Light Modulation of Cellular cAMP by a Small Bacterial Photoactivated Adenylyl Cyclase, bPAC, of the Soil Bacterium Beggiatoa

Manuela Stierl,Patrick Stumpf,Daniel Udwari,Ronnie Gueta,Rolf Hagedorn,Aba Losi,Wolfgang Gärtner,Linda Petereit,Marina Efetova,Martin Schwarzel,Thomas G Oertner,Georg Nagel,Peter Hegemann

doi:10.1074/jbc.m110.185496

Abstract

The recent success of channelrhodopsin in optogenetics has also caused increasing interest in enzymes that are directly activated by light. We have identified in the genome of the bacterium Beggiatoa a DNA sequence encoding an adenylyl cyclase directly linked to a BLUF (blue light receptor using FAD) type light sensor domain. In Escherichia coli and Xenopus oocytes, this photoactivated adenylyl cyclase (bPAC) showed cyclase activity that is low in darkness but increased 300-fold in the light. This enzymatic activity decays thermally within 20 s in parallel with the red-shifted BLUF photointermediate. bPAC is well expressed in pyramidal neurons and, in combination with cyclic nucleotide gated channels, causes efficient light-induced depolarization. In the Drosophila central nervous system, bPAC mediates light-dependent cAMP increase and behavioral changes in freely moving animals. bPAC seems a perfect optogenetic tool for light modulation of cAMP in neuronal cells and tissues and for studying cAMP-dependent processes in live animals.

Highlights

Is available in all animal cells, and a once transformed organism inherits the light sensitivity to the generation
Protein Purification—For purification, the bPAC SUMO fusion construct was expressed in E. coli strain BL21 DE3 at 18 °C in LB induced with 60 ␮M isopropyl-1-thio-␤-D-galactopyranoside for 48 h. bPAC was purified on cobalt-containing nitrilotriacetic acid resin (Clontech) in 50 mM NaH2PO4, 300 mM NaCl, 0.1 mM phenylmethylsulfonyl fluoride (PMSF) buffer according to the supplier’s instructions
To test the function of the protein encoded by the published bPAC sequence, we cloned bPAC into a cAMP-deficient E. coli strain in which lactose and maltose cannot be fermented [17]

Summary

EXPERIMENTAL PROCEDURES

Cyclase Activity in E. coli—E. coli-optimized synthetic DNA encoding the photoactivated cyclase bPAC of Beggiatoa sp. (accession number GU461307) was purchased from Mr Gene (Regensburg, Germany) and was cloned in-frame behind the N-terminal His tag and SUMO epitope into a pET SUMO vector (Invitrogen). The protein was expressed in E. coli strain BTH101 at 30 °C in 200 ␮M isopropyl-1-thio-␤-D-galactopyranoside for 2 h. Both transformed and non-transformed cells were plated on MacConkey agar (Difco), pH 7.5, containing 1% maltose and incubated at 30 °C overnight in darkness or in white light (average intensity: 8 W mϪ2 white light). Protein Purification—For purification, the bPAC SUMO fusion construct was expressed in E. coli strain BL21 DE3 at 18 °C in LB induced with 60 ␮M isopropyl-1-thio-␤-D-galactopyranoside for 48 h. Cyclase activity of purified recombinant protein was assessed with this assay by incubation of 10 ␮g of bPAC in 24 ␮l of a solution of 300 mM KCl, 50 mM Hepes-Tris, pH 7.4, 1 mM MgCl2, 100 ␮M MgATP at room temperature (21 °C).

RESULTS

Plotted are mean values with

DISCUSSION