In Vivo Effects on Photosynthesis Gene Expression of Base Pair Exchanges in the Gene Encoding the Light‐responsive BLUF Domain of AppA in Rhodobacter Sphaeroides

Abstract

The Rhodobacter sphaeroides protein AppA has the unique quality of sensing and transmitting light and redox signals. By acting as antirepressor to the PpsR protein, it acts as a major regulator in photosynthesis gene expression. In this study, we show that by introducing amino acid exchanges into the AppA protein, the in vivo activity as an antirepressor can be greatly altered. The tryptophan 104 to phenylalanine (W104F) base exchange greatly diminished blue-light sensitivity of the BLUF domain. From the obtained in vivo data, the difference in thermal recovery rate of the signaling state of the BLUF domain between the wild type and mutated protein was calculated, predicting an about 10-fold faster recovery in the mutant, which is consistent with in vitro data. Introduction of a tyrosine 21 to phenylalanine (Y21F) or to cysteine (Y21C) mutation led to a complete loss of AppA antirepressor activity, while additionally leading to an increase of photosynthesis gene expression after illumination with high blue-light quantities. Interestingly, this effect is not visible in a W104F/Y21F double mutant that again shows a wild-type-like behavior of the BLUF domain after blue-light illumination, thus restoring the activity of AppA.

An extended model for the repression of photosynthesis genes by the AppA/PpsR system in Rhodobacter sphaeroides

“…Circles represent experimental measurements (two repetitions) of puc inhibition taken from Metz et al. [25].…”

“…Hence, we conclude that there exists a biologically reasonable set of parameters leading to a response curve in agreement with the experimental measurements. Our mathematical model can also be used to analyze a phenotype with substantially diminished light sensitivity, as has been recently observed in an AppA mutant strain [25] ().…”

“…Dashed lines indicate how the shape and/or the position of the reference response curve would be affected by independently changing one of the parameters δ (A, C), λ or η (B, D) as indicated. Dotted lines mark the region of light irradiance over which repression of the puc gene changes from its minimal value (LI = 0.1 μmol·m −2 ·s −1 ) to its maximal value (LI = 1 μmol·m −2 ·s −1 ) as observed experimentally [23,25].…”

Photoreceptors mapping from past history till date

“…It has been found to regulate photosynthesis, phototaxis as well as mediates light induction of adenylyl cyclase activity and phosphodiesterases [4,80]. In Rhodobacter sphaeroides, AppA protein (BLUF containing protein) binds to the transcription factor, PpsR which on perceiving light is released and acts on photosynthetic gene expression [81]. Concerning to the phosphodiesterases activity, Barends et al [80] showed that BlrP1 protein and EAL phosphodiesterase which are BLUF sensor domain and output domain respectively in Klebsiella pneumoniae, after perceival of light by sensor domain there is some conformational changes that are propoagted from sensor to effector domain i.e.…”

Effects of the Cryptochrome CryB from Rhodobacter sphaeroides on Global Gene Expression in the Dark or Blue Light or in the Presence of Singlet Oxygen

“…Samples for RNA isolation were collected when the cultures reached an OD 660 nm of 0.8, exactly. Blue light experiments were performed as described elsewhere [10], [52]. Cultures inoculated to an OD 660 nm of 0.15 were kept under semiaerobic conditions (approximately 90 µM dissolved oxygen) by varying the rotation speed of the shaker.…”