Dihydroxylycopene diglucoside diesters: a novel class of carotenoids from the phototrophic purple sulfur bacteria Halorhodospira abdelmalekii and Halorhodospira halochloris

The carotenoids in Halorhodospira abdelmalekii and Halorhodospira halochloris were analyzed by spectroscopic methods. The carotenoid composition of the two species was almost the same. Both species contained substantial amounts of unusual carotenoid glycoside fatty acid esters, which have been found for the first time in phototrophic purple bacteria. Methoxy-hydroxylycopene glucoside was a major component, and dihydroxylycopene diglucoside and dihydroxylycopene diglucoside diester were also found. Lycopene, rhodopin, and 3,4,3',4'-tetrahydrospirilloxanthin were present in very small amounts. Methoxy, glucosyl, and glucosyl ester groups were observed as substituents at the positions of the two original hydroxyl groups of dihydroxylycopene and made up approximately 20, 50, and 20%, respectively, of the total end groups (100%). The fatty acid components of the three carotenoid glucoside esters were the same (C12:0 and C14:1) and were rare in the cellular lipids of the two species.     

1H-NMR studies of high-potential iron-sulfur protein from the purple phototrophic bacterium, Rhodospirillum tenue

The high-potential iron-sulfur protein (HiPIP) from Rhodospirillum tenue (strain 3761) shows only a weak (20-25%) sequence similarity to HiPIPs from Chromatium vinosum, Ectothiorhodospira halophila and Ectothiorhodospira vacuolata, including the strict conservation of only two of the twelve residues assumed to be in the 4Fe-4S cluster packing region [Tedro, S. M., Meyer, T. E. and Kamen, M. D. (1979) J. Biol. Chem. 254, 1495-1500]. In spite of these differences, the general range and distribution of hyperfine-shifted 1H-NMR peaks of oxidized and reduced R. tenue HiPIP resemble those of E. halophila HiPIP I [Krishnamoorthi, R., Markley, J. L., Cusanovich, M. A., Pryzycieki, C. T. and Meyer, T. E. (1986) Biochemistry 25, 60-67]. Temperature- and pH-dependence and longitudinal relaxation behavior were determined for hyperfine-shifted peaks of the oxidized protein. Tentative assignments of peaks to ligands and aromatic residues suggest the presence of common apoprotein-active-site interactions in these proteins. Differences occur in the pattern of paramagnetically shifted peaks attributed to hydrogens bonded to the 4Fe-4S cluster. Hyperfine-shifted peaks of R. tenue HiPIP are not perturbed by pH changes in the range 5-9. In contrast, those of the C. vinosum protein exhibit a pH-dependence of chemical shifts that has been attributed to the titration of His42 [Nettesheim, D. G., Meyer, T. E., Feinberg, B. A. and Otvos, J. D. (1983) J. Biol. Chem. 258, 8235-8239]. Since R. tenue HiPIP contains no histidine, the present observation confirms the above hypothesis.     

Electron transfer proteins of the purple phototrophic bacterium, Rhodopseudomonas rutila.

The soluble electron transfer protein content of Rhodopseudomonas rutila was found to consist of two basic cytochromes and a (4Fe-4S) ferredoxin. Cytochrome c' was easily identified by its characteristic high spin absorption spectra. The native molecular weight is 29,000 and the subunit is 14,000. Cytochrome c-550 has low spin absorption spectra and a high redox potential (376 mV) typical of cytochromes c2. The molecular weight is about 14,000. The ferredoxin is apparently a dimer (43,000) of approximately 18,000 Da subunits. There are 1.3 to 1.5 iron-sulfur clusters per monomer of 18- to 21-kDa protein. The N-terminal amino acid sequence is like the (7Fe-8S) ferredoxins of Rhodobacter capsulatus and Azotobacter vinelandii. Remarkably, there are only 2 or 3 out of 25 amino acid substitutions. Difference absorption spectra of Rps. rutila membranes indicate that there is not tetraheme reaction center cytochrome c, such as is characteristic of Rps. viridis. However, there are a high potential cytochrome c and a low potential cytochrome b in the membrane, which are suggestive of a cytochrome bc1 complex. Rps. rutila is most similar to Rps. palustris in microbiological properties, yet it does not have the cytochromes c-556, c-554, and c-551 in addition to c2 and c', which are characteristic of Rps. palustris. Furthermore, the Rps. rutila cytochrome c' is dimeric, whereas the same protein from Rps. palustris is the only one known to be monomeric. The cytochrome pattern is more like that of Rhodospirillum rubrum and Rb. capsulatus, which are apparently only able to make cytochromes c2 and c'.     

Simultaneous phototrophic and chemotrophic growth in the purple sulfur bacterium Thiocapsa roseopersicina M1

Abstract The anoxygenic phototrophic purple sulfur bacterium Thiocapsa roseopersicina was grown in illuminated continuous cultures with thiosulfate as growth limiting substrate. Aeration resulted in completely colorless cells growing chemotrophically, whereafter the conditions were changed to a 23 h oxic/1 h anoxic regime. After 11 volume changes at a dilution rate of 0.031 h⁻¹ (35% of μ_max) a time dependent equilibrium was established. During the 23 h oxic periods bacteriochlorophyll a synthesis (BChl a ) was not observed, whereas during the 1 h anoxic periods synthesis was maximal (i.e. 1.1 μg (mg protein)⁻¹ h⁻¹). As a result the BChl a concentration gradually increased from zero to an average value over 24 h of 1.9 μg (mg protein)⁻¹. Concomitantly, the protein concentration increased from 13.9 mg 1⁻¹ during continuous oxic conditions to 28.8 mg 1⁻¹. For comparison, the protein concentration during fully phototrophic growth at an identical thiosulfate concentration in the inflowing medium was 53.7 mg 1⁻¹. The specific respiration rate was 8 μmol O₂ (mg protein)⁻¹ h⁻¹ during full chemotrophic growth and gradually decreased to 3.5 μmol O₂ (mg protein)⁻¹ h⁻¹ after 11 volume changes at the regime employed. These data show that T. rosepersicina is able to simultaneously utilize light and aerobic respiration of thiosulfate as sources of energy. The ecological relevance of the data is discussed.     

Soluble cytochrome composition of the purple phototrophic bacterium, Rhodopseudomonas sphaeroides ATCC 17023

A detailed study of the soluble cytochrome composition of Rhodopseudomonas sphaeroides (ATCC 17023) indicates that there are five c-type cytochromes and one b-type cytochrome present. The molecular weights, heme contents, amino acid compositions, isoelectric points, and oxidation-reduction potentials were determined and the proteins were compared with those from other bacterial sources. Cytochromes c2 and c' have previously been well characterized. Cytochrome c-551.5 is a diheme protein which has a very low redox potential, similar to certain purple bacterial and algal cytochromes. Cytochrome c-554 is an oligomer, which is spectrally similar to the low-spin isozyme of cytochrome c' found in other purple bacteria (e.g., Rhodopseudomonas palustris cytochrome c-556). An unusual high-spin c-type heme protein has also been isolated. It is spectrally distinguishable from cytochrome c' and binds a variety of heme ligands including oxygen. A large molecular-weight cytochrome b-558 is also present which appears related to a similar protein from Rhodospirillum rubrum, and the bacterioferritin from Escherichia coli. None of the soluble proteins appear to be related to the abundant membrane-bound c-type cytochrome in Rps. sphaeroides which has a larger subunit molecular weight similar to mitochondrial cytochrome c1 and chloroplast cytochrome f.     

Occurrence of plant hormones in cells of the phototrophic purple bacterium Rhodospirillum rubrum 1R

Abstract Plant hormones from biomass of the purple non-sulfur bacterium Rhodospiririllum rubrum were isolated for the first time. These compounds show high physiological activities (300–330%) in the cytokinin bioassay. All three detected cytokinins are adenine derivatives, according to spectral analysis. One of them was identified as 6-(4-hydroxy-3-methyl-2-trans-2-bytenylamino)-9-ß-D-ribofuranosylpurine (zeatinriboside) as shown by thin-layer chromatography and reversed-phase high-performance liquid chromatography. The possible functions of bacterial cytokinins are also discussed.     

Soluble cytochromes and ferredoxins from the marine purple phototrophic bacterium, Rhodopseudomonas marina

Four soluble c-type cytochromes, the high redox potential 4-Fe-S ferredoxin known as HiPIP, a large molecular weight 2-Fe-S ferredoxin and a 4-Fe-S 'bacterial' ferredoxin, were isolated from extracts of two strains of Rps. marina. Cytochrome c-550, cytochrome c' and cytochrome c-549 were previously described, and we have extended their characterization. Cytochrome c-558, which has not previously been observed in Rps. marina, appears to be a low-spin isozyme of the more commonly observed high-spin cytochrome c'. HiPIP, which was not observed in previous work, was found to be abundant in Rps. marina. The 2-Fe-S ferredoxin, which has previously been observed only in Rps. palustris, has a native size greater than 100 kDa and a subunit size of 17 kDa. The 'bacterial' ferredoxin appears to have only a single four-iron-sulfur cluster. We examined photosynthetic membranes by difference spectroscopy and found abundant c-type cytochromes. Approximately one-quarter of the heme can be reduced by ascorbate and the remainder by dithionite. There is 2 nm difference between the high-potential heme (554 nm) and the low (552 nm). These characteristics resemble those of the tetraheme reaction center cytochrome of Rps. viridis. In addition to the electron transfer components, we found small amounts of a fluorescent yellow protein which has spectral resemblance to a photoactive yellow protein from Ec. halophila.     

Calcium ions are involved in the unusual red shift of the light-harvesting 1 Qy transition of the core complex in thermophilic purple sulfur bacterium Thermochromatium tepidum

Thermophilic purple sulfur bacterium, Thermochromatium tepidum, can grow at temperatures up to 58 degrees C and exhibits an unusual Qy absorption at 915 nm for the core light-harvesting complex (LH1), an approximately 35-nm red shift from those of its mesophilic counterparts. We demonstrate in this study, using a highly purified LH1-reaction center complex, that the LH1 Qy transition is strongly dependent on metal cations and Ca2+ is involved in the unusual red shift. Removal of the Ca2+ resulted in formation of a species with the LH1 Qy absorption at 880 nm, and addition of the Ca2+ to the 880-nm species recovered the native 915-nm form. Interchange between the two forms is fully reversible. Based on spectroscopic and isothermal titration calorimetry analyses, the Ca2+ binding to the LH1 complex was estimated to occur in a stoichiometric ratio of Ca2+/alphabeta-subunit = 1:1 and the binding constant was in 10(5) m(-1) order of magnitude, which is comparable with those for EF-hand Ca2+-binding proteins. Despite the high affinity, conformational changes in the LH1 complex upon Ca2+ binding were small and occurred slowly, with a typical time constant of approximately 6 min. Replacement of the Ca2+ with other metal cations caused blue shifts of the Qy bands depending on the property of the cations, indicating that the binding site is highly selective. Based on the amino acid sequences of the LH1 complex, possible Ca2+-binding sites are proposed that consist of several acidic amino acid residues near the membrane interfaces of the C-terminal region of the alpha-polypeptide and the N-terminal region of the beta-polypeptide.     

Diversity of extremophilic purple phototrophic bacteria in Soap Lake, a Central Washington (USA) Soda Lake

Culture-based and culture-independent methods were used to explore the diversity of phototrophic purple bacteria in Soap Lake, a small meromictic soda lake in the western USA. Among soda lakes, Soap Lake is unusual because it consists of distinct upper and lower water bodies of vastly different salinities, and its deep waters contain up to 175 mM sulfide. From Soap Lake water new alkaliphilic purple sulfur bacteria of the families Chromatiaceae and Ectothiorhodospiraceae were cultured, and one purple non-sulfur bacterium was isolated. Comparative sequence analysis of pufM, a gene that encodes a key photosynthetic reaction centre protein universally found in purple bacteria, was used to measure the diversity of purple bacteria in Soap Lake. Denaturing gradient gel electrophoresis and subsequent phylogenetic analyses of pufMs amplified from Soap Lake water revealed that a significant diversity of purple bacteria inhabit this soda lake. Although close relatives of several of the pufM phylotypes obtained from cultured species could also be detected in Soap Lake water, several other more divergent pufM phylotypes were also detected. It is possible that Soap Lake purple bacteria are major contributors of organic matter into the ecosystem of this lake, especially in its extensive anoxic and sulfidic deep waters.     

The Hfq-like protein NrfA of the phototrophic purple bacterium Rhodobacter capsulatus controls nitrogen fixation via regulation of nifA and anfA expression

A novel esterase catalyzing regioselective hydrolysis was purified from the membrane fraction of Microbacterium sp. 7-1W, and characterized. The enzyme was solubilized with Brij 58 and purified 13.8-fold to apparent homogeneity with 2.58% overall recovery. The relative molecular mass of the native enzyme as estimated by gel filtration was more than 600,000 Da, and the subunit molecular mass was 62,000 Da. The enzyme catalyzed cleavage of the terminal ester bonds of cetraxate esters and pantothenate esters. The K(m) and V(max) values for methyl cetraxate were 0.380 mM and 7.76 micromole min(-1) mg(-1) protein, respectively. The enzyme was inhibited by serine hydrolase inhibitors.     

Cloning, overproduction and characterization of cytochrome c peroxidase from the purple phototrophic bacterium Rhodobacter capsulatus.

The bacterial cytochrome c peroxidase (BCCP) from Rhodobacter capsulatus was purified as a recombinant protein from an Escherichia coli clone over-expressing the BCCP structural gene. BCCP from Rb. capsulatus oxidizes the Rhodobacter cytochrome c2 and reduces hydrogen peroxide, probably functioning as a detoxification mechanism. The enzyme binds two haem c groups covalently. The gene encoding BCCP from Rb. capsulatus was cloned through the construction of a 7-kb subgenomic clone. In comparison with the protein sequence, the sequence deduced from the gene has a 21-amino-acid N-terminal extension with the characteristics of a signal peptide. The purified recombinant enzyme showed the same physico-chemical properties as the native enzyme. Spectrophotometric titration established the presence of a high-potential (Em=+270 mV) and a low-potential haem (between -190 mV and -310 mV) as found in other BCCPs. The enzyme was isolated in the fully oxidized but inactive form. It binds calcium tightly and EGTA treatment of the enzyme was necessary to show calcium activation of the mixed valence enzyme. This activation is associated with the formation of a high-spin state at the low-potential haem. BCCP oxidizes horse ferrocytochrome c better than the native electron donor, cytochrome c2; the catalytic activities ('turnover number') are 85 800 min(-1) and 63 600 min(-1), respectively. These activities are the highest ever found for a BCCP.     

Growth of the phototrophic purple sulfur bacterium Thiocapsaroseopersicina under oxic/anoxic regimens in the light

Abstract The phototrophic purple sulfur bacterium Thiocapsa roseopersicina was grown in sulfide-limited continuous cultures exposed to oxic/anoxic regimens in continuous light. Synthesis of bacteriochlorophyll a (BChl a ) did not occur during the oxic periods, but started immediately upon the creation of anoxic conditions. In contrast, protein synthesis continued during both oxic and anoxic periods. Consequently, the specific content of BChla fluctuated. Despite the presence of oxygen and the fluctuating BChl a content, growth occurred predominantly in a phototrophic mode and respiration was virtually zero.
BChl a synthesis continued at high rates during anoxic periods, thus compensating for the lack of synthesis during oxic periods. When cultivated under regimens with oxic periods shorter than 12 h the highest specific BCh a content was 27 μg·mg⁻ protein. In contrast, when cultivated under regimens with oxic periods longer than 12 h the specific BChl a content was always lower than 27μg·mg⁻ length of the oxic periods. During the anoxic periods, BChl a synthesis occurred at the maximal velocity of 1.2 μg·mg⁻¹ protein·h⁻, but the length of the anoxic periods was not sufficient to allow the BChl a content to reach the maximum level.
Cultivation under continuously oxic conditions eventually resulted in pigmentless cells growing chemolithotrophically. The BChl a synthesizing ability was not lost during prolonged exposure to oxygen.
It was concluded that T. roseopersicina is very well adapted to oxic/anoxic cycles.     

Energy transfer and charge separation in the purple non-sulfur bacterium Roseospirillum parvum

The antenna reaction centre system of the recently described purple non-sulfur bacterium Roseospirillum parvum strain 930I was studied with various spectroscopic techniques. The bacterium contains bacteriochlorophyll (BChl) a, 20% of which was esterified with tetrahydrogeranylgeraniol. In the near-infrared, the antenna showed absorption bands at 805 and 909 nm (929 nm at 6 K). Fluorescence bands were located at 925 and 954 nm, at 300 and 6 K, respectively. Fluorescence excitation spectra and time resolved picosecond absorbance difference spectroscopy showed a nearly 100% efficient energy transfer from BChl 805 to BChl 909, with a time constant of only 2.6 ps. This and other evidence indicate that both types of BChl belong to a single LH1 complex. Flash induced difference spectra show that the primary electron donor absorbs at 886 nm, i.e. at 285 cm(-1) higher energy than the long wavelength antenna band. Nevertheless, the time constant for trapping in the reaction centre was the same as for almost all other purple bacteria: 55+/-5 ps. The shape as well as the amplitude of the absorbance difference spectrum of the excited antenna indicated exciton interaction and delocalisation of the excited state over the BChl 909 ring, whereas BChl 805 appeared to have a monomeric nature.     

The amino acid sequence of a high-redox-potential ferredoxin from the purple phototrophic bacterium, Rhodospirillum tenue strain 2761

The 61-residue amino acid sequence of Rhodospirillum tenue, strain 2761, high-redox-potential ferredoxin (HiPIP) is GTNAAMRKAFNYQDTAKNGKCSGCAQFVPGASPTAAGGCKVIPGDNEIAPGGYCDAFIVKK. It differs from that of R. tenue strain 3761 by 16 amino acid substitutions plus two single-residue deletions. This 26% sequence difference is similar to that observed among separate species of chromatiaceae such as Chromatium vinosum, C. gracile, and Thiocapsa roseopersicina, and is suprising because there are no distinguishing microbiological characteristics separating these two R. tenue strains. The most interesting amino acid substitution in R. tenue 2761 HiPIP is Gly for Asn 45 (C. vinosum numbering). Besides the four cysteines required to bind the four iron-four sulfur cluster, only Tyr 19, Asn 45, and Gly 75 are absolutely conserved in the nine previously determined HiPIP sequences. If HiPIP is used as a measure of divergence of species, then R. tenue and C. vinosum are the most distant purple bacteria examined. Quite the opposite conclusion follows based on the sequences of the cytochromes c'. It is suggested that this anomaly is more likely owing to a change in function for HiPIP with subsequently rapid evolutionary change than to a relatively recent transfer of the cytochrome c' gene between species.     

Soluble cytochromes and a photoactive yellow protein isolated from the moderately halophilic purple phototrophic bacterium, Rhodospirillum salexigens

Three soluble cytochromes were found in two strains of the halophilic non-sulfur purple bacterium Rhodospirillum salexigens. These are cytochromes C2, C and c-551. Cytochrome C2 was recognized by the presence of positive charge at the site of electron transfer (measured by laser flash photolysis), although the protein has an overall negative charge (pI = 4.7). Cytochrome C2 has a high redox potential (300 mV) and is monomeric (13 kDa). Cytochrome c was recognized from its characteristic absorption spectrum. It has a redox potential of 95 mV, an isoelectric point of 4.3, and is isolated as a dimer (33 kDa) of identical subunits (14 kDa), a property which is typical of this family of proteins. R. salexigens cytochrome c-551 has an absorption spectrum similar to the low redox potential Rb. sphaeroides cytochrome c-551.5. It also has a low redox potential (-170 mV), is very acidic (pI = 4.5), and is monomeric (9 kDa), apparently containing 1 heme per protein. The existence of abundant membrane-bound cytochromes c-558 and c-551 which are approximately half reduced by ascorbate and completely reduced by dithionite suggests the presence of a tetraheme reaction center cytochrome in R. salexigens, although reaction centers purified in a previous study (Wacker et al., Biochim. Biophys. Acta (1988) 933, 299-305) did not contain a cytochrome. The most interesting observation is that R. salexigens contains a photoactive yellow protein (PYP), previously observed only in the extremely halophilic purple sulfur bacterium Ectothiorhodospira halophila. The R. salexigens PYP appears to be slightly larger than that of Ec. halophila (16 kDa vs. 14 kDa). Otherwise, these two yellow proteins have similar absorption spectra, chromatographic properties and kinetics of photobleaching and recovery.     

Amino acid sequence of high-redox-potential ferredoxin (HiPIP) isozymes from the extremely halophilic purple phototrophic bacterium, Ectothiorhodospira halophila

The amino acid sequences of high-redox-potential ferredoxin (HiPIP) isozymes from Ectothiorhodospira halophila have been determined. These are: isozyme I, EPRAEDGHAHDYVNEAADPSHGRYQEGQLCENCAFWGEAVQDGWGRCTHPDFDEVLVKAEGWCSVYAPA S, and isozyme II, GLPDGVEDLPKAEDDHAHDYVNDAADTDHARFQEGQLCENCQFWVDYVNGWGYCQHPDFTDVLVRGEGW CSVYAPA. Isozyme II is the major form of HiPIP produced by the bacterium (65-80%) and is the most acidic of the known HiPIPs. The two isozymes are 72% identical to one another and require only a single residue deletion for alignment. Comparison of these HiPIPs with seven previously determined sequences revealed only 27% average identity. Both E. halophila HiPIP isozymes are likely to be functional since their sequences are equally distant from those of other species. The E. halophila HiPIP sequences show that H-bonding patterns recognized in Chromatium vinosum HiPIP are likely to be conserved and therefore cannot explain the unusually low redox potentials which have been reported.