FEMS Microbiology Letters 44 (1987) 221-224 221 Published by Elsevier

FEM02914

Absence of the nitrogen reserve polymer cyanophycin from marine Synechococcus species

Julie N e w m a n , Michael W y m a n and Noe l G. Car r

Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, U.K.

Received 12 June 1987 Accepted 12 June 1987

Key words: Cyanobacteria; Nitrogen reserve; Cyanophycin; Synechococcus sp.; Phycobiliproteins; Marine phytoplankton

1. SUMMARY

Cyanophycin is a high molecular weight co-poly- mer of aspartate and arginine, hitherto considered characteristic of all cyanobacteria. Chemical anal- ysis and radioisotopic incorporation procedures have shown that precipitable cyanophycin is ab- sent from the five species of Synechococcus tested, including the marine Synechococcus WH7803 (type DC2). The implications with respect to the role of cyanobacterial nitrogen reserves are discussed.

2. INTRODUCTION

Cyanobacteria are the only group of micro- organisms with clearly established macromolecu- lar reserves of nitrogen: the phycobiliproteins, and the co-polymer of arginine and aspartate, cyano- phycin. During N-starvation the light-harvesting phycobiliproteins are preferentially mobilised in support of the continued synthesis of essential cell

Correspondence to: J. Newman, Department of Biological Sci- ences, University of Warwick, Coventry CV4 7AL, U.K.

proteins. The polypeptide cyanophycin multi-L- arginyl-poly (L-aspartic acid) is a nitrogen storage compound [1] which is unique to cyanobacteria and has been considered to be of universal distri- bution among these organisms [2].

The recent discovery of small ( < 2.0/xm diame- ter) coccoid cyanobacteria as a significant fraction of marine phytoplankton [3,4] raised the im- portant possibility that nitrogen storage capacity might be available to a single component of this community. Although Synechococcus spp. are widely distributed in both neritic and oceanic waters [5] their relative contribution to primary productivity is greatest in the open ocean where other phytoplankton are less productive and where nitrogen is often considered the limiting factor for growth. The capacity for nitrogen storage in Syn- echococcus spp., therefore, has important implica- tions for marine nitrogen cycling and for phyto- plankton population dynamics. We have shown previously that the biliprotein phycoerythrin is a readily utilisable nitrogen store in the oceanic strain Synechococcus WH7803 [6]. However, the potential metabolism of the nitrogen-rich polymer cyanophycin (23% N) is also of importance since

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its specific reserve role in cyanobac te r i a is well recognised.

Genera l ly , cyanophyc in represents only a small f ract ion of the total cell n i t rogen content of the cyanobac te r ia l cell under condi t ions suppor t ing act ive growth. A c c u m u l a t i o n of cyanophyc in is p romoted , however, under env i ronmenta l condi - t ions unfavourab le for, or inh ib i to ry to, the incor- po ra t i on of ass imi la ted n i t rogen into p ro te in [7]. Since cyanophyc in synthesis is nonr ibosomal , the s t anda rd p rocedure for enr iching the cell conten t of this c o m p o u n d involves inhib i t ion of p ro te in synthesis with the an t ib io t ic ch lo ramphen ico l which p romotes the accumula t ion of ass imi la ted n i t rogen in the form of cyanophyc in [8].

To establ ish whether cyanophyc in was an im- p o r t a n t n i t rogen reserve in mar ine cyanobac te r i a we conduc ted chemical analyses of 3 c lonal iso- lates and, for compar i son , also ana lysed a range of o ther cyanobac te r i a avai lable to us in our labora- tory. The surpr is ing resul t was that cyanophyc in was absent f rom the 3 mar ine Synechococcus strains and also f rom 2 o ther f reshwater members of this cyanobac te r ia l genus.

3. M A T E R I A L S A N D M E T H O D S

3.1. Organisms and culture The organisms l is ted in Tab le 1 were ob ta ined

f rom the fol lowing sources: WH7803, WH8018 - W o o d s Hole Oceanograph ic Ins t i tu t ion, W o o d s Hole, M A 02543 U.S.A.; PCC 6301, PCC6308, PCC 7120, PCC 8009 - Pas teur Cul ture Collec- tion, Ins t i tu t Pasteur, Paris, France . Synechococcus sp. ' Syn ' was isola ted f rom the Menai Strai ts [9] and was the gift of Prof. G.E. Fogg. Synechococcus sp. D767 is a f reshwater isolate from Bangladesh he ld in the col lect ion of cyanobac te r i a at the Univers i ty of D u r h a m and was the gift of Dr. B.A. Whi t ton . Mar ine Synechococcus spp. were grown in an ar t i f ic ial seawater m e d i u m [6] at a t empera- ture of 2 5 ° C and at a l ight in tens i ty of 2 0 - 3 0 / , E . m - 2 - s - t . F re shwa te r s t rains were grown in a

C O 2 / a i r mixture (5%) in m e d i u m B G l l (plus 1 g . 1-1 N a H C O 3 ) or Med ium C (PCC 8009) at 3 2 ° C and 150 / ~ E . m 2 . s - 1 . Cul tures were harves ted b y low-speed cent r i fuga t ion in the mid- logar i thmic phase of growth or incuba ted for a fur ther 2 4 - 7 2 h in the presence of 4 ~ g . m1-1 ch lo ramphen ico l or in phospha te - f ree medium.

Table 1

Concentration of Sakaghuchi reacting material (as percent dry weight cyanophycin equivalents) in 6 uni-cellular and 2 filamentous cyanobacteria.

The data refer to analyses performed following acid hydrolysis (see MATERIALS AND METHODS).

Strain Medium Treatment Sakaghuchi reacting material (see Materials and Methods) (% dry weight)

Synechococcus marine phosphate starvation < 0.11 WH 7803 + chloramphenicol < 0.16

Synechococcus marine phosphate starvation < 0.09 WH 8018 + chloramphenicol < 0.13

Synechococcus sp. marine + chloramphenicol < 0.18 ' Syn'

Synechococcus freshwater none 0.04 PCC 6301 + chloramphenicol 0.08

Synechococcus sp. freshwater none 0.04 D767

Synechocystis freshwater + chloramphenicol 8.97 PCC 6308

Nostoc sp. MAC terrestrial + chloramphenicol 1.84 PCC 8009

A nabaena freshwater + chloramphenicol 1.61 PCC 7120

3.2. Isolation and estimation of cyanophycin Organisms were disrupted in a French Pressure

Cell (20 000 psi) or in a Braun MSK cell homo- genizer. Cyanophycin was purified following the procedure of Simon [8] as modified by Gupta and Carr [10]. Cyanophycin content was estimated di- rectly as available arginine residues using the Sakaguchi reaction and also following acid hy- drolysis (12 N HC1 for 12 h at 100°C) which maximised sensitivity. The identity of isolated cyanophycin was confirmed after hydrolysis by 2-dimensional paper chromatography [lst phase - butanol/acet ic acid/distilled water (180:45 : 75); 2nd phase - 80% phenol /ammonia (0.88 s.g.) (300 : 1.5)]. The identity of the amino acids arginine and aspartate was confirmed by co-chromatogra- phy with known standards.

3.3. Radioisotopes NaH]4CO3 (sp. act. 50-60 mCi /mmol) was

obtained from Amersham International. Incorpo- ration of label into cell material was estimated by scintillation counting of samples and radioactivity was determined to a counting accuracy of at least +9%.

4. RESULTS AND DISCUSSION

The cyanophycin content of 8 strains of cyanobacteria is presented in Table 1. For the strains other than Synechococcus spp. the values obtained are comparable with those reported in the literature. As observed by other workers, inhibition of protein synthesis leads to the accu- mulation of cyanophycin. In the Synechococcus spp. examined cyanophycin was absent (i.e. below the limits of chemical detection) and showed no response to treatment with chloramphenicol or phosphate starvation. Furthermore, chromatogra- phy of the hydrolysed material remaining after the isolation procedure (presumptive cyanophycin) showed no consistent amino acid pattern whereas that from e.g. Synechocystis 6308 yielded princip- ally arginine and aspartate. Therefore, cyanophy- cin appears to be absent from the Synechococcus ssp. analysed and the faint Sakaguchi reaction observed due to protein contamination. For the

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freshwater strain Synechococcus PCC6301 our chemical analysis is in accord with the electron- microscopic investigation of Lawry and Simon [11] who noted the apparent absence of cyanophycin from this strain.

Employing the standard isolation procedure of repeated phosphate buffer and Triton-X-100 washing, the absence of cyanophycin from the oceanic strain Synechococcu6" WH7803 was con- firmed by co-purification of putative cyanophycin from 14C-labelled cells of WH7803 and authentic cyanophycin from chloramphenicol-treated cells of Anabaena cylindrica. Total cyanophycin was purified from a mixture of 500 mg dry weight of each organism. After this initial purification cyanophycin was taken up in 0.1 M HC1 and the specific radio-activity of a sub-sample (measured as c.p.m.//~g total arginine) was determined. Cyanophycin was precipitated at neutral pH from the remaining material and a further round of purification was started from the first phosphate buffer stage [10]. The specific activity of the puri- fied material was again determined after this pro- cess. The entire procedure was repeated a further 3 times and the results are presented in Fig. 1. Throughout these further qualitative purification steps there was a rapid and progressive loss of

¢:

i¢ • - 700 Ob

:~ 6 5 0

En 6 0 0 o

; 150 :,7.

o :5 100 n -

o 5 0

o o

o 1 2 3 4 5

Fig. 1. The progressive loss of radioactivity from cyanophycin derived from a mixture of Anabaena cylindrica and ~4C-labelled Synechococcus WH 7803 during repeated rounds of purifica- tion.

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total rad ioac t iv i ty ( app rox ima te ly 2 x 105-fold) a c c o m p a n i e d b y a m u c h sma l l e r loss in cyanophyc in ( app rox ima te ly 8-fold). The specific rad ioac t iv i ty of the cyanophyc in remain ing in the mos t pur i f ied f rac t ion (stage 5) was low and auto- r a d i o g r a p h y of the c h r o m a t o g r a m of the stage 5 hydro lysa te showed that none of the remain ing act iv i ty was recovered as arginine or aspar ta te (de tec t ion l imit of this au to rad iog raph ic p rocedure = 100 c.p.m.). W e conclude, therefore, that 14C- labe l led cyanophyc in was absent f rom the mater ia l for analysis.

The results p resented show that Synechococcus WH7803 does not con ta in cyanophyc in and it is l ikely that this n i t rogen reserve is absent f rom all of the o ther Synechococcus spp. examined. The absence of this h i ther to cons idered character is t ic fea ture of cyanobac te r i a raises quest ions regard ing their compara t ive n i t rogen nut r i t ion and cer ta in ly provides a genus charac te r of t axonomic value.

R E F E R E N C E S

[1] Simon, R.D. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 265-267.

[2] Stanier, R.Y. and Cohen-Bazire, G. (1977) Ann. Rev. Microbiol. 31, 225-274.

[3] Waterbury, J.B., Watson, S.W., Guillard, R.R.L. and Brand, L.E. (1979) Nature 277, 293-294.

[4] Johnson, P.W. and Sieburth, J.M. (1979) Limnol. Oc- eanogr. 24, 928-935.

[5] Waterbury, J.B., Watson, S.W., Valois, F. and Franks, D.G. (1986) In: Photosynthetic Picoplankton (Platt, T. and Li, W.K.W., Eds.), pp. 71-120. Can. Bull. Fish. Aquat. Sci., No. 214.

[6] Wyman, M., Gregory, R.P.F. and Carr, N.G. (1985) Sci- ence 230, 818-820.

[7] Allen, M.M. (1984) Ann. Rev. Microbiol. 38, 1-25. [8] Simon, R.D. (1973) J. Bacteriol. 114, 1213-1216. [9] El-Hag and Fogg, G.E. (1986) Br. Phycol. J. 21, 45-54.

[10] Gupta, M. and Carr, N.G. (1981) J. Gen. Microbiol. 125, 17-23.

[11] Lawry, N. and Simon R.D.J. Phycol. 18, 391-399.

A C K N O W L E D G E M E N T

This research was suppor t ed by the Na tu r a l Env i ronmen t Research Council .