Characterization of Actinoplanes missouriensis Spore Flagella

Abstract

Actinoplanes missouriensis spores swim with a tuft of flagella. Flagella of newborn spores are wrapped with a membranous sheath. When the sheath is unwrapped, spores start swimming. Flagellar length is kept short, at around 1.9 μm, which covers half the circumference of the spore.

Actinoplanes species are Gram-positive, soil-inhabiting, filamentous bacteria that characteristically produce spores within a terminal sporangium (20, 21). Spores are released from sporangia upon contact with water; the process is termed dehiscence (8). Actinoplanes spores grow into mycelia and eventually form sporangia under adverse conditions (7, 8).

Interestingly, a range of genera belonging to the order Actinomycetales, such as Actinoplanes, Catenuloplanes, Kineosporia, and Spirillospora, produce spores that possess flagella and are motile (4, 8, 13). Some Actinoplanes species possess a polar tuft of flagella or a bundle of flagella originating from a narrow area of the cell wall (8), while others have peritrichous flagella. Extensive studies on bacterial flagella have been performed using Salmonella enterica serovar Typhimurium (S. Typhimurium) (14). It will be very interesting to see how closely the spore flagella of Actinomycetales, which are phylogenetically distinct from S. Typhimurium, could be related with the paradigmatic flagella. In this study, we examined spore flagella of the Actinoplanes missouriensis 431 strain (NBRC 102363).

Motility of flagellated spores.

Water is not sufficient to trigger the release of spores from sporangia of A. missouriensis, but some unknown chemicals in the soil are required (4, 7, 13). To obtain motile spores, we used “activation medium,” 10 mM phosphate buffer (pH 7.0) supplemented with soil extract, which is the water extract of the soil rich with decayed leaves. A. missouriensis mycelia were grown on 2% agar plates containing HAT medium (0.1% sucrose, 0.01% Casamino Acids, 0.05% KH₂PO₄, 0.2% humic acid, and 1% trace element solution, pH 7.5) at 30°C for 2 weeks. Trace element solution contains the following metal ions: ZnCl₂, 40 mg/liter; FeCl₃·6H₂O, 200 mg/liter; CuCl₂·2H₂O, 10 mg/liter; MnCl₂·4H₂O, 10 mg/liter; Na₂B₄O₇·10H₂O, 10 mg/liter; (NH₄)₆Mo₇O₂₄·4H₂O, 10 mg/liter. Matured sporangia formed on agar plates were collected with prewarmed phosphate buffer or the activation medium.

Cell motility was analyzed with a phase-contrast microscope, as previously described (15). Within 10 min after washing, spores were released from sporangia, and 20% of the population in a scope of the dark-field microscope started to swim. One hour later, the motile fraction was as high as 80% of the population observed and gradually decreased as time passed, and spore germination began. Three hours later, spores grew into mycelia, at which point spores lost motility. The average swimming speed of the motile spores was 135 ± 25 μm/s (mean ± standard deviation) (Table 1). In comparison, those of S. Typhimurium and Bacillus subtilis cells measured under the same conditions were 29 ± 5 μm/s and 11 ± 5 μm/s, respectively. It is known that the Kineosporia sp. motile spores can swim at a speed of 160 μm/s (17), which is as fast as Bdellovibrio cells (10). A. missouriensis spores belong to the fastest group among bacterial swimming species so far studied.

TABLE 1.

Summary of physicochemical properties of A. missouriensis spore flagella

Property	Measured value	Notes and reference(s)
Swimming speed of spores (μm/s)	135 ± 25 (n = 15)	160 for Kineosporia sp. (17), 29 ± 5 (n = 10) for S. Typhimurium, and 11 ± 5 (n = 10) for B. subtilis (this study)
Number of flagella per spore	15 ± 9 (n = 100)	>80 for Methanococcus voltae (2)
Length of flagella (μm)	1.89 ± 0.25 (n = 23)	5.55 ± 1.52 for S. Typhimurium (n = 33) (this study)
Diameter of flagellar circles (μm)	1.53 ± 0.16 (n = 48)	1.50 for coiled flagella of E. coli (5)
Percent of flagellar length versus spore circumference	40 (1.89/4.80 = 0.394)	Average length of flagella was divided by the average value of the spore circumferences

Flagellar biogenesis observed by microscopy.

A. missouriensis spores in the process of the life cycle were observed by electron microscopy in parallel with light microscopic observations. Electron microscopy was carried out as previously described (5). In brief, at 5 min after the addition of the activation medium, most of the spores released were not flagellated (Fig. 1A). At 10 min, many spores had flagella, which looked to be forming many layers around the spore. At 15 min, flagella were spread out around the spore, agreeing with microscopic observations that many spores swam at this point. Flagella in the fully spread form looked curved (Fig. 1B). In detail, at 5 min, there were two types of spores observed in the preparation of phosphotungstic acid (PTA)-stained samples: one appeared darkly stained, and the other lightly stained (Fig. 1C). The latter appeared to be a membranous empty sheath with a large crevice in the middle of the body (Fig. 1C, left). The crevice was also observed in the former spores, and a few flagella occasionally stuck out from the crevice (Fig. 1C, right). Pay attention to the picture at 10 min (Fig. 1A), which does not show a section of a cell but rather a side view of an intact spore. Thus, “many layers of flagella” does not necessarily mean that flagella overlap one over another, but that flagella may cover the surface once. Coincidentally, the sheath with a crevice was gone from those flagellated spores. There have been no reports on whether spores in sporangia have an extra sheath over the cell wall (3, 11, 12, 18, 19). We suspect that spores right after release from sporangia might still be wrapped with that newly found membranous sheath, and by unwrapping the sheath from the spore in a similar manner as ecdysis, flagella would be exposed and become free to move.

FIG. 1.

Electron micrographs of A. missouriensis spores and flagella. (A) The spore surface after 5, 10, and 15 min of incubation with the activation medium; (B) flagella fully spread out from a spore. Curved filaments on a spore were traced with circles for length measurement. (C) At 5 min, there are two types of spores: a darkly stained spore (right) and lightly stained spore (left). (D) The basal structure was occasionally seen in spontaneously broken spores. An arrow indicates the MS ring complex. Samples were stained with 2% PTA.

Flagellar basal body.

We have attempted to isolate the flagellar basal body. However, our conventional method for isolation of the basal body failed, because the spheroplast formation using lysozyme did not work (1). Instead, we looked for spontaneously lysed spores in the culture and observed the flagellar base on the membranes. As seen in Fig. 1D, the basal structure showed only two rings, which is typical for Gram-positive bacteria, as contrasted with four rings for Gram-negative bacteria (1, 6).

Number and length of flagella on a spore.

The number of flagella per spore varies from 1 to several tens among bacterial species (13). We counted the number of A. missouriensis flagella surrounding a spore in electron microscopic images of spores that were recovered from a HAT plate after 1 h of incubation with the activation medium. Spores with 10 to 19 flagella were most abundant; the average ± standard deviation was 15 ± 9 flagella per spore (Table 1).

To observe the growing position of flagella on a spore, we treated spores with osmotic shock as previously described (16), so that spores became half translucent under the electron microscope. It should be noted that A. missouriensis flagella grow from one side of the spore rather than from all over as seen for peritrichous flagella, indicating that the spore has polar flagella despite its round cell body. It is rare for cocci to retain polar flagella in eubacterial species, but another example of cocci retaining polar flagella can be seen in archaeal species, such as Methanococcus voltae, in which more than 80 flagella grow from one side of the cell body (2).

It is a hydrodynamic demand for flagella of a swimming cell to form a bundle or a tuft behind the cell body, which can be seen by dark-field microscopy. However, A. missouriensis flagella were too short to observe by light microscopy. By electron microscopy, there were no wavy filaments but only curved filaments (Fig. 2A). We did not see any fragmented filaments or any signs of degradation of filaments. The average ± standard deviation of flagellar length was 1.89 ± 0.25 μm (Table 1). It should be noted that this length distribution is much narrower than that of Salmonella flagella (Fig. 2B, Table 1), suggesting a mechanism to control the flagellar length to be short.

FIG. 2.

Numbers and lengths of flagella per spore. (A) Electron microscopic image of curved filaments on a spore; (B) distribution of flagellar length of A. missouriensis spore flagella (white bars) and S. Typhimurium flagella (black bars).

Ordinary flagella are long helices with several pitches. Spore flagella are too short to make a complete helix, and the whole length was never observed. How did we measure the complete length from part of a helix? First, we measured the diameter of circles that fit the curved filaments, assuming that the circle would be a flattened shape of an imaginary complete helix (Fig. 1B). The average diameter was 1.53 ± 0.16 μm. The circumference or periphery length of the circle is 1.53π μm, which equals 4.80 μm. We calculated that the flagellar length observed was only 40% of a circle (Table 1). Thus, we suspect that a flagellum would cover only half the spore periphery. If flagella self-assembled in a small space between the sheath and the cell wall, flagella may start growing radially from one point of the spore and stop growing when the length reaches the other side, where all the tips of growing filaments gather and clash. This physical hindrance at the poles will allow a flagellum to wrap only half the spore surface, as observed.

Flagellin.

Spore flagella were sheared off by vortexing, purified by differential centrifugation, and analyzed by SDS-PAGE as previously described (5). There was only one band, at 44 kDa, in the gel (Fig. 3), which agrees with the predicted molecular size of FliC deduced from the fliC gene (GenBank accession number AB600179), and was the only one found on the genome.

FIG. 3.

Purified flagella showed one band at 44 kDa (lane 2) in SDS gel. Lane 1 shows the low-range molecular markers. Acrylamide concentration was 12.5% (16). Gels were stained with silver (15).

Our study suggests that flagellar assembly in A. missouriensis spores might finish when spores are matured. First, we found folded flagella on spores at as early as 15 min after washing. In S. Typhimurium, it takes more than 30 min to construct the basal structure (9). Since A. missouriensis spore flagella have only two rings instead of four rings (like those of S. Typhimurium), it may take a shorter time to complete flagellar assembly. Another possibility is that flagellar basal structures may be completed in spores; flagellar filaments will be added to the basal structures upon release from sporangia. This working hypothesis awaits a direct proof.