Extra-Intestinal Virulence Status

    PubMed Central

    Willing, Benjamin P.; Croxen, Matthew A.; Dufour, Nicolas; Dion, Sara; Wachtel, Sarah; Denamur, Erick; Finlay, B. Brett

    2016-01-01

    Uropathogenic Escherichia coli (UPEC) strains live as commensals in the digestive tract of the host, but they can also initiate urinary tract infections. The aim of this work was to determine how a host detects the presence of a new UPEC strain in the digestive tract. Mice were orally challenged with UPEC strains 536 and CFT073, non-pathogenic strain K12 MG1655, and ΔPAI-536, an isogenic mutant of strain 536 lacking all 7 pathogenicity islands whose virulence is drastically attenuated. Intestinal colonization was measured, and cytokine expression was determined in various organs recovered from mice after oral challenge. UPEC strain 536 efficiently colonized the mouse digestive tract, and prior Enterobacteriaceae colonization was found to impact strain 536 colonization efficiency. An innate immune response, detected as the production of TNFα, IL-6 and IL-10 cytokines, was activated in the ileum 48 hours after oral challenge with strain 536, and returned to baseline within 8 days, without a drop in fecal pathogen load. Although inflammation was detected in the ileum, histology was normal at the time of cytokine peak. Comparison of cytokine secretion 48h after oral gavage with E. coli strain 536, CFT073, MG1655 or ΔPAI-536 showed that inflammation was more pronounced with UPECs than with non-pathogenic or attenuated strains. Pathogenicity islands also seemed to be involved in host detection, as IL-6 intestinal secretion was increased after administration of E. coli strain 536, but not after administration of ΔPAI-536. In conclusion, UPEC colonization of the mouse digestive tract activates acute phase inflammatory cytokine secretion but does not trigger any pathological changes, illustrating the opportunistic nature of UPECs. This digestive tract colonization model will be useful for studying the factors controlling the switch from commensalism to pathogenicity. PMID:27096607

  6. 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

  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. 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

  9. 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

  10. Inhibitio of flagellar coordination in Spirillum volutans.

    PubMed

    Krieg, N R; Tomelty, J P; Wells, J S

    1967-11-01

    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; MgSO(4), Mg(NO(3))(2), NiSO(4), NiCl(2), CuSO(4), and CuCl(2) 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 NiSO(4) 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 NiSO(4) and caused dual-head flagellation. Optimal concentrations of test compounds were determined with the use of pure cultures and a reproducible growth medium. PMID:6057800

  11. 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

  12. Shear Stress Transmission Model for the Flagellar Rotary Motor

    PubMed Central

    Mitsui, Toshio; Ohshima, Hiroyuki

    2008-01-01

    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 charasteristic torque-velocity relationship of the flagellar rotation. PMID:19325821

  13. 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.

  14. Laboratory adapted Escherichia coli K-12 becomes a pathogen of Caenorhabditis elegans upon restoration of O antigen biosynthesis.

    PubMed

    Browning, Douglas F; Wells, Timothy J; França, Fernanda L S; Morris, Faye C; Sevastsyanovich, Yanina R; Bryant, Jack A; Johnson, Matthew D; Lund, Peter A; Cunningham, Adam F; Hobman, Jon L; May, Robin C; Webber, Mark A; Henderson, Ian R

    2013-03-01

    Escherichia coli has been the leading model organism for many decades. It is a fundamental player in modern biology, facilitating the molecular biology revolution of the last century. The acceptance of E. coli as model organism is predicated primarily on the study of one E. coli lineage; E. coli K-12. However, the antecedents of today's laboratory strains have undergone extensive mutagenesis to create genetically tractable offspring but which resulted in loss of several genetic traits such as O antigen expression. Here we have repaired the wbbL locus, restoring the ability of E. coli K-12 strain MG1655 to express the O antigen. We demonstrate that O antigen production results in drastic alterations of many phenotypes and the density of the O antigen is critical for the observed phenotypes. Importantly, O antigen production enables laboratory strains of E. coli to enter the gut of the Caenorhabditis elegans worm and to kill C. elegans at rates similar to pathogenic bacterial species. We demonstrate C. elegans killing is a feature of other commensal E. coli. We show killing is associated with bacterial resistance to mechanical shear and persistence in the C. elegans gut. These results suggest C. elegans is not an effective model of human-pathogenic E. coli infectious disease.

  15. The SOS response is permitted in Escherichia coli strains deficient in the expression of the mazEF pathway.

    PubMed

    Kalderon, Ziva; Kumar, Sathish; Engelberg-Kulka, Hanna

    2014-01-01

    The Escherichia coli (E. coli) SOS response is the largest, most complex, and best characterized bacterial network induced by DNA damage. It is controlled by a complex network involving the RecA and LexA proteins. We have previously shown that the SOS response to DNA damage is inhibited by various elements involved in the expression of the E. coli toxin-antitoxin mazEF pathway. Since the mazEF module is present on the chromosomes of most E. coli strains, here we asked: Why is the SOS response found in so many E. coli strains? Is the mazEF module present but inactive in those strains? We examined three E. coli strains used for studies of the SOS response, strains AB1932, BW25113, and MG1655. We found that each of these strains is either missing or inhibiting one of several elements involved in the expression of the mazEF-mediated death pathway. Thus, the SOS response only takes place in E. coli cells in which one or more elements of the E. coli toxin-antitoxin module mazEF or its downstream pathway is not functioning.

  16. Molecular serotyping of Escherichia coli: A verification and reclassification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Serotyping of E. coli, based on the O- (polysaccharide side chain) and H- (flagellar) antigens using antisera is a common practice for diagnostics, outbreak investigations, and epidemiological surveillance. The full set of E. coli serogroups comprises O-groups O1 to O181, with several O...

  17. Analysis of Genome Plasticity in Pathogenic and Commensal Escherichia coli Isolates by Use of DNA Arrays

    PubMed Central

    Dobrindt, Ulrich; Agerer, Franziska; Michaelis, Kai; Janka, Andreas; Buchrieser, Carmen; Samuelson, Martin; Svanborg, Catharina; Gottschalk, Gerhard; Karch, Helge; Hacker, Jörg

    2003-01-01

    Genomes of prokaryotes differ significantly in size and DNA composition. Escherichia coli is considered a model organism to analyze the processes involved in bacterial genome evolution, as the species comprises numerous pathogenic and commensal variants. Pathogenic and nonpathogenic E. coli strains differ in the presence and absence of additional DNA elements contributing to specific virulence traits and also in the presence and absence of additional genetic information. To analyze the genetic diversity of pathogenic and commensal E. coli isolates, a whole-genome approach was applied. Using DNA arrays, the presence of all translatable open reading frames (ORFs) of nonpathogenic E. coli K-12 strain MG1655 was investigated in 26 E. coli isolates, including various extraintestinal and intestinal pathogenic E. coli isolates, 3 pathogenicity island deletion mutants, and commensal and laboratory strains. Additionally, the presence of virulence-associated genes of E. coli was determined using a DNA “pathoarray” developed in our laboratory. The frequency and distributional pattern of genomic variations vary widely in different E. coli strains. Up to 10% of the E. coli K-12-specific ORFs were not detectable in the genomes of the different strains. DNA sequences described for extraintestinal or intestinal pathogenic E. coli are more frequently detectable in isolates of the same origin than in other pathotypes. Several genes coding for virulence or fitness factors are also present in commensal E. coli isolates. Based on these results, the conserved E. coli core genome is estimated to consist of at least 3,100 translatable ORFs. The absence of K-12-specific ORFs was detectable in all chromosomal regions. These data demonstrate the great genome heterogeneity and genetic diversity among E. coli strains and underline the fact that both the acquisition and deletion of DNA elements are important processes involved in the evolution of prokaryotes. PMID:12618447

  18. A role for the membrane in regulating Chlamydomonas flagellar length.

    PubMed

    Dentler, William

    2013-01-01

    Flagellar assembly requires coordination between the assembly of axonemal proteins and the assembly of the flagellar membrane and membrane proteins. Fully grown steady-state Chlamydomonas flagella release flagellar vesicles from their tips and failure to resupply membrane should affect flagellar length. To study vesicle release, plasma and flagellar membrane surface proteins were vectorially pulse-labeled and flagella and vesicles were analyzed for biotinylated proteins. Based on the quantity of biotinylated proteins in purified vesicles, steady-state flagella appeared to shed a minimum of 16% of their surface membrane per hour, equivalent to a complete flagellar membrane being released every 6 hrs or less. Brefeldin-A destroyed Chlamydomonas Golgi, inhibited the secretory pathway, inhibited flagellar regeneration, and induced full-length flagella to disassemble within 6 hrs, consistent with flagellar disassembly being induced by a failure to resupply membrane. In contrast to membrane lipids, a pool of biotinylatable membrane proteins was identified that was sufficient to resupply flagella as they released vesicles for 6 hrs in the absence of protein synthesis and to support one and nearly two regenerations of flagella following amputation. These studies reveal the importance of the secretory pathway to assemble and maintain full-length flagella.

  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. Programmed synthesis of flagellar tubulin during cell differentiation in Naegleria.

    PubMed

    Fulton, C; Kowit, J D

    1975-06-30

    Amebae of Naegleria gruberi differentiate into flagellates when transferred from growth medium to non-nutrient buffer. This differentiation, which requires 48 min at 28 degrees C, is dependent on transcription and translation. Tubulin of the flagellar outer doublets comprises about 0.15% of the protein of flagellate, and only about 1-2% of the total tubulin. An antiserum to flagellar (outer-doublet) tubulin contains antibodies that react selectively with flagellar tubulin. Measurements using this antiserum have shown that 97-98% of the flagellar tubulin antigen appears during differentiation. The appearance of tubulin antigen is sensitive to actinomycin D and cycloheximide. Isotope dilution experiments using [35S]methione demonstrated that at least 70% of the flagellar tubulin is synthesized from amino acids during differentiation. Experiments using both the specific antiserum and isotopes have shown that flagellar tubulin synthesis begins about one-third of the way through differentiation, before any morphological change has occurred. These experiments demonstrate that most, if not all, of the flagellar tubulin is synthesized de novo during differentiation, and that cells selectively use a specific subpopulation of tubulin in assembling the outer doub)lets. The results bring into focus major unsolved questions about the synthesis and assembly of flagellar tubulin. PMID:1056749

  1. 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

  2. Locomotive consequences of non-axisymmetric flagellar configurations

    NASA Astrophysics Data System (ADS)

    Fu, Henry; Marcos, Marcos; Hyon, Yunkyong; Powers, Thomas; Stocker, Roman

    2011-11-01

    Although peritrichous bacteria can form flagellar bundles at many attachment points and directions relative to the cell body, locomotion of these bacteria is often modeled as arising from a polar bundle oriented along the cell body axis. We discuss the consequences of non-axisymmetric flagellar configurations for bacterial locomotion and implications for bacterial behavior using a boundary element method (BEM) based on the method of regularized Stokeslets. We validate our BEM by comparing to analytic results for spheres and ellipsoids, as well as results in the literature for axisymmetric flagella with spherical and ellipsoidal heads obtained from other boundary element methods and slender body theory. Non-axisymmetric flagellar configurations generically lead to wobbling cell bodies and wiggling helical cell trajectories, both of which have been observed experimentally. We compare experimental and numerically calculated wiggling trajectories to deduce information about flagellar geometries of swimming B. subtilis. We discuss the implications of off-axis flagellar geometries for bacterial rheotaxis and chemotaxis.

  3. Persistence of Escherichia coli in batch and continuous vermicomposting systems.

    PubMed

    Hénault-Ethier, Louise; Martin, Vincent J J; Gélinas, Yves

    2016-10-01

    Vermicomposting is a biooxidation process in which epigeicearthworms act in synergy with microbial populations to degrade organic matter. Vermicomposting does not go through a thermophilic stage as required by North American legislations for pathogen eradication. We examined the survival of a Green Fluorescent Protein (GFP) labeled Escherichia coli MG1655 as a model for the survival of pathogenic bacteria in both small-scale batch and medium-scale continuously-operated systems to discern the influence of the earthworm Eisenia fetida, nutrient content and the indigenous vermicompost microbial community on pathogen abundance. In batch systems, the microbial community had the greatest influence on the rapid decline of E. coli populations, and the effect of earthworms was only visible in microbially-impoverishedvermicomposts. No significant earthworm density-dependent relationship was observed on E. coli survival under continuous operation. E. coli numbers decreased below the US EPA compost sanitation guidelines of 10(3)Colony Forming Units (CFU)/g (dry weight) within 18-21days for both the small-scale batch and medium-scale continuous systems, but it took up to 51days without earthworms and with an impoverished microbial community to reach the legal limit. Nutrient replenishment (i.e. organic carbon) provided by continuous feed input did not appear to extend E. coli survival. In fact, longer survival of E. coli was noticed in treatments where less total and labile sugars were available, suggesting that sugars may support potentially antagonist bacteria in the vermicompost. Total N, pH and humidity did not appear to affect E. coli survival. Several opportunistic human pathogens may be found in vermicompost, and their populations are likely kept in check by antagonists.

  4. Persistence of Escherichia coli in batch and continuous vermicomposting systems.

    PubMed

    Hénault-Ethier, Louise; Martin, Vincent J J; Gélinas, Yves

    2016-10-01

    Vermicomposting is a biooxidation process in which epigeicearthworms act in synergy with microbial populations to degrade organic matter. Vermicomposting does not go through a thermophilic stage as required by North American legislations for pathogen eradication. We examined the survival of a Green Fluorescent Protein (GFP) labeled Escherichia coli MG1655 as a model for the survival of pathogenic bacteria in both small-scale batch and medium-scale continuously-operated systems to discern the influence of the earthworm Eisenia fetida, nutrient content and the indigenous vermicompost microbial community on pathogen abundance. In batch systems, the microbial community had the greatest influence on the rapid decline of E. coli populations, and the effect of earthworms was only visible in microbially-impoverishedvermicomposts. No significant earthworm density-dependent relationship was observed on E. coli survival under continuous operation. E. coli numbers decreased below the US EPA compost sanitation guidelines of 10(3)Colony Forming Units (CFU)/g (dry weight) within 18-21days for both the small-scale batch and medium-scale continuous systems, but it took up to 51days without earthworms and with an impoverished microbial community to reach the legal limit. Nutrient replenishment (i.e. organic carbon) provided by continuous feed input did not appear to extend E. coli survival. In fact, longer survival of E. coli was noticed in treatments where less total and labile sugars were available, suggesting that sugars may support potentially antagonist bacteria in the vermicompost. Total N, pH and humidity did not appear to affect E. coli survival. Several opportunistic human pathogens may be found in vermicompost, and their populations are likely kept in check by antagonists. PMID:27499290

  5. 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

  6. 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. PMID:25362512

  7. 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.

  8. The extracellular RNA complement of Escherichia coli

    PubMed Central

    Ghosal, Anubrata; Upadhyaya, Bimal Babu; Fritz, Joëlle V; Heintz-Buschart, Anna; Desai, Mahesh S; Yusuf, Dilmurat; Huang, David; Baumuratov, Aidos; Wang, Kai; Galas, David; Wilmes, Paul

    2015-01-01

    The secretion of biomolecules into the extracellular milieu is a common and well-conserved phenomenon in biology. In bacteria, secreted biomolecules are not only involved in intra-species communication but they also play roles in inter-kingdom exchanges and pathogenicity. To date, released products, such as small molecules, DNA, peptides, and proteins, have been well studied in bacteria. However, the bacterial extracellular RNA complement has so far not been comprehensively characterized. Here, we have analyzed, using a combination of physical characterization and high-throughput sequencing, the extracellular RNA complement of both outer membrane vesicle (OMV)-associated and OMV-free RNA of the enteric Gram-negative model bacterium Escherichia coli K-12 substrain MG1655 and have compared it to its intracellular RNA complement. Our results demonstrate that a large part of the extracellular RNA complement is in the size range between 15 and 40 nucleotides and is derived from specific intracellular RNAs. Furthermore, RNA is associated with OMVs and the relative abundances of RNA biotypes in the intracellular, OMV and OMV-free fractions are distinct. Apart from rRNA fragments, a significant portion of the extracellular RNA complement is composed of specific cleavage products of functionally important structural noncoding RNAs, including tRNAs, 4.5S RNA, 6S RNA, and tmRNA. In addition, the extracellular RNA pool includes RNA biotypes from cryptic prophages, intergenic, and coding regions, of which some are so far uncharacterised, for example, transcripts mapping to the fimA-fimL and ves-spy intergenic regions. Our study provides the first detailed characterization of the extracellular RNA complement of the enteric model bacterium E. coli. Analogous to findings in eukaryotes, our results suggest the selective export of specific RNA biotypes by E. coli, which in turn indicates a potential role for extracellular bacterial RNAs in intercellular communication. PMID:25611733

  9. Flagellar synchronization through direct hydrodynamic interactions

    PubMed Central

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

    2014-01-01

    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 PMID:25073925

  10. Flagellar synchronization through direct hydrodynamic interactions.

    PubMed

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

    2014-01-01

    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. PMID:25073925

  11. 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

  12. Differentiating enteric Escherichia coli from environmental bacteria through the putative glucosyltransferase gene (ycjM).

    PubMed

    Deng, Daiyong; Zhang, Ning; Mustapha, Azlin; Xu, Dong; Wuliji, Tumen; Farley, Mary; Yang, John; Hua, Bin; Liu, Fengjing; Zheng, Guolu

    2014-09-15

    This study is to tackle the challenge posed by the "naturalized" Escherichia coli population against the worldwide practice of E. coli-based water quality monitoring. In the literature, the putative glucosyltransferase gene (ycjM) of E. coli has been identified in silico to be one of the 114 genes specific to enteric E. coli. Based on the sequence of E. coli K-12 MG1655, a PCR assay (ycjPCR) targeting ycjM was developed in this study. As demonstrated by the ycjPCR assay using 367 E. coli strains isolated from animal feces, 97.2% of the isolates carried the ycjM with variations from 93.9% to 100% among nine different host sources, but none of the 17 strains of non-E. coli bacteria and only 23.0% of the environment-isolated cryptic Escherichia strains contained the ycjM. These data experimentally confirmed ycjM to be enteric specific. Our study also showed that the ycjPCR assay was superior to the commonly used tuf- or uidA-based PCR methods in differentiating enteric E. coli from ß-D-glucuronidase-positive environmental bacteria. Furthermore, study on 190 E. coli isolates from water samples, using EPA Method 1603 followed by bacterial identification with Biolog MicroStation™ and ycjPCR assay, indicated that the prevalence of ycjM in the E. coli water isolates had a significant (p < 0.05, odds ratio ) spatial variation from 69.6% to 93.8%. These data suggest that E. coli profile using EPA Method 1603 or other ß-D-glucuronidase-activity-based methods may need further analysis using the ycjM profile to accurately determinate fecal pollution in water.

  13. Proteomic Adaptations to Starvation Prepare Escherichia coli for Disinfection Tolerance

    PubMed Central

    Du, Zhe; Nandakumar, Renu; Nickerson, Kenneth; Li, Xu

    2015-01-01

    Despite the low nutrient level and constant presence of secondary disinfectants, bacterial re-growth still occurs in drinking water distribution systems. The molecular mechanisms that starved bacteria use to survive low-level chlorine-based disinfectants are not well understood. The objective of this study is to investigate these molecular mechanisms at the protein level that prepare starved cells for disinfection tolerance. Two commonly used secondary disinfectants chlorine and monochloramine, both at 1 mg/L, were used in this study. The proteomes of normal and starved Escherichia coli (K12 MG1655) cells were studied using quantitative proteomics. Over 60-min disinfection, starved cells showed significantly higher disinfection tolerance than normal cells based on the inactivation curves for both chlorine and monochloramine. Proteomic analyses suggest that starvation may prepare cells for the oxidative stress that chlorine-based disinfection will cause by affecting glutathione metabolism. In addition, proteins involved in stress regulation and stress responses were among the ones up-regulated under both starvation and chlorine/monochloramine disinfection. By comparing the fold changes under different conditions, it is suggested that starvation prepares E. coli for disinfection tolerance by increasing the expression of enzymes that can help cells survive chlorine/monochloramine disinfection. Protein co-expression analyses show that proteins in glycolysis and pentose phosphate pathway that were up-regulated under starvation are also involved in disinfection tolerance. Finally, the production and detoxification of methylglyoxal may be involved in the chlorine-based disinfection and cell defense mechanisms. PMID:25463932

  14. Seven gene deletions in seven days: Fast generation of Escherichia coli strains tolerant to acetate and osmotic stress.

    PubMed

    Jensen, Sheila I; Lennen, Rebecca M; Herrgård, Markus J; Nielsen, Alex T

    2015-12-08

    Generation of multiple genomic alterations is currently a time consuming process. Here, a method was established that enables highly efficient and simultaneous deletion of multiple genes in Escherichia coli. A temperature sensitive plasmid containing arabinose inducible lambda Red recombineering genes and a rhamnose inducible flippase recombinase was constructed to facilitate fast marker-free deletions. To further speed up the procedure, we integrated the arabinose inducible lambda Red recombineering genes and the rhamnose inducible FLP into the genome of E. coli K-12 MG1655. This system enables growth at 37 °C, thereby facilitating removal of integrated antibiotic cassettes and deletion of additional genes in the same day. Phosphorothioated primers were demonstrated to enable simultaneous deletions during one round of electroporation. Utilizing these methods, we constructed strains in which four to seven genes were deleted in E. coli W and E. coli K-12. The growth rate of an E. coli K-12 quintuple deletion strain was significantly improved in the presence of high concentrations of acetate and NaCl. In conclusion, we have generated a method that enables efficient and simultaneous deletion of multiple genes in several E. coli variants. The method enables deletion of up to seven genes in as little as seven days.

  15. Seven gene deletions in seven days: Fast generation of Escherichia coli strains tolerant to acetate and osmotic stress

    PubMed Central

    Jensen, Sheila I.; Lennen, Rebecca M.; Herrgård, Markus J.; Nielsen, Alex T.

    2015-01-01

    Generation of multiple genomic alterations is currently a time consuming process. Here, a method was established that enables highly efficient and simultaneous deletion of multiple genes in Escherichia coli. A temperature sensitive plasmid containing arabinose inducible lambda Red recombineering genes and a rhamnose inducible flippase recombinase was constructed to facilitate fast marker-free deletions. To further speed up the procedure, we integrated the arabinose inducible lambda Red recombineering genes and the rhamnose inducible FLP into the genome of E. coli K-12 MG1655. This system enables growth at 37 °C, thereby facilitating removal of integrated antibiotic cassettes and deletion of additional genes in the same day. Phosphorothioated primers were demonstrated to enable simultaneous deletions during one round of electroporation. Utilizing these methods, we constructed strains in which four to seven genes were deleted in E. coli W and E. coli K-12. The growth rate of an E. coli K-12 quintuple deletion strain was significantly improved in the presence of high concentrations of acetate and NaCl. In conclusion, we have generated a method that enables efficient and simultaneous deletion of multiple genes in several E. coli variants. The method enables deletion of up to seven genes in as little as seven days. PMID:26643270

  16. Seven gene deletions in seven days: Fast generation of Escherichia coli strains tolerant to acetate and osmotic stress.

    PubMed

    Jensen, Sheila I; Lennen, Rebecca M; Herrgård, Markus J; Nielsen, Alex T

    2015-01-01

    Generation of multiple genomic alterations is currently a time consuming process. Here, a method was established that enables highly efficient and simultaneous deletion of multiple genes in Escherichia coli. A temperature sensitive plasmid containing arabinose inducible lambda Red recombineering genes and a rhamnose inducible flippase recombinase was constructed to facilitate fast marker-free deletions. To further speed up the procedure, we integrated the arabinose inducible lambda Red recombineering genes and the rhamnose inducible FLP into the genome of E. coli K-12 MG1655. This system enables growth at 37 °C, thereby facilitating removal of integrated antibiotic cassettes and deletion of additional genes in the same day. Phosphorothioated primers were demonstrated to enable simultaneous deletions during one round of electroporation. Utilizing these methods, we constructed strains in which four to seven genes were deleted in E. coli W and E. coli K-12. The growth rate of an E. coli K-12 quintuple deletion strain was significantly improved in the presence of high concentrations of acetate and NaCl. In conclusion, we have generated a method that enables efficient and simultaneous deletion of multiple genes in several E. coli variants. The method enables deletion of up to seven genes in as little as seven days. PMID:26643270

  17. Undiscovered regions on the molecular landscape of flagellar assembly.

    PubMed

    Altegoer, Florian; Bange, Gert

    2015-12-01

    The bacterial flagellum is a motility structure and one of the most complicated motors in the biosphere. A flagellum consists of several dozens of building blocks in different stoichiometries and extends from the cytoplasm to the extracellular space. Flagellar biogenesis follows a strict spatio-temporal regime that is guided by a plethora of flagellar assembly factors and chaperones. The goal of this review is to summarize our current structural and mechanistic knowledge of this intricate process and to identify the undiscovered regions on the molecular landscape of flagellar assembly. PMID:26490009

  18. 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

  19. 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

  20. 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. PMID:27581279

  1. 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. PMID

  2. 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.

  3. 16S ribosomal RNA pseudouridine synthase RsuA of Escherichia coli: deletion, mutation of the conserved Asp102 residue, and sequence comparison among all other pseudouridine synthases.

    PubMed

    Conrad, J; Niu, L; Rudd, K; Lane, B G; Ofengand, J

    1999-06-01

    The gene for RsuA, the pseudouridine synthase that converts U516 to pseudouridine in 16S ribosomal RNA of Escherichia coli, has been deleted in strains MG1655 and BL21/DE3. Deletion of this gene resulted in the specific loss of pseudouridine516 in both cell lines, and replacement of the gene in trans on a plasmid restored the pseudouridine. Therefore, rsuA is the only gene in E. coli with the ability to produce a protein capable of forming pseudouridine516. There was no effect on the growth rate of rsuA- MG1655 either in rich or minimal medium at either 24, 37, or 42 degrees C. Plasmid rescue of the BL21/DE3 rsuA- strain using pET15b containing an rsuA gene with aspartate102 replaced by asparagine or threonine demonstrated that neither mutant was active in vivo. This result supports a role for this aspartate, located in a unique GRLD sequence in this gene, at the catalytic center of the synthase. Induction of wild-type and the two mutant synthases in strain BL21/DE3 from genes in pET15b yielded a strong overexpression of all three proteins in approximately equal amounts showing that the mutations did not affect production of the protein in vivo and thus that the lack of activity was not due to a failure to produce a gene product. Aspartate102 is found in a conserved motif present in many pseudouridine synthases. The conservation and distribution of this motif in nature was assessed.

  4. Plasmid DNA production with Escherichia coli GALG20, a pgi-gene knockout strain: fermentation strategies and impact on downstream processing.

    PubMed

    Gonçalves, Geisa A L; Prather, Kristala L J; Monteiro, Gabriel A; Carnes, Aaron E; Prazeres, Duarte M F

    2014-09-30

    The market development of plasmid biopharmaceuticals for gene therapy and DNA vaccination applications is critically dependent on the availability of cost-effective manufacturing processes capable of delivering large amounts of high-quality plasmid DNA (pDNA) for clinical trials and commercialization. The producer host strain used in these processes must be designed to meet the upstream and downstream processing challenges characteristic of large scale pDNA production. The goal of the present study was to investigate the effect of different glucose feeding strategies (batch and fed-batch) on the pDNA productivity of GALG20, a pgi Escherichia coli strain potentially useful in industrial fermentations, which uses the pentose phosphate pathway (PPP) as the main route for glucose metabolism. The parental strain, MG1655ΔendAΔrecA, and the common laboratory strain, DH5α, were used for comparison purposes and pVAX1GFP, a ColE1-type plasmid, was tested as a model. GALG20 produced 3-fold more pDNA (∼141 mg/L) than MG1655ΔendAΔrecA (∼48 mg/L) and DH5α (∼40 mg/L) in glucose-based fed-batch fermentations. The amount of pDNA in lysates obtained from these cells was also larger for GALG20 (41%) when compared with MG1655ΔendAΔrecA (31%) and DH5α (26%). However, the final quality of pDNA preparations obtained with a process that explores precipitation, hydrophobic interaction chromatography and size exclusion was not significantly affected by strain genotype. Finally, high cell density fed-batch cultures were performed with GALG20, this time using another ColE1-type plasmid, NTC7482-41H-HA, in pre-industrial facilities using glucose and glycerol. These experiments demonstrated the ability of GALG20 to produce high pDNA yields of the order of 2100-2200 mg/L.

  5. Effect of acetate formation pathway and long chain fatty acid CoA-ligase on the free fatty acid production in E. coli expressing acy-ACP thioesterase from Ricinus communis.

    PubMed

    Li, Mai; Zhang, Xiujun; Agrawal, Arpita; San, Ka-Yiu

    2012-07-01

    Microbial biosynthesis of fatty acid like chemicals from renewable carbon sources has attracted significant attention in recent years. Free fatty acids can be used as precursors for the production of fuels or chemicals. Wild type E. coli strains produce fatty acids mainly for the biosynthesis of lipids and cell membranes and do not accumulate free fatty acids as intermediates in lipid biosynthesis. However, free fatty acids can be produced by breaking the fatty acid elongation through the overexpression of an acyl-ACP thioesterase. Since acetyl-CoA might be an important factor for fatty acid synthesis (acetate formation pathways are the main competitive pathways in consuming acetyl-CoA or pyruvate, a precursor of acetyl-CoA), and the long chain fatty acid CoA-ligase (FadD) plays a pivotal role in the transport and activation of exogenous fatty acids prior to their subsequent degradation, we examined the composition and the secretion of the free fatty acids in four different strains including the wild type MG1655, a mutant strain with inactivation of the fatty acid beta-oxidation pathway (fadD mutant (ML103)), and mutant strains with inactivation of the two major acetate production pathways (an ack-pta (acetate kinase/phosphotransacetylase), poxB (pyruvate oxidase) double mutant (ML112)) and a fadD, ack-pta, poxB triple mutant (ML115). The engineered E. coli cells expressing acyl-ACP thioesterase with glucose yield is higher than 40% of theoretical yield. Compared to MG1655(pXZ18) and ML103(pXZ18), acetate forming pathway deletion strains such as ML112(pXZ18) and ML115(pXZ18) produced similar quantity of total free fatty acids, which indicated that acetyl-CoA availability does not appear to be limiting factor for fatty acid production in these strains. However, these strains did show significant differences in the composition of free fatty acids. Different from MG1655(pXZ18) and ML103(pXZ18), acetate formation pathway deletion strains such as ML112(pXZ18) and ML115

  6. Variability of the tandem repeat region of the Escherichia coli tolA gene.

    PubMed

    Zhou, Kai; Vanoirbeek, Kristof; Aertsen, Abram; Michiels, Chris W

    2012-06-01

    An intragenic tandem repeat (TR) region has been previously reported in the tolA gene of Escherichia coli. In silico analysis of 123 E. coli tolA sequences from Genbank and PCR analysis of the tolA TR region from 111 additional E. coli strains revealed that this TR region is highly variable. Nine different TR sizes with 8 up to 16 repeat units were found in in silico analysis and 6 of these were also found by PCR analysis. The 13-unit TR emerged as the predominant type using both approaches (47.2% and 86.5%, respectively). Remarkably, TRs in pathogenic strains appeared to be more variable than those in non-pathogens. To demonstrate the occurrence of TR variation in a clonal population, a selection system for TR deletion events was constructed by inserting the 13-unit TR region of MG1655 in frame into a plasmid-borne chloramphenicol acetyltransferase (cat) gene. The resulting cat gene no longer conferred chloramphenicol resistance unless the insert size was reduced by TR contraction. Using this system, Cm-resistant revertants with a TR contraction were recovered at a frequency of 1.1 × 10(-7), and contraction was shown to be recA-dependent and enhanced in a DNA repair-deficient mutS background. PMID:22659144

  7. Engineering Escherichia coli for selective geraniol production with minimized endogenous dehydrogenation.

    PubMed

    Zhou, Jia; Wang, Chonglong; Yoon, Sang-Hwal; Jang, Hui-Jeong; Choi, Eui-Sung; Kim, Seon-Won

    2014-01-01

    Geraniol, a monoterpene alcohol, has versatile applications in the fragrance industry, pharmacy and agrochemistry. Moreover, geraniol could be an ideal gasoline alternative. In this study, recombinant overexpression of geranyl diphosphate synthase and the bottom portion of a foreign mevalonate pathway in Escherichia coli MG1655 produced 13.3mg/L of geraniol. Introduction of Ocimum basilicum geraniol synthase increased geraniol production to 105.2mg/L. However, geraniol production encountered a loss from its endogenous dehydrogenization and isomerization into other geranoids (nerol, neral and geranial). Three E. coli enzymes (YjgB, YahK and YddN) were identified with high sequence identity to plant geraniol dehydrogenases. YjgB was demonstrated to be the major one responsible for geraniol dehydrogenization. Deletion of yjgB increased geraniol production to 129.7mg/L. Introduction of the whole mevalonate pathway for enhanced building block synthesis from endogenously synthesized mevalonate improved geraniol production up to 182.5mg/L in the yjgB mutant after 48h of culture, which was a double of that obtained in the wild type control (96.5mg/L). Our strategy for improving geraniol production in engineered E. coli should be generalizable for addressing similar problems during metabolic engineering. PMID:24269531

  8. Exchange of rotor components in functioning bacterial flagellar motor

    SciTech Connect

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

    2010-03-26

    The bacterial flagellar motor is a rotary motor driven by the electrochemical potential of a coupling ion. The interaction between a rotor and stator units is thought to generate torque. The overall structure of flagellar motor has been thought to be static, however, it was recently proved that stators are exchanged in a rotating motor. Understanding the dynamics of rotor components in functioning motor is important for the clarifying of working mechanism of bacterial flagellar motor. In this study, we focused on the dynamics and the turnover of rotor components in a functioning flagellar motor. Expression systems for GFP-FliN, FliM-GFP, and GFP-FliG were constructed, and each GFP-fusion was functionally incorporated into the flagellar motor. To investigate whether the rotor components are exchanged in a rotating motor, we performed fluorescence recovery after photobleaching experiments using total internal reflection fluorescence microscopy. After photobleaching, in a tethered cell producing GFP-FliN or FliM-GFP, the recovery of fluorescence at the rotational center was observed. However, in a cell producing GFP-FliG, no recovery of fluorescence was observed. The transition phase of fluorescence intensity after full or partially photobleaching allowed the turnover of FliN subunits to be calculated as 0.0007 s{sup -1}, meaning that FliN would be exchanged in tens of minutes. These novel findings indicate that a bacterial flagellar motor is not a static structure even in functioning state. This is the first report for the exchange of rotor components in a functioning bacterial flagellar motor.

  9. The Transcription Unit Architecture of the Escherichia Coli Genome

    SciTech Connect

    Cho, Byung-Kwan; Zengler, Karsten; Qiu, Yu; Park, Young S.; Knight, Eric M.; Barrett, Christian; Gao, Yuan; Palsson, Bernhard O.

    2009-11-01

    Under EMSL User Proposal 25660, the authors reported that bacterial genomes are organized by structural and functional elements, including promoters, transcription start and termination sites, open reading frames, regulatory noncoding regions, untranslated regions and transcription units. Here, we iteratively integrate high-throughput, genome-wide measurements of RNA polymerase binding locations and mRNA transcript abundance, 5' sequences and translation into proteins to determine the organizational structure of the Escherichia coli K-12 MG1655 genome. Integration of the organizational elements provides an experimentally annotated transcription unit architecture, including alternative transcription start sites, 5' untranslated region, boundaries and open reading frames of each transcription unit. A total of 4,661 transcription units were identified, representing an increase of >530% over current knowledge. This comprehensive transcription unit architecture allows for the elucidation of condition-specific uses of alternative sigma factors at the genome scale. Furthermore, the transcription unit architecture provides a foundation on which to construct genome-scale transcriptional and translational regulatory networks.

  10. Sequential Assembly of Flagellar Radial Spokes

    PubMed Central

    Diener, Dennis R.; Yang, Pinfen; Geimer, Stefan; Cole, Douglas G.; Sale, Winfield S.; Rosenbaum, Joel L.

    2013-01-01

    The unicellular alga Chlamydomonas can assemble two 10 μm flagella in one hour from proteins synthesized in the cell body. Targeting and transporting these proteins to the flagella are simplified by preassembly of macromolecular complexes in the cell body. Radial spokes are flagellar complexes that are partially assembled in the cell body before entering the flagella. On the axoneme, radial spokes are “T” shaped structures with a head of 5 proteins and a stalk of 18 proteins that sediment together at 20S. In the cell body, radial spokes are partially assembled; about half of the radial spoke proteins (RSPs) form a 12S complex. In mutants lacking a single radial spoke protein, smaller spoke subassemblies were identified. When extracts from two such mutants were mixed in vitro the 12S complex was assembled from several smaller complexes demonstrating that portions of the stepwise assembly of radial spoke assembly can be carried out in vitro to elucidate the order of spoke assembly in the cell body. PMID:21692193

  11. 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.

  12. 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

  13. 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

  14. 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.

  15. Active phase and amplitude fluctuations of flagellar beating.

    PubMed

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

    2014-07-25

    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.

  16. 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

  17. 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.

  18. Development of an Escherichia coli K12-specific quantitative polymerase chain reaction assay and DNA isolation suited to biofilms associated with iron drinking water pipe corrosion products.

    PubMed

    Lu, Jingrang; Gerke, Tammie L; Buse, Helen Y; Ashbolt, Nicholas J

    2014-12-01

    A quantitative polymerase chain reaction assay (115 bp amplicon) specific to Escherichia coli K12 with an ABI(TM) internal control was developed based on sequence data encoding the rfb gene cluster. Assay specificity was evaluated using three E. coli K12 strains (ATCC W3110, MG1655 & DH1), 24 non-K12 E. coli and 23 bacterial genera. The biofilm detection limit was 10(3) colony-forming units (CFU) E. coli K12 mL(-1), but required a modified protocol, which included a bio-blocker Pseudomonas aeruginosa with ethylenediaminetetraacetic acid buffered to pH 5 prior to cell lysis/DNA extraction. The novel protocol yielded the same sensitivity for drinking water biofilms associated with Fe3O4 (magnetite)-coated SiO2 (quartz) grains and biofilm-surface iron corrosion products from a drinking water distribution system. The novel DNA extraction protocol and specific E. coli K12 assay are sensitive and robust enough for detection and quantification within iron drinking water pipe biofilms, and are particularly well suited for studying enteric bacterial interactions within biofilms.

  19. Probiotic Escherichia coli Nissle 1917 reduces growth, Shiga toxin expression, release and thus cytotoxicity of enterohemorrhagic Escherichia coli.

    PubMed

    Mohsin, Mashkoor; Guenther, Sebastian; Schierack, Peter; Tedin, Karsten; Wieler, Lothar H

    2015-01-01

    Due to increased release or production of Shiga toxin by Enterohemorrhagic Escherichia coli (EHEC) after exposure to antimicrobial agents, the role of antimicrobial agents in EHEC mediated infections remains controversial. Probiotics are therefore rapidly gaining interest as an alternate therapeutic option. The well-known probiotic strain Escherichia coli Nissle 1917 (EcN) was tested in vitro to determine its probiotic effects on growth, Shiga toxin (Stx) gene expression, Stx amount and associated cytotoxicity on the most important EHEC strains of serotype O104:H4 and O157:H7. Following co-culture of EcN:EHEC in broth for 4 and 24 h, the probiotic effects on EHEC growth, toxin gene expression, Stx amount and cytotoxicity were determined using quantitative real time-PCR, Stx-ELISA and Vero cytotoxicity assays. Probiotic EcN strongly reduced EHEC numbers (cfu) of O104:H4 up to (68%) and O157:H7 to (72.2%) (p<0.05) in LB broth medium whereas the non-probiotic E. coli strain MG1655 had no effect on EHEC growth. The level of stx expression was significantly down-regulated, particularly for the stx2a gene. The stx down-regulation in EcN co-culture was not due to reduced numbers of EHEC. A significant inhibition in Stx amounts and cytotoxicity were also observed in sterile supernatants of EcN:EHEC co-cultures. These findings indicate that probiotic EcN displays strong inhibitory effects on growth, Shiga toxin gene expression, amount and cytotoxicity of EHEC strains. Thus, EcN may be considered as a putative therapeutic candidate, in particular against EHEC O104:H4 and O157:H7. PMID:25465158

  20. The flaA locus of Bacillus subtilis is part of a large operon coding for flagellar structures, motility functions, and an ATPase-like polypeptide.

    PubMed Central

    Albertini, A M; Caramori, T; Crabb, W D; Scoffone, F; Galizzi, A

    1991-01-01

    We cloned and sequenced 8.3 kb of Bacillus subtilis DNA corresponding to the flaA locus involved in flagellar biosynthesis, motility, and chemotaxis. The DNA sequence revealed the presence of 10 complete and 2 incomplete open reading frames. Comparison of the deduced amino acid sequences to data banks showed similarities of nine of the deduced products to a number of proteins of Escherichia coli and Salmonella typhimurium for which a role in flagellar functioning has been directly demonstrated. In particular, the sequence data suggest that the flaA operon codes for the M-ring protein, components of the motor switch, and the distal part of the basal-body rod. The gene order is remarkably similar to that described for region III of the enterobacterial flagellar regulon. One of the open reading frames was translated into a protein with 48% amino acid identity to S. typhimurium FliI and 29% identity to the beta subunit of E. coli ATP synthase. PMID:1828465

  1. Cranberry (Vaccinium macrocarpon) oligosaccharides decrease biofilm formation by uropathogenic Escherichia coli

    PubMed Central

    Sun, Jiadong; Marais, Jannie P. J.; Khoo, Christina; LaPlante, Kerry; Vejborg, Rebecca M.; Givskov, Michael; Tolker-Nielsen, Tim; Seeram, Navindra P.; Rowley, David C.

    2015-01-01

    The preventive effects of the American cranberry (Vaccinium macrocarpon) against urinary tract infections are supported by extensive studies which have primarily focused on its phenolic constituents. Herein, a phenolic-free carbohydrate fraction (designated cranf1b-F2) was purified from cranberry fruit using ion exchange and size exclusion chromatography. MALDI-TOF-MS analysis revealed that the cranf1b-F2 constituents are predominantly oligosaccharides possessing various degrees of polymerisation and further structural analysis (by GC-MS and NMR) revealed mainly xyloglucan and arabinan residues. In antimicrobial assays, cranf1b-F2 (at 1.25 mg/mL concentration) reduced biofilm production by the uropathogenic Escherichia coli CFT073 strain by over 50% but did not inhibit bacterial growth. Cranf1b-F2 (ranging from 0.625 - 10 mg/mL) also inhibited biofilm formation of the non-pathogenic E. coli MG1655 strain up to 60% in a concentration-dependent manner. These results suggest that cranberry oligosaccharides, in addition to its phenolic constituents, may play a role in its preventive effects against urinary tract infections. PMID:26613004

  2. Transcriptional effects of polyamines on ribosomal proteins and on polyamine-synthesizing enzymes in Escherichia coli.

    PubMed

    Huang, S C; Panagiotidis, C A; Canellakis, E S

    1990-05-01

    We find that the transcription of various ribosomal proteins can be differentially affected by polyamines and by changes in growth rates. Using strain MG1655 of Escherichia coli K-12 (F-, lambda-), we have determined the effects of polyamines and changes in growth rate on the transcription of several ribosomal genes and the polyamine-synthesizing enzymes ornithine decarboxylase (L-ornithine carboxy-lyase; EC 4.1.1.17) and arginine decarboxylase (L-arginine carboxylyase; EC 4.1.1.19). Ribosomal proteins S20 and L34 can be differentiated from the other ribosomal proteins studied; the transcription of S20 and L34 is especially sensitive to polyamines and less sensitive to changes in growth rates. In contrast, the transcription of S10, S15, S19, L2, L4, L20, L22, and L23 is insensitive to polyamines although it is particularly sensitive to changes in growth rates. Like S20 and L34, the transcription of ornithine decarboxylase and arginine decarboxylase is especially sensitive to polyamines. Polyamines specifically enhance the transcription of ribosomal proteins S20 and L34, and decrease that of ornithine decarboxylase and arginine decarboxylase. It is evident that polyamines can exert both positive and negative regulation of gene expression in E. coli that can be differentiated from the effects caused by changes in growth rates.

  3. A new custom microarray for sRNA profiling in Escherichia coli.

    PubMed

    Ruiz-Larrabeiti, Olatz; Plágaro, Ander Hernández; Gracia, Celine; Sevillano, Elena; Gallego, Lucía; Hajnsdorf, Eliane; Kaberdin, Vladimir R

    2016-07-01

    Bacterial small RNAs (sRNAs) play essential roles in the post-transcriptional control of gene expression. To improve their detection by conventional microarrays, we designed a custom microarray containing a group of probes targeting known and some putative Escherichia coli sRNAs. To assess its potential in detection of sRNAs, RNA profiling experiments were performed with total RNA extracted from E. coli MG1655 cells exponentially grown in rich (Luria-Bertani) and minimal (M9/glucose) media. We found that many sRNAs could yield reasonably strong and statistically significant signals corresponding to nearly all sRNAs annotated in the EcoCyc database. Besides differential expression of two sRNAs (GcvB and RydB), expression of other sRNAs was less affected by the composition of the growth media. Other examples of the differentially expressed sRNAs were revealed by comparing gene expression of the wild-type strain and its isogenic mutant lacking functional poly(A) polymerase I (pcnB). Further, northern blot analysis was employed to validate these data and to assess the existence of new putative sRNAs. Our results suggest that the use of custom microarrays with improved capacities for detection of sRNAs can offer an attractive opportunity for efficient gene expression profiling of sRNAs and their target mRNAs at the whole transcriptome level. PMID:27190161

  4. Metabolic Response of Escherichia coli upon Treatment with Hypochlorite at Sub-Lethal Concentrations

    PubMed Central

    Winter, Jeannette; Eisenreich, Wolfgang

    2015-01-01

    Hypochlorite is a reactive oxygen species that is worldwide as an antibacterial disinfectant. Hypochlorite exposure is known to cause oxidative damage to DNA and proteins. As a response to these effects, the metabolite profiles of organisms treated with sub-lethal doses of hypochlorite are assumed to be severely modified; however, the nature of these changes is hardly understood. Therefore, using nuclear magnetic resonance spectroscopy and gas chromatography-coupled mass spectrometry, we analyzed the time-dependent impact of hypochlorite exposure with a sub-lethal concentration (50 µM) on the metabolite profile of the Escherichia coli strain MG1655. Principle component analysis clearly distinguished between the metabolite profiles of bacteria treated for 0, 5,10, 20, 40, or 60 min. Major changes in the relative amounts of fatty acids, acetic acid, and formic acid occurred within the first 5 min. Comparative gas chromatography-coupled mass spectrometry analyses revealed that the amounts of free methionine and alanine were significantly decreased in the treated cells, demonstrating their susceptibility to hypochlorite exposure. The concentrations of succinate, urea, orotic acid, 2-aminobutyric acid, and 2-hydroxybutyric acid were also severely affected, indicating general changes in the metabolic network by hypochlorite. However, most metabolite levels relaxed to the reference values of untreated cells after 40–60 min, reflecting the capability of E. coli to rapidly adapt to environmental stress factors such as the presence of sub-lethal oxidant levels. PMID:25932918

  5. Screening of an Escherichia coli promoter library for a phenylalanine biosensor.

    PubMed

    Mahr, Regina; von Boeselager, Raphael Freiherr; Wiechert, Johanna; Frunzke, Julia

    2016-08-01

    In recent years, the application of transcription factor-based biosensors for the engineering of microbial production strains opened up new opportunities for industrial biotechnology. However, the design of synthetic regulatory circuits depends on the selection of suitable transcription factor-promoter pairs to convert the concentration of effector molecules into a measureable output. Here, we present an efficient strategy to screen promoter libraries for appropriate parts for biosensor design. To this end, we pooled the strains of the Alon library containing about 2000 different Escherichia coli promoter-gfpmut2 fusions, and enriched galactose- and L-phenylalanine-responsive promoters by toggled rounds of positive and negative selection using fluorescence-activated cell sorting (FACS). For both effectors, responsive promoters were isolated and verified by cultivation in microtiter plates. The promoter of mtr, encoding an L-tryptophan-specific transporter, was identified as suitable part for the construction of an L-phenylalanine biosensor. In the following, we performed a comparative analysis of different biosensor constructs based on the mtr promoter. The obtained data revealed a strong influence of the biosensor architecture on the performance characteristics. For proof-of-principle, the mtr sensor was applied in a FACS high-throughput screening of an E. coli MG1655 mutant library for the isolation of L-phenylalanine producers. These results emphasize the developed screening approach as a convenient strategy for the identification of effector-responsive promoters for the design of novel biosensors. PMID:27170323

  6. Acetoin Synthesis Acquisition Favors Escherichia coli Growth at Low pH

    PubMed Central

    Vivijs, Bram; Moons, Pieter; Aertsen, Abram

    2014-01-01

    Some members of the family Enterobacteriaceae ferment sugars via the mixed-acid fermentation pathway. This yields large amounts of acids, causing strong and sometimes even lethal acidification of the environment. Other family members employ the 2,3-butanediol fermentation pathway, which generates comparatively less acidic and more neutral end products, such as acetoin and 2,3-butanediol. In this work, we equipped Escherichia coli MG1655 with the budAB operon, encoding the acetoin pathway, from Serratia plymuthica RVH1 and investigated how this affected the ability of E. coli to cope with acid stress during growth. Acetoin fermentation prevented lethal medium acidification by E. coli in lysogeny broth (LB) supplemented with glucose. It also supported growth and higher stationary-phase cell densities in acidified LB broth with glucose (pH 4.10 to 4.50) and in tomato juice (pH 4.40 to 5.00) and reduced the minimal pH at which growth could be initiated. On the other hand, the acetoin-producing strain was outcompeted by the nonproducer in a mixed-culture experiment at low pH, suggesting a fitness cost associated with acetoin production. Finally, we showed that acetoin production profoundly changes the appearance of E. coli on several diagnostic culture media. Natural E. coli strains that have laterally acquired budAB genes may therefore have escaped detection thus far. This study demonstrates the potential importance of acetoin fermentation in the ecology of E. coli in the food chain and contributes to a better understanding of the microbiological stability and safety of acidic foods. PMID:25063653

  7. Acetoin synthesis acquisition favors Escherichia coli growth at low pH.

    PubMed

    Vivijs, Bram; Moons, Pieter; Aertsen, Abram; Michiels, Chris W

    2014-10-01

    Some members of the family Enterobacteriaceae ferment sugars via the mixed-acid fermentation pathway. This yields large amounts of acids, causing strong and sometimes even lethal acidification of the environment. Other family members employ the 2,3-butanediol fermentation pathway, which generates comparatively less acidic and more neutral end products, such as acetoin and 2,3-butanediol. In this work, we equipped Escherichia coli MG1655 with the budAB operon, encoding the acetoin pathway, from Serratia plymuthica RVH1 and investigated how this affected the ability of E. coli to cope with acid stress during growth. Acetoin fermentation prevented lethal medium acidification by E. coli in lysogeny broth (LB) supplemented with glucose. It also supported growth and higher stationary-phase cell densities in acidified LB broth with glucose (pH 4.10 to 4.50) and in tomato juice (pH 4.40 to 5.00) and reduced the minimal pH at which growth could be initiated. On the other hand, the acetoin-producing strain was outcompeted by the nonproducer in a mixed-culture experiment at low pH, suggesting a fitness cost associated with acetoin production. Finally, we showed that acetoin production profoundly changes the appearance of E. coli on several diagnostic culture media. Natural E. coli strains that have laterally acquired budAB genes may therefore have escaped detection thus far. This study demonstrates the potential importance of acetoin fermentation in the ecology of E. coli in the food chain and contributes to a better understanding of the microbiological stability and safety of acidic foods.

  8. Complete Genome Sequence of ER2796, a DNA Methyltransferase-Deficient Strain of Escherichia coli K-12.

    PubMed

    Anton, Brian P; Mongodin, Emmanuel F; Agrawal, Sonia; Fomenkov, Alexey; Byrd, Devon R; Roberts, Richard J; Raleigh, Elisabeth A

    2015-01-01

    We report the complete sequence of ER2796, a laboratory strain of Escherichia coli K-12 that is completely defective in DNA methylation. Because of its lack of any native methylation, it is extremely useful as a host into which heterologous DNA methyltransferase genes can be cloned and the recognition sequences of their products deduced by Pacific Biosciences Single-Molecule Real Time (SMRT) sequencing. The genome was itself sequenced from a long-insert library using the SMRT platform, resulting in a single closed contig devoid of methylated bases. Comparison with K-12 MG1655, the first E. coli K-12 strain to be sequenced, shows an essentially co-linear relationship with no major rearrangements despite many generations of laboratory manipulation. The comparison revealed a total of 41 insertions and deletions, and 228 single base pair substitutions. In addition, the long-read approach facilitated the surprising discovery of four gene conversion events, three involving rRNA operons and one between two cryptic prophages. Such events thus contribute both to genomic homogenization and to bacteriophage diversification. As one of relatively few laboratory strains of E. coli to be sequenced, the genome also reveals the sequence changes underlying a number of classical mutant alleles including those affecting the various native DNA methylation systems.

  9. A novel transducible chimeric phage from Escherichia coli O157:H7 Sakai strain encoding Stx1 production.

    PubMed

    Sváb, Domonkos; Bálint, Balázs; Maróti, Gergely; Tóth, István

    2015-01-01

    Shiga toxin-producing Escherichia coli (STEC), and especially enterohaemorrhagic E. coli (EHEC) are important, highly virulent zoonotic and food-borne pathogens. The genes encoding their key virulence factors, the Shiga toxins, are distributed by converting bacteriophages, the Stx phages. In this study we isolated a new type of inducible Stx phage carrying the stx1 gene cluster from the prototypic EHEC O157:H7 Sakai strain. The phage showed Podoviridae morphology, and was capable of converting the E. coli K-12 MG1655 strain to Shiga toxin-producing phenotype. The majority of the phage genes originate from the stx2-encoding Sakai prophage Sp5, with major rearrangements in its genome. Beside certain minor recombinations, the genomic region originally containing the stx2 genes in Sp5 was replaced by a region containing six open reading frames from prophage Sp15 including stx1 genes. The rearranged genome, together with the carriage of stx1 genes, the morphology and the capability of lysogenic conversion represent a new type of recombinant Stx1 converting phage from the Sakai strain. PMID:25445656

  10. Complete Genome Sequence of ER2796, a DNA Methyltransferase-Deficient Strain of Escherichia coli K-12

    PubMed Central

    Anton, Brian P.; Mongodin, Emmanuel F.; Agrawal, Sonia; Fomenkov, Alexey; Byrd, Devon R.; Roberts, Richard J.; Raleigh, Elisabeth A.

    2015-01-01

    We report the complete sequence of ER2796, a laboratory strain of Escherichia coli K-12 that is completely defective in DNA methylation. Because of its lack of any native methylation, it is extremely useful as a host into which heterologous DNA methyltransferase genes can be cloned and the recognition sequences of their products deduced by Pacific Biosciences Single-Molecule Real Time (SMRT) sequencing. The genome was itself sequenced from a long-insert library using the SMRT platform, resulting in a single closed contig devoid of methylated bases. Comparison with K-12 MG1655, the first E. coli K-12 strain to be sequenced, shows an essentially co-linear relationship with no major rearrangements despite many generations of laboratory manipulation. The comparison revealed a total of 41 insertions and deletions, and 228 single base pair substitutions. In addition, the long-read approach facilitated the surprising discovery of four gene conversion events, three involving rRNA operons and one between two cryptic prophages. Such events thus contribute both to genomic homogenization and to bacteriophage diversification. As one of relatively few laboratory strains of E. coli to be sequenced, the genome also reveals the sequence changes underlying a number of classical mutant alleles including those affecting the various native DNA methylation systems. PMID:26010885

  11. Simulation of the rate of transfer of antibiotic resistance between Escherichia coli strains cultured under well controlled environmental conditions.

    PubMed

    Smelt, Jan P; Hoefsloot, Huub C; de Koster, Chris G; Schuurmans, Jasper M; ter Kuile, Benno H; Brul, Stanley

    2015-02-01

    It was demonstrated that the tetracycline resistance plasmid in Escherichia coli resembling K-12 23:06 containing the E. coli plasmid DM0133 could be transferred to tetracycline sensitive E. coli K-12 MG1655 YFP. The sensitive recipient strain has a slight metabolic advantage in continuous fermentation in absence of tetracycline pressure and as a result the numbers of the resistant recipient strain increase during fermentation. In presence of tetracycline pressure the sensitive strain is eliminated, but when it acquires tetracycline resistance the strain has still the same metabolic advantage as its sensitive parent strain in absence of tetracycline. Here a model will be shown that could explain the rate of transformation of a sensitive into a resistant recipient strain and its subsequent growth during continuous fermentation. According to the model the probability of formation of mutants would be much higher at the dilution rate of 0.09 compared to 0.28, whereas the growth of mutants would be much faster at high dilution rate. The growth model shows how the recipient mutants and the donor cells behave in relation to the dilution rate and the number of mutants. Apart from a deterministic model describing the growth rate of both the donor strain and the resistant recipient strain a stochastic model was developed that is particularly useful when low numbers of mutants are formed. PMID:25500384

  12. Reduced tubulin polyglutamylation suppresses flagellar shortness in Chlamydomonas.

    PubMed

    Kubo, Tomohiro; Hirono, Masafumi; Aikawa, Takumi; Kamiya, Ritsu; Witman, George B

    2015-08-01

    Ciliary length control is an incompletely understood process essential for normal ciliary function. The flagella of Chlamydomonas mutants lacking multiple axonemal dyneins are shorter than normal; previously it was shown that this shortness can be suppressed by the mutation suppressor of shortness 1 (ssh1) via an unknown mechanism. To elucidate this mechanism, we carried out genetic analysis of ssh1 and found that it is a new allele of TPG2 (hereafter tpg2-3), which encodes FAP234 functioning in tubulin polyglutamylation in the axoneme. Similar to the polyglutamylation-deficient mutants tpg1 and tpg2-1, tpg2-3 axonemal tubulin has a greatly reduced level of long polyglutamate side chains. We found that tpg1 and tpg2-1 mutations also promote flagellar elongation in short-flagella mutants, consistent with a polyglutamylation-dependent mechanism of suppression. Double mutants of tpg1 or tpg2-1 and fla10-1, a temperature-sensitive mutant of intraflagellar transport, underwent slower flagellar shortening than fla10-1 at restrictive temperatures, indicating that the rate of tubulin disassembly is decreased in the polyglutamylation-deficient flagella. Moreover, α-tubulin incorporation into the flagellar tips in temporary dikaryons was retarded in polyglutamylation-deficient flagella. These results show that polyglutamylation deficiency stabilizes axonemal microtubules, decelerating axonemal disassembly at the flagellar tip and shifting the axonemal assembly/disassembly balance toward assembly.

  13. 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.

  14. 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

  15. Flagellar motility is necessary for Aeromonas hydrophila adhesion.

    PubMed

    Qin, Yingxue; Lin, Guifang; Chen, Wenbo; Xu, Xiaojin; Yan, Qingpi

    2016-09-01

    Adhesion to host surface or cells is the initial step in bacterial pathogenesis, and the adhesion mechanisms of the fish pathogenic bacteria Aeromonas hydrophila were investigated in this study. First, a mutagenesis library of A. hydrophila that contained 332 random insertion mutants was constructed via mini-Tn10 Km mutagenesis. Four mutants displayed the most attenuated adhesion. Sequence analysis revealed that the mini-Tn10 insertion sites in the four mutant strains were flgC(GenBank accession numbers KX261880), cytb4(GenBank accession numbers JN133621), rbsR(GenBank accession numbers KX261881) and flgE(GenBank accession numbers JQ974982). To further study the roles of flgC and flgE in the adhesion of A. hydrophila, some biological characteristics of the wild-type strain B11, the mutants M121 and M240, and the complemented strains C121 and C240 were investigated. The results showed that the mutation in flgC or flgE led to the flagellar motility of A. hydrophila significant reduction or abolishment. flgC was not necessary for flagellar biosynthesis but was necessary for the full motility of A. hydrophila, flgE was involved in both flagellar biosynthesis and motility. The flagellar motility is necessary for A. hydrophila to adhere to the host mucus, which suggests flagellar motility plays crucial roles in the early infection process of this bacterium. PMID:27432325

  16. Genetic and biochemical analysis of the flagellar hook of Treponema phagedenis.

    PubMed Central

    Limberger, R J; Slivienski, L L; Samsonoff, W A

    1994-01-01

    The periplasmic flagellum of Treponema phagedenis consists of the flagellar filament and hook-basal body. We report here a characterization of the hook gene and flagellar hook of T. phagedenis, and in the process of this analysis we found evidence that the hook polypeptide is likely cross-linked in situ. A T. phagedenis genomic library was screened with a Treponema pallidum antiserum, and the DNA segments from several positive plaques were subcloned and sequenced. DNA sequencing of two overlapping segments revealed a 1,389-nucleotide (nt) open reading frame (ORF) with a deduced amino acid sequence that was 36% identical to that of FlgE, the hook polypeptide of Salmonella typhimurium. This gene was designated T. phagedenis flgE. Beginning at 312 nt downstream from flgE was a partial ORF of 486 nt with a deduced amino acid sequence that was 33% identical to that of MotA of Bacillus subtilis, a polypeptide that enables flagellar rotation. Upstream of flgE, separated by 39 nt, was a partial (291-nt) ORF with a deduced amino acid sequence that was homologous to that of ORF8, a polypeptide of unknown function located in an operon encoding polypeptides involved in motility of B. subtilis. The T. phagedenis flgE gene was cloned into an Escherichia coli protein expression plasmid, and the purified recombinant protein was used to prepare a FlgE antiserum. Western blots (immunoblots) of whole-cell lysates probed with this antiserum revealed a 55-kDa polypeptide and a ladder of polypeptide bands with increasing molecular masses. T. phagedenis hooks were then isolated and purified, and electron microscopic analysis revealed that the morphology of the hooks resembled that in other bacteria. The hooks were slightly curved and had an average length of 69 +/- 8 nm and a diameter of 23 +/- 1 nm. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blots of purified hook preparations using the FlgE antiserum also revealed a polypeptide ladder, suggesting that the

  17. The flagellar motor of Caulobacter crescentus generates more torque when a cell swims backwards

    NASA Astrophysics Data System (ADS)

    Lele, Pushkar P.; Roland, Thibault; Shrivastava, Abhishek; Chen, Yihao; Berg, Howard C.

    2016-02-01

    The bacterium Caulobacter crescentus swims by rotating a single right-handed helical filament. These cells have two swimming modes: a pusher mode, in which clockwise (CW) rotation of the filament thrusts the cell body forwards, and a puller mode, in which counterclockwise (CCW) rotation pulls it backwards. The situation is reversed in Escherichia coli, a bacterium that rotates several left-handed filaments CCW to drive the cell body forwards. The flagellar motor in E. coli generates more torque in the CCW direction than the CW direction in swimming cells. However, C. crescentus and other bacteria with single filaments swim forwards and backwards at similar speeds, prompting the assumption that motor torques in the two modes are the same. Here, we present evidence that motors in C. crescentus develop higher torques in the puller mode than in the pusher mode, and suggest that the anisotropy in torque generation is similar in the two species, despite the differences in filament handedness and motor bias.

  18. The flagellar motor of Caulobacter crescentus generates more torque when a cell swims backward

    PubMed Central

    Lele, Pushkar P.; Roland, Thibault; Shrivastava, Abhishek; Chen, Yihao; Berg, Howard C.

    2016-01-01

    Caulobacter crescentus, a monotrichous bacterium, swims by rotating a single right-handed helical filament. CW motor rotation thrusts the cell forward 1, a mode of motility known as the pusher mode; CCW motor rotation pulls the cell backward, a mode of motility referred to as the puller mode 2. The situation is opposite in E. coli, a peritrichous bacterium, where CCW rotation of multiple left-handed filaments drives the cell forward. The flagellar motor in E. coli generates more torque in the CCW direction than the CW direction in swimming cells 3,4. However, monotrichous bacteria including C. crescentus swim forward and backward at similar speeds, prompting the assumption that motor torques in the two modes are the same 5,6. Here, we present evidence that motors in C. crescentus develop higher torques in the puller mode than in the pusher mode, and suggest that the anisotropy in torque-generation is similar in two species, despite the differences in filament handedness and motor bias (probability of CW rotation). PMID:27499800

  19. Flagellar Length Control System: Testing a Simple Model Based on Intraflagellar Transport and Turnover

    PubMed Central

    Marshall, Wallace F.; Qin, Hongmin; Brenni, Mónica Rodrigo; Rosenbaum, Joel L.

    2005-01-01

    Flagellar length regulation provides a simple model system for addressing the general problem of organelle size control. Based on a systems-level analysis of flagellar dynamics, we have proposed a mechanism for flagellar length control in which length is set by the balance of continuous flagellar assembly and disassembly. The model proposes that the assembly rate is length dependent due to the inherent length dependence of intraflagellar transport, whereas disassembly is length independent, such that the two rates can only reach a balance point at a single length. In this report, we test this theoretical model by using three different measurements: 1) the quantity of intraflagellar transport machinery as a function of length, 2) the variation of flagellar length as a function of flagellar number, and 3) the rate of flagellar growth as a function of length. We find that the quantity of intraflagellar transport machinery is independent of length, that flagellar length is a decreasing function of flagellar number, and that flagellar growth rate in regenerating flagella depends on length and not on the time since regeneration began. These results are consistent with the balance-point model for length control. The three strategies used here are not limited to flagella and can in principle be adapted to probe size control systems for any organelle. PMID:15496456

  20. 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).

  1. Nonequivalence of membrane voltage and ion-gradient as driving forces for the bacterial flagellar motor at low load.

    PubMed

    Lo, Chien-Jung; Leake, Mark C; Pilizota, Teuta; Berry, Richard M

    2007-07-01

    Many bacterial species swim using flagella. The flagellar motor couples ion flow across the cytoplasmic membrane to rotation. Ion flow is driven by both a membrane potential (V(m)) and a transmembrane concentration gradient. To investigate their relation to bacterial flagellar motor function we developed a fluorescence technique to measure V(m) in single cells, using the dye tetramethyl rhodamine methyl ester. We used a convolution model to determine the relationship between fluorescence intensity in images of cells and intracellular dye concentration, and calculated V(m) using the ratio of intracellular/extracellular dye concentration. We found V(m) = -140 +/- 14 mV in Escherichia coli at external pH 7.0 (pH(ex)), decreasing to -85 +/- 10 mV at pH(ex) 5.0. We also estimated the sodium-motive force (SMF) by combining single-cell measurements of V(m) and intracellular sodium concentration. We were able to vary the SMF between -187 +/- 15 mV and -53 +/- 15 mV by varying pH(ex) in the range 7.0-5.0 and extracellular sodium concentration in the range 1-85 mM. Rotation rates for 0.35-microm- and 1-microm-diameter beads attached to Na(+)-driven chimeric flagellar motors varied linearly with V(m). For the larger beads, the two components of the SMF were equivalent, whereas for smaller beads at a given SMF, the speed increased with sodium gradient and external sodium concentration.

  2. Nonequivalence of Membrane Voltage and Ion-Gradient as Driving Forces for the Bacterial Flagellar Motor at Low Load

    PubMed Central

    Lo, Chien-Jung; Leake, Mark C.; Pilizota, Teuta; Berry, Richard M.

    2007-01-01

    Many bacterial species swim using flagella. The flagellar motor couples ion flow across the cytoplasmic membrane to rotation. Ion flow is driven by both a membrane potential (Vm) and a transmembrane concentration gradient. To investigate their relation to bacterial flagellar motor function we developed a fluorescence technique to measure Vm in single cells, using the dye tetramethyl rhodamine methyl ester. We used a convolution model to determine the relationship between fluorescence intensity in images of cells and intracellular dye concentration, and calculated Vm using the ratio of intracellular/extracellular dye concentration. We found Vm = −140 ± 14 mV in Escherichia coli at external pH 7.0 (pHex), decreasing to −85 ± 10 mV at pHex 5.0. We also estimated the sodium-motive force (SMF) by combining single-cell measurements of Vm and intracellular sodium concentration. We were able to vary the SMF between −187 ± 15 mV and −53 ± 15 mV by varying pHex in the range 7.0–5.0 and extracellular sodium concentration in the range 1–85 mM. Rotation rates for 0.35-μm- and 1-μm-diameter beads attached to Na+-driven chimeric flagellar motors varied linearly with Vm. For the larger beads, the two components of the SMF were equivalent, whereas for smaller beads at a given SMF, the speed increased with sodium gradient and external sodium concentration. PMID:17416615

  3. Gate-controlled proton diffusion and protonation-induced ratchet motion in the stator of the bacterial flagellar motor

    PubMed Central

    Nishihara, Yasutaka; Kitao, Akio

    2015-01-01

    The proton permeation process of the stator complex MotA/B in the flagellar motor of Escherichia coli was investigated. The atomic model structure of the transmembrane part of MotA/B was constructed based on the previously published disulfide cross-linking and tryptophan scanning mutations. The dynamic permeation of hydronium/sodium ions and water molecule through the channel formed in MotA/B was observed using a steered molecular dynamics simulation. During the simulation, Leu46 of MotB acts as the gate for hydronium ion permeation, which induced the formation of water wire that may mediate the proton transfer to Asp32 on MotB. Free energy profiles for permeation were calculated by umbrella sampling. The free energy barrier for H3O+ permeation was consistent with the proton transfer rate deduced from the flagellar rotational speed and number of protons per rotation, which suggests that the gating is the rate-limiting step. Structure and dynamics of the MotA/B with nonprotonated and protonated Asp32, Val43Met, and Val43Leu mutants in MotB were investigated using molecular dynamics simulation. A narrowing of the channel was observed in the mutants, which is consistent with the size-dependent ion selectivity. In MotA/B with the nonprotonated Asp32, the A3 segment in MotA maintained a kink whereas the protonation induced a straighter shape. Assuming that the cytoplasmic domain not included in the atomic model moves as a rigid body, the protonation/deprotonation of Asp32 is inferred to induce a ratchet motion of the cytoplasmic domain, which may be correlated to the motion of the flagellar rotor. PMID:26056313

  4. SILAC-based comparative analysis of pathogenic Escherichia coli secretomes.

    PubMed

    Boysen, Anders; Borch, Jonas; Krogh, Thøger Jensen; Hjernø, Karin; Møller-Jensen, Jakob

    2015-09-01

    Comparative studies of pathogenic bacteria and their non-pathogenic counterparts has led to the discovery of important virulence factors thereby generating insight into mechanisms of pathogenesis. Protein-based antigens for vaccine development are primarily selected among unique virulence-related factors produced by the pathogen of interest. However, recent work indicates that proteins that are not unique to the pathogen but instead selectively expressed compared to its non-pathogenic counterpart could also be vaccine candidates or targets for drug development. Modern methods in quantitative proteome analysis have the potential to discover both classes of proteins and hence form an important tool for discovering therapeutic targets. Adherent-invasive Escherichia coli (AIEC) and Enterotoxigenic E. coli (ETEC) are pathogenic variants of E. coli which cause intestinal disease in humans. AIEC is associated with Crohn's disease (CD), a chronic inflammatory condition of the gastrointestinal tract whereas ETEC is the major cause of human diarrhea which affects hundreds of millions annually. In spite of the disease burden associated with these pathogens, effective vaccines conferring long-term protection are still needed. In order to identify proteins with therapeutic potential, we have used mass spectrometry-based Stable Isotope Labeling with Amino acids in Cell culture (SILAC) quantitative proteomics method which allows us to compare the proteomes of pathogenic strains to commensal E. coli. In this study, we grew the pathogenic strains ETEC H10407, AIEC LF82 and the non-pathogenic reference strain E. coli K-12 MG1655 in parallel and used SILAC to compare protein levels in OMVs and culture supernatant. We have identified well-known virulence factors from both AIEC and ETEC, thus validating our experimental approach. In addition we find proteins that are not unique to the pathogenic strains but expressed at levels different from the commensal strain, including the

  5. SILAC-based comparative analysis of pathogenic Escherichia coli secretomes.

    PubMed

    Boysen, Anders; Borch, Jonas; Krogh, Thøger Jensen; Hjernø, Karin; Møller-Jensen, Jakob

    2015-09-01

    Comparative studies of pathogenic bacteria and their non-pathogenic counterparts has led to the discovery of important virulence factors thereby generating insight into mechanisms of pathogenesis. Protein-based antigens for vaccine development are primarily selected among unique virulence-related factors produced by the pathogen of interest. However, recent work indicates that proteins that are not unique to the pathogen but instead selectively expressed compared to its non-pathogenic counterpart could also be vaccine candidates or targets for drug development. Modern methods in quantitative proteome analysis have the potential to discover both classes of proteins and hence form an important tool for discovering therapeutic targets. Adherent-invasive Escherichia coli (AIEC) and Enterotoxigenic E. coli (ETEC) are pathogenic variants of E. coli which cause intestinal disease in humans. AIEC is associated with Crohn's disease (CD), a chronic inflammatory condition of the gastrointestinal tract whereas ETEC is the major cause of human diarrhea which affects hundreds of millions annually. In spite of the disease burden associated with these pathogens, effective vaccines conferring long-term protection are still needed. In order to identify proteins with therapeutic potential, we have used mass spectrometry-based Stable Isotope Labeling with Amino acids in Cell culture (SILAC) quantitative proteomics method which allows us to compare the proteomes of pathogenic strains to commensal E. coli. In this study, we grew the pathogenic strains ETEC H10407, AIEC LF82 and the non-pathogenic reference strain E. coli K-12 MG1655 in parallel and used SILAC to compare protein levels in OMVs and culture supernatant. We have identified well-known virulence factors from both AIEC and ETEC, thus validating our experimental approach. In addition we find proteins that are not unique to the pathogenic strains but expressed at levels different from the commensal strain, including the

  6. Chemical screening methods for flagellar phenotypes in Chlamydomonas.

    PubMed

    Avasthi, Prachee; Marshall, Wallace F

    2013-01-01

    Cilia and flagella are important organelles used for sensing the external cellular environment or for motility. Abnormalities in ciliary structure or function can have devastating pathological consequences ranging from sinusitis and obesity to polycystic kidney disease, retinal degeneration, and mental retardation. Chlamydomonas flagella are excellent models to study the regulation and normal function of cilia. We utilized the 1280 compound Sigma LOPAC annotated library to screen for phenotypes in Chlamydomonas flagellar length, motility, deflagellation, and cellular toxicity. Phenotypes were assessed by quantitation from direct microscopic visualization and custom-designed motility/viability assays. Compounds were clustered based on data across all assays to facilitate the identification of novel pathways regulating flagella in Chlamydomonas. These methods can both aid our understanding of the basic biology of flagellar regulation and provide useful points of therapeutic intervention for cilia-related disorders.

  7. Regulation of flagellar biogenesis by a calcium dependent protein kinase in Chlamydomonas reinhardtii.

    PubMed

    Liang, Yinwen; Pan, Junmin

    2013-01-01

    Chlamydomonas reinhardtii, a bi-flagellated green alga, is a model organism for studies of flagella or cilia related activities including cilia-based signaling, flagellar motility and flagellar biogenesis. Calcium has been shown to be a key regulator of these cellular processes whereas the signaling pathways linking calcium to these cellular functions are less understood. Calcium-dependent protein kinases (CDPKs), which are present in plants but not in animals, are also present in ciliated microorganisms which led us to examine their possible functions and mechanisms in flagellar related activities. By in silico analysis of Chlamydomonas genome we have identified 14 CDPKs and studied one of the flagellar localized CDPKs--CrCDPK3. CrCDPK3 was a protein of 485 amino acids and predicted to have a protein kinase domain at the N-terminus and four EF-hand motifs at the C-terminus. In flagella, CrCDPK3 was exclusively localized in the membrane matrix fraction and formed an unknown 20 S protein complex. Knockdown of CrCDPK3 expression by using artificial microRNA did not affect flagellar motility as well as flagellar adhesion and mating. Though flagellar shortening induced by treatment with sucrose or sodium pyrophosphate was not affected in RNAi strains, CrCDPK3 increased in the flagella, and pre-formed protein complex was disrupted. During flagellar regeneration, CrCDPK3 also increased in the flagella. When extracellular calcium was lowered to certain range by the addition of EGTA after deflagellation, flagellar regeneration was severely affected in RNAi cells compared with wild type cells. In addition, during flagellar elongation induced by LiCl, RNAi cells exhibited early onset of bulbed flagella. This work expands new functions of CDPKs in flagellar activities by showing involvement of CrCDPK3 in flagellar biogenesis in Chlamydomonas.

  8. 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.

  9. Approaches for functional analysis of flagellar proteins in African trypanosomes.

    PubMed

    Oberholzer, Michael; Lopez, Miguel A; Ralston, Katherine S; Hill, Kent L

    2009-01-01

    The eukaryotic flagellum is a highly conserved organelle serving motility, sensory, and transport functions. Although genetic, genomic, and proteomic studies have led to the identification of hundreds of flagellar and putative flagellar proteins, precisely how these proteins function individually and collectively to drive flagellum motility and other functions remains to be determined. In this chapter we provide an overview of tools and approaches available for studying flagellum protein function in the protozoan parasite Trypanosoma brucei. We begin by outlining techniques for in vitro cultivation of both T. brucei life cycle stages, as well as transfection protocols for the delivery of DNA constructs. We then describe specific assays used to assess flagellum function including flagellum preparation and quantitative motility assays. We conclude the chapter with a description of molecular genetic approaches for manipulating gene function. In summary, the availability of potent molecular tools, as well as the health and economic relevance of T. brucei as a pathogen, combine to make the parasite an attractive and integral experimental system for the functional analysis of flagellar proteins. PMID:20409810

  10. The effect of pyrite on Escherichia coli in water: proof-of-concept for the elimination of waterborne bacteria by reactive minerals.

    PubMed

    Friedlander, Lonia R; Puri, Neha; Schoonen, Martin A A; Wali Karzai, A

    2015-03-01

    We present proof-of-concept results for the elimination of waterborne bacteria by reactive minerals. We exposed Escherichia coli MG1655 suspended in water to the reactive mineral pyrite (FeS₂) at room temperature and ambient light. This slurry eliminates 99.9% of bacteria in fewer than 4 hours. We also exposed Escherichia coli to pyrite leachate (supernatant liquid from slurry after 24 hours), which eliminates 99.99% of bacteria over the same time-scale. Unlike SOlar water DISinfection (SODIS), our results do not depend on the presence of ultraviolet (UV) light. We confirmed this by testing proposed SODIS additive and known photo-catalyst anatase (TiO₂) for antibacterial properties and found that, in contrast to pyrite, it does not eliminate E. coli under our experimental conditions. Previous investigations of naturally antibiotic minerals have focused on the medical applications of antibiotic clays, and thus have not been conducted under experimental conditions resembling those found in water purification. In our examination of the relevant literature, we have not found previously reported evidence for the use of reactive minerals in water sanitization. The results from this proof-of-concept experiment may have important implications for future directions in household water purification research.

  11. The effect of pyrite on Escherichia coli in water: proof-of-concept for the elimination of waterborne bacteria by reactive minerals.

    PubMed

    Friedlander, Lonia R; Puri, Neha; Schoonen, Martin A A; Wali Karzai, A

    2015-03-01

    We present proof-of-concept results for the elimination of waterborne bacteria by reactive minerals. We exposed Escherichia coli MG1655 suspended in water to the reactive mineral pyrite (FeS₂) at room temperature and ambient light. This slurry eliminates 99.9% of bacteria in fewer than 4 hours. We also exposed Escherichia coli to pyrite leachate (supernatant liquid from slurry after 24 hours), which eliminates 99.99% of bacteria over the same time-scale. Unlike SOlar water DISinfection (SODIS), our results do not depend on the presence of ultraviolet (UV) light. We confirmed this by testing proposed SODIS additive and known photo-catalyst anatase (TiO₂) for antibacterial properties and found that, in contrast to pyrite, it does not eliminate E. coli under our experimental conditions. Previous investigations of naturally antibiotic minerals have focused on the medical applications of antibiotic clays, and thus have not been conducted under experimental conditions resembling those found in water purification. In our examination of the relevant literature, we have not found previously reported evidence for the use of reactive minerals in water sanitization. The results from this proof-of-concept experiment may have important implications for future directions in household water purification research. PMID:25719464

  12. Flow-cytometric study of vital cellular functions in Escherichia coli during solar disinfection (SODIS).

    PubMed

    Berney, Michael; Weilenmann, Hans-Ulrich; Egli, Thomas

    2006-06-01

    The effectiveness of solar disinfection (SODIS), a low-cost household water treatment method for developing countries, was investigated with flow cytometry and viability stains for the enteric bacterium Escherichia coli. A better understanding of the process of injury or death of E. coli during SODIS could be gained by investigating six different cellular functions, namely: efflux pump activity (Syto 9 plus ethidium bromide), membrane potential [bis-(1,3-dibutylbarbituric acid)trimethine oxonol; DiBAC4(3)], membrane integrity (LIVE/DEAD BacLight), glucose uptake activity (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose; 2-NBDG), total ATP concentration (BacTiter-Glo) and culturability (pour-plate method). These variables were measured in E. coli K-12 MG1655 cells that were exposed to either sunlight or artificial UVA light. The inactivation pattern of cellular functions was very similar for both light sources. A UVA light dose (fluence) of <500 kJ m(-2) was enough to lower the proton motive force, such that efflux pump activity and ATP synthesis decreased significantly. The loss of membrane potential, glucose uptake activity and culturability of >80 % of the cells was observed at a fluence of approximately 1500 kJ m(-2), and the cytoplasmic membrane of bacterial cells became permeable at a fluence of >2500 kJ m(-2). Culturable counts of stressed bacteria after anaerobic incubation on sodium pyruvate-supplemented tryptic soy agar closely correlated with the loss of membrane potential. The results strongly suggest that cells exposed to >1500 kJ m(-2) solar UVA (corresponding to 530 W m(-2) global sunlight intensity for 6 h) were no longer able to repair the damage and recover. Our study confirms the lethal effect of SODIS with cultivation-independent methods and gives a detailed picture of the 'agony' of E. coli when it is stressed with sunlight. PMID:16735735

  13. How to become a uropathogen: Comparative genomic analysis of extraintestinal pathogenic Escherichia coli strains

    PubMed Central

    Brzuszkiewicz, Elzbieta; Brüggemann, Holger; Liesegang, Heiko; Emmerth, Melanie; Ölschläger, Tobias; Nagy, Gábor; Albermann, Kaj; Wagner, Christian; Buchrieser, Carmen; Emődy, Levente; Gottschalk, Gerhard; Hacker, Jörg; Dobrindt, Ulrich

    2006-01-01

    Uropathogenic Escherichia coli (UPEC) strain 536 (O6:K15:H31) is one of the model organisms of extraintestinal pathogenic E. coli (ExPEC). To analyze this strain's genetic basis of urovirulence, we sequenced the entire genome and compared the data with the genome sequence of UPEC strain CFT073 (O6:K2:H1) and to the available genomes of nonpathogenic E. coli strain MG1655 (K-12) and enterohemorrhagic E. coli. The genome of strain 536 is ≈292 kb smaller than that of strain CFT073. Genomic differences between both UPEC are mainly restricted to large pathogenicity islands, parts of which are unique to strain 536 or CFT073. Genome comparison underlines that repeated insertions and deletions in certain parts of the genome contribute to genome evolution. Furthermore, 427 and 432 genes are only present in strain 536 or in both UPEC, respectively. The majority of the latter genes is encoded within smaller horizontally acquired DNA regions scattered all over the genome. Several of these genes are involved in increasing the pathogens' fitness and adaptability. Analysis of virulence-associated traits expressed in the two UPEC O6 strains, together with genome comparison, demonstrate the marked genetic and phenotypic variability among UPEC. The ability to accumulate and express a variety of virulence-associated genes distinguishes ExPEC from many commensals and forms the basis for the individual virulence potential of ExPEC. Accordingly, instead of a common virulence mechanism, different ways exist among ExPEC to cause disease. PMID:16912116

  14. How to become a uropathogen: comparative genomic analysis of extraintestinal pathogenic Escherichia coli strains.

    PubMed

    Brzuszkiewicz, Elzbieta; Brüggemann, Holger; Liesegang, Heiko; Emmerth, Melanie; Olschläger, Tobias; Nagy, Gábor; Albermann, Kaj; Wagner, Christian; Buchrieser, Carmen; Emody, Levente; Gottschalk, Gerhard; Hacker, Jörg; Dobrindt, Ulrich

    2006-08-22

    Uropathogenic Escherichia coli (UPEC) strain 536 (O6:K15:H31) is one of the model organisms of extraintestinal pathogenic E. coli (ExPEC). To analyze this strain's genetic basis of urovirulence, we sequenced the entire genome and compared the data with the genome sequence of UPEC strain CFT073 (O6:K2:H1) and to the available genomes of nonpathogenic E. coli strain MG1655 (K-12) and enterohemorrhagic E. coli. The genome of strain 536 is approximately 292 kb smaller than that of strain CFT073. Genomic differences between both UPEC are mainly restricted to large pathogenicity islands, parts of which are unique to strain 536 or CFT073. Genome comparison underlines that repeated insertions and deletions in certain parts of the genome contribute to genome evolution. Furthermore, 427 and 432 genes are only present in strain 536 or in both UPEC, respectively. The majority of the latter genes is encoded within smaller horizontally acquired DNA regions scattered all over the genome. Several of these genes are involved in increasing the pathogens' fitness and adaptability. Analysis of virulence-associated traits expressed in the two UPEC O6 strains, together with genome comparison, demonstrate the marked genetic and phenotypic variability among UPEC. The ability to accumulate and express a variety of virulence-associated genes distinguishes ExPEC from many commensals and forms the basis for the individual virulence potential of ExPEC. Accordingly, instead of a common virulence mechanism, different ways exist among ExPEC to cause disease.

  15. Flow-cytometric study of vital cellular functions in Escherichia coli during solar disinfection (SODIS).

    PubMed

    Berney, Michael; Weilenmann, Hans-Ulrich; Egli, Thomas

    2006-06-01

    The effectiveness of solar disinfection (SODIS), a low-cost household water treatment method for developing countries, was investigated with flow cytometry and viability stains for the enteric bacterium Escherichia coli. A better understanding of the process of injury or death of E. coli during SODIS could be gained by investigating six different cellular functions, namely: efflux pump activity (Syto 9 plus ethidium bromide), membrane potential [bis-(1,3-dibutylbarbituric acid)trimethine oxonol; DiBAC4(3)], membrane integrity (LIVE/DEAD BacLight), glucose uptake activity (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose; 2-NBDG), total ATP concentration (BacTiter-Glo) and culturability (pour-plate method). These variables were measured in E. coli K-12 MG1655 cells that were exposed to either sunlight or artificial UVA light. The inactivation pattern of cellular functions was very similar for both light sources. A UVA light dose (fluence) of <500 kJ m(-2) was enough to lower the proton motive force, such that efflux pump activity and ATP synthesis decreased significantly. The loss of membrane potential, glucose uptake activity and culturability of >80 % of the cells was observed at a fluence of approximately 1500 kJ m(-2), and the cytoplasmic membrane of bacterial cells became permeable at a fluence of >2500 kJ m(-2). Culturable counts of stressed bacteria after anaerobic incubation on sodium pyruvate-supplemented tryptic soy agar closely correlated with the loss of membrane potential. The results strongly suggest that cells exposed to >1500 kJ m(-2) solar UVA (corresponding to 530 W m(-2) global sunlight intensity for 6 h) were no longer able to repair the damage and recover. Our study confirms the lethal effect of SODIS with cultivation-independent methods and gives a detailed picture of the 'agony' of E. coli when it is stressed with sunlight.

  16. Membrane engineering via trans unsaturated fatty acids production improves Escherichia coli robustness and production of biorenewables.

    PubMed

    Tan, Zaigao; Yoon, Jong Moon; Nielsen, David R; Shanks, Jacqueline V; Jarboe, Laura R

    2016-05-01

    Constructing microbial biocatalysts that produce biorenewables at economically viable yields and titers is often hampered by product toxicity. For production of short chain fatty acids, membrane damage is considered the primary mechanism of toxicity, particularly in regards to membrane integrity. Previous engineering efforts in Escherichia coli to increase membrane integrity, with the goal of increasing fatty acid tolerance and production, have had mixed results. Herein, a novel approach was used to reconstruct the E. coli membrane by enabling production of a novel membrane component. Specifically, trans unsaturated fatty acids (TUFA) were produced and incorporated into the membrane of E. coli MG1655 by expression of cis-trans isomerase (Cti) from Pseudomonas aeruginosa. While the engineered strain was found to have no increase in membrane integrity, a significant decrease in membrane fluidity was observed, meaning that membrane polarization and rigidity were increased by TUFA incorporation. As a result, tolerance to exogenously added octanoic acid and production of octanoic acid were both increased relative to the wild-type strain. This membrane engineering strategy to improve octanoic acid tolerance was found to require fine-tuning of TUFA abundance. Besides improving tolerance and production of carboxylic acids, TUFA production also enabled increased tolerance in E. coli to other bio-products, e.g. alcohols, organic acids, aromatic compounds, a variety of adverse industrial conditions, e.g. low pH, high temperature, and also elevated styrene production, another versatile bio-chemical product. TUFA permitted enhanced growth due to alleviation of bio-product toxicity, demonstrating the general effectiveness of this membrane engineering strategy towards improving strain robustness. PMID:26875445

  17. Probing flagellar promoter occupancy in wild-type and mutant Caulobacter crescentus by chromatin immunoprecipitation.

    PubMed

    Davis, Nicole J; Viollier, Patrick H

    2011-06-01

    In the asymmetric predivisional cell of Caulobacter crescentus, TipF and TipN mark the cellular pole for future flagellar development. TipF is essential for motility and contains a cyclic-di-GMP phosphodiesterase-like (EAL) domain that is necessary for proper function. TipN is localized to the flagellar pole before TipF and is essential for the proper placement of the flagellum in C. crescentus. Using β-galactosidase promoter-probe assays and quantitative chromatin immunoprecipitation, we investigated the influence of the C. crescentus flagellar assembly regulator TipF on flagellar gene transcription. We compared the transcriptional activity of class II-fliF-lacZ, class III-flgE-lacZ, and class IV-fljL-lacZ fusions in a ΔtipF mutant with that of other flagellar mutants and the wild-type strain. We subsequently verified the in vivo occupancy of the fliF, flgE, and fljL flagellar promoters by the flagellar regulators CtrA, FlbD, and FliX in addition to RNA polymerase. We deduce that TipF contributes to proper expression of flagellar genes in C. crescentus by acting both within and outside of the canonical flagellar gene expression hierarchy.

  18. YbiV from E. coli K12 is a HAD phosphatase

    SciTech Connect

    Roberts, Anne; Lee, Seok-Yong; McCullagh, Emma; Silversmith, Ruth E.; Wemmer, David E.

    2004-03-16

    The protein YbiV from Escherichia coli K12 MG1655 is a hypothetical protein with sequence homology to the haloacid dehalogenase (HAD) superfamily of proteins. Although numerous members of this family have been identified, the functions of few are known. Using the crystal structure, sequence analysis, and biochemical assays, we have characterized ybiV as a HAD phosphatase. The crystal structure of YbiV reveals a two domain protein, one with the characteristic HAD hydrolase fold, the other an inserted a/b fold. In an effort to understand the mechanism we also solved and report the structures of YbiV in complex with beryllofluoride (BeF3-) and aluminum trifluoride (AlF3) which have been shown to mimic the phosphorylated intermediate and transition state for hydrolysis, respectively, in analogy to other HAD phosphatases. Analysis of the structures reveals the substrate binding cavity, which is hydrophilic in nature. Both structure and sequence homology indicate ybiV may be a sugar phosphatase, which is supported by biochemical assays which measured the release of free phosphate on a number of sugar-like substrates. We also investigated available genomic and functional data in an effort to determine the physiological substrate.

  19. [Butanol as a regulatory factor of ompC gene expression in E. coli cells].

    PubMed

    Seregina, T A; Shakulov, R S; Mironov, A S

    2012-11-01

    The influence of butanol on the expression of ompC gene encoding synthesis of OmpC porin in the MG 1655 strain of E. coli and butanol-tolerant mutant ButR was studied. It was shown that in the case of wild bacteria, the addition of butanol to the growth medium results in an increased level of ompC transcription. However, OmpC porin is not detected in the membrane fraction of cells. ButR mutant exhibits a higher level of ompC gene expression. A direct correlation is observed between the level of OmpC porin expression and its content in the membrane fraction of ButR mutant cells. In the regulatory region of the ompC gene of the ButR mutant, three nucleotide substitutions located in the binding sites of OmpR and CpxR activator proteins were identified. It was shown that mutations in the regulatory region of the ompC gene in the ButR mutant are responsible for the decreased level of OmpC porin expression under normal growth conditions. However, these mutations lead to an increased level of OmpC porin synthesis in the presence of butanol. These data suggest an additional mechanism of ompC gene regulation with the participation of butanol as a positive transcription effector.

  20. Serotype-related enterotoxigenicity in Escherichia coli O6.H16 and O148.H28.

    PubMed

    Scotland, S M; Gross, R J; Rowe, B

    1977-12-01

    The ability of certain Escherichia coli strains to produce enterotoxin is determined by transmissible plasmids. It is therefore possible that any E. coli strain might be able to acquire such a plasmid and that the correlation between enterotoxigenicity and serotype might be random. However, recent studies show that the enterotoxigenic strains so far describe belong to a restricted range of serotypes. Enterotoxigenic strains of E. coli O6.H16 and E. coli O148.H28 have been associated with outbreaks of diarrhoea in several countries, therefor strains of E. coli belonging to these serotypes were selected for further study. Twenty-three strains of E. coli O6.H16 and 14 strains of E. coli O148.H28 were examined; 20 strains of E. coli O6.H16 and all 14 strains of E. coli O148.H28 were enterotoxigenic but strains of E. coli O6 wit flagellar antigens other than H16 and strains of E. coli O148 wit flagellar antigens other than H28 were not enterotoxigenic. The examination of single colony subcultures derived from the E. coli O6.H16 strains showed that in some strains loss of enterotoxigenicity had occurred in a proportional of colonies.

  1. Variation in heat and pressure resistance of verotoxigenic and nontoxigenic Escherichia coli.

    PubMed

    Liu, Yang; Gill, Alex; McMullen, Lynn; Gänzle, Michael G

    2015-01-01

    This study evaluated the heat and pressure resistance of 112 strains of Escherichia coli, including 102 strains of verotoxigenic E. coli (VTEC) representing 23 serotypes and four phylogenetic groups. In an initial screening, the heat and pressure resistance of 100 strains, including 94 VTEC strains, were tested in phosphate-buffered saline (PBS). Treatment at 60°C for 5 min reduced cell counts by 2.0 to 5.5 log CFU/ml; treatment at 600 MPa for 3 min at 25°C reduced the cell counts by 1.1 to 5.5 log CFU/ml. Heat or pressure resistance did not correlate to the phylogenetic group or the serotype. A smaller group of E. coli strains was evaluated for heat and pressure resistance in Luria-Bertani (LB) broth. Generally, the levels of heat resistance of E. coli strains in LB and PBS were similar; however, the levels of pressure resistance observed for treatments in LB broth or PBS were variable. The cell counts of pressure-resistant strains of VTEC were reduced by less than 1.5 log CFU/ml after treatment at 600 MPa for 3 min. E. coli strains were also treated with 600 MPa for 3 min in ground beef or inoculated into beef patties and grilled to 63 or 71°C. The cell counts of the VTEC E. coli O26:H11 strain 05-6544 were reduced by 2 log CFU/g by pressure treatment in ground beef. The cell counts of the heat-resistant E. coli strain AW1.7 were reduced by 1.4 and 3.4 log CFU/g in beef patties grilled to internal temperatures of 63 and 71°C, respectively. The cell counts of E. coli 05-6544 were reduced by less than 3 and 6 log CFU/g in beef patties grilled to internal temperatures of 63 and 71°C, respectively. To study whether the composition of the beef patties influenced heat resistance, E. coli strains AW1.7, AW1.7 Δ pHR1, MG1655, and LMM1030 were mixed into beef patties containing 15 or 35% fat and 0 or 2% NaCl, and the patties were grilled to an internal temperature of 63°C. The highest heat resistance of E. coli was observed in patties containing 15% fat and 2% NaCl.

  2. Application of genomic technologies for characterization, typing, and detection of E. coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Serotyping using polyclonal antibodies raised in rabbits has been the gold standard for classification of E. coli based on the O- (somatic) and H- (flagellar) antigens; however, problems associated with serotyping are that the procedure is time consuming and labor intensive, cross reactions among di...

  3. Bacterial Flagellar Microhydrodynamics: Laminar Flow over Complex Flagellar Filaments, Analog Archimedean Screws and Cylinders, and Its Perturbations

    PubMed Central

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

    2003-01-01

    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

  4. The effect of acid treatment on survival and protein expression of a laboratory K-12 strain Escherichia coli.

    PubMed

    Paul, Barbara; Hirshfield, Irvin

    2003-03-01

    Pre-exposure of log phase enteric bacteria to nonlethal acidic pH induces phenotypic changes that protect the organisms against subsequent lethal acidity. Studies have revealed that when Salmonella typhimurium is grown in minimal medium at pH 5.5 and 4.3 the organism develops a biphasic acid tolerance. This two-stage response has not been reported at present in Escherichia coli; rather it is thought that when this organism is grown in rich medium there is a single stress response throughout the pH range of 4 to 6. We believe that the evidence for such a report is lacking; therefore, in this study the acid response of log phase E. coli was examined in rich medium (LB). The pH 3.0 acid survival assays of a laboratory strain of E. coli K-12 MG1655, after cultures had been exposed to LB acidified to pH 5.5 or pH 4.3 indicate that like S. typhimurium, E. coli shows both an acid tolerance and an acid-shock response to pH 5.5 and 4.3 exposure, respectively. It was consistently found, however, that longer pre-exposure (60 min rather than 15 min) at either pH afforded better protection against the lethal pH 3.0 challenge. Analysis of polypeptide induction at pH 5.5 and 4.3 by two-dimensional gel electrophoresis clearly shows different profiles. Together the results show that in E. coli, pre-treatment between pH 4 and 6 does not result in a flat protective response.

  5. Experimental evolution of silver nanoparticle resistance in Escherichia coli

    NASA Astrophysics Data System (ADS)

    Tajkarimi, Mehrdad

    The recent exponential increase in the use of engineered nanoparticles (eNPs) means both greater intentional and unintentional exposure of eNPs to microbes. Intentional use includes the use of eNPs as biocides; unintentional exposure results from the fact that eNPs are included in a variety of commercial products (paints, sunscreens, cosmetics.) Many of these eNPs include heavy metals or metal oxides such as titanium dioxide, silver, gold, zinc and zinc oxide. The fact that early studies of the impact of metallic nanoparticles achieved approximately 90% lethality to Ag, Cu eNPs, suggests that genetic variants are already circulating in bacteria that can be co-opted to provide heavy metal eNP resistance. This project has utilized laboratory experimental evolution to evolve eNP resistance in the bacterium Escherichia coli (K12 MG1655 strain.). This is currently being validated by demonstrating the greater fitness of evolved strains versus ancestral strains in the presence of different sized and coated silver nanoparticles (10nm, 40nm, citrate-coated, PVP-coated) as well as phenotypic changes in the bacterial cell wall (as measured by Atomic Force Microscopy, AFM.). Finally, the bacterial genomes of the evolved and ancestral strains were resequenced. The genomic basis of this complex phenotype was determined. The practical application of such knowledge cannot be underestimated since nature is already evolving nanoparticle resistant bacteria. Thus knowledge of the nature of the physiological, morphological, and genomic mechanisms of resistance will be essential to deploy sustainable use of NPs as biocides, and to prevent unintentional environmental damage.

  6. Analysis of Escherichia coli mutants with a linear respiratory chain.

    PubMed

    Steinsiek, Sonja; Stagge, Stefan; Bettenbrock, Katja

    2014-01-01

    The respiratory chain of E. coli is branched to allow the cells' flexibility to deal with changing environmental conditions. It consists of the NADH:ubiquinone oxidoreductases NADH dehydrogenase I and II, as well as of three terminal oxidases. They differ with respect to energetic efficiency (proton translocation) and their affinity to the different quinone/quinol species and oxygen. In order to analyze the advantages of the branched electron transport chain over a linear one and to assess how usage of the different terminal oxidases determines growth behavior at varying oxygen concentrations, a set of isogenic mutant strains was created, which lack NADH dehydrogenase I as well as two of the terminal oxidases, resulting in strains with a linear respiratory chain. These strains were analyzed in glucose-limited chemostat experiments with defined oxygen supply, adjusting aerobic, anaerobic and different microaerobic conditions. In contrast to the wild-type strain MG1655, the mutant strains produced acetate even under aerobic conditions. Strain TBE032, lacking NADH dehydrogenase I and expressing cytochrome bd-II as sole terminal oxidase, showed the highest acetate formation rate under aerobic conditions. This supports the idea that cytochrome bd-II terminal oxidase is not able to catalyze the efficient oxidation of the quinol pool at higher oxygen conditions, but is functioning mainly under limiting oxygen conditions. Phosphorylation of ArcA, the regulator of the two-component system ArcBA, besides Fnr the main transcription factor for the response towards different oxygen concentrations, was studied. Its phosphorylation pattern was changed in the mutant strains. Dephosphorylation and therefore inactivation of ArcA started at lower aerobiosis levels than in the wild-type strain. Notably, not only the micro- and aerobic metabolism was affected by the mutations, but also the anaerobic metabolism, where the respiratory chain should not be important. PMID:24475268

  7. Rapid evolution of silver nanoparticle resistance in Escherichia coli

    PubMed Central

    Graves, Joseph L.; Tajkarimi, Mehrdad; Cunningham, Quincy; Campbell, Adero; Nonga, Herve; Harrison, Scott H.; Barrick, Jeffrey E.

    2015-01-01

    The recent exponential increase in the use of engineered nanoparticles (eNPs) means both greater intentional and unintentional exposure of eNPs to microbes. Intentional use includes the use of eNPs as biocides. Unintentional exposure results from the fact that eNPs are included in a variety of commercial products (paints, sunscreens, cosmetics). Many of these eNPs are composed of heavy metals or metal oxides such as silver, gold, zinc, titanium dioxide, and zinc oxide. It is thought that since metallic/metallic oxide NPs impact so many aspects of bacterial physiology that it will difficult for bacteria to evolve resistance to them. This study utilized laboratory experimental evolution to evolve silver nanoparticle (AgNP) resistance in the bacterium Escherichia coli (K-12 MG1655), a bacterium that does not harbor any known silver resistance elements. After 225 generations of exposure to the AgNP environment, the treatment populations demonstrated greater fitness vs. control strains as measured by optical density (OD) and colony forming units (CFU) in the presence of varying concentrations of 10 nm citrate-coated silver nanoparticles (AgNP) or silver nitrate (AgNO3). Genomic analysis shows that changes associated with AgNP resistance were already accumulating within the treatment populations by generation 100, and by generation 200 three mutations had swept to high frequency in the AgNP resistance stocks. This study indicates that despite previous claims to the contrary bacteria can easily evolve resistance to AgNPs, and this occurs by relatively simple genomic changes. These results indicate that care should be taken with regards to the use of eNPs as biocides as well as with regards to unintentional exposure of microbial communities to eNPs in waste products. PMID:25741363

  8. Rapid evolution of silver nanoparticle resistance in Escherichia coli.

    PubMed

    Graves, Joseph L; Tajkarimi, Mehrdad; Cunningham, Quincy; Campbell, Adero; Nonga, Herve; Harrison, Scott H; Barrick, Jeffrey E

    2015-01-01

    The recent exponential increase in the use of engineered nanoparticles (eNPs) means both greater intentional and unintentional exposure of eNPs to microbes. Intentional use includes the use of eNPs as biocides. Unintentional exposure results from the fact that eNPs are included in a variety of commercial products (paints, sunscreens, cosmetics). Many of these eNPs are composed of heavy metals or metal oxides such as silver, gold, zinc, titanium dioxide, and zinc oxide. It is thought that since metallic/metallic oxide NPs impact so many aspects of bacterial physiology that it will difficult for bacteria to evolve resistance to them. This study utilized laboratory experimental evolution to evolve silver nanoparticle (AgNP) resistance in the bacterium Escherichia coli (K-12 MG1655), a bacterium that does not harbor any known silver resistance elements. After 225 generations of exposure to the AgNP environment, the treatment populations demonstrated greater fitness vs. control strains as measured by optical density (OD) and colony forming units (CFU) in the presence of varying concentrations of 10 nm citrate-coated silver nanoparticles (AgNP) or silver nitrate (AgNO3). Genomic analysis shows that changes associated with AgNP resistance were already accumulating within the treatment populations by generation 100, and by generation 200 three mutations had swept to high frequency in the AgNP resistance stocks. This study indicates that despite previous claims to the contrary bacteria can easily evolve resistance to AgNPs, and this occurs by relatively simple genomic changes. These results indicate that care should be taken with regards to the use of eNPs as biocides as well as with regards to unintentional exposure of microbial communities to eNPs in waste products. PMID:25741363

  9. Both sequence and context are important for flagellar targeting of a glucose transporter

    PubMed Central

    Tran, Khoa D.; Rodriguez-Contreras, Dayana; Shinde, Ujwal; Landfear, Scott M.

    2012-01-01

    Summary Many of the cilia- and flagella-specific integral membrane proteins identified to date function to sense the extracellular milieu, and there is considerable interest in defining pathways for targeting such proteins to these sensory organelles. The flagellar glucose transporter of Leishmania mexicana, LmxGT1, is targeted selectively to the flagellar membrane, whereas two other isoforms, LmxGT2 and LmxGT3, are targeted to the pellicular plasma membrane of the cell body. To define the flagellar targeting signal, deletions and point mutations were generated in the N-terminal hydrophilic domain of LmxGT1, which mediates flagellar localization. Three amino acids, N95-P96-M97, serve critical roles in flagellar targeting, resulting in strong mistargeting phenotypes when mutagenized. However, to facilitate flagellar targeting of other non-flagellar membrane proteins, it was necessary to attach a larger region surrounding the NPM motif containing amino acids 81–113. Molecular modeling suggests that this region might present the critical NPM residues at the surface of the N-terminal domain. It is likely that the NPM motif is recognized by currently unknown protein-binding partners that mediate flagellar targeting of membrane-associated proteins. PMID:22467850

  10. Both sequence and context are important for flagellar targeting of a glucose transporter.

    PubMed

    Tran, Khoa D; Rodriguez-Contreras, Dayana; Shinde, Ujwal; Landfear, Scott M

    2012-07-15

    Many of the cilia- and flagella-specific integral membrane proteins identified to date function to sense the extracellular milieu, and there is considerable interest in defining pathways for targeting such proteins to these sensory organelles. The flagellar glucose transporter of Leishmania mexicana, LmxGT1, is targeted selectively to the flagellar membrane, whereas two other isoforms, LmxGT2 and LmxGT3, are targeted to the pellicular plasma membrane of the cell body. To define the flagellar targeting signal, deletions and point mutations were generated in the N-terminal hydrophilic domain of LmxGT1, which mediates flagellar localization. Three amino acids, N95-P96-M97, serve critical roles in flagellar targeting, resulting in strong mistargeting phenotypes when mutagenized. However, to facilitate flagellar targeting of other non-flagellar membrane proteins, it was necessary to attach a larger region surrounding the NPM motif containing amino acids 81-113. Molecular modeling suggests that this region might present the critical NPM residues at the surface of the N-terminal domain. It is likely that the NPM motif is recognized by currently unknown protein-binding partners that mediate flagellar targeting of membrane-associated proteins. PMID:22467850

  11. Visualization of the flagellar surface of protists by atomic force microscopy.

    PubMed

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

    2010-12-01

    In many cells, motility is mediated by flagellar beating. Protist parasites are capable of highly coordinated motility which contributes to their pathogenicity in mammalian hosts. Understanding the structural aspects of the flagellum may be important to the identification of novel targets for therapeutic intervention. Our group used atomic force microscopy (AFM) to examine the ultrastructure of Trypanosoma cruzi, obtaining valuable information on the organisation of the flagellar sub-structure. AFM images revealed novel flagellar components such as the presence of periodically-spaced protrusions organised along a flagellar furrow and oriented through the major flagellar axis between the axoneme and the paraflagellar rod. The nature and functional role of this structure are still unknown, although the hypothesis that the furrow might physically separate the two distinct domains of the flagellar membrane that comprise the surface of the axoneme and the paraflagellar rod (PFR) has been raised. To test whether the furrow was present or not only in PFR-bearing flagella, different protists containing or lacking the PFR, were analysed by AFM. Analysis of T. cruzi, Trypanosoma brucei and Herpetomonas megaseliae, which present distinct PFRs, showed similar and equivalent furrows along the main axis of their flagella, whereas Crithidia deanei, Giardia lamblia and Tritrichomonas foetus (in which the PFR is reduced or absent) lacked a furrow. Our results strongly suggest that the flagellar furrow is a characteristic feature of PFR-containing flagella and opens new perspectives for its functional role in the definition of sub-domains on the flagellar membrane.

  12. Narrow-spectrum inhibitors of Campylobacter jejuni flagellar expression and growth.

    PubMed

    Johnson, Jeremiah G; Yuhas, Caroline; McQuade, Thomas J; Larsen, Martha J; DiRita, Victor J

    2015-07-01

    Campylobacter jejuni is a major cause of food-borne illness due to its ability to reside within the gastrointestinal tracts of chickens. Multiple studies have identified the flagella of C. jejuni as a major determinant of chicken colonization. An inhibitor screen of approximately 147,000 small molecules was performed to identify compounds that are able to inhibit flagellar expression in a reporter strain of C. jejuni. Several compounds that modestly inhibited motility of wild-type C. jejuni in standard assays were identified, as were a number of small molecules that robustly inhibited C. jejuni growth, in vitro. Examination of similar bacterial screens found that many of these small molecules inhibited only the growth of C. jejuni. Follow-up assays demonstrated inhibition of other strains of C. jejuni and Campylobacter coli but no inhibition of the closely related Helicobacter pylori. The compounds were determined to be bacteriostatic and nontoxic to eukaryotic cells. Preliminary results from a day-of-hatch chick model of colonization suggest that at least one of the compounds demonstrates promise for reducing Campylobacter colonization loads in vivo, although further medicinal chemistry may be required to enhance bioavailability.

  13. Switching of Bacterial Flagellar Motors Is Triggered by Mutant FliG

    PubMed Central

    Lele, Pushkar P.; Berg, Howard C.

    2015-01-01

    Binding of the chemotaxis response regulator CheY-P promotes switching between rotational states in flagellar motors of the bacterium Escherichia coli. Here, we induced switching in the absence of CheY-P by introducing copies of a mutant FliG locked in the clockwise (CW) conformation (FliGCW). The composition of the mixed FliG ring was estimated via fluorescence imaging, and the probability of CW rotation (CWbias) was determined from the rotation of tethered cells. The results were interpreted in the framework of a 1D Ising model. The data could be fit by assuming that mutant subunits are more stable in the CW conformation than in the counterclockwise conformation. We found that CWbias varies depending on the spatial arrangement of the assembled subunits in the FliG ring. This offers a possible explanation for a previous observation of hysteresis in the switch function in analogous mixed FliM motors—in motors containing identical fractions of mutant FliMCW in otherwise wild-type motors, the CWbias differed depending on whether mutant subunits were expressed in strains with native motors or native subunits were expressed in strains with mutant motors. PMID:25762339

  14. 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.

  15. Efficient spatiotemporal analysis of the flagellar waveform of Chlamydomonas reinhardtii.

    PubMed

    Bayly, P V; Lewis, B L; Kemp, P S; Pless, R B; Dutcher, S K

    2010-01-01

    The 9 + 2 axoneme is a microtubule-based machine that powers the oscillatory beating of cilia and flagella. Its highly regulated movement is essential for the normal function of many organs; ciliopathies cause congenital defects, chronic respiratory tract infections and infertility. We present an efficient method to obtain a quantitative description of flagellar motion, with high spatial and temporal resolution, from high speed video recording of bright field images. This highly automated technique provides the shape, shear angle, curvature, and bend propagation speeds along the length of the flagellum, with approximately 200 temporal samples per beat. We compared the waveforms of uniflagellated wild-type and ida3 mutant cells, which lack the I1 inner dynein complex. Video images were captured at 350 fps. Rigid-body motion was eliminated by fast Fourier transform (FFT)-based registration, and the Cartesian (x-y) coordinates of points on the flagellum were identified. These x-y "point clouds" were embedded in two data dimensions using Isomap, a nonlinear dimension reduction method, and sorted by phase in the flagellar cycle. A smooth surface was fitted to the sorted point clouds, which provides high-resolution estimates of shear angle and curvature. Wild-type and ida3 cells exhibit large differences in shear amplitude, but similar maximum and minimum curvature values. In ida3 cells, the reverse bend begins earlier and travels more slowly relative to the principal bend, than in wild-type cells. The regulation of flagellar movement must involve I1 dynein in a manner consistent with these results.

  16. 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

  17. 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. PMID:26245228

  18. Flagellar waveform dynamics of freely swimming algal cells

    NASA Astrophysics Data System (ADS)

    Kurtuldu, H.; Tam, D.; Hosoi, A. E.; Johnson, K. A.; Gollub, J. P.

    2013-07-01

    We present quantitative measurements of time-dependent flagellar waveforms for freely swimming biflagellated algal cells, for both synchronous and asynchronous beating. We use the waveforms in conjunction with resistive force theory as well as a singularity method to predict a cell's time-dependent velocity for comparison with experiments. While net propulsion is thought to arise from asymmetry between the power and recovery strokes, we show that hydrodynamic interactions between the flagella and cell body on the return stroke make an important contribution to enhance net forward motion.

  19. Stoichiometry and turnover of the bacterial flagellar switch protein FliN.

    PubMed

    Delalez, Nicolas J; Berry, Richard M; Armitage, Judith P

    2014-07-01

    Some proteins in biological complexes exchange with pools of free proteins while the complex is functioning. Evidence is emerging that protein exchange can be part of an adaptive mechanism. The bacterial flagellar motor is one of the most complex biological machines and is an ideal model system to study protein dynamics in large multimeric complexes. Recent studies showed that the copy number of FliM in the switch complex and the fraction of FliM that exchanges vary with the direction of flagellar rotation. Here, we investigated the stoichiometry and turnover of another switch complex component, FliN, labeled with the fluorescent protein CyPet, in Escherichia coli. Our results confirm that, in vivo, FliM and FliN form a complex with stoichiometry of 1:4 and function as a unit. We estimated that wild-type motors contained 120 ± 26 FliN molecules. Motors that rotated only clockwise (CW) or counterclockwise (CCW) contained 114 ± 17 and 144 ± 26 FliN molecules, respectively. The ratio of CCW-to-CW FliN copy numbers was 1.26, very close to that of 1.29 reported previously for FliM. We also measured the exchange of FliN molecules, which had a time scale and dependence upon rotation direction similar to those of FliM, consistent with an exchange of FliM-FliN as a unit. Our work confirms the highly dynamic nature of multimeric protein complexes and indicates that, under physiological conditions, these machines might not be the stable, complete structures suggested by averaged fixed methodologies but, rather, incomplete rings that can respond and adapt to changing environments. Importance: The flagellum is one of the most complex structures in a bacterial cell, with the core motor proteins conserved across species. Evidence is now emerging that turnover of some of these motor proteins depends on motor activity, suggesting that turnover is important for function. The switch complex transmits the chemosensory signal to the rotor, and we show, by using single

  20. Na+ conductivity of the Na+-driven flagellar motor complex composed of unplugged wild-type or mutant PomB with PomA.

    PubMed

    Takekawa, Norihiro; Terauchi, Takashi; Morimoto, Yusuke V; Minamino, Tohru; Lo, Chien-Jung; Kojima, Seiji; Homma, Michio

    2013-05-01

    PomA and PomB form the stator complex, which functions as a Na(+) channel, in the Na(+)-driven flagellar motor of Vibrio alginolyticus. The plug region of PomB is thought to regulate the Na(+) flow and to suppress massive ion influx through the stator channel. In this study, in order to measure the Na(+) conductivity of the unplugged stator, we over-produced a plug-deleted stator of the Na(+)-driven flagellar motor in Escherichia coli. The over-production of the plug-deleted stator in E. coli cells caused more severe growth inhibition than in Vibrio cells and that growth inhibition depended on the Na(+) concentration in the growth medium. Measurement of intracellular Na(+) concentration by flame photometry and fluorescent analysis with a Na(+) indicator, Sodium Green, revealed that over-production of the plug-deleted stator increased the Na(+) concentration in cell. Some mutations in the channel region of PomB or in the cytoplasmic region of PomA suppressed both the growth inhibition and the increase in intracellular Na(+) concentration. These results suggest that the level of growth inhibition correlates with the intracellular Na(+) concentration, probably due to the Na(+) conductivity through the stator due to the mutations. PMID:23420849

  1. 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.

  2. Differential Mechanism of Escherichia coli Inactivation by (+)-Limonene as a Function of Cell Physiological State and Drug's Concentration

    PubMed Central

    Chueca, Beatriz; Pagán, Rafael; García-Gonzalo, Diego

    2014-01-01

    (+)-limonene is a lipophilic antimicrobial compound, extracted from citrus fruits' essential oils, that is used as a flavouring agent and organic solvent by the food industry. A recent study has proposed a common and controversial mechanism of cell death for bactericidal antibiotics, in which hydroxyl radicals ultimately inactivated cells. Our objective was to determine whether the mechanism of Escherichia coli MG1655 inactivation by (+)-limonene follows that of bactericidal antibiotics. A treatment with 2,000 μL/L (+)-limonene inactivated 4 log10 cycles of exponentially growing E. coli cells in 3 hours. On one hand, an increase of cell survival in the ΔacnB mutant (deficient in a TCA cycle enzyme), or in the presence of 2,2′-dipyridyl (inhibitor of Fenton reaction by iron chelation), thiourea, or cysteamine (hydroxyl radical scavengers) was observed. Moreover, the ΔrecA mutant (deficient in an enzyme involved in SOS response to DNA damage) was more sensitive to (+)-limonene. Thus, this indirect evidence indicates that the mechanism of exponentially growing E. coli cells inactivation by 2,000 μL/L (+)-limonene is due to the TCA cycle and Fenton-mediated hydroxyl radical formation that caused oxidative DNA damage, as observed for bactericidal drugs. However, several differences have been observed between the proposed mechanism for bactericidal drugs and for (+)-limonene. In this regard, our results demonstrated that E. coli inactivation was influenced by its physiological state and the drug's concentration: experiments with stationary-phase cells or 4,000 μL/L (+)-limonene uncovered a different mechanism of cell death, likely unrelated to hydroxyl radicals. Our research has also shown that drug's concentration is an important factor influencing the mechanism of bacterial inactivation by antibiotics, such as kanamycin. These results might help in improving and spreading the use of (+)-limonene as an antimicrobial compound, and in clarifying the controversy

  3. Differential mechanism of Escherichia coli Inactivation by (+)-limonene as a function of cell physiological state and drug's concentration.

    PubMed

    Chueca, Beatriz; Pagán, Rafael; García-Gonzalo, Diego

    2014-01-01

    (+)-limonene is a lipophilic antimicrobial compound, extracted from citrus fruits' essential oils, that is used as a flavouring agent and organic solvent by the food industry. A recent study has proposed a common and controversial mechanism of cell death for bactericidal antibiotics, in which hydroxyl radicals ultimately inactivated cells. Our objective was to determine whether the mechanism of Escherichia coli MG1655 inactivation by (+)-limonene follows that of bactericidal antibiotics. A treatment with 2,000 μL/L (+)-limonene inactivated 4 log10 cycles of exponentially growing E. coli cells in 3 hours. On one hand, an increase of cell survival in the ΔacnB mutant (deficient in a TCA cycle enzyme), or in the presence of 2,2'-dipyridyl (inhibitor of Fenton reaction by iron chelation), thiourea, or cysteamine (hydroxyl radical scavengers) was observed. Moreover, the ΔrecA mutant (deficient in an enzyme involved in SOS response to DNA damage) was more sensitive to (+)-limonene. Thus, this indirect evidence indicates that the mechanism of exponentially growing E. coli cells inactivation by 2,000 μL/L (+)-limonene is due to the TCA cycle and Fenton-mediated hydroxyl radical formation that caused oxidative DNA damage, as observed for bactericidal drugs. However, several differences have been observed between the proposed mechanism for bactericidal drugs and for (+)-limonene. In this regard, our results demonstrated that E. coli inactivation was influenced by its physiological state and the drug's concentration: experiments with stationary-phase cells or 4,000 μL/L (+)-limonene uncovered a different mechanism of cell death, likely unrelated to hydroxyl radicals. Our research has also shown that drug's concentration is an important factor influencing the mechanism of bacterial inactivation by antibiotics, such as kanamycin. These results might help in improving and spreading the use of (+)-limonene as an antimicrobial compound, and in clarifying the controversy about

  4. Differential mechanism of Escherichia coli Inactivation by (+)-limonene as a function of cell physiological state and drug's concentration.

    PubMed

    Chueca, Beatriz; Pagán, Rafael; García-Gonzalo, Diego

    2014-01-01

    (+)-limonene is a lipophilic antimicrobial compound, extracted from citrus fruits' essential oils, that is used as a flavouring agent and organic solvent by the food industry. A recent study has proposed a common and controversial mechanism of cell death for bactericidal antibiotics, in which hydroxyl radicals ultimately inactivated cells. Our objective was to determine whether the mechanism of Escherichia coli MG1655 inactivation by (+)-limonene follows that of bactericidal antibiotics. A treatment with 2,000 μL/L (+)-limonene inactivated 4 log10 cycles of exponentially growing E. coli cells in 3 hours. On one hand, an increase of cell survival in the ΔacnB mutant (deficient in a TCA cycle enzyme), or in the presence of 2,2'-dipyridyl (inhibitor of Fenton reaction by iron chelation), thiourea, or cysteamine (hydroxyl radical scavengers) was observed. Moreover, the ΔrecA mutant (deficient in an enzyme involved in SOS response to DNA damage) was more sensitive to (+)-limonene. Thus, this indirect evidence indicates that the mechanism of exponentially growing E. coli cells inactivation by 2,000 μL/L (+)-limonene is due to the TCA cycle and Fenton-mediated hydroxyl radical formation that caused oxidative DNA damage, as observed for bactericidal drugs. However, several differences have been observed between the proposed mechanism for bactericidal drugs and for (+)-limonene. In this regard, our results demonstrated that E. coli inactivation was influenced by its physiological state and the drug's concentration: experiments with stationary-phase cells or 4,000 μL/L (+)-limonene uncovered a different mechanism of cell death, likely unrelated to hydroxyl radicals. Our research has also shown that drug's concentration is an important factor influencing the mechanism of bacterial inactivation by antibiotics, such as kanamycin. These results might help in improving and spreading the use of (+)-limonene as an antimicrobial compound, and in clarifying the controversy about

  5. The Csr system regulates genome-wide mRNA stability and transcription and thus gene expression in Escherichia coli.

    PubMed

    Esquerré, Thomas; Bouvier, Marie; Turlan, Catherine; Carpousis, Agamemnon J; Girbal, Laurence; Cocaign-Bousquet, Muriel

    2016-04-26

    Bacterial adaptation requires large-scale regulation of gene expression. We have performed a genome-wide analysis of the Csr system, which regulates many important cellular functions. The Csr system is involved in post-transcriptional regulation, but a role in transcriptional regulation has also been suggested. Two proteins, an RNA-binding protein CsrA and an atypical signaling protein CsrD, participate in the Csr system. Genome-wide transcript stabilities and levels were compared in wildtype E. coli (MG1655) and isogenic mutant strains deficient in CsrA or CsrD activity demonstrating for the first time that CsrA and CsrD are global negative and positive regulators of transcription, respectively. The role of CsrA in transcription regulation may be indirect due to the 4.6-fold increase in csrD mRNA concentration in the CsrA deficient strain. Transcriptional action of CsrA and CsrD on a few genes was validated by transcriptional fusions. In addition to an effect on transcription, CsrA stabilizes thousands of mRNAs. This is the first demonstration that CsrA is a global positive regulator of mRNA stability. For one hundred genes, we predict that direct control of mRNA stability by CsrA might contribute to metabolic adaptation by regulating expression of genes involved in carbon metabolism and transport independently of transcriptional regulation.

  6. The Csr system regulates genome-wide mRNA stability and transcription and thus gene expression in Escherichia coli

    PubMed Central

    Esquerré, Thomas; Bouvier, Marie; Turlan, Catherine; Carpousis, Agamemnon J.; Girbal, Laurence; Cocaign-Bousquet, Muriel

    2016-01-01

    Bacterial adaptation requires large-scale regulation of gene expression. We have performed a genome-wide analysis of the Csr system, which regulates many important cellular functions. The Csr system is involved in post-transcriptional regulation, but a role in transcriptional regulation has also been suggested. Two proteins, an RNA-binding protein CsrA and an atypical signaling protein CsrD, participate in the Csr system. Genome-wide transcript stabilities and levels were compared in wildtype E. coli (MG1655) and isogenic mutant strains deficient in CsrA or CsrD activity demonstrating for the first time that CsrA and CsrD are global negative and positive regulators of transcription, respectively. The role of CsrA in transcription regulation may be indirect due to the 4.6-fold increase in csrD mRNA concentration in the CsrA deficient strain. Transcriptional action of CsrA and CsrD on a few genes was validated by transcriptional fusions. In addition to an effect on transcription, CsrA stabilizes thousands of mRNAs. This is the first demonstration that CsrA is a global positive regulator of mRNA stability. For one hundred genes, we predict that direct control of mRNA stability by CsrA might contribute to metabolic adaptation by regulating expression of genes involved in carbon metabolism and transport independently of transcriptional regulation. PMID:27112822

  7. 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. PMID:26216959

  8. 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. PMID:24528485

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

    PubMed

    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

  11. Functional state of the axonemal dyneins during flagellar bend propagation.

    PubMed Central

    Woolley, D M; Vernon, G G

    2002-01-01

    When mouse spermatozoa swim in media of high viscosity, additional waves of bending are superimposed on the primary traveling wave. The additional (secondary) waves are relatively small in scale and high in frequency. They originate in the proximal part of the interbend regions. The initiation of secondary bending happens only in distal parts of the flagellum. The secondary waves propagate along the interbends and then tend to die out as they encounter the next-most-distal bend of the primary wave, if that bend exceeds a certain angle. The principal bends of the primary wave, being of greater angle than the reverse bends, strongly resist invasion by the secondary waves; when a principal bend of the primary wave propagates off the flagellar tip, the secondary wave behind it suddenly increases in amplitude. We claim that the functional state of the dynein motors in relation to the primary wave can be deduced from their availability for recruitment into secondary wave activity. Therefore, only the dyneins in bends are committed functionally to the maintenance and propagation of the flagellar wave; dyneins in interbend regions are not functionally committed in this way. We equate functional commitment with tension-generating activity, although we argue that the regions of dynein thus engaged nevertheless permit sliding displacements between the doublets. PMID:12324433

  12. 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

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

    PubMed

    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.

  14. 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

  15. 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

  16. 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

    2014-11-01

    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.

  17. 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

  18. 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

  19. Automated methods for estimation of sperm flagellar bending parameters.

    PubMed

    Brokaw, C J

    1984-01-01

    Parameters to describe flagellar bending patterns can be obtained by a microcomputer procedure that uses a set of parameters to synthesize model bending patterns, compares the model bending patterns with digitized and filtered data from flagellar photographs, and uses the Simplex method to vary the parameters until a solution with minimum root mean square differences between the model and the data is found. Parameters for Chlamydomonas bending patterns have been obtained from comparison of shear angle curves for the model and the data. To avoid the determination of the orientation of the basal end of the flagellum, which is required for calculation of shear angles, parameters for sperm flagella have been obtained by comparison of curves of curvature as a function of length for the model and for the data. A constant curvature model, modified from that originally used for Chlamydomonas flagella, has been used for obtaining parameters from sperm flagella, but the methods can be applied using other models for synthesizing the model bending patterns.

  20. Bacterial attachment and viscoelasticity: physicochemical and motility effects analyzed using quartz crystal microbalance with dissipation (QCM-D).

    PubMed

    Gutman, Jenia; Walker, Sharon L; Freger, Viatcheslav; Herzberg, Moshe

    2013-01-01

    This investigation is focused on the combined effect of bacterial physicochemical characteristics and motility on cell adhesion and deposition using a flow-through quartz crystal microbalance with dissipation (QCM-D). Three model flagellated strains with different degrees of motility were selected, including a highly motile Escherichia coli K12 MG1655, an environmental strain Sphingomonas wittichii RW1, and a nonmotile (with paralyzed flagella) Escherichia coli K12 MG1655 ΔmotA that is incapable of encoding the motor torque generator for flagellar movement. Of the three strains, S. wittichii RW1 is highly hydrophobic, while E. coli strains are equally hydrophilic. Consideration of the hydrophobicity provides an alternative explanation for the bacterial adhesion behavior. QCM-D results show that motility is a critical factor in determining bacterial adhesion, as long as the aquatic chemical conditions are conducive for motility and the substratum and bacterial surface are similarly hydrophobic or hydrophilic. Once their properties are not similar, the contribution of hydrophobic interactions becomes more pronounced. QCM-D results suggest that during adhesion of the hydrophobic bacterium, S. wittichii RW1, the initial step of adhesion and maturation of bacteria-substratum interaction on hydrophilic surface includes a dynamic change of the viscoelastic properties of the bond bacterium-surface becoming more viscously oriented.

  1. Step-Wise Loss of Bacterial Flagellar Torsion Confers Progressive Phagocytic Evasion

    PubMed Central

    Lovewell, Rustin R.; Collins, Ryan M.; Acker, Julie L.; O'Toole, George A.; Wargo, Matthew J.; Berwin, Brent

    2011-01-01

    Phagocytosis of bacteria by innate immune cells is a primary method of bacterial clearance during infection. However, the mechanisms by which the host cell recognizes bacteria and consequentially initiates phagocytosis are largely unclear. Previous studies of the bacterium Pseudomonas aeruginosa have indicated that bacterial flagella and flagellar motility play an important role in colonization of the host and, importantly, that loss of flagellar motility enables phagocytic evasion. Here we use molecular, cellular, and genetic methods to provide the first formal evidence that phagocytic cells recognize bacterial motility rather than flagella and initiate phagocytosis in response to this motility. We demonstrate that deletion of genes coding for the flagellar stator complex, which results in non-swimming bacteria that retain an initial flagellar structure, confers resistance to phagocytic binding and ingestion in several species of the gamma proteobacterial group of Gram-negative bacteria, indicative of a shared strategy for phagocytic evasion. Furthermore, we show for the first time that susceptibility to phagocytosis in swimming bacteria is proportional to mot gene function and, consequently, flagellar rotation since complementary genetically- and biochemically-modulated incremental decreases in flagellar motility result in corresponding and proportional phagocytic evasion. These findings identify that phagocytic cells respond to flagellar movement, which represents a novel mechanism for non-opsonized phagocytic recognition of pathogenic bacteria. PMID:21949654

  2. Effects of the dynein inhibitor ciliobrevin on the flagellar motility of sea urchin spermatozoa.

    PubMed

    Wada, Yuuko; Baba, Shoji A; Kamimura, Shinji

    2015-04-01

    Ciliobrevin has recently been found to be a membrane-permeable inhibitor that is specific to AAA+ molecular motors such as cytoplasmic dyneins. In this study, we investigated how ciliobrevin inhibited the motility of sperm from sea urchins: Hemicentrotus pulcherrimus, Pseudocentrotus depressus, and Anthocidaris crassispina. After application of 100 μM of ciliobrevin A to live spermatozoa, swimming speed decreased gradually and flagellar motion stopped almost completely within 5 to 10 min. This inhibition was reversible and the frequency of flagellar beating was reduced in a concentration-dependent manner. Ciliobrevin had similar inhibitory effects on the flagellar beating of demembranated and reactivated sperm and the sliding disintegration of trypsin-treated axonemes. We also analyzed the curvature and shear angle of the beating flagella and found that the proximal region of the sperm flagellum was less sensitive to ciliobrevin compared with more distal regions, where bending motions were blocked completely. Interestingly, the shear angle analysis of flagellar motility showed that ciliobrevin induced highly asymmetric bends in the proximal region of the flagellum. These results suggest that there is heterogeneity in the inhibitory thresholds of dynein motors, which depend on the regions along the flagellar shaft (proximal or distal) and on the sites of doublets in the flagellar cross-section (doublet numbers). We expect that it will be possible to map the functional differences in dynein subtypes along and/or around the cross-sections of flagellar axonemes by analyzing the inhibitory effects of ciliobrevin.

  3. Swarming motility and the control of master regulators of flagellar biosynthesis

    PubMed Central

    Patrick, Joyce E.; Kearns, Daniel B.

    2011-01-01

    Swarming motility is the movement of bacteria over a solid surface powered by rotating flagella. The expression of flagellar biosynthesis genes is governed by species-specific master regulator transcription factors. Mutations that reduce or enhance master regulator activity have a commensurate effect on swarming motility. Here we review what is known about the proteins that modulate swarming motility and appear to act upstream of the master flagellar regulators in diverse swarming bacteria. We hypothesize that environmental control of the master regulators is important to the swarming phenotype perhaps at the level of controlling flagellar number. PMID:22092493

  4. E. Coli

    MedlinePlus

    ... E. coli is short for the medical term Escherichia coli . The strange thing about these bacteria — and lots ... cause a very serious infection. Someone who has E. coli infection may have these symptoms: bad stomach cramps and ...

  5. Activation of the Glutamic Acid-Dependent Acid Resistance System in Escherichia coli BL21(DE3) Leads to Increase of the Fatty Acid Biotransformation Activity

    PubMed Central

    Woo, Ji-Min; Kim, Ji-Won; Song, Ji-Won; Blank, Lars M.; Park, Jin-Byung

    2016-01-01

    The biosynthesis of carboxylic acids including fatty acids from biomass is central in envisaged biorefinery concepts. The productivities are often, however, low due to product toxicity that hamper whole-cell biocatalyst performance. Here, we have investigated factors that influence the tolerance of Escherichia coli to medium chain carboxylic acid (i.e., n-heptanoic acid)-induced stress. The metabolic and genomic responses of E. coli BL21(DE3) and MG1655 grown in the presence of n-heptanoic acid indicated that the GadA/B-based glutamic acid-dependent acid resistance (GDAR) system might be critical for cellular tolerance. The GDAR system, which is responsible for scavenging intracellular protons by catalyzing decarboxylation of glutamic acid, was inactive in E. coli BL21(DE3). Activation of the GDAR system in this strain by overexpressing the rcsB and dsrA genes, of which the gene products are involved in the activation of GadE and RpoS, respectively, resulted in acid tolerance not only to HCl but also to n-heptanoic acid. Furthermore, activation of the GDAR system allowed the recombinant E. coli BL21(DE3) expressing the alcohol dehydrogenase of Micrococcus luteus and the Baeyer-Villiger monooxygenase of Pseudomonas putida to reach 60% greater product concentration in the biotransformation of ricinoleic acid (i.e., 12-hydroxyoctadec-9-enoic acid (1)) into n-heptanoic acid (5) and 11-hydroxyundec-9-enoic acid (4). This study may contribute to engineering E. coli-based biocatalysts for the production of carboxylic acids from renewable biomass. PMID:27681369

  6. Biochemical properties and physiological roles of NADP-dependent malic enzyme in Escherichia coli.

    PubMed

    Wang, Baojuan; Wang, Peng; Zheng, Enxia; Chen, Xiangxian; Zhao, Hanjun; Song, Ping; Su, Ruirui; Li, Xiaoning; Zhu, Guoping

    2011-10-01

    Malic enzymes catalyze the reversible oxidative decarboxylation of L-malate using NAD(P)(+) as a cofactor. NADP-dependent malic enzyme (MaeB) from Escherichia coli MG1655 was expressed and purified as a fusion protein. The molecular weight of MaeB was about 83 kDa, as determined by SDS-PAGE. The recombinant MaeB showed a maximum activity at pH 7.8 and 46°C. MaeB activity was dependent on the presence of Mn(2+) but was strongly inhibited by Zn(2+). In order to understand the physiological roles, recombinant E. coli strains (icd (NADP)/ΔmaeB and icd (NAD)/ΔmaeB) containing NADP-dependent isocitrate dehydrogenase (IDH), or engineered NAD-dependent IDH with the deletion of the maeB gene, were constructed using homologous recombination. During growth on acetate, icd (NAD)/ΔmaeB grew poorly, having a growth rate only 60% that of the wild-type strain (icd (NADP)). Furthermore, icd (NADP)/ΔmaeB exhibited a 2-fold greater adaptability to acetate than icd (NAD)/ΔmaeB, which may be explained by more NADPH production for biosynthesis in icd (NADP)/ΔmaeB due to its NADP-dependent IDH. These results indicated that MaeB was important for NADPH production for bacterial growth on acetate. We also observed that MaeB activity was significantly enhanced (7.83-fold) in icd (NAD), which was about 3-fold higher than that in icd (NADP), when switching from glucose to acetate. The marked increase of MaeB activity was probably induced by the shortage of NADPH in icd (NAD). Evidently, MaeB contributed to the NADPH generation needed for bacterial growth on two carbon compounds.

  7. 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

  8. 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.

  9. Antiphase Synchronization in a Flagellar-Dominance Mutant of Chlamydomonas

    NASA Astrophysics Data System (ADS)

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

    2013-10-01

    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.

  10. Composition and sensory function of the trypanosome flagellar membrane

    PubMed Central

    Maric, Danijela; Epting, Conrad L.; Engman, David M.

    2010-01-01

    Summary A cilium is an extension of the cell that contains an axonemal complex of microtubules and associated proteins bounded by a membrane which is contiguous with the cell body membrane. Cilia may be nonmotile or motile, the latter having additional specific roles in cell or fluid movement. The term flagellum refers to the motile cilium of free-living single cells (e.g., bacteria, archaea, spermatozoa and protozoa). In eukaryotes, both nonmotile and motile cilia possess sensory functions. The ciliary interior (cilioplasm) is separated from the cytoplasm by a selective barrier that prevents passive diffusion of molecules between the two domains. The sensory functions of cilia reside largely in the membrane and signals generated in the cilium are transduced into a variety of cellular responses. In this review we discuss the structure and biogenesis of the cilium, with special attention to the trypanosome flagellar membrane, its lipid and protein composition and its proposed roles in sensing and signaling. PMID:20580599

  11. 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.

  12. The effects of translation and rotation on flagellar synchronization

    NASA Astrophysics Data System (ADS)

    Tu, Jonathan H.; Arcak, Murat; Maharbiz, Michel

    2014-11-01

    Synchrony is often observed in studies of swimming microorganisms. Examples include collective behavior in large populations of microswimmers, metachronal waves passing through arrays of cilia, and flagellar bundling. In this work, we focus on the hydrodynamic interactions that occur between flagella in close proximity. Specifically, we use the method of regularized Stokeslets to numerically investigate the precise mechanisms through which phase synchrony occurs in a pair of side-by-side rigid helices. Because our ``end-pinned'' model enforces restoring forces at a single end of each helix, we are able to isolate and compare the respective effects of translational and rotational motions. We find that while certain degrees of freedom promote synchrony, others promote anti-synchrony or have little effect. Funded by ONR Grant N000141310551.

  13. 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

  14. Single-file diffusion of flagellin in flagellar filaments.

    PubMed

    Stern, Alan S; Berg, Howard C

    2013-07-01

    A bacterial flagellar filament is a cylindrical crystal of a protein known as flagellin. Flagellin subunits travel from the cytoplasm through a 2 nm axial pore and polymerize at the filament's distal end. They are supplied by a pump in the cell membrane powered by a proton-motive force. In a recent experiment, it was observed that growth proceeded at a rate of approximately one subunit every 2 s. Here, we asked whether transport of subunits through the pore at this rate could be effected by single-file diffusion, which we simulated by a random walk on a one-dimensional lattice. Assuming that the subunits are α-helical, the answer is yes, by a comfortable margin.

  15. 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.

  16. The Limiting Speed of the Bacterial Flagellar Motor

    NASA Astrophysics Data System (ADS)

    Nirody, Jasmine A.; Berry, Richard M.; Oster, George

    2016-08-01

    Recent experiments on the bacterial flagellar motor have shown that the structure of this nanomachine, which drives locomotion in a wide range of bacterial species, 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 propose that, contrary to previous assumptions, the maximum speed of the motor is not universal, but rather increases as additional torque-generators are recruited. This result arises from our 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.

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

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

    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.

  18. Flagellar motility confers epiphytic fitness advantages upon Pseudomonas syringae

    SciTech Connect

    Haefele, D.M.; Lindow, S.E.

    1987-10-01

    The role of flagellar motility in determining the epiphytic fitness of an ice-nucleation-active strain of Pseudomonas syringae was examined. The loss of flagellar motility reduced the epiphytic fitness of a normally motile P. syringae strain as measured by its growth, survival, and competitive ability on bean leaf surfaces. Equal population sizes of motile parental or nonmotile mutant P. syringae strains were maintained on bean plants for at least 5 days following the inoculation of fully expanded primary leaves. However, when bean seedlings were inoculated before the primary leaves had expanded and bacterial populations on these leaves were quantified at full expansion, the population size of the nonmotile derivative strain reached only 0.9% that of either the motile parental or revertant strain. When fully expanded bean primary leaves were coinoculated with equal numbers of motile and nonmotile cells, the population size of a nonmotile derivative strain was one-third of that of the motile parental or revertant strain after 8 days. Motile and nonmotile cells were exposed in vitro and on plants to UV radiation and desiccating conditions. The motile and nonmotile strains exhibited equal resistance to both stresses in vitro. However, the population size of a nonmotile strain on leaves was less than 20% that of a motile revertant strain when sampled immediately after UV irradiation. Epiphytic populations of both motile and nonmotile P. syringae declined under desiccating conditions on plants, and after 8 days, the population size of a nonmotile strain was less than one-third that of the motile parental or revertant strain.

  19. Flagellar Motility Confers Epiphytic Fitness Advantages upon Pseudomonas syringae

    PubMed Central

    Haefele, Douglas M.; Lindow, Steven E.

    1987-01-01

    The role of flagellar motility in determining the epiphytic fitness of an ice-nucleation-active strain of Pseudomonas syringae was examined. The loss of flagellar motility reduced the epiphytic fitness of a normally motile P. syringae strain as measured by its growth, survival, and competitive ability on bean leaf surfaces. Equal population sizes of motile parental or nonmotile mutant P. syringae strains were maintained on bean plants for at least 5 days following the inoculation of fully expanded primary leaves. However, when bean seedlings were inoculated before the primary leaves had expanded and bacterial populations on these leaves were quantified at full expansion, the population size of the nonmotile derivative strain reached only 0.9% that of either the motile parental or revertant strain. When fully expanded bean primary leaves were coinoculated with equal numbers of motile and nonmotile cells, the population size of a nonmotile derivative strain was one-third of that of the motile parental or revertant strain after 8 days. Motile and nonmotile cells were exposed in vitro and on plants to UV radiation and desiccating conditions. The motile and nonmotile strains exhibited equal resistance to both stresses in vitro. However, the population size of a nonmotile strain on leaves was less than 20% that of a motile revertant strain when sampled immediately after UV irradiation. Epiphytic populations of both motile and nonmotile P. syringae declined under desiccating conditions on plants, and after 8 days, the population size of a nonmotile strain was less than one-third that of the motile parental or revertant strain. PMID:16347469

  20. Dynamics of the bacterial flagellar motor with multiple stators

    PubMed Central

    Meacci, Giovanni; Tu, Yuhai

    2009-01-01

    The bacterial flagellar motor drives the rotation of flagellar filaments and enables many species of bacteria to swim. Torque is generated by interaction of stator units, anchored to the peptidoglycan cell wall, with the rotor. Recent experiments [Yuan J, Berg HC (2008) Proc Natl Acad Sci USA 105:1182–1185] show that at near-zero load the speed of the motor is independent of the number of stators. Here, we introduce a mathematical model of the motor dynamics that explains this behavior based on a general assumption that the stepping rate of a stator depends on the torque exerted by the stator on the rotor. We find that the motor dynamics can be characterized by two timescales: the moving-time interval for the mechanical rotation of the rotor and the waiting-time interval determined by the chemical transitions of the stators. We show that these two timescales depend differently on the load, and that their cross-over provides the microscopic explanation for the existence of two regimes in the torque-speed curves observed experimentally. We also analyze the speed fluctuation for a single motor by using our model. We show that the motion is smoothed by having more stator units. However, the mechanism for such fluctuation reduction is different depending on the load. We predict that the speed fluctuation is determined by the number of steps per revolution only at low load and is controlled by external noise for high load. Our model can be generalized to study other molecular motor systems with multiple power-generating units. PMID:19234112

  1. Differential substrate specificity and kinetic behavior of Escherichia coli YfdW and Oxalobacter formigenes formyl coenzyme A transferase.

    PubMed

    Toyota, Cory G; Berthold, Catrine L; Gruez, Arnaud; Jónsson, Stefán; Lindqvist, Ylva; Cambillau, Christian; Richards, Nigel G J

    2008-04-01

    The yfdXWUVE operon appears to encode proteins that enhance the ability of Escherichia coli MG1655 to survive under acidic conditions. Although the molecular mechanisms underlying this phenotypic behavior remain to be elucidated, findings from structural genomic studies have shown that the structure of YfdW, the protein encoded by the yfdW gene, is homologous to that of the enzyme that mediates oxalate catabolism in the obligate anaerobe Oxalobacter formigenes, O. formigenes formyl coenzyme A transferase (FRC). We now report the first detailed examination of the steady-state kinetic behavior and substrate specificity of recombinant, wild-type YfdW. Our studies confirm that YfdW is a formyl coenzyme A (formyl-CoA) transferase, and YfdW appears to be more stringent than the corresponding enzyme (FRC) in Oxalobacter in employing formyl-CoA and oxalate as substrates. We also report the effects of replacing Trp-48 in the FRC active site with the glutamine residue that occupies an equivalent position in the E. coli protein. The results of these experiments show that Trp-48 precludes oxalate binding to a site that mediates substrate inhibition for YfdW. In addition, the replacement of Trp-48 by Gln-48 yields an FRC variant for which oxalate-dependent substrate inhibition is modified to resemble that seen for YfdW. Our findings illustrate the utility of structural homology in assigning enzyme function and raise the question of whether oxalate catabolism takes place in E. coli upon the up-regulation of the yfdXWUVE operon under acidic conditions. PMID:18245280

  2. Chlamydomonas alpha-tubulin is posttranslationally modified in the flagella during flagellar assembly

    PubMed Central

    1983-01-01

    The principal alpha-tubulin within Chlamydomonas reinhardtii flagellar axonemes differs from the major alpha-tubulin in the cell body. We show that these two isoelectric variants of alpha-tubulin are related to one another since posttranslational modification of the cell body precursor form converts it to the axonemal form. During flagellar assembly, precursor alpha-tubulin enters the flagella and is posttranslationally modified within the flagellar matrix fraction prior to or at the time of its addition to the growing axonemal microtubules. Experiments designed to identify the nature of this posttranslational modification have also been conducted. When flagella are induced to assemble in the absence of de novo protein synthesis, tritiated acetate can be used to posttranslationally label alpha-tubulin in vivo and, under these conditions, no other flagellar polypeptides exhibit detectable labeling. PMID:6863393

  3. 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.

  4. The effects of upaB deletion and the double/triple deletion of upaB, aatA, and aatB genes on pathogenicity of avian pathogenic Escherichia coli.

    PubMed

    Zhu-Ge, Xiang-Kai; Pan, Zi-Hao; Tang, Fang; Mao, Xiang; Hu, Lin; Wang, Shao-Hui; Xu, Bin; Lu, Cheng-Ping; Fan, Hong-Jie; Dai, Jian-Jun

    2015-12-01

    Autotransporters (ATs) are associated with pathogenesis of Avian Pathogenic Escherichia coli (APEC). The molecular characterization of APEC ATs can provide insights about their relevance to APEC pathogenesis. Here, we characterized a conventional autotransporter UpaB in APEC DE205B genome. The upaB existed in 41.9 % of 236 APEC isolates and was predominantly associated with ECOR B2 and D. Our studies showed that UpaB mediates the DE205B adhesion in DF-1 cells, and enhances autoaggregation and biofilm formation of fimbria-negative E. coli AAEC189 (MG1655Δfim) in vitro. Deletion of upaB of DE205B attenuates the virulence in duck model and early colonization in the duck lungs during APEC systemic infection. Furthermore, double and triple deletion of upaB, aatA, and aatB genes cumulatively attenuated DE205B adhesion in DF-1 cells, accompanying with decreased 50 % lethal dose (LD50) in duck model and the early colonization in the duck lungs. However, DE205BΔupaB/ΔaatA/ΔaatB might "compensate" the influence of gene deletion by upregulating the expression of fimbrial adhesin genes yqiL, yadN, and vacuolating autotransporter vat during early colonization of APEC. Finally, we demonstrated that vaccination with recombinant UpaB, AatA, and AatB proteins conferred protection against colisepticemia caused by DE205B infection in duck model.

  5. mRNA abundance changes during flagellar regeneration in Chlamydomonas reinhardtii.

    PubMed Central

    Schloss, J A; Silflow, C D; Rosenbaum, J L

    1984-01-01

    Flagellar amputation in Chlamydomonas reinhardtii induces the accumulation of a specific set of RNAs, many of which encode flagellar proteins. We prepared a cDNA clone bank from RNA isolated from cells undergoing flagellar regeneration. From this bank, we selected clones that contain RNA sequences that display several different patterns of abundance regulation. Based on quantitation of the relative amounts of labeled, cloned cDNAs hybridizing to dots of RNA on nitrocellulose filters, the cloned sequences were divided into five regulatory classes: class I RNAs remain at constant abundance during flagellar regeneration; classes II, III, and IV begin to increase in abundance within a few minutes after deflagellation, reach maximal abundance at successively later times during regeneration, and return to control cell levels within 2 to 3 h; and class V RNA abundance decreases during flagellar regeneration. Alpha- and beta-tubulin mRNAs are included in regulatory class IV. The abundance kinetics of alpha-tubulin mRNAs differ slightly from those of beta-tubulin mRNAs. The availability of these clones makes possible studies on the mechanisms controlling the abundance of a wide variety of different RNA species during flagellar regeneration in Chlamydomonas. Images PMID:6546968

  6. Complex flagellar motions and swimming patterns of the flagellates Paraphysomonas vestita and Pteridomonas danica.

    PubMed

    Christensen-Dalsgaard, Karen K; Fenchel, Tom

    2004-03-01

    Most flagellates with hispid flagella, that is, flagella with rigid filamentous hairs (mastigonemes), swim in the direction of the flagellar wave propagation with an anterior position of the flagellum. Previous analysis was based on planar wave propagation showing that the mastigonemes pull fluid along the flagellar axis. In the present study, we investigate the flagellar motions and swimming patterns for two flagellates with hispid flagella: Paraphysomonas vestita and Pteridomonas danica. Studies were carried out using normal and high-speed video recording, and particles were added to visualize flow around cells generating feeding currents. When swimming or generating flow, P. vestita was able to pull fluid normal to, and not just along, the flagellum, implying the use of the mastigonemes in an as yet un-described way. When the flagellum made contact with food particles, it changed the flagellar waveform so that the particle was fanned towards the ingestion area, suggesting mechano-sensitivity of the mastigonemes. Pteridomonas danica was capable of more complex swimming than previously described for flagellated protists. This was associated with control of the flagellar beat as well as an ability to bend the plane of the flagellar waveform.

  7. Analysis of the motA flagellar motor gene from Rhodobacter sphaeroides, a bacterium with a unidirectional, stop-start flagellum.

    PubMed

    Shah, D S; Sockett, R E

    1995-09-01

    Rhodobacter sphaeroides swims by unidirectional rotation of a single medial flagellum, re-orienting randomly by Brownian motion when flagellar rotation stops and restarts. Previously we identified a mutant with a paralysed flagellum, which was complemented by a Rhodobacter gene that had homology to motB of Escherichia coli, a bacterium with bidirectional flagella. In the current work, interposon mutagenesis upstream of the Rhodobacter motB gene gave rise to another paralysed mutant, RED5. DNA sequence analysis of this upstream region showed one open reading frame, the predicted polypeptide sequence of which shows homology to the MotA protein of E. coli. MotA is thought to be a proton 'pore' involved in converting proton-motive force into flagellar rotation. Several potential proton-binding amino acids were conserved between putative membrane-spanning regions of R. sphaeroides and E. coli MotA sequences, along with a highly charged cytoplasmic linker region. Complementation studies with mutant RED5 showed the presence of an active promoter upstream from motA which was found to be necessary for expression of both motA and motB. Examination of the upstream DNA sequence showed only one putative promoter-like sequence which resembled a sigma 54-type promoter, including a potential enhancer binding site. The overall similarities between the R. sphaeroides MotA protein and those from other bacteria suggest that, despite the novel unidirectional rotation of the R. sphaeroides flagellum, the function of the MotA protein is similar to that in bacteria with bidirectional flagella.

  8. Transcriptomic analysis of Escherichia coli O157:H7 and K-12 cultures exposed to inorganic and organic acids in stationary phase reveals acidulant- and strain-specific acid tolerance responses.

    PubMed

    King, Thea; Lucchini, Sacha; Hinton, Jay C D; Gobius, Kari

    2010-10-01

    The food-borne pathogen Escherichia coli O157:H7 is commonly exposed to organic acid in processed and preserved foods, allowing adaptation and the development of tolerance to pH levels otherwise lethal. Since little is known about the molecular basis of adaptation of E. coli to organic acids, we studied K-12 MG1655 and O157:H7 Sakai during exposure to acetic, lactic, and hydrochloric acid at pH 5.5. This is the first analysis of the pH-dependent transcriptomic response of stationary-phase E. coli. Thirty-four genes and three intergenic regions were upregulated by both strains during exposure to all acids. This universal acid response included genes involved in oxidative, envelope, and cold stress resistance and iron and manganese uptake, as well as 10 genes of unknown function. Acidulant- and strain-specific responses were also revealed. The acidulant-specific response reflects differences in the modes of microbial inactivation, even between weak organic acids. The two strains exhibited similar responses to lactic and hydrochloric acid, while the response to acetic acid was distinct. Acidulant-dependent differences between the strains involved induction of genes involved in the heat shock response, osmoregulation, inorganic ion and nucleotide transport and metabolism, translation, and energy production. E. coli O157:H7-specific acid-inducible genes were identified, suggesting that the enterohemorrhagic E. coli strain possesses additional molecular mechanisms contributing to acid resistance that are absent in K-12. While E. coli K-12 was most resistant to lactic and hydrochloric acid, O157:H7 may have a greater ability to survive in more complex acidic environments, such as those encountered in the host and during food processing.

  9. Real-time observation of Escherichia coli cells under irradiation with a 2-MeV H{sup +} microbeam

    SciTech Connect

    Kato, Mikio; Meissl, Walter; Ikeda, Tokihiro; Yamazaki, Yasunori; Umezawa, Kenji

    2012-05-07

    A high-energy H{sup +} microbeam generated by tapered glass capillary optics was applied to a single Escherichia coli cell, in order to evaluate the effects of irradiation on the activity of the flagellar motor and cell growth in real time. The flagellar motor of the tethered cells was stopped by irradiation with an average ion fluence of 2.0 x 10{sup 12} protons/cm{sup 2}. When a lower dose was applied to the cells attached to the substrate, an elongated cell, which seemed ready to divide, divided into two daughter cells; however, the daughter cells did not elongate, neither did further cell division occur.

  10. Genome Sequence and Analysis of Escherichia coli MRE600, a Colicinogenic, Nonmotile Strain that Lacks RNase I and the Type I Methyltransferase, EcoKI

    PubMed Central

    Kurylo, Chad M.; Alexander, Noah; Dass, Randall A.; Parks, Matthew M.; Altman, Roger A.; Vincent, C. Theresa; Mason, Christopher E.; Blanchard, Scott C.

    2016-01-01

    Escherichia coli strain MRE600 was originally identified for its low RNase I activity and has therefore been widely adopted by the biomedical research community as a preferred source for the expression and purification of transfer RNAs and ribosomes. Despite its widespread use, surprisingly little information about its genome or genetic content exists. Here, we present the first de novo assembly and description of the MRE600 genome and epigenome. To provide context to these studies of MRE600, we include comparative analyses with E. coli K-12 MG1655 (K12). Pacific Biosciences Single Molecule, Real-Time sequencing reads were assembled into one large chromosome (4.83 Mb) and three smaller plasmids (89.1, 56.9, and 7.1 kb). Interestingly, the 7.1-kb plasmid possesses genes encoding a colicin E1 protein and its associated immunity protein. The MRE600 genome has a G + C content of 50.8% and contains a total of 5,181 genes, including 4,913 protein-encoding genes and 268 RNA genes. We identified 41,469 modified DNA bases (0.83% of total) and found that MRE600 lacks the gene for type I methyltransferase, EcoKI. Phylogenetic, taxonomic, and genetic analyses demonstrate that MRE600 is a divergent E. coli strain that displays features of the closely related genus, Shigella. Nevertheless, comparative analyses between MRE600 and E. coli K12 show that these two strains exhibit nearly identical ribosomal proteins, ribosomal RNAs, and highly homologous tRNA species. Substantiating prior suggestions that MRE600 lacks RNase I activity, the RNase I-encoding gene, rna, contains a single premature stop codon early in its open-reading frame. PMID:26802429

  11. Genome Sequence and Analysis of Escherichia coli MRE600, a Colicinogenic, Nonmotile Strain that Lacks RNase I and the Type I Methyltransferase, EcoKI.

    PubMed

    Kurylo, Chad M; Alexander, Noah; Dass, Randall A; Parks, Matthew M; Altman, Roger A; Vincent, C Theresa; Mason, Christopher E; Blanchard, Scott C

    2016-03-01

    Escherichia coli strain MRE600 was originally identified for its low RNase I activity and has therefore been widely adopted by the biomedical research community as a preferred source for the expression and purification of transfer RNAs and ribosomes. Despite its widespread use, surprisingly little information about its genome or genetic content exists. Here, we present the first de novo assembly and description of the MRE600 genome and epigenome. To provide context to these studies of MRE600, we include comparative analyses with E. coli K-12 MG1655 (K12). Pacific Biosciences Single Molecule, Real-Time sequencing reads were assembled into one large chromosome (4.83 Mb) and three smaller plasmids (89.1, 56.9, and 7.1 kb). Interestingly, the 7.1-kb plasmid possesses genes encoding a colicin E1 protein and its associated immunity protein. The MRE600 genome has a G + C content of 50.8% and contains a total of 5,181 genes, including 4,913 protein-encoding genes and 268 RNA genes. We identified 41,469 modified DNA bases (0.83% of total) and found that MRE600 lacks the gene for type I methyltransferase, EcoKI. Phylogenetic, taxonomic, and genetic analyses demonstrate that MRE600 is a divergent E. coli strain that displays features of the closely related genus, Shigella. Nevertheless, comparative analyses between MRE600 and E. coli K12 show that these two strains exhibit nearly identical ribosomal proteins, ribosomal RNAs, and highly homologous tRNA species. Substantiating prior suggestions that MRE600 lacks RNase I activity, the RNase I-encoding gene, rna, contains a single premature stop codon early in its open-reading frame. PMID:26802429

  12. Escherichia coli nfuA is essential for maintenance of Shiga toxin phage Min27 lysogeny under iron-depleted condition.

    PubMed

    Cao, Dongmei; Ji, Wenhui; Fu, Qiang; Lu, Chenping; Wang, Hengan; Sun, Jianhe; Yan, Yaxian

    2015-10-01

    It has been earlier hypothesized that lysogenic infection with Stx-encoding phages influences protein expression in the bacterial host, and therefore, some differentially expressed proteins could affect survival characteristics and pathogenicity. We compared the protein expression profiles of the host MG1655 and lysogens by 2D electrophoresis. Four different genes identified were all related to Fe/S subunit production, namely, nfuA, fdoH, sdhB and ftnA. To explore the role of nfuA in the biology of Stx prophage lysogeny, gene knockout experiments and phage lysogenic conversion were performed. The inactivation of nfuA caused the prophage to enter its lytic life cycle, especially under an iron-depleted condition. A similar activity was also detected in the Escherichia coli O157:H7 strain from which the Stx phage Min 27 was originally isolated. NfuA might be the positive regulator of genes controlling lysogenic cycle such as cI, cII and cIII since their transcriptional level was significantly reduced in nfuA deletion mutant as shown by qRT-PCR. We conclude that NfuA is essential for maintenance of Stx phage lysogeny in host's genetic reservoir under iron-deficient condition. PMID:26337151

  13. The EcoKI Type I Restriction-Modification System in Escherichia coli Affects but Is Not an Absolute Barrier for Conjugation

    PubMed Central

    Aarestrup, Frank M.; Hasman, Henrik

    2014-01-01

    The rapid evolution of bacteria is crucial to their survival and is caused by exchange, transfer, and uptake of DNA, among other things. Conjugation is one of the main mechanisms by which bacteria share their DNA, and it is thought to be controlled by varied bacterial immune systems. Contradictory results about restriction-modification systems based on phenotypic studies have been presented as reasons for a barrier to conjugation with and other means of uptake of exogenous DNA. In this study, we show that inactivation of the R.EcoKI restriction enzyme in strain Escherichia coli K-12 strain MG1655 increases the conjugational transfer of plasmid pOLA52, which carriers two EcoKI recognition sites. Interestingly, the results were not absolute, and uptake of unmethylated pOLA52 was still observed in the wild-type strain (with an intact hsdR gene) but at a reduction of 85% compared to the uptake of the mutant recipient with a disrupted hsdR gene. This leads to the conclusion that EcoKI restriction-modification affects the uptake of DNA by conjugation but is not a major barrier to plasmid transfer. PMID:25384481

  14. The EcoKI type I restriction-modification system in Escherichia coli affects but is not an absolute barrier for conjugation.

    PubMed

    Roer, Louise; Aarestrup, Frank M; Hasman, Henrik

    2015-01-01

    The rapid evolution of bacteria is crucial to their survival and is caused by exchange, transfer, and uptake of DNA, among other things. Conjugation is one of the main mechanisms by which bacteria share their DNA, and it is thought to be controlled by varied bacterial immune systems. Contradictory results about restriction-modification systems based on phenotypic studies have been presented as reasons for a barrier to conjugation with and other means of uptake of exogenous DNA. In this study, we show that inactivation of the R.EcoKI restriction enzyme in strain Escherichia coli K-12 strain MG1655 increases the conjugational transfer of plasmid pOLA52, which carriers two EcoKI recognition sites. Interestingly, the results were not absolute, and uptake of unmethylated pOLA52 was still observed in the wild-type strain (with an intact hsdR gene) but at a reduction of 85% compared to the uptake of the mutant recipient with a disrupted hsdR gene. This leads to the conclusion that EcoKI restriction-modification affects the uptake of DNA by conjugation but is not a major barrier to plasmid transfer. PMID:25384481

  15. 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

  16. 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. PMID:26873312

  17. A design-constraint trade-off underpins the diversity in ecologically important traits in species Escherichia coli.

    PubMed

    Phan, Katherine; Ferenci, Thomas

    2013-10-01

    Bacterial species are internally diverse in genomic and multi-locus gene comparisons. The ecological causes of phenotypic and genotypic diversity within species are far less well understood. Here, we focus on the competitive fitness for growth on nutrients within Escherichia coli, an internally rich species. Competition experiments in nutrient-limited chemostats revealed that members of the ECOR collection exhibited a wide continuum of competitive abilities, with some fitter and some less fit than the lab strain MG1655. We observed an inverse relationship between competitiveness and the resistance of strains to detergent and antibiotic, consistent with the notion that membrane permeability and competitive fitness are linked by a trade-off between self-preservation and nutritional competence (SPANC); high permeability has a postulated cost in antibacterial sensitivity whereas a low permeability has a cost in nutrient affinity. Isolates moved along the markedly nonlinear trade-off curve by mutational adaptation; an ECOR strain sensitive to antibacterials and a good competitor was easily converted by mutation into a mutant with higher resistance but poorer competition in the presence of low antibiotic concentrations. Conversely, a resistant ECOR strain changed into a better competitor after a short period of selection under nutrient limitation. In both directions, mutations can affect porin proteins and outer membrane permeability, as indicated by protein analysis, gene sequencing and an independent assay of outer membrane permeability. The extensive, species-wide diversity of E. coli in ecologically important traits can thus be explained as an evolutionary consequence of a SPANC trade-off driven by antagonistic pleiotropy.

  18. The Gearbox of the Bacterial Flagellar Motor Switch.

    PubMed

    Pandini, Alessandro; Morcos, Faruck; Khan, Shahid

    2016-07-01

    Switching of flagellar motor rotation sense dictates bacterial chemotaxis. Multi-subunit FliM-FliG rotor rings couple signal protein binding in FliM with reversal of a distant FliG C-terminal (FliGC) helix involved in stator contacts. Subunit dynamics were examined in conformer ensembles generated by molecular simulations from the X-ray structures. Principal component analysis extracted collective motions. Interfacial loop immobilization by complex formation coupled elastic fluctuations of the FliM middle (FliMM) and FliG middle (FliGM) domains. Coevolved mutations captured interfacial dynamics as well as contacts. FliGM rotation was amplified via two central hinges to the FliGC helix. Intrinsic flexibility, reported by the FliGMC ensembles, reconciled conformers with opposite FliGC helix orientations. FliG domain stacking deformed the inter-domain linker and reduced flexibility; but conformational changes were not triggered by engineered linker deletions that cause a rotation-locked phenotype. These facts suggest that binary rotation states arise from conformational selection by stacking interactions. PMID:27345932

  19. 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

  20. The Gearbox of the Bacterial Flagellar Motor Switch.

    PubMed

    Pandini, Alessandro; Morcos, Faruck; Khan, Shahid

    2016-07-01

    Switching of flagellar motor rotation sense dictates bacterial chemotaxis. Multi-subunit FliM-FliG rotor rings couple signal protein binding in FliM with reversal of a distant FliG C-terminal (FliGC) helix involved in stator contacts. Subunit dynamics were examined in conformer ensembles generated by molecular simulations from the X-ray structures. Principal component analysis extracted collective motions. Interfacial loop immobilization by complex formation coupled elastic fluctuations of the FliM middle (FliMM) and FliG middle (FliGM) domains. Coevolved mutations captured interfacial dynamics as well as contacts. FliGM rotation was amplified via two central hinges to the FliGC helix. Intrinsic flexibility, reported by the FliGMC ensembles, reconciled conformers with opposite FliGC helix orientations. FliG domain stacking deformed the inter-domain linker and reduced flexibility; but conformational changes were not triggered by engineered linker deletions that cause a rotation-locked phenotype. These facts suggest that binary rotation states arise from conformational selection by stacking interactions.

  1. [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

  2. 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.

  3. Role of porins in sensitivity of Escherichia coli to antibacterial activity of the lactoperoxidase enzyme system.

    PubMed

    De Spiegeleer, Philipp; Sermon, Jan; Vanoirbeek, Kristof; Aertsen, Abram; Michiels, Chris W

    2005-07-01

    Lactoperoxidase is an enzyme that contributes to the antimicrobial defense in secretory fluids and that has attracted interest as a potential biopreservative for foods and other perishable products. Its antimicrobial activity is based on the formation of hypothiocyanate (OSCN-) from thiocyanate (SCN-), using H2O2 as an oxidant. To gain insight into the antibacterial mode of action of the lactoperoxidase enzyme system, we generated random transposon insertion mutations in Escherichia coli MG1655 and screened the resultant mutants for an altered tolerance of bacteriostatic concentrations of this enzyme system. Out of the ca. 5,000 mutants screened, 4 showed significantly increased tolerance, and 2 of these had an insertion, one in the waaQ gene and one in the waaO gene, whose products are involved in the synthesis of the core oligosaccharide moiety of lipopolysaccharides. Besides producing truncated lipopolysaccharides and displaying hypersensitivity to novobiocin and sodium dodecyl sulfate (SDS), these mutants were also shown by urea-SDS-polyacrylamide gel electrophoresis analysis to have reduced amounts of porins in their outer membranes. Moreover, they showed a reduced degradation of p-nitrophenyl phosphate and an increased resistance to ampicillin, two indications of a decrease in outer membrane permeability for small hydrophilic solutes. Additionally, ompC and ompF knockout mutants displayed levels of tolerance to the lactoperoxidase system similar to those displayed by the waa mutants. These results suggest that mutations which reduce the porin-mediated outer membrane permeability for small hydrophilic molecules lead to increased tolerance to the lactoperoxidase enzyme system because of a reduced uptake of OSCN-.

  4. Role of Porins in Sensitivity of Escherichia coli to Antibacterial Activity of the Lactoperoxidase Enzyme System

    PubMed Central

    De Spiegeleer, Philipp; Sermon, Jan; Vanoirbeek, Kristof; Aertsen, Abram; Michiels, Chris W.

    2005-01-01

    Lactoperoxidase is an enzyme that contributes to the antimicrobial defense in secretory fluids and that has attracted interest as a potential biopreservative for foods and other perishable products. Its antimicrobial activity is based on the formation of hypothiocyanate (OSCN−) from thiocyanate (SCN−), using H2O2 as an oxidant. To gain insight into the antibacterial mode of action of the lactoperoxidase enzyme system, we generated random transposon insertion mutations in Escherichia coli MG1655 and screened the resultant mutants for an altered tolerance of bacteriostatic concentrations of this enzyme system. Out of the ca. 5,000 mutants screened, 4 showed significantly increased tolerance, and 2 of these had an insertion, one in the waaQ gene and one in the waaO gene, whose products are involved in the synthesis of the core oligosaccharide moiety of lipopolysaccharides. Besides producing truncated lipopolysaccharides and displaying hypersensitivity to novobiocin and sodium dodecyl sulfate (SDS), these mutants were also shown by urea-SDS-polyacrylamide gel electrophoresis analysis to have reduced amounts of porins in their outer membranes. Moreover, they showed a reduced degradation of p-nitrophenyl phosphate and an increased resistance to ampicillin, two indications of a decrease in outer membrane permeability for small hydrophilic solutes. Additionally, ompC and ompF knockout mutants displayed levels of tolerance to the lactoperoxidase system similar to those displayed by the waa mutants. These results suggest that mutations which reduce the porin-mediated outer membrane permeability for small hydrophilic molecules lead to increased tolerance to the lactoperoxidase enzyme system because of a reduced uptake of OSCN−. PMID:16000755

  5. Enforced ATP futile cycling increases specific productivity and yield of anaerobic lactate production in Escherichia coli.

    PubMed

    Hädicke, Oliver; Bettenbrock, Katja; Klamt, Steffen

    2015-10-01

    The manipulation of cofactor pools such as ATP or NAD(P)H has for long been recognized as key targets for metabolic engineering of microorganisms to improve yields and productivities of biotechnological processes. Several works in the past have shown that enforcing ATP futile cycling may enhance the synthesis of certain products under aerobic conditions. However, case studies demonstrating that ATP wasting may also have beneficial effects for anaerobic production processes are scarce. Taking lactic acid as an economically relevant product, we demonstrate that induction of ATP futile cycling in Escherichia coli leads to increased yields and specific production rates under anaerobic conditions, even in the case where lactate is already produced with high yields. Specifically, we constructed a high lactate producer strain KBM10111 (= MG1655 ΔadhE::Cam ΔackA-pta) and implemented an IPTG-inducible overexpression of ppsA encoding for PEP synthase which, together with pyruvate kinase, gives rise to an ATP consuming cycle. Under induction of ppsA, KBM10111 exhibits a 25% higher specific lactate productivity as well as an 8% higher lactate yield. Furthermore, the specific substrate uptake rate was increased by 14%. However, trade-offs between specific and volumetric productivities must be considered when ATP wasting strategies are used to shift substrate conversion from biomass to product synthesis and we discuss potential solutions to design optimal processes. In summary, enforced ATP futile cycling has great potential to optimize a variety of production processes and our study demonstrates that this holds true also for anaerobic processes.

  6. Flagellar apparatus absolute orientations and the phylogeny of the green algae.

    PubMed

    O'Kelly, C J; Floyd, G L

    The absolute orientation of the flagellar apparatus in green algal motile cells is a feature of considerable value in studies of green algal systematics and phylogeny. The absolute orientation patterns found in those algae for which this feature is known or can be deduced are reviewed. Counterclockwise absolute orientation occurs in all classes except the Chlorophyceae and is considered primitive, while the clockwise absolute orientation present in most members of the Chlorophyceae is the result of progressive clockwise rotation of components during evolution. Extant intermediates documenting this rotation include Hafniomonas vegetative cells, which show counterclockwise absolute orientation, and Chaetopeltis quadriflagellate zoospores, in which the flagellar apparatus is strictly cruciate except for a slight clockwise offset of the microtubular rootlets. The V-shaped arrangement of the basal bodies in the flagellar apparatus, as well as the presence of proximal sheaths and of two layers of scales on the cell body, further identifies the Chaetopeltis zoospore as a primitive cell type within the Chlorophyceae . Trends towards the exsertion of basal bodies from a flagellar pit, either apically or laterally, the elimination of quadriflagellate cells, and, in the Chlorophyceae , an increasing amount of basal body offset, indicate advancement within the classes. Absolute orientation is conserved during flagellar apparatus replication and development. Events after flagellar apparatus division in the algae studied may be subdivided into component assembly, which is universal and preserves phylogenetically-useful features, and component reorientation, which occurs in relatively few green algae and adapts the flagellar apparatus to specialized functions. From these flagellar apparatus orientation studies, a major reevaluation of evolution within the Chlorophyceae is proposed, with weakly- thalloid algae possessing desmoschisis (e.g. Chaetopeltis ) considered primitive, and

  7. 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

  8. Comparative analysis of the secretion capability of early and late flagellar type III secretion substrates

    PubMed Central

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

    2016-01-01

    Summary A remarkable feature of the flagellar-specific type III secretion system (T3SS) is the selective recognition of a few substrate proteins among the many thousand cytoplasmic proteins. Secretion substrates are divided into two specificity classes: early substrates secreted for hook-basal body (HBB) construction and late substrates secreted after HBB completion. Secretion was reported to require a disordered N-terminal secretion signal, mRNA secretion signals within the 5′-untranslated region (5′-UTR) and for late substrates, piloting proteins known as the T3S chaperones. Here, we utilized translational β-lactamase fusions to probe the secretion efficacy of the N-terminal secretion signal of fourteen secreted flagellar substrates in Salmonella enterica. We observed a surprising variety in secretion capability between flagellar proteins of the same secretory class. The peptide secretion signals of the early-type substrates FlgD, FlgF, FlgE and the late-type substrate FlgL were analysed in detail. Analysing the role of the 5′-UTR in secretion of flgB and flgE revealed that the native 5′-UTR substantially enhanced protein translation and secretion. Based on our data, we propose a multicomponent signal that drives secretion via the flagellar T3SS. Both mRNA and peptide signals are recognized by the export apparatus and together with substrate-specific chaperones allowing for targeted secretion of flagellar substrates. PMID:24946091

  9. Cytoplasmic Dynein Heavy Chain 1b Is Required for Flagellar Assembly in Chlamydomonas

    PubMed Central

    Porter, Mary E.; Bower, Raqual; Knott, Julie A.; Byrd, Pamela; Dentler, William

    1999-01-01

    A second cytoplasmic dynein heavy chain (cDhc) has recently been identified in several organisms, and its expression pattern is consistent with a possible role in axoneme assembly. We have used a genetic approach to ask whether cDhc1b is involved in flagellar assembly in Chlamydomonas. Using a modified PCR protocol, we recovered two cDhc sequences distinct from the axonemal Dhc sequences identified previously. cDhc1a is closely related to the major cytoplasmic Dhc, whereas cDhc1b is closely related to the minor cDhc isoform identified in sea urchins, Caenorhabditis elegans, and Tetrahymena. The Chlamydomonas cDhc1b transcript is a low-abundance mRNA whose expression is enhanced by deflagellation. To determine its role in flagellar assembly, we screened a collection of stumpy flagellar (stf) mutants generated by insertional mutagenesis and identified two strains in which portions of the cDhc1b gene have been deleted. The two mutants assemble short flagellar stumps (<1–2 μm) filled with aberrant microtubules, raft-like particles, and other amorphous material. The results indicate that cDhc1b is involved in the transport of components required for flagellar assembly in Chlamydomonas. PMID:10069812

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

    PubMed

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

    2013-11-19

    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

  11. Identification of a palmitoyl acyltransferase required for protein sorting to the flagellar membrane.

    PubMed

    Emmer, Brian T; Souther, Christina; Toriello, Krista M; Olson, Cheryl L; Epting, Conrad L; Engman, David M

    2009-03-15

    Protein palmitoylation has diverse effects in regulating protein membrane affinity, localization, binding partner interactions, turnover and function. Here, we show that palmitoylation also contributes to the sorting of proteins to the eukaryotic flagellum. African trypanosomes are protozoan pathogens that express a family of unique Ca(2+)-binding proteins, the calflagins, which undergo N-terminal myristoylation and palmitoylation. The localization of calflagins depends on their acylation status. Myristoylation alone is sufficient for membrane association, but, in the absence of palmitoylation, the calflagins localize to the pellicular (cell body) membrane. Palmitoylation, which is mediated by a specific palmitoyl acyltransferase, is then required for subsequent trafficking of calflagin to the flagellar membrane. Coincident with the redistribution of calflagin from the pellicular to the flagellar membrane is their association with lipid rafts, which are highly enriched in the flagellar membrane. Screening of candidate palmitoyl acyltranferases identified a single enzyme, TbPAT7, that is necessary for calflagin palmitoylation and flagellar membrane targeting. Our results implicate protein palmitoylation in flagellar trafficking, and demonstrate the conservation and specificity of palmitoyl acyltransferase activity by DHHC-CRD proteins across kingdoms. PMID:19240115

  12. 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

  13. Flagellar pocket restructuring through the Leishmania life cycle involves a discrete flagellum attachment zone.

    PubMed

    Wheeler, Richard J; Sunter, Jack D; Gull, Keith

    2016-02-15

    Leishmania promastigote parasites have a flagellum, which protrudes from the flagellar pocket at the cell anterior, yet, surprisingly, have homologs of many flagellum attachment zone (FAZ) proteins--proteins used in the related Trypanosoma species to laterally attach the flagellum to the cell body from the flagellar pocket to the cell posterior. Here, we use seven Leishmania mexicana cell lines that expressed eYFP fusions of FAZ protein homologs to show that the Leishmania flagellar pocket includes a FAZ structure. Electron tomography revealed a precisely defined 3D organisation for both the flagellar pocket and FAZ, with striking similarities to those of Trypanosoma brucei. Expression of two T. brucei FAZ proteins in L. mexicana showed that T. brucei FAZ proteins can assemble into the Leishmania FAZ structure. Leishmania therefore have a previously unrecognised FAZ structure, which we show undergoes major structural reorganisation in the transition from the promastigote (sandfly vector) to amastigote (in mammalian macrophages). Morphogenesis of the Leishmania flagellar pocket, a structure important for pathogenicity, is therefore intimately associated with a FAZ; a finding with implications for understanding shape changes involving component modules during evolution.

  14. Flagellar pocket restructuring through the Leishmania life cycle involves a discrete flagellum attachment zone.

    PubMed

    Wheeler, Richard J; Sunter, Jack D; Gull, Keith

    2016-02-15

    Leishmania promastigote parasites have a flagellum, which protrudes from the flagellar pocket at the cell anterior, yet, surprisingly, have homologs of many flagellum attachment zone (FAZ) proteins--proteins used in the related Trypanosoma species to laterally attach the flagellum to the cell body from the flagellar pocket to the cell posterior. Here, we use seven Leishmania mexicana cell lines that expressed eYFP fusions of FAZ protein homologs to show that the Leishmania flagellar pocket includes a FAZ structure. Electron tomography revealed a precisely defined 3D organisation for both the flagellar pocket and FAZ, with striking similarities to those of Trypanosoma brucei. Expression of two T. brucei FAZ proteins in L. mexicana showed that T. brucei FAZ proteins can assemble into the Leishmania FAZ structure. Leishmania therefore have a previously unrecognised FAZ structure, which we show undergoes major structural reorganisation in the transition from the promastigote (sandfly vector) to amastigote (in mammalian macrophages). Morphogenesis of the Leishmania flagellar pocket, a structure important for pathogenicity, is therefore intimately associated with a FAZ; a finding with implications for understanding shape changes involving component modules during evolution. PMID:26746239

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

    PubMed

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

    2013-11-19

    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.

  16. A MORN Repeat Protein Facilitates Protein Entry into the Flagellar Pocket of Trypanosoma brucei

    PubMed Central

    2015-01-01

    The parasite Trypanosoma brucei lives in the bloodstream of infected mammalian hosts, fully exposed to the adaptive immune system. It relies on a very high rate of endocytosis to clear bound antibodies from its cell surface. All endo- and exocytosis occurs at a single site on its plasma membrane, an intracellular invagination termed the flagellar pocket. Coiled around the neck of the flagellar pocket is a multiprotein complex containing the repeat motif protein T. brucei MORN1 (TbMORN1). In this study, the phenotypic effects of TbMORN1 depletion in the mammalian-infective form of T. brucei were analyzed. Depletion of TbMORN1 resulted in a rapid enlargement of the flagellar pocket. Dextran, a polysaccharide marker for fluid phase endocytosis, accumulated inside the enlarged flagellar pocket. Unexpectedly, however, the proteins concanavalin A and bovine serum albumin did not do so, and concanavalin A was instead found to concentrate outside it. This suggests that TbMORN1 may have a role in facilitating the entry of proteins into the flagellar pocket. PMID:26318396

  17. Structural flexibility of the periplasmic protein, FlgA, regulates flagellar P-ring assembly in Salmonella enterica

    PubMed Central

    Matsunami, Hideyuki; Yoon, Young-Ho; Meshcheryakov, Vladimir A.; Namba, Keiichi; Samatey, Fadel A.

    2016-01-01

    A periplasmic flagellar chaperone protein, FlgA, is required for P-ring assembly in bacterial flagella of taxa such as Salmonella enterica or Escherichia coli. The mechanism of chaperone-mediated P-ring formation is poorly understood. Here we present the open and closed crystal structures of FlgA from Salmonella enterica serovar Typhimurium, grown under different crystallization conditions. An intramolecular disulfide cross-linked form of FlgA caused a dominant negative effect on motility of the wild-type strain. Pull-down experiments support a specific protein-protein interaction between FlgI, the P-ring component protein, and the C-terminal domain of FlgA. Surface plasmon resonance and limited-proteolysis indicate that flexibility of the domain is reduced in the covalently closed form. These results show that the structural flexibility of the C-terminal domain of FlgA, which is related to the structural difference between the two crystal forms, is intrinsically associated with its molecular chaperone function in P-ring assembly. PMID:27273476

  18. Gliding movement in Peranema trichophorum is powered by flagellar surface motility.

    PubMed

    Saito, Akira; Suetomo, Yasutaka; Arikawa, Mikihiko; Omura, Gen; Khan, S M Mostafa Kamal; Kakuta, Soichiro; Suzaki, Etsuko; Kataoka, Katsuko; Suzaki, Toshinobu

    2003-08-01

    A colorless euglenoid flagellate Peranema trichophorum shows unique unidirectional gliding cell locomotion on the substratum at velocities up to 30 micro m/s by an as yet unexplained mechanism. In this study, we found that (1) treatment with NiCl(2) inhibited flagellar beating without any effect on gliding movement; (2) water currents applied to a gliding cell from opposite sides caused detachment of the cell body from the substratum. With only the anterior flagellum adhering to the substratum, gliding movement continued along the direction of the anterior flagellum; (3) gentle pipetting induced flagellar severance into various lengths. In these cells, gliding velocity was proportional to the flagellar length; and (4) Polystyrene beads were translocated along the surface of the anterior flagellum. All of these results indicate that a cell surface motility system is present on the anterior flagellum, which is responsible for cell gliding in P. trichophorum.

  19. A genome-scale metabolic flux model of Escherichia coli K–12 derived from the EcoCyc database

    PubMed Central

    2014-01-01

    Background Constraint-based models of Escherichia coli metabolic flux have played a key role in computational studies of cellular metabolism at the genome scale. We sought to develop a next-generation constraint-based E. coli model that achieved improved phenotypic prediction accuracy while being frequently updated and easy to use. We also sought to compare model predictions with experimental data to highlight open questions in E. coli biology. Results We present EcoCyc–18.0–GEM, a genome-scale model of the E. coli K–12 MG1655 metabolic network. The model is automatically generated from the current state of EcoCyc using the MetaFlux software, enabling the release of multiple model updates per year. EcoCyc–18.0–GEM encompasses 1445 genes, 2286 unique metabolic reactions, and 1453 unique metabolites. We demonstrate a three-part validation of the model that breaks new ground in breadth and accuracy: (i) Comparison of simulated growth in aerobic and anaerobic glucose culture with experimental results from chemostat culture and simulation results from the E. coli modeling literature. (ii) Essentiality prediction for the 1445 genes represented in the model, in which EcoCyc–18.0–GEM achieves an improved accuracy of 95.2% in predicting the growth phenotype of experimental gene knockouts. (iii) Nutrient utilization predictions under 431 different media conditions, for which the model achieves an overall accuracy of 80.7%. The model’s derivation from EcoCyc enables query and visualization via the EcoCyc website, facilitating model reuse and validation by inspection. We present an extensive investigation of disagreements between EcoCyc–18.0–GEM predictions and experimental data to highlight areas of interest to E. coli modelers and experimentalists, including 70 incorrect predictions of gene essentiality on glucose, 80 incorrect predictions of gene essentiality on glycerol, and 83 incorrect predictions of nutrient utilization. Conclusion Significant

  20. 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

  1. Isolation and characterization of flagellar filaments from Bacillus cereus ATCC 14579.

    PubMed

    Tagawa, Yuichi

    2014-12-01

    Isolated flagellar filaments from the type strain of Bacillus cereus, ATCC 14579, were shown to consist of 34, 32 and 31 kDa proteins in similar proportions as judged by band intensities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequences of these three proteins of strain ATCC 14579 were identical with the deduced sequences of three flagellin genes BC1657, BC1658 and BC1659 in the whole genome sequence. Strain ATCC 14579 was classified into serotype T2 by a flagellar serotyping scheme for B. cereus strains that are untypeable into known flagellar serotypes H1 to H23. Flagellar filaments from a reference strain of serotype T2 contained two protein bands at 34 and 32 kDa, but a single protein band at 39 kDa was detected in flagellar filaments of a reference strain of serotype H1. Two murine monoclonal antibodies, 1A5 and 2A5, which recognize both the 34 and 32 kDa flagellins and a single flagellin of 32 kDa, respectively, were specifically reactive with B. cereus strains ATCC 14579 and serotype T2 in whole-cell ELISA and bacterial motility inhibition tests. In immunoelectron microscopy with monoclonal antibodies 1A5 and 2A5, colloidal gold spheres were shown to localize almost evenly over the entire part of flagellar filaments. Since strain ATCC 14579, and presumably strain serotype T2, are unusual among B. cereus strains in possessing multiple genes that encode flagellin subunits, a possible unique mechanism may contribute to assembly of multiple flagellin subunits into the filament over its entire length.

  2. Knockdown of Inner Arm Protein IC138 in Trypanosoma brucei Causes Defective Motility and Flagellar Detachment

    PubMed Central

    Wilson, Corinne S.; Chang, Alex J.; Greene, Rebecca; Machado, Sulynn; Parsons, Matthew W.; Takats, Taylor A.; Zambetti, Luke J.; Springer, Amy L.

    2015-01-01

    Motility in the protozoan parasite Trypanosoma brucei is conferred by a single flagellum, attached alongside the cell, which moves the cell forward using a beat that is generated from tip-to-base. We are interested in characterizing components that regulate flagellar beating, in this study we extend the characterization of TbIC138, the ortholog of a dynein intermediate chain that regulates axonemal inner arm dynein f/I1. TbIC138 was tagged In situ-and shown to fractionate with the inner arm components of the flagellum. RNAi knockdown of TbIC138 resulted in significantly reduced protein levels, mild growth defect and significant motility defects. These cells tended to cluster, exhibited slow and abnormal motility and some cells had partially or fully detached flagella. Slight but significant increases were observed in the incidence of mis-localized or missing kinetoplasts. To document development of the TbIC138 knockdown phenotype over time, we performed a detailed analysis of flagellar detachment and motility changes over 108 hours following induction of RNAi. Abnormal motility, such as slow twitching or irregular beating, was observed early, and became progressively more severe such that by 72 hours-post-induction, approximately 80% of the cells were immotile. Progressively more cells exhibited flagellar detachment over time, but this phenotype was not as prevalent as immotility, affecting less than 60% of the population. Detached flagella had abnormal beating, but abnormal beating was also observed in cells with no flagellar detachment, suggesting that TbIC138 has a direct, or primary, effect on the flagellar beat, whereas detachment is a secondary phenotype of TbIC138 knockdown. Our results are consistent with the role of TbIC138 as a regulator of motility, and has a phenotype amenable to more extensive structure-function analyses to further elucidate its role in the control of flagellar beat in T. brucei. PMID:26555902

  3. Knockdown of Inner Arm Protein IC138 in Trypanosoma brucei Causes Defective Motility and Flagellar Detachment.

    PubMed

    Wilson, Corinne S; Chang, Alex J; Greene, Rebecca; Machado, Sulynn; Parsons, Matthew W; Takats, Taylor A; Zambetti, Luke J; Springer, Amy L

    2015-01-01

    Motility in the protozoan parasite Trypanosoma brucei is conferred by a single flagellum, attached alongside the cell, which moves the cell forward using a beat that is generated from tip-to-base. We are interested in characterizing components that regulate flagellar beating, in this study we extend the characterization of TbIC138, the ortholog of a dynein intermediate chain that regulates axonemal inner arm dynein f/I1. TbIC138 was tagged In situ-and shown to fractionate with the inner arm components of the flagellum. RNAi knockdown of TbIC138 resulted in significantly reduced protein levels, mild growth defect and significant motility defects. These cells tended to cluster, exhibited slow and abnormal motility and some cells had partially or fully detached flagella. Slight but significant increases were observed in the incidence of mis-localized or missing kinetoplasts. To document development of the TbIC138 knockdown phenotype over time, we performed a detailed analysis of flagellar detachment and motility changes over 108 hours following induction of RNAi. Abnormal motility, such as slow twitching or irregular beating, was observed early, and became progressively more severe such that by 72 hours-post-induction, approximately 80% of the cells were immotile. Progressively more cells exhibited flagellar detachment over time, but this phenotype was not as prevalent as immotility, affecting less than 60% of the population. Detached flagella had abnormal beating, but abnormal beating was also observed in cells with no flagellar detachment, suggesting that TbIC138 has a direct, or primary, effect on the flagellar beat, whereas detachment is a secondary phenotype of TbIC138 knockdown. Our results are consistent with the role of TbIC138 as a regulator of motility, and has a phenotype amenable to more extensive structure-function analyses to further elucidate its role in the control of flagellar beat in T. brucei. PMID:26555902

  4. 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

  5. Multiplex PCR for distinguishing the most common phase-1 flagellar antigens of Salmonella spp.

    PubMed

    Herrera-León, Silvia; McQuiston, John R; Usera, Miguel A; Fields, Patricia I; Garaizar, Javier; Echeita, M Aurora

    2004-06-01

    Most Salmonella serotypes alternatively express either phase-1 or phase-2 flagellar antigens, encoded by the fliC and fljB genes, respectively. Flagellar phase reversal for the identification of both flagellar antigens is not necessary at the genetic level. Variable internal regions of the fliC genes encoding the H:i, H:r, H:l,v, H:e,h, H:z(10), H:b, and H:d antigens have been sequenced; and the specific sites for each antigen in selected Salmonella serotypes have been determined. These results, together with flagellar G-complex variable internal sequences obtained by the Foodborne and Diarrheal Diseases Branch at the Centers for Disease Control and Prevention in Atlanta, GA, have been used to design a multiplex PCR to identify the G-complex antigens as well as the H:i, H:r, H:l,v, H:e,h, Hz(10), H:b, and H:d first-phase antigens. These antigens are part of the most common Salmonella serotypes possessing first-phase flagellar antigens. Salmonella enterica serotype Enteritidis is identified by adding a specific primer pair published previously. This multiplex PCR includes 13 primers. A total of 161 Salmonella strains associated with 72 different serotypes were tested. Each strain generated one first-phase-specific antigen fragment ranging from 100 to 500 bp; Salmonella serotype Enteritidis, however, generated two amplicons of 500 bp that corresponded to the G complex and a 333-bp serotype-specific amplicon, respectively. Twenty-three strains representing 19 serotypes with flagellar genes different from those targeted in this work did not generate any fragments. The method is quick, specific, and reproducible and is independent of the phase expressed by the bacteria when they are tested.

  6. Flagellar regeneration in Chlamydomonas: a model system for studying organelle assembly.

    PubMed

    Johnson, K A; Rosenbaum, J L

    1993-05-01

    How do the many different components of an organelle assemble into a functional structure at an appropriate place and time? Flagellar regeneration by the biflagellate green alga Chlamydomonas is one experimental system in which genetics, biochemistry and ultrastructural analysis are being combined to investigate the assembly of a microtubule-containing organelle. Recent advances in the molecular biology of this 'green yeast' have made possible several new approaches to the problem of flagellar assembly; insights from these new approaches are the focus of this review.

  7. A protein thermometer controls temperature-dependent transcription of flagellar motility genes in Listeria monocytogenes.

    PubMed

    Kamp, Heather D; Higgins, Darren E

    2011-08-01

    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

  8. Characterization of Calflagin, a Flagellar Calcium-Binding Protein from Trypanosoma congolense

    PubMed Central

    Eyford, Brett A.; Kaufman, Laura; Salama-Alber, Orly; Loveless, Bianca; Pope, Matthew E.; Burke, Robert D.; Matovu, Enock; Boulanger, Martin J.; Pearson, Terry W.

    2016-01-01

    Background Identification of species-specific trypanosome molecules is important for laboratory- and field-based research into epidemiology and disease diagnosis. Although Trypanosoma congolense is the most important trypanosome pathogen of cattle in Africa, no species-specific molecules found in infective bloodstream forms (BSF) of the parasites have been identified, thus limiting development of diagnostic tests. Methods Immuno-mass spectrometric methods were used to identify a protein that is recognized by a T. congolense-specific monoclonal antibody (mAb) Tc6/42.6.4. The identified molecule was expressed as a recombinant protein in E. coli and was tested in several immunoassays for its ability to interact with the mAb. The three dimensional structure of the protein was modeled and compared to crystal- and NMR-structures of the homologous proteins from T. cruzi and T. brucei respectively, in order to examine structural differences leading to the different immunoreactivity of the T. congolense molecule. Enzyme-linked immunosorbent assays (ELISA) were used to measure antibodies produced by trypanosome-infected African cattle in order to assess the potential for use of T. congolense calflagin in a serodiagnostic assay. Results The antigen recognized by the T. congolense-specific mAb Tc6/42.6.4 was identified as a flagellar calcium-binding protein, calflagin. The recombinant molecule showed immunoreactivity with the T. congolense-specific mAb confirming that it is the cognate antigen. Immunofluorescence experiments revealed that Ca2+ modulated the localization of the calflagin molecule in trypanosomes. Structural modelling and comparison with calflagin homologues from other trypanosomatids revealed four non-conserved regions on the surface of the T. congolense molecule that due to differences in surface chemistry and structural topography may form species-specific epitopes. ELISAs using the recombinant calflagin as antigen to detect antibodies in trypanosome

  9. 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.

  10. 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. PMID:26943926

  11. 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

  12. 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.

  13. Advances in Molecular Serotyping and Subtyping of Escherichia coli.

    PubMed

    Fratamico, Pina M; DebRoy, Chitrita; Liu, Yanhong; Needleman, David S; Baranzoni, Gian Marco; Feng, Peter

    2016-01-01

    Escherichia coli plays an important role as a member of the gut microbiota; however, pathogenic strains also exist, including various diarrheagenic E. coli pathotypes and extraintestinal pathogenic E. coli that cause illness outside of the GI-tract. E. coli have traditionally been serotyped using antisera against the ca. 186 O-antigens and 53 H-flagellar antigens. Phenotypic methods, including bacteriophage typing and O- and H- serotyping for differentiating and characterizing E. coli have been used for many years; however, these methods are generally time consuming and not always accurate. Advances in next generation sequencing technologies have made it possible to develop genetic-based subtyping and molecular serotyping methods for E. coli, which are more discriminatory compared to phenotypic typing methods. Furthermore, whole genome sequencing (WGS) of E. coli is replacing established subtyping methods such as pulsed-field gel electrophoresis, providing a major advancement in the ability to investigate food-borne disease outbreaks and for trace-back to sources. A variety of sequence analysis tools and bioinformatic pipelines are being developed to analyze the vast amount of data generated by WGS and to obtain specific information such as O- and H-group determination and the presence of virulence genes and other genetic markers.

  14. Advances in molecular serotyping and subtyping of Escherichia coli

    DOE PAGES

    Fratamico, Pina M.; DebRoy, Chitrita; Liu, Yanhong; Needleman, David S.; Baranzoni, Gian Marco; Feng, Peter

    2016-05-03

    Escherichia coli plays an important role as a member of the gut microbiota; however, pathogenic strains also exist, including various diarrheagenic E. coli pathotypes and extraintestinal pathogenic E. coli that cause illness outside of the GI-tract. E. coli have traditionally been serotyped using antisera against the ca. 186 O-antigens and 53 H-flagellar antigens. Phenotypic methods, including bacteriophage typing and O- and H- serotyping for differentiating and characterizing E. coli have been used for many years; however, these methods are generally time consuming and not always accurate. Advances in next generation sequencing technologies have made it possible to develop genetic-based subtypingmore » and molecular serotyping methods for E. coli, which are more discriminatory compared to phenotypic typing methods. Furthermore, whole genome sequencing (WGS) of E. coli is replacing established subtyping methods such as pulsedfield gel electrophoresis, providing a major advancement in the ability to investigate food-borne disease outbreaks and for trace-back to sources. Furthermore, a variety of sequence analysis tools and bioinformatic pipelines are being developed to analyze the vast amount of data generated by WGS and to obtain specific information such as O- and H-group determination and the presence of virulence genes and other genetic markers.« less

  15. Specific Growth Rate Determines the Sensitivity of Escherichia coli to Thermal, UVA, and Solar Disinfection

    PubMed Central

    Berney, Michael; Weilenmann, Hans-Ulrich; Ihssen, Julian; Bassin, Claudio; Egli, Thomas

    2006-01-01

    Knowledge about the sensitivity of the test organism is essential for the evaluation of any disinfection method. In this work we show that sensitivity of Escherichia coli MG1655 to three physical stresses (mild heat, UVA light, and sunlight) that are relevant in the disinfection of drinking water with solar radiation is determined by the specific growth rate of the culture. Batch- and chemostat-cultivated cells from cultures with similar specific growth rates showed similar stress sensitivities. Generally, fast-growing cells were more sensitive to the stresses than slow-growing cells. For example, slow-growing chemostat-cultivated cells (D = 0.08 h−1) and stationary-phase bacteria from batch culture that were exposed to mild heat had very similar T90 (time until 90% of the population is inactivated) values (T90, chemostat = 2.66 h; T90, batch = 2.62 h), whereas T90 for cells growing at a μ of 0.9 h−1 was 0.2 h. We present evidence that the stress sensitivity of E. coli is correlated with the intracellular level of the alternative sigma factor RpoS. This is also supported by the fact that E. coli rpoS mutant cells were more stress sensitive than the parent strain by factors of 4.9 (mild heat), 5.3 (UVA light), and 4.1 (sunlight). Furthermore, modeling of inactivation curves with GInaFiT revealed that the shape of inactivation curves changed depending on the specific growth rate. Inactivation curves of cells from fast-growing cultures (μ = 1.0 h−1) that were irradiated with UVA light showed a tailing effect, while for slow-growing cultures (μ = 0.3 h−1), inactivation curves with shoulders were obtained. Our findings emphasize the need for accurate reporting of specific growth rates and detailed culture conditions in disinfection studies to allow comparison of data from different studies and laboratories and sound interpretation of the data obtained. PMID:16597961

  16. 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.

  17. The Role of Preassembled Cytoplasmic Complexes in Assembly of Flagellar Dynein Subunits

    PubMed Central

    Fowkes, Mary Elizabeth; Mitchell, David Rees

    1998-01-01

    Previous work has revealed a cytoplasmic pool of flagellar precursor proteins capable of contributing to the assembly of new flagella, but how and where these components assemble is unknown. We tested Chlamydomonas outer-dynein arm subunit stability and assembly in the cytoplasm of wild-type cells and 11 outer dynein arm assembly mutant strains (oda1-oda11) by Western blotting of cytoplasmic extracts, or immunoprecipitates from these extracts, with five outer-row dynein subunit-specific antibodies. Western blots reveal that at least three oda mutants (oda6, oda7, and oda9) alter the level of a subunit that is not the mutant gene product. Immunoprecipitation shows that large preassembled flagellar complexes containing all five tested subunits (three heavy chains and two intermediate chains) exist within wild-type cytoplasm. When the preassembly of these subunits was examined in oda strains, we observed three patterns: complete coassembly (oda 1, 3, 5, 8, and 10), partial coassembly (oda7 and oda11), and no coassembly (oda2, 6, and 9) of the four tested subunits with HCβ. Our data, together with previous studies, suggest that flagellar outer-dynein arms preassemble into a complete Mr ≃ 2 × 106 dynein arm that resides in a cytoplasmic precursor pool before transport into the flagellar compartment. PMID:9725897

  18. Self-Sustained Oscillatory Sliding Movement of Doublet Microtubules and Flagellar Bend Formation.

    PubMed

    Ishijima, Sumio

    2016-01-01

    It is well established that the basis for flagellar and ciliary movements is ATP-dependent sliding between adjacent doublet microtubules. However, the mechanism for converting microtubule sliding into flagellar and ciliary movements has long remained unresolved. The author has developed new sperm models that use bull spermatozoa divested of their plasma membrane and midpiece mitochondrial sheath by Triton X-100 and dithiothreitol. These models enable the observation of both the oscillatory sliding movement of activated doublet microtubules and flagellar bend formation in the presence of ATP. A long fiber of doublet microtubules extruded by synchronous sliding of the sperm flagella and a short fiber of doublet microtubules extruded by metachronal sliding exhibited spontaneous oscillatory movements and constructed a one beat cycle of flagellar bending by alternately actuating. The small sliding displacement generated by metachronal sliding formed helical bends, whereas the large displacement by synchronous sliding formed planar bends. Therefore, the resultant waveform is a half-funnel shape, which is similar to ciliary movements. PMID:26863204

  19. Dual stator dynamics in the Shewanella oneidensis MR-1 flagellar motor.

    PubMed

    Paulick, Anja; Delalez, Nicolas J; Brenzinger, Susanne; Steel, Bradley C; Berry, Richard M; Armitage, Judith P; Thormann, Kai M

    2015-06-01

    The bacterial flagellar motor is an intricate nanomachine which converts ion gradients into rotational movement. Torque is created by ion-dependent stator complexes which surround the rotor in a ring. Shewanella oneidensis MR-1 expresses two distinct types of stator units: the Na(+)-dependent PomA4 B2 and the H(+)-dependent MotA4 B2. Here, we have explored the stator unit dynamics in the MR-1 flagellar system by using mCherry-labeled PomAB and MotAB units. We observed a total of between 7 and 11 stator units in each flagellar motor. Both types of stator units exchanged between motors and a pool of stator complexes in the membrane, and the exchange rate of MotAB, but not of PomAB, units was dependent on the environmental Na(+)-levels. In 200 mM Na(+), the numbers of PomAB and MotAB units in wild-type motors was determined to be about 7:2 (PomAB:MotAB), shifting to about 6:5 without Na(+). Significantly, the average swimming speed of MR-1 cells at low Na(+) conditions was increased in the presence of MotAB. These data strongly indicate that the S. oneidensis flagellar motors simultaneously use H(+) and Na(+) driven stators in a configuration governed by MotAB incorporation efficiency in response to environmental Na(+) levels.

  20. 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

  1. Organization and temporal expression of a flagellar basal body gene in Caulobacter crescentus.

    PubMed Central

    Hahnenberger, K M; Shapiro, L

    1988-01-01

    Caulobacter crescentus assembles a single polar flagellum at a defined time in the cell cycle. The protein components of the flagellar hook and filament are synthesized just prior to their assembly. We demonstrated that the expression of a gene, flaD, that is involved in the formation of the flagellar basal body is under temporal control and is transcribed relatively early in the cell cycle, before the hook and flagellin genes are transcribed. Thus, the order of flagellar gene transcription reflects the order of assembly of the protein components. A mutation in the flaD gene results in the assembly of a partial basal body which is missing the outermost P and L rings as well as the external hook and filament (K.M. Hahnenberger and L. Shapiro, J. Mol. Biol. 194:91-103, 1987). The flaD gene was cloned and characterized by nucleotide sequencing and S1 nuclease protection assays. In contrast to the protein components of the hook and filament, the protein encoded by the flaD gene contains a hydrophobic leader peptide. The predicted amino acid sequence of the leader peptide of flaD is very similar to the leader peptide of the flagellar basal body P ring of Salmonella typhimurium (M. Homma, Y. Komeda, T. Iino, and R.M. Macnab, J. Bacteriol. 169:1493-1498, 1987). Images PMID:2842303

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  3. The Flagellar Arginine Kinase in Trypanosoma brucei Is Important for Infection in Tsetse Flies

    PubMed Central

    Ooi, Cher-Pheng; Rotureau, Brice; Gribaldo, Simonetta; Georgikou, Christina; Julkowska, Daria; Blisnick, Thierry; Perrot, Sylvie; Subota, Ines; Bastin, Philippe

    2015-01-01

    African trypanosomes are flagellated parasites that cause sleeping sickness. Parasites are transmitted from one mammalian host to another by the bite of a tsetse fly. Trypanosoma brucei possesses three different genes for arginine kinase (AK) including one (AK3) that encodes a protein localised to the flagellum. AK3 is characterised by the presence of a unique amino-terminal insertion that specifies flagellar targeting. We show here a phylogenetic analysis revealing that flagellar AK arose in two independent duplication events in T. brucei and T. congolense, the two species of African trypanosomes that infect the tsetse midgut. In T. brucei, AK3 is detected in all stages of parasite development in the fly (in the midgut and in the salivary glands) as well as in bloodstream cells, but with predominance at insect stages. Genetic knockout leads to a slight reduction in motility and impairs parasite infectivity towards tsetse flies in single and competition experiments, both phenotypes being reverted upon expression of an epitope-tagged version of AK3. We speculate that this flagellar arginine kinase is important for T. brucei infection of tsetse, especially in the context of mixed infections and that its flagellar targeting relies on a system equivalent to that discovered for calflagins, a family of trypanosome flagellum calcium binding proteins. PMID:26218532

  4. 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

  5. The flagellar apparatus of Breviata anathema, a eukaryote without a clear supergroup affinity.

    PubMed

    Heiss, Aaron A; Walker, Giselle; Simpson, Alastair G B

    2013-08-01

    Breviata anathema is an anaerobic amoeboid flagellate that does not branch within any established 'supergroup'. Molecular phylogenies suggest affinities to Amoebozoa, Opisthokonta, or apusomonads. Here we describe its flagellar apparatus ultrastructure. Breviata has two basal bodies. The flagellated anterior basal body (AB) is associated with a fan of ∼18 microtubules and a short singlet microtubular root. Three microtubular roots associate with the posterior basal body. One, the right root (RR), is initially a triplet that splits into two parts. The other two are singlets: the left root (LR), and the middle root (MR), which arises on the posterior side of the basal body. The MR, LR and smaller part of RR support the left ventral side of the cell, while the larger part of RR runs down the right. Outer dynein arms were not observed on the flagellar axoneme. The mitochondrion-like organelle sometimes contains some tubular cristae. The posterior flagellar apparatus resembles that of several eukaryotic lineages, particularly apusomonads, ancyromonads, excavates, and myxogastrid amoebozoans. This comparison suggests that the complex flagellar apparatus of myxogastrids is actually plesiomorphic within Amoebozoa. The widely distributed splitting right root and posterior singlet (MR in Breviata) may be plesiomorphies in many eukaryotic lineages, and thus could be features of the last eukaryotic common ancestor.

  6. Self-Sustained Oscillatory Sliding Movement of Doublet Microtubules and Flagellar Bend Formation

    PubMed Central

    Ishijima, Sumio

    2016-01-01

    It is well established that the basis for flagellar and ciliary movements is ATP-dependent sliding between adjacent doublet microtubules. However, the mechanism for converting microtubule sliding into flagellar and ciliary movements has long remained unresolved. The author has developed new sperm models that use bull spermatozoa divested of their plasma membrane and midpiece mitochondrial sheath by Triton X-100 and dithiothreitol. These models enable the observation of both the oscillatory sliding movement of activated doublet microtubules and flagellar bend formation in the presence of ATP. A long fiber of doublet microtubules extruded by synchronous sliding of the sperm flagella and a short fiber of doublet microtubules extruded by metachronal sliding exhibited spontaneous oscillatory movements and constructed a one beat cycle of flagellar bending by alternately actuating. The small sliding displacement generated by metachronal sliding formed helical bends, whereas the large displacement by synchronous sliding formed planar bends. Therefore, the resultant waveform is a half-funnel shape, which is similar to ciliary movements. PMID:26863204

  7. 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

  8. 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.

  9. S-adenosyl homocysteine hydrolase (SAHH) accelerates flagellar regeneration in Dunaliella salina.

    PubMed

    Li, Qinghua; Zhu, Liqiang; Yan, Yunmeng; Chai, Dandan; Li, Jie; Xue, Lexun

    2013-08-01

    S-adenosylhomocysteine hydrolase (SAHH) is an enzyme, which catalyzes the hydrolysis of S-adenosylhomocysteine (SAH) which is formed after the donation of the methyl group of S-adenosylmethionine (SAM) to a methyl acceptor in methylation reaction. As a potent regulator of methylation, SAHH plays a critical role in methylation reaction in the cells. Here we cloned the SAHH gene from unicellular green alga Dunaliella salina (dsSAHH) and investigated its effects on flagellar regeneration of D. salina, and found that dsSAHH was upregulated both at the protein and the transcription levels during pH shock-triggered flagellar regeneration of D. salina. The flagellar regeneration was accelerated when dsSAHH was overexpressed, but it was inhibited by SAHH inhibitor 3-deazaadenosine (DZA). Moreover, a receptor for activated C kinase 1 from D. salina (dsRACK1), which was identified to interact with dsSAHH, was increased when dsSAHH was overexpressed in D. salina as shown by real-time PCR. The findings of this study suggest that dsSAHH may participate in the regulation of flagellar regeneration of D. salina.

  10. 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

  11. 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

  12. 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. PMID:27251063

  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

    2016-01-01

    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

  14. Roles of Lon protease and its substrate MarA during sodium salicylate-mediated growth reduction and antibiotic resistance in Escherichia coli.

    PubMed

    Bhaskarla, Chetana; Das, Mrinmoy; Verma, Taru; Kumar, Anujith; Mahadevan, S; Nandi, Dipankar

    2016-05-01

    The cellular proteolytic machinery orchestrates protein turnover and regulates several key biological processes. This study addresses the roles of Lon, a major ATP-dependent protease, in modulating the responses of Escherichia coli strain MG1655 to low and high amounts of sodium salicyclate (NaSal), a widely used clinically relevant analgesic. NaSal affects several bacterial responses, including growth and resistance to multiple antibiotics. The loss of lon reduces growth in response to high, but not low, amounts of NaSal. From amongst a panel of Lon substrates, MarA was identified to be the downstream target of Lon. Thus, stabilization of MarA in the absence of lon lowers growth of the strain in the presence of higher amounts of NaSal. The steady-state transcript levels of marA and its target genes, acrA, acrB and tolC, are higher in the Δlon strain compared with the WT strain. Consequently, the resistance to antibiotics, e.g. tetracycline and nalidixic acid, is enhanced in Δlon in a marA-dependent manner. Furthermore, the target genes of MarA, i.e. acrB and tolC, are responsible for NaSal-mediated antibiotic resistance. Studies using atomic force microscopy demonstrated that ciprofloxacin led to greater cell filamentation, which is lower in the Δlon strain due to higher levels of MarA. Overall, this study delineates the roles of Lon protease, its substrate MarA and downstream targets of MarA, e.g. acrB and tolC, during NaSal-mediated growth reduction and antibiotic resistance. The implications of these observations in the adaptation of E. coli under different environmental conditions are discussed. PMID:26944926

  15. Roles of Lon protease and its substrate MarA during sodium salicylate-mediated growth reduction and antibiotic resistance in Escherichia coli.

    PubMed

    Bhaskarla, Chetana; Das, Mrinmoy; Verma, Taru; Kumar, Anujith; Mahadevan, S; Nandi, Dipankar

    2016-05-01

    The cellular proteolytic machinery orchestrates protein turnover and regulates several key biological processes. This study addresses the roles of Lon, a major ATP-dependent protease, in modulating the responses of Escherichia coli strain MG1655 to low and high amounts of sodium salicyclate (NaSal), a widely used clinically relevant analgesic. NaSal affects several bacterial responses, including growth and resistance to multiple antibiotics. The loss of lon reduces growth in response to high, but not low, amounts of NaSal. From amongst a panel of Lon substrates, MarA was identified to be the downstream target of Lon. Thus, stabilization of MarA in the absence of lon lowers growth of the strain in the presence of higher amounts of NaSal. The steady-state transcript levels of marA and its target genes, acrA, acrB and tolC, are higher in the Δlon strain compared with the WT strain. Consequently, the resistance to antibiotics, e.g. tetracycline and nalidixic acid, is enhanced in Δlon in a marA-dependent manner. Furthermore, the target genes of MarA, i.e. acrB and tolC, are responsible for NaSal-mediated antibiotic resistance. Studies using atomic force microscopy demonstrated that ciprofloxacin led to greater cell filamentation, which is lower in the Δlon strain due to higher levels of MarA. Overall, this study delineates the roles of Lon protease, its substrate MarA and downstream targets of MarA, e.g. acrB and tolC, during NaSal-mediated growth reduction and antibiotic resistance. The implications of these observations in the adaptation of E. coli under different environmental conditions are discussed.

  16. KHARON1 mediates flagellar targeting of a glucose transporter in Leishmania mexicana and is critical for viability of infectious intracellular amastigotes.

    PubMed

    Tran, Khoa D; Rodriguez-Contreras, Dayana; Vieira, Danielle P; Yates, Phillip A; David, Larry; Beatty, Wandy; Elferich, Johannes; Landfear, Scott M

    2013-08-01

    The LmxGT1 glucose transporter is selectively targeted to the flagellum of the kinetoplastid parasite Leishmania mexicana, but the mechanism for targeting this and other flagella-specific membrane proteins among the Kinetoplastida is unknown. To address the mechanism of flagellar targeting, we employed in vivo cross-linking, tandem affinity purification, and mass spectrometry to identify a novel protein, KHARON1 (KH1), which is important for the flagellar trafficking of LmxGT1. Kh1 null mutant parasites are strongly impaired in flagellar targeting of LmxGT1, and trafficking of the permease was arrested in the flagellar pocket. Immunolocalization revealed that KH1 is located at the base of the flagellum, within the flagellar pocket, where it associates with the proximal segment of the flagellar axoneme. We propose that KH1 mediates transit of LmxGT1 from the flagellar pocket into the flagellar membrane via interaction with the proximal portion of the flagellar axoneme. KH1 represents the first component involved in flagellar trafficking of integral membrane proteins among parasitic protozoa. Of considerable interest, Kh1 null mutants are strongly compromised for growth as amastigotes within host macrophages. Thus, KH1 is also important for the disease causing stage of the parasite life cycle.

  17. KHARON1 mediates flagellar targeting of a glucose transporter in Leishmania mexicana and is critical for viability of infectious intracellular amastigotes.

    PubMed

    Tran, Khoa D; Rodriguez-Contreras, Dayana; Vieira, Danielle P; Yates, Phillip A; David, Larry; Beatty, Wandy; Elferich, Johannes; Landfear, Scott M

    2013-08-01

    The LmxGT1 glucose transporter is selectively targeted to the flagellum of the kinetoplastid parasite Leishmania mexicana, but the mechanism for targeting this and other flagella-specific membrane proteins among the Kinetoplastida is unknown. To address the mechanism of flagellar targeting, we employed in vivo cross-linking, tandem affinity purification, and mass spectrometry to identify a novel protein, KHARON1 (KH1), which is important for the flagellar trafficking of LmxGT1. Kh1 null mutant parasites are strongly impaired in flagellar targeting of LmxGT1, and trafficking of the permease was arrested in the flagellar pocket. Immunolocalization revealed that KH1 is located at the base of the flagellum, within the flagellar pocket, where it associates with the proximal segment of the flagellar axoneme. We propose that KH1 mediates transit of LmxGT1 from the flagellar pocket into the flagellar membrane via interaction with the proximal portion of the flagellar axoneme. KH1 represents the first component involved in flagellar trafficking of integral membrane proteins among parasitic protozoa. Of considerable interest, Kh1 null mutants are strongly compromised for growth as amastigotes within host macrophages. Thus, KH1 is also important for the disease causing stage of the parasite life cycle. PMID:23766511

  18. Centrin-mediated microtubule severing during flagellar excision in Chlamydomonas reinhardtii

    PubMed Central

    1989-01-01

    Chlamydomonas cells excise their flagella in response to a variety of experimental conditions (e.g., extremes of temperature or pH, alcohol or detergent treatment, and mechanical shear). Here, we show that flagellar excision is an active process whereby microtubules are severed at select sites within the transition zone. The transition zone is located between the flagellar axoneme and the basal body; it is characterized by a pair of central cylinders that have an H shape when viewed in longitudinal section. Both central cylinders are connected to the A tubule of each microtubule doublet of the transition zone by fibers (approximately 5 nm diam). When viewed in cross section, these fibers are seen to form a distinctive stellate pattern characteristic of the transition zone (Manton, I. 1964. J. R. Microsc. Soc. 82:279- 285; Ringo. D. L. 1967. J. Cell Biol. 33:543-571). We demonstrate that at the time of flagellar excision these fibers contract and displace the microtubule doublets of the axoneme inward. We believe that the resulting shear force and torsional load act to sever the axonemal microtubules immediately distal to the central cylinder. Structural alterations of the transition zone during flagellar excision occur both in living cells and detergent-extracted cell models, and are dependent on the presence of calcium (greater than or equal to 10(-6) M). Immunolocalization using monoclonal antibodies against the calcium- binding protein centrin demonstrate the presence of centrin in the fiber-based stellate structure of the transition zone of wild-type cells. Examination of the flagellar autotomy mutant, fa-1, which fails to excise its flagella (Lewin, R., and C. Burrascano. 1983. Experientia. 39:1397-1398), demonstrates that the fa-1 lacks the ability to completely contract the fibers of the stellate structure. We conclude that flagellar excision in Chlamydomonas involves microtubule severing that is mediated by the action of calcium-sensitive contractile fibers

  19. Multiplex PCR for Distinguishing the Most Common Phase-1 Flagellar Antigens of Salmonella spp.

    PubMed Central

    Herrera-León, Silvia; McQuiston, John R.; Usera, Miguel A.; Fields, Patricia I.; Garaizar, Javier; Echeita, M. Aurora

    2004-01-01

    Most Salmonella serotypes alternatively express either phase-1 or phase-2 flagellar antigens, encoded by the fliC and fljB genes, respectively. Flagellar phase reversal for the identification of both flagellar antigens is not necessary at the genetic level. Variable internal regions of the fliC genes encoding the H:i, H:r, H:l,v, H:e,h, H:z10, H:b, and H:d antigens have been sequenced; and the specific sites for each antigen in selected Salmonella serotypes have been determined. These results, together with flagellar G-complex variable internal sequences obtained by the Foodborne and Diarrheal Diseases Branch at the Centers for Disease Control and Prevention in Atlanta, Ga., have been used to design a multiplex PCR to identify the G-complex antigens as well as the H:i, H:r, H:l,v, H:e,h, Hz10, H:b, and H:d first-phase antigens. These antigens are part of the most common Salmonella serotypes possessing first-phase flagellar antigens. Salmonella enterica serotype Enteritidis is identified by adding a specific primer pair published previously (P. G. Agron, R. L. Walker, H. Kinde, S. J. Sawyer, D. C. Hayes, J. Wollard, and G. L. Andersen, Appl. Environ. Microbiol. 67:4984-4991, 2001). This multiplex PCR includes 13 primers. A total of 161 Salmonella strains associated with 72 different serotypes were tested. Each strain generated one first-phase-specific antigen fragment ranging from 100 to 500 bp; Salmonella serotype Enteritidis, however, generated two amplicons of 500 bp that corresponded to the G complex and a 333-bp serotype-specific amplicon, respectively. Twenty-three strains representing 19 serotypes with flagellar genes different from those targeted in this work did not generate any fragments. The method is quick, specific, and reproducible and is independent of the phase expressed by the bacteria when they are tested. PMID:15184437

  20. Coproduction of Acetaldehyde and Hydrogen during Glucose Fermentation by Escherichia coli ▿ †

    PubMed Central

    Zhu, Huilin; Gonzalez, Ramon; Bobik, Thomas A.

    2011-01-01

    Escherichia coli K-12 strain MG1655 was engineered to coproduce acetaldehyde and hydrogen during glucose fermentation by the use of exogenous acetyl-coenzyme A (acetyl-CoA) reductase (for the conversion of acetyl-CoA to acetaldehyde) and the native formate hydrogen lyase. A putative acetaldehyde dehydrogenase/acetyl-CoA reductase from Salmonella enterica (SeEutE) was cloned, produced at high levels, and purified by nickel affinity chromatography. In vitro assays showed that this enzyme had both acetaldehyde dehydrogenase activity (68.07 ± 1.63 μmol min−1 mg−1) and the desired acetyl-CoA reductase activity (49.23 ± 2.88 μmol min−1 mg−1). The eutE gene was engineered into an E. coli mutant lacking native glucose fermentation pathways (ΔadhE, ΔackA-pta, ΔldhA, and ΔfrdC). The engineered strain (ZH88) produced 4.91 ± 0.29 mM acetaldehyde while consuming 11.05 mM glucose but also produced 6.44 ± 0.26 mM ethanol. Studies showed that ethanol was produced by an unknown alcohol dehydrogenase(s) that converted the acetaldehyde produced by SeEutE to ethanol. Allyl alcohol was used to select for mutants with reduced alcohol dehydrogenase activity. Three allyl alcohol-resistant mutants were isolated; all produced more acetaldehyde and less ethanol than ZH88. It was also found that modifying the growth medium by adding 1 g of yeast extract/liter and lowering the pH to 6.0 further increased the coproduction of acetaldehyde and hydrogen. Under optimal conditions, strain ZH136 converted glucose to acetaldehyde and hydrogen in a 1:1 ratio with a specific acetaldehyde production rate of 0.68 ± 0.20 g h−1 g−1 dry cell weight and at 86% of the maximum theoretical yield. This specific production rate is the highest reported thus far and is promising for industrial application. The possibility of a more efficient “no-distill” ethanol fermentation procedure based on the coproduction of acetaldehyde and hydrogen is discussed. PMID:21803884

  1. The structure of the archeabacterial flagellar filament of the extreme halophile Halobacterium salinarum R1M1 and its relation to eubacterial flagellar filaments and type IV pili.

    PubMed

    Cohen-Krausz, Sara; Trachtenberg, Shlomo

    2002-08-16

    Although the phenomenology and mechanics of swimming are very similar in eubacteria and archaeabacteria (e.g. reversible rotation, helical polymorphism of the filament and formation of bundles), the dynamic flagellar filaments seem completely unrelated in terms of morphogenesis, structure and amino acid composition. Archeabacterial flagellar filaments share important features with type IV pili, which are components of retractable linear motors involved in twitching motility and cell adhesion. The archeabacterial filament is unique in: (1) having a relatively smooth surface and a small diameter of approximately 100A as compared to approximately 240A of eubacterial filaments and approximately 50A of type IV pili; (2) being glycosylated and sulfated in a pattern similar to the S-layer; (3) being synthesized as pre-flagellin with a signal-peptide cleavable by membrane peptidases upon transport; and (4) having an N terminus highly hydrophobic and homologous with that of the olygomerization domain of pilin. The synthesis of archeabacterial flagellin monomers as pre-flagellin and their post-translational, extracellular glycosylation suggest a different mode of monomer transport and polymerization at the cell-proximal end of the filament, similar to pili rather than to eubacterial flagellar filaments. The polymerization mode and small diameter may indicate the absence of a central channel in the filament. Using low-electron-dose images of cryo-negative-stained filaments, we determined the unique symmetry of the flagellar filament of the extreme halophile Halobacterium salinarum strain R1M1 and calculated a three-dimensional density map to a resolution of 19A. The map is based on layer-lines of order n=0, +10, -7, +3, -4, +6, and -1. The cross-section of the density map has a triskelion shape and is dominated by seven outer densities clustered into three groups, which are connected by lower-density arms to a dense central core surrounded by a lower-density shell. There is

  2. 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.

  3. Actin-interacting and flagellar proteins in Leishmania spp.: Bioinformatics predictions to functional assignments in phagosome formation

    PubMed Central

    2009-01-01

    Several motile processes are responsible for the movement of proteins into and within the flagellar membrane, but little is known about the process by which specific proteins (either actin-associated or not) are targeted to protozoan flagellar membranes. Actin is a major cytoskeleton protein, while polymerization and depolymerization of parasite actin and actin-interacting proteins (AIPs) during both processes of motility and host cell entry might be key events for successful infection. For a better understanding the eukaryotic flagellar dynamics, we have surveyed genomes, transcriptomes and proteomes of pathogenic Leishmania spp. to identify pertinent genes/proteins and to build in silico models to properly address their putative roles in trypanosomatid virulence. In a search for AIPs involved in flagellar activities, we applied computational biology and proteomic tools to infer from the biological meaning of coronins and Arp2/3, two important elements in phagosome formation after parasite phagocytosis by macrophages. Results presented here provide the first report of Leishmania coronin and Arp2/3 as flagellar proteins that also might be involved in phagosome formation through actin polymerization within the flagellar environment. This is an issue worthy of further in vitro examination that remains now as a direct, positive bioinformatics-derived inference to be presented. PMID:21637533

  4. Actin-interacting and flagellar proteins in Leishmania spp.: Bioinformatics predictions to functional assignments in phagosome formation.

    PubMed

    Diniz, Michely C; Costa, Marcília P; Pacheco, Ana C L; Kamimura, Michel T; Silva, Samara C; Carneiro, Laura D G; Sousa, Ana P L; Soares, Carlos E A; Souza, Celeste S F; de Oliveira, Diana Magalhães

    2009-07-01

    Several motile processes are responsible for the movement of proteins into and within the flagellar membrane, but little is known about the process by which specific proteins (either actin-associated or not) are targeted to protozoan flagellar membranes. Actin is a major cytoskeleton protein, while polymerization and depolymerization of parasite actin and actin-interacting proteins (AIPs) during both processes of motility and host cell entry might be key events for successful infection. For a better understanding the eukaryotic flagellar dynamics, we have surveyed genomes, transcriptomes and proteomes of pathogenic Leishmania spp. to identify pertinent genes/proteins and to build in silico models to properly address their putative roles in trypanosomatid virulence. In a search for AIPs involved in flagellar activities, we applied computational biology and proteomic tools to infer from the biological meaning of coronins and Arp2/3, two important elements in phagosome formation after parasite phagocytosis by macrophages. Results presented here provide the first report of Leishmania coronin and Arp2/3 as flagellar proteins that also might be involved in phagosome formation through actin polymerization within the flagellar environment. This is an issue worthy of further in vitro examination that remains now as a direct, positive bioinformatics-derived inference to be presented. PMID:21637533

  5. Impact of cranberry on Escherichia coli cellular surface characteristics

    SciTech Connect

    Johnson, Brandy J.; Malanoski, Anthony P.; Ligler, Frances S.

    2008-12-19

    The anti-adhesive effects of cranberry have been attributed to both interactions of its components with the surface of bacterial cells and to inhibition of p-fimbriae expression. Previous reports also suggested that the presence of cranberry juice changed the Gram stain characteristics of Escherichia coli. Here, we show that the morphology of E. coli is changed when grown in the presence of juice or extract from Vaccinium macrocarpon (cranberry). Gene expression analysis indicates the down regulation of flagellar basal body rod and motor proteins. Consistent with this finding and previous reports, the SEM images indicate a decrease in the visible p-fimbriae. The iodine used in Gram-staining protocols was found to interact differently with the bacterial membrane when cells were cultured in spiked media. Slight alterations in the Gram stain protocol demonstrated that culturing in the presence of cranberry juice does not change the Gram stain characteristics contradicting other reports.

  6. Impact of cranberry on Escherichia coli cellular surface characteristics.

    PubMed

    Johnson, Brandy J; Lin, Baochuan; Dinderman, Michael A; Rubin, Robert A; Malanoski, Anthony P; Ligler, Frances S

    2008-12-19

    The anti-adhesive effects of cranberry have been attributed to both interactions of its components with the surface of bacterial cells and to inhibition of p-fimbriae expression. Previous reports also suggested that the presence of cranberry juice changed the Gram stain characteristics of Escherichia coli. Here, we show that the morphology of E. coli is changed when grown in the presence of juice or extract from Vaccinium macrocarpon (cranberry). Gene expression analysis indicates the down regulation of flagellar basal body rod and motor proteins. Consistent with this finding and previous reports, the SEM images indicate a decrease in the visible p-fimbriae. The iodine used in Gram-staining protocols was found to interact differently with the bacterial membrane when cells were cultured in spiked media. Slight alterations in the Gram stain protocol demonstrated that culturing in the presence of cranberry juice does not change the Gram stain characteristics contradicting other reports.

  7. Subpellicular and flagellar microtubules of Trypanosoma brucei brucei contain the same alpha-tubulin isoforms

    PubMed Central

    1987-01-01

    The cytoskeleton of the parasitic hemoflagellate Trypanosoma brucei brucei essentially consists of two microtubule-based structures: a subpellicular layer of singlet microtubules, which are in close contact with the cell membrane, and the flagellar axoneme. In addition, the cells contain a small pool of soluble tubulin. Two-dimensional gel electrophoretic analysis of the tubulins present in these subcellular compartments revealed two distinct electrophoretic isoforms of alpha- tubulin, termed alpha 1 and alpha 3. alpha 1-Tubulin most likely represents the primary translation product, while alpha 3-tubulin is a posttranslationally acetylated derivative of alpha 1-tubulin. In the pool of soluble cytoplasmic tubulin, alpha 1 is the predominant species, while the very stable flagellar microtubules contain almost exclusively the alpha 3-tubulin isoform. The subpellicular microtubules contain both isoforms. Neither of the two alpha-tubulin isoforms is organelle specific, but the alpha 3 isoform is predominantly located in stable microtubules. PMID:3818788

  8. Rotational speed control of Na +-driven flagellar motor by dual pipettes.

    PubMed

    Nogawa, Kousuke; Kojima, Masaru; Nakajima, Masahiro; Kojima, Seiji; Homma, Michio; Fukuda, Toshio

    2009-12-01

    Single cell analysis has attracted much attention to reveal the detailed and localized biological information. Local environmental control technique is desired to analyze the detailed and localized properties of single cells. In this paper, we propose the local environmental control system with nano/micro dual pipettes to control the local reagent concentration dynamically and arbitrarily. Local environmental control by dual pipettes is applied to the rotational speed control of bacterial flagellar motor, which is a rotary molecular machine. We demonstrate quick response and iterative rotational speed control of Na (+)-driven flagellar motor in both accelerating and relaxing directions by switching the local spout between Na (+)-containing and Na (+) -free solutions with dual pipettes. It is shown that the rotational speed might be controllable by changing the spouting velocity of Na (+)-containing solution with multiplying the applied dc voltage. PMID:19887330

  9. 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

  10. 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.

  11. 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.

  12. Nonlinear instability in flagellar dynamics: a novel modulation mechanism in sperm migration?

    NASA Astrophysics Data System (ADS)

    Gadelha, H.; Gaffney, E.; Smith, D.; Kirkman-Brown, J.

    2010-11-01

    Throughout biology, cells and organisms use flagella and cilia to propel fluid and achieve motility. While the mechanics of flagellum-fluid interaction has been the subject of extensive mathematical studies, these models have been restricted to being geometrically linear or weakly nonlinear. In this talk, we study the effect of geometrical nonlinearity, focusing on the spermatozoon flagellum. For a wide range of physiologically relevant parameters, the nonlinear model predicts that flagellar compression by the internal forces initiates an effective buckling behaviour, leading to a symmetry-breaking bifurcation that causes profound and complicated changes in the waveform and swimming trajectory, as well as the breakdown of the linear theory. The emergent waveform also induces curved swimming in an otherwise symmetric system, with the swimming trajectory being sensitive to head shape - no signalling or asymmetric forces are required. We conclude that non-linear models are essential in understanding the flagellar waveform in migratory human sperm.

  13. Mechanisms of flagellar motility deduced from backward-swimming bull sperm.

    PubMed

    Phillips, D M; Kalay, D

    1984-07-01

    Under certain conditions of cryopreservation, bull spermatozoa undergo an interesting structural alteration. The sperm tail becomes bent back on itself to form a hairpin shape. The bend in the tail occurs at a very precise point, 11 microns behind the neck, and it causes the tail to become kinked. Flagellar microtubules and dense fibers become broken and the ninefold symmetry of the flagellum is greatly distored. Although the portion of the flagellum between the kink and the sperm head does not propagate a wave, the distal portion of the flagellum propagates a base-to-tip wave, causing the spermatozoan to progress backward. These observations suggest that the mammalian spermatozoon does not need basal structures to propagate a flagellar wave.

  14. 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

  15. 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

  16. 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

  17. Structure of the torque ring of the flagellar motor and the molecular basis for rotational switching.

    PubMed

    Lee, Lawrence K; Ginsburg, Michael A; Crovace, Claudia; Donohoe, Mhairi; Stock, Daniela

    2010-08-19

    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. PMID:20676082

  18. 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.

  19. 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

    2012-03-01

    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.

  20. The counterbend phenomenon in flagellar axonemes and cross-linked filament bundles.

    PubMed

    Gadêlha, Hermes; Gaffney, Eamonn A; Goriely, Alain

    2013-07-23

    Recent observations of flagellar counterbend in sea urchin sperm show that the mechanical induction of curvature in one part of a passive flagellum induces a compensatory countercurvature elsewhere. This apparent paradoxical effect cannot be explained using the standard elastic rod theory of Euler and Bernoulli, or even the more general Cosserat theory of rods. Here, we develop a geometrically exact mechanical model to describe the statics of microtubule bundles that is capable of predicting the curvature reversal events observed in eukaryotic flagella. This is achieved by allowing the interaction of deformations in different material directions, by accounting not only for structural bending, but also for the elastic forces originating from the internal cross-linking mechanics. Large-amplitude static configurations can be described analytically, and an excellent match between the model and the observed counterbend deformation was found. This allowed a simultaneous estimation of multiple sperm flagellum material parameters, namely the cross-linking sliding resistance, the bending stiffness, and the sperm head junction compliance ratio. We further show that small variations on the empirical conditions may induce discrepancies for the evaluation of the flagellar material quantities, so that caution is required when interpreting experiments. Finally, our analysis demonstrates that the counterbend emerges as a fundamental property of sliding resistance in cross-linked filamentous polymer bundles, which also suggests that cross-linking proteins may contribute to the regulation of the flagellar waveform in swimming sperm via counterbend mechanics. PMID:23824293

  1. 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

  2. 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. PMID:26976588

  3. Bimodal rheotactic behavior reflects flagellar beat asymmetry in human sperm cells.

    PubMed

    Bukatin, Anton; Kukhtevich, Igor; Stoop, Norbert; Dunkel, Jörn; Kantsler, Vasily

    2015-12-29

    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

  4. 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.

  5. Studies on the serology of flagellar antigens of Yersinia enterocolitica and related Yersinia species.

    PubMed

    Aleksić, S; Bockemühl, J; Lange, F

    1986-05-01

    A total of 1242 strains of Y. enterocolitica, 104 strains of Y. frederiksenii, 95 strains of Y. kristensenii and 85 strains of Y. intermedia were serotyped with antisera against 56 O antigens and 19 H antigens according to the extended antigenic scheme of Wauters, and with additional antisera against 4 somatic and 19 flagellar antigens not previously described. H antigens of Y. frederiksenii, Y. kristensenii, and Y. intermedia turned out to be rather homogeneous without distinct subfactors. In these species the scope of identified serovars was narrow. Flagellar antigens of Y. enterocolitica were mostly composed of several subfactors, leading to a total of 117 serovars identified in the species. A number of cross-reactions between Yersinia H antigens were observed which could be avoided by absorption without significant lowering of the titre. Flagellar antigens of Yersinia were monophasic, and species specific. The antigens remained stable after storage in agar stabs and repeated subcultures. The epidemiological value of serotyping is demonstrated by strains from three different sources. It is suggested serotyping of Yersinia strains should be performed in three steps: O typing of the prevailing enteropathogenic Y. enterocolitica serogroups in the medical routine laboratory; O and H typing of Y. enterocolitica by National Reference Centres applying a typing scheme reduced to this species; and O and H typing of Y. enterocolitica, Y. frederiksenii, Y. kristensenii and Y. intermedia by specialized International Centres using an extended typing scheme. The need for international standards comparable to those established for Salmonella is emphasized.

  6. 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

  7. Bimodal rheotactic behavior reflects flagellar beat asymmetry in human sperm cells.

    PubMed

    Bukatin, Anton; Kukhtevich, Igor; Stoop, Norbert; Dunkel, Jörn; Kantsler, Vasily

    2015-12-29

    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.

  8. 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

    2012-03-01

    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. PMID:22245577

  9. Transcriptional activation of the aldehyde reductase YqhD by YqhC and its implication in glyoxal metabolism of Escherichia coli K-12.

    PubMed

    Lee, Changhan; Kim, Insook; Lee, Junghoon; Lee, Kang-Lok; Min, Bumchan; Park, Chankyu

    2010-08-01

    The reactive alpha-oxoaldehydes such as glyoxal (GO) and methylglyoxal (MG) are generated in vivo from sugars through oxidative stress. GO and MG are believed to be removed from cells by glutathione-dependent glyoxalases and other aldehyde reductases. We isolated a number of GO-resistant (GO(r)) mutants from Escherichia coli strain MG1655 on LB plates containing 10 mM GO. By tagging the mutations with the transposon TnphoA-132 and determining their cotransductional linkages, we were able to identify a locus to which most of the GO(r) mutations were mapped. DNA sequencing of the locus revealed that it contains the yqhC gene, which is predicted to encode an AraC-type transcriptional regulator of unknown function. The GO(r) mutations we identified result in missense changes in yqhC and were concentrated in the predicted regulatory domain of the protein, thereby constitutively activating the product of the adjacent gene yqhD. The transcriptional activation of yqhD by wild-type YqhC and its mutant forms was established by an assay with a beta-galactosidase reporter fusion, as well as with real-time quantitative reverse transcription-PCR. We demonstrated that YqhC binds to the promoter region of yqhD and that this binding is abolished by a mutation in the potential target site, which is similar to the consensus sequence of its homolog SoxS. YqhD facilitates the removal of GO through its NADPH-dependent enzymatic reduction activity by converting it to ethadiol via glycolaldehyde, as detected by nuclear magnetic resonance, as well as by spectroscopic measurements. Therefore, we propose that YqhC is a transcriptional activator of YqhD, which acts as an aldehyde reductase with specificity for certain aldehydes, including GO.

  10. E. Coli and Pregnancy

    MedlinePlus

    ... care provider. What is E. coli? E. coli (Escherichia coli) is a bacterium that lives in your colon ( ... 10):1411-1413. Jones B, et al. 2004. Escherichia coli: a growing problem in early onset neonatal sepsis. ...

  11. E. Coli Infection

    MedlinePlus

    ... is E. coli? E. coli is short for Escherichia coli -- bacteria (germs) that cause severe cramps and diarrhea. E. ... and especially in people who have another illness. E. coli infection is more common during the summer months and ...

  12. Extragenic suppressors of paralyzed flagellar mutations in Chlamydomonas reinhardtii identify loci that alter the inner dynein arms

    PubMed Central

    1992-01-01

    We have analyzed extragenic suppressors of paralyzed flagella mutations in Chlamydomonas reinhardtii in an effort to identify new dynein mutations. A temperature-sensitive allele of the PF16 locus was mutagenized and then screened for revertants that could swim at the restrictive temperature (Dutcher et al. 1984. J. Cell Biol. 98:229- 236). In backcrosses of one of the revertant strains to wild-type, we recovered both the original pf16 mutation and a second, unlinked suppressor mutation with its own flagellar phenotype. This mutation has been identified by both recombination and complementation tests as a new allele of the previously uncharacterized PF9 locus on linkage group XII/XIII. SDS-PAGE analysis of isolated flagellar axonemes and dynein extracts has demonstrated that the pf9 strains are missing four polypeptides that form the I1 inner arm dynein subunit. The primary effect of the loss of the I1 subunit is a decrease in the forward swimming velocity due to a change in the flagellar waveform. Both the flagellar beat frequency and the axonemal ATPase activity are nearly wild-type. Examination of axonemes by thin section electron microscopy and image averaging methods reveals that a specific domain of the inner arm complex is missing in the pf9 mutant strains (see accompanying paper by Mastronarde et al.). When combined with other flagellar defects, the loss of the I1 subunit has synergistic effects on both flagellar assembly and flagellar motility. These synthetic phenotypes provide a screen for new suppressor mutations in other loci. Using this approach, we have identified the first interactive suppressors of a dynein arm mutation and an unusual bypass suppressor mutation. PMID:1387404

  13. Escherichia Coli

    ERIC Educational Resources Information Center

    Goodsell, David S.

    2009-01-01

    Diverse biological data may be used to create illustrations of molecules in their cellular context. I describe the scientific results that support a recent textbook illustration of an "Escherichia coli cell". The image magnifies a portion of the bacterium at one million times, showing the location and form of individual macromolecules. Results…

  14. E. coli

    MedlinePlus

    ... sure that ground beef has reached a safe internal temperature of 160° F. Wash hands before preparing food, after diapering infants, and after contact with cows, sheep, or goats, their food or treats, or their living environment . General Information E. coli Infections (NIH MedlinePlus) Trusted ...

  15. Intraflagellar transport particle size scales inversely with flagellar length: revisiting the balance-point length control model

    PubMed Central

    Engel, Benjamin D.; Ludington, William B.

    2009-01-01

    The assembly and maintenance of eukaryotic flagella are regulated by intraflagellar transport (IFT), the bidirectional traffic of IFT particles (recently renamed IFT trains) within the flagellum. We previously proposed the balance-point length control model, which predicted that the frequency of train transport should decrease as a function of flagellar length, thus modulating the length-dependent flagellar assembly rate. However, this model was challenged by the differential interference contrast microscopy observation that IFT frequency is length independent. Using total internal reflection fluorescence microscopy to quantify protein traffic during the regeneration of Chlamydomonas reinhardtii flagella, we determined that anterograde IFT trains in short flagella are composed of more kinesin-associated protein and IFT27 proteins than trains in long flagella. This length-dependent remodeling of train size is consistent with the kinetics of flagellar regeneration and supports a revised balance-point model of flagellar length control in which the size of anterograde IFT trains tunes the rate of flagellar assembly. PMID:19805630

  16. KHARON Is an Essential Cytoskeletal Protein Involved in the Trafficking of Flagellar Membrane Proteins and Cell Division in African Trypanosomes.

    PubMed

    Sanchez, Marco A; Tran, Khoa D; Valli, Jessica; Hobbs, Sam; Johnson, Errin; Gluenz, Eva; Landfear, Scott M

    2016-09-16

    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

  17. Multiplex PCR-based detection and identification of the most common Salmonella second-phase flagellar antigens.

    PubMed

    Echeita, M Aurora; Herrera, Silvia; Garaizar, Javier; Usera, Miguel A

    2002-03-01

    Most Salmonella serotypes alternatively express phase 1 or phase 2 flagellar antigens encoded by fliC and fljB genes respectively. Flagellar phase reversal to identify both flagellar antigens is not necessary at the genetic level. Variable internal regions of the fljB genes encoding H:1,w, H:e,n,x and H:e,n,z15 antigens have been sequenced and the specific sites for each antigen determined in selected Salmonella serotypes. These results, together with flagellar H1 complex variable internal sequences previously published, have been used to design a multiplex-PCR to identify H:1,2, H:1,5, H:1,6, H:1,7, H:1,w, H:e,n,x and H:e,n,z15 second-phase antigens. These antigens are part of the most common Salmonella serotypes possessing second-phase flagellar antigens. This multiplex-PCR includes 10 primers. A total of 140 Salmonella strains associated with 49 different serotypes were tested. Each strain generated one second-phase-specific antigen fragment, ranging between 50 and 400 bps. Twenty-five strains associated with 17 serotypes, with no second-phase antigen or with an antigen different from those tested in this work, did not generate any fragments. The method is quick, specific and reproducible and is independent of the phase expressed by the bacteria when tested.

  18. Mating in Chlamydomonas: a system for the study of specific cell adhesion. I. Ultrastructural and electrophoretic analyses of flagellar surface components involved in adhesion

    PubMed Central

    1976-01-01

    To determine the ultrastructural and biochemical bases for flagellar adhesiveness in the mating reaction in Chlamydomonas, gametic and vegetative flagella and flagellar membranes were studied by use of electron microscope and electrophoretic procedures. Negative staining with uranyl acetate revealed no differences in gametic and vegetative flagellar surfaces; both had flagellar membranes, flagellar sheaths, and similar numbers and distributions of mastigonemes. Freezecleave procedures suggested that there may be a greater density of intramembranous particles on the B faces of gametic flagellar membranes than on the B faces of vegetative flagellar membranes. Gamone, the adhesive material that gametes release into their medium, was demonstrated, on the basis of ultrastructural and biochemical analyses, to be composed of flagellar surface components, i.e., membrane vesicles and mastigonemes. Comparison of vegetative (nonadhesive) and gametic (adhesive) "gamones" by use of SDS polyacrylamide gel electrophoresis showed both preparations to be composed of membrane, mastigoneme, and some microtubule proteins, as well as several unidentified protein and carbohydrate-staining components. However, there was an additional protein of approximately 70,000 mol wt in gametic gamone which was not present in vegetative gamone. When gametic gamone was separated into a membrane and a mastigoneme fraction on CSCl gradients, only the membrane fraction had isoagglutinating activity; the mastigoneme fraction was inactive, suggesting that mastigonemes are not involved in adhesion. PMID:1245545

  19. Synthetic brominated furanone F202 prevents biofilm formation by potentially human pathogenic Escherichia coli O103:H2 and Salmonella ser. Agona on abiotic surfaces

    PubMed Central

    Vestby, LK; Johannesen, KCS; Witsø, IL; Habimana, O; Scheie, AA; Urdahl, AM; Benneche, T; Langsrud, S; Nesse, LL

    2014-01-01

    Aims Investigate the use of a synthetic brominated furanone (F202) against the establishment of biofilm by Salmonella ser. Agona and E. coli O103:H2 under temperature conditions relevant for the food and feed industry as well as under temperature conditions optimum for growth. Methods and Results Effect of F202 on biofilm formation by Salmonella ser. Agona and E. coli O103:H2 was evaluated using a microtiter plate assay and confocal microscopy. Effect of F202 on bacterial motility was investigated using swimming and swarming assays. Influence on flagellar synthesis by F202 was examined by flagellar staining. Results showed that F202 inhibited biofilm formation without being bactericidal. F202 was found to affect both swimming and swarming motility without, however, affecting the expression of flagella. Conclusions F202 showed its potential as a biofilm inhibitor of Salmonella ser. Agona and E. coli O103:H2 under temperature conditions relevant for the feed and food industry as well as temperatures optimum for growth. One potential mode of action of F202 was found to be by targeting flagellar function. Significance and Impact of the Study The present study gives valuable new knowledge to the potential use of furanones as a tool in biofilm management in the food and feed industry. PMID:24118802

  20. Transcription Profiling of the Stringent Response in Escherichia coli▿ †

    PubMed Central

    Durfee, Tim; Hansen, Anne-Marie; Zhi, Huijun; Blattner, Frederick R.; Jin, Ding Jun

    2008-01-01

    The bacterial stringent response serves as a paradigm for understanding global regulatory processes. It can be triggered by nutrient downshifts or starvation and is characterized by a rapid RelA-dependent increase in the alarmone (p)ppGpp. One hallmark of the response is the switch from maximum-growth-promoting to biosynthesis-related gene expression. However, the global transcription patterns accompanying the stringent response in Escherichia coli have not been analyzed comprehensively. Here, we present a time series of gene expression profiles for two serine hydroxymate-treated cultures: (i) MG1655, a wild-type E. coli K-12 strain, and (ii) an isogenic relAΔ251 derivative defective in the stringent response. The stringent response in MG1655 develops in a hierarchical manner, ultimately involving almost 500 differentially expressed genes, while the relAΔ251 mutant response is both delayed and limited in scope. We show that in addition to the down-regulation of stable RNA-encoding genes, flagellar and chemotaxis gene expression is also under stringent control. Reduced transcription of these systems, as well as metabolic and transporter-encoding genes, constitutes much of the down-regulated expression pattern. Conversely, a significantly larger number of genes are up-regulated. Under the conditions used, induction of amino acid biosynthetic genes is limited to the leader sequences of attenuator-regulated operons. Instead, up-regulated genes with known functions, including both regulators (e.g., rpoE, rpoH, and rpoS) and effectors, are largely involved in stress responses. However, one-half of the up-regulated genes have unknown functions. How these results are correlated with the various effects of (p)ppGpp (in particular, RNA polymerase redistribution) is discussed. PMID:18039766

  1. Adenine Nucleotide Metabolism and a Role for AMP in Modulating Flagellar Waveforms in Mouse Sperm1

    PubMed Central

    Vadnais, Melissa L.; Cao, Wenlei; Aghajanian, Haig K.; Haig-Ladewig, Lisa; Lin, Angel M.; Al-Alao, Osama; Gerton, George L.

    2014-01-01

    ABSTRACT While most ATP, the main energy source driving sperm motility, is derived from glycolysis and oxidative phosphorylation, the metabolic demands of the cell require the efficient use of power stored in high-energy phosphate bonds. In times of high energy consumption, adenylate kinase (AK) scavenges one ATP molecule by transphosphorylation of two molecules of ADP, simultaneously yielding one molecule of AMP as a by-product. Either ATP or ADP supported motility of detergent-modeled cauda epididymal mouse sperm, indicating that flagellar AKs are functional. However, the ensuing flagellar waveforms fueled by ATP or ADP were qualitatively different. Motility driven by ATP was rapid but restricted to the distal region of the sperm tail, whereas ADP produced slower and more fluid waves that propagated down the full flagellum. Characterization of wave patterns by tracing and superimposing the images of the flagella, quantifying the differences using digital image analysis, and computer-assisted sperm analysis revealed differences in the amplitude, periodicity, and propagation of the waves between detergent-modeled sperm treated with either ATP or ADP. Surprisingly, addition of AMP to the incubation medium containing ATP recapitulated the pattern of sperm motility seen with ADP alone. In addition to AK1 and AK2, which we previously demonstrated are present in outer dense fibers and mitochondrial sheath of the mouse sperm tail, we show that another AK, AK8, is present in a third flagellar compartment, the axoneme. These results extend the known regulators of sperm motility to include AMP, which may be operating through an AMP-activated protein kinase. PMID:24740601

  2. Biochemical Characterization of the Flagellar Rod Components of Rhodobacter sphaeroides: Properties and Interactions

    PubMed Central

    Osorio-Valeriano, Manuel; de la Mora, Javier; Camarena, Laura

    2015-01-01

    ABSTRACT The flagellar basal body is a rotary motor that spans the cytoplasmic and outer membranes. The rod is a drive shaft that transmits torque generated by the motor through the hook to the filament that propels the bacterial cell. The assembly and structure of the rod are poorly understood. In a first attempt to characterize this structure in the alphaproteobacterium Rhodobacter sphaeroides, we overexpressed and purified FliE and the four related rod proteins (FlgB, FlgC, FlgF, and FlgG), and we analyzed their ability to form homo-oligomers. We found that highly purified preparations of these proteins formed high-molecular-mass oligomers that tended to dissociate in the presence of NaCl. As predicted by in silico modeling, the four rod proteins share architectural features. Using affinity blotting, we detected the heteromeric interactions between these proteins. In addition, we observed that deletion of the N- and C-terminal regions of FlgF and FlgG severely affected heteromeric but not homomeric interactions. On the basis of our findings, we propose a model of rod assembly in this bacterium. IMPORTANCE Despite the considerable amount of research on the structure and assembly of other flagellar axial structures that has been conducted, the rod has been barely studied. An analysis of the biochemical characteristics of the flagellar rod components of the Fla1 system of R. sphaeroides is presented in this work. We also analyze the interactions of these proteins with each other and with their neighbors, and we propose a model for the order in which they are assembled. PMID:26574514

  3. The effect of cell growth phase on the regulatory cross-talk between flagellar and Spi1 virulence gene expression.

    PubMed

    Mouslim, Chakib; Hughes, Kelly T

    2014-03-01

    The flagellar regulon controls Salmonella biofilm formation, virulence gene expression and the production of the major surface antigen present on the cell surface: flagellin. At the top of a flagellar regulatory hierarchy is the master operon, flhDC, which encodes the FlhD₄C₂ transcriptional complex required for the expression of flagellar, chemotaxis and Salmonella pathogenicity island 1 (Spi1) genes. Of six potential transcriptional start-sites within the flhDC promoter region, only two, P1(flhDC) and P5(flhDC), were functional in a wild-type background, while P6(flhDC) was functional in the absence of CRP. These promoters are transcribed differentially to control either flagellar or Spi1 virulent gene expression at different stages of cell growth. Transcription from P1(flhDC) initiates flagellar assembly and a negative autoregulatory loop through FlhD₄C₂-dependent transcription of the rflM gene, which encodes a repressor of flhDC transcription. Transcription from P1(flhDC) also initiates transcription of the Spi1 regulatory gene, hilD, whose product, in addition to activating Spi1 genes, also activates transcription of the flhDC P5 promoter later in the cell growth phase. The regulators of flhDC transcription (RcsB, LrhA, RflM, HilD, SlyA and RtsB) also exert their control at different stages of the cell growth phase and are also subjected to cell growth phase control. This dynamic of flhDC transcription separates the roles of FlhD₄C₂ transcriptional activation into an early cell growth phase role for flagellar production from a late cell growth phase role in virulence gene expression.

  4. 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.

  5. 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.

  6. 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. PMID:27193546

  7. Transcriptional organization of the region encoding the synthesis of the flagellar filament in Pseudomonas fluorescens.

    PubMed

    Redondo-Nieto, Miguel; Lloret, Javier; Larenas, Javiera; Barahona, Emma; Navazo, Ana; Martínez-Granero, Francisco; Capdevila, Silvia; Rivilla, Rafael; Martín, Marta

    2008-06-01

    Pseudomonas fluorescens F113 is motile by means of type b flagella. Analysis of the region encoding the synthesis of the flagellar filament has shown a transcriptional organization different from that of type a flagella. Additionally to the promoters driving fliC, fliD, and fleQ expression, we have found promoters upstream of the flaG gene and the fliST operon. These promoters were functional in vivo. Both promoters have been mapped and appear to be dependent on the vegetative sigma factor and independent of FleQ, the master regulator of flagellum synthesis.

  8. A new automated method of image analysis: comparison of ciliary and flagellar beats.

    PubMed

    Schoevaert, D; Marano, F; Serres, C; Berrebah, H

    1990-01-01

    A new automated method of image analysis of sperm flagellar (human) and cilia (Dunaliella) bends is developed. This method permits an automatic determination of the line characterizing the flagellum. Two dynamic parameters are measured: the wave propagation velocity and the wave curvature radius. The data reveal similar patterns in the propagation of the principal and reverse waves between flagelated and ciliated cells. Conversely, differences are seen in principal wave curvature due perhaps to the presence of periaxonemal structures in the flagellum, absent in cilium. The identical patterns of reverse wave curvaturei in both systems may be linked to axonemal limitations.

  9. Bioinformatics, genomics and evolution of non-flagellar type-III secretion systems: a Darwinian perspective.

    PubMed

    Pallen, Mark J; Beatson, Scott A; Bailey, Christopher M

    2005-04-01

    We review the biology of non-flagellar type-III secretion systems from a Darwinian perspective, highlighting the themes of evolution, conservation, variation and decay. The presence of these systems in environmental organisms such as Myxococcus, Desulfovibrio and Verrucomicrobium hints at roles beyond virulence. We review newly discovered sequence homologies (e.g., YopN/TyeA and SepL). We discuss synapomorphies that might be useful in formulating a taxonomy of type-III secretion. The problem of information overload is likely to be ameliorated by launch of a web site devoted to the comparative biology of type-III secretion ().

  10. Characterization of non-Shiga-toxin-producing Escherichia coli O157 strains isolated from dogs.

    PubMed

    Bentancor, A; Vilte, D A; Rumi, M V; Carbonari, C C; Chinen, I; Larzábal, M; Cataldi, A; Mercado, E C

    2010-01-01

    Shiga toxin-negative Escherichia coli O157 strains of various H types have been associated with diarrhea in children and are considered potentially pathogenic for humans. In this study, we describe non-Shiga toxin-producing E. coli O157 E. coli strains previously obtained from dogs in Argentina. Different E. coli phylogenetic lineages corresponding to flagellar types H16, H29 and H45 were identified. E. coli serotypes O157:H16 and O157:H45 contained intimin subtypes epsilon and alpha 1, respectively. Serotype O157:H45 carried the bfp gene encoding the bundle-forming pilus. Localized adherence-like patterns to HEp-2 cells were observed in O157:H16 strains, while O157:H45 adhered in a typical localized pattern. A total of eight different XbaI-pulse field electrophoresis patterns with more than 74 % similarity were identified among the nine E. coli O157:H16 strains. Our data emphasized the fact that dogs may harbor human pathogenic E. coli O157 which do not correspond to Shiga toxin-producing strains and whose potential human health hazard should not be underestimated. PMID:20461294

  11. Electrostatic interactions between rotor and stator in the bacterial flagellar motor

    PubMed Central

    Zhou, Jiadong; Lloyd, Scott A.; Blair, David F.

    1998-01-01

    Bacterial flagellar motors rotate, obtaining power from the membrane gradient of protons or, in some species, sodium ions. Torque generation in the flagellar motor must involve interactions between components of the rotor and components of the stator. Sites of interaction between the rotor and stator have not been identified. Mutational studies of the rotor protein FliG and the stator protein MotA showed that both proteins contain charged residues essential for motor rotation. This suggests that functionally important electrostatic interactions might occur between the rotor and stator. To test this proposal, we examined double mutants with charged-residue substitutions in both the rotor protein FliG and the stator protein MotA. Several combinations of FliG mutations with MotA mutations exhibited strong synergism, whereas others showed strong suppression, in a pattern that indicates that the functionally important charged residues of FliG interact with those of MotA. These results identify a functionally important site of interaction between the rotor and stator and suggest a hypothesis for electrostatic interactions at the rotor–stator interface. PMID:9600984

  12. Electrostatic interactions between rotor and stator in the bacterial flagellar motor.

    PubMed

    Zhou, J; Lloyd, S A; Blair, D F

    1998-05-26

    Bacterial flagellar motors rotate, obtaining power from the membrane gradient of protons or, in some species, sodium ions. Torque generation in the flagellar motor must involve interactions between components of the rotor and components of the stator. Sites of interaction between the rotor and stator have not been identified. Mutational studies of the rotor protein FliG and the stator protein MotA showed that both proteins contain charged residues essential for motor rotation. This suggests that functionally important electrostatic interactions might occur between the rotor and stator. To test this proposal, we examined double mutants with charged-residue substitutions in both the rotor protein FliG and the stator protein MotA. Several combinations of FliG mutations with MotA mutations exhibited strong synergism, whereas others showed strong suppression, in a pattern that indicates that the functionally important charged residues of FliG interact with those of MotA. These results identify a functionally important site of interaction between the rotor and stator and suggest a hypothesis for electrostatic interactions at the rotor-stator interface. PMID:9600984

  13. Electrostatic Interactions between Rotor and Stator in the Bacterial Flagellar Motor

    NASA Astrophysics Data System (ADS)

    Zhou, Jiadong; Lloyd, Scott A.; Blair, David F.

    1998-05-01

    Bacterial flagellar motors rotate, obtaining power from the membrane gradient of protons or, in some species, sodium ions. Torque generation in the flagellar motor must involve interactions between components of the rotor and components of the stator. Sites of interaction between the rotor and stator have not been identified. Mutational studies of the rotor protein FliG and the stator protein MotA showed that both proteins contain charged residues essential for motor rotation. This suggests that functionally important electrostatic interactions might occur between the rotor and stator. To test this proposal, we examined double mutants with charged-residue substitutions in both the rotor protein FliG and the stator protein MotA. Several combinations of FliG mutations with MotA mutations exhibited strong synergism, whereas others showed strong suppression, in a pattern that indicates that the functionally important charged residues of FliG interact with those of MotA. These results identify a functionally important site of interaction between the rotor and stator and suggest a hypothesis for electrostatic interactions at the rotor-stator interface.

  14. Nonlinear instability in flagellar dynamics: a novel modulation mechanism in sperm migration?

    PubMed Central

    Gadêlha, H.; Gaffney, E. A.; Smith, D. J.; Kirkman-Brown, J. C.

    2010-01-01

    Throughout biology, cells and organisms use flagella and cilia to propel fluid and achieve motility. The beating of these organelles, and the corresponding ability to sense, respond to and modulate this beat is central to many processes in health and disease. While the mechanics of flagellum–fluid interaction has been the subject of extensive mathematical studies, these models have been restricted to being geometrically linear or weakly nonlinear, despite the high curvatures observed physiologically. We study the effect of geometrical nonlinearity, focusing on the spermatozoon flagellum. For a wide range of physiologically relevant parameters, the nonlinear model predicts that flagellar compression by the internal forces initiates an effective buckling behaviour, leading to a symmetry-breaking bifurcation that causes profound and complicated changes in the waveform and swimming trajectory, as well as the breakdown of the linear theory. The emergent waveform also induces curved swimming in an otherwise symmetric system, with the swimming trajectory being sensitive to head shape—no signalling or asymmetric forces are required. We conclude that nonlinear models are essential in understanding the flagellar waveform in migratory human sperm; these models will also be invaluable in understanding motile flagella and cilia in other systems. PMID:20462879

  15. Dynamics of a tightly coupled mechanism for flagellar rotation. Bacterial motility, chemiosmotic coupling, protonmotive force.

    PubMed

    Meister, M; Caplan, S R; Berg, H C

    1989-05-01

    The bacterial flagellar motor is a molecular engine that couples the flow of protons across the cytoplasmic membrane to rotation of the flagellar filament. We analyze the steady-state behavior of an explicit mechanical model in which a fixed number of protons carries the filament through one revolution. Predictions of this model are compared with experimentally determined relationships between protonmotive force, proton flux, torque, and speed. All such tightly coupled mechanisms produce the same torque when the motor is stalled but vary greatly in their behavior at high speed. The speed at zero load predicted by our model is limited by the rates of association and dissociation of protons at binding sites on the rotor and by the mobility of force generators containing transmembrane channels that interact with these sites. Our analysis suggests that more could be learned about the motor if it were driven by an externally applied torque backwards (at negative speed) or forwards at speeds greater than the zero-load speed. PMID:2720081

  16. The Trypanosome Flagellar Pocket Collar and Its Ring Forming Protein-TbBILBO1.

    PubMed

    Perdomo, Doranda; Bonhivers, Mélanie; Robinson, Derrick R

    2016-03-02

    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.

  17. Architecture of a flagellar apparatus in the fast-swimming magnetotactic bacterium MO-1

    PubMed Central

    Ruan, Juanfang; Kato, Takayuki; Santini, Claire-Lise; Miyata, Tomoko; Kawamoto, Akihiro; Zhang, Wei-Jia; Bernadac, Alain; Wu, Long-Fei; Namba, Keiichi

    2012-01-01

    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. PMID:23184985

  18. 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.

  19. Common Evolutionary Origin for the Rotor Domain of Rotary Atpases and Flagellar Protein Export Apparatus

    PubMed Central

    Kishikawa, Jun-ichi; Ibuki, Tatsuya; Nakamura, Shuichi; Nakanishi, Astuko; Minamino, Tohru; Miyata, Tomoko; Namba, Keiichi; Konno, Hiroki; Ueno, Hiroshi; Imada, Katsumi; Yokoyama, Ken

    2013-01-01

    The V1- and F1- rotary ATPases contain a rotor that rotates against a catalytic A3B3 or α3β3 stator. The rotor F1-γ or V1-DF is composed of both anti-parallel coiled coil and globular-loop parts. The bacterial flagellar type III export apparatus contains a V1/F1-like ATPase ring structure composed of FliI6 homo-hexamer and FliJ which adopts an anti-parallel coiled coil structure without the globular-loop part. Here we report that FliJ of Salmonella enterica serovar Typhimurium shows a rotor like function in Thermus thermophilus A3B3 based on both biochemical and structural analysis. Single molecular analysis indicates that an anti-parallel coiled-coil structure protein (FliJ structure protein) functions as a rotor in A3B3. A rotary ATPase possessing an F1-γ-like protein generated by fusion of the D and F subunits of V1 rotates, suggesting F1-γ could be the result of a fusion of the genes encoding two separate rotor subunits. Together with sequence comparison among the globular part proteins, the data strongly suggest that the rotor domains of the rotary ATPases and the flagellar export apparatus share a common evolutionary origin. PMID:23724081

  20. Defective flagellar assembly and length regulation in LF3 null mutants in Chlamydomonas

    PubMed Central

    Tam, Lai-Wa; Dentler, William L.; Lefebvre, Paul A.

    2003-01-01

    Four long-flagella (LF) genes are important for flagellar length control in Chlamydomonas reinhardtii. Here, we characterize two new null lf3 mutants whose phenotypes are different from previously identified lf3 mutants. These null mutants have unequal-length flagella that assemble more slowly than wild-type flagella, though their flagella can also reach abnormally long lengths. Prominent bulges are found at the distal ends of short, long, and regenerating flagella of these mutants. Analysis of the flagella by electron and immunofluorescence microscopy and by Western blots revealed that the bulges contain intraflagellar transport complexes, a defect reported previously (for review see Cole, D.G., 2003. Traffic. 4:435–442) in a subset of mutants defective in intraflagellar transport. We have cloned the wild-type LF3 gene and characterized a hypomorphic mutant allele of LF3. LF3p is a novel protein located predominantly in the cell body. It cosediments with the product of the LF1 gene in sucrose density gradients, indicating that these proteins may form a functional complex to regulate flagellar length and assembly. PMID:14610061

  1. Building a radial spoke: flagellar radial spoke protein 3 (RSP3) is a dimer.

    PubMed

    Wirschell, Maureen; Zhao, Feifei; Yang, Chun; Yang, Pinfen; Diener, Dennis; Gaillard, Anne; Rosenbaum, Joel L; Sale, Winfield S

    2008-03-01

    Radial spokes are critical multisubunit structures required for normal ciliary and eukaryotic flagellar motility. Experimental evidence indicates the radial spokes are mechanochemical transducers that transmit signals from the central pair apparatus to the outer doublet microtubules for local control of dynein activity. Recently, progress has been made in identifying individual components of the radial spoke, yet little is known about how the radial spoke is assembled or how it performs in signal transduction. Here we focus on radial spoke protein 3 (RSP3), a highly conserved AKAP located at the base of the radial spoke stalk and required for radial spoke assembly on the doublet microtubules. Biochemical approaches were taken to further explore the functional role of RSP3 within the radial spoke structure and for control of motility. Chemical crosslinking, native gel electrophoresis, and epitope-tagged RSP3 proteins established that RSP3 forms a dimer. Analysis of truncated RSP3 proteins indicates the dimerization domain coincides with the previously characterized axoneme binding domain in the N-terminus. We propose a model in which each radial spoke structure is built on an RSP3 dimer, and indicating that each radial spoke can potentially localize multiple PKAs or AKAP-binding proteins in position to control dynein activity and flagellar motility. PMID:18157907

  2. Calcium ion-mediated assembly and function of glycosylated flagellar sheath of marine magnetotactic bacterium.

    PubMed

    Lefèvre, Christopher T; Santini, Claire-Lise; Bernadac, Alain; Zhang, Wei-Jia; Li, Ying; Wu, Long-Fei

    2010-12-01

    Flagella of some pathogens or marine microbes are sheathed by an apparent extension of the outer cell membrane. Although flagellar sheath has been reported for almost 60 years, little is known about its function and the mechanism of its assembly. Recently, we have observed a novel type of sheath that encloses a flagellar bundle, instead of a single flagellum, in a marine magnetotactic bacterium MO-1. Here, we reported isolation and characterization of the sheath which can be described as a six-start, right-handed helical tubular structure with a diameter of about 100 nm, and a pitch of helix of about 260 nm. By proteomic, microscopic and immunolabelling analyses, we showed that the sheath of MO-1 consists of glycoprotein with an apparent molecular mass > 350 kDa. This protein, named sheath-associated protein (Sap), shows homology with bacterial adhesins and eukaryotic calcium-dependent adherent proteins (cadherin). Most importantly, we showed that calcium ions mediate the assembly of the tubular-shaped sheath and disintegration of the sheath was deleterious for smooth swimming of MO-1 cells. The disintegrated sheath was efficiently reconstituted in vitro by adding calcium ions. Altogether, these results demonstrate a novel bacterial Ca(2+) -dependent surface architecture, which is essential for bacterial swimming. PMID:21091512

  3. 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

  4. 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

  5. Serotyping of Serratia marcescens: current status of seven recently described flagellar (H) antigens.

    PubMed

    Traub, W H; Fukushima, P I

    1979-07-01

    The slightly revised, current scheme of 20 flagellar (H) antigens of Serratia marcesens was examined. The seven new H antigens were demonstrated to be antigenically distinct as determined with Le Minor's H-immobilization test. The H-immobilization antibodies of rabbit anti-H immune sera proved resistant to treatment with 2-mercaptoethanol and dithiothreitol, respectively. On the other hand, dual absorptions of rabbit anti-H immune sera with killed cells of Staphylococcus aureus strain Cowan I, i.e., protein A, failed to reduce significantly H-immobilization titers of rabbit sera, although human immunoglobulins G and M were bound by protein A. It was tentatively concluded that the 2-mercaptoethanol- and dithiothreitol-refractory H-immobilizing rabbit antibodies belonged to the immunoglublin M class. H-antigen (phase) variation was not demonstrable in several extramural, clinical isolates of S. marcescens for which this phenomenon had been claimed. Rather, four of these six isolates were found to consist of cell populations of two distinct serotypes, as also borne out by bacteriocin typing; the flagellar H-antigens of the remaining two isolates were stable, with minor, hterologous H-antigen cross-reactivity.

  6. Flagellar localization of a novel isoform of myosin, myosin XXI, in Leishmania.

    PubMed

    Katta, Santharam S; Sahasrabuddhe, Amogh A; Gupta, Chhitar M

    2009-04-01

    Leishmania major genome analysis revealed the presence of putative genes corresponding to two myosins, which have been designated to class IB and a novel class, class XXI, specifically present in kinetoplastids. To characterize these myosin homologs in Leishmania, we have cloned and over-expressed the full-length myosin XXI gene and variable region of myosin IB gene in bacteria, purified the corresponding proteins, and then used the affinity purified anti-sera to analyze the expression and intracellular distribution of these proteins. Whereas myosin XXI was expressed in both the promastigote and amastigote stages, no expression of myosin IB could be detected in any of the two stages of these parasites. Further, myosin XXI expression was more predominant in the promastigote stage where it was preferentially localized in the proximal region of the flagellum. The observed flagellar localization was not dependent on the myosin head region or actin but was exclusively determined by the myosin tail region, as judged by over-expressing GFP conjugates of full-length myosin XXI, its head domain and its tail domain separately in Leishmania. Furthermore, immunofluorescence and immuno-gold electron microscopy analyses revealed that this protein was partly associated with paraflagellar rod proteins but not with tubulins in the flagellar axoneme. Our results, for the first time, report the expression and detailed analysis of cellular localization of a novel class of myosin, myosin XXI in trypanosomatids. PMID:19121339

  7. Metachronal waves in the flagellar beating of Volvox and their hydrodynamic origin.

    PubMed

    Brumley, Douglas R; Polin, Marco; Pedley, Timothy J; Goldstein, Raymond E

    2015-07-01

    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 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. PMID:26040592

  8. 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

  9. Differentiation of the major flagellar antigens of Pseudomonas aeruginosa by the slide coagglutination technique.

    PubMed

    Ansorg, R A; Knoche, M E; Spies, A F; Kraus, C J

    1984-07-01

    Antisera against the two major flagellar antigens of Pseudomonas aeruginosa were obtained by immunization of rabbits with isolated flagella and absorption of contaminating antisomatic antibodies. In the conventional slide agglutination test, the pure H antisera did not agglutinate the flagellated cells of the homologous strains. The addition of protein A-bearing staphylococci to H antiserum and homologous flagellated cells, the so-called slide coagglutination, results in a rapid development of flaky clumps. H coagglutination tests of reference strains, which formerly have been H typed by long-term tube agglutination and by the indirect fluorescent-antibody technique, yielded exactly the same subdivision of the strains in H type a and H type b as the more laborious and time-consuming methods. O grouping and H typing of 181 isolates from clinical specimens revealed a free combination of the somatic and flagellar antigens. 25 OH serovars were found. The simple and rapid coagglutination technique can promote the serovar determination of P. aeruginosa, particularly for the purpose of hospital infection control. PMID:6430957

  10. 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

  11. NgUNC-119, Naegleria homologue of UNC-119, localizes to the flagellar rootlet.

    PubMed

    Chung, Sunglan; Kang, Seungmin; Paik, Soonyoung; Lee, Joohun

    2007-03-01

    The UNC-119 family of proteins is ubiquitous in animals. The expression of UNC-119 is prominent in neural tissues including photoreceptor cells. Homologues of UNC-119 are also found in ciliated (or flagellated) single-celled organisms; however, the cellular distribution of this protein in protists is unknown. We cloned and characterized a homologue of unc-119 from the ameboflagellate Naegleria gruberi (Ngunc-119) and identified the cellular distribution of the protein. The Ngunc-119 open reading frame contained 570 nucleotides encoding a protein of 189 amino acids with a predicted molecular weight of 22.1 kDa, which is similar to that of Paramecium UNC-119 and Trypanosoma UNC-119. These three proteins are 46-48% identical in their amino acid sequences. The smaller NgUNC-119 corresponds to the conserved C-terminal 3/4 of the UNC-119 from multi-cellular organisms. The amino acid sequence of NgUNC-119 is 43-50% identical to that of the conserved C-terminal regions. NgUNC-119 was not found in growing amoebae but accumulated rapidly after the initiation of differentiation into flagellates. Indirect immunofluorescence staining of differentiating N. gruberi showed that NgUNC-119 begins to concentrate at a spot near the nucleus of differentiating cells and then elongates into a filamentous structure. Purification and indirect immunofluorescence staining of the Naegleria flagellar rootlet suggested that NgUNC-119 is a component of the flagellar rootlet.

  12. Metachronal waves in the flagellar beating of Volvox and their hydrodynamic origin.

    PubMed

    Brumley, Douglas R; Polin, Marco; Pedley, Timothy J; Goldstein, Raymond E

    2015-07-01

    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 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.

  13. E. coli enteritis

    MedlinePlus

    Traveler's diarrhea - E. coli ; Food poisoning - E. coli ; E. coli diarrhea; Hamburger disease ... properly reheated Fish or oysters Raw fruits or vegetables that have not been washed well Raw vegetable ...

  14. Failed Escape: Solid Surfaces Prevent Tumbling of Escherichia coli

    NASA Astrophysics Data System (ADS)

    Molaei, Mehdi; Barry, Michael; Stocker, Roman; Sheng, Jian

    2014-08-01

    Understanding how bacteria move close to surfaces is crucial for a broad range of microbial processes including biofilm formation, bacterial dispersion, and pathogenic infections. We used digital holographic microscopy to capture a large number (>103) of three-dimensional Escherichia coli trajectories near and far from a surface. We found that within 20 μm from a surface tumbles are suppressed by 50% and reorientations are largely confined to surface-parallel directions, preventing escape of bacteria from the near-surface region. A hydrodynamic model indicates that the tumble suppression is likely due to a surface-induced reduction in the hydrodynamic force responsible for the flagellar unbundling that causes tumbling. These findings imply that tumbling does not provide an effective means to escape trapping near surfaces.

  15. Seroprevalence in Chickens against Campylobacter jejuni Flagellar Capping Protein (FliD) in Selected Areas of the U.S.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Campylobacter jejuni, a Gram-negative rod, is a zoonotic pathogen associated with human acute bacterial gastroenteritis. Poultry products are regarded as a major source for human infection with this microorganism. We have demonstrated that the flagellar capping protein (FliD) of C. jejuni is highl...

  16. 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.

  17. The Structure of the Salmonella typhimurium Type III Secretion System Needle Shows Divergence from the Flagellar System

    PubMed Central

    Galkin, Vitold E.; Schmied, Wolfgang H.; Schraidt, Oliver; Marlovits, Thomas C.; Egelman, Edward H.

    2010-01-01

    The Type III Secretion System (T3SS) is essential for the infectivity of many pathogenic Gram-negative bacteria. The T3SS contains proteins that form a channel in the inner and outer bacterial membranes, as well as an extracellular needle that is used for transporting and injecting effector proteins into a host cell. The homology between the T3SS and the bacterial flagellar system has been firmly established, based upon both sequence similarities between respective proteins in the two systems and the structural homology of the higher-order assemblies. It has previously been shown that the Shigella flexneri needle has a helical symmetry of ~ 5.6 subunits per turn, which is quite similar to that of the most intensively studied flagellar filament, from Salmonella typhimurium, which has ~ 5.5 subunits per turn. We now show that the S. typhimurium needle, expected by homology arguments to be more similar to the S. typhimurium flagellar filament than is the needle from Shigella, actually has ~ 6.3 subunits per turn. It is not currently understood how host cell contact, made at the tip of the needle, is communicated to the secretory system at the base. In contrast to the S. typhimurium flagellar filament, which shows a nearly crystalline order, the S. typhimurium needle has a highly variable symmetry, which could be used to transmit information about host cell contact. PMID:20060835

  18. Flagellar Kinematics and Swimming Behavior of Algal Cells in Viscoelastic Fluids

    NASA Astrophysics Data System (ADS)

    Arratia, Paulo; Yang, Jing; Gollub, Jerry

    2013-11-01

    The motility behavior of microorganisms can be significantly affected by the rheology of their fluidic environment. In this talk, we experimentally investigate the effects of fluid elasticity on both the flagella kinematics and swimming dynamics of the microscopic alga Chlamydomonas reinhardtii. We find that the flagellar beating frequency and wave speed are both enhanced by fluid elasticity. Interestingly, the swimming speeds during the alga power and recovery strokes are enhanced by fluid elasticity for De>1. Despite such enhancements, however, the alga net forward speed is hindered by fluid elasticity by as much as 30% compared to Newtonian fluids of similar shear viscosities. The motility enhancements could be explained by the mechanism of stress accumulation in the viscoelastic fluid. This work was supported by the National Science Foundation - DMR-1104705.

  19. 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

  20. From Organelle to Protein Gel: A 6-Wk Laboratory Project on Flagellar Proteins

    PubMed Central

    Graziano, Mary R.

    2006-01-01

    Research suggests that undergraduate students learn more from lab experiences that involve longer-term projects. We have developed a one-semester laboratory sequence aimed at sophomore-level undergraduates. In designing this curriculum, we focused on several educational objectives: 1) giving students a feel for the scientific research process, 2) introducing them to commonly used lab techniques, and 3) building skills in both data analysis and scientific writing. Over the course of the semester, students carry out two project-based lab experiences and write two substantial lab reports modeled on primary literature. Student assessment data indicate that this lab curriculum achieved these objectives. This article describes the first of these projects, which uses the biflagellate alga Chlamydomonas reinhardtii to introduce students to the study of flagellar motility, protein synthesis, microtubule polymerization, organelle assembly, and protein isolation and characterization. PMID:17012215

  1. Ciliary and flagellar structure and function--their regulations by posttranslational modifications of axonemal tubulin.

    PubMed

    Konno, Alu; Setou, Mitsutoshi; Ikegami, Koji

    2012-01-01

    Eukaryotic cilia and flagella are evolutionarily conserved microtubule-based organelles protruding from the cell surface. They perform dynein-driven beating which contributes to cell locomotion or flow generation. They also play important roles in sensing as cellular antennae, which allows cells to respond to various external stimuli. The main components of cilia and flagella, α- and β-tubulins, are known to undergo various posttranslational modifications (PTMs), including phosphorylation, palmitoylation, tyrosination/detyrosination, Δ2 modification, acetylation, glutamylation, and glycylation. Recent identification of tubulin-modifying enzymes, especially tubulin tyrosine ligase-like proteins which perform tubulin glutamylation and glycylation, has demonstrated the importance of tubulin modifications for the assembly and functions of cilia and flagella. In this chapter, we review recent work on PTMs of ciliary and flagellar tubulins in conjunction with discussing the basic knowledge. PMID:22364873

  2. Ciliary and flagellar structure and function--their regulations by posttranslational modifications of axonemal tubulin.

    PubMed

    Konno, Alu; Setou, Mitsutoshi; Ikegami, Koji

    2012-01-01

    Eukaryotic cilia and flagella are evolutionarily conserved microtubule-based organelles protruding from the cell surface. They perform dynein-driven beating which contributes to cell locomotion or flow generation. They also play important roles in sensing as cellular antennae, which allows cells to respond to various external stimuli. The main components of cilia and flagella, α- and β-tubulins, are known to undergo various posttranslational modifications (PTMs), including phosphorylation, palmitoylation, tyrosination/detyrosination, Δ2 modification, acetylation, glutamylation, and glycylation. Recent identification of tubulin-modifying enzymes, especially tubulin tyrosine ligase-like proteins which perform tubulin glutamylation and glycylation, has demonstrated the importance of tubulin modifications for the assembly and functions of cilia and flagella. In this chapter, we review recent work on PTMs of ciliary and flagellar tubulins in conjunction with discussing the basic knowledge.

  3. Restriction fragment length polymorphism of the periplasmic flagellar flaA1 gene of Serpulina species.

    PubMed Central

    Fisher, L N; Mathiesen, M R; Duhamel, G E

    1997-01-01

    Forty-one reference and field isolates of intestinal spirochetes representing Serpulina hyodysenteriae, Serpulina innocens, Serpulina pilosicoli, Brachyspira aalborgi, and nonclassified weakly beta-hemolytic intestinal spirochetes were compared by restriction fragment length polymorphism (RFLP) of the periplasmic flagellar (PF) flaA1 gene. Six genetically distinct groups (I through VI), each with a unique RFLP fingerprint pattern, were identified by Southern blotting analysis of EcoRV chromosomal DNA digests with a PCR-amplified digoxigenin-labeled 1-kb fragment of the S. hyodysenteriae isolate B78 PF flaA1 gene. The RFLP fingerprint patterns corresponded to known DNA homology differences between Serpulina species and to provisionally designated species described previously by using phenotypic and genotypic classification schemes. RFLP fingerprinting of the PF flaA1 gene provides a relatively simple genotypic method for identification of intestinal spirochetes without the use of radioisotopes. PMID:9384289

  4. Single-molecule imaging of electroporated dye-labelled CheY in live Escherichia coli

    PubMed Central

    Di Paolo, Diana; Afanzar, Oshri; Armitage, Judith P.; Berry, Richard M.

    2016-01-01

    For the past two decades, the use of genetically fused fluorescent proteins (FPs) has greatly contributed to the study of chemotactic signalling in Escherichia coli including the activation of the response regulator protein CheY and its interaction with the flagellar motor. However, this approach suffers from a number of limitations, both biological and biophysical: for example, not all fusions are fully functional when fused to a bulky FP, which can have a similar molecular weight to its fused counterpart; they may interfere with the native interactions of the protein and the chromophores of FPs have low brightness and photostability and fast photobleaching rates. A recently developed technique for the electroporation of fluorescently labelled proteins in live bacteria has enabled us to bypass these limitations and study the in vivo behaviour of CheY at the single-molecule level. Here we show that purified CheY proteins labelled with organic dyes can be internalized into E. coli cells in controllable concentrations and imaged with video fluorescence microscopy. The use of this approach is illustrated by showing single CheY molecules diffusing within cells and interacting with the sensory clusters and the flagellar motors in real time. This article is part of the themed issue ‘The new bacteriology’. PMID:27672145

  5. Single-molecule imaging of electroporated dye-labelled CheY in live Escherichia coli.

    PubMed

    Di Paolo, Diana; Afanzar, Oshri; Armitage, Judith P; Berry, Richard M

    2016-11-01

    For the past two decades, the use of genetically fused fluorescent proteins (FPs) has greatly contributed to the study of chemotactic signalling in Escherichia coli including the activation of the response regulator protein CheY and its interaction with the flagellar motor. However, this approach suffers from a number of limitations, both biological and biophysical: for example, not all fusions are fully functional when fused to a bulky FP, which can have a similar molecular weight to its fused counterpart; they may interfere with the native interactions of the protein and the chromophores of FPs have low brightness and photostability and fast photobleaching rates. A recently developed technique for the electroporation of fluorescently labelled proteins in live bacteria has enabled us to bypass these limitations and study the in vivo behaviour of CheY at the single-molecule level. Here we show that purified CheY proteins labelled with organic dyes can be internalized into E. coli cells in controllable concentrations and imaged with video fluorescence microscopy. The use of this approach is illustrated by showing single CheY molecules diffusing within cells and interacting with the sensory clusters and the flagellar motors in real time.This article is part of the themed issue 'The new bacteriology'. PMID:27672145

  6. A simple mapping between cell swimming behavior and single-motor state in multi-flagellated E. coli

    NASA Astrophysics Data System (ADS)

    Mears, Patrick; Koirala, Santosh; Rao, Christopher; Golding, Ido; Chemla, Yann

    2014-03-01

    We present new data that resolve a long-standing question on bacterial motility: How does the cell's swimming behavior depend on the number and state of the flagella that propel it? Addressing this question brings us closer to a full understanding of bacterial chemotaxis, arguably still our best paradigm for the way cells modulate their behavior based on signals from the environment. This new is enabled by technical innovation: we combine optical traps, fluorescence microscopy, and microfluidics to simultaneously track the swimming behavior and flagellar rotation state of individual, immobilized E. coli cells. We reveal a simple mathematical relationship between the number of flagella on the cell, their rotational bias, and the resulting probability of tumbling. Importantly, inter-flagella correlations result in E. colibehaving as if they possess a smaller number of effectively independent flagella than the actual number of flagella. Data from a chemotaxis mutant and stochastic modeling of the network suggest that temporal fluctuations of the key regulator CheY-P are the source of the observed flagellar correlations. A consequence of inter-flagellar correlations is that a cell's run/tumble behavior is only weakly dependent on number of flagella. The work was supported primarily by NSF Grant 082265, Physics Frontiers Center: Center for the Physics of Living Cells.

  7. Flagellar hydrodynamics. A comparison between resistive-force theory and slender-body theory.

    PubMed

    Johnson, R E; Brokaw, C J

    1979-01-01

    This paper investigates the accuracy of the resistive-force theory (Gray and Hancock method) which is commonly used for hydrodynamic analysis of swimming flagella. We made a comparison between the forces, bending moments, and shear moments calculated by resistive-force theory and by the more accurate slender-body theory for large-amplitude, planar wave forms computed for a flagellar model. By making an upward empirical adjustment, by about 35%, of the classical drag coefficient values used in the resistive-force theory calculations, we obtained good agreement between the distributions of the forces and moments along the length of the flagellum predicted by the two methods when the flagellum has no cell body attached. After this adjustment, we found the rate of energy expenditure calculated by the two methods for the few typical test cases to be almost identical. The resistive-force theory is thus completely satisfactory for use in analysis of mechanisms for the control of flagellar bending, at the current level of sophistication of this analysis. We also examined the effects of the presence of a cell body attached to one end of the flagellum, which modifies the flow field experienced by the flagellum. This interaction, which is not considered in resistive-force theory, is probably insignificant for small cell bodies, such as the heads of simple spermatozoa, but for larger cell bodies, or cell bodies that have large-amplitude motions transverse to the swimming direction, use of slender-body theory is required for accurate analysis. PMID:262381

  8. Evidence for loss of a partial flagellar glycolytic pathway during trypanosomatid evolution.

    PubMed

    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

  9. Flagellar membrane fusion and protein exchange in trypanosomes; a new form of cell-cell communication?

    PubMed Central

    Imhof, Simon; Fragoso, Cristina; Hemphill, Andrew; von Schubert, Conrad; Li, Dong; Legant, Wesley; Betzig, Eric; Roditi, Isabel

    2016-01-01

    Diverse structures facilitate direct exchange of proteins between cells, including plasmadesmata in plants and tunnelling nanotubes in bacteria and higher eukaryotes.  Here we describe a new mechanism of protein transfer, flagellar membrane fusion, in the unicellular parasite Trypanosoma brucei. When fluorescently tagged trypanosomes were co-cultured, a small proportion of double-positive cells were observed. The formation of double-positive cells was dependent on the presence of extracellular calcium and was enhanced by placing cells in medium supplemented with fresh bovine serum. Time-lapse microscopy revealed that double-positive cells arose by bidirectional protein exchange in the absence of nuclear transfer.  Furthermore, super-resolution microscopy showed that this process occurred in ≤1 minute, the limit of temporal resolution in these experiments. Both cytoplasmic and membrane proteins could be transferred provided they gained access to the flagellum. Intriguingly, a component of the RNAi machinery (Argonaute) was able to move between cells, raising the possibility that small interfering RNAs are transported as cargo. Transmission electron microscopy showed that shared flagella contained two axonemes and two paraflagellar rods bounded by a single membrane. In some cases flagellar fusion was partial and interactions between cells were transient. In other cases fusion occurred along the entire length of the flagellum, was stable for several hours and might be irreversible. Fusion did not appear to be deleterious for cell function: paired cells were motile and could give rise to progeny while fused. The motile flagella of unicellular organisms are related to the sensory cilia of higher eukaryotes, raising the possibility that protein transfer between cells via cilia or flagella occurs more widely in nature. PMID:27239276

  10. BILBO1 Is a Scaffold Protein of the Flagellar Pocket Collar in the Pathogen Trypanosoma brucei

    PubMed Central

    Florimond, Célia; Sahin, Annelise; Vidilaseris, Keni; Dong, Gang; Landrein, Nicolas; Dacheux, Denis; Albisetti, Anna; Byard, Edward H.; Bonhivers, Mélanie; Robinson, Derrick R.

    2015-01-01

    The flagellar pocket (FP) of the pathogen Trypanosoma brucei is an important single copy structure that is formed by the invagination of the pellicular membrane. It is the unique site of endo- and exocytosis and is required for parasite pathogenicity. The FP consists of distinct structural sub-domains with the least explored being the annulus/horseshoe shaped flagellar pocket collar (FPC). To date the only known component of the FPC is the protein BILBO1, a cytoskeleton protein that has a N-terminus that contains an ubiquitin-like fold, two EF-hand domains, plus a large C-terminal coiled-coil domain. BILBO1 has been shown to bind calcium, but in this work we demonstrate that mutating either or both calcium-binding domains prevents calcium binding. The expression of deletion or mutated forms of BILBO1 in trypanosomes and mammalian cells demonstrate that the coiled-coil domain is necessary and sufficient for the formation of BILBO1 polymers. This is supported by Yeast two-hybrid analysis. Expression of full-length BILBO1 in mammalian cells induces the formation of linear polymers with comma and globular shaped termini, whereas mutation of the canonical calcium-binding domain resulted in the formation of helical polymers and mutation in both EF-hand domains prevented the formation of linear polymers. We also demonstrate that in T. brucei the coiled-coil domain is able to target BILBO1 to the FPC and to form polymers whilst the EF-hand domains influence polymers shape. This data indicates that BILBO1 has intrinsic polymer forming properties and that binding calcium can modulate the form of these polymers. We discuss whether these properties can influence the formation of the FPC. PMID:25822645

  11. Evidence for loss of a partial flagellar glycolytic pathway during trypanosomatid evolution.

    PubMed

    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.

  12. 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

  13. Flagellar membrane fusion and protein exchange in trypanosomes; a new form of cell-cell communication?

    PubMed

    Imhof, Simon; Fragoso, Cristina; Hemphill, Andrew; von Schubert, Conrad; Li, Dong; Legant, Wesley; Betzig, Eric; Roditi, Isabel

    2016-01-01

    Diverse structures facilitate direct exchange of proteins between cells, including plasmadesmata in plants and tunnelling nanotubes in bacteria and higher eukaryotes.  Here we describe a new mechanism of protein transfer, flagellar membrane fusion, in the unicellular parasite Trypanosoma brucei. When fluorescently tagged trypanosomes were co-cultured, a small proportion of double-positive cells were observed. The formation of double-positive cells was dependent on the presence of extracellular calcium and was enhanced by placing cells in medium supplemented with fresh bovine serum. Time-lapse microscopy revealed that double-positive cells arose by bidirectional protein exchange in the absence of nuclear transfer.  Furthermore, super-resolution microscopy showed that this process occurred in ≤1 minute, the limit of temporal resolution in these experiments. Both cytoplasmic and membrane proteins could be transferred provided they gained access to the flagellum. Intriguingly, a component of the RNAi machinery (Argonaute) was able to move between cells, raising the possibility that small interfering RNAs are transported as cargo. Transmission electron microscopy showed that shared flagella contained two axonemes and two paraflagellar rods bounded by a single membrane. In some cases flagellar fusion was partial and interactions between cells were transient. In other cases fusion occurred along the entire length of the flagellum, was stable for several hours and might be irreversible. Fusion did not appear to be deleterious for cell function: paired cells were motile and could give rise to progeny while fused. The motile flagella of unicellular organisms are related to the sensory cilia of higher eukaryotes, raising the possibility that protein transfer between cells via cilia or flagella occurs more widely in nature.

  14. Structure of the Chlamydomonas agglutinin and related flagellar surface proteins in vitro and in situ

    PubMed Central

    1985-01-01

    Using the quick-freeze, deep-etch technique, we compare the structure of the cane-shaped plus and minus sexual agglutinin molecules purified from gametes of Chlamydomonas reinhardi. We also describe the structure of three additional gamete-specific fibrillar molecules, called short canes, loops, and crescents, which are structurally related to the agglutinins. Four non-agglutinating mutant strains are found to produce the three latter fibrils but not canes, supporting our identification of the cane-shaped molecule as the agglutinin. The heads of the plus and minus canes are shown to differ in morphology. Moreover, two treatments that inactivate the plus agglutinin in vitro--thermolysin digestion and disulfide reduction/alkylation--bring about detectable structural changes only in the head domain of the cane, suggesting that the head may play an indispensible role in affecting gametic recognition/adhesion. We also present quick-freeze, deep-etch images of the flagellar surfaces of gametic, vegetative, and mutant cells of Chlamydomonas reinhardi. The gametic flagella are shown to carry the canes, short canes, loops, and crescents present in in vitro preparations. The cane and crescent proteins self-associate on the flagellar surface into stout fibers of uniform caliber, and they align along the longitudinal axis of the flagellum. The short canes and loops co-purify with flagella but, in the presence of mica, dissociate so that they lie to the sides of the flagella. The agglutinin canes of both mating types are oriented with their hooks at the membrane surface and their heads directed outward, where they are positioned to participate in the initial events of sexual agglutination. PMID:4030899

  15. Single-Cell E. coli Response to an Instantaneously Applied Chemotactic Signal

    PubMed Central

    Sagawa, Takashi; Kikuchi, Yu; Inoue, Yuichi; Takahashi, Hiroto; Muraoka, Takahiro; Kinbara, Kazushi; Ishijima, Akihiko; Fukuoka, Hajime

    2014-01-01

    In response to an attractant or repellant, an Escherichia coli cell controls the rotational direction of its flagellar motor by a chemotaxis system. When an E. coli cell senses an attractant, a reduction in the intracellular concentration of a chemotaxis protein, phosphorylated CheY (CheY-P), induces counterclockwise (CCW) rotation of the flagellar motor, and this cellular response is thought to occur in several hundred milliseconds. Here, to measure the signaling process occurring inside a single E. coli cell, including the recognition of an attractant by a receptor cluster, the inactivation of histidine kinase CheA, and the diffusion of CheY and CheY-P molecules, we applied a serine stimulus by instantaneous photorelease from a caged compound and examined the cellular response at a temporal resolution of several hundred microseconds. We quantified the clockwise (CW) and CCW durations immediately after the photorelease of serine as the response time and the duration of the response, respectively. The results showed that the response time depended on the distance between the receptor and motor, indicating that the decreased CheY-P concentration induced by serine propagates through the cytoplasm from the receptor-kinase cluster toward the motor with a timing that is explained by the diffusion of CheY and CheY-P molecules. The response time included 240 ms for enzymatic reactions in addition to the time required for diffusion of the signaling molecule. The measured response time and duration of the response also revealed that the E. coli cell senses a similar serine concentration regardless of whether the serine concentration is increasing or decreasing. These detailed quantitative findings increase our understanding of the signal transduction process that occurs inside cells during bacterial chemotaxis. PMID:25099812

  16. Diarrheagenic Escherichia coli

    PubMed Central

    Nataro, James P.; Kaper, James B.

    1998-01-01

    Escherichia coli is the predominant nonpathogenic facultative flora of the human intestine. Some E. coli strains, however, have developed the ability to cause disease of the gastrointestinal, urinary, or central nervous system in even the most robust human hosts. Diarrheagenic strains of E. coli can be divided into at least six different categories with corresponding distinct pathogenic schemes. Taken together, these organisms probably represent the most common cause of pediatric diarrhea worldwide. Several distinct clinical syndromes accompany infection with diarrheagenic E. coli categories, including traveler’s diarrhea (enterotoxigenic E. coli), hemorrhagic colitis and hemolytic-uremic syndrome (enterohemorrhagic E. coli), persistent diarrhea (enteroaggregative E. coli), and watery diarrhea of infants (enteropathogenic E. coli). This review discusses the current level of understanding of the pathogenesis of the diarrheagenic E. coli strains and describes how their pathogenic schemes underlie the clinical manifestations, diagnostic approach, and epidemiologic investigation of these important pathogens. PMID:9457432

  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. 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

  19. Tubulin polyglutamylation regulates flagellar motility by controlling a specific inner-arm dynein that interacts with the dynein regulatory complex.

    PubMed

    Kubo, Tomohiro; Yagi, Toshiki; Kamiya, Ritsu

    2012-12-01

    The tpg1 mutant of Chlamydomonas lacks the tubulin polyglutamylase TTLL9 and is deficient in flagellar tubulin polyglutamylation. It exhibits slow swimming, whereas the double mutant with oda2 (a slow-swimming mutant that lacks outer-arm dynein) is completely nonmotile. Thus, tubulin polyglutamylation must be important for the functioning of inner-arm dynein(s). In this study, we show that the tpg1 mutation only slightly affects the motility of mutants that lack dynein "e," one of the seven species of major inner-arm dyneins, whereas it greatly reduces the motility of mutants lacking other inner-arm dynein species. This suggests that dynein e is the main target of motility regulation by tubulin polyglutamylation. Furthermore, the motility of various mutants in the background of the tpg1 mutation raises the possibility that tubulin polyglutamylation also affects the dynein regulatory complex, a dynein e-associated key regulator of flagellar motility, which possibly constitutes the interdoublet (nexin) link. Tubulin polyglutamylation thus may play a central role in the regulation of ciliary and flagellar motility. © 2012 Wiley Periodicals, Inc.

  20. Computer simulation of flagellar movement X: doublet pair splitting and bend propagation modeled using stochastic dynein kinetics.

    PubMed

    Brokaw, Charles J

    2014-04-01

    Experimental observations on cyclic splitting and bending by a flagellar doublet pair are modeled using forces obtained from a model for dynein mechanochemistry, based on ideas introduced by Andrew Huxley and Terrill Hill and extended previously for modeling flagellar movements. The new feature is elastic attachment of dynein to the A doublet, which allows movement perpendicular to the A doublet and provides adhesive force that can strain attached dyneins. This additional strain influences the kinetics of dynein attachment and detachment. Computations using this dynein model demonstrate that very simple and realistic ideas about dynein mechanochemistry are sufficient for explaining the separation and reattachment seen experimentally with flagellar doublet pairs. Additional simulations were performed after adding a "super-adhesion" elasticity. This elastic component is intended to mimic interdoublet connections, normally present in an intact axoneme, that would prevent visible splitting but allow sufficient separation to cause dynein detachment and cessation of shear force generation. This is the situation envisioned by Lindemann's "geometric clutch" hypothesis for control of dynein function in flagella and cilia. The simulations show abrupt disengagement of the "clutch" at one end of a bend, and abrupt reengagement of the "clutch" at the other end of a bend, ensuring that active sliding is only operating where it will cause bend propagation from base to tip.

  1. Monoclonal antibodies for detection of the H7 antigen of Escherichia coli.

    PubMed Central

    He, Y; Keen, J E; Westerman, R B; Littledike, E T; Kwang, J

    1996-01-01

    Two murine monoclonal antibodies (MAbs) (2B7 and 46E9-9) reactive with the H7 flagellar antigen of Escherichia coli were produced and characterized. A total of 217 E. coli strains (48 O157:H7, 4 O157:NM, 23 O157:non-H7, 22 H7:non-O157, and 120 non-O157:nonH7), 17 Salmonella serovars, and 29 other gram-negative bacteria were used to evaluate the reactivities of the two MAbs by indirect enzyme-linked immunosorbent assay (ELISA). Both MAbs reacted strongly with all E. coli strains possessing the H7 antigen and with H23- and H24-positive E. coli strains. Indirect ELISA MAb specificity was confirmed by inhibition ELISA and by Western blotting (immunoblotting), using partially purified flagellins from E. coli O157:H7 and other E. coli strains. On a Western blot, MAb 46E9-9 was more reactive against H7 flagellin of E. coli O157:H7 than against H7 flagellin of E. coli O1:K1:H7. Competition ELISA suggested that MAbs 2B7 and 46E9-9 reacted with closely related H7 epitopes. When the ELISA reactivities of the MAbs and two commercially available polyclonal anti-H7 antisera were compared, both polyclonal antisera and MAbs reacted strongly with E. coli H7 bacteria. However, the polyclonal antisera cross-reacted strongly both with non-H7 E. coli and with many non-E. coli bacteria. The polyclonal antisera also reacted strongly with H23 and H24 E. coli isolates. The data suggest the need to define serotype-specific epitopes among H7, H23, and H24 E. coli flagella. The anti-H7 MAbs described in this report have the potential to serve as high-quality diagnostic reagents, used either alone or in combination with O157-specific MAbs, to identify or detect E. coli O157:H7 in food products or in human and veterinary clinical specimens. PMID:8795222

  2. Insights into the mechanism of ADP action on flagellar motility derived from studies on bull sperm.

    PubMed

    Lesich, Kathleen A; Pelle, Dominic W; Lindemann, Charles B

    2008-07-01

    Adenosine diphosphate (ADP) is known to have interesting effects on flagellar motility. Permeabilized and reactivated bull sperm exhibit a marked reduction in beating frequency and a greatly increased beat amplitude in the presence of 1-4 mM ADP. In this study we examined the force production of sperm reactivated with 0.1 mM ATP with and without 1 mM ADP and found that there is little or no resulting change in the stalling force produced by a bull sperm flagella in response to ADP. Because bull sperm bend to a higher curvature after ADP treatment we explored the possibility that ADP-treated sperm flagella are more flexible. We measured the stiffness of 50 muM sodium vanadate treated bull sperm in the presence of 4 mM ADP, but found no change in the passive flagellar stiffness. When we analyzed the torque that develops in ADP-treated sperm at the point of beat reversal we found that the torque developed by the flagellum is significantly increased. Our torque estimates also allow us to calculate the transverse force (t-force) acting on the flagellum at the point of beat direction reversal. We find that the t-force at the switch-point of the beat is increased significantly in the ADP treated condition, averaging 0.7 +/- 0.29 nN/microm in 0.1 mM ATP and increasing to 2.9 +/- 1.2 nN/microm in 0.1 mM ATP plus 4 mM ADP. This suggests that ADP is exerting its effect on the beat by increasing the tenacity of dynein attachment at the B-subtubule. This could be a direct result of a regulatory effect of ADP on the binding affinity of dynein for the B-subtubule of the outer doublets. This result could also help to explain a number of previous experimental observations, as discussed. PMID:18375503

  3. Insights into the Mechanism of ADP Action on Flagellar Motility Derived from Studies on Bull Sperm

    PubMed Central

    Lesich, Kathleen A.; Pelle, Dominic W.; Lindemann, Charles B.

    2008-01-01

    Adenosine diphosphate (ADP) is known to have interesting effects on flagellar motility. Permeabilized and reactivated bull sperm exhibit a marked reduction in beating frequency and a greatly increased beat amplitude in the presence of 1–4 mM ADP. In this study we examined the force production of sperm reactivated with 0.1 mM ATP with and without 1 mM ADP and found that there is little or no resulting change in the stalling force produced by a bull sperm flagella in response to ADP. Because bull sperm bend to a higher curvature after ADP treatment we explored the possibility that ADP-treated sperm flagella are more flexible. We measured the stiffness of 50 μM sodium vanadate treated bull sperm in the presence of 4 mM ADP, but found no change in the passive flagellar stiffness. When we analyzed the torque that develops in ADP-treated sperm at the point of beat reversal we found that the torque developed by the flagellum is significantly increased. Our torque estimates also allow us to calculate the transverse force (t-force) acting on the flagellum at the point of beat direction reversal. We find that the t-force at the switch-point of the beat is increased significantly in the ADP treated condition, averaging 0.7 ± 0.29 nN/μm in 0.1 mM ATP and increasing to 2.9 ± 1.2 nN/μm in 0.1 mM ATP plus 4 mM ADP. This suggests that ADP is exerting its effect on the beat by increasing the tenacity of dynein attachment at the B-subtubule. This could be a direct result of a regulatory effect of ADP on the binding affinity of dynein for the B-subtubule of the outer doublets. This result could also help to explain a number of previous experimental observations, as discussed. PMID:18375503

  4. New mutations in flagellar motors identified by whole genome sequencing in Chlamydomonas

    PubMed Central

    2013-01-01

    Background The building of a cilium or flagellum requires molecular motors and associated proteins that allow the relocation of proteins from the cell body to the distal end and the return of proteins to the cell body in a process termed intraflagellar transport (IFT). IFT trains are carried out by kinesin and back to the cell body by dynein. Methods We used whole genome sequencing to identify the causative mutations for two temperature-sensitive flagellar assembly mutants in Chlamydomonas and validated the changes using reversion analysis. We examined the effect of these mutations on the localization of IFT81, an IFT complex B protein, the cytoplasmic dynein heavy chain (DHC1b), and the dynein light intermediate chain (D1bLIC). Results The strains, fla18 and fla24, have mutations in kinesin-2 and cytoplasmic dynein, respectively. The fla18 mutation alters the same glutamic acid (E24G) mutated in the fla10-14 allele (E24K). The fla18 strain loses flagella at 32?C more rapidly than the E24K allele but less rapidly than the fla10-1 allele. The fla18 mutant loses its flagella by detachment rather than by shortening. The fla24 mutation falls in cytoplasmic dynein and changes a completely conserved amino acid (L3243P) in an alpha helix in the AAA5 domain. The fla24 mutant loses its flagella by shortening within 6 hours at 32?C. DHC1b protein is reduced by 18-fold and D1bLIC is reduced by 16-fold at 21?C compared to wild-type cells. We identified two pseudorevertants (L3243S and L3243R), which remain flagellated at 32?C. Although fla24 cells assemble full-length flagella at 21?C, IFT81 protein localization is dramatically altered. Instead of localizing at the basal body and along the flagella, IFT81 is concentrated at the proximal end of the flagella. The pseudorevertants show wild-type IFT81 localization at 21?C, but proximal end localization of IFT81 at 32?C. Conclusions The change in the AAA5 domain of the cytoplasmic dynein in fla24 may block the recycling of IFT

  5. Intact Flagellar Motor of Borrelia burgdorferi Revealed by Cryo-Electron Tomography: Evidence for Stator Ring Curvature and Rotor/C-Ring Assembly Flexion▿ †

    PubMed Central

    Liu, Jun; Lin, Tao; Botkin, Douglas J.; McCrum, Erin; Winkler, Hanspeter; Norris, Steven J.

    2009-01-01

    The bacterial flagellar motor is a remarkable nanomachine that provides motility through flagellar rotation. Prior structural studies have revealed the stunning complexity of the purified rotor and C-ring assemblies from flagellar motors. In this study, we used high-throughput cryo-electron tomography and image analysis of intact Borrelia burgdorferi to produce a three-dimensional (3-D) model of the in situ flagellar motor without imposing rotational symmetry. Structural details of B. burgdorferi, including a layer of outer surface proteins, were clearly visible in the resulting 3-D reconstructions. By averaging the 3-D images of ∼1,280 flagellar motors, a ∼3.5-nm-resolution model of the stator and rotor structures was obtained. flgI transposon mutants lacked a torus-shaped structure attached to the flagellar rod, establishing the structural location of the spirochetal P ring. Treatment of intact organisms with the nonionic detergent NP-40 resulted in dissolution of the outermost portion of the motor structure and the C ring, providing insight into the in situ arrangement of the stator and rotor structures. Structural elements associated with the stator followed the curvature of the cytoplasmic membrane. The rotor and the C ring also exhibited angular flexion, resulting in a slight narrowing of both structures in the direction perpendicular to the cell axis. These results indicate an inherent flexibility in the rotor-stator interaction. The FliG switching and energizing component likely provides much of the flexibility needed to maintain the interaction between the curved stator and the relatively symmetrical rotor/C-ring assembly during flagellar rotation. PMID:19429612

  6. YjjQ Represses Transcription of flhDC and Additional Loci in Escherichia coli

    PubMed Central

    Wiebe, Helene; Gürlebeck, Doreen; Groß, Jana; Dreck, Katrin; Pannen, Derk; Ewers, Christa; Wieler, Lothar H.

    2015-01-01

    ABSTRACT The presumptive transcriptional regulator YjjQ has been identified as being virulence associated in avian pathogenic Escherichia coli (APEC). In this work, we characterize YjjQ as transcriptional repressor of the flhDC operon, encoding the master regulator of flagellar synthesis, and of additional loci. The latter include gfc (capsule 4 synthesis), ompC (outer membrane porin C), yfiRNB (regulated c-di-GMP synthesis), and loci of poorly defined function (ybhL and ymiA-yciX). We identify the YjjQ DNA-binding sites at the flhDC and gfc promoters and characterize a DNA-binding sequence motif present at all promoters found to be repressed by YjjQ. At the flhDC promoter, the YjjQ DNA-binding site overlaps the RcsA-RcsB DNA-binding site. RcsA-RcsB likewise represses the flhDC promoter, but the repression by YjjQ and that by RcsA-RcsB are independent of each other. These data suggest that YjjQ is an additional regulator involved in the complex control of flhDC at the level of transcription initiation. Furthermore, we show that YjjQ represses motility of the E. coli K-12 laboratory strain and of uropathogenic E. coli (UPEC) strains CFT073 and 536. Regulation of flhDC, yfiRNB, and additional loci by YjjQ may be features relevant for pathogenicity. IMPORTANCE Escherichia coli is a commensal and pathogenic bacterium causing intra- and extraintestinal infections in humans and farm animals. The pathogenicity of E. coli strains is determined by their particular genome content, which includes essential and associated virulence factors that control the cellular physiology in the host environment. However, the gene pools of commensal and pathogenic E. coli are not clearly differentiated, and the function of virulence-associated loci needs to be characterized. In this study, we characterize the function of yjjQ, encoding a transcription regulator that was identified as being virulence associated in avian pathogenic E. coli (APEC). We characterize YjjQ as transcriptional

  7. LrhA as a new transcriptional key regulator of flagella, motility and chemotaxis genes in Escherichia coli.

    PubMed

    Lehnen, D; Blumer, C; Polen, T; Wackwitz, B; Wendisch, V F; Unden, G

    2002-07-01

    The function of the LysR-type regulator LrhA of Escherichia coli was defined by comparing whole-genome mRNA profiles from wild-type E. coli and an isogenic lrhA mutant on a DNA microarray. In the lrhA mutant, a large number (48) of genes involved in flagellation, motility and chemotaxis showed relative mRNA abundances increased by factors between 3 and 80. When a representative set of five flagellar, motility and chemotaxis genes was tested in lacZ reporter gene fusions, similar factors for derepression were found in the lrhA mutant. In gel retardation experiments, the LrhA protein bound specifically to flhD and lrhA promoter DNA (apparent K(D) approximately 20 nM), whereas the promoters of fliC, fliA and trg were not bound by LrhA. The expression of flhDC (encoding FlhD(2)C(2)) was derepressed by a factor of 3.5 in the lrhA mutant. FlhD(2)C(2) is known as the master regulator for the expression of flagellar and chemotaxis genes. By DNase I footprinting, LrhA binding sites at the flhDC and lrhA promoters were identified. The lrhA gene was under positive autoregulation by LrhA as shown by gel retardation and lrhA expression studies. It is suggested that LrhA is a key regulator controlling the transcription of flagellar, motility and chemotaxis genes by regulating the synthesis and concentration of FlhD(2)C(2). PMID:12123461

  8. A SAS-6-like protein suggests that the Toxoplasma conoid complex evolved from flagellar components.

    PubMed

    de Leon, Jessica Cruz; Scheumann, Nicole; Beatty, Wandy; Beck, Josh R; Tran, Johnson Q; Yau, Candace; Bradley, Peter J; Gull, Keith; Wickstead, Bill; Morrissette, Naomi S

    2013-07-01

    SAS-6 is required for centriole biogenesis in diverse eukaryotes. Here, we describe a novel family of SAS-6-like (SAS6L) proteins that share an N-terminal domain with SAS-6 but lack coiled-coil tails. SAS6L proteins are found in a subset of eukaryotes that contain SAS-6, including diverse protozoa and green algae. In the apicomplexan parasite Toxoplasma gondii, SAS-6 localizes to the centriole but SAS6L is found above the conoid, an enigmatic tubulin-containing structure found at the apex of a subset of alveolate organisms. Loss of SAS6L causes reduced fitness in Toxoplasma. The Trypanosoma brucei homolog of SAS6L localizes to the basal-plate region, the site in the axoneme where the central-pair microtubules are nucleated. When endogenous SAS6L is overexpressed in Toxoplasma tachyzoites or Trypanosoma trypomastigotes, it forms prominent filaments that extend through the cell cytoplasm, indicating that it retains a capacity to form higher-order structures despite lacking a coiled-coil domain. We conclude that although SAS6L proteins share a conserved domain with SAS-6, they are a functionally distinct family that predates the last common ancestor of eukaryotes. Moreover, the distinct localization of the SAS6L protein in Trypanosoma and Toxoplasma adds weight to the hypothesis that the conoid complex evolved from flagellar components.

  9. Stochastic and Deterministic Flagellar Dynamics Provide a Mechanism for Eukaryotic Swimming Reorientation

    NASA Astrophysics Data System (ADS)

    Polin, Marco; Tuval, Idan; Drescher, Knut; Goldstein, Raymond

    2009-03-01

    The biflagellated alga Chlamydomonas reinhardtii is a good model organism to study the origin of flagellar synchronization. Here we employ high-speed imaging to study the beating of the two flagella of Chlamydomonas, and show that a single cell can alternate between two distinct dynamical regimes: asynchronous and synchronous. The asynchronous state is characterized by a large interflagellar frequency difference. In the synchronous state, the flagella beat in phase for lengthy periods, interrupted episodically by an extra beat of either flagellum. The statistics of these events are consistent with a model of hydrodynamically coupled noisy oscillators. Previous observations have suggested that the two flagella have well separated intrinsic beat frequencies, and are synchronized by their mutual coupling. Our analysis shows instead that the synchronized state is incompatible with coupling-induced synchronization of flagella with those intrinsic frequencies. This suggests that the beat frequencies themselves are under the control of the cell. Moreover, high-resolution three-dimensional tracking of swimming cells provides strong evidence that these dynamical states are related to non-phototactic reorientation events in the trajectories, yielding a eukaryotic equivalent of the ``run and tumble'' motion of peritrichously flagellated bacteria.

  10. Investigation of the Swimming and Flagellar Dynamics of Shear guided Motile Alga

    NASA Astrophysics Data System (ADS)

    Chengala, Anwar; Hondzo, Miki; Sheng, Jian

    2011-11-01

    We examine the behavior of force-free swimming cell in a shear flow having spheroidal bodies with two flagella located at the anterior part of the cell that uses breast-like motion for its propulsion. The cell, Dunaliella primolecta, displays a unique behavior of propelling along the local vorticity direction in a linear shear flow. The cell rotation is absent during this display and the flagella beating is observed to be asynchronous. The forces and moments generated by the flagella are estimated numerically. Based on the Resistive Force Theory approach, we attempt to demonstrate that the moments generated by beating flagella and their alignment to flow are necessary to counter-act the vorticity of the flow. In addition, we explore the various mechanisms that cell may use to re-orient while in a shear flow as well as how critical the variations in the flagellar beating pattern are to a cell's swimming dynamics. NSF (No: CBET-0844647), Minnesota Futures Grant.

  11. Flagellar antigen based CI-ELISA for sero-surveillance of surra.

    PubMed

    Ligi, M; Sengupta, P P; Rudramurthy, G R; Rahman, H

    2016-03-30

    Trypanosoma evansi causes a disease known as 'surra' in wide range of domesticated and wild animals including cattle, buffaloes, horses, camels etc. The disease is transmitted through the bites of haematophagous tabanid flies and is characterized by undulating fever, chronic progressive weakness, and hypoglycemia leading to low productivity in animals. In the present study, monoclonal antibodies (MAbs) have been produced (IgG3 sub-type) against purified flagellar (FLA) protein of T. evansi and its immunoreactivity was evaluated by serological tests. MAb and purified protein were then exploited in the development of CI-ELISA and the diagnostic potentiality of the new ELISA test has been evaluated using 1230 sera samples from field animals including cattle, buffaloes, camels, horses and donkeys. The statistical analysis of the data showed optimum combination of sensitivity and specificity at 95.8 and 94.4, respectively. The positive-negative cut off percentage inhibition (PI) value was found to be >55, with a Cohen's Kappa value of 0.83. The study showed that the new assay has potential for application in sero-diagnosis as well as sero-surveillance of surra. PMID:26921034

  12. Coupling between Switching Regulation and Torque Generation in Bacterial Flagellar Motor

    NASA Astrophysics Data System (ADS)

    Bai, Fan; Minamino, Tohru; Wu, Zhanghan; Namba, Keiichi; Xing, Jianhua

    2012-04-01

    The bacterial flagellar motor plays a crucial role in both bacterial locomotion and chemotaxis. Recent experiments reveal that the switching dynamics of the motor depend on the rotation speed of the motor, and thus the motor torque, nonmonotonically. Here we present a unified mathematical model which treats motor torque generation based on experimental torque-speed curves and the torque-dependent switching based on the conformational spread model. The model successfully reproduces the observed switching rate as a function of the rotation speed, and provides a generic physical explanation independent of most details. A stator affects the switching dynamics through two mechanisms: accelerating the conformational flipping rate of individual rotor-switching units, which contributes most when the stator works at a high torque and thus a low speed; and influencing a larger number of rotor-switching units within unit time, whose contribution is the greatest when the motor rotates at a high speed. Consequently, the switching rate shows a maximum at intermediate speed, where the above two mechanisms find an optimal output. The load-switching relation may serve as a mechanism for sensing the physical environment, similar to the chemotaxis mechanism for sensing the chemical environment. It may also coordinate the switch dynamics of motors within the same cell.

  13. Mechanics of the eukaryotic flagellar axoneme: Evidence for structural distortion during bending.

    PubMed

    Lesich, Kathleen A; dePinho, Tania G; Pelle, Dominic W; Lindemann, Charles B

    2016-05-01

    The sliding doublet mechanism is the established explanation that allows us to understand the process of ciliary and flagellar bending. In this study, we apply the principles of the sliding doublet mechanism to analyze the mechanics of the counterbend phenomenon in sea urchin sperm flagella. When a passive, vanadate-treated, flagellum is forced into a bend with a glass microprobe, the portion of the flagellum distal to the probe exhibits a bend of opposite curvature (counterbend) to the imposed bend. This phenomenon was shown to be caused by the induction of inter-doublet shear and is dependent on the presence of an inter-doublet shear resistance. Here we report that in sea urchin flagella there is systematically less shear induced in the distal flagellum than is predicted by the sliding doublet mechanism, if we follow the assumption that the diameter of the flagellum is uniform. To account for the reduced shear that is observed, the likeliest and most direct interpretation is that the portion of the axoneme that is forced to bend undergoes substantial compression of the axoneme in the bending plane. A compression of 30-50 nm would be sufficient to account for the shear reduction from a bend of 2 radians. A compression of this magnitude would require considerable flexibility in the axoneme structure. This would necessitate that the radial spokes and/or the central pair apparatus are easily compressed by transverse stress. © 2016 Wiley Periodicals, Inc. PMID:27001352

  14. A molecular brake, not a clutch, stops the Rhodobacter sphaeroides flagellar motor

    PubMed Central

    Pilizota, Teuta; Brown, Mostyn T.; Leake, Mark C.; Branch, Richard W.; Berry, Richard M.; Armitage, Judith P.

    2009-01-01

    Many bacterial species swim by employing ion-driven molecular motors that power the rotation of helical filaments. Signals are transmitted to the motor from the external environment via the chemotaxis pathway. In bidirectional motors, the binding of phosphorylated CheY (CheY-P) to the motor is presumed to instigate conformational changes that result in a different rotor-stator interface, resulting in rotation in the alternative direction. Controlling when this switch occurs enables bacteria to accumulate in areas favorable for their survival. Unlike most species that swim with bidirectional motors, Rhodobacter sphaeroides employs a single stop-start flagellar motor. Here, we asked, how does the binding of CheY-P stop the motor in R. sphaeroides—using a clutch or a brake? By applying external force with viscous flow or optical tweezers, we show that the R. sphaeroides motor is stopped using a brake. The motor stops at 27–28 discrete angles, locked in place by a relatively high torque, approximately 2–3 times its stall torque. PMID:19571004

  15. Mechanism and kinetics of a sodium-driven bacterial flagellar motor

    PubMed Central

    Lo, Chien-Jung; Sowa, Yoshiyuki; Pilizota, Teuta; Berry, Richard M.

    2013-01-01

    The bacterial flagellar motor is a large rotary molecular machine that propels swimming bacteria, powered by a transmembrane electrochemical potential difference. It consists of an ∼50-nm rotor and up to ∼10 independent stators anchored to the cell wall. We measured torque–speed relationships of single-stator motors under 25 different combinations of electrical and chemical potential. All 25 torque–speed curves had the same concave-down shape as fully energized wild-type motors, and each stator passes at least 37 ± 2 ions per revolution. We used the results to explore the 25-dimensional parameter space of generalized kinetic models for the motor mechanism, finding 830 parameter sets consistent with the data. Analysis of these sets showed that the motor mechanism has a “powerstroke” in either ion binding or transit; ion transit is channel-like rather than carrier-like; and the rate-limiting step in the motor cycle is ion binding at low concentration, ion transit, or release at high concentration. PMID:23788659

  16. Microtubule-severing proteins are involved in flagellar length control and mitosis in Trypanosomatids.

    PubMed

    Casanova, Magali; Crobu, Lucien; Blaineau, Christine; Bourgeois, Nathalie; Bastien, Patrick; Pagès, Michel

    2009-03-01

    Microtubules are key players in the biology of Trypanosomatid parasites, not only as classical components of the mitotic spindle, microtubule-organizing centres and flagellum but also as the essential constituent of the cytoskeleton. Their length dynamics are regulated by, among others, microtubule-severing proteins. Four and six genes encoding microtubule-severing proteins can be found bioinformatically in the Leishmania major and Trypanosoma brucei genome respectively. We investigated all these proteins in these organisms, which include the katanin, katanin-like, spastin and fidgetin, and looked at their subcellular localization as well as their putative function by examining 'loss-of-function' phenotypes. The katanin-like KAT60b was found implicated in flagellar length reduction, but not in its size increase, while the katanin p80 subunit appeared clearly involved in cytokinesis. Fidgetin and spastin homologues were both localized in the nucleus: the first as a discrete and variable number of dots during most of the cell cycle, redistributing to the spindle and midbody during mitosis; the second concentrated as < or = 5 perinucleolar punctuations, similar to the electron-dense plaques identified in T. brucei, which were assimilated to kinetochores. This first study of microtubule-severing proteins in 'divergent' eukaryotes gives further insight into the multiple functions of these proteins identified in the hitherto studied models. PMID:19183280

  17. Bacterial flagellar motility on hydrated rough surfaces controlled by aqueous film thickness and connectedness.

    PubMed

    Tecon, Robin; Or, Dani

    2016-01-01

    Recent studies have shown that rates of bacterial dispersion in soils are controlled by hydration conditions that define size and connectivity of the retained aqueous phase. Despite the ecological implications of such constraints, microscale observations of this phenomenon remain scarce. Here, we quantified aqueous film characteristics and bacterial flagellated motility in response to systematic variations in microhydrological conditions on porous ceramic surfaces that mimic unsaturated soils. We directly measured aqueous film thickness and documented its microscale heterogeneity. Flagellar motility was controlled by surface hydration conditions, as cell velocity decreased and dispersion practically ceased at water potentials exceeding -2 kPa (resulting in thinner and disconnected liquid films). The fragmentation of aquatic habitats was delineated indirectly through bacterial dispersal distances within connected aqueous clusters. We documented bacterial dispersal radii ranging from 100 to 10 μm as the water potential varied from 0 to -7 kPa, respectively. The observed decrease of flagellated velocity and dispersal ranges at lower matric potentials were in good agreement with mechanistic model predictions. Hydration-restricted habitats thus play significant role in bacterial motility and dispersal, which has potentially important impact on soil microbial ecology and diversity. PMID:26757676

  18. Mechanics of the eukaryotic flagellar axoneme: Evidence for structural distortion during bending.

    PubMed

    Lesich, Kathleen A; dePinho, Tania G; Pelle, Dominic W; Lindemann, Charles B

    2016-05-01

    The sliding doublet mechanism is the established explanation that allows us to understand the process of ciliary and flagellar bending. In this study, we apply the principles of the sliding doublet mechanism to analyze the mechanics of the counterbend phenomenon in sea urchin sperm flagella. When a passive, vanadate-treated, flagellum is forced into a bend with a glass microprobe, the portion of the flagellum distal to the probe exhibits a bend of opposite curvature (counterbend) to the imposed bend. This phenomenon was shown to be caused by the induction of inter-doublet shear and is dependent on the presence of an inter-doublet shear resistance. Here we report that in sea urchin flagella there is systematically less shear induced in the distal flagellum than is predicted by the sliding doublet mechanism, if we follow the assumption that the diameter of the flagellum is uniform. To account for the reduced shear that is observed, the likeliest and most direct interpretation is that the portion of the axoneme that is forced to bend undergoes substantial compression of the axoneme in the bending plane. A compression of 30-50 nm would be sufficient to account for the shear reduction from a bend of 2 radians. A compression of this magnitude would require considerable flexibility in the axoneme structure. This would necessitate that the radial spokes and/or the central pair apparatus are easily compressed by transverse stress. © 2016 Wiley Periodicals, Inc.

  19. Bacterial flagellar motility on hydrated rough surfaces controlled by aqueous film thickness and connectedness

    PubMed Central

    Tecon, Robin; Or, Dani

    2016-01-01

    Recent studies have shown that rates of bacterial dispersion in soils are controlled by hydration conditions that define size and connectivity of the retained aqueous phase. Despite the ecological implications of such constraints, microscale observations of this phenomenon remain scarce. Here, we quantified aqueous film characteristics and bacterial flagellated motility in response to systematic variations in microhydrological conditions on porous ceramic surfaces that mimic unsaturated soils. We directly measured aqueous film thickness and documented its microscale heterogeneity. Flagellar motility was controlled by surface hydration conditions, as cell velocity decreased and dispersion practically ceased at water potentials exceeding –2 kPa (resulting in thinner and disconnected liquid films). The fragmentation of aquatic habitats was delineated indirectly through bacterial dispersal distances within connected aqueous clusters. We documented bacterial dispersal radii ranging from 100 to 10 μm as the water potential varied from 0 to –7 kPa, respectively. The observed decrease of flagellated velocity and dispersal ranges at lower matric potentials were in good agreement with mechanistic model predictions. Hydration-restricted habitats thus play significant role in bacterial motility and dispersal, which has potentially important impact on soil microbial ecology and diversity. PMID:26757676

  20. Bacterial Flagellar Motor Switch in Response to CheY-P Regulation and Motor Structural Alterations.

    PubMed

    Ma, Qi; Sowa, Yoshiyuki; Baker, Matthew A B; Bai, Fan

    2016-03-29

    The bacterial flagellar motor (BFM) is a molecular machine that rotates the helical filaments and propels the bacteria swimming toward favorable conditions. In our previous works, we built a stochastic conformational spread model to explain the dynamic and cooperative behavior of BFM switching. Here, we extended this model to test whether it can explain the latest experimental observations regarding CheY-P regulation and motor structural adaptivity. We show that our model predicts a strong correlation between rotational direction and the number of CheY-Ps bound to the switch complex, in agreement with the latest finding from Fukuoka et al. It also predicts that the switching sensitivity of the BFM can be fine-tuned by incorporating additional units into the switch complex, as recently demonstrated by Yuan et al., who showed that stoichiometry of FliM undergoes dynamic change to maintain ultrasensitivity in the motor switching response. In addition, by locking some rotor switching units on the switch complex into the stable clockwise-only conformation, our model has accurately simulated recent experiments expressing clockwise-locked FliG(ΔPAA) into the switch complex and reproduced the increased switching rate of the motor.

  1. Structure and protein composition of the striated flagellar rootlets of some protists.

    PubMed

    Dingle, A D; Larson, D E

    1981-01-01

    The striated rootlets of different protists are extremely diverse and, on the basis of structural organization, can be assigned to no fewer than four major types. In light of this extreme variation in fine-structure