RESUMEN
The α-1,3-glucanase Agl-EK14 from Flavobacterium sp. EK-14 comprises a signal peptide (SP), a catalytic domain (CAT), a first immunoglobulin-like domain (Ig1), a second immunoglobulin-like domain (Ig2), a ricin B-like lectin domain (RicinB), and a carboxy-terminal domain (CTD). SP and CTD are predicted to be involved in extracellular secretion, while the roles of Ig1, Ig2, and RicinB are unclear. To clarify their roles, domain deletion enzymes Agl-EK14ΔRicinB, Agl-EK14ΔIg2RicinB, and Agl-EK14ΔIg1Ig2RicinB were constructed. The insoluble α-1,3-glucan hydrolytic, α-1,3-glucan binding, and fungal cell wall hydrolytic activities of the deletion enzymes were almost the same and lower than those of Agl-EK14. Kinetic analysis revealed that the Km values of the deletion enzymes were similar and uniformly higher than those of Agl-EK14. These results suggest that the deletion of RicinB causes a decline in binding and hydrolytic activity and increases the Km value. To confirm the role of RicinB, Ig1, Ig2, and RicinB were fused with green fluorescent protein (GFP). As a result, RicinB-fused GFP (GFP-RicinB) showed binding to insoluble α-1,3-glucan and Aspergillus oryzae cell walls, whereas Ig1- and Ig2-fused GFP did not. These results indicated that RicinB is involved in α-1,3-glucan binding. The fusion protein GFP-Ig1Ig2RicinB was also constructed and GFP-Ig1Ig2RicinB showed strong binding to the cell wall of A. oryzae compared to GFP-RicinB. Gel filtration column chromatography suggested that the strong binding was due to GFP-Ig1Ig2RicinB loosely associated with itself.
Asunto(s)
Pared Celular , Flavobacterium , Glucanos , Dominios Proteicos , Flavobacterium/enzimología , Flavobacterium/genética , Flavobacterium/metabolismo , Pared Celular/metabolismo , Glucanos/metabolismo , Hidrólisis , Dominio Catalítico , Cinética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Glicósido Hidrolasas/metabolismo , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/química , Señales de Clasificación de ProteínaRESUMEN
Monolignols and their derivatives exhibit various pharmaceutical and physiological characteristics, such as antioxidant and anti-inflammatory properties. However, they remain difficult to synthesize. In this study, we engineered several whole-cell bioconversion systems with carboxylate reductase (CAR)-mediated pathways for efficient synthesis of p-coumaryl, caffeyl, and coniferyl alcohols from l-tyrosine in Escherichia coli BL21 (DE3). By overexpressing the l-tyrosine ammonia lyase from Flavobacterium johnsoniae (FjTAL), carboxylate reductase from Segniliparus rugosus (SruCAR), alcohol dehydrogenase YqhD and hydroxylase HpaBC from E. coli, and caffeate 3-O-methyltransferase (COMT) from Arabidopsis thaliana, three enzyme cascades FjTAL-SruCAR-YqhD, FjTAL-SruCAR-YqhD-HpaBC, and FjTAL-SruCAR-YqhD-HpaBC-COMT were constructed to produce 1028.5 mg/L p-coumaryl alcohol, 1015.3 mg/L caffeyl alcohol, and 411.4 mg/L coniferyl alcohol from 1500, 1500, and 1000 mg/L l-tyrosine, with productivities of 257.1, 203.1, and 82.3 mg/L/h, respectively. This work provides an efficient strategy for the biosynthesis of p-coumaryl, caffeyl, and coniferyl alcohols from l-tyrosine.
Asunto(s)
Escherichia coli , Ingeniería Metabólica , Tirosina , Tirosina/metabolismo , Tirosina/química , Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Metiltransferasas/metabolismo , Metiltransferasas/genética , Arabidopsis/metabolismo , Arabidopsis/genética , Flavobacterium/metabolismo , Flavobacterium/enzimología , Flavobacterium/genética , Oxidorreductasas/metabolismo , Oxidorreductasas/genética , Lignina/metabolismo , Lignina/química , Amoníaco-Liasas/metabolismo , Amoníaco-Liasas/genética , Amoníaco-Liasas/química , FenolesRESUMEN
Environmental pollution stands as one of the significant global challenges we face today. Polycyclic aromatic hydrocarbons (PAHs), a class of stubborn organic pollutants, have long been a focal point of bioremediation research. This study aims to explore the impact and mechanisms of graphene oxide (GO) on the phytoremediation effectiveness of PAHs. The results underscore the significant efficacy of GO in accelerating the degradation of PAHs. Additionally, the introduction of GO altered the diversity and community structure of endophytic bacteria within the roots, particularly those genera with potential for PAH degradation. Through LEfSe analysis and correlation studies, we identified specific symbiotic bacteria, such as Mycobacterium, Microbacterium, Flavobacterium, Sphingomonas, Devosia, Bacillus, and Streptomyces, which coexist and interact under the influence of GO, synergistically degrading PAHs. These bacteria may serve as key biological markers in the PAH degradation process. These findings provide new theoretical and practical foundations for the application of nanomaterials in plant-based remediation of polluted soils and showcase the immense potential of plant-microbe interactions in environmental restoration.
Asunto(s)
Bacterias , Biodegradación Ambiental , Grafito , Hidrocarburos Policíclicos Aromáticos , Microbiología del Suelo , Contaminantes del Suelo , Grafito/química , Hidrocarburos Policíclicos Aromáticos/metabolismo , Contaminantes del Suelo/metabolismo , Bacterias/efectos de los fármacos , Bacterias/metabolismo , Endófitos/metabolismo , Raíces de Plantas/microbiología , Sphingomonas/metabolismo , Plantas/microbiología , Plantas/metabolismo , Mycobacterium/efectos de los fármacos , Mycobacterium/metabolismo , Flavobacterium/efectos de los fármacos , Flavobacterium/metabolismo , Streptomyces/metabolismo , Microbacterium/metabolismoRESUMEN
Plant root-associated environments such as the rhizosphere, rhizoplane, and endosphere, are notably different from non-root-associated soil environments. However, the microbial dynamics in these spatially divided compartments remain unexplored. In this study, we propose a combinational analysis of single-cell genomics with 16S rRNA gene sequencing. This method enabled us to understand the entire soil microbiome and individual root-associated microorganisms. We applied this method to soybean microbiomes and revealed that their composition was different between the rhizoplane and rhizosphere in the early growth stages, but became more similar as growth progressed. In addition, a total of 610 medium- to high-quality single-amplified genomes (SAGs) were acquired, including plant growth-promoting rhizobacteria (PGPR) candidates while genomes with high GC content tended to be missed by SAGs. The whole-genome analyses of the SAGs suggested that rhizoplane-enriched Flavobacterium solubilizes organophosphate actively and Bacillus colonizes roots more efficiently. Single-cell genomics, together with 16S rRNA gene sequencing, enabled us to connect microbial taxonomy and function, and assess microorganisms at a strain resolution even in the complex soil microbiome.
Asunto(s)
Glycine max , Microbiota , Raíces de Plantas , ARN Ribosómico 16S , Rizosfera , Análisis de la Célula Individual , Microbiología del Suelo , Glycine max/microbiología , Raíces de Plantas/microbiología , ARN Ribosómico 16S/genética , Bacterias/clasificación , Bacterias/genética , Bacterias/metabolismo , Bacterias/aislamiento & purificación , Flavobacterium/genética , Flavobacterium/clasificación , Flavobacterium/metabolismoRESUMEN
The type IX secretion system (T9SS) is a large multi-protein transenvelope complex distributed into the Bacteroidetes phylum and responsible for the secretion of proteins involved in pathogenesis, carbohydrate utilization or gliding motility. In Porphyromonas gingivalis, the two-component system PorY sensor and response regulator PorX participate to T9SS gene regulation. Here, we present the crystal structure of PorXFj, the Flavobacterium johnsoniae PorX homolog. As for PorX, the PorXFj structure is comprised of a CheY-like N-terminal domain and an alkaline phosphatase-like C-terminal domain separated by a three-helix bundle central domain. While not activated and monomeric in solution, PorXFj crystallized as a dimer identical to active PorX. The CheY-like domain of PorXFj is in an active-like conformation, and PorXFj possesses phosphodiesterase activity, in agreement with the observation that the active site of its phosphatase-like domain is highly conserved with PorX.
Asunto(s)
Proteínas Bacterianas , Flavobacterium , Proteínas Bacterianas/metabolismo , Flavobacterium/metabolismo , Bacteroidetes/metabolismo , Actividad Motora , Sistemas de Secreción Bacterianos/genética , Porphyromonas gingivalis/metabolismoRESUMEN
No disponible
Asunto(s)
Humanos , Masculino , Anciano , Chryseobacterium/metabolismo , Chryseobacterium/fisiología , Chryseobacterium/patogenicidad , Flavobacterium/metabolismo , Flavobacterium/fisiología , Flavobacterium/patogenicidad , Respiración Artificial/instrumentación , Respiración Artificial/métodos , Respiración Artificial , Neumonía/inducido químicamente , Neumonía Asociada al Ventilador/diagnóstico , Neumonía Asociada al Ventilador/prevención & control , Neumonía Asociada al Ventilador/terapia , Infección Hospitalaria/inducido químicamente , Infección Hospitalaria/patología , Infección Hospitalaria/terapia , Antibacterianos/efectos adversos , Antibacterianos/uso terapéutico , Diagnóstico Diferencial , AncianoRESUMEN
Thirty nine isolates of Flavobacterium columnare from Brazilian fish farms had their carbohydrate composition of EPS evaluated by high efficiency liquid chromatography, using the phenol-sulfuric acid method of EPS. The occurrence of capsules on F. columnare cells was not directly related to biofilm formation, and the predominant monosaccharide is glucose.
Asunto(s)
Animales , Peces/microbiología , Flavobacterium/aislamiento & purificación , Flavobacterium/metabolismo , Monosacáridos/análisis , Polisacáridos Bacterianos/química , Brasil , Cromatografía LiquidaRESUMEN
Colonies of the fish pathogen Flavobacterium psychrophilum have gliding motility in media with low agar concentrations. Although gliding motility, particularly in Flavobacterium johnsoniae, has been well-studied, little is known about its regulation by environmental factors. The work described here shows that the ability of F. psychrophilum to spread over surfaces depends on nutrient availability. In fact, as the nutrient contents of the medium decreased, spreading was favored and the diameter of the colonies increased. Macroscopy examination revealed modifications in colony morphology as nutrient depletion increased: from a dense and defined colony to the formation of microcolonies inside a general colony structure. Additionally, colony expansion dynamics and population density across the colony radius varied inversely with bacterial biomass production. Motility was an immediate response when bacteria were transferred from a rich to a more diluted medium. Our results suggest that, when nutrients are limiting, F. psychrophilum activates a specific growth mode that enables it to colonize surfaces by means of gliding motility. The use of diluted media allowed the differentiation, among previously isolated F. psychrophilum non-gliding mutants, of those completely unable to glide and those with only partially impaired gliding ability (AU)
No disponible