A host-vector system for gene cloning in the cyanobacterium Anacystis nidulans R2

PLASMID 10, 156-163 (1983)

A Host-Vector System for Gene Cloning in the Cyanobacterium Anacystis nidulans R2 C. J. KUHLEMEIER,’ A. A. M. THOMAS,’ A. VAN DER ENDE, R. W. VAN LEEN, W. E. BORRIAS, C. A. M. J. J. VAN DEN HONDEL,~ AND G. A. VAN ARKEL Department qf Molecular

Cell Biology. University of Utrecht. P.O. Bo.x 80.056, 3508 TB Utrecht. The Netherlands

Received December 12, 1982 We describethe construction of a seriesof vectorssuitable for genecloning in the cyanobacterium Anacystis nidulans R2. From the indigenous plasmid pUH24, derivatives were constructed with

streptomycin as the selective marker; one of these plasmids was used to construct pUC303. a shuttle vector capable of replication in A. nidulans R2 as well as in Escherichia coli K12. It has two markers, streptomycin and chloramphenicol resistance, and three unique restriction sites. Instability of recombinant plasmids was overcome by using a derivative of A. nidulans R2cured of the indigenous plasmid pUH24. This strain, R2-SPc, can be transformed stably and at high frequency by the plasmids described in this paper. The combination of the cured strain R2-SPc and the new plasmid pUC303 serves as a suitable host-vector system for gene cloning in cyanobacteria.

Severalgenesfrom cyanobacteria have been isolated with the aid of recombinant DNA techniques. Nitrogen fixation genes were selected from an Anabaena gene library by hybridization with heterologous DNA probes (Mazur et al., 1980; Fisher et al., 1981). Ribosomal DNA genesfrom Anacystis nidulans were found using the corresponding RNAs as probes (Tomioka et al., 198I). We recently described the cloning of a gene involved in methionine biosynthesis in A. nidulans R2 (Tandeau de Marsac et al., 1982). However, in these systems direct selection for a given gene in the homologous host is not possible. A system in which a gene could be introduced into a cyanobacterium and maintained on an autonomous replicon would be helpful not only for shotgun cloning but also for studying cloned genes in their natural environment. Our aim is to use a cloning strategy in which wild-type chromosomal DNA is spliced into

a suitable vector and transformed into cyanobacterial mutants. After selection for both the vector and the wild-type gene the recombinant plasmid DNA is then transferred to Escherichia co/i to allow further analysis of the particular gene. Our previous efforts have been devoted to the characterization of the highly transformable strain A. nidufans R2 and the construction of vectors (Van den Hondel et al., 1980; Kuhlemeier et al.. 1981). A drawback is the presenceof the small plasmid pUH24 in the host strain, which interferes with vector stability (Kuhlemeier et al.. 1981). Furthermore, all vectors which are currently in use rely on ampicillin resistance as the selective marker. This marker, however, is less suitable in our cloning strategyfor two reasons. First, isolation of stable mutants has up till now been performed by transposon mutagenesis using the ampicillin resistance encoding transposon Tn90 1,thus eliminating ampicillin resistance as a marker for transformation. ’ To whom reprint requests should be addressed. Second,ampicillin resistant transformants are z Present address: Laboratory of Microbiology, Free obtained at a rather low frequency (at least in University of Brussels,Laarbekelaan 103, 1090 Brussels, our hands) probably due to the nature of the Belgium. ampicillin selection (Kuhlemeier et al., 198I). ’ Presentaddress:Medical Biological Laboratory TNO. To overcome these difficulties we have isoP.O. Box 45, 2288 GJ Rijswijk, The Netherlands. 0147-619X/83 $3.00 Copyri%r 0 1983 by Academx Press. Inc. All rights of reproduction in any form resened.

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CYANOBACTERIAL

lated a host strain cured of the indigenous plasmid pUH24 and constructed vectors with a streptomycin resistance marker. MATERIALS

AND

METHODS

Strains and Media The cyanobacterial strains used in this study were A. nidufans R2 (PCC 7942) and its derivative R2-SPc, cured of the small plasmid pUH24. The latter strain was isolated by a modification of the method of Inuzuka et al. (1969). A R2 derivative in which the indigenous plasmid pUH24 was replaced by the ampicillin-resistant (AP~)~ plasmid pUC 1 (Van den Hondel et al., 1980) was diluted to lo3 cells/ml and grown in BG-11 medium in the presence of 0.003% SDS until a cell concentration of lo* was reached. A dilution was spread on BG-11 agar plates without ampicillin and colonies were replica-plated on plates with and without ampicillin. Of 122 colonies tested, 1 had lost ampicillin resistance. This strain, R2-SPc, has lost its small plasmid by the following criteria: (i) the ApR marker of pUCl was lost, (ii) no plasmid of 5.5 MDa was detected on agarose gels, and (iii) on a Southern blot of R2 and R2-SPc DNA digests probed with the indigenous small plasmid pUH24, no bands were detected in the R2SPcdigest, suggestingthat pUC 1 had not been integrated into the chromosome. Transformation Transformation of A. nidufans R2 and R2SPc was performed as described previously (Van den Hondel et al., 1980). For transformation to E. coli K12, strain 803 (lac gal met recA h.sdSsupE supF) was used. Recombinant DNA Procedures Recombinant DNA manipulations were performed according to standard procedures. Partial digestions with HpaI and BgfiI were 4 Abbreviations used: ApR, ampicillin resistance;SmR, streptomycin resistance;CmR, chloramphenicol resistance; MIC, minimal inhibitory concentration.

157

CLONING VECTORS

done with excessenzyme in the presence of 22 and 80 pg/ml ethidium bromide, respectively (Parker et al., 1977). Electron microscopy of heteroduplexes was according to Van der Ende et al. (1982). Containment Experiments involving recombinant DNA were performed under CI conditions according to the guidelines of the Dutch Committee on Recombinant DNA. RESULTS

Construction of Vectors with Streptomycin Resistance as the Selective Marker The ampicillin resistance-encoding cyanobacterial vector pUC1 (Van den Hondel et al., 1980)wasconverted into a seriesof vectors with streptomycin resistance as the selective marker (Fig. 1). Insertion of a fragment containing the streptomycin resistance(SmR)gene into pUCl gave rise to pUC12; deletion of the ApR gene from pUC 12 resulted in pUC 13 and pUC14. Plasmids pUC13 and pUC14 have unique restriction sites for Bg/II and XhoI, which are available for gene cloning. To enable pUC 13, the smallest SmRvector, to replicate in E. cofi, it was coupled to the E. coli vector pACYC 184 (Chang and Cohen, 1978). BgfiI-digested pUC13 was ligated to BarnHI-digested pACYC 184 and transformed to E. co/i. Sixteen streptomycin (SmR) and chloramphenicol (CmR) resistant transformants were found and were analyzed further. One orientation would produce Hind111fragments of 6.4 and 0.8 MDa, the other orientation would give Hind111 fragments of 4.2 and 3.0 MDa. One plasmid, pUC301, gave fragments of 5.6 and 0.8 MDa, indicating the first orientation with a deletion in the large fragment. Further analysis showed that a deletion of approximately 0.65 MDa is located between the EcoRI and SalI sites in the pACYC184 part, covering the two BglI sites in that region (Fig. 2). The sizeand the location of this deletion were confirmed by hetero-

158

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ET AL.

HmdIU

A”.31

FIG. I. Construction of plasmids with SmRas the selective marker. The HpaI-PvuII fragment of pRI477S was inserted into partially HP&digested pUCI, resulting in pUC 12. Around the site of insertion of pUCl at least six additional HpaI fragments (90, 70, 55, and 36 bp plus two smaller ones) are present. In pUCl2 the fragments of 90, 70, and 36 bp appeared to be missing, whereas at least the 36-bp fragment was still present (data not shown). The existence of a deletion in pUCl2 compared to pUCl was confirmed by heteroduplex analysis (see Fig. 3). The ApR gene was removed from pUCI2 in two ways, giving rise to pUCl3 and pUCl4. BglII/BamHI denotes a hybrid site recognized by neither enzyme.

15.9/14.5-

6.34.5x5/&2.3-

=g:;;

1.51.3vi,=

-0.58 -0.40

l.l0.88-0.21 -0.19/c

I.18

-0.16 -0.13

,l

23,,456,,789 EcoRI

/-/indIJ.I

10

11,

Bgl I

SGI FIG. 2. Restriction enzyme analysis of pUC30 I and pUC303. Plasmids were digested with EcoRI + SafI, HindIII, and Bg/l and run on a I% agarose gel (lanes l-9) or on a 3-10s polyacrylamide gel (lanes IO and I I). Lanes I, 4, 7: pACYC184, lanes 2, 5, 8, IO: pUC301; lanes 3, 6, 9, I I: pUC303. Molecular weight markers were a lambda ~1857Hind111digest combined with a Hind111+ EcoRI double digest for the left hand panel and a @XI74 Hue111digest for the right-hand panel. In I5 casesthe pattern of pUC303 was found. In I casethe pattern of pUC30 I was found, which can only be in agreement with the other orientation if a deletion of 0.65 MDa between EcoRI and &/I covering the two Bgfi sites in that region is assumed.

CYANGBACTERIAL

duplex analysis (Fig. 3). The other 15 plasmids all had the opposite orientation and apparently contained a full length copy of both constit-

CLONING VECTORS

159

uents (Fig. 2). One of them, pUC303, was used for further experiments, A restriction map of pUC303 is shown in Fig. 4.

FIG. 3. Analysis of heteroduplexes between pUCIO5 and pUC301. Restriction enzyme analysis indicated that a deletion of Hpal fragments had occurred during the construction of pUC 12 (seeFig. 1). To confirm this observation pUClO5 and pUC301 were linearized with EcoRI and prepared for heteroduplex analysis under the electron microscope. Plasmid pUClO5 is a derivative of pUC1 having the same HpaI sites (Kuhlemeier et al., 1981),pUC301 has retained the same HpaI sites as pUCl2. In addition, the 0.65MDa deletion in the pACYCl84 part of pUC301 is visualized. Twenty heteroduplexes were measured and the following length values (in megadaltons) were found for the single strand regions: (a) 0.70 f 0.06, (b) 1.10 f 0.15, (c) 0.15 f 0.04, (d) 1.80 f 0.25; and for the double stranded regions: O-a) 0.56 f 0.05, a-b) 0.43 f 0.05, b-d) 3.23 + 0.26, d-0) 1.10 + 0.12. 6X174 DNA was used as a standard. The bar indicates 0.2 pm. The data are interpreted as follows. Loop a representsthe deletion in pUC301 which was formed after transformation to E. co/i. Loop b represents the Smx fragment of pUC301, and loop c the small HpuI fragments deleted from pUC1 during the insertion of the SmR fragment. Loop d represents the sequence containing the Apa gene which is present in pUClO5 and not in pUC301.

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KUHLEMEIER

ET AL.

pucxn

BglI

725 Mdal WI HindP :I

5.0

FIG. 4. Restriction enzyme map of pUC303. The part derived from pACYC184 has been drawn in bold line, the pUC 13 part is shown with the thin line. The orientation of the CmR and SmR genes is according to Chang and Cohen (1978) and Barth and Grinter (1974). respectively.

Transformation to A. nidulans R2 and R2-SPc The plasmid pUC 12 could be transformed to R2 aswell asto R2-SPc,the cured derivative of R2. In R2 the plasmid tended to lose its nonselected marker, presumably as a result of interaction with the resident plasmid (Kuhlemeier et al., 1981); in R2-SPc pUC 12 was stable (Tables 1 and 2). Both pUC13 and pUC14 transformed R2 as well as R2-SPc at high frequency (Table 1); plasmids of the expected molecular weights were recovered in all casesindicating that they are stable in both hosts. Interestingly, pUC 13 has a minimal inhibitory concentration (MIC) for streptomycin of 40 pg/ml versus 80 pg/ml for pUC14 (for

comparison: R2 < 1 pg/ml), although they contain the same SmR gene in the same site and orientation. Both pUC30 1 and pUC303 transformed E. coli to SmR and CmR at a frequency comparable to that of pRI477S and pACYC184 (data not shown). Plasmid pUC301 transformed R2 to SmRat a low frequency and all transformants were found to be CmS; after chloramphenicol selection no transformants were found. However, pUC301 could not be transformed into R2-SPc.In contrast, pUC303 transformed R2 as well as R2-SPc at high frequency. After transformation in R2 the transformants tended to lose the nonselected marker; in R2-SPc, pUC303 did not lose its nonselected marker and a plasmid of the ap-

161

CYANOBACT ERIAL CLONING VECTORS

DISCUSSION

TABLE I TRANSFORMATION OFA. nidulans R2-SPc Transformation frequency Sm

Plasmid


no DNA puc12 puc13 puc14 puc30 I puc303 puc301 + puc303

Cm 10’” lo6 IO6 IO6 IO’ IO6 IO5

<3 x IO2

<3 x IO2 9x IO5 9 x IO5

Note. Cultures were grown to 5. IO’ cells/ml and concentrated to l-2 X IO9cells/ml. Plasmid DNA was added at a final concentration of approximately 35 rig/ml. Streptomycin (Sm) and chloramphenicol (Cm) (0.5 ml) were added underneath the agar at concentrations of 0.5 and 0.75 mg/ml, respectively. Transformation frequenciesare expressedas colonies per microgram of DNA. a With Sm as selection some colonies may arise on control plates, these do not grow when streaked on fresh plates containing Sm.

propriate molecular weight was always recovered (Tables 1 and 2). Plasmid pUC303 has a MIC for Sm of at least 100 pg/ml, which is considerably higher than that of pUC13.

Plasmid pUC303 is a hybrid of the cyanobacterial plasmid pUH24 and the E. coli vector pACYC184. It is an efficient shuttle vector carrying streptomycin and chloramphenicol resistance markers. It has unique restriction sites for EcoRI, SalI, and XhoI. The EcoRI site is located within the CmR gene, allowing screening of recombinants by insertional inactivation (Fig. 4). Plasmid pUC303 and the plasmids from which it originated are stable in A. nidulans R2-SPc, a derivative of R2 cured of the indigenous plasmid pUH24 (Table 2). On the other hand, pUC 12 and pUC303 tend to lose their nonselectedmarkers after transformation to R2. The same phenomenon has previously beendescribedfor the shuttle plasmid pUC 105 (Kuhlemeier et al., 1981). It was then argued that recombination with the resident plasmid pUH24 occurs whenever the incoming plasmid harbors two regions homologous with pUH24 separatedby two nonhomologous regions. This situation is also found with pUC 12 and pUC303. In R2-SPc, no sequences homologous with the recombinant vectors exist and hence segregation cannot occur. Therefore, R2-SPc is a preferred host in cloning experiments.

TABLE 2 STABILITYOFTHE NONSELECTED MARKER OFpUC I2 AND pUC303 AFTERTRANSFORMATION TO A. nidulans R2 AND R2-SPc

Selection

Number of transformants tested

Percentage resistant against nonselected antibiotic

R2

Sm AP

100 100

30 21

R2-SPc

Sm AP

99 100

100 loo

R2

Sm Cm

191 80

40 74

R2-SPc

Sm Cm

183 100

100 100

Plasmid

Strain

pUCI2

puc303

Note. Transformant colonies were streaked onto BG-I 1 plates containing ampicillin (Ap) (0.5 &ml), streptomycin (Sm) (10 &ml), or chloramphenicol (Cm) (5 &ml).

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KUHLEMEIER

Hybrids between pUC13 and pACYC184 can be formed in two orientations and these orientations are expected to occur in equal numbers. After transformation of the pUC 13pACYC 184 ligation mixture to E. cob, however, one orientation, the pUC303 type, was found predominantly, while only a single transformant with the other orientation was isolated. In the latter case a deletion had occurred downstream of the CmR gene which is located exclusively in the pACYC 184 part of the molecule. It could be that, in this orientation, transcription of the SmR gene is only possible from the CmR promoter, as the SmR gene probably is not preceded by a promoter sequence(Van Embden, 1978; Rubens ef al., 1976). In that casea terminator-like sequence had to be deleted to allow expression of the SmR gene in E. co/i. The deleted area in pUC301 is known to be involved in the formation of an inverted repeat structure (Stuber and Bujard, 1981). Moreover. it should be noted that the deleted area comprises a sequence which was accidentally duplicated during the construction of the vector pBR325 (Bolivar, 1978; Prentki et al., 1981). In contrast to pUC303, pUC30 I transforms R2 poorly and R2-SPc not at all. Colonies arising by transformation with pUC301 are most likely the result of recombination with the resident plasmid pUH24. In preliminary experiments we mapped the cyanobacterial replication functions between coordinates 5.7 and 3.4 (see Fig. 4). It could be that transcription from the tetracycline promoter interferes with expression of the replication functions. Using pUC13, a gene involved in nitrate assimilation has been isolated by complemet-nation of the corresponding mutant (to be published). With pUC303 asa vector, shotgun cloning in A. nidulans has the additional advantage that, after selection of the desired clone, rapid transfer to E. co/i is possible, which greatly facilitates the structural analysis of the particular gene.Screening different mutants with chromosomal DNA inserted in pUC303 offers a powerful extension of the strategies currently available for the cloning and analysis of cyanobacterial genes.

ET AL. ACKNOWLEDGMENTS We are indebted to Ms. J. H. Huizingh for technical assistance.Plasmid pRI477S was a gift of J. D. A. van Embden. This work was supported in part by the Netherlands Foundation for Chemical Researchwith financial aid from the Netherlands Organization for the Advancement of Pure Research. Nofe added in prooJ Two additional single cleavage

sitesweremapped in pUC303: an XbaI site at 0.9 MDa and a SstI site at I.55 MDa. REFERENCES BARTH, P. T., AND GRINTER,N. J. (1974). Comparison of the deoxyribonucleic acid molecular weights and homologies of plasmids conferring linked resistance to streptomycin and sulphonamides.J. Eacferiol. 120,6 I8630. BOLIVAR, F. (1978). Construction and characterization of new cloning vehicles. III. Derivatives of pBR322 carrying unique EcoRI sites for selection of EcoRI generated recombinant molecules. Gene 4, I2 I- 136. CHANG,A. C. Y., ANDCOHEN,S. N. (1978). Construction and characterization of amplifiable multicopy DNA cloning vehiclesderived from the Pl5A cryptic plasmid. J. Eucferiol. 134, 1141-1156. FISHER,R., TULI, R., AND HASELKORN,R. ( 198I). A cloned cyanobacterial gene for glutamine synthetase functions in Escherichiu co/i, but the enzyme is not adenylated. Proc. Narl. Acad. Sci. CLSA78, 3393-3397. INUZUKA, N., NAKAMURA, S.. INUZUKA, M., AND TOMOEDA,M. ( 1969).Specificaction of sodium dodecyl sulphate on the sex factor of E. coli K-12 and Hfr strains. J. Baderio/. 101, 827-839. KUHLEMEIER,C. J., BORRIAS,W. E.. VAN DENHONDEL, C. A. M. J. J., AND VAN ARKEL, G. A. ( 198I ). Vectors for cloning in cyanobacteria: construction and characterization of two recombinant plasmids capable of transformation to Escherichia coli K12 and Anacyssris nidulans R2. Mol. Gen. Gene!. 184, 249-254. MAZUR, B. J., RICE. D., AND HASELKORN,R. (1980). Identification of blue-green algal nitrogen fixation genes by using heterologousDNA hybridization probes. Proc. Natl. Acad. Sci. US.4 77, 186-190. PARKER, R. C.. WATSON, R. M., AND VINOGRAD, J. (1977). Mapping of closed circular DNAs by cleavage with restriction endonucleasesand calibration by agarose gel electrophoresis.Proc. Natl. ‘-icad. Sci. USA 74, 85 I855. PRENTKI,P.. KARCH,F.. IIDA, S., AND MEYER,J. ( 198I ). The plasmid cloning vector pBR325 contains a 482 base-pair long inverted duplication. Gene 14,289-299. RUBENS,C., HEFFRON,F., AND FALKOW, S. (1976). Transposition of a plasmid deoxyribonucleic acid sequence that mediates ampicillin resistance: independence from host recA functions and orientation of insertion. J. Bacterial. 128, 425-434. STUBER,D., AND BUJARD, H. (1981). Organization of transcriptional signals in plasmids pBR322 and

CYANOBACTERIAL pACYC184. Proc. Natl. Acad. Sci. USA 78, 167-171. TANDEAUDEMARSAC,N., BORRLU,W. E., KUHLEMEIER, C. J., CASTETS,A. M., VAN ARKEL, G. A., AND VAN DEN HONDEL,C. A. M. J. J. (1982). A new approach for molecular cloning in cyanobacteria: cloning of an Anacystis nidulans met gene using a Tn901-induced mutant. Gene 20, I1 1-I 19. TOMIOKA,N., SHINOZAKI,K., AND SUGIURA,M. (198 I). Molecular cloning and characterization of ribosomal RNA genesfrom a blue-green alga, .4nacystis nidulans. Mol. Gen. Genet. 184, 359-363.

VAN DENHONDEL,C. A. M. J. J., KEEGSTRA,W., BORRIAS,W. E., AND VAN ARKEL, G. A. ( 1979).Homology of plasmids in strains of unicellular cyanobacteria. Plasmid 2, 323-333.

VAN DEN HONDEL, C. A. M. J. J., VERBEEK,S., VAN

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DERENDE,A., WEISBEEK,P. J., BORRIAS,W. E., AND VAN ARKEL, G. A. (1980). Introduction of transposon Tn90 1 into a plasmid of Anacystis nidulans: preparation for cloning in cyanobacteria. Proc. Natl. .4cad. Sci. USA 77, 1570-1574. VAN EMBDEN,J. D. A. (1978). Translocation of an ampicillin resistant determinant within an R-factor aggregate in Salmonella Panama. Antonie van Leeuwenhoek J. Microbial.

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VAN DER ENDE, A., TEERTSTRA,R., AND WEISBEEK, P. J. (1982). Initiation and termination of the bacteriophage $X I74 rolling circle DNA replication in vivo: packaging of plasmid single-stranded DNA into bacteriophage $X I74 coats. Nucleic Acids Rex 10, 68496863.