Photosynthesis Gene Expression Research Articles

Complex regulatory activities associated with the histidine kinase PrrB in expression of photosynthesis genes in Rhodobacter sphaeroides 2.4.1.

Rhodobacter sphaeroides 2.4.1 synthesizes a specialized photosynthetic membrane upon reduction of the O2 tension below threshold levels. The genes prrB and prrA encode a sensor kinase and a response regulator, respectively, of a two-component regulatory system that presumably is involved in transduction of the signal(s) that monitors alterations in oxygen levels. A third gene, prrC, is also involved in this cascade of events. Previously, we described a mutant form of PrrB, namely, PrrB78 (J. M. Eraso and S. Kaplan, J. Bacteriol. 177:2695-2706, 1995), which results in aerobic expression of the photosynthetic apparatus. Here we examine three mutated forms of the prrB gene that have the potential to encode truncated polypeptides containing the N-terminal 6, 63, or 163 amino acids, respectively. The resulting mutant strains showed residual levels of the light-harvesting spectral complexes and had diminished photosynthetic growth rates at high light intensities with no discernible growth under intermediate or low light conditions. When either lacZ transcriptional fusions or direct mRNA determinations were used to monitor specific photosynthesis gene expression, all the mutant strains showed unexpectedly high levels of gene expression when compared to mutant strains affected in prrA. Conversely, when translational fusions were used to monitor photosynthesis gene expression in these mutant strains, expression of both puc and puf operons was reduced, especially puf expression. In light of these studies and those of the PrrB78 mutant, the data suggest that PrrA can be activated in situ by something other than PrrB, and it also appears that PrrB can function as a negative regulator acting through PrrA. Finally, we consider the role of the Prr regulatory system in the posttranscriptional control of photosynthesis gene expression.

Photosynthesis gene expression in Rhodobacter sphaeroides is regulated by redox changes which are linked to electron transport

The effect of the respiratory electron acceptor nitrous oxide on the synthesis of the photosynthetic apparatus of Rhodobacter sphaeroides was examined. In phototrophically grown cells, the addition of nitrous oxide caused a reduction in the level of light-harvesting complex I and light-harvesting complex II under conditions of high light intensity (200 W m−2) and low light intensity (16 W m−2). 5-Aminolaevulinate synthase activity was decreased during growth in the presence of nitrous oxide and this limited production of spectral complexes since addition of exogenous 5-aminolaevulinic acid partially suppressed the effect of nitrous oxide. The effect of nitrous oxide on the expression of the operons encoding the pigment-binding proteins of light-harvesting complex I (puf), light-harvesting complex II (puc) and the two isoenzymes of 5-aminolaevulinate synthase (hemA and hemT) were measured using transcriptional fusions to lacZ. Nitrous oxide caused a decrease in puf and hemA transcription. However, there was an apparent increase in the expression of puc and hemT transcriptional fusions. The level of light-harvesting complexes in cells grown in the dark with different electron acceptors was also examined. Cells grown anaerobically with DMSO had a higher level of light-harvesting complexes than those grown anaerobically with nitrous oxide as electron acceptor. Cells grown aerobically had the lowest level of light-harvesting complexes. It is proposed that FnrL-dependent and FnrL-independent expression of photosynthesis genes is modulated by redox changes elicited by nitrous oxide respiration.

Evolution and expression of C4 photosynthesis genes.

Evolution and expression of C4 photosynthesis genes.

Directed mutagenesis of the Rhodobacter capsulatus puhA gene and orf 214: pleiotropic effects on photosynthetic reaction center and light-harvesting 1 complexes.

Rhodobacter capsulatus puhA mutant strains containing either a nonpolar, translationally in-frame deletion or a polar insertion of an antibiotic resistance cartridge were constructed and evaluated for their photosynthetic growth properties, absorption spectroscopy profiles, and chromatophore protein compositions. Both types of mutants were found to be incapable of photosynthetic growth and deficient in the reaction center (RC) and light-harvesting 1 (LH1) complexes. The translationally in-frame puhA deletion strains were restored to the parental strain phenotypes by complementation with a plasmid containing the puhA gene, whereas the polar puhA mutants were not. Analogous nonpolar and polar disruptions of orf 214 (located immediately 3' of the puhA gene) were made, and the resultant mutant strains were evaluated as described above. The strain containing the nonpolar deletion of orf 214 exhibited severely impaired photosynthetic growth properties and had greatly reduced levels of the RC and LH1 complexes. Complementation of this strain with a plasmid that expressed orf 214 from the nifHDK promoter restored photosynthetic growth capability, as well as the RC and LH1 complexes. The polar disruption of orf 214 yielded cells that were incapable of photosynthetic growth and had even lower levels of the RC and LH1 complexes, and complementation in trans with orf 214 only marginally improved these deficiencies. These results indicate that orf 214 and at least one additional gene located 3' of orf 214 are required to obtain the RC and LH1 complexes, and transcription read-through from the puhA superoperon is necessary for optimal expression of these new photosynthesis genes.

Cloning and characterization of senC, a gene involved in both aerobic respiration and photosynthesis gene expression in Rhodobacter capsulatus

The purple nonsulfur photosynthetic eubacterium Rhodobacter capsulatus is a versatile organism that can obtain cellular energy by several means, including the capture of light energy for photosynthesis as well as the use of light-independent respiration, in which molecular oxygen serves as a terminal electron acceptor. In this study, we have identified and characterized a novel gene, senC, mutations in which affect respiration as well as the induction of photosynthesis gene expression. The protein coded by senC exhibits 33% sequence identity to the yeast nucleus-encoded protein SCO1, which is thought to be a mitochondrion-associated cytochrome c oxidase assembly factor. Like yeast SCO1, SenC is required for optimal cytochrome c oxidase activity in aerobically grown R. capsulatus cells. We further show that senC is required for maximal induction from the puf and puh operons, which encode the structural polypeptides of the light-harvesting and reaction center complexes.

A Sensory Transducer Homologous to the Mammalian Peripheral-type Benzodiazepine Receptor Regulates Photosynthetic Membrane Complex Formation in Rhodobacter sphaeroides 2.4.1

The Rhodobacter sphaeroides 2.4.1 tryptophan-rich sensory protein gene, tspO (formerly crtK, ORF160) encodes a 17-kDa protein which has an unusually high content of aromatic amino acids in general and of L-tryptophan in particular. The TspO protein was localized to the outer membrane of aerobically grown R. sphaeroides 2.4.1 by use of a polyclonal antibody against the purified protein. This protein is present in severalfold higher levels in photosynthetic as opposed to aerobic grown cells. Although tspO lies within the crt gene cluster, null mutations have an intact carotenoid biosynthetic pathway. In the TSPO1 mutant there was an increased production of carotenoids and bacteriochlorophyll relative to the wild type, particularly when cells were grown aerobically or semiaerobically. When present in trans the tspO gene restored "normal" pigment production to TSPO1. The effect of the tspO gene on pigment production was shown to take place at the level of gene expression. Because the tspO gene product of R. sphaeroides 2.4.1 shows significant sequence homology and similarity to the peripheral-type benzodoazepine receptor from mammalian sources, TspO-specific antibodies when probed against liver and kidney mitochondrial protein showed strong cross-reactivity. The role of TspO in R. sphaeroides 2.4.1 and its relation to photosynthesis gene expression are discussed.

AppA, a novel gene encoding a trans-acting factor involved in the regulation of photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1

A new gene, the product of which is involved in the regulation of photosynthesis gene expression in the anoxygenic photosynthetic bacterium Rhodobacter sphaeroides 2.4.1, has been identified. The isolation of this gene, designated appA (activation of photopigment and puc expression), was based on its ability, when provided in extra copies, to partially suppress mutations in the two-component PrrB-PrrA regulatory system. The presence of extra copies of the appA gene in either prrB, prrA, or wild-type strains resulted in an activation of puc::lacZ expression under aerobic conditions. Constructed AppA null mutants did not grow photosynthetically and were impaired in the synthesis of both bacteriochlorophyll and carotenoids, as well as the structural proteins of the photosynthetic spectral complexes. When grown anaerobically in the dark, these mutants accumulated bacteriochlorophyll precursors. The expression of lacZ fusions to several photosynthesis genes and operons, including puc, puf, and bchF, was decreased in the AppA mutant strains in comparison with the wild type. To examine the role of AppA involvement in bacteriochlorophyll biosynthesis, we inactivated an early gene, bchE, of the bacteriochlorophyll pathway in both wild-type and AppA- mutant backgrounds. The double mutant, AppA- BchE-, was found to be severely impaired in photosynthesis gene expression, similar to the AppA- BchE+ mutant and in contrast to the AppA+ BchE- mutant. This result indicated that AppA is more likely involved in the regulation of expression of the bch genes than in the biosynthetic pathway per se. The appA gene was sequenced and appears to encode a protein of 450 amino acids with no obvious homology to known proteins.

Isolation of regulatory mutants in photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1 and partial complementation of a PrrB mutant by the HupT histidine-kinase

The photosynthetic bacterium Rhodobacter sphaeroides responds to the transition from aerobiosis to anaerobic photosynthesis by increasing the expression of the photosynthesis genes. Mutants have been isolated based on their inability, following such a transition, to increase transcription of the puc operon encoding the apoproteins of the light-harvesting complex II. Mutant D5, a representative of one mutant class, described here, although remaining photosynthetically competent, produced only low levels of the photosynthetic spectral complexes. Complementation analysis revealed that either the gene for the photosynthesis response regulator prrA or the gene encoding its cognate sensor kinase, prrB, was capable of rescuing this mutant. However, partial complementation of this mutant was achieved by placing in trans additional copies of other defined genes from the cosmid library of R. sphaeroides. We describe this effect in detail, attributable to the hupT gene, which has been proposed to encode a histidine-kinase for the hydrogen uptake system in Rhodobacter capsulatus. The effect of HupT on the expression of the photosynthesis genes was mediated through PrrA and independent of a functioning hydrogen uptake system. Thus, we raise the possibility that HupT can participate in phosphorylation of the heterologous response regulator PrrA by so-called cross-talk and therefore partially compensate for the defect in the mutant described. The observation of cross-talk, together with the complementation analysis, allowed us to assign the original mutation to the prrB gene; this was confirmed by DNA sequencing. Analysis of cross-talk in the wild-type, prrB and prrA genetic backgrounds suggested that besides kinase activity, PrrB may possess phosphatase activity toward PrrA. We also report the cloning, organization and structure of some of the hup genes from R. sphaeroides and construction of a Hup- strain.

Oxygen-insensitive synthesis of the photosynthetic membranes of Rhodobacter sphaeroides: a mutant histidine kinase

Two new loci, prrB and prrC, involved in the positive regulation of photosynthesis gene expression in response to anaerobiosis, have been identified in Rhodobacter sphaeroides. prrB encodes a sensor histidine kinase that is responsive to the removal of oxygen and functions through the response regulator PrrA. Inactivation of prrB results in a substantial reduction of photosynthetic spectral complexes as well as in the inability of cells to grow photosynthetically at low to medium light intensities. Together, prrB and prrA provide the major signal involved in synthesis of the specialized intracytoplasmic membrane (ICM), harboring components essential to the light reactions of photosynthesis. Previously, J. K. Lee and S. Kaplan (J. Bacteriol. 174:1158-1171, 1992) identified a mutant which resulted in high-level expression of the puc operon, encoding the apoproteins giving rise to the B800-850 spectral complex, in the presence of oxygen as well as in the synthesis of the ICM under conditions of high oxygenation. This mutation is shown to reside in prrB, resulting in a leucine-to-proline change at position 78 in mutant PrrB (PRRB78). Measurements of mRNA levels in cells containing the prrB78 mutation support the idea that prrB is a global regulator of photosynthesis gene expression. Two additional mutants, PRRB1 and PRRB2, which make two truncated forms of the PrrB protein, possess substantially reduced amounts of spectral complexes. Although the precise role of prrC remains to be determined, evidence suggests that it too is involved in the regulatory cascade involving prrB and prrA. The genetic organization of the photosynthesis response regulatory (PRR) region is discussed.

Identification and molecular genetic characterization of a sensor kinase responsible for coordinately regulating light harvesting and reaction center gene expression in response to anaerobiosis

Our laboratory recently demonstrated that anaerobic induction of light harvesting and reaction center structural gene expression involved a trans-acting factor, RegA, which exhibits sequence similarity to the class of prokaryotic sensory transduction proteins known as response regulators (M. W. Sganga and C. E. Bauer, Cell 68:945-954, 1992). In this study, we performed a screen for additional genes involved in inducing anaerobic expression of light harvesting and reaction center structural genes. This search resulted in the isolation of four strains that were shown by complementation and marker rescue analysis to harbor mutations allelic to the originally described regA mutation and one strain with a mutation found to be linked but nonallelic to regA. Sequence analysis indicated that this additional gene, regB, codes for a polypeptide that exhibits sequence similarity to the prokaryotic family of histidine sensor kinases. Analysis of photosynthesis gene expression in regB mutants indicates that the disruption of regB results in a phenotype that is very similar to that described for regA mutants, namely, a failure to trans activate anaerobic expression of the puf, puh, and puc operons. In analogy to other prokaryotic sensory transduction systems, we propose that RegB functions as a membrane-spanning sensor kinase that controls the anaerobic phosphorylation state of RegA, which in turn controls the induction of light harvesting and reaction center structural genes.

Erratum: Differential Expression of Photosynthesis Genes in Leaf Tissue Layers of Peperomia as Revealed by Tissue Printing

Erratum: Differential Expression of Photosynthesis Genes in Leaf Tissue Layers of Peperomia as Revealed by Tissue Printing

Separate photosensory pathways co-regulate blue light/ultraviolet-A-activated psbD-psbC transcription and light-induced D2 and CP43 degradation in barley (Hordeum vulgare) chloroplasts.

We studied the effects of spectral quality and fluence on the expression of several chloroplast-encoded photosynthesis genes and on the stability of their protein products in barley (Hordeum vulgare). During light-dependent chloroplast maturation, mRNA levels for psbD-psbC and psbA were maintained at higher levels compared with mRNAs encoding proteins for other photosynthesis functions (atpB, rbcL). Maintenance of psbD-psbC mRNA levels was accounted for by differential activation of the psbD-psbC light-responsive promoter by high-irradiance blue light and, secondarily, ultraviolet A (UV-A) radiation. Promoter activation was fluence dependent and required continuous illumination for 2 h at threshold fluences of 1.3 (blue light), 7.5 (white light), or 10 (UV-A) mumol m-2 s-1. From immunoblot analysis experiments, we showed that the psbD-psbC gene products D2 and CP43 undergo light-mediated turnover similar to light-labile D1. Other photosynthesis proteins such as the beta subunit of ATP synthase and the large subunit of ribulose-1,5-bisphosphate carboxylase were relatively stable. In the absence of protein synthesis, D2 degradation paralleled the degradation of D1 (relative half-lives, 9.5-10 h). CP43 decay was about half of D2 and D1 decay. In contrast with activation of the light-responsive promoter, the fluence-dependent degradation of D1, D2, and CP43 required 50- to 100-fold higher fluences of photosynthetically active white, red, blue, or UV-A irradiation. We interpret the different fluence and wavelength requirements to indicate that separate photosensory systems regulate activation of psbD-psbC transcription and turnover of D1, D2, and CP43. We propose that a blue light/UV-A photosensory pathway activates the psbD-psbC light-responsive promoter, differentially maintaining the capacity of mature chloroplasts to synthesize D2 and CP43, which are damaged and turned over in illuminated plants.

Differential Expression of Photosynthesis Genes in Leaf Tissue Layers of Peperomia as Revealed by Tissue Printing

Differential Expression of Photosynthesis Genes in Leaf Tissue Layers of Peperomia as Revealed by Tissue Printing

Differential expression of photosynthesis genes in leaf tissue layers of Peperomia as revealed by tissue printing

Peperomia has species that may be C3, show Crassulacean acid metabolism (CAM), or CAM‐cycling. Species that show CAM progress from C3 to CAM through CAM‐cycling during leaf development. In CAM and CAM‐cycling species, CAM metabolism is predominately in the upper multiple epidermis and lower spongy mesophyll, whereas C3 metabolism is localized mostly in the palisade mesophyll. Using specific protein and cDNA probes prepared from P‐enolpyruvate carboxylase (PEPc) and ribulose‐1,5‐bisphosphate carboxylase (Rubisco), we have now studied the differential distribution of photosynthetic metabolism in Peperomia leaves using the technique of tissue printing. The tissue printing studies detected Rubisco protein in leaves of C3 P. orba, but not PEPc. Young C3 leaves of P. scandens and P. camptotricha showed Rubisco protein, but not PEPc; however, the mature leaves of these two species that have CAM showed PEPc protein and RNAs in both the multiple epidermis and spongy mesophyll. In contrast, Rubisco protein and RNAs were present throughout the leaf. The tissue printing data are consistent with our previously published data showing the differential distribution of photosynthetic metabolism in leaves of Peperomia. Although the tissue printing technique is qualitative, coupled with quantitative data it has proven useful for the study of function related to structure.

PrrA, a putative response regulator involved in oxygen regulation of photosynthesis gene expression in Rhodobacter sphaeroides

A new locus, prrA, involved in the regulation of photosynthesis gene expression in response to oxygen, has been identified in Rhodobacter sphaeroides. Inactivation of prrA results in the absence of photosynthetic spectral complexes. The prrA gene product has strong homology to response regulators associated with signal transduction in other prokaryotes. When prrA is present in multiple copies, cells produce light-harvesting complexes under aerobic growth conditions, suggesting that prrA affects photosynthesis gene expression positively in response to oxygen deprivation. Analysis of the expression of puc::lacZ fusions in wild-type and PrrA- cells revealed a substantial decrease in LacZ expression in the absence of prrA under all conditions of growth, especially when cells were grown anaerobically in the dark in the presence of dimethyl sulfoxide. Northern (RNA) and slot blot hybridizations confirmed the beta-galactoside results for puc and revealed additional positive regulation of puf, puhA, and cycA by PrrA. The effect of truncated PrrA on photosynthesis gene expression in the presence of low oxygen levels can be explained by assuming that PrrA may be effective as a multimer. PrrA was found to act on the downstream regulatory sequences (J. K. Lee and S. Kaplan, J. Bacteriol. 174:1146-1157, 1992) of the puc operon regulatory region. Finally, two spontaneous prrA mutations that abolish prrA function by changing amino acids in the amino-terminal domain of the protein were isolated.

Regulation of expression of photosynthesis genes in anoxygenic photosynthetic bacteria

Regulation of expression of photosynthesis genes in anoxygenic photosynthetic bacteria

The role of mRNA degradation in the regulated expression of bacterial photosynthesis genes

Regulation of gene expression in bacteria, as in eukaryotic cells, is often achieved by variation of mRNA levels. Since the steady state levels of mRNA depend on both the rate of synthesis and the rate of decay, both mechanisms are important for gene regulation. After considerable effort undertaken over many years to understand the regulation of transcription, mRNA degradation has recently gained increasing attention as an important step in the regulation of some bacterial genes, and many investigations have addressed the mechanisms involved in mRNA decay. The puf mRNA of Rhodobacter capsulatus encoding pigment binding proteins has become a model system to study decay of a polycistronic mRNA and the role of mRNA degradation in gene expression. Individual segments of the polycistronic puf mRNA display extremely different half-lives. These differences in stability of mRNA segments are involved in the differential expression of puf encoded genes and consequently contribute to the stoichiometry of light-harvesting I and reaction centre complexes that results in optimal growth. In addition, control of mRNA stability is involved in the oxygen-dependent regulation of photosynthesis genes. High oxygen tension results in decreased stability of the reaction-centre specific puf mRNA segment, most likely by affecting the rate of endonucleolytic cleavage within the reaction centre coding region. The results obtained from studying puf mRNA degradation in R. capsulatus and Escherichia coli suggest that a specific distribution of decay promoting and decay impeding mRNA elements along the polycistronic mRNA is responsible for the different half-lives of individual puf segments.

Regulation of expression of photosynthesis genes in anoxygenic photosynthetic bacteria.

Regulation of expression of photosynthesis genes in anoxygenic photosynthetic bacteria.

Regulatory factors controlling photosynthetic reaction center and light-harvesting gene expression in Rhodobacter capsulatus

Most species of photosynthetic bacteria synthesize their photosynthetic apparatus only under conditions of reduced oxygen tension. To a large extent, this phenomenon is dependent upon anaerobic induction of photosynthesis gene expression. Here we report an example of a regulatory gene, regA, that is involved in transactivating anaerobic expression of the photosynthetic apparatus. We show that RegA is itself responsible for differential induction of light-harvesting and reaction center gene expression relative to operons for photopigment biosynthesis. Surprisingly, strains disrupted for regA were found to retain normal photosynthetic growth capabilities under high light intensities. We further show that photosynthetic growth in the absence of transactivating structural gene expression is a consequence of the superoperonal organization of the photosynthetic gene cluster.

Expression of chloroplast photosynthesis genes during leaf senescence

Expression of chloroplast photosynthesis genes during leaf senescence