Phospholipids in DNA
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7/23/2019 Phospholipids in DNA http://slidepdf.com/reader/full/phospholipids-in-dna 1/5 Proc. Natl. Acad. Sci. USA Vol. 92, pp. 783-787, January 1995 Biochemistry In vivo evidence for the involvement of anionic phospholipids in initiation of DNA replication in Escherichia coli WEIMING XIA AND WILLIAM DowHAN* Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston, TX 77225 Communicated by Eugene P. Kennedy, Harvard Medical School, Boston, MA, October 31, 1994 ABSTRACT In vitro, anionicphospholipids can reactivate inactivated DnaA protein, which is essential for initiation of DNA replication at the oriC site of Escherichia coli [Sekimizu, K. Kornberg, A. (1988) J. Biol. Chem.263, 7131-7135]. Mutations in the pgs4 gene encodingphosphatidylglycero- phosphate synthase)limit the synthesis of the majoranionic phospholipids and lead to arrest of cellgrowth. We report herein that a mutation in the rnhAgene encoding RNase H) that bypasses the need for the DnaAproteinthrough induction of constitutive stable DNA replication [Kogoma, T. von Meyenburg, K. (1983) EMBO J. 2, 463-468] also suppressed the growtharrest phenotype of a pgsAmutant. The mainte- nance of plasmidsdependent on an oriC site for replication, and therefore DnaA protein, was also compromised under conditions of limiting anionic phospholipid synthesis. These results providesupport for the involvement ofanionic phos- pholipids in normalinitiation of DNA replication at oriC in vivo by the DnaA protein. Substantial evidence indicates that DNA replication in Esch- erichia coli is associated with the cell membrane and may require anionic phospholipids (1-3). From electron micros- copy and cell fractionation studies, there appear to be non- covalent interactions among the membrane, the origin of replication, the replication fork, and the termination site ofthe chromosome (4). DnaA, the protein necessary for initiation of DNA replication at the oriC site, is peripherally bound to membranes in vitro, copurifies with phospholipids, and spe- cifically binds anionic phospholipids (5, 6). Furthermore, anionic phospholipids facilitate the conversion of the inactive ADP-bound form of the DnaA protein to the active ATP- bound form in vitro, resulting in regeneration of the initiation activity of previously inert DnaAprotein (2, 3, 7). Only anionic phospholipids containing unsaturated fatty acids are func- tional in stimulating DnaAfunction in vitro (7), an observation that is also consistent with the observation that initiation of chromosome replication in vivo appears to be dependent on a lipid binding site in the membrane containing unsaturated fattyacids (8). However, whetheranionic phospholipids are involved in initiating DNA synthesis in vivo has not been established. Initiation of DNA replication requires formation of an RNA primer-DNA hybrid duplex at the initiation site. RNase H, the rnhA gene product, restricts DNA replication in vivo to the oriC site by preferentially degrading these RNA primers at sites other than the DnaA protein-dependent oriC initiation site (9). When the rnhAgene (10) is inactivated, constitutive stable DNA replication cSDR) can be initiated at several DnaA protein-independent oriK sites by utilizing the unde- graded RNA primers (11, 12). Introduction of an rnhA mu- tation into strains lacking the dnaA gene or the oriC site bypasses the need for these genetic loci for cell viability (13, 14). RecA protein encoded by the recA gene) is required for cSDR at the oriKsites (15), but this requirement is suppresse in a lexA Def) mutant(16). If anionic phospholipids are essential for DnaA protein function in vivo, then mutations that suppress the need fo DnaA protein-dependent initiation of DNA replication at the oriC site may also suppress the growtharrest phenotype of mutantswith a limiting level of anionic phospholipids The phospholipidcomposition of the E. coli cell membrane is 70-80 phosphatidylethanolamine (zwitterionic), 15-20 phosphatidylglycerol PG, anionic), and 2-5 cardioli (CL, anionic) (17). Phosphatidylglycerophosphate syntha encoded bythepgsAgene) catalyzes the committed step in the biosynthesis of PG and CL (18) and is essential for cell growth (19). To systematically study the specific roles of anionic phospholipids in cell functions, a strain bearing a D[lac pgsA+] gene fusion in a pgsA::kan null allele background was constructed (20). In this strain, anionicphospholipid conten gene expression, and cell growth rate can be exogenous controlled by the inducer of the lac operon isopropyl P-D thiogalactopyranoside IPTG). In the absence of IPTG, the basal level of the pgsA gene product is insufficient to suppor cell growth, although cells remain viable, as indicated by resumption of growth uponaddition of IPTG.Failure to los viability indicates that anionic phospholipids are essential fo certain cell functions independent of a membrane structu requirement (21). Basedon studies of the suppression of the growtharres phenotype of the abovepgsA mutantunder conditions of cSDR,we report results that support the involvement of anionic phospholipids in the initiation of DNA replicatio by the DnaA protein at the oriC site in vivo. MATERIALS AND METHODS Strains, Plasmids, and Cell Growth. Table 1 summarizes the strains and plasmids used in this work and outlines the schem used to construct strains by P1 transduction (23). To construct strain MRLA12, the malG::TnlO locus was first -introdu into strain MR12 and used to monitor its replacement with the malG+ locus alongwith the closely linked (0.2 min)lexA(D allele (24,25). Strain MDL12 was constructed by successiv P1 transductions of strain MG1655 (no known mutations by using strain HDL1001 as donor.Plasmids were introduced int cells by electroporation(Electro Cell Manipulator 600). Cell were grown at 37°C (unless otherwiseindicated) in liqu cultures or on agar plates in LB medium supplemented with 100 ,M IPTG where indicated). Antibiotics (26) werepresen during the growth of plasmid-containing strains unless oth wise indicated and were used only for the initial selection of strains carrying chromosomally located antibiotic-resi markers. Abbreviations: PG, phosphatidylglycerol; CL, cardiolipin; cSDR, con- stitutive stable DNA replication; IPTG,isopropyl f-D-thiogala ranoside. *To whom reprint requests should be addressed. 783 The publication costs of thisarticle were defrayed in part by page charge payment. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. §1734 solely to indicate thisfact.
Transcript of Phospholipids in DNA
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I n v i v o e v i d e n c ef o r t h e i n v o l v e m e n to f a n i o n i c p h o s p h o l i p i d si ni n i t i a t i o n o f DNA r e p l i c a t i o n i n E s c h e r i c h i ac o l i
W E I M I N GX I AAND W I L L I A MD o w H A N *D e p a r t m e n to f B i o c h e m i s t r ya n d M o l e c u l a rB i o l o g y , U n i v er s i t yo f T e x as M ed i c a lS c h o o l ,H o u s t o n ,TX 7 7 2 2 5
C o m m u n i c a t e db yE u g e n eP . K e n n e d y ,H a r v a r dM e d i c a lS c h o o l ,B o s t o n ,MA , O c t o b e r3 1 ,1 9 9 4
A B S T R A C T In v i t r o ,a n i o n i cp h o s p h o l i p i d sc a n r e a c t i v a t ei n a c t i v a t e dDnaA p r o t e i n ,w h i c hi s e s s e n t i a l f o ri n i t i a t i o no fD N Ar e p l i c a t i o na t t h eo r i C s i t eo f E s c h e r i c h i ac o l i[ S e k i m i z u ,K . K o r n b e r g ,A . ( 1 9 8 8 )J . B i o l .C h e m .2 6 3 ,7 1 3 1 - 7 1 3 5 ] .M u t a t i o n si n t h e p g s 4 g e n e e n c o d i n gp h o s p h a t i d y l g l y c e r o -p h o s p h a t es y n t h a s e )l i m i tt h es y n t h e s i so f t h em a j o ra n i o n i cp h o s p h o l i p i d sa n d l e a dt o a r r e s to f c e l lg r o w t h .W e r e p o r th e r e i n t h a ta m u t a t i o ni n t h e r n h Ag e n e e n c o d i n gR N a s eH )t h a tb y p a s s e st h e n e e df o rt h e DnaAp r o t e i nt h r o u g hi n d u c t i o no f c o n s t i t u t i v es t a b l eD N Ar e p l i c a t i o n[ K o g o m a ,T . v o nM e y e n b u r g ,K . ( 1 9 8 3 )E M B OJ . 2 , 4 6 3 - 4 6 8 ]a l s os u p p r e s s e dt h e g r o w t ha r r e s tp h e n o t y p eo f a p g s Am u t a n t .T h e m a i n t e -n a n c eo f p l a s m i d sd e p e n d e n to n a no r i C s i t ef o rr e p l i c a t i o n ,a n d t h e r e f o r eDnaA p r o t e i n ,w a s a l s oc o m p r o m i s e du n d e rc o nd i t i o ns o fl i m i t i n ga n i o n i c p h o s p h o l i p i ds y n t h e s i s .T h e s er e s u l t sp r o v i d es u p p o r tf o rt h ei n v o l v e m e n to f a n io n i cp h o s -p h o l i p i d si n n o r m a li n i t i a t i o no fD N Ar e p l i c a t i o na t o r i C i nv i v ob y t h e DnaA p r o t e i n .
S u b s t a n t i a le v i d e n c ei n d i c a t e st h a tDNA r e p l i c a t i o ni nE s c h -e r i c h i ac o l ii s a s s o c i a t e dw i t h t h ec e l lm e m b r a n ea n d m ayr e q u i r ea n i o n i c p h o s p h o l i p i d s( 1 - 3 ) .From e l e c t r o nm i c r o s -c o p ya n d c e l lf r a c t i o n a t i o ns t u d i e s ,t h e r ea p p e a rt o b e n o n -c o v a l e n t i n t e r a c t i o n sa m o n gt h e m e m b r a n e ,t h e o r i g i no fr e p l i c a t i o n ,t h er e p l i c a t i o nf o r k ,a n d t h et e r m i n a t i o ns i t eo f t h ec h r o m o s o m e( 4 ) .D n a A ,t h ep r o t e i nn e c e s s a r yf o ri n i t i a t i o no fDNA r e p l i c a t i o na t t h eo r i C s i t e ,i s p e r i p h e r a l l yb o u n dt om e m b r a n e si n v i t r o ,c o p u r i f i e sw i t h p h o s p h o l i p i d s ,a n d s p e -c i f i c a l l yb i n d s a n i o n i cp h o s p h o l i p i d s( 5 , 6 ) . F u r t h e r m o r e ,a n i o n i cp h o s p h o l i p i d sf a c i l i t a t et h ec o n v e r s i o no f t h ei n a c t i v eA D P - b o u n df o r m o f t h eD n a A p r o t e i nt o t h ea c t i v eATP-b o u n df o r mi nv i t r o ,r e s u l t i n gi nr e g e n e r a t i o no ft h ei n i t i a t i o na c t i v i t yo f p r e v i o u s l yi n e r tD n a Ap r o t e i n( 2 ,3 ,7 ) .O n l ya n i o n i cp h o s p h o l i p i d s c o n t a i n i n gu n s a t u r a t e d f a t t ya c i d s a r ef u n c -t i o n a li ns t i m u l a t i n gD n a Af u n c t i o ni nv i t r o( 7 ) ,a n o b s e r v a t i o nt h a ti s a l s oc o n s i s t e n tw i t h t h eo b s e r v a t i o nt h a ti n i t i a t i o no fc h r o m o s o m er e p l i c a t i o ni nv i v oa p p e a r st o b e d e p e n d e n to n al i p i db i n d i n gs i t ei n t h em e m b r a n ec o n t a i n i n gu n s a t u r a t e df a t t ya c i d s( 8 ) .H o w e v e r ,w h e t h e ra n i o n i c p h o s p h o l i p i d sa r ei n v o l v e di n i n i t i a t i n gDNA s y n t h e s i si n v i v oh a s n o t b e e ne s t a b l i s h e d .
I n i t i a t i o no fDNA r e p l i c a t i o nr e q u i r e sf o r m a t i o no fa n RNA
p r i m e r - D N Ah y b r i dd u p l e xa t t h ei n i t i a t i o ns i t e .R N a s eH , t h er n h A g e n e p r o d u c t ,r e s t r i c t sDNA r e p l i c a t i o ni n v i v o t ot h eo r i C s i t eb y p r e f e r e n t i a l l yd e g r a d i n gt h e s eRNA p r i m e r sa ts i t e so t h e r t h a n t h eD n a A p r o t e i n - d e p e n d e n to r i C i n i t i a t i o ns i t e( 9 ) .When t h er n h Ag e n e ( 1 0 )i s i n a c t i v a t e d ,c o n s t i t u t i v es t a b l eDNA r e p l i c a t i o n c S D R )c a n b e i n i t i a t e da t s e v e r a lD n a A p r o t e i n - i n d e p e n d e n to r i K s i t e sb y u t i l i z i n gt h eu n d e -g r a d e dRNA p r i m e r s( 1 1 , 1 2) .I n t r o d u c t i o no f a n r n h Am u -t a t i o ni n t o s t r a i n sl a c k i n gt h ed n a Ag e n e o r t h eo r i C s i t eb y p a s s e st h en e e d f o rt h e s eg e n e t i cl o c if o rc e l lv i a b i l i t y( 1 3 ,1 4 ) .RecA p r o t e i n e n c o d e db y t h er e c Ag e n e )i sr e q u i r e df o r
cSDR a t t h eo r i Ks i t e s( 1 5 ) ,b u t t h i sr e q u i r e m e n ti ss u p p r e s s ei n a l e x A D e f )m u t a n t( 1 6 ) .
I f a ni o ni c p h o s ph o li p i d sa r ee s s e n t i a lf o r D n a A p r o t e i nf u n c t i o ni n v i v o ,t h e n m u ta t i o n st h a ts u p p r e s st h en e e d f oD n a Ap r o t e i n - d e p e n d e n ti n i t i a t i o no f DNA r e p l i c a t i o na t t h eo r i C s i t emay a l s os u p p r e s st h eg r o w t ha r r e s tp h e n o t y p eo fm u t a n t sw i t h a l i m i t i n gl e v e lo f a n i o n i c p h o s p h o l i p i d sT h ep h o s p h o l i p i dc o m p o s i t i o no f t h eE . c o l ic e l lm e m b r a n ei s7 0 - 8 0 p h o s p h a t i d y l e t h a n o l a m i n e( z w i t t e r i o n i c ) ,1 5 - 2 0p h o s p h a t i d y l g l y c e r o l P G , a n i o n i c ) ,a n d 2 - 5 c a r d i o l i( C L ,a n i o n i c )( 1 7 ) .P h o s p h a t i d y l g l y c e r o p h o s p h a t es y n t h a e n c o d e db y t h e p g s A g en e)c a t a l y z e st h ec o m m i t t e ds t e pi nt h eb i o s y n t h e s i so f P G a n d CL ( 1 8 )a n d i se s s e n t i a lf o rc e l lg r o w t h( 1 9 ) .To s y s t e m a t i c a l l ys t u d y t h e s p e c i f i cr o l e so f a n i o n i cp h o s p h o l i p i d si n c e l lf u n c t i o n s ,a s t r a i nb e a r i n ga D [ l a cp g s A + ]g e n e f u s i o ni n a p g s A : : k a nn u l la l l e l eb a c k g r o u n dw a sc o n s t r u c t e d( 2 0 ) .I n t h i ss t r a i n ,a n i o n i cp h o s p h o l i p i dc o n t e ng e n e e x p r e s s i o n ,a n d c e l lg r o w t h r a t ec a n b e e x o g e n o u sc o n t r o l l e db y t h ei n d u c e r o f t h el a co p e r o n i s o p r o p y lP - Dt h i o g a l a c t o p y r a n o s i d e I P T G ) . I n t h ea b s e n c eo f I P T G , t h eb a s a ll e v e lo f t h ep g s Ag e n e p r o d u c ti s i n s u f f i c i e n tt o s u p p o rc e l lg r o w t h ,a l t h o u g hc e l l sr e m a i nv i a b l e ,a s i n d i c a t e db yr e s u m p t i o no f g r o w t hu p o na d d i t i o no f I P T G .F a i l u r et o l o sv i a b i l i t yi n d i c a t es t h a ta n i o n i cp h o s p h o l i p i d sa r e e s s e n t i a lf oc e r t a i nc e l lf u n c t i o n si n d e p e n d e n to f a m e m b r a n es t r u c t ur e q u i r e m e n t( 2 1 ) .
B a s e d o ns t u d i e so f t h e s u p p r e s s i o no f t h eg r o w t ha r r e sp h e n o t y p eo f t h e a b o v ep g s A m u t a n t u n d e r c o n d i t i o n s o fc S D R ,we r e p o r tr e s u l t st h a ts u p p o r t t h e i n v o l v e m e n to fa n i o n i cp h o s p h o l i p i d si n t h ei n i t i a t i o no fDNA r e p l i c a t i ob yt h eD n a A p r o t e i na t t h eo r i C s i t ei n v i v o .
M AT E R I A L SA N DMETHODS
S t r a i n s ,P l a s m i d s ,a n d C e l lG r o w t h .Ta b l e1 s u m m a r i z e st h es t r a i n sa n d p l a s m i d su s e d i nt h i sw o r ka n d o u t l i n e st h es c h e mu s e dt oc o n s t r u c ts t r a i n sb y P 1 t r a n s d u c t i o n( 2 3 ) .To c o n s t r u c ts t r a i nMRLA12, t h em a l G : : T n l Ol o c u sw a s f i r s t- i n t r o d ui n t os t r a i nM R 1 2a n du s e dt om o n i t o ri t sr e p l a c e m e n tw i t ht h em a l G +l o c u sa l o n gw i t h t h ec l o s e l yl i n k e d( 0 . 2m i n )l e x A ( Da l l e l e( 24 , 2 5 ) .S t r a i nM D L 1 2w a sc o n s t r u c t e db ys u c c e s s i vP 1
t r a n s d u c t i o n so f s t r a i nMG1655 ( n ok n o w nm u t a t i o n sb yu s i n gs t r a i nHDL1001a sd o n o r .P l a s m i d sw e r e i n tr o du c edi n tc e l l sb y e l e c t r o p o r a t i o n ( E l e c t r oC e l lM a n i p u l a t o r6 0 0 ) .C e l lw e r e g r o w na t 3 7 ° C( u n l e s so t h e r w i s ei n d i c a t e d )i n l i q uc u l t u r e so r o n a g a r p l a t e si n LB medium s u p p l e m e n t e dw i t h1 0 0 ,M IPTG w h e r ei n d i c a t e d ) .A n t i b i o t i c s( 2 6 )w e r ep r e s e nd u r i n gt h eg r o w t ho fp l a s m i d - c o n t a i n i n gs t r a i n su n l e s s o t hw i s e i n d i c a t e da n d w e r e u s e d o n l yf o rt h ei n i t i a ls e l e c t i o no fs t r a i n sc a r r y i n gc h r o m o s o m a l l yl o c a t e da n t i b i o t i c - r e s im a r k e r s .
A b b r e v i a t i o n s :P G ,p h o s p h a t i d y l g l y c e r o l ;C L ,c a r d i o l i p i n ;c S D R ,c o n -s t i t u t i v es t a b l eDNA r e p l i c a t i o n ;I P T G ,i s o p r o p y lf - D - t h i o g a l ar a n o s i d e .* T owhom r e p r i n tr e q u e s t s s h o u l db e a d d r e s s e d .
7 8 3
T h e p u b l i c a t i o nc o s t so f t h i sa r t i c l ew e r ed e f r a y e di np a r tb y p a g e c ha rg ep a y m e n t .T h i s a r t i c l em u s tt h e r e f o r eb e h e r e b ym a r k e d a d v e r t i s e m en ti na c c o r d a n c ew i th 1 8U . S . C .§ 1 7 3 4s o l e l yt o i n d i c a t et h i sf a c t .
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Ta b l e 1 . S t r a i n sa n d p l a s m i d s
S t r a i n so rp l a s m i d R e l e v a n tc h a r a c t e r i s t i c ( s ) S o u r c eo r r e f .
S t r a i nM G 1 6 5 5 W i l d t y p e G . W e i n s t o c kKA2 186 A r e c As r l : : T n l O G . We i n s t o c kGE4 50 m a l G : : T n l O G . We i n s t o c kGE1 52 l e x A D e f ) 5 1 G . We i n s t o c k
K H G 7 1 2 r n h A l : : T n 3 T . H o r i u c h iHDL1001* p g s A 3 0 : : k a nt 4 l a c O P - p g s A + ] Il a c Z 2 0l a c Y:: T n 9r e c As r l : : T n l O
HDL11 p g s A 3 0 : : k a n4 D l a c O P - p g s A + ] 1l a c Z 2 1l a c Y : : T n 9l p p 2z d g : : T n l O
H D 3 0 t p g s A 3 0 : : k a n 1 9H L 3 0 t H D 3 0 ,I p p 2 z d g : : T n l O ;P 1 :HD30 x HDL11 T h i s w o r kH R 3 0 t H D 3 0 ,r n h A l : : T n 3 ;P 1 :HD30 x K H G 7 1 2 T h i s w o r kH L R 3 0 t H L 3 0 ,r n h A l : : T n 3 ;P 1 :HL30 x K H G 7 1 2 T h i s w o r kMDL12* p g s A 3 0 : : k a nD [ l a c O P - p g s A + ] 1l a c Z T h i s w o r k
l a c Y : : Tn 9 ; P 1 :MG1655x HDL1001M R 1 2 MDL12, r n h A l : : T n 3 ;P 1 :M D L 1 2x K H G 7 1 2 T h i s w o r kM R A 1 2 * M R 1 2 ,A r e c As r l : : T n l O ;P 1 :M R 1 2 x KA2186 T h i s w o r kMRM12 MR12, m a l G : : T n l O ;P 1 :M R 1 2 x GE450 T h i s w o r kM R L 1 2 M R 1 2 ,l e x A ( D e f ) 5 1 ;P 1 :M R M 1 2x GE152 T h i sw o r kM R L A 1 2 MRL12, A r e c As r l : : T n l O ;P 1 :M R L 1 2x KA2186 T h i s w o r k
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p M M 5 p B R 3 2 2 ,r n h A +a m p rt e t r 2 2pAC11 o r i C m i o C +s p e c r p r J . Z y s k i n dpAC12 o r i C m i o C -s p e c rs t r e p r J . Z y s k i n d
R e l e v a n tc h a r a c t e r i s t i c sa r e a p a r t i a ll i s to f g e n e t i cm a r k e r s .G . We i n s t o c ki s a t U n i v e r s i t yo f T e x a sM e d i c a lS c h o o l ,H o u s t o n ;T .H o r i u c h ii sa t K y u s h uU n i v e r s i t y ,F u k u o k a ,J a p a n ;J . Z y s k i n di sa t S a nD i e g oS t a t eU n i v e r s i t y ,S a n D i e g o .* R e q u i r e sIPTG f o rv i a b i l i t y .t R e q u i r e sp l a s m i dp H D 1 0 2f o rv i a b i l i t y .
M e a s u r e m e n to f D e p e n d e n c eo n a F u n c t i o n a lp g s AG e n e .IPTG w a s w a s h e do u t o f c u l t u r e so f s t r a i nM D L 1 2a n d i t sd e r i v a t i v e sa f t e rg r o w t ho v e r n i g h ti n t h ep r e s e n c eo f IPTG( 2 0 ) .C e l l sw e r e d i l u t e da n d p l a t e di n e q u a l p o r t i o n so n LBa g a r p l a t e s ,e i t h e rw i t h o r w i t h o u t I P T G . C o l o n yn u m b e r s 1 0 - 5 0 0c o l o n i e sp e r p l a t e )w e r e s c o re da f t e ri n c ub a t i o n o ft h ep l a t e sf o r 2 4 - 3 6 h r , a n d t h e p e r c e n t a g eo f c e l l sf o r m i n gc o l o n i e si n t h ea b s e n c eo fIPTG I P T Gi n d e p e n d e n c ei n d e x ,a v e r a g eo f t h r e ee x p e r i m e n t s )w a sc a l c u l a t e db yc o m p a r i n gt h ec o l o n yn u m b e ri nt h ea b s e n c eo f IPTG t o t h a ti nt h ep r e s e n c eo fI P T G .A s i m i l a ra p p r o a c hw a s u s e d t o t e s tt h es u p p r e s s i o no f t h e t e m p e r a t u r e - s e n s i t i v eg r o w t h p h e n o t y p eo f s t r a i n sHR30 a n d HLR30 ( b o t hc a r r y i n gp l a s m i dp H D 1 0 2 ) ,e x c e p tw i t h t h e s es t r a i n st h e p e r m i s s i v ec o n d i t i o n sw e r e g r o w t ha t3 0 ° C a n d t h e r e s t r i c t i v ec o n d i t i o n sw e r e g r o w t h a t 4 2 ° C .G r o w t ho f a l lt h e a b o v es t r a i n su n d e rr e s t r i c t i v ec o n d i t i o n sf o rp e r i o d s l o n g e rt h a n 3 6 h r r e s u l t e di n f o r m a t i o no f a d d i t i o n a lc o l o n i e sd u e t oc o n s t i t u t i v em u t a t i o n si nt h el a cp r o m o t e r( 2 0 )o r t e m p e r a t u r e - r e s i s t a n tm u t a n t so fp l a s m i dpHD102 ( 1 9 )a s
r e p o r t e d .A s s a yf o rP l a s m i dS t a b i l i t y .A n o v e r n i g h tc u l t u r eg r o w ni nt h ep r e s e n c eo f IPTG a n d t h e a p p r o p r i a t ea n t i b i o t i c[ s p e c t i -n o m y c i n( 5 0g g / m l )a n ds t r e p t o m y c i n( 5 0, t g / m l )f o rp l a s m i d spAC11 a n d pAC12 o r t e t r a c y c l i n e( 1 0j i g / m l )f o r p l a s m i dp B R 3 2 2 ]w a s d i l u t e di n t oa n t i b i o t i c - f r e eLB medium e i t h e rw i t h o r w i t h o u tI P T G .T h ec u l t u r e sw e r e k e p t i n e x p o n e n t i a lg r o w t hf o r6 - 1 4g e n e r a t i o n sb yd i l u t i o nw i t hf r e s hm e d i u m .A tt h e i n d i c a t e dt i m e p o i n t s ,d i l u t i o n so ft h ec u l t u r ew e r e s p r e a do n LB a g a r p l a t e si n t h ep r e s e n c e o f IPTG e i t h e rw i t h o rw i t h o u t t h ea p p r o p r i a t ea n t i b i o t i c ( s )t o d e t e r m i n et h ep e r -c e n t a g eo f c e l l sr e t a i n i n gt h ec o r r e s p o n d i n gp l a s m i d ( s ) .
O t h e rP r o c e d u r e s .P h o s p h o l i p i dc o m p o s i t i o no f c e l l su n i -f o r m l yl a b e l e d w i t h 3 2 p O 4wa s d e t e r m i n e da s d e s c r i b e d( 2 0 ) ,e x c e p t t h e r a d i o l a b e lw a s d e t e ct eda n d q u a n t i f i e du s i n ga
B e t a s c o p e B e t a g e n e ,W a l t h a m ,M A ) .P h o s p h a t i d y l g lp h o s p h a t es y n t h a s ea c t i v i t yw a sd e t e r m i n e da sd e s c r i b e d( 1 8 )
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f o r m a t i o nb y s t r a i nM D L 1 2 o n LB a g a r p l a t e si s I P T G -d e p e n d e n td u e t o a l a c ko f s u f f i c i e n ta n i o n i cp h o s p h o l is y n t h e s i st o s u s t a i nc e l lg r o w t hu n d e rr e p r e s s e dc o n d i t i o n sf o rt h e t 4 l a c O P - p g s A + ]g e n e ( 2 0 ) .A n i o n i cp h o s p h o l i p i d si n t h ep r e s e n c eo fA TPw e r ed e m o n s t r a t e dt o r e a c t i v a t et h ei n a c t i vADP f o r mo f D n a Ap r o t e i nf o rc o n t i n u i n gr o u n d so f i n i t i a t i oo fDNA r e p l i c a t i o na t t h eo r i C s i t ei n v i t r o( 2 ) .S i n c ea n r n h Am u t a t i o ns u p p r e s s e st h er e q u i r e m e n tf o ra f u n c t i o n a lD n a Ap r o t e i n ,s u c h a m u t a t i o nm ay a l s os u p p r e s st h eg r o w t ha r r e s tp h e n o t y p eo f t h er e p r e s s e d( I [ l a c O P - p g s A + ]m u t a n tt h e r e bl i n k i n ga n a n i o n i cp h o s p h o l i p i dr e q u i r e m e n tt o D n a Ap r o t e if u n c t i o ni n v i v o .
W i t hs t r a i nM D L 1 2( r n h A + )a s c o n t r o l ,t h eIPTG i n d e p ed e n c ei n d e xo ft h es t r a i nM R 1 2( r n h A )w a s d e t e r m i n e d( F i g1 ) ;8 5 o f t h eM R 1 2c e l l sw e r e I P T G - i n d e p e n d e n ta n d o n l y2 o f t h eM D L 1 2c e l l sw e r e I P T G - i n d e p e n d e n t .T h e IPTGi n d e p e n d e n c ei n d e x o f s t r a i nM R 1 2w a s v e r y s i m i l a rt o t h ep l a t i n ge f f i c i e n c y 8 0 ) o f d n a A 4 6 f s - c o n t a i n i n gc e l l sa t t h er e s t r i c t i v et e m p e r a t u r e( 4 2 ° C )v s .t h ep e r m i s s i v et e m p e r a t u r e( 3 0 ° C )a f t e ri n t r o d u c t i o no f a n r n h Am u t a t i o n( 9 ) .T h e2 o fM D L 1 2c e l l st h a tf o r m e dc o l o n i e si n t h ea b s e n c eo f IPTGs h o w e da l a c ko fr e p r e s s i o no f t h el a c O Pp r o m o t e r ,w h i c hi st h em o s t c o m m o n s p o n t a n e o u sm u t a t i o nl e a d i n gt o IPTG i n d ep e n d e n c e( 2 0 ) .T h u s ,t h eg r o w t h - a r r e s tp h e n o t y p ed u e t o am u t a t i o ni nt h ea b i l i t yt o m a i n t a i na n o r m a ll e v e lo f t h em a j o ra n i o n i cp h o s p h o l i p i d sc a n b e s u p p r e s s e db y a m u t a t i o nr e n -d e r i n gc e l l si n d e p e n d e n to f D n a A p r o t e i na n d t h e o r i C s i t ei . e . ,b yu t i l i z i n gc S D R .I n t r o d u c t i o no f p l a s m i dp M M 5 r n h A + )
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M D L 1 2 - 6 9 . 3 1 6 . 6 2 . 8 8 4 . 4 5 . 8 0 . 8M R 1 2 + 6 9 . 9 1 7 . 7 2 . 1 8 5 . 2 4 . 5 0 . 6M R 1 2 / p M M 5 - 6 9 . 1 1 9 . 1 2 . 5 8 3 . 4 5 . 0 0 . 7M R A 1 2 - 7 0 . 7 1 8 . 72 . 0 8 5 . 8 2 . 7 0 . 4M R L A 1 2 + 6 9 . 2 1 8 . 7 2 . 5 8 6 . 1 3 . 7( D e v i a t i o n ) ( ± 2 . 8 )( ± 1 . 9 ) ( ± 0 . 2 )( ± 1 . 7 )( ± 0 . 4 ) ( ± 0
P E , p h o s p h a t i d y l e t h a n o l a m i n e .
F I G .1 . IPTG d e p e n d e n c eo f s t r a i nM D L 1 2a n d i t sd e r i v a t i v e s .A l ls t r a i n sw e r e f i r s tg r o w ni n t h ep r e s e n c e o f I P T G , a n d t h e IPTGi n d e p e n d e n c ei n d e xw a sd e t e r m in e d . E r ro rb a r ss h o w i n gt h eS E Ma r ei n d i c a t e d .
i n t o s t r a i nM R 1 2r e t u r n e d t h eIPTG i n d e p e n d e n c ei n d e xt o4 ( F i g .1 ) ,t h e r e b yv e r i f y i n gt h a ts u p p r e s s i o ni n s t r a i nM R 1 2w a s d i r e c t l yr e l a t e dt o t h er n h Am u t a t i o n .
I n i t i a t i o no f DNA r e p l i c a t i o na t t h eo r i K s i t e si n a s t r a i nc a r r y i n ga n r n h Aa l l e l ei sR e c A - p r o t e i n - d e p e n d e n t( 1 5 ) .RecAp r o t e i ni se s s e n t i a lf o rr e c o m b i n a t i o na n d t h eS OS DNA r e p a i rs y s t e m( 2 7 ,2 8 )t h r o u g h i t s i n t e r a c t i o nw i t h s i n g l e - s t r a n d e dD N A .T h eRecA p r o t e i nr e q u i r e m e n tf o rcSDRm ay s i m i l a r l yi n v o l v et h e b i n d i n ga n d s t a b i l i z a t i o no f s i n g l e - s t r a n d e dDNAa t t h eo r i Ks i t e sf o ra s s e m b l yo f t h er e p l i c a t i o nc o m p l e xi nt h ea b s e n c eo f f u n c t i o n a lD n a Ap r o t e i n( 2 9 ) .I nt r o du c t io n o ft h eA r e c Aa l l e l ei n t os t r a i nM R 1 2 r e d u c e d t h eIPTG i n d e p e n -d e n c ei n d e xt o 0 . 4 ( F i g .1 ) ,f u r t h e rs u p p o r t i n gt h e i n d u c t i o no f cSDR a s t h e m e c h a n i s mf o r s u p p r e s s i o no f t h ep g s Am u t a t i o n .
B e s i d e s R e c A - p r o t e i n - d e p e n d e n tc S D R , T o r r e y a n dKogoma ( 1 6 )h a v e r e p o r t e d t w o R e c A - p r o t e i n - i n d e p e n d e n tcSDR m o d e s ,o n e o fw h i c ho c c u r s i nc e l l sw i t h t h ef o l l o w i n gg e n o t y p e :r n h Ar e c A l e x A ( D e f ) .T h e LexA p r o t e i n ,w h i c hi s
i n a c t i v a t e db y t h eRecA p r o t e i ni n r e s p o n s et o DNA d a m a g e ,n o r m a l l ya c t st o s u p p r e s sg e n e si n d u c e di n t h eS OS r e s p o n s eo f c e l l st o DNA d a m a g e ;i n a l e x A D e f )s t r a i nt h e s eg e n e sa r ec o n s t i t u t i v e l ye x p r e s s e d( 2 8 ) .A l t h o u g ht h ee x a c t m e c h a n i s mo f r e c A + - i n d e p e n d e n tcSDR i s n o t k n o w n ,i t i s p o s s i b l et h a tamong t h e d e r e p r e s s e dSOS g e n e s , o n e g e n e p r o d u c t i se x p r e s s e dt h a tc r e a t e s a RecA p r o t e i ns u b s t i t u t et ob y p a s st h er e q u i r e m e n tf o rRecA p r o t e i ni ncSDR ( 1 6 ) .S t r a i nM R L A 1 2[ r n h Ar e c Al e x A ( D e f ) ]s h o w e da n IPTG i n d e p e n d e n c ei n d e xo f8 6 ( F i g . 1 )c o n s i s t e n tw i t h a p r e d i c t e dr e c A + - i n d e p e n d e n tcSDR s u p p r e s s i o no f t h er e q u i r e m e n tf o rD n a A p r o t e i n .
A n i o n i cp h o s p h o l i p i dm e t a b o l i s mi n a l lt h eM D L 1 2d e r i v -a t i v e sw a si n v e s t i g a t e dt o a s s u r e t h a ti n d u c t i o no f cSDRh a dn oe f f e c to n p h o s p h a t i d y l g l y c e r o p h o s p h a t es y n t h a s ea c t i v i t yl e v -e l s( T a b l e2 )o r p h o s p h o l i p i dc o m p o s i t i o n( T a b l e3 )f o ra l lt h es t r a i n s .C e l l sg r o w ni n t h e a b s e n c eo f IPTG f o r5 h r s h o w e d
Ta b l e 2 . P h o s p h a t i d y l g l y c e r o s p h o s p h a t es y n t h a s ea c t i v i t i e so fs t r a i n sd e r i v e df r o mM D L 1 2
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S p e c i f i ca c t i v i t y :1 u n i to f e n z y m ea c t i v i t yi s d e f i n e da s t h eamounto f e n z y m et h a tc o n v e r t s 1 n m o l o f g l y c e r o l3 - p h o s p h a t ei n t oac h l o r o f o r m - s o l u b l ep r o d u c ti n 1 m i n .
t h e e x p e c t e dr e d u c t i o ni n enzyme a c t i v i t ya n d a n i o n i cp h o s -p h o l i p i dc o n t e n t .
To t e s tw h e t h e rt h e i n i t i a t i o no f DNA r e p l i c a t i o ni s t h e o n l ye s s e n t i a lc e l lp r o c e s s d e p e n d e n ton a n i o n i cp h o s p h o l i p i d s ,a nr n h A m u t a t i o nwas i n t r o d u c e di n t ot wo p g s A : : k a nn u l la l l e ls t r a i n sl ac k i ng t h e4 F [ l a c O P - p g s A + ]a l l e l e( 1 9 , 2 1 ) ,r e s u l t i n gi ns t r a i n sHR30 a n d HLR30. Th e p a r e n t s t r a i n sa r e d e p e n d e nf o rg r o w t hon p l a s m i dpHD 10 2 p g s A + ) ,w h i c hi s i t s e l ft e m -p e r a t u r e - s e n s i t i v ef o rr e p l i c a t i o n .The I pp m u t a t i o n[ n o r m a l l ye n c o d e st h e m a j o ro u t e r membrane l i p o p r o t e i n( 3 0 ) ]i ns t r a iHLR30 i s a s u p p r e s s o r o f t h e I P T G d e p e n d e n c eo f t h eD [ l a c O P - p g s A + ]a l l e l eb u t n o t o f a n u l la l l e l eo f t h e p g s Ag e n e( 2 1 ) .E x p e r i m e n t a lr e s u l t ss h o w e dt h a ts t r a i n sHR30 a n dHLR30r e m a i n e dt e m p e r a t u r e - s e n s i t i v ef o rg r o w t ha n d , t h e r ef o r e ,d e p e n d e n ton a f u n c t i o n a l p g s A g e n e t h a tc a n n o t b eb y p a s s e db y i n d u c t i o no f c S D R d a t a n o t s h o w n ) .T h e r e f o r et h e r e m u s t b e e s s e n t i a lr e q u i r e m e n t sf o ra n i o n i cp h o s p h o l i p i do t h e r t h a n D n a A p r o t e i n f u n c t i o n even i n t h e p re sence o fa n o t h e rknown p a r t i a ls u p p r e s s o r ( I p p )o f t h e pgsA g e n e ( 3 1 )
D e p e n d e n c eo f o r i CP l a s m i dM a i n t e n a n c eon A n i o n i cP h o s -p h o l i p i d s .A s a n i n d e p e n d e n tm e a s u r e o f t he r e q u i r e m e n tf o ra n i o n i cp h o s p h o l i p i d sf o rD n a A - p r o t e i n - d e p e n d e n ti n i t i a t ia t t h e o r i C s i t e ,t h e s t a b i l i t yo f p l a s m i d s p A C 1 1a n d pAC12)d e p e n d e n ton o r i C f o r r e p l i c a t i o nwas e x a m i n e du n d e r c o n -
d i t i o n so f l i m i t i n ga n i o n i c p h o s p h o l i p i ds y n t h e s i s .P l a s m i dpBR322 w a s u s e das a c o n t r o ls i n c ei t sr e p l i c a t i o ni nv i v oi sn o td e p e n d e n to n t h e D n a A p r o t e i n ( 3 2 ) .B o t ho r i C p l a s m i d sa n dpBR322 ar e p a r t i t i o n e d r a n d o m l ya t c e l ld i v i s i o n ,a n d t h e is t a b i l i t yo f i n h e r i t a n c ei s p r o p o r t i o n a lt o t h e c o p y number i nt h e c e l l( 3 3 ) .P l a s m i dpAC11 h a s a h i g h e r c o p y number t h a np l a s m i dp A C 1 2 3 0 v s . 6 c o p i e s ;A . C h i a r a m e l l oa n d J . W .Z ys k i nd , p e r so n a lc o m m u n i c a t i o n )d u e t o t h e mioC+ l o c u s( 3 4 ,3 5 ) a n d , t h e r e f o r e ,s h o u l d p a r t i t i o ns i m i l a r t o p l a s m i dpBR322 a t d i v i s i o n .
N e i t h e r p l a s m i d pAC11 n o r p A C 1 2 y i e l d e d s i g n i f i cn u m b e r so f t r a n s f o r m a n t s( 2 ± 2 or f e w e rt r a n s f o r m a n t sper1 0 n g o f p l a s m i dD N A )o f s t r a i nM R 1 2( IPTG i n d e p e n d e n twhen c e l l sw e r e p l a t e d i n t h e a b s e n c eo f IPTG. B o t hp l a s m i d sr e s u l t e d i ns i m i l a r l yh i g h n u m b e r so f t r a n s f o r m a n t s 1 7 0 ± 2 0
t r a n s f o r m a n t sp er 1 0 n g o f p l a s m i dD N A ) when c e l l swerep l a t e d i n t h e presence o f I P T G . I n c o n t r a s t , t h e t r a n s f o r m at i o ne f f i c i e n c yo f s t r a i nM R 1 2b y p l a s m i dpBR322 was s i m i lu n d e rb o t h p l a t i n gc o n d i t i o n s( p l u sI P T G ,8 8 1 6 t r a n s f o r -m a n t s p er 1 0 p g o f p l a s m i dD N A ; m i n u s I P T G , 7 5 ± 1 5t r a n s f o r m a n t sper 1 0 p g o f p l a s m i dDNA).
Th e s t a b i l i t yo f p l a s m i d spAC11 a n d p A C 1 2 v s . p l a s m i dpBR322 w a s f u r t h e r e x a m i n e di n s t r a i n sM D L 1 2a n d M R 1 2grown i nl i q u i dm e d i u m i n t h e p re sence a n d a b s e n c eo f IPTG.S t r a i n M D L 1 2w i l lc o n t i n u e t o g r o w s l o w l yi n l i q u i dm e d i u meven a f t e ri t r e a c h e s i t s m i n i m u ma n i o n i c p h o s p h o l i p i dc o n -
t e n t ,w h i c hr e q u i r e sa b o u tseven g e n e r a t i o n so f g r o w t hi n t h ea b s e n c eo f I P T G ( 2 0 ) .Du e t o t h e r n h Am u t a t i o nt h e g r o w t ho f s t r a i nM R 1 2i s I P T G - i n d e p e n d e n ti n l i q u i dm e d i u m .W i t h -o u t d r u g s e l e c t i o n ,p l a s m i dpBR322 i s l e s ss t a b l e i n r n h Amutants t h a n i nw i l d - t y p ec e l l s ,b u t t h i so n l yb e c o m e sa p p a r e n t
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a f t e rg r o w t hf o rmany m o r eg e n e r a t i o n st h a n e m p l o y e dh e r e( 9 ) . A l t h o u g ht h e p e r c e n t a g eo f c e l l sr e t a i n i n gt h e o r i Cp l a s m i d s i n t h e a b s e n c e o f d r u g s e l e c t i o nd e c r e a s e da s af u n c t i o no fg e n e r a t i o nn u m b e re v e ni n t h e p r e s e n c eo f I P T G ,c e l l so f b o t hs t r a i n sM D L 1 2a n d M R 1 2g r o w i n gw i t h o u tI P T Gl o s tt h eo r i Cp l a s m i d sa ta s i g n i f i c a n t l y f a s t e rr a t e( F i g .2 )w h i l ep l a s m i d pBR322 w a s s t a b l eu n d e r a l l g r o w t h c o n d i t i o n s .T h e r e f o r e , t h e r e i s a c l e a rd e p e n d e n c eo f o r i C p la s mi d sf o r
s t a b l em a i n t e n a n c e o ne i t h e rt h el e v e lo r r a t eo f s y n t h e s i so fa n i o ni c p h o s ph o li p i d s.
DISCUSSIONT h ed e p e n d e n c yo f c e l li n t e g r i t yo n membrane p h o s p h o l i p i d sa n d t h er e q u i r e m e n tf o rs p e c i f i cp h o s p h o l i p i d sf o rm a n y c e l lp r o c e s s e sa r e t w o s e p a r a t eb u t r e l a t e df u n c t i o n so f p h o s p h o -l i p i d s .Du e t o t h ed i f f i c u l t yi n s y s t e m a t i c a l l ya l t e r i n gm e m -b r a n e p h o s p h o l i p i dc o m p o s i t i o nw i t h o u t c a u s i n gc e l ld e a t h ,t h em a j o r i t yo f e v i d e n c ef o rs p e c i f i cr o l e sf o rp h o s p h o l i p i d si nm o s tc e l l u l a rp r o c e s s e sh a s come f r o mr e s u l t so b t a i n e db y i nv i t r os t u d i e sw i t h l i t t l ei n v i v od a t a a v a i l a b l et o s u p p o r tt h e s ei n v i t r or e s u l t s .I n E . c o l i ,a m u t a n ti nw h i c ht h eP G - p l u s - C Lc o n t e n to fc e l l sc a n b e s y s t e m a t i c a l l yr e g u l a t e da sa f u n c t i o no f
IPTG ( 2 0 )h a s b e e nu s e d t o e s t a b l i s ht h ei n v i v or e q u i r e m e n tf o r a n i o n i cp h o s p h o l i p i d si n t h e S e c A - p r o t e i n - d e p e n d e n tt r a n s l o c a t i o no fp r o t e i n sa c r o s s t h e i n n e r m e m b r a n e( 3 6 ,3 7 ) .S e c Ap r o t e i nh a s l a t e n tA T P a s ea c t i v i t yt h a ti s a c t i v a t e di nv i t r ou p o na s s o c i a t i o no f t h i sc y t o p la s m i c p r o te i nw i t h a n i o n i cp h o s p h o l i p i d - c o n t a i n i n gm e m b r a n e s( 3 6 , 3 7 ) .T h eD n a A p r o -t e i ni s a l s oa c y t o p l a s m i cATPase t h a ti s d r a m a t i c a l l ya l t e r e di n i t si n t e r a c t i o nw i t h A TP b y a s s o c i a t i o nw i t h a n i o n i cp h o s -p h o l i p i d si n v i t r o( 2 ,3 ) .B a s e d o nt h e s es i m i l a r i t i e s ,t h ea b o v em u t a t i o ni n a n i o n i c p h o s p h o l i p i ds y n t h e s i s( s t r a i nMDL12)w a s u t i l i z e dt o o b t a i n i n v i v oe v i d e n c et o s u p p o r tt h ei n v i t r oe v i d e n c ef o r t h ed e p e n d e n c eo f D n a A p r o t e i nf u n c t i o n o na n i o n i cp h o s p h o l i p i d s .
S i n c ea m u t a t i o ni nt h er n h Ag e n e a l l o w si n i t i a t i o no f DNAr e p l i c a t i o ni n d e p e n d e n to f t h e D n a Ap r o t e i na n d t h eo r i Cs i t e( 1 4 ) ,a s t r a i nc a r r y i n ga n r n h Aa l l e l es h o u l d b y p as st h en e e df o ra l lc e l l u l a rc o m p o n e n t ss p e c i f i c a l l yr e q u i r e df o ri n i t i a t i o na tt h eo r i C s i t e .Ou r r e s u l t sc l e a r l yd e m o n s t r a t et h a ta n r n h Am u t a t i o nc a n s u p p r e s st h eg r o w t ha r r e s tp h e n o t y p eo f m u t a n t sd e f e c t i v ei n a n i o n i cp h o s p h o l i p i ds y n t h e s i sa n d t h u ss t r o n g l y
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s u p p o r tt h e h y p o t h e s i st h a ta n i o n i cp h o s p h o l i p i d sa r e r e q u i r e df o rt h ei n i t i a t i o no f DNA r e p l i c a t i o na t t h eo r i C s i t ei n a nr n h A +s t r a i n .T h e s u p p r e s s i o nb y t h e r n h A 4m u t a t i o nh a d t h ep r o p e r t i e se x p e c t e df r o m t h e c S D R - d e p e n d e n tb yp a s s o ft h eD n a Ap r o t e i na n d t h e o r i Cs i t e .T h es u p p r e s s i o nw a sr e v e r s e db y a p l a s m i dc a r r y i n g t h er n h 4+ g e n e ,w a sd e p e n d e n to n RecAp r o t e i n i n a l e x A+ b a c k g r o u n d ,a n d w a s R e c A - p r o t e i ni n d e p e n d e n ti n a l e x A D e f )b a c k g r o u n d .In a n i n d e p e n d e n
m e a s u r eo f i n v i v oD n a A p r o t e i n f u n c t i o n ,i t w a s s h o w nt h a tp l a s m i d sd e p e n d e n to n a n o r i C s i t ef o rr e p l i c a t i o n ,a n d t h e r ef o r eD n a Ap r o t e i n ,w e r eh i g h l yd e p e n d e n to n n o r m a la n i o n i cp h o s p h o l i p i ds y n t h e s i s f o rs t a b l em a i n t e n a n c ei n t h e a b s e n c eo f o t h e r s e l e c t i v ef o r c e s .F i n a l l y ,s u p p r e s s i o no fIPTG d e p e n -d e n c eb y a n r n h Am u t a t i o nw a s n o t t h er e s u l to f a r e t u r n t on o r m a la n i o n i c p h o s p h o l i p i dm e t a b o l i s m .
T h em o d e li nF i g .3 s u m m a r i z e st h e i nv i v oa n d i nv i t r od a t ao n t h e i n v o l v e m e n to f a n i o ni cp h o s p h o l i p i d si nD n a Ap r o t e if u n c t i o na n d t h e p r o p o s e dm e c h a n i s mo f s u p p r e s s i o no f t h ep g s Am u t a t i o nb y a n r n m h Am u t a t i o n .B a s e do n t h i sm o d e l ,i ts h o u l db e p o s s i b l et oi s o l a t em u t a n t si nt h eD n a Ap r o t e i ni t s e lt h a ta l s os u p p r e s st h eIPTG r e q u i r e m e n tf o rs t r a i nMDL12.Inv i t r o ,s u c h m u t a t e dD n a A p r o t e i n ss h o u l de i t h e rb e i n d e p e n -d e n t o f a n i o n i c p h o s p h o l i p i d sf o ra c t i v a t i o no r b e a l t e r e di nt h e i ri n t e r a c t i o np r o p e r t i e sw i t h a n i o n i c p h o sp h o l i p i dI n i t i am u t a n ts c r e e n i n g sh a v eg e n e r a t e da c o l l e c t i o no f m u t a t i o n si na p l a s m i d b o r n e c o p y o f t h e d n a A g e n e t h a ta p p e a r s t os u p p r e s st h eI PTG r e q u i r e m e n tf o rs t r a i nM D L 1 2( d a t an o ts h o w n ) .
T h e i n a b i l i t yt o s u p p r e s sa n u l la l l e l eo f t h ep g s Ag e n ev i ai n d u c t i o no f cSDR i s s i m i l a rt o t h e r e s u l t sw i t h a m u t a t i o ni nt h eI p pg e n et h a tc a n o n l ys u p p r e s s p g s Aa l l e l e sc a p a b l eo f somer e s i d u a ll e v e lo fa n i o n i cp h o s p h o l i p i ds y n t h e s i s( 21 , 3 1 ) .T h el i m i t i n gl e v e lo f a n i o n i c p h o s p h o l i p i da t t h ep o i n to f c e l lg r o w t ha r r e s ti n a p g s An u l l s t r a i n( 1 9 )i s v e r y s i m i l a rt o t h eb a s a ll e v e lo f a n i o n i c p h o s p h o l i p i di n t h e r e g u l a t e ds t r a i ng r o w ni n t h e a b s e n c eo f I P T G ,w h i c hd o e s n o t r e s u l ti n c e l ld e a t hb u t s i m p l ya r e d u c t i o ni nt h eg r o w t hr a t eo f t h e c e l l( 2 0 )T h e r e f o r e ,t h e r ea p p e a rt ob e m u l t i p l ee s s e n t i a lr e q u i r e m e n tf o ra n i o n i cp h o s p h o l i p i d sw i t h d i f f e r e n tm i n imumt h r e s h o lf o re a c h e s s e n t i a lf u n c t i o n .
T h e e f f e c to f r e d u c e d a n i o n i c p h o s p h o l i p i do n p r o t e it r a n s l o c a t i o ni s n o t a b s o l u t eb u t i s r e f l e c t e di n a g r e a t lr e d u c e dr a t eo f t r a n s l o c a t i o ni n p r o p o r t i o nt o t h er e m a i n i n ga n i o n i cp h o s p h o l i p i d s( 3 8 ,3 9 ) . S i m i l a r l y ,t h ed i r ec t r e l a t is h i pb e t w e e nt h e r a t e o f s y n t h e s i so f a n i o n i cp h o s p h o l i( i . e . ,t h el e v e lo f t h ep g s Ag e n e p r o d u c t )a n d t h eg r o w t hr a t ef o rI P T G - d e p e n d e n tc e l l s( 2 0 )m i g h tb e a r e s u l to ft h e r e d u c e dr a t eo f i n i t i a t i o nb y t h eD n a A p r o t e i n ,w h i c hw o u l da f f e c tt h e
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F I G . 2 . S t a b i l i t yo f o r i C p l a s m i d si n t h e s t r a i n sl a c k i n g a n i o n i cp h o s p h o l i p i d s .S t r a i n s M D L 1 2 A a n d B ) a n d M R 1 2 C a n d D)c a r r y i n gp l a s m i dpBR322 ( - , p l u s I P T G ;L o ,m i n u sI P T G ) ,pAC11 ( 0 ,p l u s I P T G ;0, m i n u sI P T G ) ,or pAC12( - , p l u s I P T G ; A m i n u sIPTG)w e r e g r o w n i nLB m e d i u mw i t h o u ta n t i b i o t i c s .Th e p e r c e n t a g eo f c e l l ss t i l lc a r r y i n g a p l a s m i dwas d e t e r m i n e da f t e rt h e i n d ic a t ednumber o fg e n e r a t i o n s .
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F I G .3 . M o d e lf o r t h e i n i t i a t i o no f DNA r e p l i c a t i o ni n E . c o l iU n d e rn o r m a lc o n d i t i o n si n i t i a t i o no f DNA r e p l i c a t i o ni sr e s t r i c t e dt ot h eo r i Cs i t ea n d d e p e n d e n to n t h eDnaAp r o t e i nd u e t o t h ed i g e s t io f RNA p r i m e r sa t t h eo r i Ks i t e sb y RNase H . A n i o n i cp h o s p h o l i( s y n t h e s i z e db y t h ep g s Ag e n ep r o d u c t )f a c i l i t a t et h eg e n e r a t i o no f t h ea c t i v eA TP f o r mo f t h e D n a Ap r o t e i n .T h ep r o p o s e dr e q u i r e m e n tf o ra n i o n i cp h o s p h o l i p i d si nm a i n t a i n i n gf u n c t i o n a lD n a Ap r o t e i nc a n b es u p p r e s s e dt h r o u g hi n d u c t i o no f cSDR i n a n r n h / Am u t a n tb y p e r m it i n gi n i t i a t i o na t t h eo r i Ks i t e sw h e r eRecA p r o t e i nm a y s u bs t i t u t eD n a A p r o t e i n .
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g r o w t hr a t eo f t h e c e l l( 4 0 ) .B y p a s s i n gt h en e e d f o rD n a Ap r o t e i nm ay r e m o v et h eg r o w t hr a t ed e p e n d e n c eo f t h e c e l lo nD n a A p r o t e i nf u n c t i o na n d p r e v e n tc e l lg r o w t ha r r e s te v e na ta r e d u c e dl e v e lo f a n io n i c p h o s p h ol ip i d .T h e m e c h a n i s mo fs u p p r e s s i o no f t h e g r o w t h a r r e s tp h e n o t y p eo f some p g s Am u t a n t sb y t h el p pm u t a t i o nm i g h tb e t h r o u g hi n c r e a s i n gt h er a t ea t w h i c hb u l k a n i o n i cp h o s p h o l i p i di s made a v a i l a b l ef o rm e m b r a n ef o r m a t i o na n d t os u s t a i nt h e g r o w t hr a t eo f t h ec e l ld e p e n d e n to n D n a A - p r o t e i n - d i r e c t e di n i t i a t i o n .M a t u r a t i o n
o f t h e l p pg e n e p r od u c tc o n s u m e s7 x 1 0 5m o l e c u l e so f P G p e rg e n e r a t i o ni na p o s t t r a n s l a t i o nm o d i f i c a t i o ne v e n t ( 4 1 ) .A t t h er e l a t i v e l yd e f i c i e n tl e v e lo f 5 P G , t h e r e a r e a b o u t 1 0 6P Gm o l e c u l e sd i s t r i b u t e dm a i n l yb e t w e e nt h et w o l e a f l e t so f t h ei n n e rm e m b r a n eo fE . c o l i( 4 2 ) .T h e r e f o r e ,a n y p r o c e s s ,s u c ha s i n i t i a t i o no f DNA r e p l i c a t i o n ,d e p e n d e n to n t h e m a i n t e -n a n c eo f a t h r e s h o l dl e v e lo f f r e ea n i o n i cp h o s p h o l i p i do n t h ei nn e r s ur fa ce o f t h e membrane m i g h t b e a f f e c t e db y t h ed i v e r s i o no f n e w l ys y n t h e s i z e dP G t o t h i sp o s t t r a n s l a t i o n a le v e n t .Any f u r t h e r d e c l i n ei na n i o n i cp h o s p h o l i p i d s ,s u c ha si na p g s A n u l l s t r a i ne v e n when c a r r y i n gt h e l p p a n d r n h As u p p r e s s o rm u t a t i o n s ,m i gh t b e g int o c o m p r o m i s ea f u n c t i o nw i t h a l o w e r t h r e s h o l dr e q u i r e m e n tf o ra n i o n i cp h o s p h o l i p i d sa n d ,t h e r e f o r e ,s t i l lr e s u l ti n a r r e s to f c e l lg r o w t h .
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c e l l u l a rp r o c e s s e sc a n n o tb e m a d e f r o me i t h e ri n v i t r oo r i nv i v os t u d i e sa lo ne b u tc a n b e m a d e b y c o m b i n i n gs u p p o r t i n gd a t af r o m b o t h t y p e so f s t u d i e s .I t i s now c l e a rt h a tp r o t e i nt r a n s l o c a t i o n a c r o s st he i nn erm e m b r a n ei nE . c o l ii s d e p e n -d e n t o n a n i o n i c p h o s p h o l i p i d s .T h e r e s u l t sr e p o r t e d h e r es t r o n g l ys u p p o r ta s i m i l a ri n v o l v e m e n to fa n i o n i cp h o s p h o l i p -i d si n i n i t i a t i o no f DNA r e p l i c a t i o n .O t h e re x a m p l e se x i s to ft h ea s s o c i a t i o no f c y t o p l a s m i cp r o t e i n s 4 3 - 4 5 )w i t h p h o s p h o -l i p i d s ,i n c l u d i n g p r o t e i n st h a ta l s oi n t e r a c tw i t h n u c l e i ca c i d s( 4 6 , 4 7 ) ,r e s u l t i n gi n b i o l o g i c a l l y s i g ni f i c a n tc h a n g e s .T h e r e -f o r e ,a s p e c i f i cr e q u i r e m e n tf o rp h o s p h o l i p i d si n t h e o r g a n i -z a t i o no f p r o t e i n sa t t h e m e m b r a n es u r f a c e i n f u n c t i o n a lc o m p l e x e sa p p e a r st o b e w i d e s p r e a d .
We t h a n kD r . J u d i t hZ y s k i n df o rp r o v i d i n gu s w i t h p l a s m i d sp A C 1 1a n d pAC12b e f o r e t h e i rp r o p e r t i e sw e r e r e p o r t e d .We a l s ot h a n kD r .
E l l i o t tC r o o k ef o rh e l p f u ld i s c u s s i o n sd u r i n gt h e s es t u d i e s .T h i s w o r kw a s s u p p o r t e db y N a t i o n a lI n s t i t u t e so f H e al th G r a n tG M - 2 0 4 8 7 .
1 . F i r s h e i n ,W. ( 1 9 8 9 )A n n u .R e v .M i c r o b i o l .4 3 ,8 9 - 1 2 0 .2 . S e k i m i z u ,K . K o r n b e r g ,A . ( 1 9 8 8 )J . B i o l .C h e m .2 6 3 ,7 1 3 1 -
7 1 3 5 .3 . C r o o k e ,E . ,C a s t u m a ,C .E . K o r n b e r g ,A . ( 1 9 9 2 )J . B i o l .C h e m .
2 6 7 ,1 6 7 7 9 - 1 6 7 8 2 .4 . Ry t e r , A . ( 1 9 6 8 )B a c t e r i o l .R e v .3 2 ,3 9 - 5 2 .5 . S e k i m i z u ,K . ,Yu n g ,B .Y . K o r n b e r g ,A . ( 1 9 8 8 )J . B i o l .C h e m .
2 6 3 ,7 1 3 6 - 7 1 4 0 .6 . H w a n g ,D . S . ,C r o o k e ,E . K o r n b e r g ,A . ( 1 9 9 0 )J . B i o l .C h e m .
2 6 5 ,1 9 2 4 4 - 1 9 2 4 8 .7 . Yu n g ,B .Y . K o r n b e r g ,A . ( 1 9 8 8 )P r o c .N a t l .A c a d .S c i .USA8 5 ,
7 2 0 2 - 7 2 0 5 .8 . F r a l i c k ,J . A . L a r k ,K . G . ( 1 9 7 3 )J . M o l . B i o l .8 0 ,4 5 9 - 4 7 5 .9 . H o r i u c h i ,T . ,M a k i ,H . S e k i g u c h i ,M . ( 1 9 8 4 )M o l . G e n . G e n e t .
1 9 5 ,1 7 - 2 2 .1 0 . K a n a y a ,S . C r o u c h ,R . ( 1 9 8 3 )J . B i o l .C h e m .2 5 8 ,1 2 7 6 - 1 2 8 1 .1 1 . M a s s y , B . , F a y e t ,0 . K o g o m a ,T . ( 1 9 8 4 )J . M o l .B i o l .1 7 8 ,
2 2 7 - 2 3 6 .
1 2 . To r r e y ,T . ,A t l u n g ,T . K o g o m a ,T .( 1 9 8 4 )M o l . G e n .G e n e t .1 9 63 5 0 - 3 5 5 .
1 3 . F r e y ,J . ,C h a n d l e r ,M . C a r o , L . ( 1 9 8 1 )M o l. G e n.G e n e t .1 8 23 6 4 - 3 6 6 .
1 4 . K o g o m a ,T . v o n M e y e n b u r g ,K . ( 1 9 8 3 )EMBO J . 2 , 4 6 3 - 4 6 81 5 . K o g o m a ,T . ,S k a r s t a d ,K . ,B o y e ,E . ,v o n M e y e n b u r g ,K . S t e e n
H . B . ( 1 9 8 5 )J . B a c t e r i o l .1 6 3 ,4 3 9 - 4 4 4 .1 6 . To r r e y , T . K o g o m a ,T . ( 1 9 8 7 )M o l. G e n.G e n e t .2 0 8 ,4 2 0 - 4 2 71 7 . R a e t z ,C .R . D o w h a n ,W. ( 1 9 9 0 ) J .B i o l .C h e m .2 6 5 , 1 2 3 5 - 1 2 31 8 . G o p a l a k r i s h n a n ,A . S . ,C h e n ,Y . ,T e m k i n ,M . D o w h a n ,W .
( 1 9 8 6 )J . B i o .C h e m .2 6 1 ,1 3 2 9 - 1 3 3 8 .1 9 . H e a c o c k ,P .N . D o w h a n ,W. ( 1 9 8 7 )J . B i o l .C h e m .2 6 21 3 0 4 4 - 1 3 0 4 9 .
2 0 . H e a c o c k ,P .N . D o w h a n ,W. ( 1 9 8 9 )J . B i o l .C h e m . 2 6 41 4 9 7 2 - 1 4 9 7 7 .
2 1 . D o w h a n ,W. 1 9 9 2N AT OASI S e r .H 6 3 ,1 1 - 3 2 .2 2 . H o r i u c h i ,T . , M a k i ,H . ,M a r u y a m a ,M . S e k i g u c h i ,M . ( 1 9 8
P r o c .N a t l .A c a d .S c i .U S A7 8 ,3 7 7 0 - 3 7 7 4 .2 3 . S i l h a v y ,T . , B e r m a n ,M . E n q u i s t ,L . ( 1 9 8 4 )E x p e r i m e n t sw i t
G e n eF u s i o n C o l d S p r i n gH a r b o rL a b .P r e s s ,P l a i n v i e w ,N Y ) ,p p .1 0 7 - 1 1 2 .
2 4 . B a c h m a n n ,B . L o w , K . B . ( 1 9 8 0 )M i c r o b i o l .R e v .4 4 ,1 - 5 6 .2 5 . M i k i ,T . , E b i n a , Y . ,K i s h i ,F . N a k a z a w a ,A . ( 1 9 8 1 )N u c l e i c
A c i d sR e s .9 , 5 2 9 - 5 4 3 .2 6 . M a n i a t i s ,T . , F r i t s c h ,E .F . S a m b r o o k ,J . ( 1 9 8 2 )M o l e c u l a
C l o n i n g :A L a b o r a t o r yM a n u a l C o ld S p ri ngH a r b o rL a b . P r e s sP l a i n v i e w ,N Y ) ,p p .4 4 0 - 4 4 4 .
2 7 . W i t k i n ,E. ( 1 9 9 1 )B i o c h i m i e7 3 ,1 3 3 - 1 4 1 .2 8 . C o x ,M . L e h m a n ,I . ( 1 9 8 7 )A n n u .R e v .B i o c h e m .5 6 ,2 2 9 - 2 6 22 9 . S k a r s t a d ,K . B o y e ,E . ( 1 9 8 8 )J . B a c t e r i o l . 1 7 0 ,2 5 4 9 - 2 5 5 4 .3 0 . W u , H . C . ,T o k u n a g a ,M. , T o k u n a g a ,H . ,H a y a s h i ,S . G i a m
C . - Z .( 1 9 8 3 )J . C e l l .B i o c h e m .2 2 ,1 6 1 - 1 7 1 .3 1 . A s a i ,Y . ,K a t a y o s e ,Y . ,H i k i t a ,C . ,O h t a ,A . S h i b u y a ,I . ( 1 9 8
J . B a c t e r i o l .1 7 1 ,6 8 6 7 - 6 8 6 9 .3 2 . A b e , M . ( 1 9 8 0 )J . B a c t e r i o l .1 4 1 ,1 0 2 4 - 1 0 3 0 .3 3 . K o r n b e r g ,A . B a k e r , T . ( 1 9 9 2 )D N AR e p l i c a t i o n F r e e m a n
N ew Y o r k ) ,p p .6 4 7 , 7 5 9 .3 4 . S t u i t j e ,A . R . M e i j e r ,M . ( 1 9 8 3 )N u c l e i cA c i d sR e s .1 1 ,5 7 7 5 -
5 7 9 1 .3 5 . L o b n e r - O l e s e n ,A . ,A t l u n g ,T . R a s m u s s e n ,K . ( 1 9 8 7 )J . B a c -
t e r i o l .1 6 9 ,2 8 3 5 - 2 8 4 2 .3 6 . L i l l ,R . ,D o w h a n ,W. W i c k n e r ,W . ( 1 9 9 0 )C e l l6 0 ,2 7 1 - 2 8 0 .3 7 . H e n d r i c k ,J . P . W i c k n e r , W. ( 1 9 9 1 )J . B i o l .C h e m . 2 6 6
2 4 5 9 6 - 2 4 6 0 0 .3 8 . d e Vr i j e ,T . ,d e S w a r t ,R . L . ,D o w h a n ,W . , T o m m a s s e n ,J . d e
K r u i j f f ,B . ( 1 9 8 8 )N a t u r e L o n d o n )3 3 4 ,1 7 3 - 1 7 5 .3 9 . K u s t e r s ,R . ,D o w h a n ,W . d e ,K . B . ( 1 9 9 1 )J . B i o l .C h e m .2 6 6
8 6 5 9 - 8 6 6 2 .4 0 . H e l m s t e t t e r ,C .( 1 9 8 7 )i nE s c h e r i c h i ac o l ia n d S a l m o n e l l aT y p h
m u r i u m :C e l l u l a ra n dM o l e c u l a rB i o l o g y ,e d s .I n g r a h a m ,J . ,L o w ,K . B . ,M a g a s a n i k ,B . ,S c h a e c h t e r ,M. U m b a r g e r ,H . E . A m .S o c .M i c r o b i o l . ,Wa s h i n g t o n ,D C ) ,Vo l .2 , p p .1 5 9 4 - 1 6 0 5 .
4 1 . S a n k a r a n ,K . W u , H . C . ( 1 9 9 4 )J . B i o l .C h e m .2 6 9 ,1 9 7 0 11 9 7 0 6 .
4 2 . N e i d h a r d t ,F . C . ( 1 9 8 7 )i n E s c h e r i c h i ac o l ia n d S a l m o n e l l aT yp h i m u r i u m :C e l l u l a ra n d M o l e c u l a rB i o l o g y ,e d s .I n g r a h a m ,J . ,L o w , K . B . ,M a g a s a n i k ,B . ,S c h a e c h t e r ,M . U m b a r g e r ,H . E. A m .S o c .M i c r o b i o l . ,Wa s h i n g t o n ,D C ) ,Vo l . 1 , p . 4 .
4 3 . R a n d o ,R . R . ( 1 9 8 8 )FA S E BJ . 2 , 2 3 4 8 - 2 3 5 5 .4 4 . B e l l ,R . M . B u r n s ,D . J . ( 1 9 9 1 )J . B i o l .C h e m .2 6 6 ,4 6 6 1 - 4 6 6 44 5 . Yu l i ,W . , M a c D o n a l d ,J . I S . K e n t , C . ( 1 9 9 3 )J . B i o l .C h e m
2 6 8 ,5 5 1 2 - 5 5 1 8 .4 6 . Yo s h i d a ,S . ,Ta m i y a ,K . K . K o j i m a ,K . ( 1 9 8 9 )B i o c h i m .B i o p h
A c t a 1 0 0 7 ,6 1 - 6 6 .4 7 . P a r k s ,W. A . ,C o u p e r ,C .L . L o w , R . L . ( 1 9 9 0 )J . B i o l .C h e m
2 6 5 ,3 4 3 6 - 3 4 3 9 .
B i o c h e m i s t r y :X i a a n dDowhan
