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1.
Bioresour Technol ; 318: 124217, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33096440

RESUMO

Myxobacteria are soil microorganisms with the ability to break down biological macromolecules due to the secretion of a large number of extracellular enzymes, but there has been no research report on myxobacterial lytic polysaccharide monooxygenases (LPMOs). In this study, two LPMO10s, ViLPMO10A and ViLPMO10B, from myxobacterium Vitiosangium sp. GDMCC 1.1324 were characterized. Of which, ViLPMO10B is a C1-oxidizing cellulose-active LPMO. Moreover, ViLPMO10B could decrease the degree of polymerization of crop straws cellulose and synergize with commercial cellulase to promote the saccharification. When the weight ratio of commercial cellulase to ViLPMO10B was 9:1, the conversion efficiency of corn stalk, sugarcane bagasse, and rice straw into reducing sugar was improved by 17%, 16%, and 22%, respectively, compared with commercial cellulase without ViLPMO10B. These results indicate that ViLPMO10B has the potential to be a component of a high-efficient cellulase cocktail and has application value in the saccharification of agricultural residual biomasses.


Assuntos
Celulase , Myxococcales , Biomassa , Celulase/metabolismo , Celulose/metabolismo , Produtos Agrícolas/metabolismo , Hidrólise , Myxococcales/metabolismo , Oxirredução
2.
Appl Environ Microbiol ; 86(16)2020 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-32532868

RESUMO

Biological nitrogen fixation is an essential reaction in a major pathway for supplying nitrogen to terrestrial environments. Previous culture-independent analyses based on soil DNA/RNA/protein sequencing could globally detect the nitrogenase genes/proteins of Anaeromyxobacter (in the class Deltaproteobacteria), commonly distributed in soil environments and predominant in paddy soils; this suggests the importance of Anaeromyxobacter in nitrogen fixation in soil environments. However, direct experimental evidence is lacking; there has been no research on the genetic background and ability of Anaeromyxobacter to fix nitrogen. Therefore, we verified the diazotrophy of Anaeromyxobacter based on both genomic and culture-dependent analyses using Anaeromyxobacter sp. strains PSR-1 and Red267 isolated from soils. Based on the comparison of nif gene clusters, strains PSR-1 and Red267 as well as strains Fw109-5, K, and diazotrophic Geobacter and Pelobacter in the class Deltaproteobacteria contain the minimum set of genes for nitrogenase (nifBHDKEN). These results imply that Anaeromyxobacter species have the ability to fix nitrogen. In fact, Anaeromyxobacter PSR-1 and Red267 exhibited N2-dependent growth and acetylene reduction activity (ARA) in vitro Transcriptional activity of the nif gene was also detected when both strains were cultured with N2 gas as a sole nitrogen source, indicating that Anaeromyxobacter can fix and assimilate N2 gas by nitrogenase. In addition, PSR-1- or Red267-inoculated soil showed ARA activity and the growth of the inoculated strains on the basis of RNA-based analysis, demonstrating that Anaeromyxobacter can fix nitrogen in the paddy soil environment. Our study provides novel insights into the pivotal environmental function, i.e., nitrogen fixation, of Anaeromyxobacter, which is a common soil bacterium.IMPORTANCE Anaeromyxobacter is globally distributed in soil environments, especially predominant in paddy soils. Current studies based on environmental DNA/RNA analyses frequently detect gene fragments encoding nitrogenase of Anaeromyxobacter from various soil environments. Although the importance of Anaeromyxobacter as a diazotroph in nature has been suggested by culture-independent studies, there has been no solid evidence and validation from genomic and culture-based analyses that Anaeromyxobacter fixes nitrogen. This study demonstrates that Anaeromyxobacter harboring nitrogenase genes exhibits diazotrophic ability; moreover, N2-dependent growth was demonstrated in vitro and in the soil environment. Our findings indicate that nitrogen fixation is important for Anaeromyxobacter to survive under nitrogen-deficient environments and provide a novel insight into the environmental function of Anaeromyxobacter, which is a common bacterium in soils.


Assuntos
Myxococcales/metabolismo , Ciclo do Nitrogênio , Fixação de Nitrogênio , Microbiologia do Solo , Myxococcales/classificação , Myxococcales/isolamento & purificação , Fixação de Nitrogênio/genética
3.
J Biol Chem ; 295(6): 1587-1597, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-31914416

RESUMO

The heme-based oxygen sensor protein AfGcHK is a globin-coupled histidine kinase in the soil bacterium Anaeromyxobacter sp. Fw109-5. Its C-terminal functional domain exhibits autophosphorylation activity induced by oxygen binding to the heme-Fe(II) complex located in the oxygen-sensing N-terminal globin domain. A detailed understanding of the signal transduction mechanisms in heme-containing sensor proteins remains elusive. Here, we investigated the role of the globin domain's dimerization interface in signal transduction in AfGcHK. We present a crystal structure of a monomeric imidazole-bound AfGcHK globin domain at 1.8 Å resolution, revealing that the helices of the WT globin dimer are under tension and suggesting that Tyr-15 plays a role in both this tension and the globin domain's dimerization. Biophysical experiments revealed that whereas the isolated WT globin domain is dimeric in solution, the Y15A and Y15G variants in which Tyr-15 is replaced with Ala or Gly, respectively, are monomeric. Additionally, we found that although the dimerization of the full-length protein is preserved via the kinase domain dimerization interface in all variants, full-length AfGcHK variants bearing the Y15A or Y15G substitutions lack enzymatic activity. The combined structural and biophysical results presented here indicate that Tyr-15 plays a key role in the dimerization of the globin domain of AfGcHK and that globin domain dimerization is essential for internal signal transduction and autophosphorylation in this protein. These findings provide critical insights into the signal transduction mechanism of the histidine kinase AfGcHK from Anaeromyxobacter.


Assuntos
Proteínas de Bactérias/química , Globinas/química , Histidina Quinase/química , Myxococcales/química , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Globinas/metabolismo , Histidina Quinase/metabolismo , Modelos Moleculares , Myxococcales/metabolismo , Fosforilação , Conformação Proteica , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Multimerização Proteica , Transdução de Sinais
4.
Appl Environ Microbiol ; 86(2)2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31676482

RESUMO

Corallococcus spp. are common soil-dwelling organisms which kill and consume prey microbes through the secretion of antimicrobial substances. Two species of Corallococcus have been described previously (Corallococcus coralloides and Corallococcus exiguus). A polyphasic approach, including biochemical analysis of fatty acid methyl esters, substrate utilization, and sugar assimilation assays, was taken to characterize eight Corallococcus species strains and the two type strains. The genomes of all strains, including that of C. exiguus DSM 14696T (newly reported here), shared an average nucleotide identity below 95% and digital DNA-DNA hybridization scores of less than 70%, indicating that they belong to distinct species. In addition, we characterized the prey range and antibiotic resistance profile of each strain, illustrating the diversity of antimicrobial activity and, thus, the potential for drug discovery within the Corallococcus genus. Each strain gave a distinct profile of properties, which together with their genomic differences supports the proposal of the eight candidate strains as novel species. The eight candidates are as follows: Corallococcus exercitus sp. nov. (AB043AT = DSM 108849T = NBRC 113887T), Corallococcus interemptor sp. nov. (AB047AT = DSM 108843T = NBRC 113888T), Corallococcus aberystwythensis sp. nov. (AB050AT = DSM 108846T = NBRC 114019T), Corallococcus praedator sp. nov. (CA031BT = DSM 108841T = NBRC 113889T), Corallococcus sicarius sp. nov. (CA040BT = DSM 108850T = NBRC 113890T), Corallococcus carmarthensis sp. nov. (CA043DT = DSM 108842T = NBRC 113891T), Corallococcus llansteffanensis sp. nov. (CA051BT = DSM 108844T = NBRC 114100T), and Corallococcus terminator sp. nov. (CA054AT = DSM 108848T = NBRC 113892T).IMPORTANCE Corallococcus is a genus of predators with broad prey ranges, whose genomes contain large numbers of gene clusters for secondary metabolite biosynthesis. The physiology and evolutionary heritage of eight Corallococcus species strains were characterized using a range of analyses and assays. Multiple metrics confirmed that each strain belonged to a novel species within the Corallococcus genus. The strains exhibited distinct patterns of drug resistance and predatory activity, which mirrored their possession of diverse sets of biosynthetic genes. The breadth of antimicrobial activities observed within the Corallococcus genus highlights their potential for drug discovery and suggests a previous underestimation of both their taxonomic diversity and biotechnological potential. Taxonomic assignment of environmental isolates to novel species allows us to begin to characterize the diversity and evolution of members of this bacterial genus with potential biotechnological importance, guiding future bioprospecting efforts for novel biologically active metabolites and antimicrobials.


Assuntos
Cadeia Alimentar , Genoma Bacteriano , Myxococcales/classificação , Myxococcales/genética , Myxococcales/metabolismo , Filogenia
5.
Mar Drugs ; 17(12)2019 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-31842310

RESUMO

To date, studies describing myxobacterial secondary metabolites have been relatively scarce in comparison to those addressing actinobacterial secondary metabolites. This realization suggests the immense potential of myxobacteria as an intriguing source of secondary metabolites with unusual structural features and a wide array of biological activities. Marine-derived myxobacteria are especially attractive due to their unique biosynthetic gene clusters, although they are more difficult to handle than terrestrial myxobacteria. Here, we report the discovery of two new pyrazinone-type molecules, enhypyrazinones A and B, from a marine-derived myxobacterium Enhygromyxa sp. Their structures were elucidated by HRESIMS and comprehensive NMR data analyses. Compounds 1 and 2, which contain a rare trisubstituted-pyrazinone core, represent a unique class of molecules from Enhygromyxa sp.


Assuntos
Produtos Biológicos/isolamento & purificação , Indóis/isolamento & purificação , Myxococcales/metabolismo , Pirazinas/isolamento & purificação , Produtos Biológicos/química , Indóis/química , Imagem por Ressonância Magnética , Pirazinas/química , Metabolismo Secundário
6.
Proc Natl Acad Sci U S A ; 116(49): 24808-24818, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31744876

RESUMO

Myxobacteria are an example of how single-cell individuals can transition into multicellular life by an aggregation strategy. For these and all organisms that consist of social groups of cells, discrimination against, and exclusion of, nonself is critical. In myxobacteria, TraA is a polymorphic cell surface receptor that identifies kin by homotypic binding, and in so doing exchanges outer membrane (OM) proteins and lipids between cells with compatible receptors. However, TraA variability alone is not sufficient to discriminate against all cells, as traA allele diversity is not necessarily high among local strains. To increase discrimination ability, myxobacteria include polymorphic OM lipoprotein toxins called SitA in their delivered cargo, which poison recipient cells that lack the cognate, allele-specific SitI immunity protein. We previously characterized 3 SitAI toxin/immunity pairs that belong to 2 families. Here, we discover 4 additional SitA families. Each family is unique in sequence, but share the characteristic features of SitA: OM-associated toxins delivered by TraA. We demonstrate that, within a SitA family, C-terminal nuclease domains are polymorphic and often modular. Remarkably, sitA loci are strikingly numerous and diverse, with most genomes possessing >30 and up to 83 distinct sitAI loci. Interestingly, all SitA protein families are serially transferred between cells, allowing a SitA inhibitor cell to poison multiple targets, including cells that never made direct contact. The expansive suites of sitAI loci thus serve as identify barcodes to exquisitely discriminate against nonself to ensure populations are genetically homogenous to conduct cooperative behaviors.


Assuntos
Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Myxococcales/genética , Myxococcales/metabolismo , Receptores de Superfície Celular/metabolismo , Alelos , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Toxinas Bacterianas/classificação , Toxinas Bacterianas/imunologia , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Lipoproteínas , Myxococcus xanthus/genética , Myxococcus xanthus/metabolismo , Filogenia , Análise de Sequência
7.
Protein Expr Purif ; 164: 105481, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31470096

RESUMO

The lamC gene encoding a novel ß-(1,3)-glucanase was cloned from Corallococcus sp. EGB and successfully expressed in the industrial yeast Pichia pastoris. The mature protein without the initial 26 residues of signal peptide, designated LamC27, was found to be composed of fascin-like module and laminarinase-like catalytic module. The purified recombinant enzyme (rLamC27) with a calculated molecular mass of 45.3 kDa displays activities toward a broad range of ß-linked polysaccharides, including laminarin, curdlan, pachyman, lichenan, and CMC. Enzymological characterization showed that rLamC27 performes its optimal activity under the condition of 45 °C and pH 7.0, respectively, and preferentially catalyzes the hydrolysis of glucans with a ß-1,3-linkage, which is similar to the LamC previously expressed in E. coli. TherLamC27 enzyme was activated by Mn2+ and Ba2+, while it was inhibited by Cu2+, Zn2+, and Co2+. Moreover, rLamC27 was strongly inhibited by 10 mM EDTA with 7.5% of its original activity remiaining, and weakly by SDS and Triton X-100. In antifungal assay, rLamC27 was conformed to possess lytic and antifungal activity against rice blast fungus. Specifically, a significant decrease germ tube and appressorium formation ratios from 94% to 59% and 97%-51%, respectively, were observed following exposure to rLamC27. H2DCFDA and CFW staining further demonstrated that the fungistasis capability of rLamC27 could be contributed by its ability to hydrolyze components of the cell wall. All these favorable properties indicate a promising potential for using rLamC27 as a biological antifungal agent in areas such as plant protection and food preservation.


Assuntos
Endo-1,3(4)-beta-Glucanase/metabolismo , Myxococcales/enzimologia , Clonagem Molecular , Endo-1,3(4)-beta-Glucanase/genética , Endo-1,3(4)-beta-Glucanase/farmacologia , Fungicidas Industriais/metabolismo , Fungicidas Industriais/farmacologia , Expressão Gênica , Metais/metabolismo , Myxococcales/genética , Myxococcales/metabolismo , Oryza/microbiologia , Pichia/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Especificidade por Substrato
8.
Org Lett ; 21(14): 5407-5412, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31184172

RESUMO

The structures of five linear lipopeptides, thaxteramides A1, A2, B1, B2, and C isolated from the myxobacterium Jahnella thaxteri, were elucidated. They have a C-terminal common tetrapeptidic Tyr-Gly-ß-Ala-Tyr core but differ in the stereochemistry of the tyrosine units, methylations, the remaining amino acids, and the N-terminal polyketide. In silico analysis of the genome sequence complemented with feeding experiments revealed two distinct hybrid PKS/NRPS gene clusters. Three semisynthesized cyclic analogues were found to inhibit the growth of Gram-positive bacteria.


Assuntos
Lipopeptídeos/biossíntese , Myxococcales/metabolismo , Sequência de Aminoácidos , Simulação por Computador , Lipopeptídeos/química
9.
Chem Biol Drug Des ; 93(4): 539-543, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30480356

RESUMO

Epothilone, the macrolide compound produced by Sorangium cellulosum, has antitumor activity. Its anti-tumor mechanism is similar to that of paclitaxel, which promotes the polymerization of tubulin and induces apoptosis. Herein, 7-O-ß-d-galactosyl-polyethylene glycol-epothilone B 6 was synthesized. It showed that the toxicity of the synthesized compound was 1/182 of the epothilone B. In addition, compound 6 also had significant anticancer activity under the action of enzyme.


Assuntos
Antineoplásicos Fitogênicos/síntese química , Epotilonas/química , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Epotilonas/síntese química , Epotilonas/farmacologia , Galactose/química , Humanos , Células MCF-7 , Myxococcales/química , Myxococcales/metabolismo , Polietilenoglicóis/química
10.
Microb Biotechnol ; 12(2): 377-391, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30592153

RESUMO

One of the major challenges in chemical synthesis is the selective oxyfunctionalization of non-activated C-H bonds, which can be enabled by biocatalysis using cytochrome P450 monooxygenases. In this study, we report on the characterization of the versatile CYP109Q5 from Chondromyces apiculatus DSM436, which is able to functionalize a wide range of substrates (terpenes, steroids and drugs), including the ring of ß-ionone in non-allylic positions. The crystal structure of CYP109Q5 revealed flexibility within the active site pocket that permitted the accommodation of bulky substrates, and enabled a structure-guided approach to engineering the enzyme. Some variants of CYP109Q5 displayed a switch in selectivity towards the non-allylic positions of ß-ionone, allowing the simultaneous production of 2- and 3-hydroxy-ß-ionone, which are chemically challenging to synthesize and are important precursors for carotenoid synthesis. An efficient whole-cell system finally enabled the production of up to 0.5 g l-1 hydroxylated products of ß-ionone; this system can be applied to product identification in further biotransformations. Overall, CYP109Q5 proved to be highly evolvable and active. The studies in this work demonstrate that, using rational mutagenesis, the highly versatile CYP109Q5 generalist can be progressively evolved to be an industrially valuable specialist for the synthesis of specific products.


Assuntos
Engenharia Genética , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Myxococcales/enzimologia , Myxococcales/metabolismo , Terpenos/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Oxigenases de Função Mista/química , Modelos Moleculares , Conformação Proteica
11.
Neuroscience ; 399: 1-11, 2019 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-30496822

RESUMO

Astrocytes, the main non-neuronal cells in the brain, have significant roles in the maintenance and survival of neurons. Oxidative stress has been implicated in various neurodegenerative disorders such as Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and Parkinson's disease (PD). Myxobacteria produce a wide range of bioactive metabolites with notable structures and modes of action, which introduce them as potent natural product producers. In the present study, we evaluated the effects of myxobacterial extracts on hydrogen peroxide (H2O2)-mediated toxicity on primary human astrocytes. We showed that myxobacterial extracts could decrease the formation of reactive oxygen species (ROS), nitric oxide (NO) production, and cell death assessed by the release of lactate dehydrogenase (LDH). Myxobacterial extracts were also able to reduce the nitric oxide synthase (NOS) activity. The extracts reduced the oxidative effect of H2O2 on over-activation of poly (ADP-ribose) polymerase (PARP1), therefore preventing the cell death by restoring the NAD+ levels. In addition, myxobacterial extracts ameliorated the oxidative stress by increasing the glutathione level in cells. The overall results showed myxobacterial extracts, especially from the strains Archangium sp. UTMC 4070 and Cystobacter sp. UTMC 4073, were able to protect human primary astrocytes from oxidative stress.


Assuntos
Astrócitos/efeitos dos fármacos , Produtos Biológicos/farmacologia , Myxococcales/metabolismo , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Astrócitos/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Glutationa/metabolismo , Humanos , Peróxido de Hidrogênio/toxicidade , L-Lactato Desidrogenase/metabolismo , NAD/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Estresse Oxidativo/fisiologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Espécies Reativas de Oxigênio/metabolismo
12.
J Ind Microbiol Biotechnol ; 46(3-4): 319-334, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30506464

RESUMO

Type III polyketide synthases (PKSs) are comparatively small homodimeric enzymes affording natural products with diverse structures and functions. While type III PKS biosynthetic pathways have been studied thoroughly in plants, their counterparts from bacteria and fungi are to date scarcely characterized. This gap is exemplified by myxobacteria from which no type III PKS-derived small molecule has previously been isolated. In this study, we conducted a genomic survey of myxobacterial type III PKSs and report the identification of uncommon alkylpyrones as the products of type III PKS biosynthesis from the myxobacterial model strain Myxococcus xanthus DK1622 through a self-resistance-guided screening approach focusing on genes encoding pentapetide repeat proteins, proficient to confer resistance to topoisomerase inhibitors. Using promoter-induced gene expression in the native host as well as heterologous expression of biosynthetic type III PKS genes, sufficient amounts of material could be obtained for structural elucidation and bioactivity testing, revealing potent topoisomerase activity in vitro.


Assuntos
Produtos Biológicos/química , Genoma Bacteriano , Myxococcales/genética , Policetídeo Sintases/genética , Aciltransferases , Bactérias/genética , Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Vias Biossintéticas/genética , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica , Variação Genética , Família Multigênica , Myxococcales/metabolismo , Policetídeo Sintases/metabolismo , Conformação Proteica , Análise de Sequência de DNA
13.
Sci Rep ; 8(1): 16600, 2018 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-30413766

RESUMO

Comparative genomic/metabolomic analysis is a powerful tool to disclose the potential of microbes for the biosynthesis of novel specialized metabolites. In the group of marine myxobacteria only a limited number of isolated species and sequenced genomes is so far available. However, the few compounds isolated thereof so far show interesting bioactivities and even novel chemical scaffolds; thereby indicating a huge potential for natural product discovery. In this study, all marine myxobacteria with accessible genome data (n = 5), including Haliangium ochraceum DSM 14365, Plesiocystis pacifica DSM 14875, Enhygromyxa salina DSM 15201 and the two newly sequenced species Enhygromyxa salina SWB005 and SWB007, were analyzed. All of these accessible genomes are large (~10 Mb), with a relatively small core genome and many unique coding sequences in each strain. Genome analysis revealed a high variety of biosynthetic gene clusters (BGCs) between the strains and several resistance models and essential core genes indicated the potential to biosynthesize antimicrobial molecules. Polyketides (PKs) and terpenes represented the majority of predicted specialized metabolite BGCs and contributed to the highest share between the strains. BGCs coding for non-ribosomal peptides (NRPs), PK/NRP hybrids and ribosomally synthesized and post-translationally modified peptides (RiPPs) were mostly strain specific. These results were in line with the metabolomic analysis, which revealed a high diversity of the chemical features between the strains. Only 6-11% of the metabolome was shared between all the investigated strains, which correlates to the small core genome of these bacteria (13-16% of each genome). In addition, the compound enhygrolide A, known from E. salina SWB005, was detected for the first time and structurally elucidated from Enhygromyxa salina SWB006. The here acquired data corroborate that these microorganisms represent a most promising source for the detection of novel specialized metabolites.


Assuntos
Proteínas de Bactérias/genética , Genoma Bacteriano , Metaboloma , Myxococcales/metabolismo , Água do Mar/microbiologia , Biologia Computacional , Família Multigênica , Myxococcales/classificação , Myxococcales/genética , Filogenia
14.
BMC Genomics ; 19(1): 757, 2018 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-30340510

RESUMO

BACKGROUND: An efficient signal transduction system allows a bacterium to sense environmental cues and then to respond positively or negatively to those signals; this process is referred to as taxis. In addition to external cues, the internal metabolic state of any bacterium plays a major role in determining its ability to reside and thrive in its current environment. Similar to external signaling molecules, cytoplasmic signals are also sensed by methyl-accepting chemotaxis proteins (MCPs) via diverse ligand binding domains. Myxobacteria are complex soil-dwelling social microbes that can perform a variety of physiologic and metabolic activities ranging from gliding motility, sporulation, biofilm formation, carotenoid and secondary metabolite biosynthesis, predation, and slime secretion. To live such complex lifestyles, they have evolved efficient signal transduction systems with numerous one- and two-component regulatory system along with a large array of chemosensory systems to perceive and integrate both external and internal cues. RESULTS: Here we report the in silico characterization of a putative energy taxis cluster, Cc-5, which is present in only one amongst 34 known and sequenced myxobacterial genomes, Corallococcus coralloides. In addition, we propose that this energy taxis cluster is involved in oxygen sensing, suggesting that C. coralloides can sense (either directly or indirectly) and then respond to changing concentrations of molecular oxygen. CONCLUSIONS: This hypothesis is based on the presence of a unique MCP encoded in this gene cluster that contains two different oxygen-binding sensor domains, PAS and globin. In addition, the two monooxygenases encoded in this cluster may contribute to aerobic respiration via ubiquinone biosynthesis, which is part of the cytochrome bc1 complex. Finally, we suggest that this cluster was acquired from Actinobacteria, Gammaproteobacteria or Cyanobacteria. Overall, this in silico study has identified a potentially innovative and evolved mechanism of energy taxis in only one of the myxobacteria, C. coralloides.


Assuntos
Quimiotaxia/genética , Simulação por Computador , Myxococcales/genética , Myxococcales/metabolismo , Citoplasma/genética , Evolução Molecular , Genoma Bacteriano/genética , Genômica , Myxococcales/citologia
15.
Mar Drugs ; 16(9)2018 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-30189599

RESUMO

Prior to 2005, the vast majority of characterized myxobacteria were obtained from terrestrial habitats. Since then, several species of halotolerant and even obligate marine myxobacteria have been described. Chemical analyses of extracts from these organisms have confirmed their ability to produce secondary metabolites with unique chemical scaffolds. Indeed, new genera of marine-derived myxobacteria, particularly Enhygromyxa, have been shown to produce novel chemical scaffolds that differ from those observed in soil myxobacteria. Further studies have shown that marine sponges and terrestrial myxobacteria are capable of producing similar or even identical secondary metabolites, suggesting that myxobacterial symbionts may have been the true producers. Recent in silico analysis of the genome sequences available from six marine myxobacteria disclosed a remarkably versatile biosynthetic potential. With access to ever-advancing tools for small molecule and genetic evaluation, these studies suggest a bright future for expeditions into this yet untapped resource for secondary metabolites.


Assuntos
Organismos Aquáticos/metabolismo , Biodiversidade , Produtos Biológicos/farmacologia , Myxococcales/metabolismo , Poríferos/microbiologia , Animais , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/metabolismo , Vias Biossintéticas/genética , Simulação por Computador , Genoma Bacteriano/genética , Myxococcales/genética , Filogenia , Microbiologia do Solo , Simbiose
16.
ACS Chem Biol ; 13(9): 2699-2707, 2018 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-30179448

RESUMO

Polyketide synthases (PKS) are a rich source of natural products of varied chemical composition and biological significance. Here, we report the characterization of an atypical dehydratase (DH) domain from the PKS pathway for gephyronic acid, an inhibitor of eukaryotic protein synthesis. Using a library of synthetic substrate mimics, the reaction course, stereospecificity, and tolerance to non-native substrates of GphF DH1 are probed via LC-MS analysis. Taken together, the studies establish GphF DH1 as a dual-function dehydratase/isomerase that installs an odd-to-even double bond and yields a product consistent with the isobutenyl terminus of gephyronic acid. The studies also reveal an unexpected C2 epimerase function in catalytic turnover with the native substrate. A 1.55-Å crystal structure of GphF DH1 guided mutagenesis experiments to elucidate the roles of key amino acids in the multistep DH1 catalysis, identifying critical functions for leucine and tyrosine side chains. The mutagenesis results were applied to add a secondary isomerase functionality to a nonisomerizing DH in the first successful gain-of-function engineering of a PKS DH. Our studies of GphF DH1 catalysis highlight the versatility of the DH active site and adaptation for a specific catalytic outcome with a specific substrate.


Assuntos
Alcenos/metabolismo , Hidroliases/metabolismo , Myxococcales/enzimologia , Policetídeo Sintases/metabolismo , Alcenos/química , Vias Biossintéticas , Domínio Catalítico , Ácidos Graxos Monoinsaturados/química , Ácidos Graxos Monoinsaturados/metabolismo , Hidroliases/química , Isomerases/química , Isomerases/metabolismo , Modelos Moleculares , Myxococcales/química , Myxococcales/metabolismo , Policetídeo Sintases/química , Domínios Proteicos , Especificidade por Substrato
17.
Angew Chem Int Ed Engl ; 57(43): 14270-14275, 2018 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-30088846

RESUMO

Soil-living microbes are an important resource for the discovery of new natural products featuring great structural diversity that are reflective of the underlying biosynthetic pathways as well as incorporating a wide range of intriguing small-molecule building blocks. We report here the full structural elucidation, total synthesis, and biosynthesis of chloromyxamides, a new class of tetrapeptides that display an unprecedented 6-chloromethyl-5-methoxypipecolic acid (CMPA) substructure. Chemical synthesis-including an approach to access the CMPA unit-was pursued to confirm the structure of the chloromyxamides and enabled determination of the absolute configuration in the CMPA ring. A model for the nonribosomal assembly of chloromyxamides was devised on the basis of the combined evaluation of the biosynthetic gene cluster sequence and the feeding of stable isotope-labeled precursors. This provided insight into the formation of the various chloromyxamide derivatives and the biogenesis of the CMPA unit.


Assuntos
Amidas/química , Amidas/síntese química , Myxococcales/metabolismo , Oligopeptídeos/biossíntese , Ácidos Pipecólicos/química , Amidas/metabolismo , Filogenia
18.
PLoS One ; 13(7): e0201605, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30063768

RESUMO

The identification of inhibitors of eukaryotic protein biosynthesis, which are targeting single translation factors, is highly demanded. Here we report on a small molecule inhibitor, gephyronic acid, isolated from the myxobacterium Archangium gephyra that inhibits growth of transformed mammalian cell lines in the nM range. In direct comparison, primary human fibroblasts were shown to be less sensitive to toxic effects of gephyronic acid than cancer-derived cells. Gephyronic acid is targeting the protein translation system. Experiments with IRES dual luciferase reporter assays identified it as an inhibitor of the translation initiation. DARTs approaches, co-localization studies and pull-down assays indicate that the binding partner could be the eukaryotic initiation factor 2 subunit alpha (eIF2α). Gephyronic acid seems to have a different mode of action than the structurally related polyketides tedanolide, myriaporone, and pederin and is a valuable tool for investigating the eukaryotic translation system. Because cancer derived cells were found to be especially sensitive, gephyronic acid could potentially find use as a drug candidate.


Assuntos
Fator de Iniciação 2 em Eucariotos/antagonistas & inibidores , Myxococcales/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Ácidos Graxos Monoinsaturados/farmacologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Técnicas Microbiológicas , Myxococcales/genética , Myxococcales/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
19.
Mar Drugs ; 16(9)2018 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-30158489

RESUMO

Over the last two decades, halophilic (organisms that thrive at high salt concentrations) and halotolerant (organisms that have adapted to high salt concentrations) myxobacteria emerged as an important source of structurally diverse secondary metabolites from the marine environment. This review explores the advance of metagenomics analysis and 16S rRNA gene phylogeny of the cultured and uncultured myxobacteria from marine and other salt-environments up to July 2018. The diversity of novel groups of myxobacteria in these environments appears unprecedented, especially in the Sorangiineae and Nannocystineae suborders. The Sandaracinaceae related clade in the Sorangiineae suborder seems more widely distributed compared to the exclusively marine myxobacterial cluster. Some of the previously identified clones from metagenomic studies were found to be related to the Nannocystineae suborder. This understanding provides the foundation for a vital, unexplored resource. Understanding the conditions required to cultivate these yet "uncultured" myxobacteria in the laboratory, while a key next step, offers a significant potential to further expand access to diverse secondary metabolites.


Assuntos
Organismos Aquáticos/genética , Produtos Biológicos/farmacologia , Metagenômica , Myxococcales/genética , RNA Ribossômico 16S/genética , Organismos Aquáticos/metabolismo , Biodiversidade , Produtos Biológicos/metabolismo , DNA Bacteriano/genética , Myxococcales/metabolismo , Filogenia , Tolerância ao Sal/genética , Análise de Sequência de DNA
20.
Appl Environ Microbiol ; 84(14)2018 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-29752267

RESUMO

The gene encoding the novel amylolytic enzyme designated CoMA was cloned from Corallococcus sp. strain EGB. The deduced amino acid sequence contained a predicted lipoprotein signal peptide (residues 1 to 18) and a conserved glycoside hydrolase family 13 (GH13) module. The amino acid sequence of CoMA exhibits low sequence identity (10 to 19%) with cyclodextrin-hydrolyzing enzymes (GH13_20) and is assigned to GH13_36. The most outstanding feature of CoMA is its ability to catalyze the conversion of maltooligosaccharides (≥G3) and soluble starch to maltose as the sole hydrolysate. Moreover, it can hydrolyze γ-cyclodextrin and starch to maltose and hydrolyze pullulan exclusively to panose with relative activities of 0.2, 1, and 0.14, respectively. CoMA showed both hydrolysis and transglycosylation activities toward α-1,4-glycosidic bonds but not to α-1,6-linkages. Moreover, glucosyl transfer was postulated to be the major transglycosidation reaction for producing a high level of maltose without the attendant production of glucose. These results indicated that CoMA possesses some unusual properties that distinguish it from maltogenic amylases and typical α-amylases. Its physicochemical properties suggested that it has potential for commercial development.IMPORTANCE The α-amylase from Corallococcus sp. EGB, which was classified to the GH13_36 subfamily, can catalyze the conversion of maltooligosaccharides (≥G3) and soluble starch to maltose as the sole hydrolysate. An action mechanism for producing a high level of maltose without the attendant production of glucose has been proposed. Moreover, it also can hydrolyze γ-cyclodextrin and pullulan. Its biochemical characterization suggested that CoMA may be involved the accumulation of maltose in Corallococcus media.


Assuntos
DNA Bacteriano/isolamento & purificação , Glicosídeo Hidrolases/genética , Myxococcales/genética , Oligossacarídeos/metabolismo , Clonagem Molecular , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica , Glucanos/metabolismo , Glicosídeo Hidrolases/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Maltose/metabolismo , Myxococcales/metabolismo , Análise de Sequência de DNA , Amido/metabolismo , Temperatura , alfa-Amilases/genética , alfa-Amilases/metabolismo , gama-Ciclodextrinas/metabolismo
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