RESUMEN
Bioinformatic analysis revealed that the genomes of ubiquitous Penicillium spp. might carry dozens of biosynthetic gene clusters (BGCs), yet many clusters have remained uncharacterized. In this study, a detailed investigation of co-culture fermentation including the basidiomycete Armillaria mellea CPCC 400891 and the P. brasilianum CGMCC 3.4402 enabled the isolation of five new compounds including two bisabolene-type sesquiterpenes (arpenibisabolanes A and B), two carotane-type sesquiterpenes (arpenicarotanes A and B), and one polyketide (arpenichorismite A) along with seven known compounds. The assignments of their structures were deduced by the extensive analyses of detailed spectroscopic data, electronic circular dichroism spectra, together with delimitation of the biogenesis. Most new compounds were not detected in monocultures under the same fermentation conditions. Arpenibisabolane A represents the first example of a 6/5-fused bicyclic bisabolene. The bioassay of these five new compounds exhibited no cytotoxic activities in vitro against three human cancer cell lines (A549, MCF-7, and HepG2). Moreover, sequence alignments and bioinformatic analysis to other metabolic pathways, two BGCs including Pb-bis and Pb-car, responsible for generating sesquiterpenoids from co-culture were identified, respectively. Furthermore, based on the chemical structures and deduced gene functions of the two clusters, a hypothetic metabolic pathway for biosynthesizing induced sesquiterpenoids was proposed. These results demonstrated that the co-culture approach would facilitate bioprospecting for new metabolites even from the well-studied microbes. Our findings would provide opportunities for further understanding of the biosynthesis of intriguing sesquiterpenoids via metabolic engineering strategies. KEY POINTS: ⢠Penicillium and Armillaria co-culture facilitates the production of diverse secondary metabolites ⢠Arpenibisabolane A represents the first example of 6/5-fused bicyclic bisabolenes ⢠A hypothetic metabolic pathway for biosynthesizing induced sesquiterpenoids was proposed.
Asunto(s)
Armillaria , Técnicas de Cocultivo , Fermentación , Penicillium , Metabolismo Secundario , Sesquiterpenos , Armillaria/metabolismo , Armillaria/genética , Penicillium/metabolismo , Penicillium/genética , Penicillium/química , Sesquiterpenos/metabolismo , Sesquiterpenos/química , Humanos , Familia de Multigenes , Línea Celular Tumoral , Vías Biosintéticas/genética , Policétidos/metabolismo , Policétidos/química , Policétidos/aislamiento & purificación , Células Hep G2RESUMEN
Actinomycetota have been widely described as valuable sources for the acquisition of secondary metabolites. Most microbial metabolites are produced via metabolic pathways encoded by biosynthetic gene clusters (BGCs). Although many secondary metabolites are not essential for the survival of bacteria, they play an important role in their adaptation and interactions within microbial communities. This is how bacteria isolated from extreme environments such as Antarctica could facilitate the discovery of new BGCs with biotechnological potential. This study aimed to isolate rare Actinomycetota strains from Antarctic soil and sediment samples and identify their metabolic potential based on genome mining and exploration of biosynthetic gene clusters. To this end, the strains were sequenced using Illumina and Oxford Nanopore Technologies platforms. The assemblies were annotated and subjected to phylogenetic analysis. Finally, the BGCs present in each genome were identified using the antiSMASH tool, and the biosynthetic diversity of the Micrococcaceae family was evaluated. Taxonomic annotation revealed that seven strains were new and two were previously reported in the NCBI database. Additionally, BGCs encoding type III polyketide synthases (T3PKS), beta-lactones, siderophores, and non-ribosomal peptide synthetases (NRPS) have been identified, among others. In addition, the sequence similarity network showed a predominant type of BGCs in the family Micrococcaceae, and some genera were distinctly grouped. The BGCs identified in the isolated strains could be associated with applications such as antimicrobials, anticancer agents, and plant growth promoters, among others, positioning them as excellent candidates for future biotechnological applications and innovations. KEY POINTS: ⢠Novel Antarctic rare Actinomycetota strains were isolated from soil and sediments ⢠Genome-based taxonomic affiliation revealed seven potentially novel species ⢠Genome mining showed metabolic potential for novel natural products.
Asunto(s)
Sedimentos Geológicos , Familia de Multigenes , Filogenia , Microbiología del Suelo , Regiones Antárticas , Sedimentos Geológicos/microbiología , Metabolismo Secundario/genética , Actinobacteria/genética , Actinobacteria/metabolismo , Actinobacteria/clasificación , Genoma Bacteriano , Biotecnología/métodos , Vías Biosintéticas/genética , Péptido Sintasas/genética , Péptido Sintasas/metabolismo , Sintasas Poliquetidas/genética , Sintasas Poliquetidas/metabolismoRESUMEN
Mycolic acids, a hallmark of the genus Mycobacterium, are unique branched long-chain fatty acids produced by a complex biosynthetic pathway. Due to their essentiality and involvement in various aspects of mycobacterial pathogenesis, the synthesis of mycolic acids-and the identification of the enzymes involved-is a valuable target for drug development. Although most of the core pathway is comparable between species, subtle structure differences lead to different structures delineating the mycolic acid repertoire of tuberculous and some nontuberculous mycobacteria. We here report the characterization of an α'-mycolic acid-deficient Mycobacterium smegmatis mutant obtained by chemical mutagenesis. Whole-genome sequencing and bioinformatic analysis identified a premature stop codon in MSMEG_4301, encoding an acyl-CoA synthetase. Orthologs of MSMEG_4301 are present in all mycobacterial species containing α'-mycolic acids. Deletion of the Mycobacterium abscessus ortholog MAB_1915 abrogated synthesis of α'-mycolic acids; likewise, deletion of MSMEG_4301 in an otherwise wild-type M. smegmatis background also caused loss of these short mycolates. IMPORTANCE Mycobacterium abscessus is a nontuberculous mycobacterium responsible for an increasing number of hard-to-treat infections due to the impervious nature of its cell envelope, a natural barrier to several antibiotics. Mycolic acids are key components of that envelope; thus, their synthesis is a valuable target for drug development. Our results identify the first enzyme involved in α'-mycolic acids, a short-chain member of mycolic acids, loss of which greatly affects growth of this opportunistic pathogen.
Asunto(s)
Mycobacterium abscessus , Mycobacterium , Vías Biosintéticas/genética , Ácidos Grasos/metabolismo , Mycobacterium/metabolismo , Mycobacterium abscessus/genética , Mycobacterium abscessus/metabolismo , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Ácidos Micólicos/metabolismo , Micobacterias no TuberculosasRESUMEN
Mycotoxins are secondary metabolites that are known to be toxic to humans and animals. On the other hand, some mycotoxins and their analogues possess antioxidant as well as antitumor properties, which could be relevant in the fields of pharmaceutical analysis and food research. Omics techniques are a group of analytical tools applied in the biological sciences in order to study genes (genomics), mRNA (transcriptomics), proteins (proteomics), and metabolites (metabolomics). Omics have become a vital tool in the field of mycotoxins, especially contributing to the identification of biomarkers with potential use for the detection of mycotoxigenic species and the gathering of information about the biosynthetic pathways of mycotoxins in different environments. This approach has provided tools for the development of prevention strategies and control measures for different mycotoxins. Additionally, research has revealed important information about the impact of global warming and climate change on the prevalence of mycotoxin issues in society. In the context of foodomics, the aim is to apply omics techniques in order to ensure food safety. The objective of the present review is to determine the state of the art regarding the development of analytical techniques based on omics in the identification of biosynthetic pathways related to mycotoxin synthesis.
Asunto(s)
Vías Biosintéticas , Micotoxinas , Animales , Vías Biosintéticas/genética , Inocuidad de los Alimentos , Humanos , Metabolómica , Micotoxinas/análisis , ProteómicaRESUMEN
Technologies based on synthetic biology to produce bacterial natural carotenoids depend on information regarding their biosynthesis. Although the biosynthetic pathway of common carotenoids is known, there are carotenoids whose pathways are not completely described. This work aimed to mine the genome of the deep-sea bacterium Erythrobacter citreus LAMA 915, an uncommon bacterium that forms yellow colonies under cultivation. This work further explores the potential application of the carotenoids found and low-cost substrates for bacterial growth. A combined approach of genome mining and untargeted metabolomics analysis was applied. The carotenoid erythroxanthin sulfate was detected in E. citreus LAMA 915 cell extract. A proposal for carotenoid biosynthesis by this bacterium is provided, involving the genes crtBIYZWG. These are responsible for the biosynthesis of carotenoids from the zeaxanthin pathway and their 2,2'-hydroxylated derivatives. E. citreus LAMA 915 extracts showed antioxidant and sun protection effects. Based on the high content of proteases and lipases, it was possible to rationally select substrates for bacterial growth, with residual oil from fish processing the best low-cost substrate selected. This work advances in the understanding of carotenoid biosynthesis and provides a genetic basis that can be further explored as a biotechnological route for carotenoid production.
Asunto(s)
Vías Biosintéticas , Carotenoides , Sphingomonadaceae , Vías Biosintéticas/genética , Carotenoides/metabolismo , Sphingomonadaceae/metabolismoRESUMEN
Azospirillum brasilense is a non-photosynthetic member of the family Rhodospirillaceae. Some strains of this bacterium are reported to produce bacterioruberin type of carotenoids, which are generally produced by halophilic or psychrophilic bacteria. Since no other member of Rhodospirillaceae produces bacterioruberin type of carotenoids, we investigated the presence of genes involved in bacterioruberin and spirilloxanthin biosynthetic pathways in A. brasilense Cd. Although genes encoding the spirilloxanthin pathway were absent, homologs of several genes (crtC and crtF) involved in the biosynthesis of bacterioruberins were present in the genome of A. brasilense Cd. However, the homolog of CruF responsible for the final step in bacterioruberin biosynthesis could not be found. We also characterized the carotenoids of A. brasilense Cd using thin-layer chromatography, high-performance liquid chromatography, absorption spectra and high-resolution mass spectrometry (HRMS). Resolution of the methanol extract of carotenoids in ultra-performance liquid chromatography showed nine peaks, out of which six peaks showed absorption spectra characteristic of carotenoids. HRMS of each peak produced 1-14 fragments with different m/z values. Two of these fragments were identified as 19'-hydroxyfucoxanthinol and 8'-apoalloxanthinal, which are the carotenoids found in aquatic microalgae.
Asunto(s)
Azospirillum brasilense/metabolismo , Carotenoides/metabolismo , Azospirillum brasilense/genética , Proteínas Bacterianas/genética , Vías Biosintéticas/genética , Genoma Bacteriano/genética , Xantófilas/metabolismoRESUMEN
Acidithiobacillus species are fundamental players in biofilm formation by acidophile bioleaching communities. It has been previously reported that Acidithiobacillus ferrooxidans possesses a functional quorum sensing mediated by acyl-homoserine lactones (AHL), involved in biofilm formation, and AHLs naturally produced by Acidithiobacillus species also induce biofilm formation in Acidithiobacillus thiooxidans. A c-di-GMP pathway has been characterized in Acidithiobacillus species but it has been pointed out that the c-di-GMP effector PelD and pel-like operon are only present in the sulfur oxidizers such as A. thiooxidans. PEL exopolysaccharide has been recently involved in biofilm formation in this Acidithiobacillus species. Here, by comparing wild type and ΔpelD strains through mechanical analysis of biofilm-cells detachment, fluorescence microscopy and qPCR experiments, the structural role of PEL exopolysaccharide and the molecular network involved for its biosynthesis by A. thiooxidans were tackled. Besides, the effect of AHLs on PEL exopolysaccharide production was assessed. Mechanical resistance experiments indicated that the loss of PEL exopolysaccharide produces fragile A. thiooxidans biofilms. qRT-PCR analysis established that AHLs induce the transcription of pelA and pelD genes while epifluorescence microscopy studies revealed that PEL exopolysaccharide was required for the development of AHL-induced biofilms. Altogether these results reveal for the first time that AHLs positively regulate pel genes and participate in the molecular network for PEL exopolysaccharide biosynthesis by A. thiooxidans.
Asunto(s)
Acidithiobacillus thiooxidans/genética , Acil-Butirolactonas/metabolismo , Extremófilos/genética , Regulación Bacteriana de la Expresión Génica , Polisacárido Liasas/genética , Acidithiobacillus thiooxidans/metabolismo , Biopelículas/crecimiento & desarrollo , Vías Biosintéticas/genética , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Extremófilos/metabolismo , Operón , Polisacárido Liasas/metabolismo , Polisacáridos Bacterianos/biosíntesis , Percepción de QuorumRESUMEN
BACKGROUND: Acinetobacter baumannii outbreaks have been associated with pandemic International Clones (ICs), but the virulence factors involved with their pathogenicity are sparsely understood. Pigment production has been linked with bacterial pathogenicity, however, this phenotype is rarely observed in A. baumannii. OBJECTIVES: This study aimed to characterise the reddish-brown pigment produced by A. baumannii strains, and to determine its biosynthetic pathway by genomic approaches. METHODS: Pigment characterisation and antimicrobial susceptibility were conducted by phenotypic tests. The clonal relationship was obtained by pulsed field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). The genome of an A. baumannii was obtained for characterisation of genes involved with pigment production. FINDINGS: The pyomelanin was the pigment produced by A. baumannii. Strains were extensively drug resistant and belonged to the IC-5/ST79. The pyomelanin biosynthetic pathway was determined and presented a particular architecture concerning the peripheral (tyrB, phhB and hpd) and central (hmgB, hmgC and hmgR) metabolic pathway genes. The identification of a distant HmgA homologue, probably without dioxygenase activity, could explain pyomelanin production. Virulence determinants involved with adherence (csuA/BABCDE and a T5bSS-carrying genomic island), and iron uptake (basABCDEFGHIJ, bauABCDEF and barAB) were characterised. MAIN CONCLUSION: There is a biosynthetic pathway compatible with the pyomelanin production observed in persistent A. baumannii IC-5 strains.
Asunto(s)
Infecciones por Acinetobacter/microbiología , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/genética , Vías Biosintéticas/genética , Melaninas , Acinetobacter baumannii/aislamiento & purificación , Antibacterianos/farmacología , Electroforesis en Gel de Campo Pulsado , Humanos , Pruebas de Sensibilidad Microbiana , Tipificación de Secuencias Multilocus , Pandemias , beta-LactamasasRESUMEN
Carotenoids are terpenoid pigments synthesized by all photosynthetic and some non-photosynthetic organisms. In plants, these lipophilic compounds are involved in photosynthesis, photoprotection, and phytohormone synthesis. In plants, carotenoid biosynthesis is induced by several environmental factors such as light including photoreceptors, such as phytochromes (PHYs) and negatively regulated by phytochrome interacting factors (PIFs). Daucus carota (carrot) is one of the few plant species that synthesize and accumulate carotenoids in the storage root that grows in darkness. Contrary to other plants, light inhibits secondary root growth and carotenoid accumulation suggesting the existence of new mechanisms repressed by light that regulate both processes. To identify genes induced by dark and repressed by light that regulate carotenoid synthesis and carrot root development, in this work an RNA-Seq analysis was performed from dark- and light-grown carrot roots. Using this high-throughput sequencing methodology, a de novo transcriptome model with 63,164 contigs was obtained, from which 18,488 were differentially expressed (DEG) between the two experimental conditions. Interestingly, light-regulated genes are preferably expressed in dark-grown roots. Enrichment analysis of GO terms with DEGs genes, validation of the transcriptome model and DEG analysis through qPCR allow us to hypothesize that genes involved in photomorphogenesis and light perception such as PHYA, PHYB, PIF3, PAR1, CRY2, FYH3, FAR1 and COP1 participate in the synthesis of carotenoids and carrot storage root development.
Asunto(s)
Vías Biosintéticas/genética , Carotenoides/metabolismo , Biología Computacional/métodos , Daucus carota/genética , Daucus carota/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/metabolismo , Daucus carota/crecimiento & desarrollo , Perfilación de la Expresión Génica , Pigmentación , Proteínas de Plantas/genéticaRESUMEN
Despite the existence of considerable research on somatic embryogenesis (SE), the molecular mechanism that regulates the biosynthesis of auxins during the SE induction process remains unknown. Indole-3-acetic acid (IAA) is an auxin that is synthesized in plants through five pathways. The biosynthetic pathway most frequently used in this synthesis is the conversion of tryptophan to indol-3-pyruvic acid (IPA) by tryptophan aminotransferase of Arabidopsis (TAA) followed by the conversion of IPA to IAA by enzymes encoded by YUCCA (YUC) genes of the flavin monooxygenase family; however, it is unclear whether YUC-mediated IAA biosynthesis is involved in SE induction. In this study, we report that the increase of IAA observed during SE pre-treatment (plants in MS medium supplemented with 1-naphthaleneacetic acid (NAA) 0.54 µM and kinetin (Kin) 2.32 µM for 14 days) was due to its de novo biosynthesis. By qRT-PCR, we demonstrated that YUC gene expression was consistent with the free IAA signal found in the explants during the induction of SE. In addition, the use of yucasin to inhibit the activity of YUC enzymes reduced the signal of free IAA in the leaf explants and dramatically decreased the induction of SE. The exogenous addition of IAA restored the SE process in explants treated with yucasin. Our findings suggest that the biosynthesis and localization of IAA play an essential role during the induction process of SE in Coffea canephora.
Asunto(s)
Coffea/embriología , Coffea/metabolismo , Ácidos Indolacéticos/metabolismo , Reguladores del Crecimiento de las Plantas/biosíntesis , Vías Biosintéticas/efectos de los fármacos , Vías Biosintéticas/genética , Coffea/genética , Inhibidores Enzimáticos/farmacología , Perfilación de la Expresión Génica , Genes de Plantas , Oxigenasas de Función Mixta/antagonistas & inhibidores , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Familia de Multigenes , Reguladores del Crecimiento de las Plantas/genética , Proteínas de Plantas/antagonistas & inhibidores , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Técnicas de Embriogénesis Somática de Plantas , Triazoles/farmacologíaRESUMEN
Benzenoids are compounds associated with floral and fruity flavours in flowers, fruits and leaves and present a role in hormonal signalling in plants. These molecules are produced by the phenyl ammonia lyase pathway. However, some yeasts can also synthesize them from aromatic amino acids using an alternative pathway that remains unknown. Hanseniaspora vineae can produce benzenoids at levels up to two orders of magnitude higher than Saccharomyces species, so it is a model microorganism for studying benzenoid biosynthesis pathways in yeast. According to their genomes, several enzymes have been proposed to be involved in a mandelate pathway similar to that described for some prokaryotic cells. Among them, the ARO10 gene product could present benzoylformate decarboxylase activity. This enzyme catalyses the decarboxylation of benzoylformate into benzaldehyde at the end of the mandelate pathway in benzyl alcohol formation. Two homologous genes of ARO10 were found in the two sequenced H. vineae strains. In this study, nine other H. vineae strains were analysed to detect the presence and per cent homology of ARO10 sequences by PCR using specific primers designed for this species. Also, the copy number of the genes was estimated by quantitative PCR. To verify the relation of ARO10 with the production of benzyl alcohol during fermentation, a deletion mutant in the ARO10 gene of Saccharomyces cerevisiae was used. The two HvARO10 paralogues were analysed and compared with other α-ketoacid decarboxylases at the sequence and structural level.
Asunto(s)
Derivados del Benceno/metabolismo , Vías Biosintéticas/genética , Hanseniaspora/genética , Piruvato Descarboxilasa/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcriptoma , Benzaldehídos/metabolismo , Alcohol Bencilo/metabolismo , Fermentación , Hanseniaspora/metabolismoRESUMEN
Brasiliamides are a class of piperazine-containing alkaloids produced by Penicillium brasilianum with a range of pharmaceutical activities. The mechanism of brasiliamide biosynthesis, including piperazine ring formation and multiple tailoring modifications, still remains unclear. In this study, the biosynthetic gene cluster of brasiliamides, brs, was identified from the marine-derived fungal strain Penicillium brasilianum WZXY-M122-9. Deletion of a histone deacetylase-encoding gene using a CRISPR/Cas9 gene editing system led to the production of a new compound, namely brasiliamide I (1). The brs-encoded single-module nonribosomal peptide synthetase (NRPS) BrsA is involved in the formation of the piperazine skeleton of brasiliamides. Full-length BrsA protein (113.6 kDa) was purified, and reconstitution of enzymatic activity in vitro confirmed that BrsA stereoselectively accepts L-phenylalanine as the substrate. Multiple deletion of tailoring genes and analysis of purified proteins in vitro enabled us to propose a brasiliamide biosynthetic pathway. In the tailoring steps, an α-ketoglutarate (KG)-dependent nonheme iron dioxygenase, BrsJ, was identified to catalyze piperazine ring cleavage during biosynthesis of brasiliamide A (2). KEY POINTS: The gene cluster encoding brasiliamide biosynthesis, brs, is identified. Deletion of a histone deacetylase-encoding gene produces brasiliamide I. BrsA catalyzes brasiliamide piperazine skeleton formation. BrsJ catalyzes piperazine ring cleavage to produce brasiliamide A. Graphical abstract.
Asunto(s)
Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismo , Dioxoles/metabolismo , Proteínas Fúngicas/metabolismo , Péptido Sintasas/metabolismo , Piperazina/metabolismo , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/genética , Vías Biosintéticas/genética , Catálisis , Dioxoles/química , Dioxoles/aislamiento & purificación , Proteínas Fúngicas/genética , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Estructura Molecular , Familia de Multigenes , Mutación , Penicillium/genética , Penicillium/metabolismo , Péptido Sintasas/genética , Piperazina/química , Piperazina/aislamiento & purificaciónRESUMEN
KEY MESSAGE: Successful orange rust development on sugarcane can potentially be explained as suppression of the plant immune system by the pathogen or delayed plant signaling to trigger defense responses. Puccinia kuehnii is an obligate biotrophic fungus that infects sugarcane leaves causing a disease called orange rust. It spread out to other countries resulting in reduction of crop yield since its first outbreak. One of the knowledge gaps of that pathosystem is to understand the molecular mechanisms altered in susceptible plants by this biotic stress. Here, we investigated the changes in temporal expression of transcripts in pathways associated with the immune system. To achieve this purpose, we used RNA-Seq to analyze infected leaf samples collected at five time points after inoculation. Differential expression analyses of adjacent time points revealed substantial changes at 12, 48 h after inoculation and 12 days after inoculation, coinciding with the events of spore germination, haustoria post-penetration and post-sporulation, respectively. During the first 24 h, a lack of transcripts involved with resistance mechanisms was revealed by underrepresentation of hypersensitive and defense response related genes. However, two days after inoculation, upregulation of genes involved with immune response regulation provided evidence of some potential defense response. Events related to biotic stress responses were predominantly downregulated in the initial time points, but expression was later restored to basal levels. Genes involved in carbohydrate metabolism showed evidence of repression followed by upregulation, possibly to ensure the pathogen nutritional requirements were met. Our results support the hypothesis that P. kuehnii initially suppressed sugarcane genes involved in plant defense systems. Late overexpression of specific regulatory pathways also suggests the possibility of an inefficient recognition system by a susceptible sugarcane genotype.
Asunto(s)
Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Hojas de la Planta/microbiología , Puccinia/fisiología , Saccharum/genética , Saccharum/microbiología , Vías Biosintéticas/genética , Pared Celular/metabolismo , Susceptibilidad a Enfermedades , Genotipo , Estrés Oxidativo/genética , Fotosíntesis/genética , Hojas de la Planta/genética , Reproducibilidad de los Resultados , Saccharum/inmunología , Estrés Fisiológico/genética , Factores de Tiempo , Factores de Transcripción/metabolismoRESUMEN
The phenylpropanoid pathway is an important route of secondary metabolism involved in the synthesis of different phenolic compounds such as phenylpropenes, anthocyanins, stilbenoids, flavonoids, and monolignols. The flux toward monolignol biosynthesis through the phenylpropanoid pathway is controlled by specific genes from at least ten families. Lignin polymer is one of the major components of the plant cell wall and is mainly responsible for recalcitrance to saccharification in ethanol production from lignocellulosic biomass. Here, we identified and characterized sugarcane candidate genes from the general phenylpropanoid and monolignol-specific metabolism through a search of the sugarcane EST databases, phylogenetic analysis, a search for conserved amino acid residues important for enzymatic function, and analysis of expression patterns during culm development in two lignin-contrasting genotypes. Of these genes, 15 were cloned and, when available, their loci were identified using the recently released sugarcane genomes from Saccharum hybrid R570 and Saccharum spontaneum cultivars. Our analysis points out that ShPAL1, ShPAL2, ShC4H4, Sh4CL1, ShHCT1, ShC3H1, ShC3H2, ShCCoAOMT1, ShCOMT1, ShF5H1, ShCCR1, ShCAD2, and ShCAD7 are strong candidates to be bona fide lignin biosynthesis genes. Together, the results provide information about the candidate genes involved in monolignol biosynthesis in sugarcane and may provide useful information for further molecular genetic studies in sugarcane.
Asunto(s)
Vías Biosintéticas/genética , Lignina/biosíntesis , Proteínas de Plantas/genética , Propanoles/metabolismo , Saccharum/genética , Saccharum/metabolismo , Regulación de la Expresión Génica de las Plantas , Genotipo , Lignina/genética , Propanoles/química , Saccharum/clasificación , Saccharum/crecimiento & desarrolloRESUMEN
Zincphyrin IV is a potential organic photosensitizer which is of significant interest for applications in biomedicine, materials science, agriculture (as insecticide), and chemistry. Most studies on Zincphyrin are focused on Zincphyrin III while biosynthesis and application of Zincphyrin IV is comparatively less explored. In this study, we explored Zincphyrin IV production in Streptomyces venezuelae ATCC 15439 through combination of morphology engineering and "One strain many compounds" approach. The morphology engineering followed by change in culture medium led to activation of cryptic Zincphyrin IV biosynthetic pathway in S. venezuelae with subsequent detection of Zincphyrin IV. Morphology engineering applied in S. venezuelae increased the biomass from 7.17 to 10.5 mg/mL after 48 h of culture. Moreover, morphology of engineered strain examined by SEM showed reduced branching and fragmentation of mycelia. The distinct change in color of culture broth visually demonstrated the activation of the cryptic biosynthetic pathway in S. venezuelae. The production of Zincphyrin IV was found to be initiated after overexpression ssgA, resulting in the increase in titer from 4.21 to 7.54 µg/mL. Furthermore, Zincphyrin IV demonstrated photodynamic antibacterial activity against Bacillus subtilis and photodynamic anticancer activity against human ovarian carcinoma cell lines.
Asunto(s)
Antibacterianos/biosíntesis , Antineoplásicos/metabolismo , Coproporfirinas/biosíntesis , Ingeniería Metabólica/métodos , Fármacos Fotosensibilizantes/metabolismo , Streptomyces/crecimiento & desarrollo , Streptomyces/metabolismo , Antibacterianos/farmacología , Antineoplásicos/farmacología , Bacillus subtilis/efectos de los fármacos , Vías Biosintéticas/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Fenómenos Químicos , Coproporfirinas/farmacología , Medios de Cultivo/química , Humanos , Microscopía Electrónica de Rastreo , Fármacos Fotosensibilizantes/farmacología , Streptomyces/genética , Streptomyces/ultraestructuraRESUMEN
BACKGROUND Acinetobacter baumannii outbreaks have been associated with pandemic International Clones (ICs), but the virulence factors involved with their pathogenicity are sparsely understood. Pigment production has been linked with bacterial pathogenicity, however, this phenotype is rarely observed in A. baumannii. OBJECTIVES This study aimed to characterise the reddish-brown pigment produced by A. baumannii strains, and to determine its biosynthetic pathway by genomic approaches. METHODS Pigment characterisation and antimicrobial susceptibility were conducted by phenotypic tests. The clonal relationship was obtained by pulsed field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). The genome of an A. baumannii was obtained for characterisation of genes involved with pigment production. FINDINGS The pyomelanin was the pigment produced by A. baumannii. Strains were extensively drug resistant and belonged to the IC-5/ST79. The pyomelanin biosynthetic pathway was determined and presented a particular architecture concerning the peripheral (tyrB, phhB and hpd) and central (hmgB, hmgC and hmgR) metabolic pathway genes. The identification of a distant HmgA homologue, probably without dioxygenase activity, could explain pyomelanin production. Virulence determinants involved with adherence (csuA/BABCDE and a T5bSS-carrying genomic island), and iron uptake (basABCDEFGHIJ, bauABCDEF and barAB) were characterised. MAIN CONCLUSION There is a biosynthetic pathway compatible with the pyomelanin production observed in persistent A. baumannii IC-5 strains.
Asunto(s)
Humanos , Infecciones por Acinetobacter/microbiología , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/genética , Vías Biosintéticas/genética , Melaninas , beta-Lactamasas , Pruebas de Sensibilidad Microbiana , Electroforesis en Gel de Campo Pulsado , Acinetobacter baumannii/aislamiento & purificación , Tipificación de Secuencias Multilocus , Pandemias , Antibacterianos/farmacologíaRESUMEN
The geographic mosaic theory of coevolution (GMC) posits that coevolutionary dynamics go beyond local coevolution and are comprised of the following three components: geographic selection mosaics, coevolutionary hot spots, and trait remixing. It is unclear whether the GMC applies to bacteria, as horizontal gene transfer and cosmopolitan dispersal may violate theoretical assumptions. Here, we test key GMC predictions in an antibiotic-producing bacterial symbiont (genus Pseudonocardia) that protects the crops of neotropical fungus-farming ants (Apterostigma dentigerum) from a specialized pathogen (genus Escovopsis). We found that Pseudonocardia antibiotic inhibition of common Escovopsis pathogens was elevated in A. dentigerum colonies from Panama compared to those from Costa Rica. Furthermore, a Panama Canal Zone population of Pseudonocardia on Barro Colorado Island (BCI) was locally adapted, whereas two neighboring populations were not, consistent with a GMC-predicted selection mosaic and a hot spot of adaptation surrounded by areas of maladaptation. Maladaptation was shaped by incongruent Pseudonocardia-Escovopsis population genetic structure, whereas local adaptation was facilitated by geographic isolation on BCI after the flooding of the Panama Canal. Genomic assessments of antibiotic potential of 29 Pseudonocardia strains identified diverse and unique biosynthetic gene clusters in BCI strains despite low genetic diversity in the core genome. The strength of antibiotic inhibition was not correlated with the presence/absence of individual biosynthetic gene clusters or with parasite location. Rather, biosynthetic gene clusters have undergone selective sweeps, suggesting that the trait remixing dynamics conferring the long-term maintenance of antibiotic potency rely on evolutionary genetic changes within already-present biosynthetic gene clusters and not simply on the horizontal acquisition of novel genetic elements or pathways.IMPORTANCE Recently, coevolutionary theory in macroorganisms has been advanced by the geographic mosaic theory of coevolution (GMC), which considers how geography and local adaptation shape coevolutionary dynamics. Here, we test GMC in an ancient symbiosis in which the ant Apterostigma dentigerum cultivates fungi in an agricultural system analogous to human farming. The cultivars are parasitized by the fungus Escovopsis The ants maintain symbiotic actinobacteria with antibiotic properties that help combat Escovopsis infection. This antibiotic symbiosis has persisted for tens of millions of years, raising the question of how antibiotic potency is maintained over these time scales. Our study tests the GMC in a bacterial defensive symbiosis and in a multipartite symbiosis framework. Our results show that this multipartite symbiotic system conforms to the GMC and demonstrate that this theory is applicable in both microbes and indirect symbiont-symbiont interactions.
Asunto(s)
Aclimatación/fisiología , Actinobacteria/metabolismo , Antibacterianos/metabolismo , Coevolución Biológica , Simbiosis/fisiología , Actinobacteria/genética , Animales , Antibacterianos/farmacología , Hormigas/microbiología , Vías Biosintéticas/genética , Costa Rica , Interacciones Microbiota-Huesped/fisiología , Hypocreales/efectos de los fármacos , Hypocreales/patogenicidad , Metabolismo Secundario/genética , Simbiosis/genéticaRESUMEN
OBJECTIVE: To evaluate the induction of monoterpenoid indole alkaloids (MIA) and phenolic compound production by yeast extract (YE) and its relationship with defense responses in Uncaria tomentosa (Rubiaceae) root cultures. RESULTS: Root cultures were elicited by YE at three concentrations. The 0.5 mg YE ml-1 treatment did not affect cell viability but increased the hydrogen peroxide concentration by 5.7 times; guaiacol peroxidase activity by twofold; and the glucoindole alkaloid 3α-dihydrocadambine (DHC) content by 2.6 times (to 825.3 ± 27.3 µg g-1). This treatment did not affect the contents of monoterpenoid oxindole alkaloids or chlorogenic acids. In response to 0.5 mg YE ml-1 treatment, the transcript levels of MIA biosynthetic genes, TDC and LAMT, increased 5.4 and 1.9-fold, respectively, that of SGD decreased by 32%, and that of STR did not change. The transcript levels of genes related to phenolic compounds, PAL, CHS and HQT, increased by 1.7, 7.7, and 1.2-fold, respectively. Notably, the transcript levels of Prx1 and Prx encoding class III peroxidases increased by 1.4 and 2.5-fold. CONCLUSION: The YE elicitor induced an antioxidant defense response, increased the transcript levels of genes encoding enzymes related to strictosidine biosynthesis precursors and class III peroxidases, and decreased the transcript level of SGD. Thus, YE could stimulate antifungal DHC production in root cultures of U. tomentosa.
Asunto(s)
Antioxidantes/metabolismo , Uña de Gato/metabolismo , Medios de Cultivo/química , Raíces de Plantas/metabolismo , Alcaloides de Triptamina Secologanina/metabolismo , Levaduras/química , Vías Biosintéticas/genética , Ácido Clorogénico/metabolismo , Mezclas Complejas/metabolismo , Perfilación de la Expresión Génica , Genes de Plantas , Peróxido de Hidrógeno/metabolismo , Fenoles/metabolismoRESUMEN
Cryptococcosis is a fungal disease caused by C. neoformans. To adapt and survive in diverse ecological niches, including the animal host, this opportunistic pathogen relies on its ability to uptake nutrients, such as carbon, nitrogen, iron, phosphate, sulfur, and amino acids. Genetic circuits play a role in the response to environmental changes, modulating gene expression and adjusting the microbial metabolism to the nutrients available for the best energy usage and survival. We studied the sulfur amino acid biosynthesis and its implications on C. neoformans biology and virulence. CNAG_04798 encodes a BZip protein and was annotated as CYS3, which has been considered an essential gene. However, we demonstrated that CYS3 is not essential, in fact, its knockout led to sulfur amino acids auxotroph. Western blots and fluorescence microscopy indicated that GFP-Cys3, which is expressed from a constitutive promoter, localizes to the nucleus in rich medium (YEPD); the addition of methionine and cysteine as sole nitrogen source (SD-N + Met/Cys) led to reduced nuclear localization and protein degradation. By proteomics, we identified and confirmed physical interaction among Gpp2, Cna1, Cnb1 and GFP-Cys3. Deletion of the calcineurin and GPP2 genes in a GFP-Cys3 background demonstrated that calcineurin is required to maintain Cys3 high protein levels in YEPD and that deletion of GPP2 causes GFP-Cys3 to persist in the presence of sulfur amino acids. Global transcriptional profile of mutant and wild type by RNAseq revealed that Cys3 controls all branches of the sulfur amino acid biosynthesis, and sulfur starvation leads to induction of several amino acid biosynthetic routes. In addition, we found that Cys3 is required for virulence in Galleria mellonella animal model.
Asunto(s)
Aminoácidos Sulfúricos/biosíntesis , Vías Biosintéticas , Calcineurina/metabolismo , Cryptococcus neoformans/metabolismo , Proteínas Fúngicas/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Vías Biosintéticas/genética , Cryptococcus neoformans/genética , Cryptococcus neoformans/crecimiento & desarrollo , Cryptococcus neoformans/patogenicidad , Regulación Fúngica de la Expresión Génica , Ontología de Genes , Proteínas Fluorescentes Verdes/metabolismo , Modelos Biológicos , Estado Nutricional , Transporte de Proteínas , Proteómica , Azufre/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Virulencia/genéticaRESUMEN
Secondary metabolites play an important role in the avocado fruit defense system. Phenolic compounds are the main biosynthesized metabolites of this system response. Our objective in this investigation was to evaluate the induction of specific metabolic pathways using chitosan as an elicitor. Extracts obtained from avocado in intermediate and consumption maturity stages treated with chitosan exhibited an increase in antifungal activity, which caused inhibition of mycelial growth and a decrease in sporulation as well as spore germination of Colletotrichum gloeosporioides. Additionally, RNA from epicarp of the fruits treated and untreated with chitosan was obtained in order to evaluate the expression of genes related to phenylpropanoids and the antifungal compound 1-acetoxy-2-hydroxy-4-oxo-heneicosa-12,15-diene biosynthesis. An increased in gene expression of genes that participates in the phenylpropanoids route was observed during the stage of physiological fruit maturity, others genes such as Flavonol synthase (Fls), increased only in samples obtained from fruit treated with chitosan at consumption maturity. Our results reveal a new molecular mechanism where chitosan induces a specific accumulation of phenylpropanoids and antifungal diene; this partially explains avocado's resistance against fungal pathogens. Finally, we discuss the molecular connections between chitosan induction and gene expression to explain the biological events that orchestrate the resistance pathways in fruits.