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Toni Greene

Advisor: John Spudich, Ph.D.

Accepted position as high school science teacher after receiving MS

The genome of the freshwater cyanobacterium Anabaena sp. PCC 7120 was sequenced in 2001 and a chromosomal gene encoding the photosensor Anabaena Sensory Rhodopsin (ASR) was found. A single copy of the aop gene is located in an operon with a second gene (asrt) encoding its putative functional partner, ASR Transducer (ASRT). Photochemical studies in our laboratory on ASR heterologously expressed in E. coli have shown ASR is a green light-activated photosensory receptor that interacts with, and has moderate binding to, ASRT. Its putative use of a soluble transducer is novel for microbial rhodopsins. Although our laboratory solved the x-ray crystal structures of both ASR and ASRT, their physiological functions are still unknown.
Bioinformatic analysis of the aop and asrt sequences revealed ribosomal binding sites and a predicted mRNA hairpin in the 16bp region separating the two genes. Also, primary sequence motifs on the ASR cytoplasmic tail and an ASRT accessible loop have positive and negative residue pattern that correspond to a possible anti-parallel ionic interaction. A predicted serine/threonine phosphorylation site is located near this charge motif on ASRT. Using a polyclonal antibody, we proved by quantitative immunoblotting that ASRT is synthesized in wild-type Anabaena. The quantity of ASRT was calculated to be 600 molecules per cell and the expression level was constant over 36 hours of continuous growth.
We made single (Δaop, Δasrt) and double (Δaop/Δasrt) gene knockouts in Anabaena, and several phenotypes were examined for light responsive physiological functions. A consistent phenotypic difference seen between wild-type and mutants was the ratio of chlorophyll to phycobiliproteins (Chl:PBP). Growth in high or low light incubators induced a change in wild-type Chl:PBP but did not elicit a change in the mutants. These results suggest that ASR and ASRT signal through protein:protein interactions between positive/negative motif patches, and that this interaction is differentially regulated according to the phosphorylation state of ASRT. The pathway for ASR-ASRT regulation is not known, but this signaling pair appears to directly or indirectly regulate the levels of photosynthetic pigments in the cell.

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Research Info

ASR and ASRT: Bioinformatics, Biosynthesis and Photophysiology