Vol. 158, No. 2JOURNAL OF BACTERIOLOGY, May 1984, p. 597-6020021-9193/84/050597-06$02.00/0Copyright © 1984, American Society for Microbiology

Attachment of Diaminopimelic Acid to Bdelloplast PeptidoglycanDuring Intraperiplasmic Growth of Bdellovibrio bacteriovorus 109J

E. G. RUBY'* AND S. C. RITTENBERG2

Department of Biological Sciences, University of Southern California, Los Angeles, California 90089,1 and Department ofMicrobiology, University of California, Los Angeles, California 900242

Received 2 November 1983/Accepted 19 January 1984

An early event in the predatory lifestyle of Bdellovibrio bacteriovorus 109J is the attachment ofdiaminopimelic acid (DAP) to the peptidoglycan of its prey. Attachment occurs over the first 60 min of thegrowth cycle and is mediated by an extracellular activity(s) produced by the bdellovibrio. Some 40,000 DAPresidues are incorporated into the Escherichia coli bdelloplast wall, amounting to ca. 2 to 3% of the totalinitial DAP content of its prey cells. Incorporation of DAP occurs when E. coli, Pseudomonas putida, orSpirillum serpens are the prey organisms. The structurally similar compounds lysine, ornithine, citrulline,and 2,4-diaminobutyric acid are not attached. The attachment process is not affected by heat-killing the preynor by the addition of inhibitors of either energy generation (cyanide, azide, or arsenate), protein or RNAsynthesis (chloramphenicol and rifamycin), or de novo synthesis of cell wall (penicillin or vancomycin).Approximately one-third of the incorporated DAP is exchangeable with exogenously added unlabeled DAP,whereas the remaining incorporated DPA is solubilized only during the lysis of the bdelloplast wall.Examination of DAP incorporation at low prey cell densities suggests that bdellovibrios closely couple theincorporation to an independent, enzymatic solubilization of DAP by a peptidase. The data indicate thatDAP incorporation is a novel process, representing the second example of the ability of the bdellovibrio tobiosynthetically modify the wall of its prey.

The predatory lifestyle of the bdellovibrios is character-ized by a number of unique biochemical and physiologicaladaptations, a most striking and distinctive example ofwhich is the modification of the prey cell into the bdelloplast(16). The structural modifications that lead to the formationof the bdelloplast are responsible for converting the prey cellinto a secured environment in which the intracellular bdello-vibrio carries out the growth phase of its life cycle (8).Predominant among these modifications are degradativeattacks on the peptidoglycan of the prey cell by severalbdellovibrio-specified enzymatic activities including an N-deacetylase, a protease, a glycanase, and a peptidase (12,13). Of particular significance to the work reported here isthe peptidase, which cleaves diaminopimelic acid (DAP)residues from the peptidoglycan (13). In addition to degrada-tive modifications, the bdellovibrios also mediate biosyn-thetic work including the covalent attachment of fatty acids(14), perhaps as complete phospholipids (M. Thomashow,H. Aitchison, and S. C. Rittenberg, unpublished data), tothe modified bdelloplast. The study reported here, whichgrew out of preliminary observations by M. Thomashow,presents further evidence of the capacity of the bdellovibriofor synthetic modification of its growth chamber, an attach-ment ofDAP residues to the peptidoglycan of the bdelloplastwall.

MATERIALS AND METHODSOrganisms and general culturing procedure. Bdellovibrio

bacteriovorous 109J was maintained on Escherichia coliML35 as previously described (12). Pregrown E. coli ML35or W7M5, a lysine and DAP double auxotroph unable tocatabolize glucosamine (12), were used as the substrate cells

* Corresponding author.

for bdellovibrio cultures. To obtain synchronous bdellovi-brio cultures, equal volumes of suspensions of bdellovibrios(2 to 3 x 1010 cells per ml) and E. coli W7M5 or ML35 (1 x1010 cells per ml) were mixed in HM buffer (1 mM N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid adjustedto pH 7.6 with NaOH plus 1 mM CaCl2 and 0.1 mM MgC92)and incubated with vigorous shaking at 30°C. The resultingsingle cycle of growth took about 2.5 to 3 h to complete.

Labeling of E. coli peptidoglycan. When appropriate, E.coli W7M5 peptidoglycan was labeled with [3H]DAP aspreviously described (12). For some experiments, high-specific-activity DAP-labeled cells were produced by thefollowing modifications. One hundred microliters of an over-night culture of E. coli W7M5 were inoculated into 5.5 ml ofnutrient broth medium, which contained 5 g of nutrient broth(Difco Laboratories, Detroit, Mich.) per liter and 3 g of yeastextract (Difco Laboratories) per liter, adjusted to pH 7.6with NaOH, that was made 0.2 mM with lysine and 0.09 mMwith [G-3H]DAP (final specific activity, 3 ,uCi/ml). After 6 hof incubation with shaking at 30°C, the labeled cells (ca. 3 x109 cells per ml) were harvested, washed twice with coldnutrient broth medium, suspended in 13 ml of nutrient brothmedium supplemented with lysine (0.2 mM) and unlabeledDAP (0.1 mM), and shaken at 30°C for another 90 min tochase any free (pool) labeled DAP. The cells were thenharvested, washed in HM buffer, and suspended to 8.3 x1010 cells per ml (8.9 x 107 cpm/ml) in HM buffer.

Chemicals. Radiolabeled [G-3H]DAP and [U-_4C]D-ala-nine were obtained from Amersham Corp., ArlingtonHeights, Ill., and [1-14C]glucosamine was obtained fromNew England Nuclear Corp., Boston, Mass. Penicillin G,vancomycin, bacitracin, cycloserine, and chloramphenicolwere obtained from Sigma Chemical Co., St. Louis, Mo.Rifamycin (stock solution made daily as 5 mg/ml in 70%ethanol) was obtained from Calbiochem-Behring Corp., LaJolla, Calif.

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FIG. 1. Incorporation of [3H]DAP into bdelloplasts duringperiplasmic growth. A synchronous culture containing bdebrios, heat-treated E. coli ML35, and labeled DAP (1.0 VM;105 cpm/ml) was ipitiated. At 30 min into the cycle (arrow), addiwere made to separate portions of the culture as follow<additions (0); 100 pug of chloramphenicol per ml (O); 150 1rifamycin per ml (A); or, 100 pig of vancomycin per ml (0)amount of label incorporated into SDS-EDTA-insoluble ma

was determined on samples of culture removed at intervals. Ccsuspensions contained only bdellovibrios (U) or only heat-treatcoli (A).

Incorporation of radioactive label. The kinetics of inc(ration of labeled DAP into cold trichloroacetic acid (Tinsoluble macromolecules during bdelloplast formationdetermined as follows. Bdellovibrios were added to a

pension of heat-inactivated prey cells (65°C for 3 min)initiate a synchronous culture as described above. At50 min after initiation of attack, [G-3H]DAP was added tsuspension. Periodically, 50- to 75-pul samples of cu

were spotted onto Whatman 3MM filter paper (WhaInc., Clifton, N.J.) previously saturated with 10% TCdiethyl ether and air dried. These samples were fuprocessed by the method of Kimchi and Rosenbergfollows. One hour after spotting, the sheets of paperwashed three times (15 min each) in aqueous 10%(0°C). This was followed by sequential washing inethanol, ethanol-ether (1:1), and ether. The sheets werinto squares, which were then inserted into vials contairml of PCS solubilizer fluid (Amersham Corp., ArliiHeights, Ill.) for scintillation counting.

Label incorporation into peptidoglycan, defined o

tionally as sodium dodecyl sulfate (SDS)- and JEDTA-inble material, was determined after isolation of the perglycan by the procedure of Thomashow and RittenbergTo 5 volumes of cell culture, 1 volume of a 20% (wSDS-0.05 M EDTA solution, pH 7.6, was added.suspension was placed in a boiling-water bath for 10 miuthen held at 37°C overnight. The preparation was son]disrupted with a W200P sonifier (Branson Sonic PowerDanbury, Conn.) to fragment the nucleic acid, ancinsoluble material was sedimented by centrifugation (14x g, 1 h). The sedimented material was washed free ofEDTA by repeated suspension in and sedimentation frc

mM NaCI. The final sediments were suspended in 100 ,ul ofwater and transferred to PCS solubilizer fluid.

a Solubilization of labeled DAP from prey peptidoglycan.o Release of [3H]DAP residues from prelabeled prey cellpeptidoglycan during bdelloplast formation was determinedas previously described (12). At intervals during the first 60min, samples were removed from a synchronous culture,mixed with an equal volume of 10% TCA, and held at 0°C for60 min. Particulate material was sedimented by centrifuga-tion at 14,000 x g for 15 min in a Microfuge (Fisher ScientificCo., Pittsburgh, Pa.), and samples of the supernatant fluid,which contained the solubilized DAP, were transferred to

o Bray's Solution (Research Products International) for scin-tillation counting.Enzymatic digestion of the bdelloplast wall. The peptidogly-

can of the bdelloplast wall was solubilized with a lyticenzyme concentrate, prepared as previously described (10).Treatment of bdelloplasts with 10 mM EDTA in 120 mM

*~w Tris-hydrochloride buffer, pH 7.6, for 3 min at 30°C followed150 by dilution in 10 vol of lytic enzyme concentrate and

incubation for 30 min at 30°C solubilized over 80% of thebdelloplast peptidoglycan without affecting that of the bdel-

intra- lovibrios (10).Ilovi-4.9 x RESULTSitions Addition of [3H]DAP to a culture of B. bacteriovorus

s:g of growing synchronously on E. coli prey cells led to a rapid. The incorporation of label into the resulting bdelloplasts (Fig. 1).iterial Essentially identical results were obtained when either heat-)ntrol treated or unheated E. coli were used as prey. The formerLed E. type was generally employed to diminish the possibility that

catalytic enzymes of the E. coli influenced the experimentalresults. The amount of label incorporated into the peptido-glycan fraction (hot SDS-EDTA insoluble) of the culture was

)rpo- equivalent to that present in the total macromolecular frac-CA)- tion (cold TCA insoluble); thus, essentially all of the incor-were porated DAP was in the peptidoglycan fraction (Table 1).sus- The incorporation did not occur in suspensions containing

(3) to either bdellovibrios or prey cells alone. Use of bacterial cells20 to of any of several distinct genera (Escherichia, Pseudomo-:o the nas, or Spirillum) as prey for bdellovibrio attack led to theilture formation of bdelloplasts with similar patterns of DAPtman incorporation, thus indicating that incorporation was not a'A in property of a particular prey organism (data not shown).irther5) as

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TABLE 1. Incorporation of [3H]DAP into total cellularmacromolecules and peptidoglycan during the first 60 min of a

synchronous bdellovibrio cultureaIncorporated radioactivity (cpm/ml)

Time (min)Total Peptidoglycanb

0 345 2,630 2,390 (91)

10 4,230 4,010 (95)20 6,600 6,580 (100)30 7,770 6,540 (84)60 8,080 8,180 (102)

a Bdellovibrios and heat-treated E. coli ML35 were combined inHM buffer containing [3H]DAP (total radioactivity, 29,000 cpn/ml;total added DAP, 58 nM). At the indicated times, duplicate sampleswere taken, and the radioactivity that was incorporated into eithercold TCA-insoluble material (total) or hot SDS-EDTA-insolublematerial (peptidoglycan) was determined.

b Numbers in parentheses indicate the percentage of total macro-molecular radioactivity accountable as peptidoglycan.

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ATTACHMENT OF DAP TO BDELLOPLAST PEPTIDOGLYCAN 599

In synchronous cultures growing on E. coli, incorporationreached linear kinetics within 20 min of mixing bdellovibriosand prey cells (Fig. 1). Depending on the prey used, maxi-mum net incorporation occurred between 60 and 100 min.After a plateau period, solubilization of the label begancoincident with the onset of bdelloplast lysis and the exit ofprogeny bdellovibrios at the end of the growth phase. Aftercomplete release of progeny, at about 200 min, less than 7%of the incorporated label was still in the macromolecularfraction, and none of this material was SDS-EDTA insolu-ble.There are two possible sites of attachment of DAP to

peptidoglycan in the bdelloplast, the growing cell wall of thebdellovibrio or the modified peptidoglycan of the bdelloplastwall or both. The observation that the solubilization of labelcoincided with lysis of the bdelloplast and release of progenybdellovibrios suggested that the incorporation of DAP (Fig.1) was into the bdelloplast wall. However, solubilization ofincorporated label was also coincident with fragmentation ofthe bdellovibrio filament at the end of growth. This lattercoincidence, combined with the unusual characteristic ofrapid turnover of the peptidoglycan of freshly releasedbdellovibrios (15), argued for the incorporation of DAP intothe peptidoglycan of the bdellovibrio. To directly determinethe location of attachment, we used a lytic enzyme prepara-tion that specifically solubilizes bdelloplast peptidoglycanand not bdellovibrio peptidoglycan (10), thus making itpossible to differentiate between these two potential sites ofattachment. A 120-min bdelloplast suspension, to which[3H]DAP had been added at the initiation of attack, wasextensively washed to remove residual unincorporated labeluntil 99% of the total labeled DAP was in peptidoglycan-associated material of the bdelloplasts (Table 2). Treatmentof these bdelloplasts with a lytic enzyme concentrate solubi-lized most of the bdelloplast wall and released the growingbdellovibrio cells so that they were intact and viable. Only9% of the label was associated with these released bdellovib-rios, whereas the balance was present in a solubilized form.Thus, DAP incorporated into bdelloplasts was almost exclu-

TABLE 2. Distribution of incorporated [3H]DAP after treatmentof bdelloplasts with lytic enzyme concentrate"

RadioactivitySuspension or Distribution'supernatant %siuo% Total

Before treatment:Supernatant fluid 200 1Bdelloplasts 20,000 99

After treatment:Supernatant fluid 17,900 91Bdellovibrios 1,710 9

a Heat-treated E. coli cells and bdellovibrios were combined inHM buffer containing [3H]DAP (1 ,uM; 5.0 x 105 cpm/ml). After 120min of incubation, the labeled bdelloplasts were collected by centrif-ugation and washed several times. The bdelloplasts were suspendedin buffer and centrifuged a final time. The distribution of labelbetween the sedimented bdelloplasts and supernatant fluid wasdetermined. The bdelloplasts were then treated sequentially withEDTA and lytic enzyme concentrate to solubilize bdelloplast cellwall and release intracellular bdellovibrios. The treated suspensionwas then centrifuged, and the label distribution was determined.

b Per 200 l. of starting bdelloplast suspension; average of dupli-cate samples.

sively localized in the bdelloplast wall peptidoglycan and notin the cell wall of the bdellovibrio.The addition, at 30 min after attack, of inhibitors of either

protein synthesis (chloramphenicol and, in other experi-ments, streptomycin) or RNA synthesis (rifamycin) did notinterfere with the incorporation process itself (Fig. 1). How-ever, these additions inhibited further morphogenic develop-ment of the bdellovibrios (4, 17), and phase-contrast micro-scopic observation of the cultures revealed that the normalgrowth cycle was not completed and that the bdelloplasts didnot lyse. Similarly, the release of incorporated DAP label didnot occur, but instead the plateau value was maintainedindefinitely (Fig. 1). Sodium azide, cyanide, and arsenatehave been reported to be inhibitors of bdellovibrio respira-tion and ATP formation (1). Control experiments showedthat these inhibitors also were effective on bdellovibrioslocated within the bdelloplast; however, inhibitor additionduring the early stages of bdelloplast formation had littleeffect on the rate of incorporation, suggesting that theprocess ofDAP incorporation was independent of continuedenergy generation by the bdellovibrios.

In addition to the lack of effect of metabolic inhibitors, twoadditional observations indicated that the incorporation ofDAP into peptidoglycan did not reflect activities associatedwith normal cell wall synthesis. First, the uptake of either oftwo other typical cell wall precursors, glucosamine or D-alanine, into bdelloplast peptidoglycan did not accompanythe DAP incorporation; i.e., added exogenously, radiola-beled substrates were not incorporated into SDS-EDTA-insoluble material during the growth cycle of bdellovibrios.Secondly, although de novo synthesis of both bdellovibrioand E. coli peptidoglycan was inhibited by cell wall antibiot-ics that are effective against gram-negative bacteria, incorpo-ration of labeled DAP into peptidoglycan was unaffected bythe addition of 100 ,ug of vancomycin per ml (Fig. 1), as wellas by either penicillin G (125 ,ug/ml), bacitracin (50 p.g/rnl), orcycloserine (400 pug/ml).During bdellovibrio attack and penetration of the prey

cell, there occurred not only the DAP incorporation shownhere (Fig. 1) but also a release of free DAP into the culturefluid by an as yet uncharacterized bdellovibrio peptidaseactivity (12). This release resulted in a pool of soluble DAPin the culture fluid. For interpretation of the DAP incorpo-ration studies, it was important to know to what extent thispool of solubilized DAP decreased the specific activity of thelabeled DAP added to the culture. As a means of calculatingthe actual specific activity of DAP, the initial rates ofincorporation of radioactivity were measured for parallelbdelloplast cultures containing 1 ,uM [3H]DAP, made 2, 3,10, or 30 ,uM with unlabeled carrier DAP. The concentrationof carrier DAP needed to decrease the initial rate of incorpo-ration by 50% was assumed to be equal to the effective DAPconcentration in the carrier-free culture. For the experimentdepicted in Fig. 2, an estimated concentration of 4.5 ,uM forthe carrier-free culture was indicated. A graphical compari-son of the predicted percent decrease in incorporation rateresulting from the addition of carrier DAP to a cultureassumed to already be 4.5 ,uM in DAP and the actualmeasured rates is given (Fig. 2 inset). The linear relationshipthat was observed satisfied the prediction that the effectiveconcentration of DAP in the carrier-free culture was about4.5 ,uM. In this experiment, then, the specific activity oflabeled DAP was ca. 140 mCi/mmol, and from this value itwas calculated that about 40,000 DAP residues were incor-porated per bdelloplast. Based on the reported peptidogly-can content of E. coli ML35 (5 p.g/1010 cells [7]), this

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FIG. 2. Rates of incorporation into bdelloplasts of [3H]DAP atdifferent specific activities. Synchronous cultures contained 1 ,uM[3H]DAP (9 x 105 cpm/ml) to which either no additions (@) or 1(0),3 (A), 10 (Fl), or 30 (A) nmoles of unlabeled carrier DAP per ml hadbeen added. The amount of label appearing in TCA-insolublematerial was determined during the growth cycle. Initial linearportions of the curves are drawn in solid lines. Inset: comparison ofthe actual measured decrease in initial rate to that predicted for anaddition of 0, 1, 3, 10, or 30 nmoles of carrier DAP per ml to a

suspension assumed to have attained a concentration of 4.5 puMDAP by the onset of DAP incorporation.

incorporation was equivalent to about 2% of the totalnumber of DAP residues in the original E. coli wall and toabout 3% of the remaining DAP residues in the bdelloplastwall at 60 min (12).The attachment of DAP to the bdelloplast peptidoglycan

exhibited characteristics of a partially reversible reaction.Exogenous addition of a high concentration of unlabeledDAP, but not lysine, ornithine, citrulline, or 2,4-diarninobu-tyric acid, to a bdelloplast suspension incorporating DAPresulted in an immediate and rapid release from the bdello-plast peptidoglycan of approximately one-third of the radio-activity already incorporated (Fig. 3). The remaining radio-activity was not exchangeable with the added unlabeledDAP and was not solubilized until lysis of the bdelloplastenvelope at the end of the normal bdellovibrio growth cycle.The simultaneous addition of chloramphenicol with theunlabeled DAP did not significantly affect the initial solubili-zation of label, but it did prevent the secondary releaseassociated with bdelloplast lysis.Because DAP solubilization (12) and DAP incorporation

into prey cell peptidoglycan occur concomitantly early inbdellovibrio attack, the question arose as to whether thesetwo activities were directly related. As previously men-tioned, the DAP-incorporating activity, which is expressedbetween 30 and 60 min after attack, is not diminished by thepresence of either chloramphenicol (Fig. 1) or NaCN. How-ever, such was not the case with the simultaneously occur-ring net release of DAP, which was significantly curtailed bythe addition of these inhibitors (Table 3). Thus, these twoprocesses do not appear to be the result of a common andreversible enzyme activity but instead are apparently meta-bolically distinct activities.High concentrations of prey, ca. 5 x 1Q9 cells per ml, were

employed in the experiment described above, and the DAPreleased during bdellovibrio attack and bdelloplast formation

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FIG. 3. Effect of exogenously added unlabeled DAP on thesolubilization of [3H]DAP that had been previously incorporatedinto bdelloplast peptidoglycan. A synchronous culture was initiatedby combining bdellovibrios with heat-treated E. coli ML35. Twentyminutes after initiation, [3H]DAP (0.51 ,uM; 2.5 x 106 cpm/ml) wasadded, and its incorporation into TCA-insoluble material was deter-mined periodically (@). Forty minutes into the cycle, portions of thesuspension received additions of either 500 nmoles of unlabeledDAP (0) per ml, 100 jsg of chloramphenicol (E) per ml, or bothunlabeled DAP and chloramphenicol (O), after which the presenceof labeled DAP in TCA-insoluble cell material was determinedperiodically.

attained concentrations in the culture fluid (ca. 4.5 ,uM) thatwere undoubtedly many fold higher than the concentrationsin the natural environment from which bdellovibrios havebeen isolated (18). An attempt was made, therefore, todetermine whether incorporation could be measured underconditions of low prey densities in which the only source ofDAP was that solubilized by the peptidase reaction duringbdellovibrio attack on its prey. E. coli W7M5 was grownunder conditions that achieved the highest practical specific

TABLE 3. Effect of metabolic inhibitors on the relative rates of[3H]DAP incorporation and release in synchronous cultures of

bdelloplastsDAP Net DAP

Addition incorporationa releaseb (%)

100 100Chloramphenicol 100 53

(100 ,ug/ml)NaCN (50 mM) 94 0

a Bdellovibrios and heat-treated E. coli W7M5 were combined inHM buffer containing [3H]DAP as described in the legend to Fig. 1.The effect of the addition of inhibitors, at 25 min after attack, onlabel incorporation was measured during subsequent incubation.The relative rates were calculated from the initial linear portions ofthe label incorporation curves (25 to 50 min). A relative rate of 100%was equivalent to ca. 300 cpm incorporated per 70 ,ul per 10 min.

b Bdellovibrios and heat-treated E. coli W7M5 prelabeled with[3H]DAP were combined in HM buffer as described previously (14).The effect of the addition of inhibitors, at 25 min after attack, on thenet release of DAP label was measured durinrg subsequent incuba-tion. The relative rates were calculated from the initial linearportions of the label release curves (25 to 60 min). A relative rate of100% was equivalent to about 250 cpm released per 70 ,u per 10 min.

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activity of incorporated [3H]DAP. Two synchronous cul-tures of bdellovibrios growing on E. coli W7M5 were theninitiated that differed only in their use of these radiolabeledcells as prey in one culture and unlabeled cells in the other.After 30 min the cultures, now in the bdelloplast stage, werecooled to 0°C and centrifuged. The supernatant fluid derivedfrom the labeled culture, which contained about 25% of theinitial radioactivity, was then used to suspend the bdelloplastpellet from the unlabeled culture. Samples of this suspensionwere diluted up to 2,000-fold with HM buffer and reincubat-ed. The kinetics of incorporation of the solubilized, prey-derived, labeled DAP into the unlabeled bdelloplasts weredetermined. The data showed that DAP incorporation couldbe detected at bdelloplast concentrations as low as 3.1 x 106per ml (Fig. 4). However, it was evident that the efficiency ofincorporation of this external DAP was considerably re-duced when the label and bdelloplasts were both present atlower absolute concentrations. That is, the amount of labelincorporated into each bdelloplast was less in the morediluted suspensions although the total amount of label addedper bdelloplast was the same in each suspension.

DISCUSSIONWe report here a novel biochemical activity of bdellovib-

rios that results in the biosynthetic modification of the cellwall of its prey. The incorporation of DAP residues into theprefabricated peptidoglycan of another bacterium is withoutprecedent and complements the report of a similar attach-ment of fatty acids to the bdelloplast peptidoglycan bybdellovibrios (14). The existence of two such biosyntheticmodifications of the bdelloplast wall suggests that the bdello-vibrio carefully controls the characteristics of its growthchamber.The DAP attachment process is not a function of the

particular prey cell attacked but instead occurs over a rangeof prey genera, indicating that it is a programed, generalfacet of the attack process. Heating the prey cells to 65°Cbefore attack has no effect on subsequent activity, butsimilarly heating a 30-min bdelloplast culture during theperiod of incorporation eliminates continued activity. Theseresults indicate that DAP incorporation is mediated by abdellovibrio enzymes(s) and not by an activity that is intrin-sic to the prey cell. Because a number of structurally similaramino acids like lysine and 2,4-diaminobutyric acid wereunable to compete with DAP, the attachment process exhib-its a degree of specificity. Incorporation does not result fromthe processes of normal peptidoglycan synthesis. In fact,unlike the more typical intracellular enzymes of DAP metab-olism (2), this activity is extracellular, i.e., completelyoutside of the bdellovibrio cell, although within the confinesof the bdelloplast. Taken together, the above propertiessuggest that DAP incorporation is brought about by a novelextracellular enzyme(s) produced by bdellovibrios early indevelopment.DAP incorporation is not significantly inhibited by chlor-

amphenicol or NaCN as is the DAP-releasing peptidaseactivity reported by Thomashow and Rittenberg (12). Thus,DAP incorporation relies upon neither continued proteinsynthesis nor metabolic energy generation. One possibleexplanation for this insensitivity to metabolic inhibition isthat the attack-phase bdellovibrios carry preformed theDAP-incorporating activity, perhaps as a proenzyme thatis released as an active extracellular enzyme during the earlystages of penetration and bdelloplast formation. The rapidburst of DAP-incorporating activity appears only during thefirst 60 min, that is, during attack and stabilization of the

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FIG. 4. Incorporation of solubilized [3H]DAP into bdelloplasts atlow cell density. Two suspensions of heat-treated E. coli W7M5 (6.2x 109 cells per ml), one of which was labeled with [3H]DAP to a highspecific activity (1.5 x 107 cpm/ml) as described in the text, were

incubated with bdellovibrios (1.2 x 1010 cells per ml). After 30 min,the suspensions of bdelloplasts were cooled to 0°C and centrifuged,producing cell-free supernatant fluids. The supernatant fluid fromthe labeled suspension containing solubilized DAP (3.7 x 106 cpm/ml) was used to suspend the unlabeled bdelloplast pellet. Thissuspension was diluted 20-, 200-, and 2,000-fold with HM buffer todensities of 3.1 x 108 (0), 3.1 x 107 (d), and 3.1 x 106 (A)bdelloplasts per ml, and the resulting dilutions were brought to 30°Cfor continued incubation. At intervals, samples containing 6.2 x 108bdelloplasts were filtered through 0.2-,um membrane filters (Milli-pore Corp., Bedford, Mass.) and washed with HM buffer. The filterswere then dried and counted in scintillation cocktail.

bdelloplast. In this way it is similar to other bdellovibrioextracellular enzyme activities such as their glycanase,whose activity is regulated by an elegant and unusualmechanism of substrate modification (12).The incorporation process requires the presence of DAP

in the surrounding milieu, and because growing bdellovibriosdo not incorporate extracellular DAP into their peptidogly-can, their own processes of cell wall synthesis do notcompete for any external DAP that is present. Free DAP isgenerated by the degradative peptidase activity that releasesDAP from the peptidoglycan tetrapeptide of gram-negativebacteria (9), the broad taxon to which all known prey

organisms belong (6). Under natural conditions of bdellovi-brio growth, there will be no experimentally supplied source

of DAP, and concentrations of prey cells will rarely, if ever,be as high as the 5 x 109 cells per ml used in most of thelaboratory cultures described herein. In experiments inwhich only DAP released from the prey wall by peptidaseactivity was available, incorporation of this prey-derived,extracellular DAP was detected in bdelloplast suspensions atdensities as low as ca. 3 x 106 bdelloplasts per ml. Experi-ments at lower bdelloplast concentrations were not techni-cally feasible.

This incorporation was proportionately less at higherdilutions of bdelloplasts even though the total amount ofextracellular labeled DAP available per bdelloplast was the

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same in each case. If we assume that a fixed amount of DAPis incorporated into a bdelloplast during its development,then at higher dilutions of bdelloplasts, in which the incorpo-ration of extracellular (labeled) DAP is proportionately less,a greater percentage of the total DAP incorporated perbdelloplast must be coming from the unlabeled DAP poolthat is being continuously produced from solubilization ofthe bdelloplast peptidoglycan. Thus, bdellovibrios may tight-ly couple the enzymatic release of DAP to the incorporationprocess, preferentially utilizing the solubilized DAP before itcan diffuse out of the bdelloplast envelope and be lost bydilution. Such coupling may be overwhelmed by experimen-tally introduced, unusually high external DAP concentra-tions, leading to the linear relationship observed in the insetof Fig. 2. Under the more natural conditions of low concen-trations of extracellular DAP, the coupling may be importantin retaining and utilizing solubilized DAP before it is lostfrom the bdelloplast. In fact, bdellovibrios have been previ-ously shown to exhibit just such an efficient incorporation ofother monomeric breakdown products (amino acids, nucleicacids, etc.) of the prey cell with only a minimal loss to theexterior (8).Although the chemical nature of the attachment to the

peptidoglycan is unknown, the fact that the product with-stands extraction with boiling SDS and EDTA and multiplewashes with 10 mM NaCl indicates that it is a strong,probably covalent bonding. Presumably such an attachmentinvolves a synthetic, endergonic reaction and, thus, wouldneed to be coupled to an energy-releasing process of as yetunknown nature. It is possible that the incorporation of DAPinto bdelloplast peptidoglycan consists of more than a singletype of attachment because only about one-third of thelabeled DAP was solubilized when unlabeled DAP wasadded (Fig. 3). These data may alternatively be interpretedas suggesting a two-step process, the second of which leavesthe incorporated DAP unavailable for exchange. This DAP isfreed from the peptidoglycan only upon the natural lysis ofthe bdelloplast wall during release of bdellovibrio progeny(Fig. 3) or by artificial treatment with a lytic enzymeconcentrate (Table 2).

Until the structural location of the attached DAP isascertained, no function can be ascribed to the incorpo-ration. The similar kinetics of attachment to and release frompeptidoglycan described for fatty acids (14) and DAP (Fig. 1)suggest that the two processes may be connected. Regard-less of its actual function, the previously described attach-ment of fatty acids and the DAP incorporation reported hereindicate that biosynthetic work is occurring on the bdello-plast wall; thus, the bdellovibrios are capable of conductingtemporally controlled enzymatic synthesis on a structuretotally external to themselves. This constitutes a level ofextracellular anabolic control that is potentially even moresophisticated than that of normal outer-envelope synthesis,because here the catalytic site is not an enzyme that is fixedin the organized cytoplasmic membrane. To our knowledge,such an ability has not been previously described within theprocaryotes, although investigations of vesicular membranegrowth during chlamydial infection (11) suggest that similarprocesses might well be occurring undiscovered in intracel-lular host-parasite interactions.

ACKNOWLEDGMENTThis research was supported by grant PCM80-13467 from the

National Science Foundation to S.C.R.

LITERATURE CITED1. Gadkeri, D., and H. Stolp. 1975. Energy metabolism of

Bdellovibrio bacteriovorous. I. Energy production, ATP pool,energy charge. Arch. Microbiol. 102:179-185.

2. Ghuysen, J. M., and G. D. Shockman. 1973. Biosynthesis ofpeptidoglycan, p. 37-130. In L. Leive (ed.), Bacterial mem-branes and walls. Marcel Dekker, Inc., New York.

3. Hespell, R. B. 1978. Intraperiplasmic growth of Bdellovibriobacteriovorous on heat-treated Escherichia coli. J. Bacteriol.133:1156-1162.

4. Hespell, R. B., R. A. Rosson, M. F. Thomashow, and S. C.Rittenberg. 1973. Respiration of Bdellovibrio bacteriovorousstrain 109J and its energy substrates for intraperiplasmicgrowth. J. Bacteriol. 113:1280-1288.

5. Kimchi, A., and E. Rosenberg. 1976. Linkages between deoxyri-bonucleic acid synthesis and cell division in Myxococcus xan-thus. J. Bacteriol. 128:69-79.

6. Rittenberg, S. C. 1982. Bdellovibrios-intraperiplasmic growth,p. 379-391. In R. G. Bums and J. H. Slater (ed.), Experimentalmicrobial ecology. Blackwell Scientific Publishers, Oxford,England.

7. Rittenberg, S. C., and R. B. Hespell. 1975. Energy efficiency ofintraperiplasmic growth of Bdellovibrio bacteriovorous. J. Bac-teriol. 121:1158-1165.

8. Rittenberg, S. C., and M. F. Thomashow. 1979. Intraperiplasmicgrowth-life in a cozy environment, p. 80-86. In D. Schles-singer (ed.), Microbiology-1979. American Society for Micro-biology, Washington, D.C.

9. Rodgers, H. J. 1974. Peptidoglycans (mucopeptides): structure,function, and variations. Ann. N.Y. Acad. Sci. 235:29-51.

10. Ruby, E. G., and S. C. Rittenberg. 1983. Differentiation afterpremature release of intraperiplasmically growing Bdellovibriobacteriovorus. J. Bacteriol. 154:32-40.

11. Stokes, G. V. 1973. Formation and destruction of internalmembranes in L cells infected with Chlamydia psittaci. Infect.Immunol. 7:173-177.

12. Thomashow, M. F., and S. C. Rittenberg. 1978. Intraperiplasmicgrowth of Bdellovibrio bacteriovorus 109J: solubilization ofEscherichia coli peptidoglycan. J. Bacteriol. 135:998-1007.

13. Thomashow, M. F., and S. C. Rittenberg. 1978. Intraperiplasmicgrowth of Bdellovibrio bacteriovorus 109J: N-deacetylation ofEscherichia coli peptidoglycan amino sugars. J. Bacteriol.135:1008-1014.

14. Thomashow, M. F., and S. C. Rittenberg. 1978. Intraperiplasmicgrowth of Bdellovibrio bacteriovorus 109J: attachment of long-chain fatty acids to Escherichia coli peptidoglycan. J. Bacteriol.135:1015-1023.

15. Thomashow, M. F., and S. C. Rittenberg. 1978. Penicillin-induced formation of osmotically stable spheroplasts in non-growing Bdellovibrio bacteriovorus. J. Bacteriol. 133:1484-1491.

16. Thomashow, M. F., and S. C. Rittenberg. 1980. The intraperi-plasmic growth cycle-the lifestyle of the bdellovibrios, p. 115-138. In J. H. Parish (ed.), Developmental biology of prokary-otes. University of California Press, Berkeley.

17. Varon, M., and M. Shilo. 1968. Interaction of Bdellovibriobacteriovorus and host bacteria. I. Kinetic studies of attach-ment and invasion of Escherichia coli B by Bdellovibrio bacte-riovorus. J. Bacteriol. 95:744-753.

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