Unfolding of tertiary structure ofHalobacterium halobium flagellins does not result in flagella destruction

The structure ofHalobacterium halobium R₁M₁ flagella is investigated by the methods of scanning microcalorimetry, circular dichroism, and electron microscopy. It is shown that melting curves of flagella in solutions with a different concentration of NaCl display only one peak of heat capacity that corresponds to one cooperatively melting domain. It is found that flagella do not dissociate after melting. The possible structural organization of archaebacterial flagella is discussed.     

Effect of temperature on the in vitro assembly of bacterial flagella

The temperature dependence for the rate of reconstitution or polymerization (k₊) at neutral pH of the protein, flagellin, to flagella was measured using Ostwald-type viscometers. Similarly, the kinetics for the reverse process, the thermally-induced depolymerization of flagella filaments to the flagellin monomer (k_?) was measured. The temperature at which k_? equals zero was used to define the thermal dissociation temperature or melting point of flagella filaments. The remarkable similarity of melting points obtained (36.8 ± 0.2 deg. C) for flagella isolated from three Salmonella strains (SJ670, SJ25 and SJ30 bearing H-antigen types i, 1.2 and e, n, x, respectively) suggests that the structural stability of these different protein filaments is also similar.On increasing the temperature between 12 and 28°C, k₊ increased smoothly and had a Q₁₀ of 1.8. Above 28.0, k₊ decreased rapidly and fell to zero at a temperature near 37°C, its precise value varying with the bacterial strain. This result supports the prior hypothesis (Gerber &; Noguchi, 1967) that on heating, a reversible co-operative transconformation occurs between different states of the protein; in one state, flagellin (M) can polymerize to flagella, whereas its conformational isomer(s) may do so with difficulty or not at all.For strains SJ25 and SJ30 the rates of polymerization and depolymerization both fall to zero near 37°C. Therefore, mixtures of monomer and flagella fragments (short polymers or “seeds”), in all ratios, appear to be in equilibrium at temperatures near this critical temperature, and neither polymerization of flagellin to flagella nor melting of polymers is apparent.Measurements made on flagella from strain SJ670 showed that k₊ and k_? approached zero at 45 and 37°C, respectively. Within this temperature range the conc entration of monomer in equilibrium with filaments was determined. By a null -point type experiment, solutions of monomer and seed were mixed to find the ratio that showed neither increases (polymerization) nor decreases (depolyme rization) in viscosity with time. An unexpected finding was that the temperature defines a critical monomer concentration, which exists in equilibrium with any concentration of filaments (and not the ratio of monomer-to-filament concentrations). Thus, the polymerization of fiagellin to flagella corresponds to a phase change akin to either crystallization or condensation.Application, of the Clapeyron-Clausius equation to the results obtained yields a heat of condensation of 70 kcal/mol of monomeric protein. The enthalpy change associated with M ? M_i is estimated as 110 kcal/mol of protein. Since the heat content of these various forms of flagella protein lies in the order M_i > F > M, by difference we estimate the enthalpy change for the conversion of monomers to polymers to be 40 kcal/mol of monomer.     

Effect of Flagellar Mutations on Yersinia enterocolitica Biofilm Formation

Yersinia enterocolitica biovar 1B is one of a number of strains pathogenic to humans in the genus Yersinia. It has three different type III secretion systems, Ysc, Ysa, and the flagella. In this study, the effect of flagella on biofilm formation was evaluated. In a panel of 31 mutant Y. enterocolitica strains, we observed that mutations that abolish the structure or rotation of the flagella greatly reduce biofilm formation when the bacteria are grown under static conditions. These results were further evaluated by assessing biofilm formation under continuous culture using a flow cell chamber. The results confirmed the important contribution of flagella to the initiation of biofilm production but indicated that there are differences in the progression of biofilm development between static growth and flow conditions. Our results suggest that flagella play a critical role in biofilm formation in Y. enterocolitica.     

Functional interaction between information processing pathways in Escherichia coli and the nature of Δ-H+ (redox)-sensing

Glucose taxis and O₂-taxis in Escherichia coli signal to flagella via a pathway that includes PTS^glc and adenylate cyclase. Information from a number of attractants and repellents is focused at the level of methy-accepting chemotaxis proteins (MCPs) and information is passed to flagella by a separate pathway. Mutants defective in adenylate cyclase (Δcya) had a residual taxis to glucose that was eliminated by preincubating the cells with MCP attractants, or by depleting the -CH₃ donor. A methyltransferase mutant had a decreased sensitivity to MCP repellents and this response was completely blocked by preincubating the cells with glucose. Likewise, the response of cells, depleted for -CH₃, towards repellents, was blocked if bacteria carried a pts mutation. It is concluded that PTS and MCP pathways exchange information. In cya cells, O₂ taxis was restored in the presence of maltose, an MCPII attractat. It is suggested that MCPII is an additional protonmotive force (pmf) sensor.     

Vanadium ion inhibition of alkaline phosphatase-catalyzed phosphate ester hydrolysis.

The intracellular location of guanylate cyclase was examined in sperm from two species of sea urchin, Strongylocentrotus purpuratus and Lytechinus pictus, and from the tube worm Chaetopterus variopedatus. Cells suspended in a medium isotonic with sea water were passed repeatedly through a 23-gauge hypodermic needle to break flagella from heads. This preparation was then fractionated by two methods, one based on centrifugation over a 25% sucrose medium and the other involving repeated differential centrifugation, to resolve flagella from heads. Guanylate cyclase specific activity was increased 3.5–4.5-fold in the flagellar fraction relative to the starting sperm homogenate. Relatively little activity was present in the head fraction where specific activity was $\text{1}\text{10}\text{–}\text{1}\text{100}$ that of the flagella. Plasma membranes were separated from axonemal microtubules by dialyzing flagella against low ionic strength buffer, followed by centrifugation over a 40% sucrose medium. Although the overall recovery of guanylate cyclase was low, the specific activity in the plasma membrane fraction was increased two- to threefold over the dialyzed flagella, and over 90% of the recovered activity resided in this fraction. Thus the flagellar plasma membrane is a site rich in guanylate cyclase. It could not be determined, however, whether this is the only intracellular locale of the enzyme.     

A NIMA-Related Kinase Suppresses the Flagellar Instability Associated with the Loss of Multiple Axonemal Structures

CCDC39 and CCDC40 were first identified as causative mutations in primary ciliary dyskinesia patients; cilia from patients show disorganized microtubules, and they are missing both N-DRC and inner dynein arms proteins. In Chlamydomonas, we used immunoblots and microtubule sliding assays to show that mutants in CCDC40 (PF7) and CCDC39 (PF8) fail to assemble N-DRC, several inner dynein arms, tektin, and CCDC39. Enrichment screens for suppression of pf7; pf8 cells led to the isolation of five independent extragenic suppressors defined by four different mutations in a NIMA-related kinase, CNK11. These alleles partially rescue the flagellar length defect, but not the motility defect. The suppressor does not restore the missing N-DRC and inner dynein arm proteins. In addition, the cnk11 mutations partially suppress the short flagella phenotype of N-DRC and axonemal dynein mutants, but do not suppress the motility defects. The tpg1 mutation in TTLL9, a tubulin polyglutamylase, partially suppresses the length phenotype in the same axonemal dynein mutants. In contrast to cnk11, tpg1 does not suppress the short flagella phenotype of pf7. The polyglutamylated tubulin in the proximal region that remains in the tpg1 mutant is reduced further in the pf7; tpg1 double mutant by immunofluorescence. CCDC40, which is needed for docking multiple other axonemal complexes, is needed for tubulin polyglutamylation in the proximal end of the flagella. The CCDC39 and CCDC40 proteins are likely to be involved in recruiting another tubulin glutamylase(s) to the flagella. Another difference between cnk11-1 and tpg1 mutants is that cnk11-1 cells show a faster turnover rate of tubulin at the flagellar tip than in wild-type flagella and tpg1 flagella show a slower rate. The double mutant shows a turnover rate similar to tpg1, which suggests the faster turnover rate in cnk11-1 flagella requires polyglutamylation. Thus, we hypothesize that many short flagella mutants in Chlamydomonas have increased instability of axonemal microtubules. Both CNK11 and tubulin polyglutamylation play roles in regulating the stability of axonemal microtubules.     

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

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

Electron spin resonance melting of chemically spin-labeled nucleic acids.

Conformational transitions of nitroxide labeled and unlabeled nucleic acids were analyzed by esr and uv spectroscopy to evaluate potential perturbation effects caused by chemical modifications of nucleic acids with spin labels. The melting temperature (T_m) determined by uv or esr melting profiles of 2 → 1 or 3 → 1 transitions is similar for labeled and unlabeled polyadenylic acid [(A)_n] and polyuridylic acid [(U)_n] complexes provided spin-labeled (A)_n with a nitroxide to nucleotide ratio of 0.002 is used. Complexes formed with spin-labeled (A)_n of greater spin-labeling extent display a noticeable perturbation of their thermal melting profiles. The studies reconfirm the existence of a low temperature esr transition at about 20 °C with calf thymus and T4 DNA duplexes spin-labeled with a nitroxide to nucleotide ratio of about 0.006. The uv melting profiles of the spin-labeled duplexes reveal no low-temperature discontinuity, but the T_m values reflecting the 2 → 1 transitions were reduced by several degrees versus those of the unlabeled duplexes. Thus, these studies suggest that with homopolymers, chemically modified to a low extent with nitroxides, the monitoring of local conformational transitions of duplexes or triplexes reflect the overall 2 → 1 or 3 → 1 transitions. In the case of the heteropolymers the possibility that the chemical modification is responsible for the low-temperature phenomenon cannot be ruled out.     

Non-motile mini-transposon mutants of Bordetella bronchiseptica exhibit altered abilities to invade and survive in eukaryotic cells

Non-motile mutants of Bordetella bronchiseptica were generated after mini-transposon mutagenesis. One non-motile mutant (designated VMM1) was derived from the bvg-positive strain BB7865 and four mutants (designated AMM1–4) were derived from the isogenic bvg-negative strain BB7866. Southern hybridisation analysis indicated that loss of motility was not due to the disruption of the flagellin subunit gene. Western blot and transmission electron microscopic analysis indicated that three of the five mutants expressed neither the flagellin subunit (40 kDa) nor flagella whereas one mutant expressed intact flagella under all conditions tested. One unique bvg-negative mutant, AMM4, exhibited temperature-dependent repression of flagella biosynthesis and motility at 37°C. The ability of AMM4 to invade and survive in HeLa cells was significantly decreased.     

Polar, peritrichous, and lateral flagella belong to three distinguishable flagellar families

The bacterial flagellum transforms its shape into several distinguishable helical shapes (polymorphs) under various environmental conditions. Polymorphs of each type of flagellum stay on a circle in the pitch-diameter (P versus πD) plot, indicating that they all belong to one family. Previously, we showed that the flagellar family of a marine bacterium Idiomarina loihiensis (Family II) differed from the conventional flagellar family of Salmonella typhimurium (Family I). The pitch and diameter of Family II flagella are half those of Family I flagella. We have suggested that Family I encompasses peritrichous flagella, while Family II forms a polar flagellum. In this study, we have surveyed the polymorphs of flagella from 18 other species and categorized their family types. Previous observations were confirmed; Family I form peritrichous flagella and Family II form polar flagella. Furthermore, we found that lateral flagella had helical parameters much smaller than those of the other two Families and thus belong to a new family (Family III).     

Equilibrium and kinetics of thermal unfolding of yeast 5.8S ribosomal RNA in aqueous salt solutions

Equilibrium and kinetics of thermal melting of yeast 5.8S ribosomal RNA in aqueous NaCl were investigated by differential thermal melting and temperature jump methods. Two peaks were observed in each of the melting curves at 1 mM-1 M Na⁺ and linearity between each melting temperature T_m and log[Na⁺] was found at [Na⁺> 10 mM. From the difference spectrum ratio, $\text{d}\text{A}{}_{280}\text{d}\text{A}{}_{260}$, the G-C content in the local structures was calculated to be 91 and 56%. The temperature jump to 70–85°C in aqueous 30 mM Na⁺ of the RNA solution induced first-order kinetics, from which the kinetically determined melting curve was calculated. The curve could be approximately described in a Gaussian form with a T_m which agrees well with the high T_m in the static melting curve at 30 mM Na⁺. The kinetic properties of the reaction indicated a double helix-coil transition. However, the temperature jump to 20–60°C did not induce monophasic kinetics. The kinetic amplitude of the slow component showed a T_m which corresponded to the low T_m in the static melting curve at 30 mM Na⁺. The slow relaxation had the characteristics of a double helix-to-coil transition. However, contributions from very fast processes including single strand unstacking, were most noticeable in the low temperature melting region of the static curve. The thermodynamic parameters of both transitions from double helix to coil were analysed in detail. Both activation energies for helix formation were negative, and the nucleation is thought to follow a process similar to that in oligonucleotides. Values of T_m and enthalpy change of both helix-coil transitions indicated the cloverleaf model as the most plausible one for some limited regions of yeast 5.8S RNA among the previously proposed models: burp gun, cloverleaf and Rubin's models.     

Role of the Helicobacter hepaticus flagellar sigma factor FliA in gene regulation and murine colonization

The enterohepatic Helicobacter species Helicobacter hepaticus colonizes the murine intestinal and hepatobiliary tract and is associated with chronic intestinal inflammation, gall stone formation, hepatitis, and hepatocellular carcinoma. Thus far, the role of H. hepaticus motility and flagella in intestinal colonization is unknown. In other, closely related bacteria, late flagellar genes are mainly regulated by the sigma factor FliA (σ²⁸). We investigated the function of the H. hepaticus FliA in gene regulation, flagellar biosynthesis, motility, and murine colonization. Competitive microarray analysis of the wild type versus an isogenic fliA mutant revealed that 11 genes were significantly more highly expressed in wild-type bacteria and 2 genes were significantly more highly expressed in the fliA mutant. Most of these were flagellar genes, but four novel FliA-regulated genes of unknown function were identified. H. hepaticus possesses two identical copies of the gene encoding the FliA-dependent major flagellin subunit FlaA (open reading frames HH1364 and HH1653). We characterized the phenotypes of mutants in which fliA or one or both copies of the flaA gene were knocked out. flaA_1 flaA_2 double mutants and fliA mutants did not synthesize detectable amounts of FlaA and possessed severely truncated flagella. Also, both mutants were nonmotile and unable to colonize mice. Mutants with either flaA gene knocked out produced flagella morphologically similar to those of wild-type bacteria and expressed FlaA and FlaB. flaA_1 mutants which had flagella but displayed reduced motility did not colonize mice, indicating that motility is required for intestinal colonization by H. hepaticus and that the presence of flagella alone is not sufficient.     

Transposon insertion into a chromosomal copy of flhB gene is concurrent with defects in the formation of polar and lateral flagella in the bacterium Azospirillum brasilense Sp245

Based on an example of Azospirillum brasilense Sp245, it was shown that, in bacteria with mixed flagellation, insertional mutagenesis of one of the copies of the flhB gene, which encodes a component of the flagellar protein export apparatus, may be concurrent with defects in the formation of both a constitutive polar flagellum and inducible lateral flagella. Despite the presence of a second copy of flhB in the plasmid-located gene cluster, which seems necessary for the formation of lateral flagella, the flhB1::Omegon-Km Sp245 mutant completely lost the ability to produce them. The described effect of the inactivation of flhB1 might be explained by the use of FlhBl for the assembly of both types of flagella. Since the open reading frame AZOBR_150176, which is transcribed in the same direction and codes for a hypothetical multisensor hybrid histidine kinase/response regulator, adjoins to the 3’-end of flhB1, the participation of the latter protein in the induction of the lateral flagellar synthesis in response to the increase in the density of the environment was not excluded.     

Three Mutations in Escherichia coli That Generate Transformable Functional Flagella

Hydrodynamics predicts that swimming bacteria generate a propulsion force when a helical flagellum rotates because rotating helices necessarily translate at a low Reynolds number. It is generally believed that the flagella of motile bacteria are semirigid helices with a fixed pitch determined by hydrodynamic principles. Here, we report the characterization of three mutations in laboratory strains of Escherichia coli that produce different steady-state flagella without losing cell motility. E. coli flagella rotate counterclockwise during forward swimming, and the normal form of the flagella is a left-handed helix. A single amino acid exchange A45G and a double mutation of A48S and S110A change the resting flagella to right-handed helices. The stationary flagella of the triple mutant were often straight or slightly curved at neutral pH. Deprotonation facilitates the helix formation of it. The helical and curved flagella can be transformed to the normal form by torsion upon rotation and thus propel the cell. These mutations arose in the long-term laboratory cultivation. However, flagella are under strong selection pressure as extracellular appendages, and similar transformable flagella would be common in natural environments.     

Three-dimensional reconstruction and averaging of 50 S ribosomal subunits of Escherichia coli from electron micrographs

The thermal stability and melting kinetics of the α-helical conformation within several regions of the rabbit myosin rod have been investigated. Cyanogen bromide cleavage of long myosin subfragment-2 produced one coiled-coil α-helical fragment corresponding to short subfragment-2 with molecular weight 90,000 (M_r = 45,000) and two fragments from the hinge region with molecular weights of 32,000 to 34,000 (M_r = 16,000 to 17,000) and 24,000 to 26,000 (M_r = 12,000 to 13,000). Optical rotation melting experiments and temperature-jump kinetic studies of long subfragment-2 and its cyanogen bromide fragments show that the hinge and the short subfragment-2 domains melt as quasi-independent co-operative units. The α-helical structure within the hinge has an appreciably lower thermal stability than the flanking short subfragment-2 and light meromyosin regions of the myosin rod. Two relaxation processes for helix-melting, one in the submillisecond range (τ_f) and the other in the millisecond range (τ_s), are observed in the light meromyosin and short subfragment-2 regions of the rod, but melting in the hinge domain is dominated by the fast (τ_f) process. Results suggest that the hinge domain of the subfragment-2 link may be the locus of force generation in a cycling cross-bridge.     

Characterization of Chemotactic Responses and Flagella of Hyphomicrobium Strain W1-1B

Motile swarmer cells of Hyphomicrobium strain W1-1B displayed positive chemotactic responses toward methylamine, dimethylamine, and trimethylamine but did not display significant chemotactic responses towards methanol and arginine. Electron micrographs of negatively stained intact flagellar filaments indicated a novel striated surface pattern. The flagella were composed of two proteins of 39 and 41 kDa. Neither protein was a glycoprotein as determined by Schiff’s staining and by enzyme immunoassay. Protein fingerprints visualized from silver-stained polyacrylamide gels and Western blots of protease-digested samples indicated that the two proteins were similar but not identical. Monoclonal antibodies prepared to the complex flagella of Rhizobium meliloti cross-reacted with the striated flagella of Hyphomicrobium strain W1-1B; however, these antibodies did not cross-react with smooth-surface flagella. These results suggest that complex and striated flagella possess homologous epitope regions.     

Sex-specific binding and inactivation of agglutination factor in Chlamydomonas eugametos

Gametes of opposite mating type (mt ⁺ and mt ^-) of the green alga Chlamydomonas eugametos agglutinate via their flagella as a prelude to sexual fusion. To quantitate sexual agglutination, an in vitro assay has been developed using ³⁵S-labeled flagella and the isolated mt ^-agglutination factor. It is shown that not only isolated flagella, but also the mt ^-agglutination factor rapidly bind to the flagella of intact gametes of the opposite mating type. This confirms the role of the mt ^-agglutination factor in determining the sexual agglutinability of mt ^-gametes. As a function of binding, the agglutinative power of the flagella of both mating types is destroyed by a temperature-sensitive process. Likewise, the mt ^-agglutination factor can be completely inactivated.Abbreviations Mt ^+/- mating type plus or minus - PAS periodic-acid Schiff-reagent - Hepes 4-(2-hydroxyethyl)-1-piperazineethansulfonic acid - HMC buffer Hepes buffer (10 mM. pH 7.2, containing 1 mM MgCl₂ and 1 mM CaCl₂)     

Kharon1 Null Mutants of Leishmania mexicana Are Avirulent in Mice and Exhibit a Cytokinesis Defect within Macrophages

In a variety of eukaryotes, flagella play important roles both in motility and as sensory organelles that monitor the extracellular environment. In the parasitic protozoan Leishmania mexicana, one glucose transporter isoform, LmxGT1, is targeted selectively to the flagellar membrane where it appears to play a role in glucose sensing. Trafficking of LmxGT1 to the flagellar membrane is dependent upon interaction with the KHARON1 protein that is located at the base of the flagellar axoneme. Remarkably, while Δ kharon1 null mutants are viable as insect stage promastigotes, they are unable to survive as amastigotes inside host macrophages. Although Δ kharon1 promastigotes enter macrophages and transform into amastigotes, these intracellular parasites are unable to execute cytokinesis and form multinucleate cells before dying. Notably, extracellular axenic amastigotes of Δ kharon1 mutants replicate and divide normally, indicating a defect in the mutants that is only exhibited in the intra-macrophage environment. Although the flagella of Δ kharon1 amastigotes adhere to the phagolysomal membrane of host macrophages, the morphology of the mutant flagella is often distorted. Additionally, these null mutants are completely avirulent following injection into BALB/c mice, underscoring the critical role of the KHARON1 protein for viability of intracellular amastigotes and disease in the animal model of leishmaniasis.     

Peroxiredoxin 1 is involved in disassembly of flagella and cilia

Cilia/flagella are evolutionarily conserved cellular organelles. In this study, we demonstrated that Dunaliella salina Peroxiredoxin 1 (DsPrdx1) localized to the flagella and basal bodies, and was involved in flagellar disassembly. The link between DsPrdx1 and flagella of Dunaliella salina (D. salina) encouraged us to explore the function of its human homologue, Homo sapiens Peroxiredoxin 1 (HsPrdx1) in development and physiology. Our results showed that HsPrdx1 was overexpressed, and cilia were lost in esophageal squamous cell carcinoma (ESCC) cells compared with the non-cancerous esophageal epithelial cells Het-1A. Furthermore, when HsPrdx1 was knocked down by short hairpin RNA (shRNA) lentivirus in ESCC cells, the phenotype of cilia lost can be reversed, and the expression levels of tumor suppressor genes LKB1 and p-AMPK were increased, and the activity of the oncogene Aurora A was inhibited compared with those in cells transfected with scrambe-shRNA lentivirus. These findings firstly showed that Prdx1 is involved in disassembly of flagella and cilia, and suggested that the abnormal expression of the cilia-related gene including Prdx1 may affect both ciliogenesis and cancernogenesis.     

Analysis of a Spontaneous Non-Motile and Avirulent Mutant Shows That FliM Is Required for Full Endoflagella Assembly in Leptospira interrogans

Pathogenic Leptospira strains are responsible for leptospirosis, a worldwide emerging zoonotic disease. These spirochetes are unique amongst bacteria because of their corkscrew-like cell morphology and their periplasmic flagella. Motility is reported as an important virulence determinant, probably favoring entry and dissemination of pathogenic Leptospira in the host. However, proteins constituting the periplasmic flagella and their role in cell shape, motility and virulence remain poorly described. In this study, we characterized a spontaneous L. interrogans mutant strain lacking motility, correlated with the loss of the characteristic hook-shaped ends, and virulence in the animal model. Whole genome sequencing allowed the identification of one nucleotide deletion in the fliM gene resulting in a premature stop codon, thereby preventing the production of flagellar motor switch protein FliM. Genetic complementation restored cell morphology, motility and virulence comparable to those of wild type cells. Analyses of purified periplasmic flagella revealed a defect in flagella assembly, resulting in shortened flagella compared to the wild type strain. This also correlated with a lower amount of major filament proteins FlaA and FlaB. Altogether, these findings demonstrate that FliM is required for full and correct assembly of the flagella which is essential for motility and virulence.