Vol.
149,
No.
1
JOURNAL
OF
BACTERIOLOGY,
Jan.
1982,
p.
59-64
0021-9193/82/010059-06$02.00/0
Use
o
Plasmid
R68.45
o
Cons uc ing
a
Ci cula
Linkage
Map
o
he
Rhizobium
i olii
Ch omosome
MANUEL
MEGIAS,1
MIGUEL
A.
CAVIEDES,'
ANTONIO
J.
PALOMARES,l*
AND
JULIO
PEREZ-
SILVA2
Depa amen o
de
Mic obiologia,
Facul ad
de
Fa macia,'
and
Depa amen o
de
Mic obiologia,
Facul ad
de
Biologia,2
Uni e sidad
de
Se illa,
Se illa,
Spain
Recei ed
10
Ma ch
1981/Accep ed
29
June
1981
Plasmid
R68.45
was
used
o
p omo e
conjugal
ans e
o
ch omosomal
ma ke s
in
Rhizobium
i olii
RS55.
Analysis
o
wo- ac o
and
h ee- ac o
c osses
among
R.
i olii
s ains
enabled
cons uc ion
o
a
ci cula
linkage
map
o
he
R.
i olii
ch omosome,
con aining
17
nu i ional
and
esis ance
ma ke s.
The
economic
ad an ages
ha
may
acc ue
om
he
imp o emen
o
ni ogen- ixing
sys-
ems
ha e
become
inc easingly
e iden
du ing
he pas
decade.
One
o
he
mos
in e es ing
ni ogen- ixing
sys ems
is
he
Rhizobium-legume
symbiosis
because
o
i s
high
ag icul u al
impo -
ance.
The
pas
se e al
yea s
ha e
wi nessed
impo an
de elopmen s
in
Rhizobium
gene ics
(3,
7),
and
a
numbe
o
sui able
p ocedu es
o
he
gene ic
manipula ion
o
hizobia
ha e
e-
cen ly
been
de eloped
(4-6,
8,
9,
17, 18,
20,
23,
24).
Fo
many
yea s,
one
o
he
majo
limi a ions
o
gene ic
analysis
o
hizobia
was
he
lack
o
known
indigenous
ch omosome-mobilizing
plas-
mids
ha
would
allow
gene
ans e
s udies.
This
di icul y
has
been
o e come
by
ans e -
ing
b oad-hos - ange
plasmids
wi h
ch omo-
some-mobilizing
abili y
(Cma)
o
Rhizobium
spp.
To
da e,
he
Pseudomonas
ae uginosa
d ug-
esis ance
plasmids
R68.45
(13)
and
RP4
(12)
ha e
been
he
plasmids
o
choice
o
ob aining
gene
ans e
in
Rhizobium.
R68.45
is
e y
e i-
cien
o
mobilizing
ch omosomal
ma ke s
in
R.
melilo i
(10,
21)
and
R.
leguminosa um
(5)
as
well
as
in
o he
g am-nega i e
bac e ia
(14,
26).
In
bo h
R.
melilo i
and
R.
leguminosa um,
R68.45
p omo es
nonpola ized
ch omosomal
ans e
om
a
numbe
o
o igins
(4,
5,
10,
21).
In
hose
species
o
Rhizobium
so
a
examined,
R68.45
can
ans e
agmen s
o
ch omosome
long
enough
o
enable
accu a e
mapping
o
a
a ie y
o
ma ke s
and
o
es ablish
ch omosome
ci cula i y
(5,
10,
21).
RP4
is
a
less
e icien
sex
ac o
(15).
Howe -
e ,
i
has
been
success ully
used
o
media ing
ans e
o
ch omosomal
ma ke s
in
R.
melilo i
(22).
To
da e,
he
only
epo
on
linkage
mapping
s udies
in
R.
i olii
was
an
a emp
a
using
co-
mu a ion
echniques
(29).
As
an
al e na i e
ap-
p oach,
we
desc ibe
he e
he
use
o
plasmid
R68.45
o
p omo ing
ch omosomal
ans e
in
R.
i olii.
Linkage
mapping
expe imen s
epo -
ed
he e
ha e
led
o
he
cons uc ion
o
a
ci cula
linkage
map
o
R.
i olii
con aining
17
nu i ion-
al
and
esis ance
ma ke s.
MATERIALS
AND
METHODS
Bac e ial
sains.
The
s ains
used
a e
lis ed
in
Table
1.
Plasmid.
R68.45,
belonging
o
he
P1
incompa ibili y
g oup
and
media ing
esis ance
o
ampicillin,
kanamy-
cin,
and
e acycline,
was
used
(13).
Media
and
g ow h
condi ions.
Esche ichia
coli
was
cul u ed
on
nu ien
b o h
(Oxoid);
when
equi ed
in
solidi ied
o m,
aga
(Oxoid)
was
added
a
10
g/li e .
Fo
R.
i olii
s ains
comple e
medium
was
YT
(2)
and
minimal
medium
was
ha
o
Vogel
and
Bonne
(27)
supplemen ed
wi h
19
g
o
glucose
pe
li e
and
12
,ug
o
bio in
pe
ml.
When
equi ed,
amino
acid
and
base
supplemen s
we e
used
a
a
inal
concen a ion
o
1
mM.
E.
coli
was
always
g own
a
35°C.
R.
i olii
was
always
g own
a
28(C.
An ibio ics.
Excep
o
i ampin,
which
was
dis-
sol ed
in
me hanol
and
added
un il e ed,
an ibio ics
we e
added
o
aga
media
as
il e -s e ilized
aqueous
solu ions.
Concen a ions
used
we e
as
ollows:
s ep-
omycin
sul a e
(Sigma),
1
mg/ml;
kanamycin
sul a e
(Sigma),
25
pg/ml;
oxy e acycline
(Sigma),
10
.g/ml;
i ampin
(Lepe i ),
20
,ug/ml;
ampicillin
(Sigma),
10
Ag/
ml,
and
cana anine
sul a e
(Sigma),
80
,ug/ml.
Ma ings.
Ma ings
we e
pe o med
on
Sa o ius
il-
e s
(0.45-,m
po e
size)
as
desc ibed
by
Jacob
e
al.
(16).
One
millili e
o
log-phase
dono and
1
ml
o
s a iona y-phase
ecipien
(bo h
con aining
a
leas
108
cells
pe
ml)
we e
ma ed
on
he
memb ane.
Mem-
b anes
we e
placed
on
he
su ace
o
nu ien
aga
pla es
and
incuba ed
o
abou
20
h
a
28°C.
Then
ma ing
mix u es
we e
esuspended
in
S
ml
o
T is-sal s
bu e ,
pH
7.2,
dilu ed,
and
pla ed
on
selec i e
media.
Linkage
mapping
o
he
R. I oii
ch omosome.
Recombinan
colonies
o
a
selec ed
ma ke
we e
picked
on o
selec i e
media
o
de e mine
cohe i ance
o
unselec ed
ma ke s.
Each
pai
o
ma ke s
was
analyzed
by
sco ing
150
o
300
colonies.
I
was
as-
sumed
ha
linkage
alues
we e
in e sely
ela ed
o
59
TABLE
1.
Bac e ial
s ainsa
S ain
Rele an
cha ac e is ics
Re e ence
o
sou ce
E.
coli
1230
P o-
Me -
(R68.45)
J.
E.
Be inge
R.
i olii
RS55
Wild- ype
Isola ed
om
clo e
oo
nodules
RS176
s -l
This
pape
RS225
his-6
s -l
This
pape
RS235
his-6
me -36
s -l
This
pape
RS238
his-6
phe-18
s -l
This
pape
RS241
i -6
ade-17
This
pape
RS246
i4 -6
ade-29
This
pape
RS271
s -l
leu-23
This
pape
RS288
his-6
y -28
s -l
This
pape
RS290
his-6
h -14
s -l
This
pape
RS292
his-6
pdx-3
s -l
This
pape
RS293
i -6
ade-29
y -13
This
pape
RS294
i -6
ade-29
h -27
This
pape
RS295
i -6
ade-29
a g-19
This
pape
RS296
his-6
me -31
s -I
can-3
This
pape
RS230
(R68.45)
his-6
s -l
C oss
1230
x
RS225
RS278
(R68.45)
i -6
ade-17
C oss
1230
x
RS241
I
Allelle
numbe s
a e
a bi a y.
Gene
symbols
a e
hose
o
Bachmann
and
Low
(1).
All
he
R.
i olii
s ains
a e
de i ed
om
he
wild- ype
RS55,
isola ed
in
ou
labo a o y
om
T i olium
epens
L.
nodules.
Auxo ophic
mu a ions
we e
induced
by
ei he
N-me hyl-N'-ni o-N-ni osoguanidine
o
e hyl
me hane
sul ona e
mu agene-
sis.
Ni osoguanid ne
ea men s
we e
ca ied
ou
as
ollows:
a
log
cul u e
con aining
abou
10'
cells
pe
ml
was
cooled,
washed
wice
wi h
T is-malea e
bu e ,
pH
7.5,
and
esuspended
in
500
mg
o
ni osoguanidine
(Sigma)
pe
li e
o
30
min.
Then
he
cells
we e
ha es ed
and
ca e ully
washed
wi h
bu e
be o e
pla ing
on
YT
aga .
Fo
e hyl
me hane
sul ona e
mu agenesis,
a
cul u e
con aining
abou
4
x
10'
cells
pe
ml
was
washed
wice
wi h
phospha e
bu e ,
pH
8.0,
and
suspended
in
0.1
M
e hyl
me hane
sul ona e
(Sigma).
A e
incuba ion
a
30°C
o
45
min,
6%
(w / ol)
Na2S203
was
added.
Cells
we e
ha es ed,
washed,
and
pla ed
on
YT.
D ug- esis ance
mu a ions
we e
spon aneous.
physical
dis ances
be ween
genes.
Linkage
alues
we e
ans o med
in o
addi i e
map
dis ances
by
using
he
equa ion
de i ed
by
Kempe
(19):
C
=
(1
-
)
+
(Qn
)
whe e
C
is
he
linkage
equency
alue
and
is
he
map
dis ance.
RESULTS
T anse
o
plasd
R68.45
om
E.
coli
o
R.
& ol.
Plasmid
R68.45
was
easily
ans e ed
om
E.
coli
1230
o
R.
i olii
RS176
in
mem-
b ane
ma ings.
T ansconjugan s
we e
selec ed
on
minimal
medium
wi h
an ibio ics.
When
he
ecipien
s ain
was
one
o
he
auxo ophic
de-
i a i es
o
RS176
desc ibed
abo e,
selec i e
medium
was
supplemen ed
wi h
he
equi e-
men s
o
he
ecipien
s ain.
In
hese
c osses
he
equency
o
plasmid
ans e
was
abou
10-
pe
dono
bac e ium.
This
equency
is
sligh ly
lowe
han
hose
e-
TABLE
2.
R68.45
ans e
and
ch omosome
mobiliza ion
in
R.
i olii
RS176
Dono
Recipien
F equency
o
Selec ed
F equency
o
R
ans e 0
pheno ype
ecombinan s'
RS230
RS241
2.8
x
10-1
Ade+
2.7
x
10-4
S
8.9
x
1o-4
RS230
RS294
4.3
x
10-1
Th +
1.1
X
1O-4
Ade+
1.1-
xi10-5
RS278
RS235
4.2
x
10-1
Me +
3.0
x
1O-5
His+
9.2
x
10-4
RS278
RS288
8.3
x
10-1
His+
1.1
x
10-4
Ty +
1.6
x
1O-4
Ri
6.0
x
10-5
RS278
RS271
5.8
x
10-1
Leu+
1.1
x
1O-4
Ri
7.0
x
1O-4
RS278
RS292
3.3
x
10-1
Pdx+
1.7
x
1O-4
a
Pe
dono
bac e ium.
(O
MEGIAS
ET
AL.
J.
BACTERIOL.
CIRCULAR
LINKAGE
MAP
OF
R.
TRIFOLII
TABLE
3.
De ec ion
o
R68.45
ma ke s
in
ansconjugan s
ha
had
ecei ed
ch omosomal
ma ke s
C oss
~~Selec ed
No.
o R
R%R
C oss
pheno ype
clones
es eda
%
R
RS230
x
RS293
Ade+
204
198
6
3
RS278
x
RS288
Ty '
163
160
3
2
RS278
x
RS238
His+
113
112
1
1
RS230
x
RS271
S
199
197
2
1
a
Replica
pla ed
on
an ibio ic
medium.
po ed
o
E.
coli
x
R.
leguminosa um
and
E.
R68.45
was
able
o
p omo e
ch omosome
ans-
coli
x
R.
melilo i
c osses
(5,
10).
e
a
a
a ie y
o
o igin
si es
as
p e iously
F om
hese
c osses
R'
ansconjugan s
o
R.
epo ed
o
o he
hizobia
(4,
5,
10,
21).
i olii
we e
isola ed
o
be
used
as
u he
dono s
When
he
p esence
o
plasmid
R68.45
was
in
in aspeci ic
c osses.
All
isola es
we e
pu i-
in es iga ed
among
he
ecombinan s,
mos
o
ied
wo
o
h ee
imes
be o e
being
s o ed.
hem
p o ed
o
be
R'
a
leas
o
esis ance
In aspeci ic
ma ings:
ans e
o
plasmid
ma ke s
(see
Table
3).
This
sugges ed
ha
ch o-
R68.45
and
mobiliza ion
o
ch omosomal
ma ke s
mosomal
ans e
media ed
by
plasmid
R68.45
in
R.
i ol
i.
As
shown
in
Table
2,
he
equency
was
F'-like
a he
han
H -like.
The
same
phe-
o
plasmid
ans e
in
in aspeci ic
ma ings
was
nomenon
has
been
desc ibed
in
R.
legumino-
highe
han
in
in e gene ic
c osses.
T ans e
sa um
and
R.
melilo i
(4,
10,
21).
equencies
desc ibed
he e
a e
simila
o
hose
Linkage
analysis.
The
a ionale
o
linkage
epo ed
o
o he
Rhizobium
species,
such
as
R.
s udies
based
upon
R68.45-media ed
gene
ans-
melilo i
(9,
21),
R.
leguminosa um
(4,
5),
and
e
can
be
summa ized
as
ollows:
since
any
in e speci ic
c osses
o
R.
leguminosa um
x
R.
ma ke
has
a
ela i ely
de ined
equency
o
melilo i
(17).
ans e ,
he
chance
o
a
pai
o
ma ke s
being
Mobiliza ion
o
single
ch omosomal
ma ke s
co ans e ed
in
he
same
c oss
will
depend
on
occu ed
a
equencies
anging
om
abou
5
x
he
dis ance
be ween
hem.
10-5
o
5
x
10-3
pe
dono
cell.
Absence
o
Table
4
shows
he
esul s
ob ained
in
a
se ies
g ea
di e ences
among
he
equencies
o
mobi-
o
wo- ac o
c osses.
In
each
ma ing
ini ial
liza ion
o
di e en
ma ke s
indica ed
ha
selec ion
was
ca ied
ou
o
a
single
ma ke .
TABLE
4.
Linkage
analysis
o
se e al
ma ke
pai s
o
R.
i olii
RS176
Dono
Recipien
Selec ed
Ma ke s
Linkage
Dis ance
pheno ype
pai s
(C)
( )
RS230
RS246
j
i -6
ade-29
0.41
0.24
i -6
s -l
0.21
0.42
Ade+
ade-29
s -1
0.04
0.73
RS230
RS293
ade-29
y -13
0.39
0.26
Ri F
i -6
y -13
0.79
0.05
Ty '
y -13
s -l
0.17
0.47
RS230
RS294
Th +
h -18
ade-29
0.75
0.06
h -18
i -6
0.69
0.09
RS230
RS295
Ade+
ade-29
a g-19
0.65
0.10
RM
i -6
a g-19
0.25
0.38
RS278
RS235
His+
his-6
me -36
0.40
0.25
Me +
me -36
j -6
0.03
0.76
RS278
RS238
Phe+
phe-18
his-6
0.04
0.73
phe-18
i -6
0.16
0.49
RS278
RS271
Leu+
leu-23
i -6
0.04
0.73
Ade+
ade-29
leu-23
0.47
0.20
RS278
RS290
Th +
h -27
his-6
0.42
0.23
S
s -l
h -27
0.09
0.60
Ri T
n -6
h -27
0.70
0.08
RS278 RS292
Pdx+
pdx-3
s -l
0.27
0.36
pdx-3
his-6
0.03
0.76
RS278
RS296
Can
can-3
his-6
0.16
0.49
can-3
me -31
0.54
0.16
His+
his-6
me -31
0.35
0.29
Me +
me -31
i -6
0.12
0.55
61
VOL.
149,
1982
TABLE
5.
Th ee- ac o
c osses
be ween
R.
i olii
s ains
Dono
Recipien
Selec ed
Ma ke s
Linkage
Dis ance
pheno ype
pai s
(C)
( )
RS278
RS288
His'
his-6
y -28
0.43
0.23
his-6ade-17
0.06 0.67
Ty '
y -28
his-6
0.46
0.21
y -28
ade-17
0.28
0.35
RS278
RS238
His'
his-6
phe-18
0.05
0.70
his-6
ade-17
0.07
0.65
Phe+
phe-18
his-6
0.06
0.67
RS278
RS271
S
s -l
ade-17
0.19
0.45
Leu+
leu-23
ade-17
0.03
0.76
RS230
RS293
Ty '
y -13
ade-29
0.28
0.35
y -13
his-6
0.23
0.40
Ade+
ade-29
y -13
0.26
0.37
ade-29
his-6
0.03
0.76
Co ans e
o
unselec ed
ma ke s
was
de ec ed
RP1,
RP4,
Rld dl9,
and
R68.45
(M.
Meglas,
by
eplica
pla ing
on
app op ia e
selec i e
me-
Ph.D.
hesis,
Uni e si y
o
Se illa,
Se illa,
dia.
Linkage
equencies
lowe
han
0.03
we e
Spain,
1981).
Howe e ,
as
in
o he
Rhizobium
disca ded.
Dis ances
we e
calcula ed
by
using
species,
he
mos
e icien
plasmid
wi h
ch omo-
he
Kempe
equa ion
as
desc ibed
abo e.
some-mobilizing
abili y
is
R68.45
(15;
Megias,
Resul s
ob ained
in
h ee- ac o
c osses
a e
Ph.D.
hesis,
1981).
summa ized
in
Table
5.
Usual
equencies
o
mobiliza ion
o
ch omo-
A
diag am
ha
summa izes
he
linkage
ela-
somal
ma ke s
by
R68.45
a e
highe
han
e e -
ionships
among
17
ch omosomal
ma ke s
is
sion
a es.
Fo
ins ance,
all
he
auxo ophic
p esen ed
in
Fig.
1.
Numbe s
abo e
he
a ows
mu a ions
epo ed
in
his
pape
had
e e sion
indica e
linkage
equencies
ound
in
indepen-
equencies
lowe
han
10-6,
whe eas
he
den
expe imen s.
This
p elimina y
linkage
map
chance
o
de ec ing
single-ma ke
ans e
in
an
can
be
ci cula ized
and
ep oduced
o
scale
as
R68.45-media ed
c oss
was
10-4
o
10-5
pe
shown
in
Fig.
2.
dono
cell.
Thus,
accu a e
measu es
o
ans e
DISCUSSION
equencies
may
be
eadily
ob ained.
F equency
anges
desc ibed
in
his
pape
a e
simila
o
Mobiliza ion
o
he
R.
i olii
ch omosome
can
hose
epo ed
o
R.
melilo i
(10,
21)
and
o
he
be
achie ed
by
using
se e al
plasmids,
such
as
o iginal
hos
o
R68.45,
P.
ae uginosa
(13).
Iu-23
ad.-17
pdx-3
can-3
me -36
me -31
y -28
s -1
his-6
hi-l
y -13
i -0
h -27
h -18
ade-29
. g-19
phe-18
leu-23
116
LI
S
L
LII
6~~~~~~~~~~~~~~~~~~~.73
6.76
gg
s.11
176
6.66
6.23;6.21
6.37;
6.26
:..35
126
6~~~~~~~~~~~~.36
6.26
6.24
6.55
6.42
6.76;
6.67;
6.73
6.76
6.23
6.76
6.73
FIG.
1.
P elimina y
linkage
map
o
he
R.
i olii
RS176
ch omosome.
Numbe s
beside
gene
symbols
indica e
allele
numbe s.
Numbe s
on
he
a ows
indica e
conjugal
linkage
pe cen ages.
62
MEGIAS
ET
AL.
J.
BACTERIOL.
CIRCULAR
LINKAGE
MAP
OF
R.
TRIFOLII
FIG.
2.
Ci cula
linkage
map
o
he
R.
i olii
RS176
ch omosome.
Dis ances
a e
abou
in e sely
p opo ional
o
he
linkage equencies.
Minimal
link-
age
alues
(3%)
would
ep esen ,
a
leas ,
one- ou h
o
he
o al
leng h
o
he
ch omosome.
Ano he
ad an age
o
using
plasmid
R68.45
o
mapping
pu poses
in
Rhizobium
spp.
is
he
la ge
size
o
ch omosomal
agmen s
mobilized
by
he
plasmid.
Subsequen ly,
simul aneous
ans e
o
dis an
ma ke s
may
be
eadily
de-
ec ed
in
app op ia e
c osses.
Fo
ins ance,
ma ke
pai s
showing
low
linkage
equencies,
such
as
his-61phe-18,
me -361 i -6,
and
his-61pdx-
3,
may
be
loca ed
as
a
apa
as
one- ou h
o
he
o al
leng h
o
he
ch omosome.
These
e-
sul s
ag ee
wi h
hose
epo ed
o
o he
Rhizo-
bium
species
(4,
10,
21).
T ans o ma ion
o
linkage
measu es
in o
addi-
i e
map
dis ances
is
easily
ob ained
by
use
o
o mulas
such
as
ha
o
Wu
(28)
o
ha
o
Kempe
(19).
These
equa ions
we e
i s
p o-
posed
o
use
in
co ansduc ion
expe imen s,
bu
hey
a e
also
sui able
o
conjugal
mapping
(21;
Meg as,
Ph.D.
Thesis,
1981).
We
ha e
cho-
sen
o
use
Kempe 's
equa ion
because
i
does
no
equi e
any
assessmen
o
he
leng h
o
ans e ed
ch omosomal
segmen s.
Unlike
R.
melilo i
(10,
11,
25)
and
R.
legumin-
osa um
(8)
gene al
ansduc ion
sys ems
ha e
no
ye
been
desc ibed
o
R.
i olii.
Thus,
conjugal
mapping
media ed
by
plasmid
R68.45
is
he
only
me hod
now
a ailable
o
ex ensi e
mapping
s udies
in
R.
i olii.
We
hope
i
will
be
sui able
o
u he
gene ic
analysis
o
ni ogen-
ixing
clo e
symbiosis.
ACKNOWLEDGMENTS
We
a e
g a e ul
o
John
Be inge
o
p o iding
plasmid
R68.45
and
o
Josep
Casades s
o
help ul
sugges ions
du ing
he
p epa a ion
o
he
manusc ip .
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