Spirilloxanthin is released by detergent from Rubrivivax gelatinosus reaction center as an aggregate with unusual spectral properties

A preparation containing spirilloxanthin has been isolated from Rubrivivax gelatinosus SC2, a mutant devoid of the reaction center-associated tetraheme cytochrome c, after solubilisation of membranes with lauryl-di-methyl-amine oxide. It was purified by ammonium sulfate precipitation and gel filtration, and analyzed by SDS-gel electrophoresis. Spirilloxanthin was shown to be aggregated in large particles (apparent M_W > 600 kDa) and was not associated with a specific protein. This aggregate was characterized by absorption, circular dichroism and resonance Raman spectroscopies. The absorption spectrum contained two UV bands at 370 and 300 nm, and did not present the visible bands of spirilloxanthin, which however reappeared when spirilloxanthin was extracted from the aggregate with organic solvents. Resonance Raman spectra indicated that at least four different populations of spirilloxanthin were present in the preparation as a mixture of different trans and cis configurations. These properties are similar to those described for a so-called carotenoprotein solubilized with sodium dodecyl sulfate from Rhodospirillum rubrum membranes [Schwenker et al. (1974) Biochim Biophys Acta 351: 246-260; Kito et al. (1983) Photochem Photobiophys 5: 209-217]. We further observed absorption spectra of pure spirilloxanthin dissolved in mixtures of water, polar solvents and detergent, in the absence of protein, resembling those of the.aggregate. We conclude that the aggregate is not a carotenoprotein, but rather an artefact due to the release of spirilloxanthin from the reaction center, leading to the isomerization and association of spirilloxanthin molecules in a detergent particle. We propose the same interpretation for the complex isolated from Rhodospirillum rubrum.     

High resolution crystal structure of Rubrivivax gelatinosus cytochrome c'

The structure of the cytochrome c′ from the purple non-sulfur phototrophic bacterium Rubrivivax gelatinosus was determined using two crystals grown independently at pH 6.3 and pH 8. The resolution attained for the two structures (1.29 Å and 1.50 Å for the crystals at high and low pH, respectively) is the highest to date for this class of proteins. The two structures were compared in detail in an attempt to investigate the influence of pH on the geometry of the haem and of the coordination environment of the Fe(III) ion. However, while the results suggest some small propensity for the movement of the metal atom out of the plane of the haem ring upon pH increase, the accuracy of the measurements at these two pH below the pK of the axial histidine is not sufficient to provide hard evidence of a shift in the iron position and associated changes.     

Responses of Neurons in the Rat Nucleus Submedius to Noxious and Innocuous Mechanical Cutaneous Stimulation

Extracellular recordings were used to characterize responses to cutaneous mechanical stimulation of 78 neurons in the rat nucleus submedius (SM). Thirty-nine of these units were activated by some type of cutaneous mechanical stimulation. Eighteen cells were activated exclusively by noxious stimuli. In 13 of these cells, responses were of swift onset and relatively rapid termination following stimulus application. In contrast, in three neurons responses were delayed both in onset and termination, and in two the response was immediate, but the markedly increased evoked activity outlasted stimulus application by 13 min. Receptive fields (RFs) of these nociceptive neurons were generally large, although none were bilateral. Four SM neurons were activated by innocuous stimuli, but their maximal response was obtained only after noxious stimulation. Responses of all of these neurons were of immediate onset and recovery, and their RFs were large (two were bilateral). Twelve SM neurons were activated maximally by innocuous stimuli. Responses of seven of these cells were immediate in onset and termination, while that of three were delayed in both onset and termination. Two of the 12 innocuous-only neurons quickly became unresponsive to repeated stimulus applications, and could be reactivated only after a rest period during which no stimuli were applied. RFs of these units were also generally large, and in three cases were bilateral. Five SM neurons responded by decreasing, or completely ceasing, their firing subsequent to noxious-only (n = 2), or innocuous-only (n = 3) stimulation. Four of these units had large RFs (two were bilateral). The remaining 39 SM neurons could not be activated by any type of mechanical cutaneous stimulation we tried.

Electrical stimulation of the ventrolateral orbital cortex (VLO) was employed to examine frontal cortical projections of 21 SM neurons. Ten of these units were activated, although all of them synaptically rather than antidromically, and two were inhibited. There was no clear-cut relationship between neuronal location, physiological type, RF site, or VLO stimulation effects among the 39 SM neurons.

These results provide further support for the involvement of SM neurons in nociceptive information signaling, and suggest additionally that the role of the nucleus is not limited to nociception but encompasses a wider range of cutaneous sensations.     

Purification and Characterization of Alkaline Serine Proteinase from Photosynthetic Bacterium,Rubrivivax gelatinosus KDDS1

In order to reduce the protein content of wastewater, photosynthetic bacteria producing proteinases were screened from wastewater of various sources and stocked in culture. An isolated strain, KDDS1, was identified as Rubrivivax gelatinosus, a purple nonsulfur bacterium that secretes proteinase under micro-aerobic conditions under light at 35°C. Molecular weight of the purified enzyme was estimated to be 32.5 kDa. The enzyme showed the highest activity at 45°C and pH 9.6, and the activity was completely inhibited by phenylmethyl sulfonyl fluoride (PMSF), but not by EDTA. The amino-terminal 24 amino acid sequence of the enzyme showed about 50% identity to those of serine proteinases from Pseudoalteromonas piscicida strain O-7 and Burkholderia pseudomallei. Thus, the enzyme from Rvi. gelatinosus KDDS1 was thought to be a serine-type proteinase. This was the first serine proteinase characterized from photosynthetic bacteria.     

Crystal structure of cytochrome c' from Rhodocyclus gelatinosus and comparison with other cytochromes c'

Cytochromes c' are heme proteins found in photosynthetic and denitrifying bacteria, where they are presumably involved in electron transport. The cytochrome c' isolated from the bacterium Rhodocyclus gelatinosus (RGCP) forms a homodimer with each polypeptide containing 129 residues. It has been crystallised in ammonium sulfate at pH?6. Crystals belong to space group P3₁21 with cell parameters a?=?70.2?Å and c?=?126.8?Å, which corresponds to a dimer in the asymmetric unit (VM?=?3.5?ų?/?Da). The crystal structure of RGCP was solved by the molecular replacement method and refined using data to 2.5-Å resolution. The final crystallographic R factor was 17.9% for all reflections (above 2?σ) in the resolution range 27.4 to 2.5?Å. The refined model includes 1876 non-hydrogen protein atoms and 56 water molecules. As typical of c–type cytochromes, the heme group is covalently bound to Cys-X-Y-Cys-His through thio-ether bonds, and His123 occupies the fifth axial coordination position. On the vacant "distal" site, Phe16 blocks the direct access to the sixth coordination site, which is in a predominantly hydrophobic environment. In spite of the low sequence homology among cytochromes c' the overall fold is similar. The monomer structure consists of 4 anti-parallel α-helices and has random coils in the loops between the helices, and at the N- and C-termini. The subunits cross each other to form an X shape.     

Phosphorylation and nucleotidylation of proteins in Rhodocyclus gelatinosus

Abstract Cells of Rhodocyclus gelatinosus were radioactively labeled by addition of [³²P]orthophosphate, [¹⁴C]inosine or [¹⁴C]orotic acid during anaerobic growth on citrate in the light. Protein analysis by two-dimensional gel electrophoresis and autoradiography of the gels revealed the presence of several radioactively labeled protein species in this organism. The molecular mass and the isoelectric point of all these proteins were determined. Treatment of the ³²P-labeled protein fractions with acid and alkaline phosphatase clearly showed that at least 8 protein species were modified by phosphorylation. The experiments conducted with the ¹⁴C-labeled precursors of purines and pyrimidines indicated the presence of 4 protein species which were modified by a compound containing a purine and phosphate, and a single protein simultaneously being labeled with pyrimidine and phosphate.     

New records of Aphyonidae (Teleostei: Ophidiiformes) from the south-western Atlantic

During austral summer and winter of 2003, three specimens of Aphyonus gelatinosus and one specimen of Barathronus bicolor (Ophidiiformes: Aphyonidae) were trawled off Rio de Janeiro, Brazil.     

Regulation of anaerobic carbon monoxide oxidation activity in Rhodocyclus gelatinosus

Rhodocyclus gelatinosus grows anaerobically at the expense of carbon monoxide (CO). The CO-oxidation system was substrate-induced and in CO/light, cells grew at an exponential rate with ever increasing amounts of CO:MV oxidoreductase activity (the measure of CO oxidation). Once strain 1 reached a high cell density, the concentration of CO became limiting and gas oxidation activity suddenly decreased. Cell growth continued unaffected. To help explain this, it appeared that strain 1 variably used both CO oxidation and photometabolism to support growth in CO/light. Light intensity determined the upper limit of amounts of CO:MV oxidoreductase in a culture, while intermediate amounts were regulated by CO concentration. Thus, in darkness, cells produced the maximum CO oxidation activity, whereas in growth-saturating light, the minimum limit occurred. The lower the levels of CO:MV oxidoreductase in cells, the greater the content of bacteriochlorophyll. In this manner, strain 1 grew with a generation time of 6.7 independent of light intensity.     

Phototrophic growth on n-fatty acids by species of the genus Rhodocyclus sensu Imhoff et al.

Abstract Type strains of Rhodocyclus purpureus, R. gelatinosus and R. tenuis along with three local isolates of R. gelatinosus were tested for growth in the light on n -fatty acids ranging in chain length from C₅ (valerate) to C₂₂ (docosanoate).
R. purpureus , the type species of the genus, was anomalous in its limited ability to grow on n -fatty acids; no fatty acids of chain length greater than C₉ (nonanoate) were utilized. R. gelatinosus and R. tenuis , on the other hand, utilized all fatty acids in the range C₅ to C₁₈ inclusive. R. gelatinosus showed some restricted ability to use C₂₀ (eicosanoate) and C₂₂ (docosanoate) fatty acids.