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1.
Photochem Photobiol Sci ; 20(12): 1645-1656, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34796467

RESUMO

Light-oxygen-voltage (LOV) domains are common photosensory modules that found many applications in fluorescence microscopy and optogenetics. Here, we show that the Chloroflexus aggregans LOV domain can bind different flavin species (lumichrome, LC; riboflavin, RF; flavin mononucleotide, FMN; flavin adenine dinucleotide, FAD) during heterologous expression and that its physicochemical properties depend strongly on the nature of the bound flavin. We show that whereas the dissociation constants for different chromophores are similar, the melting temperature of the protein reconstituted with single flavin species varies from ~ 60 °C for LC to ~ 81 °C for FMN, and photobleaching half-times vary almost 100-fold. These observations serve as a caution for future studies of LOV domains in non-native conditions yet raise the possibility of fine-tuning various properties of LOV-based fluorescent probes and optogenetic tools by manipulating the chromophore composition.


Assuntos
Chloroflexus , Oxigênio , Mononucleotídeo de Flavina , Flavina-Adenina Dinucleotídeo , Riboflavina
2.
Biomed Res Int ; 2021: 9050026, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34307671

RESUMO

Chloroflexus aurantiacus is a thermophilic bacterium that produces a multitude of proteins within its genome. Bioinformatics strategies can facilitate comprehending this organism through functional and structural interpretation assessments. This study is aimed at allocating the structure and function through an in silico approach required for bacterial protein biosynthesis. This in silico viewpoint provides copious properties, including the physicochemical properties, subcellular location, three-dimensional structure, protein-protein interactions, and functional elucidation of the protein (WP_012256288.1). The STRING program is utilized for the explication of protein-protein interactions. The in silico investigation documented the protein's hydrophilic nature with predominantly alpha (α) helices in its secondary structure. The tertiary-structure model of the protein has been shown to exhibit reasonably high consistency based on various quality assessment methods. The functional interpretation suggested that the protein can act as a translation initiation factor, a protein required for translation and protein biosynthesis. Protein-protein interactions also demonstrated high credence that the protein interconnected with 30S ribosomal subunit involved in protein synthesis. This study bioinformatically examined that the protein (WP_012256288.1) is affiliated in protein biosynthesis as a translation initiation factor IF-3 of C. aurantiacus.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Chloroflexus/metabolismo , Simulação por Computador , Biossíntese de Proteínas , Sequência de Aminoácidos , Domínio Catalítico , Modelos Moleculares , Anotação de Sequência Molecular , Mapas de Interação de Proteínas , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Frações Subcelulares/metabolismo
3.
Photosynth Res ; 149(3): 313-328, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34138452

RESUMO

The pigment composition of isolated reaction centers (RCs) of the green filamentous bacterium Chloroflexus (Cfl.) aurantiacus was changed by chemical exchange of native bacteriopheophytin a (BPheo) molecules with externally added pheophytin a (Pheo) or [3-acetyl]-Pheo upon incubation of RC/pheophytin mixtures at room temperature and 45 °C. The modified RCs were characterized by Vis/NIR absorption spectroscopy, and the effect of pigment exchange on RC photochemical activity was assessed by measuring the photoaccumulation of the reduced pigment at the binding site HA. It is shown that both pheophytins can be exchanged into the HA site instead of BPheo by incubation at room temperature. While the newly introduced Pheo molecule is not active in electron transfer, the [3-acetyl]-Pheo molecule is able to replace functionally the photoreducible HA BPheo molecule with the formation of the [3-acetyl]-Pheo- radical anion instead of the BPheo-. After incubation at 45 °C, the majority (~ 90%) of HA BPheo molecules is replaced by both Pheo and [3-acetyl]-Pheo. Only a partial replacement of inactive BPheo molecules with pheophytins is observed even when the incubation temperature is raised to 50 °C. The results are discussed in terms of (i) differences in the accessibility of BPheo binding sites for extraneous pigments depending on structural constraints and incubation temperature and (ii) the effect of the reduction potential of pigments introduced into the HA site on the energetics of the charge separation process. The possible implication of Pheo-exchanged preparations for studying early electron-transfer events in Cfl. aurantiacus RCs is considered.


Assuntos
Chloroflexus/química , Chloroflexus/metabolismo , Transporte de Elétrons , Feofitinas/química , Feofitinas/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/química , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo
4.
Biochem Biophys Res Commun ; 567: 143-147, 2021 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-34153684

RESUMO

LOV domains are widespread photosensory modules that have also found applications in fluorescence microscopy, optogenetics, and light-driven generation of reactive oxygen species. Many of these applications require stable proteins with altered spectra. Here, we report a flavin-based fluorescent protein CisFbFP derived from Chloroflexus islandicus LOV domain-containing protein. We show that CisFbFP is thermostable, and its absorption and fluorescence spectra are red-shifted for ∼6 nm, which has not been observed for other cysteine-substituted natural LOV domains. We also provide a crystallographic structure of CisFbFP at the resolution of 1.2 Å that reveals alterations in the active site due to replacement of conservative asparagine with a serine. Finally, we discuss the possible effects of presence of cis-proline in the Aß-Bß loop on the protein's structure and stability. The findings provide the basis for engineering and color tuning of LOV-based tools for molecular biology.


Assuntos
Proteínas de Bactérias/química , Chloroflexus/química , Flavinas/química , Proteínas Luminescentes/química , Cristalografia por Raios X , Modelos Moleculares , Conformação Proteica , Domínios Proteicos
5.
Microb Biotechnol ; 14(4): 1671-1682, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34081409

RESUMO

The use of the methylotrophic yeast Pichia pastoris (Komagataella phaffi) to produce heterologous proteins has been largely reported. However, investigations addressing the potential of this yeast to produce bulk chemicals are still scarce. In this study, we have studied the use of P. pastoris as a cell factory to produce the commodity chemical 3-hydroxypropionic acid (3-HP) from glycerol. 3-HP is a chemical platform which can be converted into acrylic acid and to other alternatives to petroleum-based products. To this end, the mcr gene from Chloroflexus aurantiacus was introduced into P. pastoris. This single modification allowed the production of 3-HP from glycerol through the malonyl-CoA pathway. Further enzyme and metabolic engineering modifications aimed at increasing cofactor and metabolic precursors availability allowed a 14-fold increase in the production of 3-HP compared to the initial strain. The best strain (PpHP6) was tested in a fed-batch culture, achieving a final concentration of 3-HP of 24.75 g l-1 , a product yield of 0.13 g g-1 and a volumetric productivity of 0.54 g l-1  h-1 , which, to our knowledge, is the highest volumetric productivity reported in yeast. These results benchmark P. pastoris as a promising platform to produce bulk chemicals for the revalorization of crude glycerol and, in particular, to produce 3-HP.


Assuntos
Benchmarking , Glicerol , Chloroflexus , Ácido Láctico/análogos & derivados , Pichia/genética , Proteínas Recombinantes/genética , Saccharomycetales
6.
Phys Chem Chem Phys ; 23(22): 12761-12770, 2021 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-34042141

RESUMO

In photosynthetic green bacteria, chlorosomes provide light harvesting with high efficiency. Chlorosomal carotenoids (Cars) participate in light harvesting together with the main pigment, bacteriochlorophyll (BChl) c/d/e. In the present work, we studied the excited-state dynamics in Cars from Chloroflexus (Cfx.) aurantiacus chlorosomes by near infrared pump-probe spectroscopy with 25 fs temporal resolution at room temperature. The S2 state of Cars was excited at a wavelength of ∼520 nm, and the absorption changes were probed at 860-1000 nm where the excited state absorption (ESA) of the Cars S2 state occurred. Global analysis of the spectroscopy data revealed an ultrafast (∼15 fs) and large (>130 nm) red shift of the S2 ESA spectrum together with the well-known S2 → S1 IC (∼190 fs) and Cars → BChl c EET (∼120 fs). The S2 lifetime was found to be ∼74 fs. Our findings are in line with earlier results on the excited-state dynamics in Cars in vitro. To explain the extremely fast S2 dynamics, we have tentatively proposed two alternative schemes. The first scheme assumed the formation of a vibrational wavepacket in the S2 state, the motion of which caused a dynamical red shift of the S2 ESA spectrum. The second scheme assumed the presence of two potential minima in the S2 state and incoherent energy transfer between them.


Assuntos
Carotenoides/metabolismo , Chloroflexus/química , Carotenoides/química , Chloroflexus/metabolismo , Processos Fotoquímicos , Espectroscopia de Luz Próxima ao Infravermelho , Fatores de Tempo
7.
J Biol Chem ; 296: 100662, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33862085

RESUMO

Photoactive biological systems modify the optical properties of their chromophores, known as spectral tuning. Determining the molecular origin of spectral tuning is instrumental for understanding the function and developing applications of these biomolecules. Spectral tuning in flavin-binding fluorescent proteins (FbFPs), an emerging class of fluorescent reporters, is limited by their dependency on protein-bound flavins, whose structure and hence electronic properties cannot be altered by mutation. A blue-shifted variant of the plant-derived improved light, oxygen, voltage FbFP has been created by introducing a lysine within the flavin-binding pocket, but the molecular basis of this shift remains unconfirmed. We here structurally characterize the blue-shifted improved light, oxygen, voltage variant and construct a new blue-shifted CagFbFP protein by introducing an analogous mutation. X-ray structures of both proteins reveal displacement of the lysine away from the chromophore and opening up of the structure as instrumental for the blue shift. Site saturation mutagenesis and high-throughput screening yielded a red-shifted variant, and structural analysis revealed that the lysine side chain of the blue-shifted variant is stabilized close to the flavin by a secondary mutation, accounting for the red shift. Thus, a single additional mutation in a blue-shifted variant is sufficient to generate a red-shifted FbFP. Using spectroscopy, X-ray crystallography, and quantum mechanics molecular mechanics calculations, we provide a firm structural and functional understanding of spectral tuning in FbFPs. We also show that the identified blue- and red-shifted variants allow for two-color microscopy based on spectral separation. In summary, the generated blue- and red-shifted variants represent promising new tools for application in life sciences.


Assuntos
Proteínas de Bactérias/química , Chloroflexus/metabolismo , Flavinas/metabolismo , Proteínas Luminescentes/química , Proteínas Mutantes/química , Mutação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Chloroflexus/crescimento & desenvolvimento , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Simulação de Dinâmica Molecular , Mutagênese , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fotoquímica , Conformação Proteica , Teoria Quântica
8.
Biochim Biophys Acta Bioenerg ; 1862(6): 148396, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33581107

RESUMO

Chlorosomes of photosynthetic green bacteria are unique molecular assemblies providing efficient light harvesting followed by multi-step transfer of excitation energy to reaction centers. In each chlorosome, 104-105 bacteriochlorophyll (BChl) c/d/e molecules are organized by self-assembly into high-ordered aggregates. We studied the early-time dynamics of the excitation energy flow and energy conversion in chlorosomes isolated from Chloroflexus (Cfx.) aurantiacus bacteria by pump-probe spectroscopy with 30-fs temporal resolution at room temperature. Both the S2 state of carotenoids (Cars) and the Soret states of BChl c were excited at ~490 nm, and absorption changes were probed at 400-900 nm. A global analysis of spectroscopy data revealed that the excitation energy transfer (EET) from Cars to BChl c aggregates occurred within ~100 fs, and the Soret â†’ Q energy conversion in BChl c occurred faster within ~40 fs. This conclusion was confirmed by a detailed comparison of the early exciton dynamics in chlorosomes with different content of Cars. These processes are accompanied by excitonic and vibrational relaxation within 100-270 fs. The well-known EET from BChl c to the baseplate BChl a proceeded on a ps time-scale. We showed that the S1 state of Cars does not participate in EET. We discussed the possible presence (or absence) of an intermediate state that might mediates the Soret â†’ Qy internal conversion in chlorosomal BChl c. We discussed a possible relationship between the observed exciton dynamics and the structural heterogeneity of chlorosomes.


Assuntos
Proteínas de Bactérias/metabolismo , Bacterioclorofilas/metabolismo , Chloroflexus/metabolismo , Transferência de Energia , Luz , Organelas/metabolismo , Fotossíntese , Chloroflexus/efeitos da radiação , Cinética , Organelas/efeitos da radiação
9.
ACS Synth Biol ; 10(1): 72-83, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33325704

RESUMO

Protein-fragment complementation assays are used ubiquitously for probing protein-protein interactions. Most commonly, the reporter protein is split in two parts, which are then fused to the proteins of interest and can reassemble and provide a readout if the proteins of interest interact with each other. The currently known split fluorescent proteins either can be used only in aerobic conditions and assemble irreversibly, or require addition of exogenous chromophores, which complicates the design of experiments. In recent years, light-oxygen-voltage (LOV) domains of several photoreceptor proteins have been developed into flavin-based fluorescent proteins (FbFPs) that, under some circumstances, can outperform commonly used fluorescent proteins such as GFP. Here, we show that CagFbFP, a small thermostable FbFP based on a LOV domain-containing protein from Chloroflexus aggregans, can serve as a split fluorescent reporter. We use the available genetic and structural information to identify three loops between the conserved secondary structure elements, Aß-Bß, Eα-Fα, and Hß-Iß, that tolerate insertion of flexible poly-Gly/Ser segments and eventually splitting. We demonstrate that the designed split pairs, when fused to interacting proteins, are fluorescent in vivo in E. coli and human cells and have low background fluorescence. Our results enable probing protein-protein interactions in anaerobic conditions without using exogenous fluorophores and provide a basis for further development of LOV and PAS (Per-Arnt-Sim) domain-based fluorescent reporters and optogenetic tools.


Assuntos
Proteínas de Bactérias/metabolismo , Flavinas/metabolismo , Corantes Fluorescentes/química , Proteínas de Bactérias/genética , Cálcio/química , Chloroflexus/metabolismo , Endopeptidases/metabolismo , Escherichia coli/metabolismo , Flavinas/química , Transferência Ressonante de Energia de Fluorescência , Domínios Proteicos/genética , Dobramento de Proteína , Mapas de Interação de Proteínas
10.
Bioresour Technol ; 320(Pt A): 124362, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33186840

RESUMO

Acetate can be used as carbon feedstock for the production of 3-hydroxypropionic acid (3-HP), but the production level was low due to inefficient cell growth on acetate. To better utilize acetate, a two-stage strategy, whereby glucose is used for cell growth and acetate for 3-HP formation, was attempted. Dissected malonyl-CoA reductase of Chloroflexus aurantiacus, alone or along with acetyl-CoA carboxylase and/or transhydrogenase, was overexpressed, and by-products formation pathway, glyoxylate shunt (GS) and gluconeogenesis were modified. When GS or gluconeogenesis was disrupted, cell growth on glucose was not hampered, while on acetate it was completely abolished. Consequently, 3-HP production, at production stage, were low, though 3-HP yield on acetate was increased, especially in the case of aceA deletion. In two-stage bioreactor, strain with upregulated GS produced 7.3 g/L 3-HP with yield of 0.26 mol/mol acetate. This study suggests that two-stage cultivation is a good strategy for 3-HP production from acetate.


Assuntos
Escherichia coli , Glucose , Acetatos , Chloroflexus , Escherichia coli/genética , Ácido Láctico/análogos & derivados , Engenharia Metabólica
11.
ISME J ; 14(11): 2649-2658, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32647310

RESUMO

Diverse aerobic bacteria persist by consuming atmospheric hydrogen (H2) using group 1h [NiFe]-hydrogenases. However, other hydrogenase classes are also distributed in aerobes, including the group 2a [NiFe]-hydrogenase. Based on studies focused on Cyanobacteria, the reported physiological role of the group 2a [NiFe]-hydrogenase is to recycle H2 produced by nitrogenase. However, given this hydrogenase is also present in various heterotrophs and lithoautotrophs lacking nitrogenases, it may play a wider role in bacterial metabolism. Here we investigated the role of this enzyme in three species from different phylogenetic lineages and ecological niches: Acidithiobacillus ferrooxidans (phylum Proteobacteria), Chloroflexus aggregans (phylum Chloroflexota), and Gemmatimonas aurantiaca (phylum Gemmatimonadota). qRT-PCR analysis revealed that the group 2a [NiFe]-hydrogenase of all three species is significantly upregulated during exponential growth compared to stationary phase, in contrast to the profile of the persistence-linked group 1h [NiFe]-hydrogenase. Whole-cell biochemical assays confirmed that all three strains aerobically respire H2 to sub-atmospheric levels, and oxidation rates were much higher during growth. Moreover, the oxidation of H2 supported mixotrophic growth of the carbon-fixing strains C. aggregans and A. ferrooxidans. Finally, we used phylogenomic analyses to show that this hydrogenase is widely distributed and is encoded by 13 bacterial phyla. These findings challenge the current persistence-centric model of the physiological role of atmospheric H2 oxidation and extend this process to two more phyla, Proteobacteria and Gemmatimonadota. In turn, these findings have broader relevance for understanding how bacteria conserve energy in different environments and control the biogeochemical cycling of atmospheric trace gases.


Assuntos
Hidrogenase , Acidithiobacillus , Bactérias , Chloroflexus , Hidrogênio , Hidrogenase/genética , Hidrogenase/metabolismo , Oxirredução , Filogenia
12.
Microbes Environ ; 35(2)2020.
Artigo em Inglês | MEDLINE | ID: mdl-32418929

RESUMO

Filamentous anoxygenic photosynthetic bacteria grow by photosynthesis and aerobic respiration. The present study investigated the effects of light and O2 on bacteriochlorophyll contents and the transcription levels of photosynthesis-related genes in Chloroflexus aurantiacus J-10-fl T. Under aerobic conditions, C. aurantiacus produced marked amounts of bacteriochlorophylls in the presence of light, although their production was strongly suppressed in the dark. The transcription levels of genes related to the synthesis of bacteriochlorophylls, photosystems, and chlorosomes: bchM, bchU, pufL, pufBA, and csmM, were markedly increased by illumination. These results suggest that C. aurantiacus continuously synthesizes ATP by photophosphorylation even in the presence of O2.


Assuntos
Bacterioclorofilas/biossíntese , Chloroflexus/metabolismo , Luz , Oxigênio/metabolismo , Trifosfato de Adenosina/biossíntese , Proteínas de Bactérias/genética , Bacterioclorofilas/genética , Regulação Bacteriana da Expressão Gênica , Fotossíntese , Complexo de Proteínas do Centro de Reação Fotossintética/genética
13.
Metab Eng ; 59: 142-150, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32061966

RESUMO

We engineered a type II methanotroph, Methylosinus trichosporium OB3b, for 3-hydroxypropionic acid (3HP) production by reconstructing malonyl-CoA pathway through heterologous expression of Chloroflexus aurantiacus malonyl-CoA reductase (MCR), a bifunctional enzyme. Two strategies were designed and implemented to increase the malonyl-CoA pool and thus, increase in 3HP production. First, we engineered the supply of malonyl-CoA precursors by overexpressing endogenous acetyl-CoA carboxylase (ACC), substantially enhancing the production of 3HP. Overexpression of biotin protein ligase (BPL) and malic enzyme (NADP+-ME) led to a ∼22.7% and ∼34.5% increase, respectively, in 3HP titer in ACC-overexpressing cells. Also, the acetyl-CoA carboxylation bypass route was reconstructed to improve 3HP productivity. Co-expression of methylmalonyl-CoA carboxyltransferase (MMC) of Propionibacterium freudenreichii and phosphoenolpyruvate carboxylase (PEPC), which provides the MMC precursor, further improved the 3HP titer. The highest 3HP production of 49 mg/L in the OB3b-MCRMP strain overexpressing MCR, MMC and PEPC resulted in a 2.4-fold improvement of titer compared with that in the only MCR-overexpressing strain. Finally, we could obtain 60.59 mg/L of 3HP in 42 h using the OB3b-MCRMP strain through bioreactor operation, with a 6.36-fold increase of volumetric productivity compared than that in the flask cultures. This work demonstrates metabolic engineering of type II methanotrophs, opening the door for using type II methanotrophs as cell factories for biochemical production along with mitigation of greenhouse gases.


Assuntos
Proteínas de Bactérias , Chloroflexus/genética , Ácido Láctico/análogos & derivados , Engenharia Metabólica , Metano/metabolismo , Methylosinus trichosporium , Oxirredutases , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ácido Láctico/metabolismo , Methylosinus trichosporium/genética , Methylosinus trichosporium/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo
14.
Sci Rep ; 10(1): 228, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31937882

RESUMO

Early-time dynamics of absorbance changes (light minus dark) in the long-wavelength Qy absorption band of bacteriochlorophyll dimer P of isolated reaction centers (RCs) from thermophilic green bacterium Chloroflexus (Cfx.) aurantiacus was studied by difference pump-probe spectroscopy with 18-fs resolution at cryogenic temperature. It was found that the stimulated emission spectrum gradually moves to the red on the ~100-fs time scale and subsequently oscillates with a major frequency of ~140 cm-1. By applying the non-secular Redfield theory and linear susceptibility theory, the coherent dynamics of the stimulated emission from the excited state of the primary electron donor, bacteriochlorophyll dimer P*, was modeled. The model showed the possibility of an extremely fast transition from the locally excited state P1* to the spectrally different excited state P2*. This transition is clearly seen in the kinetics of the stimulated emission at 880 and 945 nm, where mostly P1* and P2* states emit, respectively. These findings are similar to those obtained previously in RCs of the purple bacterium Rhodobacter (Rba.) sphaeroides. The assumption about the existence of the second excited state P2* helps to explain the complicated temporal behavior of the ΔA spectrum measured by pump-probe spectroscopy. It is interesting that, in spite of the strong coupling between the P1* and P2* states assumed in our model, the form of the coherent oscillations is mainly defined by pure vibrational coherence in the excited states. A possible nature of the P2* state is discussed.


Assuntos
Chloroflexus/fisiologia , Transporte de Elétrons , Fotossíntese , Complexo de Proteínas do Centro de Reação Fotossintética/química , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Transdução de Sinais , Temperatura
15.
Photosynth Res ; 146(1-3): 95-108, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31939070

RESUMO

Chlorosomes of green photosynthetic bacteria are the most amazing example of long-range ordered natural light-harvesting antennae. Chlorosomes are the largest among all known photosynthetic light-harvesting structures (~ 104-105 pigments in the aggregated state). The chlorosomal bacteriochlorophyll (BChl) c/d/e molecules are organized via self-assembly and do not require proteins to provide a scaffold for efficient light harvesting. Despite numerous investigations, a consensus regarding the spatial structure of chlorosomal antennae has not yet been reached. In the present work, we studied hyperchromism/hypochromism in the chlorosomal BChl c Q/B absorption bands of the green photosynthetic bacterium Chloroflexus (Cfx.) aurantiacus. The chlorosomes were isolated from cells grown under different light intensities and therefore, as we discovered earlier, they had different sizes of both BChl c antennae and their unit building blocks. We have shown experimentally that the Q-/B-band hyperchromism/hypochromism is proportional to the size of the chlorosomal antenna. We explained theoretically these findings in terms of excitonic intensity borrowing between the Q and B bands for the J-/H-aggregates of the BChls. The theory developed by Gülen (Photosynth Res 87:205-214, 2006) showed the dependence of the Q-/B-band hyperchromism/hypochromism on the structure of the aggregates. For the model of exciton-coupled BChl c linear chains within a unit building block, the theory predicted an increase in the hyperchromism/hypochromism with the increase in the number of molecules per chain and a decrease in it with the increase in the number of chains. It was previously shown that this model ensured a good fit with spectroscopy experiments and approximated the BChl c low packing density in vivo. The presented experimental and theoretical studies of the Q-/B-band hyperchromism/hypochromism permitted us to conclude that the unit building block of Cfx. aurantiacus chlorosomes comprises of several short BChl c chains.This conclusion is in accordance with previous linear and nonlinear spectroscopy studies on Cfx. aurantiacus chlorosomes.


Assuntos
Bacterioclorofilas/metabolismo , Chloroflexus/metabolismo , Fotossíntese , Proteínas de Bactérias/metabolismo , Chloroflexus/efeitos da radiação , Luz , Organelas/metabolismo , Análise Espectral
16.
J Phys Chem B ; 123(46): 9786-9791, 2019 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-31660744

RESUMO

Recently, a hybrid approach combining solid-state NMR spectroscopy and cryo-electron microscopy showed that the baseplate in green sulfur bacterium Chlorobaculum tepidum is a 2D lattice of BChl a-CsmA dimers [Nielsen, J. T.; et al., Nat. Commun. 2016, 7, 12454-12465]. While the existence of the BChl a-CsmA subunit was previously known, the proposed orientations of the BChl a pigments had only been elucidated from spectral data up to this point. Regarding the electronic structure of the baseplate, two models have been proposed. 2D electronic spectroscopy data were interpreted as revealing that at least four excitonically coupled BChl a might be in close contact. Conversely, spectral hole burning data suggested that the lowest energy state was localized, yet additional states are sometimes observed because of the presence of the Fenna-Matthews-Olson (FMO) antenna protein. To solve this conundrum, this work studies the chlorosome-baseplate complex from Chloroflexus aurantiacus, which does not contain the FMO protein. The results confirm that in both C. tepidum and C. aurantiacus, excitation energy is transferred to a localized low-energy trap state near 818 nm with similar rates, most likely via exciton hopping.


Assuntos
Proteínas de Bactérias/química , Bacterioclorofilas/química , Chloroflexus/metabolismo , Transferência de Energia , Proteínas de Bactérias/metabolismo , Bacterioclorofilas/metabolismo , Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Temperatura
17.
Microbes Environ ; 34(3): 304-309, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31391357

RESUMO

Chloroflexus aggregans is a thermophilic filamentous anoxygenic phototrophic bacterium frequently found in microbial mats in natural hot springs. C. aggregans often thrives with cyanobacteria that engage in photosynthesis to provide it with an organic substrate; however, it sometimes appears as the dominant phototroph in microbial mats without cyanobacteria. This suggests that C. aggregans has the ability to grow photoautotrophically. However, photoautotrophic growth has not been observed in any cultured strains of C. aggregans. We herein attempted to isolate a photoautotrophic strain from C. aggregansdominated microbial mats in Nakabusa hot spring in Japan. Using an inorganic medium, we succeeded in isolating a new strain that we designated "ACA-12". A phylogenetic analysis based on 16S rRNA gene and 16S-23S rRNA gene internal transcribed spacer (ITS) region sequences revealed that strain ACA-12 was closely related to known C. aggregans strains. Strain ACA-12 showed sulfide consumption along with autotrophic growth under anaerobic light conditions. The deposited elemental sulfur particles observed by microscopy indicated that sulfide oxidation occurred, similar to that in photoautotrophic strains in the related species, C. aurantiacus. Moreover, we found that other strains of C. aggregans, including the type strain, also exhibited a slight photoautotrophic growing ability, whereas strain ACA-12 showed the fastest growth rate. This is the first demonstration of photoautotrophic growth with sulfide in C. aggregans. The present results strongly indicate that C. aggregans is associated with inorganic carbon incorporation using sulfide as an electron donor in hot spring microbial mats.


Assuntos
Chloroflexus/metabolismo , Processos Fototróficos , Sulfetos/metabolismo , Proteínas de Bactérias/genética , Chloroflexus/classificação , Chloroflexus/genética , Chloroflexus/efeitos da radiação , Meios de Cultura/química , DNA Bacteriano/genética , DNA Espaçador Ribossômico/genética , Fontes Termais/microbiologia , Japão , Luz , Oxirredução , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Sulfetos/análise , Enxofre/metabolismo
18.
FEMS Microbiol Lett ; 366(10)2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31158281

RESUMO

The genus Chloroflexus is a deeply branching group of thermophilic filamentous anoxygenic phototrophic bacteria. The bacteria in this genus have been shown to grow well heterotrophically under anaerobic photosynthetic and aerobic respiratory conditions. We examined autotrophic growth in new isolates of Chloroflexus strains from hot springs in Nakabusa, Japan. The isolates belonging to Chloroflexus aggregans (98.7% identity of 16S rRNA gene sequence to the respective type strain) and Chloroflexus aurantiacus (99.9% identity to the respective type strain) grew photoautotrophically under a 24% H2 atmosphere. We also observed chemolithotrophic growth of these isolates under 80% H2 and 5% O2 conditions in the dark. This is the first report showing that Chloroflexus grew under both photoautotrophic and chemolithotrophic conditions in addition to photoheterotrophic and aerobic chemoheterotrophic conditions.


Assuntos
Processos Autotróficos , Chloroflexus/crescimento & desenvolvimento , Chloroflexus/isolamento & purificação , Fontes Termais/microbiologia , Hidrogênio/metabolismo , Aerobiose , Proteínas de Bactérias/genética , Crescimento Quimioautotrófico , Chloroflexus/genética , Escuridão , Oxigênio/metabolismo , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
19.
Photochem Photobiol Sci ; 18(7): 1793-1805, 2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31116222

RESUMO

Light-Oxygen-Voltage (LOV) domains are conserved parts of photoreceptors in plants, bacteria and fungi that bind flavins as chromophores and detect blue light. In the past, LOV domain variants have been developed as fluorescent reporter proteins (called flavin-based fluorescent proteins; FbFPs), which due to their ability to fluoresce under anaerobic conditions, fast folding kinetics and a small size of ∼12-16 kDa are a promising reporter system for quantitative real-time analysis of biological processes. Here, we present a small thermostable flavin-based fluorescent protein CagFbFP derived from a soluble LOV domain-containing histidine kinase from the thermophilic bacterium Chloroflexus aggregans. CagFbFP is composed of 107 amino acids with a molecular weight of 11.6 kDa and consists only of the conserved LOV core domain. The protein is thermostable with a melting point of about 68 °C. It crystallizes easily and its crystals diffract to 1.07 Å. Both the crystal structure and small angle scattering data show that the protein is a dimer. Unexpectedly, glutamine 148, which in LOV photoreceptor proteins is the key residue responsible for signal transduction, occupies two conformations. Molecular dynamics simulations show that the two conformations interconvert rapidly. The crystal structure of the wild-type Chloroflexus aggregans LOV domain determined at 1.22 Å resolution confirmed the presence of two alternative conformations of the glutamine 148 side chain. Overall, this protein, due to its stability and ease of crystallization, appears to be a promising model for ultra-high resolution structural studies of LOV domains and for application as a fluorescent reporter.


Assuntos
Proteínas de Bactérias/química , Chloroflexus/metabolismo , Flavinas/química , Proteínas Luminescentes/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Peso Molecular , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Espalhamento a Baixo Ângulo , Alinhamento de Sequência , Espectrometria de Fluorescência , Temperatura de Transição , Difração de Raios X
20.
Biotechnol Lett ; 41(3): 419-426, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30666483

RESUMO

OBJECTIVE: This study was conducted to characterize recombinant α-L-rhamnosidase from Chloroflexus aurantiacus and apply the enzyme in the production of isoquercitrin from rutin. RESULTS: The α-L-rhamnosidase from C. aurantiacus was cloned and expressed in Escherichia coli BL21 and purified as a soluble enzyme. α-L-rhamnosidase purified from C. aurantiacus has a molecular mass of approximately 105 kDa and is predicted to exist as a homodimer with a native enzyme of 200 kDa. The purified enzyme exhibited the highest specific activity for rutin among the reported isoquercitrin producing α-L-rhamnosidases and was applied in the production of isoquercitrin from rutin. Under the optimised conditions of pH 6.0, 50 °C, 0.6 U mL-1 α-L-rhamnosidase, and 30 mM rutin, α-L-rhamnosidase from C. aurantiacus produced 30 mM isoquercitrin after 2 h with a 100% conversion yield and productivity of 15 mM h-1. CONCLUSIONS: We achieved a high productivity of isoquercitrin from rutin. Moreover, these results suggest that α-L-rhamnosidase from C. aurantiacus is an effective isoquercitrin producer.


Assuntos
Chloroflexus/enzimologia , Glicosídeo Hidrolases/metabolismo , Quercetina/análogos & derivados , Proteínas Recombinantes/metabolismo , Rutina/metabolismo , Biotransformação , Chloroflexus/genética , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/isolamento & purificação , Concentração de Íons de Hidrogênio , Peso Molecular , Quercetina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Temperatura
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