BACTERIOLOGICAL REVIEWS, Mar., 1967, p. 54-64 Vol. 31, No. ICopyright © 1967 American Society for Microbiology Printed In U.S.A.

Bacterial Phosphatides and Natural Relationships,MIYOSHI IKAWA

Department of Biochemistry, University ofNew Hampshire, Durham, New Hampshire

INTRODUCTION........................................................TYPEs AND DISTRIBUTION OF PHOSPHATIDES IN BACTERIA ...............

Phosphatidylcholine and Phosphatidylethanolamine...........Phosphatidylinositol .Lipids Containing Ether Linkages... ..................................

Phosphatidylglycerol, Diphosphatidylglycerol (Cardiolipin), and LipoaminoAcids ...........................................................

HISTOCHEMISTRY OF BACTERIAL LiPIDs ................................PHOSPHATIDES AND PHYLOGENETIC RELATIONSHIPS.........................

LITERATURE CITED .................................................

5454575858

58595961

INTRODUCTIONEven though a very high degree of biochemical

unity does exist among the various forms of lifeon earth from the simple to the complex, it hasbeen becoming increasingly apparent that a greatdeal of biochemical diversity also is present. Thediversity found in the realm of the bacteria is sogreat that it is clear they comprise a very hetero-geneous group. A working classification of bac-teria has been devised which relies on grossmorphology and also upon biochemical andchemical differences. Progress in unraveling thephylogenetic relationships among the bacteriahas been hampered by their simple gross mor-phology and the inability of the light microscopeto reveal much of their fine structure, and also bythe inability to apply paleontological methods andobserve ontogenetic development. Establishmentof phylogenetic relationships will undoubtedlyalso have to rely on biochemical and chemicalproperties and on any theories of biochemicalevolution which can be deduced from them. Onearea in which striking chemical differences havebeen noted among the various types of bacteriaincludes the phosphatides. These compounds oc-cur in living organisms in a number of relatedstructures. and differences in these constituentsmanifest themselves in either a qualitative orquantitative manner. The phosphatides com-monly encountered include phosphatidic acid,phosphatidylcholine, phosphatidylethanolamine,phosphatidylserine, phosphatidylinositol, phos-phatidylglycerol, and disphosphatidylglycerol(cardiolipin). Phosphatidylcholine is the prin-cipal phosphatide of both higher plants andanimals (2, 11). However, quantitative differences

I Published with the approval of the Director of theNew Hampshire Agricultural Experiment Station(Scientific Contribution no. 375).

54

in some of the other phosphatide constituents doseem to be present between these two majorkingdoms; e.g., larger amounts of phosphatidyl-ethanolamine occur in animals than in plants, andlarger amounts of phosphatidylglycerol occur inplants than in animals.The subject of bacterial lipids in general has

been recently reviewed (5, 6, 52). In general,bacteria appear to contain no sterols, sphingo-lipids, or polyunsaturated acids; smaller amountsof neutral glycerides than other organisms; andmore branched-chain, cyclopropane, hydroxy,and free fatty acids, and perhaps more glycolipidsthan other organisms.

TYPES AND DISTRIBUTION OF PHOSPHATIDESiN BACTERIA

The major groups of bacteria consist of theeubacteria (orders Pseudomonadales, Chlamydo-bacteriales, Eubacteriales, Actinomycetales, Car-yophanales), budding bacteria (Hyphomicro-biales), gliding bacteria (Beggiatoales, Myxo-bacterales), spirochetes (Spirochaetales), andpleuropneumonia group (Mycoplasmatales). Theorder Rickettsiales should be considered with thebacteria. Information concerning bacterial phos-phatides is restricted almost entirely to the eubac-teria and to the orders Pseudomonadales, Eubac-teriales, and Actinomycetales. However, thesethree orders include most of the currently de-scribed bacteria. This paper deals only with theseorganisms.

Table 1 lists the phosphatide constituents whichhave been shown to be present in bacteria or pre-sumed to be present or absent for one reason oranother. Certain inconsistencies are apparent.Some of these may be due to differences in criteriaused to determine whether a constituent is presentor absent or to differences in conditions under

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TABLE 1. Phosphatide constituents of bacteriaa (continued on niext page)

Organism PC PE PS PI PH PG DPG Other Method (ref)

PseudomonadalesAthiorhodaceaeRhodopseudomonas spheroides

(chromatophores) .............R. spheroides....................Rhodospirillum rubrum (chro-matophores) ..................

R. rubrum (chromatophores).....Thiobacteriaceae

Thiobacillus thiooxidans......T. thiooxidans (extracellular

lipid) .........................T. thiooxidans (extracellular

lipid) .........................PseudomonadaceaePseudomonas aeruginiosaP. aeruginosa.......P. aeruginosa....................P. denitrificans..................Halobacterium cutirubrum........H. cutirubrum.

SpirillaceaeVibrio cholerae..................V. comma.

EubacterialesAzotobacteraceae

Azotobacter agilis...............A. agilis (walls) ...............

RhizobiaceaeAgrobacterium radiobacter.A. rhizogenes....................A. tumefaciens......A. tumefaciens........

AchromobacteraceaeAlcaligenes faecalis..........A. faecalis......................

En terobacteriaceaeEscherichia coli.E. coli..........................E. coli..........................

63b22c

30c4d

25c

17c

3c

14c8c+13'

10040

12+

3

0

+90

+

76f+

+

74

80f+

+-

+

15+

0

1

+

24

0

29

8014

42+

72

49

6c

0

5

10

73e+g

+g

+9+0

C, H (34)C (63)

C, R (13)C, R (12)

C, R (49)

C, R (49)

C (85)

H (33)H (88)C (89)H (27)C (86)C, H (26)

H (14)H (7)

C, H (51)H (22)

C, R, H (33)C, R, H (33)H (29, 65, 99)C, H (51)

H (33)C, H (84)

C, H (39)C, H (51)C, H (64)

a Abbreviations: PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine;PI, phosphatidylinositol; PH, phosphatidic acid; PG, phosphatidylglycerol; DPG, diphosphatidyl-glycerol (cardiolipin). DPG refers also to diphosphatidylglycerols in general. The methods used were:C, chromatographic methods including isolation by chromatography; H, hydrolysis products; R,radioisotope; I, isolation procedures not involving chromatography.

bMillimicromoles per milligram of chromatophore.c Percentage of total phospholipid phosphorus.d Symbols: +, present; -, absent; 4, present in small amounts or traces.e The main phosphatide is a long-chain ether analogue of diphosphatidyl glycerol (see text).f Percentage of total lipid.g Evidence for the presence of phosphatidyl-N-(monomethyl)-ethanolamine and phosphatidy-

N, N-(dimethyl)-ethanolamine.h PG = 21% of the total phospholipids from log-phase cells and only 7% from stationary-phase cells.Percentage of total phospholipid.Lipids containing phosphorylated sugars.

k Phosphatidylinositol mannosides.1 A total of 55% of the ethanolamine phosphatides, 9% of the phosphatidylglycerol, and 78% of the

N-monomethyl-ethanolamine phosphatides was present in plasmalogen form.

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TABLE 1 .-(Continued)

PC P'E PiS Pi PH IPG I)PG Other MIethod (ref)

E. coli..

Aerobacter aerogeiiesSerratia marcescens..

S. marcesces ..

S. marcesces..Proteus mirabilis P-18Proteus P-18...P. morgaiiP. va/gunis.Salmoniella paratyphi..S. typhimuriumS. typhosa.

BrucellaceaeBrucella abortus. .

B. abortus +

MicrococcaceaeMicrococcus Iloaeleiitr-ificaiisM. lysodeikticuisM. lysodeikticis.M. lysodeikticus (membrane).M. lysodeikticuis (membrane).Staphylococcus albus....S. aureus (small particle fraction).;S. aureus.

S. aureus

S. aureus

Sarcina lutea.S. lutea. IfSarciniasp.

NeisseriaceaeNeisseria gotorrIoeae. .+

LactobacillaceaeStreptococcus BS. faecalis.S. faecalis (membrane).S. faecalis (membrane).S. faecalis (membrane).Streptococcus sp. (Leuconiostoc

mesenteroides P-60).S. lactis..... +Pediococcus cerevisiae.Lactobacillus acidophiluts....±.....-4lL. casei......................... -

L casei -

L casei +

L. plantarumL. plaitaru.

CorynebacteriaceaeCorynebacterium diphtheriae..C. diphtheriae.C. diphtheriae.

BacillaceaeBacillus cereusB. cereus ........................

B. cereus ........................ 7c

Bacillus MB. megaterium -

B. megaterium.B. megateriuim (membrane)..

+

63c

70i+

84c

+

+

+ + +-' C (50)R (33)H (33)C (111)

8 4 6 C (53)+ C, H (83)

-V+.4-+ C (78)

+ C, H (82)C, R, H (33)H (18)

I 0 C (71)

l + +' H (19, 21)

H (90)+ C (108)

13

5c

+

5I

i~~~~

40i

46C50

+

+4k

+

I+ C (56)C (33)

72 4 C (70)80f H, low N (31)

+; 68 C, no N (69)R (33)H (27)

+ + C (71)90C + C (42)

+ + H,lowN (81)R (33)

51 4 14 17 C,H (44)

+ I, lowN (1)

H (92)

R (33)

H (46)low N (87)

54f C (45)I±i +;+ C (104)

H (46)

C, H (74)H (46)

III H (23)R (33)H (46)

+ C (97)R (33)H (46)

I#+ 1 (37)H (20)C, H (4)

+ C (38)43 + C (40)

26 C (54)+ H (105)

R (33)

II+ C (38)

I C, H (39)

Orgainism

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57BACTERIAL PHOSPHATIDES

TABLE 1 -(Continiued)

Organism

B. megaterium (membrane).B. polymyxa................IB. stearothermophilus...

B. subtilis.Clostridium acetobutylicumC. butyricum ....................

C. butyricum ....................

C. histolyticum

C. propionicum ................

C. tetanomorphum..............C. welchii.....................

ActinomycetalesMycobacteriaceaeMycobacterium avium............M. avium........M. fortuitum ...................

M. leprae.M. marinum..............M. phlei..

M. phlei........................M. phlei.........................M. phlei....M. phlei........................M. tuberculosis (bovine)........

PC PE PS

- ±

+ +

14cl1 :±

15c!

+k+ i+K_ I_ +k

3CI

M. tuberculosis (bovine).........M. tuberculosis.................

M. tuberculosis.................M. tuberculosis.................M. tuberculosis.................M. tuberculosis.................

ActinomycetaceaeNocardia brasiliensis ............

12c

PI 1 PH PG DPG OtherI

Method (ref)

! ~H (I110)+ + + IHC (75)

H (68)

R (33)R (33)C, R, H (32, 33)

i 261 78w1 C (10)R (33)R (33)R (33)

+ + C (72)

C (94)+ C (25)

C (103)H (3)C, H (76)H, low N (79)C, R, H (33)C, H (101)

i+ C (25)C (94)H (17); C, H

(100)+ C (25)

C, no N (16,101)

C (94)+ C, H (102)

+ 1 (15)+ I (73); C (25,

93)

+ + C (61)

which organisms were grown and the lipids iso-lated. At any rate, these inconsistencies give an

indication of which components are of less quan-titative importance, since they would most likelyarise with constituents present in relatively smallamounts.

Phosphatidylcholine and Phospha-tidylethanolamine

One of the most obvious conclusions to bedrawn from the data presented in Table 1 is thatphosphatidylcholine, the most abundant of thephosphatides in tissues of higher plants andanimals, is generally absent in the bacteria studiedexcept among the Pseudomonadales and Rhizo-biaceae. On the other hand, phosphatidylethanol-amine, which occurs widely in the plant andanimal worlds, is also a major phosphatide inmost of the bacteria studied. However, even thiscomponent is either absent or present only in

minor amounts in the Lactobacillaceae. Micro-coccaceae, Corynebacteriaceae, and Mycobac-teriaceae. The presence of phosphatidylethanol-amine but not necessarily of phosphatidylcholineis a situation which might be expected to exist,since the endogenous synthesis of the cholinemoiety involves three successive methylations ofthe ethanolamine moiety. It has been shown thatstrains of Proteus vulgaris and Clostridium butyri-cum, which do not synthesize phosphatidylcholine,do contain phosphatidyl-N-monomethylethanola-mine and phosphatidyl-N,N-dimethylethanola-mine. in addition to phosphatidylethanolamine(33). Phosphatidylcholine-containing Agrobacte-rium species were also found to contain thesepartially methylated phosphatides.

In addition to the occurrence of ethanolaminein free phospholipid, ethanolamine has also beenidentified in hydrolysates of the endotoxins ofEscherichia coli (47, 60, 95, 107), Salmonella

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+ k

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abortivoequina (95, 107), S. typhosa (95), andNeisseria gonorrhoeae (95). However, accordingto Grollman and Osborn (35), who have identifiedO-phosphorylethanolamine as a major compo-nent in the lipopolysaccharides of S. typhimurium,S. abortivoequina, S. enteritidis, S. typhosa,Shigella flexneri, and E. coli, the ethanolamine isnot present in the endotoxins in the form ofphosphatidylethanolamine but is a major com-ponent of a phosphorylated "backbone" poly-saccharide.

Phosplhatid,ylinositol

Due to the very limited amount of informationavailable, it is difficult to assess the importance ofphosphatidylinositol in the bacteria in general.However, among the corynebacteria and myco-bacteria the unusual situation exists where themajor phosphatides are mannosides of phos-phatidylinositol. A multiplicity of related struc-tures ranging from the monomannoside throughthe hexamannoside have been reported (see 67).Of these, the dimannoside seems to occur in themost abundance and has been isolated the mostfrequently. In a brilliant series of investigationsby Ballou and his co-workers (8, 9, 66, 67) thedetailed structures of these complex phospho-lipids have been established. These have beenshown to be 2- and 6-oa-D-mannopyranosides of1-phosphatidyl-L-myoinositol (Structure I). Evi-dence based on hydrolysis and infrared spectra in-dicates a similar type of compound occurring inthe corynebacteria (4).

64

0

O-P-O(- H,

HO CHOCOR

CH O(COR'

Structure I

Mannoside A BMono- M-* H-Di- M- M-Tri- M- M-(1-6)-M-Tetra- M- M-(1-6)-M-(1-6)-M-Penta M- M-(1-2)-M-(1-6)-M-(1-6)-M-*M- = a-D-Mannopyranosyl-

Lipids Containing Ether Linkages

Ether-containing lipids occur widely in theanimal world in the form of ether analogues of

phosphatides (96), vinyl ether analogues ofphosphatides (plasmalogens), and glycerol ethers(batyl, selachyl, and chimyl alcohols in elasmo-branch fishes). Oddly, the pseudomonad Halo-bacterium cutirubrum, although it contains limitedamounts of phosphatidylcholine, phosphatidyl-ethanolamine, and phosphatidylinositol, containsas its major phosphatide component an etheranalogue of cardiolipin for which structures II,containing two dihydrophytyl groups (55, 57),and III, containing other higher fatty alcohols(26), have been proposed.

CH,O(CH 2CH2CHCH)CH,),HCHO(CH,CH,CHCH CH2)4HC H2OPO(OH)OCH CHOHCH OPO(OH)

S'tructure' II

('H2O(CH,),,CHRCHO(CH,),,( H

C(H OPO(O)H)O( Hl ( H{OH( 11 OH

Struciure /I1

Plasmalogen-type lipids have been reported inexponential-phase cells of Clostridiuni butyricum(10), where they made up 55%,o of the ethanol-amine phosphatides, 78% of the N-monomethyl-ethanolamine phosphatides, and 9%O of the phos-phatidylglycerol.

Phosphatidylglycerol, Diphosphatidylglycerol(Cardiolipin), and Lipoanzino Acids

Although information is still limited, the majorproportion of the phosphatides of most bacteriastudied appears to be nitrogen-free in its basicstructure and to consist of phosphatidylglyceroland diphosphatidylglycerol (cardiolipin). Never-theless, nitrogen in the form of amino acids otherthan serine has often been encountered in con-siderable quantity in phospholipid fractions frombacteria, and these complexes have been desig-nated as lipoamino acids. Amino acids are veryfrequently encountered as impurities of phospho-lipid preparations from natural sources (36), andthe artifact nature of such combinations has beendemonstrated (109). The possible nature of suchartifacts as esters has been shown by the isolationof 'y-(1-glycerol)-glutamate, a compound readilyformed from glutamic acid and glycerol underacidic conditions (48). However, since only alimited number of amino acids, notably lysine,omithine, and alanine (14, 30, 41, 42, 46, 72, 76,98, 104), have been generally encountered inbacterial lipoamino acids, these compounds ap-

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pear to have a biochemical significance and notto be simply artifacts. Phosphorus-containinglipoamino acids have been isolated from bacterialsources. These compounds are esters of phos-phatidylglycerol. Structure IV has been proposedfor the compound isolated from C. welchii (72),and a similar structure has been proposed for theornithine-containing compound from Bacilluscereus (41).

CH2OPO(OH)OCH2CHOCOR' CH- -OH

CH20COR' H2- -OCOCHNH2R

Structure IV

The formation of the phosphatidylglycerolester of lysine by Staphylococcus aureus has beenshown to be pH-dependent (42). During the post-exponential phase of growth, when the pH of themedium has dropped, or during growth in amedium of lowered pH, the lysine ester is formedat the expense of phosphatidylglycerol. If the pHis maintained near neutrality during and aftergrowth, very little lysine ester is formed. It hastherefore been suggested that the lysine ester isformed at low pH to maintain a proper chargeof the membrane lipid layer. Thus, the presenceof lipoamino acids in bacteria does not appear tobe a necessary and constant feature.

Present evidence indicates that lipoamino acidsother than those containing phosphorus are alsopresent in the phospholipid fractions of bacteria.Thus, the lipoamino acids present in the phospho-lipid fraction of B. megaterium have been frac-tionated by chromatography on diethylamino-ethyl cellulose into phosphorus-containing andphosphorus-free fractions (43). The amino acidsassociated with mycobacterial phospholipidshave been shown to be present as phosphorus-freelipid (80); one such compound containing theamino acid ornithine has been isolated and itsstructure proposed as a N,N'-diacylornithine(Structure V) (62). Such a structure would beentirely analogous to N-fatty acyl derivatives ofphenylalanine formed by a soluble enzyme systemfrom rat liver (28). The omithine-containinglipid in the phospholipid fraction of Rhodopseudo-monas spheroides showed no radioactivity whenthe cells were grown on P32 and therefore alsoappears not to be a phospholipid (34).

CH2CH2CH2CHCOOHNHCOR NHCOR

Structure V

HISTOCHEMISTRY OF BACTERIAL LiPiDsWith the development of methods for the isola-

tion of particulate fractions of the bacterial cellin a pure state, it has been possible to demonstratethe localization of high concentrations of lipid inthe cell membrane fraction. In B. megateriummembranes, lipid contents of 16 to 21% (106)and 22.9% (110) have been reported; in BacillusM, 13 to 21% (105); in Micrococcus lysodeikticus,28% (31); in S. aureus, 22.5% (77); and in Strep-tococcus faecalis, 28 to 40% (87), 21% (45), and30.4% (104). Furthermore, studies indicate thatmost of the lipid of the cell is concentrated in themembrane. In Bacillus M, 65% of the total lipidand 93% of the free lipid was located in the mem-brane (105), and, in S. faecalis, 94% of the totalcell lipid was in the membrane fraction (104). Ex-periments with S. faecalis have also shown nolipid phosphorus in the cell wall, no more than4% of the lipid phosphorus in the cyloplasmicfraction, and the majority in the membrane (58).In R. spheroides, over 90% ofthe lipid phosphoruswas located in the membrane fraction (63).

Nitrogen values for membrane lipid prepara-tions have been variable but generally low. Thus,in S. faecalis membrane lipid, 0.12 to 0.26%nitrogen (87) and no choline or amino nitrogen(45) have been reported; in M. Iysodeikticus, nonitrogen (69) and 0.22% nitrogen (31) have beenreported. These results would certainly excludethe presence of nitrogen bases and amino acidsin great amounts in the preparations tested. Onthe other hand, nitrogen-containing membranelipids have also been reported. A B. megateriummembrane lipid preparation contained a molarratio of a-amino nitrogen to phosphorus of 1.25,of which the ratio of ethanolamine to phosphoruswas 0.97 (110). There was no choline, but smallamounts of several amino acids were present. Amembrane lipid preparation of S. faecalis con-tained 1.5% nitrogen and oontained lysine, gly-cine, and alanine in ester link (104).

Phosphatidylglycerol and diphosphatidyl-glycerol comprise most of the membrane lipidsof M. lysodeikticus (31, 69), BacillusM (105), andS. faecalis (104). Phosphatidylinositol has beenfound in M. lysodeikticus (69), and non-phos-phorus-containing lipids such as monoglucosyl-glyceride and glucosyl- and galactosyl-diglycerideshave been found in S. faecalis (104). These find-ings are consistent with the generaUy low nitrogenvalues found in membrane lipid preparations.

PHOSPHATIDES AND PHYLOGENETICRELATIONSHIPS

Although information on the phosphatidecomposition of bacteria is still largely incomplete,

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it is apparent from what knowledge is availablethat members of a single bacterial family, as mightbe expected, generally contain similar phospha-tides (52). It may furthermore be generalized thatthe gram-negative organisms contain morephosphatidylethanolamine and certainly more ofcomplex phosphorus-containing lipopolysac-charides than the gram-positive bacteria, and thatthe latter organisms may contain larger quan-tities of the basically non-nitrogenous phos-phatidylglycerol and di- and polyphosphatidyl-glycerols (52). Since some gross differences inphosphatide composition are apparent among thevarious bacteria which have been studied, it ispossible that phosphatide composition maysomehow be even more closely related to phyloge-netic relationships among the bacteria than isgenerally realized.One hypothesis which has been presented on the

evolution of the groups of bacteria (see 91) placesprimary importance on morphology and postu-lates a more or less polyphyletic origin of threemain classes of bacteria: the eubacteria, the myxo-bacteria, and the spirochetes. Of these, the ear-liest eubacterium is conceived of as having had amorphologically simple coccus shape and ofhaving been in turn the ancestor for several mainlines of eubacterial differentiation. This hy-pothesis suggests, therefore, several relativelyindependent phylogenetic units among the eu-bacteria. These will be considered in the follow-ing paragraphs.A natural relationship into an independent

taxonomic unit has been suggested for the non-sporeforming gram-positive Eubacteriales (Micro-coccaceae, Lactobacillaceae, Propionibacteriaceae,and Corynebacteriaceae) and the Actinomycetales(59, 91). The unique absence or minor occurrenceof phosphatidylethanolamine among the Micro-coccaceae, Lactobacillaceae, Corynebacteriaceae,and Mycobacteriaceae (representing the Actino-mycetales) would certainly strengthen on bio-chemical grounds the view that these bacteriaare indeed phylogenetically closely related.The gram-positive endospore-producing Bacil-

laceae (Bacillus and Clostridium), on the otherhand, contain appreciable amounts of phos-phatidylethanolamine and are in this respectunique from the other gram-positive organisms.This group is indeed considered a separate eubac-terial taxonomic unit (91) on other grounds whichdifferentiate this group fundamentally from theother gram-positive eubacteria.

Phosphatidylethanolamine also seems to be acommon constituent of the gram-negative eu-bacteria, occurring in the Pseudomonadales andthe gram-negative families of the order Eubac-teriales (Azotobacteraceae, Rhizobiaceae, Achro-mobacteraceae, Enterobacteriaceae, Brucellaceae,

and Neisseriaceae). On the basis of other prop-erties, a close natural relationship has been sug-gested between the gram-negative Eubacterialesand the also gram-negative Pseudomonadales (24).

Phosphatidylcholine, which may be considereda biochemically more advanced form of thephosphatide molecule, judging from its pre-ponderant occurrence in the tissues of higherplants and animals, occurs, on the other hand, inrelatively few of the eubacteria studied. Theseinclude members of the orders Pseudomonadales(Athiorhodaceae, Thiobacteriaceae) and Eubac-teriales (Rhizobiaceae). The hypothesis that thepseudomonads containing photosynthetic pig-ments constitute an additional taxonomic unitapart from the nonpigmented organisms (91), aswell as the occurrence of phosphatidylcholine inthe photosynthetic Athiorhodaceae, suggests thatthis phosphatide might be widely occurring in andcharacteristic of this group. Higher concentrationsof phospholipids have been found in pigmented R.spheroides than in strains lacking the photosyn-thetic pigments, and it has been suggested thatthe phospholipids might play an important rolein electron transport systems connected withbacterial photosynthesis just as phospholipidsappear to be an integral part of the electron trans-port system in mitochondria (63). However, theoccurrence of phosphatidylcholine in the non-photosynthetic sulfur-oxidizing pseudomonadThiobacillus and pigmentless Agrobacterium(Eubacteriales) indicates that this molecule prob-ably evolved before the photosynthetic apparatusbecame a reality. The presence of phosphatidyl-choline in Agrobacterium certainly makes thisgenus seem out of place among the Eubacterialesand strengthens the speculations which have beenmade relating certain gram-negative members ofthe Eubacteriales to the pseudomonads (24). Abroader common feature among the phos-phatidylcholine-containing organisms might beefficient electron transport systems, such as areexemplified by photosynthetic ability and aerobicmetabolism. Since all aerobic organisms do notcontain phosphatidylcholine (e.g., Azotobacterand Bacillus among the Eubacteriales and Pseudo-monas among the Pseudomonadales) or, for thatmatter, may not even contain phosphatidyl-ethanolamine, phosphatidylcholine is not a neces-sary feature for aerobic metabolism, although itmay be better adapted for it.Even though phosphatidylethanolamine may

occur as a minor lipid component among certainbacteria, its presence, even in these minoramounts, cannot be ignored. If its presence insmall amounts is not due simply to its havingbeen present in the culture medium, then a systemcapable of its synthesis is implied. Factors limitingthe synthesis of phosphatidylethanolamine or

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Nonspore-forming

- Gram(±)

LSpore-forming

_ Anaerobic andaerobic

Gram-(-)

Efficient-electron

transporting

± PE- PC

+PEI PCJ

PE- PC

{+PE+ PC

MicrococcaceaeLactobacillaceaeCorynebacteriaceaeActinomycetales

Bacillaceae

EubacterialesPseudonionadales

EubacterialesPseudomonadales

SCHEME 1. Relationship between phosphatide constitution and classification. PE, phosphatidylethanzolaminie;PC, phosphatidylcholine.

failure of this product to accumulate may accountfor the low levels present in some organisms.When one considers that phosphatidylethanol-amine is an important constituent of higherplants and animals and of bacteria which havedeveloped biochemically advanced features suchas aerobic respiration and photosynthesis, one can

surmise that phosphatidylethanolamine is not themost primitive phosphatide and that it probablyevolved from a more primitive type, perhaps a

non-nitrogenous one such as phosphatidic acidor phosphatidylglycerol, both of which seem tooccur in most bacteria. There has been evidencefor considering the group comprised of the Micro-coccaceae, Lactobacillaceae, Propionibacteria-ceae, Corynebacteriaceae, and Actinomycetalesas an independent taxonomic unit and as one ofthe early ancestral lines of eubacteria. The findingof small amounts of phosphatidylethanolaminein this group might indicate that the phosphatidyl-ethanolamine-synthesizing mechanism came intoexistence in a primitive form at an early stage ofbiochemical evolution and that perhaps it neverdid fully develop here as in other ancestral lines.The relationships between the distribution of

phosphatidylethanolamine and phosphatidyl-choline in eubacteria and other properties basicto their classification are summarized in Scheme 1.These emphasize the possible importance of thesephospholipids in the evolution of the present-dayeubacterial types.

ACKNOWLEDGMENTS

I thank William R. Chesbro of the Department ofMicrobiology for his helpful criticisms and sugges-tions.

This investigation was supported by Public Health

Service Research Grant GM-12582 from the NationalInstitute of General Medical Sciences.

LITERATURE CITED

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