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Proc. Nat. Acad. Sci. USA Vol. 69, No. 5, pp. 1073-1076, May 1972 Catabolite Repression of Bacterial Bioluminescence: Functional Implications (cyclic AMP/inducible enzyme/luciferase/arginine/glucose) KENNETH H. NEALSON*, ANATOL EBERHARDt AND J. W. HASTINGSt The Biological Laboratories, Harvard University, 16 Divinity Avenue, Cambridge, Massachusetts 02138 Communicated by Boris Magasanik, February 14, 1972 ABSTRACT The synthesis of the bioluminescent sys- plete plus glycerol-5.0 g tryptone, 0.5 g yeast extract, and tem of the marine luminous bacterium Photobacteriurn 3.0 ml glycerol. fischeri (strain MAV) is subjectto both transient and catab- olite repression by glucose, and this repression can be Experiments were done in 4-ml liquid shake cultures in reversed by adenosine 3':5'-cyclic monophosphate. Cata- 120-mm screw cap tubes at 25 i 20. Cell growth was moni- bolite repression is a mechanism that characteristically tored by reading relative optical density in these tubes at controls the synthesis of inducible enzymes involved in 660 nm in a Coleman Jr. II spectrophotometer. The relative energy metabolism. The fact that luciferase synthesis is subject to this control suggests that whatever its role(s) optical density values presented in the figures can be con- may be, it cannot be considered a nonfunctional or vesti- verted to true optical densities by multiplying by about 2. gial enzyme system as previously hypothesized, and may In vivo bioluminescence was likewise monitored in the same actually have some more direct role in metabolic processes. tubes by placing the tubes in a special light-tight adapter, which exposed them to the photomultiplier (8). In vivo ac- Bacterial operons may be classified into two groups: those tivity is expressed in the figures in terms of intensity in light with catabolite-insensitive promoters and those with, catabo- units where each unit is equal to 1.1 X 1011 quanta sec' (9). lite-sensitive promoters (1). Those operons with catabolite- In vitro determinations of extractable luciferase were done sensitive promoters have the common characteristics that in as One ml of reduced flavin mono- of previously described (3). the presence glucose or certain other rapidly metabolized nucleotide (5 X 10-5 M) was rapidly mixed with a cell extract carbon sources they are repressed, and that this repression in the presence of oxygen and dodecanal, with 0.01 M phos- can be reversed by the addition of cyclic AMP (cAMP). Most buffer in a final volume of 2 ml. The initial in- of the members of phate (pH 7) this sensitive group are inducible enzyme tensity of this reaction is an accurate measure of the relative systems concerned with energy metabolism (2). of The is in the Luciferase of the marine bacterium amount luciferase present. intensity expressed Photobacteriumfischeri, figure in light units (where each unit is equal to 2.2 X 1010 strain MAV (3), is also an inducible enzyme, but induction is ml of culture from which the lu- not yet it quanta sec-) per original understood; requires the presence of arginine to- ciferase was extracted. gether with the production of a compound produced by the where avail- themselves Chemical reagents were of analytical quality cells (4, 5). able. Flavin mononucleotide and cAMP were obtained from We have found that the bioluminescent system is also sensi- and tive to catabolite repression. The presence of glucose in the Sigma Chemical Co., St. Louis, Mo.; 2-deoxyglucose do- medium (either complex or minimal and either with or with- decanal from Aldrich Chemical Co., Milwaukee, Wisconsin. out another carbon source) causes a marked diminution both RESULTS in the light emission of the cells and in the amount of luciferase 1 by synthesized. This repression is reversed by the addition of Fig. illustrates the repression of luciferase synthesis glu- cAMP. cose and its reversal by cAMP during the growth of cells in a complete liquid medium. The data are presented as a plot of MATERIALS AND METHODS luciferase content (measured by extractable activity) as a Photobacterium fischeri (strain MAV) has been described else- function of cell density. As with any inducible system, two where (3, 4, 6) and was used for all experiments. A basic mini- factors combine to determine the final level of enzyme at- mal salts solution was prepared according to Farghaly (7), tained: (1) the rate and (2) the duration of enzyme synthesis. containing 30 g NaCl, 7 g Na2HPO4, 1 g KH2PO4, 0.5 g As shown in Fig. 1, for glucose repression of luciferase, the (NH4)2PO4, and 0.1 g MgSO4 per liter of distilled water. For effect involves primarily a decrease in the rate of luciferase the various media, the following additions were made, per synthesis resulting in a diminution of the final level reached liter of medium: minimal medium-3 ml glycerol; minimal by a factor of 10 (Table 1, A and B). The effect of cAMP in plus arginine-3 ml glycerol plus 0.5 g arginine; complete- the presence of glucose is to restore the final level of extract- 5.0 g Difco bacto tryptone and 0.5 g Difco yeast extract; com- able luciferase nearly to normal (Table 1, A and B). As with repression, its reversal might occur by increasing either the rate or duration of enzyme synthesis. Fig. 1 demonstrates * Postdoctoral fellow, National Institutes of Health. that a major effect of cAMP is at the level of the rate of lu- t Present address: Department of Chemistry, Fairleigh Dickin- ciferase synthesis, although effects upon the duration of syn- son University, Teaneck, N. J. 07666. thesis do occur. To whom reprint requests may be addressed. Fig. 2 illustrates the same type of experiment, but the in 1073 Downloaded by guest on September 27, 2021 1074 Microbiology: Nealson et al. Proc. Nat. Acad. Sci. USA 69 (1972) luminescence; it also slightly decreases the rate of growth. This effect on growth occurs in either the presence or absence of glucose. The luminescent system is also sensitive to transient re- pression, as is characteristic of systems sensitive to catabolite repression. This is illustrated in Fig. 3, where glucose, 2- deoxyglucose, and cAMP in various combinations were added to a culture after induction had occurred in the complete medium. Subsequent to the addition of glucose a severe, but transient (-30 min) decrease in light intensity occurred, followed by a resumption of synthesis at the characteristically decreased rate. Nonmetabolizable sugars, such as 2-deoxyglu- cose, are known to cause this type of transient repression with- out causing the continued (catabolite) repression (10). As shown in Fig. 3, this was also observed for the luminescent 02 0.4 0.6 0 D 660 system, with both types of repression being reversed in the presence of cAMP. FIG. 1. Effect of glucose and cAMP on the luciferase content of luminous bacteria growing in a complete medium plus glycerol. DISCUSSION With an exponentially growing culture of strain MAV as an ino- The luminescent system thus exhibits features characteristic culum, cells were diluted into fresh medium to an OD of about of an operon sensitive to catabolite repression. The induction 0.005, and 4-ml aliquots of this inoculum was dispensed into of the luciferase system is unusual because it involves a condi- 16 X 125 mm screw cap culture tubes containing the indicated tioning of the medium in which the cells themselves synthe- and the additions and shaken vigorously. Tubes were removed a substance that is involved in the induction Be- cells were harvested and assayed for luciferase content. The data size (4, 5). from several experiments are plotted on a single graph. Luciferase cause of this, the time course of luciferase synthesis cannot content, expressed in light units (1 unit = 2.2 X 1010 quanta be readily manipulated. Nevertheless, it is possible to show sec-), is plotted (ordinate) as a function of the cell density that both transient and catabolite repression by glucose will (abscissa). Symbols: X -X, control, no glucose or cAMP; occur at any time after induction has occurred. O-O, glucose; * , glucose plus 0.5 mg/mi cAMP. A further complication in the control of the bioluminescence is that the inducer-like substance produced by the cells is active in stimulating luciferase synthesis only when exogenous arginine is added. Or, conversely, one might say that arginine vivo bioluminescence was plotted instead of the extractable is active in stimulating luciferase synthesis only in the pres- luciferase. Here the same pattern can be seen; glucose de- ence of the substance produced by the cells. creases the rate of appearance of the luminescence and cAMP reverses this effect. Table 1 (A and B) presents data for the effects of glucose and cAMP on the in vivo and in vitro levels of bioluminescence in complete medium both with and without added glycerol. It also shows that although the growth rate is slightly faster 10 in the presence of glucose, this effect does not appear to be related to catabolite repression, since relief of repression by cAMP has no effect on growth. 1.0 With cells growing in a minimal medium with added argi- I nine there is a similar pattern of repression of luminescence by glucose, and its relief by cAMP (Table 1, C). Glucose addi- 0.1 tion results in a diminution of the rate of development and of the final level of both in vivo luminescence and extractable luciferase. Addition of cAMP results in the restoration of 0.01 luciferase to control levels or above; it stimulates in vivo light production to levels considerably above the control level. With cells growing in minimal medium in the absence of added arginine there is very little light emission and correspondingly OD 660 low levels of luciferase (Table 1, D).

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