Studies on the Membrane Lipids of Bacillus Amyloliquefaciens And

ro.la ¡-

STUDIES ON TI]E MEMBRANE LIPIDS OF

BAC.ILLUS 4YELL]9gEFACIENS AND TIIEIR RELATION TO Ð"TRACELLULAR PROTEIN SECRETION

A Thesis Subrnitted for the Degree of Doctor of Philosophy in the University of Adel-eide by James Cleland Paton, B. Sc. Hons, (A<1e1aide)

DEPARTMENT OF BIOCFïEþIISTR)i FEBRUARY, 7979

r4.2,./t , .lz J /) fa,'o'"^¡ 'n /î ? ? TABLE OF CONTENTS

, !l r !ì 1- -l Page

SUMMARY 1_

STATEMENT r_v

ABBREVIATIONS V

AC KNOI{TL ED GEX4ENT S V1

CHAPTER ONE

INTRODUCTION

A GENERAL INTRODUCTION 1

B PROTEIN SECRETION IN EUKARYOTES J

C EXTRACELLULAR ENZYME SECRETION BY BACTERIA 10

D AIMS OF THE I\IORK IN THIS THESIS 15

CFIAPTER TWO

MATERIALS AND METHODS

A MATERIALS 1.7

B METI.IODS 22

CFTAPTER THREE

STUDIES ON THE Mi]I,IBRANE PHOSPHOLIPIÐS OF

B . AMYI,OL I FAC IENS A. INTRODUCTION 35

B. RESULTS

1. Phospliolipid composition of ce11s and pro topl as t s 36 ') T'he use of phospholipases as a probe for phospholipid distribution 38 Pa_g€.

3, Treatment of intact protoplasts u¡ith phospholipases 3B 4. The effect of pìrospholipases on the integrity of protoplasts 40 5. Digestion of protoplasts with sepharose phospholipase C 42

6 . l['ree,tlnent of permeab iTized protoplast s with phospholipases 42 7 , Digestion of membrane vesicles with pho spho 1 ipas e s 43 8 . Attenptecl exper iments with phospholipid exchange proteins 46

9. Chemical modification of phospholipids 47

10. Extent of penetration of TNBS into ce11s 48

11. lr{odif ícation of PË by TNBS at 4oC 50

1,2, Phospholipase i digestion of intact ce11s 51

C DI SCUSSION 52

CI{APTER FOUR

STUDIES ON TFIE FATTY ACID COMPOSITION OF TFIE

MDMBRANE OF B. A}IYLOL I UEFACIENS AND TTS

RELATION TO THE OCCURRINCE OF COLD SHOCK

A INTRODUCT ION 64 ts RESULTS

1 F a t tv acid composition of ce1ls of B. 1 iquefac iens 66 ? Fatty acid analysis of cel1s grown at different tenperatur CS 66

3 Cold shock of cel1s grown at 30oC 68

4 The effect o fg rowth temperature on the critical telnper ature zone for cold shoclc 70

5 The effect of Trveen-80 on the critical ternperature zorLe f or cold shock 71 Page

C. DISCUSSION 72

CHAPTER F IVE

STUDIES ON THE RELATIONSHIP BETIVEEN LIPID

SYNTFIESIS AND PROTEIN SECRETION

A. INTRODUCTION 78

B. RESULTS

1 Inhibition of oc-amylase and protease secretion by cerulenin 79

2 Inhibition of total protein secretion by cerulenin 79

J Effect ,.lf cerulenin on lipid synthesi-s 79

4 Effect of cerulenin on general protein synthe s i s 80

5 Effect of cerulenin on RNA synthesis 80

6 Recoverl.' of x-amylase secretion after removal of cerulenin 80

7 Reversal of cerulenin i.nhibition of o<- anylase ancl protease secretion with fatty acids 81_

8 Attenpt to detect forms of o<-a:itylase and protease with attached phospholipid 83

9 Test to see whether cerulenin causes intracei-lu1a,r a.ccurnulation of enzymes nornalll' secre ted 84 C. DISCUSSION 85

CHAPTER STX

ATTEMPTS TO DETECT PRECURSORS TO O{-AÞ{YLASE ANl)

PROTEASE

A INTRODUCTION E2

B RESULTS Page

1 Does a precursor to ó(-amylase exist in the external medium of B. amYlo- efac iens 93

') Cell a ssoci-ated protease activitY in B. amy 1ol- iquef aciens 94 C. DISCUSSION 96

CHAPTER SEVEN

FINAL DISCUSSION AND SUMMARY 99

CFTAPTER EIGHT

REFERENCES 101

APPENDICES

APPENDIX A - PUBLICATIONS 11_ 1 1

SUT{¡{ARY

1. The major phospholipids extracted from Bacillus anyloli qile - faciens \vere cardiol ipin, phosphaticylglycerol and phosphatidylethanolamine.

2. The distribution of these phospirolipids between the trvo halves of the cytoplasmic membrane bi.layer ì/\ias studied using phospholipase C (8. cereus , phospholipase A, (Cr o talus I ai-.,d the non-penetrating chemical probe trinitlobenzenesulphonj c aci<1 (TNBS). Af ter treatment of intact protoplasts of B. g4ytqlijge-

&Sl""t rvith either phospholi.pase, approximately 7 0% of total mernblane phospholipid was hydrolysed; specifically approxiniately 90%, 90% ancl 30% of phosphatidylethanolamine, phosphatidylglycerol and carcLiolipin respectively. Under these conditions, protoplasts re¡nained intact and sealed. However, rvhen protoplasts that were perneabilized by cold shock treatment were incubated with either of the phospholipases, up to 80% of cardiolipin was hydrolysed and phosphatidylglycerol and phosphatidylethanolamine were hyciro- lysed virtually to completion. rn intact cells, gz% of the phosphatidylethanolamine could be 1abe1led rvith TNBS under conditions in which the reagent did not penetrate the membrane to aîy significant extent..

3. These results suggest that 70% of total phospholipid of this bacillus exists in the outer half of the bilayer. The

4. The fatty acid composition of cells grown at different temperatures r,r¡as investigated. I{hen cells hlere grown at 3goc, branched-chain saturated fatty acids made up over 80% of the total fatty acids. Saturated straight-chain f.atty acids made up the bulk of the rernainder. Less than 7% of the total fal-ty acids were unsaturated. Decrease in grovrth temperature 'uJas accornpanied by an increase in the ratio of branched to straight- chain f.atty acids and a marked increase in the level of unsaturation of branched-chain fatty acids.

5. When ce1ls of this organism, grown at 30oC, were cold shocked, viability and ability to secrete extracellular protease were lost. Growth of this organism at lower ternperatures or addition of Tween-80 to cel1s caused the critical temperature zorLe for cold shocking to be significantly lowered. These results suggest a direct correlation between membrane fluidity and the susceptibility to cold shock.

6. The role of lipids in the process of extracellular enzyne secretion u¡as studied using cerul-enin, an antibiotic knov¡n to inhibit f.atty acid synthesis in nicroorganisrns. Ceruleltin inhibited the secretion of oc-amylase and protease in rvashed ce11 suspensions by 80% and 75% respectively over 3 hours. The effect rvas a general one since secretion of all protein species into the nedium was drastically reduced by the antibiotic. At the concentration of cerulenin used (7oo//s/nl) , Ito.] - acetate incorporation into ce1lu1ar lipid rvas inhibited by approxinately 50% but total cel1u1ar protei-n and RNA synthesis were virtually unaffected. The inhibitory effect of cerulenin on oc-amylase and protease secretion could be partially reversed if cel1 suspensions r,vere supplemented rvith either fatty ac.ids prepared frorn 111 the li.pids extracted from B. arnylol iquef ac iens or var ].ous individual pure fattY acids. These results suggest that f.attY acid synthesis may be required for protein secretion by this organisn.

7 . Atternpts were rnade to detect precursors to extracellular enzymes either associated with the ce11s or in the culture rnedium, êmploying immunological techniques. These experinents, however, were not successful. lV.

STATEMENT

This thesis contains no naterial which has been accepted for the award of any other degree or diplorna in any university.

The rvork was done entirely by nyself, except that of Table 4-7 which was carried out in collaboration with Mr. E.J. McMurchie.

To the best of my knorvledge and belief, this thesis contains no rnateriel previously published or written, except where due reference is rnade in the text.

Signed:

JAMES CLELAND PATON. \r

ABBREVIAT TONS

The abbreviations used in this thesis are acceptable to the Journal of Molecular Biology, or a-Te def ined in the text. vl.

ACKNOWLEDGEMENTS

I ,wish to sincerely thank Professor W.H. Elliott and Dr. B.K. May for supervision, encouragement and rewarding discussions throughout the course of this work.

I also wi'sh to thank the other members of our research group for their advice and cooperation.

I am particularly indebted to Mrs.L. Mercer, who performed the electron microscopy, and Miss J. Bielicki for assistance in conjunction with the column chromatography work.

I also wish to thank Miss A. Heylen, Miss J. Rosie and Miss J. Thornpson for competent technical assistance.

The financial support of a Comrnonwealth Post-graduate Research Award is also acknowledged. CTIAPTER ONE

INTRODUCTION 1

A GENERAL INTRODUCTION

The work c¿rried out in this research group is ained at gaining a greater knowledge of r,he mechanism of secretion of extracellular enzymes through membranes. The secretion of oc-amylase and protease by Bac i'l1us amylo l iquef ac iens r,{as chosen as the nodel system to be studied, as this gram positive organisn secretes large amounts of these and other enzymes into the external nediun of both grorving and non- grorving cell cultures. The work described in this thesis is prinarily concerned with the cytoplasmic membrane lipi.ds of this organism, since as will be discussed later, it hras clear that a cornplete understanding of protein secretion required a thorough and detailed study of the lipids and their role.

Bacterial extracellular proteins have been defined by Pollock (L962) as proteins which: . ". . . . exist in the nedium around the ce11, having originated from the ce11 v¡ithout any alteration to the cell structure greater than the maximum compatible with the ce1lfs normal processes of growth and reproduction. " These proteins are predominantly degradative enz)¡nes and their principal role appears to be the breakciown of large molecules in the environment into smaller compounds that can be readily taken up and utilized by the organism (Mandelstarn, 1969). The notable exception to this is penicí11inase whose function is presumably protective. The problem of how bacterial ce1ls synthesize often vast amounts of these enzymes (nany of which would be potentiaLly 1etha1 if active inside the cells) and transport them fron the cytoplasm to the external mediun has been the subject of intensive study in recent years (Glenn,

7s7 6) . )

Although the bacterial ce1l wall has been shown to limit both the release of secreted proteins into the external mediuin and the accerr'itility of the cell membrane to extracellular enzymes (Gou1d et 4L.r 1975), it is generally agreed that the cell membrane itself is the najor permeability barrier that separates secreted proteins from the cytoplasn (G1enn, T976). Consequently, a study of the ce11 membrane pel se r{as important, and since recent reports (which will be discussed later) implicate the direct involvement of lipid in the proc.ess of protein secretion, information on the lipids of the nembralte was considered to be of particular relevance. Accordingly, the work described in this thesis is centred upoil the lipids of the nembrane of B. amyloliquefaciens and their role in the process of enzyme secretion by this organism. Literature relevant to these aspects is discussed more fully in the appropriate chapters.

The large field of bacterial enzyme secretion has been comprehcnsively covered in two recent reviev¡s (G1enn, I976; Priest, 1,977) and consequently, this introduction will be Iargely confined to more recent publ ications and- points of particular importance that deserve emphasis. Protein secretion in eukaryotic ce11s, rqhich raises similar problems ío the process in microorganisms, rvi1l be discussed f irst, f o11ov¡ed by a revierv of extracellular e\zpe secretion in bactei'ia. The specific aims of the rvork presented in this thesj.s will then be stated. 3

B PROTEIN S]]CRETION IN EUKAP.YOTES

certain eu.kp.r.yotic cells, such as those of the liver ancl pancreas of vertebrates, are activc in protein secretion. In these ce11s, extracellular proteins are secreted across the membrane of the rough endoplasnic reticulun into the cisternal space from whence they aïe transported, via the smooth endoplasmic reticulum, to the golgi complex. Here the proteins ar:e

further processed and packaged into secretory vesicles luhi c.h subsequently fuse with the plasma membrane, releasing their contents outside the cel1 by reverse pinocytosis (Andrern-s and Tata, 1,97I; Castle et ã7., I972; Caro and Palade, 1964; Jamieson and Palacle, L967 a,b) Peters et aL, f97Ð. The process of secretion of these proteins across the endoplasmic reticulum membrane into the cisternal space (thereby isolating the secreted protein from the cytoplasm) rnay be analogous to the process of secretion of bacterial extracellular enzyrnes through the cytoplasnic membrane into the surrounding nedi.um.

There is a large anount of evidence that suggests that in eukaryotic ce11s, exportable proteins are synthesized by ribosomes bound to the menbrane of the endoplasmic reticulum, whereas proteins for the cytosol are synthesized by free ribosomes (Borgese et ãI. , 797 3; Ganoza and Wi1lia,ns, 1969; Hicks et al., 1969; Me1chers,1971"; Redrnan, 19ó9; Takagi et âI., L970; Rolleston, L974). Redman and sabatini (1966) were the first to propose that the synthesis of secretory proteins on membrane- bouncl ribosomes and their secretion across the membrane occurr- jmultaneousl ed. s y. These workers studied _in vitro synthesis of secretory proteins by guinea pig liver rough microsones (seal-ed nrembrane vesicles rvith ri bosomes bound to the outsicle 4 that Ìfere derived from the rough endcplasmic reti-cu1um). The lumen of the microsome corresponds with the cisternal space of intact endoplàsmic reticulum. They showed that completed proteins, or nascent polypeptide cl:ains released from the ribosomes by treatnent r,vith puromycin, remained associated with tlre nicrosomes, and cou1cl only be released af ter solub ilization of the nenbrane with detergent. Although the possibility existed that the released polypeptides \^/ere non-spec.ifically adsorbed to the outside of the nicrosomes, as was subsequently pointed out by Sauer and Burrow (L972) and Burke and Redman (1,973), they proposed, on the basis of these and other studi-es (Reclnan, L967; Sabatini and Blobel , 1970), the nodel that proteins secreted by the pancreas were \rectorially extruded across the membrane during their synthesis on ribosomes bound to the rough endoplasmic reticulum.

The relationship between membrane-bound and free ribosontes has been clarified in recent years by Blobel and his colleagues (B1obe1 and Dobberstein, 1975 a,b) who isolated mRNA specific for imrnunoglobulin light chains. I¡lhen this hlas translated in vitro in heterologous cell free systems in the presence of ribosome-free nicrosomes, secretion of the proteins into these vesicles was achieved. When globin nRNA was used, globin chains were synthesized, but these were not secreted into the nicrosomes. These resul Es indicated that mernbrane-bound ribosones were derived from the pool of free ribo-somes of the in vitro translation system, and the clestination of completed proteins was deternined by the nRNA. Milstein et a1. (1972) observerl that inmunoglobulin light chain was synthesized on detatched polysomes as a precursor prot.ein rvith an extr:a amj.no terminai 5

sequence and proposed that this extra sequence was responsible for binding of the polysomes to the nenbrane. ; i'; i,i:- B1obe1 and sabatini (79TL) independently proposed. a similar model which has been expanded in recent years to form the "signal hypothesis" (B1obe1 and Dobberstein, rgzs a,bi Devillers-Thiery et al.,1975). They proposed that during synthesis, an amino ter:minal "signal sequencet' of the grolving secretory polypeptide chain emerges from the large subunit of a free ribosone and directs the ribosome to a specific protein receptor site on the endoplasnic reticulum membrane, which billds the signal sequence and perniits secretion. The signal sequence is subsequently removed by a mernbrane-bound protease prior to conpletion of the polypeptide chain. Nascent proteins which are not destined for secretion, such as globin, do not contain

such signal sequences. when the ribosome attaches to the mem-

brane a junction capable of permitting secïetion is formed. To account for the passage of hydrophilic seguences across the bilayer it was further proposed that this junction consists of a protein lined tunnel spanning the bilayer (formed by aggregation of specific membrane proteins) through which the growing polypeptide chain is extruded, amino terrninus first, into the intra-cisternal space. After chain lermination and release of the riboscme from the membrane, the secretory protein is completely segregated rvithiu the lumen of i.he endoplasmic retj.cu- lum, while dissociation and diffusion of the nembïane receptor proteins elininates the membrane tunnel.

,4. large body of evidence exists in support of the s ignal hypothesis. A large number of secretory proteins have nolv been ó

translated in in vitro systems lacking nembranes to form higher molecular weight precursors lvith extra amino terminal anino ; ì t acid sequences (B1obe1'' and Dobberstein, L975 a,b, Dobberstein and Blobel, 7977; Shields and BlobeI 7977, L97B; Lingappa et aI.r7977). In each case, the presence of microsomal menbranes resulted in the correct co-translc-tional processing of these precursors and concurrent segregation into the nicrosomal lunen. However, if intact microsomes hrere added to the in vitro system after the conpletion of the polypeptide chains, the precuïsoïs were neither processed nor segregated (Lingappa et ãI., I977).

The length of the extra amino terminal sequences of the precursors to various secretory proteins, which are all enriched in hydlophobic amino acids, varies from about 77 to 30 residues (Habener et ãL., 1978). The amino acid sequence of these extra peptides has been shorvn to be partially conserved for several pancreatic secretory protein precursors (Devillers- Thiery et al., 1975). In addition, there appears to be 1itt1e species specificity, as many of the reports in support of the signal hypothesis employed heterologous reconstituted systens. For example, B1obel and Dobberstein (1975b) sho'.ied that ribo- somal subunits from rabbit reticulocytes could function with dog pancreatic microsomes stripped of ribosomes, in a protein synthesis systern derived from Krebs ascites cel1s to translate nRNA for inrnunoglobulin light chains isolated from murine myeloma ce11s. The translation product was proteolyticaLly processed to the same si-ze as the authentic protein and was segregated into the lumen of the microsome (as determined by insensitivity to proteases). 7

Blobel (1"976) suggested that the signal hypothesis night also be applicable to the incorporation of integral membrane proteins into thë.1ipid bilayer. J. Rothnan and Lodish (LgT7) studied the in vitro synthesis of the riembrane glycoprotein of vesicular stomatitis virus. rn virio_, this protein is synthesized on the rough endoplasmic reticulum of infected cells and the amino terninal portion, which is secreted through the membrane and exposed on the cisternal side, is subsequently glycosylated. The carboxyl terminal portion however, remains exposed o1ì the cytoplasnic side of the endoplasnic reticulun membrane. when the glycoprotein was synthesized in vitro by wheat germ ribosomes in the presence of pancreatic rough microsomes, the resulting polypeptide was incorporated into the membrane, but this did not occur rvhen the glycoprotein rvas added to microsomes after synthesis. Using techniques to synchronise the growth of nascent polypeptide chains, it was shorvn that no more than one quarter of the glycoprotein chain could be synthesized in the absence of microsomes and stil1 cros5 the lipid bilayer rvhen chains were subsequently completed in the presence of microsomes. These findings indicate that the amino terminal residues are important in the insertion of the protein into the menbralte, and that this process is tightly coupled to protein synthesis. This is perhaps the best evidence so rar presented for vectorial translational extrusion of nascent peptides.

Not all pubrlished evidence, Ìrowever, is consistent with arl aspects of t-he signal hypothesis. The egg white proteins lysozyme, ovomucoid and. conalbumin, which are secreted by glands in the oviduct have been synthesizecl i.n vitro as precur- (to sors with extra amino terrninal signal sequences of !8^23 atnino acids. However similar studies with ovalbunin, which is secreted by the same ce11s, has shol'¿n that it does not posess a signal peptide, yet it is secreted norrnally in vivo (Pa1ni-ter et ãI. , 1978) . In addition the f irst 20 N- terminal amino acids l^Iere less hydrophobic than those of transient signal peptides.

Highfield and E11is (1978) have reported that the snal1 subunit of ribulose biphosphate carboxylase was synthesized in vitro as a high molecular weight precursor with an extra amino terminal sequence enriched in acidic rather than hydrophobic amino acids. This precursor r\ras tahen up by isolated chloroplasts and cleaved to its final size (presumably by a membrane-bound enzyme) wi-'thout concomitant protein synthesis. These workers proposed that a specific carrier protein in the chloroplast membrane was responsible for the observed uptake of ribulose biphosphate carboxylase precursor. Ohashi and Sinohara (1978) have recently reported tentativc evidence that cornpleted rat liver 5-aminolevulinate synthetase can traverse the nembrane of mitochondria. It has been suggested by B1obe1 (1-976) that a model other than the signal hlpotnesis rnay have to be proposed to explain the transfer of cytoplasmically synthesized proteins across two membranes into the innermost compartment of rnitochondria and chloroplasts.

S.S. Rothman and coworker-c haye proposed that intact digestive enzymes secreted by the pancreas are specifically reabsorbed by the snall intestine and returned via the blood- stream to the pancreas, from where they are resecreterl into the duodenuln (Rothman, 7975) , An analogous enterohepatic circula- 9

tion is knclrvn to exist for the bile salts secreted by the 1iver. The eyídence in support of this enteropancreatic circulation of digestive ettzymes (which has been summarized by Diamond (1978) ) is as fo11ot4/s: - Bidirectional f luxes of digestive enzymes including chymotrypsinogen and amylase across the zynogen granule membrane, the basolateral membrane and the apical rnembrane were reported for rabbit pancreatic ce1ls (i,eibow and Rothnan I972, L974; fsenman and Rothnan L977). These claims are in direct conflict with the vieiv of Palade (1-975) that the transport of secretoty proteins is vectorial, with only the apical membrane of pancreatic ce11s being permeable, and then only in the cell-to-duct direction. Palade (1,976) has critic;sed Rothnants rvork on the basis of his cell fractionation techniques. However this criticism cannot be applied to experiments in which the circulation of digestive enzymes was studied in perfused intact rabbit pancrees and whole rabbits. These experiments showed that [3H].ny*o- trypsinogen added to the external bathing nediurn of whole rabbit pancrgâs¡ in vitro, is taken up by the ce11s at a much greater rate than albunin and subsequentiy appears in the ductal secîetion (Leibow and Rothnan, 19 75). nf r" [3f!- chymotrypsinogen instilled into the intestine or injected in- to the bloodstrean of whole rabbits rvas subsequently recovered in pancreatic secretions (Leibow and Rothman, I975; Götze and Rothnan, L975). The efficiency of recirculation of total pancreatic enzymes was estimated by Götze and Rothnan (1975) to be 80-90%. The manner in which the tissues are protected from attack by such la-rge amounts of digestive enzyr.es in both the bloodstreant and rvj-thin ce1ls is not explained. 10.

rn our laboratory, it has been found that active d-_ amylase and ribonuclease are released from intact, right side out, tat pancreatic nticrosomes during incubation (pearce et al., 1978). This release was not due to leakage through damaged membranes or to release of protein adsorbecl to the exterior of the rnicrosomes, The release was bloclced. by physiological concentrations of Mg2+ or in the presence of proteases. In the latter case, the ,r-amylase and ribonuclease vrere not destroyed and could subsequently be released from the microsomes by Cetergent treatment.

It therefore seems possible that the signal hypothesi.s may not be the only ¡nechanism by which proteins are transport_ ed across mernbranes.

c EXTRACELLULAR ENZYME SECRETTON BY BACTERIA

The translational extrusion model for enzyme secretion in bacteria was proposed by l{ay and. Elliott (1g6g). Accord._ ing to this model (which is similar to that jnitiarry propos_ ed by Rednan and sabatini (196ó) for protein secretion in animal ce11s), extracellular proteins are synthesized on ribosomes associated rvith the ce11 membrane and the nascent chains are dircctly extruded through the bilayer, and take up their f inal three dimensional conl:ormation ou.tside the ce11. The basis of this model was their observation that in B. am),lc- liquefaciens, the extracellular enzynes protease, o._arnylase and ribonuclease h/ere not found in detectable alnounts in the cytoplasn and l-he appearance of these enzymes in the external medium was tight.ly coupled to protein synthesis. The exist- ance of an ilrtracellular ribonuclease inhibitor rt¡hich irrever- 11. sibly binds the extracellular ribonuclease (Srneaton and El1iott, 1967 ) nakes it unl ikely that this enzyme ever exists in colnplet - ed form in the cytoplasm. In further support of this model, Sanders and ltlay (197 5) reported that êt-amyl.ase and protease energec1 from protoplasts in a conformation different to that of the active enzymes, as determined by sensitivity to degradation by trypsin. Smith et al. (L977 ) treated E. coli sphero- plasts rvith acetyl [tttJ - methionl,l rnethyl phosphate sulphone, a reagent rvhich reacts with amino groups, but does not cross the inembrane. About 6e; of the label in the menbrane-polysone fraction was attached to the polysomes via peptidyl-IRNA. A signif icant proportion of thi s v/as identif ied irnmunologically as alkaline phosphatase (a periplasmic protei-n in this or.gan- isn) after itt ti_ltg chain conpletion. This shows that nascent alkaline phosphatase chains can be labe11ed from outside the cell rvhile stil1 attached to the ribosome, âS predicted by the translational extrusion model.

A requirement of the translational extrusion model j s that proteins destined for secretion are translated on ribosomes associated with -,he menbrane. The existence of membrane- bound ribosotnes in bacteria was initially tìte sub j ect of nuch argument (see discussion in Glenn, (1976) ). I4any early reports riray have been complicated by entrapment of ribosones r,vithin membrane vesicles, and artifactual binding mediated by lysozyme which was commonly usecl in the isolation of menrbranes (Patterson et aI., 1970). Nevertheless, a functional differ- ence betlveen menrbrane-bound and free polysomes has been dentonstratecl for E. coli by Randall and Llardy (7977) . In y.!lrp. protein synthesis studies showed that the menbrane-bound I¿.

polysomes (which weïe pr.epared without the use of lysozyne) synthesized outer membïane proteins and a secreted periplasmic protein- the maÍtose bincl ing protein. The ma j or protein syn- thesizedbyfreepoll'soneswast]reelongationfactorTu,a soluble cytoplasnic protein. Also, Cancedda. and Schlesinger (7g74) showed that polysomes containing nascent alkaline phosphatase chains (detected immunologically) were associated with the cell menbrane of E. coli. It therefore seems probable that, like eukaryotes, bacterial exportable proteins are Syn- thes ]-zed on membrane-bound ribosomes.

This raises the problen of how the nRNAs for extracellular enzymes (which have been shown to be unstable for B. .eryfg-- liquefaciens (May and Elliott, 1968; Both et â1.,1972) ) migrate from the chromosome to the translation sites on the membrane. on the basis of studies of the effec.ts of various antibiotics on protease secretion by B. amyl o 1 iquefaciens, it has been proposed (Both et al.,!97?; OrConnor et al., 1978)

that a 1 arge excess of nRNA for extracellular enzymes is transcribed such that sufficient intact nRNA eventually reaches the mernbrane to saturate the translation sites- even after many half Life decay periods. Confirmation of this hypothesis, however, will depend on the isolation and direct measure,nent of tire nRNA species.

The proposed spatial separation of transcription and tra.nslation of extracellular protein nRNA, if it occurs, would suggest that there is a means of recognizing these species such that they are not translated on free cytoplasmic libosomes, at least tc¡ a fu11 extent. It has been suggested by Lanpen L3.

(1974) that extracellular protein nRNA nolecules have a commcn St sequence that prevents such premature initiation and only a11ows recognitiott by membrane bound ribosomes. Evid,ence in support of this suggestion is discussed by Glenn (1976).

Recent work, involving the in vitro translation of bacterial extracellular protein nRNA has shown that these proteins nay be initially synthesized lvith amino terminal "signal sequences", sinjlar to those reported for pancreatic secretory protejns by Devillers-Thiery et aI. (1-975), and these rnay be important in interaction with the membrane. Arnbler and Scott (1978) showecl that the penicillinase coded for by _E_._ eo_lr plasrnid R6K was synthesized in vit__ro as a precursor with aî extra amirro terninal sequence of 23 predorninantly hydrophobic amino acids. In extended form, a sequence of this length lvould be capable of spanning the bilayer. The outer membrane lipoprotein of E. coli was shown by S Inouye et al, (1977) to be synthesized in vitro as a higher molecular lveight precursor with an addition- a1 aminc terninal amino acid sequence. This extra sequence contained 20 amino acids, 60% of lvhich hlere hydrophobic, rvhercas in the compl-eted protein, only 38% of the amino acid residues are hydrophobic.

Not all reported bacterial signal sequenrles r however ,a.Te enriched for hydrophobic amino acids. The extracellular penicillinase of !_ licheniformis is synthes ized in vitro as a precursor rvith an extra anino tèrminal amino acid sequence. This extra sequence contained hydrophilic residues, but a hydrophobic phosphatidic acid molecule I^/-as attached to the

amino terninal serine resiclue (Dancer and Lampen, 1975) " 1.4 .

This form of the enzyme remains Lround. to the menbrane and the extracellular form is released from it þy proteolytic c1 eavage 25 or 27 residues fron the amino terminus (Yamanoto and Lampen, I976). H,.Inouye and Beckwith (I977 ) have also reported that E. coli alkaline plr.osphatase is also synthes ized as a precursor rvhich is larger and more hydrophobic than the completed protein. The nature of this extra segnrent (which was shown to be removed by an enzyme associated with the outer nernbrane) was not further investigated, and it is not known whether the hydrophobicity was a resûl.t of its amlno acid composition or possible associatecl lipid. Nevertheless, hydrophobicity appears to be a conìmon feature of signal sequences in bacteria. From the amino acid composition of the signal sequence of B. licheniformis penicillinase, Yanamoto and Lampen (1,976) deduced that the segment of nRNA coding for this sequence rvould have a purine content of approximately 80%. This may be structura1-1-y similar to the poly-A segment found at the 3t end of eukaryotic nRNAs which has been reported to have an affinity for the membrane (Milcarek and Penman, 7974; Lande et al-. , L975). It is possible, therefore, that both the 5r end of the nRNA and the nascent polypeptidt> chain may fascilitate interation with the membrane in bacteria.

The finding of Dancer and Lampen (1975) that the B. licheniformis penicillinase rrlas synthesized with a phospholipid covalently attached to the amino terminus, wãs the first report of direct involvement of lipid in the secretion of extracellular enzymes. Phospholipids have also been implicated in association rvíth extracellular 1ipase of Acínetobacter 016 (Breuil and Kushner, 1975). In adclition Glenn and Gould (1973) 15.

have suggested that Pho spholipd may be cosecreted lvith extracellular enzymes in B, arnyloliquef ac.iens. It is theref ore possible that'Ii'pids are directlf involvecl in the initial interaction of polypeptide chains witli the nelnblrane, or their transport across the bilaYer.

Despite the advances in this field achi eved in recent years, no clear expla-nation of the rnechanism of bacterial (or eukaryotic) protein secretion presents itsel f, and indeed. more than one mechanistn nay be operating. I t hras hoped that a study of the lipids of the rnembrane of B. arnyloliquefaciens (which seems warranted, from the above findings) would contribute to an increased understanding of these mechanisms

D A]MS OF THE WORK IN THIS THESIS

The initial ai-m of this work was to gather infornation on the membr:ane lipids of B. amyloliquefaciens. In Chapter 3, the phospholipid composition was determined. l{hen this work started, the distribution of phospholipids between the inner and outer halves of the membrane bilayer had not been investi- gatecl" in bacteria, and So this feature lvas exanined in B. anryi oliquefaciens. Chapter 4 describes a study of the latty acids of this organism. The aim of this study tvas to determine wþether the menbrane fatty acid compcsition (and helce fluidity) could be altered by changes in grolvth temperature. It tvas hoped that such alterations might overcome the effects of cold shoc-k, which occurs in this organism when ce1ls are rapì.d1y chi11ed. The occurrence of cold shock hacl harnpered previous attenpts in this laboraLoty to achieve it tit1t s1'nthes i-s ol. extracel l.u1ar enzyme.s and a means of ortercoming 16. the problem hras therefore of great inportance.

After the completion of these studies, experiments (Chapter 5) I,rere ained at deterrnining whether or not lipid rt¡as directly involved in the process of protein secretion in B. anyloliquefaciens. Subsequent experiments (Chapter 6) were ained at detecting precursors to the secreted enzymes, but these studies were not fruitful. CFTAPTER TIVO

MATERIALS AND METHODS 77.

A MATERIALS

1 Bacterial Strain

An unclassified strain of Bacillus am 1o1 i uefaciens I^Ias obtained from the Takamine Laboratories Inc., Clifton, N.J., U. S.A. Tiris organism was previously described as a strain of Bacillus subtilis, but has since been iclentif ied as B. amylo-

liquefaciens (Welker and Carnpbell, 1967) on the basis of DNA base conposition and hybridization studies. A mutant strain of this organism that did not secrete "surfactin" (May and Elliott, I97 0) was used in this worl<.

? Liquid Growth l4edium

(NH4) 1-mM The liquid grorvth mediurn contained 34n14 ZHPO4, MgSOO , 5rnM KCl, 4.2SnM sodiurn citrate, 0.125mM CaCL2, 0.0125ruV ZnSOO, 0.smM FeClr, 0.5% (w/v) Bacto Casarnino acids, 0.05% 0¡/v) Bacto yeast extract, trace metal solution (0.25m1/1) and I% (w/v) maltose. The medium was adju.sted to pH 7.3 rvith H3P04. The trace metal solution contained 0.5ng CoClZ.6HZ0, 0.5mg

ammonium molybdate, 5 . Omg MnCl Z.4HZ0 and 0 . 01ng CuSOO . sHZ0 , dissolved in 1 litre ot water. The medium was sterilised by autoclaving at I2OoC for 25 minutes. Maltose r^/as autoclaved separately and was ad.led just before use.

3 Washed Ce11 Suspension l{edium (WCSM)

This medium rvas the same as the liquid grolrth medium, except that FeCl, and Bacto yeast extract were onitted, there-

by liniting cel1 growth to an increase in dry weight of L5% in 3 hours. In some experiments (Chapter 4) , the concentration 18.

of Bacto Casamino acids was reduced to 0.025eo (w/v) and is referred to as WCSM 1ow aa.

4 Protop last Mediun

BmM (NH+) srnll This mediun cc¡ntained 2 5ml4 Tr is , 5 . ,UeOo , KCl, 0 .1,25ruV CaCl r, SrnM MgSOO, 0 . 2 5n1 per l itre of the trace rnetal solution described above , I% (w/v) rnaltose, 0,025% (rv/v) Bacto Casamino acids and 22% (w/v) sucr'ose. The nedium was adjusted to pH 7.3 with HCl.

5 Antibiotics

Chlorarnphenicol was obtained from Parke Davis and Co' , Sydney, Aust'raLia, as "chloromycetin" and wrs stored as a Zmg per ml aqueous solution Ir{akor Chernicals, Jerusalem, Israel, and was stored as a 2Oing per ml solution in ethanol at -20oC.

6 Radioisotopes oa] Ir Acetate (96.8nci/nrnole) , t- Lt*al - phenylalanine (46onrci/mrnole) and z- V4d uracil (55mCi/rnnole) were purchased from Schrvarz Mann, Orangeburg, New York. [ttr] - Methionine lzzf (77}Ci/mmo1e) was obtained from Amersham. L yJ - lnorganlc phosphate (carrier froe) was obtained from the Aust,:a1ian Atonic Energy Commission, Lucas Heights, N.S.W.

7 . Enzy.ne s

Lysozyme (three times crystallized from egg rvhite) and a -chymotrypsin lvere purchased frorn Sigma Chenical Co. Phospholipase C (8. cereus) type V was purchased as a suspen^ L9. sion in 3.2M(NH4) ZS0+ from Sigrna Chemical Co. Phospholipase Az Cro ta lus was purchased as a solution in 50% (w/v) glycerol from Boehringel: l4annhein Corp. TPCK (",-1-tosylamido- 2-phenyl- ethyl chloromethyl ketone) treated trypsin was obtained from Worthington Biochenical Corp.,Freehold, N.J. B. amylolique_- faciens extracellular protease anci o<-amylase r^/ere purified by the nethods of Both et al. (L972) and Grant (1967) respecrively.

I Enzyme Substrates

(a) Protease substrate: Remazol Brilliant Blue (RBB) was a gift from Farbwerke Hoechst AG, Frankfurt and Hicle Powder was purchased from Calbiochem, San Diego, Callfornia. The RBB- Ilide Powder complex rvas prepared according to the methocl of Rinderknecht et a1. (1968). trPhadebas" (b) "<-Arnylase substrate: tablets were purchased from Pharmacia (South Seas)Pty. Ltd., Lane Cove, N.S.W.

9 Thin Layer Chromatography Plates

Flastj.c-backed silica ge1 plates (KieselgeI 60 FZS4) I^/ef e purchased from E. Merck, Darmstadt.

10. Gas Liquid Chroma.tography Co lumns

Four separate stainless steel 6 foot x 7/8 " 0.D. columns containing 3% Apiezon L, 5% Apiezon L, 70% EGSS-X (ethylene succinate-methyl silicone polymer, 1ow silicone) or 25eo DEGS (diethylene g1ycol succinate), coated onto 100-120 mesh, acid washed, Siranized Chromosorb P were purchased prepacked from Applied Science Laboratories Inc., Pa., U.S.A. 20.

11. Lipid Standards

The following phospholipid standards vüere purchased from Sigrna Chenical Co. : - phosphatidylserine, Phosphatidylethancl- arnine, phosphatidyl gIycerol, cardiolipin, phosphatidylcholine, lysophosphatidylethanolamine. These had a stated purity of greater than 98%.

The saturated iso fatty acids of 14, L6,18 and 20 carbon atoms, the saturated anteiso fatty acids of 15, 77,19 and 27 carbon atoms, the saturated normal f.atty acids of !0-22 carbon atoms and the cis-9-nonounsaturated fatty acids of 1-6 and 18 carbon atoms were all purchased as the methyl esters from Applied Science Lahoratories Inc. Pa., U.S.A. either individu- aILy or as a prepared rnix. Standards had a stated purity of greater than 96%.

12. Scintillation Fluid

Scintillation fluid contained 3g of 2,5'diphenyl oxazole (PPO) and 0.3g of L,4-bis- 2^(4 nethylphenyl oxazolyl) benzene (POPOP) (which wcre both supplied by the Packard fnstrument Co.) per litre of toluene. t3. Detergents

Sodium dodecylsulphate (SDS), Polyoxyethylene sorbitan mono - oleate (l'ween- B 0) , sodium deoxycliolate and t - octyl phenoxy polyethoxyethanol (Triton X-405) were aII purchased from Sigma Chemical Co. 27.

1.4 . Immunoglobul ins

Rabbit anti protease and rabbit anti<^amylase gamma globulins were prepared as described by Sanders (1974) and characterized by Ouchterlony imnunodiffusion and titration exper inents .

15. Other Chenicals

Bacto-Agar, Bacto Casamino acids and Bacto ¡reast extract were purchased from Difco Laboratories, Detroit, Mich. Acrylanide (E. Merck, Darmstadt) and Bis-actylanide (Nt ,Nt- rnethylenebisacrylamide) (Sigma Chernical Co.) \tlere recrystall^ ized by the method of Loening (1967). Sephadex G-25 and G-

100 r^/ere purchased from Pharmacia (South Seas) Pty. Ltd. , Lane Cove, N.S.W. DEAE cel1ulose (DE-23) was purchased from Whatman. TLCK (1-chloro-3-tosylanide-7-amino-2-heptanone- HCl) was obtained from Cyclo Chemical Corp., Los Angeles, California. Glutaraldehyde, osmium tetroxide, TNBS (2- 4- 6- trinitrobenzenesulphonic acid), PMSF (Phenylrnet.hylsulphonyl f luoride) and EGTA (ethyleneglycol-bis - W- aminoethyl ether) NrN'-tetraacetic acid) were purchased front .3igma Chenjcal Co. TEMED (N,N,Nt,Nt - tetramethylethylenediamine) was purchased from Eastman KoCak Co. , Rochester, N.Y. Tris (hydr-oxymethyl) aminomethane was purchased as "Trizmatt base from Signa Chemi- ca1 Co.

1_6. So lyent s

Chloroform and methanol were supplied by J.T. Baker Chenical Co. , Philli.psburg, N.J the latter being redistilled over anhydrous CaCI, before use. Diethyl ether v/as redistill - 22. ed bef ore use. Petroleum ether (B.D.H. ) 40'60o fraction, Í/as first stirred overnight rvith concentrated HZS04, dried using NaOII pellets and redistilled over NaOH. Hexane, rvhich rvas purclìased frolrr Ajax Chemical Co., Sydney, wâs redistilled and stored in the presence of sodium wj-re. Carbon disulphide l{as obtained frorn May and Baker Ltd., England, and dried using anhydrous CaCIr, 74% (w/v) boron trifluoride in methanol was purchased from Appl-ied Science La-boratories Inc. Pa. U. S.A. With the exception of the latter , ãI1 solvents were deaerated with N, before use.

1"7 . Glassware

All glassware used for lipid analysis v,ras washed in ethanolic KOH and rinsed in solvent before use.

L8. Buffers and Solutions

All buffers and solutions were prepared in double- distilled water, except the liquid grortth medium which was prepared in mono-distilled water.

B METHODS

1_ Grolth of 0rganism

B. amyloliquefaciens was gro\^/n fron a spore inocultrm rn the liquid growth med.ium. Flasks were incubated at 30oC in an orbital shaking incubator (Paton, mode1- 461),osci11ating at 300rpm. The cells hlere harvested after l8h of growth when they had reached late logarithnic phase (absorbance at 60Onm=

3.6) . 23.

2 Pre aration of Ploto lasts

Cel1s urere harvested, rvashed orce in 50n1,{ Tris-FICI (pH 7 .3) and resuspended in their initial volume of protoplast mediun. The cells were then gently swirled at 50oC in an orbital shaking water bath (Paton, model OlV L4LZ) in the presence of L33tg of lyso zyme per ml. Protoplast formation was complete within 60 minutes, as judged by phase contrast microscopy.

3 Incubation with Phospholipase A ', and C

Protoplasts weTe resuspended in fresh protoplast medium such that a 1 nl aliquot contained approximately 700f9 of phospholipid. These aliquots were incubated at 37oC with either 4 I.U. of phospholipas. Az Crotalus or 30 I.U. of phospholipase C (Br_ cereus . Control protoplasts were incubated with the addition of a volume of the buffer in which the enzyme hras suspended equal to the volume of respective erlzyme added to the other aliquots. When phospholipas" ÃZ was used, the incubation nedium was supplemented rvith I0 ¡c1 of 0.1 M CaClr. At the end of the incubation period, the cnz)rrnes were inhibited by addition of 7C0y1 of 1M EDTA prior to lipid extrac.tion.

When intact ce1ls or membrane vesicles hlere incubated with phospholipases, they were suspended in 5OmM Tris-HC1, 0.2SnM CaCLr, 0.25mM MgCI., 0.1mlr{ ZnCLZ (pH 7.3') such that a 1nl aliquot contained approximateLy I00Fg of phospholipid. These aliquots were then incubated with 30 I.U. of phospholipase C or 4 I.U. of phospholipase A, as described above. 24.

4 As sa for Leaka e of Intracellular Ribonuclease Inhib i tor from Proto'pl.asts

Protoplastr'*"t" incubated with or without added phospholipases and then centrifuged (4,000 x E, 5 ninutes). A fixed amount of B. arnyloliquefaciens ribonuclease was added and the mixture IVas then assayed for ribonuclease activity. The arnount of inhibitor present was directly proportional to the number of units of ribonuclease inactivated. The total arnount of ribonuclease inhibitor inside the protoplasts \^/as determined by lysing then in an Aminco French pressure ce1l at 15,000 Ib/inL,') centrifuging at 4r000 x g for 5 ninutes and then assaying the supernatant fraction for ribonuclease inhibit.or as described above.

5 Electron I'licroscopy of Protoplasts

Protoplasts tvere treated with protoplast medium containing 4% glutaraldehyde for 60 minutes at room temperature. The protoplasts were then pelleted by centrifugation (4,000 x 8, 5 minutes) and washed in protoplast rnedium for 30 minutes. The pellets were then treated v¡ith I% osmium tetroxide in protoplast medium at 4oC o',¡ernight, progressively dehydrated in ethanol and final1y embedded in "Ara1dite". Thin sections were cut on an LKB nicrotome and examined using a Siemens Elrniskop 702 rnicroscope operated at 8Okv with a tOf objective aperture.

6 Li id Techni ues

[a) Ext.raction of l ipids: Two methods were enployed. In the work described in Chapters 3 and 5, lipids hrere extracted 25. by the rnethod of Kates (1972) (except that the final lipid extract rvas dehydrated with NarS0O (anhydrous) rather than with benzene, prior to being clried under a stream of nitrogen) . This procedure involved 2 extraction steps with chloroform/ methanoL (!:2 v/v), 99% of total phospholipid being extracted by the first. However, in order to obtain complete extraction of cardiolipin fron intact cel1s, it lvas necessary to first disrupt the cells in an Aminco French pressure ce11 operated at 15,0 00 Ib/ inZ .

In the work described in Chapter 4, harvested cel1s were resuspended in 50nM Tris-HCl (pH7.5) and l volume of cel1 suspension was extracted with 1 volume of chloroform and 2.17 volumes of nethanol by shaking at ZoC for 2 int. Undissolved rnaterial t^ras pel1et ed by, centrifugation and re-extracted trvo more times. Extracts were pooled and 0,2 volumes of L0% (rv/v) NaCl was added to form 2 phases. The chloroform phase was removed and the aqueous phase lvas re-extracted with an equal volune of chloroform. The chloroform extracts were pooled, dehydrated oVeI anhydrous sodium sulphate, concentrated in a rotary evaporator and dried under a stream of Nr. Lipid extracts were redissolved in chlorofolm and stored under N2 at -20oC. Poly-/l-hyCroxybuty¡¿1" (inclu<1ed as en anti- oxidant) was removed from the lipid extract by the:rrethod cf Bislrop et al . i7967). (b) Thin Lay er chromatographY and quantitation of phospho- l jpids: Phospholipids I\Iere separated by tr^¡o-dimensional thin layer chromatography on plastic baclced silica ge1 plates (Merck,Kieselgel 60 FZ54). Samples (70yr) of total lipid extract in CHCI' l{ere loaded on to 10cm x 10cn plates which 26. after drying under NZ were deyeloped in chloroforrn/nethanol-/ water (65:25:4 v/v) in the first dirnension, then dried uncler NZ and developeá'in chloroform/methano1-/acetic acid (65:25:4 v/v) in the second dinension. The location of radioactive phospholipid spots r^/as determined by overnight autoradiography. The spots l\rere then scraped off an,J counted by liquid scintillation. In all cases, the recovery of 32p-counts from the chromatograms Ì^¡as greater than 95%. Phospholi.pid species were identified by comparing their nigration with that of phospholipid standards.

(c) Preparation and analysis of fatty acids: After removal of the solvent in rvhich they were dissolved, lipids were saponified by refluxing for 3 hours in 1- N NaOH in 50% (v/v) methan- o1 under NZ. Non-saponifiable material was removed by extract- ing three tines with hexane. The aqueous Iayer hras acidified with 20 N H2S04 and the fatty acids extracted three tines with diethyl ether. The extract was dehydrated over anydrous sodium sulphate and then dried under a stream of NZ. Fatty acids ü/ere then converted to their rnethyl esters by heating at 75oC for 1 hr in the presence of 74% (w/v ) BFS ir methanol. The sample was then cooled on ice, 3rn1 of water added and the methyl esters were extracted with petroleum ether (+OoC to 6OoC frac- tit-rn). Extracts were therr dried under NZ and taken up in a small volume of CS 2'

Fatty acid methyl esters were analysed on a Perkin-Elrner 880 gas chromatograph equipped with a flame-ionization detect- or, connected to a Rikadenlci mi1livo1t recorder. The flame- ionization detector was operated at floru rates of 30tn1 /min 27. and 450m1/nin for HZ and Air respectively. Four separate columns were used for the identification of fatty acid rnethyl esters: 3% Apiezon L, 5% Apiezon L, 10% EGSS-X, and 25eo DEGS.

The 3% and 5% Apiezon L columns weïe operated at 190oC with a carrier gas (N2), flow rate of 5Onl/nin, the temperature of the injector block and the detector being 2400C and 2000C respectively. DEGS and EGSS-X columns weïe operated at 165oC with a carrier gas flow rate of 40m1/min, the injector block and detector temperature being 215oC and 17soc respectively.

Fatty acid rnethyl esters were identified by running un- knowns anci rnethyl ester standards on both polar and non-po1ar columns, determining their R, values relative to rnethyl palmitate and plotting the logarithn of the R, value against the carbon number. Unsaturated Latty acids were identified by their different behaviour on the two types of column together with their disappearance after hydrogenation, using palladium on charcoal as catalyst.

Quantitative analysis was perforrne

In experiments clescribed in Chapter 5, fatt¡, acids were added exogenously to ce11 suspensions. These fatty acids Ì{ere prepared either by saponification of total cellu1ar lipic1 extracts as described above, or by hydrolysis of pure fatty acid nethyl esters for 2 hours at 80oC in 1 N NaOFI in 50% (v/v) methanol, acidif ication r,vith HZS04 and extraction with diethylether. 28.

with NII OH, These f.atty acicl preparations were neutralized 4 dr ied under N,, and taken up in 5OnM Tris-HC1, pr1 7 .3 ¿ , ì I ,. ' : 7 Procedure for Cooling C el1s

Cells were harvested, washed and resuspended in IVCSI'I 1ov/ aa Thick suspensions of these celIs naintained at their grorvth tenperature were rapidly cooled by squirting 0.5n1 samples into 19.5m1 lots of stirred üICSM 1ow aa. maintained at diff erent temperatures. (This addition increased the temperature of the medium by not more than 0.soc. ) Alternatively, ce1ls u¡ere slowly cooled by shaking flasks of cells in an ice-water bath (the rate of temperatuïe decrease being approxirnately +oC/min) until the desired temperature was reached.

I As say for the Occurrence of Cold Shock

(a) Protease secretion: Aliquots (20n1) of ce11s that had been cooled were returned to an o rbital shaking water bath naintained at 30oC and allorved five minutes to equilibrate. Incubaticn was continued for 30 nin and samples were withdrawn at 10 min intervals. These h¡ere centrifuged (4000 x E, Snin) and the amount of protease present in the supernatant fraction was assayed.

(b) viability: I{ashed cel1s were shearecl in a sorvall Omninix at 18,000rpn. for 3 nin which rt:sulted in the breakage of the normal B. anyloliquefaciens chains of cells into single cel1s. Ce1ls u¡ere either rapidly or s1ow1y cooled as described above. Cel1s t{ere then serially diluted in gror,vth nediun at 5goc ancl 0.1m1 samples of the appropriate dilutions were spread on prewarmed plates contailing grorvth medíum and 1.5% (rv/v) 29.

Bacto-agar. Triplicate plates for eaclr. temperature sample were scored for colonies after overnight incubation at 37oC

9 tf :ay s for Enz)'rn e Activity

(a) x -Arnyl ase was assayed as f o11oÌ\¡s: One "Phadebasil tab- let was suspended in 7mI of buffer, PH 6.2, containing 10nü KZI-1P04, 25nM NaCl and 0.4nM CaCl , and 1.4n1 aliquots were dislrensed into tubes. Enzyne samples (700¡11-) were added at 0 nin and the tubes were incubated at 37oC. The reaction rvas stopped by adding 0.2m1 of 0.5 M NaOH and Lhe absorbance at 620 nm of the supernatant was measured after centrifugation (4000 x E, l_0 ninutes). A unit of o<-amylase activity is defined aS that amount of enzyme that produces an increase in absorbance at 620 nm of 1.0 in 30 min at 37oC.

(b) Protease activity rvas determined by the Remazol bri11i- ant blue hi-de polvder assaY described by Rinderknecht et al. (1968). A unit of protease activity is defined as that amount of enzyme which produces an increase in absorbance at 595 nm of 5.7 in 40 ninutes at 37oC.

(c) Ribonuciease activity lvas determined using the nethod described by Coleman and Elliott (1-965). A unit of ribonuclease is defined as that amount of enz)rnìe which produces an increase in absorbance at 260 nm of 0.8 in 30 minutes at zsoc.

10. Measurement of Total F'atty Acid Synthesis

Washed ce1ls in IVCSM rvere incubated with 2.5 7,Ci of F-d -acetate (e6 B mCi/mmole) per ml. The h/CSlt{ was supple- 30. mented r\rith 10mlvl sodium acetate as carrier. Suspensions were incubated at 3OoC with shaking and at intervals 0.25n1 samples were withdrawr::ârd added to 4n1 of ice-cold 1 M sodium acetate. The cel1s t{ere pelleted iry centrifugation, taken up in 1rnl of 1 M sodiun acetate and the lipids were then extracted as previously described. The total radioactive lipid extract of each sample r\ras counted by liquid scintillation in a Packard Tri-Carb spectrometer.

11. Measurement of Total Protein Synthesis

Cells in WCSM vrere incubated with 0.25 yCi of t FOd phenylalanine (46OnCi/mmo1e) per ml. Samples (0.1m1) were withdrawn at intervals and added to 3n1 of ice-cold 70% (w/v) trichloroacetic acid containing 7% (r,v/v) casamino acids. The samples were chilled on ice for 30 minutes, then heated at gSoC for 20 minutes and then chilled on ice for a further 30 minutes. The sarnples were then f iltered on lVhatman GF/A filters and washed five times with 5n1 of 70% (w/v) trichloroacetic acid containing I% (w/v) casanino acids and then three times with 5ml of I% (v/v) acetic acid. The f ilters were then dried and counted by liquid scintillation.

]-2. Measurement of Total RNA Synthesis

Cells in I^ICSM were incubated with Z.ilyci of , ÍOJ:- uracil (55 nCi/nmole) per nl with 50¡S/nT of unlabelled uracil as carrier. Sanples (0.1n1) were withdralvn at various times and added to 3n1 of ice-cold 1"0% (w/v) trichloroacetic acid containing 0.1% (rv/v) uracil. 'fhe sa-mp1es $¡ere chilled on ice

f or 3 0 minutes and then f iltered on l{hatnian GF/A f ilters . The filters r.,fere washed five times with 5n1 of L0% (w/v) trichloro- 3I acetic acid containing 0,1,% (w/v) uracil and then three tines I^Iith Sml of 1% (v /v ) acetic acid. The f ilters u/ere then dried and counted by, 1Íquid scintillation.

L3, SDS-Polyacrylamide Ge1 Electrophoresis

Slab gels (1 .5mrn thick) u/ere prepared by the nethod of Laemmli (1970), which incorporates a stacking ge1 and a separating gel. The stacking ge1 was ?cm long and was composed of 3% (w/v) acrylamide, 0.I% SDS and 0.I25 M Tris-HCl, pH 6. B. The separating ge1 was 10cm long and was composed of 72.5% or L5% (w/v) acrylanide, 0.1% (w/v) SDS and 0.375 I,{ Tris-FICI, pH 8.8. Electrophoresis was perforned at 4SmA for 30 minutes to stack the proteins which rn¡ere then run through the separating gel at 1,20 V f or ZL, hours.

To detect protein, gels were stained overnigltt in 25% isopropanol, 70% acetic acid, 0.05% coomassie blue and then destained f or' 6- B hours in I0% isopropanol, IOeo acetic acid.

Alternatively to detect radioactivity, gels were prepared for fluorography as follows: Inmediately after electrophoresis, tlre ge1 was fixed in 70% isopropanol, 70% acetic acid for at least t hour. The gel was then soaked in DMSO (dirnethyl sulphoxide ) f or t hour (the DMSO ivas changed arcter 5 0 rninutes ) and then in 50m1 of DMSO containing 12g of PPO for i hours. The ge1 v\¡as then washed in water for \14 hours, dried and then fluorographed at -7 0o C.

For molecular weight determinaticns, the following proteins were used as markers: bovine serum albumin, glutamate dehydrogenase 2 glyceraldehyde- 3 -phosphate dehydrogenase , 32.

cucumber mosaic virus coat protein and globin.

To elute proteins from preparative slab gels, the gels were first briefly stained and destained (1 hour for each step) to visualize the bands. The appropriate band was then sliced out, finely rninced using a glass/teflon homogenizer and eluted overnight by gently swirling in buffer. Fragments of the gel were removed by filtration.

14. Non-denaturinq Polyacrylanide Gels

Non-clenaturing gels (7 % polyacrylanide, Tris/glycine buf - fer system, pH 8.3) were prepared in glass tubes of 5mm internal diameter according to the method of Davies (1964). Electro- phoresis, at a constant current of 3nA per tube, I{aS perforned at 4oC for 2, hours.

To detect protease activity, frozen gels Ìvere cut into Zmn slices using a Mickle ge1 slicer. These were eluted overnight in 50nM Tris-HCl, pH 7 .8, and assayed for protease activity.

15. Sp ec if ic Proteolytic Cleavaqe

Approxinately 50fg of purified protein was boiled for f ive minutes in 125nlr{ Tris-HC1, pH 6.8 containing 0 -I% (w/v) SDS. To these samples, 5/g of o<-chymotrypsin o,: TPCK-treated trypsin was aclded, and the mixture hlas incubated at 37oC for 30 minutes. The concentration of SDS rvas then increased to 2% (w/v) and the mixture was boiled for a further 5 minutes p::ior to electrophoresis oll SDS-15% poLyactylamide slab gels. 33.

16. C ano en Bronide Cleava e

Approxirnately 30019 of purified protein was treated overnight at room temperature rvith 3Orng of cyanogen bromide dissolved in lnl of 70eo HCOOH. This was then diluted to 5rn1 with water and lyophilized. The dried material r,rias dissolved in 5n1 of water and then lyophiLized twice more. The material was then taken up in 2% (w/v) SDS containing 70eo (v/v) 2-nercaptoethanol and boiled for 5 minutes prior to electrophoresis on SDS-15% polyacrylamide ge1s.

1.7 . Purification of the fntracellular Protease

Late logarithnic ce1ls (from a 100n1 culture) were harvested,washed and suspended in WCSM low aa. After incubation for 75 ninutes at 3OoC rvith shaking, the cel1s \vere v/ashed twice to remove extracellular protease and suspended in 5OnM Tris-HC1 pÍ17.8 and then lysed by treatment in a French pressure ce1l at ') 15,000 Lb/in". This, and all subsequent steps were performed at 4oC. The lysate was centrifuged at 35r000 x g for 20 minutes and the supernatant was freeze dried, taken up in 5rn1 of 50mM Tris-HCl, pH 7 I and applied to a Sephadex G-100 column (98crn x 1.7crn) eq-urilibrated with the same buff er. Eluted f.ractions containing protease activity were applied to a DEAE- ce1lu1ose column (DE-23) (21cn x 1.1cn), equilibrated and developed with the same buffer. After a periocl of flolv, elu- tion was changed to a linear gradient, formed by 300m1 each of 50nlr{ Tris-HCl, pH 7.8 and the same buffer containing0.S l'''l NaCl.

Intracellular protease activity eluted between 0,2 and 0.3 M NaCl . Fractions containing this actívity v,rere pooled, and after removal of salt by. chrornatography on a sephadex G-zs s4.

column, the extract was electrophoresed on non-denaturing 7% polyacrylanide gels, pH 8,3. The gels were sliced and those slices containing intracellular protease activity were eluted overnight, dialysed against 50nI4 Tris-HC1, pH 7.8, and stored at - 2ooc. CFTAPTER THREE

STUD IES ON THE },IEMBRANE PHOSPHOLIP IDS OF B. AMYLOLIQUEFACIENS 35.

A INTRODUCT I ON

this chapter was aimed initially at The r,uork 1"_t::ibed in determining the phospholipid composjtion of the rnembrane of B. amy loliquefaciens. Subsequent experiments wer'e aimed at deterrnining the distribution of these phospholipids between the two halves of the rnembrane bilayer, emLrloying specif ic phospholipases and chenical labe11ing techniques.

The reasons for studying the phospholipids and their distribution were twofold. First1-y, information on the phospholipids of the cytoplasmic membrane was potentiaLly useful in understandi-ng the mechani-sm of protein secretion in this organism, especially since it has been reported by Dancer and Larnpen (1975 ) that the penicillinase of B. lichenifornis was synthesized with attached phospholipid. As well as this, studies (reported later) have inplicated the involvement of lipid in the process of secretion in B. amyloliquefaciens . Secondly, ãIthough asymmetric distributions of phospholipids between the trvo halves of the membrane bilayer had been report- ecl f or eukaryotes (Bretscher ,I97 2; Verklei j et a1. , L973; Whiteley and Berg , 797 4) and viruses (Tsai and Lenard , 197 5; Fong et al. , 7976; Rothnan et al ., L976), ãt the tirne this rvork r^ras commenced, there had been no reports of such asymmetric distributions in bacteria and it seemed worthlvhile to examine this.

Previous work in this laboratory (McMurchie, 1"977) showed that the nembrane of B. amyloliquefaciens was made up of approx r - rnately 40% lipid and 60% protein (by weight) . Mcl,lurchie fur ther fractionated the lipids of the nenrbrane by sj licic acid chrone - 36. tography and found that they were comprised of approxinately 75% ptrospholipid, 14% neutral lipid and 1I'o glycolipid. Virtually all of the cel1ular phospholipid was associated rvith

the rnembTane "

B. RESULTS

1 Pho spho 1 ipid Composit ion of Ce11s and Protoplasts

To unif orrnly 1abe1 cel1ular phospholipids, ce11s l{rere 32p' grown in 10nI cultures in the presence of 200yCi of labelled inorganic phosphate. The phospholipids were extracted from late logaritlmic ce11s using chloroform/nethanor. (I:2 vol/vo1) and separated by two dimensional thin layer chromatography (see "Lipid Techniques", Chapter 2). For complete extraction of cardiolipin frorn the cel1s, it was found necessary to first disrupt them in a French pressure cel1 operated

at 15 r 000 Ib/ ittz, prior to lipid extraction. The radioactive phospholipid spots were detected by autoradiography, then scraped off and counted by liquid scintillation. A typical autoradiogram is shown in Plate 3-1. Alternatively, ='n 1abel1ed ce11s l{ere coirverted to protoplasts by incubation rvith lyso zyme (as described in Chapter 2) before lipid ext.raction. Pþospholipid species werc identified by conrparing their rnigration on the chromatography plates rvith that of phospholipid standards. The phosphoiipid conposition of both ce1ls and protoplasts is given in Table 3-1. The three major phospholipids were found to be cardiolipin (CLP), Phosphatidyl- glycerol (PG) and phosphatidylethanolamine (PE). The respective proportions of total phospholipid (on a molar basis) for CLP,

PG an<1 PE rvere 9%, 52% and 30% for ce1.ls, and 30%, 29% and 32% r: r: ¡ ¡: r. -: '

PLATE 3.7

AUTORADIOGRA]4 OF B. A}'IYLOLIQUEFACIENS PHOSPFIOLIPIDS

SEPARATED 3Y TWO DIMENSIONAL THIN LAYER CHROMOTOGRAPHY. Lipids hrere extracted, separated and identified as described in Chapter 2. Solvent !, chloroforn/methanol/water (65:25:4, v/v). Solvent 2, chloroform/nethanol/acetic acid (65:25:4, v/v). 1. phosphatidylethanolamine. 2. phosphatidylglycerol.

3. cardiolipin . 4. unknown, (the probable identity of these species is referred to in the text). \ solverur FRONTS \

2 (D 4

1 z,- ¡rl J o Ø .ORlGlN

SOLV ENT 2 + TABLE 3-L

. ¡- : : ii PHOSPHOLIPID COMPOSITION OF CELLS AND PROTOPLASTS OF

BACILLUS AMYLOL]QUEFACI ENS

COMPONENT CELLS PROTOPLASTS

Cardio 1 ip in 9 (L37 s) 30 (3801)

Phosphatidylglycero 1 52 (3e4e) 29 (182s) z) Phosphat i dyl e thano 1 amine 30 (2248) (20s3)

Renainder 9 (67 z) 9 (s82)

Phospholipids weTe extracted from whole ce11s imned- iately after lysis in a French pressure cell, or from cel1s that weïe converted to protoplasts. The phospholipids were separated and quantitated as described in Chapter 2 and the proportions of each phospholipid expressed as a molar per- 32p-counts/min centagc of total phospholipid. The original above background are given in parentheses. Since CLP has two phosphate groups per molecule, CLP counts have been halved before calculation of the molar percentages. 37. for protoplasts. The remaining phospholipid in both cel1s and pïotoplasts (9% of total phospholipid in both cases) consisted of several snialï cómponents that did not migrate far from the origin on the thin layer chromatography plates. These rninor components were not identified, but were assumed to be lipoamino acids since they contained a phosphate group, were ninhydrin positive and did not co-migrate with PE, phospliatidyl- serine or lysophosphatidylethanolarnine. Lipoamino acids, particularly lysylphosphatidylglycerol, have been found ín similar proportions in B. subtilis (Bishop et al.,1967; Cp den Kanp 9I aI., 7972).

It is apparent from Table 3-1 that rvhen cells are converted to protoplasts, a marked alteration in the relative amounts of PG and CLP occurS. The respective proportions of total phospholipid for CLP and PG change from 9% and 5?% to 30eo and 29%. The 32p total loss of -counts from PG was approxirnately equal to the increase in 32p-counts in CLP. Tine couïse experiments showed that after the initial 60 ninute incubation with lysozyme, ro further significant alteration in the relative proportions of the phospholipid species occurred in the protopl-ast membrane (Figure 3-La). I{hen lysozpe r\¡as onitted from the incubation medium, increase in CLP was not observed (Figure 3-1b).

It has been reported by Hirschberg and Kennedy. (7972) that PG can be directly converted to CLP in E. co1i, and poss: ibly such a direct conversion occurs in B. amyloliquefaciens . 0p den Kamp et ?]-. (7972) have reported an apparently simì.lar, but less narl

THE EFFECT OF INCUBATION IVITH LYSOZYME ON THE PHOSPHO-

LIPID COMPOSITION OF B. AMYLOL I UEFAC IENS . t'n Ce11s, 1abe11ed with , vrere suspend.ed in proto - plast nedium and incubated at 3ToC, in the presence (a) or absence (b) of 733 ys/nr of lyso zwe. At the indicated times, samples hrere withdr=arvn and l ipid extracted. phos - pholipids were then separated by 2D-thin raye,' chromatography and quantitated, âs.described in Chapter Z. The amount of cardiolipin (c), phosphatidylglycerol (O), phosphatidylethanolamine (a) and the remainde:: (r) is expressed as a molar percentage of total phospholipid. I rorAL PnosPHoL¡PtD o oo()19Ào. o oN) ooÀO. o !,

(, o { 3 m

o, C)

3 z o\o

ol\) 38.

2 The Use of Phospholipases as a Probe for Phospholipid Distribution

Specific phospholipases have been successfully used by several workers to establish the existence of asymmetrical phospholipid distributions between the inner and outer halves of various aninal cel1 nembranes, particularly the erythrocyte plasrna membrane (Verkleij et a1., I973) and viral membranes (Rothrnan et al., 1976). As alteady mentioned, when the present work started there had been no reports as to whether phospholipids of bacterial nenbranes were also asynmetrical1-y distributed. Phospholipases, being large molecules, are not expected to cross rnembrane bilayers. Consequently, if a mernbrane exists in a continuous, sealed form (such as the plasma membrane of the erythrocyte or the cytoplasmic rnembrane of bacteria ) then only phospholipids in the outer half of the nenblane bilayer would be accessible to the enzyme. The proportion of each phospholipid species that is hydrolysed by these enzymes therefore would be expected to reflect the proportion of that species in the outer monolayer of the membrane lipid bilayer. This is on the assumption that reactions proceed to comPletion and that transmembrane movement (f 1ip-f lop) of phospìro1i pids is not occurring (see discussion).

3 Treatment of Intact Protoplasts with Phospholipases

Two Phospho lipases, phospholipase C (!_,- c er eus and Phospholipase A, (Crota.l-us) were used to probe the phosphol ipid distribution in B. amyloliquef aciens, Since the pr'esence of the cel1 wa1l night hinder the acc-ess of these enzymes to the membrane, initial studies were performed on protoplasts. 39.

These were prepared from 32 P-1abe11ed cel1s, suspended in protoplast rnedium and incubated with either phospholipase C or AZ (as descrlbecl in Chapter 2). The action of the enzymes on the phospholipids rvas deterntined by following the loss in radioactivity in the individual phospholipids isolated by thin layer chromatography. The tine course of hydrolysis of the three rnajor phospholipid species by these enzymes is shown in Figure 3-2. In the presence of phospholipase C about 70% of the total phospholipid in the protoplast membrane was degraded after t hour at 37oC. About 90 % of total PE and PG hras degraded compared with only 30% of CLP. A similar result r\ras obtained rvhen protoplasts were incubated rvith phospho- 1ipas. AZ although the initial rates of degradation were slightly less for PE and PG.

When 32p-labe11ed protoplasts lvere incubated for t hour at 37oC in the absence of added phospholipases, and the phospholipids were isolated at different times, there was no alteration in the arnount of any of the phospholipid species.

Incubation of protoplasts at 37oC rvith twice the amount of enzyme (Aror C) and for longer time periods did not result in arry increased degracLation of arry of the phospholipid species. During the incubation with the phospholipases, the protoplasts remained intact as judged by phase contrast microscopy.

fnterestingly, when protoplasts I{êr€ incubated with lOunits/nl of N. naja phospholipase A2 (Sigrna) at 37oC for 1 hour, nearly 90% of total phospholipid was hydrolysed (80% of CLP, 93% of PG ancl 95% of PE) " However, examination of these FIGURE 3-2

THE EFFECT OF PIIOSPHOLIPASES A2 AND C ON THE PI]OSPHO- LIPIDS OF B. AMYLOL IQUEFAC IENS PROTOPLASTS. 3zp- Aliqucts (1 rnl) of label1ed B. any 1o1 iquefac iens protoplasts in protoplast medium, each containing approxinately 100 fg of phospholipid, 'h¡ere incubated at 37oC with (a) 30 I.U. PhosPholiPase C ( B. cereus (b) 4 I.U. phospholipase A, (Crotalus) + t0 yL of 0 ' 1 M CaCL r. At the indicated times, the reaction was stopped by the adclition of 0.1 nl of 1M EDTA, the lipids extracted and the phosph.olipids quantitated as described in Chapter 2. The arnount of cardiolipin (e), phosphatidylglycerol (o) and phosphatidylethanolanine (¡) degraded is expressed as a percentage of the total amount of each phospholipid in protoplasts that rvere incubated for the same time in the absence of added phospholipase. ¡00 a I

50 o l¡J Ø o o É, o J.

0 I b I 00 À o x À c, ¡ o o ô- 50 o-{

o o o

0 I I 0 r5 30 45 ó0 75

TIME (ru lru¡ 40. protoplasts under the phase contrast rnicroscope revealed that substantial lysis had occurred during the incubation period. It is not knowrf why this preparation behaved differently from the other phospholipases.

4 The Effect of Phospholipases on the Integrity of Protoplasts

The fact that approxirnately 30% of CLP' 90% of PE and 90% of PG was accessible to phospholipase C or CrglgluE phospholipas. ÃZ suggeststhat sinilar proportions of these phospholipids exist in the outer monoJ-ayet of the protoplast membrane. This conclusion, however, depends on the assumption that the inner monolayer phospholipids wer'e inaccessible to the enzymes, The protoplasts appeared to remain intact (as judged by phase contrast rnicroscopy) during incubation f.or up to 75 minutes at 37oC with either of these two enzymes. This was examined further by turbidity measurements (Figure 5-3). When protoplasts were lysed by resuspension in buffer without sucrose, the AOOO decreased to about 5% of the initial value.

The AOOO of protoplast suspensions (in protoplast nedium) did not decrease during 60 ninutes incubation at 37oC with each phospholipase. There was a gradual increase in the AU'O of the suspensions whether the phospholipases ï/eÌ:e present or not, due to swelling of protoplasts resulting from glutarnate uptake from the nediun (8. K. May, unpublished data).

The integrity of the protoplast rnembrane after phospho- f.ipase digestion rvas also investigated using electron rnicroscopy. Protoplasts were incubated at 37oC for 75 ninutes with or without phospholipase C or qI"t=I: phospholipase 42, and were then fixed and sectioned as described in Chapter Z. The FIGURE 3-3

TI-IE EFFECT OF PHOSPHOLIPASES A2 AND C ON THE ABSORBANCE AT 600 nn OF PROTOPLAST SUSPENSIONS. Suspensions of protoplasts in protoplast mediun containing approximately 700 ¡rg of phospholipid/nl were incubated at 37oC in the presence of 4 I.U. of phospholipase A2 Crotalus per m1 (^) , 50 I.U. of phospholipase C (8. ceTeus I per rnl (o) , or with no added enzyme r.tf) . Sarnples rvere rvithdrawn at intervals and the AU00 was measured in an llitachi model 101- spectrophotometer. As a comparison, the Aooo of protoplasts suspended in buffer without sucrose v¿as followed (¡), total lysis having occurred within 10 minutes. 1-2

A o tr ç o 1.0 \oo

0.8 F

o.ó

t¡¡ (J z É 0.4 É. o C, æ o.2 u^. !-t-! E t ! 0 I o r0 20 30 40 50 ó0

T¡ME (ru lrrl) 4.1, . electron micrograplr.s of these thin sections a.re shown in Plate 3^2, It can be seen that the protoplasts remain intact in the presence of the phospholipases and no discontinuities in the menbrane structure of any of the protoplasts lvere observed.

As a further check on the inaccessibility of inner non- Iayer phospholipids to exogenous phospholipases, the pellnea- bility of the protoplast membrane to sma1l proteins vlas studied The leakage of an intracellular ribonuclease inhibitor (a protein with a molecular weight of 12,000 daltons) in the presence of either phospholipase C or Az Cro talu s I\rAS eXam].n- ed over a 60 ninute time period. This inhibitor (which binds irreversibly with the ribonuclease secreted by B. e4y]-9-ligg-q- fac iens has previously been shornrn by Smeaton and Elliott (19ó7) to leak from ce11s permeabiltzed by cold shock treatment. protoptasts were incubated for 60 ninutes at 37oC with or without either phospholipase. The protoplasts 14/ere then pelleted by centrifugation and the amount of ribonuclease inhibitor present in the supernatant was assayed as described in Chapter 2. The total amount of inhibitor present in the protoplasts was determined by lysing them in a French pressure cell at ) 15,0001b/in", centrifuging as bcfore, and assaying the supel- natant fraction for libonuclease inhibitor. It was found that about 4eo of the total intracellular inhibitor llras released from intact protoplasts in the presence or absence of either phospholipase. It is therefore clear that the phospholipases do not promote protoplast lysis or significant alteration in the pel'm- eability properties of the protoplast that míght permit free passage of proteins acloss the membratre during the period of incubation. PLATE 3.2

COMPOSITE ELECTRON }{ICROGRAPH OF SECTIONED SPECIMENS OF B.AMYLOLIQUEFACIENSPROTOPLASTSTREATEDWITHPHOSPHO- LIPASES. Protoplasts, suspended in protoplast medium, were enzyme (A) incubated at 37oC for 75 minutes r^¡ith no ' 30 I.U. phospholiPase C (8. cereus (B), or 4 I.U. phos- protoplasts were then pholipaser¡L A, (Crotalus) (C). The fixed and sectioned as described in Chapter 2' Bar marker = 0.L/. ,r f!f,

o .j*¿ I 42.

'Sepharo 5 Di estion of Proto 1as t s with s e. Pho s pholipase C

To further demonstràte that the phospholipases hydrolysed only external phospholipid, ãttenpts l,,/ere made to degrade the phospholipids of protoplasts rvi-th phospholipase c linked to sepharose beads. Although phospholipid degradation patterns similar to that observed rvith free enzyme resulted, control experiments revealed that significant amounts of enzyme had separated from the beads during the incubation period. This line of approach was therefore discarded.

6 Treatment of Permeabilized Protoplasts with Phospholipases

When intact protoplasts hrere incubated with phospholipase C or Ar, 30% of. CLP, 90eo of PG and 90% of PE were hydrolysed. If this reflects the proportions of these phospholipicls in the outer monolayer of the rnenbrane, then it rnight be expected tha.t incubation with phospholipases under conditions in lvhich the enzymes had access to both sides of the membTane would result in total degradation of all phospholipids. When protoplast s of B. loli uefaciens are rapidly chi11- ed (cold shocked) , by diluti-on in ice-cold media, there j s a breakdcwn in the permeabili-ty barrier of the cel1. This breakdown permits sma11 proteins such as the intracelluiar ribonuclease inhibitor to leak out, although the protoplasts remain intact as judged by phase contrast microscopy. Up to B5% of total intracellular inhibitor could be released in this manner (Paton, 1975).

To make protoplasts 1eaky, 10m1 suspensions were pelleted by centrifugation (4 ,000 x g, 5 rninutes) ¡ taken up in 0.5m1 of 43. protoplast medi.um at 3OoC andigentty added to 9,5m1 of ice- cold protoplast medium. To investigate rvhether additional phospholipid could be degraded in these leaky protoplasts, they were inct,bated lvith either phospholipase C or AZ Cro talus at 37oC for 60 minutes. The phospholipids were extracte<1, separated and quanti-tated as bef ore. The results (Table 3-2) show that nearly three times more CLP was exposed to phospholipase C in cold-shocked, âs compared with normal protoplasts. In addition, PG and PE were further degraded to 98% and 99eo respectively. I{hen phospholipase A, rsas used, again PG and PE were degraded almost to completion while CLP was digested to 77%.

7 Digestion of li{embrane Vesicles ivith Phospholipases

The eff ect of phospholipases on cytoplasrnic. membrane vesicles of B. anyloliquefaciens was studied. The ultimate aim was to obtain homogeneous preparations of vesicles with either the same or opposite orientation as that of intact cells. Digestion of these two preparations with phospliolipases would therefore be expected to yietd complementary phospholipid degr adation patterlìs .

To prepare membrane vesiclcs of normal orientation, 10m1 suspensions of protoplasts l\rere pelleted by centrif'-rgation (4,000 x E, 5 ninutes), and osmotically lysed by resuspension in 10m1 of 50mI4 Tris-HCl, 0.25mM CaCI2, 0.25m1{ MgCL2, 0. hrù4 ZnCIZ (pU 7.3). Membrane vesicles were pelleted by centrifugation (35r000 x E, 20 rninutes) and resuspended in the same buffer. It has been shown by several workers that vesicles prepared by osmotjc lysis of protoplasts have the same orienta- TABLE 3-Z

DIGÈSTION OF PIJOSPI]OLTPIDS OF INTACT AND

PERMEABILIZED PROTOPLASTS BY PHOSPHOLIPASES A ) AND C

PHOSPHOLIPASE C PIIOSPHOLIPASE A 2 COMPONENT INTACT PERMEABILIZED INTACT PERMEABILIZED

Cardi o1 ip in 50 BO 29 7L

Phosphatidyl - glyce ro 1 91 98 87 96 Phosphatidyl - ethanol amine 93 99 89 99

Aliquots (1 nl) of 32p-labe11ed intact protoplasts or protoplasts permeabilized by cold shock treatment (each containing approxinately 100 pg phospholipid) r.\Iere incubated in protoplast rnedium with either 30 I.U. phospholipase C, or 4 I.U. phospholipase with 10 pl of 0.1 \[ CaCI2, for ó0 min- ^Z utes at 37"C. The reaction hras then stopped by the addition of 0.1 ml of 1 M EDTA and the phospholipids were extracted, separated and quantitated as described in Chapter 2. The Cegradation is expresseC as a percentage of the total amount of each phospholipid species present jn aliquots of intact or perneabíIized protoplasts that were incubated in the absence of added eîzyme. 44. tion as that of intact cells, ( Konings et al . , 1,97 3; Short et ?!. , 797 4; Altendorf and Staehe 1in, 79741 Futai and Tanaka,

197 5; Koning s, 1- 97 5) . Electron microscopy of thin sections of such vesicie s llrepared from B. arnylol iquefac iens (McMurchie, 1977) has shown them to be intact (no discontinuities in the membrane 't^rer e observed) and rvith a diameter of approxirnately 0.3p. They have also been shown to be capable of actively transpor t ing FOa] leucine (Paton, unpublished data).

trVhen these vesicles r^rere incubated with phospholipase 42, the rate of digestion of the three major phospholipids r{as slow in comparison with that in protoplasts (Figure 3-4). Even after 2 hours at 37oC, d,igestion v¡as not complete ancL only 22% of CLP, 62eo of PE and 75% of PG were hydrolysed. It was been shown by Denel et aL. (1975) that the activity of various phospholipases towards monomolecular filrns of phospholipids , spread at an air-water interface, is dependent upon the lateral surface pressure. These findings were used by ZwaaL et aI. (1975) to explain the different effects of eight purified phospholipases on human erythrocytes and ghosts. The differ- ence in activity of phospholipase A, towards protoplasts and membrane vesicles of B. amyloliquefacíens could possibly be explained by differences in the lateral pressure in these two membrane systems.

l\lhen phospholipase C hras incubated with the vesicles, the degradation of the phospholipids (Figure 3-Sa) was sjmilar to t}rrat observed in protoplasts except that the rates were somewhat faster. Approximately g}eo of PE, 90% of PG and 34% of CLP were hydrolysed. 1: \ì c !: il I

FIGURE 3^4

THE EIìFECT OF PHOSPHOLIPASE A2 ON TFIE PHOSPHOLIPIDS OF

MEMBRANE VESICLES PREPARED BY OSMOTIC LYSIS OF PROTOPLASTS. Membrane vesicles were prepared from 32p-labe1led protoplasts as described in the text. 4 I.U. of phospho- l ipas. Az Crotalus was incubated at 37oC with 1 nl aliquots of these vesicles (containing approximately 100f9 of phospholipid). At the indicated times, the reaction was stopped, the lipids extracted and the phospholipids separated and quantitated, âs described in Chapter 2. The amount of cardiolipin (O), phosphatidylglycerol (O) and phosphatidylethanolamine (¡) degraded was determined as described in the legend to Figure 3-2. "l PHosPHoLrPrD HYDRoLySED (rl I o o o o

oC^t

J =m ect)

L=- (0o

.¡ e¡\) FIGURE 5.5

THE EFFECT OF PHOSPHOLIPASE C ON THE PHOSPHOLIPIDS OF

MEMBRANE VESICLES PREPARED BY OSMOTIC LYSIS OF PROTOPLASTS. Phospholipase C (30 I.U. /1,00 fg phospholipid) was added to protoplasts after (a), or before (b), lysis in 50 nM Tris-HCl , 0.25 nM CaCL2t 0.25 rnM MgC12, 0.1 nM ZnCL, (pH 7.3). After incubation at 37oC, the percentage of the total amount of cardiolipin (O), phosphatidylglycerol (O) and phosphatidylethanolarnine (r) degraded was deterrnined as described in the legend to Figure 3^2. 7. PHOSPHOLTPTD HYDROLYSED Ur o (¡ o o o o o o o

o v I I Ð

(¡r J o o g OE trt

(.) o

E ¿. OE c O¡

(¡N

6 o o OE @ 45.

In another experiment phospholipase C lvas added to protoplasts immediately prior to osmotic lysis in the hope of putting enzyme ron:'both sides of the rnembrane. However the rates and extents of phospholipid hydlolysis (Figure 3-5b) ü/ere sinilar to those observed when phosphollpase C rvas added after lysis, suggesting that the cnzyme was not entrapped within these vesicles. In an attenìpt to confirm the results obtained in section 3-6, vesicles r¡ere incubated with phospholipase C in the presence of 0.05% deoxycholate. Similar concentrations of deoxycholate have been shown by Nilsson and Dallner (7977 ) to render rat liver microsomes permeable to macromolecules but without disrupting the menbrane (analogous to cold shock-permeabilízed protoplasts). Although the degradation of CLP, PG and PE increased to 49%, 96% and 100% respectively after 60 minutes at 37oC, the detergent interfer- ed with the efficiency of the lipid extraction procedure, naking interpretation of the results difficult.

To test the effect of phospholipases when only inner monolayer phospholipids are exposed to the enzymes, atternpts Ì\Iere made to prepare inside-out membrane vesicles. The sonication procedure used by Rothrnan and Kennedy (7977a) to prepare these from B. megaterium failed to significantly disrupt ce11s of B. amylo 1 iquefac iens , even af.ter 10 minutes treatrnent at maxiumum pc)rveï of a Branson Sonif ier (model 830) . When vesicles r\rere prepared by treating cells in a French ? pressure ce11 at 15r0001b/in" according to the method of Futai

(797 4) f or g. g_g.1_i, v€sicles approxirnately 0. 0Srcc in diameter r.{ere obtained, but these had lost 80% of membrane protein (determined by comparing the protein and lipid content of 46. these vesicles with that of osmotically prepared vesicles). l{hen these vesicles IVere incubated with phospholipase C, a phospholipid deþiadation pattern similar to that observed in osmotically prepaled vesicles and protoplasts resulted. It therefore seems 1ikely that the vesicles obtained were of the same orientation as protoplasts, though no attempt was made to confirm this, siuce the effect of sonication and French pressure ce11 treatment on subsequentvesicle orientation is perhaps not as clear cut as was once believed (I{. Salton, personal communication) .

B Attemp ted Exper iments with Phospholipid Exchange Proteins

In an attempt to obtain independent confirmation of the results obtained with phospholipases' accessibility of protoplast phospholipids to a phospholipid exchange protein was examined. Unlike phospholipases, these proteins (which catalyse a one for one exchange of phospholipids between the exposed monoLayers of membranes (Wi-rtz, 797 4)) do not covalently alter the phospholipids of the outer monolayer, which might conccivably have caused artifacts. A phospholipid exchange protein capable of transferring CLP, PG and PE was partially purifiecl from rat liver according to the methcd of Bloj and Zi)-versmit (I977). The proportion of each phospholipid species 32p-labe11ed that coulcl be transferred from protoplasts to an excess of unlabelled artificial liposomes would be expected to reflect the proportion of that species in the outer half of the protoplast membrane. FIowever, during incubation of B. amyloliquefaqiel:qE protoplasts and liposomes (prepared by 47. sonication of previously isolated B. amylol iquefac iens pho spho - lipids) rvith the exchange protein, non specific aggregation and seclimentat,i-oh,of sorne of the liposomes occurred, making interpretation of the results irnpossibl-e. It was suggested by Zilversnit (personal communication) that EDTA is needed to prevent such aggregation. Since EDTA lyses B. anylo1 iquefac iens protoplasts (Sanders, I974) this could not be used. Further experiments with exchange proteins were abandoned.

9 Chemical Modification of Phospholipids

Tr in i tr o b en z ene su lphon ic acid (TNBS), a reagent that con- verts aminophosphatides to their trinitrophenyl derivatives, has been used to selectivel y labe]- PE in the outer monolayer of the ce11 membrane of B. megater ium (Rothman and Kennedy, 7977a), fn an attempt to obtain independent confirmation of the phospholipase results, at least for PE, cells of B. amyloliquefaciens were 1abel1ed with the reagent.

The procedure used l^ras essentially the same as that of 32P Rothman and Kennedy (7977a) . Cells (5rn1) grorvn in t"t" harvested, rvashed once in 0.1 M KCl containing 50nM potassuim phosphate (pH 7.9) and taken up in 5n1 of the same buffer. The cel1s were then adjusted to the temperature at which the l¿rbe11ing was tc be carried out (37oC or +oC). In the latter case it was necessary to slow1y cool the ce1ls to avoid the occurrence of cold-shock (see Chapter 4). Sufficient TNBS (ZBnM in freshly prepared 5% (w/v) NaHCO, (pl-l 8.5) ) was then added to achieve the desired f inal concentlation of TNBS (3ml'{ for most experiments). The ce1ls were then incubated at the appropriate tenperature in an orbital shaking water bath, 48. during which tine, the pl{ of tlle suspension rvas maintained at pH 8.1. Experiments with TNBS were perforrned with intact cel1s since protoplasts are not stable under the prolonged incubation conditions ïequired for TNBS nodification at 4oC.

10. Extent of Penetration of TNBS into Cells

If TNBS was to be used to labe1 externaLLy localized PE, it rvas important to establ ish condit j-ons in which it did not penetrate the cell membrane. The extent of entry of TNBS into the ce1ls r^ras estimated by neasuring the extent of labelling of a crude cytoplasmic protein fraction. Labelling of protein hras detected by the yelloh/ colour of the trinitrophenyl group.

Aliquots of cells (10m1) rvere incubated wi-th SmM TNBS at 37oC for 30 minutes oï at 4oC for 4 hours. The cells were then washed with 40m1 of 50nM Tris-HC1 containing 50mM KCl and 20mM

2-rnercaptoethanol (pH 8.5) and resuspended in 10m1 of 5Omlt{ potassium phosphate containing 100mlt{ KCl (pH 7.0). The ce11s hrere then lysed in a French pïessure ce11 at 15,000 Ib/ inZ and centrifuged (105,000 x g, 60 ninutes). Supernatant protein was precipitated with I.ZmI of ice-co1d 30% (w/v) trichloroacetic acid and 0.4n1 of 5mg/m1 BÍiA. The pellet was then colfected by centrìfugation, taken up in 10rn1 of 1 M Tris free base contai ning 5"4 (w/v) sodium dodecyl sr-rlphate and the AOrO rneasured in an Hitachi nodel 101 Spectrophoto- meter. The absorbances were compared with those of trinitrophenylated crude cytoplasmic protein f.ractions of ce11s that r,üere lysed in the French pressure ce1l prior to labe11ing with TNIIS under: the respective conditions. In each case, the A+tO of the crude cytoplasmic protein fractions from control 49. cells,that were not trinitrophenylated, was subtracted from

the readings '

Frorn Table 3-3 it is clear that at 37oC cells are readily permeable to TNBS, since greater than 60% of ce1lular cytoplasmic protein was modified. However, when the modification *ras carried out at 4oC (for 4 hours) the entry of TNBS into of the cel1s was consider ab1y less (Table 3-3). The fraction cytoplasmic protein isolated from labe11ed cel1s had approxi- groups found mately Z0% of the colour due to trinitrophenyl in previously isolated cytoplasmic protein that was incubated with TNBS. These results are quantitatively sinilar to those reported by Rothman and Kennedy (I977a) v¡hen B' megater ium ce11s were treated with TNBS at 3oC. These authors suggested for that at 37oC a membrane .transport protein rvas responsible the rapid entrY of TNBS'

However, even at 4oC, there is labelling of protein of intact ce11s treated with rNBs (20% of that of previously isolated cytoplasmic protein). This apparent 1abe11ing nay be due to ïeaction rvith periplasmic proteins which r^Iould be released by French pressuTe cell treatment and would not be separated from the cytoplasmic protein by the nethod ernployed' It may also be due Lo membrane protein shed from the bilayer during French pressure ce1l treatment or to ninute fragments of ce11 u¡a1l materiaL. To investigate this possibility, instead of lysing ce11s in a French pressure cel1 after 1abe11i-ng, the 1abe11ed rvashed ce11s were suspended in protopl a.st neclium and convertecl to protoplasts as described in Chapter 2. The protoplastS v,Iere tlashed witlr protoplast TABLE 3-3

EXTENT OF PENETRATION OF TNBS INTO CELLS

A+tO of crude cytoplasrnic A41g of cytoplasnic protein fra.ction protein of jntact ce1ls Lysis Incr.rbation as a % of A41g of the procedure conditions isolated protein fraction Intact Isolated protein labelled rn-rder the same ce1ls fraction conditions

French 37o rnin. 0. 708 1.108 6s.9% pressuTe ,30 ce11 lysis 4o 14 hours 0.227 1.017 22.3% f,smotic lysis 4"14 hours 0.025 0. 850 2.9%

Ce1ls were incubated with 3 nM TNBS under various conditions, washed and then lysed either by French pressure ce11 treatrnent, or by conversion to protoplasts followed by osmotic lysis. The lysates were then centrifuged (105r000 x E, 60 minutes) and the absorbance at 410 nm of supernatant protein rvas determined as described in the text. This value was compared with that of a previously isolated cytoplasmic protein fraction that was labelled with TNBS under the same conditions. The A+tO of the cytoplasmic protein fraction of ce11s not labelled with TNBS was 0.010 and rvas subtracted from all readings to yield the above values. 50. nedium and lysed oslnotically by dilution into buffer lacking sucrose; phase contra.st microscopy shor.¿ed that no intact protoplasts remained. The lysate v\ras centrifuged, the degree of label1ing of supernatant (cytoplasmic) proteins determíned as before, and compared with the extent of labe11ing of a c1'toplasniic protein fraction isolated by this latter procedure and then trinitrophenylated. Less than 3eo of cytoplasrnic protein was labe11ed by TNBS in intact cells (Table 3-3), suggesting that at 4oC there is very 1itt1e, if ãîy, penetration of TNBS into the ce1ls.

11 Modification of PE b TNBS at 4OC

32 Suspensions of ce11s (5n1) uniformly 1abe1led with P were incubated with SnM TNBS at 4oC as previously described. Samples (0.5m1) were withdrawn at intervals and the reaction stopped by addition of 70077 of ice-cold 30% (w/v ) trichloroacetic acid and 25 yI of 5mg/n1 bovine serum albunin (BSA). After 5 minutes on ice, the precipitate was collected by centrifugation, taken up in 1nl of 0.1. À{ KClr 0.1 N HCI and then lipid extracted as described in Chapter 2. The PE and trinitrophenyl-PE spots Írere separated by two-dimensio;ra1 thin Layer chromatography and quantitated as described in Chapter 2. PE and trinitrophenyl-PE were well separated by this techníque, their respective R, values being 0.54 and 0.61 for the first dimension and 0.33 and 0.88 for the second dimension. The chenical nodification was fo1lowecl by the loss of radioactivity from the PE spot and its appearance in the trinitrophenyl-PE spot. It can be seen in Figure 3-6 that the rea-ction is virtually complete after 3 hours and a maximum of 92% of FIGURE 3-6

MODIFICATION OF CELLUTAR PHOSPHATIDYLETFIANOLA}4INE BY TNBS. 32p-1abe11ed ce11s urere incubated with 3 nM TNBS at 4oC. Samples viere withdrawn at the indicated times, the reaction stopped, and the % of the total amount of PE that had been trinitrophenylated was determined as described in the text. 7. PE MODIFIED o o(,r o ô

{ 3 m

l\'

(.,

¡ 51.

the total ce1lu1ar PE is tri.nitrophenyla.tecl '

To ensure that there is sufficient TNBS to react rvith all available PE, 32p^1abe11ed ce11s l{ere incubated lvith various concentrations of TNBS for 4 hours at 4oC ' It can be seen from Figure 3-7 that the extent of PE 1abel1ing increases with TNBS concentration until a saturation point is reached at approximately 2mM TNBS. The maximum amount of PE that could be nodified, even in the presence of 6mlt4 TNBS was 93%.

When ce11s lvere permeabilized with 7% (v/v) toluene, which would render PE on both sides of the rnembrane accessible to TNBS at 4oC, 99. ieo of. cellular PE was labelled in the presence of 3mM TNBS after 4 hours at 4oC (result not shown).

72. Phospholipase C Digestion of Intact Cel1s

As nentioned previousll, experiments rvith TNBS weTe performed on cells rather than protoplasts owing to the f.act that protoplasts were not stable under the conditions required for TNBS nodification. The unlil

MODIFICATION OF CELLULAR PE BY TNBS AT DIFFERENT

CONCENTRATIONS. Aliquots of 32p-label1ed ce11s rqere incubated with different amounts of TNBS at 4oC. After 4 hours, the reaction was stopped and the % of the total amount of PE that had been nodified was deternined as described in the text. 7" PE MOD IF IED (¡ o o o o

l\) o Tz tþ \ J¿ (, o

I ¡¡ À, o =

(Jt

o. o FIGURE 3- 8

THE EFFECT OF PHOSPHOLIPASE C ON TI]E PIIOSPHOLIPIDS OF ]NTACT CELLS OF B. AMYLOLIQUEFACIENS. Aliquots (1 m1) of ce1ls, suspended in 50 nM Tris-

HCl, 0. 25 mM CaCTrt 0.25 mM MgClr, 0.1 rnM ZtClZ (pH 7 .3) , and containing approximately I00 /g of phospholipid, hrere incubated with 30 I.U. of phospholipase C (8. cereus at 37oC. The percentage of the total amount of cardiolipin (o), phosphatidylglycerol (o) and phosphatidylethanolamine (r) degraded was determineã as described in the legend to Figure 3-2, 7. PHOSPHO L tP tD HYDROLYSED (¡r o o o o

J 3 (, m o T

o, =z o o o

o\o o I 52.

C D I SCUSS ION

The major phospholipids extracted f::orn B' anyloliquefaciens are CLP, PG and PE. Since the mesosomal f.taction represents only a small propor:tion of the total and has the same phospholipid composition as the cytoplasmic membrane (Greenawalt ancl ÌVhiteside, 797 5) , it nay be assuined that these phospholipids are the predominant conponents of the cytoplasmic rnembrane. The results are sinilar to that reported for other bacilli; these three phospholipids (rvhich make up more than 90% of the total phospholipids in B. am 1o1i uefac iens were also found to be the rnajor phospholipids of B: subtilis l-68 (Bishop et a1., 1967), !_._ subtilis lt{arbur g (0p den l(amp et q!. , 197 2) , B. megaterium (0p den Kanp et aI., 1965) and B. stearothernophilus 865 and Z1-B4 (0o and Lee, I972, Card et a1., 1969). The relative proportions of the phospholipids of both B. subtilis Marburg and B_. megater ium (0p den Karnp et ãI., t972 and 1965) were narkedly affected by growth conditions.

The increase in CLP during protoplast formation in B. amyloliquefaciens, tvhich also occurs to a lesser extent in B. subtilis (Op den Kamp et al., 7972) is probably due to a conversion of PG to CLP. This has been sholvn to occur in E. coli (Hirschberg and Kennedy, 1972). If this is the case, and one considers the distribution of these two phospholipids in the protoplast rnernbrane (as will be discussed later), then a transnembrane rearrangenent of phospholipid nay be occurring during protoplast formation in B. =ry!1i_quefaciens. However, as the distribution of these tr,,'o phospholipids in the membrane of whole cells is not c1ear, to definite conclusions can be 53. made.

Studies with phospholipases suggested that in the protoplast membrane, there was an asynmetric distribution of total phospholipid and individual phospholipid species. More than twice as rnuch phospholipid exists in the outer compared rvith the inner monolayer. It has previously been shown in this laboratory (I4cMurchie, 7977) that the membrane protein particles of this organism nake up approximately 60% of the membrane by weight and are highly asymrnetrically distributed, as revealed by f.reeze fracture electron microscopy studies on protoplasts. The inner monolayer contained protein particles r.vith an average diameter of 120R at a density of 2740 t 224 partictes/yZ. In the outer monolayer their average diameter was 175R and they r,üere present at a density of 338 t 74 ) particle sþ". Calculations then showed that on a volume basis (assuning spherical particles) there was approximately three tines more protein associated ivith the inner monolayer. This would necessitate a highly asymmetric distribution of membrane 1ipid, and hence the observed overall phospholipid distribution is not unexpected.

The conclusi-on that the amount of each phospholipid species hydrolysed by the phospholipases in protoplasts reflects the amount of that phospholipid in the outer haif of tlre nembrane, depends on the assumption that the enz)rynes could not penetrate the protoplasts. This is unlikely since no lysis of protoplasts occurred and they did not leak the ribo.nuclease inhibitor rvhich is of smaller molecular weight than the phosphoLipases. 54.

Although the tirne course of hydrolysis of protoplast phospholipids suggested that the reaction with phospholipases had proceeded to completion, it lras possibl e that some phospholipid in tire outer half of the bilayer may have been protected perhaps by shielding by proteins. This possibility was eliminated for PG and PE since exposure of the inner mono- Iayer, by cold-shock permeabl-l-ization of protoplasts resulted in almost cornplete degradation of these two phospholipids. All ce1lu1ar PE could also be labelled by TNBS in toluene permeabil ized cel1s . Horvever a smal1 proportiorr of the CLP was not lrydrolysed by the enzymes. This nay have been due to a smal1 fraction of protoplasts that escaped cold shocking, or rapid

Tesealing of some protoplasts after perneabi-Lization (only 85% of total intracellular inhibitor is released (Paton, 1975)). Shielding of phospholipiá by proteins (rvhich has been observed

by Barsukov et a1 (L97 6) for Micrococcus 1 ys ode ikt icus ') 1S also a possible explanation. Since the localization of this protected fraction of phospholipid is not known, the conclusion that 30% of the CLP is externally oriented nay possibly be a slight underestimate.

The interpretation of results in terms of lipid aslanmetr¡ that involve accessibility of outer monolayer phospholipids to enzynres or chemical reagents, also depends on the at'sence of transbiLayer lipid movement (flip-f1op) during incubation.

The rate of phosphol ipid f lip-f 1op in lipid bilayers l\¡as initially thought to be extremely sma11. Measurements of the rates of decay of radioisotopic phospholipid asymrnetries in artif.icial phospholipicl vesicles (Rothrnan and Dalvidowicz, 55.

1975; Johnson et aL, 1975) and influenza virions (Rothman et a!., L976; Tsai and Lenard, 1'975; Lenard and Rothman, 1976) showed that flip-flop occurred rvith a half time of greater than 10 days.

Rates of flip-flop very much faster than these have recently been r.epor'ted, however, for various membranes under certain conditions in rvhich the equilibriurn distribution of phospholipids is disturbed. De Kruijff and Baken (l-978) treated pure sonicated PC vesicles with phospholipase D, con- verting aIL externally oriented PC to phosphatidic acid. A transmembrane rearrangement of both PC and phosphatidic acid then occurred, the half time of flip-f1op of both phospholipids being approxirnately 30 ninutes. De Kruijff et a1 . (l-978) have also reported that the rate of PC flip-flop in sonicated lipid vesicles was enhanced by at least two orders of magnitude by the incorporation of glycophorin in the bilayer. Although the rapid rates of flip-flop in these highly curved artificial vesicles can be entirely accounted for by consid- erations of thernodynamics, interaction free energy and rnolecular geometry (Israelachvili et a1.., L977) the possibility that glycophorin was catalytically inportant in the latter example can¡rot be elininated at present. Indeed there is evidence to suggest thai: rnembrane protein rnay be directly involved in the process of flip-flop in natural membranes, which aïe generally not as highly curved. Bevers et aL. (7977) reported that the total amount of PG that was accessible to pig pancreas phospholipas. Az in Acholeplasma laidlaivii was narkedly reduced at lorver incubation temperatuTes, even though the membrane was sti11 in the liquid-crystalline state. 56.

This raised the possibility that a ternperature dependent protein was catalysing the movement of PG from the inner to the outer monolayer in response to attack by the pho spho - lipase. Rothnan and Kennedy (7977b) showed that PE was initially synthesized on the cytoplasmic side of the cytoplasmic membrane bilayer in grorr'ing cel1s of B. rnegater iuln and was then translocated to the outer monolayer with a half time of only 3 minutes. This rate of transmembrane movement r¡as nearly five orders of rnagnitude greater than that shown for PE in artificial lipotot"s at equilibriun (Roseman et aI., 7975) and Rothrnan and Kennedy suggested that a membrane protein night catalyse this process. They also proposed that the translocation process was reversible, âS the complete equilibration of the specific radioactivities of the internal and external pools of PE, observed in pulse label1ed ce11s, could not be explained by an irreversible outward movement of PE. They point out that this depends on the assumption that the translocation mechanism cannot distinguish newly synthesized PE from old PE.

In animal ce11s phospholipid synthesis occurs primarily on tlre endoplasmic reticulum (lvlcMurray and Magee, I972; Gatt and Barenholz, 7973; Van den Bosch, 1974). Interestingly Van den BesseLaar et al. (197i ) have shown that although all PC of rat liver microsomes (which has been shown to be symmetri- ca1ly ilistributed (Nilsson and Da11ner, L977) ) was accessible to a PC-exchange protein, only 60% of the PC in vesicles prepared by sonication of microsonal lipids r+'as exchangeable (see Footnote, p57). Zilversnit and llughes (7977) reported that in intact rat liver microsomes 85eo'95% of aLI phospho- 57. lipids were accessible to phospholipid exchange proteins within 1*2 hours. These latter two studies are of particular importance because rapid transbilayer movement of phospholipids occurred even though the net distribution of phospholipids had nct been disturbed. I{oderate rates of flip-flop of PC have also been reported for membranes that are not capable of growth. Half tirnes for loss of radioisotopic PC asymmetry of only a few hours have been reported for whole erythrocytes (Renooij et al., 1,976) and erythrocyte ghosts (81oj and Zilversmit, 1976).

It therefore is possible that flip-flop may occur quite rapidly in some natural membranes, even in the absence of bilayer perturbation (assuming that the phospholipid exchange proteins do not somehow disturb the menbrane). Great care must therefore be taken when drawing conclusions about the phospholipid distribution in nembranes from studies on the accessibility of externally oriented phospholipids to phospholipases, chenical Teagents or phospholipid exêhange proteins.

Despite the above possibilities, some conclusions can be drawn fron the present study. The asymmetric distribution of PE in the membrane of B. arnyloliquefaciens was confirmed by TNBS 1abe1ling studies. These experiments were perforned at

Footnote: If phospholipids of artificial vesicle bilayers h¡ere randornly distributed, 50% of the PC rvould be expected to be e>cchangeable (assurning the absence of f 1ip-ffoþ). Never- theless, the accessibility of 60% of the PC to the exchange protein is not surprising since mixtures.of phospholipids spon taneously form asymnetric bilayers in these highly curved vesicles (Berden et a1. , 7975; Bergelson and Barsukov, 1977). 58.

4oC at which temperature the cytoplasmic membrane of this organism is in the solid state, as indicated by differential scanning calorinetry (Mcl4urchie, 1,977). This elirninates the possibility that PE flip-flop (which would be expected to require a fluid bilayer) occurs during nodification with TNBS. Since the amount of PE label1ed by TNBS under conditions in which there was 1ittle or no penetration of the reagent into the cells, is identical to the amount hydrolysed by phospholipases in intact protoplasts, it seems reasonable to conclude that PE flip-f1op does not occur during phospholipase treatrnent. However, although the phospholipase results also suggest the existence of asymmetric distributiors of CLP and PG, the possibility that flip-flop of these phospholipids occurs during -enzyrhic digestion cannot be- eliminated.

Interestingly, Van den Besselaar et a1. (1977 ) reported that although all PC of rat liver microsomes hras rapidly accessible to a PC exchange protein as discussed previously, only 80% could be hydrolysed by phospholipase A, and the remaining 20%

was then not rapidly exchangeable. To explain this they . suggested that hydrolysis of a large proportion of membrane phospholipids nay result in a change in the membrane structure with a concommitant loss of the facility of presumed protein- nediated transmembrane movement of phospholipids. The apparent lack of PE flip-f1op in B. amyloliquefaciens during phospholipase digestion might possibly be explained by such a theory.

Since the completion of this work, Bishop et a1. (7977) reported a similar susceptibility of approxirnately 90% of PE to phospholipase C digestion in intact B. subtilis protoplasts. 59.

How ever only 60% o f the PE could be 1abe1led with TNBS. To acc ount for this d iscrepancy, they suggested that 30% of the

.': I .. ttex ternal" PE was the result of flip-flop caused by phospholip ase treatment, No direct evidence for this proposal Ì\Ias pre sented and it i s conceivable that the TNBS reaction had not gone to comple tion under their conditions. Nevertheless, the overall conclu sion was that at least 60% of the PE in the B. subtilis membra ne r^ras in the outer monolayet. Barsul

Rothnan and Kennedy (I977a); this phospholipid being 33% externally and 67% internally oriented, as shown be TNBS labe1- ling studies. This PE distribution differs markedly from that reported in the present work for B. anyloliquefaciens.

Phospholipid asymmetry is a widespread phenomenon and asymmetric phospholipid distributions have also been reported for viruses (Fong et a1., L976; Rothman et aI., 1976; Tsai and Lenard, 1975), bacteriophages (Schafer et al. , L974) and a number of animal cel1 membranes (Renooij et ãL,,7974; Bloj and ZiIver snit , 797 6; Chap .et al . , 1977; Va1e, 7977; Nilsson and llallner, 7977; Sandra and Pagano, 1978); the nost detailed study being performed on the human erythrocyte (Bretscher,1972:' Whiteley and Berg, 7974; Marinetti and Love, L976; Verkleij et aI., L973). Some of the above studies, however, may be in doubt as the authors did not consider the possibility that fl.ip-f1op 60. may be occurring during the course of their experiments.

Although. pþospholipid asymmetry appears to occur in many biological membranes, its functional significance is not c1ear. One possibility is that phosphlipid asymmetry, in conjunction with differences in the fatty acid composition of phospholipid classes, might inpart different fluidities in the two monolayers. Sandra and Pagano (1978) reported that in the plasma membrane of mouse LM ce1ls, there r,trere no major differences in the transbilayer distribution of fatty acyl chains within a phospholipid class. However, differences in the acyl chains betv¡een phospholipid classes were detected, and since there was an asymmetric distribution of the phospholipid polar headgroups, this resulted in an enrichment of unsaturated fatty acids on the cytoplasnic side of the bilayer. Patzer et a1. (1978) showed that in vesicular stomatitis virus, amino- phospholipids contained almost all of the polyunsaturated fatty acids of the membrane, and these phospholipids Ì{ere greatly enriched in the inner monolayer. However, in addition to this PE derived from the inner nonolayer of this virus contained a higher proportion of unsaturated f.atty acids than PE derived from the outer monolayer (Fong and Brown, 1978). Fatty acyl chain asymmetry a1one, however, does not irnply that there is a "fluidity asymmetry" as the nature of the polar headgroup has been shown to greatll' affect the melting temperatures of phospholipids (Ladbrooke and Chapman, 1969). Interestingly, McMurchie (7977) produced tentatir¡e evidence (based on ESR studies) that trvo lipid compartments of different fluidity exist in the cytoplasmic membrane of B. anyloliquefaciens, but, the existence and localization of these 61. compartments was not shown by independent means.

Another possibi lity is that membra ne proteins, which are all asymnetrically d istributed (Rothman and Lenard, 1977), may require specific 1ip ids for activity. Although only a smal1 number of phospholip id nolecules may be required for each protein mo1ecu1e, in B. anyloliquefaciens, protein comprises approximately 60eo of the nembrane (lr{cMurchie, 7977), and hence a considerable proportion of membrane lipid rnay be in close association with protein particles. A number of mernbrane bound enzymes have been shown to require specific lipids for maximum activity (Fourcans and Jain, 1.974; Roelofsen and Schatzmann, 1,977); the lipid headgroup and the f.atty acid ccjmposition are both important (Vik and Capaldi, L977).

Charge separations bêtween the two sides of the nenbrane bilayer produced by asymmetric distributions of particular phospholipids may also be important. The asymmetric distribution of anionic phospholipids has been used to explain the effects of some membrane active drugs on the shape of eryrhro- cytes (Sheetz and Singer , L97 4) . A higher proportion of negative charge on the cytoplasnic side may also facilitate interaction with extrinsic membrane proteins (Bergelson and Barsukov, 1977),

It is also possible that regions of acute curvature in membranes (e.g. the mitochondrial cristae) are produced by areas of localized lipid asymmetry that give the inner and outer monolayers different surface tensions. Some rnechanism for liniting lateral diffusion of lipids would be required to stabiLíze these regions and interaction r^¡ith membrane protein 62. may account for this.

One specific biological role for phospholipid asymnetry has, however, been reported. Zwaal 9t 31. (7977 ) have shown that negatively charged phospholipids such as phosphatidylserine (PS) are catal-ytically important in the process of blood coagulation. Hence the asymrnetric distribution of phospholipids, particular1-y PS, in the platelet plasma membrane (Chap et ãI., 7977) nay contribute to maintaining the balance between regulation of haemostasis and avoidance of thrombosis. A specific biological purpose for phospholipid asymmetry lnas yet to be found for other membranes.

The view that phospholipid asymnetry serves no b io log ical

purpose has been put forward by Rothnan and his col 1 eague s (Rothrnan and Kennedy, 1977 a; Rothnan and Lenard, 19 77). They have shown (Rothrnan and Kennedy, 1977b) that in B. megater ium,- PE is synthes ized on the cytoplasrnic side of the membrane and is then translocated to the outer monolayet. They suggest that if this rvere true for all phospholipids, then the distribution of each species in the nenbrane rvould presumably reflect its relative net rates of synthesis and translocation. Unless the net rate of translocation was precisely half that of synthesis, art asymmetric menbrane would always result.

It is possible that the net rates of synthesis and translocation of a given phospholipid species are deliberately controlled to achieve a particular asymmetry. It is also poss' ib1e, however, that the rates are essential-Ly fortuitous and the asynrnetry equally without fundamental significance as suggested by these workers. This latter proposal is supported 63. by the present finding that the PE distribution in the c/to= plasnic membrane of B, o1i uÞfaciens differs markedly fron ìm'egateiiurn that found in" 81.' (Rothnan and Kenned y, 1,977a) , indicating that there is no over-riding need for a particular orientation of PE in bacterial membranes. CHAPTER FOUR

STUDIES ON THE FATTY ACID COT{POSITION OF THE MEMBRANE OF B. AMYLOLIQUEFACIENS AND ]TS RELATION TO THE OCCURRENCE OF COLD SHOCK 64.

A INTRODUCTION

Informatigl,,g.n the f.atty acids of the membrane of B. arnyloliquefaciens was important to an understanding of the mechanism of protein secretion. Knou¡ledge of the identity of the fatty acids of the nenbrane r^ras of particular irnportance since itwas discovered, âs will be discussed in Chapter 5, that fatty acid synthesis is required for protein secretion in this organism. The fatty acid composition of the membrane has been shown to affect extracellular enzyme secretion in E. coli (Kirnura and Izui, 797 6) and B. caldolyticus (Lauwers and

Heinen, 1973) .

Another reason for studying the latty acids was that alteration to the fluidity of the mernbrane may overcome the effects of cold shock. This phenomenon, which occurs when ce11s of B. amyloliquefaciens are rapidly chilled (Sneaton and El1iott, L967), results in a breakdown of the permeability barrier of the cell, and loss of the ability to synthesize proteins. It has been shcrn¡n by Mclnnes (1974) that in order to prepare polysomes from this organism (rvhich would be irnportant for in vitro studies on the synthes is of extracellular enzyrnes ) it I^Ias necessary to rapidly chill cel1s to prevent run-off of ribosomes. How- ever, this resulted in cold shock and in vitrc translation systems derived from such cel1s were inactive.

The cold shock phenomenon is not confined to B. amyloliquefaciens and has also been reported in E. gg_!i (Leder, L972;

Haest s! al. , 1,972; Meynel-1, 1958; Sato and Takahashi, 1968), Strep tomyces hydrogenans (Ring, 1965), Serratia marcescens 65.

(strange and Ness, 1963), Aerobacter aero enes (Strange and Dark, 7962), Salnonella t inurium (Gorri1l and McNei11, 1960), pseudomonas aeruginosa (Farre11 and Rose, 7967;lvlacKelvie et ãI. , r_e68 ) , Pseudomonas fluorescens (Sato and Takahashi, l-969) and Baci subtilis (Sato and Takahashi, 1969; Henneberry and Freese, 7973). The principal manifestations of the cold shock phenomenon in all these organÍsms were increases in the permeability of the ce11s and loss of viability.

It has been suggested by Haest et al' (L972) and Leder (L972) that the cold shock phenomenon in E. coli was caused by solidification of the lipids of the cytoplasmic membrane. If this was the cause cf the phenomenon observed in B. arnylolique- fac iens then alterations to the fatty acid composition might be expected to alter the temperature at which the nembrane lipid solidified, and hence cold shock might be avoided, thereby fascilitating studies on the in vitro synthesis of extracellular enzymes.

The experiments described in this chapter were aimed initially at deternining the fatty acid composition of the rnembrane of B. amyloliquefaciens, and then altering this composition by changes in growth temperature. Subsequent experiments were aimed at further^ investigating the cold shock phenomenon and. testing the effects of the above alterations to the f.atty acid composition on its occurrence. 66.

B RESULTS

1 Fatty Acid Comp osition of Cells of B. amyloliquefaciens

It has been shown by Bishop 9t al., (1967) that essentially al1 lipid of B. suÞti1is was associated with the rnembraTLe. For this ïeason the fatty acid composition of cel1s, rather than isolated membranes of B. amyloliquefaciens was deternined and was assumed to reflect the f.atty acid composition of the cytoplasrnic membrane .

Cel1s were grown at 30oC and lipid extracted. The lipids were saponified and the f.atty acids converted to methyl esters which h¡ere analysed by gas-liquid chromatography as described in chapter 2, The results are included in Table 4-L. The most abundant fatty acids ü¡ere.normal C16:0 (1'2% of total fatty acids by weight), i.so CL4:0 (7 .Seo) , isoand anteiso C15:0 (43 '5% collectively), isocl6:0 (I2.8%) and iso and anteiso c17:0 (20.4% collectively) . There were smaller amounts of the straight chain saturated fatty acids norrnal C14:0 (0.9%), normel C15:0 (1..7e"), normal CI7:0 (7.2e") and normal c18:0 (0.4e0), as well as trace amounts of thc unsaturated acids ncrmal C18:1 (0 .2%) , iso cl6:1 (0.l-%) and iso and anteiso c77 :I (0.t% collectively) . Branched chain saturated f.atty acids collectively represented g4.0% of total fatty acids, rvhile straight chain saturated f.atly acids and unsaturated fatty acids replesented 15.6% and 0.4% respectively.

2 Fatty Ä.ci d Analysis of Ce11s Grown at Different

Temp er a tur e s

A major ain of this study of the fatty acids of L amvlo- TABLE 4-L

FATTY ACID COMPOSITION OF CELLS GROWN AT DIFFERENT

TEMPERATURES

FATTY ACID GROWTH TEMPERATURE

METHYL ESTERS 37"C 300c 250C 20" c

i-C14:0 4.4 7.3 8.3 8.7 n-C14:0 1.0 0.9 0.1 0.2

i+a-C15:0* 39.6 43.5 48. s 58.0 n-C15:0 0.7 1.1 0.3 0.1 i-C16:0 14.1 12 .8 15.9 tL.2 n-C16:0 L3 .3 12.0 4.s 0.9 i+a-C17:0* 23.8 20.4 19.8 11.5

n-C17:0 0.5 L.7, 0.7 0

n-C18:1 0.4 0.2 0 0

n-C18:0 2.2 0.4 0 0

i-C16:1 0 0.1 1.1 4.3

i+a-C17:1* 0 0.1 0.8 5.1

Fatty acid rnethyl esters were analysed as described in Chapter 2. Values are expressed as a % of total fatty acids. Fatty acid methyl esters are denoted Cx:y where x = no. of carbon atoms and y = no. of double bonds; n, i and a denote normal, iso and anteíso latty acids respectively. *Some iso and anteiso compounds weTe not sufficiently separated to allow indivj.dual quantitation. 67. liquefaciens was to see whether the f,atty acid composition and hence the nenbrane fluidity could be altered, with a view to preventing:,the;.occurrence of cold shock. Alteration of the growth tenperature has previously been shorvn to affect the fatty acid composition of other bacilli, notably B. cereus (Kaneda, 7972) and B. stearo thermophilus (McElhaney and Souza, 1,976) and consequently the f.atty acids from cel1s of B. amyloliquefaciens grown at different temperatures Ì\Iere analysed.

Ce11s were grohrn from spores at 37oC, 50oC , zSoC or 20oC and the lipids were extracted when cell growth had reached late- logarithmic phase (4600 = 3.6). Cell cultures grown at these temperatures took 72, 18, 34 and 65 hours respectively, to reach the required cell density. The dry weights of the cells grov/n at the different tempeTatures 'h/ere the Same when the ce11s h¡eïe harvested at the same absorbance value. The f.atty acid rnethyl esters derived from the extracted lipids hlere analysed by gas liquid chromatography and the results are Shown in Table 4-L. When the growth temperature was lowered from 37oC to 25oC, the proportion of normal saturated fatty acids decreased from 17,7% to 5.7% while the proportion of saturated branched-chain fatty acids increased from 81.5% to 92.9%, hlo unsaturated branched-chain latty acids l^Iere detected in ce11s grown at 37oC, but the mono:unsatuTated norrnal C1B:1 acid was present in small a,nounts (0.4%). However, when the growth temperature rvas lowered to 25oC, this C18:1 acid could no longer be detected, but the monounsaturated branched- chain fatty acids iso C16:1 and iso and anteiso C77:l- now appeared (trace amounts were detected in 3OoC ce11s), compris- 68. ing L.geo of total cellular f,atty acids. Further reduction in the growth temperature to 2OoC resulted in a further decrease of normal saturated f.atty acids from 5,1,% to 7,2% and a marked increase in the monounsaturated branched-chain fatty acids (1,9% to 9,4e"). The proportion of saturated branched-chain fatty acids decreased slightly from 92.geo to 89.4eo, presunably as a result of desaturation of the branched-chain acids containing 16 and 77 carbon atoms.

3 Cold Shock of Ce1ls Grown at 30oC

Alteration of the growth temperature has therefore caused a narked change in the fatty acid cornposition of the menbrane of B. amyloliquefaciens . Before investigating the effects of this alteration on the occurrence of cold shock, aspects of the phenornenon were studied in cel1s grown at the normal ternperature (3OoC).

In previous studies of the cold shock phenomenon in this organism, release of intracell-u1ar ribonuclease inhibitor was employed as an assay for the occurrence of cold shock (Srneaton and E11iott, 1967). A more convenient alternative procedure was used in the present study; loss of ability of cel1s to secrete extracellular protease following rapid cooling was examined.

Cultures of ce11s grown at 3OoC were washed, resuspended in fresh nediun and cooled (rapidly or slowly) to various temperatures. The ce11 suspensions were then shaken at 3OoC and after 5 nin re-equilibration time, the rate of protease secretion over the following 30 nin was measured as described 69. in Chapter 2,

Protease production by washed cells hlas linear for 30 nin and the rates of protease secretion (expressed as a percentage of that of ce1ls which were not cooled ) were plotted as a function of the temperature to which the ce1ls were cooled (Figure 4-1)

When cel1s were rapidly cooled from 3OoC to 1oC they completely lost the ability to synthesize protease. The require- ments for this cold shock effect were rather precise. The critical temperature zorle through which the ce1ls must be cooled to obtain the effect is about 16oC-11oC and as pïeviously observed (Sneaton and E11iott, 1967) rapid chilling was essential for this phenomenon. When cell suspensions ü¡ere slowIy cooled from 5OoC to as low as 1oC subsequent protease secretion after return to 30oC was normal (Figure 4-1).

When cel1 viability was measured, âs described in Chapter Z, as an assay for the occurrence of cold shock, similar results to those found for protease secretion were obtained (Figure 4-2). Cel1s that hlere rapidly cooled from 5OoC to 1oC completely lost viability, the critical temperature range measured in this way being 1,7oC-15oC. When cel1s were slowly cooled through this ternperature range, the viability loss was less than 25% (Figure 4-2).

The cold shock effect is not dependent on the nagnitude of the temperature decrease, nor on the starting temperature, (pro- vided it is above the critical zone). Cells slowly cooled to lgoc from 3goc and then rapidly cooled to temperatures belorv FIGURE 4-T

THE EFFECT OF COOLING RATE ON SUBSEQUENT PROTEASE

SECRETION BY CELLS COOLED TO VARIOUS TEMPERATURES. Cells of B. amyloliquefaciens grown at 3OoC were washed and resuspended in WCSM. They l^/ere then rapidly (o), or slowly (o) cooled to the indicated temperatures as described in Chapter 2. Aliquots of ce11s (20 nl) were returned. to a 30oC shaking rvater bath and after 5 rnin equilibration, the rate of protease secretion was measured over the following 30 min as described in Chapter 2. The rate of protease secretion is expressed as a % of that of cells that were not cooled. 7" PRoTEASE SECRETION o o o o tsà 8t

6 o trl{ p.

(,r o.o oÀt o

t Or

o..¡ FIGURE 4.2

THE EFFECT OF COOLING RATE ON THE VIABILITY OF CELLS

COOLED TO VARIOUS TEMPERATURES. Ce11s of B. anyloliquefaciens grown at 3OoC were washed and resuspended in WCSM and then sheared in a Sorvall Onnimix for 3 min at 18r 000 r.p.Í1. Aliquots u¡ere then rapidly (O), or slowly (O) cooled and their viability deternined as described in Chapter 2. The viability is expressed as a % of. the viability of ce11s that were not cooled. 40 o\

0 o 5 r0 15 20 25 30 TEMP. (oc ) 70.

1soc were completely inactive (result not shown).

The critical temperature range deterrnined here is soÍìêr what broader than the 16oC-14oC range reported by Smeaton and Elliott (7967 ) for this organism when rneasuring the selective release of ribonuclease inhibitor. Ilowever, measurement of cold shock by release of ribonuclease inhibitor in the present work gave 1,7oC-13oC as the critical zone (result not shown).

4 The Effect of Growth Temperature on the Critical Temperature Zone for Cold Shock

Cel1s were grown from a spore inoculum at SZoC, SOoC , ZS0C and 20oc and harvested when the Aooo of the culture v/as 3.6 in each case, corresponding to the end of the logarithrnic phase of growth. The effect of rapid cooling on viability or the ability to subsequently secrete protease at 3OoC was measured in cel1s grown at these tenperatures, âS described in chapter 2. (cel1s grown at these different temperatures, when resuspended in WCSM 1ow aa., synthesized protease linearly for at least s0 min and at conparable rates such that the final level of protease after 30 min was about 50 units/ml. ) From Figure 4-3 it is clear that growing cel1s at temperatures lower than 3Ooc has a d.rarnatic effect on the critical temperature zone for the cold shock phenomenon, as measured by protease secretion. cerls grown at zooc can be rapidly chilled to temperatures as 1ow as 7oc with no i1l effect on the ce11sr ability to secrete, while chilling to 1oC stil1 results in a subsequent 50% rate of protease secretion, lVith cel1s grown at 25oc, somewhat intermediate results were obtained for protease secretion, while with those grohrn at 37oC there was only a slight shift in the critical FIGURE 4-3

THE EFFECT OF GROWTH TEN{PERATURE ON THE LOSS OF ABILITY

TO SECRETE PROTEASE AFTER RAPID COOLING TO VARIOUS :

TEMPERATURES. Cells of B. any1o1 iquefaciens grown at 37oc (o), SgoC (o), ZsoC (r) or z6oc (o) h¡eïe washed and resuspended in WCSM and then rapidly cooled to the indicated tempera- tuie. Their ability to secrete protease (expressed as a % of that of cel1s that were not cooled) was determined as described in Chapter 2. 7" PRoTEAsE sEcRETtoN o r.¡rôo6ooooo o

lJl

o {tl 3 !

l¡

'\)o

o o

À) l¡

t, 7L. zone to higher ternperatures (Figure 4"3).

When ce11s grown at 20oC were rapidly cooled to temperatures as low as 1oC, there hlas no significant loss of viability (Figure 4-4). However, there u/as alrnost total loss of viability when cells grown at 3OoC hlere similarly cooled to 1oC (Figure 4-4).

5. The Effect of Tween-80 on the Critical Temp erature Zone for Cold Shock

The non ionic detergent Tween-80 (polyoxyethylene sorbitan mono-oleate) has been shown by McMurchie (1,977 ) to alter the response of menbrane-bound Tespiratory enzymes to changes in ternperature in membrane vesicles of B. anyloliquefaciens, but without altering the norphology of these vesicles. The detergent which perturbates the lipid bilayer, thereby promoting disorder (Raison et a1_. , 7977), would not be expected to enter the cel1s owing to the large hydrophilic sorbitan moeity (lr{cMurchie, 1977) , It hlas theref ore decided to investigate the effect of Tween-80 on the occurrence of cold shock.

Cells grown at 3OoC or 20oC r,íere washed and resuspended

in WCSlvl 1ow aa. with (or without) Tween-8O (1e" v/v) and rapidly chilled to various temperatures in WCSM low aa. with (or without) 7% (v/v) Tween-80. The occurrence of cold shock was assayed by the ability of the cells to secrete protease after return to 30oC as described in Chapter Z. The presence of the detergent caused no inhibition of protease secretion in control ce1ls, It can be seen from Figure 4-5 that for 30oC grown cells, the pTesence of Tween.80 in the cooling nediurn caused FIGURE 4.4.

THE EFFECT OF GROWTH TEMPERATURE ON THE LOSS OF VIABILITY

AFTER RAPID COOLING. Cel1s of B. amyloliquefaciens grown at 3OoC (o), or 20oC (o) were washed and resuspended in WCSM and sheared in a Sorvall Onninix (18,000 r.P.fl., 5 rnin). They r4rere then rapidly chilled to the indicated tenperature, and their viability determined as described in Chapter 2 (expressed as a % of. the viability of ce1ls that were not cooled). o o too

Þ J gõóo

40 o ox

o o 5 to 15 20 25 30 TEMP. (oc) FIGURE 4 - 5

THE EFFECT OF TWEEN.SO ON THE CRITICAL TEMPERATURE ZONE

FOR COLD SHOCK AS DETERMINED BY DECREASE IN THE RATE OF

PROTEASE SECRETION. Ce1ls grown at 50oC were washed, rêsuspended and rapidly cooled to the indicated temperature in WCSM (o) or WCS},I + 7% (v/v) Tween-80 (o). Cel1s grown at 20oC were sinilarly treated in IVCSM (r) or WCSM + 1'% (v/v) Tween-80 (O). The subsequent rate of protease secretion (expressed as a % of that of ce1ls that were not cooled) was deternined after return to 3OoC, âs described in Chapter 2. 7" PRoTEASE SECRETION ll, I o oo oo 8t

(¡l

o { m 3 ! (¡ oo ol\,

N t¡

(, o .t ., a depression of the critical temperature for cold shock of about soc. Howeyer, for 20oC grown cells, no such depression was observed.

The effect of Tween-80 observed was not due to the presence of contaminating free oleate. Separate experiments, in which oleate was added to the I^¡csM low aa. during washing and incubation of ce11s, established that ce1lular protein synthesis and protease production were significantly inhibited by concentrations of oleate as 1ow as 0.9 ¡e/n1-. Concentrations of oleate below this figure did not affect the critical ternperature for cold shock (results not shown).

C DI SCUSSION

The najor fatty acids of B. anyloliquefaciens cells glown ät 30oC were saturated branched chain Latty acids of \4,15, 16 or 77 carbon atoms which collectively rnade up 84% of the total. Although anteiso as well as iso compounds were detected for the branched chain f.atty acids of 15 and t7 carbon atoms' they 1,/eïe not sufficiently separated to allow individual quantitation. Most of the rernaining fatty acids of B. anyloliquefaciens (15.6%) IVeïe saturated straight chain fatty acids (principally normal C16:0) though trace amounts (0.4%) of monounsaturated f.atty acids weïe detected. A high proportion of branched chain fatty acids, together with a low proportion of unsaturated fatty acids appears to be a general feature for bacilli (reviewed by Kaneda (I977 ) ). Branched chain fatty acids have also been shown to be the major constituents in other gram positive organisms, including Micrococcus lysodeik- ticus and staplz]gsgrglrs (Macfarlane, 1-961; Kates, 1'964; 73.

Kaneda, 1967),

Although sma11 amounts of unsaturated fatty acids were detected in B. arnYloliq uefaciens, none were detected by Bishop et a1.. (7967 ) for B. subtilis. Small amounts have been found in B. purnilis, B. licheniformis and Þ. stearothermophilus (Shen et al., 1970) and larger arnounts (up to 27%) in B. cereus (Kaneda, 1972).

Growth tenperature was found to have a marked effect on the fatty acid composition of g. gylo1iq""f aci""s. Reduction of the growth ternperature from 37oC to 20oC resulted in a 15 fold reduction in the proportion of straight chain saturated fatty acids. There was a 1,0% increase in the proportion of branched chain saturated latty acids, ãs well as a slight decrease in the average number of carbon atoms in these acids (fron 1,5.7 to 15.2). In addition there was a Z0 fold increase in the level of unsaturation, thì-s increase being confined to monounsaturated branched chain fatty acids.

This response of B. arnyloliquefaciens to lowered growth ternperature is different from that reported for other baci11i. Kaneda (1,972) found for B. cereus that lowered growth temperature resulted in no change in the ratio of straight to branched- chain f.atty acids, but a 5 fold increase in the 1eve1 of unsaturation of both normal and branched-chain fatty acids ' McElhaney and Souza (L97 6) found that when the growth temperature of B. stearothermophilus was lowered, there was a significant increase in the ratio of branched to straight-chain fatty acids and an increase in straight-chain unsaturated fatty acids, but no unsaturated branched-chain fatty acids lrere detected. 74.

The most important finding in the present work is that the cold shock phenomenon it L amyfoliquefaciens can be avoided if cells are first grown at low temperatures. There is an apparent correlation between the critical temperature zoîe required for cold shock and the fatty acid composition of cells grown at different temperatures. The alterations in the fatty acids in response to lowered growth temperature described above would all result in a membrane with increased lipid fluidity. Correspondingly, the temperature zones for cold shock are significantly reduced. With cells grown at 2OoC compared with SQoC, the zone is shifted from 1-6oC - 11oC to soc - less than 1oC. In practice this means that although cells grofin at 30oC and instantly chilled to soc totally lose viability and ability to secrete protease, those grown at 20oC can be chilled to this temperature with almost no effect on these parameters. This finding is of essential inportance to future in vitro studies on extracellular enzyme synthesis. Cel1s grown at 2soc exhibit an intermediate fatty acid composition and an internediate critical ternperature zorLe for cold shock.

Attempts were made to further alter the fatty acid composition by growth at temperatures lower than 2OoC. Although cel1s grow at i-SoC, the latty acid composition was identical to those grown at 20oC and the critical temperature zoîe for cold shock was unaltered. Apparently there is a lirnit to which the fatty acid composition (and hence the critical cold shock temperature) can be altered by lowering growth tenperature.

That the nembrane lipid fluidity is inportant for deternining the cold shock effect was further substantiated by the finding that Tween-80, which presumably perturbates the lipid bi- 75.

layer thereby promoting disorder, lowered the critical temperature zoîe for cold shock by soc. Since the Tween-80 effect occurs rapidly and does not involve cell growth in the presence of the detergent it seems likely that its effect is on the membrane itself. Combination of growth at low temperature and Tween-80 treatment again failed to reduce the critical temperature for cold shock beyond the reduction already achieved by growth at 1ow temperatures a1one.

Conpatible with the findings of the present study are those of Farrell and Rose (1-967) who found differences in the fatty aci-d composition between mesophilic and psychrophilic pseudomonads that correlated with the different effects of rapid chilling on these two organisms. Haest et al. (797?) also reported that the critical temperature for cold shock in an unsaturated Latty acid requiring auxotroph of E. cóli was dependent upon the nature of the unsaturated fatty acid supple- ment. These workers, as well as Leder (7972) proposed that the temperature for cold shock in E. coli was that at which the hydrocarbon core solidified. They suggested that upon rapid chi11ing, the solidification gave rise to discontinuities in the membrane packing, rêsulting in the release of sma1l molecules. Studies of the thermal properties of B. anyloliquefaciens mernbranes using differential scanning calorinetry (McMurchie, Lg77) have revealed that in cel1s grown at 3OoC, the nenbrane lipids are solid below approximately 13oC. This appears to correspond to the critical cold shock temperature found in the present study for 3OoC gror^rn cells. Unfortunately, owing to the unavailability of a differential scanning calori- meter, thermal analysis of the nernbranes of cel1s grown at 76.

'different tenperatures was not possible. Nevertheless, it seems 1ike1y that solidification of membrane lipid is also the cause of the cold shock phenomenon in B, loli uefaciens. The nechanism by which this rapid solidification gives rise to permanent permeability increases can be speculated uponrbut is not understood. CHAPTER FIVE

STUDIES ON THE RELATIONSHIP BETI,VEEN L IP ID SYNTHESI S

AND PROTEIN SECRETION 78.

A INTRODUCT ION

The work described in the previous two chapters provides information on the membrane lipids of B. amyl oliquef aciens. The question of rvhether these lipids are involved in the process of extracellular enzyme Secretion was investigated in the work described in this chapter.

It has previously been reported (Yarnamoto and Lampen, lg76; Dancer and Lampen, \975) that the penicillinase of B' enzyne, 1 icheniformi s , which exists in part as a rnenbrane bound is synthesized with a phospholipid attached to its amino terminus. This phospholipoprotein remains firrnly attached to the membrane and the extracellular form of the enzyme is released by proteolytic cleavage 25 or 27 residues from the amino terminus. The purpose of the phospholipid is not understood, but one possibility is that it anchors the enzyme to the membrane. If this were the case, Phospholipid would only be associated with secreted proteins that remain bound to the rnembrane. Alternatively, Phospholipid may act as a "signa1" (see Chapter one) to trigger binding of nascent penicillinase chains to the membrane, thereby fascilitating their secretion, in which case phospholipid may be important in the secretion of all extracellular enzymes.

To investigate the role of lipid in the process of secretion in B. amyloliquefaciens, cerulenin was used' This antibiotic specifically inhibits /3-ketoacyl-acyl carrier protein synthetase (ntAgnolo 9! al., 1'973) and thereby prevents fatty acid synthesis in a wide range of organisms (onura, 7g76). The effect of inhibiting Latty acid synthesis on the 79.

secretion of o<"aflylase and protease Ì\Ias studied '

B RESUTTS

1 Inhibition of o(- Amylase and Protease Secretion by Cerulenin

When tvashed ce1l suspensions of B. amyloliquefaciens were incubated with cerulenin at a concentration of 700yï/mL, the secretion of o<-amylase (Figure 5-1) and protease (Figure 5-2) were inhibited over a 3 h period by 80% and 75% Iespec- tively. The residual enzyme secretion in the presence of cerulenin depended on protein synthesis since it was inhibited by chloramphenicol (result not shown). A separate experiment 'showed that cerulenin did not inhibit the enzyme activities under the experinental conditions used. z Inhibition of Tota 1 Protein Secretion bv Cerulenin

It rvas possible that cerulenin caused the procluction of inactive enzyme molecules rather than inhibiting secretion of protein. To test this, cells in WCSM were label1ed for 3 h with [ttt]-methionine in the presence or absence of the antibiotic; the ce11s were removed by centrifugation and the proteins secreted into the rnedium run on SDS-polyacryLamide gels and fluorographed (see Chapter 2), Plate 5-1 shows that the amount of each protein secreted by the ce1ls is greatly reduced by the presence of cerulenin.

3 Effect of Cerulejnin on- Lipid Synthes is

The incorporation of [toa]-acerate into total cellular lipid was measured oyer 3 h. In the presence of 1,00 ye/nL FIGURE 5-1

EFFECT OF CERUTENIN ON O(.AIVIYLASE SECRETION BY B.

AMYLOL IQUEFAC IENS . Cells l\tere washed and resuspended in WCSM. Aliquots (2 ¡nl) of ce11s were incubated at 50oC with (O), or without (c) :-00 rg/mL of cerulenin. At the indicated tines, 0.15 n1 samples were withdrawn, centrifuged at 4r000 x g for 5 min and the supernatants assayed for <-amylase as described in ChaPter 2. 400

300 =tr f

200

1 00

0 0 1 2 3 TIME (h) FIGURE S^Z

EFFECT OF CERULENIN ON PROTEASE SECRETION BY B.

AMYLOLIQUEFAC IENS. Ce1ls were washed and resuspended in WCSM. Aliquots (2 rnl) of cells were incubated at 30oC with (o), or without (o) 100 fg/nI of cerulenin. At the indicated times, 0.15n1 samples were withdrawn, centrifuged at 4,000 x g for 5 rnin and the supernatant assayed for protease as described in Chapter 2. 200

150 c f

100

IJJ C) t¡¡ l- o É. o- 50

0 0 1 2 3 TIME (h) PLATE 5-1

EFFECT OF CERULENIN ON EXTRACELLULAR PROTEIN SECRETION

BY B. AMYLOL IQUEFAC IENS. suspensions of cells were incubated for 3 h with absence (a) or L0 ¡ßi/mL of [ttt]-methionine in the presence (b) of L00 pg/nl of cerulenin. The suspensions were then centrifuged at 4 r 000 x g for 5 min and the proteins in the supernatant were separated by sDS- t2.5% polyacrylamide gel electrophoresis and fluorographed as described in chapter 2. The nobilities of x-amylase and protease are indicated- a b ongln Ð, ^*_

c - amylase + Ë protease + 80. of cerulenin the incorporation of [t-d- acetate over the first L5 nin was almost totally inhibited, butsubsequently recovered to approxinately 50% of the control rate (Figure 5-3). The addition of a further 700 yA/nl of cerulenin after 90 nin (arrow, Figure 5-5) caused further inhibition of [toa]-acetate incorporation, suggesting that the observed recovery was due to the drug being destroyed. It has previously been reported (Onura, 7976) that cerulenin is unstable inside anirnal cel1s.

4 Effect of Cerulenin on General Protein Synthesis

Incorporation of [tOd-phenylalanine into total trichloroacetic acid insoluble material was slightly reduced by the drug (Figure 5-4). Production of extracellular proteins collectively represents approximately 5% of total cel1u1ar protein synthesis by this organism (May, unpublished), and it is possible that the small inhibition of total protein synthesis observed in the presence of cerulenin was due to inhibition of extracellular enzyme production.

5 Effect of Cerulenin on RNA Synthesis

Incorporation of F4c]-uracil over 3 h was only slightly affected by cerulenin (Figure 5-5). These results contrast with those of Wi1le et aL. (1975) for B. subtilis, where cerulenin inhibited total protein and RNA synthesis by 50% and 90% respectively within 40 nin of addition.

6 Recovery of o<-Anylase Secretion After Removal of Cerulenin

The inhibitory effect of cerulenin on enzyme secretion FIGURE 5-3

EFFECT OF CERULENIN ON r-.1 - ACETATE INCORPORATION INTO LIP ID BY B. AMYLOLIQUEFACIENS. Suspensions of cells (5 n1) were incubated with [to. acetate (2.5/Ci/nL) at 3OoC, in the absence (O) or presence (o) of 100 tr,/nL of cerulenin. After 90 rnin incubation (arrow), a further 100 tE/mL of cerulenin was added to a portion of the cerulenin treated ce1ls (tr). At the indicated times , 0.25 ml sanples were withdrawn and the incorporation of label into ce1lu1ar lipid was deternined as described in Chapter Z. l4C.ACETATE INCORPORATION (cem x 1o-') -¡l o N cÉ, o 5 or o Ì\Ð

=¿rn

---+

lÞ

C^) FIGURE 5.4

EFFECT OF CERULENIN ON - rNCoRpoRAr roN [t-.] 'HEN'LALANTNE BY B. AMYLOL IQUEFAC IENS. Suspensions of cell s (Z ml) hrere incubated at 50oC wirh 0.25 yci/n\ of Lt-a]-phenylalanine in the absence (o) or presence (O) of t00 ¡tg/ml of cerulenin. At the indicated tirnes, 0.1 nl samples were withdrawn and the incorporation of label into trichloroacetic acid - insoluble material was determined as described in Chapter 2. lac- pHE tNcoRPoRATloN (cPM x 10-3 ) (rr e ¿ Í\¡ C^) È ct¡ o

-{¿ =m

CÐ FIGURE 5- 5

EFFECT OF CERULENIN ON - URACIL INCORPORATION BY B.

AMYLOL IQUEFAC IENS . Suspensions of ce1ls (2 rn1) were incubated at 5ooc with 2.5 yCí/nI of F-a]-uracil in the absence (o) or presence (O) of L00 7g/nL of cerulenin. At the indicated tines, 0.1 n1 samples were withdrawn and the incorporation of 1abe1 into trichloroacetic acid-insoluble material was deternined as described in. Chapter 2. 1ac - uRActL rNcoRpoRATtoN (ceur x to-3 ) ¿ ¿ á o N è ct) æ o È o ^)

¡¡ l =rt|

C.¡ 81.

was found to be reversible. Remoyal of the drug by washing the ce1ls resulted in a gradual resumption of or-amylase secr et ion (Figure 5-6).

7 Reversal of Cerulenin Inhibition of o<-Arnylase and Protease Secretion with Fatty Acids.

The inhibi-tion of o(-amylase and protease secretion by cerulenin was signíficantry reversed. by the addition of fatty acids, obtained by hydrolysis of total ce1lular lipids as described in chapter 2. rt was reported in chapter 4 that the principal ratty acids in this organism are the branched chain acids of 15, 16 and 1,7 carbon atoms and palnitic acid, which collectively make up 90% of total fatty acids. when the total fatty acid mixture and the drug were added sirnul- taneously to ce11 suspensions, the rate of "<-amylase secretion over three hours was more than twice that in the presence of cerulenin alone (Figure 5-7). The fatty acid mixture (final concentration r00ye/nL) had a slight inhibitory effect on the rate of x-amylase secretion in the absence of cerulenin. sinilar results Ìrere obtained for protease secretion (Figure 5-8), but the amount of protease secreted

in the presence of cerulenin was stinulated only by about 50eo by the addition of f.atty acids at zero tine. rn both cases, additional experiments (not shown) established that the increased secretion in the presence of fatty acids was inhibited by chlorarnphenicol. Further reversal of cerulenin inhibition was not achieved by the addition of greater amounts of fatty acid mixture; concentrations greater than 250 yg/nr caused significant FIGURE 5.6

REVERSAL OF CERULENIN INHIBITION OF O(-AMYLASE SECRETION

BY REMOVAL OF THE DRUG. Suspensions of cel1s hrere incubated at 3goc in the absence (o) or presence (o) of 100 ¡s/ml of cerulenin. Af ter t hour (arrow) , a portion (f) hlas withdrawn frorn the cerulenin treated cel1s. This was washed and resuspended in fresh WCSI,{, without cerulenin and returned to the bath. At the indicated times, samples welre withdrawn, centrifuged and the supernatant assayed for "<-anylase as described in ChaPter 2 - AMYLASE ( u /rut) I N C.¡o o o o o o

{ <.¡ + m

1\¡

C^J FIGURE 5.7

REVERSAL OF CERULENIN INHIBITION OF o(.AMYIASE SECRETION BY FATTY ACIDS. Cell supensions (2 ml) were 'incubated in the absence (closed synbols) or presence (open syrnbols) of 1'00 yg/nL of cerulenin, with (squares) or without (circles) l-00 fg/nt of B. amyloliquefaciens Latty acids. At the indícated times, 0.1-5 n1 sanples were withdrawn, centrifuged at 4,000 x g for 5 min and the supernatants assayed for o<- amylase as described in Chapter Z. AMYLASE (u/ ML ) J N C.) o o o e o o o o

lr =m

C^t FIGURE 5.8

REVERSAL OF CERULENIN INHIBITION OF PROTEASE SECRETION BY FATTY ACIDS. Ce1l suspensions (2 ml) were incubated in the absence (closed synbols) or presence (open symbols) of 1,00 yg/nL of cerulenin, with (squares) or without (circles) 100 ¡g/nl of B. amyloliquefaciens fatt y acids. At the indicated times, 0.15 nl samples were withdrawn, centrifuged at 41000 x g for 5 min and the supernatants assayed for protease as described in ChaPter 2, 175

150

ìtzs E f

100

-, 75

¡¡¡ ot-- É, o' so

0 0 1 2 3 TIME (n) 82. inhibitions of the control rates of protease and o<-amylase secretion.

The reversal of cerulenin inhibition of enzyme secretion by exogenous latty acids was not due to a reversal of the inhibition of f.atty acid synthesis by the drug. Experiments showed that the inhibition of the incorporation of [toa]-"."rare into lipid in the presence of cerulenin was unaffected by the addition of the fatty acid mixture (Figure 5-9). The time of addition of the fatty acid mixture to cerulenin-treated cel1s was, nevertheless, important since addition 60 nin after the addition of cerulenin did not cause significant reversal of the inhibition of enzyme secretion (result not shown).

Individual fatty acids added singly were also effective. Anteiso C15:0, iso C16:0, anteiso CI7:0 and palrnitate (normal C16:0) added at concentrations of 50, 100, 100 and 50¡*e/nI respectively (these concentrations were optimal) restored er'zyme secretion in the presence of cerulenin (Table 5-1). With the ex'ception of anteiso C15:0 the reversal by the in- dividuaL latty acids was of the same order as that observed for the total cel1u1ar latty acid mixture. At concentrations greater than those quoted as the optimun for each fatty acid, the rate of enzyme secretion both in the presence and absence of cerulenin was inhibited. The ce11s were particularly sensitive to anteiso C15:0 which at 1,00 Ve/nl inhibited c(-amylase and protease production by greater than 90% with or without cerulenin. Similarly, iso C16:0, anteiso C17:0 and palmitate, at concentrations of 250ye/nI, each caused approxirnately 50% inhibition of o<- arnylase and protease secretion. A FIGURE 5 - 9

EFFECT OF FATTY ACIDS ON CERULENIN INHIBITION OF FO.] - ACETATE INCORPORATION INTO LIPID. Suspensions of cells (2 nl) were labelled with -d z.s yci/nr of f acetate in the absence (closed symbols) or presence (open symbols) of 1,00 pS/nl of cerulenin, with (squares) or without (circles) 100 ,ag/nL of B. amyloliquefaciens ratty acids. At the indicated times, 0.25 nl sanples were withdrawn and the incorporation of labe1 into cellular lipid was determined as described in Chapter z. 14c- AcETATE tNcoRPoRATloN (cervl x t0-3) å qtl o-¡ (tr oo

I 3 J m

(.J TABLE 5-1

EFFECT OF PURE FATTY ACIDS ON THE SECRETION OF EXTRACELLULAR

ENZYMES BY B. AMYLOLIQUEFACIENS IN THE PRESENCE OF CERULENIN

% o-Arnylase % Protease Supp lement secretion secretion

No supplernent 22 .8 38.2

50 ugln1 a-C15:0 35 .4 39. 0 100ug/m1 i-Cl6:0 4r.2 52.0

100u E/nt a-CI7 : 0 59.0 49.1 50 uglm1 n-C16:0 39.7 43 .4

Suspensions of ce11s (1 n1) were incubated in the presence of 100 vg/mI of cerulenin. The suspensions were supplernented with individual fatty acid species (prepared as described in Chapter Z) at the indicated optimal concentrations.

After 3 h the suspensions hrere centrifuged at 41000 x g for 5 min and the supernatants assayed for cx,-amylase and protease as described in Chapter 2. The total amount of enzyme secreted is expressed as a percentage of that of control ce1ls that were incubated in the absence of cerulenin. Fatty acids are denoted Cx:y where x is the number of carbon atoms and y is the nurnber of double bonds; n, i and a denote norrnal, iso and anteiso fatty acids respectively. 83, separate experiment (not shown) revealed that these f.atty acids inhibited total cellular protein synthesis, implying a general toxic effect.

I Attenpt to Detect Forms of o(- Amylase and Protease with Attached PhosP holipid

Since lipid synthesis aPPeared necessary for protein secretion in B. amyloliquefaciens, attempts r.iere made to detect phospholipoprotei-n intermediates of o<-amylase or protease.

presence of sL,d;../ml Cells hrere grolvn in the "t F'{- inorganic phosphate, harvested and suspended in ice-co1d phosphate buffered saline containing 0.5% (v/v) Triton X405, 5mM PMSF (phenylnethylsulphonyl fluoride), 1% (w/v) sodium deoxycholate, and 1OmM orthophenanthroline. The latter two compounds are inhibitors of the penicillinase releasing protease described by Traficante and Lampen (L977). The ce1ls were lysed in a French pressure ce11 and ce11 debris hlas removed by centrifugation at 8,000 x g for 10 ininutes. The lysates were incubated with 200¡e/nL of rabbit anti-protease or rabbit anti- x-amylase gamma globulins and after 3 hours at 4oC,40 units/nl of purified protease or 55 units/nl of purified a<-amylase hlere aclcled to the respective sarnples and 1ef t overnight at 4oC. Immunoprecipitates were collected by centrifugation and washed four times with 1m1 of phosphate buffered saline containing 0.5% (v/v) Triton X405 to remove non-specifically adsorbed material. After treatment with trichloroacetic acid, they were then washed four times by suspension in 4rn1 of chloroform/ nethanoL (7:2 v/v) to lemove non-covalently attached lipid. The precipitates were finally rvashed with ethanol and then 84. diethylether, boiled in 2% (w/v) sodiun doilecylsulphate (SDS) with 10% (v/v) 2^mercaptoethanol and electrophoresed on SDS- 72.5% polyacrylamide gels, âs described in Chapter 2. The gels were dried and autoradiographed for 72 hours. Even though the sensitivity of this technique is such that as little as 50 3Zp-counts per rninute could be detected after overnight exposuïer to 32p counts could be found associated with the immunoprecipitated mater ial .

9 Test to See Whether Cerulenin Causes Intracellular Accumulation of Enzym es Normally Secreted

Cel1s were incubated with or rvithout 700¡S/nI of cerulenin for i hours in the presence of 70¡uci/m1 of L3s$-metfrionine. The ce1ls were then washed and resuspended in ice-cold phosphate buffered saline containing 0.5% (v/v) Triton X405 and 0.5% (w/v) sodium deoxycholate. The cells hrere 1ysed, centrifuged and incubated with antibodies to a<-amylase and protease, as described above. Immunoprecipitates were washed four times with l-rnl of phosphate buffered saline containing 0.5% (v/v) Triton X405, boiled in Z% (w/v) SDS with L0% (v/v) 2- rnercaptoethanol and electrophoresed on SDS-72.5% po1-yacrylarnide ge1s, which weïe fluorographed for 7 days (see Chapter 2). Accord- 55S ing to Bonner and Laskey (7974),60 counts per minute can be detected after overnight exposure. Although some cLarity has been lost in reproduction, it c.an be seen from Plate 5-2 that only trace anounts of 1abe1led c<-amylase and protease were immunoprecipitated fron the control ce11 lysates. However, even smaller amounts were precipitated from the lysates of the cerulenin treated ce11s, indicating that the drug did not cause PLATE 5-Z

SLAB GEL ELECTROPHORESIS OF ANTIBODY PRECIPITABLE

MATERIAL FROM NORNÍAL AND CERULENIN TREATED CELL LYSATES. Suspensions of cells were incubated for 3 h with 1.0 vci/nL of ["t] -methionine in the presence or absence of 100 þg/nL of cerulenin. The ce11s were then washed, lysed and treated with antibodies to o(-amylase or protease as described in the text. The immunoprecipitates were then washed, êlectrophoresed on a SDS-1,2.5% polyactylamide slab gel and fluorographed, âs described in Chapter 2. A sample of trichloroacetic acid precipitated culture supernatant is included as a marker. The rnobilities of o.-amylase and protease in this sample are indicated. a. Trichloroacetic acid precipitated culture supernatant. b. Material precipitated from normal cel1 lysate by o<-amylase antibody. c. Material precipitated from cerulenin treated ce11 lysate by x-anYlase antibodY. d. Material precipitated from normal ce11 lysate by protease antibodY. e. Material precipitated from cerulenin treated ce11 lysate by protease antibody. a bc d e ongln +

c-amylase + -l) <- protease -- 85. intracellular accumulation of immunoprecipitable material.

C DISCUSSION

The work described in this chapter has shown that cerulenin inhibits the appearance of extracellular o¿-amylase and protease in non-growing suspensions (see Chapter 2) of B. amyloliquefaciens. The effect is a general one, with appearance of all newly synthesized protein species found in the external nediun being drastically reduced by the antibiotic. The inhibition rvas specific for secreted proteins since total ce11u1ar protein synthesis, as measured by F-d phenylalanine incorporation, was only slightly inhibited' Under these conditions, cerulenin reduced the incorporation "f F4d-acetate into lipid by over 50%. Since the addition of f.atty acids partially reversed the inhibition of protein secretion, the question must now be considered whether lipid synthesis is essential for synthesis andfot secretion of extracellular proteins by this organism, as discussed below.

Inhibition of protease and o,-amylase secretion by cerulenin becomes maximal after approximately one hour, by which time the rate of f.atty acid synthesis in the presence of the drug has recovered to approximately 50% of the control rate. A similar del-ay before inhibition becomes maximal and partial recovery of f.atty acid synthesis after cerulenin treatment, has been recently reported by Fishnan et al. (1978) for penicillinase secretion by B. licheniformis (see later). It is possible that a pool of f.atty acids exists in B-'- amylol iquefaciens that perrnits secretion in the absence of fatty acid synthesis and that this pool becomes exhausted 30-60 minutes after the 86. addition of the drug. This night explai-n the delay in the onset of inhibition of secretion. The f.act that secretion of o(-amylase and protease remains inhibited after partial recovery of fatty acid synthesis is difficult to explain. One possibility is that during the first 30 minutes, when FOa]-acetate incorporation was completely inhibited, irreversible membrane damage rnay occur. This, however, seems unlikely since rvhen cerulenin rvas washed away after 60 minutes, o(-amylase secretion recovered after a further 60 minute tine lag. A second possibility is that inhibition of protein secretion is a secondary effect of the drug, not concerned with f.atty acids. The fact that addition of exogenous fatty acids partially reverses this inhibition argues against this being the complete explanation. A third possibility is that even though f.atty acid synthesis has partially recovered, a certain critical intracellular concentration of f.atty acids or derived complex lipids rnay be required to support secretion.

As mentioned above, addition of exogenous f.atty acids only partially reversed the inhibition of enzyme secretion by cerulenin. This incomplete response is worrying, but may be due to the third possibility described above, namely t'he necess- ity to build up a critical intracellular concentration of 1ipid. The effect of adding higher concentrations of f.atty acids could not be tested as they inhibited total cell.ular protein synthesis (approxinately 50qø inhibition at Z50yC/nL).

To summar ize, the results in this chapter do not unequivo- ca l1y prove the involvement of lipid in protein secretion by B. anyloliquefaciens. Nevertheless, the fact that addition 87. of fatty acids did give significant reversal of cerulenin inhibition of secïetion and the fact that cerulenin did not inhibit intracellular protein synthesis (as judged by total cel1u1ar protein synthesis) strongly supports this possibility.

At the tine this work was started, there had been no reports of cerulenin inhibiting bacterial protein secretion. However, Fishman et q!. (1978) recently reported that penicillinase secretion in B. licheniformis was inhibited by the drug, a finding that correlates well rvith the observation of Dancer and Lanpen (1975) that this enzyme hlas synthesized with a phospholipid attached it its amino terminus. In addition, inhibition by cerulenin of the secretion of enterotoxins B and Cin Staphylococcus aureus (Altenbern, I977) and levansucrase in B. subtilis (Caulfield et a1.,L976) was reported during the course of this work. The involvement of lipid in enzlme secretion was also implicated by the finding of Beacham et al. , (1976) that alkaline phosphatase secretion was inhibited in the absence of phospholipid synthesis in a glycerol-requiring auxotroph of E. co1i.

It was initially thought possible that the role of the phospholipid night be to anchor proteins such as B' 1 icheni - formis penícillinase to the membrane. If this wer.e the case' then only those proteins destined to become significantly membrane bound would have an attached phospholipid and hence represent a class of proteins, the synthesis of which would be sensitive to cerulenin. This possibility was eliminated in the present work. The secretion of B. any1ol iquefac iens o<-amylase and protease was sensitive to cerulenin, but only 88. trace amounts of these enzymes could be d etected immunologi- ca1lyr âssociated with the ce11s, Previo us rvork in this laboratory (Gould, !973) also failed to detect ce1l associated, imnunoprecipitable x-amylase or protease beyond trace amounts. No immunoprecipitable material was found associated with protoplasts (May, unpublished) and it is there fore possible that the trace amounts detected in ce1ls was asso ciated with the cell wall. Membrane bound active o<^amylase with different electrophoretic mobility fron that of ext racellular ô(-amylase, has, however, been reported by Fernandez- Rivera Rio and Arroyo-

Begovich (1975) for another strain of B. amyloliquefaciens . These workers pointed out however, that these proteins nay be the product of 2 different genes.

There are several possible explanations to account for the effects of cerulenin on bacterial protein secretion. One possibility is that the presence of the drug causes non-specific alterations to the membrane (Fishrnan et aI. (1978) shorved that cerulenin decreased the lipid to protein ratio in membranes of

B. l ichen-i:lam:Lå) . Such non-specific alterations night in some way prevent either synthesis of extracellular polypeptides or their passage across the membrane bilayer. Hotvever, such a general phenomenon rnight be expected to also affect other membrane transport processes, which in turn would cause inhibition of protein and RNA synthesis. This was not observed in B. amyloliquefaciens, but nonetheless this possibility cannot be rej ected at present.

A second possibility is that synthesis of all secreted bacterial proteins occurs with a phospholipid initíaILy attached to the amino terminus, possibly to direct the nascent polypep- 89. tide chains to the nenbrane, and cerulenin prevents synthesis of this phospholipid. Phospholipoprotein interrnediates of o(-anylase and protease were not detectecl in B. arnylol.iquefaciens, but this does not eliminate this possibility. It has previously been shown in this laboratory (Both et al. , 1972) that the synthesis and secretion of extracellular proteins in this organism are tightly coupled. If the phospholipid was removed either during or inrnediately after the secretion process, the number of molecules of phospholipoprotein intermediates existing at any one tine could be so smal1 as to make detection difficult. It is conceivable that if the role of the phospholipid is to act as a signal to trigger binding of nascent polypeptide chains to the menrbrane, it is rernoved before the chains are sufficiently completed to perrnit reaction with antibodY. B. 1 icheniformi s penicillinase (the only conf irmed phospholipoprotein so f.ar reported in bacteria (Lampen, 1978) may be unique in that the phospholipid is not removed during synthesis because of an additional function; that of anchoring the enzyme to the cytoplasmic membrane. The intportance of this anchorage in protecting the ce1l has al-teady been suggested by Lanpen (1978). The tight coupling of the processes of synthesis and secretion of extracellular proteins may also account for the failure of cerulenin to cause an accurnulation, within the cel1s, of o(-amylase and protease antibody precipitable naterial. Blockage of the secretion process may somehorv prevent the synthesis of nerv polypeptide chains. However, this observation could also be explained by rapid degradation of non secreted enzynìes.

A third consideration is that B. licheniformis penì-cil1- 90. inase is initially made with an attached phospholipid, so1e1y because of the requirement to anchor the enzyme to the membrane. Other bacterial secretory proteins nay be synthesízed without arL atlached phospholipid and only contain an amino terminal t'signal peptide" (B1obe1 and Dobberstein, 7975a) to trigger binding to the rnembrane. In this situation, cerulenin may directly inhibit B. I icheniformis penicillinase sYnthesis by preventing phospholipid attachment and indirectly inhibit synthesis of other bacterial secretory proteins by altering the general lipid content of cytoplasmic nembranes, as discussed above.

At present, there is insufficient evidence at the nolecular level to distinguish between the various possibilities.

It is of interest to note that for E. co1i, the periplasmic protein, penicillinase (Anbler and scott, 1978) and the outer membrane lipoprotein (S. Inouye et a1., I977) have been synthesized in vitro with arnino terminal "signal sequences". These sequences did not contain phospholipid, but were enriched in hydrophobic aniino acids. The extra peptide contained 23 and 20 amino acids for penicillinase and lipoprotein respectively and in extended form, they would be capable of spanning the membrane bilayer. By comparison, sequence analysis has shown that the amino terminal leader sequence of B' l icheniformis penicillinase (which contains 25 or 27 amino acids) is not enriched in hydrophobic amino acids, but owing to the presence of the phosphatidic acid attached to the N-terminal serine residue, it is nevertheless hydrophobic overall (Yarnanoto and Lampen, 1g76), These authors also suggested that owing to the 91_ . high purine content of the segment of the penicillinase nRNA that codes for this amino acid sequence, this region of the rnRNA would also be hydrophobic. H. Inouye and Beckwith (1977) have also reported that 8.. cgli alkaline phosphatase is initially synthe sized as a larger rnolecular weight precursor that is more hydrophobic than the completed enztsme, but the reason for the increased hydrophobicity was not deternined. It would be of great interest to see whether cerulenin inhibited the secretion of proteins for which precursors had been shown not to contain phospholipid. J¡. vitro synthesis of o<-amylase and protease frqn B. arnyloliquefaciens nay provide useful infornation on the role of lipid in protein secretion and possibly the nolecular cause of the cerulenin effect. Such work is currently in progress in this laboratory.

ADDENDUM

Experiments perforrned subsequent to the submission of this thesis have shown that rather than "non*growing", the ce1ls are in fact growing s1owly (approximately 30% of the normal growth rate). This finding, however, has no bearing on the interpretation of the work described in this chapter since cerulenin had no effect on this growth. CFTAPTER S IX

ATTEMPTS TO DETECT PRECURSORS TO O<-AMYLASE AND PROTEASE 92.

A INTRODUCTION

Experiments presented in chapter 5 showed that only trace amounts of rnaterial precipitable by antibodies to o<-amylase or protease could be detected in detergent treated lysates of B. amy1o1 iquefaciens . During these experiments, the cell culture supernatant hlas treated with the antibodies as a control for their activity. The protease antibody precipitated only one species of protein from the culture supernatant rvhich coelectrophoresed with purified protease. Rernarkably, however, the o<-arnylase antibody precipitated z species of protein. The possibility that these two species were in some tvay connected, perhaps by a precursor-product relationship r,\ras investigated in this chapter.

As already discussed, several eukaryotic and bacterial secretory proteins are initially synthesized as precursors with a molecular weight higher than that of the conpleted protein. Although stable intracellular forms of secretory proteins have not been reported in bacteria, s. Rothman and coworkers have suggested that active digestive enzynes can exist in the cytoplasrn of pancreatic cel1s (see chapter 1). Attempts were therefore nade to detect active intracellular precursors to o(-amylase and protease in B. @.

Neither of these studies proved fruitful, however, and accordingly the results will be only briefly summarized. 93.

B RESULTS

.a L Does precursor to or-Anylase Exist in the External

Ir{edium of B. anyloliq uefac iens ?

Ce1l culture supernatant was incubated with stg of rabbit anti-a.-amylase gamma globulin per unit of enzyme activity. After overnight incubation at 4oC, the immunoprecipitates hlere collected by centrifugation, washed and electrophoresed on SDS- polyacrylamide gels as described in the previous chapter. These were stained for protein as described in Chapter 2. The a<-amylase antibody precipitated two species (Plate 6-1); one exhibited an apparent rnolecular weight of 63,000 daltons and coelectrophoresed with purified o<-amylasen, while the other exhibited an apparent molecular weight of precisely half this value (51,500 daltons). The smaller species was not a result of protease cleavage during inmunoprecipitation, aS it was also present in gels of trichloroacetic acid precipitated culture supernatant.

* The nolecular rveig ht of B. am 1o 1i efaciens o<-amylase has previously been reported bY B oÏgra a amp e 978) to be app roximately 48,000 daltons on the basi-s o f amino acid composi- tio n. The figu re of 63,000 daltons obtained in the Present study b v SDS polyacry lanide ge1 electrophoresis is probabLy an overest 1ma te. It has been reported that B . subtil is x-amYlase does not bind as much SDS per unit weigh t as-ã-ores BSA, and therefore has reduced rnobility on SDS po Iyacrylamide gels (M itchell et ãL, 7973). In the p1' esent study , a value of 48 ,000 daltõnE-wa s obtained for the mo 1 ecul ar rve ight of B. an 1o1i uefac iens x-amylase when the purified enzyme was chroma - tograp e onaco lumn of BiogeJ A- 5 M in the pTesence of 614 guanid ine-HC1. The purified "<-amylase c hromat ographed as a single peak, iuhich tvhen subsequentlY e1e ctrophoresed on SDS po lyacrylarni deg e1s again yielded a mole cular weight of 63,000 daltons. No sma 1ler molecular weight sp ecies was observed after guanidine-HC1 treatment (results not shown). PLATE 6 - 1.

GEL ELECTROPHORESIS OF O(-AMYLASE ANTIBODY PRECIPITABLE MATERIAL FROM B. AMYLOLIQUEFACIENS CULTURE SUPERNATANT

B. amy loliquefaciens culture supernatant was treated with oc-amylase antibody as described in the text. The imnunoprecipitate was electrophoresed on a sDS-t2.5% polyacrylamide slab gel and stained for protein as described in chapter z. The rnobility of various marker proteins of known molecular weight is indicated. The narker serum albumin (68 000), glutamate proteins used weÏ.e bovine ' dehydrogenase (53, 000), glyceraldehyde-3-phosphate dehydrogenase (36,000) and globin (15,5 00)' a. Pure B. amy 1o1 iquefac iens o<- amYlase . b Material precipitated fron the culture supernatant with rabbit anti o<-amylase gamma-g1obu1in' c Rabbit anti "<-amylase gamna-globulin' a b c or¡g¡n

68,000 + t *,t

53,000 +

15,500 94.

This finding raised the possibility that the smaller species was related in Some way to the active o<-anylase' To investigate this possibility, purified c'(-amylase and the sma11- er species (eluted frorn preparative SDS'poLyactyLalnide gels as described in chapter 2) were digested with chyrnotrypsin or TpCK treated trypsin and the products were electrophoresed on

SDS- 1,5% polyacrylarnide gels which lvere stained for protein (see Chapter 2). No sinilarities in the digestion products of the two species were observed. As a further check, samples of the two proteins were cleaved by treatment with cyanogen bromide as described in Chapter 2. Analysis of the products on SDS-polyacrylanide gels again revealed no similarities ' It was therefore concluded that the two proteins r^Iere not related. The function of the smaller moleculaÏ. weight species and the reason for its reaction with the o<-arnylase antibody is not under stood.

2 Cel1 Associated Protease ActivitY in B. anYl olique- fac iens

In a further attempt to detect precursors to extracellular enzymes, lysates prepared by treatrnent of rvashed cel1s in a French pressure cel1, vleTe assayed for c<-amylase oT protease activity. Only small amounts of the latter were detected (approximately 0.5 units per ml of culture strength cel1 lysate) ' This protease, when electrophoresed on nondenaturing polyacrylanide gels (see Chapter Z) did not comigrate with extracellular protease^?k (Rf values weïe 0.61 and 0.t7 respectively). The

n There are at amylol iquef acien5; optinurn, whi1e the with a neutral PFI olì this particular ge1 system. 95. ce11 associated proteaSe activity was not released into the medium when cells were converted to protoplasts (as described in Chapter 2), but was released when the protoplasts wele 1ysed. Activity was not sedimented during centrífugation of ce11 lysates at 150r000 x g for 60 minutes, suggesting that the enzyme is a soluble cytoplasmic protein.

To investigate the possibility that this intracellular protease was a precursor of one of the secreted proteases, it was partiaLly purified as described in Chapter Z and its properties were compared with that of the extracellular enzytnes.

The nolecular weight of the intracellular protease lvas determined by chromatography on a Sephadex G-100 column and by SDS-polyacrylanide ge1 electrophoresis. The molecular weight estimates obtained were 59,500 and 50,000 daltons respectively, suggesting that the active enzyme rnay be a diner, consisting of two 50,000 dalton subunits. The nolecular weight of both extracellular proteases has been shown to be approximately 27 r500 daltons, as deterrnined by SDS-poLyacrylamide ge1 electrophoresis (J. Bielicki, unpublished data).

The intracellular protease was inhibited by EDTA, EGTA, and Pl,lSF, but was unaffected by TLCK and orthophenanthroline. In contrast, the alkaline extracellular protease is inhibited only by PMSF while the neutral extracellular protease is inhibited by EDTA and orthophenanthroline (D. R. Love, unpublished data).

When radioactÌvely labelled intracellular protease I\Ias incubated with antibody to the neutral extracellular protease (as described in Chapter 5), no immunoprecipitation of radio- 96 act ivity occurred. Unfortunately antibody to the alkaline protease was not available ¡

These latter results make it unlikely that the intracellular protease is related to one of the extracellular enzymes.

As a f inal check on this, cells weïe pulse labelled with [tOa ]- leucine for a period of 90 seconds. Radioactivity rernained associated with the intracellular protease for at least 30 ninutes after incorporation had been stopped by addition of a 5000 fold excess of unlabelled leucine. Since pulse labe11ed intracellular protease did not leave the ce11s, the possibility that it is a precursor of one of the secreted proteases can be rej ected.

C DISCUSSION

Attenpts to detect precurSors to extracellular "<-amylase and protease were not successful. Two proteins were precipitated by antibody to <-amylase frorn the culture nediun of B.

amylo 1 iquefaciens and it was initially thought that these species rnight be related in some manner (one perhaps being a precursor. of the other). However, comparison of their primary structure, by analysis of the products of proteolytic or chemical cleavage, showed that this was not the case. The srnaller rnolecular weight species, that did not coelectrophorese with purified <-amylase was not further characterized and its function and the reason for its precipitation by the x-amylase antibody are not understood.

An active intracellular protease was detected in ce11

lysate s of B. amyloliquefaci.ens and at f irst it was considered 97. possible that this protein nay haye been a precursor of one of the extracellular proteases. However, this possibility was rejected for the following reasons:^ L. The effect of various inhibitors on the activity of the intracellular protease was different to their effect on the

extracellular proteases . 2. The intracellular protease was not precipitated by antibody to the neutral extracellular protease. (Antibody to the alkaline extracellular protease was not available, but it has been reported by Hagernan and Carlton (7973) that a sinilar intracellular serine protease was not precipitated by antibody

to the alkaline extracellular protease in B. subt i1 i s 3. In a pulse chase experirnent, labelled intracellular protease did not leave the cells.

fnterestingly, ãIthough the intracellular protease is clearly not a precursor of either extracellular protease, its production was stinulated by transfer into a low amino acid medium and was insensitive to rifampicin (experiments not presented). These effects are sinilar to those reported for the production of extracellular protease by this organism

(0'Connor et aI. , 1978) .

Intracellular serine proteases with sirnilar propertíes have been found in other baci1li, including B. megaterium (Set1ow, L976;Chaloupka et a1., 1977), B. cereus (Cheng and Aronson,7977) and B. subtilis (Hageman and Carlton, 1,973; Hiroishi and Kadota, L976; Reysset and Mi11et, 1972; Szulmajster and Keryer, 1975). There was general agreement among these workers that the intracellular protease was involved in protein rnodif ication or tur:n- 98. over prior to sporulation. The intracellular protease from B. subtilis 450 has recently been purif ied (Strongin e.t ?L-., 1973) and the first 50 anino acids frorn the anino terminus have been sequenced. This sequence from the intracellular protease was greater than 50% homologous not only to the alkaline serine protease secreted by this organism, but also to that published by Kurihara et al. (1,972) for secretory subtilisins from other baci1li. (This included the subtilisins from B. amyloliquefaciens BPN!, B. subtilis Carlsberg and B. amylo sacchar it icusl Strongin e-t a1. (1978) concluded that the B. subtilis 450 genome contains at least two homologous structural genes for serine proteases (intracellular protease and secretory subti- lisin) that alose as a result of ancestor gene duplj-cation.

Since the intracellular protease detected in the present study was shown not to be a precursor of one of the extracellular enzymes, it was not further investigated. CHAPTER SEVEN

F INAL D I SCUSS ION AND SUMT,IARY 99.

F INAL DISCUSSION AND SUMMARY

Inthepastfewyearsthefieldofextracellularenzyme secretion has developed in a remarkable fashion' Following group' the early and now almost classical work of Palade and his a cinderella there was a period in which the aTea became almost of leader research topic. In eukaryote studies, the existence caused a Spectacu- Sequences and their relationship to Secretion of B. lar advance in our knowledge. The para1le1 discovery licheniformis extracellular penicillinase synthesis occurring viaaphospholipoproteinintermediatewasinportantinanew direction.

Despite the advances, QUêstions are appearing aS rapidly molecular as solutions. we stil1 know 1itt1e about the precise 0n the one rnechanisrn by which proteins traverse membranes' hand,pancTeaticSecTetoryproteinsaTebelievedtotraverse but the endoplasmic reticulum membrane aS nascent peptides, to be ribulose biphosphate carboxylase of chloroplasts seems with able to cross the membrane as a cornpleted protein, sti11 in the a leader Sequence. Ovalburnin Secretion, which occurs absenceofaleaderSequence,posesfurtherquestions.l\Ie picture stil1 need to knorv more of the position in the overall oflipoproteinsassecretoryintermediates.Westj.llneed resolution of the controversial theories of s' Rothrnan which appeartoconflictwiththoseofthePaladeschool.

In the research group in this laboratory, direct confirna- nRNA for tion is still needed for the existence of a pool of extracellular Proteins in !. amylo liq uefaciens. Further work in this group will be concerned rvith this and with direct 100. studies on the mechanism of enzyme synthesis and secretion. The work in this thesis, apatt frorn suggesting the involvenent of lipid in the process of secretion it I. gy.loliquefaciensr provides usefulr Possibly essential, background data on the structure and composition of the nembrane of this organism and more directly, gives a potentially useful tool in methods to rapidly "freeze" polysones in cells by cooling without the occurrence of rnembrane cold shock. CFTAPTER E IGHT

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APPENDIX A

PUBLICATIONS

J.C. Paton, B.K. May and w.H. El1iott. (1978). I'Phospholipid asymmetry in the menbrane of Bac.i1lus_ amyloliquefaciens".

Proc. Aust. Biochen. Soc. 1-1 , t07 .

J.C. Paton, B.K. May and I\r.H. E11iott. (1978). "Membrane phospholipid asymmetry in Bacillus gmyloliquefaciens". J Bacteriol. 135, 393- 401.

J.C. Paton, E.J. McMurchie, B.K. May and I4I.H. El1iott. (1978). "Effect of growth tenperature on membrane fatty acid composition and susceptibility to cold shock in Bacillus amyloliquefaciens". J. Bacteriol. 155, 754-759.

J.C. Paton, B.K. May and w.H. El1iott. (1979). "Selective inhibition of Bacillus amyloliquefaciens protein secretion by cerulenin". l4anuscript submitted to Biochinica et Biophysica Acta.