Differing isoforms of the cobalamin binding photoreceptor AerR oppositely regulate photosystem expression.

Phototrophic microorganisms adjust photosystem synthesis in response to changes in light intensity and wavelength. A variety of different photoreceptors regulate this process. Purple photosynthetic bacteria synthesize a novel photoreceptor AerR that uses cobalamin (B12) as a blue-light absorbing chromophore to control photosystem synthesis. AerR directly interacts with the redox responding transcription factor CrtJ, affecting CrtJ's interaction with photosystem promoters. In this study, we show that AerR is translated as two isoforms that differ by 41 amino acids at the amino terminus. The ratio of these isoforms was affected by light and cell growth phase with the long variant predominating during photosynthetic exponential growth and the short variant predominating in dark conditions and/or stationary phase. Pigmentation and transcriptomic analyses show that the short AerR variant represses, while long variant activates, photosynthesis genes. The long form of AerR also activates many genes involved in cellular metabolism and motility.

Utilization of distillery wastewater for hydrogen production in one-stage and two-stage processes involving photofermentation.

In this study, distillery wastewater was treated by dark fermentation or photofermentation alone, and by sequential dark and photofermentation processes using anaerobic saccharolytic consortium and purple nonsulfur bacteria. Combination of dark and photofermentation resulted in the maximal H2 yield of 17.6L/L of distillery waste with chemical oxygen demand 40g/L. It is equivalent to 205kJ/L distillery wastewater and corresponds to recovery of approximately 4-8% of energy consumed during ethanol production. Optimal performance of photofermentation was observed at 20% concentration of pre-fermented distillery waste. In photofermentation, the range of the suitable distillery waste concentrations was extended and the H2 yield was improved by choosing the tolerant strain of purple bacteria Rhodobacter sphaeroides B-3059. After two stages, organic acids and sugars were completely consumed that means wastewater treatment concomitant to H2 production.

Stoichiometry of ATP hydrolysis and chlorophyllide formation of dark-operative protochlorophyllide oxidoreductase from Rhodobacter capsulatus.

Dark-operative protochlorophyllide (Pchlide) oxidoreductase (DPOR) is a nitrogenase-like enzyme catalyzing a reduction of the C17 = C18 double bond of Pchlide to form chlorophyllide a (Chlide) in bacteriochlorophyll biosynthesis. DPOR consists of an ATP-dependent reductase component, L-protein (a BchL dimer), and a catalytic component, NB-protein (a BchN-BchB heterotetramer). The L-protein transfers electrons to the NB-protein to reduce Pchlide, which is coupled with ATP hydrolysis. Here we determined the stoichiometry of ATP hydrolysis and the Chlide formation of DPOR. The minimal ratio of ATP to Chlide (ATP/2e(-)) was 4, which coincides with that of nitrogenase. The ratio increases with increasing molar ratio of L-protein to NB-protein. This profile differs from that of nitrogenase. These results suggest that DPOR has a specific intrinsic property, while retaining the common features shared with nitrogenase.

Optimizing multi-step B-side charge separation in photosynthetic reaction centers from Rhodobacter capsulatus.

Using high-throughput methods for mutagenesis, protein isolation and charge-separation functionality, we have assayed 40 Rhodobacter capsulatus reaction center (RC) mutants for their P(+)QB(-) yield (P is a dimer of bacteriochlorophylls and Q is a ubiquinone) as produced using the normally inactive B-side cofactors BB and HB (where B is a bacteriochlorophyll and H is a bacteriopheophytin). Two sets of mutants explore all possible residues at M131 (M polypeptide, native residue Val near HB) in tandem with either a fixed His or a fixed Asn at L181 (L polypeptide, native residue Phe near BB). A third set of mutants explores all possible residues at L181 with a fixed Glu at M131 that can form a hydrogen bond to HB. For each set of mutants, the results of a rapid millisecond screening assay that probes the yield of P(+)QB(-) are compared among that set and to the other mutants reported here or previously. For a subset of eight mutants, the rate constants and yields of the individual B-side electron transfer processes are determined via transient absorption measurements spanning 100 fs to 50 µs. The resulting ranking of mutants for their yield of P(+)QB(-) from ultrafast experiments is in good agreement with that obtained from the millisecond screening assay, further validating the efficient, high-throughput screen for B-side transmembrane charge separation. Results from mutants that individually show progress toward optimization of P(+)HB(-)→P(+)QB(-) electron transfer or initial P*→P(+)HB(-) conversion highlight unmet challenges of optimizing both processes simultaneously.

High yield single stage conversion of glucose to hydrogen by photofermentation with continuous cultures of Rhodobacter capsulatus JP91.

Photofermentative hydrogen (H(2)) production from glucose with the photosynthetic bacterium Rhodobacter capsulatus JP91 (hup(-)) was examined using a photobioreactor operated in continuous mode. Stable and high hydrogen yields on glucose were obtained at three different retention times (HRTs; 24, 48 and 72 h). The H(2) production rates, varying between 0.57 and 0.81 mmol/h, and optical densities (OD(600 nm)) were similar for the different HRTs examined. However, the rate of glucose consumption was influenced by HRT being greater at HRT 24h than HRTs 48 and 72 h. The highest hydrogen yield, 9.0 ± 1.2 mol H(2)/mol glucose, was obtained at 48 h HRT. These results show that single stage photofermentative hydrogen production from glucose using photobioreactors operated in continuous culture mode gives high, nearly stoichiometric yields of hydrogen from glucose, and thus is considerably more promising than either two stage photofermentation or co-culture approaches.

Optimization of the hydrogen yield from single-stage photofermentation of glucose by Rhodobacter capsulatus JP91 using response surface methodology.

Hydrogen production from glucose via single-stage photofermentation was examined with the photosynthetic bacterium Rhodobacter capsulatus JP91 (hup-). Response surface methodology with Box-Behnken design was used to optimize the independent experimental variables of glucose concentration, glutamate concentration and light intensity, as well as examining their interactive effects for maximization of molar hydrogen yield. Under optimal condition with a light intensity of 175W/m(2), 35mM glucose, and 4.5mM glutamate, a maximum hydrogen yield of 5.5 (±0.15)molH(2)/molglucose, and a maximum nitrogenase activity of 246 (±3.5)nmolC(2)H(4)/ml/min were obtained. Densitometric analysis of nitrogenase Fe-protein expression under different conditions showed significant variation in Fe-protein expression with a maximum at the optimized central point. Even under optimum conditions for hydrogen production, a significant fraction of the Fe-protein was found in the ADP-ribosylated state, suggesting that further improvement in yields might be possible.

Enhanced photo-fermentative hydrogen production by Rhodobacter capsulatus with pigment content manipulation.

High content of pigment in purple nonsulfur photosynthetic bacteria hinders its photo-hydrogen production rate under intense light irradiation. In order to alleviate the light shielding effect and improve its photo-fermentative hydrogen production performance, pufQ, which is the regulatory gene of bacteriochlorophyll biosynthesis in Rhodobacter capsulatus, was cloned and relocated in the genome under cbb3 promoter by homologous recombination. The UV-vis spectra indicated that the light absorption of the mutant between 300 and 900 nm was reduced. Photo-hydrogen production experiments by the recombinant and wild type strain were carried out in 350 mL photo bioreactors using acetic and butyric acid as substrate. The results showed that the hydrogen production of recombinant with reduced pigment was 27% higher than that of its parental strain, indicating that it is effective on enhancing photo-fermentative hydrogen production by manipulating pigment biosynthesis in purple nonsulfur photosynthetic bacteria.

Photoproduction of hydrogen by Rhodobacter capsulatus from thermophilic fermentation effluent.

Rhodobacter capsulatus was used for the phototrophic hydrogen production on effluent solution derived from the thermophilic fermentation of Miscanthus hydrolysate by Thermotoga neapolitana. Pretreatments such as centrifugation, dilution, buffer addition, pH adjustment and sterilization were suggested for the effluent before being fed to the photofermentation. Batch-wise experiments showed that R. capsulatus grows and produces hydrogen on the pretreated effluent solution. Moreover, it was found that the hydrogen yield increased from 0.3 to 1.0 L/L(culture) by addition of iron to the effluent solution.

[Effect of growth conditions on electrophysical properties of Rhodobacter capsulatus PG cells].

Electrophysical characteristics of cells of the phototrophic bacterium Rhodobacter capsulatus PG grown in complete Hutner medium in light or dark were found to differ depending on the composition of their lipopolysaccharides (LPS). Under dark cultivation, the cells synthesized LPS with a shortened structure that determined the electrophoretic properties of cell surfaces. The observed decrease in the effective high-frequency electroconductivity of the dark-grown cells is assumed to be due to a decrease in the intracellular K+ concentration resulting from increased permeability of cytoplasmic membranes of the cells grown under these conditions.

The photosynthetic deficiency due to puhC gene deletion in Rhodobacter capsulatus suggests a PuhC protein-dependent process of RC/LH1/PufX complex reorganization.

Optimal photosynthetic reaction centre (RC) and core antenna (LH1) levels in the purple bacterium Rhodobacter capsulatus require the puhC gene. Deletion of puhC had little effect on RC and LH1 assembly individually, but significantly inhibited the photosynthetic growth of RC+ LH1- strains, suggesting that maximal RC catalytic activity is PuhC-dependent. Consistent with post-assembly reorganization of the RC/LH1/PufX core complex by PuhC to include latecomer proteins, spatial separation of pufX from the RC/LH1 genes inhibited PufX accumulation and photosynthetic growth only in PuhC- strains. Photosynthetic activity improved to different degrees when PuhC homologues from three other species were expressed in PuhC- R. capsulatus, indicating that PuhC homologues function similarly but may interact inefficiently with a heterologous core complex. Anaerobic photosynthetic growth of PuhC- strains was affected by the duration of prior semiaerobic growth, and by two genes that modulate bacteriochlorophyll production: pufQ and puhE. These observations agree with a speculative model in which reorganization of the core complex is an important regenerative process, accelerated by PuhC.

Lasers-an effective artificial source of radiation for the cultivation of anoxygenic photosynthetic bacteria.

The laser diode (LD) is a unique light source that can efficiently produce all radiant energy within the narrow wavelength range used most effectively by a photosynthetic microorganism. We have investigated the use of a single type of LD for the cultivation of the well-studied anoxygenic photosynthetic bacterium, Rhodobacter capsulatus (Rb. capsulatus). An array of vertical-cavity surface-emitting lasers (VCSELs) was driven with a current of 25 mA, and delivered radiation at 860 nm with 0.4 nm linewidth. The emitted light was found to be a suitable source of radiant energy for the cultivation of Rb. capsulatus. The dependence of growth rate on incident irradiance was quantified. Despite the unusual nearly monochromatic light source used in these experiments, no significant changes in the pigment composition and in the distribution of bacteriochlorophyll between LHII and LHI-RC were detected in bacterial cells transferred from incandescent light to laser light. We were also able to show that to achieve a given growth rate in a light-limited culture, the VCSEL required only 30% of the electricity needed by an incandescent bulb, which is of great significance for the potential use of laser-devices in biotechnological applications and photobioreactor construction.

Photoresponsive flagellum-independent motility of the purple phototrophic bacterium Rhodobacter capsulatus.

We report the discovery of photoresponsive, flagellum-independent motility of the alpha-proteobacterium Rhodobacter capsulatus, a nonsulfur purple phototrophic bacterium. This motility takes place in the 1.5% agar-glass interface of petri plates but not in soft agar, and cells move toward a light source. The appearances of motility assay plates inoculated with wild-type or flagellum-deficient mutants indicate differential contributions from flagellar and flagellum-independent mechanisms. Electron microscopy confirmed the absence of flagella in flagellar mutants and revealed the presence of pilus-like structures at one pole of wild-type and mutant cells. We suggest that R. capsulatus utilizes a flagellum-independent, photoresponsive mechanism that resembles twitching motility to move in a line away from the point of inoculation toward a light source.

[Effect of environmental factors on the composition of lipopolysaccharides released from the Rhodobacter capsulatus cell wall].

The goal of this study was to compare the environmental factors that determine viability of the gram-negative photosynthesizing bacteria Rhodobacter capsulatus (specifically, light and growth medium composition) and to assess the effect of these factors on the synthesis and composition of lipopolysaccharides released from the cell wall. Depletion of medium resulted in the release of lipopolysaccharides with a truncated polysaccharide fragment. It is concluded that, due to high viability of these bacteria, the main factor that induced lipopolysaccharide release from the cell wall, irrespective of culturing conditions, is cell division rather than cell death.

Tributyl phosphate degradation by Rhodopseudomonas palustris and other photosynthetic bacteria.

Tributyl phosphate (TBP) is widely used in nuclear fuel processing and other waste generating chemical industries. Although TBP is bacteriostatic, some microbes are resistant to it and may degrade it. Under dark aerobiosis, purple non-sulfur photosynthetic bacteria degraded up to 0.6 mM TBP, initially present at 2 mM, within 3 weeks and under photosynthetic conditions, Rhodopseudomonas palustris degraded 1.6 mM TBP within 3 weeks. The curing of the Rhodopseudomonas palustris endogenous plasmid demonstrated that the genes involved in the TBP degradation are chromosomal.

Improvement of growth stability of photosynthetic bacterium Rhodobacter capsulatus.

Using a semicontinuous culture method, in which operational parameters such as cell concentration and light intensity distribution were maintained almost constant, instability of the specific growth rate of Rhodobacter capsulatus B-100, a purple bacterium, was observed to be similar to that of R. capsulatus ST-410 when cultivated under high ratios of light intensity on the illuminated side to that of the transmitted light. Such instability was not observed in the cultivation of Chlorella vulgaris, a eukaryotic green alga, even at higher cell concentrations. Under the same conditions, the increase in only the ferrous concentration from 43 microM, the concentration in the original RCV medium, to 172 microM sustained a stable growth, whereas Fe(2+) was slightly consumed during the cultivation. Supplemental illumination with a fluorescent lamp on the transmitted side of a flat plate photobioreactor sustained a moderate level of stable growth, while a halogen lamp slightly affected the growth stability. Our results showed that an increase in Fe(2+) concentration or supplemental illumination improves the growth stability of R. capsulatus.

Effects of light intensity distribution on growth of Rhodobacter capsulatus.

For cultivation of photosynthetic cells under defined light intensity distributions, the repeated batch culture, in which a part of culture broth containing grown cells was repeatedly replaced at predetermined time intervals with a fresh medium to keep the cell concentration constant at an initial value, was employed. By use of this method the effects of the light intensity distribution on the growth characteristics of Rhodobacter capsulatus were studied. Unexpected decreases in the specific growth rate were observed in culture of R. capsulatus at high cell concentrations and a long light path length. Big differences in the light intensities of lightly and darkly illuminated portions in photobioreactors, which reflects the light intensity distribution, seemed to cause this phenomenon, which must be taken into consideration for stable growth of photosynthetic cells.

Effect of light and oxygen and adaptation to changing light conditions in a photosynthetic mutant in which the LHII complex of Rhv. sulfidophilum was heterologously expressed in a strain of Rb. capsulatus whose puc operon was deleted.

In this paper we show the effect of oxygen and light on the expression of the photosynthetic apparatus of a mutant heterologously expressing the puc operon. This mutant was obtained by introducing in trans an expression plasmid, bearing the puc A, B, and C genes of Rhv. sulfidophilum, as well as its own promoter, in an LHII(-) mutant of Rb. capsulatus. The results showed that oxygen and light repressed LHII expression. Even low-light intensities lowered the LHII content to undetectable levels by spectrophotometry or by SDS-PAGE. In high-light grown cells, where the relative ratios of LHI and LHII complexes were significantly diminished, we were able to detect LHII complexes. Under the latter condition, the absorption spectrum showed that some pigment accumulated in the membrane even in the absence of cell division. These pigments were used in a later step to assemble LHII complexes, when the high-light grown cells were transferred to semiaerobiosis in the dark. Transition of high-light grown cells to low-light conditions allowed us to study the adaptability of these heterologous mutant cells. We observed that adaptation never occurred, in part probably owing to energy limitation.

Flash-induced turnover of the cytochrome bc1 complex in chromatophores of Rhodobacter capsulatus: binding of Zn2+ decelerates likewise the oxidation of cytochrome b, the reduction of cytochrome c1 and the voltage generation.

The effect of Zn2+ on the rates of electron transfer and of voltage generation in the cytochrome bc1 complex (bc1) was investigated under excitation of Rhodobacter capsulatus chromatophores with flashing light. When added, Zn2+ retarded the oxidation of cytochrome b and allowed to monitor (at 561-570 nm) the reduction of its high potential heme b(h) (in the absence of Zn2+ this reaction was masked by the fast re-oxidation of the heme). The effect was accompanied by the deceleration of both the cytochrome c(1) reduction (as monitored at 552-570 nm) and the generation of transmembrane voltage (monitored by electrochromism at 522 nm). At Zn2+ <100 microM the reduction of heme b(h) remained 10 times faster than other reactions. The kinetic discrepancy was observed even after an attenuated flash, when bc1 turned over only once. These observations (1) raise doubt on the notion that the transmembrane electron transfer towards heme b(h) is the main electrogenic reaction in the cytochrome bc1 complex, (2) imply an allosteric link between the site of heme b(h) oxidation and the site of cytochrome c1 reduction at the opposite side of the membrane, and (3) indicate that the internal redistribution of protons might account for the voltage generation by the cytochrome bc1 complex.

Molecular analysis and identification of the radC gene from the phototrophic bacterium Rhodobacter capsulatus B10.

The radC gene, whose product plays a role in the prokaryotic repair of DNA damage after UV and X-ray irradiation, was cloned and sequenced from the phototrophic bacterium Rhodobacter capsulatus B10. The gene codes for a protein of 214 amino acids with a molecular mass of 23,792 Da. The deduced amino acid sequence showed significant homology with the RadC proteins of Escherichia coli, Bacillus subtilis and Haemophilus influenzae. Northern blot analysis indicated that under both chemotrophic and phototrophic growth conditions the radC gene was relatively highly expressed and was induced about five-fold after UV-irradiation. Primer extension analysis revealed that transcription was initiated from the same position before and after UV treatment. Mutants (radC negative) have a low survival rate and a slower growth rate than the wild type.

In bacterial reaction centers, a key residue suppresses mutational blockage of two different proton transfer steps.

In reaction centers of Rhodobacter (Rb.) capsulatus, the M43Asn --> Asp substitution is capable of restoring rapid rates for delivery of the second proton to QB in a mutant that lacks L212Glu. Flash-induced absorbance spectroscopy was used to show a nearly native rate for transfer of the second proton to QB (approximately 700 s-1) in the L212Gln+M43Asp double-mutant reaction center; this rate was shown to decrease more than 1000-fold in the photoincompetent L212Glu --> Gln mutant [Miksovska, J., Kálmán, L., Maróti, P., Schiffer, M., Sebban, P., and Hanson, D.K. (1997) Biochemistry 36, 12216-12226]. In Rb. sphaeroides, the equivalent M44Asn --> Asp mutation was reported to restore the rate of transfer of the first proton to a wild-type level when it is added to the L213Asp --> Asn photoincompetent mutant [Rongey, S.H., Paddock, M.L., Feher, G., and Okamura, M.Y. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 1325-1329]. It is remarkable that the same second-site mutation can compensate for both of these mutations which severely impair reaction center function by blocking two different proton-transfer reactions. We suggest that residue M43Asp is situated in a key position which can link pathways for delivery of both the first and second protons (involving structured water molecules) to QB.