Metabolism and function of the membrane phospholipids of Escherichia coli

25

BIOCHIMICA ET BIOPHYSICA ACTA BBA 85102 METABOLISM OF

AND

FUNCTION

OF

THE

MEMBRANE

PHOSPHOLIPIDS

ESCHERICHL4 COLI

J O H N E CRONAN, JR and P R O Y VAGELOS

Department of Moleculat Biophysics and Btochemtstly, Yale University, New Haven, Conn (U S A ) and Department of Biological Chemistry, Washmgton Umverstty School of Medicine, St Louts, Mo ( U S A ) (Received August 3rd, 1971)

CONTENTS I Introduction

27

II Intracellular location of phosphohplds

27

III Phosphohpid composition and structure

28

A The fatty acids of the phosphohplds 1 Saturated fatty acids 2 Unsaturated fatty acids 3 Cyclopropane fatty acids 4 Miscellaneous fatty acids 5 Unsaturated fatty acid auxotrophs

28 28 28 29 30 30

B Phospholipzd species 1 Phosphatldylethanolarmne 2 Phosphatldylglycerol 3 Cardiohpm 4 PhosphatldlC acid 5 Phosphatidylserlne 6 Other phospholiplds

33 33 33 33 33 35 35

IV Metabohsm of the phosphohplds

36

A Synthesis 1 Synthesis of phosphatldlc acid 2 Synthesis of CDP-dlglycerIde 3 Synthesis of phosphatldylglycerol 4 Synthesis of cardlohpm 5 Synthesis of phosphatldylethanolamlne 6 Anomalous enzymes

36 36 42 42 43 43 44

B Effect of culture condlUons 1 Effect of temperature 2 Effect of growth phase 3 Changes in energy metabolism

44 44 45 46

Abbreviations ACP, acyl carrier protein, TMG, methyl-l-thlO-#-D-galactoslde

Blochlm Blophys Acta, 265 (1972) 25-60

26

J E CRONAN, P R VAGELOS C Regulation of phospholip~d biosynthesis

46

D Degradation of the hplds

47

V Functions of the phospholiplds

48

A Requirement for enzymatic activity

48

B Structural integrity of the cell

50

C Active transport

51

D Growth and macromolecule biosynthesis

55

VI Conclusion

56

Acknowledgements

58

References

58

Blochtm Btophys Acta, 265 (1972) 25-60

MEMBRANE PHOSPHOLIPIDS OF E colt

27

I INTRODUCTION The flexible permeablhty b a m e r which bounds the cellular cytoplasm is composed mainly of liplds and proteins It is currently thought that at is the Interaction of these components which gives the membrane its characterastlc and vital properties A requisite step toward the understanding of biological membranes is the understanding of the contribution of the lipid component of the complex to the structure and function of the membrane It seems timely to concentrate on the role of the lipid component as methods to deal effectively with the intractable protein component of the membrane are not yet available The eubacterla possess many advantages as organisms in which to study membranes These cells have no lntracellular membranes, and their lipid compositions are much less complex than the compositions of eucaryotac cells The obvious choice among the eubacterla is Eschenchta coh, as the wealth of genetic, blochemacal and physiological data concernang this organism is unsurpassed in biology The llpad composition of this organism is at least as simple as that of other bacterm and the bulk of the data concerning lipid metabolism m bacteria has employed this organism In the last ten years the study of various aspects of the E cob membrane hpids has increased tremendously The avowed purpose of the present communication is to deal critically with the literature on the structure, metabolism, and function of the phosphohpids of the E cob envelope An emphasis wall be placed on the results that genetac manipulation has contributed to the understanding of the metabolism and structure of the phosphollplds II INTRACELLULAR LOCATION OF THE PHOSPHOLIPIDS The entire lipid content of E coh IS found in the cell envelope 1 and comprises about one-tenth the dry weight of the cell 2-7 The hplds consist mainly of phosphohpids A small amount of fatty acid is found covalently bound to galactosamIne moieties of the polysaccharlde s,9 This is called lipid A or bound lipid and will not be dascussed in this revaew It has been demonstrated very recently that phosphohplds are found in two discrete structures of the cell envolope 1°-13 The fractions are defined by their density as detected by lsopycnlc centrlfugatlon The more dense fraction, called the outer membrane, is derived from the cell wall, as indicated by a high content of llpopolysaccharlde, the low content of cytochromes, and by its morphology The fraction of lesser density is composed of vesacles derived from the cytoplasmic membrane and thus is called the inner membrane This fraction is enriched m cytochromes, in the enzymes of the electron transport system and in other membrane-bound enzymes and proteins The morphology and staining properties of these vesicles are very similar to those of the cytoplasmic membrane Both fractions contain phosphohpad and the distribution of phosphohpId species found in either of these fractions is very similar to that of intact cells or cell envelopes (M J Osborn, personal communication) Prehmlnary X-ray diffraction data of D M Engelman Btochtm Btophys Acta, 265 (1972) 25-60

28

J E CRONAN, P R VAGELOS

(personal communication) indicate that the bulk of the lipid component of both of these fractions is structured into a lipid bilayer such as that found m the membrane of Mycoplasma latdlawu 14,1s Thus, the phosphohplds of E colt are found in more complex and diverse structures than was formerly beheved Until the recent appearance of these data it was thought that all the cellular phospholipld was found in the cytoplasmic membrane Therefore the phrase membrane lipid has been used where the term envelope (wall plus membrane) lipid is more correct It appears that the site of synthesis of the phosphohplds is within the cell envelope rather than m the cytoplasm of the cell The phosphohpld biosynthetic enzymes are found in the particulate (envelope) fraction of disrupted cells (see below), and there is autoradxographic evidence that the acyl carrier protein (ACP) component of the fatty acid synthetic system is localized on the inner surface of the cell envelope 16 Lin, Hlrota and Jacob (E C C Lln, personal communication) have reported autoradlographlc analyses of growing cells labeled with specific phosphohpld precursors which indicate that the insertion of new phosphohpid molecules into the cell envelope occurs randomly over all the envelope surface

III PHOSPHOLIPID COMPOSITION AND STRUCTURE Ilia

The fatty acids of the phosphohptds

1 Saturated fatty aczds The saturated fatty acids consist of palmitlC (hexadecanotc) and myrlstlc (tetradecanoic) acids with traces of stearlc (octadecanolc) and lauric (dodecanoic) acids 2-6,17-24 The structures of these and the other E coh fatty acids are given in Table I The identification of these acids is based on their gas chromatographic behavior, the fact that this behavior is unchanged by hydrogenation, and comparison of their melting points with authentic standards PalmltiC acid comprises about half of the total fatty acid of the cell, and it is found esterlfied almost exclusively to position l of the glycerol backbone of the phosphohplds, while the other saturated fatty acids are distributed between both positions 17 19 23 2 Unsaturated fatty acids The unsaturated fatty acids found m E coh are all monoenes of the czs conformation 2-6,17 27 Structure determination is based on gas-hqutd2-6,17-27 and argentation 24-27 chromatographic behavior, and on the infrared spectra of the a o d s 6 Acids of chain lengths of sixteen and eighteen carbons are found by gas chromatography, and their chain lengths have been further estabhshed by gas chromatography after hydrogenation to the saturated derivative ~a,22 The hexadecenolc acid has invariably 22,25-27 been identified as palmltoleJc (c~-9hexadecenoic) acid, whde the octadecenoic acid fraction has now been shown by several techniques to consist solely of cts-vaccenic (cts-ll-octadecenolc) acid The first analysis of the composition of the octadecenoic acid fraction reported the fraction to consist of a mixture of cts-vaccenlc and olelc (cts-9-octadecenoic) 22 acids These workers used a strain of bacteria isolated and identified by their laboratory (A G Btochlm Btophys Acta, 265 (1972) 25-60

MEMBRANE PHOSPHOLIPIDS OF E coh

29

TABLE I THE STRUCTURES OF THE FATTY ACIDS OF E coh Structural type General structure

Systematic names

Trzwal names

Saturated

Dodecanolc acid (x = 10) Tetradecanolc acid (x = 12) Hexadecanolc acid (x = 14) Octadecanolc acid (x = 16)

Laurie acid Myrlstlc acid Palmmc acid Steanc acid

Unsaturated

CH3-(CH2)rCOOH

H

H

i

I

CHa-(CH2)5 C = C-(CH2)~r-COOH cts-9-Hexadecenolc acid

Palrmtolelc acid

(x = 7)

cls-11-Octadecenolc acid (x = 9)

cts-Vaccenlc acid

CH~ A

Cyclopropane CHz-(CH2)5-C-C-(CH2)~-COOH cts-9, 10-Methylene hexaI4 H decanolc acid (x = 7) cts-ll, 12-Methyleneoctadecanolc acid (x = 9)

none

Hydroxy

3-Hydroxyrnyrlstlc acid

CH ~(CH 2)~-CHOH-CH~-COOH D(--)-3-Hydroxytetradecanoac acid (x = 10)

Lactobacdhc acid

M a r r , p e r s o n a l c o m m u n i c a t i o n ) r a t h e r than a s t a n d a r d l a b o r a t o r y strata This might a c c o u n t for their d a t a W e l n b a u m a n d P a n o s 28 have also r e p o r t e d the presence o f oleic acid in E cob, b u t a later r e p o r t f r o m the same l a b o r a t o r y using the same techniques d e m o n s t r a t e d that olelc acid was n o t present z9 S c h e u e r b r a n d t a n d Bloch 2s first r e p o r t e d that cts-vaccenlc acid is the sole octadecenolc acid f o u n d m E colt Their identification was based u p o n the identification o f the f r a g m e n t s p r o d u c e d b y p e r m a n g a n a t e o x i d a t i o n o f the d o u b l e b o n d Several o t h e r such analyses using o x i d a t i o n 24 o r ozonolysls la o f the d o u b l e b o n d have given the same answer A r e p o r t 26 a d d r e s s e d solely to this question s e p a r a t e d the m e t h y l ester o f the isomeric acids b y c h r o m a t o g r a p h y on A g N O z - l m p r e g n a t e d slhca gel thin layers The acids were labeled biosynthetically d u r i n g g r o w t h o f the b a c t e r i a on u n i f o r m l y labeled glucose as sole c a r b o n source 26 This r e p o r t u n e q u i v o c a l l y showed that ClS-Vaccemc acid is the sole o c t a d e c e n o l c acid in the three E cob strains e x a m i n e d (strains K12, B, a n d W) A similar analysis has subsequently a p p e a r e d giving the same conclusion z7 These r e p o r t s plus the s u p p o r t given by the genetic a n d e n z y m o logical analysis (see below) establish t h a t olelc acid is n o t synthesized by E coh Palmltolelc a n d cts-vaccentc acids are f o u n d esterlfied p r e d o m i n a t e l y to p o s i t i o n 2 o f the sn-glycerol-3-phosphate b a c k b o n e o f the p h o s p h o l l p i d s 3 Cyclopropanefatty actds The c y c l o p r o p a n e fatty acids o f E cob are f o r m e d by the m e t h y l a t l o n o f the u n s a t u r a t e d fatty acids o f the p h o s p h o l l p l d s 5,3°,31 The methyl g r o u p is d o n a t e d by S - a d e n o s y l m e t h l o n m e a n d the m e t h y l a t l o n is believed to occur at the level o f the p h o s p h o l i p i d s (see below) These fatty acids consist o f cts-9, 10-methylenehexadecanolc acid z°,z2 a n d cts-11,12-methyleneoctadecanolc (lactoBtochtm Btophys Acta, 265 (1972) 25-60

30

J E CRONAN, P R VAGELOS

bacllhc) acid 29,31 The identification of these acids is based on (1) the structures of the monoenoic acids from which they are derived (see above), (n) chromatographic analysis of the acids b e f o r e 2°,22,29 31 and after degradation 22 of the cyclopropane ring, and (m) mass 32 and infrared spectroscopy 2°,22 The cts conformation of these acids has not been directly determined as the physical techniques used to date do not discriminate between ClS and trans Indirect data indicate the configuration is cls Only cts unsaturated acids are converted to their cyclopropane derivatives m vti'o 24,33,34, and the molecular cross section of E colt phosphatidylethanolamme containing cyclopropane fatty acids is that of a cts acid rather than a trans acid a~,36 The presence of lactobacllllc acid as the only methyleneoctadecanoic acid isomer in the cell confirms the absence of oleic acid in E colt, since any oleic acid would be converted 2~ az,3a to C l S - 9 , 1 0 - m e t h y l e n e o c t a d e c a n o i c (dlhydrostercuhc) acid which is not found in E colt 29 4 M i s c e l l a n e o u s f a t t y acid3 An acid possessing a gas chromatographic behavior of a fifteen-carbon cyclopropane fatty acid has been mentioned in several papers s,2a,37 To date this acid has not been identified DeSiervo 37 has reported the presence of a large number of fatty acids (identified as gas-liquid chromatography peaks) not recorded by other workers It seems likely that many of these peaks are due to impurities in the unusual solvents he used for extraction and chromatography Other fatty acids also present in the cell are the acyl moieties of the "qlpld A" component of the cell wall Lipid A consists of a mixture of laurlc, mynstic, palmitlC and /~-hydroxymyristlc aods bound to glucosamlne through amlde and ester linkages a,9 /~-Hydroxymyristic acid is a specific marker for lipid A and is not found in phosphohpld z,a,9 The function of lipid A in the cell wall is unknown 5 U n s a t u r a t e d f a t t y a c t d a u x o t r o p h s The study of the function of fatty acids as a component of the phospholiplds of the cell envelope has been greatly faclhtated by the isolation of auxotrophs requiring unsaturated fatty acids for growth The first unsaturated fatty acid auxotroph was isolated by Sllbert and Vagelos 38 This mutant could grow on glycerol as carbon source only when the medium was supplemented with an appropriate unsaturated or cyclopropane fatty acid The composition of the unsaturated fatty acids in the mutant, unlike the wild type, reflected the unsaturated fatty acid provided in the medium This is illustrated in FJg 1 which shows the gashquld chromatographic pattern of fatty acid methyl esters derived from the hpids of the wild-type parent and the auxotroph grown in medium supplemented with palmitoleate Both wild type (Fig 1A) and mutant (Fig 1B) contained mynstate and palmltate as the major saturated fatty acids As unsaturated fatty acids the wdd type contained palmltoleate and c t s - v a c c e n a t e and their cyclopropane derivatives, whereas the mutant contained only the palmltoleate provided in the medium together with its cyclopropane derivative When the mutant was grown m medium supplemented with palmitoleate and tracer amounts of [l*C]acetate, the saturated fatty acids derived from the hpids were radioactive, while the unsaturated and cyclopropane fatty acids contained essentially no radioactivity These m i,tl,O experiments, which indicated that the mutant strain did not synthesize unsaturated fatty acids, were corroborated by m vttro experiments which showed that extracts of the mutant Btochlm Btophys Acta, 265 (1972) 25-60

M E M B R A N E PHOSPHOLIPIDS OF E colt

31

A P,)

(al

(e)

18

Anpa0rI 6

5"L II

5

25

Fig 1 Gas-llquld chromatography of fatty acid methyl esters derived from stationary cultures of wdd-type and mutant stratus 3s A Fatty acid esters from wdd type grown on glycerol and palmltoleate B Esters from unsaturated fatty acid auxotroph grown on glycerol and palmltoleate The peaks are identified as follows (a) myrlstate, (b) palmltate, (c) palrmtoleate, (d) 9, 10-methylenehexadecanoate, (e) cts-vaccenate, (f) lactobac]llate For each peak the corresponding contrlbutxon to total fatty acids is recorded m % catalyzed the synthesis of saturated but not unsaturated fatty acids from acetyl-CoA and m a l o n y l - C o A The lnabdlty of the m u t a n t to synthesize unsaturated fatty acids was shown to be due to a defective fl-hydroxydecanoyl thloester dehydrase, the enzyme studied by Bloch and coworkers a9,4°,4~ who postulated that this enzyme is responsible for the introduction o f a cts-3 double b o n d in the pathway o f unsaturated fatty acid synthesis Fig 2 illustrates, in abbreviated form, the fatty aczd biosynthetic scheme o f E cob (see ref 42 for a recent general review o f fatty acid biosynthesis) and demonstrates that fl-hydroxydecanoyl-ACP is the intermediate at the branch point between the pathways to saturated and unsaturated fatty acids /~-Hydroxydecanoyl th~oester dehydrase, catalyzing reaction A, is very specific m the formation o f cts-3-decenoylA C P wluch is then elongated to f o r m palmltolelc and cts-vaccenlc acids 41 Dehydration o f ~-hydroxydecanoyl-ACP by pathway B leads to the introduction o f a trans double bond, and trans-2-decenoyl-ACP is an lntermedmte m saturated fatty Bwchtm Bwphys Acta, 265 (1972) 25-60

32

J E CRONAN, P R VAGELOS Acetyl-CoA + Malonyl- CoA

/A

CH3(CH2)sCH2CHOHCH2CO-S-ACP

HH S

I

CH3(CH2)sC=CCH2CO-S-ACP

H I

CH3(CH2)sCH2C=qCO-S-ACP H

+3C 2

INADPH

H H 1 t

CH3(CH2)5C=C(CH2)7CO-S-ACP PALMITOLEATE

+ C2

CH3(CH2)sCHzCH2CHzCO-S-ACP + 3 C2

HH I

I

CH3(CH2)sC=C(CH2)9CO-S-ACP

PALMITATE

cts-VACCENATE

Fig 2 Fatty acid bxosyntheslsm E colt ReactmnA lmttates the pathway to unsaturated acids, and Reaction B the pathway to saturated aods acid synthesis .3 Thus, organisms containing a mutation at A are unable to synthesize any unsaturated fatty acids either m l'ti'o or in v t t r o and are totally dependent on a supply of an appropriate exogenous fatty acid a3,as,*5 These mutants, however, synthesize saturated fatty acids normally even in the absence of unsaturated fatty acids 38,44 The isolation of the mutant defective in fl-hydroxydecanoyl thloester dehydrase, and the synthesis of a specific inhibitor of this enzyme which functions both m v t v o . 4 and m v i t r o 4L strongly support the proposed function of fl-hydroxydecanoyl thioester dehydrase as the enzyme speofically responsible for the introduction of cts double bonds in unsaturated fatty acids The later isolation of temperature-sensmve auxotrophlc mutants .5 possessing an enzyme of increased thermolabfllty has further confirmed this conclusion (J E Cronan and P R Vagelos, unpublished data, and K Bloch, personal communication) Recent analysis of several unsaturated fatty acid auxotrophs by enzymologlcal and genetic techniques has shown that there are at least two classes of mutants one of these lacks the fl-hydroxydecanoyl thloester dehydrase as discussed above, the other contains an enzymatic defect which has not yet been identified .5 It has been possible to supplement the unsaturated fatty acid auxotrophs with a surprisingly broad spectrum of fatty acids In addition to the normal constituents, cts-monoenoic and cyclopropane fatty acids, growth IS supported by t r a n s - m o n o e n o l c , c t s - d i e n o i c , c t s - t r i e n o i c , and bromine-substituted fatty acids as well as a broad spectrum of positional isomers of various chain lengths 11,2.,33,34 Thus it has been possible to manipulate the fatty acid composition and thereby the physical properties of the membrane phosphohplds to a great extent Btochtm Btophy~ Acta,

265 (1972) 25-60

MEMBRANE PHOSPHOLIPIDS OF E cob

33

I I I B Phosphohptd species

The species of phosphohpld found in E cob are all derivatives of phosphatldlC acid The structures of the three major species of phosphohpid found in E cob are g~ven in Fig 3 The data establishing these structures are given below All of the hp~ds discussed are found to be labeled with radioactive precursors and are found m cells grown in defined medium, thus estabhshlng their synthesis by the bacterium 1 Phosphattdylethanolamlne This lipid is the major phosphohpld found in E coh and comprises 7 0 - 8 0 ~ of the cellular phospholipld 2,4-6 37,4-6 51 That the structure of this lipid IS identical to that shown in Fig 1 has been established by (1) cochromatography with authentic phosphatidylethanolamlne of both synthetic and natural origin in many chromatographic systems 4-6'~8'19'23'a7'46-51 , ( n ) t h e presence of ethanolamlne or amino group, phosphate, glycerol and acyl ester in a molar ratio of 1 1 1 24'37'46'48, (Ill) the production of glycerolphosphorylethanolamme upon mdd alkaline hydrolysis4,5,~8,46- 51,5~,55, (iv) the degradation with specific phosphohpases to the expected products 18,19,37 , and (v) infrared spectral analysis 6 2 Phosphatldylglycerol This hpld comprises about 5 - 1 5 ~ of the cellular phosphohpid and has the structure given in Fig 3 The structure of this lipid was identified by the following criteria (1) cochromatography with authentic synthetic and natural phosphatldylglycerols 46,49, (n) presence of glycerol, phosphate, and acyl ester in a 2 1 2 ratio 4,3v,46,~s, (in) presence of Vlclnal hydroxyl groups in the intact lipid 46, (iv) formation of 1 mole of formaldehyde per mole of phosphate 46 upon perlodate oxidation of the intact purified lipid, (v) formation of glycerolphosphorylglycerol by mild alkahne hydrolysis of the lipid 4,5,~s,46-Sa , and (Vl) production of the predicted products upon degradation by specific phosphohpases (J E Cronan and P R Vagelos, unpublished data) 3 Cardtohpln The cardlollpln which comprises about 5-15 ~ of the phospholipid of E colt has a structure identical with that of dlphosphatldylglycerol and bovine heart cardlohpin The diphosphatldylglycerol structure of the E cob hpld was demonstrated by (l) cochromatography with bovine heart cardlollpln 4,5,a7,46-49, (n) the finding of glycerol, phosphate, and acyl ester in a 3 2 4 ratio 4,aT,46,4a, (lU) the formation of the barium salt 46, (iv) the formation of dl(glycerolphosphoryl)-glycerol 4,s,18,46-a9 upon mild alkahne hydrolysis, and (v) the production of predicted products upon degradation by specific phosphollpases 52 4 Phosphattdtc acid Phosphatldlc acid is found in trace amounts (less than I of the total phosphohpld phosphorus) in E cob 4'46'4a'53 Although Randle et a149 have reported values of 5 ~ of the total phosphohpld, this result was based on the analysis of the products of mild alkaline hydrolysis of the mixture of hpids extracted from the cell The hydrolysis procedure used by Randle et a149 has been shown to give rise to spurious sn-glycerol phosphate by the degradation of the other glycerolphosphoryl derivates during hydrolysis 54 Another report from the same laboratory does not report phosphatldlC acid in an analysis of E coh hplds using another technique s 5 Blochtm Btophys Acta, 265 (1972) 25-60

34

J E CRONAN, P R VAGELOS Fig 3 The biosynthesis of the phosphohplds of E colt

0 0 II II 2 RC-S-CoA or 2 RC-S-ACP

CH20H I

HOCH

O I II CH20-P-OttoHI~

sn-Glycero-3-Phosphate

~H2OOCR RCOOCH 0 I II CH2-O-P-OH I OH

PhosphatlchcAcid

PP]

~

H2OOCR RCOOCH O 0 I II II CH2-O-PIo-HO-PIo-HO-Cyt

/

3

L Serme

sn-Glycero-~ \

/

~

3-Phosphate " ~

/

X ~

/ ~

~

CDP-Dtgly~ende

_ CH20OCR

Phosphattdylglycerol Phosphate

I

LP

o

CH2-O-I~o-HO-CH2CH(OH)CH2- O-P- OH

CH2OOCR RCOOCH O I II CH2-O-P-O-CHaCHCOOH I I OH NH2

6

CH2OOCR I RCOOCH 0 I II CH2-O-~-O-CIt2CIt(OH)CH~OH Oil

Phosphandylserlne 4 "~C02

Phosphattdylglyc~rol

~

H2OOCR RCOOCH O II CH2-O-~-O-CH2CH2NH2 OH

CDP-D~gly¢ende

7

~CMP

PhosphaUdylethanolarmae

CH200CR RCOOCH2 I I RCOOCH O O HCOOCR I II II I CH2-O-P-O-CH~CH(OH)CH2-O-P-O-CH2 OH OH Catd~ohpm(DtphosphatadylglyceroI)

Btochtm BIophys Acta, 265 (1972) 25-60

MEMBRANE PHOSPHOLIPIDS OF E cob

35

The identity of phosphatidlc acid has been confirmed by (1) cochromatography with authentic synthetic and natural standards 4,1a,46-51,53, (n) degradation with phosphatldlc acid phosphatase53, and (in) formation of sn-glycerolphosphate *6-s 1,s 3,56 upon mild alkaline hydrolysis The glycerolphosphate identified chromatographically was shown to be sn-glycero-3-phosphate by its reaction with sn-glycero-3-phosphate N A D oxldoreductase s6 The phosphatldlc acid fraction extracted from E coh is not an artifact of the degradation of the other phosphollplds since (1) exposing cells for a very short time to radioactive P1s3 or radioactive glycerol s6 results in phosphatldlc acid being the only labeled phosphatlde, and (11) the label in the phosphatldlc a o d is very quickly lost 5a,56 when the radioactive compound is chased by adding a large excess of non-radioactive precursor 5 Phosphattdylserme Very small amounts of this hpid are found in E cob 4°'5°,51 The criteria used for its identification are (1) cochromatography with authentic phosphatidylserine 46,47, (li) labeling with radioactive phosphate and L-serine 46,4v,s°,sl, 0il) production of glycerolphosphorylserine by mild alkaline hydrolysis of the lipid 46'5°'51 , and (iv) reaction with nlnhydrin 46 6 Other phosphohptds Lysophosphatldylethanolamme has often been reported m trace amounts in E coh 18'4a,s7,sS,s9 In many of these cases sufficient care was not taken to dlstmgmsh this hpld from phosphatldylserlne In other reports extraction of the cells with HC104 before lipid extraction was probably responsible for the formation of large amounts of lysophosphatidylethanolamlne from phosphatldylethanolamme 53,6° In view of the ease of reduction of phosphohpase action (see below) ~t appears likely that in the other reports of more than traces of this hpid, its presence may be due to activation of a phosphohpase during the handling of the cells prior to lipid extraction s9 Phosphatidylglycerol phosphate has been reported twice in E coh 37'.9 In the first report .9 the criterion used for identification of this hpid was the presence of glycerolphosphorylglycerol phosphate detected after mild alkahne hydrolysis Since glycerolphosphorylglycerol phosphate is readily formed from cardlohpln by this hydrolysis procedure 61, the results must be reevaluated The other report 37 contains few data and is hence difficult to evaluate A d~rect search for this lipid has failed to disclose it s° Amino acid esters of phosphatldylglycerol have not been reported in E colt although they are readdy separable from the other hplds and should be easily detected by ninhydrin A systematic search of several stratus of E colt did not disclose even traces of such hplds 62 (J E Cronan, unpublished data) One report of the presence of phosphatldyhnositol m E coh exists 63 This lipid was probably phosphatidylglycerol, since the test used to identify lnosltol would also react with glycerol Cytidlne dlphosphate dJglycerlde has been reported in E coh sa Unfortunately, no detads of its identification were given in the paper and therefore the identification must be considered tentative The following liplds have not been detected in E coh, although they were Btochtrn Btophys Acta, 265 (1972) 25-60

36

J E CRONAN, P R VAGELOS

searched for by sensitive methods phosphatldylchohne6, 49, N-methylated derivatives of phosphatldylethanolamlne 6,49, and phosphatMyhnosltol 6,49 No plasmalogens have yet been detected in E coh 6,6'* A minor phosphohpld designated Y was reported by Ames 46 in both Salmonella typhtmurtum and E cob Although the structure of hpM Y is unknown, it contains phosphate and fatty acid m the ratio expected for a phosphatlde 6s The Mentificatlon of this lipid has been hampered by the elusiveness of its producnon Cultures grown under seemingly identical conditions do or do not possess this hpld depending on some unknown factor (G F Ames, personal communication)

IV METABOLISM OF THE PHOSPHOLIPIDS IVA

Synthests

The current scheme of phosphohpld biosynthesis in E colt is given in Fig 3 This scheme is based on results from several laboratories, especially that of Kennedy and coworkers Information concerning all of the reactions of this pathway except for the formation of phosphatldlc acid is based on the identification and study of enzymatic activities in cell extracts The synthesis of phosphatidic acid is understood m greater detail since enzyme studms have been supplemented with data derived from m vtvo and genetic experiments All of the enzymes thought to be involved in phosphohpld biosynthesis are found in the particulate (cell envelope) fraction of cells broken by a variety of methods 6 6 - 7 3 Although ACP and the enzymes of fatty acid biosynthesis are found in the soluble fraction of such extracts, it has been shown that m vti'o ACP and hence probably the fatty acid biosynthetic enzymes are located close to the tuner surface of the cell envelope 16 Thus it seems apparent that the site of the synthesis of the envelope llplds is the cell envelope ~tself Th~s is consistent with the reports of several workers that phosphohpid synthesis from inorganic phosphate 6a,74-76 and from L-serlne 77 and glycme 77 can be observed in membrane vesicle preparations 1 Synthests ofphosphattchc actd sn-Glycero-3-phosphate is the precursor of the phospholiplds of E coh This has been shown directly by the incorporation of sn-glycero-3-phosphate into the phosphohplds of growing cells Experiments using the approprmte radioactive labels show that both the glycerol 78-8° and phosphate moieties (J E Cronan and P R Vagelos, unpubhshed observations) can be incorporated as a unit into the phosphohplds These studms show that sn-glycero-3phosphate and not free glycerol is the form in which the glycerol moiety is incorporated into the phosphohplds These results have been confirmed by the isolation of mutants requiring sn-glycero-3-phosphate for normal growth The first such mutant, which was isolated by Kito, Lubln, and Plzer a~, dM not show an absolute growth requirement for sn-glycero-3-phosphate, but the rate of growth was greatly snmulated by exogenous sn-glycero-3-phosphate This phenotype was shown to be due to a Btochtm Blophvs Acta, 265 (1972) 25-60

MEMBRANE PHOSPHOLIPIDS OF E colt

37

defect in the sn-glycero-3-phosphate acyltransferase sl This defect was in the affimty of the acyltransferase for sn-glycero-3-phosphate which was 10-fold lower in the m u t a n t enzyme than in the normal enzyme Hsu and Fox 47 have reported a m u t a n t with an absolute reqmrement for glycerol This m u t a n t has an inactive biosynthetic s2 sn-glycero-3-phosphate dehydrogenase 47 Other mutants with a defect in this dehydrogenase have also been isolated including one which is supersensitive to p r o d u c t inhibition (L I Plzer, personal communication) The acylatlon o f sn-glycero-3-phosphate to form phosphatldlc acid (Fig 3, Reaction 1) has been the subject o f much recent work 71-73 so,as-as The Isolation o f temperature-sensitive mutants o f E cob unable to undergo the complete acylatlon sequence has contributed greatly to the understanding of these reactions The first acyltransferase mutants were reported by Cronan, R a y and Vagelos so and were isolated by a selection technique based on kllhng of n o n - m u t a n t cells with tritiated sn-glycero-3-phosphate The selection procedure is illustrated m he experiment shown in Fig 4 The wild-type parent, Strain 8, was defective m alkaline phosphatase and

I 0 ..---...~..~

o Non-Ra&oact~ve

10 "1

I0 |

10 ~

NO

)6~ I0 I

)o~

w i

i

i

DAYS OF STORAGE

Fig 4 Loss of viability of Strain 8 cells, mutagemzed with nltrosoguamdlne, due to the incorporation of all-radioactive or non-radloactxve sn-glycero-3-phosphate for 70 man The cultures were stored at 4°C and periodically plated for survivors at 25°C The ordinate is expressed as the number (N) of cells/ml at a given time dlvtded by the number (No) of cells/ml at the begmnmg of storage 8° the catabohc sn-glycero-3-phosphate dehydrogenase and was constitutive for the transport of sn-glycero-3-phosphate Because o f these characteristics over 9 8 ~ o f sn-[3H]glycero-3-phosphate taken up from the growth medium was incorporated into the cellular phosphohplds The incorporation of highly radioactive (4 7 C1/mmole) sn-[aH]glycero-3-phosphate by wild-type cells led to cell death, presumably due to radioactive isotope decomposition Thus, after Strata 8 was mutagenlzed with Btochtm Btophys Acta, 265 (1972) 25-60

38

J E CRONAN, P R VAGELOS

nttrosoguanldlne, a radioactive pulse was performed for 70 m]n with sn-[3H]glycero 3-phosphate at 37°C, the restrictive temperature The culture was then filtered, washed, a n d stored at 4°C, a n d allquots were plated dally at 25°C, the permissive temperature, in order to assess the n u m b e r of survivors The procedure was aimed at o b t a i n i n g m u t a n t s defecttve in phosphollp]d b]osynthesls at 37°C but n o r m a l at 25°C by selecting the cells able to grow at 25°C b u t u n a b l e to grow at 37°C Fig 4 shows that less t h a n one in 107 cells which were exposed to sn-[3H]glycero-3-phosphate at 37°C remained viable after storage for 22 days Approximately 75 ° o of the survtvors at 22 days were temperature sensitive in their growth behavior, a n d essentially all of these were defective in p h o s p h o h p i d biosynthesis as measured by [32p]Pi i n c o r p o r a t i o n into the phosphohptd at 37°C Thus thts technique ts very effective m selection of phosphohpld mutants A p p r o x i m a t e l y half of these phosphollpzd m u t a n t s were shown to c o n t a i n a tempelature-sensltlve sn-glycero-3-phosphate acyltransferase, the m u t a t i o n s in the rem a l m n g phosphohpld m u t a n t s have not yet been ]dentlfied The t h e r m o l a b l h t y of the acyltransferase of Strain 8 and of one of the acyltransferase mutants, CVI 5, is compared in Fig 5, where particulate fractions dertved from the two strains grown at

$ 30"

lO0

~.. 8C

~

~

~ 35"

60 " ~ ~

~42 °

•

4C

5 ~ 37"

I

'

,5

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0

I

,o

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I

~

MINUTESOF PREINCUBATIONAT GIVEN TEMPERATURE

'

3o

Fig 5 Thermolablhty of the ~n-glycero-3-phosphate acyltransferase activities of Strain 8 (left panel) and mutant CVI 5 (right panel) Particulate fractions of Strata 8 and CV15 cells grown at 25°C were incubated m phosphate buffer at the indicated temperatures After various time intervals, samples were removed and their initial rates of acyltransferase actlvlty were measured spectrophotometrlcally at 22cC The data are expressed relative to an unheated sample of the same extract ( ~ 100~) 8° 25°C were m c u b a t e d at 30-42°C for various time intervals before assay at 22°C It is a p p a r e n t that the acyltransferase activity decreased m u c h faster in the m u t a n t at every temperature Reversion analysis a n d enzymatic analyses of temperatureresistant revertants have indicated that the phenotype of th=s m u t a n t is due to a single m u t a t i o n All the m-glycero-3-phosphate acyltransferase m u t a n t s isolated by this Btochtm Btoph:,a Acta, 265 (1972) 25-60

MEMBRANE PHOSPHOLIPIDS OF E colt

39

technique have contained normal l-acyl-sn-glycero-3-phosphate acyltransferase activity On the other hand, a temperature-sensitive mutant possessing the converse phenotype has been reported by Hechemy and Goldfine a8 This mutant contains a thermolablle monoacyl-sn-glycero-3-phosphate acyltransferase, but It has a normal sn-glycero-3-phosphate acyltransferase The availability of these mutants has estabhshed that at least two different enzymes are required for the conversion of sn-glycero3-phosphate to phosphat~dic acid as shown in Reactions 1 and 2 sn-Glycero-3-phosphate + CoA

+

acyl-CoA

-~ monoacyl-sn-glycero-3-phosphate (1)

Monoacyl-sn-glycero-3-phosphate q- acyl-CoA ~ phosphatldlc acid [ CoA (2) In most naturally occurring phosphoglycerldes unsaturated fatty acids are preferentially esterlfied at position 2 and saturated fatty acids at position 1 of the glycerol molecule (see ref 35 for a comprehensive review) This asymmetric fatty acid distribution is thought to be of major importance in the functional and structural role of phosphoglycertdes m membrane processes, and thus the enzymatic mechamsms conferring such positional specificity of fatty acid residues are of central importance As mentioned above the fatty acids of the E colt phosphohplds are asymmetrically distributed with palmltate esterlfied to position 1 and unsaturated fatty acids prlmardy esterified to position 2 of the glycerol backbone t7,19,23 The fatty acid compositions of the various phosphohpid species are quite slmdar 4,18 as IS to be expected if they are all derived from phosphatldlc acid At what level does this specificity arise 9 Evidence from studies with mutants auxotrophlc for unsaturated fatty acids indicates that control does not reside at the level of the supply of the fatty acid species A likely origin of the asymmetric distribution of fatty acids would be m the esterlfication of sn-glycero-3-phosphate to form phosphatldlc acid Results of van den Bosch and Vagelos T2, later confirmed by Ray et a184 and Smensky a5 have shown that both the acylatlon of sn-glycero-3-phosphate in Reaction 1 and the acylatlon of 1-acyl-snglycero-3-phosphate m Reaction 2 proceed with great specifimty As noted in Table II, incubation of an envelope fraction with sn-glycero-3-phosphate and palmltyl-CoA resulted m the formation of monoacyl-sn-glycero-3-phosphate in which 92 ~o of the palmltate was m the 1-position 84 In contrast, when the acyl donor was oleyl-CoA, palmltoleyl-CoA or cls-vaccenyl-CoA, the fatty a o d of the monoacyl-sn-glycero-3phosphate produced was mainly in the 2-position Myrlstate was found to be d~stributed between both positions with position 2 predominating Slmdar specificity studies have been carried out in which 1-acyl-sn-glycero-3-phosphate acyltransferase was tested w~th saturated and unsaturated thloesters In this instance, acylatlon proceeded at much h~gher rates w~th unsaturated thloesters than with saturated thioesters ~2 These tn vitro reactions result m products 84 a5 which are remarkably consistent with the composition found in the intact cells 17,1s,23 Taylor and Heath s9 have shown a very selective transfer of long chain/~-hydroxy fatty acids from ACP thloesters to the l-position of sn-phosphatldylethanolamlne However, the slgmfiBtochtm Btophys Acta, 265 (1972) 25-60

40

J E CRONAN, P R VAGELOS

T A B L E II S P E C I F I C I T Y IN A C Y L A T I O N O F s n - G L Y C E R O - 3 - P H O S P H A T E BY A P A R T I C U L A T E P R E P A R A T I O N F R O M E colt The sn-glycero-3-phosphate acyltransferase assay rmxtures contained 0 1 M Trls-HCl, p H 8 3, 5 raM MgC1.., 1 5 m M umformly labeled sn-[laC]glycero-3-phosphate (specific actxvlty 1 mC1/ramole), 1 mg/ml of fatty acid-free bovine serum albumin, 100 ug of enzyme protein, and the v i n o u s acyl-CoA's at the indicated concentrations, which were found to give maximal actw~ty After incubation for 10 m m at 25°C the reacUon m~xtures were extracted and the monoacyl-sn-glycero-3-phosphate fractions were isolated, dephosphorylated with phosphatld~c acid phosphatase, and the resulting isomeric monoglycerldes were separated b) borate-impregnated sd~ca gel thin-layer chromatography~a

Acyl d o n o r

Palmltyl-CoA Oleyl-CoA Palmltoleyl-CoA cts-Vaccenyl-CoA Mynstyl-CoA

Ac)l-CoA concentration ( uM )

Total rnonoaol-snglycero-3-phosphate treated with phosphatase (nmoles)

30 30 5 5 5

17 55 I 53 1 49 1 38

Recovered monoglycertde (nmoles ) 1-Acyl- 2-Ao'Iglycerol glycerol 14 0 41 0 23 0 15 0 47

0 12 45 1 14 1 14 0 81

Percentage o] total t ecovered monoglvce~ tde as l-acylglvcetol ( °o)

92 8 17 12 37

cance of this reaction is not clear as 3-hydroxy fatty aods are not found in phosphohplds m I,/Vo 2,8,9 The availability of the temperature-sensitive sn-glycero-3-phosphate acyltransferase mutants described above, and the strict positional specificity demonstrated m this reaction with regards to saturated vs unsaturated acyl groups, permitted the posing of another question How many enzymes are involved in the specific acylatlon of sn-glycero-3-phosphate9 Fig 6 shows the kinetics of thermal inactivation of s n glycero-3-phosphate acyltransferase activities of particulate preparations derived from two d~fferent sn-glycero-3-phosphate mutants, CV15 and CV31, when tested with either palmltyl-CoA or oleyl-CoA as acyl donors 84 It is apparent that the rate of thermal lnactwatlon of the enzyme was similar m these two mutants (half-life of 17 mm for CV15, 18 5 mln for CV31) More important, however, was the finding that the thermolabdlty of the actlvlty with either palmatyl-CoA or oleyl-CoA as acyl donor was identical m enzyme preparations from a given mutant S~mdar results have been obtained with additional temperature-sensmve sn-glycero-3-phosphate acyltransferase mutants tested with several saturated and unsaturated acyl th~oesters as donors Since it is known that under the conditions of these acyltransferase reactions oleate ~s predominantly transferred to poslt~on 2 of 6n-glycero-3-phosphate whereas palmltate ~s transferred almost exclusively to position 1, the finding that the enzyme activity xs heat inactivated at the same rate when tested with these acyl donors suggests that a single enzyme catalyzes the transfer of unsaturated acyl groups to posmon 2 or palmltate to position 1 of ~n-glycero-3-phosphate S~mllar studies to determine the number of enzymes involved m the acyl transfer to monoacyl-~n-glycero-3-phosphate have not yet been reported Btochm7 Btophvs Acta, 265 (1972) 25-60

MEMBRANE PHOSPHOLIPIDS OF E colt

41

These a c y l a t l o n r e a c t i o n s are also t h o u g h t to be the level at whxch the t e m p e r a t u r e a d a p t a t i o n o f the p h o s p h o h p l d c o m p o s i t i o n is achieved (see below) S m e n s k y as has shown t h a t the species o f p h o s p h a t l d l c acid synthesized m vltro when the p a m c u l a t e enzyme p r e p a r a t i o n was i n c u b a t e d at various t e m p e r a t u r e s with sn-glycero-3p h o s p h a t e a n d a m~xture o f s a t u r a t e d a n d u n s a t u r a t e d acyl C o A derlvatxves was q m t e consistent with the species o f p h o s p h o l l p l d s f o u n d m cells grown at the same temp e r a t u r e s Esfahanl et a133,90 have shown that an u n s a t u r a t e d fatty acid a u x o t r o p h i n c o r p o r a t e s exogenous fatty acids into its m e m b r a n e llplds m such a m a n n e r as to minimize variations in the physical p r o p e r t i e s o f the p h o s p h o h p J d s Sflbert 23 also

100 80

, . . . . ~ ~ ~ ~ i

60

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o

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,

..\ a \\ o

40

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~_. 2O

'

1;

' 2; MINUTES AT 37" '

3;

Fig, 6 Thermolablhty of the sn-glycero-3-phosphate acyltransferase achwty from mutants CVI5 and CV31 with palmltyl-CoA or oleyl-CoA as acyl donors 84 Enzyme preparations from the mutants were incubated for various t~me periods at 37°C, chdled to 0°C, and assayed spectrophotometrlcally for 5 mm at 25°C with either palrmtyl-CoA (30 F,M) or oleyl-CoA (30 aM) The data are expressed relative to an unheated sample of the same extract ( = 100%) ©--©, CV15, palrrutyl-CoA, D - E3, CVI5, oleyl-CoA, 0 - - 0 , CV31, palmltyl-CoA, l - l , CV31, oleyl-CoA r e p o r t e d similar d a t a I n view o f the lack o f c o n t r o l f o u n d at the fatty acid b~osynthetlc level a n d the extreme c o n t r o l d e m o n s t r a t e d for the a c y l a t l o n reactions, it is very likely t h a t these results m a y also be explained by the specificity o f the acylatlon reactions The identity o f the m e r c a p t a n m o i e t y o f the thloesters which function as acyl d o n o r s in R e a c t i o n s 1 a n d 2 m vtvo has n o t yet been established The discovery that fatty acids are synthesized d e n o v o as thloesters o f A C P 42 p r o m p t e d the c o m p a r i s o n o f a c y l - A C P ' s with a c y l - C o A ' s in the two acyltransferase reactions 71,72,a6 s7 Both a c y l - A C P a n d a c y l - C o A have similar activities a n d specifiCitles with regards to the Btochtm Blophys Acta, 265 (1972) 25-60

42

J E CRONAN, P, R VAGELOS

acyl group in the sn-glycero-3-phosphate acyltransferase reaction In the 1-acyl-snglycero-3-phosphate acyltransferase reaction there appears to be somewhat greater speoficity with regards to the acyl group when ACP thJoesters, as opposed to CoA thloesters, are substrates Since thloesters of both ACP and CoA are active substrates in the synthesis of phosphatidate m vmo, It IS impossible to conclude from these studies which cofactor functions under physiological conditions It is possible that both types of thloesters function under different condmons For instance, thioesters of ACP might be the substrates when phosphatldate is synthesized from newly synthesized fatty acids, whereas thioesters of CoA could be the substrates during renewal of phosphohpid acyl chains with pre-existing fatty a o d s or for the incorporation of exogenous fatty acids taken up from the medium In support of this proposal are the experiments of Overath et ct191 which have indicated that the first enzyme of the fl-oxldatlon pathway, acyl-CoA synthetase, IS required for the lncorporation of exogenous fatty acids into phosphohplds m vtvo Mutants defioent in this enzyme incorporate endogenously produced fatty acids into phosphohplds normally but are unable to incorporate exogenous fatty a c i d s 79 2 Synthe~l~ of CDP-dlglycerMe This hpld IS regarded as an activated phosphatidic a o d The evidence for involvement of CDP-dlglyceride in the pathway is clrcumstantml since the hpld has not been isolated from any organism This evidence is based upon the presence of an enzyme system m the particulate fraction of the cell which catalyzes the synthesis of CDP-diglycerlde from CTP and phosphatldic a c i d 69 (Fig 3, Reaction 2), and the presence of enzymes catalyzing the condensation of CDP-diglycerlde with L-serlne and with ~n-glycero-3-phosphate to form phosphatldylserme 66 and phosphatldylglycerol phosphate 67 (Fig 3, Reactions 3 and 5), respecttvely Although these latter two enzymes were not tested to see if phosphatldic acid could function as the phosphatidyl donor, the finding that the reverse of the reaction which catalyzes the synthesis of phosphatldylglycerol phosphate is greatly stimulated b y C M P 67 indicates that the nucleot~de is required for the forward reaction 3 Synthest~ of phosphattdylglycetol Although the reaction between CDPdlglycerlde and sn-glycero-3-phosphate is believed to yield phosphatldylglycerol phosphate as an intermediate, this mtermedmte has not been demonstrated in E colt The rationale for its inclusion m the pathway is based on the presence in this orgamsm of a particulate enzyme that catalyzes its synthes~s from CDP-dlglycerlde and snglycero-3-phosphate 6~ (Fig 3, Reaction 5) and the existence of a separate particulate enzyme which catalyzes the dephosphorylation 68 of phosphatidylglycerol phosphate to yield phosphatidylglycerol (Fig 3, Reaction 6) Both of these enzymes have been extracted from the particulate fraction and partially purified 67 68 The sn-glycero-3phosphate CMP phosphatldyltransferase is rather reactive compared with most of the other phosphohpld biosynthetic enzymes It is stimulated by the detergent Triton X-100, and ~t is inactive with 6n-glycero-l-phosphate, glycerol, myo-inosltol, serlne and sn-glycero-2-phosphate It requires Mn -'+ or Mg 2+ for activity 67 The phosphatJdylglycerol phosphate phosphatase is much more active than the preceding enzyme 68 This enzyme is also stimulated by Triton X-100, requires Mg 2+, and is Btochtm Btophys Acta, 265 (1972) 25-60

MEMBRANE PHOSPHOLIPIDS OF E. cob

43

specific for phosphatldylglycerol phosphate The enzyme does not hydrolyze snglycero-3-phosphate or phosphatldIc acid 4 Synthests o f cardwhpm Fig 3, Reaction 7, demonstrates the synthesis of cardlohpln from phosphatidylglycerol and CDP-dlglycerlde, however, unequivocal evidence that this is the major reaction producing cardiohpin in E cob has not yet been provided The preliminary report by Stanacev et a173 of an enzyme catalyzing this step and some evidence for a precursor-product relationship between phosphat~dylglycerol and cardlohpin in i,lVO lend support to this hypothesis (see below) On the other hand, the results of Stanacev et a173 showed only a moderate stimulation of cardlohpln biosynthesis by the addition of CDP-dlglycende It seems unlikely that the synthesis occurring in the absence of added CDP-diglycerlde was due to endogenous CDP-diglycerlde, as other enzymes in identical preparations show an absolute requirement for exogenous CDP-dlglycende 66.67 Ramplnl et al92 have postulated another pathway for the synthesis of cardiohpin from phosphatldylglycerol in E eoh which stipulates that 2 molecules ofphosphatldylglycerol react to form 1 molecule of cardlohpin plus free glycerol This postulate is based on the conversion of phosphatidylglycerol to cardJollpln in whole cells in which energy metabolism and phosphohpld synthes~s (hence presumably CDPdlglyceride synthesis) is defective92 Under such circumstances the decrease in the phosphate moieties of phosphatldylglycerol was stolchlometrlc with the increase in phosphate of cardiohpm This postulate is chemically quite reasonable, as Stanacev and Stuhne-Sekalec 93 demonstrated that cabbage phosphohpase D will catalyze the formation of cardiohpln from two molecules of phosphatldylglycerol Thus, there may be another pathway for cardlohptn biosynthesis in E cob 5 Synthesis o_[ phosphattdylethanolamlne The reformation concerning the biosynthesis of this predominant E colt hpld, is also rather fragmentary The involvement of phosphatldylserme as an intermediate in this synthesis Js predicated on the presence of a small pool of phosphatJdylserine which has a rapid synthetic rate and turnover rate 46,5° and on the demonstration of L-serJne-CMP phosphatldyltransferase and phosphatidylserine decarboxylase activities (Fig 3, Reactions 3 and 4) in the cell 66 The L-serme-CMP phosphatidyltransferase activity differs from the other phosphohpld btosynthetlc enzymes in that it is found m the soluble fraction of the cell 66 However, we (J E Cronan and P R Vagelos, unpublished data) have found that a considerable portion of the activity is present m the washed envelope fraction when the cells are gently lysed Phosphatldylserlne decarboxylase is much more active than the phosphatidyltransferase activity 66, and this is consistent with the lack of accumulation of phosphatldylserlne m i,tvo The phosphatidylserlne decarboxylase has recently been extracted from the membrane and purified to homogeneity 94 Its enzymatic actiwty is inhibited by hydroxylamlne and 4-bromo-3-hydroxylbenzyloxyamme, indicating a possible pyrldoxal cofactor 94 Treatment of growing cells with hydroxylamlne results in the decreased synthesis of phosphatldylethanolamine and a marked accumulation of phosphatidylserlne (J E Cronan, unpubhshed results) Btochtm Bwphys Acta, 265 (1972) 25-60

44

J E CRONAN, P R VAGELOS

Kanfer and Kennedy 66 have shown that crude extracts E colt do not contain the enzymes of the CDP-ethanolamine pathway of biosynthesis of phosphatldylethanolamine found in mammalian cells 6 Anomalous enzymes Two enzymes have been described in E coll which are potentially part of the biosynthetic pathway but for whxch no role is postulated The first enzyme is a dlglyceride klnase activity first described by Pierlnger and Kunnes 9s This enzyme catalyzes the phosphorylatlon of monoglyceride or dlglycerlde by ATP to form lysophosphatldic acid or phosphatldlc acid The enzyme has recently been solublllzed 96 and shown to have an absolute specificity for 1,2-dlglycendes 97 of the L configuration 56 This enzyme is not assigned a role in the biosynthetic pathway as Chang and Kennedy s6 have shown that no rapidly metabolized pool of dlglycerlde is found in growing cells such as would be expected for a biosynthetic intermediate The second anomalous enzyme catalyzes the dephosphorylatlon of phosphatldlc acid or lysophosphatldlc acid to diglyceride or monoglycerlde respectively 72 This enzyme, which is also particulate, is distinct from phosphatldylglycerol phosphate phosphatase and alkaline phosphatase A role for this enzyme is unassigned as no active pool of glyceride is found in cells s6

IVB

Effect o f culture condmons

1 Effect of temperature It was first noted by Marr and Ingraham 98 and later by many other workers 36'aS,aa 99,100 that E ~coh adJusts the fatty acid composition of its phosphohplds in response to growth temperature As the temperature of growth is lowered the proportion of unsaturated fatty acids (chiefly cts-vaccenlc acid) in the membrane increases The hpxds isolated from E coh cultures grown at various temperatures have quite different physical characteristics a6 The permeability of hposomes made from these hpids decreases markedly when the phosphollpld used is from bacteria grown at higher temperatures Such studies suggest that when the temperature of growth is increased, the bacteria compensate for the increase in permeablhty (caused by the temperature change) by reducing the degree of unsaturatlon of the phosphohpld paraffin chains This as the result of changes in fatty acid composition of the phosphohplds, as the relative proportion of the phosphohplds does not change at different temperatures ~6 (and J E Cronan and P R Vagelos, unpublished data) Okuyama 1°1 has shown that the metabolism of the phosphohpids of E cob is dramatically altered by a shift in temperature from 37 to 10°C The data suggest that temperature acclimatization involves the reshuffling of the fatty acid components of the hpids in order to produce hpids of the proper composition The best explanation for the changes in phosphohpld fatty acid composition with changes m growth temperatures comes from the work of Slnensky 8s, who, as mentioned above, demonstrated that the m vttro synthesis of lysophosphatidate and phosphatldate from sn-glycero-3-phosphate and a mixture of palmltyl-CoA and oleyl-CoA at different temperatures is consxstent with the composition of the phosphohplds of the cells E

Blochlm Btophys Acta, 265 (1972) 25-60

MEMBRANE PHOSPHOLIPIDS OF E colt

45

grown at the same temperatures These experiments suggest that the primary enzymes responsible for these temperature adaptations are the sn-glycero-3-phosphate and the monoacyl-sn-glycero-3-phosphate acyltransferases What advantage to the cell is a mechanism to alter the fatty acid composition of its phosphohplds in response to temperature9 Recent physical studies on mycoplasmal membranes indicate that the membrane lipid m vtvo exists in a somewhat "fluid" state 14.is This "fluid" state is dependent on the species of fatty acids of the membrane hplds Saturated fatty acids tend to render the membrane less fluid while unsaturated fatty acids have the opposite effect Lowering the temperature of the environment of the membrane renders a membrane of given fatty acid composition less fluid Thus the increase in unsaturatlon may be vtewed as a means to ensure that the proper degree of membrane fluidity IS maintained Cells with membranes which have lipid components of excessive or too httle fluidity are expected to function in an aberrant manner This is observed when unsaturated fatty acid auxotrophs are supplemented with abnormal fatty acids Such cells divide abnormally (F Ruch, D F Sllbert and P R Vagelos, unpublished data) and lyse when shifted in temperature a3'9° 2 Effect o f growth phase Four pronounced changes in the phosphohpld composition occur during the transition of E colt cultures from exponential growth to the stationary growth phase These changes are an increase in cardiolipln 5,18,49.6°, a decrease in phosphatldylglycerol s,ls,49.5°,6°, an increase in cyclopropane fatty acids 5"1a'3°'99'1°2'1°3, and a decrease in unsaturated fatty acids s'la'a° 100,a02,103 The first two changes are thought to be related as phosphattdylglycerol is a precursor of

cardlohpln 46,92, and the decrease in phosphatldylglycerol phosphorus is almost stolchiometrlc with the increase in cardlohpin phosphorus s,la,6° This Stolchlometry has been questioned by Randle and coworkers 49, but their report indicating the presence of a relatively large amount of phosphatldlC acid renders their data difficult to mterpret The increase in cyclopropane fatty acids is accompanied by a concomitant StOlch~ometric loss of unsaturated fatty acids An enzyme which converts the unsaturated fatty acid moieties of intact phosphohpids to their cyclopropane derivatives has been described in several bacteria including E colt 5.3o,lo3,1o4. The methyl group is donated by methlonine through S-adenosyl methionlne 5.3°.~°4,1°7 The enzyme from Clostrtdturn butyrtcum, which actively catalyzes the methylation of unsaturated fatty a o d of phosphohplds, is unable to use unsaturated free fatty acids or their CoA esters as substrates ~°6,~°7 Thus, an intact phosphohpld containing an unsaturated fatty acid is considered to be the substrate tn i,tvo This Is consistent with the finding that the phosphohplds of E colt are methylated to various extents and at differing rates tn VlVO4'5'1s Phosphatldylethanolamlne is much richer in cyclopropane fatty acids during exponential growth than are phosphatidylglycerol and cardlohpin The rate of methylatlon of phosphatldylethanolamme m vtvo is constant throughout growth, whereas the rate of methylation of the other two phosphohpids shows a dramatic increase during the transition from exponential to stationary growth s.~8 Several rationalizations for the conversion of unsaturated to cyclopropane fatty acids Btochtm Btophys Acta, 265 (1972) 25-60

46

J E CRONAN, P R VAGELOS

have been put forward It has been postulated that this conversion is a mechanism to prevent the oxidation of double bonds 3°, to prevent phosphohpld turnover 5 or to play a structural role in the cell envolope 28 All these postulates assume that the conversion gives an advantage to stationary phase cells but would be a disadvantage to growing cells This is consistent with the finding that cyclopropane fatty acids are poorer growth supplements than their unsaturated homologs for the unsaturated fatty acid auxotrophs 2°'34 Also interesting m thls context is the finding that variants of Vtbrlo cholerae, which have unusual resistance to adverse environmental conditions, contain cyclopropane fatty acids, whereas the parent of these variants does not l°s 3 Changes m energy metabohsm The changes in the proportions of phosphatidylglycerol and cardlohpln which occur during the transition from exponential to stationary phase also occur during the following manipulations addition of cohcln K 57, dlnltrophenol sT, penicillin 5s 60, or cyanide 1°9, and infection with bacteriophage T411°-112 These manipulations, although very diverse, have one character m common, they all result in decreased cellular phosphorylatlng ability The production of lysophosphatldylethanolamine has also been reported under some of the same conditions Except for the case of bacteriophage infection 59,j12,113, where sensitive controls for cellular lysls were used a~3, the production of lysophosphatldylethanolamlne can be attributed to phosphohpase digestion caused by lysls of the cell (see below). Changes in the amount of cellular cyclopropane fatty acid can be engendered by changes in the medium ~°°.~°x, although the data are not explained by a simple hypothesis Decreased oxygen tension dramatically increases the amount of cyclopropane fatty acid 1°°'1°3, while limitation of carbon source decreases it, and phosphate starvation has no effect 1°2 The amount of hpid in E COil has been shown to be Independent of the growth medium used 7'76 (J E Cronan, unpublished data) Many carbon sources have been tested to see if the amount or the proportion of the phosphohpld fraction of E coh can be altered, but no sigmficant differences were seen Some of the carbon sources tested were glucose, glycerol, acetate, fatty acids, succlnate and amino acids The same variation in carbon source also has no effect on the fatty acid composition of the cells76 9a Starvation or chemostatlc limitation of phosphate or of carbon source does not decrease the amount of phospholIpld 7 and produces no changes in the fatty acid composition of the phosphollplds 1°2

1VC Regulatton of phosphohpld biosynthesis As seen above, the lipid composition of E COil is remarkably constant and is largely independent of the culture conditions As mentioned above, phosphohpid and fatty acid biosynthesis in normal growing E eoh do not appear to be regulated at the level of small molecule precursors It has been reported that the addition of fatty acids to the growth medium does not depress endogenous fatty acid biosynthesis aS, Btochtm Btophys Acta, 265 (1972) 25-60

MEMBRANE PHOSPHOLIPIDS OF E coh

47

although more recent data indicating the opposite conclusion have been presented ss, and the differences in these results are probably due to strain differences (D F. Sllbert, personal communication) A d d m o n of intact phosphohplds to the medium appears to have no effect on the cellular phosphohplds (although the added hplds are rapidly degraded m the medium (J E Cronan and P R Vagelos, unpublished data)) Thus, the lipid composition of the cell envelope appears to be rigidly controlled, but the mechanism(s) of this control ~s completely unknown Sokawa et aL 114 have shown that lipid synthesis is controlled by amino acid starvation in qualitatively the same way as R N A synthes~s, although the quanntatlve effect is not as profound as that observed on R N A synthesis Amino acid starvation of a stringent strain of E cob results in a large decrease in the rate of phosphollpld biosynthesis This inhibition is overcome by genetic mutation of the rel locus or by treatment with chloramphenlcol, as is the mhlblnon of R N A synthesis 11s Experiments in our laboratories (J E. Cronan and P R Vagelos, unpubhshed) and in that of G L Powell (personal commumcatlon) have confirmed the results of Sokawa et a1114 and have shown that phosphohplds are made in normal propornons in amino acid deprived cells T r o p p et a1116 on the other hand, have reported that hpld synthesis continued at the same rate in the presence or absence of a reqmred amino acid in both relaxed control and stringent control cells However, recent experiments from Tropp's laboratory (personal commumcatlon) mdlcate that the differential effect in stringent and relaxed strams reported by Sokawa et a1114 is observed only under conditions of high aeration When the rate of aeration is limited, no difference in hpld synthesis is observed between relaxed and stringent strains starved for a required amino acid. The degree of aeration under these conditions affects only the differential effects seen in lipid synthesis The differential effect seen in R N A synthesis in the relaxed and stringent strains does not depend on the rate of aeration of the culture Therefore, ~t seems that the relaxed phenotype has an effect on hpld synthesis only under certain conditions, and this effect is probably secondary to the effect on R N A synthesis.

IVD

Degradatton o f the hptds

The degradation of the phosphohplds of E colt has been studied to a hm~ted extent In growing cultures the phosphate atom of phosphatidylethanolamme is completely stable while the phosphate moieties ofphosphandylglycerol and cardlollpm turn over at a moderate rate (half-hfe of about one generation time) 4,46,s°.117 The non-acylated glycerol of phosphatldylglycerol turns over at the same rate as the phosphate atom 1iv Some of the apparent turnover of phosphatldylglycerol is thought to be due to its conversion to cardlohpm TM, but the situation is complicated by the turnover of cardlohpm 4,46 Although the turnover of phospyatidylglycerol appears to reqmre energy 117, this turnover does not appear to reqmre de novo lipid synthesis as it occurs when phosphohpld synthesis ~s halted by glycerol starvation of a glycerol auxotroph 47 or by inactivation of a thermolabde sn-glycero-3-phosphate acyltransBtochtrn Btophys Acta, 265 (1972) 25-60

48

J E CRONAN, P R VAGELOS

ferase (J E C r o n a n and P R Vagelos, unpublished results) Phosphatidylethanolamine shows no turnover of phosphorus during the absence of hpld synthesis 47 (J E C r o n a n and P R Vagelos, unpublished results) The fatty acid moieties o f the phosphollplds are apparently exceedingly stable N o turnover of fatty acids is seen in growing cells s,3° or even in chilled cells 1°° (in which the turnover of phosphatldylethanolamlne phosphorus is observed) Several phosphohpase activities 59'tla'121 have been detected m E colt but the functions of these enzymes are u n k n o w n Phosphohpase A159'121, A2121, and C 1Is activities have all been reported It is supposed that these enzymes are compartmentalized in the cell since cellular phosphohpids are quite stable (see above), the phosphohpases are apparently activated if the cells are ruptured 5s,59 or heated 59 i22 The phosphohpase A1 has been extracted from the particulate fraction of the cell and purified to near homogeneity The enzyme was extracted from the cell envelope and purified in the presence of sodium dodecyl sulfate to which it is extremely stable s9 It seems probable that th~s enzyme IS responsible for the p h o s p h o h p l d hydrolysis seen during phage T4 Infection s 9.1 i1 112 and cohcln K action 57 The enzyme is localized on the outer m e m b r a n e of the cell and may be thus compartmentalized In contrast with phosphollpld degradation, the catabolism of fatty acids m E colt is well understood The catabolism proceeds by the classical fl-oxidatlon sequence found lm mammalian mitochondrla and results in the complete degradation of the fatty acid to acetyl-CoA which then can be used as a carbon source 9L123 The levels of the oxidative enzymes found in stratus o f E cob growing in medium not containing fatty acids is quite low U p o n addition of fatty acids to the medium the levels of the enzymes are coordinately increased at least 10-fold 9I 123 Thus this pathway is reducible The inducing fatty acids must be at least twelve carbons long Several other enzymatic activities required in the degradation of rlcinoleic acid have also been described I24

v FUNCTIONS OF THE PHOSPHOLIPIDS Phosphollplds are beheved to have at least two distinct roles m biological membranes The first role is structural The hpld c o m p o n e n t of the m e m b r a n e ~s believed to impart much o f the uniqueness o f membranes (e g permeability) as well as providing a matrix in which m e m b r a n e proteins are imbedded A n o t h e r clearly delineated role for hpids is their requirement for the function o f certain membraneassociated enzymes

VA

Requtrement f o r enzvmatle acttvtty

Enzymes which require phosphohpld for activity have been described from m a n y sources xzS, and these represent the m e m b r a n e - b o u n d enzymes that have been soluBtochtm Bwphys Acta, 265 (1972) 25-60

MEMBRANE PHOSPHOLIPIDS OF E coh

49

blllzed, or at least delipldated to the point where a hpM requirement is demonstrable One of the systems best studied is the galactosyl transferase of S typhtmurtum studied by Rothfield and coworkers (see ref 126 for a recent review) These investigators have shown that the galactosyltransferase enzyme involved in hpopolysaccharlde synthesis forms a tertiary complex which is needed for activity The complex is composed of the enzyme, llpopolysaccharlde and phosphollpld Although the phospholipid requirement is satisfied by phosphatldylethanolamlne, phosphatldylglycerol, or cardlohpm, phosphatldylchohne is not active in this system This system also displayed specificity in regard to the fatty acids since only phosphohplds which contained at least one double bond or cyclopropane fatty acid were active t26'a27 Another example of a membrane-bound enzyme, which has a phosphohpld requirement after ~t is solubdlzed, is Enzyme II of the phosphotransferase system studied by Roseman and coworkers 128"~29 The phosphotransferase system, consisting of three protein fractions, Enzymes I and II, and a low molecular weight histldmecontaining protein designated HPr, functions in the following reactions Mg2+ Enzyme I

phosphoenolpyruvate 6- HPr

~

phospho-HPr + pyruvate

Mg2+ Enzyme II phospho-HPr ÷ sugar --~ sugar-P 6- HPr Enzyme I, a soluble enzyme, catalyzes the phosphorylatlon of an lmldazole nitrogen of a hxstidlne residue of HPr The transfer of phosphate from phospho-HPr to carbohydrate is catalyzed by Enzyme II which is a membrane component Although the details of action of Enzyme II are complex, since it has been fractxonated into a number of protein components, of major interest is the fact that solubilization of this fraction caused it to become inactive in the absence of crude E colt lipid Phosphatldylglycerol, which ~s a minor component of this lipid, was found to be the hpld factor which reactivates purified Enzyme II The phosphotransferase system is of particular significance since recent evidence 12s suggests that it is involved in the active transport of carbohydrates into bacterial cells An attempt has been made by Mdner and Kaback TM to demonstrate that phosphatIdylglycerol is required for both the phosphorylatlon and uptake of a-methylglucoslde mediated by the phosphotransferase of E colt membrane preparations Treatment of membranes with a crude phospholipase D preparation was shown to inhibit completely both the phosphorylatlon and uptake of a-methylglucoside Under these conditions approximately 5 0 ~ of the membrane phosphatldylglycerol was hydrolyzed, whereas other phosphohpids were much less degraded The authors also demonstrated that transport activity returned spontaneously to normal within 15 mm after removal of the phospholipase, and that phosphatidylglycerol was specifically synthesized by the membrane preparation during that interval Although the quantity of phosphatidylglycerol synthesized after removal of the phosphollpase was extremely small (compared to that amount lost during the phospholipase hydrolysis), and a Btochtm Btophys Acta, 265 (1972) 25-60

50

J E CRONAN, P R VAGELOS

very crude phosphohpase preparation was used, the experimental results are consistent with the involvement of phosphatldylglycerol in the phosphotransferase system m membrane particles However, it should be mentioned that reshuffhng of the remaining membrane phosphatldylglycerol could also account for phosphotransferase reactivation even in the absence of hpld synthesis Cunnmgham and Hager 1~°.131 have studied the E colt pyruvate oxldase which binds both thiamine pyrophosphate and F A D as prosthetic groups and catalyzes the oxidative decarboxylatlon of pyruvate to acetate and CO2 Although this enzyme is found m the soluble fraction when cells are ruptured, the natural electron acceptor for the reduced enzyme is the cell envelope-associated electron transport system which includes both ublqulnone-6 and cytochrome bl, and this suggests that the enzyme tn vtvo is probably closely associated with the membrane This hypothesis is supported by the discovery that activity of crystalline pyruvate oxldase is stimulated 15-100-fold in the presence of phosphollp~ds or long-chain fatty a o d s Water-soluble forms of any of the naturally occurring phosphohplds are equally active m this system Phosphatides dramatically effect the kinetic parameters of the enzyme the Km values for pyruvate and thmmlne pyrophosphate decrease 13-fold and 4-fold, respectively, and thiamine pyrophosphate is bound cooperatively to the enzyme On the bas~s of these findings, the authors have suggested that phosphohp~ds m~ght act as allosteric activators of this enzyme m vtvo

VB

Structural mtegrlty o f the cell

Several hnes of evidence suggest that unsaturated fatty acids are needed to support growth and cellular integrity The supplementation of unsaturated fatty acid auxotrophs of E colt with exogenous fatty a o d s of unusual geometry causes altered growth 24 The rate and extent of growth at 37°C is affected and the cells tend to lyse and grow in non-septated filaments when fatty acids which support growth poorly are supphed (D F Sllbert, F Ruch and P R Vagelos, unpublished results) When such auxotrophs are starved of an appropriate fatty acid, the cell continues to grow and divide for about a generation before lysls and cell death occur 132 It has been shown that growth must occur m order that the cell be lysed during unsaturated fatty acid starvation 9°'132 These findings suggest that lysls Js due to the production of phosphollplds lacking an appropriate unsaturated fatty acid 2a and that such phosphohplds are not sufficient for cell integrity 132 The starvation of glycerol auxotrophs 47 or the shift of temperature-sensitive phosphohpld mutants to non-permissive conditions does not cause cellular lysm 1a3 However, the latter conditions result in cell death, probably due to the l r r e v e r s l b l h t y of the enzyme inactivation In contrast to the situation m uninfected cells, the shift of a temperaturesensitive sn-glycero-3-phosphate acyltransferase mutant to a non-permisswe temperature after infection with bacteriophage T 4 results Jn cell lysls 133 This result has been lhochtm Btophys Acta, 265 (1972) 25-60

MEMBRANE PHOSPHOLIPIDS OF E colt

51

interpreted as showing a requirement of host phosphohpld metabolism for the successful infection by T a Several workers have reported that various phosphollpase preparations can lyse E cob cells la4'~3s, and the reported data have been interpreted as demonstrating a role for phosphohpld in the rigidity of the cell envelope A recent finding 136 that the observations of at least one of these reports 134 were due to the &gest~on of the polysaccharlde moiety of the envolope by an enzyme contaminating the phosphollpase preparation suggests that these experiments should be reevaluated since they were carried out with crude phosphohpase preparations 13s

VC

Active transport

Nlkaldo la7, later Tarlov and Kennedy 51 demonstrated that phosphollpld synthes~s is stimulated during the functioning of the/~-galactoslde transport system. The latter authors 51 reported that a general increase involving all species of phosphollplds occurred, and they suggested that this might result from a transient release of the inhibition of phosphohpld biosynthesis due to the fact that nitrogen-starved cells were used in these experiments To date, the study of active transport using mutants defective in hpld synthesis has been concerned solely with sugar transport, specifically the fl-glucoslde transport system and the ~-galactoslde transport system The fl-galactoside transport system is specified by the lac operon y gene The protein product of this gene, called the M (for membrane) protein, has been identified and isolated by Kennedy, Fox and coworkers13S Fox and coworkers 47'139-141 have utilized the unsaturated fatty acid auxotrophs described above (Section I l i A 5) and a glycerol auxotroph to probe the function of phosphohplds in the synthesis of the/%galactoslde and #-glucoslde transport systems In the first series of experiments they investigated the induction of the lac operon in the unsaturated fatty acid auxotroph grown in the presence or absence of the required fatty acid Cells were grown Initially m the presence of oleate, then washed free of the fatty acid, and ahquots were grown and induced for the proteins of the lac operon either in the presence or absence of oleate Fig 7 indicates that for the first 30 mln of reduction, ~-galactosldase and galactoside acetylase, two soluble protein products of the lac operon, were synthesized at the same rates in the presence and absence of oleate Induction of the transport system, measured as the uptake of 14C-labeled methyl-l-thlo-fl-D-galactoslde (TMG), however, did not occur when oleate was omitted from the medium When inducer was removed and oleate was added to the culture lacking fatty acid after 30 mln of induction, transport activity was not recovered In addition, removal of oleate from the induced culture did not affect the capacity of those cells for transport of galactoslde These experiments demonstrated that an unsaturated fatty acid, and therefore normal phosphohpld synthesis, is required for induction of the fl-galactoslde transport system F r o m the results of additional Btochtm Btophys Acta, 265 (1972) 25-60

52

J E CRONAN, P R VAGELOS 25

*m e

o

Ole0te J5

~o ,o ~

5

2500

/ Oleat/

& u

o/

2000

ISO0

J"

Z"

IOO0 i

Omit .~,Z~~t~r'" Ol eale

500 o i-.

c o

2b

,b

6b

0

20

o 40

60

0

-~'

Induchon Period (Minutes)

Fig 7 Induction of the lac operon m an unsaturated fatty acid auxotroph A culture of the auxotroph defective in 3-hydroxydecanoyl thioester dehydrase was grown at 37°C to late exponential phase in standard medium supplemented with 100 l~g/ml of olelc acid The cells were harvested, washed, resuspended, and divided equally between two cultures containing standard medium Olelc acid (100 ~,g/ml) was added to one but omitted from the second After 15 mm of Incubation at 37°C, mopropyl-l-thlO-t~-galactoslde was added to both at a final concentration of 0 5 mM Samples were withdrawn and the cells were harvested, washed, and processed for the assay of ~-galactosidase ([], II), galactoslde acetylase ('~, A), and uptake of [14C]TMG The activities of ~-galactosldase and acetylase are expressed as per cent of specific activity of a culture induced for three generations in standard medium supplemented with 100 i,g/ml of olelc acid and lsopropyl-l-thlo-3-galactoslde At point A, the cells of a sample of the culture with olelc acid were collected, washed, and suspended in medium with oleic acid and ~sopropyl-l-thlo-O-galactoslde omitted (~ -- 01) At point B, cells of the culture with olelc acid ormtted were collected, washed as above for A, and suspended m medmm containing olelc acid but no lsopropyl-l-thlo-t~-galactoslde ( ~ - ~ ) The subcultures A and B were incubated in parallel with the parent cultures, and samples were assayed for TMG uptake From F O X 130

experiments the a u t h o r c o n c lu d e d that unsaturated fatty acids are also required for the l o n g - t e r m m a i n t e n a n c e o f the t r a n s p o r t system once it has been induced

How-

ever, this i n t e r p r e t a t i o n is c o m p h c a t e d by the fact that a u x o t r o p h s deprived o f the essential fatty acid are generally unhealthy and tend to lyse 132 H su and F o x 47 utlhzed a glycerol a u x o t r o p h to study the effect o f blocking o f hp]d synthesis on the i n d u c t i o n of the lactose t r a n s p o r t system

R e m o v a l o f glycerol f r o m the m e d i u m

caused an i m m e d i a t e cessation o f p h o s p h o h p I d synthesis in this organism, whereas D N A and protein synthesis c o n ti n u e d for a short period Th e kinetics o f i n d u c t i o n o f 3-galactosidase were unusual in cells newly deprived o f glycerol This m ay be related to the fact that a p o o r e r c a r b o n and energy source was used in those cultures deprived o f glycerol H o w e v e r , as in the case o f the u n s a t u r a t e d fatty acid a u x o t r o p h , where p h o s p h o l l p l d synthesis was blocked by r e m o v a l o f the required u n sat u r at ed fatty acid, blocking o f hpid synthes]s by glycerol d e p r i v a ti on caused a d r a m a t i c decrease m the reducibility o f the 3-galactoslde t r a n s p o r t activity relative to 3-galactosldase activity In these experiments the M protein was assayed and f o u n d to be present in n o r m a l c o n c e n t r a t i o n m the m e m b r a n e s o f cells which were deprived o f glycerol Thus it IS a p p a r e n t that the defect m t r a n s p o r t was n o t due to a defect in the reduction o f the M Biochtm Blophys Acta, 265 (1972) 25-60

M E M B R A N E PHOSPHOLIPIDS OF E colt

53

protein Although experiments to assess the possible passive leakage of accumulated T M G during glycerol starvation were not reported, these experiments together suggest that the synthests of certain membrane proteins as physiologically funcUonal structures requires the simultaneous synthesis of membrane phosphohpld On the other hand, experiments of Overath, Hdl and Lamnek t42 suggest that fl-galactosIde transport activity can be induced after the interruption of overall lipid synthesis These authors used 3-decynoyl-N-acetyl cysteamine, a specific inhibitor of the ¢/-hydroxydecanoyl thloester dehydrase, to block unsaturated fatty acid synthesis Coordinate synthesis of transport activity and fl-galactosidase activity was noted for half a generation In addition these authors found that a functional //-galactoside transport system could be induced after removal of the required unsaturated fatty actd from an unsaturated fatty acid auxotroph Since the results of Fox and co4000 X

s:

2000

I

:\

I

I

oN

I000 600

-6 E

4OO

C

200 o

=

I00

\

60

4e I

,,k

32

I

I

I

34 (l/T)

[ 36

x 103

Fig 8 Temperature dependence ofp-nJtrophenyl-~-glucoside transport Cultures of an unsaturated fatty amd auxotroph were grown m standard medium supplemented with 0 1 mM phenyl-l-thlo-flglucoslde (an reducer of the fl-glucoslde system) and 0 02 ~ hnolelc (O - - 0 ) or elaldlC (©--©) acids At a culture density of 109 cells/ml, chloramphemcol was added at 50 F,g/ml, and the cells were processed for the assay of ~-glucoslde transport From Wilson and Fox x4°

workers 47,139 are in conflict with those of Overath, Hill and Lamnek 142, addmonal studies must be done to resolve these differences It should be pointed out that a dependence on phosphohpld synthesis for the formation of functional transport systems IS not a general phenomenon Mlndlch 143 has shown that net lipid synthesis is not required for the induction of lactose permease m Staphylococcus aureus In other experiments, Wlllecke and Mlndlch 144 have shown that citrate transport can be induced in the absence of phosphohpld synthesis Another type of membrane study utlhzlng the unsaturated fatty acid auxotrophs took advantage of the fact that the unsaturated fatty acids of the phosphohplds can be manipulated by varying the fatty acid supplement added to the growth medium First Schalrer and Overath 34, and then Wilson, Rose and Fox 141 investigated the effect of varying the phosphohpld fatty acid composition on membrane transport Fig 8 demonstrates the effect of temperature on fl-glucoslde transport by an unsaturated Btochlm Btophys Acta, 265 (1972) 25-60

54

J E CRONAN, P R VAGELOS

fatty acid auxotroph grown in medtum supplemented with either hnolelc or elaldic acids It is noted that the Arrhenlus plots for transport are blphaslc, the slopes extrapolating to an intersection at umque transmon temperatures The effects of five fatty acid supplements, including dlhydrosterculate (cts-9,10-methyleneoctadecanoate), cts-vaccenate, oleate, hnoleate, (cts-cts-9, 12-octadecadlenoate), and elaJdate, (trans-9-octadecenoate) were tested m similar temperature studies of both/%glucoslde and {/-galactosxde transport systems The transition temperatures varied wldel2~ from 7°C for hnoleate to 30°C for elaldate, but membranes containing each particular fatty acid showed the same transition temperature for both transport systems Since the two transport systems are independent, the authors suggested that the transition temperatures are an indication of a transition in the hpld environment of transport sites of the membrane Experiments reported by Overath et al145 suggest that the temperature profiles noted with different fatty acids may reflect a general change in cellular permeability and physiology rather than a specific change m membrane transport alone These workers found that the efflux of T M G as well as cell respiration and growth showed thermal transition points s~mllar to those found in the reflux studies, and that these transition points were similar to those exhibited by the phosphohplds extracted from the cells Wilson and Fox 14° have exploited the specification of the temperature profile of permease actwlty by membrane fatty acid composition to study the assembly of the transport system The effect of fatty acid compostt~on on transport was ascertained by assaying for transport at two different temperatures, one above and one below the transition temperature When the auxotroph was grown and induced on oleate or hnoleate, it was found that the 10°C/28°C transport activity ratio was characteristic for each fatty acid When cells were grown on a supplement of oleate and then shifted to medmm supplemented with hnoleate during a brief period of mduct~on of the transport system, the transport actw~ty ratio was characteristic of ceils grown entirely in medium supplemented with llnoleate On the other hand, when cells were induced for transport during growth m oleate medium and then shifted and grown for up to one generation m hnoleate medium, the transport actwity ratio remained characteristic of cells grown entirely with the fatty acid supplement present during the induction period These results led the authors to conclude that newly synthesized proteins associate preferentially with newly synthesized hplds A comphcatmg result in these experiments was the failure to observe the transport temperature profile charactensuc of oleate when cells were induced in oleate medium after prior growth m hnoleate medium This was rationalized by arguing that {~-galactoslde transport sites, reduced for short periods in oleate lipid, may not contain sufficment mass of oleate-derlved lipid to assume, between the two transition temperatures, the more ordered structure characteristic of a membrane containing only oleate It should be noted that the use of the thermal profile as a probe of transport system biogenesis is not comphcated by the finding of Overath et a1145 that other cellular processes are also affected Wdson and Fox ~4° grew the cells at a temperature such that the hpids formed from either supplement could be assumed to Btochtm Btophy~ Acta, 265 (1972) 25-60

MEMBRANE PHOSPHOLIPIDS OF E colt

55

be in a disordered state independent of the fatty acid composition The growth rates of oleate- and hnoleate-grown cells were identical as were the final extents of induction t 39 The results of the above studies differ from those of Overath, Hill and Lamnek 142 who studied the temperature characteristics of the /~-galactoslde transport system in an E cob unsaturated fatty acid auxotroph grown in medium supplemented with oleate and palmitelaldate These investigators grew the auxotroph in medium supplemented with one fatty acid and then shifted the organism to medium supplemented with the other fatty acid for various periods of growth prior to induction of the lac operon In addition cells growing with oleate or palmltelaldate were induced and subsequently shifted to media lacking inducer but containing palmitelaldate or oleate, respectively Temperature studies of the /~-galactos~de transport system in all instances indicated that the transition points observed in the Arrhenlus plots were dependent upon the average fatty acid compositions rather than the fatty acid composition of the phosphohpid synthesized during reduction of the transport system Thus additional experiments will be reqmred to explain the differences between the experimental results obtained in these two laboratories It should be mentioned that the inflection points of the thermal transitions observed for the above transport systems are much (15-20°C) lower than the inflection points observed in the same membranes by X-ray diffraction 146 (D M Engleman, personal communication) Also the range of temperatures over which the X-ray transition occurs is much greater than the abrupt transition range seen in transport Hence the temperature transitions for transport occur at or slightly below the beginning of the thermal transition observed by X-ray techniques X-ray methods observe the average lipid phase transition This suggests that transport systems occupy the most mobile or hquld sites of the membrane or that the transport p~oteins contribute to a lowering of the thermal transition point of the fatty acyl groups

VD

Growth and macromolecule biosynthesis

Danlels 147 has reported that phosphohpld synthesis is a cyclic phenomenon Using cells synchronized by a methionine starvation technique, he found that a large transient increase in the rate of phosphohpld synthesis occurred at the time of cell division These results must be considered prehmlnary Jn light of the effects of amino acid starvation on phosphohpld synthesis (see above) and should be repeated using less perturbing methods of synchronization For simdar reasons, the data of Ballestra and Schaechter 117 are difficult to interpret These authors used shift-down experiments and a mutant of E cob defective in an unknown step in cell division to conclude that phosphatldylethanolamine (and not phosphatidylglycerol) synthesis has a structural role related to growth and cell division Phosphohpld synthesis appears to have a vital role in cell growth and division Halting of phosphohpld synthesis by use of temperature-sensitive acyltransferase mutants 8°,ss or of a glycerol auxotroph 47 both Btochtm Btophys Acta, 265 (1972) 25-60

56

J E CRONAN, P R VAGELOS

cause an abrupt halt in cell growth Agents which inhibit cell division also change the relative amounts of the phosphohpJds to resemble the amounts found m stationary phase cells 148 The coupling of macromolecule synthesis to phosphohpld biosynthesis or vtce versa has only been established for the case of relaxed control (discussed above) When phosphohpld synthesis is halted in temperature-sensitive acyltransferase 8° s8 mutants or by starvation of a glycerol auxotroph 47, macromolecule synthesis declines at a rate less than the rate of decay of phosphohpld biosynthesis Differential effects among the various macromolecules have been noted, but these data should probably be classified as prehmlnary as the lntracellular pools and transport of the precursors used to monitor synthesis may seriously affect the results We have used a temperature-sensitlve acyltransferase mutant 8° to test the hypothesis that membrane lipid and membrane protein synthesis might be tightly coupled We found no indication that a halt in membrane lipid synthesis produced any preferential effect on membrane protein synthesis (J E Cronan and P R Vagelos, unpublished data) A similar result was found during glycerol starvation of a glycerol auxotroph 47 Two mutants of E coll selected as defective in D N A synthesis also show abnormahtles in phosphohpld biosynthesis 18 149 The composition of the phosphohplds of these mutant strains are quite abnormal These mutants also have a grossly altered morphology There are no data to indicate whether altered phospholipid synthesis is the cause or the result of the other abnormahtles vI CONCLUSION At the time of writing of this review the structures of the major phosphohplds of E colt are established, and the mechanisms of biosynthesis are understood to some degree Detailed analysis of the enzymatic mechanisms involved in phosphohpld metabohsm has not yet been possible because most of the enzymes are localized in the cell envelope Only recently has one of these enzymes, phosphatldylserlne decarboxylase 94, been solubJhzed and obtained as a homogeneous protein, therefore, more sophisticated studies will undoubtedly follow Although the hope exists that general procedures can be developed for the isolation of membrane proteins, at th~s t~me most membrane proteins cannot be obtained pure in an active form, and therefore new methods must be sought From the wealth of information that has been derived from the few membrane enzymes that have yielded to purification procedures, such as the galactosyl transferase involved in hpopolysaccharide synthesis 125, and Enzyme II of the phosphotransferase system I28, it is apparent that a serious effort in this direction is warranted In addition to the enzymes with obvious functions in phosphohpld synthesis, there exist also the dlglyceride kinase 9-"and the lysophosphatldate phosphatase 72, enzymes whose function in the metabolism of phosphohpids are not understood Other enzymes with functions which must yet be delineated are the phosphohpases which have been identified in this organism The recent purification of one of these, phosphohpase A1, to near-homogeneity 59 should allow more fruitful investigations into the function of this class of enzymes Btochtm Btoph)s Acta, 265 (1972) 25-60

MEMBRANE PHOSPHOL1PIDS OF E colt

57

Emphasis has been placed in this review on the use of lipid mutants in studies of the metabolism and function of phosphohplds Isolation of the unsaturated fatty acid auxotrophs has helped to elucidate the reactions at the branch-point between saturated and unsaturated fatty acid biosynthesis The critical function of the unsaturated fatty acids of phosphohplds was illustrated by the isolation of auxotrophs More important, the avadabihty of these auxotrophs has made possible the manipulation of the fatty acid composition of the phosphohpJds Thus, the effects of blocking phosphohpld synthesis either by deprivation of the required unsaturated fatty acid in the unsaturated fatty acid auxotroph or by deprivation of glycerol in the glycerol auxotroph have been studied in order to determine whether simultaneous phosphohpld synthesis IS required during the synthesis of the lactose transport system Although conflicting experimental results have been reported 47 139,142 It IS apparent that this experimental system should yield a decisive answer to this question In addition, the ability to vary the unsaturated fatty acld component of the membrane phosphohpids has permitted studies which have indicated that a number of physiological membrane functions, including transport, reflect the physical properties of the phosphohplds In order to study the biogenesis of the transport system, Wilson and Fox 14°,~41 extended the observation of Schalrer and Overath 34 indicating that the temperature profile of transport activity IS specified by the fatty acid composition of the phosphohplds These workers found that properties of both the fl-galactoslde and the fl-glucoslde transport systems are characteristic of the phosphohpld being synthesized at the time of transport system induction, rather than the predominant phosphohpid of the membrane, and they concluded that newly synthesized proteins associate preferentially with newly synthesized hpids However, Sm~llar experiments by Overath, Hill and Lamnek ~42 have led these investigators to conclude that an induced transport system assocmtes with the average hplds present at the time of induction rather than the hplds specifically synthesized at the time of induction Additional experiments will certainly clarify th~s issue Although much has been learned concerning the function of unsaturated fatty acids in biological membranes by use of the unsaturated fatty acid auxotrophs of E cob, attempts to isolate auxotrophs reqmring saturated fatty acids have not yet succeeded, and therefore the function of saturated fatty acids has not been similarly investigated It is apparent that the functions of the different phosphohpid classes are largely unknown, and mutants that are blocked In the synthesis of a specific phosphohpld class would be enormously useful in this regard At this time the only phosphohpld mutants available are blocked early in the pathway at the level of phosphat~dic acid synthesls s°'sl'ss Valuable information concerning the mechamsm by which the saturated and unsaturated fatty acids are specifically positioned in the phosphohplds has been derived from these mutants s4 Moreover, it is hoped that modifications of the procedure used to select these early mutants will allow the selection of mutants defectwe in the synthesis of specific phosphohpld classes Such mutants should permit the deduction of the specific roles of various phosphohplds m membrane structure and function Btochtm Blophys Acta, 265 (1972) 25-60

58

J E CRONAN, P R VAGELOS

ACKNOWLEDGEMENTS We are indebted to Dr E C

C

Lm, A

C F

Kornberg, and P

F o x f o r a l l o w i n g us to use F i g s 7 a n d 8, t o D r s Overath for preprints, and to our colleagues for

permission to quote their unpubhshed

results

The unpubhshed

experimental work

from our laboratories and the preparation of this article have been assisted by grants f r o m t h e N a t i o n a l I n s t i t u t e s o f H e a l t h ( I R O 1 - A I - 1 0 1 8 6 to J E C , 5 - S O 5 - R R - 0 7 0 1 5 to Yale University, ROI-HEI0406

to P R V ) and the National Science Foundation

(GB-5142X to P R V )

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