Genetic analysis of the bacterial flagellum

Escherichia coli and Salmonella typhimurium invest considerable resources in making flagella, motor organelles that function much like the propellers on a ship. Both classical and molecular genetic studies have begun to reveal how flagellar genes are regulated and how their products build and operate these remarkable devices.     

Novel conserved assembly factor of the bacterial flagellum

TP0658 (FliW) and its orthologs, conserved proteins of unknown function in Treponema pallidum and other species, interact with a C-terminal region of flagellin (FlaB1-3 in T. pallidum; FliC in most other species). Mutants of orthologs in Bacillus subtilis and Campylobacter jejuni (yviF, CJ1075) showed strongly reduced motility. TP0658 stabilizes flagellin in a way similar to FliS, suggesting that TP0658 is a conserved assembly factor for the bacterial flagellum.     

Noise underlies switching behavior of the bacterial flagellum

We report the switching behavior of the full bacterial flagellum system that includes the filament and the motor in wild-type Escherichia coli cells. In sorting the motor behavior by the clockwise bias, we find that the distributions of the clockwise (CW) and counterclockwise (CCW) intervals are either exponential or nonexponential with long tails. At low bias, CW intervals are exponentially distributed and CCW intervals exhibit long tails. At intermediate CW bias (0.5) both CW and CCW intervals are mainly exponentially distributed. A simple model suggests that these two distinct switching behaviors are governed by the presence of signaling noise within the chemotaxis network. Low noise yields exponentially distributed intervals, whereas large noise yields nonexponential behavior with long tails. These drastically different motor statistics may play a role in optimizing bacterial behavior for a wide range of environmental conditions.     

Self-assembly and type III protein export of the bacterial flagellum

The bacterial flagellum is a supramolecular structure consisting of a basal body, a hook and a filament. Most of the flagellar components are translocated across the cytoplasmic membrane by the flagellar type III protein export apparatus in the vicinity of the flagellar base, diffuse down the narrow channel through the nascent structure and self-assemble at its distal end with the help of a cap structure. Flagellar proteins synthesized in the cytoplasm are targeted to the export apparatus with the help of flagellum-specific chaperones and pushed into the channel by an ATPase, whose activity is controlled by its regulator to enable the energy of ATP hydrolysis to be efficiently coupled to the translocation reaction. The export apparatus switches its substrate specificity by monitoring the state of flagellar assembly in the cell exterior, allowing this huge and complex macromolecular assembly to be built efficiently by a highly ordered and well-regulated assembly process.     

Coarse-grained molecular dynamics simulations of a rotating bacterial flagellum

Many types of bacteria propel themselves using elongated structures known as flagella. The bacterial flagellar filament is a relatively simple and well-studied macromolecular assembly, which assumes different helical shapes when rotated in different directions. This polymorphism enables a bacterium to switch between running and tumbling modes; however, the mechanism governing the filament polymorphism is not completely understood. Here we report a study of the bacterial flagellar filament using numerical simulations that employ a novel coarse-grained molecular dynamics method. The simulations reveal the dynamics of a half-micrometer-long flagellum segment on a timescale of tens of microseconds. Depending on the rotation direction, specific modes of filament coiling and arrangement of monomers are observed, in qualitative agreement with experimental observations of flagellar polymorphism. We find that solvent-protein interactions are likely to contribute to the polymorphic helical shapes of the filament.     

Interaction of bacterial flagellum filaments with skeletal muscle myosin

Interaction of isolated bacterial flagellum filaments (BFF) and intact flagella from E. coli MS 1350 and B. brevis G.-B.p+ with rabbit skeletal myosin was studied. BFF were shown to coprecipitate with myosin (but not with isolated myosin rod) at low ionic strength, that is, under conditions of myosin aggregation. The data of electron microscopy indicate that filaments of intact bacterial flagella interact with isolated myosin heads (myosin subfragment 1, S1), and this interaction is fully prevented by addition of Mg2+ -ATP. Addition of BFF inhibited both K+ -EDTA- and Ca2+ -ATPase activity of skeletal muscle myosin, but had no effect on its Mg2+ -ATPase activity. Monomeric flagellin did not coprecipitate with myosin and had no effect on its ATPase activities. BFF were shown to compete with F-actin in myosin binding. It is concluded that BFF interact with myosin heads and affect their ATPase activity. Thus, BFF composed of a single protein flagellin are in many respects similar to actin filaments. Common origin of actin and flagellin may be a reason for this similarity.     

Signatures of antagonistic pleiotropy in a bacterial flagellin epitope

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Oligomerization and activation of the FliI ATPase central to bacterial flagellum assembly

FliI is the peripheral membrane ATPase pivotal to the type III protein export mechanism underlying the assembly of the bacterial flagellum. Gel filtration and multiangle light scattering showed that purified soluble native FliI protein was in a monomeric state but, in the presence of ATP, FliI showed a propensity to oligomerize. Electron microscopy revealed that FliI assembles to a ring structure, the yield of which was increased by the presence of a non-hydrolysable ATP analogue. Single particle analysis of the resulting electron micrograph images, to which no symmetry was applied, showed that the FliI ring structure has sixfold symmetry and an external diameter of approximately 10 nm. The oligomeric ring has a central cavity of 2.5-3.0 nm, which is comparable to the known diameter of the flagellar export channel into which export substrates feed. Enzymatic activity of the FliI ATPase showed positive co-operativity, establishing that oligomerization and enzyme activity are coupled. Escherichia coli phospholipids increased enzyme co-operativity, and in vitro cross-linking demonstrated that they promoted FliI multimerization. The data reveal central facets of the structure and action of the flagellar assembly ATPase and, by extension, the homologous ATPases of virulence-related type III export systems.     

Modeling the bacterial flagellum by an elastic network of rigid bodies

Bacteria such as Escherichia coli propel themselves by rotating a bundle of helical filaments, each driven by a rotary motor embedded in the cell membrane. Each filament is an assembly of thousands of copies of the protein flagellin which assumes two different states. We model the filament by an elastic network of rigid bodies that form bonds with one another according to a scheme suggested by Namba and Vondervistz (1997 Q. Rev. Biophys. 30 1-65) and add additional binding sites at the inner part of the rigid body. Our model reproduces the helical parameters of the 12 possible polymorphic configurations very well. We demonstrate that its energetical ground state corresponds to the normal helical form, usually observed in nature, only when inner and outer binding sites of the rigid body have a large axial displacement. This finding correlates directly to the elongated shape of the flagellin molecule. An Ising Hamiltonian in our model directly addresses the two states of the flagellin protein. It contains an external field that represents external parameters which allow us to alter the ground state of the filament.     

Membrane targeting and translocation of bacterial hydrogenases

Periplasmic or membrane-bound bacterial hydrogenases are generally composed of a small subunit and a large subunit. The small subunit contains a peculiar N-terminal twin-arginine signal peptide, whereas the large subunit lacks any known targeting signal for export. Genetic and biochemistry data support the assumption that the large subunit is cotranslocated with the small subunit across the cytoplasmic membrane. Indeed, the signal peptide carried by the small subunit directs both the small and the large subunits to the recently identified Mtt/Tat pathway, independently of the Sec machinery. In addition, the twin-arginine signal peptide of hydrogenase is capable of directing protein import into the thylakoidal lumen of chloroplasts via the homologous deltapH-driven pathway, which is independent of the Sec machinery. Therefore, the translocation of hydrogenase shares characteristics with the deltapH-driven import pathway in terms of Sec-independence and requirement for the twin-arginine signal peptide, and with protein import into peroxisomes in a "piggyback" fashion.     

Theoretical and computational models of ion channels

Computational studies can make meaningful contributions to our understanding of biological ion channels. A wide variety of methods, at different levels of approximation, can be used. Over the past few years, progress in the experimental determination of three-dimensional structures has given a fresh impetus to the theorists. Noteworthy progress has been made in carefully constructing realistic models of a number of complex biological channels to address important questions about their function.     

Stenotrophomonas maltophilia flagellin is involved in bacterial adhesion and biofilm formation

Stenotrophomonas maltophilia is an emerging drug-resistant pathogen and an important opportunistic pathogen. S. maltophilia flagellin was purified using serial ultracentrifugation. The purity of flagellin was checked by SDS-PAGE. The antibodies were raised in rabbits. The presence of anti-flagellin and the titer of flagellin were detected by immunoblotting and bacterial agglutination techniques. Two methods (viable bacterial count and spectrophotometric methods) were applied to evaluate bacterial adhesion and biofilm formation. Pretreatment of S. maltophilia with dilutions of anti-flagellin (from 1/40 to 1/640) reduced the ability of S. maltophilia to adhere and form biofilms on polystyrene (P < 0.05). In the present study, the inhibition of bacterial adhesion to polystyrene was dose-dependent. The positive correlation was observed between the antibody dilutions and bacterial adhesion (CFU/mL) (r > +0.5, P < 0.05), while, the negative correlation (r < ?0.5, P < 0.05) was observed between the percentage of adhesion inhibition and anti-flagellin dilutions. The current study proved the direct role of S. maltophilia flagellin in bacterial adhesion to and biofilm formation on polystyrene.     

Cloning,sequencing and expression of the flagellin core protein and other genes encoding structural proteins of the Vibrio cholerae flagellum

Vibrio cholerae is a Gram-negative bacterium with a single polar flagellum. Motility is an important virulence factor for this non-invasive pathogen. We cloned and sequenced a locus in V. cholerae V86 (El Tor, Inaba) that contained five different structural genes of the flagellum. The cloned genes and their products were assigned names and functions based on homology with sequences of similar genes and their products from other related bacteria. All of these genes of V. cholerae V86, namely, flgI, J, M, L and flaA, were transcribed in the same direction. These genes respectively encoded the P- and L-ring proteins, the hook-associated proteins 1 and 3 and the flagellin core protein of the flagellum. Our data indicated the presence of more than one flagellar locus in V. cholerae which could provide a means of immunoavoidance during infection. When compared with homologs in other bacteria, the flagellin core protein of V. cholerae exhibited conservation in the N- and C-termini, but had diverged in the central region.     

“细菌移位”还是“细菌易位”？

关于“Bacterial translocation”的研究虽已有几十年的历史,但是国内文献对将“Bacterial Translocation”翻译为“细菌移位”还是“细菌易位”还一直存在广泛争议。为对“Bacterial Translocation”的准确翻译提供理论依据,本文阐明了其研究背景及定义、发生机制及生物学意义;系统总结了国内文献中“细菌移位”和“细菌易位”的使用现状,从中文词义和生物学过程2个角度探讨了“Bacterial Translocation”对应的中文翻译;最终认定翻译为“细菌移位”更准确、认可度更高、更有利于推进相关研究的规范化。     

Genomic investigation of phenotypic variation in Campylobacter jejuni flagellin.

Western blots of whole-cell sonicates of 10 different clones of a faecal isolate of Campylobacter jejuni 533 detected the expression of flagella antigens of either 59 or 62 kDa. Other antigenic proteins appeared identical both in the parent and all the clones. The mechanism for this phenotypic variation was studied using Southern blotting with a flagellin-specific gene probe and products of a polymerase chain reaction (PCR) using flagellin-gene primers. Restriction-enzyme digestion and Southern blotting did not detect any genomic rearrangements in the flagellin genes of the different phenotypes nor did restriction-enzyme analysis of the PCR products.     

Calorimetric determination of polymerization enthalpy for bacterial flagellin (Salmonella)

The polymerization of bacterial flagellin protein (Salmonella strain SJ814) into flagellar filaments has been found by direct calorimetric measurement to be $\text{exothermic}$ at 25° in .15M KCl, pH 6.8 with a ΔH of ?12.7 ± 0.6 kcal per mole of monomer polymerized. The calorimetric result at 25° contrasts sharply with the endothermic ΔH of +38 kcal/mole inferred from temperature dependence of the critical monomer concentration near 40°C. Comparison between these two values implies that unless a different mechanism of polymerization prevails at the two temperatures the heat capacity change for flagellin polymerization may be as large as 3.3 kcal/mole deg.