JOURNAL OF BACTERIOLOGY, Mar. 1978, p. 1156-1162 Vol. 33, N0021-9193/78/0133-1156$02.00/0Copyright © 1978 American Society for Microbiology Printed in U..

Intraperiplasmic Growth of Bdellovibrio bacteriovorus onHeat-Treated Escherichia coli

ROBERT B. HESPELLDepartment ofDairy Science, University of Illinois, Urbana, Illinois 61801

Received for publication 8 September 1977

Heat treatment (55°C for 40 min) of cell suspensions in buffer (ca. 3 x 109 cellsper ml) of Escherichia coli ML35 caused a 4- to 4.5-log loss of cell viability.Similar results were found for several other E. coli strains that were examined.As a result of this heat treatment, 260-nm- and 280-nm-absorbing materials werereleased into the suspending buffer, along with about 10% of the total cellularradioactivity, when cells uniformly labeled with 14C were used. In comparisonwith untreated cells, heat-treated E. coli ML35 cells showed (i) no significantchanges in macromolecular composition other than ca. 22% less RNA content, (ii)an increased permeability to o-nitrophenyl-,8-D-galactopyranoside (a compoundto which untreated cells are impermeable), (iii) almost complete loss ofrespiratorypotential, and (iv) substantial losses of numerous glycolytic enzyme activities incell extracts prepared from these cells. Intraperiplasmic development of Bdello-vibrio bacteriovorus 109J with heat-treated E. coli ML35 as substrate cellsappeared normal when observed microscopically, although bdellovibrio attach-ment and resultant bdelloplast formation were slightly retarded. No significantchanges were observed in cell yields or in the ratios and contents of DNA, RNA,or protein between bdellovibrios harvested from untreated cells and those fromheat-treated substrate cells after single-developmental-cycle growth on thesecells. The average YATP values for intraperiplasmic growth on untreated and heat-treated substrate cells were 16.0 and 17.9, respectively. It is concluded thatintraperiplasmic bdellovibrio growth on gently heat-treated E. coli substrate cellsis very similar to growth on untreated substrate cells, even though the formersubstrate cells are nonviable and substantially impaired in many metabolicactivities.

The periplasmic region of a suitable gram-negative bacterium (substrate cell) is a normalgrowth environment for Bdellovibrio bacterio-vorus. The apparently regulated utilization ofsubstrate cell components (5, 11-13) and highenergy efficiency of the process (YATP, 18 to 26)(14) indicate that intraperiplasmic bdellovibriogrowth involves substantial biochemical com-

plexity. Although it is known that after bdello-vibrio penetration the substrate cell is nonviable(18) and has lost some metabolic activities (6,15), some substrate cell enzymes are functionaluntil inactivated or degraded (or both) by bdel-lovibrio enzymes (3). Thus, to establish that anyone biochemical or physical event of intraperi-plasmic growth can be specifically caused bybdellovibrio enzymes alone, it must be clearlyshown that homologous substrate cell enzymesare not involved. One way to establish this latterpoint would be to grow bdellovibrio intraperi-plasmically on heat-treated substrate cells. Pre-sumably, these heat-treated substrate cells

would have a variety of enzymes that would bepartially or completely inactive as a result of the

heat treatment. In the case of complete inacti-vation, bdellovibrio enzymes would be com-

pletely responsible during intraperiplasmicgrowth for the occurrence of biochemical eventshomologous to those caused by the inactive sub-strate cell enzymes. However, a major assump-tion made in this experimental approach is thatintraperiplasmic bdellovibrio growth on heat-killed substrate cells is not significantly alteredfrom growth on normal substrate cells. The stud-ies reported here indicate that this assumptionis largely valid for the severity of heat treatmentused here (55°C for 40 min).

MATERLALS AND METHODSOrganisms and culture conditions. B. bacterio-

vorus 109J was the experimental organism; it was

grown on Escherichia coli in dilute nutrient broth (15,19). E. coli ML35 (lacI lacY) and E. coli B were

grown at 30°C in a glucose-mineral salts medium, as

described previously (7). E. coli strains K-10 BJ565(11), K-12 AB1157-1-5A-1, K-12 57A-12, and K-12 4K-26 (22) were grown on glucose-mineral salts mediumsupplemented with 0.2% (wt/vol) casein hydrolysate,plus ribose, tryptophan, methionine, and thiamine at

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BDELLOVIBRIO GROWTH ON HEAT-KILLED CELLS

concentrations of 10, 20, 20, and 2 ,g/ml, respectively.Cells were harvested by centrifugation, and cell sus-pensions were made in 5 x 10- M N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid containing 10-3 MCaCl2 and MgCl2 (HM buffer; pH 7.6) after beingwashed twice in this buffer by centrifugation.

Cell numbers in cell suspensions were determinedby turbidity measurements (15, 19). Viable cell num-bers were determined for E. coli as colony counts onnutrient agar plates and for B. bacteriovorus asplaque-forming units.Heat treatment. Unless indicated otherwise, the

standard heat treatment procedure used was as fol-lows. All E. coli cultures were harvested in mid-expo-nential growth phase (optical density, 0.5 to 0.7 at 660am). After being washed twice by centrifugation (5min, 4°C, 6,000 x g), the harvested cells were concen-trated about 10-fold in HM buffer (3 x 109 to 9 x 109cells per ml), and the suspension was divided into twoequal portions. One portion was then heat treated.The washed cell suspension (15 to 30 ml) was rapidlywarmed to 30°C, followed by incubation with gentleshaking at 55°C for 40 min in a water-bath shaker.The culture was then immediately cooled in an icewater bath. Both the heat-treated and untreated cellswere suspended in HM buffer to the same volumeafter being washed twice by centrifugation.Growth experiments. Single-cycle and multicycle

growth experiments, in which E. coli served as thesole source of nutrients for intraperiplasmic growth ofbdellovibrios, were done by standard procedures (6,15). Experimental determinations of YATP (grams [dryweight] of cell material formed per mole of ATP) forintraperiplasmic bdellovibrio growth were calculatedas described previously (14). The oxygen consumptionof cell suspensions were measured with an 02 elec-trode, and respiratory quotients for intraperiplasmicgrowth were determined by standard methods (6, 15).Chemical and enzyme assays. Previously pub-

lished procedures (5) were used for the fractionationof cells into their major constituents and for analysesof protein, RNA, and DNA. Enzyme activities in cellextracts were measured (1, 3, 4) immediately afterpreparation ofthe extract obtained by French pressurecell disruption of whole cells (5,000 lb/in2 [3.5 x 10rkg/m2] at 5°C, twice).

Radioactivity and miscellany. All radioactivitymeasurements were made by scintillation countingwith PCS solubilizer fluid (Amersham/Searle, Arling-ton Heights, Ill.). All biochemicals and reagents usedwere of reagent or biological grade.

RESULTSEffect of heat treatment on E. coli viabil-

ity. The loss of viability in HM buffer of E. coliML35 cells harvested from the mid-logarithmicgrowth phase was dependent on the temperatureand length of heat treatment (Fig. 1). A temper-ature of about 55°C seemed critical in affectingthe viability of E. coli. The data of other exper-iments indicated that, at a slightly higher (ca.57°C) or lower (ca. 53°C) temperature, the rateof viability loss was significantly increased ordecreased, respectively. When individual cellsuspensions containing initial cell concentrations

1-qR

4 107

; 60°C11

10 20 30 40 50 60Time (min)

FIG. 1. Loss of viability of E. coli ML35 cell sus-pensions incubated at various temperatures. All sus-pensions in HM buffer were incubated at the indi-cated temnperature, and the viable ceU counts in thesesuspensions were determined as described in the text.

over the range of 6 x 10' to 11 x 109 cells per mlwere heat treated at 55°C for 40 min, the residualviable cell counts of the suspensions were 1 x104 to 2.5 x 105 cells per ml, respectively.

Cell suspensions (ca. 3 x 109 cells per ml) ofother E. coli strains (see Materials and Meth-ods) were also subjected to heat treatment (550Cfor 40 min). Both the E. coli K-10 and K-12strains showed a 4-log or greater loss in viability,whereas E. coli strain B viability declined onlyby 2 logs. With all E. coli strains examined, therate of viability loss at 55°C slowed substantiallyafter the viable count dropped to ca. 105 cells perml. This suggested that a very small fraction ofthe initial population (ca. 3 x 109 cells per ml)was relatively more heat resistant. Nevertheless,a standard heat treatment of 55°C for 40 minwas used routinely in the subsequent experi-ments, as this procedure yielded a 4- to 4.5-logloss in cell viability with no substantial inhibi-tion of bdellovibrio attachment to these heat-treated cells (see below).

Effects of heat treatment on whole celisofE. coli When U-_4C-labeled E. coli cells wereheat-treated, 10% or less of the initial whole-cellradioactivity was released into the suspendingbuffer (Table 1). In addition, 260-nm- and 280-nm-absorbing (UV) materials appeared in thesuspending buffer. The rates of appearance inthe suspending buffer ofUV material and radio-activity paralleled one another. About 80% ofthe total released material was lost from the

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TABLE 1. Effects of heat treatment on E. coli cell compositiona

Suspend- Whole Cold RNA DNA Protein residueSuspend Whole HC104 S0-E. coli cells ing buffer cells uble(cpm/ml) (cpm/ml) (cpm/nl) cpm/ml pg/Ml cpm/ml pg/ml cpm/ml pg/ml

Untreated 256 44,418 1,504 19,644 428 6,442 21.4 19,490 824Heat treated 4,604 41,198 1,168 13,171 340 6,372 19.8 20,400 830a A cell suspension containing 5 X 101 U-_4C-labeled E. coli ML35 cells per ml (45,406 cpm/ml) was divided

equally. One portion was kept at 40C, and the other was heated at 55°C for 40 min. The suspensions werecentrifuged, and the cell pellets were fractionated into their major constituents (see text).

cells within the first 12 min of incubation at550C. These UV and radioactive materials were

not precipitated by treatment of the suspendingbuffer with trichloroacetic acid or HC104 (finalconcentration, 5% [vol/vol]), but dialysis treat-ment removed 80% or more of this material fromthe suspending buffer.Untreated and heat-treated E. coli ML35 cells

were fractionated into their major cell compo-nents. Analyses of these fractions from the twotypes of cells indicated no great differences inthe radioactivity or DNA content of the DNAfraction (Table 1), but the RNA fraction fromheat-treated cells decreased about 25%. In theprotein fraction from heat-treated cells, a smallincrease in radioactivity (5 to 8%) and no in-crease in protein content was consistently ob-served. Similar results were obtained when cellsof other E. coli strains were analyzed in an

analogous manner.When cell lipids were extracted with chloro-

form-methanol (11), the resultant extract ob-tained from heat-treated E. coli ML35 usuallycontained about 15% less radioactivity than thatobtained from untreated cells. These data, inconjunction with the above data on released cell

materials, suggested that heat-treated cells weremore permeable than normal cells because ofcell membrane damage. Untreated E. coli ML35whole cells showed less than 2% of the ,B-galac-tosidase activity present in cell extracts (Table2), as expected, because E. coli ML35 (lacI lacY(is constitutive for fl-galactosidase synthesis butis impermeable to lactose and o-nitrophenyl-fl-D-galactopyranoside. In contrast, heat-treatedwhole E. coli ML35 cells showed about 25% ofthe ,B-galactosidase activity present in cell ex-tracts (Table 2). In agreement with previousstudies (3, 15), more than 95% of the cellular /8-galactosidase activity was unmasked duringbdellovibrio attachment and bdelloplast (spher-ical substrate cell with intraperiplasmic bdello-vibrio) formation, not only on untreated cells,but also on heat-treated cells (Table 2). In ad-dition, bdeliovibrio cells and cell extracts showedonly very low o-nitrophenyl-fl-D-galactopyrano-side hydrolysis activities, as found previously(15).

TABLE 2. Effects of heat treatment and/or B.bacteriovorus on permeability of E. coli ML35'

,B-Galactosidase activityb

Cell suspension Whole cells Cell extracts

0 min 45 min 0 min 45 min

Untreated E. coli 14 40 770 800Untreated E. coli + 12 740 790 730

bdellovibrio

Heat-treated E. coli 160 200 590 610Heat-treated E. coli 180 580 560 600+ bdellovibrio

Bdellovibrio 5 6 20 18a Cell suspensions were prepared as indicated to

contain 3.3 X 109 untreated or heat-treated E. coliML35 cells per ml or 5.0 X 109 B. bacteriovorus cellsper ml, or both. o-Nitrophenyl-,8-galactoside hydroly-sis activity was measured from whole cells or cellextracts obtained from the cultures at 0 min or after45 min incubation at 30°C, at which time all E. colicells were bdelloplasts if bdellovibrios were addedinitially.

b Expressed as nanomoles of o-nitrophenyl-f,-galac-toside hydrolyzed per minute per milliliter of culture,divided by 10.

Effect ofheat treatment on E. coli enzymeactivities. The levels of various enzyme activi-ties in cell extracts prepared from untreated andheat-treated E. coli cells were measured (Table3). Most of the enzyme activity levels in cellextracts prepared from heat-treated cells were10% or less of those in cell extracts from un-treated cells. The decreases in the activities ofthe glucose-6-phosphate and glyceraldehyde-3-phosphate dehydrogenases (Table 3) and fl-ga-lactosidase (Table 2) indicate that these en-zymes are not drastically affected by the stan-dard heat treatment of whole cells. The ,8-galac-tosidase activity level in extracts of heat-treatedE. coli or of bdelloplasts from heat-treated cellswas similar to the level observed with wholebdelloplasts (Table 2). These latter data indicatethat the procedures used in cell extract prepa-ration were not the cause of any observableinactivation of enzyme activity. When cell ex-tracts from untreated and heat-treated cellswere mixed, the activity levels of various en-

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BDELLOVIBRIO GROWTH ON HEAT-KILLED CELLS 1159

TABLE 3. Inactivation of E. coli ML35 enzymeactivities by heat treatment

Enzyme activityaLoss of ac-

Enzyme Untreated Heat- tivityUtetdtreated (%)bcells cells

Hexokinase ... 46.7 3.8 93Glucose-6-phosphate de-hydrogenase. 157.3 72.1 54

Fructose-diphosphate al-dolase.266.0 5.4 98

Glyceraldehyde-3-phos-phate dehydrogenase 540.9 447.4 17

Triose phosphate isomer-ase.. 134.5 7.7 94

Pyruvate kinase. 201.3 11.4 93Pyruvate dehydrogenase 309.2 31.8 90Lactic acid dehydrogenase 872.4 61.8 93a-Ketoglutarate dehydro-

genase 134.2 3.6 97a Expressed as change in nanomoles of substrate per minute

per milligram of protein.bCalculated as follows: [1 - (heat-treated cell activity +

untreated cell activity)] x 100.

zymes in the resultant extract were approxi-mately (±4%) equal to the sum of the levelspresent in the individual cell extracts, suggestingthe absence of inhibitors in extracts from heatedcells.Because the level of reduced nicotinamide

adenine dinucleotide-oxidizing activity in cellextracts of heat-treated cells was found to beless than 5% of that present in cell extracts ofuntreated cells, it seemed plausible that heat-treated cells might have an impaired respiratorypotential. Measurement of oxygen consumptionby untreated cells indicated an endogenous res-piration rate of 20 to 25 nmol of 02 per min per1010 cells, whereas no endogenous respirationcould be detected with heat-treated cells. In thepresence of glucose, untreated cells and heat-treated cells had respiration rate ranges of 280to 275 and 8 to 10 nmol of 02 per min per 1010cells, respectively. In cell suspensions supple-mented with fructose, lactate, pyruvate, or suc-cinate, heat-treated cells always displayed a res-piration rate of less than 5% of the rate foundwith untreated cells.Bdellovibrio attachment and bdelioplast

formation with heat-treated celis. Single-cy-cle developmental cultures were initiated bymixing suspensions of bdellovibrios and sub-strate cells to yield an input ratio of 1.8 to 1.2bdellovibrios per substrate cell. These cultureswere periodically examined by phase-contrastmicroscopy and assayed for fi-galactosidase ac-tivity. The results showed that virtually all ofthe substrate cells were converted to bdello-plasts, and full unmasking of fl-galactosidaseactivity occurred within 20 to 25 min (untreated

cells) and 40 to 45 min (heat-treated cells) whenE. coli strains K-10 BJ565, ML35, or K-12 4K-26 were used as substrate cells. When thesestrains of E. coli were heat treated longer (550Cfor 60 min) and used as substrate cells, bdello-vibrio attachment appeared to be reduced, asindicated by microscopic observations. Conse-quently, at 60 min after initiation of the devel-opmental culture, less than 50% of the substratecells were bdelloplasts. After 100 min of incu-bation, little or no bdellovibrio attachment andfew or no bdelloplasts were observed with sus-pensions of bdellovibrios and E. coli cells thathad been heat treated at 70°C or higher for 15to 60 min.Bdellovibrio growth on heat-treated

cells. When viable cell counts of bdellovibriocell suspensions were determined by standardplating procedures (17, 19) with lawns of un-treated or heat-treated E. coli ML35 substratecells, no discernible differences in the numbers,morphology, or rate of appearance of plaqueswere observed between the two types of sub-strate cells employed. With single-cycle devel-opmental cultures, observations by phase-con-trast microscopy indicated no morphological dif-ferences in the intraperiplasmic growth of bdel-lovibrios on untreated and heat-treated E. colicells. However, the complete lysis of the heat-treated substrate cells with concomitant releaseof progeny bdellovibrios was delayed. On theaverage, a 240- to 260-min developmental cycleoccurred with heat-treated substrate cells, anda 200- to 220-min cycle occurred with untreatedcells. The number of viable bdellovibrios presentat the complete lysis of the substrate cells wasdetermined by plaque counts. The data indi-cated no significant decrease in bdellovibrio cellyields from growth on heat-treated substratecells (Table 4). Furthermore, analysis of the cell

TABLE 4. Cell yield and ceU composition of B.bacteriovorus 109J after intraperiplasmic growth on

untreated and heat-treated E. coli ML35a

Organism Cells per DNA RNA Proteinml (mg/ml) (pg/ml) (jg/mi)

Initial B. Bacterio-vorus ...... ... 7.1 x 10' 23.9 61.0 221.0

Initial E. coliUntreated ....... 4.4 x 109 34.3 372.0 713.0Heat-treated 4.4 x 109 33.1 243.0 705.0

Progeny B. Bacter-iovorus

Untreated E. coli 15.1 x 10' 51.0 152.0 513.0Heat-treated E.

coli.14.2 x 10' 48.6 142.0 508.0

a Single-cycle developmental cultures were set up; whenmicroscopic observations indicated that complete lysis of E.coli had occurred (220 to 260 min), the bdellovibrios wereharvested and analyzed. The data are averages of four inde-pendent experiments.

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1160 HESPELL

composition of bdellovibrios harvested at lysisshowed no differences in the DNA, RNA, andprotein ratios and less than a 5% decrease in theabsolute amounts of these cell components withbdellavibrios grown on heat-treated E. coliML35 substrate cells as compared with bdello-vibrios grown on untreated E. coli ML35 sub-strate cells (Table 4). Similar results were foundwhen E. coli strains B, K-10 BJ565, and K-12 1-5A-1 were used as substrate cells, but a decreaseof about 30% in bdellovibrio DNA, RNA, andcell numbers was observed with growth on heat-treated E. coli K-12 57A-12 cells.

YATP for intraperiplasmic growth on

heat-treated cells. It has been previously re-

ported (14) that bdellovibrios have a high energyefficiency for intraperiplasmic growth, as YATP

values of 18 to 26 were demonstrated. TheseYATP values were determined from the distribu-tion of radioactivity in the bdellovibrios and inthe respired CO2 after growth on U-14C-labeledE. coli (14) and from the respiratory quotientfor intraperiplasmic growth (6). Because the res-

piratory quotients for intraperiplasmic growthon untreated and heat-treated substrate cellswere found to be similar, YATP values were de-termined for bdellovibrios grown on untreatedand heat-treated cells. The data show that theYATP for intraperiplasmic growth on heat-treated E. coli ML35 cells was only slightlyhigher (17.9) than that on untreated cells (16.0).

DISCUSSIONThe data show that the synchronous single-

cycle intraperiplasmic growth of B. bacteriovo-rus 109J on heat-treated (55°C for 40 min) E.coli substrate cells is very similar, if not identi-cal, to growth on untreated cells. The conclusionis supported by a comparison of the data ob-tained from measurement of several parametersof intraperiplasmic bdellovibrio growth on thetwo types of substrate cells. The data showed no

substantial differences in (i) progeny bdellovi-brio yields, (ii) progeny bdellovibrio cell com-

position, and (iii) YATP values from intraperi-plasmic bdellovibrio growth. In addition, micro-scopic observations indicated no major differ-ences in bdellovibrio attachment and penetra-tion per se, or in morphological aspects of intra-periplasmic bdellovibrio development.

Several studies have indicated that bdellovi-brios are capable of growth on heat-treated sub-strate cells (2, 8, 16, 17, 20), but, unlike our study,the bdellovibrio growth observed in these stud-ies was extracellular or occurred by asynchro-nous multicycle intraperiplasmic development,or both. Multicycle intraperiplasmic growth can

occur on Spirillum serpens (16) or E. coli cells

(this study) that have been subjected to a pro-

longed, low-temperature heat treatment (55 to56°C for 60 to 90 min). We observed little or nobdellovibrio attachment to E. coli cells that wereheat-treated at 98°C (5 min or more), and, inagreement with other studies (2, 16), only extra-cellular bdellovibrio growth occurred with thesecells, provided the substrate cells were notwashed after heat treatment. The substrate-cell-derived bdellovibrio growth initiation factor (7,9) and other materials released into the suspend-ing fluid during heat treatment provide the nu-trients necessary for extracellular bdellovibriogrowth, as has been observed for B. bacteriovo-rus 6-5-S (2). Whether bdellovibrio grows intra-periplasmically or extracellularly may dependon the extent of damage to the substrate cell.Presumably, some of the substrate cell compo-nents that are affected by heat treatment areassociated with (or are a part of) the suggestedbdellovibrio attachment sites (20) in the outermembrane layers of the gram-negative substratecell.

In a study examining intraperiplasmic growthconditions, Varon and Shilo (21), using multi-cycle developmental cultures (initial bdellovi-brios per substrate cell, 0.1 or less), reported thatB. bacteriovorus 109 grew intraperiplasmicallyon heat-treated (70°C for 15 min) E. coli B-2262substrate cells. Their data showed reduced prog-eny bdellovibrio yields as compared with growthon untreated cells. Our data, obtained with sin-gle-cycle developmental cultures with B. bacter-iovorus 109J and heat-treated (55°C for 40 min)E. coli ML35 (and other strains), indicated nolarge reductions in bdellovibrio cell yields. Nodefinitive explanation can be readily offered toaccount for the differences between our resultsand those of Varon and Shilo (21). However,neither the exact substrate cell growth or heattreatment conditions were clearly given in thelatter study, and these factors, as our data haveshown, can significantly influence bdellovibriogrowth on heat-treated cells.Our data show that cell suspensions of various

E. coli strains after heat treatment at 55°C for40 min have a 4-log decrease in viable cells.These heat-treated cells are substantially im-paired in many physiological functions. The cellsshow little or no capacity for endogenous orsubstrate-based respiration, and numerous gly-colytic and tricarboxylic acid cycle enzyme ac-tivities have been reduced to trace levels.Clearly, heat-treated cells are incapable of sig-nificant energy generation by substrate or oxi-dative phosphorylation. E. coli ML35 is nor-mally impermeable to o-nitrophenyl-,f-D-galac-topyranoside, but the cell membrane in heat-treated whole cells has been slightly damagedbecause ca. 30% of the total cellular f-galacto-

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sidase activity was observed with intact, heat-treated cells. Our data indicate that normal in-traperiplasmic B. bacteriovorus 109J growth oc-curred on these heat-treated E. coli ML35 cells.Obviously, then, substrate cell energy genera-tion, respiration, and permeability control arenonessential for any phase of bdellovibriogrowth, including attachment, as expected, be-cause respiration and permeability control wereshown to be rendered nonfunctional very earlyin the developmental cycle (6, 15).

After bdellovibrio penetration into normal,untreated substrate cells, a wide variety of sub-strate cell enzymes could remain functionallyactive to some degree; specifically, this has beenshown to exist for certain substrate cell glyco-lytic enzyme activities (3). Therefore, many sub-strate cell enzymes could be capable of partici-pation to some degree in various biochemicalprocesses occurring during intraperiplasmicbdellovibrio growth. To establish that any oneof these biochemical processes can be caused bybdellovibrio-specific enzymes alone would bedifficult. In this regard, the use of heat-treatedsubstrate cells would be of value, as it could beconcluded that bdellovibrio enzymes are respon-sible for biochemical processes homologous tothose catalyzed by substrate cell enzymes thatwere totally inactivated by heat treatment.Thus, it can be concluded, for instance, thatsubstrate cell glycolytic enzyme activities thatpersist after penetration (3) are not needed forbdellovibrio growth, since these activities arealmost completely absent from heat-treated cells(Table 3).

Currently, we are using heat-treated substratecells to study the reported (5) catabolism ofnucleotide sugars, and the preliminary data sug-gest that bdellovibrio enzymes could be mostlyresponsible for the loss of ribose during intra-periplasmic growth.The use of heat-treated substrate cells to in-

vestigate various aspects of intraperiplasmicbdellovibrio growth is a good method, but be-cause it is an indirect approach, the techniquedoes have certain limitations. First of all, it doesnot allow one to totally exclude the possibleinvolvement during growth on normal, un-treated substrate cells of the inactivated en-zymes observed with heat-treated cells. Sec-ondly, in the case of an only slightly or partiallyinactivated enzyme, the residual activity may besufficient for bdellovibrio growth. Thirdly, thetechnique can only be used to draw conclusionsabout enzymes that are susceptible to heat in-activation. However, the major advantage ofusing heat-treated substrate cells is that it is arelatively simple method for obtaining substratecells that are partially or completely deficient in

one or more enzymes, but for which no suitableE. coli mutant strains deficient in these enzymesare readily available. By comparing differencesin specific growth parameters between bdellovi-brio growth on normal and heat-treated sub-strate cells in conjunction with other appropriatesupporting data, one can clearly establishwhether the particular substrate cell function isessential to bdellovibrio growth.

ACKNOWLEDGMENTSThis research was supported by grant PCM 75-23110 from

the National Science Foundation and by grant ILLU-35-349from the Agricultural Experiment Station of the University ofIllinois.

LITERATURE CITED1. Carls, R. A., and R. S. Hanson. 1971. Isolation and

characterization of tricarboxylic acid cycle mutants ofBacillus subtilis. J. Bacteriol. 106:848-855.

2. Crothers, S. F., H. B. Fackrell, J. C. C. Huang, and J.Robinson. 1972. Relationship between Bdellovibriobacteriovorus 6-5-S and autoclaved host bacteria. Can.J. Microbiol. 18:1941-1948.

3. Hespell, R. B. 1976. Glycolytic and tricarboxylic acidcycle enzyme activities during intraperiplasmic growthof Bdellovibrio bacteriovorus on Escherichia coli. J.Bacteriol. 128:677-680.

4. Hespell, R. B., and E. Canale-Parola. 1970. Carbohy-drate metabolism in Spirochaeta stenostrepta. J. Bac-teriol. 103:216-226.

5. Hespell, R. B., G. F. Miozzari, and S. C. Rittenberg.1975. Ribonucleic acid destruction and synthesis duringintraperiplasmic growth of Bdellovibrio bacterioorus.J. Bacteriol. 123:481-491

6. Hespell, R. B., R. A. Rosson, M. F. Thomashow, andS. C. Rittenberg. 1973. Respiration of Bdellovibriobacteriovorus strain 109J and its energy substrates forintraperiplasmic growth. J. Bacteriol. 113:1280-1288.

7. Horowitz, A. T., M. Kessel, and M. Shilo. 1974. Growthcycle of predacious bdellovibrios in host-free extractsystem and some properties of the host extract. J.Bacteriol. 117:270-282.

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