RESUMEN
Natural products have provided many molecules to treat and prevent illnesses in humans, animals and plants. While only a small fraction of the existing microbial diversity has been explored for bioactive metabolites, tens of thousands of molecules have been reported in the literature over the past 80 years. Thus, the main challenge in microbial metabolite screening is to avoid the re-discovery of known metabolites in a cost-effective manner. In this perspective, we report and discuss different approaches used in our laboratory over the past few years, ranging from bioactivity-based screening to looking for metabolic rarity in different datasets to deeply investigating a single Streptomyces strain. Our results show that it is possible to find novel chemistry through a limited screening effort, provided that appropriate selection criteria are in place.
Asunto(s)
Bacterias/metabolismo , Productos Biológicos/metabolismo , Biblioteca de Genes , Animales , Bacterias/química , Bacterias/genética , Productos Biológicos/química , Investigación Biomédica , Evaluación Preclínica de Medicamentos , HumanosRESUMEN
NAI-112, a glycosylated, labionine-containing lanthipeptide with weak antibacterial activity, has demonstrated analgesic activity in relevant mouse models of nociceptive and neuropathic pain. However, the mechanism(s) through which NAI-112 exerts its analgesic and antibacterial activities is not known. In this study, we analyzed changes in the spinal cord lipidome resulting from treatment with NAI-112 of naive and in-pain mice. Notably, NAI-112 led to an increase in phosphatidic acid levels in both no-pain and pain models and to a decrease in lysophosphatidic acid levels in the pain model only. We also showed that NAI-112 can form complexes with dipalmitoyl-phosphatidic acid and that Staphylococcus aureus can become resistant to NAI-112 through serial passages at sub-inhibitory concentrations of the compound. The resulting resistant mutants were phenotypically and genotypically related to vancomycin-insensitive S. aureus strains, suggesting that NAI-112 binds to the peptidoglycan intermediate lipid II. Altogether, our results suggest that NAI-112 binds to phosphate-containing lipids and blocks pain sensation by decreasing levels of lysophosphatidic acid in the TRPV1 pathway.
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Analgésicos/farmacología , Farmacorresistencia Bacteriana/efectos de los fármacos , Péptidos/farmacología , Staphylococcus aureus/metabolismo , Animales , Masculino , Ratones , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/metabolismoRESUMEN
Covering: up to February 2019Actinomycetes are Gram positive bacteria of the phylum Actinobacteria. These organisms are one of the most important sources of structurally diverse, clinically used antibiotics and other valuable bioactive products, as well as biotechnologically relevant enzymes. Most strains were discovered by their ability to produce a given molecule and were often poorly characterized, physiologically and genetically. The development of genetic methods for Streptomyces and related filamentous actinomycetes has led to the successful manipulation of antibiotic biosynthesis to attain structural modification of microbial metabolites that would have been inaccessible by chemical means and improved production yields. Moreover, genome mining reveals that actinomycete genomes contain multiple biosynthetic gene clusters (BGCs), however only a few of them are expressed under standard laboratory conditions, leading to the production of the respective compound(s). Thus, to access and activate the so-called "silent" BGCs, to improve their biosynthetic potential and to discover novel natural products methodologies for genetic manipulation are required. Although different methods have been applied for many actinomycete strains, genetic engineering is still remaining very challenging for some "underexplored" and poorly characterized actinomycetes. This review summarizes the strategies developed to overcome the obstacles to genetic manipulation of actinomycetes and allowing thereby rational genetic engineering of this industrially relevant group of microorganisms. At the end of this review we give some tips to researchers with limited or no previous experience in genetic manipulation of actinomycetes. The article covers the most relevant literature published until February 2019.
Asunto(s)
Actinobacteria/genética , Productos Biológicos/metabolismo , Ingeniería Metabólica , Actinobacteria/metabolismo , Clonación Molecular , Ingeniería Metabólica/métodos , Familia de Multigenes/genéticaRESUMEN
Mechanotransduction in the mammalian auditory system depends on mechanosensitive channels in the hair bundles that project from the apical surface of the sensory hair cells. Individual stereocilia within each bundle contain a core of tightly packed actin filaments, whose length is dynamically regulated during development and in the adult. We show that the actin-binding protein epidermal growth factor receptor pathway substrate 8 (Eps8)L2, a member of the Eps8-like protein family, is a newly identified hair bundle protein that is localized at the tips of stereocilia of both cochlear and vestibular hair cells. It has a spatiotemporal expression pattern that complements that of Eps8. In the cochlea, whereas Eps8 is essential for the initial elongation of stereocilia, Eps8L2 is required for their maintenance in adult hair cells. In the absence of both proteins, the ordered staircase structure of the hair bundle in the cochlea decays. In contrast to the early profound hearing loss associated with an absence of Eps8, Eps8L2 null-mutant mice exhibit a late-onset, progressive hearing loss that is directly linked to a gradual deterioration in hair bundle morphology. We conclude that Eps8L2 is required for the long-term maintenance of the staircase structure and mechanosensory function of auditory hair bundles. It complements the developmental role of Eps8 and is a candidate gene for progressive age-related hearing loss.
Asunto(s)
Células Ciliadas Auditivas/patología , Pérdida Auditiva/genética , Proteínas de Microfilamentos/deficiencia , Análisis de Varianza , Animales , Audiometría de Respuesta Evocada , Células Ciliadas Auditivas/fisiología , Células Ciliadas Auditivas/ultraestructura , Ratones , Ratones Noqueados , Proteínas de Microfilamentos/genética , Microscopía Electrónica , Técnicas de Placa-ClampRESUMEN
In the search for structurally novel metabolites with antibacterial activity, innovative approaches must be implemented to increase the probability of discovering novel chemistry from microbial sources. Here we report on the application of metabolomic tools to the genus Actinoallomurus, a poorly explored member of the Actinobacteria. From examining extracts derived from 88 isolates belonging to this genus, we identified a family of cyclodepsipeptides acylated with a C20 polyketide chain, which we named allopeptimicins. These molecules possess unusual structural features, including several double bonds in the amino-polyketide chain and four non-proteinogenic amino acids in the octapeptide. Remarkably, allopeptimicins are produced as a complex of active and inactive congeners, the latter carrying a sulfate group on the polyketide amine. This modification is also a mechanism of self-protection in the producer strain. The structural uniqueness of allopeptimicins is reflected in a biosynthetic gene cluster showing a mosaic structure, with dedicated gene cassettes devoted to formation of specialized precursors and modular assembly lines related to those from different pathways.
RESUMEN
The glycopeptide A40926, produced by the actinomycete Nonomuraea gerenzanensis, is the precursor of dalbavancin, a second-generation glycopeptide antibiotic approved for clinical use in the USA and Europe in 2014 and 2015, respectively. The final product of the biosynthetic pathway is an O-acetylated form of A40926 (acA40926). Glycopeptide biosynthesis in N. gerenzanensis is dependent upon the dbv gene cluster that encodes, in addition to the two essential positive regulators Dbv3 and Dbv4, the putative members of a two-component signal transduction system, specifically the response regulator Dbv6 and the sensor kinase Dbv22. The aim of this work was to assign a role to these two genes. Our results demonstrate that deletion of dbv22 leads to an increased antibiotic production with a concomitant reduction in glycopeptide resistance. Deletion of dbv6 results in a similar phenotype, although the effects are not as strong as in the Δdbv22 mutant. Consistently, quantitative RT-PCR analysis showed that Dbv6 and Dbv22 negatively regulate the regulatory genes (dbv3 and dbv4), as well as some dbv biosynthetic genes (dbv23 and dbv24), whereas Dbv6 and Dbv22 positively regulate transcription of the single, cluster-associated resistance gene. Finally, we demonstrate that exogenously added acA40926 and its precursor A40926 can modulate transcription of dbv genes but with an opposite extent: A40926 strongly stimulates transcription of the Dbv6/Dbv22 target genes while acA40926 has a neutral or negative effect on transcription of those genes. We propose a model in which glycopeptide biosynthesis in N. gerenzanensis is modulated through a positive feedback by the biosynthetic precursor A40926 and a negative feedback by the final product acA40926. In addition to previously reported control systems, this sophisticated control loop might help the producing strain cope with the toxicity of its own product. This work, besides leading to improved glycopeptide producing strains, enlarges our knowledge on the regulation of glycopeptide biosynthesis in actinomycetes, setting N. gerenzanensis and its two-component system Dbv6-Dbv22 apart from other glycopeptide producers.
Asunto(s)
Actinobacteria/metabolismo , Antibacterianos/metabolismo , Teicoplanina/análogos & derivados , Actinobacteria/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Vías Biosintéticas , Regulación Bacteriana de la Expresión Génica , Genes Reguladores , Familia de Multigenes , Teicoplanina/metabolismoRESUMEN
Signaling from receptor tyrosine kinases (RTKs)* requires the sequential activation of the small GTPases Ras and Rac. Son of sevenless (Sos-1), a bifunctional guanine nucleotide exchange factor (GEF), activates Ras in vivo and displays Rac-GEF activity in vitro, when engaged in a tricomplex with Eps8 and E3b1-Abi-1, a RTK substrate and an adaptor protein, respectively. A mechanistic understanding of how Sos-1 coordinates Ras and Rac activity is, however, still missing. Here, we demonstrate that (a) Sos-1, E3b1, and Eps8 assemble into a tricomplex in vivo under physiological conditions; (b) Grb2 and E3b1 bind through their SH3 domains to the same binding site on Sos-1, thus determining the formation of either a Sos-1-Grb2 (S/G) or a Sos-1-E3b1-Eps8 (S/E/E8) complex, endowed with Ras- and Rac-specific GEF activities, respectively; (c) the Sos-1-Grb2 complex is disrupted upon RTKs activation, whereas the S/E/E8 complex is not; and (d) in keeping with the previous result, the activation of Ras by growth factors is short-lived, whereas the activation of Rac is sustained. Thus, the involvement of Sos-1 at two distinct and differentially regulated steps of the signaling cascade allows for coordinated activation of Ras and Rac and different duration of their signaling within the cell.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteína SOS1/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Proteínas ras/metabolismo , Animales , Células COS , Proteínas Portadoras/metabolismo , Línea Celular , Proteínas del Citoesqueleto , Activación Enzimática , Proteína Adaptadora GRB2 , Péptidos y Proteínas de Señalización Intracelular , Cinética , Sustancias Macromoleculares , Modelos Biológicos , Unión Proteica , Proteínas/metabolismo , Transducción de SeñalRESUMEN
The exponential increase in available microbial genome sequences coupled with predictive bioinformatic tools is underscoring the genetic capacity of bacteria to produce an unexpected large number of specialized bioactive compounds. Since most of the biosynthetic gene clusters (BGCs) present in microbial genomes are cryptic, i.e. not expressed under laboratory conditions, a variety of cloning systems and vectors have been devised to harbor DNA fragments large enough to carry entire BGCs and to allow their transfer in suitable heterologous hosts. This minireview provides an overview of the vectors and approaches that have been developed for cloning large BGCs, and successful examples of heterologous expression.
Asunto(s)
Bacterias/genética , Cromosomas Artificiales/genética , Clonación Molecular/métodos , Genómica/métodos , Proteínas Bacterianas/genética , Biología Computacional , Vectores Genéticos/genéticaRESUMEN
In screening for novel antibiotics, an attractive element of novelty can be represented by screening previously underexplored groups of microorganisms. We report the results of screening 200 strains belonging to the actinobacterial genus Actinoallomurus for their production of antibacterial compounds. When grown under just one condition, about half of the strains produced an extract that was able to inhibit growth of Staphylococcus aureus. We report here on the metabolites produced by 37 strains. In addition to previously reported aminocoumarins, lantibiotics and aromatic polyketides, we described two novel and structurally unrelated polyethers, designated α-770 and α-823. While we identified only one producer strain of the former polyether, 10 independent Actinoallomurus isolates were found to produce α-823, with the same molecule as main congener. Remarkably, production of α-823 was associated with a common lineage within Actinoallomurus, which includes A.fulvus and A.amamiensis. All polyether producers were isolated from soil samples collected in tropical parts of the world.
RESUMEN
We report the genome sequence of Planobispora rosea ATCC 53733, a mycelium-forming soil-dweller belonging to one of the lesser studied genera of Actinobacteria and producing the thiopeptide GE2270. The P. rosea genome presents considerable convergence in gene organization and function with other members in the family Streptosporangiaceae, with a significant number (44%) of shared orthologs. Patterns of gene expression in P. rosea cultures during exponential and stationary phase have been analyzed using whole transcriptome shotgun sequencing and by proteome analysis. Among the differentially abundant proteins, those involved in protein metabolism are particularly represented, including the GE2270-insensitive EF-Tu. Two proteins from the pbt cluster, directing GE2270 biosynthesis, slightly increase their abundance values over time. While GE2270 production starts during the exponential phase, most pbt genes, as analyzed by qRT-PCR, are down-regulated. The exception is represented by pbtA, encoding the precursor peptide of the ribosomally synthesized GE2270, whose expression reached the highest level at the entry into stationary phase.
Asunto(s)
Actinomycetales/genética , Actinomycetales/metabolismo , Genoma Bacteriano , Péptidos Cíclicos/biosíntesis , Proteoma/análisis , Transcriptoma , Actinomycetales/crecimiento & desarrollo , Proteínas Bacterianas/análisis , Proteínas Bacterianas/metabolismo , Genómica , Glucosa/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Redes y Vías Metabólicas/genética , Familia de Multigenes , ARN Bacteriano/análisis , Análisis de Secuencia de ARN , TiazolesRESUMEN
GE2270 is a thiopeptide antibiotic generated by extensive posttranslational modifications of a ribosomally generated precursor peptide. Thiopeptides are especially active against Gram-positive bacteria, including methicillin resistant Staphylococcus aureus (MRSA). In this study the GE2270 biosynthetic gene cluster (pbt) from Planobispora rosea ATCC 53733 was successfully expressed in the heterologous host strain Streptomyces coelicolor M1146. Notably, exconjugants containing the pbt gene cluster could only be obtained after deletion of the major part of the ribosomal genes flanking the gene cluster. This is a striking example that genes belonging to primary metabolism can prevent the successful conjugative transfer of DNA from phylogenetic distant species and thus complicate heterologous expression of secondary metabolite gene clusters. GE2270 production in the heterologous producer strain increased after introduction of the constitutive ermE* promoter upstream of the GE2270 resistance gene tuf from P. rosea. Insertion of the inducible tcp830 promoter resulted in inducible GE2270 production. When the regulatory gene pbtR was deleted, the resulting strain ceased to produce GE2270, suggesting an essential role of PbtR as a putative transcriptional activator of GE2270 expression.
Asunto(s)
Actinomycetales/genética , Antibacterianos/biosíntesis , Proteínas Bacterianas/biosíntesis , ADN Ribosómico/genética , Péptidos Cíclicos/biosíntesis , Streptomyces coelicolor/genética , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/farmacología , Clonación Molecular , Cósmidos/genética , Pruebas Antimicrobianas de Difusión por Disco , Farmacorresistencia Bacteriana , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Tipificación Molecular , Familia de Multigenes , Péptidos Cíclicos/genética , Péptidos Cíclicos/farmacología , Filogenia , Regiones Promotoras Genéticas , ARN Ribosómico 16S/genética , Streptomyces coelicolor/efectos de los fármacos , Tiazoles/farmacología , Activación TranscripcionalRESUMEN
Thiopeptides are ribosomally synthesized, posttranslationally modified peptides with potent activity against Gram-positives. However, only GE2270 has yielded semisynthetic derivatives under clinical investigations. The pbt gene cluster from the GE2270 producer Planobispora rosea was successfully expressed in the genetically tractable Nonomuraea ATCC39727. Gene deletions established that PbtO, PbtM1, PbtM2, PbtM3, and PbtM4 are involved in regiospecific hydroxylation and methylations of GE2270, leading to the generation of various derivatives with altered decorations. Further deletions established that PbtH and PbtG1 are involved in C-terminal amide and oxazoline formation, respectively. Surprisingly, preventing either step resulted in the accumulation of linear precursors in which the pyridine-generated macrocycle failed to form, and only one of the pyridine-forming serine residues had been dehydrated. Often, these linear precursors present a shortened C terminus but retain the full set of methylation and hydroxylation decorations.
Asunto(s)
Actinomycetales/genética , Actinomycetales/metabolismo , Antibacterianos/metabolismo , Péptidos Cíclicos/metabolismo , Tiazoles/metabolismo , Secuencia de Aminoácidos , Antibacterianos/química , Genes Fúngicos , Datos de Secuencia Molecular , Familia de Multigenes , Oxazoles/química , Oxazoles/metabolismo , Péptidos Cíclicos/química , Péptidos Cíclicos/genética , Piridinas/química , Piridinas/metabolismo , Tiazoles/químicaAsunto(s)
Antibacterianos/aislamiento & purificación , Descubrimiento de Drogas , Macrólidos/aislamiento & purificación , Micromonosporaceae/metabolismo , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacología , Cromatografía Líquida de Alta Presión , Depsipéptidos/agonistas , Depsipéptidos/farmacología , Agonismo de Drogas , Macrólidos/química , Macrólidos/metabolismo , Macrólidos/farmacología , Espectrometría de Masas , Resistencia a la Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/crecimiento & desarrollo , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Reproducibilidad de los Resultados , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/crecimiento & desarrollo , Estereoisomerismo , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pneumoniae/crecimiento & desarrollo , Streptococcus pyogenes/efectos de los fármacos , Streptococcus pyogenes/crecimiento & desarrolloRESUMEN
BACKGROUND: In a variety of organisms, including mammals, caloric restriction improves metabolic status and lowers the incidence of chronic-degenerative diseases, ultimately leading to increased lifespan. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that knockout mice for Eps8, a regulator of actin dynamics, display reduced body weight, partial resistance to age- or diet-induced obesity, and overall improved metabolic status. Alteration in the liver gene expression profile, in behavior and metabolism point to a calorie restriction-like phenotype in Eps8 knockout mice. Additionally, and consistent with a calorie restricted metabolism, Eps8 knockout mice show increased lifespan. The metabolic alterations in Eps8 knockout mice correlated with a significant reduction in intestinal fat absorption presumably caused by a 25% reduction in intestinal microvilli length. CONCLUSIONS/SIGNIFICANCE: Our findings implicate actin dynamics as a novel variable in the determination of longevity. Additionally, our observations suggest that subtle differences in energy balance can, over time, significantly affect bodyweight and metabolic status in mice.
Asunto(s)
Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/fisiología , Proteínas del Citoesqueleto/fisiología , Mucosa Intestinal/metabolismo , Células 3T3-L1 , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Peso Corporal , Células CACO-2 , Restricción Calórica , Proteínas del Citoesqueleto/genética , Metabolismo Energético , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microvellosidades/metabolismoRESUMEN
Dynamic modulation of the actin cytoskeleton is critical for synaptic plasticity, abnormalities of which are thought to contribute to mental illness and addiction. Here we report that mice lacking Eps8, a regulator of actin dynamics, are resistant to some acute intoxicating effects of ethanol and show increased ethanol consumption. In the brain, the N-methyl-D-aspartate (NMDA) receptor is a major target of ethanol. We show that Eps8 is localized to postsynaptic structures and is part of the NMDA receptor complex. Moreover, in Eps8 null mice, NMDA receptor currents and their sensitivity to inhibition by ethanol are abnormal. In addition, Eps8 null neurons are resistant to the actin-remodeling activities of NMDA and ethanol. We propose that proper regulation of the actin cytoskeleton is a key determinant of cellular and behavioral responses to ethanol.
Asunto(s)
Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Conducta Animal/efectos de los fármacos , Depresores del Sistema Nervioso Central/farmacología , Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Etanol/farmacología , Proteínas Adaptadoras Transductoras de Señales/genética , Consumo de Bebidas Alcohólicas , Animales , Conducta Animal/fisiología , Células Cultivadas , Cerebelo/citología , Proteínas del Citoesqueleto/genética , Citoesqueleto/efectos de los fármacos , Femenino , Humanos , Masculino , Ratones , Ratones Noqueados , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal/fisiologíaRESUMEN
EPS8 codes for a protein essential in Ras to Rac signaling leading to actin remodeling. Three genes highly homologous to EPS8 were discovered, thereby defining a novel gene family. Here, we report the genomic structure of EPS8 and the EPS8-related genes in human and mouse. We performed BLASTN searches against the Celera Human Genome and Mouse Fragments Database. The mouse fragments were manually assembled, and the organization of both human and mouse genes was reconstructed. The gene structures in Celera annotations of the human and mouse genomes were compared to outline correspondences and divergences. We also compared the EPS8 family gene structures predicted by Celera with those predicted by NCBI. Moreover, we performed a virtual analysis of the expression of the EPS8 gene family members by using the SAGEmap Database in NCBI. Finally, we analyzed the domain organization of the gene products and their evolutionary conservation to define novel putative domains, thereby helping to predict novel modality of action for the members of this gene family. The data obtained will be instrumental in directing further experimental functional characterization of these genes.