Wolk CP, Vonshak A, Kehoe P, Elhai J.. Construction of shuttle vectors capable of conjugative transfer from Escherichia coli to nitrogen-fixing filamentous cyanobacteria. Proc Natl Acad Sci USA 81: 1561-1565

Abstract

Wild-type cyanobacteria of the genus Anabaena are capable of oxygenic photosynthesis, differentiation of cells called heterocysts at semiregular intervals along the cyanobacterial filaments, and aerobic nitrogen fixation by the heterocysts. To foster analysis of the physiological processes characteristic of these cyanobacteria, we have constructed a family of shuttle vectors capable of replication and selection in Escherichia coli and, in unaltered form, in several strains of Anabaena. Highly efficient conjugative transfer of these vectors from E. coli to Anabaena is dependent upon the presence of broad host-range plasmid RP-4 and of helper plasmids. The shuttle vectors contain portions of plasmid pBR322 required for replication and mobilization, with sites for Anabaena restriction enzymes deleted; cyanobacterial replicon pDU1, which lacks such sites; and determinants for resistance to chloramphenicol, streptomycin, neomycin, and erythromycin.

Full text

Proc.
Natl.
Acad.
Sci.
USA
Vol.
81,
pp.
1561-1565,
March
1984
Microbiology
Construction
of
shuttle
vectors
capable
of
conjugative
transfer
from
Escherichia
coli
to
nitrogen-fixing
filamentous
cyanobacteria
(Anabaena/plasmid
RP4/plasmid
pBR322/restriction
sites/photosynthesis)
C.
PETER
WOLK,
AVIGAD
VONSHAK*,
PATRICIA
KEHOE,
AND
JEFFREY
ELHAI
MSU-DOE
Plant
Research
Laboratory,
Michigan
State
University,
East
Lansing,
MI
48824
Communicated
by
Charles
J.
Arntzen,
November
16,
1983
ABSTRACT
Wild-type
cyanobacteria
of
the
genus
Ana-
baena
are
capable
of
oxygenic
photosynthesis,
differentiation
of
cells
called
heterocysts
at
semiregular
intervals
along
the
cyanobacterial
filaments,
and
aerobic
nitrogen
fixation
by
the
heterocysts.
To
foster
analysis
of
the
physiological
processes
characteristic
of
these
cyanobacteria,
we
have
constructed
a
family
of
shuttle
vectors
capable
of
replication
and
selection
in
Escherichia
coli
and,
in
unaltered
form,
in
several
strains
of
Anabaena.
Highly
efficient
conjugative
transfer
of
these
vec-
tors
from
E.
coli
to
Anabaena
is
dependent
upon
the
presence
of
broad
host-range
plasmid
RP-4
and
of
helper
plasmids.
The
shuttle
vectors
contain
portions
of
plasmid
pBR322
required
for
replication
and
mobilization,
with
sites
for
Anabaena
re-
striction
enzymes
deleted;
cyanobacterial
replicon
pDUl,
which
lacks
such
sites;
and
determinants
for
resistance
to
chloramphenicol,
streptomycin,
neomycin,
and
erythromycin.
Many
filamentous
cyanobacteria
fix
dinitrogen
aerobically
within
specialized
cells
called
heterocysts
that
differentiate
at
semiregular
intervals
along
the
filaments.
All
cyanobac-
teria
are
capable
of
oxygenic
photosynthesis.
Genetic
meth-
ods
usable
for
study
of
nitrogen
fixation,
differentiation,
pat-
tern
formation,
and
photosynthesis
in
these
organisms
have
long
been
sought.
Several
unicellular
cyanobacteria
can
be
transformed
by
DNA
in
the
growth
medium
(1-3).
Shuttle
vectors,
plasmids
able
to
replicate
in
Escherichia
coli
and
in
an
alternative
host,
have
been
constructed
for
two
such
strains,
Anacystis
nidulans
strain
R2
and
Agmenellum
quadruplicatum
PR-6
(4-6).
An
Anacystis
gene
cloned
in
a
shuttle
vector
in
E.
coli
was
returned
to
the
cyanobacterium
by
transformation
(7).
To
date,
no
reproducible
transformation
system
is
known
for
filamentous
cyanobacteria.
Conjugation
provides
an
alternative
approach
to
transfer
of
cloned
DNA.
RP-4
and
related
plasmids
can
transfer
themselves
or
derivatives
of
themselves
into
a
wide
range
of
Gram-negative
bacteria
(8-10).
Cyanobacteria
have
the
structure
and
wall
composition
of
Gram-negative
bacteria
(11).
Delaney
and
Reichelt
(12)
have
described
a
very
low
frequency
transfer
of
R68.45,
closely
related
to
RP-4,
into
a
unicellular
cyanobacterium,
but
establishment
of
RP-4
in
a
filamentous
cyanobacterium
has
not
been
observed.
One
can
utilize
the
conjugal
properties
of
RP-4
without
demanding
that
that
plasmid
replicate
in
a
new
host.
Several
conjugative
plasmids
can
promote
the
transfer
of
derivatives
of
pBR322
between
strains
of
E.
coli
(13)
or
from
E.
coli
to
other
Gram-negative
bacteria
(14,
15)
so
long
as
the
bom
(ba-
sis
of
mobility)
region
of
the
transferred
plasmid
is
left
intact
and
requisite
trans-acting
factors
are
present.
Such
factors
are
provided
by
pDS4101
(ColK::Tnl)
or
pGJ28
(ColD
Kmr;
ref.
13).
Shuttle
vectors
based
on
pBR322
may
thereby
as-
sume
the
wide
conjugal
range
of
RP-4.
We
report
the
construction
of
a
hybrid
between
pBR322
and
plasmid
pDU1
(16)
from
the
filamentous
cyanobacter-
ium
Nostoc.
Because
restriction
endonucleases
present
in
strains
of
cyanobacteria
(17)
apparently
reduce
retention
of
DNA
transferred
into
those
cyanobacteria
(4,
18),
the
hybrid
plasmid
was
restructured
to
eliminate
sites
for
restriction
en-
zymes
present
in
several
strains
of
Anabaena.
Additional
antibiotic-resistance
determinants
were
added,
lest
an
orga-
nism
be
unable
to
use
any
one
such
determinant.
The
deriva-
tives
of
the
hybrid
plasmid
proved
to
be
shuttle
vectors,
ca-
pable
of
RP-4-mediated
transfer
into
several
strains
of
Ana-
baena
and
of
replication
in
those
strains.
MATERIALS
AND
METHODS
Anabaena
sp.
PCC
7120,
Anabaena
sp.
U.
Leningrad
strain
458
(PCC
7118),
Anabaena
sp.
U.
Tokyo
M-131,
and
Nostoc
sp.
PCC
7524
were
grown
with
nitrate
as
described
(19).
An-
acystis
nidulans
strain
R2
was
grown
in
medium
BG-11
(20).
E.
coli
was
grown
in
L
broth,
supplemented
as
appropriate
with
none,
one,
or
two
of
the
following:
25
jig
of
chloram-
phenicol
(Cm)
per
ml,
10-25
pg
of
streptomycin
(Sm)
per
ml,
50
,ug
of
kanamycin
(Km)
per
ml,
50
,g
of
ampicillin
(Ap)
per
ml.
Plasmids
were
isolated
from
cyanobacteria
by
established
methods
(5,
21,
22)
or
minor
variations
thereof,
and
from
E.
coli
strains
HB101
and
Gm48
(dam-
dcm-)
essentially
as
described
(23).
Plasmids
were
digested
with
restriction
en-
zymes
from
New
England
BioLabs
and
Bethesda
Research
Laboratories
in
buffers
recommended
by
the
suppliers,
or
slight
variations
thereof.
Recombinant
DNA
techniques
were
standard
(23).
For
mating
experiments,
E.
coli
HB101
containing
a
cyanobacterial
hybrid
plasmid
and
either
pGJ28
or
pDS4101,
and
E.
coli
J53(RP-4)
were
grown
in
shaken
test
tubes,
over-
night,
at
37°C,
then
separately
diluted
0.25
ml:
10
ml
of
antibi-
otic-supplemented
L
broth.
After
approximately
2.5
hr
of
growth
in
shaken
flasks,
0.75-ml
portions
were
harvested
(12,000
x
g,
1
min).
The
bacteria
were
washed
with
L
broth,
mixed
or
not
with
other
strains,
centrifuged
as
before,
and
resuspended
in
60
,ul
of
L
broth,
permitting
unselected
trans-
fer
of
RP-4
to
HB101
to
take
place.
Anabaena
strains
becom-
ing
light-limited
at
30°C,
but
still
growing
actively,
were
con-
centrated
20-fold
(12,000
x
g,
1
min);
Anacystis
was
concen-
trated
to
109
cells
per
ml.
Nuclepore
filters
sterilized
in
water
(24)
were
set
atop
solidified
cyanobacterial
media
(19,
20)
supplemented
with
5%
(vol/vol)
L
broth,
in
Petri
plates.
Twenty-microliter
portions
of
each
of
a
series
of
1:10
dilu-
tions
of
the
cyanobacterial
suspensions
were
then
streaked
onto
the
filters,
and
the
streaks
were
dried.
Two-microliter
portions
of
bacterial
suspensions
were
spotted
atop
the
dried
Abbreviations:
Ap,
ampicillin;
Cm,
chloramphenicol;
Em,
erythro-
mycin;
Km,
kanamycin;
Nm,
neomycin;
Sm,
streptomycin;
Tc,
tet-
racycline;
r,
resistant
(resistance);
s,
sensitive
(sensitivity);
bp,
base
pair(s);
kb,
kilobase
pair(s).
*Present
address:
The
Jacob
Blaustein
Institute
for
Desert
Re-
search,
Sede
Boqer
Campus,
Israel
84990.
1561
The
publication
costs
of
this
article
were
defrayed
in
part
by
page
charge
payment.
This
article
must
therefore
be
hereby
marked
"advertisement"
in
accordance
with
18
U.S.C.
§1734
solely
to
indicate
this
fact.

Proc.
NatL
Acad
Sci.
USA
81
(1984)
streaks
of
cyanobacteria,
and
the
Petri
plates
were
incubated
at
ca.
230C
and
low
light
intensity
for
4-6
hr.
The
filters
were
then
transferred
to
unsupplemented,
solidified
cyanobacter-
ial
medium
and
were
set
at
30'C
and
high
light
intensity
(ca.
3500
erg
cm2
s-).
When
inducible
resistance
to
erythromy-
cin
(Em)
was
to
be
transferred,
the
medium
contained
a
sub-
inhibitory
concentration
(0.1
4g/ml)
of
Em.
Twenty-four
hours
after
the
filters
were
spotted
with
bacteria
[this
period
of
time
provided
for
expression
of
antibiotic
resistance
(24)],
the
filters
were
transferred
to
solidified
cyanobacterial
medi-
um
supplemented
with
selective
concentrations
of
antibiot-
ics
and
returned
to
30'C
and
high
light
intensity.
RESULTS
Cloning
Plasmid
pDUl
from
Nostoc
PCC
7524.
Plasmid
pDU1,
linearized
by
partial
digestion
with
EcoRV,
was
in-
serted
into
the
EcoRV
site
of
plasmid
pBR322
and
used
to
transform
E.
coli
HB101.
One
clone
contained a
10.6-kilo-
base-pair
(kb)
plasmid,
denoted
pVW1,
which
was
chosen
for
detailed
study.
The
three
EcoRV
fragments,
4.1,
1.2,
and
0.98
kb,
of
pDU1
are
retained
in
pVW1
(Fig.
1A)
without
rearrangement
(Fig.
1B);
pBR322
has
been
inserted
within
the
largest
HindIII
fragment
(Fig.
1B,
lanes
1
and
4).
Plasmid
pVW1
and
derivatives
of
it
(see
below)
were
test-
ed
for
restriction
by
additional
endonucleases.
Cloned
pDU1
contained
restriction
sites
shown
in
Fig.
2,
plus
at
least
4
sites
for
Aha
III
(from
a
cyanobacterium)
and
at
least
10
sites
for
Hha
I.
Apa
I,
Ava
I,
Ava
II,
Avr
II,
Bal
I,
BamHI,
Ban
II,
Bgl
II,
BstEII,
Cla
I,
EcoRI,
HgiAI,
Kpn
I,
Nae
I,
Nar
I,
Nco
I,
Nde
I,
Nru
I,
Pst
I,
Sac
II,
Sal
I,
Sau96I,
Sph
I,
Sst
I,
and
Stu
I
failed
to
cut
the
pDU1
portion
of
pVW1.
Enzymes
that
failed
to
cut
pVW1
did
cut
X
DNA
when
X
DNA
was
added
to
the
pVW1
restriction
reaction
mixtures.
Cloned
pDU1
was
found
to
contain
sites
for
Bcl
I,
Pvu
I
(Xor
II),
Pvu
II,
and
Hae
III
(because
the
plasmid
is
cut
by
Xma
III)
although
these
enzymes
did
not
cut
pDU1
from
Nostoc
(16).
Construction
of
Conjugal
Shuttle
Vectors.
Plasmid
pVW1
has
two
disadvantages
for
use
as
a
cloning
vector.
First,
it
has
eight
Ava
II
sites
and
one
Ava
I
site
at
which
it
could
be
restricted
upon
transfer
to
numerous
strains
of
Anabaena
(17).
Second,
its
selectable
markers,
resistance
to
tetracy-
cline
(Tc)
and
Ap,
are
not
well
suited
for
conjugal
transfer
to
cyanobacteria:
Tc
breaks
down
rapidly
in
the
light
used
for
growth
of
cyanobacteria;
and
resistance
to
Ap
is
mediated
by
a
1-lactamase,
secretion
of
which
by
a
donor
strain
could
protect
a
recipient
strain.
Resistance
to
Cm
encoded
by
pBR328
(25)
has
neither
of
these
disadvantages.
A
Sau3AI
fragment
containing
the
en-
tire
structural
gene
for
Cm
acetyltransferase
was
therefore
ligated
into
the
BamHI
site
of
pVW1
(Figs.
3
and
4).
Plas-
mids
in
three
Cmr
Apr
Tcs
transformants
of
E.
coli
all
con-
tained
the
same
insert
in
the
same
orientation
(transcription
antiparallel
to
that
of
the
Apr
gene).
One
of
the
plasmids
was
designated
pVWlC.
To
remove
the
Ava
I
and
Ava
II
sites
from
pVWlC,
this
plasmid
was
digested
with
Ava
II,
treated
with
alkaline
phos-
phatase,
extracted
with
phenol,
digested
with
Sau96I,
ligat-
ed,
and
used
to
transform
E.
coli;
cells
were
selected
for
resistance
to
Cm.
The
5'
extensions
of
the
Sau96I
sites
are
trinucleotides
and
therefore
not
self-complementary.
Theo-
retically,
possible
repolymerizations
(see
refs.
25-27)
must
contain
Sau96I
fragments
[in
pBR322
coordinates
(26,
27)]
from
bp'173
to
bp
524,
containing
the
EcoRV
site,
hence
the
cloned
pDU1,
and
the
erstwhile
BamHI
site,
hence
the
Cmr
determinant;
from
bp
1951
to
bp
3411,
containing
bom
and
the
vegetative
origin;
and
either
bp
4346
through
bp
0
to
bp
172
(containing
a
unique
site
for
Cla
I)
or
bp
3412
to
bp
3490,
either
providing
a
necessary
linker.
Plasmid
pRL1,
used
for
subsequent
constructions,
has
the
structure
shown
in
Fig.
4.
It
lacks
sites
for
Ava
I,
Ava
II,
and
Avr
II.
Determinants
for
2
3
kb
Or.
-48.50
-23.13
9.42
6.56
4.36
2.32
2.02
1
.63
0.51
A
approx.
bp
4800-
4590-
4130-
3420-
2010-
1240-
1110
-
910
-
810
-
500-
430
-
2
3
4
5
6
7
standards
bp
-
9419
-
6559
-4363
*
2319
2335
-2023
-
1810
-
1746
-
1632
-
1434
-1064
-745
*
506,517
-396
B
FIG.
1.
Electrophoretograms
(agarose
0.6%
in
A,
1.2%
in
B)
of
products
of
digestion
of
pDU1
(A,
lane
1;
B,
lanes
1-3),
pVW1
(A,
lane
2,
B,
lanes
4-7),
and
pBR322
(A,
lane
3).
The
positions
of
mo-
lecular
weight
markers,
derived
by
digestion
of
pBR322
with
Hinfl,
Bgl
I,
and
Ava
II
and
of
X
DNA
with
HindIII,
are
shown.
(A)
Re-
striction
by
EcoRV.
(B)
Restriction
by
HindIII
[lanes
1
(partial
di-
gest)
and
4],
EcoRV
(lane
5),
HindIII
and
EcoRV
[lanes
2
(partial
digest)
and
6],
and
Bgl
I
and
EcoRV
(lanes
3
and
7).
Bands
at
ca.
3420,
1110,
810,
500,
and
430
base
pairs
(bp)
(underlines)
in
lane
1
of
B
are
seen
in
limit
digests.
Bands
at
ca.
4590
(-
3420
+
1110),
4130
(a
3420
+
500),
2010
(a
810
+
1110),
1240
(=
810
+
430),
and
910
(a
500
+
430)
bp
confirm
the
sequence
(ref.
16;
Fig.
2)
of
HindIII
frag-
ments
in
pDU1.
Equality
of
certain
fragment
lengths
for
pDU1
and
pVW1
(lanes
1
and
4,
2
and
6,
and
3
and
7)
is
possible
only
if
the
sequence
and
relative
orientations
of
the
EcoRV
fragments
of
pDU1
are
retained
in
pVW1.
1562
Microbiology:
Wolk
et
aL

Proc.
NatL
Acad.
Sci.
USA
81
(1984)
1563
.?
4
Ptu
lU
,
Hind
m
FIG.
2.
Restriction
map
of
pDU1.
A,
Site
of
insertion
of
pBR322
in
pVW1.
The
orientation
of
this
map
matches
that
in
ref.
16
but
is
the
reverse
of
the
orientation
in
Fig.
4.
Smr
(28)
in
pRL5,
Kmr/neomycin
(Nm)r
(29,
30)
in
pRL6,
and
Emr
(31)
in
pRL8
(Figs.
3
and
4)
introduce
sites
for
Ava
I
(Sm9,
Ava
II
(Smr,
Kmr,
Emr),
and
Ava
III
(EmD
but
not
for
Avr
II.
Conjugal
Transfer
of
Vectors
to
Cyanobacteria.
Three
ele-
ments
suffice
for
the
conjugal
transfer
of
pBR322
and
de-
rived
plasmids:
a
suitable
conjugative
plasmid-e.g.,
RP-4;
a
plasmid
such
as
pGJ28
or
pDS4101
to
provide
necessary
transfer
functions;
and
the
pBR322
derivative
(13).
The
latter
pDU
1
pBR322
6.28
kb
4.363
kb
partial\
Eco
RV
Eco
RV
ligate
pVW1
(Apr)
10.64
kb
pBR328
Bam
HI
Sau
3A\
CIA
P
ligate
pVW1C
(AprrCmr)
11.62
kb
1.Ava
H
2.
CIA
P
3.Sau
961
fl4.
Iigate
pRL
1
(Cmr)
9.26
kb
B
Nae
I
Nae
I
Col
El::Tn5
HpaI
1.Ava
I
+
2.
SI
nuclease
Pvu
H1
ligate
ligate
pRL5
pRL6
11.1
kb
11.3
kb
(CmrSmr)
(CmrKmr/Nmr)
Cla
I,
CIAP
pE194
<Taq
I
ligate.
Pst
I.screen
transformants
pRL8
1
1.5
kb
(CmrEmr)
FIG.
3.
Construction
of
pRL1,
pRL5,
pRL6,
and
pRL8.
CIAP,
calf
intestinal
alkaline
phosphatase.
FIG.
4.
Maps
of
plasmids
pRL1,
pRL5,
pRL6,
and
pRL8,
show-
ing
potential
cloning
sites;
the
portions
derived
from
pDU1
(solid
bars)
and
pBR322
(lines),
the
Cmr
fragment
from
pBR328
(cross-
hatched
bars),
the
Smr
fragment
from
R300B
(lined
bars),
the
Kmr
fragment
from
TnS
(hatched
bars),
and
the
Emr
fragment
from
pE194
(empty
bars),
and
the
positions
of
the
origin
of
vegetative
replication,
oriV
(stippled
bars)
and
the
bom
region
(arrows).
two
elements
were
combined
prior
to
mating
by
using
pRL1,
pRL5,
and
pRL8
to
transform
HB101(pGJ28)
and
using
pRL6
to
transform
HB101(pDS4101).
Triparental
matings
were
then
performed
(8)
with
strains
of
cyanobacteria,
E.
coli
J53(RP-4),
and
E.
coli
HB101
containing
pRLi
or
deriva-
tive
and
the
helper
plasmid.
As
illustrated
in
Fig.
5,
only
matings
in
which
all
elements
were
present
resulted
in
sub-
stantial
growth
of
cyanobacteria
different
from
unmated
controls.
At
high
concentrations
of
cyanobacterial
inoculum,
the
cyanobacteria
often
grew
in
the
presence
of
antibiotic
(Fig.
5D).
Occasional
colonies
that
developed
in
the
pres-
ence
of
only
one
or
two
plasmids
were
not
studied
further.
Upon
attempted
conjugal
transfer
of
pRL1V,
pRL5V,
or
pRL6V
(i.e.,
pRL1,
-5,
or
-6
from
which
the
pDU1
portion
had
been
excised)
to
strains
of
Anabaena,
and
of
pRL1,
-5,
or
-6
to
Anacystis,
growth
was
observed
only
so
long
as
via-
ble
donor
bacteria
were
present
(see
Discussion).
The
pRL1
progenitor,
pVWlC,
containing
one
Ava
I
and
eight
Ava
II
sites,
was-unlike
pRL1-not
transferred
efficiently
(Fig.
5D).
To
characterize
their
content
of
plasmids,
the
presump-
tively
exconjugant
cyanobacteria
were
freed
of
the
auxotro-
phic
strains
of
E.
coli
used,
by
streaking
on
metabolite-free
cyanobacterial
medium.
Portions
of
single
colonies
were
transferred
to
L
agar.
Colonies
appearing
axenic
were
grown
in
liquid
medium
with
antibiotic.
Plasmids
were
isolated
from
dense
suspensions,
the
purity
of
which
was
confirmed
by
transfer
to
L
broth
and
L
agar.
The
presumptive
exconju-
gants
showed
plasmid
profiles
that
included
bands
corre-
sponding
to
the
hybrid
plasmids
(Fig.
6).
Transformation
of
E.
coli
HB101
by
plasmid-containing
extracts
yielded
a
large
number
of
transformants.
The
transformed
plasmids
were
isolated,
restricted,
and
subjected
to
electrophoresis
(Fig.
7).
We
found
that
pRL1,
-5,
and
-6
could
be
transferred
from
E.
coli
to
Anabaena,
and
back,
without
alteration;
pRL8
may
be
less
stable.
Evidence
of
plasmid
transfer
and
replication
was
obtained
with
combinations
of
cyanobacteria,
plasmids,
and
selective
agents
shown
in
Table
1.
Resistances
to
Sm,
R300
Microbiolo'gy:
Wolk
et
aL
a

Proc.
Natl.
Acad.
Scd
USA
81
(1984)
1
2
3
4
5
6
7
8
9
10
11
i_
_
_ _ _E._
tg _
_
_
_
_
__
__
_
_
_
*
__
___
*
,
_S____
-
-
_
|
_
s
i
|
FIG.
5.
Typical
initial
results
of
mating
experiments.
Nuclepore
filters
were
streaked
(from
top
to
bottom)
with
successive
1:10
dilu-
tions
of
suspensions
of
Anabaena
strains
7120
(A),
458
(B),
and
M-
131
(C
and
D).
The
streaks
in
A,
B,
and
C
were
dotted
with
2-M1
portions
of
(from
left
to
right)
suspensions
of
E.
coli
strains
contain-
ing
plasmids
RP-4
(spots
1);
pRL6
(spots
2);
pDS4101
(spots
3);
RP-
4,
pRL6,
and
pDS4101
(spots
4);
pRL6
and
pDS4101
(spots
5);
RP-4
and
pDS4101
(spots
6);
RP-4
and
pRL6
(spots
7);
RP-4,
pDS4101,
and
pRL6V
(which
is
pRL6
lacking
pDU1)
(spots
8);
and
only
L
broth
(spots
9).
The
streaks
in
D
were
dotted
with
2-,l
portions
of
suspensions
of
E.
coli
strains
containing
RP-4,
pGJ28,
and
either
pVW1C
(spots
1)
or
pRL1
(spots
2).
The
media
contained
Nm
at
25
(A
and
C)
or
10
(B)
pg/ml,
or
Cm
at
20
jtg/ml
(D).
D
is
magnified
x
1.24
relative
to
A,
B,
and
C.
Nm,
and
Em
characteristic
of
pRL5,
-6,
and
-8
were
not
con-
ferred
by
pRL1.
In
experiments
such
as
Fig.
5
A-C,
the
ratio
of
exconju-
gant
cyanobacterial
colonies
to
the
number
of
cyanobacterial
cells
subtended
by
a
spot
of
bacteria
was
about
10-3.
How-
ever,
when
Anabaena
M-131
was
fragmented
by
cavitation
to
an
average
length
of
1.3
cells
per
filament
before
conjugal
transfer
of
pRL1
(for
which
no
restriction
is
expected),
the
ratio
of
exconjugant
colonies
(developing
in
the
presence
of
Cm
at
20
txg/ml)
to
total
colonies
(developing
in
the
absence
of
Cm)
was
approximately
0.03.
DISCUSSION
We
have
demonstrated
RP-4-
and
helper
plasmid-dependent
transfer
of
pBR322-based
plasmids
across
the
wide
taxo-
nomic
gap
from
E.
coli
to
Anabaena.
It
was
fortunate
that
our
hybrid
vectors,
with
a
Nostoc
replicon,
were
able
to
rep-
licate
in
strains
of
Anabaena.
They
apparently
cannot
repli-
A
B
C
D
1
2
3
4
5
1
2
3
4
1
2
3
4
5
5
1
2
3
4
FIG.
7.
Electrophoretograms
of
EcoRV
digests
of
DNA
extract-
ed
from
E.
coli
after
transformation
with
extracts
of axenic
Ana-
baena
cultures
that
had
been
mated
with
E.
coli
strains
bearing
pRL1
(lanes
2
and
3),
pRL5
(lane
5),
and
pRL6
(lanes
7-9).
Lanes
4,
5,
and
10,
authentic
pRL1,
-5,
and
-6;
lanes
1
and
11,
HindIII
digest
of
X
DNA.
Anabaena
strains:
lanes
2
and
7,
7120;
lanes
3,
5,
and
8,
M-131;
and
lane
9,
458.
cate
in
Anacystis.
When
matings
are
performed
from
E.
coli
to
Anacystis
with
pRL1,
-5,
or
-6,
or
when
matings
are
per-
formed
to
Anabaena
in
which
pRL1,
-5,
or
-6
is
replaced
by
pRL1V,
pRL5V,
or
pRL6V-i.e.,
without
inclusion
of
pDUl-growth
of
the
cyanobacteria
is
often
seen.
However,
once
freed
of
E.
coli,
the
cyanobacteria
no
longer
form
colo-
nies
in
the
presence
of
antibiotic.
A
mobilizable
derivative
of
shuttle
vector
pSG111
(32),
which
contains
an
Anacystis
rep-
licon,
could
be
transferred
to
Anacystis
by
conjugation
and
could
replicate
there
(unpublished
data).
It
appears
that
plas-
mids
transferred
to
the
cyanobacteria
by
conjugation
can
confer
antibiotic
resistance
upon
them,
but
that
only
when
the
resistance-determinant
is
on
a
replicon
functional
in
the
particular
cyanobacterium
is
resistance
maintained
in
the
ab-
sence
of
further
infusion
of
plasmids.
Our
success
was
apparently
dependent
in
part
on
the
re-
duction
or
elimination
of
restriction
as
an
impediment
to
re-
tention
of
conjugally
transferred
DNA
(see
Fig.
SD).
This
was
possible
because
the
specificities
of
restriction
enzymes
of
numerous
strains
of
cyanobacteria
have
been
identified,
and
M-131,
in
particular,
appears
to
have
only
isoschizomers
of
Ava
I
and
Ava
II;
because
the
Cmr
gene
from
pBR328
and
essential
parts
of
pBR322
lack
sites
for
Ava
I
and
Ava
II;
and
because
the
cyanobacterial
replicon
pDU1
present
in
our
hy-
brid
vector
lacks
sites
for
Ava
I
and
Ava
II.
It
may
not
be
a
matter
of
chance
that
pDU1
lacks
such
sites.
Nostoc
sp.
PCC
7524,
from
which
pDU1
is
derived,
has
five
restriction
endonucleases,
Nsp(7524)I
through
V
(33).
Nsp(7524)III
(which
cuts
CIY-C-G-R-G;
Y
=
pyrimi-
dine;
R
=
purine)
is
an
isoschizomer
of
Ava
I.
The
specific-
F
G
H
1
2
345
5
12
34
5
1234
FIG.
6.
Electrophoretogramsof
DNA
extracted
from
Anabaena
(strain
7120
in
A,
D,
and
G;
strain
M-131
in
B,
C,
E,
and
H;
strain
458
in
F)
both
unmated
(lanes
1)
and
mated
(lanes
2)
with
E.
coli
bearing
pRL1
(A
and
B),
pRL5
(C),
pRL6
(D,
E,
and
F),
or
pRL8
(G
and
H)
and
then
rendered
axenic.
Lanes
3,
DNA
extracted
from
E.
coli
after
transformation
with
a
portion
of
the
extract
in
lanes
2;
lanes
4,
authentic
pRL1
(A
and
B),
pRL5
(C),
pRL6
(D,
E,
and
F),
or
pRL8
(G
and
H);
and
lanes
5,
HindIII
digest
of
X
DNA.
1564
Microbiology:
Wolk
et
aL
1
2
E3
4

Proc.
Natl.
Acad.
Sci.
USA
81
(1984)
1565
Table
1.
Selective
conditions
for
plasmid
transfer
to
cyanobacteria
Plasmid
and
antibiotic,
,.g/ml
pRL1
pRL5
pRL6
pRL8
Anabaena
strain
Cm
Sm
Nm
Em
M-131
20,
30
3,
5,
10
10,
25
2,
5
458
10
7120
5,
10 25
5
ity
of
Nsp(7524)IV
(GIG-N-C-C;
ref.
33),
an
isoschizomer
of
Sau961, includes
all
Ava
II
sites
(G!GA-C-C).
Unlike
Hpa
II
(COC-G-G;
ref.
16),
Hae
III
(G-GIC-C;
see
above),
and
Hha
I
(G-C-GIC;
see
above),
Sau96I
fails
to
cut
pDU1
(P
-
10-11).
Furthermore,
Ban
II,
HgiAI,
and
Sph
I,
the
specificities
of
which
are
subsumed
by
those
of
Nsp(7524)I
and
-II,
all
fail
to
cut
unmodified
pDU1
(P
<
0.004).
Finally,
cloned
chromo-
somal
DNA
from
Anabaena
ATCC
29413
(unpublished
data)
and
Anabaena
7120
(34-38)
is
statistically
deficient
in
sites
for
Ava
I,
and
Ava
I
and
II,
respectively,
for
which
they
have
isoschizomers
(17).
It
must
be
considered
that
an
organism
may
evolve
to
minimize
the
number
of
target
sites
in
its
DNA
for
its
own
restriction
endonucleases.
Demonstration
of
high-frequency
genetic
transfer
to
Ana-
baena
opens
the
way
to
analysis
of
cyanobacterial
nitrogen
fixation
and
development
by
the
techniques
of
modem
bio-
chemical
genetics.
In
addition,
it
provides
a
major
tool
for
study
of
oxygenic
photosynthesis.
We
are
very
grateful
to
J.
Reaston
for
unpublished
data
about
restriction
of
pDU1;
to
E.
Rosenvold
for
Avr
II;
to
D.
Sherratt
for
pGJ28
and
pDS4101;
to
L.
Sherman
for
pSG111;
to
him
and
to
J.
Williams
for
Anacystis
strain
R2;
to
L.
Snyder
forE.
coli
Gm48;
and
to
P.
Barth
for
R300B.
A.V.
acknowledges
receipt
of
a
research
fel-
lowship
from
the
Rothschild
Foundation.
This
work
was
supported
by
the
U.S.
Department
of
Energy
under
Contract
DE-AC02-
76ER01338.
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Microbiology:
Wolk
et
aL