Identification of Proteins Involved in Aldehyde Production for Bioluminescence

Transcription

1 JOURNAL OF BACTERIOLOGY, Aug. 1984, p /84/ $02.00/0 Copyright C) 1984, American Society for Microbiology Vol. 159, No. 2 In Vivo and In Vitro Acylation of Polypeptides in Vibrio harveyi: Identification of Proteins Involved in Aldehyde Production for Bioluminescence LEE A. WALL, DAVID M. BYERS, AND EDWARD A. MEIGHEN* McGill University, Department of Biochemistry, Montreal, Quebec H3G I Y6, Canada Received 14 February 1984/Accepted 21 May 1984 Incubation of soluble extracts from Vibrio harveyi with [3H]tetradecanoic acid (+ATP) resulted in the acylation of several polypeptides, including proteins with molecular masses near 20 kilodaltons (kda), and at least five polypeptides in the 30- to 60-kDa range. However, in growing cells pulse-labeled in vivo with [3HJtetradecanoic acid, only three of these polypeptides, with apparent molecular masses of 54, 42, and 32 kda, were specifically labeled. When extracts were acylated with [3H]tetradecanoyl coenzyme A, on the other hand, only the 32-kDa polypeptide was labeled. When luciferase-containing dark mutants of V. harveyi were investigated, acylated 32-kDa polypeptide was not detected in a fatty acid-stimulated mutant, whereas the 42- kda polypeptide appeared to be lacking in a mutant defective in aldehyde synthesis. Acylation of both of these polypeptides also increased specifically during induction of bioluminescence in V. harveyi. These results suggest that the role of the 32-kDa polypeptide is to supply free fatty acids, whereas the 42-kDa protein may be responsible for activation of fatty acids for their subsequent reduction to form the aldehyde substrates of the bioluminescent reaction. Light emission by bioluminescent marine bacteria is catalyzed by luciferase in a reaction which oxidizes reduced flavin mononucleotide and a long-chain aliphatic aldehyde to flavin mononucleotide and the corresponding fatty acid (2). Recently, we have reported a fatty acid reductase activity in extracts of the bioluminescent strain Photobacterilum phosphoreum which is capable of supplying the aldehyde substrates for the luminescent reaction of luciferase (6, 11). Purification of the P. phosphoreium fatty acid reductase has shown that it is a high-molecular-weight complex consisting of three different polypeptides (the three proteins of the P. phosphoreum fatty acid reductase complex have been designated 58,000 [58K], 50K, and 34K as previously described [14, 18], whereas proteins in Vibrio harveyi, to prevent confusion, have been designated by the apparent molecular masses on sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE] in kilodaltons [kda]): an acyl protein synthetase component (50K) which, in the presence of ATP, activates fatty acids to an acyl-sok intermediate, an acyl-coenzyme A (CoA) reductase subunit (58K) which catalyzes the reduction of acyl-50k or acyl-coa with NADPH, and a third polypeptide (34K) whose role in fatty acid utilization remains unknown (13, 14). In recent studies, it has also been shown that these polypeptides can be specifically identified in extracts of P. phosphoreum by acylation with [3H]tetradecanoic acid (+ATP), which labels the 50K and 34K polypeptides, or [3H]tetradecanoyl-CoA, which labels the 34K and 58K polypeptides (14, 18). In V. harveyi, the luminescent bacterium which has been studied in the greatest detail, the mechanism of the luciferase reaction has been well characterized, whereas the biosynthesis of the aldehyde substrate in this strain is only poorly understood. The existence of a luciferase-containing dark mutant of V. harveyi, which can be stimulated to produce light by the addition of exogenous fatty acids, has suggested that aldehyde biosynthesis may also proceed via fatty acid reduction (16). However, despite repeated attempts, fatty * Corresponding author. acid reductase activity has not yet been measured in vitro in lysates of V. harveyi, and therefore, the identity of the proteins involved remains unknown. In the present study, lysates of V. harveyi and dark mutants (whose luminescence is stimulated by fatty aidehyde) were acylated in vitro with [3H]tetradecanoic acid and [3H]tetradecanoyl-CoA in an attempt to establish which polypeptides may be involved in aldehyde biosynthesis. These techniques were supplemented by fatty acid labeling in vivo and resulted in the identification of polypeptides involved in supplying aldehyde for the bioluminescent system of V. harveyi. MATERIALS AND METHODS Materials. Acrylamide, N,N-methylenebisacrylamide, ATP, NADPH (type III), flavin mononucleotide, and,bmercaptoethanol were all obtained from Sigma Chemical Co. SDS-PAGE molecular mass standards (94 kda, phosphorylase b; 67 kda, bovine serum albumin; 43 kda, ovalbumin; 30 kda, carbonic anhydrase; 20 kda, soybean trypsin inhibitor; and 14 kda, a-lactalbumin) were obtained from Pharmacia Fine Chemicals. [3H]tetradecanoic acid (21 Ci/mmol) was prepared by New England Nuclear Corp. by reduction of cis-9-tetradecenoic acid with tritium gas in the presence of a 5% palladium-on-carbon catalyst. [3H]tetradecanoyl-CoA (1 Ci/mmol) was prepared from the radioactive fatty acid as previously described (13). Fatty acids and aldehydes were stored at -20 C as stock solutions in ethanol or isopropanol. Phosphate buffers were made by mixing the appropriate amounts of K2HPO4 and NaH2PO4. The bioluminescent bacterial strains used in these studies were P. phosphoreum NCMB 844 and V. harveyi B392. The dark mutants derived from V. harveyi by mutagenesis with nitrosoguanidine were a generous gift from J. W. Hastings (Harvard University). M17 is a dark mutant which can be stimulated to produce light by addition of exogenous aidehydes or fatty acids, as has been described previously (16). The acylation patterns of three different aldehyde synthesis mutants, which respond only to aldehyde and not fatty acids, 720

2 VOL. 159, 1984 were examined and found to be identical; hence, experiments on only one of these mutants (designated Aldl6) are presented in this report. Cell growth and lysis. P. phosphoreum was grown at 19 to 20 C in 3% NaCI complex media (11), whereas the V. harveyi strains were grown in 1% NaCI complex media at 26 to 27 C. Cultures (50 to 300 ml) were inoculated at an optical density of 660 nm (Aw) of 0.05 and grown in Erlenmeyer flasks in a reciprocatory shaker. The increase in A60 and in vivo luminescence of 1-ml samples was followed with growth. For M17 and Aldl6 cells, the maximum in vivo bioluminescence was measured after addition of 100,uM tetradecanoic acid or 100,M decanal, respectively. One light unit (LU) equals 6 x 109 quanta per s based on the standard of Hastings and Weber (4). For in vitro analyses, a constant amount of cells (A60 x volume [in milliliters] = 20) was isolated by centrifugation (15,000 x g, 15 min) and frozen at -20 C. P. phosphoreum cells were thawed and sonicated (20 s, three times) into 250 RI of 1 mm phosphate (ph 7.0) containing 1 mm EDTA and 1 mm 3-mercaptoethanol and then centrifuged to remove cellular debris. The supernatant was made 50 mm in phosphate (ph 7.0)-4-mercaptoethanol by addition of 1 M phosphate (ph 7.0) containing 1 M,B-mercaptoethanol. V. harveyi cells were extracted by sonication directly into 250 Rl of 50 mm phosphate (ph 7.0) containing 20 mm,-mercaptoethanol, followed by centrifugation. In vivo acylation. A constant amount of growing cells (A660 x volume [in milliliters] = 1.0) was removed and placed in a 1.5-mi Eppendorf tube with 2.5,uM [3H]tetradecanoic acid (21 Ci/mmol) added. The cells were incubated by shaking for 5 min at the proper growth temperature and then isolated immediately by centrifugation, washed twice with cold medium, and stored at -70 C. The labeled cells were extracted by sonication (20 s, three times) into 50 RI of 1 mm phosphate (ph 7.0) containing 1 mm f-mercaptoethanol. The cellular debris was removed by centrifugation, and after removal of a small aliquot for protein determination, the supernatant was diluted (1:1) with SDS-PAGE sample buffer containing 0.12 M Tris-hydrochloride (ph 6.8), 25% glycerol (vol/vol), 2.5% SDS, 0.35 M,B-mercaptoethanol, and 0.01% (wt/vol) bromophenol blue as tracking dye. The samples were then boiled for 5 min. In vitro acylation. For fatty acid acylation, 25 RI of the extracts was made up to a total volume of 100 RI containing 50 mm phosphate (ph 7.0), 5 mm ATP, 12.5,uM [3H]tetradecanoic acid (21 Ci/mmol), and 10 mm P-mercaptoethanol, with 10 mm MgSO4 for V. harveyi samples or without MgSO4 for P. phosphoreum samples. After 10 min, the reaction was stopped by mixing with an equal volume of SDS-PAGE sample buffer and boiling for 5 min. Proteins were acylated with acyl-coa by incubation with 10,uM [3H]tetradecanoyl-CoA (instead of tetradecanoic acid and ATP) for 30 s under the conditions described above. This short labeling period was used, as it resulted in the maximum incorporation into extracts from V. harveyi strains. Longer incubation times resulted in a decreased incorporation, apparently due to a loss of acyl-coa by high thioesterase activities in the extracts. Enzyme assays. The purification and the standard assay procedures for luciferase have been previously described (3, 7), with P. phosphoreum luciferase being measured in the presence of 0.01% dodecanal and V. harveyi luciferase being assayed with 0.002% dodecanal. Acyl-CoA reductase activity was measured by the luciferase-coupled assay in 50 mm phosphate (ph 7.0), containing ACYLATION OF POLYPEPTIDES IN V. HARVEYI mm,b-mercaptoethanol and 10 mm MgSO4 as previously described (13). Extracts were assayed in the presence of 10,uM NADPH and 5,uM tetradecanoyl-coa for 2 min, with 5,ug of P. phosphoreum luciferase added. Under these conditions, 1 LU corresponds to 4 pmol of aldehyde produced in the assay. SDS-PAGE. SDS-PAGE was performed by the system described by Laemmli (5) with 12% polyacrylamide resolving gels (8.5 cm) and 5% stacking gels. Gels were stained with Coomassie brilliant blue R (Aldrich Chemical Co.), destained, soaked in En3Hance (New England Nuclear Corp.), dried under vacuum, and fluorographed for 5 to 8 days with Kodak X-OMAT AR film as previously described (14). Protein assays. Protein concentrations were determined by the Bio-Rad dye binding assay (1), with bovine serum albumin used as a standard. RESULTS AND DISCUSSION We have previously demonstrated that the 50K (acyl protein synthetase) and the 34K subunits of the P. phosphoreum fatty acid reductase complex can be specifically labeled with [3H]tetradecanoic acid (+ATP) in cell extracts (14) with similar acylation patterns being observed in two other Photobacterium strains. In V. harveyi extracts, however, the relative intensity of acylated polypeptides and their molecular masses were found to be quite different from P. phosphoreum. The majority of the fatty acid label migrated at a position on SDS-PAGE (20 kda) which was close to that expected for the acyl-acyl carrier protein (acyl-acp), which migrates anomalously on this gel system (12). As polypeptides of higher molecular masses were only labeled to a low degree in extracts of V. harveyi, a clear relationship between the acylated polypeptides in V. harveyi and those of the fatty acid reductase complex in P. phosphoreum was not established. In the present investigation, experimental conditions were modified to enhance the labeling of V. harveyi proteins in vitro; these modifications included the omission of EDTA from the cell lysis buffer and the addition of Mg2+ during the incubation of the extracts with [3H]tetradecanoic acid (+ATP). The results are presented in Fig. la, lane 1. In contrast to the labeling pattern seen in P. phosphoreum (Fig. lb, lane 1), a number of acylated polypeptides are observed in V. harveyi extracts. In addition to the intensely labeled 20- kda bands, labeled polypeptides can be observed at 32 and 50 kda (two bands) as can lighter bands at 54 and 42 kda. The in vitro acylation with fatty acid of all these V. harveyi proteins was found to be ATP dependent, and in some experiments, the acylated 54-kDa band was found to be very low, or at an undetectable level, relative to the other labeled bands. The relatively large number of fatty acid-labeled polypeptides observed in extracts of V. harveyi could partially arise from nonspecific acylation under the in vitro conditions employed, thus raising the question of which of these acylated polypeptides, if any, are involved in aldehyde biosynthesis. Since V. harveyi cells are capable of taking up fatty acids from the growth medium (17),- growing cells were labeled with fatty acids to determine which of the acyl polypeptides are present in significant amounts in vivo. After pulse-labeling with [3H]tetradecanoic acid, SDS- PAGE of the soluble proteins, followed by fluorography, revealed essentially three labeled polypeptides (Fig. la, lane 2). These proteins corresponded in molecular mass to the 54-, 42-, and 32-kDa bands acylated in vitro with [3H]tetrade-

3 722 WALL, BYERS, AND MEIGHEN 54kDa- 5OkDa- 42 kda- - a m 32kDa- _oo.m 20kDa- Front- b -50K K FIG. 1. In vitro and in vivo acylation of V. harveyi and P. phosphoreiin. Cells were grown to near peak luminescence (A~(,,( 1.8, 400 LU/ml and A(,6) = 3.3, 3,000 LU/ml for V. Ihanievi and P. phosphorcien, respectively). In vivo and in vitro acylation followed by separation of the polypeptides by SDS-PAGE and fluorography were performed as described in the text. The fluorogram of the gel is presented. (a) V. hari'evi; each lane contains 50 p.g of protein. (b) P. phlosplhori-ei'n:1 each lane contains 25 p.g of protein. Lanes 1. in vitro acylation with V3H]tetradecanoic acid (-+ATP); lanes 2. in vivo acylation with 3Hltetradecanoic acid; lane 3. in vitro acylation with [3H ]tetradecanoyl-coa. canoic acid (+ATP) (Fig. la, lane 1), although the relative degree of labeling of the 32-kDa polypeptide was found to be much lower in vivo. Very minor in vivo-acylated bands near 50 kda were sometimes also detected; however, their level was always found to be low in comparison with the 54-, 42-, and 32-kDa acyl derivatives. The heavily acylated 20-kDa bands observed in vitro in V. harveyi extracts may be absent from samples labeled in vivo, but this conclusion must be viewed with caution since polypeptides in this region are only partially resolved from the large lipid band seen near the front of the gel in in vivo-labeled samples. An apparent lack of in vivo-labeled ACP derivatives in V. htiirvevi would be consistent with reports suggesting that Esclerichia coli does not activate exogenous fatty acids to acyl-acp in vivo (15), even though acyl-acp synthetase activity can be detected in E. coli extracts (10). Acylation of P. phosphoreum proteins was also investigated by labeling in vivo with [3H]tetradecanoic acid. The results (Fig. lb, lane 2) indicate that only 50K, the acylprotein synthetase subunit, is labeled in vivo with exogenous fatty acid in this bacteria. The absence of label associated with the 34K subunit suggests that the acylation of this polypeptide by [3H]tetradecanoic acid (+ATP) in vitro is J. BACTERIOL. different than its acylation in vivo under physiological conditions. Possible relationships between bioluminescence-specific proteins in P. pliospiworeiii and V. harieyi were further examined in vitro by acylation with [3H]tetradecanoyl-CoA. We have prevously shown that the 34K component and, to a lesser extent, the 58K (acyl-coa reductase) component of P. phosplioreiuml fatty acid reductase can be labeled with this substrate (18). Only the 32-kDa polypeptide was highly labeled when V. harlevi extracts were incubated with [3H]tetradecanoyl-CoA for short periods of time (Fig. la, lane 3). The strong, relatively specific labeling of the V. hanreyi 32-kDa polypeptide and the P. phosphoreiumw1 34K subunit by tetradecanoyl-coa (18) suggests that these two proteins may perform analogous roles in their respective bacterial strains. This conclusion is supported not only by their similarity in molecular weight, but also by the tendency of both proteins to be much more readily acylated in vitro than in vivo by fatty acid (Fig. 1). It is interesting in this regard that the most highly labeled polypeptides with tetradecanoyl-coa in a variety of other luminescent bacteria are also in the 30- to 35-kDa range (unpublished data). The bacterial bioluminescence system is growth dependent, as it is induced only at later stages of exponential growth (9). It is reasonable to expect that any enzyme activities associated with this system will be coinduced with in vivo luminescence, as has been demonstrated for luciferase (8) and for each of the fatty acid reductase components of P. phosphoreium (18). Thus, to establish a direct relationship between bioluminescence and acylated proteins in V. hari'evi, the in vivo acylation patterns from V. hanr'evi cells pulse-labeled before the onset of luminescence were compared with cells labeled near peak luminescence. In vivo acylation of the 54-, 42-, and 32-kDa polypeptides is significantly higher in induced cells (Fig. 2a). In vitro acylation with 3H-labeled fatty acid (Fig. 2b) also demonstrates induced acylation of the 42- and 32-kDa polypeptides, whereas labeling of the 20-kDa bands, as well as the 50-kDa polypeptides, appears to be growth independent. The increase in the 32-kDa polypeptide is also clearly seen by labeling extracts with [3H]tetradecanoyl-CoA (Fig. 2c). These results provide evidence that the three polypeptides which are acylated in vivo (54, 42, and 32 kda) are involved in V. harivevi bioluminescence and raise the question of what role these proteins play in this system. To investigate possible functions of the 54-, 42-, and 32- kda polypeptides, the acylation patterns of two different types of dark mutants (M17 and Aldl6) were investigated. Under normal growth conditions, the V. harvevi M17 mutant does not emit light, although it can be stimulated to luminesce by the addition of tetradecanoic acid or tetradecanal (16). Presumably, M17 possesses the ability to reduce exogenous fatty acids to the corresponding aldehydes, but it is incapable of aldehyde biosynthesis de novo. When cells of M17. which reached the same level as wild-type cells in terms of growth and extractable luciferase activity (Table 1), were incubated with [33H]tetradecanoic acid in vivo, both the 42- and 54-kDa polypeptides were acylated, although at a lower level than in wild-type cells (Fig. 3a). However, acylated 32-kDa polypeptide was not detected in the labeled M17 cells. Similarly, in vitro labeling of M17 extracts with [3H]tetradecanoic acid and ATP resulted in a very low level of acylation of the 32-kDa protein, whereas the labeling of all other polypeptides remained relatively constant with respect to wild type (Fig. 3b). The difference between wild-type and M17 strains is even more dramatically illustrated by labeling

4 VOL. 159, 1984 ACYLATION OF POLYPEPTIDES IN V. HARVEYI 723 a b C a b C 54kDa- 42kDa- 32 kda 54 kda- 42 k Da kda FIG. 2. Induction of acylation of polypeptides in V. harveyi. Cells of V. harveyi before induction of luminescence (Aw = 0.35, 0.5 LU/mI) or near peak luminescence (A6w = 1.5, 230 LU/ml) were labeled in vivo and in vitro. Protein from each sample (50,ug) was separated by SDS-PAGE, and the gel was fluorographed as described in the text. (a) In vivo or (b) in vitro labeling with [3H]tetradecanoic acid; (c) in vitro labeling with tetradecanoyl-coa. Lanes 1, preinduced cells; lanes 2, induced cells. with [3H]tetradecanoyl-CoA in vitro; the label associated with the 32-kDa polypeptide in wild-type extracts was absent in M17 (Fig. 3c). These results suggest that the 32-kDa polypeptide is either nonfunctional or absent in M17 cells and thus provide a clear link between this protein and bioluminescence in V. harveyi. Unlike M17, the Aldl6 dark mutant of V. harveyi is not capable of converting fatty acids to aldehydes and can therefore only be stimulated to luminesce if aldehyde is added exogenously. When Aldl6 cells (Table 1) were labeled with [3HJtetradecanoic acid in vivo, acylation of the 54- and 42-kDa polypeptides was extremely low, whereas the 32- kda protein was found to be acylated at a higher level than in wild-type cells (Fig. 3a). Fatty acid labeling of Aldl6 extracts in vitro revealed the absence of the acylated 42-kDa polypeptide only (Fig. 3b), whereas no significant difference in the [3HJtetradecanoyl-CoA labeling pattern was observed TABLE 1. Growth of V. hari'evi and dark mutants Inuvivos In vitro InvtoA reduc- Cell type A6w lumines- luciferase tasedumo cence (LU/mg) tase (pmol (U(LU/ m LUm) min-' mg-') Wild type M " Ald b "Maxirmum luminescence after addition of 100 FM tetradecanoic acid. b Maximum luminescence after addition of 100 FM decanal. w..._ ' FIG. 3. in vivo and in vitro acylation of dark mutants of V. harveyi. Wild-type, M17, and Aldl6 cells were all grown to equivalent levels as depicted in Table 1. The samples were labeled in vivo and in vitro, and 50,ug of protein was separated by SDS-PAGE as described in the text. The fluorogram of the SDS gel is presented. (a) In vivo or (b) in vitro acylation with [3H]tetradecanoic acid; (c) in vit,ro acylation with [3H]tetradecanoyl-CoA. Lanes 1, wild type; lanes 2, M17; lanes 3, Aldl6. between Aldl6 and wild-type extracts (Fig. 3c). Although it is not understood at present why the 54-kDa polypeptide in Aldi6 cells is labeled at wild-type levels in vitro, but not in vivo, the observations described above indicate that the 42- kda (and, perhaps, the 54-kDa) protein may be involved in the biosynthesis of long-chain aldehydes. Although the specific functions of these proteins cannot be directly assessed from the experiments described above, the phenotypic characteristics of the M17 and Aldl6 mutants do provide some clues regarding their roles in V. hars'evi bioluminescence. In M17 cells, the inability to provide the endogenous fatty acids required for aldehyde synthesis appears to be related to the lack of a functional 32-kDa polypeptide. A direct role of this protein in fatty acid synthesis can be ruled out since M17 cells grow as well as wild-type cells and both strains exhibit similar fatty acid synthetase activities in vitro (unpublished data). On the other hand, it is quite possible that this protein functions by supplying free fatty acids from an activated or storage form. This hypothesis could explain the increased acylation of the 32-kDa protein in the Aldl6 mutant in vivo (Pig. 3a), in which fatty acid conversion to aldehyde is blocked. Additional support for this idea comes from recent observations in our laboratory which indicate that the analogous 34K subunit purified from P. phosphoreum fatty acid reductase exhibits acyl esterase activity with a number of activated fatty acyl compounds (L. Carey, A. Rodriguez, and E. A. Meighen, J. Biol. Chem., in press). The function of the 42-kDa polypeptide in V. harv'eyi bioluminescence is probably more closely involved with the reduction of fatty acid to aldehyde, since both this activity and the acylated 42-kDa protein appear to be missing in Aldl6 cells. It is not likely that the 42-kDa protein corresponds to the acyl-coa reductase component of P. phosphoreum fatty acid reductase because we have recently

5 724 WALL, BYERS, AND MEIGHEN demonstrated that an aldehyde dehydrogenase, of higher molecular weight, is responsible for this activity in V. harveyi (D. M. Byers and E. A. Meighen, J. Biol. Chem., in press). In any case, it is illustrated in Table 1 that acyl- CoA reductase activity was similar in the wild-type and mutant strains. There is some evidence that the V. hars'eyi 42-kDa polypeptide may, in fact, be analogous to the acyl protein synthetase component (50K) of P. phosphoreium. Both proteins are labeled by [3HJtetradecanoic acid in vivo and in vitro, but not by [3H]tetradecanoyl-CoA (Fig. 1). Moreover, the increased in vivo acylation of the 42-kDa polypeptide in the wild type relative to M17 cells, in which the 32-kDa polypeptide is absent, could relate to the stimulation of P. phosphorelum 50K fatty acid acylation by the 34K subunit (14). The relationship between the aldehyde stimulatable mutants and the 32-, 42-, and 54-kDa acylated polypeptides is shown in the following pathway of aldehyde biosynthesis in V. harveyi: Mutant M17 Mutant Aldl6 Fatty acid precursor Fatty acid 32 kda 42 kda 54 kda? Aldehyde -. Fatty acid + light Luciferase Experiments are currently under way in our laboratory to further characterize the 42- and 32-kDa proteins, as well as the 54-kDa polypeptide, and their relationship to the fatty acid reductase reaction. ACKNOWLEDGMENTS This work was supported by a Medical Research Council Grant (MT-4314), a Medical Research Council Studentship (to L.A.W.), and a Medical Research Council Fellowship (to D.M.B.). We would like to thank J. W. Hastings (Harvard University) for supplying the V. harveyi mutants and Rose Szittner for technical assistance. LITERATURE CITED 1. Bradford, M. M A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: Dunn, D. K., G. A. Michalizyn, I. G. Bogacki, and E. A. Meighen Conversion of aldehyde to acid in the bioluminescent reaction. Biochemistry 12: J. BACTERIOL. 3. Gunsalus-Miguel, A., E. A. Meighen, M. Z. Nicoli, K. H. Nealson, and J. W. Hastings Purification and properties of bacterial luciferases. J. Biol. Chem. 247: Hastings, J. W., and G. Weber Total quantum flux of isotropic sources. J. Opt. Soc. Am. 53: Laemmli, U. K Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227: Meighen, E. A Biosynthesis of aliphatic aldehydes for the bacterial bioluminescent reaction, stimulation by ATP and NADPH. Biochem. Biophys. Res. Commun. 87: Meighen, E. A., and i. Bartlet Complementation of subunits from different bacterial luciferases. J. Biol. Chem. 255: Michaliszyn, G. A., and E. A. Meighen Induced polypeptide synthesis during the development of bacterial bioluminescence. J. Biol. Chem. 251: Nealson, K. H., T. Platt, and J. W. Hastings Cellular control of the synthesis and activity of the bacterial luminescent system. J. Biol. Chem. 104: Ray, T. K., and J. E. Cronan, Jr Activation of long chain fatty acids with acyl carrier protein. Proc. Natl. Acad. Sci. U.S.A. 73: Riendeau, D., and E. Meighen Evidence for a fatty acid reductase catalyzing the synthesis of aldehydes for the bacterial bioluminescent reaction. J. Biol. Chem. 254: Rock, C. O., and J. E. Cronan, Jr Re-evaluation of the solution structure of acyl-carrier protein. J. Biol. Chem. 254: Rodriguez, A., D. Riendeau, and E. Meighen Purification of the acyl coenzyme A reductase component from a complex responsible for the reduction of fatty acids in bioluminescent bacteria. J. Biol. Chem. 258: Rodriguez, A., L. Wall, D. Riendeau, and E. Meighen Fatty acid acylation of proteins in bioluminescent bacteria. Biochemistry 22: Silbert, D. F., F. Ruch, and P. R. Vagelos Fatty acid replacement in a fatty acid auxotroph of Escherichia coli. J. Bacteriol. 95: Ulitzur, S., and J. W. Hastings Myristic acid stimulation of bacterial bioluminescence in 'aldehyde" mutants. Proc. Natl. Acad. Sci. U.S.A. 75: Ulitztar, S., and J. W. Hastings Reversible inhibition of bacterial bioluminescence by long chain fatty acids. Curr. Microbiol. 3: Wall, L., A. Rodriguez, and E. Meighen Differential acylation in vitro with tetradecanoyl-coa and tetradecanoic acid (+ATP) of three polypeptides shown to have induced synthesis in Photobacterium phosphoniirn. J. Biol. Chem. 259: