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1.
Nat Biotechnol ; 37(10): 1149-1154, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31501558

RESUMO

Actinobacteria, which are one of the largest bacterial phyla and comprise between 13 and 30% of the soil microbiota, are the main source of antibiotic classes in clinical use1. During screens for antimicrobials, as many as 50% of actinomycete strains are discarded because they produce a known antibiotic (Supplementary Fig. 1) (ref. 2). Despite each strain likely having the capacity to produce many compounds, strains are abandoned because the already characterized antibiotic could interfere with screening for, or purification of, newly discovered compounds3. We applied CRISPR-Cas9 genome engineering to knockout genes encoding two of the most frequently rediscovered antibiotics, streptothricin or streptomycin, in 11 actinomycete strains. We report that this simple approach led to production of different antibiotics that were otherwise masked. We were able to rapidly discover rare and previously unknown variants of antibiotics including thiolactomycin, amicetin, phenanthroviridin and 5-chloro-3-formylindole. This strategy could be applied to existing strain collections to realize their biosynthetic potential.


Assuntos
Antibacterianos/biossíntese , Streptomyces/metabolismo , Sistemas CRISPR-Cas , DNA Bacteriano/genética , Escherichia coli , Regulação Bacteriana da Expressão Gênica , Família Multigênica , Mutação , Streptomyces/genética
2.
J Agric Food Chem ; 67(26): 7266-7273, 2019 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-31244199

RESUMO

Chemical investigation of fungus Pochonia chlamydosporia strain 170, derived from rice fermentation sediment samples, afforded seven radicicol analogues, including two new compounds, monocillin VI (1) and monocillin VII (2), and five known compounds, monocillin II (3), monorden D (4), monocillin IV (5), monocillin V (6), and pochonin M (7). The structures of compounds 1-7 were established primarily by analysis of nuclear magnetic resonance data, and the absolute configurations of the secondary alcohol in compounds 1 and 2 were assigned by the modified Mosher method. All seven compounds have modest antibacterial activities, with a minimal inhibitory concentration (MIC) of 25.6 µg/mL for compounds 1 and 3-7 and 51.2 µg/mL for compound 2, on inhibition of the growth of the plant pathogen Xanthomonas campestris (the positive control ampicillin showed a MIC value of 12.8 µg/mL), indicating that the fungus has the potential to control bacterial disease. The biosynthetic gene cluster and putative biosynthetic pathways of these radicicol analogues in the P. chlamydosporia genome were proposed. These findings increase our knowledge of the chemical potential of P. chlamydosporia and may allow us to better utilize the fungus as a biological control agent.


Assuntos
Antibacterianos/química , Hypocreales/metabolismo , Macrolídeos/química , Antibacterianos/biossíntese , Antibacterianos/farmacologia , Vias Biossintéticas , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hypocreales/química , Hypocreales/genética , Macrolídeos/metabolismo , Macrolídeos/farmacologia , Testes de Sensibilidade Microbiana , Família Multigênica , Xanthomonas campestris/efeitos dos fármacos , Xanthomonas campestris/crescimento & desenvolvimento
3.
Microb Pathog ; 135: 103609, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31247255

RESUMO

This article reports the utilization of Malus domestica for the synthesis of silver nanoparticles (AgNPs) with cytotoxic activity against the Michigan Cancer Foundation-7 (MCF-7) cell line as well as their antibacterial and radical scavenging potential. The biosynthesized AgNPs were confirmed using various analytical characterization techniques. The cytotoxic effect of Malus domestica-AgNPs (M.d-AgNPs) was studied by MTT assay and scavenging efficacy was assessed by DPPH, nitric oxide radical and phosphomolybdate assays. Furthermore, green synthesized nanoparticles were evaluated for their antibacterial activity against multidrug resistant-clinical isolates. M.d-AgNPs were observed to be almost spherical in shape with an average diameter from 50 to 107.3 nm as assessed by TEM and DLS. M.d-AgNPs revealed the dose-dependent antioxidant activity and antimicrobial activity against multidrug-resistant bacterial strain viz. Enterobacter aerogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Escherichia coli. Also, in vitro studies revealed dose-dependent cytotoxic effects of M.d-AgNPs treated MCF-7 cell line. The data strongly suggest that M.d-AgNPs had a potential antioxidant, antimicrobial and cytotoxicity activity.


Assuntos
Antibacterianos/biossíntese , Antibacterianos/farmacologia , Química Verde/métodos , Células MCF-7/efeitos dos fármacos , Malus/metabolismo , Nanopartículas Metálicas/química , Prata/farmacologia , Antineoplásicos/farmacologia , Antioxidantes/análise , Biofilmes/efeitos dos fármacos , Análise Custo-Benefício , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Estabilidade de Medicamentos , Depuradores de Radicais Livres , Química Verde/economia , Células HEK293/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Tamanho da Partícula , Compostos Fitoquímicos/farmacologia , Difração de Raios X
4.
PLoS Genet ; 15(6): e1008195, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31181062

RESUMO

To characterize the consequences of eliminating essential functions needed for peptidoglycan synthesis, we generated deletion mutations of Acinetobacter baylyi by natural transformation and visualized the resulting microcolonies of dead cells. We found that loss of genes required for peptidoglycan precursor synthesis or polymerization led to the formation of polymorphic giant cells with diameters that could exceed ten times normal. Treatment with antibiotics targeting early or late steps of peptidoglycan synthesis also produced giant cells. The giant cells eventually lysed, although they were partially stabilized by osmotic protection. Genome-scale transposon mutant screening (Tn-seq) identified mutations that blocked or accelerated giant cell formation. Among the mutations that blocked the process were those inactivating a function predicted to cleave murein glycan chains (the MltD murein lytic transglycosylase), suggesting that giant cell formation requires MltD hydrolysis of existing peptidoglycan. Among the mutations that accelerated giant cell formation after ß-lactam treatment were those inactivating an enzyme that produces unusual 3->3 peptide cross-links in peptidoglycan (the LdtG L,D-transpeptidase). The mutations may weaken the sacculus and make it more vulnerable to further disruption. Although the study focused on A. baylyi, we found that a pathogenic relative (A. baumannii) also produced giant cells with genetic dependencies overlapping those of A. baylyi. Overall, the analysis defines a genetic pathway for giant cell formation conserved in Acinetobacter species in which independent initiating branches converge to create the unusual cells.


Assuntos
Acinetobacter/genética , Genes Essenciais/genética , Glicosiltransferases/genética , Peptidoglicano/genética , Acinetobacter/enzimologia , Antibacterianos/biossíntese , Ciclo Celular/genética , Divisão Celular/genética , Parede Celular/enzimologia , Parede Celular/genética , Elementos de DNA Transponíveis/genética , Escherichia coli/genética , Deleção de Genes , Genoma Bacteriano/genética , Peptidoglicano/biossíntese , Peptidil Transferases/genética , Deleção de Sequência/genética
5.
PLoS Comput Biol ; 15(5): e1007109, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31150382

RESUMO

Understanding the mechanisms that promote the assembly and maintenance of host-beneficial microbiomes is an open problem. Empirical evidence supports the idea that animal and plant hosts can combine 'private resources' with the ecological phenomenon known as 'community bistability' to favour some microbial strains over others. We briefly review evidence showing that hosts can: (i) protect the growth of beneficial strains in an isolated habitat, (ii) use antibiotics to suppress non-beneficial, competitor strains, and (iii) provide resources that only beneficial strains are able to translate into an increased rate of growth, reproduction, or antibiotic production. We then demonstrate in a spatially explicit, individual-based model that these three mechanisms act similarly by selectively promoting the initial proliferation of preferred strains, that is, by acting as a private resource. The faster early growth of preferred strains, combined with the phenomenon of 'community bistability,' allows those strains to continue to dominate the microbiome even after the private resource is withdrawn or made public. This is because after a beneficial colony reaches a sufficiently large size, it can resist invasion by parasites without further private support from the host. We further explicitly model localized microbial interactions and diffusion dynamics, and we show that an intermediate level of antibiotic diffusion is the most efficient mechanism in promoting preferred strains and that there is a wide range of parameters under which hosts can promote the assembly of a self-sustaining defensive microbiome. This in turn supports the idea that hosts readily evolve to promote host-beneficial defensive microbiomes.


Assuntos
Interações entre Hospedeiro e Microrganismos/fisiologia , Microbiota/fisiologia , Animais , Antibacterianos/biossíntese , Biologia Computacional , Ecossistema , Modelos Biológicos , Simbiose/fisiologia
6.
Microb Pathog ; 132: 129-136, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31054367

RESUMO

Deep Sea sediment cores were collected from the surrounding of active volcanic Barren Island, Andaman & Nicobar Islands. A total of 123 cultivable marine actinobacteria were isolated and identified based on their biochemical and 16S rDNA sequences. The isolates were categorized under 10 genera, of which Streptomyces sp., Dietzia sp. and Brevibacterium sp. are the dominant genera. Of 123 isolates, 73 isolates exhibited appreciable inhibitory activity against both Gram positive and Gram negative bacteria. High frequencies of positive PCR amplification were obtained for PKS-I (39.13%), PKS-II (56.52%) and NRPS (69.57%). The highest levels of biosynthetic systems were observed for NRPS and PKS-II. Majority of the actinobacterial isolates revealed excellent potential for bioprospecting of novel byproducts with industrial and pharmaceutical importance.


Assuntos
Actinobacteria/classificação , Actinobacteria/isolamento & purificação , Actinobacteria/metabolismo , Antibacterianos/biossíntese , Biodiversidade , Sedimentos Geológicos/microbiologia , Água do Mar/microbiologia , Actinobacteria/genética , DNA Bacteriano/genética , DNA Ribossômico/genética , Bactérias Gram-Negativas/patogenicidade , Bactérias Gram-Positivas/patogenicidade , Ilhas , Filogenia , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genética , Metabolismo Secundário , Streptomyces/classificação , Streptomyces/genética
7.
MBio ; 10(3)2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31088917

RESUMO

Actinobacteria have long been the main source of antibiotics, secondary metabolites with tightly controlled biosynthesis by environmental and physiological factors. Phosphorylation of exogenous glucosamine has been suggested as a mechanism for incorporation of this extracellular material into secondary metabolite biosynthesis, but experimental evidence of specific glucosamine kinases in Actinobacteria is lacking. Here, we present the molecular fingerprints for the identification of a unique family of actinobacterial glucosamine kinases. Structural and biochemical studies on a distinctive kinase from the soil bacterium Streptacidiphilus jiangxiensis unveiled its preference for glucosamine and provided structural evidence of a phosphoryl transfer to this substrate. Conservation of glucosamine-contacting residues across a large number of uncharacterized actinobacterial proteins unveiled a specific glucosamine binding sequence motif. This family of kinases and their genetic context may represent the missing link for the incorporation of environmental glucosamine into the antibiotic biosynthesis pathways in Actinobacteria and can be explored to enhance antibiotic production.IMPORTANCE The discovery of novel enzymes involved in antibiotic biosynthesis pathways is currently a topic of utmost importance. The high levels of antibiotic resistance detected worldwide threaten our ability to combat infections and other 20th-century medical achievements, namely, organ transplantation or cancer chemotherapy. We have identified and characterized a unique family of enzymes capable of phosphorylating glucosamine to glucosamine-6-phosphate, a crucial molecule directly involved in the activation of antibiotic production pathways in Actinobacteria, nature's main source of antimicrobials. The consensus sequence identified for these glucosamine kinases will help establish a molecular fingerprint to reveal yet-uncharacterized sequences in antibiotic producers, which should have an important impact in biotechnological and biomedical applications, including the enhancement and optimization of antibiotic production.


Assuntos
Actinobacteria/enzimologia , Actinobacteria/genética , Glucosamina/análogos & derivados , Glucose-6-Fosfato/análogos & derivados , Fosfotransferases/genética , Fosfotransferases/metabolismo , Antibacterianos/biossíntese , Impressões Digitais de DNA , Glucosamina/metabolismo , Glucose-6-Fosfato/metabolismo , Fosforilação , Ligação Proteica , RNA Ribossômico 16S/genética , Microbiologia do Solo
8.
Appl Microbiol Biotechnol ; 103(11): 4539-4548, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30997553

RESUMO

The MtrA-MtrB two-component regulatory system is highly conserved in Actinobacteria and plays crucial roles in cell cycle progression, cell morphology, antibiotic resistance, and osmoprotection. Previously, we revealed that the MtrA protein of Saccharopolyspora erythraea E3 strain (a high erythromycin-producing strain) had a two amino acid (H197 and V198) deletion in the DNA recognition helices of the C-terminal domain compared to the wild type S. erythraea strain NRRL2338. Here, we identified mepA (encoding a membrane protein related to metalloendopeptidases) as an MtrA target gene, and found that deleting the two amino acids in MtrA (MtrAdel) resulted in the loss of its DNA-binding activity for the mepA gene. The mutant MtrAdel lost its regulatory activity and affected various physiological functions consistent with mtrA deletion, including increased erythromycin biosynthesis, enhanced antibiotic resistance, deregulated osmoprotection, and improved transport of substances. The introduction of the wild type mtrA gene into the S. erythraea E3 strain with the mtrAdel gene decreased the erythromycin yield by approximately 50%, confirming that MtrA repressed erythromycin production. These findings demonstrate that MtrA is an important pleiotropic regulator of erythromycin biosynthesis, antibiotic resistance, osmoprotection, and substance transport in S. erythraea and provide new insights for improving erythromycin production. Future studies linking the molecular effects of MtrA to these phenotypes will improve our understanding of the MtrA-MtrB two-component regulatory system in Actinobacteria.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Antibacterianos/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Eritromicina/biossíntese , Saccharopolyspora/enzimologia , Saccharopolyspora/metabolismo , Deleção de Sequência , Transporte Biológico , Farmacorresistência Bacteriana , Regulação Bacteriana da Expressão Gênica , Teste de Complementação Genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fenótipo , Saccharopolyspora/crescimento & desenvolvimento
9.
Org Lett ; 21(9): 3148-3152, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-30990701

RESUMO

Feeding studies indicate a possible synthetic pattern for the N-terminal cis-aminocyclopentane carboxylic acid (ACPC) and suggest an unusual source of the high-carbon sugar skeleton of amipurimycin (APM). The biosynthetic gene cluster of APM was identified and confirmed by in vivo experiments. A C9 core intermediate was discovered from null mutants of ACPC pathway, and an ATP-grasp enzyme (ApmA8) was reconstituted in vitro for ACPC loading. Our observations allow a first proposal of the APM biosynthetic pathway.


Assuntos
Antibacterianos/biossíntese , Família Multigênica , Nucleosídeos/biossíntese , Purinas/biossíntese , Açúcares/química , Trifosfato de Adenosina/metabolismo , Antibacterianos/química , Vias Biossintéticas/genética , Cicloleucina/química , Enzimas/genética , Enzimas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Mutação , Nucleosídeos/química , Purinas/química , Streptomyces/genética , Streptomyces/metabolismo
10.
Mar Drugs ; 17(4)2019 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-30934741

RESUMO

Vibrio diabolicus A1SM3 strain was isolated from a sediment sample from Manaure Solar Saltern in La Guajira and the produced crude extracts have shown antibacterial activity against methicillin-resistant Staphylococcus aureus and cytotoxic activity against human lung cell line. Thus, the aim of this research was to identify the main compound responsible for the biological activity observed and to systematically study how each carbon and nitrogen source in the growth media, and variation of the salinity, affect its production. For the characterization of the bioactive metabolites, 15 fractions obtained from Vibrio diabolicus A1SM3 crude extract were analyzed by HPLC-MS/MS and their activity was established. The bioactive fractions were dereplicated with Antibase and Marinlit databases, which combined with nuclear magnetic resonance (NMR) spectra and fragmentation by MS/MS, led to the identification of 2,2-di(3-indolyl)-3-indolone (isotrisindoline), an indole-derivative antibiotic, previously isolated from marine organisms. The influence of the variations of the culture media in isotrisindoline production was established by molecular network and MZmine showing that the media containing starch and peptone at 7% NaCl was the best culture media to produce it. Also, polyhydroxybutyrates (PHB) identification was established by MS/MS mainly in casamino acids media, contributing to the first report on PHB production by this strain.


Assuntos
Antibacterianos/biossíntese , Antibacterianos/farmacologia , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Técnicas Bacteriológicas/métodos , Vibrio/química , Vibrio/metabolismo , Alcaloides/biossíntese , Alcaloides/isolamento & purificação , Alcaloides/farmacologia , Antibacterianos/isolamento & purificação , Antineoplásicos/isolamento & purificação , Organismos Aquáticos/microbiologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Misturas Complexas/química , Misturas Complexas/isolamento & purificação , Meios de Cultura , Humanos , Hidroxibutiratos/química , Hidroxibutiratos/farmacologia , Isoindóis/isolamento & purificação , Isoindóis/metabolismo , Modelos Moleculares , Poliésteres/química , Poliésteres/farmacologia , Poli-Hidroxialcanoatos/química , Poli-Hidroxialcanoatos/farmacologia , Salinidade
11.
Appl Microbiol Biotechnol ; 103(11): 4467-4481, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30989253

RESUMO

Locillomycins are cyclic lipononapeptides assembled by a nonlinear hexamodular NRPS and have strong antibacterial activity. In this study, we genetically engineered Bacillus velezensis FZB42 as a surrogate host for the heterologous expression of the loc gene cluster for locillomycins. The fosmid N13 containing whole loc gene cluster was screened from the B. velezensis 916 genomic library. Subsequently, a spectinomycin resistance cassette, and the cassette fused with an IPTG inducible promoter Pspac, was introduced in the fosmid N13 using λ Red recombination system, respectively. The resulting fosmids, designated N13+Spec and N13+PSSpec, were used for the transformation of B. velezensis FZB42 to obtain derivative strains FZBNPLOC and FZBPSLOC. RT-PCR and qRT-PCR results revealed the efficient heterologous expression of the loc gene cluster in both derivative strains. Particularly, there was positive correlation between the derivative FZBPSLOC strain and the enhanced production of locillomycins upon addition of the inducer IPTG with the highest production of locillomycins at 15-fold more than that of B. velezensis 916. This overproduction of locillomycins was also related to the enhancement of antibacterial activity against methicillin-resistant Staphylococcus aureus, and exhibited moderate changes in its hemolytic activity. Together our findings demonstrate that the nonlinear hexamodular NRPS, encoded by the loc gene cluster from B. velezensis 916, is sufficient for the biosynthesis of cyclic lipononapeptide locillomycins in the surrogate host B. velezensis FZB42. Moreover, the FZBPSLOC strain will also be useful for further development of novel locillomycins derivatives with improved antibacterial activity.


Assuntos
Antibacterianos/biossíntese , Bacillus/metabolismo , Proteínas de Bactérias/biossíntese , Vias Biossintéticas/genética , Lipopeptídeos/biossíntese , Engenharia Metabólica/métodos , Peptídeos Cíclicos/biossíntese , Proteínas Recombinantes/biossíntese , Bacillus/genética , Proteínas de Bactérias/genética , Expressão Gênica , Lipopeptídeos/genética , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Família Multigênica , Peptídeos Cíclicos/genética , Proteínas Recombinantes/genética
13.
Microb Pathog ; 131: 87-97, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30951817

RESUMO

Occasionally, endophytic fungal species cognize as a hidden prospective source of plant secondary metabolites. In this study, a potent Penicillium setosum sp. nov. was explored for its detailed antibacterial action on Escherichia coli and Staphylococcus aureus through different in vitro and in silico assays. Fluorescence based viability assay determined increase in the number of dead cells in course of time with the continual exposure of extract during a 4 h period. Scanning electron micrographs reflect the distinguishable morphological changes in treated cells, namely shortening of size, bubbles, and blisters on the surface of E. coli, as well as open holes and deep craters on the surface of S. aureus, ultimately leading to rupture of cells. Significant intracellular changes in bacteria were remarkably noticed through different membrane permeabilization assays. The rate of Na+ and K+ leakage with respect to time, intracellular material and cytoplasmic ß-galactosidase release were measured spectroscopically. The results indisputably prove that membrane disruption of S. aureus cells occurs within 2 h and in E.coli occurs in between 2 and 4 h of exposure. Crude extract of P. setosum was fractioned using semi-preparative HPLC and the separated antibacterial active fraction showed antibacterial efficacy with the minimum inhibitory concentration of 8 µg/mL against both organisms. Active fraction contains four well-known plant metabolite belongs to the polyphenolic group (Leucodelphinidin, dihydroquercetin, kaempferol, and quercetin) and one polyketide (patulin) familiar as fungal metabolite, identified through high resolution LC-MS. Interaction mechanisms of identified compounds with nine important antimicrobial drug targets showed highest binding affinity by leucodelphinidin followed by dihydroquercetin > kaempferol > quercetin. This is the first instance of using leucodelphinidin and dihydroquercetin for detailed interaction study with multiple targets, and it was found that they showed more effective interaction than quercetin, which was earlier utilized for antibacterial studies.


Assuntos
Antibacterianos/farmacologia , Simulação por Computador , Simulação de Acoplamento Molecular , Penicillium/metabolismo , Antibacterianos/biossíntese , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Flavonoides/biossíntese , Flavonoides/farmacologia , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Potássio/metabolismo , Quercetina/análogos & derivados , Quercetina/biossíntese , Quercetina/farmacologia , Metabolismo Secundário , Sódio/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , beta-Galactosidase/metabolismo
14.
Appl Microbiol Biotechnol ; 103(10): 4089-4102, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30937499

RESUMO

Teicoplanin is a frontline glycopeptide antibiotic produced by Actinoplanes teichomyceticus. It is used to treat complicated cases of infection, including pediatric ones, caused by Gram-positive pathogens. There is a steady interest in elucidating the genetic mechanisms determining teicoplanin production, as they would help overproduce known teicoplanins and discover novel glycopeptides. Herein, we investigate the transcriptional organization of the tei biosynthetic gene cluster and the roles of the cluster-situated regulatory genes in controlling teicoplanin production and self-resistance in A. teichomyceticus. We demonstrate that the tei cluster is organized into nine polygenic and nine monogenic transcriptional units. Most of tei biosynthetic genes are subjected to StrR-like Tei15* control, which, in turn, appears to be regulated by LuxR-type Tei16*. Expression of the genes conferring teicoplanin self-resistance in A. teichomyceticus is not co-regulated with antibiotic production. The gene tei31*, coding for a putative DNA binding protein, is not expressed under teicoplanin producing conditions and is dispensable for antibiotic production. Finally, phylogenesis reconstruction of the glycopeptide cluster-encoded regulators reveals two main clades of StrR-like regulators. Tei15* and close orthologues form one of these clades; the second clade is composed by orthologues of Bbr and Dbv4, governing the biosynthesis of balhimycin and teicoplanin-like A40926, respectively. In addition, the LuxR-type Tei16* appears unrelated to the LuxR-like Dbv3, which is controlling A40926 biosynthesis. Our results shed new light on teicoplanin biosynthesis regulation and on the evolution of novel and old glycopeptide biosynthetic gene clusters.


Assuntos
Antibacterianos/biossíntese , Vias Biossintéticas/genética , Regulação Bacteriana da Expressão Gênica , Genes Reguladores , Micromonosporaceae/genética , Micromonosporaceae/metabolismo , Teicoplanina/biossíntese , Farmacorresistência Bacteriana , Perfilação da Expressão Gênica , Ordem dos Genes , Óperon
15.
J Photochem Photobiol B ; 194: 119-127, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30953913

RESUMO

'Go green' has also been implied to nanotechnology by harbouring eco-benign principle for a cleaner production of silver nanoparticles (AgNPs). This was achieved using a nitrate reducing Bacillus subtilis L1 (KT266579.1) inhabiting rhizosphere soil under optimized laboratory conditions, highlighting on its antibacterial modus operandi. Nano-characteristics and antimicrobial mechanism were investigated using spectroscopic and electron microscopic studies. Spectroscopic and microscopic characterization revealed typical surface plasmon resonance (SPR) with λmax 420 nm showing mean particle size of ~28.30 nm and spherical shaped nanoparticles. Antimicrobial susceptibility pattern of clinically important pathogens (n = 15) exposed to AgNPs at 10 µg, 15 µg and 20 µg/mL for 18 h was found significant in a dose dependent fashion. Electron and atomic force microscopic (AFM) studies have demonstrated the typical bactericidal effect of AgNPs (<25 µg/mL) associated with 'pitting effect', cell shrinkage and increase in surface roughness. The EDX spectrum of the control and treated bacteria showed the intrusion of AgNPs inside the bacterial cells endorsing the event of bacterial paralysis. DNA fragmentation assay demonstrated significant DNA damage in the form of smear, indicative of genotoxicity at ≤32 µg and ≤16 µg/mL of AgNPs respectively for Gram positive and negative strains in <12 h. These results suggest that AgNPs possess excellent antimicrobial activity, providing a potential lead for developing a broad spectrum antibacterial agent and extending its therapeutic modalities targeting antibiotic resistant strains at gene level.


Assuntos
Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Bioengenharia , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Nanopartículas Metálicas , Prata/metabolismo , Prata/farmacologia , Antibacterianos/biossíntese , Antibacterianos/química , Antibacterianos/farmacologia , Análise Custo-Benefício , Fragmentação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Concentração de Íons de Hidrogênio , Imagem Molecular , Prata/química , Temperatura Ambiente
16.
Biotechnol Lett ; 41(4-5): 495-502, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30927135

RESUMO

Mupirocin is an antibiotic from monocarboxylic acid class used as antibacterial agent against methicillin-resistant Staphylococcus aureus (MRSA) and can be obtained as a mixture of four pseudomonic acids by Pseudomonas fluorescens biosynthesis. Nowadays improving antibiotics occupies an important place in the pharmaceutical industry as more and more resistant microorganisms are developing. Mupirocin is used to control the MRSA outbreaks, for infections of soft tissue or skin and for nasal decolonization. Due to its wide use without prescription, the microorganism's resistance to Mupirocin increased from up to 81%, thus becoming imperative its control or improvement. As the biotechnological production of Mupirocin has not been previously reviewed, in the present paper we summarize some consideration on the biochemical process for the production of pseudomonic acids (submerged fermentation and product recovery). Different strains of Pseudomonas, different culture medium and different conditions for the fermentation were analysed related to the antibiotics yield and the product recovery step is analysed in relation to the final purity. However, many challenges have to be overcome in order to obtain pseudomonic acid new versions with better properties related to antibacterial activity.


Assuntos
Antibacterianos/biossíntese , Antibacterianos/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Mupirocina/biossíntese , Mupirocina/farmacologia , Pseudomonas fluorescens/metabolismo , Infecções Estafilocócicas/tratamento farmacológico , Portador Sadio/tratamento farmacológico , Portador Sadio/microbiologia , Farmacorresistência Bacteriana , Fermentação , Humanos , Infecções dos Tecidos Moles/tratamento farmacológico , Infecções dos Tecidos Moles/microbiologia , Infecções Estafilocócicas/microbiologia , Infecções Cutâneas Estafilocócicas/tratamento farmacológico , Infecções Cutâneas Estafilocócicas/microbiologia , Tecnologia Farmacêutica/métodos
17.
Nat Commun ; 10(1): 1115, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30846700

RESUMO

The genome of the thermophilic bacterium, Aeribacillus pallidus 8, encodes the bacteriocin pallidocin. It belongs to the small class of glycocins and is posttranslationally modified, containing an S-linked glucose on a specific Cys residue. In this study, the pallidocin biosynthetic machinery is cloned and expressed in Escherichia coli to achieve its full biosynthesis and modification. It targets other thermophilic bacteria with potent activity, demonstrated by a low minimum inhibitory concentration (MIC) value. Moreover, the characterized biosynthetic machinery is employed to produce two other glycopeptides Hyp1 and Hyp2. Pallidocin and Hyp1 exhibit antibacterial activity against closely related thermophilic bacteria and some Bacillus sp. strains. Thus, heterologous expression of a glycocin biosynthetic gene cluster including an S-glycosyltransferase provides a good tool for production of hypothetical glycocins encoded by various bacterial genomes and allows rapid in vivo screening.


Assuntos
Bacillaceae/metabolismo , Bacteriocinas/biossíntese , Sequência de Aminoácidos , Antibacterianos/biossíntese , Antibacterianos/química , Antibacterianos/farmacologia , Bacillaceae/genética , Bacteriocinas/genética , Bacteriocinas/farmacologia , Clonagem Molecular , Dissulfetos/química , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos , Glicopeptídeos/biossíntese , Glicopeptídeos/genética , Glicopeptídeos/farmacologia , Testes de Sensibilidade Microbiana , Família Multigênica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos
18.
Org Lett ; 21(5): 1287-1291, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30735051

RESUMO

The burnettramic acids are a new class of antibiotics from an Australian fungus Aspergillus burnettii. The rare bolaamphiphilic scaffold consists of ß-d-mannose linked to a pyrrolizidinedione unit via a 26-carbon chain. The most abundant metabolite displayed potent in vitro antifungal activity. Comparative genomics identified the hybrid PKS-NRPS bua gene cluster, which was verified by heterologous pathway reconstitution in Aspergillus nidulans.


Assuntos
Antibacterianos/síntese química , Aspergillus/química , Compostos Heterocíclicos com 2 Anéis/química , Antibacterianos/biossíntese , Aspergillus/metabolismo , Austrália , Isomerismo , Manose/química , Estrutura Molecular , Família Multigênica , Oxirredução , Pirrolidinas/química , Metabolismo Secundário
19.
Fish Shellfish Immunol ; 87: 410-413, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30703553

RESUMO

NK-lysin, despite being a direct effector of cytotoxic T and natural killer cells, is an antimicrobial peptide (AMP) with known antibacterial function in vertebrates and so in fish. Its presence has been described in different tissues of teleost fish. One of the strongest antimicrobial barriers in fish is skin-secreted mucus; however, this mucus has been found to contain only a small number of AMPs. The present study describes for the first time the constitutive expression of NK-lysin in Atlantic salmon (Salmo salar) mucus produced by the skin, recording the AMP at a higher concentration than in serum with greater bacteriostatic activity. Hepcidin may be involved to a greater extent in systemic responses since it was expressed to a higher degree in serum which was more potent for alternative complement and peroxidase activities.


Assuntos
Antibacterianos/imunologia , Hepcidinas/imunologia , Muco/imunologia , Proteolipídeos/imunologia , Salmo salar/imunologia , Animais , Antibacterianos/biossíntese , Hepcidinas/biossíntese , Hepcidinas/sangue , Imunidade Inata , Proteolipídeos/biossíntese , Pele/metabolismo
20.
Mol Cell ; 73(4): 749-762.e5, 2019 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-30661981

RESUMO

The introduction of azole heterocycles into a peptide backbone is the principal step in the biosynthesis of numerous compounds with therapeutic potential. One of them is microcin B17, a bacterial topoisomerase inhibitor whose activity depends on the conversion of selected serine and cysteine residues of the precursor peptide to oxazoles and thiazoles by the McbBCD synthetase complex. Crystal structures of McbBCD reveal an octameric B4C2D2 complex with two bound substrate peptides. Each McbB dimer clamps the N-terminal recognition sequence, while the C-terminal heterocycle of the modified peptide is trapped in the active site of McbC. The McbD and McbC active sites are distant from each other, which necessitates alternate shuttling of the peptide substrate between them, while remaining tethered to the McbB dimer. An atomic-level view of the azole synthetase is a starting point for deeper understanding and control of biosynthesis of a large group of ribosomally synthesized natural products.


Assuntos
Antibacterianos/biossíntese , Proteínas de Bactérias/metabolismo , Bacteriocinas/biossíntese , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Complexos Multienzimáticos/metabolismo , Ribossomos/enzimologia , Inibidores da Topoisomerase II/metabolismo , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Bacteriocinas/química , Bacteriocinas/farmacologia , Sítios de Ligação , Cristalografia por Raios X , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Modelos Moleculares , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Quaternária de Proteína , Ribossomos/efeitos dos fármacos , Ribossomos/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Relação Estrutura-Atividade , Inibidores da Topoisomerase II/química , Inibidores da Topoisomerase II/farmacologia , Difração de Raios X
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