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1.
Proc Natl Acad Sci U S A ; 117(29): 17195-17203, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32606248

RESUMO

The vast majority of intracellular protein targets are refractory toward small-molecule therapeutic engagement, and additional therapeutic modalities are needed to overcome this deficiency. Here, the identification and characterization of a natural product, WDB002, reveals a therapeutic modality that dramatically expands the currently accepted limits of druggability. WDB002, in complex with the FK506-binding protein (FKBP12), potently and selectively binds the human centrosomal protein 250 (CEP250), resulting in disruption of CEP250 function in cells. The recognition mode is unprecedented in that the targeted domain of CEP250 is a coiled coil and is topologically featureless, embodying both a structural motif and surface topology previously considered on the extreme limits of "undruggability" for an intracellular target. Structural studies reveal extensive protein-WDB002 and protein-protein contacts, with the latter being distinct from those seen in FKBP12 ternary complexes formed by FK506 and rapamycin. Outward-facing structural changes in a bound small molecule can thus reprogram FKBP12 to engage diverse, otherwise "undruggable" targets. The flat-targeting modality demonstrated here has the potential to expand the druggable target range of small-molecule therapeutics. As CEP250 was recently found to be an interaction partner with the Nsp13 protein of the SARS-CoV-2 virus that causes COVID-19 disease, it is possible that WDB002 or an analog may exert useful antiviral activity through its ability to form high-affinity ternary complexes containing CEP250 and FKBP12.


Assuntos
Actinobacteria/genética , Genoma Bacteriano , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Proteína 1A de Ligação a Tacrolimo/química , Proteína 1A de Ligação a Tacrolimo/metabolismo , Actinobacteria/metabolismo , Sequência de Aminoácidos , Antibacterianos/química , Antibacterianos/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Calcineurina/genética , Calcineurina/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Evolução Molecular , Células HEK293 , Humanos , Modelos Moleculares , Conformação Proteica , Homologia de Sequência , Sirolimo/química , Sirolimo/metabolismo , Bibliotecas de Moléculas Pequenas/química , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
2.
PLoS One ; 15(7): e0236165, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32697804

RESUMO

In order to define the diversity and composition of the microbial communities colonizing of the soil microbiome of the Jinsha earthen relic, we used high-throughput sequencing technology to identify and characterize the microbiota in 22 samples collected from the Jinsha earthen relic in China during 2017 and 2018. We compared the taxonomy of the microbial communities from samples taken at different times and different sites. Our results showed that the identity of the dominant bacterial phyla differed among the samples. Proteobacteria (23-86.2%) were the predominant bacterial phylum in all samples taken from site A in both 2017 and 2018. However, Actinobacteria (21-92.3%) were the most popular bacterial phylum in samples from sites B and C in 2017 and 2018. Ascomycota were identified as the only fungal phyla in samples in 2017. However, the group varied drastically in relative abundance between 2017 and 2018. Functional analysis of the soil bacterial community suggested that abundant members of the microbiota may be associated with metabolism and the specific environment. This report was the first high-throughput sequencing study of the soil of the Jinsha earthen relic microbiome. Since soil microbiota can damage soil and archeological structures, comprehensive analyses of the microbiomes at archeological sites may contribute to the understand of the influence of microorganisms on the degradation of soil, as well as to the identification of potentially beneficial or undesirable members of these microbial communities in archeological sites. The study will be helpful to provide effective data and guidance for the prevention and control of microbial corrosion of the Jinsha earthen relic.


Assuntos
Arqueologia , Microbiota/genética , Microbiologia do Solo , Actinobacteria/genética , Actinobacteria/isolamento & purificação , Ascomicetos/genética , Ascomicetos/isolamento & purificação , China , DNA Bacteriano/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala , Proteobactérias/genética , Proteobactérias/isolamento & purificação , RNA Ribossômico 16S/genética
3.
Arch Microbiol ; 202(8): 2197-2205, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32519020

RESUMO

During the course of isolating rare actinobacteria from unexplored habitats, strain CH32T was obtained from an arid soil sample in eastern Anatolia, Turkey. Polyphasic characterization and comprehensive genome analyses showed that the strain is a member of the genus Nonomuraea and it is closely related to Nonomuraea gerenzanensis ATCC 39727T, Nonomuraea polychroma DSM 43925T and Nonomuraea maritima FXJ7.203T with gene identity level of 98.7%, 98.2% and 98.1%, respectively. The whole-cell hydrolysates contain meso-diaminopimelic acid as diagnostic diaminoacid and glucose, ribose, galactose, mannose and madurose as whole cell sugars. The predominant menaquinones are MK-9(H4), MK-9(H6) and MK-9(H2) while MK-9 exists as minor component. The polar lipid profile consists of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, glycolipid, glycophospholipids, phospholipids and unidentified lipids. The major cellular fatty acids are iso-C16:0 and C17:0 10-methyl. The total genome size is about 9.6 Mb and the G + C content is 71.0%. The genome contains biosynthetic gene clusters encoding for terpenes, siderophores, a type III polyketide synthase, a non-ribosomal polypeptide synthetase and a bacteriocin. The genome-based comparisons of the strain with its phylogenetic neighbours, as indicated by digital DNA-DNA hybridization and average nucleotide identity analyses, reveal that strain CH32T (= JCM 33876T = KCTC 49368T) is a novel member of the genus Nonomuraea, for which Nonomuraea terrae sp. nov. is proposed.


Assuntos
Actinomycetales/classificação , Microbiologia do Solo , Actinobacteria/classificação , Actinobacteria/genética , Actinomycetales/química , Actinomycetales/genética , Actinomycetales/isolamento & purificação , Composição de Bases , Ácidos Graxos/análise , Genoma Bacteriano/genética , Fosfolipídeos/análise , Filogenia , Solo/química , Turquia
4.
Braz Oral Res ; 34: e042, 2020 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-32401932

RESUMO

A few investigations of caries biofilms have identified Scardovia spp.; however, little is known about its involvement in caries pathogenesis. The purpose of this study was to assess the gene expression profile of Scardovia spp. in root caries, and compare it with other microorganisms. Clinical samples from active root caries lesions were collected. Microbial mRNA was isolated and cDNA sequenced. The function and composition of the Scardovia were investigated using two methods: a) de novo assembly of the read data and mapping to contigs, and b) reads mapping to reference genomes. Pearson correlation was performed (p < 0.05). Proportion of Scardovia inopinata and Scardovia wiggsiae sequences ranged from 0-6% in the root caries metatranscriptome. There was a positive correlation between the transcriptome of Lactobacillus spp. and Scardovia spp. (r = 0.70; p = 0.03), as well as with other Bifidobacteriaceae (r = 0.91; p = 0.0006). Genes that code for fructose 6-phosphate phosphoketolase (the key enzyme for "Bifid shunt"), as well as ABC transporters and glycosyl-hydrolases were highly expressed. In conclusion, "Bifid shunt" and starch metabolism are involved in carbohydrate metabolism of S. inopinata and S. wiggsiae in root caries. There is a positive correlation between the metabolism abundance of Lactobacillus spp., Bifidobacteriaceae members, and Scardovia in root caries.


Assuntos
Actinobacteria/genética , Expressão Gênica , Cárie Radicular/microbiologia , Actinobacteria/isolamento & purificação , Adulto , Idoso , Idoso de 80 Anos ou mais , Biofilmes , Mapeamento Cromossômico , DNA Bacteriano , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Valores de Referência , Análise de Sequência de DNA , Estatísticas não Paramétricas , Transcriptoma
5.
Int J Syst Evol Microbiol ; 70(5): 3226-3233, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32375929

RESUMO

A novel actinomycete, designated strain NEAU-C151T, was isolated from soil collected from Mount Song and characterized using a polyphasic approach. Analysis of the 16S rRNA gene sequence indicated that strain NEAU-C151T belongs to the genus Streptomyces and exhibited 97.5, 97.4 and 97.4 % similarities to Streptomyces lincolnensis NRRL 2936T, Streptomyces coacervatus AS-0823T, and Streptomyces longisporus ISP 5166T, respectively. The assignment of strain NEAU-C151T to the genus Streptomyces was confirmed by chemotaxonomic data: anteiso-C15 : 0, C16 : 0, iso-C16 : 0, C16 : 1 (ω7c) and anteiso-C17 : 0 as the major cellular fatty acids; whole-cell sugars contained ribose and glucose; phospholipid profile consisted of diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), unidentified phospholipid (PL), unidentified lipids (L) and phosphatidylinositol mannoside (PIM); the menaquinones were MK-9(H4), MK-9(H6), MK-10(H2) and MK-9(H8). However, multilocus sequence analysis based on five other house-keeping genes (atpD, gyrB, recA, rpoB, and trpB), DNA-DNA relatedness and phenotypic data showed that strain NEAU-C151T could be distinguished from its closest relatives. Consequently, strain NEAU-C151T represents a novel species of the genus Streptomyces, for which the name Streptomyces montanus sp. nov. is proposed. The type strain is NEAU-C151T (=CGMCC 4.7498T=DSM 107808T).


Assuntos
Filogenia , Microbiologia do Solo , Streptomyces/classificação , Actinobacteria/genética , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Genes Bacterianos , Tipagem de Sequências Multilocus , Hibridização de Ácido Nucleico , Fosfolipídeos/química , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Streptomyces/isolamento & purificação , Vitamina K 2/análogos & derivados , Vitamina K 2/química
6.
PLoS One ; 15(5): e0232699, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32374760

RESUMO

The metal hyperaccumulator Azolla filiculoides is accompanied by a microbiome potentially supporting plant during exposition to heavy metals. We hypothesized that the microbiome exposition to selected heavy metals will reveal metal tolerant strains. We used Next Generation Sequencing technique to identify possible metal tolerant strains isolated from the metal-treated plant (Pb, Cd, Cr(VI), Ni, Au, Ag). The main dominants were Cyanobacteria and Proteobacteria constituting together more than 97% of all reads. Metal treatment led to changes in the composition of the microbiome and showed significantly higher richness in the Pb-, Cd- and Cr-treated plant in comparison with other (95-105 versus 36-44). In these treatments the share of subdominant Actinobacteria (0.4-0.8%), Firmicutes (0.5-0.9%) and Bacteroidetes (0.2-0.9%) were higher than in non-treated plant (respectively: 0.02, 0.2 and 0.001%) and Ni-, Au- and Ag-treatments (respectively: <0.4%, <0.2% and up to 0.2%). The exception was Au-treatment displaying the abundance 1.86% of Bacteroidetes. In addition, possible metal tolerant genera, namely: Acinetobacter, Asticcacaulis, Anabaena, Bacillus, Brevundimonas, Burkholderia, Dyella, Methyloversatilis, Rhizobium and Staphylococcus, which form the core microbiome, were recognized by combining their abundance in all samples with literature data. Additionally, the presence of known metal tolerant genera was confirmed: Mucilaginibacter, Pseudomonas, Mycobacterium, Corynebacterium, Stenotrophomonas, Clostridium, Micrococcus, Achromobacter, Geobacter, Flavobacterium, Arthrobacter and Delftia. We have evidenced that A. filiculoides possess a microbiome whose representatives belong to metal-resistant species which makes the fern the source of biotechnologically useful microorganisms for remediation processes.


Assuntos
Cádmio/farmacologia , Cromo/farmacologia , Gleiquênias/microbiologia , Chumbo/farmacologia , Microbiota/efeitos dos fármacos , Microbiota/genética , Poluentes do Solo/farmacologia , Actinobacteria/efeitos dos fármacos , Actinobacteria/genética , Actinobacteria/metabolismo , Bacteroidetes/efeitos dos fármacos , Bacteroidetes/genética , Bacteroidetes/metabolismo , Biodegradação Ambiental , Cádmio/metabolismo , Cromo/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Firmicutes/efeitos dos fármacos , Firmicutes/genética , Firmicutes/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Chumbo/metabolismo , RNA Ribossômico 16S/genética , Microbiologia do Solo
7.
Arch Microbiol ; 202(7): 1701-1708, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32296869

RESUMO

Responses to sunlight exposure of the oil-degrading Dietzia cinnamea P4 strain were evaluated by transcriptional levels of SOS genes, photoreactivation and genes involved in tolerance to high levels of reactive oxygen species. The P4 strain was exposed for 1 and 2 h and the magnitude of level changes in the mRNA was evaluated by qPCR. The results described the activation of the SOS system, with the decline of the repressor lexA gene levels and the concomitant increase of recA and uvrAD genes levels. The genes that participate in the photoreactivation process were also responsive to sunlight. The phrB gene encoding deoxyribodipyrimidine photo-lyase had its expression increased after 1-h exposure, while the phytAB genes showed a progressive increase over the studied period. The protective genes against reactive oxygen species, catalases, superoxides, peroxidases, and thioredoxins, had their expression rates detected under the conditions validated in this study. These results show a fast and coordinated response of genes from different DNA repair and tolerance mechanisms employed by strain P4, suggesting a complex concerted protective action against environmental stressors.


Assuntos
Actinobacteria/genética , Actinobacteria/efeitos da radiação , Regulação Bacteriana da Expressão Gênica/efeitos da radiação , Luz Solar , Adaptação Fisiológica , Proteínas de Bactérias/genética , Reparo do DNA/genética , Hidrolases/genética , Oxirredutases/genética , Reação em Cadeia da Polimerase em Tempo Real
8.
J Biosci Bioeng ; 130(1): 106-113, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32253091

RESUMO

Monoclonal antibodies (mAbs) are active pharmaceutical ingredients in antibody drugs, produced mainly using recombinant Chinese hamster ovary (CHO) cells. The regulation of recombinant CHO cell proliferation can improve the productivity of heterologous proteins. Chemical compound approaches for cell cycle regulation have the advantages of simplicity and ease of use in industrial processes. However, CHO cells have genetic and phenotypic diversity, and the effects of such compounds might depend on cell line and culture conditions. Increasing the variety of cell cycle inhibitors is a promising strategy to overcome the dependency. Marine microorganisms are a vast and largely undeveloped source of secondary metabolites with physiological activity. In this study, we focused on secondary metabolites of marine microorganisms and evaluated their effectiveness as cell cycle inhibitory compounds. Of 720 extracts from microorganisms (400 actinomycetes and 320 filamentous fungi) collected from the Okinawan Sea, we identified nine extracts that decreased the specific growth rate and increased the specific production rate without reducing cell viability. After fractionating the extracts, the components of active fractions were estimated using time-of-flight mass spectrometry analysis. Then, four compounds, including staurosporine and undecylprodigiosin were deduced to be active compounds. These compounds have been reported to exert a cell cycle inhibitory effect on mammalian cells. These compounds might serve as additives to improve mAb production in CHO cells. This study indicates that secondary metabolites of marine microorganisms are a useful source for new cell cycle inhibitory compounds that can increase mAb production in CHO cells.


Assuntos
Actinobacteria/química , Ciclo Celular/efeitos dos fármacos , Fungos/química , Inibidores do Crescimento/farmacologia , Água do Mar/microbiologia , Actinobacteria/genética , Actinobacteria/isolamento & purificação , Actinobacteria/metabolismo , Animais , Células CHO , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Avaliação Pré-Clínica de Medicamentos , Fungos/genética , Fungos/isolamento & purificação , Fungos/metabolismo , Inibidores do Crescimento/metabolismo , Prodigiosina/análogos & derivados , Prodigiosina/metabolismo , Prodigiosina/farmacologia , Estaurosporina/metabolismo , Estaurosporina/farmacologia
9.
PLoS One ; 15(4): e0231083, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32255799

RESUMO

Plant-associated microbial communities have diverse phenotypic effects on their hosts that are only beginning to be revealed. We hypothesized that morpho-physiological variations in the tropical tree Tabebuia heterophylla, observed on different geological substrates, arise in part due to microbial processes in the rhizosphere. We characterized the microbiota of the rhizosphere and soil communities associated with T. heterophylla trees in high and low altitude sites (with varying temperature and precipitation) of volcanic, karst and serpentine geologies across Puerto Rico. We sampled 6 areas across the island in three geological materials including volcanic, serpentine and karst soils. Collection was done in 2 elevations (>450m and 0-300m high), that included 3 trees for each site and 4 replicate soil samples per tree of both bulk and rhizosphere. Genomic DNA was extracted from 144 samples, and 16S rRNA V4 sequencing was performed on the Illumina MiSeq platform. Proteobacteria, Actinobacteria, and Verrucomicrobia were the most dominant phyla, and microbiomes clustered by geological substrate and elevation. Volcanic samples were enriched in Verrucomicrobia; karst was dominated by nitrogen-fixing Proteobacteria, and serpentine sites harbored the most diverse communities, with dominant Cyanobacteria. Sites with similar climates but differing geologies showed significant differences on rhizobiota diversity and composition demonstrating the importance of geology in shaping the rhizosphere microbiota, with implications for the plant's phenotype. Our study sheds light on the combined role of geology and climate in the rhizosphere microbial consortia, likely contributing to the phenotypic plasticity of the trees.


Assuntos
Raízes de Plantas/microbiologia , Rizosfera , Microbiologia do Solo , Tabebuia/genética , Actinobacteria/classificação , Actinobacteria/genética , Geologia , Filogenia , Raízes de Plantas/genética , Proteobactérias/classificação , Proteobactérias/genética , Porto Rico , RNA Ribossômico 16S/genética , Tabebuia/crescimento & desenvolvimento , Tabebuia/microbiologia , Clima Tropical
10.
Arch Microbiol ; 202(6): 1535-1543, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32236722

RESUMO

A Gram-stain-positive, aerobic, spore-forming actinobacterial strain, designated 160415T, was isolated from a surface soil sample, which was formed on basaltic parent material, collected from Samsun, Turkey. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain 160415T clustered closely with species of the genus Nonomuraea, and showed the highest sequence similarity to Nonomuraea zeae NEAU-ND5T, Nonomuraea candida HMC10T and Nonomuraea turkmeniaca DSM 43926T with 99.1%, 98.9% and 98.7%, respectively. Chemotaxonomic properties including major menaquinones, diaminopimelic acid, sugar and phospholipid profiles also confirmed the affiliation of the strain to the genus Nonomuraea. The DNA G+C content of strain 160415T was 69.6 mol%. DNA-DNA hybridization and average nucleotide identity values between the strain and closely related type strains were less than the recommended cut-off values. On the basis of phylogenetic relationships, genotypic and phenotypic characterizations, strain 160415T represents a novel species of the genus Nonomuraea, for which the name Nonomuraea basaltis sp. nov. is proposed. The type strain is 160415T (= KCTC 39875T = DSM 104309T).


Assuntos
Actinobacteria , Actinobacteria/classificação , Actinobacteria/genética , Actinobacteria/isolamento & purificação , Actinomycetales/genética , Técnicas de Tipagem Bacteriana , Composição de Bases/genética , DNA Bacteriano/genética , Ácidos Graxos/análise , Genoma Bacteriano/genética , Fosfolipídeos/análise , Filogenia , RNA Ribossômico 16S/genética , Sideróforos/análise , Sideróforos/metabolismo , Solo , Microbiologia do Solo , Turquia
11.
J Biosci Bioeng ; 130(2): 187-194, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32334990

RESUMO

Carotenoids serve as one of the most important group of naturally-occurring lipid-soluble pigments which exhibit great biological activities such as antioxidant, anti-inflammatory and provitamin A activities. Owing to their advantageous health effects, carotenoids are widely applied in various industries. Microbial carotenoids synthesis therefore has attracted increasing attention in recent years. In the present study, a marine microorganism originally isolated from seawater in northern Taiwan was determined to be a strain of Gordonia terrae based on its 16S rRNA gene sequence. The strain G. terrae TWRH01 has the ability to synthesize and accumulate the intracellular pigments was identified by gas chromatography-mass spectrometry (GC-MS). The biochemical production characteristics of this strain were studied by employing different fermentation strategies. Findings suggested that G. terrae TWRH01 can actively grow and efficiently synthesize carotenoids in medium adjusted to pH 7 containing 16 g L-1 sucrose as the carbon source, 16 g L-1 yeast extract as the nitrogen source, 0.6 M NaCl concentration, and supplemented with 0.45% (v/v) 1 M CaCl2. Results revealed that the optimization of fermentation yielded 15.29 g L-1 dry biomass and 10.58 µmol L-1 relative ß-carotene concentration. According to GC-MS analysis, the orange-red colored pigments produced were identified as carotenoid derivatives, mainly echinenone and adonixanthin 3'-ß-d-glucoside. Therefore, the new bacterial strain showed a highly potential bioresource for the commercial production of natural carotenoids.


Assuntos
Actinobacteria/metabolismo , Carotenoides/metabolismo , Fermentação , Microbiologia Industrial , Actinobacteria/genética , Biomassa , Nitrogênio/metabolismo , RNA Ribossômico 16S/genética , Taiwan
12.
Arch Microbiol ; 202(6): 1563-1569, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32172289

RESUMO

Cellulosimicrobium sp. JZ28, a root endophytic bacterium from the desert plant Panicum turgidum, was previously identified as a plant growth-promoting bacterium. The genome of JZ28 consists of a 4378,193 bp circular chromosome and contains 3930 CDSs with an average GC content of 74.5%. Whole-genome sequencing analysis revealed that JZ28 was closely related to C. aquatile 3 bp. The genome harbors genes responsible for protection against oxidative, osmotic and salinity stresses, such as the production of osmoprotectants. It also contains genes with a role in the production of volatiles, such as hydrogen sulfide, which promote biotic and abiotic stress tolerance in plants. The presence of three copies of chitinase genes indicates a possible role of JZ28 as biocontrol agent against fungal pathogens, while a number of genes for the degradation of plant biopolymers indicates potential application in industrial processes. Genome sequencing and mining of culture-dependent collections of bacterial endophytes from desert plants provide new opportunities for biotechnological applications.


Assuntos
Actinobacteria , Endófitos/isolamento & purificação , Panicum/microbiologia , Desenvolvimento Vegetal/fisiologia , Actinobacteria/classificação , Actinobacteria/genética , Actinobacteria/isolamento & purificação , Genoma Bacteriano/genética , Plantas/microbiologia , Estresse Fisiológico
13.
J Biosci Bioeng ; 130(1): 36-47, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32179024

RESUMO

Ansamitocin P-3 (AP-3) shows strong anticancer effects and has used as a payload for antibody-drug conjugates. Our previous study have shown that although genetically engineered Actinosynnema pretiosum strains with enhanced UDP-glucose (UDPG) biosynthesis displayed improved AP-3 production compared to the wild-type strain, the increase in yield was far from meeting the industrial demand. In this study, comparative metabolomics analysis complemented with quantitative real-time PCR analysis was performed for the wild-type strain and two mutants (OpgmOugp, ΔzwfΔgnd) to identify possible metabolic bottlenecks and non-intuitive targets for further enhancement of AP-3 production. We observed that enhancing intracellular UDPG availability facilitated the accumulation of intracellular N-demethyl-AP-3 and AP-3, where the transporting of them outside the cell still needs to be developed. We also found that the UDPG biosynthesis was closely associated with the availability of fructose in the medium and a suitable fructose feeding strategy could promote the further improvement of AP-3 titer. In addition, pathway abundance analysis revealed that undesired fatty acid accumulation and down-regulation of amino acid metabolism may be unfavorable for ansamitocin biosynthesis in later stage of production. These results indicate that genetic modification of the UDPG biosynthetic pathways may have pleiotropic effects on AP-3 production. Efforts must be made to eliminate these newly identified metabolic bottlenecks to boost AP-3 production in A. pretiosum.


Assuntos
Actinobacteria/metabolismo , Maitansina/análogos & derivados , Uridina Difosfato Glucose/metabolismo , Actinobacteria/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Vias Biossintéticas/genética , Frutose/metabolismo , Maitansina/biossíntese , Metabolômica
14.
Enzyme Microb Technol ; 135: 109494, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32146933

RESUMO

Glucose isomerase (GIase), an efficient enzyme in the isomerization of d-glucose to d-fructose, has been widely used in food processing. In this study, an efficient expression system for a Thermobifida fusca GIase (GIaseTfus) in Escherichia coli was firstly designed via a two-stage feeding strategy for improving expression level. The cultivation strategy was performed at an exponential feeding rate during the pre-induction phase, followed by a gradient-decreasing feeding rate at the induction phase in a 3-L fermenter. During this process, the effect of induction conditions and the complex nitrogen supplementation in feeding solutions on GIaseTfus production were investigated and optimized. Under the optimal conditions, the yield of GIaseTfus reached 124.1 U/mL, which is the highest expression level of GIase by recombinant E. coli reported to date. Additionally, the obtained GIaseTfus was performed to produce high fructose corn syrup (HFCS) with conversion approacing 55 % from glucose (45 %, w/v) to fructose. According to the molecular dynamic simulation, a number of hydrogen bonds existed in the enzyme-substrate complex could stablilize the transient states, and a appreciate reaction distance of M1 catalytic site and oxygen atom of glucose make the reaction proceed easily, thus resulting in the efficient biosynthesis of HFCS. The function of GIaseTfus renders it a valuable catalyst for HFCS-55 (containing 55 % d-fructose) manufacturing, the most favorable industrial product of HFCS. The efficient expression of GIaseTfus and its efficient HFCS production lays the foundation for its proming industrial application.


Assuntos
Actinobacteria/enzimologia , Aldose-Cetose Isomerases/metabolismo , Proteínas de Bactérias/metabolismo , Xarope de Milho Rico em Frutose/metabolismo , Actinobacteria/genética , Aldose-Cetose Isomerases/química , Aldose-Cetose Isomerases/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Domínio Catalítico , Escherichia coli/genética , Escherichia coli/metabolismo , Frutose/metabolismo , Expressão Gênica , Glucose/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
15.
Enzyme Microb Technol ; 135: 109510, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32146935

RESUMO

An enzyme, l-ribose isomerase (l-RI), mostly catalyzes the isomerization of l-ribose and l-ribulose. These so-called rare sugars are essential for the treatment of cancer and other viral diseases. In the present study, l-ribose isomerase produced from a bacterium, Mycetocola miduiensis (Mm-LRIse), by using l-ribose as a carbon source. The recombinant l-ribose isomerase gene was cloned and overexpressed from M. miduiensis and purified with an exclusive band of 32 kDa by nickel-affinity chromatography. This gene possessed 267 amino acids protein having an estimated molecular weight of 29,568.17 Da. The native molecular weight of Mm-LRIse estimated by HPLC was 134.84 kDa. The recombinant l-ribose isomerase was highly active in sodium phosphate (50 mM) buffer at 40 °C and pH 7.5, showing the specific activity up to 47.40 U mg-1. Mm-LRIse showed no significant enhancement in activity with metallic ions except Mn2+ and Co2+. The values of Km, Kcat, Kcat/Km and Vmax of Mm-LRIse against l-ribose substrate were 42.48 mM, 9259.26 min-1, 217.43 min-1 mM-1, and 277.78 U mg-1 respectively. At equilibrium, the l-ribulose transformation rate was nearly 32 % (6.34 g L-1) using 20 g L-1 of l-ribose. The results revealed that the Mm-LRIse enzyme has a potential for L-ribulose production from l-ribose.


Assuntos
Actinobacteria/enzimologia , Aldose-Cetose Isomerases/química , Proteínas de Bactérias/química , Pentoses/metabolismo , Actinobacteria/genética , Aldose-Cetose Isomerases/genética , Aldose-Cetose Isomerases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Estabilidade Enzimática , Cinética , Pentoses/química , Ribose/metabolismo , Especificidade por Substrato
16.
Appl Environ Microbiol ; 86(9)2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32169935

RESUMO

Conjugation is one of the main mechanisms involved in the spread and maintenance of antibiotic resistance in bacterial populations. We recently showed that the emerging macrolide resistance in the soilborne equine and zoonotic pathogen Rhodococcus equi is conferred by the erm(46) gene carried on the 87-kb conjugative plasmid pRErm46. Here, we investigated the conjugal transferability of pRErm46 to 14 representative bacteria likely encountered by R. equi in the environmental habitat. In vitro mating experiments demonstrated conjugation to different members of the genus Rhodococcus as well as to Nocardia and Arthrobacter spp. at frequencies ranging from ∼10-2 to 10-6 pRErm46 transfer was also observed in mating experiments in soil and horse manure, albeit at a low frequency and after prolonged incubation at 22 to 30°C (environmental temperatures), not 37°C. All transconjugants were able to transfer pRErm46 back to R. equi Conjugation could not be detected with Mycobacterium or Corynebacterium spp. or several members of the more distant phylum Firmicutes such as Enterococcus, Streptococcus, or Staphylococcus Thus, the pRErm46 host range appears to span several actinobacterial orders with certain host restriction within the Corynebacteriales All bacterial species that acquired pRErm46 expressed increased macrolide resistance with no significant deleterious impact on fitness, except in the case of Rhodococcus rhodnii Our results indicate that actinobacterial members of the environmental microbiota can both acquire and transmit the R. equi pRErm46 plasmid and thus potentially contribute to the maintenance and spread of erm(46)-mediated macrolide resistance in equine farms.IMPORTANCE This study demonstrates the efficient horizontal transfer of the Rhodococcus equi conjugative plasmid pRErm46, recently identified as the cause of the emerging macrolide resistance among equine isolates of this pathogen, to and from different environmental Actinobacteria, including a variety of rhodococci as well as Nocardia and Arthrobacter spp. The reported data support the notion that environmental microbiotas may act as reservoirs for the endemic maintenance of antimicrobial resistance in an antibiotic pressurized farm habitat.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Transferência Genética Horizontal , Genes Bacterianos , Macrolídeos/farmacologia , Rhodococcus equi/genética , Actinobacteria/genética , Plasmídeos/genética
17.
Nature ; 578(7796): 582-587, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32051588

RESUMO

Addressing the ongoing antibiotic crisis requires the discovery of compounds with novel mechanisms of action that are capable of treating drug-resistant infections1. Many antibiotics are sourced from specialized metabolites produced by bacteria, particularly those of the Actinomycetes family2. Although actinomycete extracts have traditionally been screened using activity-based platforms, this approach has become unfavourable owing to the frequent rediscovery of known compounds. Genome sequencing of actinomycetes reveals an untapped reservoir of biosynthetic gene clusters, but prioritization is required to predict which gene clusters may yield promising new chemical matter2. Here we make use of the phylogeny of biosynthetic genes along with the lack of known resistance determinants to predict divergent members of the glycopeptide family of antibiotics that are likely to possess new biological activities. Using these predictions, we uncovered two members of a new functional class of glycopeptide antibiotics-the known glycopeptide antibiotic complestatin and a newly discovered compound we call corbomycin-that have a novel mode of action. We show that by binding to peptidoglycan, complestatin and corbomycin block the action of autolysins-essential peptidoglycan hydrolases that are required for remodelling of the cell wall during growth. Corbomycin and complestatin have low levels of resistance development and are effective in reducing bacterial burden in a mouse model of skin MRSA infection.


Assuntos
Antibacterianos , Descoberta de Drogas , Peptídeos Cíclicos , Peptidoglicano/efeitos dos fármacos , Peptidoglicano/metabolismo , Actinobacteria/química , Actinobacteria/genética , Actinobacteria/metabolismo , Animais , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Vias Biossintéticas/genética , Parede Celular/metabolismo , Clorofenóis/química , Clorofenóis/metabolismo , Clorofenóis/farmacologia , Modelos Animais de Doenças , Resistência Microbiana a Medicamentos/efeitos dos fármacos , Resistência Microbiana a Medicamentos/genética , Feminino , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Camundongos , Testes de Sensibilidade Microbiana , Família Multigênica , N-Acetil-Muramil-L-Alanina Amidase/antagonistas & inibidores , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Peptídeos Cíclicos/farmacologia , Filogenia , Pele/microbiologia , Infecções Estafilocócicas/microbiologia
18.
Nat Microbiol ; 5(4): 642-650, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32042128

RESUMO

Although Clostridium difficile is widely considered an antibiotic- and hospital-associated pathogen, recent evidence indicates that this is an insufficient depiction of the risks and reservoirs. A common thread that links all major risk factors of infection is their association with gastrointestinal disturbances, but this relationship to C. difficile colonization has never been tested directly. Here, we show that disturbances caused by diarrhoeal events trigger susceptibility to C. difficile colonization. Using survey data of the human gut microbiome, we detected C. difficile colonization and blooms in people recovering from food poisoning and Vibrio cholerae infections. Carriers remained colonized for year-long time scales and experienced highly variable patterns of C. difficile abundance, where increased shedding over short periods of 1-2 d interrupted week-long periods in which C. difficile was undetectable. Given that short shedding events were often linked to gastrointestinal disturbances, our results help explain why C. difficile is frequently detected as a co-infecting pathogen in patients with diarrhoea. To directly test the impact of diarrhoea on susceptibility to colonization, we developed a mouse model of variable disturbance intensity, which allowed us to monitor colonization in the absence of disease. As mice exposed to avirulent C. difficile spores ingested increasing quantities of laxatives, more individuals experienced C. difficile blooms. Our results indicate that the likelihood of colonization is highest in the days immediately following acute disturbances, suggesting that this could be an important window during which transmission could be interrupted and the incidence of infection lowered.


Assuntos
Infecções por Clostridium/microbiologia , Clostridium difficile/efeitos dos fármacos , Clostridium difficile/patogenicidade , Diarreia/microbiologia , Microbioma Gastrointestinal/efeitos dos fármacos , Laxantes/efeitos adversos , Polietilenoglicóis/efeitos adversos , Actinobacteria/genética , Actinobacteria/crescimento & desenvolvimento , Actinobacteria/isolamento & purificação , Animais , Bacteroidetes/genética , Bacteroidetes/crescimento & desenvolvimento , Bacteroidetes/isolamento & purificação , Infecções por Clostridium/complicações , Clostridium difficile/crescimento & desenvolvimento , Contagem de Colônia Microbiana , Diarreia/induzido quimicamente , Diarreia/complicações , Modelos Animais de Doenças , Fezes/microbiologia , Firmicutes/genética , Firmicutes/crescimento & desenvolvimento , Firmicutes/isolamento & purificação , Fusobactérias/genética , Fusobactérias/crescimento & desenvolvimento , Fusobactérias/isolamento & purificação , Humanos , Masculino , Camundongos , Proteobactérias/genética , Proteobactérias/crescimento & desenvolvimento , Proteobactérias/isolamento & purificação , RNA Ribossômico 16S/genética
19.
Gene ; 733: 144379, 2020 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-31972312

RESUMO

An actinobacterial strain designated Kitasatospora sp. MMS16-BH015, exhibiting high level of heavy metal resistance, was isolated from soil of an abandoned metal mining site, and its potential for metal resistance and secondary metabolite production was studied. The strain was resistant to multiple heavy metals including zinc (up to 100 mM), nickel (up to 2 mM) and copper (up to 0.8 mM), and also showed antimicrobial potential against a broad group of microorganisms, in particular filamentous fungi. The genome of strain MMS16-BH015 was 8.96 Mbp in size with a G + C content of 72.7%, and contained 7270 protein-coding genes and 107 tRNA/rRNA genes. The genome analysis revealed presence of at least 121 metal resistance related genes, which was prominently higher in strain MMS16-BH015 compared to other genomes of Kitasatospora. The genes included those for proteins representing various families involved in the transport of heavy metals, for example dipeptide transport ATP-binding proteins, high-affinity nickel transport proteins, and P-type heavy metal-transporting ATPases. Additionally, 43 biosynthetic gene clusters (BGCs) for secondary metabolites, enriched with those for non-ribosomal peptides, were detected in this multiple heavy metal resistant actinobacterium, which was again the highest among the compared genomes of Kitasatospora. The pan-genome analysis also identified higher numbers of unique genes related to secondary metabolite production and metal resistance mechanism in strain MMS16-BH015. A high level of correlation between the biosynthetic potential and heavy metal resistance could be observed, thus indicating that heavy metal resistant actinobacteria can be a promising source of bioactive compounds.


Assuntos
Actinobacteria/efeitos dos fármacos , Actinobacteria/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/genética , Genoma Bacteriano , Metais Pesados/farmacologia , Actinobacteria/classificação , Actinobacteria/isolamento & purificação , Mineração , Família Multigênica , Filogenia , Microbiologia do Solo
20.
PLoS One ; 15(1): e0227559, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31910230

RESUMO

A multiplex real-time PCR method based on fluorescent TaqMan® probes was developed for the simultaneous detection of the tomato pathogenic bacteria Clavibacter michiganensis subsp. michiganensis, Pseudomonas syringae pv. tomato and bacterial spot-causing xanthomonads. The specificity of the multiplex assay was validated on 44 bacterial strains, including 32 target pathogen strains as well as closely related species and nontarget tomato pathogenic bacteria. The designed multiplex real-time PCR showed high sensitivity when positive amplification was observed for one pg of bacterial DNA in the cases of Clavibacter michiganensis subsp. michiganensis and Pseudomonas syringae pv. tomato bacteria and 100 pg for bacterial spot-causing xanthomonads. The reliability of the developed multiplex real-time PCR assay for in planta detection was verified by recognition of the target pathogens in 18 tomato plants artificially inoculated by each of the target bacteria and tomato samples from production greenhouses.


Assuntos
Actinobacteria/isolamento & purificação , Lycopersicon esculentum/microbiologia , Pseudomonas syringae/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Xanthomonas/isolamento & purificação , Actinobacteria/genética , Actinobacteria/fisiologia , Ambiente Controlado , Lycopersicon esculentum/crescimento & desenvolvimento , Pseudomonas syringae/genética , Pseudomonas syringae/fisiologia , Xanthomonas/genética , Xanthomonas/fisiologia
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