Photochemistry and Photobiology, 1978, Vol. 27, pp. 465-469.
Pergamon Press.
Printed in Great Britain
SIMILAR SPECTRA FOR THE INACTIVATION BY MONOCHROMATIC LIGHT OF TWO DISTINCT LEUCINE TRANSPORT SYSTEMS IN ESCHERICHIA COLI F. T. ROBB,*J. H. HAUMAN*and M. J. PEAK? Departments of Microbiology* and Zoology and Entomologyt, Rhodes University, Grahamstown 6140, South Africa (Received 14 July 1977; accepted 28 September 1977)
Abstract-Kinetics of inactivation of two separate leucine transport systems (leucine specific and LIV) in E. coli by seven wavelengths of monochromatic light have been studied. Loss of leucine uptake is not due to generalized membrane damage causing non-specific leucine leakage. Inactivations are usually exponential but some wavelengths show shoulders at low doses. Two-component spectra between 254 and 435 nm occur for both transport systems. Inactivation is most efficient at 290 nm and a second peak occurs at 365 nm. Both leucine transport systems are inactivated similarly.
INTRODUCTION
Gram-negative bacteria possess several transport systems for the uptake of branched chain amino acids (Penrose et al., 1968; Furlong and Weiner, 1970; Rahmanian and Oxender, 1972; Rahmanian et al., 1973; Guardiola et al., 1974; Wood, 1975). Molecular mechanisms for this active transport are currently being elucidated. For instance, periplasmic amino acid binding proteins have been implicated as components of several uptake systems (Ames and Lever, 1970; Penrose, 1968; Furlong and Weiner, 1970). This is reviewed by Halpern (1974) who quotes further references. Leucine uptake by E. coli has been demonstrated to exist as at least three transport systems whose relationship with each other is unclear (Rahmanian et al., 1973). One system, leucine specific (the leu system) is sensitive to osmotic shock, has a high affinity for leucine, and fails to recognize isoleucine. Two other leucine uptake systems (LIV I and LIV 11, known together as LIV) are competitively inhibited by isoleucine and valine. The distinction between LIV I and LIV I1 is that the former (90% of the total LIV activity) is sensitive to osmotic shock. Thus it seems possible that both the leu system and LIV I are dependent upon binding proteins, removable by osmotic shock. Amino acid uptake has been shown to be inhibited by visible and near-UV light. For instance, Sprott et al. (1976), who quote other literature, have shown that light from wavelengths 366 nm to 578 nm inhibits the uptake of several amino acids and that the uptake of certain of these amino acids (e.g. gly) may be dependent upon oxidative metabolism. They also present evidence that this inhibition is not due simply to a non-specific increase in permeability, because different systems exhibit varied photosensitivity. Also, Barran et al. (1974) and Koch et al. (1976) showed that
near-UV inactivates the galactoside permease system. It also interferes with metabolic energy production or transfer, and causes increased passive membrane permeability, but only after large fluences (Koch et at., 1976). Wavelength dependence for photoinhibition of various uptake systems was studied by Sprott et al. (1976) between 578 and 366nm. Efficiency increased with shorter wavelength but no data below 366nm were presented, and the spectral regions for maximal efficiency for any uptake system have not been fully elucidated. Thus, in order to attempt to separate and subsequently elucidate individual molecular events in systems with binding protein activity, we have measured the wavelength dependence of the inactivation of leu and LIV transport systems separately.
MATERIALS AND METHODS
Bacterial growth. E. coli K12 EO 0319 was originally obtained from Lh. C. E. Furlong. This mutant has trpgenotype and is derepressed for leu specific and LIV transport whereas LIV I1 transport is minor (Rahmanian et at., 1973). A loop inoculum was grown for 18 h at 37°C on a reciprocating shaker in minimal A medium (Miller, 1972) supplemented with 0.05% glycerol and 5 0 p g of trplmd. Glycerol was then added to the cultures to a final concentration of 0.5% and the cells allowed to grow as before for 2-3 h. The cells were then washed twice by centrifugation and resuspension at room temperature in buffer (0.05 M potassium phosphate pH 6.9 containing 0.1 mM MgS04). Cell density was always adjusted turbidometrically to 5 x lo9 cells/mC for irradiation. Irradiation of the cells was always performed within 3 h of harvesting, during which period the leu uptake activity was stable. Assay for leucine transport. Leu and LIV uptake systems were assayed essentially as described by Rahmanian et al. (1973). Reproducibility was improved by adding the 50 pf aliquots of irradiated cells to 50 pt buffer and holding for 6 0 s before starting the reaction by addition of 50pf I4C-leu (final concentration 10 p M ) . Initial rates of uptake were linear at 20°C for 6 0 s and reactions were stopped
465
F. T. ROBB, J . H. HAUMAN and M. J . PEAK
Jhh
at 30 s by filtration of 100 pl samples and washing with buffer at 20°C on 0.45 p n membranes (Gelman Instrument Co.). The dry weight of cells filtered for each assay was 0.031 mg. Unirradiated cells normally had 8-10,000 cpm of 14C-leu. Each nanomol of leu was represented by 40,000cpm. Leu and LIV uptake systems were distinguished as follows. Both systems were assayed together in the absence of ile and the leu system then measured in parallel by assaying in the presence of 200 p M ile which completely inhibits the LIV system (Rahmanian et al., 1973). The LIV activity was determined by difference. All assay mixtures contained 50 pg/m/ chloramphenicol, but chloramphenical was absent during irradiation. Energy source was 0.5% glycerol. Chloramphenicol and glycerol were present in the assay buffer. Cells were irradiated at 20°C in 1 cm spectrophotometer cuvettes made of quartz or Pyrex glass where appropriate. The suspension was stirred vigorously during irradiation by a stream of air. Irradiation procedures. UV radiation at 254 nm was obtained from a Philips 15 W germicidal lamp. Radiation at all other wavelengths was obtained from a Hg-Xe Hanovia 2.5 kW lamp in coniunction with a Schoeffel predispersion prism and a Bausch and Lomb grating monochromator. Filters to reduce scattered light were as follows: 290 nm (Corning 0-56), 313 nm (Corning &54), 334 nm, LOF (kindly lent by Dr. R. M. Tyrrell), 365 (Corning 0-52), 405 and 434 (Corning 3-75). Fluences were measured at the front of the vessel with a YSI-Kettering 65A radiometer calibrated against an Eppley 12-junction bismuth silver thermopile. Fluences for 254, 290, 313, 334, 365, 405 and 435nm were 14, 10, 25, 100, 50, 300 and 400 Jm-2s- respectively. Where necessary, fluences were reduced to the required rate with a 10% neutral density filter. Starting volumes of 0.9 mC of cell suspensions were used so that the entire front face of the suspension was uniformly irradiated. Aliquots (50 p/) were sampled at intervals before and during irradiation, and were assayed for amino acid uptake. Correction for light scattering and cellular shielding. It is necessary to irradiate at high cell densities in order to obtain sufficient cells in each sample to assay uptake within the period (1 min) during which uptake is linear. Due to light scatter and cell shielding (self absorption) the average dose received by a cell is less than the measured dose especially at short wavelengths (Jagger, 1967; Jagger et al., 1975). However, most of the light scattered by a bacterial suspension is deflected only a few degrees (Koch, 1961) and should be considered as transmitted light for the purposes of correction. As shown by Jagger (1975). the use of a spectrophotometer provides a true Morowitz (1950) correction, providing its design allows for maximal collection of scattered light by the photomultiplier. He showed that the Aminco-Chance duochromator is a suitable instrument because of two criteria which increase the solid angle subtended to the photomultiplier from the cuvette. These are (1) short cuvette-photomultiplier distance, (2) large photomultiplier. We have used a Beckman Acta MVI spectrophotometer which follows these criteria and was designed specifically for the purpose of collecting forward-directed scattered light. Further, it has an accessory for collecting scattered transmission (Beckman catalog number 569599). Correction factors were obtained at each wavelength from the relationships between nine cell concentrations (ranging from 5 x lo9 to 5 x 10’) and absorbance measured with the MVI spectrophotometer adjusted to
’
*Irradiation of different suspensions of this strain at longer wavelengths results in complex kinetics lacking constant dose reduction between dense and thin suspensions. These artifacts may be caused by leakage of photosensitizing cellular constituents in the dense suspensions.
Table 1. Morowitz correction factors for high cell density Wavelength nm
Correction factor
254 290 313 334 365 405 435
0.10 0.13 0.31 0.39 0.45 0.52 0.66
capture scattered light, with the scattered transmission accessory installed. These are shown by Table 1, and are specifically for cell densities of 5 x 10’ cells/m/. Attempts were made to obtain true Morowitz correction factors by measuring the lethal effect of different wavelengths at different cell concentrations, as described by Jagger (1975). This was successful for the shorter wavelengths only where irradiation times are short but was not successful for the longer wavelengths.* Thus, in order to maintain a consistent correction throughout the action spectrum we used the Morowitz corrections obtained from our absorbance measurements for all the wavelengths (Table 1). Chromatography. Samples containing ‘‘C-labelled and carrier leu were chromatographed on 20 cm Whatman no. 1 paper strips using two solvent systems (ethanol:NH,: water, 80:4:16 and butano1:acetic acid:water, 12:5:3). Action spectra. The relative efficiency per quantum was calculated using the method described by Jagger (1967), using the corrected fluence at which 10% of the original activity remained, derived from full inactivation curves.
RESULTS
In our normal assay procedure, 2.5 x lo8 cells ( 5 0 p / of a suspension containing 5.0 x lo9 cells m/ - *) accumulate u p t o 16000 cpm of I4C-leu in 30 s. Under identical conditions with the addition of 10 m M sodium azide, approximately 200-500 cpm were associated with the cells. Thus about 97% of the I4C-leu associated with the cells represented energy dependent uptake, and about 3% may have been found, without uptake, outside the cells. Figure 1 shows the effects of two U V wavelengths upon the ability of E. coli to retain radioactivity after loading the cells with 14C-leu under standard conditions. Chloramphenical was present during loading and cells were washed with buffer immediately prior to irradiation. Both wavelengths induce leakage (open circles) and the radioactivity released co-chromatographed with control 14C-leu, using the two solvent systems as described in Materials and Methods. Thus the leaked radioactivity represents leu and 80% of the accumulated material was not metabolized during irradiation since i t was recovered unchanged from the cell supernate (Fig. 1). Figure 1 shows the effects of 365 and 290nm light upon the ability of E. coli t o retain leu. Also, in parallel experiments, the kinetics of the inactivation of the cells’ leu specific uptake system are shown. Leucine uptake is rapidly inhibited by both 365 and 290 n m radiation, whereas in control experiments not shown by Fig. I. unirradiated cells have stable leu
467
Photoinactivation of leucine transport
50
T
-
w
z
u 3
-J W
0
z-
$ W K
4
365 nrn
2
Figure 1. Cells were loaded with radioactive leucine under standard assay conditions, washed in buffer, diluted to 5 x to9 cells/m/ and then exposed to the radiation. Parallel aliquots of control (unirradiated) and irradiated cells were filtered, washed and counted as described in Materials and Methods. In counterflow experiments, started at the time shown by arrows, aliquots of both control and irradiated cells were exposed to 0.6 mM of unlabelled L-leu in the dark. After 5 min exposure to 0.6 mM leu the cells were filtered, washed and counted as before. The fluence was not corrected for cell shielding and scattering. Control, unirradiated cells, loss of leu, M; irradiated cells loss of leu, o--
Pergamon Press.
Printed in Great Britain
SIMILAR SPECTRA FOR THE INACTIVATION BY MONOCHROMATIC LIGHT OF TWO DISTINCT LEUCINE TRANSPORT SYSTEMS IN ESCHERICHIA COLI F. T. ROBB,*J. H. HAUMAN*and M. J. PEAK? Departments of Microbiology* and Zoology and Entomologyt, Rhodes University, Grahamstown 6140, South Africa (Received 14 July 1977; accepted 28 September 1977)
Abstract-Kinetics of inactivation of two separate leucine transport systems (leucine specific and LIV) in E. coli by seven wavelengths of monochromatic light have been studied. Loss of leucine uptake is not due to generalized membrane damage causing non-specific leucine leakage. Inactivations are usually exponential but some wavelengths show shoulders at low doses. Two-component spectra between 254 and 435 nm occur for both transport systems. Inactivation is most efficient at 290 nm and a second peak occurs at 365 nm. Both leucine transport systems are inactivated similarly.
INTRODUCTION
Gram-negative bacteria possess several transport systems for the uptake of branched chain amino acids (Penrose et al., 1968; Furlong and Weiner, 1970; Rahmanian and Oxender, 1972; Rahmanian et al., 1973; Guardiola et al., 1974; Wood, 1975). Molecular mechanisms for this active transport are currently being elucidated. For instance, periplasmic amino acid binding proteins have been implicated as components of several uptake systems (Ames and Lever, 1970; Penrose, 1968; Furlong and Weiner, 1970). This is reviewed by Halpern (1974) who quotes further references. Leucine uptake by E. coli has been demonstrated to exist as at least three transport systems whose relationship with each other is unclear (Rahmanian et al., 1973). One system, leucine specific (the leu system) is sensitive to osmotic shock, has a high affinity for leucine, and fails to recognize isoleucine. Two other leucine uptake systems (LIV I and LIV 11, known together as LIV) are competitively inhibited by isoleucine and valine. The distinction between LIV I and LIV I1 is that the former (90% of the total LIV activity) is sensitive to osmotic shock. Thus it seems possible that both the leu system and LIV I are dependent upon binding proteins, removable by osmotic shock. Amino acid uptake has been shown to be inhibited by visible and near-UV light. For instance, Sprott et al. (1976), who quote other literature, have shown that light from wavelengths 366 nm to 578 nm inhibits the uptake of several amino acids and that the uptake of certain of these amino acids (e.g. gly) may be dependent upon oxidative metabolism. They also present evidence that this inhibition is not due simply to a non-specific increase in permeability, because different systems exhibit varied photosensitivity. Also, Barran et al. (1974) and Koch et al. (1976) showed that
near-UV inactivates the galactoside permease system. It also interferes with metabolic energy production or transfer, and causes increased passive membrane permeability, but only after large fluences (Koch et at., 1976). Wavelength dependence for photoinhibition of various uptake systems was studied by Sprott et al. (1976) between 578 and 366nm. Efficiency increased with shorter wavelength but no data below 366nm were presented, and the spectral regions for maximal efficiency for any uptake system have not been fully elucidated. Thus, in order to attempt to separate and subsequently elucidate individual molecular events in systems with binding protein activity, we have measured the wavelength dependence of the inactivation of leu and LIV transport systems separately.
MATERIALS AND METHODS
Bacterial growth. E. coli K12 EO 0319 was originally obtained from Lh. C. E. Furlong. This mutant has trpgenotype and is derepressed for leu specific and LIV transport whereas LIV I1 transport is minor (Rahmanian et at., 1973). A loop inoculum was grown for 18 h at 37°C on a reciprocating shaker in minimal A medium (Miller, 1972) supplemented with 0.05% glycerol and 5 0 p g of trplmd. Glycerol was then added to the cultures to a final concentration of 0.5% and the cells allowed to grow as before for 2-3 h. The cells were then washed twice by centrifugation and resuspension at room temperature in buffer (0.05 M potassium phosphate pH 6.9 containing 0.1 mM MgS04). Cell density was always adjusted turbidometrically to 5 x lo9 cells/mC for irradiation. Irradiation of the cells was always performed within 3 h of harvesting, during which period the leu uptake activity was stable. Assay for leucine transport. Leu and LIV uptake systems were assayed essentially as described by Rahmanian et al. (1973). Reproducibility was improved by adding the 50 pf aliquots of irradiated cells to 50 pt buffer and holding for 6 0 s before starting the reaction by addition of 50pf I4C-leu (final concentration 10 p M ) . Initial rates of uptake were linear at 20°C for 6 0 s and reactions were stopped
465
F. T. ROBB, J . H. HAUMAN and M. J . PEAK
Jhh
at 30 s by filtration of 100 pl samples and washing with buffer at 20°C on 0.45 p n membranes (Gelman Instrument Co.). The dry weight of cells filtered for each assay was 0.031 mg. Unirradiated cells normally had 8-10,000 cpm of 14C-leu. Each nanomol of leu was represented by 40,000cpm. Leu and LIV uptake systems were distinguished as follows. Both systems were assayed together in the absence of ile and the leu system then measured in parallel by assaying in the presence of 200 p M ile which completely inhibits the LIV system (Rahmanian et al., 1973). The LIV activity was determined by difference. All assay mixtures contained 50 pg/m/ chloramphenicol, but chloramphenical was absent during irradiation. Energy source was 0.5% glycerol. Chloramphenicol and glycerol were present in the assay buffer. Cells were irradiated at 20°C in 1 cm spectrophotometer cuvettes made of quartz or Pyrex glass where appropriate. The suspension was stirred vigorously during irradiation by a stream of air. Irradiation procedures. UV radiation at 254 nm was obtained from a Philips 15 W germicidal lamp. Radiation at all other wavelengths was obtained from a Hg-Xe Hanovia 2.5 kW lamp in coniunction with a Schoeffel predispersion prism and a Bausch and Lomb grating monochromator. Filters to reduce scattered light were as follows: 290 nm (Corning 0-56), 313 nm (Corning &54), 334 nm, LOF (kindly lent by Dr. R. M. Tyrrell), 365 (Corning 0-52), 405 and 434 (Corning 3-75). Fluences were measured at the front of the vessel with a YSI-Kettering 65A radiometer calibrated against an Eppley 12-junction bismuth silver thermopile. Fluences for 254, 290, 313, 334, 365, 405 and 435nm were 14, 10, 25, 100, 50, 300 and 400 Jm-2s- respectively. Where necessary, fluences were reduced to the required rate with a 10% neutral density filter. Starting volumes of 0.9 mC of cell suspensions were used so that the entire front face of the suspension was uniformly irradiated. Aliquots (50 p/) were sampled at intervals before and during irradiation, and were assayed for amino acid uptake. Correction for light scattering and cellular shielding. It is necessary to irradiate at high cell densities in order to obtain sufficient cells in each sample to assay uptake within the period (1 min) during which uptake is linear. Due to light scatter and cell shielding (self absorption) the average dose received by a cell is less than the measured dose especially at short wavelengths (Jagger, 1967; Jagger et al., 1975). However, most of the light scattered by a bacterial suspension is deflected only a few degrees (Koch, 1961) and should be considered as transmitted light for the purposes of correction. As shown by Jagger (1975). the use of a spectrophotometer provides a true Morowitz (1950) correction, providing its design allows for maximal collection of scattered light by the photomultiplier. He showed that the Aminco-Chance duochromator is a suitable instrument because of two criteria which increase the solid angle subtended to the photomultiplier from the cuvette. These are (1) short cuvette-photomultiplier distance, (2) large photomultiplier. We have used a Beckman Acta MVI spectrophotometer which follows these criteria and was designed specifically for the purpose of collecting forward-directed scattered light. Further, it has an accessory for collecting scattered transmission (Beckman catalog number 569599). Correction factors were obtained at each wavelength from the relationships between nine cell concentrations (ranging from 5 x lo9 to 5 x 10’) and absorbance measured with the MVI spectrophotometer adjusted to
’
*Irradiation of different suspensions of this strain at longer wavelengths results in complex kinetics lacking constant dose reduction between dense and thin suspensions. These artifacts may be caused by leakage of photosensitizing cellular constituents in the dense suspensions.
Table 1. Morowitz correction factors for high cell density Wavelength nm
Correction factor
254 290 313 334 365 405 435
0.10 0.13 0.31 0.39 0.45 0.52 0.66
capture scattered light, with the scattered transmission accessory installed. These are shown by Table 1, and are specifically for cell densities of 5 x 10’ cells/m/. Attempts were made to obtain true Morowitz correction factors by measuring the lethal effect of different wavelengths at different cell concentrations, as described by Jagger (1975). This was successful for the shorter wavelengths only where irradiation times are short but was not successful for the longer wavelengths.* Thus, in order to maintain a consistent correction throughout the action spectrum we used the Morowitz corrections obtained from our absorbance measurements for all the wavelengths (Table 1). Chromatography. Samples containing ‘‘C-labelled and carrier leu were chromatographed on 20 cm Whatman no. 1 paper strips using two solvent systems (ethanol:NH,: water, 80:4:16 and butano1:acetic acid:water, 12:5:3). Action spectra. The relative efficiency per quantum was calculated using the method described by Jagger (1967), using the corrected fluence at which 10% of the original activity remained, derived from full inactivation curves.
RESULTS
In our normal assay procedure, 2.5 x lo8 cells ( 5 0 p / of a suspension containing 5.0 x lo9 cells m/ - *) accumulate u p t o 16000 cpm of I4C-leu in 30 s. Under identical conditions with the addition of 10 m M sodium azide, approximately 200-500 cpm were associated with the cells. Thus about 97% of the I4C-leu associated with the cells represented energy dependent uptake, and about 3% may have been found, without uptake, outside the cells. Figure 1 shows the effects of two U V wavelengths upon the ability of E. coli to retain radioactivity after loading the cells with 14C-leu under standard conditions. Chloramphenical was present during loading and cells were washed with buffer immediately prior to irradiation. Both wavelengths induce leakage (open circles) and the radioactivity released co-chromatographed with control 14C-leu, using the two solvent systems as described in Materials and Methods. Thus the leaked radioactivity represents leu and 80% of the accumulated material was not metabolized during irradiation since i t was recovered unchanged from the cell supernate (Fig. 1). Figure 1 shows the effects of 365 and 290nm light upon the ability of E. coli t o retain leu. Also, in parallel experiments, the kinetics of the inactivation of the cells’ leu specific uptake system are shown. Leucine uptake is rapidly inhibited by both 365 and 290 n m radiation, whereas in control experiments not shown by Fig. I. unirradiated cells have stable leu
467
Photoinactivation of leucine transport
50
T
-
w
z
u 3
-J W
0
z-
$ W K
4
365 nrn
2
Figure 1. Cells were loaded with radioactive leucine under standard assay conditions, washed in buffer, diluted to 5 x to9 cells/m/ and then exposed to the radiation. Parallel aliquots of control (unirradiated) and irradiated cells were filtered, washed and counted as described in Materials and Methods. In counterflow experiments, started at the time shown by arrows, aliquots of both control and irradiated cells were exposed to 0.6 mM of unlabelled L-leu in the dark. After 5 min exposure to 0.6 mM leu the cells were filtered, washed and counted as before. The fluence was not corrected for cell shielding and scattering. Control, unirradiated cells, loss of leu, M; irradiated cells loss of leu, o--
