Optimization of the cell envelope disruption of extremely halophilic bacteria

Journal of Biochemical and Biophysical Methods, 14 (1987) 19-28

19

Elsevier BBM 00579

Optimization of the cell envelope disruption of extremely halophilic bacteria F.J.G. Muriana, M.C. S~nchez, J.D. Rodulfo, M.C. Alvarez-Ossorio and A.M. Relimpio Departamento de Bioqulmica, Facultad de Farmacia, Unwersidad de Seoilla, Seville, Spain (Received 27 August 1986) (Accepted 10 November 1986)

Summary This paper describes an examination of the cell envelope stability opposite to disruption by chemical and physical methods of extremely halophihc bacteria. The following methods of cell treatment were studied: solvent and chelating agents; pressure shearing at several pressures; ultrasonic disintegration for various times; ballistic disintegration; grinding with cold alumina; lysozyme digestion; osmotic shock; and freezing and thawing. The procedure is based on the determination of three cytoplasmic enzymes released by the cell treatment. Menadione reductase was also used as convenient marker enzyme for damage to the permeability barrier. Of all the methods, only pressure sheafing and ultrasonic disintegration yielded a crude extract with high halophilic enzyme activities. These procedures are suitable in designing a cell fractionation scheme for halophilic enzyme purifications. Key words: Cell envelope; Disruption methods; Halophile bacteria; Halophilic enzymes.

Introduction Cells of extreme halophiles have been shown to depend for their structural and morphological stability on sufficient concentrations of suitable ions in their environment [1]. Chemically, cell envelopes from the obligately halophilic bacteria of the genus H a i o b a c t e r i u m are characterized by the lack of the peptidoglycan layer, the high content of acidic amino acids, the high ratio of nitrogen to carbon, and the absence of diaminopimelic acid, muramic acid, and esterified fatty acids [2,3]. Despite the lack of a rigid peptidoglycan layer, these organisms are able to maintain a rod-shaped Correspondence address: Francisco J.G. Muriana, Departamento de Bioquimica, Facultad de Farmacia, Tramontana s / n , 41012 Seville, Spain. 0165-022X/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

20 morphology when grown under optimal conditions, showing resilient thick cell walls. However, when the salt concentration is lowered, the individual microorganisms suddenly lyse [4,5]. Lysis procedure has been used to obtain cell envelopes [6,7] and to release halophific enzymes [8-11]. The latter without positive results, essentially because a close connection exists between salt requirement for optimum growth and maximum enzyme activity [9,12]. In general a decrease of the salt concentration is followed by a loss of activity, either reversible or irreversible. To our knowledge, none of the breakage procedure optimizations has been reported for an extreme halophile. In an attempt to determine the choice of an appropriate method, a systematic study was made of the stability of the cell envelope from one halobacteria resistant to disruption by chemical and physical methods.

Material and Methods

Organism A new carbohydrate-utilizing extreme halophile, Halobacterium mediterranei, strain ATCC 33500, was used in all experiments [13]. This strain possesses a thick compact layer of electron-dense material which may be responsible for the greater resistance to disruption than that of other Halobacterium species.

Growth conditions The organisms were grown with humidified aeration in 2 ! batches at 37°C in 5 1 Erlenmeyer flasks. The growth medium used had been previously described by Rodriguez-Valera et al. [14].

Organism collection The bacteria were harvested after 72 h of growth by centrifugation at 16 300 × g for 45 min at 15°C. After centrifugation the medium was discarded, the packed cells were resuspended in 4.3 M NaCl-0.01 M sodium phosphate (Standard Buffer Solution, SBS), adjusted to pH 7.2, and washed once to remove nutrients adsorbed by the cell surface.

Enzymatic assays Glutamate dehydrogenase (GDH). G D H activity was assayed in 0.3 mM N A D P H , 100 mM NH4CI, 5 mM ct-cetoglutarate, 3.87 M NaCI and 10 mM sodium phosphate, pH 7.2, in a final volume of 1 ml. Malate dehydrogenase (MDH). M D H activity was assayed in 0.3 mM N A D H , 4 mM oxaioacetate, 3.87 M NaCI and 10 mM sodium phosphate, pH 7.2, in a final volume of 1 ml. Aspartate-aminotransferase (AAT). AAT activity was assayed in 200 mM Laspartate, 0.3 mM NADH, 240 units of malate dehydrogenase from pig heart, 5 mM a-cetoglutarate, 3.87 M NaCi and 10 mM sodium phosphate, pH 7.2, in a final volume of 1 ml.

21

Menadione reductase (MDR). M D R activity was assayed in 0.4 mM menadione, 2 mM KCN, 0.3 mM N A D H , 4 M NaCI and 10 mM sodium phosphate, pH 7.2, in a final volume of 1 ml. All manipulations involving this enzyme were carried out in semidarkened rooms, the enzyme being light-sensitive in dilute solution. All enzyme-activity measurements and rate studies were made at room temperature. The oxidation of NAD(P)H was followed at 340 nm with a Bausch & Lomb Spectronic 2000 spectrophotometer. Throughout this paper, one unit of activity is defined as the amount of enzyme catalysing conversion of 1 #mol of substrate per min. G D H , M D H and AAT were used to evaluate the enzymes released by various permeabilization or breakage techniques. MDR was used to monitor cell suspensions for lysis, this enzyme being accessible only from the cytoplasm. Therefore M D R can serve as a convenient marker enzyme for assessing damage to the permeability barrier [15]. Protein determination The method of Lowry was used routinely, using bovine serum albumin as standard.

Permeabilization techniques Treatment with solvent (S). The suspension of organisms diluted 10-fold with SBS was placed in a reciprocal shaker maintained at 37°C, and toluene (2, 4, 5 and 6%, v / v ) was added. After 10 and 45 min, G D H , MDH, AAT and M D R activities were assayed in samples. Treatment with chelating agent (CH). The suspension of organisms was placed in a reciprocal shaker maintained at 37°C, and EDTA (0.6 and 1.2 mM, final concentration) was added. Ten minutes later, the cells were diluted 10-fold with SBS. Samples were assayed for G D H , MDH, AAT and MDR activities. The envelope integrity of EDTA-treated cells was measured as follows. Aliquots of EDTA-treated cells, diluted with SBS as described above, were either shaken for 45 rain at 37°C with toluene (2%, v/v), or were not treated with toluene. Both samples were assayed for G D H , MDH, AAT and M D R activities. The envelope integrity is defined as enzyme activity without toluene treatment enzyme activity with toluene treatment

Breakage techniques Pressure shearing (PS).

X

I00.

The organisms were broken by transferring the suspension through a French Pressure Cell 40000 lb/inch 2 Assembly (AMINCO, Silver Spring, MD) at 4°C. The French pressure cell was experimentally operated at various pressures: 19200, 14400, 9600 and 4800 lb/inch z. MDR activity was directly assayed on broken cell suspensions, and after removing unbroken cells and cell debris by centrifugation at 167000 × g for 45 rain at

22 15°C, G D H , M D H and AAT activities were assayed on the resulting dark-red supernatant fractions. Ultrasonic disintegration (UD). Ultrasonic disintegration was carried out at 4°C using a Sonifier B-12 (Darburg, CT) ultrasonic disintegrator equipped with a microtip ( at 20 kHz and 90-100 W). The suspension of organisms was experimentally sonicated: for one period of 4 min, for one period of 8 min, for three periods of 1 rain with a 30 s pause between successive sonications, or for three periods of 3 min with a 1 min pause between successive sonications. M D R activity was directly assayed on broken cell suspensions, and then centrifuged at 167000 × g for 45 rain at 15°C. G D H , M D H and AAT activities were assayed on supernatant fluids. Ballistic disintegration (BD). The suspension of organisms was placed in a precooled beaker, and a ratio of 1:1.5 ( v / v ) of beads (Glasperlen, B. Braun Melsengen, 0.10-0.11 mm ~) to cells was added. After mixing, the cell paste was transferred to the sample container of an Edmund Btihler Shaker (Type Vi 2, Ttibingen). The container was shaken at full speed for 5 min at 4°C. M D R activity was directly assayed on the diluted paste, and G D H , M D H and AAT activities were assayed on the supernatant obtained after centrifugation at 167 000 × g for 45 min at 15°C. Solid shearing (SS). The suspension of organisms was placed in a precooled mortar, and a ratio of 1 : 2 parts by weight of precooled alumina to cell paste was added. The cells were disrupted by grinding vigorously with a pestle for 5 min at 4°C. MDR activity was directly assayed on the diluted paste. The extract was centrifuged at 167000 × g for 45 min at 15°C, and G D H , M D H and AAT activities were assayed on the supernatant. Lysozyme digestion (LD). The suspension of organisms was treated with EDTA as above (see CH) and then lysozyme (1 mg/ml, final concentration) was added. Lysis was conducted at 37°C with occasional stirring. Forty min later, the sphaeroplasts formed were gently sonicated for two periods of 30 s with a 10 s pause between successive sonications (at 20 kHz and 70-80 W). M D R activity was directly assayed, and unbroken cells and cell debris were then removed by centrifugation at 167000 x g for 45 min at 15°C. G D H , M D H and AAT activities were assayed on the supernatants. Osmotic lysis (OL). The suspension of organisms was diluted with 0.05 M sodium phosphate (Buffer Unsaline), pH 7.2, until 1 M NaCl concentration was reached (4-fold dilution). After stirring vigorously at room temperature for 30 rain, M D R activity was directly assayed, and the suspension was then centrifuged at 167000 × g for 45 min at 15°C. G D H , M D H and AAT activities were assayed in the supernatant ('salt-low supernatant'). Reactivation of enzyme activities from the salt-low state was carried out by the following procedure. Salt-low supernatants were dialyzed against 25 vol of solution 2 M NaCl-0.01 M sodium phosphate, pH 7.2, or against 25 vol of SBS; twice, each time for 10 h. Both dialyzed samples were assayed for G D H , M D H and AAT activities.

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Freezing and thawing (FT). The suspension of organisms was placed in a metal tube covered by a thick insulating material and shell frozen by inmersion in liquid nitrogen overnight. The metal tube was then thawed at r o o m temperature until the ice was slightly melted. M D R activity was directly assayed. Subsequently, the disrupted cells were centrifuged at 167000 × g for 1 h at 15°C. After centrifugation, three fractions were obtained: a reddish hard pellet; a clear, red viscous liquid (soft pellet) at the b o t t o m of the tubes; and a colourless supernatant. The soft pellet and the supernatant were collected and treated with deoxyribonuclease (1 m g / m l ) , and the suspension was stirred for 1 h at room temperature. The resulting suspension was centrifuged once again at the same speed as above. G D H , M D H and A A T activities were assayed on the supernatant.

Results

and Discussion

Permeabilization techniques By exposing H. mediterranei cells to toluene, an effective permeabilization takes place. At 10 min incubation G D H activity rose in proportion to the increase of toluene concentration. However, when the cells were incubated for 45 min, in the presence of 2, 4, 5 or 6% toluene, G D H yield decreased concomitantly with the solvent concentration (Fig. 1). Apparently, the effects of salts on halophilic enzymes ascribed to h y d r o p h o b i c interactions are decreased by denaturing agents, such as toluene [16]. O n the other hand, cells in the presence of 0.6 m M E D T A showed 0nly G D H activity. W h e n the cells were incubated in the presence of 1.2 m M E D T A , M D H activity was also present (Table 1). Thus, like eubacteria, archaebacterial micro-

TABLE 1 ACTIVITIES OF GDH, MDH, AAT AND MDR AFTER TREATMENT OF H. MEDITERRANEI CELLS BY CHELATING AGENT (CH) PERMEABILIZATION TECHNIQUE Treatment a

Activity b

CHO,6 mM d C H 12raM CHO,6 mM, 2%

GDH 3.6 9.5 24.9 30.5

C H 1 2mM'2~'

MDH 0.0 3.8 10.6 13.3

AAT 0.0 0.0 3.5 5.7

MDR c n.d. n.d. n.d. n.d.

a Procedure is designed in Material and Methods. t, mU/mg protein. c By this technique MDR was inaccessible, due to the inability of the substrates to reach the enzyme; n.d. = not detectable. a Upper indexes express EDTA (mM) and toluene (%) concentrations, respectively. Control cells did not show any enzyme activity. Each value represents the mean of triplicate determinations.

24

I0C 9C 8C 7(3

~

B

6O

~ ~c ~

4o

sg 3C

f

A

~- 2C

10 I Toluene

(*/.)

Fig. 1. Release of G D H from toluene-treated cells. Procedure is described under Material and Methods. 100% activity, obtained in 2% toluene treatment for 45rain, was 56.0 m U / m g protein. (A) 10rain incubation; (B) 45 min incubation. MDH and A A T presented similar behaviour. MDR was not possible detected. Control cells did not show any enzyme activity. Each point represents the mean of triplicate determinations. Note the semilogarithmic scale of the ordinata.

organisms are also sensitive to chelating agent. This is in accordance with the McClare model [17] which implies magnesium ions for maintaining the structure and function of the Halobacterium cell envelope. The strain in question was treated with EDTA, and exposed to toluene for 45 min with shaking. Measurements of the envelope integrity by this procedure showed the cell permeability increased to an average of 15% for G D H at 0.6 mM EDTA, and averages of 30% for G D H and M D H at 1.2 mM EDTA were detected. EDTA treatment, with toluene, increases permeability. However, these cells do not show greater permeability than with toluene alone. It therefore appears that more systematic studies are required to evaluate the contribution of EDTA and toluene to the permeability change.

Breakage techniques The most effective procedures were Pressure Shearing and Ultrasonic Disintegration. The highest activities were obtained by gradual extrusion of the cells at 19 200 lb/inch 2 pressure and by ultrasound at 90-100 W for one period of 4 min, respectively (Figs. 2 and 3). In both techniques, the enzymes released were found to

25

1oof ill// a.

i

6

I

I

French p r e s s

pressure

chomber

( × 1 0 3 PS [ }

Fig. 2. Release of halophilic enzymes from French Press-treated cells. Procedure is described under Material and Methods. 100% activities, obtained at 19200 lb/inch 2 pressure, were 72.5,316.2, 147.4 and 22.0 mU/mg protein, for MDR (O), GDH (e), MDH (zx) and AAT (,,), respectively. Each point represents the mean of quadruplicate determinations.

be coincident with cell disruption. But at low pressure or for short sonic energy treatment time, M D H is released more slowly than the mean of the other enzymes' release rate. M D H is probably found compartmented within the cell. From Fig. 2, it is also possible to deduce that G D H and AAT are found located in different cellular 'pools', one of easy disruption (at 4 800 I b / i n c h 2) and another of most difficult one (at 14400 lb/inch2). Lysozyme Digestion and Ballistic Disintegration were also suitable as breakage techniques (Table 2), but the results were somewhat less effective than Pressure Shearing and Ultrasonic Disintegration. Lysozyme specifically catalyses the hydrolysis of bonds in the mucopeptide moiety of bacterial cell walls. But this method had limited use unless the cell envelope was first weakened by addition of EDTA. Therefore, although this method is soft-treatment in breaking, it will not be available when polyvalent cation dependent enzymes are extracted. Osmotic Shock was an effective cell disruption technique, and not all cytoplasmic halophilic enzymes were irreversibly inactivated (Table 2). When the salt-low preparation was then dialyzed against 2 or 4.3 M sodium chloride, averages of 127 and 150% from the initial value of G D H activity were restored; averages of 116 and 132% from the initial value of M D H activity were also recovered; and A A T activity remained the same. All M D R activity was lost. It has been postulated that by this

26

1oo

9o

2 g g I

|

T r e a t m e n t t~me (m~n)

Fig. 3. Release of halophilic enzymes from Ultrasonic Disintegration-treated cells. Procedure is described under Material and Methods. 100% activities, obtained at sonication for one period of 4 min, were 68.9, 304.5, 109.2 and 20.2 m U / m g protein, for M D R (O), G D H (O), M D H (4) and A A T (A), respectively. Each point represents the mean of quadruplicate determinations.

TABLE 2 ACTIVITIES OF G D H , M D H , A A T A N D M D R A F T E R D I S R U P T I O N OF H. M E D I T E R R A N E I CELLS BY D I F F E R E N T B R E A K A G E T E C H N I Q U E S Treatment

BD SS L D o 6 mM " L D l'2mM~ OL OL2.O M d OL3.4 M d FT

a

Supernatant activity b

Broken cells activity b

GDH

MDH

AAT

MDR

122.1 66.5 218.7 222.5 89.6 113.9 133.9 74.7

47.4 30.4 75.4 77.4 26.1 34.7 50.3 38.6

11.8 8.8 13.2 13.5 4.7 4.1 4.8 10.1

46.8 26.8 24.6 34.4 n.d.

a Symbols and procedures are designed in Material and Methods. b m U / m g protein. c Upper indexes express E D T A (mM) concentration. a Upper indexes express molar concentration of the supernatant after dialysis. Control cells showed less than 5% of the activity of treated cells. n.d. = not detected. Each value represents the mean of triplicate determinations.

35.1

27

procedure the cell loses certain specific enzymes, essentially, those that exist just within the cell surface, either bound to the surface or between the membrane and cell wall [18]. The formation of intracellular and extracellular ice crystals by Freezing and Thawing technique did not result in further damage to the halophile cells (Table 2). Freezing-Thawing and Solid Shearing were the worst of all disruption techniques. Since the strain H. mediterranei possesses a thick compact layer, about 25-85 nm thick, which gives greater resistance to disruption [13], this microorganism is useful for the evaluation of cell envelope stability, not only for other halobacteria but also for other nonhalophilic bacteria with similar membrane properties [19-21]. In conclusion, the chemical and physical methods used can be ranked according to their disruptive force in the following sequence: PS > UD > LD > OL > BD > F T > SS> S > CH.

Simplified description of the method and its applications Various chemical and physical methods for the evaluation of the cell envelope stability of extremely halophilic bacteria arc described. For this purpose, three cytoplasmic enzymes and one enzyme accessible only from the cytoplasm were assayed to monitor cell suspensions for lysis. Pressure Shearing and Ultrasonic Disintegration, at 19200 Ib/inch 2 and for one period of 4 min (at 20 kHz and 90-100 W), respectively, were necessary to obtain a crude extract with high halophilic enzyme activities. The various methods ranked according to their disruptive force are also discussed. This study has been made due to the fact that none of the breakage procedure optimizations, which is the first stage of enzyme purification or cell envelope isolation techniques, has been reported for an extreme halophile.

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