Journal of Microbiological Methods 15 (1992) 7 - 15

© 1992 Elsevier Science Publishers B.V. All rights reserved 0167- 7012/92/$ 5.00

MIMET 00474

A simplified method for screening and in cyanobacteria I,,,,llOl, I OI.~.,,LK,,I I L a t l O i l

Dinesh Goyal Department of Microbiology, RLA College, University o f Delhi, New Delhi, India

(Received 25 July 1991 revision received 5 November 1991; accepted 7 November 1991)

Summary

Plasmid distribution among 14 different cyanobacteria was studied using a simplified method which involved direct agarose gel d, ectrophoresis of heat-treated, ethanol-precipitated, plasmid preparations from the cleared lysates witho~at requiring ultracentrifugation. The method is sensitive and can be effective- ly used to determine the number of different plasmid species and their molecular weights from the agarose gel patterns. The results m.'npare well with those obtained by the CsCI-EtBr equilibrium density centrifu- gation technique. Out of t4 cyanobacteria, eight were found to contain plasmid DNA of size r.a.nging from 0.9 to 35.0 MDa. All the ,:yanobacteria. viz., Lyngbya, Piectonema and Phormidium, belonging to the LPP group sho,,ed different plasmid profiles, supporting the view that these strains are independent isolates of different speci~:s. Anabaena azollae did not contain any plasmid DNA.

Key words: CCC DNA; ~Syanobac'~erium; Gel electrophoresis

Introduction

Because of the s~ructural simplicity combined with the biochemical complexity of the higher organisms, cyanobacteria serve as attractive model organisms for studying many fundamental biological problems at molecular level [1, 2]. The ever-expanding potential of eco,aomic exploitation of cyanobacteria has generated interest in geneti- cally manipul.J, ting these organisms [3, 41 to develop tailored strains which can per- form the desired functions.

Plasmids have been reported in most of the unicellular [5 - 14] and many of the filamentous [7, 8, 13, 14- 18] cyanobacteria. All plasmids so far characterized ap- pear to be genetically cryptic, and their role in physiology, ecology and evolution of cyanobacteria is still unknown [7, 9, 11, 19]. To explore the possibilities of using plasmids as vehicles to shuttle the genetic material amongst cyanobacteria [3, 4, 20],

Correspondence to: D. Goyal, Pocket B-3, Flat No. 13-A, Lawrence Road, Delhi 110 035, India.

it is essential to ascertain and characterize their distributional pattern in this group of organisms. The present communication embodies the results of a simple agarose gel electrophoresis method used to study the plasmid content of 14 cyanobacteria belonging to four typological groups.

Materials and Methods

Strains and growth conditions All cyanobacterial strains (Table 1) used in the present investigation were obtained

from the National Facility for Blue-Green Algal Collections, Indian Agricultural Research Institute, New Delhi. They were grown as stationary batch cultures in BG-11 medium [21], at 30_+ 1 °C and 3000-4000 lux light intensity provided by cool daylight white fluorescent tube lamps.

The culture of Bacillus thuringiensis ssp. kurstaki BA82B, which harbours plas- mids of 150, 47, 30, 9.6, 4.9 and 1.3 MDa size [22] was obtained from the Department of Biological Sciences, University of Warwick, UK. The strain of Escherichia coli CA1830, having the plasmid pBR322 (2.8 MDa), was procured from Biotechnology Centre, Indian Agricultural Research Institute, New Delhi and that of E. coil JMC 18 containing RP-4 (40 MDa) was obtained from the Department of Bacteriology, Uni- versity of California, Davis, California, USA. These cultures were grown as shake cultures in Luria-Bertani (LB) broth [23], at 37_+ 1 °C with or without appropriate antibiotics (35 #g-ml -~ of Amp, 12.5 #g.ml -~ of Tet).

Isolation of plasmid DNA

Method 1 Algal cells from 100- 200 ml of exponentially growing (14 days old) cultures were

harvested by centrifugation, washed twice in SE buffer (sodium chloride, 0.15 M; EDTA, 0.05 M), pH 8, and resuspended in STE buffer (sucrose, 25°7o; Tris base, 0.05 M; EDTA, 0.1 M), pH 8 containing lysozyme (5 mg.ml -~) and kept at 37°C for 1 h. Sodium dodecyl sulphate (SDS) was added to a final concentration of 2°7o and again kept at 37°C for 30 min [24]. Cold 5 M sodium chloride was added to the final concentration of 1 M and kept overnight at 4°C. The cell debris was removed by centrifugation at 17000 × g for 35 min at 4°C and to the supernatant, equal volume of TE buffer (Tr is- HCI, 10 mM; EDTA, 1 mM), pH 8, was added. After this, one volume of TE saturated phenol was added and the tube inverted gently several times and spun at 10000 rpm for 15 min at 20°C to obtain a clear aqueous phase, which was then treated twice with equal volume of phenol : chloroform (1 : 1) mixture and gently mixed and centrifuged. The aqueous phase was then treated once with cold chloro- form : isoamyl alcohol (24 • 1, v/v).

To the clear aqueous phase, 2 vols. of cold ethanol (95%) were added and kept overnight at - 20°C. The precipitated DNA was recovered by centrifugation at 10000 rpm at 4°C for 10 min. Ethanol was then drained off completely from the tube. The pellet of DNA was washed with 70°7o ethz, r.ol, dried in vacuum dessicator (or at room temperature for 30 -45 min) and suspended in 100-200 ~tl of TE buffer, pH 7.5.

The DNA samples were analysed immediately by agarose gel electrophoresis or stored at 4°C.

Method 2 The cells were harvested and washed once with GTE buffer (glucose, 50 mM;

T r i s -HCI , 25 raM; EDTA, 10 raM), pH 8 [25], and again suspcnded in the same buffer containing lysozyme (5 mg,ml-z) and kept at 37°C for I h. After this, 1/4 of the initial volume of freshly prepared solution II (10070 SDS in 0.2 N NaOH) was added and kept at 37°C for 1 5 - 3 0 min. Then, 5 M NaCl was added to a final concentration of 1 M and kept overnight at 4°C. The ceil debris was removed by centrifugation at 17000 × g for 30 rain at 4°C and the supernatant was extracted twice with phenol and once with chloroform • isoamyi alcohol (24 • i, ~ 0. The DNA was precipitated as described in Method 1.

Method 3 The plasmids from the bacterial cultures were isolated following the mini-prep

alkaline extraction method [26]. Plasmids from the cleared lysates obtained by all the above methods were purified

by CsCI - EtBr (cesium chloride-ethidium bromide) densi ty- gradient centrifuga- tion after precipitation by polyethylene glycol 6000 [27].

Heat treatment o f plasmid DNA Plasmid preparation (25 - 50 #1) prior to gel electrophoresis, was heat treated for

2 min at 100°C in presence of 0.1% sarkosyl solution followed by fast cooling in ice water [10, 11]. The sample was then immediately loaded on the gel to prevent any renaturation.

Gel electrophoreMs

Standard method of horizontal agarose slab gel electrophoresis (0.5 x 9.5 x 10.0 cm} was emnloyed to ~enar:ate and id,~ntiFy the ,-,lo~m~n r~MA Th,~ r~TA . . . . ,-~ , J r - - - - l . . , . u o a i x a u L I i ~ E I . . l i l t . , J L J ~ . N / ' ~ 3 a l l l l . Y l ~ 3

(20 -50 #l) were subjected to electrophoresis as described by Maniatis et al. [28], using various percentages of agarose gels (0 .5-0 .8%) . The gel and running buffer contained 0.04 M Tris - acetate and 0.001 M EDTA (TAE), pH 8. The samples mixed with 6 x gel loading buffer (bromophenol blue, 0.25%; sucrose, 40% ), were loaded into slot (0.5 cm) of the submerged gel. E|ectrophoresis was carried out at room

- 1 temperature for 5 - 6 h or until the dye reached the bottom of the gel, at 5 v.cm using LKB 2301 Macrodrive l power supply. The gel was stained in the dark in TAE buffer containing 2 #g EtBr, ml-~ for 30 min. Fluorescent D N A - E t B r complexes were then observed in transmitted UV light and photographed through a Fotodyne transilluminator using B/W polaroid type 667 film.

Molecular weights

Molecular weights of the plasmid DNAs were calculated by comparing them with standard reference covalently closed circular (CCC) DNAs after running them on the same gel. These CCC marker DNAs were the plasmids isolated from Bacillus thurin- giensis ssp. kurstaki BA82B. Following the alkaline extraction method [26], only four bands of CCC DNAs of 47, 30, 9.6 and n..9 MDa size were obtained. These CCC

10

DNAs along with pBR322 (2.8 MDa) and RP-4 (40 MDa) from E. coli were used as standard markers.

Results and Discussion

Out of 14 different strains of cyanobacteria examined, eight were found to contain p!asmid DNAs of size ranging from 0 .9 -35 .0 MDa (Table 1). The presence of covalently closed circular DNAs was confirmed following purification of plasmid DNA from the cleared lysates with polyethylene glycol 6000 and CsCI - EtBr density- gradient centrifugation [11, 27]. In Anacystis nidulans, Plectonema notatum and Anabaena variabilis, two DNA bands were obtained in the CsCI-EtBr gradient whereas only one band was observed in Anabaena azollae. The lower band of DNA was further analysed by agarose gel electrophoresis after heat treatment. Anacystis nidulans and Plectonema notatum were found to have two CCC DNAs each whereas Anabaena variabilis had only one (Fig. 1A).

Since CsCI- EtBr density-gradient centrifugation requires an extensive period of centrifugation at high speed, an al*ernative method was developed involving gel electrophoresis of heat-treated ethanol precipitated plasmid preparation from the

TABLE 1

PLASMID DISTRIBUTION IN CYANOBACTERIA

Family Cyanobacteria Strain Morphology No. of Size (MDa) plasmid DNA

Chroococcaceae Anacystis nidulans ARM 336 Unicellular 2 35, 6 Gloeocapsa rupestris ARM 338 Colonial 0 -

Oscillatoriaceae Lyngbya versicoior ARM 348 Non-Het, Fil i Piectonema notatum ARM 73 Non-Het, Fil 2 Phormidium foveolarum ARM 695 Non-Het, Fil 3

Nostoceae Anabaena variabilis Anabaena variabilis Anabaena variabilis Anabaena azollae (Azolla filiculoides) A nabaena azollae (,4 zolla mexicana) A nabaena azoilae (,4 zolla pinnata) A nabaena azollae (Azolla caroliniana) Nostoc spongiaeforme

Scytonemataceae Tolypothrix tenuis

ARM 394 I-iet, Fil 1 ARM 310 Het, Fil 1 ARM 668 Het, Fil 1 ARM 664 Het, Fil Symbiotic 0 ARM 665 Het, Fil Symbiotic 0 ARM 666 Het, Fil Symbiotic 0 ARM 667 Het, Fil Symbiotic 0 ARM 401 Het, Fil l

ARM 76 Het, Fil 0

4 27, 8.2 34, 10, 0.9

31 31 31

m

30

Het, Heterocystous; Fil, Filamentous.

a b c d a h a b h b

11

S$

.o¢/L

A B Fig. 1. (A) 0.7% agarose gel electrophoresis of plasmid DNA. Plasmid RP-4 (40 MDa) from JMC 18 (Lane a); Plasmid DNA purified by CsC1-EtBr centrifugation and heat-treated from Piectonema notatum ARM 73 (Lane b); Anabaena variabilis ARM 394 (Lane c); AnatTstis nidulans ARM 336 (Lane d). (B) 0.5% agarose gel electrophoresis of heat-treated (Lane a n) and unheated (Lane a) ethanol-precipitated plasmid DNA from cleared lysates of Anacystis nidulans ARM 336.0.7°70 agarose gel electrophoresis of heat-treated (Lane b h) and unheated (Lane b) plasmid pBR322. Arrows indicate positions of CCC DNA;

C, chromosomal DNA; SS, single-stranded DNA; and OC/L, open circular/linear DNA.

cleared lysates. Electrophoresis of such plasmid preparation from Anacystis nidulans after heat treatment showed the disappearance of OC/L (open circle/linear) DNA and simultaneous appearance of a faster-migrating band of single-strand (SS) DNA [10] whereas no change occurred in the position of the CCC DNA bands (Fig. 1B, Lane ah). The large plasmid (35 MDa) comigrated along with the chromosomal DNA, which upon heat treatment remained at its original position whereas the chromoso- mal DNA was completely denatured. This method can also be effectively employed to determine the number of different plasmid species from the agarose gel patterns after verifying the CCC character of the DNA bands by heat treatment.

To demonstrate the validity of this method, the standard plasmid pBR322 from E. coli was taken and heat treated. The OC/L DNA band disappeared after heat treat- ment and in fact was completely denatured whereas, the CCC DNA did not change its position (Fig. 1 B, lane bh). This technique was used to analyse the plasmid content

12

rn a a h b b h c c h d d h e e h

m f fh h h ghg h i j

2.8

Fig. 2. Agarose gel electrophoresis of plasmid DNA from filamentous cyanobacterial strains, Anabaena variabilis ARM 394 (Lane a); Anabaena azollae ARM 664 (Lane b); Anabaena variabilis ARM 310 (Lane c); Tolypothrix tenuis ARM 76 (Lane d); Nostoc spongiaeforme ARM 401 (Lane e); Piectonema notatum ARM 73 (Lane f); Lyngbya versicolor ARM 348 (Lane g); Phormidium foveolarum ARM 695 (Lane h). Marker plasmid DNA (MDa) from Bacillus tkuringiensis ssp. kurstaki BA82B (Lane m); Anacystis (Lane i); and pBR322 (Lane j). Arrows indicate position of CCC DNA; superscript h, represents heat-treated

plasmid preparation as described under Materials and Methods.

of Piectonema notatum (Fig. 2, lane fh) Anabaena variabilis and A. azollae (Fig. 2, lane a h, bh). Results obtained by this method were comparable to those obtained with the DNA purified by the CsCI -E tBr density-gradient centrifugation method. The plasmid content of the other filamentous cyanobacteria was also analysed using this method. Amongst the heterocystous cyanobacteria, Anabaena variabilis had only one plasmid of 31 MDa size, Tolypothrix tenuis [7] (Fig. 2, lane dh), all the strains of Anabaena azollae and colonial nonheterocystous Gloeocapsa rupestris, did not show the presence of any extra chromosomal DNA even with different protocols and their modifications.

13

While isolating plasmid from different cyanobacteria, the major difficulty is wo achieve gentle and efficient lysis of the cells [8]. This could be due to the presence of a thick mucilagenous sheath of undetermined composition [29]. Efficient lysis of the ceils in the case of Anacystis, Gloeocapsa, strains of Anabaena, Nostoc and Toly- pothrix was obtained only by using Method I. However, plasmid content of non- heterocystous filamentous cyanobacteria (LPP group) could be studied only by using Method 2 which is a modification of Methods 1 and 3 and is simpler than the earlier method [29]. Attempts to use other methods and their various modifications like varying the time of incubation with lysozyme or detergent, harvesting cells at their different growth stages and taking varying ceil populations in the suspension failed [14].

All the members of the LPP group of cyanobacteria showed different p!asmid profiles (Fig. 2, Lanes gh, fh, hh), supporting the view that these strains are independent isolates of different species [17]. The protocol standardised during the present study is a simplified method over the existing m-thods as it does not require extensive period of ultracentrifugation and the heat treatment step directly establishes the CCC char- acter of the DNA bands observed on the gels, giving clear information about the various plasmid species present in a cyanobacterial strain. The method is distinct from the in-gel lysis technique [13] as it can be easily adopted for the large scale purification of plasmid DNA without any contamination of chromosomal DNA. This method has also been found to be effective in obtaining sufficient plasmid DNA for several restriction enzyme digests in Phormidiumfoveolarum. The smallest plas- mid (0.9 MDa) from this cyanobacterium was found to have two restriction sites for HindlIl, one for HinfI and three for KBnl restriction enzymes.

The'molecular weights of the different plasmid species were estimated by compar- ison of their relative migration to that of the plasmids isolated from Bacillus thurin- giensis ssp. kurstaki BA82B. The present study reveals that the presence of CCC DNA is apparently a common phenomenon in cyanobacteria and their numl)er may vary

u l , ~ . t u t , t l ~ ¢ . • l l g l J I U t U k . U l 3 t a t l L l a l L l l b C L l in the present study is less time con- suming and inexpensive and useful for carrying out a systematic survey of plasmid DNAs and their characterization in large number of cyanobacteria. So far, no corre- lations have been found between the presence of CCC DNAs and the range of meta- bolic activities studied and the purpose of their existence in the cyanobacterial cells is still unknown. Possib!v the small plasmid of 0.9 MDa size from Phormidium foveolarum can be developed as a shuttle vector between a suitable bacterial system and cyanobacteria. This will not only help in ascertaining the role of this plasmid DNA but also enable us to develop a protocol for gene cloning in filamentous non- heterocystous cyanobacteria.

Acknowledgements

The author is grateful to the Project Director, National Facility for Blue-Green Algal Collections, for facilities and to the Director, Indian Agricultural Research Institute, New Delhi, for financial support.

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