APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1976, p. 146-149
Copyright C 1976 American Society for Microbiology
Vol. 31, No. 1
Printed in U.S.A.
Rickettsial Cell Water and Membrane Permeability Determined by a Micro Space Technique HERBERT H. WINKLER
Department of Microbiology, University of Virginia Medical School, Charlottesville, Virginia 22901 Received for publication 14 October 1975
A micro space technique for determining membrane permeability in Rickettsia prowazeki is described and justified. The cell water, cell wall plus periplasmic volume, and glutamate, ethylene glycol, and adenosine diphosphate permeabilities were determined by this method. The effect of nonionic detergents on rickettsial permeability was examined: Triton X-100 destroyed the permeability barrier, whereas Lubrol-WX left it intact.
The assessment of membrane permeability by "space" measurements has a long tradition in both animal and microbial cell physiology (1, 3, 6, 7, 10, 11). In such measurements one determines the volume, or space, occupied by an isotopically labeled test substance relative to the total water in the system. A large impermeant molecule is accessible to only the extracellular space, a permeant but not accumulated substance will have the same space as the total water, and an accumulated (or metabolized molecule) will have a space which appears greater than the total water in the system since the molecule is concentrated within the cell. Investigations of active transport in single-cell systems, such as bacteria, have often employed more rapid and manipulatable techniques such as filtration of the cells on microporous filters followed by a quick wash to remove extracellular material. The isotope remaining associated with the filter is assumed to be that which entered the cell. However, studies on nonaccumulating systems and passive permeability have made extensive use of the space technique (1, 3, 6, 7, 10, 11). In addition, sucrose and inulin space measurements in both gram-positive and gram-negative bacteria have been used to determine cell water (4, 12, 13, 17, 18). Myers et al. have used similar techniques with Rickettsia mooseri to determine periplasmic volume changes after procedures that may lead to plasmolysis (8). The space measurement should have a very important role in studies of permeability in rickettsiae. These obligatory intracellular parasites have a membrane that is labile once isolated from the host cytoplasm and which has often been assumed to be "leaky," i.e., to have a greatly increased passive permeability. The permeability properties of an organism which
normally has cytoplasm on both sides of the cell membrane should be very interesting and may supply important clues concerning the nature of its parasitism. If one were to attempt to measure the uptake of material by membrane filtration in such cells, one could introduce a serious artifact by leaching the substrate out of the cell during the wash procedure. For example, a substance to which the cell is freely permeable could appear to be impermeant due to this artifact. The space method does not have this objection, since no wash is involved. Another limitation of work on rickettsial permeability is the amount of material available. In our hands, using the yolk sac for cultivation of rickettsia, we typically obtain in the rickettsial fraction 1 mg of protein per gram (wet weight) of yolk sac (15). Hence, economic considerations demand that space measurements be done on the smallest amount of material suitable for consistent results. The most serious caveat in working with rickettsiae is that biologically active rickettsial preparations are not pure rickettsiae but are contaminated with host cell fragments and organelles. This makes absolute values for rickettsiae unobtainable, and meaningful experiments must be done with controls from the same preparation. There are disadvantages of the space measurements. For example, binding of the test substance to the surface of the cells is a problem when very dense suspensions are used in which the receptor capacity of the cell surface becomes large related to the amount of test substance in the suspension. Furthermore, very short incubation times are not possible and one must work with dense suspensions where oxygen and energy sources can become exhausted. Other limitations of the space technique, while germane to sophisticated studies of the hydrody-
146
VOL. 31, 1976
namic properties of membranes, are not serious drawbacks in the simple studies described. Thus, with this technique a permeant substance will appear permeant and an impermeant substance impermeant, although an actively transported species may not be accumulated to the maximal gradient. Once having obtained this basic information one may progress to other techniques to study those compounds that are of interest, especially those substrates that are accumulated. In this study I describe a micro space method in which more than 20 space determinations can be done on the rickettsiae derived from 10 yolk sacs. This method will be justified by comparison of values for Escherichia coli obtained herein with those in the literature obtained by macro space techniques. A measure of the cell water in preparations of R. prowazeki, an examination of the permeability of cells to some representative compounds, and the effects of two nonionic detergents upon rickettsial permeability will also be presented. To determine the excluded volume of any compound by the micro space technique, rickettsiae were resuspended in the sucrose-phosphate-glutamate buffer of Bovarnick et al. (2) to approximately 10 mg of protein/ml and incubated in a tube (6 by 10 mm) with a pair of radiolabeled space markers, for example, 3Hlabeled water and '4C-labeled sucrose. Substances labeled with 14C were present at 1 ,uCi/ ml, and 3H-labeled compounds were present at 8 uCi/ml; chemical concentrations are indicated in the tables. Three 30-,il disposable volumetric capillary tubes (Drummond) were filled to 25 ,Iu with the suspension, and the end not containing cells was sealed with a pinpoint flame from a propane torch. (Experiments using 10-lI tubes filled with 8 ,ul have also been successful.) After incubation for 10 min (similar values are obtained after a 30-min incubation) the tubes were centrifuged for 5 min in a microhematocrit centrifuge (15,000 x g). Longer centrifugations result in a more tightly packed pellet; however, since the extracellular volume is a measured quantity, the 5-min centrifugation schedule was found more convenient and just as precise. The outside of the tubes were rinsed three times in distilled water and once in ethanol, and the total volume and pellet volume were measured to the nearest 0.1 mm with a ruler under a magnifying glass. Packed cells usually occupied 5% of the total volume. Each tube was scored and broken off immediately above the packed cell pellet. The extracellular volume contributed by the small layer of fluid on top of the cell pellet is a known quantity and does not
NOTES
147
interfere with the permeability measurements since it contains both 3H-labeled water and the other marker, and it is the difference between these quantities that is of interest. All operations were carried out at room temperature (22 C). At these temperatures and times the biological activity of the rickettsiae was not decreased by these procedures since the cells from the pellet, when suspended with erythrocytes, were able to lyse sheep erythrocytes (9) as well as uncentrifuged material. The portion of the tube containing the cells was placed in a Minivial (Nuclear Associates) containing 0.3 ml of formic acid. Two 5-,ul portions of the incubated suspension and a 5-,pd portion of the supernatant fluid from each tube were also placed in vials with formic acid. The Minivials were capped and incubated overnight at room temperature. Aquasol (5 ml) (New England Nuclear Corp.) was then added to the vials, and they were counted by liquid scintillation spectrometry. When 14C-labeled inulin was used, the protocol was modified in two ways: the inulin solution was centrifuged at 10,000 x g for 10 min to remove any particulate material and water (1.5 ml) was added to the Minivial with the Aquasol to make a gel. The volume (microliters) accessible to each marker in the pellet was calculated from the counts per minute in the pellet divided by the counts per minute/microliter in the supernatant fluid (which was essentially the same as the counts per minute/microliter in the incubated suspension). The excluded volume of any substance was calculated by subtracting the volume of the substance from the total aqueous volume as measured with 3H-labeled water. The values were then normalized to the pellet volume (cubic millimeters). A pellet volume of 1 mm3 (1 ,ul) contains about 0.2 mg of protein measured by the procedure of Lowry et al. (5) with bovine albumin as standard; the portion of this total protein which represents rickettsial protein (usually about 75%) is, of course, dependent on the purity of the preparation. A comparison of micro and macro space determinations is shown in Table 1, which gives the results of four exeriments (each in triplicate) in which the cell water, or inulin-excluded volume, was determined by the micro space techniques in E. coli under the same conditions used subsequently for R. prowazeki. A value of 0.60 ,ul per pellet volume of 1 mm3 or 2.3 + 0.2 ,ul per mg (dry weight) was determined using the micro space method. This value is in excellent agreement with the values (2.7 + 0.6 Mul per mg [dry weight]) obtained by macro space measurements in which the weight of the cell
148
APPL. ENVIRON. MICROBIOL.
NOTES
water was determined from the wet weight of the cells minus the dry weight as reported by four laboratories in the literature (12, 13, 17, 18). The permeability of rickettsiae to selected compounds by micro space measurements was determined and is shown in Table 2. Freshly isolated rickettsiae have an excluded volume with inulin of 0.31 ,ul per pellet volume of 1 mm3. Inulin is a polysaccharide with an average molecular weight of 5,000; the cell wall can be assumed to be impermeable to such a compound. Ethylene glycol, on the other hand, is TABLE 1. Comparison of micro and macro estimates of E. coli cell watera Inulin-excluded vol Pellet vol Dry wt (p1/mm3)5 (jllmg)
Expt
Micro 1 Micro 2 Micro 3 Micro 4 Macro 1 Macro 2 Macro 3 Macro 4
0.67 0.56 0.63 0.53
+ 0.02 ± 0.08 ± 0.03 ± 0.03
2.0c 2.4c
2.5e 2.4c 2.7 2.2 3.5 2.3c
(17)d (18) (12) (13)
a Strain DF2000 was grown in casein hydrolysate to exponential stage as previously described (14) for the micro experiments, and cells were grown as described in the references for the macro experiments. b Values are expressed as the mean and standard deviation for three 30-gl capillary tube measurements. c Calculated using the relationship: 1 ml of culture at a turbidity of 100 Klett units = 222 iLg (dry weight). d Reference number.
TABLE 2. Excluded volumes of R. prowazekia Test substance Inulin (1 mg/ml) Sucrose (0.22 M) Sucrose (0.22 M) Sucrose (0.22 M) Ethylene glycol (2 mM) Glutamate (5 mM) ADPc (25 MM) ADP (25 jM)
Treatment None None Lubrol-WX
Excluded vol5
(Elud/M3)
0.31 + 0.24 ± 0.20 + Triton-X100 0.02 + None 0.06 + None -0.41 + None -6.68 ± Lubrol-WX -5.32±
0.06 (4) 0.08 (25) 0.07 (13) 0.05 (8) 0.01 (4) 0.04 (3) 2.97 (3) 2.84 (4)
a Rickettsiae Madrid E strain were prepared from infected yolk sacs by differential centrifugation and Celite, albumin, and Mg+ adsorption as previously described (15). b Excluded volume = [(Al of pellet 3H-labeled water) (MlI of pellet "4C-labeled test substance)]/(pellet volume [mm3]). Values are expressed as the mean of the determinations plus or minus one standard deviation, with the number of experiments in parentheses. c ADP, Adenosine diphosphate.
very permeant in rickettsiae, as it is in many cell types, since it occupies almost the same volume as water in the cell pellet. Sucrose, which is commonly used as the osmotic stabilizer of rickettsiae and, hence, the cell membrane must be essentially impermeable to this molecule, has an excluded volume of 0.24 ,ull mm3. This value is less than that for inulin, indicating that the cell wall is permeable to sucrose and that sucrose has access to the periplasmic space between the wall (or outer membrane) and cell membrane as well as to the cell wall volume itself. The sucrose-impermeable space is thus the cytoplasmic cell water of the organism. It is this sucrose-excluded volume to which all tests of permeability are made relative. If a neutral compound has an excluded volume greater than or equal to sucrose it is not permeant to the cell membrane, whereas if the value is less than sucrose the compound is permeant. Glutamate is known to be metabolized (and perhaps is accumulated) by rickettsiae and, hence, has an excluded volume less than zero. Adenosine diphosphate is accumulated to a large extent and has a large negative excluded volume. The effects of nonionic detergents on rickettsial permeability were investigated. Rickettsiae in sucrose-phosphate-glutamate buffer were treated with the nonionic detergents Lubrol-WX or Triton X-100 for 1 h at 0 C at a protein concentration of 0.6 mg/ml and a detergent concentration of 0.1%, centrifuged at 10,000 x g for 10 min, washed, and resuspended in sucrose-phosphate-glutamate buffer. Triton X-100 destroys the permeability barrier of the rickettsiae since sucrose becomes totally permeant (Table 2). Rickettsaie treated with Triton X-100 also lose all biological activity, as indicated by their inability to lyse sheep erythrocytes. These effects of Triton X-100 are in accord with its wide usage as an agent to make intracellular enzymes accessible to normally inaccessible substrates. On the other hand, Lubrol-WX at this concentration (0.1%) leaves both the permeability of rickettsiae and its biological activity, as measured by hemolysis and the ability to accumulate adenosine diphosphate, intact. Lubrol-WX has been used to form biologically active vesicular preparations of mitochondria (16), and the same concentration of detergent was useful in removing contaminating mitochondria from rickettsial preparations. This work was supported by Public Health Service research grant AI-10164 from the National Institute of Allergy and Infectious Diseases and research career development award 5-KO4-GM-13737 from the National Institute of General Medical Sciences. I thank Beth Miller for her expert technical assistance.
VOL. 31, 1976 LITERATURE CITED 1. Black, S. H., and P. Gerhardt. 1961. Permeability of bacterial spores. I. Characterization of glucose uptake. J. Bacteriol. 82:743-749. 2. Bovarnick, M. R., J. C. Miller, and J. C. Snyder. 1950. The influence of certain salts, amino acids, sugars, and proteins on the stability of rickettsiae. J. Bacteriol. 59:509-522. 3. Conway, E. J., and M. Downey. 1950. An outer metabolic region of the yeast cell. Biochem. J. 47:347-355. 4. Harold, F. M., E. Pavlasova, and J. R. Baarda. 1970. A transmembrane pH gradient in Streptococcus faecalis: origin, and dissipation by proton conductors and N,N'-dicyclohexylcarbodiimide. Biochim. Biophys. Acta 196:235-244. 5. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 6. MacDonald, R. E., and P. Gerhardt. 1958. Bacterial permeability: the uptake and oxidation of citrate by Escherichia coli. Can. J. Microbiol. 4:109-124. 7. Mitchell, P. 1959. Biochemical cytology of microorganisms. Annu. Rev. Microbiol. 13:407-440. 8. Myers, W. F., P. J. Provost, and C. L. Wisseman, Jr. 1967. Permeability properties of Rickettsia mooseri. J. Bacteriol. 93:950-960. 9. Ramm, L. E., and H. H. Winkler. 1973. Rickettsial hemolysis: adsorption of rickettsiae to erythrocytes. Infect. Immun. 7:93-99.
NOTES
149
10. Scherrer, R., and P. Gerhardt. 1964. Molecular sieving by cell membranes of Bacillus megaterium. Nature (London) 204:649-650. 11. Scherrer, R., and P. Gerhardt. 1971. Molecular sieving by the Bacillus megaterium cell wall and protoplast. J. Bacteriol. 107:718-735. 12. Schultz, S. G., and A. K. Solomon. 1961. Cation transport in E. coli. I. Intracellular Na and K concentrations and net cation movement. J. Gen. Physiol. 45: 355-369. 13. Winkler, H. H. 1971. Efflux and steady state in afmethylglucoside transport in Escherichia coli. J. Bacteriol. 106:362-368. 14. Winkler, H. H. 1973. Energy-coupling of the hexose phosphate transport system in Escherichia coli. J. Bacteriol. 116:203-209. 15. Winkler, H. H. 1974. Inhibitory and restorative effects of adenine nucleotides on rickettsial adsorption and hemolysis. Infect. Immun. 9:119-126. 16. Winkler, H. H., and A. L. Lehninger. 1968. The atractyloside-sensitive nucleotide binding site in a membrane preparation from rat liver mitochondria. J. Biol. Chem. 243:3000-3008. 17. Winkler, H. H., and T. H. Wilson. 1966. The role of energy coupling in the transport of p-galactosides by E. coli. J. Biol. Chem. 241:2200-2211. 18. Zwaig, N., W. S. Kistler, and E. C. C. Lin. 1970. Glycerol kinase, the pacemaker for the dissimilation of glycerol inEscherichia coli. J. Bacteriol. 102:753-759.
Copyright C 1976 American Society for Microbiology
Vol. 31, No. 1
Printed in U.S.A.
Rickettsial Cell Water and Membrane Permeability Determined by a Micro Space Technique HERBERT H. WINKLER
Department of Microbiology, University of Virginia Medical School, Charlottesville, Virginia 22901 Received for publication 14 October 1975
A micro space technique for determining membrane permeability in Rickettsia prowazeki is described and justified. The cell water, cell wall plus periplasmic volume, and glutamate, ethylene glycol, and adenosine diphosphate permeabilities were determined by this method. The effect of nonionic detergents on rickettsial permeability was examined: Triton X-100 destroyed the permeability barrier, whereas Lubrol-WX left it intact.
The assessment of membrane permeability by "space" measurements has a long tradition in both animal and microbial cell physiology (1, 3, 6, 7, 10, 11). In such measurements one determines the volume, or space, occupied by an isotopically labeled test substance relative to the total water in the system. A large impermeant molecule is accessible to only the extracellular space, a permeant but not accumulated substance will have the same space as the total water, and an accumulated (or metabolized molecule) will have a space which appears greater than the total water in the system since the molecule is concentrated within the cell. Investigations of active transport in single-cell systems, such as bacteria, have often employed more rapid and manipulatable techniques such as filtration of the cells on microporous filters followed by a quick wash to remove extracellular material. The isotope remaining associated with the filter is assumed to be that which entered the cell. However, studies on nonaccumulating systems and passive permeability have made extensive use of the space technique (1, 3, 6, 7, 10, 11). In addition, sucrose and inulin space measurements in both gram-positive and gram-negative bacteria have been used to determine cell water (4, 12, 13, 17, 18). Myers et al. have used similar techniques with Rickettsia mooseri to determine periplasmic volume changes after procedures that may lead to plasmolysis (8). The space measurement should have a very important role in studies of permeability in rickettsiae. These obligatory intracellular parasites have a membrane that is labile once isolated from the host cytoplasm and which has often been assumed to be "leaky," i.e., to have a greatly increased passive permeability. The permeability properties of an organism which
normally has cytoplasm on both sides of the cell membrane should be very interesting and may supply important clues concerning the nature of its parasitism. If one were to attempt to measure the uptake of material by membrane filtration in such cells, one could introduce a serious artifact by leaching the substrate out of the cell during the wash procedure. For example, a substance to which the cell is freely permeable could appear to be impermeant due to this artifact. The space method does not have this objection, since no wash is involved. Another limitation of work on rickettsial permeability is the amount of material available. In our hands, using the yolk sac for cultivation of rickettsia, we typically obtain in the rickettsial fraction 1 mg of protein per gram (wet weight) of yolk sac (15). Hence, economic considerations demand that space measurements be done on the smallest amount of material suitable for consistent results. The most serious caveat in working with rickettsiae is that biologically active rickettsial preparations are not pure rickettsiae but are contaminated with host cell fragments and organelles. This makes absolute values for rickettsiae unobtainable, and meaningful experiments must be done with controls from the same preparation. There are disadvantages of the space measurements. For example, binding of the test substance to the surface of the cells is a problem when very dense suspensions are used in which the receptor capacity of the cell surface becomes large related to the amount of test substance in the suspension. Furthermore, very short incubation times are not possible and one must work with dense suspensions where oxygen and energy sources can become exhausted. Other limitations of the space technique, while germane to sophisticated studies of the hydrody-
146
VOL. 31, 1976
namic properties of membranes, are not serious drawbacks in the simple studies described. Thus, with this technique a permeant substance will appear permeant and an impermeant substance impermeant, although an actively transported species may not be accumulated to the maximal gradient. Once having obtained this basic information one may progress to other techniques to study those compounds that are of interest, especially those substrates that are accumulated. In this study I describe a micro space method in which more than 20 space determinations can be done on the rickettsiae derived from 10 yolk sacs. This method will be justified by comparison of values for Escherichia coli obtained herein with those in the literature obtained by macro space techniques. A measure of the cell water in preparations of R. prowazeki, an examination of the permeability of cells to some representative compounds, and the effects of two nonionic detergents upon rickettsial permeability will also be presented. To determine the excluded volume of any compound by the micro space technique, rickettsiae were resuspended in the sucrose-phosphate-glutamate buffer of Bovarnick et al. (2) to approximately 10 mg of protein/ml and incubated in a tube (6 by 10 mm) with a pair of radiolabeled space markers, for example, 3Hlabeled water and '4C-labeled sucrose. Substances labeled with 14C were present at 1 ,uCi/ ml, and 3H-labeled compounds were present at 8 uCi/ml; chemical concentrations are indicated in the tables. Three 30-,il disposable volumetric capillary tubes (Drummond) were filled to 25 ,Iu with the suspension, and the end not containing cells was sealed with a pinpoint flame from a propane torch. (Experiments using 10-lI tubes filled with 8 ,ul have also been successful.) After incubation for 10 min (similar values are obtained after a 30-min incubation) the tubes were centrifuged for 5 min in a microhematocrit centrifuge (15,000 x g). Longer centrifugations result in a more tightly packed pellet; however, since the extracellular volume is a measured quantity, the 5-min centrifugation schedule was found more convenient and just as precise. The outside of the tubes were rinsed three times in distilled water and once in ethanol, and the total volume and pellet volume were measured to the nearest 0.1 mm with a ruler under a magnifying glass. Packed cells usually occupied 5% of the total volume. Each tube was scored and broken off immediately above the packed cell pellet. The extracellular volume contributed by the small layer of fluid on top of the cell pellet is a known quantity and does not
NOTES
147
interfere with the permeability measurements since it contains both 3H-labeled water and the other marker, and it is the difference between these quantities that is of interest. All operations were carried out at room temperature (22 C). At these temperatures and times the biological activity of the rickettsiae was not decreased by these procedures since the cells from the pellet, when suspended with erythrocytes, were able to lyse sheep erythrocytes (9) as well as uncentrifuged material. The portion of the tube containing the cells was placed in a Minivial (Nuclear Associates) containing 0.3 ml of formic acid. Two 5-,ul portions of the incubated suspension and a 5-,pd portion of the supernatant fluid from each tube were also placed in vials with formic acid. The Minivials were capped and incubated overnight at room temperature. Aquasol (5 ml) (New England Nuclear Corp.) was then added to the vials, and they were counted by liquid scintillation spectrometry. When 14C-labeled inulin was used, the protocol was modified in two ways: the inulin solution was centrifuged at 10,000 x g for 10 min to remove any particulate material and water (1.5 ml) was added to the Minivial with the Aquasol to make a gel. The volume (microliters) accessible to each marker in the pellet was calculated from the counts per minute in the pellet divided by the counts per minute/microliter in the supernatant fluid (which was essentially the same as the counts per minute/microliter in the incubated suspension). The excluded volume of any substance was calculated by subtracting the volume of the substance from the total aqueous volume as measured with 3H-labeled water. The values were then normalized to the pellet volume (cubic millimeters). A pellet volume of 1 mm3 (1 ,ul) contains about 0.2 mg of protein measured by the procedure of Lowry et al. (5) with bovine albumin as standard; the portion of this total protein which represents rickettsial protein (usually about 75%) is, of course, dependent on the purity of the preparation. A comparison of micro and macro space determinations is shown in Table 1, which gives the results of four exeriments (each in triplicate) in which the cell water, or inulin-excluded volume, was determined by the micro space techniques in E. coli under the same conditions used subsequently for R. prowazeki. A value of 0.60 ,ul per pellet volume of 1 mm3 or 2.3 + 0.2 ,ul per mg (dry weight) was determined using the micro space method. This value is in excellent agreement with the values (2.7 + 0.6 Mul per mg [dry weight]) obtained by macro space measurements in which the weight of the cell
148
APPL. ENVIRON. MICROBIOL.
NOTES
water was determined from the wet weight of the cells minus the dry weight as reported by four laboratories in the literature (12, 13, 17, 18). The permeability of rickettsiae to selected compounds by micro space measurements was determined and is shown in Table 2. Freshly isolated rickettsiae have an excluded volume with inulin of 0.31 ,ul per pellet volume of 1 mm3. Inulin is a polysaccharide with an average molecular weight of 5,000; the cell wall can be assumed to be impermeable to such a compound. Ethylene glycol, on the other hand, is TABLE 1. Comparison of micro and macro estimates of E. coli cell watera Inulin-excluded vol Pellet vol Dry wt (p1/mm3)5 (jllmg)
Expt
Micro 1 Micro 2 Micro 3 Micro 4 Macro 1 Macro 2 Macro 3 Macro 4
0.67 0.56 0.63 0.53
+ 0.02 ± 0.08 ± 0.03 ± 0.03
2.0c 2.4c
2.5e 2.4c 2.7 2.2 3.5 2.3c
(17)d (18) (12) (13)
a Strain DF2000 was grown in casein hydrolysate to exponential stage as previously described (14) for the micro experiments, and cells were grown as described in the references for the macro experiments. b Values are expressed as the mean and standard deviation for three 30-gl capillary tube measurements. c Calculated using the relationship: 1 ml of culture at a turbidity of 100 Klett units = 222 iLg (dry weight). d Reference number.
TABLE 2. Excluded volumes of R. prowazekia Test substance Inulin (1 mg/ml) Sucrose (0.22 M) Sucrose (0.22 M) Sucrose (0.22 M) Ethylene glycol (2 mM) Glutamate (5 mM) ADPc (25 MM) ADP (25 jM)
Treatment None None Lubrol-WX
Excluded vol5
(Elud/M3)
0.31 + 0.24 ± 0.20 + Triton-X100 0.02 + None 0.06 + None -0.41 + None -6.68 ± Lubrol-WX -5.32±
0.06 (4) 0.08 (25) 0.07 (13) 0.05 (8) 0.01 (4) 0.04 (3) 2.97 (3) 2.84 (4)
a Rickettsiae Madrid E strain were prepared from infected yolk sacs by differential centrifugation and Celite, albumin, and Mg+ adsorption as previously described (15). b Excluded volume = [(Al of pellet 3H-labeled water) (MlI of pellet "4C-labeled test substance)]/(pellet volume [mm3]). Values are expressed as the mean of the determinations plus or minus one standard deviation, with the number of experiments in parentheses. c ADP, Adenosine diphosphate.
very permeant in rickettsiae, as it is in many cell types, since it occupies almost the same volume as water in the cell pellet. Sucrose, which is commonly used as the osmotic stabilizer of rickettsiae and, hence, the cell membrane must be essentially impermeable to this molecule, has an excluded volume of 0.24 ,ull mm3. This value is less than that for inulin, indicating that the cell wall is permeable to sucrose and that sucrose has access to the periplasmic space between the wall (or outer membrane) and cell membrane as well as to the cell wall volume itself. The sucrose-impermeable space is thus the cytoplasmic cell water of the organism. It is this sucrose-excluded volume to which all tests of permeability are made relative. If a neutral compound has an excluded volume greater than or equal to sucrose it is not permeant to the cell membrane, whereas if the value is less than sucrose the compound is permeant. Glutamate is known to be metabolized (and perhaps is accumulated) by rickettsiae and, hence, has an excluded volume less than zero. Adenosine diphosphate is accumulated to a large extent and has a large negative excluded volume. The effects of nonionic detergents on rickettsial permeability were investigated. Rickettsiae in sucrose-phosphate-glutamate buffer were treated with the nonionic detergents Lubrol-WX or Triton X-100 for 1 h at 0 C at a protein concentration of 0.6 mg/ml and a detergent concentration of 0.1%, centrifuged at 10,000 x g for 10 min, washed, and resuspended in sucrose-phosphate-glutamate buffer. Triton X-100 destroys the permeability barrier of the rickettsiae since sucrose becomes totally permeant (Table 2). Rickettsaie treated with Triton X-100 also lose all biological activity, as indicated by their inability to lyse sheep erythrocytes. These effects of Triton X-100 are in accord with its wide usage as an agent to make intracellular enzymes accessible to normally inaccessible substrates. On the other hand, Lubrol-WX at this concentration (0.1%) leaves both the permeability of rickettsiae and its biological activity, as measured by hemolysis and the ability to accumulate adenosine diphosphate, intact. Lubrol-WX has been used to form biologically active vesicular preparations of mitochondria (16), and the same concentration of detergent was useful in removing contaminating mitochondria from rickettsial preparations. This work was supported by Public Health Service research grant AI-10164 from the National Institute of Allergy and Infectious Diseases and research career development award 5-KO4-GM-13737 from the National Institute of General Medical Sciences. I thank Beth Miller for her expert technical assistance.
VOL. 31, 1976 LITERATURE CITED 1. Black, S. H., and P. Gerhardt. 1961. Permeability of bacterial spores. I. Characterization of glucose uptake. J. Bacteriol. 82:743-749. 2. Bovarnick, M. R., J. C. Miller, and J. C. Snyder. 1950. The influence of certain salts, amino acids, sugars, and proteins on the stability of rickettsiae. J. Bacteriol. 59:509-522. 3. Conway, E. J., and M. Downey. 1950. An outer metabolic region of the yeast cell. Biochem. J. 47:347-355. 4. Harold, F. M., E. Pavlasova, and J. R. Baarda. 1970. A transmembrane pH gradient in Streptococcus faecalis: origin, and dissipation by proton conductors and N,N'-dicyclohexylcarbodiimide. Biochim. Biophys. Acta 196:235-244. 5. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 6. MacDonald, R. E., and P. Gerhardt. 1958. Bacterial permeability: the uptake and oxidation of citrate by Escherichia coli. Can. J. Microbiol. 4:109-124. 7. Mitchell, P. 1959. Biochemical cytology of microorganisms. Annu. Rev. Microbiol. 13:407-440. 8. Myers, W. F., P. J. Provost, and C. L. Wisseman, Jr. 1967. Permeability properties of Rickettsia mooseri. J. Bacteriol. 93:950-960. 9. Ramm, L. E., and H. H. Winkler. 1973. Rickettsial hemolysis: adsorption of rickettsiae to erythrocytes. Infect. Immun. 7:93-99.
NOTES
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