AUTOINDUCTION AND ALDEHYDE CHAIN‐LENGTH EFFECTS ON THE BIOLUMINESCENT EMISSION FROM THE YELLOW PROTEIN ASSOCIATED WITH LUCIFERASE IN Vibrio fischeri STRAIN Y‐1b

Abstract

Abstract— A new strain of the yellow‐light emitting bacterium Vibrio fischeri Y‐lb, possessing a higher fraction of its emission in the yellow, was isolated and characterized. The in vivo yellow to blue ratio (Y/B) increases during growth in parallel with the autoinduction of luciferase, reaching a value above 5 at 17°C. This Y/B increase is attributed to concomitant increases in the intracellular levels of both luciferase and the specific protein associated with yellow emission, the yellow fluorescent protein (YFP), and to the association of the two proteins at higher concentrations. The yellow emission is rapidly but reversibly lost by brief exposures to a temperature of 27°C (5 s), and irreversibly so at 40°C, attributed to dissociation of the two proteins. A concentrated crude extract emits almost exclusively yellow light in the in vitro reaction, and theY/B ratio decreases with dilution. The Y/B ratio in vitro is also dependent on the aldehyde chain length; experiments with aldehyde analogs suggest that the aldehyde binding site of luciferase must be occupied for YFP to stimulate light emission.

Effectiveness of the Accessory Yellow Fluorescent Protein in the Bacterial Luciferase Reaction Correlates with the Lifetime of the Peroxyhemiacetal Intermediate: The Stereochemistry of the Reaction

“…= 9 with 10 FM YFP) and about six times smaller with tetradecanal ( Fig. 3); this trend, consistent with a previous report (28), was the same at the three YFP concentrations tested. Figure 4 shows the effect of YFP on k,, the rate constant for the decay of light emission, as a function of aldehyde chain length.…”

“…At two temperatures, 4 and 24"C, both the initial light intensity, I,, and its rate of decay, kdr increase sharply with aldehydes of 12 or more carbons. The data on kd at 24°C show the same trend as reported by Hastings et al (26) and are also in good agreement with the results of Cho et al (28). Note that these effects are not monotonic.…”

“…It is well known, if not fully explained, that the chain length of the aliphatic aldehyde determines both the intensity of emission and its rate of decay (26), with the natural aldehyde, tetradecanal (27), generating the highest intensities and hexanal the lowest. Interestingly, this dependence is essentially reversed in the presence of yellow fluorescent protein (YFP), the accessory yellow fluorescence protein present in V. jischeri strain Y-1 (28). In the appropriate temperature range, the bioluminescence spectrum of this bacterium shows two spectral bands, one peaking at 490 nm, as in the case of other bacteria, the other at 540 nm; when the latter predominates, the bioluminescence appears yellow.…”

A Blue Fluorescent Protein From a Yellow‐emitting Luminous Bacterium

“…It is interesting that the effects of fluorescent proteins such as YFP, Y1-BFP and Lump are dependent on the type of luciferase. The association between a fluorescent protein and a luciferase reaction intermediate is thus an essential factor in its mode of action (Eckstein et al, 1990;Cho et al, 1989). In earlier studies on the V. ficheri Y 1 bioluminescence (Ruby and Nealson, 1977;Daubner and Baldwin, 1989), the color change in the light emission was postulated to be due to energy transfer from the excited emitter generated in the normal luciferase reaction, Eq. 1, to the Auorophore of YFP.…”

A time-dependent bacterial bioluminescence emission spectrum in an in vitro single turnover system: energy transfer alone cannot account for the yellow emission of Vibrio fischeri Y-1.

“…A V. fischeri strain Y-lb with a high yellow/blue ratio in vivo (Y/B = 5.5) at 17'C was obtained by selection of a single colony for brightness (9) on seawater complete agar medium (750 ml of seawater, 3 g of yeast extract, 5 g of bactotryptone, 2 ml of glycerol, and 25 g of agar per liter of medium; ref. 10).…”