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29. PRINCSHEIM, E. G. 1946. Pure cultures of Algae. Their Preparation and Mnintenance. Cambridge Univ. Press, 119 PP.

30. PROVUOLI, L. & PINTNER, I. J. 1953. Ecological implica- tions of in v i t r o nutritional requirements of algal flagel- lates. Ann. N . Y . Acad. .Sci. 56:839-51.

31. RIS, H. 8c PLAUT, W. 1962. Ultrastructure of DNA-con- taining areas in the chloroplast of Chlamydomonns. J . Cell B i d . 13583-92.

32. - & SINGH, R. N. 1961. Electron microscope studies on blue-green algae. J . Biophys. Biochem. Cytol. 9:63-80.

33. SCHNEPF, E. 1964. Zur Feinstruktur von Geosiphon pyri-

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/. Plrycol. 3, 31-32 (1967) Notulae

EFFECT OF LYSOZYME (MURAMIDASE) ON MARINE AND FRESHWATER BLUE-GREEN ALGAE'

Li ic ia F ~ i l c o , ~ P. Karfi inkel,3 and S. Aa?.onson Biology Dcpnrtmcnt, Queens College, City University of New York, Flushing, New York 11365,

and Haskins Laboratories, New York, New York 10017

SUMMARY

iblarine blrie-green algae, Lyngbya Lagerheimii, Microcoleus chthonoplastes, Plectonema terebrans, &penellurn quadruplicatum, and freshwater blzie- green algae, Anacystis nidulans, Anabaena variabilis, Nostoc muscorum, and Oscillatoria sp. treated with lysozyme (moiramidase) formed spheroplasts but not protoplasts. T h e time needed for spheroplast induc- tion uaried with the species. Approximate internal osmotic pressures of the blue-green algae were de- termined. Marine algae generally had a higher osmotic ;bresszire than freshwater algae.

INTRODUCTION

Protoplasts or spheroplasts have been obtained Erom freshwater blue-green algae (2-4) by means of

1 Aided by a grant from the City University of New York to S . Aaronson. Received November 3, 1966; revised FeOruary 21, 1967.

2 Present address: Biology Department, Brandeis University, Waltham, Massachusetts.

9 Present address: Biology Department, Yale University, New Haven, Connecticut.

lysozyme treatment. N o one to our knowledge has obtained protoplasts or spheroplasts from marine blue-green algae. Lysozyme treatment of marine and freshwater blue-green algae might indicate whether the cell walls of the marine algae are the same as the freshwater forms, and might give some idea of the internal osmotic pressure of marine as contrasted with that of freshwater algal cells.

METHODS

The marine forms Lyngbya Lagerheimii, iVlicrocolezis ch tho- noplnstes (2 strains), Plectonema terebrans, and Agmenelluin qz~adrn;Dlicatum, obtained through the kindness of Dr. C. van Baalen, Institute of Marine Science, Port Aransas, Texas, were grown in a modified ASP-2 medium (8), i.e., minus Na SiO,.9H,O and vitamin mix S 3 at p H 7.5 at 25 C in fluores- cent light. T h e freshwater blue-green algae Anacystis nidulnns, AntrDnena uariabilis, and Nostoc muscorum, obtained through the generositv of Dr. M. B. Allen, Kaiser Foundation, Rich- mond, California, were grown on Kratz-Myers (KM) medium (7) at p H 7.8 at 25 C in fluorescent light. Oscillatorin sp. was obtained from commercial sources and used immediately. Lysozynie (niuramidase), 2 X recrystallized, was purchased from

52 LIICI:\ FULCO, P. KARFC'NKEL. .ASD S. .4.4RONSO.U

TABLE 1. green ([lgae.

Ef ler t of ~s . toz~vre OJI rplreroplast formation in blue-

Days required Sugnr concentration (1%)

formation 3 6 10 15 20 Organism for spheroplast

Marine: L . Lagerheimii A . quadruplicatiim P. terebrans M. chthonoplastes strain

Jamaica 111. ch t h onoplost es strain

Puerto Rico Freshwater:

A . nidulans A . variabilis N. muscoriim Oscillatorin sp.

+ - _ _ _ 2

+ + - - - f-12

+ = Spheroplasts formed. - = No spheroplasts formed after l2-dny incubation.

the Worthington Riocheniic;il Corporation, Freehold, Sew Jcrsey.

The lysozyme ex-periniciits w c ~ e clone i n t u l m containing -1 ml of ASP-2 or K S I medium. a coiiccntratell suspension of I)luc-grccn algae, 0.05% lysozyine. and sucrose (0, 3, 6, 10, 15, o r 20%). Control series w i t h o u t Icsozyiiie were also run. Cell suspensions were iiicubatetl for 2-14 days at room tcmpcrature ant1 cxaminetl with a Sikon binocular phase microscope each clay. Lysoryme-treated cells in sucrose mcdia were diltitcd with tlistillcil w;iter to see wlicthcr the rounclecl forms burst (proto- plasts) or retained rlicir slial)e (splieroplasts).

RESU LI'S

Lysoqme treatment ol' blue-green algae resulted in the disruption of filaments and rounding of cells (Table 1) . Xone of the lysozyne-treated algae burst on dilution of the suspending medium with distilled water; we assume from this that the rounded forms were spheroplasts rather than protoplasts. T h e sphero- plasts in some cases did not, however, maintain them- selves in hypoosmotic media for long time periods (Table 1). The marine species required somewhat more time than the freshwater species for lysozyme to form spheroplasts.

T h e approximate internal osmotic pressure of marine blue-green algae except for that of N . mzis- corzim is expectedly higher than that of freshwater algae and conforms generally to the observations of others as reviewed by Guillard ( 5 ) (Table 2). T h e unusually high osmotic pressure of N. miiscorzim re- mains unexplained.

T. iB1.E 2. .-lppro.xiinate osmotic pressure of blue-green algae.

Approximate osmotic pressure Organism in atmosphrresll

Marine: L. Lagerheimii 4.3 A . quadrirplicat 1oU 10.7 P . terebrans 14.3 JL. chthonoplastes (Jamaica strain) 10.7 .%I. chthonoplastes (Puerto Rico strain) 9.1

A , nidirlans 2.4 A . variabilis 2.4

Oscillatoria sp. 2.4

Freshwater:

iv. nl lLSCO~l lJ71 14.3

a Osmohc pressure calculated as: osmotic pressure = C (moles/liter) x R (0.082 liter ntmospheres/degree/ mole) X T (absolute temperature) using lowest concentration of sugar in Tnble 1 which permitted spheroplast formation.

DISCUSSION

T h e cell walls of different species of blue-green algae vary in sensitivity to lysozyme (2,3). This is confirmed here by the different intervals of exposure to lysozyme needed for spheroplast formation. Like Gram-negative bacteria (9), blue-green algal cell walls may consist of varying amounts of mucopeptitle ma- trix associated with other materials, e.g., carbohydrate (6). Future work may show that some blue-green algal cell walls resemble the cell walls of other algae and thus may indicate more about the relationship between blue-green and other algae ( I ) .

REFERENCES

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