Vol. 147, No. 3JOURNAL OF BACTERIOLOGY, Sept. 1981, p. 869-8740021-9193/81/090869-06$02.00/0

Partial Characterization of Lipid A of IntraperiplasmicallyGrown Bdellovibrio bacteriovorusDAVID R. NELSONt AND SYDNEY C. RITTENBERG*

Department ofMicrobiology, University of California, Los Angeles, California 90024

Received 2 March 1981/Accepted 10 June 1981

The lipid A components of substrate cell origin incorporated by Bdellovibriobacteriovorus during intraperiplasmic growth (D. R. Nelson and S. C. Rittenberg,J. Bacteriol. 147:860-868, 1981) were shown to be integrated into its lipopolysac-charide structure. Lipid A isolated from bdellovibrios grown on Escherichiacoli was resolved into two fractions by thin-layer chromatography. Fraction 2 hadthe same Rf as the single lipid A fraction of axenically grown bdellovibrios, andboth stained identically with aniline-diphenylamine reagent. Fraction 1 resem-bled, in Rf and staining reaction, the slower migrating of two lipid A fractionsobtained from the E.coli used as the substrate cell. Both fractions 1 and 2contained glucosamine, a substrate cell-derived compound. Greater than 65% ofthe fatty acids in fraction 1 were derived from the substrate cell, whereas morethan 60% of the fatty acids of fraction 2 were synthesized by the bdellovibrio.Nevertheless, each fraction contained significant amounts of fatty acid of bothorigins. The substrate cell-derived fatty acids had the same distribution of N-acyland 0-acyl linkages as in E. coli lipid A. The data indicate that the two lipid Amoieties in lipopolysaccharide of intraperiplasmically grown bdellovibrios arehybrids of substrate cell-derived and bdellovibrio-synthesized components. Thedata also suggest that disaccharide units and N- and O-acyl linkages preexistingin the substrate cell lipid A may be conserved. A possible explanation for theunequal distribution of substrate cell-derived material in the two lipid A fractionsof the bdellovibrio is suggested.

In the preceding paper (6) we reported thatsubstrate cell lipid A components are incorpo-rated into Bdellovibrio bacteriovorus lipopoly-saccharide (LPS) during intraperiplasmicgrowth. The units used are taken up in verynearly the proportion in which they occur in thesubstrate cell and are conserved in bdellovibrioLPS for at least two intraperiplasmic culturegenerations. The data suggested that the incor-porated units might be taken up as intact lipidA moieties which serve as the base upon whicha complete LPS is synthesized. Alternatively,one could postulate that the substrate cell lipidA was degraded to its component units, whichentered in equal proportion into the pool fromwhich bdellovibrio LPS is synthesized. Thesealternatives can be formulated in another way,i.e., is the lipid A of intraperiplasmically grownB. bacteriovorus a hybrid of bdellovibrio-syn-thesized and substrate cell-derived materials, ordoes it exist as two distinct moieties havingdifferent compositions and structures related totheir origins? The experiments reported in this

t Present address: Department of Microbiology and Im-munology, University of California, Berkeley, CA 94720.

paper were an attempt to answer these ques-tions. They show the heterogeneous nature ofthe lipid A of intraperiplasmically grown bdel-lovibrios.

MATERIALS AND METHODSB. bacteriovorus 109J and 109JS, a wild-type and

facultative mutant capable of axenic growth, respec-tively, were the experimental organisms. Escherichiacoli W7M5 served as the substrate cell for the growthof strain 109J. A description of the organisms used anddetails of culture procedures, growth experiments, pu-rification of cell suspensions, and extraction of LPSare given in the preceding paper (6).

Preparation of lipid A from LPS. Lipid A wasobtained by hydrolysis of LPS in 1% acetic acid at100°C for 90 to 120 min (14). The hydrolysis releasesthe lipid A and renders it soluble in nonpolar solvents.Hydrolysates were extracted with chloroform threetimes. The chloroform extracts were combined,washed with distilled water, and then dried undernitrogen gas. Lipid A was also obtained from hydrol-ysates by centrifugation (20,000 x g, 20 min at 400)which sedimented the desired fraction. The sedi-mented material was washed twice with distilled waterand then extracted with chloroform. The chloroformextract was dried under nitrogen gas. Dried lipid Awas stored at -20°C.

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870 NELSON AND RITTENBERG

Fatty acid analyses. Fatty acids were obtainedafter hydrolysis of LPS or lipid A in vacuo in 4 N HCIfor 5 h at 100°C. Nonadecanoic acid (19:0) was addedto samples before hydrolysis as an internal standard.Fatty acid residues were methylated with BF3-meth-anol reagent, and the resulting fatty acid methyl esterswere identified and quantified by gas-liquid chroma-tography. The specific details for these procedureshave been described previously (6).To determine N-acyl and O-acyl fatty acids, a mod-

ification of the procedure described by Rietschel et al.(8) was followed. O-Acyl fatty acids were removed bytreatment with alkaline hydroxylamine, freshly pre-pared as described by Rapport and Alonzo (7). About3 mg of lyophilized LPS was treated with 3 ml ofalkaline hydroxylamine at 65°C for 2 min. The samplewas cooled to 0°C and centrfuged at 20,000 x g for 60min. The supernatant, which contained the O-acylfatty acids as hydroxamates, was acidified with 0.25 mlof concentrated HCl and heated at 100°C for 1 h toregenerate the free fatty acids. The sediment, whichcontained the N-acyl fatty acids of the de-O-acylatedLPS, was resuspended in 1.5 ml of 4 N HCl, nona-decanoic acid was added as an internal standard, andthe preparation was hydrolyzed in vacuo for 5 h at100°C. The fatty acids in the O-acyl and N-acyl prep-arations were extracted, methylated with BF3-metha-nol, and separated by gas-liquid chromatography as

described (6).TLC. Thin-layer chromatography (TLC) of lipid A

employed polysilicic acid-impregnated glass fibersheets (ITLC SA, Gelman Instruments Co., Ann Ar-bor, Mich.) (3). The TLC plates were activated byheating at 110°C for 2 h before use. Samples of lipidA dissolved in chloroform were applied to the plate 2cm above the bottom and allowed to dry. Plates weredeveloped in chloroform-methanol-water-ammoniumhydroxide (100:50:8:4) for about 2 h and were driedovernight at room temperature. Lipid A fractions weredetected either by staining or by measuring radioac-tivity. For visualization by staining, the chromatogramwas sprayed with a solution of aniline (4 ml), diphen-ylamine (4 g), and 85% phosphoric acid (20 ml) in 200ml of acetone and then heated at 100°C for 10 to 20min (3). The procedure yields brown or blue spots,presumably depending on the glycosidic bond in thelipid A disaccharide (11). Radioactive ([1-_4C]gluco-samine-labeled) lipid A fractions were located by cut-ting the chromatogram into 0.5-by-1.0-cm pieceswhose radioactivity was measured by liquid scintilla-tion counting.

Scintillation counting. Radioactive samples werecounted in 2.0 ml of PCS (Amersham, ArlingtonHeights, Ill.) solubilizer scintillation fluid with a Beck-man LS200 scintillation spectrometer.

RESULTS

Chloroform solubility of substrate cell-derived fatty acids in bdellovibrio LPS. LPSfrom intraperiplasmically grown bdellovibrioswas extracted with chloroform, and free lipid Awas isolated from the residue after its mild acidhydrolysis. The total fatty acids of the starting

LPS, the extracted LPS, the chloroform-extract,and free lipid A, were then released by morevigorous acid hydrolysis, methylated, and ana-lyzed by gas-liquid chromatography. An LPSsample isolated from the E. coli strain used asthe substrate was similarly analyzed as a control.The data show (Table 1) that no significantamount of fatty acid-containing material waspresent in the chloroform extracts of either theexperimental or control samples. Essentially allfatty acids in the initial LPS sample were re-tained during extraction and were found in thefree lipid A released by mild acid hydrolysis.These results show that the lipid A componentsderived from the substrate cell are integratedinto the bdellovibrio LPS and do not remain asfree lipid A.Qualitative analysis of lipid A by TLC.

The lipid A portion of B. bacteriovorus LPS iscomposed of de novo-synthesized and substratecell-derived components (6). Three possibilitiesfor its structure were considered. First, the totalsubstrate cell lipid A structure was conservedand incorporated as a unit into the bdellovibrioLPS. Second, substrate cell lipidA was degradedinto its component parts, which entered a com-mon pool of precursors for synthesis of bdello-vibrio lipid A. Third, some combination of thefirst two possibilities occurred; e.g., substrate celllipid A structure was partially conserved andmodified by addition of components synthesizedde novo. These possibilities were expected tolead to different results by TLC analysis, whichgives chromatographic patterns characteristic ofspecific lipid A structures (3). For example, ifthe first possibility above were the case, thechromatographic pattern for intraperiplasmi-cally formed lipid A should be the same as thatfrom a mixture of lipid As from the substratecells and from axenically grown bdellovibrios.

Lipid A preparations isolated from B. bacter-iovorus 109J grown intraperiplasmically on E.coli W7M5 (a K-12 mutant), from axenically

TABLE 1. Effect of chloroforn extraction on thefatty acid content ofLPS of intraperiplasmically

grown bdellovibriosaFatty acid content(pg/mg of starting

Sample LPS)B. bacter- E. coliiovorus

Starting LPS 109 120Chloroform-extracted LPS 104 88Chloroform extract 0.6 0.2Lipid A 84 126

a See text for experimental protocols.

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LIPID A OF B. BACTERIOVORUS 871

grown strain 109JS, and from E. coli W7M5were chromatographed on thin-layer plates ofpolysilicic acid and visualized with an aniline-diphenylamine stain. The results (Table 2)showed that lipid A from both intraperiplasmi-cally grown bdellovibrios and from E. coliyielded two widely separated chromatographicfractions. The result for the E. coli strain wasconsistent with the recent findings of Rosner etal. (9, 10), who showed that E. coli K-12 D31M4,a heptose-less LPS strain, possesses two distinctclasses of lipid A. Both E. coli fractions gave abrown color with the aniline-diphenylamine re-agent, whereas the slower-moving bdellovibriofraction stained brown and the faster-movingone stained blue. In contrast, lipid A from ax-enically grown B. bacteriovorus yielded a singlefast-running blue-stained component that co-migrated with the faster-moving fraction fromthe intraperiplasmically grown bdellovibrios.These data suggested that lipid A of intraperi-plasmically grown bdellovibrios was composedof two distinct moieties or classes of moieties.One, the slower-moving fraction, was E. coli-likein both its Rf value and stained color (see Table2). In contrast, the faster-migrating fraction wasmore bdellovibrio-like, with its Rf and stainedcolor similar to that of the axenic bdellovibriolipid A.When axenically grown strain 109JS lipid A

was mixed with E. coli lipid A and chromato-graphed, three spots were observed (Table 2).Two fractions moved with Rf values and color(brown) identical to those of E. coli lipid A. Thethird component stained blue and had an Rfvalue the same as that of strain 109JS lipid A.These data show that the two fractions observedin lipid A from intraperiplasmically grown bdel-lovibrios is not a result of simple mixing of alllipid A subunits found in E. coli and in 109JS.

[1-_4C]Glucosamine-labeled lipid A from E.coli W7M5 and from B. bacteriovorus 109Jgrown on the labeled substrate cells were chro-matographed as described above, and the chro-matogram was analyzed for radioactivity. Tworadioactive fractions were observed with eachlipid A (Fig. 1). The locations of the radioactivepeaks corresponded to the positions of thestained fractions found in the previous experi-ment (Table 2). Since we have previously shownthat all radioactivity incorporated into bdello-vibrio LPS under these experimental conditionsremains as unaltered glucosamine residues (6),the data show that both the slow-moving andfast-moving bdellovibrio lipid A fraction con-tained glucosamine residues, a substrate cell-de-rived material. Thus, the lipid A fraction ofintraperiplasmically grown bdellovibrios, which

TABLE 2. TLC of lipid A from axenically andintraperiplasmically grown B. bacteriovorus and

from E. colia* ~~~~~~~~ColorofLipid A source Rf spot

E. coli W7M5 0.54 Brown0.97 Brown

B. bacteriovorus 109JS (axenic) 0.91 Blue

B. bacteriovorus 109J (Ipb on E. 0.60 Browncoli W7M5) 0.92 Blue

E. coli + B. bacteriovorus 109JS 0.54 Brown(axenic)c 0.91 Blue

0.97 Brown

aApproximately 3 mg each of LPS from E. coliW7M5, B. bacteriovorus 109J, and B. bacteriovorus109JS were hydrolyzed to yield lipid A. The lipid Awas extracted into chloroform. The chloroform extractwas dried under N2, and the lipid A was dissolved in100 pl of chloroform. Ten microliters of each lipid Asolution was spotted onto the silicic acid plate. Theplate was developed in a solvent system of chloroform-methanol-water-ammonium hydroxide (100:50:8:4) forabout 90 min. The chromatogram was dried overnight,and the spots were visualized with aniline-diphenyla-mine.

b IP, Intraperiplasmic.C Ten niicroliters of each lipid A were spotted to-

gether.

0-11E

C-)

0. I

0._

I

Fraction NumberFIG. 1. Chromatography of [1-'4C]glucosamine-

labeled lipidA on silicic acid thin-layer plates. LipidA from [1-'4Clglucosamine-labeled E. coli W7M5(0) and from B. bacteriovorus 109J grown on thelabeled E. coli (0). Chromatography was as de-scribed in Table 2.

resembled axenically grown bdellovibrio LPS inRf and color reactions, in fact contained bothsubstrate cell-derived and de novo synthesizedcomponents.

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872 NELSON AND RITTENBERG

Analysis of fatty acids from lipid A frac-tions. The results of a typical fatty acid analysisof lipid A fractions separated by TLC are pre-sented in Table 3. Axenically grown B. bacter-iovorus lipid A, which migrated as a single frac-tion, had a fatty acid composition essentiallyidentical to that of axenic bdellovibrio LPS (6).The predominant species of fatty acid was 19:1,which made up almost 90% of the residues. Theslower-migrating fraction 1 of E.coli lipid A hadslightly decreased amounts of saturated fattyacids (12:0, 14:0, 16:0) and an elevated quantityof ,BOH14 as compared to either the faster-mi-grating fraction 2 or to the total E. coli LPSfatty acids (6). These data, indicating a non-

homogeneous distribution of individual fattyacids in the two E. coli lipid A fractions, are

consistent with the findings of Rosner et al. (9,10).The two intraperiplasmically grown bdellovi-

brio lipid A fractions were very different in fattyacid composition (Table 3). The more rapidlymigrating fraction 2 contained fatty acids pre-dominantly synthesized by the bdellovibrio;greater. than 60% of the fatty acid residues were19:1. The fatty acids of the slower-migratingfraction 1 were mostly derived from the sub-strate cell, with 8OH14 making up 67% of thetotal. The ratio of the two dominant fatty acids,19:1 and ,BOH14, was 3.4:1 in fraction 2 and 0.2:1 in fraction 1. Nevertheless, both fractions con-

tained significant amounts of fatty acid of eachorigin.Fatty acid linkages in B. bacteriovorus

and E. coli lipid A. Lipid A fatty acids fromboth axenic and intraperiplasmically grown B.bacteriovorus and from E.coli W7M5 were lib-erated in a stepwise fashion by successive treat-ments of LPS samples with alkaline hydroxyl-amine and 4 N hydrochloric acid. The former

treatment releases only O-acyl groups, whereasthe latter hydrolyzes the remaining N-acylgroups (8). The fatty acids in each preparationwere methylated and analyzed by gas-liquidchromatography. These results were comparedto the total fatty acids released by acid hydrol-ysis.The data for E. coli (Table 4) closely resem-

bled those reported by previous investigators (2,12). The only amide-linked fatty acid present insignificant amounts was 8OH14. About 60% ofthe hydroxy acid was in this linkage; the remain-ing ,B0H14 and virtually all of the saturatedfatty acids were present as O-acyl groups.The major lipid A fatty acid of axenically

grown B. bacteriovorus 109JS, 19:1, was about60% amide bound while the remaining 40% was

present as esters (Table 4). Hydroxy fatty acids,which were present in small and variable quan-

tities (6), were amide linked. In the experimentshown, only f8OH13 was found. The other fattyacids usually found in trace amounts in axenicbdellovibrio LPS were present primarily as es-

ters.As in axenically grown cells, about 60% of the

19:1 and all of the ,fOH13 in intraperiplasmicallygrown bdellovibrios were amide linked (Table4). The major E. coli-derived fatty acids had thesame distribution of linkages in the bdellovibriolipid A as they did in the donor cells. That is,about 60% of the ,8OH14 was amide bound, andalmost all of the 14:0 was O-acyl. Thus, thelinkages of the substrate cell-derived fatty acidswere to a large degree conserved.

DISCUSSION

The data presented in this and the precedingpaper (6) permit some conclusions and infer-ences about the structure of the lipid A of intra-

TABLE 3. Fatty acid composition of lipid A fractions separated by silicic acid TLCaFatty acid composition (% of total)

Sample Rf12:0 14:0 16:0 19:1 ,BOH14

B. bacteriovorus 1O9JR 0.91 NDb 0.7 2.5 87.5 1.5

E. coli W7M5Fraction 1 0.54 10.2 16.2 9.9 ND 61.7Fraction 2 0.97 11.5 25.2 8.5 ND 54.8

B. bacteriovorus 109JFraction 1 0.60 2.4 1.7 7.7 12.7 67.0Fraction 2 0.92 5.2 8.2 6.2 61.4 18.1a Lipid A samples from 3 to 5 mg of LPS were applied to silicic acid thin-layer plates (one sample per plate)

and chromatographed as described in Table 2. Lipid A fractions were located by staining or radioactivity asdescribed in the text. Fractions were cut out and extracted with chloroform-methanol (2:1). Extracted lipid Awas hydrolyzed to give free fatty acids, which were methylated and analyzed by gas-liquid chromatography.

b ND, Not detected.

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LIPID A OF B. BACTERIOVORUS 873

TABLE 4. Nature offatty acid linkages in B. bacteriovorus and E. coli lipid A a

Fatty acid linkages (% of total)

E. coli W7M5 B. bacteriovorus grown:Fatty acid

Axenically IntraperiplasmicallyO-Acyl N-Acyl

O-Acyl N-Acyl O-Acyl N-Acyl

12:0 5.0 0.5 NDb ND 0.2 0.714:0 15.0 0.7 2.5 2.0 7.4 0.716:0 5.4 1.6 2.0 ND 2.5 2.319:1 ND ND 33.8 47.2 17.2 22.3fiOH13 ND ND 0.7 11.8 ND 3.3/iOH14 29.4 42.3 ND ND 17.8 25.6

aAbout 3 mg of each type of LPS was first treated with alkaline NH20H and then with 4 N HCI to liberateO-acyl and N-acyl fatty acids, respectively, and the released fatty acids were methylated and analyzed by gas-liquid chromatography.

b ND, Not detected.

periplasmically grown B. bacteriovorus 109J.First, the non-extractability by chloroform ofthe substrate cell-derived lipid A fatty acidsfrom unhydrolyzed LPS of intraperiplasmicallygrown bdellovibrios shows that these compo-nents are not present as free lipid A. Consideringthat they partition into the aqueous phase dur-ing a phenol-water extraction of bdellovibriocells, they must be part ofa fairly polar molecule.The partitioning behavior of this molecule ascompared to LPS derived from various roughmutants of E. coli and Salmonella (5), and theobservation that the ratio of neutral sugars toamino sugars is about the same in LPS frombdellovibrios grown axenically or intraperi-plasmically (6), suggest that the substrate cell-derived components are integrated into a com-plete LPS.

Second, the lipid A of intraperiplasmicallygrown bdellovibrios is heterogeneous in beingcomposed of two distinct fractions separable byTLC. One of these resembles, in Rf and in colorreaction with aniline-diphenylamine, the slow-moving fraction of E. coli lipid A, whereas theother is similar in these properties to the lipid Aof axenically grown bdellovibrios, which mi-grates as a single, fast-moving spot. However,both of these fractions are, in turn, heteroge-neous in terms of the origin of their subunits,some obviously being derived from the substratecell (e.g., fiOHl4 and glucosamine) whereas oth-ers are synthesized de novo by the bdellovibrio.This manifestation of heterogeneity stronglysuggests that each TLC fraction consists of asingle type of hybrid molecule. The altemateexplanation, that each TLC fraction is a mixtureof two kinds of molecules, one derived exclu-sively from the substrate cell and the othersynthesized de novo, appears much less likely.This is because the E. coli fraction similar to the

fast-moving bdellovibrio fraction does not co-migrate with it and has a different staining re-action, and because the lipid A of axenic bdel-lovibrios lacks a slow-moving fraction.

If, as suggested, both lipid A fractions of intra-periplasmically grown bdellovibrios are hybridmolecules, then the substrate cell lipid A mustbe processed into smaller units that are com-bined with units synthesized de novo to makethe lipid As. The question then arises as to whyone lipid fraction is so predominantly E. coli-like and the other so predominantly bdellovi-brio-like in fatty acid compositions and stainingreactions with aniline-diphenylamine. One pos-sible explanation is that both lipid As are syn-thesized from a common pool of precursors thatchanges in composition during the course of theintraperiplasmic growth cycle. It has been re-ported (10) that changes in the growth mediuminfluence the composition and structure of E.coli lipid As. With respect to the intraperi-plasmic growth cycle, we have shown (13) thatLPS components (as measured by the release ofradioactivity from [1-'4C]glucosamine-labeled E.coli) are solubilized during the penetration ofthe bdellovibrio into its substrate cell, and thatthis solubilization of radioactivity ceases afterthe first 20 to 30 min of the growth cycle. Thissuggests that the substrate cell-derived precur-sors may be the major components of the poolof biosynthetic units early in bdellovibrio growthand that the slow-moving lipid A fraction issynthesized at that time. Consistent with thissuggestion is the observation that the LPS iso-lated from purified sheathed flagella of B. bac-teriovorus grown on E. coli has a fatty acidcomposition similar to that of LPS from axeni-cally grown bdellovibrios (Linda Thomashow,Ph.D. Thesis, University of California, Los An-geles, 1979). It is known that the flagellum is

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874 NELSON AND RITTENBERG

made at the end of the growth cycle (1).A second possible explanation for the unequal

distribution of the E. coli-derived fatty acidsbetween the two bdellovibrio lipid A fractions isthat the assimilated subunits are, in part, par-tially conserved lipid A structures. The obser-vation that these fatty acids have about thesame distribution of linkages, amide and ester,in the parent and product lipid A gives somesupport to this suggestion. However, this simi-larity could also result from a similar substratespecificity of the N-acylating enzymes of E. coliand the bdellovibrio.The brown staining reaction of the E. coli-like

fraction also suggests conservation of the glyco-sidic linkage of the substrate cell lipid A disac-charide. The chemical basis for the developmentof a brown or blue color with the aniline-diphen-ylamine reagent is not completely understood.Schwimmer and Benvenue (11) reported that ina series of unsubstituted di- and oligosaccha-rides, a blue color with this reagent correlatedwith the presence of a glycosyl bond involvingthe 4-OH of the reducing end of the molecule. Abrowning reaction was given by those saccha-rides having a free OH at this position. It hasbeen shown that the glucosamine residues of E.coli lipid A are linked ,f(l -> 6) (4), leaving the4-OH free or O-acylated. The brown stainingreaction of the E. coli-like (slow-moving) lipid Afraction of the bdellovibrio suggests conserva-tion of this bond. One would predict from thisargument that no fucosamine should be presentin the slow-moving fraction. Unfortunately,amino sugar analyses of separate lipid A frac-tions were not done. Parenthetically, since fu-cosamine (a 6-deoxy sugar) is the only aminosugar in the LPS of axenic bdellovibrios, theblue color given by its lipid A with aniline-di-phenylamine suggests a fB(1-) 4) linkage of itsdisaccharide.

It is apparent that more detailed chemicalstudies of the lipid A of intraperiplasmicallygrown bdellovibrios will be required to deter-mine the precise structures of the two fractionsand the extent to which subunits ofthe substratecell lipid A are conserved. Although this is anintriguing question per se, there is perhaps amore compelling reason why the bdellovibriosshould be of interest to those concerned withlipid A biochemistry. Our data show that thebdellovibrio must catalyze a variety of degrada-tive cleavages of the lipid A moiety. It is notunreasonable to speculate that as a preliminaryto these processes it cleaves the lipid A from theLPS molecule. Considering that there is appar-ently as yet no available procedure which une-

quivocally yields unaltered lipid A from LPS (4),the search for such an enzyme in the bdellovibriocould prove very rewarding.

ACKNOWLEDGMENTISThis investigation was supported by grant PCM75-18883

from the National Science Foundation. D.R.N. held a fellow-ship from the Upjohn Corp. during part of the period of thisstudy.

LITERATURE CITED1. Burnham, J. C., T. Hashimoto, and S. F. Conti. 1970.

Ultrastructure and cell division of a facultatively para-sitic strain of Bdellovibrio bacteriovorus. J. Bacteriol.101:997-1004.

2. Burton, A. J., and H. E. Carter. 1964. Purification andcharacterization of the lipid A component of the lipo-polysaccharides from Escherichia coli. Biochemistry 3:411-418.

3. Buttke, T. M., and L. 0. Ingram. 1975. Comparison oflipopolysaccharides from Agmenellum quadruplicatumto Escherichia coli and Salmonella typhimurium byusing thin-layer chromatography. J. Bacteriol. 124:1566-1573.

4. Galanos, C., 0. Luderitz, E. T. Rietachel, and 0.Westphal. 1977. Newer aspects of the chemistry andbiology of bacterial lipopolysaccharides with specialreference to their lipid A component, p. 239-335. In T.W. Goodwin (ed.), International review of biochemistry,biochemistry of lipids II, vol. 14. University Park Press,Baltimore.

5. Galanos, C., 0. Luderitz, and 0. Westphal. 1969. Anew method for the extraction ofR lipopolysaccharides.Eur. J. Biochem. 9:245-249.

6. Nelson, D. R., and S. C. Rittenberg. 1981. Incorporationof substrate cell lipid A components into the lipopoly-saccharide of intraperiplasmically grown Bdellovibriobacteriovorus. J. Bacteriol. 147:860-868.

7. Rapport, M. M., and N. Alonzo. 1955. Photometricdetermination of fatty acid ester groups in phospholip-ids. J. Biol. Chem. 217:193-198.

8. Rietschel, E. T., H. Gottest, 0. Luderitz, and 0. West-phal. 1972. Nature and linkages of the fatty acidspresent in the lipid A component of Salmonella lipo-polysaccharides. Eur. J. Biochem. 28:166-173.

9. Rosner, M. R., H. G. Khorana, and A. C. Satterthwait.1979. The structure of lipopolysaccharide from a hep-tose-less mutant of Escherichia coli K-12. II. The ap-plication of 31p NMR spectroscopy. J. Biol. Chem. 254:5918-5925.

10. Rosner, M. R., J. Tang, I. Barzilay, and H. G. Khor-ana. 1979. Structure of the lipopolysaccharide from anEscherichia coli heptose-less mutant. I. Chemical deg-radations and identification of products. J. Biol. Chem.264:5906-5917.

11. Schwimmer, S., and A. Bevenue. 1956. Reagent fordifferentiation of 1,4- and 1,6-linked glucosaccharides.Science 123:543-544.

12. Taylor, A., K. W. Knox, and E. Work. 1966. Chemicaland biological properties ofan extracellular lipopolysac-charide from Escherichia coli grown under lysine-lim-iting conditions. Biochem. J. 99:53-61.

13. Thomashow, M. F., and S. C. Rittenberg. 1978. Intra-periplasmic growth of Bdellovibrio bacteriovorus 109J:solubilization ofEscherichia coli peptidoglycan. J. Bac-teriol. 135:998-1007.

14. Wilkinson, S. G., and L. Galbraith. 1975. Studies oflipopolysaccharides from Pseudomonas aeruginosa.Eur. J. Biochem. 52:331-343.

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