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Azospirillum spp. are nitrogen-fixing rhizobacteria with the potential to increase the yield of economically important cereals and grasses (18). Motility and chemotaxis are thought to be important factors for efficient plant colonization (21). Azospirillum spp. move using flagella (for a review on flagellar motility, see reference 12). Within the genus Azospirillum, Azospirillum brasilense, A. lipoferum, and A. irakense have mixed flagellation: a single polar flagellum when grown in a liquid medium and additional lateral flagella when grown on a solid medium (10, 20). The polar flagellum of A. brasilense rotates in both clockwise and counterclockwise directions and is required for swimming in liquid media (22). The lateral flagella are thinner in diameter and have a shorter wavelength (20) and are required for movement across solid surfaces, i.e., swarming (6). Mixed flagellation is typical of several proteobacterial species which can both swim and swarm (for a review, see reference 14).

A. lipoferum 4B produces a stable variant form, 4V_I, at high frequencies as a result of a phenotypic switching process similar to phase variation in other bacteria (2). Compared to the parental strain, the 4V_I variant shows pleiotropic modifications of metabolic and morphologic characteristics, including changes in motility. The 4V_I variant is not capable of swimming. In the present study, we analyzed the motility and the flagellation pattern of the 4V_I variant of A. lipoferum in comparison with its parental strain, 4B.

A. lipoferum 4B and 4V_I cells (2) were grown in tryptone-yeast extract medium at 30°C. Swimming motility was observed by using a phase-contrast microscope (Zeiss, Jena, Germany). Flagellation of bacteria was observed by using a Philips CM 120 transmission electron microscope. Grids coated with Formvar and carbon were incubated for 30 s in a drop of a bacterial suspension and then for 20 to 30 s in 1% sodium silicotungstate. Spreading of bacteria through the semisoft medium or across the surface of the solid medium was tested on plates with different agar concentrations. Cell fractionation was performed as described previously (5). Induction of lateral flagella was achieved by incubating liquid bacterial cultures (2 × 10⁸ cells/ml) with different concentrations of antiflagellum antibodies as described previously for A. brasilense Sp7 (17). Lateral flagellin was then detected by using the As-Laf polyclonal antibody (see below) on whole-cell protein extracts. Polar and lateral flagella were obtained by the method of Alberti and Harshey (1). Protein concentration was determined by the bicinchoninic acid protein assay (Pierce, Rockford, Ill.). Polar (Fla) and lateral (Laf) flagellins were purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (11). Gels were stained with copper (Copper Stain and Destain kit; Bio-Rad, Richmond, Calif.). Bands corresponding to polar and lateral flagellins were cut and destained. Each protein was electroeluted (Electro-eluter 422; Bio-Rad) and then lyophilized. Purified lyophilized flagellins were injected into New Zealand White rabbits. Antisera were collected 2 weeks after the final injection. The specificity of each antiserum (As-Laf and As-Fla) was checked on Western blots of whole-cell protein extracts from A. lipoferum 4B and 4V_I. No cross-reaction was detected for the As-Laf antibody; the As-Fla antibody cross-reacted slightly with lateral flagellin. For immunodetection of flagellins, samples were subjected to SDS-PAGE and electroblotting (Cera Labo, Aubervilliers, France) onto a nitrocellulose membrane. The membranes were incubated in a 1:100,000 (As-Fla) or 1:50,000 (As-Laf) dilution of each flagellin-specific antiserum, followed by a goat anti-rabbit immunoglobulin G-peroxidase conjugate. Blotted proteins were sugar stained by using the periodic acid-Schiff technique as described previously for A. brasilense Sp7 (16).

Motility of A. lipoferum 4B and its variant, 4V_I, in agar media. In liquid media, A. lipoferum 4B is motile whereas the 4V_I variant is not (2). In agar-containing media, two types of motility were observed in A. lipoferum: swimming (spreading through the semisoft medium) and swarming (spreading across the surface) (Table 1). Low agar concentrations (0.1 to 0.2%) were optimal for swimming of the wild type, whereas only very limited spreading was observed for the variant (Table 1). No typical swimming motility (22) was observed in variant cells recovered from 0.1 or 0.2% agar. Both wild-type and variant cells showed optimal swarming on media containing 0.4 to 0.6% agar (Table 1). Thus, the 4V_I variant was capable of swarming but not of swimming.

Flagellation of A. lipoferum 4V_I. The lack of swimming motility can be due to a paralysis of the flagellar motor (Mot) or a deficiency in flagellar synthesis or assembly (Fla). First, electron microscopy was used in order to distinguish between these possibilities. The wild type, A. lipoferum 4B, had a single polar flagellum when grown in liquid medium and mixed flagellation when grown on solid medium (Fig. 1A and B). No polar flagellum was observed in the 4V_I variant either in liquid or on solid medium, whereas lateral flagella were constitutively expressed (Fig. 1C and D). Therefore, the variant has a Fla phenotype. The variant produced more lateral flagella when grown on solid medium than when grown in liquid medium. In contrast to the best-studied mixed flagellated bacterium, Vibrio parahaemolyticus (3, 13, 14), A. lipoferum hyperflagellated swarming cells were not elongated (Fig. 1B and D). The presence of constitutively expressed lateral flagella in the 4V_I cells grown in liquid media may explain the ability of these cells to spread through the medium at low agar concentrations (Table 1).

View larger version (155K): [in this window] [in a new window]   FIG. 1.   Transmission electron micrographs of A. lipoferum 4B (A and B) and 4VI (C and D) cells. Cells were grown in liquid (A and C) and on solid (B and D) media. The lateral flagella are thinner in diameter and have a shorter wavelength. Abbreviations: F, polar flagellum; L, lateral flagella. Bars, 1 µm (A, B, and D) and 0.7 µm (C).

Polar and lateral flagellins. The lateral flagellin, Laf, of A. lipoferum 4B had an apparent molecular size of approximately 45 kDa (Fig. 2), which is similar to that of A. brasilense Sp7 (17). However, the apparent molecular size of the polar flagellin, Fla, of A. lipoferum 4B was slightly higher; 110 kDa (Fig. 2), versus 100 kDa in A. brasilense Sp7 (16). As in A. brasilense Sp7 (16), Fla appeared to be glycosylated, as revealed by periodic acid-Schiff staining (data not shown).

View larger version (39K): [in this window] [in a new window]   FIG. 2.   Detection of polar and lateral flagellins in intracellular protein extracts of A. lipoferum 4B (lanes 1) and 4VI (lanes 2) cells grown in liquid medium (A and B) and on solid medium (C and D) by using polyclonal As-Fla (A and C) and As-Laf (B and D) antibodies. The same number of wild-type and variant cells was recovered (108 cells/ml from liquid medium and 109 cells/ml from solid medium). Equal amounts of total protein (260 µg) were loaded in each sample on the gel. Arrows indicate positions of the 45-kDa lateral flagellin (Laf) and the 110-kDa polar flagellin (Fla). Molecular mass markers (in kilodaltons) are indicated on the left. The weak signal corresponding to Laf observed in 4B liquid-grown cells (panel B, lane 1) may be due to a small fraction of cells for which the rotation of the polar flagellum was impaired due to cell-cell aggregation or adherence to the surface of the tube (see text for details).

Laf expression in the wild type and the 4V_I variant. Expression of lateral flagella in mixed flagellated bacteria is controlled by a polar flagellum (3, 13, 17). V. parahaemolyticus mutants defective in the polar flagellum constitutively expressed lateral flagella, indicating that this control is negative (13). The polar flagellum senses a physical constraint of its movement, such as increased viscosity of the medium or agglutination with antiflagellum antibody (13, 14). As in other mixed flagellated bacteria, the polar flagellum of A. lipoferum 4B appeared to negatively control expression of lateral flagella. Addition of the As-Fla polyclonal antibody to the 4B cultures caused cell agglutination, several cells being bound together at one pole and rotating (data not shown). Under these conditions, Laf induction correlated with the concentration of the antiserum in the medium (Fig. 3A). Impeded movement of the polar flagellum by the specific As-Fla antibody led to lateral flagellum expression in liquid medium (data not shown), as it did on solid medium (Fig. 1B). The same results were obtained for A. brasilense (17), the most closely related species of the same genus. Since the polar flagellum may negatively control lateral-flagellum expression in A. lipoferum 4B, we propose that the lack of a polar flagellum in the 4V_I variant led to a lack of the control, and thus lateral flagella were expressed constitutively.

View larger version (77K): [in this window] [in a new window]   FIG. 3.   Induction of expression of lateral flagellin in A. lipoferum 4B (A) and 4VI (B) cells by antibody agglutination of the polar or lateral flagella. Polar flagellin (As-Fla) or lateral flagellin (As-Laf) polyclonal antibodies diluted in ultrapure water were added to exponential-phase-grown cultures of A. lipoferum 4B and 4VI, respectively. Whole-cell protein extracts were used for immunodetection of lateral flagellin. Equal amounts of total protein (260 µg) were loaded in each sample on the gel. Incubation with preimmune antisera led to the same result (data not shown) as that in the control experiment, in which cells were incubated with ultrapure water (panels A and B, lanes 1). Antibodies (As-Fla [A] or As-Laf [B]) were added to final dilutions of 100 (lane 2), 101 (lane 3), 102 (lane 4), 103 (lane 5), 104 (lane 6), and 105 (lane 7).