Genetic studies on a heat stable surface antigen of Escherichia coli K12
by Aletha Sylvia Markusen
A thesis submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY in Genetics
Montana State University
© Copyright by Aletha Sylvia Markusen (1963)
Abstract:
The flocculent agglutination produced by many strains of E. coli K12 in response to antiserum is due in
part to the presence of flagellar antigen and in part to a heat stable antigen (HSA) not associated with
flagella.
Presence of HSA is not obviously correlated with F status. Appropriate crosses show its genetic basis
not to be closely linked to the TLB1 region.
Occasional mutations from HSA+ to HSA- and the reverse have occurred simultaneously with
mutations of other genes not obviously related physiologically. GENETIC STUDIES ON A HEAT STABLE SURFACE ANTIGEN
OF ESCHERICHIA GOLI K12
by
ALETHA So MRKUSEN
A t h e s i s s u b m i t te d t o t h e G r a d u a te F a c u l t y i n p a r t i a l
f u l f i l l m e n t o f t h e r e q u i r e m e n ts f o r t h e d e g r e e
of
DOCTOR OF PHILOSOPHY
in
G e n e tic s
A pproved 8 •
H ea^y M a jo r D e p a rtm e n t
- T
rD
C h a irm a n , E x a m in in g C om m ittee
D ean , G ra d u a te D i v i s i o n
MONTANA STATE COLLEGE
Bozem an, M ontana
J u n e , 1963
ACKNOWLEDGMENTS
G r a t e f u l ack n o w led g m e n t i s made t o D r. P 0 D. S k a a r f o r g u id a n c e
d u r i n g t h e s t u d y a n d i n 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 , a n d t o D r. L . HS.
S m ith a n d D r. J . R. S c h a e f f e r f o r v a l u a b l e a s s i s t a n c e a n d e n c o u ra g e m e n t.
The a u t h o r i s i n d e b t e d t o D r s . E . an d J . L e d e r b e r g who f u r n i s h e d
some o f t h e b a c t e r i a l c u l t u r e s u s e d i n t h e i n v e s t i g a t i o n .
T h is w o rk h a s b e e n s u p p o r t e d i n p a r t b y a g r a n t ( C -3 9 0 2 ) fro m
t h e N a t i o n a l C a n c e r I n s t i t u t e , P u b l i c H e a l th S e r v i c e , t o D r. P . D.
S k a a r ; i n p a r t b y a f e l l o w s h i p aw a rd ed b y t h e N o rth w e s t R e g io n o f
S o r o p t i m i s t s j an d i n p a r t b y a N a t i o n a l E d u c a tio n A c t F e l l o w s h i p .
.
,
iii
TABLE- OF CONTENTS
P age
INTRODUCTION
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MATERIALS AND METHODS. . .
2
A.
B a c t e r i a l a n d B a c te r io p h a g e S t r a i n s .
B.
C u l t u r e M edia and S u p p le m e n ts .
C.
A g g lu tin a tio n P ro c e d u re s . . . .
D.
.
. . „
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P r o d u c ti o n o f A n tis e r u m .
. . . .
4
2.
P r e p a r a t i o n o f V a c c in e . . . . .
5
3.
P r e p a r a t i o n o f A b s o rb in g A n tig e n
5
4.
Serum A b s o r p tio n . . . . . . . .
.5
5.
A g g l u t i n a t i o n T e s ts
6.
P r e p a r a t i o n o f A n tig e n s f o r H e a t S t a b i l i t y T e s ts
>
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P r o c e d u r e s I n v o lv i n g C r o s s e s .
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6
1.
F I n f e c tio n P ro c ed u re s
2.
C ro ss P ro c e d u re s
3.
S c o r in g o f R e c o m b in a n t M a rk e rs
7
4«
P r e p a r a t i o n o f Lambda L y s a te 's .
. 7
5.
T e s ts f o r L y s o g e n i c i t y
0
0
9
0
9
6
9
0
0
0
0
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0
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0
7
8
B e ' In c u b a tio n o f C u ltu re s
RESdLTSo o o o o
O
O
O
o
o
o
o
o
o
o
o
o
9
0
0
0
0
0
A.
S e r o l o g i c a l A n a l y s is o f t h e S u r f a c e A n tig e n s o f C S l l .
B.
P h y s i c a l P r o p e r t i e s o f t h e A n tig e n o f MSC$6. .
C.
O c c u r re n c e o f HSA Among K12 S t r a i n s .
>
iv
0
9
0
0
0
0
9
9
12
0
0
16
P age
D0
M u ta tio n o f t h e HSA P r o p e r t y "in D e r i v a t i v e s o f
W ilW o o o o o
o
e
e
o
o
a
o
o
o
e
o
o
o
o
o
o
19
1.
M u ta tio n t o S tr e p to m y c in R e s i s t a n c e . . . . . . . .
22
2.
C o n v e r s io n o f F - S t r a i n s t o F+ . . . . . .
3„
HSA M u ta tio n s A s s o c ia t e d w i t h C hanges i n Lambda
R e l a te d P r o p e r t i e s
O
O
O
O
O
O
o
o
o
o
, 0 . .
24
28
b
Eo
M u ta tio n o f HSA A s s o c ia t e d w i t h E n h an ced M o t i l i t y .
31
Fo
L o c a l i z a t i o n o f t h e HSA Gene on t h e B a c t e r i a l
o
o
o
o
o
o
o
Chromosome . .
32
DISCUSSION . . . .
SUMMARY. . . . .
.
.
O
O
O
.
.
.
O
.
O
0
O
O
O
O
O
O
. . . * 0 0
O
O
O
’
O
o
O
O
o . o o o o .
O
O
O
O
0 *
40
45
46
REFERENCES CITED . . . .
v
LIST OF TABLES
T a b le
I.
II.
III.
17.
7.
71.
7 II-.
P age
B a c te ria l S tra in s
. . . . . . . . . . . . . . . . . . .
3
S e r o l o g i c a l R e l a t i o n s h i p s Among C S l l i 115039*
a n d 11S05 ^ . .
. . . . . . . . . . . . . . . . . . . .
10
H eat S t a b i l i t y o f A n tig e n s .
15
. . . . . . . . . . . . . .
HSA S t a t u s o f S e l e c t e d K12 S t r a i n s .
. . . . . . . . . .
17
S e r o l o g i c a l R e l a t i o n s h i p s Among F - s F + , a n d H f r
S tra in s . . . . . . . . . . . . . . . . . . . . . . .
26
R e l a t i v e E f f i c i e n c y o f F+ C o n v e r s io n o f MSC501*MSC503, and MSC505 b y M ixed G row th w i t h W6 . . . . . .
29
S e g r e g a t i o n o f HSA and O th e r U n s e l e c t e d M a rk e rs
■ Among P r o t o t r o p h i c R e c o m b in a n ts R e s u l t i n g From
C r o s s e s B etw een W6 (F + , T^s i I a c + , x y l + . m a l+ ,
m t l + o S s 5 HSA+ ) a n d W U77' C F -, V1 1*, XaoT,
m al , ' HltX , HSA ) ^ o e e » e o o o » e o o o » o
I
V
vi
o
0
0
3^*
L IST OF FIGURES
F ig u re
I.
P ag e
Genomes o f W6 a n d EL177 . . . . . . . . . . . . . . . .
:■ •
v ii
r
37
ABSTRACT
The f l o c c u l e n t a g g l u t i n a t i o n p r o d u c e d b y many s t r a i n s o f E, c o l i
K12 i n r e s p o n s e t o a n t i s e r u m i s due i n p a r t t o t h e p r e s e n c e o f f l a g e l l a r
a n t i g e n a n d i n p a r t t o a h e a t s t a b l e a n t i g e n (HSA) n o t a s s o c i a t e d w i t h
fla g e lla .
P r e s e n c e o f HSA i s n o t o b v i o u s l y c o r r e l a t e d w i t h F s t a t u s .
A p p r o p r ia t e c r o s s e s show i t s g e n e t i c b a s i s n o t t o b e c l o s e l y l i n k e d t o
t h e TLBi r e g i o n .
O c c a s io n a l m u ta ti o n s fro m HSA"*" t o HSA- and t h e r e v e r s e h av e o c­
c u r r e d s i m u l t a n e o u s l y w i t h m u ta ti o n s o f o t h e r g e n e s n o t o b v i o u s l y r e ­
la te d p h y s io lo g ic a lly .
t. ••
..
i
•-
v iii
INTROroCTION
I n a n e a r l i e r i n v e s t i g a t i o n (M a rk u s e n 5 1 9 6 0 ) 5 a n E= c o l i h y b r id
b e tw e e n s t r a i n K l2 ( m o t i l e ) and s t r a i n B ( h o n - m o t i l e ) w as f o u n d t o be
n o n - m o t il e e v e n a f t e r r e p e a t e d p a s s a g e s on m o t i l i t y m edium .
Upon exam in­
a t i o n b y l i g h t a n d e l e c t r o n m ic r o s c o p y , i t w as fo u n d a l s o t o be n o n fla g e lla te d .
N o n e t h e l e s s , c u l t u r e s o f t h i s h y b r id s t r a i n
(MSG56) w e re
a g g l u t i n a t e d b y h i g h d i l u t i o n s o f a n ti s e r u m w h ic h h a d b e e n p ro d u c e d i n
r e s p o n s e t o i t s K12 p a r e n t .
The a g g r e g a te fo rm e d i n t h e a g g l u t i n a t i o n
r e s e m b le d f l a g e l l a r a g g l u t i n a t i o n b o t h i n a p p e a r a n c e a n d i n t h e k i n e t i c s
of its
f o rm a tio n .
C lo s e s e r o l o g i c a l s i m i l a r i t y b e tw e e n f u n c t i o n l e s s m o le c u le s and
t h e i r n o rm a l h o m o lo g u es h a s b e e n d e m o n s tr a te d i n o t h e r s t u d i e s , f o r ex­
am ple t h e s i m i l a r i t y b e tw e e n a c r o s s - r e a c t i n g p r o t e i n (CRM) and t r y p t o p h a n
s y n t h e t a s e ( Y a n o fs k y , i 9 6 0 ) .
F u r th e r m o r e i n S a lm o n e lla t h e g e n e t i c l o c i
d e te r m i n in g f l a g e l l a r a n t i g e n s p e c i f i c i t y ( t h e H l o c i ) a r e s e p a r a b l e fro m
t h e g e n e s f o r f l a g e l l a r s t r u c t u r e ( S t o c k e r e t a l , 1 953)«
T h e refo re, i t
w as p o s t u l a t e d t h a t MSC56 w as an h i t h e r t o u n d e s c r i b e d re c o m b in a n t ty p e
w h ic h c a r r i e d ( a n d e x p r e s s e d ) t h e f l a g e l l a r a n t i g e n s p e c i f i c i t y g e n e s
o f K12 b u t l a c k e d t h e g e n e s f o r f l a g e l l a r s t r u c t u r e .
The p r e c e d in g w o r k in g h y p o t h e s i s h a s b e e n ex am in ed a n d fo u n d t o
be u n lik e ly .
I n s t e a d , t h e a n t i g e n c a r r i e d b y 1SE56 a p p e a r s t o b e n o t
o f t h e c l a s s i c a l "H" t y p e , b u t r a t h e r a n a n t i g e n w i t h n o v e l p r o p e r t i e s .
The f i n d i n g t h a t a t l e a s t one K12 m u ta n t l a c k s t h i s a n t i g e n
p e rm its i n v e s t i g a t i o n o f i t s
g e n e tic b a s is .
MATERIALS AND, METHODS
Ae
B a c t e r i a l a n d B a c te r io p h a g e S t r a i n s
A ll b a c t e r i a l s t r a i n s u se d in th e in v e s tig a tio n a re d e s c rib e d in
T a b le I e
W ild ty p e fo rm s o f b o t h t h e v i r u l e n t p h ag e T5 an d t h e te m p e r­
a t e p h a g e la m b d a (A dam s, 1 9 5 9 ) w e re u s e d .
Be
C u l t u r e M edia a n d S u p p le m e n ts
A l l f i n a l c o n c e n t r a t i o n s g iv e n b e lo w a r e i n g ram s p e r l i t e r o f
d i s t i l l e d w a t e r e x c e p t w h e re i n d i c a t e d !
A l l c u l t u r e m e d ia w e re s t e r i ­
l i z e d i n t h e a u t o c l a v e a t 15 p o u n d s p r e s s u r e f o r 20 m i n u t e s .
N u tr ie n t A g ars
D if c o n u t r i e n t b r o t h 8 , NaCl 5> a g a r 1 5 .
M o t i l i t y . M edium s
B a c to C a s i to n e I O 9 y e a s t e x t r a c t 3 , a g a r 4 ,
D if c o g e l a t i n SO9 NaCl 5 .
The pH w as a d j u s t e d t o 7 .0 a f t e r s t e r i l i z a ­
tio n .
P e n n a s s a y B r o th ( B a c to A n t i b i o t i c M edium 3 ) s
B a c to -B e e f E x t r a c t
1 . 5 , B a c to - Y e a s t E x t r a c t 1 . 5 , B a c to - P e p to n e 5 . 0 , B a c to D e x tro s e I eO9
NaCl 3 . 5 , W2HPO4 ., 3 . 6 , KH2PO4 1 . 3 .
E o s in M e th y le n e B lu e Medium ( EMB')s
C a s e in d i g e s t IO eO9 y e a s t
e x t r a c t I eO9 NaCl 5 .0 , K2 HPO4 2 . 0 9 E o s in Y 0 . 4 , M e th y le n e B lu e 0 .0 6 5 ,
a g a r 15. 0 , su g a r 10. 0 .
D a v is M in im al A gar (DMA)s
E 2HPO4 7 . 0 , KH2PO4 2 . 0 , c r y s t a l l i n e
^ so d iu m c i t r a t e 0 . 5 , c r y s t a l l i n e MgS04 0 . 1 , c r y s t a l l i n e (NH4 ) 2 SO4 1 . 0 ,
g lu c o s e 1 . 0 , and a g a r 1 5 . 0 .
and s t e r i l i z e d .
i .-v
The s a l t s w e re ad d ed t o 500 m l o f w a t e r
The g lu c o s e a n d a g a r w e re s t e r i l i z e d
t o g e t h e r i n 500
3
T a b le 1»
B a c te ria l S tr a ia s .
S tr a in d e s ig n a tio n
¥677
¥1177
-C Sll
MSC503
MSC501
¥1603
¥2736
MSC505
CS19
¥ 6 ( 58- 1 6 1 )
IS C 69
¥1895
C S lO l
MSC70
MSC18
IMN60
S o u rc e
Ei c o l i K12 -M*
¥677
¥1177
¥677
¥1177
¥1177
¥1603
ZSC501
E. c o l i K12**
, E . c o l i K12**
¥6
¥ 6'
¥1895
’ MSC69
¥6
MSC39
E , c o l i BHBIN60
BQJ 64
MSC56
Eo c o l i B*
C S ll x IMN64
MSC500
MSC56
New g e n e t i c m a rk e rs
T-» I f 9 B1- 5 l a c “ 5 . m a l" ,
m t l " , VjT
Sr
F+ ( S o u r c e o f Fg ¥ 6 )
m a l+
m a l+ , m tl+ , Bl +
M T* I +
&E2 S
m tlp a " , m at+
Vt
FH fr C a v a lli
Vir
F+ ( S o u rc e o f F s C S l l )
P ro"'
try " , I l a r
made m o t+ , f l a + b y tra n s d u c t i o n w i t h P l grow n on
MSC18
• s h i " , Vl 1"
p r o to tr o p h ic , la c + , x y l” ,
m tl” , f l a " , m o t" , Sa '
sr ■
* E . c o l i B i s m o t" , fla ~ ~, I a c + , m al , x y l + , m t l + , Ss } ij_a s s
p ro to tro p h ic .
c o l i K12 i s m o t+ , f I a + , I a c + , m a l+ , x y l + , m t l + , Sp9 Vj_a 3 L p^+ ,
L poS , m a t - , I i a s j p r o t o t r o p h i c .
N o t a t i o n s f o r g e n e t i c m a rk e rs a r e a s f o l l o w s $ T> t h r e o n i n e j L 5
l e u c i n e 5 B^ 5 t h ia m i n e ; £ a 5 p h e n y l a l a n i n e ; t r y , t r y p t o p h a n e ; s h i ,
s h ik i m ic a c i d ; Mj m e t h io n i n e ; A5 a r g i n i n e ; l a c ', l a c t o s e ; m a l, m a l to s e ;
x y l , x y l o s e ; m t l , m a n n i t o l ; S j s tr e p to m y c in s 7 i , s e n s i t i v i t y t o T l
and T5; V ia , s e n s i t i v i t y t o T l n o t T5 ; L p i., I y s o g e n i c i t y f o r lam b d a ;
h p p , a b i l i t y t o a d s o r b la m b d a ; m o t, m o t i l i t y ; f l a , p r e s e n c e o f f l a g e l l a ;
m a t, m u t a b i l i t y .
4
ml o f w a te r and added to th e s a l t s o lu t i o n a f t e r s t e r i l i z a t i o n
(L e d er-
b e r g , 1950)o
S a lin e :
WaGl 8 . 6 .
S tre p to m y c in ;
D ih y d r o s tr e p to m y c in s u l f a t e was u s e d i n a l l t e s t s
f o r s tr e p to m y c in r e s i s t a n c e .
A l l s tr e p to m y c in b r o t h an d a g a r m ed ia
w e re p r e p a r e d t o g i v e a f i n a l c o n c e n t r a t i o n o f 2 5 0 > ’g /m l.
G row th F a c t o r s s
The f o l l o w i n g s u p p le m e n ts w e re a d d e d t o m in im a l
a g a r p l a t e s w h e re v er a p p r o p r ia te to g iv e th e s e f i n a l c o n c e n tr a tio n s :
T h ia m in e .0 0 0 1 , j> -am inobem zoic a c i d .0 0 1 , a r g i n i n e „ 0 2 , m e th io n in e .0 2 ,
l e u c i n e „ 0 2 , p h e n y l a l a n i n e „ 0 2 , t y r o s i n e „ 0 2 , tr y p t o p h a n e „ 0 2 , t h r e o n i n e
. 0 4 , p r o l i n e „0 2 ,
C.
A g g lu tin a tio n P ro c e d u re s
I.
P r o d u c ti o n o f A n tis e r u m s
F i f t y m l o f b lo o d w e re draw n fro m
th e m a rg in a l v e in o f th e e a r o f a r a b b i t .
The b lo o d was c e n t r i f u g e d
a n d t h e se ru m d e c a n te d a n d s t o r e d i n t h e d e e p f r e e z e f o r f u t u r e u s e a s
a c o n tro l in a g g lu tin a tio n t e s t s .
V a c c in e w as a d m i n i s t e r e d i n t r a v e n o u s ­
l y t o t h e r a b b i t a c c o r d in g t o t h e f o l l o w i n g s c h e d u le ( C a r p e n t e r , 1 9 5 6 ) :
F ir s t day
0 . 1 .ml
F o u rth d ay .
0 „ 3 ml
E i g h th d a y .
0 .5 ml
E le v e n th d a y .
1 . 0 ml
F if te e n th day .
..................................... 2 .0 ml
T h re e m ore 1 . 0 m l i n j e c t i o n s w e re g iv e n a t w e e k ly i n t e r v a l s and
t h e r a b b i t w as b l e d on t h e f i f t y - s e c o n d d a y .
The b lo o d w as c e n t r i f u g e d .
5
t h e serum, d e c a n t e d a n d f r o z e n f o r f u t u r e u s e .
2.
P r e p a r a t i o n o f V a c o in e :
K b l l e f l a s k s o f n u t r i e n t a g a r w e re
i n o c u l a t e d w i t h n u t r i e n t b r o t h c u l t u r e s o f a p p r o p r i a t e s t r a i n s and in c u ­
b a te d o v e r n i g h t a t 37 C.
s te rile
Grow th was rem oved c a r e f u l l y by w a s h in g w ith
s a li n e c o n ta in in g 0 .2 p e r c e n t f o rm a lin .
N u trie n t a g a r p la te s
w e re s t r e a k e d w i t h t h e c e l l s u s p e n s i o n an d i n c u b a t e d a t 37 C t o t e s t
f o r v i a b i l i t y o f c e ll s in th e f o rm a lin -tre a te d c u ltu r e .
Tubes o f b r a i n
h e a r t i n f u s i o n a g a r a l s o w e re i n o c u l a t e d a n d in c u b a t e d a t 37 C t o c h eck
f o r t h e p r e s e n c e o f v i a b l e a n a e r o b ic c o n ta m in a n t s .
No g ro w th e v e r was
o b s e r v e d on e i t h e r p l a t e s o r s t a b s a f t e r s e v e n d ay s i n c u b a t i o n .
The c e l l s w e re s h a k e n b y h a n d i n s c r e w - c a p t e s t t u b e s c o n t a i n i n g
g l a s s b e a d s i n o r d e r t o o b t a i n a hom ogeneous s u s p e n s i o n .
T h is ' was
f i l t e r e d th r o u g h s t e r i l e g a u z e a n d s t e r i l e g l a s s w ool and s t a n d a r d i z e d
w i t h a M c F a r la n d 's n e p h e lo m e te r t o a d e n s i t y o f a p p r o x i m a t e ly I x IO^
c e l l s p e r m l.
3«
P r e p a r a t i o n o f A b s o rb in g A n t i g e n s
N u t r i e n t a g a r p l a t e s w e re
i n o c u l a t e d w i t h 0 .1 m l q u a n t i t i e s o f a n o v e r n i g h t P e n n a s s a y b r o t h c u l ­
tu re .
The n u t r i e n t a g a r p l a t e s w e re i n c u b a t e d t w e n t y - f o u r h o u r s a t 37 C
and t h e g ro w th was rem oved b y w a s h in g w i t h 5 .0 ml o f f o h m a lin iz e d . ( 0 . 5J>)
s a lin e .
4.
Serum A b s o r p t i o n s
A m o d i f i c a t i o n o f t h e m eth o d s recom m ended
b y t h e M ack ie a n d M c C a rtn e y Handbook ( C r u ic k s h a n k 5 i 9 6 0 ) w as f o llo w e d .
The f o r m a l i n i z e d c e l l s u s p e n s i o n s w e re c e n t r i f u g e d a n d r e s u s p e n d e d i n
s u c c e s s i v e v o lu m e s o f t h e s a l i n e s u s p e n s i o n s e v e r a l tim e s i n o r d e r t o
6
o b ta in a s u ita b le c e ll d e n s ity .
The c o n c e n t r a t e d s u s p e n s i o n w as ad d ed
t o an e q u a l volum e o f a 1?5 d i l u t i o n o f c o m p le te a n t i s e r u m an d t h e m ix­
t u r e was i n c u b a t e d a t 55 'C i n t h e w a t e r b a t h f o r t w e n ty h o u r s .
The
c e l l s w e re a g a i n c e n t r i f u g e d and t h e s u p e r n a t a n t f l u i d w as t e s t e d f o r
p r e s e n c e o f hom ologous a n t i b o d y b y t i t r a t i o n w i t h P e n n a s s a y b r o t h c u l ­
tu re s .
I f t h e s e ru m w as fo u n d t o b e i n c o m p l e t e l y a b s o r b e d , i t w as r e ­
i n c u b a t e d w i t h p a c k e d c e l l s i n o r d e r t o rem ove r e m a in in g a n t i b o d y w i t h ­
o u t f u r t h e r d i l u t i n g th e a n tis e ru m .
When t h e a n t i s e r u m w as fo u n d t o
b e c o m p l e te l y a b s o r b e d , i t w as f i l t e r e d
5.
A g g lu tin a tio n T e s ts s
th ro u g h a S e itz f i l t e r .
F o r q u a n ti ta t iv e t e s t s , tw o -fo ld s e r i a l
d i l u t i o n s o f a n ti s e r u m w e re made w i t h f o r m a l in i z e d s a l i n e .
A l i q u o ts
o f 0 .5 ml o f t w e n t y - f o u r h o u r P e n n a s s a y b r o t h c u l t u r e s w e re a d d e d t o
0 . 5 m l o f t h e se ru m d i l u t i o n i n e a c h t u b e .
A l l t e s t s w e re in c u b a t e d
i n t h e w a t e r b a t h a t 55 C f o r tw o h o u r s an d r e a d m a c r o s c o p i c a l l y by
o b l i q u e l i g h t on a Kahn v i e w e r .
6.
P r e p a r a t i o n o f A n tig e n s f o r H e a t S t a b i l i t y T e s t s s
P en n assay
b r o t h . c u l t u r e s w e re h e a t e d f o r t h e a p p r o p r i a t e l e n g t h o f tim e i n th e
w a t e r b a t h w hen t e m p e r a t u r e s b e lo w 100 C w e re i n d i c a t e d .
F o r te m p e ra ­
t u r e s o f 100 C and h i g h e r , c u l t u r e s w e re h e a t e d i n t h e a u t o c l a v e .
D„
P ro c e d u re s I n v o lv in g C ro sse s
1.
F In fe c tio n P ro c e d u re s;
2.
C ro s s P r o c e d u r e s :
See s e c t i o n D, p a r t 2 o f R e s u l t s .
F i v e xm l q u a n t i t i e s o f s i x t e e n h o u r
P e n n a s s a y b r o t h c u l t u r e s w e re c e n t r i f u g e d , w ash ed i n s a l i n e , an d r e s u s ­
p e n d e d i n 1 .0 m l o f s a l i n e .
The s a l i n e s u s p e n s i o n s w e re m ixed w e l l
7
and 0 .1 m l o f t h e s u s p e n s i o n o f e a c h c u l t u r e in v o lv e d i n t h e c r o s s was
p i p e t t e d t o a p l a t e o f DMA.
The two s u s p e n s i o n s w e re m ixed w e l l w i t h a
g l a s s s p r e a d e r and a llo w e d t o d r y .
C o n tro ls c o n s is te d o f
001 .m l.i.
q u a n t i t i e s o f e a c h c u l t u r e p l a t e d s e p a r a t e l y on DMA p l a t e s .
A ll c u l­
t u r e s w e re i n c u b a t e d f o r f o r t y - e i g h t h o u r s and s c o r e d f o r p r o t o t r o p h i c
r e c o m b in a n t s .
3«
S c o r in g o f R e c o m b in a n t M a r k e r s ; . To s c o r e f o r p r o t o t r o p h y , an
in o c u lu m fro m w e l l - p u r i f i e d r e c o m b in a n ts was grown i n b r o t h a n d s t r e a k e d
t o . . p l a t e s o f DMA.
To s c o r e f o r f e r m e n t a t i v e m a r k e r s , s i m i l a r b r o t h
c u l t u r e s w e re s t r e a k e d on a p p r o p r i a t e p l a t e s o f M B .
S tr e p to m y c in r e ­
s i s t a n c e w as t e s t e d by s t r e a k i n g b n n u t r i e n t a g a r p l a t e s c o n t a i n i n g
s tre p to m y c in .
S e n s i t i v i t y t o b a c t e r i o p h a g e w as t e s t e d a s f o l l o w s s
N u t r i e n t a g a r p l a t e s w e re s t r e a k e d o n c e w i t h a l a r g e l o o p f u l o f a s u s ­
p e n s io n o f t h e a p p r o p r i a t e p h a g e and a llo w e d t o d r y .
B r o th c u l t u r e s t o
be t e s t e d w e re t h e n s t r e a k e d a c r o s s t h e p h a g e in o c u lu m .
P l a t e s w ere
i n c u b a t e d f o r t w e n t y - f o u r h o u r s and e x a m in ed f o r ' l y s i s .
4»
P r e p a r a t i o n o f Lambda L y s a t e s :
A tw e n ty -fo u r h o u r P en n assay
b r o t h c u l t u r e o f a l y s o g e n i c s t r a i n w as c e n t r i f u g e d t o s e d im e n t t h e
The s u p e r n a t a n t f l u i d w as r e t a i n e d a n d 0 .1 ml o f CBCl3 was
c e lls .
ad d e d t o
$ .0 m l o f t h e f l u i d
t o k i l l a n y r e m a in in g c e l l s .
The m ix tu re
w as a llo w e d t o s t a n d a t room t e m p e r a t u r e f o r t w e n ty - f o u r h o u r s b e f o r e
use.
5.
T e s ts f o r L y s o g e n i c i t y t
S ee s e c t i o n D, p a r t 3 o f R e s u l t s .
11 Il
8
E„
In c u b a tio n o f C u ltu re s
A l l c u l t u r e s w e re i n c u b a t e d a t 37 C u n l e s s o t h e r w i s e s t a t e d .
RESULTS
A.
S e r o l o g i c a l A n a l y s is o f t h e S u r f a c e A n tig e n s o f G S ll
A n tis e r u m p ro d u c e d i n r e s p o n s e t o f o r m a l i n - k i l l e d w h o l e - c e l l
s u s p e n s i o n s o f t h e E. c o l i s u b - s t r a i n , G S l l , a g g l u t i n a t e s c u l t u r e s o f
t h e n o n - f l a g e l l a t e d K12 x B h y b r i d , HSC56 ( s e e T a b le I I ) .
B oth MSO56
an d C S ll a r e a g g l u t i n a t e d by h i g h ( I $1280) d i l u t i o n s o f C S ll a n ti s e r u m j
b u t when t h e a n ti s e r u m i s a b s o rb e d w i t h d e n s e c e l l s u s p e n s io n s o f MSC56
u n til th is s tr a in
no l o n g e r i s a g g l u t i n a t e d , C S ll c o n ti n u e s t o r e a c t
w i t h 1$160 d i l u t i o n s , s u g g e s t i n g t h a t t h e a n ti s e r u m c o n t a i n s two a n t i ­
b o d y c o m p o n e n ts, one o f w h ic h d o e s n o t h a v e an a n t i g e n i c c o u n t e r p a r t
i n MSC56 .
T h i s , i n t u r n , i n d i c a t e s t h a t C S ll p o s s e s s e s a t l e a s t two
g e n e t i c a l l y s e p a r a b l e a n t i g e n i c c o m p o n e n ts .
A m o t i l e K12 x B h y b r i d , 1SC 39, a l s o i s a g g l u t i n a t e d by h ig h
d i l u t i o n s o f C S ll a n t i s e r u m .
L ik e MSC5 6 , MSC39 a l s o o n l y p a r t i a l l y
a b s o r b s C S ll a g g l u t i n i n s fro m a n ti s e r u m p r e p a r e d a g a i n s t C S l l .
Ab­
s o r p t i o n by MSC39 i s n o t n e a r l y a s e f f e c t i v e a s a b s o r p t i o n b y MSC56 .
R e s u l t s g iv e n i n T a b le I I show t h a t a n t i s e r u m a b s o rb e d b y MSC39 a g g l u t ­
i n a t e s C S ll a t a I s 640 d i l u t i o n .
N o n e t h e le s s , t h e p o s s i b i l i t y a r i s e s
t h a t t h e two h y b r i d s a r e a n t i g e n i c a l l y a l i k e .
To t e s t t h i s p o s s i b i l i t y ,
a n t i s e r u m w as p r e p a r e d w i t h HSC39 and t e s t e d f o r i t s a b i l i t y t o a g g l u t ­
in a te th e th re e s t r a i n s .
R e s u l t s g iv e n i n T a b le I I show t h a t h ig h
d i l u t i o n s o f t h i s a n t i s e r u m a g g l u t i n a t e d b o th C S ll and MSC39.
was n o t a g g l u t i n a t e d e v e n a t a d i l u t i o n . o f I s 2 0 .
MSC56
T hese r e s u l t s demon­
s t r a t e d a n a n t i g e n i c r e l a t i o n s h i p b e tw e e n MSC39 and C S ll b u t n o t b e tw e e n
10
T a b le I I .
T est
a n tig e n
S e r o l o g i c a l R e l a t i o n s h i p s Among G S ll 3 MSC393 a n d MSCg6 .
R e c i p r o c a l o f a g g l u t i n a t i n g t i t e r i n C S ll a n tis e r u m ?
U n a b s o rb e d
A b so rb e d w i t h MSG39
A b so rb e d w i t h MSCgb
maximum d e t e c t a b l e
maximum d e t e c t a b l e
maximum d e t e c t a b l e
a g g lu t.
a g g lu t.
a g g lu t.
a g g lu t.
a g g lu t.
a g g lu t.
G S ll
1280
2 g 60
640
1280
80
IbO
MSCgb
1280
2 g60
640
1280
0
0
MSC39
640
1280
0
0
IbO
320
R e c i p r o c a l o f a g g l u t i n a t i n g t i t e r i n MSC39 a n t i s e r u m s
maximum d e t e c t a b l e
a g g lu t.
a g g lu t.
640
1280
MSCgb
0
0
MSC39
b 40
1280
G S ll
H0 n m eans n e g a t i v e i n a serum , d i l u t i o n o f 1 : 2 0 .
t u b e s w e re a l l 0 .
C o n t r o l serum
11
MSC39 a n d MSC56.
T hese r e l a t i o n s h i p s w e re c l a r i f i e d f u r t h e r when i t
w as f o u n d t h a t ' a n t i s e r u m a b s o r b e d w i t h MSC56 c o n tin u e d t o a g g l u t i n a t e
I
MSC39 a n d t h a t a n t i s e r u m a b s o r b e d w i t h MSC39 c o n ti n u e d t o a g g l u t i n a t e
MSC56.
The a b i l i t y o f MSC56 t o lo w e r t h e a g g l u t i n a t i n g t i t e r o f C S ll
a n t i s e r u m f o r MSC39 i s u n e x p l a i n e d .
When C S ll a n ti s e r u m w as a b s o rb e d
s e q u e n t i a l l y w i t h MSC56 a n d ESC39, i t no l o n g e r a g g l u t i n a t e d C S l l . .
It
was c o n c lu d e d t h a t C S ll a n t i s e r u m c o n t a i n s tw o a n t i b o d y c o m p o n e n ts, one
o f w h ic h c o r r e s p o n d s t o t h e f l a g e l l a r a n t i g e n o f ESC39$ t h e o t h e r t o
an a n t i g e n c a r r i e d b y MSC56 and n o t c l e a r l y a s s o c i a t e d w i t h f l a g e l l a .
R e s u l t s o f t h e f o l l o w i n g t e s t show t h a t t h e a n t i g e n o f MSC56 , when
c a r r ie d b y C S ll, i s n o t in tim a te ly a s s o c ia te d w ith th e f l a g e l l a .
When
m o t i l i z e d c u l t u r e s a r e i n o c u l a t e d i n t o t h e c e n t e r o f a p l a t e o f sem i­
s o l i d c u l t u r e m edium , t h e c e l l s w i l l m i g r a t e th r o u g h t h e medium p ro d u c ­
i n g a v i s i b l e a n d i n c r e a s i n g l y l a r g e r c i r c l e o f g ro w th e x t e n d i n g to w a rd
th e p e r ip h e r y o f th e p l a t e .
I f hom ologous f l a g e l l a r a n t i b o d y i s i n ­
c o r p o r a t e d i n t o t h e m edium , t h i s m i g r a t i o n o f c e l l s i s im p ed ed and
g ro w th i s
c o n f in e d t o t h e s i t e o f i n o c u l a t i o n .
To d e te r m in e w h e th e r
t h e a n ti b o d y r e m a in in g i n C S ll a n ti s e r u m a b s o rb e d w i t h MSC39 i s a s s o c i ­
a t e d w i t h f l a g e l l a r a n t i g e n , a s o f t a g a r p l a t e c o n t a i n i n g a I §50 d i l u ­
t i o n o f t h i s a n ti s e r u m w as i n o c u l a t e d w i t h a n a c t i v e l y m o t i l e c u l t u r e
o f C S ll.
A t t h e same t i m e , a p l a t e c o n t a i n i n g t h e same d i l u t i o n o f
u n a b s o r b e d C S ll a n t i s e r u m was s i m i l a r l y i n o c u l a t e d .
A t h i r d in o c u la te d
p l a t e c o n ta in in g , n o rm a l se ru m s e r v e d a s a c o n t r o l .
The c u l t u r e swarmed
t h r o u g h t h e m edium c o n t a i n i n g t h e a b s o r b e d a n ti s e r u m a s w e l l a s th r o u g h
J
12
t h a t c o n t a i n i n g c o n t r o l se ru m .
c o n t a i n i n g u n a b s o rb e d s e ru m .
No sw a rm in g o c c u r r e d i n t h e medium
I t is
i n f e r r e d fro m t h i s d a t a t h a t t h e
a n t i b o d y r e m a in in g i n t h e a b s o rb e d s e ru m d o e s n o t c o r r e s p o n d t o an
a n tig e n o f s t r u c t u r a l C d ll f l a g e l l a .
The f l o c c u l e n t n a t u r e o f th e . MSC56 a g g l u t i n a t i o n s u g g e s t s t h a t ,
as in
"H" ( f l a g e l l a r ) a g g l u t i n a t i o n , t h e r e a c t i n g a n t i g e n i s s i t u a t e d
a t some d i s t a n c e fro m t h e m a in c o n to u r o f t h e c e l l .
I n f a c t , th e
p o s s i b i l i t y a r i s e s t h a t t h e a n t i g e n o f MSC56 i s a " s e c r e t i o n " n o t p h y s i ­
c a ll y in c o n ta c t w ith th e c e l l .
T h is s i t u a t i o n seem s u n l i k e l y i n v ie w
o f r e s u l t s o b t a i n e d fro m t h e f o l l o w i n g e x p e r im e n t.
A tw e n ty -fo u r h o u r
P e n n a s s a y b r o t h c u l t u r e o f ^ K12 s u b - s t r a i n (MSC501) known t o l a c k t h e
a n t i g e n p o s s e s s e d b y MSC56 ( s e e l a t e r s e c t i o n ) - and a s i m i l a r c u l t u r e o f
MSC56 w e re c e n t r i f u g e d , d e c a n t e d , w a sh e d i n s t e r i l e b r o t h , a n d r e s u s ­
pended in th e r e c ip r o c a l s u p e rn a ta n t b r o th c u ltu r e f l u i d s .
The c e l l s
o f MSC56 r e s u s p e n d e d i n t h e s u p e r n a t a n t f l u i d o f t h e MSC501 c u l t u r e
w e re a g g l u t i n a t e d b y a I s lb O d i l u t i o n o f a b s o r b e d a n t i s e r u m j no a g g l u t ­
i n a t i o n w as s e e n i n th e. s u s p e n s i o n o f MSC501 c e l l s .
Thus i t a p p e a r s
t h a t t h e MSC56 a n t i g e n i s a n a d h e r e n t p a r t o f t h e c e l l s u r f a c e .
B.
P h y s i c a l P r o p e r t i e s o f t h e A n tig e n o f MSS5 6
The a n t i g e n - a n t i b o d y a g g r e g a te fo rm e d by MSC56 a n d b y C S ll i n
re s p o n s e to a n tis e ru m i s v e ry s im i la r t o , b u t n o t i d e n t i c a l w ith , t h a t
p ro d u c e d b y MSG39«.
I n b o t h c a s e s , t h e r e s p o n s e i s r a p i d | a g g l u t i n a t i o n ..
u s u a l l y i s v i s i b l e i n t h e f i r s t tu b e w i t h i n t e n m in u te s a n d i s
w i t h i n an h o u r .
c o m p le te
B o th g iv e t h e f l o c c u l e n t a p p e a r a n c e c h a r a c t e r i s t i c o f
13
f l a g e l l a r a g g l u t i n a t i o n b u t t h e a g g r e g a te fo rm e d b y MSC39 i s v e r y f r a g i l e
a n d e a s i l y d i s p e r s e d by s h a k in g .
The a g g r e g a t e s fo rm e d b y MSC56 and b y
C S ll a r e n o t c o m p l e te l y d i s p e r s e d b y s h a k in g b u t re m a in s u s p e n d e d a s
d i s c r e t e p a r t i c l e s w h ic h a r e r e a d i l y v i s i b l e w hen v ie w e d u n d e r t h e
d i s s e c t i n g m ic r o s c o p e .
W e ib u ll ( 1 9 4 9 ) h a s d o n e an e x t e n s i v e c h e m ic a l a n a l y s i s o f t h e
f l a g e l l a o f some o f t h e E n t e r o b a c t e r i a c e a e a n d fo u n d them t o be m a in ly ,
i f n o t c o m p l e te l y , p r o t e i n .
The a n t i g e n i c s t a b i l i t y o f t h e f l a g e l l a
o f t h i s g ro u p i s d e s c r i b e d ( C r u i c k s h a n k , i 9 6 0 ) a s b e in g d e s t r o y e d a t
t e m p e r a t u r e s o f fro m 80 C t o 100 C f o r a b o u t tw e n ty m in u t e s .
l a b i l i t y , a lo n g w ith th e f la k y n a tu r e o f th e a g g lu t in a t io n , i s
te ris tic
T h is h e a t
c h a ra c ­
o f t h e f l a g e l l a r a n t i g e n - a n t i b o d y r e a c t i o n and s e r v e s t o d i s ­
t i n g u i s h i t fro m o t h e r s e r o l o g i c a l r e a c t i o n s commonly o b s e r v e d f o r t h e
s u r f a c e a n t i g e n s i n t h i s g ro u p o f b a c t e r i a .
I f t h e a n t i g e n o f MSC56
i s , i n f a c t , a f l a g e l l a r p r o t e i n , i t s h o u ld be h e a t l a b i l e l i k e th e
a n tig e n o f s t r u c t u r a l f l a g e l l a .
T h is p o i n t i s o f m ore t h a n i n c i d e n t a l
i n t e r e s t f o r th e fo llo w in g re a s o n .
M o ti l e s t r a i n s o f E . c o l i . u n l i k e
t h e c l o s e l y - r e l a t e d S a l m o n e ll a , a r e m o n o p h a s ic .
No s t r a i n s h a v e b e e n
d e s c r i b e d w h ic h h a v e m ore t h a n one f l a g e l l a r s e r o t y p e .
I t may b e ,
h o w e v e r, t h a t E. c o l i i s n o r m a lly d i p h a s i c , one p h a s e r e p r e s e n t e d by a
u n iq u e s t a t e i n w h ic h f l a g e l l a r p r o t e i n i s p r e s e n t b u t n o t o r g a n iz e d a s
s tru c tu ra l f la g e lla .
T h is w o u ld be c o n s i s t e n t w i t h t h e f a i l u r e o f
s t a n d a r d m eth o d s t o d e t e c t a t y p i c a l d i p h a s i c c o n d i t i o n .
^ rsk o v
a n d 0 r s k o v ( i 9 6 0 ) r e j e c t t h e K12 and B s t r a i n s o f E . c o l i
14
a s s u i t a b l e s u b j e c t s f o r s e r o l o g i c a l s t u d y o f s u r f a c e a n t i g e n on th e
b a s is t h a t th e y a re a u to - a g g lu tin a b le ,
H a c c a c a ro and Colom bo ( 1 9 56 )
r e p o r t t h e d e t e c t i o n o f K a n d O a n t i g e n s i n K12 s t r a i n s u s i n g s a l i n e
s u s p e n s i o n s o f c e l l s i n a g g l u t i n a t i o n t e s t s d e s ig n e d t o com pare a n t i ­
g e n ic p r o p e r t i e s o f F - a n d F+ c u l t u r e s .
O ur e x p e r i e n c e h a s b e e n t h a t
c u ltu r e s su sp en d ed in s a lin e a re r e a d i ly a u to - a g g lu tin a b le e s p e c ia lly
u pon h e a t i n g .
S i m i l a r l y , s l i d e a g g l u t i n a t i o n t e s t s u s i n g g ro w th fro m
s o l i d m e d ia s u s p e n d e d i n s a l i n e h a v e b e e n fo u n d t o b e u n r e l i a b l e .
•However, b y m eans o f tu b e a g g l u t i n a t i o n s c a r r i e d o u t w i t h P e n n a s s a y
b ro th c u ltu r e s , i t has been p o s s ib le to o b ta in r e l i a b l e s e r o lo g ic a l
d a ta .
The h e a t s t a b i l i t i e s o f C S l l , MSE39, a n d MSO56 w e re ex am in ed b y
t h i s m e th o d .
R e s u l t s a p p e a r i n T a b le I I I .
A l l t h r e e s t r a i n s w e re h e a te d t o T l C f o r f i f t e e n m in u te s and
C S ll an d MSC56 con­
s u b s e q u e n tly t e s t e d f o r r e a c ti o n w ith a n tis e ru m .
t i n u e d t o be a g g l u t i n a t e d b y 1 :6 4 0 d i l u t i o n s o f C S ll a n t i s e r u m .
MSC39 show ed no a g g l u t i n a t i o n w i t h C S ll a n t i s e r u m .
N e i t h e r MSC39 n o r
C S ll w e re a g g l u t i n a t e d b y MSC39 a n t i s e r u m e v e n a t a 1 :2 0 d i l u t i o n .
As t h e c u l t u r e s o f C S ll and MSC56 w e re e x p o s e d t o i n c r e a s e s i n te m p e r­
a t u r e , t h e i r a b i l i t y t o be a g g l u t i n a t e d d e c l i n e d .
B o th , h o w e v e r,
g a v e a s t r o n g r e a c t i o n i n a 1 :2 0 d i l u t i o n o f a n ti s e r u m e v e n a f t e r h a v in g
b e e n h e a te d t o H O ti f o r a n h o u r .
No a p p r e c i a b l e d i f f e r e n c e i n a g g l u t -
i n a b i l i t y was n o t e d f o r a n y g iv e n t e m p e r a t u r e i f t h e tim e o f h e a t i n g
was i n c r e a s e d fro m f i f t e e n m in u te s t o an h o u r .
'F o r e x a m p le , C S ll
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100 C f o r f i f t e e n m in u te s and r e a c t e d t o a p p r o x i m a t e ly t h e same d i l u ­
t io n a f t e r h e a tin g f o r a f u l l h o u r.
T hese e x p e r im e n ts i n d i c a t e c l e a r l y t h a t th e a n t i g e n s o f MSC39
and MSC56 h a v e d i s t i n c t and d i f f e r e n t h e a t s t a b i l i t i e s .
The b e h a v io r
o f t h e MSC39 a n t i g e n i s c o m p l e te l y c o m p a tib le w i t h t h e a c c e p t e d c r i t e r ­
i o n f o r 11H11 a n t i g e n i c i t y .
T h a t o f MSC56 i s n o t .
I t is n e c e ssa ry ,
t h e r e f o r e , t o e x c lu d e t h e h y p o t h e s i s t h a t t h e MSG56 h e a t s t a b l e a n t i g e n
i s a n o n -fu n c tio n in g f l a g e l l a r a n tig e n .
I n s u b s e q u e n t s e c t i o n s , t h i s a n t i g e n w i l l be r e f e r r e d t o a s t h e
H e a t S t a b l e A n tig e n (HSA) and G S ll a n t i s e r u m a b s o rb e d w i t h MSC39 a s
HSA a n t i s e r u m .
C.
O c c u r re n c e o f HSA Among K12 S t r a i n s
.
S in c e no a n t i g e n l i k e HSA h a s b e e n d e s c r i b e d f o r E , c o l i K12,
i t was a p p r o p r i a t e t o d e te r m in e w h e th e r C S ll was an u n u s u a l a n t i g e n i c
m u ta n t o f t h e s t r a i n .
in our c o lle c tio n .
A s u r v e y w as made fro m among t h e s t o c k c u l t u r e s
N in e s u b - s t r a i n s w e re ex am ined f o r p r e s e n c e o f t h e
a n t i g e n a s w e l l a s f o r a l l o t h e r known m a r k e r s .
T a b le IV*
R e s u l t s a r e shown i n
The f o l l o w i n g s e v e n c u l t u r e s r e a c t e d w i t h HSA a n ti s e r u m and
f a i l e d t o a g g l u t i n a t e when t e s t e d q u a n t i t a t i v e l y w i t h c o n t r o l serum s
MSG503, G S l l , MSC69, MSC70, W6, CS19, a n d MSC18.
The H f r s t r a i n , C S lO l,
i s a g g l u t i n a t e d b u t g i v e s a l e s s s t r i k i n g r e a c t i o n w i t h a n t i s e r u m th a n
do t h e c u l t u r e s c i t e d a b o v e .
A n o th e r H f r c u l t u r e , TM1895, a g g l u t i n a t e s
s p o n ta n e o u s ly b o t h i n t e s t se ru m a n d i n c o n t r o l s e ru m .
MSC501 a lo n e
i s s t a b l e i n c o n t r o l seru m and f a i l s t o r e a c t w i t h a n t i s e r u m , and i s .
17
T a b le IV .
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9 L-
^N um bers g i v e n I n t h i s colum n r e p r e s e n t t h e r e c i p r o c a l o f t h e
h i g h e s t d i l u t i o n o f a n t i s e r u m w h ic h g i v e s a g g l u t i n a t i o n .
"A" i n t h i s
colum n m eans a u t o - a g g l u t i n a t i o n . A ll. o t h e r seru m c o n t r o l s w e re n eg a­
tiv e .
nOn i n t h i s colum n m eans n e g a t i v e i n a serum d i l u t i o n o f 1 :2 0 .
R o r S i n d i c a t e r e s i s t a n c e o r s e n s i t i v i t y t o s tr e p to m y c in
( colum n 3 ) o r t o T5 b a c t e r i o p h a g e ( colum n V]_). + o r - i n d i c a t e a b i l i t y
o r la c k o f a b i l i t y to fe rm e n t th e fo llo w in g c a rb o h y d ra te s : l a c t o s e ,
m a l t o s e , x y l o s e , m a n n i t o l . N o t a ti o n f o r a u x o tr o p h i c m a r k e rs i s a s
f o l l o w s : A, a r g i n i n e I L , l e u c in e | T, t h r e o n i n e j B]_, t h ia m i n e j M,
m e t h io n i n e j p a , p h e n y l a l a n i n e „I
I
J
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h e n c e HSA- «
As c a n b e s e e n fro m T a b le IV s no o b v io u s c o r r e l a t i o n e x i s t s
b e tw e e n t h e HSA p r o p e r t y a n d p o s s e s s i o n o f a n y o t h e r known m a r k e r .
No
c o r r e l a t i o n b e tw e e n c o l o n i a l m o rp h o lo g y a n d HSA h a s b e e n o b s e r v e d .
Some
o f t h e HSA+ s t r a i n s
c o n s i s t e n t l y p ro d u c e r o u g h c o l o n i e s w h i l e o t h e r s
p ro d u c e sm o o th c o l o n i e s .
P o s s e s s i o n o f HSA can n o t be c o r r e l a t e d w i t h
t h e m u co id a p p e a r a n c e w h ic h K12 c u l t u r e s o f t e n p r e s e n t .
C S lls f o r
e x a m p le s p r o d u c e s l a r g e am ounts o f m ucus a n d c o n s i s t e n t l y r e a c t s w i t h
h i g h d i l u t i o n s o f HSA a n t i s e r u m .
ESC501 a l s o t e n d s t o p ro d u c e l a r g e
am o u n ts o f m ucus b u t d o e s n o t r e a c t w i t h HSA a n ti s e r u m e v e n when t e s t
a n t i g e n s a r e p r e p a r e d fro m p a r t i c u l a r l y m u co id g ro w th .
P e n n a ssa y b ro th
c u l t u r e s o f HSA+ s t r a i n s t y p i c a l l y p ro d u c e a n a d h e r e n t r e s i d u e a ro u n d
t h e i n s i d e o f t h e t e s t tu b e w h ic h can b e d e m o n s tr a te d b y t i l t i n g t h e
c u l t u r e tu b e t o d i s p l a c e t h e b r o t h fro m t h e s i d e o f t h e t u b e .
The
p r e s e n c e o f t h i s r e s i d u e i s r e l i a b l e e n o u g h t o s e r v e a s a c o n v e n ie n t
s c r e e n i n g d e v ic e i n s e a r c h i n g f o r HSA+ c u l t u r e s .
h o w e v e r.
I t is not in fa llib le
B r o th c u l t u r e s o f some s t r a i n s w h ic h p ro d u c e ro u g h c o lo n i e s
on s o l i d m edium a r e o f t e n c l e a r w i t h m q st o f t h e c e l l s s e d im e n te d a t
t h e b o tto m o f t h e t u b e .
T h ese b r o t h c u l t u r e s do n o t p ro d u c e th e a d h e r ­
e n t r e s i d u e c h a r a c t e r i s t i c o f HSA+ s t r a i n s b u t may g i v e t y p i c a l a g g l u t ­
i n a t i o n when t e s t e d w i t h a n t i s e r u m .
The r e s u l t s o f t h i s l i m i t e d s u r v e y o f s t r a i n s
c l e a r l y shows
t h a t p o s s e s s i o n o f HSA i s n o t a u n iq u e p r o p e r t y o f C S ll b u t i s
c h a ra c ­
t e r i s t i c o f many K12 s u b - s t r a i n s a n d s f u r t h e r , t h a t t h i s a n t i g e n i c
19
p ro p e rty i s
n o t a p l e i o t r o p i c e f f e c t o f a n y o f t h e known g e n e t i c m a r k e r s „
The c o n s i s t e n t an d u n if o r m r e a c t i o n o f m o st c u l t u r e s when t e s t e d w i t h
a n t i s e r u m show s t h a t HSA i s a s t a b l e c h a r a c t e r i s t i c n o t r e a d i l y i n f l u ­
e n c e d b y v a r i a t i o n i n g e n e t i c o r e n v ir o n m e n ta l b a c k g ro u n d .
T h a t th e
HSA“ p r o p e r t y o f MSGfiOl i s l i k e w i s e s t a b l e i s i n f e r r e d fro m two y e a r s
o b s e rv a tio n o f t h i s
s tra in .
Even m ore d r a m a ti c e v id e n c e o f t h e s t a b i l ­
i t y o f HSA~ w as p r o v id e d b y e x a m in a tio n o f a new c u l t u r e o f THTll77
p r o v id e d b y D r. J . L e d e r b e r g .,
MSCfJOl i s a c u l t u r e o f 1 1 1 7 7 W hichs i n
t h e c o u r s e o f s t o r a g e i n D r. S k a a r t S l a b o r a t o r y , h a s becom e m a l+, m t l +,
and
The L e d e r b e r g c u l t u r e o f 1 1 1 7 7 h a s a c q u ir e d a new r e q u ir e m e n t
f o r p h e n y la la n in e .
I n s p i t e o f t e n y e a r s i s o l a t i o n , t h e s e tw o 1 1 1 7 7
d e r i v a t i v e s a r e b o t h HSAy.
I n f r e q u e n t r e v e r s i o n s o f HSA- t o HSA+ and
t h e r e v e r s e among v a r i a n t s o f 1 1 1 7 7 a n d MSGfJOl w i l l b e t h e s u b j e c t o f
th e n e x t s e c tio n .
D.
M u ta tio n o f t h e HSA P r o p e r t y i n D e r i v a t i v e s o f 1 1 1 7 7
The HSA- n a t u r e o f MSCfJOl h a s b e e n known f o r a b o u t two y e a r s .
D u rin g t h i s p e r i o d t h e c u l t u r e h a s b e e n p r o p a g a te d on n u t r i e n t a g a r
s l a n t s and h a s been r e p e a te d ly s u b c u ltu r e d .
B ro th c u l t u r e s i n o c u l a t e d
fro m t h e a g a r s l a n t s h a v e c o n s i s t e n t l y f a i l e d t o r e a c t w i t h HSA a n t i ­
seru m 3 h o w e v e r, on one o c c a s i o n ,, when b r o t h c u l t u r e s w e re d i l u t e d and
p l a t e d on s o l i d m edium t o g iv e i s o l a t e d c o l o n i e s w h ic h , i n t u r n , w ere
t r a n s f e r r e d t o b r o t h , a few HSA+ r e v e r t a n t s w e re d e t e c t e d .
A ll o f
t h e s e r e v e r t a n t s a r o s e on p l a t e s o f EMB l a c t o s e w h ic h h a d b e e n s p r e a d
w i t h h i g h d i l u t i o n s o f o n e P e n n a s s a y b r o t h c u l t u r e o f MSGfJOl.
F iv e o f
20
f o r t y c o l o n i e s p ic k e d w e re fo u n d t o b e HSA4-.
A ll o f th e s e c u ltu r e s
h a v e b e e n r e t a i n e d on n u t r i e n t a g a r s l a n t s a n d c o n tin u e t o b e s t a b l e
f o r t h e HSA+ p r o p e r t y and g i v e r e a c t i o n s i n a n ti s e r u m w h ic h a r e com­
p a ra b le w ith t h a t o f C S ll.
One o f t h e s e c u l t u r e s i s MSC505.
I t seem s u n l i k e l y t h a t g ro w th on EMB l a c t o s e h a d in d u c e d t h e
c h an g e i n a n t i g e n i c i t y ; b u t , t o t e s t t h i s p o s s i b i l i t y , a n o t h e r in o c u lu m
fro m t h e same MSC501 s l a n t w as grown u p i n b r o t h , d i l u t e d , and p l a t e d
on EMB l a c t o s e ,
A s e c o n d p l a t i n g w as made fro m an HSA+ r e v e r t a n t .
F o r t y c o l o n i e s fro m e a c h p l a t i n g w e re e x a m in ed f o r m u ta ti o n o f HSA.
No m u ta ti o n i n e i t h e r d i r e c t i o n was o b s e r v e d .
s a m p lin g i s o f l i t t l e
O b v io u s ly , t h i s s m a ll
v a l u e i n d e te r m i n in g t h e r a t e o f m u ta ti o n o f t h e
HSA c h a r a c t e r , b u t t h e r e s u l t s s t r e n g t h e n o u r i m p r e s s i o n t h a t th e
c h an g e i s r a r e a n d o c c u r s w i t h a f r e q u e n c y t h a t i s m ore c o m p a tib le w i t h
a t r u e m u ta ti o n t h a n w i t h a m ore n a r r o w ly o s c i l l a t i n g c h a n g e s u c h a s
p hase v a r i a t i o n .
H i s t o r i c a l l y , W ll77 w as o b t a i n e d b y D r, L e d e r b e r g a s a s t r e p t o ­
m y cin r e s i s t a n t m u ta n t o f ¥ 6 7 7 ,
O ur c u l t u r e o f ¥ 67 ? (w h ic h h a s b een
re n u m b e re d MSC503 b e c a u s e o f a m u ta ti o n fro m m al~ t o m a l+ ) i s s t a b l y
HSA+ ,
S in c e t h e c u l t u r e o f ¥ 1 1 7 7 r e c e n t l y o b t a i n e d fro m D r, L e d e rb e r g
g i v e s an HSA~. r e a c t i o n , i t i s b e l i e v e d t h a t t h e HSA+' c h a r a c t e r was l o s t
s i m u l t a n e o u s l y w i t h t h e l o s s o f s tr e p to m y c in s e n s i t i v i t y .
C S ll i s a
d e r i v a t i v e o f ¥ 1 1 7 7 w h ic h w as made F+ b y D r. P . D. S k a a r b y t r e a t m e n t
w i t h s t r a i n 5 8 - 1 6 1 , ' C S ll c o n s i s t e n t l y g i v e s an HSA+ r e a c t i o n .
Thus
i t a p p e a r s t h a t t h e HSA+ t o HSA"" c h an g e may h a v e a c c o m p a n ie d t h e m u ta ti o n
21
t o s tr e p to m y c in r e s i s t a n c e and t h a t t h e HSA- t o HSA+ r e v e r s i o n may h av e
a c c o m p a n ie d t h e a c q u i s i t i o n o f t h e s e x f a c t o r .
I n a d d i t i o n t o t h e W ll77 c u l t u r e s two d e r i v a t i v e s o f W ll77 w h ic h
d i f f e r i n t h e i r p r o p e r t i e s r e l a t i n g t o t h e te m p e r a te b a c t e r i o p h a g e ,
lam bda, w e re o b t a i n e d f ro m D r. L e d e r b e r g a n d t e s t e d f o r t h e i r r e a c t i o n
w i t h HSA a n t i s e r u m .
The lambda-^ r e l a t e d p r o p e r t i e s o f K12 s t r a i n s a r e
c o n t r o l l e d b y g e n e s a t two l o c i .
The L p i l o c u s c o n t r o l s p o s s e s s i o n o f
t h e p r o p h a g e , t h e Lpp l o c u s c o n t r o l s p o s s e s s i o n o f t h e r e c e p t o r s f o r
a d s o r p tio n o f phage ( L e d e rb e rg and L e d e rb e rg , 1 9 5 3 )°
L por c u l t u r e s
c a n n o t a d s o r b lam b d a p h a g e a n d a r e t h e r e f o r e immune t o i n f e c t i o n e v e n
th o u g h t h e y may b e n o n - l y s o g e n i c .
phage a n d , i f
phage.
Lpps c u l t u r e s a r e a b l e t o a d s o r b
n o n -ly s o g e n ic , a re s e n s i t i v e to i n f e c ti o n by e x t r i n s i c
The g e n o ty p e o f t h e HSA- s t r a i n , W ll7 7 , i s L p^+ , L por .
w h ic h i s a n Lpi
L ppr - .d e r i v a t i v e o f W ll7 7 , i s HSA+ .
an L p i~ 0 Lp o S d e r i v a t i v e o f W1603 i s HSA- .
Wl60 3 ,
W 2736, w h ic h i s
The a p p e a r a n c e , i n r a p i d
s u c c e s s i o n , o f tw o r e v e r s i o n s i n t h e HSA p r o p e r t y o c c u r r i n g s im u l ta n e ­
o u s l y w i t h m u t a t i o n a l c h a n g e s i n t h e lam b d a p r o p e r t i e s r a i s e s t h e p o s ­
s i b i l i t y t h a t c h a n g e s o f t h e HSA s t a t e may b e i n v a r i a b l y a c c o m p a n ie d b y
changes o f o th e r , n o t o b v io u s ly r e l a t e d , p r o p e r tie s .
A l l o f t h e f i v e HSA- t o HSA+ r e v e r t a n t s o f MSC501 w e re d i f f e r e n t
fro m t h e p a r e n t s t r a i n w hen grow n on EMB m a n n i to l .
When MSC503 i s
grown on EMB m a n n i t o l , t h e c o l o n i e s w h ic h a r i s e h a v e a t y p i c a l m tlap p e ara n ce .
The a p p e a r a n c e o f MSCgOl c o l o n i e s on EMB m a n n i to l i s
i n t e r m e d i a t e b e tw e e n m tl** and m t l + .
E ach o f t h e f i v e r e v e r t a n t s gave a
22
t y p i c a l m tl~ r e a c t i o n .
The f a c t t h a t a l l n in e o f t h e o b s e r v e d m u ta tio n s
o f t h e HSA c h a r a c t e r h a v e o c c u r r e d s i m u l t a n e o u s l y w i t h o t h e r g e n e t i c
c h a n g e s a f f e c t i n g p r o p e r t i e s w h ic h h a v e no o b v io u s p h y s i o l o g i c a l r e l a ­
t i o n s h i p t o HSA o r t o e a c h o t h e r made i t seem a p p r o p r i a t e t o d e te r m in e
w h e th e r t h e c o n c u r r e n t m u ta ti o n s w e re s im p l y f o r t u i t o u s e v e n t s .
B e-.
c a u s e o f t h e l a c k o f s u i t a b l e m eth o d s f o r s e l e c t i n g HSA m u t a n t s 9 t h e
p ro b le m was a p p ro a c h e d b y s t u d y i n g t h e a s s o c i a t e d m u t a t i o n s .
The
c h a n g e s i n c o l o n i a l a p p e a r a n c e on EMB m a n n i to l was n o t i n v e s t i g a t e d
b e c a u s e t h i s m u ta ti o n a l s o d o e s n o t l e n d i t s e l f t o s e l e c t i v e m e th o d s .
The c h a n g e s i n s tr e p to m y c in r e s i s t a n c e , lam b d a c h a r a c t e r i s t i c s , and
m a tin g p r o p e r t i e s ( g a i n o r l o s s o f s e x f a c t o r ) can be r e a d i l y s t u d i e d .
I.
M u ta tio n t o S tr e p to m y c in R e s i s t a n c e s
C h r o n o l o g i c a l l y , th e
f i r s t o b s e r v e d m u ta ti o n t o HSA- a p p e a r e d a t t h e tim e o f o r i g i n o f
H i7 7 .
The f i r s t p o s s i b i l i t y t o b e c o n s id e r e d i s t h a t HSA“ and S2”
a re p le io tr o p ic e f f e c ts o f a s in g le gene.
T h is i s r u l e d o u t b y t h e
o c c u r r e n c e o f HSA+ d e r i v a t i v e s o f MSC501 w h ic h a r e s t i l l Sr .
A re ­
m a in in g e x p l a n a t i o n i s t h a t , i n t h e g e n e t i c b a c k g ro u n d o f MSC503 CHSA+ .
Ss ) , m u ta ti o n t o HSA- a lw a y s a c c o m p a n ie s t h e Ss t o Sr m u t a t i o n .
To
t e s t t h i s s e c o n d p o s s i b i l i t y , new s tr e p to m y c in r e s i s t a n t m u ta n ts o f
MSC503 w e re s o u g h t b y t h e f o l l o w i n g p r o c e d u r e .
O n e - te n t h m l q u a n t i t i e s
o f a n o v e r n i g h t P e n n a s s a y b r o t h c u l t u r e o f MSC503 w e re t r a n s f e r r e d t o
f o r t y 5 .0 m l q u a n t i t i e s o f P e n n a s s a y b r o t h and in c u b a t e d t w e n t y - f o u r
h o u r s a t 37 0 .
T h ese c u l t u r e s w e re c e n t r i f u g e d a n d t h e p e l l e t r e s u s ­
p e n d e d i n b r o t h c o n t a i n i n g s tr e p to m y c in and r e i n c u b a t e d .
F o u r o f th e
23
f o r t y t u b e s show ed t u r b i d g ro w th w i t h i n f o r t y - e i g h t h o u r s .
T h ese c u l ­
t u r e s w e re s t r e a k e d on n u t r i e n t a g a r c o n t a i n i n g s tr e p to m y c in and in c u ­
b a t e d u n t i l w e l l - d e v e l o p e d i s o l a t e d c o l o n i e s a p p e a r e d w h ic h w e re th e n
p ic k e d a n d t r a n s f e r r e d t o t u b e s o f P e n n a s s a y b r o t h .
A f te r tw e n ty -fo u r
h o u r s i n c u b a t i o n s t h e c u l t u r e s w e re t e s t e d f o r HSA and f o r s tr e p to m y c in
re s is ta n c e .
A l l w e re fo u n d t o be HSA+ l i k e MSC503 a n d w e re s tr e p to m y ­
c in r e s i s t a n t .
Thus i t i s e v i d e n t t h a t t h e m u ta ti o n t o s tr e p to m y c in
r e s i s t a n c e and t h e m u t a t i o n t o HSA- a r e n o t i n v a r i a b l y a s s o c i a t e d i n
MSC503,
O b v io u s ly , t h e s m a l l num ber o f m u ta n ts ex am in ed i n t h i s e x p e r i ­
m en t o f f e r s l i t t l e
i n d i c a t i o n o f t h e f r e q u e n c y o f s i m i l a r s im u lta n e o u s
m u ta ti o n s among K12 s t r a i n s i n g e n e r a l .
To o b t a i n a l a r g e r s a m p lin g
o f m u t a n t s , t h e same s e l e c t i o n w as made u s i n g s t r a i n CS19.
s u b - c u l t u r e o f t h e m u ta b le s t r a i n ,
5 8 -2 7 8 ,
CS19 i s a
T h is c u l t u r e m u ta te s from
s tr e p to m y c in s e n s i t i v i t y t o s tr e p to m y c in r e s i s t a n c e a t a f r e q u e n c y
w h ic h i s a b o u t a h u n d re d t im e s g r e a t e r t h a n t h a t o f o t h e r K12 s t r a i n s
( 'T r e f f e r s , e t a l , 1 9 5 4 , S k a a r , 1 9 5 6 ) ,
Among 100 s u c h s tr e p to m y c in
r e s i s t a n t m u ta n ts o b t a i n e d , a l l w e re f o u n d t o b e HSA „
Thus i t a p p e a r s
t h a t t h e HSA m u ta ti o n o c c u r s a t a f r e q u e n c y w h ic h i s l e s s th a n one p e r
one h u n d re d m u ta ti o n s t o s tr e p to m y c in r e s i s t a n c e .
I f t h e g e n e t i c b a s i s f o r t h e HSA p r o p e r t y i s c y t o p l a s m i c , th e
s im u lta n e o u s o c c u r r e n c e o f Sr an d HSA- m u ta ti o n s m ig h t b e a t t r i b u t e d t o
th e s t r e p t o m y c i n t r e a t m e n t em ployed i n s e l e c t i n g t h e Sr m u ta n t, Tfifll??,
The a b i l i t y o f s t r e p t o m y c i n t o d i s i n f e c t c e r t a i n c e l l s o f c y to p la s m ic
g e n e s h a s b e e n d e m o n s tr a te d i n a l g a l f l a g e l l a t e s ( P r o v a s o l i , e t a l ,
24
1 9 5 1 ) , i n s e e d p l a n t s ( B o g o ra d s 1 9 5 0 ) , and i n E a g le n a ( P r o v a s o l i 3 e t a l ,
1 9 4 8 ),
I n o r d e r t o t e s t t h i s p o s s i b i l i t y , c u l t u r e s o f MSC5003 w h ic h
i s a s tr e p to m y c in r e s i s t a n t v a r i a n t o f MSC56, and o f C S ll w e re su b ­
c u ltu r e d d a i l y f o r f o u r te e n days in P e n n a ssa y b r o th c o n ta in in g s tr e p to ­
m y c in .
The c u l t u r e s w e re t e s t e d d a i l y f o r r e a c t i o n w i t h HSA a n ti s e r u m .
B o th c u l t u r e s c o n tin u e d t o r e a c t w i t h h i g h d i l u t i o n s o f a n t i s e r u m w h ich
m e a n t t h a t t h e y w e re n o t m a s s i v e l y c o n v e r t e d t o a n HSA"" s t a t e .
2.
C o n v e r s io n o f P - S t r a i n s t o F+g
G e n e tic r e c o m b in a tio n by
c o n j u g a t i o n i n E . c o l i d e p e n d s u p o n a s e x u a l d i f f e r e n t i a t i o n o f th e
s t r a i n s in v o lv e d .
G enes a r e t r a n s f e r r e d u n i - d i r e c t i o n a l l y fro m an F+
d o n o r t o an F - r e c i p i e n t .
T h is d i f f e r e n c e i s b a s e d on t h e p r e s e n c e o f
a s e x f a c t o r w h ic h i s p o s s e s s e d b y F+ s t r a i n s b u t n o t b y F - s t r a i n s .
The sex factor is independent of the b acterial chromosome and can be
transferred at high frequency from an F+ to F - Culture3 thereby con­
ferring upon the la tte r the a b ility to act as a gene donor ('Cavalli3
e t a l 3 1 9 5 3 ),
'
The s e x f a c t o r i s assu m ed t o h a v e two e f f e c t s upon t h e c e l l i t
in h a b its .
The f i r s t e f f e c t i s .t o b r i n g a b o u t a s u r f a c e c h a n g e w h ic h
f a c i l i t a t e s p a i r i n g w i t h c o m p a tib le F - c e l l s | th e s e c o n d i s t o e f f e c t
t h e m i g r a t i o n o f chrom osom al g e n e s ( I f a k e l a 3 et_ a l . 1 9 6 2 ) .
I t has been
p r o p o s e d t h a t 3 s i n c e u n l i k e c e l l m e t a b o l i t e s 3 en zy m es, o r v e g e t a t i v e
p h a g e , t h e s e x f a c t o r i s t r a n s f e r r e d a t h i g h f r e q u e n c y i n F+ x F c r o s s e s , t h e s e x f a c t o r s t r u c t u r e i s n o t ra n d o m ly d i s t r i b u t e d i n th e
c y to p la s m b u t o c c u p ie s a s p e c i f i c s i t e c l o s e t o t h e b a c t e r i a l c e l l w a l l
25
(Ja c o b , e t a l , I9 6 0 ).
C e r t a i n d i f f e r e n c e s , b e tw e e n t h e p h y s i c a l p r o p e r t i e s o f F+ c e l l
s u r f a c e s a n d t h o s e o f F - c e l l s h a v e b e e n r e p o r t e d b y M a c c a c a ro (1 9 5 5 )»
S n e a th a n d L e d e r b e r g (1 9 6 1 ) h a v e -fo u n d t h a t t r e a t m e n t o f F+ c e l l s w i t h
m e t a p e r i o d a t e m akes them p h e n o t y p i c a l l y u n a b le t o a c t a s g e n e d o n o r s ,
p r e s u m a b ly b y t h e o x i d a t i o n o f s u r f a c e c a r b o h y d r a te s w h ic h f u n c t i o n
in th e p a ir in g p r o c e s s .
D i f f e r e n c e s b e tw e e n F - and F+ c u l t u r e s o f K12
s t r a i n s w i t h r e s p e c t t o t h e i r a g g l u t i n a b i l i t y w i t h h o m ologous a n ti s e r u m
h a v e b e e n r e p o r t e d b y M a c c a c a ro a n d Colom bo ( 1 9 5 6 ) .
0 r s k o v an d 0 r s k o v ( i 9 6 0 ) r e p o r t a n >ef +,r a n t i g e n w h ic h o c c u r s
i n s t r a i n s o f E . c o l i w h ic h h a v e b e e n c o n v e r t e d t o t h e d o n o r s t a t e by
t r e a t m e n t w i t h F+ K12 s t r a i n s .
A l l c o n v e r t e d s t r a i n s t e s t e d w e re shown
t o p o s s e s s t h e f + a n t i g e n b u t n o t a l l ( f + ) + - s t r a i n s w e re f o u n d t o be
F + . - Some s t r a i n s o f E . c o l i h a v e , i n f a c t , b e e n f o u n d t o h a v e th e
c a p a c i t y f o r c o n v e r t i n g o t h e r s t r a i n s t o t h e F+ s t a t e e v e n th o u g h t h e y
th e m s e lv e s c o u ld n o t a c t a s d o n o rs f o r chrom osom al g e n e s ( F u r n e s s and
R ow ley, 1 9 5 7 ) .
fu lfills
The 0 o r s k o v s h y p o t h e s i z e a " d e f e c t i v e " s e x f a c t o r w h ic h
th e f u n c tio n o f p a ir in g an d o f a n t i g e n i c i t y b u t n o t o f p ro ­
m o tin g g en e t r a n s f e r .
I t i s c l e a r t h a t n o t a l l F - s t r a i n s a r e HSA” .
MSC503 w h ic h i s
F - i s HSA+ and i s a b l e t o a b s o r b a l l d e t e c t a b l e a n t i b o d y fro m t h e
hom ologous a n tis e ru m - o f t h e F+ s t r a i n , C S ll ( s e e T a b le 7 ) .
H ow ever,
t h e f a c t t h a t MSC501 ( F - , HSA- ) when made F+ ( C S l l ) a l s o becam e HSA^
r a i s e s t h e p o s s i b i l i t y t h a t t h e HSA a n t i g e n i s i n d i s p e n s a b l e ( b u t n o t
26
T a b le V.
T est
a n tig e n
S e r o l o g i c a l R e l a t i o n s h i p s Among F - , F + s and H f r S t r a i n s .
R e c i p r o c a l o f a g g l u t i n a t i n g t i t e r i n C S ll a n t i s e r u m s
a b so rb e d w ith
a b so rb e d w ith
a b so rb e d w ith
GS101
•MSC503
MSC39
C S ll F+
640
0
0
MSC503 F -
640
0
0
C S lO l H f r
640
0
0
!,0 i! m eans n e g a t i v e i n a se ru m d i l u t i o n o f I g 2 0 . C o n t r o l seru m
t u b e s w e re a l l 0 . F i g u r e s a r e f o r maximum a g g l u t i n a t i o n .
27
s u f f i c i e n t ) f o r t h e m a tin g p r o c e s s „' On t h i s b a s i s , a l l F+ s t r a i n s
s h o u ld b e HSAjf ,
To t e s t t h i s , F+ d e r i v a t i v e s o f MSC501 w e re o b t a i n e d
b y m ix ed g ro w th w i t h tw o F+ d o n o r s , MSC70 and W6„
O v e r n ig h t c u l t u r e s
o f t h e t h r e e s t r a i n s w e re grow n u p s e p a r a t e l y i n P e n n a s s a y b r o t h t o a
c o n c e n t r a t i o n o f a b o u t I x IO ^ c e l l s p e r m l.
One h u n d re d f o l d d i l u ­
t i o n s w e re made o f a l l c u l t u r e s and t h e y w e re in c u b a t e d a g a i n u n t i l
t h e y r e a c h e d c o n c e n t r a t i o n s o f a b o u t I x IO ^ c e l l s p e r m l.
T hen, 2 ,5
ml q u a n t i t i e s o f M5C501 w e re a d d e d t o 5 . 0 m l q u a n t i t i e s o f e a c h o f t h e
F+ c u l t u r e s , and t h e m ix t u r e s w e re i n c u b a t e d i n t h e 37 Gw a t e r b a th f o r
one h o u r.
D i l u t i o n s w e re made o f t h e c u l t u r e s and 0 ,1 m l s a m p le s w e re
s p r e a d on p l a t e s o f EMB l a c t o s e t o g iv e a p p r o x im a te ly 100 c o l o n i e s p e r
p la te .
The p l a t e s w e re in c u b a t e d a t 37 0 f o r 30 h o u r s ,
W e ll-is o la te d
l a c " c o l o n i e s w e re t r a n s f e r r e d t o P e n n a s s a y b r o t h , i n c u b a t e d 16 h o u rs
a t 37 C an d th e n c r o s s e s w e re made w i t h IMN64 on DMA p l a t e s w i t h s e l e c ­
t io n f o r t r a n s f e r o f t h e s h ik im ic a c id l o c u s .
The DMA p l a t e s w ere
i n c u b a t e d f o r 48 h o u r s a t 37 C and s c o r e d f o r t h e num ber o f p r o t o t r o p h i c
r e c o m b in a n t s .
S ev en c u l t u r e s o f MSC501 fro m t h e m ix in g w i t h W6 gave
l a r g e num bers o f r e c o m b in a n ts w i t h IMM64 .
F o u r c u l t u r e s o f MSC501 fro m
t h e m ix in g w i t h MSC70 a l s o g av e l a r g e nu m b ers o f r e c o m b in a n ts .
p l a t i n g s o f t h e c r o s s co m p o n en ts a lo n e w e re b a r r e n .
C o n tr o l
None o f t h e b r o t h
c u l t u r e s fro m th e . n e w ly - tr a n s f o r m e d MSC501 F+ s t r a i n s r e a c t e d w i t h HSA
a n t i s e r u m , i n d i c a t i n g t h a t t h e HSA a n t i g e n i s n o t a n e s s e n t i a l a t t r i ­
b u t e o f F+ K12 s t r a i n s .
To t e s t t h e p o s s i b i l i t y t h a t HSA- c e l l s m ig h t d i f f e r fro m HSA"*
28
c e l l s i n t h e r e a d i n e s s w i t h w h ic h t h e y a r e c o n v e r te d t o F + s t h e f o llo w ­
in g e x p e r im e n t was p e r f o r m e d .
C u l t u r e s o f t h r e e F - s t r a i n s -(MSCSbljl
MSC503) a n d MSGSOS) w e re e x p o s e d t o W6 a s i n t h e p r e c e d in g e x p e r im e n t^
and t h e p u r i f i e d p r o g e n y o f t h e e x p o s e d c e l l s w e re t e s t e d f o r F- s t a t u s .
MSC503 ( t h e m a l- d e r i v a t i v e o f ¥ 6 7 7 ) i s HSA+ ; MSC501 ( t h e m a l+ d e r i v a ­
t i v e o f ¥ 1 1 7 7 ) i s HSA"s MSCSOS i s t h e HSA+ d e r i v a t i v e o f MSC501.
As
s e e n i n T a b le V I ( F i r s t G r o s s ) , a l l t h r e e s t r a i n s w e re c o n v e r t e d t o
F+ w i t h e q u a l f a c i l i t y .
To d e te r m in e w h e th e r t h e F+ s t a t e w as s t a b l e i n t h e n e w ly con­
v e r t e d s t r a i n s , e a c h w as s t r e a k e d t o a s e c o n d s e t o f EMB l a c t o s e p l a t e s .
T h ese p l a t e s w e re i n c u b a t e d 30 h o u r s a s b e f o r e a n d b r o t h c u l t u r e s
d e r i v e d fro m i s o l a t e d c o l o n i e s w e re t e s t e d f o r t h e i r a b i l i t y t o a c t a s
g e n e t i c d o n o rs f o r IMN64 (S e c o n d C r o s s , T a b le V I ) .
I n a l l c a s e s , th e
F+ p r o p e r t y o f t h e n e w ly c o n v e r te d s t r a i n s w as r e t a i n e d i n t h e s u b c u l­
tu re .
No e x p l a n a t i o n can be o f f e r e d a t t h e p r e s e n t tim e f o r t h e i n ­
f e r i o r f e r t i l i t y o f MSC501 an d MSC505 d e r i v a t i v e s , a s c o n t r a s t e d w i t h
t h o s e o f MSC503.
A g a in , a l l n e w ly c o n v e r t e d F+ d e r i v a t i v e s o f MSG501 w e re s t i l l
HSA" a t t h e tim e o f b o th t h e f i r s t a n d s e c o n d c r o s s e s .
The p r e c e d in g r e s u l t s
w i t h o u t HSA.-
c l e a r l y show t h a t K12 s t r a i n s may be, F+
I n a d d i t i o n , t h e y show t h a t , i f HSA i s e p i s o m a l l y d e t e r ­
m in e d , t h e i n f e c t i v i t y o f t h a t e p is o m e i s lo w e r th a n i s t h a t o f t h e
sex f a c to r .
3.
HSA M u ta tio n s A s s o c ia t e d w i t h C hanges i n Lam bda B e l a te d
29
T a b le 7 1 .
R e l a t i v e E f f i c i e n c y o f F+ C o n v e r s io n o f MSCSOls MSC503s. and
MSCSOS b y M ixed G row th w i t h W6.
P r o t o t r o p h i c r e c o m b in a n ts p e r p l a t e i n c r o s s e s w i t h IMM64f
F i r s t c ro ss
_______ S eco n d c r o s s
T r e a t e d s t r a i n s . MSC501,
MSC501
MSC503
MSC505
MSC505
MSC503
E x t r a c t e d c lo n e s
I
100
0
0
100
0
0
2
100
100
> 1000
200
> 1000
>1000
' 3
40
160
>1000
100
200
>1000
4
100
0
0
60
5
0
100
0
0
400
0
6
0
0
0
0
0
0
7
0
300
0
0
0
8
70
100
> 1000
100
400
X
120
>1000
9
0
0
>1000
0
0
> 1000
10
500
140
> 1000
200
200
•
'
0 ■
0
>1000 .
C u l t u r e s o f MSCSOls ESCSOSs an d MSC503 ( a l l l a c " ) w e re grow n w i t h
W6 ( I a c + ) .
Ten l a c ~ c lo n e s w e re t h e n e x t r a c t e d fro m e a c h m i x t u r e .
T hese w e re im m e d ia te ly t e s t e d f o r F s t a t u s b y c r o s s i n g t o HEN64 ( f i r s t
c r o s s ) . E ach c lo n e w as s t r e a k e d a n d a new c u l t u r e i n i t i a t e d f ro m .a h .,is o ­
l a t e d c o lo n y .
T h is new c u l t u r e w as th e n t e s t e d f o r , F s t a t u s (s e c o n d
c r o s s ) . A c o n t r o l c r o s s b e tw e e n W6 a n d IMN64 g a v e > 1 0 0 0 p r o t o t r o p h i c
r e c o m b in a n t s .
C o n t r o l p l a t i n g s o f a l l c o m p o n e n ts w e re n e g a t i v e .
'r
- r
30
P ro p e rtie s :
1/51177 i s HSA- 3 L p ^ + , L p^r a a n d m al]_~„
The c o r r e s p o n d in g
n ia l+ s t r a i n , MSC501, i s HSA- , Lp]_+ , a n d p re s u m a b ly L pgs a c c o r d in g t o
L e d e r b e r g f S c o n c lu s i o n s t h a t L pgg a n d mal-]~ a r e u n d e r t h e p l e i o t r o p i c
c o n t r o l o f a s i n g l e g en e ( L e d e r b e r g , 1 9 5 5 ) .
m al]_"o
Wl603 i s HSA+ , L p ^ " . Lpg r 0
I t s d e r i v a t i v e , W2736, i s HSA+, L p i- , Lpo5 . mal-] + »
The HSA c h a r a c t e r an d t h e p r o p e r t i e s r e l a t e d t o lam b d a a r e n o t
u n d e r p l e i o t r o p i c c o n t r o l s i n c e W1177 a n d ¥ 2 7 3 6 a r e a l i k e f o r HSA b u t
d i f f e r e n t f o r Lpg w h i l e ¥ 1 1 7 7 an d Wl603 a r e a l i k e f o r Lpj_ b u t d i f f e r e n t
f o r HSA.
To d e te r m in e w h e th e r a m u ta ti o n a t e i t h e r t h e Lpi o r t h e L po
l o c u s c a n b e c o r r e l a t e d w i t h c h a n g e s i n t h e HSA p r o p e r t y , m u ta n ts o f
¥ 2 6 3 7 w e re s o u g h t f o r s t u d y b y t h e f o l l o w i n g m eth o d '* :
A n u trie n t agar
p l a t e w as s p r e a d w i t h a 0 ,1 m l o v e r n i g h t P e n n a s s a y b r o t h c u l t u r e o f
¥ 2 6 3 7 a n d a llo w e d t o d r y .
A lam b d a s u s p e n s i o n was p r e p a r e d fro m 1SC503
( s e e M a t e r i a l s a n d M e th o d s) and a l o o p f u l o f t h i s s u s p e n s i o n was s p o t t e d
on t h e W2637 p l a t e .
The p l a t e w as i n c u b a t e d f o r 24 h o u r s and ex am in ed f o r l y s i s .
'
la w n o f g ro w th a p p e a r e d o v e r th e e n t i r e p l a t e e x c e p t f o r a n a r e a o f
A
n e a r l y c o n f l u e n t l y s i s i n t h e r e g i o n w h e re t h e phage h a d b e e n a p p l i e d .
S t r e a k i n g s w ere, made fro m t h i s l y s e d a r e a t o s e v e r a l p l a t e s o f EMB m al­
t o s e w h ic h h a d b e e n s p r e a d w i t h s t r e p t o m y c i n t o k i l l a n y c o n ta m in a tin g
MSC503 c e l l s „
The p l a t e s w e re i n c u b a t e d f o r 24 h o u r s .
F ifty -fo u r
c o l o n i e s w e re p ic k e d a n d t r a n s f e r r e d t o t u b e s o f P e n n a s s a y b r o t h , i n c u ­
b a t e d f o r 24 h o u r s and t e s t e d w i t h a 1 :4 0 d i l u t i o n o f HSA a n t i s e r u m .
31
None w e re a g g l u t i n a t e d b y t h e a n t i s e r u m .
Among t w e n t y - e i g h t o f t h e c o l o n i e s p ic k e d fro m t h e m a l to s e
s tr e p to m y c in p l a t e ,, tw e n ty - o n e w e re maL+ a n d immune t o la m b d a .
o f t h e s e w e re d e m o n s tr a te d t o b e l y s o g e n i c .
F ifte e n
The r e m a in in g c o lo n i e s
w e re m al~ a n d ( s u r p r i s i n g l y ) s e n s i t i v e t o la m b d a .
The a p p e a r a n c e o f
m al~ lam b d a s e n s i t i v e m u ta n ts i s i n t e r e s t i n g i n i t s e l f a n d d e s e r v e s
'
f u r t h e r s tu d y .
The i m p o r t a n t p o i n t h e r e , h o w e v e r, i s t h a t m u ta tio n
o f t h e HSA' p r o p e r t y i s n o t i n v a r i a b l y c o r r e l a t e d w i t h m u ta ti o n fro m
lam b d a s e n s i t i v i t y t o lam b d a l y s o g e n i c i t y n o r w i t h t h e m u ta ti o n fro m .
n ia l+ t o m a l~ .
11
I
E.
M u ta tio n o f .HSA A s s o c ia t e d w i t h E n h an ced M o t i l i t y
The l o s s o f f e r t i l i t y a s s o c i a t e d w i t h p r o lo n g e d p a s s a g e on
m o t i l i t y medium h a s b e e n i n t e r p r e t e d a s due t o s e l e c t i o n f o r a l l e l e s
w i t h p l e i o t r o p i c e f f e c t s on f e r t i l i t y
e t a l , 1 9 5 7 ).
a n d r a p i d i t y o f sw arm in g ( S k a a r ,
T h ese a u t h o r s s u g g e s t t h a t c e l l s n o t e n cu m b ered b y a
s u r f a c e com ponent n e c e s s a r y f o r f e r t i l i t y m ay be th u s f o r t u i t o u s l y
ii
s e l e c t e d a s r a p i d s w a rm e rs ,
F o r th e same r e a s o n , i t m ig h t b e p r e d i c t e d
;!
t h a t s e l e c t i o n f o r h i g h m o t i l i t y m ig h t r e s u l t i n c o r r e l a t e d s e l e c t i o n
f o r HSA~.
T h is p o s s i b i l i t y w as t e s t e d a s f o ll o w s t
A c u l t u r e o f W6
w as m o t i l i z e d b y t e n s u c c e s s i v e t r a n s f e r s on m o t i l i t y m edium u n t i l t h e
e n t i r e p o p u l a t i o n w as h i g h l y m o tile , an d g av e a d e l i c a t e sw arm .
.
An
; ;j
;i
.
in o c u lu m fro m t h e ed g e o f t h e swarm on t h e t e n t h p l a t e w as t r a n s f e r r e d
to P e n n a ssa y b r o th and in c u b a te d .
A t t h e sam e t im e , a n in o c u lu m fro m
t h e s t o c k c u l t u r e a g a r s l a n t w as s i m i l a r l y t r a n s f e r r e d to . b r o t h an d
i;
32
i n cub a t e d o
At th e end o f f o r t y - e i g h t h o u rs in c u b a tio n th e b r o th c u l­
t u r e s w e re t e s t e d q u a n t i t a t i v e l y f o r a g g l u t i n a t i o n b y HSA a n t i s e r u m .
The c u l t u r e d e r i v e d fro m t h e a g a r s l a n t w as a g g l u t i n a t e d s t r o n g l y b y a
1 :6 4 0 d i l u t i o n o f a n t i s e r u m ; t h e h i g h l y m o t i l e c u l t u r e show ed no de­
t e c t a b l e a g g lu t in a t io n even w ith a 1 :2 0 d i l u t i o n o f a n tis e ru m .
The
b r o t h c u l t u r e s w e re th e n s u b c u l t u r e d i n t o f r e s h b r o t h an d a t h i r d b r o t h
c u l t u r e w as i n i t i a t e d fro m an in o c u lu m o b t a i n e d d i r e c t l y fro m a swarm
on m o t i l i t y m edium .
A l l t h r e e c u l t u r e s w e re t e s t e d f o r t h e i r a b i l i t y
t o p ro d u c e p r o t o t r o p h i c r e c o m b in a n ts w i t h W ll7 7 .
The m eth o d u s e d i n
t h i s c r o s s w as i d e n t i c a l w i t h t h a t d e s c r i b e d i n s e c t i o n F o f R e s u l t s
e x c e p t t h a t t h e DMA p l a t e s w e re s p r e a d w i t h p h e n y la l a n i n e ( s e e M a t e r i a l s
a n d M e th o d s) t o r e l a x s e l e c t i o n f o r t r a n s f e r o f t h e P a + g e n e .
A f t e r tw o d a y s i n c u b a t i o n , t h e DMA p l a t e s w e re s c o r e d f o r p r o to ­
t r o p h i c r e c o m b in a n t s .
The n o n - m o t i l i z e d c u l t u r e gave m ore t h a n 1000
r e c o m b in a n t s , t h e c r o s s w i t h t h e f i r s t m o t i l i z e d c u l t u r e show ed an
a p p r o x i m a t e ly t e n - f o l d d e c r e a s e i n num ber o f r e c o m b in a n ts , a n d th e
c u l t u r e w h ic h w as i n o c u l a t e d d i r e c t l y fro m t h e swarm show ed a n a p p r o x i­
m a t e ly t w e n t y - f o l d d e c r e a s e i n num ber o f r e c o m b in a n ts .
T h ese r e s u l t s
s u g g e s t t h a t l o s s o f HSA+ a n d l o s s o f F+ may accom pany t h e s e l e c t i o n o f
t h e h i g h l y m o t i l e g e n o ty p e .
The r e s u l t s a l s o show , h o w e v e r, t h a t HSA+
and F+ do n o t h a v e a common g e n e t i c b a s i s , s u p p o r t i n g c o n c l u s i o n s '
re a c h e d i n a p re v io u s s e c t i o n .
••
F-.
-
L o c a l i z a t i o n o f t h e HSA Gene on t h e B a c t e r i a l Chromosome
S t r a i n 1 1 1 7 7 w as i s o l a t e d a s a s tr e p to m y c in r e s i s t a n t m u ta n t o f
33
¥677.
MSC501 i s a m a l+ m u ta n t o f ¥ 1 1 7 7 an d MSG503 i s a m a l+ m u ta n t o f
¥677.
MSC503 i s a g g l u t i n a t e d b y a 1 :6 4 0 d i l u t i o n o f HSA a n t i s e r u m .
A g g lu tin a tio n a t t h i s d i l u t i o n i s s tr o n g 5 t h a t i s , a la r g e f la k y a n t i ­
g e n - a n t ib o d y a g g r e g a te i s fo rm e d an d t h e s u p e r n a t a n t l i q u i d i s
c le a r.
MSC501 g i v e s no v i s i b l e r e a c t i o n w i t h HSA a n ti s e r u m an d a b s o r b s o n ly
f l a g e l l a r a n t i b o d y fro m C S ll a n t i s e r u m .
The a g g l u t i n a t i o n o f HSA+
r e v e r t a n t s o f MSC501 i s i n d i s t i n g u i s h a b l e fro m t h a t o f MSC503.
These
f a c t s s u g g e s t t h a t t h e g e n e t i c b a s i s o f t h e HSA d i f f e r e n c e s among t h e
s tr a in s re s id e s a t a s in g le lo c u s .
is
I f t h i s i s s o , and i f t h e HSA l o c u s
c h ro m o so m a l, i t s h o u ld b e p o s s i b l e t o e s t a b l i s h t h e p o s i t i o n o f t h e
lo c u s by s ta n d a rd c r o s s e s .
W ith t h i s en d i n v ie w , a c r o s s w as made
b e tw e e n ¥ 6 (F + HSA+ ) and ¥ 1 1 7 7 ( F - , HSA"').
The c r o s s was made on m in im a l m edium w i t h s e l e c t i o n f o r p r o t o ­
tro p h y .
¥ 6 i s d e f i c i e n t f o r m e t h io n i n e , ¥ 1 1 7 7 i s d e f i c i e n t f o r t h r e o ­
n i n e , l e u c i n e , and t h i a m i n e .
A l a r g e num ber o f p r o t o t r o p h i c c o l o n i e s
a p p e a r in g on t h e c r o s s p l a t e s w e re s t r e a k e d on DMA3 t h e n s i n g l e c o lo n i e s
fro m t h e s e p l a t e s w e re s t r e a k e d on c o m p le te m edium .
I s o l a t e d c o lo n ie s
fro m t h e c o m p le te m edium w e re t r a n s f e r r e d t o 5 .0 m l q u a n t i t i e s o f P e n n a s s a y b r o th and in c u b a te d f o r tw e n ty - f o u r h o u r s .
T hese b r o t h c u l t u r e s
w e re t e s t e d f o r a g g l u t i n a t i o n b y 1 :4 0 d i l u t i o n s o f HSA a n t i s e r u m , and
f o r a u t o - a g g l u t i n a t i o n i n i d e n t i c a l d i l u t i o n s o f c o n t r o l s e ru m .
At
t h e sam e tim e t h e b r o t h c u l t u r e s w e re v e r i f i e d a s p r o t o t r o p h s b y s p o t t i n g
on p l a t e s o f DMA and w e re a l s o t e s t e d f o r s i x u n s e l e c t e d m a r k e r s in v o lv e d
in th e c r o s s .
R e s u l t s a r e g iv e n i n T a b le V I I .
The d a t a o f L e d e r b e r g ,
34
T a b le V I I 0
A.
S e g r e g a t i o n o f HSA a n d O th e r U n s e l e c t e d M a rk e rs Among P ro ­
t o t r o p h i c R e c o m b in a n ts R e s u l t i n g fro m G r o s s e s B etw een W6'
CF+, F1 S , l a c + , x y l + , m a l+ , m t l ¥ , Ss , HSA+) a n d TCL177 ( F - ,
F1 1", l a c ~ , x y l - , m a l- , m t l - , HSA- ) .
F r e q u e n c y o f T r a n s f e r o f F+ A l l e l e s
T o t a l num ber
ex am in ed
%t r a n s f e r o f F+ a l l e l e s o f t
S
Il l a c H l m al m tl
M ark u sen
(1 9 6 3 )
106
73
42
L e d e rb e rg
e t a l ' (1 9 5 1 )
100
67
28
B,
10
9
8
21
17
HSA- s t a t u s o f *
■ r e c o m b in a n ts
HSA±
HSA+
16
15
12
15.
R e l a t i o n s h i p B etw een t h e T r a n s f e r o f .HSA+ and t h e T r a n s f e r o f O th e r
U n s e l e c t e d F+ A l l e l e s
HSA
s ta tu s *
T o t a l num ber
e x a m in ed
+
* 17
+
13
76
'
'%t r a n s f e r o f F+ a l l e l e s o f S
S
Il l a c x y l m al m tl
76
53
12
12
6
12
77
62
23
15
23
31
71
36
8
8"
7
'
13'
* I n t h e s e c o lu m n s , + m eans s t r o n g a g g l u t i n a t i o n i n a l s 4 0 d i l u t i o n
o f HSA a n t i s e r u m , — m eans p a r t i a l a g g l u t i n a t i o n a t t h e same d i l u t i o n o f
a n tis e ru m .
35
a t aJL (1 9 5 1 ) a r e i n c l u d e d i n p a r t A t o show t h a t t h e p r o p o r t i o n o f un­
s e l e c t e d m a r k e r s o b s e r v e d i n t h e p r e s e n t c r o s s i s d i s t o r t e d som ew hat
fro m t h a t u s u a l l y o b t a i n e d i n a c r o s s b e tw e e n an F + s M- s t r a i n and an
F - s T , L"” s
s tra in .
S p e c if i c a l ly , th e fre q u e n c y o f l a c t r a n s f e r
i s h i g h e r t h a n e x p e c t e d , w h i l e t h e f r e q u e n c y o f t r a n s f e r o f m al a n d m tl
a r e lo w e r t h a n e x p e c t e d .
The d i s t o r t i o n may f a l l w i t h i n t h e r a n g e o f
v a r i a t i o n among d i f f e r e n t c r o s s e s .
On t h e o t h e r h a n d , i t may b e due
t o t h e c o m p l ic a t io n t h a t W ll77 h a s a c q u i r e d an a d d i t i o n a l n u t r i t i o n a l
re q u ire m e n t f o r p h e n y la la n in e .
n o t been w e l l s tu d i e d .
The p h e n y l a l a n i n e l o c u s ( o r l o c i ) h av e
One l o c u s i s known n o t t o be c l o s e l y l i n k e d t o
TLB-] ( J a c o b an d W bllm an, 1 9 6 1 ) .
The h ig h i n c i d e n c e o f I a c + a p p e a r in g
among t h e r e c o m b in a n ts s u g g e s t s t h a t t h e new pa_ m u ta ti o n i s n e a r l a c .
S in c e t h e f r e q u e n c y o f V^s (b e tw e e n TLB-^ and l a c ) i s n o t n o t a b l y h i g h ,
i t is u n lik e ly th a t pa is s im ila rly s itu a te d .
On t h i s b a s i s , t h e f r e q u e n c y o f HSA+ p r o t o t r o p h s i n d i c a t e s t h a t
t h e r e s p o n s i b l e l o c u s f o r HSA i s e i t h e r i n t h e v i c i n i t y o f TLB] o r o f
th e pa lo c u s .
H ow ever, b e f o r e d i s c u s s i n g t h i s p o i n t f u r t h e r , a n o t h e r
u n e x p e c te d f e a t u r e o f t h i s c r o s s n e e d s t o b e c o n s i d e r e d .
T h is w as t h e
a p p e a r a n c e o f f o u r re c o m b in a n t p o p u l a t i o n s w h ic h w e re h e te r o g e n e o u s
f o r th e l a c m a rk e r.
R e c o m b in a n ts o b t a i n e d fro m K12 x K12 c r o s s e s
u s u a lly a re s ta b le .
The mer o z y g o te q u i c k l y s e g r e g a t e s an d g i v e s r i s e
to s ta b l e h a p lo id c lo n e s .
E x c e p tio n s h a v e b e e n o b s e r v e d 5 o c c a s i o n a l
c r o s s e s p ro d u c e p e r s i s t e n t h e t e r o z y g o t e s ( L e d e r b e r g , 1949)<>
The d e t e c ­
t i o n o f s e g r e g a t i n g I a c + c lo n e s i n t h e p r e s e n t ..c ro ss, i n s p i t b o f
36
s t a n d a r d p r e c a u t i o n s o f r e c o m b in a n t p u r i f i c a t i o n , r e v e a l s a n o t h e r imrp o r t a n t c o m p l i c a t i o n a l s o a t t r i b u t a b l e , p e r h a p s , t o t h e new p a m a r k e r .
T w e n ty - e ig h t p e r c e n t o f t h e p r o t o t r o p h i c r e c o m b in a n ts w e re
a g g l u t i n a t e d b y a- 1 :4 0 d i l u t i o n o f HSA a n t i s e r u m 5 h o w e v e r, t h e y d i d
n o t r e a c t u n i f o r m l y a s w as e x p e c t e d .
r e a c t i o n l i k e t h a t o f $[30503.
S e v e n te e n c u l t u r e s g a v e a s t r o n g
The r e m a in in g t h i r t e e n w e re d e f i n i t e l y
a g g l u t i n a t e d , b u t i n t h e s e t h e s u p e r n a t a n t l i q u i d w as n o t c l e a r .
c u l t u r e s a g g l u t i n a t e d i n c o n t r o l se ru m .
c o u ld be e x p la i n e d i f
Ho
The p a r t i a l a g g l u t i n a t i o n
some r e c o m b in a n ts h a d r e c e i v e d o n l y a p o r t i o n o f
an HSA c o m p le x , b u t t h i s e x p l a n a t i o n i s i n c o m p a ti b le w i t h o u r o b s e r v a ­
t i o n s on t h e HSA^HSA+ m u ta ti o n s and w i t h t h e u n i f o r m i t y o f t h e HSA+
c h a r a c t e r o f s t r a i n s h a v in g v e r y d i v e r s e g e n e t i c b a c k g ro u n d s ( s e e
T a b le I T ) .
A n o th e r e x p l a n a t i o n i s t h a t t h e c lo n e s w i t h w h ic h some o f
t h e b r o t h c u l t u r e s w e re i n o c u l a t e d w e re h e te r o g e n e o u s f o r HSA.
T h is
p o s s i b i l i t y i s s t r e n g t h e n e d b y t h e p r e s e n c e o f c lo n e s d e s c r i b e d above
w h ic h w e re m ix e d f o r l a c .
E x i s t e n c e o f a new s e l e c t e d m a rk e r p l u s t h e o c c u r r e n c e o f s e g ­
r e g a tin g d ip lo id s c o n fu se s a tte m p ts to i n t e r p r e t th e d a ta w ith re g a rd
t o l o c a t i o n o f t h e HSA g e n e .
c o n f id e n c e s
One i m p o r t a n t s t a t e m e n t c a n be made w i t h
I f t h e HSA d i f f e r e n c e b e tw e e n W6 and 1 1 1 7 7 i s d u e t o a.'
s in g le lo c u s , t h a t lo c u s d o es n o t l i e
i n t h e se g m e n t o f t h e J a c o b -
W ollm an c i r c u l a r l i n k a g e map e x te n d i n g fro m :B]_. t o l a c ( s e e F i g u r e I ) .
T r a n s f e r o f t h e l i n k e d b lo c k , o f g e n e s , Bq+ , %+ , L + i s o b l i g a t o r y i n
th is c ro ss.
Any s e l e c t e d m a r k e r s l y i n g b e tw e e n Bq and L , o r c l o s e l y t o
v ].ae
m l
^
try
«
F i g u r e I . Genomes o f ¥ 6 a n d W llTT B a s e d on t h e L in k a g e Map o f J a c o b a n d W o llm a n '(1 9 ,6l ) .
Sym bols u s e d c o r r e s p o n d t o s y n t h e s i s o f t h r e o n i n e ( T ) # th ia m i n e ( B ^ ) 5 l e u c i n e ( l ) ,
t r y p t o p h a n ( t r y ) ^ h i s t i d i n e ( h i s ) , m e th io n in e (M ); s e n s i t i v i t y o r r e s i s t a n c e t o s t r e p ­
to m y c in ( s ) , s e n s i t i v i t y o r r e s i s t a n c e t o T5 (Y1 )^ f e r m e n t a t i o n o f m a l to s e ( m a l ) , x y lo s e
( x y l ) , m a n n i to l ( m tl) j, l a c t o s e ( l a c ) ,
38
t h e r i g h t , w i l l a p p e a r i n t h e p r o t o t r o p h i c r e c o m b in a n ts i n h i g h f r e ­
quency.
The g e n e s , V i3 a hd I a c + . known t o l i e
L and i n t h a t o r d q r , i l l u s t r a t e t h i s .
c lo s e ly to th e r ig h t o f
Even i f b o th t h o s e re c o m b in a n ts
g i v i n g a s t r o n g r e a c t i o n w i t h HSA a n t i s e r u m a n d t h o s e g i v i n g o n l y a
p a r t i a l r e a c t i o n a r e s c o r e d a s i n s t a n c e s o f p a s s a g e s o f t h e F+ HSA
a l l e l e , t h e p e r c e n ta g e t r a n s f e r o f t h a t a l l e l e
p e r c e n ta g e t r a n s f e r o f I a c + ( 4 2 ) .
( 2 8 ) d o e s n o t e q u a l th e
F u r th e r m o r e , t h e o n l y p o s s i b l e com­
p l i c a t i o n o f t h e new F - s e l e c t i v e m a r k e r , p a " , ( r e g a r d l e s s o f i t s l o c a ­
t i o n ) w o u ld b e t o h a v e i n c r e a s e d t h e f r e q u e n c y o f t r a n s f e r o f HSA+ .
T h is n e g a t i v e s t a t e m e n t c o n c e r n in g t h e l o c a t i o n o f HSA i s i m p o r t a n t ,
s i n c e t h e l o c u s d e t e r m i n i n g t h e som ew hat s i m i l a r f i m b r i a l a n t i g e n h a s
b e e n l o c a t e d i n t h e T L . Bq r e g i o n (M a c c a c a ro , e t a l , 1 9 5 9 )°
Beyond t h i s , o n l y t e n t a t i v e s t a t e m e n t s c a n b e m ade.
S i n g le
c o lo n y i s o l a t e s o f W1177 a lo n e h a v e a l l p r o v e d t o b e HSA"". b u t s u c h
t e s t s have been l im ite d .
F u r th e r m o r e , no s u c h e x a m in a tio n w as made o f
t h e same c u l t u r e u s e d i n t h e c r o s s .
I t is
c o n c e i v a b le , t h e r e f o r e ,
t h a t t h e c r o s s e d c u l t u r e w as a " j a c k p o t " p o p u l a t i o n c o n t a i n i n g a l a r g e
num ber o f HSA+ m u t a n t s .
I f s o , t h e c r o s s p r o v id e s no e v id e n c e t h a t
th e gene i s even t r a n s f e r r e d by c o n ju g a tio n .
H ow ever, on t h e p r e c e d in g h y p o t h e s i s , th e d i s t r i b u t i o n o f HSA+
c e l l s s h o u ld o c c u r among t h e r e c o m b in a n ts ra n d o m ly w i t h r e s p e c t t o
o th e r u n s e le c te d m a rk e rs.
As shown i n T a b le V I I , t h e r e i s a s u g g e s tio n ,
t h a t t h i s i s n o t s o , t h a t , i n f a c t , an i n t e r a c t i o n w i t h l a c e x i s t s .
The d a t a on HSA+ r e c o m b in a n ts a lo n e i s i n s u f f i c i e n t t o w a r r a n t c o n 'f i-
39'
dence in t h i s i n te r a c t io n .
I f t h e f r e q u e n c y o f I a c + among' b o t h s t r o n g l y
a g g l u t i n a t i n g an d p a r t i a l l y a g g l u t i n a t i n g r e c o m b in a n ts i s
com pared t o
t h e f r e q u e n c y o f I a c + among t h e HSA- r e c o m b in a n t s , h o w e v e r, t h e i n t e r ­
a c t i o n i s s i g n i f i c a n t a t t h e 5 p e r c e n t l e v e l (X^ *= 5 ) .
I f t h i s i n t e r a c t i o n i s r e a l , i t show s n o t o n l y t h a t HSA+ i s
t r a n s f e r r e d , b u t a l s o t h a t , i f t h e HSA l o c u s i s
som ew here t o t h e r i g h t o f l a c ;
ch ro m o so m al, i t l i e s
I f th e new p a " g en e i s r e s p o n s i b l e f o r
t h e e x c e s s o f .I a c + among a l l r e c o m b in a n t s , i t a l s o w o u ld a p p e a r t o l i e
t o t h e r i g h t o f l a c , a p p a r e n t l y beyond HSA.
B ut S i s n o t c l o s e l y l i n k e d
t o p a , p a i s n o t c l o s e l y l i n k e d t o HSA0 n o r i s HSA c l o s e l y l i n k e d t o
la c .
T e n t a t i v e p o s i t i o n i n g , t h e r e f o r e , w o u ld p l a c e p a n e a r h i s ^ , HSA
n e a r t r y 2 (se e F ig u re I ) .
^ R e f e r s t o t h e m a rk e r f o r h i s t i d i n e .
S R e f e rs t o t h e m a rk e r f o r t r y p t o p h a n .,
DISOJ SSION
The r o l e o f HSA i n t h e c e l l i s d i f f i c u l t t o a s c e r t a i n .
Its
p h y s i c a l p r o p e r t i e s do n o t p e r m i t a s s i g n i n g i t t o a n y o f t h e c a t e g o r i e s
o f t h e c l a s s i c a l and w e l l - s t u d i e d Os KT3 a n d H a n t i g e n s o f E . c o l i .
s o m a tic o r 0 a n t i g e n i s h e a t s t a b l e . ' I t s
by h e a t i n g t o 100 C f o r tw o h o u r s .
an d t h e a g g r e g a te p ro d u c e d i s
The
a n t i g e n i c i t y i s n o t d e s tr o y e d
The a g g l u t i n a t i n g r e a c t i o n i s slo w
g r a n u la r and f ir m .
The H o r f l a g e l l a r
a n t i g e n fo rm s a l o o s e , f l o c c u l a r a g g r e g a te b u t t h e a n t i g e n i s h e a t
la b ile
(K a u ffm a n , 1 9 5 1 ) .
I t s a n t i g e n i c i t y , a c c o r d in g t o H a c k ie and
M c C a rtn e y ( C r u i c k s h a n k , i 9 6 0 ) i s d e s t r o y e d by h e a t i n g t o 60 C f o r a b o u t
tw e n ty m in u t e s .
The K a n t i g e n s a r e a g ro u p o f e n v e lo p e o r c a p s u l a r a n t i g e n s w h ic h
a re re s p o n s ib le f o r th e Q - in a g g lu tin a b ility o f liv in g b a c te r ia .
The
L a n t i g e n o f t h i s g ro u p i s d e s t r o y e d b y h e a t i n g t o 100 C f o r a n h o u r .
■The c a p s u l a r A a n t i g e n i s t h e r m o s t a b l e b u t i s a g g l u t i n a t e d o n l y b y
lo w ( I g lO o r l e s s ) d i l u t i o n s o f a n t i s e r u m . 1 The B a n t i g e n o c c u p ie s an
in te r m e d ia te p o s itio n in t h i s g ro u p .
h eat la b ile but i t s
I t s a g g lu tin a tin g a n tig e n i s
a n ti b o d y - b i n d i n g c a p a c i t y r e s i s t s h e a t i n g t o 100 C
f o r tw o and o n e - h a l f h o u r s .
The Ii (m u c u s) a n t i g e n a l s o i s a g g l u t i n a t e d
o n l y by lo w d i l u t i o n s o f a n tis e r u m ..
I t s a g g l u t i n a b i l i t y i s d e s tr o y e d
b y h e a t i n g t o 100 C f o r two and o n e - h a l f h o u r s (K a u ffm an , 1 9 5 1 ) .
■
'A
0 r s k o v a n d 0 r s k o v ( i 9 6 0 ) d e s c r i b e t h e f + a n t i g e n a s b e in g d e s tr o y e d
by h e a t i n g t o 100 C f o r a n h o u r .
The a g g l u t i n a t i n g r e a c t i o n i s slo w a n d
b o t h t h e a p p e a r a n c e o f t h e a g g r e g a te fo rm e d an d t h e s t r e n g t h o f th e
.3
41
a g g l u t i n a t i o n a r e v a r i a b l e d e p e n d in g upon e n v ir o n m e n ta l f a c t o r s .
No
a t t e m p t w as m ade, d u r in g t h e c o u rs e o f t h e p r e s e n t i n v e s t i g a t i o n , t o
d e m o n s tr a te a K12 a n t i g e n c o m p a ra b le t o f + .
F a lk o w and B aro n (1 9 6 2 )
r e p o r t a g g l u t i n a t i o n o f K12 F+ and H fr s t r a i n s b y f + a n t i s e r u m b u t no
d a t a a r e g iv e n c o n c e r n in g t h e s o u r c e o f t h e a n t i s e r u m .
Many o f t h e E n t e r o b a c t e r i a c e a e p o s s e s s f i m b r i a e ( p i l l ) .
A g g lu t­
i n a t i o n o f f i m b r i a t e d c u l t u r e s by a n ti s e r u m i s s i m i l a r t o f l a g e l l a r
a g g l u t i n a t i o n a n d i s a common s o u r c e o f c o n f u s io n ( G r u ic k s h a n k , i 9 6 0 ) .
The f i m b r i a l a n t i g e n s a r e m ore h e a t s t a b l e t h a n a r e f l a g e l l a b u t a r e
d e s t r o y e d b y h e a t i n g t o 100 C f o r a n h o u r ( G i l l i e s a n d D u g u id , 1 9 5 8 ) .
I n v e s tig a tio n s in to th e g e n e tic b a s is o f s u rf a c e a n tig e n s o f
t h o s e b a c t e r i a l s t r a i n s w h ic h l e n d th e m s e lv e s t o s t a n d a r d re c o m b in a ­
tio n s tu d ie s have been v e ry lim ite d .
L in k e d t r a n s f e r o f g e n e s f o r
f l a g e l l a r s t r u c t u r e , f u n c t i o n , and a n t i g e n i c s p e c i f i c i t y i n c r o s s e s
b e tw e e n E . c o l i K12 m ot+ and E . c o l i B m o t" h a s b e e n r e p o r t e d b y F u r n e s s
and R ow ley ( 1 9 5 5 ) .
R e s u l t s fro m s u c h c r o s s e s r e v e a l e d a l o c u s f o r th e
m o t i l i t y g e n e n e a r a t y r o s i n e m a rk e r ( F u r n e s s , 1 9 5 8 ) .
P h a g e -m e d ia te d
t r a n s f e r o f m o t i l i t y b e tw e e n K l2 a n d B s t r a i n s (M a rk u se h , i 9 6 0 ) a l s o
i n d i c a t e s t h a t i n t h e s e s t r a i n s , a l l a t t r i b u t e s o f m o t i l i t y may b e con­
t r o l l e d b y a s i n g l e gen.e, o r b y c lo s f e ly l i n k e d g e n e s .
I n a c r o s s em ploy­
i n g a n o th e r n o n - f l a g e l l a t e d s t r a i n , ,and u s i n g a K12 s t r a i n a s t h e m o t i l e
d o n o r , 0 r s k o v and 0 r s k o v (1 9 6 2 ) r e p o r t t h e t r a n s f e r o f a f l a
in d e p e n d e n t o f g e n e s f o r a n t i g e n i c s p e c i f i c i t y . ,
o f t h i s gene in th e h i s r e g io n .
gene t o b e
They p l a c e t h e s i t e
T r a n s f e r o f a gene f o r 0 a n t i g e n wfts
42
d e m o n s tr a te d a s w e l l a s t h o s e f o r tw o o f t h e K a n t i g e n s .
T h e ir d a ta
in d ic a te t h a t th e gene f o r th e 0 a n tig e n i s s i t u a t e d ' c lo s e to th e h is
l o c u s a s i s t h e H g en e d e s c r i b e d a b o v e .
A c c o rd in g t o t h e 0 r s k o v s j
m a r k e r s f o r b o t h t h e A an d B t y p e s o f E a n t i g e n a r e c l o s e l y l i n k e d to
t h e 0 l o c u s b u t a r e s e p a r a b l e fro m i t .
The L a n t i g e n c o u ld a l s o be
t r a n s f e r r e d b y c o n ju g a t io n b u t i t s e x p r e s s i o n a p p e a r e d t o b e c o n t r o l l e d
by g e n e s a t no l e s s t h a n tw o l o c i .
R e s u l t s o f r e c o m b in a tio n e x p e r i ­
m e n ts i n d i c a t e d t h a t t h e p o t e n t i a l f o r e x p r e s s i n g t h e L a n t i g e n can be
p r e s e n t w ith o u t e x p re s s io n .
I n c r o s s e s i n w h ic h t h e d o n o r p a r e n t
c a r r ie d th e l a t e n t a b i l i t y to e x p re s s th e L a n tig e n and th e r e c e p to r
p a r e n t c a r r i e d a n L a n t i g e n o f a n o th e r s p e c i f i c i t y , r e c o m b in a n ts w e re
o b t a i n e d w h ic h h a d t h e same s p e c i f i c i t y a s t h a t o f t h e d o n o r p a r e n t .
The m u co id p r o p e r t y o f r e c o m b in a n ts o b t a i n e d i n K12 x B c r o s s e s by
C a l e f an d C a v a l l i ( o f 0 r s k o v and 0 r s k o v , i 9 6 0 ) a l s o was th o u g h t t o b e
c o n t r o l l e d b y a p a i r o f c o m p le m e n ta ry g e n e s . . 0 r s k o v a n d 0 r s k o v (1 9 6 1 )
d e s c r i b e an F+ ’s t r a i n w h ic h , when c o n v e r t i n g o t h e r s t r a i n s t o t h e F+
s t a t e , a ls o t r a n s f e r s th e g e n e tic d e te rm in a n t f o r th e L a n tig e n .
The f i m b r i a t e p r o p e r t i e s o f E . c o l l B and o f E. c o l i K12 h a v e
b e e n s t u d i e d e x t e n s i v e l y b y B r i n to n ( 1 9 5 9 )0
F im b ria tio n in th e s e
s t r a i n s w as f o u n d t o be c o r r e l a t e d w i t h c o l o n i a l m o rp h o lo g y b u t n o t
w ith p re se n c e o r ab sen ce o f f l a g e l l a n o r w ith F s t a t u s .
w as fo u n d t o b e f i m b r i a t e d w h i l e 5 8 -1 6 1 F+ w as n o t .
S tr a in ¥677 F -
F o u r te e n o f n in e ­
t e e n s t r a i n s e x a m in ed w e re f o u n d t o h a v e t h e p o t e n t i a l i t y f o r f i m b r i a tio n .
T h ese s t r a i n s d e m o n s tr a te d a p h a s e v a r i a t i o n b e tw e e n a f i m b r i a t e d
43
and a n o n -fim b ria te d s t a t e .
R e v e r s io n s o f t h e f i m b r i a t e d s t a t e w ere
f o u n d t o o c c u r a t a f r e q u e n c y o f a b o u t 4 x 10“ 4 p e r c e l l p e r g e n e r a t i o n .
C r o s s e s b e tw e e n a f i m b r i a t e d K12 s t r a i n a n d a p e rm a n e n tly n o n - f i m b r i a t e d
K12 m u ta n t h a v e r e v e a l e d a chrom osom al l o c u s f o r a g e n e fim “ a t a s i t e
i n t h e T L r e g i o n (M a c c a c a ro 5 e t a l s 1 9 5 9 ) .
D a ta fro m t h e s e c r o s s e s
s u g g e s t e d t h a t e x p r e s s i o n o f t h e f im + g e n e may d e p e n d i n p a r t o n a
c y to p la s m ic f a c t o r .
The f i m b r i a t e p r o p e r t y a l s o h a s b e e n t r a n s f e r r e d
b y c o n j u g a t i o n fro m a n H f r s t r a i n o f -E. c o l i K12 t o a n o n - f i m b r i a t e d
S a lm o n e lla s p e c i e s ( B r i n to n and B a ro n 5 I 9 6 0 ) .
The a p p a r e n t lo w f e r t i l i t y o f F+ d e r i v a t i v e s o f MSC501 o b t a i n e d
i n t h e p r e s e n t i n v e s t i g a t i o n ( s e e T a b le T l ) d e s e r v e s f u r t h e r s tu d y .
S uch d i f f e r e n c e s i n f e r t i l i t y
o b s e r v e d e ls e w h e r e h a v e b e e n a t t r i b u t e d
t o a m u ta n t F+ f a c t o r ( C l a r k a n d A d e lb e r g 5 1 9 6 2 ) b u t t h i s e x p l a n a t i o n
seem s u n l i k e l y i n t h e p r e s e n t c a s e s i n c e t h e s o u r c e o f F w as i d e n t i c a l
fo r a l l th re e c u ltu r e s .
F+ a n d H f r s t r a i n s w h ic h h a v e l o s t t h e i r
a b i l i t y t o a d s o r b a m a l e - s p e c i f i c b a c t e r i o p h a g e h a v e a l s o b e e n demon­
s t r a t e d t o show a l o s s i n d e g r e e o f f e r t i l i t y
( C la r k a n d A d e lb e r g 5
1962).
The n e g a t i v e c o r r e l a t i o n o f h i g h m o t i l i t y w i t h p r e s e n c e o f HSA+
i n s t r a i n W6 ( s e e s e c t i o n E) i s p r e c e d e n te d by r e p o r t e d i n s t a n c e s i n
w h ic h t h e r e p l i c a t i o n o f c y to p la s m ic e le m e n ts h a s b e e n shown t o b e r e ­
p r e s s e d i n c e r t a i n g e n e t i c b a c k g ro u n d s .
An F f a c t o r c a n i n c o r p o r a t e a
.m a rk e r fro m t h e b a c t e r i a l chrom osom e a n d t h u s t r a n s f e r t h i s f a c t o r fro m
c e l l to
c e l l a t h ig h f re q u e n c y .
An F I a e + f a c t o r 5 w h ic h t r a n s f e r s t h e
44
I a o g e n e i s u n a b l e t o r e p l i c a t e n o r m a lly i n c e l l s w h ic h c o n t a i n , i n
a d d i t i o n , a n o rm a l s e x f a c t o r .
R e p lic a tio n o f F I a c + i s
i n h i b i t e d i n H f r c e l l s ( S c a i f e an d G r o s s , 1 9 6 2 ) .
i s e v id e n c e d i n t h e c a s e o f a c o l i c i n f a c t o r .
c o m p le te ly
A s im ila r in flu e n c e
The g e n e t i c b a s i s o f
C o l i c i n I , when p r e s e n t i n s t r a i n s o f E. c o l i K12 i s a b l e t o i n i t i a t e
its
own t r a n s f e r a s w e l l n s t h e t r a n s f e r o f chrom osom al m a r k e rs i n t h e
absence o f F .
The k i n e t i c s o f c o l i c i n f a c t o r t r a n s f e r i s i n f l u e n c e d
by th e p re s e n c e o f F in e i t h e r th e c o l I + d o n o r o r th e c o ll " r e c i p ie n t
(M onk, 1 9 6 2 ) .
SUMMARY
The f l o e e u l e n t a g g l u t i n a t i o n p r o d u c e d b y m any s t r a i n s o f E» c o l i
K12 i n r e s p o n s e t o a n t i s e r u m i s d u e i n p a r t t o t h e p r e s e n c e o f f l a g e l l a r
a n t i g e n a n d i n p a r t t o a h e a t s t a b l e a n t i g e n (HSA) n o t a s s o c i a t e d w i t h
fla g e lla .
P r e s e n c e o f HSA i s n o t o b v i o u s l y c o r r e l a t e d w i t h F . s t a t u s .
p r o p r i a t e c r o s s e s show i t s
Ap­
g e n e tic b a s is n o t to be c lo s e l y lin k e d to
t h e TLB^ r e g i o n .
O c c a s io n a l m u ta ti o n s fro m .HSA+ t o .HSA- an d t h e r e v e r s e h a v e o c­
c u r r e d s i m u l t a n e o u s l y w i t h m u ta ti o n s o f o t h e r g e n e s n o t o b v i o u s l y r e ­
la te d p h y s io lo g ic a lly .
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