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Some optical properties of luminous bacteria
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Some Optical Properties
86, 391-408
(1969)
of Luminous
Bacteria
Bernard L. Strehler From the Gerontology Branch, National Public Health Service, Department Maryland, and the Baltimore Received
Heart Institute,
National Institutes of Health, and Welfare, Bethesda, Baltimore, Maryland
of Health, Education City Hospitals, April
30, 1959
INTRODUCTION
The light-producing system of luminous bacteria, like that of fireflies and some other luminous organisms, consists of a cyanide-insensitive respiratory pathway. This system, which has been extracted and characterized with respect to the nature of the diffusible components involved (l-lo), appears to be a flavine autoxidase. Specifically, hydrogen is bransferred from reduced pyridine nucleotides to flavine mononucleotide. The reduced flavine is then oxidized by molecular oxygen with the intervention in some ancillary function of a long-chain aldehyde (6). In contrast to its insensitivity in viva and in vitro to cyanide, the luminescent reaction is remarkably sensitive to naphthoquinone inhibition (1). Cormier and Totter (3) have shown that this capacity of naphthoquinones to inhibit luminescence is probably due to a shunting of hydrogen from flavine through the quinone to a cytochrome pathway. From this and from earlier in viva studies (11, la), it appears that there is a linkage between the cyanide-sensitive and the luminescent respiration pathways in these bacteria. However, the nature of the interrelationship in viva, the identity of the heme system involved, and its relation to the energy economy of the cells have not been established. The present study was undertaken to describe the characteristics of the chromophores associated with hydrogen transport in Photobacterium jischeri. The difference spectra and some kinetic aspects of the spectral changes associated with the transition from aerobic to anaerobic conditions is described. In addition, the discovery of a respiration- and luminescence-dependent change in transmission which cannot be ascribed to a specific hydrogen or electron carrier and the experiments leading to the elucidation of its nature are described. It has been found that there is a general increase in transmission at all wavelengths under aerobic conditions. Conversely, under
392
STREHLER
anaerobic conditions, there is a decrease in transmission which takes approximately 30 min. to approach completion. Paralleling this effect of anaerobiosis, there is a gradual inhibition of the capacity to exhibit luminescence when oxygen is readmitted. Chemical, physical, and optical factors entering into this phenomenon have been examined. It is concluded that the effect is due to a scattering change rather than to a change in absorption or a change in metallic reflectivity and that the phenomenon is probably not due to the presence of conduction-band electrons (13). MATERIAL
AND METHODS
A. Culture of Luminous Bacteria Photobacterium fischeri’ and a luminous diplococcus were grown in a liquid complete medium as described by Farghaly (14). The bacteria were grown at 25°C. in 250-ml portions in Erlenmeyer flasks on a shaker. After harvesting by rapid centrifugation of suspensions chilled to O”C., the bacteria were resuspended in nitrogen-free basal medium, washed once, and kept at 0°C. in suspension until used.
B. Spectrophotometry All spectral measurements here described were carried out with a Cary model 14 spectrophotometer. The following accessories were developed for use in this study: 1. Cuvettes for aerating samples without direct interference with the light path. Regular Cary cuvettes (4 ml.) were fitted with small Teflon inserts, mounted parallel to the direction of light transmission. These inserts were rectangular and were mounted about 1.5 mm. from one side of the cuvette. They extended from approximately 2 mm. above the bottom of the cuvette to approximately 2 mm. below the liquid level. A small plastic tube was placed between the cuvette wall and the Teflon insert, and purified gases or air from a small pump was bubbled through the side chamber. This not only aerated the bacteria but also circulated them through the cuvette. See also (33). 2. In order to determine whether there is a change in scatter as well as absorption, a device was constructed to measure changes in scatter perpendicular to the light path. This consisted of a Lucite cuvette holder with attached mirrors. This arrangement is shown in Fig. 1 which also illustrates the aerating cuvette. Light incident upon the cuvette is prevented from passing directly to the detector. Rather, only the light scattered at right angles to the incident beam is picked up by the mirrors and transmitted to the radiation detector. 3. The absorption spectrum, free of first-order scattering artifacts, can be obtained by the use of light integrators or diffusers as described elsewhere (13) [see also (2932)]. We have constructed such a device. In principle, it operates as follows. All of the incident light that is not absorbed is scattered several times, either within the sample cuvette or by the white reflecting surface of the integrating box. Light emerging from the boxes is then transmitted to the detector. 1 P. fischeri kindly furnished by Dr. W. D. McElroy; through the courtesy of Dr. M. Cormier.
the diplococcus
supplied
393
LUMINOUS BACTERIA
Aerating Tube
hastic Baffle
4 ,Mzr M3 = Front Surface
Mirrors
FIG. 1. Optical arrangement of cuvette and mirrors for measuring changes in side scatter of suspensions of bacteria or other scattering particles. Light from the monochromator incident upon the suspension is scattered by it. A small fraction of the scattered light is reflected successively off mirrors Mr , M, , and Ma and thence into the detector. By contrast, light that is transmitted through the sample is intercepted by mirror M, and prevented from reaching the detector.
C. Luminescence Light emission was measured concurrently with absorption at several wavelengths by mounting a mirror beside the cuvette. Light from the side of the cuvette was reflected into a photomultiplier mounted on a lid to the spectrophotometer chamber. The measuring beam did not, by itself, produce measurable signal in the luminescence detector. The bacteria were observed under aerobic and anaerobic conditions with light and dark field microscopy. EXPERIMENTAL
RESULTS
Wavelength-Dependent Changes in Transmission (a) Absorption Spectrum. The absorption spectra of P. jkcheri under aerobic and anaerobic conditions are illustrated in Fig. 2. These tracings were obtained using the light-integrating devices described above. Corrections in relative peak heights produced by the second-order effect of scatter on effective optical path length have not been made. (b) Aerobic-Anaerobic Difference Spectra. The oxidized-reduced difference spectra for P. fischeri were measured as follows: Bacteria were introduced into both the sample and reference chamber cuvettes. On a regular time schedule the air was circulated into the sample cuvette and a tracing of the time course of transmission was obtained. This method is similar to that of Smith (23). The 04.1 O.D.U. slide-wire
394
STREHLER
Reduced
< I 370
I 430
I
I 490
I
I 560
,I
I 610
I
I 670
I
FIG. 2. Absorption spectra of oxidized and of reduced luminous bacteria as measured in the light-integrating chambers described elsewhere. The wavelength-dependent scatter does not introduce spurious signals, but it does modify relative heights of bands by changing effective optical path length. Numbers along the ordinate represent millimicrons whereas the range of absorbancy is O-l O.D.U.
scale of the Cary model 14 spectrophotometer was used. By measuring the magnitude and sign of the tracing deflection at various wavelengths at the instant of oxygen exhaustion (as measured by the disappearance of luminescence due to endogenous respiration) and plotting these deflections against wavelength, the difference spectrum shown in Fig. 3 was obtained. Also shown is the difference spectrum obtained by the use of the integrating boxes described above. In this case two cuvettes (one kept anaerobic, the other aerated) were used. An examination of Figs. 2 and 3 reveals typical cytochrome peaks in the 550-560-rnp region. There is no typical cytochrome a f a3 band at 600-605 rnp. There is, rather, a weak band at about 628 rnp, which is quite similar to the spectrum described for the a2 type cytochrome (16, 27). A band due to flavine reduction is clearly visible as is one due to reduced DPN. Wavelength-Independent
Transmission Changes
(a) General Properties. There is, as indicated above, evidence of spectral behavior similar to that observed in other organisms when 02 disappears. However, in addition to these wavelength specific changes, there are
LUMINOUS
395
BACTERIA
changes of considerable magnitude which occur after oxygen depletion, but which are not localized to any specific spectral region. Rather, they appear to be due to (a) a generalized change in absorption at all wavelengths, (b) to a change in scatter irrespective of wavelength, or (c) to a combination of these two (Fig. 4). A number of experiments were performed in order to eliminate various optical artifacts, physical effects, etc. and to establish the relationship of this transmission change to scatter and/or absorption. These are summarized in Table I. DIFFERENCE
SPECTRUM
A. Fischeri
+ ,035, 030. ;p ,025. 020. 5 .Ol!iL o ,010 2z: ,005. t 03 s 2 2
. ,t
fl-\
Y,
\ \ 4 \ I : : : 350 : -4 250 1 : : : :: : : 'I\ ‘"00 ,010. b ,015. ,020. .025. 030~ ,035' \ wo . ,' P'
-
Aulomatic recording using “spheres”
---
Spectrum obtwwd point for point wthout “spheres”
FIG. 3. Reduced-oxidized difference spectra for P. jischeri as determined by two separate methods. Solid line = direct tracing using integrating chambers. The control sample was allowed to become anaerobic. The experimental sample was aerated at 5°C. and the difference spectrum then determined. Dashed line = difference spectrum determined by plotting the change in O.D. at each wavelength during the transition from aerobic to anaerobic conditions. The control sample was permitted to become anaerobic. The experimental was aerated, as described in the text, and the time course of transmission was followed as the sample became anaerobic. The Iight-integrating boxes were not used in this second method. Note that the two methods yield differences in relative peak heights. These differences are due to differences in effective optical path length.
396
STREHLER
I 0
t I ,
I 2
I 3
I 4
I 5
I 6
I 7 TIME
I I 6 9 (minutes)
I 0
11 II
12
II, 13
14
I5
FIG. 4. Difference between effect of Nz gas and air on scatter. The sample was aerated between A and B. The rapid decrease in transmission is due to the presence of bubbles in the side chamber of the cuvette and occurs irrespective of the kind of gas bubbled through the sample (see D to E). Following a brief period of aeration, the optical density continues to decrease until the oxygen is exhausted at C, at which time decrease in transmission begins to occur. Note particularly that bubbling Nz through the sample does not produce the same effect as bubbling air. A = 600 mp.
(b) Kinetics of Transmission Chwge. Figure 5 illustrates the time course of the transmission change at several wavelengths during a period of anaerobiosis. (c) Wavelength Independence of Transmission Change. The difference spectrum for this slow change in transmission was determined by repeated scans of the same suspension before, during, and after the transition from aerobic to anaerobic conditions. Figure 6 illustrates the results obtained in a typical series of repeat scans. Note that the difference between successive scans is largely independent of wavelength over the region measured except for the first tracing which includes wavelength specific changes. The flatness of the slow change is shown by the fact that the distance between the lines representing the 3- and 5-min. tracings are approximately constant at all wavelengths. (d) Association of Transmission Change with Luminescence. We had earlier noted that bacteria which had been centrifuged in the cold anaerobically required some few minutes of aeration in order to attain their full luminescent intensity. It was of interest to determine whether the change in transmission described above is related to this inhibition of luminescence by anaerobiosis of longer duration. Therefore we measured the time course of both luminescence and transmission concurrently. Typical results are shown in Fig. 7. Figure 8 illustrates the per cent of luminescence in-
TABLE General
Properties
-
of the Wavelength
I Unspecijk
Transmission
Change
Possible source of effect
Control
1. Photometric errors due to luminescence of bacteria
la. Effect continues atter luminescence ceases lb. Base line is not shifted by inserting incandescent source in one sample compartment lc. Effect is observable using infrared detector light and “reversed path” of Cary model 14
1. No artifact bacterial cence
2. Settling
2a. No settling of bacteria overnight 2b. Stirring with Nz without effect (see Fig. 4)
2. Effect not due to set,tling of bacteria
3. Not tion
due to aggrega-
due
of bacteria
Conclusion
due to lumines-
3. Aggregation teria
of
bac-
3. Microscopic tion
4. Orientation teria
of
bac-
4a. Both cocci and rods show effect 4b. No effect of NS stirring
4. Not tion
5. pH change
5. pH was measured and no change as great as 0.01 pH unit was found after 2 hr.
5. Not due to pH change (externally)
6. Temperature difference between bacteria and medium due to metabolic activity
6. On basis of cell mass and respiratory rate measurement the temdifference perature must be less than O.Ol”C.
6. Not ture
7. Mass heating or cooling of bacterial suspension
7. No appreciable change in transmission between 6 and 30°C.
7. Not due to gross changes in temperature of suspension
8. Absorption
8. See following sections (e) and (f) in text
8. Effect due to scatter, not absorption
or scatter
-
observa-
to
orienta-
due to temperadifferencea
a Oxygen dissolves in water to the extent of 0.008 g./l. = 0.00025 moles/l. Since approximately 110 kcal. are liberated/mole, the temperature rise would be 0.0275”C. Assuming 1% bacteria by volume and no heat transfer between bacteria and media during the time 02 is consumed, the maximum temperature differential between bacteria and medium would be 2.75”C. 397
t
LUM.DlSAPPEARS
I
3
L
4 TIME
5(MIN.)6
5. Time course of transmission changes at two wavelengths. Note that there is a rapid change during the transition to anaerobiosis which may be of either positive or negative sign (depending on the spectral properties of the hydrogen carriers) followed by a slower change which is always of the same sign (i.e., decreased transmission). FIG.
I 320
I 360
I 440
I 500
WAVELENGTH FIG. 6. Successive tracings of difference spectra Note relative lack of specific effects and approximate lengths.
398
I 560
I 620
(rnb) following equality
a period of aeration. of effects at all wave-
LUMINOUS
--I%+ t 0
399
BACTERIA
I
,ttt,
I
2
, 3
4
5
T I M E (minuted
FIG. 7. Time course of luminescence and transmission change, after various periods of anaerobiosis. Note particularly that there is almost no immediate luminescence when air is admitted after a long period of anaerobiosis, but that the luminescence approaches peak value almost immediately when the anaerobic conditions are brief. Note also that the degree of initial inhibition of luminescence approximately parallels the magnitude of the scattering change.
hibition and the per cent change in absorption after various periods of anaerobicity. Thus, it appears that the decreased ability of the bacteria to luminesce is correlated with the change in their transmission. It remained to be determined whether the transmission change is due to changes in absorption or scattering or both. The marked inhibition of luminescence under anaerobic conditions, which parallels the optical effects, suggested the possibility that some essential component of the luminescent pathway, specifically the aldehyde cofactor, might be reduced under these conditions. McElroy (15) has shown that certain mutants of P. jischeri, which normally cannot luminesce, do so when a small amount of aldehyde is added to the medium. To test the above explanation of the luminescence inhibition, dodecyl aldehyde was added to inhibited suspensions. No shortening of the induction time was noted and there certainly was no immediate effect as is the case with the mutants. It, therefore, appears that the inhibition of luminescence and also probably the transmission changes are not due to aldehyde reduction. (e) Evidence That the E$ect Is Primarily Due to Scattering Changes. Although it is certain that this phenomenon was not an instrumental artifact or attributable to some simple chemical alteration such as reduction of aldehyde, it was not clear whether it represented primarily a change in absorption or of scatter. That there was a large scattering component to the phenomenon was demonstrated as follows: If there is a large scattering component, it follows that the light appear-
400
STREHLER
l
lniiial
0
Scatter
A Sea tter
,c 0
6
12 DURATION
IS
Luminescence (!+ time =4.0min.)
(‘4 time
24
OF ANAEROBICITY
= 3.4 min)
30 (min.)
FIG. 8. Initial luminescence intensity and scattering change vs. duration of anaerobicity. The values of luminescence, when oxygen was admitted, were measured after a series of runs consisting of various lengths of exposure to anaerobicity. The scattering values represent the difference (on two individual runs) between the asymptote approached after a long exposure to anoxia and the measured value. A small correction for a gradual change in base line was made. This correction was estimated from the Bnal slope of the transmission curve. Note that the half-times for both phenomena are of the order of 4 min., lending credence to the view that they may be united by some common underlying process.
the sides of the cuvettes should behave in an opposite direction from the transmitted ‘(beam” since the untransmitted light (due to scatter) emerges at other angles. Therefore, we measured the time course of transmission and side scatter at various wavelengths through the use of the special cuvette holder and mirror arrangement described in Material and Methods. Typical results are illustrated in Fig. 9. Since the direction of the effect changes, depending on whether transmitted or scattered light is meassured, whereas the sign of the wavelength specific changes is unaltered, it appears that scattering changes are a major factor in the phenomenon.
ing at
LUMINOUS
I
I
I
I
I I I I I I I I
I I I I I
1 I I I I
401
BACTERIA
I I
I I I I I
I I i
430 mu
A. FISCHERI AEROBIC + ANAEROBIC KINETICS OF TRANSMITTED AND SIDE SCATTERED RADIATION
FIG. 9. Evidence that the nonwavelength specific transmission change is primarily due to a change in scatter. Note that the immediate change in transmission (due to specific absorption bands) which occurs upon exhaustion of 02 is similar in both the side scattered and transmitted beams, but that the subsequent changes are opposite in sign depending on whether the transmitted or scattered component is measured.
These results demonstrated that there is a large scattering component in the effect, but they do not rule out absorption changes of a somewhat lesser magnitude. Therefore, the “integrating boxes” described above were constructed. With both samples inside these “spheres,” no wavelength unspecific changes could be observed during anaerobic conditions (see Fig. 10). It is, therefore, concluded that all of the measurable effect is due to scattering changes. The data shown in Fig. 11 make understandable the “wavelength independence” of the phenomenon. Evidence That Transmission Changes Are Due to Changes in Geometry and/or Refractive Index Rather Than to Changes in Metallic ReJlectivity. Three possibilities existed: The scattering change is due to (a) change in refractive index of two contiguous phases; (b) change in size (or shape) of scattering objects; and (c) change in metallic reflectivity of particles or parts thereof. The first two possibilities could be tested by a measurement of effective size of the bacteria or by alterations in the refractive index of the suspending medium (assuming that the bacterial medium interface is the determining one). The third possibility, which is the most interesting one because of the potential role of semiconductors or pseudometallic objects in biological
402
STREHLER I
I
I
I
I
I /I
WAVELENGTH-6300°A ---
Regular cuvettes
-
In scattering
i
chamber : 4’ /’
--lZsec.---)3mhl.-
I--Lc--
I
0
I
I
I
I
I
I
12
24
36
46
60
72
TIME
(seconds)
10. Tracing of absorption changes at 630 my within and without light-integrating chamber. A to B: aerobic conditions. B to C: 02 disappears. Note increase in absorption due to reduced cytochrome band at 630 rnp. C to D: subsequent changes in scatter (do not occur in light integrators). This indicates that the transmission changes are due to scattering changes. D to E: compressed time scale. Note lack of change in chamber as compared to large change using regular cuvettes. The abscissa represents changes in optical density. The total deflection equals about 0.05 and 0.01 O.D.U., respectively. FIG.
electron transport (17-21), was tested through the courtesy and suggestion of Dr. Wm. A. Arnold of the Oak Ridge National Laboratory, Biology Division. A metallic reflector will interact differently with polarized light than one in which no highly mobile electrons are involved. In the case of the metallic reflector, the electrical and magnetic vectors will generally be thrown out of phase with each other, and the reflected light will be eliptitally polarized. Thus, if one starts with planepolarized light, it will be much more effectively depolarized by metallic reflection (from the point of view of the analyzer) than by an equal amount of nonmetallic scatter. To distinguish between these alternatives, the degree of depolarization of scattered light under aerobic and anaerobic conditions was compared using a Gaertner polarimeter. The results obtained were negative; that is, the additional scatter was not more highly depolarized than the original scatter (which presumably arises from differences in refractive index). (See Table II.) Thus, we must conclude that, the phenomenon is not, due to changes in the numbers of
LUMINOUS
3QO
360
420
403
BACTERIA
460 WAVELENGTH
540
600
660
(m/d
FIG. 11. Differences in scatter spectra of bacteria as a function of concentration differences. The numbers represent number of units of bacteria in reference and sample cuvettes, respectively. One unit equals approximately 0.25% bacteria by volume. Note that for constant differences in bacterial density, e.g., Cl, l-2,2-3, and 34 units, the scattering difference spectra exhibit extremely broad maxima of decreasing prominence which occur at successively longer wavelengths. The scattering changes reported showed very slight wavelength dependence. These present observations make it clear that a scattering change may appear to be unspecific with respect to wavelength even though the scattering objects, in greater dilution, would exhibit scatter with a strong wavelength dependence. The effect illustrated in Fig. 11 is explicable in the following terms: If one starts with moderately scattering suspensions, the addition of further scattering material does not attenuate the transmission at any wavelength according to Beer’s law, but the scatter at the red end of the spectrum approaches Beer’s law more closely than at the blue end. In the blue region scatter is almost complete, and therefore additional scattering material does not appreciably change the transmission. At the far red end of the spectrum, Beer’s law is still being approximated, but the efficiency of scatter is less so the apparent increase in O.D. is less than at some shorter wavelength. At some intermediate wavelength, the two effects are maximized. The above considerations imply that the wavelength of maximum transmission change will be shifted to the red region of the spectrum as the absolute concentration is increased, while a constant difference in concentration is maintained. The observations confirm this expectation.
404
STREHLER
TABLE Depolarization
.9
A. Thin Air (15 min.) Nf (10 min.) Nz (45 min.) Air (5 min.)
(10 min.) (10 min.) (25 min.) (28 min.) (5 min.)
Air Ns N? Nz Air
(5 min.) (1 min.) (17 min.) (30 min.) (5 min.)
suspension: 26.38 26.30 27.07 26.78 26.93
C. Thin
suspension: 61.15 61.5 61.5 61.45 60.92
D. Thin Air (10 min.) Nz (15 min.) Nz (30 min.) Air (5 min.)
suspension: 4.11 4.10 4.135 4.14
B. Heavy Air Nz N.P Nz Air
II
of Scattered Light by Bacteria under Aerobic Anaerobic (Nz) Conditions
suspension: 17.69 17.23 17.50 17.32
P
D
(Air)
and
“$
x 100
XQ = 17”, Yb = 45” 0.98971 0.98977 0.98958 0.98956
0.01029 0.01023 0.01042 0.01044
-0.58309 +1.2633 $1.4577
Xa = O”, Yb = 0” 0.6050 0.6072 0.5858 0.5938 0.5896
0.3950 0.3928 0.4142 0.4062 0.4104
-0.5569 +4.860 +2.835 $3.898
Xa = 9”, Yb = 90” 0.5342 0.54450 0.54450 0.5430 0.5274
0.4658 0.4555 0.4555 0.4570 0.4726
-2.211 -2.211 -1.8892 f1.460
Xa = 90”, Yb = 0” 0.81535 0.82465 0.8192 0.82270
0.18465 0.17535 0.18080 0.17730
-5.0365 -2.0850 -3.9805
0 X = angle in degrees between incident beam and measured beam. 6 Y = axis of polarization of incident beam in degrees to right of vertical. c The sample was not agitated during period preceding this measurement. Otherwise all measurements in Expts. A and B included stirring by an appropriate gas (pure Nz or air). Experiments C and D did not involve stirring during anaerobiosis. It thus appears likely that such slight differences as exist represent effects due to changes in cell orientation or distribution as a result of stirring. d 0 = degrees from vertical of movable analyzing prism at balance. P = polarization of scattered light as fraction of original polarization. D = fractional depolarization. AD = difference between initial depolarization and subsequent depolarization. AD - D X 100 = percentage change of depolarization as compared to initial depolarization.
LUMINOUS
405
BACTERIA
freely mobile electrons but rather either to changes in refractive to particle geometry or both.
index or
DISCUSSION
Signijkance
of Di$erence Spectra
Except for the absence of a cytochrome oxidase with an absorption maximum at around 600-605 rnp and the presence of a weak band around 630 rnp, the oxidized minus reduced spectrum does not appear to be remarkable. The recordings do not readily permit the assignment of a kinetic sequence of electron transport among the various absorbing components. However, it is probable that the sequence is similar to that demonstrated by Chance et al. (22) in other systems by more refined methods of kinetic measurement at several wavelengths, e.g., luciferase ____) hv 02 \ Quinone I --+ B type ---f C type + 630 rnp * cytochrome cytochrome compound
AH2 -+ DPN
BHz
-+ &vine
\\
02
Signijicance of Generalized Scattering Change The lack of wavelength specificity for the scattering change and its apparent association with the function of the luminescent system are the most striking characteristics of this phenomenon. The former property is explicable in the following terms. It can be shown that an apparent nonspecificity of scatter with respect to wavelength using the apparatus as described is obtained if the suspension is already sufficiently dense to provide multiple scatter for each quantum. This is illustrated in Fig. 11 in which the changes in absorption of bacterial suspensions which differ from each other by a constant amount of scattering material are compared. That is, the reference and sample cuvettes were filled with suspensions of bacteria that differed from each other by a constant amount (O-l, 1-2, 2-3 units, etc.). Note that as the turbidity increases, the additional scatter becomes less and less wavelength dependent until it becomes practically independent of wavelength. It is thus probable that the increase or decrease in scatter at all wavelengths such as observed with the bacteria is really due to an underlying wavelength-dependent scatter which is masked by the fact that multiple scatterings take place. There are several possible explanations of the apparent relationship between the scattering changes and the inhibition of the luminescent reaction.
406
STREHLER
Since both changes are effected by anaerobiosis, it is tempting to postulate that the scattering change occurs in particles connected with respiration. One possibility is that the luminescent system coalesces with the regular mitochondrial system under anaerobic conditions. If the luminescent enzymes are too small to be effective scatterers by themselves, they could nevertheless increase the scatter of the existing particulates by enlarging them. If the passage of electrons to the particulate terminal oxidases is facilitated by such an association, it would possibly result in an inhibition analogous to the naphthoquinone effect (5). An alternative explanation may be that all of the respiratory enzymes and particulates move peripherally (i.e., toward the cell wall) under conditions of anaerobiosis. This would increase the refractive-index difference at the cell wall-medium interface, thereby increasing the scatter. At the same time, a closer coupling between the luminescent and nonluminescent respiratory pathways could produce an inhibition as mentioned above. These present results are reminiscent of several earlier sets of observations. Smith (23) has observed changes in the transmission of suspensions of bacteria which she regards as scattering changes. Strehler and Lynch (24) observed a wavelength nonspecific transmission change in illuminated Chlorella which had earlier been treated at 51°C. for a few minutes. The most directly analogous observations are those of Cleland, Slater, et al. (25-27) on the change in size of mitochondria under various conditions of respiration, toxicity, substrate depletion, etc. Since the actual volume of mitochondria has been observed to change (by mitocrit measurements) (28) under at least some of these conditions, it is tempting to extrapolate these results to the present case. Chance and Packer (26) have recently demonstrated that a transmission change occurring when adenosine diphosphate (ADP) is added to mitochondria is probably due to a scattering change attendant on size alteration of isolated mitochondria. These changes were too small to permit mitocrit measurements, and it could not be said with certainty that this change represents swelling, rearrangement, or an actual change in a broad absorption band. If, by analogy with Chance and Packer’s observation, the change in the luminous bacteria is due to a change in the size or shape of respiratory organelles, it would appear that the most likely explanation of the inhibition of luminescence is that the swelling of the particle exposes certain acceptor sites to the reduced flavine and thus places the luminescent reaction in a poor competitive position for substrate. Or, finally, it may be that the anaerobic system accumulates phosphate acceptors so that during the first moments after anaerobiosis the cytochromes are capable of handling all of the endogenous reducing equivalents.
LUMINOUS
BACTERIA
407
As the phosphate acceptor pool is depleted and coupling of respiration with phosphorylation begins to exert control, however, the luminescent system may perhaps receive the surplus electrons or hydrogens. SUMMARY
AND
CONCLUSIONS
The oxidized-reduced difference spectrum of the luminous bacterium, P. fischeri, has been determined. The spectrum consists of b and c type cytochromes, flavine components, some bands probably ascribable to reduced diphosphopyridine nucleotide and a component absorbing at about 630 rnp, perhaps cytochrome a2. See also Smith (27). No cytochrome a3 band was apparent. A new phenomenon was discovered, namely, that there occurs during anaerobiosis a gradual decrease in transmission of light through the bacteria which is paralleled by an inhibition of their capacity to luminesce when oxygen is readmitted. It was shown that this transmission change is not due to the appearance of an absorbing component. Rather, it appears to be due to an increase in scatter. Moreover, this increased scatter is probably not of the metallic type to be expected of semiconductors since it does not depolarize the scattered light any more effectively than does the normal scatter of these bacteria. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
HARVEY, E. N., “Bioluminescence.” Academic Press, New York, 1952. STREHLER, B. L., AND CORMIER, M. J., Arch. Biochem. Biophys. 47, 16 (1953). CORMIER, M. J., AND TOTTER, J. R., J. Am. Chem. Sot. 76, 4744 (1954). MCELROY, W. I)., HASTINGS, J. W., SONNENFELD, V., AND COULOMBRE, J., Science 118, 385 (1953). MCELROY, W. D., AND GREEN, A. A., Arch. Biochem. Biophys. 66, 240 (1955). STREHLER, B. L., AND CORMIER, M. J., J. Biol. Chem. 21, 213 (1954). STREHLER, B. L., in “The Luminescence of Biological Systems” (F. H. Johnson, ed.), p. 209 ff. Amer. Assoc. Adv. Sci., Washington, D. C., 1955. STREHLER, B. L., HARVEY, E. N., CHANG, J. J., AND CORMIER, M. J., PI-OC. Natl. Acad. Sci. U. S. 40, 10 (1954). STREHLER, B. L., AND JOHNSON, F. H., Proc. N&Z. Acad. Sci. U. S. 40, 606 (1964). CORMIER, M. J., AND TOTTER, J. R., Biochim. et Biophys. Acta 26, 229 (1957). STREHLER, B. L., AND MCELROY, W. D., Bacterial. Revs. 18, 177 (1954). JOHNSON, F. H., VAN SCHOUWENBURG, K. L., AND VAN DER BURG, A., Enzymologia 7, 195 (1939). STREHLER, B. L., Federation Proc. 17, (l), 1256 (1958). MCELROY, W. D., AND FARGHALY, A. H. M., Arch. Biochem. 17, 379 (1948). ROGERS, P., AND MCELROY, W. I)., Proc. Natl. Acad. Sci. U. S. 41, 67 (1955). NEGELEIN, E., AND GERISCHER, W., Naturwissenschuften 21, 884 (1933). SZENT-GY~RGYI, A., “Chemistry of Muscular Contraction.” Academic Press, New York, 1947. ARNOLD, W. A., AND SHERWOOD, H. K., Proc. Natl. Acad. Sci. U. S. 43, 106 (1967).
408
STREHLER
19. COMMONER, B., HEISE, J. J., AND TOWNSEND, J., Proc. Natl. Acad. Sci. U. S. 42, 710 (1956). 20. BRADLEY, D. F., AND CALVIN, M., Proc. Natl. Acad. Sci. U. S. 41, 563 (1955). 21. STREHLER, B. L., in “Rhythmic and Synthetic Processes in Growth” (D. Rudnick, ed.), p. 171 ff. Princeton Univ. Press, Princeton, N. J., 1957. 22. CHANCE, B., J. Biol. Chem. 202, 397 (1953). 23. SMITH, L., Arch. Biochem. Biophys. 60, 299 (1954). 24. STREHLER, B. L., AND LYNCH, V. A., Arch. Biochem. Biophys. 70, 527 (1957). 25. CLELAND, K. W., AND SLATER, E. C., Quart. J. Microscop. Sci. 94, 329 (1953). 26. CHANCE, B., AND PACKER, L., Biochem. J. 68, 295 (1958). 27. SMITH, L., Bacterial. Revs. 18, 106 (1954). 28. TAPLEY, D. F., J. Biol. Chem. 222, 325 (1956). 29. BATEMAN, J. B., AND MONK, G. W., Science 121, 441 (1955). 30. GIOVANELLI, R. G., Australian J. Exptl. Biol. Med. Sci. 36, 143 (1957). 31. SHIBATA, K., J. Biochem. (Tokyo) 46, 599 (1958). 32. KEILIN, D., AND HARTREE, E. F., Biochim. et Biophys. Acta 27, 173 (1958). 33. LIJNDEGARDH, H. Acta Chem. Stand. 10, 1083 (1956).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Some Optical Properties
86, 391-408
(1969)
of Luminous
Bacteria
Bernard L. Strehler From the Gerontology Branch, National Public Health Service, Department Maryland, and the Baltimore Received
Heart Institute,
National Institutes of Health, and Welfare, Bethesda, Baltimore, Maryland
of Health, Education City Hospitals, April
30, 1959
INTRODUCTION
The light-producing system of luminous bacteria, like that of fireflies and some other luminous organisms, consists of a cyanide-insensitive respiratory pathway. This system, which has been extracted and characterized with respect to the nature of the diffusible components involved (l-lo), appears to be a flavine autoxidase. Specifically, hydrogen is bransferred from reduced pyridine nucleotides to flavine mononucleotide. The reduced flavine is then oxidized by molecular oxygen with the intervention in some ancillary function of a long-chain aldehyde (6). In contrast to its insensitivity in viva and in vitro to cyanide, the luminescent reaction is remarkably sensitive to naphthoquinone inhibition (1). Cormier and Totter (3) have shown that this capacity of naphthoquinones to inhibit luminescence is probably due to a shunting of hydrogen from flavine through the quinone to a cytochrome pathway. From this and from earlier in viva studies (11, la), it appears that there is a linkage between the cyanide-sensitive and the luminescent respiration pathways in these bacteria. However, the nature of the interrelationship in viva, the identity of the heme system involved, and its relation to the energy economy of the cells have not been established. The present study was undertaken to describe the characteristics of the chromophores associated with hydrogen transport in Photobacterium jischeri. The difference spectra and some kinetic aspects of the spectral changes associated with the transition from aerobic to anaerobic conditions is described. In addition, the discovery of a respiration- and luminescence-dependent change in transmission which cannot be ascribed to a specific hydrogen or electron carrier and the experiments leading to the elucidation of its nature are described. It has been found that there is a general increase in transmission at all wavelengths under aerobic conditions. Conversely, under
392
STREHLER
anaerobic conditions, there is a decrease in transmission which takes approximately 30 min. to approach completion. Paralleling this effect of anaerobiosis, there is a gradual inhibition of the capacity to exhibit luminescence when oxygen is readmitted. Chemical, physical, and optical factors entering into this phenomenon have been examined. It is concluded that the effect is due to a scattering change rather than to a change in absorption or a change in metallic reflectivity and that the phenomenon is probably not due to the presence of conduction-band electrons (13). MATERIAL
AND METHODS
A. Culture of Luminous Bacteria Photobacterium fischeri’ and a luminous diplococcus were grown in a liquid complete medium as described by Farghaly (14). The bacteria were grown at 25°C. in 250-ml portions in Erlenmeyer flasks on a shaker. After harvesting by rapid centrifugation of suspensions chilled to O”C., the bacteria were resuspended in nitrogen-free basal medium, washed once, and kept at 0°C. in suspension until used.
B. Spectrophotometry All spectral measurements here described were carried out with a Cary model 14 spectrophotometer. The following accessories were developed for use in this study: 1. Cuvettes for aerating samples without direct interference with the light path. Regular Cary cuvettes (4 ml.) were fitted with small Teflon inserts, mounted parallel to the direction of light transmission. These inserts were rectangular and were mounted about 1.5 mm. from one side of the cuvette. They extended from approximately 2 mm. above the bottom of the cuvette to approximately 2 mm. below the liquid level. A small plastic tube was placed between the cuvette wall and the Teflon insert, and purified gases or air from a small pump was bubbled through the side chamber. This not only aerated the bacteria but also circulated them through the cuvette. See also (33). 2. In order to determine whether there is a change in scatter as well as absorption, a device was constructed to measure changes in scatter perpendicular to the light path. This consisted of a Lucite cuvette holder with attached mirrors. This arrangement is shown in Fig. 1 which also illustrates the aerating cuvette. Light incident upon the cuvette is prevented from passing directly to the detector. Rather, only the light scattered at right angles to the incident beam is picked up by the mirrors and transmitted to the radiation detector. 3. The absorption spectrum, free of first-order scattering artifacts, can be obtained by the use of light integrators or diffusers as described elsewhere (13) [see also (2932)]. We have constructed such a device. In principle, it operates as follows. All of the incident light that is not absorbed is scattered several times, either within the sample cuvette or by the white reflecting surface of the integrating box. Light emerging from the boxes is then transmitted to the detector. 1 P. fischeri kindly furnished by Dr. W. D. McElroy; through the courtesy of Dr. M. Cormier.
the diplococcus
supplied
393
LUMINOUS BACTERIA
Aerating Tube
hastic Baffle
4 ,Mzr M3 = Front Surface
Mirrors
FIG. 1. Optical arrangement of cuvette and mirrors for measuring changes in side scatter of suspensions of bacteria or other scattering particles. Light from the monochromator incident upon the suspension is scattered by it. A small fraction of the scattered light is reflected successively off mirrors Mr , M, , and Ma and thence into the detector. By contrast, light that is transmitted through the sample is intercepted by mirror M, and prevented from reaching the detector.
C. Luminescence Light emission was measured concurrently with absorption at several wavelengths by mounting a mirror beside the cuvette. Light from the side of the cuvette was reflected into a photomultiplier mounted on a lid to the spectrophotometer chamber. The measuring beam did not, by itself, produce measurable signal in the luminescence detector. The bacteria were observed under aerobic and anaerobic conditions with light and dark field microscopy. EXPERIMENTAL
RESULTS
Wavelength-Dependent Changes in Transmission (a) Absorption Spectrum. The absorption spectra of P. jkcheri under aerobic and anaerobic conditions are illustrated in Fig. 2. These tracings were obtained using the light-integrating devices described above. Corrections in relative peak heights produced by the second-order effect of scatter on effective optical path length have not been made. (b) Aerobic-Anaerobic Difference Spectra. The oxidized-reduced difference spectra for P. fischeri were measured as follows: Bacteria were introduced into both the sample and reference chamber cuvettes. On a regular time schedule the air was circulated into the sample cuvette and a tracing of the time course of transmission was obtained. This method is similar to that of Smith (23). The 04.1 O.D.U. slide-wire
394
STREHLER
Reduced
< I 370
I 430
I
I 490
I
I 560
,I
I 610
I
I 670
I
FIG. 2. Absorption spectra of oxidized and of reduced luminous bacteria as measured in the light-integrating chambers described elsewhere. The wavelength-dependent scatter does not introduce spurious signals, but it does modify relative heights of bands by changing effective optical path length. Numbers along the ordinate represent millimicrons whereas the range of absorbancy is O-l O.D.U.
scale of the Cary model 14 spectrophotometer was used. By measuring the magnitude and sign of the tracing deflection at various wavelengths at the instant of oxygen exhaustion (as measured by the disappearance of luminescence due to endogenous respiration) and plotting these deflections against wavelength, the difference spectrum shown in Fig. 3 was obtained. Also shown is the difference spectrum obtained by the use of the integrating boxes described above. In this case two cuvettes (one kept anaerobic, the other aerated) were used. An examination of Figs. 2 and 3 reveals typical cytochrome peaks in the 550-560-rnp region. There is no typical cytochrome a f a3 band at 600-605 rnp. There is, rather, a weak band at about 628 rnp, which is quite similar to the spectrum described for the a2 type cytochrome (16, 27). A band due to flavine reduction is clearly visible as is one due to reduced DPN. Wavelength-Independent
Transmission Changes
(a) General Properties. There is, as indicated above, evidence of spectral behavior similar to that observed in other organisms when 02 disappears. However, in addition to these wavelength specific changes, there are
LUMINOUS
395
BACTERIA
changes of considerable magnitude which occur after oxygen depletion, but which are not localized to any specific spectral region. Rather, they appear to be due to (a) a generalized change in absorption at all wavelengths, (b) to a change in scatter irrespective of wavelength, or (c) to a combination of these two (Fig. 4). A number of experiments were performed in order to eliminate various optical artifacts, physical effects, etc. and to establish the relationship of this transmission change to scatter and/or absorption. These are summarized in Table I. DIFFERENCE
SPECTRUM
A. Fischeri
+ ,035, 030. ;p ,025. 020. 5 .Ol!iL o ,010 2z: ,005. t 03 s 2 2
. ,t
fl-\
Y,
\ \ 4 \ I : : : 350 : -4 250 1 : : : :: : : 'I\ ‘"00 ,010. b ,015. ,020. .025. 030~ ,035' \ wo . ,' P'
-
Aulomatic recording using “spheres”
---
Spectrum obtwwd point for point wthout “spheres”
FIG. 3. Reduced-oxidized difference spectra for P. jischeri as determined by two separate methods. Solid line = direct tracing using integrating chambers. The control sample was allowed to become anaerobic. The experimental sample was aerated at 5°C. and the difference spectrum then determined. Dashed line = difference spectrum determined by plotting the change in O.D. at each wavelength during the transition from aerobic to anaerobic conditions. The control sample was permitted to become anaerobic. The experimental was aerated, as described in the text, and the time course of transmission was followed as the sample became anaerobic. The Iight-integrating boxes were not used in this second method. Note that the two methods yield differences in relative peak heights. These differences are due to differences in effective optical path length.
396
STREHLER
I 0
t I ,
I 2
I 3
I 4
I 5
I 6
I 7 TIME
I I 6 9 (minutes)
I 0
11 II
12
II, 13
14
I5
FIG. 4. Difference between effect of Nz gas and air on scatter. The sample was aerated between A and B. The rapid decrease in transmission is due to the presence of bubbles in the side chamber of the cuvette and occurs irrespective of the kind of gas bubbled through the sample (see D to E). Following a brief period of aeration, the optical density continues to decrease until the oxygen is exhausted at C, at which time decrease in transmission begins to occur. Note particularly that bubbling Nz through the sample does not produce the same effect as bubbling air. A = 600 mp.
(b) Kinetics of Transmission Chwge. Figure 5 illustrates the time course of the transmission change at several wavelengths during a period of anaerobiosis. (c) Wavelength Independence of Transmission Change. The difference spectrum for this slow change in transmission was determined by repeated scans of the same suspension before, during, and after the transition from aerobic to anaerobic conditions. Figure 6 illustrates the results obtained in a typical series of repeat scans. Note that the difference between successive scans is largely independent of wavelength over the region measured except for the first tracing which includes wavelength specific changes. The flatness of the slow change is shown by the fact that the distance between the lines representing the 3- and 5-min. tracings are approximately constant at all wavelengths. (d) Association of Transmission Change with Luminescence. We had earlier noted that bacteria which had been centrifuged in the cold anaerobically required some few minutes of aeration in order to attain their full luminescent intensity. It was of interest to determine whether the change in transmission described above is related to this inhibition of luminescence by anaerobiosis of longer duration. Therefore we measured the time course of both luminescence and transmission concurrently. Typical results are shown in Fig. 7. Figure 8 illustrates the per cent of luminescence in-
TABLE General
Properties
-
of the Wavelength
I Unspecijk
Transmission
Change
Possible source of effect
Control
1. Photometric errors due to luminescence of bacteria
la. Effect continues atter luminescence ceases lb. Base line is not shifted by inserting incandescent source in one sample compartment lc. Effect is observable using infrared detector light and “reversed path” of Cary model 14
1. No artifact bacterial cence
2. Settling
2a. No settling of bacteria overnight 2b. Stirring with Nz without effect (see Fig. 4)
2. Effect not due to set,tling of bacteria
3. Not tion
due to aggrega-
due
of bacteria
Conclusion
due to lumines-
3. Aggregation teria
of
bac-
3. Microscopic tion
4. Orientation teria
of
bac-
4a. Both cocci and rods show effect 4b. No effect of NS stirring
4. Not tion
5. pH change
5. pH was measured and no change as great as 0.01 pH unit was found after 2 hr.
5. Not due to pH change (externally)
6. Temperature difference between bacteria and medium due to metabolic activity
6. On basis of cell mass and respiratory rate measurement the temdifference perature must be less than O.Ol”C.
6. Not ture
7. Mass heating or cooling of bacterial suspension
7. No appreciable change in transmission between 6 and 30°C.
7. Not due to gross changes in temperature of suspension
8. Absorption
8. See following sections (e) and (f) in text
8. Effect due to scatter, not absorption
or scatter
-
observa-
to
orienta-
due to temperadifferencea
a Oxygen dissolves in water to the extent of 0.008 g./l. = 0.00025 moles/l. Since approximately 110 kcal. are liberated/mole, the temperature rise would be 0.0275”C. Assuming 1% bacteria by volume and no heat transfer between bacteria and media during the time 02 is consumed, the maximum temperature differential between bacteria and medium would be 2.75”C. 397
t
LUM.DlSAPPEARS
I
3
L
4 TIME
5(MIN.)6
5. Time course of transmission changes at two wavelengths. Note that there is a rapid change during the transition to anaerobiosis which may be of either positive or negative sign (depending on the spectral properties of the hydrogen carriers) followed by a slower change which is always of the same sign (i.e., decreased transmission). FIG.
I 320
I 360
I 440
I 500
WAVELENGTH FIG. 6. Successive tracings of difference spectra Note relative lack of specific effects and approximate lengths.
398
I 560
I 620
(rnb) following equality
a period of aeration. of effects at all wave-
LUMINOUS
--I%+ t 0
399
BACTERIA
I
,ttt,
I
2
, 3
4
5
T I M E (minuted
FIG. 7. Time course of luminescence and transmission change, after various periods of anaerobiosis. Note particularly that there is almost no immediate luminescence when air is admitted after a long period of anaerobiosis, but that the luminescence approaches peak value almost immediately when the anaerobic conditions are brief. Note also that the degree of initial inhibition of luminescence approximately parallels the magnitude of the scattering change.
hibition and the per cent change in absorption after various periods of anaerobicity. Thus, it appears that the decreased ability of the bacteria to luminesce is correlated with the change in their transmission. It remained to be determined whether the transmission change is due to changes in absorption or scattering or both. The marked inhibition of luminescence under anaerobic conditions, which parallels the optical effects, suggested the possibility that some essential component of the luminescent pathway, specifically the aldehyde cofactor, might be reduced under these conditions. McElroy (15) has shown that certain mutants of P. jischeri, which normally cannot luminesce, do so when a small amount of aldehyde is added to the medium. To test the above explanation of the luminescence inhibition, dodecyl aldehyde was added to inhibited suspensions. No shortening of the induction time was noted and there certainly was no immediate effect as is the case with the mutants. It, therefore, appears that the inhibition of luminescence and also probably the transmission changes are not due to aldehyde reduction. (e) Evidence That the E$ect Is Primarily Due to Scattering Changes. Although it is certain that this phenomenon was not an instrumental artifact or attributable to some simple chemical alteration such as reduction of aldehyde, it was not clear whether it represented primarily a change in absorption or of scatter. That there was a large scattering component to the phenomenon was demonstrated as follows: If there is a large scattering component, it follows that the light appear-
400
STREHLER
l
lniiial
0
Scatter
A Sea tter
,c 0
6
12 DURATION
IS
Luminescence (!+ time =4.0min.)
(‘4 time
24
OF ANAEROBICITY
= 3.4 min)
30 (min.)
FIG. 8. Initial luminescence intensity and scattering change vs. duration of anaerobicity. The values of luminescence, when oxygen was admitted, were measured after a series of runs consisting of various lengths of exposure to anaerobicity. The scattering values represent the difference (on two individual runs) between the asymptote approached after a long exposure to anoxia and the measured value. A small correction for a gradual change in base line was made. This correction was estimated from the Bnal slope of the transmission curve. Note that the half-times for both phenomena are of the order of 4 min., lending credence to the view that they may be united by some common underlying process.
the sides of the cuvettes should behave in an opposite direction from the transmitted ‘(beam” since the untransmitted light (due to scatter) emerges at other angles. Therefore, we measured the time course of transmission and side scatter at various wavelengths through the use of the special cuvette holder and mirror arrangement described in Material and Methods. Typical results are illustrated in Fig. 9. Since the direction of the effect changes, depending on whether transmitted or scattered light is meassured, whereas the sign of the wavelength specific changes is unaltered, it appears that scattering changes are a major factor in the phenomenon.
ing at
LUMINOUS
I
I
I
I
I I I I I I I I
I I I I I
1 I I I I
401
BACTERIA
I I
I I I I I
I I i
430 mu
A. FISCHERI AEROBIC + ANAEROBIC KINETICS OF TRANSMITTED AND SIDE SCATTERED RADIATION
FIG. 9. Evidence that the nonwavelength specific transmission change is primarily due to a change in scatter. Note that the immediate change in transmission (due to specific absorption bands) which occurs upon exhaustion of 02 is similar in both the side scattered and transmitted beams, but that the subsequent changes are opposite in sign depending on whether the transmitted or scattered component is measured.
These results demonstrated that there is a large scattering component in the effect, but they do not rule out absorption changes of a somewhat lesser magnitude. Therefore, the “integrating boxes” described above were constructed. With both samples inside these “spheres,” no wavelength unspecific changes could be observed during anaerobic conditions (see Fig. 10). It is, therefore, concluded that all of the measurable effect is due to scattering changes. The data shown in Fig. 11 make understandable the “wavelength independence” of the phenomenon. Evidence That Transmission Changes Are Due to Changes in Geometry and/or Refractive Index Rather Than to Changes in Metallic ReJlectivity. Three possibilities existed: The scattering change is due to (a) change in refractive index of two contiguous phases; (b) change in size (or shape) of scattering objects; and (c) change in metallic reflectivity of particles or parts thereof. The first two possibilities could be tested by a measurement of effective size of the bacteria or by alterations in the refractive index of the suspending medium (assuming that the bacterial medium interface is the determining one). The third possibility, which is the most interesting one because of the potential role of semiconductors or pseudometallic objects in biological
402
STREHLER I
I
I
I
I
I /I
WAVELENGTH-6300°A ---
Regular cuvettes
-
In scattering
i
chamber : 4’ /’
--lZsec.---)3mhl.-
I--Lc--
I
0
I
I
I
I
I
I
12
24
36
46
60
72
TIME
(seconds)
10. Tracing of absorption changes at 630 my within and without light-integrating chamber. A to B: aerobic conditions. B to C: 02 disappears. Note increase in absorption due to reduced cytochrome band at 630 rnp. C to D: subsequent changes in scatter (do not occur in light integrators). This indicates that the transmission changes are due to scattering changes. D to E: compressed time scale. Note lack of change in chamber as compared to large change using regular cuvettes. The abscissa represents changes in optical density. The total deflection equals about 0.05 and 0.01 O.D.U., respectively. FIG.
electron transport (17-21), was tested through the courtesy and suggestion of Dr. Wm. A. Arnold of the Oak Ridge National Laboratory, Biology Division. A metallic reflector will interact differently with polarized light than one in which no highly mobile electrons are involved. In the case of the metallic reflector, the electrical and magnetic vectors will generally be thrown out of phase with each other, and the reflected light will be eliptitally polarized. Thus, if one starts with planepolarized light, it will be much more effectively depolarized by metallic reflection (from the point of view of the analyzer) than by an equal amount of nonmetallic scatter. To distinguish between these alternatives, the degree of depolarization of scattered light under aerobic and anaerobic conditions was compared using a Gaertner polarimeter. The results obtained were negative; that is, the additional scatter was not more highly depolarized than the original scatter (which presumably arises from differences in refractive index). (See Table II.) Thus, we must conclude that, the phenomenon is not, due to changes in the numbers of
LUMINOUS
3QO
360
420
403
BACTERIA
460 WAVELENGTH
540
600
660
(m/d
FIG. 11. Differences in scatter spectra of bacteria as a function of concentration differences. The numbers represent number of units of bacteria in reference and sample cuvettes, respectively. One unit equals approximately 0.25% bacteria by volume. Note that for constant differences in bacterial density, e.g., Cl, l-2,2-3, and 34 units, the scattering difference spectra exhibit extremely broad maxima of decreasing prominence which occur at successively longer wavelengths. The scattering changes reported showed very slight wavelength dependence. These present observations make it clear that a scattering change may appear to be unspecific with respect to wavelength even though the scattering objects, in greater dilution, would exhibit scatter with a strong wavelength dependence. The effect illustrated in Fig. 11 is explicable in the following terms: If one starts with moderately scattering suspensions, the addition of further scattering material does not attenuate the transmission at any wavelength according to Beer’s law, but the scatter at the red end of the spectrum approaches Beer’s law more closely than at the blue end. In the blue region scatter is almost complete, and therefore additional scattering material does not appreciably change the transmission. At the far red end of the spectrum, Beer’s law is still being approximated, but the efficiency of scatter is less so the apparent increase in O.D. is less than at some shorter wavelength. At some intermediate wavelength, the two effects are maximized. The above considerations imply that the wavelength of maximum transmission change will be shifted to the red region of the spectrum as the absolute concentration is increased, while a constant difference in concentration is maintained. The observations confirm this expectation.
404
STREHLER
TABLE Depolarization
.9
A. Thin Air (15 min.) Nf (10 min.) Nz (45 min.) Air (5 min.)
(10 min.) (10 min.) (25 min.) (28 min.) (5 min.)
Air Ns N? Nz Air
(5 min.) (1 min.) (17 min.) (30 min.) (5 min.)
suspension: 26.38 26.30 27.07 26.78 26.93
C. Thin
suspension: 61.15 61.5 61.5 61.45 60.92
D. Thin Air (10 min.) Nz (15 min.) Nz (30 min.) Air (5 min.)
suspension: 4.11 4.10 4.135 4.14
B. Heavy Air Nz N.P Nz Air
II
of Scattered Light by Bacteria under Aerobic Anaerobic (Nz) Conditions
suspension: 17.69 17.23 17.50 17.32
P
D
(Air)
and
“$
x 100
XQ = 17”, Yb = 45” 0.98971 0.98977 0.98958 0.98956
0.01029 0.01023 0.01042 0.01044
-0.58309 +1.2633 $1.4577
Xa = O”, Yb = 0” 0.6050 0.6072 0.5858 0.5938 0.5896
0.3950 0.3928 0.4142 0.4062 0.4104
-0.5569 +4.860 +2.835 $3.898
Xa = 9”, Yb = 90” 0.5342 0.54450 0.54450 0.5430 0.5274
0.4658 0.4555 0.4555 0.4570 0.4726
-2.211 -2.211 -1.8892 f1.460
Xa = 90”, Yb = 0” 0.81535 0.82465 0.8192 0.82270
0.18465 0.17535 0.18080 0.17730
-5.0365 -2.0850 -3.9805
0 X = angle in degrees between incident beam and measured beam. 6 Y = axis of polarization of incident beam in degrees to right of vertical. c The sample was not agitated during period preceding this measurement. Otherwise all measurements in Expts. A and B included stirring by an appropriate gas (pure Nz or air). Experiments C and D did not involve stirring during anaerobiosis. It thus appears likely that such slight differences as exist represent effects due to changes in cell orientation or distribution as a result of stirring. d 0 = degrees from vertical of movable analyzing prism at balance. P = polarization of scattered light as fraction of original polarization. D = fractional depolarization. AD = difference between initial depolarization and subsequent depolarization. AD - D X 100 = percentage change of depolarization as compared to initial depolarization.
LUMINOUS
405
BACTERIA
freely mobile electrons but rather either to changes in refractive to particle geometry or both.
index or
DISCUSSION
Signijkance
of Di$erence Spectra
Except for the absence of a cytochrome oxidase with an absorption maximum at around 600-605 rnp and the presence of a weak band around 630 rnp, the oxidized minus reduced spectrum does not appear to be remarkable. The recordings do not readily permit the assignment of a kinetic sequence of electron transport among the various absorbing components. However, it is probable that the sequence is similar to that demonstrated by Chance et al. (22) in other systems by more refined methods of kinetic measurement at several wavelengths, e.g., luciferase ____) hv 02 \ Quinone I --+ B type ---f C type + 630 rnp * cytochrome cytochrome compound
AH2 -+ DPN
BHz
-+ &vine
\\
02
Signijicance of Generalized Scattering Change The lack of wavelength specificity for the scattering change and its apparent association with the function of the luminescent system are the most striking characteristics of this phenomenon. The former property is explicable in the following terms. It can be shown that an apparent nonspecificity of scatter with respect to wavelength using the apparatus as described is obtained if the suspension is already sufficiently dense to provide multiple scatter for each quantum. This is illustrated in Fig. 11 in which the changes in absorption of bacterial suspensions which differ from each other by a constant amount of scattering material are compared. That is, the reference and sample cuvettes were filled with suspensions of bacteria that differed from each other by a constant amount (O-l, 1-2, 2-3 units, etc.). Note that as the turbidity increases, the additional scatter becomes less and less wavelength dependent until it becomes practically independent of wavelength. It is thus probable that the increase or decrease in scatter at all wavelengths such as observed with the bacteria is really due to an underlying wavelength-dependent scatter which is masked by the fact that multiple scatterings take place. There are several possible explanations of the apparent relationship between the scattering changes and the inhibition of the luminescent reaction.
406
STREHLER
Since both changes are effected by anaerobiosis, it is tempting to postulate that the scattering change occurs in particles connected with respiration. One possibility is that the luminescent system coalesces with the regular mitochondrial system under anaerobic conditions. If the luminescent enzymes are too small to be effective scatterers by themselves, they could nevertheless increase the scatter of the existing particulates by enlarging them. If the passage of electrons to the particulate terminal oxidases is facilitated by such an association, it would possibly result in an inhibition analogous to the naphthoquinone effect (5). An alternative explanation may be that all of the respiratory enzymes and particulates move peripherally (i.e., toward the cell wall) under conditions of anaerobiosis. This would increase the refractive-index difference at the cell wall-medium interface, thereby increasing the scatter. At the same time, a closer coupling between the luminescent and nonluminescent respiratory pathways could produce an inhibition as mentioned above. These present results are reminiscent of several earlier sets of observations. Smith (23) has observed changes in the transmission of suspensions of bacteria which she regards as scattering changes. Strehler and Lynch (24) observed a wavelength nonspecific transmission change in illuminated Chlorella which had earlier been treated at 51°C. for a few minutes. The most directly analogous observations are those of Cleland, Slater, et al. (25-27) on the change in size of mitochondria under various conditions of respiration, toxicity, substrate depletion, etc. Since the actual volume of mitochondria has been observed to change (by mitocrit measurements) (28) under at least some of these conditions, it is tempting to extrapolate these results to the present case. Chance and Packer (26) have recently demonstrated that a transmission change occurring when adenosine diphosphate (ADP) is added to mitochondria is probably due to a scattering change attendant on size alteration of isolated mitochondria. These changes were too small to permit mitocrit measurements, and it could not be said with certainty that this change represents swelling, rearrangement, or an actual change in a broad absorption band. If, by analogy with Chance and Packer’s observation, the change in the luminous bacteria is due to a change in the size or shape of respiratory organelles, it would appear that the most likely explanation of the inhibition of luminescence is that the swelling of the particle exposes certain acceptor sites to the reduced flavine and thus places the luminescent reaction in a poor competitive position for substrate. Or, finally, it may be that the anaerobic system accumulates phosphate acceptors so that during the first moments after anaerobiosis the cytochromes are capable of handling all of the endogenous reducing equivalents.
LUMINOUS
BACTERIA
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As the phosphate acceptor pool is depleted and coupling of respiration with phosphorylation begins to exert control, however, the luminescent system may perhaps receive the surplus electrons or hydrogens. SUMMARY
AND
CONCLUSIONS
The oxidized-reduced difference spectrum of the luminous bacterium, P. fischeri, has been determined. The spectrum consists of b and c type cytochromes, flavine components, some bands probably ascribable to reduced diphosphopyridine nucleotide and a component absorbing at about 630 rnp, perhaps cytochrome a2. See also Smith (27). No cytochrome a3 band was apparent. A new phenomenon was discovered, namely, that there occurs during anaerobiosis a gradual decrease in transmission of light through the bacteria which is paralleled by an inhibition of their capacity to luminesce when oxygen is readmitted. It was shown that this transmission change is not due to the appearance of an absorbing component. Rather, it appears to be due to an increase in scatter. Moreover, this increased scatter is probably not of the metallic type to be expected of semiconductors since it does not depolarize the scattered light any more effectively than does the normal scatter of these bacteria. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
HARVEY, E. N., “Bioluminescence.” Academic Press, New York, 1952. STREHLER, B. L., AND CORMIER, M. J., Arch. Biochem. Biophys. 47, 16 (1953). CORMIER, M. J., AND TOTTER, J. R., J. Am. Chem. Sot. 76, 4744 (1954). MCELROY, W. I)., HASTINGS, J. W., SONNENFELD, V., AND COULOMBRE, J., Science 118, 385 (1953). MCELROY, W. D., AND GREEN, A. A., Arch. Biochem. Biophys. 66, 240 (1955). STREHLER, B. L., AND CORMIER, M. J., J. Biol. Chem. 21, 213 (1954). STREHLER, B. L., in “The Luminescence of Biological Systems” (F. H. Johnson, ed.), p. 209 ff. Amer. Assoc. Adv. Sci., Washington, D. C., 1955. STREHLER, B. L., HARVEY, E. N., CHANG, J. J., AND CORMIER, M. J., PI-OC. Natl. Acad. Sci. U. S. 40, 10 (1954). STREHLER, B. L., AND JOHNSON, F. H., Proc. N&Z. Acad. Sci. U. S. 40, 606 (1964). CORMIER, M. J., AND TOTTER, J. R., Biochim. et Biophys. Acta 26, 229 (1957). STREHLER, B. L., AND MCELROY, W. D., Bacterial. Revs. 18, 177 (1954). JOHNSON, F. H., VAN SCHOUWENBURG, K. L., AND VAN DER BURG, A., Enzymologia 7, 195 (1939). STREHLER, B. L., Federation Proc. 17, (l), 1256 (1958). MCELROY, W. D., AND FARGHALY, A. H. M., Arch. Biochem. 17, 379 (1948). ROGERS, P., AND MCELROY, W. I)., Proc. Natl. Acad. Sci. U. S. 41, 67 (1955). NEGELEIN, E., AND GERISCHER, W., Naturwissenschuften 21, 884 (1933). SZENT-GY~RGYI, A., “Chemistry of Muscular Contraction.” Academic Press, New York, 1947. ARNOLD, W. A., AND SHERWOOD, H. K., Proc. Natl. Acad. Sci. U. S. 43, 106 (1967).
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19. COMMONER, B., HEISE, J. J., AND TOWNSEND, J., Proc. Natl. Acad. Sci. U. S. 42, 710 (1956). 20. BRADLEY, D. F., AND CALVIN, M., Proc. Natl. Acad. Sci. U. S. 41, 563 (1955). 21. STREHLER, B. L., in “Rhythmic and Synthetic Processes in Growth” (D. Rudnick, ed.), p. 171 ff. Princeton Univ. Press, Princeton, N. J., 1957. 22. CHANCE, B., J. Biol. Chem. 202, 397 (1953). 23. SMITH, L., Arch. Biochem. Biophys. 60, 299 (1954). 24. STREHLER, B. L., AND LYNCH, V. A., Arch. Biochem. Biophys. 70, 527 (1957). 25. CLELAND, K. W., AND SLATER, E. C., Quart. J. Microscop. Sci. 94, 329 (1953). 26. CHANCE, B., AND PACKER, L., Biochem. J. 68, 295 (1958). 27. SMITH, L., Bacterial. Revs. 18, 106 (1954). 28. TAPLEY, D. F., J. Biol. Chem. 222, 325 (1956). 29. BATEMAN, J. B., AND MONK, G. W., Science 121, 441 (1955). 30. GIOVANELLI, R. G., Australian J. Exptl. Biol. Med. Sci. 36, 143 (1957). 31. SHIBATA, K., J. Biochem. (Tokyo) 46, 599 (1958). 32. KEILIN, D., AND HARTREE, E. F., Biochim. et Biophys. Acta 27, 173 (1958). 33. LIJNDEGARDH, H. Acta Chem. Stand. 10, 1083 (1956).