Transition of bacterial flagella from helical to straight forms with different subunit arrangements.

We have found that several kinds of helical flagella from Salmonella and Escherichia become straight in the presence of 0·5 m-citric acid at pH values below 4·0, while the straight flagella from a mutant Salmonella (SJ814) are transformed into a helical shape under the same conditions. These transformations are reversible and transitional.Current models of bacterial flagella (Calladine, 1976,1978; Kamiya, 1976) predict that the family of distinct wave-forms should include two types of straight flagella, which have either an extreme right-handed twist (about 7 ° at the surface of the flagellum) or an extreme left-handed twist (2 ° to 3 °). As the inclination of the near-longitudinal rows of subunits in the Salmonella SJ814 flagellum (O'Brien &; Bennett, 1972) agrees closely with the degree of twisting predicted for the right-handed type, this flagellum has been considered to be the right-handed type. We have determined that the basic (1-start) helix in flagella is right-handed, using the method of Finch (1972). This fact, together with the selection rule (O'Brien &; Bennett, 1972), strongly suggests that the near-longitudinal rows in an SJ814 flagellum are right-handed, in agreement with the prediction. However, our optical diffraction and X-ray diffraction studies have revealed that the near-longitudinal rows of subunits in the citric acid-induced straight flagella and in the straight flagella from a mutant E. coli (Kondoh &; Yanagida, 1975) tilt at an angle of 2 ° to 3 ° with respect to the flagellar axis. This inclination is probably left-handed. Thus the predicted presence of the two types of straight flagella seems to be proved.     

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.     

Formation of a flagella-like but straight polymer of Salmonella flagellin

Salmonella flagellin (monomer) polymerizes into flagellar filaments with the addition of (NH₄)₂SO₄ (Ada et al., 1963; Wakabayashi et al., 1969). When, however, this process was allowed to take place in the presence of a high concentration of NaCl (about 1.5 m), the product consisted of flagella-like but straight filaments. This phenomenon was common to four kinds of flagellins derived from strains SJ670, SJ25, SJ30 and SJ814. When the straight filament, suspended in 0.15 m-NaCl, was heated, it depolymerized to the monomer, which could in turn be polymerized into flagellar filaments by the addition of short fragments of flagella at room temperature. Nevertheless, attempts at direct transformation between the two types of filaments were unsuccessful. In 0.15 m-NaCl, straight filaments prepared from the four kinds of flagellins had markedly different heat stabilities, which were much lower than that of any kind of flagella. When monomeric flagellin dissolved in 3.5 m-NaCl was seeded with short fragments of straight filaments, the monomer polymerized onto the ends of the short fragments, which consequently grew into long straight filaments. In this type of experiment, monomers and seeds derived from the four strains were able to interact in any combination, suggesting that straight filaments consisting of the four kinds of flagellins have the same substructures. Whether the concentration of added NaCl was 0.15 m or 3.5 m, fragments of flagella (or straight filaments) were unable to act as seeds for the formation of straight filaments (or flagellar filaments). From this and other experimental results, it was concluded that in the two filamentous structures, flagellin molecules may be packed in different ways.     

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.     

Thermal transition in helical forms of Salmonella flagella

Flagellar filaments from three strains of Salmonella undergo reversible structural transitions between discrete helical forms when the temperature is changed under appropriate aqueous conditions. These transitions can be quantified by viscometry of concentrated flagellar solutions. A transition resulting in an increase in the right-handed structural twist of the filament is always exothermic. The change in van't Hoff enthalpy accompanying the transformation ranged between 90 kcal and 250 kcal per co-operative unit depending on the type of transformation. From the relation between the transition temperature and the pH, it is inferred that binding of five to six protons to a co-operative unit is involved in the transformations of two kinds of flagella.When small amounts of flagellins from straight mutants are incorporated into normal flagellar filaments by copolymerization, the transition temperature either increases or decreases, depending on the species of the mutant flagellins, as compared with that in the normal homopolymers. These results are discussed in terms of two-state models of flagellar polymorphism (Calladine, 1978; Kamiya et al., 1979).     

Stimulation of Ligninolytic Peroxidase Activity by Nitrogen Nutrients in the White Rot Fungus Bjerkandera sp. Strain BOS55

Bjerkandera sp. strain BOS55, a newly isolated wild-type white rot fungus, produced lignin peroxidase (LiP) in nitrogen (N)-sufficient glucose-peptone medium, whereas no LiP was detectable in N-limited medium. The production of LiP was induced by the peptide-containing components of this medium and also by soy bean protein. Furthermore, the production of manganese-dependent peroxidase was stimulated by organic N sources, although lower production was also evident in N-limited medium. Further research showed that the induction of LiP depended on the combination of pH and the type of N source. An amino acid mixture and ammonium induced LiP only at either pH 6 or 7.3, respectively. Peptone induced LiP activity at all pH values tested; however, the highest activity was observed at pH 7.3. The results presented here indicate that Bjerkandera spp. are distinct from the model white rot fungus, Phanerochaete chrysosporium, which produces ligninolytic peroxidases in response to N limitation.     

A Novel dnaJ Family Gene,sflA, Encodes an Inhibitor of Flagellation in Marine Vibrio Species

The marine bacterium Vibrio alginolyticus has a single polar flagellum. Formation of that flagellum is regulated positively and negatively by FlhF and by FlhG, respectively. The ΔflhF mutant makes no flagellum, whereas the ΔflhFG double-deletion mutant usually lacks a flagellum. However, the ΔflhFG mutant occasionally reverts to become motile by forming peritrichous flagella. We have isolated a suppressor pseudorevertant from the ΔflhFG strain (ΔflhFG-sup). The suppressor strain forms peritrichous flagella in the majority of cells. We identified candidate suppressor mutations by comparing the genome sequence of the parental strain, VIO5, with the genome sequences of the suppressor strains. Two mutations were mapped to a gene, named sflA (suppressor of ΔflhFG), at the VEA003730 locus of the Vibrio sp. strain EX25 genome. This gene is specific for Vibrio species and is predicted to encode a transmembrane protein with a DnaJ domain. When the wild-type gene was introduced into the suppressor strain, motility was impaired. Introducing a mutant version of the sflA gene into the ΔflhFG strain conferred the suppressor phenotype. Thus, we conclude that loss of the sflA gene is responsible for the suppressor phenotype and that the wild-type SflA protein plays a role in preventing polar-type flagella from forming on the lateral cell wall.     

Optimized BlaM-transposon shuttle mutagenesis of Helicobacter pylori allows the identification of novel genetic loci involved in bacterial virulence

Helicobacter pylori is an important etiologic agent of gastroduodenal disease in humans. In this report, we describe a general genetic approach for the identification of genes encoding exported proteins in H. pylori. The novel TnMax9 mini-blaM transposon was used for insertion mutagenesis of a H. pylori gene library established in Escherichia coli. A total of 192 E. coli clones expressing active β-lactamase fusion proteins (BlaM⁺) were obtained, indicating that the corresponding target plasmids carry H. pylori genes encoding putative extracytoplasmic proteins. Natural transformation of H. pylori P1 or P12 using the 192 mutant plasmids resulted in 135 distinct H. pylori mutant strains (70%). Screening of the H. pylori collection of mutant strains allowed the identification of mutant strains impaired in motility, in natural transformation competence and in adherence to gastric epithelial cell lines. Motility mutants could be grouped into distinct classes: (i) mutant strains lacking the major flagellin subunit FlaA and intact flagella (class I); (ii) mutant strains with apparently normal flagella, but reduced motility (class II), and (iii) mutant strains with obviously normal flagella, but completely abolished motility (class III). Two independent mutations that exhibited defects in natural competence for genetic transformation mapped to different genetic loci. In addition, two independent mutant strains were isolated by their failure to bind to the human gastric carcinoma cell line Katoill. Both mutant strains carried a transposon in the same gene, 0.8 kb apart, and showed decreased autoagglutination when compared to the wild-type strain.     

Transformation of Acinetobacter calco-aceticus (Bacterium anitratum)

A highly efficient transformation system has been demonstrated in a strain of Acinetobacter calco-aceticus (Bacterium anitratrum). During mixed growth of various stable, unencapsulated, mutant strains, deoxyribonucleic acid (DNA) is liberated and fully encapuslated transformants can be isolated. Purified DNA preparations have been used to transform suitable recipient mutant strains for ability to synthesize capsules, ability to dispense with a growth factor requirement, and resistance to streptomycin. When the wild-type strain is deprived of its capsule, either by mechanical stripping or by mutation, the unencapsulated cells tend to form large clumped masses. A nonclumping mutant of an unencapsulated strain has been isolated. When ability to synthesize capsules is transformed into this nonclumping strain, the resultant cells no longer form chains, unlike the wild-type encapsulated strain. It appears likely that the occurrence of transformation during growth of mixed cultures, with glucose or gluconate as the carbon source, may be the result of osmotic rupture resulting from the inability of unencapsulated strains to oxidize triose phosphates as fast as they are formed. The finding of transformation in Acinetobacter may provide an additional useful organism for the study of this mode of genetic transfer since this strain grows well in a simple mineral medium containing a single oxidizable source of carbon. Furthermore, no special supplementary factors seem to be required for transformation to take place.     

Gains of Bacterial Flagellar Motility in a Fungal World

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.     

Evidence That an Internal Carbonic Anhydrase Is Present in 5% CO(2)-Grown and Air-Grown Chlamydomonas

Inorganic carbon (C_i) uptake was measured in wild-type cells of Chlamydomonas reinhardtii, and in cia-3, a mutant strain of C. reinhardtii that cannot grow with air levels of CO₂. Both air-grown cells, that have a CO₂ concentrating system, and 5% CO₂-grown cells that do not have this system, were used. When the external pH was 5.1 or 7.3, air-grown, wild-type cells accumulated inorganic carbon (C_i) and this accumulation was enhanced when the permeant carbonic anhydrase inhibitor, ethoxyzolamide, was added. When the external pH was 5.1, 5% CO₂-grown cells also accumulated some C_i, although not as much as air-grown cells and this accumulation was stimulated by the addition of ethoxyzolamide. At the same time, ethoxyzolamide inhibited CO₂ fixation by high CO₂-grown, wild-type cells at both pH 5.1 and 7.3. These observations imply that 5% CO₂-grown, wild-type cells, have a physiologically important internal carbonic anhydrase, although the major carbonic anhydrase located in the periplasmic space is only present in air-grown cells. Inorganic carbon uptake by cia-3 cells supported this conclusion. This mutant strain, which is thought to lack an internal carbonic anhydrase, was unaffected by ethoxyzolamide at pH 5.1. Other physiological characteristics of cia-3 resemble those of wild-type cells that have been treated with ethoxyzolamide. It is concluded that an internal carbonic anhydrase is under different regulatory control than the periplasmic carbonic anhydrase.     

Tn5 Insertion Mutants of Pseudomonas fluorescens Defective in Adhesion to Soil and Seeds

Tn5 insertion mutants of a soil isolate, Pseudomonas fluorescens Pf0-1, were selected for decreased ability to adhere to quartz sand in a column assay. Three adhesion-deficient mutants that differed in the location of the Tn5 insertion in the chromosome were isolated and compared with the wild-type strain. One mutant, Pf0-5, was described previously as an adhesion-defective, nonmobile, flagellumless mutant (M. F. DeFlaun, A. S. Tanzer, A. L. McAteer, B. Marshall, and S. B. Levy, Appl. Environ. Microbiol. 56:112-119, 1990). Another insertion mutant, Pf0-10, was also missing flagella and the 34-kDa outer membrane protein that was absent in Pf0-5 but present in the wild-type strain. The third mutant (Pf0-15) had increased amounts of this 34-kDa outer membrane protein and more flagella than the wild-type strain. These mutants also displayed decreased ability to adhere to sterile and natural (live) soil and to a variety of plant seeds. In kinetics studies, the wild-type strain showed an initial rapid binding to seeds followed by a later slow phase of binding. The mutant strains were defective in the initial stages of attachment but did show the later slow binding. The findings indicate that the same mutations that affect binding to sand and soil also affect adhesion to plant seeds.     

Effect of temperature,salts, pH and other factors on the development of peritrichous flagella in Vibrio alginolyticus

The development of peritrichous flagella and, consequently, swarming of Vibrio alginolyticus depend on a complex relationship between temperature, salt concentrations and pH. At temperatures above 28°C V. alginolyticus did not develop peritrichous flagella unless certain minimal concentrations of NaCl are present: the higher the temperature, the higher the NaCl concentrations required for peritrichous flagella synthesis. This requirement for NaCl at high temperatures is much more pronounced at pH 9 than at pH 6. High temperatures and low concentrations of NaCl also inhibited swarming of cells already armed with peritrichous flagella. Other cations, such as Li⁺, K⁺ and Mg²⁺, replaced NaCl only at temperatures below 28°C.     

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.     

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.     

Role of flagellins from A and B loci in flagella formation of Halobacterium salinarum

Haloarchaeal flagella are composed of a number of distinct flagellin proteins, specified by genes in two separate operons (A and B). The roles of these flagellins were assessed by studying mutants of H. salinarum with insertions in either the A or the B operon. Cells of the flgA- mutant produced abnormally short, curved flagella that were distributed all over the cell surface. The flgA2- strain produced straight flagella, mainly found at the poles. The flgB- mutant had flagella of the same size and spiral shape as wild-type cells, but these cells also showed unusual outgrowths, which appeared to be sacs filled with basal body-like structures. In broth cultures of this mutant, the medium accumulated flagella with basal body-like structures at their ends.     

Minimum Requirements of Flagellation and Motility for Infection of Agrobacterium sp. Strain H13-3 by Flagellotropic Bacteriophage 7-7-1

The flagellotropic phage 7-7-1 specifically adsorbs to Agrobacterium sp. strain H13-3 (formerly Rhizobium lupini H13-3) flagella for efficient host infection. The Agrobacterium sp. H13-3 flagellum is complex and consists of three flagellin proteins: the primary flagellin FlaA, which is essential for motility, and the secondary flagellins FlaB and FlaD, which have minor functions in motility. Using quantitative infectivity assays, we showed that absence of FlaD had no effect on phage infection, while absence of FlaB resulted in a 2.5-fold increase in infectivity. A flaA deletion strain, which produces straight and severely truncated flagella, experienced a significantly reduced infectivity, similar to that of a flaB flaD strain, which produces a low number of straight flagella. A strain lacking all three flagellin genes is phage resistant. In addition to flagellation, flagellar rotation is required for infection. A strain that is nonmotile due to an in-frame deletion in the gene encoding the motor component MotA is resistant to phage infection. We also generated two strains with point mutations in the motA gene resulting in replacement of the conserved charged residue Glu98, which is important for modulation of rotary speed. A change to the neutral Gln caused the flagellar motor to rotate at a constant high speed, allowing a 2.2-fold-enhanced infectivity. A change to the positively charged Lys caused a jiggly motility phenotype with very slow flagellar rotation, which significantly reduced the efficiency of infection. In conclusion, flagellar number and length, as well as speed of flagellar rotation, are important determinants for infection by phage 7-7-1.     

Na<Superscript>+</Superscript> and flagella-dependent swimming of alkaliphilic <Emphasis Type="Italic">Bacillus pseudofirmus</Emphasis> OF4: a basis for poor motility at low pH and enhancement in viscous media in an “up-motile” variant

Flagella-based motility of extremely alkaliphilic Bacillus species is completely dependent upon Na⁺. Little motility is observed at pH values < ∼8.0. Here we examine the number of flagella/cell as a function of growth pH in the facultative alkaliphile Bacillus pseudofirmus OF4 and a derivative selected for increased motility on soft agar plates. Flagella were produced by both strains during growth in a pH range from 7.5 to 10.3. The number of flagella/cell and flagellin levels of cells were not strongly dependent on growth pH over this range in either strain although both of these parameters were higher in the up-motile strain. Assays of the swimming speed indicated no motility at pH < 8 with 10 mM Na⁺, but significant motility at pH 7 at much higher Na⁺ concentrations. At pH 8–10, the swimming speed increased with the increase of Na⁺ concentration up to 230 mM, with fastest swimming at pH 10. Motility of the up-motile strain was greatly increased relative to wild-type on soft agar at alkaline pH but not in liquid except when polyvinylpyrrolidone was added to increase viscosity. The up-motile phenotype, with increased flagella/cell may support bundle formation that particularly enhances motility under a subset of conditions with specific challenges.