JOURNAL
OF
BACTERIOLOGY,
No .
1973,
p.
527-530
Copy igh
i
1973
Ame ican
Socie y
o
Mic obiology
Vol.
116,
No.
2
P in ed
in
U.S.A.
Ni osoguanidine
Assay
o
Episomal
In eg a ion
in
Esche ichia
coli
CRISTINA
P.
LLOVERES
AND
E.
CERDA-OLMEDO
Depa amen o
de
Gene ica,
Facul ad
de
Ciencias,
Uni e sidad
de
Se illa,
Spain
Recei ed
o
publica ion
10
July
1973
Mu a ions
a ec ing
u iliza ion
o
lac ose
and
esis ance
o
he
male-speci ic
phages
l,
2,
and
Q8
end
o
occu
simul aneously
mo e
o en
han
expec ed
by
chance
in
H
s ains
whose
o igin
o
ans e
is
close
o
he
genes
o
lac ose
u iliza ion,
bu
no
in
F+
s ains.
S ains
de i ed
om
he
H ,
bu
exhibi ing
poo
abili y
o
ans e
ea ly
ch omosomal
genes,
may
o
may
no
show
his
comu a ion
phenomenon.
These
esul s
suppo
he
concep
ha
he
F
ac o
is
in eg a ed
in o
he
H
ch omosome
du ing
ege a i e
g ow h,
bu
is
au onomous
in
he
F+
s ains
and
could
se e
as
an
assay
o
episomal
localiza ion.
N-me hyl-N'-ni o-N-ni osoguanidine
is
an
e ec i e
mu agen
in
Esche ichia,coli
(2,
11)
and
in
many
o he
o ganisms.
The
inac i a ion
o
he
F
ac o
by
ni osoguanidine,
as
we
will
abb e ia e
i s
name,
has
been
desc ibed
(13),
and
many
mu a ions
induced
by
his
agen
ha e
been
used
in
he
gene ic
analysis
o
F
unc ions
( e iew
in
e e ence
1).
Al hough
all
genes
a e
suscep ible
o
mu a ion
by
ni osoguanidine,
he
mu a ions
in
any
pa icula
cell
a e
no
andomly
dis ibu ed
o e
he
ch omosome
bu
c owded
oge he
in
small
egions,
ex ending
o e
less
han
2
min
o
he
E.
coli
ch omosome
(6),
and
placed
a
he
posi ions
occupied
by
he
eplica ion
poin s
a
he
ime
o
he
ea men
(5).
Gene ic
si es
close
han
2
min
exhibi
comu a ion,
ha
is,
hey
end
o
mu a e
simul-
aneously
mo e
o en
han
expec ed
by
chance.
Comu a ion
may
be
a
use ul
ool
in
some
p oblems
o
gene ic
mapping,
such
as
he
ecog-
ni ion
o
he
in eg a ed
and
au onomous
s a es
o
episomes.
We
ha e
used
he
F
ac o
as
a
model
sys em,
de ining
i
as
an
episome
con e -
ing
sensi i i y
o
male-speci ic
phages,
such
as
he
ibonucleic
acid
(RNA)
phages
Q,B
and
2
and
he
deoxy ibonucleic
acid
(DNA)
phage
l.
MATERIALS
AND
METHODS
Bac e ia
and
phages.
X15
is
a
p o o ophic
F+
s ain
o
Esche ichia
coli,
de i ed
om
he
F+
s ain
W1485;
X493
is
a
p o o ophic
H
s ain,
de i ed
om
X15
and
ans e ing
i s
ch omosome
in
he
o de
O-p oB-p oA-leu- h -...
-lac-F.
Bo h
we e
ecei ed
om
R.
Cu iss
(3).
DF73
is
an
F-
s ain
ca ying
he
ma ke s
s A,
hyA,
h ,
leu,
hi,
p o,
a g,
lac,
gal,
a a,
xyl,
m l,
de i ed
om
he
s ain
AB1157
and
ecei ed
om
K.
B ooks.
The
wild- ype
RNA
phages
Q
and
2
we e
ecei ed
om
A.
J.
Cla k,
and
he
wild- ype
DNA
phage
l
was
om
N.
D.
Zinde .
Cul u e
condi ions.
Bac e ia
we e
g own
a
37
C
in
is(hyd oxyme hyl)aminome hane
(T is)-sal s
mini-
mal
medium
(10),
wi h
D-glucose
a
2
g/li e
as
ca bon
and
ene gy
sou ce.
Solid
media
we e
p epa ed
in
he
same
way
wi h
25
g
o
aga
(Di co)
pe
li e .
When
app op ia e,
amino
acids
and
hymine
we e
added
a
20
gg/ml,
hiamine
a
1
zg/ml,
and
s ep omycin
a
100
ug/ml,
and
,-lac ose
was
subs i u ed
o
glucose
a
2
g/li e .
Ni osoguanidine
ea men .
Cells
om
exponen-
ially
g owing
cul u es
we e
ea ed
wi h
100
yg
o
ni osoguanidine
(Se a,
Heidelbe g,
Ge many)
pe
ml
o
T is-malea e
bu e ,
pH
7.5,
a
37
C
o
30
min
(4);
hey
we e
washed
wice
by
cen i uga ion
bo h
be o e
and
a e
he
ea men .
Tes
o
phage
esis ance.
Phage
s ocks
we e
p oduced
in
b o h-g own
E.
coli
s ain
X493.
A
d op
o
phage
s ock,
con aining
o e
109
plaque- o ming
uni s
o
he
indica ed
phage
o
phage
mix u e,
was
ex-
ended
in
a
diame ical
band
on
an
aga
pla e
wi h
a
wi e
loop.
Bac e ia
om
single
colonies
we e
s eaked
ac oss
he
phage-con aining
band
wi h
a
s e ile
oo h-
pick.
A
colony
was
conside ed
esis an
when
bac e-
ial
g ow h
was
he
same
on
bo h
sides
o
he
phage-con aining
band,
as
obse ed
a e
o e nigh
incuba ion
a
37
C.
RESULTS
New
s ains.
Two
Lac-
de i a i es
o
he
H
s ain
x493,
unable
o
u ilize
lac ose,
we e
isola ed
a e
ni osoguanidine
ea men
and
called
SE12
and
SE13.
To
his
end,
exponen-
ially-g owing
cells
o
s ain
x493
we e
ea ed
wi h
ni osoguanidine,
allowed
o
g ow
in
glu-
cose
liquid
medium
o
3
h,
and
pla ed
on
glucose
solid
medium;
indi idual
colonies
we e
hen
es ed
o
g ow h
on
lac ose
solid
medium.
The
conjuga ional
abili y
and
he
o de
o
gene
527
LLOVERES
AND
CERDA-OLMEDO
ans e
we e
checked
in
hese
s ains
by
in e -
up ed
ma ing
(9)
wi h
DF73,
and
hey
we e
ound
o
be
he
same
as
in
he
o iginal
s ain.
Two
Lac-
de i a i es
o
he
F+
s ain
X15
we e
isola ed
by
he
same
p ocedu e
as
o
he
H ,
and
called
SE20
and
SE21.
They
we e
checked
o
be
s ill
F+
by
obse ing
he
appea -
ance
o
esis ance
o
he
male-speci ic
phages
l,
2,
and
QB
a e
p olonged
incuba ion
wi h
ac idine
o ange
(7).
S ains
exhibi ing
poo
conjuga ional
abili y
we e
isola ed
om
he
Lac-
H .
Bac e ia
om
SE12
colonies
we e
spo ed
on o
pla es
co e ed
wi h
some
109
cells
o
s ain
DF73
and
con ain-
ing
adequa e
supplemen s
o
he
selec ion
o
Leu+
Th +
S +
ecombinan s
( ha
is,
con ain-
ing
s ep omycin,
hiamine,
hymine,
p oline,
and
a ginine).
In
mos
cases
a
hick
g ow h
o
ecombinan s
appea ed,
bu
some
colonies,
con-
s i u ing
abou
1%
o
he
o al,
did
no
p oduce
any
ecombinan s.
A e
eisola ion
and
e es -
ing,
ou
o
hem
we e
called
SE18,
SE19,
SE22,
and
SE23.
The
same
p ocedu e
was
applied
o
he
s ain
SE13,
and
s ains
SE15,
SE16,
and
SE17
we e
isola ed.
These
s ains
we e
all
ound
o
be
sensi i e
o
male-speci ic
phages.
Comu a ion.
The
lac
mu a ions
in
s ains
SE12,
SE13,
SE20,
and
SE21
we e
ob ained
independen ly.
The
su i al
o
hese
s ains
a e
ni osoguanidine
ea men
and
he
e-
quencies
o
Lac+
e e an s
a e
indica ed
in
Table
1,
which
gi es
he
esul s
o
ypical
expe imen s
in
which
bac e ial
samples,
aken
jus
be o e
o
jus
a e
he
ea men
wi h
ni osoguanidine,
we e
pla ed
on
glucose
and
ac ose
media.
Highe
su i al
a es
can
be
ob ained
by
dec easing
he
pH
a
he
ime
o
he
ea men
o
5.5.
I
can
be
no ed
ha
ni-
osoguanidine
usually
inc eases
he
equency
o
e e an s
mo e
han
1,000- old.
The
s ains
de i ed
om
SE12
and
SE13
beha e
like
hei
pa en s
in
hese
espec s.
When
un ea ed
samples
we e
pla ed
on
ei he
glucose
o
lac ose,
he
esul ing
colonies
we e
always
phage
sensi i e.
No
a
single
phage- esis an
colony
was
ound
a e
es ing
a
o al
o
8,000
colonies
belonging
o
all
he
s ains
ob ained
in
he
p e ious
sec ion.
A e
he
mu agenic
ea men ,
a
sizable
p opo ion
o
phage- esis an
colonies
was
ound
bo h
among
he
gene al
popula ion
(glu-
cose
pla es)
and
among
he
Lac+
e e an s
(lac ose
pla es).
Table
2
gi es
he
incidence
o
esis ance
o
phages
QB
and
2,
and
Table
3
gi es
he
incidence
o
esis ance
o
phage
l.
The
colonies
esis an
o
l
we e
ound
o
be
esis an
o
Q,
and
2,
bu
he
e e se
was
o en
un ue.
As
a
consequence,
he
p opo ions
in
Table
2
end
o
be
highe
han
hose
in
Table
3.
TABLE
1.
Su i al
and
e e sion
o
Lac+
a e
ni osoguanidine
ea men
F equency
o
Lac+
e e an s
S ain
S
ing
(pe
colony- o ming
uni )
Su i ing
cin
Be o e
he
A e
he
ea men
ea men
SE12
(H )
0.15
2.2
x
10'
2.7
x
10-'
SE13
(H )
0.050
7.5
x
10-7
9.4
x
10-4
SE20
(F+)
0.096
<
10-8
4.1
x
10-4
SE21
(F+)
0.072
<
10-8
2.8
x
10-
In
he
H
s ains
he
mu a ion
o
esis ance
o
male-speci ic
phages
was
always
mo e
e-
quen
among
he
Lac+
e e an s
han
among
he
gene al
popula ion;
hus,
bo h
kinds
o
mu a ions
ended
o
occu
simul aneously
in
he
same
cells.
No
such
associa ion
was
ound
in
he
F+
s ains.
whe e
bo h
kinds
o
mu a ions
occu ed
as
independen
e en s.
One
o
he
se en
s ains
isola ed
om
he
H
because
o
hei
poo
abili y
o
ans e
ea ly
ch omosomal
genes
showed
comu a ion
be ween
phage
esis -
ance
and
Lac+
e e sion,
bu
he
o he s
did
no .
DISCUSSION
The
wo
kinds
o
mu a ions
in es iga ed
he e
we e
chosen
because
one
is
p esumed
o
depend
on
ch omosomal
genes
and
he
o he
is
p e-
sumed
o
depend
on
F- ac o
genes.
The
expe i-
men al
esul
is
ha
in
he
H
he
wo
kinds
o
mu a ions
occu
simul aneously
mo e
o en
han
expec ed
by
chance
a e
ni osoguanidine
ea men ,
whe eas
his
does
no
happen
in
F+
s ains.
This
esul
indica es
ha
he
genes
e-
sponsible
o
he
wo
mu a ions
eplica e
a
abou
he
same
ime
in
he
H
bu
no
in
he
F+.
This
is
in
excellen
ag eemen
wi h
he
hy-
po hesis
(8)
ha
he
F
ac o
o
he
H
is
in-
eg a ed
wi h
he
ch omosome
and
eplica es
as
a
pa
o
i ,
e en
du ing
ege a i e
g ow h
un ela ed
o
conjuga ion.
The
s ains
isola ed
om
he
H
because
o
hei
poo
abili y
o
ans e
ea ly
ch omosomal
genes
may
be
expec ed
a
p io i
o
all
in o
wo
g oups,
one
lacking
comu a ion
be ween
episo-
mal
and
ch omosomal
genes
(p esumably
due
o
de achmen
om
he
ch omosome
o
an
F
ac o
ha
does
no
ca y
he
ele an
ch omo-
somal
genes)
and
ano he
exhibi ing
comu a-
ion
(de achmen
o
an
F
ac o
ca ying
he
ele an
ch omosomal
genes,
o
mu a ional
loss
o
conjuga ional
abili y
wi hou
de achmen
o
acquisi ion
o
esis ance
o
male-speci ic
phages).
Six
s ains
o
he
i s
g oup
and
one
o
he
second
g oup
we e
isola ed.
Gue ola
e
al.
(6)
de ined
he
comu a ion
index
as
he
equency
o
double
mu an s
ela-
528
J.
BACTERIOL.
EPISOMAL
INTEGRATION
IN
E.
COLI
TABLE
2.
Resis ance
o
phages
QpS
and
2
a e
ni osoguanidine
ea men
Pla ed
on
glucose
Pla ed
on
lac ose
S ain
S a is ical
Colonies
Pe cen
Colonies
Pe cen
signi icance"
es ed
esis an
es ed
esis an
H
s ains
SE12
800
4.6
800
9.2
P
<
0.001
SE13
1825
4.8
1825
7.9
P
<
0.001
F+
s ains
SE20
700
5.0
800
5.1
NS
SE21
400
4.7
300
5.3
NS
Conjuga ion-de ec i e
s ains
de i ed
om
H
SE15
400
3.5
400
12.5
P
<
0.001
SE16
400
4.7
400
5.0
NS
SE17
400
5.2
400
6.7
NS
SE18
400
4.0
400
4.0
NS
SE19
400
5.0
400
7.0
NS
SE22
500
5.0
500
5.6
NS
SE23
400
5.0
400
5.4
NS
a
The
p obabili y
o
a
chance
de ia ion
equal
o
o
la ge
han
he
expe imen al
one
Was
calcula ed
a e
he
a Csin
ans o ma ion
and
he
es
(12).
All
"no
signi ican "
(NS)
cases
ga e
P
>
0.2.
TABLz
3.
Resis ance
o
phage
l
a e
ni osoguanidine
ea men
S ain
H
s ains
SE12
SE13
F+
s ains
SE21
Conjuga ion-de ec i e
s ains
de i ed
om
H
SE17
SE18
Pla ed
on
glucose
Colonies
es ed
500
375
500
500
500
Pe cen
esis an
3.2
2.1
2.6
2.4
1.4
Pla ed
on
lac ose
Colonies
es ed
500
375
400
500
500
Pe cen
esis an
8.6
7.2
3.0
2.0
1.4
i e
o
he
p oduc
o
he
equencies
o
he
single
mu an s;
hey
ound
an
index
o
15
o
genes
placed
1
min
away
in
he
ch omosome
map.
The
comu a ion
index
o
Lac+
e e sion
and
l
esis ance
is
abou
3
in
he
H
s ains.
By
compa ison
wi h
Gue ola's
esul s,
he
ele-
an
genes
may
be
placed
some
1.5
min
away
in
he
H
ch omosome.
Comu a ion
is
clea e
and
he
index
is
sligh ly
highe
when
l
is
used
o
assay
o
esis ance
han
when
Q i
and
2
a e
used.
On
he
o he
hand,
bac e ia
esis an
o
l
a e
usually
esis an
o
Q i
and
2,
bu
no
ice
e sa.
This
may
be
due
o
mu a ions
con e ing
esis ance
o
Q8
and
2,
bu
no
l,
loca ed
in
ch omosomal
genes
a
om
lac
and
he
F
ac o ,
and
is
p ob-
ably
ela ed
o
he
di e en
pa icipa ion
o
bac-
e ial
genes
in
he
de elopmen
o
he
wo
ypes
o
phage.
Comu a ion
s udies
ashioned
in
di e en
ways
may
be
used
o
es
o
he
in eg a ion
o
an
episome
in o
a
ch omosome.
Such
a
es
is
independen
o
whe he
he
episome
con e s
conjuga ional
abili y,
o
whe he
a
gene ic
sys-
em
has
been
de eloped
in
he
bac e ial
species
unde
s udy,
and
o
whe he
he
episome
ca ies
any
indispensable
genes
(and
hus
canno
be
los
upon
incuba ion
wi h
ac idine
dyes).
Fo
example,
a
mu a ional
change
in
he
episome
would
be
selec ed,
ei he
in
he
o wa d
o
S a is ical
signi icance
P
<
0.001
P
<
0.001
NS
NS
NS
VOL.
116,
1973
529
530
LLOVERES
AND
e e se
di ec ion,
a e
ea men
wi h
ni-
osoguanidine,
and
he
esul ing
mu an s
would
be
assayed
o
he
p esence
o
ch omo-
somal
mu a ions.
Auxo ophic
mu a ions
a e
con enien
o
assay
and
a e
usually
sp ead
o e
he
ch omosome.
I
he
episomal
mu a ion
is
equen ly
associa ed
wi h
a
pa icula
g ow h
equi emen ,
he
implica ion
would
be
ha
he
episome
is
close
o
he
genes
esponsible
o
ha
kind
o
auxo ophy.
In
ac
comu a ion
only
means
ha
he
wo
gene ic
s uc u es
eplica e
simul aneously.
Co-
mu a ion
migh
be
ound
be ween
genes
in
wo
independen
eplicons
i
he e
is
a
p ecise
syn-
ch oniza ion
in
hei
eplica ion.
Au onomous
episomes
a e
known
o
eplica e
a
ce ain
imes
o
he
cell
cycle
(14,
15),
bu
he e
is
no
indica ion
ha
he
synch oniza ion
is
good
enough
o
show
app eciable
comu a ion
wi h
ch omosomal
genes.
Re e sion
o
wild- ype
pheno ype
may
some-
imes
be
due
mo e
o
in e genic
supp ession
han
o
in agenic
e e sion
and
he
comu a ion
esul s
would
hen
be
misin e p e ed.
In
ou
expe ience
such
high
ela i e
incidence
o
in e -
genic
supp ession
is
uncommon
and
e o s
can
be
p e en ed
by
he
s udy
o
di e en
o iginal
mu a ions
o
he
same
ype.
ACKNOWLEDGMENTS
We
hank
K.
B ooks,
A.
J.
Cla k,
R.
Cu iss
Ill,
and
N.
D.
Zinde
o
he
o iginal
s ains
o
bac e ia
and
phages,
and
J.
Yeb a
o
expe
echnical
help.
LITERATURE
CITED
1.
Ach man,
M.
1973.
Gene ics
o
he
F
sex
ac o
in
.ERDA-OLMEDO
J.
BACTERIOL.
En e obac e iaceae.
Cu .
Top.
Mic obiol.
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60:79-123.
2.
Adelbe g,
E.
A.,
M.
Mandel,
and
G.
C. C.
Chen.
1965.
Op imal
condi ions
o
mu agenesis
by
N-me hyl-N'-
ni o-N-ni osoguanidine
in
Esche ichia
coli
K12.
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chem.
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Res.
Commun.
18:788-795.
3.
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C.
M.,
and
R.
Cu iss.
1967.
T ansposi ion
de i a-
i es
o
an
H
s ain
o
Esche ichia
coli
K-12.
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56:503-525.
4.
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E.,
and
P.
C.
Hanawal .
1968.
Diazome-
hane
as
he
ac i e
agen
in
ni osoguanidine
mu a-
genesis
and
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Mol.
Gen.
Gene .
101:191-202.
5.
Ce da-Olmedo,
E.,
P.
C.
Hanawal ,
and
N.
Gue ola.
1968.
Mu agenesis
o
he
eplica ion
poin
by
ni-
osoguanidine:
Map
and
pa e n
o
eplica ion
o
he
Esche ichia
coli
ch omosome.
J.
Mol.
Biol.
33:705-719.
6.
Gue ola,
N.,
J.
L.
Ing aham,
and
E.
Ce da-Olmedo.
1971.
Induc ion
o
closely
linked
mul iple
mu a ions
by
ni osoguanidine.
Na u e
(London)
230:122-125.
7.
Hi o a,
Y.
1960.
The
e ec
o
ac idine
dyes
on
ma ing
ype
ac o s
in
Esche ichia
coli.
P oc.
Na .
Acad.
Sci.
U.S.A.
46:57-64.
8.
Jacob,
F.,
and
E.
L.
Wollman.
1957.
Analyse
des
g oupes
de
liaison
gene ique
de
di e en es
souches
dona ices
d'Esche ichia
coli
K12.
C.
R.
Acad.
Sci.
245:1840-1843.
9.
Low,
B.,
and
T.
H.
Wood.
1965.
A
quick
and
e icien
me hod
o
in e up ion
o
bac e ial
conjuga ion.
Ge-
ne .
Res.
6:300-303.
10.
Maaloe,
O.,
and
P.
C.
Hanawal .
1961.
Thymine
de i-
ciency
and
he
no mal
DNA
eplica ion
cycle.
J.
Mol.
Biol.
3:144-155.
11.
Mandell,
J.
D.,
and
J.
G eenbe g.
1960.
A
new
chemical
mu agen
o
bac e ia:
1-me hyl-3-ni o-1-ni osoguani-
dine.
Biochem.
Biophys.
Res.
Commun.
3:575-578.
12.
Sokal,
R.
R.,
and
F.
J.
Rohl .
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Biome y,
p.
607.
W.
H.
F eeman
and
Co,
San
F ancisco.
13.
Takahashi,
I.,
and
R.
A.
Ba na d.
1967.
E ec
o
N-
me hyl-N'-ni o-N-ni osoguanidine
on
he
F
ac o
o
Esche ichia
coli.
Mu a .
Res.
4:111-117.
14.
Yabe,
Y.,
and
S.
Mi suhashi.
1971.
Replica ion
and
ans e
o
he
R
ac o
in
a
synch onized
cul u e
o
Esche ichia
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15:21-27.
15.
Zeu hen,
J.,
and
M.
L.
Pa o.
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o
he
F'lac
sex
ac o
in
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cell
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o
Esche ichia
coli.
Mol.
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111:242-255.
10
c