Transformation of a bop-hop-sop-I-sop-II-Halobacterium halobium mutant to bop+: effects of bacteriorhodopsin photoactivation on cellular proton fluxes and swimming behavior.
We have transformed Pho81, a Halobacterium halobium mutant strain which does not contain any of the four retinylidene proteins known in this species, with the bop gene cluster to create Pho81BR, a BR+HR-SR-I-SR-II-strain. The absorption spectrum, pigment reconstitution process, light-dark adaptation and photochemical reaction cycle of the expressed protein are indistinguishable from those of native bacteriorhodopsin (BR) in purple membrane of wild type strains. Strain Pho81BR permits for the first time characterization of effects of BR photoactivation alone on cell swimming behavior and energetics in the absence of the spectrally similar phototaxis receptor sensory rhodopsin I (SR-I) and electrogenic chloride pump halorhodopsin (HR). A non-adaptive upward shift in spontaneous swimming reversal frequency occurs following 3 s of continuous illumination of Pho81BR cells with green light (550 +/- 20 nm). This effect is abolished by low concentrations of the proton ionophore carbonylcyanide m-chlorophenylhydrazone. Although BR does not mediate phototaxis responses in energized Pho81BR cells under our culture conditions, proton pumping by BR in Pho81BR cells partially deenergized by inhibitors of respiration and adenosine triphosphate synthesis results in a small attractant response. Based on our measurements, we attribute the observed effects of BR photoactivation on swimming behavior to secondary consequences of electrogenic proton pumping on metabolic or signal transduction pathways, rather than to primary sensory signaling such as that mediated by SR-I. Proton extrusion by BR activates gated proton influx ports resulting in net proton uptake in wild-type cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Yan
B
B
Department of Microbiology and Molecular Genetics, University of Texas Medical School, Houston 77030.
Cline
S W
SW
Doolittle
W F
WF
Spudich
J L
JL
D001436:Bacteriorhodopsins / Q000378:metabolism*; D002465:Cell Movement / Q000187:drug effects / Q000528:radiation effects*; D003001:Cloning, Molecular; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D005798:Genes, Bacterial; D019617:Halobacterium salinarum / Q000235:genetics / Q000502:physiology* / Q000528:radiation effects; D008027:Light; D005810:Multigene Family; D010957:Plasmids; D011522:Protons; D014170:Transformation, Genetic
D011522:Protons; D001436:Bacteriorhodopsins; D004024:Dicyclohexylcarbodiimide