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1.
J Chem Phys ; 151(19): 195101, 2019 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-31757137

RESUMO

The solid-state photochemically induced dynamic nuclear polarization (photo-CIDNP) effect has been studied in a quinone-depleted uniformly (u-)13C,15N-labeled photosynthetic reaction center (RC) protein from purple bacterium Rhodobacter (R.) sphaeroides wild type (WT). As a method for investigation, solid-state 15N NMR under magic-angle spinning (MAS) is applied under both continuous illumination (steady state) and nanosecond-laser flashes (time-resolved). While all previous 15N photo-CIDNP MAS NMR studies on the purple bacterial RC used the carotenoid-less mutant R26, this is the first using WT samples. The absence of further photo-CIDNP mechanisms (compared to R26) and various couplings (compared to 13C NMR experiments on 13C-labeled samples) allows the simplification of the spin-system. We report 15N signals of the three cofactors forming the spin-correlated radical pair (SCRP) and, based on density-functional theory calculations, their assignment. The simulation of photo-CIDNP intensities and time-resolved 15N photo-CIDNP MAS NMR data matches well to the frame of the mechanistic interpretation. Three spin-chemical processes, namely, radical pair mechanism, three spin mixing, and differential decay, generate emissive (negative) 15N polarization in the singlet decay channel and absorptive (positive) polarization in the triplet decay channel of the SCRP. The absorptive 15N polarization of the triplet decay channel is transiently obscured during the lifetime of the triplet state of the carotenoid (3Car); therefore, the observed 15N signals are strongly emissive. Upon decay of 3Car, the transiently obscured polarization becomes visible by reducing the excess of emissive polarization. After the decline of 3Car, the remaining nuclear hyperpolarization decays with nuclear T1 relaxation kinetics.


Assuntos
Ressonância Magnética Nuclear Biomolecular , Complexo de Proteínas do Centro de Reação Fotossintética/química , Rhodobacter sphaeroides/metabolismo , Modelos Moleculares , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Conformação Proteica
2.
Biochemistry (Mosc) ; 84(9): 1107-1115, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31693470

RESUMO

Using absorption spectroscopy in the visible/near-IR and mid-IR regions, spectral and photochemical properties of isolated reaction centers (RCs) from Rhodobacter sphaeroides R-26 were studied in dried films on the inorganic support surface (quartz or CaF2 plates) under vacuum dehydration conditions (10-2 or 7·10-5 mm Hg). Three detergents, N,N-dimethyldodecylamine N-oxide (LDAO), Triton X-100 (TX100), and n-dodecyl-ß-D-maltoside (DM), were tested for their ability to stabilize the RC-detergent complexes in the vacuum-dried state. It was shown that in the presence of LDAO, RC complexes underwent destruction in vacuum. In contrast, DM provided an environment that minimized irreversible disruptive changes in the RCs in vacuum. The effects of vacuum dehydration on the RC-DM films included a small increase in the content of α-helices in the RC protein, a short-wavelength reversible shift in the optical transitions of pigments, and minor changes in the electronic structure of the P+ dimer. The films retained their photochemical activity upon excitation with high-intensity light (200 mW/cm2). TX100 also helped to maintain spectral and functional properties of the RCs in vacuum; however, in this case, the stabilizing effect was less pronounced than in the presence of DM, especially, at high detergent concentrations. The results are discussed within the framework of a model suggesting that the detergent-protein interactions and the properties of detergent micelles play a dominant role in maintaining the structure of the RCs upon vacuum dehydration of the RC complexes. The obtained data can be useful for developing hybrid photoconverting systems based on bacterial RCs.


Assuntos
Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Rhodobacter sphaeroides/metabolismo , Vácuo , Processos Fotoquímicos , Rhodobacter sphaeroides/isolamento & purificação , Espectrofotometria Ultravioleta , Espectroscopia de Infravermelho com Transformada de Fourier , Propriedades de Superfície
3.
J Agric Food Chem ; 67(34): 9560-9568, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31368704

RESUMO

ß-Carotene is a precursor of vitamin A and a dietary supplement for its antioxidant property. Producing ß-carotene by microbial fermentation has attracted much attention owing to consumers' preference for the natural product. In this study, an engineered photosynthetic Rhodobacter sphaeroides producing ß-carotene was constructed by the following strategies: (1) five promoters of different strengths were used to investigate the effect of the expression level of crtY on ß-carotene content. It was found that PrrnB increased the ß-carotene content by 109%. (2) blocking of the branched pentose phosphate pathway by zwf deletion, and (3) overexpressing dxs could restore the transcriptional levels of crtE and crtB. Finally, the engineered RS-C3 has the highest ß-carotene content of 14.93 mg/g dry cell weight (DCW) among all of the reported photosynthetic bacteria and the ß-carotene content reached 3.34 mg/g DCW under light conditions. Our results will be available for industrial use to supply a large quantity of natural ß-carotene.


Assuntos
Proteínas de Bactérias/genética , Liases Intramoleculares/genética , Rhodobacter sphaeroides/genética , Rhodobacter sphaeroides/metabolismo , beta Caroteno/biossíntese , Proteínas de Bactérias/metabolismo , Fermentação , Liases Intramoleculares/metabolismo , Luz , Engenharia Metabólica , Regiões Promotoras Genéticas , Rhodobacter sphaeroides/efeitos da radiação
4.
J Ind Microbiol Biotechnol ; 46(8): 1179-1190, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31187318

RESUMO

Rhodobacter sphaeroides is a metabolically versatile bacterium capable of producing terpenes natively. Surprisingly, terpene biosynthesis in this species has always been investigated in complex media, with unknown compounds possibly acting as carbon and nitrogen sources. Here, a defined medium was adapted for R. sphaeroides dark heterotrophic growth, and was used to investigate the conversion of different organic substrates into the reporter terpene amorphadiene. The amorphadiene synthase was cloned in R. sphaeroides, allowing its biosynthesis via the native 2-methyl-D-erythritol-4-phosphate (MEP) pathway and, additionally, via a heterologous mevalonate one. The latter condition increased titers up to eightfold. Consequently, better yields and productivities to previously reported complex media cultivations were achieved. Productivity was further investigated under different cultivation conditions, including nitrogen and oxygen availability. This novel cultivation setup provided useful insight into the understanding of terpene biosynthesis in R. sphaeroides, allowing to better comprehend its dynamics and regulation during chemoheterotrophic cultivation.


Assuntos
Processos Heterotróficos , Rhodobacter sphaeroides/metabolismo , Carbono/metabolismo , Eritritol/análogos & derivados , Eritritol/metabolismo , Nitrogênio/metabolismo , Rhodobacter sphaeroides/genética , Fosfatos Açúcares/metabolismo
5.
Chemosphere ; 233: 597-602, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31195264

RESUMO

Carbaryl wastewater treatment and the resource recycling of biomass as sludge by Rhodopseudomonas sphaeroides (R. sphaeroides) with the assistance of starch processing wastewater (SPW) was investigated in this research. It was observed that carbaryl was not degraded under the 100, 500 mg/L COD groups. The addition of SPW assisted R. sphaeroides to degrade carbaryl efficiently. Carbaryl removal reached 100% after 5 days under the optimal group (3500 mg/L). Interestingly, carbaryl in the mixed wastewater began to be degraded after day 1. Further research indicated that cehA gene was expressed after day 1. Subsequently, carbaryl hydrolase was synthesized under gene regulation. Analysis revealed that cehA and carbaryl hydrolase were adaptive gene expressions and enzymes. Carbaryl as stimulus signal started cehA gene expression through signal transduction pathway. This process took one day for R. sphaeroides. However, organics in 100, 500 mg/L COD groups were deficient, which could not maintain R. sphaeroides growth for over one day. Organics in SPW provided sufficient carbon sources for R. sphaeroides under other groups. The method could complete the mixed (SPW and carbaryl) wastewater treatment, carbaryl removal, the resource recycling of R. sphaeroides biomass as sludge simultaneously.


Assuntos
Carbaril/metabolismo , Rhodobacter sphaeroides/metabolismo , Eliminação de Resíduos Líquidos/métodos , Biodegradação Ambiental , Biomassa , Esgotos , Águas Residuárias/microbiologia , Purificação da Água
6.
Microb Cell Fact ; 18(1): 98, 2019 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-31151455

RESUMO

BACKGROUND: Farnesol is an acyclic sesquiterpene alcohol present in the essential oils of various plants in nature. It has been reported to be valuable in medical applications, such as alleviation of allergic asthma, gliosis, and edema as well as anti-cancerous and anti-inflammatory effects. Coenzyme Q10 (CoQ10), an essential cofactor in the aerobic respiratory electron transport chain, has attracted growing interest owing to its clinical benefits and important applications in the pharmaceutical, food, and health industries. In this work, co-production of (E,E)-farnesol (FOH) and CoQ10 was achieved by combining 3 different exogenous terpenes or sesquiterpene synthase with the RNA interference of psy (responsible for phytoene synthesis in Rhodobacter sphaeroides GY-2). RESULTS: FOH production was significantly increased by overexpressing exogenous terpene synthase (TPS), phosphatidylglycerophosphatase B (PgpB), and sesquiterpene synthase (ATPS), as well as RNAi-mediated silencing of psy coding phytoene synthase (PSY) in R. sphaeroides strains. Rs-TPS, Rs-ATPS, and Rs-PgpB respectively produced 68.2%, 43.4%, and 21.9% higher FOH titers than that of the control strain. Interestingly, the CoQ10 production of these 3 recombinant R. sphaeroides strains was exactly opposite to that of FOH. However, CoQ10 production was almost unaffected in R. sphaeroides strains modified by psy RNA interference. The highest FOH production of 40.45 mg/L, which was twice as high as that of the control, was obtained from the TPS-PSYi strain, where the exogenous TPS was combined with the weakening of the phytoene synthesis pathway via psy RNA interference. CoQ10 production in TPS-PSYi, ATPS-PSYi, and PgpB-PSYi was decreased and lower than that of the control strain. CONCLUSIONS: The original flux that contributed to phytoene synthesis was effectively redirected to provide precursors toward FOH or CoQ10 synthesis via psy RNA interference, which led to weakened carotenoid synthesis. The improved flux that was originally involved in CoQ10 production and phytoene synthesis was redirected toward FOH synthesis via metabolic modification. This is the first reported instance of FOH and CoQ10 co-production in R. sphaeroides using a metabolic engineering strategy.


Assuntos
Carotenoides/metabolismo , Farneseno Álcool/metabolismo , Engenharia Metabólica/métodos , Rhodobacter sphaeroides/metabolismo , Ubiquinona/análogos & derivados , Alquil e Aril Transferases/genética , Vias Biossintéticas , Escherichia coli , Proteínas de Escherichia coli/genética , Geranil-Geranildifosfato Geranil-Geraniltransferase/genética , Fosfatidato Fosfatase/genética , Interferência de RNA , Rhodobacter sphaeroides/enzimologia , Rhodobacter sphaeroides/genética , Ubiquinona/biossíntese , Ubiquinona/metabolismo
7.
Biochemistry (Mosc) ; 84(5): 520-528, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31234766

RESUMO

In our recent X-ray study, we demonstrated that substitution of the natural leucine residue M196 with histidine in the reaction center (RC) from Rhodobacter (Rba.) sphaeroides leads to formation of a close contact between the genetically introduced histidine and the primary electron donor P (bacteriochlorophylls (BChls) PA and PB dimer) creating a novel pigment-protein interaction that is not observed in native RCs. In the present work, the possible nature of this novel interaction and its effects on the electronic properties of P and the photochemical charge separation in isolated mutant RCs L(M196)H are investigated at room temperature using steady-state absorption spectroscopy, light-induced difference FTIR spectroscopy, and femtosecond transient absorption spectroscopy. The results are compared with the data obtained for the RCs from Rba. sphaeroides pseudo-wild type strain. It is shown that the L(M196)H mutation results in a decrease in intensity and broadening of the long-wavelength Qy absorption band of P at ~865 nm. Due to the mutation, there is also weakening of the electronic coupling between BChls in the radical cation P+ and increase in the positive charge localization on the PA molecule. Despite the significant perturbations of the electronic structure of P, the mutant RCs retain high electron transfer rates and quantum yield of the P+QA- state (QA is the primary quinone acceptor), which is close to the one observed in the native RCs. Comparison of our results with the literature data suggests that the imidazole group of histidine M196 forms a π-hydrogen bond with the π-electron system of the PB molecule in the P dimer. It is likely that the specific (T-shaped) spatial organization of the π-hydrogen interaction and its potential heterogeneity in relation to the bonding energy is, at least partially, the reason that this type of interaction between the protein and the pigment and quinone cofactors is not realized in the native RCs.


Assuntos
Proteínas de Bactérias/metabolismo , Histidina/metabolismo , Leucina/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Rhodobacter sphaeroides/metabolismo , Proteínas de Bactérias/genética , Cristalografia por Raios X , Transporte de Elétrons , Histidina/genética , Cinética , Leucina/genética , Mutagênese Sítio-Dirigida , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Espectroscopia de Infravermelho com Transformada de Fourier
8.
Biochemistry (Mosc) ; 84(5): 570-574, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31234771

RESUMO

Studying pigment-protein interactions in the photosynthetic reaction centers (RCs) is important for the understanding of detailed mechanisms of the photochemical process. This paper describes spectral and photochemical characteristics, pigment composition, and stability of the Rhodobacter sphaeroides RCs with the I(L177)Y and I(M206)Y amino acid substitutions. The obtained data are compared with the properties of I(L177)H, I(L177)D, and I(M206)H RCs reported previously. It is shown that the I(L177)Y and I(M206)Y mutations cause a similar shift of the QYP band in the absorption spectra of the mutant RCs and do not affect the distribution of the electron spin density within the photo-oxidized P+ dimer. The differences in the position and amplitude of the QYB band in the I(L177)Y and I(M206)Y RCs were determined. The results indicate the possibility of new pigment-protein interactions in the vicinity of monomeric bacteriochlorophylls in the A and B chains, which might be of interest for future research.


Assuntos
Proteínas de Bactérias/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Bacterioclorofilas/química , Bacterioclorofilas/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Mutagênese Sítio-Dirigida , Complexo de Proteínas do Centro de Reação Fotossintética/química , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Estrutura Terciária de Proteína , Rhodobacter sphaeroides/metabolismo , Espectrofotometria
9.
Biochemistry (Mosc) ; 84(4): 370-379, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31228928

RESUMO

This review focuses on recent experimental data obtained by site-directed mutagenesis of the reaction center in purple nonsulfur bacteria. The role of axial ligation of (bacterio)chlorophylls in the regulation of spectral and redox properties of these pigments, as well as correlation between the structure of chromophores and nature of their ligands, are discussed. Cofactor ligation in various types of reaction centers is compared, and possible reasons for observed differences are examined in the light of modern ideas on the evolution of photosynthesis.


Assuntos
Proteínas de Bactérias/metabolismo , Bacterioclorofilas/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Proteobactérias/metabolismo , Proteínas de Bactérias/genética , Bacterioclorofilas/química , Evolução Molecular , Ligantes , Mutagênese Sítio-Dirigida , Fotossíntese , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Rhodobacter sphaeroides/metabolismo
10.
BMC Genomics ; 20(1): 358, 2019 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-31072330

RESUMO

BACKGROUND: In natural environments, bacteria must frequently cope with extremely scarce nutrients. Most studies focus on bacterial growth in nutrient replete conditions, while less is known about the stationary phase. Here, we are interested in global gene expression throughout all growth phases, including the adjustment to deep stationary phase. RESULTS: We monitored both the transcriptome and the proteome in cultures of the alphaproteobacterium Rhodobacter sphaeroides, beginning with the transition to stationary phase and at different points of the stationary phase and finally during exit from stationary phase (outgrowth) following dilution with fresh medium. Correlation between the transcriptomic and proteomic changes was very low throughout the growth phases. Surprisingly, even in deep stationary phase, the abundance of many proteins continued to adjust, while the transcriptome analysis revealed fewer adjustments. This pattern was reversed during the first 90 min of outgrowth, although this depended upon the duration of the stationary phase. We provide a detailed analysis of proteomic changes based on the clustering of orthologous groups (COGs), and compare these with the transcriptome. CONCLUSIONS: The low correlation between transcriptome and proteome supports the view that post-transcriptional processes play a major role in the adaptation to growth conditions. Our data revealed that many proteins with functions in transcription, energy production and conversion and the metabolism and transport of amino acids, carbohydrates, lipids, and secondary metabolites continually increased in deep stationary phase. Based on these findings, we conclude that the bacterium responds to sudden changes in environmental conditions by a radical and rapid reprogramming of the transcriptome in the first 90 min, while the proteome changes were modest. In response to gradually deteriorating conditions, however, the transcriptome remains mostly at a steady state while the bacterium continues to adjust its proteome. Even long after the population has entered stationary phase, cells are still actively adjusting their proteomes.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Variação Genética , Proteoma/análise , Rhodobacter sphaeroides/crescimento & desenvolvimento , Transcriptoma , Rhodobacter sphaeroides/genética , Rhodobacter sphaeroides/metabolismo
11.
Biochim Biophys Acta Bioenerg ; 1860(6): 461-468, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30974094

RESUMO

In contrast to plants, algae and cyanobacteria that contain glycolipids as the major lipid components in their photosynthetic membranes, phospholipids are the dominant lipids in the membranes of anoxygenic purple phototrophic bacteria. Although the phospholipid compositions in whole cells or membranes are known for a limited number of the purple bacteria, little is known about the phospholipids associated with individual photosynthetic complexes. In this study, we investigated the phospholipid distributions in both membranes and the light-harvesting 1-reaction center (LH1-RC) complexes purified from several purple sulfur and nonsulfur bacteria. 31P NMR was used for determining the phospholipid compositions and inductively coupled plasma atomic emission spectroscopy was used for measuring the total phosphorous contents. Combining these two techniques, we could determine the numbers of specific phospholipids in the purified LH1-RC complexes. A total of approximate 20-30 phospholipids per LH1-RC were detected as the tightly bound lipids in all species. The results revealed that while cardiolipin (CL) exists as a minor component in the membranes, it became the most abundant phospholipid in the purified core complexes and the sum of CL and phosphatidylglycerol accounted for more than two thirds of the total phospholipids for most species. Preferential association of these anionic phospholipids with the LH1-RC is discussed in the context of the recent high-resolution structure of this complex from Thermochromatium (Tch.) tepidum. The detergent lauryldimethylamine N-oxide was demonstrated to selectively remove phosphatidylethanolamine from the membrane of Tch. tepidum.


Assuntos
Membrana Celular/metabolismo , Chromatiaceae/metabolismo , Complexos de Proteínas Captadores de Luz/metabolismo , Fosfolipídeos/metabolismo , Cromatóforos Bacterianos/química , Cromatóforos Bacterianos/metabolismo , Membrana Celular/química , Chromatiaceae/química , Escherichia coli/química , Escherichia coli/metabolismo , Hyphomicrobiaceae/química , Hyphomicrobiaceae/metabolismo , Complexos de Proteínas Captadores de Luz/química , Ressonância Magnética Nuclear Biomolecular , Fosfolipídeos/química , Rhodobacter sphaeroides/química , Rhodobacter sphaeroides/metabolismo , Rhodospirillum rubrum/química , Rhodospirillum rubrum/metabolismo , Espectrofotometria Atômica
12.
Microb Cell Fact ; 18(1): 20, 2019 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-30704481

RESUMO

BACKGROUND: Long-chain free fatty acids (FFAs) are a type of backbone molecule that can react with alcohol to produce biodiesels. Various microorganisms have become potent producers of FFAs. Efforts have focused on increasing metabolic flux to the synthesis of either neutral fat or fatty acyl intermediates attached to acyl carrier protein (ACP), which are the source of FFAs. Membrane lipids are also a source of FFAs. As an alternative way of producing FFAs, exogenous phospholipase may be used after heterologous production and localization in the periplasmic space. In this work, we examined whether Rhodobacter sphaeroides, which forms an intracytoplasmic membrane, can be used for long-chain FFA production using phospholipase. RESULTS: The recombinant R. sphaeroides strain Rs-A2, which heterologously produces Arabidopsis thaliana phospholipase A2 (PLA2) in the periplasm, excretes FFAs during growth. FFA productivity under photoheterotrophic conditions is higher than that observed under aerobic or semiaerobic conditions. When the biosynthetic enzymes for FA (ß-ketoacyl-ACP synthase, FabH) and phosphatidate (1-acyl-sn-glycerol-3-phosphate acyltransferase, PlsC) were overproduced in Rs-A2, the FFA productivity of the resulting strain Rs-HCA2 was elevated, and the FFAs produced mainly consisted of long-chain FAs of cis-vaccenate, stearate, and palmitate in an approximately equimolar ratio. The high-cell-density culture of Rs-HCA2 with DMSO in two-phase culture with dodecane resulted in an increase of overall carbon substrate consumption, which subsequently leads to a large increase in FFA productivity of up to 2.0 g L-1 day-1. Overexpression of the genes encoding phosphate acyltransferase (PlsX) and glycerol-3-phosphate acyltransferase (PlsY), which catalyze the biosynthetic steps immediately upstream from PlsC, in Rs-HCA2 generated Rs-HXYCA2, which grew faster than Rs-HCA2 and showed an FFA productivity of 2.8 g L-1 day-1 with an FFA titer of 8.5 g L-1. CONCLUSION: We showed that long-chain FFAs can be produced from metabolically engineered R. sphaeroides heterologously producing PLA2 in the periplasm. The FFA productivity was greatly increased by high-cell-density culture in two-phase culture with dodecane. This approach provides highly competitive productivity of long-chain FFAs by R. sphaeroides compared with other bacteria. This method may be applied to FFA production by other photosynthetic bacteria with similar differentiated membrane systems.


Assuntos
Alcanos/química , Ácidos Graxos não Esterificados/biossíntese , Periplasma/enzimologia , Fosfolipases A2/metabolismo , Rhodobacter sphaeroides/metabolismo , Lipídeos de Membrana/metabolismo , Engenharia Metabólica , Rhodobacter sphaeroides/genética
13.
Biochim Biophys Acta Bioenerg ; 1860(4): 317-324, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30707884

RESUMO

In the native and most mutant reaction centers of bacterial photosynthesis, the electron transfer is coupled to proton transfer and is rate limiting for the second reduction of QB- → QBH2. In the presence of divalent metal ions (e.g. Cd2+) or in some ("proton transfer") mutants (L210DN/M17DN or L213DN), the proton delivery to QB- is made rate limiting and the properties of the proton pathway can be directly examined. We found that small weak acids and buffers in large concentrations (up to 1 M) were able to rescue the severely impaired proton transfer capability differently depending on the location of the defects: lesions at the protein surface (proton gate H126H/H128H + Cd2+), beneath the surface (M17DN + Cd2+, L210DN/M17DN) or deep inside the protein (L213DN) could be completely, partially or to very small extent recovered, respectively. Small zwitterionic acids (azide/hydrazoic acid) and buffers (tricine) proved to be highly effective rescuers consistent with their enhanced binding affinity and access to any of the proton acceptors (including QB- itself) in the pathway. As a consequence, back titration of the protons at L212Glu could be observed as a pH-dependence of the rate constant of the charge recombination in the presence of azide or formate. Model calculations support the collective influence of the acid cluster on the change of the protonation states upon extension of the cluster with the bound small acid. In proton transfer mutants, the rescuing agents decreased the free energy of activation together with their enthalpic and entropic components. This is in agreement with the hypothesis that they function as protein-penetrating protonophores delivering protons into the chain and select dominating paths out of many alternate routes. We estimate that the proton delivery will be accelerated in one pathway out of 100-200 alternate pathways. The implications for design of the chemical recovery of impaired intra-protein proton transfer pathways in proton transfer mutants are discussed.


Assuntos
Proteínas de Bactérias/metabolismo , Mutação de Sentido Incorreto , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Prótons , Rhodobacter sphaeroides/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Transporte Biológico/genética , Concentração de Íons de Hidrogênio , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Rhodobacter sphaeroides/genética
14.
mBio ; 10(1)2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30782656

RESUMO

Cardiolipin (CL) is an anionic phospholipid that plays an important role in regulating protein biochemistry in bacteria and mitochondria. Deleting the CL synthase gene (Δcls) in Rhodobacter sphaeroides depletes CL and decreases cell length by 20%. Using a chemical biology approach, we found that a CL deficiency does not impair the function of the cell wall elongasome in R. sphaeroides; instead, biosynthesis of the peptidoglycan (PG) precursor lipid II is decreased. Treating R. sphaeroides cells with fosfomycin and d-cycloserine inhibits lipid II biosynthesis and creates phenotypes in cell shape, PG composition, and spatial PG assembly that are strikingly similar to those seen with R. sphaeroides Δcls cells, suggesting that CL deficiency alters the elongation of R. sphaeroides cells by reducing lipid II biosynthesis. We found that MurG-a glycosyltransferase that performs the last step of lipid II biosynthesis-interacts with anionic phospholipids in native (i.e., R. sphaeroides) and artificial membranes. Lipid II production decreases 25% in R. sphaeroides Δcls cells compared to wild-type cells, and overexpression of MurG in R. sphaeroides Δcls cells restores their rod shape, indicating that CL deficiency decreases MurG activity and alters cell shape. The R. sphaeroides Δcls mutant is more sensitive than the wild-type strain to antibiotics targeting PG synthesis, including fosfomycin, d-cycloserine, S-(3,4-dichlorobenzyl)isothiourea (A22), mecillinam, and ampicillin, suggesting that CL biosynthesis may be a potential target for combination chemotherapies that block the bacterial cell wall.IMPORTANCE The phospholipid composition of the cell membrane influences the spatial and temporal biochemistry of cells. We studied molecular mechanisms connecting membrane composition to cell morphology in the model bacterium Rhodobacter sphaeroides The peptidoglycan (PG) layer of the cell wall is a dominant component of cell mechanical properties; consequently, it has been an important antibiotic target. We found that the anionic phospholipid cardiolipin (CL) plays a role in determination of the shape of R. sphaeroides cells by affecting PG precursor biosynthesis. Removing CL in R. sphaeroides alters cell morphology and increases its sensitivity to antibiotics targeting proteins synthesizing PG. These studies provide a connection to spatial biochemical control in mitochondria, which contain an inner membrane with topological features in common with R. sphaeroides.


Assuntos
Cardiolipinas/metabolismo , Parede Celular/metabolismo , Rhodobacter sphaeroides/citologia , Rhodobacter sphaeroides/metabolismo , Uridina Difosfato Ácido N-Acetilmurâmico/análogos & derivados , Proteínas da Membrana Bacteriana Externa/metabolismo , Vias Biossintéticas , Deleção de Genes , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Uridina Difosfato Ácido N-Acetilmurâmico/biossíntese
15.
Nat Commun ; 10(1): 902, 2019 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-30796237

RESUMO

Photosynthetic proteins have been extensively researched for solar energy harvesting. Though the light-harvesting and charge-separation functions of these proteins have been studied in depth, their potential as charge storage systems has not been investigated to the best of our knowledge. Here, we report prolonged storage of electrical charge in multilayers of photoproteins isolated from Rhodobacter sphaeroides. Direct evidence for charge build-up within protein multilayers upon photoexcitation and external injection is obtained by Kelvin-probe and scanning-capacitance microscopies. Use of these proteins is key to realizing a 'self-charging biophotonic device' that not only harvests light and photo-generates charges but also stores them. In strong correlation with the microscopic evidence, the phenomenon of prolonged charge storage is also observed in primitive power cells constructed from the purple bacterial photoproteins. The proof-of-concept power cells generated a photovoltage as high as 0.45 V, and stored charge effectively for tens of minutes with a capacitance ranging from 0.1 to 0.2 F m-2.


Assuntos
Fontes de Energia Bioelétrica/microbiologia , Fenômenos Eletromagnéticos , Complexos de Proteínas Captadores de Luz/metabolismo , Fotossíntese/fisiologia , Rhodobacter sphaeroides/metabolismo , Eletroquímica , Transporte de Elétrons/fisiologia
16.
Microb Cell Fact ; 18(1): 40, 2019 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-30808422

RESUMO

BACKGROUND: Due to various environmental problems, biodegradable polymers such as poly (3-hydroxybutyrate) (PHB) have gained much attention in recent years. Purple non-sulfur (PNS) bacteria have various attractive characteristics useful for environmentally harmless PHB production. However, production of PHB by PNS bacteria using genetic engineering has never been reported. This study is the first report of a genetically engineered PNS bacterial strain with a high PHB production. RESULTS: We constructed a poly (3-hydroxyalkanoate) depolymerase (phaZ) gene-disrupted Rhodobacter sphaeroides HJ strain. This R. sphaeroides HJΔphaZ (pLP-1.2) strain showed about 2.9-fold higher volumetric PHB production than that of the parent HJ (pLP-1.2) strain after 5 days of culture. The HJΔphaZ strain was further improved for PHB production by constructing strains overexpressing each of the eight genes including those newly found and annotated as PHB biosynthesis genes in the KEGG GENES Database. Among these constructed strains, all of gene products exhibited annotated enzyme activities in the recombinant strain cells, and HJΔphaZ (phaA3), HJΔphaZ (phaB2), and HJΔphaZ (phaC1) showed about 1.1-, 1.1-, and 1.2-fold higher volumetric PHB production than that of the parent HJΔphaZ (pLP-1.2) strain. Furthermore, we constructed a strain that simultaneously overexpresses all three phaA3, phaB2, and phaC1 genes; this HJΔphaZ (phaA3/phaB2/phaC1) strain showed about 1.7- to 3.9-fold higher volumetric PHB production (without ammonium sulfate; 1.88 ± 0.08 g l-1 and with 100 mM ammonium sulfate; 0.99 ± 0.05 g l-1) than those of the parent HJ (pLP-1.2) strain grown under nitrogen limited and rich conditions, respectively. CONCLUSION: In this study, we identified eight different genes involved in PHB biosynthesis in the genome of R. sphaeroides 2.4.1, and revealed that their overexpression increased PHB accumulation in an R. sphaeroides HJ strain. In addition, we demonstrated the effectiveness of a phaZ disruption for high PHB accumulation, especially under nitrogen rich conditions. Furthermore, we showed that PNS bacteria may have some unidentified genes involved in poly (3-hydroxyalkanoates) (PHA) biosynthesis. Our findings could lead to further improvement of environmentally harmless PHA production techniques using PNS bacteria.


Assuntos
Ácido 3-Hidroxibutírico/biossíntese , Proteínas de Bactérias/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Nitrogênio/metabolismo , Rhodobacter sphaeroides/genética , Proteínas de Bactérias/genética , Hidrolases de Éster Carboxílico/genética , Engenharia Genética , Polímeros , Rhodobacter sphaeroides/metabolismo
17.
Nat Commun ; 10(1): 933, 2019 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-30804346

RESUMO

Understanding the mechanism behind the near-unity efficiency of primary electron transfer in reaction centers is essential for designing performance-enhanced artificial solar conversion systems to fulfill mankind's growing demands for energy. One of the most important challenges is distinguishing electronic and vibrational coherence and establishing their respective roles during charge separation. In this work we apply two-dimensional electronic spectroscopy to three structurally-modified reaction centers from the purple bacterium Rhodobacter sphaeroides with different primary electron transfer rates. By comparing dynamics and quantum beats, we reveal that an electronic coherence with dephasing lifetime of ~190 fs connects the initial excited state, P*, and the charge-transfer intermediate [Formula: see text]; this [Formula: see text] step is associated with a long-lived quasi-resonant vibrational coherence; and another vibrational coherence is associated with stabilizing the primary photoproduct, [Formula: see text]. The results show that both electronic and vibrational coherences are involved in primary electron transfer process and they correlate with the super-high efficiency.


Assuntos
Rhodobacter sphaeroides/química , Transporte de Elétrons , Elétrons , Cinética , Complexo de Proteínas do Centro de Reação Fotossintética/química , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Rhodobacter sphaeroides/genética , Rhodobacter sphaeroides/metabolismo , Análise Espectral , Vibração
18.
J Biosci Bioeng ; 127(6): 698-702, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30709705

RESUMO

Coenzyme Q10 (CoQ10) plays an important role in the human respiratory chain and is widely used as medicine and dietary supplement. To improve the fermentation efficiency of CoQ10, a modified version of atmospheric and room temperature plasma (ARTP) treatment was used to mutate Rhodobacter sphaeroides. Meanwhile, Vitamin K3, a structural analog of CoQ10, was used as an inhibitor for mutant selection. In the first round of screening in 24-well plates, three mutants were obtained, with the production of CoQ10 at 311 mg/L, 307 mg/L, and 309 mg/L, which were increased from the parent's production at 265 mg/L. Furthermore, a second round of mutation and screening was performed based on the mutant strain with the highest production in the first round, leading to the identification of a mutant AR01 with the production of CoQ10 at ∼330 mg/L. Finally, 590 mg/L CoQ10 was obtained for AR01 after 100 h fermentation, which was ∼25.5% higher than that of the original parent strain. It is the first report of ARTP treatment usage for the selection of CoQ10 producing bacteria and the results show that plasma jet, driven by helium-based ARTP, can be a feasible strategy for mutation feeding.


Assuntos
Atmosfera , Mutagênese , Gases em Plasma/farmacologia , Rhodobacter sphaeroides/efeitos dos fármacos , Rhodobacter sphaeroides/genética , Temperatura Ambiente , Ubiquinona/análogos & derivados , Fermentação/efeitos dos fármacos , Mutação , Rhodobacter sphaeroides/metabolismo , Ubiquinona/biossíntese
19.
Environ Microbiol Rep ; 11(2): 118-128, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30451391

RESUMO

As a free-living bacterium Rhodobacter sphaeroides needs to respond to many environmental stresses. Oxidative stress, membrane stress or heat stress induce the ompR-1 gene encoding a protein of the OmpR family. Overexpression of OmpR-1 results in increased resistance to organic peroxides and diamide. Our data demonstrate that OmpR-1 positively affects expression of several sRNAs with an established role in R. sphaeroides stress defences and negatively affects the promoter of the rpoHI gene. The RpoHI sigma factor has a main role in the activation of many stress responses. Thus OmpR-1 has a balancing effect on the activation of the RpoHI regulon. We present a model with OmpR-1 as part of a regulatory network controlling stress defences in R. sphaeroides.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Estresse Oxidativo , Rhodobacter sphaeroides/fisiologia , Transativadores/metabolismo , Proteínas de Bactérias/genética , Expressão Gênica , Estresse Oxidativo/efeitos dos fármacos , Peróxidos/farmacologia , Regiões Promotoras Genéticas , Pequeno RNA não Traduzido/genética , Regulon , Rhodobacter sphaeroides/efeitos dos fármacos , Rhodobacter sphaeroides/genética , Rhodobacter sphaeroides/metabolismo , Fator sigma/genética , Fator sigma/metabolismo , Transativadores/genética
20.
Appl Microbiol Biotechnol ; 103(2): 917-927, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30421110

RESUMO

The present study aimed to evaluate the in vitro antioxidant activities and the protective effect of Rhodobacter sphaeroides on H2O2-induced oxidative stress in Caco-2 cells. The results showed that the antioxidant action of R. sphaeroides varied with different cell concentrations and treatments. Also, the intact cells and intracellular cell-free extracts showed better antioxidant activities. Caco-2 cell-based oxidative stress model was developed by optimizing H2O2 concentration and culture time with the half lethal dose and methyl thiazolyl tetrazolium. By increasing the activity of endogenous antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase, upregulating the antioxidant ability of the anti-superoxide anion and anti-hydroxyl radical, R. sphaeroides, especially the mutant strain R. sphaeroides (CGMCC No. 8513), exhibited significant protective activity against H2O2-induced oxidative stress in Caco-2 cells. Taken together, R. sphaeroides (CGMCC No. 8513) exhibits strong antioxidant activities and is a candidate to be investigated as a potential probiotic in the future.


Assuntos
Antioxidantes/metabolismo , Células Epiteliais/fisiologia , Oxidantes/toxicidade , Rhodobacter sphaeroides/metabolismo , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/toxicidade , Estresse Oxidativo
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