Phototrophic Bacterium Rhodobacter Sphaeroides Research Articles

Nickel ion extracellular uptake by the phototrophic bacterium Rhodobacter sphaeroides: new insights from Langmuir modelling and X-ray photoelectron spectroscopic analysis

The presence of anionic functional groups on the surface of microbial cells makes the bacterial biomass very attractive as metal cation removal tool for environmental bioremediation. To go insight to the heavy metal sorption abilities of Gram-negative bacteria, we characterized the extracellular uptake of Ni2+ ions by the phototrophic bacterium Rhodobacter sphaeroides, whose ability to sequester toxic metal ions has been widely documented. The sorption process proved to be well described by the Langmuir model, suggesting that the cell envelope behaves as an ideal adsorbent surface, showing a layer of chemically equivalent binding sites with high affinity for Ni2+. X-ray photoelectron spectroscopic data reveal that, at binding site saturation, Ni2+ ions fully displace alkali metal ions from the bacterial surface, while they are unable to displace Ca2+ and Mg2+ from their tight binding sites. The analysis of carbon, nitrogen and oxygen speciation, as arisen from high resolution spectra fitting, highlights that the Ni2+/K+ cation exchange affects the surface distribution of main chemical components, with increased contribution by peptides and phosphatidylcholines. A role of Ni2+ ions in favoring the stacking and/or packing of lipopolysaccharide sugar chains by interaction with carboxylate groups is proposed and discussed.

Experimental Optimization of Green Hydrogen Production from Phototrophic Bacteria Rhodobacter sphaeroides

Background and Objective: This study utilizes Rhodobacter sphaeroides bacteria for the photoproduction of hydrogen under various cultural conditions. R. sphaeroides was isolated from sewage water. We have examined different carbon and nitrogen sources for hydrogen production and further established the conditions for optimum hydrogen production by R. sphaeroides. Methods: The cumulative hydrogen produced by the bacteria at various intervals of time was measured using a Gas Chromatograph. Initially, by classical one factor at a time method, it was found that Benzoate and Glycine promote higher amounts of hydrogen production under anaerobic light conditions after 96 h. Results: The production was also observed to be enhanced in the presence of growth factors B12. Further, the Response Surface Methodology (RSM) was employed to optimize the hydrogen production. The first level of optimization was done using Box-Behnken Design (BBD) followed by Central Composite Design (CCD) method. The maximum production of hydrogen achieved by BBD and CCD was 6.8 ml/30 ml and 8.12 ml/30 ml, respectively. The significant model predicted is a quadratic model with R2 value 0.9459. Conclusion: Moreover, work presented here suggests an environment-friendly approach of harvesting H2, which could meet energy demand as clean fuel via the green route.

Engineering of B800 bacteriochlorophyll binding site specificity in the Rhodobacter sphaeroides LH2 antenna

The light-harvesting 2 complex (LH2) of the purple phototrophic bacterium Rhodobacter sphaeroides is a highly efficient, light-harvesting antenna that allows growth under a wide-range of light intensities. In order to expand the spectral range of this antenna complex, we first used a series of competition assays to measure the capacity of the non-native pigments 3-acetyl chlorophyll (Chl) a, Chl d, Chl f or bacteriochlorophyll (BChl) b to replace native BChl a in the B800 binding site of LH2. We then adjusted the B800 site and systematically assessed the binding of non-native pigments. We find that Arg−10 of the LH2 β polypeptide plays a crucial role in binding specificity, by providing a hydrogen-bond to the 3-acetyl group of native and non-native pigments. Reconstituted LH2 complexes harbouring the series of (B)Chls were examined by transient absorption and steady-state fluorescence spectroscopies. Although slowed 10-fold to ~6 ps, energy transfer from Chl a to B850 BChl a remained highly efficient. We measured faster energy-transfer time constants for Chl d (3.5 ps) and Chl f (2.7 ps), which have red-shifted absorption maxima compared to Chl a. BChl b, red-shifted from the native BChl a, gave extremely rapid (≤0.1 ps) transfer. These results show that modified LH2 complexes, combined with engineered (B)Chl biosynthesis pathways in vivo, have potential for retaining high efficiency whilst acquiring increased spectral range.

Agrobacterial ipt gene for cytokinin biosynthesis is found in phototrophic non-sulfur purple bacteria Rhodobacter sphaeroides and Rhodopseudomonas palustris

Phototrophic non-sulfur bacteria (PNSB) are close relatives of cytokinin producing agrobacteria. However, the sequenced PNSB genomes do not have the ipt gene sequences involved in the cytokinin biosynthesis. In Rhodobacter sphaeroides and Rhodopseudomonas palustris transformed with ipt, we previously observed the substantial morphological and physiological changes as compared with the original strains. To find associations of these changes with the ipt function, its expression levels were studied in the transformants and the parent strains. Unexpectedly, the ipt transcripts were found in both the transformants and the original untransformed strains, although the ipt expression level was several orders of magnitude lower in the latter compared to the transformants. The ipt sequences amplified from the plasmid DNA templates of the parent PNSB strains showed that they were identical to each other but differed in four nucleotides compared to the ipt gene from the Agrobacterium tumefaciens pTiBo 542 plasmid. The in silico comparison amongst the ipt sequences from the other agrobacterial plasmids, as well as from the other PNSB, demonstrated their low identity. This is the first report on the active gene of the cytokinin biosynthesis found in Rba. sphaeroides and Rps. palustris which is homologous to the agrobacterial ipt gene.

Modification of Gold Electrodes with Bacterial Reaction Centres Immobilized by Laser Induced Forward Transfer (LIFT) Technique for Amperometric Herbicide Detection

The functionalization of screen-printed electrodes (SPEs) with a thin film of reaction centre (RC) proteins from the phototrophic bacterium Rhodobacter (R.) sphaeroides, by means of laser induced forward transfer (LIFT) technique, allowed the fabrication of robust and sensitive bio-hybrid devices for terbutryn detection and analysis. The optimal wiring between RCs and the gold electrode surface, achieved by LIFT, led to the generation of cathodic photocurrents sustained by a direct electron transfer (DET) mechanism, which were attenuated by addition of the herbicide inhibitor.

New insights into the photochemistry of carotenoid spheroidenone in light-harvesting complex 2 from the purple bacterium Rhodobacter sphaeroides

Light-harvesting complex 2 (LH2) from the semi-aerobically grown purple phototrophic bacterium Rhodobacter sphaeroides was studied using optical (static and time-resolved) and resonance Raman spectroscopies. This antenna complex comprises bacteriochlorophyll (BChl) a and the carotenoid spheroidenone, a ketolated derivative of spheroidene. The results indicate that the spheroidenone-LH2 complex contains two spectral forms of the carotenoid: (1) a minor, “blue” form with an S2 (11Bu+) spectral origin band at 522 nm, shifted from the position in organic media simply by the high polarizability of the binding site, and (2) the major, “red” form with the origin band at 562 nm that is associated with a pool of pigments that more strongly interact with protein residues, most likely via hydrogen bonding. Application of targeted modeling of excited-state decay pathways after carotenoid excitation suggests that the high (92%) carotenoid-to-BChl energy transfer efficiency in this LH2 system, relative to LH2 complexes binding carotenoids with comparable double-bond conjugation lengths, derives mainly from resonance energy transfer from spheroidenone S2 (11Bu+) state to BChl a via the Qx state of the latter, accounting for 60% of the total transfer. The elevated S2 (11Bu+) → Qx transfer efficiency is apparently associated with substantially decreased energy gap (increased spectral overlap) between the virtual S2 (11Bu+) → S0 (11Ag−) carotenoid emission and Qx absorption of BChl a. This reduced energetic gap is the ultimate consequence of strong carotenoid–protein interactions, including the inferred hydrogen bonding.

Characteristics of Pos19 - A Small Coding RNA in the Oxidative Stress Response of Rhodobacter sphaeroides.

The phototrophic bacterium Rhodobacter sphaeroides induces several small RNAs (sRNAs) when singlet oxygen (1O2) levels are elevated, a situation also referred to as photo-oxidative stress. An RNA-seq study identified the RSs0019 sRNA, which is renamed Pos19 (photo-oxidative stress induced sRNA 19). Pos19 is part of the RpoE regulon and consequently induced upon 1O2 and peroxide stress. The 219 nt long Pos19 transcript contains a small open reading frame (sORF) of 150 nt, which is translated in vivo. Over-expression of Pos19 results in reduced mRNA levels for several genes, of which numerous are involved in sulfur metabolism. The negative effect on the potential targets is maintained even when translation of the sORF is abolished, arguing that regulation is entailed by the sRNA itself. Reporter studies further revealed that regulation of the most affected mRNA, namely RSP_0557, by Pos19 is Hfq-dependent. Direct binding of Pos19 to Hfq was shown by co-immunoprecipitation. Physiological experiments indicated Pos19 to be involved in the balance of glutathione biosynthesis. Moreover, a lack of Pos19 leads to elevated reactive oxygen species levels. Taken together our data identify the sRNA Pos19 as a coding sRNA with a distinct expression pattern and potential role under oxidative stress in the phototrophic bacterium R. sphaeroides.

Absence of the cbb3 Terminal Oxidase Reveals an Active Oxygen-Dependent Cyclase Involved in Bacteriochlorophyll Biosynthesis in Rhodobacter sphaeroides.

ABSTRACTThe characteristic green color associated with chlorophyll pigments results from the formation of an isocyclic fifth ring on the tetrapyrrole macrocycle during the biosynthesis of these important molecules. This reaction is catalyzed by two unrelated cyclase enzymes employing different chemistries. Oxygenic phototrophs such as plants and cyanobacteria utilize an oxygen-dependent enzyme, the major component of which is a diiron protein named AcsF, while BchE, an oxygen-sensitive [4Fe-4S] cluster protein, dominates in phototrophs inhabiting anoxic environments, such as the purple phototrophic bacterium Rhodobacter sphaeroides. We identify a potential acsF in this organism and assay for activity of the encoded protein in a strain lacking bchE under various aeration regimes. Initially, cells lacking bchE did not demonstrate AcsF activity under any condition tested. However, on removal of a gene encoding a subunit of the cbb3-type respiratory terminal oxidase, cells cultured under regimes ranging from oxic to micro-oxic exhibited cyclase activity, confirming the activity of the oxygen-dependent enzyme in this model organism. Potential reasons for the utilization of an oxygen-dependent enzyme in anoxygenic phototrophs are discussed.IMPORTANCE The formation of the E ring of bacteriochlorophyll pigments is the least well characterized step in their biosynthesis, remaining enigmatic for over 60 years. Two unrelated enzymes catalyze this cyclization step; O2-dependent and O2-independent forms dominate in oxygenic and anoxygenic phototrophs, respectively. We uncover the activity of an O2-dependent enzyme in the anoxygenic purple phototrophic bacterium Rhodobacter sphaeroides, initially by inactivation of the high-affinity terminal respiratory oxidase, cytochrome cbb3. We propose that the O2-dependent form allows for the biosynthesis of a low level of bacteriochlorophyll under oxic conditions, so that a rapid initiation of photosynthetic processes is possible for this bacterium upon a reduction of oxygen tension.

Dimerization of core complexes as an efficient strategy for energy trapping in Rhodobacter sphaeroides

In the purple phototrophic bacterium Rhodobacter sphaeroides, light harvesting LH2 complexes transfer absorbed solar energy to RC–LH1–PufX core complexes, which are mainly found in the dimeric state. Many other purple phototrophs have monomeric core complexes and the basis for requiring dimeric cores is not fully established, so we analysed strains of Rba. sphaeroides that contain either native dimeric core complexes or altered monomeric cores harbouring a deletion of the first 12 residues from the N-terminus of PufX, which retains the PufX polypeptide but removes the major determinant of core complex dimerization. Membranes were purified from strains with dimeric or monomeric cores, and with either high or low levels of the LH2 complex. Samples were interrogated with absorption, steady-state fluorescence, and picosecond time-resolved fluorescence kinetic spectroscopies to reveal their light-harvesting and energy trapping properties. We find that under saturating excitation light intensity the photosynthetic membranes containing LH2 and monomeric core complexes have fluorescence lifetimes nearly twice that of membranes with LH2 plus dimeric core complexes. This trend of increased lifetime is maintained with RCs in the open state as well, and for two different levels of LH2 content. Thus, energy trapping is more efficient when photosynthetic membranes of Rba. sphaeroides consist of RC–LH1–PufX dimers and LH2 complexes.

Extract from a mutant Rhodobacter sphaeroides as an enriched carotenoid source

BackgroundThe extract Lycogen™ from the phototrophic bacterium Rhodobacter sphaeroides (WL-APD911) has attracted significant attention because of its promising potential as a bioactive mixture, attributed in part to its anti-inflammatory properties and anti-oxidative activity.ObjectiveThis study aims to investigate the components of Lycogen™ and its anti-inflammatory properties and anti-oxidative activity.Design and resultsThe mutant strain R. sphaeroides (WL-APD911) whose carotenoid 1,2-hydratase gene has been altered by chemical mutagenesis was used for the production of a new carotenoid. The strain was grown at 30°C on Luria–Bertani (LB) agar plates. After a 4-day culture period, the mutant strain displayed a 3.5-fold increase in carotenoid content, relative to the wild type. In the DPPH test, Lycogen™ showed more potent anti-oxidative activity than lycopene from the wild-type strain. Primary skin irritation test with hamsters showed no irritation response in hamster skins after 30 days of treatment with 0.2% Lycogen™. Chemical investigations of Lycogen™ using nuclear magnetic resonance (NMR) 1H, 13C, and COSY/DQCOSY spectra have identified spheroidenone and methoxyneurosporene. Quantitative analysis of these identified compounds based on spectral intensities indicates that spheroidenone and methoxyneurosporene are major components (approximately 1:1); very small quantities of other derivatives are also present in the sample.ConclusionsIn this study, we identified the major carotenoid compounds contained in Lycogen™, including spheroidenone and methoxyneurosporene by high-resolution NMR spectroscopy analysis. The carotenoid content of this mutant strain of R. sphaeroides was 3.5-fold higher than that in normal strain. Furthermore, Lycogen™ from the mutant strain is more potent than lycopene from the wild-type strain and does not cause irritation in hamster skins. These findings suggest that this mutant strain has the potential to be used as an enriched carotenoid source.

A cluster of four homologous small RNAs modulates C1 metabolism and the pyruvate dehydrogenase complex in Rhodobacter sphaeroides under various stress conditions.

In bacteria, regulatory RNAs play an important role in the regulation and balancing of many cellular processes and stress responses. Among these regulatory RNAs, trans-encoded small RNAs (sRNAs) are of particular interest since one sRNA can lead to the regulation of multiple target mRNAs. In the purple bacterium Rhodobacter sphaeroides, several sRNAs are induced by oxidative stress. In this study, we focused on the functional characterization of four homologous sRNAs that are cotranscribed with the gene for the conserved hypothetical protein RSP_6037, a genetic arrangement described for only a few sRNAs until now. Each of the four sRNAs is characterized by two stem-loops that carry CCUCCUCCC motifs in their loops. They are induced under oxidative stress, as well as by various other stress conditions, and were therefore renamed here sRNAs CcsR1 to CcsR4 (CcsR1-4) for conserved CCUCCUCCC motif stress-induced RNAs 1 to 4. Increased CcsR1-4 expression decreases the expression of genes involved in C1 metabolism or encoding components of the pyruvate dehydrogenase complex either directly by binding to their target mRNAs or indirectly. One of the CcsR1-4 target mRNAs encodes the transcriptional regulator FlhR, an activator of glutathione-dependent methanol/formaldehyde metabolism. Downregulation of this glutathione-dependent pathway increases the pool of glutathione, which helps to counteract oxidative stress. The FlhR-dependent downregulation of the pyruvate dehydrogenase complex reduces a primary target of reactive oxygen species and reduces aerobic electron transport, a main source of reactive oxygen species. Our findings reveal a previously unknown strategy used by bacteria to counteract oxidative stress. Phototrophic organisms have to cope with photo-oxidative stress due to the function of chlorophylls as photosensitizers for the formation of singlet oxygen. Our study assigns an important role in photo-oxidative stress resistance to a cluster of four homologous sRNAs in the anoxygenic phototrophic bacterium Rhodobacter sphaeroides. We reveal a function of these regulatory RNAs in the fine-tuning of C1 metabolism. A model that relates oxidative stress defense to C1 metabolism is presented.

Inoculum Sizes of Locally Isolated Phototrophic Bacterium on the Utilization of Palm Oil Mill Effluent

To produce Rhodobacter sphaeroides strain UMSPSB3 biomass with the reduction of chemical oxygen demand (COD) from palm oil mill effluent. Locally isolated phototrophic bacterium with different inoculum levels were used in Palm Oil Mill effluent (POME). Collected POME was characterized before used as substrate. Inoculum of bacterium was grown in synthetic media and 48 hours inoculum was used to utilize the substrate. Biotechnological laboratory, Borneo Marine Research Institute, University Malaysia Sabah, Kota Kinabalu, Sabah, Malaysia, between February 2014 to April 2014. Growth characteristics of bacterium Rhodobacter sphaeroides strain UMSPSB3 was monitored at different light intensities. Later phototrophic bacterium Rhodobacter sphaeroides strain UMSPSB3 was grown in settled non-sterilized Palm Oil Mill effluent (POME). The growth characteristics of bacterium in term of dry cell weight and total carotenoids production, and reduction of COD were compared using 10%, 20% and 30% (v/v) levels of inoculum developed in synthetic 112 media.

Aberrant Assembly Complexes of the Reaction Center Light-harvesting 1 PufX (RC-LH1-PufX) Core Complex of Rhodobacter sphaeroides Imaged by Atomic Force Microscopy

In the purple phototrophic bacterium Rhodobacter sphaeroides, many protein complexes congregate within the membrane to form operational photosynthetic units consisting of arrays of light-harvesting LH2 complexes and monomeric and dimeric reaction center (RC)-light-harvesting 1 (LH1)-PufX "core" complexes. Each half of a dimer complex consists of a RC surrounded by 14 LH1 αβ subunits, with two bacteriochlorophylls (Bchls) sandwiched between each αβ pair of transmembrane helices. We used atomic force microscopy (AFM) to investigate the assembly of single molecules of the RC-LH1-PufX complex using membranes prepared from LH2-minus mutants. When the RC and PufX components were also absent, AFM revealed a series of LH1 variants where the repeating α(1)β(1)(Bchl)2 units had formed rings of variable size, ellipses, and spirals and also arcs that could be assembly products. The spiral complexes occur when the LH1 ring has failed to close, and short arcs are suggestive of prematurely terminated LH1 complex assembly. In the absence of RCs, we occasionally observed captive proteins enclosed by the LH1 ring. When production of LH1 units was restricted by lowering the relative levels of the cognate pufBA transcript, we imaged a mixture of complete RC-LH1 core complexes, empty LH1 rings, and isolated RCs, leading us to conclude that once a RC associates with the first α1β1(Bchl)2 subunit, cooperative associations between subsequent subunits and the RC tend to drive LH1 ring assembly to completion.

Metabolism of the phase variants of the phototrophic bacterium Rhodobacter sphaeroides

Growth, bacteriochlorophyll a content, electron transport chain (ETC), and activities of the tricarboxylic acid (TCA) cycle enzymes were studied in R and M phase variants of Rhodobactersphaeroides cells grown anaerobically in the light and aerobically in the dark. Under all cultivation conditions tested, bacteriochlorophyll a content was 2-3 times lower in the cells of the M. variant compared to the R variant, which therefore was predominant in the cultures grown in the light. In both variants, activity of all TCA cycle enzymes was higher for the cells grown in the dark under aerobic conditions. When grown aerobically in the dark, the R variant, unlike the M variant, did not contain cytochrome aa3, acting as. cytochrome c oxidase, in its ETC. An additional point of coupling the electron transfer to the generation of the proton gradient al the cytochrome aa3 level provided for more efficient oxidation of organic substrates, resulting in predominance of the M variant in the cultures grown in the dark under aerobic conditions.

Structual Peculiarities of Exopolysaccharides of Phototrophic Bacteria Rhodobacter Sphaeroides

For the first time, properties and structural peculiarities of exopolysaccharides produced by phototrophic bacteria Rhodobacter sphaeroides isolated from hydrocarbonate mineral waters in Armenia with a degree of mineralization in limits of 3.5-5.0g/l, pH 7.2 have been studied. It is noteworthy that despite membership of the isolated strains in one and the same genus, these strains are significantly different by the nature of the produced exooligosaccharide. The studies implemented show that phototrophic bacteria are able to synthesize exopolysaccharides with different compositions and structures: cyclic and linear, depending on the cultivation conditions.

Direct transition of pGA482:ipt plasmid bearing the cytokinin biosynthesis gene into the cells of phototrophic purple bacteria Rhodobacter sphaeroides and Rhodopseudomonas palustris by electroporation

Direct transition of pGA482:ipt plasmid bearing the cytokinin biosynthesis gene into the cells of phototrophic purple bacteria Rhodobacter sphaeroides and Rhodopseudomonas palustris by electroporation

A ternary AppA–PpsR–DNA complex mediates light regulation of photosynthesis-related gene expression

The anoxygenic phototrophic bacterium Rhodobacter sphaeroides uses different energy sources depending on environmental conditions including aerobic respiration or, in the absence of oxygen, photosynthesis. Photosynthetic genes are repressed at high oxygen tension, but at intermediate levels their partial expression prepares the organism for using light energy. Illumination, however, enhances repression under semi-aerobic conditions. Here, we describe molecular details of two proteins involved in oxygen- and light-control of photosynthesis gene expression, the light-sensing anti-repressor AppA and the transcriptional repressor PpsR. We combine information from crystal structures of both proteins and their complex with hydrogen-deuterium exchange data to show that light-activation of AppA–PpsR2 affects the PpsR effector region within the complex. DNA-binding studies demonstrate the formation of a light-sensitive ternary AppA–PpsR–DNA complex. Implications of these results for light- and oxygen-regulation are discussed, highlighting new insights into blue-light-mediated signal transduction.

Identification of an 8-vinyl reductase involved in bacteriochlorophyll biosynthesis in Rhodobacter sphaeroides and evidence for the existence of a third distinct class of the enzyme

The purple phototrophic bacterium Rhodobacter sphaeroides utilises bacteriochlorophyll a for light harvesting and photochemistry. The synthesis of this photopigment includes the reduction of a vinyl group at the C8 position to an ethyl group, catalysed by a C8-vinyl reductase. An active form of this enzyme has not been identified in R. sphaeroides, but its genome contains two candidate ORFs (open reading frames) similar to those reported to encode C8-vinyl reductases in the closely related Rhodobacter capsulatus (bchJ), and in plants and green sulfur bacteria (rsp_3070). To determine which gene encodes the active enzyme, knock-out mutants in both genes were constructed. Surprisingly, mutants in which one or both genes were deleted still retained the ability to synthesize C8-ethyl bacteriochlorophyll. These genes were subsequently expressed in a cyanobacterial mutant that cannot synthesize C8-ethyl chlorophyll a. R. sphaeroides rsp_3070 was able to restore synthesis of the WT (wild-type) C8-ethyl chlorophyll a in the mutant, whereas bchJ did not. The results of the present study demonstrate that Rsp_3070 is a functional C8-vinyl reductase and that R. sphaeroides utilises at least two enzymes to catalyse this reaction, indicating the existence of a third class, while there remains no direct evidence for the activity of BchJ as a C8-vinyl reductase.

Structural and Functional Proteomics of Intracytoplasmic Membrane Assembly in Rhodobacter sphaeroides

The results of a detailed structural and functional proteomic analysis of intracytoplasmic membrane (ICM) assembly in the model purple phototrophic bacterium Rhodobacter sphaeroides are reviewed in this report. Proteomics approaches have focused upon identification of membrane proteins temporally expressed during ICM development and spatially localized within the internal cell membranes, together with their structural and functional correlates. For the examination of temporal protein expression, procedures were established for the induction of ICM formation at low oxygen tension and for ICM remodeling in cells adapting to low intensity illumination, which permitted isolation by rate-zone sedimentation of ICM growth initiation sites (CM invaginations) in an upper-pigmented band (UPB), together with more mature ICM vesicles (chromatophores) as the main band. Nondenaturing clear native gel electrophoresis of the chromatophore fraction gave rise to four pigmented bands: the top and bottom bands contained the reaction center-light-harvesting 1 (RC-LH1) core complex and the LH2 peripheral antenna, respectively, while two bands of intermediate migration exhibited distinct associations of LH2 and core complexes. Proteomic analysis of the gel bands revealed developmental changes including increasing levels of LH2 polypeptides relative to those of core complexes as ICM development proceeded, as well as a large array of other associated proteins including high spectral counts for the F₁F_O-ATP synthase subunits, and the cytochrome bc₁ complex. High counts were also observed for RSP6124, a protein of unknown function, that were correlated with increasing LH2 levels. RC-LH1-containing clear native electrophoresis gel bands from the UPB were enriched in cytoplasmic membrane (CM) markers, including electron transfer and transport proteins, as well as general membrane assembly factors (viz., preprotein translocases YidC, YajC and SecY, bacterial type 1 signal peptidase and twin arg translocation subunit TatA), thereby confirming the origin of the UPB from both peripheral respiratory membrane and sites of active CM invagination in which preferential assembly of the RC-LH1 complex occurs. Functional aspects of the photosynthetic unit assembly process were monitored by fluorescence induction/relaxation measurements of the variable fluorescence arising from LH-bacteriochlorophyll a. Slowing of the rate of RC electron transfer turnover (τ_QA), as assessed from the relaxation phase, was correlated with the growth of the functional absorption cross section (σ) and LH2/LH1 molar ratios. This is thought to arise from the imposition of constraints upon free diffusion of ubiquinone (UQ) redox species between the RC and cytochrome bc₁ complex as the ICM bilayer becomes densely packed with LH2 rings. Such LH2 packing was confirmed in a comparison by high-resolution atomic force microscopy of ICM patches from cells grown at high and low light intensity [Adams and Hunter: Biochim Biophys Acta 2012;1817:1616-1627], in which the increasing LH2 levels form densely packed LH2-only domains, representing the light-responsive antenna complement arising under low illumination. In contrast, LH2 is initially dispersed in rows and small cluster-separating linear arrays of largely dimeric RC-LH1 core complexes, which become filled with LH2 during acclimation to reduced light intensity. In phototrophically grown cells that were transferred to oxic conditions in the dark, fluorescence induction/relaxation measurements showed that despite a growth burst independent of photosynthetic pathways, functional photosynthetic units were maintained for up to 24 h after the transition. The τ_QA was accelerated from ∼1 to 0.5 ms by 8 h, reflecting the decrease in LH2 levels, facilitating more rapid UQ redox species diffusion in the membrane bilayer as crowding by LH2 is overcome. Under these circumstances, UPB levels were elevated with significant increases in LH1/LH2 molar ratio. These changes indicate that vesiculation of CM growth initiation sites to form vesicular ICM was arrested under oxic conditions.

Culture conditions affecting H2 production by phototrophic bacterium Rhodobacter sphaeroides KD131

The effect of culture conditions on photo-H2 production was investigated using the photosynthetic bacterium Rhodobacter sphaeroides KD131. When the initial cell concentrations were either below or above a threshold of 0.56g-dcw/L, the H2 production decreased due to an imbalance between the biomass and the substrate. Malate- and succinate-fed cultures exhibited the highest substrate conversion to H2 production, whereas more than 85% of the substrate was utilized for cell growth in acetate- and butyrate-fed cultures. Compared with (NH4)2SO4, glutamate as a nitrogen source was more appropriate for the initial H2 production, but inhibited H2 evolution during extended cultivation due to released NH4+ ion. Even though the KD131 strain grew well under slightly acidic conditions, the pH value should be maintained in a neutral range in order to enhance H2 production. The highest H2 yield of 3.65mol-H2/mol-succinate was achieved when the KD131 strain grew in the succinate–glutamate medium with an initial cell concentration of 0.56g-dcw/L and the pH level controlled to 7.5.