Photosynthetic bacteria isolation

Wang S, Lin S, Lin X, Woodbury NW and Allen IP (1994) Comparative study of reaction centers from purple photosynthetic bacteria Isolation and optical spectroscopy. Photosynth Res 42 203-215... 

Unlike the photosynthetic apparatus of photosynthetic bacteria, that of cyanobacteria consits of two photosystems, PS I and II, connected by an electron transport chain. The only chlorophyll present is chlorophyll a, and, therefore, chlorophylls b—d are not of interest in this article. Chlorophyll a is the principal constituent of PS I. Twenty per cent of isolated pigment-protein complexes contain one P700 per 20—30 chlorophyll a molecules the other 80% contain only chlorophyll a20). The physical and chemical properties of chlorophyll a and its role in photosynthesis have recently been described by Meeks77), Mauzerall75), Hoch60), Butler10), and other authors of the Encyclopedia of Plant Physiology NS Vol. 5. 

DOM is derived from autochthonous sources such as phytoplankton and photosynthetic bacteria (16) at Big Soda Lake near Fallon, Nevada. This lake is alkaline (pH 9.7) and chemically stratified. It contains DOC concentrations as high as 60 mg/L and dissolved salt concentrations as high as 88,000 mg/ L (17). The DOM in this lake is colorless. The fulvic acid fraction was isolated by adsorption chromatography (Amberlite XAD-8 resin) (18) and by zeo-trophic distillation of water from N,N-dimethylformamide (19). Average molecular model synthesis was achieved in a manner similar to that used for fulvic acid from the Suwannee River. The characterization data are presented in Table I and the structural model is presented in Structure 2. 

The 14C age determination of the fulvic acid isolated rom water near the lake surface was 2300 years before the present, whereas the 14C age was 4900 years before the present for the fulvic acid isolated from water below the chemocline. These old ages for both fulvic acids from Big Soda Lake are in marked contrast to that reported for fulvic acid from the Suwannee River, less than 30 years before the present (11). The refractory nature of this type of fulvic acid derived from phytoplankton and photosynthetic bacteria is significant for carbon-cycling studies. 

Everybody knows of the spectacular success of unravelling the structure and kinetics of the photosynthetic bacteria, rhodopseudomonas sphaeroides and viridis the structure by Deisenhoffer, Huber and Michel (Deisenhofer et al., 1984) following the isolation and crystallisation by Michel (Michel, 1982) and the picosecond kinetics (which came first) by Rockley, Windsor, Cogdell and Parson (Rockley et al., 1975) and also by Dutton, Rentzepis, Netzel et al. (Netzel et al., 1977). 

The intermediate electron transfer between the pool of quinones accepting electrons from the RC, and the water soluble proteins donating electrons to the RC (bacterial RC and the PSI-RC) is always promoted, at least in the systems studied so far in detail, by a multiprotein complex containing cytochromes and Fe-S proteins, the so called h/ci complex. The universal presence of this type of complex in many redox chains of respiration and photosynthesis has been recognized only very recently [109]. As far as photosynthesis is concerned, complexes of this kind have been characterized in facultative photosynthetic bacteria [110] in cyanobacteria [111], and in higher plant chloroplasts [112]. All these preparations share common characteristics and composition these properties are also very similar to those of analogous complexes isolated from mitochondria of mammals and fungi [109]. 

In purple photosynthetic bacteria, and specifically in Rps. sphaeroides and Rps. capsulata, three cytochromes of b type have been identified by means of redox titration, in the dark, of isolated chromatophores [116]. They are characterized by midpoint potentials at pH = 7.0 equal to 0.155, 0.050 and -0.090 V (in Rps. sphaeroides)-, the of the 0.050 V species is pH dependent ( — 60 mV per pH unit) [116,117]. The presence of a cytochrome cc in these organisms, interfering spectrally with cytochrome b, makes the situation unclear as far as the existence of cyt. b E j = 0.155 V) is concerned [118]. The two other cytochromes E = 0.050 and — 0.090 V) have also been resolved kinetically in studies on the photosynthetic electron transport and on the basis of their spectral characteristics (band at 561 nm and a spht bands at 558 and 556 nm, respectively these two cytochromes will be referred to as 6-561 and 6-566 in the following) [119]. 

A second bound form of cytochrome c is an integral part of the oxidoreductase complexes. Cytochrome c, present in photosynthetic bacteria has been distinguished from cyt. C2 (the soluble electron carrier) both thermodynamically and kinetically [121,122]. It is present in the isolated oxidoreductase with a stoicheiometry of one per two cytochromes of b type, and it is associated with the 34000 Da subunit. According to kinetic evidence this cytochrome acts as immediate electron donor to cyt. C2 and electron acceptor from the high potential Fe-S protein [122]. The midpoint potential of cyt. c, is 0.285 V at pH 7 [121,122]. 

Several observations made in whole membranes or in the isolated complexes are in line with these concepts the shifts induced by antimycin A [110,137] and myxothiazol on the absorption spectra of cytochromes b and the alterations of the ESR spectrum of the FeS protein by UHDBT or DBMIB [131]. Moreover, the oxidant-induced reduction of cytochromes b, the key observation for accepting these electron transfer schemes, has been demonstrated in all h/c, complexes isolated so far from mitochondria [134], chloroplasts [111], cyanobacteria [112] and photosynthetic bacteria [110]. In the chloroplast b /f complex this reaction has been demonstrated also in the absence of any exogenously added quinol, indicating that possibly a structurally bound quinone (quinone is always present in the isolated complexes with a stoicheiometry of about 0.5-0.7 mol/mol of cyt. c, [110,111]) is sufficient to drive the reduction of cytochromes [138]. Since a detailed treatment of the genera] mechanism, as well as of the more specific problems of the mitochondrial respiratory chain, are reported in Chapter 3 of this volume, the following discussion will deal only with the specific features of the electron transfer chains in photosynthetic membranes. 

Non-sulfur, purple, photosynthetic bacteria, Rho do spirillum rub-rum and Rhodopseudomonas spheroides172 also possess a PEP-de-pendent D-fructose phosphotransferase. Two protein fractions are required for D-fructose phosphorylation. In contrast to PEP-depend-ent, phosphotransferase systems isolated from other bacteria, the aforementioned two organisms have one active protein fraction tightly associated with the membrane fraction, while another in the crude extract is solubilized by extraction with water, and has a molecular weight of about 200,000. There is no evidence for the presence of a phosphate-carrier protein of low molecular weight like HPr.171,173 The... 

The 2 Fe 2S plant type ferredoxins, MW 12,000 dal ton, Em = —430 mV, were first isolated from chloroplast and photosynthetic bacteria. Similar proteins have been purified from the bacteria E. coli (264) and Pseudomonas putida [ putidaredoxin , Em7 = —235 mV, (275)] and from mammalian adrenal cortex mitochondria [ adrenodoxin Em = — 367 mV, 13,100 dalton (165)] among other sources. 

Mathis P (1994) Electron transfer between cytochromeC2 and the isolated reaction center of purple bacterium Rhodobacter sphaeroides. Biochim Biophys Acta 1187 177-180 McDermott G, Prince SM, Freer AA, Hawthornwaite-Lawless AM, Papiz MZ, Cogdelt RJ and Isaacs NW (1995) Crystal structure of an integral membrane light-harvesting complex from photosynthetic bacteria. Nature 374 517-521 Michel H (1982) Three-dimensional crystals of a membrane protein complex. J Mol Biol 158 567-572 Michel H (1983) Crystallization of membrane proteins. Trends Biochem Sci 8 56-59... 

---