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1.
Nucleic Acids Res ; 48(6): e33, 2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-31989175

RESUMO

Light-regulated modules offer unprecedented new ways to control cellular behaviour with precise spatial and temporal resolution. Among a variety of bacterial light-switchable gene expression systems, single-component systems consisting of single transcription factors would be more useful due to the advantages of speed, simplicity, and versatility. In the present study, we developed a single-component light-activated bacterial gene expression system (eLightOn) based on a novel LOV domain from Rhodobacter sphaeroides (RsLOV). The eLightOn system showed significant improvements over the existing single-component bacterial light-activated expression systems, with benefits including a high ON/OFF ratio of >500-fold, a high activation level, fast activation kinetics, and/or good adaptability. Additionally, the induction characteristics, including regulatory windows, activation kinetics and light sensitivities, were highly tunable by altering the expression level of LexRO. We demonstrated the usefulness of the eLightOn system in regulating cell division and swimming by controlling the expression of the FtsZ and CheZ genes, respectively, as well as constructing synthetic Boolean logic gates using light and arabinose as the two inputs. Taken together, our data indicate that the eLightOn system is a robust and highly tunable tool for quantitative and spatiotemporal control of bacterial gene expression.


Assuntos
Regulação Bacteriana da Expressão Gênica/efeitos da radiação , Luz , Rhodobacter sphaeroides/citologia , Rhodobacter sphaeroides/efeitos da radiação , Proteínas de Bactérias/metabolismo , Divisão Celular/efeitos da radiação , Cinética , Lógica , Fatores de Transcrição/metabolismo
2.
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
3.
mBio ; 10(1)2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30602584

RESUMO

Coordinating chromosome duplication and segregation with cell division is clearly critical for bacterial species with one chromosome. The precise choreography required is even more complex in species with more than one chromosome. The alpha subgroup of bacteria contains not only one of the best-studied bacterial species, Caulobacter crescentus, but also several species with more than one chromosome. Rhodobacter sphaeroides is an alphaproteobacterium with two chromosomes, but, unlike C. crescentus, it divides symmetrically rather than buds and lacks the complex CtrA-dependent control mechanism. By examining the Ori and Ter regions of both chromosomes and associated ParA and ParB proteins relative to cell division proteins FtsZ and MipZ, we have identified a different pattern of chromosome segregation and cell division. The pattern of chromosome duplication and segregation resembles that of Vibrio cholerae, not that of Agrobacterium tumefaciens, with duplication of the origin and terminus regions of chromosome 2 controlled by chromosome 1. Key proteins are localized to different sites compared to C. crescentus OriC1 and ParB1 are localized to the old pole, while MipZ and FtsZ localize to the new pole. Movement of ParB1 to the new pole following chromosome duplication releases FtsZ, which forms a ring at midcell, but, unlike reports for other species, MipZ monomers do not form a gradient but oscillate between poles, with the nucleotide-bound monomer and the dimer localizing to midcell. MipZ dimers form a single ring (with a smaller diameter) close to the FtsZ ring at midcell and constrict with the FtsZ ring. Overproduction of the dimer form results in filamentation, suggesting that MipZ dimers are regulating FtsZ activity and thus septation. This is an unexpected role for MipZ and provides a new model for the integration of chromosome segregation and cell division.IMPORTANCE Cell division has to be coordinated with chromosome segregation to ensure the stable inheritance of genetic information. We investigated this coordination in the multichromosome bacterium Rhodobacter sphaeroides By examining the origin and terminus regions of the two chromosomes, the ParA-like ATPase MipZ and FtsZ, we showed that chromosome 1 appears to be the "master" chromosome connecting DNA segregation and cell division, with MipZ being critical for coordination. MipZ shows an unexpected localization pattern, with MipZ monomers interacting with ParB of the chromosome 1 at the cell poles whereas MipZ dimers colocalize with FtsZ at midcell during constriction, both forming dynamic rings. These data suggest that MipZ has roles in R. sphaeroides in both controlling septation and coordinating chromosome segregation with cell division.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Segregação de Cromossomos , Cromossomos Bacterianos , Rhodobacter sphaeroides/citologia , Rhodobacter sphaeroides/fisiologia , Microscopia Intravital , Transporte Proteico
4.
Biosci Biotechnol Biochem ; 82(1): 148-151, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29297256

RESUMO

Growth inhibition of Rhodobacter sphaeroides f. sp. denitrificans IL106 by nitrite under anaerobic-light conditions became less pronounced when the gene encoding nitrite reductase was deleted. Growth of another deletion mutant of the genes encoding nitric oxide reductase was severely suppressed by nitrite. Our results suggest that nitrite reductase increases the sensitivity to nitrite through the production of nitric oxide.


Assuntos
Nitritos/química , Rhodobacter sphaeroides/efeitos dos fármacos , Deleção de Genes , Nitrito Redutases/genética , Nitritos/farmacologia , Oxirredução , Rhodobacter sphaeroides/citologia , Rhodobacter sphaeroides/crescimento & desenvolvimento
5.
Nat Commun ; 8(1): 988, 2017 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-29042567

RESUMO

Photosynthesis transfers energy efficiently through a series of antenna complexes to the reaction center where charge separation occurs. Energy transfer in vivo is primarily monitored by measuring fluorescence signals from the small fraction of excitations that fail to result in charge separation. Here, we use two-dimensional electronic spectroscopy to follow the entire energy transfer process in a thriving culture of the purple bacteria, Rhodobacter sphaeroides. By removing contributions from scattered light, we extract the dynamics of energy transfer through the dense network of antenna complexes and into the reaction center. Simulations demonstrate that these dynamics constrain the membrane organization into small pools of core antenna complexes that rapidly trap energy absorbed by surrounding peripheral antenna complexes. The rapid trapping and limited back transfer of these excitations lead to transfer efficiencies of 83% and a small functional light-harvesting unit.During photosynthesis, energy is transferred from photosynthetic antenna to reaction centers via ultrafast energy transfer. Here the authors track energy transfer in photosynthetic bacteria using two-dimensional electronic spectroscopy and show that these transfer dynamics constrain antenna complex organization.


Assuntos
Transferência de Energia , Fotossíntese/fisiologia , Rhodobacter sphaeroides/metabolismo , Energia Solar , Proteínas de Bactérias/metabolismo , Fluorescência , Cinética , Luz , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Proteobactérias/citologia , Proteobactérias/metabolismo , Proteobactérias/efeitos da radiação , Rhodobacter sphaeroides/citologia , Rhodobacter sphaeroides/efeitos da radiação , Espectrofotometria/métodos
6.
J Bacteriol ; 199(14)2017 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-28507242

RESUMO

Under unfavorable growth conditions, bacteria enter stationary phase and can maintain cell viability over prolonged periods with no increase in cell number. To obtain insights into the regulatory mechanisms that allow bacteria to resume growth when conditions become favorable again (outgrowth), we performed global transcriptome analyses at different stages of growth for the alphaproteobacterium Rhodobacter sphaeroides The majority of genes were not differentially expressed across growth phases. After a short stationary phase (about 20 h after growth starts to slow down), only 7% of the genes showed altered expression (fold change of >1.6 or less than -1.6, corresponding to a log2 fold change of >0.65 or less than -0.65, respectively) compared to expression at exponential phase. Outgrowth induced a distinct response in gene expression which was strongly influenced by the length of the preceding stationary phase. After a long stationary phase (about 64 h after growth starts to slow down), a much larger number of genes (15.1%) was induced in outgrowth than after a short stationary phase (1.7%). Many of those genes are known members of the RpoHI/RpoHII regulons and have established functions in stress responses. A main effect of RpoHI on the transcriptome in outgrowth after a long stationary phase was confirmed. Growth experiments with mutant strains further support an important function in outgrowth after prolonged stationary phase for the RpoHI and RpoHII sigma factors.IMPORTANCE In natural environments, the growth of bacteria is limited mostly by lack of nutrients or other unfavorable conditions. It is important for bacterial populations to efficiently resume growth after being in stationary phase, which may last for long periods. Most previous studies on growth-phase-dependent gene expression did not address outgrowth after stationary phase. This study on growth-phase-dependent gene regulation in a model alphaproteobacterium reveals, for the first time, that the length of the stationary phase strongly impacts the transcriptome during outgrowth. The alternative sigma factors RpoHI and RpoHII, which are important regulators of stress responses in alphaproteobacteria, play a major role during outgrowth following prolonged stationary phase. These findings provide the first insight into the regulatory mechanisms enabling efficient outgrowth.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Rhodobacter sphaeroides/metabolismo , Proteínas de Bactérias/genética , Sequência de Bases , Divisão Celular , Sobrevivência Celular , DNA Bacteriano , Regiões Promotoras Genéticas , Rhodobacter sphaeroides/citologia , Rhodobacter sphaeroides/genética , Ativação Transcricional , Transcriptoma
7.
J Phys Chem B ; 121(15): 3787-3797, 2017 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-28301162

RESUMO

Cell doubling times of the purple bacterium Rhodobacter sphaeroides during photosynthetic growth are determined experimentally and computationally as a function of illumination. For this purpose, energy conversion processes in an intracytoplasmic membrane vesicle, the chromatophore, are described based on an atomic detail structural model. The cell doubling time and its illumination dependence are computed in terms of the return-on-investment (ROI) time of the chromatophore, determined computationally from the ATP production rate, and the mass ratio of chromatophores in the cell, determined experimentally from whole cell absorbance spectra. The ROI time is defined as the time it takes to produce enough ATP to pay for the construction of another chromatophore. The ROI time of the low light-growth chromatophore is 4.5-2.6 h for a typical illumination range of 10-100 µmol photons m-2 s-1, respectively, with corresponding cell doubling times of 8.2-3.9 h. When energy expenditure is considered as a currency, the benefit-to-cost ratio computed for the chromatophore as an energy harvesting device is 2-8 times greater than for photovoltaic and fossil fuel-based energy solutions and the corresponding ROI times are approximately 3-4 orders of magnitude shorter for the chromatophore than for synthetic systems.


Assuntos
Cromatóforos Bacterianos/química , Complexos de Proteínas Captadores de Luz/química , Simulação de Dinâmica Molecular , Rhodobacter sphaeroides/metabolismo , Trifosfato de Adenosina/biossíntese , Cromatóforos Bacterianos/metabolismo , Complexos de Proteínas Captadores de Luz/metabolismo , Conformação Proteica , Rhodobacter sphaeroides/química , Rhodobacter sphaeroides/citologia , Fatores de Tempo
8.
Dokl Biochem Biophys ; 477(1): 368-371, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29297127

RESUMO

The effect of heating at 65°C for 20 min on the absorption spectra and kinetics of the dark recombination of charges separated between photoactive bacteriochlorophyll and quinone acceptors was studied in dry films of bacterial photosynthetic reaction centers (RCs), RC films in polyvinyl alcohol, and trehalose. A pronounced protective effect of trehalose against pheophytinizaiton of molecules bacteriochlorophylls in RC structure and in maintaining their higher photochemical activity was found.


Assuntos
Temperatura Alta , Complexo de Proteínas do Centro de Reação Fotossintética/efeitos dos fármacos , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Trealose/farmacologia , Cinética , Rhodobacter sphaeroides/citologia , Rhodobacter sphaeroides/enzimologia
9.
J Phys Chem A ; 120(24): 4124-30, 2016 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-27232937

RESUMO

Light harvesting in photosynthetic organisms involves efficient transfer of energy from peripheral antenna complexes to core antenna complexes, and ultimately to the reaction center where charge separation drives downstream photosynthetic processes. Antenna complexes contain many strongly coupled chromophores, which complicates analysis of their electronic structure. Two-dimensional electronic spectroscopy (2DES) provides information on energetic coupling and ultrafast energy transfer dynamics, making the technique well suited for the study of photosynthetic antennae. Here, we present 2DES results on excited state properties and dynamics of a core antenna complex, light harvesting complex 1 (LH1), embedded in the photosynthetic membrane of Rhodobacter sphaeroides. The experiment reveals weakly allowed higher-lying excited states in LH1 at 770 nm, which transfer energy to the strongly allowed states at 875 nm with a lifetime of 40 fs. The presence of higher-lying excited states is in agreement with effective Hamiltonians constructed using parameters from crystal structures and atomic force microscopy (AFM) studies. The energy transfer dynamics between the higher- and lower-lying excited states agree with Redfield theory calculations.


Assuntos
Elétrons , Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/metabolismo , Rhodobacter sphaeroides/metabolismo , Membrana Celular/metabolismo , Fotossíntese , Rhodobacter sphaeroides/citologia
10.
Photosynth Res ; 127(1): 13-24, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25512104

RESUMO

The photosynthetic apparatus in the bacterium Rhodobacter sphaeroides is mostly present in intracytoplasmic membrane invaginations. It has long been debated whether these invaginations remain in topological continuity with the cytoplasmic membrane, or form isolated chromatophore vesicles. This issue is revisited here by functional approaches. The ionophore gramicidin was used as a probe of the relative size of the electro-osmotic units in isolated chromatophores, spheroplasts, or intact cells. The decay of the membrane potential was monitored from the electrochromic shift of carotenoids. The half-time of the decay induced by a single channel in intact cells was about 6 ms, thus three orders of magnitude slower than in isolated chromatophores. In spheroplasts obtained by lysis of the cell wall, the single channel decay was still slower (~23 ms) and the sensitivity toward the gramicidin concentration was enhanced 1,000-fold with respect to isolated chromatophores. These results indicate that the area of the functional membrane in cells or spheroplasts is about three orders of magnitude larger than that of isolated chromatophores. Intracytoplasmic vesicles, if present, could contribute to at most 10% of the photosynthetic apparatus in intact cells of Rba. sphaeroides. Similar conclusions were obtained from the effect of a ∆pH-induced diffusion potential in intact cells. This caused a large electrochromic response of carotenoids, of similar amplitude as the light-induced change, indicating that most of the system is sensitive to a pH change of the external medium. A single internal membrane and periplasmic space may offer significant advantages concerning renewal of the photosynthetic apparatus and reallocation of the components shared with other bioenergetic pathways.


Assuntos
Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Rhodobacter sphaeroides/citologia , Cromatóforos Bacterianos/metabolismo , Carotenoides/metabolismo , Citoplasma/metabolismo , Relação Dose-Resposta a Droga , Gramicidina/administração & dosagem , Gramicidina/farmacologia , Concentração de Íons de Hidrogênio , Ionóforos/administração & dosagem , Ionóforos/farmacologia , Fotossíntese , Rhodobacter sphaeroides/efeitos dos fármacos , Rhodobacter sphaeroides/metabolismo , Esferoplastos/efeitos dos fármacos
11.
J Bacteriol ; 197(21): 3446-55, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26283770

RESUMO

UNLABELLED: Cell shape has been suggested to play an important role in the regulation of bacterial attachment to surfaces and the formation of communities associated with surfaces. We found that a cardiolipin synthase (Δcls) mutant of the rod-shaped bacterium Rhodobacter sphaeroides--in which synthesis of the anionic, highly curved phospholipid cardiolipin (CL) is reduced by 90%--produces ellipsoid-shaped cells that are impaired in biofilm formation. Reducing the concentration of CL did not cause significant defects in R. sphaeroides cell growth, swimming motility, lipopolysaccharide and exopolysaccharide production, surface adhesion protein expression, and membrane permeability. Complementation of the CL-deficient mutant by ectopically expressing CL synthase restored cells to their rod shape and increased biofilm formation. Treating R. sphaeroides cells with a low concentration (10 µg/ml) of the small-molecule MreB inhibitor S-(3,4-dichlorobenzyl)isothiourea produced ellipsoid-shaped cells that had no obvious growth defect yet reduced R. sphaeroides biofilm formation. This study demonstrates that CL plays a role in R. sphaeroides cell shape determination, biofilm formation, and the ability of the bacterium to adapt to its environment. IMPORTANCE: Membrane composition plays a fundamental role in the adaptation of many bacteria to environmental stress. In this study, we build a new connection between the anionic phospholipid cardiolipin (CL) and cellular adaptation in Rhodobacter sphaeroides. We demonstrate that CL plays a role in the regulation of R. sphaeroides morphology and is important for the ability of this bacterium to form biofilms. This study correlates CL concentration, cell shape, and biofilm formation and provides the first example of how membrane composition in bacteria alters cell morphology and influences adaptation. This study also provides insight into the potential of phospholipid biosynthesis as a target for new chemical strategies designed to alter or prevent biofilm formation.


Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes , Cardiolipinas/metabolismo , Proteínas de Membrana/deficiência , Rhodobacter sphaeroides/citologia , Rhodobacter sphaeroides/enzimologia , Transferases (Outros Grupos de Fosfato Substituídos)/deficiência , Proteínas de Bactérias/genética , Proteínas de Membrana/genética , Mutação , Rhodobacter sphaeroides/genética , Rhodobacter sphaeroides/fisiologia , Transferases (Outros Grupos de Fosfato Substituídos)/genética
12.
Artigo em Inglês | MEDLINE | ID: mdl-25871207

RESUMO

The motility of swimming bacteria near solid surfaces has implications in a wide range of scenarios, including water treatment facilities, microfluidics, and biomedical implants. Using the boundary element method to numerically solve the equations of low Reynolds number fluid flow, we investigate the dynamics of a model swimmer propelled by rotating a single helical flagellum. Building on previous simulation results for swimmers near a single plane boundary, we introduce a second, parallel boundary and show that the bacterial trajectories change as the two plates are brought closer together. Analysis of this dynamical system shows that the configuration in the center of the channel and parallel to the walls is an unstable equilibrium state for large plate separations, but it becomes the only stable position for swimmers when the plate separation is reduced to three to four times the cell width. Our model also predicts that transient trajectories, i.e., those not at steady states, can exhibit curvature in the opposite sense to that expected from the well-known explanation for circular bacterial paths near a single wall.


Assuntos
Flagelos , Hidrodinâmica , Modelos Biológicos , Movimento , Rhodobacter sphaeroides/citologia , Rhodobacter sphaeroides/fisiologia
13.
PLoS One ; 10(2): e0117738, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25689864

RESUMO

Microdroplets are an effective platform for segregating individual cells and amplifying DNA. However, a key challenge is to recover the contents of individual droplets for downstream analysis. This paper offers a method for embedding cells in alginate microspheres and performing multiple serial operations on the isolated cells. Rhodobacter sphaeroides cells were diluted in alginate polymer and sprayed into microdroplets using a fingertip aerosol sprayer. The encapsulated cells were lysed and subjected either to conventional PCR, or whole genome amplification using either multiple displacement amplification (MDA) or a two-step PCR protocol. Microscopic examination after PCR showed that the lumen of the occupied microspheres contained fluorescently stained DNA product, but multiple displacement amplification with phi29 produced only a small number of polymerase colonies. The 2-step WGA protocol was successful in generating fluorescent material, and quantitative PCR from DNA extracted from aliquots of microspheres suggested that the copy number inside the microspheres was amplified up to 3 orders of magnitude. Microspheres containing fluorescent material were sorted by a dilution series and screened with a fluorescent plate reader to identify single microspheres. The DNA was extracted from individual isolates, re-amplified with full-length sequencing adapters, and then a single isolate was sequenced using the Illumina MiSeq platform. After filtering the reads, the only sequences that collectively matched a genome in the NCBI nucleotide database belonged to R. sphaeroides. This demonstrated that sequencing-ready DNA could be generated from the contents of a single microsphere without culturing. However, the 2-step WGA strategy showed limitations in terms of low genome coverage and an uneven frequency distribution of reads across the genome. This paper offers a simple method for embedding cells in alginate microspheres and performing PCR on isolated cells in common bulk reactions, although further work must be done to improve the amplification coverage of single genomes.


Assuntos
Alginatos/química , Separação Celular/métodos , Genoma Bacteriano/genética , Microesferas , Reação em Cadeia da Polimerase/métodos , Rhodobacter sphaeroides/citologia , Rhodobacter sphaeroides/genética , Cápsulas , Estudos de Viabilidade , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Análise de Sequência de DNA
14.
Photosynth Res ; 124(1): 31-44, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25527461

RESUMO

The dark relaxation of the yield of variable BChl fluorescence in the 10(-5)-10 s time range is measured after laser diode (808 nm) excitation of variable duration in intact cells of photosynthetic bacteria Rba. sphaeroides, Rsp. rubrum, and Rvx. gelatinosus under various treatments of redox agents, inhibitors, and temperature. The kinetics of the relaxation is complex and much wider extended than a monoexponential function. The longer is the excitation, the slower is the relaxation which is determined by the redox states, sizes, and accessibility of the pools of cytochrome [Formula: see text] and quinone for donor and acceptor side-limited bacterial strains, respectively. The kinetics of fluorescence decay reflects the opening kinetics of the closed RC. The relaxation is controlled preferentially by the rate of re-reduction of the oxidized dimer by mobile cytochrome [Formula: see text] in Rba. sphaeroides and Rsp. rubrum and by the rate constant of the [Formula: see text] interquinone electron transfer, (350 µs)(-1) and/or the quinol/quinone exchange at the acceptor side in Rvx. gelatinosus. The commonly used acceptor side inhibitors (e.g., terbutryn) demonstrate kinetically limited block of re-oxidation of the primary quinone. The observations are interpreted in frame of a minimum kinetic and energetic model of electron transfer reactions in bacterial RC of intact cells.


Assuntos
Bactérias/citologia , Bactérias/metabolismo , Fotossíntese , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Bactérias/efeitos da radiação , Bacterioclorofilas/metabolismo , Transporte de Elétrons/efeitos da radiação , Fluorescência , Cinética , Luz , Oxirredução/efeitos da radiação , Complexo de Proteínas do Centro de Reação Fotossintética/química , Estrutura Secundária de Proteína , Quinonas/metabolismo , Rhodobacter sphaeroides/citologia , Rhodobacter sphaeroides/metabolismo , Rhodobacter sphaeroides/efeitos da radiação , Rhodospirillum rubrum/citologia , Rhodospirillum rubrum/metabolismo , Rhodospirillum rubrum/efeitos da radiação , Temperatura
15.
Photosynth Res ; 122(3): 261-73, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25022916

RESUMO

The development of photosynthetic membranes of intact cells of Rhodobacter sphaeroides was tracked by light-induced absorption spectroscopy and induction and relaxation of the bacteriochlorophyll fluorescence. Changes in membrane structure were induced by three methods: synchronization of cell growth, adjustment of different growth phases and transfer from aerobic to anaerobic conditions (greening) of the bacteria. While the production of the bacteriochlorophyll and carotenoid pigments and the activation of light harvesting and reaction center complexes showed cell-cycle independent and continuous increase with characteristic lag phases, the accumulation of phospholipids and membrane potential (electrochromism) exhibited stepwise increase controlled by cell division. Cells in the stationary phase of growth demonstrated closer packing and tighter energetic coupling of the photosynthetic units (PSU) than in their early logarithmic stage. The greening resulted in rapid (within 0-4 h) induction of BChl synthesis accompanied with a dominating role for the peripheral light harvesting system (up to LH2/LH1 ~2.5), significantly increased rate (~7·10(4) s(-1)) and yield (F v/F max ~0.7) of photochemistry and modest (~2.5-fold) decrease of the rate of electron transfer (~1.5·10(4) s(-1)). The results are discussed in frame of a model of sequential assembly of the PSU with emphasis on crowding the LH2 complexes resulting in an increase of the connectivity and yield of light capture on the one hand and increase of hindrance to diffusion of mobile redox agents on the other hand.


Assuntos
Modelos Biológicos , Fotossíntese/fisiologia , Rhodobacter sphaeroides/metabolismo , Membranas Intracelulares/metabolismo , Membranas Intracelulares/fisiologia , Complexos de Proteínas Captadores de Luz/metabolismo , Complexos de Proteínas Captadores de Luz/fisiologia , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/fisiologia , Rhodobacter sphaeroides/citologia , Rhodobacter sphaeroides/fisiologia
16.
J R Soc Interface ; 11(97): 20140320, 2014 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-24872500

RESUMO

Most free-swimming bacteria move in approximately straight lines, interspersed with random reorientation phases. A key open question concerns varying mechanisms by which reorientation occurs. We combine mathematical modelling with analysis of a large tracking dataset to study the poorly understood reorientation mechanism in the monoflagellate species Rhodobacter sphaeroides. The flagellum on this species rotates counterclockwise to propel the bacterium, periodically ceasing rotation to enable reorientation. When rotation restarts the cell body usually points in a new direction. It has been assumed that the new direction is simply the result of Brownian rotation. We consider three variants of a self-propelled particle model of bacterial motility. The first considers rotational diffusion only, corresponding to a non-chemotactic mutant strain. Two further models incorporate stochastic reorientations, describing 'run-and-tumble' motility. We derive expressions for key summary statistics and simulate each model using a stochastic computational algorithm. We also discuss the effect of cell geometry on rotational diffusion. Working with a previously published tracking dataset, we compare predictions of the models with data on individual stopping events in R. sphaeroides. This provides strong evidence that this species undergoes some form of active reorientation rather than simple reorientation by Brownian rotation.


Assuntos
Mecanotransdução Celular/fisiologia , Modelos Biológicos , Orientação/fisiologia , Rhodobacter sphaeroides/citologia , Rhodobacter sphaeroides/fisiologia , Polaridade Celular/fisiologia , Simulação por Computador , Reologia/métodos , Rotação , Estresse Mecânico , Viscosidade
17.
Biometals ; 27(1): 65-73, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24249151

RESUMO

A detailed characterization of membrane lipids of the photosynthetic bacterium Rhodobacter (R.) sphaeroides was accomplished by thin-layer chromatography coupled with matrix-assisted laser desorption ionization mass spectrometry. Such an approach allowed the identification of the main membrane lipids belonging to different classes, namely cardiolipins (CLs), phosphatidylethanolamines, phosphatidylglycerols (PGs), phosphatidylcholines, and sulfoquinovosyldiacylglycerols (SQDGs). Thus, the lipidomic profile of R. sphaeroides R26 grown in abiotic stressed conditions by exposure to bivalent cobalt cation and chromate oxyanion, was investigated. Compared to bacteria grown under control conditions, significant lipid alterations take place under both stress conditions; cobalt exposure stress results in the relative content increase of CLs and SQDGs, most likely compensating the decrease in PGs content, whereas chromate stress conditions result in the relative content decrease of both PGs and SQDGs, leaving CLs unaltered. For the first time, the response of R. sphaeroides to heavy metals as Co(2+) and CrO4 (2-) is reported and changes in membrane lipid profiles were rationalised.


Assuntos
Cromatos/farmacologia , Cobalto/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos/análise , Rhodobacter sphaeroides/efeitos dos fármacos , Rhodobacter sphaeroides/metabolismo , Íons/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Fotossíntese , Rhodobacter sphaeroides/citologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
18.
PLoS One ; 8(11): e79520, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24223961

RESUMO

Singlet oxygen ((1)O2) is the main agent of photooxidative stress and is generated by photosensitizers as (bacterio)chlorophylls. It leads to the damage of cellular macromolecules and therefore photosynthetic organisms have to mount an adaptive response to (1)O2 formation. A major player of the photooxidative stress response in Rhodobacter sphaeroides is the alternative sigma factor RpoE, which is inactivated under non-stress conditions by its cognate anti-sigma factor ChrR. By using random mutagenesis we identified RSP_1090 to be required for full activation of the RpoE response under (1)O2 stress, but not under organic peroxide stress. In this study we show that both RSP_1090 and RSP_1091 are required for full resistance towards (1)O2. Moreover, we revealed that the DegS and RseP homologs RSP_3242 and RSP_2710 contribute to (1)O2 resistance and promote ChrR proteolysis. The RpoE signaling pathway in R. sphaeroides is therefore highly similar to that of Escherichia coli, although very different anti-sigma factors control RpoE activity. Based on the acquired results, the current model for RpoE activation in response to (1)O2 exposure in R. sphaeroides was extended.


Assuntos
Proteínas de Bactérias/metabolismo , Peptídeo Hidrolases/metabolismo , Rhodobacter sphaeroides/metabolismo , Homologia de Sequência de Aminoácidos , Fator sigma/metabolismo , Oxigênio Singlete/metabolismo , Sequência Conservada , Ativação Enzimática , Mutagênese Insercional , Estresse Oxidativo , Peptídeo Hidrolases/biossíntese , Proteólise , Rhodobacter sphaeroides/citologia , Rhodobacter sphaeroides/enzimologia , Rhodobacter sphaeroides/genética , Fator sigma/genética , Transdução de Sinais
19.
BMC Syst Biol ; 7: 89, 2013 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-24034347

RESUMO

BACKGROUND: Improving our understanding of processes at the core of cellular lifestyles can be aided by combining information from genetic analyses, high-throughput experiments and computational predictions. RESULTS: We combined data and predictions derived from phenotypic, physiological, genetic and computational analyses to dissect the metabolic and energetic networks of the facultative photosynthetic bacterium Rhodobacter sphaeroides. We focused our analysis on pathways crucial to the production and recycling of pyridine nucleotides during aerobic respiratory and anaerobic photosynthetic growth in the presence of an organic electron donor. In particular, we assessed the requirement for NADH/NADPH transhydrogenase enzyme, PntAB during respiratory and photosynthetic growth. Using high-throughput phenotype microarrays (PMs), we found that PntAB is essential for photosynthetic growth in the presence of many organic electron donors, particularly those predicted to require its activity to produce NADPH. Utilizing the genome-scale metabolic model iRsp1095, we predicted alternative routes of NADPH synthesis and used gene expression analyses to show that transcripts from a subset of the corresponding genes were conditionally increased in a ΔpntAB mutant. We then used a combination of metabolic flux predictions and mutational analysis to identify flux redistribution patterns utilized in the ΔpntAB mutant to compensate for the loss of this enzyme. Data generated from metabolic and phenotypic analyses of wild type and mutant cells were used to develop iRsp1140, an expanded genome-scale metabolic reconstruction for R. sphaeroides with improved ability to analyze and predict pathways associated with photosynthesis and other metabolic processes. CONCLUSIONS: These analyses increased our understanding of key aspects of the photosynthetic lifestyle, highlighting the added importance of NADPH production under these conditions. It also led to a significant improvement in the predictive capabilities of a metabolic model for the different energetic lifestyles of a facultative organism.


Assuntos
Metabolismo Energético , Redes e Vias Metabólicas , Rhodobacter sphaeroides/metabolismo , Biologia de Sistemas , Genômica , Modelos Biológicos , NADP/metabolismo , Fotossíntese , Reprodutibilidade dos Testes , Rhodobacter sphaeroides/citologia , Rhodobacter sphaeroides/genética , Rhodobacter sphaeroides/crescimento & desenvolvimento
20.
Mol Microbiol ; 90(2): 322-37, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23944351

RESUMO

Bacterial chemotaxis depends on signalling through large protein complexes. Each cell must inherit a complex on division, suggesting some co-ordination with cell division. In Escherichia coli the membrane-spanning chemosensory complexes are polar and new static complexes form at pre-cytokinetic sites, ensuring positioning at the new pole after division and suggesting a role for the bacterial cytoskeleton. Rhodobacter sphaeroides has both membrane-associated and cytoplasmic, chromosome-associated chemosensory complexes. We followed the relative positions of the two chemosensory complexes, FtsZ and MreB in aerobic and in photoheterotrophic R. sphaeroides cells using fluorescence microscopy. FtsZ forms polar spots after cytokinesis, which redistribute to the midcell forming nodes from which FtsZ extends circumferentially to form the Z-ring. Membrane-associated chemosensory proteins form a number of dynamic unit-clusters with mature clusters containing about 1000 CheW(3) proteins. Individual clusters diffuse randomly within the membrane, accumulating at new poles after division but not colocalizing with either FtsZ or MreB. The cytoplasmic complex colocalizes with FtsZ at midcells in new-born cells. Before cytokinesis one complex moves to a daughter cell, followed by the second moving to the other cell. These data indicate that two homologous complexes use different mechanisms to ensure partitioning, and neither complex utilizes FtsZ or MreB for positioning.


Assuntos
Proteínas de Bactérias/metabolismo , Divisão Celular , Proteínas do Citoesqueleto/metabolismo , Proteínas de Membrana/metabolismo , Rhodobacter sphaeroides/fisiologia , Proteínas de Bactérias/genética , Polaridade Celular , Quimiotaxia , Citocinese , Proteínas do Citoesqueleto/genética , Genes Bacterianos , Proteínas de Membrana/genética , Microscopia de Fluorescência , Família Multigênica , Rhodobacter sphaeroides/citologia , Homologia de Sequência de Aminoácidos
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