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Cell division in Echerichia coli : the involvement of the peptidoglycan Groves, David John 1971

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CELL DIVISION IN ESCHERICHIA COLI: THE INVOLVEMENT OF THE PEPTIDOGLYCAN BY DAVID JOHN GROVES B.Sc. ( B i o c h e m i s t r y ) , The U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1966 M.Sc. ( M i c r o b i o l o g y ) , The U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1968 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department o f M i c r o b i o l o g y We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d A u g u s t , 1971. In present ing t h i s thes is in p a r t i a l f u l f i l m e n t o f the requirements fo r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I fu r ther agree that permission for extensive copying of t h i s thes is for s c h o l a r l y purposes may be granted by the Head o f my Department or by h is represen ta t i ves . It is understood that copying or p u b l i c a t i o n o f t h i s thes is f o r f i n a n c i a l gain sha l l not be allowed without my wr i t ten permiss ion . Department of y The Un ivers i ty o f B r i t i s h Columbia Vancouver 8, Canada ^ t e f f Q fc. ( I 1 ^ 1 | i ABSTRACT The ro le of the c e l l envelope in c e l l d i v i s i o n has been examined using mutants of Escher ich ia c o l i which are temperature-sensi t ive in the process of c e l l d i v i s i o n . These mutants grow normally at 30 C but exh ib i t morphological changes at hi C. Two assays fo r p e p t i d o g l y c a n - s p e c i f i c a u t o l y t i c enzymes have been developed. One depends on the prevention of synthesis of a u t o l y t i c enzymes using chloramphenicol , whi le the a u t o l y t i c capaci ty is determined by observing the rate of c e l l l y s i s in the presence of low leve ls of a m p i c i l l i n . The second assay is based on the in  v i tro re lease of s p e c i f i c rad ioac t ive fragments from peptidoglycan prev ious ly labe l led with rad ioac t ive diaminopimelic a c i d . The major i ty of c e l l s in an exponential populat ion of E_. col i B/r/1 are not lysed by p e n i c i l l i n i f chloramphenicol is added. However, larger c e l l s are p a r t i c u l a r l y suscept ib le to l y s i s . Three types of l y s i s have been def ined by observing rates of l y s i s of synchronous cu l tures at d i f f e r e n t ages and at d i f f e r e n t growth ra tes : (A) l y s i s associa ted with c ross -wa l l formation during c e l l d i v i s i o n ; (B) l y s i s associated with i n i t i a t i o n and segregation of the r e p l i c a t i o n of DNA; and (C) l y s i s associated with general expansion of the c e l l w a l l . Filamentous mutants of E_. col i which segregate DNA exh ib i t l y s i s in excess of type C whi le f i laments formed by i n h i b i t i o n of DNA synthesis exh ib i t l y s i s only of type C. The a u t o l y t i c enzymes of E_. col i appear to be t i g h t l y bound to l o c a l i z e d areas of the c e l l envelope as determined by the in v i t r o assay. Q u a l i t a t i v e d i f fe rences between the a u t o l y t i c a c t i v i t i e s of normal c e l l s and of f i laments formed by i n h i b i t i o n of DNA synthesis are descr ibed . TABLE OF CONTENTS Page INTRODUCTION 1 I . THE GENERAL NATURE OF THE CELL WALL 1 I I . THE PEPTIDOGLYCAN 2 A. Unique P r o p e r t i e s o f t h e P e p t i d o g ! y e a n . . . 2 B. B i o s y n t h e s i s o f t h e P e p t i d o g l y c a n . . . . 4 C . A u t o l y t i c Enzymes 5 1. Involvement i n b i o s y n t h e s i s 5 2. P o s s i b l e f u n c t i o n s o f a u t o l y t i c enzymes . 7 3. A s s a y systems f o r a u t o l y t i c enzymes . . 9 I I I . EFFECT OF PENICILLIN 10 IV. CELL WALL GROWTH AND CELL DIVISION 13 A. S u r f a c e Growth L o c a l i z a t i o n i n Rod-Shaped B a c t e r i a 13 B. . - A symmetric Growth 14 V. REGULATION OF CELL DIVISION 15 A. I n t e r r e l a t i o n s h i p o f C e l l D i v i s i o n and DNA Repl i c a t i o n 15 B. I n t e r f e r e n c e w i t h C e l l D i v i s i o n 16 1 . Mutants 16 2. C h e m i c a l s 17 MATERIALS AND METHODS 18 i v Page I. BACTERIAL STRAINS AND CULTURE CONDITIONS. . . 18 I I . CONSTRUCTION OF MUTANTS 20 A. C o n s t r u c t i o n o f T e m p e r a t u r e - S e n s i t i v e (TS) F i l a m e n t o u s Mutants 20 1. M u t a g e n e s i s 20 a. E t h y l methane s u l f o n a t e (EMS) . . 21 b. Ni t r o s o g u a n i d i n e (NTG) 21 2. E n r i c h m e n t 22 a. S u c r o s e g r a d i e n t 22 b. Membrane f i l t e r . . . . . . . 23 3. S c r e e n i n g 23 B. C o n s t r u c t i o n o f A u x o t r o p h i c Mutants . . . 25 1. NTG mutagenesis 25 2. P e n i c i l l i n e n r i c h m e n t 26 3. C o n j u g a t i o n 27 k. T r a n s d u c t i o n 27 I I I . ASSAY OF AUTOLYTIC ENZYMES 29 A. In V i v o Assay o f A u t o l y t i c Enzymes . . . 29 B. In V i t r o A s s a y o f A u t o l y t i c Enzymes . . . 30 1. I s o l a t i o n o f p e p t i d o g l y c a n 30 2. P a r t i c u l a t e enzyme p r e p a r a t i o n . . . 33 3. S e p a r a t i o n and d e t e c t i o n o f c e l l f ragments by chromatography . . . . 36 Page a. P r e p a r a t i o n o f p l a t e s . . . . . . 36 b. S o l v e n t systems and c o n d i t i o n s . . . 36 c. D e t e c t i o n 37 k. A s s a y p r o c e d u r e . 38 RESULTS 40 I. IDENTIFICATION AND CLASSIFICATION OF TEMPERATURE-SENSITIVE DIVISION MUTANTS . . . . 40 I I . ACTIVITY OF AUTOLYTIC ENZYMES IN VIVO . . . . 42 A. A u t o l y t i c A c t i v i t y i n E x p o n e n t i a l C u l t u r e s . i+2 B. A u t o l y t i c A c t i v i t y i n Synchronous C u l t u r e s . 54 I I I . ACTIVITY OF AUTOLYTIC ENZYMES IN VITRO . . . . 62 DISCUSSION 76 LITERATURE CITED 88 vi LIST OF TABLES Page Table I. L i s t of bac te r i a l s t r a i n s used 19 Table II. C l a s s i f i c a t i o n of temperature-sensi t ive d i v i s i o n mutants of Escher ich ia col i 41 Table III. The r e l a t i o n s h i p between the time of c e l l d i v i s i o n , the end of a round of DNA r e p l i c a t i o n and l y s i s A 58 Table IV. Amount of peptidoglycan released as fragments by a u t o l y t i c enzymes 67 Table V. Attempts to construct dap auxotrophs of Escher ich ia col i 73 vi i LIST OF FIGURES Page Figure 1. E f f e c t of a m p i c i l l i n and chloramphenicol (CAM) on an exponential cu l tu re of Escher ich ia c o l i s t r a i n B/r/1 growing in minimal medium 43 Figure 2. The e f f e c t of treatment with a m p i c i l l i n and CAM on the s i z e d i s t r i b u t i o n of Escher ich ia  col i B/r/1 44 Figure 3- a , b. E f fec t of a m p i c i l l i n and CAM on the morphology of Escher ich ia col i B/r/1 45 Figure k. The e f f e c t of mixtures of a m p i c i l l i n and CAM or p e n i c i l l i n - G and CAM on an exponential cu l tu re of Escher ich ia col i 46 Figure 5. The e f f e c t of mixtures of D-cyc loser ine and CAM, a m p i c i l l i n and puromycin, or a m p i c i l l i n and r i fampin on exponential cu l tures of Escher ich ia col i 48 Figure 6. The e f f e c t o f a m p i c i l l i n and CAM on exponential cu l tu res of Escher ich ia c o l i B/r/1 and Escher ich ia c o l i BUG-6 growing in enriched medium MS Figure 7- The e f f e c t of a m p i c i l l i n and CAM on f i laments of Escher ich ia col? B/r/1 induced by treatment with n a l i d i x i c a c i d , and formed by Escher ich ia  col i BUG-6 grown at k2 C . 50 Figure 8. The e f f e c t of a m p i c i l l i n and CAM on c e l l s of d i f f e r e n t ages in cu l tures growing synchronously in minimal glucose medium . . 51 Figure $. The e f f e c t of a m p i c i l l i n and CAM on a synchronous cu l tu re growing in minimal medium 53 Figure 10. The e f f e c t of a m p i c i l l i n and CAM on a synchronous cu l tu re growing in enriched medium . 56 vi i i Page Figure .11. The e f f e c t of a m p i c i l l i n and CAM on synchronous c e l l s in the second generation of a " s h i f t - u p " cond i t ion 57 Figure 12. The e f f e c t of n a l i d i x i c ac id on ampici11 in-CAM l y s i s of synchronous cu l tures in m i n i ma I med i urn 60 Figure 13. a , b. The e f f e c t of n a l i d i x i c ac id on the r e l a t i v e rate of l y s i s during the second generation of " s h i f t - u p " 61 Figure 14. Chromatographic separat ion of amino ac ids from p a r t i a l l y p u r i f i e d and p u r i f i e d pepti dog 1 yean 62 Figure 15- Separat ion by chromatography of fragments released from p u r i f i e d peptidoglycan by enzyme preparat ions . . . . 65 Figure 16. incorporat ion of rad ioac t ive DAP by Escher ich ia col i ATCC 13070 l y s " ' 68 Figure 17- Release of rad ioac t ive DAP by p a r t i c u l a t e enzyme preparat ions at d i f f e r e n t pH values 70 Figure 18. Release of r a d i o a c t i v i t y by p a r t i c u l a t e enzyme preparat ions made from normal c e l l s and f i laments induced by n a l i d i x i c ac id treatment 71 Figure 19. Separation by chromatography of fragments released by p a r t i c u l a t e enzyme preparat ions l abe l l ed with rad ioac t ive DAP . . . . . . 72 ACKNOWLEDGEMENTS My s incere grat i tude is extended to Dr. D. Joseph Clark for h is superv is ion and encouragement of the research, and for h is help and const ruc t ive c r i t i c i s m in the preparat ion of the manuscript . I would l i k e to thank Dr. J . J . R . Campbell and the other members of my committee^for t h e i r ass is tance in e d i t i n g the thes i s . I would a l s o l i k e to thank my wife H a l l i e fo r her cons idera t ion and support during the research and preparat ion of the t h e s i s . L a s t l y , I o f f e r my thanks to my fe l low students , John N. Reeve and George G. Khachatourians and Clayton R. Bagwell , for help in my experiments, and to Mrs. Rita Rosbergen for the typing of the thes i s . INTRODUCTION I. THE GENERAL NATURE OF THE CELL WALL. The w a l l , as def ined by Stolp and Star r (118), includes a l l s t ruc tures outs ide the cytoplasmic membrane, less the external secre t ions such as capsu les , sl imes and gums. The topography (38, 106), chemical composition (36, 37, 103, 107) and involvement of the bac te r i a l c e l l wall in c e l l d i v i s i o n (50) have been recent ly and thoroughly reviewed. The c e l l envelope of Gram-negative bac te r ia contains a cytoplasmic membrane, var ious intermediate layers and an outer "membrane" s t ruc ture which appears as a t r i p l e - l a y e r e d s t ruc ture in e lec t ron micrographs. The wall external to the cytoplasmic membrane of Gram-negative bac te r ia contains p r o t e i n , l i p o p r o t e i n and 1 ipopolysacchar ide , as well as the pept idoglycan. Compared to the wal ls of Gram-negative organisms, the wal ls of Gram-posit ive organisms contain increased amounts of peptidoglycan and l i t t l e , i f any, l i p i d . As w e l l , they conta in var ious proport ions of t e i c h o i c a c i d s , polysacchar ides and p r o t e i n s . The major topographical change is the absence of the outer membrane found in Gram-negative envelopes; most Gram-posit ive organisms possess a s i n g l e amorphous band of wall material external to the cytoplasmic membrane. 2 II. THE PEPTIDOGLYCAN A. Unique Propert ies of the Peptidog 1 yean. The c e l l envelope has unique p r o p e r t i e s . It is d i f f e r e n t -i a l l y permeable to solutes due to the ac t ion of the membrane(s), i t is the r i g i d s t ruc ture which maintains the shape of the organism, and i t is a l i k e l y candidate for involvement in c e l l u l a r funct ions requi r ing a mechanical s t r u c t u r e , such as morphogenetic changes, anchoring of s t ructures fo r m o t i l i t y , segregat ion of nucle i and maintenance of the r i g i d c e l l shape. The r i g i d s t r u c t u r e , the R layer , was demonstrated to occur in (129) and was iso la ted from the c e l l wall (130). The R layer was shown to re ta in i ts shape and r i g i d i t y when a l l other wall components were removed. The pept idoglycan, a l s o known as murein sacculus (131) or mucopeptide only comprises up to 10% of the c e l l wall of Gram-negative organisms but can c o n s t i t u t e 90% of the dry weight of the c e l l wall of Gram-posit ive organisms (106). Although there is th is vast d i f f e r e n c e in the quant i ty of the peptidoglycan in d i f f e r e n t organisms, i t is found in v i r t u a l l y a l l bac te r ia except some extreme h a l o p h i l e s , L-forms and spherop las ts , and i ts general chemical nature is s u r p r i s i n g l y s i m i l a r in d i f f e r e n t organisms (36). The peptidoglycan is composed of glycan strands made of a l t e rna t ing -1-4 l inked N-acetyl glucosamine and N-acetyl muramic a c i d , with peptide s ide -cha ins jo ined at the N-terminal end to the l a c t y l group of the N-acetyl muramic a c i d . This ^-1-4 l inkage is the 3 basis of r i g i d s t ruc tures in many other b i o l o g i c a l systems using c h i t i n and e e l l u l o s e (113)• The te t rapept ide subunits have the general sequence R^-D-glutamic R^-D-alanine. The R^  residue is usua l ly a diamino acid such as L - o r n i t h i n e , ( . - lys ine or meso', -diaminopimel ic a c i d , and the R^  group is usua l ly L -a lan ine (36). The peptidoglycan is unique because of the use of the D-isomers of glutamic ac id and a l a n i n e , as well as diaminopimel ic ac id (DAP) which are seldom found elsewhere in nature, and the high content of amino sugars. DAP auxotrophs (dap ) have proven extremely useful for the s p e c i f i c rad ioac t ive l a b e l l i n g of the peptidoglycan (111). In b a c t e r i a , l y s i n e is synthesized by decarboxylat ion of diamino-p imel ic ac id (86), and the genet ics of var ious diaminopimelic ac id and l y s i n e auxotrophs of Escher ich ia c o l i have been studied (120, 18). Since aspartate-^-semialdehyde is a precursor of DAP and a common intermediate in the synthesis of threonine and methionine, the control mechanisms f o r these amino acids are extremely complex (86, 28). The peptidoglycans d i f f e r mainly in : (1) the composition of the peptide s ide cha in ; (2) the degree of c r o s s - l i n k a g e of these subunits and (3) the length of the glycan cha ins . The peptide subunits have been c l a s s i f i e d into f i v e groups, based on the i r chemical composition (36). The degree of c r o s s - l i n k a g e is extremely v a r i a b l e depending upon the organism and the nature of the peptide subunit (92). The average length of the glycan strands is v a r i a b l e , from 10 to 100 monosaccharide uni ts (101). k Although the peptidoglycan has been descr ibed as a s i n g l e r i g i d macromolecule ( I 3 l ) » there is no d i r e c t evidence that i t is not made up of several smal ler macromolecules. Because workers have genera l ly found r e l a t i v e l y short glycan chains in the i so la ted peptidoglycan (101 , c f . 1 3 0 . the o r i g i n a l idea of continuous glycan loops around the circumference of organisms has been changed to the concept of a looser , less r i g i d network. The dangers of in te rpre t ing changes in gross chemical composition in terms of s t r u c t u r a l models have been emphasized (50). B. Biosynthesis of the Pept idoglycan. The b iosyn the t i c steps necessary to peptidoglycan synthesis have been well character ized fo r both Gram-posit ive and Gram-negative organisms. The process has been e luc idated by Strominger and h is col leagues (37. 92), using Staphylococcus  aureus and Micrococcus lysode ik t i cus as the model systems. The process involves three main systems, the f i r s t of which is the stepwise add i t ion of the amino ac ids of the pentapeptide to a u r id ine -d i -phosphate (UDP)-muramyl group, by so lub le enzymes s p e c i f i c for both the acceptor group and the amino ac id to be added. Then the UDP-muramyl pentapeptide precursor is attached to a C-55 isoprenoid a lcohol l i p i d c a r r i e r , with subsequent add i t ion of N-acetyl glucosamine and t-RNA mediated add i t ion of c r o s s - l i n k i n g amino ac ids where necessary. F i n a l l y the complete 5 precursor molecule is t ransfer red from the l i p i d c a r r i e r and cova lent ly l inked to the peptidoglycan acceptor molecule. The react ion sequence is s i m i l a r for a l l b a c t e r i a , inc luding E_. col i . The major means of v a r i a t i o n is the synthesis of species s p e c i f i c peptide sequences. The peptide s ide chains are jo ined in a c r o s s - l i n k i n g react ion by a t ranspept idase enzyme. This c r o s s - l i n k a g e react ion has only been demonstrated in preparat ions from E_. col i . Although the formation of peptide bonds is necessary fo r peptidoglycan s y n t h e s i s , the normal route of polypept ide synthesis is not used, as shown by the continued synthesis of peptidoglycan in the presence of chloramphenicol (82). Mutants which are presumably d e f e c t i v e at var ious stages in peptidoglycan synthesis have been iso la ted (85) which accumulate nucleot ide-1 inked c e l l wall precursors (9*0 at the non-permissive temperature. C. A u t o l y t i c Enzymes. 1. Involvement in s y n t h e s i s . When Weidel and others (130, 131) descr ibed the i r model f o r a murein s a c c u l u s , they recognized the necess i ty fo r a balance between the degradative and synthe t ic mechanisms to enlarge the r i g i d macromolecule. They predicted both the usefulness of na tu ra l l y occurr ing l y t i c enzymes in determining the murein s t r u c t u r e s , and reviewed the substant ia l body of evidence fo r the ex is tence of a u t o l y t i c enzymes. Perturbat ions of growth which unbalanced precursor synthesis caused autolysis< (81). The fragments re leased from E_. c o l i during a u t o l y s i s (95) were s i m i l a r to the precursor molecules accumulated in the presence of p e n i c i l l i n (94). These observat ions led to a u n i f i e d theory of peptidoglycan synthesis according to which i n t r a c e l l u l a r h y d r o l y t i c enzymes cleaved c e r t a i n covalent l inkages of the pept idoglycan so that precursor subunits could be inserted and cova len t ly l inked to the e x i s t i n g pept idoglycan. Limited cleavage of bonds at any given time would not a f f e c t the general i n t e g r i t y of the pept idoglycan r i g i d s t ruc ture (131» 114). The only a u t o l y t i c a c t i v i t i e s which would be useful for inser t ion of precursor uni ts would be N-acetyl muramidase, which cleaves the glycan chain between the N-acetyl muramic ac id moiety and the N-acetyl glucosamine, re leas ing reducing groups on the N-acetyl muramic a c i d , and endopeptidases which s p l i t the peptide b r idge . These and other a u t o l y t i c a c t i v i t i e s might be involved in the l o c a l i z e d hydro lys is required for morphogenetic mechanisms. On the other hand, they may represent a reversal of a normally b iosyn the t i c react ion (50). A u t o l y t i c a c t i v i t i e s have been found in v i r t u a l l y every organism in which they have been sought (36, 37). For example, E. col? has been shown to have an endopeptidase, N-acetyl muramyl 7 alanine amidase, an endo-N-acety1 muramidase, D-alanine carboxy-peptidase and an exo-^-N-acety1 glucosaminidase (95); S t repto- coccus faecal is has a muramidase (117) and S_. aureus, an endo-^-glucosaminidase, an amidase and an endopeptidase (5). Bac?1 lus  subt i1 is was shown to contain amidase a c t i v i t y (138)-2. Poss ib le funct ions of a u t o l y t i c enzymes. Most attempts to c o r r e l a t e peptidoglycan metabolism with morphogenetic and other funct ions have involved studies of the a u t o l y t i c enzymes. Ear ly workers had noted the occurrence of a u t o l y t i c enzymes at maximal leve ls in exponent ia l ly growing c e l l s (88). Knowledge of the murein sacculus s t ruc ture suggested that the a u t o l y t i c enzymes were involved in c e l l growth and d i v i s i o n (88, 115, 130). Young (138) found high leve ls of the amidase during exponential growth of B_. subti 1 is and postulated i ts requirement for growth. Since th is amidase a c t i v i t y was found at higher leve ls in competent c e l l s , i t was suggested that loosening the outer s t ruc ture of the c e l l wall (139) by loosening the pept idoglycan (140) f a c i l i t a t e d entry of transforming DNA in competent s t r a i n s . A u t o l y t i c funct ions are necessary for the separat ion of c e l l s of B_. subt i 1 i s cha i ns grown at high temperature (30). These a u t o l y t i c funct ions were a lso necessary f o r a mutant s t r a i n to grow at a normal rate (31). Others (35) confirmed that B a c i l l u s ly t mutants were unable to separate t h e i r c e l l s from chains as e f f e c t i v e l y as the w i ld - type s t r a i n . Braun and 8 Schwarz.(l3) have reported a mutant of E_. col i wi th a temperature-s e n s i t i v e murein hydrolase which stops growth at the non-permissive temperature. Brown and Young (17) have demonstrated mul t ip le a u t o l y t i c a c t i v i t i e s in a sporu la t ion mutant of B_. subt i 1 is d e f i c i e n t in e x t r a c e l l u l a r protease. As mentioned, B_. subti 1 is normal ly has only an N-acetyl muramyl L - a l a n i n e amidase a c t i v i t y , and these workers postulated that the protease normally inact iva ted the extra a c t i v i t i e s , preventing c e l l death by l y s i s . The amidase is t i g h t l y bound to t e i c h o i c ac id (16) probably rendering i t r e s i s t a n t to protease a t tack . Higgins and Shockman (kS, 50) have shown the involvement of the S_. faecal is muramidase in c e l l d i v i s i o n and separat ion in that organism, and l o c a l i z e d the i n i t i a l ac t ion of the muramidase at the t ip and leading edge of the growing c r o s s - w a l l . A phage res is tan t mutant of S_. aureus H lacks t e i c h o i c ac id and a l s o exh ib i ts aberrent l y t i c a c t i v i t i e s and d i v i s i o n (21). By use of a s t r a i n of Diplococcus pneumoniae in which an a u t o l y t i c amidase can be inh ib i ted by the s u b s t i t u t i o n of ethanolamine for cho l ine in the t e i c h o i c a c i d , the amidase has been impl icated in c e l l e longa t ion , competence and a u t o l y s i s (123). However, a mutant of D_. pneumoniae which lacks the amidase a c t i v i t y but is s t i l l competent for transformation has been reported (70). 9 In sphere-rod transformations of Arthrobacter c r y s t a l l o p o i e t e s the a c t i v i t y of a muramidase increased fo r the sphere form and decreased for the rod form (68). There was a corresponding decrease in the average length of the glycan chain from 114-135 uni ts in the rod and from 15-i*0 uni ts in the sphere form (69). Peptidoglycan has been implicated in the d i f f e r e n t i a t i o n processes of s p o r u l a t i o n . T ipper and Pratt (121) have demonstrated a s p e c i f i c diaminopimelic ac id adding enzyme for synthesis of the peptide chain in B a c i l l u s sphaericus spores. At the time of sporu la t ion th is enzyme replaces the l y s i n e adding enzyme normally found in vegetat ive growth. The lactam of muramic ac id has been shown to occur in the mucopeptide of Baci11 us spores (121, 128) and the enzyme^-lactamase ( p e n i c i l l i n a s e ) has been shown, using fJ - lactamase negative mutants, to be required for s p o r u l a t i o n , re lease of spores from sporangia and a u t o l y s i s on aging (93). 3. Assay systems fo r a u t o l y t i c enzymes. Assay systems fo r a u t o l y t i c enzymes have been based on the re lease of s p e c i f i c fragments or f ree reducing sugars and amino ac ids (37). Following the release of r a d i o a c t i v i t y from labe l l ed wall preparat ions improves the s e n s i t i v i t y of th is assay (119. 111). A fur ther refinement has been to study the modi f ica t ion of rad ioac t ive low molecular weight (9*0 c e l l wall precursors by a u t o l y t i c enzyme preparat ions (125). Other less s p e c i f i c assay systems have been based on the reduct ion of t u r b i d i t y of whole c e l l s or iso la ted c e l l wal ls (44). 111. EFFECT OF PENICILLIN. The t ranspept idase enzyme which forms the peptide br idge binds p e n i c i l l i n i r r e v e r s i b l y and therefore is probably the s i t e of p e n i c i l l i n ac t ion (92, 62). A carboxypeptidase a c t i v i t y was a l s o i n h i b i t e d , but compet i t ive ly and r e v e r s i b l y . The in ter ference by p e n i c i l l i n in the c r o s s - l i n k i n g react ion had prev ious ly been proposed (133) based on the accumulation of nucleot ide-1 inked peptidoglycan precursors in the presence of p e n i c i l l i n (9*0 and the s t r u c t u r a l s i m i l a r i t y of p e n i c i l l i n to a c y l - D - a l a n y l - D - a l a n i n e (122). High concentrat ions of p e n i c i l l i n probably s p e c i f i c a l l y in te r fe re with peptidoglycan synthesis as penici11 in- induced protoplasts appear to grow normally (76, 104, k3). There is evidence that the act ion of the p e n i c i l l i n s requires a c t i v e a u t o l y t i c systems. P e n i c i l l i n is most e f f e c t i v e against rap id ly growing c e l l s (88) and the l y s i s is due to the cont inuing ac t ion of the l y t i c enzymes (133) in the absence of the terminal stages of wall s y n t h e s i s . Although there is no change in the gross accumulation of c e l l wall at the time of d i v i s i o n in Gram-negative organisms (Dr. D. T i p p e r , personal communication; 7 * 0 , there is considerable evidence, obtained using p e n i c i l l i n , that peptidoglycan metabolism and a u t o l y t i c enzymes have s p e c i f i c roles in c e l l d i v i s i o n . Staphylococci grown in the presence of p e n i c i l l i n s p l i t open (88) at the s i t e of c ross -wa l l format ion. Accumulation of f i b r e s at the d i v i s i o n s i t e in B a c i l l u s megaterium treated with p e n i c i l l i n and protected by sucrose (34) and in B a c i l l u s 1 icheniformis (51) in the presence of low leve ls of p e n i c i l l i n tend to confirm the presence of high leve ls of wall synthesis and a u t o l y s i s at the d i v i s i o n s i te . When grown in the presence of p e n i c i l l i n , B a c i l l u s cereus , E_. col ? (89) and C l o s t r idium botul ?num type A (64) formed spheroplasts near the pole of the c e l l , although the re lease of JE. c o l i protoplasts at both polar and centra l locat ions has been reported (29)• Greenwood and 0'Grady (39) observed the rapid l y s i s of a f r a c t i o n of E_. col i and Proteus mi rabi 1 is populat ions when these populat ions were exposed to p e n i c i l l i n , and cor re la ted the period of high s e n s i t i v i t y to the time of d i v i s i o n . On the other hand, Lark (22) observed the a b i l i t y of c e l l s of synchronized populat-ions of Alca1 igenes faeca1?s to overcome the l y t i c e f f e c t s of p e n i c i l l i n at the time of d i v i s i o n , and proposed that an excess of synthe t ic c a p a b i l i t y occurred at th is time. P e n i c i l l i n or chloramphenicol were shown to d i f f e r e n t i a l l y k i l l c e l l s at the time of d i v i s i o n (84). These resu l ts with chloramphenicol are p a r t i c u l a r l y d i f f i c u l t to understand in the l i g h t of the knowledge (15) that chloramphenicol is p r imar i l y a b a c t e r i o s t a t i c agent and incapable of k i l l i n g c e l l s in short incubation per iods . Several workers have observed the a b i l i t y of low leve ls of p e n i c i l l i n to s p e c i f i c a l l y i n h i b i t d i v i s i o n (59, 77, 42, 105, 110). The murein sacculus iso la ted (110) from c e l l s treated with 10 ug p e n i c i l l i n / m l exhib i ted s l i g h t bulges at the d i v i s i o n s i t e s . A higher concentrat ion of p e n i c i l l i n prevents th is bulging and r e s u l t s in o v e r a l l c e l l l y s i s . Net peptidoglycan synthesis and the composit ion of the peptidoglycan were found to be unchanged by the low-level p e n i c i l l i n treatment. The bulges were postulated to be the resu l t of excess a u t o l y t i c a c t i v i t y l o c a l i z e d at the s i t e of c ross -wa l l format ion. The low-level bulges were absent when DNA synthesis was i n h i b i t e d , ind ica t ing that there were two systems of a u t o l y t i c enzymes: one for general wall expansion which is s e n s i t i v e to high l eve ls of p e n i c i l l i n , and another for c e l l d i v i s i o n which is s e n s i t i v e to low leve ls of p e n i c i l l i n . Other workers have observed that c e l l s could be protected from p e n i c i l l i n ac t ion by preventing prote in synthesis (100, 43, 109), probably due to the prevention of synthesis of a u t o l y t i c enzymes. The requirement for a r i g i d s t ruc ture for c e l l wall growth and c e l l d i v i s i o n , e i t h e r in the form of a growth medium s o l i d i f i e d with agar or of added p a r t i c u l a t e fac to rs (25), has been demonstrated. Treatment with p e n i c i l l i n d isturbed the arrange-ment of the external 1 ipopolysacchar ide in E_. col ? (6), i nd ica t ing that the arrangement of the external st ructures ' of the Gram-negative wall were dependent on the i n t e g r i t y of the pept idoglycan. A f te r a dap-auxotroph of E_. col i was starved fo r DAP and induced to form spherop las ts , add i t ion of DAP led i n i t i a l l y to the formation of osmot ica l ly s tab le spheres fol lowed by regeneration of b a c i l l a r y form (112). The composition and extent of cross-1 inkage of the peptidoglycan was the same for the sphere and rod forms, although considerable d i f fe rences were found in the composit ion of pept idoglycan from rod forms grown in r i ch and in minimal media. Th is suggests that the chemical composit ion of the peptidoglycan is not important to the morphogenetic apparatus of the c e l l . Rather, the authors propose (112) the ex is tence of spec ia l l o c a l i z e d b iosynthe t ic mechanisms, inc luding a u t o l y t i c enzymes, as the s p e c i f i c morphogenetic mechanism (110). IV. CELL WALL GROWTH AND CELL DIVISION. A. Surface Growth L o c a l i z a t i o n in Rod-Shaped B a c t e r i a . Higgins and Shockman (50) reviewed the attempts to l o c a l i z e sur face growth of rod-shaped bac ter ia by three approaches: the d i r e c t observat ion of new c e l l growth with respect to f ixed external markers; the l a b e l l i n g of the old port ions of the envelope and noting the in t roduct ion of new envelope; and equating the p e n i c i l l i n - i n d u c e d l y s i s point as the growth po in t . The r e s u l t , due to the a p p l i c a t i o n of techniques without adequate knowledge concerning envelope turnover , s p e c i f i c i t y of the l a b e l , or the e f f e c t of growth ra te , a body of " l a r g e l y confusing and c o n f l i c t i n g data" (50). Th is repeated the warnings of e a r l i e r workers (26). The general conc lus ion is that in rod-shaped organisms at slow growth ra tes , l o c a l i z e d growth occurs at centra l and polar l o c a t i o n s . There is an increased s e n s i t i v i t y to p e n i c i l l i n at the centra l s i t e at septum format ion. Hughes and Stokes (58) used a ly t s t r a i n of B_. 1 icheni formis to prevent peptidoglycan turnover. Using a mucopept ide -spec i f ic ant iserum, they found d i s c r e t e areas of growth at the s i t e of c ross -wa l l format ion. B. Asymmetric Growth Ear ly workers (3, 40, 8)proposed a model for asymmetric growth of rod-shaped organisms, with growth occurr ing at one end of the c e l l . Clark (23) supported th is concept by cons ider ing the necess i ty for an asymmetrical mechanism of chromosome r e p l i c a t i o n and segregat ion in E_. col i . Experimental evidence has been presented by Donachie and Begg (29) in which young c e l l s of E_. col i and Pseudomonas were observed to grow unid?rect iona l ly u n t i l a c r i t i c a l s i z e was reached, a f t e r which growth proceeded b i d i r e c t i o n a l l y . V. REGULATION OF CELL DIVISION A. In te r re la t ionsh ip of 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 . The contro l of c e l l d i v i s i o n by deoxyr ibonucle ic ac id (DNA) r e p l i c a t i o n has been ex tens ive ly studied in E_. col i (23, 46). According to the Cooper and Helmstetter model (46), the r e p l i c a t i o n of the chromosome requires a constant time C, of approximately 40 min, independent of the growth ra te . Following the completion of two copies of the DNA, a constant time D of 20 min is required before septum formation is completed and d i v i s i o n takes p lace . At higher growth r a t e s , the c e l l s have m u l t i p l e r e p l i c a t i o n points (137, 46) in order to d i v i d e a f te r a d i v i s i o n period shorter than the constant 40 min C per iod . Prote in synthesis is required f o r i n i t i a t i o n of chromosome r e p l i c a t i o n (75, 127). Once i n i t i a t e d , chromosome r e p l i c a t i o n can proceed to completion without cont inuing prote in synthesis (75, 80, 135). When chromosome r e p l i c a t i o n is completed, the c e l l w i l l d i v i d e even i f fu r ther DNA synthesis has been inh ib i ted (23, 48) and may d iv ide i f prote in synthesis is a l s o inh ib i ted (7, 73, 75, 96). The i n h i b i t i o n of prote in synthesis by amino acid s ta rva t ion or chloramphenicol add i t ion has shown that a period of prote in synthesis equal in time to the C p e r i o d , i . e . the chromosome r e p l i c a t i o n p e r i o d , is necessary to permit c e l l d i v i s i o n , in add i t ion to a completed round of r e p l i c a t i o n (96). B. Interference with C e l l D i v i s i o n With so many complex s t ructures and a c t i v i t i e s involved in c e l l d i v i s i o n , i t is hardly s u r p r i s i n g that there are many ways to i n t e r f e r e with and prevent growth and d i v i s i o n . 1. Mutants. Mutant s t r a i n s which f a i l to i n i t i a t e , complete or segregate rounds of DNA r e p l i c a t i o n (66, 87, 10, 32) w i l l not d i v i d e under non-permissive c o n d i t i o n s . Other mutants (60, 61, 52) have los t the coupl ing between DNA r e p l i c a t i o n and d i v i s i o n . Many morphological mutants c lassed as " d i v i s i o n mutants" have been i s o l a t e d . Seven d i s t i n c t c lasses of mutants have been iso la ted and descr ibed by H i r o t a , Ryter and Jacob (53). Miniature E_. col i eel 1 s d e f i c i e n t in DNA (2), as well as giant c e l l s of the same organism (4), have been descr ibed . Several v a r i e t i e s of rod mutants of Baci1lus species have been descr ibed , e i ther osmot ica l ly l a b i l e (83, 102) or osmot ica l l y s tab le (11). Temperature-sensi t ive f i lamentous forms of E_. col i (124, 90) and Erwinia amylovora (56), as well as chain formers of E_. col i (91) and B_. cereus (126) have been J s o l a t e d . The Ion mutant of E_. col i forms f i laments when exposed to u l t r a v i o l e t i r r a d i a t i o n (13^, 5^). 2. Chemicals. Many fac to rs have been found to induce f i lamentous forms of w i ld - type b a c i l l i (59). Any treatment which w i l l i n h i b i t DNA s y n t h e s i s , such as removal of thymine or treatment with n a l i d i x i c ac id (23, 48) , w i l l prevent d i v i s i o n although the c e l l continues to elongate for some time. Workers have shown the induct ion of long forms by such treatments as increased pressure . (1.4.1) n u t r i t i o n a l changes (132), ion s ta rva t ion (65, 67), d i a z o u r a c i l (98, 33) c i s Dichloro diaminoplatinum II (55) 5 - f1uoro-uraci1 (108), D-ser ine and c y c l o s e r i n e (41, 42) and p e n i c i l l i n (59, 77, 110). The nature of the work to be reported here is to demonstrate that the c e l l envelope and p a r t i c u l a r l y the metabolism of the peptidoglycan is involved in the process of c e l l d i v i s i o n in E_. col i . The work is presented in three s e c t i o n s : (1) the Iso la t ion of mutants temperature-sensi t ive for d i v i s i o n ; (2) the development of assays for a u t o l y t i c enzymes, and (3) the attempts to apply these assay systems to the mutants. MATERIALS AND METHODS I. BACTERIAL STRAINS AND CULTURE CONDITIONS St ra ins of E_. col i used in th is study, the i r sources and genotypes are l i s t e d in Table I. C e l l s were grown a e r o b i c a l l y in Erlenmeyer f l asks at 30 C, 37 C or 42 C. The minimal medium was 007 (2k), supplemented with 0.2% glucose . The generation per iod in th is medium was 46 min at 37 C. Enriched medium was 007~glucose plus 0.4% w/v casamino a c i d s ; i t supported growth with a generat ion per iod of 26 min. Nutr ient broth (D i fco ) , enr iched with the appropr iate concentrat ion of <*,£-d iami nopimel ic ac id (DAP) and 50 ug thymidine per ml was used fo r studies on diaminopimelic auxotrophic s t r a i n s (dap ) and supported growth with a generation period of 22 min. Stock cul tures were maintained on nutr ient agar s lants enriched with 50 ug thymidine, 50 M9 u r a c i l , 50 fig tryptophan and 50 ug <3<,£-diaminopimel ic ac id (DAP) per ml. The sealed v i a l s were stored at 4 C a f t e r 2k hr incubation at 30 C. Synchronous cu l tures were obtained by the method of Helmstetter and Cummings (47). S tar ter cu l tures were grown overnight in two shaker f l asks to a densi ty of 1 x 10^ c e l l s per ml . Approximately 500 ml of th is cu l tu re were forced into a 150 mm M i l l i p o r e membrane of the appropriate pore s i z e (see below). The membrane with the 19 Table I. L i s t of bac te r i a l s t r a i n s used. S t ra in of Escher ich ia col i Genotype Source B/r/1 B/r/1 l y s " B/r/1 l y s " thr" B/r/1 l ys" pyr A~ AB 1157 H-2 M-2 BUG-6 BUG-6-SmS BUG-6 l y s " BUG-6 l y s " thr" BUG-6 l y s " pyr A~ B, r a d R , T R , T R , lys" B, r a d R , T R , T R , lys" B, r a d r , T j , T ^ , l ys" B , r a d r , T ^ , T^, l y s " , O r i g i n a l l y obtained from C E . Helmstetter and made T ^ - r e s i s t a n t by D . J . Clark This paper 3 thr pyr-A (arg" , ur") R K12,F ,Sm TL Arg His Pro ' B l " Mt l" xy~ ga l " This paper T I • a This paper Obtained from A . E . Adelberg K12, F + , m o t + , Sm xyl K12, F gal + mot ,Sm TS d iv TS div" TS div" TS div" mtl Derived from AB1157 by reversion of auxotrophic markers by C R . Bagwell of th is laboratory Kindly constructed from H-2 by R . J . Martinez a Sm lys l y s ' thr This paper Kindly constructed by D.J, and G.G. Khachatourians This paper 9 This paper Clark lys pyr A (arg ur )This paper 0 Fla 138 K12 2 1 ac SmR F" thr" leu" t h i + ara" g a l " x y l " Obtained from J . Adler AT 982 K12 (KL16) Hfr dap D" Obtained from A. Tay lor 15T- -555-7-321 15T" t rp" t h y - arg" met" dap" Obtained from C. Lark 15T- -555-7-321 l y s ' 15T" trp" thy" arg" met" dap" l y s -This paper 3 ATCC 13070 dap" met" Sms F Purchased from ATCC ATCC 13070 l y s " dap" met" l y s " Sms F" This paper ATCC 13070 l y s " Sm1" dap" met" l y s " Sm r F~ This paper 3 The genet ic symbols are those of Tay lor (120)• Isolated by s e l e c t i o n with p e n i c i l l i n a f t e r mutagenesis with NTG. bound c e l l s was inverted and placed in a holder in a f u l l view incubator . The loose ly bound c e l l s were washed from the membrane by adding a k cm deep pressure head of pre -condi t ioned medium above the f i l t e r . The newly d iv ided c e l l s were e luted from the membrane by maintaining a flow rate through the membrane of between 5 and 10 ml of pre-condi t ioned medium per min. A membrane of 0.22 u pore s i z e was used to bind c e l l s grown in minimal medium while a 0.65 JJ pore s i z e was used to bind c e l l s grown in enriched medium. Synchronous c e l l s were sh i f t ed from minimal medium to enriched medium by e lu t ing minimal c e l l s from the membrane d i r e c t l y into a f l a s k conta in ing a concentrated casamino acids medium. The p re -cond i t ioned , pre-warmed medium was obtained by f i l t e r i n g the c e l l s from cu l tures of the same organism in the same medium grown to 1 x 10^ c e l l s per ml and maintaining the temperature of the medium. II. CONSTRUCTION OF MUTANTS A. Construct ion of Temperature-Sensi t ive (TS) Filamentous Mutants 1. Mutagenesis. Cultures were mutated by treatment with ethyl methane sul fonate (EMS) or N-methyl, N 1 - n i t r o n i t r o s o g u a n i d i n e (NTG) according to the fo l lowing procedures. a . Ethyl methane su l fonate (EMS) This was a modi f ica t ion of the technique of Hayashi , Koch and L in (45). A cu l tu re was grown to 0.1 o p t i c a l densi ty (0D) uni ts at 660 nm in nutr ient bro th , harvested by c e n t r i f u g a t i o n and washed twice with 0.2 M tr is(hydroxymethyl) aminomethane-HCl ( T r i s ) , pH 7-4 at room temperature. The c e l l s were resuspended in 10 ml of the same buf fer and placed in a stoppered 125 nil f l a s k conta in ing 0.15 ml EMS. The suspension was incubated fo r 150 min with shaking at 37 C. F ive ml of the treated c e l l suspension was added to 100 ml of glucose minimal medium (007 glucose) and incubated for 24 hr at 30 C with shaking. b. Ni trosoguanidine (NTG) A modi f ica t ion of the technique of Kohiyama et a 1. (66) was used to mutate the parent s t r a i n . Five ml of a cu l tu re grown to 0.1 O D ^ Q was harvested by c e n t r i f u g a t i o n and washed twice with acetate buf fer (14.8 ml of 0.2 M a c e t i c a c i d , 35.2 ml of 0.2 M sodium aceta te , d i s t i l l e d water to 100 ml ) . The c e l l s were resuspended in 5 ml of acetate buf fer and added to a 50 ml f l a s k conta in ing 0.1 ml of NTG so lu t ion (4 mg/ml d i s t i l l e d water) . The suspension was incubated with the NTG f o r 130 min at 37 C with shaking, then f i l t e r e d through a 0.45 p M i l l i p o r e membrane. The c e l l s were washed three times on the f i l t e r with acetate b u f f e r , a f t e r which the f i l t e r was placed in 50 ml 007 glucose in an Erlenmeyer f l a s k . The f l ask was shaken for 2k hr at 30 C. 2. Enrichment. The cu l tures of mutagenized bac ter ia were enriched for s t r a i n s unable to d iv ide at k2 C on the bas is of increased s i z e at that temperature. Enrichment was ca r r i ed out by s i z e separat ion in a sucrose gradient and by a modi f ica t ion of the membrane technique of Van de Putte et a l . (\2k). a. Sucrose grad ient . The mutagenized cu l tu re was d i l u t e d with 007 glucose and incubated with shaking at 29 C. A f t e r a short lag the cu l tu re resumed exponential growth and was then s h i f t e d to k2 C. A f te r a fur ther 3 hr , the cu l tu re was c h i l l e d rap id ly to 0 C; 7-5 ml were harvested by c e n t r i f u g a t i o n and the c e l l s resuspended in 0.1 ml of 5% sucrose . A l l operat ions were car r ied out at k C. The suspension was layered on a 25 ml 5% to 25% sucrose gradient and spun in an Internat ional S ize One, Model C50 swinging bucket cen t r i fuge at 1,500 rpm for 10 min. Larger organisms, such as f i l aments , chains and pleomorphs which s e t t l e d fur ther into the grad ient , were recovered by puncturing the tube at the bottom, and c o l l e c t i n g the sucrose that ran out u n t i l the e a s i l y - v i s i b l e w i ld - type c e l l band approached the bottom of the tube. The c o l l e c t e d sucrose was d i l u t e d 1:1 with s t e r i l e d i s t i l l e d water and centr i fuged at 10,000 x g_ fo r 25 min. The c e l l p e l l e t was resuspended in 007 glucose and incubated for 2k hr at 30 C with shaking. The above procedure was repeated twice more and the f i n a l c u l t u r e was descr ibed as "enr iched" . b. Membrane f i1 ter The overnight c u l t u r e of mutagenized c e l l s was d i lu ted 1000-fold, grown exponent ia l ly for several generations to 5 x 10^ c e l l s per ml at 30 C and then s h i f t e d to k2 C fo r 3 hr . Five ml of th is cu l tu re was d i lu ted with s t e r i l e d i s t i l l e d water to a f i n a l volume of 20 m l , then f i l t e r e d through a membrane f i l t e r (3, 5 or 8 u pore s i z e ) . The f i l t e r was washed twice with s t e r i l e d i s t i l l e d water and placed in 10 ml of 007 g lucose . A f te r 1 hr shaking at 30 C, the cu l tu re was removed from the f i l t e r and the c e l l s washed by c e n t r i f u g a t i o n with two port ions of 007 medium. The c e l l s were resuspended in 10 ml of 007 glucose and incubated overnight at 30 C with shaking. Th is washing procedure was necessary sinee the . 1arger pore s i z e f i l t e r s inh ib i ted the growth of c e l l s . The above procedure was repeated three t imes. The f i n a l overnight cu l tu re was descr ibed as " e n r i c h e d " . 3. Screening The dens i t i es of enriched c u l t u r e s , usua l ly in s ta t ionary phase, were determined with the Coul ter Counter. Samples of appropr iate d i l u t i o n s were spread on 007 g lucose medium. The plates were incubated at 30 C u n t i l microcolonies (•50-100 per plate) jus t appeared (20-25 hr) and then sh i f t ed to 42 C fo r about 12 hr to al low the colonies to grow to maximum s i z e . The plates were screened for abnormal colony morphology, mainly microcolonies and "rough" c o l o n i e s . Individual co lonies were streaked on two 007 glucose agar plates and one nutr ient agar p l a t e . One of the minimal p la tes was incubated at 30 C and o-the other two plates were incubated at 42 C. St ra ins with temperature-sensi t ive colony morphology were t ransfer red to stock s lants fo r fur ther study. Cul tures of the p o s s i b l e d i v i s i o n mutants growing exponent ia l ly at 37 C were s h i f t e d to 42 C and observed by phase microscopy f o r abnormal morphology. St ra ins e x h i b i t i n g temperature-sensi t ive morphology were subjected to more intensive c h a r a c t e r i z a t i o n : morphology, change in number and s i z e of c e l l s as monitored on Coulter Counter, and changes in the rate of DNA s y n t h e s i s , at 30 C and a f t e r a s h i f t to 42 C. 3 DNA synthesis was measured by incorporat ion of H-thymidine over a period of 3 min. A 1 ml c e l l sample was added to 0.1 ml 3 contain ing 1.25 uCi and 0.05 ug H-thymidine. Synthesis was stopped by adding 2 ml of co ld 7-5% t r i c h l o r o a c e t i c ac id (TCA) conta in ing 200 ug thymidine/ml . Each sample was c o l l e c t e d on a 0.45 u M i l l i p o r e membrane and washed with 5 volumes of co ld TCA and 3 volumes of water at 90 C. 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 fo r count ing. B. Construct ion of Auxotrophic Mutants 1. NTG mutagenes i s A modi f ica t ion of the technique of Adelberg et a l . (1) was used. Cultures were grown in nutr ient b ro th , enriched 8 where necessary with 50 pg DAP per ml , to 1 x 10 c e l l s per ml and f i l t e r e d from the medium by using a s t e r i l e 0.45 ju membrane f i l t e r . The c e l l s were washed twice on the membrane with s t e r i l e T r is -ma lea te buf fer (1.0 g ( N H ^ S O ^ , 0.1 g MgSO^, 5 mg Ca(N0^) 2 , 0.25 mg FeSO^, 7 H 2 0 , 6.0 g T r i s and 5.8 g maleic a c i d , in 1000 m l , with the pH adjusted to 6.0 with N^  NaOrl) and the c e l l s resuspended in 3-0 ml T r is -ma lea te buf fer at 37 C. NTG was added to a f i n a l concentrat ion of 100 ug per ml and the cu l tu re ag i ta ted at 37 C for 15 min. The c e l l s were again c o l l e c t e d on a 0.45 u membrane, washed twice with the s t e r i l e T r is -ma lea te b u f f e r , then resuspended in minimal glucose medium supplemented with 50 ug per ml of the necessary amino a c i d s . The c u l t u r e was grown overnight to al low the mutant genotype to segregate and to s e l e c t against unwanted auxotrophs. Revertants of auxotrophic s t r a i n s were se lected as f o l l o w s . 8 Approximately 1 x 10 c e l l s from a cu l tu re growing exponent ia l ly were washed by c e n t r i f u g a t i o n in the cold with s t e r i l e d i s t i l l e d water and spread on plates which were s e l e c t i v e for the revertants A small c r y s t a l of NTG was placed in the middle of the p l a t e , and the p late was incubated for 48 hr at 37 C. Revertants caused by the ac t ion of the NTG were found in a r ing of growth around and some d is tance from the c r y s t a l , and were removed a s e p t i c a l l y f o r c h a r a c t e r i z a t i o n . 2. P e n i c i l l i n enrichment. 9 One ml of cu l tu re contain ing approximately 1 x 10 mutated and segregated c e l l s was centr i fuged to p e l l e t the c e l l s , and the c e l l s were washed twice by c e n t r i f u g a t i o n and resuspension in minimal glucose medium. The p e l l e t was resuspended in 100 ml of minimal glucose medium and the suspension incubated at 37 C. When the c e l l number was increasing exponent ia l l y , as measured on the Coulter Counter, s u f f i c i e n t sucrose and f r e s h l y prepared p e n i c i l l i n - G were added to give concentrat ions of 20% (w/v) and 400 I.U. per ml , r e s p e c t i v e l y . incubat ion was continued for 5 h r . Surv iv ing c e l l s were washed three times with ice c o l d , s t e r i l e d i s t i l l e d water, using c e n t r i f u g a t i o n and resuspension. These surv ivors were enriched for mutants with the des i red amino acid requirement. The enrichment c y c l e was repeated as found necessary. A mod i f i ca t ion of the method of Lederberg and S t . C l a i r (78) was used in an attempt to construct a dap auxotroph fo r E_. col i . Cul tures of s t r a i n BUG-6 which had been mutated with NTG to generate auxotrophs, was then grown in the presence of 50 ug DAP per ml to al low segregat ion of the mutat ions. Appropr iate d i l u t i o n s of these cu l tures were mixed with molten sucrose agar (78) at kS C and conta in ing 100 I .U. p e n i c i l l i n - G per ml , and poured into p l a t e s . The p lates were incubated at 30 C f o r 10 days. L-form type co lon ies were se lec ted and screened for dap auxotrophy. 3. Conjugation Cultures of donor and r e c i p i e n t s t r a i n s were grown 8 to a densi ty of 1 x 10 c e l l s per ml in Lur ia broth which contained 10 g t ryptone, 5 g yeast e x t r a c t , 5 g NaCl , 1 g g lucose , k ml of 1 N_ NaOH, and d i s t i l l e d water to 1 l i t e r . The broth was supplemented with 50 ug DAP per ml where necessary. The donor was grown in a s ta t ionary tube s ince maximum f e r t i l i t y is obtained during anaerobic growth (27). The rec ip ien t was grown a e r o b i c a l l y in f l asks held in a shaking water bath. One ml of r e c i p i e n t and 0.1 ml of donor c u l t u r e were mixed in the bottom fo f a 250 ml Erlenmeyer f l a s k , and l e f t undisturbed at 37 C for 5 h r . Samples were removed, d i lu ted with fresh medium, agi tated v igorous ly and the appropr ia te d i l u t i o n s plated on s e l e c t i v e medium. k. Transduct ion Transduct ion was ca r r i ed out by a modi f ica t ion of the techniques of Caro and Berg (19). Approximately 1 x 10 c e l l s from a s ta t ionary phase cu l tu re of the donor s t r a i n and 1 x 10 plaque-forming uni ts ( p . f . u . ) of phage P1L.4 were added to a molten agar over lay at hS C. The mixture was layered on an agar p l a t e . The over lay agar contained 10 g tryptone and 8 g agar per J i t e r of 0.01 M^CaCl,; the bottom layer contained nutr ient b ro th , 15 g agar per l i t e r and C a C ^ to 0.01 M_. A f te r 8 hr incubation at 37 C, 5 ml of phage buf fer (7 g Na^PO^, 3 g Kr^PO^ and 5 g NaCl d isso lved in 980 ml d i s t i l l e d water in the order indicated and autoc laved, s t e r i l e so lu t ions of 0.1 M MgSO^ and 0.01 M CaCl^ were prepared separate ly and 10 ml of each added to the phosphate s o l u t i o n a s e p t i c a l l y ) was added to the p l a t e , A f t e r 20 min at room temperature, the buf fer and agar over lay were removed with a broad t i p p i p e t t e , mixed thoroughly and centr i fuged at 7,000 x £ for 15 min in the c o l d . D i lu t ions of the supernatant in phage buf fer were t i t r e d against the rec ip ien t s t r a i n by the over lay method. A l l lysates were stored in the cold over chloroform to prevent b a c t e r i a l growth. To prepare a lysate for t ransduct ion , the donor s t r a i n was 8 grown to 1 x 10 c e l l s per ml in 100 ml of nutr ient broth supplemented with C a C l 2 to 10~^ M. A high t i t r e lysate of P1L.4 was added to a f i n a l concentrat ion of 1 x 10^ p . f . u . per ml . A f te r 100 min of vigorous aerat ion at 37 C, Na c i t r a t e was added to a f i n a l - 3 concentrat ion of 5 x 10 H. A f t e r 15 min, 5 ml chloroform was added, the cu l tu re agi tated for 5 min, and the c e l l debris removed by c e n t r i f u g a t i o n at 10,000 x a_ fo r 15 min. The t i t r e of the lysate was determined on the r e c i p i e n t s t r a i n . For t ransduct ion , the r e c i p i e n t bac te r ia were grown at an g exponential rate to a densi ty of from 1 to 5 x 10 c e l l s per -2 ml in Lur ia broth supplemented with CaCl_ to 10 M. The c e l l s were washed by cent r i f l iga t ion in the cold with s t e r i l e s a l i n e -2 s o l u t i o n conta in ing 10 M_ C a C ^ , and resuspended in th is s o l u t i o n . The phage lysates were shaken at 37 C to remove the chloroform, and appropr iate volumes added to the rec ip ien t c e l l s to give m u l t i p l i c i t i e s of in fec t ion (m.o . i . ) of 0.01, 0.1 and 1.0 p . f . u . per c e l l . The mixture was incubated for 20 to 30 min at 37 C to al low adsorp t ion , and appropr iate d i l u t i o n s were spread on s e l e c t i v e media. III. ASSAY OF AUTOLYTIC ENZYMES A. In Vivo Assay of A u t o l y t i c Enzymes (a) Three ml of exponential or synchronous c u l t u r e were added to a 25 ml Erlentneyer f l a s k contain ing 450 ug chloram-phenicol (CAM) in 0.25 ml water. The f l a s k was maintained at 37 C in a shaking water bath. A so lu t ion of a m p i c i l l i n was added immediately to give a f i n a l concentrat ion of 5 pg per ml . The c e l l numbers and c e l l s i zes were measured with a modif ied Coul ter Counter coupled to a pulse height analyzer (22). (b) S l ides coated with minimal glucose agar conta in ing 5 ug a m p i c i l l i n per ml and 150 ug CAM per ml were warmed in a humid atmosphere at 37 C. C e l l s were grown in minimal glucose medium and were treated with a m p i c i l l i n and CAM as in (a) above. A sample of c e l l s was pipet ted immediately onto the agar surfaces of several of the s l i d e s and the s l i d e s stored at 37 C under high humidity. S l ides were removed for observat ion at i n t e r v a l s . The lys ing c e l l s were observed and photographed by phase-contrast microscopy. B. In V i t r o Assay of A u t o l y t i c Enzymes 1 . Iso la t ion of peptidoglycan A combination of the techniques o f Blackburn ( 9 ) , Pelzer ( 9 5 ) and Schwarz et a l . ( 1 1 0 ) was used to iso lateuthe pept idoglycan from the c e l l wal ls of E_. col i . Ten gram of l y o p h i l i z e d c e l l s of E_. col i (Calbiochem, Los Angeles , C a l i f . ) were added to 6 8 0 ml of d i s t i l l e d water, fol lowed by 3 0 0 ml of 0 . 1 N_ NaOH. The mixture was s t i r r e d at room temperature for 2 0 min and then neut ra l i zed slowly with 1 H_ H C 1 , using Phenol Red as the i n d i c a t o r . One hundred L ig of deoxyribonuclease was added and the mixture s t i r r e d fo r 5 min. The c e l l s were removed by c e n t r i f u g a t i o n at 1 0 , 0 0 0 x g_ fo r 7 min at 4 C, and washed twice with d i s t i l l e d water. The p e l l e t was resuspended in a minimal volume of d i s t i l l e d water and sodium dodecyl s u l f a t e (SDS) was added to a f i n a l concentrat ion of k% w/v. The preparat ion was l e f t overnight at k C. The c e l l s were centr i fuged for 3 0 min at 2 0 , 0 0 0 x £ at room temperature, and resuspended in 0.k% w/v SDS. 0 . 6 volumes of 7 5 _ 1 0 5 u g lass beads (Sigma type 1 ) were added and the preparat ion treated in a c o l l o i d m i l l at a se t t ing of 3 0 / 1 0 0 fo r 3 min at k C. T^e beads were allowed to s e t t l e out and washed four':times with d i s t i l l e d water. The washings were pooled with the c e l l preparat ion which was washed twice with 0.4% w/v SDS and s i x times with d i s t i l l e d water. Cent r i fuga t ion required 3 hr at 14,000 x £ at 4 C on each o c c a s i o n . The f i n a l p e l l e t was extracted three times at 20 C with a l k a l i n e phenol (50 ml of 0.1 M_ sodium tetraborate conta in ing 50 g phenol ) . A fur ther three ex t ract ions were done with hot a l k a l i n e phenol at 65 C for 10 min with very vigorous shaking. A f te r each of the 20 C and 65 C phenol e x t r a c t i o n s , the c e l l s were recovered by cent r i fugat ion at 35,000 x g_ for 3 hr . The p e l l e t was washed three more times with 30% w/v phenol by c e n t r i f u g a t i o n using 15,000 x g_ for 3 hr at 4 C. F i n a l l y the p e l l e t was resuspended in a minimal volume of d i s t i l l e d water. The suspension was d ia lyzed against d i s t i l l e d water with e ight changes of volume over 44 hr . A f te r d i a l y s i s , the c e l l wa l ls were centr i fuged at 6,000 x £ and resuspended in d i s t i l l e d water. The c e l l wal ls were repeatedly washed u n t i l no opalescence was observed in the supernatant for four success ive washings. A sample was removed for amino ac id a n a l y s i s at th is time. The p e l l e t was resuspended in 50 ml of 0.02 M phosphate b u f f e r , pH 7-0, and 500 ug each of amylase and r ibonuclease were added. Five ml of toluene was added to prevent b a c t e r i a l growth, and the mixture incubated with a g i t a t i o n for 8 hr at 37 C. At t h i s t ime, 500 ^g of t ryps in were added and the preparat ion a g i t a t e d a t 37 C o v e r n i g h t . The c e l l w a l l s were c e n t r i f u g e d a t . 14,000 x £ f o r 20 min and the p e l l e t resuspended i n 0.4% w/v SDS w i t h t he a i d o f a homogenizer ( V i r t i s ) . The SDS was removed by washing s i x t i m e s w i t h d i s t i l l e d w a t e r a t room t e m p e r a t u r e u s i n g c e n t r i f u g a t i o n a t 14,000 x £.for 20 min on each o c c a s i o n . A c i d h y d r o l y s a t e s o f t h e p e p t i d o g l y c a n were p r e p a r e d t o ass a y t he c o n t e n t o f p e p t i d o g l y c a n - s p e c i f i c components. The m a t e r i a l t o be h y d r o l y z e d was suspended i n 4 N_ h y d r o c h l o r i c a c i d and s e a l e d i n vacuo i n a g l a s s ampoule. The ampoule was heated t o 100 C f o r 4 h r . The h y d r o l y s a t e was removed from t h e v i a l and s t o r e d f o r 48 h r i n a s e a l e d d e s i c c a t o r i n vacuo o v e r anhydrous s i l i c a g e l and p o t a s s i u m h y d r o x i d e p e l l e t s . The d r i e d r e s i d u e was d i s s o l v e d i n a mini m a l volume o f d i s t i l l e d w a t e r and s t o r e d f o r f u r t h e r a n a l y s i s , i The p r e s e n c e o f r e d u c i n g s u b s t a n c e s i n th e h y d r o l y s a t e s p r e p a r a t i o n s was d e t e r m i n e d by the method o f Ghuysen, T i p p e r and S t r o m i n g e r (37*). A n e u t r a l i z e d sample o f r e d u c i n g s u b s t a n c e c o n t a i n i n g r e d u c i n g power e q u i v a l e n t between 10 and 50 pmoles o f N - a c e t y l g l u c o s a m i n e was d i l u t e d t o 1 ml i n a 10 ml tube . One ml o f a f r e s h 1:1 m i x t u r e o f f e r r i c y a n i d e (0.5 g p o t a s s i u m f e r r i c y a n i d e per l i t e r w a t e r ) and c a r b o n a t e - c y a n i d e (5-3 g sodium c a r b o n a t e , 0.65 g p o t a s s i u m c y a n i d e per l i t e r w a t e r ) r e a g e n t s was added. The m i x t u r e was hea t e d f o r 15 min i n a b p i l i n g w a t e r bath and then c o o l e d . 2.5 ml o f f e r r i c i r o n reagent. (1.5 g f e r r i c ammonium s u l f a t e and 1 g sodium monolauryl s u l f a t e per l i t e r water) was added and allowed to react at room temperature for 15 min. The absorbance at 690 nm was measured. A s p e c i f i c assay for DAP, descr ibed by Work (136), was used to determine the amount of peptidoglycan Pn the mater ial hydrolyzed. The preparat ion to be assayed was d i s s o l v e d in 0.5 ml d i s t i l l e d water and 0.5 ml of g l a c i a l a c e t i c ac id added. Then 0.5 ml of f r e s h l y prepared reagent mix (250 mg ninhydrin d isso lved in 6 ml g l a c i a l a c e t i c ac id was added to k ml 6 H phosphoric acid) was added, mixed and the s o l u t i o n placed in a b o i l i n g water bath fo r 5 min. The mixture was cooled and 3-5 ml of g l a c i a l a c e t i c ac id was added immediately before measuring the absorbance at kkO nm. 2. P a r t i c u l a t e enzyme preparat ion A p a r t i c u l a t e enzyme preparat ion was prepared from growing c e l l s which had been rap id ly c h i l l e d in an ice-water bath to prevent fur ther peptidoglycan metabolism. Aerat ion was continued during the c h i l l i n g procedure. A l l fur ther operat ions were ca r r i ed out at k C. The c e l l s were washed with 0.1 ammonium acetate buf fer and a f t e r c e n t r i f u g a t i o n were resuspended in the same buf fer at 100 times the i n i t i a l c e l l concent ra t ion . The c e l l s were broken by two passages through a p r e - c h i l l e d French pressure c e l l or by k x 0.5 min treatment with a sonic probe (1.2 cm d i a . ) (Bronwi11 Biosonik) at a se t t ing of 70/100 i'n an ice-water bath. The broken c e l l suspension was centr i fuged for 20 min at 15,000 x g_ and the p e l l e t so formed was washed twice with i c e - c o l d buf fer before being resuspended in the same buf fer to serve as the enzyme source. To make a p a r t i c u l a t e enzyme preparat ion from r a d i o a c t i v e l y l abe l l ed c e l l s , a cu l tu re of a dap auxotroph growing in nutr ient broth supplemented with DAP was d i lu ted to 1 x 10^ c e l l s per ml 14 3 in 500 ml of the same medium. Twenty ul of 1,7 C or JH(G)DAP were added to give a f i n a l concentrat ion of 2 ug DAP per ml and an a c t i v i t y of 0.1 uei,3H DAP or 0.02 juCi^C-DAP per ml . The g cul tures were grown u n t i l they reached 1 x 10 c e l l s per ml , and a p a r t i c u l a t e enzyme preparat ion was made. During the 14 3 incorporat ion of C or H DAP by dap auxotrophs of E_. col i , 5 ml samples were removed at var ious t imes. Ten ml of i c e - c o l d 7-5% w/v TCA conta in ing 75 jug unlabel led DAP per ml were added to the samples with mixing and the mixture was held on ice for 15 min. The p rec ip i ta ted material was removed by f i l t r a t i o n , using a 0.45 u pore membrane, and washed s i x times on the membrane with 5 ml i c e - c o l d 5% w/v TCA conta in ing 50 ug unlabel led DAP per ml. The f i l t e r s were dr ied under a heat lamp, then placed in v i a l s conta in ing 10 ml toluene s c i n t i l l a t i o n f l u i d (40 ml L i q u i f l u o r (Nuclear Chicago, Des P l a i n e s , III.) per l i t e r toluene) and counted in a l i q u i d s c i n t i l l a t i o n spectrometer (Nuclear Chicago C o r p . , Des P l a i n e s , 111.). Other approaches were used in an attempt to s p e c i f i c a l l y label the pept idoglycan. Cul tures of E_. co 1 i s t r a i n B/r/1 lys g and BUG-6 were grown for 20 generations to a densi ty of 1 x 10 c e l l s per ml in nutr ient broth supplemented with 2 ug and 1 >uCi 3 of H-G-DAP per ml , r e s p e c t i v e l y . In a p a r a l l e l experiment, 14 10 uC i of C-U-glucose per ml was added to E_. col i B/r/1 growing exponent ia l ly in minimal medium conta in ing 0.05% w/v g lucose . g The c u l t u r e was harvested at a densi ty of 1 x 10 c e l l s per ml . P a r t i c u l a t e enzyme preparat ions were made of the three l a b e l l e d c u l t u r e s , and used in the rad ioac t ive in v i t r o a u t o l y t i c enzyme assay. Further attempts to obtain incorporat ion of exogenous DAP by dap + c e l l s included t ry ing to grow E. c o l i B/r/1 on minimal medium conta in ing 0.1% w/v DAP as e i ther the so le source of carbon or of n i t rogen , or of both. For the t r i a l s with DAP as the so le source of n i t rogen , ammonium s u l f a t e , the only ni t rogen source in the minimal medium was replaced with 2 g Na^SO^ per l i t e r . In a l l cases , the inoculum was grown for 3 sequent ia l subcultures in normal minimal medium supplemented with 0.2% w/v glucose and 0.1% w/v DAP. The c e l l s were washed twice with s t e r i l e d i s t i l l e d water by c e n t r i f u g a t i o n in the c o l d , and inoculated to a densi ty of 1 x 10** c e l l s per ml in the medium using DAP as the carbon or ni trogen source. The f l asks were incubated fo r periods up to 72 hr at 37 C and the change in c e l l number was determined using the Coulter Counter. 3. Separation and detect ion of c e l l fragments by chromatography Amino acids and c e l l wall fragments were separated by c e l l u l o s e t h i n - l a y e r chromatography. a. Preparat ion of p l a t e s . The plates were prepared by a modi f ica t ion of the technique of Jones and Heathcote (63). 18 g of MN 300 c e l l u l o s e (Machery and Nagel , Duren, Germany), 12 ml of 35% ethanol and 80 ml water were blended in a Waring blendor fo r 30 sec . The s l u r r y was spread on 20 cm square g lass p lates to a uniform thickness of 1.5 mm. The plates were dr ied at 20 C and allowed to stand at room temperature for 2k h r . b. Solvent systems and condi t ions System I of Pelzer (95) was used to separate the fragments released from c e l l wal ls by a u t o l y t i c enzymes. The solvent system was the upper phase of a mixture of n-butanol , g l a c i a l a c e t i c ac id and water (40:10:50). The best separat ion was obtained when the solvent system was allowed to e q u i l i b r a t e 2k hr before the plates were placed in the tank. The plates were allowed to run for 5 hr at room temperature, then dr ied for development. The two-dimensional system of Jones and Heathcote (63) was used to separate mixtures conta in ing several amino a c i d s . The primary solvent system was: r e - d i s t i l l e d isopropanol , f o r m i c a c i d and d i s t i l l e d w a t e r ( ^ 0 : 2 : 1 0 ) . The p l a t e s were i m m e d i a t e l y p l a c e d i n the t a n k , b e f o r e e q u i l i b r a t i o n c o u l d t a k e p l a c e and the s o l v e n t a l l o w e d t o run f o r 18 cm. The p l a t e was d r i e d and e l u t e d i n t h e second d i r e c t i o n w i t h the s o l v e n t system: t e r t - b u t a n o l , m e t h y l - e t h y l k e t o n e , ammonium h y d r o x i d e ( s p e c i f i c g r a v i t y 0.88) and d i s t i l l e d w a t e r ( 5 0 : 3 0 : 1 0 : 1 0 ) . The p l a t e s were removed and d r i e d f o r development. c. D e t e c t i o n The d e v e l o p e r was a f r e s h l y p r e p a r e d m i x t u r e o f 5 0 ml s o l u t i o n 1 ( 5 0 ml 0 . 2 5 % w/v anhydrous e t h a n o l i c n i n h y d r i n s o l u t i o n , 1 0 ml g l a c i a l a c e t i c a c i d , and 2 ml 2 , 4 , 6 - c o l 1 i d i n e ) and 3 ml o f s o l u t i o n 2 ( U w/v Cu ( N O ^ ) 2 - 3 H 2 0 i n anhydrous e t h a n o l ) . The d e v e l o p e r was s p r a y e d on the chromatogram, w h i c h was then heated t o 1 0 0 C f o r 1 5 min. R a d i o a c t i v e fragments on chromatograms were d e t e c t e d by p l a c i n g the e l u t e d and d r i e d chromatograms i n an X-ray f i l m h o l d e r a g a i n s t a sheet o f X-ray f i l m f o l l o w e d by e x p o s u r e o f the f i l m f o r the a p p r o p r i a t e l e n g t h o f t i m e , w h i c h v a r i e d from 2 days t o 2 weeks, depending upon the t o t a l r a d i o a c t i v i t y a p p l i e d t o the chromatograph. Then the f i l m was removed and d e v e l o p e d f o r 5 min a t 2 0 C, washed f o r 2 min i n r u n n i n g w a t e r , f i x e d f o r 3 min a t 2 0 C, and washed f o r 1 hr i n r u n n i n g w a t e r . A l l m a n i p u l a t i o n s were c a r r i e d o u t i n t o t a l d a r k n e s s . h. Assay procedure The assay system was based on that o f Pe lzer (95). The p a r t i c u l a t e enzyme preparat ion was added to a suspension of the p u r i f i e d pept idog lycan . 0.5 ml toluene was added to prevent growth of contaminating b a c t e r i a , and the mixture was ag i ta ted at 37 C. Samples were taken at 0 time and at regular i n t e r v a l s and added to an equal volume of i c e - c o l d 95% e thano l . A f t e r c e n t r i f u g a t i o n at 9,000 x a_ f o r 10 min in the cold to remove the la rger wal l fragments, the supernatant was removed by evaporat ing to dryness at k$ C in vacuo. The residue was resuspended in a minimal volume of water fo r fu r ther a n a l y s i s . The fragments of pept idoglycan were separated by one-dimensional t h i n - l a y e r chromatography. A s i m i l a r technique was used to detect the s o l u b i l i z a t i o n of pept idoglycan from r a d i o a c t i v e l y l a b e l l e d p a r t i c u l a t e enzyme p r e p a r a t i o n s . At 0 t ime, the p a r t i c u l a t e preparat ion was warmed to 37 C and held under to luene , with a g i t a t i o n . Samples o f 100 jul were removed at 0 time and at regular in te rva ls therea f te r and mixed with 100 JJ 1 of i c e - c o l d 95% ethanol in a microfuge tube. The mixture was cen t r i fuged (Beckman microfuge) for k min in the co ld and the supernatant was removed with a pasteur p i p e t t e and spotted on a membrane f i l t e r in a s c i n t i l l a t i o n v i a l . The f i l t e r was dr ied by a current of warm a i r and a heat lamp and the r a d i o a c t i v i t y i t contained measured. Larger samples taken for q u a l i t a t i v e ana lys is of the fragments were added to equal volumes of i c e - c o l d 95% ethanol and cent r i fuged at 9,000 x g_ in the co ld for 10 min. The supernatants were removed and evaporated to dryness and the residues suspended in a minimal known volume of d i s t i l l e d water for fur ther a n a l y s i s . Chloramphenicol , Puromycin and lysozyme were purchased from Sigma Chemical C o . , S t . L o u i s , Mo.; A m p i c i l l i n (Penbr i t in from Ayerst Labs, S t . Laurent , P.O..; D -cyc loser ine from N u t r i t i o n a l Biochemicals C o r p . , C leve land , Ohio; n a l i d i x i c ac id from S t e r l i n g Winthrop L a b s . , Aurora , Ont . ; p e n i c i l l i n - G from Connaught Medical Research Labora tor ies , Toronto, Ont . ; X-ray f i l m , developer and f i x e r from Kodak L t d . , Rochester, N.Y amylase and r i fampin from Calbiochem, Los Angeles, C a l i f . ; Ik 1,7 - C-diaminopimel ic ac id from T r a c e r l a b , Waltham, Mass. ; (G)-diaminopimel ic ac id and ^ C - ( U ) - D - g l u c o s e from Amersham-S e a r l e , A r l ing ton Heights, 111. RESULTS I. IDENTIFICATION AND CLASSIFICATION OF TEMPERATURE-SENSITIVE DIVISION MUTANTS Temperature-sensi t ive mutants which were aberrant for c e l l growth or de fec t i ve in c e l l d i v i s i o n at kl C were i so la ted as described under Methods. Each mutantswas examined f o r : (1) s i z e at 30 C and s i z e at kl C fo l lowing a s h i f t from 30 C to kl C; (2) increase in c e l l numbers a f t e r the s h i f t from 30 C to kl C; (3) the morphology of the c e l l (at 30 C and kl C ) ; and (k) the pattern of DNA synthesis at kl C. The types o f c e l l considered inc luded: f i l aments , m i n i - c e l l producers, s i n g l e c e l l s , double ts , cha in - formers . Mutants were a lso grouped according to the synthesis of DNA at the non-permissive temperature; i . e . whether DNA synthesis continued i n d e f i n i t e l y , stopped immediately, had stopped a f t e r 80 min at the non-permissive temperature or continued at a lowered rate than in the cont ro l c u l t u r e . The mutants were t e n t a t i v e l y c l a s s i f i e d as indicated in Table I I. The values of the parameters fo r each mutant were coded and placed on computer punch-cards. A c a r d - s o r t e r was used to s e l e c t s t r a i n s e x h i b i t i n g p a r t i c u l a r combinations of character -i s t i c s . One mutant, laboratory s t r a i n BUG-6, which f e l l into .41 Table II. C l a s s i f i c a t i o n of temperature-sensi t ive d i v i s i o n mutants of Escher ich ia c o l i . Parameter Express ion 1. S i ze II. C e l l number fo l lowing s h i f t from 30 C to 42 C III. Morphology IV. DNA A. Permissive (30C) 1. Average 2. Small 3. Large B. Non-permissive (42 C) 1. Average 2. Smal1 3. Large A. No increase B. Increase 1. Less than 10% 2. Less than 50% 3. cMore than 100% C. Decrease 1. Less than 10% 2. Less than 50% 3. More than 100% A. Type at permissive temperature (30 C) A. B. C. 1 . 2. 3-4. 5. Normal Filament Cha i n Min i -ce l1 Pleomorph B. Type at non-permissive temperature (42 C) 1. Normal 2. Filament 3. Chain 4. M i n i - c e l l 5. Pleomorph No synthes is at 42 C No synthesis a f te r 80 min at 42 C Continued DNA r e p l i c a t i o n a f te r 80 min. the category IA1, IB3, IIA, IIIA1, IIIB2, IVC, as def ined by Table II, has been extens ive ly studied by others (99) • BUG-6 and several other mutants temperature-sensi t ive for d i v i s i o n but normal for a l l other growth parameters such as macromolecule s y n t h e s i s , volume increase and o p t i c a l densi ty which have been measured, were se lected for fur ther study as true " d i v i s i o n " mutants. II. ACTIVITY OF AUTOLYTIC ENZYMES IN VIVO A. A u t o l y t i c A c t i v i t y in Exponential Cultures Exponent ia l ly growing cu l tu res of E_. col i B/r/1 in minimal glucose were exposed to 5 ug a m p i c i l l i n per ml , 150 ug CAM per ml , or a mixture of both. The numbers were monitored as a funct ion of time and the resu l ts are shown in F i g . 1. The c u l t u r e treated with CAM stopped d i v i d i n g wi th in 30 min.and the c e l l number remained constant for at least 3 h r . The c u l t u r e treated with a m p i c i l l i n stopped d i v i d i n g almost immediately. Twenty min a f t e r the add i t ion of a m p i c i l l i n , the c e l l number decreased at a rate of 50% loss in 60 min. The c u l t u r e t reated with a mixture of CAM and a m p i c i l l i n a l s o stopped d i v i d i n g almost immediately. However, there was only a gradual loss of about 25% in c e l l number over a period of 120 min. A f t e r 120 min of slow l y s i s , the remainder of the c u l t u r e lysed at the same rate observed fo r the c u l t u r e conta in ing ampici11 in a lone. 0 40 80 120 160 ZOO 240 MINUTES Figure 1. E f f e c t of a m p i c i l l i n and chloramphenicol (CAM) on an exponential cu l ture of Escher ich ia c o l i s t r a i n B / r / 1 . The cu l ture was grown in minimal medium for several generations at 37 C (0). At 70 min, subcultures were treated wi th: a m p i c i l l i n (5 /Jg / m l f i n a l concentrat ion) and CAM (150 p /ml ( • ) ) , a m p i c i l l i n alone ( ° ) and CAM alone (A) . SIZE F i g u r e 2. The e f f e c t o f t r e a t m e n t w i t h ampici11 i n and CAM on the s i z e d i s t r i b u t i o n o f E s c h e r i c h i a c o l i B / r / 1 . S i ze d i s t r i b u t i o n s o f an e x p o n e n t i a l l y growing c u l t u r e (•) and o f a sample a f t e r t r e a t m e n t f o r 50 min w i t h a m p i c i l l i n (5 pg/ml) and CAM (150/ug/ml) (0), were o b t a i n e d u s i n g a m o d i f i e d C o u l t e r Counter a t t a c h e d t o a p u l s e - h e i g h t a n a l y z e r (22). Figure 3. a , b. E f f e c t of a m p i c i l l i n and CAM on the morphology of Escher ich ia c o l i s t r a i n B / r/1. C e l l s were removed from a cu l tu re t reated with a m p i c i l l i n (5 yg/ml) and CAM ( 1 5 0 yg/ml) and incubated at 37 C. S l ides were removed at h0 min and examin-ed using a Ze iss phase-contrast photomicroscope. Magni f ica t ion of these photographs is approxi -mately 4,000 x. Examples are presented of a centra l (a) and a polar (b) p e n i c i l l i n " b l e b " . 45 Figure 4. The e f f e c t of mixtures of a m p i c i l l i n and CAM or p e n i c i l l i n - G and CAM on an exponential cu l tu re of Escher ich ia c o l i . The cu l tu re was grown in minimal medium for several generations at 37 C ( ).. At 0 min, subcultures were treated with a m p i c i l l i n (5 pg/ml f i n a l concentrat ion) and CAM (150/jg/ml) (•), or pen i c i 11 i n-G (100 U. I . /ml) and CAM (150 ug/ml) (0). A comparison of the s i z e d i s t r i b u t i o n s of an exponent ia l ly growing c u l t u r e is shown in F i g . 2. It is c l e a r that there is a s e l e c t i v e l y s i s of large c e l l s from the populat ion of the treated c u l t u r e . Microscopic examination of the cu l tu re 40 min a f t e r the treatment with the mixture of a n t i b i o t i c s revealed a number of c e l l s with centra l and terminal p e n i c i l l i n b lebs ' ( F i g . 3). The k i n e t i c s of l y s i s of exponential cu l tures treated with 100 ug p e n i c i l l i n - G and 150 jug CAM per ml were s i m i l a r to those treated with ampici11in-CAM ( F i g . 4 ) . CAM was a lso able to protect from l y s i s c e l l s pre - t reated for 15 min with 30 jug cyc loser ine per ml ( F i g . 5); c y c l o s e r i n e caused rapid l y s i s in the absence of CAM. Puromycin at 120 jug per ml was capable of pro tect ing cu l tures treated with 5 jug a m p i c i l l i n per ml and gave k i n e t i c s of l y s i s s i m i l a r to ampici11in-CAM ( F i g . 5)• .E_. c o l i s t r a i n 982 dap D was starved for DAP fo r 15 min under condi t ions which would normally resu l t in tota l l y s i s of the cu l tu re wi th in 30 min. A f te r 15 min DAP s t a r v a t i o n , 150 ug CAM per ml was added. The c e l l s formed clumps, making counting by Coul ter Counter impossible . However, the cu l tu re was observed m i c r o s c o p i c a l l y to lyse p a r t i a l l y a f t e r 20 min. The major i ty of the populat ion remained s tab le fo r at least a fur ther 60 min. Figure 5- The e f f e c t of mixtures of D-cyc loser ine and CAM, a m p i c i l l i n and puromycin, and a m p i c i l l i n and r i fampin on an exponential cu l tu re of E_. col ?. The cu l tu re was grown in minimal medium for several generations at 37 C ( ) . At -15 min, sub-cu l tures were treated with D-cyc loser ine (30 ug/ml) (0, •) and r i fampin (20 jjg/ml) (A). At 0 min: one of the sub-cu l tures prev ious ly t reated with D-cyc loser ine was treated with CAM (150 ug/ml) (0); the subcul ture prev ious ly treated with r i fampin was treated with a m p i c i l l i n (5 ug/ml) (A); and another subcul ture was treated with ampici11 in (5 ug/ml) and puromycin (120 ug/ml) . MINUTES Figure 6. E f f e c t of a m p i c i l l i n and CAM on an exponential cu l tu re of Escher ich ia col i B/r/1 and E_. col ? BUG-6 growing in enriched medium. The cu l tures were grown in minimal medium for several generations at 30 C ( ) . At 0 min subcultures of E_. col i B/r/1 (•) and E_. col i BUG-6 (0) were treated with a m p i c i l l i n (5 ug/ml) and CAM (150 ug /ml ) . Figure 7- E f f e c t of a m p i c i l l i n and CAM on f i laments of Escher ich ia col B/r/1 induced by treatment with n a l i d i x i c ac id and of E_. col i BUG-6 formed by growth at kl C. Cul tures grown fo r several generations in enriched medium at 30 C ( ) . At -60 min, subcultures were s h i f t e d to kl C and the subcul ture of E_. co 1 i B/r/1 was treated with n a l i d i x i c ac id (10 ug /ml ) . At 0 min both the E_. col i BUG-6 (0) and the n a l i d i x i c ac id treated E. c o l i B/r/1 (•) were treated with a m p i c i l l i n (5 ug/ml) and CAM (150 ug /ml ) . 51 4 0 60 MINUTES I00 Figure 8. The e f f e c t of a m p i c i l l i n and CAM on c e l l s of d i f f e r e n t ages in cu l tures growing synchronously in minimal glucose medium. Synchronous cul tures of Escher ich ia col? s t r a i n B/r/1, se lected by the membrane f i l t e r technique (2) from cu l tures growing in minimal glucose medium, were treated with a mixture of a m p i c i l l i n (5 ug/ml) and CAM (150 ;ug/ml) at var ious times a f te r c o l l e c t i o n from the membrane, and then incubated at 37 C. The rates of l y s i s were determined by measuring d i f fe rences between the number of c e l l s at the time when the o lder c e l l s , t reated at 30 min (•) s tar ted to lyse (15 min) and the time when the younger c e l l s , treated at 0 min (0) , s tar ted rapid general l y s i s (70 min). The rates of l y s i s for c e l l s treated at many d i f f e r e n t ages were compared to the most rapid rate of l y s i s to give a r e l a t i v e rate of l y s i s . Stra in B/r/1 and mutant s t r a i n BUG-6 were grown in nut r ient broth and exposed to a mixture of 5 ug a m p i c i l l i n and 150 ug CAM per ml (F ig . 6). The k i n e t i c s of l y s i s of B/r/1 in nutr ient broth were s i m i l a r to those found in minimal medium ( F i g . 1). The l y s i s of the larger c e l l s of the populat ion occurred in two stages, at approximately 30 min and 90 min and with a 33% loss in numbers over 160 min. BUG-6 lysed to the extent of 50% over 130 min, with corresponding times of l y s i s at 10 min and 50 min. When BUG-6 exponent ia l ly growing at 30 C in nutr ient broth was s h i f t e d to 42 C, i t stopped d i v i d i n g immediately ( F i g . 7). At 42 C, BUG-6 grows normally in a l l known respects except fo r d i v i s i o n . Thus i t elongates exponent ia l ly into a f i lament (99). Af te r 60 min at 42 C, the cu l tu re was treated with 5 ug a m p i c i l l i n and 150 jug CAM per ml . The cu l tu re lysed completely a f te r 30 min exposure to the a n t i b i o t i c s . A f te r 40 min, no intact c e l l s were observable by phase microscopy. S imi la r resu l ts were found for other mutants of the BUG-6 c l a s s . E_. col i B/r/1 growing exponent ia l ly at 30 C in enriched medium was treated with 10 ug n a l i d i x i c acid per ml . A f t e r 60 min the 0D of the cu l tu re was s t i l l increasing exponent ia l ly but d i v i s i o n has slowed and the number of c e l l s had reached a p la teau . The cu l tu re was observed micro-s c o p i c a l l y to contain c e l l s approximately four times as long as normal. When the c u l t u r e of n a l i d i x i c ac id induced long forms was exposed to 5 ug a m p i c i l l i n and 150 ug CAM per ml , the numbers remained constant for approximately 80 min, before the cu l tu re Figure 9- The e f f e c t of a m p i c i l l i n and CAM on a synchronous cu l tu re growing in minimal medium. Samples c o l l e c t e d from a membrane into minimal medium at 37 C and growing with a generation time of kl min, were treated at d i f f e r e n t ages with a mixture of a m p i c i l l i n (5 L ig /ml ) and CAM (150 jug/ml) and the i r r e l a t i v e rates of c e l l l y s i s ca lcu la ted as in F i g . k (0). Samples were a lso treated with CAM (150 ;jg/ml) at various ages and a m p i c i l l i n (5 pg/ml) was added to a l l samples at kO min (•.)'. lysed (F ig . 7). B. A u t o l y t i c A c t i v i t y in Synchronous Cultures Synchronous cu l tures of E_. col i B/r/1 were treated with a mixture of a m p i c i l l i n and CAM at d i f f e r e n t ages. The e f f e c t on c e l l number of add i t ion of these a n t i b i o t i c s at age = 5 min and age = 35 min is shown in F i g . 8. A r e l a t i v e comparison of the d i f f e r e n t i a l e f f e c t of the a n t i b i o t i c mixture on c e l l s of d i f f e r e n t ages is obtained by e s t a b l i s h i n g a " r e l a t i v e rate of l y s i s " . The cu l tu re which shows the greatest loss in c e l l number is given a value of 1.0. A l l measurements were completed 80 min a f t e r ampici11in-CAM a d d i t i o n . A l l other cu l tures are compared with th is maximum l o s s . A cu l tu re which has los t only 1/5 as many c e l l s as the standard cu l tu re is given a value of 0 .2. A summary of the r e l a t i v e rates of l y s i s fo r c e l l s of many d i f f e r e n t ages grown in minimal medium is shown in F i g . 9. The r e l a t i v e rate of l y s i s is observed to be constant for the f i r s t 20 min. It increases slowly at 25 min, forms a plateau and then increases to a rapid rate at 35 min. We have designated the most rapid rate of l y s i s , seen between age 30-40 min, as " l y s i s A" and the lower rate of l y s i s , seen between age 20-30 min, as " l y s i s B". In a control experiment, CAM was added at d i f f e r e n t ages and the a m p i c i l l i n was added at 40 min. The resu l ts were • identical to those obtained when a m p i c i l l i n and CAM were added s imultaneously. S i m i l a r experiments were performed on synchronous cu l tures which were grown in enriched medium with a generation time of 26 min. The r e l a t i v e rates of l y s i s were ca lcu la ted as f o r the minimal cu l tu re above and are presented in F i g . 10. The rate of l y s i s decreased between 12 and 16 min and then increased to a maximum rate of l y s i s at approximately 20 min. The e f f e c t of a m p i c i l l i n and CAM on a synchronous cu l tu re which was sh i f t ed from minimal medium to enriched medium was observed during the second d i v i s i o n period (age 48-74). The r e l a t i v e rates of l y s i s during the second generation are presented in F i g . 11. The r e l a t i v e rate of l y s i s was constant from age 48-56, then l y s i s B occurred between 57~64 min and f i n a l l y l y s i s A occurred between 64-69 min. The manner in which DNA r e p l i c a t i o n patterns were a l t e red during th is s h i f t - u p condi t ion is known. A comparison of the generat ion time, the age at which the end of a round of r e p l i c a t i o n occurs and the time at which l y s i s A occurs is tabulated in Table III. For a d i v i s i o n period of 47 min, l y s i s A occurred at 36 min, the end o f the round of DNA r e p l i c a t i o n at 25 min. The time interva l between l y s i s A and c e l l d i v i s i o n was 11 min. The time in terva l between l y s i s A and the end of the round of DNA r e p l i c a t i o n was a l s o 11 min. In the enriched c o n d i t i o n , the generat ion time was 26 min and l y s i s A occurred at 19 min, 12 16 20 A G E ( M I N U T E S ) 24 Figure 10. The e f f e c t of a m p i c i l l i n and CAM on a synchronous cu l tu re growing in enriched medium.' Samples c o l l e c t e d from a membrane with enriched medium at 37 C and growing with a generation time of 26 min, were treated at d i f f e r e n t ages with a mixture of a m p i c i l l i n (5 ug/ml) and CAM (150 jug/ml) and the r e l a t i v e rates of l y s i s were measured as in F i g . k. Results of two separate t r i a l s are presented (0, • ) . Figure 11. The e f f e c t of a m p i c i l l i n and CAM on synchronous c e l l s in the second generat ion of a " s h i f t - u p " c o n d i t i o n . Samples eluted from a membrane f i l t e r conta in ing c e l l s grown in minimal medium were c o l l e c t e d in a f l a s k conta in ing s u f f i c i e n t concentrated so lu t ion of casamino acids to make enriched medium. A m p i c i l l i n (5 L i g / m l ) and CAM (150 ug/ml) were added at var ious ages during the second generat ion, from 50 to 70 min (A), and the r e l a t i v e rates of l y s i s determined as in F i g . 8. t 1/2 A A ED.R. MEDIUM DIV LYSIS A E.O.R. DIV. MINIMAL 47 36 25 II 1 1 CASAMINO ACIDS 26 1 9 8 7 II SHIFT UP (2nd GEN.) 76 (26) 67 55 9 12 Table III. The r e l a t i o n s h i p between the time of c e l l d i v i s i o n , the end of a round of DNA r e p l i c a t i o n and l y s i s A. The times of the end of a round of DNA r e p l i c a t i o n (23, 46), c e l l d i v i s i o n and l y s i s A ( F i g . 5, 6 and 7) for synchronous c e l l s grown in minimal and enriched medium, as well as in the second generation of a cu l tu re s h i f t e d to enriched medium, are compared. 0 time is the time of c o l l e c t i o n of the c e l l s from the membrane. while the end of the round was at age = 8 min. There was an in terva l of 7 min between l y s i s A and c e l l d i v i s i o n and of 11 min between l y s i s A and the end of the round of DNA r e p l i c a t i o n . Consider ing the second d i v i s i o n during the s h i f t - u p of a synchronous c u l t u r e , the second d i v i s i o n occurred at 76 min which was 26 min a f t e r the f i r s t d i v i s i o n . Lys is A was observed at 67 min and the end of the round was found to be at 55 min. Thus there was an in terva l of 9 min between l y s i s A and c e l l d i v i s i o n and of 12 min between the end of the round and l y s i s A. Synchronous cu l tures in minimal medium were treated with n a l i d i x i c ac id at d i f f e r e n t ages and ampici11in-CAM was added to a l l f l a s k s at 36 min. The r e l a t i v e rate of l y s i s was determined by measuring c e l l number as a funct ion of time for each age. The r e l a t i v e rate of l y s i s in the presence of n a l i d i x i c ac id as a funct ion of c e l l age is shown in F i g . 12. There was an increase in the rate of l y s i s at age = 25 min. When n a l i d i x i c ac id was added at d i f f e r e n t ages of the " s h i f t - u p " c u l t u r e and ampici11in-CAM added at 67 min, the changes in the r e l a t i v e rate of l y s i s occurred in two stages: one at 41 min to a low rate and one at 55 min to a high rate ( F i g . 13). More carefu l examination of the period at h] min confirmed the increase to a sub-maximal rate of l y s i s well above the basal rate (F ig . 13b). 60 24 26 28 30 AGE (MINUTES) 32 Figure 12. The e f f e c t of n a l i d i x i c ac id on ampici11in-CAM l y s i s of synchronous cu l tu re in minimal medium. Samples c o l l e c t e d from a membrane f i l t e r with minimal medium were treated with n a l i d i x i c ac id (10 jug/ml) at 10 min (•) and at various times between 20 and 30 min (0). A l l samples, inc luding a control without n a l i d i x i c ac id (A) were treated with CAM (150 ug/ml) and a m p i c i l l i n (5 ug/ml) at 36 min. 1 1 1 1 1 1 1 1 I 1.0 — A — CO CO >--•0.8 O £0.6 CC U J > >- 0.4 •< _ J — — * * * CC 0.2 | | 1 ° l 1 1 1 1 1 10 20 30 40 50 60 70 80 90 AGE (MINUTES) Figure 13- a , b. The e f f e c t of n a l i d i x i c ac id on the r e l a t i v e rate of l y s i s during the second generat ion of " s h i f t - u p " . Cultures were prepared in the s h i f t - u p condi t ion as in F i g . A and treated with n a l i d i x i c ac id at var ious times between 30 and 70 min (a) (0) . A l l f l a s k s , inc luding a contro l without n a l i d i x i c ac id (A) were treated with, a m p i c i l l i n (5 ug/ml) and CAM (150/jg/ml) at 67 min, and the i r rates of l y s i s compared. The experiment was repeated with n a l i d i x i c ac id added at ages from 30 to 55 min (b) (0). Figure 13b. III. ACTIVITY OF AUTOLYTIC ENZYMES IN VITRO The peptidoglycan of l y o p h i l i z e d c e l l s of E_. col ? B was iso la ted by the c e l l wall preparat ion before t ryps in treatment and of the p u r i f i e d peptidoglycan were a c i d - h y d r o l y z e d . The hydrolysates were analyzed for amino ac id composit ion by two-dimensional chromatography ( F i g . 14). The t r y p s i n treatment removed nine n i n h y d r i n - p o s i t i v e compounds, so that the f i n a l preparat ion contained only diaminopimelic a c i d , D-glutamic a c i d , DL-a lan ine , glucosamine and one neutral u n i d e n t i f i e d amino aci d. The unknown amino ac id may have been a hydro lys is product of a component of the pept idoglycan, or may have been used to cova len t ly l ink other wall polymers to the pept idoglycan, s i m i l a r to the funct ion of l y s i n e in binding a l i p o p r o t e i n to the pept idoglycan in E. c o l i (14). However, an i n s i g n i f i c a n t amount of prote in of normal amino ac id composit ion remained in the p u r i f i e d peptidoglycan ( F i g . 14). The hydrolyzed wall preparat ion was assayed fo r diaminopimel a c i d , which is s p e c i f i c for pept idoglycan, a lso assayed for reducing sugar. The preparat ion contained 0.23 mg pept idoglycan per mg dry weight of p repara t ion , by reducing sugar assay using N-acetyl glucosamine as the standard. The preparat ion contained 0.49 mg .mucopeptide per mg preparat ion as ca lcu la ted from the content of diaminopimelic a c i d . One poss ib le explanat ion for 0 0 « 0 & 0 < > . glu g 0<p P 63 O oc - w • . glucose-NH2 o fl° 0 0 dap b ' 2° DIRECTION Figure 14. Chromatographic separat ion of amino acids from p a r t i a l l y p u r i f i e d and p u r i f i e d pept idoglycan. Samples of peptidoglycan before treatment with t ryps in (a) and a f te r f i n a l p u r i f i c a t i o n (b) were ac id -hydro lyzed and samples spotted on c e l l u l o s e t h i n - l a y e r p l a t e s . The plates were f i r s t run in the v e r t i c a l d i r e c t i o n with i -propanol : formic acidiH^O (40:2:10) and in the hor izonta l d i r e c t i o n with a second solvent t e r t - b u t a n o l : methyl ethyl • ketone: HH^Ortrr^O (50:30:10:10). The amino ac id spots were detected by spraying with n inhydr in . th is discrepancy may be a d i f f e r e n t i a l s e n s i t i v i t y of the reducing sugar to degradation under the condi t ions of ac id h y d r o l y s i s , which would resu l t in a decreased value for peptidoglycan content compared to the value ca lcu la ted from the content of DAP. A crude c e l l envelope preparat ion from exponent ia l ly 8 growing E_. col i eel 1 s at 1 x 10 c e l l s per ml contained 0.833 umoles of diaminopimelic ac id or 0.833 mg of mucopeptide per l i t e r of c e l l suspension. This value was used in determining the pept idoglycan content of p a r t i c u l a t e enzyme preparat ions . The a b i l i t y of enzyme preparat ions from E_. col i to s o l u b i l i z e p u r i f i e d mucopeptide was assayed by measuring the re lease of fragments as ou t l ined by Pelzer (95). The r e s u l t s , along with those of a test of the a b i l i t y of lysozyme to s o l u b i l i z e the p u r i f i e d pept idoglycan, are shown in F i g . 15-The wall preparat ion and enzyme preparat ion at 0 t ime, and the lysozyme a f t e r 48 hr incubat ion , d id not contain s i g n i f i c a n t amounts of components which migrated in the chromatography sy,stem. The wall exposed to lysozyme for 48 hr released fragments numbered 1 to 7 which corresponded to those descr ibed by others (95). The peptidoglycan fragments released from p u r i f i e d peptidoglycan by lysozyme or by a p a r t i c u l a t e enzyme preparat ion were separated by t h i n - l a y e r chromatography ( F i g . 15). They migrated a d is tance s i m i l a r to the migrat ion of the fragments descr ibed by Pelzer (95), when compared to the migrat ion of a lanine (R , ) . o 1 O a g LL. O O O o 7 o o o ^ ^ 5 £ S t »•* • "2 O 2 0 6 , § 1 f a b Figure 15- Separat ion by chromatography of fragments released from p u r i f i e d peptidoglycan by enzyme preparat ions . P u r i f i e d peptidoglycan was exposed to p a r t i c u l a t e a u t o l y t i c enzyme preparat ions (a) and lysozyme (d) . A f te r 48 hr , samples were treated with equal volumes of i c e - c o l d 95% ethanol and centr i fuged at 10,000 x £ fo r 10. min in the c o l d . The supernatant was removed, evaporated to dryness, and the residue resuspended in a minimal volume of d i s t i l l e d water. An a l iquo t was spotted on a c e l l u l o s e t h i n -layer chromatography p la te and run in the solvent system n -bu tano l :ace t ic a c i d : H 20 (40:10:50). The d is tance of migrat ion of peptidoglycan fragments released from: pept idoglycan exposed to a p a r t i c u l a t e enzyme preparat ion (a); the p a r t i c u l a t e enzyme preparat ion alone (b); the p u r i f i e d peptidoglycan alone (c ) ; p u r i f i e d peptidoglycan exposed to lysozyme (d); and from lysozyme alone (e) was compared to the migrat ion of a lanine ( f ) . The plates were developed by spraying with n inhydr in . P e l z e r ' s fragment C-3 contains 2 peptidoglycan subuni ts , each of which contains an N-acetyl glucosamine and N-acetyl muramic acid residue with a pentapeptide s ide c h a i n . The two s ide chains are jo ined by a peptide bond from the terminal a lan ine of one chain to the DAP of the other . C-4 is character i zed as C-3 with the muramyl group of one peptidoglycan subunit jo ined by a (3.-1,4 l inkage to the N-acetyl glucosamine of the second subuni t . C~5 is a peptide glycan subunit with a te t rapept ide which ends at DAP. C-6 is t>5 with a pentapeptide s ide chain which ends with D-a lanine. By comparing the R , values f o r the fragments 1 to 4 3 1 d released by lysozyme ( F i g . 15) to those descr ibed by P e l z e r , fragments 1 to 4 appear to correspond to fragments C-3, C-4, C~5 and C-6 r e s p e c t i v e l y . The lower molecular weight fragments have not been charac ter i zed but are probably products of fur ther degradation of fragments C~3 to C-6. The samples contain ing p a r t i c u l a t e enzyme preparat ion released fragments s i m i l a r to those found a f t e r the lysozyme treatment. Fragments 1 to 3, found at low leve ls a f te r lysozyme treatment, were not detectable in the samples conta in ing the enzyme prepara t ion . However, three d i f f e r e n t fragments of low molecular weight appeared in the whole enzyme prepara t ions , which were absent a f t e r lysozyme treatment. The fragments released by treatment of the p u r i f i e d mucopeptide by lysozyme and by the p a r t i c u l a t e enzyme prepara t ion , were Table IV. Amount of peptidoglycan released as fragments by a u t o l y t i c enzymes. Source Mg peptidoglycan fragments/ml Added as substrate 1.38 Added as enzyme 0.08 Released from p u r i f i e d peptidoglycan by enzyme preparat ion 0.08 to 0.16 Released by lysozyme 1.3 400 -?200 2 CL O QL < Q I X ro 100 80 60 Q £ 40 < o tr o o 20 1 1 1 1 1 — /° — — V — / 1 1 1 1 1 1 20 40 60 80 MINUTES 100 120 Figure 16. Incorporation of rad ioac t ive DAP by E. c o l i ATCC 13070 lys dap . A c u l t u r e of E_. col i ATCC 13070 lys growing exponent ia l ly in 3 enriched medium conta in ing 2 jug and 0.1 uCi H-DAP per ml was assayed for incorporat ion of r a d i o a c t i v i t y at var ious times a f te r the cu l tu re densi ty reached 1 x 10^ c e l l s per ml . Five ml samples were added to 7.5 ml of 7-5% i c e - c o l d t r i c h l o r o a c e t i c ac id (TCA) conta in ing 75 ug DAP per ml , mixed and held on ice for 20 min. The treated samples were f i l t e r e d onto a membrane of 0.45 M pore s i z e , and the f i l t e r ' w a s washed.6 times with i c e - c o l d 5% TCA conta in ing 50 /jg DAP per ml. The f i l t e r s were dr ied and the r a d i o a c t i v i t y measured by s c i n t i l l a t i o n counter . analyzed for reducing a b i l i t y and the resu l ts expressed as mg pept idoglycan per ml are compared to the amounts of peptidoglycan added as substrate .and enzyme (Table IV). A d iaminopimel ic ac id auxotroph of E_. col i , s t r a i n ATCC 13070, was made auxotrophic for l y s i n e by NTG mutagenesis, fol lowed by 3 p e n i c i l l i n treatment. H-DAP with a s p e c i f i c a c t i v i t y of 0.1 uCi per ml was added to an exponent ia l ly growing c u l t u r e of th is lys dap organism in nutr ient broth supplemented with 50 jug l ys ine per ml and 2 ug DAP per ml . The incorporat ion of label into i c e - c o l d 5% TCA p r e c i p i t a b l e mater ial was exponential for at least 100 min (F ig . 16). The label incorporated into cold TCA p r e c i p i t a b l e material was found by one-dimensional ana lys is of an ac id hydrolysate to be 88.2% DAP, with less than 10% of the label in the region of l y s i n e . A p a r t i c u l a t e preparat ion of l abe l l ed wal ls from the dap auxotroph was allowed to autolyze for 24 hr . and the fragments released were c o l l e c t e d , a c i d - h y d r o l y z e d , and the amino acids released then analyzed by two-dimensional chromatography. Some 89% of the r a d i o a c t i v i t y was found as diaminopimelic a c i d . The 14 commercial preparat ion of C-diaminopimel ic ac id was analyzed by two-dimensional t h i n - l a y e r chromatography, and found to contain 1 major spot contain ing 84.5% of the r a d i o a c t i v i t y , and 5 minor spots contain ing the remainder of the r a d i o a c t i v i t y . HOURS Figure 17. Release of rad ioac t ive DAP by p a r t i c u l a t e enzyme preparat ions at d i f f e r e n t l eve ls of pH. C e l l s of E_. c o l i l abe l l ed with ^H-DAP were broken and used to make p a r t i c u l a t e enzyme preparat ions by d i f f e r e n t i a l c e n t r i f u g a t i o n . During incubation in phosphate buf fers of var ious pH l e v e l s , 100 ul samples were taken at i n t e r v a l s , added to 100 ul of i c e - c o l d 95% e thano l , and centr i fuged in the cold using a Beckman microfuge. The supernatant was removed q u a n t i t a t i v e l y , spotted on a membrane f i l t e r i n a s c i n t i l l a t i o n v i a l and d r i e d . The r a d i o a c t i v i t y of the fragments released at pH 8.0 ( ), 7-5 ( ), 7.0 ( ), 6.5 (0) and 6.0 (0) at vanious times was measured by s c i n t i l l a t i o n counter . 0 4 8 12 16 20 24 28 HOURS 18. Release of r a d i o a c t i v i t y by p a r t i c u l a t e enzyme preparat ions made from normal c e l l s and f i laments induced by n a l i d i x i c ac id treatment. A subculture of c e l l s on c o l i ATCC 13070 l y s - growing in enriched medium conta in ing 2 ug and 0.1 uCi 3H-DAP per ml was treated with 10 ug n a l i d i x i c ac id per ml for 60 min. This n a l i d i x i c ac id treated subculture and an untreated subcul ture were used to make p a r t i c u l a t e enzyme prepara t ions . The re lease of rad ioac t ive fragments was measured as in F i g . 17 for the preparat ion made from n a l i d i x i c a c i d - t r e a t e d (0) and untreated (0) c e l l s . <r ^ O V © o (7 0 a +nal - a Figure 19. Separation by chromatography of fragments released by p a r t i c u l a t e enzyme preparations l abe l l ed with 1,7 - 1 ^C-DAP. P a r t i c u l a t e enzyme preparat ions of n a l i d i x i c ac id t reated and untreated c e l l s were made as in F i g . 18, and incubated for 2k h r . The rad ioact ive fragments were recovered and separated by chromatography as in F i g . 15- The rad ioac t ive fragments released by preparat ions made from n a l i d i x i c ac id treated (na l + ) and untreated (-) c e l l s were detected by autoradiography. Alanine (a) (Rg^ ) was detected by spraying with n inhydr in . T a b l e V. A t t e m p t s t o c o n s t r u c t dap a u x o t r o p h s o f E s c h e r i c h i a c o l I . Method o f c o n s t r u c t i o n Donor R e c i p i e n t Recombinant t y p e and number s c r e e n e d s e l e c t e d c o - s e l e c t e d A. C o n j u g a t i o n f o l l o w e d by 3 ju membrane f i l t e r e n r i c h m e n t f o r l o n g forms B. T r a n s d u c t i o n - s e l e c t e d f o r growth on t h r " p l a t e s - s e l e c t e d f o r growth on a r g " u r " p l a t e s BUG-6 Sm s F + T S d | v d a p + m e t + 13070 l y s " 13070 l y s " 13070 VyT F~ dap" met Sm BUG-6 l y s " t h r " BUG-6 l y s " p y r A* Sm r m e t + 3 t r i a l s o f 1000 each t h r ,8 3 t r i a l s 1x10 / t r i a l 8 C. NTG i s o l a t i o n f o l l o w e d by BUG-6 l y s p e n i c i l l i n e n r i c h m e n t t n medium w i t h 50 ug DAP/ml BUG-6 l y s -- B/r/1 l y s — B/r/1 l y s " D. NTG r e v e r s i o n by p l a t e method — BUG-6 l y s " t h r " — BUG-6 l y s " p y r A — B/r/1 l y s " t h r " B/r/1 l y s " p y r A 3 t r i a l s 1x10 (between 100 r e c o m b i n a n t s t r a n s d u c t i o n t h r " 2 t r i a l s , 300 p y r A" ( a r g 1 t r i a l 500 t h r " 3^5 p y r A' ( a r g 200 t h r + 2 t r i a l s 350 each p y r A + 2 t r i a l s kSO each t h r + 275 p y r A 250 / t r i a l and 300 f o r each t r i a l ) each u r " ) u r " ) T S d I v dap" 0 T S d l v dap" dap" t h r " d ap" 0 p y r A" dap" 0 T S d I v 0 0 0 0 The re lease of rad ioac t ive fragments from H-DAP labe l l ed c e l l s of E_. col i ATCC 13070 lys dap was assayed. Resuspension of the p a r t i c u l a t e p e l l e t in 0.2 M phosphate buf fers of varying pH fol lowed by incubation gave the resu l ts shown in F i g . 17. The samples incubated at pH 8.0 and 7-5 released v i r t u a l l y no r a d i o a c t i v i t y , whi le those incubated at pH 7.0 and 6.5 rap id ly released 8% and 12% of the to ta l r a d i o a c t i v i t y , r e s p e c t i v e l y . The sample incubated at pH 6.0 gave a l i nea r re lease for approximately 50 h r , u n t i l 28% of the tota l r a d i o a c t i v i t y was 3 re leased . If the H-DAP labe l led p a r t i c u l a t e envelope preparat ion was resuspended in 0.1 M_ ammonium ace ta te , pH about 6.0, 3% of the label was released in 10 hr ( F i g . 18). In a p a r a l l e l experiment, the l abe l l ed c e l l s were treated with n a l i d i x i c ac id for 60 min before harves t ing . At the time of harves t ing , the o p t i c a l densi ty of the cu l tu re and an untreated contro l c u l t u r e were i d e n t i c a l . The re lease of rad ioac t ive fragment from the n a l i d i x i c ac id treated f i laments was s i m i l a r to that of the untreated c u l t u r e (F ig . 15). The fragments were recovered and separated by one-dimensional t h i n - l a y e r chromatography ( F i g . 19) The fragment patterns were detected by autoradiography. The preparat ion of untreated c e l l s released two major fragments whi le the n a l i d i x i c ac id treated preparat ion released 3 fragments, two of which were of lower molecular weight than any of those released from the untreated prepara t ion . P a r t i c u l a t e enzyme preparat ions der ived from cu l tures of BUG-6 lys or B/r/1 l abe l l ed with C-U-glucose or JH-DAP released v i r t u a l l y no r a d i o a c t i v i t y in the rad ioac t ive in vi tro assay system. Repeated attempts to grow col ? B/r/1 or BUG-6 with DAP as the sole source of carbon or ni trogen or both resul ted in no detectable increase in c e l l numbers of o p t i c a l densi ty during 72 hr incubation at 30 C. S imi la r attempts to grow cu l tures of E_. col ? BUG-6 or B/r/1 in these media a f t e r mutagenesis with NTG were a l s o unsuccess fu l . These resu l ts indicated the necess i ty of using dap s t r a i n s in order to s p e c i f i c a l l y label the peptidoglycan with a level of r a d i o a c t i v i t y s u f f i c i e n t for detect ion of the re lease of pept ido-glycan fragments. The resu l ts of var ious attempts to construct dap s t r a i n s of E_. col i BUG-6 lys and JE. col i B/r/1 lys are summarized in Table V. Attempts to t ransfer known dap markers into E^. col i BUG-6 by the conventional genet ic techniques of conjugat ion and t ransduct ion were unsuccess fu l . Attempts to produce dap s t r a i n s in JE. col i BUG-6 and E_. col i B/r/1 by mutation with NTG and s e l e c t i o n for auxotrophic markers very c l o s e l y associa ted (120) with the known dap genes were equal ly unsuccessfu1. DISCUSSION Although i t would be d i f f i c u l t to prove c o n c l u s i v e l y that changes in peptidoglycan s t ruc ture or metabolism are d i r e c t l y res-ponsib le for the morphogenesis of the septum during c e l l d i v i s i o n , i t has been poss ib le to c o r r e l a t e the a c t i v i t i e s of the pept idoglycan-s p e c i f i c a u t o l y t i c enzymes with 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 pa t te rns . The 25% of the exponential populat ion which was d i f f e r e n t i a l l y s e n s i t i v e to a m p i c i l l i n in the presence of CAM appeared by s i z e d i s t r i b u t i o n and microscopic observat ions to have been large c e l l s c lose to d i v i s i o n (F ig . 1 and F i g . 2 ) . That the rapid l y s i s ( l y s i s A) is assoc ia ted with eel 1 i d i v i s i o n is supported by experiments which show: ( l ) the expression of l y s i s A at a c e l l age which corresponds to septat ion (F ig . 9); (2) a requirement f o r completion of a round of DNA r e p l i c a t i o n (F ig . 12); and (3) the formation of p e n i c i l l i n "b lebs" at the locat ion of c ross -wa l l formation (F ig . 3). From the data in Table 1, i t appears that l y s i s A occurs approximately one-ha l f way through the D p e r i o d , as defined by Helmstetter et al (46), of 20 min between the end of 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 and is independent of the growth rate . Controls ind icate that CAM in te r fe res with l y t i c funct ions in c e l l s of d i f f e r e n t ages s ince the add i t ion of CAM alone at d i f f e r e n t times with the add i t ion of a m p i c i l l i n at a constant time thereaf ter gives s i m i l a r resu l ts ( F i g . 9). Other i n h i b i t o r s of prote in synthe-s i s e . g . puromycin or r i fampin , gave s i m i l a r p ro tec t ion from l y s i s for younger c e l l s ( F i g . 5). Theprevention of l y s i s by i n h i b i t i o n of prote in synthesis might be due to d i r e c t prevention of the synthesis of the a u t o l y t i c enzymes. However, other workers (Shockman, Thompson, Conover) have demonstrated the presence of proteinases which ac t i va te a u t o l y t i c enzymes from zymogens. If the synthesis of a s p e c i f i c p ro te inase , or any other prote in fac tor necessary fo r the a u t o l y t i c a c t i v i t y , was i n h i b i t e d , th is would have the same e f f e c t on the a c t i v i t y of the a u t o l y t i c enzymes as the d i r e c t i n h i b i t i o n of the synthesis of the enzyme. That l y s i s caused by a m p i c i l l i n is due to in ter ference in the resynthesis of mucopeptide a f te r a u t o l y s i s rather than to some other s p e c i f i c property of the drug is indicated by the observat ion that s i m i l a r k i n e t i c s of l y s i s , were obtained when CAM was added to E. co1i s t r a i n AT 982 dap , starved fo r diaminopimelic a c i d , or to s t r a i n B/r/1 t reated with c y c l o s e r l n e ( F i g . 5). Although l y s i s B is less c l e a r l y def ined than l y s i s A , I am convinced that they are qui te d i s t i n c t because of : ( l) the s e n s i -t i v i t y to l y s i s as a funct ion of c e l l age shows a b iphas ic mode of l y s i s ( F i g . 9, 10, 11; (2) the existence of two types of p e n i c i l l i n "b lebs" one at the s i t e of c ross -wa l l formation and a second very near the pole of the c e l l ( F i g . 3); (3) the requirement for completion of the end of a round of r e p l i c a t i o n for the expression of l y s i s A while l y s i s B occurs in the absence of completion of the end of a round of DNA r e p l i c a t i o n (F ig . 12 and 13); (4) the requirement for i n i a t i o n of a new round of r e p l i c a t i o n fo r the expression of l y s i s B ( F i g . 13). The most convincing evidence that l y s i s A and l y s i s B are d i s t i n c t is that p e n i c i l l i n "b lebs" at the s i t e of the cross-wal1 < are found at the time of rapid l y s i s ( l y s i s A) while terminal pen i -c i l l i n "b lebs" are found at the time of slower l y s i s designated l y s i s B. A fur ther c l e a r d i s t i n c t i o n between the two types of l y s i s a l s o resu l ts from an examination of t h e i r expression during the second generat ion of s h i f t - u p of a synchronous populat ion of c e l l s , which allows for the separat ion of the end of a round of DNA r e p l i c a t i o n from the i n i t i a t i o n of a new round of r e p l i c a t i o n . Newly d iv ided c e l l s growing in minimal medium have chromosomes which are approximately 1/2 rep l i ca ted (23, 46). Since the r e p l i c a t i o n per iod is 40 min and the doubling time is approximately 40 min, the end of the round of r e p l i c a t i o n and the beginning of the new round both occur around 20.min. C e l l s growing with f a s t e r generat ion times maintain the 40 min r e p l i c a t i o n per iod but accommodate the fas ter doubling time by the in t roduct ion of mul t ip le r e p l i c a t i o n points (46, 137). If synchronous eel Is growing in minimal medium are s h i f t e d to a r ich medium at age = 0, they w i l l i n i t i a t e new DNA r e p l i c a t i o n points to adjust to the f a s t e r growth rate . Since c e l l d i v i s i o n is dependent on the end of the round of r e p l i c a t i o n , the time at which the f i r s t d i v i s i o n occurs w i l l be unaffected because the r e p l i c a t i o n cyc le in progress at the time of the s h i f t - u p w i l l continue at the same ra te . Completion of th is cyc le at 25 min (23) w i l l be the s igna l for the f i r s t d i v i s i o n . On the other hand, s h i f t - u p w i l l cause the premature i n i t i a t i o n of new rounds which w i l l be completed 40 min fo l lowing i n i t i a t i o n . Our measurements ind icate that new rounds are i n i t i a t e d 10 - 15 min a f t e r the s h i f t - u p . The second c e l l d i v i s i o n w i l l occur 20 min a f t e r completion of th is premature DNA r e p l i c a t i o n c y c l e , or 70 - 75 min a f t e r s h i f t - u p . I n i t i a t i o n of subsequent r e p l i c a t i o n cyc le w i l l occur at regular in te rva ls of one generat ion per iod (46) a f t e r i n i t i a t i o n of the second r e p l i c a -t ion c y c l e . If r e p l i c a t i o n is f i r s t i n i t i a t e d 15 min a f te r the " s h i f t - u p " , the next round must be i n i t i a t e d 26 min l a te r or at 41 min. Thus, in the second generat ion , we have es tab l ished a broad separat ion between the end of a round of r e p l i c a t i o n (55 = 45 + 10; min) and the i n i t i at ion of a round of rep l i cat ion (41 mi n) . The resul ts obtained by examining the second generation of a synchronous cu l ture sh i f t ed -up to a f a s t e r growth medium ( F i g . 11) show c l e a r l y that l y s i s B preceeds l y s i s A. From Table III, the time between l y s i s A and the end of the round of DNA r e p l i c a t i o n is 12 min. Lys is B is co inc ident with the end of the round (55 min) s i m i l a r to that seen in the non-sh i f t -up condi t ions (F ig . 9)• However, a level of l y s i s s i m i l a r to l y s i s B is not observed i f DNA r e p l i c a t i o n is blocked p r i o r to the i n i t i a t i o n of a new round of r e p l i c a t i o n (41 min) under the s h i f t - u p , condi t ions ( F i g . 13). L,ysis B is observed i f DNA r e p l i c a t i o n is blocked a f te r i n i t i a t i o n , of DNA r e p l i c a t i o n (41 min) but p r i o r to the end of a round of r e p l i c a t i o n (55 min). I in terpre t th is to mean that l y s i s B is assoc ia ted with the i n i t i a t i o n of a new round of r e p l i c a t i o n but is independent of the completion of a round of r e p l i c a t i o n . Since i t occurs co inc ident with the end of the round and not the i n i t i a t i o n , l y s i s B may a l s o depend upon some metabol ic funct ion which continues independent of the r e p l i c a t i o n of DNA but which is completed at about the same time as the end of a round of r e p l i c a t i o n . In r i c h medium, chromosome r e p l i c a t i o n occurs by mul t ip le r e p l i c a t i o n points (46, 137). With a generat ion time of 26 min, there is a change in the number of r e p l i c a t i o n points from 3 to 2 at age = 5 min. At age 18 min, there is the add i t ion of 4 more r e p l i c a t i o n points to give a t o t a l of 6. During the in terva l of 12 to 18 min we not ice a change in the rate of l y s i s from 0.6 to o.4 ( F i g . 10). This r e s u l t , coupled with the observat ion of terminal p e n i c i l l i n " b l e b s " , leads me t o suspect that l y s i s B may be associated with a segregat ion mechanism producing asymetric growth (23, 29, 50). Growth of cu l tures in condi t ions where they would have an increased growth rate or an increased number of growing s i t e s per c e l l increases t h e i r sens i t i v i . t v to l y s i s by ampici 11 in-CAM. In enr iched medium, a larger f r a c t i o n of c e l l s lysed soon a f t e r add i t ion of the a n t i b i o t i c s (F ig . 7) than in cu l tures grown in minimal medium ( F i g . 1). The time between the end of the round of 'DNA r e p l i c a t i o n and the time of l y s i s A , as well as the time between l y s i s A and the time of d i v i s i o n , appear to be independent of the growth ra te . (Table IV). The shor ter generation time in enriched medium would make the c e l l s suscept ib le to l y s i s A over a larger f r a c t i o n of the generat ion time, and in tu rn , a larger f r a c t i o n of the c e l l s would be suscept ib le to l y s i s A. j^dly- mutants would be expected to be less s u s c e p t i b l e to immediate l y s i s by ampici11in-CAM in exponential cu l ture at 42 C, due to lack of septum formation. However, a l l TS^-^- mutants tested exhib i ted rapid tota l l y s i s of f i lamentous forms (F ig . 7)• One p o s s i b l e explanat ion of t h i s is based on the fact that DNA r e p l i c a t i o n and segregat ion are normal in a l l the mutants tes ted . This would resu l t in every f i lamentous c e l l having more than one segregat ing mechanism at any given time and an increased suscep-t i b i l i t y to l y s i s B. An equal ly poss ib le explanat ion is that l y s i s A a c t i v i t y continues in the mutant f i l a m e n t s , even though septum formation is i n h i b i t e d . It would appear that l y s i s A is under the control of the DNA r e p l i c a t i o n mechanism that cont ro ls c e l l d i v i s i o n , and may be an a u t o l y t i c funct ion necessary to the formation of the cross w a l l . Lys is B on the other hand, is assoc ia ted with the i n i t i a t i o n of DNA during asymmetrical c e l l growth. General c e l l expansion may be f a c i l i t a t e d by the less a c t i v e l y s i s C. There are several l ines of evidence presented for the binding of the a u t o l y t i c enzymes of E_. c o l i to the c e l l envelope, inc luding 82 the i n a b i l i t y of added p a r t i c u l a t e enzymes to s o l u b i 1 i z e the p u r i -f i e d pept idoglycan (Table IV), and the i n a b i l i t y of p a r t i c u l a t e preparat ions conta in ing rad ioact ive DAP to release more than 28% of the r a d i o a c t i v i t y . Further evidence for the l o c a l i s a t i o n of the a u t o l y t i c enzymes is the formation of s p e c i f i c breaks in the c e l l wall in the presence of a m p i c i l l i n and CAM (Fig 3 ) . The l o c a l i z a t i o n of a u t o l y t i c enzymes would serve as an e f f e c t i v e means of regulat ing t h e i r a c t i v i t y . They could be l imi ted to ac t ion in areas of l o c a l i z e d growth for wall expansion or for septum format ion. S t r i c t l o c a l i z a t i o n of the three auto-l y t i c systems found in the i n vi vo assay systems could f a c i l i t a t e the separate control of t h e i r a c t i v i t i e s . Other workers have reported the l o c a l i z a t i o n of a u t o l y t i c enzymes by t igh t binding to the c e l l envelope components ( 3 6 ) . .Muramidase a c t i v i t y in S. f a e c a l i s has been demonstrated to be l o c a l i z e d in recent ly synthesized wall (116) and to be released only by treatment with high concentrat ions of s a l t ( 9 7 ) . The enzymes could be bound cova lent ly to the peptidoglycan or other wall polymers by mechanisms s i m i l a r to the covalent bonding of the l i p o p r o t e i n to the muramic ac id resudies in E. c o l i ( 13 , 14) or through a phosphodiester l inkage to the muramic a c i d , as reported for the polysacchar ides in Staph aureus and Micrococcus lysod iek t icus ( 36 , 5 7 ) . The a u t o l y t i c a c t i v i t i e s of E. c o l i must a l s o contain enzymes other than B-1,A N-acetyl muramidase, or lysozyme, as the fragments released by the p a r t i c u l a t e enzyme preparat ions were of lower average molecular weight, and therefore migrated fur ther in the chromatography system (F ig . 14), than those released by the ac t ion of lysozyme on p u r i f i e d pept idoglycan. In add i t ion to a muramidase a c t i v i t y , Pe lzer (95) descr ibed the presence of g lucosaminidase, amidase, endopeptidase, and carboxypeptidase a c t i v i t i e s associa ted with the c e l l envelope of E_. col i . These a c t i v i t i e s would be s u f f i c i e n t to degrade the peptodoglycan subunits released by muramidase a c t i v i t y to the i r const i tuent amino sugars and amino a c i d s . The degradation is not complete, s ince the fragment c o r r e s -ponding to lysozyme fragment 4 ( F i g . 14) which in turn corresponds to P e l z e r ' s (95) fragment C-6, a complete peptodoglycan subunit with penta-pept ide s ide c h a i n , is present a f te r extensive incubat ion. In p a r t i c u l a t e enzyme preparat ions made from exponent ia l ly growing n a l i d i x i c ac id- induced f i l aments , the degradation is even more extensive than in the normal c e l l s . Only one of the two major rad ioac t ive fragments released by normal c e l l s ( F i g . 19) is present in the fragments released by the preparat ions made from the n a l i d i x i c ac id treated c e l l s . Two products of fur ther degrada-t ion make up the remainder of the r a d i o a c t i v i t y re leased. The f a i l u r e to re lease more than 10% of the r a d i o a c t i v i t y (F ig . 19) confirms that the a u t o l y t i c enzymes responsible for peptidoglycan degradation are t i g h t l y bound, with only a f r a c t i o n of the rad io -a c t i v i t y in both n a l i d i x i c ac id treated and untreated c e l l s a v a i l a b l e to t h e i r a c t i o n . The mechanism of ac t ion of n a l i d i x i c ac id in i n h i b i t i o n of DNA r e p l i c a t i o n is poorly understood ( 1 2 ) . However, i f n a l i d i x i c ac id were to i n h i b i t DNA r e p l i c a t i o n by changing the s t ruc ture of the membrane attachment s i t e for DNA, i t might e a s i l y change the environment of one of the a u t o l y t i c enzymes other than the muramidase, such as the amidase, g lucosaminidase, endopeptidase, or carboxypeptidase so as to cause more extensive degradation of the peptidoglycan in p a r t i c u l a t e preparat ions made from n a l i d i x i c ac id treated eel 1s. To use the in v i t r o assay technique to assay the a u t o l y t i c a c t i v i t y of the TS-^-^- mutants i so la ted (Table II) would require the const ruc t ion of dap- auxotrophs of the TS^-^- mutant s t r a i n s . Despite repeated attempts by various approaches, th is proved extremely d i f f i c u l t , and no TS^-^-" dap- s t r a i n s were formed (Table V) . Although i t is dangerous to construct hypotheses on the basis of the f a i l u r e to i s o l a t e a c e r t a i n type of mutant, one p o s s i b l e explanat ion for the f a i l u r e to i s o l a t e a BUG-6 dap s t r a i n may be that the BUG-6 l es ion is involved with DAP metabolism. This is u n l i k e l y s ince BUG-6 grown at kl C on plates conta in ing 50 ug DAP per ml e x h i b i t s the c h a r a c t e r i s t i c f i lamentous phenotype. The combination of the BUG-6 and dap- mutations may be l e t h a l , even at 30 C. Extreme d i f f i c u l t y in the s p e c i f i c rad ioact ive l a b e l l i n g of the peptidoglycan has been encountered because there appears to be no normal active transport system for diaminopimelic acid (79). Although dap auxotrophic mutants have been isolated by use of the pen ic i l l in enrichment technique (28) i t is d i f f i cu l t to see how this can possibly enrich for dap auxotrophs as cel ls starved for DAP do not stop growth as in the normal stringent response to amino starvation, but rather form spheroplasts and lyse ( 2 8 ) . Low levels of pen ic i l l in have been reported to select for cel l wall mutants, includi ng dap auxotrophs ( 7 8 ) . Other workers have isolated dap mutants but were not able to use any enrichment techniques (C. Lark, U. Henning, personal communication; 1 8 ) . Because NTG has been reported to act at the DNA replication point (20) and closely linked genetic markers are replicated at nearly the same time, mutagenesis by NTG might be used as a method of co-selecting closely linked markers, and of mapping closely linked markers. The fai lure to find dap auxotrophs after selecting auxotrophic (or prototrophic reversions of) markers which are closely linked to dap, from cultures of BUG-6 lys- or B/r /1 lys mutagenized with NTG (Table V), argues against the effectiveness of co-mutagenesis with NTG to select or map genetic markers. However, the quality of the NTG available for these experiments was suspect, and the technique might prove valuable with a different preparation of NTG in future studies. The synthesis of the autolytic enzymes has been correlated with the c e l l c y c l e , although the value of the in v ivo assay in i d e n t i f y i n g the les ions in the TS-^-^ mutants have been l i m i t e d . This is la rge ly due to the i n a b i l i t y to d i f f e r e n t i a t e between l y s i s A and l y s i s B in f i laments larger than twice noamrl. The preparat ions of cu l tures of TS^-^- synchronous for DNA r e p l i c a t i o n might al low separat ion between l y s i s A and B on a bas is s i m i l a r to that used to separate the i r t iming in synchronous cul tures of normally d i v i d i n g c e l l s . The in vi t ro assay shows promise in determining the ro le of a u t o l y t i c systems in the T S d ' v s t r a i n s . E i ther a l t e r n a t i v e methods to construct TS^-^- dap s t r a i n s must be found or TS-^-^- cu l tures must be i so la ted from dap s t r a i n s . The a b i l i t y to prepare synchronous cu l tures of dap s t r a i n s would a l s o be valuable to observe f luc tua t ions of the a u t o l y t i c enzymes during the c e l l c y c l e . A quan t i t a t i ve measurement of the a c t i v i t i e s of gene products involved in c e l l d i v i s i o n is essen t ia l i f recent ly developed con-cepts of control mechanisms are to be appl ied to understanding the contro l of c e l l d i v i s i o n . The involvement of the pept idoglycan and a u t o l y t i c enzymes in c e l l d i v i s i o n has been demonstrated in th is d i s s e r t a t i o n . The fur ther c h a r a c t e r i z a t i o n of the a u t o l y t i c enzymes and other enzymes involved in peptodoglycan metabolism would appear to be an ideal approach by which to charac te r i ze the gene products involved in c e l l d i v i s i o n . In summary, i t has been shown that the major i ty of the c e l l s 87 in exponential cu l tures of E_. col i can be protected from immediate l y s i s , due to in ter ference with peptidoglycan s y n t h e s i s , by i n h i b i t i o n of prote in s y n t h e s i s . This i n h i b i t i o n of l y s i s is thought to be due to the prevention of synthesis of the a u t o l y t i c enzymes, or of a prote in fac tor necessary for the i r a c t i v i t y , associated with septum formation and d i v i s i o n . Using synchronous cu l tures of E_. coj_i_, growing at various ra tes , and exposing them to chloramphenicol and a m p i c i l l i n at various ages, i t has been poss ib le to show that there are at least three d i f f e r e n t a u t o l y t i c systems, separable on a temporal and quant i t a t i ve b a s i s . One system is associated with general ex-pansion of the c e l l w a l l , the second with the formation of the cross-wa l l during d i v i s i o n , and the th i rd with i n i t i a t i o n and perhaps segregat ion of the r e p l i c a t i n g copies of DNA. Exponent ia l ly growing mutant long forms of E. col? have at least the segregating and wall expansion f u n c t i o n s , while long forms induced by i n h i b i t i o n of DNA synthesis have only the general wall expansion a u t o l y t i c system. S t r i c t b c a l i z a t i o n by t ight binding could f a c i l i t a t e separate control of these three d i f f e r e n t a u t o l y t i c systems. Using the in v i t r o assay system, the a u t o l y t i c enzymes of E_. co 1 ? we re shown to be t i g h t l y bound to the c e l l envelope. They were l o c a l i z e d to the extent that they produced defined les ions during p e n i c i l l i n induced l y s i s , and that they were able to release only a f r a c t i o n of the incorporated rad ioact ive DAP from the peptodoglycan. 88 LITERATURE CITED 1. Ade lberg , E . A . , M. Mandel, and G . C . C . Chen. 1965. Optimal condi t ions f o r mutagenesis by N-methyl, N ' n i t r o , N -n i t ro -soguanidine in Escher ich ia col? K12. 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