Vol. 172, No. 6JOURNAL OF BACTERIOLOGY, June 1990, p. 3138-31450021-9193/90/063138-08$02.00/0Copyright C 1990, American Society for Microbiology

Use of a Conditionally Lethal Gene in Anabaena sp. Strain PCC7120 To Select for Double Recombinants and To Entrap

Insertion SequencesYUPING CAI AND C. PETER WOLK*

MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824-1312

Received 8 September 1989/Accepted 23 February 1990

Use of the sacB gene (J. L. RBed and A. Coilmer, Gene 57:239-246, 1987) provides a simple, effective,positive selection for double recombinants in Anabaena sp. strain PCC 7120, a filamentous cyanobacterium.This gene, which encodes the secretory levansucrase of BaciUus subtlis, was inserted into the vector portion ofa suicide plasmid bearing a mutant version of a chromosomal gene. Cells of colonies in which such a plasmidhad integrated into the Anabaena chromosome through single recombination were plated on solid mediumcontaining 5% sucrose. Under this condition, the presence of the sacB gene is lethal. A small fraction of the cells

from initially sucrose-sensitive colonies became sucrose resistant; the majority of these sucrose-resistantderivatives had undergone a second recombinational event in which the sacB-containing vector had been lostand the wild-tpe form of the chromosomal gene had been replaced by the mutant form. By the use of thistechnique, we mutated two selected genes in the chromosome of Anabaena sp. strain PCC 7120. Theconditionally lethal nature of the sacB gene was also used to detect insertion sequences from this Anabaenastrain. Sucrose-resistant colonies derived from cells bearing a sacB-containing autonomously replicatingplasmid were analyzed. Five different, presumed insertion sequences were found to have inserted into the sacBgene of the plasmids in these colonies. One of them, denoted IS892, was characterized by physical mapping. Itis 1.7 kilobases in size and is present in at least five copies in the genome of Anabaena sp. strain PCC 7120.

Cyanobacteria constitute a highly diverse group of pro-caryotes that perform oxygenic photosynthesis. Some fila-mentous species are capable of differentiation and aerobicnitrogen fixation (34). Molecular genetic studies of cyano-bacteria have been greatly facilitated by recent advances intechniques for genetic transfer (28, 37). However, site-directed modification of the chromosome, a powerful tool forstudy of gene function, has been applied nearly exclusivelyto unicellular cyanobacteria (28) because of the ease withwhich double recombinants can be isolated from theseorganisms.

Isolation of double recombinants from filamentous cyano-bacteria such as Anabaena species has been difficult for tworeasons. First, when homologous sequences of DNA withinplasmids are transferred to Anabaena sp. by conjugation,single-crossover events (integration recombinations) occurfar more frequently than double-crossover events (replace-ment recombinations) (18; this study). Second, because cellsof Anabaena sp. have multiple genomic equivalents ascalculated from the genetic complexity (3, 22) and theamount of DNA per cell (7) and are linked, isolation ofrecessive mutants requires both segregation of mutant andwild-type forms of the genome and physical disjunction ofadjacent cells of a filament bearing the two genomic forms(8).Golden and Wiest (18) first introduced an insertional

mutation into the chromosome ofAnabaena sp. by screeningfor double-recombinant derivatives of selected single recom-binants. Their screening experiment made use of a 17-kilobase (kb) homologous fragment of DNA with an antibi-

* Corresponding author.

otic resistance-conferring inactivation cassette (19) insertednear the middle. Quantitative work with Escherichia coli hasshown that the rate of recombination decreases greatly asthe length of homology decreases (33). Recombination inAnabaena sp. may have a similar size dependence. InAnabaena sp. strain PCC 7120, exhaustive screens havefailed to isolate double recombinants when the size of thehomologous region was below 4 kb (J. Elhai, personalcommunication; this study). For this reason, we sought toisolate double recombinants by positive selection for loss ofthe vector portions of plasmids that had integrated into thechromosome. Such positive selection can be achieved byinclusion of a conditionally lethal gene within the vectorportion of the plasmid.A conditionally lethal gene, sacB, has been used to isolate

double recombinants in the gram-negative bacterium Er-winia chrysanthemi (30). This gene, from the gram-positivebacterium, B. subtilis, encodes levansucrase, a 50-kilodaltonsecretory protein, production of which is induced by sucrose(14). The gene has been cloned (15) and sequenced (35), andits expression has been well studied (2, 24). Expression ofthe gene is lethal to such gram-negative bacteria as E. coli,Agrobacterium tumefaciens, Rhizobium meliloti, and E.chrysanthemi in the presence of 5% sucrose in solid medium(14, 15, 30). Growth is inhibited and cells lyse within as littleas 1 h after the induction by sucrose (15). Lethality may bedue to transfructosylation from sucrose to various metabol-ically important acceptors (15) or to accumulation of unse-creted protein in the cell membranes because of inadequatecleavage and export (compare reference 5). Because cyano-bacteria have the peripheral structure characteristic of gram-negative bacteria, we tested two strains ofAnabaena sp. andfound both of them susceptible to sucrose when bearing the

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TABLE 1. Strains and plasmids used in this study

Strain or plasmid Marker Other relevant propertiesa Reference or source

Escherichia coliDH5a

HB101

Nxr

Smr

480dlacZAM15A(lacZYA-argF)U169derivative of strain DH5

recA

21; Bethesda Research Laboratories

Bethesda Research Laboratoriescatalog

Anabaena sp.EF116M-131PCC 7120PCC 7120::pRL256PCC 7120::pRL263PS250-1 to -10

PS256-5

PS256-17PS263-1 (also -2,

-50)PS263-42 (also -53,

-54)

PlasmidspDU1

pRL1pRL44pRL52

pRL57

pRL61pRL61M

pRL171PSmpRL256pRL256pRL263pRL272pRL351

pRL393

pRL517b

pRL528

pUCD800pUM24'rrvx

Smr/SprSmr/Spr EmrNmr

Smr/Spr

Smr/SprSm/Spr Emr

Smr/Spr Emr

CmrKmr/Nmr Smr/SprKmrfNmr

Cmr KmrfNmr Smr/Spr

Kmr/Nmr Smr/SprKmr/Nmr Smr/Spr

Apr Smr/SprCmr KmrNmr SucsSmr/Spr SucsCmr Emr Smr/Spr SucsCmr Kmr/NmrApr

KmrINmr

Cmr Smr/Spr

Cmr

Kmr SucsApr Kmr Suc'Apr

hetA Nif- (aerobically)Het-Nif+Suc' Nif+Sucs Nif+Sucr variants of strain PCC 7120(pRL250); Sucr

Sucr variant of PCC 7120::pRL256;Sucr Nif+

hetA::C.S4; Sucr Nif- (aerobically)nijD::C.S4; Suc' Nif-

Sucr variant of PCC 7120::pRL263;Sucr Nif+

Confers capacity to replicate inAnabaena sp.

Shuttle vectorS.K3 + L.HEH2 + C.S3(Previously denoted 41B9 subclone

17) Anabaena sp. strain 7120 hetAin pRL25C

S.K5 + L.HEH2 + C.S3; positiveselection shuttle (pDU1) cloningvector

hetA::C.S4 derivative of PRL52MluI deletion of pRL61; unable to

replicate autonomously in Ana-baena sp.

S.A1 + L.HEH1 + C.S4pRL57-based shuttle, contains sacBhetA::C.S4, contains sacBnijD::C.S4, contains sacBsacB::IS892 derivative of pRL250hetA in pUC8

nifH and part of nipD in pRL19B

nifH and part of nifD, from pAn154,with C.S4 inserted in the KpnIsite of nifD, in a pRL1V-basedplasmid

Helper plasmid for conjugal transfer;contains methylase genes for AvaIand Eco47II sites

Contains sacBContains sacB-nptI cartridgeSource of polylinker

3637See reference 37This studyThis studyThis study

This study

This studyThis study

This study

See reference 37

37This study23, 36

10; this study

This studyThis study

This studyThis studyThis studyThis studyThis studyD. Holland and C. P. Wolk

(unpublished data)10; G. Schmetterer and C. P. Wolk

(unpublished data)29, 37; J. Elhai and C. P. Wolk

(unpublished data)

11

143032

a Ap, Ampicillin; Cm, chloramphenicol; Em, erythromycin; Km, kanamycin; Nm, neomycin; Nx, nalidixic acid; Sm, streptomycin; Sp, spectinomycin; Suc,5% sucrose. We used neomycin in Anabaena sp. and kanamycin in E. coli to select resistance conferred by the npt gene (37). For plasmids pRL44, pRL57, andpRL171P5m, see reference 10 for nomenclature. C.S4 is described in reference 4.

sacB gene. We further found that the conditional lethality ofsacB in Anabaena sp. enables direct selection for doublerecombinants on sucrose-containing solid medium.The conditional lethality of the sacB gene has also been

used to isolate insertion sequence (IS) elements from gram-negative bacteria (14). IS elements are mobile genetic ele-ments that are generally about 1 to 2 kb in size and containonly genes related to transposition (13). Direct genetic

selection for transposition of these elements is generally notpossible. However, IS elements can be detected indirectly asa consequence of their transposition into and inactivation ofa marker gene (12, 26). The sacB gene is a good example ofsuch a marker gene; insertion of an IS element abolishes thelethality of the sacB gene and thus permits growth on solidmedium containing sucrose. By this means, we detected atleast five putative insertion sequences from Anabaena sp.

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3140 CAI AND WOLK

EcoRV lulC

strain PCC 7120. One of these, 1.7 kb in size, was restrictionmapped.

MATERIALS AND METHODS

The bacterial strains and plasmids used are described inTable 1. Anabaena sp. was grown in AA-based media (1),supplemented with nitrate when so indicated. Filter-steril-ized 50% sucrose was added to autoclaved LB or AA plusN03 agar medium to a final concentration of 5%.Enzymes purchased from Bethesda Research Laborato-

ries, Inc. (Gaithersburg, Md.) or New England BioLabs,Inc. (Beverly, Mass.) were used essentially according to therecommendations of the supplier. DNA was labeled with therandom primer labeling kit purchased from Bethesda Re-search Laboratories. Cloning (27), Southern transfer (9) andhybridization (27), and conjugation with plasmid pRL528 asthe helper plasmid (11, 37) followed standard procedures. Toselect for simultaneous resistance ofAnabaena sp. to strep-tomycin (Sm') and spectinomycin (Spr) (18), we added 2.5,ug/ml of each to nitrate-containing solid medium and 1.5

1 kb

FIG. 1. Essential features of plasmids pRL250 (a), pRL256 (b),and pRL263 (c). The construction of these plasmids is described inMaterials and Methods. The horizontal line in panels b and crepresents a portion of the chromosome ofAnabaena sp. strain PCC7120; the bold region represents the portion subcloned into thecorresponding plasmids. The position and direction of transcriptionof the hetA gene shown are based on reference 23. The same scalemarker is valid for panels a, b, and c.

,ug/ml of each to nitrate-containing liquid medium and ni-trate-free solid medium. Other antibiotics were used atconcentrations described previously (37).

Total DNA was isolated from Anabaena sp. by the fol-lowing modification of a published technique (17; D. Hol-land, personal communication). Cells ofAnabaena sp. in themid-log phase of growth were harvested from 25 to 50 ml ofliquid culture and suspended in a final volume of 400 ,ul in amicrocentrifuge tube with 10 mM Tris-0.1 mM EDTA, pH7.5. Then, 150 j,l of sterile glass beads (catalog no. G-9143;Sigma Chemical Co., St. Louis, Mo.), 20 ,ul of 10%o sodiumdodecyl sulfate, and 450 ,ul of phenol-chloroform (1:1, vol/vol) were added. The mixture was subjected to a cycle ofvigorous vortexing for 1 min followed by cooling on ice for 1min for a total of four times. The resulting suspension wascentrifuged at 15,000 x g for 15 min, and the clear superna-tant solution was transferred to a new microcentrifuge tubeand phenol extracted, and its DNA was ethanol precipitated.To isolate double recombinants, initial exconjugants were

suspended in 50 ml of AA/8 plus N03 medium, shakenunder growth conditions for 4 to 6 h, subjected to cavitationin a sonic cleaning bath for 5 to 10 min, and washed twicewith the same medium. Cells (106 to 107) were then plated onAA plus N03 plus 1% purified (6) agar containing strepto-mycin, spectinomycin, and 5% sucrose. We refer to thisprocedure as direct plating. In some experiments, excon-jugants were also subcultured in 50 ml of AA/8 plus N03

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ISOLATION OF DOUBLE RECOMBINANTS FROM ANABAENA SPECIES

plus streptomycin and spectinomycin liquid medium, withcavitation of the culture for 5 min at 4- to 7-day intervals for1 month. The cells were then washed once and plated asdescribed for direct plating but at 106, 105, and 104 cells perplate. Cell density was estimated from methanolic extractsof cells (25) by assuming a content of 0.4 pg of chlorophyll aper cell.

Plasmid pRL57, a positive selection shuttle cloning vec-tor, was constructed by ligation of the pDU1-containingClaI-NdeI fragment of pRL1 (37) with the npt-containingNdeI-AsuII fragment of pRL44 (Table 1). Plasmid pRL250(Fig. la) was constructed by cloning the 3,881-base-pair (bp)BamHI fragment containing the sacB-nptl cartridge frompUM24 (30) between the two BamHI sites of pRL57. Suicide(integration) plasmid pRL256 (Fig. lb) was constructed asfollows. Smr/Spr cassette C.S4 (4), bounded by SmaI sites,from plasmid pRL171PSm (Table 1) was inserted into theNruI site of the 3.5-kb partial Sau3AI fragment ofAnabaenasp. DNA in plasmid pRL52, inactivating the hetA genewhich is required for complementation of mutant EF116 (23,36). The resulting plasmid, pRL61, was then stripped of itsability to replicate autonomously in Anabaena sp. by delet-ing a 1.7-kb MluI fragment from the pDUl portion (31),yielding plasmid pRL61M. Finally, the 2,597-bp BamHI-sacB-PstI fragment from plasmid pUCD800 (14) was in-serted between the BgtII and PstI sites in the npt region ofpRL61M. Suicide plasmid pRL263 (Fig. lc) was constructedas follows. The cat gene of plasmid pRL517b (Table 1),excised with NheI and AsuII, was replaced by the 3,851-bpXbaI-sacB-nptI-AccI fragment from pUM24. The nptl genewas then cut out with PstI and replaced by cassette C.CE1(10) from NsiI to HindIlI supplemented with the 42-bpfragment of 'rvx (32) from HindIII to PstI.

RESULTS

Effect of sacB expression on Anabaena cells. The two strainsof Anabaena sp. that we tested, PCC 7120 and M-131, grewas well, and often better, on media containing 5% sucrosethan on media lacking sucrose. When bearing the multiple-copy plasmid pRL250 that contains the sacB gene (Fig. la),both strains became extremely sensitive to 5% sucrose insolid medium. After plating of 107 cells of newly derivedexconjugants, filaments decomposed and cells bleachedwithin 12 h and few or no colonies subsequently arose. Asimilar phenotype was observed when the sacB gene waspresent as a single copy in the chromosome ofAnabaena sp.strain PCC 7120 (see below, strains PCC 7120::pRL256 andPCC 7120::pRL263). Thus, despite the complexity of theregulatory region of the sacB gene from B. subtilis (35), thatgene functions in Anabaena sp.

Site-directed inactivation of nifD gene in chromosome ofAnabaena sp. strain PCC 7120. Although plasmid pRL263(Fig. lc) cannot replicate in Anabaena sp. strain PCC 7120,the cyanobacterium can acquire resistance to streptomycinand spectinomycin by homologous recombination withcloned Anabaena sp. DNA that flanks the drug resistancecassette. Single recombinants would be predicted to showthe phenotype Smr/Spr Emr Sucs Nif+ and double recombi-nants to show the phenotype Smr/Spr Ems Sucr Nif. Aftermating (11), exconjugants arose at a frequency of ca. 1i-' ofcells plated. All 200 Smr/Spr exconjugants that we testedwere Nif+ and were therefore provisionally considered sin-gle recombinants and denoted PCC 7120::pRL263. Another200 colonies derived from a 2-month-old culture of a single-recombinant colony (see Fig. 2, lane B) were screened for

A B C O E F

10.89.0- a,

6.0 4111 a l

4.1-

FIG. 2. Southern analysis ofDNA from PS263 colonies. Markersindicate size of DNA in kilobases. Total DNA from wild-type PCC7120 (lane A), single recombinant PCC 7120::pRL263 (lane B),double recombinants PS263-1, -2, and -50 (lanes C, D, and F,respectively), and pseudo-double recombinant PS263-42 (lane E)was digested with ClaI and probed with linearized plasmid pRL393.Figure lc elucidates the origins of the bands observed. The 4.1-kbband in the wild type containing the nifH gene and part of the niff)gene is replaced by a 6.0-kb band in all other colonies, in which the1.9-kb cassette C.S4 is inserted within the nifD gene. Single recom-binant PCC 7120::pRL263 (lane B) and PS263-42 (lane E), a pseudo-double recombinant, have a 9.0-kb band generated from thejunctionto the vector, and lane B shows also a 10.8-kb band which resultedfrom duplication of the whole plasmid. In the double recombinants(lanes C, D, and F), the 6.0-kb band which contains the nijD genewith the C.S4 insert is present, but the 9.0-kb band to which thevector contributes has been lost.

Nif- phenotype arising as a result of double recombination;that phenotype was not observed.We selected for double recombinants by plating excon-

jugants on solid medium containing 5% sucrose. Positivelyselected sucrose-resistant colonies (denoted PS263) ap-peared at a frequency of ca. 10-5 in experiments employingdirect plating and ca. l0' when initial exconjugants weregrown for 1 month in liquid medium before cavitation andplating. Of 20 PS263 colonies resulting from direct plating,all but one, PS263-42, had an Smr/Spr Ems Sucr Nif-phenotype, suggestive of double recombination. Replace-ment of the wild-type nifD gene by the nifD: :C.S4 derivativeas a result of double recombination was confirmed bySouthern analysis (Fig. 2). PS263-42 had an Smr/Spr EmrSucr Nif+ phenotype and showed a pattern of hybridizationpredicted for a single recombinant (Fig. 2). Presumably, thesacB gene had been inactivated, rather than lost as inauthentic double recombinants. We refer to such strains aspseudo-double recombinants. Possible mechanisms for theiroccurrence are discussed below. Thirty PS263 coloniesisolated after extra cycles of growth in liquid and cavitationwere examined, and 28 proved to have the phenotype of adouble recombinant. Two, PS263-53 and PS263-54, resem-bled PS263-42 in phenotype.

PS263-1, a representive, authentic double recombinant,was indistinguishable from wild-type PCC 7120 in bothgrowth rate and morphology when grown in medium con-taining fixed nitrogen. When transferred to medium free offixed nitrogen, in which it cannot grow, PS263-1 developedheterocysts that were not distinguishable by light micros-copy from those of the wild-type strain. On the average,heterocysts were separated by fewer vegetative cells in the

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3142 CAI AND WOLK

A B C 0 E 1 2 3 4 5 6 7 8 9 10 '1 12

4.0-

3,4- Nola

-s

Oe.aa

2.4- :W _

2.1--6"' OM iiii4I

1.7-

FIG. 3. Southern analysis ofDNA from PS256 colonies. Markersindicate size of DNA in kilobases. Total DNA was digested withEcoRV and probed with linearized plasmid pRL351. The bands of2.4, 2.1, and 5.8 kb correspond to the three EcoRV segments in thehorizontal line of Fig. lb (the 5.8-kb segment has ca. 0.9 kb 5' fromhetA that is homologous to the probe; this A+T-rich region [Hollandand Wolk, unpublished data] often gives rise to only a faint bandafter autoradiography). The bands of 3.4 and 1.7 kb, which appearonly in single and pseudo-double recombinants, arise from thevector-insert junctions on the left and right sides of Fig. lb,respectively. Wild-type PCC 7120 (lane A) and mutant EF116 (laneB) have bands of equal size, indicating that there is no significantvariation in the hetA region of the chromosome of the mutant. Indouble-recombinant PS256-17 (lane E), the 2.1-kb band is replacedby a 4.0-kb band, reflecting the insertion of C.S4 into the hetA gene.Pseudo-double-recombinant PS256-5 (lane D) has the same patternof hybridization as single-recombinant PCC7120::pRL256 (lane C).

mutant than in the wild-type strain (data not shown), pre-sumably because the mutant is more deficient in nitrogen.

Site-directed inactivation of hetA gene in chromosome ofAnabaena sp. strain PCC 7120. The hetA gene is a differen-tially regulated gene that affects the biosynthesis of thepolysaccharide layer of the heterocyst envelope (23). A hetAmutant, EF116, generated by UV irradiation (36) can becomplemented by a 3.5-kb sequence of chromosomal DNAthat harbors the hetA gene. Complementation is abolishedwhen a drug cassette is inserted into the NruI site withinhetA (23, 36). Using sacB-mediated positive selection andthe suicide plasmid pRL256 (Fig. lb), the wild-type hetAgene in the chromosome of PCC 7120 was replaced throughdouble recombination by a mutant hetA gene in whichcassette C.S4 had been inserted into its NruI site. ResultinghetA mutants, represented by PS256-17, were Smr/Spr and,under aerobic conditions, Nif-. Pseudo-double recombi-nants, represented by PS256-5, were identified by theirphenotype (Smr/Spr Nif7) and by Southern analysis (Fig. 3).Single and double recombinants arose in these experimentswith frequencies slightly lower than those observed in theniJD inactivation experiments described above.

In medium free of fixed nitrogen, mutant PS256-17 devel-oped cells that had the shape and spacing of heterocysts butthat showed no deposition of heterocyst envelope polysac-charide. In contrast, heterocyst envelope polysaccharide isdeposited irregularly in mutant EF116 (36).

Isolation of IS elements from Anabaena sp. strain PCC7120. Anabaena sp. strain PCC 7120 bearing plasmid

a

9.5

4.3

3- -2.6

-1.2

- 0.5

FIG. 4. Southern analysis ofDNA from PS250 colonies. Markersindicate size of DNA in kilobases. Total DNA from coloniesPS250-1 to PS250-10 (lanes 1 to 10) and from PCC 7120(pRL250)(lane 12) and DNA of plasmid pRL250 (lane 11) were digested withPstI and probed with plasmid pRL250. The bands of 2.6, 1.2, 0.5,and 9.5 kb correspond, respectively, to the PstI segments bearingsacB, npt, the inverted repeats, and the rest of the plasmid (Fig. la).In sucrose-sensitive Anabaena sp. strain PCC 7120(pRL250), thehybridization pattern (lane 12) is the same as that of pRL250 (lane11), indicating that there is no obvious change of the plasmid withinthe cells of Anabaena sp. In the sucrose-resistant colonies (lanes 1to 10), the 2.6-kb band has been replaced by bands of larger sizes,suggesting that transposable elements have been inserted into it.Other bands are unchanged in size. Because the inserts in colonies4 to 8 differ in size, they may represent different insertion se-quences.

pRL250 cannot grow on sucrose-containing solid mediumbecause of the presence of the sacB gene in the plasmid (Fig.la). One such sucrose-sensitive colony was subculturedfor 2 months in AA/8 plus N03 plus neomycin liquidmedium, and then about 107 cells were plated on AA plusN03 plus neomycin plus sucrose solid medium. About 300colonies had grown on the plate after 10 days. Ten of thesecolonies (denoted PS250-N, where N = 1, 2 ... , 10),randomly selected, were analyzed. Each showed insertion ofa presumed transposable element within the sacB region ofpRL250 (Fig. 4), accounting for the viability of the colony inthe presence of sucrose. Insertion sequences of at least fivedifferent sizes appeared to be represented. A plasmid,pRL272, recovered by transformation of E. coli DH5a(Table 1) with total DNA from colony PS250-3 was furtherstudied. In this plasmid, a 1.7-kb sequence ofDNA had beeninserted into the open reading frame of sacB within the 57-bpregion between EcoRI and PvuII (35), presumably by trans-

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ISOLATION OF DOUBLE RECOMBINANTS FROM ANABAENA SPECIES

mys--1200bp

ovs

Im

lis

E e

1389111341hp m

a0

LU

am

IS8921700bp

SS Z a

===

Ew as E'MES

_ _

E EI I I I

a a

_

Do L

sGI

aK

m u~tla

aa

VI) = = .al1 1 . I

I I I

= __

= L Ecs

FIG. 5. Physical maps of elements mys (Alam and Curtis, 1st Int. Congr. Plant Mol. Biol.; Curtis, personal communication), IS892 fromAnabaena sp. strain PCC 7120, and IS891 (4) from Anabaena sp. strain M-131.

position. A restriction map of the 1.7-kb element, denotedIS892, is shown in Fig. 5.The 1.4-kb DraI-EcoRV internal fragment of IS892 was

used to probe total DNA of Anabaena sp. strain PCC 7120.The results (data not presented) showed that there are atleast five copies of the IS element in the genome. Althoughthe probe also hybridized to the genomic DNA ofAnabaenasp. strain M-131, it did not hybridize to IS891, an IS elementrecently isolated from this strain (4) (Fig. 5), indicating theabsence of significant homology between these insertionsequences from two Anabaena strains. The 1.4-kb internalfragment was also used to probe total DNA of E. coliHB101(pRL528) and DH5a, transient hosts of plasmidpRL250 during conjugation and transformation. Under ourstandard, high-stringency conditions, no hybridization wasobserved, thus excluding the possibility that this IS elementwas derived from cells of E. coli during conjugal transfer.

DISCUSSION

Application of the technique of Ried and Collmer (30)permits positive selection for double recombinants in thefilamentous cyanobacterium Anabaena sp. strain PCC 7120and presumably in any other cyanobacterial strains thatshow similar susceptibility to sucrose when bearing the sacBgene. Using this technique with Anabaena sp. strain PCC7120, we inserted an Smr/Spr cassette (C.S4) into the nifDgene in the chromosome to create a nifD mutant, PS263-1,and into the hetA gene to create a hetA mutant, PS256-17,that is defective in heterocyst formation and phenotypicallyNif- under aerobic conditions.

This technique has significant advantages relative toscreening. Double recombinants can be obtained within lessthan 1 month after conjugation. As in the experiment withplasmid pRL263, pseudo-double recombinants can be easilydistinguished by testing for the antibiotic resistance (Em')conferred by the vector. As little as 0.75 kb of homologous

DNA bordering the inactivation drug cassette is sufficient forisolating double recombinants. By contrast, it is very dif-ficult, if at all possible, to isolate double recombinants byscreening when there is such a small amount of bordering,homologous DNA. Because the size of homologous DNArequired is small, it is relatively easy to find a unique site atwhich to insert a cassette into the gene of interest. More-over, use of sacB should allow an unmarked mutation to beintroduced into the chromosome (30).One problem in the use of the sacB system is the produc-

tion of pseudo-double recombinants as a result of inactiva-tion of the sacB gene. Spontaneous mutations of sacB incells of Anabaena sp., including point mutations, deletions(our unpublished observations), and the insertion of ISelements, can certainly contribute to the appearance ofpseudo-double recombinants. Our results upon plating cellsofAnabaena sp. strain PCC 7120(pRL250) in the presence of5% sucrose suggest that spontaneous mutations of sacB inAnabaena cells accumulate with time of cell culture.

Nonetheless, we consider that inactivation of the sacBgene in E. coli cells before conjugation is probably the majorcause of the appearance of pseudo-double recombinants. Weobserved that an overnight liquid culture inoculated with a

single small sucrose-sensitive colony of E. coli DH5a(pUM24) gave rise to large and small sucrose-resistantcolonies at a total frequency of 10'-. Restriction analysis ofplasmid pUM24 from these sucrose-resistant colonies sug-

gested that in 1% of the total, and mostly in large colonies,the plasmid had experienced insertion of transposable ele-ments. IS2- and ISJO-like elements, tentatively identified bysize and by restriction analysis (13, 16, 20), were found in thesacB gene in this test. If sacB-bearing cells of E. coli aresubcultured repeatedly before conjugation, the percentage ofpseudo-double recombinants among sucrose-resistant colo-nies of Anabaena sp. can exceed 50%. However, if care istaken in choosing sucrose-sensitive colonies for inoculation

U-

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3144 CAI AND WOLK

of plasmid-donating cultures of E. coli and in their length ofculture, the percentage of pseudo-double recombinants isusually less than 5%. Similarly, although a longer period ofculturing single-recombinant colonies increases the absolutenumber of double recombinants, such subculture of singlerecombinants has also been observed to increase the per-centage of pseudo-double recombinants among sucrose-resistant colonies, presumably due to an accumulation ofspontaneous mutations of the sacB gene in the cells ofAnabaena sp.

Surprisingly, Anabaena sp. strain PCC 7120::pRL256 andPCC 7120: :pRL263, both of which have a functional copy ofsacB in the chromosome, grew well in AA/8 plus N03- plus5% sucrose liquid medium, so that culture in sucrose-containing liquid medium did not enrich double recombi-nants. We cannot account for this difference in resultsbetween solid and liquid media. Similarly, UV irradiation ofsingle recombinants in liquid (with the intent of increasingthe frequency of a second recombinational event beforeselection on sucrose plates) only increased the percentage ofpseudo-double recombinants, possibly by increasing themutation rate of the sacB gene in the cells of Anabaena sp.A family (denoted mys) of elements having a structure

characteristic of insertion sequences has been reported fromAnabaena sp. strain PCC 7120 (J. Alam and S. E. Curtis,Abstr. 1st Int. Congr. Plant Mol. Biol., abstr. OR-22-07,1985; S. Curtis, personal communication), but transpositionof the mys elements has not been observed. Their size is ca.500 bp less than the size of the IS element, IS892, that wemapped, and none has a set of restriction sites resemblingthat of IS892 (Fig. 5). We are further characterizing theelements that we have identified as being mobile in Ana-baena sp.

ACKNOWLEDGMENTS

We thank A. Collmer for the gift of plasmid pUM24, C. I. Kadofor plasmid pUCD800, M. Steinmetz for information about the sacBgene, S. E. Curtis for information about the mys family of IS-likeelements from Anabaena sp. strain PCC 7120, J. Elhai for helpfuldiscussions, D. Holland for information about hetA and for helpfuldiscussions, and Elaine Oren for technical assistance.

This work was supported by a fellowship from the Nitrogen-Availability Program at Michigan State University to Y.C. and bythe U.S. Department of Energy under contract DE-AC02-76ER0-1338.

LITERATURE CITED

1. Allen, M. B., and D. I. Arnon. 1955. Studies on nitrogen-fixingblue-green algae. I. Growth and nitrogen fixation by Anabaenacylindrica Lemm. Plant Physiol. 30:366-372.

2. Aymerich, S., G. Gonzy-Treboul, and M. Steinmetz. 1986. 5'-Noncoding region sacR is the target of all identified regulationaffecting the levansucrase gene in Bacillus subtilis. J. Bacteriol.166:993-998.

3. Bancroft, I., C. P. Wolk, and E. V. Oren. 1989. Physical andgenetic maps of the genome of the heterocyst-forming cyano-bacterium Anabaena sp. strain PCC 7120. J. Bacteriol. 171:5940-5948.

4. Bancroft, I., and C. P. Wolk. 1989. Characterization of aninsertion sequence (IS891) of novel structure from the cyano-bacterium Anabaena sp. strain M-131. J. Bacteriol. 171:5949-5954.

5. Beckwith, J., and T. J. Silhavy. 1983. Genetic analysis of proteinexport in Escherichia coli. Methods Enzymol. 97:3-11.

6. Braun, A. C., and H. N. Wood. 1962. On the activation ofcertain essential biosynthetic systems in cells of Vinca rosea L.

Proc. Natl. Acad. Sci. USA 48:1776-1782.7. Craig, I. W., C. K. Leach, and N. G. Carr. 1969. Studies with

deoxyribonucleic acid from blue-green algae. Arch. Mikrobiol.65:218-227.

8. Currier, T. C., J. F. Haury, and C. P. Wolk. 1977. Isolation andpreliminary characterization of auxotrophs of a filamentouscyanobacterium. J. Bacteriol. 129:1556-1562.

9. Davis, L. G., M. D. Dibner, and J. F. Battey. 1986. Basicmethods in molecular biology, p. 62-65. Elsevier Science Pub-lishing, Inc., New York.

10. Elhai, J., and C. P. Wolk. 1988. A versatile class of positive-selection vectors based on the nonviability of palindrome-containing plasmids that allows cloning into long polylinkers.Gene 68:119-138.

11. Elhai, J., and C. P. Wolk. 1988. Conjugal transfer of DNA tocyanobacteria. Methods Enzymol. 167:747-754.

12. Fiandt, M., W. Szybalski, and M. H. Malamy. 1972. Polarmutations in lac, gal and phage X consist of a few IS-DNAsequences inserted with either orientation. Mol. Gen. Genet.119:223-231.

13. Galas, D. J., and M. Chandler. 1989. Bacterial insertion se-quences, 109-162. In D. E. Berg and M. M. Howe (ed.), MobileDNA. American Society for Microbiology, Washington, D.C.

14. Gay, P., D. Le Coq, M. Steinmetz, T. Berkelman, and C. I.Kado. 1985. Positive selection procedure for entrapment ofinsertion sequence elements in gram-negative bacteria. J. Bac-teriol. 164:918-921.

15. Gay, P., D. Le Coq, M. Steinmetz, E. Ferrari, and J. A. Hoch.1983. Cloning structural gene sacB, which codes for exoenzymelevansucrase of Bacillus subtilis: expression of the gene inEscherichia coli. J. Bacteriol. 153:1424-1431.

16. Ghosal, D., H. Sommer, and H. Saedler. 1979. Nucleotidesequence of the transposable DNA-element IS2. Nucleic AcidsRes. 6:1111-1122.

17. Golden, J. W., S. J. Robinson, and R. Haselkorn. 1985. Rear-rangement of nitrogen fixation genes during heterocyst differen-tiation in the cyanobacterium Anabaena. Nature (London)314:419-423.

18. Golden, J. W., and D. R. Wiest. 1988. Genome rearrangementand nitrogen fixation in Anabaena blocked by inactivation ofxisA gene. Science 242:1421-1423.

19. Golden, S. S. 1988. Mutagenesis of cyanobacteria by classicaland gene-transfer-based methods. Methods Enzymol. 167:714-727.

20. HaDling, S. M., R. W. Simons, J. C. Way, R. B. Walsh, and N.Kleckner. 1982. DNA sequence organization of IS10-right ofTnlO and comparison with ISlO-left. Proc. Natl. Acad. Sci.USA 79:2608-2612.

21. Hanahan, D. 1985. Techniques for transformation of E. coli, p.109-135. In D. M. Glover, (ed.), DNA cloning, vol. 1. IRLPress, Oxford.

22. Herdman, M., M. Janvier, R. Rippka, and R. Y. Stanier. 1979.Genome size of cyanobacteria. J. Gen. Microbiol. 111:73-85.

23. Holland, D., and C. P. Wolk. 1990. Identification and charac-terization of hetA, a gene that acts early in the process ofmorphological differentiation of heterocysts. J. Bacteriol. 172:3131-3137.

24. Klier, A., A. Fouet, M. Debarbouile, F. Kunst, and G. Papoport.1987. Distinct control sites located upstream from the levansu-crase gene of Bacillus subtilis. Mol.' Microbiol. 1:233-241.

25. Mackinney, G. 1941. Absorption of light by chlorophyll solu-tions. J. Biol. Chem. 140:315-322.

26. Malamy, M. J. 1970. Some properties of insertion mutations inthe lac operon, p. 359-373. In J. R. Beckwith and D. Zipser(ed.), The lactose operon. Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y.

27. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecularcloning: a laboratory manual. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

28. Porter, R. D. 1986. Transformation in cyanobacteria. Crit. Rev.Microbiol. 13:111-132.

29. Rice, D., B. Mazur, and R. Haselkorn. 1982. Isolation andphysical mapping of nitrogen fixation genes from the cyanobac-

J. BACTERIOL.

on March 30, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

ISOLATION OF DOUBLE RECOMBINANTS FROM ANABAENA SPECIES

terium Anabaena 7120. J. Biol. Chem. 257:13157-13163.30. Ried, J. L., and A. Colimer. 1987. An nptI-sacB-sacR cartridge

for constructing directed, unmarked mutations in Gram-nega-tive bacteria by marker exchange-eviction mutagenesis. Gene57:239-246.

31. Schmetterer, G., and C. P. Wolk. 1988. Identification of theregion of cyanobacterial plasmid pDU1 necessary for replica-tion in Anabaena sp. strain M-131. Gene 62:101-109.

32. Seed, B. 1983. Purification of genomic sequences from bacterio-phage libraries by recombination and selection in vivo. NucleicAcids Res. 11:2427-2445.

33. Shen, P., and H. V. Huang. 1986. Homologous recombination inEscherichia coli: dependence on substrate length and homol-ogy. Genetics 112:441-457.

34. Stanier, R. Y., and G. Cohen-Bazire. 1977. Phototrophic prokar-

3145

yotes: the cyanobacteria. Annu. Rev. Microbiol. 31:225-274.35. Steimmetz, M., D. Le Coq, S. Aymerich, G. Gonzy-Treboul, and

P. Gay. 1985. The DNA sequence of the gene for the secretedBacillus subtilis enzyme levansucrase and its genetic controlsites. Mol. Gen. Genet. 200:220-228.

36. Wolk, C. P., Y. Cai, L. Cardemil, E. Flores, B. Hohn, M. Murry,G. Schmetterer, B. Schrautemeier, and R. Wilson. 1988. Isola-tion and complementation of mutants of Anabaena sp. strainPCC 7120 unable to grow aerobically on dinitrogen. J. Bacteriol.170:1239-1244.

37. Wolk, C. P., A. Vonshak, P. Kehoe, and J. Elhai. 1984. Con-struction of shuttle vectors capable of conjugative transfer fromEscherichia coli to nitrogen-fixing filamentous cyanobacteria.Proc. Natl. Acad. Sci. USA 81:1561-1565.

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