Introduction of transposon Tn901 into a plasmid of Anacystis nidulans: Preparation for cloning in cyanobacteria

Abstract

We have used the TEM beta-lactamase transposon Tn901, located on Escherichia coli plasmid pRI46, to introduce in vivo a genetic marker into plasmid pUH24, present in the cyanobacterial strain Anacystis nidulans R-2. Restriction enzyme analysis and heteroduplex studies of the 8.3 x 10(6)-dalton plasmids pCH1-pCH5, present in the ampicillin-resistant A. nidulans R-2 colonies obtained after transformation with pRI46, demonstrated that these plasmids consist of the complete sequence of Tn901 inserted at different places into plasmid pUH24. The pUH24::Tn901 recombinant plasmids transform A. nidulans R-2 with a frequency of 10(-4)--10(-5) per microgram of plasmid DNA and contain a single cleavage site for the restriction enzyme Xho I. From pCH1 a plasmid of 5.5 x 10(6) daltons,pUC1, was constructed with only a part of the Tn901 sequence and an additional single cleavage site for the restriction enzyme BamHI. This plasmid, as well as plasmids pCH1-pCH5, are potentially useful as vectors for cloning genes in cyanobacteria and for studying cyanobacterial plasmid biology.

Full text

Proc.
Natl.
Acad.
Sci
USA
Vol
77,
No.
3,
pp.
1570-1574,
March
1980
Genetics
Introduction
of
transposon
Tn901
into
a
plasmid
of
Anacystis
nidulans:
Preparation
for
cloning
in
cyanobacteria
(plasmid
transformation/deletion
plasmid/transposable
element/ampicillin
resistance)
CEES
A.
M.
J.
J.
VAN
DEN
HONDEL*,
SJEF
VERBEEK*,
ARIE
VAN
DER
ENDEt,
PETER
J.
WEISBEEK*,
WILHELMINA
E.
BORRIAS*,
AND
GERARD
A.
VAN
ARKEL*
*Department
of
Molecular
Cell
Biology
and
tInstitute
of
Molecular
Biology,
State
University
of
Utrecht,
Utrecht,
The
Netherlands
Communicated
by
Roger
Y.
Stanier,
December
17,
1979
ABS1RACT
We
have
used
the
TEM
,-lactamase
transposon
Tn9OM,
located
on
Escherichia
coli
plasmid
pRI46,
to
introduce
in
vivo
a
genetic
marker
into
plasmid
pUH24,
present
in
the
cyanobacterial
strain
Anacystis
nidulans
R-2.
Restriction
en-
zyme
analysis
and
heteroduplex
studies
of
the
8.3
X
106-dalton
plasmids
pCH1-pCH5,
present
in
the
ampicillin-resistant
A.
nidulans
R-2
colonies
obtained
after
transformation
with
pRI46,
demonstrated
that
these
plasmids
consist
of
the
complete
se-
quence
of
Tn901
inserted
at
different
places
into
plasmid
pUH24.
The
pUH24::Tn9Ol
recombinant
plasmids
transform
A.
nidulans
R-2
with
a
frequency
of
10-4-10-5
per
yg
of
plasmid
DNA
and
contain
a
single
cleavage
site
for
the
restriction
en-
zyme
Xho
I.
From
pCHl
a
plasmid
of
5.5
X
106
daltons,
pUC1,
was
constructed
with
only
a
part
of
the
Tn9O1
sequence
and
an
additional
single
cleavage
site
for
the
restriction
enzyme
BamHI.
This
plasmid,
as
well
as
plasmids
pCH1-pCH5,
are
potentially
useful
as
vectors
for
cloning
genes
in
cyanobacteria
and
for
studying
cyanobacterial
plasmid
biology.
The
presence
of
a
system
for
converting
light
energy
similar
to
that
in
chloroplasts
makes
the
cyanobacteria
(1,
2)
attractive
organisms
for
molecular
studies
of
photosynthesis
and
associated
phenomena.
For
a
better
understanding
of
the
molecular
biology
of
these
processes
it
is
necessary
to
analyze
the
function,
organization,
and
regulation
of
the
genes
involved.
An
appro-
priate
method
for elucidation
of
the
number
and
character
of
such
genes
is
the
induction
and
analysis
of mutations.
Moreover,
the
development
of
molecular
cloning
in
Escherichwi
coli
il-
lustrates
the
feasibility
of
handling
DNA
fragments
that
contain
genes
that
are
correlated
with
given
mutations,
thus
providing
an
approach
to
the
isolation,
mapping,
and
analysis
of
genes
on
a
DNA
level
and
to
the
identification
of
the
corresponding
gene
products
(3).
The
availability
of
a
comparable
system
in
cy-
anobacteria
is
desirable.
Such
a
system
would
be
profitable
for
the
study
not
only
of
photosynthesis,
but
also
of
other
processes
in
these
organisms
such
as
nitrogen
fixation
and
the
develop-
ment
of
differentiated
cells
such
as
heterocysts
and
akinetes
(2,
4).
Suitable
mutations
have
been
isolated
and
characterized
(5-9),
but
their
genetic
basis
is
largely
unknown
because
it
has
been
almost
impossible
to
analyze
them
by
the
classical
methods
of
bacterial
genetics
(10).
Having
been
confronted
with
this
difficulty,
we
decided
to
investigate
whether
molecular
cloning
could
offer
new
possibilities
for
molecular
genetic
analysis
in
cyanobacteria.
In
the
development
of
a
cloning
system,
at
least
two
condi-
tions
have
to
be
fulfilled.
First,
a
suitable
host
must
be
available,
preferably
one
that
can
be
used
in
a
"self-cloning"
system,
which
means
that
mutations
and
phenotypes
can
be
correlated
directly
with
genes.
Several
unicellular
cyanobacterial
strains
seemed
promising
as
a
host
because
they
show
transformation
by
chromosomal
DNA
(11-14).
Second,
cloning
requires
an
appropriately
marked
vector
that
can
be
propagated
in
the
host.
Many
cyanobacterial
strains
contain
plasmids,
but
because
all
have
proved
to
be
cryptic
thus
far
(15-18),
at
least
one
marker
had
to
be
introduced
to
make
the
potential
vectors
traceable.
The
present
paper
reports
the
construction
of
a
class
of
ge-
netically
marked
plasmids
by
the
in
vvo
introduction
of
the
ampicillin
(Ap)
resistance
transposon
Tn901
into
the
small
plasmid
of
Anacystis
nidulans
R-2.
These
marked
plasmids
transform
A.
nidulans
R-2
efficiently.
Analyses
with
different
restriction
endonucleases
showed
that
Tn901
can
be
inserted
at
different
sites
and
that
the
new
plasmids
[8.3
X
106
daltons
(8.3
MDal)]
contain
a
single
cleavage
site
for
the
restriction
endonuclease
Xho
I
for
cloning
chromosomal
DNA.
After
a
part
of
the
DNA
was
deleted,
a
smaller
plasmid
(5.5
MDal)
with
an
immobilized
transposon
and
single
cleavage
sites
for
the
re-
striction
enzymes
BamHI
and
Xho
I
was
obtained.
MATERIALS
AND
METHODS
Bacterial
Strains
and
Plasmids.
A.
nidulans
R-2,
obtained
from
S.
Shestakov
(Moscow),
was
used
as
the
recipient
strain
for
the
plasmid
transformations
(12).
Plasmids
pRI477S
and
pRI46
were
obtained
from
J.
van
Embden
(Bilthoven).
pRI477S
is
a
plasmid
that
encodes
streptomycin
(Sm)
and
sulfonamide
resistance
(19).
pRI46
is
a
pRI477S::Tn901
isolate
of
9.3
MDal
(19).
pJN60,
a
CloDF13::Tn90l
isolate
(20),
was
obtained
from
E.
Veltkamp
(Amsterdam).
Tn901
is
a
3-MDal
transposable
element
that
encodes
resistance to
Ap
through
synthesis
of
TEM
f3-lactamase
(19).
pBR322
was
a
gift
of
C.
van
Sluis
(Leiden).
Plasmid
Isolation.
The
covalently
closed
circular
DNA
of
pRI477S,
pRI46,
pBR322,
and
pJN60
was
isolated
from
clear
lysates
and
purified
by
CsCI/ethidium
bromide
equilibrium
centrifugation
(21).
The
DNA
was
precipitated
with
ethanol
and
dissolved
in
10
mM
Tris-HCl/0.1
mM
EDTA,
pH
7.6.
Plasmid
DNA
from
A.
nidulans
R-2
and
A.
nidulans
R-2
transformants
was
purified
as
described
(17).
Transformation.
Transformation
of
A.
nidulans
R-2
was
based
on
the
method
of
Shestakov
and
Khyen
(22).
A.
nidulans
R-2
was
grown
at
30'C
in
a
250-ml
erlenmeyer
flask
containing
50
ml
of
BGl
medium
(23),
illuminated
by
banks
of
white
fluorescent
tubes
that
provided
an
intensity
of
4000-5000
lux
(lm/m2)
at
the
surface
of
the
flask.
Samples
of
exponentially
growing
cells
at
a
concentration
of
about
1-2
X
108
cells
per
ml
were
incubated
with
1
,tg
of
plasmid
DNA
per
ml
at
30°C
under
the
same
light
conditions.
After
30
min
of
incubation,
samples
of
0.1
ml
were
plated
on
BG11
agar
(Difco,
1%)
plates
without
the
antibiotic.
After
1
day
of
culturing,
0.5
ml
of
Ap
(30-100
gg/ml)
was
added
underneath
the
agar.
The
concen-
Abbreviations:
Tn901,
transposable
DNA
sequence
encoding
TEM
,B-lactamase;
Ap,
ampicillin;
Sm,
streptomycin;
MDal,
106
daltons.
1570
The
publication
costs
of
this
article
were
defrayed
in
part
by
page
charge
payment.
This
article
must
therefore
be
hereby
marked
"ad-
vertisement"
in
accordance
with
18
U.
S.
C.
§1734
solely
to
indicate
this
fact.

Proc.
Natl.
Acad.
Sci.
USA
77
(1980)
1571
tration
of
the
antibiotic
after
diffusion
throughout
the
entire
volume
was
0.5-1.6
jig/ml.
After
5-6
days,
resistant
colonies
were
scored.
For
transformation
experiments
with
the
plasmid
pUC1,
a
concentration
of
30
,gg
of
Ap
per
ml
was
used
for
se-
lection.
The
transformation
efficiencies
under
the
conditions
described
were
10-4-10-5
per
pug
of
cyanobacterial
plasmid
DNA.
Restriction
Enzyme
Analysis
and
In
Vitro
Construction
of
Recombinant
Plasmids.
The
restriction
endonuclease
HindII
was
a
gift
of
P.
Baas
(Utrecht).
The
other
restriction
endonucleases
were
purchased
from
Bethesda
Research
(Rockville,
MD)
or
Biolabs
(Beverly,
NE).
They
were
used
ac-
cording
to
the
manufacturer's
instructions
or
as
described
(17).
Electrophoresis
was
performed
as
described
(17).
After
sepa-
ration
on
sucrose
gradients,
the
fragments
of
BamHI-cleaved
pCH1
plasmid
were
ligated
either
separately
or
mixed
in
the
presence
of
E.
coli
DNA
ligase
(Bethesda
Research)
under
the
same
conditions
as
recommended
by
the
manufacturer
except
that
the
fragments
were
incubated.for
18
hr
at
40C.
Electron
Microscopy.
Heteroduplexes
of
pUH24
and
pCH1
DNA
or
pCHl
and
pCH2
DNA
were
prepared
by
heating
the
Xho
I-cleaved
plasmid
mixtures
in
70%
formamide/10
mM
EDTA,
pH
8.0,
for
5
min
at
700C
The
solution
was
then
cooled
on
ice.
Reannealing
was
carried
out
for
30
min
at
370C
in
final
concentrations
of
50%
formamide,
0.1
M
ammonium
acetate,
and
10
mM
EDTA
at
pH
8.0.
DNA
was
spread
for
electron
microscopy
by
the
formamide
modification
of
the
protein
monolayer
technique
(24).
Spreading
conditions
and
length
measurements
were
as
described
(17).
RESULTS
Introduction
of
Transposon
Tn9O1
into
Plasmid
pUH24.
A.
nidulans
R-2,
a
strain
that
can
be
transformed
by
chromo-
somal
DNA
with
a
high
frequency
(12),
contains
two
plasmid
species
of
5.3
MDal
(pUH24)
and
33
MDal
(pUH25)
(Fig.
la).
Restriction
enzyme
analysis
(unpublished
data)
revealed
that
they
are
similar
to
or
identical
with
the
two
plasmid
species
previously
found
in
several
other
cyanobacterial
strains
(16,
17).
The
analysis
also
showed
that
pUH24
possesses
a
limited
number
of
cleavage
sites
for
restriction
enzymes
producing
cohesive
ends,
which
are
useful
in
cloning
experiments
(3).
This
property,
together
with
its
small
size,
makes
pUH24
attractive
as
a
potential
vector
plasmid.
Because
plasmid-encoded
prop-
erties
could
not
be
detected,
we
made
the
plasmid
traceable
through
the
in
vivo
introduction
of
a
selective
marker
into
pUH24
by
a
method
analogous
to
those
described
for
marking
E.
coli
plasmids
(20,25).
For
this
purpose,
E.
coli
plasmid
pRI46
was
transformed
into
cells
of
A.
nidulans
R-2.
In
addition
to
the
antibiotic
resistance
genes
against
Sm
and
sulfonamide,
pRI46
contains
the
transposable
element
Tn901,
which
carries
a
f-lactamase
gene
for
Ap
resistance
(19).
If
transposition
of
Tn901
(3
MDal)
to
pUH24
(5.3
MDal)
were
to
occur,
a
new
plasmid
of
about
8.3
MDal
would
be
formed,
which
would
be
genetically
marked.
After
transformation
with
pRI46,
Ap-
and
Sm-resistant
colonies
of
A.
nidulans
R-2
were
found
with
a
very
low
fre-
quency
of
10-8-10-9/,ug
of
plasmid
DNA
(transformation
frequency
of
chromosomal
DNA
was
10-4-10-5/Ag
of
DNA).
Analysis
of
the
plasmid
content
of
five
Ap-resistant
colonies
that.
were
obtained
after
direct
selection
with
Ap
(strains
XIAlb
and
XIAle)
or
obtained
after
Ap
selection
after
a
first
cycle
of
Sm
selection
(IVS4A1,
IVS4A2,
and
XIIS4s34)
showed
that
pUH24
was
absent
and
was
replaced
by
a
new
plasmid
of
8.3
MDal;
pUH25
was
present
but
unchanged
(Fig.
1
a
and
b).
This
finding
suggests
that
recombinant
plasmids
of
the
type
pUH24::Tn9Ol
have
actually
been
formed.
Analysis
of
strain
33-
8.3-
5.3-
i-33
_-8.3
-_5.5
_5.3
pUH24M5
M12M13
M20
M1
FIG.
1.
Agarose
gel
electrophoresis
of
plasmid
DNA
isolated
from
A.
nidulans
R-2
and
Ap-resistant
A.
nidulans
R-2
transformants.
Plasmid
DNAs
were
purified
by
CsCl/ethidium
bromide
centrifu-
gation
and
analyzed
by
electrophoresis
on
a
0.6%
horizontal
agarose
gel
at
200
V
for
3
hr
in
the
presence
of
2
,ug
of
ethidium
bromide
per
ml.
The
sizes
of
the
plasmids
are
indicated
in
MDal.
(a
and
b)
Ap-
resistant
transformants
(IVS4A1
not
shown)
obtained
after
trans-
formation
with
pRI46;
A.
nidulans
R-2
(R-2)
given
as
a
reference.
(c)
Ap-resistant
transformants
obtained
after
transformation
with
ligated
5.5-MDal
BamHI
fragments
from
pCH1
(Ml
and
M4;
M2
and
M3
not
shown)
or
with
the
ligated
mixture
of
the
5.5-MDal
and
2.8-MDal
BamHI
fragments
from
pCH1
(M5/M12
and
M13/M20;
strains
M5-M12
and
M13-M20
were
analyzed
together).
pUH24
and
pCH1
were
run
on
the
same
gel
as
references.
XIAle
revealed
that,
in
addition
to
the
8.3-MDal
plasmid,
a
plasmid
of
5.5
MDal
was
present
(Fig.
lb),
which
is
a
deletion
mutant
of
the
8.3-MDal
plasmid
(unpublished
data).
Subsequent
transformation
of
one
of
the
8.3-MDal
plasmids
into
A.
nidulans
R-2
gave
rise
to
Ap-resistant
colonies
with
a
frequency
of
10-4-10-5/,g
of
plasmid
DNA.
Plasmid
analysis
of
two
of
these
colonies
(strains
VIIA1
and
VIIA2)
showed
that
pUH25
and
a
plasmid
of
8.3
MDal
were
present
whereas
pUH24
had
disappeared
(results
not
shown).
Analysis
of
these
8.3-MDal
plasmids
with
several
restriction
enzymes
that
pro-
duce
a
fair
number
of
fragments
(such
as
HindII
and
Pst
I,
see
Table
1)
indicated
that
they
are
identical
to
the
8.3-MDal
plasmid
used
for
the
transformation
experiment.
From
these
results
it
can
be
concluded
that
the
information
for
Ap
resis-
tance
is
really
located
on
the
8.3-MDal
plasmid.
Restriction
Enzyme
Analysis
of
8.3-MDal
Plasmids.
Evi-
dence
that
the
8.3-MDal
plasmids
(designated
pCHl-pCH5)
of
the
five
Ap-resistant
colonies
described
above
do
consist
of
Tn901
inserted
at
different
sites
into
pUH24
was
obtained
from
the
analysis
of
these
plasmids
with
several
restriction
enzymes
(Table
1).
As
an
example,
the
digestion
patterns
of
plasmids
pCH1-pCH5
after
cleavage
by
HindII
are
shown
(Fig.
2).
The
presence
of
the
pUH24
sequence
in
the
8.3-MDal
plasmids
would
be
indicated
by
the
presence
of
all
the
pUH24
fragments
in
the
digests
except
the
fragment
in
which
the
transposon
was
inserted.
Comparison
of
the
cleavage
patterns
of
two
Tn9Ol-
containing
marker
plasmids
pRI46
and
pJN60
with
the
patterns
of
pCH1-pCH5
revealed
that
the
two
HindII
fragments
lo-
cated
within
Tn901
were
also
present
in
the
patterns
of
the
8.3-MDal
plasmids
(Fig.
2),
showing
that
Tn901
was
also
present
in
pCH1-pCH5.
In
addition,
two
"bridge-fragments"
Genetics:
van
den
Hondel
et
al.

1572
Genetics:
van
den
Hondel
et
al.
Table
1.
Number
of
cleavage
sites
of
different
restriction
enzymes
in
pUH24,
pCH1-pCH5,
pRI46
(pRI477S::Tn9Ol),
pRI477S,
and
Tn9O1
Enzyme
pUH24
pCH1
pCH2-5
pRI46
pRI477S
Tn9Ol*
HindII
11
14
14
6
3
3t
Pst
I
7
10
NDt
5
2
3t
BglI
4
7
ND
ND
ND
3t
Hpa
I
¢4 ¢4
ND
1
1
ot
Kpn
I
3
3 3
0
0
ot
Pvu
II
2
4
ND
4
2
2
HindIII
2
2
ND
0
0
0
BgI
II
2
2
2
0
0
0
Xho
I
1 1
ND
0
0
of
BamHI
1
2
2
1
0
lt
Xba
I
00
ND
ND
ND
ot
EcoRI
00
ND
1
1
ot
Sma
I
0
0
ND
ND
ND
of
*
The
number
of
cleavage
sites
within
Tn901
was
determined
by
comparing
the
digests
of
pRI477S
with
those
of
pRI46.
The
same
number
of
cleavage
sites
has
been
found
previously
(ref.
20
and
E.
Veltkamp,
personal
communication).
ND,
not
determined.
containing
a
part
of
the
pUH24
DNA
and
a
part
of
the
Tn9OW
DNA
were
also
found
(Fig.
2).
From
these
results,
together
with
those
obtained
from
the
analysis
with
the
other
restriction
en-
zymes
(Table
1),
we
conclude
that
the
8.3-MDal
plasmids
pCH1-pCH5
are
pUH24::Tn9Ol
recombinants.
Four
different
insertion
sites
have
been
found
(Fig.
2),
three
in
HinA
(insertion
of
pCH1,
pCH2, pCH3,
and
pCH5)
and
one
in
HinD
(pCH4).
By
combination
of
the
data
from
the
size
of
the
bridge-fragments,
the
digest
patterns
obtained
with
other
restriction
enzymes,
and
the
physical
maps
of
Tn9OW
(20)
and
pUH24,
the
position
and
the
orientation
of
the
transposon
(the
latter
is
the
same
in
all
five
insertions)
were
established
(Fig.
3).
The
procedure
and
data
for
determination
of
the
physical
map
of
pUH24
as
well
as
the
insertion
sites
of
Tn9O1
will
be
reported
in
detail
elsewhere.
Heteroduplex
Analysis.
The
insertion
of
Tn901
into
pUH24
was
demonstrated
by
visualization
of
the
transposon
as
an
in-
sertion
loop
in
a
heteroduplex
molecule
formed
after
hybrid-
pUH24
pCH3
pCH2
pJN6O
pRI46
pCH1
pRI46
pJN60
pCH5
pCH4
pCH3
pCH1
pUH24
A-_l
-_
B
-_
AA
C
B
C)-~~~~~~~~~~~~~~~
D-
_
-C
FIG.
2.
Restriction
endonuclease
patterns
of
the
-8.3-MDal
plasmids
pCH1-pCH5.
Plasmids
pCH1-pCH5
(present
in
the
Ap-
resistant
strains
IVS4A1,
XIAle,
XIAlb,
IVS4A2,
and
XIIS4s34,
respectively),
together
with
marker
plasmids
pJN60
(CloDF13::
Tn901)
and
pRI46
(pRI477S::Tn9Ol),
were
purified
and
digested
with
Hindul.
Digests
were
electrophoretically
separated
on
1.6%
agarose
gels
at
40
V
for
18
hr
in
the
presence
of
2
Ag
of
ethidium
bromide
per
ml.
A-D
refer
to
the
HindII
fragments
from
pUH24;
fragments
E-K
(0.
15-<0.05
MDal)
cannot
be
seen
on
this
gel.
Arrows
indicate
internal
HindII
fragments
from
Tn901;
stars
indicate
the
"bridge-fragments"
from
pCH1-pCH5,
which
contain
a
part
of
pUH24
and
Tn9O1
DNA.
The
faint
extra
bands
in
several
lanes
are
due
to
incomplete
digestion
or
to
the
presence
of
a
small
amount
of
33-MDal
plasmid
pUH25
in
the
DNA
preparations.
£
b
3
I
Tn
9Ol|
I
aX
FIG.
3.
Restriction
maps
of
pUH24
and
Tn901
and
the
sites
of
transposon
insertion.
Locations
of
the
restriction
recognition
sites
of
BamHI,
BgI
I,
Bgl
II,
HindII,
HindIII,
Kpn
I,
Pvu
II,
and
Xho
I
in
pUH24,
indicated
by
arrows
outside
the
circle,
were
determined
by
digesting
pUH24
with
one
or
more
restriction
enzymes
and
ana-
lyzing
the
size
and
number
of
the
fragments
produced.
The
positions
of
only
four
of
the
eleven
HindII
cleavage
sites
were
determined
and
are
therefore
in
parentheses.
The
different
sites
of
Tn9Ol
in
pUH24
are
indicated
by
arrows
inside
the
circle.
The
orientation
of
the
cleavage
map
of
Tn9OW,
given
on
the
part
of
the
circle
outside
the
map
of
pUH24,
is
as
found
in
plasmids
pCH1-pCH5.
ization
of
pUH24
DNA
and
pUH24::Tn9Ol
DNA.
For
this
purpose,
plasmid
pCH1
was
cleaved
by
Xho
I
to
a
linear
mol-
ecule,
heteroduplexed
with
Xho
I-digested
pUH24,
and
ex-
amined
by
electron
microscopy
(Fig.
4A).
Examination
of
25
heteroduplex
molecules
showed
that
each
molecule
contained
a
single-stranded
insertion
loop
in
a
perfectly
matched
linear
heteroduplex.
The
finding
in
all
the
heteroduplexes
of
a
short
double-stranded
stem
at
the
junction
between
the
insertion
loop
and
the
other
part
of
the
molecule
indicates
the
presence
of
an
inverted
repeat
DNA
sequence
at
both
ends
of
the
transposon.
Contour
length
measurements
of
the
heteroduplex
molecules
showed
that
the
linear
duplex
has
a
length
equivalent
to
a
size
of
5.2
MDal
(equal
to
the
size
of
pUH24)
and
the
insertion
loop
has
a
length
equivalent
to
a
size
of
3.1
MDal,
a
value
that
matches
the
size
of
Tn901
(19).
From
both
observations
it
can
be
concluded
that
the
entire
transposon
Tn901
is
present
in
plasmid
pCH1.
From
the
heteroduplex
analysis,
.onclusions
can
also
be
drawn
about
the
location
of
the
Tn901
insertion.
The
distance
from
one
end
of
the
Xho
I-cleaved
molecule
to
the
Tn901
in-
sertion
site
was
measured
and
appeared
to
be
equivalent
to 1.3
MDal.
It
agrees
with
the
value
of
1.4
MDal
determined
by
re-
striction
enzyme
analysis.
Analysis
of
heteroduplexes
prepared
between
two
different
pUH24::Tn901
isolates
gives
further
information
on
the
or-
ientation
of
the
transposons
and
on
the
location
of
the
insertions
relative
to
one
another
and
to
the
Xho
I
cleavage
site.
This
has
been
worked
out
for
the
combination
pCH1/pCH2
(Fig.
4
B
and
C).
Molecules
of
the
type
shown
in
Fig.
4B,
in
which
the
DNA
strands
of
the
transposons
have
not
interacted,
are
in-
dicative
of
the
distance
between
the
insertion
sites
and
the
distances
from
insertion
to
the
ends
of
the
molecule.
From
Proc.
Natl.
Acad.
Sci.
USA
77
(1980)

Genetics:
van
den
Hondel
et
al.
'A
';,<
11
*-
'
S
.a'
*
r.
;
;'24CH
0am"
5
k''
,r~ffe-+tF''f-A
p0t%
-b
6
i
Proc.
Natl.
Acad.
Sci.
USA
77
(1980)
1573
'ide
*
'
FIG.
4.
Heteroduplex
analysis
of
the
insertion
of
Tn901
into
pUH24
DNA.
(A)
Xho
I-cleaved
pUH24
DNA
and
the
DNA
of
pCH1
were
heteroduplexed
and
spread
for
electron
microscopy.
The
insertion
of
DNA
representing
Tn9OW
is
seen
as
a
single
loop
of
single-stranded
DNA.
The
arrow
indicates
the
short
double-stranded
stem
generated
by
the
two
inverted
repeat
sequences
of
Tn9Ol.
(B)
Heteroduplex
of
pCH1/pCH2
DNA
showing
noninteracting
insertion
loops
of
Tn9OM
at
a
distance
of
approximately
0.2
Atm.
(C)
The
same
heteroduplex
of
pCH1/pCH2
DNA
with
interaction
of
the
Tn9O1
sequences.
The
small
single-stranded
deletion
loops
are
produced
by
the
different
insertion
sites
of
the
transposon.
molecules
of
the
type
shown
in
Fig.
4C,
in
which
the
DNA
strands
of
the
transposons
are
also
annealed
(the
region
between
the
two
single-stranded
loops),
it
can
be
seen
that
the
orientation
of
Tn901
in
pCH1
and
pCH2
is
the
same.
Construction
of
Deletion
Plasmid
pUCI
from
pCH1.
The
Tn901-containing
plasmids
pCHl-pCH5
can
be
used
directly
for
cloning
chromosomal
DNA
in
their
single
Xho
I
cleavage
site.
However,
translocation
of
Tn901
can
potentially
compli-
cate
the
use
of
the
8.3-MDal
plasmid
as
a
vector.
This
can
be
circumvented
by
removing
a part
of
the
transposon.
For
this
reason
pCH1
was
treated
with
the
restriction
enzyme
BamHI,
which
produces
one
cut
inside
and
one
cut
outside
Tn901
(Fig.
3).
The
resulting
fragments
of
2.8
MDal
and
5.5
MDal,
re-
spectively,
were
isolated
and
then
separately
ligated.
As
a
control,
a
mixture
of
the
two
BamHI
fr4gments
was
ligated
too.
After
transformation
of
A.
nidulans
R-2
with
the
three
ligation
samples,
Ap-resistant
colonies
were
found
only
in
the
ligated
5.5-MDal
fragments
and
the
mixture
of
both
fragments.
Plas-
mid
analysis
of
several
Ap-resistant
colonies
(M1-M4)
obtained
after
transformation
with
the
ligated
5.5-MDal
fragments
re-
vealed
that,
in
addition
to
the
33-MDal
plasmid
pUH25,
a
5.5-MDal
plasmid
(designated
as
pUCI)
was
present
whereas
pUH24
was
absent
(Fig.
1c).
Ap-resistant
colonies
obtained
after
transformation
with
the
ligated
mixture
of
the
two
BamHI
fragments
(M5/12
and
M13/20)
also
contained
a
5.5-MDal
plasmid
(Fig.
1c).
In
addition,
a
plasmid
of
6.5
MDal
was
found,
whose
presence
cannot
be
explained
at
the
moment.
After
pUCl
was
digested
with
BamHI,
a
linear
molecule
of
5.5
MDal
was
produced,
indicating
that
pUC1
contains
a
single
cleavage
site
for
BamHI.
Moreover,
the
digestion
patterns
obtained
with
other
restriction
enzymes
support
the
conclusion
that
pUC1
consists
of
the
5.5-MDal
BamHI
fragment
circularized
after
ligation.
The
single
Xho
I
site
was
retained.
The
new
plasmid
contains
only
one
of
the
inverted
repeats
of
Tn901,
so
that
the
transposon
is
immobilized.
The
combination
of
the
character-
istics
mentioned
now
makes
pUC1
a
suitable
vector
for
the
cloning
of
DNA
fragments.
The
Ap
resistance
of
pUCi-containing
cells
was
one-fifth
to
one-third
that
of
cells
containing
pCH1.
This
decrease,
how-
ever,
did
not
interfere
with
the
selection
procedure
of
Ap-re-
sistant
transformants.
DISCUSSION
In
several
unicellular
cyanobacterial
strains
transformation
by
chromosomal
DNA
has
been
demonstrated
(11-14).
These
strains
would
be
considered
to
be
suitable
hosts
in
a
molecular
cloning
system,
provided
that
vectors
are
available
that
they
can
propagate.
Because
experiments
to
test
whether
E.
coli
cloning
vectors
(such
as
pBR322)
could
be
used with
A.
nidu-
lans
R-2
as
a
host
gave
negative
results,
a
search
was
made
for
an
indigenous
cyanobacterial
vector.
In
the
present
study
the
construction
of
genetically
marked
cyanobacterial
plasmids
is
described.
The
method
used
is
comparable
to
those
developed
to
genetically
label
E.
coli
plasmids
(20,
25).
It
is
based
on
the
introduction
of
the
Ap-
resistance-encoding
transposon
Tn901
(19)
into
the
cyano-
bacterial
plasmid
pUH24,
a
plasmid
probably
identical
to
some
of
the
cryptic
plasmids
reported
previously
(16,
17).
After
transformation
of
A.
nidulans
R-2
with
the
E.
coli
plasmid
pRI46,
the
donor
of
Tn901,
followed
by
selection
for
antibiotic
resistance,
the
original
plasmid
pUH24
was
replaced
by
a
plasmid
with
the
size
of
a
pUH24::Tn9Ol
recombinant
molecule

1574
Genetics:
van
den
Hondel
et
al.
(Fig.
1
a
and
b).
Restriction
enzyme
analysis
and
heteroduplex
studies
clearly
showed
that
the
newly
formed
plasmids
indeed
consist
of
the
insertion
of
the
entire
Tn901
into
pUH24
(Figs.
2
and
4).
Moreover,
the
presence
of
the
inverted repeat
se-
quences
of
Tn901
together
with
the
different
sites
of
insertion
supports
the
view
that
the
recombinant
plasmids
pCH1-pCH5
are
generated
by
transposition
of
Tn901
from
pRI46
into
pUH24.
Transformation
of
cells
of
A.
nidulans
R-2
with
pUH24::
Tn901
leads
to
Ap
resistance,
indicating
that
the
information
for
Ap
resistance
(the
3-lactamase
gene
of
Tn901)
is
really
lo-
cated
on
the
recombinant
plasmid.
At
the
same
time
this
result
shows
that
the
3-lactamase
gene
of
Tn901,
normally
functional
in
E.
ccli,
also
is
expressed
in
A.
nidulans
R-2.
Thus,
an
E.
coli
gene
is
expressed
in
a
cyanobacterial
cell.
Based
on
the
supposed
similarities
in
the
genetic
organization
of
the
transposons
Tn901,
Tnl,
and
Tn3
(26-28),
one
may
expect
that
the
functions
nec-
essary
for
transposition
of
Tn901
will
also
be
expressed.
This
would
open
the
possibility
in
cyanobacteria
for
use
of
Tn901
in
exploiting
the
methods
of
genetic
engineering
in
vivo
as
described
for
E.
coli
(29).
The
fact
that
after
the
transformation
of
A.
nidulans
R-2
with
E.
coli
plasmid
pRI46
the
transposon
Tn901
was
trans-
posed
into
the
indigenous
plasmid
pUH24
proves
that
pRI46
was
taken
up
by
the
cells
and
that
the
transposition
functions
(26-28),
were
expressed.
Nevertheless,
in
Ap-resistant
cells
pRI46
appears
to
be
absent
(Fig.
1
a
and
b).
It
seems
reasonable
to
assume
that
this
plasmid
cannot
maintain
itself
in
cyano-
bacterial
cells.
A
possible
explanation
for
the
rapid
segregation
of
pRI46
may
be
that
the
level
of
gene
expression
is
too
low
for
its
maintenance
or
that
Anacystis
lacks
a
proper
replication
system
.for
E.
coli
plasmids:
On
the
other
hand,
we
cannot
rule
out
the
possibility
that
the
method
for
selection
of
resistant
cells
after
transformation
plays
a
role
in
the
elimination
of
a
resident
plasmid.
The
selection
pressure
applied
might
lead
to
a
situation
in
which
those
plas-
mids
prevail
that
show
the
highest
rate
of
expression
for
resis-
tance
or
the
highest
copy
number
(or
both).
It
would
explain
the
complete
replacement
of
pUH24
by
pUH24::Tn9Ol
or
by
pUCl
in
the
experiment
described.
Restriction
enzyme
analysis
of
plasmids
pCH1-pCH5
shows
that
these
plasmids
contain
a
single
Xho
I
site,
which
can
be
used
for
cloning
chromosomal
DNA.
By
deleting
a
part
of
pCH1,
a
smaller
plasmid
(pUC1)
was
constructed
that
contains
a
single
BamHI
site
as
well.
Because
the
deleted
part
of
pCHI
includes
one
end
of
the
Tn901
DNA,
the
possible
transposition
property
of
Tn901,
which
may
complicate
the
use
of
the
pUH24::Tn9Ol
plasmids
as
a
vector,
was
inactivated
in
pUC1.
Cells
carrying
pUC1
show
a
decreased
phenotypic
expression
of
Ap
resistance
when
compared
with
cells
containing
pCH1.
Although
a
difference
in
the
copy
numbers
of
both
plasmids
cannot
completely
be
ruled
out,
it
seems
more
likely
that
a
strong
promoter
is
located
in
the
deleted
part
of
pCH1.
If
so,
it
is
not
yet
clear
whether
this
is
a
promoter
of
Tn901
itself
or
one
originally
present
in
pUH24
and
pCH1.
Our
recent
isolation
of
a
recombinant
plasmid
that
consists
of
pUH24
in
which
a
5-MDal
part
of
pRI46
(including
its
Sm
resistance
gene)
has
been
inserted
opens
the
way
for
construc-
tion
of
a
plasmid
that
carries
two
resistance
genes
(Ap
and
Sm)
and,
therefore,
can
be
used
in
cloning
procedures
that
are
based
on
insertional
inactivation.
We
thank
Dr.
S.
V.
Shestakov
for
his
advice
on
cyanobacterial
transformation
and
his
generous
gift
of
transformable
strains,
Dr.
J.
D.
A.
van
Embden,
Dr.
E.
Veltkamp,
and
Dr.
C.
A.
van
Sluis
for
their
gifts
of
plasmids,
and
Dr.
P.
D.
Baas
for
his
gift
of
HindII.
C.A.M.J.J.v.d.H.
obtained
a
travel
grant
from
the
Netherlands
Orga-
nization
for
the
Advancement
of
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