Dietary supplementation with probiotic Rhodobacter sphaeroides SS15 extract to control acute hepatopancreatic necrosis disease (AHPND)-causing vibrio parahaemolyticus in cultivated white shrimp.

Cultivation of Penaeus vannamei (Pacific white shrimp) is faced with the serious problem of acute hepatopancreatic necrosis disease (AHPND), caused by Vibrio parahaemolyticus that carries plasmids containing binary toxin genes. The disease is typically moderated by the use of antibiotics. To investigate the control of AHPND and maintenance of water quality without the use of antibiotics, the supplementation of shrimp feed with anti-vibrio compounds from a crude extract of probiotic Rhodobacter sphaeroides SS15 was evaluated. The experimental design comprised four treatments: two that were challenged with AHPND-causing V. parahaemolyticus SR2 at a density of 6.0 × 105 cells mL-1 and two that were not challenged. The unchallenged groups comprised a control group that received commercial feed only (CF) and a group that received CF supplemented with 0.27% (w/w) of the extract of R. sphaeroides SS15 (modified CF: MCF). The treatments challenged with V. parahaemolyticus SR2 comprised a challenge group that received CF only (challenge CF: CF-SR2) and a challenge group that received modified CF (challenge MCF: MCF-SR2). V. parahaemolyticus SR2 was inoculated at the start of cultivation and at day 48 at the same cell density. No significant difference in growth performance was found among all treatments. All water quality parameters were better in the two treatments that received modified CF but excess nitrite, due to overfeeding in low salinity (5-8 ppt), caused shrimp mortality in all treatments. Vibrio populations were much higher in the CF treatments than in the modified CF treatments. After the first challenge, the survival rate was about 67% in both the CF-SR2 and MCF-SR2 treatments, compared with approximately 83% in the unchallenged treatments. One day after the second challenge, mortality in the CF-SR2 treatment was 100%, whereas 16.67% survived in the MCF-SR2 treatment. The survival rate was roughly 27% higher in the MCF treatment than in the CF treatment. The hepatopancreas and gut of both modified CF treatments showed no sign of AHPND. Via better water quality and trained immunity, the anti-vibrio compounds in the modified CF have great potential to increase the survival of cultivated shrimp infected with AHPND-causing strain SR2.

Polychromatic solar energy conversion in pigment-protein chimeras that unite the two kingdoms of (bacterio)chlorophyll-based photosynthesis.

Natural photosynthesis can be divided between the chlorophyll-containing plants, algae and cyanobacteria that make up the oxygenic phototrophs and a diversity of bacteriochlorophyll-containing bacteria that make up the anoxygenic phototrophs. Photosynthetic light harvesting and reaction centre proteins from both kingdoms have been exploited for solar energy conversion, solar fuel synthesis and sensing technologies, but the energy harvesting abilities of these devices are limited by each protein's individual palette of pigments. In this work we demonstrate a range of genetically-encoded, self-assembling photosystems in which recombinant plant light harvesting complexes are covalently locked with reaction centres from a purple photosynthetic bacterium, producing macromolecular chimeras that display mechanisms of polychromatic solar energy harvesting and conversion. Our findings illustrate the power of a synthetic biology approach in which bottom-up construction of photosystems using naturally diverse but mechanistically complementary components can be achieved in a predictable fashion through the encoding of adaptable, plug-and-play covalent interfaces.

Electron-Transfer Secondary Reaction Matrices for MALDI MS Analysis of Bacteriochlorophyll a in Rhodobacter sphaeroides and Its Zinc and Copper Analogue Pigments.

Bacteriochlorophyll a (BChl a), a photosynthetic pigment performing the same functions of chlorophylls in plants, features a bacteriochlorin macrocycle ring (18 π electrons) with two reduced pyrrole rings along with a hydrophobic terpenoid side chain (i.e., the phytol residue). Chlorophylls analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is not so straightforward since pheophytinization (i.e., release of the central metal ion) and cleavage of the phytol-ester linkage are invariably observed by employing protonating matrices such as 2,5-dihydroxybenzoic acid, sinapinic acid, and α-cyano-4-hydroxycinnamic acid. Using BChl a from Rhodobacter sphaeroides R26 strain as a model system, different electron-transfer (ET) secondary reaction matrices, leading to the formation of almost stable radical ions in both positive ([M]+•) and negative ([M]-•) ionization modes at m/z 910.55, were evaluated. Compared with ET matrices such as trans-2-[3-(4-t-butyl-phenyl)-2-methyl-2-propenylidene]malononitrile (DCTB), 2,2':5',2''-terthiophene (TER), anthracene (ANT), and 9,10-diphenylanthracene (DP-ANT), 1,5-diaminonaphthalene (DAN) was found to provide the highest ionization yield with a negligible fragmentation. DAN also displayed excellent ionization properties for two metal ion-substituted bacteriochlorophylls, (i.e., Zn- and Cu-BChl a at m/z 950.49 and 949.49), respectively. MALDI MS/MS of both radical charged molecular species provide complementary information, thus making analyte identification more straightforward. Graphical Abstract ᅟ.

Occurrence of oleic and 18:1 methyl-branched acyl chains in lipids of Rhodobacter sphaeroides 2.4.1.

The fatty acids (FAs) composition of lipids extracted from Rhodobacter sphaeroides 2.4.1 was investigated by gas chromatography-mass spectrometry (GC-MS) analysis of the corresponding FA methyl esters (FAMEs), obtained through trans-esterification of the original lipid species. A GC stationary phase based on a highly polar ionic liquid (IL) was selected, aimed to enhance the separation of isomeric FAMEs with particular emphasis on positional and geometrical isomers of monounsaturated 16:1 and 18:1 fatty acyl chains. The occurrence of 18:1 cis-Δ(9) (oleic) acid, a positional isomer of the well-known and most predominant 18:1 cis-Δ(11) (cis-vaccenic) acid, has been demonstrated here for the first time. Furthermore a methyl branched 18:1 FA was also identified and its structure tentatively assigned as 11-methyl-Δ(12)-octadecenoic acid (most likely as trans isomer). The unprecedented observation about 18:1 cis-Δ(9) FA occurrence in R. sphaeroides 2.4.1 is, even indirectly, supported by a biosynthetic pathway postulated with the aid of the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The concurrent presence of 16:1 cis-Δ(7) and 18:1 cis-Δ(9) FAs suggested the existence of parallel and/or complementary processes to those invoked for the formation of most common 16:1 cis-Δ(9) and 18:1 cis-Δ(11) FAs. A further route was hypothesized for the trans FAs biosynthesis in wild-type cells of R. sphaeroides.

Extension of the broadband single-mode integrated optical waveguide technique to the ultraviolet spectral region and its applications.

We report here the fabrication, characterization, and application of a single-mode integrated optical waveguide (IOW) spectrometer capable of acquiring optical absorbance spectra of surface-immobilized molecules in the visible and ultraviolet spectral region down to 315 nm. The UV-extension of the single-mode IOW technique to shorter wavelengths was made possible by our development of a low-loss single-mode dielectric waveguide in the UV region based on an alumina film grown by atomic layer deposition (ALD) over a high quality fused silica substrate, and by our design/fabrication of a broadband waveguide coupler formed by an integrated diffraction grating combined with a highly anamorphic optical beam of large numerical aperture. As an application of the developed technology, we report here the surface adsorption process of bacteriochlorophyll a on different interfaces using its Soret absorption band centred at 370 nm. The effects of different chemical compositions at the solid-liquid interface on the adsorption and spectral properties of bacteriochlorophyll a were determined from the polarized UV-Vis IOW spectra acquired with the developed instrumentation. The spectral extension of the single-mode IOW technique into the ultraviolet region is an important advance as it enables extremely sensitive studies in key characteristics of surface molecular processes (e.g., protein unfolding and solvation of aromatic amino-acid groups under surface binding) whose spectral features are mainly located at wavelengths below the visible spectrum.

Practical removal of radioactivity from soil in Fukushima using immobilized photosynthetic bacteria combined with anaerobic digestion and lactic acid fermentation as pre-treatment.

Practical removal of radioactivity from polluted soil in Fukushima, Japan was done using a photosynthetic bacterium, Rhodobacter sphaeroides SSI, immobilized in alginate beads. The beads were put in a mesh bag and soaked in which soil was suspended (5 kg of soil/10 L of tap water). The radioactivity of the broth decreased by 31% after 15 d of aerobic treatment. When lactic acid bacterial culture broth was added to the suspend broth, about 50% of the radioactivity was transferred to a suspend broth fraction consisting of small particles from the soil after 3 d of fermentation and 20 s of sedimentation. The results suggest that organic matter in the soil was decomposed by anaerobic digestion and lactic acid fermentation simultaneously, and was then transferred into the liquid as small particles. With combined treatment by anaerobic digestion and lactic acid fermentation for 5 d and immobilized bead aerobic treatment for an additional 19 d, the radioactivity of suspend broth decreased by 66%. The radioactivity of the original soil (10.56 µSv/h) ultimately decreased by 67% (3.52 µSv/h) after the combined treatment.

Primary radical pair P(+)H(-) lifetime in rhodobacter sphaeroides with blocked electron transfer to Q(A). Effect of o-phenanthroline.

Transient absorption spectroscopy with a time resolution of approximately 1 ns was applied to study the decay of the primary radical pair P+H- in Rhodobacter sphaeroides R-26 reaction centers with blocked electron transfer from H- to QA. The block in the electron transfer was realized in two ways: by either reducing or removing QA. We found very different kinetics of the P+H- decay in these two cases. Convolution of the multiexponential decay with the instrument response function allowed resolution of as many as three kinetic components of <1-, 3-4-, and 9-12-ns lifetimes in chromatophores with QA reduced and in isolated reaction centers both with QA either reduced or removed (with or without o-phenanthroline) but with variable relative amplitudes. Removing QA or adding o-phenanthroline to isolated reaction centers increased the amplitude of the slowest decay phase relative to that of the fastest phase. On the basis of these observations, we propose that reaction centers adopt three conformational states characterized by different decay kinetics of P+H-. These conformational states appear to be controlled by the charges in the vicinity of the QA site as revealed by the effects of QA reduction and o-phenanthroline-mediated protonation of the sites close to QA.

High-field EPR spectroscopy applied to biological systems: characterization of molecular switches for electron and ion transfer.

The last decade witnessed a tremendous growth in combined efforts of biologists, chemists and physicists to understand the dominant factors determining the specificity and directionality of transmembrane transfer processes in proteins. A large variety of experimental techniques is being used including X-ray and neutron diffraction, but also time-resolved optical, infrared and magnetic resonance spectroscopy. This is done in conjunction with genetic engineering strategies to construct site-specific mutants for controlled modification of the proteins. As a general perception of these efforts, the substantial influence of weak interactions within the protein and its membrane interfaces is recognized. The weak interactions are subject to subtle changes during the reaction cycle owing to the inherent flexibility of the protein-membrane complex. Specific conformational changes accomplish molecular-switch functions for the transfer process to proceed with optimum efficiency. Characteristic examples of time varying non-bonded interactions are specific H-patterns and/or polarity effects of the microenvironment. The present perception has emerged from the coupling of newly developed spectroscopic techniques - and advanced EPR certainly deserves credit in this respect - with newly developed computational strategies to interpret the experimental data in terms of protein structure and dynamics. By now, the partners of this coupling, particularly high-field EPR spectroscopy and DFT-based quantum theory, have reached a level of sophistication that applications to large biocomplexes are within reach. In this review, a few large paradigm biosystems are surveyed which were explored lately in our laboratory. Taking advantage of the improved spectral and temporal resolution of high-frequency/high-field EPR at 95 GHz/3.4 T and 360 GHz/12.9 T, as compared to conventional X-band EPR (9.5 GHz/0.34 T), three biosystems are characterized with respect to structure and dynamics: (1) Light-induced electron-transfer intermediates in wild-type and mutant reaction-centre proteins from the photosynthetic bacterium Rhodobacter sphaeroides, (2) light-driven proton-transfer intermediates of site-specifically nitroxide spin-labelled mutants of bacteriorhodopsin proteins from Halobacterium salinarium, (3) refolding intermediates of site-specifically nitroxide spin-labelled mutants of the channel-forming protein domain of Colicin A bacterial toxin produced in Escherichia coli. The detailed information obtained is complementary to that of protein crystallography, solid-state NMR, infrared and optical spectroscopy techniques. A unique strength of high-field EPR is particularly noteworthy: it can provide highly desired detailed information on transient intermediates of proteins in biological action. They can be observed and characterized while staying in their working states on biologically relevant time scales. The review introduces the audience to origins and basic experiments of EPR in relation to NMR, describes the underlying strategies for extending conventional EPR to high-field/high-frequency EPR, and highlights those details of molecular information that are obtained from high-field EPR in conjunction with genetic engineering and that are not accessible by "classical" spectroscopy. The importance of quantum-chemical interpretation of the experimental data by DFT and advanced semiempirical molecular-orbital theory is emphasized. A short description of the laboratory-built 95 GHz and 360 GHz EPR/ENDOR spectrometers at FU Berlin is also presented. The review concludes with an outlook to future opportunities and challenges of advanced bio-EPR in interdisciplinary research.

Synthetic analogues of the histidine-chlorophyll complex: a NMR study to mimic structural features of the photosynthetic reaction center and the light-harvesting complex.

Mg(II)-porphyrin-ligand and (bacterio)chlorophyl-ligand coordination interactions have been studied by solution and solid-state MAS NMR spectroscopy. (1)H, (13)C and (15)N coordination shifts due to ring currents, electronic perturbations and structural effects are resolved for imidazole (Im) and 1-methylimidazole (1-MeIm) coordinated axially to Mg(II)-OEP and (B)Chl a. As a consequence of a single axial coordination of Im or 1-MeIm to the Mg(II) ion, 0.9-5.2 ppm (1)H, 0.2-5.5 ppm (13)C and 2.1-27.2 ppm (15)N coordination shifts were measured for selectively labeled [1,3-(15)N]-Im, [1,3-(15)N,2-(13)C]-Im and [1,3-(15)N,1,2-(13)C]-1-MeIm. The coordination shifts depend on the distance of the nuclei to the porphyrin plane and the perturbation of the electronic structure. The signal intensities in the (1)H NMR spectrum reveal a five-coordinated complex, and the isotropic chemical shift analysis shows a close analogy with the electronic structure of the BChl a-histidine in natural light harvesting 2 complexes. The line broadening of the ligand responses support the complementary IR data and provide evidence for a dynamic coordination bond in the complex.