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 r k e y K i r k p a t r i c k
A th e s i s su b m itted in 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 t h e d e g re e
of
M aster o f S c ie n c e
in
B iology
MONTANA STATE UNIVERSITY
Bozeman, Montana
November, 1985
<3o^ su
ii
APPROVAL
o f a t h e s i s s u b m it te d by
Mary Ann S ta r k e y K i r k p a t r i c k
T h i s t h e s i s h a s been r e a d by each member of th e t h e s i s c o m m ittee
a n d h a s b e e n f o u n d t o 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, f o r m a t , c i t a t i o n s , b i b l i o g r a p h i c s t y l e , and c o n s i s t e n c y , and i s
r e a d y f o r s u b m i s s i o n t o th e C o lle g e o f G ra d u a te S tu d ie s .
Date
/
C h a ir p e r s o n , G /a d u a te Committee
Approved f o r th e Major D epartm ent
///ZL S/X .IS
Date
Head, B iology
Approved f o r th e C o lle g e o f G ra d u a te S t u d i e s
('I - r ^ t ^
Date
Graduate Dean
iii
STATEMENT OF PERMISSION TO USE
In
p re se n tin g
th is
th e sis
in
p a rtia l
fu lfillm e n t
of th 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 o n ta n a 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 n d e r
ru le s
of
a llo w a b le
th e
L ib rary .
w ith o u t
B rie f
sp e c ia l
q u o ta tio n s
p e rm issio n ,
from
th is
p ro v id e d
th e sis
are
th a t a c c u ra te
acknow ledgm ent of s o u r c e i s made.
P e r m i s s i o n f o r e x t e n s i v e q u o t a t i o n from o r r e p r o d u c t i o n o f t h i s
t h e s i s may be g r a n t e d by my m a jo r p r o f e s s o r , o r i n h i s / h e r ab sen ce , by
t h e D i r e c t o r o f L i b r a r i e s when, i n t h e o p i n i o n o f e i t h e r ,
u se o f t h e m a t e r i a l i s f o r s c h o l a r l y p u rp o s e s .
t h e proposed
Any co p y in g or use o f
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 t h o u t my w r i t t e n p e r m i s s i o n .
V
' ACKNOWLEDGMENT
I w o u ld l i k e t o e x p r e s s my g r a t i t u d e t o th e f o l l o w i n g p e o p le .
Dr.
Guylyn W arren f o r h e r s t i m u l a t i n g d i s c u s s i o n ,
e d ito ria l
c o m m e n ts
d u rin g
th e
p r e p a r a t i o n o f t h e m a n u s c r i p t.
course
of
th e se
g u id a n c e ,
and
ex p e rim e n ts
an d
Her s u g g e s t i o n o f t h e t h e s i s problem ,
a s w e l l a s th e s c i e n t i f i c and p e r s o n a l i n t e r s ! i n my d e v e lo p m e n t a s a
p r o f e s s i o n a l woman, a r e g r e a t l y a p p r e c i a t e d .
Dr. E r n e s t Vyse f o r h i s d e d i c a t i o n t o th e d ev elo p m en t o f so many
stu d e n ts,
space,
in c lu d in g m y self,
a s w e l l a s th e use of h i s l a b o r a t o r y
eq u ip m en t, s u p p l i e s , and e x t e n s i v e p r o f e s s i o n a l tim e .
My G rad u ate C om m ittee:
Dr. Samuel R ogers 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 r e a o f m o l e c u l a r g e n e t i c s and Dr. David Cameron f o r d i s c u s s i o n
o f a b ro a d e r view o f B io lo g y and G e n e ti c s .
Sue
Zaske f o r
her
p a tie n t
te a c h in g
m anner
and
sh a rin g
of
e x p e r t i s e w ith t h e e l e c t r o n m ic ro s c o p e .
Dr. N e ls N elso n f o r d i s c u s s i o n and g u id a n c e 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 m ethods f o r B a c i l l u s s t e a r o t h e r m o p h i l u s .
vi
TABLE OF CONTENTS
Page
APPROVAL..........................................................................................................
STATEMENT OF PERMISSIONTO U S E .........................................................................
VITA ................
ACKNOWLEDGMENTS....................................
TABLE OF CONTENTS..................................
LIST OF TABLES..................
LIST OF FIGURES.........................
ii
iii
iv
v
vi
v iii
ix
LIST OF PLATES ...........................................................................................................
x
ABSTRACT................................................................................. .......................................
. xi
INTRODUCTION ...............
B io c h e m is tr y o f T herm ophily
F a t t y a c i d s and membranes........................................................................
P r o t e i n s ..............................................................................
Ribosom es..............................................................
N u c le i c a c i d s ....................................................................................................
DNA DAMAGE AND REPAIR ..................................
DNA D a m a g e ....................
1 . R a d i a t i o n ...........................................................
2.
Chemical m u tag en s ..............................
3 . T r a n s p o s o n s ...............................................................................
DNA Damage: UV and MC .......................................................
Types o f DNA R e p a i r ..................
1. P h o t o r e a c t i v a t i o n ................. ................ .........................................
2. E x c i s i o n r e p a i r ...............................................................................
3.
P o s t r e p l i c a t i o n a l r e p a i r ..........................................................
4 . The a d a p t i v e r e s p o n s e ...............................................................
5. SOS r e p a i r .............................................................................'............
6 . SOS d e f i c i e n t m u ta n ts o f JL c o l i .........................................
7. H eat s h o c k ...........................................................................................
8. B a c t e r i a u sed f o r r e s e a r c h ......................................................
9. S ta te m e n t o f r e s e a r c h pro b lem s ...............................................
I
2
3
4
5
7
7
8
8
9
9
10
12
13
14
14
15
17
19
21
21
vii
TABLE OF OOMTENTS-Continued
Page
MATERIALS AND METHODS ...........................................................................................
S o u rce o f o rg a n ism s .........
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 .................
B a c t e r i a l grow th c o n d i t i o n s .............................................................
M icroscopy .........................................................
DNA damaging t r e a t m e n t s ......................................................................
Exposure t o UV ................................................................................
E x posure t o MG ....................................................................................
R e p a ir a s s a y s ......................................................................................
S u r v i v a l ..............................................................
23
23
23
24
27
28
28
28
28
30
RESULTS ...........................................................................................................................
A. The o rg a n is m s o f t h e stu d y .............................................. ................
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 therm op h i l u s ...........
Morphology ................................
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 .................................................
C h a r a c t e r i s t i c s o f Thermus ...................
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 byi r r a d i a t i o n ...................
C. E xposure t o UV and M G.................................. ................................... ..
D. Q u a n t i t a t i v e r e s u l t s o f p h o t o r e a c t i v a t i o n and d i r e c t
l i g h t r e p a i r f o l l o w i n g UV t r e a t m e n t ....................
31
3031
31
31
35
39
41
DISCUSSION ...................................................................................................
47
REFERENCES CITED ......................................................................................................
57
43
viii
i LIST OF TABLES
T a b le s
1.
Page
Sum m ary
of
C h a r a c te r is tic s of
2.
P h o to re a c tiv a tio n :
and EL. c o l i
3.
C o n d it io n s
T h e rm o p h ile s
B a c illu s ste a ro th e rm o p h ilu s
o f A tte m p te d D i r e c t L i g h t R e p a ir
37
45
46
ix
LIST OF FIGURES
Page
F ig u res
1.
S t r u c t u r e o f a Thymine d im er
11
2.
S t r u c t u r e o f M itom ycin C
11
3.
UV S u r v i v a l :
JL c o l i . Thermus 55 C,
Thermus 70 C
40
4.
UV S u r v i v a l :
B a c i l l u s s t e a r o t h e r m o n h i l u s 55 C
42
5.
UV S u r v i v a l :
Thermus. 1 , 2 , 3 h o u r s a f t e r
t r a n s f e r from 55 C t o 70 C
44
X
LIST OF PLATES
P la te s
Page
1.
B a c i l l u s . G ra m sta in
32
2.
B a c i l l u s . T erm inal e l l i p t i c a l s p o r e
33
3.
B a c illu s . F la g e lla
34
4.
. Thermus 55 C and Thermus 70 C
36
xi
ABSTRACT
Two t h e r m 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 r m o o h i l u s and
Thermus T2 w ere o b s e r v e d f o r r e s p o n s e t o known DNA-dam a g i n g a g e n ts , UV
r a d i a t i o n and t h e c h e m ic a l mutagen, M itom ycin C.
The e x i s t e n c e o f 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 s y ste m was d i s c o v e r e d i n B a c i l l u s
s t e a r o t h e r m o p h i I u s . U n l i k e Eh c o l i w hose d a r k DNA r e p a i r - i s UVi n d u c i b l e , T h e rm u s w a s n o t f o u n d t o h a v e a U V - i n d u c i b l e r e p a i r
mechanism.
However t h e p re s e n c e o f a DNA r e p a i r sy ste m i n d u c i b l e by
e i t h e r h e a t or c h e m i c a l s was o b s e r v e d i n Thermus, r e l a t i n g t e m p e r a t u r e a s s o c i a t e d DNA r e p a i r w i t h s u r v i v a l a t h ig h te m p e r a t u r e s .
1
INTRODUCTION
B io c h e m is tr y o f T herm oohilv
M e so p h ilic
m ic ro o rg a n sim s
have
a
maximum
g ro w th
ra te
at
t e m p e r a t u r e s around 30-37 C; i n th e ca se o f Et c o l i T g ro w th c e a s e s a t
45—48 C w h i l e t e m p e r a t u r e s o f 50-52 C o r g r e a t e r cause c e l l death.
T h e r m o p h ilic m ic r o o r g a n is m s can t h r i v e a t t e m p e r a t u r e s up t o 85
C.
They a r e f o u n d i n g e o t h e r m a l l y a c t i v e a r e a s s u c h 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 , as c o n ta m in a n ts of canned fo o d and d a i r y
p ro d u c ts,
in h o t w a te r h e a te r s ,
an d 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 s m s h a v e p i q u e d t h e i n t e r e s t o f t h e s c i e n t i f i c c o m m u n ity f o r
reasons of
th e ir
th e rm o sta b ility
th e rm o p h ily
is
p ro life ra tio n
of
a t e le v a te d
t h e i r m a c ro m o le c u le s.
a p p lic a b le
to
stu d ie s
of
t e m p e r a t u r e s and t h e
B a sic
e v o lu tio n
k n o w le d g e
of
and eco lo g y ,
m o l e c u l a r b io lo g y and b i o c h e m i s t r y , and c o u ld l e a d t o u t i l i z a t i o n f o r
i n d u s t r i a l e n z y m i c p r o d u c t i o n o f m a r k e t a b l e p r o d u c ts (Amelunxen and
Murdoch,
1977).
T e m p e r a tu re i s o n ly one of t h e v a r i a b l e s i n f l u e n c i n g t h e g row th
o f l i v i n g o rg a n is m s.
O ther e n v i r o n m e n 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 , a n d l i g h t i n t e r a c t t o i n f l u e n c e t h e
o p t i m a l and maximum g ro w th t e m p e r a t u r e s .
Under v a r y i n g c o n d i t i o n s th e
i n t e r r e l a t i o n s h i p o f c h e m ic a l s t r u c t u r e , c o n f o r m a tio n , 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 o c h a c h k a a n d
Somero,
1973).
Thus,
a tte m p ts 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
2
a t h ig h
te m p e r a tu r e have e v o lv e d from
th e o b s e r v a tio n of g e n e ra l
p h y s io lo g y t o a b io c h e m ic a l a p p ro a c h w i t h c o n c o m ita n t e x a m in a t io n a t
th e m o lecu la r le 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 a n d 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 t a t i o n ,
are re sp o n sib le f o r ra p id re p la c e ­
ment o f h e a t-d a m a g e d p r o t e i n s w as advanced by A lle n (1953).
However,
when Brock (1967) p u b l i s h e d g row th r a t e d a t a o f v a r i o u s m e s o p h i l i c and
t h e r m o p h i l i c b a c t e r i a a t t h e i r optimum t e m p e r a t u r e s A l l e n 's h y p o t h e s i s
was d is c o u n te d .
Brock (1967) fo u n d t h a t th e r m o p h i l e s do not grow a s
f a s t a t t h e i r o p tim a a s p r e d i c t e d by p u r e l y
o f e f f e c t of t e m p e r a t u r e on p h y s io lo g y .
th e o re tic a l c a lc u la tio n s
When U l r i c h (1971) combined
p h y s i o l o g i c a l and b i o c h e m i c a l a p p r o a c h e s t o exam ine a Thermus- I i k e
o rg a n is m f o r m o r p h o l o g ic a 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 m echanism , and
r e g u l a t i o n o f enzyme s y n t h e s i s ,
he found no m a jo r d i f f e r e n c e s betw een
m e s o p h i le and th e r m o p h i l e , e x c e p t t h e r m o s t a b i l i t y .
B io c h e m ic a l
s tu d ie s
of
t h e r m o p h i l e s have d ete rm in e d
th e
p r o p e r t i e s o f s p e c i f i c c e l l com ponents o r m o l e c u le s and com pared t h e s e
to t h e ir
c o u n t e r p a r ts i n m e so p h ile s.
F a t t y a c i d s and m em branes,
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 ll a s n u c l e ic
a c i d s o f s e v e r a l t h e r m o p h i l e s have been i s o l a t e d and exam ined.
F a t t y a c i d s and m em branes:
The membranes o f t h e r m o p h i l e s a r e exposed
t o t h e e n v iro n m e n t so t h i s component o f t h e c e l l was one o f th e f i r s t ,
t o be exam ined f o r h e a t s t a b i l i t y .
branes i s
c o n sta n tly
The f l u i d i t y o f b a c t e r i a l biomem­
m a in ta in e d i n
g ro w in g c e l l s .
V a ria tio n in
3
com plex l i p i d c o n t e n t and s t r u c t u r a l ch an g e s i n f a t t y a c i d com ponents
a r e s u g g e s te d a s m e ch an ism s to a c h i e v e
th e f l u i d i t y
i m p o r t a n t f o r m e m b ra n e f u n c t i o n s ( C r o n a n ,
c o n te n t of v a r io u s m ic ro o rg a n ism s i s
1 9 7 8 ).
know n t o
The f a t t y
known t o be a f f e c t e d
t e m p e r a t u r e a t w hich th e y a r e grown (Oshima, 1978).
be
acid
by t h e
High p r o p o r t i o n s
o f u n s a t u r a t e d a c i d s a r e fo u n d a t lo w e r t e m p e r a t u r e s ,
w h ile s a tu r a te d
f a t t y a c id s in c re a s e w ith in c re a s in g te m p e ra tu re .
The p r e s e n c e o f
h i g h l y bran ch e d ,
l o n g e r c h a in ,
sa tu ra te d f a tty
a c i d s i n membranes o f
th e r m o p h i l e s h a s been c o n f ir m e d (Oshima e t a l . , 1976).
Also,
a novel
g l y c o l i p i d c o n s t i t u t i n g up t o 70% o f th e t o t a l l i p i d o f two s t r a i n s o f
T herm us
has
been
id e n tifie d
( O s h im a
and
A rig a ,
1976).
It
is
c o n j e c t u r e d t h a t th e u n iq u e l i p i d c o n t e n t o f th e t h e r m o p h i l i c membrane
i s r e s p o n s i b l e f o r s u c c e s s f u l membrane f u n c t i o n a t h ig h te m p e r a tu r e .
P ro te in s:
S in c e th e p rim a ry ,
secondary,
te rtia ry
and q u a te 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 v a r y a s much betw een p r o t e i n s o f th e same
f u n c t i o n o b t a i n e d fro m v a r i o u s m e s o p h i l i c o r g a n is m s a s b e tw e e n therm o­
p h i l i c and m e s o p h i l i c p r o t e i n s o f t h e same f u n c t i o n ,
i t is d iffic u lt
t o e x p l a i n t h e i n v a r i a b l e s t a b i l i t y t o d e n a t u r i n g c o n d i t i o n s ( c h e m ic a l
d e n a tu ra n ts as w e ll as h eat) of th e rm o p h ilic p ro te in s.
1
t i g a t o r s have p ro p o sed t h a t enhanced s t a b i l i t y i s
(Ohta,
1966), h y d rogen (B arn es and S t e l l w a g e n ,
and R a i d t ,
V a rio u s i n v e s -
due t o
h y drophobic
1973)? o r i o n i c ( P e r u tz
1975) bonding p ro d u c in g c o n f o r m a tio n s w i t h l a r g e r o r more
d e n s e l y p a c k e d 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)> m ore o r
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 l w a g e n and B a r n e s ,
1 9 7 6 ), m o r e
e x t e n s i v e l y l a c e d m a c r o m o le c u la r s u r f a c e s ( P e ru tz and R a i d t ,
1975), or
4
m o re c o m p l e m e n t a r y I n t e r s u b u n i t c o n t a c t s
(T h is
to p ic
has
been re v ie w e d
( B i e s e c k e r e t a l . , I 9 7 7 ).
by Z u b e r ,
1 976;
F rie d m a n ,
1978;
Amelunxen and Murdock, 1977; S i n g l e t o n and Amelunxen, 1973.)
H e a t-re s is ta n c e i s o fte n
conferred
by o n l y a few a m in o a c i d
c h a n g e s a s s h o w n by M e r k l e r e t a l . (1 9 8 1 ) who c o m p a r e d 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 o f p r o t e i n s o f c l o s e l y r e l a t e d m e s o p h i l i c and th e rm o ­
p h ilic
b a c illi.
Argos e t a l .
(1979),
amino a c i d s i n c r e a s e d i n t e r n a l
p o la rity .
found s t r a t e g i c a l l y
su b stitu te d
h y d r o p h o b ic i ty and i n c r e a s e d e x t e r n a l
H ydrophobic bonds a r e more s t a b l e a t h ig h t e m p e r a t u r e th a n
a t low t e m p e r a t u r e .
A p p a re n tly m o l e c u l a r i n t e r a c t i o n s w i t h i n p o ly p e p r
t i d e c h a i n s a r e s u f f i c i e n t t o cause t h e r m o s t a b i l i t y .
Ribosomes:
Ribosomes and th e o t h e r com ponents a s s o c i a t e d w i t h p r o t e i n
s y n th e s is a re a ls o th e rm o s ta b le i n th erm o p h ile s.
R ibosom al s u b u n i t s
( p r o t e i n a n d RNA) h a v e b e e n f o u n d t o be h e a t s t a b l e ( Y a g u c h i e t a l . ,
1 9 7 8 ).
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 i c h d e l i v e r an d c a t a l y z e t h e
b in d i n g o f c h a rg e d t-RNAs t o t h e rib o s o m e a r e r e q u i r e d f o r e l o n g a t i o n
o f t h e p o l y p e p t i d e c h a i n i n p r o t e i n s y n t h e s i s i n p r o k a r y o te s .
These
f a c t o r s have been p u r i f i e d fro m Thermus t h e r m o p h il u s and compared t o
E. c o l i (A ra l e t a l . , 1978).
ex tre m ely
d e n atu ran ts.
sta b le
The t h e r m o p h i l i c e l o n g a t i o n f a c t o r s a r e
a g a in st h eat,
acid ,
a lk a li,
and o th e r
p ro te in
Thermus e l o n g a t i o n f a c t o r s showed a l a c k o f s u l f h y d r y l
gro u p s in c o n t r a s t to
t h o s e o f EL c o l i w h e r e s u l f h y d r y l s
e s s e n tia l r o le in c a t a ly t ic fu n c tio n .
p la y a n
I n c o n t r a s t t o t h e m o n o m e r ic
f o r m s fo u n d i n t h e m e s o p h ile th e e x i s t e n c e o f m u l t i m e r i c f o r m s w ere
5
d e m o n s t r a te d i n th e th e r m o p h il e .
I t i s a p p a r e n t t h a t no s i n g l e m e c h a n is m o r c e l l c o m p o n e n t i s
r e s p o n s i b l e f o r th e rm o p h ily .
As th e t h e r m o s t a b i l i t y o f membranes was
s u g g e s te d t o be due to p r e s e n c e o f n ovel g l y c o l i p i d s a s w e l l a s d eg ree
of
s a tu ra tio n
of i t s
fa tty
a c id s
and
th e
v a ria tio n
in
p ro te in
s t r u c t u r e a l l o w i n g f u n c t i o n a t h ig h t e m p e r a t u r e was d e m o n s tr a te d ,
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 a s a l s o f o u n d t o be h e a t
sta b le .
N u c le ic a c i d s :
The n u c l e i c a c i d s o f t h e r m o p h i l e s have been s t u d i e d i n
a v a r i e t y o f w ays i n c l u d i n g base c o m p o s i tio n ;
p r e s e n c e and a c t i o n o f
a s s o c i a t e d p o ly a m in e s; i s o l a t i o n and s tu d y o f enzym es in v o lv e d i n
s y n t h 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 i o n of t h e r m o p h i l i c n u c l e i c a c i d 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 s m i d s ,
a s th e clo n in g o f
th e rm o p h ilic genes w ith
as w e ll
s u b se q u e n t e x p r e s s i o n and
c h a r a c t e r i z a t i o n i n m e s o p h ile s .
H eat s t a b i l i t y
of th e rm o p h ilic n u c le ic a c id s i n c r e a s e s w ith
i n c r e a s e d G-C c o n t e n t p r o d u c i n g h i g h e r c o r r e s p o n d i n g i n c r e a s e i n
m e l t i n g t e m p e r a t u r e (Oshima e t a l . , 1976).
may a r i s e
T h e r m o p h ilic DNA s t a b i l i t y
p a r t i a l l y from a s s o c i a t i o n w i t h d i v a l e n t
c a tio n s as i t
know n 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 an d RN A.
n u c le ic a c id s in c re a s e s w ith te m p e r a tu r e and i s
T h io la tio n of
d ire c tly
c o rre la te d
w i t h t h e r m o s t a b i l i t y o f t-RNA i n t h e c e l l (Q uigley and R ich,
P o ly am in es a r e g e n e r a l l y
b io c h e m ic a l
p ro cesses
such
c o n s id e r e d t o
as
is
1976).
be i n v o l v e d i n i m p o r t a n t
sta b iliz in g
DNA a n d RNA,
p ro te in
6
b io sy n th e is,
DNA and RNA b i o s y n t h e s e s ,
t o e n v i r o n m e n ta l s t r e s s .
th e rm o p h ile,
c e ll d iv isio n ,
and a c c l i m a t i o n
Novel poly am in es a r e produced by th e e x tre m e
Thermus th e r m o o h i l u s ( Oshima, I 975 and 1982).
Alan Malcolm h a s p ro p o s e d i n a t h e o r e t i c a l p a p e r (1981) t h a t th e
i n c r e a s e d G-C c o n t e n t o f m-RNA w i t h co n s e q u e n t i n c r e a s e d s t a b i l i t y o f
s e c o n d a ry s t r u c t u r e s h o u ld a l s o be c o n s id e r e d a s a s e l e c t i o n p r e s s u r e
i n t h e t h e r m o p h i l i c e n v iro n m e n t and t h a t t h e m ost common amino a c i d
changes b etw ee n m e s o p h i l e s and th e rm o p h i l e s r e c o r d e d by Argos e t a l.
(1979) a r e c o n s i s t e n t w i t h t h i s h y p o t h e s i s .
S i n g l e base changes found
i n t h e m-RNA codons of t h e r m o p h i l e s e i t h e r i n c r e a s e
secondary
s tru c tu re
or
have l i t t l e
effect
th e s t a b i l i t y
(n o n e
w o u ld
of
d ecrease
s e c o n d a ry s t r u c t u r e ) .
S t e n i s h and M a d is o n (19 7 9 ) c o m p a r e d t h e s t a b i l i t y o f m-RNA i n
m e s o p h i le s and t h e r m o p h i l e s , fo u n d t h e h a l f - l i f e o f m-RNA t o d e c r e a s e
a s g ro w th
te m p e ra tu re in c re a s e d but d isco v ere d th e " s t a b i l i t y
index"
( h a l f - l i f e o f m R N A /d o u b lin g t i m e o f c e l l s ) t o be c o n s t a n t f o r e a c h
o rg a n ism
r e g a r d le s s of te m p eratu re.
T h is s u p p o r t s t h e c o n c e p t 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 th e r m o p h il y ; th e
h a l f - l i f e o f th e m-RNA i s a f i x e d f r a c t i o n o f t h e d o u b lin g tim e.
N u c le i c a c i d s of t h e r m o p h i l e s and m e s o p h i le s have been shown t o
v a r y i n n u c l e o t i d e c o n t e n t , p r e s e n c e o f n ovel p o ly a m in e s , s t r u c t u r e of
enzym es in v o lv e d i n
enzymes.
However,
sy n th e sis,
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
t h e r e a r e many s i m i l a r i t i e s i n b a s i c o r g a n i z a t i o n
o f g e n e t i c m a t e r i a l and e x p r e s s i o n o f i n f o r m a t i o n .
T h e r m o p h ilic DNA
a n d RNA p o l y m e r a s e s ( K a l e d i n e t a l . , I 980; C h i e n e t a l . , 1 976; D a t e ,
7
1975),
DNA m e t h y la s e ( S a to e t a l . , 1980),
and DNA r e s t r i c t i o n enzymes
( S a to e t a l . , 1977) have been i s o l a t e d and c h a r a c t e r i z e d .
tie s
The p r o p e r ­
d e m o n s t r a t e d by t h e s e e n z y m e s a r e s i m i l a r t o t h o s e o r o t h e r
th e rm o sta b le p ro te in s .
T h is i s a l s o t r u e o f enzym es p ro d u ced from
g e n e s c l o n e d f r o m t h e r m o p h i l e s ( N a g a h a r i e t a l . , 1 9 8 0 ).
d ic h r o i s m
A c irc u la r
s tu d y o f th e complex betw een p ro m o te r DNA and Thermus RNA
p o l y m e r a s e ( T s u j i , 1980) s h o w e d o n l y m o re m e l t i n g i n t h e p r o m o t e r
r e g i o n t h a n fo u n d i n El c o l i r c o n f ir m in g th e s i m i l a r i t i e s of th e two
o rg a n is m s.
R e s e a r c h on DNA r e p a i r a n d m u t a g e n e s i s i n m e s o p h i l i c b a c t e r i a ,
i n i t i a l o b s e r v a t i o n s o f t h e f i l a m e n t o u s h i g h l y t h e r m o p h i l i c b a c te r iu m ,
Therm us. 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 r e s p o n s e t o 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 a n d e u k a r y o t e s by a g e n t s
t h a t can in d u c e changes i n DNA r e p a i r a c t i v i t y
a s s o c ia te d w ith f ! l a ­
m e n t a t i o n s u g g e s t e d t o Dr. G uylyn W arren a p o s s i b l e n a t u r a l a s s o c i a ­
t i o n b etw ee n g row th a t h ig h t e m p e r a t u r e , DNA damage and r e p a i r .
DNA Damage and R e p a ir
DNA D amage:
been s t u d i e d :
F o u r m a i n t y p e s o f DNA a l t e r a t i o n s o r dam age h a v e
I) D i m e r i z a t i o n o f two a d j a c e n t p y r i m i d i n e s on the same
DNA s t r a n d w h en t h e p y r i m i d i n e s beco m e c o n n e c t e d by a c y c l o b u t a n e
r i n g , 2) c h e m ic a l a l t e r a t i o n o f b a s e s by d e a m in a t io n o r 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 l e n t c r o s s l i n k s b etw ee n b a s e s on two s t r a n d s ,
4) b re a k s i n one or both s t r a n d s .
and
Any o f t h e s e 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 c o d in g p r o p e r t y o r m u ta tio n .
M u t a t i o n s c a n 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 r o m e n z y m a t i c
8
d y s f u n c t i o n d u r in g DNA r e p l i c a t i o n o r r e c o m b in a tio n .
M u ta tio n ,
in a
broad s e n s e , a lth o u g h a h e r i t a b l e change, may n o t a f f e c t th e phenotype
o r be r e c o g n iz e d .
However,
to r e f e r to a h e rita b le
t h e te rm w i l l be used i n t h i s m a n u s c r i p t
change i n n u c l e o t i d e sequence o f an o rganism
w hich i s r e c o g n i z e d by i t s e f f e c t on t h e phenotype o f t h e organism .
A m u tag en i s an a g e n t w hich c a u s e s changes,
as. d e s c r i b e d above,
i n g e n o m ic n u c l e i c a c i d a n d i n c r e a s e s t h e m u t a t i o n r a t e a b o v e t h e
s p o n ta n e o u s l e v e l a s o b s e r v e d phendt y p i c a l l y .
in
th e
e n v iro n m en t
m u ta g e n s
(a lk y la tin g
i n t e r c a l a t i n g ag en ts,
1.
in c lu d e
and
ra d ia tio n
Known m u tag en s p r e s e n t
(UV and X - r a y s ) ,
d e a m in a ti n g
a g e n ts,
base
c h e m ic a l
a n a lo g s,
and c r o s s - l i n k i n g a g e n t s ) and t r a n s p o s e ns.
R ad iatio n .
a)
U l t r a v i o l e t (UV) l i g h t c a u s e s f o r m a t i o n o f d im e r s betw een
p y r i m i d i n e b a s e s on th e same s t r a n d ( i n t r a s t r a n d ) o f DNA.
d i n e s b eco m e c o n n e c t e d by a f o u r
carb o n c y c lo b u ta n e
The p y r i m i ­
rin g .
( S ee
diagram and d e t a i l i n UV s e c t i o n below.)
b) X - r a y s cause b re a k s i n t h e p h o sp h o d ie s t e r backbone i n one
o r both s t r a n d s o f DNA.
2.
Chem ical m utagens e f f e c t m o d i f i c a t i o n s i n DNA b a s e s i n s i t u
by d e a m i n a t i o n ,
ad d u cts.
a lk y la tio n ,
or th e a d d i t i o n of a v a r i e t y
C h e m ic a l m u t a g e n s a l s o i n c l u d e :
of b u lk y
s t r u c t u r a l a n a lo g s w hich
v a r y i n b o n d i n 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 ic h
i n s e r t d u rin g r e p l i c a t i o n , d i s t o r t th e base p a i r i n g and le 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 o r an added base i n
sized s tra n d ;
th e new ly s y n th e ­
and c r o s s - l i n k i n g a g e n t s w hich form i n t e r s t r a n d c r o s s ­
9
l i n k s p o s in g a n a b s o l u t e b lo c k t o 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 ransposons,
o r u n i t s o f DNA t h a t h a v e t h e c a p a b i l i t y o f
moving from one DNA m o le u le t o a n o t h e r ,
r e s u l t i n r e a r r a n g e m e n t s and
d e l e t i o n s i n t h e m o l e c u le t h a t was l e f t and i n s e r t i o n and d i s t u r b a n c e
o f DNA c o i l i n g i n t h e m o le c u le e n t e r e d .
DNA Damage - UV and MG:
UV and M ito m y cin C w e re chosen f o r exam ina­
t i o n o f th e t h e r m o p h i l e s ' r e s p o n s e t o m utagens.
caused
by UV i s
th e
best
research ed
le sio n .
The p y r im i d i n e d im er
The c r o s s - l i n k i n g
mechanism o f M itom ycin C p r o v id e d a second mechanism o f DNA damage f o r
o b se rv a tio n .
UV Damage:
DNA e f f i c i e n t l y
a b s o r b s l i g h t i n t h e ra n g e o f 240-300 nm
r e s u l t i n g i n e x c i t e d e n e rg y s t a t e s o f th e b a s e s and c a u s in g a v a r i e t y
o f p h o t o c h e m i c a l r e a c t i o n s (Wang,
1 9 7 6 ).
The p r i n c i p l e p r o d u c t ,
p y r i m i d i n e d im e r s , c a u s in g th e p r i n c i p l e b i o l o g i c a l e f f e c t s , l e t h a l i t y
a n d m u t a g e n i s i s , i s f o r m e d w h en tw o a d j a c e n t p y r i m i d i n e b a s e s o n 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 i g .
I).
The tw o
b a s e s a r e p u l l e d o u t o f a l ig n m e n t , t h e h y d ro g en bonds t o com plem entary
b a s e s a r e b r o k e n , a n d t h e DNA b a c k b o n e i s d i s t o r t e d , p r e v e n t i n g t h e
c o r r e c t p a i r i n g o f t h e tw o b a s e s o n e a c h s i d e o f t h e d i m e r .
The
p r e s e n c e of a s i n g l e d im er can i n t e r r u p t t r a n s c r i p t i o n o r r e p l i c a t i o n .
E v en i f r e p l i c a t i o n r e s u m e s o n t h e o t h e r s i d e o f t h e d i m e r , a gap i s
l e f t i n t h e n e w ly s y n t h e s i z e d s t r a n d , b l o c k i n g t r a n s c r i p t i o n o f t h e
e n tire
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 t h e n e x t c y c le
(H an aw alt e t a l . , 1 979).
10
M ito m y c in
C ( F ig .
2) i s
m e ta b o lic a lly
red u ced
by a q u i n o n e
r e d u c t a s e i n th e c e l l t o a h ydroquinone d e r i v a t i v e w hich a l k y l a t e s and
ex ten siv ely
c r o s s - l i n k s DNA ( I y e r and S z y b a l s k i , 1963).
The b i o l o g i ­
c a l s i g n i f i c a n c e o f 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 i d e n t from s t u d i e s
o n t r a n s f o r m i n g DNA a n d b a c t e r i a l v i r u s e s (Kohn e t a l . , I 963; B e c k 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 t o cause th e
i n a c t i v a t i o n o f a t l e a s t 3 ,0 0 0 b a s e p a i r s w i t h i n a DNA m o l e c u l e ,
p resu m ab ly a s a consequence o f b lo c k in g co m p le te s t r a n d s e p a r a t i o n f o r
re p lic a tio 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 m o r e 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 m o re s e n s i t i v e t o M i t o m y c i n C t h a n w i l d t y p e
stra in s.
rep air,
W hile s i m p l e a l k y l a t i o n damage i s
n o t r e p a i r e d by e x c i s i o n
c r o s s - l i n k i n g cau sed by th e b i f u n c t i o n a l M itom ycin 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 l e e t a l . , 1 9 7 6 ).
DNA R e p a ir :
For every o rg a n is m ,
life
and c o n t i n u i t y fro m g e n e r a t i o n
to g e n e r a t i o n depend on th e lo n g - te r m
m a te ria l,
th e
DNA.
S in c e
a ll
c e lls
s t a b i l i t y of i t s h e re d ita ry
are
s e n sitiv e
r a d i a t i o n and c h e m ic a l a g e n t s i n t h e e n v iro n m e n t,
to
dam age
by
a s y ste m o f rem oval
o f l e s i o n s a n d 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 h a v e b e e n
ad o p ted .
I t i s n o t p o s s i b l e f o r DNA p o ly m e ra s e I I I t o r e p l i c a t e a r e a s
o f DNA c o n t a i n i n g d im e r s o r c r o s s - l i n k s , a lth o u g h i t can r e s t a r t a f t e r
t h e damaged r e g i o n h a s been passed.
H o w a rd -F la n d e rs (19.75) h a s 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 r o m U V -dam aged DNA c o n t a i n
g ap s a p p r o x im a te ly t h e s i z e o f one o r more Okazaki f r a g m e n t i n d i c a t i n g
11
O
d Rib
F ig u re I .
S t r u c t u r e o f a th y m in e d im e r r e s u l t i n g fro m 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 a v i s , B.D., Dul b e c c o , R.,
Bi s e n , H.N., G i n s b e r g , H.S. M i c r o b i o l o g y , T h i r d E d i t i o n ,
H arper and Row, M aryland, 1980).
F ig u r e 2 .
S t r u c t u r e o f M i t o m y c i n C ( b a s e d on F i s h b e i n , L., W.G.
Flam m , a n d H.L. F a l k , e d s . , C h e m ic a l M u ta g e n s .
A c a d e m ic
P r e s s , N.Y., 1 9 7 0 ).
12
re s u m p tio n of r e p l i c a t i o n does occur w ith c o n c o m ita n t gaps i n th e
d a u g h te r s t r a n d .
DNA r e p a i r i n v o l v 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
in itia te
th e
b io c h e m ic a l r e a c t i o n s w h ich l e a d to
c irc u m v e n tio n of th e le s io n .
e lim in a tio n
or
W h i le s p e c i f i c e n z y m e s r e p a i r som e
s p e c i f i c base m o d i f i c a t i o n s or c o r r e c t a c h e m i c a l a l t e r a t i o n ,
o th e r
t y p e s o f r e p a i r a r e m o re g e n e r a l i n n a t u r e an d c a n 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 o r a llo w a second chance a t s p e c i f i c r e p a i r th ro u g h
r e co m b i n a t i o n a l p r o c e s s e s .
The m a j o r t y p e s o f DN A r e p a i r known i n
b a c te ria are p h o to re a c tiv a tio n ,
th e
a d a p tiv e resp o n se
rep air.
base s u b s t i t u t i o n ,
and r e c o m b i n a t i o n a l
Except f o r p h o t o r e a c t i v a t i o n ,
or
ex c isio n re p a ir,
p o st-re p lin a tio n a l
s p e c if ic p ro o f-re a d in g fu n c­
t i o n s i n DNA r e p l i c a t i o n an d 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
error-prone.
I.
. .
P h o to re a c tiv a tio n .
P h o t o r e a c t i v a t i o n w as d is c o v e re d a s a r e d u c t i o n i n th 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 s u b s e q u e n t e x p o s u re t o l o n g e r wave­
le n g th s.
I t h a s been d e m o n s t r a te d i n many c e l l u l a r s y s t e m s i n c l u d i n g
b a c t e r i a l and human and a c t s on p y r im i d i n e d im e r s only.
The p h o to r e ­
a c t i v a t i n g enzyme b in d s t o t h e d i m e r - c o n t a i n i n g r e g i o n o f t h e DNA th u s
g e n e r a t i n g a DNA -enzym e c h r o m o p h o r e 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 t h o u t b r e a k i n g any phosphod ie s t e r bonds.
P h o to m e d ia te d r e c o v e r y from UV damage was d i s c o v e r e d i n b a c t e r i a
an d 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 co o ).
The m e c h a n is m w a s
13
c h a ra c te riz e d i n
1962 (R u p e rt).
R e c e n tly ,
an a l t e r n a t e r o l e f o r th e
p h o t o r e a c t i v a t i n g enzyme was s u g g e s te d by Yamamoto e t a l .
(1983) who
d i s c o v e r e d a n E. c o l i r e c A m u t a 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
presence of a p h o t o r e a c t i v a t i n g enzym e i n th e d a rk .
P h o to re a c tiv a ­
t i o n , how ever, can be masked by an e f f i c i e n t e x c i s i o n r e p a i r system .
2.
E x c i s i o n R e p a ir .
D i f f e r e n t m o d e s o f e x c i s i o n r e p a i r o f d a m a g e d DNA h a v e b e e n
d i s c o v e r e d i n d i f f e r e n t o rg a n is m s.
I n El c o l i t h r e e d i s t i n c t l o c i a r e
known t o be r e q u i r e d f o r e x c i s i o n o f damage i n U V - i r r a d i a t e d DNA.
The
u l t r a v i o l e t l i g h t d am ag e 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 S e k ig u c h i ,
1981) t o make
t h e UVRABC e n z y m e , now c l o n e d a n d c h a r a c t e r i z e d by S a n c a r a n d 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++ a n d ATP, f o r
m aking two c u t s ,
one on each s i d e o f th e damaged DNA,
t h u s rem o v in g a
1 2 -1 3 n u c l e o t i d e l o 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.
th e n f i l l e d
lig a se .
The g ap i s
t h r o u g h a c t i o n o f DNA p o l y m e r a s e I a n d s e a l e d
by DNA
I t i s s u g g e s t e d t h a t t h e e n zy m e may b i n 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 1 2 - 1 3 b a s e p a i r f r a g m e n t r e s u l t i n g f r o m DNA
damage, t h e i n s t a b i l i t y enhanced by b in d i n g o f UVRA and UVRC t o s i n g l e
s t r a n d e d DNA ( S e e b e r g a n d S t e i m e m , 1 9 8 2 ; S a n c a r a n d Rupp, 1979) a n d
t h a t t h e DNA p o ly m e ra s e I e x o n u c le a s e a c t i v i t y may enhance rem oval of
th e e x c i s e d s t r a n d .
The c u t s , d i s p l a c e d fro m th e s i t e s o f damage, can
r e p a i r a broad sp e c tru m of dam ages w ith o u t p r e c i s e r e c o g n i t i o n of
p a r t i c u l a r ad d u cts.
w ith
The e n z y m e a c t s o n DNA w h ic h h a s b e e n t r e a t e d
U V -i r r a d i a t i o n ,
Pt
(II)
com pounds,
p so ra le n
p lu s
n ear­
14
u ltra v io le t,
n i t r o u s a c i d o r M itom ycin C (Brash and B a s e l t i n e ,
1982).
Kenyon a n d W a l k e r (19 81) h a v e s u g g e s t e d t h a t t h e UVRA a n d UVRB
p r o t e i n s a r e p r o d u c e d 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 i n d u c e d t o
h i g h e r l e v e l s of p r o d u c t i o n by th e c o n t r o l e l e m e n t s o f t h e i n d u c i b l e
"SOS" r e s p o n s e 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 h a s n o t o c c u r r e d and
DNA r e p l i c a t i o n i s b lo c k e d a t th e 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 rep air.
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 a n t a g e o f O k a z a k i - s i z e d gaps
w hich a r e l e f t by p o ly m e ra s e I I I i n th e new ly s y n t h e s i z e d DNA o p p o s i t e
t h e d am a g e d r e g i o n .
re trie v a l,
used to
P o s t r e p l i c a t i o n a l r e p a i r , th e n , i s a sy stem of
whereby m a t e r i a l from one s t r a n d o f a d u p le x o f DNA can be
re p a ir
th e gap i n a n o th e r .
The m e c h a n is m
of cro sso v ers
b etw ee n two d a u g h t e r m o l e c u le s , whose gaps do not u s u a l l y c o i n c i d e i s
r e c o g n i z a b l e by e l e c t r o n m icro sco p y o f DNA (Rupp and H o w a rd -F la n d e rs ,
I 9 6 8 ); Rupp a n d c o w o r k e r s ( 1 9 7 1 ) d e m 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 r e f o r m e d i n DNA i n
an e x c i s i o n - d e f e c t iv e
s tra in .
The l e s i o n
r e m a i n s o n one s t r a n d ; h o w e v 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 c a n be
made a n d 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 o m b i n a n t
m o le c u le .
The d a u g h t e r
stra n d
gap r e p a i r r e q u i r e s
a fu n c tio n a l
r e c combin a t i o n r e c A gene a s w e l l as p o ly m e ra se , p o l A o r p o l C.
4.
The A d a p tiv e Response.
When exposed t o low c o n c e n t r a t i o n s o f m e t h y l a t i n g o r e t h y l a t i n g
a g e n t s , Et c o l i becomes r e s i s t a n t t o t h e m u ta g e n 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 sa m e a g e n t s ( J e g g o e t a l , I 977 a n d 1 978).
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 t h w a y w a s f i r s t d i s c o v e r e d by S am so n a n d
15
C a i r n s (1977) and h a s been te rm e d t h e a d a p t i v e r e s p o n s e (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 t w o r k i s unknow n.
tiv e ly
a c tin g re g u la to ry
e lem en t of th e ad a p tiv e ,
The p o s i ­
ada.
lo c u s i s
s u g g e s te d by W alker (1984) t o be s p e c i f i c a l l y in d u c e d by th e a d d u c t o f
th e
a l k y l a t i o n o f g u an in e.
The a d a l o c u s c o n s i s t s o f an o p e r o n
c o d in g f o r two p r o t e i n s ( L i n d a h l,
th e
a d a p tiv e
response
is
th e
1982).
Also known t o be in v o l v e d i n
u n id e n tifie d
a lk y Ig u an in e -D N A a l k y l t r a n s f e r a s e
lo c u s
(L in d ah l
et
a l,
co d in g f o r
1982)
Ch­
(w h ic h
c a t a l y z e s t h e t r a n s f e r o f t h e m e th y l o r e t h y l group from t h e a l k y l a t e d
g u a n in e t o th e 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 t h y l a d e n i ne-DNA g l y c o s y I a s e I I w h ic h 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 b e rg ,
1982) and an unknown number of o t h e r
genes.
A lthough m e t h y l a t i n g and a l k y l a t i n g a g e n t s can i n 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 a n d 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 i n d u c e a n o t h e r mode,
nSOSn r e p a i r ,
w h ic h r e s u l t s i n a c t i v e
m u t a g e n e s is .
5.
SOS R e p a ir.
The te rm nSOS f u n c t i o n s " h a s been d e s i g n a t e d 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 .
In c lu d e d
in
th e se
resp o n ses
are
in h ib itio n
of c e ll
d iv isio n ,
f i l a m e n t o u s g ro w th i n r e p a i r - d e f i c i e n t m u t a n t s a s s o c i a t e d w i t h in d u c ­
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 ,
in d u c e d b a c t e r i a l m u ta g e n e s is , i n d u c t i o n of p ro p h ag e,
W eig le r e a c t i v a t i o n and W eig le m u ta g e n e s is .
a s w e ll as
The l a t t e r two r e s p o n s e s
16
w ere d i s c o v e r e d by W eigle (1953) when he o b s e rv e d t h a t U V - i r r a d i a t e d
b a c t e r i o p h a g e y i e l d e d more p la q u e s and a h i g h e r p r o p o r t i o n o f m u ta n ts
w hen p l a t e d on l i g h t l y
u n irra d ia te d c e lls.
U V - i r r a d i a t e d JL c o l i t h a n w h en p l a t e d on
The same g e n e t i c and p h y s i o l o g i c a l r e q u i r e m e n t s
a p p l i e d t o p r o p h a g e i n d u c t i o n ( D e f a i s e t a l . , 1971) w h ic h r e q u i r e s
s i t e s p e c i f i c r e c o m b in a t io n .
DNA a s w e l l .
la te d
The m u t a g e n ic r e s p o n s e o p e r a t e d on h o s t
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 th e r e g u ­
fu n c tio n s
rep resen t
an in d u c ib le
response
of
b a c te ria
u n r e p a i r e d damage i n t h e i r DNA o r an "SOS" s i g n a l (Radman,
to
1975).
I n d u c t i o n o f t h e SOS r e s p o n s e s i s accom panied by t h e a p p e a r a n c e
o f a p r o m in e n t 40 Kd p r o t e i n (Inouye,
1971) now known t o be ( c o n s t i t u -
t i v e l y p r o d u c e d i n lo w l e v e l s ) t h e r e c A g e n e p r o d u c t .
S y n th e sis of
t h e r e c k p r o t e i n i s r e g u l a t e d by t h e gene p r o d u c t o f I e x Af a r e p r e s s o r
o f r e c A a n d a n u m b e r o f o t h e r g e n e s , a n d 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 form ,
1975) .
a p ro te a s e or a cleav ag e
stim u la to r
(G e o r g e e t a l . ,
DNA d e g r a d a t i o 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 f o r k s may r e s u l t i n i n d u c t i o n o f t h e SOS re s p o n s e .
The s i g n a l
may be a c o m p le x o f s i n g l e - s t r a n d e d DNA, 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 a n o l i g o n u c l e t i d e ( O i s h i , 1 9 7 8 ).
W itk in (1974, 1975,
1976) s u g g e s t e d t h a t SOS c o u ld o p e r a t e a s ( I ) a n e r r o r - p r o n e v a r i a n t
o f reco m b i 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 i o n a l r e p a i r system
w h i c h p o l y m e r i z e s DNA p a s t t h e p y r i m i d i n e d i m e r o r o t h e r n o n c o d i n g
le sio n s
in
th e
te m p la te
stra n d .
C ooper
and H a n a w a l t
( 1 9 7 2 a , b)
p r e s e n t e d b i o c h e m i c a l e v i d e n c e t h a t t h e r e a r e tw o t y p e s o f r e p a i r
w hich f u n c t i o n i n c l o s u r e o f e x c i s i o n gaps,
one o f w hich i s dependent
17
on r e c gene p r o d u c ts .
The p r i n c i p a l
The e v id e n c e s u p p o r t s W i tk in 's p r o p o s a l.
m odel 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 is
p r o p o s e s " t r a n s d i m e r s y n t h e s i s " ( C la r k and V o l k e r t ,
1978) a s a r e s u l t
o f m o d i f i c a t i o n o f norm al DNA p o ly m e r a s e s ( p e rh a p s t h e 3 '— 5' e d i t i n g
e x o n u clease a c t i v i t y )
by a n i n d u c i b l e
p ro te in
th 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 .
In
c a s e s o f m a s s i v e dam ag e 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 o n
o p p o site s tra 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 o u ld s t o p a t a s e c o n d c l o s e l y - s p a c e d l e s i o n o n t h e o p p o s i t e s t r a n d
and w ould r e q u i r e t r a n s d i m e r s y n t h e s i s f o r c o m p le ti o n r e s u l t i n g i n a
lo n g r e p a i r p a tc h and p r o d u c t i o n o f m u t a t i o n s .
M u t a t i o n i n JSjl c o l i i s d e p e n d e n t u p o n t h e g e n e p r o d u c t s o f t h e
recA+ and I e x A+ g e n o ty p e and t h e f u n c t i o n o f o t h e r g e n e s, d e s c r i b e d i n
th e next s e c tio n .
M u ta tio n i s m e d ia te d by a l l th e p a th w a y s d e s c r i b e d
f o r SOS r e p a i r :
a)
tra n sd im e r
sy n th e sis
due
to
in d u c ib le
su p p ressed
3 ' — 5*
e d i t i n g a c t i v i t y o f p o ly m e r a s e s ;
b)
d a u g h t e 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 l u s t r a n s d i m e r s y n t h e s i s i n c a s e s o f m a s s iv e
damage r e s u l t i n g i n lo n g p a t c h r e p a i r .
M u ta t io n r a t e s a r e d e c r e a s e d i n t h 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 i c i e n t M u ta n ts o f E s c h e r i c h i a c o l i .
G e n e ti c a n a l y s i s o f r e p a i r m u t a n t s h a s r e v e a l e d a m a jo r p o r t i o n
o f w hat i s known a b o u t t h e m o l e c u l a r b a s i s o f SOS r e p a i r and m utagene­
sis.
Many o f t h e m u t a n ts s t u d i e d w e re i s o l a t e d f o r o t h e r t r a i t s and
18
s u b s e q u e n t l y i d e n t i f i e d a s a f f e c t i n g m u t a g e n e s is .
a.
rec
A l l e l e s o f r e c A ( r e c o m b i n a t i o n - d e f i c i e n t I and l e x A showed
A and le x
A to
be r e q u i r e d
fo r
SOS a n d m u t a g e n e s i s ,
to
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 an d s u g g e s t e d t h e
sy ste m had t o be in d u c e d (W alker, 1984).
b.
T e m p e r a tu r e - in d u c e d .f! l a m e n t a t i o n ,
o r t i f m u t a n ts ,
show t h 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 c o n c o m ita n t h ig h l e v e l s o f r e c A p r o t e i n and s u b s e q u e n 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 ic h a c t e d on r e p r e s s o r s of th e
v a r i e t y o f SOS f u n c t i o n s d e s c r i b e d e a r l i e r ( Gudas and Mount, 1977).
c.
When n o n -m u ta b le ( d e f e c t i v e i n SOS) m u t a n ts w ere s c r e e n e d f o r
and i d e n t i f i e d ,
m u ta n ts o th e r th a n o f th e in d u c tio n p ro c e s s i t s e l f
w e re i d e n t i f i e d .
I n t h i s way umu C and umu D m u t a n ts w e re d is c o v e r e d
( K a to
and
S h in o u ra ,
1 9 7 7 ).
Umu m u t a n t s
are
s till
c a p a b le
of
e x p r e s s i n g a v a r i e t y o f SOS r e s p o n s e s and t h e r e f o r e code f o r p r o d u c ts
r e q u i r e d f o r SOS p r o c e s s i n g and n o t e s s e n t i a l t o t h e c e l l .
and D l o c i
have
now
been
c lo n e d
C h a ra c te riz a tio n of th e a c t i v i t y
( E lle d g e
and
The umu C
W a lk e r,
1983).
o f t h e gene p r o d u c t so f a r c o n f ir m s
t h e i n f e r e n c e s from e a r l i e r w ork w i t h m u ta n ts .
d.
S t r a i n s c a r r y i n g t h e . s i n g l e . s t r a n d DNA b in d i n g ( s s b ) m u t a ti o n
a r e a l s o d e f i c i e n t i n SOS p r o c e s s e s .
s tro n g ly
to
The s s b gene p r o d u c t w hich b in d s
s i n g l e - s t r a n d e d DNA i s
a p p a re n tly p la y in g a p o s itiv e
c o n t r o l l i n g r o l e i n th e s y ste m (Myer e t a l . , 1979).
e. The l o n g f o r m 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 lls .
The I o n g e n e p r o d u c t i s
an A T P -d ep en d en t p r o t e a s e w h ich
19
re g u la te s
th e
am ount
of
f !la m e n ta tio n
by a f f e c t i n g
th e r a te
of
d e g r a d a t i o n o f t h e su p p r e s s o r o f I o n o r s u l gene p r o d u c t ( C h a r e t t e e t
a l.,.
1981;
Chung
and
G o ld b e rg ,
1981).
Lon+ c e l l s
fila 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 r e 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 a n be i n 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 S O S -in d u ced .
The I o n g e n e p r o d u c t a l s o a f f e c t s t h e
d e g r a d a t i o n o f a m u ta n t sig m a s u b u n i t o f RNA p o ly m e ra se .
. te m p e r a tu r e . p r o t e i n Cjrtp) m u ta n t i s
sim ila r,
t h e sig m a s u b u n i t o f RNA p o ly m e r a s e ;
S in c e a h ig h
slo w in g d e g r a d a tio n of
(W alker, 1984) s u g g e s t s t h a t th e
h tp R may be in v o l v e d i n c o n t r o l l i n g t h e a c t i v i t y o f t h e Io n p r o t e a s e
and o t h e r p r o t e a s e s . .
7.
H eat
Shock.
A nother i n d u c i b l e p r o t e c t i v e m e a su re f o r c e l l s i s t h e " h e a t shock"
p r o t e c t i v e r e s p o n s e a g a i n s t th e r m a l k i l l i n g .
te m p e ra tu re
e lic its
p ro te in s,
some
(Y am am o ri
et
phenom ena w as
changes in
cease
a l.,
th e
b ein g
1 9 7 8 ).
m ad e,
p ro d u c tio n of
som e
The o r i g i n a l
h e a t-in d u c e d
c h r o m o s o m e s ( R i t o s a , 1 9 6 2 ).
th e
I n JL c o l i a s h i f t up i n
p u ffin g
are
tra n sie n tly
d isc o v e ry
of
m ost c e l l u l a r
of
in d u c e d
heat
D ro so p h ila
shock
p o ly te n e
" P u ff in 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 t h e o o lv t e n e chromosomes.
S u b s e q u e n tly an a n a l o g o u s r e s p o n s e
t o h e a t was found i n many o t h e r s p e c i e s i n c l u d i n g b a c t e r i a ,
c e l l s , and p l a n t s ( S c h l e s i n g e r , 1982).
mammalian
I n 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 i c h h a v e b e e n f o u n d by N e i d h a r d t e t a l .
(1981,
1 9 8 2 , 1983) t o c o n s t i t u t e a H ig h T e m p e r a t u r e R e g u lo n (HTR) w h ic h 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 i v e p r o t e i n s had been found ( N e id h a r d t e t a l , 1984).
The h e a t
(H ig h to w er,
shock re s p o n s e i s
1 9 8 0 ).
a u b iq u ito u s
response
to. s t r e s s
A v a r i e t y of a g e n ts can in d u c e c h an g e s i n gene
a c t i v i t y s i m i l a r t o th o s e caused by h e a t shock.
D r o s o p h 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 th e s y n th e s is o f th r e e s m a ll h e a t shock
p r o t e i n s w i t h e i g h t o f t e n t e r a t o g e n t r e a t m e n t s w h ile sev en d ru g s 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
(B u zin e t
a l.,
1 9 8 2 ).
A ls o ,
in
do n o t i n d u c e h e a t s h o c k p r o t e i n s
D ro so p h ila ,
a m in o a c i d
an alo g s,
s u lf h y d ry I - r e a c tin g re a g e n ts , t r a n s i t i o n m e ta l io n s , u n c o u p le rs of
o x id a tiv e p h o sp h o x y la tio n , v i r a l i n f e c tio n ,
a n tib io tic s ,
som e
c h e la to rs
e th a n o l,
and i o n o p h o r e s i n d u c e
a v a rie ty of
th e response.
K r u e g e r a n d W a l k e r (1 9 8 4 ) f o u n d t h a t h e a t s h o c k 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 ,
I n d u c t i o n was fo u n d t o be c o n t r o l l e d
(h to
R) g e n e
p ro d u c t,
by t h e h ig h t e m p e r a t u r e . p r o t e i n
a p o sitiv e ly
a c tin g
e x p r e s s i o n o f h e a t shock g en es i n JSl c o l i
1 9 8 1 ).
So,
UV and n a l a d i x i o
in d e p e n d e n t r e g u l a t o r y s y ste m ,
elem en t
re q u ire d
fo r
(N e ih a r d t and Van B ogelen,
a c id in d u c e
t h e SOS s y s t e m
t h e h e a t shock r e s p o n s e .
t h e i n d u c i n g s i g n a l i s s t i l l unknow a
th e h e a t
UV an d n a l a d i x i o a c i d .
and t h e
The n a t u r e o f
The f u n c t i o n a l s i g n i f i c a n c e of
shock r e s p o n s e i s a l s o unknow a
However,
t h e r e i s e v id e n c e
t h a t D N A -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
in d u c tio n (P e llo n e t a l,
th e n ,
1 9 8 0 , 1981 , 1982).
H eat sh o ck p r o t e i n s ,
c o u ld be r e q u i r e d f o r t h e s t a b i l i t y o f chromosome s t r u c t u r e or
in v o l v e d i n r e p a i r o f h e a t damaged DNA.
Walker (1984) s p e c u l a t e s t h a t
t h e h t o R-c o n t r o l l e d gene p r o d u c ts a r e i n v o l v e d i n d e g r a d a t i o n o f SOS-
21
i n d u c e d p r o t e i n s w h i c h w o u ld be d e l e t e r i o u s
to th e c e l l
if
th e y
p e r s i s t e d a f t e r c o m p le ti o n of th e SOS r e s p o n s e .
8.
The b a c t e r i a u s e d f o r r e s e a r c h w o rk a r e t h e i n t e r m e d i a t e
th e r m o p h i l e , B a c i l l u s s t e a r o t h e r m o p h i l u s . and th e e x tre m e th e r m o p h il e ,
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 r m o p h i l u s ( G ordon,
I 923) i s a g r a m - v a r i a b l e ,
m o t i l e , s t r a i g h t r o d - s h a p e d o rg a n is m w hich i s c a p a b le o f g ro w th a t 65700 w i t h o p tim u m g r o w t h a t 55 0; a n d w h i c h p r o d u c e s h e a t - r e s i s t a n t
e n d o s p o re s .
S p o re s a r e fo rm e d i n s o i l i n a l l c l i m a t i c zones.
V eg eta­
t i v e g r o w t h i s r a p i d i n many f o o d s o f pH a b o v e 5.0 ( i f h e l d a t a n
a p p r o p r i a t e e l e v a t e d t e m p e r a t u r e ) , i n h e a t i n g com post, and i n c o n ta m i­
n a te d ,
im p r o p e r ly p r o c e s s e d ,
canned fo o d s.
B1. s t e a r o t h e r m o p h i l u s h a s
been o f t e n u sed i n l a b o r a t o r y r e s e a r c h of th e rm o p h ily .
Thermus a a u a t i c u s . a n o n - m o t i l e o b l i g a t e a e ro b e w hich 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 r o c k and
F r e e z e (1 9 6 9 ) 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 6 5 -7 0 C a n d s t a t i o n a r y
phase c u ltu r e s .
E l e c t r o n m i c r o g r a p h s t u d i e s o f e x t r e m e l y t h e r m o p h i l i c b a c t e r i a show
JThermu s ,
in
c o n tra st
to
Et c o l i . t o h a v e a r e g u l a r
s c a llo p -lik e
c o n n e c t i o n t o t h e i n n e r m e m b r a n e /c e ll w a l l g i v i n g i s o l a t e s an a n n e l i d ­
l i k e a p p e a r a n c e (Ramaly e t a l . , 1978).
w a te r h e a t e r s
(Brock and Boylen,
The genus h a s a p p e a r e d i n h o t
1973) and n a t u r a l l y - o c c u r r i n g w a t e r
t h a t h a s been c o n ta m in a te d by th e r m a l e f f l u e n t (D egryse e t a l . , 1978).
9.
S ta te m e n t o f R esearch Problem s.
The p u rp o s e of th e r e s e a r c h r e p o r t e d h e r e was t o combine c u r r e n t
22
k n o w l e d g e o f t h e f i e l d s o f t h e r m o p h i l y a n d DNA r e p a i r i n a s t u d y o f
two t h e r m o p h i l i c m i c r o o r g a n is m s i n o r d e r t o advance know ledge i n t h e
a r e a o f s u r v i v a l o f l i f e a t h ig h t e m p e r a t u r e .
a.
The s tu d y in c lu d e d :
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 t h e r m o p h i l i c s t r a i n
by m o r p h o l o g ic a l e x a m in a t io n and b io c h e m ic a l t e s t i n g .
b.
D e v e lo p m e n t o f a p p r o p r i a t e m edium f o r
stu d y
of th e
tw o
t h e r m o p h i l e s a t 55C and 70C.
c.
E xposure o f B a c i l l u s s t e a r o t h e r m o o h i l u s and Therm us T2 to t h e
known DNA-damaging a g e n t s , UV and M itom ycin C, f o r d e t e r m in a ­
tio n of th e ir su rv iv a l p a tte rn s.
d.
E x a m in a tio n o f t h e two s t r a i n s f o r th e e x i s t e n c e o f c o n s t i t u ­
tiv e
p h o to re a c tiv a tio n
re p a ir
sy stem s
sim ila r
to
th e ir
m e so p h ilic c o u n te rp a rts.
e.
D e t e r m i n a t i o n o f t h e e x i s t e n c e of an i n d u c i b l e r e p a i r system
(SOS) i n t h e two o rg an ism s.
f.
E x a m in a tio n
of
th e
re la tio n sh ip
of h e a t-to le ra n c e
i n d u c i b l e r e p a i r and f ! l a m e n t a t i o n i n Thermus.
to
MATERIALS AND METHODS
S o u rce o f o rg a n ism s
1. T h e rm u s w as 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 r o m a n a l k a l i n e
t h e r m a l s p r i n g i n Yellow s t o n e Park.
S t r a i n T2 i s a m u t a n t l a b o r a t o r y
s t r a i n w hich i s non-m ucoid and c o n s e q u e n t ly e a s i e r t o w ork w i t h i n t h e
la b o ra to ry .
The c u l t u r e u s e d f o r t h e s e s t u d i e s was o b t a i n e d from t h e
A m erican Type C u l t u r e C o l l e c t i o n (ATCC #27737).
2.
The s t r a i n o f B a c i l l u s s t e a r o t h e r m o n h i l u s u s e d i n
w as o r i g i n a l l y
t h i s s tu d y
i s o l a t e d a s a c o n t a m i n 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 i v e r s i t y B io c h e m i s t r y D epartm ent
and was i d e n t i f i e d d u r i n g t h e c o u r s e o f t h i s study.
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 i m e n t s w a s
s t r a i n H53 i s o l a t e d f r o m
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 l a n t P a th o lo g y D ep artm en t, MSU).
4.
C o n tr o l E1. c o l i f o r UV s u r v i v a l e x p e r i m e n t s w as s t r a i n AB1157
X-(K12) (B achm an, I 9 7 2 ).
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 a n d M a l a c h i t e G r e e n s p o r e
s t a i n w e r e p e r f o r m e d a c c o r d i n g t o C o n n '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 tin g agar,
o f M ic ro b io lo g y ,
Gordon e t a l .
o b t a i n e d fro m Dr.
Montana S t a t e U n i v e r s i t y ,
N els N elso n ,
D epartm ent
was p r e p a r e d a c c o r d i n g t o
(1973), i n o c u l a t e d w i t h a lo o p and i n c u b a t e d a t 55 C f o r
24
18 h o u r s .
3.
R e d u c t i o n o f n i t r a t e an d n i t r i t e .
N i t r a t e r e d u c t i o n medium
w as o b t a i n e d from Dr. N e ls N elson, D epartm ent o f M ic ro b io lo g y ,
S ta te U n iv e rsity ,
and was p r e p a r e d a c c o r d i n g t o Gordon (1973).
Montana
Medium
t o d e t e c t p r o d u c t i o n o f Ng g a s was p r e p a r e d by a d d in g a t r a c e o f Zn t o
t h e n i t r a t e r e d u c t i o n m edium and p l a c i n g i t i n a n I n v e r t t u b e w h i c h ,
d u r in g a u t o c l a v i n g p r o c e d u r e ,
tu b e w i t h " l i q u i d ,
e x p e l l s t h e g a s and f i l l s
th e in v e rte d
th e r e b y a l l o w i n g any g a s p r o d u c t i o n by t h e org an ism
t o be o b s e r v e d a s b u b b le s i n th e i n v e r t e d tu b e .
B a c t e r i a l Growth C o n d it io n s
I.
Media
A.
The r o u t i n e
stu d ie s
of B a c illu s
e m p lo y e d a medium
c o n t a i n i n g 0.5% T r y p t o n e ( D i f c o ) a n d 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 l u t i o n (10 ml each o f S o l u t i o n s I ,
II,
I I I per l i t e r ) .
The pH
was a d j u s t e d t o 7-7 p r e a u t o c l a v i n g w hich s h i f t e d t o a f i n a l pH o f 7.5
d u r i n g t h e a u t o c l a v e p ro c e d u re .
S o lu tio n I
(NH21) 2SO4
( 3 .9 6 g / 1 )
KCl
( 7-46 g /1 )
NaCl
( 2 3 .3 3 g / 1 )
Na2 HPO4 . 2H2 0
( 4 6 .2 7 g / D
S o lu tio n I I
Ca(NO3 ) 2 . 4H20
( I .18 g / 1 )
S o lu tio n I I I
MgSO4 . 7H2 0
or
MgSO4
( 9 .8
g /1 )
( 4 .8
g /1 )
The a g a r c o n t e n t (D ifco , M i c r o b i o l o g i c a l g ra d e ) o f s o l i d medium was 2%
25
(20 gm /1).
p l a t e s w e re d r i e d a t room t e m p e r a t u r e f o r s e v e r a l days o r
a t 70°C f o r I 1/2 h o u r s b e f o r e use.
B. Media u sed f o r th d c u l t u r e o f Thermus w ere:
I) t h e 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 a n d 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
( 1 9 7 1 ) ; o r 2) a s i m p l e r medium w o r k e d o u t by Dr. Emmet J o h n s o n a t
T u lan e U n i v e r s i t y
(personal
c o m m u n ic a tio n ) s p e c i f i c a l l y
fo r
th e
c u l t u r e o f Therm us.
Johnson* s Thermus medium:
S a l t s S o l u t i o n I (100X)
(NH4 ) 2 SO4
4 . 0 :g
KCL
7 .0 g
Ca (NO3 ) 2 -H2 O
1 .2 g
b ro u g h t t o I ,000 ml w ith w a te r
S a l t s S o l u t i o n I I (100X)
NaCl
20 g
Na2HPO4 '7 H2 O
30 g
MgSO4 -7 H2 O
10 g
b ro u g h t t o I ,000 ml w i t h w a te r
Thermus medium
S a lts S o lu tio n
I
10 ml
S a lts S o lu tio n
I I -10 ml
FeSO4 (0.05%)
I ml
Y east e x t r a c t
2g 1
D ifco t r y p t o n e
2g
added t o 1,000 ml w a te r t o p r e v e n t p r e c i p i t a t i o n o f s a l t s .
s •
26
Two g e l l i n g a g e n t s w ere used i n c o n j u n c t i o n w i t h t h e two m edia;
2% B a c t o - a g a r o r 0.8% G e l r i t e w e r e c o m p a r e d i n e a c h r e c i p e .
G e l r i t e was used,
fo llo w e d
m a g n e s iu m
When
I g CaClg°2 HgO was d i s s o l v e d i n one l i t e r o f w a t e r
by 8 g G e l r i t e a n d f i n a l l y
th e
o th e r in g re d ie n ts .
c o n t e n t o f J o h n s o n ’ s m edium w a s a l s o
MgSOij'7 HgO p e r l i t e r
fo r
use
of G e lrite .
The
in c re a s e d to I g
F in a lly ,
th e
pH w as
a d j u s t e d w i t h NaOH t o a p r e a u t o c l a v e pH o f 8.1 w h ic h 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 t o c l a v i n g .
Thermus X-Gal Medium
CaClg’2g0
0 .5 gm
MgSO4 -I H2 O
0 . 5 gm
i n I l i t e r o f w a te r .
Then add
G e l-rite
4 . 0 gm
f o llo w e d by
J o h n s o n 's S o l u t i o n I
5 .0 ml
. ■I I
5 .0 ml
Casamino a c i d s
5.Q gm
1.0 ml each
V ita m in s
(b io tin , lip o ic
a c i d , B1 g , p a r a
am inobenzoic a c i d )
A d ju s t pH t o 8 .1
( a b o u t 0 . 7 . ml 4M NaOH) .
A u to c la v e b e f o r e a d d in g t h e f o l l o w i n g :
L a c to s e (10%)
10.0 ml
X-Gal
2 0 .0 mg ( d i s s o l v e d i n d i m e t h y l
formam ide)
IPTG
6 0 .0 mg
: 2
7
2.
In c u b a tio n .
Covered s h a k in g w a t e r b a t h s 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 s w ere
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 .
s o lid
m e d ia w e re in c u b a t e d a t :
C u l t u r e s on
a ) 58 C i n a H o t p a c k h u m i d 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 a n d 3 - 4 d a y s f o r T herm us
w e r e r e q u i r e d f o r g r o w t h ; 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 b a g s
w i t h a b la n k 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 t e r p la c e d
i n bag b o t t o m t o p r e v e n t d e h y d r a t i o n ;
■
g ro w th o f Thermus.
3.
2 -3 d a y s w e r e r e q u i r e d f o r
B uffers
B a c t e r i a l w ash es and 1 0 -f o l d 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 w e re
made i n a p p r o p r i a t e l i q u i d medium o r t h e f o l l o w i n g p h o s p h a te b u f f e r s :
Thermus
NaCl
8 .5 gm
K2HPO1,
5 *7 gm
KH2 POlt
gm
NaCl
LO
CO
b)
B a c illu s
^=T
CO
a)
gm
pH = 7 .2
pH = 7 .8
K2HPO1,
M icroscopy
A ll l i g h t m i c r o s c o p i c o b s e r v a t i o n s and p h o to g r a p h s w e re made w ith
a W ild
(H eerb ru g )
a t ta c h m e n t.
M-20 m i c r o s c o p e
fitte d
w ith
a 3.5
mm c a m e r a
Kodak h ig h c o n t r a s t copy f i l m was used and d e v elo p ed w i t h
Kodak d e v e l o p e r D-19.
E l e c t r o n m ic r o s c o p e e x a m i n a t i o n s r e q u i r e d b a c t e r i a grow n i n
s t a n d a r d g r o w t h m edium t o be w a s h e d i n p h o s p h a t e b u f f e r .
E ith e r a
28
drop of 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 l o n y fr o m s o l i d m edium
su sp e n d ed i n w a t e r was p la c e d on a 300 mesh copper g r i d c o a te d w i t h 0.2%
Form var i n c h lo ro fo rm .
P la tin u m
shadow c a s t i n g w a s do n e w i t h p la tin u m - p a la d i u m w ire .
O b s e r v a tio n s w e re made w i t h a Z e i s s (EM 952) e l e c t r o n m ic r o s c o p e and
a s s o c i a t e d cam era.
DNA Damaging T r e a tm e n ts
1.
E xposure t o UV.
The S y l v a n i a GTE 8 w a t t g e r m i c i d 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 e n e r a t e d a dosage o f 190 u w a tt s / c m ^ a s
m easu red by a S p e c t r o l i n e DRC-IOOx d i g i t a l r a d io m e te r .
A.
C u l t u r e s on s o l i d m e d i a w e r e i r r a d i a t e d a c c o r d i n g t o
G re e n b e rg (1 967).
B. L iq u id C u l t u r e s .
p e r ml,
At a d e n s i t y o f a p p r o x im a te ly IO^ c e l l s
two ml a l i q u o t s o f w ashed c e l l s su sp e n d ed i n t h e a p p r o p r i a t e
p h o s p h a t e b u f f e r w e r e 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 t h UV a s above.
The c e l l s c o u ld th e n be d i l u t e d and p l a t e d
d i r e c t l y o r u sed f o r a p h o t o r e a c t i v a t i o n t r e a t m e n t .
2.
E x p o s u r e t o M i t o m y c i n C (MG).
T est of s u s c e p t i b i l i t y
of
Thermus t o MC was a c h ie v e d by p l a t i n g a s u s p e n s io n o f t h e organism on
s o l i d medium and i n c u b a t i n g a t th e a p p r o p r i a t e t e m p e r a t u r e (58 C o r 70
C) f o r a p p r o x im a te ly 24 h o u r s b e f o r e 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
u p o n w h i c h w e r e 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 ru g .
A fte r
a n o t h e r one to two days o f grow th' t h e zone o f i n h i b i t i o n s u r r o u n d in g
th e d i s c was m easured.
3.
R e p a ir a s s a y s .
29
D ire c t
L ig h t
R ep air.
P h o to re a c tiv a tio n
e x p e rim e n ts
w ere
v a r i a t i o n s i n th e p r o t o c o l f o r t e a c h i n g l a b o r a t o r y e x p e r i m e n t s w i t h E.
c o l i ( S e e le y and Van Demark, 1981).
C e l l s i n l o g p h a s e w e re 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 o rg an ism th e n r e s u s p e n d e d 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 e re p la c e d i n s t e r i l e d i s p o s ­
a b l e 60 x 15 P e t r i
d is h e s and i r r a d i a t e d f o r v a r y in g tim e s .
The
p l a t e s w e re i m m e d i a te l y p l a c e d i n p l a s t i c b i n s and c o v e re d w i t h 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 r h e a d l i g h t s .
c e l l s w ere t r a n s f e r r e d
t e m p e r a t u r e b a th t o
re p a ir.
to
ste rile
As s o o n a s p o s s i b l e t h e
t u b e s and p l a c e d i n a c o n s t a n t
p r e v e n t o v e r h e a t i n g from t h e l i g h t b u l b u sed f o r
The b u l b s u s e d w e r e 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 lu e 250 w a t t S y lv a n i a BCA p la c e d i n a p h o t o r e f l e c t o r shade s i x i n c h e s
above t h e tu b e s.
C o n tr o l s in c l u d e d :
N o n -irria te d
c e lls:
T re a tm e n t J.
1
C o n s ta n t te m p e r a t u r e b a t h ; d a r k ( f o i l - c o v e r e d )
2
C o n s ta n t t e m p e r a t u r e b a t h ; l i g h t
3
Room t e m p e r a t u r e b a t h ; d a r k
and i r r a d i a t e d c e l l s :
T re a tm e n t ±
4
C o n s ta n t t e m p e r a t u r e b a t h ; d a r k
5
Room t e m p e r a t u r e b a t h ; d a rk
6
C o n s ta n t te m p e r a t u r e b a t h ; l i g h t
P e r c e n t r e c o v e r y was c a l c u l a t e d by t h e f o l owing e q u a t io n :
reco v ery =
S u r v i v o r s ( T re a tm e n t #6)
T o t a l # (T re a tm e n t #1)
30
S u rv iv a l
D i l u t i o n s o f a p p r o p r i a t e c u l t u r e s w e re p l a t e d and t h 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 of tim e.
C o lo n y c o u n t i n g w as d o n e w i t h a New
B ru n sw ic k B i o t r a n I I a u to m a tic co lo n y c o u n te r.
S p ectro p h o to m etric
d e t e r m i n a t i o n o f c e l l d e n s i t y i n b r o t h was made u s i n g LKB u l t r o s p e c
4050 a t a w a v e l e n g t h o f 550 mu.
S u r v i v a l r a t e w as c a l c u l a t e d a s
number o f c e l l s s u r v i v i n 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 c o u n t) d i v i d e d
by number o f c e l l s i r r a d i a t e d ( p l a t e c o u n t, no i r r a d i a t i o n ) .
31
RESULTS
A.
The Organisms o f t h e Study
I.
I d e n ti f ic a tio n o f B a c illu s ste a ro th e rm o n h ilu s
I n i t i a l o b se rv a tio n s.
The c u l t u r e o b t a i n e d f r o m t h e M o n ta n a
S t a t e U n i v e r s i t y B i o c h e m i s t r y D ep artm en t and i n i t i a l l y
th o u g h t t o be
T h e rm u s d e v i a t e d f r o m n o r m a l T h erm u s b e h a v i o r i n g e n e r a t i o n t i m e ,
o p tim u m pH, and g r o w t h t e m p e r a t u r e .
No g r o w t h on m i n i m a l l a c t o s e
m edium 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 r o n .
was n o t c o n c l u s i v e a s c e l l s from a
Gram n e g a t i v e , e l o n g a t e d r o d s .
t h e o rg an ism
Gram s t a i n i n g
colony on s o l i d m e d ia ap p ea red a s
F u r t h e r i n v e s t i g a t i o n , how ever, showed
to be a s p o r e - f o r m e r w i t h f l a g e l l a ,
not fila m e n 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.
M orphology.
Gram s t a i n s o f f r e s h
t o be G r a m - v a r i a b le s h o r t r o d s ( P l a t e
c u l t u r e s showed t h e b a c te r iu m
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 r o d u c e d many s p o r e s w h i c h , w hen s t a i n e d w i t h
M a l a c h i t e Green, w ere e a s i l y d e t e c t e d un d er th e l i g h t m ic ro s c o p e .
The
s p o r e s w e re e l i p t i c a l i n shape and l o c a t e d i n t h e t e r m i n a l r e g i o n o f
th e
c e ll.
Shadow
c a ste d
sp e c im e n s o b se rv e d
m ic ro sc o p e in d i c a te d e l i p t i c a l
under
th e
e le c tro n
t e r m i n a l s p o r e s ( P l a t e 2) and f l a g e l l a
( P l a t e 3).
P h y sio lo g ic a l C h a r a c te r is tic s .
Optimum pH f o r g ro w th on b a s a l
s a l t s p l u s T ry p to n e and y e a s t e x t r a c t i s 7.5.
g r o w t h i s 55 C.
Optimum t e m p e r a t u r e f o r
G e n e ra tio n tim e (d o u b lin g tim e ) under th e s e co n d i-
32
P la te I.
B a c i l l u s ( G ra m s ta in ) 50X
P la te 2.
B a c illu s te rm in a l e l l i p t i c a l spore.
E l e c t r o n m ic r o g r a p h 3 1 ,OOOX
34
P l a t e 3• B a c i l l u s F la g e llu m .
E l e c t r o n m ic r o g r a p h 186 ,OOOX
35
n i t r a t e and n i t r i t e
t e s t s w e re made,
t i o n s i s a b o u t 15 m in u te s .
gave
a
p o sitiv e
m o rp h o lo g ic a l
E.
To f u r t h e r c h a r a c t e r i z e t h e B a c i l l u s ^ c o l i
re a c tio n
stu d ie s,
b o th o f which w ere n e g a tiv e .
as
and
a
c o n tro l.
th ro u g h
use
F ro m
of
th e se
B ergevt s
te sts,
M anual
of
D e t e r m i n a t i v e B a c t e r i o l o g y ( 1957) a n d The G enus B a c i l l u s ( G ordon,
I 973) i t w as d e t e r m i n e d t h a t t h e o r g a n i s m o f s t u d y i s a s t r a i n o f B.
ste a ro th e rm o p h ilu s.
T ab le I p r e s e n t s
c o m p a r a tiv e c h a r a c t e r i s t i c s
of
Thermus and Bi. s t e a r o t h e r m o p h i l u s .
C u ltu re of B a c illu 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 m edium (pH 7 .5 ) w e r e u s e d r o u t i n e l y f o r Bi. s t e a r o t h e r m o ­
p h ilu s.
N u t r i e n t a g a r gave co m p a ra b le r e s u l t s i n one e x p e r im e n t and
would be a s i m p l e r p r e p a r a t i o n .
I t was found t h a t t h e m o s t r e p r o d u c ­
i b l e r e s u l t s w ere o b t a i n e d when t h e p l a t e s w ere d r i e d w i t h 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 ­
t o r y bench a t room t e m p e r a t u r e f o r s e v e r a l days b e f o r e use.
C h a r a c t e r i s t i c s o f Thermus.
M orphology. . Thermus was o r i g i n a l l y
its
o p tim u m t e m p e r a t u r e o f 70 C.
f !la m e n ta tio n decreases.
d e sc rip tio n ,
d esc rib e d a s fila m e n to u s a t
H o w e v e r,
a t lo w e r te m p e ra tu re s
E l e c t r o n m i c r o g r a p h s ( p l a t e 4) c o n firm
E l e c t r o n m ic ro s c o p y
also
th is 1
showed a l a c k o f f l a g e l l a and
s p o r e s and c o n f ir m e d t a n g l i n g and f ! l a m e n t a t i o n a t h ig h te m p e r a t u r e s .
C u l t u r e s o f Thermus.
P o i n t s o f c o n s i d e r a t i o n i n th e developm ent
o f s o l i d medium f o r g ro w th o f Thermus a t 70 C i n c l u d e d :
a)
R e q u ire m e n t f o r a f i r m ,
• 7 0 C.
n o n - m e l t i n g medium f o r i n c u b a t i o n a t
36
37
T a b le
I.
Sum m ary 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 r m o o h i l u s and
Thermus T2.
B a c illu s
G r a m - s ta i n
S p o res
F la g e lla
P r e s e n c e o f f u n c t i o n a l l a c o p e ro n
F i l a m e n t a t i o n a t 58 C
F i l a m e n t a t i o n a t 70 C
Optimum Growth T em p eratu re
Optimum Growth pH
G e n e r a t i o n Time
'
Thermus
V
+
+
—
+
+
70 C
7 .8
I h o u r (70 C)
2 .5 h o u r s (55 C)
-
55 C
7 .5
15 min
V - V ariab le
+ = P resence
- = Absence
b)
P re v e n tio n
of
d ry in g
at
th e
h ig h
te m p e ra tu re
is
i n d i s p e n s a b l e , y e t m edia t h a t i s t o o m o i s t a l l o w s c o l o n i e s t o
ru n t o g e t h e r ,
c)
p r e v e n t i n g a c c u r a t e co u n tin g .
C le a r medium t o a llo w u se o f t h e a u t o m a t i c co lo n y c o u n t e r was
d e sira b le .
d)
G e lrite
an d
th e
re q u ire d f o r i t s
h ig h
c o n c e n tra tio n s
of
CaClg
and MgSOjj
p o l y m e r i z a t i o n p r e c i p i t a t e s h ig h s a l t from
a:ny medium i n w hich b o th 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 g row th o f Thermus.
f)
M edia c o n t a i n i n g G e l r i t e
s o l i d i f i e s a t a h i g h e r te m p e r a t u r e
t h a n a g a r and m u st be p oured above 60 C.
c a n n o t be m e lte d a g a in .
Once s o l i d i f i e d , i t
38
The medium o f c h o ic e f o r g ro w th o f Thermus on s o l i d s u p p o r t a t 70
C became J o h n s o n 's w i t h G e l r i t e a s th e s a l t c o n t e n 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 a n d y e t low en o u g h t o a l l o w
th e use of 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 C aC l2 a n d MgS Cfy r e q u i r e d f o r u s e o f Ge l r i t e .
In
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 m edium h ad t o be p o u r e d a t 65 C,
G e l r i t e p ro 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 llo w e d u se o f th e
a u t o m a t i c colony c o u n te r .
B e t t e r g ro w th (co lo n y number) was o b s e rv e d
w i t h G e l r i t e a s a g e l l i n g a g e n t by L in and C asida (1984).
The r e s u l t s
o f t h i s s tu d y d id n o t c o r r o b o r a t e t h e L in and C a s id a r e s u l t s a s to an
i n c r e a s e i n colony number i n c r e a s e .
G r o w th o f T h e r m u s i n b r o t h a t 70 C c o u l d 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 lr ic h 's
b u t o n ly
in
shaken
c u ltu re .
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 c o u n ts w e re o n ly s u c c e s s f u l when d i l u e n t
w a s a t pH 7.8.
A ty p ic a l
g ro w th
cu rv e
d e te rm in e d s p e c tr o p h o to m e tr ic a lly
i n d i c a t e d a g e n e r a t i o n tim e o f a p p r o x i m a t e l y 150 m i n u 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 t o o b s e r v a t i o n s o f U l r i c h ( 1 9 7 1 ) .
tu rb id ity
or a b s o rb a n c e and p l a t e
H o w e v er,
c o u n t s sh o w e d 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 se p e rh 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 re v e n tio n of c e l l d iv i s i o n or ta n g lin g .
R e p ro d u c ib le r e s u l t s i n
e x p e r i m 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 l y w i t h O.D. <.
0.500 and w i t h 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 i o n s (pH, medium, p l a t i n g ) .
With g e n e r a t i o n t i m e o f one hour and a lo n g l a g p h a s e f o r Thermus
an u n r e a s o n a b le tim e w as r e q u i r e d to a c h ie v e lo g p h a se a t a c e l l
d e n sity
a p p ro p ria te
fo r
e x p e r i m e n t a l w o rk .
T h is d i f f i c u l t y
w as
39
overcom e by i n o c u l a t i n g b r o th from a s i n g l e colony and a l l o w i n g t h e
n e w ly
in o c u la te d
c u ltu re
to
grow
o v e rn ig h t
at
th 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 i n g (d a y # 1 ).
I f g r o w t h h ad
o c c u r r e d , a d i l u t i o n w a s m ade i n g r o w t h m e d iu m , t h e c u l t u r e g ro w n
a g a in a t th e a p p r o p r ia te te m p e ra tu re w ith o p ti c a l d e n s ity
m o n ito re d ,
and th e c u l t u r e rem oved d u r in g l o g p hase t o t h e r e f r i g e r a t o r (day #2).
The
c u ltu re
co u ld
th e n
be
ta k en
from
th e
co ld
e x p e r i m e n t (d ay #3)> d i l u t e d a n d i n c u b a t e d a g a i n .
th e
day
of
th e
W ith v e r y l i t t l e
l a g p h ase t h e c u l t u r e q u i c k l y r e - e n t e r e d l o g phase.
B.
Q u a n t i t a t i v e R e s u 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 ic ro o rg a n is m
as th e l o s s of i t s a b i l i t y
i s d e fin e d f o r th e se ex p erim en ts
t o i n i t i a t e a colony.
W ith in a p o p u l a t i o n
t h e i n d i v i d u a l s t h a t h av e e x p e r i e n c e d enough c h e m ic a l e v e n t s t o r e s u l t
i n u n r e p a i r e d changes a r e i n a c t i v a t e d or " k i l l e d " .
th e
d am a g in g
e v e n ts
occur
ran d o m ly
and
I t i s assum ed t h a t
in d e p e n d e n tly
in th e
s u s c e p t i b l e g ro u p s w i t h th e p r o b a b i l i t y o f such an e v e n t p r o p o r t i o n a l
to th e dose of r a d i a t i o n per u n i t tim e .
T arget s iz e i s g e n e ra lly
i n d i c a t e d by t h e s l o p e o f t h e k i l l i n g c u r v e on s e m i - l o g p a p e r .
lo n g a s t h e dose r a t e i s
As
c o n s t a n t o r g a n i s m s w i t h t h e s am e t a r g e t
(g e n o m e ) s i z e can be c o m p a r e d a n d t h e r e l a t i o n s h i p b e t w e e n d o s e a n d
k i l l i n g a n a ly z e d .
The r e f e r e n c e
Ei. c o l i
r e s i s t a n t w i l d ty p e s t r a i n .
curve
w as
o b ta in e d
under
s tra in
( AB1157)
of
F ig u re
3 is
a UV
Data u s e d f o r g e n e r a t i o n o f th e r e f e r e n c e
id e n tic a l
c o n d itio n s
u sin g
th e
sam e
f a c i l i t i e s a s t h e Thermus and B a c i l l u s s t e a r o t h e r m o o h i l u s i r r a d i a t i o n
40
NUMBER OF SURVIVORS/TOTAL CELLS PLATED
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 r v i v 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 i m e n t s i n t h e l a b o r a t o r y o f G u y ly n W a r r e n , MStL
F ig u re B a lso
shows t h e s u r v i v a l c u r v e s g e n e r a t e d 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 t o 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 c u r v e w i t h e x p o n e n t i a l decay a s does Thermus grown a t
55 C.
H o w e v er, T h e rm u s g r o w n 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 rv e w ith a s h o u ld e r w ith th e
e x p o n e n tia l decay i n d i c a t i v e of k i l l i n g
e x p o s u r e t o UV.
o n l y a f t e r 30 s e c o n d s o f
F u r t h e r m o r e , t h e e x p e r i m e n t s i n w h i c h T h e rm u s w a s
grown a t one t e m p e r a t u r e and s w i t c h e d t o t h e o t h e r t e m p e r a t u r e p r i o 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 th e s w i t c h fro m lo w e r t o h i g h e r te m p e ra ­
t u r e i n d u c e d t h e m e c h a n is m 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 r v e ,
t h a t t h e i n d u c t i o n was c o m p le te w i t h i n two h o u r s a f t e r th e t e m p e r a t u r e
s w i t c h ( F i g u r e 4 ), and t h a t t h i s mechanism re m a in e d a c t i v e th ro u g h th e
f o u r h o u r s m o n i to r e d and shown h e r e .
(D ata n o t p r e s e n t e d d e m o n s tr a te d
t h a t t h e a c q u i r e d 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 l o g t o s t a t i o n ­
ary
phase
mechanism
o v e rn ig h t
c u ltu re
a p p e a re d a c t i v e
f r o m 70 C t o 55 C.)
ex perim ents,
for
grow n a t
at le a st
70
C.
A d d itio n a lly ,
th r e e hours a f t e r
th e
sw itch in g
G raphs p r e s e n t an a v e ra g e o f t h r e e r e p l i c a t i v e
each e x p e r im e n t i n c l u d i n g t h r e e p l a t e c o u n t s p e r p o in t.
E r r o r b a r s r e p r e s e n t h ig h and low v a l u e s a t each p o in t .
C.
E xposure t o UV and MG.
One e x p l a n a t i o n o f t h e m u l t i p l e - h i t s u r v i v a l c u r v e o f Thermus T2
co u ld
be i n d u c t i o n
o f a DNA r e p a i r
sy stem .
T h is w as
te ste d
by
e x p o s in g d i l u t i o n s o f Thermus on p l a t e s t o UV and t h e n i n c u b a t i n g t h e
42
NUMBER OF SURVIVORS/TOTAL CELLS PLATED
BACILLUS 55°C
o.oo I -
0 .0 0 0 I
o .o o o o i
F ig u re 4.
20
30
40
50
UV IRRADIATION (SECONDS)
UV S u r v i v a l : Thermus 1 , 2 , 3
55 C t o 70 C.
h o u rs a f t e r t r a n s f e r from
43
p l a t e s w i t h M ito m y c in C i n d i s c s and o b s e r v in g , a f t e r c o l o n i e s had
grown up,
th e d i a m e t e r o f th e k i l l zone s u r r o u n d in g th e d i s c s .
e x p o s u r e in d u c e d a DNA r e p a i r s y ste m ,
c i n C would be e x p e c te d .
A lte rn a te ly ,
I f UV
i n c r e a s e d r e s i s t a n c e to Mitomy­
a l o g phase c u l t u r e o f Thermus
was exposed t o M itom ycin C f o r a s h o r t d e te r m in e d l e n g t h o f tim e ,
m utag en w ashed o u t ,
d o s e s o f UV.
c e l l s p la te d ,
th e
and th e n i r r a d i a t e d w i t h i n c r e a s i n g
The r e s u l t i n g s u r v i v a l c u r v e w o u ld i n d i c a t e w h e t h e r
p r e t r e a t m e n t w i t h M itom ycin C i n c r e a s e d r e s i s t a n c e t o UV.
I n one e x p e r im e n t w i t h MG d i s c s t h e r e was n o t a c o n c l u s i v e zone
siz e d iffe re n c e .
In one e x p e r im e n t w ith t r i p l i c a t e
s a m p l e s o f MG
p r e t r e a t m 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 l o p e d a
s h o u ld e r s i m i l a r t o t h e 70 C c u l t u r e s ( F ig u r e 5).
D.
Q u a n t i t a t i v e R e s u l t s o f P h o t o r e a c t i v a t i o n and D i r e c t L i g h t R e p a ir
F o llo w in g UV T r e a tm e n t.
T a b le 3 p r e s e n t s t h e r e s u l t s o f p h o t o r e a c t i v a t i o n e x p e r i m e n t s on
a w ild
ty p e
EL. c o l i
and
B a c illu s
ste a ro th e rm o p h ilu s
c o n d i t i o n s d e s c r i b e d i n t h e Methods s e c t i o n o f t h i s t h e s i s .
under
th e
Thermus 55o-70°C
NUMBER OF SURVIVORS/TOTAL CELLS PLATED
1 hour
2 hour
3 hour
O.OOI
0 .0 0 0 1
0 .0 0 0 0 I
F ig u re 5.
20
30
40
50
UV IRRADIATION (SECONDS)
M itomycin C + UV S u r v i v a l :
Thermus 55 C.
P h o to re a c tiv a tio n :
and Ej . c o l i
T rea tm e n t #
# S u r v iv o r s
B a c illu s ste a ro th e rm o o h ilu s
# S u r v i v o r s / # T o ta l*
P h o to re a c tiv a tio n :
1.1
1 .2
9 .7
4 .7
5 .3
9 .4
I
2
3
4
5
6
X
107
X
ioJ«
X
X
X
X
P h o to re a c tiv a tio n :
E.
X
X
X
X
IO8
IO7 *
IO8
IO7
IO7
IO7
0 .7
0.71
0 .2 4
0 .2 4
0.6 7 9
E. c o l i
97%
CU
6
X
X
( ± 3%)
+1^
1 .4
9 .8
1 .0
3 .3
3 .3
9 .5
I
2
3
4
5
B a c illu s
I .00
0.88
0 .0 4
0 .0 5
0 .8 5
IO6
IO5
10->
IO6
% P h o to re a c tiv a tio n
VO
Ov
T ab le 2.
c o l i . a f t e r 30 s e c o n d s o f i r r a d i a t i o n and s u b s e q u e n t s u r v i v a l
o f 2% o f th e p o p u l a t i o n w i t h no p h o t o r e a c t i v a t i o n t r e a t m e n t show 97%
r e a c t i v a t i o n a f t e r 30 m i n u t e s o f e x p o s u r e t o a w h i t e i n c a n d e s c e n t
p h o to g r a p h e r 's b u lb p la c e d s i x i n c h e s from th e c u l t u r e .
B a c illu s w ith
20 s e c o n d s o f i r r a d i a t i o n and 5% s u r v i v a l of th e n o n p h o t o r e a c t i v a t e d
p o p u l a t i o n d e m o n s t r a t e s a 96% r e a c t i v a t i o n i n r e s p o n s e t o e x p o s u re to
a p h o t o g r a p h e r 's blu e l i g h t
bulb.
The d a t a p r e s e n te d i n each case i s
t h e a v e ra g e o f two e x p e r i m e n t s w ith s a m p le s ta k e n 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 U V -dosed T herm us was a l s o a t t e m p t e d (T ab le
46
3).
D isa p p o in tin g ly ,
d e sp ite
som e
v a ria tio n
of
UV d o s a g e
and
te m p e r a t u r e d u r i n g p h o t o r e a c t i v a t i o n a s w e l l a s use o f l i g h t c o n d i t i o n s
th a t
w ere
su ccessfu l
ste a ro th e rm o o h ilu s.
in
p h o to re a c tiv a tin g
p h o to re a c tiv a tio n
of
Ei
c o li
and
B.
T herm us
c o u ld
not
be
d e m o n s tr a te d .
T ab le 3 .
C o n d it io n s o f A ttem pted D i r e c t l i g h t R e p a ir
Irra d ia tio n
(s e c o n d s )
T em p e ra tu re
L i g h t ( tim e i n
m in u te s )
S u c c e s s f u l R e p a ir
E. c o l i
B a c illu s
Thermus
30
20
20, 30
40 G
I c e , 37 C, 50 C ,
Blue ( 1 5 ,3 0 )
B lue ( 1 5 ,3 0 )
+
-
Ice
-
White ( 3 0 ,6 0 )
+
.
47
DISCUSSION
T h e r m a l l y i n j u r e d c e l l s o f a n y o r g a n i s m may d i e o r r e p a i r t h e
damage d ep en d in g on t h e e x t e n t o f damage and e n v i r o n m e n ta l c o n d i t i o n s .
DNA-damage can be r e p a i r e d ,
t h e mechanism c a l l e d upon d ep ending upon
t h e ty p e and e x t e n t o f damage and t h e e n v i r o n m e n ta l c o n d i t i o n s a s w e l l
a s th e c a p a b i l i t i e s o f th e o rg an ism .
The r e l a t i v e r o l e s o f s t r u c t u r a l
ch an g e s and i n d u c i b l e o r n o n - i n d u c i b l e b io c h e m ic a l ch a n g e s a r e b e in g
e l u c i d a t e d i n t h e h i g h l y s t u d i e d m e s o p h i le s .
I exam ined t h e r m o p h i l i c
B a c i l l u s a n d T h e r m u s u s i n g v a r i a t i o n s o n 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 ith t h e i r m e so p h ilic c o u n te rp a rts .
Once t h e t e c h n i c a l
a s p e c t was a f f i r m a t i v e l y e s t a b l i s h e d f o r t h e th e r m o p h i l e s ,
an a t t e m p t
was made t o compare th e d a t a o b t a i n e d w i t h t h a t p u b l i s h e d f o r therm o­
p h i l e s and c o m p a r a ti v e m e s o p h ile s .
The
c h a lle n g e
of
o b ta in in g
re p ro d u c ib le
f i l a m e n t o u s Thermus was m et p r i m a r i l y
a p p ro p ria te
m edium
c o n d itio n s
in c lu d in g :
m a n ip u la tio n s,
fo r
p la te
a)
w ith
th e
th ro u g h th e d e v e lo p m e n t o f an
c o u n ts
tim e
re su lts
and th e p r e c i s e
re q u ire d
fo r
c o n tro l of
e x p e rim e n ta l
b) optimum pH and t e m p e r a t u r e o f d i l u t i o n and p l a t i n g
medium, and c) o f c r i t i c a l im p o r ta n c e ,
c e l l d e n s it y .
w i t h CXD^q above 0.5 p roduced h i g h l y e r r a t i c r e s u l t s .
Use o f c u l t u r e s
The d e g re e o f
f ! l a m e n t a t i o n and t a n g l i n g s e e n i n t h e s e c u l t u r e s c o u ld i n t e r f e r e w i t h
any p l a t e c o u n t a s s a y which r e q u i r e d dev elo p m en t o f c l o n e s from s i n g l e
48
c e lls.
C o m p a r i s o n s o f t h e r m o p h i l i c a n d m e s o p h i l i c membrane p r o t e i n s ,
rib o s o m e s , and n u c l e i c a c i d s have shown th e b io c h e m ic a l m echanism s o f
o r g a n i s m s g r o w i n g i n t h e tw o 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 r m o s t a b i l i t y t o be i n h e r e n t i n m o l e c u l a r s t r u c t u r a l d i f f e r e n c e s i n
t h e b io c h e m ic a l com ponents o f t h e c e l l s .
each a r e a c t i v e on t h e DNA o f th e o t h e r .
The r e s t r i c t i o n enzymes o f
DNA p o ly m e ra s e i s o l a t e d from
Thermus and com pared t o t h e JS1. c o l i p o ly m e r a s e i n d i c a t e s t h e s u b u n i t s
to
be
sim ila r
except
T h e r m u s DNA c l o n e d
fo r
in to
th e rm o sta b ility
Ejs. c o l i
p roduces
(Tanaka, 1981; N a g a h a ri e t a l . , 1980).
( C h ie n e t
a l.,
th e rm o sta b le
1976).
p ro te in s
I t i s l i k e l y t h a t th e sp e c tru m
o f DNA r e p a i r m echanism s p r e s e n t i n t h e r m o p h i l e s w i l l a l s o be s i m i l a r
t o t h a t o b s e r v e d among m e s o p h ile s .
We c o u ld e x p e c t t h a t p h o t o r e a c t i ­
v a t i o n c o u l d be q u a n t i t a t e d i n t h e t h e r m o p h i l e s by r e c o v e r y fro m UV
a f t e r v i s i b l e l i g h t tre a tm e n t.
C e r t a i n l y i f a n i n d u c i b l e S O S - ty p e
r e p a i r sy ste m w ere p r e s e n t i n a th e r m o p h i l e , we co u ld e x p e c t a " m u l t i h i t " UV s u r v i v a l curve.
re p a ir
u sin g
ag e n ts
A p o s i t i v e i n d i c a t i o n o f SO S-type i n d u c i b l e
know n t o
in d u c e
th e
heat
shock re sp o n se in
m e s o p h i l e s a t t h e h i g h e r o f two t e m p e r a t u r e s w i t h i n t h e t h e r m o p h i l i c
ran g e w ould a l s o d e m o n s t r a t e th e p o s s i b i l i t y f o r a h e a t shock re s p o n s e
sim ila 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
th e r m o p h il e .
DNA r e p a i r ,
in
th e
D e m o n s tr a tio n o f t h e p r e s e n c e o f h e a t sh o ck p r o t e i n s i n
t h e r m o p h i l e s w ould a l s o d e m o n s t r a te th e c a p a b i l i t y .
S t u 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 ­
gated,
o n ly two showed good p h o t o r e a c t i v a t i o n ,
four
showed m o d e ra te
49
p h o t o r e a c t i v a t i o n and t h e o t h e r s w e re n o t p h o t o r e a c t i v a b l e .
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 a n d
u sed i n t h e s e e x p e r i m e n t s was shown t o be p h o t o r e a c t i v a b l e .
showed t h a t th e s t r a i n can s u r v i v e a t 70 C.
We a l s o
D i r e c t l i g h t r e p a i r c o u ld
n o t be d e m 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 T h e r m u s w h i l e t h e
e x p e r i m e n t s w ere s u c c e s s f u l w i t h JEL. c o l i and B a c i l l u s
ste a ro th e rm o -
p h ilu s.
T h e r e i s t h e q u e s t i o n o f w h e t h e r U V - in d u c e d p h o t o p r o d u c t s a r e
s t a b l e a t 70 C.
A p p a r e n 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 w hich
can be r e p a i r e d by p h o t o r e a c t i v a t i o n ,
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
b u t I have a s y e t ,
a t 70 C.
no in f o r m a ­
A p o s itiv e r e s u l t w ith
p h o t o r e a c t i v a t i o n i n T h e r m u s w o u ld h a v e c o n f i r m e d t h e p r e s e n c e o f
d im e r s a s w e l l as d e m o n s t r a te d r e p a i r
by t h e i r d is a p p e a r a n c e .
W hile
som e r e s e a r c h e r s c o n s i d e r UV dam age t o DNA t o be i n c l u d e d i n o n e
c a t e g o r y ( Hanawa l t e t a l . , 1979), o t h e r s s tu d y more e x t e n s i v e fo rm s o f
s tru c tu ra l
d am age
(B rash
W hatever th e e f f e c t i n
and B a s e l t i n e ,
198 2 ;
H a se ltin e ,
T h e r m u s f UV d o e s " k i l l " t h e
s i m i l a r t o t h a t of o t h e r b a c t e r i a .
1 983) •
c e l l in doses
I t seem s v a l u a b l e t o c o n tin u e t h e
search f o r a p p ro p ria te co n d itio n s fo 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.
C om parison o f Thermus and JL_ c o l i s u r v i v a l a f t e r e x p o s u r e t o UV
o r th e c r o s s - l i n k i n g a g e n t ,
M itom ycin C, o r M itom ycin C co u p led w i t h
i r r a d i a t i o n showed t h a t d o s a g e s r e q u i r e d f o r " k i l l " w e re s i m i l a r f o r
both o rg a n is m s.
When a s u r v i v a l o r k i l l i n g c u rv e i s
a s tra ig h t lin e
p a s s in g th ro u g h th e o r ig i n , i t i s c a l le d a " s i n g l e - h i t " cu rv e w ith a
50
s in g le event r e s p o n s ib le f o r th e d e s tr u c t io n of th e v i a b i l i t y of th e
o rg a n ism .
" M u l t i p l e - h i t " c u rv e s have a s h o u ld e r n ear th e o r i g i n
b e f o r e b e c o m in g 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 u m u l a t e i n a
v ia b le c e l l (u n it) b efore i t i s in a c tiv a te d .
of ev en ts re q u ire d f o r in a c tiv a tio n ,
(To d e t e r m i n e th e number
th e s t r a i g h t p a r t of th e su rv iv a l
cu rv e i s e x t r a p o l a t e d back to m eet th e o r d i n a t e a x i s .)
The s l o p e o f
t h 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 c u rv e h a s t h e same m eaning a s f o r
a s i n g l e - h i t c u rv e (D ulbecco,
F ifty -fiv e
C B a c illu s
" s in g le -h it" s u rv iv a l
" m u ltip le -h it"
ty p e
in
1980).
a n d 55 C T h e r m u s e x h i b i t e d
curve
w h ile
th e se
70
C T h e r mu s
ex p erim en ts.
a ty p ic a l
d isp la y e d
th e
The 70 C T h e rm u s UV
s u r v i v a l c u r v e was fo u n d to be s i m i l a r i n shape t o t h a t o f w i l d ty p e
E.
c o l i ( AB1157) and t y p i c a l o f i n d u c i b l e r e p a i r .
The " m u l t i p l e - h i t "
cu rv e shows a s h o u ld e r w hich d e c l i n e s w i t h UV k i l l a f t e r 30 seco n d s o f
tre a tm e n t.
However, when Thermus i s i n c u b a t e d a t 55 C a " s i n g l e - h i t "
s u r v iv a l curve i s g e n e ra te d ,
s u g g e s t i n g t h a t th e i n d i c a t e d i n d u c i b l e
r e p a i r i s n o t in d u c e d by UV a t t h e lo w e r t e m p e r a t u r e o r i s in d u c ed by
te m p e r a t u r e o n ly .
To f u r t h e r exam ine t h i s problem , Thermus was grown
a t each t e m p e r a t u r e ,
a t v a r i o u s tim e s .
s w i tc h e d t o t h e o t h e r c o n d i t i o n ,
and i r r a d i a t e d
The r e s u l t s of t h e s e e x p e r i m e n t s i n d i c a t e t h a t th e
mechanism 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 i s f u l l y in d u c ed by
tw o h o u r s
a fte r
a sw itc h
up t o
h ig h e r
te m p e ra tu re and
th a t
th e
mechanism r e m a in s a c t i v e a t t h a t t e m p e r a t u r e ( c o n f ir m e d by i r r a d i a t i n g
an o v e rn ig h t c u ltu r e ) .
I t w a s a l s o show n ( d a t a n o t p r e s e n t e d ) t h a t
t h e mechanism r e m a in s a c t i v e f o r a t l e a s t t h r e e h o u r s f o l l o w i n g t h e
51
s w i t c h f r o m 70 C t o 55 C.
S i n c e t h e T h e r m u s UV s u r v i v a l c u r v e i s
r e l a t e d t o th e t e m p e r a t u r e a t w hich th e org an ism i s g ro w in g , we have
q u e s t i o n s c o n c e r n i n 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 S O S - i n d u c i n g
a g e n ts
at
55
C and
70
C and
th e
re la tio n sh ip ,
if
any,
of
th e
f ! l a m e n t a t i o n a l s o s e e n a t 70 C w i t h t h i s o rg an ism .
M itom ycin C was used a s a p r e t r e a t m e n t f o l l o w e d by t h e s t a n d a r d
UV t r e a t m e n t o f both 55 C and 70 C c u l t u r e s t o p r o v id e c o n f i r m a t i o n o f
in d u c tio n
of
DNA r e p a i r .
H e re
th e
70 C T h erm u s
wm u l t i p i e - h i t ”
s u r v i v a l c u r v e r e m a in e d unchanged w h i l e th e 5 5 C Thermus s u r v i v a l c u rv e
i n d i c a t e s i n d u c i b i l i t y t o be p r e s e n t .
A p p a re n tly ,
th e i n d u c i n g s i g n a l
c a n n o t be g e n e r a t e d by UV damage un d er ou r c o n d i t i o n s a t 55 C w h i l e a
p o s i t i v e r e s u l t i s o b t a i n e d w i t h th e c r o s s - l i n k i n g mutagen.
N u c le o id
H o w e v e r,
stru c tu re
L o ssiu s e t
a l.
of
T herm os
has
not
(.1983) h a v e r e p o r t e d
yet
been
e x a m in e d .
d iffe re n c e s in
th e
s e d im e n ta tio n c o e f f i c i e n t s o f th e e n v e lo p e -fre e n u c le o id s of se 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 u v r A . u v rB , a n d r e c A
g e n e s when in d u c e d by M itom ycin C and p ro p o s e t h a t t h e s e s t r u c t u r e s
are re p a ir in te rm e d ia te s.
The e x a m i n a t i o n a n d 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 T h e r m u s m u t a n t s w i l l be a n e x c i t i n g a r e a o f
research,
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 of th e r e l a t i o n s h i p of
h e a t t o l e r a n c e and i n d u c i b l e DNA r e p a i r .
The c o m p le x s e t o f o p e r o 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
sh ift
to h ig h e r
te m p e ra tu re d e m o n stra te s th e p r in c i p le
T h e rm u s may i n d u c e DNA r e p a i r a n d p r o t e c t i v e
p r o te i n s i n resp o n se to heat.
by w h ic h
s t r u c t u r a l n u c le o id
Thermus grow s w e l l o v er t h e t e m p e r a t u r e
52
ra n g e s t u d i e d ,
p re s u m a b ly a d j u s t i n g p r o t e i n s y n t h e s i s and d e g r a d a t i o n
i n r e s p o n s e t o t e m p e r a t u r e change.
A lthough t h e t e m p e r a t u r e ra n g e i s
d i f f e r e n t f o r EU. e o l i and T herm usf t h e ch anges f o r each w i l l p ro b a b ly
be shown t o be a continuum o v er th e e n t i r e r a n g e a t w h ich th e o rg an ism
su rv iv e s,
i . e . , we can e x p e c t t h e p r o t e i n s a s s o c i a t e d w i t h th e DNA t o
change w i t h h e a t i n c r e a s e s .
p r o t e i n s i s unknown.
The f u n c t i o n a l s i g n i f i c a n c e o f h e a t shock
N u c le ic a c i d s t a b i l i t y ,
DNA r e p a i r ,
and d eg ra d a ­
t i o n o f d e l e t e r i o u s p r o t e i n s have been p o s t u l a t e d a s f u n c t i o n s .
For
T h e r m u s 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 i n 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 ig h t e m p e r a t u r e r e q u i r e s th e p r e s e n c e of a s s o c i a t e d p r o t e i n s
in d u c e d by h e a t a s w e l l a s t h e o t h e r s t a b i l i z i n g f a c t o r s s u g g e s te d i n
th e l i t e r a t u r e .
p o ssib le .
DNA r e p a i r a n d 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
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
T h e rm u s a t
tw o
te m p e ra tu re s
co u ld
be t h a t f o r
som e r e a s o n
th e
i n d u c i b l e r e p a i r system i s n o t i n d u c i b l e by UV a t t h e lo w e r te m p e ra ­
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 to th e shape of th e
curve.
P erhaps a b e t t e r e x p la n a tio n i s t h a t th e h ig h e r te m p e ra tu re
i t s e l f i n d u c e s a r e p a i r sy ste m c a p a b le o f 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 ,
m u t a t i o n s w h ich
in tric a te
co nfer
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
s e n sitiv ity
to
many m u t a g e n s .
How t h 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 l e c u l a r c o n t r o l i n
Io 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 e m a in s
cloudy.
I n t h a t o rg a n is m ,
ta tio n .
C e r t a i n l y te m p o r a r y f i l a m e n t a t i o n can be in d u c e d a lo n g w i t h
SOS t r a i t s ( W i t k i n , 1 9 7 6 ).
t h e r e i s m ore th a n one pathw ay t o f i l a m e n ­
I n T h e r m u s f i l a m e n t a t i o n w a s f o u n d w hen
53
t h e o rg an ism was grown a t 70 C; UV i n d u c t i o n o f t h e . r e p a i r system we
are
o b se rv in g
is
not re q u ire d .
If
fila m e n ta tio n
is
a su rv iv a l
mechanism a t h ig h t e m p e r a t u r e , t h e c e l l may have more t i m 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 ls o , 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 a m e n t o u s form may be a b l e t o u t i l i z e t h e lo w e r am ounts 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 t u d i e s o f c o n s t i t u t i v e and i n d u c i b l e r e p a i r , te m p e r a t u r e - i n d u c e d
f ila m e n ta tio n , d is c o v e rie s of th e s i n g l e s tr a n d b in d in g p r o t e i n and
t h e p r i n c i p l e u v r p r o t e i n s a s w e l l as r e g u l a t o r y mechanism o f th e r e c
A a n d l e x A g e n e p r o d u c t s o f SOS r e p a i r s y s t e m s , a n d t h e h e a t s h o c k
r e s p o n s e h a v e b e e n p r o d u c t i v e i n JjL1. c o l i b e c a u s e o f i s o l a t i o n a n d
s tu d y o f m u t a n ts d e m o n s t r a t i n g t h e s e c o o r d i n a t e l y in d u c e d 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 T h e rm u s i s e s s e n t i a l .
The s u i c i d e m ethod once em ployed f o r i s o l a t i n g a u x o t r o p h i c m u ta n ts o f
E. c o l i (D avis,
1948) h a s been em ployed by S an car and R u p e r t (1978) i n
t h e i s o l a t i o n o f p h o t o r e a c t i v a t i o n - and d a r k - r e p a i r - d e f i c i e n t m u t a n ts
from E1. c o l i and c o u ld p ro d u ce r e p a i r - d e f i c i e n t m u t a n t s from Thermus
f o r th e c o n t i n u a t i o n o f t h i s l i n e
o f s tu d y .
Too, a u x o t r o p i c m u t a n ts
c o u l d 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 an d t h e m u t a t i o n r a t e
o b serv ed as a r a t e of r e v e r s io n to p ro to tro p h y .
Dr. Emmet J o h n s o n
( p e r s o n a l co m m u n ica tio n ) h a s o b s e r v e d a v e r y high m u t a t i o n r a t e w i t h i n
t h e l a c o p e r o n o f Thermus a t 70 C ( n o t exam ined a t 55 C).
ti o n of th e r a te
c la rify
D ete rm in a ­
o f m u t a b i l i t y i n T h e r m u s a t 55 C a n d 70 C w o u ld
t h e q u e s t i o n o f in v o lv e m e n t o f e r r o r - p r o n e DNA r e p a i r i n th e
70 C T h e r m u s r e s u l t .
W a rn e r (1 9 8 3 ) 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 e m o n s t r a t e r e p a i r i n uracil-D N A g l y c o s y l a s e and a p u r i n i c
en d o n u clease a c t i v i t i e s ,
b o t h o f w h i c h a r e known t o h a v e r o l e s i n
h e a t-d a m a g e d DNA r e p a i r i n Et c o l i b u t t h a t t h e l e v e l s d e t e c t e d a r e no
g r e a t e r th a n t h a t fo u n d i n Et c o l i .
M u ta tio n i n th e Io n gene i s r e s p o n s i b l e f o r o v er p r o d u c tio n o f
c a p s u la 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 ll a s
d e fe c tiv e
for
b a c te rio p h a g e
I y so g e n iz a tio n
p la s m id s .
Lon m u t a n t s a r e a l s o d e f i c i e n t i n d e g r a d a t i o n o f a b n o rm a l
p o l y p e p t i d e s ( G o tte sm an and Z i p s e r ,
m u l t i p l e i n v iv o p h en o ty p es a re
d e f i c i e n t p ro p erty .
and i n h e r i t a n c e
1978) and i t i s
d e r i v e d from
of F
th o u g h t t h a t th e
th e ir
d e g ra d a tio n -
I t h a s been shown t h a t m u t a t i o n s i n I o n d e c r e a s e
t h e r a t e o f d e g r a d a t i o n o f a m u ta n t sigm a s u b u n i t o f RNA p o ly m e ra s e i n
v i v o ( G r o s s m a n e t a l , I 985) ( a n d 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
affect
th e
ra te
of
th e
sig m a
s y n t h e s i s .as w e l l ) .
T ra sle r
and
G o t t e s m a n (1 9 8 4 ) 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 r m a t i o n f o u n d i n I o 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 iti v e r e g u la to r of ca p su le s y n th e s is .
I t i s now known t h a t t h e I o n
gene p r o d u c t o f E s c h e r i c h i a c o l i i s a h e a t shock p r o t e i n (G off e t a l . ,
1984; P h i l l i p s e t a l . , 1 9 8 4 ).
The i n d u c i n g s i g n a l o f h e a t shock i n o t h e r o r g a n is m s i s unknown.
Why a l l th e phenomena a s s o c i a t e d w i t h S O S -re p a ir,
pro p h ag e i n d u c t i o n ,
and t h e h e a t shock r e s p o n s e can be p roduced by such d i f f e r e n t a g e n t s
a s UV i r r a d i a t i o n , e t h a n o l , o r t h y m i n e s t a r v a t i o n h a s n o t y e t b e e n
e x p l a in e d .
I n h i b i t i o n o f DNA s y n t h e s i s (Radman,
1975) and p r o d u c t i o n
o f low m o l e c u l a r w e ig h t p r o d u c ts o f DNA d e g r a d a t i o n ( P a r d e e , 1975) a r e
55
s u g g e ste d
e x p la n a tio n s.
That
know n in d u c in g f a c t o r 's
c a u sin g
r e l a x a t i o n o f DNA m o l e c u le s ( s u p e r h e l i c a l i n l i v i n g o r g a n is m s ) th ro u g h
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 dam age r e s u l t i n g i n s i n g l e
s t r a n d b re a k s (UV and M itom ycin C) o r i n h i b i t i o n o f DNA s y n t h e s i s and
p r e v e n t i o n o f l i g a t i o n o f s i n g l e s t r a n d b r e a k s c o u ld cau se 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 t o d i s s o c i a t e f r o m t h 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 c h a n g e ( 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 of th e r e p r e s s e d g en es h a s a ls o been s u g g e s te d as an
e x p l a n a t i o n (Luchhik,
1979).
The b i n d i n g 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 l y m e r a s e ) t o
p ro v id e f o r a r a p i d re s p o n s e to change i n e n v iro n m n e t a l c o n d itio n s .
By t h i s th e o r y s u p e r h e l i c i t y would c o n t r o l b in d in g o f t h e E2. c o l i l e 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
w ould p e r m i t t r a n s c r i p t i o n o f t h e SOS g e n e s ;
i n d u c e d i n t h e sam e f a s h i o n .
o t h e r f u n c t i o n s c o u ld be
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 c a u s e s 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 t e n t o f DNA w in d in g i s t e m p e r a t u r e - d e p e n d e n t and a 15 C i n c r e a s e i n
t e m p e r a t u r e ( h e a t shock) unw inds DNA one base p a i r p e r 200 base p a i r s
( T r a v e r s a n d M ace, 1982) ( w h ic h 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 t h 1 ,500 p o ly m e ra s e m o l e c u le s unw inding
t h e 15,000 b a s e p a i r genome).
i n JL c o l i .
T r a v e r s and Mace i n v e s t i g a t e d t h i s i d e a
T h e ir s t u d i e s showed t h a t i n h i b i t i o n o f t h e B s u b u n i t o f
DNA t o p o i s o m e r a s e I I i n d u c e s p r o t e i n s w hich 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 know n h s p s .
The h e a t s h o c k p h en o m e n o n 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 .
H o w e v er, t h e y c o n -
56
e l u d e d t h a t r e l a x a t i o n p e r s e i s u n l i k e l y a s a s i g n a l an d 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 ic h a f f e c t s t h e . a b i l i t y o f t h e
to p o iso m e r a s e t o s u p e r c o i l th e chromosome.
In c o n c lu s io n , r e s e a r c h h a s i n d i c a t e d th e b a s i s o f l i f e a t h ig h
te m p e ra tu re
to
be
c o m p le x .
E lu c id a tio n
th ro u g h
a
v a rie ty
of
i n t e g r a t e d o b s e r v a t i o n s w i l l c e r t a i n l y in c l u d e t h e e s s e n t i a l a r e a of
DNA r e p a i r .
The f i n d i n g s of t h i s s tu d y a r e i n d i c a t i v e o f th e p r e s e n c e
o f two t y p e s o f DNA r e p a i r i n t h e r m o p h i l i c b a c t e r i a .
D h o to reac tiv a-
t i o n w as fo u n d i n B a c i l l u s but n o t i d e n t i f i e d i n Thermus.
However, i n
T h e rm u s t h e p r e s e n c e o f a h e a t a n d c h e m i c a l l y i n d u c i b l e DNA r e p a i r
sy ste m was fo u n d to be a c t i v e a t 55 C w h i l e a t 70 C f u l l i n d u c t i o n was
o b s e r v e d i n d i c a t i n g a r o l e f o r t e m p e r a t u r e - a s s o c i a t e d DNA r e p a i r i n
s u r v i v a l a t h ig h te m p e r a t u r e .
C o n tin u a tio n o f th e stu d y th ro u g h e x a m in a tio n o f th e r m o p h il ic
b a c t e r i a l n u c l e o i d r a t e s o f m u t a b i l i t y , and b e h a v io r o f m u ta n t s t r a i n s
s h o u ld p r o v id e a more com p lete view o f DNA r e p a i r i n t h e r m o p h il e s .
57
REFERENCES CITED
58
REFERENCES CITED
A lle n , M. B. 1953. The t h e r m o p h i l i c s p o r e - f o r m i n g b a c t e r i a . Bact. Rev.
1 7 :1 2 5 -1 7 3 .
A m e lu n x e n , R. E., a n d A. L. M urdock. 1 977. P r o p o s e d m o l e c u l a r m ech­
a n ism s of s t a b i l i t y fo r th e r m o p h ilic enzym es.
J n L . A.
U n d e r k o f le r (ed.).
D evelopm ents I n I n d u s t r i a l M ic ro b io lo g y .
Vol
18. S o c i e t y f o r I n d u s t r i a l m i c r o b io lo g y . A r l i n g t o n , VA.
A r a l, K., S. N a k a m u ra , N. A r a l , a n d Y. K a z i r o . I 978. P o l y p e p t i d e c h a i n
e l o n g a t i o n f a c t o r s f r o m a n e x t r e m e t h e r m o p h i l e , HB8. J n S.M.
F r i e d m a n ( e d . ) . B i o c h e m i s t r y o f T h e r m o p h i l e s . A c a d e m ic P r e s s ,
NY. pp. I 9 3 - 2 1 0 .
A r g o s , P., M. R o ssm an , U. G rau , H. Z u b e r , G. F r a n k , a n d J . T r a t s c h i n .
1979. Therm al s t a b i l i t y and p r o t e i n s t r u c t u r e . Biochem. 18:56985703.
Bachman, B. J.
c o l i K12.
1972. P e d i g r e e s o f some m u ta n t s t r a i n s o f E s c h e r i c h i a
B a c t. Rev. 3 6 :5 2 5 -5 5 7 .
B a r n e s , L., and E. S t e l l w a g e n . 1 973. E n o l a s e f r o m t h e t h e r m o p h i l e
Thermus X - I . Biochem. 12 :1 5 5 9 -1 565.
B e c k e r , E. F. J r . , K. B u r k e , B. K. Z im m erm an, E. P. G u id u s h e k . 1 964.
S t r u c t u r e and f u n c t i o n o f c r o s s - l i n k e d DNA. J. Mol. B i o l . 8:377*
B e r g e y 1S M a n u al o f D e t e r m i n a t i v e B a c t e r i o l o g y .
W i tk in s Co., B a l tim o r e .
19 5 7 . W i l l i a m s a n d
B i e s e c k e r , G., J. I. H a r r i s , j . C. T h i e r r y , J. E. W a l k e r , and A. J.
W o n a c o t t . 1 977. S e q u e n c e a n d s t r u c t u r e o f 0 - g l y c e r o l d e h y d e 3 p h o s p h a te d eh y d ro g en a se from B a c i l l u s s t e a r o t h e r m o u h i l u s . N atu re
266:328.
B r a s h , D. E., a n d W. A. H a s e l t i n e . I 982. U V - in d u c e d m u t a t i o n h o t s p o t s
o c c u r a t DNA damage h o t s p o t s . N a tu r e 198:189-192.
Brock,
T. D. 1967. L i f e a t h ig h te m p e r a t u r e s .
S c ie n c e 158:1012-1019.
B r o c k , T. D., a n d H. F r e e z e . 1 969. T h e r m u s a q u a t l c u s g e n . n. a n d sp_.,
n. a n o n s p o r u l a t i n g e x tre m e th e r m o p h il e . J. Bact. 98:289-297.
59
B r o c k , T. D., a n d K. L. B o y le n . 1 973* P r e s e n c e o f t h e r m o p h i l i c b a c ­
t e r i a i n la u n d r y and d o m e s tic h o t w a t e r h e a t e r s . Appl. M ic r o b io l.
2 5 :7 2 - 7 6 .
B u l l , H. B., a n d K. B r e e s e . 1 973. T h e r m a l t r a n s i t i o n s o f p r o t e i n s .
Arch. Biochem. Biophys. 156:604-612.
B u z i n , C. H., an d N. Bow i n i a s - Var d i a b a s i s. I 982. The i n d u c t i o n o f a
s u b s e t o f h e a t - s h o c k p r o t e i n s by d ru g s t h a t i n h i b i t d i f f e r e n t i a ­
t i o n i n D r o s o p h i la em bryonic c e l l c u l t u r e s . Jjn M.J. S c h l e s i n g e r ,
M. A shburn e r , and A. T i s s i e r e s (eds.).
H eat Shock, From B a c t e r i a
To Man. Cold S p r in g H arbor L a b o r a to r y , New York.
C h a r e t t e , M. I . , G. H. H e n d e r s o n , a n d A. M a r k o v i t z . 19 81. ATP
h y d ro ly s is -d e p e n d e n t p ro te a s e a c t i v i t y of th e Ion p r o te in of
E s c h e r i c h i a c o l i K-12. PNAS 78:4728-4732'. .
C h ie n , A., D. B. E d g a r , an d J . M. T r e l a . 1976. D e o x y r i b o n u c l e i c a c i d
p o l y m e r a s e f r o m t h e e x t r e m e t h e r m o p h i l e T h e r m u s a c u a t i c u s . J.
B a c t . I 2 7 ( 3 ) : 1550-1557.
Chung, C. H., an d A. L. G o i l d b e r g . 1981. The p r o d u c t o f t h e I o n g e n e
i n E s c h e r i c h i a c o l i i n t h e ATP-dependent p r o t e a s e , p r o t e a s e La.
PNAS 7 8:4931-4935.
C l a r k , A. J . , and M. R. V o l k e r t . 1 978. I n H a n a w a l t , P.C., e t a l . ,
e d s ., DNA r e p a i r m echanism s. Academic P r e s s , New York, pp 57-72.
Cole, S. T. 1976. Removal o f p s o r a l a n i n t e r s t r a n d c r o s s l i n k s from DNA
o f EL. c o l i .
M e c h a n ism a n d G e n e t i c C o n t r o l .
J. Mol. B i o l .
1 0 3 :3 9 .
C o o p e r , P ., an d P. C. H a n a w a l t . 1 9 7 2a. H e t e r o g e n i c i t y o f p a t c h s i z e
i n r e p a i r r e p l i c a t e d DNA i n E. c o l i . J. Mol. B i o l . 6 7 :1 .
C o o p e r , P ., an d P. C. H a n a w a l t . I 972b. R o le o f DNA p o l y m e r a s e I an d
t h e r e c sy stem i n e x c i s i o n - r e c p i a r i n E. c o l i . PNAS 69:1156.
Cooper, P., and P. C. H anaw alt.
1972c. Exchanges b e tw e e n DNA s t r a n d s
i n U V - i r r i d a t e d E. c o l i . J. Mol. B i o l . 6 1 : 2 5 - 4 4 .
C r o n a n , J. E. J r . 1 978. M o l e c u l a r b i o l o g y
l i p i d s . Ann. Rev. B io c h e m . 4 7 :1 6 3 .
of b a c t e r ia l
m em brane
D ate, T., K. S u z u k i , a n d K. I m a h o r i . 1 9 7 5 . P u r i f i c a t i o n a n d som e
p r o p e r t i e s o f DNA -dependent RNA p o ly m e r a s e from an e x tre m e t h e r ­
m o p h ile , Thermus t h e r m o p h i l u s , HB8. J. Biochem. 78:845-858.
D a v i s , B. D. 1 948. I s o l a t i o n o f b i o c h e m i c a l l y d e f i c i e n t m u t a n t s o f
b a c t e r i a by p e n i c i l l i n .
J. Amer. Chem. Soc. 7 0 : 4 2 6 7 .
60
D e f a i s , M., P. T a u q u e t , M. Radman, a n d M. E v e r a . 1971. U l t r a v i o l e t
r e a c t i v a t i o n and u l t r a v i o l e t m u t a g e n e s i s o f lam bda i n d i f f e r e n t
g e n e t i c s y ste m s .
V iro lo g y 43:495-503.
D egryse, E., N. G l a n s d o r f f , and A. P i e r a r d . 1978. A c o m p a r a tiv e a n a l y ­
s i s o f ex tre m e th e rm o p h ilic b a c t e r i a b e lo n g in g to th e genus
Thermu s . Arch. M ic r o b i o l. 1 17:189-196.
D ulb ecco, R. 1949. R e a c t i v a t i o n o f u l t r a v i o l e t i n a c t i v a t e d
phage by v i s i b l e l i g h t .
N a tu r e 163:949.
b a c te rio ­
D u l b e c c o , R. 1 980. O r g a n i z a t i o n , a l t e r a t i o n , a n d e x p r e s s i o n o f t h e
g e n e t i c i n f o r m a t i o n . J n B. D a v i s , R. D u lb e c c o , H. E i s e n , a n d H.
G in s b e rg ( e d s . ) , M i c r o b i o l o g y , T h i r d E d i t i o n , pp. 183-213.
E l l i d g e , S. J . , a n d G. C. W a l k e r . 1983. P r o t e i n s r e q u i r e d f o r u l t r a ­
v i o l e t l i g h t and c h e m ic a l m u ta g e n e s is : i d e n t i f i c a t i o n o f th e
p r o d u c t s o f t h e umu C l o c u s o f E s c h e r i c h i a c o l i . J. Mol. B i o l .
164: 175-192.
Evanson, G., and E. S eeberg. 1982. A d a p ta t io n t o a l k y l a t i o n r e s i s t a n c e
i n v o l v e s t h e i n d u c t i o n o f a DNA g l y c o s y l a s e . N a tu r e 296:773-775.
F i s h b e i n , L., W. G. Flam m , an d H. L. F a l k , e d s . 1970. Chem. M u ta g e n s .
Academic P r e s s , New York.
F r i e d m a n , S. M., ( e d . ) . 19 7 8 . B i o c h e m i s t r y o f T h e r m o p h i l y . A c a d e m ic
P r e s s , New York.
G e o r g e , J . , M. C a s t e l l a z z i , and G.
a n d c e l l d i v i s i o n i n Ei. c o l i
r e s t o r e d i v i s i o n i n t i f and I o n
of m u ta to r p r o p e r ti e s o f t i f .
B u t t i n . 1975. P r o p h a g e i n d u c t i o n
III.
M u ta tio n s s f i A and s f i B
s t r a i n s and p e r m i t t h e e x p r e s s i o n
Mol. Gen. Genet. 140:309-332. .
G o ff, A. A., L. C a s s o n , a n d A. G o ld b e r g . I 984. H e a t s h o c k r e g u l a t o r y
gene htpR i n f l u e n c e s r a t e s of p r o t e i n d e g r a d a t i o n and e x p r e s s i o n
o f t h e I o n gene i n E. c o l i . PNAS 8 1 :6 6 4 7 -6 6 5 1 .
G o rd o n , R. E., W. C. H a y n e s , P. C. H orN ay. 1 973. The g e n u s B a c i l l u s .
Ag. H andbook No. 4 2 7 . Ag. Res. S e r v . U.S.D. A., W a s h i n g t o n , D.C.
G o t t e s m a n , S., a n d D. Z i p s e r .
1 978.
Peg p h e n o t y p e o f E s c h e r i c h i a
c o l i I o n m u ta n ts .
J . B a c t. 133:844-851 .
G r e e n b e r g , J . I 967. L o c i f o r r a d i a t i o n s e n s i t i v i t y i n E s c h e r i c h i a
c o l i s t r a i n Bg^ . G e n e tic s 5 5 :1 9 3 - 2 0 1 .
6
1
G r o s s m a n , A., Y. Zhou, C. G r o s s , J. H e i l i g e , G. C h r i s t i e , and R.
C a le n d a r.
1985.
M u ta t io n s i n t h e r o o H (htpR) gene o f E s c h e r i ­
c h i a c o l i K l2 p h en o t y p i c a l Iy s u p p r e s s a t e m p e r a t u r e - s e n s i t i v e
m u ta n t d e f e c t i v e i n t h e
70 s u b u n i t o f RNA p o ly m e ra s e .
J. B act.
1 6 1 :9 3 9 -9 4 3 .
G u d as, L. J . , a n d D. W. M ount. I 977. I d e n t i f i c a t i o n o f t h e r e c A ( t j l f )
gene p r o d u c t o f J L c o l i . PNAS 7 4 :5 2 8 0 - 5 2 8 4 .
Gudas, L. J. and A. B. Pa rdee. 19__ . Model f o r r e g u l a t i o n o f E s c h e r i ­
c h ia c o l i DNA r e p a i r f u n c t i o n s . PNAS 72:2330-2334.
H anaw a l t , P. C., P. C o o p e r , A. G a n e s a n , an d C. S m it h . 1 9 7 9 . DNA r e p a i r
i n b a c t e r i a and mammalian c e l l s .
Ann. Rev. Biochem. 48:783-836.
Harm, W. 1 978. E v i d e n c e f o r p h o t o e n z y m a t i c a l l y r e p a i r a b l e , l e t h a l
"nondim er" p h o to p r o d u c t s fo rm ed i n JL c o l i c e l l s by n e a r UV l i g h t
I n DNA R e p a ir Mechanisms, pp. 147-150.
H a s e l t i n e , W. A. 1983. U l t r a v i o l e t l i g h t r e p a i r
r e v i s i t e d . C e ll 3 3:13-17.
and m u ta g e n e sis
H i g h t o w e r , L. E. 1 980. C u l t u r e d a n i m a l c e l l s e x p o s e d t o am in o a c i d
a n a l o g s o r p u ro m y e in r a p i d l y s y n t h e s i z e s e v e r a l p o l y p e p t i d e s . J.
C e l l P h y s io lo g y 102:407-427.
H o c h a c h k a , P ., an d G. S om ero.
1 973.
S a u n d e rs C o ., N o rth C a r o l in a .
B io lo g ic a l A d a p ta tio n .
W.B.
Howa r d - F l a n d e r s , P. 1975. R e p a ir by g e n e t i c r e c o m b i n a t i o n i n b a c t e r i a
In P. H an aw alt and R. B. S e tl o w , (e d s .), M o le c u la r M echanisms f o r
R e p a ir o f DNA, P a r t A. Plenum P r e s s I n c ., New York.
I n o u y e , M. 1971. P l e i o t r o p i c e f f e c t o f t h e r e c A g e n e o f E s c h e r i c h i a
c o l i : U n c o u p lin g o f c e l l d i v i s i o n from d e o x y r i b o n u c l e i c a c i d
re p lic a tio n .
J. B a c t . I 0 6 :5 3 9 - 5 4 2 .
I y e r , V. N., and W. S z y b a ls k i. 1963. A m o l e c u l a r mechanism o f Mitomy­
c i n a c t i o n : L in k in g o f com plem entary DNA s t r a n d s . PNAS 50:355.
J e g g o , P ., e t a l . I 977. An a d a p t i v e r e s p o n s e o f E. c o l i t o low l e v e l s
o f a l k y l a t i n g a g e n t : C om parison w i t h p r e v i o u s l y c h a r a c t e r i z e d DNA
r e p a i r pathw ays. Mol. Gen. Genet. 15 7 :1 -9 .
J e g g o , P., e t a l . I 978. The a d a p t i v e r e s p o n s e o f E. c o l i t o lo w l e v e l s
o f a l k y l a t i n g a g e n t : The r o l e o f p o l A i n k i l l i n g a d a p t a t i o n .
Mol. Gen. G e n e t . 1 6 2 :2 9 9 - 3 0 5 .
J o h n so n , E.
1984.
T ulane U n i v e r s i t y , New O rle a n s , LA.
62
Kagawa, Y., and Z. A r i z a. 1977. D e t e r m i n a t i o n o f m o l e c u la r s p e c i e s o f
p h o s p h o l i p i d s from t h e r m o p h i l i c b a c t e r iu m PS3 by m ass chrom atog­
r a p h y . J. B io c h em . 8 1 :1 1 6 1 .
K a l e d i n , A. S ., A. G. S l y u s a r e n k o , a n d S. I. G o r o d e t s k i i . 1980. I s o l a ­
t i o n and p r o p e r t i e s o f DNA p o ly m e ra s e from th e e x t r e m e l y therm o­
p h i l i c b a c t e r i u m T h e rm u s a o u a t i c u s YT-I. B io k h im iy a. 4 5 ( 4 ) :6 M 651.
K a r r a n , P ., e t a l . 1982. The a d a p t i v e r e s p o n s e t o a l k y l a t i n g a g e n t s :
The re m o v a l o f C r - m e th y lg u a n in e fro m DNA i s n o t d e p en d e n t on DNA
p o ly m e r a s e - 1 . M utat. Res. 104:67-73.
Kato, T., a n d T. S h i n o u r a . 1977. I s o l a t i o n a n d c h a r a c t e r i z a t i o n o f
m u tan ts o f E s c h e ric h ia c o l i d e f i c i e n t i n in d u c tio n o f m u tatio n s
by u l t a r v i o l e t l i g h t . Mol. Gen, G enet. 156:121-131.
K e ln e r, A. 1949. P h o t o r e a c t i v a t i o n o f u l t r a v i o l e t - i r r a d i a t e d E s c h e r i ­
c h i a c o l i w i t h s p e c i a l r e f e r e n c e t o th e dose r e d u c t i o n p r i n c i p l e
and t o u l t r a v i o l e t - i n d u c e d m u t a ti o n .
J. Bact. 58:511.
K e l n e r , A. 1949.
E f f e c t o f v i s i b l e l i g h t on th e re c o v e ry o f
S t r e p t o m v c e s g r i s e u s c o n d i a f r o m UV i r r a d i a t i o n i n j u r y . PNAS
3 5 :7 3 .
K enyon, C. J . , a n d G. C. W a lk e r . 1 980. D N A -dam aging a g e n t s s t i m u l a t e
g e n e e x p r e s s i o n a t s p e c i f i c l o c i i n E s c h e r i c h i a c o l i . PNAS
7 7 :2 8 1 9 -2 8 2 3 .
K enyon, C., an d G. W a lk e r . 1981. E x p r e s s i o n o f t h e E. c o l i uvrA g e n e
i s i n d u c i b l e . N a tu r e 289:808-810.
Kohn, K., D. G r e e n , a n d P. D oty. 19 6 3 . I n t e r s t r a n d c r o s s - l i n k i n g o f
DNA by n i t r o g e n m u s t a r d (HN2) a n d i t s e f f e c t o n t r a n s f o r m i n g
a c tiv ity .
Fed. P ro c. 2 2 :5 8 2 .
L i l l i e , R. D.
1977.
H. J . C o n n 's B i o l o g i c a l S t a i n s N i n t h E d i t i o n .
W illia m s and W ilk in s C o ., B a l tim o r e .
L in ,
C. C., an d L. E. C a s i d a . 1984. G e l r i t e a s a g e l l i n g a g e n t i n
m e d ia f o r th e g ro w th o f t h e r m o p h i l i c m ic r o o r g a n is m s , A pplied and
E n v i r o n . M i c r o b i o l . 47 (2 ):4 2 7 -4 2 9 .
L i n d a h l,
T. 1982. DNA r e p a i r enzymes. Ann. Rev. Biochem. 51:61-87.
L i n d a h l , T., B. D em p le, a n d P. R o b in s . 19 8 2 . S u i c i d e i n a c t i v a t i o n o f
t h e JL. c o l i 0 " -m e t h y I g u a n i n e - D N A m e t h y l t r a n s f e r a s e . EMBO J.
1:1359-1363-
6
3
L o s s i u s , I . , P. K r u g e r , R. M ole, and K. K le p p e . 1983. M i t o m y e i n - C in d u c e d changes i n th e n u c l e o i d o f E s c h e r i c h i a Kl2. M u ta t io n Res.
10 9 : 13- 2 0 .
L u c h n i k , A.
1979." On t h e m e c h a n is m
in d u c tio n : R e la x a tio n h y p o th e sis.
o f S O S -re p a ir and p ro p h ag e
J . Theor. B i o l . 7 7 :2 2 9 -2 3 1 .
M a lc o lm , A. D., B. 1981. T h e r m o p h i l e s a n d RNA s t a b i l i t y .
B io l . 9 2 : 4 7 9 - 4 8 1 .
J. T h e o r.
M e r k l e r , D. J . , G. K. F a r r i n g t o n , an d F. C. W e d l e r . 1981. P r o t e i n
th e rm o s ta b ility . C o r r e la tio n s b etw een c a l c u l a t e d m a c ro sc o p ic
p a r a m e te r s and g ro w th te m p e r a tu r e f o r c l o s e l y r e l a t e d th e rm o ­
p h i l i c and m e s o p h i l i c b a c i l l i . I n t . J. P ept. P r o t e i n Res. 18:430432.
Myer, R. R., J. G l a s s b e r g , a n d A. K o r n b e r g . I 979. An E s c h e r i c h i a c o l i
m u ta n t d e f e c t i v e i n s i n g l e - s t r a n d e d b in d in g p r o t i e n i s d e f e c t i v e
i n DNA r e p l i c a t i o n . PNAS 76:1702-1705.
N a g a h a r i , K., T. K o s h ik a w a, and K. S a k a g u c h i . 1980. C l o n i n g and
e x p r e s s i o n o f t h e l e u c i n e ge n e from Thermus t h e r m o p h i l u s i n
E s c h e r i c h i a c o l i . Gene 10:137-145.
Nakabeppu, Y., and M. S e k ig u c h i. 1981. P h y s i c a l a s s o c i a t i o n o f p y r i m i ­
d in e d im er DNA g l y co sy I a s e and a p u r i n i c - a p y r i m i d i n i c DNA endonu­
c l e a s e e s s e n t i a l f o r r e p a i r o f u l t r a v i o l e t d a m a g e d DNA. PNAS
7 8 :2 4 7 2 -2 7 4 6 .
N e i d h a r d t , F. C., a n d R. A. Van B o g e l e n . 19 8 1 . P r o t e i n r e g u l a t o r y g e n e
f o r t e m p e r a t u r e - c o n t r o l l e d p r o t e i n s i n E s c h e r i c h i a c o l i . Biochem.
Biophy s, Res. Commun. 100:894-900.
N e i d h a r d t , F. C., a n d R. A. Van B o g e l e n . 1982. The h i g h t e m p e r a t u r e
r e g u l o n o f E s c h e r i c h i a c o l i . J n M. S c h l e s i n g e r , M. A s h b u r n e r ,
a n d A. T i s s i e r e s ( e d s . ) , H e a t S h o c k , From B a c t e r i a t o Man. C o ld
S p r in g H arbor L a b o r a to r y , New York.
pp. 139-145.
N e i d h a r d t , F. C., R. A. Van B o g e l e n , a n d E. T. L au . 1983. M o l e c u l a r
c l o n i n g and e x p r e s s i o n o f a gene t h a t c o n t r o l s t h e h ig h te m p e ra ­
t u r e r e g u l o n o f E s c h e r i c h i a c o l i . J. B act. 153:597-603.
N e i d h a r d t , F. C., R. A. Van B o g e l e n , an d V. Vaughn. 1 984. G e n e t i c s a n d
r e g u l a t i o n o f h e a t shock p r o t e i n s . Ann. Rev. G en et, pp. 894-900.
O hta, Y., Y. O g u ra, a n d A. Wada. 1 966.
T h e r m o s ta b le p r o t e a s e from
th e rm o p h ilic b a c te ria .
J. B io l. Chem. 241:5919.
64
O is h i , M., C. S m i t h , an d B. F r i e f e l d .
1 979.
M o le c u la r e v e n ts and
m o l e c u l e s t h a t l e a d t o i n d u c t i o n o f prophage and SOS f u n c t i o n s .
CSH S y m p o s ia on Quant. B iol. 43:897-907.
Oshima, M. 1978. S t r u c t u r e and f u n c t i o n o f membrane l i p i d s i n th e rm o ­
p h i l i c b a c t e r i a . JEn S. F r i e d m a n ( e d . ) , B i o c h e m i s t r y o f T h e rm o p h ily .
O s h im a , M., an d T. A r i g a . 1976. A n a l y s i s o f t h e a n o m e r i c c o n f i g u r a ­
t i o n o f a g a l a c t o f u r a n o s e c o n t a i n i n g g l y c o l i p i d fro m an e x tre m e
th erm o p h ile.
FEBS L e t t e r s 64:440.
O s h im a , T. 1975. T h e r m i n e : a new p o l y a m in e f r o m a n e x t r e m e t h e r m o p h i l e . Biochem. Biophys. Res. Comm. 63:1093-1098.
Oshima, T. 1982. A p e n ta a m in e i s p r e s e n t i n an e x tr e m e th e r m o p h il e .
B i o l . Chem. 2 5 7 ( 17):9913-9914.
J.
O sh im a , T., Y. S a k a k i , N. Wakayama, K. W a ta n a b e , Z. O h a s h i, and S.
N is h im u r a. 1976. B io c h e m ic a l s t u d i e s on a n e x t r e m e t h e r m o p h i l e
T h e rm u s t h e r m o p h i l u s : t h e r m a l s t a b i l i t i e s o f c e l l c o n s t i t u e n t s
a n d a b a c t e r i o p h a g e . J n H. Z u b e r , ( e d . ) , E n z y m e s a n d P r o t e i n s
From T h e r m o p h ilic M ic ro o rg a n ism s. B erkhduser V e r la g , B asel, pp.
317-331.
P e l l o n , J. R., K. M. U l l m e r , and R. G. Gomez. I 980. H e a t dam age t o t h e
f o l d e d chromosome o f E s c h e r i c h i a c o l i K12. Appl. E n v iro n . M icro­
b i o l . 4 0 :3 5 8 - 3 6 4 .
P e l l o n , J. R., an d R. G. Gomez. 1981. R e p a i r o f t h e r m a l dam age t o t h e
E s c h e r i c h i a c o l i n u c l e o id . J. B a c t. 145:1456-1458.
P e l l o n , J. R., R. F. Gomez, and A. J. S i n s k e y . 1 982. A s s o c i a t i o n o f
th e E s c h e r ic h ia c o l i n u c le o id w ith p r o te in s y n th e s iz e d d u rin g
t h e r m a l t r e a t m e n t s . J n M. S c h l e s i n g e r , M. A s h b u r n e r , and A.
T i s s i e r e s (e d s.), H eat Shock, FrOm B a c t e r i a t o Man.
Cold S p r in g
H arbor L a b o r a t o r y , New York. pp. 121-130.
P e r u t z , M. J . , a n d H. R a i d t . 1 975.
S te re o c h e m ic a l b a s is of h e a t
s t a b i l i t y i n b a c t e r i a l f e r r e d o x i n s and i n h a e m o g lo b in A2. N a tu re
2 5 5 :2 5 6 .
P h i l l i p s , T., R. Van B o g e l e n , and F. C. N e i d h a r d t .
19 8 4 . Lon g e n e
p r o d u c t o f E. c o l i i s a h e a t - s h o c k p r o t e i n .
J. B a c t . 1 5 9 :2 8 3 287.
Q u i g l e y , G. S ., a n d A. R ic h . 19 7 6 . S t r u c t u r a l d o m a i n s o f t r a n s f e r RN A
m o l e c u le s . S c ie n c e 194:796-806.
65
Radman, M. 1 975. In Hanawa l t , P. C., and R. B. S e t l o w , e d s , M o l e c u l a r
M ec ha n ism s f o r R e p a i r of. DMA, Plenum P r e s s , New York, pp. 3 5 5 367.
R a m a ly , R. F., F. R. T u r n e r , L. E. M a l i c k , an d R. B„ W i ls o n . 1978. The
m orphology and s u r f a c e s t r u c t u r e o f some e x t r e m e l y t h e r m o p h i l i c
b a c t e r i a f o u n d i n s l i g h t l y a l k a l i n e h o t s p r i n g s . J n S. F r i e d m a n
(ed.), B io c h e m i s t r y o f Thermo p h i ly .
Academic P r e s s , New york.
R i t o s a , F. I 962. A new p u f f i n g p a t t e r n i n d u c e d by t e m p e r a t u r e s h o c k
and DNP i n D r o s o p h ila . E x p e r i e n t i a 18:571-573.
R u p e r t , C. S. 19__ . P h o t o e n z y m a t i c r e p a i r o f u l t r a v i o l e t dam age i n
DNA. I. K i n e t i c s o f t h e r e a c t i o n . J. Gen. P h y s i o l . 4 5 : 7 0 3 - 7 2 4 .
R u p e r t , C. S. I 9__ . P h o t o e n z y m a t i c r e p a i r o f u l t r a v i o l e t dam age i n
DNA. I I . F o r m a t i o n o f a n e n z y m e - s u b s t r a t e c o m p le x . J. Gen.
P h y s i o l . 4 5 :7 2 5 -7 4 1 .
Rupp, W. D., and P. H o w a rd -F la n d e rs. 1968. D i s c o n t i n u i t i e s i n t h e DNA
s y n t h e s i z e d i n a n e x c i s i o n - d e f e c t i v e s t r a i n o f Ei, c o l i f o l l o w i n g
UV i r r i d a t i o n .
J . Mol. B io l. 3 1 :2 9 1 .
Rupp, W. D., C. E. W i ld e I I I , D. L. R eno, a n d P. H o w a r d - F l a n d e r s .
1971. E x c h a n g e s b e t w e e n DNA s t r a n d s i n u l t r a v i o l e t - i r r a d i a t e d
E s c h e r i c h i a c o l i . J. Mol. B i o l . 61 : 2 5 - 4 4 .
S am so n , L., a n d J . C a i r n s . 1977. A new p a th w a y f o r DNA r e p a i r i n
E s c h e r i c h i a c o l i . N a tu r e 267:281-282.
S a n c a r , A., an d C. R u p e r t . 1 978. A g e n e r a l m e th o d f o r 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 ta n ts . J n P. C. Hanaw a l t , E. C. F r i e d berg, and
C. F. Fox ( e d s . ) , DNA R e p a i r M e c h a n i s m s , A c a d e m ic P r e s s , New
Y ork, pp. 1 0 1 - 1 0 4 .
S a n c a r , A., an d W. D. Rupp. 1 979. C l o n i n g o f uvrA , I e x C a n d s s b g e n e s
o f E s c h e r i c h i a c o l i . Biochem. Biophys. Res. Comm. 90( I ):1 23-129.
S a n c a r , A., an d W. D. Rupp. 1 983. A n o v e l r e p a i r e n z y m e : UVRABC e x c i ­
s i o n n u c l e a s e o f E. c o l i c u t s a DNA s t r a n d on b o t h s i d e s o f t h e
damaged r e g io n . C e l l 33:249-260.
S a to , S., K. N a k a z a w a , a n d T. S h i n o m i y a. 1980. A DNA m e t h y l a s e f r o m
Thermus t h e r m o n h i l u s HB8. J. Biochem. 88:737-747.
S a t o , S., C. A. H u t c h i s o n , a n d J. I. H a r r i s . 1977. A t h e r m o s t a b l e
s e q u e n c e - s p e c i f i c e n d o n u c l e a s e f r o m T h e rm u s a o u a t i c u s . PNAS
7 4 :5 4 2 -5 4 6 .
66
S c h l e s i n g e r , M. J . , M. Ash b u r n e r , a n d A. T i s s i e r e s ( e d s ) . 1982. H e a t
Shock, From B a c t e r i a t o Maa Cold S p r in g H arbor L a b o r a to r y , New
York.
S e e b e r g , E., and A. L. S t e i n u m . 1982. P u r i f i c a t i o n a n d p r o p e r t i e s o f
t h e uvrA p r o t e i n from E s c h e r i c h i a c o l i . PNAS 7 9 :9 8 8 -9 9 2 .
S e e l e y , H., a n d P. Van D em ark. 1981. L e t h a l a c t i o n o f u l t r a v i o l e t
l i g h t : p h o t o r e a c t i v a t i o n . J n W. H. F r e e m a n , a n d P. Van D em ark
( e d s . ) , M i c r o b e s i n A c t i o n , L a b o r a t o r y M anual o f M i c r o b i o l o g y ,
pp. 7 4 - 7 7 .
S i n g l e t o n , R., and R. E. A m elunxen. I 973. P r o t e i n s f r o m t h e r m o p h i l i c
m ic r o o r g a n is m s . B act. Rev. 37(3):320-342.
S t e n i s h , J ., and J. B. Madison. 1979. S t a b i l i t y o f b a c t e r i a l m essenger
RNA i n m e s o p h i l e s a n d t h e r m o p h i l e s . B io c h im . e t B i o p h y s. A c t a
5 6 5 :1 5 4 -1 6 0 .
S t i l l w a g e n, E., and L. D. B a r n e s . 1 976. I n Z u b e r , H., ed. E n zy m es a n d
(
P r o t e i n s fro m T h e r m o p h ili c M ic ro o rg a n ism s, p. 223.
S t u y , R. 1956.
S t u d i e s on t h e m echanism o f r a d i a t i o n i n a c t i v a t i o n o f
m ic r o o r g a n is m s . I I . P h o t o r e a c t i v a t i o n o f some B a c i l l i and o f t h e
s p o r e s o f tw o B a c i l l u s c e r e u s s t r a i n s . B io c h im . B io p h y s . A c ta
2 2 :2 3 8 -2 4 0 , 2 4 1 - 2 4 6 .
Tanaka, T., N. Kawa m , and T. O shim a 1981. C loning o f 3 - i s o p ro p y I m a la t e d e h y d ro g e n a se gene o f an e x tre m e th e r m o p h ile and p a r t i a l
p u r i f i c a t i o n o f t h e gene p ro d u c t. Biochem. 89:677-682.
T r i s l e r , G., and T. G o tte sm an . 1984. Lon t r a n s c r i p t i o n a l r e g u l a t i o n o f
genes n ecessary to c a p su la r p o ly sa c c h a rid e s y n th e s is i n E sc h e ri­
c h i a c o l i K -12. J. B a c t . 1 6 0 : 1 8 4 - 1 9 1 .
T r a v e r s A., and H. Mace. 1982. The h e a t - s h o c k
A p r o t e c t i o n a g a i n s t DNA r e l a x a t i o n ?
A s h b u r n e r , and A. T i s s i e r e s ( e d s . ) H e a t
Man. Cold S p r in g H arbor L a b o r a t o r y , New
phenomenon i n b a c t e r i a JEn M. S c h l e s i n g e r , M.
S h o ck , From B a c t e r i a t o
York, p p. 127-130.
T suj i , S., M. Takanami, and K. Im ah o r i . 1980. A s tu d y o f t h e i n t e r a c ­
t i o n betw een p r o m o te r DNA and JL. th e r m o p h il u s DNA-dependent RNA
p o ly m e ra s e .
J. Biochem. 88:715-724.
U l r i c h , J. T. 1971. A s t u d y o f some b i o c h e m i c a l a n d p h y s i o l o g i c a l
p r o p e r t i e s o f an e x tr e m e th e r m o p h ile . T h e s is : MSU ( M ic r o b io lo g y ) .
U l r i c h , J. T., G. A. M c F e t e r s , a n d K. L. T e m p le . 1972. I n d u c t i o n a n d
c h a r a c t e r i z a t i o n o f B - g a l a c t o s i d a s e i n 'an e x tre m e th e r m o p h ile . J.
B a c t. 1 1 0 ( 2 ) : 6 9 1 - 6 9 8 .
67
W alker, G. C. 1984. M u ta g e n e s is and i n d u c i b l e r e s p o n s e s t o d e o x y rib o ­
n u c l e i c a c i d d am ag e i n E s c h e r i c h i a c o l l . M i c r o b i o l . Rev.
4 8 (0 :6 0 -9 3 .
Wang, S. Y. ( e d . ). 1976. P h o t o c h e m i s t r y an d P h o t o b i o l o g y o f n u c l e i c
a c i d s . Academic P r e s s , New York. p. 11.
W a r n e r , H. R. 1 983. B ase e x c i s i o n r e p a i r i n t h e t h e r m o p h i l e , T h e rm u s
s p . s t r a i n X - I . J. B a c t . 15 4 ( 3 ) : 14 5 1 - 1 4 5 4 .
W e i g l e , J. J. I 953. I n d u c t i o n o f m u t a t i o n s i n a b a c t e r i a l v i r u s . PNAS
3 9 :6 2 8 -6 3 6 .
W i t k i n, E. M. 1946. G e n e ti c s o f r e s i s t a n c e t o r a d i a t i o n i n E s c h e r i c h i a
c o l i . G e n e ti c s 3 2 : 2 2 1 - 2 4 8 .
W itk in , E. M. 1974. Therm al enhancem ent o f u l t r a v i o l e t m u t a b i l i t y i n
a t i f I uvr d e r i v a t i v e o f El c o l i BK: E vidence t h a t UV m utagene­
s i s depends upon an i n d u c i b l e f u n c t i o n .
PNAS 71:1930-1934.
W i t k i n , E. M. 1975.
E le v a te d m u t a b i l i t y o f pol A and uvr A pol A
d e r i v a t i v e s o f E s c h e r ic h ia c o l i B /r a t s u b l e t h a l d o ses o f
u ltra v io le t lig h t:
E vidence f o r an i n d u c i b l e e r r o r - p r o n e r e p a i r
s y s t e m ("SOS r e p a i r " ) a n d i t s a n o m a l o u s e x p r e s s i o n i n t h e s e
s t r a i n s . G e n e ti c s 79:1 9 9 - 2 1 3 .
W itk in , E. M. 1976. U l t r a v i o l e t m u t a g e n e s i s and i n d u c i b l e DNA r e p a i r
i n E. c o l i . B a c t. Rev. 4 0 :8 6 9 -9 0 7 .
Y a g u c h i, M., L. D. V i s e n t i n , R. N. N a z a r , a n d A. T. M a t h e s o n. 1978.
S t r u c t u r e and t h e r m a l s t a b i l i t y o f r ib o s o m a l co m p o n en ts fro m
t h e r m o p h i l i c b a c t e r i a . J n S. F r i e d m a n ( e d . ) , B i o c h e m i s t r y o f
Thermo p h i I y , Academic P r e s s , New York. pp. 169-191.
Y am am o ri, T., K. I t o , Y. N ak am u ra , an d T. Y ura. 19 7 8 . T r a n s i e n t r e g u ­
l a t i o n o f p r o t e i n s y n t h e s i s i n E s c h e r i c h i a c o l i upon s h i f t - u p o f
g ro w th te m p e r a t u r e . J. Bact. 134:1133-1140.
Y am am o ri, T., a n d T. Y ra. 1980. T e m p e r a t u r e i n d u c e d s y n t h e s i s o f
s p e c i f i c p r o t e i n s i n E s c h e r i c h i a c o l i : e v id e n c e f o r t r a n s c r i p ­
t i o n a l c o n t r o l . J. B a c t. 142:843-851.
Yamamori, T. 1982. G e n e tic c o n t r o l o f h e a t shock p r o t e i n s y n t h e s i s and
i t s b e a r i n g on g ro w th and th e r m a l r e s i s t a n c e i n E s c h e r i c h i a c o l i .
PNAS 7 9 :8 6 0 -8 6 4 .
Y am am oto, Y., M. K a t s u k i , M. S e k i g u i c h i , an d N. O t s u j i . I 078. E s c h e r ­
i c h i a c o l i gene t h a t c o n t r o l s s e n s i t i v i t y t o a k l y l a t i n g a g e n t s .
J. B a c t. 1 3 5 : 1 4 4 - 1 5 2 .
68
Y am am oto, K., M. S a t a k e , H. S h i n e g a w a, a n d Y. F u j i w a r a. I 983. A m e li­
o r a t i o n o f t h e UV s e n s i t i v i t y o f a n E s c h e r i c h i a c o l i r e c A m u ta n t
i n t h e d a r k by p h o t o r e a c t i v a t i n g en zy m e. Mol. Gen. G e n e t;
1 9 0 :5 1 1 -5 1 5 .
Zuber, H. (ed.) 1976. Enzymes and P r o t e i n s from T h e r m o p h ili c M ic ro o r­
g a n ism s, B e r k h a u s e r V e r la g , B a s e l.
MONTANA STATE UNIVERSITY LIBRARIES
I 'a in
N3T8
K6 3 6
cop. 2
DATE
K i r k p a t r i c k , M.A.S.
An e x a m in a tio n o f c o n s titu tiv e d ire c t l i g h t . ..
I S S U E D TO
-
Main
rl J fO
K6 3 6
cop. 2