Isolation of the in vivo emitter in bacterial bioluminescence

Abstract

A blue fluorescence protein has been isolated and purified from extracts of the luminous bacterium Photobacterium phosphoreum. It is a single polypeptide of molecular weight 22,000 with absorption maxima at 274 and 418 nm. It is efficiently fluorescent (OF 0.45), with a fully corrected spectral maximum (476 nm) and distribution identical to the in vivo bioluminescence from this same type of bacterium. At low concentration this fluorescence shifts towards the red and becomes identical to the in vitro bioluminescence emission. This spectral shift apparently results from a change in the protein pulled by dissociation of the chromophore . If the blue fluorescence protein is included in the in vitro bioluminescence reaction with reduced FMN, oxygen, aldehyde, and luciferase (P. phosphoreum), the bioluminescence spectrum is shifted towards the blue from its maximum at 490 nm to one at 476 nm, where it is again identical in all respects to the in vivo bioluminescence spectrum. This is accompanied by an increase in the initial light intensity by an order of magitude at saturating levels of blue fluorescence protein, and the specific light yield of the luciferase is increased 4-fold. It is suggested that the blue fluorescence protein acts as a sensitizer of the bacterial bioluminescence reaction. (14) claimed that the emitter must be some sort of flavin-derived species (2), three proposals for its structure were put forward, supported almost solely by the similarity of the fluorescence of model compounds to the bioluminescence spectra. Eley et al. (12) (17) proposed an FMNH2 molecule substituted in the 4a-position.It had been tacitly assumed that the mechanism of reaction and identity of the emitter are the same in vivo as in vitro. The possibility of a difference has been raised by the recent discovery of a bacterial type emitting at 545 nm (18). In this paper we show that a protein-bound chromophore can be isolated from extracts of the bioluminescent bacteria P. phosphoreum that is closely associated with luciferase and that fulfills all the conditions to qualify it as the in vivo emitter. MATERIALS AND METHODSThe bacterium Beneckea harveyi, strain 392 in the classification scheme of Reichelt and Baumann (19), previously designated "MAV,"' was obtained from J. W. Hastings (Harvard University). The type "A-13" was isolated from the light organ of the "silver macrourid" fish by J. Paxton (Australian Museum) and has been identified as Photobacterium phosphoreum (J. Fitzgerald, private communication). The type Photobacterium fischeri, strain 399, was obtained from F. H. Johnson (Princeton University). The bacteria were grown and the luciferase and FMN were purified as described (20,21). The blue fluorescence protein was routinely assayed by its fluorescence intensity at 470 nm when excited at 420 nm, with an Aminco-Bowman Spectrofluorimeter. Luciferase activity was determined with a digital photometer, designed and constructed by G. J. Faini, which was calibrated for absolute photon sensitivity with the luminol ch...

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