Phototrophic Bacterium Rhodobacter Sphaeroides Research Articles

Study of phototrophic purple bacterium Rhodobacter sphaeroides cell morphology of wild-type and ipt-transformant by atomic force and electron microscopy

A comparative study of phototrophic purple bacterium Rhodobacter sphaeroides cell morphology of wild-type and ipt-transformant was done by atomic force and electron microscopy. It was shown that transformation led to a decrease in the number or total disappearance of the flagella, as well as to changes in the structure of the outer membrane of the bacteria cell wall. On the wild-type cell surface phage-like structures were found, and in transformed cells at their places hollows were identified. This study significantly extends an understanding of the changes occurring in the ipt-transformants of phototrophic purple bacterium Rhodobacter sphaeroides. This investigation not only confirmed earlier obtained data about the differences in the wild-type and ipt-transformant phototrophic purple bacteria cell wall, but also showed fine changes in the structure of its outer membrane.

Experimental Investigation on Biological Hydrogen Production Using Different Biomass

Hydrogen is a clean and efficient fuel, considered as a potential and more sustainable energy substitute for fossil fuels. Biological hydrogen production stands out as an eco friendly process carried out under mild operating conditions with renewable resources. In the current work laboratory scale production of hydrogen using phototrophic purple non-sulphur bacteria Rhodobacter sphaeroides, anaerobic dark fermentative bacteria’s Clostridium pasteurinum, Bacillus licheniformis and Enterobacter clocae with different substrates were investigated. The study investigated the potential of biological conversion of different substrates to produce hydrogen by studying various experimental parameters like temperature, pH and optical density (OD) of cells. It was found that the Rhodobacter sphaeroides took relatively longer duration (48 hrs) for hydrogen production. The optimum temperature and pH for maximum production of hydrogen in case of Rhodobacter sphaeroides were found to be 32°C and 7.5 respectively, 32°C and 7 for Clostridium pasteurinum respectively, 30°C and 6.8 respectively for Bacillus licheniformis and Enterobacter clocae. Results of the batch tests showed that Rhodobacter sphaeroides produced maximum amount of hydrogen (35%) as compared to 21% by Clostridium pasteurinum, 16% by Bacillus licheniformis and 8% by Enterobacter clocae. However the quantity of hydrogen production in case of Rhodobacter sphaeroides was relatively lower compared to Clostridium pasteurinum. Key words: Hydrogen; biomass, microbial bioconversion, dark fermentation, photofermentation, non-sulphur bacteria, anaerobic

Salt Stress-Induced Changes in the Transcriptome, Compatible Solutes, and Membrane Lipids in the Facultatively Phototrophic Bacterium Rhodobacter sphaeroides

Responses to NaCl stress were investigated in phototrophically grown Alphaproteobacterium Rhodobacter sphaeroides by transcriptome profiling, mutational analysis, and measurements of compatible solutes and membrane phospholipids. After exposure to salt stress, genes encoding two putative glycine betaine uptake systems, proVWX and betS, were highly upregulated. Mutational analysis revealed that BetS, not ProVWX, was the primary transporter of this compatible solute. Upon the addition of salt, exogenous glycine betaine was taken up rapidly, and maximal intracellular levels were reached within minutes. In contrast, synthesis of another important compatible solute in R. sphaeroides, trehalose, increased slowly following salt stress, reaching maximal levels only after several hours. This accumulation pattern was consistent with the more gradual increase in salt-induced transcription of the trehalose biosynthesis operon otsBA. Several genes encoding putative transcription factors were highly induced by salt stress. Multiple copies of one of these factors, crpO (RSP1275), whose product is a member of the cyclic AMP receptor protein/fumarate and nitrate reduction regulator (CRP/FNR) family, improved NaCl tolerance. When crpO was provided in multicopy, expression of genes for synthesis or transport of compatible solutes was unaltered, but the membrane phospholipid composition became biased toward that found in salt-stressed cells. Collectively, this study characterized transcriptional responses to salt stress, correlated changes in transcription with compatible solute accumulation rates, identified the main glycine betaine transporter and trehalose synthase, characterized salt-induced changes in phospholipid composition, and uncovered a transcription factor associated with changes in phospholipids. These findings set the stage for deciphering the salt stress-responsive regulatory network in R. sphaeroides.

Triplet state of the primary donor in reaction centers of the phototrophic bacterium Rhodobacter sphaeroides R26 with active photoinduced electron transfer

EPR characteristics of transient paramagnetic states photoinduced in isolated reaction centers of Rhodobacter sphaeroides R26 with intact electron transfer have been studied. It was demonstrated that the detected weak triplet state EPR signal belongs to the primary donor molecule and is populated via the conventional mechanism of radical pair S-T0 mixing. The distortion of the spectral shape of this signal is explained by the triplet quantum yield anisotropy brought about by the short lifetime of precursor radical pairs. The angular dependence of the anisotropy was evaluated. It was shown that the spectral shape of the triplet state of photosystem II reaction center observed in the case of singly-reduced primary quinone acceptor can also be described by the anisotropic quantum yield of the triplet, with practically the same angular dependence. These properties confirm the conclusions on the mechanism of photoinduced electron transfer in photosystem II, made in previous publications. The peculiarities in the functioning of photosystem II reaction centers are probably determined by strict limitations on the triplet state generation.

Enhanced biohydrogen generation from organic wastewater containing NH 4 + by phototrophic bacteria Rhodobacter sphaeroides AR-3

NH4+ is typically an inhibitor to hydrogen production from organic wastewater by photo-bacteria. In this experiment, biohydrogen generation with wild-type anoxygenic phototrophic bacterium Rhodobacter sphaeroideswas found to be sensitive to NH4+ due to the significant inhibition of NH4+ to its nitrogenase. In order to avoid the inhibition of NH4+ to biohydrogen generation by R. sphaeroides, a glutamine auxotrophic mutant R. sphaeroides AR-3 was obtained by mutagenizing with ethyl methane sulfonate. The AR-3 mutant could generate biohydrogen efficiently in the hydrogen production medium with a higher NH4+ concentration, because the inhibition of NH4+ to nitrogenase of AR-3 was released. Under suitable conditions, AR-3 effectively produced biohydrogen from tofu wastewater, which normally contains 50–60 mg/L NH4+, with an average generation rate of 14.2 mL/L·h. This generation rate was increased by more than 100% compared with that from wild-type R. sphaeroides.

Characterization of an Unusual LOV Domain Protein in the α‐Proteobacterium Rhodobacter sphaeroides

The facultatively phototrophic purple bacterium Rhodobacter sphaeroides 2.4.1 harbors a LOV (light, oxygen and voltage) domain protein, which shows a particular structure. LOV domains perceive blue light by a noncovalently bound flavin and transmit the signal to various coupled output domains. Proteins, that harbor a LOV core, function e.g. as phototropins or circadian clock regulators. Jalpha helices, which act as linker between the LOV core and the output domain, were shown to be involved in the light-dependent activation of the output domain. Like PpSB2 from Pseudomonas putida, the LOV domain protein of R. sphaeroides is not coupled to an effector domain and harbors an extended C-terminal alpha helix. We expressed the R. sphaeroides LOV domain recombinantly in Escherichia coli. The protein binds an FMN as a cofactor and shows a photocycle typical for LOV domain containing proteins. In R. sphaeroides, we detected the protein as well in the cytoplasm as in the membrane fraction, which was not reported for other bacterial LOV domain proteins.

Isolation and purification of malate dehydrogenase isoforms from phototrophic purple bacteria Rhodobacter sphaeroides and Rhodopseudomonas palustris

A five-step procedure was used to obtain electrophoretically pure preparations of malate dehydrogenase (EC 1.1.1.37) from Rhodobacter sphaeroides and Rhodopseudomonas palustris. The procedure included extraction, ammonium sulfate fractionation, gel filtration, and ion exchange and gel permeation chromatography. The enzyme was found to exist in two isoforms, dimeric and tetrameric, formed by the oligomerization of identical subunits. The isoforms are assumed to be involved in different metabolic processes.

No single way to understand singlet oxygen signalling in plants

When plant cells are under environmental stress, several chemically distinct reactive oxygen species (ROS) are generated simultaneously in various intracellular compartments and these can cause oxidative damage or act as signals. The conditional flu mutant of Arabidopsis, which generates singlet oxygen in plastids during a dark-to-light transition, has allowed the biological activity of singlet oxygen to be determined, and the criteria to distinguish between cytotoxicity and signalling of this particular ROS to be defined. The genetic basis of singlet-oxygen-mediated signalling has been revealed by the mutation of two nuclear genes encoding the plastid proteins EXECUTER (EX)1 and EX2, which are sufficient to abrogate singlet-oxygen-dependent stress responses. Conversely, responses due to higher cytotoxic levels of singlet oxygen are not suppressed in the ex1/ex2 background. Whether singlet oxygen levels lower than those that trigger genetically controlled cell death activate acclimation is now under investigation.

EFFECTS OF THE HERBICIDE, BUTACHLOR, ON NITROGEN FIXATION IN PHOTOTROPHIC NONSULFUR BACTERIA

In an effort to identify possible microbes for seeking bioagents for remediation of herbicide-contaminated soils, seven species of phototrophic nonsulfur bacteria (Rhodobacter capsulatus and sphaeroides, Rhodospirillum rubrum, Rhodopseudomonas acidophila, blastica and viridis, Rhodomicrobium vannielii) were grown in the presence of the herbicide, butachlor, and bacterial growth rates and nitrogen fixation were measured with different carbon sources. Under general conditions, all species showed 17-53% reductions in growth rate following butachlor treatment. Under nitrogen-fixing conditions, Rb. capsulatus and Rs. rubrum showed 1-4% increases in the growth rates and 2-10% increases in nitrogen-fixing abilities, while the other 5 species showed decreases of 17-47% and 17-85%, respectively. The finding that Rp. acidophila, Rp. blastica, Rp. viridis and Rm. vannielii showed stronger inhibitions of nitrogenase activity seems to indicate that species in genera Rhodobacter and Rhodospirillum are less influenced by butachlor than those in Rhodopseudomonas and Rhodomicrobium in terms of nitrogen-fixing ability. Overall, nitrogenase activity was closely correlated with both growth rate and glutamine synthetase activity (representing nitrogen metabolism). When the carbon sources were compared, pyruvate (three carbons) was best for all species in terms of growth rate and nitrogen fixation, with malate (four carbons) showing intermediate values and ribose (five carbons) showing the lowest; these trends did not change in response to butachlor treatment. We verified that each of the 7 species had a plasmid (12.2~23.5 Kb). We found that all 7 species could use butachlor as a sole carbon source and 3 species were controlled by plasmid-born genes, but it is doubtful whether plasmid-born genes were responsible to nitrogen fixation.

Protein Synthesis Patterns Reveal a Complex Regulatory Response to Singlet Oxygen inRhodobacter

Singlet oxygen (1O2) is a stress factor and signal in the facultative phototrophic bacterium Rhodobacter sphaeroides. In vivo protein labeling with L-[35S]-methionine and analysis by two-dimensional gel electrophoresis revealed that the synthesis of 61 proteins was changed in response to 1O2. After 1O2 treatment, protein synthesis patterns were distinct from those after H2O2 treatment but similar to those after high light exposure. This indicates regulatory mechanisms selective for different reactive oxygen species (ROS) and a response to light partly mediated by 1O2. Analysis of mutant strains support that the response to 1O2 is regulated mainly by rpoE (sigma E), but also a modulation of the sigma E dependent response by other factors and the existence of sigma E independent responses. The involvement of the RNA chaperon Hfq in the 1O2 response implies a role of small regulatory RNAs.

An Unorthodox Bacteriophytochrome from Rhodobacter sphaeroides Involved in Turnover of the Second Messenger c-di-GMP

Bacteriophytochromes are bacterial photoreceptors that sense red/far red light using the biliverdin chromophore. Most bacteriophytochromes work as photoactivated protein kinases. The Rhodobacter sphaeroides bacteriophytochrome BphG1 is unconventional in that it has GGDEF and EAL output domains, which are involved, respectively, in synthesis (diguanylate cyclase) and degradation (phosphodiesterase) of the bacterial second messenger c-di-GMP. The GGDEF-EAL proteins studied to date displayed either diguanylate cyclase or phosphodiesterase activity but not both. To elucidate the function of BphG1, the holoprotein was purified from an Escherichia coli overexpression system designed to produce biliverdin. The holoprotein contained covalently bound biliverdin and interconverted between the red (dark) and far red (light-activated) forms. BphG1 had c-di-GMP-specific phosphodiesterase activity. Unexpectedly for a photochromic protein, this activity was essentially light-independent. BphG1 expressed in E. coli was found to undergo partial cleavage into two species. The smaller species was identified as the EAL domain of BphG1. It possessed c-di-GMP phosphodiesterase activity. Surprisingly, the larger species lacking EAL possessed diguanylate cyclase activity, which was dependent on biliverdin and strongly activated by light. BphG1 therefore is the first phytochrome with a non-kinase photoactivated enzymatic activity. This shows that the photosensory modules of phytochromes can transmit light signals to various outputs. BphG1 is potentially the first "bifunctional" enzyme capable of both c-di-GMP synthesis and hydrolysis. A model for the regulation of the "opposite" activities of BphG1 is presented.

Induction of stress proteins in the phototrophic bacterium Rhodobacter sphaeroides

The stress response of the phototrophic bacterium Rhodobacter sphaeroides was investigated by two-dimensional gel electrophoresis. Oxygen, 0.5% and 4% ethanol, UV radiation, heat shock at 42°C and 0.01% phenanthrene were tested as stress factors. The protein pattern on two-dimensional gels of stressed as compared to control cells revealed that all stress factors applied caused modifications in the protein pattern of R. sphaeroides. The intensity of particular spots increased or decreased as a consequence of altered protein synthesis. Specific and general stress responses were observed.

Structural evidence for a proton transfer pathway coupled with haem reduction of cytochrome c″ from Methylophilus methylotrophus

The crystal structures of the oxidized and reduced forms of cytochrome c" from Methylophilus methylotrophus were solved from X-ray synchrotron data to atomic resolution. The overall fold of the molecule in the two redox states is very similar and is comparable to that of the oxygen-binding protein from the purple phototrophic bacterium Rhodobacter sphaeroides. However, significant modifications occur near the haem group, in particular the detachment from axial binding of His95 observed upon reduction as well as the adoption of different conformations of some protonatable residues that form a possible proton path from the haem pocket to the protein surface. These changes are associated with the previously well characterized redox-Bohr behaviour of this protein. Furthermore they provide a model for one of the presently proposed mechanisms of proton translocation in the much more complex protein cytochrome c oxidase.

Nucleotide sequence based characterizations of two cryptic plasmids from the marine bacterium Ruegeria isolate PR1b

Two plasmids, 76 and 148 kb in size, isolated from Ruegeria strain PR1b were entirely sequenced. These are the first plasmids to be characterized from this genus of marine bacteria. Sequence analysis revealed a biased distribution of function among the putative proteins encoded on the two plasmids. The smaller plasmid, designated pSD20, encodes a large number of putative proteins involved in polysaccharide biosynthesis and export. The larger plasmid, designated pSD25, primarily encodes putative proteins involved in the transport of small molecules and in DNA mobilization. Sequence analysis revealed uncommon potential replication systems on both plasmids. pSD25, the first repABC-type replicon isolated from the marine environment, actually contains two repABC-type replicons. pSD20 contains a complex replication region, including a replication origin and initiation protein similar to iteron-containing plasmids (such as pSW500 from the plant pathogen Erwinia stewartii) linked to putative RepA and RepB stabilization proteins of a repABC-type replicon and is highly homologous to a plasmid from the phototrophic bacterium Rhodobacter sphaeroides. Given the nature of the putative proteins encoded by both plasmids it is possible that these plasmids enhance the metabolic and physiological flexibility of the host bacterium, and thus its adaptation to the marine sediment environment.

Effect of selenite on growth and protein synthesis in the phototrophic bacterium Rhodobacter sphaeroides.

The effect of selenite on the growth rate and protein synthesis has been investigated in Rhodobacter sphaeroides. This photosynthetic bacterium efficiently reduces selenite with intracellular accumulation under both dark aerobic and anaerobic photosynthetic conditions. Addition of 1 mM selenite under these two growth conditions does not affect the final cell density, although a marked slowdown in growth rate is observed under aerobic growth. The proteome analysis of selenite response by two-dimensional gel electrophoresis shows an enhanced synthesis of some chaperones, an elongation factor, and enzymes associated to oxidative stress. The induction of these antioxidant proteins confirms that the major toxic effect of selenite is the formation of reactive oxygen species during its metabolism. In addition, we show that one mutant unable to precipitate selenite, selected from a transposon library, is affected in the smoK gene. This encodes a constituent of a putative ABC transporter implicated in the uptake of polyols. This mutant is less sensitive to selenite and does not express stress proteins identified in the wild type in response to selenite. This suggests that the entry of selenite into the cytoplasm is mediated by a polyol transporter in R. sphaeroides.

Mobile cytochrome c2 and membrane-anchored cytochrome cy are both efficient electron donors to the cbb3- and aa3-type cytochrome c oxidases during respiratory growth of Rhodobacter sphaeroides.

We have recently established that the facultative phototrophic bacterium Rhodobacter sphaeroides, like the closely related Rhodobacter capsulatus species, contains both the previously characterized mobile electron carrier cytochrome c2 (cyt c2) and the more recently discovered membrane-anchored cyt cy. However, R. sphaeroides cyt cy, unlike that of R. capsulatus, is unable to function as an efficient electron carrier between the photochemical reaction center and the cyt bc1 complex during photosynthetic growth. Nonetheless, R. sphaeroides cyt cy can act at least in R. capsulatus as an electron carrier between the cyt bc1 complex and the cbb3-type cyt c oxidase (cbb3-Cox) to support respiratory growth. Since R. sphaeroides harbors both a cbb3-Cox and an aa3-type cyt c oxidase (aa3-Cox), we examined whether R. sphaeroides cyt cy can act as an electron carrier to either or both of these respiratory terminal oxidases. R. sphaeroides mutants which lacked either cyt c2 or cyt cy and either the aa3-Cox or the cbb3-Cox were obtained. These double mutants contained linear respiratory electron transport pathways between the cyt bc1 complex and the cyt c oxidases. They were characterized with respect to growth phenotypes, contents of a-, b-, and c-type cytochromes, cyt c oxidase activities, and kinetics of electron transfer mediated by cyt c2 or cyt cy. The findings demonstrated that both cyt c2 and cyt cy are able to carry electrons efficiently from the cyt bc1 complex to either the cbb3-Cox or the aa3-Cox. Thus, no dedicated electron carrier for either of the cyt c oxidases is present in R. sphaeroides. However, under semiaerobic growth conditions, a larger portion of the electron flow out of the cyt bc1 complex appears to be mediated via the cyt c2-to-cbb3-Cox and cyt cy-to-cbb3-Cox subbranches. The presence of multiple electron carriers and cyt c oxidases with different properties that can operate concurrently reveals that the respiratory electron transport pathways of R. sphaeroides are more complex than those of R. capsulatus.

Axenic cultivation of anoxygenic phototrophic bacteria, cyanobacteria, and microalgae in a new closed tubular glass photobioreactor.

A low-cost closed tubular glass photobioreactor allowing axenic cultivation of phototrophic microorganisms was constructed. Standard glass tubes were arranged in a helical array providing a working volume of 80 1. The glass tubes were connected with a degassing chamber, which also provided ports for measuring and regulating oxygen supply, pH, foam, and optical density and for adding substrates and antifoam agents as well as disposing of vent gas. A pump module allowed agitation of the medium in the bioreactor at a laminar flow rate of 1.5 m/s. Upstream of the pump module a gas inlet was located, allowing efficient mixing of the used gases with the medium. The temperature of the medium was controlled by a Pt-100 sensor and by a heat exchanger with an effective surface of 0.12 m2 connected to an external thermostat. Irradiation was provided by three light panels each consisting of ten fluorescent tubes. The entire photobioreactor - apart from the light panels and motor - could be sterilized at 121 degrees C in an autoclave. In addition to a detailed description of this photobioreactor, we report on first experiments to cultivate the anoxygenic phototrophic bacteria Rhodobacter sphaeroides and Rhodospirillum rubrum, the oxygenic phototrophic cyanobacterium Synechocystis sp. strain PCC6803, and the microalga Chlorella sp. in this photobioreactor.

Morphological changes in phototrophic bacteria induced by metalloid oxyanions

The phototrophic bacteria Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodospirillum rubrum, Rhodobacter blasticus and Rhodocyclus tenuis alter their cell size when exposed to the metalloid oxyanions chromate, selenate and arsenate at concentrations which only slightly reduce the growth rate of the organisms. Dependent on the oxyanions the cells increased in size up to tenfold and more. Selenate and arsenate have the biggest impact on the morphology of the bacteria. These microscopic observations were confirmed by counting and sizing the cells in a Neubauer counting chamber and by flow cytometry. In addition comparison of the protein pattern of arsenate stressed cells with the proteins from control cells showed that the morphological changes are paralleled by changes on the protein level.

Cytochrome c″ from the obligate methylotroph Methylophilus methylotrophus, an unexpected homolog of sphaeroides heme protein from the phototroph Rhodobacter sphaeroides

The complete primary structure of an unusual soluble cytochrome c isolated from the obligate methylotrophic bacterium Methylophilus methylotrophus has been determined to contain 124 amino acids and to have an average molecular mass of 14 293.0 Da. The sequence has two unusual features: firstly, the location of the heme-binding cysteines is far downstream from the N-terminus, namely at positions 49 and 52; secondly, an extra pair of cysteine residues is present near the C-terminus. In both respects, cytochrome c″ is similar to the oxygen-binding heme protein SHP from the purple phototrophic bacterium Rhodobacter sphaeroides. In contrast to SHP, cytochrome c″ changes from low-spin to high-spin upon reduction, due to dissociation of a sixth heme ligand histidine which is identified as His-95 by analogy to the class I cytochromes c. The distance of His-95 from the heme (41 residues) and the presence of certain consensus residues suggests that cytochrome c″ is the second example of a variant class I cytochrome c.

Hydrogen production from tofu wastewater by Rhodobacter sphaeroides immobilized in agar gels

Hydrogen production from the wastewater of tofu factory was examined by using anoxygenic phototrophic bacterium Rhodobacter sphaeroides immobilized in agar gels. The maximum rate of hydrogen production observed from the wastewater was 2.1 l h −1 m 2 gel which was even slightly higher than that from a glucose medium (as control). The hydrogen production lasted up to 50 h. The yield of hydrogen was 1.9 ml⧸mlwastewater or 0.24 ml⧸mg carbohydrates contained in the wastewater. This yield corresponds to53% or 65% of that from the glucose medium, according to the different expressions of the yield. The TOC (total organic carbon) removal ratio in 85 h reached 41% which was comparable to that from the glucose medium. The immobilization protected the bacterium from the inhibitory effect of ammonium ion. © 1999 International Association for Hydrogen Energy. Published by Elsevier Science Ltd. All rights reserved.