An overlap between operons involved in carotenoid and bacteriochlorophyll biosynthesis in Rhodobacter capsulatus

A new example of superoperonal gene arrangement has been documented in the Rhodobacter capsulatus photosynthetic gene cluster. The promoter for the operon initiated by the bchI gene is embedded within an upstream operon for carotenoid synthesis. The stop codon for the crtA gene, the only gene in the first operon, overlaps the start codon of the downstream bchI gene. As a consequence of this overlap, the promoter(s) for the bch operon must be located within the crtA structural gene. The bchI gene is shown here for the first time to be required for the conversion of protoporphyrin IX to subsequent intermediates in bacteriochlorophyll biosynthesis.     

Control of bacteriochlorophyll accumulation by light in Rhodobacter capsulatus.

Oxygen levels which control induction of the assembly of the pigment-protein photosynthetic polypeptides in dark-grown Chloroflexus aurantiacus were determined. The induction signal by low-oxygen tension is not directly related to the respiratory competence of these photosynthetic cells. Cytochrome c554, the primary electron donor to P865+ of the reaction center, is not present in dark-grown respiratory cells but is induced in parallel with bacteriochlorophylls a and c and at similar oxygen partial pressure. The development of these components of the photosynthetic apparatus and its electron transport chain is completely independent of the presence of any detectable light or bacteriochlorophyll c or a pigments in C. aurantiacus.     

The PufX protein of Rhodobacter capsulatus affects the properties of bacteriochlorophyll a and carotenoid pigments of light-harvesting complex 1

A pufX gene deletion in the purple bacterium Rhodobacter capsulatus causes a severe photosynthetic defect and increases core light-harvesting complex (LH1) protein and bacteriochlorophyll a (BChl) levels. It was suggested that PufX interrupts the LH1 alpha/beta ring around the reaction centre, allowing quinone/quinol exchange. However, naturally PufX(-) purple bacteria grow photosynthetically with an uninterrupted LH1. We discovered that substitutions of the Rhodobacter-specific LH1 alpha seryl-2 decrease carotenoid levels in PufX(-)R. capsulatus. An LH1 alphaS2F mutation improved the photosynthetic growth of a PufX(-) strain lacking the peripheral LH2 antenna, although LH1 BChl absorption remained above wild-type, suggesting that Rhodobacter-specific carotenoid binding is involved in the PufX(-) photosynthetic defect and LH1 expansion is not. Furthermore, PufX overexpression increased LH1-like BChl absorption without inhibiting photosynthetic growth. PufX(+) LH1 alphaS2-substituted mutant strains had wild-type carotenoid levels, indicating that PufX modulates LH1 carotenoid binding, inducing a conformational change that favours quinone/quinol exchange.     

Coregulation of the syntheses of bacteriochlorophyll and pigment-binding proteins in Rhodobacter capsulatus

We quantified the expression of the puf and puc operons, which encode pigment binding proteins, in a number of strains of Rhodobacter capsulatus that have defects in genes affecting different steps of bacteriochlorophyll biosynthesis. Our results show that these mutations have a very similar effect on puf and puc expression. This suggests that the reduced expression of genes encoding pigment-binding proteins is due neither to the accumulation of a specific intermediate of bacteriochlorophyll synthesis nor to the lack of an early intermediate, but is rather the result of the lack of a very late intermediate or the bacteriochlorophyll molecule itself. Received: 14 September 1998 / Accepted: 29 December 1998     

The RegB/RegA two-component regulatory system controls synthesis of photosynthesis and respiratory electron transfer components in Rhodobacter capsulatus

Recently, we demonstrated that the RegB/RegA two-component regulatory system from Rhodobacter capsulatus functions as a global regulator of metabolic processes that either generate or consume reducing equivalents. For example, the RegB/RegA system controls expression of such energy generating processes as photosynthesis and hydrogen utilization. In addition, RegB/RegA also control nitrogen and carbon fixation pathways that utilize reducing equivalents. Here, we use a combination of DNase I protection and plasmid-based reporter expression studies to demonstrate that RegA directly controls synthesis of cytochrome cbb3 and ubiquinol oxidases that function as terminal electron acceptors in a branched respiratory chain. We also demonstrate that RegA controls expression of cytochromes c2, c(y) and the cytochrome bc1 complex that are involved in both photosynthetic and respiratory electron transfer events. These data provide evidence that the RegB/RegA two-component system has a major role in controlling the synthesis of numerous processes that affect reducing equivalents in Rhodobacter capsulatus.     

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).     

Electron transfer in reaction centers of Rhodobacter sphaeroides and Rhodobacter capsulatus monitored by fluorescence of the bacteriochlorophyll dimer

Spectral and kinetic characteristics of fluorescence from isolated reaction centers of photosynthetic purple bacteria Rhodobacter sphaeroides and Rhodobacter capsulatus were measured at room temperature under rectangular shape of excitation at 810 nm. The kinetics of fluorescence at 915 nm reflected redox changes due to light and dark reactions in the donor and acceptor quinone complex of the reaction center as identified by absorption changes at 865 nm (bacteriochlorophyll dimer) and 450 nm (quinones) measured simultaneously with the fluorescence. Based on redox titration and gradual bleaching of the dimer, the yield of fluorescence from reaction centers could be separated into a time-dependent (originating from the dimer) and a constant part (coming from contaminating pigment (detached bacteriochlorin)). The origin was also confirmed by the corresponding excitation spectra of the 915 nm fluorescence. The ratio of yields of constant fluorescence over variable fluorescence was much smaller in Rhodobacter sphaeroides (0.15±0.1) than in Rhodobacter capsulatus (1.2±0.3). It was shown that the changes in fluorescence yield reflected the disappearance of the dimer and the quenching by the oxidized primary quinone. The redox changes of the secondary quinone did not have any influence on the yield but excess quinone in the solution quenched the (constant part of) fluorescence. The relative yields of fluorescence in different redox states of the reaction center were tabulated. The fluorescence of the dimer can be used as an effective tool in studies of redox reactions in reaction centers, an alternative to the measurements of absorption kinetics.Abbreviations Bchl bacteriochlorophyll - Bpheo bacteriopheophytin - D electron donor to P⁺ - P bacteriochlorophyll dimer - Q quinone acceptor - Q_A primary quinone acceptor - Q_B secondary quinone acceptor - RC reaction center protein - UQ₆ ubiquinone-30     

Rhodobacter capsulatus genes involved in early steps of the bacteriochlorophyll biosynthetic pathway.

Three open reading frames in the Rhodobacter capsulatus photosynthesis gene cluster, designated F0, F108, and F1025, were disrupted by site-directed mutagenesis. Mutants bearing insertions in these reading frames were defective in converting protoporphyrin IX to magnesium-protoporphyrin monomethyl ester, protochlorophyllide to chlorophyllide a, and magnesium-protoporphyrin monomethyl ester to protochlorophyllide, respectively. These results demonstrate that the genes examined most likely encode enzyme subunits that catalyze steps common to plant and bacterial tetrapyrrole photopigment biosynthetic pathways. The open reading frames were found to be part of a large 11-kilobase operon that encodes numerous genes involved in early steps of the bacteriochlorophyll a biosynthetic pathway.     

bchFNBH bacteriochlorophyll synthesis genes of Rhodobacter capsulatus and identification of the third subunit of light-independent protochlorophyllide reductase in bacteria and plants.

We present the nucleotide and deduced amino acid sequences of four contiguous bacteriochlorophyll synthesis genes from Rhodobacter capsulatus. Three of these genes code for enzymes which catalyze reactions common to the chlorophyll synthesis pathway and therefore are likely to be found in plants and cyanobacteria as well. The pigments accumulated in strains with physically mapped transposon insertion mutations are analyzed by absorbance and fluorescence spectroscopy, allowing us to assign the genes as bchF, bchN, bchB, and bchH, in that order. bchF encodes a bacteriochlorophyll alpha-specific enzyme that adds water across the 2-vinyl group. The other three genes are required for portions of the pathway that are shared with chlorophyll synthesis, and they were expected to be common to both pathways. bchN and bchB are required for protochlorophyllide reduction in the dark (along with bchL), a reaction that has been observed in all major groups of photosynthetic organisms except angiosperms, where only the light-dependent reaction has been clearly established. The purple bacterial and plant enzymes show 35% identity between the amino acids coded by bchN and chlN (gidA) and 49% identity between the amino acids coded by bchL and chlL (frxC). Furthermore, bchB is 33% identical to ORF513 from the Marchantia polymorpha chloroplast. We present arguments in favor of the probable role of ORF513 (chlB) in protochlorophyllide reduction in the dark. The further similarities of all three subunits of protochlorophyllide reductase and the three subunits of chlorin reductase in bacteriochlorophyll synthesis suggest that the two reductase systems are derived from a common ancestor.     

Bacterial regulatory networks include direct contact of response regulator proteins: interaction of RegA and NtrX in Rhodobacter capsulatus

The formation of photosynthetic complexes in facultatively photosynthetic bacteria is controlled by the oxygen tension in the environment. In Rhodobacter capsulatus the two-component system RegB/RegA plays a major role in the redox control of photosynthesis genes but also controls other redox-dependent systems. The response regulator RegA is phosphorylated under low oxygen tension and activates the puf and puc operons, which encode pigment binding proteins, by binding to their promoter regions. Data from a yeast two-hybrid analysis as well as an in vitroanalysis indicate that RegA interacts with the NtrX protein, the response regulator of the NtrY/NtrX two-component system which is believed to be involved in regulation of nitrogen fixation genes. Our further analysis revealed that NtrX is indeed involved in the regulation of the puf and puc operons. Furthermore, we showed that an altered NtrX protein, which is predicted to adopt the conformation of phosphorylated NtrX protein, binds within the puf promoter region close to the RegA binding sites. We conclude that a direct interaction of two response regulators connects the regulatory systems for redox control and nitrogen control.     

Autophosphorylation, phosphotransfer, and DNA-binding properties of the RegB/RegA two-component regulatory system in Rhodobacter capsulatus.

In the purple, photosynthetic bacterium, Rhodobacter capsulatus, the RegB/RegA two-component system is required for activation of several anaerobic processes, such as synthesis of the photosynthetic apparatus and assimilation of CO2 and N2. It is believed that RegB is an integral membrane histidine kinase that monitors the external environment. Under anaerobic growth conditions, it transduces a signal through phosphorylation of the response regulator, RegA, which then induces target gene expression. We used an in vitro assay to characterize the phosphorylation of wild-type RegA and a mutant variant (RegA*) that is responsible for abnormally high photosynthesis gene expression under both aerobic and anaerobic growth conditions. Phosphorylation assays indicate that phosphorylated RegA* (RegA* approximately P) is much more stable than RegA approximately P, indicating that it may be locked in a conformation that is resistant to dephosphorylation. DNase I footprint assays also indicate that unphosphorylated RegA* has a much higher affinity for specific DNA binding sites than the wild-type protein. Phosphorylation of RegA* increases DNA binding 2. 5-fold, whereas phosphorylation of RegA increases DNA binding more than 16-fold. Collectively, these results support the hypothesis that RegA* is a constitutively active variant that does not require phosphorylation to assume a structural conformation required to bind DNA.     

The hupTUV operon is involved in negative control of hydrogenase synthesis in Rhodobacter capsulatus.

The hupT, hupU, and hupV genes, which are located upstream from the hupSLC and hypF genes in the chromosome of Rhodobacter capsulatus, form the hupTUV operon expressed from the hupT promoter. The hupU and hupV genes, previously thought to belong to a single open reading frame, encode HupU, of 34.5 kDa (332 amino acids), and HupV, of 50.4 kDa (476 amino acids), which are >/= 50% identical to the homologous Bradyrhizobium japonicum HupU and HupV proteins and Rhodobacter sphaeroides HupU1 and HupU2 proteins, respectively; they also have 20 and 29% similarity with the small subunit (HupS) and the large subunit (HupL), respectively, of R. capsulatus [NiFe]hydrogenase. HupU lacks the signal peptide of HupS and HupV lacks the C-terminal sequence of HupL, which are cleaved during hydrogenase processing. Inactivation of hupV by insertional mutagenesis or of hupUV by in-frame deletion led to HupV- and Hup(UV)- mutants derepressed for hydrogenase synthesis, particularly in the presence of oxygen. These mutants were complemented in trans by plasmid-borne hupTUV but not by hupT or by hupUV, except when expressed from the inducible fru promoter. Complementation of the HupV- and Hup(UV)- mutants brought about a decrease in hydrogenase activity up to 10-fold, to the level of the wild-type strain B10, indicating that HupU and HupV participate in negative regulation of hydrogenase expression in concert with HupT, a sensor histidine kinase involved in the repression process. Plasmid-borne gene fusions used to monitor hupTUV expression indicated that the operon is expressed at a low level (50- to 100-fold lower than hupS).