THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 264, No. 21, Issue of July 25, pp. 12546-12553, 1989 Printed in U. S. A.

Novel Water Stress Protein from a Desiccation-tolerant Cyanobacterium PURIFICATION AND PARTIAL CHARACTERIZATION*

(Received for publication, February 14, 1989)

Siegfried SchererS and Malcolm Potts From the Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061

A desiccation-tolerant cyanobacterium Nostoc com- mune accumulates a novel group of acidic proteins when colonies are subjected to repeated cycles of drying and rehydration. The proteins occur in high concentrations; they have isoelectric points between 4.3 and 4.8 and apparent molecular masses between 30 and 39 kDa. The purification of three of these proteins with molecular masses of 33, 37, and 39 kDa is described. The amino-terminal sequence of the 39- kDa protein is Ala-Leu-Tyr-Gly-Tyr-Thr-Ile-Gly-Glu. Peptide mapping of the 39- and the 33-kDa proteins, using different proteases, gave similar patterns of digestion fragments. The amino acid compositions of the proteins isolated were similar, and each cross- reacted with a polyclonal antibody raised against the largest (39-kDa) protein. The results indicate that the microheterogeneity observed was generated by in vivo proteolysis of the 39-kDa protein. It is suggested that this protein is a water stress protein with a protective function on a structural level.

Desiccation tolerance is a widespread and long known phe- nomenon, occurring over a wide range of taxa including bac- teria, plants, and animals (Crowe and Clegg, 1978). However, as Leopold (1986) stated, “until recently, the question of how organisms can tolerate desiccation has been almost a cryptic one, potentially interesting but undeciphered.” Important progress toward the elucidation of the mechanisms of desic- cation tolerance was made by demonstrating that both phos- pholipid bilayers and proteins can be stabilized during water stress by sugars, especially by trehalose (for review, see Crowe et al., 1987). Trehalose is found in a variety of microorganisms including cyanobacteria when they are subjected to drying (matric water stress) or osmotic stress (e.g. Reed et al., 1984; McBride and Ensign, 1987).

Less is known about the possible effects of desiccation on the synthesis of novel proteins. The desiccation-tolerant moss Tortula ruralis, when rehydrated, synthesizes “rehydration proteins” (Oliver and Bewley 1984b), which apparently allow this plant to survive the rehydration process. In desiccation- intolerant mosses, rehydration rather than desiccation seems to be the fatal event (Bewley, 1979; Oliver and Bewley, 1984a). The synthesis of specific proteins in response to desiccation, however, has been reported only recently for seeds of maize

* The research reported in this study was supported by National Science Foundation Grant DCB 883068 (to M. P.).

2 Supported by Deutscher Akademischer Austauschdienst, Bonn, FRG (Sonderprogramm “Gentechnologie”) and by a travel grant from Volkswagen Stiftung allowing for field work in the People’s Republic of China. On leave from Lehrstuhl Physiologie und Biochemie der Pflanzen, Universitat Konstanz, D-7750 Konstanz, FRG.

(Gomez et al., 1988) and rice (Mundy and Chua, 1988). The function(s) of these proteins remains unknown.

The cosmopolitan terrestrial cyanobacterium Nostoc corn- mune is able to tolerate acute water stress and can survive in the air-dry state for many years. Under natural conditions, the cells are embedded and immobilized in a water-absorbing sheath composed of carbohydrates. Growth results in the formation of macroscopic colonies (0.5-3 mm thick) which cover areas of several square centimeters. Desiccation toler- ance in N. commune has been studied on the structural (Potts and Bowman, 1985; Peat and Potts, 1987), physiological (Cox- son and Kershaw, 1983; Scherer et al., 1984; Potts and Bow- man, 1985), and biochemical levels (Olie and Potts, 1986; Potts and Morrison, 1986; Scherer et al., 1986). Due to its procaryotic cell organization and capacity for the higher plant type of photosynthesis, N. commune lends itself as a suitable model system for the study of desiccation tolerance at the molecular level. As a first step in this direction, we describe the isolation and partial characterization of novel water stress protein(s) (Wsp)’ from N. commune.

EXPERIMENTAL PROCEDURES

Organisms N. commune var. commune Vauch. was collected in China in 1981

(Hunan province, Henyong district), August 1986 (Wuhan), Septem- ber 1987 (Heibei province, Yu county), and June 1988 (Konstanz, Federal Republic of Germany). Desiccated colonies were kept at 22- 25 “C in plastic bags in the dark until needed. Extended desiccation, storage, and subsequent rehydration of field material does not lead to any major discernible structural damage to either vegetative cells or heterocysts (Peat et al., 1988). The functional integrity of cells is also maintained during prolonged desiccation (Scherer et al., 1984, 1986). Field material was washed several times in distilled water and then subsequently dried and rewetted with BG11, (Rippka et al., 1979) 6-10 times at room temperature. After washing, the field material of Nostoc retained only very few bacterial contaminants at the outer surface of colonies. The interior of colonies was found to be axenic (Jager and Potts, 1988a, 1988b; Peat et al., 1988). Desiccation of colonies in air (approximately 50% relative humidity, equivalent to -95.2 megapascals) was achieved after 6-12 h, depending on the thickness of the colony. Field material of Nostoc was cultivated in the laboratory as macroscopic spherical colonies of 2-10-mm diameter as described (Scherer et al., 1988). N. commune UTEX 584 was grown in axenic culture in BGll,, at 32 “C in an airlift fermentor as described (Potts, 1985).

Methods Extraction of Soluble Protein-Dry Nostoc was frozen in liquid

nitrogen and ground under liquid nitrogen to a very fine powder. In a typical experiment, 15 g of this powder was suspended in 150 ml of

The abbreviations used are: Wsp, water stress protein(s); IEF, isoelectric focusing; SDS, sodium dodecyl sulfate; PAGE; polyacryl- amide gel electrophoresis; UTEX, University of Texas Culture Col- lection.

12546

Water Stress Protein 12547

ice-cold cracking buffer (50 mM Tris-Hcl, pH 7.8, 10 mM MgClz, 20 mM KCl, 1 mM NaN3, 1 mM phenylmethylsulfonyl fluoride, 1 mM p- mercaptoethanol) and passed immediately through a precooled French pressure cell at 130 megapascals. After the addition of 50-100 ml of cracking buffer, the brie was passed through the French press two more times. For analytical purposes, the suspension was centri- fuged for 20 min at 12,000 X g. The supernatant fraction was precip- itated by trichloroacetic acid (10% (w/v) final concentration), washed twice in 5% (w/v) trichloroacetic acid, and washed once each with 50% (v/v) acetone and ether. After evaporation of the ether, the proteins were solubilized in IEF sample buffer and analyzed by two- dimensional electrophoresis (see Fig. 1, A, B, and E and Potts, 1986). For preparatiue purposes (compare Fig. 1, C and D, as well as the following experiments), unbroken cells, cell debris, membranes, and macromolecular carbohydrate complexes originating from the sheath material were removed by ultracentrifugation (4 'C, 2 h, 70,000 rpm, Beckman Ti-70 rotor). The supernatant fraction was subjected to ammonium sulfate fractionation, the 35-60% precipitate resuspended in 20 mM Tris-HC1, pH 7.8, dialyzed exhaustively against this buffer, and centrifuged at 100,000 X g for 1 h at 4 "C.

Column Chromatography-This was performed with fast perform- ance liquid chromatography (Pharmacia LKB Biotechnology Inc.) at 22-25 "C. After separation, fractions were kept on ice. For analytical separations, a Mono Q HR 5/5 anion exchanger, a Mono S HR 5/5 cation exchanger, a Mono P 5/20 chromatofocusing column, a Phen- ylsuperose HR 5/5 hydrophobic interaction column, or a Superose 12 HR 10/30 gel exclusion chromatography column was used. Prepara- tive chromatography was performed on a Mono Q HR 10/10 anion exchanger. The urea used for gel exclusion chromatography was obtained from Sigma and was recrystallized from ethanol prior to use.

Ultrafiltration-Ultrafiltration was performed at 4 "C using an ultrafiltration cell (Amicon Corp., Danvers, MA) operated under 4-5 megapascals of nitrogen with a membrane YM-30 (exclusion limit M, = 30,000). Desalting and buffer changes were performed with PD-10 columns (Pharmacia).

Electrophoresis-Analytical SDS-PAGE (Laemmli, 1970) was per- formed in 1.5- or 0.75-mm minigels, using either a self-built device or a Mighty Small I1 SE 250 chamber (Hoefer Scientific Instruments, San Francisco). For preparative SDS-PAGE, 1.5 X 160 X 200-mm gels (12% (w/v) acrylamide) were developed in a Protean I1 cell (Bio- Rad). Nondenaturing PAGE was performed according to Bryan (1977) and Davis (1964) using a series of gels between 5 and 10% (w/ v) acrylamide, following the protocol given in the 1986 Sigma tech- nical bulletin MKR-137. Calibration was performed with Sigma MW- ND-500 molecular weight markers. Two-dimensional electrophoresis was accomplished using the procedure described by O'Farrel (1975), either using 110 X 2.5-mm IEF tube gels and 1.5 X 150 X 200-mm SDS slab gels (Potts, 1986) or 1.5 X 65-mm IEF tube gels and 1.5 X 55 X 80-mm SDS slab gels in the Mighty Small I1 chamber. Biolytes (5% (w/v), Bio-Rad) were used as described in the figure legends.

Electroelution-Bands of protein were excised from Coomassie- stained preparative SDS gels and electroeluted using the electroelu- tion chamber of the Isco model 1750 electrophoretic concentrator (Isco, Inc., Lincoln, NE). Gels used for this procedure were stained 5 min and destained 30 min. Electroelution was performed for 3-5 h in 192 mM glycine and 25 mM Tris-HC1, pH 8.3, at a constant current of 2 A. Proteins were eluted in volumes of 200 p1 and were dialyzed exhaustively against water.

Peptide Mapping-Electroeluted proteins were lyophilized, resol- ubilized in 125 mM Tris-HC1, pH 6.8, 0.5% (w/v) SDS, 10% (w/v) glycerol, 0.001% (w/v) bromphenol blue to a final concentration of 0.5-0.7 mg ml-', and then boiled for 2 min. After cooling on ice, either chymotrypsin (Sigma, type 11), papain (Sigma, type IV) or Staphylo- coccus aureus protease (Sigma, type XVII-S) was added to a concen- tration of 0.1 mg ml-', and the solutions were incubated for 5 min at 32 "C. After adding 0-mercaptoethanol and SDS to final concentra- tions of 5% (w/v) and 2% (w/v), respectively (Cleveland et al., 1977), cleavage products were resolved by SDS-PAGE, using 21% (w/v) acrylamide gels, and analyzed after staining with Coomassie Blue as well as silver stain (Oakley et al., 1980).

Carbohydrate Analysis-Purified proteins (2 pg) were dot blotted using nitrocellulose and assayed for concanavalin A-binding carbo- hydrates according to Clegg (1982). The staining of glycoproteins (separated by SDS-PAGE) with periodic acid-Schiff reagent or alcian blue was performed following the protocols of Zacharias et al. (1969) and Kunicki et al. (1981), respectively. Fluorometric carbohydrate analysis of proteins electroeluted from SDS-PAGE gels was per-

formed according to Perrini and Peters (1982). Neuraminidase treat- ment was carried out as described by Chen et al. (1985) using neura- minidase type X isolated from Clostridium perfringens (Sigma).

Amino Acid Composition-After acid hydrolysis (20 h, 110 "C, HC1 atmospher-), the amino acids were quantified using a Waters Picotech amino acid analyzer system (Millipore, Waters Chromatographic Division, Bedford, MA). Assays were run in duplicate. The amino acid composition divergence D was estimated according to Harris and Teller (1973) as

D = [{mol % ( i ~ ) - mol %(i~)]']'''

with M%(~A) and ~ % ( i ~ ) being the molar fraction of the ith amino acid in protein A or B, respectively.

Amino-terminal Sequence Analysis-The 39-kDa form of Wsp was excised from an SDS-PAGE gel, electroeluted as described above, and dot blotted to a polyvinylidene difluoride membrane (Immobilon P, Millipore) which was then washed extensively in distilled water. Edman degradation of the protein bound to the membrane (Matsu- daira, 1987) was performed in a gas-phase protein sequenator (Ap- plied Biosystems, model 470A) according to Hewick et al. (1981). The experiment was performed twice and gave identical results.

Generation of Antibodies-After electroelution from SDS-PAGE gels and extensive dialysis against distilled water, a solution of purified proteins was made 40 mM with phosphate buffer, pH 7.5, mixed 1:l with complete Freund's adjuvant, and injected into 3-4- week-old C3H-HEJ mice (approximately 10 pg of protein/mouse in 0.2 ml). After 2 weeks, a booster with the same amount of protein mixed 1:l with incomplete Freund's adjuvant was given, and the mice were bled after another 10 days. In addition, antibodies directed against the complete water stress protein fraction were raised in rabbits. A sample of 0.1 mg was dissolved in 0.5 ml of phosphate buffer, mixed with 0.5 ml of complete Freund's adjuvant, and injected subcutaneously at different sites. After 2 and 4 weeks, the immune response was stimulated by injecting 0.1 mg of protein dissolved in incomplete Freund's adjuvant and phosphate buffer. After precipita- tion of the blood clot, the serum was used without further treatment. Preimmune serum was collected before injection.

Western Blotting-After separation on SDS-PAGE, proteins were transferred (24 V, 1 A, 1 h) to nitrocellulose (Bio-Rad) using 25 mM Tris, 192 mM glycine, 20% (v/v) methanol. Immunodetection was performed according to standard techniques, using mouse antiserum diluted 1:500 and an alkaline phosphatase-conjugated sheep anti- mouse IgG antibody (Sigma) diluted 1:1,000, following the protocol described in the Promega Biotec technical manual (Madison, WI). Rabbit antiserum was diluted 1:400, and bound antibodies were visualized using the protein A-horseradish peroxidase Immunoblot kit (Bio-Rad), following the manufacturer's instructions.

RESULTS

Presence of Water Stress Proteins in Desiccated Cells-The two-dimensional protein index of field-grown N. commune collected in China is shown in Fig. L4 for a sample that has been kept in the dry state for 1 year and in Fig. 1B for a sample that had been desiccated for 5 years. A conspicuous cluster of acidic proteins with isoelectric points of 4.5-5 and molecular masses between 30 and 40 kDa, designated Wsp, was found in the desiccated field materials but not in the liquid-grown laboratory cultures (Fig. 1E). Colonies from the field usually contained much less phycobiliproteins (Pbp) compared with laboratory-grown colonies (desiccation of liq- uid-grown N. commune UTEX 584 caused phycobilisomes to dissociate from thylakoids and induced light-dependent phy- cobiliprotein degradation (Potts, 1985)). After prolonged des- iccation, Wsp appear to be more persistent than other soluble proteins (compare Fig. 1, A and B ) . Protein extracts of colo- nies collected from different geographic regions (Europe, Ant- arctica, North America, China, Aldabra Atoll) all contain the Wsp. Although present in varying relative amounts in colonies collected from these different locations, Wsp accounts always for a considerable fraction of soluble protein.

Wsp were nearly absent in laboratory-grown liquid cultures that were derived from desiccated field material (compare Fig. 1, C and D ) . Laboratory-grown colonies of field material

12548 Water Stress Protein

pH 6.2 5.4 4.5

- -

C d

D

- t,.:lr-.l I. . ....*

..’ m wsp: , ,. , ., a < 39 koa

< 33 koa W-.pk”.ll

. .

2 9

66

45

2 9

- 14

FIG. 1. Water stress proteins are present only in desiccated N. commune cells. A, soluble proteins of a sample collected at Wuhan, after 1 year of desiccation; B, soluble proteins of a sample collected at Hunan, after 5 years of desiccation; C, soluble proteins of a laboratory-grown culture derived from the Heibei sample, which had not been subjected to desiccation; D, soluble proteins of desic- cated field-grown Nostoc collected at Heibei; E, soluble proteins of N. commune UTEX 584 grown in liquid culture. A-D, Coomassie stain; E, Coomassie and silver stains. Pbp, phycobiliproteins. B, D, and E, protein constellations used for identification (Potts, 1986). For IEF, Bio-Lyte 5-7 (2%, w/v), 6-8 (2%, w/v), and 3-10 (1% w/v) were used. Note that different preparations of proteins were used for the experiments shown in A, B, and E compared with C and D (for details, see “Methods”).

subjected to one cycle of drying contain the Wsp but in lower concentrations than in desiccated field material (compare with Fig. 7).

Isolation of Water Stress Proteins-Although SDS-PAGE (Fig. 1, C and D) revealed several differences between desic- cated and nondesiccated cells, further efforts were focused on the purification of the Wsp cluster with apparent M , values of 30,000-40,000. For this purpose, field-grown colonies col- lected in the desiccated state in Heibei province, China (stored for 5 months) were used. An ultracentrifugation step after breakage of the cells was essential in order to remove high molecular weight carbohydrate complexes that otherwise re- duced column efficiency. Unfortunately, this centrifugation step also removed a considerable fraction of the water stress proteins (compare Fig. 1, A and D). After ammonium sulfate fractionation and extensive dialysis, the highly viscous extract was diluted when necessary and applied t o a Mono Q anion exchange column (Fig. 2). The flow-through contained the Wsp. Although the phycobiliprotein content of field material was not as high as that of liquid cultures, the major compo- nents of fractions C and D were the biliproteins C-phycocy- anin and R-phycoerythrin, respectively. Reliable protein de- terminations were not possible at this stage since all fractions

1.4

1.2

- I -

0.8 E 0 aJ (Y

m 0 0

+

5 0.4 g v) n U

0

A B

Urn-

I m-= * * ”_

e

*- e-

l

I

I I

~~ ~~

0 4 8 12 Elution volume (ml)

0.9

0.7

I I I I I - P)

0.5 ? 0 c 0 - E a .- v) v) m 0 c)

0.3 L - Q

2”

0.1

FIG. 2. Water stress proteins do not bind to an anion ex- change resin. A typical elution profile from a Mono Q HR 5 /5 column, operated in 20 mM Tris-HCI, pH 7.8, with a flow rate of 1 ml/min is shown. Inset, Coomassie-stained SDS-PAGE (12% (w/v) acrylamide) of fractions A (flow-through, containing Wsp; u, upper band; I , lower band) and C and D as well as molecular mass markers (B, arrows indicate 67-, 36-, 29-, 24-, 20-, and 14-kDa markers, Pharmacia). Fractions A and C-E were concentrated prior to electro- phoresis, which did not permit estimation of the relative amount of single proteins.

still contained high amounts of carbohydrate which also caused the comparatively low resolution on the Mono Q column when developed with the KC1 gradient.

The flow-through of the anion exchange column was con- centrated by ultrafiltration, which resulted in a further puri- fication of Wsp since an abundant UV-A/J3 protecting pig- ment (Scherer et al., 1988), carbohydrates, and small proteins all passed the ultrafiltration membrane. The concentrated Wsp fraction was then loaded onto a gel filtration column in the presence of 5 M urea (Fig. 3A). The bulk of Wsp eluted from the column in the first fraction at a high apparent molecular weight. SDS-PAGE revealed two major bands at M , = 33,000 (lower band, I ) , M , = 39,000 (upper band, u), and a minor band at M, = 37,000 (middle band, m). In fraction 11, the same Wsp were found, enriched with the band at M , = 33,000, and were contaminated with several low molecular mass proteins. By raising the urea concentration to 8 M, most of the Wsp were eluted in fraction IIs (Fig. 3B) but were still contaminated with low molecular weight proteins. By cali- brating the column in the presence of 8 M urea with molecular

Water Stress Protein 12549

0.2

E 0 E

co (v

c 0.1

P, 0 C m -2 0, a U

0

Elution volume (ml)

I I I 1 1 1

I

0 4 a 12 16 20 Elution volume (ml)

c

B

A L ~~

0 4 a 12 16 20 Elution volume (ml)

FIG. 3. Water stress proteins elute at high apparent molecular mass from gel exclusion chromatog- raphy. A, a typical elution profile from a Superose 12 HR 10/30 column operated in 20 mM Tris, pH 7.8, and 5 M urea a t a flow rate of 0.3 ml/min is shown. Fraction A shown in Fig. 2 was applied to the column after concentration. Inset, Coomassie-stained SDS-PAGE (15% (w/v) acrylamide) of molecular mass markers (arrows indicate 200-, 97-, 66-, 43-, and 26-kDa marker proteins) and the main Wsp fraction. u, upper band rn, middle band 1, lower band. B, gel exclusion chromatography of another Wsp preparation in the presence of 8 M urea. The apparent molecular masses of fractions 1s and 11s were determined from the calibration slope depicted in Fig. 3C, using molecular mass markers of 669,443,200, 66,45, 29,20, and 14 kDa, operating the column in the presence of 8 M urea.

weight markers (Fig. 3C), apparent molecular masses of 130- 170 kDa for fraction Is and 20-30 kDa for fraction 118 were determined. The optimum purification of Wsp, as judged from SDS-PAGE, was thus achieved by collecting fraction I (Fig. 3A) from gel exclusion chromatography in the presence of 5 M urea. This fraction was desalted and transferred into dis- tilled water by using PD-10 columns, lyophilized subse- quently, and stored desiccated at -20 "C. This preparation was used for the further characterization of Wsp.

Efforts to separate the three bands in the Wsp fraction, either using Phenylsuperose (hydrophobic interaction chro- matography) or chromatofocusing on Mono P, were not suc- cessful (data not shown). Therefore, single proteins were isolated by preparative SDS-PAGE and subsequent electroe- lution of the bands excised from gels stained with Coomassie Blue (see above).

Characterization of Water Stress Proteins-The apparent

molecular masses of Wsp were estimated by using nondena- turing PAGE. Interestingly, the Wsp hardly entered the gel prior to treatment with urea, using the fraction derived from anion exchange. After purification, however, the bulk of Wsp migrated as a single major band (Fig. 4C) with a very small satellite band. However, as revealed by subsequent SDS- PAGE, both bands were composed of similar proteins, al- though being present in different ratios. For each of the bands, the slope of the plots of relative mobility uersus acrylamide concentration (Fig. 4A), and thus the values for apparent molecular weights of proteins in the bands, were identical, yielding an apparent M , value between 30,000 and 40,000 (Fig. 4B).

As illustrated in Fig. 4C, lane b, the Wsp fraction exhibits a certain degree of microheterogeneity with respect to the apparent molecular weight. By analyzing the fraction by two- dimensional electrophoresis, this microheterogeneity became

Water Stress Protein 12550

1.4

1.2

1.0 s! X

u P

In - - - 0.0

I

0.6

m c ii a b c d

I I I I 1 I J 4.2 4.6 5.0 5.4

log (molecular weight)

FIG. 4. Apparent molecular mass of Wsp is between 30,000 and 40,000. A, the purified fraction of Wsp (compare with Fig. 3) was analyzed by a series of six nondenaturing PAGE gels with acrylamide concentrations ranging between 5.5 and 10% (w/v). The slope of the semilogarithmical plot of retention values against acryl- amide concentrations was used to determine the native molecular mass as shown in B. B, double logarithmical plot of the slope of marker proteins as determined in series of nondenaturing PAGE gels (compare with Fig. 4A) against molecular masses. The position of purified Wsp is shown together with its apparent molecular mass by the arrows. C, the band of Wsp as running in a nondenaturing gel (top of Fig. 4C) was excised and split into upper (lane a) and lower ( l a n e b) parts, which were subjected subsequently to SDS-PAGE (arrows, 15% (w/v) acrylamide, with lane d exhibiting molecular mass markers of 200, 97, 66, 43, and 26 kDa). As a control, the purified Wsp fraction is shown in lane c.

more obvious (Fig. 5). Eight to 10 spots were visible on the Coomassie-stained two-dimensional gel, not only similar in molecular weight but also in PI (Fig. 5B). Efforts to visualize the Wsp by the more sensitive method of silver staining failed since these proteins cannot be stained by this method (data not shown).

Efforts to demonstrate convincingly that carbohydrate is bound to the Wsp using either concanavalin A, periodic acid- Schiff stain, or alcian blue were unsuccessful. The fluoro- metric analysis of an electroeluted sample of the 39-kDa protein indicated very low amounts of carbohydrate with a maximum of 1 mol of glucosamine x mol of protein-'. There was no apparent change in the isoelectric point of Wsp following treatment with neuraminidase.

The largest form of the Wsp, M, = 39,000, was subjected to Edman degradation. Nine residues of the amino-terminal sequence were determined unequivocally (Table I), the partial sequence being Ala-Leu-Tyr-Gly-Tyr-Thr-Ile-Gly-Glu. In no experiment was methionine found at the amino terminus.

The two main Wsp proteins seen in the SDS-PAGE with M, = 33,000 and 39,000 (compare Fig. 3A) were isolated by

IEF, pH 8-4 -

A

B

SDS

I - 97

- 68

-43

-26

I

FIG. 5. Acidic Wsp occur in microheterogeneic forms. The purified Wsp have been subjected to IEF (pH 8-4) in the first and SDS-PAGE (12% (w/v) acrylamide) in the second dimension. Panel A shows the entire two-dimensional gel, whereas panel B enlarges the region in which Wsp have been focused to demonstrate the micro- heterogeneity. The arrows indicate positions where spots were visible after Coomassie staining.

TABLE I Amino-terminal sequence of the 39-kDa Wsp

Cycle Amino acid Yield pmol

1 Alanine 115 2 Leucine 94 3 Tyrosine 107 4 Glycine 110 5 Tyrosine 106 6 Threonine 32 7 Isoleucine 104 8 Glycine 109 9 Glutamic acid 111

electroelution and subjected to proteolytic digestion. The po- sition of the isolated upper (u) and lower ( I ) band is indicated in Fig. 6. Papain, S. aureus protease, and chymotrypsin yielded strikingly similar patterns of fragments, although the patterns were not completely identical. Peptide mapping of the middle (m) band using S. aureus protease produced a very similar pattern to that found for the upper band. As was mentioned above, nondigested Wsp could not be stained by silver staining, but some of the proteolytic fragments could. Generally, as indicated by the black circles in Fig. 6, the smaller the size of a fragment, the greater was the capacity to be silver stained.

The amino acid compositions of the Wsp bands electroe- luted from SDS-PAGE gels are presented in Table 11. The overall patterns of amino acid composition are very similar, although not identical. In order to quantify the relatedness, the composition divergence was calculated. The values ob- tained were 1.8 between the upper and middle, 5.7 between the upper and lower, and 6.1 between the middle and lower bands (compare Fig. 3A, inset). For comparison, the compo- sition divergence between the upper band of the Wsp fraction and the y-subunit of the DNA-dependent RNA polymerase from N. commune UTEX 584 (Xie et al., 1989) is 18.8. The close relationship between the three Wsp fractions was fur-

Water Stress Protein 12551

FIG. 6. Water stress proteins with different molecular masses yield similar fragments after limited proteolytic deg- radation. The position of single Wsp isolated by electroelution is shown by arrows (left side of panel; u and 1 stand for upper and lower bands, respectively; compare with Fig. 2, inset, lane A ) . For compar- ison, the sample prior to electroelution is shown in lane 3. Filled circles (right side of the panel) identify bands that are stained by silver staining (three circles, heavily stained; one circle, weakly stained). Lane 6 shows molecular mass markers of 43, 26, 18, and 14 kDa. Staphyl., S. aureus protease; Chymo., chymotrypsin.

TABLE I1 Amino acid compositions of different Wsp are similar

Figures are based on amino acids asparagine/aspartic acid through phenylalanine. Tyrosine and lysine are present only in low concen- trations and could not be quantified reliably with the small amounts of protein available. Isoleucine is present but could not be quantified since its signal was partly superimposed by a signal probably origi- nating from carbohydrate. Methionine is present but partly destroyed by hydrolysis. Tryptophan and cysteine are partly destroyed by hydrolysis and were not detectable (ND).

Isoform

39 kDa 31 kDa 33 kDa (upper band) (middle band) (lower band)

mol % Asx 12.1 11.9 11.5 Glx 8.0 8.4 6.2 Ser 9.4 9.0 7.0 GlY 13.2 13.8 15.8 His 0 0 0 Arg 8.1 8.7 5.5 Thr 11.2 10.3 10.9 Ala 10.0 10.2 9.1 Pro 6.0 6.1 6.0 Val 4.8 5.9 6.2 Leu 21.9 22.8 19.2 Phe 2.9 3.1 3.0 TYr + + + LYS + + + Ile + + + Met + + + Trp ND ND ND CYS ND ND ND

ther supported by immunological evidence. A polyclonal an- tibody raised against the upper band cross-reacted strongly with both the middle and lower bands after blotting proteins from SDS-PAGE gels to nitrocellulose and probing the pri- mary antibody with phosphatase-conjugated sheep anti-

A B C

FIG. 7. Isoforms of Wsp cross-react immunologically and are subjected to proteolysis in vivo. A, 60 ng of the Wsp were separated by 15% (w/v) SDS-PAGE and transferred to nitrocellulose, which was probed with an antibody raised in a mouse against the upper band. R, dry powder of N. commune collected in Wuhan was extracted rapidly (6 mg in 0.1 ml) without previous rewetting in 8% (w/v) SDS, 5% (w/v) fi-mercaptoethanol, 0.5 mM phenylmethylsul- fonyl fluoride, 50 mM Tris-HCI, pH 6.8, and 0.001% bromphenol blue by boiling and freezing the sample five times. After separation on a 15% (w/v) polyacrylamide-SDS gel and transfer to nitrocellulose, proteins were detected using antibodies raised in rabbits and directed against the complete Wsp fraction. C, a laboratory-grown colony derived from field material collected in Heibei province was dried once and treated as described for lane R. Experiments with preim- mune serum gave no color reactions.

mouse antibodies (Fig. 7, lane A). A population of antibodies directed against the mixture of Wsp (all three bands) was used to probe the Wsp in a field-grown colony (Fig. 7, lane B). A variety of bands was detected, but the apparent M , values of all of them were below 40,000. In contrast, when laboratory-grown colonies derived from field material were dried once and probed with the antibodies, only one band with an M, value of 39,000 was found. Occasionally a fainter second band with an M , of 24,000 was detected. Protein extracts from cells grown in liquid culture in a fermentor gave no signals with the antibody.

DISCUSSION

The Proteins Isolated Are Water Stress Proteins-Although the function(s) of the Wsp remains unknown, their involve- ment in the water stress response of cells is supported by the following findings. (i) Wsp are present in cells subjected to drying and desiccation but not in cells grown without water stress (Fig. 1). Laboratory-grown colonies, when subjected to drying, synthesized the 39,000-kDa band of Wsp. (ii) The Wsp protein fraction withstands prolonged desiccation more than other soluble proteins (compare Fig. 1, A and B ) despite the extensive proteolysis that occurs after prolonged desicca- tion of N. commune UTEX 584 (Potts 1985, 1986). In this respect, it might be significant that the Wsp show only low levels of tyrosine, methionine, and lysine (Table 11), amino acids that are sensitive to oxidative damage or amino group modification. (iii) Wsp form a substantial fraction of the cellular proteins of Nostoc, often more than the phycobilipro- teins of photosystem 11’ (Fig. 1).

Since the Wsp were isolated from desiccated field material with an essentially unknown history, it is worth considering the possibility that the proteins studied in this work might have been partly damaged by (photo)oxidation or nonspecific proteolysis during desiccation and storage. This, however, seems to be unlikely for the following reasons. (i) Colonies

* S. Scherer and M. Potts, unpublished data.

12552 Water Stress Protein

grown in the laboratory and desiccated once contained the protein with the same maximum molecular mass (39 kDa) as found in the field material (Fig. 7). (ii) The pattern of Wsp on Western blots (see Fig. 7) was found to be very similar for materials collected over a period of 1 4 years in China, Europe, North America, Aldabra Atoll (Indian Ocean), and Antarctica (data not shown).

Molecular Weight of Wsp-With an isoelectric point of approximately 4.5, the Wsp should bind to an anion exchange column at pH 7.8; but as is shown in Fig. 2, this is not the case, indicating that the charged groups are shielded. The Wsp elute from gel filtration columns at urea concentrations below 5 M with apparent molecular masses of 130-170 kDa (Fig. 3). Interestingly, the elution volume changed consider- ably when comparing different preparations, which prohibited the elucidation of a certain molecular weight. Furthermore, Wsp hardly entered native PAGE gels unless treated with urea concentrations higher than 4 M. On the other hand, SDS-PAGE as well as native PAGE analysis (Fig. 4) of purified Wsp indicated approximate apparent molecular masses between 30 and 40 kDa. High urea concentrations led to an elution of Wsp from gel filtration columns with approx- imate molecular masses of 20-30 kDa, apparently splitting the Wsp fraction into smaller products. From these data, we conclude that Wsp occur i n vivo, as high molecular weight complexes composed of microheterogeneous smaller units. A certain stoichiometry, however, cannot yet be established.

Microheterogeneity of Wsp-The occurrence of Wsp in mi- croheterogeneous forms is evident from the different molec- ular masses (Fig. 4C) and isoelectric points (Figs. 1 and 5). The different forms appear to be very similar as judged from peptide mapping (Fig. 6) and cross-reaction of monospecific antibodies (Fig. 7A). The composition divergence values as derived from the very similar amino acid compositions (Table 11) demonstrate the close relatedness of the Wsp (compare Harris and Teller (1973) with Cornish-Bowden (1983)) but also clearly show that they are different.

Glycoproteins are known to occur in different isoforms (e.g. Butler and Bond, 1988; Ziltner et al., 1988). A major carbo- hydrate content of Wsp, however, can be excluded by the data reported. If the different forms had been generated by differ- ential glycosylation, the amino acid composition should be identical, which is clearly not the case. Also, contrary to the data obtained (Fig. 6), peptide mapping should yield identical patterns, except if carbohydrate is bound to Wsp at the cleavage sites of all of the proteases tested, which seems rather unlikely. Since neuraminidase treatment did not change the isoelectric point of Wsp, it is assumed that sialic acid is not present.

Alternatively, the microheterogeneity can be explained by either a proteolytic digestion of Wsp by cellular proteases i n vivo, by proteolysis during preparation, or may reflect highly homologous genes encoding different Wsp forms. Multigene families have been reported occasionally for procaryotes (e.g. Golden et al., 1986; White et al., 1988); and in N. commune UTEX 584, multiple nifH sequences are present (Defrancesco and Potts, 1988). However, Western blots of total cellular extracts of heterogenous field material revealed a variety of proteins with molecular masses below, but not higher than, 39 kDa reacting with the antibodies against Wsp. In contrast, in water-stressed laboratory cultures, a Wsp at 39 kDa, but not at apparent molecular masses of 37 and 33 kDa, was present (Fig. 7). Since the microheterogeneity in molecular weight was also found in field material boiled without previous rewetting in buffer containing high concentrations of SDS, mercaptoethanol, and phenylmethylsulfonyl fluoride (Fig.

7B), proteolysis during preparation appears to be unlikely. We suggest, therefore, that the microheterogeneity of Wsp most probably is due to in vivo proteolytic cleavage. The largest fragments, which are still similar in size and ionic properties, copurified during the preparation procedure.

According to the amino-terminal sequence of the 39-kDa form, an amino-terminal methionine is missing. It has been reported that the distribution of amino termini of proteins from microorganisms is highly nonrandom. The presence of, among others, alanine in the second position (Table I) does allow the post-translational removal of methionine (Tsuna- sawa et al., 1985). Furthermore, highly expressed genes most frequently contain GCU (alanine) in the second position (Gold and Stormo, 1987). Since a Wsp with a molecular mass higher than 39 kDa was not detected, we assume that methi- onine is removed by proteolytic cleavage during maturation of the Wsp.

Protective Function of Wsp?-Liquid-grown cells of N. com- mune UTEX 584 maintain the protein synthesis machinery intact after subjection to drying (Angeloni and Potts, 1986); but under the conditions of rapid desiccation applied in these studies, the mRNA pool did not change significantly (Jager and Potts, 1988a, 1988b), and no novel proteins accumulated as shown by 35S pulse labeling (Potts, 1986). It has been reported frequently that rapid drying does not allow sufficient time for the expression of desiccation tolerance (Bewley, 1979; Leopold, 1986; Dhindsa, 1987). The presence of high amounts of Wsp in field material after numerous slow desiccation events and the smaller amounts produced after one desicca- tion event in laboratory-grown cells may suggest that Wsp accumulate during the growth of a colony in order to protect the cellular structure. Interestingly, fast dried N. commune UTEX 584 filaments do not preserve structurally intact het- erocysts (Peat and Potts, 1987), in contrast to field material that does and that contains the Wsp in high amounts (Peat et al., 1988).

The Wsp isolated from N. commune differ markedly from the proteins synthesized in seeds of wheat, which contain 69.4% glycine and which are formed during preparation of the embryo for dormancy (Gomez et al., 1988). Similarities of N. commune Wsp to the proteins synthesized in rice embryos are evident only in that the latter are found as microheterogenous forms in both molecular weight and isoelectric point. How- ever, the rice proteins are basic and, as found for the wheat proteins, are rich in glycine (Mundy and Chua, 1988). It was noted that the proteins found in dry rice embryos disappeared quickly after germination and exhibited some similarity to DNA-binding proteins such as histones (Mundy and Chua, 1988). In contrast, it has been suggested that the “E,-protein” isolated from wheat embryos is a water-binding protein, main- taining a certain level of hydration in the cells (McCubbin et al., 1985). The high amounts of Wsp in N. commune (up to 20% of cellular protein) may suggest a structural function. Since cyanobacteria are readily accessible to genetic manip- ulation, the isolation of Wsp from N. commune provides a promising tool for the investigation of both the action of water on gene expression and the possible protective nature of Wsp in photosynthetic cells.

Acknowledgments-We are indebted to Dr. J. S. Bond for critical comments on the manuscript and Dr. G. Schurig for help in raising antibodies in mice. Dr. T. W. Chen and Dr. Z. P. Zhong assisted in the collection of N. commune in China. The amino-terminal sequence was determined in the laboratory of Dr. R. Harris; amino acid analysis was performed in the laboratory of Dr. K. E. Webb, and Dr. F. Perrini performed the fluorometric carbohydrate analysis.

Water Stress Protein 12553

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