Three novel proteins co-localise with polyhydroxybutyrate (PHB) granules in Rhodospirillum rubrum S1.

Polyhydroxybutyrate (PHB), a biodegradable polymer accumulated by bacteria is deposited intracellularly in the form of inclusion bodies often called granules. The granules are supramolecular complexes harbouring a varied number of proteins on their surface, which have specific but incompletely characterised functions. By comparison with other organisms that produce biodegradable polymers, only two phasins have been described to date for Rhodosprillum rubrum, raising the possibility that more await discovery. Using a comparative proteomics strategy to compare the granules of wild-type R. rubrum with a PHB-negative mutant housing artificial PHB granules, we identified four potential PHB granules' associated proteins. These were: Q2RSI4, an uncharacterised protein; Q2RWU9, annotated as an extracellular solute-binding protein; Q2RQL4, annotated as basic membrane lipoprotein; and Q2RQ51, annotated as glucose-6-phosphate isomerase. In silico analysis revealed that Q2RSI4 harbours a Phasin_2 family domain and shares low identity with a single-strand DNA-binding protein from Sphaerochaeta coccoides. Fluorescence microscopy found that three proteins Q2RSI4, Q2EWU9 and Q2RQL4 co-localised with PHB granules. This work adds three potential new granule associated proteins to the repertoire of factors involved in bacterial storage granule formation, and confirms that proteomics screens are an effective strategy for discovery of novel granule associated proteins.

Fluorescence relaxation in intact cells of photosynthetic bacteria: donor and acceptor side limitations of reopening of the reaction center.

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.

Modelled microgravity cultivation modulates N-acylhomoserine lactone production in Rhodospirillum rubrum S1H independently of cell density.

The photosynthetic alphaproteobacterium Rhodospirillum rubrum S1H is part of the Micro-Ecological Life Support System Alternative (MELiSSA) project that is aiming to develop a closed life support system for oxygen, water and food production to support human life in space in forthcoming long-term space exploration missions. In the present study, R. rubrum S1H was cultured in a rotating wall vessel (RWV), simulating partial microgravity conditions on Earth. The bacterium showed a significant response to cultivation in simulated microgravity at the transcriptomic, proteomic and metabolic levels. In simulated microgravity conditions three N-acyl-l-homoserine lactones (C10-HSL, C12-HSL and 3-OH-C14-HSL) were detected in concentrations that were twice those detected under normal gravity, while no differences in cell density was detected. In addition, R. rubrum cultivated in modelled microgravity showed higher pigmentation than the normal gravity control, without change in culture oxygenation. When compared to randomized microgravity cultivation using a random positioning machine, significant overlap for the top differentially expressed genes and proteins was observed. Cultivation in this new artificial environment of simulated microgravity showed new properties of this well-known bacterium, including its first, to our knowledge, complete quorum-sensing-related N-acylhomoserine lactone profile.

Near-IR absorbing solar cell sensitized with bacterial photosynthetic membranes.

Current interest in natural photosynthesis as a blueprint for solar energy conversion has led to the development of a biohybrid photovoltaic cell in which bacterial photosynthetic membrane vesicles (chromatophores) have been adsorbed to a gold electrode surface in conjunction with biological electrolytes (quinone [Q] and cytochrome c; Magis et al. [2010] Biochim. Biophys. Acta 1798, 637-645). Since light-driven current generation was dependent on an open circuit potential, we have tested whether this external potential could be replaced in an appropriately designed dye-sensitized solar cell (DSSC). Herein, we show that a DSSC system in which the organic light-harvesting dye is replaced by robust chromatophores from Rhodospirillum rubrum, together with Q and cytochrome c as electrolytes, provides band energies between consecutive interfaces that facilitate a unidirectional flow of electrons. Solar I-V testing revealed a relatively high I(sc) (short-circuit current) of 25 µA cm(-2) and the cell was capable of generating a current utilizing abundant near-IR photons (maximum at ca 880 nm) with greater than eight-fold higher energy conversion efficiency than white light. These studies represent a powerful demonstration of the photoexcitation properties of a biological system in a closed solid-state device and its successful implementation in a functioning solar cell.

Molecular assembly of artificial photosynthetic antenna core complex on an amino-terminated ITO electrode.

Bacterial photosynthetic membrane proteins, light-harvesting antenna complex (LH1), reaction center (RC), and their combined 'core' complex (LH1-RC) are functional elements in the primary photosynthetic events, i.e., capturing and transferring light energy and subsequent charge separation. These photosynthetic units (PSUs) isolated from Rhodospirillum rubrum (Rs. rubrum) were assembled onto an ITO electrode modified with 3-aminopropyltriethoxysilane (APS-ITO). The near IR absorption spectra of PSUs on the assembled electrodes were identical to those of solutions, indicating that the LH1 and LH1-RC core complexes were native on the electrode. Photocurrent response of PSUs on the electrode was examined upon illumination of the LH1 complex at 880 nm. The LH1-RC and a mixed assembly of LH1 and RC exhibited photocurrent response, but not LH1 only, consistent with the function of these PSUs, capturing light energy and transferring electron. This result provides useful methodology for building an artificial fabrication of PSUs on the electrode.

Rhodospirillum rubrum has a family I pyrophosphatase: purification, cloning, and sequencing.

The cytoplasmic pyrophosphatase of the photosynthetic bacterium Rhodospirillum rubrum was purified to electrophoretic homogeneity. The enzyme is a homohexamer of 20-kDa monomers. The gene was cloned and sequenced. Alignment of the deduced 179-amino-acid protein with known bacterial pyrophosphatases revealed conservation of all residues in the active site. Attempts to obtain an insertion mutant of the cytoplasmic pyrophosphatase gene did not yield any cell completely devoid of cytoplasmic pyrophosphatase activity. The mutants obtained showed 50% of the enzymatic activity and grew in twice the generation time of wild-type cells. This suggests that the membrane-bound pyrophosphatase of Rsp. rubrum is not sufficient for a normal growth rate, whereas the cytoplasmic enzyme is essential for growth. The characteristics of the gene and the encoded protein fit those of prokaryotic family I pyrophosphatases.

[Effect of serotonin (5-hydroxytryptamine) on the growth and differentiation of microorganisms]. / Deistvie serotonina (5-oksitriptamina) na rost i differentsiatsiiu mikroorganizmov.

Serotonin (5-hydroxytryptamine), a neurotransmitter and social behavior factor in higher animals, accelerates culture growth and induces cell aggregation in Escherichia coli and Rhodospirillum rubrum at concentrations of 2 x 10(-7)-2 x 10(-5)M. In the myxobacterium Polyangium sp., 10(-6)-10(-5)M serotonin stimulates cell aggregation and myxospore formation. At concentrations over 20 microM, serotonin induces the opposite effect: it inhibits cell aggregation and microbial culture growth. Serotonin at these concentrations also inhibits the light-dependent membrane potential generation in Rsp. rubrum (the data were obtained by the method of penetrating ions). Therefore, the above effects can be due to the elimination of the transmembrane electrical gradient by serotonin. As for micromolar serotonin concentrations, their effects presumably result from the specific action of serotonin as an intercellular communication agent accelerating and possibly synchronizing the development of the cell population.

Cell-cycle-specific fluctuation in cytoplasmic membrane composition in aerobically grown Rhodospirillum rubrum.

Aerobic growth with synchronous cell division was induced in Rhodospirillum rubrum by starvation methods. Cells were harvested at different points in the cell cycle. Analysis of the composition of the cell envelope prepared by differential centrifugation or density gradient-purified cytoplasmic membrane obtained from cells at different times indicated that the protein/phospholipid ratio fluctuated with the cell cycle. The protein/phospholipid ratio of cell envelope from selection-synchronized cells also fluctuated with the cell cycle. These studies indicate that the phenomenon of cell-cycle-dependent fluctuation in membrane composition is not restricted to the intracytoplasmic chromatophore membrane of phototrophic cells.

In vivo interaction between nitrogenase molybdenum-iron protein and membrane in Azotobacter vinelandii and Rhodospirillum rubrum.

Oriented whole cell multilayers of Azotobacter vinelandii and Rhodospirillum rubrum were analyzed by electron spin resonance (ESR) spectroscopy to detect possible structural associations between nitrogenase molybdenum-iron (MoFe) protein and cytoplasmic or intracytoplasmic membrane. Initially, protocols were designed to obtain strong molybdenum-iron protein ESR signals in whole cell samples of each organism. Then, two-dimensional orientation of whole cell membranes was demonstrated in whole cell multilayers using doxyl stearate spin label in A. vinelandii and the bacteriochlorophyll a dimer triplet signal, (BCHl a)T2, from the intracytoplasmic membrane-bound photosynthetic apparatus of R. rubrum. Subsequent analysis of the low-field signals, g = 4.3 and g = 3.6, of molybdenum-iron protein in whole cell multilayers of each organism showed orientation-dependent characteristics, although the properties of each were different. Specifically, as the normal to the membrane plane was rotated from perpendicular to parallel with the ESR magnetic field, the amplitude of the g = 3.6 signal decreased from maximum to about 37% of maximum in A. vinelandii and from maximum to about 88% of maximum in R. rubrum. The angular dependence of the g = 4.3 peak during rotation varied in A. vinelandii, but decreased from maximum to about 63% of maximum in R. rubrum. These data suggest that the molybdenum-iron protein of nitrogenase was oriented in response to the physical orientation of cellular membranes and that a structural association may exist between this nitrogenase component and membrane in these organisms.

Measurement by a flow dialysis technique of the steady-state proton-motive force in chromatophores from Rhodospirillum rubrum. Comparison with phosphorylation potential.

1. In the light a transmembrane electrical potential of 100 mV has been estimated to occur in chromatophores from Rhodospirillum rubrum. The potential was determined by measuring the steady-state distribution of the permeant SCN- across the chromatophore membrane using a flow dialysis technique. The potential was not observed in the dark, nor in the presence of antimycin. It was dissipated on the addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The potential was reduced by between 15 and 20 mV when ADP and Pi were added. Hydrolysis of ATP by the chromatophores generated a membrane potential of about 80 mV. 2. Using a flow dialysis technique light-dependent uptake of methylamine was observed only in the presence of concentrations of SCN- that were 500-fold higher than were used to measure the membrane potential. It is concluded that the pH gradient across the illuminated chromatophore membrane is insignificant except in the presence of relatively high concentrations of a permeant anion like thiocyanate. Further evidence that a negligible pH gradient was generated by the chromatophores is that addition of K+ and nigericin to illuminated chromatophores did not stimulate uptake of SCN-. 3. In the light of chromatophores established and maintained a phosphorylation potential of up to 14 kcal/mol. If a phosphorylation potential of this magnitude is to be poised against a proton-motive force that comprises solely a membrane potential of approx. 100 mV, then at least five protons must be translocated for each ATP synthesised via a chemiosmotic mechanism.

Phototactic response of aerobically cultivated Rhodospirillum rubrum.

Motile cells of aerobically cultivated Rhodospirillum rubrum, containing no detectable bacteriochlorophyll, assembled at a spot of strong light projected through a dark-field condenser. Far-red light was not effective, indicating that bacteriochlorophyll and thus photosynthetic metabolism was not responsible for the phenomenon. Bacteria moving towards the centre of the light spot changed direction less frequently than those moving towards the margin. They also responded to temporal changes in the intensity of light, altering their swimming direction more frequently after a sudden decrease in light intensity than after an abrupt increase.

Motility in normal and filamentous forms of Rhodospirillum rubrum.

By suitable choice of medium, Rhodospirillum rubrum has been grown both in normal (length 2 mum) and filamentous (length up to 60 mum) forms. Both forms were highly motile, and negatively-stained preparations showed bipolar flagellated cells, with an average of seven flagella at each pole. Motion consisted of a series of runs and tumbles, the ditribution of run time-lengths being Poissonian. Both forms tumbled in response to dark shock and showed negative chemotaxis to oxygen. The observation that the motility pattern was very similar in normal and filamentous forms makes chemical control of tumbling unlikely and favours a system involving membrane potentials.

Characterization of two cell-envelope fractions from chemotrophically grown Rhodospirillum rubrum.

Two cell-envelope fractions were isolated from chemotrophically grown cells of Rhodospirillum rubrum. On the basis of electron-microscopic investigations, chemical analysis, distribution of components involved in respiration, and poly-acrylamide gel electrophoresis, the heavy fraction (rho20 = 1.246 g per cm3) was identified as cell-wall, and the light fraction (rho = 1.145 g per cm3) as cyto-plasmic-membrane fragments. Electron micrographs showed cell-wall fragments as open structures while cytoplasmic-membrane preparations were composed of closed membrane vesicles. With respect to the main classes of chemical compounds, cell wall could be distinguished from cytoplasmic membranes by a rather low ratio of phospholipids per protein and a high ratio of carbohydrates per protein. The relative proportion of individual neutral sugars as well as phospholipids (except for lysophosphatidyl ethanolamine) revealed no significant differences between both envelope fractions. Fatty acid analysis demonstrated a higher proportion of saturated fatty acids in cell-wall than in cyto-plasmic-membrane fractions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the fractions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the fractions showed distinct protein compositions. While in cell-wall preparations polypeptides of 43,000 and 14,000 daltons predominated, 56,000- and 52,000-dalton polypeptides were the main protein subunits of cytoplasmic membranes. Cross contaminations of both cell-envelope fractions were defined.