RECOMBINATION IN BACT.COLE K 12: UNE~DIRECTIONAL
TRANSVER OF GENETIC MeTERLAL.

- Phve years ago Lederberg and Tatum! demonstrated that. when
teo mutant strains of Bect.cold K 12 heving double and complementary
grewth factor reculrewents were slated together in « basal synthetic
medium, colonies appeared in the retio of one to every 106 or 107
cells seeded, elthough colonies did not crise by back mutction shen
the sutents were slated seperately. These colonies were
protetrophic end similar to the wild type in thelr independence of
ecued growth factors, Not only did they breed true, but it was
also shown thet if cach mutant was further denoted by complenentary
differences in choracters not seleeted by the besel medium (sueh cs
fermentative canselty for verlous carbohydrates and sensitivity or
resistence to spceifie wirus infeetion), the pattern of these
cheracters in protetroph cultures wes usually different from thet
in either mutent. It was clear, therefore, thet prototroph
eolonles crose from the transfer of genetic material between the
matants enc not from complementary exchange of growth factors.
meunococcus or the "directed mutation"
of similar mecheniss in Bect.sold, growth of one K 12 sutent in
eulture filtrcetes of the other fsiledé to stimulate srototroph

Unlike type trensfornation in Pheu

 

formition, This, in conjunction with recent evidence for the
eceasionsl oceurrence of diploid heterozygous prototrophs*, strongly

support the wlew thet genetic recombination 1s mediated by the

 
classicel mechenism of sexual conjug:tion, Aéttexpts to

reproduce the phenomenon by similar techni:ves in other strains
of Bact. cold und in Sulm.tyobtuuriym hewe failed, though Cevelil
end Heslot succseded in out-crossing K 12 mutents with « naturally
nutritionelly exacting struin of Bect.agidd Lucticl.

In the following experiments, Best. cold K 12 mutants 50-161,
revuiring biotin and nethionine, end ¥ 677, requiring leucine,
threonine ond cneurin, were exployed. The aneurin re uirenent
of 677 wes neutrelised by the addition of this substence to the
besel medium. An ettecpt was made to investigate the dy

 

of reconbination by sprecding « mixture of 55-161 and s streptomycin-
roaistent sutent of F 677 (" 677/57) on a series of plates of
begal nedium, enti then adding streptomycin to each plate in turn
at ¢ifforent intervals cfter secding. It sus enticipeted thet
the streptomycin would recidly block the recoubination mechan sn
by insetiveting 58-161, while alloving rcesistunt prototroph cells
formed prior to its sddition to develop into colonies. It was
found in orsetice thet the number of srotetroyh colonies did not
differ greatly whether atreotomyein was incorporated in the basal
medium before pluting, or wcs added up to four hours Luter.

Since similar results cere obtained «hen the outents were mixed
for the first tice during pleting, the ocevrrence of recombins tion
in wixtures before eontect with streptemyein was excluded. Rither

protetrophs arose before the setion of streptomycin on the

 
sensitive mutant bocane effective, or else those functions of the
cell affeeted by streptomycin were not involved in reconbinetion,

Logerithete phose broth cultures of 56-161 were treated vith
either 1000 or 2000 pg./ml. straptomycin, under conditions
optinel for beetericidal effect, for from 4 to 18 hours, After
weshing and concentration in seline, volumes of suspension
turbimstrically ecuivelent to from 1.5 £ 109 to 4.0 x 108 eelis
(i.e. double the inoculum used in recombinution experiments)
fre-uently proved sterile and rarely yielded more then 100 colonies
on nutriont agar after 72 hours ct 3%7°C. Nevertheless such
susyensions (5@-161/5*) invariably etinulcted prototroph formation
when mixed with % 677/s¥ on b cal sedium contelning 200 pss /nl.
streptomycin. Tho rcecombinetion rete, however, was usuelly
appreeiebly lesa, end showed much more marked variation between
experiments then when viable, untreated 58-161 suspensions were
used. “henever streptomycin treatzent failed to produce sterility,
control reconstruction experiments shoved thet about 1000 times is
many untrested 58-161 cells as those which ned survived tresetmont
failed to yield protetrosh colonies vhen nixed with % 677/s*
suspension under similar conditions.

Wixtures of strectonycin-trected ¥ 677 (F 677/s*) and 58—161/s",
on the other rand, izmveriebly felled to produce prototrophs cl&hough
comperable recoubination rates were given by the aixtures
(7 677 3g + 58-1412) and and (58~161/83+ 7677). In these experinents

 
streptomycin wes not incorporated in the besel medium since
previous anolysia of proven prototroshs hed shown thet ibout

95 cor cent carried the s° or 3° cherecter of ¥ 677. The
clearcut distinction between 58-161/s* ena " 677/s* in <bility
to pertlelpcte in recombinction was shown to be independant both
of the presence of the S’ cherecter in the complemontury wutent
and of the basal medinw enviroment. Thus (58-161/st + 7 677)
produced prototronn colonies on every ocension whether cultured
directly oh bose medium or initielly on nutrient cger. The
mixture (v 677/s* + 58161), however, fulled to do so repextedly
on besel medium and, in « single experimant, when seeded on
mitrient acar.

These findings require « revision of the current concept of
reconbinetion, It is unlizely that sensitive cells which heave
been acted unon for 18 hours by very high coneentrations of
atrectomyein are still oble to perticipete in conjugetion in
the continued oresence of the drug. Moreover, if conjugction
under these conditions was possible for G1 61/st it might also
revsonsbly be .ssumed for 7 677/6" wnten 15 ecually sensitive to
streptomycin, Yet suspensions of the latter are inuctive to
reconbinc tion, It is simsler to suppose thet recombination is
mediated by genetic clenents which cre extruded by the viable
cell but remain cdherent to the cell well and which, like viruses,

are unaffeeted by streptomycin, Thus the call could be regarded

 
merely as a pessive carrier for its genctic clexents after their
expulsion, o function ehieh could be performed esually well by
decd or by living cells. The incompetence of ¥ 677/3* becomes
intelligible only if we suppose that the role of 7 677 is the
vital one of cecepting genes end incorsorsting then inte ite
genotic structure, It 1s possible that © 677 1s also « gene
donator but, if this is so, then 56-161 is inherently incepuble
of seeepting then.

It hes recently been shown thet symbioetie bacterial viruses
ean transfer hereditary chracters to heterologous cells which
they infect®. It is ulso known that K 12 carries a symbtotie
Virus which is liberated from the cells in uppreelable mabers
only efter UV irreciction. This virus cen infeet and lyse
meture culturos of the K 12 mutant "5" elthough young cultures
of "5? display the peevliar property of rezlseting lysia while
still sbsorbing the virus’. ‘The existence of « latent virus
in 56-161 has been unmesked by K~radiation (personsl observstion).
It seem possible thet suck a virus might be the agent of genetle
trensfer, Phe apserent restriction of the recombinstion
phenomenon to one strain of Bact.coli, its relatively low rate
end the inability of culture filtrates to Induce it, the
stimulction of recombination by doses of OV Light too low to
increase the mutetion rate®, the competence of streptonycin~

"sterilised" cultures of 58161 end the one-way transfer of the

 
bn

genetic agent ore cll in conformity with such - concept. “ork

is now in progress to detercine whether filtrates of SE-161,

‘rou which l.tent virus has been released by UV irradiation,

can induce pretotrosh foruetion in ¥ 677.

Depurtment of Bacterlology
Postgraducte Medical School of Lonion.

YW. Heyes,

Gth November, 1951.

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Lederberg, J. and T:tum, B.L., (Mature), 158, 558 (1946).
Lederberg, Jo, Proc Natl» icud-SeieUrde,» 35, 178 (1949).
Cavalli, Leb. ond Heslot, H., Nature, 164, 1057 (1949).
Folix, 4. and Anderson, £.5., Nature, 167, 603, (1951).
Weigle, J.J. and Delbrtick, M., J. Baet., 62, 301(1952),

Hees, Fe, “yss, 0. and Stone, .5., PEG Mists hts Seb niie»
> a» 4740)»