An examination of constitutive direct light DNA repair and inducibility of DNA repair in two thermophilic bacteria by Mary Ann Starkey Kirkpatrick A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Biology Montana State University © Copyright by Mary Ann Starkey Kirkpatrick (1985) Abstract: Two thermophilic bacteria, Bacillus stearothermophilus and Thermus T2 were observed for response to known DNA-damaging agents, UV radiation and the chemical mutagen, Mitomycin C. The existence of a constitutive direct light DNA repair system was discovered in Bacillus stearothermophiIus. Unlike E. coli whose dark DNA repair is UV-inducible, Thermus was not found to have a UV-inducible repair mechanism. However the presence of a DNA repair system inducible by either heat or chemicals was observed in Thermus, relating temperature-associated DNA repair with survival at high temperatures.  AN EXAMINATION OF CONSTITUTIVE DIRECT LIGHT DNA REPAIR AND INDUCIBILITY OF DNA REPAIR IN TWO THERMOPHILIC BACTERIA by Mary Ann S ta rk ey K i r k p a t r i c k A t h e s i s s u bm it te d i n p a r t i a l f u l f i l lm e n t o f th e requ irem en ts f o r th e degree of Master of Sc ience i n Biology MONTANA STATE UNIVERSITY Bozeman, Montana November, 1985 <3o^ su i i APPROVAL o f a t h e s i s subm it ted by Mary Ann S ta rk ey K i rk p a t r i c k Th is t h e s i s has been re ad by each member of th e t h e s i s committee and h a s b een fo u n d to be s a t i s f a c t o r y r e g a r d i n g c o n t e n t , E n g l i s h usage, fo rm a t , c i t a t i o n s , b ib l io g r a p h i c s t y l e , and c o n s is te n cy , and i s ready f o r subm is s io n to th e College o f G raduate S tud ie s . Date / C ha irpe rson , G /aduate Committee Approved f o r th e Major Department Date ///ZLS/X.IS Head, Biology Approved f o r the College o f G raduate S tu d ie s ( ' I - r ^ t ^ Date Graduate Dean iii STATEMENT OF PERMISSION TO USE I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l l m e n t o f t h e r e q u i r e m e n t s f o r a m a s t e r ’s d e g r e e a t M ontana S t a t e U n i v e r s i t y , I a g r e e t h a t t h e L i b r a r y s h a l l make i t a v a i l a b l e t o b o r r o w e r s u nde r r u l e s o f t h e L i b r a r y . B r i e f q u o t a t i o n s f rom t h i s t h e s i s a r e a l l o w a b l e w i t h o u t s p e c i a l p e rm i s s i o n , p r o v id e d t h a t a c c u r a t e acknowledgment of sou rce i s made. P e rm is s io n f o r e x te n s iv e q u o ta t io n from or r e p ro d u c t io n of t h i s t h e s i s may be g ran te d by my majo r p ro f e s s o r , o r i n h i s / h e r absence, by th e D i r e c to r of L i b r a r i e s when, i n th e op in io n o f e i t h e r , th e proposed use of th e m a t e r i a l i s f o r s c h o la r l y purposes . Any copying or use of t h e m a t e r i a l i n t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i th o u t my w r i t t e n pe rm iss ion . V' ACKNOWLEDGMENT I would l i k e t o e x p re s s my g r a t i t u d e to the f o l low in g peop le . Dr. Guylyn Warren f o r her s t im u l a t i n g d is c u s s io n , gu idance , and e d i t o r i a l comments d u r i n g th e c o u r s e o f t h e s e e x p e r im e n t s and p r e p a r a t io n o f th e m anu sc r ip t . Her s u g g e s t io n of th e t h e s i s problem, a s w e l l a s the s c i e n t i f i c and p e rson a l i n t e r s ! i n my development a s a p r o f e s s io n a l woman, a r e g r e a t l y a p p re c ia te d . Dr. E rn e s t Vyse f o r h i s d e d ic a t io n to the development o f so many s t u d e n t s , i n c l u d i n g m y s e l f , a s w e l l a s t h e u se o f h i s l a b o r a t o r y space , equipment, s u p p l i e s , and e x te n s iv e p r o f e s s io n a l t im e. My G raduate Committee: Dr. Samuel Rogers f o r o v e r a l l i n s p i r a t i o n i n th e a re a o f m o le cu la r g e n e t i c s and Dr. David Cameron f o r d i s c u s s io n o f a b roader view of B iology and G ene t ic s . Sue Z aske f o r h e r p a t i e n t t e a c h i n g m anner and s h a r i n g o f e x p e r t i s e w ith th e e l e c t r o n m icroscope . Dr. N e ls Nelson f o r d i s c u s s io n and gu idance i n i d e n t i f i c a t i o n and c h a r a c t e r i z a t i o n methods f o r B a c i l l u s s te a r o th e rm o p h i lu s . v i TABLE OF CONTENTS Page APPROVAL.......................................................................................................... i i STATEMENT OF PERMISSION TO U SE ......................................................................... i i i VITA ................ iv ACKNOWLEDGMENTS.................................... v TABLE OF CONTENTS.................................. v i LIST OF TABLES.................. v i i i LIST OF FIGURES......................... i x LIST OF PLATES ........................................................................................................... x ABSTRACT................................................................................. ....................................... . x i INTRODUCTION ............... I B iochem is try o f Thermophily F a t ty a c id s and membranes........................................................................ 2 P r o t e i n s .............................................................................. 3 Ribosomes.............................................................. 4 N uc le ic a c i d s .................................................................................................... 5 DNA DAMAGE AND REPAIR .................................. 7 DNA D amage .................... 7 1 . R a d i a t i o n ........................................................... 8 2. Chemical mutagens .............................. 8 3 . T r a n s p o s o n s ............................................................................... 9 DNA Damage: UV and MC ....................................................... 9 Types o f DNA R e p a i r .................. 10 1. P h o t o r e a c t i v a t i o n ................. ................ ......................................... 12 2. E x c is io n r e p a i r ............................................................................... 13 3. P o s t r e p l i c a t i o n a l r e p a i r .......................................................... 14 4. The a d a p t iv e re sponse ............................................................... 14 5. SOS r e p a i r .............................................................................' ............ 15 6. SOS d e f i c i e n t mu tan ts o f JL c o l i ......................................... 17 7. Heat s h o c k ........................................................................................... 19 8. B a c t e r i a used f o r r e s e a r c h ...................................................... 21 9. S ta tem en t of r e s e a r c h problems ............................................... 21 TABLE OF OOMTENTS-Continued v i i Page MATERIALS AND METHODS ........................................................................................... 23 Source of organism s ......... 23 I d e n t i f i c a t i o n and d i f f e r e n t i a t i o n o f B a c i l l u s ................. 23 B a c t e r i a l growth c o n d i t io n s ............................................................. 24 Microscopy ......................................................... 27 DNA damaging t r e a tm e n t s ...................................................................... 28 Exposure t o UV ................................................................................ 28 Exposure to MG .................................................................................... 28 Repair a s s a y s ...................................................................................... 28 S u rv iv a l .............................................................. 30 RESULTS ........................................................................................................................... 31 A. The o rganism s o f th e study .............................................. ................ 30- I d e n t i f i c a t i o n o f B a c i l l u s s t e a r o thermop h i lu s ........... 31 Morphology ................................ 31 P h y s io lo g ic a l c h a r a c t e r i s t i c s ................................................. 31 C h a r a c t e r i s t i c s o f Thermus ................... 35 B. Q u a n t i t a t i v e r e s u l t s of k i l l i n g by i r r a d i a t i o n ................... 39 C. Exposure to UV and MG.................................. ................................... .. 41 D. Q u a n t i t a t i v e r e s u l t s of p h o to r e a c t i v a t io n and d i r e c t l i g h t r e p a i r fo l low in g UV t r e a tm en t .................... 43 DISCUSSION ................................................................................................... 47 REFERENCES CITED ...................................................................................................... 57 v i i i i LIST OF TABLES Tab les Page 1. Summary o f C h a r a c t e r i s t i c s o f T h e rm o p h i l e s 37 2. P h o to r e a c t iv a t io n : B a c i l l u s s te a ro th e rm o p h i lu s 45 and EL. c o l i C ond it ion s o f A ttempted D i r e c t L ig h t R epa ir 463. i x LIST OF FIGURES F igu re s 1. S t r u c tu r e o f a Thymine d imer 2 . S t r u c tu r e of Mitomycin C 3. UV S u rv iv a l : JL c o l i . Thermus 55 C, Thermus 70 C 4 . UV S u rv iv a l : B a c i l l u s s te a ro th e rm onh i lu s 55 C UV S u rv iv a l : Thermus. 1 , 2 , 3 hou rs a f t e r t r a n s f e r from 55 C to 70 C Page 11 11 40 42 5. 44 XLIST OF PLATES P la t e s Page 1. B a c i l l u s . G ramstain 32 2. B a c i l l u s . Terminal e l l i p t i c a l sp o re 33 3. B a c i l l u s . F l a g e l l a 34 4. . Thermus 55 C and Thermus 70 C 36 x i ABSTRACT Two t h e rm o p h i l i c b a c t e r i a , B a c i l l u s s t e a r o t h e rm o o h i l u s and Thermus T2 were ob se rved f o r re spon se to known DN A-dam ag in g agen ts , UV r a d i a t i o n and th e chem ica l mutagen, M itomycin C. The e x i s t e n c e of a c o n s t i t u t i v e d i r e c t l i g h t DNA r e p a i r system was d is cov e re d i n B a c i l l u s s t e a r o th e rm o p h i I u s . U n l ik e Eh c o l i whose d a rk DNA r e p a i r - i s UV- i n d u c i b l e , Thermus was n o t f o u n d t o h av e a U V - in d u c ib l e r e p a i r mechanism. However th e p resence o f a DNA r e p a i r system in d u c ib le by e i t h e r h e a t or c h em ica ls was ob se rv ed i n Thermus, r e l a t i n g tem p e ra tu r e - a s s o c i a t e d DNA r e p a i r w i th s u r v iv a l a t h igh tem pe ra tu re s . 1INTRODUCTION B iochem is try o f Thermoohilv M e s o p h i l i c m i c r o o r g a n s im s h av e a maximum g row th r a t e a t t em p e ra tu r e s around 30-37 C; i n the case o f Et c o l i T grow th cea se s a t 45—48 C w h i le t em p e ra tu r e s o f 50-52 C o r g r e a t e r cause c e l l death. T he rm oph il ic m ic roo rgan ism s can t h r i v e a t t em p e ra tu r e s up t o 85 C. They a r e f o u n d i n g e o th e rm a l l y a c t i v e a r e a s such a s h o t s p r i n g s , s o l a r - h e a t e d d e s e r t s o i l , a s c o n t a m in a n t s o f c anned f o o d and d a i r y p r o d u c t s , i n h o t w a t e r h e a t e r s , and i n i n d u s t r i a l e f f l u e n t . Such o r g a n i sm s h av e p iq u e d t h e i n t e r e s t o f th e s c i e n t i f i c community f o r r e a s o n s o f t h e i r p r o l i f e r a t i o n a t e l e v a t e d t e m p e r a t u r e s and t h e t h e r m o s t a b i l i t y o f t h e i r m a c r o m o l e c u l e s . B a s i c k n o w le d g e o f t h e rm o p h i l y i s a p p l i c a b l e t o s t u d i e s o f e v o l u t i o n and e c o lo g y , m o le cu la r b io logy and b io c h em is t ry , and cou ld le a d to u t i l i z a t i o n f o r i n d u s t r i a l en zym ic p r o d u c t i o n o f m a rk e tab le p roduc ts (Amelunxen and Murdoch, 1977). Tempera tu re i s on ly one of th e v a r i a b l e s in f l u e n c in g th e growth o f l i v i n g organism s. O ther env ironm en ta l f a c t o r s such a s pH, n u t r i e n t q u a l i t y and q u a n t i t y , s a l i n i t y , and l i g h t i n t e r a c t t o i n f l u e n c e th e o p t im a l and maximum grow th tem pe ra tu re s . Under v a ry in g c o n d i t io n s the i n t e r r e l a t i o n s h i p o f chem ica l s t r u c t u r e , con fo rm a tion , and f u n c t i o n of b i o l o g i c a l m o l e c u l e s may v a r y i n a d a p t i n g t o s t r e s s (H ochachka and Somero, 1973). Thus, a t t em p t s to e x p la in th e s p e c ia l a b i l i t y to l i v e 2a t h ig h t e m p e r a t u r e h av e e v o lv e d f rom th e o b s e r v a t i o n o f g e n e r a l phys io logy to a b io chem ica l approach w i th concom itan t e x am in a t io n a t t h e m o le cu la r l e v e l . The h y p o t h e s i s t h a t i n c r e a s e d r a t e s o f s y n t h e s i s and t u r n o v e r , e i t h e r by a d a p t a t i o n o r m u ta t io n , a r e r e s p o n s ib l e f o r r a p id r e p l a c e ­ ment of heat-damaged p r o t e i n s was advanced by A llen (1953). However, when Brock (1967) p ub l i sh ed growth r a t e d a ta o f v a r io u s m e so p h i l ic and th e rm o p h i l ic b a c t e r i a a t t h e i r optimum tem p e ra tu r e s A l le n 's h y p o th e s i s was d iscoun ted . Brock (1967) found t h a t th e rm oph i le s do not grow a s f a s t a t t h e i r op tim a a s p r e d ic te d by p u re ly t h e o r e t i c a l c a l c u l a t i o n s of e f f e c t of tem pe ra tu re on physio logy . When U lr ic h (1971) combined p h y s i o l o g i c a l and b io c h em ic a l a p p r o a c h e s to examine a Thermus- I i k e o rgan ism f o r m o rpho log ica l c h a r a c t e r i s t i c s , r e s p i r a t o r y mechanism, and r e g u l a t i o n o f enzyme s y n th e s i s , he found no major d i f f e r e n c e s between m esoph i le and th e rm oph i le , ex cep t t h e rm o s t a b i l i t y . B i o c h e m i c a l s t u d i e s o f t h e r m o p h i l e s h av e d e t e rm in e d th e p r o p e r t i e s of s p e c i f i c c e l l components o r m o lecu le s and compared th e se t o t h e i r c o u n t e r p a r t s i n m e s o p h i l e s . F a t t y a c i d s and m embranes , p r o t e i n s and th e p r o t e i n - s y n t h e s i z i n g m a c h in e ry a s w e l l a s n u c l e i c a c id s of s e v e r a l th e rm oph i le s have been i s o l a t e d and examined. F a t ty a c i d s and membranes: The membranes of th e rm oph i le s a re exposed to th e env ironment so t h i s component of th e c e l l was one of th e f i r s t , to be examined f o r h e a t s t a b i l i t y . The f l u i d i t y of b a c t e r i a l biomem­ b r a n e s i s c o n s t a n t l y m a in t a i n e d i n g row in g c e l l s . V a r i a t i o n i n 3complex l i p i d co n ten t and s t r u c t u r a l changes i n f a t t y a c id components a r e s u g g e s t e d a s m e ch an ism s t o a c h i e v e t h e f l u i d i t y known to be im p o r t a n t f o r membrane f u n c t i o n s (C ronan , 1978). The f a t t y a c i d c o n t e n t o f v a r i o u s m i c r o o r g a n i sm s i s known to be a f f e c t e d by th e t em p e ra tu r e a t which they a r e grown (Oshima, 1978). High p ro p o r t io n s of u n s a tu r a t e d a c id s a r e found a t low er t em p e ra tu re s , w h i le s a tu r a t e d f a t t y a c i d s i n c r e a s e w i t h i n c r e a s i n g t e m p e r a t u r e . The p r e s e n c e o f h ig h ly branched, lo n g e r cha in , s a t u r a t e d f a t t y a c id s i n membranes o f th e rm o p h i l e s h as been con firm ed (Oshima e t a l . , 1976). Also, a novel g l y c o l i p id c o n s t i t u t i n g up to 70% of th e t o t a l l i p i d o f two s t r a i n s o f Therm us h a s b een i d e n t i f i e d (O sh im a and A r ig a , 1976 ) . I t i s c o n je c tu re d t h a t the un ique l i p i d co n ten t of the th e rm o p h i l i c membrane i s r e s p o n s ib l e f o r s u c c e s s fu l membrane f u n c t i o n a t h igh tem pera tu re . P r o t e i n s : S in c e th e p r im a r y , s e c o n d a r y , t e r t i a r y and q u a t e r n a r y s t r u c t u r e o f p r o t e i n s o f t e n vary as much between p r o t e i n s o f th e same f u n c t i o n o b ta in e d from v a r io u s m e so p h i l i c o rgan ism s a s between thermo­ p h i l i c and m e so p h i l ic p r o t e i n s of th e same fu n c t io n , i t i s d i f f i c u l t t o e x p l a in th e i n v a r i a b l e s t a b i l i t y to d en a tu r in g c o n d i t io n s (chem ical d e n a tu r a n ts a s w e l l a s h e a t ) o f th e rm o p h i l ic p ro te in s . V arious in v e s - 1 t i g a t o r s have proposed t h a t enhanced s t a b i l i t y i s due t o hydrophobic (Ohta, 1966), hydrogen (Barnes and S te l lw ag en , 1973)? o r i o n i c (Peru tz and R a id t , 1975) bonding p roducing con fo rm a tion s w i th l a r g e r o r more d e n s e ly p a ck ed p r o t e i n i n t e r i o r s (B u l l and B r e e s e , 1973)> more or d i f f e r e n t s e c o n d a ry s t u r c t u r e ( S t e l lw a g e n and B a r n e s , 1976), m ore e x t e n s iv e ly la ced m acromolecu la r s u r f a c e s (Peru tz and R a id t , 1975), or 4more c om p lem en ta ry I n t e r s u b u n i t c o n t a c t s (B i e s e c k e r e t a l . , I 977). (T h i s t o p i c h a s b een r e v i e w e d by Z u b e r , 1 976; F r ie dm an , 1978; Amelunxen and Murdock, 1977; S in g le to n and Amelunxen, 1973.) H e a t - r e s i s t a n c e i s o f t e n c o n f e r r e d by o n ly a few am ino a c i d c h a n g e s a s shown by M e rk le r e t a l . (1981) who com pared t h e p h y s i c a l c h a r a c t e r i s t i c s of p r o t e i n s o f c lo s e ly r e l a t e d m e so p h i l i c and thermo­ p h i l i c b a c i l l i . Argos e t a l . (1979), found s t r a t e g i c a l l y s u b s t i t u t e d amino a c id s in c r e a s e d i n t e r n a l hyd rophob ic i ty and in c r e a s e d e x t e r n a l p o l a r i t y . Hydrophobic bonds a r e more s t a b l e a t h igh tem p e ra tu re th an a t low tem pe ra tu re . A pparen tly m o le cu la r i n t e r a c t i o n s w i t h i n polypepr t i d e ch a in s a r e s u f f i c i e n t t o cause t h e rm o s ta b i l i t y . Ribosomes: Ribosomes and th e o th e r components a s s o c i a t e d w i th p r o t e i n s y n th e s i s a r e a l s o th e rm o s ta b le i n th e rm oph i le s . Ribosomal s u b u n i ts ( p r o t e i n and RNA) h av e b een fo u n d to be h e a t s t a b l e ( Y aguch i e t a l . , 1978). P r o t e i n e l o n g a t i o n f a c t o r s w h ich d e l i v e r and c a t a l y z e th e b ind ing of charged t-RNAs to th e ribosome a re r e q u i r e d f o r e lo n g a t io n o f th e po ly p ep t id e ch a in i n p r o t e i n s y n th e s i s i n p rok a ryo te s . These f a c t o r s have been p u r i f i e d from Thermus th e rm oph ilu s and compared to E. c o l i (A ral e t a l . , 1978). The th e rm o p h i l ic e lo n g a t io n f a c t o r s a r e e x t r e m e l y s t a b l e a g a i n s t h e a t , a c i d , a l k a l i , and o t h e r p r o t e i n d e n a tu ran ts . Thermus e l o n g a t io n f a c t o r s showed a l a c k o f s u l fh y d ry l g ro u p s i n c o n t r a s t t o t h o s e o f EL c o l i w he re s u l f h y d r y l s p la y a n e s s e n t i a l r o l e i n c a t a l y t i c f u n c t i o n . I n c o n t r a s t t o t h e m onom eric fo rm s found i n th e m esoph ile the e x i s t e n c e o f m u l t im e r ic fo rm s were 5dem onstra ted i n the th e rm oph ile . I t i s a p p a r e n t t h a t no s i n g l e m echan ism o r c e l l componen t i s r e s p o n s ib l e f o r the rm oph ily . As th e t h e rm o s t a b i l i t y o f membranes was sugges ted to be due to p resence of novel g ly c o l i p id s a s w e l l as degree o f s a t u r a t i o n o f i t s f a t t y a c i d s and th e v a r i a t i o n i n p r o t e i n s t r u c t u r e a l low in g f u n c t i o n a t h igh tem p e ra tu re was dem onstra ted , th e n u c l e i c a c i d p o r t i o n o f p r o t e i n s y n t h e s i s w as a l s o f o u n d to be h e a t s t a b l e . N uc le ic a c id s : The n u c le ic a c id s o f th e rm oph i le s have been s tu d ie d i n a v a r i e t y o f ways in c lu d in g base com pos i t ion ; p resence and a c t i o n of a s s o c i a t e d p o ly am in e s ; i s o l a t i o n and s tu d y o f en zym es i n v o l v e d i n s y n th e s i s , r e s t r i c t i o n and m o d i f i c a t io n of th e rm o p h i l ic n u c l e i c a c id s ; i s o l a t i o n and c h a r a c t e r i z a t i o n o f mRNA, t-RNA, r-RNA and DNA, i s o l a ­ t i o n of a n t i b i o t i c r e s i s t a n c e - c a r r y i n g and c r y p t i c p la sm id s , a s w e l l a s th e c lo n in g o f th e rm o p h i l ic genes w i th subsequen t e x p r e s s io n and c h a r a c t e r i z a t i o n i n m esoph ile s . H ea t s t a b i l i t y o f t h e rm o p h i l i c n u c l e i c a c i d s i n c r e a s e s w i t h i n c r e a s e d G-C c o n t e n t p r o d u c in g h i g h e r c o r r e s p o n d in g i n c r e a s e i n m e l t in g tem p e ra tu r e (Oshima e t a l . , 1976). Therm oph il ic DNA s t a b i l i t y may a r i s e p a r t i a l l y from a s s o c i a t i o n w i th d iv a le n t c a t i o n s a s i t i s known t h a t d i v a l e n t c a t i o n s s t a b i l i z e DNA and RN A. T h i o l a t i o n o f n u c l e i c a c id s i n c r e a s e s w i th t e m p e r a t u r e and i s d i r e c t l y c o r r e l a t e d w i th t h e rm o s t a b i l i t y of t-RNA i n th e c e l l (Quigley and Rich, 1976). Polyam ines a re g e n e r a l ly con s id e red to be in v o lv ed i n im p o r ta n t b i o c h em ic a l p r o c e s s e s su ch a s s t a b i l i z i n g DNA and RNA, p r o t e i n 6b io s y n th e i s , DNA and RNA b io sy n th e se s , c e l l d iv i s io n , and a c c l im a t io n to en v i ronm en ta l s t r e s s . Novel poly am ines a re produced by the ex treme th e rm oph i le , Thermus th e rm ooh i lu s (Oshima, I 975 and 1982). Alan Malcolm has proposed i n a t h e o r e t i c a l paper (1981) t h a t th e in c r e a s e d G-C c o n ten t of m-RNA w i th consequen t in c r e a s e d s t a b i l i t y of secondary s t r u c t u r e shou ld a l s o be con s id e red a s a s e l e c t i o n p r e s s u re i n th e th e rm o p h i l ic env ironm en t and t h a t th e most common amino a c id changes between m esoph i le s and thermop h i l e s reco rd ed by Argos e t a l . (1979) a r e c o n s i s t e n t w i th t h i s h y po th e s is . S in g le base changes found i n th e m-RNA codons of th e rm oph i le s e i t h e r i n c r e a s e the s t a b i l i t y of s e c o n d a ry s t r u c t u r e o r h av e l i t t l e e f f e c t (none w ou ld d e c r e a s e secondary s t r u c t u r e ) . S t e n i s h and M ad ison (1979) com pa red t h e s t a b i l i t y o f m-RNA i n m e soph i le s and th e rm oph i le s , found th e h a l f - l i f e of m-RNA to d ec rease as grow th tem p e ra tu re in c r e a s e d but d is cov e red th e " s t a b i l i t y index" ( h a l f - l i f e o f mRNA/doubling t im e o f c e l l s ) to be c o n s t a n t f o r e a ch o rg a n ism r e g a r d l e s s o f t e m p e r a t u r e . Th is s u ppo r ts t h e concept t h a t k i n e t i c c o n s i d e r a t i o n s p la y a s i g n i f i c a n t r o l e i n t h e rm o p h i l y ; th e h a l f - l i f e of th e m-RNA i s a f ix e d f r a c t i o n of th e doub ling time. N uc le ic a c id s of th e rm oph i le s and m esoph i le s have been shown to v a ry i n n u c le o t id e c o n ten t , p resence of novel po lyam ines , s t r u c t u r e of en zym es i n v o l v e d i n s y n t h e s i s , and r e s t r i c t i o n and m o d i f i c a t i o n enzymes. However, t h e r e a re many s i m i l a r i t i e s i n b a s ic o r g a n iz a t io n of g e n e t i c m a t e r i a l and e x p r e s s io n o f in fo rm a t io n . Therm oph il ic DNA and RNA p o ly m e r a s e s (K a le d in e t a l . , I 980; C h ie n e t a l . , 1 976; D a te , 71975), DNA m e thy la se (Sa to e t a l . , 1980), and DNA r e s t r i c t i o n enzymes (Sato e t a l . , 1977) have been i s o l a t e d and c h a r a c te r i z e d . The p rop e r ­ t i e s d em o n s t r a t e d by t h e s e enzym es a r e s i m i l a r t o t h o s e o r o t h e r t h e rm o s t a b l e p r o t e i n s . T h is i s a l s o t r u e o f enzym es p ro d u c ed f rom g e n e s c lo n e d f rom t h e rm o p h i l e s (N ag a h a r i e t a l . , 1 980). A c i r c u l a r d ich ro ism s tudy o f the complex between p romoter DNA and Thermus RNA p o lym e r a s e ( T s u j i , 1980) showed o n ly more m e l t i n g i n t h e p rom o te r r e g io n th an found i n AN c o l i r c o n f irm ing th e s im i l a r i t i e s of the two o rgan ism s. R e s e a r c h on DNA r e p a i r and m u t a g e n e s i s i n m e so p h i l ic b a c t e r i a , i n i t i a l o b s e rv a t io n s of th e f i l am e n to u s h ig h ly th e rm o p h i l ic b ac te r ium , Thermus. and r e c e n t r e p o r t s t h a t a p r o t e c t i v e re sponse to h e a t s t r e s s can be i n d u c e d i n m e s o p h i l i c p r o k a r y o t e s and e u k a r y o t e s by a g e n t s t h a t can induce changes i n DNA r e p a i r a c t i v i t y a s s o c i a t e d w i th f ! l a ­ m en ta t io n su g g e s te d to Dr. Guylyn Warren a p o s s ib le n a tu r a l a s s o c i a ­ t i o n between growth a t h igh tem pe ra tu re , DNA damage and r e p a i r . DNA Damage and Repair DNA Damage: Fou r m a in t y p e s o f DNA a l t e r a t i o n s o r damage h av e been s tu d ie d : I) D im e r iz a t io n of two a d j a c e n t p y r im id in e s on the same DNA s t r a n d when t h e p y r im i d i n e s become c o n n e c te d by a c y c l o b u t a n e r i n g , 2) chem ica l a l t e r a t i o n of bases by d eam in a t io n or a k l y l a t i o n , 3) i n t r o d u c t i o n o f c o v a le n t c r o s s l i n k s between bases on two s t r a n d s , and 4) b reaks i n one or both s t r a n d s . Any of th e se damages can r e s u l t i n l e t h a l i t y o r an a l t e r e d coding p ro p e r ty o r mu ta tion . M u ta t i o n s can be s p o n t a n e o u s , p o s s i b l y a r i s i n g f rom e n z ym a t i c 8d y s fu n c t io n du r ing DNA r e p l i c a t i o n o r recom b ina t ion . Mutation , i n a broad sense , a l though a h e r i t a b l e change, may no t a f f e c t the phenotype o r be recogn ized . However, th e te rm w i l l be used i n t h i s m anu sc r ip t t o r e f e r t o a h e r i t a b l e change i n n u c l e o t id e sequence of an organism which i s re cogn iz ed by i t s e f f e c t on th e phenotype of th e organism. A mutagen i s an a g en t which causes changes, as. d e s c r ib e d above, i n g enom ic n u c l e i c a c i d and i n c r e a s e s t h e m u t a t i o n r a t e above th e spon taneous l e v e l a s ob se rved phendty p i c a l l y . Known mutagens p re s e n t i n t h e e n v i r o nm e n t i n c l u d e r a d i a t i o n (UV and X - r a y s ) , c h em ic a l m u t a g e n s ( a l k y l a t i n g a n d d e a m i n a t i n g a g e n t s , b a s e a n a l o g s , i n t e r c a l a t i n g ag en ts , and c r o s s - l i n k i n g ag en ts ) and t r a n s p o s e ns. 1. R ad ia t io n . a) U l t r a v i o l e t (UV) l i g h t cau ses f o rm a t io n of d im e rs between p y r im id in e base s on th e same s t r a n d ( i n t r a s t r a n d ) of DNA. The p y r im i­ d i n e s become c o n n e c t e d by a f o u r c a r b o n c y c l o b u t a n e r i n g . ( See diagram and d e t a i l i n UV s e c t i o n below.) b) X -rays cause b reaks i n th e phosphodie s t e r backbone i n one or both s t r a n d s o f DNA. 2. Chemical mutagens e f f e c t m o d i f i c a t io n s i n DNA b a se s i n s i t u by d e am in a t i o n , a l k y l a t i o n , o r t h e a d d i t i o n o f a v a r i e t y o f b u lk y a d d u c t s . C h em ica l m u tag en s a l s o in c lu d e : s t r u c t u r a l ana logs which v a r y i n b o n d in g w i t h t h e p a r t n e r b a s e ; i n t e r c a l a t i n g a g e n t s w h ich i n s e r t d u r i n g r e p l i c a t i o n , d i s t o r t t h e b a se p a i r i n g and l e a v e a f t e r r e p l i c a t i o n r e s u l t i n g i n a gap or an added base i n the newly syn the ­ s i z e d s t r a n d ; and c r o s s - l i n k i n g a g e n t s which form i n t e r s t r a n d c ro s s ­ 9l i n k s po s ing an a b s o lu t e b lock to r e p l i c a t i o n and t r a n s c r i p t i o n . 3. T r a n s p o s o n s , o r u n i t s o f DNA t h a t h av e t h e c a p a b i l i t y o f moving from one DNA mo leu le t o a n o th e r , r e s u l t i n re a r ra n g em en ts and d e l e t i o n s i n th e m o lecu le t h a t was l e f t and i n s e r t i o n and d is tu rb a n c e of DNA c o i l i n g i n th e m o lecu le e n te red . DNA Damage - UV and MG: UV and M itomycin C were chosen f o r examina­ t i o n of the th e rm oph i le s ' r e spon se to mutagens. The py r im id in e d imer c a u s e d by UV i s t h e b e s t r e s e a r c h e d l e s i o n . The c r o s s - l i n k i n g mechanism o f Mitomycin C p rov ided a second mechanism of DNA damage f o r o b s e rv a t io n . UV Damage: DNA e f f i c i e n t l y ab so rb s l i g h t i n th e range of 240-300 nm r e s u l t i n g i n e x c i t e d energy s t a t e s of the bases and c au s ing a v a r i e t y o f p h o to c h em ic a l r e a c t i o n s (Wang, 1976). The p r i n c i p l e p r o d u c t , p y r im id in e d im ers , cau s ing the p r i n c i p l e b io lo g ic a l e f f e c t s , l e t h a l i t y and m u t a g e n i s i s , i s f o rm ed when two a d j a c e n t p y r im id i n e b a s e s on a s t r a n d a r e l i n k e d t o g e t h e r by a f o u r - c a r b o n r i n g (F ig . I ) . The two bases a r e p u l l e d ou t o f a l ignm en t , th e hydrogen bonds to complementary b a s e s a r e b ro k en , and th e DNA backbone i s d i s t o r t e d , p r e v e n t i n g th e c o r r e c t p a i r i n g o f t h e two b a s e s on e a c h s i d e o f t h e d im e r . The p resence of a s in g l e d imer can i n t e r r u p t t r a n s c r i p t i o n or r e p l i c a t i o n . Even i f r e p l i c a t i o n r e s um e s on t h e o t h e r s i d e o f t h e d im e r , a gap i s l e f t i n th e new ly s y n t h e s i z e d s t r a n d , b l o c k in g t r a n s c r i p t i o n o f th e e n t i r e t r a n s c r i p t i o n u n i t and a b o r t i n g r e p l i c a t i o n i n th e next cy c le (Hanawalt e t a l . , 1 979). 10 M itom y c in C (F ig . 2) i s m e t a b o l i c a l l y r e d u c e d by a q u in o n e r e d u c ta s e i n the c e l l t o a hydroquinone d e r i v a t i v e which a l k y l a t e s and e x t e n s iv e ly c r o s s - l i n k s DNA ( Iy e r and S z y b a l s k i , 1963). The b io lo g i ­ c a l s i g n i f i c a n c e of i n t e r s t r a n d c r o s s - l i n k i n g i s e v id e n t from s tu d ie s on t r a n s f o rm i n g DNA and b a c t e r i a l v i r u s e s (Kohn e t a l . , I 963; B eck e r e t a l . , 1964)). One i n t e r s t r a n d c r o s s - l i n k i s s u f f i c i e n t to cause the i n a c t i v a t i o n o f a t l e a s t 3 ,000 b a s e p a i r s w i t h i n a DNA m o le c u le , p resumably a s a consequence of b lo ck ing complete s t r a n d s e p a r a t io n f o r r e p l i c a t i o n . EL. c o l i m u t a n t s d e f e c t i v e i n one o r more u v r g e n e s ( e x c i s i o n r e p a i r ) a r e more s e n s i t i v e t o M itom yc in C t h a n w i l d ty p e s t r a i n s . While s im p le a l k y l a t i o n damage i s no t r e p a i r e d by e x c i s io n r e p a i r , c r o s s - l i n k i n g caused by the b i f u n c t io n a l Mitomycin C r e q u i r e s e x c i s i o n r e p a i r ( F i s h b e i n e t a l . , I 970; C o le e t a l . , 1 976). DNA R epa ir : For every o rgan ism , l i f e and c o n t in u i ty from g e n e ra t io n t o g e n e r a t i o n depend on t h e l o n g - t e rm s t a b i l i t y o f i t s h e r e d i t a r y m a t e r i a l , t h e DNA. S in c e a l l c e l l s a r e s e n s i t i v e t o damage by r a d i a t i o n and chem ica l a g en ts i n th e env ironment, a system of removal o f l e s i o n s and r e s t o r a t i o n o f t h e i n t a c t DNA a p p e a r s t o have been adopted. I t i s no t p o s s i b l e f o r DNA po lymerase I I I t o r e p l i c a t e a r e a s of DNA c o n ta in in g d im ers o r c r o s s - l i n k s , a l though i t can r e s t a r t a f t e r th e damaged r e g io n has been passed. Howard-F landers (19.75) h as shown t h a t d a u g h t e r DNA m o l e c u l e s r e p l i c a t e d f rom UV-damaged DNA c o n t a i n gaps app rox im a te ly th e s i z e of one or more Okazaki f ragm en t i n d i c a t i n g 11 O d Rib F igu re I . S t r u c t u r e o f a th ym in e d im e r r e s u l t i n g f rom u l t r a v i o l e t i r r a d i a t i o n o f DNA (b a s e d on D av is , B.D., Dul b ecco , R., Bi s en , H.N., G in s b e rg , H.S. M ic r o b io lo g y , T h i r d E d i t i o n , Harper and Row, Maryland, 1980). F igu re 2 . S t r u c t u r e o f M i tom yc in C (b a s e d on F i s h b e i n , L., W.G. Flamm, and H.L. F a lk , e d s . , C h em ica l M u tagens . A cadem ic P r e s s , N.Y., 1 970). 12 r e s u m p t i o n o f r e p l i c a t i o n do e s o c c u r w i t h c o n c om i t a n t g ap s i n t h e daugh te r s t r a n d . DNA r e p a i r in v o lv e s r e c o g n i t i o n o f a l e s i o n by a p r o t e i n t h a t can i n i t i a t e t h e b i o c h em ic a l r e a c t i o n s w h ich l e a d t o e l i m i n a t i o n o r c i r c u m v e n t i o n o f t h e l e s i o n . W h ile s p e c i f i c en zym es r e p a i r some s p e c i f i c base m o d i f i c a t io n s or c o r r e c t a c h em ic a l a l t e r a t i o n , o t h e r t y p e s o f r e p a i r a r e more g e n e r a l i n n a t u r e and c an e i t h e r r e p a i r a v a r i e t y o f l e s i o n s or a l low a second chance a t s p e c i f i c r e p a i r th rough r e combi n a t i o n a l p r o c e s s e s . The m a jo r t y p e s o f DN A r e p a i r known i n b a c t e r i a a re p h o to r e a c t i v a t i o n , b a se s u b s t i t u t i o n , e x c i s i o n r e p a i r , t h e a d a p t i v e r e s p o n s e and r e c o m b i n a t i o n a l o r p o s t - r e p l i n a t i o n a l r e p a i r . Excep t f o r p h o t o r e a c t i v a t i o n , s p e c i f i c p r o o f - r e a d i n g f u n c ­ t i o n s i n DNA r e p l i c a t i o n and e x c i s i o n r e p a i r , DNA r e p a i r i s l a r g e l y e r r o r - p r o n e . . . I . P h o to r e a c t iv a t io n . P h o t o r e a c t i v a t i o n w as d i s c o v e r e d a s a r e d u c t i o n i n t h e l e t h a l e f f e c t of f a r - UV i r r a d i a t i o n by a sub sequen t exposure t o lo n g e r wave­ le n g th s . I t h a s been dem ons tra ted i n many c e l l u l a r sy s tem s in c lu d in g b a c t e r i a l and human and a c t s on py r im id in e d im ers only. The pho to re ­ a c t i v a t i n g enzyme b inds to th e d im e r - c o n ta in in g r e g io n o f th e DNA thu s g e n e r a t i n g a DNA-enzyme ch rom opho re t h a t a b s o r b s v i s i b l e l i g h t t o c a t a l y z e c l e a v a g e o f t h e j o i n e d b a s e s w i th o u t b reak ing any phospho- d i e s t e r bonds . Pho tom ed ia ted re cove ry from UV damage was d is cove red i n b a c t e r i a and b a c t e r i o p h a g e i n I 94 9 (K e l n e r ; Dul be coo). The m echan ism was 13 c h a r a c t e r i z e d i n 1962 (Rupert) . R ecen tly , an a l t e r n a t e r o l e f o r th e p h o to r e a c t i v a t in g enzyme was sugges ted by Yamamoto e t a l . (1983) who d i s c o v e r e d an E. c o l i r e c A m u ta n t t o be l e s s s e n s i t i v e t o UV i n t h e p resence o f a p h o t o r e a c t i v a t i n g enzyme i n th e d a rk . P h o t o r e a c t i v a ­ t i o n , however, can be masked by an e f f i c i e n t e x c i s io n r e p a i r system. 2. E x c is io n R epa ir . D i f f e r e n t modes o f e x c i s i o n r e p a i r o f damaged DNA h av e been d is cov e red i n d i f f e r e n t organ ism s. In AN c o l i th re e d i s t i n c t l o c i a re known to be r e q u i r e d f o r e x c i s io n o f damage i n U V - i r ra d ia te d DNA. The u l t r a v i o l e t l i g h t damage r e p a i r , o r u v r T g e n e s a r e r e s p o n s i b l e f o r t h r e e p r o t e i n s t h a t a s s o c i a t e (Nakabeppu and Sek iguch i , 1981) t o make t h e UVRABC enzyme, now c lo n e d and c h a r a c t e r i z e d by S a n c a r and Rupp (1983 ) . UVRABC i s r e s p o n s i b l e , i n t h e p r e s e n c e o f Mg++ and ATP, f o r making two c u t s , one on each s id e of the damaged DNA, th u s removing a 12 -13 n u c l e o t i d e lo n g , s i n g l e - s t r a n d e d f r a g m e n t o f DNA. The gap i s t h e n f i l l e d t h r o u g h a c t i o n o f DNA p o lym e r a s e I and s e a l e d by DNA l i g a s e . I t i s s u g g e s t e d t h a t t h e enzyme may b in d t o t h e r e l a t i v e l y u n s t a b l e s e c t i o n o f t h e 12 -13 b a se p a i r f r a g m e n t r e s u l t i n g f rom DNA damage, th e i n s t a b i l i t y enhanced by b ind ing o f UVRA and UVRC to s in g le s t r a n d e d DNA (S e e b e rg and S te im em , 1982; S a n c a r and Rupp, 1979) and t h a t th e DNA po lymerase I exonuc lease a c t i v i t y may enhance removal of th e e x c i s e d s t ran d . The c u t s , d i s p la c e d from the s i t e s o f damage, can r e p a i r a b ro a d s p e c t r um o f d am ages w i t h o u t p r e c i s e r e c o g n i t i o n o f p a r t i c u l a r a d d u c t s . The enzyme a c t s on DNA w h ich h a s b een t r e a t e d w i t h UV-i r r a d i a t i on , P t ( I I ) c om p o u n d s , p s o r a l e n p l u s n e a r ­ 14 u l t r a v i o l e t , n i t r o u s a c id o r M itomycin C (Brash and B ase l t i n e , 1982). Kenyon and W alke r (19 81) have s u g g e s t e d t h a t t h e UVRA and UVRB p r o t e i n s a r e p ro d u c ed a t c o n s t ! t u t i v e l y low l e v e l s b u t in d u c e d t o h ig h e r l e v e l s of p ro du c t io n by the c o n t ro l e lem en ts o f th e in d u c ib le "SOS" re sp on se o f b a c t e r i a a c t i v a t e d when r e p a i r has no t o ccu rred and DNA r e p l i c a t i o n i s b locked a t the s i t e o f damage. 3. P o s t r e p l i c a t i o n a l r e p a i r . P o s t r e p l i c a t i o n a l r e p a i r t a k e s a d v an tag e of O kazak i-s iz ed gaps which a r e l e f t by po lymerase I I I i n the newly s y n th e s iz e d DNA o ppo s i te t h e damaged r e g i o n . P o s t r e p l i c a t i o n a l r e p a i r , t h e n , i s a s y s tem o f r e t r i e v a l , whereby m a t e r i a l from one s t r a n d of a dup lex o f DNA can be u s e d t o r e p a i r t h e gap i n a n o t h e r . The m echan ism o f c r o s s o v e r s between two daugh te r m o lecu le s , whose gaps do not u s u a l ly c o in c id e i s r e c o g n iz a b le by e l e c t r o n m icroscopy o f DNA (Rupp and Howard-F landers , I 968); Rupp and c ow o rk e r s (1971 ) d em o n s t r a t e d t h a t d i s c o n t i n u i t i e s w e re fo rm ed i n DNA i n an e x c i s i o n - d e f e c t i v e s t r a i n . The l e s i o n r em a in s on one s t r a n d ; h ow ev e r , a n o t h e r a t t e m p t t o r e p l i c a t e can be made and may be s u c c e s s f u l w i t h r e p l i c a t i o n o f t h e new r e c om b in a n t m o le c u le . The d a u g h t e r s t r a n d gap r e p a i r r e q u i r e s a f u n c t i o n a l r e c combin a t io n r e c A gene a s w e l l as po lymerase , po l A o r po l C. 4. The Adaptive Response. When exposed to low c o n c e n t r a t i o n s o f m e th y la t in g o r e t h y la t i n g a g en ts , Et c o l i becomes r e s i s t a n t t o th e mu tagen ic and l e t h a l e f f e c t s o f h i g h e r d o s e s o f t h e same a g e n t s ( J eggo e t a l , I 977 and 1978). The i n d e p e n d e n t l y r e g u l a t e d p a thw ay w as f i r s t d i s c o v e r e d by Samson and 15 C a irn s (1977) and has been te rm ed th e a d a p t iv e re sponse (Jeggo e t a l . , 1 977). The i n d u c i n g s i g n a l f o r t h e n e tw o rk i s unknown. The p o s i ­ t i v e l y a c t i n g r e g u l a t o r y e l em e n t o f t h e a d a p t i v e , a d a . l o c u s i s sugges ted by Walker (1984) to be s p e c i f i c a l l y induced by th e adduc t of t h e a l k y l a t i o n o f g u a n in e . The ad a l o c u s c o n s i s t s o f an o p e ro n coding f o r two p r o t e i n s (L indah l, 1982). Also known to be in vo lv ed i n t h e a d a p t i v e r e s p o n s e i s t h e u n i d e n t i f i e d l o c u s c o d in g f o r Ch­ a lk y Ig u an in e -DN A a l k y l t r a n s f e r a s e ( L in d a h l e t a l , 1982) (w h ic h c a t a ly z e s th e t r a n s f e r o f th e m e thy l or e t h y l group from th e a lk y la te d guan ine t o the p r o t e i n i t s e l f ) , a p r o t e i n o f broad s p e c i f i c i t y , th e 3 - m e th y l a d e n i ne-DNA g l y c o s y I a s e I I w h ich i s t h e p r o d u c t o f t h e a l k A gene (Evenson, G. and E. See berg , 1982) and an unknown number of o th e r genes . Although m e th y la t in g and a l k y l a t i n g a g en ts can in t r o d u c e l e s i o n s t h a t m i s p a i r and r e s u l t i n m u t a t i o n , o t h e r l e s i o n s c a u s e d by t h e s e a g e n t s in d u c e a n o t h e r mode, nSOSn r e p a i r , w h ich r e s u l t s i n a c t i v e m u tagenes is . 5 . SOS Repair . The term nSOS f u n c t io n s " has been d e s ig n a ted f o r a complex group o f r e s p o n s e s i n JL_ c o l i t h a t a p p e a r t o be c o o r d i n a t e l y r e g u l a t e d . I n c l u d e d i n t h e s e r e s p o n s e s a r e i n h i b i t i o n o f c e l l d i v i s i o n , f i l am en to u s grow th i n r e p a i r - d e f i c i e n t m u tan ts a s s o c i a t e d w i th induc ­ t i o n o f SOS r e p a i r , i n h i b i t i o n o f p o s t i r r a d i a t i o n DNA d e g r a d a t i o n , i n d u c e d b a c t e r i a l m u t a g e n e s i s , i n d u c t i o n o f p ro p h a g e , a s w e l l a s Weigle r e a c t i v a t i o n and Weigle m u tagenes is . The l a t t e r two re sp on se s 16 were d is cov e red by Weigle (1953) when he observed t h a t U V - i r ra d ia te d b a c te r io p h ag e y i e ld e d more p laques and a h ig h e r p ro p o r t i o n o f m u tan ts when p l a t e d on l i g h t l y U V - i r r a d i a t e d JL c o l i t h a n when p l a t e d on u n i r r a d i a t e d c e l l s . The same g e n e t i c and p h y s io lo g ic a l r e q u i rem en ts a p p l i e d t o p ro ph ag e i n d u c t i o n (D e f a i s e t a l . , 1971) w h ich r e q u i r e s s i t e s p e c i f i c re com b in a t io n . The m u tagen ic re spon se o p e ra te d on h o s t DNA a s w e l l . These o b s e r v a t i o n s l e d t o t h e p r o p o s a l t h a t t h e r e g u ­ l a t e d f u n c t i o n s r e p r e s e n t an i n d u c i b l e r e s p o n s e o f b a c t e r i a t o u n rep a i r e d damage i n t h e i r DNA o r an "SOS" s ig n a l (Radman, 1975). In d u c t io n o f th e SOS r e sp o n se s i s accompanied by th e appearance o f a p rom inen t 40 Kd p r o t e i n (Inouye, 1971) now known to be ( c o n s t i t u - t i v e l y p ro d u c ed i n low l e v e l s ) t h e r e c A gene p r o d u c t . S y n t h e s i s o f th e re c k p r o t e i n i s r e g u la t e d by th e gene p roduc t of I e xAf a r e p r e s s o r o f r e c A and a number o f o t h e r g e n e s , and by t h e r e c A p r o t e i n i n t h e a c t i v e fo rm , a p r o t e a s e o r a c l e a v a g e s t i m u l a t o r (G eorge e t a l . , 1975) . DNA d e g ra d a t io n a t i n c i s i o n s i t e s a s w e l l a s a t s t a l l e d r e p l i ­ c a t i o n fo rk s may r e s u l t i n i n d u c t io n o f th e SOS response . The s ig n a l may be a com p lex o f s i n g l e - s t r a n d e d DN A, a s i n g l e - s t r a n d - b i n d i n g p r o t e i n , p l u s an o l i g o n u c l e t i d e (O i s h i , 1978). W i tk in (1974 , 1975, 1976) sugg e s ted t h a t SOS cou ld o p e r a t e a s ( I ) an e r r o r - p r o n e v a r i a n t o f recombi n a t i o n a l r e p a i r o r (2) a s a no n r ecombi n a t io n a l r e p a i r system w h ich p o l y m e r i z e s DNA p a s t t h e p y r im id i n e d im e r o r o t h e r n on co d in g l e s i o n s i n t h e t e m p l a t e s t r a n d . Cooper and H a n aw a l t ( 1 9 7 2 a ,b) p r e s e n t e d b io c h em ic a l e v id e n c e t h a t t h e r e a r e two t y p e s o f r e p a i r which f u n c t i o n i n c lo s u r e of e x c i s io n gaps, one of which i s dependent 17 on re c gene p roduc ts . The ev idence s u p p o r t s W itk in 's p ropo sa l . The p r i n c i p a l model f o r W e ig le r e a c t i v a t i o n and m u t a g e n e s i s p roposes " t ra n sd im e r s y n th e s i s " (C la rk and V o lk e r t , 1978) a s a r e s u l t o f m o d i f i c a t io n o f normal DNA po lym erases (perhaps t h e 3 '— 5' e d i t i n g e x o n u c l e a s e a c t i v i t y ) by a n i n d u c i b l e p r o t e i n t h u s f a c i l i t a t i n g r e p l i c a t i o n p a s t l e s i o n s and i n c r e a s i n g p r o b a b i l i t y o f e r r o r . I n c a s e s o f m a s s iv e damage w i t h r e s u l t a n t c l o s e l y - s p a c e d l e s i o n s on o p p o s i t e s t r a n d s , e x c i s i o n and r e s y n t h e s i s i n i t i a t e d a t one l e s i o n w ou ld s t o p a t a s e co n d c l o s e l y - s p a c e d l e s i o n on th e o p p o s i t e s t r a n d and would r e q u i r e t r a n s d im e r s y n th e s i s f o r com p le t ion r e s u l t i n g i n a lo ng r e p a i r p a tch and p ro d u c t io n o f m u ta t io n s . M u ta t i o n i n EXMN c o l i i s d e p e n d e n t upon t h e gene p r o d u c t s o f t h e recA+ and I e xA+ genotype and th e f u n c t i o n of o th e r genes, d e s c r ib e d i n th e next s e c t io n . M u ta t ion i s m ed ia ted by a l l th e pathways d e sc r ib ed f o r SOS r e p a i r : a) t r a n s d im e r s y n t h e s i s due t o i n d u c i b l e s u p p r e s s e d 3 '— 5* e d i t i n g a c t i v i t y of po lym erases ; b) d augh te r s t r a n d gap r e p a i r by s i s t e r s t r a n d exchange; c ) e x c i s i o n r e p a i r p lu s t r a n sd im e r s y n th e s i s i n c a se s o f m ass ive damage r e s u l t i n g i n long p a tch r e p a i r . M u ta t io n r a t e s a re d ec re a sed i n th e r e c A” and l e x A” s t r a i n s . 6 . SOS R e p a i r -D e f ic ie n t Mutants o f E sc h e r i c h ia c o l i . G ene t ic a n a l y s i s of r e p a i r m u tan ts h a s r e v e a le d a m a jo r p o r t i o n o f what i s known about th e m o le cu la r b a s i s o f SOS r e p a i r and mutagene­ s i s . Many of th e m u tan ts s tu d ie d were i s o l a t e d f o r o th e r t r a i t s and 18 sub sequ en t ly i d e n t i f i e d a s a f f e c t i n g m u tagenes is . a. A l l e l e s o f r e c A ( r e c om b in a t i o n - d e f i c i e n t I and l e x A showed r e c A and l e x A t o be r e q u i r e d f o r SOS and m u t a g e n e s i s , t o be c o n t r o l l i n g a v a r i e t y o f p h y s i o l o g i c a l r e s p o n s e s and s u g g e s t e d t h e system had to be induced (Walker, 1984). b. T em pera tu re - induced .f! l am e n ta t io n , o r t i f m u tan ts , show th e SOS i n d u c i b l e r e s p o n s e s t o be i n d u c i b l e by t e m p e r a t u r e w i t h o u t DNA damage, w ith concom itan t h igh l e v e l s o f r e c A p r o t e i n and subsequen t " a c t i v a t i o n " o f i t t o th e p r o t e a s e w h ich a c t e d on r e p r e s s o r s o f t h e v a r i e t y of SOS f u n c t i o n s d e s c r ib e d e a r l i e r ( Gudas and Mount, 1977). c. When non-mutab le ( d e f e c t iv e i n SOS) m u tan ts were sc re en ed f o r and i d e n t i f i e d , m u t a n t s o t h e r t h a n o f t h e i n d u c t i o n p r o c e s s i t s e l f w ere i d e n t i f i e d . In t h i s way umu C and umu D m u tan ts were d iscove red (K a to and S h in o u r a , 1977). Umu m u t a n t s a r e s t i l l c a p a b l e o f e x p r e s s in g a v a r i e t y o f SOS re sp on se s and th e r e f o r e code f o r p roduc ts r e q u i r e d f o r SOS p ro c e s s in g and no t e s s e n t i a l to th e c e l l . The umu C a n d D l o c i h a v e now b e e n c l o n e d ( E l l e d g e a n d W a l k e r , 1 9 8 3 ) . C h a r a c t e r i z a t i o n o f th e a c t i v i t y o f th e gene p roduc t so f a r con f irm s th e i n f e r e n c e s from e a r l i e r work w i th mu tan ts . d. S t r a i n s c a r r y in g th e . s in g le . s t ra n d DNA b ind ing (s sb ) m u ta t io n a re a l s o d e f i c i e n t i n SOS p ro ce sse s . The ssb gene p roduc t which b inds s t r o n g l y t o s i n g l e - s t r a n d e d DNA i s a p p a r e n t l y p l a y i n g a p o s i t i v e c o n t r o l l i n g r o l e i n th e system (Myer e t a l . , 1979). e. The lo n g fo rm m u t a t i o n ( I o n ) a l l o w s f ! l a m e n t a t i o n i n E. c o l i c e l l s . The Io n gene p r o d u c t i s an ATP -dependen t p r o t e a s e w h ich 19 r e g u l a t e s t h e am oun t o f f ! l a m e n t a t i o n by a f f e c t i n g t h e r a t e o f d e g ra d a t io n o f th e supp r e s s o r o f Io n o r s u l gene p roduc t (C h a re t te e t a l . , . 1 9 8 1 ; Chung a n d G o l d b e r g , 1 9 8 1 ) . Lon+ c e l l s f i l a m e n t t r a n s i e n t l y ; JLon- c e l l s f i l a m e n t i n d e f i n i t e l y and a re s e n s i t i v e t o UV. The s u l A p r o t e i n c an be in d u c e d t o i n h i b i t f ! l a m e n t a t i o n i n I o n + c e l l s ; s u l A i s SOS - induced . The Io n gene p r o d u c t a l s o a f f e c t s t h e d e g ra d a t io n o f a mutan t s igma su b un i t of RNA polymerase . S ince a h igh . tem pe ra tu re .p ro te in Cjrtp) m u tan t i s s i m i l a r , s l o w in g d e g r a d a t i o n o f th e s igma s u b un i t o f RNA po lym erase ; (Walker, 1984) s u g g e s t s t h a t th e h tp R may be in vo lv ed i n c o n t r o l l i n g th e a c t i v i t y of th e Ion p ro te a s e and o th e r p ro te a se s . . 7. H ea t Shock. Another in d u c ib l e p r o t e c t i v e measure f o r c e l l s i s th e "h ea t shock" p r o t e c t i v e re sp on se a g a i n s t th e rm a l k i l l i n g . In JL c o l i a s h i f t up i n t e m p e r a t u r e e l i c i t s c h a n g e s i n t h e p r o d u c t i o n o f m o s t c e l l u l a r p r o t e i n s , some c e a s e b e in g made, some a r e t r a n s i e n t l y in d u c e d (Yamamori e t a l . , 1978). The o r i g i n a l d i s c o v e r y o f h e a t sho ck phenomena was t h e h e a t - i n d u c e d p u f f i n g o f D r o s o p h i l a p o ly t e n e ch rom osom es ( R i t o s a , 1962). " P u f f i n g " i s i n d i c a t i v e o f a c t i v e gene l o c i i n th e o o lv ten e chromosomes. Subsequen tly an ana logou s re sponse to h e a t was found i n many o th e r s p e c ie s in c lu d in g b a c t e r i a , mammalian c e l l s , and p l a n t s (S c h le s in g e r , 1982). In E. c o l i a group o f 13 h e a t - i n d u c e d p r o t e i n s w h ich h av e been fo u n d by N e id h a r d t e t a l . (1981 , 1982 , 1983) t o c o n s t i t u t e a H igh T em p e r a tu r e R egu lon (HTR) w h ich i s d e p e n d e n t upon a p o s i t i v e r e g u l a t o r y p r o t e i n . By 1985 a t o t a l o f 17 ' 20 h e a t shock r e s p o n s iv e p r o t e i n s had been found (N eidhard t e t a l , 1984). The h e a t sh o ck r e s p o n s e i s a u b i q u i t o u s r e s p o n s e to . s t r e s s (H ig h tow e r , 1980). A v a r i e t y o f a g e n t s can in d u c e c h an g e s i n gene a c t i v i t y s im i l a r to tho se caused by h e a t shock. D rosoph ila c e l l s show a s i g n i f i c a n t i n c r e a s e i n t h e s y n t h e s i s o f t h r e e s m a l l h e a t sho ck p r o t e i n s w i th e ig h t of t e n te r a to g e n t r e a tm e n t s w h ile seven d rugs t h a t do n o t i n h i b i t d i f f e r e n t i a t i o n do n o t in d u c e h e a t s h o ck p r o t e i n s (B u z in e t a l . , 1982). A lso , i n D r o s o p h i l a , am ino a c i d a n a l o g s , s u l f h y d r y I - r e a c t i n g r e a g e n t s , t r a n s i t i o n m e t a l i o n s , u n c o u p l e r s o f o x i d a t i v e p h o s p h o x y l a t i o n , v i r a l i n f e c t i o n , e t h a n o l , a v a r i e t y o f a n t i b i o t i c s , some c h e l a t o r s and i o n o p h o r e s i n d u c e t h e r e s p o n s e . K ru eg e r and W alke r (1984) fo u n d t h a t h e a t sho ck p r o t e i n s i n E.. c o l i a r e i n d u c e d by SOS r e p a i r - i n d u c i n g a g e n t s , UV and n a l a d i x i o a c id . I n d u c t io n was found to be c o n t r o l l e d by th e h igh tem p e ra tu r e .p ro te in ( h t o R) g en e p r o d u c t , a p o s i t i v e l y a c t i n g e l em e n t r e q u i r e d f o r e x p r e s s io n of h e a t shock genes i n EXN c o l i (N e ih a rd t and Van Bogelen, 1981). So, UV and n a l a d i x i o a c i d i n d u c e t h e SOS s y s tem and th e in d ep end en t r e g u la to r y system , th e h e a t shock response . The n a tu re of th e in du c in g s ig n a l i s s t i l l unknow a The f u n c t i o n a l s ig n i f i c a n c e of th e h e a t shock re sp on se i s a l so unknow a However, t h e r e i s ev idence t h a t DNA-associated p r o t e i n s i n Et. c o l i a r e c l e a r l y a l t e r e d w ith h e a t i n d u c t i o n ( P e l l o n e t a l , 1980 , 1981 , 1982). H ea t s h o ck p r o t e i n s , then , could be r e q u i r e d f o r th e s t a b i l i t y of chromosome s t r u c t u r e or in v o lv ed i n r e p a i r o f h e a t damaged DNA. Walker (1984) s p e c u la t e s t h a t th e h to R-c o n t r o l l e d gene p roduc ts a r e in vo lv ed i n d e g ra d a t io n of SOS- 21 i n d u c e d p r o t e i n s w h ich w ou ld be d e l e t e r i o u s t o t h e c e l l i f th e y p e r s i s t e d a f t e r com p le t ion of th e SOS response . 8. The b a c t e r i a u sed f o r r e s e a r c h work a r e t h e i n t e r m e d i a t e th e rm oph i le , B a c i l l u s s t e a r o th e rm o p h i lu s . and the ex trem e the rm oph ile , Thermus ( s t r a i n T2). B a c i l l u s s t e a r o t h e rm o p h i l u s ( Gordon, I 923) i s a g ram -v a r ia b le , m o t i l e , s t r a i g h t ro d -sh ap ed organ ism which i s capab le of grow th a t 65- 700 w i t h op timum g row th a t 55 0; and w h ich p r o d u c e s h e a t - r e s i s t a n t endospores . Spores a r e fo rmed i n s o i l i n a l l c l im a t i c zones. Vegeta­ t i v e g row th i s r a p i d i n many fo o d s o f pH above 5.0 ( i f h e l d a t an a p p ro p r i a t e e l e v a te d tem p e ra tu re ) , i n h e a t in g compost, and i n con tam i­ na ted , im p rope r ly p ro cessed , canned foods. B1. s te a ro th e rm o p h i lu s has been o f t e n used i n l a b o r a to r y r e s e a r c h of the rmoph ily . Thermus a a u a t i c u s . a n on -m o t i le o b l i g a t e aerobe which i s a gram­ n e g a t i v e r o d r e s e m b l i n g E. c o l i . was' f i r s t d e s c r i b e d by B rock and F r e e z e (1969) who r e p o r t e d t h e i r i n i t i a l i s o l a t e s a s f i l a m e n t o u s , commonly f i n d i n g f i l a m e n t s i n 65-70C and s t a t i o n a r y p h a se c u l t u r e s . E l e c t r o n m ic r o g r a p h s t u d i e s o f e x trem e ly th e rm o p h i l ic b a c t e r i a show JThermu s , i n c o n t r a s t t o Et c o l i . t o h av e a r e g u l a r s c a l l o p - l i k e connec t io n to th e in n e r m em brane /ce ll w a l l g iv in g i s o l a t e s an a n n e l id ­ l i k e appearance (Ramaly e t a l . , 1978). The genus h a s appea red i n h o t w a te r h e a t e r s (Brock and Boylen, 1973) and n a tu r a l l y - o c c u r r i n g w a te r t h a t has been con tam ina ted by th e rm a l e f f l u e n t (Degryse e t a l . , 1978). 9 . S ta tem en t o f Research Problems. The purpose of th e r e s e a r c h r e p o r te d h e re was to combine c u r r e n t 22 know led g e o f t h e f i e l d s o f t h e rm o p h i l y and DNA r e p a i r i n a s tu d y o f two th e rm o p h i l ic m ic roo rgan ism s i n o rd e r t o advance knowledge i n th e a r e a o f s u r v iv a l o f l i f e a t h igh tem pe ra tu re . The s tudy in c luded : a. I d e n t i f i c a t i o n and d i f f e r e n t i a t i o n o f one th e rm o p h i l i c s t r a i n by m o rpho log ica l ex am in a t io n and b iochem ica l t e s t i n g . b. D eve lopm en t o f a p p r o p r i a t e medium f o r s t u d y o f th e two th e rm oph i le s a t 55C and 70C. c. Exposure o f B a c i l l u s s te a ro th e rm o o h i lu s and Thermus T2 to th e known DNA-damaging ag en ts , UV and Mitomycin C, f o r d e te rm in a ­ t i o n o f t h e i r s u r v iv a l p a t t e r n s . d. Exam ina t ion o f th e two s t r a i n s f o r the e x i s t e n c e o f c o n s t i t u ­ t i v e p h o t o r e a c t i v a t i o n r e p a i r s y s t em s s i m i l a r t o t h e i r m e so p h i l ic c o u n te rp a r ts . e. D e te rm in a t io n o f th e e x i s t e n c e of an in d u c ib l e r e p a i r system (SOS) i n th e two organisms. f . E x a m i n a t i o n o f t h e r e l a t i o n s h i p o f h e a t - t o l e r a n c e to i n d u c ib l e r e p a i r and f ! l am e n ta t io n i n Thermus. MATERIALS AND METHODS Source o f organism s 1. Thermus was o r i g i n a l l y i s o l a t e d by U l r i c h f rom an a l k a l i n e th e rm a l s p r in g i n Yellow s ton e Park. S t r a i n T2 i s a m u tan t l a b o r a to r y s t r a i n which i s non-mucoid and con sequen t ly e a s i e r to work w i th i n th e la b o ra to ry . The c u l t u r e used f o r th e s e s t u d i e s was o b ta in e d from th e American Type C u l tu re C o l l e c t io n (ATCC #27737). 2. The s t r a i n o f B a c i l l u s s te a ro th e rm o n h i lu s used i n t h i s s tudy was o r i g i n a l l y i s o l a t e d a s a c o n t am in a n t i n t h e l a b o r a t o r y o f Dr. Gordon J u l i a n o f th e Montana S t a t e U n iv e r s i t y B io ch em is t ry Department and was i d e n t i f i e d d u r in g th e cou rse o f t h i s s tudy. 3. W i l d - t y p e E. c o l i u s e d i n p h o t o r e a c t i v a t i o n e x p e r im e n t s was s t r a i n H53 i s o l a t e d f rom c h i c k e n caecum i n t h e l a b o r a t o r y o f Dr. David Sands (P la n t Pathology Department, MSU). 4. C on tro l E1. c o l i f o r UV s u r v iv a l expe r im en ts was s t r a i n AB1157 X-(K12) (Bachman, I 972). I d e n t i f i c a t i o n and D i f f e r e n t i a t i o n o f B a c i l l u s 1. S t a i n i n g p r o c e d u r e s . Gram s t a i n and M a l a c h i t e G reen s p o r e s t a i n w e re p e r f o rm e d a c c o r d i n g t o C onn 's B i o l o g i c a l S t a i n s ( L i l l i e , 1936). 2. S p o ru la t in g aga r , o b ta in ed from Dr. Nels Nelson , Department o f M icrob io logy , Montana S t a t e U n iv e r s i t y , was p rep a red a cco rd in g to Gordon e t a l . (1973), in o c u la te d w i th a loop and in c u b a te d a t 55 C f o r 24 18 h o u r s . 3. R e d u c t io n o f n i t r a t e and n i t r i t e . N i t r a t e r e d u c t i o n medium was o b ta in e d from Dr. Nels Nelson, Department o f M icrob io logy , Montana S t a t e U n iv e r s i ty , and was p repa red a c co rd in g to Gordon (1973). Medium to d e t e c t p ro d u c t io n o f Ng gas was p rep a red by add ing a t r a c e o f Zn to t h e n i t r a t e r e d u c t i o n medium and p l a c i n g i t i n an I n v e r t t u b e w h ich , d u r in g a u to c la v in g p rocedu re , e x p e l l s th e gas and f i l l s th e in v e r t e d tube w i t h " l i q u id , th e reby a l low in g any gas p ro d u c t io n by th e organism to be observed as bubb les i n the i n v e r t e d tube. B a c t e r i a l Growth Cond it ion s I . Media A. The r o u t i n e s t u d i e s o f B a c i l l u s em p loyed a medium c o n t a i n i n g 0.5% T ry p to n e (D ifc o ) and 0.5% y e a s t e x t r a c t i n a b a s a l s a l t s s o lu t i o n (10 ml each o f S o lu t io n s I , I I , I I I p e r l i t e r ) . The pH was a d ju s t e d to 7-7 p r e a u to c la v in g which s h i f t e d to a f i n a l pH o f 7.5 d u r in g th e a u to c la v e p rocedure . S o lu t io n I (NH21)2SO4 ( 3 .96 g /1 ) KCl ( 7-46 g /1 ) NaCl (23 .33 g /1 ) Na2HPO4 . 2H20 (46 .27 g /D S o lu t io n I I Ca(NO3 ) 2 . 4H20 ( I .18 g /1 ) S o lu t io n I I I MgSO4 . 7H2 0 or MgSO4 ( 9 .8 g /1 ) ( 4 .8 g /1 ) The aga r c o n ten t (D ifco, M ic ro b io lo g ic a l g rade) o f s o l i d medium was 2% 25 (20 gm/1). p l a t e s were d r ie d a t room tem pe ra tu re f o r s e v e r a l days o r a t 70°C f o r I 1/2 hou rs b e fo re use. B. Media used f o r thd c u l t u r e o f Thermus were: I) th e b a sa l s a l t s p l u s y e a s t e x t r a c t and t r y p t o n e , pH 7 .8 , d e s c r i b e d by U l r i c h (1971 ) ; o r 2) a s im p l e r medium w o rked o u t by Dr. Emmet J o h n so n a t T u la n e U n i v e r s i t y ( p e r s o n a l c om m un ic a t io n ) s p e c i f i c a l l y f o r t h e c u l t u r e o f Thermus. Johnson* s Thermus medium: S a l t s S o lu t io n I (100X) (NH4 )2SO4 4 . 0 :g KCL 7 .0 g Ca (NO3 )2 -H2O 1 .2 g brough t to I ,000 ml w ith w a te r S a l t s S o lu t io n I I (100X) NaCl 20 g Na2HPO4 '7 H2O 30 g MgSO4 -7 H2O 10 g brough t t o I ,000 ml w i th w a te r Thermus medium S a l t s S o lu t io n I 10 ml S a l t s S o lu t io n I I -10 ml FeSO4 (0.05%) I ml Yeast e x t r a c t 2 g 1 Difco t r y p to n e 2 g added t o 1,000 ml w a te r to p rev en t p r e c i p i t a t i o n o f s a l t s . 26 s • Two g e l l i n g a g en t s were used i n co n ju n c t io n w i th th e two media; 2% B a c t o - a g a r o r 0.8% G e l r i t e w e re com pared i n e ach r e c i p e . When G e l r i t e was used, I g CaClg°2 HgO was d i s s o lv e d i n one l i t e r of w a te r f o l l o w e d by 8 g G e l r i t e and f i n a l l y th e o t h e r i n g r e d i e n t s . The m agnesium c o n t e n t o f J o h n so n ’s medium was a l s o i n c r e a s e d t o I g MgSOij'7 HgO p e r l i t e r f o r u se o f G e l r i t e . F i n a l l y , t h e pH was a d j u s t e d w i t h NaOH to a p r e a u t o c l a v e pH o f 8.1 w h ich r e s u l t e d i n t h e f i n a l pH 7.8 a f t e r a u to c lav in g . Thermus X-Gal Medium CaClg’2g0 0 .5 gm MgSO4 -I H2O 0 .5 gm i n I l i t e r o f w a te r . Then add G e l - r i t e 4 .0 gm fo llow ed by Jo h n so n 's S o lu t io n I 5 .0 ml . ■ I I 5 .0 ml Casamino a c id s 5.Q gm V itam ins 1.0 ml each ( b i o t i n , l i p o i c a c id , B1 g , p a ra aminobenzoic a c id ) Adjust pH to 8 .1 (abou t 0 . 7 .ml 4M NaOH) . Autoclave b e fo re add ing th e fo l low in g : L ac to se (10%) 10.0 ml X-Gal 20 .0 mg (d is s o lv e d i n d im e th y l formamide) IPTG 60 .0 mg : 27 2. I n c u b a t io n . Covered shak ing w a te r b a th s a t th e a p p ro p r ia t e tem p e ra tu re s were s e t t o r o t a t e o r s h a k e a t 100 rpm f o r l i q u i d C u l t u r e s . C u l t u r e s on s o l i d m ed ia w e re i n c u b a t e d a t : a ) 58 C i n a H o tp a ck h u m id i f i e d i n c u b a t o r ; t w e n t y - f o u r h o u r s f o r B a c i l l u s and 3 -4 d ay s f o r Thermus w e re r e q u i r e d f o r g r ow th ; b) 70 C i n s e a l e d p l a s t i c P e t r i d i s h bag s w i th a b lank p l a t e a s a s p a c e r ,and 5 ml s t e r i l e d i s t i l l e d w a te r p laced i n bag b o t tom to p r e v e n t d e h y d r a t i o n ; 2 -3 d ay s w e re r e q u i r e d f o r ■ grow th o f Thermus. 3. B u f fe r s B a c t e r i a l washes and 10-f o ld d i l u t i o n s o f b a c t e r i a l c u l t u r e s were made i n a p p ro p r i a t e l i q u i d medium or th e fo l low in g phosphate b u f f e r s : a) B a c i l l u s NaCl 8 .5 gm K2HPO1, 5 * 7 gm KH2POlt =^T CO gm b) Thermus NaCl LOCO gm K2HPO1, pH = 7 .2 pH = 7 .8 Microscopy A ll l i g h t m ic ro scop ic o b s e rv a t io n s and pho tographs were made w ith a W ild (H e e rb ru g ) M-20 m ic r o s c o p e f i t t e d w i t h a 3.5 mm cam era a t tachm en t . Kodak h igh c o n t r a s t copy f i lm was used and developed w i th Kodak deve lope r D-19. E l e c t r o n m ic r o s c o p e e x am in a t i o n s r e q u i r e d b a c t e r i a g rown i n s t a n d a r d g row th medium to be w ash ed i n p h o s p h a te b u f f e r . E i t h e r a 28 d ro p o f t h i s s u s p e n s i o n o r o n e .made o f a c o lony from s o l i d medium suspended i n w a te r was p la ced on a 300 mesh copper g r i d coa ted w i th 0.2% Formvar i n ch lo ro fo rm . P l a t i n um shadow c a s t i n g w as done w i t h p la t in um -pa lad ium w ire . O bse rv a t ion s were made w i th a Z e is s (EM 952) e l e c t r o n m icroscope and a s s o c i a t e d camera. DNA Damaging T rea tm en ts 1. Exposure to UV. The S y lv an ia GTE 8 w a t t g e rm ic id a l lamp used f o r u l t r a v i o l e t i r r a d i a t i o n g en e ra te d a dosage o f 190 u w a tts /cm ^ as measured by a S p e c t r o l in e DRC-IOOx d i g i t a l rad iom e te r . A. C u l t u r e s on s o l i d m ed ia w e re i r r a d i a t e d a c c o r d i n g t o G reenberg (1 967). B. L iqu id C u l tu re s . At a d e n s i ty o f app rox im a te ly IO^ c e l l s pe r ml, two ml a l i q u o t s o f washed c e l l s suspended i n t h e a p p ro p r ia t e p h o s p h a te b u f f e r w e re p l a c e d i n d i s p o s a b l e 6 0 x 1 5 P e t r i d i s h e s and t r e a t e d w i th UV a s above. The c e l l s cou ld then be d i l u t e d and p la t e d d i r e c t l y o r used f o r a p h o to r e a c t i v a t io n t r e a tm en t . 2. E x p o su re t o M itom yc in C (MG). T e s t o f s u s c e p t i b i l i t y o f Thermus to MC was ach ieved by p l a t i n g a su spens ion o f th e organism on s o l i d medium and in c u b a t in g a t the a p p ro p r ia t e tem p e ra tu re (58 C o r 70 C) f o r app rox im a te ly 24 hours b e fo re a d d i t i o n o f s t e r i l e f i l t e r d i s c s upon w h ich w e re p l a c e d v a r y i n g c o n c e n t r a t i o n s o f t h e d rug . A f t e r a n o th e r one to two days o f growth ' th e zone of i n h i b i t i o n su rround ing the d i s c was measured. 3 . R epair a s say s . 29 D i r e c t L i g h t R e p a i r . P h o t o r e a c t i v a t i o n e x p e r im e n t s w e re v a r i a t i o n s i n the p ro to c o l f o r te a c h in g l a b o r a to r y ex p e r im en ts w i th E. c o l i (See ley and Van Demark, 1981). C e l l s i n lo g phase were washed i n b u f f e r o f pH a p p r o p r i a t e f o r each organism then re su spended i n b u f f e r a t 1/1,0 volume. Two m i l l i t e r a l i q u o t s w ere p laced i n s t e r i l e d isp o s ­ a b l e 60 x 15 P e t r i d i s h e s and i r r a d i a t e d f o r v a r y i n g t im e s . The p l a t e s were imm ed ia te ly p la c ed i n p l a s t i c b in s and covered w i th f o i l t o p r e v e n t r e p a i r due t o o v e rh e a d l i g h t s . As soon a s p o s s i b l e t h e c e l l s w e re t r a n s f e r r e d t o s t e r i l e t u b e s and p l a c e d i n a c o n s t a n t tem pe ra tu re ba th to p rev en t o v e rh e a t in g from th e l i g h t b u l b used f o r r e p a i r . The b u lb s u s ed w e re e i t h e r w h i t e 250 w a t t S y l v a n i a BBAS o r b lue 250 w a t t S y lv an ia BCA p la ced i n a p h o to r e f l e c t o r shade s i x in c h e s above th e tubes. C on tro ls in c lu d ed : N o n - i r r i a t e d c e l l s : T rea tment J. 1 Cons tan t tem pe ra tu re b a th ; da rk ( f o i l - c o v e r e d ) 2 Cons tan t tem pera tu re b a th ; l i g h t 3 Room tem pe ra tu re b a th ; d a rk and i r r a d i a t e d c e l l s : T rea tment ± 4 Constan t tem pe ra tu re b a th ; da rk 5 Room tem pe ra tu re ba th ; dark 6 Cons tan t tem pe ra tu re b a th ; l i g h t P e rc en t re cove ry was c a l c u l a t e d by th e f o l owing equa t io n : recove ry = S u rv iv o rs ( Trea tment #6) T o ta l # (T rea tment #1) 30 S u rv iv a l D i lu t io n s o f a p p ro p r ia t e c u l t u r e s were p la t e d and th e n i r r a d i a t e d f o r v a r i o u s l e n g t h s o f t im e . Colony c o u n t i n g was done w i t h a New B ru n sw ic k B i o t r a n I I a u t o m a t i c c o lo n y c o u n te r . S p ec t ro p h o tom e tr ic d e t e rm in a t io n o f c e l l d e n s i ty i n b ro th was made u s in g LKB u l t r o s p e c 4050 a t a w a v e le n g th o f 550 mu. S u r v i v a l r a t e was c a l c u l a t e d a s number of c e l l s s u r v iv in g i r r a d i a t i o n ( i r r a d i a t e d p l a t e coun t) d iv id ed by number o f c e l l s i r r a d i a t e d ( p l a t e coun t, no i r r a d i a t i o n ) . 31 RESULTS A. The Organisms o f th e Study I . I d e n t i f i c a t i o n o f B a c i l l u s s te a ro th e rm o n h i lu s I n i t i a l o b s e r v a t i o n s . The c u l t u r e o b t a i n e d f rom th e Montana S t a t e U n iv e r s i ty B io ch em is t ry Department and i n i t i a l l y though t to be Thermus d e v i a t e d f rom n o rm a l Thermus b e h a v i o r i n g e n e r a t i o n t im e , op timum pH, and g row th t e m p e r a t u r e . No g row th on m in im a l l a c t o s e medium i n d i c a t e d a l a c k o f a f u n c t i o n i n g l a c o p e ro n . Gram s t a i n i n g was no t c o n c lu s iv e as c e l l s from a colony on s o l i d m edia appeared a s Gram n eg a t iv e , e lo ng a ted rods . F u r th e r i n v e s t i g a t i o n , however, showed th e organism to be a spo re - fo rm e r w i th f l a g e l l a , no t f i l am en to u s i n l i q u i d c u l t u r e a t 55 C o r 70 C. Morphology. Gram s t a i n s o f f r e s h c u l t u r e s showed th e bac te r ium to be G ram -va r iab le s h o r t ro d s ( P l a t e I). When grown on s p o r u l a t i n g a g a r f o r I 8 h o u r s i t p ro d u c ed many s p o r e s w h ich , when s t a i n e d w i t h M a la ch i te Green, were e a s i l y d e t e c te d under th e l i g h t m icroscope. The spo re s were e l i p t i c a l i n shape and lo c a t e d i n th e t e rm in a l r e g io n o f t h e c e l l . Shadow c a s t e d s p e c im e n s o b s e r v e d u n d e r t h e e l e c t r o n m ic r o s c o p e i n d i c a t e d e l i p t i c a l t e r m i n a l spo re s ( P la t e 2) and f l a g e l l a ( P l a t e 3). P h y s i o l o g i c a l C h a r a c t e r i s t i c s . Optimum pH f o r grow th on b a sa l s a l t s p lu s T ryptone and y e a s t e x t r a c t i s 7.5. Optimum tem p e ra tu re f o r g row th i s 55 C. G e n e r a t i o n t im e ( d o u b l i n g t im e ) u n d e r t h e s e c o n d i - 32 P la t e I . B a c i l l u s (G ram sta in ) 50X P la t e 2 . B a c i l l u s te rm in a l e l l i p t i c a l spo re . E le c t ro n m icrog raph 3 1 ,OOOX 34 P la t e 3• B a c i l l u s F lage llum . E le c t r o n m icrograph 186 ,OOOX 35 n i t r a t e and n i t r i t e t e s t s were made, both of which were nega t iv e . E. t i o n s i s abou t 15 m inu tes . To f u r t h e r c h a r a c t e r i z e th e B a c i l l u s ^ c o l i g a v e a p o s i t i v e r e a c t i o n a s a c o n t r o l . F rom t h e s e t e s t s , m o r p h o l o g i c a l s t u d i e s , and t h r o u g h u se o f B e rg ev t s Manual o f D e t e rm i n a t i v e B a c t e r i o l o g y ( 1957) and The Genus B a c i l l u s ( Gordon, I 973) i t was d e t e rm in e d t h a t t h e o rg a n i sm o f s tu d y i s a s t r a i n o f B. s t e a r o th e rm o p h i lu s . Table I p r e s e n t s com para tiv e c h a r a c t e r i s t i c s of Thermus and Bi. s te a ro th e rm o p h i lu s . C u l t u r e o f B a c i l l u s . The b a s a l s a l t s p l u s y e a s t e x t r a c t and t r y p t o n e medium (pH 7 .5 ) w e re u s ed r o u t i n e l y f o r Bi. s t e a r o t h e rm o ­ p h i l u s . N u t r ie n t aga r gave comparable r e s u l t s i n one exper im en t and would be a s im p le r p r e p a ra t io n . I t was found t h a t th e most rep roduc ­ i b l e r e s u l t s were ob ta in ed when th e p l a t e s were d r ie d w i th l i d s a j a r a t 70 C f o r a b o u t I 1 /2 h o u r s o r l e f t w i t h o u t s e a l i n g o n ' t h e l a b o r a ­ to r y bench a t room tem pe ra tu re f o r s e v e r a l days b e fo re use. C h a r a c t e r i s t i c s o f Thermus. Morphology. . Thermus was o r i g i n a l l y d e s c r ib e d a s f i l am en to u s a t i t s op tim um t e m p e r a t u r e o f 70 C. However, a t l o w e r t e m p e r a t u r e s f ! l am e n ta t io n d ec rease s . E le c t ro n m ic rog raphs ( p l a t e 4) confirm t h i s 1 d e s c r ip t io n , E le c t r o n m icroscopy a l s o showed a l a c k o f f l a g e l l a and sp o re s and con firm ed t a n g l in g and f ! l am e n ta t io n a t h igh tem pe ra tu re s . C u l tu re s o f Thermus. P o in ts of c o n s id e r a t io n i n th e development o f s o l i d medium f o r grow th of Thermus a t 70 C in c lu d ed : a ) Requirement f o r a f i rm , n on -m e l t in g medium f o r in c u b a t io n a t • 7 0 C. 36 37 Table I . Summary o f c h a r a c t e r i s t i c s o f B. s t e a r o t h e rm o o h i l u s and Thermus T2. B a c i l l u s ' Thermus G ram -s ta in V — Spores + - F l a g e l l a + P resence o f f u n c t i o n a l l a c operon - + F i lam en ta t io n a t 58 C - - F i lam en ta t io n a t 70 C - + Optimum Growth Temperature 55 C 70 C Optimum Growth pH 7.5 7 .8 G ene ra t io n Time 15 min I hour (70 C) 2 .5 hours (55 C) V - V a r ia b le + = P resence - = Absence b) P r e v e n t i o n o f d r y i n g a t t h e h i g h t e m p e r a t u r e i s in d i s p e n s a b le , y e t media t h a t i s to o m o is t a l low s c o lo n ie s to run to g e th e r , p re v en t in g a c c u r a te counting . c ) C lea r medium to a l low use o f th e a u tom a t ic colony coun te r was d e s i r a b l e . d) G e l r i t e and t h e h ig h c o n c e n t r a t i o n s o f CaClg and MgSOjj r e q u i r e d f o r i t s p o lym e r iz a t io n p r e c i p i t a t e s h igh s a l t from a:ny medium i n which both a r e used, e s p e c i a l l y a t pH above 7; e ) S a l t s and pH 7.8 a r e r e q u i r e d f o r growth o f Thermus. f ) Media c o n ta in in g G e l r i t e s o l i d i f i e s a t a h ig h e r tem p e ra tu re th an ag a r and must be poured above 60 C. Once s o l i d i f i e d , i t cannot be m e lted again . 38 The medium of cho ice f o r grow th of Thermus on s o l i d su p p o r t a t 70 C became Johnson 's w i th G e l r i t e a s the s a l t con ten t was n u t r i t i o n a l l y s u f f i c i e n t and y e t low enough to a l l o w t h e u se o f a d d i t i o n a l h ig h c o n c e n t r a t i o n o f CaCl2 and MgS Cfy r e q u i r e d f o r u se o f G e l r i t e . I n s p i t e o f t h e d i s a d v a n t a g e t h a t t h e medium had t o be p o u re d a t 65 C, G e l r i t e p r o v id e d th e c l e a r e s t p r o d u c t and th u s a l l o w e d u se o f t h e a u tom a t ic colony coun te r . B e t te r grow th (colony number) was observed w i th G e l r i t e a s a g e l l i n g agen t by L in and Casida (1984). The r e s u l t s o f t h i s s tudy d id no t c o r ro b o ra te th e L in and Casida r e s u l t s as to an i n c r e a s e i n colony number in c re a s e . G row th o f Thermus i n b r o t h a t 70 C c o u ld be a t t a i n e d i n e i t h e r J o h n s o n 's o r U l r i c h ' s b u t o n ly i n sh ak en c u l t u r e . The t e n - f o l d d i l u t i o n s r e q u i r e d f o r p l a t e coun ts w ere only s u c c e s s fu l when d i l u e n t was a t pH 7.8. A t y p i c a l g r o w t h c u r v e d e t e rm in e d s p e c t r o p h o t o m e t r i c a l l y i n d i c a t e d a g e n e r a t io n t im e of a p p ro x im a te ly 150 m inu te s a t 55 C o r 60 m i n u t e s a t 70 C s i m i l a r to o b s e r v a t i o n s o f U l r i c h (1971) . However, t u r b i d i t y o r a b s o r b a n c e and p l a t e c o u n t s showed a d i s c r e p a n c y i n l o g a r i t h m i c p h a s e p e r h a p s b e c a u se o f f i l a m e n t a t i o n and c o n s e q u e n t p r e v e n t i o n o f c e l l d i v i s i o n o r t a n g l i n g . R e p r o d u c i b l e r e s u l t s i n e x p e r im e n t s i n v o l v i n g p l a t e c o u n t s w e r e o b t a i n e d o n ly w i th O.D. <. 0.500 and w i th p r e c i s e l y c o n t r o l l e d c o n d i t io n s (pH, medium, p la t in g ) . With g e n e r a t io n t im e o f one hour and a long l a g phase f o r Thermus an u n r e a s o n a b l e t im e w as r e q u i r e d t o a c h i e v e l o g p h a s e a t a c e l l d e n s i t y a p p r o p r i a t e f o r e x p e r im e n t a l work. T h i s d i f f i c u l t y was 39 overcome by i n o c u la t i n g b ro th from a s i n g l e colony and a l low in g th e n ew ly i n o c u l a t e d c u l t u r e t o grow o v e r n i g h t a t t h e ' a p p r o p r i a t e t e m p e r a t u r e (55 C o r 70 C) w i t h s h a k in g (day #1). I f g row th had o c c u r r e d , a d i l u t i o n was made i n g row th medium , t h e c u l t u r e grown ag a in a t th e a p p ro p r ia t e t em p e ra tu re w i th o p t i c a l d e n s i ty monito red , and th e c u l tu r e removed d u r ing lo g phase to th e r e f r i g e r a t o r (day #2). The c u l t u r e c o u ld t h e n be t a k e n f rom th e c o ld t h e day o f t h e e x p e r im e n t (day #3)> d i l u t e d and i n c u b a t e d a g a in . W ith v e r y l i t t l e l a g phase th e c u l t u r e q u ic k ly r e - e n t e r e d lo g phase. B. Q u a n t i t a t i v e R e su l t s o f K i l l i n g bv I r r a d i a t i o n The k i l l i n g o f a m icroorgan ism i s d e f in e d f o r th e se expe r im en ts a s th e l o s s of i t s a b i l i t y to i n i t i a t e a colony. W ith in a p o p u la t io n th e i n d i v i d u a l s t h a t have expe r ien ced enough chem ica l e v en ts to r e s u l t i n u n re p a i r e d changes a re i n a c t i v a t e d or "k i l le d " . I t i s assumed t h a t t h e d am a g in g e v e n t s o c c u r r a n d o m l y a n d i n d e p e n d e n t l y i n t h e s u s c e p t i b l e g roups w i th the p r o b a b i l i t y of such an even t p ro p o r t i o n a l t o t h e do se o f r a d i a t i o n p e r u n i t t im e . T a r g e t s i z e i s g e n e r a l l y i n d i c a t e d by th e s l o p e o f th e k i l l i n g c u rv e on s e m i - l o g p ap e r . As lo n g a s t h e dose r a t e i s c o n s t a n t o r g a n i sm s w i t h t h e same t a r g e t (genome) s i z e can be com pared and t h e r e l a t i o n s h i p b e tw e e n do se and k i l l i n g ana lyzed . The r e f e r e n c e Ei. c o l i s t r a i n (AB1157) o f F i g u r e 3 i s a UV r e s i s t a n t w i ld type s t r a i n . Data used f o r g e n e r a t io n of th e r e f e r e n c e c u r v e was o b t a i n e d u n d e r i d e n t i c a l c o n d i t i o n s u s i n g t h e same f a c i l i t i e s a s th e Thermus and B a c i l l u s s te a ro th e rm o o h i lu s i r r a d i a t i o n NU M BE R OF S U R VI VO R S/ TO TA L CE LL S PL A TE D 40 E. co Ii 7 0 eC Thermus 55°C Thermus O.OOI 0.000 I 0 .0 0 0 0 1 UV IRRADIATION (SECONDS) F ig u re 3 . UV S u rv iv a l : Jgj . s o l i , B a c i l l u s , Thermus 55 C and Thermus 70 C. 41 e x p e r im e n t s i n t h e l a b o r a t o r y o f G uy lyn W arren , MStL F i g u r e B a l s o shows th e s u r v iv a l cu rves g en e ra ted f o r Thermus grown a t 55 C and 70 C. B a c i l l u s grown a t 55 C p r i o r to i r r a d i a t i o n shows a c h a r a c t e r i s ­ t i c s i n g l e - h i t cu rve w i th e x p o n en t ia l decay as does Thermus grown a t 55 C. However, Thermus g rown a t 70 C p r i o r t o i r r a d i a t i o n d i s p l a y s th e c h a r a c t e r i s t i c " m u l t i p l e - h i t " c u r v e w i t h a s h o u l d e r w i t h th e e x p o n e n t i a l d ecay i n d i c a t i v e o f k i l l i n g o n ly a f t e r 30 s e c o n d s o f e x p o s u r e t o UV. F u r t h e rm o r e , th e e x p e r im e n t s i n w h ic h Thermus was grown a t one tem p e ra tu re and sw i tch ed t o th e o th e r tem p e ra tu re p r io r t o i r r a d i a t i o n i n d i c a t e d t h a t the sw i tc h from low er t o h ig h e r tempera­ t u r e i n d u c e d t h e m echan ism r e s p o n s i b l e f o r t h e m u l t i p l e - h i t c u rv e , t h a t th e in d u c t io n was comple te w i t h i n two hou rs a f t e r the tem p e ra tu re sw i tc h (F ig u re 4 ), and t h a t t h i s mechanism remained a c t i v e through th e f o u r h ou rs m on i to red and shown he re . (Data not p re s en te d d em ons tra ted t h a t th e a cq u i red c h a r a c t e r i s t i c was a c t i v e i n a l a t e lo g to s t a t i o n ­ a r y p h a s e o v e r n i g h t c u l t u r e g rown a t 70 C. A d d i t i o n a l l y , t h e mechanism appeared a c t i v e f o r a t l e a s t t h r e e hours a f t e r sw i tc h in g f rom 70 C t o 55 C.) G raph s p r e s e n t a n a v e r a g e o f t h r e e r e p l i c a t i v e ex p e r im en ts , each exper im en t in c lu d in g th r e e p l a t e c oun ts per po in t . E r ro r b a rs r e p r e s e n t h igh and low v a lu e s a t each po in t . C. Exposure to UV and MG. One e x p la n a t io n o f th e m u l t i p l e - h i t s u r v iv a l cu rve o f Thermus T2 c o u ld be i n d u c t i o n o f a DNA r e p a i r s y s tem . T h is w as t e s t e d by expos ing d i l u t i o n s o f Thermus on p l a t e s to UV and th e n in c u b a t in g th e N U M B ER O F SU R VI VO R S/ TO TA L C E LL S PL A TE D 42 BACILLUS 55°C o.oo I - 0 .000 I o . o o o o i 20 30 40 50 UV IRRADIATION (SECONDS) F igu re 4 . UV S u rv iv a l : Thermus 1 , 2 , 3 hours a f t e r t r a n s f e r from 55 C to 70 C. 43 p l a t e s w i t h M itom yc in C i n d i s c s and o b s e r v i n g , a f t e r c o l o n i e s had grown up, the d iam e te r of the k i l l zone su r round ing the d is c s . I f UV exposu re induced a DNA r e p a i r system , in c re a s e d r e s i s t a n c e to Mitomy­ c in C would be expec ted . A l t e rn a te l y , a lo g phase c u l t u r e o f Thermus was exposed to M itomycin C f o r a s h o r t d e te rm ined le n g th of time, th e mutagen washed o u t , c e l l s p la te d , and th en i r r a d i a t e d w i th in c r e a s in g d o s e s o f UV. The r e s u l t i n g s u r v i v a l c u r v e wou ld i n d i c a t e w h e th e r p r e t r e a tm e n t w i th Mitomycin C in c r e a s e d r e s i s t a n c e t o UV. In one exper im en t w i th MG d i s c s t h e r e was no t a c o n c lu s iv e zone s i z e d i f f e r e n c e . In one e x p e r im e n t w i t h t r i p l i c a t e s am p le s o f MG p r e t r e a tm e n t f o l l o w e d by UV, t h e 55 C s u r v i v a l c u r v e d e v e lo p e d a shou ld e r s im i l a r t o th e 70 C c u l t u r e s (F igu re 5). D. Q u a n t i t a t i v e R e su l t s o f P h o to r e a c t iv a t io n and D i r e c t L igh t Repair Fo llow ing UV T rea tm en t. Tab le 3 p r e s e n t s th e r e s u l t s o f p h o to r e a c t i v a t io n ex p e r im en ts on a w i l d t y p e EL. c o l i and B a c i l l u s s t e a r o t h e rm o p h i l u s u nd e r t h e c o n d i t io n s d e s c r ib e d i n th e Methods s e c t i o n of t h i s t h e s i s . NU M BE R OF S UR VI VO RS /T O TA L CE LL S PL AT ED Thermus 55o-70°C 1 hour 2 hour 3 hour O.OOI 0 .0 0 0 1 0 .0 0 0 0 I 20 30 40 50 UV IRRADIATION (SECONDS) F igu re 5 . Mitomycin C + UV S u rv iv a l : Thermus 55 C. Table 2. P h o to r e a c t i v a t i o n : B a c i l l u s s te a ro th e rm ooh i lu s and Ej . c o l i T reatment # # S u rv ivo rs # S u rv iv o r s /# To ta l* % P h o to r e a c t iv a t io n P h o to r e a c t iv a t io n : B a c i l l u s I 1.1 X 107 2 1 .2 X i o J « I .00 3 9.7 X IO6 0.88 4 4 .7 X IO5 0.04 5 5 .3 X 10-> 0 .05 6 9 .4 X IO6 0.85 VO Ov (± 3%) P h o to r e a c t iv a t io n : E. c o l i I 1 .4 X IO8 2 9.8 X IO7* 0.7 3 1.0 X IO8 0.71 4 3 .3 X IO7 0.24 5 3 .3 X IO7 0.24 6 9.5 X IO7 0.679 97% CU +1^ E. c o l i . a f t e r 30 seconds o f i r r a d i a t i o n and subsequen t s u rv iv a l o f 2% o f the p o p u la t io n w i th no p h o to r e a c t i v a t io n t r e a tm e n t show 97% r e a c t i v a t i o n a f t e r 30 m in u t e s o f e x p o s u r e to a w h i t e i n c a n d e s c e n t p ho tog raphe r 's bu lb p laced s ix in c h e s from the c u l tu r e . B a c i l l u s w i th 20 seconds o f i r r a d i a t i o n and 5% s u r v iv a l of th e n onpho to re a c t iv a te d p o p u la t io n d em on s t ra te s a 96% r e a c t i v a t i o n i n re sponse to exposure to a p ho to g raph e r 's blue l i g h t bulb. The d a ta p re sen te d i n each case i s th e average of two e x p e r im en ts w ith samp les taken i n t r i p l i c a t e . P h o t o r e a c t i v a t i o n o f UV-dosed Thermus was a l s o a t tem p ted (Table 46 3) . D i s a p p o i n t i n g l y , d e s p i t e some v a r i a t i o n o f UV do sag e and tem p e ra tu re d u r ing p h o to r e a c t i v a t io n a s w e l l a s use o f l i g h t c o n d i t io n s t h a t w e r e s u c c e s s f u l i n p h o t o r e a c t i v a t i n g Ei c o l i and B. s t e a r o t h e r m o o h i l u s . p h o t o r e a c t i v a t i o n o f T h e rm u s c o u l d n o t be demonstra ted . Table 3 . C ond it ion s o f A ttempted D i r e c t l i g h t R epa ir E. c o l i B a c i l l u s Thermus I r r a d i a t i o n (seconds) 30 20 20, 30 Temperature Ice - 40 G I c e , 37 C, 50 C , L igh t ( t im e i n m inu tes ) White (30 ,60 ) . Blue (15 ,30 ) Blue (15 ,30 ) S u c c e s s fu l R epair + + - 47 DISCUSSION T h e rm a l ly i n j u r e d c e l l s o f any o rg a n i sm may d i e o r r e p a i r t h e damage depending on th e e x t e n t of damage and env ironm en ta l c o nd i t io n s . DNA-damage can be r e p a i r e d , th e mechanism c a l l e d upon depending upon th e type and e x t e n t o f damage and th e env ironm en ta l c o n d i t io n s as w e l l a s th e c a p a b i l i t i e s o f th e organism . The r e l a t i v e r o l e s of s t r u c t u r a l changes and in d u c ib l e or n o n - in d u c ib le b iochem ica l changes a r e being e l u c id a t e d i n th e h ig h ly s tu d ie d m esoph i le s . I examined th e rm oph i l ic B a c i l l u s and Therm us u s i n g v a r i a t i o n s on t e c h n i c a l p r o c e d u r e s now s u c c e s s f u l w i t h t h e i r m e s o p h i l i c c o u n t e r p a r t s . Once t h e t e c h n i c a l a s p e c t was a f f i rm a t i v e l y e s t a b l i s h e d f o r th e th e rm oph i le s , an a t tem p t was made to compare th e d a ta o b ta in e d w i th th a t p u b l i s h e d f o r thermo­ p h i l e s and com para tiv e m esoph iles . The c h a l l e n g e o f o b t a i n i n g r e p r o d u c i b l e r e s u l t s w i t h t h e f i l am e n to u s Thermus was met p r im a r i ly th rough th e development o f an a p p r o p r i a t e medium f o r p l a t e c o u n t s and t h e p r e c i s e c o n t r o l o f c o n d i t i o n s i n c l u d i n g : a ) t i m e r e q u i r e d f o r e x p e r i m e n t a l m a n ip u la t io n s , b) optimum pH and tem p e ra tu r e of d i l u t i o n and p l a t i n g medium, and c) of c r i t i c a l im po rtance , c e l l d en s i ty . Use of c u l tu r e s w i th ITOF C above 0.5 produced h ig h ly e r r a t i c r e s u l t s . The degree of f ! l am e n ta t io n and t a n g l in g seen i n th e s e c u l t u r e s cou ld i n t e r f e r e w i th any p l a t e count a ssay which r e q u i r e d development o f c lo n e s from s in g le 48 c e l l s . C om p a r is o n s o f t h e rm o p h i l i c and m e s o p h i l i c membrane p ro te in s , r ibo som es , and n u c l e i c a c id s have shown th e b iochem ica l mechanisms o f o r g a n i sm s g row in g i n t h e two t e m p e r a t u r e r a n g e s t o be s i m i l a r and t h e rm o s t a b i l i t y to be i n h e r e n t i n m o le cu la r s t r u c t u r a l d i f f e r e n c e s i n th e b io chem ica l components o f th e c e l l s . The r e s t r i c t i o n enzymes o f each a r e a c t i v e on th e DNA o f th e o th e r . DNA polymerase i s o l a t e d from Thermus and compared to th e JS1. c o l i po lym erase i n d i c a t e s th e s u b u n i ts t o be s i m i l a r e x c e p t f o r t h e r m o s t a b i l i t y ( Ch ien e t a l . , 1976). Therm us DNA c lo n e d i n t o Ejs. c o l i p r o d u c e s t h e rm o s t a b l e p r o t e i n s (Tanaka, 1981; Nagahari e t a l . , 1980). I t i s l i k e l y t h a t the spectrum o f DNA r e p a i r mechanisms p r e s e n t i n th e rm oph i le s w i l l a l s o be s im i l a r t o t h a t ob se rved among m esoph ile s . We cou ld expec t t h a t p h o to r e a c t i ­ v a t i o n c o u ld be q u a n t i t a t e d i n t h e t h e rm o p h i l e s by r e c o v e r y from UV a f t e r v i s i b l e l i g h t t r e a tm e n t . C e r t a i n l y i f an i n d u c i b l e SOS-type r e p a i r system were p r e s e n t i n a th e rm oph i le , we could expec t a "m u l t i - h i t " UV s u r v iv a l curve. A p o s i t i v e i n d i c a t i o n o f SOS-type in d u c ib le r e p a i r u s i n g a g e n t s known t o in d u c e t h e h e a t s h o ck r e s p o n s e i n m e soph i le s a t th e h ig h e r of two tem p e r a tu r e s w i t h i n th e th e rm o p h i l ic range would a l s o d em on s t ra te th e p o s s i b i l i t y f o r a h e a t shock response s i m i l a r t o .Ei. c o l i , i n c l u d i n g h e a t - i n d u c i b l e DNA r e p a i r , i n t h e th e rm oph ile . D em ons tra t ion o f th e p re sence of h e a t shock p r o t e i n s i n th e rm oph i le s would a l s o d em on s tra te th e c a p a b i l i t y . S tu y ( 1956) h a s r e p o r t e d t h a t o f 15 B a c i l l u s s t r a i n s i n v e s t i ­ g a ted , on ly two showed good p h o to r e a c t i v a t io n , fo u r showed modera te 49 p h o to r e a c t i v a t i o n and th e o th e r s were not p h o to r e a c t iv a b le . However, t h e s t r a i n o f B a c i l l u s a p p e a r i n g i n Dr. J u l i a n ' s MSU l a b o r a t o r y and used i n th e s e ex p e r im en ts was shown t o be p h o to r e a c t iv a b le . We a l s o showed t h a t the s t r a i n can s u rv iv e a t 70 C. D ire c t l i g h t r e p a i r could n o t be d em o n s t r a t e d u n d e r t h e s e c o n d i t i o n s w i t h Therm us w h i l e t h e ex p e r im en ts were s u c c e s s f u l w i th JEL. c o l i and B a c i l l u s s te a ro th e rm o - p h i l u s . T h e re i s t h e q u e s t i o n o f w h e th e r UV -induced p h o to p r o d u c t s a r e s t a b l e a t 70 C. A pparen tly , B a c i l l u s i n c u r s 55 C-s t a b l e l e s i o n s which can be r e p a i r e d by p h o to r e a c t i v a t io n , bu t I have as y e t , no in fo rm a­ t i o n c o n c e r n in g d im e r s t a b i l i t y a t 70 C. A p o s i t i v e r e s u l t w i t h p h o t o r e a c t i v a t i o n i n Therm us wou ld h av e c o n f i rm e d t h e p r e s e n c e o f d im ers a s w e l l as d em ons tra ted r e p a i r by t h e i r d isappea rance . While some r e s e a r c h e r s c o n s i d e r UV damage t o DNA to be i n c l u d e d i n one c a teg o ry (Hanawa l t e t a l . , 1979), o th e r s s tudy more e x t e n s iv e fo rm s o f s t r u c t u r a l damage (B ra sh and B a s e l t i n e , 1982; H a s e l t i n e , 1983) • W ha tev e r t h e e f f e c t i n T he rm us f UV does " k i l l " t h e c e l l i n d o s e s s im i l a r to t h a t of o th e r b a c t e r i a . I t seems v a lu a b le to con t inue th e s e a rch f o r a p p ro p r ia t e c o n d i t io n s f o r d i r e c t l i g h t r e p a i r a t 55 C a s w e l l a s 70 C. Comparison o f Thermus and JL_ c o l i s u rv iv a l a f t e r exposu re to UV or th e c r o s s - l i n k i n g agen t , M itomycin C, o r Mitomycin C coupled w i th i r r a d i a t i o n showed t h a t dosages r e q u i r e d f o r " k i l l " w ere s im i l a r f o r both organ ism s. When a s u r v iv a l or k i l l i n g curve i s a s t r a i g h t l i n e p a s s i n g t h r o u g h t h e o r i g i n , i t i s c a l l e d a " s i n g l e - h i t " c u r v e w i t h a 50 s i n g l e even t r e s p o n s ib l e f o r th e d e s t r u c t i o n o f th e v i a b i l i t y o f th e o rg a n ism . " M u l t i p l e - h i t " c u r v e s h av e a s h o u ld e r n e a r t h e o r i g i n b e f o r e b ecom ing l i n e a r b e c a u s e s e v e r a l e v e n t s m u s t a c c um u la t e i n a v i a b l e c e l l ( u n i t ) b e fo re i t i s i n a c t i v a t e d . (To d e te rm in e th e number o f e v en ts r e q u i r e d f o r i n a c t i v a t i o n , th e s t r a i g h t p a r t o f th e s u rv iv a l c u r v e i s e x t r a p o l a t e d back t o m e e t t h e o r d i n a t e a x i s . ) The s lo p e o f th e s t r a i g h t p a r t o f a m u l t i p l e - h i t curve has th e same meaning a s f o r a s i n g l e - h i t curve (Dulbecco, 1980). F i f t y - f i v e C B a c i l l u s and 55 C Thermus e x h i b i t e d a t y p i c a l " s i n g l e - h i t " s u r v i v a l c u rv e w h i l e 70 C T h e rmu s d i s p l a y e d t h e " m u l t i p l e - h i t " t y p e i n t h e s e e x p e r im e n t s . The 70 C Thermus UV s u r v iv a l cu rve was found to be s im i l a r i n shape to t h a t o f w i ld type E. c o l i (AB1157) and t y p i c a l of in d u c ib l e r e p a i r . The "m u l t i p l e - h i t " curve shows a shou ld e r which d e c l in e s w i th UV k i l l a f t e r 30 seconds o f t r e a tm e n t . However, when Thermus i s in c u b a te d a t 55 C a " s i n g l e - h i t " s u r v iv a l cu rve i s g en e ra te d , s u g g e s t in g t h a t th e i n d i c a t e d in d u c ib le r e p a i r i s no t induced by UV a t th e low er t em p e ra tu re o r i s induced by tem p e ra tu re only . To f u r t h e r examine t h i s problem , Thermus was grown a t each tem p e ra tu re , sw i tch ed to th e o th e r c o n d i t io n , and i r r a d i a t e d a t v a r io u s t im es . The r e s u l t s of th e se ex p e r im en ts i n d i c a t e th a t the mechanism r e s p o n s ib l e f o r th e "m u l t i p l e - h i t " curve i s f u l l y induced by two h o u r s a f t e r a s w i t c h up t o h i g h e r t e m p e r a t u r e and t h a t t h e mechanism rem a in s a c t i v e a t t h a t t em p e ra tu r e (confirm ed by i r r a d i a t i n g an o v e r n i g h t c u l t u r e ) . I t was a l s o shown ( d a t a n o t p r e s e n t e d ) t h a t th e mechanism rem a in s a c t i v e f o r a t l e a s t th r e e hou rs f o l low in g th e 51 s w i t c h f rom 70 C t o 55 C. S in c e t h e Thermus UV s u r v i v a l c u rv e i s r e l a t e d to the tem p e ra tu re a t which th e organism i s grow ing, we have q u e s t i o n s c o n c e r n in g i n d u c i b i l i t y o f r e p a i r by o t h e r SO S - in d u c in g a g e n t s a t 55 C and 70 C and t h e r e l a t i o n s h i p , i f any , o f th e f ! l am e n ta t io n a l s o seen a t 70 C w i th t h i s organism . M itomycin C was used a s a p r e t r e a tm e n t fo l low ed by th e s ta n d a rd UV t r e a tm e n t o f both 55 C and 70 C c u l t u r e s to p rov ide c o n f i rm a t io n o f i n d u c t i o n o f DNA r e p a i r . H ere t h e 70 C Thermus wm u l t i p i e - h i t ” s u r v iv a l cu rve rem ained unchanged w h i le th e 55C Thermus s u r v iv a l curve i n d i c a t e s i n d u c i b i l i t y to be p re s en t . A pparen tly , th e in du c in g s ig n a l cannot be g e n e ra te d by UV damage under our c o n d i t io n s a t 55 C w h i le a p o s i t i v e r e s u l t i s o b ta in e d w ith the c r o s s - l i n k i n g mutagen. N u c le o id s t r u c t u r e o f Thermos h a s n o t y e t b e en exam ined . However, L o s s i u s e t a l . (.1983) h av e r e p o r t e d d i f f e r e n c e s i n t h e s e d im en ta t io n c o e f f i c i e n t s o f th e e n v e lo p e - f r e e n u c le o id s o f s e v e ra l s t r a i n s o f EL c o l i c a r r y i n g m u t a t i o n s i n t h e uv rA . uv rB , and r e c A genes when induced by M itomycin C and propose t h a t th e s e s t r u c t u r e s a r e r e p a i r i n t e r m e d i a t e s . The e x am in a t i o n and i d e n t i f i c a t i o n o f n u c l e o i d s t r u c t u r e s i n Thermus m u t a n t s w i l l be an e x c i t i n g a r e a o f r e s e a r c h , an i n t e g r a l p a r t of th e i l l u c i d a t i o n o f th e r e l a t i o n s h i p o f h e a t to l e r a n c e and in d u c ib l e DNA r e p a i r . The com p lex s e t o f o p e ro n s c o o r d i n a t e l y i n d u c e d i n E. c o l i by a s h i f t t o h i g h e r t e m p e r a t u r e d e m o n s t r a t e s t h e p r i n c i p l e by w h ich Thermus may i n d u c e DNA r e p a i r and p r o t e c t i v e s t r u c t u r a l n u c l e o i d p r o t e i n s i n re spon se to hea t . Thermus grows w e l l over th e tem pe ra tu re 52 range s tu d ie d , p resumably a d ju s t in g p r o t e i n s y n th e s i s and d eg rad a t io n i n re spon se to tem p e ra tu re change. Although th e tem p e ra tu r e range i s d i f f e r e n t f o r EU. e o l i and Thermusf th e changes f o r each w i l l p robably be shown to be a continuum over the e n t i r e range a t which the organism su rv iv e s , i . e . , we can expec t th e p r o t e i n s a s s o c ia t e d w i th th e DNA to change w i th h e a t in c r e a s e s . The f u n c t i o n a l s ig n i f i c a n c e o f h e a t shock p r o t e i n s i s unknown. N uc le ic a c id s t a b i l i t y , DNA r e p a i r , and degrada­ t i o n of d e l e t e r i o u s p r o t e i n s have been p o s tu la t e d a s f u n c t io n s . For Thermus i t i s r e a s o n a b l e t o e x p e c t t h a t m a in t e n a n c e o f a s t a b l e DNA h e l i x a t h igh tem p e ra tu r e r e q u i r e s th e p resence of a s s o c i a t e d p r o t e i n s induced by h e a t a s w e l l a s th e o th e r s t a b i l i z i n g f a c t o r s sugges ted i n t h e l i t e r a t u r e . DNA r e p a i r and d e g r a d a t i o n o f p r o t e i n s may a l s o be p o s s i b l e . E x p l a n a t i o n s f o r t h e v a r i a t i o n i n UV s u r v i v a l c u r v e s f o r Thermus a t two t e m p e r a t u r e s c o u ld be t h a t f o r some r e a s o n t h e in d u c ib le r e p a i r system i s no t in d u c ib l e by UV a t th e low er tempera­ t u r e o r t h a t f ! l a m e n t a t i o n i t s e l f c o n t r i b u t e s t o t h e sh ap e o f t h e c u rv e . P e rh a p s a b e t t e r e x p l a n a t i o n i s t h a t t h e h i g h e r t e m p e r a t u r e i t s e l f in d u c e s a r e p a i r system capab le of r e p a i r i n g UV l e s i o n s . As d i s c u s s e d e a r l i e r , f i l a m e n t a t i o n i n E. c o l i i s i n v o l v e d i n m u t a t i o n s w h ich c o n f e r s e n s i t i v i t y t o many m u ta g en s . How th e i n t r i c a t e 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 i t s m o le cu la r c o n t ro l i n I o n and s u l m u t a n t s i s r e l a t e d t o r e p a i r and r e c o m b i n a t i o n r em a in s cloudy. In t h a t organ ism , t h e r e i s more th an one pathway to f i l am en ­ t a t i o n . C e r t a in ly temporary f i l a m e n t a t i o n can be induced a long w i th SOS t r a i t s (W i tk in , 1 976). In Therm us f i l a m e n t a t i o n w as fo u n d when 53 th e organism was grown a t 70 C; UV in d u c t io n o f t h e . r e p a i r system we a r e o b s e r v i n g i s n o t r e q u i r e d . I f f i l a m e n t a t i o n i s a s u r v i v a l mechanism a t h igh tem pe ra tu re , th e c e l l may have more t im e f o r r e p a i r b e f o r e a n o t h e r c e l l d i v i s i o n . ( A lso , t h e l a r g e r s u r f a c e a r e a o f t h e f i l am en to u s form may be a b le to u t i l i z e th e lower amounts o f oxygen i n 70 C f l u i d more e f f i c i e n t l y . ) S tu d ie s o f c o n s t i t u t i v e and i n d u c ib l e r e p a i r , tem p e ra tu re - in d u c ed f i l am e n t a t i o n , d i s c o v e r i e s o f t h e s i n g l e s t r a n d b in d i n g p r o t e i n and th e p r i n c i p l e uv r p r o t e i n s a s w e l l as r e g u la to r y mechanism of th e r e c A and l e x A gene p r o d u c t s o f SOS r e p a i r s y s t em s , and t h e h e a t sh o ck r e s p o n s e h av e b een p r o d u c t i v e i n JjL1. c o l i b e c a u se o f i s o l a t i o n and s tudy o f m u tan ts d em on s t ra t in g th e se c o o rd in a te ly induced a c t i v i t i e s . The i s o l a t i o n o f r e p a i r - d e f i c i e n t m u t a n t s o f Thermus i s e s s e n t i a l . The s u i c i d e method once employed f o r i s o l a t i n g au x o t ro p h ic m u tan ts o f E. c o l i (Davis, 1948) h a s been employed by Sancar and R uper t (1978) i n th e i s o l a t i o n of p h o to r e a c t i v a t io n - and d a r k - r e p a i r - d e f i c i e n t m u tan ts from E1. c o l i and cou ld produce r e p a i r - d e f i c i e n t m u tan ts from Thermus f o r the c o n t in u a t io n o f t h i s l i n e of s tudy. Too, a u x o t ro p ic m u tan ts c o u ld be i s o l a t e d f o l l o w i n g m u t a g e n i z a t i o n and t h e m u t a t i o n r a t e o b s e r v e d a s a r a t e o f r e v e r s i o n t o p r o t o t r o p h y . Dr. Emmet J o h n so n (p e rso n a l communica tion) h a s observed a very high m u ta t io n r a t e w i th in th e l a c operon o f Thermus a t 70 C (no t examined a t 55 C). Determ ina­ t i o n o f t h e r a t e o f m u t a b i l i t y i n Therm us a t 55 C and 70 C w ou ld c l a r i f y th e q u e s t i o n o f invo lvem en t o f e r r o r - p ro n e DNA r e p a i r i n the 70 C Thermus r e s u l t . W arner (1983) r e c e n t l y o b s e r v e d t h a t 70 C 54 Thermus does d em on s t ra te r e p a i r i n uracil-DNA g ly c o sy la s e and a p u r in i c e n d o n u c l e a s e a c t i v i t i e s , b o th o f w h ich a r e known t o h a v e r o l e s i n hea t-damaged DNA r e p a i r i n Et c o l i bu t t h a t th e l e v e l s d e t e c te d a r e no g r e a t e r th an t h a t found i n Et c o l i . M u t a t i o n i n t h e I o n gene i s r e s p o n s i b l e f o r o v e r p r o d u c t i o n o f c a p s u l a r p o l y s a c c h a r i d e f i l a m e n t a t i o n upon i r r a d i a t i o n a s w e l l a s d e f e c t i v e f o r b a c t e r i o p h a g e I y s o g e n i z a t i o n and i n h e r i t a n c e o f F p la sm id s . Lon m u tan ts a r e a l so d e f i c i e n t i n d e g ra d a t io n o f abnormal p o ly p e p t id e s (Gottesman and Z ip se r , 1978) and i t i s th o ugh t t h a t th e m u l t i p l e i n v iv o p h e n o ty p e s a r e d e r i v e d f rom t h e i r d e g r a d a t i o n - d e f i c i e n t p ro p e r ty . I t h as been shown t h a t m u ta t io n s i n Io n d ec rease th e r a t e of d e g ra d a t io n o f a mu tan t sigma s u b u n i t o f RNA polymerase i n v iv o (G ro ssm an e t a l , I 985) (and s u g g e s t e d t h a t t h e l o n ~ a l l e l e ,may a f f e c t t h e r a t e o f t h e s igm a s y n t h e s i s .as w e l l ) . T r a s l e r and G o t te sm an (1984) s u g g e s t t h a t t h e d e f e c t i n r e g u l a t i o n o f c a p s u l e f o rm a t i o n f o u n d i n Io n c e l l s may be t h e r e s u l t o f d e g r a d a t i o n o f a p o s i t i v e r e g u l a t o r o f c a p su le s y n th e s i s . I t i s now known t h a t th e Ion gene p roduc t o f E s c h e r i c h ia c o l i i s a h e a t shock p r o t e i n (Goff e t a l . , 1984; P h i l l i p s e t a l . , 1 984). The in du c in g s ig n a l of h e a t shock i n o th e r o rg an ism s i s unknown. Why a l l the phenomena a s s o c i a t e d w i th SOS-repa ir , prophage in d u c t io n , and th e h e a t shock re spon se can be produced by such d i f f e r e n t a g en ts a s UV i r r a d i a t i o n , e t h a n o l , o r th ym in e s t a r v a t i o n h a s n o t y e t been exp la in ed . I n h i b i t i o n o f DNA s y n th e s i s (Radman, 1975) and p ro d u c t io n o f low m o le c u la r w e igh t p roduc ts o f DNA d eg rad a t io n (Pardee , 1975) a re 55 s u g g e s t e d e x p l a n a t i o n s . T h a t known i n d u c i n g f a c t o r ' s c a u s in g r e l a x a t i o n o f DNA m o lecu le s ( s u p e r h e l i c a l i n l i v i n g o rgan ism s) th rough s i n g l e s t r a n d b r e a k s ( x - r a y s ) , e x c i s a b l e damage r e s u l t i n g i n s i n g l e s t r a n d b reaks (UV and Mitomycin C) o r i n h i b i t i o n o f DNA s y n th e s i s and p r e v e n t io n o f l i g a t i o n o f s in g l e s t r a n d b reaks cou ld cause r e p r e s s o r p r o t e i n s r e a c t i n g w i t h s u p e r h e l i c a l DNA to d i s s o c i a t e f rom th e DNA m o l e c u l e w i t h a DNA c o n f i g u r a t i o n a l change ( r e l a x a t i o n ) p e r m i t t i n g t r a n s c r i p t i o n o f t h e r e p r e s s e d g e n e s h a s a l s o been s u g g e s t e d a s an e x p la n a t io n (Luchhik, 1979). The b ind ing o f p r o t e i n s t o DNA i s f i n e l y b a l a n c e d ( t o p o i s o m e r a s e v s . l o c a l m e l t i n g f o r RNA p o lym e r a s e ) t o p r o v i d e f o r a r a p i d r e s p o n s e t o change i n e n v i r o nm n e t a l c o n d i t i o n s . By t h i s th eo ry s u p e r h e l i c i t y would c o n t ro l b ind ing o f th e E2. c o l i le x A p r o d u c t t o t h e o p e r a t o r s i t e i n t h e r e c A o p e ro n and d i s s o c i a t i o n would p e rm i t t r a n s c r i p t i o n o f th e SOS genes; o th e r f u n c t i o n s could be i n d u c e d i n t h e same f a s h i o n . One e x p l a n a t i o n f o r o u r d a t a i s t h a t h e a t cau ses th e s u p e r h e l i c a l r e l a x a t i o n r e q u i r e d f o r i n d u c t i o n The e x te n t o f DNA w ind ing i s tem p e ra tu re -d ep end en t and a 15 C in c r e a s e i n t em p e ra tu re (h e a t shock) unwinds DNA one base p a i r p e r 200 base p a i r s ( T r a v e r s and Mace, 1982) (w h ich i s e q u i v a l e n t t o t h e maximum t r a n ­ s c r i p t i o n a l l e v e l i n E2. c o l i w i th 1 ,500 po lymerase m o lecu le s unwinding th e 15,000 base p a i r genome). T rave rs and Mace i n v e s t i g a t e d t h i s id e a i n JL c o l i . Their s t u d i e s showed t h a t i n h i b i t i o n of th e B su b un i t o f DNA to po isom era se I I in d u c e s p r o t e i n s which a r e h e a t shock p r o t e i n s b u t n o t t h e f u l l s e t o f known h s p s . The h e a t sh o ck phenomenon i s s u g g e s t e d t o be p r o t e c t i o n a g a i n s t r e l a x a t i o n . However, th e y con - 56 e l u d e d t h a t r e l a x a t i o n p e r se i s u n l i k e l y a s a s i g n a l and s u g g e s t i n s t e a d t h a t t h e i n t e r a c t i o n o f many p r o t e i n s w i t h t h e DNA i s v e r y s e n s i t i v e t o i o n i c s t r e n g t h o r pH, w h ich a f f e c t s t h e . a b i l i t y o f t h e topoisom e r a s e to super c o i l the chromosome. I n c o n c l u s i o n , r e s e a r c h h a s i n d i c a t e d t h e b a s i s o f l i f e a t h ig h t e m p e r a t u r e t o be complex . E l u c i d a t i o n th r o u g h a v a r i e t y o f i n t e g r a t e d o b s e rv a t io n s w i l l c e r t a i n l y in c lu d e th e e s s e n t i a l a rea of DNA r e p a i r . The f i n d i n g s of t h i s s tudy a r e i n d i c a t i v e of th e p resence o f two ty p e s o f DNA r e p a i r i n th e rm o p h i l ic b a c t e r i a . D ho to re a c t iv a - t i o n was found i n B a c i l l u s but no t i d e n t i f i e d i n Thermus. 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