RESUMEN
The survival strategy of members of the bacterial genus Actinoplanes is fascinating from morphological and evolutionary perspectives. A brief motile phase is incorporated in the filamentous and resting stages of the life cycle of Actinoplanes missouriensis. Spores either lie dormant or swim under different external conditions. This review presents microscopic observations and molecular genetic analyses of A. missouriensis morphological development. Selected examples of the characterization of developmental genes and their products are also introduced.
Asunto(s)
Actinoplanes , Actinoplanes/genética , Actinoplanes/metabolismo , Esporas Bacterianas/genética , Esporas Bacterianas/crecimiento & desarrollo , MicroscopíaRESUMEN
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 (ΔatsA) strain formed irregular sporangia of various sizes. Transmission electron microscopy revealed that some ΔatsA sporangiospores did not mature properly. Phase-contrast microscopy revealed that sporangium dehiscence did not proceed properly in the abnormally small sporangia of the ΔatsA strain, whereas apparently normal sporangia opened to release the spores. Consistently, the number of spores released from ΔatsA sporangia was lower than that released from wild-type sporangia. These phenotypic changes were recovered by introducing atsA with its own promoter into the ΔatsA 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.IMPORTANCEActinoplanes 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.
Asunto(s)
Actinoplanes , Aciltransferasas , Esporangios , Actinoplanes/genética , Actinoplanes/metabolismo , Actinoplanes/crecimiento & desarrollo , Actinoplanes/enzimología , Aciltransferasas/genética , Aciltransferasas/metabolismo , Esporangios/crecimiento & desarrollo , Esporangios/genética , Esporangios/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Esporas Bacterianas/genética , Esporas Bacterianas/crecimiento & desarrollo , Esporas Bacterianas/enzimología , Esporas Bacterianas/metabolismo , Lípidos de la Membrana/metabolismoRESUMEN
Glycopeptide antibiotics (GPAs) are among the most clinically successful antimicrobials. GPAs inhibit cell-wall biosynthesis in Gram-positive bacteria via binding to lipid II. Natural GPAs are produced by various actinobacteria. Being themselves Gram-positives, the GPA producers evolved sophisticated mechanisms of self-resistance to avoid suicide during antibiotic production. These self-resistance genes are considered the primary source of GPA resistance genes actually spreading among pathogenic enterococci and staphylococci. The GPA-resistance mechanism in Actinoplanes teichomyceticusthe producer of the last-resort-drug teicoplaninhas been intensively studied in recent years, posing relevant questions about the role of Tei3 sensor histidine kinase. In the current work, the molecular properties of Tei3 were investigated. The setup of a GPA-responsive assay system in the model Streptomyces coelicolor allowed us to demonstrate that Tei3 functions as a non-inducible kinase, conferring high levels of GPA resistance in A. teichomyceticus. The expression of different truncated versions of tei3 in S. coelicolor indicated that both the transmembrane helices of Tei3 are crucial for proper functioning. Finally, a hybrid gene was constructed, coding for a chimera protein combining the Tei3 sensor domain with the kinase domain of VanS, with the latter being the inducible Tei3 ortholog from S. coelicolor. Surprisingly, such a chimera did not respond to teicoplanin, but indeed to the related GPA A40926. Coupling these experimental results with a further in silico analysis, a novel scenario on GPA-resistance and biosynthetic genes co-evolution in A. teichomyceticus was hereby proposed.
Asunto(s)
Actinoplanes , Actinoplanes/efectos de los fármacos , Actinoplanes/genética , Antibacterianos/farmacología , Glicopéptidos , Teicoplanina/farmacología , Factores de TranscripciónRESUMEN
The knowledge on sulfur incorporation mechanism involved in sulfur-containing molecule biosynthesis remains limited. Chuangxinmycin is a sulfur-containing antibiotic with a unique thiopyrano[4,3,2-cd]indole (TPI) skeleton and selective inhibitory activity against bacterial tryptophanyl-tRNA synthetase. Despite the previously reported biosynthetic gene clusters and the recent functional characterization of a P450 enzyme responsible for C-S bond formation, the enzymatic mechanism for sulfur incorporation remains unknown. Here, we resolve this central biosynthetic problem by in vitro biochemical characterization of the key enzymes and reconstitute the TPI skeleton in a one-pot enzymatic reaction. We reveal that the JAMM/MPN+ protein Cxm3 functions as a deubiquitinase-like sulfurtransferase to catalyze a non-classical sulfur-transfer reaction by interacting with the ubiquitin-like sulfur carrier protein Cxm4GG. This finding adds a new mechanism for sulfurtransferase in nature.
Asunto(s)
Antibacterianos/biosíntesis , Proteínas Bacterianas/metabolismo , Sulfurtransferasas/metabolismo , Actinoplanes/genética , Actinoplanes/metabolismo , Antibacterianos/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Indoles/análisis , Indoles/química , Indoles/metabolismo , Familia de Multigenes , Pyrococcus/enzimología , Pyrococcus/genética , Azufre/metabolismo , Sulfurtransferasas/química , Sulfurtransferasas/genética , Ubiquitinación , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMEN
Echinocandin B deacylase (ECBD) from Actinoplanes utahensis can be applied to produce echinocandin B nucleus (ECBN), an essential intermediate of the echinocandins antifungal drugs such as anidulafungin. To date, the expression of ECBD has been limited to Streptomyces. To achieve the active expression of ECBD in Escherichia coli (E. coli), we constructed a plasmid carrying two subunits of ECBD for T7 RNA polymerase driven transcription of dicistron messenger after codon optimization. Subsequently, the introduction of peptide tags in the recombinant ECBD was adopted to reduce the formation of inclusion bodies and enhance the ECBD solubility. The peptide tags with the opposite electrostatic charge, hexa-lysine (6K) and GEGEG (GE), exhibited the best positive effect, which was verified by activity assay and structural simulation. After that, optimization of culture conditions and characterization of ECBD were conducted, the optimal pH and temperature were 7.0 and 60 °C. It is the first report concerning the functional expression of ECBD in the host E. coli. Our results reported here can provide a reference for the high-level expression of other deacylases with respect to a possible industrial application.
Asunto(s)
Actinoplanes/enzimología , Amidohidrolasas/metabolismo , Proteínas Bacterianas/metabolismo , Equinocandinas/metabolismo , Escherichia coli/enzimología , Proteínas Fúngicas/metabolismo , Actinoplanes/genética , Amidohidrolasas/genética , Anidulafungina/metabolismo , Antifúngicos/metabolismo , Proteínas Bacterianas/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Concentración de Iones de Hidrógeno , Oligopéptidos/genética , Oligopéptidos/metabolismo , Solubilidad , Especificidad por Sustrato , TemperaturaRESUMEN
The biosynthesis of the glycopeptide antibiotics (GPAs) demonstrates the exceptional ability of nonribosomal peptide (NRP) synthesis to generate diverse and complex structures from an expanded array of amino acid precursors. Whilst the heptapeptide cores of GPAs share a conserved C terminus, including the aromatic residues involved cross-linking and that are essential for the antibiotic activity of GPAs, most structural diversity is found within the N terminus of the peptide. Furthermore, the origin of the (D)-stereochemistry of residue 1 of all GPAs is currently unclear, despite its importance for antibiotic activity. Given these important features, we have now reconstituted modules (M) 1-4 of the NRP synthetase (NRPS) assembly lines that synthesise the clinically relevant type IV GPA teicoplanin and the related compound A40926. Our results show that important roles in amino acid modification during the NRPS-mediated biosynthesis of GPAs can be ascribed to the actions of condensation domains present within these modules, including the incorporation of (D)-amino acids at position 1 of the peptide. Our results also indicate that hybrid NRPS assembly lines can be generated in a facile manner by mixing NRPS proteins from different systems and that uncoupling of peptide formation due to different rates of activity seen for NRPS modules can be controlled by varying the ratio of NRPS modules. Taken together, this indicates that NRPS assembly lines function as dynamic peptide assembly lines and not static megaenzyme complexes, which has significant implications for biosynthetic redesign of these important biosynthetic systems.
Asunto(s)
Actinobacteria/metabolismo , Actinoplanes/metabolismo , Antibacterianos/biosíntesis , Biosíntesis de Péptidos Independientes de Ácidos Nucleicos , Péptido Sintasas/genética , Teicoplanina/análogos & derivados , Teicoplanina/biosíntesis , Actinobacteria/genética , Actinoplanes/genética , Secuencia de Aminoácidos , Antibacterianos/química , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Ingeniería Genética/métodos , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Estructura Molecular , Péptido Sintasas/metabolismo , Dominios Proteicos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Teicoplanina/químicaRESUMEN
Actinoplanes sp. SE50/110 (ATCC 31044) is the wild type of industrial producer strains of acarbose. Acarbose has been used since the early 1990s as an inhibitor of intestinal human α-glucosidases in the medical treatment of type II diabetes mellitus. The small secreted protein Cgt, which consists of a single carbohydrate-binding module (CBM) 20-domain, was found to be highly expressed in Actinoplanes sp. SE50/110 in previous studies, but neither its function nor a possible role in the acarbose formation was explored, yet. Here, we demonstrated the starch-binding function of the Cgt protein in a binding assay. Transcription analysis showed that the cgt gene was strongly repressed in the presence of glucose or lactose. Due to this and its high abundance in the extracellular proteome of Actinoplanes, a functional role within the sugar metabolism or in the environmental stress protection was assumed. However, the gene deletion mutant ∆cgt, constructed by CRISPR/Cas9 technology, displayed no apparent phenotype in screening experiments testing for pH and osmolality stress, limited carbon source starch, and the excess of seven different sugars in liquid culture and further 97 carbon sources in the Omnilog Phenotypic Microarray System of Biolog. Therefore, a protective function as a surface protein or a function within the retainment and the utilization of carbon sources could not be experimentally validated. Remarkably, enhanced production of acarbose was determined yielding into 8-16% higher product titers when grown in maltose-containing medium.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Acarbosa/metabolismo , Actinoplanes/genética , Actinoplanes/metabolismo , Proteínas Bacterianas/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/genética , Sistemas CRISPR-Cas , Metabolismo de los Hidratos de Carbono , Eliminación de Gen , Familia de Multigenes , Unión Proteica , Proteoma/metabolismo , Almidón/metabolismoRESUMEN
Teicoplanin (Tcp) is a clinically relevant glycopeptide antibiotic (GPA) that is produced by the actinobacterium Actinoplanes teichomyceticus. Tcp is a front-line therapy for treating severe infections caused by multidrug-resistant Gram-positive pathogens in adults and infants. In this review, we provide a detailed overview of how Tcp is produced by A. teichomyceticus by describing Tcp biosynthesis, regulation, and resistance. We summarize the knowledge gained from in vivo and in vitro studies to provide an integrated model of teicoplanin biosynthesis. Then, we discuss genetic and nutritional factors that contribute to the regulation of teicoplanin biosynthesis, focusing on those that have been successfully applied for improving teicoplanin production. A current view on teicoplanin self-resistance mechanisms in A. teichomyceticus is given, and we compare the Tcp biosynthetic gene cluster with other glycopeptide gene clusters from actinoplanetes and from unidentified isolates/metagenomics samples. Finally, we provide an outlook for further directions in studying Tcp biosynthesis and regulation.
Asunto(s)
Actinoplanes/genética , Actinoplanes/metabolismo , Antibacterianos/biosíntesis , Regulación Bacteriana de la Expresión Génica , Familia de Multigenes , Teicoplanina/biosíntesis , Antibacterianos/química , Bacterias/efectos de los fármacos , Vías Biosintéticas , Teicoplanina/químicaRESUMEN
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.
Asunto(s)
Actinoplanes/genética , Actinoplanes/metabolismo , Proteínas Bacterianas/genética , Factor sigma/genética , Esporangios/metabolismo , Esporas Bacterianas/metabolismo , Actinoplanes/crecimiento & desarrollo , Regulación Bacteriana de la Expresión Génica/genética , Regiones Promotoras Genéticas/genética , Transcripción Genética/genéticaRESUMEN
The urgent need for discovering new bioactive metabolites prompts exploring novel actinobacterial taxa by developing appropriate tools for their genome mining and rational genetic engineering. One promising source of new bioactive natural products is the genus Actinoplanes, a home to filamentous sporangia-forming actinobacteria producing many important specialized metabolites such as teicoplanin, ramoplanin, and acarbose. Here we describe the development of a gene expression system for a new Actinoplanes species, A. rectilineatus (NRRL B-16090), which is a potential producer of moenomycin-like antibiotics. We have determined the optimal conditions for spore formation in A. rectilineatus and a plasmid transfer procedure for its engineering via intergeneric E. coli-A. rectilineatus conjugation. The φC31- and pSG5-based vectors were successfully transferred into A. rectilineatus, but φBT1- and VWB-based vectors were not transferable. Finally, using the glucuronidase reporter system, we assessed the strength of several heterologous promoters for gene expression in A. rectilineatus.
Asunto(s)
Actinoplanes/genética , Expresión Génica , Plásmidos/genética , Actinoplanes/ultraestructura , Regulación de la Expresión Génica , Ingeniería Genética , Vectores Genéticos/genética , Regiones Promotoras Genéticas , Esporas Bacterianas/ultraestructuraRESUMEN
Moenomycin-type antibiotics are phosphoglycolipids that are notable for their unique modes of action and have proven to be useful in animal nutrition. The gene clusters tchm from Actinoplanes teichomyceticus and moe from Streptomyces are among a limited number of known moenomycin-biosynthetic pathways. Most genes in tchm have counterparts in the moe cluster, except for tchmy and tchmz, the functions of which remain unknown. Sequence analysis indicates that TchmY belongs to the isoprenoid enzyme C2-like superfamily and may serve as a prenylcyclase. The enzyme was proposed to be involved in terminal cyclization of the moenocinyl chain in teichomycin, leading to the diumycinol chain of moenomycin isomers. Here, recombinant TchmY protein was expressed in Escherichia coli and its crystal structure was solved by SIRAS. Structural analysis and comparison with other prenylcyclases were performed. The overall fold of TchmY consists of an (α/α)6-barrel, and a potential substrate-binding pocket is found in the central chamber. These results should provide important information regarding the biosynthetic basis of moenomycin antibiotics.
Asunto(s)
Actinoplanes/enzimología , Proteínas Bacterianas/química , Teicoplanina/biosíntesis , Actinoplanes/genética , Transferasas Alquil y Aril/química , Antibacterianos/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Familia de Multigenes , Oligosacáridos/química , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Teicoplanina/químicaRESUMEN
Fidaxomicin, an 18-membered macrolide antibiotic, is highly active against Clostridium difficile, the most common cause of diarrhea in hospitalized patients. Though the biosynthetic mechanism of fidaxomicin has been well studied, little is known about its regulatory mechanism. Here, we reported that FadR1, a LAL family transcriptional regulator in the fidaxomicin cluster of Actinoplanes deccanensis Yp-1, acts as an activator for fidaxomicin biosynthesis. The disruption of fadR1 abolished the ability to synthesize fidaxomicin, and production could be restored by reintegrating a single copy of fadR1. Overexpression of fadR1 resulted in an approximately 400 % improvement in fidaxomicin production. Electrophoretic mobility shift assays indicated that fidaxomicin biosynthesis is under the control of FadR1 through its binding to the promoter regions of fadM, fadA1-fadP2, fadS2-fadC, and fadE-fadF, respectively. And the conserved binding sites of FadR1 within the four promoter regions were determined by footprinting experiment. All results indicated that fadR1 encodes a pathway-specific positive regulator of fidaxomicin biosynthesis and upregulates the transcription levels of most of genes by binding to the four above intergenic regions. In summary, we not only clearly elucidate the regulatory mechanism of FadR1 but also provide strategies for the construction of industrial high-yield strain of fidaxomicin.
Asunto(s)
Actinoplanes/metabolismo , Antibacterianos/biosíntesis , Proteínas Bacterianas/metabolismo , Fidaxomicina/metabolismo , Proteínas Represoras/metabolismo , Actinoplanes/genética , Proteínas Bacterianas/genética , Vías Biosintéticas , Clostridioides difficile/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica , Proteínas Represoras/genéticaRESUMEN
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 σFliA-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 (ΔpilA) 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 ΔpilA 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.
Asunto(s)
Actinoplanes/genética , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/metabolismo , Esporas Bacterianas/fisiología , Actinoplanes/fisiología , Proteínas Fimbrias/genética , Fimbrias Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Esporas Bacterianas/genéticaRESUMEN
Fogacin and two novel fogacin derivatives, fogacinsâ B and C, were isolated from the rare actinomycete Actinoplanes missouriensis. Biosynthesis of fogacinâ C apparently requires ßâ alkylation of a polyketide chain. The fogacin biosynthetic typeâ II polyketide synthase (PKS) gene cluster contains a hydroxymethylglutaryl-coenzymeâ A synthase (HCS) cassette, which is usually responsible for ßâ alkylation in the typeâ 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.â 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 ß-alkylated starter and the other preferred a normal acetyl starter. This study expands knowledge of HCS cassette-dependent ßâ alkylation into the typeâ II PKS system and provides a natural example of combinatorial biosynthesis for producing diverse polyketides from different starter substrates.