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 m, 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% KH 2 PO 4 , 0.2% humic acid, and 1% trace element solution, pH 7.5) at 30°C for 2 weeks. 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 Ϯ 25 m/s (mean Ϯ standard deviation) (Table 1). In comparison, those of S. Typhimurium and Bacillus subtilis cells measured under the same conditions were 29 Ϯ 5 m/s and 11 Ϯ 5 m/s, respectively. It is known that the Kineosporia sp. motile spores can swim at a speed of 160 m/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.Flagellar biogenesis observed by microscopy. A. missouriensis spores in the process of the life...

Genetic and Transcriptional Analyses of the Flagellar Gene Cluster in Actinoplanes missouriensis

“…In our previous study, the flagellar filament was purified and analyzed via sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Only one band was observed at 44 kDa in the gel, and this size was in agreement with the predicted molecular size of the filament protein (flagellin, FliC) encoded by AMIS76190 (fliC) (6). To confirm that fliC encodes the filament protein, we constructed a fliC deletion mutant (⌬fliC), which formed sporangia normally.…”

“…Moreover, this strain produces hundreds of spherical spores within each of the terminal sporangia. The spores are encased within a membranous substance called a "spore sheath" (6). The spores are released from the sporangia upon contact with water through a process known as dehiscence.…”

“…However, pure distilled water cannot induce the dehiscence of sporangia, and thus an unknown compound(s) in soil extract is required (7). After the release of spores from the sporangia, the spore sheaths are removed to generate zoospores, which begin swimming using the flagella at an amazingly rapid speed (6). Zoospores have chemotactic properties for several types of substrates, including sugars, amino acids, aromatic compounds, and mineral ions (7).…”

“…Zoospores can swim for a short period and stop swimming to germinate within a few hours. Therefore, the flagellation of A. missouriensis is temporally regulated during its life cycle (6).…”

Regulation of Sporangium Formation by BldD in the Rare Actinomycete Actinoplanes missouriensis

“…The RNA-Seq analyses were performed using RNA samples prepared from the cells cultivated on HAT agar for 1, 3, 6, and 40 days (29). c Orthologues in S. coelicolor A3 (2). When the organization of each gene locus is conserved in S. coelicolor A3(2), the gene is regarded as an orthologue of the corresponding S. coelicolor A3(2) gene.…”