INFECTION AND IMMUNITY, Feb. 1981, p. 668-673 Vol. 31, No. 20019-9567/81/020668-06$02.00/0

Use of Cycloheximide to Study Independent Lipid Metabolismof Chlamydia trachomatis Cultivated in Mouse L Cells Grown

in Serum-Free MediumSTELLA I. REED, LLOYD E. ANDERSON, AND HOWARD M. JENKIN*

The Hormel Institute, University ofMinnesota, Austin, Minnesota 55912

A system for measuring chlamydial lipid synthesis was developed with mouseL cells grown in serum-free modified Waymouth 752/1 medium in a shakerculture. Host lipid synthesis was reduced -90% when cells were incubated for 24h in medium containing cycloheximide (2 ug/ml). Lipid metabolism was moni-tored by measuring the incorporation of [3H]isoleucine into the total lipid ofnormal and infected cells. The results suggested that lipid synthesis of Chlamydiatrachomatis lymphogranuloma venereum (LGV-404L) was not inhibited by cy-cloheximide treatment when the chlamydiae were grown in L cells, whereas hostlipid synthesis was inhibited. Chlamydial lipid metabolism began about 6 to 12 hafter infection when the noninfectious reticulate body was found and continuallyincreased until the beginning of the appearance of intracellular infectious elemen-tary bodies at 24 to 30 h.

Cycloheximide (Cx), a glutarimide antibiotic,has been used to study different aspects of themetabolism of obligate intracellularly grownchlamydiae (1, 11, 20). Cx inhibits deoxyribo-nucleic acid and protein synthesis in eucaryotes(7), but not in procaryotes such as chlamydiae(1, 2). Ripa and Mirdh (19) have proposed theuse ofCx-treated cells for a more efficient systemof detecting and isolating Chlamydia tracho-matis strains from clinical specimens. Our stud-ies have been directed toward investigating chla-mydial lipid synthesis. When Cx is supple-mented in medium to grow L cells, induction offatty acid synthesis is prevented (18), which mayprovide a sensitive system to study chlamydiallipid metabolism to differentiate host and para-site lipid synthesis. Such a system would allowmonitoring of chlamydial lipid synthesisthroughout the developmental cycle. Past stud-ies on chlamydial lipid synthesis were done byusing a mixture of reticulate and elementarybodies isolated from embryonated chicken eggs(9) or in cells with ongoing host lipid metabolism(8).Cx-treated L cells grown in modified Way-

mouth 752/1 (WO5) medium (17) without serumor undefined exogenous lipids provides an idealsystem for studying chlamydial lipid synthesis.The shaker culture system provides a means ofobtaining high yields of cells and chlamydiaewith a minimal chance of contamination (10).We were able to monitor chlaymdial lipid syn-thesis by measuring the incorporation of [3H]-

isoleucine, an amino acid required for L-cell andchlamydial growth (11), into total lipids.

MATERIALS AND METHODSCells. Mouse L cells, clone 5b, originally obtained

from J. Moulder (University of Chicago, Chicago, Ill.)and routinely grown in our laboratory in spinner cul-ture in W05 serum-free medium (17), were adapted togrow in a shaker culture with the same medium. Theserum-free medium W05 contains a premixed combi-nation of 5 jig of sodium oleate per ml and 2 mg offatty acid-free bovine serum albumin per ml (Pentex,Inc., Division of Miles Laboratories, Inc., Kankakee,Ill.). Viable and nonviable cell counts were made byusing the trypan blue dye exclusion test with a Cyto-graf cell counter (Ortho Instruments, Westwood,Mass.).

Monolayers of monkey kidney (LLC-MK-2) cells(12) were grown in Eagle minimum essential mediumsupplemented with 5% newborn calf serum (MEM6),as described by Fan and Jenkin (8).

Preparation of chlamydial seed in L cellsgrown in shaker culture. C. trachomatis LGV-404Lwas kindly supplied by J. Schachter (University ofCalifornia, San Francisco). LGV-404L (multiplicity ofinfection, -3) was mixed with L cells and centrifugedat 600 x g for 20 min at room temperature. Theabsorption period was continued for 45 min at 37°C ina New Brunswick Gyrotory shaker (125 rpm). Theinfected cell suspension (106 cells per ml) was trans-ferred to a screw-cap Erlenmeyer flask containing W05medium and incubated at 37°C in the Gyrotory shakerbefore harvesting at 42 h. The infected cells werecentrifuged for 10 min at 300 x g to sediment the cells.After the supernatant fluid was discarded, the infectedcells were suspended in a sucrose-potassium-gluta-

668

VOL. 31, 1981

mate diluent (5) (_107 cells per ml) at 4°C and soni-cated to release the chlamydiae from the cells. Thissuspension was again centrifuged to sediment the cell

debris, and the supernatant fluid containing chlamy-diae was rapidly frozen in alcohol-dry ice and storedat -76°C.

Cell infection and chlamydial titration. L cellswere infected by the procedure described above forthe preparation of the chlamydial seed. Chlamydiaewere titrated in MK-2 cells by the procedure of Kuoet al. (15). The inoculum was centrifuged onto cells

(106 cells per vial) at 600 x g for 30 min. After centrif-ugation, 1 ml of enriched MEMlo (8) containing strep-tomycin and vancomycin (100 ug of each per ml) was

added to each shell viaL Cells were incubated at 370Cfor 48 h. The cells on the cover glasses were stainedwith May-Grunwald Giemsa and mounted on slides.The inclusion-forming units (IFU) per milliliter were

determined microscopically as the percentage of in-fected cells from 500 to 1,000 cells per cover glass. Todetermine the percentage of infected cells after ab-sorption, 106 cells were added in enriched MEMio toshell vials. The cultivation and counting procedureswere performed as described above.

Lipid extraction. Lipids from uninfected and in-fected LGV-404L cells were extracted by the methodof Bligh and Dyer (4). Two samples from each lipidextraction were transferred to preweighed aluminum

pans and dried to constant weight in a vacuum desic-cator over KOH pellets, and the gravimetric weightwas determined on a Cahn microbalance (Cahn In-strument Co., Paramount, Calif.). Each pan was placedin a scintillation vial, and 0.7 ml of methanol-water (2:1) was added to remove dried lipid from the pan beforethe scintillant was added.

Protein extraction. Protein was extracted, andthe amount of [3H]isoleucine incorporated into proteinwas determined by the procedure of Alexander (1).Protein was measured by the method of Lowry et al.(13).

Radioactivity determination. L-[4,5-3H]isoleu-cine (97.6 Ci/mmol) was purchased fromNew EnglandNuclear Corp. (Boston, Mass.). The radioactivity ofeach lipid sample was determined in a toluene-basedscintillant containing Triton X-100 (33%) preparedwith Omnifluor (New England Nuclear Corp.). Theamount of radioactivity present in the protein sampleswas measured in a dioxane-based scintillant preparedwith Permablend I (Packard Instrument Co., Inc.,Downers Grove, ill.) with a Packard liquid scintillationcounter (model 2405). Specific activities were ex-

pressed as the amount of radioactivity in the lipidsample per weight of the lipid sample or as the amountof radioactivity in the protein sample per weight of theprotein sample.

Effect of Cx on L-celi viability. Cells from thesame population, grown in shaker culture, were usedfor each set of experiments. Cell populations of 2 x106 and 5 x 101 cells per ml were each distributed to12 flasks, for a total of 24 flasks. Three flasks fromeach group of 12 were incubated with either 0, 2, 5, or

10 pg of Cx per ml in WO5 medium. Duplicate 0.5-mlsamples were removed from each flask at 0, 24, 48, 72,and 96 h. Total and nonviable cell counts were deter-

CHLAMYDIAL LIPID METABOLISM 669

mined as described above.Effect ofCx on L-celi lipid and protein synthe-

sis. Lipid synthesis was monitored by measuring theincorporation of [9H]isoleucine into the total lipid ofL cells. Cells were divided into 3 groups and treatedwith 0, 2, or 5 ,ug of Cx per ml. Each group wassubdivided into six flasks containing -2.0 x 107 cells(2 x 106 cells per ml) and incubated at 370C until timefor the 6-h pulse with [3H]isoleucine. Cells were pulsedby using 4 ,uCi per flask of [3H]isoleucine for 18 to 24h, 42 to 48 h, and 66 to 72 h. At the end of each pulseperiod, the cells were harvested and washed threetimes with phosphate-buffered saline (pH 7.4), and thelipid was extracted. Lipid and protein specific activitieswere determined as described above.To determine at what time lipid synthesis was in-

hibited with respect to protein synthesis, cells weredivided into two groups and treated with 0 or 2 pg ofCx per ml. Cells were manipulated as described above.Lipid synthesis was monitored in cells pulsed for 0 to4 h, 0 to 6 h, 6 to 12 h, 12 to 18 h, and 18 to 24 h.Protein synthesis was monitored in cells pulsed for 0to 4 h, 6 to 12 h, and 12 to 18 h. Lipid and proteinspecific activities were determined as described above.LGV-404L infectivity in L celis pretreated with

Cx. Cells (2 x 106/ml) were incubated with Cx (2 ug/ml) for 0, 2, 4, 12, 18, and 24 h before infection withLGV-404L to determine whether Cx treatment af-fected the susceptibility of L celLs to LGV-404L infec-tivity. Cells were infected with a multiplicity of infec-tion of 3 to produce '100% infection, as describedabove. At the end of the absorption period, the per-centage of infected cells was determined. Cells fromeach Cx time treatment were diluted to 105 cells perml in enriched MEM,o, and 1 ml of cell suspensionfrom each duplicate set of time treatments was placedin triplicate shell vials. After 48 h of incubation, coverglasses were removed and stained, and the number ofIFU per milliliter was measured as described earlier.Growth cycle of LGV-404L in Cx-treated L

celis. Cells were treated with Cx for 24 h and infectedas described above. After absorption with LGV-404L,cells were suspended (2 x 106 cells per ml) in W05medium containing 2 pg of Cx per ml and incubated at370C. Samples (2 ml) were removed from duplicateflasks at 0, 24, 30, 36, 48, and 96 h for chlamydialtitration and cell counts. Cell-associated infectivityand supernatant fluid infectivity were determined foreach sample.

Effect of Cx treatment on lipid synthesis ininfected and uninfected L cells grown in shakerculture. L cells (2 x 106 cells per ml) were incubatedwith either 0 or 2 pg of Cx per ml at 370C for 24 h.Half of the Cx-treated group and the untreated groupwas infected with LGV-404L, and the other half wasleft uninfected: (i) Cx-treated, infected; (ii) Cx-treated,uninfected; (iii) no Cx, infected, (iv) no Cx, uninfected.Each group was subdivided into 10 flasks (2 x 107 cellsper flask) and pulsed with [3H]isoleucine (4 pCi perflask) for 6-h periods as described above. Cells were

pulsed for 0 to 6 h, 6 to 12 h, 12 to 18 h, 18 to 24 h, and24 to 30 h. Cells were harvested and washed, the lipidwas extracted, and specific activities were determinedas described above.

670 REED, ANDERSON, AND JENKIN

RESULTSEffect of Cx on L-cell viability. The toxic

effect of various concentrations of Cx on L-cellviability was determined at differen.t cell densi-ties. No cell growth was observed in any culturestreated with 2, 5, or 10 ,ug of Cx per ml (Fig. 1).A decrease of less than 10% in the number ofviable cells was observed in low- and high-den-sity cell cultures, 5 x 105 and 2 x 106 cells perml, respectively, treated with 2 or 5 ,ug of Cx perml between 0 and 48 h of incubation. All culturestreated with 5 or 10 ,ug of Cx per ml showed adecrease in the number of viable cells after 48 hof incubation. The greater the concentration ofCx, the greater the decrease in cell viability.Percentage of LGV-404L-infected cells

after Cx treatment. L cells were pretreated for24 h with 2 jig of Cx per ml followed by infectionwith C. trachomatis. Cx was added to the me-dium again for the rest of the infection. Fromthe fact that 87 to 94% of the cells containedinclusions when six separate samples were pe-riodically taken between 8 and 24 h of infection,

6- '----- 0,iq0'uq106 5

2 ,uq

_ 5 Jq

U05

one can conclude that Cx does not inhibit thenumber of chlamydial inclusions.LGV-404L growth cycle in Cx-treated L

cells grown in serum-free medium. A typicalgrowth curve of LGV-404L cells cultivated inthe presence ofCx is shown in Fig. 2, correspond-ing to the growth curve of Bernkopf et al. (3) forcultivation of untreated cells infected with C.trachomatis. The first measurable intracellularinfectivity of LGV-404L was observed at 24 hpostinfection. Extracellular infectivity was notobserved until 30 h. A 500-fold increase in intra-cellular infectivity occurred by 36 h, and a 100-fold increase in extracellular infectivity was ob-served by 48 h of infection. Intracellular (36 h)and extracellular (48 h) infectivity decreasedrapidly after reaching a maximum titer. Thedecrease in the number of viable L cells infectedwith LGV-404L coincided with the decrease inintracellular infectivity. Experiments were per-formed to determine differences in the yield ofinfectious LGV-404L with and without supple-mentation of 2 ug of Cx per ml. After 48 h of

107

E(IOd 106

V

C3105

.E

2

1

0 24 48 72 96Hours

FIG. 1. Effect of Cx on viability of cells grown inshaker culture at high- and low-density populations.Cells were treated with 0, 2, 5, or 10 pg of Cx per mlin W06 medium. See text for experimental conditions.Viable cell counts were performed on duplicate sam-ples from triplicate flasks (n = 3). The data arerepresentative of two independent experiments.

L cells

- L"eL0~L cells-LGV

24 48 72 96TIME AFTER INFECTION (hrs)

FIG. 2. LGV-404L growth cycle in Cx-treated Lcells grown in serum-free medium. Infectivity is ex-pressed as IFUper milliliter. Viable cell counts wereperformed on duplicate samples from duplicateflasks(n = 2). See text for experimental conditions.

INFECT. IMMUN.

CHLAMYDIAL LIPID METABOLISM 671

incubation at 37CC, we found that the infectivitywas 1.6 X 107 IFU/ml with Cx and 2.3 x 107IFU/ml without Cx. These results showed no

appreciable difference in the infectious yield ofLGV-404L from inoculum prepared from twoserial passages in L cells. This growth curve issinilr to results we obtained with untreatedLGV-404L-infected L cells as well as resultsfrom LGV-440L-infected MK-2 cells reportedearlier by Fan and Jenkin (8). The extracellularinfectivity found after 48 h of incubation withoutCx for LGV-404L was 3 x 106 IFU/ml, which isconsidered to be an average yield for C. tracho-matis LGV-404L.Lipid and protein synthesis in L cells

grown in serum-free medium. Lipid synthe-sis, monitored by [3H]isoleucine incorporationinto total lipid, was reduced to the same extent(-89%) in L cells treated with 2 or 5 Ag of Cx per

ml by 24 h of incubation. Therefore, in all sub-sequent experiments a concentration of 2 ug ofCx per ml was used. No further inhibition oflipid synthesis occurred between 24 and 48 hwith 5 or 10 ,ug of Cx, but some toxicity of cells

was seen with 10 itg ofthe antibiotic. By decreas-ing the time of Cx treatment to less than 24 h,we observed that after 4 h of treatment lipidsynthesis in cells was inhibited only 51% (Table1), whereas protein synthesis was inhibited 95%by this time. Lipid synthesis in L cells was

inhibited -85 to 90% after 18 to 24 h of incuba-tion.Lipid metabolism and protein synthesis

in LGV-404L-infected L cells treated withCx. Since our method of cultivation of C. tra-chomatis agents was different from those usedin earlier investigations (1, 11), because it wasserum free, utilized a Gyrotory shaker ratherthan a spinner culture, and used a differentchlamydial agent, we decided to run a growthcurve to determine the dose response of Cx on

LGV-404L. In an earlier study by Alexander (1),

TABLE 1. Inhibition of lipid synthesis' in Cx-treatedb mouse L cells grown in serum-free medium

[3H]isoleucine Lipid synthesis in: % Inhibi-pulse (h) Treated cells Untreated cells tion

0to4 1.5±0.1O 3.1 ±0.9c 510to6 1.8±0.1 4.1±0.4 566to 12 1.2±0.3 5.1 0.7 7712to18 0.9±0.1 6.3±0.1 8618to24 0.8±0.1 7.3±0.7 89

a Monitored by [3H]isoleucine incorporation into

total lipid.b A Cx concentration of 2 ug/ml of W05 medium

was maintained throughout this experiment.c (Disintegrations per minute x 103 per milligram of

total lipid) standard deviation (n = 3).

2 h was the maximum time for pretreatment ofthe cells with Cx before an obvious lethal effectwas observed. In our system of cultivation wefound that pretreatment of cells 24 h beforeinfection did not appear to affect the viability ofL cells, which allowed inhibition of lipid synthe-sis (89%). The inhibition of host protein synthe-sis preceded lipid inhibition by 18 to 24 h.Our data on protein inhibition of host cells in

the presence of Cx were essentially the same asreported by Alexander (1) and Hatch (11), ex-cept our experimental time of observation with-out observing a lethal effect was longer thantheirs. In another series of experiments, proteinsynthesis was monitored as a control with lipidsynthesis of normal and infected cells in thepresence of 2 itg of Cx per ml. The amount of[3H]isoleucine incorporated into total lipid dif-fered between untreated LGV-404L-infectedcelLs and untreated uninfected celLs during the30-h observation period (Table 2). The amountof [3H]isoleucine incorporated into total lipid inLGV-404L-infected cells treated with Cx in-creased about twofold during the 6- to 12-h pulse(Table 2). LGV-404L-infected cells pretreatedwith Cx showed a continual increase in the in-corporation of [3H]isoleucine into lipid until 24h after infection. Treated LGV-404L-infectedcells in contrast to the treated uninfected cellsincorporated 100-fold more [3H]isoleucine 24 hafter infection (Table 2). The untreated LGV-404L-infected cells incorporated only -16-foldmore [3H]isoleucine into total lipid than diduntreated uninfected cells 24 h after infection.Cx treatment inhibited protein synthesis in

LGV-404L-infected and uninfected L cellswithin 0 to 4 h, as determined by using a [3H]-isoleucine probe. Cells were incubated in me-

TABLE 2. Lipid synthesis' in LGV-404L-infected Lcells treated with Cx

Lipid synthesis in:

Time of CX-treated cellsb Untreated cellsincuba-tion (h) Unin- Unin-

Infected fce Inected fce

6 5.0 ± 0.2c 1.7 ± 0.1 13.1 ± 0.1 14.8 ± 0.712 13.1 ± 0.4 1.7 ± 0.2 13.3 ± 3.6 7.6 + 0.218 107.0 ± 4.7 1.5 + 0.2 99.3 ± 8.8 7.9 ± 0.624 182.2 + 47.5 1.5 + 0.5 100.4 + 5.8 6.0 + 0.230 207.0 ± 10.6 2.7 ± 2.0 21.2 + 5.9 5.6 ± 0.1

aMonitored by [3H]isoleucine incorporation intototal lipid after a 6-h pulse.bL cells were pretreated for 24 h with 2 ,Lg of Cx per

ml of W05 medium.(Disintegrations per minute x 103 per milligram of

total lipid) + difference from the mean. Duplicateanalyses from each of two independent samples (n =2).

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672 REED, ANDERSON, AND JENKIN

dium containing Cx for 24 h before and 12 hafter infection. Chlamydial protein synthesis inthe presence of Cx was evident 12 h after infec-tion as shown by a twofold increase in the incor-poration of [3H]isoleucine into the protein frac-tion of LGV-404L-infected cells compared withuninfected cells.

DISCUSSIONAlexander (1) observed the usefulness of Cx

in monitoring chlamydial protein synthesis inhost L cells. Based on the results observed inFig. 1 and Table 1, tbe addition of Cx (2 ,ug/ml)to the medium 24 h before infection was used asa routine procedure for monitoring lipid metab-olism in chlamydia-infected cells. We examinedthe effects of Cx on L cells uninfected and in-fected with LGV-404L and grown in serum-freemedium in a shaker culture, since the dimen-sions of our system were different from those ofpreviously reported work (1, 11). The differencesobserved in L-cell toxicity in the presence of Cxbetween Alexander's observation and this reportcannot be explained at this time and are imma-terial for the interpretation of this work. Cellviability in the presence of Cx (2 jig/ml) wasmaintained for 72 h, well within the experimen-tal time frame of this study. Cell cultures wereroutinely started at -2 x 106 cells per ml (sta-tionary phase) to minimize the difference in themetabolic states of cells in each treatment con-dition, since no differences were observed in cellviability in the presence of 0 or 2 ,tg of Cx (Fig.1).

Isoleucine was chosen as a label for monitoringlipid metabolism because it is a branched-chainamino acid required for chlamydial metabolism(11) and can be transaminated to a branched-chain fatty acid (14) or degraded and used as asubstrate for phospholipid synthesis (6, 13).Gaugler et al. (9) observed that chlamydiae la-beled with isoleucine showed radioactivity inphospholipids. Chlamydiae contain procaryotic-type branched-chain fatty acids (9, 16), whichare absent in L cells, thereby distinguishing lipidsynthesis between eucaryotes and procaryotesin the presence of Cx.

In a study on the competition between Chia-mydia psittaci and L cells for isoleucine, Hatch(11) suggested that chlamydiae inefficientlycompete with the host cell for precursors ofmacromolecular synthesis. He postulated thatthe addition ofCx may spare essential precursorsin the soluble pools of the host for the biosyn-thetic needs of chlamydiae. There is a 16-folddifference 24 h after infection in the incorpora-tion of [3H]isoleucine between infected and un-infected cells. Use of [3H]isoleucine could very

INFECT. IMMUN.

well magnify lipid synthesis events in chlamy-diae since the high amount of branched-chainfatty acid synthesis (8) occurs as well as proteinsynthesis, whereas the host would incorporatethe label primarily in the protein and does notnormally utilize isoleucine to synthesize lipid.Also, since the cells are near the stationaryphase, host metabolic synthesis can be markedlyinhibited. The results are further magnified inthe treated infected and uninfected cells (-100-fold, Table 2). A concomitant increase in theinfectivity of C. trachomatis was not observedby us in treated uninfected cells, as Hatch (11)reported for a C. psittaci strain. Further inves-tigation is necessary to account for these differ-ences. Inhibition of host lipid synthesis with Cxnow provides a means to monitor early chlamyd-ial lipid synthesis.

Chlamydial lipid synthesis, as measured by[3H]isoleucine accumulation and pulse-labelingexperiments, was similar and appeared early inthe infection (at 6 to 12 h). Lipid synthesisincreased rapidly from 12 to 24 h during the timeof division of the reticulate bodies and ap-proached a steady state (at -30 h) when ele-mentary bodies were found, as measured byintracellular infectivity (Fig. 1, Table 2). Fanand Jenkin (8), using [14C]acetate, observed adecrease in lipid synthesis in chlamydial-in-fected cells and postulated that chlamydiae re-duce host lipid synthesis to the level observed incells in the stationary phase of growth. Thereduction observed in their data (8) was not seenin our system, which contained Cx in the me-dium, thereby more directly unmasking procar-yotic lipid synthesis. This suggests that theamount of host lipid synthesis from cells in thestationary phase of L-cell growth may alreadybe reduced which can account for the differencesobserved. Fan and Jenkin (8) associated a sud-den increase (at 30 to 36 h) followed by a rapiddecrease (at 36 to 42 h) in lipid synthesis, witha sharp increase in intracellular infectivity ofLGV-440L between 30 and 60 h. One can spec-ulate that this is the same time that reticulatebodies also are dividing asynchronously between30 and 48 h. From our results we believe thatlipid synthesis is associated with chlamydialmultiplication (reticulate bodies) and not nec-essarily linked to chlamydial maturation (ele-mentary bodies).

Since a 24-h pretreatment of cells with Cx (2,ug/ml) inhibited host lipid synthesis and did notinhibit LGV-404L multiplication in L cells, chla-mydial lipid synthesis could be studied in theintact host (Table 2). Detailed chlamydial lipidmetabolism can now be investigated. Therefore,it may be postulated that lipid synthesis in pre-

CHLAMYDIAL LIPID METABOLISM 673

treated, infected L cells in the presence of Cx isprobably associated with the biosynthetic activ-ities of the chlamydial agent. This does notexclude any possible activities the agent mayinduce the cell to perform.Our results extended the use of L cells grown

in a serum-free medium in a spinner culture (17)to their successful use in a shaker culture (10).With this culture system we were able to obtainequal or higher titers of C. trachomatis LGV-404L than those observed with other methods ofcultivation, as well as organisms free of serumcontamination and adventitious agents from se-rum. The comparatively high yield of extracel-lular C. trachomatis organisms was possibly re-leased by mechanical agitation of the infectedcells in the shaker culture. This serum-free sys-tem may provide a method to obtain clean an-tigens for performing antigenic analyses and di-agnostic studies and reducing some of the addi-tional added steps of purification and cell frac-tionation currently in use.

ACKNOWLEDGMENTSThis investigation was supported in part by research grant

HL 08214 from the Program Projects Branch, ExtramuralPrograms, National Heart, Lung, and Blood Institute, and byThe Hormel Foundation.We thank Tze-ken Yang for his technical assistance.

LITERATURE CITED1. Alexander, J. J. 1968. Separation of protein synthesis in

meningopneumonitis agent from that in L cells by dif-ferential susceptibility to cycloheximide. J. Bacteriol.95:327-332.

2. Alexander, J. J. 1969. Effect of infection with the menin-gopneumonitis agent on deoxyribonucleic acid and pro-tein synthesis by its L-cell host. J. Bacteriol. 97:653-657.

3. Bernkopf, H. P. Mashiah, and Y. Becker. 1962. Cor-relation between morphological and biochemicalchanges and appearance of infectivity in FL culturesinfected with trachoma agent. Ann. N. Y. Acad. Sci. 98:62-81.

4. Bligh, E. G., and W. J. Dyer. 1959. A rapid method oftotal lipid extraction and purification. Can. J. Biochem.Physiol. 37:911-917.

5. Bovarnick, M. R., J. C. Miller, and J. C. Snyder. 1950.The influence of certain salts, amino acids, sugars, and

protein on the stability of rickettsiae. J. Bacteriol. 59:509-522.

6. Coon, M. J. 1955. Enzymatic synthesis of branched chainacids from amino acids. Fed. Proc. 13:762-764.

7. Ennis, H. L., and M. Lubin 1964. Cycloheximide aspectsof inhibition of protein synthesis in mammalian cells.Science 146:1474-1476.

8. Fan, V. S. C., and H. M. Jenkin. 1974. Lipid metabolismofmonkey kidney cells (LLC-MK-2) infected with Chia-mydia trachomatis strain lymphogranuloma venereum.Infect. Immun. 10:4684470.

9. Gaugler, R. W., E. M. Neptune, Jr., G. M. Adams, T.L. Sallee, E. Weiss, and N. W. Wilson. 1969. Lipidsynthesis by isolated Chiamydia psittaci. J. Bacteriol.100:823-826.

10. Guskey, L E., and H. ML Jenkin. 1976. The serialcultivation of suspended BHK-21/13 cells in serum-freeWaymouth medium. Proc. Soc. Exp. Biol. Med. 151:221-224.

11. Hatch, T. P. 1975. Competition between Chlamydia psit-taci and L cells for host isoleucine pools: a limitingfactor in chlamydial multiplication. Infect. Immun. 12:211-220.

12. Hull, R. N., W. R. Cherry, and 0. J. Tritch. 1962.Growth characteristics of monkey kidney cell strainsLLC-MK1, LLC-MK2 and LLC-MK2 (NCTC-3196)and their utility in virus research. J. Exp. Med. 115:903-918.

13. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275.

14. Kaneda, T. 1977. Fatty acids of the genus Bacillus: anexample of branched-chain preference. Bacteriol. Rev.41:391-418.

15. Kuo, C. C., S. P. Wang, B. B. Wentworth, and J. T.Grayston. 1972. Primary isolation of TRIC organismsin HeLa 229 cells treated with DEAE-dextran. J. Infect.Dis. 125:665-668.

16. Makino, S., H. M. Jenkin,H. M. Yu, and D. Townsend.1970. Lipid composition of Chlamydiapsittaci grown inmonkey kidney cells in defined medium. J. Bacteriol.103:62-70.

17. Morrison, S. J., and H. M. Jenkin. 1972. Growth ofChiamydia psittaci strain meningopneumonitis inmouse L cells cultivated in a defined medium in spinnercultures. In Vitro 8:94-100.

18. Raff, R. A. 1970. Induction of fatty acid synthesis incultured mammalian celis: effects of cycloheximide andx-rays. J. Cell Physiol. 75:341-351.

19. Ripa, K. T., and P. Mhrdh. 1977. Cultivation of Chia-mydia trachomatis in cycloheximide-treated McCoycells. J. Clin. Microbiol. 6:328-331.

20. Stokes, G. 1974. Cycloheximide-resistant glycosylation inL cells infected with Chlamydiapsittaci. Infect. Immun.9:497-499.

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