11 results on '"Tezuka, T."'
Search Results
2. Involvement of a putative acyltransferase gene in sporangium formation in Actinoplanes missouriensis .
- Author
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Hu S, Maeda S, Tezuka T, and Ohnishi Y
- Subjects
- Bacterial Proteins genetics, Bacterial Proteins metabolism, Spores, Bacterial genetics, Spores, Bacterial growth & development, Spores, Bacterial enzymology, Spores, Bacterial metabolism, Membrane Lipids metabolism, Actinoplanes genetics, Actinoplanes metabolism, Actinoplanes growth & development, Actinoplanes enzymology, Acyltransferases genetics, Acyltransferases metabolism, Sporangia growth & development, Sporangia genetics, Sporangia metabolism
- Abstract
The actinomycete Actinoplanes missouriensis forms branched substrate mycelia during vegetative growth and produces terminal sporangia, each of which contains a few hundred spherical flagellated spores, from the substrate mycelia through short sporangiophores. Based on the observation that remodeling of membrane lipid composition is involved in the morphological development of Streptomyces coelicolor A3(2), we hypothesized that remodeling of membrane lipid composition is also involved in sporangium formation in A. missouriensis . Because some acyltransferases are presumably involved in the remodeling of membrane lipid composition, we disrupted each of the 22 genes annotated as encoding putative acyltransferases in the A. missouriensis genome and evaluated their effects on sporangium formation. The atsA ( AMIS_52390 ) null mutant (Δ atsA ) strain formed irregular sporangia of various sizes. Transmission electron microscopy revealed that some Δ atsA sporangiospores did not mature properly. Phase-contrast microscopy revealed that sporangium dehiscence did not proceed properly in the abnormally small sporangia of the Δ atsA strain, whereas apparently normal sporangia opened to release the spores. Consistently, the number of spores released from Δ atsA sporangia was lower than that released from wild-type sporangia. These phenotypic changes were recovered by introducing atsA with its own promoter into the Δ atsA strain. These results demonstrate that AtsA is required for normal sporangium formation in A. missouriensis , although the involvement of AtsA in the remodeling of membrane lipid composition is unlikely because AtsA is an acyltransferase_3 (AT3) protein, which is an integral membrane protein that usually catalyzes the acetylation of cell surface structures.IMPORTANCE Actinoplanes missouriensis goes through a life cycle involving complex morphological development, including mycelial growth, sporangium formation and dehiscence, swimming as zoospores, and germination to mycelial growth. In this study, we carried out a comprehensive gene disruption experiment of putative acyltransferase genes to search for acyltransferases involved in the morphological differentiation of A. missouriensis . We revealed that a stand-alone acyltransferase_3 domain-containing protein, named AtsA, is required for normal sporangium formation. Although the molecular mechanism of AtsA in sporangium formation, as well as the enzymatic activity of AtsA, remains to be elucidated, the identification of a putative acyltransferase involved in sporangium formation is significant in the study of morphological development of A. missouriensis . This finding will contribute to our understanding of a complex system for producing sporangia, a rare multicellular organism in bacteria., Competing Interests: The authors declare no conflict of interest.
- Published
- 2024
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3. Identification of a putative cell wall-hydrolyzing amidase involved in sporangiospore maturation in Actinoplanes missouriensis .
- Author
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Tan Z, Tezuka T, and Ohnishi Y
- Subjects
- Spores, Hydrolases, Cell Wall, N-Acetylmuramoyl-L-alanine Amidase, Actinoplanes
- Abstract
Actinoplanes missouriensis is a filamentous bacterium that differentiates into terminal sporangia, each containing a few hundred spores. Previously, we reported that a cell wall-hydrolyzing N -acetylglucosaminidase, GsmA, is required for the maturation process of sporangiospores in A. missouriensis ; sporangia of the gsmA null mutant (Δ gsmA ) strain released chains of 2-20 spores under sporangium dehiscence-inducing conditions. In this study, we identified and characterized a putative cell wall hydrolase (AsmA) that is also involved in sporangiospore maturation. AsmA was predicted to have a signal peptide for the general secretion pathway and an N -acetylmuramoyl-l-alanine amidase domain. The transcript level of asmA increased during the early stages of sporangium formation. The asmA null mutant (Δ asmA ) strain showed phenotypes similar to those of the wild-type strain, but sporangia of the Δ gsmA Δ asmA double mutant released longer spore chains than those from the Δ gsmA sporangia. Furthermore, a weak interaction between AsmA and GsmA was detected in a bacterial two-hybrid assay using Escherichia coli as the host. Based on these results, we propose that AsmA is an enzyme that hydrolyzes peptidoglycan at septum-forming sites to separate adjacent spores during sporangiospore maturation in cooperation with GsmA in A. missouriensis .IMPORTANCE Actinoplanes missouriensis produces sporangiospores as dormant cells. The spores inside the sporangia are assumed to be formed from prespores generated by the compartmentalization of intrasporangium hyphae via septation. Previously, we identified GsmA as a cell wall hydrolase responsible for the separation of adjacent spores inside sporangia. However, we predicted that an additional cell wall hydrolase(s) is inevitably involved in the maturation process of sporangiospores because the sporangia of the gsmA null mutant strain released not only tandemly connected spore chains (2-20 spores) but also single spores. In this study, we successfully identified a putative cell wall hydrolase (AsmA) that is involved in sporangiospore maturation in A. missouriensis ., Competing Interests: The authors declare no conflict of interest.
- Published
- 2024
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4. The ssgB gene is required for the early stages of sporangium formation in Actinoplanes missouriensis .
- Author
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Akutsu T, Tezuka T, Maruko M, Hirata A, and Ohnishi Y
- Subjects
- Sporangia metabolism, Bacterial Proteins metabolism, Spores, Bacterial genetics, Spores, Bacterial metabolism, Actinoplanes, Streptomyces genetics, Streptomyces coelicolor genetics, Streptomyces coelicolor metabolism, Actinobacteria metabolism
- Abstract
In Streptomyces , multiple paralogs of SsgA-like proteins (SALPs) are involved in spore formation from aerial hyphae. However, the functions of SALPs have not yet been elucidated in other actinobacterial genera. Here, we report the primary function of an SsgB ortholog (AmSsgB) in Actinoplanes missouriensis , which develops terminal sporangia on the substrate mycelia via short sporangiophores. Importantly, AmSsgB is the sole SALP in A. missouriensis . The transcription of AmssgB was upregulated during sporangium formation, consistent with our previous findings that AmssgB is a member of the AmBldD regulon. The AmssgB null mutant (Δ AmssgB ) strain formed non-globose irregular structures on the substrate mycelium. Transmission electron microscopy revealed that the irregular structures contained abnormally septate hypha-like cells, without an intrasporangial matrix. These phenotypic changes were restored by complementation with AmssgB . Additionally, analysis of the heterologous expression of seven SALP-encoding genes from Streptomyces coelicolor A3(2) ( ssgA-G ) in the Δ AmssgB strain revealed that only ssgB could compensate for AmSsgB deficiency. This indicated that SsgB of S. coelicolor A3(2) and AmSsgB have comparable functions in A. missouriensis . In contrast to the Δ AmssgB strain, the ftsZ -disrupted strain showed a severe growth defect and produced small sporangium-like structures that swelled to some extent. These findings indicate that AmSsgB is crucial for the early stages of sporangium formation, not for spore septum formation in the late stages. We propose that AmSsgB is involved in sporangium formation by promoting the expansion of the "presporangium" structures formed on the tips of the substrate hyphae., Importance: SsgB has been proposed as an archetypical SsgA-like protein with an evolutionarily conserved function in the morphological development of spore-forming actinomycetes. SsgB in Streptomyces coelicolor A3(2) is involved in spore septum formation. However, it is unclear whether this is the primary function of SsgBs in actinobacteria. This study demonstrated that the SsgB ortholog (AmSsgB) in Actinoplanes missouriensis is essential for sporangium expansion, which does not seem to be related to spore septum formation. However, the heterologous expression of ssgB from S. coelicolor A3(2) restored morphological abnormalities in the Δ AmssgB mutant. We propose that the primary function of SsgB is to initiate sporulation in differentiating cells (e.g., aerial hyphae in Streptomyces and "presporangium" cells in A. missouriensis ) although its molecular mechanism remains unknown., Competing Interests: The authors declare no conflict of interest.
- Published
- 2024
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5. Architecture of Actinoplanes missouriensis sporangia and zoospores visualized using quick-freeze deep-etch electron microscopy.
- Author
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Hu S, Tahara YO, Tezuka T, Miyata M, and Ohnishi Y
- Subjects
- Microscopy, Electron, Flagella, Sporangia, Actinoplanes
- Abstract
The architecture of sporangia and zoospores of Actinoplanes missouriensis was analyzed at a high resolution using quick-freeze deep-etch replica electron microscopy. This analysis revealed that (i) sporangia were surrounded by at least 2 membranous layers with smooth surfaces, (ii) zoospores were enclosed by a fibrillar layer, and (iii) flagella were generated in a restricted area on the zoospore surface., (© The Author(s) 2023. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)
- Published
- 2024
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6. A unique sigma/anti-sigma system in the actinomycete Actinoplanes missouriensis.
- Author
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Tezuka T, Mitsuyama K, Date R, and Ohnishi Y
- Subjects
- Sigma Factor genetics, Bacterial Proteins genetics, Bacterial Proteins metabolism, Actinobacteria metabolism, Actinoplanes metabolism
- Abstract
Bacteria of the genus Actinoplanes form sporangia that contain dormant sporangiospores which, upon contact with water, release motile spores (zoospores) through a process called sporangium dehiscence. Here, we set out to study the molecular mechanisms behind sporangium dehiscence in Actinoplanes missouriensis and discover a sigma/anti-sigma system with unique features. Protein σ
SsdA contains a functional sigma factor domain and an anti-sigma factor antagonist domain, while protein SipA contains an anti-sigma factor domain and an anti-sigma factor antagonist domain. Remarkably, the two proteins interact with each other via the anti-sigma factor antagonist domain of σSsdA and the anti-sigma factor domain of SipA. Although it remains unclear whether the SipA/σSsdA system plays direct roles in sporangium dehiscence, the system seems to modulate oxidative stress responses in zoospores. In addition, we identify a two-component regulatory system (RsdK-RsdR) that represses initiation of sporangium dehiscence., (© 2023. The Author(s).)- Published
- 2023
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7. Regulation of Sporangium Formation, Spore Dormancy, and Sporangium Dehiscence by a Hybrid Sensor Histidine Kinase in Actinoplanes missouriensis: Relationship with the Global Transcriptional Regulator TcrA.
- Author
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Hashiguchi Y, Tezuka T, Mouri Y, Konishi K, Fujita A, Hirata A, and Ohnishi Y
- Subjects
- Actinoplanes physiology, Bacterial Proteins genetics, Gene Expression Regulation, Bacterial, Histidine Kinase genetics, Sequence Deletion, Spores, Bacterial enzymology, Actinoplanes enzymology, Bacterial Proteins metabolism, Histidine Kinase metabolism, Spores, Bacterial physiology, Transcription Factors metabolism
- Abstract
The rare actinomycete Actinoplanes missouriensis forms terminal sporangia containing a few hundred flagellated spores. In response to water, the sporangia open and release the spores into external environments. The orphan response regulator TcrA functions as a global transcriptional activator during sporangium formation and dehiscence. Here, we report the characterization of an orphan hybrid histidine kinase, HhkA. Sporangia of an hhkA deletion mutant contained many distorted or ectopically germinated spores and scarcely opened to release the spores under sporangium dehiscence-inducing conditions. These phenotypic changes are quite similar to those observed in a tcrA deletion mutant. Comparative RNA sequencing analysis showed that genes controlled by HhkA mostly overlap TcrA-regulated genes. The direct interaction between HhkA and TcrA was suggested by a bacterial two-hybrid assay, but this was not conclusive. The phosphorylation of TcrA using acetyl phosphate as a phosphate donor markedly enhanced its affinity for the TcrA box sequences in the electrophoretic mobility shift assay. Taking these observations together with other results, we proposed that HhkA and TcrA compose a cognate two-component regulatory system, which controls the transcription of the genes involved in many aspects of morphological development, including sporangium formation, spore dormancy, and sporangium dehiscence in A. missouriensis IMPORTANCE Actinoplanes missouriensis goes through complex morphological differentiation, including formation of flagellated spore-containing sporangia, sporangium dehiscence, swimming of zoospores, and germination of zoospores to filamentous growth. Although the orphan response regulator TcrA globally activates many genes required for sporangium formation, spore dormancy, and sporangium dehiscence, its partner histidine kinase remained unknown. Here, we analyzed the function of an orphan hybrid histidine kinase, HhkA, and proposed that HhkA constitutes a cognate two-component regulatory system with TcrA. That HhkA and TcrA homologues are highly conserved among the genus Actinoplanes and several closely related rare actinomycetes indicates that this possible two-component regulatory system is employed for complex morphological development in sporangium- and/or zoospore-forming rare actinomycetes., (Copyright © 2020 American Society for Microbiology.)
- Published
- 2020
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8. Involvement of three FliA-family sigma factors in the sporangium formation, spore dormancy and sporangium dehiscence in Actinoplanes missouriensis.
- Author
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Hashiguchi Y, Tezuka T, and Ohnishi Y
- Subjects
- Actinoplanes growth & development, Gene Expression Regulation, Bacterial genetics, Promoter Regions, Genetic genetics, Transcription, Genetic genetics, Actinoplanes genetics, Actinoplanes metabolism, Bacterial Proteins genetics, Sigma Factor genetics, Sporangia metabolism, Spores, Bacterial metabolism
- Abstract
The rare actinomycete Actinoplanes missouriensis forms sporangia, which open up and release zoospores in response to water. Here, we report a genetic and functional analysis of four FliA-family sigma factors, FliA1, FliA2, FliA3 and FliA4. Transcription of fliA1, fliA2 and fliA3 was directly activated by the global transcriptional activator TcrA during sporangium formation and dehiscence, while fliA4 was almost always transcribed at low levels. Gene disruption analysis showed that (a) deletion of fliA2 reduced the zoospore swimming speed by half, (b) the fliA1-fliA2 double-deletion mutant formed abnormal sporangia in which mutant spores ectopically germinated and (c) deletion of fliA3 induced no phenotypic changes in the wild-type and mutant strains of fliA1 and/or fliA2. Comparative RNA-Seq analyses among the wild-type and gene deletion mutant strains showed probable targets of each FliA-family sigma factor, indicating that FliA1- and FliA2-dependent promoters are quite similar to each other, while the FliA3-dependent promoter is somewhat different. Gene complementation experiments also indicated that the FliA1 regulon overlaps with the FliA2 regulon. These results demonstrate that A. missouriensis has developed a complex transcriptional regulatory network involving multiple FliA-family sigma factors for the accomplishment of its characteristic reproduction process, including sporangium formation, spore dormancy and sporangium dehiscence., (© 2020 John Wiley & Sons Ltd.)
- Published
- 2020
- Full Text
- View/download PDF
9. Identification and Characterization of a Cell Wall Hydrolase for Sporangiospore Maturation in Actinoplanes missouriensis.
- Author
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Mitsuyama K, Tezuka T, and Ohnishi Y
- Subjects
- Actinoplanes physiology, Actinoplanes enzymology, Cell Wall enzymology, Hydrolases physiology, Spores, Bacterial physiology
- Abstract
The rare actinomycete Actinoplanes missouriensis grows as substrate mycelium and forms terminal sporangia containing a few hundred spores as dormant cells. Upon contact with water, the sporangia open up and release spores to external environments. Here, we report a cell wall hydrolase, GsmA, that is required for sporangiospore maturation in A. missouriensis The gsmA gene is conserved among Actinoplanes species and several species of other rare actinomycetes. Transcription of gsmA is activated in the late stage of sporangium formation by the global transcriptional activator TcrA, which is involved in sporangium formation and dehiscence. GsmA is composed of an N-terminal signal peptide for the twin arginine translocation pathway, two tandem bacterial SH3-like domains, and a glucosaminidase domain. Zymographic analysis using a recombinant C-terminal glucosaminidase domain protein showed that GsmA is a hydrolase able to digest cell walls extracted from the vegetative mycelia of A. missouriensis and Streptomyces griseus A gsmA deletion mutant (Δ gsmA ) formed apparently normal sporangia, but they released chains of 2 to 20 spores under sporangium dehiscence-inducing conditions, indicating that spores did not completely mature in the mutant sporangia. From these results, we concluded that GsmA is a cell wall hydrolase for digesting peptidoglycan at septum-forming sites to separate adjacent spores during sporangiospore maturation in A. missouriensis Unexpectedly, flagella were observed around the spore chains of the Δ gsmA mutant by transmission electron microscopy. The flagellar formation was strictly restricted to cell-cell interfaces, giving an important insight into the polarity of the flagellar biogenesis in a spherical spore. IMPORTANCE In streptomycetes, an aerial hypha is compartmentalized by multiple septations into prespores, which become spores through a series of maturation processes. However, little is known about these maturation processes. The rare actinomycete Actinoplanes missouriensis produces sporangiospores, which are assumed to be formed also from prespores generated by the compartmentalization of intrasporangium hyphae via septation. The identification of GsmA as a cell wall hydrolase for the separation of adjacent spores sheds light on the almost unknown processes of sporangiospore formation in A. missouriensis Furthermore, the fact that GsmA orthologues are conserved within the genus Actinoplanes but not in streptomycetes indicates that Actinoplanes has developed an original strategy for the spore maturation in a specific environment, that is, inside a sporangium., (Copyright © 2019 American Society for Microbiology.)
- Published
- 2019
- Full Text
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10. Characterization of Zoospore Type IV Pili in Actinoplanes missouriensis.
- Author
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Kimura T, Tezuka T, Nakane D, Nishizaka T, Aizawa SI, and Ohnishi Y
- Subjects
- Actinoplanes physiology, Fimbriae Proteins genetics, Fimbriae, Bacterial genetics, Gene Expression Regulation, Bacterial, Spores, Bacterial genetics, Actinoplanes genetics, Fimbriae Proteins metabolism, Fimbriae, Bacterial metabolism, Spores, Bacterial physiology
- Abstract
The rare actinomycete Actinoplanes missouriensis produces terminal sporangia containing a few hundred flagellated spores. After release from the sporangia, the spores swim rapidly in aquatic environments as zoospores. The zoospores stop swimming and begin to germinate in niches for vegetative growth. Here, we report the characterization and functional analysis of zoospore type IV pili in A. missouriensis The pilus gene ( pil ) cluster, consisting of three apparently σ
FliA -dependent transcriptional units, is activated during sporangium formation similarly to the flagellar gene cluster, indicating that the zoospore has not only flagella but also pili. With a new method in which zoospores were fixed with glutaraldehyde to prevent pilus retraction, zoospore pili were observed relatively easily using transmission electron microscopy, showing 6 ± 3 pili per zoospore ( n = 37 piliated zoospores) and a length of 0.62 ± 0.35 μm ( n = 206), via observation of fliC -deleted, nonflagellated zoospores. No pili were observed in the zoospores of a prepilin-encoding pilA deletion (Δ pilA ) mutant. In addition, the deletion of pilT , which encodes an ATPase predicted to be involved in pilus retraction, substantially reduced the frequency of pilus retraction. Several adhesion experiments using wild-type and Δ pilA zoospores indicated that the zoospore pili are required for the sufficient adhesion of zoospores to hydrophobic solid surfaces. Many zoospore-forming rare actinomycetes conserve the pil cluster, which indicates that the zoospore pili yield an evolutionary benefit in the adhesion of zoospores to hydrophobic materials as footholds for germination in their mycelial growth. IMPORTANCE Bacterial zoospores are interesting cells in that their physiological state changes dynamically: they are dormant in sporangia, show temporary mobility after awakening, and finally stop swimming to germinate in niches for vegetative growth. However, the cellular biology of a zoospore remains largely unknown. This study describes unprecedented zoospore type IV pili in the rare actinomycete Actinoplanes missouriensis Similar to the case for the usual bacterial type IV pili, zoospore pili appeared to be retractable. Our findings that the zoospore pili have a functional role in the adhesion of zoospores to hydrophobic solid surfaces and that the zoospores use both pili and flagella properly according to their different purposes provide an important insight into the cellular biology of the zoospore., (Copyright © 2019 American Society for Microbiology.)- Published
- 2019
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11. Involvement of β-Alkylation Machinery and Two Sets of Ketosynthase-Chain-Length Factors in the Biosynthesis of Fogacin Polyketides in Actinoplanes missouriensis.
- Author
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Sato K, Katsuyama Y, Yokota K, Awakawa T, Tezuka T, and Ohnishi Y
- Subjects
- Actinoplanes genetics, Alkylation, Dimerization, Genes, Bacterial, Multigene Family, Substrate Specificity, Actinoplanes metabolism, Polyketide Synthases metabolism, Polyketides metabolism
- Abstract
Fogacin and two novel fogacin derivatives, fogacins B and C, were isolated from the rare actinomycete Actinoplanes missouriensis. Biosynthesis of fogacin C apparently requires β alkylation of a polyketide chain. The fogacin biosynthetic type II polyketide synthase (PKS) gene cluster contains a hydroxymethylglutaryl-coenzyme A synthase (HCS) cassette, which is usually responsible for β alkylation in the type I PKS system. Another characteristic of the fog cluster is that it encodes two sets of ketosynthase (KS) and chain-length factor (CLF). Inactivation of either of the two KS genes in A. missouriensis and heterologous expression of the HCS cassette with either of the two KS-CLF genes in Streptomyces albus indicated that each KS-CLF had a different starter substrate specificity: one preferred an unusual β-alkylated starter and the other preferred a normal acetyl starter. This study expands knowledge of HCS cassette-dependent β alkylation into the type II PKS system and provides a natural example of combinatorial biosynthesis for producing diverse polyketides from different starter substrates., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2019
- Full Text
- View/download PDF
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