Cyanide Oxygenase and Cyanase Activities of Pseudomonas

FEMS Microbiology Letters 60 (1989) 289-294 289 Published by Elsevier

FEM 03645

Cyanide oxygenase and cyanase activities of Pseudomonas fluorescens NCIMB 11764 Downloaded from https://academic.oup.com/femsle/article/60/3/289/452954 by guest on 30 September 2021

Patrick K. Dorr and Christopher J. Knowles *

Biological Laboratoo'. Universi(v of Kent. Canterbury, U.K.

Received 21 March 1989 Accepted 3 April 1989

Key words: Pseudomonas fluorescens; Nickel cyanide; (Nitrogen source)

1. SUMMARY limited continuous culture as well as in batch culture with Ni(CN)]- as the nitrogen source Pseudomonas fluorescens NCIMB 11764 is able [1,2]. Under all these growth conditions, cyanide to utilise cyanide (both KCN and Ni(CN) 2-) as a oxygenase activity is induced. An intracellular, nitrogen source for growth. Under such conditions multicomponent, soluble enzyme system is re- cyanide oxygenase activity is induced. When sponsible for cyanide oxygenase activity [3]. The potassium cyanate (KOCN) is supplied as the sole overall stoichiometry of the process is consistent nitrogen source for growth, cyanase activity is with the enzyme system being a dioxygenase: induced. It has been demonstrated that these two enzymic activities are physiologically distinct, and HCN + 0 2 ~ ~t~, CO 2 + NH 3 (1) are not co-induced under any of the growth condi- NAD' .- H+NAD ' tions tested. In a search for possible intermediates of this pathway a number of chemicals were tested. Low 2. INTRODUCTION amounts of cyanase activity were found in both ammonia and cyanide limited fed-batch cultures. Pseudomonas fiuorescens NCIMB 11764 was This suggested that cyanide oxygenase could be a isolated by a cyanide vapour plate technique, de- monoxygenase: signed to select for organisms capable of utilising HCN + 0 2 ~m°n°xygenase--....,,)HOCN + H~O. (2) cyanide (KCN = HCN) as the sole source of nitrogen for growth [1]. Growth of this organism NAD-4 H" NAD" has been demonstrated in fed-batch culture with HOCN + H20 cyanasc~co2 + NH 3 (3) KCN as the growth limiting nutrient and in N- It is reported here that induction of cyanide oxygenase by KCN or Ni(CN),~- does not induce cyanase activity. Similarly induction of cyanase by Correspondence to." C.J. Knowles, Biological Laboratory, Uni- cyanate does not induce cyanide oxygenase activ- versity of Kent, Canterbury, Kent CT2 7N J, U.K. ity. 0378-1097/89/$03.50 ~ 1989 Federation of European Microbiological Societies 290

3. MATERIALS AND METHODS 3.8. Assay of cyanate Cyanate in the cell-free culture medium was 3.1. Growth conditions determined using a modification of the spectro- lnocula for all growth experiments were pre- photometric assay described by Guilloton and pared as previously described [1], except 2 mM Karst [8]. In their paper, they reported that al- NHaCI was used as the nitrogen source. kaline or strongly buffered solutions interfered with the assay. By lowering the pH of the culture 3.2. Growth of P. fluorescens on different nitrogen medium to 6.4, no interference of the cyanate Downloaded from https://academic.oup.com/femsle/article/60/3/289/452954 by guest on 30 September 2021 sources in batch culture assay was observed. Ammonia formation due to All cultures were grown in 250-ml conical flasks acid hydrolysis of cyanate was not observed at this containing 100 ml medium with 10 mM glucose as pH. carbon source, M9 salts [4], 1 ml trace metals 1 3.9. Assay of Nickel cyanide [5], and either 2 mM NH4C1, 2 mM KOCN or 0.5 Ni(CN) 2- in the cell-free culture medium was mM Ni(CN)4z- as the nitrogen source. All flasks assayed by the spectrophotometric assay as de- were incubated at 30°C in a gyratory shaker at scribed by Rollinson et al. [2]. 250 rev/min.

3. 3. Fed-batch culture growth conditions 4. RESULTS Cyanide (KCN --- HCN) limited fed batch cultures were set up as previously described [1]. 4.1. Growth of P. fluorescens in batch culture As reported previously [1-3], P. fluorescens grew in batch culture on 10 mM glucose with 2 3. 4. Harvesting bacteria mM NH4Ci or 0.5 mM Ni(CN)]- included in the Bacteria from all cultures were harvested by growth medium; growth on the latter compound centrifugation and resuspension in 5") mM- gave a somewhat lower growth yield. The Na2HPO4/NaHzPO 4 pH 7.0 buffer as previously bacterium was also found to grow on 10 mM described [2]. glucose plus 2 mM KOCN, with a similar growth yield to growth on NH4CI. In each case growth 3.5. Assay of ~yanide oxygenase activity terminated due to nitrogen depletion from the Cyanide oxygenase activity was assayed by the growth medium. oxygen uptake method as previously described [2], Fig, 1. shows growth on KOCN as the source except that KCN was added to a final concentra- of nitrogen. KOCN depletion from the medium tion of 200 #M in the oxygen electrode. occurred prior to growth and was associated with formation of ammonia, which was utilized during 3.6. Assay of cyanase activity growth. Cyanase activity was induced to a maxi- Suspensions of bacteria were added to an assay mum of about 2.34 ~mol NH 3 formed/min/mg mixture in a magnetically stirred chamber with a dry wt cells, and was lost once the cyanate had 30°C water jacket to give a final A6oo of 0.2. The been depleted from the medium. Cyanide oxy- assay mixture was as described by Anderson [6]. genase activity was absent throughout the growth Cyanase activity was determined by measuring the cycle. initial rate of ammonia formation from cyanate Fig. 2. shows growth on Ni(CN) 2 " as the source hydrolysis in the assay mixture, after bacteria had of nitrogen. This compound was utilized through- been removed by centrifugation. out growth. Cyanide oxygenase activity was induced by Ni(CN)]- up to a maximum of about 3. 7. Assay of ammonia 38 nmol 0 2 consumed/min/mg dr 5' wt cells. Ac- Ammonia in the cell-free culture medium, and tivity was lost after depletion of Ni(CN)24 - from in the cyanase assays was measured colorimetri- the medium. Cyanase activity was not induced tally by the method of Fawcett and Scott [7]. (> 43 nmoi NH 3 formed/min/mg dry wt cells). 291

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0 .O 16 Time from inoculation (h) Fig. 1. The growth of P. fluorescens on cyanate as the source of nitrogen. Growth (o), cyanate (ll), ammonia (zx) and cyanase activity (A)

4.2. Induction of cyanide oxygenase and cyanase monia were detectable in the medium. To induce actioities in stationary phase batch cultures cyanide oxygenase activity either 0.25 mM Cultures were grown for 24 h in batch culture Ni(CN)~- or 1 mM KCN were when added to the when 10 mM glucose and 2 mM NH4CI as the medium. It was found that Ni(CN)42- induced sources of carbon and nitrogen, respectively. At higher specific activity of cyanide oxygenase than the end of this period neither glucose nor am- KCN, although an equivalent concentration of

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m ~IZI F-I .O -1,5 " 4 (~ 8 10 12 1~4 16 ~ t8 Time from inoculation (h) Fig. 2. The growth of P. fluorescens on Ni(CN)~- as the source of nitrogen. Growth (o), Ni(CN)~- (v) and cyanide oxygenase activity (D). 292

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,I" i O -~--=---=~,&- --- ~11 LO 0 20 4O 60 80 100 120 3 .time from addition of KOCN (n)in) (h~] 5 6 Fig. 3. Induction of cyanase activity in stationary phase culture. P. fluorescens was grown [or 24 h on glucose/N}la('l and 2 mM KOCN then added. KOCN (1), growth to). ammonia (a), and cyanasc activity (,,). cyanide had been added. The optimum concentra- ': :5O tion of Ni(CN) 4 for induction of cyanide o,3. I oxygenase activity was found to be 0.5 mM. Activ- jo .... ©! ity was at its highest level five hours after the /, / addition of Ni(CN)] . No induction of cyanase o 0.1 "~ [] iO ~-4o occurred under these conditions. For induction of / cyanase activity, 2 mM KOCN was added to // / / . i parallel cultures grown for 24 h. (Fig. 3). No ._= induction of cyanide oxygenase activity occurred -o.~1 E under these conditions. Once cyanate had been [] 11-1-1 [ .30 E lost from the medium, cyanase activity decreased. ,' [~ / C) , / ': A / ,, z 4.3. Cyanide oxygenase and (yanase actit,ities in -0.3~ KCN-limited fed-batch culture !' [] ' •i @ A 500 ml KCN-limited fed-batch culture of P. Fo ~ .. / [] fluorescens was set up, cyanide oxygenase and !, ,:2 , i cyanase activities were measured throughout - 0,5:' ,' growth (Fig. 4). I / ! ' ,~.) Cyanide oxygenase was induced to a maximum /' c I activity of 40 nmol O 2 consumed/min/mg dry wt :! 0 .lo -~ I iff- A cells at the mid-exponential phase of growth. Only - 0.71 &-Jl, ( • very low levels of cyanase were observed, which i showed no correlation with cyanide oxygenase 'Lr~'/'' / ~ I activity. Indeed, when cyanide oxygenase activity / ~ i "\ / £/) was at its highest to cyanase activity was detected. ~ O ..... _.___ ...... 10 15 20 A variable low basal level of cyanase activity Time from inoculation (hi was present in bacteria grown under such condi- Fig. 4. Cyanase and cyanide oxygenase activities m KCN- tions. This activity was at its highest at the start of limited fed-hatch culture. Growth (o), cyanide oxygenase ac- growth, and after growth had terminated. tivity (1) and cyanase activity O'). 293

Pulsing mid exponential phase (A550 = 1.0) In this paper we have shown that a separate KCN-limited fed-batch cultures with 1 mM KCN cyanase enzyme is probably not involved in thc resulted in rapid loss of cyanide and consequent utilisation of cyanide by P. fluorescens NCIMB formation of ammonia in the culture medium. No 11764. This suggests that the pathway given in cyanate was detected prior to ammonia formation equations (2) and (3) is probably not a route for from cyanide. cyanide utilisation. However it is possible that cyanatc is an intermediate of cyanide utilisation, but is present as a bound intermediate of the Downloaded from https://academic.oup.com/femsle/article/60/3/289/452954 by guest on 30 September 2021 5. DISCUSSION cyanide oxygenase activity without release, thereby not inducing a separate cyanase activity. Cyanase and cyanide oxygenase activities were never co-induced. Cyanide (either NI(CN) 2- or KCN) induced cyanide oxygenase activity, but not ACKNOWLEDGEM ENTS cyanase activity. Conversely, cyanate induced cyanase activity not cyanide oxygenase activity. It is noteworthy that the induced activity of cyanase We wish to thank Dr. Steve Bungard (I.C.I. was much higher than the induced cyanide BioProducts) for his helpful discussion and en- oxygenase activity. couragement. This work was supported by the The growth of ammonia and cyanate contain- Science and Engineering Research Council and ing batch cultures were strikingly similar, the major Imperial Chemical Industries BioProducts Busi- difference being the extended lag period of KOCN ness via a C.A.S.E. award to P.K.D. grown cultures. This extended lag period is probably due to the induction of cyanase activity to provide ammonia as the nitrogen source for REFERENCES growth. During the exponential phase of growth, both KOCN and NHaCI grown cultures utilised [1] Harris, R.E. and Knowles. C.J. (1983) J. Gen. Microbiol. ammonia as the nitrogen source. The growth pro- 129, 1005-1011. file of cultures utilising Ni(CN) 4 as nitrogen [2] Rollinson, G., Jones, R., Meadows, M.P., Harris. R.E., source bears little similarity. Ni(CN) 2- was and Knowles, C.J. (1987) FEMS Microbiol. Lett. 40, 199- 205. utilized during growth, with no formation of am- [3] Harris, R.E. and Knowles, C.J. (1983) FEMS Microbiol. monia in the medium. Cyanide oxygenase activity Lett. 337-341. was low during the lag phase of growth, but [41 Miller, J.H. (1972) in Experiments in Molecular Genetics, reaches a maximum at mid exponential phase. At p. 431 Cold Spring Harbor Laboratory, Cold Spring this phase of growth, cyanase activity in cyanate Harbor, N.Y. [5] Bauchop, T. and Elsden, S.R. (1960) J. Gen. Microbiol. grown cultures was at a basal level. 23. 457-469. Induction of cyanide oxygenase and cyanase [6] Anderson. P.M. (1980) Biochemistry 19. 2882 2888. activities in stationary phase cultures were also [7] Fawcett, J.K. and Scott, J.E. (1960) J. Clin. Pathol. 13, mutually exclusive. Due to the difficulty in ob- 156-160. taining large quantities of cells induced for cyanide 18] Guilloton, M. and Karst. F. (1985) Anal. Biochem. 149, 291-295. oxygenase activity, such conditions for induction [9] Taussig, A. (1960) Biochim. Biophys. Acta 44, 510-519. may prove useful for purification purposes. [10] Taussig. A. (1965) Can. J. Biochem. 43, 1062-1069. Cyanase has been extensively studied in [11] Taussig, A., Normen, A. (1970) (7an. J. Biochem. 48, Escherichia coli [9-11]. It has been purified and 790-798. characterised fully [6,12-15], cloned [16] and [12] Chin, C.C.Q., Anderson, P.M., and Wald, F. (1983) J. Biol. Chem. 258, 276-287. studied at a genetic level [17]. Kunz and Nagap- [13] Anderson, P.M. and Little, R.M. (1986) Biochemistry 25, pan [18] using P. fluorescens NC1MB 11764 have 1621 - 1626. recently confirmed our earlier observation of the [14] Anderson, P.M., Johnson, W.V., Endrizzi. J.A.. Little, presence of cyanase activity in this bacterium [3]. R.M., and Korte, J.J. (1987) Bi~v,:hemistry 262, 3938-3943. 294

[15] Johnson. W.V. and Anderson, P.M. (1987) J. Biol. Chem. [17] Sung, Y.C., Anderson, P.M., Fuchs, J.A. (1987) J. 262, 9021-9025. Bacteriol. 169, 5224-5230. [16] Sung, Y.C., Parsell, D., Anderson, P.M., and Fuchs, J.A. [18] Kunz, D.A. and Nagappan, D. (1989) Appl. Env. Micro- (1987) J. Bacteriol. 169, 2639-2642. biol. 55, 257-258. Downloaded from https://academic.oup.com/femsle/article/60/3/289/452954 by guest on 30 September 2021