Stable integration of foreign DNA into the chromosome of the cyanobacterium Synechococcus R2

37

Gene, 24 (1983) 37-5 1 Elsevier

GEN 00815

Stable integration of foreign DNA into the chromosome of the cyanobacterium Synechococcus R2 (Genetic

engineering;

John G.K. Williams

blue-green

photosynthesis)

* and Aladar A. Szalay **

Boyce Thompson Institute, (Received

algae; gene library;

Cornell University,

Tower Road, Ithaca, NY 148.53 (U.S.A.)

Tei. (607) X7- 2030

March 4th. 1983)

(Revised

April

(Accepted

15th, 1983)

April

18th, 1983)

SUMMARY

The blue-green aiga, Synechococcus R2, is transformed to antibiotic resistance by chimeric DNA molecules consisting of ~~~ee~ocoec~~ R2 chromosomal DNA linked to antibiotic-resistance genes from Escherichia coii. Chimeric DNA integrates into the 5’ynec~ococcus R2 chromosome by homologous recombination, The efficiency of transformation, as well as the stability of integrated foreign DNA, depends on the position of the foreign genes relative to Sy’nechococcus R2 DNA in the chimeric molecule. When the Qnechococcus R2 DNA fragment is interrupted by foreign DNA, integration occurs through replacement of chromosomal DNA by homologous chimeric DNA containing the foreign insert; transformation is efficient and the foreign gene is stable. Mutagenesis in some cases attends integration. depending on the site of insertion. Foreign DNA linked to the ends of Sl’nechococcus R2 DNA in a circular molecule, however, integrates less efficiently. Integration results in duplicate copies of Synechococcus R2 DNA flanking the foreign gene and the foreign DNA is unstab!e. Transformation in Synechococcus R2 can be exploited

to modify

precisely

and extensively

the genome

INTRODUCTION

Cyanobacteria (blue-green algae) are photosynthetic prokaryotes which occur as unicellular or * Present address: MSU-DOE Lansing, **To

Plant Research

Laboratory,

East

MI 48823 {U.S.A.). whom

correspondence

and

reprint

requests

shouid

be

addressed. Abbreviations: phatase; kilobase

Ap, ampicillin:

BAPF,

Cm, chloramphenicol;

EtBr,

or 1000 nucleotide

0378-I 119/83/$03.00

bacterial ethidium

alkaline bromide;

phoskb,

pairs of DNA.

SC 1983 Eisevier Science Publishers

B.V.

of this photosynthetic

microorganism.

filamentous organisms (Stanier and Cohen-Bazire, 1977). Their photosynthetic system, similar to that found in plants, consists of two photosystems, PSI and PSII, which cooperate to supply reductant and ATP for fixation of carbon dioxide. Reductant is obtained from water by photooxidation, accompanied by the evolution of molecular oxygen. These properties distinguish cyanobacteria from the other photosynthetic prokaryotes, the green and purple bacteria, which have single, non-oxygenic photosystems and require reduced sulfur

38

compounds, pounds

molecular

hydrogen,

as sources of reductant

or organic (Clayton,

com-

1980).

Several cyanobacterial species have a naturally occurring ability to take up and assimilate into their

genomes

(Shestakov her,

homologous

and Khyen,

1976; Devilly

and Porter,

and

Houghton,

R2 (Anacystis by recombinant

1977; Stevens cyanobacterium

nidulans

R2) can be

plasmid

DNA

con-

sisting of a native Synechococcus R2 plasmid linked to E. coli plasmid DNA that encodes resistance to various antibiotics (Van den Hondel et al., 1980; Kuhlemeier et al., 1981; Sherman and Van de Putte, 1982). The hybrid plasmid replicates extrachromosomally in the antibiotic resistant transformants. We were interested in developing a method for integrating foreign DNA mosome of Synechococcus method ifications

would permit

stably into the chroR2. Use of such a

more extensive

than those achievable

ing vehicles. combination

Previous studies in bacteria and

genetic mod-

with plasmid

from

Worthington.

All other

en-

zymes were from Boehringer-Mannheim. (b) Organisms and culture conditions

DNA

1970; Astier and Espardel-

1980). The unicellular

Synechococcus transformed

chromosomal

E. cofi BAPF

clon-

of homologous rein fungi (Contente

and Dubnau, 1979; Iglesias et al., 1981; Ruvkun and Ausubel, 1981; Jackson and Fink, 198 1) suggested to us that foreign DNA joined to Synechococcus R2 chromosomal DNA might integrate into the cyanobacterial chromosome during the transformation process. Here we report the results of transformation experiments with foreign DNA linked in two distinct configurations to a fragment of

E. coli HB 10 1 (Bolivar and Backman, used as host cell in all recombinant structions.

Transformation

out basically

con-

with DNA was carried

as described

Synechococcus

1979) was

plasmid

(Maniatis

et al.. 1982).

R2 (or A. nidulans R2; Shestakov

and Khyen,

1970) was obtained

from Drs. C. van

den Hondel

and G. van Arkel. and was cultivated

in BG-11 mineral salts (Rippka et al., 1979). BG- 11 solidified with 1.5% Difco Bacto agar was prepared as described (Allen, 1968). Synechococcus R2 was cultivated at 37°C in 1600 lux of “warm white” fluorescent light supplemented in some cases with 250 lux from a 60 W tungsten bulb. Liquid

cultures,

cultivated

in erlenmeyer

flasks

topped with silicone rubber caps (Bellco), were perfused with air (about 20 ml/min per 100 ml of culture); the air was moistened and sterilized by passage through a solution of 1% CuSO,. a filter of activated charcoal (Gelman 12122) and a membrane filter (0.3 pm pore; Gelman 4210). As determined by microscopic examination, a culture of Synechococcus R2 containing 1 X 10’ cells/ml has an absorbance (A,,,) of 0.25. Between 30% and 50% of cells in liquid suspension develop into colonies

on solid

medium

under

our conditions.

Synechococcus R2 DNA. Foreign DNA was found to integrate into the cyanobacterial chromosome

Since Synechococcus sp. cells are killed by cold temperature (Rao et al., 1977) we kept cells between 20°C and 40°C in all procedures. Cultures were checked for contamination as described (Rip-

by homologous

pka et al.. 1979).

recombination.

(c) Purification of DNA MATERIALS

AND

METHODS

(a) Materials and enzymes Polycillin-N (ampicillin) was purchased from Bristol Lab and chloramphenicol was from Sigma. Enzymes were used as described by Bethesda Research Laboratories (BRL). Restriction endonucleases Sau3A, Hue11 and HaeIII were purchased from BRL, E. coli DNA polymerase large fragment from New England BioLabs, and

DNA concentrations tion with diphenylamine

were determined by reac(Giles and Myers, 1965).

Synechococcus R2 plasmid and chromosomal DNA were purified by CsCl-EtBr equilibrium centrifugation. A 400 ml culture (A,,,, = 1.5 to 2.0) was harvested by centrifugation and suspended in 4 ml of 10% sucrose in 50 mM Tris . HCl. pH 8.5, 50 mM NaCl, 5 mM EDTA. The suspension was mixed with 0.1 ml lysozyme (35 mg/ml in water), frozen in a dry-ice ethanol bath, thawed. mixed with 0.1 ml lysozyme and 20 pl ribonuclease

39

A (10 mg/ml min),

and

incubated

spheroplasts sodium

in water;

heated

at 37°C

at 75°C

sarcosinate

15

for 1 to 2 h. The

were lysed by addition

N-lauryl

for

of 4 ml of 2%

in the above

buffer.

tion, up to about 0.3 pg/ml;

few additional

formants are obtained with higher DNA concentrations. The efficiency of transformation varies at

least

20-fold

between

different

amounts

of DNA,

experiments.

Using

saturating

served

at most

Deonier, 1979); the upper band in the gradient comprised broken plasmid and chromosomal DNA

(Table

I).

while

the lower

(e) Southern hybridization analysis of DNA

DNA

only.

The lysate was sheared and centrifuged to equilibrium in CsCl-EtBr as described (Hadley and

scribed

band

Both

(Maniatis

To prepare except

intact processed

DNA

plasmid

amplification

E. coli, host

(Maniatis was

et al., achieved

(450 pg/ml; Sigma). The cells and centrifuged (Hadley and

1979).

(d) Transformation

one

transformant

we have obper

200 cells

as deSamples

from

as described

that

using spectinomycin were lysed, sheared, Deonier,

were

et al., 1982). plasmid

cells were cultivated 1982a),

comprised

bands

trans-

of Synechococcus R2

Synechococcus R2 was transformed by a modification of the procedure of Van den Hondel et al. (1980). Cells from a young culture (A 730 less than 1.5) were diluted into fresh BG-11 medium (at A 730 = 0.06). The fresh culture was cultivated overnight (until A,,, = 0.25 to 0.35), harvested at room temperature by centrifugation, and suspended in l/l0 vol. of fresh BG-11. Competent cells (1 vol.) were mixed in a test tube with DNA (in l/50 vol., or less, of 10 mM Tris . HCl, 0.1 mM EDTA). Transformation mixtures were incubated under growth conditions and the tubes were agitated intermittently to keep the cells in suspension. After incubation for 6 h, aliquots were spread onto membrane filters (Nuclepore Membra-Fil, 0.45 pm pore size; autoclaved in water prior to use) resting on the surface of solid BG-11 medium in polystyrene petri plates. After incubation under growth conditions for 20 h, the filters were transferred to solid BG-11 medium containing the appropriate antibiotics (0.2 pg Ap/ml; 5 pg Cm/ml) and incubation was continued for at least 5 days to select for colonies of transformed cells. The yield of transformants is maximal after 4-5 h incubation of the transformation mixture. Incubation in darkness decreases the efficiency of transformation by at least IO-fold. The yield of transformants produced by the DNA molecules shown in Fig. 1 increases with DNA concentra-

of

Synechococcus

DNA (1 pg), Synechococcus

R2

chromosomal

R2 plasmid

DNA (50

ng), and pKWl075 DNA (50 ng), were digested exhaustively with restriction endonucleases and fractionated by electrophoresis in a 1% agarose gel; phage X DNA cleaved with Hind111 was included as molecular size standard. The gel was stained with EtBr, photographed, and transferred bidirectionally to nitrocellulose membranes (Smith and Summers, 1980). Hybridization conditions were as described by Compton et al. (1982); each membrane was incubated with 1 pg (10s cpm) of probe DNA labeled in vitro with 32P (Rigby et al., 1977). 32P bound to the membrane was detected by autoradiography. (f) Construction of the structural isomers pKW 1064 and pKW1065 pBR322 DNA (Bolivar and Backman, 1979) was cleaved with BamHI and spliced, using T4 DNA ligase, to fragments of Synechococcus R2 DNA generated by partial digestion with Sau3A. E. co/i HBlOl

was transformed

to Ap resistance

with the ligated DNA. From the resulting “gene library” we selected the plasmid pKWl006 (8.9 kb) by its ability to complement the thi-1 mutation in the host cell. Analysis of pKW 1006 by Southern hybridization indicated that it comprised a contiguous fragment of Synechococcus R2 DNA (4.5 kb) spliced to pBR322 (not shown). To construct pKWl064 (Fig. I), a 1.3-kb DNA fragment encoding resistance to Cm was spliced into pKWl006 at the unique BamHI cleavage site located at one of the junctions between pBR322 and the cyanobacterial DNA fragment. The Cm-resistance gene, excised from E. co/i plasmid pACYC184 (Chang and Cohen, 1978) by cleavage with HueII. was provided with BamHI “sticky ends” before use by

40

treatment with E. coli DNA polymerase large fragment to blunt the ends, followed by ligation to

RESULTS

BumHI

(a) Transformation tic resistance

linkers

(BioLogicals;

Like pKWl006, mutation

method

Bahl et al., 1976). complements

in E. coli. To construct

l), about cleavage

pKW1064

0.7 kb of DNA, site, was deleted

pKWl065

including

mid, which retained Hue111

to generate

ligated

to BamHI

the ability

to complement

was cleaved full-length

linkers,

the BumHI

the deletion partially

linear

cleaved

pBR322

DNA

of

pKW1072,

was cleaved

pKW1074

with

the with

BumHI,

and then ligated to the 1.3-kb Cm-resistance (see above). pKW1065 does not complement E. coli thi-1 mutation. (g) Construction pKW 1075

plas-

molecules,

with

BumHI

of Synechococcus R2 to antibio-

(Fig.

from pKW 1006 by the

of Covey et al. (1976);

E. coli thi-1 mutation,

the Uri-1

To construct transformation pared

DNA

a gene library

fragments

molecules

in Synechococcus

in E. cob, comprising

from Synechococcus

E. coli plasmid

for the study

pBR322.

R2 inserted

pBR322

of

R2, we first preDNA into the

specifies

resis-

tance to Ap. From the gene library we selected the recombinant plasmid pKW 1006 by its ability to

gene the

complement the thi-1 mutation in E. co/i HBlOl. Construction was completed by inserting into the cyanobacterial DNA a 1.3 kb DNA fragment which specifies resistance to Cm; this fragment

and

was derived from the E. cofi plasmid pACYCl84. The new plasmid, pKWl065 (Fig. 1). is unable to

and

complement the E. coli thi-1 mutation insertion of the Cm-resistance gene.

because

of

joined to fragments of Synechococcus R2 DNA generated by exhaustive digestion with BglII. The ligated DNA was used to transform E. coli to Ap

It is important to note that the continuity of the cyanobacterial DNA is interrupted by the Cm-resistance gene. but not by pBR322 DNA. The

resistance. The mixture of recombinant plasmids was isolated from E. coli, cleaved with BumHI (which, by design of the procedure, cleaves only in the cyanobacterial DNA), and ligated to the 1.3-kb

linkage arrangement determines the capacity of the foreign E. cob DNA to transform Synechococcus R2. Both antibiotic resistance genes are expressed in Synechococcus R2. a circumstance that enabled us to trace the different fates of each foreign DNA element during transformation. To transform Synechococcus R2, cells from an actively growing culture are suspended in fresh

Cm-resistance gene (see above). The ligated DNA was used to transform E. coli to Ap plus Cm resistance; the transformants were pooled; the mixture of recombinant plasmids was isolated and used to transform Synechococcus R2. This procedure selects for DNA structures capable of transformation. To cyanobacterial Cm (primarily chromosomal BglII, ligated

recover these structures from the cells, all transformants resistant to type I transformants) were pooled; DNA was extracted, digested with to pBR322 cleaved with BumHI,

and the ligated DNA was used to transform E. coli to Ap plus Cm resistance. We examined plasmid DNA from 30 individual E. coli Gansformants and found that only three different structures were represented: These are pKWl072, pKWl074, and pKW 1075 (Fig. 1). Southern hybridization analysis indicated that all three molecules contained Synechococcus R2 DNA of chromosomal origin (data not shown for pKW 1072 and pKW1074; see Fig. 2 for pKW1075).

growth medium and mixed with donor DNA. The transformation mixture is incubated in the light for 6 h. Aliquots of cells are spread onto membrane filters resting on solid medium. The cells are incubated for 20 h, the filters are transferred to solid medium that contains Cm, Ap, or both antibiotics, and incubation is continued for at least five days by which time resistant transformants have formed colonies. Transformation of Synechococcus R2 by pKWl065 results in three types of transformant (Table I): those resistant to Cm only (type I). to Ap only (type II), and to both antibiotics (type III). The predominant transformant was of type 1, there being 107 of these for each 8 of type II and 1 of type III. The transformation process was efficient, producing 2 x 10’ type I transformants per pg of DNA. Plasmid pACYCl84, the source of the

41

Fig. 1. Restriction

maps of donor

lines, the Cm-resistance indicate

continuity

Numbers

indicate

DNA molecules

gene from pACYCl84;

of Synechococcus length,

and pKWl065

contain

(MATERIALS

AND

R2 DNA;

in kb. Restriction the same fragment

METHODS,

sections

used for transformation. and open double and a dashed

segment

sites are: R, EcoRI; of cyanobacterial

Thin lines represent

lines, pBR322 of arrow

H, HrndIII;

DNA,

signifies

B. BarnHI;

while the other

Transformation

upon

The number

of foreign

DNA

interruption

Arrows

by the Cm resistance

P, Purl. The structural

donor

solid thick

a gene for Ap resistance.

molecules

contain

gene.

isomers

pKWl064

unrelated

fragments

f and g)

Cm-resistance gene in pKW1065, was unable to transform Synechococcus R2 to Cm resistance (Table I), which suggests that transformation depends linkage

R2 DNA;

Synechococcus

which contains

to cyanobacterial

DNA and indicates that the bacterial plasmid does not replicate autonomously in Synechococcus R2. The majority of transformants growing in the presence of Cm formed unusually small colonies, whereas transformants resistant to Ap (types II and III) formed colonies of normal size (Table I). The few normal colonies growing in the presence of Cm were generated at the same frequency as type III transformants (resistant to both Cm and Ap), which suggests that these colonies are type III transformants. Thus, the mutant phenotype is associated with acquisition of Cm resistance alone, but not in conjunction with Ap resistance. The mutant colonies grew no larger when supplemented with thiamine. We are therefore unable to explain the mutant phenotype as a defect in thia-

TABLE

I

antibiotics

of Synechococcur of normal

size colonies

are tabulated,

in the presence of each donor

of transformation

of antibiotics.

Plasmid

Transformants

per ml of

DNA

transformation

mixture, X 10m3

Cm pACYC 184 pKW 1065

Cm+Ap

AP 0

0

0

17

170

18 210

2 100(S) a pKW 1064

200

140

pKW 1072

190

4

2

pKW1074

46

1

0

pKW1075

1500

40

5

a Small colonies

(S) were generated

of

mixDNA

on M,). There were a total

units/ml

in the absence

resistance

The transformation

5 x IO9 molecules

50 ng, depending

of 3.8 X lOa colony-forming ture, determined

arising

as indicated.

tures (0.5 ml) contained species (or approx.

R2 to antibiotic

by pKWl065

DNA.

mix-

42

mine

biosynthesis

in the type I transformants,

might be expected

from the behavior

in E. coli (see above). that

mutagenesis

DNA

in the donor

relationship

Data presented

depends

as

of pKW 1065 below show

on the cyanobacterial

molecule,

and on its linkage

to the Cm-resistance

gene.

formation: pKW 1072, pKW 1074. and pKW 1075 (Fig. 1). Each DNA species differed in the efficiency

with which transformants

ranging

from 2-70s

as well as in the ratios of type I, type II, and type Ill transformants transformants

(b) Segregation and equal expression of Cm and Ap resistance The disparity resistant

in the number

to Cm and Ap suggests

tic-resistance tion, possibly

disparity

in all cases

in the greatest

and there were at least as many transof type

of the new DNA

tively

could also be explained

I). However,

formants

none

and homoloHowever, the

(Table

of type I occurred

behavior that the antibio-

cyanobacterial DNA in pKWl065 gous DNA in the recipient cell. observed

abundance,

of transformants

genes segregate during transformaas a result of recombination between

were produced.

of the yield with pKW 1065.

similar

II as of type

to that

of pKW1065,

of the new transformants

Ill.

Thus,

molecules

the

is qualitaexcept

have mutant

that phe-

notypes. (d) DNA structure in Synechococcw mants

R2 transfor-

by more For further

analysis,

we isolated

several

trans-

efficient expression of Cm resistance compared to Ap resistance. To investigate these possibilities, we constructed pKWl064 (Fig. 1), a structural isomer of pKWl065 in which the Cm-resistance gene is linked directly to pBR322; the absence of intervening cyanobacterial DNA should prevent segregation of the antibiotic resistance genes by homologous recombination. When Synechococcus R2 was

formants of each type produced by pKWl075. Transformants were purified by streaking on solid medium containing appropriate antibiotics (Cm or Ap or both); we verified that the type I transformants were killed by Ap, and type II by Cm. Each isolate was then cultivated in liquid medium with antibiotics, and its DNA was isolated.

transformed transformed

Initially, we analyzed DNA from only one example of each type of transformant, by Southern

antibiotics

with pKWl064, equal numbers of colonies grew in the presence of both together

and of Cm alone,

while two-

thirds as many grew in the presence of Ap alone (Table I). No mutant colony types were observed. These results indicate that expression of both resistance phenotypes is nearly equal, as measured by transformation. Furthermore, the differences between pKW1064 and pKWl065 show that the outcome of transformation depends on the configuration of the donor DNA, and that the resistance genes as they are arranged in pKWl065 probably segregate by recombination in Synechococcus R2. (c) Transformation

with non-mutagenic

DNA

The mutant type I transformants produced by pKWl065 could not be analyzed further because they died when subcultured. Therefore, the transformation process itself was used to aid construction of several non-mutagenic DNA molecules having the same general structure as pKW1065 (see MATERIALS AND METHODS, section g). We obtained three new DNA molecules capable of trans-

blot hybridization. DNA samples were cleaved with restriction endonucleases; the restriction fragments were fractionated by electrophoresis in an agarose gel, transferred to nitrocellulose membranes, and hybridized with two different radioactive DNA probes. Restriction fragments hybridizing to the probes were detected by autoradiography (Fig. 2). One of the probes was the donor DNA, pKWl075. and the other a mixture of pBR322 and pACYCl84. The cyanobacterial DNA fragment in pKWl075 is termed “insertional DNA”, because it mediates insertion of foreign DNA

into

the genome

of the recipient

cell (see

below). Although the pBR322 and pACYC184 probes failed to hybridize to DNA from the recipient (Fig. 2; lanes 1, 2, 7, 12) they hybridized strongly to DNA from the transformants (all other lanes), indicating that the transformants contain foreign DNA. The 3.5-kb insertional DNA fragment in pKW1075 was originally isolated from the

43

endonuclease: lane: DNA:

Barn

Hi

Eco Rt

6

78

910

'II 12 13 14 I5

D

R

il III

0

Bgl II 12345

R

pR 1 II III

I

R

I

II III

Probe

pBR322

f

Fig. 2. Southern

hybridization

analysis

of DNA isolated

type (I, II, and III), as well as from the untransformed endonucleases were derived

BglII,

BarnHI,

and EcoRI,

as indicated.

to reveal bands of low radioactivity,

lanes

a mixture

recipient

of chromosomal

(pR). See MATERIALS

AND

blotting

lighter exposures

and plasmid METHODS.

DNA,

R2 transformants.

and the donor

The top and bottom

from a single agarose gel using a bidirectional

was necessary contain

from Synechococcus recipient(R)

panels,

procedure.

for lane 2 which

from each transformant

(D), were cleaved with restriction

which show hybridization

Whereas

(not shown) enabled

except

DNA samples

DNA pKW1075

with different

the dark autoradiographic

us to resolve the more radioactive contains

purified

plasmid

probes,

exposure DNA

shown

bands.

All

from

the

section e. for details.

cyanobacterial genome by cleavage with RglII. Accordingly, the insertional DNA fragment is seen in DNA from the recipient as a 3.5-kb BglII restriction fragment that hybridizes to pKW1075, but not to pBR322 or pACYC184 (Fig. 2; lane 1). In contrast, plasmid DNA from the recipient failed to hybridize to either probe, demonstrating that the insertional DNA fragment is of chromosomal origin (Fig. 2; lane 2). The 3.5-kb insertional DNA fragment is not detected among BglII restriction fragments from any of the transformants. Instead, there are novel restriction fragments of larger sizes-4.8 kb from type I, 11.4 kb from type II, and 12.7 kb from type

III (Fig. 2; lanes 3-5)-suggesting that the transformants contain specific lengths of foreign DNA integrated into the chromosomal copy of insertional DNA. Indeed, all three novel Bg/II restriction fragments hybridize to the pBR322 and pACYC184 probes as well as to the pKW1075 probe. In constructing pKW 1075, the 1.3-kb Cm-resistance gene was spliced into a unique BanzHI cleavage site in the insertional DNA. Therefore, if this gene is present in a transformant, it should be excised precisely by cleavage with BarnHI. As expected, both probes hybridized to BamHI restriction fragments of 1.3 kb in the type I and III

44

transformants

only

(Fig.

2; lanes

8, lo),

which

The restriction

indicates that the Cm resistance gene is present in both Cm-resistant transformants and is absent

tify the foreign

from the type II transformant

integrated

and III transformants, striction BarnHI

fragments

(lane 9). The type II

however,

fragment

(Fig. 2;

by cleavage

of pBR322

plus in-

by assuming

in both Ap-resistant

that pBR322

transformants,

from

the

recipient

yields

two

produced

of foreign DNA is by

recombination,

we

deduced

the

arrangement of foreign DNA in the chromosome of each transformant (Fig. 3). In the type I trans-

not in the type I transformant. DNA

patterns

(Fig. 2; lanes 1 l- 15). and

that integration

homologous

but

at a site

DNA in pKW 1075.

the hybridization with EcoRI

in each

chromosome

to the insertional

By analyzing

above iden-

present

that the foreign DNA is

into the recipient

homologous

to the 7.9-kb

DNA (Fig. 1). This indicates

is present

re-

analyzed

elements

and indicate

of pKW1075

lanes 6, 9, 10) which consists sertional

yield BumHI

corresponding

restriction

transformant

fragments DNA

formant,

BumHI

the

chromosomal

copy

of insertional

restriction fragments, 2.5 kb and 12.5 kb in length, which hybridizes only to the pKW 1075 probe (Fig. 2; lane 7). These must be junction fragments comprising insertional DNA plus neighboring chromosomal DNA. The presence of the junction fragments in all three transformants (Fig. 2; lanes

DNA was replaced by homologous DNA from pKW1075 containing the Cm-resistance gene. In the type II transformant, all of pKW1075 except for the Cm-resistance gene was added to the chro-

8, 9, 10) indicates that additions to the recipient DNA occur so as to preserve the junction frag-

pKW1075 was added to the chromosome of the type III transformant, resulting in an integrated copy of pBR322 flanked by two copies of inser-

mosome, resulting in duplicate tional DNA flanking pBR322.

ments.

Recipient

Type 1

copies of inserFinally, all of

R I

R(B) L I

It 1

B II II

R(B) 1 1

II

B B :R: II (B)

(8) 1

R

-Cm-

Type

Type

D

R (B) 1 i

II I 4

n~

R(B) 1 I

II 1

B I

B B :R : Ap

Fig. 3. Arrangement (recipient)

parentheses fragments

of foreign

with pKW1075.

Fig. 2. Arrows for BglII;

1 IO

I 0

indicate

and other

I .----

the extent of Synechococcus

cleavage

(B); we show only one of the two possible of 2.5 and 12.5 kb (“junction

fragments”,

(B)

arrangements, see RESULTS,

generated

by transformation

in pKW1075

of two of the BamHI

which in recipient section d).

I

I 30

from the Southern

DNA) present

in the location

R I

I

I

sites were deduced

R2 DNA (insertional

are as in Fig. 1. Uncertainty

R 1

I 20

I

endonuclease

(B) I

B II 1,

DNA in type I, type II and type III transformants

Restriction

symbols

R 1

I

+-Cm-

kb

B II II

R I J A~I -

I

of Synechococcus

hybridization

patterns

(see Fig. 1). (II), cleavage cleavage

and transformants,

sites is designated

yields BarnHI

R2

shown in site by

restriction

45

tional

DNA,

one of which contains

mediated

the Cm resis-

duplicate

tance gene. The structures

diagrammed

probes,

found only in the type II and III transfor-

mants.

Two

of these

ments (approx.

are BglII

restriction

III) which are larger than the strongly respectively) there

may

minority

fragments

Several

frag-

analyzed

of foreign

DNA

lanes

is

l-5).

among cells in seemed tandem,

supported by the other weakly hybridizing fragments, 7.9 kb in both the type II and III transformants, which were produced by cleavage with EcoRI (Fig. 2; lanes 14, 15). Since 7.9 kb is the length of pKW1075 minus the Cm resistance gene,

ment during ent cultures.

1

Fig. 4. Diversity EcoRI

2

4

of DNA structure

and analyzed

by Southern

5

6

7

in the transformants. hybridization.

as indicated

sam-

and hybridized

with

DNA

probe.

When

we

was some

diversity,

however,

had a detectable

suggesting

amount

of the 7.9-kb

that the degree of rearrange-

cultivation

can be different

in differ-

Diversity was most evident among the type III transformants. None of the three additional trans-

Ill 8

9

DNA samples

The hybridization

DNA from the same type I, II and III transformants transformants,

fragment,

II

3

were

kb EcoRI restriction fragment (Fig. 4; lane 6). None of the three additional type II transformants

tandem. Whereas the cultures of each transformant analyzed originated from a single cell, we suggest that rearrangements occur during cultivation of type II and III transformants, probably

Lance:

type DNA

the type II transformants. A minority of the type II transformant analyzed above to have two copies of DNA integrated in as indicated by a weakly-hybridizing 7.9-

we analyzed

I

each

hybridization.

pKW1075

There

we believe that in some of the cells two copies of this portion of donor DNA are integrated in

Type:

I of

transformants by Southern

compared four additional type I transformants to the one analyzed above, all were identical (Fig. 4;

in a

this interpretation

of DNA structure in the transfomants

a radioactive

12.7 kb,

(Fig. 2; lanes 4, 5). This suggests that

of the cells. Indeed,

between

DNA.

ples were cleaved with EcoRI

hybridizing

(11.4 kb and

be two copies

crossing-over

copies of insertional

(e) Diversity

20 kb in type II, and 22 kb in type

restriction

unequal

in Fig. 3 account

for all of the hybridization data except for a few restriction fragments that hybridize weakly to the

BglII

by

11

12

13

from several type I, II and III transformants

probe was the donor

that were analyzed

in the figure (Type I, II or III). Arrows

10

DNA pKW1075.

in Fig. 2; the remaining

point to faint bands,

Lanes

lanes contain

7.9 kb in length.

were cleaved with 1, 6, and 10 contain

DNA from additional

46

formants

analyzed

were identical

to one another

or

graphs taken earlier. The results of this experiment

to the one analyzed above (Fig. 4; lanes lo- 13). Of the four type III transformants, two contained

(Table

II) reveal a striking

among

the transformants.

the weakly-hybridizing of rearrangement,

7.9-kb

fragment

two

contained

and

restriction

fragment,

which corresponds

the EcoRI

restriction

fragments

that in these two clones

resistance

resides

gene

on

mant

Cm resistance

a 3.3-kb

tance

was detected

to one of

tested.

in pKW1075

1) and indicates rather

indicative

the

(Fig.

left of pBR322,

analysis

of antibiotic

Transformants

resistance

analyzed

hybridization

Nuclepore

membrane

had developed; 0.4 pg/ml). period

Control

(g) Transformation

III

to be as stable as native

with linear DNA

colonies

was continued

by comparison

to liquid

without

antibiotics.

transferred

for a second

taken

and cultivated

dilutions

containing Colonies

after

the first period:

all colonies

culture

during

dead colonies

of type I transformants

conditions.

Transformant

First incubation

type

(no antibiotics) Number

of colonies

(Cm at 5 pg/ml: the second

of

Antibiotics

1025

1025

283

243

III

307

226

III

292

219

incubation

III

292

217

Cm

0

AP Cm

14

AP Cm+Ap

Ap at

incubation

lose their green color

colonies

II

onto

and

are killed by Ap, and type II

killed in second Number

of 3 x lo9

were spread

% of colonies

Second incubation

at The

for six days until small colonies

as indicated

killed by antibiotics

of antibiotics antibiotics;

to a final density

of each liquid

antibiotics

without

under non-selective

The filters were incubated

to medium

six day period.

that under these conditions

medium

resistance,

in the presence

on solid medium

are defined as being of the first generation

with the photographs

showed

were streaked

by Cm, as expected.

I

type

Synechococcus R2 can be transformed by chromosomal DNA isolated from each type of trans-

To test cells for antibiotic

filters resting on solid medium

experiments

II and

recombination between duplicate copies of insertional DNA. The type

in a type I transformant DNA.

of each type was transferred

then the filters were photographed,

were identified

disappear.

One colony

of 45 generations.

and incubation

Type

(the same as in Fig. 2) were grown on solid medium

cells resting on the solid medium

for a total

above.

phenotypes

by Southern

cells grew into colonies. cells/ml,

we how-

I transformant, which contains no duplication, is stable. Accordingly, we would expect foreign DNA

(Cm for type I; Ap for type II; Cm + Ap for type III). The resulting this point, individual

II and type III transformants,

described

homologous chromosomal

antibiotic resistance genes. Cells from each culture were spread onto membrane filters resting on solid medium and were incubated in the absence of antibiotics for 6 days until small colonies had formed. The filters were photographed, transferred to medium containing antibiotics, and incubated for a second 6-day period. Colonies killed by period antibiotics during the second incubation were identified by comparison with the photo-

Stability

no loss of resis-

the 1025 colonies

transformants are phenotypically unstable, probably due to structural rearrangements mediated by

The three transformants analyzed in Fig. 2 were cultivated for 45 generations in the absence of antibiotics to avoid killing cells that lose their

II

was stable: among

one-quarter of those from the type III culture had lost resistance to Cm and Ap. These results correlate well with the structural

in Fig. 3.

(f) Stability of antibiotic resistance phenotypes

TABLE

in stability

ever, were unstable. Of the colonies from the type II culture 14% were killed by Ap, and about

the Cm

than on the right, as diagrammed

Type

difference

In the type I transfor-

26 25 25

formant.

Chromosomal

pose was isolated pKW1075

DNA

used

for this pur-

tance

generated

resistance

from transformants

and

consisted

of random

by

cells to Cm resistance

type II, to Ap resis-

all of the

Exp. 1: Transformation

mixtures

DNA (M, 2.7 X 109; Herdman

chromosomal

or 200 ng of pKWl075

ments

as indicated;

generated.

complete

in both

agarose

in pBR322 tained

diagrams

Symbols samples

are

(Bolivar

forming

as judged

and Backman,

of antibiotics.

with BamHI frag-

1. Cleavage

was

by electrophoresis

in an

of the Ap resistance

gene

1979); cleavage, mixtures

per se, does (1.2 ml) con-

There were a total of 4.3 X 10’ colony

units per ml of transformation

the absence

1974).

show the restriction

this gene. Transformation

120 ng of DNA.

exhaustively

as in Fig.

gel. Hind111 cleaves outside

not destroy

and Carr,

DNA.

Exp. 2: pKW 1065 DNA was digested or HindIII,

120 ng of

Diagram

DNA

mixture, symbols

determined

are as in Fig. 1.

Transformants

per

ml of transformation mixture, Cm Exp. 1

X IO-’

Ap

1). Similar

re-

with chromosomal

DNA from

analyzed

hybridization

in Fig. 4 (data not shown).

Whereas

the antibiotic

resistance

phenotypes

in transformation

were

tightly

linked

DNA

Ap resistance

conjunction

from

were found

DNA,

in transformation

with

the type

III

with Cm resistance.

may be related to the structural

context of pBR322.

ever,

pBR322

cyanobacterial

interrupts DNA

the

(see type

continuity

ent forms of linear pKW1065 with restriction endonucleases Table III; Exp. 2). pKW1065

0

produced no Cm-resistant transformants, Ap-resistant transformants arose at

0

2.3 0

nearly

type III chromosome

4.6

1.2 I.4

Opposite results were observed for cleaved with HindIII: no Ap-resistant

38

1.7

Cm

0

the same frequency

as with circular

DNA.

pKW 1065 transfor-

mants were formed, while Cm-resistant transformants appeared at a frequency only three times lower than with circular DNA. These results show that linear DNA can transform Synechococcus R2 with roughly the same efficiency as can the corresponding circular DNA, providing that the antibiotic resistance genes are linked, on both ends, to cyanobacterial DNA.

AP

cut

the

DNA by cleavage (see diagrams in DNA cleaved with

6.8

1.8

of

III transformant;

Fig. 3) and integrates at a frequency more like that of the Cm-resistance gene. To investigate transformation with linear molecules of defined structure, we prepared two differ-

although

500(S) a

to the ends and it in-

tegrates with much lower efficiency than the Cmresistance gene. In the chromosomal DNA, how-

BamHI

Cm

in

This observation

In pKW1075, pBR322 DNA is linked of the cyanobacterial DNA fragment

Cm+Ap

1.1 0.1

transfor-

was nearly always acquired

0 65

to

with pKWl075

type II chromosome

pKW 1065

pKWl065

Experiment

Cm and

type I chromosome

pKW1075 Exp. 2

Ap

in

to both

Southern

mant:

(0.3 ml) contained

III;

transformants

chromosomal of Synechococcus with linear DNA

III,

by

they III

Transformation

type

(Table

segregate TABLE

and

sults were observed

fragments

produced by shearing during purification. DNA from a type I transformant transformed recipient only;

only;

46

0

with Barn HI DISCUSSION

AP Cm

0.07 with Hind111

150(S) = AP

a see Table I.

0

0

Synechococcus R2 has a natural ability to take up and be transformed by exogenous DNA. Natural transformation, rare among living organisms, has been studied extensively in a number of bacteria (Smith et al., 1981). We found that foreign DNA, when linked to Synechococcus R2 chro-

48

mosomal

DNA,

bination

is inserted

by homologous

into the cyanobacterial

though

nearly

all organisms

recom-

chromosome.

are capable

each

exogenous

donor

pKW 1072, pKW1074, the most frequently tion

of type

placement DNA

and

containing type

(pKWl065,

pKWl075;

detected

which

involves

DNA by homologous

the Cm-resistance

re-

gene (Fig. 3).

will result

only

the cyanobacterial

of

into the recipient

the

another

Cm-resistance

gene,

but

chromosome

cross-over

on the right,

occurs

not

providing on the left,

of the Cm-resistance

donor

molecule

to the recipient

would

generate

a type III transformant

if

chromosome

and

(Fig.

5;

that of a double cross-over, which implies that type III transformants should be produced in

DNA fragment.

TYPE

- __+

RATIO’ MUTANT’ STABLE2

-w-1

!

could

by donor

bottom pathway). According to genetic theory, the probability of a single cross-over is greater than

so as

- ---

DNA

gene. The occurrence of only a single cross-over, however, would result in addition of the entire

donor

foreign DNA is linked in the donor molecule to interrupt

I),

event was the genera-

I transformants

transfer pBR322, and

Table

mechanisms

of recipient

that one reciprocal

tested

I transformants

of recipient

Clearly,

DNA.

molecule

recombination

replacement

DNA (type I transformant). A reciprocal exchange between recipient and donor DNA would result in

of de-

in which Synechococ-

pends on the specific manner For

mediate

of homol-

ogous recombination, the particular distribution transformation products we observed probably cus R2 processes

Two known

Al-

107

S

M

Cm

chromosome

donor

DNA

h

- --+

-

-1

1

FT- Cm Ap - = WT

Fig. 5. Model for integration designated To generate because DNA.

The non-replicating

transformant,

except

subsequent

reciprocal

single reciprocal donor

occurs

DNA

wild-type.

cross-over

molecule

are as in Fig. 1. Homologous

on the left). The paired DNA is replaced

We found

is eliminated

from

of pBR322.

generated

donor

DNA

exchange instead

(Bottom):

donor

and recipient

in three different

subsequently

Type III transformants

resistant

a type I1

to the recipient. can be generated

either on the left or right of pBR322

’ Determined

transformants).

(see Table II). S. stable;

gene;

and recipient

in generating

transferred

gene.

DNA is

ways: (Top):

the Cm-resistance

in both donor

is also involved of being

of the Cm-resistance

to 18 Cm+Ap

by pKWl075

between

DNA that contains

gene is present

gene can integrate

to 170 Ap resistant,

in transformants

pairing

can be resolved

Non-reciprocal the donor

on the left or right, respectively,

I: 2100 Cm resistant,

was measured

by homologous

is finally lost. (Middle):

that the Cm-resistance

occurs

intermediate

the Cm-resistance

event (X) leads to integration

the cross-over

(see Table

’ Stability

Symbols

exchange,

the Cm-resistance

cross-over.

on whether

chromosomal

by non-reciprocal donor

that

DNA.

dashes (diagram

a type I transformant,

replacement

depends

of foreign

by a series of vertical

U. unstable.

A by a

(Fig. 4); this for pKWl065

(M) mutant.

(WT)

49

greater

yield than

observed

type I transformants.

just the opposite,

transformants

we suggest

are generated

Since we that type I

by a different

process,

non-reciprocal exchange (Fig. 5; top pathway); in fungi, non-reciprocal exchange occurs more frequently Fink,

than reciprocal

crossing-over

198 1; Radding,

(Jackson

1978). Non-reciprocal

change could result either in transfer sistance

gene

postulated

into

the recipient

for the type

versely, in elimination DNA

(see Radding,

ex-

of the Cm-re-

chromosome

I transformant,

as

or con-

of this gene from the donor 1979). The latter

conjunction with a single reciprocal would generate a type II transformant middle pathway). that Ap resistance

and

It is interesting was transferred

reaction

in

cross-over (Fig. 5;

in this regard from donor to

recipient with the same efficiency whether or not the insertional DNA was interrupted by the Cmresistance gene (compare the structural isomers pKW1064 and pKW1065; Table I). According to our model, this suggests that the occurrence of a non-reciprocal exchange event had little effect on the probability of reciprocal crossing-over in neighboring DNA. Linear DNA was found to be nearly as efficient

DNA.

A type I transformant

equally

well with linear

obtain could

could be generated

or circular

DNA;

type II and III transformants, be repaired

gous interaction

cules, ranging formants

form by homolo-

with the recipient

chromosome.

R2 was transformed

indicates

DNA

(Table

that insertional

of the donor

mosome structure in the recipient cell: some regions may be more accessible or recombinogenic than others. Also, uptake of donor DNA might depend on the presence of certain nucleotide sequences, bacteria

as shown for the naturally H.

influenzae

and

H.

transformable parainfluenzae

(Danner et al., 1980). A mutant colony type was associated only with type I transformants generated by pKW1065, but not with type II or type III transformants (Table I). This pattern is explained by the molecular structures

deduced

for each transformant

Type I transformants because their single

echococcus R2 and B. subtilis suggests that the cyanobacterium also may cleave donor DNA concomitant with the transformation process. Cleavage of donor DNA, particularly within the foreign elements, could strongly influence the distribution of transformant types. models diAlthough the recombination agrammed in Fig. 5 show the donor DNA in circular

form,

the

models

also

apply

to linear

to

or to chro-

naturally (Duncan

than circular DNA. The increased activity of linear DNA seen in yeast is thought to be related to easier access of recombination enzymes to DNA at the ends of linear molecules (Orr-Weaver et al., 1981). This idea is consistent with transformation in B. subtilis, since donor DNA is cleaved by the bacterium itself concomitant with uptake (Smith et al., 1981). The similarity of transformation in Syn-

of

could be related

molecules

Type II and III transformants are normal both contain intact copies of insertional

cially induced transformation of yeast, where linear donor DNA integrates by homologous recombination several thousand times more efficiently

frag-

insertion

as circular DNA in transformation (Table III; Exp. 2). This is in accord with observations in the transformable bacterium B. subtihs et al., 1978) but contrasts with the artifi-

I). This

DNA

to mediate

DNA. Such differences

the structure

mole-

from 5 X lo5 to 2 X 10’ type I trans-

ments differ in their ability foreign

with differ-

by each of the four donor

per pg of donor

observation

DNA

to a circular

Synechococcus ent efficiencies

and to

linear

(Fig. 3). because DNA.

have a mutant phenotype copy of insertional DNA is

interrupted by foreign DNA. Generation of mutants by pKW1065, but not by the other donor molecules, implies that insertion of the Cm-resistance gene always occurs at the site of homology with insertional DNA, a conclusion supported by the physical analysis of 13 independent transformants (Fig. 4). We have shown that foreign DNA can be inserted efficiently and stably into the chromosome of Synechococcus R2. Transformation can be used to mutagenize, identify, and isolate cyanobacterial genes. In addition, a multitude of foreign genes can be added to the cyanobacterial chromosome at a variety of sites. Type I transformants can be generated with foreign DNA fragments as large as 20 kb (K.S. Kolowsky, J.G.K. Williams and A.A. Szalay; manuscript in preparation). This technology should prove useful in studying the molecular genetics of photosynthesis and other processes, as well as for genetic engineering in a photosynthetic organism.

50

ACKNOWLEDGEMENTS

Hadley,

R.G. and Deonier,

type II F’ plasmids.

The authors

gratefully

acknowledge the generR2 from Dr. Cees van

ous gift of Synechococcus den Hondel Russ

striction

for assistance

endonuclease

DNA. grant

and Dr. G. van Arkel. We thank

MacDonald This

cleavage

research

was

PCM-8106755

by the National

sites

in

supported

awarded

Dr.

in mapping

re-

donor

in part

to Aladar

by

A. Szalay

Herdman,

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Tn90f

Intro-

Communicated

by H.O. Smith.

of Anacysfis

in cyanobacteria.

Sci. USA 77 (1980) 1570-1574.

S.. Van der Ende, A..

W.E. and Van Arkel. G.A.:

into a plasmid

for cloning

Proc.