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Romanian Biotechnological Letters Vol. 15, No.3, 2010 Copyright © 2010 University of Bucharest Printed in Romania. All rights reserved

ORIGINAL PAPER

5361

Isolation of permeaplasts and spheroplasts from Spirulina platensis

Received for publication, September 15, 2009 Accepted, May 20, 2010

ALEXANDRU FILIP VLADIMIRESCU National Institute of Research-Development for Microbiology and Immunology“Cantacuzino”, 103 Splaiul Independentei, 050096, Bucharest, Romania, tel. +40726170840 E-mail: [email protected]

Abstract From the point of view of cyanobacteria enormous biotechnological potential, the use of

genetic transformation and engineering in these organisms can break new, completely unimagined grounds, for the intensive exploitation of this natural resource. A natural barrier against the genetic transformation experiments in cyanobacteria is represented by the cell-wall itself. The experiments conducted showed that 4 hours of lysozyme treatments in Spirulina platensis lead to the advent of permeaplasts displaying a normal internal structure, certified by Transmission Electron Microscopy (TEM) studies. However, prolonged lysozyme treatments had a destructive effect on the bacterial cells. Enzymatic degradation was not identical in all the cells, some of them being more resistant than others to lysozyme attack. The results presented here plead for the use of genetic manipulation in Spirulina platensis via permeaplasts, instead of spheroplasts fusion.

Keywords: Spirulina, cyanobacteria, spheroplast, permeaplast, lysozyme, TEM Introduction

From the point of view of cyanobacteria enormous biotechnological potential, the use of genetic transformation and engineering in these organisms can break new, completely unimagined grounds, for the intensive exploitation of this natural resource. A natural barrier against the genetic transformation experiments in cyanobacteria is represented by the cell-wall itself.

Cyanobacteria have a cell-wall that is similar in structure to that of Gram-negative bacteria [1]. This cell-wall contains peptidogycan – a heteropolymer resistant to lysozyme treatment.

The term of spheroplast used in this article designates (according to the established terminology for microorganisms) a cell whose the cell-wall was vastly removed by lysozyme treatment and the remaining undigested bits adhere to the plasmalemma that is osmotically sensitive and protects the rest of the cellular constituents which are still intact.

The term of permeaplast, encountered in an article by Daniel et al. [2], refers to spheroplasts that present only a slight enzymatic degradation of the cell-wall, under the form of breeches that allow some macromolecules (eg. DNA: pBR322) to pass through the plasmalemma into the cell. Unlike spheroplasts, permeaplasts expound a larger reversion (cell-wall reconstruction) percentage and, as a consequence of this, the destructive effect of long term lysozyme incubation is superseded.

Spheroplasts yielding in cyanobacteria was the subject of numerous studies [3, 4, 5, 6, 7, 8, 9]. Attempts to obtain Spirulina spheroplasts were made by Robinson et al. [10], Ciferi [11], Abo-Shady et al. [12]), Wu [13], Peng Gao et al. [14], Gan et al. [15] and Yi et al. [16].

The current paper proposes to monitor lysozyme attack on S. platensis trichomes, using both optical and electron microscopy techniques.


5362 Romanian Biotechnological Letters, Vol. 15, No. 3, 2010

Material and methods Biological material

Spirulina platensis (Gom.) Geitleri (Fig. 1) strain used in this study was obtained by the amiability of Prof. Dr. Marioara Godeanu (Applied Ecology Institute) and maintained axenically in Zarrouk [17] liquid culture medium, with natural photoperiodicity and air bubbling (using an aquarium air pump provided with a cotton filter).

Figure 1. Microscopical aspect of Spirulina platensis suspension cultivated on Zarrouk medium (Ob 40x)

Obtaining spheroplasts - permeaplasts in Spirulina platensis cyanobacteria Following numerous trials I adopted a formula (protoplastization solution) for

isolating cyanobacterial sphero-/permeaplasts consisting of 50 mM glucose + 40 mM Tris HCl, pH = 8.0 and 1 mM EDTA, pH = 8,0 + 4 mg/ml lysozyme (Merck), the latter being added extemporaneously, as powder.

Cyanobacterial suspensions were centrifuged at 3000-6000 rpm for 10 minutes. The sediment was washed three times in fresh culture medium [17] and thereupon it was resuspended into an equal volume of protoplastization solution. The incubation was at room temperature (avg. 20 °C) for 4 to 24 hours. Samples were collected at 4, 7 and 24 hr intervals and were examined with the optical microscope.

The samples collected at 4 and 7 hr respectively were washed in fresh culture medium, added with 50 mM glucose, and centrifuged in preparation for electron microscopy (TEM).

The cyanobacteria were prefixed in 2.5% glutaraldehyde in 0.1 M cacodilate buffer, pH = 7.2, for 2 hr, at + 4 °C. Following three successive washes (20 minutes each, in cacodilate buffer pH = 7.2) postfixation occurred in 1% osmium tetroxide (OsO4) in cacodilate buffer, pH = 7.2, for 2 hr, at + 4 °C.

Dehydration was performed by an ascending series of acetone dilutions (30%, 50%, 70%, 90%, 100%), of 30 minutes each, followed by 3 times treatment with absolute acetone, 30 minutes each.

The inclusion was performed in DER and EPON resins in 5:1 g/g ratio. The serial sections were obtained with a LKB ultramicrotome and they had a golden-

yellow colour and an approximate thickness of 800 Å. Those sections were placed on formval-coated, copper grills and subsequently

coloured with uranyl acetate (13% alcoholic solution) for 10 min and lead citrate for 5 min.

ALEXANDRU FILIP VLADIMIRESCU

Romanian Biotechnological Letters, Vol. 15, No. 3, 2010 5363

Examination of the sections was performed under a Phillips 201 electron microscope, with a magnification power of 200-200000x, an electron accelerating voltage of 40, 60, 80 and 100 kV, and a 5 Å resolution. Optical microscopy observation of spheroplasts

This was performed on living bacteria, using a MC5 light microscope equipped with 10, 20 and 90x objectives.

Figure 2. Optical microscopy observations (A-D) on Spirulina platensis lysozyme trated trichomes and cells (Ob. 40x , 90x) The breaking of trichomes into 4-5 cells fragments can be observed (A, B, E); the appearance of spheroplasts (A-E) that can subsequently generate minicells (B,D,E, arrows); a spontaneous spheroplasts aggregation tendency can also be noticed (D şi E); F = longitudinal section through Spirulina platensis trichome, examined by TEM – the integrity of the cell-wall (CW) and that of the cellular constituents is visible.

Results

Enzymatic attack in Spirulina platensis became obvious (by optical microscopy) after 4 hr of incubation with lysozyme and started by fragmenting the trichome into pieces of filament containing on average 5 connected cells (Fig. 2 A, B, E). Further on, these fragments began to generate spheroplasts and some of the cells (from the endings as well as from the centre) acquired the aspect of budding cells (Fig. 2 B, E), due to the protoplasm effusion and minicells production. This phenomenon was also encountered in Spirulina spherical protoplasts in which such minicells were observed (Fig. 2 C, D, E).

Following lysozyme treatment an inflation of the affected cells could be observed, even though these cells did not detach from the trichome (Fig. 2 B).



In few cases spontaneous fusion of pairwise Spirulina spheroplasts was observed (Fig. 2 D). Microscopic aspect of lysozyme digested trichomes and spheroplasts indicated that

they suffered color degradation, from intense green to yellow-green, and sometimes even an apparent depletion from the cellular content (Fig2 A-E). Corroboration of these observations with the ones derived from electron microscopy showed an obvious destructive effect of the lysozyme treatment on thylakoids (Fig. 4 C). After 24 hr the number of those degraded trichomes and spheroplasts increased considerably.

Even after 24 hr of incubation with lysozyme some healthy green cells trichomes were still present.

Some elder cells were more refractory to lysozyme tratment. TEM studies on Spirulina platensis cyanobacterial spheroplasts were performed.

Figure 3. TEM studies on Spirulina platensis cyanobacterial spheroplasts (A, B) were performed using samples collected at 4 h of lysozyme incubation. In Spirulina platensis cells displaying an not advanced (A) and an advanced protoplastization stage (B), LIV layer appears as a far more electron-transparent material halo [CW] (arrow) comparing to its normal electron microscopic aspect (CW, arrow); P = pores; Tl =Tylakoids

After 4hr of lysozyme treatment Spirulina cells displayed different stages of

enzymatic attack which may indicate the existence of differences in enzymatic sensitivity (Fig. 3 A). Spirulina cells that were in an incipient lysozyme degradation stage (Fig. 3 A-B) still had a 4 layered cell-wall (LI – LIV) [18,19]: LI layer was electron-transparent and situated right over the plasmalemma, LII layer was electron-dense, LIII layer was electron-transparent and LIV layer was electron-dense and rippled (Fig. 2 F, 4 A); the cell-wall would suffer various preliminary digestion:

- LIV displayed ruptures and a larger number of discontinuities than it normally did on untreated material; it was rippled and generally rough;

- LIII layer, which is usually modestly represented, was then better marked out, probably due to a lysozyme induced inflation process;

- LII layer started to lose its integrity, particularly in the vicinity of the areas where LIV was also interrupted;

- LI,layer remained closely associated to the plasma membrane.



Figure 4. Electron microscopy aspects in Spirulina platensis: A = longitudinal section through a trichome that was not treated with lysozyme – the integrity of the cell-walls is obvious; B = detail of a permeaplast that was produced by lysozyme treatment – numerous pores (P) surrounding the cell; C = Spirulina – after 7 hr of lysozyme digestion a “refractory” cell was produced, whose spheroplast remained attached to the trichome (with partially degraded cell-wall: CW); in the area where the layers were digested the protoplasm effused and, unless stopped by LIII and LIV intact layers, it would have been released outside (arrow). Prolonged lysozyme treatment determines its penetration into the cell, with consecutive degradation of the cellular constituents (Tl = Tylacoids)

Cells that underwent advanced lysozyme treatment (Fig. 3 B) displayed the following

changes: - LIV layer appeared as a far more electron-transparent material halo comparing to its

normal electron microscopic aspect. It had a continuous disposition around the cell and was composed of slightly electron-dense fibers that were embedded into a more electron-transparent matrix;

- LIII layer was electron-transparent and heavily inflated; - LII layer appeared to be less electron-dense due to its lysis under lysozyme action; in

some cases it displayed pores all around the cell (Fig. 3 B, 4 B); - The whole cell-wall thickness was increased from 0.5 to 1-1.8 μm following

lysozyme treatment; - After 4 hr of lysozyme digestion Spirulina permeaplasts and spheroplasts presented

thylakoidal lamellae with normal conformation and size just like the rest of cellular constituents (Fig. 3 A-B).

After 7 hr of lysozyme digestion Spirulina cells still displayed a non-uniform conformation, with numerous destroyed cells and cells presenting spheroplasts that remained



attached to the trichome (with partially degraded cell-walls). In such cells, refractory to the the lysozyme treatment (Fig. 4 C), cell-wall layers presented the following characteristics:

- LIV layer was electron-dense, fairly intact, without inflations, and with some scattered breeches; it was less rippled and without a rough surface;

- LIII expounded adherences to LIV layer and to a lesser extent to LII layer; - LII and LI layers appeared to be quite frequently continuous and intact. In the area

where these layers were digested the protoplasm effused (Fig. 4 C, arrow) and, unless stopped by LIII and LIV intact layers, it would have been released outside. This is how the occurrence of some minicells and the presence of some depleted cells in optical microscopy could be explained.

Prolonged lysozyme treatment determined its penetration into the cell with consecutive degradation of cellular constituents. The cytoplasm appeared to be granular and interspersed with numerous electron-transparent zones, determined by the lysis of cellular components.

Thylakoids seemed to be the most sensitive organelles; if normally they have the aspect of thylakoidal lamellae fascicles, more or less parallel to the longitudinal ax of the trichome, after prolonged lysozyme treatment their number decreased and the distance between two lamellae increased.

Thylakoidal lamellae degradation (rich in chlorophyll a) was observed in optical microscopy as a discoloration of the blue-green Spirulina trichomes as the lysozyme attack was extended (Fig. 2 E).

Discussion

Following the lysozyme treatment an inflation of affected cells occurs, even if they did not detach from the trichome. This observation stands in with those made by Dwain et al. [7] regarding the behavior of Microcystis aeruginosa, Anacystis nidulans and Anabaena floss-aquae protoplasts to the lysozyme attack, using optical microscopy. Dwain et al. [7] determined that after a few hours of enzymatic treatment cyanobacteria undergo shape changes as it follows: a) the cells became spherical and even some polar plasmolysis fenomena can be noted (Anacystis nidulans); b) the inflation of the cells during lysozyme treatment leads to some more or less spherical casts (Microcystis aeruginosa); c) in the case of Anabaena cyanobacteria (the most similar in trichome structure with Spirulina) lysozyme treatment affects the shape of cells, even though they are still attached to the trichome; these become spherical and easily detachable from the trichome. Spirulina platensis cells/spheroplasts don’t detach from the trichome that easily, fact that was noted by Yi et al. [16]; they noticed that lysozyme treatment was unable to disintegrate cells of Spirulina efficiently, because the cell-wall of Spirulina was thicker.

Even after 24 hr of lysozyme incubation there are still present some trichomes with healthy green cells. This observation is in non-compliance with the researches made by Biggins [5], Vance and Ward [20] and Rogers [8] on Anacystis nidulans cells treated with lysozyme for 12 hr, whereby it was observed that the cell age does not affect the process of obtaining spheroplasts. The result obtained here for Spirulina platensis agrees with the research Lindsey et al. [9] made on synchronized Anacystis nidulans cultures, showing that elder cells are more refractory to lysozyme treatment. In the present case trichome growth can be achieved either from a single cell (seldom) or from a trichome composed of few cells, detaching from the maternal trichome (more often).

The different behavior to enzymatic treatment can be explained by these cells metabolism which varies the content of β-1, 4-N-acetilglucosamine polymers (lysozyme specific substrate) in elder areas of trichome’s cell-wall, comparing to the youer areas of the



same trichome. This could be a plausible explanation for Spirulina trichome breakage into 4-5 cells units under lysozyme treatment, fact that was also evidenced by Peng Gao et al. [14] and Yi et al. [16] in ultrasonication experiments on Spirulina platensis.

Spirulina cells that are in an incipient stage of lysozyme degradation (Fig. 3 A-B) have a 4 layered cell-wall (LI –LIV) as it was described by Jost [18] in Oscillatoria rubescens and later on evidenced by Ionescu and Titu [19] through TEM studies on Spirulina platensis.

The appearance and size of TEM highlighted pores are very much alike those described by Ionescu and Tiţu [19] in Spirulina platensis.

Thylakoidal lamellae (containing chlorophyll a) degradation is observed in optical microscopy as a fading of Spirulina trichomes blue-green color as lysozyme attack is extended.

This aspect is in compliance with the observations Lindsey et al. [9] made on Anacystis nidulans. At these cyanobacteria the cell-wall appears to be less rigid following 1 hr of lysozyme treatment, the cytoplasm is less dense and the thylakoidal system is not so much compressed against the internal cell-wall (but the cohesion of thylakoidal lamellae is still maintained towards the central part of the cell and their orientation appear to be random). After 3 hr of lysozyme treatment in Anacystis nidulans the cyanobacterial cells display randomly distributed thylakoids and increased interthylakoidal spaces. This fact is also in compliance with Wu [13] finding that Spirulina platensis spheroplasts obtained by lysozyme treatment have a reduced photosynthesis and a weak lipid metabolism.

The current paper pleads for the use of genetic manipulation in Spirulina platensis via permeaplasts due to the fact that spheroplasts number is extremely low (according also to the research made by Gan et al. [15]).

Conclusions

4 hr lysozyme treatment in Spirulina platensis lead to permeaplsts issuance, with normal internal structure (verified by TEM). Prolonged lysozyme treatment has a destructive effect on the cells.

Enzymatic degradation is not occurring evenly in all the cells – some cells are more sensitive than others to lysozyme attack.

The results presented here plead for the use of genetic manipulation in Spirulina platensis via permeaplasts, instead of spheroplasts fusion. Acknowledgements

I express my gratitude to Dr. Mircea Dan Ionescu and Cornel Pârvu (Cantacuzino Institute) as well as to Prof. Dr. Lucian Gavrilă (Faculty of Biology, University of Bucharest) for their scientific help and advice. I also give special thanks to Miss Elena Claudia Coipan for the help in this manuscript’s work out.

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