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.     

Redox properties of the Rhodobacter sphaeroides transcriptional regulatory proteins PpsR and AppA

Redox properties of the photosynthetic gene repressor PpsR and the blue-light photoreceptor/antirepressor AppA from Rhodobacter sphaeroides have been characterized. Redox titrations of PpsR reveal the presence of a two-electron couple, with an E (m) value of -320 mV at pH 7.0, which is likely to arise from the reversible conversion of two cysteine thiols to a disulfide. This E (m) value is very much more negative than the E (m) = -180 mV value measured previously at pH 7.0 for the disulfide/dithiol couple in CrtJ, the homolog for PpsR in the closely related bacterium Rhodobacter capsulatus. AppA, a flavin-containing blue-light receptor that is also involved in the regulation of gene expression in R. sphaeroides, contains multiple cysteines in its C-terminal region, two of which function as a redox-active dithiol/disulfide couple with an E (m) value of -325 mV at pH 7.0 in the dark. Titrations of this dithiol/disulfide couple in illuminated samples of AppA indicate that the E (m) value of this disulfide/dithiol couple is -315 mV at pH 7.0, identical to the value obtained for AppA in the dark within the combined experimental uncertainties of the two measurements. The E (m) values of AppA and PpsR demonstrate that these proteins are thermodynamically capable of electron transfer for their activity as an anti-repressor/repressor in R. sphaeroides.     

Use of transcriptomic data for extending a model of the AppA/PpsR system in <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

Background

Photosynthetic (PS) gene expression in Rhodobacter sphaeroides is regulated in response to changes in light and redox conditions mainly by PrrB/A, FnrL and AppA/PpsR systems. The PrrB/A and FnrL systems activate the expression of them under anaerobic conditions while the AppA/PpsR system represses them under aerobic conditions. Recently, two mathematical models have been developed for the AppA/PpsR system and demonstrated how the interaction between AppA and PpsR could lead to a phenotype in which PS genes are repressed under semi-aerobic conditions. These models have also predicted that the transition from aerobic to anaerobic growth mode could occur via a bistable regime. However, they lack experimentally quantifiable inputs and outputs. Here, we extend one of them to include such quantities and combine all relevant micro-array data publically available for a PS gene of this bacterium and use that to parameterise the model. In addition, we hypothesise that the AppA/PpsR system alone might account for the observed trend of PS gene expression under semi-aerobic conditions.

Results

Our extended model of the AppA/PpsR system includes the biological input of atmospheric oxygen concentration and an output of photosynthetic gene expression. Following our hypothesis that the AppA/PpsR system alone is sufficient to describe the overall trend of PS gene expression we parameterise the model and suggest that the rate of AppA reduction in vivo should be faster than its oxidation. Also, we show that despite both the reduced and oxidised forms of PpsR binding to the PS gene promoters in vitro, binding of the oxidised form as a repressor alone is sufficient to reproduce the observed PS gene expression pattern. Finally, the combination of model parameters which fit the biological data well are broadly consistent with those which were previously determined to be required for the system to show (i) the repression of PS genes under semi-aerobic conditions, and (ii) bistability.

Conclusion

We found that despite at least three pathways being involved in the regulation of photosynthetic genes, the AppA/PpsR system alone is capable of accounting for the observed trends in photosynthetic gene expression seen at different oxygen levels.

     

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.     

紫细菌光合机构及光合作用基因的表达调控

紫细菌是研究细菌光合作用的重要生物.介绍了紫细菌光合机构捕光色素蛋白复合体Ⅰ(light-harvestingⅠ)、捕光色素蛋白复合体Ⅱ(1ight-harvesting Ⅱ)和光化学反应中心(reaction center)的结构,并探讨了其光合作用基因的转录调控机制,重点阐述了PpsR/AppA系统对紫细菌光合作用基因的转录调控.     

紫细菌光合机构及光合作用基因的表达调控

紫细菌是研究细菌光合作用的重要生物。介绍了紫细菌光合机构捕光色素蛋白复合体Ⅰ(light-harvesting I)、捕光色素蛋白复合体Ⅱ(light-harvesting II)和光化学反应中心(reaction center)的结构, 并探讨了其光合作用基因的转录调控机制, 重点阐述了PpsR/AppA系统对紫细菌光合作用基因的转录调控。     

Light and redox control of photosynthesis gene expression in Bradyrhizobium: dual roles of two PpsR

The two closely related bacteria Bradyrhizobium and Rhodopseudomonas palustris show an unusual mechanism of regulation of photosystem formation by light thanks to a bacteriophytochrome that antirepresses the regulator PpsR. In these two bacteria, we found out, unexpectedly, that two ppsR genes are present. We show that the two Bradyrhizobium PpsR proteins exert antagonistic effects in the regulation of photosystem formation with a classical repressor role for PpsR2 and an unexpected activator role for PpsR1. DNase I footprint analysis show that both PpsR bind to the same DNA TGTN12ACA motif that is present in tandem in the bchC promoter and the crtED intergenic region. Interestingly, the cycA and aerR promoter regions that contain only one conserved palindrome are recognized by PpsR2, but not PpsR1. Further biochemical analyses indicate that PpsR1 only is redox sensitive through the formation of an intermolecular disulfide bond, which changes its oligomerization state from a tetramer to an octamer under oxidizing conditions. Moreover, PpsR1 presents a higher DNA affinity under its reduced form in contrast to what has been previously found for PpsR or its homolog CrtJ from the Rhodobacter species. These results suggest that regulation of photosystem synthesis in Bradyrhizobium involves two PpsR competing for the binding to the same photosynthesis genes and this competition might be modulated by two factors: light via the antagonistic action of a bacteriophytochrome on PpsR2 and redox potential via the switch of PpsR1 oligomerization state.