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F o li a M i c ro b io l. 4 7 ( 4) , 3 6 5 - 3 7 0 ( 20 0 2) h e l p : / / i , - , , - , . b : i .o m a d , c a , , . c z / m b u / f o l s

Electrorelease of Escher ichia col i N u c l e o i d sE . S O L E Y M A N O O L U *Biophysics and Microscopy Group, Section of Molecular Cytology, Institute fo r Molecular Cell Biology, BioCentrum Amsterdam,University of Amsterdam, 1098 SM Amsterdam, The Netherlandse-mail ethan@berlin.corn

Received 3 December 2001

AB ST RA CT . Bacterial chromosome is assemb led and folded into one or several nucleoids, depending onthe metabol ic s tatus o f the cell . Developm ent of rel iable nucleoid isolat ion protocols has a lways been anobject ive for researchers . A rapid and reprod ucible procedure for isolat ion of E. coli nucleoids is describedhere, while the cel l envelope is maintained. Mem brane dispersions and vesicles were prepared b y lysoz ym e-ED TA treatment with subseq uent rupture o f the spheroplasts b y electric field. Un der these conditions theyield of electroreleased nucleoids was around 9 0 %. The extent of DN A-en velope contacts was determinedby l ight microsc opy employing phase contrast and fluorescence modes.

Despi te the fact that bacterial chrom osomes are appreciated sometimes also as prok aryot ic counter-parts of euka ryotic chromatin and nucle osom es (Sharpe and Errington 1999), the E. coil chromosome i sa unique nucleic acid-protein machinery highly co mp acted in the cell. Fo r a long time, the understanding o fthe structure-function relationships o f the E. coli nucleoids rel ied on data coming from pioneering electronmicroscopy work ( for rev iew see Ro bino w and Kellenberger 1994) and sedimentation a nalysis (W oree l andBurgy 1972). The former experimental approach visual ized the nucleoids as a large dense and nonviscousribosome-free mass occupying the center of E. coli cytoplasm, the remaining space being filled with poly-ribosomes. H ow eve r, the published picture s of the nucle oid structures va ried largely dep end ing on thepreparative me thods, thus rendering the bacterial nucleo id as a structure w ithout defined boundaries. In addi-tion, different methodologies used to visualize the nucleoids resulted in either membrane-bound (Pontalierand W orcel 1976), or membrane-free (K ave noff and Bow en 1976) s t ructures depending on experimentalconditions applied. These two structures were distinguished afterwards according to their different sedimen-tation rates. Mo st of the labo ratory procedu res app lied for the purification and further characterization of thebacterial n ucleo ids either used physical disruption or mild lysis with various detergents, which resulted inDN A breakage or in appearance of v i scous DNA par t ic les , ref lec t ing the unfo ld ing o f the chromo some,most ly due to charge repulsion of phosphate groups in the DN A (Hirschbein and Guillen 1982). Thus,depending o n the purification method use d nuc leoid components diff ered from one another.

De spite its immature status and poor ly understood m olecu lar mechanisms, the use of electric fieldson biological cel ls , organelles and t issues is now adays widely appl ied in cel l biophy sics and b iomedicine(Fre y 19 94). The im pulse f or this has originated from the practical nee d to achieve stable and nondestructivetransformation o f bacteria, yeasts, animal and p lant cells with further potential in the fields o f gene tic engi-neering, nonviral g ene transfer and therapy, as well as in agrobiotechnology. In this contex t, the obje ctive isto achieve direct transfer o f nucleic acids (D NA and R NA ), proteins and oth er important pharmaceuticalsand biom acro mo lecule s by means o f electroporation into various recipient ceils, the main adv antag e of thisapproach being that cell suspensions are manipulated in chemically intact maner (Szostkov~ et al. 1999). Thestrength o f the app lied electric field and the duration o f the pulse(s) are ad justed acc ording to the cell sizeand type, density of the cell suspension, m edium composition, etc. (Tsong 1990; Neumann 1992; Sowers1992). Interestingly, electric field effe cts we re applied m ostly to perm eabilize c ell me mb rane s for variousgenetic and pharmacological purposes (Orlow ski and Mir 1993), whereas their u se in electrorelease of cel-lular com pon ents with their subse quen t characterization was not studied in detail until now. H avin g cons ide-red the abo ve drawbacks o f the experimental procedures applied s o far for the isolation of bacterial nucleoids(see also Bendich 2001), the latter possibility opens new avenues for isolating and further characterizingthem as a m odel system for s tudying the in vivo propert ies of bacterial DN A.

Mem brane perm eabil i ty has a natural bioelectroehemical origin (Hon g 1994), both in terms o f regu-lation of gene expre ssion (Matzke and M atzke 1991) and cell signaling (O livotto et al. 1996). Electrical

*Present address: Biom edical Centre for Nanotechnology, U pper Austrian Research Gm bH, St.Peter-Strafle 25, A-4021 Linz, Austria.

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fields induce either irreversible or reversible effects on bilayer permeability. The former case involves modi-fication of lipid asymmetric structures and proteins within it, leading to disruption of cytoplasmic consti-tuents. When electrical parameters and certain medium characteristics, such as temperature and ionicstrength, are suitably chosen, reversible and nondestructive cell membrane permeabilization is achieved,with viable self-assembling of the electro-broken cellular constituents (Zimmermann and Neil 1996). Sincethe importance of lipid-nucleic acid associations are well established in terms of various models for the ori-gin of protocells (Deamer 1997; Stileymano~lu 1999), evolution of prokaryotes (Smith and Szathmary 1995),metabolic activities (Hellingwerf and Palmen 1996; Funnell 1996), transcriptional activity of prokaryoticchromosomes (Gorke and Rak 2001), as well as in the field of development of novel tools of non-viral genetherapy vectors, obtaining of membrane-bound nucleoids is emphasized. Molecular details of these self-assemblies and their functional implications are not understood. This work describes the electric-field-indu-ced release of envelope-bound E. coli nucleoids, which can be used further as models of relevant in vivomacromolecular assemblages.

MATERIALS AND METHODSChemicals and sources. Low-melting point agarose, egg white lysozyme (EC 3.2.1.17), NaC1,

EDTA were purchased from Sigma (USA) 4',6-diamidino-2-phenylindole (DAPI) and FM 4-64 were obtai-ned from Molecular Probes (USA). Glycerol and formaldehyde were purchased from Aldrich. All otherchemicals used were of highest analytical grade. Unless otherwise stated, all experiments were performed atroom temperature (26-28 ~Bacterial s train and gr owth condit ions. Glycerol stock o f E. col i strain MC 1000 (kindly providedby M. Wery, Labo ratoire de Physiologie Bacterienne, IBPC , Paris, France), was grown under vigorousshaking at 30 *C in glucose minimal medium. Growth was followed turbidimetrically at 450 nm. A singlecolony of agar plates was inoculated into 5 mL fresh glucose minimal medium, supplemented with ampi-cillin, pH = 7.2; osmotic pressure 300 mmol/L (determined osmometrically). During early stationary phasethe culture was diluted appropriately into prewarmed medium and incubated at 30 ~ with shaking at 3.3 Hzin a rotary shaker (N ew B r u n s w ick G-25D) in a-500 mL Erleumeyer flask for better aeration. The culturewas diluted several times to reach exponential phase. Determination of the total number of cells was donewith Coulter Counter (Coulter Electronics , U K) in a-30 lma orifice and in a 100-~tL volume. Cells with con-stant average cell mass were used as the criterion for steady-state culture. Since bacterial cell size and DNA(Akerlund et al. 1995; Gasol et al. 1999), as well as protein content (Azam et al. 1999) is growth-phase-dependent, it was important to use steady-state cultures.

Elect ro lys i s proced ure . Ten mL of the exponential-phase cells were centrifuged (83 Hz, 2 min) in10 Eppendorf tubes. The pellets were collected into a single Eppendorf tube and kept on ice, into which200 laL lysozyme buffer (10 g/L lysozyme, 10x phosphate buffer saline (PBS), pH = 7.2; 0.25 mol/L EDTA,1 mol/L sucrose) was added. The resulting turbid mixture was leR for 5 min at room temperature and rechil-led. Spheroplast suspension was subjected to electromanipulation as foliows. One-mm cuvettes were keptat -20 ~ and put on ice before use. E. coli pulser (Bio-Rad Laboratories , USA) current was adjusted to1.8 kV/cm. One-mL 10 % ice-cold glycerol was added into each cuvette, along with 50 ~tL of spheroplastsuspension with careful mixing. A single pulse was used for electrical breakdown of the spheroplasts. BothDAPI and FM 4-64 stains were added afterwards.

Light microscopy and image analysis . Both DAPI and FM 4-64 were added to spheroplast sus-pension at final concentration of 1 g/L (Pogliano et al. 1999). Glass cover slips were placed immediatelyonto 10 ~tL sample and sealed with nail polish, to prevent the movement of electroreleased nucleoids. Theseslides were left for 10 min and examined by phase contrast, fluorescence, or combined phase contrast-fluo-rescence modes of O l ym p u s BH-2 fluorescence microscope, equipped with Princeton CCD camera, using100x SPlan PL phase contrast objective. The microscopy samples can be used during the next 1-2 d withoutany substantial damage of the patterns o f the electroreleased E. coli nucleoids in terms o f their subsequentquantification.

RESULTS AND DISCUSSIONIsolatiOn of E. coli nucleoids has always been approached by research groups. Despite the fact thateven nowadays different viewpoints concerning the term "isolated nucleoids" are shared, a great success has

been achieved with regard to reproducibility o f isolation procedures. The difficulty arises from the fact that

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t h e i s o l a t e d n u c l e o i d s i z e , m o r p h o l o g y a n d m o l e c u l a r p r o p e r t i e s d o n o t n e c e s s a r i l y c o r r e s p o n d t o t h o s eex i s t i n g in v ivo . T h e l a t t e r i s s t i l l c o n t r o v e r s i a l l y d o c u m e n t e d b y v a r i o u s r e s e a r c h g r o u p s , d u e t o m e t h o d o -l o g ic a l l i m i t a t io n s , s u c h a s c r y o f i x a t io n a r t i fa c t s o f e le c t r o n m i c r o s c o p y p r o t o c o l s , a s w e l l a s t h o s e c r e a t e db y t h e u s e o f v a r i o u s d e t e r g e n t s i n t h e l y s i s s t e p . i n a d d i ti o n , t h e n u c l e o i d s d e c o m p a c t a f l e r l y s is o f th eb a c t e r i a l c e l l s a n d t h u s t h e v i s u a l iz e d s t r u c t u r e s a r e f a r f r o m b e i n g r e l i a b l e s u b s t i t u t e s o f t h o s e i n l i v in gE. co i l c e l l s ( M u r p h y a n d Z i m m e r m a n 2 0 0 1 ) . H e n c e , to a p p r o a c h c l o s e l y t h e li v i n g li q u i d a n d c r o w d e d s t a teo f b a c t e r i a l c y t o p l a s m , t h e r e i s a n e e d t o v i s u a l i z e t h e n u c l e o i d s d i r e c t l y b o t h i n g r o w i n g c e l l s a n d u p o n t h e i rl y s is , w h i l e a v o i d i n g t h e u s e o f i o n ic a n d n o n i o n i c d e t e r g e n t s a n d d e s t r u c t i v e m i c r o s c o p y a r t i fa c t s . O n ea l t e r n a ti v e o f th e u s e o f c h e m i c a l s i s th e a p p l i c a t i o n o f p h y s i c a l f o r c e s , w h i c h p e r m i t w o r k i n a c o n t r o l la b l ec h e m i c a l - fr e e w a y . T h e w i d e u s e o f e l e c t r ic f i e l d s i n b i o t e c h n o l o g y i s m a i n l y t o p e r m e a b i l i z e t h e m e m b r a n eo f t h e h o s t c e l l f o r in t r o d u c t io n o f v a r io u s m a c r o m o l e c u l e s w i th c o m m e r c i a l a n d t h e r a p e u t i c b e n e f i t , a n de v e n o r g a n e l l e s , b u t th e u s e o f e le c t r i c f i e ld s f o r is o l a t io n o f b i o m o l e c u l e s o f in t e r e s t f r o m t h e c r o w d e d c y t o -p l a s m i s o n l y r u d i m e n t a r y .

F i g . 1 A i s a p h a s e - c o n t r a s t i m a g e o f v i a b l e E. co i l c e l l s . T h e n u c l e o i d w a s v i s u a l i z e d h e r e w i t h i nl i vi n g c e l ls a s a b o d y o f u n d e f i n e d e d g e s b y u s i n g p h a s e c o n t r a s t a n d D A P I f l u o r e s c e n c e i m a g e s ( F i g . I B ) .I n th e s e e x p o n e n t i a l - p h a s e c e l l s D N A a p p e a r s t o o c c u p y m o s t o f th e c e l l v o l u m e , b e i n g i r r e g u l a r i n s h a p ea n d w i t h e d g e s r e a c h i n g t h e m e m b r a n e , v i s u a l i z e d h e r e w i t h F M 4 - 6 4 s t a i n i n g ( F ig . 1 C ) . F M 4 - 6 4 i s a ne n v e l o p e - i m p e r m e a n t d y e , w h i c h b i n d s t o t h e o u t e r l e a f le t o f li p i d b i l a y e r s ( P o g l i a n o et al . 1 9 9 9 ) . To d e t e r -m i n e t h e e x t e n t o f p h o s p h o l i p i d - D N A a s s o c i a t i o n s , e x p o n e n t i a l - p h a s e c u l t u r e s o f E. co i l M C 1 0 0 0 s t r a i nw e r e s t a i n e d w i t h b o t h F M 4 - 6 4 a n d D A P I , e i t h e r d u r in g c u l t i v a ti o n o f b a c t e r i a o r i m m e d i a t e l y b e f o r e m i -c r o s c o p i c e x a m i n a t io n . B o t h F M 4 - 6 4 a n d D A P I d o n o t in t e r f e re w i t h g r o w t h r a t e s o f c e l l s a n d c a n b e u s e df o r th e i r s ta i n i ng a t d i f f e r e n t p o i n ts o f th e i r g r o w t h c u r v e f o r m i c r o s c o p i c o b s e r v a t i o n . F i g . 1 C s h o w s t h es t a in i n g p a t te r n o f F M 4 - 6 4 o n l i v in g b a c t e r i a l c e ll s . T h e i r m e m b r a n e s a p p e a r a s i n t e n s e l y s t a i n e d m a s ss u r r o u n d i n g d a r k c y t o p l a s m , w h i c h is o c c u p i e d b y n u c le o i d , a s s e e n b y D A P I i m a g e i n F ig . l B . T h u s , c o m -b i n at io n o f D A P I a n d F M 4 - 6 4 i m a g e s b r i n g s o u t a m e m b r a n e - n u c l e o i d a s s o c i a ti o n p a tt e r n w h i c h i s a l soa v a l i d m o d e l f o r l i v in g c e l l s o f E. co i l ( F u n n e l l 1 9 9 6 ) . P h a s e - c o n t r a s t a n d f l u o r e s c e n c e - m i c r o s c o p i c i m a g e so f th e s p h e r o p l a s t s a r e s h o w n i n F i g s 1 D - F . T h e D N A c o n t e n t w i t h i n t h e s p h e r o p l a s t s ( F ig . 1 E ) h a s a m u c hg rea t e r i n t en s i t y t h an n u c l eo id s i n l i v in g E. co l i c e l l s (F i g . 1 B ) , w h i c h i s a n i n d i c a t io n o f t h e i r c o m p a c t i o nw i t h i n a s m a l l e r v o l u m e o f v e s i c l e s . S o m e a g g r e g a t i o n o f s p h e r o p l a s t o n t h e r i g h t - h a n d s i d e ( F i g . 1E , F ) i sd u e t o t h e p r e s e n c e o f i n o r g a n i c b i v a l e n t c a t i o n s . T h e i n t e n s it y o f t h e F M 4 - 6 4 s i g n a l o f s p h e r o p l a s t s u s p e n -s i o n ( F ig . I F ) m o r e o r l e s s m a t c h e s t h a t o f l iv i n g e x p o n e n t i a l p h a s e E . c o l i M C l O 0 0 ce l l s . Th e r esu l t s a r e i na g r e e m e n t w i th p r e v i o u s o b s e r v a t io n s o f P o g l ia n o et al . ( 1 9 9 9) . T h e m o r p h o l o g y o f th e n u c l e o i d s c h a n g e su p o n e l e c t r i c b r e a k d o w n o f t h e s p h e ro p l a s t s ( F i g . 1 G ) . E l e c t r o l y s i s e f f e c t s a r e s e e n h e r e i n t h e d is a p p e a r a n c eo f th e n u c le o i d s f r o m t h e p h a se - c o n tr a s t m o d e o f t he m i c r o s c o p e . T h e r e l e a s e d D N A s o m e h o w d e c o m p a c t s( a s d e s c r i b e d b y M u r p h y a n d Z i m m e r m a n 2 0 0 1 ) ; a s i n d i c a t e d b y t h e d e c r e a s e d i n t e n s it y o f th e D A P I s i gn a l.I n a d di ti o n , n u c l e o i d s c o l l a p s e o n t h e s u r f a c e o f t h e m i c r o s c o p i c g l a s s s l i d e s. T h e a m o u n t o f D N A r e l e a s e df r o m d i f f e r e n t i n d i v i d u a l s p h e r o p l a s t s a l s o v a r i e s , i n d i c a ti n g p r o b a b l y a c e r t a i n d e g r e e o f e l e c tr i c - f ie l d - i n d u -c e d f u s i o n o f s e v e r a l a g g r e g a t e d E. co l i v e s i c l e s p r i o r t o r e l e a s e o f t h e n u c l e i c a c i d c o n t e n t .

N u c l e o i d s i n l y s o z y m e - i n d u c e d s p h e r o p l a s t s w e r e i n a c c e s s i b l e t o n u c l e a s e s a n d o t h e r a g e n t s d i s -r u p t in g D N A - m e m b r a n e c o n t a c t s , a s w e l l a s to S D S t r e a tm e n t s . A l l t h e s e a g e n t s d i s a g g r e g a t e t h e s e c o m -p l e x e s ( F i g . 1 G , H ) , u p o n e l e c t r o l y s i s ( d a t a n o t s h o w n ) . S p h e r o p l a t s s w e l l d u e t o t h e a c t i o n o f th e c o l l o i do s m o t i c p r e s s u r e o f th e c y t o p l a s m i c m a c r o m o l e c u l e s , b e c a u s e o f e i t h e r d i e l e c t r ic b r e a k a g e o r e l e c t r o o s m o s i sw i t h s u b s e q u e n t h y d r o d y n a m i c e n t r a n c e o f w a t e r a n d i n o r g a n ic i o n s in t o th e v e s i c l e s ( T s o n g 1 9 9 0 ; D e u t i c k ea n d S c h w i s t e r 1 9 9 2 ) . T h e s i z e o f t h e i n d u c e d e l e c t r o p o r e s a l s o i n c r e a s e s s t e a d i l y , l e a d i n g t o c o l l o i d o v e r -s w e l l i n g a n d f i n a l l y t o r u p t u r e o f th e v e s i c l e m e m b r a n e s a n d l y s i s a f t e r t h e i r c r i t i c a l i n n e r v o l u m e h a sb e e n e x c e e d e d . A p p a r e n t l y , m e m b r a n e b l e b s a r e c r e a t e d a t t h i s s t a g e a n d l a r g e r b r o k e n l i p i d p i e c e s m a y b er e l e a s e d f r o m t h e s p h e r o p l a s ts ( T s o n g 1 9 9 0 ) . S u c h a D o n n a n - o s m o t i c c y t o l y s i s ( G l a s e r 2 0 0 1 ) i n v o l v e s a ne l e c t r o - i n d u c e d e f f e c t o n b o t h m e m b r a n e p r o t e i n s a n d l i p id s ( D e u t i c k e a n d S c h w i s t e r 1 9 9 2 ; A k i n l a j a a n dS a c h s 1 9 9 8 ) . S i n c e F M 4 - 6 4 c a n n o t d i s t i n g u is h b e t w e e n v a r io u s l i p i d d o m a i n s a n d s t a i n s w i t h e q u a l e f fi -c i e n c y t h e w h o l e b i l a y e r s t r u c t u r e , th e o b s e r v e d f l u o r e s c e n c e i n t e n s it i e s o f d i ff e r e n t m e m b r a n e r e g i o n sw o u l d i n d ic a t e t h e ir n u m b e r a n d t o p o l o g y ( P o g l i a n o et al . 1 9 9 9 ) . I t i s i n t e r e s t i n g t o n o t e t h e m u c h g r e a t e ri n t e n s it y o f t h e g h o s t s ( F i g . 1 H ) , a s c o m p a r e d w i t h t h e u n l y s e d E. co i l c e l l s ( F ig . 1 C ), w i t h a n o b v i o u s e f f e c to f d e l a y e d l i p i d a s y m m e t r y o f t h e o ri g in a l c e l l e n v e l o p e . E. co l i s p h e r o p l a s t s w e r e o b t a i n e d b y l y s o z y m e -E D T A - s u c r o s e p r o c e d u r e ( T a k e t o a n d K u n o 1 9 6 9) . P h a s e c o n tr a s t (F i g. I D ) , D A P I f l u o r e s c e n c e (F i g. 1 E )a n d F M 4 - 6 4 ( F i g . I F ) s h o w t h e m a t c h o f a ll th r e e m i c r o s c o p i c i m a g e s o f t h e s a m e s p h e r o p l a s t s , w h i l e t h ei n t e n s it y o f th e D A P I s i g n a l i s m u c h h i g h e r t h a n F M 4 - 6 4 . M e m b r a n e d y e s h o w s a s t a in i n g p a t t e r n o f th eg h o s t s u r r o u n d i n g t h e n u c l e o i d . T h e s e s p h e r o p l a s t s a r e v e r y c l e a r l y d e f i n e d a l s o b y p h a s e - c o n t r a s tm i c r o s c o p y ( F i g . 1 D ) , w h i c h i s i n a g r e e m e n t w i t h r e c e n t o b s e r v a t i o n s ( Z i m m e r m a n a n d M u r p h y 2 0 0 1 ) . T h e

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D

,~ ~ Fi g, 1. Light microscopy of E. coil cells and electroteleased nucleoids from ED TA-ly sozym e sphero-plasts. Exponential-phase E. coli MCI000 cells are shown as phase contrast (A), DAPI- (B) and FM 4-64-stained (C) images; spheroplasts were obtained by classical ED TA-ly sozym e procedure (see Materials andMethods); D - phase contrast, g: - DAPI-treated, F - FM 4-64-stained E. coil vesicles; electric-field-inducedshrinkage o f the spheroplasts with resulting envelope-bound nu cleoids is shown as DAPI (O) signal fornucleoids and as FM 4-64 (H) pattern for broken membrane dispersions, surrounding the DNA; ba r- 5 pro .

amount o f lysozyme to be used depends on the cell type. A low lysozyme amount was used here to preventits well-known effec t on nonspecific association between bacterial mem branes and DN A (Silberstein andInouye 1974 ). Lys ozym e acts as N-acetylmuramidase by degrading the bacterial polysacchar ide chains todisaccharide fi'agments. Com plete breakdow n of the bacterial cell is prevented by doing lysis in a wea khypertonic or isotonic solutio n o f sucrose. Un der these conditions, spheroplasts highly sensitive to osmoticmanipulations are form ed (Fig. 1D-F), w hich can be ruptured only if place d in a hypotonic med ium leavingthen remnants o f plasma membranes. Spheroplasts are structures in which permeability is increased butcellular metabolism is not altered (Hageumaier et al. 1997). The release of nucleoids fi 'om these lys ozym e-ED TA spheroplasts was achiev ed by applying electric fields (Fig. 1G, H), instead of osmotic shock, to avoidthe drawbacks o f chemical reagents, such as salt effects (Baumgarten and Feh er 1995). Application of anelectric pulse to living E. coli cells does not lead to their breakdown (data not shown), because of thestrength and rigidity of the peptidoglycan layer of the envelope. Und er these circumstances the sequ ence o fbacterial respons es followin g a single square wav e pulse o f the electropulsator is orientation o f the bacte-rium with the long axis in the direction of the external field and, sub sequently, electro -induced permeabili-zation o f the envelope, in which the lipid compon ent is affected (Neumann 1992; Eynard et al. 1998). How-ever, this permeabilization is not sufficient for the release o f cytop lasm ic contents. In order to obtain functi-onal cells with weak ened peptidoglycan layer of the bacterial envelope, ly sozy me -ED TA spheroplasts wereprepared, as described above (Fig. 1D-F). Electric pulses can now be applied to rupture the spheroplasts,thus releasing nucleoids. Physico-chemical parameters, such as osm otic pressure, ionic strength o f the con-ducting media, etc . influence the induced perm eability of the spheroplasts (Orlow ski and Mir 1993). In this

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context, it is important to avoid high fields, wh ich give rise to partial strand denaturation o f DN A and fo rm:ation of DNA clots, where viscous medium is used to avoid the osmotic shock. Obviously, the pulse durationis a critical parameter in such cases (Eynard et aL 1998). L iving cell mem branes poss ess natural dielectricproperties. Upon overloading with an external electric field creating a potential exceeding their dielectricstrength, the membran e resistance to permeation breaks dow n (Tsong 1990). Therefo re, followin g thisapproach, the po ssibility exists to achieve controlled electrorelease o f bacterial nucleoids (Fig. 1G, H), aswell as oth er cyto solic products. How ever, structural information o f the ctescribed nucleoids m ust be relatedto their functional implications for living bacterial cells and the information obtaine d and m odels bu ilt in thiscontext remain to b e tested.

Besid es the bro ad range of applications of the isolated nucleo ids, mentioned by Hirsch bein andGuillen (1982 ) and Zimmerman and M urph y (2001), it is o f particular interest to emp loy them as biochem i-cal and biophy sical mo dels of self-assembling nucleic acid- lipid particles with respec t to their in vivo com-paction and con densatio n properties, and to stud y how the folding of the bacterial c hrom osom e fiber withinthe macromolecularly crowded E. coli cytoplasm is related to physico-chemical and bio-inorganic regulationof prokaryotic gene expression.

Thanks are due to Dr. A. Zaritsky and Dr. I. Fishov (Department ofLife Sciences, Ben-Gurion University of he Negev, Beer-Sheva, Israel) for many info rmal discussions and helpful suggestions during their short visit to our laboratory at The University ofAmsterdam. This work was supported by the physics (FOM) and biolog y (ALW ) branches of the Dutch Foundation of ScientificResearch (NWO ) in the context of the special interdisciplinary p rogram Physical Biology.REFERENCESAKERLUNDT., NORDSTROMK., BERNANDERR.; Analysis of cell size and DANN content in exponentially growing and stationary-

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