Vol. 121, No. 3 Printed in U.S.A.
JOURNAL OF BACrERIOLOGY, Mar. 1975, p. 1158-1165 Copyright © 1975 American Society for Microbiology
Energy Efficiency of Intraperiplasmic Growth of Bdellovibrio bacteriovorus SYDNEY C. RITTENBERG* AND ROBERT B. HESPELL' Department of Bacteriology, University of California, Los Angeles, California 90024 Received for publication 15 October 1974
The YATP (energy efficiency) of intraperiplasmic growth of Bdellovibrio bacteriovorus was determined from the distribution of radioactivity of the substrate organism ([U- 14C ]Escherichia coli) between CO2 and bdellovibrio cells at the end of growth. A "best" YATP value of 18.5 was obtained from single growth cycle experiments and an average value of 25.9 from multicycle experiments. Both values are much higher than the usual value of 10.5 for bacteria growing in rich media. The bases for the unusual energy efficiency for growth of B. bacteriovorus are discussed.
Accumulated information indicates that the normal growth environment for Bdellovibrio bacteriovorus is the intraperiplasmic space of an appropriate substrate bacterium (24, 26). Growing in this environment, the bdellovibrio has available a potential source of all major monomeric subunits necessary for the synthesis of its cellular macromolecules. We have shown that exogenous substrates are used only to a minor extent by the bdellovibrio during intraperiplasmic growth (8), and that there is an efficient utilization of deoxyribonucleic acid (DNA) (13) and ribonucleic acid (RNA) (unpublished data) precursors preexisting in the substrate organism for biosynthesis of the homologous polymer by the bdellovibrio. These data make it likely that little biosynthesis of monomers occurs during intraperiplasmic growth. This conclusion was strengthened by the demonstration that this mode of growth is insensitive to the inhibitor methotrexate, whereas axenic growth of the bdellovibrio is strongly inhibited by this compound (16). We have also demonstrated that the bdellovibrio conserves phosphate bond energy by using nucleoside monophosphates and probably glycerol phosphate (17) per se from the substrate organism as precursors for nucleic acid and lipid synthesis. The bdellovibrio also conserves energy by incorporating intact fatty acid molecules from the same source (11). The above listed properties of B. bacteriovorus suggest that its intraperiplasmic development should occur with minimum energy expenditure. Preliminary experiments
briefly mentioned elsewhere (S. C. Rittenberg, Abstr. 1st Int. Congr. Microbiol. 1:108, 1973) indicated a YATP (grams [dry weight] of cell material formed per mole of adenosine triphosphate [ATP] [2]) for intraperiplasmic growth of between 20 and 30, values that approach the theoretical maximum (5, 6, 27). Results of more critical experiments that are reported here confirm this unusual efficiency of energy expendi-
I Present address: Department of Dairy Science, University of Illinois, Urbana, Ill. 61801.
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ture. MATERIALS AND METHODS Organisms and growth procedures. B. bacteriovorus 109J was the experimental organism, and Escherichia coli ML35 (lacI, lacY) served as its substrate. The bdellovibrios were grown on the E. coli growing in dilute nutrient broth (28). E. coli was grown at 37 C in glucose-salts medium (8). Overnight cultures were diluted to 108 to 1.5 x 108 cells/ml in fresh medium and incubated with shaking for 3 to 3.5 h to give a cell density of about 1.5 x 109/ml. To obtain uniformly labeled [U-14CE. coli, the glucose-salts medium was prepared with [U"4C]glucose (0.02 M, 0.2 to 1 MCi/ml). To obtain doubly labeled E. coli cells, the [U-_4C]glucose medium was supplemented with [methyl-3H]thymidine (10-5 M, 1 uCi/ml) and deoxyadenosine (10-3 M) (3, 13). Over 90'c of the tritium label in the resulting [U- 14Cj-[9H]thymidine-labeled E. coli was in its DNA. The dilute nutrient broth and the glucose cultures were harvested by centrifugation. Cell suspensions of both organisms were made in 5 x 10-3 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid containing MgCl2 and CaCl2 (HEPES buffer, pH 7.8) after two washes in the buffer. The salts were at 10-4 M in single-cycle experiments and 10W3 M in multicycle experiments. Cell numbers in the suspensions were determined from turbidity measurements by reference to standard curves based on plaque counts for B. bacteriovorus and colony counts for E. coli.
VOL. 121, 1975
YATP OF BDELLOVIBRIO INTRAPERIPLASMIC GROWTH
Growth experiments. The E. coli cells served as the exclusive source of all nutrients for the growth of B. bacteriovorus. The buffer suspensions of the two organisms were added (5 to 10 ml, total volume) to a 250-ml Erlenmeyer flask with a glass center well, and the flask was sealed with a rubber serum stopper. In single-cycle experiments an input ratio (B. bacteriovorus to E. coli cells) of 1.4 to 1.8:1 was generally used, and growth of the bdellovibrios was complete in 210 to 240 min. In multicycle experiments an input ratio of about 10-4:1 was used, and an incubation time of 14 to 18 h was required for complete lysis of the E. coli. Flasks were shaken at 30 C until lysis of the E. coli was essentially complete as determined by phase microscope observations of parallel cultures in unsealed flasks. Then the cultures were cooled to 4 C, 2.5 ml of M KOH and 0.2 ml of 2 N H2S04 were injected into the center well and culture fluid, respectively, with hypodermic syringes, and the flasks were shaken at 4 C for an additional 20 h before opening. Radioactivity in CO2 was determined on samples removed from the center well. Bdellovibrio cells were harvested from the cultures, washed twice in HEPES buffer by centrifugation (5 min, 4 C, 27,000 x g), and resuspended in HEPES buffer for determination of radioactivity. To obtain optimal development of the bdellovibrio cultures and reproducible results in the growth experiments, the following conditions had to be met. The E. coli were harvested from the glucose cultures during exponential phase and B. bacteriovorus were harvested from the dilute nutrient broth cultures as soon as lysis of E. coli cells was complete. Cell suspensions of both organisms were used within 3 h of preparation and were kept in ice until used. The final concentration of substrate cells was 3 x 109 to 5 x 109/ml. The bdellovibrios from the growth experiments were harvested immediately after culture development was complete to minimize "CO2 release from the oxidation of compounds in the E. coli debris or from endogenous respiration of free bdellovibrios (8). The last precaution was particularly crucial for obtaining reliable data for YATP calculations. Radioactivity. All radioactivity measurements were made by scintillation counting in PCS solubilizer scintillation fluid (Amersham-Searle, Arlington Heights, Ill.). Cell composition. Chemical analyses were made of whole cells for protein (12), DNA (4), RNA (21), and polysaccharides (1) by standard procedures. Washed cells were fractionated into their major constituents as described by Roberts et al. (19). The lipid contents were determined from the radioactivity of the ethanol ether extracts of U-'4C-labeled cells. The polysaccharide and peptidoglycan contents of E. coli ML35 are estimates based on general values for gram-negative organisms (14, 19, 29). The peptidoglycan content of B. bacteriovorus 109J was determined from the weight of isolated material. Purification of B. bacteriovorus on Ficoll gradients. One milliliter of washed cell suspension (1010 to 3 x 1010 cells) was layered onto 25 ml of a 5 to 20% linear Ficoll gradient (24) and centrifuged at
1159
27,000 x g for 5 min at 4 C. The visible band of bdellovibrio cells, 25 to 30 mm beneath the meniscus, was removed with a Pasteur pipette, diluted 10-fold in HEPES buffer, and washed three times in buffer by
centrifugation. RESULTS Experimental determination of YATP. YATP values were calculated (Table 1) from the distribution of radioactivity in respired CO2 and in the bdellovibrios after growth of B. bacteriovorus on [U- 14C ]E. coli as the exclusive source of all nutrients and from a previously determined respiratory quotient (RQ) of 1.05 for intraperiplasmic growth (8). The calculation makes only three assumptions: (i) cell carbon is 50% of cell dry weight (D, Table 1); (ii) the P/O ratio for B. bacteriovorus is 3 (E, Table 1); and (iii) substrate level phosphorylation is negligible during intraperiplasmic growth. These assumptions are considered in the discussion. Independent of the assumptions, for the YATP determinations to be valid two general conditions had to be achieved experimentally; (i) the harvested bdellovibrios at the end of growth had to be free of E. coli debris, or else the YATP values would be in error on the high side; (ii) the radioactive CO2 collected either had to be derived exclusively from the respiration of the bdellovibrios growing intraperiplasmically or had to be corrected for radioactive CO2 from other sources, or else the calculated YATP values would be spuriously low. There were only two possible additional sources of radioactive CO2 in these experiments: from the endogenous respiration of the [U- 14C ]E. coli before bdellovibrio attack, and from extraperiplasmic bdellovibrios oxidizing materials released from attacked or lysed E. coli. These two conditions dictated the experiments that follow. Purity of harvested bdellovibrios. B. bacteriovorus was grown on [U- 14C I- [2H ]thymidine-labeled E. coli as the substrate organism in single-cycle growth experiments. Progeny bdellovibrios were harvested and washed twice as described above and the distribution of the two radioactive tracers between the progeny cells, the buffer, and the CO2 was determined (Table 2). On the average, about 52% of the initial E. coli carbon and 59% of its thymidine radioactivity were present in the washed bdellovibrio suspensions. Portions of these suspensions were treated with deoxyribonuclease I (5 jg/ml, 20 min, 30 C), centrifuged, washed, and recounted. No significant loss in the quantity of 3H was ob-
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served. Thus, all harvested DNA was protected from enzyme attack (i.e., was intracellular). This result is consistent with the previous finding that during bdellovibrio attack the DNA of the substrate organism is rapidly and completely degraded (13). The data indicate that the tritium label is a reliable measure of bdellovibrio material in these experiments. The harvested bdellovibrios were then "purified" on Ficoll gradients as described in Materials and Methods. There was little change in the initial '4C/3H ratio after this treatment (Table 2). The changes measured, +1.3% to -2.9%, fell within the average counting error (+3%) and are not significant. It may be concluded that the harvested bdellovibrios are not contaminated with significant amounts of miscellaneous E. coli debris. The alternate possibility is that E. coli debris of constant composition and quantity is not separated from the bdellovibrios by the Ficoll procedure. Critical tests reported in another connection (11) make the second alternative highly unlikely.
YATP for single-cycle growth experiments. Table 3 shows the distribution of the radioactivity initially in the substrate organisms after single-cycle growth of B. bacteriovorus on [U"C ]E. coli. The YATP values were calculated from these data as shown in Table 1. The experimental design largely satisfied the condition that little of the "'CO, came from the endogenous respiration of the starting [UJ- "4C ]E. coli since at input ratios of B. bacteriovorus to E. coli of 1.4 to 1.8, E. coli respiration is eliminated within a very short time after addition of the bdellovibrios (18). However, a significant (and unknown) number of bdellovibrios in these experiments remain extracellular and do not grow. They do, nevertheless, oxidize material released from E. coli and liberate '4CO,. The YATP values should therefore be in error on the low side. To obtain some indication of the magnitude of this effect, experiments were done in which cultures were initiated with variable numbers of bdellovibrios and constant numbers of E. coli to give input ratios of 0.1 to 2.8. All cultures were incubated for 240 min, at which time biological activity TABLE 1. Determination of YATP for intraperiplasmic was terminated by addition of acid. Microscopic growth of B. bacteriovorus on [U-"C]E. coli from distribution of radioactivity between the bdellovibrios observations showed that by this time all E. coli in cultures with starting input ratios above 1.3 and CO, to 1.4 had been lysed, whereas E. coli remained 14C counts/min in cells Moles of carbon assimilated in cultures initiated with lower input ratios. A A. Moles of carbon respired 14C counts/min in COM plot of "4CO, release against initial input ratios Moles of carbon assimilated showed a sharp discontinuity in the results at A x RQ = A x 1 .05 B. Mole of 0, consumed an input ratio of about 1.2 (Fig. 1). The points below and above the transition approximate Grams of carbon assimilated C B Mole of 02 consumed two straight lines having slopes of 9.05 0.29 D Dry wt of cells formed C 2 and 1.01 0.23 (percentage of initial radioacMole of 0, consumed tivity/input ratio). Dry wt of cells formed E. YATP= =D-6 The of the experiment makes it likely design Moles of ATP produced that the greater slope is a measure of the effect =
12
TABLE 2. Distribution of radioactivity after growth of B. bacteriovorus 1O9Jon [U-'C]-['H]thymidine-labeled E. coli ML 35a Initial radioactivity (%) in:
Expt
Isotope
1
"4C
2
3H 14C 3H
3
"4C
4
3H 14C 3H
Cells
Buffer
CO2
53 57 54 54 50 66
32 48 38 48 32 38 34 40
16
51 59
16
15 16
Recovery (%)
101 105 108 102 97 104 101 99
aCultures initially contained 11 x 109 B. bacteriovorus and 6 x 109
"4C/3H in cells' Harvested
Purified
0.458
0.464
0.481
0.467
0.694
0.678
0.674
0.669
[U-_4C]-[3H]thymidine-labeled
E.
coli/ml ('H, 1.2 x 10' to 1.5 x 10' counts/min; 4C, 6 x 104 to 8 x 104 counts/min). Input ratio = 1.8. 'After lysis, the bdellovibrio cells were washed twice by centrifugation (harvested) and then banded on Ficoll gradients (purified).
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YATP OF BDELLOVIBRIO INTRAPERIPLASMIC GROWTH
TABLE 3. YATP for intraperiplasmic development determined from distribution of radioactivity after single-cycle growth of B. bacteriovorus on [U-'4CJE. colia Initial E. coli radioactivity (%) in:
(YAIl) B. bac-
ratio Determination Input (Bd/Ec)b
Buffer CO2
teriovorus
14 31 1.6 Avg ......... Range ......... 1.4-1.8 24-38 12-16 Standard deviation .5.3 + 1.7
56 49-64
17 14-20
±6.1
I2.8
coli in the bdellovibrio culture is 74% of that released by the same number of E. coli shaken in buffer for the same time. Applying this 0.75 x 2,260; Table 4), correction (10,600 the "4CO2 released by bdellovibrio respiration during growth was 7.8% of the initial, and the corrected YATP was 28.2. The above calculation assumes that the number of attacked, nonviable (and nonrespiring) E. coli is equal to the number of bdellovibrio 1614-
a Data are average of 11 experiments; the cultures initially contained between 5 x 10' and 8 x 109 B. bacteriovorus and 3 x 10' and 5 x 10' [U-4C JE. coli (70,000 to 550,000 counts/ min) per ml. ° Bd, B. bacteriovorns; Ec, E. coli.
of increasing numbers of growing intraperiplasmic bdellovibrios (i.e., E. coli excess), and the lesser slope is a measure of the effect of increasing numbers of nongrowing extracellular bdellovibrios, on CO2 release (i.e., bdellovibrio excess). If so, the intercept represents the balance point at which essentially all E. coli are attacked and few bdellovibrios are extracellular. The radioactivity of CO2 released from such a culture at the end of growth is about 12.5% of the initial (Fig. 1). The YATP of the balanced culture, which we believe gives the best estimate from single-cycle growth experiments, is about 18.5. The value is on the low side since some, probably small, amount of '4CO2 must come from E. coli endogenous respiration. YATP for multicycle growth experiments. Table 4 shows the results of a typical multicycle growth experiment in which the input ratio of B. bacteriovorus to [U-14CJE. coli was about 0.0027. In this particular experiment, some 52% of the starting E. coli carbon (radioactivity) was converted to bdellovibrio cell material and 9% was released as CO2. The uncorrected data, calculated as shown in Table 1, give a YATP Of 23.3 for the experiment. The experimental design satisfies the condition that little 14CO2 is derived from nongrowing bdellovibrios. However, because of the low input ratio, most E. coli cells remained unattacked until very late in culture development. Some 14CO2 must have been derived from the endogenous respiration of these E. coli and the YATP value is therefore low. An E. coli survival curve was calculated from the input ratio and a doubling time of 1.6 h for bdellovibrio plaque-forming units, a value typical of multicycle cultures grown under the conditions of this experiment (16). From the area under the curve, the 14C2O released by E.
S>
~
~
Slope - 0/
12
So
10
0
=9.05 >
6.
0 4-
r-5 2
0
.5
15
10
2.0
35
bacteriovorus. E.coli FIG. 1. Relation between CO, release and input ratio in single-cycle growth of B. bacteriovorus on [U-14CJE. coli. Initial cultures contained 4 x 109 [U-'4CJE. coli/ml (83,000 counts/min per ml) and B. bacteriovorus to give the indicated input ratios. All cultures were incubated for 240 min, at which time all E. coli had been lysed in cultures having an input ratio of about 1.3 or higher. INPUT
RATIO-B
TABLE 4. Distribution of radioactivity after multicycle growth of B. bacteriovorus on [U-14CIE. colia Component
Radioactivity (counts/min per ml) in: E. coli CO B. bacterio-
CO,
vorus
Culture .... 10,600 (9.3) 60,000 (52) 102,000 (89) Control .... 2,260 (2.0) a The initial culture contained 8 x 10' B. bacteriovorus and 3 x 109 [U- 14C ]E. coli (counts/min = 115,000/ml) and was incubated for 17 h until all E. coli had lysed. The control consisted of the same number of E. coli in buffer incubated for the same time. Values in parentheses are percentage of initial counts.
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plaque-forming units in the culture at any time until the total plaque-forming units essentially equals the input E. coli. Direct plating of samples of a multicycle culture on nutrient agar with time gave E. coli colony counts somewhat lower but approximating those calculated (Table 5). The differences can be accounted for by small differences in doubling time and/or input ratio and do not alter the correction appreciably. The calculation also assumes that the respiration of unattacked E. coli in the culture is neither stimulated nor inhibited by materials released from attacked and/or lysed E. coli or from the bdellovibrios. To check this point, the respiration rates of E. coli cells suspended in buffer and in the supernatant fluid obtained by centrifuging 10- and 18-h (complete lysis) multicycle cultures were measured. The values obtained for the three suspensions, 9.8, 8.5, and 7.3 ul of 0/min per 1010 cells, were similar and supported the assumpitions. Table 6 summarizes the results of 14 indeTABLE 5. Calculated and observed surviving E. coli in a multicycle B. bacteriovorus culturea Surviving E. coli (% initial) Time (h)
0 10 12 14 16
Calculated
Found
100
100
90 76 43 0
94 51 5
JOURNAL OF BACrERIOLOGY, Mar. 1975, p. 1158-1165 Copyright © 1975 American Society for Microbiology
Energy Efficiency of Intraperiplasmic Growth of Bdellovibrio bacteriovorus SYDNEY C. RITTENBERG* AND ROBERT B. HESPELL' Department of Bacteriology, University of California, Los Angeles, California 90024 Received for publication 15 October 1974
The YATP (energy efficiency) of intraperiplasmic growth of Bdellovibrio bacteriovorus was determined from the distribution of radioactivity of the substrate organism ([U- 14C ]Escherichia coli) between CO2 and bdellovibrio cells at the end of growth. A "best" YATP value of 18.5 was obtained from single growth cycle experiments and an average value of 25.9 from multicycle experiments. Both values are much higher than the usual value of 10.5 for bacteria growing in rich media. The bases for the unusual energy efficiency for growth of B. bacteriovorus are discussed.
Accumulated information indicates that the normal growth environment for Bdellovibrio bacteriovorus is the intraperiplasmic space of an appropriate substrate bacterium (24, 26). Growing in this environment, the bdellovibrio has available a potential source of all major monomeric subunits necessary for the synthesis of its cellular macromolecules. We have shown that exogenous substrates are used only to a minor extent by the bdellovibrio during intraperiplasmic growth (8), and that there is an efficient utilization of deoxyribonucleic acid (DNA) (13) and ribonucleic acid (RNA) (unpublished data) precursors preexisting in the substrate organism for biosynthesis of the homologous polymer by the bdellovibrio. These data make it likely that little biosynthesis of monomers occurs during intraperiplasmic growth. This conclusion was strengthened by the demonstration that this mode of growth is insensitive to the inhibitor methotrexate, whereas axenic growth of the bdellovibrio is strongly inhibited by this compound (16). We have also demonstrated that the bdellovibrio conserves phosphate bond energy by using nucleoside monophosphates and probably glycerol phosphate (17) per se from the substrate organism as precursors for nucleic acid and lipid synthesis. The bdellovibrio also conserves energy by incorporating intact fatty acid molecules from the same source (11). The above listed properties of B. bacteriovorus suggest that its intraperiplasmic development should occur with minimum energy expenditure. Preliminary experiments
briefly mentioned elsewhere (S. C. Rittenberg, Abstr. 1st Int. Congr. Microbiol. 1:108, 1973) indicated a YATP (grams [dry weight] of cell material formed per mole of adenosine triphosphate [ATP] [2]) for intraperiplasmic growth of between 20 and 30, values that approach the theoretical maximum (5, 6, 27). Results of more critical experiments that are reported here confirm this unusual efficiency of energy expendi-
I Present address: Department of Dairy Science, University of Illinois, Urbana, Ill. 61801.
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ture. MATERIALS AND METHODS Organisms and growth procedures. B. bacteriovorus 109J was the experimental organism, and Escherichia coli ML35 (lacI, lacY) served as its substrate. The bdellovibrios were grown on the E. coli growing in dilute nutrient broth (28). E. coli was grown at 37 C in glucose-salts medium (8). Overnight cultures were diluted to 108 to 1.5 x 108 cells/ml in fresh medium and incubated with shaking for 3 to 3.5 h to give a cell density of about 1.5 x 109/ml. To obtain uniformly labeled [U-14CE. coli, the glucose-salts medium was prepared with [U"4C]glucose (0.02 M, 0.2 to 1 MCi/ml). To obtain doubly labeled E. coli cells, the [U-_4C]glucose medium was supplemented with [methyl-3H]thymidine (10-5 M, 1 uCi/ml) and deoxyadenosine (10-3 M) (3, 13). Over 90'c of the tritium label in the resulting [U- 14Cj-[9H]thymidine-labeled E. coli was in its DNA. The dilute nutrient broth and the glucose cultures were harvested by centrifugation. Cell suspensions of both organisms were made in 5 x 10-3 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid containing MgCl2 and CaCl2 (HEPES buffer, pH 7.8) after two washes in the buffer. The salts were at 10-4 M in single-cycle experiments and 10W3 M in multicycle experiments. Cell numbers in the suspensions were determined from turbidity measurements by reference to standard curves based on plaque counts for B. bacteriovorus and colony counts for E. coli.
VOL. 121, 1975
YATP OF BDELLOVIBRIO INTRAPERIPLASMIC GROWTH
Growth experiments. The E. coli cells served as the exclusive source of all nutrients for the growth of B. bacteriovorus. The buffer suspensions of the two organisms were added (5 to 10 ml, total volume) to a 250-ml Erlenmeyer flask with a glass center well, and the flask was sealed with a rubber serum stopper. In single-cycle experiments an input ratio (B. bacteriovorus to E. coli cells) of 1.4 to 1.8:1 was generally used, and growth of the bdellovibrios was complete in 210 to 240 min. In multicycle experiments an input ratio of about 10-4:1 was used, and an incubation time of 14 to 18 h was required for complete lysis of the E. coli. Flasks were shaken at 30 C until lysis of the E. coli was essentially complete as determined by phase microscope observations of parallel cultures in unsealed flasks. Then the cultures were cooled to 4 C, 2.5 ml of M KOH and 0.2 ml of 2 N H2S04 were injected into the center well and culture fluid, respectively, with hypodermic syringes, and the flasks were shaken at 4 C for an additional 20 h before opening. Radioactivity in CO2 was determined on samples removed from the center well. Bdellovibrio cells were harvested from the cultures, washed twice in HEPES buffer by centrifugation (5 min, 4 C, 27,000 x g), and resuspended in HEPES buffer for determination of radioactivity. To obtain optimal development of the bdellovibrio cultures and reproducible results in the growth experiments, the following conditions had to be met. The E. coli were harvested from the glucose cultures during exponential phase and B. bacteriovorus were harvested from the dilute nutrient broth cultures as soon as lysis of E. coli cells was complete. Cell suspensions of both organisms were used within 3 h of preparation and were kept in ice until used. The final concentration of substrate cells was 3 x 109 to 5 x 109/ml. The bdellovibrios from the growth experiments were harvested immediately after culture development was complete to minimize "CO2 release from the oxidation of compounds in the E. coli debris or from endogenous respiration of free bdellovibrios (8). The last precaution was particularly crucial for obtaining reliable data for YATP calculations. Radioactivity. All radioactivity measurements were made by scintillation counting in PCS solubilizer scintillation fluid (Amersham-Searle, Arlington Heights, Ill.). Cell composition. Chemical analyses were made of whole cells for protein (12), DNA (4), RNA (21), and polysaccharides (1) by standard procedures. Washed cells were fractionated into their major constituents as described by Roberts et al. (19). The lipid contents were determined from the radioactivity of the ethanol ether extracts of U-'4C-labeled cells. The polysaccharide and peptidoglycan contents of E. coli ML35 are estimates based on general values for gram-negative organisms (14, 19, 29). The peptidoglycan content of B. bacteriovorus 109J was determined from the weight of isolated material. Purification of B. bacteriovorus on Ficoll gradients. One milliliter of washed cell suspension (1010 to 3 x 1010 cells) was layered onto 25 ml of a 5 to 20% linear Ficoll gradient (24) and centrifuged at
1159
27,000 x g for 5 min at 4 C. The visible band of bdellovibrio cells, 25 to 30 mm beneath the meniscus, was removed with a Pasteur pipette, diluted 10-fold in HEPES buffer, and washed three times in buffer by
centrifugation. RESULTS Experimental determination of YATP. YATP values were calculated (Table 1) from the distribution of radioactivity in respired CO2 and in the bdellovibrios after growth of B. bacteriovorus on [U- 14C ]E. coli as the exclusive source of all nutrients and from a previously determined respiratory quotient (RQ) of 1.05 for intraperiplasmic growth (8). The calculation makes only three assumptions: (i) cell carbon is 50% of cell dry weight (D, Table 1); (ii) the P/O ratio for B. bacteriovorus is 3 (E, Table 1); and (iii) substrate level phosphorylation is negligible during intraperiplasmic growth. These assumptions are considered in the discussion. Independent of the assumptions, for the YATP determinations to be valid two general conditions had to be achieved experimentally; (i) the harvested bdellovibrios at the end of growth had to be free of E. coli debris, or else the YATP values would be in error on the high side; (ii) the radioactive CO2 collected either had to be derived exclusively from the respiration of the bdellovibrios growing intraperiplasmically or had to be corrected for radioactive CO2 from other sources, or else the calculated YATP values would be spuriously low. There were only two possible additional sources of radioactive CO2 in these experiments: from the endogenous respiration of the [U- 14C ]E. coli before bdellovibrio attack, and from extraperiplasmic bdellovibrios oxidizing materials released from attacked or lysed E. coli. These two conditions dictated the experiments that follow. Purity of harvested bdellovibrios. B. bacteriovorus was grown on [U- 14C I- [2H ]thymidine-labeled E. coli as the substrate organism in single-cycle growth experiments. Progeny bdellovibrios were harvested and washed twice as described above and the distribution of the two radioactive tracers between the progeny cells, the buffer, and the CO2 was determined (Table 2). On the average, about 52% of the initial E. coli carbon and 59% of its thymidine radioactivity were present in the washed bdellovibrio suspensions. Portions of these suspensions were treated with deoxyribonuclease I (5 jg/ml, 20 min, 30 C), centrifuged, washed, and recounted. No significant loss in the quantity of 3H was ob-
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RITTENBERG AND HESPELL
served. Thus, all harvested DNA was protected from enzyme attack (i.e., was intracellular). This result is consistent with the previous finding that during bdellovibrio attack the DNA of the substrate organism is rapidly and completely degraded (13). The data indicate that the tritium label is a reliable measure of bdellovibrio material in these experiments. The harvested bdellovibrios were then "purified" on Ficoll gradients as described in Materials and Methods. There was little change in the initial '4C/3H ratio after this treatment (Table 2). The changes measured, +1.3% to -2.9%, fell within the average counting error (+3%) and are not significant. It may be concluded that the harvested bdellovibrios are not contaminated with significant amounts of miscellaneous E. coli debris. The alternate possibility is that E. coli debris of constant composition and quantity is not separated from the bdellovibrios by the Ficoll procedure. Critical tests reported in another connection (11) make the second alternative highly unlikely.
YATP for single-cycle growth experiments. Table 3 shows the distribution of the radioactivity initially in the substrate organisms after single-cycle growth of B. bacteriovorus on [U"C ]E. coli. The YATP values were calculated from these data as shown in Table 1. The experimental design largely satisfied the condition that little of the "'CO, came from the endogenous respiration of the starting [UJ- "4C ]E. coli since at input ratios of B. bacteriovorus to E. coli of 1.4 to 1.8, E. coli respiration is eliminated within a very short time after addition of the bdellovibrios (18). However, a significant (and unknown) number of bdellovibrios in these experiments remain extracellular and do not grow. They do, nevertheless, oxidize material released from E. coli and liberate '4CO,. The YATP values should therefore be in error on the low side. To obtain some indication of the magnitude of this effect, experiments were done in which cultures were initiated with variable numbers of bdellovibrios and constant numbers of E. coli to give input ratios of 0.1 to 2.8. All cultures were incubated for 240 min, at which time biological activity TABLE 1. Determination of YATP for intraperiplasmic was terminated by addition of acid. Microscopic growth of B. bacteriovorus on [U-"C]E. coli from distribution of radioactivity between the bdellovibrios observations showed that by this time all E. coli in cultures with starting input ratios above 1.3 and CO, to 1.4 had been lysed, whereas E. coli remained 14C counts/min in cells Moles of carbon assimilated in cultures initiated with lower input ratios. A A. Moles of carbon respired 14C counts/min in COM plot of "4CO, release against initial input ratios Moles of carbon assimilated showed a sharp discontinuity in the results at A x RQ = A x 1 .05 B. Mole of 0, consumed an input ratio of about 1.2 (Fig. 1). The points below and above the transition approximate Grams of carbon assimilated C B Mole of 02 consumed two straight lines having slopes of 9.05 0.29 D Dry wt of cells formed C 2 and 1.01 0.23 (percentage of initial radioacMole of 0, consumed tivity/input ratio). Dry wt of cells formed E. YATP= =D-6 The of the experiment makes it likely design Moles of ATP produced that the greater slope is a measure of the effect =
12
TABLE 2. Distribution of radioactivity after growth of B. bacteriovorus 1O9Jon [U-'C]-['H]thymidine-labeled E. coli ML 35a Initial radioactivity (%) in:
Expt
Isotope
1
"4C
2
3H 14C 3H
3
"4C
4
3H 14C 3H
Cells
Buffer
CO2
53 57 54 54 50 66
32 48 38 48 32 38 34 40
16
51 59
16
15 16
Recovery (%)
101 105 108 102 97 104 101 99
aCultures initially contained 11 x 109 B. bacteriovorus and 6 x 109
"4C/3H in cells' Harvested
Purified
0.458
0.464
0.481
0.467
0.694
0.678
0.674
0.669
[U-_4C]-[3H]thymidine-labeled
E.
coli/ml ('H, 1.2 x 10' to 1.5 x 10' counts/min; 4C, 6 x 104 to 8 x 104 counts/min). Input ratio = 1.8. 'After lysis, the bdellovibrio cells were washed twice by centrifugation (harvested) and then banded on Ficoll gradients (purified).
VOL. 121, 1975
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YATP OF BDELLOVIBRIO INTRAPERIPLASMIC GROWTH
TABLE 3. YATP for intraperiplasmic development determined from distribution of radioactivity after single-cycle growth of B. bacteriovorus on [U-'4CJE. colia Initial E. coli radioactivity (%) in:
(YAIl) B. bac-
ratio Determination Input (Bd/Ec)b
Buffer CO2
teriovorus
14 31 1.6 Avg ......... Range ......... 1.4-1.8 24-38 12-16 Standard deviation .5.3 + 1.7
56 49-64
17 14-20
±6.1
I2.8
coli in the bdellovibrio culture is 74% of that released by the same number of E. coli shaken in buffer for the same time. Applying this 0.75 x 2,260; Table 4), correction (10,600 the "4CO2 released by bdellovibrio respiration during growth was 7.8% of the initial, and the corrected YATP was 28.2. The above calculation assumes that the number of attacked, nonviable (and nonrespiring) E. coli is equal to the number of bdellovibrio 1614-
a Data are average of 11 experiments; the cultures initially contained between 5 x 10' and 8 x 109 B. bacteriovorus and 3 x 10' and 5 x 10' [U-4C JE. coli (70,000 to 550,000 counts/ min) per ml. ° Bd, B. bacteriovorns; Ec, E. coli.
of increasing numbers of growing intraperiplasmic bdellovibrios (i.e., E. coli excess), and the lesser slope is a measure of the effect of increasing numbers of nongrowing extracellular bdellovibrios, on CO2 release (i.e., bdellovibrio excess). If so, the intercept represents the balance point at which essentially all E. coli are attacked and few bdellovibrios are extracellular. The radioactivity of CO2 released from such a culture at the end of growth is about 12.5% of the initial (Fig. 1). The YATP of the balanced culture, which we believe gives the best estimate from single-cycle growth experiments, is about 18.5. The value is on the low side since some, probably small, amount of '4CO2 must come from E. coli endogenous respiration. YATP for multicycle growth experiments. Table 4 shows the results of a typical multicycle growth experiment in which the input ratio of B. bacteriovorus to [U-14CJE. coli was about 0.0027. In this particular experiment, some 52% of the starting E. coli carbon (radioactivity) was converted to bdellovibrio cell material and 9% was released as CO2. The uncorrected data, calculated as shown in Table 1, give a YATP Of 23.3 for the experiment. The experimental design satisfies the condition that little 14CO2 is derived from nongrowing bdellovibrios. However, because of the low input ratio, most E. coli cells remained unattacked until very late in culture development. Some 14CO2 must have been derived from the endogenous respiration of these E. coli and the YATP value is therefore low. An E. coli survival curve was calculated from the input ratio and a doubling time of 1.6 h for bdellovibrio plaque-forming units, a value typical of multicycle cultures grown under the conditions of this experiment (16). From the area under the curve, the 14C2O released by E.
S>
~
~
Slope - 0/
12
So
10
0
=9.05 >
6.
0 4-
r-5 2
0
.5
15
10
2.0
35
bacteriovorus. E.coli FIG. 1. Relation between CO, release and input ratio in single-cycle growth of B. bacteriovorus on [U-14CJE. coli. Initial cultures contained 4 x 109 [U-'4CJE. coli/ml (83,000 counts/min per ml) and B. bacteriovorus to give the indicated input ratios. All cultures were incubated for 240 min, at which time all E. coli had been lysed in cultures having an input ratio of about 1.3 or higher. INPUT
RATIO-B
TABLE 4. Distribution of radioactivity after multicycle growth of B. bacteriovorus on [U-14CIE. colia Component
Radioactivity (counts/min per ml) in: E. coli CO B. bacterio-
CO,
vorus
Culture .... 10,600 (9.3) 60,000 (52) 102,000 (89) Control .... 2,260 (2.0) a The initial culture contained 8 x 10' B. bacteriovorus and 3 x 109 [U- 14C ]E. coli (counts/min = 115,000/ml) and was incubated for 17 h until all E. coli had lysed. The control consisted of the same number of E. coli in buffer incubated for the same time. Values in parentheses are percentage of initial counts.
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J. BACTERIOL.
RITTENBERG AND HESPELL
plaque-forming units in the culture at any time until the total plaque-forming units essentially equals the input E. coli. Direct plating of samples of a multicycle culture on nutrient agar with time gave E. coli colony counts somewhat lower but approximating those calculated (Table 5). The differences can be accounted for by small differences in doubling time and/or input ratio and do not alter the correction appreciably. The calculation also assumes that the respiration of unattacked E. coli in the culture is neither stimulated nor inhibited by materials released from attacked and/or lysed E. coli or from the bdellovibrios. To check this point, the respiration rates of E. coli cells suspended in buffer and in the supernatant fluid obtained by centrifuging 10- and 18-h (complete lysis) multicycle cultures were measured. The values obtained for the three suspensions, 9.8, 8.5, and 7.3 ul of 0/min per 1010 cells, were similar and supported the assumpitions. Table 6 summarizes the results of 14 indeTABLE 5. Calculated and observed surviving E. coli in a multicycle B. bacteriovorus culturea Surviving E. coli (% initial) Time (h)
0 10 12 14 16
Calculated
Found
100
100
90 76 43 0
94 51 5
