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1.
Nat Commun ; 11(1): 2137, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358514

RESUMO

The light-driven sodium-pumping rhodopsin KR2 from Krokinobacter eikastus is the only non-proton cation active transporter with demonstrated potential for optogenetics. However, the existing structural data on KR2 correspond exclusively to its ground state, and show no sodium inside the protein, which hampers the understanding of sodium-pumping mechanism. Here we present crystal structure of the O-intermediate of the physiologically relevant pentameric form of KR2 at the resolution of 2.1 Å, revealing a sodium ion near the retinal Schiff base, coordinated by N112 and D116 of the characteristic NDQ triad. We also obtained crystal structures of D116N and H30A variants, conducted metadynamics simulations and measured pumping activities of putative pathway mutants to demonstrate that sodium release likely proceeds alongside Q78 towards the structural sodium ion bound between KR2 protomers. Our findings highlight the importance of pentameric assembly for sodium pump function, and may be used for rational engineering of enhanced optogenetic tools.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Flavobacteriaceae/metabolismo , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/metabolismo , Cristalografia por Raios X , Escherichia coli/metabolismo , Simulação de Dinâmica Molecular , Dobramento de Proteína , Rodopsina/química , Rodopsina/metabolismo , Sódio/metabolismo , Difração de Raios X
2.
Biochim Biophys Acta Bioenerg ; 1861(7): 148190, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32194062

RESUMO

Krokinobacter rhodopsin 2 (KR2) was discovered as the first light-driven sodium pumping rhodopsin (NaR) in 2013, which contains unique amino acid residues on C-helix (N112, D116, and Q123), referred to as an NDQ motif. Based on the recent X-ray crystal structures of KR2, the sodium transport pathway has been investigated by various methods. However, due to complicated structural information around the protonated Schiff base (PRSB) region in the dark state and lack of structural information in the intermediates with sodium bound in KR2, detailed sodium pump mechanism is still unclear. Here we applied comprehensive low-temperature light-induced difference FTIR spectroscopy on isotopically labeled KR2 WT and site-directed mutant proteins (N112A, D116E, R109A, and R109K). We assigned the N-D stretching vibration of the PRSB at 2095 cm-1 and elucidate the hydrogen bonding interaction with D116 (a counter ion for the PRSB). We also assigned strongly hydrogen-bonded water (2333 cm-1) near R109 and D251, and found that presence of a positive charge at the position of R109 is prerequisite for the pumping function of KR2.


Assuntos
Luz , Retinaldeído/química , Rodopsina/química , Bases de Schiff/química , ATPase Trocadora de Sódio-Potássio/metabolismo , Cristalografia por Raios X , Flavobacteriaceae/metabolismo , Ligação de Hidrogênio , Isomerismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Isótopos de Nitrogênio , Espectroscopia de Infravermelho com Transformada de Fourier , Vibração , Água/química
3.
Molecules ; 25(1)2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31906348

RESUMO

The synthesis of bioplastic from marine microbes has a great attendance in the realm of biotechnological applications for sustainable eco-management. This study aims to isolate novel strains of poly-ß-hydroxybutyrate (PHB)-producing bacteria from the mangrove rhizosphere, Red Sea, Saudi Arabia, and to characterize the extracted polymer. The efficient marine bacterial isolates were identified by the phylogenetic analysis of the 16S rRNA genes as Tamlana crocina, Bacillus aquimaris, Erythrobacter aquimaris, and Halomonas halophila. The optimization of PHB accumulation by E. aquimaris was achieved at 120 h, pH 8.0, 35 °C, and 2% NaCl, using glucose and peptone as the best carbon and nitrogen sources at a C:N ratio of 9.2:1. The characterization of the extracted biopolymer by Fourier-transform infrared spectroscopy (FTIR), Nuclear magnetic resonance (NMR), and Gas chromatography-mass spectrometry (GC-MS) proves the presence of hydroxyl, methyl, methylene, methine, and ester carbonyl groups, as well as derivative products of butanoic acid, that confirmed the structure of the polymer as PHB. This is the first report on E. aquimaris as a PHB producer, which promoted the hypothesis that marine rhizospheric bacteria were a new area of research for the production of biopolymers of commercial value.


Assuntos
Biopolímeros/biossíntese , Biopolímeros/química , Hidroxibutiratos/química , Hidroxibutiratos/metabolismo , Poliésteres/química , Poliésteres/metabolismo , Sphingomonadaceae/química , Sphingomonadaceae/metabolismo , Avicennia/microbiologia , Bacillus/química , Bacillus/genética , Bacillus/metabolismo , Biopolímeros/análise , Carbono/química , Carbono/metabolismo , Fermentação , Flavobacteriaceae/química , Flavobacteriaceae/genética , Flavobacteriaceae/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Halomonas/química , Halomonas/genética , Halomonas/metabolismo , Hidroxibutiratos/análise , Espectroscopia de Ressonância Magnética , Nitrogênio/química , Nitrogênio/metabolismo , Filogenia , Poliésteres/análise , RNA Ribossômico 16S/genética , Rizosfera , Salinidade , Arábia Saudita , Água do Mar/microbiologia , Espectroscopia de Infravermelho com Transformada de Fourier , Sphingomonadaceae/genética , Sphingomonadaceae/isolamento & purificação , Temperatura
4.
Mar Drugs ; 17(10)2019 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-31597240

RESUMO

Ulvan lyases can degrade ulvan to oligosaccharides with potent biological activity. A new ulvan lyase gene, ALT3695, was identified in Alteromonas sp. A321. Soluble expression of ALT3695 was achieved in Escherichia coli BL21 (DE3). The 1314-bp gene encoded a protein with 437 amino acid residues. The amino acid sequence of ALT3695 exhibited low sequence identity with polysaccharide lyase family 25 (PL25) ulvan lyases from Pseudoalteromonas sp. PLSV (64.14% identity), Alteromonas sp. LOR (62.68% identity), and Nonlabens ulvanivorans PLR (57.37% identity). Recombinant ALT3695 was purified and the apparent molecular weight was about 53 kDa, which is different from that of other polysaccharide-degrading enzymes identified in Alteromonas sp. A321. ALT3695 exhibited maximal activity in 50 mM Tris-HCl buffer at pH 8.0 and 50 °C. ALT3695 was relatively thermostable, as 90% activity was observed after incubation at 40 °C for 3 h. The Km and Vmax values of ALT3695 towards ulvan were 0.43 mg·mL-1 and 0.11 µmol·min-1·mL-1, respectively. ESI-MS analysis showed that enzymatic products were mainly disaccharides and tetrasaccharides. This study reports a new PL25 family ulvan lyase, ALT3695, with properties that suggest its great potential for the preparation of ulvan oligosaccharides.


Assuntos
Alteromonas/metabolismo , Polissacarídeo-Liase/metabolismo , Polissacarídeos/metabolismo , Sequência de Aminoácidos , Clonagem Molecular/métodos , Flavobacteriaceae/metabolismo , Concentração de Íons de Hidrogênio , Oligossacarídeos/metabolismo , Pseudoalteromonas/metabolismo
5.
mBio ; 10(4)2019 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-31337718

RESUMO

The factors and processes that influence the behavior and functionality of ecosystems inhabited by complex microbiomes are still far from being clearly understood. Synthetic microbial communities provide reduced-complexity models that allow an examination of ecological theories under defined and controlled conditions. In this study, we applied a multiphasic approach to study synthetic methane-oxidizing communities and species interactions as proxies to the natural communities. Our results confirm that, under selective pressures, natural-sediment communities of high complexity simplify rapidly, selecting for several major functional guilds, the major partners in methane oxidation being the Methylococcaceae methanotrophs and the Methylophilaceae methylotrophs, along with minor but persistent partners, members of Burkholderiales and Flavobacteriales As a proof of concept, we established minimalist synthetic communities that were representative of the four functional guilds to demonstrate the dependency of the non-methane-utilizing species on the methanotrophs as the primary carbon-providing species. We observed that in communities consisting of multiple representatives of the key guilds, members of the same guild appeared to compete for resources. For example, when two methanotrophs of the same family were present, the two expressed similar key methanotrophy pathways and responded similarly to changing environmental conditions, suggesting that they perform a similar keystone function in situ Similar observations were made for the Methylophilaceae However, differences were noted in the expression of auxiliary and unique genes among strains of the same functional guild, reflecting differential adaptation and suggesting mechanisms for competition. At the same time, differences were also noted in the performances of partners with specific metabolic schemes. For example, a mutant of Methylotenera mobilis impaired in nitrate utilization behaved as a more efficient cooperator in methane consumption, suggesting that the loss of function may lead to changes in communal behavior. Overall, we demonstrate the robust nature of synthetic communities built of native lake sediment strains and their utility in addressing important ecological questions while using a simplified model.IMPORTANCE The metabolism of methane is an important part of the global carbon cycle. While deciphering the community function and the potential role of the different functional guilds is very difficult when considering native complex communities, synthetic communities, built of species originating from a study site in question, present a simplified model and allow specific questions to be addressed as to carbon, nitrogen, and other nutrient transfer among species in a controlled system. This study applies an ecophysiological approach, as a proof of principle, to an already well-studied model system, contributing to a better understanding of microbial community function and microbial ecosystem processes.


Assuntos
Sedimentos Geológicos/microbiologia , Lagos/microbiologia , Metano/metabolismo , Interações Microbianas , Microbiota , Burkholderiales/genética , Burkholderiales/metabolismo , Carbono/metabolismo , Ciclo do Carbono , Flavobacteriaceae/genética , Flavobacteriaceae/metabolismo , Methylococcaceae/genética , Methylococcaceae/metabolismo , Nitrogênio/metabolismo , Oxirredução
6.
Environ Microbiol ; 21(11): 4002-4019, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31314938

RESUMO

This study determines the natural product biosynthesis and full coding potential within the bacterial genus Aquimarina. Using comprehensive phylogenomics and functional genomics, we reveal that phylogeny instead of isolation source [host-associated (HA) vs. free-living (FL) habitats] primarily shape the inferred metabolism of Aquimarina species. These can be coherently organized into three major functional clusters, each presenting distinct natural product biosynthesis profiles suggesting that evolutionary trajectories strongly underpin their secondary metabolite repertoire and presumed bioactivities. Aquimarina spp. are highly versatile bacteria equipped to colonize HA and FL microniches, eventually displaying opportunistic behaviour, owing to their shared ability to produce multiple glycoside hydrolases from diverse families. We furthermore uncover previously underestimated, and highly complex secondary metabolism for the genus by detecting 928 biosynthetic gene clusters (BGCs) across all genomes, grouped in 439 BGC families, with polyketide synthases (PKSs), terpene synthases and non-ribosomal peptide synthetases (NRPSs) ranking as the most frequent BGCs encoding drug-like candidates. We demonstrate that the recently described cuniculene (trans-AT PKS) BGC is conserved among, and specific to, the here delineated A. megaterium-macrocephali-atlantica phylogenomic clade. Our findings provide a timely and in-depth perspective of an under-explored yet emerging keystone taxon in the cycling of organic matter and secondary metabolite production in marine ecosystems.


Assuntos
Bacteroidetes/metabolismo , Flavobacteriaceae/metabolismo , Sedimentos Geológicos/microbiologia , Água do Mar/microbiologia , Metabolismo Secundário/genética , Organismos Aquáticos/classificação , Organismos Aquáticos/genética , Organismos Aquáticos/metabolismo , Bacteroidetes/classificação , Bacteroidetes/genética , Carbono/metabolismo , Ecossistema , Flavobacteriaceae/classificação , Flavobacteriaceae/genética , Genômica/métodos , Glicosídeo Hidrolases/genética , Família Multigênica , Peptídeo Sintases/genética , Filogenia , Policetídeo Sintases/genética
7.
J Appl Microbiol ; 127(3): 713-723, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31211899

RESUMO

AIM: Study is focused on the influence of cadmium addition to growth media on production yield, their size and molecular mass of exopolysaccharides (EPS) synthesized by three rhizosphere bacteria strains. Inhibition of bacterial growth by increasing concentrations of Cd2+ was also analysed. METHODS AND RESULTS: The highest impact of Cd2+ was noticed on the growth of Arthrobacter sp. and Rhizobium metallidurans. Chryseobacterium sp. and Arthrobacter sp. produced significantly lower when compared to R. metallidurans amounts of EPS under the influence of Cd2+ . In all bacterial strains both size and molecular mass decreased after addition of Cd2+ to growth media. It causes a change in EPS conformation to more planar, which minimizes the volume of liquid in the interglobular space next to the bacterial wall. Results confirmed strong effect of Cd2+ on the structure and synthesis of bacterial EPS what can be a key factor in the interactions between rhizosphere bacteria and host plants in heavy metal polluted soils. CONCLUSION: This work proves that due to the presence of cadmium ions, the size and conformation of EPS produced by selected bacterial strains is changed to minimize their impact on cell. We suggest that shifting in EPS conformation from bigger globular particles to the smaller planar ones could be one of the probable mechanisms of Cd resistance in metallotolerant bacteria, and finally explain increased efficiency of heavy metal phytoextraction by EPS-producing plant growth-promoting micro-organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: One of the most promising remediation technique for Cd-contaminated areas is the phytoremediation in which rhizosphere bacteria play an important role by protecting plants' roots from toxic condition thus enhancing efficiency of intake. EPS secretion by bacteria is one of the most common mechanisms to protect the cell from impact of unpleasant environmental conditions, for example, toxicity of heavy metals like Cd.


Assuntos
Bactérias/efeitos dos fármacos , Cádmio/farmacologia , Polissacarídeos Bacterianos/biossíntese , Poluentes do Solo/farmacologia , Arthrobacter/efeitos dos fármacos , Arthrobacter/metabolismo , Biodegradação Ambiental , Flavobacteriaceae/efeitos dos fármacos , Flavobacteriaceae/metabolismo , Polissacarídeos Bacterianos/química , Rhizobium/efeitos dos fármacos , Rizosfera
8.
Science ; 364(6445)2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31196985

RESUMO

Chemical defense against predators is widespread in natural ecosystems. Occasionally, taxonomically distant organisms share the same defense chemical. Here, we describe an unusual tripartite marine symbiosis, in which an intracellular bacterial symbiont ("Candidatus Endobryopsis kahalalidefaciens") uses a diverse array of biosynthetic enzymes to convert simple substrates into a library of complex molecules (the kahalalides) for chemical defense of the host, the alga Bryopsis sp., against predation. The kahalalides are subsequently hijacked by a third partner, the herbivorous mollusk Elysia rufescens, and employed similarly for defense. "Ca E. kahalalidefaciens" has lost many essential traits for free living and acts as a factory for kahalalide production. This interaction between a bacterium, an alga, and an animal highlights the importance of chemical defense in the evolution of complex symbioses.


Assuntos
Clorófitas , Flavobacteriaceae/metabolismo , Gastrópodes , Glicosídeos/metabolismo , Comportamento Predatório , Simbiose , Triterpenos/metabolismo , Animais , Evolução Biológica , Flavobacteriaceae/química
9.
Curr Microbiol ; 76(9): 975-981, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31144108

RESUMO

A novel Gram-stain-negative bacterium, designated strain BH-SD17T, was isolated from a marine sediment sample in the Bohai Gulf, Yellow Sea, China. Cells of strain BH-SD17T are aerobic, yellow-colored, non-flagellated rods. Growth occurs between 15 and 40 °C (optimum, 30 °C), at pH 6.0-8.5 (optimum, 7.5) and with 1.0-8.0% (w/v) NaCl (optimum, 3.0%). Strain BH-SD17T contains phosphatidylethanolamine and two unidentified lipids as the major polar lipids. The predominant fatty acids are iso-C15:0 (28.5%), iso-C15:1 G (24.4%), and iso-C17:0 3-OH (12.3%). The major respiratory quinone is MK-6. Strain BH-SD17T shows moderate 16S rRNA gene sequence similarity to existing identified strains, is most closely related to the genera Lutimonas (92.1-92.4%), Lutibacter (91.6-92.3%), and Taeania (91.9%). Phylogenetic trees based on 16S rRNA gene sequences show that strain BH-SD17T forms a distinct lineage within the family Flavobacteriaceae. Based on the results of phenotypic, chemotaxonomic and phylogenetic analysis, strain BH-SD17T is considered to represent a novel genus and species in the family Flavobacteriaceae, for which the name Aureibaculum marinum is proposed. The type strain is BH-SD17T (=CCTCC AB 2017072T=KCTC 62204T).


Assuntos
Flavobacteriaceae/isolamento & purificação , Sedimentos Geológicos/microbiologia , Técnicas de Tipagem Bacteriana , DNA Bacteriano , Ácidos Graxos/metabolismo , Flavobacteriaceae/classificação , Flavobacteriaceae/genética , Flavobacteriaceae/metabolismo , Filogenia , RNA Ribossômico 16S/genética , Água do Mar/microbiologia , Cloreto de Sódio/metabolismo
10.
Biophys J ; 116(10): 1941-1951, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-31036257

RESUMO

Light-driven sodium pumps (NaRs) are microbial rhodopsins that utilize light energy to actively transport sodium ions out of the cell. Here, we used targeted mutagenesis and electrophysiological methods in living cells to demonstrate that NaRs can be converted into light-activated cation channels by molecular engineering. Specifically, introduction of the R109Q mutation into the sodium ion pump of Dokdonia eikasta (KR2) results in passive ion conductance, with a high preference for potassium over sodium ions. However, in this mutant, residual active outward pumping of sodium ions competes with passive inward transport of potassium. Channel-like behavior could also be achieved by introduction of other mutations into the KR2 counterion complex, and further, these modifications were transferrable to other NaRs. Combining the R109Q replacement with modifications at position S70 removed the residual sodium pumping and greatly enhanced the channel-like activity. However, passive photocurrents were only observed in leak mutants if the KR2 counterions, D116 and D251, were deprotonated, which was only observed under alkaline conditions. Overall, our results reveal that interactions between R109 and the nearby residues, L75, S70, D116, and D251, prevent passive backflow during ion transport in NaRs.


Assuntos
Flavobacteriaceae/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Membrana Celular , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Transporte de Íons , Luz , Camundongos , Proteínas Mutantes/metabolismo , Potássio , Ligação Proteica , Conformação Proteica , Ratos , Rodopsinas Microbianas , Sódio
11.
J Agric Food Chem ; 67(16): 4595-4602, 2019 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-30907589

RESUMO

Aflatoxin B1 (AFB1), is a type I carcinogen that is one of the strongest naturally occurring aflatoxins and can be injurious to humans and livestock upon ingestion, inhalation, or skin contact, with carcinogenic and mutagenic effects. It causes significant hazardous effects to the food- and animal-production industries. We found a bacterial strain, 3J2MO, that degraded AFB1 well, and here we tested and characterized its AFB1-degradation ability. The strain degraded about 93.82% of the AFB1 after incubation for 48 h in Luria-Bertani (LB) medium at 37 °C with a final concentration of 100 ppb and an inoculation quantity of 1 × 107 cfu/mL. High-performance liquid chromatography-fluorescence detection (HPLC-FLD) was used to determine AFB1 amounts. The maximum degradation rates were 89.23% at pH 8.5; 55.78% at an inoculation quantity of 1 × 108 cfu/mL; and 71.50 and 71.21% at 34 and 37 °C, respectively. Treatment with sucrose and soluble starch as carbon sources and beef extract and ammonium acetate as nitrogen sources stimulated the degradation rate. Mg2+ and Ca2+ ions were activators for AFB1 degradation; however, Mn2+, Fe3+, Zn2+, and Cu2+ were strong inhibitors. This bacterial strain has potential in bioremediation and the detoxification of aflatoxin contamination for biocontrol strategies in both agricultural products and food-industry matrices.


Assuntos
Aflatoxina B1/metabolismo , Flavobacteriaceae/metabolismo , Aflatoxina B1/análise , Biodegradação Ambiental , Cromatografia Líquida de Alta Pressão , Meios de Cultura/química , Meios de Cultura/metabolismo , Flavobacteriaceae/química , Concentração de Íons de Hidrogênio , Sacarose/análise , Sacarose/metabolismo
12.
Antonie Van Leeuwenhoek ; 112(7): 1077-1085, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30729398

RESUMO

A Gram-stain negative, aerobic, rod-shaped, and non-motile bacterium, designated strain CCMM003T, was isolated from a culture of the green alga Ulva prolifera. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain CCMM003T belongs to the family Flavobacteriaceae and exhibits a close relationship to Pseudozobellia thermophila DSM 19858T (92.5%). Optimal growth occurred in the presence of 4% (w/v) NaCl, at pH 7.0 and 30 °C. The polar lipids of strain CCMM003T consisted of phosphatidylethanolamine and six unidentified lipids. The predominant isoprenoid quinone was MK-6. The major fatty acids were iso-C15:0, iso-C15:1 G, iso-C17:0 3-OH and summed feature 3 (C16:1ω7c and/or iso-C15:0 2-OH). The DNA G + C content of strain CCMM003T calculated on the basis of the genome sequence was 41.2 mol% and the genome size was 5.9 Mbp. On the basis of data from this polyphasic study, strain CCMM003T is considered to represent a novel genus and species of the family Flavobacteriaceae, for which the name Ulvibacterium marinum gen. nov., sp. nov. is proposed. The type strain is CCMM003T (= MCCC 1K03244T =KCTC 52639T).


Assuntos
Flavobacteriaceae/isolamento & purificação , Água do Mar/microbiologia , Ulva/microbiologia , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Flavobacteriaceae/classificação , Flavobacteriaceae/genética , Flavobacteriaceae/metabolismo , Filogenia , RNA Ribossômico 16S/genética , Ulva/crescimento & desenvolvimento
13.
Microbiologyopen ; 8(8): e00808, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30793504

RESUMO

The production and release of extracellular vesicles (EVs) is a common process occurring in various types of bacteria. However, little is known regarding the functions of EVs derived from marine bacteria. We observed that during cell growth, Sediminicola sp. YIK13, a proteorhodopsin (PR)-containing marine flavobacterium, produces EVs (S13EVs). Transmission electron microscopy showed that Sediminicola sp. YIK13 released two spherical vesicle types, with mono- and/or bi-layered membranes, in the culture. Interestingly, the S13EVs have an orange pigment, indicating the presence of putative carotenoid and PR pigments ascribed to the parental cells. The S13EVs demonstrated the same PR-derived absorption peak spectrum and light-induced proton pump activity as the parental cells. Western blot (immunoblot) analysis of the S13EVs revealed the presence of PR. We confirmed the 16S rRNA gene, pro gene, and genes required for chromophore retinal synthesis, namely blh and crtI, in the DNA packaged into these vesicles. In addition, by metagenomic sequencing, we found microbial rhodopsin-related genes in vesicles derived from natural aquatic environments. Our results suggest that EVs as well potentially pursue horizontal gene transfer of diverse microbial rhodopsin genes in marine ecosystems.


Assuntos
Organismos Aquáticos/metabolismo , Vesículas Extracelulares/metabolismo , Flavobacteriaceae/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos da radiação , Luz , Bombas de Próton/metabolismo , Rodopsinas Microbianas/metabolismo , Organismos Aquáticos/efeitos da radiação , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Vesículas Extracelulares/ultraestrutura , Flavobacteriaceae/efeitos da radiação , Redes e Vias Metabólicas/genética , Microscopia Eletrônica de Transmissão , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
14.
Syst Appl Microbiol ; 42(3): 263-274, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30773292

RESUMO

Flavobacteriia are abundant in many marine environments including hadal waters, as demonstrated recently. However, it is unclear how this flavobacterial population adapts to hadal conditions. In this study, extensive comparative genomic analyses were performed for the flavobacterial strain Euzebyella marina RN62 isolated from the Mariana Trench hadal water in low abundance. The complete genome of RN62 possessed a considerable number of carbohydrate-active enzymes with a different composition. There was a predominance of GH family 13 proteins compared to closely related relatives, suggesting that RN62 has preserved a certain capacity for carbohydrate utilization and that the hadal ocean may hold an organic matter reservoir distinct from the surface ocean. Additionally, RN62 possessed potential intracellular cycling of the glycogen/starch pathway, which may serve as a strategy for carbon storage and consumption in response to nutrient pulse and starvation. Moreover, the discovery of higher glycoside hydrolase dissimilarities among Flavobacteriia, compared to peptidases and transporters, suggested variation in polysaccharide utilization related traits as an important ecophysiological factor in response to environmental alterations, such as decreased labile organic carbon in hadal waters. The presence of abundant toxin exporting, transcription and signal transduction related genes in RN62 may further help to survive in hadal conditions, including high pressure/low temperature.


Assuntos
Metabolismo dos Carboidratos/genética , Ecossistema , Flavobacteriaceae/genética , Flavobacteriaceae/metabolismo , Água do Mar/microbiologia , Adaptação Fisiológica/genética , Flavobacteriaceae/classificação , Genes Bacterianos/genética , Genoma Bacteriano/genética , Glicogênio/metabolismo , Filogenia , Polissacarídeos/metabolismo
15.
Phys Chem Chem Phys ; 21(8): 4461-4471, 2019 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-30734791

RESUMO

We report a comparative study on the structural dynamics of the light-driven sodium pump Krokinobacter eikastus rhodopsin 2 wild type under sodium and proton pumping conditions by means of time-resolved IR spectroscopy. The kinetics of KR2 under sodium pumping conditions exhibits a sequential character, whereas the kinetics of KR2 under proton pumping conditions involves several equilibrium states. The sodium translocation itself is characterized by major conformational changes of the protein backbone, such as distortions of the α-helices and probably of the ECL1 domain, indicated by distinct marker bands in the amide I region. Carbonyl stretch modes of specific amino acid residues helped to elucidate structural changes in the retinal Schiff base moiety, including the protonation and deprotonation of D116, which is crucial for a deeper understanding of the mechanistic features in the photocycle of KR2.


Assuntos
Flavobacteriaceae/metabolismo , Rodopsinas Microbianas/metabolismo , Canais de Sódio/metabolismo , Membrana Celular/metabolismo , Escherichia coli/genética , Flavobacteriaceae/efeitos da radiação , Transporte de Íons , Cinética , Luz , Modelos Moleculares , Estrutura Molecular , Processos Fotoquímicos , Rodopsinas Microbianas/efeitos da radiação , Canais de Sódio/efeitos da radiação , ATPase Trocadora de Sódio-Potássio/metabolismo , Espectrofotometria Infravermelho , Termodinâmica
16.
FEMS Microbiol Ecol ; 95(3)2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30689834

RESUMO

Interactions between photoautotrophs and heterotrophs are central to marine microbial ecosystems. Synechococcus are dominant marine phototrophs, and they are frequently associated with heterotrophic bacteria. These co-cultures provide a useful research system to investigate photoautotroph-heterotroph interactions in marine systems. Bacteria within the Roseobacter clade and Flavobacteria are two of the main bacterial lineages that exhibit intimate associations with Synechococcus populations. We conducted metagenomic analyses of a Synechococcus culture, followed by genomic binning of metagenomic contigs, and recovered five nearly complete genomes, including members of the Roseobacter clade (i.e. Marivita sp. XM-24) and Flavobacteria (i.e. Fluviicola sp. XM-24). Marivita sp. XM-24 is an ecological generalist of the Roseobacter clade and displays diverse metabolic capacities for the acquisition of nutrients and energy sources. Specifically, the genome contained numerous gene complements involved in the uptake and metabolism of nitrogen- and phosphorus-containing inorganic and organic compounds, in addition to the potential for aerobic anoxygenic photosynthesis, oxidation of carbon monoxide, inorganic sulfur oxidation, DMSP demethylation and PHA metabolism. The genome of the Flavobacteria representative, Fluviicola sp. XM-24, contained numerous peptidases, glycoside hydrolases, adhesion-related proteins and genes involved in gliding motility. Fluviicola sp. XM-24 likely specialize in the degradation of high molecular weight compound exudates from Synechococcus cells, including polysaccharides and polypeptides via attachment to particles, surfaces or cells. The distinct metabolic strategies identified within several heterotrophic bacteria that are associated with Syneochococcus cells provide insights into their lifestyles and nutrient utilization patterns, in addition to their interactions with photoautotrophs. Biological interactions, including mutualism, competition and antagonism, shape the microbial community structure of marine environments and are critical for understanding biogeochemical cycling in the ocean. These results provide valuable insights into the nature of interactions between dominant marine photoautotrophs and associated bacterial heterotrophs.


Assuntos
Estuários , Processos Heterotróficos/fisiologia , Água do Mar/microbiologia , Synechococcus/fisiologia , Flavobacteriaceae/classificação , Flavobacteriaceae/genética , Flavobacteriaceae/metabolismo , Flavobacteriaceae/fisiologia , Genômica , Processos Heterotróficos/genética , Redes e Vias Metabólicas/genética , Interações Microbianas , Microbiota/genética , Roseobacter/classificação , Roseobacter/genética , Roseobacter/metabolismo , Roseobacter/fisiologia , Synechococcus/genética
17.
Environ Microbiol ; 21(1): 374-388, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30411473

RESUMO

Fungi living in sediments ('mycobenthos') are hypothesized to play a role in the degradation of organic matter deposited at the land-sea interface, but the environmental factors influencing the mycobenthos are poorly understood. We used mock community calibrated Illumina sequencing to show that the mycobenthos community structure in a coastal lagoon was significantly changed after exposure to a lignocellulose extract and subsequent development of benthic anoxia over a relatively short (10 h) incubation. Saprotrophic taxa dominated and were selected for under benthic anoxia, specifically Aquamyces (Chytridiomycota) and Orbilia (Ascomycota), implicating these genera as important benthic saprotrophs. Protein encoding genes involved in energy and biomass production from Fungi and the fungal-analogue group Labyrinthulomycetes had the highest increase in expression with the added organic matter compared with all other groups, indicating that lignocellulose stimulates metabolic activity in the mycobenthos. Flavobacteria dominated the active bacterial community that grew rapidly with the lignocellulose extract and crashed sharply upon O2 depletion. Our findings indicate that the diversity, activity and trophic potential of the mycobenthos changes rapidly in response to organic matter and decreasing O2 concentrations, which together with heterotrophic Flavobacteria, undergo 'boom and bust' dynamics during lignocellulose degradation in estuarine ecosystems.


Assuntos
Ascomicetos/crescimento & desenvolvimento , Quitridiomicetos/crescimento & desenvolvimento , Substâncias Húmicas/microbiologia , Lignina/metabolismo , Micobioma/fisiologia , Estramenópilas/crescimento & desenvolvimento , Anaerobiose , Ascomicetos/isolamento & purificação , Biomassa , Quitridiomicetos/isolamento & purificação , Ecossistema , Flavobacteriaceae/crescimento & desenvolvimento , Flavobacteriaceae/metabolismo , Processos Heterotróficos , Oxigênio/metabolismo , Estramenópilas/metabolismo
18.
Nat Prod Res ; 33(24): 3559-3562, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29882431

RESUMO

Luteoloside (luteolin-7-O-glucoside), the biomarker of Lonicera japonica, was efficiently bio-synthetized from its cheaper precursor luteolin. The structure of luteoloside was characterized by LC-MS and NMR analyses. Compared to the significant inhibitory effect of luteolin on human hepatocyte cell line LO2 at high doses, luteoloside did not show obvious cytotoxic effects at any test dose. Moreover, luteoloside exhibited obvious promotive effects on human hepatocyte cells, suggesting a potential application in hepatoprotective therapies.


Assuntos
Bactérias/metabolismo , Flavobacteriaceae/química , Flavonas/isolamento & purificação , Glucosídeos/isolamento & purificação , Lonicera/química , Luteolina/isolamento & purificação , Rizosfera , Linhagem Celular , Flavobacteriaceae/metabolismo , Flavonas/química , Glucosídeos/biossíntese , Glucosídeos/química , Glucosídeos/farmacologia , Humanos , Lonicera/microbiologia , Luteolina/biossíntese , Luteolina/química , Luteolina/farmacologia , Estrutura Molecular , Substâncias Protetoras
19.
J Microbiol ; 57(1): 23-29, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30456754

RESUMO

A novel Gram-stain-negative, aerobic, motile by means of gliding, and short rod-shaped bacterium, designated strain SH35T, was isolated from the dry surface of a tidal flat in Hwasung-si, South Korea. Growth occurred at 10-40°C (optimum 30°C), at pH 6.0-8.0 (optimum pH 7.0), in 1-12% NaCl (optimum 2%), and was inhibited in the absence of NaCl and Ca2+ ions. Phylogenetic analysis based on the 16S rRNA gene sequences showed that strain SH35T belonged to the genus Gramella and was a member of the family Flavobacteriaceae with highest sequence similarity to Gramella flava JLT2011T (96.1%), followed by Gramella oceani CCAMSZ-TT (95.6%), and 93.0-94.9% to other recognized Gramella species. The major cellular fatty acids (> 5% of the total) of strain SH35T were iso-C15:0, Iso-C16:0, anteiso-C15:0, iso-C17:0 3-OH and summed feature 9 (C16:0 10-methyl and/or C17:1 iso ω9с). The major polar lipids were phosphatidylethanolamine, two unidentified aminolipids and nine unidentified polar lipids. The major respiratory quinone and the predominant polyamine were menaquinone-6 (MK-6) and symhomospermidine, respectively. The DNA G + C content was 40.5 mol% (39.7% based on total genome calculations). Based on phylogenetic analysis and physiological and biochemical characterization, strain SH35T represents a novel species of the genus Gramella, for which the name Gramella fulva sp. nov. is proposed. The type strain is SH35T (= KACC 19447T = JCM 32369T).


Assuntos
Flavobacteriaceae/isolamento & purificação , Água do Mar/microbiologia , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Flavobacteriaceae/classificação , Flavobacteriaceae/genética , Flavobacteriaceae/metabolismo , Filogenia , RNA Ribossômico 16S/genética , República da Coreia , Água do Mar/análise , Cloreto de Sódio/análise , Cloreto de Sódio/metabolismo
20.
ISME J ; 13(1): 76-91, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30111868

RESUMO

Marine algae convert a substantial fraction of fixed carbon dioxide into various polysaccharides. Flavobacteriia that are specialized on algal polysaccharide degradation feature genomic clusters termed polysaccharide utilization loci (PULs). As knowledge on extant PUL diversity is sparse, we sequenced the genomes of 53 North Sea Flavobacteriia and obtained 400 PULs. Bioinformatic PUL annotations suggest usage of a large array of polysaccharides, including laminarin, α-glucans, and alginate as well as mannose-, fucose-, and xylose-rich substrates. Many of the PULs exhibit new genetic architectures and suggest substrates rarely described for marine environments. The isolates' PUL repertoires often differed considerably within genera, corroborating ecological niche-associated glycan partitioning. Polysaccharide uptake in Flavobacteriia is mediated by SusCD-like transporter complexes. Respective protein trees revealed clustering according to polysaccharide specificities predicted by PUL annotations. Using the trees, we analyzed expression of SusC/D homologs in multiyear phytoplankton bloom-associated metaproteomes and found indications for profound changes in microbial utilization of laminarin, α-glucans, ß-mannan, and sulfated xylan. We hence suggest the suitability of SusC/D-like transporter protein expression within heterotrophic bacteria as a proxy for the temporal utilization of discrete polysaccharides.


Assuntos
Flavobacteriaceae/metabolismo , Fitoplâncton/metabolismo , Polissacarídeos Bacterianos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Flavobacteriaceae/genética , Regulação Bacteriana da Expressão Gênica , Genômica , Mar do Norte , Proteômica
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