Molecular genetic analysis suggesting interactions between AppA and PpsR in regulation of photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1.

The AppA protein plays an essential regulatory role in development of the photosynthetic apparatus in the anoxygenic phototrophic bacterium Rhodobacter sphaeroides 2.4.1 (M. Gomelsky and S. Kaplan, J. Bacteriol. 177:4609-4618, 1995). To gain additional insight into both the role and site of action of AppA in the regulatory network governing photosynthesis gene expression, we investigated the relationships between AppA and other known regulators of photosynthesis gene expression. We determined that AppA is dispensable for development of the photosynthetic apparatus in a ppsR null background, where PpsR is an aerobic repressor of genes involved in photopigment biosynthesis and puc operon expression. Moreover, all suppressors of an appA null mutation thus far isolated, showing improved photosynthetic growth, were found to contain mutations in the ppsR gene. Because ppsR gene expression in R. sphaeroides 2.4.1 appears to be largely independent of growth conditions, we suggest that regulation of repressor activity occurs predominately at the protein level. We have also found that PpsR functions as a repressor not only under aerobic but under anaerobic photosynthetic conditions and thereby is involved in regulating the abundance of the light harvesting complex II, depending on light intensity. It seems likely therefore, that PpsR responds to an integral signal (e.g., changes in redox potential) produced either by changes in oxygen tension or light intensity. The profile of the isolated suppressor mutations in PpsR is in accord with this proposition. We propose that AppA may be involved in a redox-dependent modulation of PpsR repressor activity.     

A single flavoprotein,AppA, integrates both redox and light signals in Rhodobacter sphaeroides

Anoxygenic photosynthetic proteobacteria exhibit various light responses, including changing levels of expression of photosynthesis genes. However, the underlying mechanisms are largely unknown. We show that expression of the puf and puc operons encoding structural proteins of the photosynthetic complexes is strongly repressed by blue light under semi-aerobic growth in Rhodobacter sphaeroides but not in the related species Rhodobacter capsulatus. At very low oxygen tension, puf and puc expression is independent of blue light in both species. Photosynthetic electron transport does not mediate the blue light repression, implying the existence of specific photoreceptors. Here, we show that the flavoprotein AppA is likely to act as the photoreceptor for blue light-dependent repression during continuous illumination. The FAD cofactor of AppA is essential for the blue light-dependent sensory transduction of this response. AppA, which is present in R. sphaeroides but not in R. capsulatus, is known to participate in the redox-dependent control of photosynthesis gene expression. Thus, AppA is the first example of a protein with dual sensing capabilities that integrates both redox and light signals.     

Redox and light regulation of gene expression in photosynthetic prokaryotes

All photosynthetic organisms control expression of photosynthesis genes in response to alterations in light intensity as well as to changes in cellular redox potential. Light regulation in plants involves a well-defined set of red- and blue-light absorbing photoreceptors called phytochrome and cryptochrome. Less understood are the factors that control synthesis of the plant photosystem in response to changes in cellular redox. Among a diverse set of photosynthetic bacteria the best understood regulatory systems are those synthesized by the photosynthetic bacterium Rhodobacter capsulatus. This species uses the global two-component signal transduction cascade, RegB and RegA, to anaerobically de-repress anaerobic gene expression. Under reducing conditions, the phosphate on RegB is transferred to RegA, which then activates genes involved in photosynthesis, nitrogen fixation, carbon fixation, respiration and electron transport. In the presence of oxygen, there is a second regulator known as CrtJ, which is responsible for repressing photosynthesis gene expression. CrtJ responds to redox by forming an intramolecular disulphide bond under oxidizing, but not reducing, growth conditions. The presence of the disulphide bond stimulates DNA binding activity of the repressor. There is also a flavoprotein that functions as a blue-light absorbing anti-repressor of CrtJ in the related bacterial species Rhodobacter sphaeroides called AppA. AppA exhibits a novel long-lived photocycle that is initiated by blue-light absorption by the flavin. Once excited, AppA binds to CrtJ thereby inhibiting the repressor activity of CrtJ. Various mechanistic aspects of this photocycle will be discussed.     

Identification of nitrogenase and carboxylase genes in the photosynthetic bacteria and cloning of a carboxylase gene from Rhodopseudomonas sphaeroides

The presumptive genes for the ribulose 1,5-bisphosphate carboxylase large subunit and for nitrogenase-specific components from Rhodopseudomonas sphaeroides and several other photosynthetic bacteria were identified and located by interspecific probing. Restriction digests of R. sphaeroides genomic DNA were hybridized under stringent conditions to cloned DNA from Rhodospirillum rubrum (plasmid pRR2119 carrying the carboxylase gene) and Klebsiella pneumoniae (pSA30 carrying the nitrogenase genes). The nitrogenase probe hybridized with different signal intensities to several distinct HindIII, BglII, EcoRI, BamHI and PvuII fragments of R. sphaeroides 2.4.1.DNA. The carboxylase probe hybridized to only single R. sphaeroides 2.4.1.DNA fragments produced with all five restriction enzymes. A 3000-bp EcoRI-BamHI R. sphaeroides 2.4.1.DNA fragment carrying the presumptive gene for the large subunit of ribulose 1,5-bisphosphate carboxylase was cloned into pBR322 and positively identified by probing with a 32P-labeled internal PstI fragment of the Rhodospirillum carboxylase gene.     

Principles of redox control in photosynthesis gene expression

Light is one of the most important environmental factors influencing gene expression in photosynthetic organisms. In particular, genes for components of the photosynthetic machinery show light-dependent expression. In recent years, it has become clear that photosynthesis itself contributes important signals to this light control of gene expression by means of changes in the reduction/oxidation (redox) state of signalling molecules. Such changes in redox state are induced by changes in quality and quantity of the incident light. Redox signalling mechanisms therefore provide photosynthesis with the possibility of acclimational changes in the structure of the photosynthetic apparatus via a feedback control of photosynthesis gene expression. The great variety of these signalling mechanisms is summarised under the term 'redox control'. In some cases, oxygen acts as a different environmental, light-independent stimulus of photosynthetic gene expression, providing an additional redox signal and a different kind of redox control. In this review, we summarise present knowledge about such redox control mechanisms and analyse common properties as well as differences in the various signalling pathways. We suggest that there is an urgent need for a clear distinction between different kinds of redox control. Accordingly, we propose a categorisation into perceptional and transductional redox control. These categories are defined and examples given. The generalisation and comparability of results obtained in different physiological test systems and species are critically discussed.     

Plasmid rearrangements in the photosynthetic bacterium Rhodopseudomonas sphaeroides

Mu d1(Ap lac) was introduced into the photosynthetic bacterium Rhodopseudomonas sphaeroides 2.4.1. via the R-plasmid R751 in an attempt to isolate fusion derivatives involving photosynthetic operons. The selection system is potentially very powerful since R. sphaeroides is normally Lac negative. Among the exconjugants, photosynthesis-deficient mutants were recovered, some of which had elevated beta-galactosidase levels. Among the mutants examined, beta-galactosidase expression was linked exclusively to R751 . Many of the photosynthesis-deficient mutants were found to have alterations in their indigenous plasmids which apparently involved the exchange of DNA from one plasmid to another. Southern blot analysis revealed that there are extensive DNA sequences which are shared by the two plasmids that are involved in the rearrangements and that no exogenous DNA sequences appear to be involved. It was further discovered that plasmid rearrangement is a general phenomenon which can occur spontaneously in R. sphaeroides 2.4.1 and shows a high correlation with a photosynthesis minus phenotype.     

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.     

Generalized approach to the regulation and integration of gene expression

The volume of electron flow through the cbb3 branch of the electron transport chain and the redox state of the quinone pool generate signals that regulate photosynthesis gene expression in Rhodobacter sphaeroides. An inhibitory signal is generated at the level of the catalytic subunit of the cbb3 cytochrome c oxidase and is transduced through the membrane-localized PrrC polypeptide to the PrrBA two-component activation system, which controls the expression of most of the photosynthesis genes in response to O2. The redox state of the quinone pool is monitored by the redox-active AppA antirepressor protein, which determines the functional state of the PpsR repressor protein. The antirepressor/repressor system as well as a modulator of AppA function, TspO, together with FnrL and PrrA stringently control photopigment gene expression. These regulatory elements, together with spectral complex-specific assembly factors, control the ultimate cellular levels and composition of the photosynthetic membrane.     

Evolutionary constraints and expression analysis of gene duplications in Rhodobacter Sphaeroides 2.4.1

ABSTRACT: BACKGROUND: Gene duplication is a major force that contributes to the evolution of new metabolic functions in all organisms. Rhodobacter sphaeroides 2.4.1 is a bacterium that displays a wide degree of metabolic versatility and genome complexity and therefore is a fitting model for the study of gene duplications in bacteria. A comprehensive analysis of 234 duplicate gene-pairs in R. sphaeroides was performed using structural constraint and expression analysis. RESULTS: The results revealed that most gene-pairs in in-paralogs are maintained under negative selection (omega [less than or equal to] 0.3), but the strength of selection differed among in-paralog gene-pairs. Although in-paralogs located on different replicons are maintained under purifying selection, the duplicated genes distributed between the primary chromosome (CI) and the second chromosome (CII) are relatively less selectively constrained than the gene-pairs located within each chromosome. The mRNA expression patterns of duplicate gene-pairs were examined through microarray analysis of this organism grown under seven different growth conditions. Results revealed that that ~62% of paralogs have similar expression patterns (cosine [greater than or equal to] 0.90) over all of these growth conditions, while only ~7% of paralogs are very different in their expression patterns (cosine < 0.50). CONCLUSIONS: The overall findings of the study suggest that only a small proportion of paralogs contribute to the metabolic diversity and the evolution of novel metabolic functions in R. sphaeroides. In addition, the lack of relationships between structural constraints and gene-pair expression suggests that patterns of gene-pair expression are likely associated with conservation or divergence of gene-pair promoter regions and other coregulation mechanisms.     

Analysis of the role of the nnrR gene product in the response of Rhodobacter sphaeroides 2.4.1 to exogenous nitric oxide.

Rhodobacter sphaeroides 2.4.1, which is incapable of denitrification, has been found to carry nnrR, the nor operon, and nnrS, which are utilized for denitrification in R. sphaeroides 2.4.3. The gene encoding nitrite reductase was not found in 2.4.1. Expression of beta-galactosidase activity from a norB-lacZ fusion was activated when cells of 2.4.1 were incubated with NO-producing bacteria. This result indicates that the products of nnrR and the genes flanking it are utilized when 2.4.1 is growing in an environment where denitrification occurs.