JOURNAL
OF
BAC ERIOLOGY,
Feb.
1994,
p.
1093-1098
Vol.
176,
No.
4
0021-9193/94/$04.00
+
0
Copy igh
©
1994,
Ame ican
Socie y
o
Mic obiology
T ans e
o
a
Gene ic
Ma ke
om
a
Megaplasmid
o
Anabaena
sp.
S ain
PCC
7120
o
a
Megaplasmid
o
a
Di e en
Anabaena
S ain
ALICIA
M.
MURO-PASTOR,'
TANYA
KURITZ,2
ENRIQUE
FLORES,l*
ANTONIA
HERRERO,'
AND
C.
PETER
WOLK2
Ins i u o
de
Bioquimica
Vege al
y
Fo os n esis,
Uni e sidad
de
Se illa-CSIC,
Facul ad
de
Biologia,
Apa ado
1113,
E-41080
Se ille,
Spain,'
and
MSU-DOE
Plan
Resea ch
Labo a o y
and
Cen e
o
Mic obial
Ecology,
Michigan
S a e
Uni e si y,
Eas
Lansing,
Michigan
488242
Recei ed
4
Augus
1993/Accep ed
7
Decembe
1993
The
410-kb
a
megaplasmid
o
he
he e ocys - o ming
cyanobac e ium
Anabaena
sp.
s ain
PCC
7120
was
ound
o
bea
he
nucA
gene
ha
encodes
a
suga -nonspeci ic
nuclease.
Tha
gene
was
mu a ed
by
inse ion
o
a
casse e
ha
con e s
esis ance
o
neomycin.
The
esul ing
s ain,
AMP2,
was
ma ed
wi h
a
s ep omycin-
esis an
de i a i e
o
Anabaena
sp.
s ain
PCC
7118,
a
s ain
ha
does
no
o m
he e ocys s.
Cells
esis an
o
bo h
neomycin
and
s ep omycin
ha
we e
de i ed
om
such
ma ings
we e
ound
o
bea
he
neomycin
esis ance
casse e
o
he
dono
s ain
in
a
la ge
megaplasmid
cha ac e is ic
o
he
ecipien
s ain
and
did
no
o m
he e ocys s.
This
is
he
i s
example
o
ans e
o
a
gene ic
ma ke
di ec ly
be ween
s ains
o
cyanobac e ia
in
which
incon o e ible
physical
e idence
o
ans e
has
been
ob ained.
DNA
sequences
homologous
o
he
nucA
gene
we e
p esen
in
13
he e ocys - o ming
cyanobac e ia
ha
we e
es ed
bu
in
none
o
six
di e se
unicellula s ains
ha
we e
examined.
The
cyanobac e ia
a e
pho o ophic
eubac e ia
ha
can
ca y
ou
oxygenic
(plan - ype)
pho osyn hesis.
Many
ilamen-
ous
cyanobac e ia
a e
able
o
ix
dini ogen
in
specialized
cells
called
he e ocys s
ha
de elop
a
semi egula
in e als
along
he
ilamen s.
We
ha e
ecen ly
shown
ha Anabaena
sp.
s ain
PCC
7120,
a
he e ocys - o ming
cyanobac e ium,
p oduces
an
exocy oplasmic
suga -nonspeci ic
nuclease
ha
is
able
o
de-
g ade
linea
o
co alen ly
closed
ci cula
double-s anded
DNA
as
well
as
single-s anded
DNA
o
RNA
(16).
The
gene,
nucA,
encoding
his
enzyme
has
been
cloned
and
sequenced
(16).
In
his
epo ,
we
show
ha
nucA
is
loca ed
on
he
o
megaplasmid,
whose
p esence
in
s ain
PCC
7120
had
been
epo ed
p e iously
(4).
A
casse e
con e ing
esis ance
o
neomycin
(NM),
when
in oduced
in o
he
nucA
gene
o
s ain
PCC
7120,
could
he eupon
be
ans e ed
o
a
la ge
megaplasmid
o
a
s ep omycin
(SM)- esis an
de i a i e
o
Anabaena
sp.
s ain
PCC
7118.
We
ha e
also
ound
ha
he
nucA
gene
is
p esen
in
nume ous
he e ocys - o ming
cya-
nobac e ia.
MATERIALS
AND
METHODS
O ganisms
and
cul u e
condi ions.
Cyanobac e ial
s ains
we e
g own
pho oau o ophically
a
30°C
in
BG11
medium
(18)
wi h
shaking.
Whe e
speci ied,
BG11
medium
was
solid-
i ied
by
he
addi ion
o
1%
sepa a ely
au ocla ed
Bac o
Aga
(Di co).
Fo
es ing
g ow h
on
dini ogen,
BG11
lacking
NaNO3
was
used.
Fo
g ow h
o
de i a i e
AMP2
o
Anabaena
sp.
s ain
PCC
7120,
liquid
BG11
medium
was
supplemen ed
wi h
5
jig
o
NM
pe
ml
and
solidi ied
BG11
medium
was
supplemen ed
wi h
25
jig
o
NM
pe
ml.
De i a i e
NW18
o
Anabaena
sp.
s ain
PCC
7118
was
g own
in
BG11
medium
*
Co esponding
au ho .
Telephone:
34-5-455.70.86.
Fax:
34-5-
462.01.54.
Elec onic
mail
add ess:
[email p o ec ed].
P esen
add ess:
Depa men
o
Mic obiology,
Uni e si y
o
Illi-
nois
a
U bana-Champaign,
U bana,
IL
61801.
supplemen ed
wi h
20
jig
o
SM
pe
ml.
Nm
Sm
de i a i es
o
s ain
NW18
we e
g own
in
BG11
medium
supplemen ed
wi h
a
combina ion
o
20
jig
o
SM
pe
ml
and
NM
ei he
a
5
jig/ml
in
liquid
medium
o
25
,ug/ml
in
solid
medium.
Chlo ophyll
a
was
de e mined
in
me hanolic
ex ac s
(13).
Esche ichia
coli
DH5o ,
used
o
all
plasmid
cons uc ions,
was
g own
in
Lu ia
b o h
as
desc ibed
p e iously
(14).
P epa a ion
and
manipula ion
o
DNA.
To al
DNA
om
cyanobac e ia
was
isola ed
as
desc ibed
by
Cai
and
Wolk
(6).
DNA
o
high
molecula
weigh
om
Anabaena
sp.
s ains
PCC
7120
and
PCC
7118
and
hei
de i a i es
was
p epa ed
and
cu
wi h
es ic ion
endonucleases,
and
he
esul ing
plasmids
we e
subjec ed
o
pulsed
homogeneous
o hogonal
ield
gel
elec o-
pho esis
(PHOGE)
(2)
as
desc ibed
by
Ku i z
e
al.
(11).
Bands
wi h
a
size
o
less
han
50
kb
we e
measu ed
by
using
HindIll
diges s
o
coliphage
lambda
DNA
as
s anda ds
in
pulsed- ield
gels
(11).
Plasmid
isola ion
om
E.
coli,
ans o ma ion
o
E.
coli,
es ic ion,
and
liga ion
wi h
T4
DNA
ligase
we e
pe o med
by
s anda d
p ocedu es
(1,
14).
DNA
agmen s
we e
pu i ied
om
aga ose
gels
wi h
he
GeneClean
ki
(Bio
101,
Inc.).
The
nucA-con aining
hyb idiza ion
p obes
we e
ei he
plasmid
pRLA1,
which
ca ies
12
kb
o
s ain
PCC
7120
genomic
DNA
including
he
nucA
gene
and
lanking
sequences,
o
a
473-bp
HindIII-Bs NI
agmen
in e nal
o
nucA,
isola ed
om
pCSAM29
(16).
The
neomycin
phospho ans e ase
(np )
p obe
used
was
casse e
C.K3
(7)
excised
wi h
BamHI.
DNA
p obes
we e
32P
labeled
wi h
[oL-32P]dCTP
by
using
a
nick
ansla ion
ki
(Boeh inge
Mannheim)
o
wi h
[a_-32P]dATP
by
using
a
andomly
p imed
DNA
labeling
sys em
ki
(Be hesda
Re-
sea ch
Labo a o ies)
o
we e
digoxigenin
labeled
wi h
a
Ge-
nius
II
ki
and
he
signal
was
enhanced by
Lumi-Phos
530
(Boeh inge
Mannheim).
Sou he n
blo ing
o
o al
DNA
om
cyanobac e ia
o
Hybond-N+
memb anes
(Ame sham)
was
pe o med
unde
alkaline
condi ions
(14).
Hyb idiza ion
was
ca ied
ou
unde
low-s ingency
condi ions
a
55°C
as
de-
sc ibed
p e iously
(1)
o
unde
high-s ingency
condi ions
a
1093
on July 25, 2017 by USE/BTCA.GENERAL UNIVERSITARIA Se illah p://jb.asm.o g/Downloaded om
1094
MURO-PASTOR
ET
AL.
J.
BAC ERIOL.
65°C
as
desc ibed
in
he
ecommenda ions
o
he
manu ac u e
o
Hybond-N+
memb anes
by
using
5
mM
KHP04
bu e
(pH
7.2)
con aining
1
mM
EDTA
and
0.2%
sodium
dodecyl
sul a e
(SDS)
o
wash
he
il e s.
PHOGE- esol ed
agmen s
o
DNA
we e
blo ed
o
GeneSc een
memb anes
(DuPon ),
and
hy-
b idiza ion
was
ca ied
ou
as
desc ibed
p e iously
(4,
11).
Plasmid
RP-4
was
diges ed
wi h
EcoRV
be o e
labeling
wi h
32p.
Hyb idiza ion
o
2P-labeled
RP-4
wi h
HindIII
o
EcoRV
diges s
o
genomic
DNA
om
s ains
AMP2
and
PCC
7120
was
ca ied
ou
unde
high-s ingency
condi ions.
Ma ings
be ween
cyanobac e ia.
Cells
o
s ain
NW18
and
o
a
dono
s ain
ca ying
he
np
gene
in eg a ed
in
i s
genome
we e
ha es ed
and
washed
wi h
BG11
medium
by
low-speed
cen i uga ion,
mixed,
and
sp ead
on
Nuclepo e
REC-85
il e s
se
a op
pla es
o
solid
BG11
medium.
The
dono
s ain
used
in
mos
expe imen s
was
AMP2
(16),
bu
s ain
EFI
16(pRL52)
(9)
was
also
ied.
An
amoun
o
cells
con aining
abou
4
,ug
o
chlo ophy!l
o
he
dono
s ain
and
8
,ug
o
chlo ophyll
o
s ain
NW18
was
inocula ed
pe
pla e
(1
,ug
o
chlo ophyll
co e-
sponds
o
ca.
3.3
x
106
cells
o
hese
cyanobac e ia).
A e
48
h
o
incuba ion
unde
g ow h
condi ions,
he
il e s
we e
ans e ed
o
pla es
o
BG11
medium
supplemen ed
wi h
25
,ug
o
NM
pe
ml
and
20
p,g
o
SM
pe
ml
and
u he
incuba ed
o
2
o
4
weeks.
Nuclease
assays.
Nuclease
assays
in
DNA-con aining
SDS-
polyac ylamide
gel
elec opho esis
(SDS-PAGE)
gels
we e
pe o med
essen ially
as
desc ibed
p e iously
(19)
bu
wi h
modi ica ions
(16).
Samples
we e
subjec ed
o
elec opho esis
in
gels
con aining
15
,ug
o
cal
hymus
DNA
( ype
IV;
Sigma)
pe
ml.
A e
elec opho esis,
p o eins
we e
ena u ed
by
gen le
agi a ion
o
he
gels
in
nuclease
bu e ,
and
gels
we e
incuba ed
o e nigh
in
he
same
bu e
o
allow
enzyma ic
deg ada ion
o
embedded
nucleic
acids.
The
gels
we e
hen
s ained
wi h
e hidium
b omide
and
pho og aphed
unde
UV
illumina ion.
Fo
p epa a ion
o
cell
ex ac s
o
cyanobac e ia,
cells
(ca.
200
,ug
o
chlo ophyll)
we e
suspended
in
0.25
ml
o 10
mM
T is-HCl
bu e
(pH
7.5)
con aining
1
mM
phenylme hylsul o-
nyl
luo ide
and
hen
subjec ed
o
eezing
in
liquid
ai
in
a
p echilled
mo a
and
g inding.
The
homogena e
was
cen i-
uged
a
10,000
x
g
o
10
min.
The
esul ing
supe na an
solu ion
cons i u ed
he
cell
ex ac .
P o ein
concen a ion
in
cell
ex ac s
was
es ima ed
as
desc ibed
p e iously
(5).
P o ein
concen a ion
in
cell
suspensions
was
es ima ed
by
he
me hod
o
Ma kwell
e
al.
(15).
RESULTS
AND
DISCUSSION
Loca ion
o
nucA
in
he
a
megaplasmid.
S ain
AMP2
is
a
de i a i e
o
Anabaena
sp.
s ain
PCC
7120
in
which
he
nucA
gene
had
been
inac i a ed
by
he
inse ion
o
C.K3,
a
casse e
bea ing
he
np
gene
om
Tn5
(16).
DNA
o
high
molecula
weigh
was
isola ed
om
PCC
7120
and
AMP2
and
subjec ed
o
diges ion
wi h
Ps I
o
Sall,
and
he
es ic ion
agmen s
hus
gene a ed
we e
esol ed
as
desc ibed
p e iously
(2).
The
pa e n
o
bands
(4,
11)
ob ained
in
each
o
he
wo
diges ions
was
iden ical
o
PCC
7120
and
AMP2,
excep
ha
he
band
co esponding
o
he
o
megaplasmid
(Fig.
1,
a ow
a),
a
plasmid
o
410
kb
p esen
in
Anabaena
sp.
s ain
PCC
7120
ha
bea s
a
single
si e
o
Sall
(3,
4),
was
eplaced
by
bands
o
ca.
390
(Fig.
1,
a ow
c)
and
19
(no
seen
in
Fig.
1)
kb
in
he
Sall
diges
o
AMP2
because
o
he
p esence
o
a
Sall
si e
nea
he
end
o
he
np -con aining
casse e
inse ed
in
nucA
in
AMP2.
In
addi ion,
he
a
megaplasmid
om
AMP2
is
linea -
ized
by
Ps I
(Fig.
1,
a ow
b)
because
o
he
in oduc ion
o
a
Ps I
es ic ion
si e
on
he
inse ed
casse e.
These
esul s
1
2
3
4
b
c
-500
*-
a
-400
-300
-200
100
FIG.
1.
Al e a ion
o
Anabaena
sp.
s ain
PCC
7120
a
megaplasmid
by
he
inse ion
o
casse e
C.K3
in o
he
nucA
gene
encoding
a
suga -nonspeci ic
nuclease.
DNA
o
high
molecula
weigh
om
s ains
AMP2
(lanes
1
and
2)
o
PCC
7120
(lanes
3
and
4)
was
subjec ed
o
diges ion
wi h
Ps I
(lanes
1
and
3)
o
Sall
(lanes
2
and
4)
and
esol ed
by
he
PHOGE
sys em.
A ow
a
poin s
o
plasmid
ao
(410
kb)
in
Sall-diges ed
s ain
PCC
7120
DNA,
whe eas
a ow
c
poin s
o
a
diges ion
p oduc
o
his
plasmid
in
Sall-diges ed
DNA
o
s ain
AMP2.
A ow
b
poin s
o
plasmid
a
in
Ps I-diges ed
DNA
o
s ain
AMP2.
The
posi ions
o
se e al
size
ma ke s
(in
kilobases),
as
deduced
om
a
48.5-kb
lambda
ladde ,
a e
shown
o
he
igh
o
he
igu e.
The
e ec
o
DNA
concen a ion
on
he
dis ance
o
mig a ion
was
dis-
cussed
p e iously
(11).
sugges ed
ha
he
nucA
gene
was
loca ed
in
he
a
megaplas-
mid.
To
es
his
possibili y
u he ,
labeled
plasmid
pRLA1,
which
ca ies
he
nucA
gene
and
lanking
DNA,
was
used
o
p obe
high-molecula -weigh
DNA
o
s ain
PCC
7120
ha
had
been
diges ed
wi h
BlnI,
Ps I,
o
Sall,
esol ed
by
PHOGE,
and
blo ed.
A
s ong
hyb idiza ion
signal
esul ed
in
he
band
ha ep esen s
he
linea ized
a
megaplasmid
(Fig.
2).
Megaplasmid
a,
poo ly
seen
on
he
PHOGE
gels
o
ei he
Ps I
o
BlnI
diges s
o
DNA
om
wild- ype
PCC
7120
( he e
a e
no
si es
o
Ps I
o
BlnI
in
he
(x
megaplasmid),
is
isualized
in
Sou he n
hyb idiza ions
because
some
megaplasmid
linea ized
by
nicking
en e s
he
gel.
Plasmids
anging
in
size
om
ca.
1.5
kb
o
1
Mb
ha e
been
desc ibed
in
he
cyanobac e ia
(10,
17,
26).
All
cyanobac e ial
plasmids
he e o o e
s udied
emain
c yp ic;
i.e.,
beyond
epli-
ca ion
unc ions
(20,
24-26,
30),
he e
a e
no
known
unc ions
encoded
by
hem
excep
ha
a
no mally
silen
copy
o
psbG
has
been
ound
o
be
plasmid
localized
in
Synechocys is
sp.
s ain
PCC
6803
(22).
Ou
da a
show
ha
nucA
ep esen s
he
i s
exp essed
ma ke
o
an
indigenous
cyanobac e ial
plasmid
(11).
T ans e
o
nucA::C.K3
be ween
cyanobac e ial
s ains.
Many
la ge,
bac e ial
plasmids
a e
sel - ansmissible
by
conju-
ga ion
(27).
Conjugal
ans e
o
plasmid
DNA
om
E.
coli
o
Anabaena
sp.
p oceeds
e icien ly
(29),
bu
gene ic
ans e
be ween
cyanobac e ial
cells
(see,
e.g.,
e e ence
23)
has
no
been
unequi ocally
demons a ed
p e iously.
The
nucA
gene,
and
he e o e
he
nucA
::C.K3
cons uc ion
p esen
in
s ain
AMP2,
ep esen s
a
ma ke
o
he
a-
megaplasmid
o
Anabaena
sp.
s ain
PCC
7120.
Conjuga ion
be ween
cya-
nobac e ia
was
sough
by
using
his
ma ke .
We
sough
o
ans e
nucA::C.K3
om
s ain
AMP2
o
NW18,
an
Sm
de i a i e
o
Anabaena
sp.
s ain
PCC
7118.
Like
i s
pa en
s ain
PCC
7118,
s ain
NW18
is
unable
o
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B
1
2
3
4
5
a
a
11.38-
4.4-
2.61-
1.7-
FIG.
2.
Localiza ion
o
he
nucA
gene
in
he
a
megaplasmid.
(A)
High-molecula -weigh
DNA
om
Anabaena
sp.
s ain
PCC
7120
was
diges ed
wi h
BlnI
(lane
1),
Ps I
(lane
2),
o
Sall
(lane
3),
and
he
agmen s
we e
esol ed
by
PHOGE.
Lane
4
con ains
lambda
DNA
oligome s.
(B)
Hyb idiza ion
o
he
PHOGE- esol ed
agmen s
wi h
32P-labeled
nucA-con aining
plasmid
pRLA1
is
shown.
The
posi ion
o
he
linea ized
a
megaplasmid
is
indica ed.
The
posi ions
o
se e al
size
ma ke s
(in
kilobases),
as
deduced
om
he
48.5-kb
lambda
ladde ,
a e
shown
o
he
igh
o
he
igu e.
de elop
he e ocys s
o
o
ix
dini ogen
unde
ae obic
condi-
ions,
whe eas
AMP2,
like
i s
pa en
PCC
7120,
makes
he e o-
cys s
and
can
g ow
on
dini ogen
unde
ae obic
condi ions.
Anabaena
sp.
s ain
PCC
7120
and
i s
de i a i e
AMP2
ha e
he
410-kb
megaplasmid
men ioned
abo e,
whe eas
de i a i e
NW18
o
Anabaena
sp.
s ain
PCC
7118
bea s
a
510-kb
plasmid,
de ec ed
by
pulsed- ield
gel
elec opho esis
o
un e-
s ic ed
DNA
(da a
no
shown).
Cells
o
s ains
AMP2
and
NW18
we e
mixed
and
sp ead
on
il e s
se
a op
pla es
o
solid
cyanobac e ial
medium
(see
Ma e ials
and
Me hods
o
de-
ails).
F om
a
o al
amoun
o
ca.
6.6
x
108
cells
o
s ain
NW18
and
ca.
3.3
x
108
cells
o
s ain
AMP2,
in
se e al
expe imen s,
117
Nm
Sm
colonies
we e
ob ained.
Se en y-six
o
hese
colonies
we e
es ed
o
he
abili y
o
g ow
on
dini ogen
unde
ae obic
condi ions
(an
unselec ed
ma ke ),
and
all
we e
ound
o
be
unable
o
do
so,
implying
ha
hey
we e
Nm
de i a i es
o
s ain
NW18
a he
han
Sm
de i a-
i es
o
s ain
AMP2.
Because
we
ha e
ound
ha
spon aneous
Nm
mu an s
appea
a
a
equency
o
less
han
10-9
pe
cell,
Nm
de i a i es
o
s ain
NW18
we e
expec ed
o
ha e
e-
cei ed
he
C.K3
casse e
om
s ain
AMP2
a he
han
o
ha e
a isen
om
spon aneous
mu a ion.
Sou he n
blo
analysis
o
o al
DNA
isola ed
om
se e al
Nm
Sm
de i a i es
ha
was
p obed
wi h
a
32P-labeled,
C.K3-con aining
DNA
agmen
showed
ha
hose
de i a i es
con ained
he
C.K3
casse e
(Fig.
3A).
In
addi ion,
a
Sou he n
blo
o
o al
DNA
isola ed
om
s ains
AMP2
and
NW18
and
i e
Nm
Sm
de i a i es
was
p obed
wi h
a
32P-labeled
in e nal
agmen
o
he
nucA
gene
(Fig.
3B).
The
esul s
ob ained
(see
lowe
band
in
each
lane)
indica e
ha
he
nucA::C.K3
cons uc
o iginally
p esen
in
s ain
AMP2
( ha
esul s
in
a
Clal
band
o
ca.
5.7
kb)
had
eplaced
he
wild- ype
nucA
gene
o
s ain
NW18
( ha
esul s
in
a
ClaI
band
o
ca.
4.4
kb)
in
he
i e
Nm
Sm
de i a i es
ha
we e
examined.
Mo eo e ,
unlike
NW18,
hese
s ains
exhib-
i ed
no
ac i i y
o
he
29-kDa
nuclease
in
SDS-PAGE
assays
(Fig.
4).
The
uppe
nucA-hyb idizing
bands
obse ed
in
AMP2
FIG.
3.
Sou he n
analysis
o
Nm
Sm
de i a i es.
To al
DNA
om
dono
s ain
AMP2,
ecipien
s ain
NW18,
and
i e
Nm
Sm
de i a-
i es
(lanes
1
o
5)
was
diges ed
wi h
Clal
and
hyb idized
wi h
a
1.1-kb
BamHI
DNA
agmen
con aining
he
TnS
np
gene
in
casse e
C.K3
(A)
o
a
473-bp
HindIlI-Bs NI
DNA
agmen
in e nal
o
he
nucA
gene
o
Anabaena
sp.
s ain
PCC
7120
(B).
Hyb idiza ion
was
ca ied
ou
unde
condi ions
o
high
s ingency
(see
Ma e ials
and
Me hods).
The
posi ions
o
se e al
size
s anda ds
(in
kilobases)
a e
shown
o
he
le
o
he
igu e.
and
NW18
in
Fig.
3B
do
no
co espond
o
a
gene
ha
encodes
an
ac i e
29-kDa
nuclease
(16)
(Fig.
4).
Figu e
5
p esen s
a
Sou he n
blo
o
PHOGE- esol ed
high-molecula -weigh
DNA
om
s ains
PCC
7120,
AMP2,
and
NW18,
and
wo
Nm
Sm
de i a i es,
p obed
wi h
pRLA1
(nucA).
The
a
megaplasmid
o
Anabaena
sp.
s ain
PCC
7120
has
single
es ic ion
si es
o
SphI
and
Sall
ha
a e
abou
105
kb
dis an
om
one
ano he
(4).
In oduc ion
o
C.K3
in o
nucA
added
a
second
Sall
si e
ca.
19
kb
dis an
om
he
i s
(sizing
da a
no
shown),
a
Ps I
si e
a
which
he
plasmid
can
be
linea ized,
and
a
second
SphI
si e
ca.
126
kb
dis an
om
he
i s
(Fig.
5,
lanes
4
and
5;
Fig.
1).
The
510-kb
megaplasmid
o
s ain
NW18
also
appea s
o
ha e
single
si es
o
SphI
and
Sall
(no
shown)
as
well
as
a
locus
ha
hyb idizes
wi h
pRLA1
(Fig.
0
N
co
<
Z
1
2
3
4
5
29-kDa
nuclease
FIG.
4.
Nuclease
ac i i y
o
cell
suspensions
om
wild- ype
Anabaena
sp.
s ain
PCC
7120,
dono
s ain
AMP2,
ecipien
s ain
NW18,
and
se e al
Nm
Sm
de i a i es
(lanes
1
o
5).
Cul u e
samples
con aining
5
o
10
,ug
o
p o ein
we e
used.
The
loca ion
o
he
29-kDa
nuclease
(NucA)
is
indica ed.
A
1
234
A
1
234
!
I:
N
Co
0z
2
3
4
5
VOL.
176,
1994
1
im.
I-
7717.
`
77"77777..
I
on July 25, 2017 by USE/BTCA.GENERAL UNIVERSITARIA Se illah p://jb.asm.o g/Downloaded om
1096
MURO-PASTOR
ET
AL.
Sphl
Ps l
Sall
1
23
4
5
X1
23
4
5
X
1
23
4
5
510
kb
**
***
440
0;
i*
0
i
*
i
i
;
X;0i-
410kb
FIG.
5.
Sou he n
analysis
o
PHOGE
gel
o
NM
SM
de i a i es.
Aga ose
beads
con aining
DNA
de i ed
om
NW18
(lanes
1)
NM
SM
de i a i e
2
(lanes
2),
NM
Sm
de i a i e
(lanes
3),
AMP2
(lanes
4),
and
wild- ype
PCC
7120
(lanes
5)
we e
subjec ed
o
PHOGE
analysis
as
desc ibed
p e iously
(11)
a e
diges ion
wi h
Sphl
(pa ial
diges
[
1
1]),
Ps l,
o
Sall.
A
Sou he n
blo
o
he
gel
was
p obed
wi h
pRLAL.
X,
beads
con aining
conca eme s
o
DNA
om
coliphage
lambda;
*,
size
o
his
agmen
measu ed
sepa a ely.
5,
lanes
1).
We
shall
e e
o
his
locus
as
he
nucA
gene
o
NW18.
The
NM
Sm
de i a i es
con ain
plasmids
ha
a e
app oxima ely
equal
in
size
o
he
ca.
510-kb
megaplasmid
o
NW18
(Fig.
5,
lanes
1
o
3;
Ps l
diges ion).
Upon
pa ial
diges ion
wi h
Sphl,
hese
plasmids
as
well
as
he
smalle
a.
megaplasmid
o
AMP2
gi e
ise
o
a
agmen
o
abou
126
kb
(Fig.
5,
lanes
2
o
4;
SphI
diges ion)
ha
measu es
he
dis ance
om
SphI
in
CdK3
(in
nucA)
o
he
unique
SphI
o
he
megaplasmid.
SaIl
cu s
he
same
megaplasmids
and
gi es
ise
o
agmen s
wi h
a
size
close
o
19
kb
(sizing
da a
no
shown).
(Bands
p esen
in
lanes
3
ha
a e
absen
om
lanes
2
a e
o
unclea
o igin.)
Acco ding
o
hese
esul s,
he
Sphl
and
Sail
si es
a e
as
a
om
nucA
in
he
la ges
megaplasmid
o
he
NM
SM
de i a i es
o
s ain
NW18
as
hey
a e
in
he
a
megaplasmid
o
PCC
7120.
These
esul s
sugges ed
ha ,
in
he
wo
NM
SM
de i a i es
in es iga ed,
he
nucA::C.K3
ma ke
had
been
inco po a ed
by
ecombina ion
in
he
megaplasmid
o
s ain
NW18.
The
p esence
o
he
CK3
ma ke
in
he
510-kb
plasmid
o
he
NM
SM
de i a i es
o
s ain
NW18
was
con i med
by
hyb idiza ion
using
casse e
CaK3
as
a
p obe.
When
DNA
diges ed
wi h
Sall
was
es ed,
Cu3
was
obse ed
o
hyb idize
(unde
he
condi ions
o
hyb idiza ion
used
o
he
PHOGE
gels)
only
o
he
19-kb
band
(da a
no
shown).
T ans e
o
he
C
a3
casse e
om
s ain
AMP2
o
s ain
NW18
was
no
a ec ed
by
he
p esence
o
bo ine
panc ea ic
DNase
(g ade
II;
Boeh inge
Mannheim),
which
was
added
a
7.5
s g/ml
bo h
o
he
mix u e
o
cells
be o e
hey
we e
sp ead
in
he
solid
medium
and
o
he
medium
in
he
pla es.
The e-
o e,
he
ans e
p esumably
did
no
in ol e
DNA
ee
in
he
medium.
T ans e
by
conjuga ion
is
possible,
al hough
o he
mechanisms
a e
no
excluded
(21).
Because
he
dono
s ain
used
in
he
ma ings,
AMP2,
is
i sel
an
exconjugan ,
de i ed
om
Anabaena
sp.
s ain
PCC
7120,
which
o igina ed
in
an
RP-4-p omo ed
conjuga ion
wi h
E.
coli,
i
was
possible
ha
s ain
AMP2
bo e
he
a
genes
om
RP-4
and
ha
hese
genes
we e
esponsible
o
he
phenom-
enon
o
DNA
ans e
be ween
cyanobac e ia
ha
we
ha e
obse ed.
The e o e,
we
es ed
by
hyb idiza ion
he
p esence
o
DNA
sequences
om
RP-4
in
s ain
AMP2.
No
e idence
o
he
p esence
o
RP-4
o
RP-4
agmen s
in
his
s ain
was
ound
(da a
no
shown).
To
es
whe he
ans e
occu s
speci ically
o
genes
in
he
a
megaplasmid,
we
sough
o
ans e
he
np
gene
om
s ain
EF116(pRL52)
o
s ain
NW18.
EF116
is
a
mu an
o
Anabaena
sp.
s ain
PCC
7120
ha
is
unable
o
ix
dini ogen
unde
ae obic
condi ions,
and
pRL52
is
an
np -bea ing
shu le
plasmid
whose
inse
complemen s
he
mu a ion
in
EF116
(9,
28).
In
ma ings
in ol ing, in
se e al
expe imen s,
a
o al
amoun
o
ca.
2.3
x
108
cells
o
s ain
NW18
and
ca.
1.15
x
108
cells
o
s ain
EF116(pRL52),
no
Nm
Sm
colonies
we e
ob ained.
These
nega i e
esul s
p o ide
no
e idence
ha
genes
loca ed
ou side
he
ao
megaplasmid
can
be
ans e ed.
Widesp ead
occu ence
o
nucA
in
he e ocys - o ming
cya-
nobac e ia.
I
was
o
in e es
o
es
how
widesp ead
he
nucA
gene
is
in
cyanobac e ia
o he
han
s ain
PCC
7120.
Judging
by
DNA-DNA
eassocia ion
expe imen s,
Anabaena
sp.
s ains
010
u
C)~~~~~~~~~"
.
C
qj
UC
a^
qb
s
.
U
xu
e~
xu
xu
x
Cu
2
0
o0
CQ
11.38
*
10.50'
*
4.40-
2.61
-
2.06
-
1.70
-
1.11
-
0.66
-
FIG.
6.
Hyb idiza ion
o
DNA
om
se e al
he e ocys - o ming
cyanobac e ia
wi h
he
nucA
gene
om
Anabaena
sp.
s ain
PCC
7120.
To al
DNA
om
each
o
he
cyanobac e ia
was
diges ed
wi h
EcoRV
and
hyb idized
o
a
473-bp
HindIII-Bs NI
agmen
in e nal
o
he
nucA
gene
o
Anabaena
sp.
s ain
PCC
7120.
Hyb idiza ion
was
ca ied
ou
unde
condi ions
o
high
s ingency
(see
Ma e ials
and
Me hods).
The
posi ions
o
size
s anda ds
(in
kilobases)
a e
indica ed
o
he
le
o
he
igu e.
J.
BACTERIOL.
on July 25, 2017 by USE/BTCA.GENERAL UNIVERSITARIA Se illah p://jb.asm.o g/Downloaded om
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TRANSFER
BETWEEN
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1097
TABLE
1.
P esence
o
absence
o
nucA
gene-hyb idizing
DNA
and
a
nuclease
o
abou
29
kDa
in
some
unicellula
and
ilamen ous,
he e ocys - o ming
cyanobac e ia
S aina
Hyb idi-
29-kDa
za ionb
nucleasec
G oup
A
Anabaena
a iabilis
ATCC
29413-FD
+
+
Anabaena
sp.
s ain
PCC
6411
(ATCC
27898)
+
+
Anabaena
sp.
s ain
PCC
7118
(ATCC
27892)
+
+
Anabaena
sp.
s ain
PCC
7119
(ATCC
29151)
+
+
Nos oc
sp.
s ain
PCC
6705
(ATCC
29131)
+
+
Nos oc
sp.
s ain
PCC
6719
(ATCC
29105)
+
+
G oup
B
Calo h ix
sp.
s ain
PCC
7601
+
+
Fische ella
muscicola
UTEX
1829
+
Nos oc
ellipsospo um
B
1453-7d
+
+
Nos oc
sp.
s ain
ATCC
43237
+
NDe
Nos oc
sp.
s ain
PCC
7107
(ATCC
29150)
+
Nos oc
sp.
s ain
PCC
7422
(ATCC
29132)
+
+
Nos oc
sp.
s ain
PCC
7413
(ATCC
29106)
+
+
G oup
C
Gloeobac e
sp.
s ain
PCC
7421
(ATCC
29082)
-
ND
Synechococcus
sp.
s ain
PCC
7202
(ATCC
29140)
-
-
Synechococcus
sp.
s ain
PCC
7425
(ATCC
29141)
-
-
Synechococcus
sp.
s ain
PCC
7942
-
-
Synechocys is
sp.
s ain
PCC
6308
(ATCC
27150)
-
-
Synechocys is
sp.
s ain
PCC
6803
(ATCC
27184)
-
-
a
G oup
A
includes
de i a i e
FD
o
A.
a iabilis
(6a)
and
cyanobac e ia
ha ,
as
desc ibed
by
Lachance
(12),
a e
closely
ela ed
o
Anabaena
sp.
s ain
PCC
7120.
a
G oup
B
includes
he e ocys - o ming
cyanobac e ia
ha
a e
no
(see
oo no e
d
below)
as
closely
ela ed
o
Anabaena
sp.
s ain
PCC
7120.
G oup
C
includes
unicellula
s ains.
ATCC,
Ame ican
Type
Cul u e
Collec ion;
PCC,
Pas eu
Cul u e
Collec ion;
UTEX,
Uni e si y
o
Texas
Cul u e
Collec ion.
N.
ellipsospo um
B
1453-7
was
ob ained
om
he
Uni e si y
o
Go ingen.
b
Hyb idiza ion
was
ca ied
ou
a
high
s ingency
o
s ains
in
g oup
A
and
a
low
s ingency
o
s ains
in
g oups
B
and
C
(see
Ma e ials
and
Me hods).
c
P esence
o
a
nuclease
o
abou
29
kDa
as
shown
by
he
SDS-PAGE
nuclease
assay
(see
Ma e ials
and
Me hods)
o
cell
ex ac s
(30
,ug
o
p o ein
o
s ains
in
g oup
A
and
90
,ug
o
p o ein
o
s ains
in
g oups
B
and
C).
dN.
ellipsospo um
ga e
a
pa icula ly
s ong
hyb idiza ion
signal
and,
as
p e iously
discussed
(8),
may
be
closely
ela ed
o
s ains
in
g oup
A.
'ND,
no
de e mined.
PCC
6411,
PCC
7118,
and
PCC
7119
and
Nos oc
sp.
s ains
PCC
6719
and
PCC
6705
a e
phylogene ically
e y
closely
ela ed
o
s ain
PCC
7120
(12).
We
ha e
in es iga ed,
by
means
o
Sou he n
analysis,
whe he
he
nucA
gene
is
p esen
in
hese
cyanobac e ia.
A
32P-labeled
in e nal
p obe
o
he
nucA
gene
(co esponding
o
amino
acids
52
o
210
o
he
NucA
p o ein)
was
used
o
hyb idiza ion
wi h
o al
DNA
om
he
cyanobac e ia
men ioned
abo e
unde
condi ions
o
high
s ingency.
The
esul s
in
Fig.
6
show
ha
all
o
hese
s ains,
as
well
asA.
a iabilis
ATCC
29413,
ca y
DNA
sequences
ha
exhibi
homology
o
nucA.
DNA
om
o he
he e ocys - o m-
ing
cyanobac e ia
ha
a e
no
as
closely
ela ed
o
s ain
PCC
7120
and
om
six
di e se
unicellula
cyanobac e ia
was
es ed
o
hyb idiza ion
wi h
he
nucA
p obe
om
s ain
PCC
7120
unde
condi ions
o
low
s ingency.
All
o
he
ilamen ous,
he e ocys - o ming
s ains
es ed,
bu
none
o
he
unicellula
s ains,
showed
hyb idiza ion
o
he
nucA
p obe
(da a
no
shown).
The
esul s
ob ained
a e
summa ized
in
Table
1.
Table
1
also
shows
he
esul s
o
es ing,
by
means
o
a
DNA-
con aining,
SDS-PAGE
assay
(see
Ma e ials
and
Me hods),
whe he
some
o
he
cyanobac e ial
s ains
con ain
a
nuclease
o
abou
29
kDa,
which
is
he
size
o
he
p o ein
encoded
by
he
nucA
gene
o
Anabaena
sp.
s ain
PCC
7120.
Wi h
he
excep-
ion
o
Fische ella
muscicola
and
Nos oc
sp.
s ain
PCC
7107,
in
which
no
nuclease
ac i i y
was
de ec ed,
all
o
he
s ains
es ed
whose
DNA
hyb idized
wi h
he
nucA
p obe
also
exhibi ed
a
nuclease
o
ca.
29
kDa.
I
is
possible
ha
he
nucA-hyb idizing
sequences
in
F.
muscicola
and
Nos oc
sp.
s ain
PCC
7107
do
no
encode
an
ac i e
nuclease
o
ha
assay
by
SDS-PAGE
is
no
sui able
o
he
nuclease
om
hose
pa icula
cyanobac-
e ia.
We
conclude
ha
he
nucA
gene
is
commonly
p esen
in
he
he e ocys - o ming
cyanobac e ia
bu
no
in
some
o he
cyanobac e ia.
I
he
gene
ans e
ha
we
obse ed
was
due
o
conjuga ion,
conjuga ion
could
accoun
o
la e al
ans e
o
a
megaplasmid,
and
hus
o
nucA,
be ween
s ains.
Concluding
ema ks.
The
esul s
p esen ed
in
his
wo k
show
ha
he
nucA
gene
is
loca ed
in
he
a.
megaplasmid
o
Anabaena
sp.
s ain
PCC
7120
and
ha
a
ma ked
e sion
o
nucA
(nucA::np )
can
be
ans e ed
om
s ain
PCC
7120
di ec ly
o
ano he
s ain
o
Anabaena
sp.
Al hough
he
e iciency
o
ans e
plus
ecombina ion
appea s
low,
i
may
be
amenable
o
inc ease
by
mu a ion
(27).
The
inding
o
a
na u ally
occu ing
ans e
sys em
ep esen s
a
no el
aspec
o
he
biology
o
cyanobac e ia.
Fu he
esea ch
will
be
neces-
sa y
i
he
p ocess
is
o
be
made
use ul
as
a
gene ic
ool.
The
ac
ha
he
a
megaplasmid
om
PCC
7120
ecom-
bined
wi h
he
megaplasmid
om
PCC
7118
a he
han
eplacing
i
may
sugges
ha
he
a
megaplasmid
is
no
ans e ed
en i ely,
al hough
o he
in e p e a ions
a e
possi-
ble.
T ans e
by
conjuga ion
migh
be
abo ed
be o e
he
plasmid
is
comple ely
ans e ed
so
ha
(i)
he
equency
o
ans e
o
a
plasmid-bo ne
gene ic
ma ke
would
depend
on
i s
dis ance
om
he
o igin
o
ans e ,
and
(ii)
acquisi ion
o
a
ma ke
would
depend
upon
i s
inco po a ion
in o
an
endoge-
nous
eplicon.
I
ecombina ion
is
a
low- equency
e en ,
as
i
is
upon
conjugal
ans e
om
E.
coli
(ou
unpublished
obse -
a ions),
ans e
o
nucA::np
o
NW18
may
be
a
much
mo e
equen
e en
han
we
obse ed.
ACKNOWLEDGMENTS
We
hank
Nancy
Wood
o
Anabaena
sp.
s ain
NW18
and
Ma ia
Isabel
Mu o-Pas o
o
help
wi h
some
hyb idiza ions.
Wo k
in
Se ille
was
suppo ed
by
he
Jun a
de
Andalucia
(g oup
no.
3057)
and
by
he
Di ecci6n
Gene al
de
In es igaci6n
Cien i ica
y
Tecnica
(g an
no.
PB90-0114),
Spain.
Wo k
in
Eas
Lansing
was
suppo ed
by
he
U.S.
Depa men
o
Ene gy
g an
DE-FG02-
90ER20021
and
he
Cen e
o
Mic obial
Ecology.
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