Plasmid distribution among unicellular and filamentous cyanobacteria: occurrence of large and mega-plasmids

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1 FEMS Microbiology Letters 37 (1986) Published by Elsevier FEM02603 Plasmid distribution among unicellular and filamentous cyanobacteria: occurrence of large and mega-plasmids (Cyanobacterium; plasmid; distribution) Marie-Christine Rebirre **, Anne-Marie-Castets, Jean Houmard, and Nicole Tandeau de Marsac * Institut Pasteur, Unitd de Physiologie Microbienne, Ddpartement de Biochimie et Gdn~tique Moldculaire, 28 rue du Docteur Roux, Paris Cedex 15, France Received 15 August 1986 Accepted 18 August SUMMARY The plasmid content of 7 unicellular and 4 filamentous cyanobacterial strains has been analyzed. All strains were found to carry small plasmids while some strains harbor large plasmids up to approx. 400 kb. In addition, at least one megaplasmid of about 1000 kb was detected in the unicellular strain Synechococcus PCC7942. In the filamentous strain Calothrix PCC7601, 2 different distribution patterns of plasmids were observed in different subcultures, suggesting the presence of mobile DNA elements in this strain. 2. INTRODUCTION Cyanobacteria constitute the only group of oxygen-evolving photosynthetic prokaryotes [1]. The presence of small plasmids in cyanobacteria is well documented, especially in those strains which have been considered suitable for genetic manipulations [2]. In some instances, such plasmids have been used to construct shuttle cloning vectors [2]. * To whom correspondence should be addressed. So far, however, no coding functions have been attributed to any of these plasmids. A systematic search for the presence of large plasmids or megaplasmids in cyanobacteria has not been performed, except in the collection of 30 Ananaeba- Nostoc strains isolated from tropical soils [3]. Since large plasmids have been found to carry information for important functions in, some bacterial strains [4-7], we have started an investigation of the plasmid content of some of the strains of the Pasteur Culture Collection. In the present paper, we report the size distribution of plasmids found in 7 unicellular and 4 filamentous cyanobacterial species. Although this study is primarily aimed at the identification of large plasmids, we have also included the characterization of small plasmids, particularly for previously uncharacterized strains or when our results differed from those previously reported. 3. MATERIALS AND METHODS 3.1. Strains and growth conditions The strain Rhizobium mefiloti GMIll was obtained from C. Rosenberg (CNRS-INRA, /86/$ Federation of European Microbiological Societies

2 270 Castanet-Tolosan, France). This strain, derived from R. meliloti L5-30, harbors the following plasmids: prme L5-30 (136 kb); prme41 from R. meliloti Rm41 (225 kb); pgmi4142, a RP-4 derivative carrying a 47-kb insert from prme41 (107 kb); and 2 megaplasmids ( kb) ([8,9] and C. Rosenberg, personal communication). Cultures were grown in 6 ml of LB medium [10] at 30 C. All cyanobacterial strains were from the Pasteur Culture Collection (PCC). The strains Synechococcus PCC7335 and 7336 were grown in ASN-III medium and Synechococcus PCC7002 (=Agmenellum quadruplicatum PR-6) in ASN-III medium supplemented with vitamin B12 (10/~g/1) [11]. The strains Synechococcus PCC7942 (=Anacystis nidulans R-2), Synechocystis PCC6701, 6803 and 6808, Pseudanabaena PCC6802 and 7409 (previously classified in the L.P.P. group) [11], Calothrix PCC7601 (= Fremyella diplosiphon UTEX481) and Nostoc PCC8009 (= Nostoc MAC) were grown in BG-11 medium [11]. Stock cultures were grown in 50 ml of medium at C without stirring under a cool white fluorescent illumination of 10 /~E. m -2. s -1. When required, 3-1 cultures were grown in the appropriate medium with 0.2 g/1 of Na2CO 3 and continuous stirring and bubbling with 1% CO2/99% air, under an illumination of 50/~E. m -2. S Plasmid isolation and gel electrophoresis Method 1: Large plasmids were isolated according to Rosenberg et al. [12] from ml of cultures grown to stationary phase ( Klett units at 660 nm). For the unicellular strains, penicillin G (1 U/Klett unit at 660 nm/ml of culture) was added about h before cell harvesting in order to improve cell lysis. For the filamentous strains Pseudanabaena PCC7409 and Calothrix PCC7601, the step in which the cell pellets were washed in the presence of sarkosyl was omitted, due to easy cell lysis. In parallel, plasmids were extracted from 0.5 ml of an exponentially growing culture of R. meliloti GMIll whose plasmids were used as size markers. Electrophoreses of the DNA samples were performed on a vertical 0.7% agarose gel in Tris-acetate buffer, as described by Rosenberg et al. [12]. Method 2: The small-scale isolation procedure of Kuhlemeier et al. [13] was used on 50 ml early stationary growth phase cultures (approx. 200 Klett units at 660 nm). Prior to electrophoresis, samples were incubated with RNase (0.5 /~g/ml) for 30 min at 37 C. Electrophoresis was performed on a horizontal 0.7% agarose slab gel in Tris-acetate buffer, as described by van den Hondel et al. [14]. The plasmids from Synechococcus PCC7002 were used as size markers [15]. Method 3: Large scale plasmid isolation was performed on 3 1 of a culture collected in early stationary growth phase (2 g of cells, wet wt.) using the procedure of van den Hondel et al. [14] modified as follows. For the filamentous strains, Pseudanabaena PCC7409 and Calothrix PCC7601, which lyse easily and spontaneously during repeated centrifugations, the first washing step of the cell pellet was omitted. In general, the composition of the lysis buffer was identical to the one used in method 2 and the incubation in this buffer was performed for 2 h at 37 C. After polyethylene glycol precipitation, the pellet was resuspended in 10 mm Tris-HC1/0.1 mm EDTA ph 7.5, to give a final volume of 6.5 ml. Then, 6.9 g CsC1 and 0.7 ml ethidium bromide (5 mg/ml) were added to the concentrated clear lysate. This mixture was clarified by a precentrifugation at x g for 10 min at 4 C. Aggregates on top of the tube were removed and the supernatant was centrifuged at g for 48 h at 15 C. The DNA collected was treated with NaCl-saturated isopropanol to remove the ethidium bromide, extensively dialyzed against 10 mm Tris-HC1/0.1 mm EDTA, ph 7.5, and ethanol-precipitated. To determine whether the bands visible on the agarose gel were cccdna, the plasmid preparations were heat treated in the presence of 0.1% (w/v) sarkosyl for 2 min at 100 C as described previously [14]. Electrophoresis was performed on either 0.35% or 0.7% agarose gels as in Method 2. Throughout this paper, the following abbreviations will be used: ccc, covalently closed circular DNA; oc, open-circular DNA and ss, singlestranded DNA.

3 RESULTS AND DISCUSSION The main physiological characteristics of the 11 strains that we have analyzed for their plasmid content are reported in Table 1. Using the 'in gel' lysis technique of Eckhardt as modified by Rosenberg et al. [12], we have detected the presence of large plasmids in each of the strains tested (Fig. 1). The approximate sizes of the different plasmid species were estimated by comparison of their relative migration to that of the plasmids extracted from R. meliloti GMIll using the same procedure. In Pseudanabaena PCC7409, Calothrix PCC7601 and Nostoc PCC8009, large plasmids up to about 400 kb were present. However, only one strain, Synechococcus PCC7942, harbors a megaplasmid of approx kb. The largest pasmids ( kb) found in cyanobacteria have been reported by Franche and Reynaud [3] for 2 tropical Nostoc strains, but no megaplasmids were detected. Since, even in the case of R. rneliloti GMIll, the bacterial strain for which this technique of plasmid extraction has been developed, it is difficult to reproducibly extract the megaplasraids, some of the cyanobacterial strains tested may well contain megaplasmids that were not detected under the conditions of extraction used. When plasmids were extracted from Synechocystis PCC6701 and Calothrix PCC7601, more than 10 bands were visible on the gels (Fig. 1). According to Casse et al. [16], only the ccc forms of large plasmids can be resolved on the agarose gel, while the oc forms do not enter the gel and the linear DNAs migrate with chromosomal DNA. Therefore, the slow-migrating bands observed in the gel probably correspond to ccc DNAs. For small plasmids ( < 30 kb), the oc forms can enter the gel and it cannot be excluded that some of the bands observed for Synechocystis PCC6701 correspond to such forms of DNA molecules. Surprisingly, plasmids smaller than 30 kb were only rarely visible in the other strains tested by the Eckhardt procedure. To have a more complete pattern of the plasmid content of the cyanobacterial strains chosen for this study, we also extracted plasmids by Methods 2 and/or 3. These procedures permitted extraction of plasmids up to approx. 100 kb. Our results, together with the previously reported sizes of plasmids for some of the strains, are presented in Table 1. With the exception of Calothrix PCC7601 (see below), only few discrepancies were observed between our results and those previously reported. These discrepancies could be due to spontaneous plasmid loss, as previously observed for small plasmids ([17,18,34]. The use of Method 3 allowed a precise determination of the number of ccc plasmid forms after heat treatment of the DNA samples. Following this treatment, oc and linear forms are converted into ss forms with different electrophoretic mobilities, while ccc forms are not altered and thus DNAs migrate at the same positions whether treated or not [14]. For example, Fig. 2 shows the migration of the different plasmid species extracted from Synechococcus PCC7002 before and after heat treatment. In this figure, it is clear that Synechococcus PCC7002 cells contain 5 ccc plasmid species of differing molecular sizes, 4.5, 15.4, 30, 36.9 and 112 kb, the 9.7-kb species mentioned by Roberts and Koths [15] being the oc form of the 4.5-kb plasmid. We succeeded in identifying plasmids in Calothrix PCC7601 only by using Method 3. All our attempts to use Method 2 and several modifications of that method failed (e.g., time of incubation with lysozyme or with detergent, as well as cell harvesting at different growth states or varying concentration of the cell suspension). Unexpectedly, when DNA was isolated from 8 subcultures grown under the same conditions, 2 different plasmid patterns were obtained. Pattern A (7 subcultures) and B (1 subculture), shown in Fig. 3, reveal the presence of 4 plasmids differing in molecular size. These different plasmid patterns could not be related to any phenotypic differences such as cell morphology, cell pigmentation, generation time, heterotrophy or molecular nitrogen fixation. However, it is interesting to note that none of these plasmid patterns correspond to those found by either Simon [19] or Bogorad et al. [20]. The collective results obtained in different laboratories, together with the unusually high frequency of spontaneous mutations in cell pigmentation [21], suggest that mobile DNA elements are present in this particular strain. Although both small and large plasmids have

4 Table 1 Plasmid content of unicellular and filamentous cyanobacteria Cyanobacterial strain Main physiological characteristics Plasmid content Designation PCCnumber Morphology a Chromatic b Heterotrophic adaptation Synechococcus 7002 U - Glycerol U III Fructose U III Fructose U Number Sizes in kb c Previously reported Reference 4.5, 15.4, 30, 36.9*, 112" 32*, , 32, , 50.8*, , 9.7, 15.4, 30, 36.9, 112 [15] 5.2, 9.3, 15.8, 38.8, /> 74.6 [17] 7.9, 49.3 [24] 8.0, 48.7 [25-27] Synechocystis 6701 U II > U - Glucose U II ", 6.4", 8.4*, 14.9", 18.1", 22.7* + others , 5.2, 50", 100" 3.8, 22, 26, 50, [17] 5, 7.5, 13.5, bands [28] 227, 5.2 [29] 2.1, 45, 90 [30] Pseudanabaena 6802 _+ U III F III 6 2.5, 37, 40, , 4.2, 6.6, 10, 130, 180 Calothrix d 7601 F II Glucose > 10 >4 9.1, 1.5, 19.1 *, 90* + others ~< 200 (A) 11.9, 15, 16.7, 19.7 (B) 35.8, 47.8, 60, ,15.9, others [19] [20] Nostoc 8009 F III Glucose 6 7.9, 30, 38, 40*, 225, approx ,30,60 [31,32] U, unicellular; F, filamentous. b Groups of chromatic adaptation to which strains have been assigned according to [33]. c Plasmid sizes are indicated in normal characters when extractions were performed using Method 2, in italics when using Method 3, and in bold type when using Method 1. Asteriks indicate plasmids also visible when Methods 2 or 3 were used. d Formerly called Fremyella diplosiphon, UTEX481 [18,20]. A and B refer to the 2 distinct patterns as discussed in the text. Method 1 has not been tested for the B-type subculture.

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6 274 Fig. 2. Plasmids from Synechococcus PCC7002 in 0.7% agarose gel. Lane 1, no heat treatment; lane 2, heat treatment in the presence of 0.1% (w/v) sarkosyl; C, cccdna; O, oc DNA; S, ss DNA. Sizes of plasmids in kb. Fig. 3. Plasmids from Calothrix PCC7601 in 0.35% agarose gel. Lanes A and B, plasmid patterns obtained with subcultures of type A and type B, respectively. Arrows indicate ccc, and asterisks denote oc forms of the plasmids. Sizes of plasmids in kb. ACKNOWLEDGEMENTS been found in all the cyanobacterial strains tested, no coding function has been demonstrated for any plasmid. No hybridization was obtained when the plasmids from Calothrix PCC7601 were probed with DNA fragments carrying the structural nif genes from Anabaena PCC7120 [22] or the gene encoding the gas vesicle protein of Calothrix [23]. However, a better knowledge of the plasmid content of cyanobacterial strains and mutants for which physiological properties are well characterized, as well as localization in the genome of cloned genes, should help to elucidate the genetic roles of plasmids. In this respect, it is desirable to conduct a systematic survey with available DNA probes. We wish to thank Drs. G. Cohen-Bazire, I. Saint-Girons and S. Das Sarma for critical comments on this manuscript. We are grateful to Dr. C. Franche for her technical advice during the analysis of large plasmids and to Dr. C. Rosenberg for providing the Rhizobium strain. This work was supported by an ATP-CNRS grant (955353, 1984) and by funds allocated to the UA-CNRS REFERENCES [1] Startler, R.Y. and Cohen-Bazire, G. (1977) Annu. Rev. Microbiol. 31,

7 275 [2] Tandeau de Marsac, N. and Houmard, J. (1986) in Cyanobacteria: Current Research, Elsevier/North- Holland, Amsterdam, in press. [3] Franche, C. and Reynaud, P.A. (1986) Ann. Inst. Pasteur/Microbiol. 137A, [4] Banfalvi, Z., Sakanyan, V., Koncz, C., Kiss, A., Dusha, I. and Kondorosi, A. (1981) Mol. Gen. Genet. 184, [5] D6nari6, J., Rosenberg, C., Boistard, P., Truchet, G., Casse-Delbart, F. (1981) in Current Perspectives in Nitrogen Fixation (Gibson, A.H. and Newton, W.E., Eds.) pp Elsevier/North-Holland, Amsterdam. [6] Kondorosi, A., Banfalvi, Z., Sakanyan, V., Koncz, C., Dusha, I. and Kiss, A. (1981) in Current Perspectives in Nitrogen Fixation (Gibson, A.H. and Newton, W.E., Eds.), p Elsevier/North-Holland, Amsterdam. [7] Rosenberg, C., Boistard, P., D6nari6, J. and Casse-Delbart, F. (1981) Mol. Gen. Genet. 184, [8] Huguet, T., Rosenberg, C., Casse-Delbart, F., De Lajudie, P., Jouanin, L., Batut, J., Boistard, P., Julliot, J.-S. and Drnarir, J. (1983) in Molecular Genetics of the Bacteria- Plant Interaction (Pithier, A., Ed.) pp Springer- Verlag, Berlin and Heidelberg. [9] Burkardt, B. and Burkardt, H.J. (1984) J. Mol. Biol. 175, [10] Miller, J.H. (1972) in Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [11] Rippka, R., Deruelles, J., Waterbury, J.B., Herdman, M. and Stanier, R.Y. (1979) J. Gen. Microbiol. 111, [12] Rosenberg, C., Casse-Delbart, F., Dusha, I., David, M., Boucher, C. (1982) J. Bacteriol. 150, [13] Kuhlemeier, C.J., Borrias, W.E., van den Hondel, C.A.M.J.J. and van Arkel, G.A. (1981) Mol. Gen. Genet. 184, [14] Van den Hondel, C.A.M.J.J., Keegstra, W., Borrias, W.E. and van Arkel, G.A. (1979) Plasmid 2, [15] Roberts, T.M. and Koths, K.E. (1976) Cell, 9, [16] Casse, F., Boucher, C., Julliot, J.S., Michel, M. and Dfnarir, J. (1979) J. Gen. Microbiol. 113, [17] Lau, R.H., Sapienza, C. and Doolittle, W.F. (1980) Mol. Gen. Genet. 178, [18] Lau, R.H. and Doolittle, W.F. (1979) J. Bacteriol. 137, [19] Simon, R.D. (1978) J. Bacteriol. 136, [20] Bogorad, L., Gendel, S.M., Haury, J.H., Koller, K.P. (1983) in Photosynthetic Prokaryotes: Cell Differentiation and Function (Papageorgiou, G.C. and Packer, L., Eds.) pp Elsevier, New York. [21] Tandeau de Marsac, N..(1983) Bull. Inst. Pasteur 81, [22] Kallas, T., Rebirre, M.C., Rippka, R. and Tandeau de Marsac, N. (1983) J. Bacteriol. 155, [23] Tandeau de Marsac, N., Mazel, D., Bryant, D.A., Houmard, J. (1985) Nucl. Acids Res. 13, [24] Van den Hondel, C.A.M.J.J., Verbeek, S., van der Ende, A., Weisbeek, P.J., Borrias, W.E. and van Arkel, G.A. (1980) Proc. Natl. Acad. Sci. USA 77, [25] Engwall, K.S. and Gendel, S.M. (1985) FEMS Microbiol. Lett. 26, [26] Gendel, S., Straus, N., Pulleyblank, D. and Williams, J. (1983) J. Bacteriol. 156, [27] Laudenbach, D.E., Straus, N.A., Gendel, S. and Williams, J.P. (1983) Mol. Gen. Genet. 192, [28] Anderson, L.K. and Eiserling, F.A. (1985) FEMS Microbiol. Lett. 29, [29] Chauvat, F., de Vries, L., van der Ende, A. and van Arkel, G.A. (1986) Mol. Gen. Genet., in press. [30] Shestakov, S., Elanskaya, I. and Bibikova, M. (1985) in V Int. Symp. Photosynthetic Prokaryotes, Grindelwald, Switzerland, Abstr., p [31] Lambert, G.R. and Carr, N.G. (1982) Arch. Microbiol. 133, [32] Lambert, G.R., Scott, J.G. and Scott, N.G. (1984) FEMS Microbiol. Lett. 21, [33] Tandeau de Marsac, N. (1977) J. Bacteriol. 130, [34] Castets, A.M., Houmard, J. and Tandeau de Marsac, N. (1986) FEMS Microbiol. Lett. 37,