J. Bacteriol.-1984-Schoemaker-551-61
11
8/10/2019 J. Bacteriol.-1984-Schoemaker-551-61 http://slidepdf.com/reader/full/j-bacteriol-1984-schoemaker-551-61 1/11 Vol. 158, No. 2 OURNAL OF BACTERIOLOGY, May 1984, p. 551-561 0021-9193/84/050551-11 02.00/0 Copyright C) 1984, American Society for Microbiology Regulation of Cell Division in Escherichia coli: SOS Induction and Cellular Location of the SulA Protein, a Key to lon-Associated Filamentation and Death JOYCE M. SCHOEMAKER,t RANDALL C. GAYDA,t AND ALVIN MARKOVITZt* Department ofMicrobiology, The University of Chicago, Chicago, Illinois 60637 Received 7 February 1983/Accepted 7 February 1984 Mutations in sulA (sfiA) block the filamentation and death of capR (ion) mutants that occur after treatments that either damage DNA or inhibit DNA replication and thereby induce the SOS response. Previous suiA-iacZ gene fusion studies showed that sulA is transcriptionally regulated by the SOS response system (lexAlrecA). SulA protein has been hypothesized to be additionally regulated proteolytically through the capR (lon) protease, i.e., in lon mutants lacking a functional ATP-dependent protease there would be more SulA protein. A hypothesized function for SulA protein is an inhibitor of cell septation. To investigate aspects of this model, we attempted to construct Ion, I on sulA, and I on sulB strains containing multicopy plasmids specifying the suiA+ gene. Multicopy suIA+ plasmids could not be established in I on strains because more SulA protein accumulates than in a Ion strain. When the sulA gene was mutated by a mini- Mu transposon the plasmid could be established in the I on strains. In contrast, suiA+ plasmids could be established in lon+, lon sulA, and I on sulB strains. The suiA+ plasmids caused I on sulA and I on sulB cells to exist as filaments without SOS induction and to be sensitive to UV light and nitrofurantoin. Evidence implicated higher basal levels of SulA protein in these I on plasmid suiA + strains as the cause of filamentation. We confirmed that the SulA protein is an 18-kilodalton polypeptide and demonstrated that it was induced by treatment with nalidixic acid. The SulA protein was rapidly degraded in a lon+ strain, but was comparatively more stable in vivo in a lon sulB mutant. Furthermore, the SulA protein was localized to the membrane by several techniques. The major phenotypic property of cell division mutants in Escherichia coli is the production of long, nonseptated filamentous cells. Why these mutants filament is a major query in the understanding of cell division in E. coli. One cell division mutant is the lon (or capR) mutant (1, 21, 32, 35, 39, 54). Ion strains filament as long, nonseptated cells after treatments that damage DNA (UV irradiation, nitrofurantoin [NF]) or inhibit DNA replication (nalidixic acid, thymine starvation) or even by shifting them from minimal to com- plex medium (14). Most of these inducing treatments corre- late with the induction of a cellular response termed the SOS response and imply that the cell division defect associated with the lon mutation is mediated through the SOS response. This response is characterized by enhanced capacity for DNA repair and mutagenesis, prophage induction, and inhi- bition of cell division (filamentation). Regulation of the SOS response involves two proteins: LexA, the repressor of the SOS inducible operons, and RecA, a protease that is activat- ed by the SOS-inducing signal and specifically cleaves the LexA protein (reviewed in reference 29). In addition to filamentation, Ion strains show other pheno- typic effects such as the overproduction of capsular polysac- charide, i.e., mucoidy (32, 33) and decreased lysogenization of lambda (12, 14, 55) and P1 phages (53). The Ion mutation also affects the stability of certain abnormal (6, 18, 19, 28, 29, 36, 48) and normal (11, 16, 38, 46) polypeptides. The lon+ gene has been cloned (47, 57), and the protein has been purified to homogeneity (9, 56). It is a tetramer (8) with subunits of 94 kilodaltons (kdal) that has multiple * Corresponding author. t Present address: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637. enzymatic activities: an ATP hydrolysis-dependent protease activity (9, 10; M. F. Charette, Ph.D. thesis, University of Chicago, 1981), DNA stimulated ATPase activity (7a), and nucleic acid-binding activity (56; Charette, Ph.D. thesis). In addition, a defective CapR protein (capR9 allele) has also been purified. The CapR9 protein has retained the general nucleic acid affinity (9, 56), b ut h as lost the protease activity 8, 9). In Ion mutants, second site mutations in sul (suppressor of Ion) prevent the filamentation and UV sensitivity without affecting the mucoid phenotype of the cells (14, 17, 26, 27). These mutants were isolated as UV-, nitro-NF-, or methyl methanesulfonate-resistant derivatives of lon strains. The sul mutations are located at two loci on the E. coli chromo- some; sulA is near pyrD (22 min), and sulB is near leu (2 min). sfiA and sfiB sfi for suppressor of filament induction) are identical to sulA and suiB, respectively, and were isolated as spontaneous thermoresistant revertants of tif-1 (recA441) Ion strains (15, 23, 24). The tif-l Ion strains filament and die at 41°C. To account for their data, George et al. (15) proposed that a division inhibitor (productof the sulA or sulB gene?) w as induced by UV and that it might be more stable in Ion strains. By means of a Mu d(amp-lac) operon fusion that linked the structural gene for 3-galactosidase (lacZ) to the promoter of the sulA operon (22; A. McPartland, J. Yamashita, and M. Villarejo, Abstr. Annu. Meet. Am. Soc. Microbiol. 1981, K58, p. 147), it w as demonstrated that sulA gene transcrip- tion is under SOS (LexA repressor) control. This finding further strengthened the hypothesis that the cell division (septation) defect is mediated through the SOS response. The induction of sulA (sfiA) by UV irradiation was identical in lon+ and Ion mutant sfiA::lacZ fusion strains, indicating that lon does not affect the transcription of sulA 22). Thus, 551
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Transcript of J. Bacteriol.-1984-Schoemaker-551-61
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V o l . 1 5 8 ,
N o . 2
OURNAL
O F
B A C T E R I O L O G Y , May 1 9 8 4 , p .
5 5 1 - 5 6 1
0 0 2 1 - 9 1 9 3 / 8 4 / 0 5 0 5 5 1 - 1 1 0 2 . 0 0 / 0
C o p y r i g h t C )
1 9 8 4 ,
A m e r i c a n
S o c i e t y
f o r
M i c r o b i o l o g y
R e g u l a t i o n o f
C e l l
D i v i s i o n i n E s c h e r i c h i a
c o l i :
SO S I n d u c t i o n
a n d
C e l l u l a r
L o c a t i o n
o f t h e
S u l A
P r o t e i n ,
a
Key
t o
l o n - A s s o c i a t e d
F i l a m e n t a t i o n a n d D e a t h
JOYCE M.
SCHOEMAKER,t RANDALL
C .
GAYDA,t
AND
ALVIN
MARKOVITZt*
D e p a r t m e n t
o f M i cr o b i o l o g y , T h e
U n i v e r s i t y o f C h i c a g o , C h i c a g o ,
I l l i n o i s
6 0 6 3 7
R e c e i v e d
7
F e b r u a r y 1 9 8 3/ A cc e p t e d
7 F e b r u a r y 1 9 8 4
M u t a t i o n s i n s u l A
( s f i A )
b l o c k
t h e
f i l a m e n t a t i o n a n d d ea t h o f capR ( i o n )
mutants t h a t
oc c u r
a f t e r
t r e a t m e n t s t h a t e i t h e r d a m a g e
DNA
or i n h i b i t
DNA
r e p l i c a t i o n
a n d
t h e r e b y
i n d u c e t h e
SO S
response.
P r e v i o u s s u i A -i a c Z
gene
f u s i o n s t u d i e s
s h o w e d t h a t s u l A
i s t r a n s c r i p t i o n a l l y
r e g u l a t e d
b y
t h e
SO S
res pon s e
system
( l e x A l r e c A ) . S u l A
p r o t e i n
h a s
b e e n h y p o t h e s i z e d t o b e
a d d i t i o n a l l y
r e g u l a t e d p r o t e o l y t i c a l l y
t h r o u g h
t h e
capR ( l o n ) p r o t e a s e ,
i . e . ,
i n
l o n
mutants l a c k i n g a f u n c t i o n a l A T P - d e p e n d e n t p r o t e a s e t h e r e w o u l d
b e
more
S u l A
p r o t e i n . A h y p o t h e s i z e d f u n c t i o n f o r S u l A p r o t e i n
i s an i n h i b i t o r
o f c e l l
s e p t a t i o n . To i n v e s t i g a t e
a s p e c t s o f t h i s
m o d e l ,
we
a t t e m p t e d t o c o n s t r u c t Ion, Ion
s u l A ,
a n d Ion s u l B
s t r a i n s
c o n t a i n i n g
m u l t i c o p y
p l a s m i d s
s p ec i f y i n g t h e s u i A +
gene.
M u l t i c o p y s u IA + p l a s m i d s c o u l d n o t
b e e s t a b l i s h e d i n Ion s t r a i n s
b e c a u s e more S u l A p r o t e i n
a c c u m u l a t e s
t h a n
i n
a
I o n
s t r a i n .
When t h e s u l A g e n e was
m u t a t e d b y
a
m i n i -
Mu
t r a n s p o s o n t h e p l a s m i d c o u l d b e e s t a b l i s h e d i n t h e Ion s t r a i n s .
I n
c o n t r a s t ,
s u i A +
p l a s m i d s c o u l d
b e
e s t a b l i s h e d
i n
l o n + ,
l o n
s u l A ,
a n d
Ion
s u l B
s t r a i n s .
T h e
s u i A + p l a s m i d s c a u s e d
Ion
s u l A
a n d
Ion
s u l B
c e l l s
t o
e x i s t
as
f i l a m e n t s
w i t h o u t
SO S i n d u c t i o n a n d t o b e s e n s i t i v e
t o UV
l i g h t a n d
n i t r o f u r a n t o i n .
E v i d e n c e
i mp l i ca t ed h i g h e r b as a l
l e v e l s
o f S u l A
p r o t e i n
i n
t h e s e
Ion
p l a s m i d s u i A
+
s t r a i n s
as
t h e
cause
o f
f i l a m e n t a t i o n . We c o n f i r m e d t h a t t h e S u l A p r o t e i n i s a n 1 8 - k i l o d a l t o n p o l y p e p t i d e
a n d d e m o n s t r a t e d t h a t i t
was i n d u c e d b y t r e a t m e n t w i t h
n a l i d i x i c
a c i d . The
S u l A
p r o t e i n
was r a p i d l y d e g r a d e d i n a
l o n +
s t r a i n , b u t
was
c o m p a r a t i v e l y
more
s t a b l e
i n v i v o i n a
l o n
s u l B
m u t a n t . F u r t h e r m o r e , t h e
S u l A
p r o t e i n
was l o c a l i z e d t o
t h e
membrane b y s e v e r a l
t e c h n i q u e s .
T h e
m a j o r p h e n o t y p i c p r o p e r t y
o f c e l l
d i v i s i o n
m u t a n t s
i n
E s c h e r i c h i a
c o l i
i s t h e
p r o d u c t i o n
o f l o n g ,
n o n s e p t a t e d
f i l a m e n t o u s
c e l l s .
Why t h e s e
m u t a n t s
f i l a m e n t
i s
a m a j o r
q u e r y
i n
t h e u n d e r s t a n d i n g
o f
c e l l d i v i s i o n
i n E .
c o l i . O n e
c e l l
d i v i s i o n
m u t a n t
i s t h e l o n ( o r c a p R )
m u t a n t
( 1 ,
2 1 , 3 2 ,
3 5 ,
3 9 ,
5 4 ) .
I o n
s t r a i n s
f i l a m e n t
a s l o n g ,
n o n s e p t a t e d
c e l l s
a f t e r
t r e a t m e n t s t h a t d a m a g e DNA (UV
i r r a d i a t i o n ,
n i t r o f u r a n t o i n
[ N F ] )
o r
i n h i b i t
DNA
r e p l i c a t i o n
( n a l i d i x i c
a c i d ,
t h y m i n e
s t a r v a t i o n ) o r
e v e n
b y
s h i f t i n g t h e m
f r o m
m i n i m a l t o c o m -
p l e x
medium
( 1 4 ) . M o s t
o f t h e s e i n d u c i n g t r e a t m e n t s c o r r e -
l a t e w i t h
t h e i n d u c t i o n o f a c e l l u l a r r e s p o n s e t e r m e d
t h e
SOS
r e s p o n s e
a n d
i m p l y
t h a t
t h e c e l l d i v i s i o n d e f e c t a s s o c i a t e d
w i t h
t h e l o n m u t a t i o n i s
m e d i a t e d t h r o u g h
t h e SOS r e s p o n s e .
T h i s
r e s p o n s e
i s
c h a r a c t e r i z e d
b y
e n h a n c e d
c a p a c i t y
f o r
DNA
r e p a i r a n d m u t a g e n e s i s , p r o p h a g e
i n d u c t i o n ,
a n d i n h i -
b i t i o n o f
c e l l
d i v i s i o n
( f i l a m e n t a t i o n ) .
R e g u l a t i o n
o f t h e SOS
r e s p o n s e i n v o l v e s t w o p r o t e i n s :
L e x A ,
t h e
r e p r e s s o r
o f
t h e
SOS
i n d u c i b l e
o p e r o n s ,
a n d
R e c A ,
a
p r o t e a s e
t h a t
i s
a c t i v a t -
e d
b y
t h e
S O S - i n d u c i n g
s i g n a l
a n d
s p e c i f i c a l l y
c l e a v e s
t h e
LexA
p r o t e i n
( r e v i e w e d
i n r e f e r e n c e
2 9 ) .
I n
a d d i t i o n
t o f i l a m e n t a t i o n ,
I o n
s t r a i n s s h o w
o t h e r
p h e n o -
t y p i c e f f e c t s
s u c h a s
t h e
o v e r p r o d u c t i o n
o f
c a p s u l a r
p o l y s a c -
c h a r i d e ,
i . e . ,
m u c o i d y
( 3 2 , 3 3 )
a n d d e c r e a s e d
l y s o g e n i z a t i o n
o f l a m b d a
( 1 2 , 1 4 ,
5 5 )
a n d
P 1
p h a g e s
( 5 3 ) .
T h e
I o n
m u t a t i o n
a l s o
a f f e c t s
t h e
s t a b i l i t y
o f
c e r t a i n
a b n o r m a l
( 6 , 1 8 ,
1 9 ,
2 8 , 2 9 ,
3 6 , 4 8 )
a n d
n o r m a l
( 1 1 ,
1 6 , 3 8 ,
4 6 )
p o l y p e p t i d e s .
T h e
l o n +
g e n e
h a s
b e e n c l o n e d
( 4 7 ,
5 7 ) ,
a n d t h e
p r o t e i n
h a s
b e e n
p u r i f i e d
t o
h o m o g e n e i t y
( 9 ,
5 6 ) .
I t
i s
a t e t r a m e r
( 8 )
w i t h s u b u n i t s o f 9 4 k i l o d a l t o n s ( k d a l )
t h a t
h a s
m u l t i p l e
*
C o r r e s p o n d i n g
a u t h o r .
t
P r e s e n t a d d r e s s :
D e p a r t m e n t
o f
B i o c h e m i s t r y
a n d M o l e c u l a r
B i o l o g y ,
U n i v e r s i t y
o f
C h i c a g o , C h i c a g o ,
I L
6 0 6 3 7 .
e n z y m a t i c a c t i v i t i e s :
a n A TP
h y d r o l y s i s - d e p e n d e n t
p r o t e a s e
a c t i v i t y ( 9 ,
1 0 ;
M.
F .
C h a r e t t e ,
P h . D . t h e s i s , U n i v er s i t y o f
C h i c a g o ,
1 9 8 1 ) ,
DNA
s t i m u l a t e d
A T P a s e a c t i v i t y
( 7 a ) , a n d
n u c l e i c
a c i d - b i n d i n g
a c t i v i t y
( 5 6 ; C h a r e t t e ,
P h . D . t h e s i s ) . In
a d d i t i o n , a
d e f e c t i v e
CapR
p r o t e i n ( c a p R 9
a l l e l e ) h a s a l s o
b e e n
p u r i f i e d .
T h e
CapR9 p r o t e i n
h a s
r e t a i n e d t h e g e n e r a l
n u c l e i c a c i d a f f i n i t y ( 9 ,
5 6 ) ,
b u t h a s l o s t
t h e
p r o t e a s e a c t i v i t y
8 ,
9 ) .
I n
I o n
m u t a n t s ,
s e c o n d
s i t e
m u t a t i o n s i n
s u l
( s u p p r e s s o r o f
I o n )
p r e v e n t t h e
f i l a m e n t a t i o n
a n d
UV s e n s i t i v i t y
w i t h o u t
a f f e c t i n g t h e m u c o i d p h e n o t y p e o f
t h e
c e l l s ( 1 4 , 1 7 ,
2 6 ,
2 7 ) .
T h e s e m u t a n t s w e r e
i s o l a t e d
a s U V - , n i t r o - N F - , o r
m e t h y l
m e t h a n e s u l f o n a t e - r e s i s t a n t d e r i v a t i v e s o f l o n
s t r a i n s . T h e
s u l
m u t a t i o n s
a r e
l o c a t e d
a t
t w o
l o c i o n t h e E .
c o l i
c h r o m o -
s o m e ;
s u l A
i s n e a r
pyrD
( 2 2
m i n ) ,
a n d
s u l B
i s
n e a r
l e u
( 2
m i n ) . s f i A
a n d
s f i B
s f i
f o r s u p p r e s s o r
o f f i l a m e n t i n d u c t i o n )
a r e
i d e n t i c a l
t o
s u l A
a n d
s u i B ,
r e s p e c t i v e l y ,
a n d w e r e
i s o l a t e d
a s
s p o n t a n e o u s t h er m o re s i s t an t r e v e r t a n t s
o f
t i f - 1
( r e c A 4 4 1 )
I o n
s t r a i n s ( 1 5 ,
2 3 ,
2 4 ) .
T h e t i f - l
I o n s t r a i n s
f i l a m e n t
a n d d i e a t 4 1 ° C .
To
a c c o u n t f o r
t h e i r
d a t a ,
G e o r g e
e t
a l .
( 1 5 ) p r o p o s e d t h a t a
d i v i s i o n
i n h i b i t o r ( p r o d uc t o f t h e
s u l A
o r
s u l B g e n e ? )
was i n d u c e d
b y
UV
a n d t h a t
i t
m i g h t
b e
m o r e
s t a b l e i n
I o n
s t r a i n s .
By
means
o f
a Mu
d ( am p - l ac ) o p e r o n f u s i o n
t h a t
l i n k e d
t h e
s t r u c t u r a l
g e n e f o r
3 - g a l a c t o s i d a s e
( l a c Z )
t o
t h e
p r o m o t e r
o f t h e
s u l A
o p e r o n ( 2 2 ;
A .
M c P a r t l a n d ,
J .
Y a m a s h i t a ,
a n d M.
V i l l a r e j o ,
A b s t r . A n n u .
M e e t . Am. S o c .
M i c r o b i o l .
1 9 8 1 ,
K 5 8 , p . 1 4 7 ) ,
i t was d e m o n s t r a t e d
t h a t
s u l A g e n e t r a n s c r i p -
t i o n
i s
u n d e r
SO S ( L e x A
r e p r e s s o r )
c o n t r o l .
T h i s
f i n d i n g
f u r t h e r s t r e n g t h e n e d
t h e
h y p o t h e s i s
t h a t
t h e c e l l
d i v i s i o n
( s e p t a t i o n )
d e f e c t
i s m e d i a t e d
t h r o u g h
t h e SO S
r e s p o n s e .
T h e i n d u c t i o n
o f
s u l A
( s f i A )
b y
U V
i r r a d i a t i o n
w as i d e n t i c a l
i n
l o n +
a n d I o n
m u t a n t
s f i A : : l a c Z
f u s i o n
s t r a i n s ,
i n d i c a t i n g
t h a t
l o n d o e s n o t
a f f e c t
t h e
t r a n s c r i p t i o n
o f
s u l A
2 2 ) .
T h u s ,
5 5 1
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a n d
p R G C 1 3 .
C l o n e d
c h r o m o s o m a l genes are a c c e n t e d
b y
t h e h e a v y l i n e .
( A )
p T U 3 0 2 h a s
a 1 . 7 8 - k i l o b a s e
( k b ) BamHI f r a g m e n t e n c o d i n g t h e
s u l A +
( 2 , 3 8 ) gene
a n d
t h e N H 2 - t e r m i n a l
e n d
o f an
ompA
a m b e r
mutant
[ o m p A 3 1 ]
c l o n e d i n t h e m u l t i c o p y v e c t o r p B R 3 2 2 ( 2 ,
5 ,
2 0 ) .
A n
e n l a r g e m e n t o f t h e
s u l A
ompA
DNA i s
i n d i c a t e d
w i t h
t h e
d a s h e d
l i n e s . ( B )
p T U 3 0 2 : : m i n i - M u ( p R G C 1 3 ) h a s an 1 1 - k i l o b a s e
m i n i - M u
i n s e r t i o n w i t h i n
t h e s u l A g en e
o f p T U 3 0 2 . T h e m i n i - M u
i n s e r t i o n
s i t e i n s u l A
( ± 5 0 b a s e s )
i s p r e s e n t e d as a l o l l i p o p
s t r u c t u r e .
( C )
pRGC11 c o n t a i n s t h e
BamHI t o H a e I I I
DNA
f r a g m e n t
o f
pTU302
c o n t a i n i n g
t h e
s u i A + g en e
( - ) c l o n e d
i n t o
p B R 3 2 2
b y r e p l a c i n g
t h e
B a m H I - P v u I I
r e s t r i c t i o n
f r a g m e n t .
R e s t r i c t i o n
enzyme
c o d e s :
R l ,
E c o R I ;
P s t , P s t I ; B , BamHI; H , H a e I I I .
pRGC11
( F i g . 1 C )
c o n t a i n s t h e BamHI t o H a e I I I DNA
f r a g m e n t
o f p T U 3 0 2
c o n t a i n i n g
t h e
s u l A +
gene
c l o n e d
i n t o
p B R 3 2 2 b y r e p l a c i n g t h e
B a m H I - P v u I I r e s t r i c t i o n f r a g m e n t .
T h e r e c o m b i n a n t p l a s m i d was
r e c o g n i z e d as a
l o n
s u l B
( R G C 1 0 3 - 9 ) t r a n s f o r m a n t
t h a t
c o n f e r r e d NF a n d UV s e n s i -
t i v i t y
i . e . , i d e n t i c a l t o t h e
p T U 3 0 2 - c o n f e r r e d p h e n o t y p e ;
T a b l e
2 ) . R e s t r i c t i o n
enzyme a n a l y s i s o f t h e p l a s m i d DNA
c o n f i r m e d
t h a t o n l y
t h e BamHI-HaeIII f r a g m e n t was
c l o n e d .
I t
was n o t e d t h a t
pRGC11 c o n f e r r e d l e s s a m p i c i l l i n
r e s i s t -
a n c e
a n d
y i e l d e d
l e s s
p l a s m i d
DNA
t h a n d i d
p T U 3 0 2 .
S i n c e
t h e
P v u I I
r e s t r i c t i o n
s i t e
i n
p B R 3 2 2 i s
near
t h e
p l a s m i d
o r i g i n
o f
r e p l i c a t i o n ( 5 1 ) ,
i t
appears
t h a t
l i n k i n g
t h e
ompA promoter
( w h i c h
w o u l d
i n i t i a t e
t r a n s c r i p t i o n
i n a
c l o c k w i s e
d i r e c t i o n
t h r o u g h
t h e
P v u I I
r e s t r i c t i o n
s i t e )
h a s a f f e c t e d p l a s m i d
r e p l i c a t i o n ,
r e s u l t i n g
i n
l o w e r p l a s m i d
pRGC11 c o p i e s p er
c e l l
c o m p a r e d
w i t h
p T U 3 0 2 .
P h a s e - c o n t r a s t
m i c r o s c o p y .
C e l l s
f r o m
e x p o n e n t i a l l y
grow-
i n g
c u l t u r e s w ere
p h o t o g r a p h e d
on g l a s s
m i c r o s c o p e
s l i d e s
c o n t a i n i n g
a t h i n
l a y e r o f m i n i m a l
a g a r .
A
Z e i s s
s t a n d a r d
U n i v e r s a l
m i c r o s c o p e
w i t h
a 4 0 x
p h a s e o b j e c t i v e
a n d
K o -
d a k P l u s - X
P a n
f i l m
w ere
u s e d
i n
t h e
p h o t o g r a p h y .
P r o t e i n l a b e l i n g
a n d
a n a l y s i s b y PAGE-SDS. P l a s m i d -
c o n t a i n i n g
m i n i c e l l s were
i s o l a t e d a n d l a b e l e d w i t h
[ 3 5 S ] m e t h i o n i n e
as
d e s c r i b e d
p r e v i o u s l y
( 1 3 , 4 7 ) .
Whole c e l l s
w i t h
or
w i t h o u t p l a s m i d s were i n d u c e d
w i t h
n a l i d i x i c
a c i d
a n d
l a b e l e d
w i t h
[ 3 5 S ] m e t h i o n i n e
as
f o l l o w s .
C e l l s
were
grown
a t
3 7 ° C t o t h e e x p o n e n t i a l p h a s e
i n
g l u c o s e
m i n i m a l
medium
a n d
t h e n d i l u t e d i n t o
t h e same medium a t
3 7 ° C
c o n t a i n i n g
n a l i d i x i c
a c i d
t o a f i n a l c o n c e nt r a t i o n o f
4 0 , u g / m l .
S a m p l e s
were
r e m o v e d
a t
a p p r o p r i a t e
i n t e r v a l s
a n d
l a b e l e d
w i t h 1 0
t o
2 0 , u C i o f
[ 3 5 S ] m e t h i o n i n e
f o r
1 0
m i n , e x c e p t
w h e r e n o t e d
o t h e r w i s e .
L a b e l i n g
was
t e r m i n a t e d
b y c h i l l i n g
t h e
s a m p l e
i n an i c e
b a t h ,
a n d t h e c e l l s
were
washed
i n c o l d
b u f f e r
( 1 0
mM
T r i s - h y d r o c h l o r i d e
[ p H
8 . 0 ] ,
1
mM
EDTA,
1
mM
2 - m e r c a p t o e t h a n o l ,
1 0 0 mM
N a C l ) .
P r o t e i n
s a m p l e s
a n d
s t a n d a r d s
were
p r e p a r e d
a n d
s u b j e c t -
e d t o PAGE-SDS
i n
on e d i m e n s i o n
as d e s c r i b e d
p r e v i o u s l y
w i t h
a p p r o x i m a t e l y
e q u a l
q u a n t i t i e s
o f
t o t a l
r a d i o a c t i v i t y
i n
e a c h g e l
s l o t
( 1 3 ,
4 7 ) . T h e
p r e p a r a t i o n o f
c e l l s
f o r t w o -
d i m e n s i o n a l g e l e l e c t r o p h o r e s i s h a s b e e n d e s c r i b e d
p r e v i -
o u s l y
( 4 6 ) ,
a s h a v e
t h e
d e t a i l s
f o r
t h e
t w o - d i m e n s i o n a l
g e l
e l e c t r o p h o r e s i s
u s e d
h e r e
( 3 , 4 1 - 4 3 ) .
C e l l f r a c t i o n a t i o n
p r o c e d u r e s .
P r e p a r a t i o n
o f
w h o l e c e l l
e x t r a c t s , i s o l a t i o n
o f
S a r k o s y l - i n s o l u b l e m e m b r a n e s , a n d
s e p a r a t i o n
o f i n n e r a n d o u t e r m e m b r a n e s
b y
i s o p y c n i c
s u c r o s e
g r a d i e n t s
w e r e
a s
d e s c r i b e d p r e v i o u s l y
( 1 3 ) .
Mem-
b r a n e s
l a y e r e d
o n
t h e i s o p y c n i c
g r a d i e n t w e r e o b t a i n e d
b y
e i t h e r F r e n c h
p r e s s u r e
c e l l
l y s i s a t 1 6 , 0 0 0 l b / i n 2 ( 4 9 ) o r
s o n i c
d i s r u p t i o n .
T h e
q u i c k
membrane
e x t r a c t i o n p r o c e d u r e
w i t h
N a O H p r e c i p i t a t i o n ,
l e a v i n g
a s u b se t o f i n n e r a n d o u t e r
m e m b r a n e p r o t e i n s i n t h e
p e l l e t , w a s
a s d e s c r i b e d
p r e v i o u s l y
4 5 ) .
T h e f o u r
p T U 3 0 2 - c o n t a i n i n g
s t r a i n s
( R G C 1 0 3 - 2 ,
R G C 1 0 3 -
9 , M C 1 0 0 , a n d R G C 2 0 8 ) i n d u c e d w i t h
n a l i d i x i c
a c i d
f o r 9 0
m i n a n d t h e n l a b e l e d
f o r
1
m i n
w e r e
f r a c t i o n a t e d a s
f o l l o w s .
A
s a m p l e
w a s i m m e d i a t e l y
f r a c t i o n a t e d b y
N a O H
p r e c i p i t a -
t i o n ( 4 5 ) . A n o t h e r
s a m p l e w a s c h i l l e d a n d s o n i c a t e d , a n d t h e
e n v e l o p e w a s s e p a r a t e d f r o m s o l u b l e c y t o p l a s m i c
p r o t e i n s
b y c e n t r i f u g a t i o n
( 2 0 , 0 0 0 x
g f o r 1 5 m i n ) .
O n e
h a l f o f t h e
e n v e l o p e f r a c t i o n w a s t h e n f u r t h e r
s o l u b i l i z e d
w i t h
1%
S a r k o s y l
f o r
o u t e r
m e m b r a n e
l o c a l i z a t i o n .
T h e
p r o t e i n s
w e r e
e x a m i n e d b y
PAGE-SDS a n d a u t o r a d i o g r a p h y .
RESULTS
E f f e c t s
o f h i g h s u l A g e ne d o s a g e o n
v a r i o u s l o n
s t r a i n s .
W e
a t t e m p t e d t o c o n s t r u c t
l o n ,
l o n
s u l A ,
a n d
I o n s u l B s t r a i n s
c o n t a i n i n g
t h e
m u l t i c o p y
s u i A +
p l a s m i d , p T U 3 0 2
( T a b l e 2 ) .
T h e
a b i l i t y t o e s t a b l i s h t h e m u l t i c o p y
s u l A +
p l a s m i d
pTU302
i n
a l o n
m u t a n t m i g h t
b e e x p e c t e d
t o d e p e n d o n w h e t h e r
t h e
S u l A
p r o t e i n i s
m o r e s t a b l e i n
t h e l o n c e l l a n d , i f
s o , t h e
p h e n o t y p i c e f f e c t s o f
i n c r e a s e d s u l A+ g e n e d o s a g e
i . e . ,
w h e t h e r m u l t i p l e
c o p i es o f s u l A a r e l e t h a l
t o
t h e
c e l l ) . A l l
s t r a i n s
w e r e g r o w n i n
m i n i m a l m e d i u m b e c a u s e i n
c o m p l e x
m e d i u m s o m e
l o n s t r a i n s t e n d
t o
f i l a m e n t
a n d
d i e ( 1 4 ) .
A f t e r
t r a n s f o r m a t i o n , c e l l s
w e r e
p l a t e d
o n m i n i m a l a g a r a n d
c o m -
p l e x a g a r
w i t h
c a r b e n i c i l l i n a n d w i t h o r w i t h o u t t h e
d r u g
NF.
N F
m i m i c s
t h e
e f f e c t s
o f
UV
i r r a d i a t i o n
o n
I o n
c e l l s ;
i t
i n v o k e s
t h e
SO S r e s p o n s e i n t h e s e
c e l l s
( 2 2 ) , l e a d i n g
t o c e l l
f i l a m e n t a t i o n
a n d d e a t h
( 1 4 ) .
p T U 3 0 2
t r an s f o r ma nt s o f
t h e
l o n + s t r a i n
MC100 w e r e
o b t a i n e d
o n a l l m e d i a w i t h
n o
a p p a r e n t
e f f e c t s o f
t h e
i n c r e a s e d
s u l A +
g e n e d o s a g e
( c o m -
T A B L E 2 .
C h a r a c t e r i z a t i o n o f
a
m u l t i c o p y p l a s m i d c o n t a i n i n g
s u l A ( p T U 3 0 2 )
b y
t r a n s f o r m a t i o n a
T r a n s f o r m a n t s
p e r n g
o f DNA
R e c i p i e n t
b a c t e r i a l
P l a s m i d
p T U 3 0 2
( s u l A + ) P l a s m i d p B R 3 2 2
( c o n t r o l )
s t r a i n ___________________________________
M i n i m a l Y E T C Y ET - N F 2 d
M i n i m a l
YET
YET-NF2
MC100
( c a p R + ) 9 7 8 7 8 9 7 9 0
5 4 0 4 2 0
R G C 1 0 3
( c a p R 9 ) 0 0
0
3 0 0 2 4 0 0
R G C 1 0 3 - 2
3 6 e 2 7 e 0
2 7 0 3 5 0
3 5 0
( c a p R 9
s u l A 2 )
R G C 1 0 3 - 9 2 8
2 6 0 1 9 0 1 2 0 2 2 0
( c a p R 9
s u I B 9 )
a
A l l
a g a r
m e d i a
a l s o
c o n t a i n e d
2 5
, u g
o f c a r b e n i c i l l i n
p e r
m l .
b M i n i m a l
medium
i s M9 w i t h o u t
C a C l 2
a n d
w i t h o u t
s t r e p t o m y c i n
5 7 ) .
C
YET
i s
y e as t e x t r ac t - t r y p t o n e ( 1 4 ) .
d
YET-NF2
i s YET s u p p l e m e n t e d w i t h
2
, u g
o f n i t r o f u r a n t o i n
p e r
m l .
eThe c o l o n y
s i z e o f t h e s e t r a n s f o r m a n t s
was s m a l l e r
t h a n t h a t o f
c o m p a r a b l e
t r a n s f o r m a n t s
w i t h
p B R 3 2 2 .
V O L .
1 5 8 ,
1 9 8 4
8/10/2019 J. Bacteriol.-1984-Schoemaker-551-61
http://slidepdf.com/reader/full/j-bacteriol-1984-schoemaker-551-61 4/11
5 5 4 SCHOEMAKER,
GAYDA,
AND M A R K O V I T Z
p a r e w i t h a n d
w i t h o u t
N F ) ,
w h e r e a s
pTU302
t r a n s f o r m a n t s
o f
t h e i s o g e n i c I o n
s t r a i n RGC103
w e r e n o t
o b t a i n e d o n a n y
o f
t h e m e d i a
( T a b l e
2 ) .
S t r a i n
RGC103
w a s t r a n s f o r m a b l e
w i t h t h e c l o n i n g
v e c t o r p B R 3 2 2 o n b o t h
m i n i m a l a n d c o m -
p l e x m e d i a
i n t h e s a m e
e x p e r i m e n t s , a l t h o u g h t h e I o n
s t r a i n
d i d n o t s u r v i v e o n m e d i u m
w i t h N F a s
e x p e c t e d ( 5 7 ) .
p T U 3 0 2
t r a n s f o r m a n t s
w e r e
o b t ai n e d w i t h
t h e
l o n
s u l A a n d
l o n
s u l B
s t r a i n s
( T a b l e 2 ) . p T U 3 0 2
t r a n s f o r m a n t s
o f t h e I o n
s u l A
s t r a i n
( R G C 1 0 3 - 2 ) g r e w
s l o w l y ,
p r o d u c i n g
s m a l l
c o l o -
n i e s o n b o t h
m i n i m a l a n d c o m p l e x
a g a r
c o m p a r e d
t o
p B R 3 2 2 - t r a n s f o r m a n t s .
I n c o n t r a s t p T U 3 0 2 - t r a n s f o r m a n t s
o f t h e I o n s u l B
s t r a i n
( R G C 1 0 3 - 9 )
g r e w
a l m o s t
a s w e l l a s t h e
c o n t r o l
p B R 3 2 2 - t r a n s f o r m a n t s .
N o
p T U 3 0 2 t r a n s f o r m a n t s
o f
e i t h e r
I o n
s u l A
o r l o n s u l B
w e r e o b t a i n e d o n a g a r
c o n t a i n i n g
N F , a l t h o u g h
t h e s e
s t r a i n s a r e
t h e m s e l v e s
r e s i s t a n t
t o
N F
( 1 4 ;
T a b l e
2 ) .
S i n c e t h e
s u l A
g e n e i s
i n d u c i b l e b y
S O S -
i n d u c i n g t r e a t m e n t s ( 2 2 ) ,
t h i s
s e n s i t i v i t y
o f
p T U 3 0 2 - t r a n s -
f o r m e d
l o n s u l
s t r a i n s
t o
NF i s
c o n s i s t e n t
w i t h t h e
i n t e r p r e t a -
t i o n t h a t
t h e l e t h a l e f f e c t s
o f a
h i g h
d o s a g e
o f
s u I A +
g e n e
a f t e r SOS
i n d u c t i o n
c a n n o t
b e
c o m p e n s a t e d
f o r
b y
a
s i n g l e
c h r o m o s o m a l
m u t a t i o n i n e i t h e r
s u l A
o r
s u l B .
S i m i l a r t r a n s f o r m a t i o n
r e s u l t s w i t h
p l a s m i d p T U 3 0 2
w e r e
o b t a i n e d
f o r
a n o t h e r s e t
o f
i s o g e n i c
l o n + , l o n ,
a n d l o n
s u l A
s t r a i n s w i t h o u t s u p p r e s s o r s
( X 7 1 0 2 ,
R G C 1 2 3 , R G C 1 2 3 - 2 ;
T a b l e
3 ) .
T h e
r e s u l t s o f
t r a n s f o r m a t i o n
w i t h
p l a s m i d s
pRGC11
a n d
pRGC13
( T a b l e
3 )
s h o w
t h a t
t h e
i n a b i l i t y
t o t r a n s f o r m i n t o a
c a p R ( l o n )
s t r a i n
i s
a r e s u l t
o f
t h e
p l a s m i d - l o c a l i z e d
s u l A +
a l l e l e .
T h e s u b c l o n e d
s u l A + - c o n t a i n i n g p l a s m i d
p R G C 1 1
s p e c i f i e s
t h e
s u I A + g e n e
p l u s o n l y
t h e
f i r s t
2 0 a m i n o
a c i d s
o f
t h e
ompA p r o t e i n
s t i l l r e t a i n i n g
t h e
o m p A 3 1
a m b e r
m u t a t i o n
a t
p o s i t i o n
7 o f t h e a m i n o
a c i d
s e q u e n c e ;
F i g .
1 C ) .
N o
p R G C 1 1
t r a n s f o r m a n t s
w e r e
o b t a i n e d
w i t h
t h e
c a p R
( l o n )
s t r a i n ,
a s
w i t h
p T U 3 0 2 .
I n
c o n t r a s t ,
m u t a t i n g
t h e
s u l A
g e n e
o f
p T U 3 0 2
b y i n s e r t i n g
a
m i n i - M u
t r a n s p o s o n , g e n e r a t i n g
p l a s m i d pRGC13
( F i g . 1 B ) ,
r e s u l t e d
i n v i a b l e t r a n s f o r m a n t s
o f
t h e
c a p R ( I o n )
s t r a i n
( T a b l e
3 ) .
W e
n e x t
a s k e d
t h e
q u e s t i o n :
c a n we
i n t r o d u c e
t h e
c a p R 9
a l l e l e
b y
P 1 t r a n s d u c t i o n
i n t o
a
c a p R +
s t r a i n
a l r e a d y
c o n -
t a i n i n g
p T U 3 0 2 . T h e
i n t r o d u c t i o n
o f
t h e
c a p R 9
m u t a t i o n
was
a t t e m p t e d b y
P 1
p h a g e
t r a n s d u c t i o n
i n t o
a
c a p R + ( X 7 1 0 2 )
s t r a i n c o n t a i n i n g
p l a s m i d
p T U 3 0 2
b y s e l e c t i n g
f o r
p r o C +
t r a n s d u c t a n t s
a n d
s c o r i n g
f o r
c a p R 9 ( m u c o i d p h e n o t y p e ) .
I n s u c h a n
e x p e r i m e n t
i n
w h i c h
3 8 0
p r o C +
t r a n s d u c t a n t s
w e r e s c r e e n e d ,
n o
m u c o i d
p r o C +
t r a n s d u c t a n t s o f
X 7 1 0 2 ( p T U 3 0 2 )
w e r e
o b t a i n e d ,
w h e r e a s w i t h s t r a i n s X 7 1 0 2
a n d
X 7 1 0 2 ( p B R 3 2 2 ) ,
10% o f t h e
p r o C +
t r a n s d u c t a n t s
w e r e
m u c o i d .
T h u s
i t
a p p e a r s
t h a t e v e n when
t h e
p l a s m i d
p T U 3 0 2
i s s t a b l e
t h e i n t r o d u c t i o n
o f a
c a p R 9
a l l e l e
p r o d u c e s
a
n o n a c c e p t a b l e
c o m b i n a t i o n .
A
f u r t h e r c h a r a c t e r i z a t i o n o f
t h i s
p h e n o m e n o n
i s
p r e s e n t e d
i n a
l a t e r s e c t i o n .
T A B L E
3 . T r a n s f o r m a t i o n
a b i l i t y
o f s u l A
p l a s m i d s
i n t o
c a p R
m u t a n t s a
T r a n s f o r m a t i o n
a b i l i t y b
R e c i p i e n t
b a c t e r i a l s t r a i n
p T U 3 O 2
p R G C l l
p R G C 1 3
X 7 1 0 2
( c a p R + )
++ ++ ++
RGC123
( c a p R 9 )
-
-
+
R G C 1 2 3 - 2
( c a p R 9
s u l A 1 2 )
+
+ +
a
A l l
a g a r
m e d i a
c o n t a i ne d 2 5 , u g o f
c a r b e n i c i l l i n p e r m l .
b
++,
A p p r o x i m a t e l y
1 0 0
t r a n s f o r m a n t s
p e r n g
o f D N A ;
+ ,
a p p r o x i m a t e l y
2 5
t o 5 0 t r a n s fo r m a nt s p e r
n g o f
D N A ;
- ,
n o
t r a n s f o r m a n t s
o b t a i n e d .
P l a s m i d
t r a n s f o r m a t i o n
w a s d o n e
w i t h
m i n i m a l
m e d i u m - g r o w n
a n d
p l a t e d
c e l l s
( s e e T a b l e
2 , f o o t n o t e b ) .
P h e n o t y p i c e f f e c t s
o f t h e s u l A U p l a s m i d . I f
t h e S u l A p r o t e i n
b y i t s e l f
i s a n i n h i b i t o r
o f
c e l l d i v i s i o n
a n d
i s
m o r e
s t a b l e i n
I o n
s t r a i n s , t h e n t h e
i n t r o d u c t i o n o f a h i g h - c o p y s u i A +
p l a s m i d m i g h t m i m i c
UV
i r r a d i a t i o n
o r o t h e r S O S - i n d u c i n g
t r e a t m e n t s
b y c a u s i n g I o n
c e l l s t o f i l a m e n t .
S i n c e
p T U 3 0 2 -
t r a n s f o r m a n t s o f I o n
m u t a n t s a r e n o t v i a b l e , we e x a m i n e d
p T U 3 0 2
t r a n s f o r m a n t s o f l o n s u l A
( R GC 1 03 - 2 ) a n d
l o n s u l B
( R G C 1 0 3 - 9 ) a s
w e l l a s l o n + ( M C 1 0 0 ) c e l l s .
p B R 3 2 2 t r a n s -
f o r m a n t s
o f
t h e s e
s t r a i n s
w e r e
a l s o
e x a m i n e d .
A l l t r a n s -
f o r m e d
s t r a i n s w e r e g r o w n i n
m i n i m a l
m e d i u m t o t h e m i d e x -
p o n e n t i a l p h a s e .
A s
a
f i l a m e n t a t i o n c o n t r o l
a
s a m p l e
o f
e a c h
c u l t u r e
w a s
a l s o
t r e a t e d
w i t h
n a l i d i x i c a c i d
f o r
1 2 0
m i n
t o
i n d u c e
t h e SOS
r e s p o n s e a n d
f i l a m e n t s .
T h e c e l l s
w e r e
e x a m i n e d
b y
p h a s e - c o n t r a s t m i c r o s c o p y .
E v e n w i t h o u t
n a l i -
d i x i c
a c i d t r e a t m e n t ,
t h e
p T U 3 0 2 - t r a n s f o r m e d
l o n s u l A
a n d
l o n
s u l B p o p u l a t i o n s ( F i g .
2 B a n d
D , r e s p e c t i v e l y )
c o n t a i n e d
many
l a r g e
f i l a m e n t o u s c e l l s c o m p a r e d
w i t h t h e
c o n t r o l
p o p u l a t i o n s t r a n s f o r m e d
w i t h
p B R 3 2 2
( F i g . 2A
a n d
C ) .
T h e
l o n
s u l B m u t a n t
w i t h ( F i g . 2 C ) o r w i t h o u t
p B R 3 2 2
( d a t a
n o t s h o w n )
i s
a l w a y s
e l o n g a t e d
c o m p a r e d
w i t h t h e
l o n
s u l A m u t a n t ( F i g .
2 A )
i n t h e a b s e n c e o f a n
S O S - i n d u c i n g
t r e a t m e n t
w h e n s a l t c o n c e n t r a t i o n
i s l o w
( 1 7 , 2 6 )
a s
i n
m i n i m a l m e d i u m . p T U 3 0 2 - t r a n s f o r m e d
s t r a i n s t r e a t e d w i t h
n a l i d i x i c a c i d
f o r 1 2 0 m i n
w e r e n o t s u b s t a n t i a l l y
m o r e
f i l a m e n t o u s
t h a n
t h e
u n t r e a t e d
c e l l s
( d a t a
n o t
s h o w n ) ,
a l -
t h o u g h t h i s t y p e
o f t r e a t m e n t i s
l e t h a l .
T h u s ,
t h e
i n t r o d u c t i o n
o f a m u l t i c o p y
s u l A +
p l a s m i d
c a u s e s c e l l
f i l a m e n t a t i o n
i n I o n
s u l A
a n d
l o n
s u l B
s t r a i n s w i t h o u t a n y
a p p a r e n t SOS
r e -
s p o n s e .
I d e n t i f i c a t i o n
o f t h e S u l A
p r o t e i n
s p e c i f i e d
b y
p l a s m i d
p T U 3 0 2 .
P l a s m i d
p T U 3 0 2 w a s t r a n s f o r m e d i n t o
t h e m i n i c e l l -
p r o d u c i n g
s t r a i n D S 4 1 0
t o i d e n t i f y
t h e s u l A p r o d u c t s p e c i f i e d
b y p T U 3 0 2 .
W e
w e r e a l s o
a b l e
t o
o b t a i n
v i a b l e p T U 3 0 2
t r a n s f o r m a n t s
o f
a l o n ( c a p R 6 )
s t r a i n t h a t
p r o d u c e s
m i n i c e l l s
( d e s i g n a t e d
L o n M i n )
b e c a u s e
t h e
c a p R 6 m u t a t i o n
h a s
r e s i d u a l
l o n f u n c t i o n ( m o r e
UV r e s i s t a n c e )
a s c o m p a r e d
w i t h
t h e
c a p R 9 a l l e l e ( 3 4 ) .
M i n i c e l l s
w e r e i s o l a t e d ,
l a b e l e d
f o r
1 0
m i n
a t 3 7 ° C
w i t h [ 3 5 S ] m e t h i o n i n e , a n d
t h e n
d i l u t e d
i n t o c o l d
b u f f e r
c o n t a i n i n g e x c e s s
L - m e t h i o n i n e
a n d t h e p r o t e a s e
i n h i b i t o r
p h e n y l m e t h y l s u l f o n y l
f l u o r i d e .
A
s h o r t
l a b e l i n g
t i m e w a s c h o s e n
b e c a u s e : i ) t h e S u l A
p r o t e i n i s l e s s s t a b l e
i n
w i l d - t y p e
b a c t e r i a l
s t r a i n s
( 3 8 ) , a n d
i i ) t h e S u l A p r o t e i n
w a s
n o t d e t e c t e d
w h e n
pTU100
p l a s m i d - c o n t a i n i n g m i n i c e l l s
w e r e l a b e l e d f o r
2 h
( 2 ,
5 ) . I n F i g . 3 ,
t h e
S u l A
p r o t e i n
c a n
b e
i d e n t i f i e d a s t h e m a j o r
u n i q u e
b a n d
s p e c i f i e d
b y p l a s m i d
p T U 3 0 2 ( F i g .
3 ,
c o m p a r e
l a n e s 1 , 2 ) . I t h a s a m o l ec u l ar
m a s s
o f 1 8
k d a l .
T h e
o t h e r
m a j o r p o l y p e p t i d e b a n d s
a r e
s p e c i f i e d
b y
t h e
c l o n i n g
v e c t o r p B R 3 2 2
a n d i n c l u d e t h e 3 - l a c t a m a s e
t h a t m i g r a t e s a s 3 1 - a n d 2 8 - k d a l
p o l y p e p t i d e s . We f o u n d n o
e n h a n c i n g e f f e c t s
o f t h e S O S - i n d u c i n g
d r u g s n a l i d i x i c
a c i d o r
b l e o m y c i n o n
t h e e x p r e s s i o n o f t h e
s u l A g e n e i n i s o l a t e d
m i n i c e l l s ( F i g . 3 ,
l a n e s 1 , 3 , a n d
4 ) . T h e
p r e s e n c e o f
PMSF
d u r i n g t h e
l a b e l i n g p e r i o d
d i d n o t i n c r e a s e t h e
r e l a t i v e
a m o u n t o f S u l A
p r o t e i n ( F i g . 3 ,
l a n e s
1
a n d 5 ) . I n a d d i t i o n ,
t h e
S u l A
p r o t e i n
w a s s y n t h e s i z e d
i n Lon
M i n
m i n i c e l l s
c o n t a i n i n g
p T U 3 0 2
( F i g .
3 ,
l a n e s 6 a n d
7 ) a n d
i n
D S 4 1 0
m i n i c e l l s c o n t a i n i n g
t h e p T U 1 0 0
p l a s m i d ( F i g . 3 ,
l a n e
8 ) ,
w h i c h a l s o
s p e c i f i e s
t h e
c o m p l e t e O m p A p r o t e i n ,
p r o t e i n
I I * .
T h i s l a t t e r
p r o t e i n
i s
v i s i b l e ( 3 3 k d a l )
a l o n g w i t h
a
s l i g h t l y l a r g e r
p o l y p e p t i d e t h a t i s t h e
p r ec u r s o r o f p r o t e i n
I I *
( 2 0 ) .
T h e Lon
M i n ( p T U 3 0 2 )
s t r a i n s h o w s s e v e r a l
a d d i t i o n a l
p o l y p e p t i d e
b a n d s
i n c l u d i n g t w o
p o l y p ep t i d e s w i t h
m o l e c u -
l a r
m a s s e s
o f a p p r o x i m a t e l y
2 9 k d a l ( j u s t a b o v e
t h e
2 8 - k d a l
, - l a c t a m a s e
p o l y p e p t i d e )
a n d 2 4 k d a l t h a t
a r e
n o t
v i s i b l e
i n
D S 4 1 0 ( p T U 3 0 2 ) ( F i g .
3 ,
c o m p a r e
l a n e s
5
a n d 6 ) .
T h e y a r e
J .
B A C T E R I O L .
8/10/2019 J. Bacteriol.-1984-Schoemaker-551-61
http://slidepdf.com/reader/full/j-bacteriol-1984-schoemaker-551-61 5/11
R EG U L A T I O N
O F C E L L
DIVISION I N
E . COLI 5 5 5
t
~~~~~~~~~~~~~~~~~~~~~~-
s W ~~~~~j~~
~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ,
~~~----~~~~
~~ N ~~~
V-.
F
~ ~~~YN
F I G . 2 .
E f f e c t o f
m u l t i c o p y
s u i A
p l a s m i d
o n
m o r p h o l o g y
o f b a c t e r i a l m u t a n t s
g r o w n
i n
m i n i m a l
m e d i u m .
( A ) R G C 1 0 3 - 2 i o n s u l A )
w i t h
c l o n i n g
v e c t o r
p B R 3 2 2 ; ( B )
R G C 1 0 3 - 2
w i t h s u i A
p l a s m i d p T U 3 0 2 ;
( C )
R G C 1 0 3 - 9
( I o n
s u i B )
w i t h
p B R 3 2 2 ; ( D )
R G C 1 0 3 - 9
w i t h
p T U 3 0 2 .
p o l y p e p t i d e s
r e s u l t i n g
f r o m
s u p p r e s s i o n
o f
t h e
a m b e r a l l e l e
o f t h e
ompA
c l o n e d
f r a g me n t b e c a u s e i )
t h e
L o n
M i n
s t r a i n
c o n t a i n s
a s u p p r e s s o r ( 1 1 ) , i i ) t h e p o l y p e p t i d e s a r e
n o t
v i s i b l e
i n
t h e c o n t r o l , Lon M i n ( p B R 3 2 2 ) ( F i g .
3 ,
l a n e
7 ) ,
a n d
i i i )
B r e m e r
e t
a l .
r e p o r t e d
t h e
a p p e a r a n c e
o f p o l y p e p t i d e s
w i t h
a p p a r en t m o l e c u l ar
m a s s e s o f
3 0
a n d 2 4
k d a l i n
t h e
e n v e l o p e s
o f
p T U 3 0 2 - t r a n s f o r m e d s u p D o r s u p F s t r a i n s t h a t
w e r e
a b s e n t
i n
n o n s u p p r e s s i n g s t r a i n s
( 5 ) .
T h e y
a l s o
p r o v i d -
e d
e v i d e n c e t h a t t h e
l a r g e r
p o l y p e p t i d e i s a
p r e c u r s o r o f t h e
2 4 - k d a l
p o l y p e p t i d e ( 5 ) .
I d e n t i f i c a t i o n
o f t h e S u l A p r o t e i n
i n
w h o l e
c e l l s .
T h e
i d e n t i f i c a t i o n o f
t h e
S u l A
p r o t e i n s p e c i f i e d b y
p T U 3 0 2 i n
m i n i c e l l s
p r o v i d e d
a s t a n d a r d
t o
u s e
t o i d e n t i f y
t h e S u l A
p r o t e i n
i n
w h o l e c e l l s .
S i n c e
t h e s u l A
p r o m o t e r i s SOS
i n d u c i b l e
( 2 2 ) ,
S u l A
p r o t e i n s h o u l d b e
i n
h i g h a m o u n t s i n
c e l l s
t r e a t e d w i t h n a l i d i x i c
a c i d
( a n S O S - r e s p o n s e i n d u c e r ) .
We
i n i t i a l l y
c h o s e
p l a s m i d
t r a ns f o r ma n t s o f t h e
l o n s u l B
a n d
l o n s u l A
s t r a i n s
a s
t h e
b e s t
s t r a i n s
f o r
o b s e r v i n g
S u l A
p r o t e i n
b e c a u s e
o f
p r e v i o u s l y c i t e d
e v i d e n c e
s u g g e s t i n g
t h a t
t h i s
p r o t e i n
i s
m o r e
s t a b l e
i n
a
l o n
b a c k g r o u n d .
A n
e x p o n e n t i a l l y
g r o w i n g
c u l t u r e i n
m i n i m a l m e d i u m w a s
t r e a t e d w i t h
n a l i d i x -
i c a c i d
f o r v a r i o u s
l e n g t h s
o f
t i m e b e t w e e n
0
a n d
4
h .
S a m p l e s
o f t h e
c u l t u r e s
w e r e
l a b e l e d w i t h
[ 3 5 S ] m e t h i o n i n e
f o r
1 0 m i n . C e l l
p r e p a r a t i o n s
w e r e
t h e n
e x a m i n e d
f o r
S u l A
p r o t e i n ,
a s
i d e n t i f i e d
b y
c o m p a r i s o n
w i t h
t h e
l a b e l e d
m i n i c e l l
p r e p a r a t i o n
( F i g .
4 ,
l a n e
1 0 ) .
S u l A
p r o t e i n
w a s
n o t
d e t e c t e d
i n
t h e
a b s e n c e o f n a l i d i x i c a c i d t r e a t m e n t o f
I o n
s u l B
o r I o n
s u l A
s t r a i n s
c o n t a i n i n g
t h e
s u i A + p l a s m i d p T U 3 0 2 ( F i g . 4 ,
l a n e s 5 a n d
6 ;
F i g .
5 ,
l a n e
7 ) .
H o w e v e r ,
i t
was v i s i b l e
i n
t h e s e
s t r a i n s a f t e r
t h e 1
h
o f
n a l i d i x i c
t r e a t m e n t
[ F i g .
4 ,
l a n e s
7
t h r o u g h
9 ,
I o n
s u l B ( p T U 3 0 2 ) ;
F i g . 5 ,
l a n e s 8 a n d
9 ,
I o n
s u l A ( p T U 3 0 2 ) ] .
S u l A p r o t e i n w a s a l s o v i s i b l e a f t e r n a l i d i x i c
a c i d t r e a t m e n t
o f
t h e I o n s u l B
s t r a i n
c o n t a i n i n g
t h e o t h e r
s u I A + p l a s m i d , pTU100 ( F i g . 4 , l a n e s
1 1
t h r o u g h 1 4 ) .
T h e
O m p A p r o t e i n ( p r o m i n e n t
b a n d
b e t w e e n
2 4 a n d 4 5
k d a l )
i n
31
....
8
2 4
-
1
8
( s . I A )
4 n , .
1
2 3 4 5 6 7 8
F I G .
3 . P o l y p e p t i de s s y n t h es i z ed
i n
p l a s m i d - c o n t a i n i n g
m i n i c e l l s
a n d
s e p a r a t e d
o n a 1 5 t o 30%
p o l y a c r y l a m i d e g r a d i e n t
i n
S D S .
L a n e s :
1 ,
D S 4 1 0 ( p T U 3 0 2 ) ; 2 ,
D S 4 1 0 ( p B R 3 2 2 ) ; 3 , D S 4 1 0 ( p T U 3 0 2 )
t r e a t e d w i t h n a l i d i x i c
a c i d
( 4 0
p . g / m l )
f o r 3 0
m i n
b e f o r e
[ 3 5 S ] m e t h i o -
n i n e
l a b e l i n g ; 4 ,
p T U 3 0 2
t r e a t e d
w i t h
b l e o m y c i n
( 5 0
, g / m l )
f o r
3 0
m i n b e f o r e
l a b e l i n g ; 5 , D S 4 1 0 ( p T U 3 0 2 )
l a b e l e d
i n t h e
p r e s e n c e
o f
t h e
p r o t e a s e
i n h i b i t o r
p h e n y l m e t h y l s u l f o n y l
f l u o r i d e
( 2 0 0
p g / m l ) ;
6 ,
Lon
M i n
( p T U 3 0 2 ) ; 7 ,
Lon
M i n
( p B R 3 2 2 ) ;
8 ,
D S 4 1 0
( p T U 1 0 0 ) .
V O L .
1 5 8 ,
1 9 8 4
v f . .
S I
..,
4 01
- O . w w
IM P
W . ,
. -
- i M 1 7 7 :
- 4 1 0 O W :
8/10/2019 J. Bacteriol.-1984-Schoemaker-551-61
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5 5 6
SCHOEMAKER, GAYDA,
AND
M A R K O V I TZ
-
a
w:M
v 6
P
q
Te_eH a
5
c i
o
ns
;
t
_
g
24
~ ~ ~ ~
;
1
2 3 4 5 6
7 8
9
1 0
1
1
1 2
1 3
1 4
F I G . 4 . I n d u c t i o n o f
S u l A
p r o t e i n b y
n a l i d i x i c
a c i d i n
s t r a i n
R G C 1 0 3 - 9
( I o n
s u i B )
c o n t a i n i n g
s u i A +
p l a s m i d s .
E l e c t r o p h o r e s i s
was
as i n
F i g . 2 . L a n e s :
1 t o 4 , R G C 1 0 3 - 9 ( p B R 3 2 2 )
i n c u b a t e d
w i t h
n a l i d i x i c a c i d
f o r
0 , 1 ,
2 , a n d
4
h , r e s p e c t i v e l y ,
b e f o r e [ 35 S ]m e th i o n -
i n e
l a b e l i n g ; 5 t o 9 , R G C 1 0 3 - 9 ( p T U 3 0 2 )
i n c u b a t e d w i t h o u t
n a l i d i x i c
a c i d f o r
2 h
( l a n e 5 )
a n d 0 h
( l a n e
6 )
a n d
w i t h n a l i d i x i c a c i d f o r
1 ,
2 ,
a n d
4
h ( l a n e s
7 , 8 , 9 ,
r e s p e c t i v e l y )
b e f o r e l a b e l i n g ; 1 0 ,
m i n i c e l l
D S 4 1 0 ( p T U 3 0 2 ) as a s t a n d a r d
f o r s u l A
s p e c i f i e d p r o t e i n ;
1 1 t o
1 4 ,
R G C 1 0 3 - 9 ( p T U 1 0 0 )
i n c u b a t e d
w i t h
n a l i d i x i c
a c i d f o r
0 , 1 ,
2 ,
a n d 4
h ,
r e s p e c t i v e l y ,
b e f o r e l a b e l i n g .
t h i s s t r a i n
d o e s
n o t
appear
t o b e
s y n t h e s i z e d
i n
h i g h e r
amounts ( F i g .
4 , co m p are
l a n e
1 1 w i t h
l a n e s 1 2
t h r o u g h
1 4 ) ,
i n d i c a t i n g
t h a t
t r a n s l a t i o n
o f
t h e
O m p A
p r o t e i n
i s n o t
SO S
i n d u c e d .
.
.n
r.
. . . . ...
q W . .
. . . T
. . . .
.:. :.;
S u l A p r o t e i n
w a s
a l s o
o b s e r v e d , a l b e i t t o
a
l e s s e r
e x t e n t ,
i n t h e
n a l i d i x i c
a c i d - t r e a t e d I o n s u l B
h a p l o i d
s t r a i n c o n t a i n -
i n g o n l y t h e
c o n t r o l p l a s m i d
p B R 3 2 2 ( F i g .
4 , l a n e s 2 t h r o u g h
4 ;
F i g . 5 ,
l a n e s
2
a n d 3 ) .
T h i s
o b s e r v a t i o n a l l o w e d
u s t o
e x a m i n e
a n a l i d i x i c
a c i d - i n d u c e d , h a p l o i d l o n
s u l A s t r a i n f o r
e i t h e r
l a c k
o f
S u l A p r o t e i n o r
a n a l t e r e d S u l A
p r o t e i n .
A l t h o u g h a n
SOS r e s p o n s e
a p p e a r s
t o
h a v e
o c c u r r e d ,
s i n c e
n e w l y
i n d u c e d
p r o t e i n s
w e r e
v i s i b l e
( F i g . 5 , a r r o w ) ,
n o S u l A
p r o t e i n
w a s
i n d u c e d
i n t h i s
s t r a i n ( F i g .
5 ,
l a n e s
4
t h r o u g h
6 ) .
T h e f a c t t h a t
a s u l A m u t a n t s t r a i n
l a c k s
a n i n d u c i b l e
1 8 - k d a l
p o l y p e p t i d e
s u p p o r t s
t h e i d e n t i f i c a t i o n
o f
t h e
1 8 - k d a l
p o l y -
p e p t i d e a s
t h e s u l A
g e n e p r o d u c t .
W h e t h e r
t h i s s u l A m u t a n t
p r o d u c e s a t r u n c a t e d
p o l y p e p t i d e
i s
u n k n o w n , a s we
d i d n o t
r e s o l v e a n y
s m a l l e r i n d u c i b l e p o l y p e p t i d e .
S t a b i l i t y
o f S u l A p r o t e i n
i n v i v o .
To
e x a m i n e
t h e h y p o t h e -
s i s
t h a t
t h e S u l A
p r o t e i n
i s
m o r e s t a b l e
i n
a I o n b a c k g r o u n d ,
we
i n d u c e d
w i t h n a l i d i x i c
a c i d a n d
l a b e l e d t h e S u l A
p r o t e i n
i n a I o n + s t r a i n
c o n t a i n i n g t h e
s u l A + p l a s m i d
p T U 3 0 2 .
By
d e c r e a s i n g t h e
l a b e l i n g
t i m e
t o 1 m i n ,
t h e
S u l A
p r o t e i n
w a s i n d ee d i n d u ce d
a n d
a m p l i f i e d i n
t h e
l o n +
s t r a i n
( F i g .
6 ,
l a n e 5 ) .
F u r t h e r m o r e ,
w h e n t h e l a b e l e d S u l A p r o t e i n
i t f t h e
l o n +
s t r a i n
w a s c h a s e d
f o r
5
m i n
b y t h e
a d d i t i o n o f m e d i u m
c o n t a i n i n g
a n e x c e s s
o f
L - m e t h i o n i n e ,
v e r y
l i t t l e S u l A p r o -
t e i n
w a s
d e t e c t e d
o n
t h e
a u t o r a d i o g r a m ,
a n d n o n e
w a s
v i s i b l e a f t e r
a c h a s e
o f 2 0 m i n . I n c o n t r a s t ,
a s i g n i f i c a n t
a m o u n t
o f
S u l A
p r o t e i n
w a s d e t e c t a b l e
i n t h e I o n
s u l B ( p T U 3 0 2 )
s t r a i n
t h a t w a s
l a b e l e d
f o r 1 m i n
a n d
c h a s e d
f o r u p
t o 2 0 m i n ( F i g . 6 ,
l a n e s 2 a n d 3 ;
i n t h e
o r i g i n a l
a u t o r a d i o g r a m
t h e
S u l A
p r o t e i n
w a s v i s i b l e
u p
t o
4 0
m i n ) .
T h u s S u l A p r o t e i n
i s v e r y
u n s t a b l e
i n
a
I o n
s t r a i n
a s
c o m p a r e d
w i t h
a
I o n
m u t a n t .
C e l l u l a r
l o c a t i o n
o f t h e
S u l A
p r o t e i n .
T h e
a b i l i t y
t o
i n d u c e
S u l A p r o t e i n
w i t h
n a l i d i x i c a c i d i n
p T U 3 0 2
t r a n s f o r m a n t s
o f
t h e
l o n
s u l B s t r a i n
a l l o w e d
u s
t o
e x a m i n e t h e
l o c a t i o n o f
t h e
S u l A p r o t e i n .
C e l l s
[ R G C 1 0 3 - 9 ( p T U 3 0 2 ) ]
w e r e i n d u c e d
w i t h
M *s :
_
_
*__l,._
.._R2 :
:
..
_E
>:
.:
:_t',
.:.
..
+ . . 4 :
*:*:.:
: . + . .
: t
.:
::
W:,...
. N S W I
.e:
S§
..
22
+ > > e
.....
* : : x : h t
_k
F I G .
5 .
N o n i n d u c i b i l i t y o f S u l A
p o l y p e p t i d e
b y
n a l i d i x i c
a c i d i n
a
h a p l o i d
s u l A
m u t a n t . E l e c t r o p h o r e s i s
was
as
i n
F i g . 2 . L a n e s :
1 t o
3 , R G C 1 0 3 - 9 [ I o n
s u I B ( p B R 3 2 2 ) ]
i n c u b a t e d w i t h
n a l i d i x i c a c i d f o r 0 ,
2 ,
a n d 4
h , r e s p e c t i v e l y ,
b e f o r e l a b e l i n g w i t h
[ 3 5 S ] m e t h i o n i n e ; 4 t o 6 ,
R G C 1 0 3 - 2
[ l o n s u l A ( p B R 3 2 2 ) ]
i n c u b a t ed w i t h
n a l i d i x i c
a c i d f o r
0 ,
2 ,
a n d
3
h ,
r e s p e c t i v e l y , b e f o r e l a b e l i n g ;
7 t o 9 , R G C 1 0 3 - 2 ( p T U 3 0 2 )
t r e a t e d w i t h
n a l i d i x i c
a c i d f o r
0 , 2 ,
a n d 3
h ,
r e s p e c t i v e l y ,
b e f o r e
l a b e l i n g .
1
2
3 4
5
6
7 8
F I G .
6 .
S t a b i l i t y
o f
t h e s u l A
p o l y p e p t i d e i n
l o n
s u l B
a n d
l o n +
s t r a i n s .
E l e c t r o p h o r e s i s w a s
a s i n F i g . 2 .
C e l l s w e r e i n c u b a t e d
w i t h
n a l i d i x i c
a c i d
f o r 1
h , l a b e l e d
w i t h [ 3 5 S ] m e t h i o n i n e
f o r 1
m i n ,
a n d
c h a s e d
w i t h
n o n r a d i o a c t i v e
m e t h i o n i n e
( 1 0 0 , u g / m l )
a s i n d i c a t e d .
L a n e s :
1
t o
4 ,
s t r a i n
R G C 1 0 3 - 9 [ I o n s u l B ( p T U 3 0 2 ) ]
l a b e l e d
f o r
1
m i n
( l a n e
1 )
a n d
c h a s e d
w i t h m e t h i o n i n e
f o r 5 , 2 0 , a n d
4 0
m i n
( l a n e s
2 ,
3 ,
a n d
4 , r e s p e c t i v e l y ) ;
5
t o
8 , s t r a i n MC100 [ l o n + ( p T U 3 0 2 )
l a b e l e d
f o r
1
m i n ( l a n e
5 )
a n d
c h a s e d
f o r 5 , 2 0 ,
a n d 4 0 m i n ( l a n e s
6 , 7 , a n d
8 ,
r e s p e c t i v e l y ) .
J . B A C T E R I O L .
i 4 t .
.,W-A-w
-N s
a
4 1 4
Ci
+1
- W . M -
8/10/2019 J. Bacteriol.-1984-Schoemaker-551-61
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REGU L A T I O N O F
CELL
DIVISION
I N E . C O LI 5 5 7
n a l i d i x i c
a c i d f o r 6 0
t o 9 0
m i n ,
a n d
t h e n t o t a l
p r o t e i n s
w e r e
l a b e l e d w i t h
[ 3 5 S ] m e t h i o n i n e f o r 1 0 m i n . T h e c e l l s
were
s o n i c a l l y
d i s r u p t e d ,
a n d
t h e
e n v e l o p e
was
s e p a r a t e d f r o m
s o l u b l e
c y t o p l a s m i c p r o t e i n s
b y c e n t r i f u g a t i o n a t 5 0 , 0 0 0
x
g
f o r 3 0 m i n . T h e
m a j o r i t y
o f t h e
S u l A p r o t e i n
r e m a i n e d w i t h
t h e
e n v e l o p e f r a c t i o n ( F i g . 7 , l a n e s
1 a n d
2 ) .
T h e l a b e l e d
c e l l
e n v e l o p e
w a s
f u r t h e r f r a c t i o n a t e d
b y t h e
c l a s s i c a l membrane
p r o c e d u r e
o f i s o p y c n i c
s u c r o s e g r ad i en t c e n t r i f u g a t i o n ( F i g .
8 ) .
I n
t h i s
p r o c e d u r e
t h e
o u t e r
m e m b r a n e
e q u i l i b r a t e s
a t
a
d e n s i t y o f 1 . 2 2
g o f s u cr o s e p e r m l , a n d t h e
i n n e r m e m b r a n e
e q u i l i b r a t e s a t
a
d e n s i t y
o f 1 . 1 7 t o
1 . 1 6 g o f s u c r o s e p er m l .
S o l u b l e p r o t e i n s f l o a t
o n
t h e
t o p o f t h e
g r a d i e n t . E l e c t r o p h o -
r e s i s
o f
t h e
l a b e l e d p r o t e i n
f r o m s u c h a m e m b r a n e
f r a c t i o n -
a t i o n
i s s h o w n i n
F i g .
7 .
L a b e l e d
S u l A p r o t e i n
i s
f o u n d i n t h e
o u t e r m e m b r a n e f r a c t i o n
( F i g .
7 , l a n e s 3
a n d
7 ) . A
s m a l l
a m o u n t
o f
S u l A p r o t e i n r e l a t i v e t o t h e
o t h e r l a b e l e d p r o t e i n s
w a s a l s o
f o u n d
i n t h e
i n n e r m e m b r a n e
f r a c t i o n
a n d
w i t h t h e
p r o t e i n s f l o a t i n g
o n
t o p
o f t h e g r a d i e n t
( F i g . 7 , l a n e s 4
t h r o u g h
6 ) .
N a l i d i x i c
a c i d - i n d u c e d
a n d l a b e l e d
R G C 1 0 3 - 9 ( p T U 3 0 2 )
c e l l s
w e r e
a l s o f r a c t i o n a t e d b a s e d
o n a l k a l i
(NaOH)
s e d i -
m e n t a t i o n . P r o t e i n s
d e r i v e d
f r o m t h e i n n e r a n d o u t e r mem -
b r a n e
r e m a i n
i n s o l u b l e f o r
t h e m o s t
p a r t ( 4 5 ) .
M o s t
p r o t e i n s
p a r t i t i o n
u n a m b i g u o u s l y
( 4 5 ) .
A
l a r g e
p o r t i o n o f
S u l A
p r o -
t e i n
w a s
N a O H p r e c i p i t a b l e ;
h o w e v e r , s o m e
S u l A p r o t e i n
w a s
a l s o
i n
t h e
s u p e r n a t a n t
f r a c t i o n .
I n
n o
c a s e w a s
t h e r e
l e s s
t h a n 50%
o f
t h e S u l A p r o t e i n
i n t h e p r e c i p i t a b l e
f r a c t i o n .
T h e
1 8 - k d a l
3 5 S - l a b e l e d
p r o t e i n b a n d
i d e n t i f i e d i n b o t h t h e
p r e c i p i t a b l e
a n d
s u p e r n a t a n t f r a c t i o ns o f
RGC103-
9 ( p T U 3 0 2 ) i s u n s t a b l e ,
s i n c e t h a t
p r o t e i n
b a n d
d e c r e a s e d
c o m p a r e d w i t h
o t h e r p r o t e i n
b a n d s a f t e r
a
2 0 - mi n c h a s e ( F i g .
9 ) .
T h i s
i s
c o n s i s t e n t w i t h
t h e i n c r e a s e d
l a b i l i t y
o f t h e
S u l A
p r o t e i n e v e n
i n
a c a p R m u t a n t .
S u l A
p r o t e i n
l o c a l i z a t i o n
i n
t h e
e n v e l o p e
w a s
a d d i t i o n a l l y
.6
I .
.4
SUIA
1
2
3
6
7
8
F I G .
7 .
E n v e l o p e
l o c a l i z a t i o n
o f
t h e S u l A
p r o t e i n .
E l e c t r o p h o r e -
s i s was a s i n
F i g .
2 . L a n e :
1 ,
s u p e r a t a n t
o f
d i s r u p t e d
c e l l s ;
2 ,
p e l l e t
f r a c t i o n
o f
d i s r u p t e d
c e l l s ;
3 ,
o u t e r
m e m b r a n e
( f r a c t i o n
a
o f
F i g .
8 ) ;
4
a n d
5 ,
i n n e r m e m b r a n e
( f r a c t i o n s
b a n d
c
o f
F i g .
8 ) ; 6 ,
s o l u b l e
p r o t e i n s
( f r a c t i o n
d
o f
F i g .
8 ) ;
7 ,
o u t e r m e m b r a n e f r o m
a
d i f f e r e n t
p r e p a r a t i o n
o b t a i n e d
b y i s o p y c n i c
s u c r o s e
g r a d i e n t
c e n t r i f u g a t i o n
( c o r r e s p o n d s
t o l a n e 3
f r a c t i o n ) ; 8 ,
m i n i c e l l s o f
D S 4 1 0 ( p T U 3 0 2 )
l a b e l e d
w i t h
[ 3 1
S ] m e t h i o n i n e .
.0-
.X =
l o 2
-
3A-...201
o~~~~~~~~~~~~~~~~~~
2~~~~~~~~~~~~~~~~~~~~~~.20
0.
z
0 .
A
F R A C T I O N N UM BE R
F I G .
8 .
S e p a r at i o n o f i n n e r
a n d
o u t e r m e m b r a n e s
b y
i s o p y c n i c
s u c r o s e
g r a d i e n t
f r a c t i o n a t i o n . S t r a i n
R G C 1 0 3 - 9 ( p T U 3 0 2 ) w a s i n -
d u c e d w i t h
n a l i d i x i c a c i d f o r
1
h a n d
t h e n l a b e l e d
w i t h
[ 3 5 S ] m e t h i o n i n e
f o r
1 0 m i n . T h e
c e l l s w e r e d i s r u p t e d a s d e s c r i b e d
i n
t h e
t e x t .
c o n f i r m e d i n t h r e e o t h e r
p T U 3 0 2 - c o n t a i n i n g
s t r a i n s
( R G C 1 0 3 - 2
[ l o n s u l A ] ,
MC100
[ l o n + ] ,
RGC208
[ompA
l o n + ] ) . T h e s e
p l a s m i d - c o n t a i n i n g
s t r a i n s
w e r e
i n d u c e d
w i t h
n a l i d i x i c
a c i d f o r 9 0
m i n
a n d
t h e n l a b e l e d w i t h
[ 3 5 S ] m e t h i o -
4 t
a_i
i b
s ~:-- Ab
. : : :
12
3 4 5
6 7
F I G .
9 .
I n s t a b i l i t y o f S u l A p r o t e i n
i n
s u p e r n a t a n t
a n d
p e l l e t
f r a c t i o n s a f t e r
N a O H
t r e a t m e n t . S t r a i n
R G C 1 0 3 - 9 ( p T U 3 0 2 ) w a s
t r e a t e d
w i t h n a l i d i x i c a c i d f o r
1
h
( o r n o t t r e a t e d )
a n d
t h e n
l a b e l e d
w i t h
[ 3 5 S ] m e t h i o n i n e f o r 1 0
m i n .
L a n e s : 1 ,
w h o l e c e l l s
t r e a t e d
w i t h
n a l i d i x i c
a c i d ;
2
t o 4 , membrane
p e l l e t
f r a c t i o n a f t e r
N a O H
t r e a t -
m e n t ; 2 ,
n o t
t r e a t e d
w i t h n a l i d i x i c
a c i d ;
3 ,
t r e a t e d
w i t h n a l i d i x i c
a c i d ; 4 ,
a s l a n e
3 a n d t h e n c h a s e d
w i t h e x c e s s
n o n r a d i o a c t i v e
m e t h i o n i n e
( 1 0 0 , u g / m l )
f o r
2 0
m i n ;
5
t o
7 , s u p e r n a t a n t
a f t e r N a O H
t r e a t m e n t ,
s a m e
s a m p l e s a s l a n e s 2 t o
4 , r e s p e c t i v e l y .
4 0 1 0 0
. . . . .
.. .
. W l l i
.
..
..
I
n
V O L .
1 5 8 ,
1 9 8 4
P
*, -t
-..
. .,
:--
:. -
Oado
.W.
su
I
A
4M
8/10/2019 J. Bacteriol.-1984-Schoemaker-551-61
http://slidepdf.com/reader/full/j-bacteriol-1984-schoemaker-551-61 8/11
5 5 8
SCHOEMAKER,
GAYDA,
AND
M A R K O V I T Z
n i n e f o r 1 m i n .
A p p r o x i m a t e l y
e q u a l
a m o u n t s
o f
S u l A
p r o t e i n
w e r e
f o u n d
i n
t h e
N a O H - s o l u b l e
a n d
- i n s o l u b l e
f r a c t i o n s .
I s S u l A p r o t e i n
a
s u b s t r a t e
o f
t h e L o n
A T P - d e p e n d e n t
p r o t e a s e ?
T h e
s i m p l e s t c u r r e n t
m o d e l o f
l o n - s u l A
i n t e r a c t i o n
p r e d i c t s
t h a t
S u l A
p r o t e i n i s a
s u b s t r a t e o f t h e
Lon
p r o t e a s e .
A l t h o u g h t h e
S u l A
p r o t e i n w a s
l o c a l i z e d
t o
t h e
c e l l u l a r
m e m b r a n e ,
we
s t i l l
t e s t e d
i t s
s u s c e p t i b i l i t y t o
t h e
s o l u b l e
L o n
p r o t e a s e
i n
v i t r o .
A s
d e s c r i b e d a b o v e ,
we
p r e p a r e d
a
c e l l e n v e l o p e
f r a c t i o n
t h a t
w a s
e n r i c h e d
f o r t h e
S u l A
p r o t e i n .
T h e e n v e l o p e
w a s
s u s p e n d e d
i n
b u f f e r
w i t h
o r
w i t h o u t
0 . 3
T r i t o n
X - 1 0 0
a n d
t h e n
a d d e d
t o a n a s s a y a l o n g
w i t h t h e
p u r i f i e d Lon
p r o t e a s e a n d
A T P . I n
t h e a s s a y
w i t h
T r i t o n
X -
1 0 0
t h e
f i n a l
c o n c e n t r a t i o n w a s
0 . 0 3 . A s
a c o n t r o l ,
t h e
e n v e l o p e
p r e p a r a t i o n s
w e r e
m i x e d i n e q u a l
p r o t e i n
c o n c e n -
t r a t i o n
w i t h
a l o n c e l l
e x t r a c t
c o n t a i n i n g
r a d i o a c t i v e
p r o t e i n
a , a n
a c i d i c 1 1 - k d a l
p o l y p e p t i d e . T h i s
p r o t e i n
i s
d e g r a d e d i n
a
s i m i l a r
i n
v i t r o
a s s a y u p o n t h e
a d d i t i o n o f
t h e
p u r i f i e d
Lon
p r o t e a s e
a n d A TP
( 4 6 ) .
T h e
a s s a y s
w e r e
i n c u b a t e d a t
3 7 ° C f o r 9 0
m i n , a n d t h e n
p r o t e i n s
w e r e
a na l y z e d b y
t w o - d i m e n s i o n a l
g e l
e l e c t r o p h o -
r e s i s .
O n l y p r o t e i n a
( t h e
c o n t r o l )
w a s
d e t e c t a b l y d e g r a d e d
( d a t a
n o t
s h o w n ) .
I n
o t h e r
e x p e r i m e n t s w i t h
o n e - d i m e n s i o n -
a l
P A G E - S D S ,
t h e
S u l A
p r o t e i n
t h a t w a s
p r e s e n t
i n
t h e
s o l u b l e f r a c t i o n
w a s
a l s o
n o t
d e g r a d e d b y
t h e
L o n
p r o t e a s e ,
a l t h o u g h t h e s e
c o n d i t i o n s
w o u l d s u p p o r t
p r o t e o l y s i s o f
c a -
s e i n b y L o n
p r o t e a s e .
S i n c e t h e S u l A
p r o t e i n
w a s
n o t
d e g r a d e d
u n d e r
c o n d i t i o n s
i n w h i c h
p r o t e i n a a n d c a s e i n
w e r e ,
we
c o n c l u d e d
t h a t
S u l A p r o t e i n
may n o t b e
a d i r e c t
s u b s t r a t e
o f
L o n
p r o t e a s e .
A l t e r n a t i v e l y ,
o t h e r
c o n d i t i o n s
t h a n
we
h a v e t e s t e d
may b e r e q u i r e d
f o r L o n
p r o t e a s e
t o
d e g r a d e t h e
S u l A
p r o t e i n
i n a d d i t i o n t o f a c t o r s
n e c e s s a r y f o r
p r o t e i n a
o r c a s e i n
d i g e s t i o n
i . e . ,
m o d i f i c a t i o n o f t h e
S u l A
p r o t e i n ,
u n i d e n t i f i e d
s m a l l m o l e c u l e
c o f a c t o r s ,
o r
o t h e r
p r o t e i n
o r
n u c l e i c a c i d f a c t o r s
o r
a b s e n c e
o f
i n h i b i t o r s ) .
F u r t h e r
s t u d i e s
a r e r e q u i r e d
t o d e c i d e
a m o n g
t h e s e
p o s s i b i l i -
t i e s .
Why
c a n y o u
e s t a b l i s h t h e
m u l t i c o p y s u U + p l a s m i d
i n a
I o n
s u L 4
s t r a i n , b u t n o t
i n a I o n
s u l A +
s t r a i n ,
w h e n
t h e
a m o u n t
o f
w i l d - t y p e
S u l A
p r o t e i n
w o u l d
b e e x p e c t e d
t o
b e
a p p r o x i m a t e l y
e q u i v a l e n t i n t h e
t w o
s t r a i n s ? F i v e
p o s s i b i l i t i e s
w e r e
c o n s i d -
e r e d .
S i n c e
s o m e
s u l A m ut a t i o n s
a r e d o m i n a n t
( 1 2 , 2 7 )
i t i s
p o s s i b l e
t h a t a p u t a t i v e
c h r o m o s o m a l S u l A
m u t a n t
p r o t e i n
i s
a n t i c o m p l e m e n t i n g ( 8 )
s o m e
o f
t h e
p l a s m i d - c o d e d
S u l A
w i l d -
t y p e
p r o t e i n ,
t h e r e b y
m o d i f y i n g
t h e l e t h a l
e f f e c t s o f
m u l t i p l e
c o p i e s o f t h e
l a t t e r .
We t e s t e d
s e v e n o t h e r
i n d e p e n d e n t
s u l A
m u t a t i o n s i n
t w o l o n
b a c k g r o u n d s ( d e r i v a t i v e s
o f
MC100
a n d
X 7 1 0 2 ) , a n d
a l l y i e l d e d
t r a n s f o r m a n t s
w i t h
p T U 3 0 2 .
I f
we
h a d
f o u n d o n e s u l A
a l l e l e
g i v i n g
n o
t r a n s f o r m a n t s
t h e d a t a
w o u l d
h a v e
s u p p o r t e d t h i s
i n t e r p r e t a t i o n . T h i s
a l t e r n a t i v e
w o u l d r e q u i r e
t h e u n l i k e l y
a s s u m p t i o n
t h a t a
c h r o m o s o m a l
( s i n g l e - c o p y ) a l l e l e
b e
d o m i n a n t
t o a
w i l d - t y p e
a l l e l e
p r e s e n t
i n
m u l t i p l e
c o p i e s .
A n o t h e r
a l t e r n a t i v e
i s
t h a t w h e n
p T U 3 0 2
( s u l A + ) i s
t r a n s -
f o r m e d
i n t o
a
c a p R 9
s u l A
s t r a i n
t h e
t r a n s f o r m a n t s t h a t
s u r v i v e a r e
i n
f a c t
h o m o g e n o t e s
o f
t h e
m u t a n t
s u l A a l l e l e
t h a t
a r o s e b y
r e c o m b i n a t i o n . T h i s
e x p l a n a t i o n
s e e m s
u n l i k e -
l y
f r o m
t h e
f o l l o w i n g
d a t a :
t h e
t r a n s f o r m a t i o n f r e q u e n c y o f
p T U 3 0 2 i n t o t h e
c a p R 9
s u l A 2 s t r a i n i s
i d e n t i c a l
t o
t h a t
o b t a i n e d
w i t h
t h e
c a p R 9
s u i B 9 s t r a i n
( T a b l e 2 ) .
F u r t h e r m o r e
t h e
p h e n o t y p e
o f t h e c a p R 9
s u I A 2 ( p T U 3 0 2 ) ( s u l A + )
i s
e a s i l y
d i s t i n g u i s h e d f r o m
t h e
c a p l ? 9
s u l A 2 ( p T U 3 0 2 ) : : m i n i - M u ( s u i A
i n s e r t e d ) ;
t h e
f o r m e r i s
f i l a m e n t o u s ( F i g . 2 ) ,
g r o w s
p o o r l y
o n
m i n i m a l
m e d i u m , a n d i s
N F
s e n s i t i v e ( T a b l e
2 ) , w h e r e a s
t h e
l a t t e r
g r o w s
w e l l o n
m i n i m a l
m e d i u m
a n d i s N F
r e s i s t a n t .
A n o t h e r
a l t e r n a t i v e i s
t h a t
c h r o m o s o m a l
s u l A
m u t a n t s
e x e r t
a
p o l a r
e f f e c t
on
t h e
f o l l o w i n g
a d j a c e n t
ompA
g e n e ,
w h i c h
i s t r a n s c r i b e d
i n
t h e
s a m e d i r e c t i o n
( F i g . 1 ) . We
p r e p a r e d ompA
m u t a n t s
b y
b a c t e r i o p h a g e K 3 h l
s e l e c t i o n
5 )
a n d
v e r i f i e d
t h a t
t h e y
were
m i s s i n g
t h e
O m p A
m e m b r a n e
p r o t e i n .
When a
c a p R 9
a l l e l e
was
t r a n s d u c e d
i n t o s u c h a
s t r a i n
i t was
m u c o i d
a n d N F
a n d UV
s e n s i t i v e .
T h e y
s h o u l d
h a v e
b e e n UV
r e s i s t a n t i f s u l A
m u t a n t s
c a u s e d
UV
r e s i s t -
a n c e
s i m p l y
b y
b l o c k i n g
Om pA
p r o t e i n
s y n t h e s i s .
F u r t h e r -
m o r e , p T U 3 0 2
t r a n s f o r m a n t s
c o u l d
n o t b e
e s t a b l i s h e d i n
s u c h
s t r a i n s .
T h e r e f o r e ,
t h e
c h r o m o s o m a l
o m p A +
a l l e l e
w as
n o t
r e q u i r e d
f o r t h e
l e t h a l
e v e n t s
t h a t
o c c u r a f t e r a n
SOS
i n d u c i n g
t r e a t m e n t
( U V )
o f t h e
I o n
s t r a i n
o r
t h a t
f o l l o w
t r a n s f o r m a t i o n o f
t h e
l o n
s t r a i n
w i t h
s u i A
p l a s m i d
p T U 3 0 2 .
A
f o u r t h
a l t e r n a t i v e
i s t h a t t h e r e
i s
g r e a t l y i n c r e a s e d
s y n t h e s i s
o f
t h e
S u l A
( w i l d - t y p e )
p r o t e i n
i n
a
h y p o t h e t i c a l
c a p R 9 s u l A + ( p T U 3 0 2 )
s t r a i n
( r e s u l t i n g
i n
l e t h a l i t y )
a s
com-
p a r e d
w i t h
a
c a p R 9 s u l A 1 2 ( p T U 3 0 2 )
s t r a i n
o r
a
c a p R + ( p T U 3 0 2 )
s t r a i n . We
e x a m i n e d t h i s
p o s s i b i l i t y
i n d i -
r e c t l y )
b y u s i n g
t h e
p l a s m i d
p R G C 1 3 ( p T U 3 0 2 : : m i n i - M u ) , i n
w h i c h
, - g a l a c t o s i d a s e
i s
t r a n s c r i b e d
f r o m t h e
s u l A
p r o m o t -
e r .
P l a s m i d
p R G C 1 3
w as
t r a n s f o r m e d i n t o
t h e
c a p R 9 s u i A 1 2
( R G C 1 2 3 - 2 ) , c a p R 9
s u l A +
( R G C 1 2 3 ) ,
c a p R 8 2
s u l A +
( X 7 1 0 2 -
8 2 ) ,
a n d
c a p R +
( X 7 1 0 2 )
s t r a i n s .
T h e s e
s t r a i n s
a r e
i s o g e n i c
a n d
s u p p r e s s o r
f r e e ,
a n d t h e
e n t i r e
l a c
o p e r o n
i s
d e l e t e d
f r o m
t h e
c h r o m o s o m e .
T h e
s p e c i f i c
e n z y m a t i c
a c t i v i t y o f
-
g a l a c t o s i d a s e
i n
e a c h s t r a i n
c o n t a i n i n g
p R G C 1 3
was
mea-
s u r e d
i n t h e
s t e a d y
s t a t e
( g r o w i n g e x p o n e n t i a l l y
i n
m i n i m a l
g l u c o s e
m e d i u m
s u p p l e m e n t e d
w i t h
0 . 5
C a s a m i n o
A c i d s ) .
The
s p e c i f i c e n z y m a t i c
a c t i v i t y
i n
s t r a i n
X7102
[ c a p R + ( p R G C 1 3 ) ]
w as 1 0 6
U / m g
o f
p r o t e i n
a t
2 8 ° C
u n d e r t h e
c o n d i t i o n s
a n d
c a l c u l a t i o n s
o f M i l l e r
( 3 7 ) .
T h e
r e l a t i v e
a c t i v i t i e s were
a s f o l l o w s :
c a p R + ( p R G C 1 3 ) ,
1 . 0 ;
c a p R 9
s u i A J 2 ( p R G C 1 3 ) ,
4 . 0 ; c a p R 9
s u i A + ( p R G C 1 3 ) ,
2 . 0 ;
c a p R 8 2 ( p R G C 1 3 ) ,
1 . 6 .
T h e r e
i s a
h i g h e r
d i f f e r e n t i a l
r a t e
o f
s y n t h e s i s
o f s u l A
p r o m o t e r - c o n t r o l l e d
, - g a l a c t o s i d a s e
i n t h e
c a p R
m u t a n t s
c o m p a r e d
w i t h t h e
c a p R
w i l d
t y p e .
H o w e v e r ,
a c c o r d i n g
t o t h e
s t a t e d
h y p o t h e s i s ,
t h e
c a p R 9 s u l A + s t r a i n
s h o u l d
h a v e b e e n
h i g h e r
t h a n
t h e
c a p R 9
s u i A 1 2
s t r a i n
t o
a c c o u n t
f o r
l e t h a l i t y ,
a n d
t h e
r e v e r s e
i s
t r u e
i f
t h e r e
i s
a n y
d i f f e r e n c e ;
t h u s
t h i s
h y p o t h e s i s
i s n o t
s u p p o r t e d
b y
t h e
d a t a .
I n c i d e n t a l l y ,
t h e
d i f f e r e n c e s
i n
3 - g a l a c t o s i d a s e
a c t i v i t y r e -
p o r t e d
h e r e
b e t w e e n
c a p R +
a n d
c a p R 9
s t r a i n s
a r e
l i k e l y
t o
r e f l e c t a
d i f f e r e n c e i n
p l a s m i d
c o p y
n u m b e r
s i n c e
H u i s m a n
a n d
D A r i
r e p o r t e d
t h a t w i t h
3 - g a l a c t o s i d a s e
i n s e r t e d
i n t o
t h e
c h r o m o s o m a l
( s i n g l e - c o p y ) s u l A
g e n e
t h e r e
i s
n o d i f f e r -
e n c e
i n
, B - g a l a c t o s i d a s e
s y n t h e s i s
i n
c a p R +
a n d
c a p R
m u -
t a n t s
2 2 ) .
S i n c e
n o n e o f
t h e
a b o v e
h y p o t h e s e s i s
s u p p o r t e d
b y t h e
d a t a ,
a n o t h e r
a l t e r n a t i v e we
c o n s i d e r
i s t h e
f o l l o w i n g .
A n -
o t h e r
u n k n o w n
p r o t e i n
i n c r e a s e s i n
c a p R 9
s u l A
m u t a n t s
i n
c o n t r a s t t o
t h e
c a p R 9 s u l A +
s t r a i n )
t h a t
s o m e h o w
o v e r -
comes t h e
l e t h a l i t y
c a u s e d
b y p l a s m i d - c o d e d
c o p i e s
o f
s u l A .
F u r t h e r
u n d e r s t a n d i n g o f t h e
m o l e c u l a r
f u n c t i o n
o f
t h e
S u l A
p r o t e i n
i s
r e q u i r e d
b e f o r e
i t
w o u l d
a p p e a r w o r t h -
w h i l e t o
i n v e s t i g a t e s u c h
a n
h y p o t h e s i s .
DISCUSSION
P r e v i o u s
s u i A - l a c Z
g e n e
f u s i o n
s t u d i e s s h o w e d
t h a t
t h e
s u l A
g e n e
i s
t r a n s c r i p t i o n a l l y
r e g u l a t e d
b y t h e
SOS
r e g p o n s e
s y s t e m ( l e x A / r e c A )
( 2 2 ) . O u r
r e s u l t s
d e m o n s t r a t e
t h a t
S u l A
p r o t e i n
s y n t h e s i s
i s
i n d u c e d b y
n a l i d i x i c a c i d .
T h u s ,
t h e
S u l A
p r o t e i n
i s
i n d u c e d
a l o n g w i t h t h e p r o d u c t s
o f t h e
o t h e r
SOS
g e n e s ( u v r A ,
u v r B , umuC,
I e x A ,
r e c A , e t c . ) u n d e r
LexA
r e p r e s s o r
c o n t r o l ( 2 9 ) . A
h y p o t h e s i s
t o
e x p l a i n
t h e
l o n c e l l
J .
B A C T E R I O L .
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R EG U L A T I O N
O F CELL
DIVISION
I N
E .
C O L I 5 5 9
d i v i s i o n d e f e c t
w o u l d
i n v o l v e t h e
SO S response ( 2 2 ,
2 9 ) ,
t h e
s u l A + gene,
a n d t h e S u l A
p r o t e i n s i n c r e a s e d
s t a b i l i t y
i n a
l o n
mutant ( 3 8 ) :
i . e . , a f t e r SOS
i n d u c t i o n
(U V
i r r a d i a t i o n ,
n a l i d i x i c
a c i d ,
e t c . )
t h e Lon p r o t e i n n e g a t i v e l y r e g u l a t e s
t h e
c o n c e n t r a t i o n
or a c t i v i t y
( o r b o t h ) o f t h e
S u l A
p r o t e i n
t h r o u g h
p r o t e o l y s i s ,
a n d
t h e S u l A p r o t e i n i n h i b i t s
s e p t a t i o n .
T h e q u e s t i o n
o f
w h e t h e r
m u l t i c o p y
s u l A +
p l a s m i d s c o u l d
b e
e s t a b l i s h e d i n Io n s t r a i n s m i g h t
b e
e x p e c t e d
t o
d e p e n d
on
t h e
e f f e c t s
o f i n c r e a s e d
c e l l u l a r
c o n c e n t r a t i o n
o f
t h e
S u l A
p r o t e i n . We
f o u n d t h a t
m u l t i c o p y s u I A +
p l a s m i d t r a n s f o r m -
a n t s
c o u l d
n o t b e
e s t a b l i s h e d
when
we
a t t e m p t e d t o
t r a n s -
f o r m Io n
s t r a i n s , b u t were
e s t a b l i s h e d i n
Ion s u l A
a n d l o n
s u l B s t r a i n s . T h e
d a t a i n d i c a t e d
t h a t
t h e l e t h a l
e f f e c t o f
i n c r e a s e d
s u l A +
gene
d o s a g e
i n
a
Ion
b a c k g r o u n d
c an b e
overcome
b y
e i t h e r
a s u l A
or s u l B m u t a t i o n , i m p l i c a t i n g
s u i B +
as w e l l
as
s u l A +
i n t h e l e t h a l
e v e n t s .
A n
e x a m i n a t i o n
o f
t h e
c e l l s o f
e i t h e r t h e Io n
s u l A or
t h e
l o n s u l B
s t r a i n s
w i t h
t h e m u l t i c o p y s u l A +
p l a s m i d s h o w e d
t h a t t h e y were
f i l a m e n -
t o u s w i t h o u t
any a p p a r e n t
i n d u c t i o n
o f
t h e SO S
res pon s e.
T h e
f i l a m e n t a t i o n
o f t h e
Ion
s u l A
a n d
l o n
s u l B
s t r a i n s
c o n t a i n i n g t h e
s u i A + p l a s m i d s
i s
very
l i k e l y
t h e
r e s u l t
o f
i n c r e a s e d
b a s a l l e v e l s
o f S u l A
p r o t e i n i n s u c h
s t r a i n s . We
m e a s u r e d t h e b a s a l l e v e l
o f
s u l A e x p r e s s i o n
b y
p l a s m i d
l o c a l i z e d
s u l A
f u s e d
t o
P - g a l a c t o s i d a s e
( p l a s m i d
pRGC13
[ s u l A : : i a c Z ] ) .
I o n
s t r a i n s
e x p r e s s e d 1 . 6 t o
4 t i m es as much
, B -
g a l a c t o s i d a s e
as d i d
a l o n +
c o n t r o l .
T h e
w i l d - t y p e
P - g a l a c t o -
s i d a s e
i s
s t a b l e
i n
l o n +
a n d
l o n
s t r a i n s .
We a t t r i b u t e
t h e
d i f f e r e n c e
i n
P - g a l a c t o s i d a s e
t o
h i g h e r
p l a s m i d
copy
number
o f Ion
c o m p a r e d
w i t h I o n
s t r a i n s ( s e e
a b o v e ) .
T h e
i n c r e a s e d
s t a b i l i t y
o f t h e
S u l A p r o t e i n i n I on s t r a i n s
( 3 8 ;
p r e s e n t
r e s u l t s )
w o u l d b e e x p e c t e d
t o exaggerate
t h e
d i f f e r -
e n c e
i n t h e
c e l l u l a r c o n c e n t r a t i o n
o f
S u l A p r o t e i n a n d c o u l d
a c c o u n t
f o r t h e f i l a m e n t a t i o n
o b s e r v e d
i n
l o n s u l A
a n d
l o n
s u l B s t r a i n s
a n d t h e i n v i a b i l i t y
o f t h e I on
s t r a i n
c o n t a i n i n g
s u i A + p l a s m i d s .
We
w e re a b l e t o
d e t e c t
t h e
S u l A
p r o t e i n
i n w h o l e c e l l s
o f
Ion s u l A a n d Io n s u l B
s t r a i n s
c o n t a i n i n g
a m u l t i c o p y
s u i A +
p l a s m i d a f t e r n a l i d i x i c
a c i d i n d u c t i o n .
We c o n f i r m e d
t h e
f i n d i n g o f M i z u s a w a
a n d
G o t t e s m a n
( 3 8 ) t h a t t h e
SulA
p r o t e i n
was
i n d e e d
u n s t a b l e
i n
t h e
I o n
s t r a i n
when
com-
p a r e d w i t h
t h e Ion
s u l B
m u t an t
i n
our
s t u d i e s .
The d e t e c t i o n
o f t h e S u l A
p r o t e i n i n t h e
I o n s t r a i n
was
d e p e n d e n t
on
i n d u c t i o n o f t h e SOS
res po n s e a n d
a
s h o r t , 1 - m i n , [ 3 5 S ] m e -
t h i o n i n e p u l s e - l a b e l i n g .
Our a b i l i t y
t o d e t e c t
t h e SulA
pro-
t e i n i n
a h a p l o i d
l o n
s u l B
s t r a i n
a f t e r n a l i d i x i c
a c i d
i n d u c t i o n ,
b u t
n o t
i n
a h a p l o i d l o n s u l A
s t r a i n ,
p r o v i d e d
a d d i t i o n a l
e v i d e n c e
t h a t
t h e
1 8 - k d a l p o l y p e p t i d e
was
i n d e e d t h e S u l A
p r o t e i n .
T h e
i n s t a b i l i t y o f
t h e S u l A
p r o t e i n i n I o n as
compared
w i t h Ion
mutant s t r a i n s
( F i g .
6 )
a n d
t h e r a r i t y
o f s p e c i f i c
p r o t e i n
i n s t a b i l i t y ( 3 9 ,
4 6 )
s t r e n g t h e n s
t h e
v i e w t h a t
t h e S u l A
p r o t e i n may f u n c t i o n
c h i e f l y as
an i n h i b i t o r
o f s e p t a t i o n
i n
c o n n e c t i o n w i t h t h e
SO S response. T h e r e
i s a t l e a s t
one
o t h e r
p r o t e i n t h a t
i s a
c a n d i d a t e
f o r a n i n h i b i t o r
o f
c e l l
d i v i s i o n ( b u t
n o t
o f
s e p t a t i o n )
t h a t
i s
n o t
c o n t r o l l e d
b y
t h e
SO S response, b u t
i s u n d e r apparent
d i r e c t c o n t r o l
o f
t h e
Lon
A T P - d e p e n d e n t protease
( 4 6 ) .
We
h a v e made
t h e s u r p r i s i n g
o b s e r v a t i o n
t h a t
t h e S u l A
p r o t e i n f r a c t i o n a t e s
w i t h t h e m e m b r a n e , a l t h o u g h
up t o
h a l f
h a s
a l s o b e e n
f o u n d i n
s o l u b l e
f o r m . F u r t h e r m o r e ,
S u l A
p r o t e i n
i s a s s o c i at e d w i t h
t h e o u t e r membrane
b y
i s o p y c n i c
sucrose
c e n t r i f u g a t i o n
a n d ,
b y t h e N a O H
t e c h n i q u e , w i t h
t h e
m e m b r a n e ; t h e
l a t t e r t e c h n i q u e
d o e s
n ot d i s t i n g u i s h
b e t w e e n
i n n e r
a n d o u t e r
m e m b r a n e
p r o t e i n s ,
b u t
h a s t h e
g r e a t
a d v a n t a g e t h a t t h e
c e l l s d o
n o t h a v e t o
b e b r o k e n
b e f o r e
t h e f r a c t i o n a t i o n
( 4 5 ) . I n o t h e r
s t u d i e s
t h e m e m b r a n e -
a s s o c i a t e d
S u l A p r o t e i n
w a s i n s o l u b l e
i n
S a r k o s y l
( d a t a
n o t
s h o w n ) , w h i c h
i s
u s u a l l y
t a k e n
a s
a d e f i n i t i v e
way
o f
l o c a l i z i n g o u t e r
m e m b r a n e p r o t e i n s . F r o m
t h e a b o v e r e s u l t s
we
m i g h t e x p e c t t h e
S u l A
p r o t e i n t o c o n t a i n
a
s i g n a l
s e q u e n c e . T h e r e i s o n l y o n e o p e n
r e a d i n g
f r a m e
i n
t h e DNA
c o n t a i n i n g t h e s u l A g e n e
( 2 ;
F i g . 1 )
t h a t c o u l d
c o d e
f o r
a n 1 8 -
k d a l
p o l y p e p t i d e .
S i n c e
t h e
S u l A
p r o t e i n
m o l e c u l a r
m a s s
i s
1 8 k d a l ( 3 8 ; p r e s e n t r e s u l t s ) , t h a t
DNA
s e q u e n c e
m u s t c o d e
f o r
t h e
S u l A
p r o t e i n .
O t h e r
g e n e t i c
d a t a s u p p o r t
h i s
c o n c l u -
s i o n
( 3 8 ) .
H o w e v e r ,
t h e r e i s
n o
a p p a r e n t
N - t e r m i n a l a m i n o
a c i d s i g n a l s e q u e n c e
s p e c i f i e d
b y t h a t s u l A
g e n e
n u c l e o t i d e
s e q u e n c e
2 ) . T h e p u l s e - c h a s e
e x p e r i m e n t s
o n t h e
i n s t a b i l i t y
o f
t h e S u l A p r o t e i n
i n
t h e
m e m b r a n e
a n d
s o l u b l e f r a c t i o n s
(NaOH
t e c h n i q u e )
d e m o n s t r a t e d
t h a t S u l A
p r o t e i n w a s
u n s t a b l e i n
b o t h
f r a c t i o n s
( F i g . 9 ) . N e v e r t h e l e s s , t h e r e w a s
n o i n d i c a t i o n
o f l a r g e r i n t e r m e d i a t e s i n
t h e
e x p e r i m e n t s
o f
F i g . 9 o r
i n t h o s e
w h e r e
t h e
l a b e l i n g
t i m e w a s a s s h o r t a s
1
m i n .
T h u s
we
c o n s i d e r
i t
n e c e s s a r y t o
l e a v e
o p e n t h e
c e l l u l a r
s i t e a t
w h i c h
t h e
S u l A
p r o t e i n
f u n c t i o n s .
T h e S u l A p r o t e i n may a c t i n t h e m e m b r a n e o r s o l u b l e
f r a c t i o n
t o i n h i b i t s e p t a t i o n . T h e s u l B g e n e
h a s r e c e n t l y
b e e n
r e p o r t e d t o b e
a n
a l l e l e o f f t s Z
( 3 0 ) .
O n l y
t e m p e r a t u r e -
s e n s i t i v e
l e t h a l m u t a n t s
o f f t s Z
h a v e
b e e n
d e s c r i b e d
p r e v i -
o u s l y ,
a n d
t h e s e
f o r m
n o n s e p t a t e
f i l a m e n t s
a t
h i g h
t e m p e r a -
t u r e a n d
d i e
( 3 1 ) . T h u s t h e s u l B
( f t s Z )
g e n e i s
a n e s s e n t i a l
g e n e . T h e S u l A
p r o t e i n c o u l d
b e f u n c t i o n i n g b y
i n h i b i t i n g
t h e
s y n t h e s i s o f t h e S u l B p r o t e i n ( S u l A
p r o t e i n
a s a
s o l u b l e
r e p r e s s o r )
o r
b y i n h i b i t i n g t h e a c t i v i t y
o f
t h e S u l B
p r o t e i n
( S u l A
p r o t e i n i n t h e
s o l u b l e o r membrane
f r a c t i o n ) .
A l -
t h o u g h
t h e S u l B p r o t e i n
i s a c a n d i d a t e
f o r a p r o t e i n
w h o s e
s y n t h e s i s
o r a c t i v i t y i s i n h i b i t e d b y t h e
S u l A p r o t e i n ,
o t h e r s
a r e
n o t e x c l u d e d
a n d a r e e q u a l l y p o s s i b l e , f o r
e x a m p l e ,
p e n i c i l l i n - b i n d i n g p r o t e i n
3 , a
m e m b r a n e - l o c a l i z e d
t r a n s p e p -
t i d a s e
a n d t r a n s g l y c o s y l a s e
( 2 5 ) a l s o known t o b e
r e q u i r e d
f o r s e p t u m
f o r m a t i o n
( 4 , 5 0 ,
5 2 ) .
A l e v e l o f
e x c e s s S u l A
p r o t e i n
t h a t
r e s u l t s
i n
c e l l
l e t h a l i t y
w a s a c h i e v e d i n b o t h
l o n
s u l A a n d
I o n
s u l B s t r a i n s
when
p T U 3 0 2 ( s u l A + )
t r a n s f o r m a n t s w e r e p l a t e d o n
n i t r o f u r a n t o i n
( T a b l e 2 ) . U n d e r t h e s a m e p l a t i n g c o n d i t i o n s t h e p l a s m i d
w a s
n o t
l e t h a l
i n
a
I o n
s t r a i n .
T h e
a b s e n c e o f
l e t h a l i t y i n
t h e
l o n +
s t r a i n i s
b e s t e x p l a i n e d i f t h e l e t h a l e x c e s s
o f S u l A
p r o t e i n
( w h i c h
a c c u m u l a t e s
i n t h e
l o n
s u l A
a n d
l o n
s u l B
m u t an t s b e c a u s e
o f
t h e
n i t r o f u r a n t o i n - i n d u c e d SO S
r e s p o n s e
a n d
m u l t i p l e c o p i e s o f t h e
s u l A + g e n e )
n e v e r
a c c u m u l a t e s
b e c a u s e i t
i s
d e s t r o y e d ,
d i r e c t l y
o r
i n d i r e c t l y , v i a
t h e
Lon
A TP h y d r o l y s i s - d e p e n d e n t
p r o t e a s e
i n t h e
I o n
s t r a i n .
We
h a v e
n o t e x c l u d e d t h e p o s s i b i l i t y t h a t o t h e r
p r o t e i n s
i n d u c e d
b y
t h e SO S
r e s p o n s e m i g h t a l s o
b e p r o t e o l y z e d
b y
t h e
Lon
p r o t e a s e a n d
c o u l d
p l a y
a
r o l e
i n t h e m u l t i c o p y s u l A +
g e n e -
d e p e n d e n t
l e t h a l i t y i n t h e a b s e n c e
o f
t h e
Lon
p r o t e a s e .
I t
may
b e t h a t t h e
l o n
a n d s u l A
g e n e
p r o d u c t s a r e
i n v o l v e d
i n
i n h i b i t i n g
c e l l d i v i s i o n
o n l y d u r i n g t h e SOS r e s p o n s e
a n d
a r e n o t i n v o l v e d
i n n o r m a l c e l l d i v i s i o n ;
t h e
i s o l a t i o n
o f
c h r o m o s o m a l
Mu
d ( a m p - l a c )
i n s e r t i o n s
i n t h e
s f i A
( s u l A )
g e n e
( 2 2 ;
M c P a r t l a n d
e t
a l . ,
A b s t r .
A n n u .
M e e t .
A m.
S o c .
M i c r o b i o l . 1 9 8 1 ,
K 5 8 , p .
1 4 7 ) a r g u e s t h a t t h e S u l A p r o t e i n
i s
d i s p e n s i b l e ;
s i m i l a r l y ,
t h e
a b s e n c e
o f t h e
Lon p r o t e i n
i n
s o m e
m u t a n t s ( 4 6 ) s u g g e s t s
t h e
Lon
p r o t e i n
i s
a l s o
d i s p e n s i -
b l e . A l t e r n a t i v e l y , b o t h
t h e
S u l A a n d
Lon
p r o t e i n s may
p a r t i c i p a t e
i n
n o r m a l
c e l l d i v i s i o n ,
b u t
h a v e b a c k u p
m e c h a -
n i s m s t h a t s u b s t i t u t e
f o r t h e m a s
i s
t h e c a s e
w i t h
o t h e r
c r u c i a l c e l l u l a r
p r o c e s s e s ( 4 0 ) . I n
e i t h e r
c a s e i t
s e e m s
l i k e l y
t h a t
t h e
S u l A
p r o t e i n
i s
i n t e r a c t i n g t o i n h i b i t s y n t h e s i s
o r
a c t i v i t y o f
p r o t e i n ( s )
i n v o l v e d i n n o r m a l s e p t a t i o n ( d u r i n g t h e
SOS r e s p o n s e )
a n d
s h o u l d t h e r e f o r e
b e
a u s e f u l t o o l
t o
f u r t h e r s t u d y t h e b i o ch e m i s t r y o f n o r m a l
c e l l d i v i s i o n .
VOL.
1 5 8 ,
1 9 8 4
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R E G U L A T I O N
O F
C E L L
DIVISION
I N
E . C O L I
5 6 1
4 2 . O F a r r e l l ,
P .
Z . ,
H .
M.
G o o d m a n ,
a n d P .
H .
O F a r r e l l .
1 9 7 7 .
H i g h r e s o l u t i o n
t w o - d i m e n s i o n a l
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o f b a s i c
as
w e l l
as
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P r o t e i n
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on
O F a r r e l l
t w o - d i m e n s i o n a l g e l s : l o c a t i o n s o f
5 5
a d d i t i o n a l
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J .
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1 9 7 7 . B a c t e r i o p h a g e i n f e c t i o n
o f m i n i c e l l s :
a
g e n e r a l
m e t h o d
f o r i d e n t i f i c a t i o n
o f
i n
v i v o b a c t e r i o p h a g e d i r e c t -
e d p o l y p e p t i d e
b i o s y n t h e s i s .
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a n
i n t e g r a l membrane
p r o t e i n : a n a l y s i s
b y
a new m e t h o d o f
m e m b r a n e
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A n E s c h e r i c h i a
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b y t h e Io n ( c a p R )
A TP
h y d r o l y s i s - d e p e n d e n t
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p o s s i b l y
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o f
t h e
gene Io n ( c a p R )
p r o d u c t as
a
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t y p h i m u r i u m :
c h e m i c a l
a n a l y s i s a n d f r e e z e - f r a c t u r e
s t u d i e s
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c e l l
d i v i s i o n
m u t a n t s
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t h e
E .
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O n t h e
p r o c e s s
o f
c e l l u l a r
d i v i s i o n
i n
E . c o l i : a
s e r i e s o f
m u t a n t s
o f E . c o l i
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B a c t e r i a l
m u t a n t s
d e f e c t i v e
i n
p l a s m i d
f o r m a -
t i o n :
r e q u i r e m e n t
f o r t h e
l o n +
a l l e l e .
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i n
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c e l l
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o f c o n d i t i o n a l c e l l d i v i s i o n
m u t a n t s
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W.
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a n d A .
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1 9 8 1 . I d e n t i f i c a t i o n a n d
p u r i f i c a t i o n o f
t h e
l o n
( c a p R + )
g e n e p r o d u c t , a D N A - b i n d i n g p r o t e i n .
P r o c .
N a t l .
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U . S . A .
7 8 : 2 0 4 3 - 2 0 4 7 .
5 7 .
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C l o n i n g o f
t h e
g e n e
I o n
( c a p R )
o f E s c h e r i c h i a c o l i K - 1 2 a n d
i d e n t i f i c a t i o n o f
p o l y -
p e p t i d e s
s p e c i f i e d
b y
t h e c l o n e d
d e o x y r i b o n u c l e i c a c i d
f r a g -
m e n t s .
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V O L .
1 5 8 ,
1 9 8 4
