Participation of p-coumaroyl glucose residue of awobanin in synthesis of the complex molecule of commelinin

In Vitro synthesis of commelinin-like metallo-anthocyanins was attempted using flavocommelin, metals and some anthocyanins having structures similar to awobanin, i.e. delphin, violanin, shisonin and cyanin. Only shisonin formed stable metallo-anthocyanins (with Mg, Zn, Cd, Mn, Ni and Co); the blue pigments were homologues of those prepared from awobanin. The ineffectiveness of delphin (deacylated awobanin) and cyanin (deacylated shisonin) suggests that the p-coumaroyl glucose residues of awobanin and of shisonin play an important role in the formation of rhamnosylglucoside) indicates that the length and nature of the sugar chain are critical factors for the formation of The ineffectiveness of violanin, similar to awobanin except for a different p-coumaroyl sugar chain (p-coumaroyl rhamnosylglucoside) indicates that the length and nature of the sugar chain are critical factors for the formation of commelinin and similar complex pigments.     

Ferric ions involved in the flower color development of the Himalayan blue poppy, Meconopsis grandis

The Himalayan blue poppy, Meconopsis grandis, has sky blue-colored petals, although the anthocyanidin nucleus of the petal pigment is cyanidin. The blue color development in this blue poppy involving ferric ions was therefore studied. We analyzed the vacuolar pH, and the organic and inorganic components of the colored cells. A direct measurement by a proton-selective microelectrode revealed that the vacuolar pH value was 4.8. The concentrations of the total anthocyanins in the colored cells were around 5mM, and ca. three times more concentrated flavonols were detected. Fe was detected by atomic analysis of the colored cells, and the ratio of Fe to anthocyanins was ca. 0.8 eq. By mixing the anthocyanin, flavonol and metal ion components in a buffered aq. solution at pH 5.0, we were able to reproduce the same blue color; the visible absorption spectrum and CD were identical to those in the petals, with Fe(3+), Mg(2+) and flavonol being essential for the blue color. The blue pigment in Meconopsis should be a new type of metal complex pigment that is different from a stoichiometric supramolecular pigment such as commelinin or protocyanin.     

Cyanosalvianin, a supramolecular blue metalloanthocyanin, from petals of Salvia uliginosa

A metalloanthocyanin, cyanosalvianin, was found in blue petals of Salvia uliginosa. Cyanosalvianin consisted of 3-O-(6-O-p-coumaroylglucopyranosyl)-5-O-(4-O-acetyl-6-O-malonylglucopyranosyl) delphinidin, 7,4′-di-O-glucopyranosylapigenin and magnesium ion. We reproduced the same blue color as the petals by mixing the three components together. An ESI-MS measurement gave a molecular weight of 9014 indicating the composition of cyanosalvianin to be six molecules of the anthocyanin component, six molecules of the flavone component and two magnesium ions. The special arrangement of the organic components in cyanosalvianin was analyzed by CD and 2D-NMR spectroscopy. It was clarified that cyanosalvianin has a similar structure to that of commelinin, a metalloanthocyanin isolated from blue dayflower, Commelina communis.     

Components of protocyanin, a blue pigment from the blue flowers of Centaurea cyanus

The components involved in the formation of protocyanin, a stable blue complex pigment from the blue cornflower, Centaurea cyanus, were investigated. Reconstruction experiments using highly purified anthocyanin [centaurocyanin, cyanidin 3-O-(6-O-succinylglucoside)-5-O-glucoside], flavone glycoside [apigenin 7-O-glucuronide-4'-O-(6-O-malonylglucoside)] and metals, Fe and Mg, showed the presence of another factor essential for the formation of protocyanin. The unknown factor was revealed to be Ca. Reconstructed protocyanin using anthocyanin, flavone, Fe, Mg, and Ca was identical with protocyanin from nature in UV-Vis and CD spectra, and was isolated as crystals for the first time. In addition, substitution of the metal components in protocyanin with other metals was also examined.     

Arthrospira (Spirulina) platensis: An effective biosorbent for nutrients

Arthrospira (Spirulina) platensis was tested for biosorption properties. Preliminary experiments concerning biosorption kinetics were performed on Cr(III) ions. Equilibrium of biosorption was tested for Cr(III), Mn(II) and Mg(II) ions, since these elements are crucial for animals with metabolic disorders. In our study, Spirulina was proposed as a feed additive for animals suffering from diseases characterized by insulin dysregulation, abnormal adipose distribution and a high risk for laminitis. Maximum biosorption capacity of A. platensis, determined from Langmuir equation, was 45.2 for Cr(III), 44.3 for Mn(II) and 42.0 mg/g for Mg(II) ions. Biosorption of Mg(II) ions by microalga has never been studied so far. Finally, the raw and enriched microalgal biomass was examined by ICP-OES to determine its multielamental analysis before and after biosorption, FTIR to indicate functional groups that participated in biosorption and SEM-EDX to illustrate the binding of metal ions on the surface of algal biomass. ICP-OES showed that the content of elements significantly increased in the enriched A. platensis. FTIR spectroscopy evidenced that biosorption of metal ions was mainly due to carboxylate groups present on the microalgal cell wall. SEM analysis clearly showed that biosorption occurred. Arthrospira platensis turned out to be a good biosorbent of metal ions.     

Kinetic analysis of the effect of zinc ion on the inorganic pyrophosphatase reaction.

A kinetic study is presented in which the effect of Zn(II) on yeast inorganic pyrophosphatase was quantitatively determined. A dual role model for metal ion effect, previously determined for the Mg(II)-pyrophosphatase system (O. A. Moe and L. G. Butler, 1972, J. Biol. Chem.247, 7308–7315), was applied successfully to the analysis of the kinetics for Zn(II)-pyrophosphatase and Zn(II), Mg(II)-pyrophosphatase systems. The model, assigning an activator role to free Zn(II) ion and a substrate role to the Zn(II)-pyrophosphate complex, gave an excellent fit to the data. Inhibition of the Mg(II)-pyrophosphatase system by Zn(II) was analyzed by a model in which competitive binding of the Mg(II)-pyrophosphate and Zn(II)-pyrophosphate complexes occurred at the enzyme active site, with both complexes undergoing reaction at different rates. Relative maximal velocities and enzymeligand dissociation constants for the Zn(II)-pyrophosphate complex were determined for the cases where the metal ion activator role was fulfilled by Zn(II) and Mg(II), respectively. The maximal velocity parameter showed a dependence on the nature of the activator metal ion, demonstrating that the role of the latter is associated both with the process of substrate binding and with the mechanism of catalysis. Values for all kinetic parameters are reported for an ionic strength of 0.2, pH 7.0, and 25.0 °C.     

An Exocellular Protein from the Oil-Degrading Microbe Acinetobacter venetianus RAG-1 Enhances the Emulsifying Activity of the Polymeric Bioemulsifier Emulsan

The oil-degrading microorganism Acinetobacter venetianus RAG-1 produces an extracellular polyanionic, heteropolysaccharide bioemulsifier termed emulsan. Emulsan forms and stabilizes oil-water emulsions with a variety of hydrophobic substrates. Removal of the protein fraction yields a product, apoemulsan, which exhibits much lower emulsifying activity on hydrophobic substrates such as n-hexadecane. One of the key proteins associated with the emulsan complex is a cell surface esterase. The esterase (molecular mass, 34.5 kDa) was cloned and overexpressed in Escherichia coli BL21(DE3) behind the phage T7 promoter with the His tag system. After overexpression, about 80 to 90% of the protein was found in inclusion bodies. The overexpressed esterase was recovered from the inclusion bodies by solubilization with deoxycholate and, after slow dialysis, was purified by metal chelation affinity chromatography. Mixtures containing apoemulsan and either the catalytically active soluble form of the recombinant esterase isolated from cell extracts or the solubilized inactive form of the enzyme recovered from the inclusion bodies formed stable oil-water emulsions with very hydrophobic substrates such as hexadecane under conditions in which emulsan itself was ineffective. Similarly, a series of esterase-defective mutants were generated by site-directed mutagenesis, cloned, and overexpressed in E. coli. Mutant proteins defective in catalytic activity as well as others apparently affected in protein conformation were also active in enhancing the apoemulsan-mediated emulsifying activity. Other proteins, including a His-tagged overexpressed esterase from the related organism Acinetobacter calcoaceticus BD4, showed no enhancement.     

Species specific plant-soil interactions influence plant distribution on serpentine soils

Where serpentine soils exist, variation in soil properties affects plant species distribution at both coarse and fine spatial scales. The New Idria (California, USA) serpentine mass has barren areas, supporting only sparse shrub and tree islands, adjacent to areas of densely-vegetated serpentine chaparral. To identify factors limiting growth on barren relative to vegetated serpentine soils, we analyzed soils from barren, shrub-island within barren, and vegetated areas and foliage from shrub-island and vegetated areas. We also grew Ceanothus cuneatus (native evergreen shrub), Achillea millefolium (native perennial forb), and Bromus madritensis ssp. rubens (invasive annual grass) in soils from barren and vegetated areas amended factorially with N, K, and Ca in a pot study. In well-watered pots, biomass was greater by 5-, 14-, and 33-fold for Ceanothus, Achillea, and Bromus, respectively, on vegetated-area-collected soils than on barren-collected soils, indicating a strong soil chemistry effect. Although field soil data suggested nutrient deficiency and not heavy metal toxicity, pot study plant data indicated otherwise for two of the three species. On barren-collected soils, only Ceanothus responded positively to added N and Ca and did not show greater foliar Mg or heavy metal (Fe, Ni, Cr, Co, Zn) concentrations than on vegetated-area-collected soils. Ceanothus maintained lower root Mg and heavy metal (Fe, Ni, Cr, Co) concentrations on barren soils and translocated less heavy metal (Fe, Ni, Cr, Co, Mn, Cu) from roots to foliage than Achillea and Bromus. Achillea and Bromus showed significant log-log biomass relationships with foliar Ca:Mg (+), Mg (-), and heavy metals (Fe, Ni, Cr, Co, Mn, Cu, Zn) (-), while Ceanothus showed relationships only with Ca:Mg (+) and Mg (-). The New Idria barren-vegetated pattern appears to be maintained by different factors for different species or functional types— low Ca:Mg ratios on barrens for all species tested, high heavy metal concentrations for Achillea and Bromus, and low macronutrient (N) concentrations for Ceanothus. Combined data from this and other studies suggest high heavy metal concentrations more strongly affect herbaceous than woody species, contributing to variation in species distribution on serpentine soils.     

Metal-Induced Production of a Novel Bioadsorbent Exopolysaccharide in a Native Rhodotorula mucilaginosa from the Mexican Northeastern Region

There is a current need to develop low-cost strategies to degrade and eliminate industrially used colorants discharged into the environment. Colorants discharged into natural water streams pose various threats, including: toxicity, degradation of aesthetics and inhibiting sunlight penetration into aquatic ecosystems. Dyes and colorants usually have complex aromatic molecular structures, which make them very stable and difficult to degrade and eliminate by conventional water treatment systems. The results in this work demonstrated that heavy metal-resistant Rhodotorula mucilaginosa strain UANL-001L isolated from the northeast region of Mexico produce an exopolysaccharide (EPS), during growth, which has colorant adsorption potential. The EPS produced was purified by precipitation and dialysis and was then physically and chemically characterized by Scanning Electron Microscopy, Fourier Transform Infrared Spectroscopy, and chemical elemental analysis. Here, the ability of the purified EPS produced to adsorb methylene blue (MB), which served as a model colorant, is studied. MB adsorption by the EPS is found to follow Langmuir Adsorption Isotherm kinetics at 25°C. Further, by calculating the Langmuir constant the adsorption capabilities of the EPS produced by the Rhodotorula mucilaginosa strain UANL-001L is compared to that of other adsorbents, both, microbially produced and from agroindustrial waste. The total adsorption capacity of the EPS, from the Rhodotorula mucilaginosa strain UANL-001L, was found to be two-fold greater than the best bioadsorbents reported in the literature. Finally, apart from determining which heavy metals stimulated EPS production in the strain, the optimal conditions of pH, heavy metal concentration, and rate of agitation of the growing culture for EPS production, was determined. The EPS reported here has the potential of aiding in the efficient removal of colorants both in water treatment plants and in situ in natural water streams.     

Free metal ion depletion by “good's” buffers: II. N-(2-acetamido)-2-aminoethanesulfonic acid (ACESH): Complexes with Calcium(II), Magnesium(II), Manganese(II), Cobalt(II), Zinc(II), Nickel(II), and Cooper(II)

Potentiometric, visible, and infrared studies of the complexation of N-(2-acetamido)-2-aminoethanesulfonic acid (ACESH) by Ca(II), Mg(II), Mn(II), Co(II), Zn(II), Ni(II), and Cu(II) are reported. Ca(II), Mg(II), and Mn(II) were found not to complex with ACES^?, while Co(II), Zn(II), Ni(II), and Cu(II) were found to form 2:1, ACES^? to M²⁺, complexes, and [Cu(ACES)₂] was found to undergo stepwise deprotonation of the amide groups to form [Cu(H_?1ACES)₂^2?]. Formation (affinity) constants for the various metal complexes are reported, and the probable structures of the various metal chelates in solution are discussed.     

Activation of rabbit kidney fructose diphosphatase by Mg-EDTA, Mn-EDTA and Co-EDTA complexes

Purified rabbit kidney fructose diphosphatase requires both a free cation and a metal-chelate when assayed at pH 8 or below. In the presence Mg²⁺ or Mn²⁺, effective metal chelates were Mn(II)-EDTA, Mg(II)-EDTA, and Co(III)-EDTA. With Mg²⁺ as the cation the affinity of the enzyme for Mn(II)-EDTA or Mg(II)-EDTA was approximately the same, and 300-fold greater than that for Co(III)-EDTA.Activation of the enzyme by the very stable Co(III)-EDTA complex, as well as failure of an ionophore antibiotic to replace EDTA as activator, exclude the possibility that the effects of EDTA are due to removal of metal inhibitors.Inhibition of fructose diphosphatase by Ca²⁺ was competitive with Mg²⁺, and noncompetitive with Mg(II)-EDTA, or Co(III)-EDTA. Conversely inhibition by Zn(II)-EDTA was competitive with Mg(II)-EDTA and noncompetitive with free Mg²⁺. The data suggest that the free metals bind to one site on the enzyme while the metal-EDTA chelates bind to a second site.     

Studying metal ion binding properties of a three-way junction RNA by heteronuclear NMR

Self-splicing group II introns are highly structured RNA molecules, containing a characteristic secondary and catalytically active tertiary structure, which is formed only in the presence of Mg(II). Mg(II) initiates the first folding step governed by the κζ element within domain 1 (D1κζ). We recently solved the NMR structure of D1κζ derived from the mitochondrial group II intron ribozyme Sc.ai5γ and demonstrated that Mg(II) is essential for its stabilization. Here, we performed a detailed multinuclear NMR study of metal ion interactions with D1κζ, using Cd(II) and cobalt(III)hexammine to probe inner- and outer-sphere coordination of Mg(II) and thus to better characterize its binding sites. Accordingly, we mapped ¹H, ¹⁵N, ¹³C, and ³¹P spectral changes upon addition of different amounts of the metal ions. Our NMR data reveal a Cd(II)-assisted macrochelate formation at the 5′-end triphosphate, a preferential Cd(II) binding to guanines in a helical context, an electrostatic interaction in the ζ tetraloop receptor and various metal ion interactions in the GAAA tetraloop and κ element. These results together with our recently published data on Mg(II) interaction provide a much better understanding of Mg(II) binding to D1κζ, and reveal how intricate and complex metal ion interactions can be.     

Purification, characterization and crystallization of the core complex from thermophilic purple sulfur bacterium Thermochromatium tepidum

A light-harvesting-reaction center (LH1-RC) core complex has been highly purified from a thermophilic purple sulfur bacterium, Thermochromatium tepidum. The bacteriochlorophyll (BChl) a molecules in the LH1 exhibit a Q_y transition at 914 nm, more than 25 nm red-shift from those of its mesophilic counterparts. The LH1-RC complex was isolated in a monomeric form as confirmed by sucrose density gradient centrifugation, blue native PAGE and size-exclusion chromatography. Four subunits (L, M, H and a tetraheme cytochrome) in RC and two polypeptides (α and β) in LH1 were identified. Spirilloxanthin was determined to be the predominant carotenoid in the core complex. The purified core complex was highly stable, no significant change in the LH1 Q_y transition was observed over 10 days of incubation at room temperature in dark. Circular dichroism spectrum of the LH1 complex was characterized by low intensity and nonconservative spectral shape, implying a high symmetry of the large LH1 ring and interaction between the BChl a and carotenoid molecules. A dimeric feature of the BChl a molecules in LH1 was revealed by magnetic circular dichroism spectrum. Crystals of the core complex were obtained which diffracted X-rays to about 10 Å.     

Blueing of sepal colour of Hydrangea macrophylla

Blue and red sepals of Hydrangea macrophylla were quantitatively analyzed for aluminium, anthocyanin (delphinidin 3-glucoside) and copigments (caffeoyl- and p-coumaroylquinic acids). All the blue sepals examined contained both Al and copigments (especially 3-caffeoylquinic acid) in considerable amounts. In in vitro experiments using 3- and 5-caffeoylquinic acids, Al and delphinidin 3-glucoside, it was shown that 3-caffeoylquinic acid and Al formed a blue complex with the anthocyanin. Absorption spectra of the blue complex were practically identical with those of the blue solutions obtained from blue hydrangea sepals by extraction with 4 M NACl. In contrast, 5-caffeoylquinic acid (chlorogenic acid) which was also present in hydrangea sepals gave only a red-purple colour with Al and the anthocyanin. Neither 3-caffeoylquinic acid nor Al independently produced blue colour when mixed with the anthocyanin in the mole ratios of 1–30, this being the range that the compounds were found in blue sepals. These results suggest that blue colour of hydrangea sepals is due mainly to the blue complex of delphinidin 3-glucoside-aluminium-3-caffeoylquinic acid. The role of aluminium may be to stabilize an interaction between the quinic ester and the anthocyanin.     

In vitro antifungal activity of metal complexes of a quaternized chitosan derivative with copper ions

Antifungal activity of synthetic metal complexes of quaternized N-(propyl) chitosan derivatives with Сu(II) against yeastlike (Saccharomyces cereviseae, Rodothorula rubra, and Candida albicans) and mycelial fungi (Fusarium oxysporum, Alternaria alternata, Cladosporium herbarum) was studied. In vitro application (at 250?500 μg/mL) of the metal complex of quaternized N-(propyl) derivative of low-molecular chitosan with 53% substitution and 1.3% copper ions proved efficient against F. оxysporum, one of ten most common fungal plant pathogens. Water-soluble quaternized N-(propyl) chitosan derivatives with 40?58% degree of substitution were synthesized using glycidyltrimethylammonium chloride under optimally adjusted conditions. Metal complexes of the chitosan derivative with 53% degree of substitution with Сu(II) ions were obtained by dialysis. The quantity of copper ions in the metal complexes was determined by atomic emission spectrometry. The structure of chitosan derivatives was confirmed by spectral analysis (IR, ¹H NMR).     

Violets of the section Melanium, their colonization by arbuscular mycorrhizal fungi and their occurrence on heavy metal heaps

Violets of the sections Melanium were examined for their colonization by arbuscular mycorrhizal fungi (AMF). Heartsease (Viola tricolor) from several heavy metal soils was AMF-positive at many sites but not at extreme biomes. The zinc violets Viola lutea ssp. westfalica (blue zinc violet) and ssp. calaminaria (yellow zinc violet) were always AMF-positive on heavy metal soils as their natural habitats. As shown for the blue form, zinc violets germinate independently of AMF and can be grown in non-polluted garden soils. Thus the zinc violets are obligatorily neither mycotrophs nor metalophytes. The alpine V. lutea, likely ancestor of the zinc violets, was at best poorly colonized by AMF. As determined by atomic absorption spectrometry, the contents of Zn and Pb were lower in AMF colonized plants than in the heavy metal soils from where the samples had been taken. AMF might prevent the uptake of toxic levels of heavy metals into the plant organs. Dithizone staining indicated a differential deposition of heavy metals in tissues of heartsease. Leaf hairs were particularly rich in heavy metals, indicating that part of the excess of heavy metals is sequestered into these cells.     

Magnesium spin state determines the energetics of an adenosinetriphosphate molecule

The molecular dynamics method (density functional theory) DFT:B3LYP (6-3IG** basis set, t = 310 K) was used to study interactions between a molecule of adenosinetriphosphate (ATP) (ATP subsystem) and the [Mg(H₂O)₆]²⁺ magnesium cofactor (Mg subsystem) in an aqueous medium simulated by 78 water molecules in the singlet (S) and triplet (T) states. Potential energy surfaces (PESs) for the S (lowest in energy) and T states (highest in energy) are significantly separated in space. Motion along them directs the Mg complex either to oxygen atoms of the γ-β-phosphate groups (O1–O2) (S state of PES) or to oxygen atoms of the β-α-phosphate groups (O2–O3) (T state of PES). Chelation of the γ-β- and β-α-phosphates leads to formation of a stable low-energy ([Mg(H₂O)₄-(OI-O2)ATP]^2?) complex or a metastable high-energy ([Mg(H₂O)₂-(O2–O3)ATP]^2?) complex, respectively, which differ in number of water molecules surrounding the Mg atom. Intersection of two T PESs is accompanied by formation of an unstable state characterized by redistribution of spins between the Mg and ATP subsystems. This state, being sensitive to interaction with the Mg nuclear spin (²⁵Mg), induces an unpaired electron spin, which initiates the ATP cleavage by the ion-radical mechanism, yielding a reactive ion radical of adenosinemonophosphate (·AMP^?), which was earlier found experimentally by the method of chemically induced dynamic nuclear polarization (CIDNP). Biological aspects of the results obtained are discussed.     

The first solvation shell of magnesium ion in a model protein environment with formate,water, and X-NH3, H2S,imidazole, formaldehyde,and chloride as ligands: An ab initio study

The first coordination shell of an Mg(II) ion in a model protein environment is studied. Complexes containing a model carboxylate, an Mg(II) ion, various ligands (NH₃, H₂S, imidazole, and formaldehyde) and water of hydration about the divalent metal ion were geometry optimized. We find that for complexes with the same coordination number, the unidentate carboxylate–Mg(II) ion is greater than 10 kcal mol^?1 more stable than the bidentate orientation. Imidazole was found to be the most stable ligand, followed in order by NH₃ formaldehyde, H₂O, and H₂S. © 1995 Wiley-Liss, Inc.     

Soil influence on Cu and Co uptake and plant size in the cuprophytes Crepidorhopalon perennis and C. tenuis (Scrophulariaceae) in SC Africa

Cuprophytes are plants that mostly occur on Cu-rich soil in SC Africa. Crepidorhopalon perennis is endemic of a single site. C. tenuis has a broader niche, from normal to Cu-rich soil. Both have been considered as Cu-Co accumulators. We examined soil factors controlling heavy metal accumulation and plant fitness in natural populations. Plant mass and element concentrations in plants and soil were determined in 153 samples from five populations of C. tenuis on copper soil (CTC), two on normal soil (CTN) and the single population of C. perennis (CP). Soil in Cu-sites had higher concentrations of Ca, Mg, P, Mn, Zn, Cu, Co. Plants from Cu-sites were larger and had higher Cu and Co content, and lower Mg, Mn and Ca. Cu in shoots was influenced positively by Cu and Mn and negatively by Ca in the soil. Co in shoots was influenced positively by Co and negatively by Mn and Fe in the soil. Shoot mass was influenced positively by Cu and Mn (CT) or by Cu and Co (both species pooled) in the soil. The results suggest that C. tenuis and C. perennis are genuinely cuprophilous species. Large variation in metal accumulation in shoots can be accounted for by synergistic and antagonistic interactions among several heavy metals, yielding specific accumulation patterns in different populations.     

Initial Characterization of the FlgE Hook High Molecular Weight Complex of Borrelia burgdorferi

The spirochete periplasmic flagellum has many unique attributes. One unusual characteristic is the flagellar hook. This structure serves as a universal joint coupling rotation of the membrane-bound motor to the flagellar filament. The hook is comprised of about 120 FlgE monomers, and in most bacteria these structures readily dissociate to monomers (∼ 50 kDa) when treated with heat and detergent. However, in spirochetes the FlgE monomers form a large mass of over 250 kDa [referred to as a high molecular weight complex (HMWC)] that is stable to these and other denaturing conditions. In this communication, we examined specific aspects with respect to the formation and structure of this complex. We found that the Lyme disease spirochete Borrelia burgdorferi synthesized the HMWC throughout the in vitro growth cycle, and also in vivo when implanted in dialysis membrane chambers in rats. The HMWC was stable to formic acid, which supports the concept that the stability of the HMWC is dependent on covalent cross-linking of individual FlgE subunits. Mass spectrometry analysis of the HMWC from both wild type periplasmic flagella and polyhooks from a newly constructed ΔfliK mutant indicated that other proteins besides FlgE were not covalently joined to the complex, and that FlgE was the sole component of the complex. In addition, mass spectrometry analysis also indicated that the HMWC was composed of a polymer of the FlgE protein with both the N- and C-terminal regions remaining intact. These initial studies set the stage for a detailed characterization of the HMWC. Covalent cross-linking of FlgE with the accompanying formation of the HMWC we propose strengthens the hook structure for optimal spirochete motility.