Purple Non-sulfur Bacterium Rhodobacter Capsulatus Research Articles

Properties of Mutant Photosynthetic Reaction Centers of Purple Non-Sulfur Bacteria Cereibacter sphaeroides with M206 Ile→Gln Substitution

Properties of Mutant Photosynthetic Reaction Centers of Purple Non-Sulfur Bacteria Cereibacter sphaeroides with M206 Ile→Gln Substitution

Reliable determination of the growth and hydrogen production parameters of the photosynthetic bacterium Rhodobacter capsulatus in fed batch culture using a combination of the Gompertz function and the Luedeking-Piret model

In this study, experimental results of hydrogen producing process based on anaerobic photosynthesis using the purple non-sulfur bacterium Rhodobacter capsulatus are scrutinized. The bacterial culture was carried out in a photo-bioreactor operated in a quasi-continuous mode, using lactate as a carbon source. The method is based on the continuous stirred tank reactors (CSTR) technique to access kinetic parameters. The dynamic evolution of hydrogen production as a function of time was accurately simulated using Luedeking-Piret model and the growth of R. capsulatus was computed using Gompertz model. The combination of both models was successfully applied to determine the relevant parameters (λ, μmax, α and β) for two R. capsulatus strains studied: the wild-type strain B10 and the H2 over-producing mutant IR3. The mathematical description indicates that the photofermentation is more promising than dark fermentation for the conversion of organic substrates into biogas.

Dark fermentation effluent as substrate for hydrogen production from Rhodobacter capsulatus highlighting the performance of different fermentation systems.

Hydrogen production by biological route is a potentially sustainable alternative. Nowadays, energy production from sustainable sources has become urgent for several countries as well as for international policies. In this perspective, hydrogen has gained substantial global attention as clean, sustainable, and versatile energy carrier. In the current work, the resulting effluent from dark fermentation, rich in organic acids, was used as substrate for the purple non-sulfur bacteria (PNS) Rhodobacter capsulatus. In the first stage, experiments were carried out in bioreactors of 50mL to check the influence of the composition of the effluent dark fermentation. The results proved that the provision of a sugar source improved bio-H2 production. The lactose and lactic acid concentrations exceeding 4.4 and 12g/L, respectively, resulted in a productivity of up to 37.14mmol H2/Ldays. Based on initial conditions obtained on the previous assays, in the second stage, a photo-fermentation in enlarged scale (1.5L) was performed with the purpose to monitor the production of hydrogen and metabolites, sugar consumption and growth cells during the process. It was observed that the maximum productivity obtained was 98.23mmol H2/Ldays in 26h of process.

Repeated batch cycles as an alternative for hydrogen production by co-culture photofermentation

The use of hydrogen as an energy source represents an alternative to reduce the environmental impact since water is the only major by-product in its combustion. It can be used as an on-board fuel for motive power through internal combustion engine or fuel cell that converts chemical energy (an electro-chemical device) and can be applied in several transportation devices. The hybrid system for hydrogen production by combining the dark fermentation followed by photofermentation has great potential. In the current work, the production of hydrogen was investigated in repeated-batch processes using dark fermentation effluent as substrate to pure culture (Rhodopseudomonas palustris) and co-culture purple non-sulfur bacteria (Rhodopseudomonas palustris and Rhodobacter capsulatus). The influence of glucose and milk whey permeate (MWP) on the hydrogen production was studied. The results showed that the system employing the co-culture and glucose kept the culture activity for a long time (679 h) and the amount of accumulated hydrogen was 0.98 ± 0.02 mol and maximum productivity reached 287.39 ± 5.75 mmol of H2/L·day. The findings from alternating feeding between glucose and milk whey permeate, showed higher results for the amount of accumulated hydrogen (1.41 ± 0.01 mol). Besides, maximum productivity was of 266.60 ± 5.33 mmol of H2/L·day.

Absence of Thiol-Disulfide Oxidoreductase DsbA Impairs cbb3-Type Cytochrome c Oxidase Biogenesis in Rhodobacter capsulatus.

The thiol-disulfide oxidoreductase DsbA carries out oxidative folding of extra-cytoplasmic proteins by catalyzing the formation of intramolecular disulfide bonds. It has an important role in various cellular functions, including cell division. The purple non-sulfur bacterium Rhodobacter capsulatus mutants lacking DsbA show severe temperature-sensitive and medium-dependent respiratory growth defects. In the presence of oxygen, at normal growth temperature (35°C), DsbA− mutants form colonies on minimal medium, but they do not grow on enriched medium where cells elongate and lyse. At lower temperatures (i.e., 25°C), cells lacking DsbA grow normally in both minimum and enriched media, however, they do not produce the cbb3-type cytochrome c oxidase (cbb3-Cox) on enriched medium. Availability of chemical oxidants (e.g., Cu2+ or a mixture of cysteine and cystine) in the medium becomes critical for growth and cbb3-Cox production in the absence of DsbA. Indeed, addition of Cu2+ to the enriched medium suppresses, and conversely, omission of Cu2+ from the minimal medium induces, growth and cbb3-Cox defects. Alleviation of these defects by addition of redox-active chemicals indicates that absence of DsbA perturbs cellular redox homeostasis required for the production of an active cbb3-Cox, especially in enriched medium where bioavailable Cu2+ is scarce. This is the first report describing that DsbA activity is required for full respiratory capability of R. capsulatus, and in particular, for proper biogenesis of its cbb3-Cox. We propose that absence of DsbA, besides impairing the maturation of the c-type cytochrome subunits, also affects the incorporation of Cu into the catalytic subunit of cbb3-Cox. Defective high affinity Cu acquisition pathway, which includes the MFS-type Cu importer CcoA, and lower production of the c-type cytochrome subunits lead together to improper assembly and degradation of cbb3-Cox.

Acetate Metabolism in the Purple Non-sulfur Bacterium Rhodobacter capsulatus.

The purple non-sulfur bacterium Rhodobacter capsulatus B10 can grow on acetate as the sole carbon source under photoheterotrophic conditions. It is known that the bacterium can use the glyoxylate cycle and, in addition to it, or alternatively to it, an unknown pathway for acetate assimilation. We analyzed the genetic potential for functioning of additional metabolic pathways of oxaloacetic acid (OAA) pool replenishment in R. capsulatus. Using published microarray data of more than 4000 transcripts of genes for R. capsulatus, the genes necessary for acetate assimilation were analyzed. The results of the analysis showed the presence of all genes necessary for functioning of the ethylmalonyl-CoA pathway, and also a combination of pathways of formation of pyruvic acid/phosphoenol pyruvate (PA/PEP) (from acetyl-CoA and formate, from acetyl-CoA and CO2, as well as from 3-phosphoglyceric acid formed in the Calvin-Benson cycle) with their subsequent carboxylation. Using expression analysis, we showed that OAA pool replenishment on acetate medium could be achieved via a combination of PA/PEP synthesis from Calvin-Benson cycle intermediates and their carboxylation (with participation of pyruvate carboxylase, two reversible malate dehydrogenases (decarboxylating) and PEP-carboxykinase) to tricarboxylic acid cycle intermediates, the glyoxylate cycle, and a modified ethylmalonyl-CoA pathway in R. capsulatus under these experimental conditions. It was found that analogs of ethylmalonyl-CoA pathway enzymes exist. These enzymes differ in their specificity for S-enantiomers.

Development of bacteriochlorophyll a-based near-infrared photosensitizers conjugated to gold nanoparticles for photodynamic therapy of cancer.

We report the synthesis and characterization of a new sulfur-containing derivative of bacteriochlorophyll a. The latter was isolated from biomass of the nonsulfur purple bacterium Rhodobacter capsulatus strain B10. The developed photosensitizer is N-aminobacteriopurpurinimide with an exocyclic amino group acylated with a lipoic acid moiety, which is a biogenic substance that acts as a cofactor of the pyruvate dehydrogenase and α-ketoglutarate dehydrogenase complexes in the body. The disulfide moiety of lipoic acid confers the compound aurophilicity, thus allowing its conjugation with gold nanoparticles (NP-Au) via S-Au bonds. The shape and the size of the resulting nanoconjugate with immobilized photosensitizer (PS-Au) were assessed by dynamic light scattering and transmission electron microscopy. The conjugated nanoparticles are spherical with hydrodynamic diameter of 100-110nm. The PS-Au conjugate absorbs light at 824nm and emits strong fluorescence at 830nm, which allowed in vivo study of its dynamic biodistribution in rats bearing sarcomaM-1. Compared to the free photosensitizer, PS loaded on the gold nanoparticles (PS-Au) showed extended circulation time in the blood and enhanced tumor uptake due to nonspecific passive targeting when the drug accumulates in tumor sites through the leaky tumor neovasculature and does not return to the circulation.

Zinc biosorption by the purple non-sulfur bacterium Rhodobacter capsulatus.

This paper presents the first report providing information on the zinc (Zn) biosorption potentialities of the purple non-sulfur bacterium Rhodobacter capsulatus. The effects of various biological, physical, and chemical parameters on Zn biosorption were studied in both the wild-type strain B10 and a strain, RC220, lacking the endogenous plasmid. At an initial Zn concentration of 10 mg·L(-1), the Zn biosorption capacity at pH 7 for bacterial biomass grown in synthetic medium containing lactate as carbon source was 17 and 16 mg Zn·(g dry mass)(-1) for strains B10 and RC220, respectively. Equilibrium was achieved in a contact time of 30-120 min, depending on the initial Zn concentration. Zn sorption by live biomass was modelled, at equilibrium, according to the Redlich-Peterson and Langmuir isotherms, in the range of 1-600 mg Zn·L(-1). The wild-type strain showed a maximal Zn uptake capacity (Qm) of 164 ± 8 mg·(g dry mass)(-1) and an equilibrium constant (Kads) of 0.017 ± 0.00085 L·(mg Zn)(-1), compared with values of 73.9 mg·(g dry mass)(-1) and 0.361 L·mg(-1) for the strain lacking the endogenous plasmid. The Qm value observed for R. capsulatus B10 is one of the highest reported in the literature, suggesting that this strain may be useful for Zn bioremediation. The lower Qm value and higher equilibrium constant observed for strain RC220 suggest that the endogenous plasmid confers an enhanced biosorption capacity in this bacterium, although no genetic determinants for Zn resistance appear to be located on the plasmid, and possible explanations for this are discussed.

A T7 RNA polymerase-based toolkit for the concerted expression of clustered genes

Bacterial genes whose enzymes are either assembled into complex multi-domain proteins or form biosynthetic pathways are frequently organized within large chromosomal clusters. The functional expression of clustered genes, however, remains challenging since it generally requires an expression system that facilitates the coordinated transcription of numerous genes irrespective of their natural promoters and terminators. Here, we report on the development of a novel expression system that is particularly suitable for the homologous expression of multiple genes organized in a contiguous cluster. The new expression toolkit consists of an Ω interposon cassette carrying a T7 RNA polymerase specific promoter which is designed for promoter tagging of clustered genes and a small set of broad-host-range plasmids providing the respective polymerase in different bacteria. The uptake hydrogenase gene locus of the photosynthetic non-sulfur purple bacterium Rhodobacter capsulatus which consists of 16 genes was used as an example to demonstrate functional expression only by T7 RNA polymerase but not by bacterial RNA polymerase. Our findings clearly indicate that due to its unique properties T7 RNA polymerase can be applied for overexpression of large and complex bacterial gene regions.

Photomixotrophic growth of Rhodobacter capsulatus SB1003 on ferrous iron.

This study investigates the role iron oxidation plays in the purple non-sulfur bacterium Rhodobacter capsulatus SB1003. This organism is unable to grow photoautotrophically on unchelated ferrous iron [Fe(II)] despite its ability to oxidize chelated Fe(II). This apparent paradox was partly resolved by the discovery that SB1003 can grow photoheterotrophically on the photochemical breakdown products of certain ferric iron-ligand complexes, yet whether it could concomitantly benefit from the oxidation of Fe(II) to fix CO(2) was unknown. Here, we examine carbon fixation by stable isotope labeling of the inorganic carbon pool in cultures growing phototrophically on acetate with and without Fe(II). We show that R. capsulatus SB1003, an organism formally thought incapable of phototrophic growth on Fe(II), can actually harness the reducing power of this substrate and grow photomixotrophically, deriving carbon both from organic sources and from fixation of inorganic carbon. This suggests the possibility of a wider occurrence of photoferrotrophy than previously assumed.

Field Evidence for the Potential of Rhodobacter capsulatus as Biofertilizer for Flooded Rice

In a previous study, we evaluated the effects of inoculating rice plants with the phototrophic purple nonsulfur bacterium Rhodobacter capsulatus (Rc) on growth and yield of rice in pots and lysimeter experiments and the results obtained have been highly encouraging. In this study, we carried out two field experiments: one in the experimental farm of the Faculty of Agriculture, Fayoum University, and the second in a farmer's field in Kafr El-sheikh, to assess the effects of Rc on growth and yield of rice in comparison and in combination with chemical nitrogen fertilizer (CNF) and farmyard manure. The results indicated that both biological and grain yields in all the Rc inoculated treatments were significantly higher than those in the uninoculated corresponding treatments in both fields. With regard to grain yield, the major factor for determining the effectiveness of any agricultural treatment, inoculation with Rc in combination with 50% of the recommended CNF rate gave a grain yield that was statistically equivalent to that obtained with 100% of the recommended CNF rate. These results provide a clear evidence for the potential of Rc as biofertilizer for flooded rice under field conditions.

Synthesis of bacteriochlorophyll a by the purple nonsulfur bacterium Rhodobacter capsulatus

The ability of purple nonsulfur bacteria Rhodobacter capsulatus B10 to synthesize bacteriochlorophyll under phototrophic and dark conditions was studied. The modes for cultivation in the dark with oxygen limitation in a continuous culture at D = 0.1 h(-1) were selected. The yield of biomass reached 20 g/l; the bacteriochlorophyll a output of the process amounted to 16.6 mg/l h(-1).

Enzymic systems proposed to be involved in the dissimilatory reduction of selenite in the purple non-sulfur bacteria Rhodospirillum rubrum and Rhodobacter capsulatus

Various enzymic systems, such as nitrite reductase, sulfite reductase and glutathione reductase, have been proposed for, or suspected to be involved in, the reduction of selenite in bacteria. As alphaproteobacteria have been shown to be highly tolerant to transition metal oxyanions, it seemed interesting to investigate the hypothetical involvement of these different enzymes in the reduction of selenite in the purple non-sulfur bacteria Rhodospirillum rubrum and Rhodobacter capsulatus. The hypothetical involvement of nitrite reductase and sulfite reductase in the reduction of selenite in these bacteria was investigated by analysing the effects of nitrite and sulfite amendments on the growth and kinetics of selenite reduction. The reduction of selenite was not concomitant with that of either sulfite or nitrite in Rs. rubrum, suggesting that the reduction pathways operate independently. In Rb. capsulatus, strong interactions were observed between the nitrite reduction and selenite reduction pathways. However, in both organisms, selenite reduction took place during both the growth phase and the stationary phase, indicating that selenite metabolism is constitutively expressed. In contrast, neither nitrite nor sulfite was transformed during stationary phase, suggesting that the metabolism of both ions is induced, which implies that identical reduction pathways for selenite and nitrite or selenite and sulfite are excluded. Buthionine sulfoximine (BSO, S-n-butyl homocysteine sulfoximine), a specific inhibitor of glutathione synthesis, was used to depress the intracellular glutathione level. In stationary-phase cultures of both Rs. rubrum and Rb. capsulatus amended with BSO, the rate of reduction of selenite was slowed, indicating that glutathione may be involved in the dissimilatory reduction of selenite in these organisms. The analysis of the headspace gases of the cultures indicated that the synthesis of methylated selenium compounds was prevented in the presence of 3.0 mM BSO in both organisms, implying that glutathione is also involved in the transformation of selenite to volatile selenium compounds.

Membrane-anchored cytochrome c as an electron carrier in photosynthesis and respiration: past, present and future of an unexpected discovery.

In the mid 1980s, it was observed that photosynthesis could still occur in the absence of the diffusible electron carrier cytochrome c (2) in the purple non-sulfur facultative phototrophic bacterium Rhodobacter capsulatus. This serendipic finding led to the discovery of a novel class of membrane-anchored electron carrier cytochromes and their associated electron transfer pathways. Studies of cytochrome c (y) of R. capsulatus (and its homologues in other species) have modified the previous dogma of electron transfer between photosynthetic and respiratory membrane protein complexes with a new paradigm, in which these proteins and their electron carriers can form 'hard-wired' structural super-complexes. Here, we reminisce on the early days of this discovery, its impacts on our understanding of cellular energy transduction pathways and the physiological roles played by the electron carrier cytochromes c, and discuss the current knowledge and emerging future challenges of this field.

A system for the heterologous expression of complex redox proteins in Rhodobacter capsulatus: characterisation of recombinant sulphite:cytochrome c oxidoreductase from Starkeya novella

The phototrophic purple non-sulfur bacterium Rhodobacter capsulatus expresses a wide variety of complex redox proteins in response to changing environmental conditions. Here we report the construction and evaluation of an expression system for recombinant proteins in that organism which makes use of the dor promoter from the same organism. A generic expression vector, pDorEX, was constructed and used to express sulphite:cytochrome c oxidoreductase from Starkeya novella, a heterodimeric protein containing both molybdenum and haem c. The recombinant protein was secreted to the periplasm and its biochemical properties were very similar to those of the native enzyme. The pDorEX system therefore seems to be potentially useful for heterologous expression of multi-subunit proteins containing complex redox cofactors.

Growth of the purple bacterium Rhodobacter capsulatus on the aromatic compound hippurate.

The purple nonsulfur bacterium Rhodobacter capsulatus strain B10 grew phototrophically on the aromatic compound hippurate (N-benzoyl-L-glycine) and related benzoyl amino acids. Absorption spectra, extraction, and GC/MS analysis of culture supernatants showed that hippurate was stoichiometrically converted to benzoate and glycine, with the latter used as a carbon or nitrogen source for growth. This conclusion was supported by detection of the enzyme hippuricase in permeabilized intact cells. Chemotrophic growth on hippurate by Rba. capsulatus, either at full or reduced oxygen tensions, was not observed. The type strain of Rhodobacter sphaeroides as well as four strains of Rhodopseudomonas palustris also grew phototrophically on hippurate, while several other aromatic-degrading species of purple bacteria did not.

FeMo cofactor biosynthesis in a nifE − mutant of Rhodobacter capsulatus

In all diazotrophic micro-organisms investigated so far, mutations in nifE, one of the genes involved in the biosynthesis of the FeMo cofactor (FeMoco), resulted in the accumulation of cofactorless inactive dinitrogenase. In this study, we have found that strains of the phototrophic non-sulfur purple bacterium Rhodobacter capsulatus with mutations in nifE, as well as in the operon harbouring the nifE gene, were capable of reducing acetylene and growing diazotrophically, although at distinctly lower rates than the wild-type strain. The diminished rates of substrate reduction were found to correlate with the decreased amounts of the dinitrogenase component (MoFe protein) expressed in R. capsulatus. The in vivo activity, as measured by the routine acetylene-reduction assay, was strictly Mo-dependent. Maximal activity was achieved under diazotrophic growth conditions and by supplementing the growth medium with molybdate (final concentration 20-50 microM). Moreover, in these strains a high proportion of ethane was produced from acetylene ( approximately 10% of ethylene) in vivo. However, in in vitro measurements with cell-free extracts as well as purified dinitrogenase, ethane production was always found to be less than 1%. The isolation and partial purification of the MoFe protein from the nifE mutant strain by Q-Sepharose chromatography and subsequent analysis by EPR spectroscopy and inductively coupled plasma MS revealed that FeMoco is actually incorporated into the protein (1.7 molecules of FeMoco per tetramer). On the basis of the results presented here, the role of NifNE in the biosynthetic pathway of the FeMoco demands reconsideration. It is shown for the first time that NifNE is not essential for biosynthesis of the cofactor, although its presence guarantees formation of a higher content of intact FeMoco-containing MoFe protein molecules. The implications of our findings for the biosynthesis of the FeMoco will be discussed.

Investigation of bacteriochlorophyll biosynthesis genes in Blastochloris viridis by functional complementation of mutants of Rhodobacter capsulatus

Blastochloris (formerly Rhodopseudomonas) viridis produces bacteriochlorophyll (BChl) b which is distinguished from BChl a by the possession of an ethylidene group at C8 instead of an ethyl. This chemical structure enables to BChl b to absorb longer wave length light (~1100 nm) than BChl a. To investigate genes involved in the BChl b biosynthesis, we have constructed genomic DNA libraries from B. viridis and introduced them to mutants of the nonsulfur purple bacterium Rhodobacter capsulatus which is deficient in BChl a biosynthesis. Total ten kinds of R. capsulatus mutants, three Mg-chelatase (BchD, H, I) mutants, three protochlorophyllide reductase (BchB, L, N) mutants, Mg-protoporphyrin IX methyltransferase (BchM) mutant, Mg-protoporphyrin IX monomethylester oxidative cyclase (BchE) mutant, bacteriochlorophyll synthase (Bch G) mutant, and 2-vinyl-bacteriochlorophyllide hydratase (BchF) mutant, were functionally complemented by B. viridis library DNA. The complemented R. capsulatus strains were demonstrated to be capable of synthesizing BChl a under photoautotrophic growth conditions.

Primary alcohols and di-alcohols as growth substrates for the purple nonsulfur bacteriumRhodobacter capsulatus†

Growth experiments were performed with the purple nonsulfur bacterium Rhodobacter capsulatus to test its ability to use aliphatic, methyl-substituted, and unsaturated alcohols, as well as di-alcohols, as carbon sources for growth. Both phototrophic and chemotrophic growth was observed on a wide variety of such alcohols. By contrast, secondary or tertiary alcohols, or primary alcohols containing an ethyl or propyl substituent, did not support growth. In addition, preculture history and serial subculturing were found to be important factors for obtaining reliable growth of R. capsulatus on alcohols. Collectively, these results suggest that the carbon nutritional diversity of Rhodobacter capsulatus is even greater than previously suspected and that besides metabolizing organic acids and fatty acids in nature, this species may also be a major consumer of alcohols.

Oxidation-reduction properties of disulfide-containing proteins of the Rhodobacter capsulatus cytochrome c biogenesis system.

Oxidation-reduction titrations for the active-site disulfide/dithiol couples of the helX- and ccl2-encoded proteins involved in cytochrome c biogenesis in the purple non-sulfur bacterium Rhodobacter capsulatus have been carried out. The R. capsulatus HelX and Ccl2 proteins are predicted to function as part of a dithiol/disulfide cascade that reduces a disulfide on the apocytochromes c so that two cysteine thiols are available to form thioether linkages between the heme prosthetic group and the protein. Oxidation-reduction midpoint potential (E(m)) values, at pH 7.0, of -300 +/- 10 and -210 +/- 10 mV were measured for the HelX and Ccl2 (a soluble, truncated form of Ccl2) R. capsulatus proteins, respectively. Titrations of the disulfide/dithiol couple of a peptide designed to serve as a model for R. capsulatus apocytochrome c(2) have also been carried out, and an E(m) value of -170 +/- 10 mV was measured for the model peptide at pH 7.0. E(m) versus pH plots for HelX, Ccl2, and the apocytochrome c(2) model peptide were all linear over the pH range from 5.0 to 8.0, with the -59 mV/pH unit slope expected for a reaction in which two protons are taken up for each disulfide that is reduced. These results provide thermodynamic support for the proposal that HelX reduces Ccl2 and that reduced Ccl2, in turn, serves as the reductant for the production of the two thiols of the CysXxxYyyCysHis heme-binding motif of the apocytochromes.