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Urease of blue-green algae (Cyanobacteria)Anabaena doliolum andAnacystis nidulans

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Page 1: Urease of blue-green algae (Cyanobacteria)Anabaena doliolum andAnacystis nidulans

CURRENT MICROBIOLOGY Vol. 16 (1987), pp. 113-117 Current Microbiology �9 Springer-Verlag New York Inc. 1987

Urease of Blue-green Algae (Cyanobacteria) Anabaena doliolum and Anacystis nidulans

Ashwani Kumar Rai and Surendra Singh

Department of Botany, Banaras Hindu University, Varanasi, India

Abstract. Urease from Anabaena doliolum and Anacystis nidulans showed maximum activity at pH 7.0-7.4 at 40~ when measured in cell-free, phosphate-buffered extracts. It is a soluble enzyme located in cytoplasm. The apparent Km for A. doliolum urease was 120 i~M. Anacystis nidulans urease exhibited biphasic kinetics (Km = 250 /~M and 1.66 mM). Enzyme, fully expressed in cells grown with urea, nitrate, or N2, was repressed in ammonia-grown cells, but ammonia did not inhibit the activity in vitro. Incubation of algal cells in N2 medium with chloramphenicol for 12 h caused degradation of urease. Cu 2+ at 1 /xM inhibited the enzyme activity by 50%, whereas Co 2+ and Ni 2+ up to 20/~M had no effect, p-Hydroxymercuribenzoate appeared to be a more powerful inhibitor of urease than acetohydroxamic acid.

The degradation of urea in microorganisms is af- fected by either of the enzymes, urease (urea ami- dohydrolase [EC 3.5.1.5]) or ATP-urea amidolyase (UALase) [16]. The latter enzymatic process, found in yeast and green algae, involves two distinct enzy- matic activities: urea carboxylase (urea: CO2 ligase [adenosine 5-diphosphate-forming], EC 6.3.4.6) and allophanate hydrolase (allophanate amidohydro- lase, EC 3.5.1.13). Blue-green algal species studied so far contain urease [3, 5, 16], as reported in higher plants, bacteria, and fungi. However, from the spores of Geotrichium candidum UALase has been reported [18]. Urease from a bacterial system is a soluble enzyme located in the cytoplasm [8]. In some bacteria, it is constitutive, but in others it is inducible [10, 23].

Nickel is an essential component of the enzyme urease [6], and some organisms effectively substi- tute Ni 2+ by a metal analogue C o 2+ [9]. At the same time, urease is highly sensitive to a variety of metals and has been used in the immobilized state as a successful indicator for the detection of metal-con- taminated environment [14]. The chelating agent, acetohydroxamic acid [7], and the sulfhydryl agent, p-hydroxymercuribenzoate [2], inhibit the enzyme. However, these agents have not been tried on urease from blue-green algae except for a single re- port [5]. Since nothing is known about the urease of blue-green algae, constitutive or inducible, its Ioca-

Address reprint requests to: c/o Prof. Robert Tabita, Department sity of Texas at Austin, Austin, TX 78712, USA.

tion, and whether it is a soluble enzyme, the present paper describes the localization, kinetics, and regu- lation of urease in blue-green algae, A. doliolum and A. nidulans.

Materials and Methods

Organisms and culture conditions. Anabaena doliolum Bharadwaja (local isolate in pure and axenic population) and Anacystis nidulans IU 625 (ATCC 27144) were grown in Chu No. 10 medium [17]. Combined nitrogen was supplemented in the medium as NH4CI, 0.5 mM; Ca(NO3h, 0.25 mM; and urea, 1 mM forA. doliolum; and as NH4CI, 0.5 mM; Ca(NO3)2, 1 mM, and urea, 5 mM for A. nidulans, since these were the optimum con- centrations of nitrogen for the respective algae. The cultures were incubated in a culture room at 24 ~ -+ 1~ and illuminated with daylight fluorescent tubes (intensity approximately 2500 lux on the surface of the vessels) for 14 h/day.

Preparation of cell-free extracts and spheroplasts. Exponentially growing algal cultures collected by centrifugation were washed repeatedly with sterile, distilled water and finally with 5 mM phosphate buffer, pH 7.3. The cells were ground in a precbilled glass mortar and pestle at 4~ in an icebath with an equal volume of acid-washed sand. Extraction of crude enzyme was done with 5 mM phosphate buffer, pH 7.3. The supernatant collected after centrifugation was used for estimation of urease activity. For preparation of spheroplasts, algal cells were treated with eth- ylenediaminetetraacetic acid-lysozyme [13] with the modifica- tion that a 20% glycerol solution was substituted for the sucrose solution to protect the spheroplasts [8].

Urease assay. Urease was estimated by determining the amount of urea decomposed within a given period of time at 37~ pH

of Microbiology, Experimental Science Building #319, The Univer-

Page 2: Urease of blue-green algae (Cyanobacteria)Anabaena doliolum andAnacystis nidulans

114 CURRENT MICROBIOLOGY Vol. 16 (1987)

Table 1. Urease activity in extracts of Anabaena doliolum and Anacystis nidulans

nmol urea hydrolyzed mg -~ protein h -~

Organisms No addition Additions

A. doliolum A. nidulans

Cell-free extract +ATP +Avidin

+ urea + buffer

498 495 498 453 468 455

+Avidin + Biotin

495 453

+ Hydroxyurea

15 16

Table 2. Effect of EDTA-lysozyme treatment on release of urease from Anabaena doliolum

Fraction

Urease activity Enzyme activity (nmol urea compared with

hydrolyzed mg -1 whole cell extract protein h 1) (%)

Whole-cell extract 652 100.00 Spheroplast lysate 591 90.64 Supernatant (glycerol- 15 2.30

Tris buffer after sedimentation of spheroplasts)

7.3, unless otherwise mentioned. The reaction mixture contained 2 ~mol of urea in a final volume of 2 ml of 5 mM pbosphate buffer (pH 7.3). The reaction was initiated by the addition of cell-free enzyme extract and terminated by the addition of 4 ml of mixed reagent used for the colorimetric assay of urea (Sigma Technical Bulletin No. 535 [19]). To distinguish between UALase and urease, the following compounds were added to the reaction mixture as required: 10/zmol ATP, 200/xg avidin, 200/xg biotin, and 20/xmol hydroxyurea.

Analytical methods. PrOtein was estimated by the method of Lowry et al. [11], with bovine serum albumin as the standard. Urea was determined by the diacetylmonoxime method [19].

Results and Discussion

To determine which of the urea-degrading enzymes, UALase or urease, is present in A. doliolum and A. nidulans, use was made of Roon and Levenberg criteria [16]: (a) UALase activity requires addition of ATP in the crude extract and is not affected by hydroxyurea; (b) avidin inhibits UALase, which is reversed by prior addition of biotin; and (c) urease activity is unaffected by the addition of ATP and avidin, and strongly inhibited by hydroxyurea. Ad-

500 p ~

2 40o 2

E �9 3 0 0

0

E C

2 0 0 ~> I . - o <t

w 1 0 0 u) <~ LU n,

I l x I

0 4 20 /~0 60

TEMPERATURE ( "C) L / / 1 I t

0 6.0 7.0 8.0 9 .0

p H

Fig. 1. Effect of temperature (O, 0) and pH (A, A) on urease activity of Anabaena doliolum (0, A) and Anacystis nidulans (@, A). Buffer used was 5 mM phosphate buffer. In the case of temperature effect, the reaction was performed at pH 7.3.

dition of ATP and biotin to the extracts of A. dolio- lum and A. nidulans produced no stimulation, nor did avidin inhibit urea hydrolysis. Hydroxyurea al- most abolished the hydrolysis of urea (Table 1). These results indicate the presence of urease as urea-hydrolyzing enzyme in A. doliolum and A. ni- dulans, as reported in blue-green algal species Phormidium luridum, Plectonema calothricoides

Page 3: Urease of blue-green algae (Cyanobacteria)Anabaena doliolum andAnacystis nidulans

A.K. Rai and S. Singh: Urease of Anabaena doliolum and Anacystis nidulans 115

300

u= 200 t-

.g

L.. e~

T E

-6 E

lOO

/ /

/ /

I I I 0 0.5 1.0 1.5 2.0

S (mM)

>

Fig. 2. Rate of urease activity (V) from Anacystis nidulans versus urea concentra- tion (S). V (�9 S/V (O--O).

[3], Nostoc muscorum [16], Aphanocapsa 6308 [21], and Spirulina maxima [5].

The cellular localization of urease in blue-green algae has not been worked out so far. Table 2 shows that urease o fA. doliolum is present in spheroplasts as a soluble enzyme, which could be released from glycerol-protected spheroplasts after osmotic shock and lysis. The enzyme activity increased drastically with increase in the temperature and reached its maximum at 40~ A further increase in the temper- ature to 60~ decreased the activity by 57% to that at 40~ The optimum pH of 7.0-7.4 for enzyme activity in phosphate buffer (Fig. 1) was in accor- dance with the observations made for urease from jack bean [20] and Brevibacterium ammoniagenes [12].

Urease from A. doliolum followed Michaelis- Menten kinetics with a Km of 120/zM, a value in accordance with those obtained for urease from the blue-green alga Spirulina maxima [5]. On the other hand, data for concentrat ion-dependent urease ac- tivity of A. nidulans showed two phases (Fig. 2),

one yielding an approximate Km value of 250/zM, and the other of 1.66 mM. The kinetics indicates that the urea-hydrolysis mechanism changes its characteristics at approximately 1 mM external concentrat ion of urea, showing a discontinuous re- lationship between urea concentrat ion and rate of urease. This discontinuity may be physiologically important for enabling A. nidulans to avoid high intracellular ammonia generation from urea hydro- lysis when the concentrat ion of urea is high in the medium, and also to grow on a lower concentrat ion of urea without much effect. Anabaena doliolum, however , cannot do so; the optimum level of urea (5 mM) for growth of A. nidulans caused lysis and death of A. doliolum.

Reports available on the nature of urease, whether constitutive or inducible, are contradic- tory. The enzyme activities in A. doliolum and A. nidulans cells grown with urea, nitrate, and dinitro- gen were almost identical (Table 3). This indicates that exogenous urea is not required as inducer, and A. doliolum and A. nidulans form urease constitu-

Page 4: Urease of blue-green algae (Cyanobacteria)Anabaena doliolum andAnacystis nidulans

tively. The presence of ammonia in the growth me- dium lowered the level of urease activity to 36.5% and 35.8% for A. doliolum and A. nidulans, respec- tively, equal to that of urea-grown cells. However, ammonia (up to 10 mM), when added to the cell- free enzyme extract (in vitro), did not inhibit the urease activity; this suggests that ammonia does not inactivate the enzyme but represses its biosyn- thesis.

Urease activity was fully expressed in urea- grown cells and cells deprived of nitrogen for 12 h (cells incubated in N2 medium); this indicated that nitrogen deprivation (for 12 h) had no effect on urease activity. Exposure of cells to chlorampheni- col (5 /zg/ml) for 12 h from the very beginning of incubation in N2 medium caused a significant de- crease in urease activity (50%) to that of cells ex- posed to chloramphenicol at the termination of the

Table 3. Urease activity in cell-free extracts of Anabaena doliolum and Anacystis nidulans cultures grown on different nitrogen sources

Nitrogen source present in the growth medium A. doliolum A. nidulans

Urease level (nmol urea hydrolyzed mg 1 protein h 1) I

30 60 90 120

T IME ( rain )

Nz 613.4 (97.4)" - - Urea 630.0 (100) 598.0 (100) a KNO3 596.8 (94.6) 568.4 (95.0) NH4C1 230.0 (36.5) 214.0 (35.8)

a Values in parentheses indicate the percent rate of urease activity.

incubation period (Fig. 3); this suggests a degrada- tion of urease. N-deprivation is reported to induce breakdown of phycobilisomes [22], cyanophycin granule polypeptides [1], and proteins [4], which in turn may make endogenous urea available through

500

400

~ 300 r

N 200

U.l

,~ 100

116 CURRENT MICROBIOLOGY Vol . 16 (1987)

Fig. 3. Effect of chloramphenicol on urease activity. Algal cells were incubated in N2 medium for 12 h. Chloramphenicol (5 Izg/ ml) was added to the algal suspensions either at the beginning of incubation (D- -D, I1--11) or at the termination of the incubation period (A--zX, A--A). Open symbols apply to Anabaena dolio- lum, and closed symbols apply to Anacystis nidulans. Control (without chloramphenicol) O - - O , I - - t .

Table 4. Effect of Cu 2+, C o 2+, and Ni z+ on the activity of urease from Anabaena doliolum and Anacystis nidulans

Per cent urease activity

Cu2+ Co 2+ Ni 2+ Concentration

(tzM) A. doliolum A. nidulans A. doliolum A. nidulans A. doliolum A. nidulans

0.0 100.0 100.0 100.0 100.0 100.0 100.0 0.05 100.0 100.0 ND ~ ND ND ND 1.0 50.0 51.6 ND ND 101.6 101.7 5.0 12.5 5.5 ND ND 101.6 100.0

10.0 0.0 0.0 103.2 100.0 101.0 101.7 20.0 0.0 0.0 100.0 100.0 100.0 100.0 50.0 ND ND 63.1 100.0 73.3 93.2

100.0 ND ND 13.1 75.0 ND ND 1000.0 ND ND 11.3 10.4 ND ND 5000.0 ND ND 5.3 0.0 ND ND

a ND, not detected.

Page 5: Urease of blue-green algae (Cyanobacteria)Anabaena doliolum andAnacystis nidulans

A.K. Rai and S. Singh: Urease of Anabaena doliolum and Anacystis nidulans 117

the catabolic pathway of arginine [21]. In cells of A. doliolum and A. nidulans incubated in N2 medium with chloramphenicol, protein synthesis, and the enzymes involved in the phycobilisomes, cyano- phycin granules and protein-degrading systems were halted. Thus, under the present set of condi- tions, urea was available neither endogenously nor exogenously and might be presumed to account for the rapid degradation of urease. A significant de- crease in ammonia release in the presence of chlor- amphenicol added at the same time as methionine sulfoximine [4] favors this assumption.

Urease from A. doliolum and A. nidulans was inhibited by 50% at 1/xM Cu 2+ and completely at 10 /xM Cu 2§ (Table 4). A comparable concentration- dependent inhibition has been obtained with jack bean urease [15]. In the present experiment, urease inactivation was apparent beyond 50 /xM Co 2§ whereas 700/xM Co 2+ was ineffective on jack bean urease [15]. Ni 2+ at a 20/xM level was at the thresh- old of "effect-no-effect" (Table 4). Urease from A. doliolum was, however, relatively more sensi- tive to the metals Cu 2§ and Co 2+ than was that ofA. nidulans urease. Like urease from the blue-green alga Spirulina maxima [5], the enzyme from A. doli- olum and A. nidulans was also inhibited by acetohy- droxamic acid and p-hydroxymercuribenzoate; however, the latter was twofold more effective than the former; for 50% inhibition of urease, acetohy- droxamic acid was required at 3/xM concentration, whereas p-hydroxymercuribenzoate did the job at only 1.5/xM.

ACKNOWLEDGMENTS

This study was supported by grants from the Council of Scien- tific and Industrial Research, New Delhi.

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