By inoculating E. coli B into the semisynthetic medium we conducted shaking culture, and observed alterations of the total phospholipid contents and the amounts of individual phospholipid components in various stages of growth. The results are briefly summarized as follows.
1. The total phospholipid content has been found to be greater during early culture period, while it decreases as the growth age advances.
2. Phosphatidyl ethanolamine gradually increase as the culture period approaches the stationary phase.
3. Phosphatidic acid and phosphatidyl glycerol decrease precipitously as growth age advances.
4. Cardiolipin shows the maximum content in the middle log phase when the growth rate is most speedy.</p

Ichikawa, Hiroyuki

Kanemasa, Yasuhiro

Takai, Kenichi

Yoshioka, Tieko

Okayama University Scientific Achievement Repository

Acta Medica OkayamaVolume 25, Issue 4 1971 Article 4AUGUST 1971Alteration of phospholipid at various growthphases of Escherichia coliYasuhiro Kanemasa∗ Tieko Yoshioka†Hiroyuki Ichikawa‡ Kenichi Takai∗∗∗Okayama University,†Okayama University,‡Okayama University,∗∗Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Alteration of phospholipid at various growthphases of Escherichia coli∗Yasuhiro Kanemasa, Tieko Yoshioka, Hiroyuki Ichikawa, and Kenichi TakaiAbstractBy inoculating E. coli B into the semisynthetic medium we conducted shaking culture, andobserved alterations of the total phospholipid contents and the amounts of individual phospho-lipid components in various stages of growth. The results are briefly summarized as follows. 1.The total phospholipid content has been found to be greater during early culture period, while itdecreases as the growth age advances. 2. Phosphatidyl ethanolamine gradually increase as theculture period approaches the stationary phase. 3. Phosphatidic acid and phosphatidyl glyceroldecrease precipitously as growth age advances. 4. Cardiolipin shows the maximum content in themiddle log phase when the growth rate is most speedy.∗PMID: 4263559 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOLActa Merl. O:ayama 25, 255-260 (1971)ALTERATION OF PHOSPHOLIPID AT VARIOUS GROWTHPHASES OF ESCHERICHIA COLIYasuhiro KANEMASA, Tieko YOSHIOKA, HiroyukiICHIKAWA and Kenichi TAKA!Department oj Microbiology, Okayama University Medical School, Okayama, japan(Director: Prof. j. T awara)Received jor publication, july 26, 1971The lipid is an object of attention because the membrane delimiting thecell, and presumably conferring to it its characteristic of impermeability,is supposed to l:e constituted largely of lipid. As such, recently muchinterest is being focussed on the analysis and dynamic aspects of bacterialphospholipids, principal constituents of the cell membrane. In previousinvestigations of the composition of the phospholipids from E. coli B, theauthors demonstrated the presence of cardiolipin, in addition to phos.phatidyl ethanolamine and phosphatidyl glycerol in the phospholipid frac.tion (1).Phospholipids, especially cardiolipin and phosphatidyl glycerol locali.zed exclusively in the cytoplasmic membrane, might reflect direct functionof the membrane in the division process. We, therefore, think it useful tosee whether any change in phospholipids occurs along with various cultureconditions (2, 3, 4). In the present study alteration of phospholipid compo-sition was investigated at various stages of growth. As a result it has beenfound that growth phase clearly gives considerable effects on the phos.pholipid composition and must be strictly controlled in investigation ofphospholipids.MATERIAL AND METHODSMicroorganism:E. coli B was grown in the semisynthetic medium at 37°0 with gentle shaking(1). In order to check the composition of phospholipid by the content of 32p inthe individual phospholipids, 20 ,uc-30,uc [32PJ labeled H3P04 per ml was addedfrom the start of incubation. Turbidity was measured periodically and sampledaliquot volume from medium at various stages. The cells were harvested bycentrifugation at 4°0.Extraction and fractionation ofphospholipids:The precipitates were placed in 40 ml of chloroform-methanol (2: 1, v/v) andallowed to stand at room temperature for several hours. The extract was separa-2551Kanemasa et al.: Alteration of phospholipid at various growth phases ofProduced by The Berkeley Electronic Press, 1971256 Y. KANEMASA, T. YOSHIOKA, H. ISHIKAWA and K. TAKAIted by filtration through paper filter. The residues were once more extracted inthe same manner overnight. The extracts were evaporated to dryness and thecarrier phospholipid from E. coli was added to the 32P-Iabeled phospholipid whenoccasion demanded. The extract thus obtained was purified by Folch's partitiondialysis procedures (5). Fractionation into the individual phospholipids was car-ried out with silica gel G thin-layer chromatography. Phospholipids were locatedby iodine vapor, and the spot corresponding to each component was scrapedfrom the plate.32P estimation :Incorporation of 32p into the total phospholipid was estimated by radioactivemeasurement of aliquot volume from the total lipid fraction. The relativeamount of individual phospholipids was determined by measurements of scrapedradioactive spots.RESULTSCharacterization of individual components of phospholipid:Since the total 'lipid fraction contains only 4 % of neutral lipid and therest being all phospholipid, the total lipid fraction is used without anytreatment as phospholipid fraction. Major components of phospholipidare phosphatidyl ethanolamine (PE), phosphatidyl glycerol (PG) andcardiolipin (CL), while minor components are phosphatidic acid (PA) andan unknown substance (assumed to be bis-phosphatidic acid, UN), asreported previously (6). Fig. I gives a radioautogram of the thin-layerchromatograph of phospholipid from cells at the late log phase.Fig. I Autoradiogram of the phospholipid of E. coli at the late logarithmic phase.The lipid was extracted from cells grown with H3 32P04 containing semisyntheticmedium. Two-dimensional chromatography was performed with solvent of chloro-form-methanol-water (70: 25: 4, v/v, first development) and chloroform-methanol-ammonia (60: 25: 4, v/v, second development).2Acta Medica Okayama, Vol. 25 [1971], Iss. 4, Art. 4http://escholarship.lib.okayama-u.ac.jp/amo/vol25/iss4/4Phospholipid in Escherichia coli 257Relation between the growth phase and the phospholipid present:The radioactivity of the extractable lipid synthesized in the cells is foundto increase closely resembling the growth curve of E. coli B during early,middle, late log and stationary phases, but more precisely it tends to de-crease as the growth phase advances (Fig. 2). Moreover, in calculating 32pper 1 mg of bacteria, it has been found that the phospholipid content atthe logarithmic phase is greater than that at the stationary phase (Fig. 3).5e$ 5'7~ 0u.... Xil) S~....0.0 UbQS2 4 6 8Incubation time (hrs)Fig. 2 Incorporation of 32p into the total phospholipid of E. coli B.Fig. 3 Changes in the amountof synthesized total phoSFholi,_ idfrom 1 mg of cells (wet wei5ht)during growth.Data were calculated fromthe 32P incorporation into thetotal phospholipid and growthcurve.til] 1.0Symbols: 0, Cell growth: _, Incorporation of 32P into the total phos;choli_:idfraction from cells in 1 ml at various growth phases. I, 2, 3, and 4 indicatethe samplin:s points individually at eary, middle, and late IOc,arithmic andstationary phases.1.5~....obQS;;--ox 0.5Sc.u3 4 5 6 7Incubation time (hrs)83Kanemasa et al.: Alteration of phospholipid at various growth phases ofProduced by The Berkeley Electronic Press, 1971258 Y. KANEMASA, T. YOSHIOKA, H. ISHIKAWA and K. TAKAIChanges in the amount of individual phospholipid components at various growthphases:Taking 32p-incorporation into phospholipid as the criterion, phospholipidcontained in the cells as each growth phase was fractionated into indivi-dual components, and the composition of these components was compared.Table shows relative amounts of individual components at each phase. Asa result it has been demonstrated that PE increases as the bacteria growTABLE PERCENTAGE OF THE INDIVIDUAL COMPONENTS OF THE PHOSPHOLIPIDOF F. COLI B AT VARIOUS GROWTH PHASESPhase Early 106 Middle 106 Late 106 StationaryPE 62.9 66.5 77 .6 82.1PC 20.1 18.3 12.3 7.0CL 3.6 4.5 4.0 2.9PA 5.0 2.7 1.0 0.4UN 2.1 3.2 3.5 2.03 15~'4)u....~~....0 PEME~6XE::l,u....5.,'4)uV~-0ME~XE0.U3 4 5 6 7 8Incubation time (hrs)Fig. 4 Changes in the absolute amount of the individual phospholipid from1 mg of cells (wet weight) during growth.Data were calculated from the 32P incorporation into each phospholipid. PE,phosphatidyl ethanolamine; PC, phosphatidyl glycerol; CL, cardiolipin;UN, unknown phospholipid; PA, phosphatidic acid,4Acta Medica Okayama, Vol. 25 [1971], Iss. 4, Art. 4http://escholarship.lib.okayama-u.ac.jp/amo/vol25/iss4/4Phospholipid in Escherichia coli 259older, while PG decreases precipitously. CL and UN increase at the stageswhen the cells undergo the most rapid fission, i. e. at the middle and latelogarithmic phases. In Fig. 4 is shown the comparison of absolute amounts of each component per 1 mg cell at a given growth phase. In thisinstance, changes in the absolute amount of individual components tendto be similar to the changes in relative amounts, but such a tendency wasmore marked. PE increases at the stationary phase, while PG and PAshow high values in the early log phase, and later as the bacteria growolder, these values decrease precipitously. CL and UN show the peakvalues in the middle to the late log phase.DISCUSSIONChanges in lipid composition of various bacteria have been reportedby many investigators to occur during the transition from the exponentialgrowth phase to the stationary (2-5).CRONAN has reported that total phospholipid content of E. coli doesnot change much in the exponential phase or in the stationary phase (4).However, the results of our analysis of phospholipids with E. coli culturedin semisynthetic medium have revealed that the absolute content of phos-pholipid is greater during the period from early log to middle log phasebut decreases as it enters the stationary phase. Since most of phospholipidis contained in the cell membrane, it is understandable that a greateramount of phospholipid is found in the younger bacteria undergoingvigorous fission.By the assays of individual phospholipids, it has been demonstratedthat as the culture age advances, PE increases gradually, but PG and PAdecrease precipitously. AsPE is the structural component and its turnoverrate is slow, it would be accumulated. On the other hand, the turnover ofPA and PG is rapid and they seem to be precursors of various phospholi.pids, so that when their growing speed slackens, the demand for theirpool will diminish, which is reflected by the decrease in their content.The turnover of CL is speedy as already reported by KANEMASA (I), andin the present experiment the content of CL is found to be greatest in themiddle log phase when the growing rate is greatest, suggesting an impor-tant role played by CL, e. g. playing a role intermediate of cell wallsynthesis or a role in active transport.Summarily, the alteration of the individual phospholipid compositionmay be thought to take place in conformance with the adaptation of thebacteria to ever-changing environmental conditions in the course of5Kanemasa et al.: Alteration of phospholipid at various growth phases ofProduced by The Berkeley Electronic Press, 1971260growth.Y. KANEMASA, T. YOSHIOKA, H. IsHIKAWA and K. TAKAISUMMARYBy inoculating E. coli B into the semisynthetic medium we conductedshaking culture, and observed alterations of the total phospholipid can.tents and the amounts of individual phospholipid components in variousstages of growth. The results are briefly summarized as follows.1. The total phospholipid content has been found to be greater duroing early culture period, while it decreases as the growth age advances.2. Phosphatidyl ethanolamine gradually increase as the culture periodapproaches the stationary phase.3. Phosphatidic acid and phosphatidyl glycerol decrease precipitouslyas growth age advances.4. Cardiolipin shows the maximum content in the middle log phasewhen the growth rate is most speedy.REFERENCES1. KANEMASA, Y., AKAMATSU, Y. and NOJIMA, S.: Composition and turnover of the phos-pholipids in Escherichia coli. Biochirn. Biophys. Acta 144, 382, 19672. KANFER, J. N. and KENNEDY, E. P.: Metabolism and function of bacterial lipids. 1.Metabolism of phospholipids in E. coli B. j. Bioi. Chern. 236, 2919, 19633. RANDLE, C. L., ALBRO, P. W. and DITTMER, J. C.: The phosphoglyceride composition ofgram-negative bacteria and the changes in composition during growth. Biochirn. Biophys.Acta 187, 214, 19694. CRONAN, J. E.: Phospholipid alterations during growth of Escherichia coli. j. Bacteriol.95, 2054, 19685. FOLCH, J., AscoLI, I., LEES, M., MEATH, J. A. and LE BARON, F. N.: Preparation oflipid extracts from brain tissue. j. Bioi. Chern. 191, 833, 19516. YAMAGAMI, A., YOSHIOKA, T. and KANEMASA, Y.: Differences in phospholipid composi-tion between wild strains and streptomycin resistant mutants of certain enteric bacteria.japan. j. Microbiol. 14, 174, 19706Acta Medica Okayama, Vol. 25 [1971], Iss. 4, Art. 4http://escholarship.lib.okayama-u.ac.jp/amo/vol25/iss4/4

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