Induced Polypeptide Synthesis during the Development of Bacterial Bioluminescence*

The dramatic increase in bioluminescence observed during the later exponential growth of Beneckea harueyi is due to the induction of luciferase activity. The mechanism by which luciferase activity is induced and the possible existence of other induced proteins was investigated in a double-labeling experiment: [4,5-‘Hlleucine was incorporated into cellular proteins synthesized during the luminescence lag period in early growth; [“Clleucine was incorporated during the later period of luminescence induction. The proteins of the cell-free extract were extensively fractionated and luciferase was purified to homogeneity. Analysis of the radioactivity incorporated into the (Y and fl subunits of luciferase showed a dramatic but equal decrease in the 3H/14C ratio for both subunits. This result proves that the synthesis of the 01 and p chains of luciferase is subject to similar controls and that the regulatory mechanism is operative at the level of gene transcription, or translation at the time of bioluminescence induction, or both. Several additional polypeptides have been found which also show a marked decrease in their SH/l’C ratio indicating that their synthesis is induced during the same period as luciferase. In addition, one polypeptide that is synthesized specifically in the bioluminescence lag period was also detected. The function and role of these new polypeptides with respect to the bioluminescent system is presently under investigation. The in

The dramatic increase in bioluminescence observed during the later exponential growth of Beneckea harueyi is due to the induction of luciferase activity. The mechanism by which luciferase activity is induced and the possible existence of other induced proteins was investigated in a double-labeling experiment: [4,5-'Hlleucine was incorporated into cellular proteins synthesized during the luminescence lag period in early growth; ["Clleucine was incorporated during the later period of luminescence induction. The proteins of the cell-free extract were extensively fractionated and luciferase was purified to homogeneity. Analysis of the radioactivity incorporated into the (Y and fl subunits of luciferase showed a dramatic but equal decrease in the 3H/14C ratio for both subunits. This result proves that the synthesis of the 01 and p chains of luciferase is subject to similar controls and that the regulatory mechanism is operative at the level of gene transcription, or translation at the time of bioluminescence induction, or both. Several additional polypeptides have been found which also show a marked decrease in their SH/l'C ratio indicating that their synthesis is induced during the same period as luciferase. In addition, one polypeptide that is synthesized specifically in the bioluminescence lag period was also detected. The function and role of these new polypeptides with respect to the bioluminescent system is presently under investigation. The development of light emission in exponentially growing cultures of luminous bacteria is delayed until considerable growth of the cells has occurred (1,2). The light-emitting reaction is catalyzed by luciferase, a heteropolymeric protein, c@ (3), and involves the oxidation of reduced FMN and a long chain aliphatic aldehyde (4)(5)(6)(7)(8)(9)(10)(11). During the luminescence lag period in early growth, the extractable luciferase activity remains constant but later increases at the same time and rate as in uiuo luminescence (2). Previous work has shown that the addition of inhibitors of RNA or protein synthesis to bioluminescent bacteria prevented any further increase in extractable luciferase activity, suggesting that the control of luciferase activity is exerted at the level of gene transcription (2,12). However, the bioluminescent system is highly sensitive to the metabolic and nutritional state of the cells (1,13) and therefore other interpretations may be possible especially since many of these drugs are not only general metabolic poisons with more than one site of action, but also, prevent further exponential growth (1,14,15). Earlier work also showed that the development of luciferase activity is paralleled by an increase in antigenic material determined by the amount of luciferase antibody necessary to inhibit luciferase activity (2). Based on the assumption that a luciferase precursor would have the same antigenic properties as the native enzyme, it was concluded that the results showed no evidence for a luciferase precursor (2). Consequently, it may be possible that the *This work was supported by Grant MA-4314 from the Medical Research Council of Canada. observed lag in the development of bioluminescence may either reflect the synthesis of one or both polypeptides of luciferase in the form of an inactive precursor with different antigenic properties than the native enzyme or reflect the repression of synthesis of one or both of the subunits of luciferase at the level of gene transcription or translation. There is now evidence to suggest that the activities of other enzymes may also be induced during the development of bioluminescence.
Studies of the growth and bioluminescence of luminous bacteria (2) have indicated that the substrates of the luciferase reaction are not present in saturating levels at all stages of cell growth. In fact, a substrate limitation occurs specifically during the luminescence lag period and becomes progressively more acute just prior to the induction of bioluminescence. Presumably, this effect arises from a "dilutingout" of substrate-producing enzymes during the luminescence lag period. Coordinate control of substrate-producing enzymes and luciferase has also been suggested from a recent investigation of temperature-sensitive bioluminescent mutants (16). In the present study, the cellular proteins were labeled with [4,S3H]leucine during the luminescence lag period and with ["Clleucine during the later luminescence induction period. This double-labeling procedure provides a direct approach for determining whether or not the polypeptide chains of luciferase are synthesized in concert with growth and if their synthesis is coordinately controlled. Furthermore a major advantage of this dotible-labeling technique is that it also permits us to investigate the possibility that the synthesis of other polypeptides is  (18), except that the leucine content was reduced to the lowest levels which did not adversely affect exponential cell growth nor the development of bioluminescence. This medium was chosen because (a) it produced good growth and high levels of bioluminescence, (b) it could be limited in leucine content, and (c) it was precisely defined.
The cells from an exponentially growing culture were inoculated into 250 ml of the above medium containing 5.0 mCi of [4, and counted on two different channels in a Packard scintillation counter. Tritium measurements have been corrected for an 8% crossover of "C counts per min into the 3H channel. The counting efficiency for 9H and "C was 8.0% and 44%, respectively.
Isotope Ratios-All 3H and "C measurements have been reported as a percentage of their respective counts in the supernatant' (O/oSH and %"C).
Isotope ratios have been presented as %"H/%"G or %"C/%3H depending on whether a given sample has shown a relative increase in 'H to "C or "C to SH, respectively, compared to the supernatant. This 1 Following exhaustive dialysis to remove unincorporated radioactivity, the supernatant or total soluble protein fraction was found to have 2.6 times as many SH counts as "C counts. The isotope ratio (%sH/%"C or %"C/%sH) is of course equal to 1.0 and is referred to as the "average" isotope ratio.
method of presentation provides equal weight to changes in the relative amount of either isotope for any given sample. For example, samples in which the 3H/"C ratio increases 3.fold relative to the supernatant (g3H/%11C = 3.0) would have the same value for the isotope ratio as samples in which the "VH ratio had increased S-fold relative to the supernatant (%"C/%$H = 3.0 The isotope ratios are presented as an increasing W3H/%"C (> 1) above the abscissa and as an increasing %"C/%SH (> 1) below the abscissa.
ratios of the gel fractions were multiplied by an appropriate correction factor such that the ratio of the total sH/l"C counts in each gel was equal to that of the applied sample. Repeated analysis of the same sample showed that this procedure gave reproducible results. All samples were counted for at least 30 min and those fractions which were low in radioactivity were counted for longer periods. Any isotope ratio with a coefficient of variation greater than 20% due to low counts was rejected.
The isotope ratios have been plotted (under "Results") as a function of electrophoretic mobility (RF), where RF is the distance of the gel slice from the top of the gel divided by the distance migrated by pyronin Y. Since no significant counts or protein staining were detected on any gel with an electrophoretic mobility greater than 0.8, gel photographs and experimental data in this region have not been presented.
Criteria for Defining a Polypeptide Whose Synthesis Is Induced or Repressed-The detection of an altered isotope ratio in a particular sample can reflect either a random deviation from the "average" isotope ratio' (1.0) or indicates the existence of a polypeptide which is synthesized specifically or predominantly during the lag period (%sH/ %'"C >> 1) or the biolummescence induction period (%'4C/%3H >> 1). We have defined those polypeptides whose isotope ratio is observed reproducibly with a value greater than 2.5 as either induced or repressed during the development of bioluminescence. This criterion was used so that it would be highly improbable, even upon analysis of a large number of samples (==lOOO), that a noninduced polypeptide would be observed with such a large deviation due to random variation2 from the "average" isotope ratio. the gel mechanically into l-mm slices and determining the isotope ratio of each slice as described under "Experimental Procedure."

RESULTS
In order to determine whether the induction of bacterial luciferase activity is regulated at the level of gene transcription-translation or by post-translational processes, a leucine auxotroph of Beneckea harveyi was incubated in the presence of [4,5-3H]leucine during the luminescence lag period in early cell growth (A,,, < 0.2) and ["Clleucine during the induction of bioluminescence (A,,, = 0.6 to 1.2) (see Fig. 1). The soluble proteins were then extracted from the doubly labeled cells and the isotope ratio of the component polypeptides analyzed after sodium dodecyl sulfate gel electrophoresis (Fig. 2). Since the majority of proteins should be synthesized in concert with growth, very little change in isotope ratio is expected. In fact, the observed change in isotope ratio between 1.0 and 1.4 simply represents the normal experimental variation. It is therefore clear that a more extensive fractionation is necessary if polypeptides with a significantly altered isotope ratio are to be detected. polypeptides have a Poisson distribution centered on the average isotope ratio of 1.0 and using Chauvenot's criterion (22) to reject data not representative of this distribution (i.e. indicative of a repressed or induced polypeptide).
The chance of observmg a noninduced polypeptide with an isotope ratio greater than 2.5 (-4 S.D. from the average) is thus less than 0.001% for a single reading and less than 1% on analysis of 1000 samples. Since the above results showed that extensive fractionation of proteins is a prerequisite for the detection of induced polypeptides, subsequent studies concentrated on the further resolution of the three remaining eluates from DEAE-cellulose (Table I). The filtrate, 0.10 M eluate, and 0.15 M eluate proteins were precipitateda by the addition of ammonium sulfate to 85% saturation and then chromatographed on DEAE-Sephadex. Fig. 6 gives the DEAE-Sephadex chromatography profile of the 0.15 M eluate which contains the majority of the FMN reductase activity. The isotope ratio changes significantly only in the region of Fraction 78. However, examination of the pooled fractions from the column after sodium dodecyl sulfate gel electrophoresis revealed the presence of induced polypeptides in three of the seven pools ( Fig. 7; Z, ZZ, and IV). Pool I, which contains luciferase activity, has two induced polypeptides with mobilities (RF) of 0.25 and 0.29, corresponding to the LY and fi subunits of luciferase. These results reconfirm the previous findings for luciferase in Pool I from the 0.35 M eluate (see Figs. 4 and 5) and consequently this pool is designated in an identical manner. Similarly, Pool II of this column (Fig. 7) contains the same induced polypeptides that were detected previously in Pool II of the 0.35 M eluate (see Fig. 5). However, Pool IV, contains an induced polypeptide with a mobility of 0.13 that has not been detected previously. Although present in small a Less than 1% of the radioactivity remained in the supernatant after addition of ammonium sulfate to 85% saturation.
fractions were combined into eight pools as indicated on the abscissa and then further resolved by sodium dodecyl sulfate gel electrophoresis. The shaded areas indicate pooled fractions in which polypeptides with altered isotope ratios were detected after electrophoresis.  Fig. 6. The isotope ratios are plotted against electrophoretic mobility.
quantities, it has been well resolved by gel electrophoresis from any noninduced proteins as indicated by the large change in isotope ratio (%1*C/%3H = 6).
In contrast, sodium dodecyl sulfate gel electrophoresis of Pool III containing the majority of the FMN reductase activity gave no indication of any polypeptides with an increase in %14C/%3H (Fig. 8). Although this enzyme was not purified further, the apparent absence of induced polypeptides in this pool would indicate that the FMN reductase is probably not specifically synthesized during the induction of bioluminescence. However, this pool does contain a component with a mobility (RF) of 0.48 that has a large increase in %3H/%14C. This was the only polypeptide detected during the analysis of the bacterial proteins which is apparently synthesized specifically during the luminescence lag and repressed later in growth during the induction of bioluminescence.
The fractionation of the 0.10 M eluate (Table I) by DEAE-Sephadex chromatography is given in Fig. 9. Although no significant change can be observed in the isotope ratio for any fraction on this column, one induced polypeptide could be detected in two adjacent pools (Fig. 9, ZVa and ZVb) after sodium dodecyl sulfate gel electrophoresis (Fig. 10). This polypeptide has the same mobility (R, = 0.14) as the induced polypeptide detected previously in Pool IV of the 0.15 M eluate (see Figs. 6 and 7). The results given in Fig. 10 for Pools IVa and IVb also show that the isotope ratio is independent of the staining intensity of the peptide band.
The Filtrate (Table I) was the last fraction to be resolved by DEAE-Sephadex chromatography (Fig. 11). No significant change in isotope ratio can be observed at any position on the column. The fractions were combined into 10 pools as indicated on the abscissa and analyzed further following sodium  Fig. 6. The isotope ratios are plotted against electrophoretic mobility.
Some of the induced polypeptides previously detected were observed in the corresponding pools (e.g. I and II). However, the change in isotope ratio was less than previously observed due to the presence of a larger proportion of noninduced protein that co-electrophoreses with the induced polypeptides. Only one induced polypeptide not observed previously was detected following resolution of the filtrate. Sodium dodecyl sulfate gel electrophoresis of Pool V (Fig. 12) shows the presence of a polypeptide with an altered isotope ratio (%14C/%63H = 4) having a mobility (RF) of 0.29. A summary of the polypeptides that have been detected with substantially altered isotope ratios is given in Table II. The experiments have shown that the synthesis of both subunits of luciferase (Pool I) is induced during the development of bacterial bioluminescence.
Furthermore, only a limited number of additional polypeptides (Pools II, IV, and V) have been detected which are synthesized specifically in this same period as well as a single polypeptide (Pool III) that is synthesized specifically during the bioluminescent lag period.

DISCUSSION
By labeling the cells of B. harueyi with [4,S3H]leucine during the luminescence lag period and [14C]leucine during luminescence induction, it has been possible to show that a differential rate of synthesis occurs for a limited number of specific polypeptides during exponential growth of the bacteria. This conclusion was based on the premise that peptides isolated with a high % 14C/%3H (> 2.5) were synthesized specifically or predominantly during the bioluminescence induction period whereas peptides with a high %3H/'% '"C (>2.5) were synthesized predominantly during the bioluminescence lag period. Extensive fractionation of the proteins was essential before polypeptides with significantly altered isotope ratios could be distinguished from the vast majority of proteins having isotope ratios ranging from 1.0 to 1.4.
A total of seven polypeptides with markedly increased %14C/%3H ratios were detected during the course of this investigation indicating that their synthesis is induced during the development of bioluminescence (Table II). Furthermore, these changes in isotope ratio are not the result of degradation of the older or 3H-labeled protein since introduction of the labels (3H and "C) into two separate cultures, harvesting both cultures immediately after their respective growth periods, and mixing the two cultures, resulted in the identification of the same polypeptides (data not given) as listed in Table II The fractions were combined into 10 pools as indicated on the abscissa. The shaded areas indicate the pooled fractions in which a polypeptide with a significantly altered isotope ratio was detected after electrophoresis. luciferase proving that the synthesis of both polypeptide chains is induced during the development of bioluminescence and is coordinately controlled. The detection of five additional polypeptides with significantly increased %14C/%3H ratios provides direct evidence that proteins other than luciferase are synthesized specifically in the bioluminescence induction period. Although it can not be definitely concluded that these proteins are involved directly in the bioluminescent system, Cline and Hastings (16) have provided evidence from the study of temperature-sensitive bioluminescence mutants that there are at least two enzymes whose activities may be induced and involved in the biosynthesis of the aldehyde substrate for the luciferase reaction. In addition, one (or more) of the above polypeptides could be involved in synthesis of FMNHz for the luminescent reaction. It should be noted however that sodium dodecyl sulfate gel electrophoresis of the major DEAE-Sephadex fraction containing FMN reductase activity (Fig. 8) provided no evidence for the presence of any induced polypeptides. This result is in agreement with evidence showing that FMN reductase activity parallels growth of the bioluminescent bacteria (24). However since this enzyme was not fully purified it is still possible that its component polypeptides may not have been sufficiently resolved to permit detection of a change in isotope ratio.
FIG. 12. Sodium dodecyl sulfate gel electrophoresis of pooled Fraction V from the DEAE-Sephadex chromatogram of the filtrate (see Fig.  11). The isotope ratio of each gel fraction is plotted against the electrophoretic mobility (RF).  Fig. 5).
*Recent experiments have shown that a long chain aldehyde dehydrogenase is present in this fraction whose activity increases upon induction of bioluminescence.' Recent evidence in this laboratory has also suggested a relationship of one of the induced polypeptides to the bioluminescent system. An enzyme involved in the oxidation of long chain aliphatic aldehydes through a nonluminescent pathway has been found which chromatographs' on DEAE-Sephadex and migrates on sodium dodecyl sulfate gels in a similar manner to the induced polypeptide observed in Pool IV (see "Results"). In addition, the activity of this enzyme is induced at the same time as luciferase. Consequently, it would seem probable that at least five of the seven polypeptides are directly involved in the bioluminescent system; two of these have been shown to be the subunits of luciferase, one may be 'E. A. Meighen, I. G. Bogacki, A. Bognar, and G. A. Michaliszyn, manuscript in preparation.