S ym p os ia B io lo g ic a H u n g a r ic a
H i
Symposia Biologica Hungarica
PROCEEDINGS OF THE
SYMPOSIUM ON BACTERIAL TRANSFORMATION AND
BACTERIOCINOGENY
P R O C E E D I N G S O F T H E S Y M P O S I U M
O N B A C T E R I A L T R ANS F O R M A T I O N
A N D
B A C T E RIO C I N O G E N Y
August, 13 — 16, 1963 B u dap est
( S y m p o s i a Biologien H u n g á riá i 6 )
T h is work co n ta in s the lec
t u r e s , debates a n d contribu
t io n s to the Sym posium organ
iz e d b y the H ungarian A cadem y o f Sciences.
T h e volume con tain s also lit e r a r y references and consists o f th r e e parts. P a r t 1 deals w i t h th e transform ation of b a c
te r ia , Part 2 w ith th e phenom e
n a o f bacterioeinogeny, and P a r t 3 with the g en etic funda
m e n ta ls of cell regulation.
A K A D É M I A I K I A B Ö B U D A P E S T
S V m |i o s i a B i o l o g i c a 1 1 u n g a r i c a
ü
S y m p o s i a B i o l o g i c a H u n g a r i c a
11 e d i g i t B. G Y Ö R F Í Y
V ol. 6
A K A D É M I A I K I A D Ó , B U D A P E S T 9 6 6
PROCEEDINGS OE THE SYMPOSIUM ON BACTERIAL
TRANSFORMATION AND BACTERIOQNOGENY
A U G U ST, 13 - 16, 1963, B U D A P E S T
A K A D É M I A I K I A D Ó , B U D A P E S T 1 9 6 6
(C) A K A D É M I A I K I A D Ó , B U D A P E S T I 9 6 0
P I M N T E I ) I N H U N G A 11 V
C O N T E N T S
F o r e w o r d ... 7 L is t o f p a r t i c i p a n t s ... 9 P h o to g r a p h s o f th e p a r tic ip a n ts o f t h e S y m p o s i u m ... 13 S tr a u b , F . B ru n o
O p en in g a d d re s s ... 15 G y ő rffy , B a r n a
I n tr o d u c to r y r e m a r k s ... IT S E C T I O N i
B A C T E R I A L T R A N S F O R M A T I O N H o tc h k is s , R o llin D .
M e ch an ism o f re c o m b in a tio n in D N A -m e d ia te d t r a n s f o r m a t i o n s ... 23 P a k u la , R o m a n
K in e tic s o f p ro v o k e d c o m p e te n c e in s tre p to c o c c a l c u ltu re s a n d its sp e c ific ity . . 33 S z y b alsk i, W a c la w a n d O p a ra -K u b in s k a , Z ofia
P h y sic o -c h e m ic a l a n d b io lo g ical p ro p e r tie s o f g e n e tic m a r k e rs in tr a n s fo r m in g D N A ... 43 Ig a li, S á n d o r
E f f e c t o f x - r a y s on th e tr a n s f o r m a tio n fre q u e n c y o¥ E scherichia c o l i... 57 D e k h ty a re n k o , T . 13.
D N A c o m p o s itio n o f tr a n s f o r m a n ts o f th e e n te r ic b a c te r ia g r o u p ... 59 K o h o u to v á , M.
In f e c tio n o f t h e re c ip ie n t cell b y tr a n s fo r m in g D N A . T h e s tim u la tio n a n d in h ib i
tio n o f i n f e c t i o n ... 65 F ö ld e s, J . a n d T r a u tn e r , T h o m a s A.
I n f e c ti v ity o f is o la te d B . su b tilis p h a g e D N A ... 73 G á b o r, M ag d a
T ra n s f o rm a tio n o f s tr e p to m y c in m a r k e r s in ro u g h s tr a in s o f R h izo b iu m lu p i n i . . 75 S ik, T ib o r
S e d im e n ta tio n s tu d ie s o f t h e d e o x y rib o n u c le ic a c id s o f R h izo b iu m m elilo ti a n d o f it s ly so g en ic f o r m ... 79
5
S E C T I O N I I
B A C T E R I O C I N O G E N Y C low es, R o y s to n C.
C olicin fa c to rs a s sex f a c t o r s ... 85 W a ta n a b e , T s u to m u
R e c e n t p ro g re ss in t h e s tu d ie s on tr a n s m is s ib le d r u g r e s is ta n c e fa c to rs in J a p a n
( p a p e r n o t re c e iv e d ) —
W a ta n a b e , T s u to m u a n d O k a d a , M o to y u k i
M a tin g in d u c e d b y co licin o g en ic f a c to r B in a n F - s tr a in o f E sch erich ia coli K 1 2 97 H o lla n d , I . B .
E ffe c t o n D N A s y n th e s is o f p r e p a r a tio n s o f a b a c te rio c in fo rm e d b y B . m egaterium ... 101 W o llm a n , E lie L .
O n t h e g e n e tic d e te r m in a tio n o f c o li c in o g e n y ... 107 P a te r s o n , A n n C.
B a c te rio c in o g e n y in P seu d o m o n a s a e r u g in o s a... 115 H a m o n , Y v e s e t P é ro n , Y v o n n e
A p ro p o s d e s b a c té rio c in e s p ro d u ite s p a r K lebsiella p n eu m o n ia e e t Aerobacter aerogenes ... 119
SEC T I O N II I
D N A S P E C I F I C I T Y A N D R E G U L A T I O N S te n t, G u n th e r S.
R e g u la tio n o f R N A s y n th e s is in b a c t e r i a ... 123 S ta h l, F r a n k lin W .
R e c o m b in a tio n in b a c te rio p h a g e T 4. H e te ro z y g o s ity a n d c ir c u la r ity ... 131 K a p la n , R . W .
I n d u c t io n o f m u t a ti o n s in th e p h a g e k a p p a ... 143 S ch aeffer, P ie r re
R e g u la tio n m e c h a n is m s c o n tro llin g b a c te r ia l s p o r u l a t i o n ... 147 Iv á n o v ic s , G.
T e m p e r a tu r e s e n s itiv ity o f m u t a n ts o f B a c illu s a n th ra cis c a u se d b y a b lo c k in th y m in e - n u c le o tid e s y n th e s is ... 153 A lfö ld i, L a jo s
R e la x a tio n a n d a m in o ac id s e n s itiv ity o f E . coli K l 2 m u t a n t s ... 155 E c k h a r t , W a lte r
A s tra in -sp e c ific p r o te in fo r th e r e p lic a tio n o f b a c te rio p h a g e s T 2 a n d T 4 ... 157
* G y ő rffy , B a r n a
C losing r e m a r k s ... 161 I n d e x e s ... 163 6
F O R E W O R D
A sym posium w ith th e title Bacterial Transform ation and Bacteriocinogeny was held in B u d ap est from A ugust 13 to 16, 1963, sponsored b y th e H u n g a ria n A cadem y o f Sciences. T he p u rp o se o f th is S ym posium w as to m ak e an a tte m p t to exam ine som e re c e n t developm ents in ce rtain avenues o f m olec
u la r genetics a n d it w as so designed to b rin g scien tists to g e th e r to re p o rt re c e n t findings. T he th re e sessions o f th e S ym posium w ere d ev o ted to t r a n s form ation, bacteriocinogeny, a n d D N A specificity an d cell re g u la tio n a n d on each o f th e sessions a u th o ritie s o f th ese top ics p re sen ted tw o in v ite d lectu res follow ed b y c o n trib u to ry papers. T h e one-day excursion to L ak e B a la to n offered a fu rth e r o p p o rtu n ity for inform al exchanges o f ideas a n d discussions. T h e p a p e rs given b y th e in v ite d speakers a n d c o n trib u to rs a n d th e discussions are p re sen ted in th is volum e. An a tte m p t has been m ade to m a in ta in th e co n v ersatio n al to n e o f th e exten siv e discussions, how ever, i t is u n fo rtu n a te , p erh ap s, t h a t th e pu b lish ed proceedings does n o t record th em com pletely. W e wish to express o u r ap p reciatio n to th e m an y people w hose co n trib u tio n s m ade th e S ym posium successful, an d especially tho se w ho g ave advice to us d u rin g th e pro g ram m in g stage, th e sp eakers an d chairm en. T h a n k s are d u e to th e co-operation o f th o se who d e v o te d efforts in arran g in g a n d ru n n in g th e p rogram m e. P a rtic u la r th a n k s are due to D r K. S zende w ho assisted in over-all e d ito rial d u ties o f th e arra n g e m e n ts for p u b licatio n o f th e proceedings. G ra titu d e is expressed to M rs É. Szelei w ho pro v id ed sec retaria l services in th e ea rly ph ase o f th e S ym posium and for helping th e indexing a n d for checking th e p age proof. A cknow ledgem ent is g ra te fu lly m ade to th e A kadém iai K iad ó , B udapest, p u b lish in g th is sym posium volum e.
The Organizing Committee B. G Y Ő R F F Y
T . S IK
L I S T 0 F P A R T I C I P A N T S
Al f ö l d i, L ajos, I n s titu te o f M icrobiology, U n iv ersity M edical School, Szeged, H u n g a ry
Bá l in t, A ndor, I n s titu te o f G enetics, H u n g a ria n A cadem y of Sciences, B udapest, H u n g a ry
Be a n, A n ita, Virus L a b o ra to ry , U n iv ersity of C alifornia, B erkeley, C ali
fornia, U.S.A.
Bé l á d y, Ilona, I n s titu te o f M icrobiology, U n iv e rsity Medical School, Sze
ged, H u n g a ry
Bö h m e, H elm ut, I n s titu t fü r K u ltu rp flan z en fo rsch u n g , D eutsche A kadem ie d er W issenschaften zu Berlin, G atersleben, D eutsche D em o k ratisch e R epublik
Cl o w e s, R o y sto n , C., M edical R esearch Council, M icrobial G enetics R e search U n it, H a m m ersm ith H o sp ital, L ondon, U .K .
De k h t y a r e n k o, T. D ., I n s titu te o f M icrobiology, A cadem y o f Sciences o f th e U k ra in ia n S .S.R ., K iev, U.S.S.R.
Ec k h a r d t, W alter, V irus L a b o ra to ry , U n iv e rsity o f C alifornia, Berkeley, C alifornia, U.S.A.
Fe j é r- Ko s s e y, Olga, I n s titu te o f G enetics, H u n g a ria n A cadem y o f S ci
ences, B u d ap est, H u n g a ry
Gá b o r, M agda, I n s titu te o f G enetics, H u n g a ria n A cadem y of Sciences, B u d ap est, H u n g a ry
GyŐr f f y, B arn a , I n s titu te o f G enetics, H u n g a ria n A cadem y o f Sciences, B u d ap est, H u n g a ry
He in c z-Cz a k o, M ária, I n s titu te o f G enetics, H u n g a ria n A cadem y of Sciences, B u d ap est, H u n g a ry
Hi l l, R ebecca, V irus L ab o rato ry , U n iv e rsity o f C alifornia, B erkeley, C ali
fornia, U.S.A.
Ho l l a n d, I. B., M icrobiology U n it, D e p a rtm e n t o f B iochem istry, U n iv e r
sity o f O xford, O xford, Ű .K .
Ho r v á t h, Is tv á n , I n s titu te o f P h arm aco lo g y , B udapest, H u n g a ry
Ho t c h k is s, R ollin, D., T he R ockefeller I n s titu te , New Y ork, N .Y ., U.S.A.
Ig a l i, S ándor, F red e ric Jolio t-C u rie N a tio n a l R esearch I n s titu te for R adio- biology a n d R adiohygiene, B udapest. H u n g a ry
Iv á n o v ic s, G yörgy, I n s titu te o f M icrobiology, U n iv ersity M edical School, Szeged, H u n g a ry
Ka p l a n, R . W ., I n s titu t fü r M ikrobiologie, J o h a n n W olfgang G oethe U n iv e rsitä t, F ra n k f u rt a. M ain, D eu tsch e B undesrep u b lik
9
Ke c s k é s, M ihály, I n s titu te o f A g rochem istry an d Soil Science, H u n g a ria n A cadem y o f Sciences, B u d ap est, H u n g a ry
Km: m en t, Z o ltán , I n s titu te o f P la n t P ro te c tio n , B u d ap est, H u n g a ry Ko h o u t o v á, M., D e p a rtm e n t o f M icrobial G enetics a n d V ariab ility , I n
s titu te o f M icrobiology, C zechoslovak A cadem y o f Sciences, P rague, C zechoslovakia
Lo v r e k o v ic s, László, I n s titu te of P la n t P ro tectio n , B u d ap est, H u n g a ry Pa k u l a, R om an, I n s titu te o f B ioch em istry a n d B iophysics o f th e P olish
A cadem y o f Sciences, W arsaw , P o la n d
Pa t e r s o n, A nn, C., A g ricu ltu ral R esearch Council U n it for M icrobiology, D e p a rtm e n t o f M icrobiology, T h e U n iv ersity , Sheffield, U .K .
Sc h a e f f e r, P ierre, I n s titu t P a ste u r, P aris, F rance
Sí k, T ibor, I n s titu te o f G enetics, H u n g a ria n A cadem y o f Sciences, B u d a p est, H u n g a ry
So l y m o s i, F erenc, I n s titu te o f P la n t P ro te c tio n , B u d a p e st, H u n g a ry St a h l, F ra n k lin , W ., D e p a rtm e n t o f Biology a n d I n s titu te o f M olecular
Biology, U n iv e rsity o f Oregon. Eugene, Oregon, U.S.A.
St e n t, G u n th e r S., V irus L a b o ra to ry , U niv ersity o f C alifornia, Berkeley, C alifornia, U.S.A.
St r a u b, F . B runo, B iological Section, H u n g a ria n A cadem y o f Sciences, B u d ap est, H u n g a ry
Sz y b a l s k i, W aclaw , Me A rdle M em orial L a b o ra to ry , U n iv ersity of W iscon
sin, M adison, W is., U.S.A.
Tr a u t n e r, T h o m as A., I n s titu t fü r G enetik d e r U n iv e rsitä t zu Köln, K ö ln , D e u tsc h e B u n d esrep u b lik
Ud v a r d y, É v a , C hem ical W orks G. R ich ter, B uclapest, H u n g a ry
Wa t a n a b e, T su to m u , D e p a rtm e n t o f M icrobiology, K eio U n iv ersity School o f M edicine, T okyo, J a p a n
Wo l l m a n, E lie L ., S ervice de P hysiologie M icrobienne, I n s t i t u t P a s te u r, P aris, F ra n c e
Ye g ia n, C harles D ., V irus L a b o ra to ry , U n iv ersity o f C alifornia, B erkeley, C alifornia, U.S.A.
10
P H O T O G R A P H S O F T H E P A R T I C I P A N T S O F T H E S Y M P O S I U M
E . L . W o llm a n G. S. S te n t F . W . S ta h l
I . B. H o lla n d T. A. T r a u tn e r R . C. C low es
R . I). H o tc h k is s
R . P a k u la W . S z y b a ls k i F . R. S tr a u b 12
j
\ f S t :
I T" ' 1 8
r * Űr- m I | g l
T . I). D e k h ty a re n k o , O. F e jé r-K o sse y , 1’. S ch aeffer, W . S z y b a ls k i a n d (íy . Ivánovics
O P E N I N G A D D R E S S By
F. Br u n o St r a u b
SECRETARY OF THE BIOLOGICAL SECTION,
HUNGARIAN ACADEMY OF SCIENCES, BUDAPEST, HUNGARY
O n b e h a lf o f th e H u n g a ria n A cadem y o f Sciences I am g lad to welcom e th e p a rtic ip a n ts o f th e Sym posium on Bacterial Transform ation and Bacterioci- nogeny.
As a biochem ist I am n o t in a p o sitio n to m ake wise in tro d u c to ry com m ents o n th e s u b je c t o f th is S ym posium , alth o u g h it is clear to a biochem ist t h a t th e analy sis o f tra n sfo rm a tio n p h en o m en a has influenced co n tem p o rary b io ch em istry q u ite p rofoundly. O ur ideas on genetic in fo rm atio n a n d on in fo rm atio n tra n s fe r are based on th is p a rtic u la r field. I am g lad to see t h a t w ell-know n scientists from differen t coun tries h av e com e to us to discuss som e aspects o f th e p re se n t s ta n d o f research in th is field. I th in k , one o f th e g re a t problem s o f science still unsolved is th e problem o f in fo rm atio n tr a n s fer b etw een scientists. M aybe, th e s tu d y of biological in fo rm atio n a n d in fo rm a tio n tra n s fe r will help us p erfec t o u r m eth o d s for scientific in fo rm a tio n tra n s fe r a n d in fo rm a tio n re trie v al.
Such a S ym posium seem s to m e like an in te lle c tu a l episom al effect, a n d I hope t h a t som e new th o u g h ts in tro d u c e d in to th ese ta lk s w ill m an ife st them selves in new m ed ia a n d p ro d u ce new resu lts. O ur A cadem y o f Sciences is giving special a tte n tio n a n d help to th e biological sciences n o w adays, as w e h av e realized th a t, in sp ite o f s c a tte re d successes in som e fields, we h av e sev eral shortcom ings in basic biological research, and, in p a rtic u la r, we did n o t do enough in th e p a s t to help basic research in genetics.
T h e convening o f th is S ym posium is one o f th e m easures to help geneticists a n d th e field o f m o d ern m icrobiology. I sincerely hope t h a t th is S ym posium will, a p a r t from fu rth e rin g in te rn a tio n a l u n d e rsta n d in g , give m uch s tim u la t
ing in telle ctu al pleasure for p a rtic ip a n ts.
T h a n k you !
15
S y m p. Hiol. H u n g . 6, 17-9 (1965)
I N T R O D U C T O R Y R E M A R K S
B y Ba r n a Gy ő r f f y DIRECTOR, INSTITUTE OF GENETICS,
HUNGARIAN ACADEMY OF SCIENCES, BUDAPEST, HUNGARY
I t is m y privilege a n d hon o u r to h e a rtily welcom e you in th e nam e o f th e O rganizing C om m ittee a n d o f th e H u n g a ria n geneticists, on th e occasion o f th e Sym posium on Bacterial Genetics held for th e first tim e in H u n g a ry .
T he ta s k o f th e opening th is S ym posium is n o t only a g re a t h o n o u r to me, b u t a source o f deep perso n al g ratification.
G enetics has com e to occupy a key positio n in m odern biology an d , re cently, to serve as a focal p o in t also for b iochem istry, physical chem istry, virology a n d so on. T h e ex tensive stu d ies m ade d u rin g th e la s t decade re su lted in a sh ift from th e classical appro ach es o f genetics to th e m olecular view o f th e m echanism o f h ered ity , w hich resid ts in re ite ra tio n o f th e d an g e r in th e u n ity o f genetics.
B efore beginning o u r sessions, we should do well to ta k e a little tim e off for considering th e to p ic o f th is Sym posium a n d its perspectives in th e g en e ra l schem e o f genetics.
I t w as alre a d y in th e la st q u a rte r o f th e 19th c e n tu ry w hen W ilson s t a t ed: “T h e tran sm issio n o f a specific su b stan ce or idioplasm , w hich we h av e seen reason to id e n tify w ith ch ro m atin , th e general role o f th e nucleus in m etabolism is o f v ita l im p o rtan ce to th e th e o ry o f in h eritan ce. T h e c y to plasm is, in a m easure, th e su b s tra tu m o f in h eritan ce, b u t it is so only by v irtu e o f its re la tio n to th e nucleus, w hich is — so to speak th e u ltim a te c o u rt o f appeal. T he nucleus c a n n o t o p e ra te w ith o u t a cytoplasm ic field in w hich its p ecu liar pow ers m ay com e in to p lay , b u t th is field is created a n d m olded b y itself. B o th are necessary to d evelopm ent, alth o u g h th e nucleus alone suffices for th e in h eritan c e o f specific possibilities o f d ev e l
o p m e n t.”
Since t h a t tim e re m a rk a b le progress has been m ade in o u r u n d e rsta n d in g o f th e basic m echanism o f h ered ity , m an y m ajo r a n d exciting problem s w ere solved along th e avenues o f th e classical genetics, an d d u rin g th e la st decade genetics has been developing sp ec ta cu larly fru itfu lly , w ith ev er increasing speed, an d b rillia n t adv an ces have been achieved, p a rtic u la rly in th e field o f m olecular genetics. W hile th is re v o lu tio n ary an d triu m p h a n t genetics (as S onneborn p arap h ra se d ) m akes an u n e x p e c te d a n d illu m in atin g su b sta n tia l progress from one week to th e o th er, th e re seem s to be also a c e rta in a m o u n t o f a n x ie ty t h a t th e re is a fu n d a m e n ta l difference betw een th e classical g ene
tics a n d th e m olecular genetics t h a t m ay cause an u n b alan c e in th e fu tu re dev elo p m en t o f genetics. I th in k , it is w rong to m ake such a c h a ra c te riz a tio n , as th e difference is fa r from being fu n d a m e n tal. T here is only a differ
ence in th e aspects o f m ethodology.
2 Symp. Biol. Hung. 0 17
I do n o t agree w ith th e som ew hat ex a g g era te d views t h a t ev e ry problem o f genetics will sooner or la te r be solved in m olecular term s a n d t h a t th e form al genetics o f th e fu tu re will consist in m atch in g in te s t tu b e s D N A s a n d m essenger R N A s from v e g e ta tiv e o r som atic cells, or t h a t th e m olecu
lar genetics is n o t genetics a t a ll b u t m uch m ore b io ch em istry or biophysics.
I am convinced t h a t th e m olecular genetics a tta c k in g first th e basic problem s by sim plifications a t th e m olecular level, will proceed fu rth e r to th e n ex t, m ore co m plicated sets o f problem s a t th e cellular, in d iv id u a l a n d even a t th e p o p u la tio n level, like it has been done b y th e classical genetics. T he fu tu re b alan ced in te g ra tio n o f b o th genetical aspects is obvious.
I t seem s v ery o ften t h a t th e problem s o f m olecular genetics p re d o m in a te a n d are m uch m ore in th e focus o f in te re st th a n an y o th ers. I firm ly believe, how ever, t h a t th e X l t h International Congress of Genetics in som e weeks a t T h e H ag u e will w itness to th e b alan c ed progress in m an y fields o f genetics.
C onsidering th e p a s t a n d p re se n t progress as well as th e p ro sp ects of genetics, th e p red ictio n seem s n o t to be im probable t h a t th e re are th re e m ain avenues along w hich th e genetics will progress in th e foreseeable fu tu re : first, th e m olecular a n d cellular level analy sin g th e fu rth e r d etails o f classi
cal chrom osom al h e re d ity as well as th e extrach ro m o so m al in h eritan ce, second, th e in d iv id u al level a tta c k in g th e still fa in tly explored genetical problem s of d iffe ren tiatio n a n d d evelopm ent, an d lastly , th e p o p u latio n level revealing th e fu n d a m e n ta l problem s o f evolution.
F ro m th e general g e n e tic ist’s p o in t o f view, I th in k , m any o f th e problem s o f m olecular genetics an d m olecular biology to be discussed in th e com ing sessions are in in te rre la tio n s w ith m an y o th e r classical fields o f genetics a n d a co rrelatio n can only c o n trib u te to th e b alan c ed u n ity o f genetics.
*
N ow I w ish to recall a few phases o f th e sto ry o f th is S ym posium . I t w a alre ad y tw o y e a rs ago w hen it w as proposed to th e H u n g arian A cadem y o f Sciences t h a t a S ym posium on b a c te ria l genetics should be held in 1962 in B u d ap est. Owing to th e severely lim ited p ossibility to sponsor such a special Sym posium , th e su b je c t to be considered w as lim ited to b ac te rial tra n sfo rm a tio n . H ow ever, th e o rg an izatio n o f th e S ym posium w as postp o n ed u n til th is y ear, w hich afforded th e o p p o rtu n ity for co rrelatin g it in som e w ay w ith th e International Congress of Genetics as well as w ith th e Third E rw in Baur-Gedächlnisvorlesungen to be held before th e Congress a t T he H ague, in G atersleben. W hen th e arra n g e m e n ts o f th e S ym posium really s ta rte d a t th e en d o f th e la s t year, it w as th o u g h t m ore p ro fitab le if th e su b je c t o f th e S ym posium w ould be e x te n d e d to bacteriocinogeny, in w hich field re su lts h av e been achieved also in H u n g a ry .
I t was a courageous step , indeed, t h a t we to o k o ver w hen we s ta rte d th is S ym posium an d we se n t o u r first inform al le tte rs to m an y o f th e research w orkers w ho h a d m ade significant adv an ces in th ese areas. H ow ever, th e response w as fa r beyond o u r ex p e ctatio n s a n d even th e in te re s t aroused m uch g re a te r th a n we could h av e im agined. T hus, ta k in g in to a c co u n t th a t, ow ing to o th e r com m itm en ts, several o f th e leading w orkers in tra n s fo rm a tio n h a d declined o u r in v ita tio n a n d as a re s u lt o f th e replies from th o se able to com e to B u d a p e st a n d p a rtic ip a te , we decided to e x te n d th e topics for discussion also to problem s o f regulation.
18
O ur id e a w as t h a t th is S ym posium should give a n o p p o rtu n ity to ex a m in e th e c u rre n t s ta te s o f research w ork in ce rtain avenues, to h ea r th e p re s e n ta tio n s o f p erso n al co n trib u tio n s to th e progress, to discuss p roblem s o f m u tu a l in te re st, a n d la st b u t n o t least, to s tre n g th e n th e links b etw een scien tists o f d ifferent n atio n s.
O ur desire, to h av e a S ym posium on b a c te ria l genetics here in B u d a p e st, is now realized an d we are p ro u d o f such an élite g ath e rin g since m an y o f o u r guests w ho ac cep ted o u r in v ita tio n a n d h av e u n d e rta k e n th e effort to com e here c o n trib u te d re m a rk a b ly to o u r u n d e rs ta n d in g o f th e basic problem s o f genetics. On th e o th e r h an d , we re g ret v ery m uch indeed t h a t P rofessor B resler, D r G oldfarb, P rofessor M aa l0 e an d P rofessor S h u g a r c a n n o t be here am ong us.
I n sp ite o f all o u r efforts, we know , th e re are also fa u lts in o u r a rra n g e m ents, th ere fo re we ea rn e stly re q u e s t you to b e a r in m in d t h a t we a re really ra n k a m a te u rs in th is k in d o f w ork a n d overlook o u r errors. W e will do o u r b e st to ensure t h a t th ese shortcom ings are outw eighed b y o u r efforts a n d good-will, a n d we hope th a t, w ith y o u r sincere co-operation and u n d ersta n d in g , th e S ym posium will be successful.
I n conclusion, I w ould like to express ap p reciatio n to th e H u n g a ria n A c ad em y of Sciences for ac cep tin g o u r proposal a n d sponsoring th is Sym posium . I should like to th a n k also all tho se w ho so graciously co llab o rated in th e arra n g e m e n ts an d are now assisting in th e ru n n in g o f th e w hole o f th e S y m posium .
So now, on b e h a lf o f th e O rganizing C om m ittee, it is m y p le a s a n t d u ty to declare th e Sym posium on Bacterial Transform ation and Bacteriocinogeny open.
T h a n k you!
2 19
S E (' TI () N
B A C T K R 1 A L T R A N S F O R M A T I O N
S y m p . Biol. H u n g . 6, 23-32 (1965) M E C H A N I S M O F R E C O M B I N A T I O N I N D N A - M E D I Á T E D
T R A N S F O R M A T I O N S Bv
Ro l l in D. Ho t c h k is s
THE ROCKEFELLER INSTITUTE, NEW YORK, U.S. A.
T h e tra n sfo rm a tio n o f re cep tiv e b ac te rial cells b y iso lated d eo x y rib o n u cleate (DNA) from re la te d stra in s provides one o f th e sim plest genetic system s.
Since th e classic w ork o f A very e t al. (1944) it has becom e increasingly clear t h a t th e ac tiv e D N A is a sim ple form o f th e genetic m a te ria l th a t , o r
ganized in to chrom osom es, is p re se n t in alm o st all living organism s. T h e d em o n stratio n s t h a t a v a rie ty o f b a c te ria l pro p erties, such as d ru g resis
tan ce s a n d vario u s en z y m a tic tra its , are sim ilarly tra n s fe rre d b y D N A ju stifie s th e e x p e c ta tio n t h a t th is d e te rm in a n t m a te ria l re p re se n ts th e functional m a tte r o f m an y genes, as well as m an y kinds o f cells.
T h e ad v a n ta g e s o f th is genetic sy stem o ver m ost o th e r ones is t h a t th e genetic m a te ria l can be m a n ip u la te d chem ically a n d physically to a fa r g re a t
er e x te n t w hen it is in th e form o f iso lated DNA th a n w hen it is p re se n t in cells, nuclei or even viruses. F o r in th ese o th e r cases, th e biological a c tiv ity can no longer be e stim a te d if th e m an ip u latio n has seriously a lte re d th e com plex co n ta in e r in w hich th e D N A is carried. T herefore, w ith tra n sfo rm a tio n system s one has th e o p p o rtu n ity to in v estig ate th e chem ical a n d physical p ro p e rtie s o f genetic m ateria l, its se n sitiv ity to chem ical o r physical change, th e creation o f m u ta tio n s, th e s tru c tu ra l req u irem en ts for its a c tiv ity . I n w h a t follows, I shall discuss som e re c e n t w ork w hich, utilizing th is app ro ach , begins to show com plex b eh a v io r p reviously n o t know n to be associated w ith single p articles o f DNA.
L e t us first recall t h a t w hen th e resu lts o f a tra n sfo rm a tio n could be m eas
u re d q u a n tita tiv e ly (H otchkiss 1951), it w as found t h a t m ost p airs of tr a its ca rried by DNA w ere se p a ra te ly in tro d u c e d in to d ifferent cells. T hus, for a p a ir o f m ark ers X an d F , in d e p e n d e n t tra n sfo rm a n ts T w ere found:
eells(wild) + t)NA(Al') gives T x and T y
T h e double tra n s fo rm a n t T XY, if recovered, ap p e ars only in th e low fre q u e n cies resu ltin g from successive single tra n sfo rm a tio n s. On th e o th e r h an d , if m a rk e r Y is linked to Z (M arm ur an d H o tch k iss 1960):
eells(w ild) + D N A (A 1 '/? ) g iv es T x + T YZ + T y + T z
I n such cases, th e linked p air Y Z seem s to be ca rried by a single D N A p a r ticle, w hich undergoes se p a ratio n b y re co m b in atio n w henever T Y or T z are produced. M ost p airs o f m ark ers b eh a v e in d e p e n d e n tly like X an d Y .
One se t o f linked m ark e rs o f Diplococcus pneumoniae, d esig n ate d Sadb, was occasionally tra n sfo rm e d to g e th e r, Sadb or adb, b u t was g enerally sepa-
23
i
ra te d in to th e sm aller u n its, Sa, ad o r d or 8 , etc. (In these, 8 rep resen ts stre p to m y c in resistance, a, d a n d b are sep a rab ly identifiable sulfonam ide re sistan ce tra its m an ifested in a folic acid sy n th e ta se .) T h e hypo th esis was, therefore, proposed t h a t th e re was a size lim ita tio n on th e a m o u n t o f g ene
tic m ateria l t h a t could be effectively tra n sfe rre d by tra n s fo rm a tio n (H o tc h kiss 1958, B alassa a n d P ré v o st 1962). R eco m b in atio n w as also being in v e sti
g a te d a t th is tim e in th e p o p u latio n o f D N A p articles resu ltin g from infection o f single cells w ith m ixed b ac te rio p h ag e s (Chase a n d D oerm ann 1958, S te in berg an d E d g a r 1962, L u ria 1962). We proposer] to ex p lain th e q u a n tita tiv e anom alies (n o n -a d d itiv ity a n d high coincidence o f m u ltip le tran sfers) in th is a n d o th e r finely a n a ly z ed system s as due sim ply to o ne-directional tra n s fe r o r copying o f sh o rt regions o f th e D N A s tra n d s. I n th is view, th e u su al recom - b in atio n e v e n t in D N A stra n d s is a double one (sw itch-over a n d n ea rb y sw itch-back) so t h a t tho se exchanges observed in th re e -fa c to r crosses, in w hich one is obliged to fo rm u late as double events, w ould still be o rd i
n a ry ones. C onsequently, th e ir fre q u e n t occurrence is ex p ected a n d th e re is little o r no a c tu a l ‘high n eg a tiv e in terfe ren c e’. B y ad h erin g to th e single e v e n t (one sw itch-over) as th e u n it reco m b in atio n al process, ph ag e gene
tic ists h av e h ad to p o s tu la te n o t only double, b u t trip le a n d q u ad ru p le ev en ts, all clu stered in, a n d occurring o ften in ac tiv e ly recom bining regions o f D N A (L u ria 1962).
Since t h a t tim e, it has seem ed m ore d o u b tfu l w h e th e r th e re is a n y sh arp basis for d istin c tio n betw een th e sm all m olecular s tra n d s o f D N A an d th e o ften fa r larger stra n d s o f chrom osom es. I f th e re is, it will p ro b a b ly be m ost im p o rta n t to learn in w h a t w ay in d iv id u a l D N A m olecules are joined to each o th e r —p erh ap s b y cations, organic bases or o th e r s u b s ta n c e s —in m aking up th e large chrom osom es. I f th e re tu rn s o u t to be no im p o rta n t d isc o n tin u ity betw een th e D N A m olecule a n d th e chrom osom e, th e n it will becom e all th e m ore significant to u n d e rs ta n d how D N A m olecules behave in th e recom b in atio n t h a t occurs in b a c te ria l tran sfo rm atio n .
B y a s tu d y o f th e effect o f D N A co n c en tratio n a n d o f tim e o f exposure to D N A on tra n sfo rm a tio n yield, it has been possible to d eterm in e how m any p articles o f D N A are re q u ire d to effect tra n sfo rm a tio n o f one cell. T his re p resen ts som e a d v a n ta g e o ver th e ph ag e reco m b in atio n s w hich occur betw een ill-defined p o p u latio n s o f phage genom es increasing b y sy n th esis a n d decreasing b y m a tu ra tio n b u t n u m b erin g p erh ap s several h u n d re d p artic le s w ith in each host cell ra n d o m ly infected w ith a few phages o f each kind.
In an early w ork w ith th e n o n -q u a n tita tiv e antig en ic m arkers, th e re w as a rough p ro p o rtio n a lity betw een D N A co n c en tratio n a n d th e nu m b ers o f sub-populations o f b a c te ria in w hich tra n s fo rm a n t cells w ere d e te c te d (M cC arthy e t al.1946, R av in 1954, S to ck er e t al. 1953). W hen individual tr a n s form ed cells could be co u n ted b y th e use o f th e q u a n tita tiv e selective tra its , it w as show n t h a t transformation yield w as sim ilarly re la te d to D N A con
c e n tra tio n (H otch k iss 1951, 1957). In th e useful cases o f s tre p to m y c in resis
ta n c e tra n sfo rm a tio n o f b o th Pneumococcus (H otchkiss 1957) a n d Hem o
philus influenzae (A lexander e t al. 1954), it w as show n t h a t th e n u m b e r of tran sfo rm ed cells w as p ro p o rtio n al to D N A co n cen tratio n o ver a lOOOfold range. I t w as concluded from such w ork t h a t a single p artic le o f D N A re a c t
ing w ith a cell is sufficient to brin g a b o u t a single tra n sfo rm a tio n for th e 24
m ark e rs w hich i t contain s. T he fa c t t h a t u n lin k ed m ark ers w ere co -tran sfo rm - ed a t a freq u en cy w hich is a p p ro x im a te ly th e p ro d u c t o f th e in d iv id u a l m a rk e r frequencies (H otch k iss 1951, M arm ur a n d H o tch k iss 1960) ind icates t h a t p ra c tic a lly all th e cells are equ ally tran sfo rm ab le. T his rela tio n sh ip w as show n b y G oodgal (1961) to a p p ly o ver a w ide c o n c e n tra tio n range.
C o -tran sfo rm atio n o f linked m arkers, on th e o th e r han d , rem ains a lin ear fu n ctio n o f co n c en tratio n in c o n s ta n t p ro p o rtio n to single tra n sfo rm a tio n s (M arm ur an d H otch k iss 1960, B alassa an d P ré v o st 1962, G oodgal 1961).
T hus, one m ay a p p ro x im a te ly ch a rac te rize a linkage re la tio n b y th e ra tio o f th e frequencies o f th e linked g roup to one o f its c o n stitu e n t m arkers.
W e m ay now re w rite th e expression given above as
w ith corresponding tw o -step tra n sfo rm a tio n s w hen DN A se is also presen t- I n a re cen t w ork w ith D r J o a n K e n t, we h av e been able to m easure t r a n s fo rm atio n yields a t a c o n sta n t D N A co n c en tratio n as a fu n c tio n o f d u ra tio n o f exposure tim e o f cells to DNA. T his ap p ro ach ta k e s th e a d v a n ta g e o f th e uniform tim e-course t h a t can be observed for th e reactio n betw een cells a n d D N A a t 30° (Fox a n d H otchkiss 1957), a te m p e ra tu re below t h a t a t w hich th e cells are n o rm ally grow n. T h e exposure is controlled b y ad d in g th e D N A a t a know n tim e an d te rm in a tin g c o n ta c t by ad d in g a t chosen tim es p a n c re a tic deoxyribonuclease to b re ak dow n th e u n u sed DN A .
L inear ra te s o f e n try are found for in d iv id u a l m arkers, a n d th ese ra te s are c h a rac te ristic o f th e m a rk e r (K e n t a n d H o tch k iss 1964). A t a given c o n c e n tra tio n o f D N A b ea rin g several m arkers, th e re la tiv e effectiveness a n d co n c en tratio n o f d ifferent m ark ers can in th is w ay be v ery a c cu ra te ly d eterm in ed . F ig u re 1 illu stra te s t h a t lin e a r ra te s o f e n try are o b ta in e d also for linked m arkers, a lth o u g h th e low est ra te s show n are p e rh a p s 3 p e r cent o f th e ra te s for single m arkers. T he re la tiv e slopes for th e linked an d single m ark e rs in a D N A solution give a m easure o f th e co -tran sfo rm a tio n effici
ency or linkage o f a m a rk e r p air, fa r m ore reliable an d ch a ra c te ristic of th e linkage g roup th a n w hen a single tim e p o in t is used.
In th is w ay, it w as possible to show t h a t from a linkage g ro u p o f th re e m arkers, all th re e , o r a n y p air, including th e tw o outside m ark e rs can be ta k e n up as a re su lt o f c o n ta c t w ith single D N A particles. An exam ple of th is la st case is th e in tro d u c tio n o f m ark ers S a n d d from g roup Sad (K e n t e t al. 1963). In all th e cases in w hich th is 1-3 in co rp o ratio n occurred, it proceeded lin early w ith tim e, a lth o u g h a t a v ery low ra te . T herefore, it seem s to be d e m o n stra te d t h a t a single D N A p artic le e n te rin g a re cip ien t cell m ay d o n a te to th e la tte r tw o se p a ra te p o rtio n s o f itself in a re co m b in a
tio n e v e n t in w hich four b re ak ag e -fu sio n ev e n ts h av e occurred. A v ery in te re stin g finding is t h a t th is m ore com plex k in d o f recom b in atio n v irtu a lly d isap p ea rs w hen th e tra n sfo rm a tio n is ca rried o u t a t th e ‘n o rm a l’ grow th te m p e ra tu re o f 37° (K e n t e t al. 1963).
cells(w ild) + D N A X - f D X A V g iv es T nn d T y Tx ~\~ l^NAy
Ty + D N A X while in th e case o f th e linked p a ir
cells(w ild) + I)N Akz g iv es T y Z + T y +
T hus, we m ay conclude t h a t a single D N A s tra n d is able to tra n s fe r n o n -a d ja ce n t m ark ers in a tra n s fo rm a tio n ev e n t; th is is one of th e w ay s D N A m ay b eh a v e .There m ay, how ever, be re stric tin g influences, fa v o red p erh ap s by evolution, w hich r e d uce th e e x te n t o f such com plex ev e n ts unless th e D N A p airin g c o n ta c t is m a in ta in e d a b n o rm a lly long b y unphysiological con d i
tions. As m entioned, th is ty p e o f e v e n t seem s to occur in p o p u latio n s o f grow ing phages, b u t it is d ifficult in t h a t case to d e te rm in e th e n u m b e r o f p a r t icles in volved a n d w h e th e r th e tru e n u m b er o f sw itch ev e n ts is th e sam e as t h a t in ferred. W hen tw o m ark ers com ing from s e p a ra te donors (m ixed DNA) are in co r
p o ra te d , th e tra n s fe r ra te is alw ays q u a d ra tic , show ing t h a t e x a c tly tw o D N A p articles are needed to p ro d uce th e p a ire d tra n s fo r
m ation. T his is also tru e for u n lin k ed m ark ers com ing from th e sam e d o n o r (K e n t an d H otch k iss 1964) (see Fig. 2) an d for p reviously linked m ark ers a f te r subcritical h ea t in activ atio n (localized d ep u rin a tio n ) (K e n t e t al. 1963).
In su m m ary , th ese new k in etic c rite ria for tra n sfo rm a tio n ra te s give stro n g in d icatio n s t h a t th e n u m b e r o f D N A m olecules involved in p a rtic u la r tra n sfo rm a tio n s o f in d iv id u a l recip ien t cells are as follows.
Transform ations effected by single D N A molecules: (1) single m arkers, (2) linked m ark e rs from single donors, (3) p a irs o f m ark e rs from linkage g ro u p even w hen in te rm e d ia te m ark ers are n o t in co rp o rated .
Transform ations requiring two D N A molecules: (1) u n lin k e d m ark e rs from single donors, (2) m ark e rs o f a lin k ag e g ro u p w hen deriv ed from d iffe re n t d o n o r stra in s (in m ixed D N A o f th e tw o stra in s, (3) a sm all p ro p o rtio n o f linked m ark e rs (p ro p o rtio n a l to single tra n s fo rm a tio n frequency) m ay e n te r by successive tra n sfo rm a tio n s by tw o m olecules from th e sam e s tra in , (negligible, ex c e p t w hen co n c en tratio n o f D N A is high or tim e o f e x p o
0 4 8 12 15 20 25 30 mm duration o f exposure to DNA
Fi g. 1 .—R a t e o f a c c u m u la tio n o f specified t r a n s fo r m a n ts a s a fu n c tio n o f e x p o su re o f d cells to S a D N A . T h e tr ip le tr a n s f o r m a n t S a + ( t h a t is S a d + o r S a D ) is p ro d u c e d a s fr e q u e n tly a s th e d o u b le S a d . T h e class S d + (o r S I ) ) in c lu d e s tw o ty p e s ; b y s u b tr a c tin g th e v a lu e s fo r th e d ire c tly m e a s u re d classes, S d + m in u s S a +, on e o b ta in s th e ‘d o u b le s w itc h re c o m b in a n ts ’, S a +d + (or S A D ) . (S = s tr e p to m y c in re s is ta n c e , a a n d d = d iffe re n t d is tin g u is h a b le su lfo n a m id e re s is
ta n c e fa c to rs)
26
sure is p rolonged), (4) linked m ark ers from th e sam e donor, w hen localized d e p u rin a tio n or o th e r d am ag e has in a c tiv a te d m an y o f th e in d iv id u a l m arkers.
One corollary o f th is finding is th a t, w hile p airin g o f D N A m olecules gives increased o p p o rtu n ity for linked m ark ers to be in co rp o rated , it does n o t s u b sta n tia lly decrease th e p ro b a b ility for a second D N A p a rtic le to d o n a te m ark ers to th e sam e or d ifferent p a r ts o f th e sam e cell genom e.
i t has been p o in te d o u t t h a t m ark ers ap p e arin g to be u n lin k ed m ight really be p a r t of th e sam e s tru c tu re b u t m erely so fa r a p a r t t h a t th e large s tru c tu re w as unab le to be in c o rp o ra te d a t once (H otchkiss 1958). B alassa a n d P ré v o s t h ave m ad e m ore d e ta ile d calculations o f such p ro b a b ilities by assum ing a size-influenced in co rp o ratio n (B alassa a n d P ré v o s t 1962) in s te a d o f a m ax im u m size-lim ited one as w as done e a rlier (H o tc h k iss 1958).
I t now seem s t h a t th e k in etic c rite ria described ab o v e are able to show t h a t m ost, if n o t all, of th e ‘u n lin k e d ’ double tra n s fo rm a tio n s we h av e o b served do indeed involve m ark e rs o f se p a ra te m olecules.
T his ty p e o f analysis is p ro v in g to be o f value in th e s tu d y o f a n o th er new fe a tu re o f tra n sfo rm a tio n , involving apparently h eterozygotic regions o f DNA. T his w ork is largely ca rried o u t in collab o ratio n w ith Dr Mihoko Abe an d is now in p re p a ra tio n for d etailed publication. A sulfonam ide resistance m arker, c, discov
ered som e tim e ago in w ork w ith Miss E v a n s (H otchkiss a n d E v a n s 1958), h a d been observed to be ‘u n s ta b le ’ w hen stra in s c o n tain in g it were re p eated ly passed for m any generatio n s. W hen reex am in ed b y D r Abe, it was found t h a t a p p ro x i
m ately one p rogeny cell from each five or six divisions (1 in 32 to 64 cells) lo st its sulfonam ide resistance.
Since th e sensitive cells p ro d uced w ere stab le a n d in d istin g u ish ab le genotypical- lyor p h en o ty p ica llv , from wild ty p e ( + ) cells, th e process ap p e ars to be as show n sch em atically in Fig.
3. T he u n sta b le c stra in s arising e ith e r by m u ta tio n or by tra n sfo rm a tio n give a m ixed p o p u latio n . T ak in g a d v a n ta g e o f specific sulfo
nam id e-related drugs, th e population m ay be selected for its p h e n o ty p ic tr a its into tw o kinds. Selected in
F i e . 2. — R a te o f a c c u m u la tio n o f u n lin k e d m a r k e rs S a n d K (s tre p to m y c in a n d m icro co ccin re sista n c e ) a t d iffe re n t D N A c o n c e n tra tio n s . A ll th e c u rv e s a re e ss e n tia lly q u a d r a tic ; t h a t is, (tra n s f o rm a n ts ) 1/2
is a lin e a r fu n c tio n o f d u r a tio n o f e x p o su re 27
sulfanilam ide (SA) th e p o p u latio n becom es ‘p u re ’ r cells, w hich, how ever, soon lead to p ro d u ctio n o f w ild ( 4 ) cells again. S elected in para-n itro b en zo ic acid (NO B), selection is a g a in st c cells, a n d a w ild ty p e p o p u latio n results w hich shows no a b ility to tu r n back in to , or recom bine to give, re sista n t cells again.
growth
© ©
©©
Fig. 3 .—S ch em e o f g ro w th , se g re g a tio n a n d selectio n o f r su lfo n a m id e r e s is ta n t cells
T his could be view ed as a m u ta tio n w ith a high reversion ra te , b u t it a p p e a re d like a segregation o f a c m a rk e r aw ay from som e h eterozygotic form w hich co n tain s b o th th e c a n d its w ild -ty p e allele C. T his h eterozygotic form could be an o v erlap p in g s tru c tu re o f som e so rt, or som e episom e-
@ ©
Fig. 4 . —S ch em e sh o w in g se g re g a tio n o f c, a n d tw o k in d s o f cd cells
b ea rin g form as suggested b y th e v ery in tere stin g re c e n t w ork o f D r W a- ta n a b e , re p o rte d a t th is S ym posium a n d elsew here (W atan ab e 1963).
T h e com bination of m a rk e r c w ith re sis ta n t stra in s a a n d b th re w no light upon th e m a tte r, fo r ac (or be) stra in s seg reg ated o r re v e rte d to a (or b) stra in s in th e sam e w ay as w hen a a n d b were n o t p resen t.
H ow ever, w hen we tra n sfe rre d m a rk e r c to a a? stra in , a closely linked p air, rd, w as produced. Now, if c m erely m u ta te s back in to C, th e n cd s tra in s should change b ack only in to d s tra in s (Cd). B u t, if a hetero zy g o tic s tru c tu re c(C)
w ere th e basis o f th e c in s ta b ility , one m ig h t ex p e c t t h a t th e D N A ca rry in g th e c m a rk e r w ould som etim es in tro d u ce its D n eighbor in to th e cd strain .
T his la tte r is w h a t a p p a re n tly happens. W hen cd s tra in s are ex am in ed by selection in th e d ru g p ara-n itro b en z o ic acid, tw o ty p e s are found.
One ty p e can prod u ce sta b le d strain s, w hile th e o th e r gives b o th stab le d a n d ( + ) ty p es. T h e recovery o f ( + ) cells [CD] m eans t h a t m a rk e r D has also been in tro d u c e d p re su m a b ly from th e c d o n o r (cD). F u rth e rm o re , w hen th e c m a rk e r leaves th e s tra in , it ap p e ars som etim es to ta k e w ith it th e d, som etim es th e D, m arker.
T he p h e n o ty p e s fo u n d are sum m arized in Fig. 4. T hese re su lts are e x p la in able if th e c stra in carries m a rk e r C unexpressed, a n d th e cd s tra in s c a rry
Fig. 5 .—O v e rla p m odel
in th e one case C a n d in th e o th e r CD, in an unex p ressed s ta te . So we e x am in ed th e D N A o f th ese stra in s for la te n t m arkers, n o t expressed in th e stra in itself, b u t tra n sfe ra b le to recip ien ts o f th e su itab le gen o ty p e. I n T ab le I are show n th e results. I t is a p p a re n t t h a t th e su sp ected la te n t m ark ers are all p resen t. B y using th e so p h istic ated k in etic te s t for th e linkage m en tio n e d above, i t is fo u n d t h a t th e p airs cd a n d C D are linked to each oth er, while c is n o t linked to D. B y an occasional reco m b in atio n , th e Cd ty p e is form ed a n d segregates o u t as an a p p a re n tly sta b le p ro d u c t. I f D is n o t p re sent, th is is th e only d e te c te d re a rra n g e m e n t; if D is p re sen t, th e CD ty p e (wild ty p e ) segregates o u t even m ore freq u en tly .
T A B L E i
T r a n s fo r m in g agents rec\
P h e n o ty p e In f e ri•ed g e n o ty p e
c
C
cd
c
c d
cd* C D
c d
S rd * C D
S c d
red fro m ed str a in s
T ra n s fo rm in g a g e n ts recognized*
c cd, c, d cd, C D , c, d, D
S rd , cd, S c, C D (an d S , c, d, D )
T hese findings in d icate t h a t m a rk e r c becom es m ore in tim a te ly associated w ith p re ex istin g b a c te ria l m ark ers (d ) th a n it w ould be if it w ere b orne on a free episom al p article. Since v irtu a lly all o f it behaves th is w ay all o f th e
29
* N o te : m a r k e r C c a n n o t be re c o g n iz e d a lo n e in tr a n s fo r m a tio n
tim e , an episom e seem s to be excluded. F u r th e r m ore, th e C D or C m ark ers so ca rried are n o t fu n c tio n al. T his w as learn ed from th e absence o f a folic acid s y n th e ta s e hav in g th e know n d ru g sen sitiv ity corresponding to these m ark e rs (u n til re co m b i
n a tio n occurs). T he D N A b ea rin g th ese m ark ers does n o t seem to be in a n y w ay p h y sically a b norm al. We will discuss th ese a n d o th e r studies on th ese stra in s in m ore d e ta il in la te r p u b lic a tions.
An a d e q u a te s tru c tu ra l m odel for th e h e te ro z y gote cd (CD) should, th e re fore, show th e following p ro p erties: (1) tw o regions hav in g d u p lic a te d genes ca rried b y (2) d o u b le-stra n d ed DNA in b o th regions, w ith (3) p ossibility for reco m b in atio n betw een th e tw o regions, b u t (4) w ith no rm al D N A replication usually repro d u cin g b o th regions a n d heterozygosity, how ever (5) th ese regions to be asy m m etric in th e ir re la tio n s to th e re s t o f th e genom e so t h a t (6) c re a tio n o f gene p ro d u c t is n o t eq u al for b o th , therefore, p re su m a b ly , sites Fi g. (i.— M odel o f p n e u m o c o c c a l ch ro m o so m e t h a t
w o u ld a c c o u n t fo r s t a b il it y a n d b e h a v io r o f cd (CD) s tr a in s , i ß = p o ssib le site s o f in itia tio n o f r e p li
c a tio n a n d o f f o r m a tio n o f m e sse n g e r, C D , cd m a r k e rs in h e te ro z y g o tic reg io n
e
Fi g. 7 .— M odel o f F ig . 6 a f t e r co m m e n c e m e n t o f p o la riz e d s in g le s tra n d (K o rn b e rg ) re p lic a tio n . N o te t h a t a t cG th e re s tin g e n d s s ta b iliz e e a c h o th e r. N o te also that, h e te ro z y g o s ity a t E w o u ld se g re g a te a t th is n e x t
d iv isio n , w h ile h e te ro z y g o s ity a t cd is re p lic a te d
o f in itia tio n o f R N A tra n s c rip tio n (in itia to r, o p erato r, p u n c tu a tio n ) being a b s e n t o r n o t fu n c tio n a l in one o f th e tw o regions.
T h e first th re e o f th ese p ro p e rtie s hold for a sim ple ov erlap m odel (Fig. 5). T he la s t th re e p ro p e rtie s seem m ore s a tisfa c to rily a c c o m o d a t
ed b y a m odel w hich we h av e been considering for som e tim e, th e one p re se n te d in F ig. 6.
D oubtless, o th e r m odels also w ould serve in v a r y ing degrees, b u t th is one has th e a d d itio n a l advan ta g e o f giving a ch ro m o som e t h a t w ould a p p e a r
3 0
to be circu lar in linkage te s ts (an d in electro n m icrographs). I t w ould allow s ta b ility o f th e loose ends also d u rin g a ‘K o rn b e rg re p lic a tio n ’ o f single stra n d s (K o rn b erg 1961), s ta rtin g from opposite ends, as suggested in F ig. 7.
I f re p lic atio n of b o th stra n d s a c tu a lly proceeded synch ro n o u sly from one end, as suggested b y re c e n t a u to ra d io g ra p h s o f d ividing Escherichia coli cells (C airns 1963), th e n th e ends could still hold each other.
M ore im p o rta n t, th e m odel gives asy m m etric positions to th e re p e a te d regions cd a n d CD. W e m ay th in k o f cd as being asso ciated w ith a site o f in itia tio n o f tra n sc rip tio n (‘in itia to r’ or ‘o p e ra to r’), p erh ap s to th e rig h t of d in Fig. 7, w hich is a b s e n t e n tire ly a t th e CD end o f th e s tra n d (a t B).
T his w ould n o t affect th e re p e a te d com plete rep lication o f th e D N A including th e overlap, b u t w ould p re v e n t fo rm atio n o f an y gene p ro d u c t (‘m essenger’) from th e (non-functioning) CD p a rt.
A lthough we w ere u n aw are o f it, th is m odel w ith te rm in a l re d u n d a n c y h a d been suggested for ph ag e h eterozygotes som e tim e before b y S treisinger a n d discussed inform ally a t vario u s tim es, being finally su m m arized in an excellent p re se n ta tio n by D r S ta h l a t th is Sym posium . W e h av e n o t begun to m ake as m any ex p e rim en tal te s ts o f th e m odel as he has ou tlin ed , b u t we m ay p e rh a p s be considered to have offered a n o th e r n o t to o rigorous ap p lica tio n o f th e m odel a n d a rg u m e n ts co n sisten t w ith it. In ad d itio n , it is o f in te re st t h a t we h ad a som ew hat different reason for considering th e m odel: th e b alan ce o f D N A rep licatio n , a n d th e im b alan ce o f D N A fu n ctio n , in th e tw o p a rts.
I n su m m ary , we can now s ta te w ith som e confidence t h a t recom bination in tra n sfo rm a tio n occurs b etw een th e b a c te ria l genom e and one o r m ore p a r ts o f a single incom ing D N A m olecule. A second DNA m olecule is n o t excluded from ac tin g on th e sam e recip ien t cell su b seq u e n tly , b u t th is p ro cess can be d istin g u ish ed from th e o th e r b y k in etic crite ria. A t le a s t one tra n sfo rm a tio n in tro d u ce s a d u p licate m a rk e r o r linked p a ir o f m ark e rs to produce a h eterozygotic s tru c tu re . T his s tru c tu re is so c o n s titu te d t h a t only one p a r t o f i t is p h en o ty p ica lly expressed in cell function. I t is rep lic ated in ta c t, how ever, ex c ep t t h a t an occasional reco m b in atio n occurs, w ith th e p ro d u c tio n o f a sta b le seg reg an t h av in g a d ifferent a sso rtm e n t o f th e m a rk ers in th e fu n c tio n a l sta te .
R E F E R E N C E S
A le x a n d e r, H . E ., L e id y , G . a n d H a h n , E . (1954) ,7. exp. M e d . 97, 797 A v e ry , O. T ., M acL eo d , C. T . a n d M c C a rth y , M . ( 1944) ,7. exp. M e d . 79, 137 B a la ss a , G . a n d P ré v o s t, G . (19G2) J . theor. B iol. 3, 315
C airn s, J . (1963) J . molec. B io l. 0, 208
C hase, M. a n d D o e rm a n n , A . H . (1958) G enetics 43, 332 F o x , M. S. a n d H o tc h k is s , R . D . (1957) N a tu re (L o n d .) 179, 61 G oodgal, S. H . (1961) .7. gen. P h y sio l. 45, 205
H o tc h k is s , R . D . (1951) Gold S p r . H arh. S y m p . q uant. B io l. 16, 457
H o tc h k is s , R . D . (1957) in C hem ical B a s is o f H ered ity, ed . W . M c E lro y a n d B . G lass, J o h n s H o p k in s U n iv . P re s s, B a ltim o re
H o tc h k is s , R . D . (1958) T h e b io lo g ical re p lic a tio n o f m a e ro m o lecu les, in S y m p . Soc.
E x p . Biol. X I I , 49
H o tc h k is s , R . D . a n d E v a n s , A . H . (1958) Cold Spr. Harb. Symp. quant. Biol. 23,58 K e n t, J . L . a n d H o tc h k is s , R . D . (1964) in p r e p a r a tio n fo r p u b lic a tio n
K e n t, J . L ., R o g e r, M . a n d H o tc h k is s , R . D . (1963) Proc. nut. Acad. Sei. ( Wash.) 50, 717 K o rn b e rg , A . (1961) E n z y m a tic S y n th e s is o f U N A , Ciha Lectures in Microbial Bio
chemistry,J o h n W iley a n d Sons, N ew Y o rk L u ria , S. E . (1962) Ann. Rev. Microbiol. IG, 205
M a rm u r, J . a n d H o tc h k is s , R . D . (1960) Proc. nat. Acad. Sei. (W ash.) 40,55
M c C a rth y , M .,T a y lo r, H . E . a n d A v e ry , O. T . (1946) Cold Spr. Harb. Symp. quant. Biol.
11, 117
R a v in , A. W . (1954) Exp. Cell Res. 7,58
S te in b e rg , C. M . a n d E d g a r, R . S. (1962) Genetics 47, 187
S to c k e r, B . A. !)., K ra u s s , M. R . a n d M acL eo d , C. T . (1952) ./. Path. Bart. GO,220 W a ta n a b e , T . (1962) Bact. Rev. 27,87
S y m p . B io l. H u n g . 6, 33-42 (1965) K I N E T I C S O F P R O V O K E D C O M P E T E N C E I N S T R E P T O C O C C A L C U L T U R E S A N D I T S
S P E C I F I C I T Y B y
Ro m a n Pa k u l a
INSTITUTE OF BIOCHEMISTRY AN D BIOPHYSICS OF THE POLISH ACADEMY OF SCIENCES, WARSAW, POLAND
T ra n sfo rm ab ility o f b a c te ria is so fa r lim ited to a sm all n u m b e r o f species a n d w ith in th ese species only to som e strain s. T h e a b ility to undergo tr a n s fo rm a tio n is defined as com petence b u t th e n a tu re o f th is phenom enon is n o t well understood.
A fte r a careful s tu d y o f com petence in strep to co ccal cu ltu res, we found t h a t th e ap p e ara n ce o f com petence resem bles in m an y resp ects th e kin etics o f ac cu m u latio n of enzym es a n d to x in s, such as strep to d o rn a se, s tr e p to kinase, hialu ro n id ase a n d stre p to ly sin 0 in cu ltu re s of th ese b ac te ria. We assum ed, therefore, t h a t streptococci do n o t develop com petence u n til a su b stan ce p ro d u c ed by th e cells them selves, w hich is capable o f co n v ertin g n o n -c o m p e ten t cells in to co m p ete n t ones, is accu m u lated . T his w as found to be tru e a n d a fa cto r prov o k in g com petence w as discovered in sterile su p e r
n a ta n ts o f tran sfo rm ab le a n d co m p ete n t strep to co ccal cultures.
B efore th e c h a ra c te riz a tio n o f th is factor, it has to be m en tio n ed th a t, u n d e r n a tu ra l conditions, com petence o f streptococci develops only in m edia su p p lem en ted w ith e ith e r serum or album in. I n th e absence o f th ese s u b sta n c es tra n sfo rm a tio n does n o t occur or, if p resen t, is ex tre m ely low. T h e onset o f com petence, th e period o f its d u ra tio n a n d th e tim e o f its d isap p earan ce are s tric tly d e p e n d e n t on th e size o f th e in itia l inoculum in th e culture. T he sm aller th e inoculum th e la te r th e a p p e ara n ce o f com petence. T hus, th e conversion to com petence o f th e n o n -c o m p e ten t b a c te ria w ith th e use o f th e pro v o k in g fa cto r can be d e m o n stra te d in tw o different conditions: (1) in cu ltu re s grow n in th e absence o f serum or album in, (2) in cu ltu re s grow n in m edia co n tain in g serum or album in. As for th e second condition, th e c u ltu re is s ta rte d w ith a sm all a m o u n t o f initial inoculum an d th e cells are exposed to th e actio n o f th e prov o k in g fa cto r fo r 2 h before n a tu ra l com pe
ten ce can be expected.
In all experim ents, ex c ep t one, th e first m eth o d o f com petence p ro v o c a
tio n was applied. T h e m ark e r used in th e tra n sfo rm a tio n ex p e rim en ts was strep to m y cin -resista n ce.T h e recipients an d donors w ere g roup H h aem olytic streptococci a n d stra in s o f Streptococcus sanguis, T y p es I, I I a n d I / I I reg ard ed to be closely re la te d to g roup H streptococci. S tra in s of S . sanguis, T y p e I / I I sh are th e C -polysaccharide ch a rac te ristic s o f g roup H streptococci.
W ith th e fa c to r prov o k in g com petence available, th e follow ing p ro ced u re o f tra n sfo rm a tio n was developed. B a c te ria grow n in th e absence o f serum or alb u m in for 2 h w ere exposed to th e ac tio n o f th e p rovoking fa cto r. A fte r 30 m in, th e cells w ere sp u n down, th e s u p e rn a ta n t co n tain in g th e prov o k in g fa c to r discarded, th e cells suspended in fresh m edium , exposed to th e actio n
3 Symp. Biol. Hung. 6 33
