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Cell division in a temperature-sensitive mutant of Escherichia coli Reeve, John N. 1971

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CELL DIVISION IN OF A TEMPERATURE-SENSITIVE MUTANT ESCHERICHIA COLI by JOHN N. REEVE B . S c , Un ivers i ty of Birmingham, 1968 A THESIS SUBMITTED IN THE REQUIREMENTS DOCTOR OF PARTIAL FULFILMENT OF FOR THE DEGREE OF PHILOSOPHY In the Department of Microbiology We accept th i s thes i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA Ju l y , 1971 In present ing th i s thes is in pa r t i a l f u l f i lmen t of the requirements fo r an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ib ra ry sha l l make it f r ee l y ava i l ab le for reference and study. I fu r ther agree that permission for extensive copying of th i s thes i s fo r s cho la r l y purposes may be granted by the Head of my Department or by h i s representat ives . It i s understood that copying or pub l i c a t i on of th i s thes i s f o r f i nanc ia l gain sha l l not be allowed without my wr i t ten permiss ion. Department of Microbiology The Un ivers i ty o f B r i t i s h Columbia Vancouver 8, Canada Date 20th J u l y , 1971 ABSTRACT A temperature-sens i t ive d i v i s i o n mutant, Escher ich ia c o l i BUG-6, has been invest igated. This organism d iv ides normally when grown at 30 C but f a i l s to d i v i de at kl C. Growth continues at kl C to produce very long, mult inuc leate fi lamentous c e l l s . When returned to 30 C, or a high osmotic environment is added at kl C, the fi lamentous c e l l s d i v ide rap id ly to produce c e l l s of normal s i z e . The k ine t i c s of c e l l d i v i s i o n of the f i laments at 30 C depends on the period at kl C. During f i lament .formation, DNA, RNA and prote in synthesis continue as measured by r ad i o - i s o top i c incorporat ion and chemical c e l l f r a c t i o n a t i o n . DNA segregation occurs as shown by autoradiography. The rapid d i v i s i o n of f i laments replaced at 30 C cannot be pre-vented by novobiocin or cyc lo se r ine but is prevented by vancomycin and p e n i c i l l i n suggesting that de novo synthesis of c e l l wa11 precursors is not required for d i v i s i o n but that pos i t ion ing and cross-1 i nki.rig of the precursors is required. Filaments growing at kl C were treated with n a l i d i x i c ac id for d i f f e r e n t lengths of time. On returning these f i laments to 30 C in n a l i d i x i c ac id the number of d i v i s i on s was proport ional to the length of time at kl C in the absence of n a l i d i x i c a c i d , i e . proport ional to the amount of DNA synthesized at kl C. Inh ib i t ion of prote in synthes is , by chloramphenicol, does not pre-vent the d i v i s i o n of f i laments on replac ing at 30 C provided that the period of f f lamentat ion at kl C was greater than 6 minutes and less than 110 minutes. The maximum amount of d i v i s i o n in the absence of prote in synthesis occurred a f t e r a longer lag and slower than in non-inh ib i ted contro l cu l t u re s . If prote in synthesis 'was inh ib i ted in f i laments at kl C the a b i l i t y of such treated c e l l s to d i v ide at 30 C was rap id ly l o s t . This l o s s ' o f ' d i v i s i o n p o t e n t i a l 1 has a h a l f - l i f e of about 0.5 minutes, i e . 0.5 minutes of prote in i n h i b i t i o n at kl C reduces the subsequent d i v i s i o n at 30 C by 50%. The normal presence of ' d i v i s i o n p o t e n t i a l ' , therefore requires the syn thet i c doubling rate to be in excess of 0 .5 minutes. Very short periods at kl C ind icate that 10 minutes incubation at kl C is required to produce th i s extremely fast synthet ic rate. A model for the production and expression of ' d i v i s i o n p o t e n t i a l ' is presented. A biochemical ana lys i s of the c e l l envelope of the fi lamentous c e l l s and of normally d i v i d i ng c e l l s is presented. The major phospho-l i p i d compositions a re ' t he same. However, the f a t t y ac id contents d i f f e r e s p e c i a l l y with regard to the c y c l i c f a t t y ac id s . When the f i laments are allowed to d iv ide by replac ing at 30 C the i r f a t t y ac id composition very rap id ly reverts to that of normally d i v i d i ng c e l l s . The rates of ind iv idua l phospholipid syntheses appear to change during the rapid c e l l d i v i s i o n phase, however th i s may be an a r t i f a c t re su l t ing from an overa l l increase in the rate of phosphol ipid synthesis during th i s per iod. An ana lys i s of the proteins with in the c e l l envelope by r ad io - ac t i ve double l a b e l l i n g techniques and followed by gel e lect rophores i s ind icates that a prote in(s ) of molecular weight 80,000 - 90,000 ex i s t s in the envelope of f i lamentous c e l l s which is not in the envelope of normal c e l l s or is made at a much slower rate in normal c e l l s . The prote is not incorporated into septa during the d i v i s i o n of f i laments at 30 C and l i t t l e turnover occurs in the major proteins synthesized at 42 C when these c e l l s are placed at 30 C. The p o s s i b i l i t y e x i s t s , however, that th i s prote in is a product of f i l amentat ion and not the temperature sen s i t i ve gene product. i v TABLE OF CONTENTS Page INTRODUCTION. ' 1 MATERIALS AND METHODS 14 I. Bacter ia l s t ra in s and c u l t u r a l c o n d i t i o n s . . . . . . . . . 14 1. Organisms 14 2. Media 14 3. Basic experimental c o n d i t i o n s . . . . 14 4. Synchronized growth... . 15 5. Removal of a n t i b i o t i c s from c u l t u r e s . . 15 It. Ce l l f r a c t i ona t i on 15 1. Dry weights of c e l l suspensions . . . 15 2. P ro te in , RNA and DNA e x t r a c t i o n . . . . 16 3. L ip id ext rac t ion 17 III. Assay procedures 18 1. Protein 18 2. Deoxyribonucleic a c i d . . 18 3. Ribonucleic a c i d ' . . .-. . . 18 4.. Phosphorus in phosphol ipid . 19 IV. Separation and i s o l a t i on of indiv idual phosphol ip ids. 20 1. Thin layer chromatography 20 2. Column chromatography 20 V Tab! e of Contents (continued) Page V. Separation of f a t t y ac ids from phospho l ip id s . . . . 2 2 VT. i d e n t i f i c a t i o n of compounds separated by chromatography . . . . . . . . . 2 3 1 . Phospho l ip ids . . ; 2 3 2 . Fatty ac id s . 2k VII. Determination of rates of macromolecular syntheses 2 5 1 . Deoxyribonucleic a c i d . 2 5 2 . R ibonucleic a c i d . . . . . . . . . . . . . . . . 2 5 3 . Prote in 2 5 k. L i p i d . 2 6 i . Total l i p i d 2 6 i i . Individual phosphol ipids 2 6 V l i t . Ana lys i s of eel 1 envelope p r o t e i n . . . . 2 7 1 . I so lat ion p r o c e d u r e s . . . . . . . . . 2 7 2 . Disaggregation of proteins . 2 9 i . From eel 1 envelope preparat ions 2 9 i i . From c e l l membrane preparations • 2 9 3 . E lectrophores i s 3 0 k. Gel s l i c i n g and c o u n t i n g . . . . 3 0 5 . Standard curve for molecular weight e s t ima t i on . . . 3 1 IX. Autoradiography 3 2 X. Microscopy 3 2 v i Table of Contents (continued) Page 1. Light microscopy 32 2. E lectron microscopy •••• 32 RESULTS 34 I. D i v i s i on k i ne t i c s of Eschertch ?a col? BUG-6 34 1. Determination of non-permissive temperature 34 2. The e f f e c t of d i f f e r e n t time periods at 42 C on recovery at 30 C 34 II. M i c ro scopy . . . . . . .. 40 1. Phase contrast l i gh t microscopy 40 2. E lectron microscopy k0 3. Autoradiography • •• hO III. Ce l l d i v i s i o n and macromolecular synthesis at 30 C and 42 C . . . . . . 44 IV. Individual phosphol ipid synthesis at 30 C and 42 C. . . 49 V. Fatty acid synthesis at 30 C and 42 C. . . . . . . 53 VI. The e f fec t of high sa l t concentrat ion on c e l l d i v i s i o n at the non-permissive temperature.. 57 VII. The e f fec t of pantoyl lactone on c e l l d i v i s i o n at the non-permissive temperature 59 V I M . The e f f e c t of i nh ib i to r s of c e l l wall synthesis on c e l l d i v i s i o n during the Recovery p e r i o d . . . . . . ^ 62 IX. The e f fec t of i nh ib i t o r s of macromolecular synthesis on c e l l d i v i s i o n during the recovery pe r i od . . 62 Table of Contents (continued) v i i Page X. The e f f e c t of i nh ib i t o r s of macromolecular synthesis on c e l l d i v i s i o n during the recovery period fo l lowing d i f f e r e n t periods at kl C 76 XI. The e f fec t of 30 C pulses on f i laments produced at kl C.i 8k XII. The e f f e c t of a short period at kl C on c e l l s of d i f f e rent ages 88 XIII. Comparison of the proteins produced at kl C with those produced at 30 C 91 1. Ce l l envelope p r o t e i n s . . . . . . . . 91 2. Non-part i cu l a te p r o t e i n s . . . . . . . . 100 DISCUSSION • . . . . . 105 BIBLIOGRAPHY. 128 LIST OF TABLES The percentages of the tota l dry weight" of c e l l s , grown at 30 C and 30 C + 60 minutes at kl C, as const i tuted by p ro te in , RNA and DNA..... The f a t t y ac id composition of E s cher i - ch ia col? BUG-6 grown at 30 C, 30 C + 60 minutes at kl C, and 30 C + 60 min-utes at kl C + 8 minutes at 30 C The f a t t y ac id composition of Escher ich ia .col i AB1157 grown at 30 C, 30 C + 60 minutes at kl C and 30 C + 60 minutes at kl C + 8 minutes at 30 C A comparison of the e f f e c t on c e l l d i v i s i o n of a k minute pulse at kl C on c e l l s of d i f f e r e n t ages i x LIST OF FtGURES Page Figure 1. The e f fec t on c e l l d i v i s i o n of s h i f t i n g Escher ich ia c o l i BUG-6 from 30 C to higher temperatures . . . . . 35 Figure 2. The e f f e c t on c e l l d i v i s i o n of s h i f t i n g Escher ich ia c o l i BUG-6 from 30' C to kl C for d i f f e r e n t t ime- in te rva l s 37 Figure 3. The va r i a t i on iii recovery time for cu l tures of Escher ich ia col 1 BUG-6 placed at 42 C for d i f f e r e n t time i n t e r v a l s . . . .......> 38 Figure 4. The e f f e c t on c e l l d i v i s i o n of s h i f t i n g Escher ich ia c o l i BUG-6 from 30 C to 42 C for d i f f e r e n t time interva l s 39 Figure 5. Fragmentation of Escher ichia c o l i BUG-6 f i laments during recovery at 30 C 41 Figure 6. E lect ron microscopy of Escher ich ia col? BUG-6 at 42 C and during recovery at 30 C . . . kl Figure 7. Nuclear segregation in Escher ich ia c o l i BUG-6 at the non-permissive temperature (42 C) as determined by autoradiography 43 Figure 8. Ce l l number (A)l and c e l l s i ze (B) of Escher ich ia  c o l i BUG-6 as a funct ion of time at the permissive (30 C) and at the non-permissive (42 C) temperature 45 Figure 9. The rate of p ro te in , RNA and DNA synthesis per c e l l number in Escher ich ia c o l i BUG-6 as a funct ion of time at the permissive (30 C) and non-permissive (42 C) temperature 46 Figure 10. The rate of l i p i d synthesis per c e l l number in ' ' E s c h e r i c h i a ' c o l i ' BUG-6 as a funct ion of time at the permissive (30 C) and non-permissive (42 C) temperature . . . . . . 48 L i s t of Figures (continued) Page Figure 1 1 . The rate of synthesis of phosphatidyl e thano l -amfne (PE) and phosphatidyl g lycero l (PG) in Escher ich ia c o l i BUG-6 as a funct ion of growth at the permissive (30 C) and non-permissive (hi C) temperatures. . . . . . . 52 Figure 12 . The e f f e c t of NaCl on Escher ich ia c o l i BUG-6 f i laments at hi C 58 Figure 13 . The e f f e c t pf NaCl on Escher ich ia c o l i BUG-6 at 30 C and hi C 60 Figure 1h. The e f f e c t of pantoyl lactone on the rate of s i ze increase of Escher ich ia c o l i BUG-6 at hi C 61 Figure 15 . The e f f e c t of c e l l wall i nh ib i to r s on c e l l d i v i s i o n of f i laments during recovery at 30 C 63 Figure 16. The e f f e c t of chloramphenicol at hi C on the c e l l d i v i s i o n of f i laments of Escher i ch ia c o l i  BUG-6 during recovery at 30 C . Sh Figure 17a . The e f fec t of chloramphenicol on res idual d i v i s i o n of f i laments during the recovery at 30 C 67 Figure 17b. Decay of d i v i s i o n potent ia l at hi C 68 Figure 18. The e f f e c t of a 10 minute treatment with chloramphenicol at hi C on d i v i s i o n of f i l a -ments during recovery at 30 C 69 Figure 19- The d i f f e r e n t i a l e f f ec t of chloramphenicol on d i v i s i o n of f i laments fo l lowing the add i t ion of NaCl versus a s h i f t from hi C to 30 C 71 Figure 20 . The e f fec t of n a l i d i x i c ac<id on c e l l d i v i s i o n of the f i laments during recovery at 30 C 73 L i s t of Figures (continued) x i Page Figure 21. Residual d i v i s i o n of Escher ichia c o l i BUG-6 fo l lowing the add i t ion of n a l i d i x i c ac id 75 Figure 22a. Fragmentation of. n a l i d i x i c a c i d ' f i l ament s during recovery at 30 C 77 Figure 22b. Fragmentation of f i laments of Escher ich ia  c o l i BUG-6 at 30 C in the presence of chloramphenicol 77 Figure 23- The e f f e c t on eel ) d i v i s i o n of s h i f t i n g Escher ich ia c o l i 'BUG-6 from 30 C to 42 C for d i f f e r e n t time in terva l s and then replac ing at 30 C in the presence of ch loramphenico l . . . 78 Figure 24. The e f f e c t of chloramphenicol on res idua.l. d i v -i s ion of f i laments during the recovery at 30 C, a f t e r d i f f e r e n t time in terva l s at k2 C 80 Figure 25. The e f fec t of n a l i d i x i c ac id or chloramphenicol on res idual d i v i s i o n of f i laments during the recovery at 30 C a f t e r d i f f e r e n t time in terva l s at k2 C . 81 Figure 26. The e f f e c t of a period of i n h i b i t i o n of DNA synthesis at k2 C on the res idual d i v i s i o n of f i laments during the recovery at 30 C in the presence of chloramphenicol 83 1Figure 27. The e f fec t on c e l l d i v i s i o n of Escher ich ia c o l i  BUG-6 of d i f f e r e n t length pulses of 30 C during h2 C growth.. 86 Figure 28. The e f f e c t on c e l l d i v i s i o n of Escher ich ia c o l i BUG-6 of d i f f e r e n t length pulses of 30 C during k2 C growth when the temperature pulse is pre-ceeded by a 3 minute chloramphenicol pu l se . . . 87 Figure 29- The e f fec t of a 4 minute.pulse at 42 C on the d i v i s i o n of c e l l s of d i f f e r e n t a g e s . . . . . . 89 Figure 30. Gel e lect rophores i s of c e l l envelope proteins prepared from Escher ich ia c o l i BUG-6 grown at 30 C and 42. C 93 L i s t of Figures (continued) x i i Page Figure 3 1 . Gel e lect rophores i s of c e l l envelope proteins prepared from Escher ten ia c o l i BUG-6 grown at 30 C and kl C . . . . . . . . . . . Sk Figure 3 2 . Gel e lect rophores i s of c e l l envelope proteins prepared from Escher ich ia c o l i A B U 5 7 grown at 30 C and kl C. . . 95 Figure 33 . Gel e lec t rophores i s of c e l l membrane proteins prepared from Escher ich ia col? BUG-6 grown' at kl C and at kl C foi lowed by recovery at 30 C 98 Figure 3**- Gel e lect rophores i s of c e l l membrane proteins prepared from Escher ich ia col? BUG-6 grown at 30 C and kl C followed by recovery at 30 C . . . 99 Figure 3 5 . Gel e lec t rophores i s of c e l l envelope proteins prepared from Escher ich ia c o l i BUG-6 grown at 30 C and grown at 30 C i n . n a l i d i x i c ac id (10 ug/ml) . . . 101 /of Figure 36 . Gel e lec t rophores i s of non-par t i cu la te proteins prepared from Escher ich ia c o l i BUG-6 grown at 30 C and at kl C . . . . . . . . . . . . 102 Figure 3 7 . Gel e lect rophores i s of non-par t i cu la te proteins prepared from Escher ich ia c o l i BUG-6 grown at 30 C and 42 C . . . 103 Figure 38 . Model for production and interconvers ion of d i v i s i o n potent ia l in Escher ich ia c o l i BUG-6. 115 For P a t r i c i a ACKNOWLEDGEMENT I would l i k e to express my s incere grat i tude to Dr. D.J. Clark for h is adv ice , help and cons t ruc t i ve c r i t i c i s m throughout th i s work. Parts of th i s work were ca r r i ed out in the laborator ies of Dr. N.H. Mendelson and Dr. D.J. Hanahan, I would l i k e to thank them for the i r he lp. F i n a l l y , I would l i k e to acknowledge the f i n a n c i a l support I have received from the Canadian Commonwealth U n i v e r s i t i e s Scholarship Committee. INTRODUCTION Studies of bac te r i a l eel 1 -d i v i s i on can be separated into i n -ves t i ga t ions of normally growing and d i v i d ing bacter ia and i n v e s t i -gations involv ing i nh ib i to r s or mutants of the c e l l d i v i s i o n process. Observat ions.of normally growing eel 1s: Studies of the f i r s t type are, of necess i ty , involved with the re l a t i on sh ip of c e l l d i v i s i o n to the whole c e l l c y c l e . Many micro-scopic invest igat ions of normally d i v i d ing c e l l s have been ca r r ied out and these have led to several conclus ions regarding c e l l growth and d i v i s i o n . Observations of bacter ia growing on agar surfaces have shown that the growth rate determines the type of growth, ie . at slow growth rates the c e l l s extend u n i d f r e c t i o n a l l y but at f a s te r growth rates they extend b i d i r e c t i o n a l l y (27). The rate of c e l l extension has been observed to be l inear for most of the c e l l cyc le and then double as c e l l d i v i s i o n approached ( 2 ) . Comparison of the growing c e l l to a f ixed external marker in the agar (27) or examination of the d i s t r i b u t i o n of young and old f l a g e l l a (10) , assuming that length is equivalent to age and f l a g e l l a are only synthesized as the c e l l wall is synthes ized, have led to the conclus ion that c e l l extension is asymmetric. The region of c e l l wall growth has been invest igated by d i r e c t microscopy. Antiserum, s p e c i f i c for the c e l l surface (19) or mucopeptide ( 4 5 ) , was combined with a f luorescent dye and used to 2 determine the locat ion of newly synthesized areas of c e l l surface or mucopeptide. The c e l l s were f i r s t coated with non-f luorescent a n t i -body so that fo l lowing growth, only newly synthesized regions would be ava i l ab l e for attachment of the f luorescent antibody and these regions would f luoresce when observed by u l t r a - v i o l e t microscopy. The re su l t s indicated that in gram-negative c e l l s , growth occurs over most of the c e l l surfape but gram-posit ive c e l l s have s p e c i f i c l o ca l i zed regions of growth (19). The resu l t for gram-negative bacter ia was corroborated by an autoradiographic invest igat ion of c e l l wall growth using rad ioact i ve dTamino-pimel1 ic acid as a s p e c i f i c label for bac te r i a l c e l l wal ls (111). The actual mechanism of c e l l d i v i s i o n d i f f e r s as has been observed by e lec t ron microscopy, ie . gram-negative c e l l s appear to d i v i de b^nconstr ict ion whereas gram-posit ive c e l l s f i r s t produce a membranous invaginat ion and then the c e l l wall grows inward as an annulus, whereas the external c e l l diameter at the area of d i v i s i o n shows l i t t l e change (107). Paulton (77) has observed, by using c e l l - w a l l s p e c i f i c s t a in ing , that Bac i l l u s sub t i l is c e l l s growing at d i f f e r e n t rates a c t u a l l y demon-s t ra te a constant period between the i n i t i a t i o n of a septum and c e l l d i v i s i o n involv ing that septum. The d i v i s i o n rate is determined by the rate of i n i t i a t i o n of septa. Escher i ch ia c o l i is not mult i septate but Adler e_t_ a_l_. (2) observed mic roscop ica l l y that septum formation is apparent between hal f and three-quarters of the way through the c e l l c y c l e . 3 Further invest igat ions of normally growing c e l l s have used synchronised populations so that change in the c e l l c yc le could 'be monitored sequent i a l l y . The growth rate has been found to change with c e l l age however, "reports d i f f e r in how the rate changes, varying from several d i f f e r e n t rates per cyc le (67) to a simple doubling in rate as a s p e c i f i c age per c e l l c yc le (59). Changes have been found for enzyme a c t i v i t y (always equated to enzyme synthesis although not proven) such that some enzymes demonstrate d i s t i n c t changes in the i r a c t i v i t y at points wi th in the c e l l c y c l e , presumably due to a doubling in the gene c o n t r o l l i n g that enzyme production (33). The nucleoside t r i -phosphate pools have been shown to f l uc tua te with c e l l age, the changes being cons istent with changes in the known pattern of discontinuous DNA synthesis.. A c o r r e l a t i o n has a l so been suggested between the level of one or more nucleot ides and the contro l for c e l l d i v i s i o n (46). This suggestion of .1 inking a DNA precursor with c e l l d i v i s i o n control is based on the knowledge, a l so obtained from synchronised cu l tu re s , that completion of a round of DNA r e p l i c a t i o n is necessary for c e l l d i v i s i o n (16, 42). Inh ib i t ion of DNA synthesis before completion blocks c e l l d i v i s i o n , but i n h i b i t i o n of DNA synthesis a f t e r completion, ie . during the next c y c l e , does not block c e l l d i v i s i o n (16, k l ) . The completion of a round of DNA r e p l i c a t i o n changes the number of DNA synthet ic regions and would be expected to a l t e r the pool leve l s of precursors . This could be the required s i t ua t i on to i n i t i a t e the d i v i s i o n processes (46). Adler et_ aj_. (2) and Paulton (77) observed microscopical ly that c e l l d i v i s i o n was i n i t i a t e d considerably before c e l l separat ion. C lark (17) used sonfcat ion and bacteriophage to produce l oca l i zed breaks in the c e l l s u r f a c e and showed that in a synchronous population an increase in survivors occurred before c e l l d i v i s i o n , ind icat ing a period of phys io log ica l d i v i s i o n before physical d i v i s i o n . This was substant iated by Daniels ( 2 2 ) , who, using the rate of incorporat ion of g l y c e r o l - 2 - H as a measure of membrane synthes i s , found a dramatic increase in the rate of synthesis of membrane s l i g h t l y before c e l l d i v i s i o n in a synchronised population presumably due to septum synthesi Recently Groves and Clark (37) have demonstrated that the timing of synthesis of a u t o l y t i c enzymes is a l so related to the c e l l c y c l e . Using chloramphenicol to block the synthesis of such enzymes at d i f -ferent c e l l ages, the subsequent c e l l l y s i s in the presence of an ip ic i l l indicates a p o s s i b i l i t y of separating the a u t o l y t i c enzymes temporari ly within the c e l l cyc le and a l so into those involved with d e f i n i t e phys io log ica l processes such as c e l l d i v i s i o n or nuclear segregation. The benef i t of a synchronized c u l t u r e , i e . enrichment of the c e l l d i v i s i o n process, can be mimicked by using a nu t r i t i ona l shift-down as descr ibed by Ba l l e s t ra and Schaechter (5). Using th i s technique they have described the changes in the ra t io s of the d i f f e r e n t phospho-l i p i d s of Escherich ia c o l i s p e c i f i c a l l y occurr ing during c e l l d i v i s i o n . The rate of synthesis of phosphatidyl-ethanolamine increased whereas that of phosphat idy l -g lycero l decreased. A second type of s h i f t , that 5 of temperature, has a l so been used on normal c e l l s and i t has been found that the c e l l d i v i s i o n process appears to be p a r t i c u l a r l y sen s i t i ve to high temperatures. Steed and Murray (107) were able to show by e lect ron microscopy a cross membrane, v i s i b l e p r i o r to d i v i s i o n , in gram-negative organisms which had been placed at 45 C, thus showing a change from the normal gram-negative d i v i s i o n by c o n s t r i c t i o n . Smith and Pardee (103) described a thermolabi le prote in synthesized ear ly in the c e l l d i v i s i o n cyc le of Escher ich ia c o l i which was inact ivated by a period of 15 minutes growth at 45 C. This prote in was required for c e l l d i v i s i o n to occur. This l a t t e r work, together with those.of Adler (2) , Groves eJ- aj_. ( 3 7 ) , Daniels ( 2 2 ) , Paulton (77) and Clark (17) described e a r l i e r , suggest that the v i s i b l e process of c e l l d i v i s i o n is only the termipation of a ser ies of metabolic events which may have started one or more c e l l generations e a r l i e r . Ce l l d i v i s i o n i n h i b i t o r s : Many i nh ib i t o r s are known to prevent c e l l d i v i s i o n and al low f t lamentation-eg. p e n i c i l l i n (30). which blocks c e l l wall synthesis (108); m-cresol (23) which in ter feres with membrane i n t e g r i t y ; mi to-mycin C ( 9 9 ) , u l t r a - v i o l e t l i gh t (6 , 78) and n a l i d i x i c ac id ( 3 4 ) , a l l i nh ib i t o r s of DNA synthes i s ; azaser ine (14) an i nh ib i t o r of purine b iosynthes i s ; 5 _ d iazpurac i1 (83) and D-serine ( 3 8 ) , whose d i r e c t act ions are not yet ascerta ined but probably a f f e c t RNA (82) and pantothenate (39) biosyntheses re spec t i ve l y ; novobiocin (70) which i nh ib i t s many biochemical steps ( 1 0 4 ) ; magnesium (114) and thymine 6 (18, 32) d e f i c i e n c i e s , and the app l i ca t i on of extreme pressure (118). Fi lamentat ion is the resu l t of i nh i b i t i on of eel 1 d i v i s i o n without complete i n h i b i t i o n of eel 1 growth. Because of the var ied s i t e s of i n h i b i t i o n of the above i n h i b i t o r s , f i lament induction cannot be a t t r i bu ted to blockage of a s ing le biochemical reac t i on , although the above inh ib i to r s could conceivably al1 a f f e c t the same react ion i n d i r e c t l y . It has been proposed, however, that changes in c e l l shape, and induction of f i l amentat ion p a r t i c u l a r l y , are due to a s p e c i f i c i n h i b i t i o n of c e l l wall synthesis (8, 76). Support is furnished by the f ind ing that very low leve l s of p e n i c i l l i n s p e c i -f i c a l l y block c e l l d i v i s i o n and higher concentrat ions p re fe ren t i a1 l y cause c e l l l y s i s at d i v i s i o n s i t e s ( 9 7 ) . When prote in synthesis is i n h i b i t e d , e.g. by add i t ion of chloram-phenicol or amino ac id s t a rva t i on , f i lamentat ion does not occur. A period of res idual c e l l d i v i s i o n r e s u l t s , fol lowed by i n h i b i t i o n of both c e l l d i v i s i o n and growth (80, 3k). < Res i dua l , d i v i s i on - a l s o occurs fo l lowing a block iii DNA synthesis e.g. n a l i d i x i c ac id add i t i on , thymine.starvat ion (16, k l ) , however f i lamentat ion then fol lows as i n -h i b i t i o n of DNA synthesis does not i nh ib i t c e l l growth. Ce l l d i v i s i o n mutations; As an a l t e r n a t i v e to the add i t ion of external i nh ib i t o r s much use has been made of " i n t e r n a l i n h i b i t o r s " , i e . mutations blocked at a stage in c e l l d i v i s i o n . Most mutations of th i s kind are cond i t iona l as the i r expression is l e t h a l , however there are at least some c e l l 7 d i v i s i o n processes in Escher ich ia col i for which mutations have been found which are not c o n d i t i o n a l . Examples are septum pos i t i on ing , which is apparently i n co r rec t l y contro l led iii the ' " m i n i - c e l l " mutation 0 ) , producing very small DNA-less c e l l s ; eel 1 separatfon, which does not occur in a mutant described by Nprmack, Bomah and Marsson ( 7 2 ) , and therefore produces chains of c e l l s ; and c e l l s i ze contro l which is lost in the mon mutat ion (4), g iv ing r i s e to abnormally large c e l l s . Condit ional mutations have added much to the knowledge of c e l l d i v i s i o n , however many of the mutations are only i n d i r e c t l y re lated to c e l l d i v i s i o n . Mutants are ava i l ab l e which under the non-permissive condi t ions do not synthesize DNA (44, 1 1 0 ) , or do not r e i n i t i a t e DNA synthesis fo l lowing completion of a round of DNA r e p l i c a t i o n ( 5 8 ) , or do not segregate completed genomes (44). From such mutants the d i r e c t l inkage between DNA synthesis and c e l l d i v i s i o n has been confirmed. Mutants in c e l l wall synthesis are a l so a va i l ab l e (68 , 1 0 9 ) , and in one case, the c e l l wall content of cho l ine and ethanolamirie can be var ied ( 1 0 9 ) . This a f f e c t s the substrate for a u t o l y t i c enzymes which in turn has shown a requirement "for the expression of a u t o l y t i c enzymes in eel 1 d iv i s ion. There a re , however, many mutants which appear to be involved ' d i r e c t l y with c e l l d i v i s i o n and i t s c o n t r o l . Most condi t iona l mutants are temperature s e n s i t i v e . Mutants of th i s type have been described which have lost the coord inat ion of DNA synthesis and c e l l d i v i s i o n such that even when DNA synthesis is i n h i b i t e d , c e l l d i v i s i o n continues 8 g iv ing r i s e to DNA-less eel 1 s .(43,47) . Other mutants appear to lose the " t r i g g e r " for d i v i s i o n at the non-permissive temperature and a l -though a l l c e l l u l a r syntheses cont inue, c e l l d i v i s i o n does not occur and long coenocytfc f i laments are formed (44, 60,.71, 110). Some mutants s t i l l d i v ide at the non-permissive temperature, but th i s d i v i s i o n is i r regu la r g iv ing r i s e to b i za r re c e l l shapes (13) or the excess ive production of membranes (115). A mutant has been described which, i f incubated at 45 C in l i q u i d media and subsequently grown on s o l i d media at e i ther 37 C or 45 C, d iv ides i f at 45. C but not i f at 37 C, i e . a period at 45 C condit ions the c e l l d i v i s i o n mechanism to funct ion on s o l i d media at 45 C but not at 37 C (102). A second group of cond i t iona l c e l l d i v i s i o n mutants ex i s t which only d i v ide normally when the external environment is supplemented. Bac? 11 us-.'subt11 is mutants have been described which grow as very misshapen c e l l s unless a high osmotic environment is suppl ied ( 9 0 ) . A mutant of Escher ich ia  c o l i is known which forms f i laments unless canavanine is added to ' the med ium (61) . A much studied condi t iona l mutant is that described as '1 on in Escher ich ia co1? Kl2 (3) and as uvs in Escher ich ia c o l i ' B ( 2 5 ) . The a b i l i t y to d i v ide is lost by th i s mutant fo l lowing an exposure to a low level of u l t r a - v i o l e t i r r a d i a t i o n , although growth is not inh ib i ted and f i laments are thus produced ( 3 ) . Comparison of d i v i d ing and non-d iv id ing c e l l s : The i n h i b i t i o n of c e l l d i v i s i o n allows the comparison of d i v id ing c e l l s with non-div id ing c e l l s . If the i n h i b i t i o n or cond i t iona l 9 mutation is rever s ib le then the requirements f o r , and the events preceding and during the onset of d i v i s i o n of the no longer inh ib i ted c e l l s , can be s tud ied. The biochemistry of non-d iv id ing eel Is, es -p e c i a l l y the membrane and c e l l wall composit ion, has been compared with that of d i v i d ing c e l l s using both i nh ib i to r s and mutations to prevent d i v i s i o n . Filaments produced by add i t ion of p e n i c i l l i n (106) have been shown to produce more c a r d i o l i p i n and less phosphatidyl g lycero l than normally d i v id ing c e l l s and on add i t ion of p e n i c i l l i n a s e , th i s s i t u a t i o n is reversed un t i l the control rates of phosphol ipid syntheses are obtained. The phosphol ipid and fa t ty ac id composition of f i laments produced by a DNA " i n i t i a t o r " mutant (unable to i n i t i a t e new rounds of DNA r e p l i c a t i o n at the non-permissive temperature) have been compared with the parent c e l l composition ( 7 9 ) . The mutant contains much more an ion ic phosphol ipids ( c a r d i o l i p i n and phosphatidyl g l ycero l ) than the parent. The f a t t y ac id composition a l so d i f f e r s in the two s t ra in s with a s i g n i f i c a n t increase in the c y c l i c f a t ty acids in the fi lamentous c e l l s . The proteins contained with in the c e l l envelope of d i v i d i ng and non-d iv id ing c e l l s , have a l so been compared ( 49 , 51, 9 8 , 100). Use has been made of a doubling label 1ing technique whereby the proteins produced by f i laments are l abe l led with one type of rad ioact ive precursor, eg. t r i t i u m l a b e l l e d , and the proteins of d i v i d i ng c e l l s are l abe l led with the same precursor compound conta in ing a d i f f e r e n t rad ioact ive l a b e l , eg. carbon-14. Extract ion of the proteins and comparison of the label of each kind in each prote in by ge l -e lec t rophores i s allows the d e f i n i t i o n 10 of proteins made p re fe ren t i a l ly by d i v id ing or non-d iv id ing c e l l s . A l l non-d iv id ing c e l l s , as reported by Inouye and co-workers (49, 5 1 ) , p r e f e r e n t i a l l y produce a prote in of molecular weight 39,000 and in.some cases a second prote in of molecular weight 80,000. If f i l amentat ion is produced by DNA i n h i b i t i o n , e i ther by add i t ion o f ' i nh ib i to r s or use of cond i t iona l DNA synthesis mutants, then the f i laments a l so lack proteins (5) which are presumed to be involved in DNA synthesis (49, 98, 100) . The c e l l wa11 s t ructure has been analysed chemical ly in d i v id ing and i nco r rec t l y d i v i d i n g c e l l s , and d i f fe rences in the degree of muco-peptide cross-1 inking have been reported (88). The abnormal c e l l s have a mucopeptide with less cross l i n k i n g . E lectron microscopy of c e l l s grown at the non-permissive temperature has demonstrated that in one case (20) the abnormal morphology can be cor re la ted d i r e c t l y with the absence of a c e l l wall l ayer , t e n t a t i v e l y i den t i f i ed as the mucopeptide layer , combined with the random l o c a l i z a t i o n of septa. Removal of eel 1 d i v i s i o n i n h i b i t i o n : ' Removal of the i n h i b i t i o n of d i v i s i o n has given the most d i r ec t information on the requirements for c e l l d i v i s i o n . Removal of i n h i - . b i t i o n can sometimes be done by removal of the i nh ib i to r orfjreplacement in the permissive cond i t i ons , but in some cases, condit ions which are not the reverse of those used to i nh ib i t d i v i s i o n , have been found to permit c e l l d i v i s i o n . The production of a 'h igh osmotic environment allows a temperature-sens i t ive f i lament-former to d i v ide at the non-permissive temperature (86). A d i f f e r e n t temperature sens i t i ve mutant, described by Kflrby, Jacob and Goldthwalte (56), d iv ides I f more s p e c i f i c addit ions are made, ie . add i t ion of guanine plus cy tos ine. Reversion to a normal morphology occurs by add i t ion of glutamine to a rod .mutant of Bac i l l u s sub t i l is ( 8 9 ) . A decrease in growth ra te , i e . s h i f t i n g from r i c h to minimal medium, has been found to induce d i v i s i o n in f i laments produced by u l t r a - v i o l e t i r r a d i a t i o n (2). Addi t ion of pantoy l - lactone a l so caused these f i laments produced by i r r a d i a t i o n , to d i v i de (38), although th i s may be a resu l t of a de le ter ious e f f ec t of th i s compound on growth rate (2), and not a s p e c i f i c e f f e c t on the c e l l d i v i s i o n mechanism. ' A complex substance, which is l ipase and temperature s e n s i t i v e , has been extracted f rom 'E scher i ch i a ' co l ? which on add i t ion to 1 on f jlaments Induces d i v i s i o n without slowing the growth rate (29) • A substance extracted from yeasts and s i m i l a r l y a f f e c t i n g c e l l d i v i s i o n pf yeasts , has a l so been described (112). When a non-d iv id ing c e l l is permitted to proceed to d i v i s i o n , proceisses d i r e c t l y involved in d i v i s i o n may be more e a s i l y demonstrated The use of DNA synthesis i nh ib i t o r s allowed the demonstration that only when DNA r e p l i c a t i o n was completed was c e l l d i v i s i o n permitted (16, 17 42). A d i r e c t c o r r e l a t i o n has been found between the r a t i o of protein:DNA in the d i v i s i o n of f i laments produced by thymine s tarvat ion i e . on resumption of DNA synthesis there "is no d i v i s i o n untjM the protein:DNA r a t i o has returned to normal (26, 28). Th is i s not true for d i v i s i o n of Ion f i l aments , which do not d i v ide unt i l cons iderably after the protein:DNA ratio has returned to normal (62). Comparison of mutants blocked in cell division by^  arginine starvation with the same cells following addition of arginine, has demonstrated that the internal ratio of spermidine to putrescine is important for cell division control (50), ie. a high putrescine to spermidine ratio is needed for division to occur. As yet no definite biochemical step has been demonstrated as a "division process" although several of the mutations have been mapped ( l , k, kk, 58). The involvement of proteins in the control of eel 1 division has been demonstrated however'by genetic means, as two specific genetic division releasing conditions have been found. An ochre suppressor mutation placed in a Jon_ strain decreases filamentation indicating that the genetic lesion is expressed as a protein which can be suppressed into a functional form ( 6 5 ) . In the second system, the presence of a recA .mutation allows the division of a temperature sensitive mutant at the non-permissive temperature (48). This suggests that the recA + product normally inhibits division by acting as a negative control factor but as yet no real knowledge of the actual proteins involved in either system is available. In the present work, I describe a reversible temperature sensitive mutation which is apparently directly involved in cell division. By use of the permissive and non-perrtiissive conditions, it is possible to produce the situations of division, non-division,' filamentation and "recovery" division of filaments, ie. division following the removal of d i v i s i o n i n h i b i t i o n . I have invest igated the contro l of d i v i s i o n as re lated to macromolecular syntheses and a l so the biochemistry of the c e l l envelope in the three d i v i s i o n s i t ua t i on s . MATERIALS AND METHODS 1. Bacter ia l s t r a i ns and cu l ture condi t ions 1. Organ isms Many temperature-sens i t ive c e l l d i v i s i o n mutants were derived by Groves (37) from Escher ich ia c o l i AB-1157 Sm/r, gal , xyl , mtl which was obtained from E.A. Adelberg. A l l mutants behave normally at the permissive temperature (30 C) and exh ib i t abnormal d i v i s i o n patterns at the non-permissive temperature (42 C). One of these d i v i s i o n mutants, laboratory s t r a i n BUG-6, was se lected for de ta i l ed analys i s . 2. Media Cultures were incubated in Erlenmeyer f l a sks contain ing nutr ient broth (3 g beef ext ract per 1, 5 g pepticase per 1 and 5 g NaCl per 1), adjusted to pH 7-3 maintained at constant temperature in a shaking water bath. For experiments in which DNA,RNA prote in or l i p i d synthesis was measured, a minimal s a l t s medium (007) prev ious ly descr ibed (16) was supplemented with 1 g pepticase per 1 and 2 g glucose per 1. For experiments in which membrane proteins were analyzed, the concentrat ion of minimal s a l t s was halved and the supplements were 80 mg pepticase per 1 and 2 g glucose per 1. The c e l l numbers and c e l l s i ze were measured with a modif ied Coulter Counter coupled to a pulse-height analyzer (16). 3. Basic experimental condit ions One of the experimental designs was used repeatedly. A cu l ture growing at the permissive temperature (30 C) was d iv ided and part of the cu l tu re placed at the non-permissive temperature (42 C). A f te r andefined time at the non-permissive temperature, the cu l tu re was again subdivided'and a port ion returned to the permissive tempera-ture (30 C). This incubation at the permissive temperature is referred to as the " recovery p e r i o d " . , 4. Synchronised growth Synchronised populations of c e l l s were obtained by using the membrane technique devised by Helmstetter and Cummings (41). ' Because the c e l l s were grown in a r i ch medium, i t was necessary to use a M i l l i p o r e membrane of pore s i ze 0.8 u. 5. Removal of a n t i b i o t i c s from cu l tures Some experiments required theradd i t ion and removal of an a n t i b i o t i c from the medium. Removal was accomplished by f i 1 t e r i n g the c e l l s from the medium plus a n t i b i o t i c s using a M i l l i p o r e membrane of pore s i ze 0.45 U, fol lowed by washing and resuspending in precondit ioned medium. (Preconditioned medium cons i s t s of the same medium as used in the experiment in which the same organism has grown to approximately the same c e l l dens i ty.as that used in the experiment. The organism has been removed by f i l t r a t i o n using a M i l l i p o r e membrane of pore s i ze 0.22 u.) II. Cel1 f r a c t ionat ion 1. Dry weights o f ' ce l l . s u spens i on s Dupl icate or t r i p l i c a t e 1 ml samples of the concentrated c e l l suspensions, in prev ious ly weighed aluminum fo i1 conta iners , were heated at 100 C un t i l the moisture had evaporated, and then were t rans -ferred to ind iv idua l screw-cap j a r s , each contain ing a layer of ac t ivated s i l i c a gel dess icant (Davidson Chemica1 Company). In th i s manner, they were dr ied to constant weight, and the resu l t s obtained were corrected for the presence of bu f fe r . 2. P ro te in , RNA and DNA ext rac t ion A mod i f i ca t ion of the procedure of Roberts et_ aj_. (87) was employed in the chemical f r a c t i ona t i on of the c e l l s . Two c e l l samples were compared, one grown at 30 C and one at 30 C and then at hi C for 60 mins. The cu l tures were rap id ly c h i l l e d and each resuspended in 6 ml of d i s t i l l e d water. F ive ml of th i s suspension were added'to 5 rmli of cold 10% t r i c h l o r o a c e t i c ac id (TCA) in a Pyrex cent r i fuge tube and the react ion mixture was held on ice for 30 min. Following cent r i fuga t ion at 7 ,500 x g_ for 15 min, the supernatant f l u i d ' ( c o l d TCA-soluble f rac t i on ) was removed and the p e l l e t was resuspended in 5 ml of 75% ethanol (pH 2.5 by add i t ion of d i l u t e t^SO^) , with the a<T>d of a Vortex mixer. The ext rac t ion was ca r r i ed out at k5 C for 15 min, a f t e r which the ethano l -so lub le f r a c t i o n was recovered by c e n t r i f u g a t i o n . Five ml of 5% TCA were added to the p e l l e t , and a f te r thorough mixing, the tube was covered and was incubated at 90 C for 10 min. A f te r coo l i n g , the tube was centrffuged and the supernatant f l u i d (hot TCA-soluble f rac t i on ) was removed. The residual p e l l e t (hot TCA- inso luble f rac t ion ) was disso lved in 5 ml of 0.1 N NaOH by heating at 50 C for 5 min. The a c i d i c - a l c o h o l so lub le f r a c t i o n and the hot TCA- inso luble f r a c t i o n were assayed for prote in (see sect ion I I 1-1). The hot TCA-so lub le f r a c t i o n was assayed for RNA and DNA (see sect ions II - 2 and 3 ) . 3 . L i p id ext ract ion The technique of Bl igh and Deyer (11) was modi f ied. Chloroform and methanol were added to the c e l l suspension in the r a t i o of 1 volume chloroform: 2 volumes methanol; 0.8 volumes c e l l suspension. This mixture was blended (Waring Blender Model /OOS) for two minutes, a fur ther 1 volume of chloroform added'and the mixture blended again for 30 seconds. One volume of water was added followed by 30 seconds blending before p lac ing the mixture in a separating funne l . The lower chloroform layer was c o l l e c t e d as the major l i p i d ex t rac t . The upper a l c o h o l i c layer was re-extracted by add i t ion of chloroform and two chloroform extracts combined, evaporated to dryness at 40 C to 50 C under vacuum and the re su l t i ng material d i s s o l v e d ' i n a small volume of chloroform: methanol: water in the ra t ios by volume 60:30 :4 .5 . This so lu t ion was placed on top of a column (diameter: height r a t i o 1:10) contain ing Sephadex G25. The Sephadex G25 was prepared by washing three times with a large volume of d i s t i l l e d water, s ix times with acetone, dry ing with suction fo l lowed'by .air drying and suspending in chloroform: methanol: water in the r a t i o by volume of 60:30:4.5 for ten minutes before placing in the column. The l i p i d extract was eluted from the column by add i t ion of more of the same mixture of chloroformrmethanol:water fol lowed by chloroformrmethanol in the r a t i o by volume 2:1. For each gram of Sephadex G25 used, 15 ml of the chloroform + methanol + water mixture and 5 ml of the chloroform + methanol mixture can be run through the column before e l u t i on of non-l.|pid materia l begins. The eluant was evaporated under vacuum and the resu l t ing material redisso lved as required for ind iv idua l l i p i d separat ion or tota l l i p i d and phosphorus assays. III. Assay Procedures 1. Prbtei n The method of Lowry et a l . (63) was employed to determine prote in concentrat ion. C r y s t a l l i n e egg albumin in concentrat ions ranging from 8 - kl ug/ml in the react ion mixture were used in preparat ion of standard curves. 2. Deoxyribonucleic ac id The method described by Schneider (96) employed diphenylamine, which reacts with purine-bound deoxyribose, was used for the determination of DNA. Standard curves were prepared from p u r i f i e d c a l f thymus DNA (Nutr i t iona l Biochemicals Corp.) in the range of concentrat ion of 18 -100 ug/ml in the react ion mixture. 3. R ibonucleic ac id Ribonucleic ac id was determined with the use of a modi f icat ion of the o rc ino l procedure of Schneider ( 9 6 ) , which measures pur ine-bound pentose. The reagent was prepared by adding 1.0 gm of o r c i n o l , in 10 ml of 35% e thano l , to 100 ml of concentrated HC1 contain ing 0.2 gm of anhydrous FeC l^ , immediately p r i o r to use. Standard or test samples contained iii 1.5 ml were added to 1.5 ml of the reagent; the tubes were covered and placed in a bo i l i n g water bath for h5 min, and a f t e r coo l i n g , the OD at 660 mu was measured on a Beckman model B spectrophotometer. Standard curves'were prepared with yeast on RNA (Nutr i t iona l Biochemicals Corp.) using concentrat ions between 3 - 18 pg/ml in the react ion mixture. Because DNA reacts s i g n i f i c a n t l y with the orc ino l reagent, standard curves using DNA were madewith each assay. Thus, with p r io r knowledge of the DNA concentrat ion in a sample, a co r rec t i on could be.made for the cont r ibut ion madeby DNA to the 0D in the o rc ino l reac t ion . k. Phosphorus in phospholipid A mod i f i ca t ion of the technique of King (55) was used. The samples were placed, in 5 ml graduated tubes, which had been prewashed {lk hours in chromic ' ac id j 5 water r in ses , 1 6N HC1 r inse and 5 d i s -t i l l e d water r i n s e s ) , and the samples evaporated to dryness on a steam bath. 0.5 ml of 70% w/v pe rch lo r i c ac id was added and the samples digested for two hours on a sand-bath at 220 C. Following coo l ing 1 ml of water, .3 ml of 0.k% w/v ammonium molybdate and 0.2 ml of an amino-napthol sulphonic ac id so lu t ion (3.0 g NaHS04 or 28.5 g Na S 20^ + 6g Na^Sp^ +0.5 g aminonapthol sulphonic a c i d , made up to 250 ml with water) were added to each ' tube. The tubes were placed at 100 C for 10 minutes and the f i n a l volume in each tube brought to 5 ml by addi t ion of water. The OD at 820 my. was read on an Hitachi -Perktn-Elmer spectrophotometer model 124. The standard curve was prepared from dr ied Kh^PO . (1.5 g KH^PO^ per 100 ml of water is 0.3414 g phosphorus per 100 ml of water) and was in the range 0.5 - 3 VgP per tube. IV. Separation and i s o l a t i on of ind iv idua l phospholipids 1 . Thin layer chromatography Separation and q u a l i t a t i v e i d e n t i f i c a t i o n of ind iv idua l phospho-l i p i d s was accomplished by un id i rec t i ona l th in layer chromatography. 27 g of S i l i c a gel G was suspended in 60 ml of water, spread on glass plates at 0.5 mm thickness. These were a i r dr ied for 20 to 30 minutes, then oven dr ied (110 -C) for two hours and stored in a des i cca tor . The solvent used for phosphol ipid separation was chloroform:methanol: water in the r a t i o by volume 95 :36:6. For demonstration of the presence and number of neutral l i p i d s the plates were prepared in the same way and the same solvents used however the i r r a t i o by volume was changed to 99 :10 :1 . (No fur ther work was done on neutral l i p i d s ) . 2. Column chromatography Preparat ive separation of ind iv idua l phospholipids was accom-pl i shed by column chromatography. The column material was Si l i e AR-CC7 (Ma 11inckrodt- Montreal) . Five g Si l i e AR-CC7 were used for each mi l l i g ram of phosphorus ( in phospholipids) to be run through the column (see sect ion I 11-4 for phosphorus assay). The S i l i c AR-CC7 was suspended in a small volume of chloroform for 10 minutes before placing in the column. Several volumes of chloroform were run through the column u n t i l even packing was obtained. The sample was placed on the top of the column in a small volume of chloroform. E lu t ion was accomplished by sequential add i t ion of increas ing ly more polar so lvents . The volume of each successive solvent added was determined by checking the re su l t ing eluant f r ac t i ons for the presence of l i p i d by the char test (see sect ion VI - 1) i e , the solvent was not changed un t i l at least 10 success ive f r ac t i ons ' con ta ined no l i p i d mate r i a l . A small sample from each f r a c t i o n was a l so appl ied to a th in layer p late (see sect ion IV-1) and the phosphol ipid(s) present i d e n t i f i e d (see sect ion VI -1). The fo l lowing e lu t i on sequence was found to be successful for separation of the major Escher ich ia col? phospho l ip ids ; -i) 1 0 0 ml of chloroform - co l l e c ted in bulk; contains neutral l i p i d s and f ree f a t t y ac id s , i t ) 150 ml of a c e t o n e - c o l l e c t e d in 5 ml f r a c t i o n s ; contains 3 un iden t i f i ed compounds which could not be fur ther resolved on S i l i c AR-CC7• i i i ) 200 ml of acetonermethanol (9:1) " c o l l e c ted in 5 ml f r a c t i o n s ; f r a c t i on s 1 to 10 contains 1 un ident i f i ed compound. Fract ions 15 to 30 contain phosphatidyl g l y c e r o l , iv) 100. ml of chloroform:methanol (9:1)- col lected in 5 ml f r a c t i o n s ; f r a c t i on s 1 to 15 contain only c a r d i o l i p i n but f ract ionss15 to 20 a l so contain traces of phosphatidyl ethanolamine. v) 200 ml of chloroform:methanol (5:1) - c o l l e c t e d in 5 ml f r a c t i o n s ; contains phosphatidyl ethanolamine only, v i ) 100 ml methanol - c o l l e c ted in bulk contains only 1 com-pound t e n t a t i v e l y i d e n t i f i e d as 1ysophosphatidyl ethanolamine. V. Separation of f a t t y acids from phospholipids The f a t t y acids were removed from the phospholipids and methylated by" treatment with the Sweeley reagent (31) cons i s t ing of 8.6 ml of concentrated HC1 and S.k ml of water made up to 100 ml with methanol. The phospholipids were placed in a tube, evaporated to dryness, the Sweeley reagent added (1 ml per mi l l i gram of sample), the tube sealed and placed in an oven at 70 C for 18 hours. The re su l t i ng material was washed into a separating funnel with 10 volumes of petroleum ether. A small volume of water was added. A f te r vigorous shaking the top ether phase was co l l e c ted and washed several times with water un t i l the aqueous phase exhib i ted a neutral pH. The washed ether phase was evaporated to dryness under vacuum. The resu l t ing methylated f a t t y acids were d i s so lved in a small ( <100 y l ) volume of r e d i s t i l l e d hexane and a port ion of th i s material was injected into a Hewlett-Packard ga s - l i qu id chromatography system for q u a l i t a t i v e separat ion of the f a t t y ac ids . The ga s - l i qu id chromatography system was equipped with a 6 f t . g lass column of 0.25" diameter conta in ing d iethy lene g l yco l succ ina te on an inert c a r r i e r . The operat ing temperature was 160 C. 23 VI. I den t i f i c a t i on of compounds s e p a r a t e d by chromatography 1. Phospholipids Following separation on a th in layer chromatography p late as described in sect ion ( IV-1), the sequential treatment of the same p la te with d i f f e r e n t reagents allows the v i s u a l i z a t i o n of NH^'Conta ining compounds, P- conta in ing compounds, carbohydrate contain ing compounds, and f i n a l l y a l l carbon containing compounds. On a l l p l a tes , standard markers were run in p a r a l l e l w i t h the unknown for i d e n t i f i c a t i o n purposes. ' A l l standards and reagents were k indly supplied by Dr. D.J. Hanahan. The NH^-containing compounds, eg. phosphatidyl ethanolamine were v i sua l i zed by spraying the p late with n inhydr in. The phosphorus con-ta in ing compounds (a l l phospholipids) were v i sua l i zed by spraying with a molybdenum reagent cons i s t ing of 25 ml of so lu t ion A + 25 ml of so lu t ion B'+ 90 ml of water, (Solution A was 40.11g MoO^ added to a l i t r e of 25N ^SO^ boi led un t i l to ta l d i s s o l u t i o n ; so lut ion B was 1.78 g of powdered molybdenum added to 500 ml of so lu t ion A, bo i led for 15 minutes, cooled and decantered from any res idue) . Carbohydrate conta in ing compounds were i d e n t i f i e d by.a pink colour react ion to napthoresorcinol a f t e r 5 minutes at 100 C. (Napthoresorcinol reagent" cons i s t s of equal parts of 20% I^SO^ and a so lut ion of 0.1 g napthores-o rc ino l in 50 ml of ethanol. ) F i n a l l y the plates were sprayed with concentrated ^SO^ and placed in a very hot sealed oven for 5 minutes. A l l carbon contain ing compounds char and appear as black spots. The plates were photographed a f te r each spray treatment so that a perma-nent record was obtained. 2 . Fatty acids , Immediately fo l lowing the ga s - l i qu id chromatography of the unknown methylated f a t t y acids standard compounds were appl ied to the column for comparison of Rf values. A standard curve, used for est imation of carbon-chain lengths of un ident i f i ed compounds, was produced by p l o t t i n g the logarithm of the d i s tance of peaks from the solvent f ront against the length of the carbon-chain of the compound producing each peak. To ensure correct i d e n t i f i c a t i o n of compounds, a sample of the unknown was hydrogenated and then compared by g a s - l i qu i d chromatography with the unknown before hydrogenation. A peak ten ta t i ve l y assigned to a compound containing an unsaturated bond should disappear and the s i ze of the peak i d e n t i f i e d as that contain ing the same carbon-chaih length compound but no unsaturated bonds, should increase. Hydro-genation was accomplished by d i s so l v ing the material in a small volume of hexane under 50 lb . of hydrogen pressure per square inch in the presence of a platinum oxide ca ta l y s t with continuous shaking for 2 hours at room temperature. The ca ta l y s t was removed by f i l t r a t i o n before the material was rechromatographed. VII. Determination of rates of macromolecular syntheses 1. Deoxyribonucleic ac id 3 DNA synthesis was measured by incorporat ion of C ^ " t h y m i d i n e over arper iod of 5 minutes. A 1 ml c e l l sample was added to 0.1 ml 3 of l abe l l ed thymidine cons i s t ing of 2.5 ul C H^-thymidine ( s p e c i f i c a c t i v i t y 20.6 Ci/mM; 1 mCi/ml; Schwarz Bioresearch Inc., Orangetown, N.Y;), d i l u ted with unlabel led thymidine to a f i n a l concentrat ion of 0.5 ug/ml thymidine. Synthesis was stopped by adding 2 ml of cold 7.5% t r i c h l o r a c e t i c ac id (TCA) conta in ing 200 ug/ml of thymidine. Each sample was f i l t e r e d through a 0.45u M i l l i p o r e membrane and washed with 5 volumes of cold TCA and 3 volumes of 90 C water. The membranes were dr ied and placed in s c i n t i l l a t i o n v i a l s and toluene based s c i n t i l l a t i o n f l u i d added. The v i a l s were counted in a Nuclear Chicago s c i n t i l l a t i o n counter. 2. R ibonucle ic ac id The same technique as used for DNA measurements was employed for RNA l a b e l l i n g with the fo l lowing mod i f i ca t ions . 0.1 ml of a 1/10 14 d i l u t i o n of u r a c i l - 2 - C ( s p e c i f i c a c t i v i t y 46.7 mCi/mM; 0.1 mCi/ml; Schwarz Bioresearch Inc., Orangeburg, N.Y.) was added to the 1 ml c e l l samples and the t r i c h l o r a c e t i c ac id contained 200 ug/ml of unlabel led urac i1 . 3. Prote in The same technique as used fo r DNA measurements was employed for prote in l a b e l l i n g with the fo l lowing mod i f i ca t ions . 0.1 ml of a 1/10 d i l u t i o n of a mixture of 15 d i f f e r e n t n o n s p e c i f i c a l l y , t r i t i um label led amino acids ( s p e c i f i c a c t i v i t y of 16.1 mCi/mg; New England Nuclear Corp., Boston, Mass.) was added to 1 ml of c e l l sample. The t r i c h l o r a c e t i c ac id contained 200 yg of pept icase/ml. 4. L ip id i) The same technique as used for DNA measurements was employed for tota l l i p i d l a b e l l i n g with the fo l lowing mod i f i ca t ions . A 1 ml c e l l sample was added to 0.1 ml of l abe l led g lycero l con-s i s t i n g of 1 y l g l y c e r o l - 2 - H ( spec i f i c a c t i v i t y 420 mCi/mM; 1 mCi/ml; Amersham/Searle, Des P la ines , 111.), d i l u ted with unlabel led g lycero l to a f i n a l concentrat ion of 0.5 yg g l y ce ro l /m l . The incorporat ion period was reduced from 5 minutes to 3 minutes and t r i c h l o r a c e t i c ac id contain ing 200 yg of g l y c e r o l / m l . i i ) When the rate of synthesis of ind iv idua l phosphol ipids was invest igated, the fo l lowing modi f icat ions were necessary. Each 3 sample cons isted of 5 ml of c e l l s and the g l y c e r o l - 2 - H, although the same concentrat ion as in i) above had double the s p e c i f i c a c t i v i t y . The M i l l i p o r e membrane plus washed c e l l s were not placed in s c i n t i l -l a t i on v i a l s but were extracted with chloroform:methanolrwater by the procedure of B l igh and Deyer (11) described in sect ion ( l l - 3 ) . The Sephadex-G25 column p u r i f i c a t i o n step was omitted. The extracted phospholipids were evaporated to dryness by bubbling a stream of dry nitrogen through the chloroform so l u t i on . The phospholipids were redisso lved in a known volume.of chloroform and 35% of each sample was chromatographed in a p a r a l l e l with the other samples on th in layer p lates as described in sect ion (IV-1). The remaining 5%'s of each sample, was spotted on a port ion of the p la te which would not be approached by the solvent system. Following chromatography, the spots were l oca l i zed under u l t r a - v i o l e t l i g h t , each spot was scraped from the p l a t e , placed in a s c i n t i l l a t i o n v i a l and counted. The 5% of each sample not a f fected by the solvent was a l so scraped from the p late and counted so that an estimate of the tota l r a d i o a c t i v i t y in each sample could be obtained. By comparison of the counts from each i d e n t i f i e d compound with the tota l number of counts app l i ed , the r e l a t i v e cont r ibut ion of each phosphol ip id, and any change in the amount of material appl ied to the p late which could not be i d e n t i f i e d could be determined. VIII. Analys i s of c e l l envelope prote in 1. I so lat ion procedures Two i s o l a t i on procedures were used so that resu l t s from tota l c e l l envelope preparations could be compared to resu l t s from pu r i f i ed cytoplasmic membrane preparat ions. .In both cases, a double l a b e l l i n g technique was employed whereby c e l l s grown under one cond i t ion were l abe l led with C-amino ac ids and c e l l s grown under the second 3 condi t ions were l abe l led with H-amino ac id s . Comparison of the per-centage of each label in a p a r t i c u l a r prote in indicated the r e l a t i v e amounts of that prote in synthesized under the two cond i t ions . i) Total c e l l envelope preparations were obtained by a modi f i cat ion of the technique of Inouye and Guthrie (49). The c e l l s were grown in the medium as described in sect ion (1 - 2 ) , the length of time and the temperature of l a b e l l i n g d i f f e r e d depending on the p a r t i c u l a r experiment. In no case did the c e l l density exceed 8 1 x 10 c e l l s / m l . For each l i t r e of medium, e i t he r 20 uCi or non-14 s p e c i f i c a l l y l abe l led C-amino acids ( s p e c i f i c a c t i v i t y 0.1 mCi/ml; 0.067 mg/ml; New England Nuclear, 575 Albany S t . , Boston, Mass.) or 3 100 uCi of n o n - s p e c i f i c a l l y l abe l led H-amino-acids ( Spec i f i c a c t i v i t y 0.5 mCi/ml; 0.0342 mg/ml, New England Nuclear, 575 Albany S t . , Boston,. Mass.) were added. On completion of the label 1ing period the cu l tures weiie rap id ly c h i l l e d by p lac ing the containing f l a sk in a bath of acetone containing s o l i d carbon d iox ide. If removal of the rad ioact ive label was required, then f i l t r a t i o n and resuspension in precondit ioned non-radioact ive medium was used as descr ibed in sect ion (1 - 5 ) . 14 The c h i l l e d c e l l suspensions, l abe l l ed with C-amino acids under 3 one condi t ion and H-amino acids under the second condi t ion were mixed and centr i fuged at 4 C at 5000 rpm for 15 minutes in a Serva l l SS-3 re f r i ge ra ted cent r i fuge . The c e l l p e l l e t was washed 3 times with 0.01 M phosphate bu f fe r , pH 7-1 and then resuspended in 10 ml of the same buf fer at 4 C. The suspension was sonicated for four 1 minute periods using a Biosonik model Bronwi11 son ica tor , at se t t ing 1 6 0 1 . The suspension was then centr i fuged for 15 minutes at 5000 rpm in a Serva l l SS-1 at 4 C. The supernatant was c o l l e c t e d . The p e l l e t was resuspended, sonFcated again using the same protocol and fo l lowing cen t r i fuga t i on th i s supernatant was mixed with the o r i g i n a l supernatant. The combined supernatants were centr i fuged at 15000 rpm in a Serva l l SS-1 at k C for 90 minutes. The resu l t ing supernatant was removed, f reeze dr ied and stored for l a ter invest igat ion by polyacrylamide d i s c - ge l e lec t rophores i s as the "non -pa r t i cu l a te f r a c t i o n " . The p e l l e t was washed twice using the same buf fer and centr i fuged se t t ing s . The f i n a l p e l l e t const i tuted the c e l l "envel-ope f r a c t i o n . ' i i ) Pu r i f i ed c e l l membranes were obtained exact ly as described by Shapiro et a 1 . (9.8). The basic d i f f e rence from the technique described in sect ion (VI I 1-1j) being the use of ethylene d i amine - te t ra -ace t i c ac id + lysozyme to digest away mucopeptide material and the use of sucrose gradients to separate the membrane from contaminating mater i a l . 2 . Disaggregation of proteins i) Ce l l envelopes as prepared by technique (i) above, were disaggregated in 0.01 M sodium phosphate bu f fe r , pH 7.1 + 1% w/v mercaptoethanol + \% w/v sodium dodecyl sulphate + 10% w/v g lycero l at 70 C for 20 minutes. (Other temperatures and times were used but l i t t l e v a r i a t i o n in f i n a l resu l t s occurred and the described condit ions appeared optimum for disaggregat ion.) i i ) Ce l l membrane preparat ions, and the "non -par t i cu l a te f r a c t i o n " were disaggregated as described by Shapiro et_ a_l_. (98) using 40 minutes at 40 C. 3 • E lectrophores i s The 7-5% polyacrylamide gel so lu t ion was prepared by mixing 6 ml of buffer (0.1 M sodium phosphate, pH 7-1 + 0.1% w/v sodium dodecyl sulphate) + 5.4 ml of acrylamide so lut ion (3-9 gm acrylamide + 0.15 gm bis -acry lamide in 25 ml of the same buffer) + 0 . 6 ml of ammonium persulphate so lut ion (15 mg/ml of water, made f r e sh l y on each occasion) + 5% of N,N,N',N-teframethylethylenediarriine (Canal Industr ia l Co. , Rockvi1le, Md.). The so lut ion was pipetted into g lass tubes and a small volume of water c a r e f u l l y layered on the surface of the polymeris ing material so that when the gel s o l i d i f i e d (approximately 20 minutes a f t e r mixing the reagents) i t would have a f l a t upper surface. The water was removed when the so lut ion had ge l l ed . The gels were placed in an e lect rophores i s apparatus, the sample plus t rack ing dye carefu l 1y placed on the top of the g e l , both reservo i r s f i l l e d with 0.1 M phosphate buf fer + 0 . 1 % sodium dodecyl sulphate and the e l e c t r o -phoresis was run for 7 hours at 5 mi 11iamps/gel. Following e l e c t r o -phores i s , the gels were f ixed in 20% w/v s u l p h o s a l i c y l i c ac id for 12 - 18 hours. 4. Gel s l i c i n g and counting The f ixed gels were washed, with water and then f rozen. The frozen gels were s l i c e d into 1 mm th ick d i scs using a mechanical gel s i i c e r (Mickle Laboratory Engineering Co., Gomshall, Surrey, England). The d i scs were placed in s c i n t i l l a t i o n v i a l s and 0.5 ml of NCS s o l u b i l i z e r so lu t ion (Amersham/Searle Co., Des P la ines , 111.) was added to each v i a l . The NCS was d i l u ted '9 volumes NCS to 1 volume water before adding to the v i a l s . The v i a l s were incubated at 50 C for 6 - 8 hours before 10 ml of s c i n t i l l a t i o n f l u i d was added. (k gm of 2,5 - diphenyloxazole +0 .05 gm of 1 ,4-(2-(5-pheny1 oxazoly i ) ) -benzene per l i t r e of to luene) . The samples were counted in a Nuclear Chicago s c i n t i l l a t i o n counter model Unilux I I adjusted so that less than 0.1% of t r i t i u m counts reg is tered in the carbon-14 channel. The f i n a l counts were corrected fo r e f f i c i e n c y of countingiain each channel and for percentage of carbon-14 counts reg i s tered in the t r i t i u m channel. Ca lcu la t ions were based on resu l t s obtained using known standards prepared by the same technique. The r a d i o a c t i v i t y in a s ing le d i sc is expressed as a percentage of the to ta l r a d i o a c t i v i t y of that isotope which entered the g e l . 5. Standard curve for molecular weight est imation A standard curve of Rf values was produced from cytochrome C (molecular weight 12,400), myoglobin (molecular weight 17,800), chymotrypsinogen (molecular weight 25,000), ovalbumin (molecular weight 45,000) and albumin (molecular weight '67,000). The standards were treated with the SDS-disaggregation so lut ion before being run in p a r a l l e l with the experiment ge l s . The standard proteins were l oca l i zed by chemical s t a in ing . IX. Autorad iography Ce l l s were grown in minimal medium with 1 gm pepticase per 1, 2 gm dextrose per 1, 2 5 0 mg deoxyadenosine per 1 to ensure incorpora-t ion of thymidine ( 1 2 ) , 5'mg thymidine per 1 and 5 ml C H^-thymidine per 1 ( s p e c i f i c a c t i v i t y 12.2 Ci/mM; 1 mCi/2 ml; Schwarz Bioresearch Inc., Orangeburg, N.Y.) at 3 0 C for a to ta l of 20 generat ions, and d i l u ted to g ive a cu l tu re contain ing 5 x 1 0 ^ c e l l s / m l . The c e l l s were f i l t e r e d from the medium and washed three times with f resh medium minus the C H^-thymidine and resuspended in th i s medium at kl C. Samples were taken a f t e r various periods at kl C and autoradiography ca r r i ed out as described by'Caro ( 1 5 ) . I l ford Lk Nuclear Emulsion was used. A f te r exposure and development, the preparations were ob-served by phase contrast using a Zeiss microscope equipped for photogra X. Microscopy 1 . Light microscopy Ce l l s were grown in nutr ient broth at kl C un t i l long f i l a -ments were produced. These were inoculated by means of a pasteur p ipet te onto nutr ient agar covered microscope s l i de s and placed in a humid atmosphere at 3 0 C. A f te r periods of time at 30 C, s l i de s were removed and the fragmenting f i laments and developing microcolonies observed and photographed by phase contrast microscopy. 2. E lectron microscopy During f i l amentat ion at kl C and subsequent recovery at 30 C, 9 ml c e l l samples were added to 1 ml of 5% v/v glutaraldehyde buffered to pH 7.2 in 0.2 M sodium cacodylate (92). The samples were immediately centr i fuged and resuspended in 1 ml of buffered 0.05% g lutaraldehyde for 10 hours. Secondary f i x a t i o n at room temperature in acetate-verona1 -buffered \% osmium te t rox ide , and subsequent washing, agar embedding and t r e a t -ment with uranyl acetate were done according to Ryter and Kellenberger ( 9 1 ) . Dehydration and Epon embedding were done'according to Luft (64). Sections were cut with a g lass kn i fe on an LKB Type 4801A u l t r a -tome and placed on carbon-formvar-coated g r id s . A l l sect ions were stained for 3 minutes with a l k a l i n e lead c i t r a t e ( 8 5 ) . Examination and photography were done with a P h i l l i p s EM 300 e lect ron microscope. RESULTS I. D i v i s i on k ine t i c s of Escher ich ia c o l i BUG-6 1 . Determination of non-permissive temperature During the i n i t i a l i s o l a t i on of the c e l l d i v i s i o n mutants (37), 42 C was always used as the non-permissive temperature, however i t was poss ib le that the known i n a b i l i t y of Escher ich ia c o l i BUG-6 to d i v i de at 42 C would extend to temperatures below 42 C, nearer the optimum temperature for growth. Escher ich ia col? BUG-6 was grown for several generations at 30 C and the cu l tu re then subdiv ided. The subcultures were placed at 36 C, 38 C, 40 C and 42 C. The 42 C cu l tu re stopped d i v i d ing almost im-mediately and the c e l l number remained constant for the remainder of the experiment. The cu l tu re at 40 C, stopped d i v i d ing a f t e r approx i -mately 10 minutes and then resumed d i v i d i ng at a rapid ra te , 35 minutes a f t e r the 30 C 40 C s h i f t . During the period of non -d i v i s i on , c e l l s i ze increased. The cu l tu re sh i f ted from 30 C to 38 C or 36 C, did not stoped d i v id ing but d iv ided at a slower rate and eventual ly assumed the rate c h a r a c t e r i s t i c of Escher ich ia c o l i BUG-6 grown at 38 C and 36 C, Figure 1. From th is r e s u l t , 42 C was subsequently used as the non-permissive temperature and 30 C as the permissive temperature. 2. The e f f e c t of d i f f e r e n t time periods at 42 C on recovery at  30 C . Escher ich ia c o l i BUG-6 was grown for several generations at 35 20 40 60 80 100 120 140 MINUTES Figure 1. The ef fect ' on eel 1 d i v i s i o n of s h i f t i n g Escher ich ia c o l i BUG-6 from 30 C to higher temperatures. E_. co 1 i BUG-6 was grown in broth for several genera-t ions at 30 C (0) and then subdivided at the time indicated by the arrow and subcultures placed at 36 C ( • ), 38 C (A), k0 C (A) and kl C (@). 30 C and then s h i f t e d to kl C f o r d i f f e r e n t lengths of t i m e . In F i g u r e 2 zero t ime i s the time when the kl C s u b c u l t u r e s were rep laced a t 30 C. The open c i r c l e s represent growth of the c o n t r o l c u l t u r e at 30 C: One minute incubat ion at kl C (F igure 2a) , has l i t t l e e f f e c t on the d i v i s i o n of the c e l l s , but a two minute pu lse (F igure 2b), at kl C, causes a lag before d i v i s i o n resumes and 3k minutes i s r e -qu i red at 30 C before the c o n t r o l va lue i s a t t a i n e d . These experiments are summarized in F igu re 3. The t ime requ i red f o r the c e l l number of the c u l t u r e incubated at kl C to reach the 30 C c o n t r o l v a l u e i s p l o t t e d a g a i n s t the length of the per iod at kl C. Th is t ime i s 3k minutes f o r pu lses of 2, 3, and k minutes (F igures 2b, 2c, 2d). The per iod decreases r a p i d l y to a lag of 22 to Ik minutes f o r temperature pu lses exceeding 10 minutes (F igure 2 i , Ij). For temperature pu lses between k and 10 minutes in te rmedia te length lags a re observed ( F i g -ures 2e - 2h). Th is recovery pe r iod remains constant u n t i l the length of the kl C pu lse exceeds 35 minutes when the recovery per iod requ i red to reach the 30 C c o n t r o l c e l l number inc reases a g a i n . In F igu re k, the recovery p a t t e r n s a t 30 C f o l l o w i n g per iods of 45, 60, 75, 90 and 105 minutes a t kl C are shown. In each c a s e , 15 minutes i s requ i red before c e l l d i v i s i o n s t a r t s and then one rap id doub l ing of c e l l number occurs fo l l owed by a slower r a t e of d i v i s i o n u n t i l the c o n t r o l r a t e of c e l l d i v i s i o n a t 30 C i s o b t a i n e d . \ — I — I — h i i i i r i i i i 0 10 Z0 30 40 0 10 20 30 40 M I N U T E S The e f f e c t oh c e l l d i v i s i o n of. s h i f t i n g Escher ich ia  c o l i BUG-6 from 30 C to kl C for d i f f e r e n t t ime- ' i n te rva l s . E_. col i BUG-6 was grown in broth for several generations at 30 C (0) and then sh i f ted to kl C ( § ) f o r , a ) l min; b) 2 min; c) 3 min; d) k min; e) 5 min; f). 6 min; g) 7 min; h) 8 min; i) 10 min; j ) 12 min before replac ing at 30 C. Time 0 min represents the time at which the kl C cu l tures were replaced at 30 C. 38 i i i i—i i — i — i — i — r n — i — i — i — [ I 1 I i 1 i i i i i i i i i i i I 0 2 4 6 8 10 12 14 16 TIME AT 42° (MINS.) Figure 3- The va r i a t i on in recovery time for cu l tures of Escher ich ia col i BUG-^ 6 placed at kl C for d i f f e r e n t ' time i n te rva l s . E_. col i BUG-6 was grown for several generations at 30 C and subcultures placed at kl C for d i f f e r e n t time in terva l s before replac ing at 30 C. The time required for the c e l l number of a subculture to a t t a i n the 30 C contro l value a f t e r being replaced at 30 C is p lotted against the time that subculture was kept at kl C. F i g u r e k. The e f f e c t on c e l l d i v i s i o n o f s h i f t i n g E s c h e r i c h i a c o l i BUG-6 from 30 C t o kl C f o r d i f f e r e n t t i m e i n t e r v a l s . E. c o l i BUG-6 was grown, i n b r o t h f o r s e v e r a l g e n e r a t i o n s a t 30 C (0) and a t 0 min p a r t o f the c u l t u r e was s h i f t e d t o kl C (©). S u b c u l t u r e s from kl C were r e p l a c e d a t 30 C a f t e r 45 min ( • ) , 60 rriin ( H ) , 75 min (A), 90 min (A) and 105 min (9) a t kl C II. Microscopy 1 . Phase contrast l i g h t microscopy The appearance of the f i laments a f t e r 3 generations of growth at the non-permissive temperature is shown in Figune 5. Photographs were taken at 10, hS, 90, 150 and 210 minutes a f t e r s h i f t i n g the c e l l s back to 30 C (Figure 5 ) . The time before f i l a -ment fragmentation occurs on the s o l i d medium is much longer than in l i q u i d medium c f . Figure h. The resu l t ing c e l l s are smaller than normal Escher ich ia c o l i BUG-6 c e l l s growing at 30 C (Figure 5) but eventua l ly return to the normal s i ze (Figure 5e, c f . Figure 8 b ) . 2. E lectron microscopy No cross -septa were found in f i lament ing c e l l s (Figure 6 a ) . Filaments were produced at hi C and then replaced at 30 C. During the recovery period samples were removed 6, 8, 10 and 12 minutes a f te r the hi C -*-30'C s h i f t . The typ i ca l s tate of the septum at each time is shown in Figures 6, b , c ,d , and e re spec t i ve l y . Twelve minutes a f te r the hi C -* 30 C s h i f t , the f i lament is d iv ided but is not yet separated. 3. Autorad iography Segregation of the nuclei was,followed by l abe l l i n g the DNA 3 at 30 C with C (-^-thymidine for 20 generations and then al lowing f i laments to form at hi C in the absence of rad ioact i ve l a b e l . Auto-radiograms of the f i laments ind icate normal segregat ion, as seen in Figure 7 . i . e . the DNA is segregated throughout the length of the f i lament. Fragmentation of BUG-6 f i laments "during recovery a t ' 30 C. A f te r three generations at 42 C, f i l a -ments were inoculated on s l i de s precoated with nutr ient agar and incubated at 30 C. S l ides were removed at 10 (a), 45 (b), 90 (c) , 150 Cd ) , and 210 (e) minutes and examined by using a Zeiss phase-contrast microscope. Magni f icat ion of these photographs, is approximately 5,000 X. Figure 5 Ca) F i g u r e 5 (b) o F i g u r e 5 Cc) Figure 5 Cd) Al b Figure 6. E lectron microscopy of Escher ich ia c o l i BUG-6 grown at hi C and during recovery at ' 30 C. A cu l tu re of E_. col i BUG-6 was grown for 60 min at hi C in broth and then replaced at 30 C. Samples were removed at a) 0 min; b) h min; c) 6 min; d) 8 min; e) 10 min, and f) 12 minutes a f t e r the hi C to 30 C s h i f t and prepared for and observed by e lec t ron microscopy as described in Methods, sect ion (X -2 ) . Magni f icat ions are approximately 6a - 3 0 , 0 0 0 ; 6b-g - 120,000 X. Nuclear segregation in BUG-6 at the non-permissive temperature (hi C) as determined by autoradiography. Ce l l s were grown at 30 C in 5 yg C^H^-thymidine per ml at a s p e c i f i c a c t i v i t y of 1 yC/yg plus 250 yg deoxyadenosine per ml for at least 20 generat ions. The c e l l s were washed, resuspended in the same medium plus unlabel led thymidine and grown at kl C. Samples removed at in terva l s were f ixed on s l i des and coated with 11 ford Lk emulsion. The s l ides were developed a f t e r an appropriate period of exposure and examined m ic ro scop i ca l l y . Magni f icat ion is 3000 X. kk II I. Ce l l d i v i s i o n and macromolecular synthesis at 30 C and kl C. Exponential c e l l s which had been grown and measured for at least 6 generations pr ior to zero time were measured c a r e f u l l y for increase in c e l l number for 25 min. At th i s time, a sample was removed from the contro l f l a sk at 30 C and sh i f ted to kl C. As seen in F igure 8a, there is an abrupt cessat ion in increase in c e l l numbers and the c e l l number remains constant for the durat ion at kl C. On the other hand, the mass continues to increase as indicated by an increase in the peak channel pos i t ion in the Coulter Counter (Figure 8 b ) . A f te r 45 min at kl C, a sample was returned to 30 C and the recovery of the c e l l s at the permissive temperature was observed. An abrupt increase in c e l l number occurs a f t e r a lag of about 10 - 15 min during the recovery period (Figure 8 b ) . The average c e l l s i ze returns to s l i g h t l y smaller than that of a c e l l during balanced growth at 30 C (Figure 8 b ) . Analys i s of the rate of tota l p ro te in , RNA and DNA synthesis during the course of the experiment is given in Figure 3. The rate of prote in synthesis per eel 1 is constant during growth at 30 C and increases exponent ia l ly during incubation at kl C. As the c e l l s are sh i f ted to 30 C during the recovery per iod , the rate qu ick ly assumes the contro l rate co inc ident with the abrupt increase in c e l l number at about 90 min. The rate of RNA synthesis as measured by the incorp-14 o ra t ion of C -urac i l during short pulses, was almost i dent i ca l to the pattern observed for prote in synthesis (Figure 9) . Figure 8. C e l l number (A) and c e l l s i z e (B) of BUG-6 as a f u n c t i o n of time a t the permissive (30 C) and at the non-perm!sslve (42 C) temperature. Part of a c u l t u r e grown f o r several generations a t 30 C (0) was s h i f t e d to kl C a t 25 min (0). A f t e r 45 min at kl C, part o f t h i s c u l t u r e was returned to 30 C ( 0 ) . C e l l s i z e changes are monitored by p l o t t i n g the p o s i t i o n of the peak of the s i z e d i s t r i b u t i o n obtained from a pulse height analyser attached to a Coulter counter. This machtne^ Is capable of separat-ing a population o f c e l l s Into 512 r e l a t i v e s i z e s and d e p i c t i n g the number of c e l l s of each s i z e . Figure 9- The rate of p ro te in , RNA and DNA synthesis per c e l l number in BUG-6 as a funct ion of time at the permissive (30 C) and non-permissive {kl C) temperature. A cu l tu re in the steady s tate at 30 C (0) was d i v i ded , and part, of the cu l tu re was placed at kl C (•).. A f ter a period of growth at kl C that cu l tu re was again divided, ' and part of i t was returned to 30 C ((B). (i) P ro te in , ( i i ) RNA, and ( i i i ) DNA synthesis were measured by 5 - min exposures of 1 ml samples to (i) 1 yCi per ml of ^H-amino ac id mixture at a s p e c i f i c a c t i v i t y of 16.1 mCi/mg to the 007 medium conta in ing 1 mg of pepticase per ml , ( i i ) 5 yg of uraci 1 -2-1 'at'-' a s p e c i f i c a c t i v i t y of 46 .7 yCi/ymole, and ( i i i ) 0 .5 yg of thymidine-^CH, at a s p e c i f i c a c t i v i t y of 3.87 Ci/mmole^ Incorporat ion was terminated by add i t ion of two volumes of 7 .5% co ld t r i c h l o r o a c e t i c ac id contain ing 200 yg of the appropriate unlabel led compound/ml. CPM C ' 4 or- no a:.-J* -CELL — 1 » q \ -t-The rate of DNA synthesis per c e l l during growth at 30 C, kl & and during the recovery period at 30 C is indicated in Figure 9- The contro l has a r e l a t i v e l y constant count of 2.5 cpm per c e l l . At the time of the s h i f t to kl C there is a sudden increase in the rate of thymidine incorporat ion to about 6 cpm per c e l l . The rate drops s i i g h t1y and then increases during incubation at kl C to about .20 cpm per c e l l at about 160 minutes. Ce l l s which are sh i f ted back to 30 C a f te r incubation at kl C show an immediate decrease in the rate of thymidine incorporat ion, which corresponds c l o se l y to the control level (3-5 cpm per c e l l count) , which increases at about 90 minutes and then, fo l lowing d i v i s i o n of the f i l aments , returns to the control level at about 160 minutes. The rate of tota l l i p i d synthesis per c e l l during growth at 30 C, kl C and during the recovery period at 30 C, measured by g lycero l-2-3 H incorporat ion, is shown in Figure 10. The rate of l i p i d synthesis per c e l l is constant during growth at 30 C and increases exponent ia l ly at kl C. The changes in rate of l i p i d synthesis during the recovery period are d i f f e r e n t from the changes in rate observed for p ro te in , RNA and DNA synthes i s . During the period between the kl C -> 30 C s h i f t and f i lament d i v i s i o n , a high rate of label incorporat ion is maintained and even a f te r the rapid d i v i s i o n phase, at 90 minutes, . a rate of incorporat ion double that of the 30 C contro l is observed. The rate then gradual ly decreases to that of the contro l by 120 minutes. These resu l t s ind icate that the f i laments produced at kl C Figure 10. The rate of l i p i d synthesis per c e l l number in Escher ich ia c o l i BUG-6.as,a funct ion of time at •the permissive (30" C) and non-permissive (42 C) temperature. An exponential cu l tu re at 30 C (0) was d iv ided and part placed at 42 C (© ) . A f t e r ; 45 minutes at 42 C that cu l tu re was again d iv ided arid part of i t returned to 30 C ((D) . L ip id synthesis was measured by 3 minute exposures of 1 ml samples to 0.5 ug of g lycero l containing 1 yCi of g l ycero l -2 -3H . Incorporation was te rmi -nated by the add i t ion of 2 volumes of 7-5% w/v cold t r i c h l o r a c e t i c ac id contain ing 200 yg g l y ce ro l /m l . continue to synthesize p ro te in , RNA, DNA and l i p i d and that on f i lament fragmentation c e l l s which synthesize these macromolecuLes at the normal 30 C rate are eventual ly produced. Because of the 3 abrupt change in the rate of C H^-thymidine incorporat ion fo l lowing the 30 C hi C s h i f t , i t was considered that perhaps th i s was an inaccurate measure of DNA synthesis and in order to check th i s pos-s i b i l i t y , chemical f r a c t i ona t i on of c e l l s grown at 30 C and 30 C plus 60 minutes at hi C, was ca r r i ed out. RNA, DNA and prote in contents, as a percentage of dry weight, were compared (Table 1 ) . Ce l l s grown at 30 C and at hi C, appear to have very s im i l a r DNA: 3 protein ra t io s supporting the suggestion that the use of C thymidine as an ind icator of DNA synthesis gives inaccurate resu l t s immediately fo l lowing a temperature s h i f t (see above and ( 1 0 3 ) ) . The chemical f r a c t i ona t i on a l so ind icates a decrease in the percentage 1h of RNA in c e l l s grown at hi C. This is supported by the u r a c i l - C incorporat ion studies (Figure 9), which indicates a slower rate of RNA synthesis than prote in synthesis at hi C, (Figure 9)• IV. Individual phospholipid synthesis at 30 C and hi C 3 The high rate of incorporat ion of g lycero l 2- H during the recovery period (Figure 10)suggested that the rate of l i p i d synthesis was elevated during th is per iod. This would be predicted i f rapid , synthesis of septa were occur r ing . Ba l l e s t ra et_ al_. (5) have shown s p e c i f i c changes in phosphol ipid composition of c e l l s undergoing d i v i s i o n . Starka et a l . (106) described changes in the ra t io s of phospholipids when Escher ich ia col? c e l l s were converted into f i laments by p e n i c i l l i n treatment. Table I. The percentages of the tota l dry weight of c e l l s , grown at 30 C and 30 C + 60 minutes at kl C, as const i tuted by p ro te in , RNA and DNA. Percentage of dry weight of eel 1s const i tuted Growth temperature Protei n RNA DNA 30 C 51 36 3.1 30 C + 60 min at kl C 56 27 3.2 The rate of synthesis of indiv idual phospholipids was therefore invest igated to determine which phospholipids were being l abe l led and whether th i s changed during f i1amentat ion or during the rapid d i v i -s ion phase. A large cu l tu re of Escher ich ia c o l i BUG-6 was grown at 30 C for several generations and then sh i f ted to kl C for kS minutes before Being returned to 30 C. F ive ml samples were taken at in terva l s 3 and pulsed for 3 minutes with g l y c e r o l - 2 - H. The phospholipids were ext racted, separated and the i r ind iv idua l r a d i o a c t i v i t y measured. The to ta l r a d i o a c t i v i t y extracted from each sample, as phosphol ip ids, was a l so measured. In Figure 1 1 , the rate of synthesis of phosph-a t i d y l ethanolamine (PE) and phosphatidyl g lycero l (PG) are depicted. The r a d i o a c t i v i t y incorporated into each ind iv idua l phosphol ipid is expressed as a percentage of the r a d i o a c t i v i t y incorporated into the material extracted by the Bl igh and Deyer (11) l i p i d ext rac t ion technique. The rates of incorporat ion of r a d i o a c t i v i t y into PE and PG remain constant at 30 C and kl C u n t i l the recovery per iod, i e . f i l amentat ion does not a l t e r the rate of synthesis of these phospho-l i p i d s . During the recovery per iod , there is at f i r s t an apparent drop in the rate o f synthesis of PE and increase in the rate of synthesis of PG. However, when the f i laments d i v i d e , at about 90 minutes, the reverse appears to occur and by-120 minutes, the same rates as before the kl C -*• 30 C s h i f t are recovered. Escher ich ia c o l i BUG-6 contains other phosphol ip ids, namely c a r d i o l i p i n (CL) and lysophosphatidyl ethanolamine. However, the amount of r a d i o a c t i v i t y incorporated into these f rac t i ons during 3 minutes is very small and at no time does i t exceed 5% of the to ta l extracted r a d i o a c t i v i t y . The f l u c tua t i on in these minor components is very small and probably with in experimental e r ro r . For th i s reason 52 MINUTES Figure 11 . The rate of synthesis of phosphatidyl ethanolamine (PE) and phosphatidyl g lycero l (PG) in. Escher ich ia  c o l i BUG-6 as a funct ion of growth at the permissive (30 C) and non-permissive (42 C) temperatures. F ive ml samples were exposed for 3 minutes to 0.5 ug g lycero l /ml containing 2 uC i of g1ycerol-2-3H/ml. The phosphol ipids were extracted and separated, as described in "Mater ia l s and Methods" and the rad io -a c t i v i t y in each ind iv idua l phospholipid was measured. At the top of the graph the "% i d e n t i f i e d " is time percentage of extracted r a d i o a c t i v i t y i d e n t i f i e d in known phospholipids for each sample. these components were omitted from Figure 11 except for c a l cu l a t i on s of the "% i d e n t i f i e d " . This is the sum of the r a d i o a c t i v i t y in each i d e n t i f i e d phosphol ipid expressed as a percentage of the tota l extracted r a d i o a c t i v i t y . The "% i d e n t i f i e d " remains above 30% throughout the growth at 30 C and f i lamentat ion at kl C but drops to about 80% during the ear ly part of the recovery per iod , co -incident with the apparent drop in the rate of synthesis of PE. During the rapid d i v i s i o n phase the "% i d e n t i f i e d " returns to above 30%. The drop in the "% i d e n t i f i e d " indicates that l abe l led material is being extracted but i t is not one of the normally extracted phosphol ipids and does not form a spot on the th i n - l a ye r chromato-graphy system employed. Poss ib le explanations w i l l be discussed l a t e r . V. Fatty ac id synthesis at 30 C and kl C Marr and Ingraham (66) demonstrated that an increase in tempera-ture of incubation increased the degree of saturat ion found in the f a t t y acids of Escher ich ia co l ? . The composition of the f a t t y acids of Escher ich ia c o l i BUG-6, extracted from the to ta l phosphol ip id, was determined for c e l l s grown at 30 C, 30 C + 60 minutes at kl C and 8 minutes a f t e r the kl C •*• 30 C s h i f t to a scer ta in whether the pro-duction of f i laments was caused by an i n a b i l i t y to regulate the degree of saturat ion found in the c e l l s ' f a t t y ac ids . The time of 8 minutes a f t e r the kl C -*• 30 C s h i f t was chosen as th is co incided with the high r a te ' o f .phospholipid synthesis as shown in Results (sect ion IV). Because the f a t t y ac id content of d i f f e r e n t phospholipids has been shown to d i f f e r markedly (24), the f a t t y acids were a l so extracted from the major ind iv idua l phospholipids i so lated from c e l l s grown at 30 C and at'30 C + 60 minutes at 42 C. This would prevent the resu l t s from tota l phospholipid f a t t y acids masking a les ion in the contro l of f a t t y ac id saturat ion in an irid ividua 1 phosphol i p i d . The resu l t s are shown in Table II, each f a t t y ac id is expressed as a percentage of the tota l f a t t y ac ids appl ied to the gas-1iquid chromatograph. The c y c l i c compounds were i d e n t i f i e d by the i r retent ion times and comparison with the published f a t t y ac id compositionsnof Escher ich ia col? (24, 79)- The compound l abe l led a, was not i d e n t i f i e d but i t s retent ion time suggested a carbon chain length longer than C-19. The resu l t s ind icate that Escher ich la c o l i BUG-6 does not regulate the degree of saturat ion of i t s f a t t y acids and in f a c t , indicates that th i s can be accomplished very r ap id l y , as 8 minutes a f te r the 42 C -> 30 C the f a t t y ac id composition is already intermediate be-tween the 30 C and 42 C compositions. The regulat ion a l so occurs ' c o r r e c t l y for the f a t t y acids contained by a l l the major ind iv idua l phosphol ip id. During growth at 42 C, the two c y c l i c f a t t y acids apparently increase markedly. For comparison, the f a t t y acids of the parent s t r a i n , Escher ich ia c o l i AB1157 were i so lated from c e l l s grown at the same temperature.(Table III). The actual percentages of 55 Total Phospholipid PE Lyso-PE PG Temp. °C 30 42 30 42 3b 30 42 30 42 30 42 cl4-0 27 4-6 3-1 1-9 2-1. 21 4-3 1-4 20 C I 6 0 47-4 52-5 49-4 44-3 477 39-6 440 407 450 C I6I 17-5 13 6 14-7 231 147 19-5 14-3 12-8 11-2 C I 8 0 21 2-6 1-9 II 17 27 3-4 17 1-5 C I8I 29-9 20-6 26-2 29-6 26-4 33-5 230 43-4 30-8 C I7V <l 1-3 2-3 <l 51 < l 7-2 <l 51 C I9V < l 3-9 2-6 < l 30 1-9 37 < l < l c? < l < l < l < l 1-3 < l < l < l 4-3 Table II. The f a t t y ac id composition of Escher ich ia c o l i BUG-6 grown at 30 C, 30 C + 60 minutes at 42 C, and 30 C + 60 minutes at 42 C,+ 8 minutes at 30 C. Each f a t t y ac id " is expressed as a percentage of the tota l f a t t y ac id composition of phosphatidyl ethanolamine (PE), 1ysophosphatidyl ethanolamine (lyso-PE) and phosph-a t i d y l g lycero l (PG) extracted from c e l l s grown at 30 C and 30 C + 60 minutes at 42 C is a l so presented. 56 Table It I. The f a t t y ac id composition of Escher ichia c o l i AB1157 grown at 30 C, 30 C + 60 minutes at 42 C and 30 C + 60 minutes at kl C + 8 minutes at 30 C. Each f a t t y ac id is expressed as a percentage of the tota l f a t t y acids appl ied to the ga s - l i qu id chromatography system. Total phospholipid Temperature °C 3 0 C 42 C 3 0 - 4 2 - 3 0 C C 1 4 . 0 k.5 4.8 4.1 C 1 6 . 0 47.5 54.2 54.4 C16.1 22.3 19.6 18.2 C 1 8 . 0 3 . 4 4.3 3 . 7 C18.1 22.9 17.1 21.6 C 1 7 v <1 <1 <1 c 19v <1 <1 <1 c* <1 <1 <1 the d i f f e r e n t f a t t y acids d i f f e r s l i g h t l y from those found for Escher ich ia c o l i BUG-6, however the saturat ion of the f a t t y ac ids is con t ro l l ed in the same manner. The most not iceable d i f fe rences between wild type and mutant is the increase in c y c l i c f a t t y acids which occurs in the mutant at 42 C but apparently does not occur in the wi ld type. (DeSiervo (24) recent ly reported that a c i d i c ' h y d r o -l y s i s of phospholipids tends to degrade c y c l i c f a t t y ac ids so that the resu l t s obtained must be regarded as minimum va lues ) . A s im i l a r v a r i a t i o n in c y c l i c C19~fatty ac id can be found in the resu l t s of Peypoux et_ al_. (79) in the i r ana lys i s of a temperature-sens i t ive DNA mutant (Escher ichia c o l i CR34T46) and i t s parent (Escherich ia c o l i  CR34). This mutant a l so forms f i laments at the non-permissive temp-erature. VI. The e f f e c t of high sa l t .concentration on c e l l d i v i s i o n at the  non-permissive temperature Escher ich ia c o l i BUG-6 resembles a temperature-sens i t ive d i v i s i o n mutant of Escher ich ia c o l i s t r a in PAT 84 ( 8 6 ) , which is able to d i v ide at the non-permissive temperature i f supplemented with high concen-t ra t i ons of NaCl or sucrose. A s im i l a r e f f e c t is observed with Escher ich ia c o l i BUG-6, as i l l u s t r a t e d in Figure 12. Escher ich ia co l ? BUG-6 was grown for several generations:,at 30 C and part was sh i f ted to 42 C. At 74 minutes a f te r the zero time, samples were removed and brought to a f i n a l concentrat ion of 5, 7, 9 and 11 g of 100 30° 50 -20 10 30° 42° 11 <B 9 NaCl added 20 60 100 MINUTES 140 180 Figure 12. The e f f e c t of NaCl on was grown at 30 C (0) in 5 ug NaCl/1. Part of the 42 C (0) at 30 minutes. A f te r BUG-6 f i laments at 42 C. BUG-6 nutr ient broth containing cu l tu re was, sh i f ted to 45 min at the hon-permissive temperature, the cu l tu re was subdivided and NaC1 was added to each cu l tu re g iv ing a f i n a l concentrat ion of 7 g/1 ( ® ) ; 9 g/1 ( 0 ) and 11 g/1 (0) NaCl per l i t e r . Ce l l d i v i s i o n occurred approximately 15 minutes a f t e r the add i t ion of s a l t (Figure 12). As the concentrat ion of NaCl is increased to 11 g/1, there is a proport ional increase in the amount of res idual d i v i s i o n . In the experiment i l l u s t r a t e d by Figure 13, the concentrat ion of NaCl was brought to 12 g/1 at the time indicated by the arrow at 30 C, and part of the cu l tu re placed at 42 C. D iv i s i on stopped at both 30 C and kl C for 20 minutes and then both cu l tures began to d i v ide rap id ly un t i l normal d i v i s i o n rates were obtained. In th i se case, d i v i s i o n continued at kl C. VII. The e f f e c t of pantoyl lactone on eel 1 .d iv i s i on -a t 42 C It has been reported that the add i t ion of pantoyl lactone to fi lamentous c e l l s causes them to d i v ide (3, 38). The e f f e c t of add i t ion of pantoyl lactone on E s c h e r i c h i a . c o l i BUG-6 at 42 C was invest igated. A cu l tu re was grown at 30 C for several generations and placed at 42 C for 45 minutes. At th i s t ime,.var ious concen-t ra t ions of pantoyl lactone were added to subcultures. In Figure 14 the e f f e c t of the pantoyl lactone on the increase in c e l l s i ze of the f i laments is dep ic ted. In no case does c e l l s i ze decrease, i n -d i ca t ing that c e l l d i v i s i o n did not occur: The rate of increase in c e l l s i ze decreases, as the concentrat ion of pantoyl lactone added increases. This confirms previous reports that pantoyl lactone has a de le ter ious e f f e c t on growth rate (3). An increase in op t i ca l density occured in the presence of pantoyl lactone but Coulter counter measurements gave no increase in c e l l d i v i s i o n . 60 Figure 13. The e f f e c t of NaCl on Escher ichia c o l i BUG-6 at 30 C and kl C. Escher ichia col ? BUG-6" was, grown in broth contain ing 5 g NaCl/1 for several generations ( 0 ) . At the time indicated by the arrow, NaCl was added g iv ing a f i n a l concentrat ion of 12 g/1. A l so , at th i s time, part of the cu l tu re was 1 sh i f ted to kl C (<D). 61 Figure 14. The e f f e c t of pantoyl lactone on the rate of s i ze "increase of Escher ichia c o l i BUG-6 at 42 C. Escher ich ia col? BUG-6 was grown for several generations at 30 C and then placed at 42 C for 45 minutes. At th i s time, 0 minutes in the graph, the cu l tu re was subdivided and pantoyl lactone added to each cul tuve g iv ing a* f i n a l concentrat ion of 0.01 M (© ) , 0.08 M ( • ), 0.06 M ( H ), 0.04 M (A) and 0.02 M (A). A c o n t r o l ' c u l t u r e was maintained at,42 C without the add i t ion of pantoyl lactone (0). VIII. The e f f e c t of . inh ib i to r s .of c e l l wall synthesis on c e l l d i v i s i o n  during the recovery per iod. In an attempt to determine the nature of the temperature-sen s i t i ve l e s i o n , i nh ib i to r s of c e l l wall synthesis were added to cu l tures during the recovery period and c e l l number recorded as a funct ion of time. Ce l l s grown at 30 C were sh i f ted to hi C to form f i laments and then returned to 30 C at 80 minutes co inc ident with the add i t ion of one of each of the fo l lowing a n t i b i o t i c s : c y c l o -se r ine , novobiocin, vancomycin and p e n i c i l l i n . The concentrat ion of each a n t i b i o t i c corresponded to an amount which blocked c e l l d i v i s i o n in Escher ichia c o l i BUG-6 at 30 C. The e f f e c t of each a n t i b i o t i c on c e l l d i v i s i o n during the recovery period is shown in Figure 15-The add i t ion of cyc lo ser ine and novobiocin have l i t t l e e f f ec t on c e l l d i v i s i o n but do a f f e c t continued growth of the c e l l s . On the other hand , penic i11 in completely i nh ib i t s c e l l d i v i s i o n during th i s per iod . . Vancomycin allows some res idual d i v i s i o n but i nh ib i t s c e l l d i v i s i o n markedly compared e i ther to the control or to those inh ib i ted with cyc lo se r ine or novobiocin. IX. The e f f e c t of i nh ib i t o r s of macromolecular synthesis on c e l l  d i v i s i o n during the recovery per iod. The requirement for prote in synthesis during the recovery period was examined using chloramphenicol (CAM) and puromycin as i n h i b i t o r s . Figure 16 shows the resu l t s of an experiment in which CAM was added 63 F igure 15- The e f f e c t of c e l l w a l l i n h i b i t o r s on c e l l d i v i s i o n of f i l a m e n t s dur ing recovery at 30. C. BUG-6 was grown at -30 C (0) in n u t r i e n t broth fo r severa l g e n e r a t i o n s . Part of the c u l t u r e was s h i f t e d to 42 C (®) . A f t e r 45 min at 42 C, the c u l t u r e was d i v i d e d and the sub -c u l t u r e s p laced at 30 C c o i n c i d e n t w i th the a d d i t i o n o f : 20 u n i t s p e n i c i l l i n / m l ( f i ) ; 400 ug vancomycin/ml (A); 100 yg novobiocin/ml (A) or 30 ug c y c l o s e r i n e / m l (0) . A c o n t r o l was returned to 30 C w i th no a d d i t i o n ( P ) . 64 J I J I _ J I 20 60 100 140 180 MINUTES Figure 16. The e f fec t of chloramphenicol at kl C on the c e l l ' d i v i s i on of f i laments of BUG-6 during the recovery at 30 C. BUG-6 was grown in nutr ient broth at 30 C ( 0 ) . Part of the cu l tu re was sh i f ted to kl C at 30 min and returned to 30 C a t 75 min (0) . Chloramphenicol (150 ug/ml) was added to subsamples of the kl C cu l tu re at:.0.min (A ) ; 5 min (A); 10 min ( • ) and 15 min ( M ) p r io r to the return to 30 C at 75 min. to ind iv idua l cu l tures at the time of the sh i f t -back to the permissive temperature or at 5, 10 and 15 minutes pr ior to the sh i f t -back to the permissive temperature. The contro l in which CAM was omitted from the cu l tu re sh i f ted back to 30 C shows an abrupt increase in c e l l number to a level which is s l i ght ly higher than the contro l which remained at 30 C. If CAM is added to the cu l tu re at the time the cu l tu re is sh i f ted from kl C to 30 C, there is a two-fold increase in c e l l number although the f i n a l plateau is cons iderably below that of the c o n t r o l . The add i t ion of CAM, 5 min before the s h i f t to the permissive temperature, de-creases the amount of res idual c e l l d i v i s i o n . If added 10, 15 min pr io r to the sh i f t -back to 30 C, there is l i t t l e or no d i v i s i o n . The resu l t s obtained with puromycin are s im i l a r to those obtained with CAM. C l e a r l y , " d i v i s i o n p o t e n t i a l " is present when the c e l l s are incubated at the non-permissive temperature s ince a sh i f t -back to 30 C resu l t s in c e l l d i v i s i o n in the absence of prote in synthesis (Figure 1 6 ) . On the other hand, th i s potent ia l is destroyed at kl C s ince a 10 min incubation at kl C in the absence of continued prote in synthesis removes the potent ia l for c e l l d i v i s i o n at the permissive cond i t i on . In an e f f o r t to descr ibe with more p rec i s i on the s t a b i l i t y of the d i v i s i o n potent ia l at kl C, a cu l tu re was treated s i m i l a r l y to that described in Figure 16, except that CAM was added at 1 min in terva l s to separate samples before and a f te r the s h i f t from kl C to 30 C. The f i n a l c e l l number at 30 C is p lot ted as a funct ion of the time at which CAM is added 1 As seen in Figure 17A, the add i t ion of CAM at 1 - 8 min pr ior to the sh i f t -back to 30 C resu l t s in no increase in c e l l number over the amount of c e l l s present in the kl C c o n t r o l . An abrupt increase in d i v i s i o n potent ia l i is observed during the period of 6 min to 2 min p r i o r to the s h i f t -back to 30 C. A f ter the sh i f t - back , there is a slower increase in the number of c e l l s obtained at the plateau increasing with a doubling time of about 20 minutes. A plot of the log percent res idual increase of the c e l l s at 30 C as a funct ion of the time of the add i t ion of CAM, gives an estimate of the s t a b i l i t y of the d i v i s i o n potent ia l at kl C. Such a plot is shown in Figure 17B. The increase in " d i v i s i o n p o t e n t i a l " at 30 C does not appear to be as rapid as at kl C. An experiment designed to determine the extent of de novo synthesis of " d i v i s i o n p o t e n t i a l " was done using a CAM pulse of 10 min at the non-permissive temperature to remove a l l d i v i s i o n p o t e n t i a l , and observing subsequent d i v i s i o n of the f i laments at 30 C. Ce l l s grown in broth for several genera-t ions were sh i f ted to kl C at 20 min. A f te r k5 min at kl C, the c e l l s were sh i f ted back to 30 C. Sub-cultures were treated with a 10 min pulse of CAM at 10, 20 and 30 min p r io r to the sh i f t -back to 30 C. The resu l t s are shown in Figure 18. A cu l tu re which is not treated with CAM d iv ides rap id ly about 12 min a f te r the sh i f t -back to 30 C. On the other hand, a cu l tu re which has been exposed to CAM 67 E F F E C T O F C A M O N R E S I D U A L D I V I S O N D U R I N G R E C O V E R Y M I N U T E S Figure 17A. The e f fec t of chloramphenicol on res idual d i v i s i o n of f i laments during the recovery at 30 C. BUG-6 was grown in broth at 30 C, sh i f ted to kl C for k$ min and then returned to 30 C. A l iquots were removed at 1 min in terva l s for 10 min before and 10. min .after the sh i f t -back and chloramphenicol (150 ug/ml) was added fo. each sample. The f i n a l c e l l number at ta ined in each sample is p lotted against the time of chloramphenicol add i t ion to that sample. Zero is the time of the. return to 30 C. 68 D E C A Y O F D I V I S I O N P O T E N T I A L AT 4 2 ° MINUTES Figure 17B. Decay of d i v i s i o n potent ia l at 42 C. The data from Figure 17A pr io r to 0 time are used to construct th i s f i g u r e . The increase in c e l l number of chloramphenicol treated samples is p lot ted as a percentage of the increase in c e l l number to which chloramphenicol ' was added'at the time of the return to 30 C. The increase in c e l l number of the cu l ture with chloramphenicol added at the time of the s h i f t from 42 C to 30 C is p lo t ted as 100% and the increase in c e l l number of other samples is p lo t ted as a percentage of this value. J I 1 I I I I I I I I L 60 80 100 120 140 160 180 200 220 240 0 20 40 MINUTES Figure 18. The e f f e c t of a 10 min treatment with chloramphenicol at kl C on d i v i s i o n of f i laments during recovery at 30 C. BUG-6 was grown at 30 C (0) for several genera-t ions in nutr ient broth. Part of the cu l tu re was sh i f ted to kl C at 20 min and returned to 30 C a t 65 min ((D). A 10 min pulse of chloramphenicol (150 fig/ml) was given to subcultures of the kl C cu l tu re at 10 min ( • ); 20 min (/j) and 30 min (|) p r io r to the return to 30 C at 65 min. for 10 min, p r io r to the sh i f t -back and then had the CAM removed, does not s ta r t to d i v ide u n t i l nearly 35 min a f te r the sh i f t -back to 30 C, and the rate of d i v i s i o n is much reduced. A 10 min pulse of CAM 20 min pr ior to a sh i f t -back to 30 C a l l o w s for a 10 min synthesis period at kl C. Ce l l d i v i s i o n s tar t s at about kO min a f te r removal of CAM. A 10 min pulse at 30 min p r i o r to a sh i f t -back to 30 C allows for a 20 min synthesis period at kl C. Ce l l d i v i s i o n s ta r t s at about 35 min a f t e r removal of CAM. With increas ing ly longer periods of prote in synthesis at kl C a f te r the removal of CAM, the recovery d i v i s i o n rates increase, supporting thessuggestion that " d i v i s i o n p o t e n t i a l " is accumulated fa s te r at kl C than at 30 C. Since high concentrat ions of s a l t were observed to reverse the block iii d i v i s i o n (Figure 12), the requirement for protein synthesis in the presence of high concentrat ions of NaCl was examined. Figure 19 shows the e f f e c t of CAM on c e l l d i v i s i o n in the presence of a high concentrat ion of s a l t compared with the e f f e c t of CAM on c e l l d i v i s i o n during the recovery period at 30 C. A cu l tu re growing exponent ia l ly under balanced growth condit ions was sh i f ted to kl C at 30 min. A f te r growth at kl C for one generat ion, samples were removed and treated (a) with high concentrat ion of s a l t and CAM and l e f t at the non-permissive temperature, or (b) sh i f ted to 30 C in the presence of CAM. As indicated in Figure 19, the c e l l s d i v i de at the permissive temperature in the presence of CAM as had 71 MINUTES Figure. 1 9 . ' The d i f f e r e n t i a l e f f e c t of chloramphenicol on d i v i s i o n of f i laments fo l lowing the add i t ion of NaCl versus a s h i f t from kl C to 30 C. BUG-6 was grown in a nutr ient broth for several generations at 30 C ( 0 ) . Part of the cu l tu re was sh i f ted to kl C. at 30 min (® ) . At 75 min, chloramphenicol (150 ug/ml) was added to subsamples of the cu l tu re at kl C, co -inc ident with the return to 30 C (0) or co inc ident with the add i t ion of NaCl (®) to a f i n a l concen-t ra t i on of 11 g/1. been seen previous ly in Figure 16. On the other hand, c e l l s which normally d i v ide in the presence of high concentrat ions of s a l t (see Figure 12), do not d i v ide in the presence of high sa l t i f CAM is added. It is well known that s p e c i f i c i nh ib i t o r s of DNA synthesis a l so block c e l l d i v i s i o n (6, 34, 78). A dependence of d i v i s i o n upon DNA r e p l i c a t i o n has been establ i shed (16, 42), although i t is poss ib le to obtain mutants in which c e l l d i v i s i o n becomes uncoupled from DNA segregation (43, 47). In the normal d i v i s i o n c y c l e , termination of the round of r e p l i c a t i o n appears to be essent ia l for d i v i s i o n (16, 42). An examination of the coupl ing between c e l l d i v i s i o n and DNA r e p l i c a t i o n was examined in Escher ich ia c o l i BUG-6 by i n h i b i t i o n of DNA synthesis at in terva l s during f i lament growth at the non-permissive temperature. Figure 20 i l l u s t r a t e s the e f f e c t of i n h i b i t i o n of DNA synthesis during f i lament formation at 42 C on subsequent c e l l d i v i s i o n during the recovery period at 30 C. A cu l tu re of Escher ich ia col? BUG-6 grown for several generations at 30 C was measured c a r e f u l l y for increase in c e l l numbers and constancy of c e l l s i ze to e s tab l i sh balanced growth of the c u l t u r e . Twenty-f ive minutes a f t e r zero time, a port ion of the cu l ture was removed and' sh i f ted to 42 C. At subsequent times of 30, 40, 50 and 60 min, samples from the cu l tu re at 42 C were each t rans ferred to a f l a sk at the same temperature (42 C) contain ing 10 ug of n a l i d i x i c ac id (NAL) per ml f i n a l concentrat ion. At 70 min, each of the cu l tures treated with NAL was returned to the permissive temperature. A control cu l tu re 73 M I N U T E S Figure 20 . The e f f e c t "of n a l i d i x i c ac id on eel 1 d i v t s i on of the f i laments during recovery at 30 C. BUG-6 was grown at 30 C in nutr ient "broth for several generat ions. Part of the cu l tu re was placed at hi C (®) at 25 min and returned to 30 C at 70 min. N a l i d i x i c ac id (10 ug/ml) was added to subsamples at hi C, 10 min (A) ; 20 min (A) ; 30 min ( • ) and hO min ( B ) p r i o r to the return to 30 C at 70 min. which had been incubated at kl C in the absence of NAL was returned to 30 C to record the increase in eel 1 numbers fo l lowing the sh i f t -back to 30 C. As indicated in Figure 20 there is a proport ional r e l a t i o n -ship between the amount of res idual d i v i s i o n during the recovery period and the time allowed for DNA synthesis at the non-permissive temperature. Thus, blocking DNA synthesis 5 min a f te r s h i f t i n g to the non-permissive temperature allows about a 50% increase in c e l l number. If DNA synthesis is allowed to continue for k5 min, there is nearly a 200% increase in the c e l l number during the recovery per iod. Inh ib i t ion of DNA synthesis in an exponential cu l tu re does not stop c e l l d i v i s i o n immediately. Apparent ly, DNA synthesis is required u n t i l the end of a round of r e p l i c a t i o n before a c e l l can d i v i d e . Af ter completion of the round, there is no further requirement for DNA synthes i s , although d i v i s i o n does not a c tua l l y occur u n t i l a cons iderable time a f t e r the end of a round ( 8 0 ) . Since there is a time lag between the end of a round of r e p l i c a t i o n and actual sep-ara t ion of the c e l l s , a large number of the c e l l s in a random popula-t ion continue to d iv ide although DNA synthesis is blocked. This is i l l u s t r a t e d in Figure 21. Ce l l s growing exponent ia l ly are treated with NAL at 30 min and res idual c e l l d i v i s i o n is measured. If a port ion of the cu l ture is treated with NAL and simultaneously placed at kl C, there is no d i v i s i o n as a re su l t of the e f f e c t of temperature. Returning th i s cu l tu re to 30 C a f te r 45 min at the non-permissive temperature allows expression of the d i v i s i o n potent ia l which was 30° I I I I I I L_ 20 60 100 140 M I N U T E S gure 21. -Residual d i v i s i o n of BUGr6 fo l lowing the add i t ion of n a l i d i x i c a c i d . BUG-6 was grown in nutr ient broth for several generat ions, at 30 C (0). N a l i d i x i c ac id (10 ug/ml) was added to part of the cu l tu re at 30 min. The cu l tu re treated with n a l i d i x i c ac id was d iv ided and one-half was l e f t at 30 C (0) . The other hal f was placed at 42 C for 45 min and then returned to 30 C (© ) . 76 present at 25 min. The manner in which the f i laments d i v ide a f te r they have been exposed to NAL.at the non-permissive temperature and then returned to 30 C is seen in Figure 22a. For comparison, f i laments treated with CAM and then returned to 30,C are shown in Figure 22b. The l a t t e r d i v ide regu lar ly into "un i t c e l l s " whereas the former, d i v i de into c e l l s of d i f f e r e n t lengths. The i r regu la r d i v i s i o n pos i t ions is cons is tent with random segregation of a subnormal amount of DNA assuming that a l l the c e l l s , so formed, contain DNA. X. The e f f e c t of i nh ib i t o r macromolecular synthesis on c e l l d i v i s i o n  during the recovery period fo l lowing d i f f e r e n t periods at kl C. In sect ion (1-2) of the re su l t s , i t was shown that the recovery d i v i s i o n k ine t i c s d i f f e r with the d i f f e r e n t periods at the non-permissive temperature. In sect ion (VI l) of the r e s u l t s , a de ta i l ed ana lys i s of e f f ec t of macromolecular i nh ib i t o r s on the recovery c e l l d i v i s i o n fo l lowing kS minutes at kl C was presented however i t was thought n e c e s s a r y t o determine the e f f e c t of these i nh ib i t o r s a f te r d i f f e r e n t periods at kl C. A cu l tu re of Escher ich ia c o l i BUG-6 was grown for several generations in broth and then a port ion sh i f ted to kl C at 0 minutes as def ined in Figure 23- A f te r 35, 50, 65, 80 and 95 minutes, sub-cu l tures were returned to 30 C in the presence of CAM (150 ug/ml f i n a l concentrat ion) . Figure k is a s im i l a r experiment with no CAM a d d i t i o n . The resu l t s in Figure 23 show a longer lag before recovery Figure 22A. Fragmentation of n a l i d i x i c ac id f i laments during recovery at 30 C. BUG-6 was grown for 3 generations at kl C with n a l i d i x i c ac id (10 ug/ml) for the last generat ion. The f i laments were inoculated onto s l i des precoated with nutr ient agar contain ing n a l i d i x i c ac id (10 ug/ml), and incubated at 30 C for 2 hrs. Figure 22B. Fragmentation of f i laments of BUG-6 at 30 C in the presence of chloramphenicol. BUG-6 was grown for 3 generations at kl C. Three minutes p r i o r to returning the f i laments to 30 C, chloramphenicol (150 ug/ml) was added and the f i laments were then inoculated onto s l i de s precoated with nutr ient agar contain ing 150 yg CAM/ml, and incubated at 30 C for 2 hr. 77 Figure 23. The e f f e c t oh c e l l d i v i s i o n of shi f t ing Escher ich?a  c o l i BUG-6 from 30 C to kl C for d i f f e r e n t time in terva l s and then replac ing at 30 C in the presence of chloramphenicol. E_. col i BUG-6 was grown in broth for several generations at 30^1) (o) and at 0 min part of the cu l tu re was sh i f ted to kl C (®) . Subcultures from kl C were replaced at 30 C and chloramphenicol added (150 ug/ml^f inal concentrat ion) , a f t e r 35 min ( • ) , 50 min . ( ' • ) , 65 min (A) ,rr,80 min (A) and 95 min ((D) at kl C. d i v i s i o n s ta r t s than shown in Figure 4, and the number of d i v i s i o n s accomplished by the f i lament decreases when the period at 42 C exceeds 50 minutes and CAM is present during the recovery. The resu l t s from Figure 4 ind icate that d i v i s i o n potent ia l is always present regardless of the length of time at 42 C as d i v i s i o n always occurs rapd i l y during the recovery per iod. However, the resu l t s from Figure 23 show that in the presence of CAM the expression of th i s potent ia l is dependent on the length of time at 42 C. A more deta i l ed ana lys i s of the expression of d i v i s i o n potent ia l iri CAM is presented in Figure 24. A growing cu l tu re of Escher ich ia  c o l i BUG-6 was placed at 42 C and at frequent in terva l s samples were removed and placed at 30 C in CAM. The f i n a l c e l l number at ta ined is p lotted against the length of time at 42 C. A maximum occurs at about 46 minutes and plateau values occur between 20 - 22 minutes and 50 - 75 minutes incubation at 42 C. No c e l l d i v i s i o n occurs i f the c e l l s are kept at 42 C for more than 110 minutes or less than 6 minutes. Samples were a l so removed from the cu l tures described in Figure 24 and at 42 C and placed at 30 C in 10 ug NAL/ml ( f ina l concentrat ion) . Figure 25a is the of the cu l tu re at 42 C, Figure 25b is the f i n a l number at ta ined by the subcultures placed at 30 C in NAL, and Figure 25c is Figure 24 repeated for ease of comparison. The OD/^o my doubling rate is 35 minutes for 70 minutes and then begins to decrease s lowly, but the i n i t i a l doubling time for the f i n a l c e l l number reached 80 Figure 24. The e f f e c t of chloramphenicol on res idual d i v i s i o n of f i laments during the recovery at 30 C a f te r d i f f e r e n t time in terva l s at 42 C. Escher ich ia c o l i  BUG-6. was grown for several generations in broth at 30 C and then placed at 42 C. Subcultures were removed and ; rep laced at 30 C in the presence of chloramphenicol. (150 ug /ml j f ina l concentrat ion) . The f i n a l c e l l number atta ined by each subculture is p lotted against the time i t was at 42 C. V 81 T 1 r 20 40 60 80 100 120 TIME AT 42°C (MINUTES) Figure 25. The e f f e c t of na1 i d i x i c ac id or chloramphenicol on res idual d i v i s i o n of f i laments during the recovery at 30 C a f t e r d i f f e r e n t time in terva l s at kl C. Escher ich ia col? BUG-6 was grown for several generations in broth at 30 C and then placed at kl C. Figure 25a is the change in OD^rj m at kl C. Subcultures were removed and returned to m ^ 30 C in the presence of n a l i d i x i c ac id (Figure 25b) ( f ina l concentrat ion 10 ug/ml) or chloramphenicol (Figure 25c) ( f ina l concentrat ion 150 yg/ml). The f i n a l c e l l number at ta ined by each subculture is p lotted against the time i t was at kl C. by c e l l s placed In NAL is 22 minutes. Filaments incubated at kl C for kS - 75 minutes d i v i d e , in NAL, to g ive the same f i n a l c e l l number. Filaments incubated for longer than 75 minutes d i v i de in NAL to give increas ing ly more c e l l s . If the f i n a l c e l l number doubling rate is summed over the f i r s t 75 minutes, i t s value is about 35 minutes, i e . the same as the 0D,or. . The major plateau of the CAM treated c e l l s and NAL treated c e l l s occurs over approx i -mately the same incubation period at kl C. It was considered that a constant level of d i v i s i o n potent ia l might be present in f i laments which were cont inua l l y increasing in length and so producing more " d i v i s i o n s i t e s " for use of th i s f ixed amount of p o t e n t i a l . If production of " d i v i s i o n s i t e s " was dependent on completed DNA r e p l i c a t i o n (16, kl), then adding NAL at kl C and al lowing f i1amentation but no production of d i v i s i o n s i t e s should produce increas ing ly longer f i laments which, with a f ixed amount of " d i v i s i o n p o t e n t i a l " and a f i xed number of 'i'd iv i s ion s i t e s " would a l l d i v ide to the same extent. An experiment was done to test th i s hypothesis. Escher ich ia col? BUG-6 was grown at 30 C for several generations and a port ion of the cu l tu re placed at kl C. A f te r 50 minutes at kl C, NAL was added to a subculture maintained at kl C and 30 minutes l a ter CAM was added to the untreated cu l tu re and the NAL treated c u l t u r e . Both cu l tures were replaced at 30 C at th i s time (Figure 2 6 ) . This produced f i laments grown for 80 minutes at kl C which in the presence of CAM would g ive submaximum recovery 83 Figure 26. The e f f e c t of a period of i n h i b i t i o n of DNA synthesis at kl C on the res idual d i v i s i o n of f i laments during the recovery at 30 C in the presence of chloramphenicol. Escher ich ia c o l i BUG-6 was grown for several genera-t ions at 30 C (0) before part of the cu l tu re was placed at kl C (® ) . The cu l tu re at kl *C was halved a f t e r 50 minutes at kl C and n a l i d i x i c ac id (10 ug/ml f i n a l concentrat ion) was added to one ha l f (A). A f te r a tota l of 80 minutes at kl C, both cu l tures were returned to 30 C in the presence of chloramphenicol (150 ug/ml f i n a l concentrat ion) . d i v i s i o n (Figure 24). However, one of the cu l tures treated with CAM was blocked fo r DNA synthesis a f t e r 50 minutes f i l amenta t ion , i e . at the time when, i f CAM had been added and the c e l l s placed at 30 C maximum recovery d i v i s i o n would have occurred. If blocking DNA synthesis prevents " d i v i s i o n s i t e " production and the number of d i v i s i o n s poss ib le with a f ixed amount o f ' d i v i s i o n p o t e n t i a l " in the presence of CAM depends on the number of d i v i s i o n s i t e s , the cu l tu re treated with NAL at 50 minutes and so having a l imi ted number of " d i v i s i o n s i t e s " should d i v ide more at 30 C than the cu l tu re which was not so t reated. The resu l t was a c tua l l y the oppos i te, i e . the combination of NAL + CAM gave less d i v i s i o n during the recovery period than the add i t ion of CAM alone. XI. The e f f e c t of 30 C pulses on f i laments produced at 42. C It has been shown in previous sect ions that Escher ich ia c o l i BUG-6 f i laments contain a high " d i v i s i o n p o t e n t i a l " which can be expressed at 30 C. A period of 15 minutes always occurs between the 42 C 30 C s h i f t and c e l l d i v i s i o n when a nutr ient broth medium is employed, ( th is period can be a l tered by use of d i f f e r e n t media g iv ing d i f f e r e n t growth ra te s ) . It was of interest to a scer ta in at which po int , i f any, during the 15 minute period the f i laments would become committed to d i v ide even i f replaced at 42 C. A cu l tu re of Escher ich ia c o l i BUG-6 was grown for several genera-t ions at 30 C and then part of the cu l tu re placed at 42 C for 45 minutes Subcultures from the kl C cu l tu re were placed at 30 C for 6, 8, 10 and 12 minutes before replac ing at kl C. A contro l cu l tu re was sh i f t ed to 30 C but no replaced at kl C. In Figure 27, the sub-sequent d i v i s i o n patterns are dep ic ted. A 6 minute pulse at 30 C allowed v i r t u a l l y no subsequent d i v i s i o n , 8 minute and 10 minute pulses allowed increasing amounts of d i v i s i o n and a 12 minute pulse allowed an amount equivalent to one doubling of eel 1 number. In Figure k i t was shown that one doubling is the maximum amount of rapid d i v i s i o n . Figure 6 indicated that d i v i s i o n was complete a f t e r 12 minutes at 30 C, but c e l l separat ion was s t i l l required. In a l l cases, d i v i s i o n started 15 minutes a f t e r the kl C to 30 C s h i f t . The resu l t s in Figure 27 suggest that the " d i v i s i o n p o t e n t i a l " in the f i laments is not expressed at one pa r t i cu l a r time point during the 15 minutes, but is required for at least the f i r s t 12 minutes of th i s period i f maximum expression is to be obtained. The amount of " d i v i s i o n p o t e n t i a l " with in a f i lament can be contro l 1ed. by CAM add i t ion and removal at kl C, i e . a three minute pulse of CAM immedia before the kl C 30 C s h i f t reduces the potent ia l by approximately 50% (Figure 17B). By such a procedure, f i laments were produced with 50% of the normal amount of " d i v i s i o n p o t e n t i a l " and the experiment described in Figure 27 repeated. The |6 minute pulse of 30 C was omitted. The resu l t s are shown in Figure 28. There is no d i v i s i o n fo l lowing an 8 minute pulse at 30 C and the d i v i s i o n fo l lowing 10 and 12 minute pulses is very much reduced as compared to Figure 27. Thus 86 20 40 6 0 80 100 120 140 MINUTES Figure 21. The e f f e c t on c e l l d i v i s i o n of Escher ich ia col? BUG-6 o f . d i f f e r e n t length pulses of 30 C during k2 C growth. E_. c o l i BUG-6 was grown for several generations in broth at 30 C (0) and at 40 min part of the cu l tu re was sh i f ted to k2 C (®) for-45 "min before replac ing a t ' 30 C. 6 min ( • ) , 8 min ( • ) , 10.min (A) and 12 min (A) a f t e r the 42 C -»- 30 C s h i f t s , subcultures were again sh i f ted to 42 C. 1 20 40 60 80 100 120 140 MINUTES Figure 28. The e f f e c t "on c e l l d i v i s i o n of Escher ichia, c o l i BUG-6 of d i f f e r e n t 1ength pulses at 30 C during 42 C growth when the temperature pulse is prece'eded fay a 3 minute chloramphenicol pulse. E_. col? BUG-6 was grown for several generations in broth at 30 C (0) and at 35 min part of the cu l tu re was sh i f ted to 42 C (®) for 45 min before replac ing at 30 C. Three minutes before replac ing at 30. C, a port ion of the cu l tu re was sub-jected to 150 ug/CAM/ml which was removed on replac ing at 30 C (A). 8 min ( • ), .KMmin ( IB ), and 12 min (A) a f t e r the 42 C •> 30 C s h i f t , subcultures of theCCAM-pu l se j cu l tu re were again shi f ted" to.42'C the amount of d i v i s i o n fo l lowing a pulse of 30 C is dependent on the amount of d i v i s i o n potent ia l within the eel 1s produced at 42 C as welt as the length of the pulse at 30 C. . Figure 28 a l so shows a cu l tu re pulsed with CAM for the last three minutes before the kl C -»•. 30 C s h i f t and then kept at 30 C. Comparison with the contro l cu l tu re not treated with CAM shows that the CAM treatment decreased 1 the amount of rapid d i v i s i o n , supporting the idea that the rapid d i v i s i o n phase is an expression of potent ia l accumulated at kl C. XII. The e f f e c t of a short period at kl C on c e l l s of d i f f e r e n t ages A synchronized cu l tu re of Escher ich ia c o l i BUG-6 was produced by the Helmstetter and Cummings (41:) membrane technique. For technica l reasons the permissive temperature was 37 C and not 30 C as usua l ly employed; however the mutant d iv ides normally at th i s temperature. Samples were removed from the synchronous cu l tu re and placed at kl C for k minutes before replac ing at 37 C. (Four minutes is adequate time at kl C to stop c e l l d i v i s i o n in an exponential population (Figure 2 ) ) . The samples were removed from the cu l tu re at k, 10, 15 and 20 minutes a f te r the c e l l s had been co l l e c ted from the Mi11ipore membrane. The parent cu l tu re began to d i v ide 22 minutes a f te r i t s c o l l e c t i o n from the membrane and the ha l f - s tep (Ti) occurred 5 minutes l a t e r . In Figure 29 the d i v i s i o n patterns of the subcu1tures, pulsed at 42 C for 4 mins is compared with the parent cu1ture. Table IV is a summary of Figure 29. I I I I ! I 0 10 20 3 0 4 0 5 0 MINUTES F igure 29. The e f f e c t of a k minute pu lse at kl C on the d i v i s i o n of c e l l s of d i f f e r e n t ages . A synchronized c u l t u r e of Escher ich ?a c o l i BUG-6 grow!ng in broth at 37 C was produced by the technique of He lmstet te r and Cummings (41) . Subcu l tures were removed at k ( 0 ) , 10 (A), 15 (A) and 20 (p) minutes a f t e r c o l l e c t i n g the c e l l s from the membrane and placed at kl C fo r k minutes before r e t u r n i n g to 37 C. The arrows i n d i c a t e the per iod over which each s u b c u l t u r e was mainta ined a t kl C. Table IV. A comparison of the e f f e c t on c e l l d i v i s i o n of a 4 minute pulse at 42 C on c e l l s of d i f f e r e n t ages. Time of hi C pulse D i v i s i on began Time between Time between a f t e r c o l l e c t i o n of c e l l s a t : - hi O 37 C and hi C -»• 37 C d i v i s i o n s ta r t and T i No pulse, i e . contro l h minutes 10 minutes 15 minutes 20 minutes 22 minutes 25 minutes 28 minutes 33 minutes 38 minutes 17 minutes 14 minutes 14 minutes 14 minutes 23 minutes 17 minutes 17 minutes 16 minutes It can be seen from Figure 29 and Table IV that d i v i s i o n is delayed by the 42 C pulse by a constant period o f ' 14 minutes for c e l l s whose age is 10 minutes or more. Younger c e l l s do not show th i s constant t iming. This suggests that a 42 C pulse inact ivates some ma te r i a l , required for d i v i s i o n , which takes at least 14 minutes to replace or which is rap id ly replaced but is used 14 minutes before c e l l d i v i s i o n occurs . That the d i v i s i o n of younger c e l l s is only delayed by the length of the pulse supports th i s ana lys i s and confirms that funct iona l material cannot be made during a short 42 C pulse. 91 The resu l t s with synchronized populations therefore support the previous resu l t s with exponential populations that under a p a r t i c u l a r set of cond i t i ons , a constant period is always found between the time of the s h i f t from non-permissive to permissive temperature,and the onset of c e l l d i v i s i o n . XIII. Compar1 son of the proteins produced at "42 C with, those produced  at 30 C 1. Ce l l envelope proteins The ana lys i s of d i v i s i o n k i ne t i c s and use of macromolecular i nh ib i to r s suggests that f i laments at 42 C contain prote in(s ) at a high concentrat ion which are used during the rapid c e l l d i v i s i o n when the f i laments are placed at 30 C. Ce l l s normally growing at 30 C would be assumed not to have th i s high concentrat ion as they d iv ide at a normal regulated rate. It was hoped that the high concentrat ion of th i s protein (s) at 42 C would be achieved by an increased rate of . synthesis as compared to 30 C and that th is would enable the detect ion and ider i t i f i ca t i onoo f the p ro te in ( s ) . Ce l l s were grown at 30 C in t r i t i u m label 1ed ami no ac ids . A second cu l tu re was grown at 30 C, then placed at 42 C for 90 minutes, with carbon-14 amino acids present during the f i n a l 60 minutes at 42 C. The two cu l tures were rap id ly c h i l l e d and mixed. Protein p r o f i l e s of the c e l l envelopes and non-p a r t i c u l a t e f r ac t i ons were produced by d i s c - ge l e lec t rophores i s . If a prote in were synthesized more rap id ly in one cu l tu re as compared to the other cu l tu re then i t should be enriched for the type of r ad io -ac t i ve label added under the condit ions of more rapid synthes i s , when compared to the to ta l amount of label incorporated. In Figure 30, the membrane p r o f i l e s are compared, and each type of r a d i o a c t i v i t y in a gel s l i c e is expressed as.a percentage of the tota l r a d i o a c t i v i t y of i t s p a r t i c u l a r isotope that entered the g e l . At the top of the f i g u r e , the AP is p lotted for each gel s l i c e . This is the percentage of r a d i o a c t i v i t y of the isotope incorporated at kl C in a gel s l i c e minus the percentageo f r a d i o a c t i v i t y of the isotope incorporated at 30 C in the same gel s l i c e . A po s i t i ve value indicates that material in that gel s l i c e was made fas ter at kl C than at 30 C and a negative value indicates the oppos i te. Figure 30 indicates that several regions of the p r o f i l e contain proteins pre-f e r e n t i a l l y synthesized under one or other cond i t i on . However; before meaningful information can be obtained, the same experiment must be repeated with the rad ioact i ve labels reversed, ie . carbon-14 at 30 C and t r i t i u m at kl C. A l so , the same experiments must be done with the parent s t r a i n . These two contro l s remove the p o s s i b i l i t y of a r t i f a c t s due to , i) the c e l l s using the d i f f e r e n t rad ioact i ve labels d i f f e r e n t l y , and i i ) changes' in protein p r o f i l e s merely due to the temperature changes and not involved with f i l amenta t ion . Figures 31 and 32 are these con t ro l s , with the labels reversed and with the wild type s t r a i n , r e spec t i ve l y . 93 i 1 1 1 1 r i i i i i i i i u 5 15 25 35 45 55 6 5 75 85 GEL SLICE F igure 30 . Gel e l e c t r o p h o r e s i s of c e l l envelope p r o t e i n s prepared from E s c h e r i c h i a c o l i BUG-6 grown at 30 C and at 42 C. The c e l l s were grown at 30 C f o r severa l generat ions w i t h ^ C - a m i n o a c i d s (0) and at 42 C f o r 90 minutes w i t h 3H-amino a c i d s (®) present f o r the l a s t 60 minutes . The p r e p a r a t i o n of the p r o t e i n s and gel e l e c t r o p h o r e s i s were as d e s c r i b e d in " M a t e r i a l s and Methods" . The r a d i o a c t i v i t y f o r each isotope in each gel s l i c e i s expressed as a percentage of the t o t a l r a d i o a c t i v i t y of that i sotope which entered the g e l . At the top of the g raph , the AP represents the d i f f e r e n c e between the percentage of r a d i o a c t i v i t y and the percentage of 3H r a d i o a c t i v i t y in each s l i c e . A p o s i t i v e va lue i n d i c a t e s m a t e r i a l s yn thes i zed more rap i d l y . a t 42 C than 30 C and a negat ive va lue i n d i c a t e s the o p p o s i t e . 94 GEL SLICE o Figure 31.. "Gel e lect rophores i s of c e l l envelope- proteins prepared from Escher ichia col? BUG-6 grown at 30 C and 42 C. The same protocol as in Figure 30, but the l i f C-amino acids were incorporated at 42 C (0) and ^H-amino acids ( 8 ) were incorporated at 30 C. The AP has been ca lcu la ted so that po s i t i ve and negative values ind icate the same as in Figure 30. 95 GEL SLICE F i g u r e 32. Gel e l e c t r o p h o r e s i s of c e l l envelope p r o t e i n s prepared from E s c h e r i c h i a c o l i AB1157 grown at 30 C and kl C. The same pro toco l as in F i g u r e 30 and the AP i n d i c a t e s the same. Comparison of F igures 3 0 , 3 1 and 3 2 i n d i c a t e that on ly one p o s i t i v e AP a r e a , i e . increased s y n t h e s i s at kl C, can be a t t r i b u t e d to the mutant which i s not present in the w i l d type and which i s not a f f e c t e d by the type of l a b e l used. Th is p r o t e i n has a molecu lar w e i g h t ' o f between 8 0 , 0 0 0 arid 9 0 , 0 0 0 when i t s Rf is compared to the standard curve p r o -duced by running known molecu lar marker p r o t e i n s (see M a t e r i a l s and Methods, s e c t i o n VI I 1 - 3 ) on a p a r a l l e l gel of the same c o n s t i t u t i o n . The marker p r o t e i n s were prepared by e x a c t l y the same procedures as used f o r the unknown p r o t e i n s (see M a t e r i a l s and Methods, s e c t i o n VI I 1 - 1 and 2 ) . The ev idence obta ined from study ing the d i v i s i o n k i n e t i c s of E s c h e r i c h i a c o l ? BUG-6, w i t h and without i n h i b i t o r s , suggested that the " d i v i s i o n p o t e n t i a l " i s " u sed -up" in the fo rmat ion of septa and t h i s suggested that a comparison of f i l a m e n t s a l lowed a per iod at 3 0 C to d i v i d e w i t h f i l a m e n t s mainta ined a t 42 C, might demonstrate an enrichment of a p r o t e i n in the fragmented f i l a m e n t s , i e . the " d i v i s i o n p o t e n t i a l " incorporated i n t o the s e p t a . E s c h e r i c h i a c o l i BUG-6 was grown at 3 0 C f o r severa l g e n e r a t i o n s , d i v i d e d in to two c u l t u r e s , and both p laced a t kl C f o r 9 0 minutes . 1 /( A f t e r 3 0 minutes at kl C, C-amino a c i d s were added to one c u l t u r e and 6 0 minutes l a t e r that c u l t u r e was stopped by rap id c h i l l i n g . A f t e r 7 5 minutes a t kl C H-amirio a c i d s were added to the second' c u l t u r e . At 9 0 minutes t h i s c u l t u r e was f i l t e r e d from the r a d i o -a c t i v e medium, washed and resuspended in the same medium, minus the r a d i o a c t i v i t y , at 30 C. Forty minutes l a t e r , th i s cul t u re 1 s growth was stopped by c h i l l i n g and was mixed with the f i r s t c u l t u r e . The c e l l membrane proteins were extracted and'analyzed by.d i sc -ge l e l ec t rophores i s . In Figure 33 a po s i t i ve AP would ind icate material synthesized at kl C but incorporated p r e f e r e n t i a l l y into c e l l septa at 30 C. There are no d i f fe rences in the proteins l abe l led under the two cond i t i ons , suggesting that the c e l l membrane prote in con-st i tut ion was v i r t u a l l y the same kO minutes a f t e r the kl C -*• 30 C s h i f t as i t was in the f i laments at kl C. To confirm th i s r e s u l t , a comparison was made of the membrane proteins of 30 C grown c e l l s with the membrane proteins of the fragmented f i l aments , l abe l led as described above (Figure 3k). If, indeed, the proteins of the c e l l membrane do not change fo l lowing the kl C 30 C s h i f t , then com-parison of 30 C c e l l s with the fragmented f i laments should g ive the same resu l t as in Figure 30 which is the comparison of 30 C c e l l s and kl C f i l aments ; The AP of Figure 3k c l o s e l y resembles the AP of Figure 30, i e . no gross changes are seen in the prote in p r o f i l e pf fragmented f i laments when compared with non-fragmented f i l aments , ie . " d i v i s i o n p o t e n t i a l " is not incorporated into septa or the amount incorporated is so small as not to be detectab le . The prote in of molecular weight 80,000 - 90,000 which was made more rap id ly at kl C than at 30 C, may or may not be the product of the genetic les ion in Escher ich ia c o l i BUG-6. It could be a resu l t of f i l amentat ion rather than a cause. An i n i t i a l inves t i ga t ion of 98 Figure 33- Gel e lect rophores i s of c e l l membrane proteins prepared from E s c h e r i c h i a . c o l i BUG-6 grown at hi C and at hi C fol lowed by recovery at 30 C. Escher ich ia c o l i -BUG-6 was grown at 30 C and then placed at hi C for 90 minutes. One subculture was treated with ^C-amino acids for the l a s t ' 6 0 minutes at hi C ( 0 ) , a second cu l tu re was treated with ^H-am.ino acids for the last 15 minutes at hi C and then placed at 30 C in the same medium minums the rad ioact ive amino ac id s . The preparat ion of c e l l membrane proteins was by the technique of Shapiro et_ aj_. ( 9 8 ) , e lect rophores i s as described in "Mater ia l s and Methods", and preparat ion of the graph as described in Figure 30 . A po s i t i ve AP indicates that material made at hi C and pre-f e r e n t i a l l y incorporated in c e l l membrane at 30 C. 99 i 1 1 1 i 1 r G E L SLICE Figure 34- Gel e lect rophores i s of c e l l membrane proteins prepared from Escher ich ia c o l i BUG-6 grown at 30 C and at kl C fol lowed by recovery at 30 C. Escher ich ia c o l i BUG-6 was grown for several generations in 1^C-amino acids at 30 C. A second cu l tu re was sh i f ted to kl C for 90 minutes with 3H-amino acids present for the f i n a l 15 minutes, .and then returned to 30 C in the same medium minus the rad ioact ive amino ac ids . The same protocol was employed a s . fo r Figure 33- A pos i t i ve AP indicates material was made at kl C more rap id ly than at 30 C. Changes from AP of Figures 30 and 31 would represent changes due to the period of recovery growth at 30 C not present in Figures 30 and 31. th i s was attempted by production of f i laments of Escher ich ia c o l i BUG-6 by an a l t e r n a t i v e method, i e . add i t ion of n a l i d i x i c ac id at 30 C. Ce l l s growing at 30 C were labe l led with C-amino acids and compared to c e l l s growing at 30 C, in the presence of 10 ug •3 NAL/ml ( f ina l concentra t ion ) , l abe l led with H-amino ac id s . In Figure 35 the c e l l envelope p r o f i l e s are compared, a positiveAAP indicates material made more rap id ly in the presence of n a l i d i x i c a c i d . A po s i t i ve peak does occur in the same region as that found when comparing 30 C c e l l s with kl C f i l aments , suggesting that th is protein(s ) is a product of f i l amentat ion and not v i c e - v e r s a . . Other AP changes are presumably re lated to the i n h i b i t i o n of DNA synthesis by n a l i d i x i c a c i d . S imi lar p r o f i l e s a f te r NAL treatment have been observed elsewhere (Shapiro. Personal communication. 1971)-2 . Non-part i.culate proteins No enrichment of " d i v i s i o n p o t e n t i a l " was found in septa although i t s involvement in septat ion was known. It was a l so known that a high level of potent ia l must ex i s t with in f i l aments , therefore a comparison of the non-par t i cu la te proteins of Escher ich ia col? BUG-6 grown at 30 C and kl C was made, using d i s c - ge l e lectrophores i s '. In Figures 36 and 37, the p r o f i l e s of these proteins are dep ic ted, the labels being reversed from Figure 36 to 37. Comparison of these graphs ind icate s i i gh t changes in the AP but because of the large number of proteins and necess i ty of adding a large amount of prote in to these ge l s , the consistency of the AP is lack ing. To continue 101 G E L SL ICE Figure 35. Gel e lect rophores i s of eel 1. envelope proteins prepared from Escher ich ia col? BUG-6 grown at 30 C and grown at 30 G i n n a l i d i x i c ac id (10 ug/ml). One cu l tu re was grown at 30 C in ^H-amino^acids and the second at 30*;C with 10 ug/ml and C-amino ac ids . The c e l l envelope proteins were prepared and the e lectrophores i s ca r r ied out ' as described in "Mater ia l s and Methods". A pos i t i ve AP indicates material made p r e f e r e n t i a l l y in the presence of n a l i d i x i c a c i d . 5 15 25 35 45 55 65 75 85 GEL SLICE F igure 36 . Gel e l e c t r o p h o r e s i s of n o n - p a r t i c u l a t e p r o t e i n s prepared from E s c h e r i c h i a c o l ? BUG-6 grown at 30 C and at 42 C. One c u l t u r e was l a b e l l e d w i t h 1 4 C -amino a c i d s at "30 C. The second c u l t u r e was grown at 30 C and then s h i f t e d to 42 C w i t h 3 H -amino a c i d s added f o r the l a s t 60 minutes at 42 C. The n o n - p a r t i c u l a t e p r o t e i n s were p r e -pared and the e l e c t r o p h o r e s i s c a r r i e d out as d e s c r i b e d in " M a t e r i a l s and Methods" . A p o s i t i v e AP i n d i c a t e s m a t e r i a l syn thes i zed p r e -f e r e n t i a l l y at 42 C. 103 GEL SLICE Figure 37 . Gel ?.electrophores is of non-part i cu la te proteins prepared from Escher ich ia c o l i BUG-6 grown at 30 C and at kl C. The protocol was the same as in Figure 36 but the ^H-amino acids were incorp-orated at 30 C and the 1 /*c-amino acids were i n -corporated at kl C. A po s i t i ve AP indicates material synthesized p r e f e r e n t i a l l y at kl C. 104 th i s work would require the subdiv i s ion of the non-par t i cu la te f r a c t i o n , eg. the column chromatography, before comparison by d i s c -gel e lec t rophores i s . DISCUSSION A deta i led ana lys i s of the c e l l envelope biochemistry and c e l l d i v i s i o n k ine t i c s of a temperature-sens i t ive c e l l d i v i s i o n mutant of Escher ich ia col? has been presented. Comparisons can ' now be made with s im i l a r published work. Escher ich ia c o l i BUG-6 grows exponent ia l ly at both 30 C and kl C and synthesizes p ro te i n , RNA, DNA and l i p i d apparently normally at both temperatures. However, i t does not d i v ide at kl C. Other mutants have been described with apparently the same gross macro-molecular propert ies (kk, 71, 110); however, these have not been invest igated in d e t a i l and as yet no information is a va i l ab l e with regard to the i r membrane biochemistry. The chemical f r a c t i ona t i on of c e l l s and chemical assay of the const i tuents indicated that the 3 use of C H^-thymidine as a means of measuring DNA synthesis is un re l i ab l e immediately fo l lowing a temperature s h i f t . This confirms the same conclus ion reached by Smith and Pardee (103). Recently, the use of thymidine by Escher ich ia c o l i as a d i r e c t precursor of normal DNA synthesis has i t s e l f been questioned (116). The chemical 1 k f r a c t i o n a t i o n of the c e l l s and the use of u rac i l-2- C both suggest that the f i l aments , at kl C, synthesize RNA slower, when compared to the rate of prote in synthes i s , than normal c e l l s growing at 30 C. The presence of mu l t ip le genomes and therefore gene copies with in o n e ' c e l l may have an e f f e c t on the rate of t r a n s c r i p t i o n . It would be of in teres t to determine what kind(s) of RNA synthesis decreases when c e l l d i v i s i o n stops. The invest igat ion of the phospholipid and f a t t y ac id changes was undertaken because i t was known that these c e l l u l a r const i tuents should show changes when the temperature of incubation was changed (Ik, 27, 66, 101), and a l so they have been cor re la ted d i r e c t l y with c e l l d i v i s i o n (5, 69, 106). The f a t t y acids do, indeed, show the expected changes, i e . at the higher temperature of incubation the degree of saturat ion of the f a t t y acids increased when compared to the f a t t y acids extracted from c e l l s grown at the lower•temperature. The change from k2 C to 30 C is accompanied by a high rate of l i p i d synthesis and i t has been shown that the f a t t y ac id composition is rap id ly changed from that typ ica l of kl C to that t yp i ca l of 30 C. This is the f i r s t ind ica t ion that Escher ich ia c o l i can accomplish th i s conversion so rapid ly.and suggests the presence of very ac t i ve enzymes and a rapid supply of f a t t y ac ids . That the composition of th i s supply is a l so regulated is shown by the d i f fe rences reported for the f a t t y ac id contents of the ind iv idua l phosphol ip ids. This agrees with published resu l t s (Ik, 101). The most in teres t ing resu l t from the work on f a t t y acids was that the f i laments of Escher ich ia col? BUG-6 contain measurable amounts of c y c l i c f a t t y acids which were not detected in normal c e l l s at 30 C nor in the parent s t r a in at'kl C (as stated in the " r e s u l t s " , the amounts of c y c l i c f a t t y acids reported should be regarded as minima as the ext ract ion procedure would tend to degrade these compounds (24)). The ro le of c y c l i c f a t t y acids is not understood but the i r importance is becoming increas ing ly apparent (24). The i r ro le in c e l l d i v i s i o n has not been invest igated but the present work and that of Peypoux and Michel (79) both suggest that these components show marked d i f fe rences when the f a t t y acids of d i v i d ing c e l l s are compared to those of non-dj.vid ing f i lament producing c e l l s . Changes have a l so been found in the c y c l i c f a t t y ac id composition of an exponent ia l ly growing and d i v i d i ng popu la t i on -o f ' ee l Is when compared to a popu-l a t ion of the same organism in the s tat ionary non-d iv id ing phase (21,24). The r e l a t i v e amounts of ind iv idua l phospholipids have a l so been found to change dependent on the growth phase (21, 52,84) and growth media (73). However, the phosphol ipid composition and the i r r e l a t i v e rates of synthes i s 'have 'a l so 'been cor re la ted d i r e c t l y to c e l l d i v i s i o n (5) or the lack of c e l l d i v i s i o n (106). Starka and Moravova (106) produced f i laments by treatment of Escher ich ia c o l i with low levels of p e n i c i l l i n and then allowed t h e i r d i v i s i o n by add i t ion of p e n i c i l l i n a s e . They found that during f i lamentat ion the c a r d i o l i p i n content increased and phosphatidyl g lycero l content decreased although the overa l l rate of phospholipid synthesis remained the same when d i v id ing and non-d i v i d i n g c e l l s were compared. When the f i laments were allowed to d i v i d e , the i r phosphol ipid composition eventual ly returned to that of the untreated Escher ich ia c o l i i Ba l l e s t ra and Schaechter (5) induced Escher ichia col? to d i v ide whi l s t other metabolic events were repressed by using a nu t r i t i ona l shift-down. They found that d i v i d i ng c e l l s increase the i r rate of phospbatidy1-ethanolamine and decrease the i r rate of phosphatidyl g lycero l synthes i s . They a l so found that Ion eel 1s a f te r u l t r a v i o l e t i r r a d i a t i o n , i e . whi l s t forming f i l aments , had a decreasing rate of phosphatidyl e thano l -amine synthesis which increased when these f i laments were induced to d i v ide by a " s h i f t down". The resu l t s with Escher ich ia c o l i  BUG-6 d i f f e r e d from both the published reports . Since the c a r d i o l i p i n content was not measured for technica l reasons, a d i r e c t comparison with the results of Starka and Moravova (106) cannot be made. Un-l i k e both published repor t s , no d i f fe rences were found in the rates of synthesis of ind iv idua l phospholipids when d i v id ing and non-d i v i d ing f i lament producing c e l l s were compared. When the f i laments were induced to d iv ide by returning the f i laments to the permissive temperature, changes in the rates of the synthesis of ind iv idua l phospholipids were apparent. The rate of synthesis of phosphatidyl g l ycero l increased and that of phosphatidylethanolamine apparently decreased when measured by a three minute incorporat ion of g l y c e r o l -3 2 - H.- This resu l t is the opposite of that reported by Ba l l e s t ra and Schaechter (5) but the resu l t may in fact be misleading because during th i s per iod, 20% of the r a d i o a c t i v i t y extracted from the c e l l s as phosphol ip id, was not i d e n t i f i e d and could conceivably have been trapped in a precursor of phosphatidyl ethanolamine. An examination of the b iosynthet ic pathways of phospholipids shows a divergence at the point of c y t i d i n e d iphosphate-d ig lycer ide in the pathway to phosphatidyl-ethanolamine and phosphatidyl g l y c e r o l / c a r d i o l i p i n ( 5 3 , 1 0 5 ) . From th i s intermediate phosphat idy l -g lycero l is pro-duced by add i t ion of g l ycero l and release of c y t i d i n e monophosphate. This step would increase the r a d i o a c t i v i t y in phosphatidyl g lycero l 3 i f g l y c e r o l - 2 - H were used to label the phosphol ip ids. Phosphat idy l -ethanolamine is produced from the CDP-dig lycer ide v i a phosphat idy l -ser ine . T h i s l a t t e r compound remains at the o r i g i n on the chroma-tography system employed in th i s work. Therefore, in a three minute pulse of r a d i o a c t i v i t y , combined with a much increased rate of synthes i s , i t is qu i te conceivable that the apparent decrease in rate of synthesis of phosphatidyl ethanolamine when compared to the rate of synthesis of phosphatidyl g lycero l can be accounted for by r a d i o a c t i v i t y trapped in phosphatidyl ser ine. If th i s is t rue , then during the rapid phase pf phosphol ipid synthes i s , presumably due to septa synthes i s , a l l phospholipids a re ' synthes ized more rap id ly and no changes re lated to c e l l d i v i s i o n can be demonstrated from th i s work. If the resu l t s are to be taken at face va lue, then during septa formation in th i s system,.the rate of phosphatidyl g lycero l synthesis is increased at the expense of phosphatidyl ethanolamine synthes i s ; and a l so a component(s) is synthesized which can be extracted by the B l igh and Deyer ( 1 1 ) ex t rac t ion technique but has not been i d e n t i f i e d . The prote in composition of the c e l l envelope of Escher ich ia  col? has been invest igated, using sodium dodecyl sulphate d i s c - g e l e l ec t rophore s i s , in several l abora to r ie s , eg. (51, 74, 93, 95, 9 8 ) , and the advantages and l im i ta t i ons of th i s and other techniques for membrane ana lys i s have been reviewed (54). It has been poss ib le to re l a te d e f i n i t e changes in the prote in composition of the c e l l envelope with mutations causing an a l t e r a t i o n in the res i s tance to a n t i b i o t i c s (74) and c o l i c i n E2 (93)- Studies re l a t i ng to mutants involv ing DNA r e p l i c a t i o n and c e l l d i v i s i o n have demonstrated that the proteins in the c e l l envelope do d i f f e r when d i v i d ing c e l l s are compared with non-d iv id ing c e l l s (51, 9 8 ) , whether the f i laments are produced by mutation (49, 98) or by use of i nh ib i t o r s (51). Inouye and Pardee (51) have demonstrated that a prote in of molecular weight 39,000 is always enriched in the c e l l envelope of f i laments and some techniques of producing f i l aments , eg. add i t ion of n a l i d i x i c a c i d , a l so produce an enrichment for a .prote in of molecular weight 80,000. It was suggested that the l a t t e r prote in may be a dimer of the 39,000 molecular weight p ro te in . In the present study, the f i laments were found to have a protein(s ) of molecular weight '80,000 - 90,000 en-riched in the i r c e l l envelope. The protein p r o f i l e s of to ta l c e l l envelope, eg. Figure 30 and p u r i f i e d c e l l membrane, eg. Figure 34, both demonstrated th i s enrichment and although there are d i f fe rences in the overa l l shape of the p r o f i l e s , there is l i t t l e d i f f e rence in the percentage change in the enriched protein peak. This suggests that p u r i f i c a t i o n of the c e l l membrane from the c e l l envelope'does-not increase the p u r i f i c a t i o n of the enriched peak. However, as discussed in the review (54), and as can be seen in the published resu l t s (51, 9 8 ) , quant i ta t ion is not r e a l l y f e a s i b l e by th i s technique. Unl ike the resu l t s with a n t i b i o t i c res i s tance ( 7 4 ) , the increased prote in in the c e l l envelope of f i laments has not been d i r e c t l y re lated to the mutation in c e l l d i v i s i o n , and the resu l t s of Inouye and Pardee (51) suggest that the enriched prote in is a resu l t of the i n h i b i t i o n of eel 1 d i v i s i o n and not v i ce - ve r s a . The present resu l t s in which a s im i l a r enriched peak was found in n a l i -d i x i c ac id induced f i laments would tend to support th i s conc lus ion. In the present work, f i lamentous eel 1s were a l so compared to the c e l l s produced when the f i laments had been induced to d i v i d e . It was known thatt the f i laments would d i v ide at the permissive temperature without prote in synthes i s , therefore the f i laments which were induced to d i v ide were l abe l led j u s t ' b e f o r e being placed at the permissive temperature. Thus the comparison would show i f p ro te ins , synthesized at the non-permissive temperature, were i n -corporated p r e f e r e n t i a l l y into the septa at the permissive temperatu No p re fe ren t i a l incorporation. was found and the resu l t s showed that the gross prote in composition remained constant up to 40 minutes a f t e r the c e l l s were returned to the permissive condit ions (Figure 3 (The method of c a l c u l a t i o n used in analyzing these resu l t s would not de tec t ' a change in a minor prote in cons t i tuent . Unfortunately 112 ca l cu l a t i on s which would detect such a change would a l so tend to exaggerate a r t i f a c t s ea s i l y produced by double l a b e l l i n g techniques.) These resu l t s therefore ind icate that the septa do not have a d i f f e r -ent protein con s t i t u t i on from the tubular part of the c e l l envelope. However, u n t i l improved methods of membrane ana lys i s and assays for proteins involved in c e l l d i v i s i o n are a v a i l a b l e , no d e f i n i t e con-c lu s ion can be made. Experiments using c e l l wall i nh ib i to r s ind icate that the block in d i v i s i o n in Escher ich ia c o l i BUG-6 is associated with a terminal step in c e l l wall synthesis (Figure 15). The act ion of cyc lo ser ine blocks the add i t ion of a l a - a l a to the N-acetyl-muramic ac id residue (108). Vancomycin has been shown to compet i t ive ly i nh i b i t the inser t ion of N-acetyl-muramic acid-N-acaty1-g1ucosamihe-pentapeptide from the phosphol ipid c a r r i e r to the acceptor in the c e l l wall ( 9 ) . P e n i c i l l i n acts at a step ju s t one stage l a t e r ; the j o i n ing of the pentapeptide uni ts (108). The act ion of novobiocin is m u l t i f o l d ; a f f e c t i n g DNA synthes i s , RNA synthesis and membrane a c t i v i t y (70, i 104). There is a suggestion that i t a l so acts in c e l l wall synthesis at some stage between the formation of the N-acetyl-muramic ac id -N -acetyl -g lucosamine-pentapeptide and i t s insert ion into the c e l l wall (108). As seen in Figure 15, cyc lo ser ine and novobiocin have l i t t l e e f f e c t on the expression of the d i v i s i o n step during the recovery per iod. These resu l t s suggest that novobiocin acts at a stage in c e l l wall synthesis which is p r i o r to that of vancomycin and p e n i c i l l i n . Evident ly Escher ich ia c o l i BUG-6 c e l l s contain c e l l wall p re -cursors at the non-permissive temperature because when c e l l d i v i s i o n is made poss ib le by reversa l of the temperature from kl C to 30 C, . d i v i s i o n occurs in the presence of c y c l o s e r i n e . The inser t ion of the precursors into cross wall does not occur at kl C s ince pen i -c i l l i n and vancomycin block d i v i s i o n at 30 C ind ica t ing precursors are not iii pos i t i on when incubated at kl C. Ce l l d i v i s i o n of Escher ich ia c o l l BUG-6 occurs during the recovery period in the presence of chloramphenicol i f the period at kl C does not exceed 110 minutes. . The a b i l i t y of f i laments to d i v i de without prote in synthesis indicates that c e l l s produce and contain a " d i v i s i o n p o t e n t i a l " at kl C which can be expressed at 30 C. When prote in synthesis is inh ib i ted at the non-permissive temperature d i v i s i o n potent ia l decays and th i s decay rate has a h a l f - l i f e of approximately 0 .5 minutes at kl C. Extrapolat ion of the decay curve, Figure 17b, indicates an o r i g i na l amount of d i v i s i o n 2 potent ia l of approximately 10 greater than that required to give d i v i s i o n . If d i v i s i o n potent ia l decays at kl C and yet there is d i v i s i o n potent ia l at any time the c e l l is sh i f ted from kl C to 30 C, the rate of synthesis of d i v i s i o n potent ia l at kl C must be greater than the rate of decay. Hence the doubling time of d i v i s i o n potent ia l at kl C must be less than the h a l f - l i f e of 0 .5 minutes. This is con-trasted to the rate of accumulation of d i v i s i o n potent ia l at 30 C equivalent to a doubling time of -20 minutes (Figure 17a), as c a l -culated from the increase in f i n a l eel 1 number for c e l l s treated with chloramphenicol during the 30 C recovery period. - It should be noted that the normal generation time of Escher ich ia c o l i BUG-6 at 30 C is 45 minutes, so that even at 30 C, d i v i s i o n potent ia l is synthesized fa s ter than required and, as a constant d i v i s i o n rate is foundj i t is necessary to postulate a contro l mechanism (see below). • . A model which is cons is tent with these observations Is shown in Figure 38. D i v i s i on potent ia l (d) necessary for the expression of c e l l d i v i s i o n is synthesized as a resu l t of metabolic a c t i v i t i e s and growth. The accumulation of substrate induces formation of d as in other standard inducib le systems. In Escher ich ia c o l i BUG-6 d is a temperature-sens i t ive compo-nent which operates at 30 C, but not at kl C. At 42.C, d changes rap id ly but reyer s ib l y to an inact ive form (X^). This conclus ion is based on the observation that Escherich?a c o l j BUG-6 stops d i v i d i ng abrupt ly upon s h i f t i n to kl C (Figure 8a). Inactivat ion must be rever s ib le s ince d i v i s i o n potent ia l is expressed as eel 1 d i v i s i o n at 30 C in the absence of prote in synthesis at 30 C (Figure 16). At kl C, X.j decays i r r e v e r s i b l y to X^, which cannot return to a c t i v e d'. The h a l f - l i f e of th i s conversion is about 0.5 minutes (Figure 17b) . SUBSTRATE ' - 5 * —— > PRODUCT Model for production and interconvers ion of d i v i s i o n potent ia l in Escher ich ia col I BUG-6. d = ac t i ve d i v i s i o n potent ia l produced at 30 C. X.j = rever s i b l y inact ive d i v i s i o n potent ia l formed by p lac ing d at hi C. X„ = i r r e v e r s i b l y inact ive d i v i s i o n potent ia l formed by decay of X at hi C. 116 If d,decays to through X^  at a very rapid ra te , and ye t ' there is always excess d i v i s i o n potent ia l at kl C in the absence of chloramphenicol, the formation of -Xj. must be in excess of the formation of X^- Since the conversion of d to v ia X^  at kl C is about 0.5 minutes, the doubling time of d and X^ is less than 0.5 minutes. This appears tp be an extreme derepressed rate fo r the formation of d, s ince the doubling time for d at 30 C is about 20 minutes (Figure 17a). As indicated, in Figure 38, the synthesis of d depends upon the concentrat ion and i n teg r i t y of d. It is proposed that d is converted to X^ at kl C, which is an inac t i ve unit with respect to feedback repress ion. Therefore, continued and derepressed rates of formation of d occur at the non-permissive temperature and i t would be predicted that a high steady state level of X^  would eventual ly be atta ined at the non-permissive temperature. This steady s tate may, however,' be constant on a per c e l l bas i s , a gene dose basis or a concentrat ion bas i s . The "amount" of d i v i s i o n potent ia l has been equated above to the number of residual d i v i s i on s occurr ing at 30 C in the presence of chloramphenicol a f t e r kS minutes at kl C, but an increase in the length of time the c e l l s were maintained at kl C would g ive both a d i f f e r e n t estimate of the high internal level and of the decay k i ne t i c s (Figure 24), so that although the model f i t s well with one p a r t i c u l a r set of cond i t i ons , i t must be considered with relevance to other cond i t i ons . If indeed, a derepression mechanism is occurr ing then very b r i e f periods at kl C should cause, (a) not a l l d to be converted to ; and (b) not a maximum amount of X^  as time is required for i t s synthesis fo l lowing derepress ion. A pulse of kl C i f less than 1 minute gives l i t t l e e f f e c t on d i v i s i o n (Figure 2a), i e . s u f f i c i e n t d is s t i l l a va i l ab le for d i v i s i o n . Pulses of 2, 3, and k minutes at kl C stop d i v i s i o n but the recovery of 30 C takes longer than the recovery of c e l l s kept at kl C for longer per iods. This would be predicted i f d were converted to X^  and some to but the rapid derepressed rate of synthesis was not yet in e f f e c t . As the period at kl C becomes longer, the rate of recovery becomes fa s te r and the very rapid d i v i s i o n phase becomes more evident (Figure 2 and 3 ) . Approximately 10 minutes at kl C gives a maximum rate of recovery. Presumably th i s is the period required to obtain a steady s tate level of X^ or the minimum time required to express d i v i s i o n p o t e n t i a l . In th i s ana l y s i s , the time required for the kl C pulsed c e l l s to a t t a i n the c e l l number of the 30 C contro l c e l l s is used as an assay of " d i v i s i o n p o t e n t i a l " . These resu l t s sub-s tan t i a te the hypothesis of a derepress ion causing an accumulation of d i v i s i o n p o t e n t i a l . One might a n t i c i p a t e that the time required to a t t a i n the control value would be independent of the length of the incubation period at the non-permissive temperature a f te r the i n i t i a l derepression stage. However, as seen in Figure k, the time required to a t t a i n the control value increases for incubation periods at kl C in excess of 35 minutes. However, the k ine t i c s of recovery are in teres t ing in two respects: (1) there seems to be an i n i t i a l burst of c e l l d i v i s i o n which cons t i tu tes exact ly one doubling at a constant of 15 minutes a f t e r the return to the permissive temperature; and (2) the rate of subsequent c e l l d i v i s i o n is more rapid than that of a normal exponential cu l tu re at 30 C. Once the contro l value is atta ined and a l l c e l l equivalents have been expressed, the growth rate re -turns to the normal 30 C;;rate. From these r e su l t s , i t is obvious that the assay of " d i v i s i o n p o t e n t i a l " described for short kl C pulses, does not.apply to long periods at kl C, suggesting i t s app l i c a t i on to short pulses may a l so be incor rec t . An a l t e r n a t i v e poss ib le assay would be the amount of very rapid d i v i s i o n occurr ing during the recovery phase. This becomes a constant as the period of f i l amentat ion -at kl C exceeds the time for one op t i ca l density doubling at kl C, i e . 35 minutes. This constant is equ iva lent " to exact ly one doubling in the c e l l number over that maintained at kl C. Using th i s as an assay of " d i v i s i o n p o t e n t i a l " then one must conclude that the f i laments contain a steady state level at less than enough for two c e l l d i v i s i on s and that i t can only be used in unit amounts therefore only on d i v i s i o n occurs. This constant of one d i v i s i o n has a l so been reported for the recovery d i v i s i o n of Ion f i laments (28) and f i laments produced by thymine s tarvat ion ( 2 6 ) . Because of th i s c o r r e l a t i o n with very d i f f e r e n t systems, i t would seem poss ib le that the common act o f d i v i d ing is c o n t r o l l i n g the amount of d i v i s i o n rather than a supply of some d i v i s i o n p re requ i s i t e , eg. separat ion of genomes which were prev ious ly in teract ing could f ea s i b l y a f f e c t the subsequent act ions of the c e l l s . If a constant amount of d i v i s i o n potent ia l were ava i l ab l e to be used at 30 C, then returning f i laments of d i f f e r e n t lengths to 30 C in the presence of chloramphenicol should g ive a constant amount of d i v i s i o n . This experiment is described in Figures 23 and 2k, and no such constant amount of d i v i s i o n occurred except over l imi ted time periods of incubation at k2 C. In f a c t , the maximum amount of d i v i s i o n gave approximately four times the number of c e l l s at k2 C. With very short periods at k2 C, no d i v i s i o n occurred at 30 C in the presence of chloramphenicol, again supporting the idea of a time required for the bui ld-up of potent ia l (Figure 2k). No d i v i s i o n occurred for c e l l s kept at k2 C for more than 110 minutes when placed at 30 C in chloramphenicol. Ce l l s treated the same way but replaced a t ' 3 0 C in the absence of chloramphenicol would have d iv ided once r ap i d l y , then at a f a s te r than normal rate u n t i l the 30 C contro l c e l l d i v i s i o n rate and number were atta ined (Figure k). One explanation of th i s paradox was to assume a f ixed amount of d i v i s i o n potent ia l per c e l l , enough for about four d i v i s i o n s ; The s ing le d i v i s i o n at 30 C when no chloramphenicol was present would be explained by creat ing a fur ther contro l system, ie . prote in synthesis at 30 C caused the p re fe ren t i a l completion of one d i v i s i o n s i t e before a second was s t a r ted . When prote in synthesis was not allowed at 30 C, th i s contro l is lost and a l l potent ia l d i v i s i o n s i t e s are a v a i l a b l e for expression of d i v i s i o n p o t e n t i a l . If a f i xed amount o f . d i v i s i o n . p o t e n t i a ] per c e l l were ava i l ab l e then an optimum c e l l length would occur when the f ixed amount of pot-, en t i a l was completely expressed by the number of a va i l ab l e " d i v i s i o n s i t e s " . As the number of s i te s increased over th i s optimum, the number of completed d i v i s i o n s would decrease as the f ixed amount of potent ia l became d i l u ted throughout the f i lament and fewer s i te s obtained s u f f i c i e n t potent ia l to d i v i d e . A f i lament length would be reached when no c e l l d i v i s i o n occurred. This system descr ibed agrees with the information in Figure 24. If the number of d i v i s i o n s i t e s could be kept constant at the optimum, even though the f i l a -ment ' increased in length, then op returning to 30 C, a f ixed optimum number of d i v i s i on s would be predicted assuming rap id , f ree mob i l i t y of d i v i s i o n p o t e n t i a l . Figure 26 is an attempt at such an experiment, assuming completed DNA r e p l i c a t i o n would t r i gger the production of d i v i s i o n s i t e s . N a l i d i x i c ac id was added a f t e r 50 minutes at 42 C and then the c e l l s returned to 30 C a f te r a fur ther 30 minutes at 42 C. The combined e f f e c t of n a l i d i x i c ac id and chloramphenicol produced less d i v i s i o n than f i laments treated with only chlorampheni-col and returned to 30 C at the same time. If, indeed, DNA r e p l i -ca t ion does regulate the number of d i v i s i o n s i t e s , th is re su l t would ind icate that d i v i s i o n s i t e s are not l i m i t i n g . On the other hand, the resu l t may i n d i c a t e . d i v i s i o n s i t e s are not con t ro l l ed by the amount of DNA rep l i ca ted or that the idea of " d i v i s i o n s i t e s " as physical e n t i t i e s , is i t s e l f untenable. The above d i scuss ion has demonstrated that the model proposed (Figure 38) for explanation of the c e l l d i v i s i o n k i ne t i c s requires fur ther assumptions, i f i t is to be extended to periods of f i l a -mentation at kl C in excess of kS minutes but other experimental r e su l t s , using. a kS min period have been presented and should be accomodated by the model. When Escher ich ia c o l i BUG-6 is sh i f ted from 30 C to kO C , ' d i v i s i o n does not cease immediately and then a f t e r a period of f i l amenta t ion , d i v i s i o n res tar t s spontaneously. Sh i f t s from 30 C to 38 C or 36 C cause a decrease in the d i v i s i o n rate fo1 lowed by an litncreased rate before rates c h a r a c t e r i s t i c of 38 C and 36 C are obtained (Figure 1). These resu l t s can be incorp-orated into the model i f the react ion d -»• occurs more slowly as the "non-permiss ive" temperature is lowered. I n i t i a l l y , d i v i s i o n takes longer to cease at kO C, s ince the rate of conversion of d "•> X is slower than at kl C and takes longer to reduce d below the threshold va lue. Once d is depleted, derepression w i l l occur and rapid synthesis of d ' w i l l ensue. Eventua l ly , the level of d w i l l exceed that required for d i v i s i o n s ince the derepressed rate of formation of d is f a s ter than the decay rate of d "^.X^ at kO C. D iv i s i on potent ia l (d) continues to be made at the derepressed rate u n t i l a l l of the d i v i s i o n s i t e s or the substrate is exhausted. This leads to the burst in c e l l d i v i s i o n seen in Figure 1.. When al 1 d i v i s i o n s i t e s have been expressed, the substrate is depleted and there is normal repress ion of the synthesis of d. The steady s tate l eveT of d, maintained by normal feedback con t ro l s , is s u f f i c i e n t to al low normal c e l l d i v i s i o n . The rate of synthesis of d at 40 C would be ant i c ipa ted to be f a s te r than that observed at 30 C because of cont inual decay of d to the inact ive forms and X^. At 36 C and 38 C, the same arguments apply, except the decay of d to X^ is never complete, but is enough for derepression to be tr iggered and cause subsequent'rapid d i v i s i o n . In order, to accommodate the resu l t s of temperature s h i f t s from 30 C to temperatures beloW 42 C, i t was necessary to suggest the d is converted to X^  at some f i n i t e rate. If th i s were t rue, then a rate of conversion of d -*• X^  should a l so occur at 42 C, and i f c e l l s could be produced with an excess of d, then on placing at 42 C c e l l d i v i s i o n should occur u n t i l the ava i l ab le d is exhausted by usage in d i v i s i o n and by conversion to X^. The model pred icts that r e -turning c e l l s to 30 C a f t e r 45 minutes at 42 C w i l l produce f i l a -ments with a high level of d (by revers ion from X^). In Figure 27" these c e l l s were again sh i f ted to 42 C a f t e r d i f f e r e n t periods at 30 C. If these c e l l s had not been sh i f ted to 42 C, they would have d iv ided 15 minutes a f t e r the 42 C s h i f t . As the length of 30 C incubation is increased before the c e l l s are returned to 42 C, the ava i l ab l e time at "42 C for d-=» X to occur before the c e l l s are due to d iv ide is decreased. This pred ic t s that with longer periods at 30 C, more res idual d i v i s i o n should occur at kl C. This resu l t was obtained. The same experiment was repeated but before the kl C 30 C s h i f t , a 3 minute pulse of chloramphenicol was given to the c e l l s . This short i n h i b i t i o n of prote in synthesis should, according to the model, reduce the d i v i s i o n potent ia l by 50% (Figure 1 7 b ) . Comparing the amounts of res idual d i v i s i o n at kl C fo r the chloramphenicol treated c e l l s with the non-treated c e l l s a f t e r the same period of 30 C incubat ion, demonstrated that the former completed very much less d i v i s i o n (Figure 2 8 ) . T h i s • r e s u l t ' is cons i s tent with a rate for d->- X.j because a lower o r i g i n a l amount of d would require less time at kl C before the internal concentrat ion of d f e l l below the threshold required for d i v i s i o n . This resu l t a l so supports the hypothesis that d i v i s i o n potent ia l decays at kl C when prote in synthesis is i n h i b i t e d . Filaments of Escher ich ia co l i ' BUG - 6 growing at kl C d i v ide when the external osmotic pressure is increased, however, th i s d i v i s i o n requires de novo prote in synthesis (Figures 12 and 1 9 ) . The same resu l t was found for a s im i l a r mutant ( 8 6 ) . Osmotic remedial mutants have been invest igated most thoroughly in yeast (7) kO) and from th i s work (7) i t has been concluded that osmotic remedial condi t ions are required to a id e i ther the polymerisat ion step in the formation of a prote in from sub-unit po lypept ides, or in the production of the cor rec t t e r t i a r y conf igurat ion of nascent polypeptides f a c i l i t a t i n g the i r conversion to an ac t i ve gene product. The inac t i va t ion of d i v i s i o n potent ia l (d) could be due to separat ion of sub-units (X^) and the add i t ion of osmotic remedial condi t ions would.enable de novo sub-units to combine at the non-permissive temperature and give d i v i s i o n . That d i v i s i o n occurs rap id ly fo l lowing the add i t ion of NaCl suggests thatt the de novo sub-units could form ac t i ve potent ia l by combination with the (X^) sub-units a lready present. The combina-t ion of de novo sub-units with X^  sub-units as compared to the r e -associated of on l y ' X j - sub -un i t s could a l so expla in the d i f f e r e n t d i v i s i o n k ine t i c s observed when c e l l s kept at kl C are returned to 30 C with or without the add i t ion of chloramphenicol. A comparison can now be made between the proposed model for control of c e l l d i v i s i o n iii Escher ich ia col i BUG-6 and models pro-posed for contro l of c e l l d i v i s i o n in other•systems. Ce l1 sd i v i s i on is dependent on DNA r e p l i c a t i o n (16, k l ) , and th i s has been found to : be true for Escher ichia. c o l i BUG-6 ( Figure 20), although Figure 25b indicates that rate of DNA r e p l i c a t i o n may osc i1 late"and not be main-tained at the same doubling rate as that found for the op t i ca l dens i ty . Donachie (26, 27, 28) has shown that f i laments produced by i n h i b i t i o n of DNA r e p l i c a t i o n do not d i v ide u n t i l the DNA/mass r a t i o is r e -turned to normal. This is not the case for Escher ich ia c o l i BUG-6. As shown in Figure 20, f i laments can be produced with less DNA than normal, but on returning to the permissive temperature, these f i laments d i v i d e . The number of d i v i s i on s dependinpnothe amount 125 of DNA with in the f i l aments , i e . in th i s system the DNA/protein r a t i o is not a d i r e c t contro l of c e l l d i v i s i o n . The resu l t s with Escher ich ia c o l i BUG-6 do however .conf i.rm Donachie's observations that fragmenting f i laments are l imi ted to one rapid d i v i s i o n (26, 2 8 ) . The model proposed here is v i r t u a l l y ident ica l to that of Adler et_'aj_. ( 3 ) . They suggested a ! 'mater ia l e s sent ia l f o r . c e l l f i s s i o n " which the c e l l produced during normal growth and which was " d e s t r o y e d or otherwise inact ivated as a funct ion of ,time, f i s s i o n occurs each time a c r i t i c a l level of th i s mater ia l is exceeded". It was suggested that f i l amentat ion occurred when th i s material never reached the c r i t i c a l l e v e l . In th i s respect Ion eel 1s were assumed to produce barely enough material and any in jur ious agent, eg. r ad i a t i on , caused the level to drop below that needed for d i v i s i o n . The les ion causing f i l amentat ion in Ion has now been determined as an e f f e c t on DNA synthesis (35, 113), but, as th i s mutation can be suppressed ( 6 5 ) , and the wi ld type 1on + is dominant in a merozygote (113), the involvement of a prote in product must be necessary. Ah a l t e r n a t i v e model for i r r a d i a t i o n f i l amentat ion is that the i r r a d i a t i o n blocks the synthesis of a repressor by a f f e c t i n g DNA i n t e g r i t y , which prevents the synthesis of an i nh ib i t o r of c e l l d i v i s i o n (117). This is a negative contro l model and a s im i l a r model has a l so been suggested by Inouye (47) in which the d i v i s i o n s i t e is normally blocked by a d i v i s i o n i nh ib i t o r (M) which is i t s e l f , inact ivated by a second a g e n t ' ( l ) . The d i v i s i o n k i ne t i c s of Escher ich ia col i 'BUG-6 could be explained i f the (t) materia l were " d i v i s i o n p o t e n t i a l " which f a i l e d to recognize (M) at 42 C. The normal pro-duct ion of ( l) would be c y c l i c , . as suggested by Inouye (47), and in th i s respect, th i s model is exact ly the same as the model of Adler et a l . and the one proposed here. The l o c a l i z a t i o n of (M) could enable the p re fe ren t i a l completion of one d i v i s i o n as described for fragmenting f i laments i f the c e l l recognized the o ldest (M) p ro te in , ie . the (M) present at the t ime-of - the 30 C -> kl C s h i f t . Paulton (77) has a l so described a system which requires the c e l l to contro l the l o c a l i z a t i o n and p re fe ren t i a l completion pf d i v i s i o n s i te s . There are several pred ic t ions for d i v i s i o n contro l which could easiMy be tested on Escher ich ia col i BUG-6. For example, B a z i l l (8) has suggested f i l amentat ion is due to an a l tered outer c e l l wall con f i g u r a t i o n . Prev.ic (81) suggested that- the absence of an enzyme re lated to d iamino-pimel ic ac id metabolism might cause f i l amentat ion A l s o , Inouye and Pardee (50) showed that the polyamine content must be cor rec t for c e l l d i v i s i o n to occur ' in the i r system and claimed th i s as a p re requ i s i t e f o r d i v i s i o n . The presence of a recA mutation has been found to al low d i v i s i o n in three systems which normally would not be expected to d i v ide (36, 48, 56). A va luable experiment"would be the production of a s t r a i n of Escher ich ia c o l i  BUG-6 contain ing the recA les ions to invest igate whether d i v i s i o n would then occur at 42.C. In summary, Escher ich ia coli 'BUG-6 appears to be a va luable tool in the inves t i ga t ion of eel 1 d i v i s i o n in Escherich ia col I. The present work has character ized the d i v i s i o n k ine t i c s of th i s mutant and a l i t t l e of i t s biochemistry. The contro l of c e l l d i v i s i o n has been shown to involve a temperature-sens i t ive prote in which is produced at a very high rate at the non-permissive temp-erature. A prote in has been i d e n t i f i e d in the c e l l envelope which is produced at a much higher rate at kl C than 30 C and i t is now necessary to determine whether or not th is prote in i s ' t h e one c o n t r o l l i n g the d i v i s i o n k ine t i c s described in th i s work. BIBLIOGRAPHY A d l e r , H.I.-, W.D. F i s h e r , , A . Cohen and A.A. H a r d i g r e e . 1967. M i n i a t u r e E s c h e r i c h i a c o l i e e l 1s d e f i c i e n t i n DNA. P r o c . Nat. Acad. S c i . U.S.A.'57: 321-326. A d l e r , H.I., W.D. F i s h e r and A.A. H a r d i g r e e . 1969- C e l l d i v i s i o n i n E s c h e r l c h l a c o l i . T r a n s ; N.Y. Acad. 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