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Regulation of methanol and methylamine dehydrogenases in Methylophilus methylotrophus
FEMS MicrobiologyLetters 68 (1990) 93-96 Published by Elsevier
93
FEMSLE 03927
Regulation of methanol and methylamine dehydrogenases in Methylophilus methylotrophus A. D a w s o n , G. S o u t h g a t e a n d P.M. G o o d w i n School of Cell and Molecular Biology. North East Surrey Collegeof TechnoloKv. Ewell. Epsom. U.I(. Received 10 October 1989 Accepted 17 November 1989 Key words: Methylophilus methylotrophus; Methanol dehydrogenase: Methylamine dehydrogenase
1. S U M M A R Y Methylophilus methylotrophus can use methylamine as sole source of carbon and nitrogen. Measurements of the specific activity of methylamine dehydrogenase ( M N D H ) in bacteria grown in batch or chemostat culture showed that M N D H was induced by methylamine and repressed when methanol or N H ~ were provided as alternative carbon or nitrogen sources. The degree of repression varied with the growth conditions. Methanol dehydrogenase ( M D H ) was present in bacteria grown on methylamine as sole carbon source, but the specific activity was low compared with that in bacteria grown on medium containing methanol, indicating that this enu.yme is induced by methanol.
2. I N T R O D U C T I O N Methylophilus methylotrophus is a restricted facuhative methylotroph capable of utilising methanol and methylated amines [1]. Methanol is
Correspondence to: P.M. Goodwin. School of Cell and Molecular Biology,North East Surrey Collegeof Technology,Reigate Road, Ewell,Epsom. Surrey. KTI7 3DS, U.K.
oxidised by methanol dehydrogenase ( M D H ) and there is evidence that synthesis of this enzyme is repressed when bacteria are grown in the presence of excess methanol [2]. Methylamine is oxidised by methylamine dehydrogenase ( M N D H ) [3]; there is little information about the regulation of this enzyme, but it appears to be induced during growth on methylated amines [4]. The aim of our work was to investigate in more detail the regulation of these two enzymes in M. methylotrophus.
3. M A T E R I A L S A N D M E T H O D S M. methylotrophus was grown in batch and continuous culture as previously described [5]. In batch culture methylamine hydrochloride was used at a final concentration of 0.2%. Methanol and methylamine oxidase activities of whole cells were estimated at 3 7 ° C using a Clark oxygen electrode. Washed cells were suspended in 2 mi of Seed2 salts medium and after measuring the endogenous rate of oxidation either methanol or methylamine was added to a final concentration of 10 raM. M N D H was assayed polarographicaily by the method of Eady and Large [6], but using phenazine ethosulphate as the electron acceptor. Residual methylamine was de-
0378-1097/90/$03.50 © 1990 Federation of European MicrobiologicalSocieties
termined enzymically using a crude extract of methylamine-grown Methylobacterium extorquens AMI, prepared according to the method of Tatra and Goodwin [7], as a source of M N D H . All other determinations were carried out as described by Southgate a n d Goodwin [5].
4. RESULTS
4.1. Growth of M. methylotrophus on methylamine as carbon and~ or nitrogen source in batch culture In batch culture M. methylotrophus grew slowly (mean generation time approx 10 hours) on medium containing methylamine as sole source of carbon and either methylamine or NH~" as the nitrogen source• In comparison, the mean generation time was 2 to 2.5 hours when methanol was the principal carbon source and either N H ~ or methylamine was the nitrogen source. Methylamine oxidase activity was similar in cells grown in media containing m~thy|a~,~ne, ut methylamine plus methanol, or methylamine plus N H ~ (Table 1). However, the specific activity of M N D H varied by more than 20-fold, being highest when methylamine was the sole carbon a n d nitrogen source, a n d lowest when methanol was present as a n alternative carbon source. M N D H activity was not detected in cells grown either on methanol plus N H ~ or o n methanol plus methylamine plus N H ~ . Methanol oxidase and M D H activities were detected in bacteria grown u n d e r all conditions, but they were relatively low in cells grown in the absence of methanol.
4.2. Growth of M. methylotrophus on methylamine as sole carbon a n d / o r nitrogen source in continuous culture. M. methylotrophus has been grown on m e t h a n o l in continuous culture at dilution rates of u p to 0.53 h - t [2]. In contrast, the highest dilution rate at which this organism could b e grown in continuous culture in medium containing methylamine as sole carbon and nitrogen source was 0.05 h - t. The growth limiting nutrient was not identified, b u t residual methylamine was detected in the medium, a n d oxygen was in excess. In extracts of cells grown under these conditions the specific activities of M D H and M N D H were 180 a n d 500 n m o l - r a i n - ~ • mg p r o t e i n - ! respectively a n d the rate of methylamine utilisation in situ was 5 m m o l • h - i . (g b i o m a s s ) - ~ (Table 2). Bacteria were also grown in m e d i u m containing methylamin¢ as sole nitrogen source a n d provided with methanol as a n alternative c a r b o n source at concentrations giving c a r b o n to nitrogen ( C : N ) molar ratios of 2.3 a n d 15.9 (Table 2). W h e n the C : N ratio was 2.3 methanol was exhausted, b u t residual methylamine was detected, d e m o n s t r a t i n g that methanol was the preferred c a r b o n source. The rate of methylamine utilisation was a b o u t half that of cells grown o n methylamine as sole c a r b o n a n d nitrogen source a n d the M N D H activity was more then ten fold lower• In contrast, M D H activity was increased about five fold. W h e n the C : N ratio was 15.9 methylamine, but not methanol, was exhausted. Methylamine w~s utilised at a very low rate a n d M N D H activity was not detectable. T h e specific activity of M D H in cell extracts was
Table 1 Oxidation of methanol and methylamine by whole cells and cell extracts of M, methylotrophus grown in batch culture Carbon and nitrogen source(s) Methanol + NH~ Methylamine Methanol + Methylamine Methylamine+ NH ~ Methanol + Methylamine+ NH~
Specific activity of MDH MNDH nmol. rain- i, (m3 protein)- i 959 ND 74 214 "/47 9.7 160 73 1123
ND
Methanol Methylamine oxidase oxidas¢ nmol, min- 1.(rag protein)- t 241 ND 43 174 292 134 29 213 -
ND, not detectable; - , not determined. Values are means of data from at least two independent cultures,
-
Table 2 Specific activities of methanol and methylamine dehydrogenases in M. methylotrophus grown in continuousculture at a dilution rate of 0.05 h - i C: N molar ratio
1.0
2.3
15.9
input output
0 0
137 ND
312 32
Methylamine input (mM) output
100 43
104 6
21 ND
Methanol (mM)
Cell dry weight(g-I- i )
0.57
1.78
2.16
Methanol dehydrogenase nmol-min- i. (mg protein)- ]
180
960
520
Methylaminedehydrogenase nmol. min- i.(mg protein)- t
500
40
ND
q mcthylami,e mmol. h-I-(g biomass)- J qmethanot retool-h- I.(g biomass)- t
5
2.75
0.49
3.85
6.48
biD: not detectable. Values are weans of data from at least two independentcultures. about half that observed in cells grown at a C : N ratio of 2.3 but the rate of methanol utilisation was increased.
5. DISCUSSION Our results have confirmed that M N D H is induced by methylamine and also show that this enzyme was repressed when methanol or N H ~ were provided in the medium in addition to methylamine. In chemostat cultures grown on limiting amounts of methylamine (as sole nitrogen source) and excess methanol the repression was so great that no M N D H activity was observed in cell extracts. U n d e r the assay conditions used this enzyme would have been detected if present at a specific activity of about 5 n m o l . m i n - ~ • (mg protein) - t . This is two to three fold less than the value which would be required to account for the rate of methylamine utilisation in situ. However, since M N D H may be operating under more favourable conditions in vivo than in vitro we cannot discount the possibility that there was sufficient activity for the assimilation of methylamine
under these growth conditions. Alternatively the function o[ M N D H may be to metabolise methylamine when it is providing the carbon source, and a different enzyme may be responsible for utilising methylamine as nitrogen source. It is well established that the activity of M D H varies with growth conditions and that there is no simple linear relationship between this and either methanol oxidase activity or the rate of methanol utilisation in situ [2,5,8]. It has been suggested that M D H is regulated by repression when the steady state concentration of methanol is high, thus minimising the potential for the overproduction of formaldehyde, the toxic product of methanol oxidation [8]. Our data confirm that M D H activity is lower in bacteria grown under conditions of methanol excess than when methanol is exhausted. However, the activity of M D H in methylaminegrown cells was less than 50% that observed when cells were grown under conditions of methanol excess, indicating that methanol does induce the enzyme. The repression observed when bacteria are grown in excess methanol is therefore unlikely to be a direct effect of this substrate, but is presumably caused by the accumulation of a product of methanol metabolism.
A C K N O W L E D G E M ENTS We thank SERC and ICI for a CASE studentship for GS and SERC for a studentship for AD.
REFERENCES [1] Jenkins, O.. Byrom. D. and Jones. D. (1987) Int. J. Syst. Bacteriol. 37. 446-448, [2] Greenwood,J.A. and Jones, C.W. (1986) J. Gen. MicrobioL 132. 1247-1256 13l Haywood. G.W., Janschke. N.S., Large. P.J. and Wallis, J.M. (1982) FEMS Microbiol. Lett. 15. 79-82. [4] Large, P.J. and Haywood. G.W. (1981) FEMS Microbiol. Lett. !1.207-209. [5] Southgate. G. and Goodwin. P.M. (1989) J. Gen. Microbiol. 135. 2859-2867. [6] Eady.R.R. and Large. P.J. (1968) Biochem.J. 106. 245-255. [71 Tatra, P.K. and Goodwin. P.M. (1985) Arch. Microbiol. 143. 169-177. t [8] Jones. C.W.. Greenwood. J.A.. Burton. S.M.. Santos, H. and Turner. D.L. (1987) J. Gen. MicrobioL133.1511-1519.
93
FEMSLE 03927
Regulation of methanol and methylamine dehydrogenases in Methylophilus methylotrophus A. D a w s o n , G. S o u t h g a t e a n d P.M. G o o d w i n School of Cell and Molecular Biology. North East Surrey Collegeof TechnoloKv. Ewell. Epsom. U.I(. Received 10 October 1989 Accepted 17 November 1989 Key words: Methylophilus methylotrophus; Methanol dehydrogenase: Methylamine dehydrogenase
1. S U M M A R Y Methylophilus methylotrophus can use methylamine as sole source of carbon and nitrogen. Measurements of the specific activity of methylamine dehydrogenase ( M N D H ) in bacteria grown in batch or chemostat culture showed that M N D H was induced by methylamine and repressed when methanol or N H ~ were provided as alternative carbon or nitrogen sources. The degree of repression varied with the growth conditions. Methanol dehydrogenase ( M D H ) was present in bacteria grown on methylamine as sole carbon source, but the specific activity was low compared with that in bacteria grown on medium containing methanol, indicating that this enu.yme is induced by methanol.
2. I N T R O D U C T I O N Methylophilus methylotrophus is a restricted facuhative methylotroph capable of utilising methanol and methylated amines [1]. Methanol is
Correspondence to: P.M. Goodwin. School of Cell and Molecular Biology,North East Surrey Collegeof Technology,Reigate Road, Ewell,Epsom. Surrey. KTI7 3DS, U.K.
oxidised by methanol dehydrogenase ( M D H ) and there is evidence that synthesis of this enzyme is repressed when bacteria are grown in the presence of excess methanol [2]. Methylamine is oxidised by methylamine dehydrogenase ( M N D H ) [3]; there is little information about the regulation of this enzyme, but it appears to be induced during growth on methylated amines [4]. The aim of our work was to investigate in more detail the regulation of these two enzymes in M. methylotrophus.
3. M A T E R I A L S A N D M E T H O D S M. methylotrophus was grown in batch and continuous culture as previously described [5]. In batch culture methylamine hydrochloride was used at a final concentration of 0.2%. Methanol and methylamine oxidase activities of whole cells were estimated at 3 7 ° C using a Clark oxygen electrode. Washed cells were suspended in 2 mi of Seed2 salts medium and after measuring the endogenous rate of oxidation either methanol or methylamine was added to a final concentration of 10 raM. M N D H was assayed polarographicaily by the method of Eady and Large [6], but using phenazine ethosulphate as the electron acceptor. Residual methylamine was de-
0378-1097/90/$03.50 © 1990 Federation of European MicrobiologicalSocieties
termined enzymically using a crude extract of methylamine-grown Methylobacterium extorquens AMI, prepared according to the method of Tatra and Goodwin [7], as a source of M N D H . All other determinations were carried out as described by Southgate a n d Goodwin [5].
4. RESULTS
4.1. Growth of M. methylotrophus on methylamine as carbon and~ or nitrogen source in batch culture In batch culture M. methylotrophus grew slowly (mean generation time approx 10 hours) on medium containing methylamine as sole source of carbon and either methylamine or NH~" as the nitrogen source• In comparison, the mean generation time was 2 to 2.5 hours when methanol was the principal carbon source and either N H ~ or methylamine was the nitrogen source. Methylamine oxidase activity was similar in cells grown in media containing m~thy|a~,~ne, ut methylamine plus methanol, or methylamine plus N H ~ (Table 1). However, the specific activity of M N D H varied by more than 20-fold, being highest when methylamine was the sole carbon a n d nitrogen source, a n d lowest when methanol was present as a n alternative carbon source. M N D H activity was not detected in cells grown either on methanol plus N H ~ or o n methanol plus methylamine plus N H ~ . Methanol oxidase and M D H activities were detected in bacteria grown u n d e r all conditions, but they were relatively low in cells grown in the absence of methanol.
4.2. Growth of M. methylotrophus on methylamine as sole carbon a n d / o r nitrogen source in continuous culture. M. methylotrophus has been grown on m e t h a n o l in continuous culture at dilution rates of u p to 0.53 h - t [2]. In contrast, the highest dilution rate at which this organism could b e grown in continuous culture in medium containing methylamine as sole carbon and nitrogen source was 0.05 h - t. The growth limiting nutrient was not identified, b u t residual methylamine was detected in the medium, a n d oxygen was in excess. In extracts of cells grown under these conditions the specific activities of M D H and M N D H were 180 a n d 500 n m o l - r a i n - ~ • mg p r o t e i n - ! respectively a n d the rate of methylamine utilisation in situ was 5 m m o l • h - i . (g b i o m a s s ) - ~ (Table 2). Bacteria were also grown in m e d i u m containing methylamin¢ as sole nitrogen source a n d provided with methanol as a n alternative c a r b o n source at concentrations giving c a r b o n to nitrogen ( C : N ) molar ratios of 2.3 a n d 15.9 (Table 2). W h e n the C : N ratio was 2.3 methanol was exhausted, b u t residual methylamine was detected, d e m o n s t r a t i n g that methanol was the preferred c a r b o n source. The rate of methylamine utilisation was a b o u t half that of cells grown o n methylamine as sole c a r b o n a n d nitrogen source a n d the M N D H activity was more then ten fold lower• In contrast, M D H activity was increased about five fold. W h e n the C : N ratio was 15.9 methylamine, but not methanol, was exhausted. Methylamine w~s utilised at a very low rate a n d M N D H activity was not detectable. T h e specific activity of M D H in cell extracts was
Table 1 Oxidation of methanol and methylamine by whole cells and cell extracts of M, methylotrophus grown in batch culture Carbon and nitrogen source(s) Methanol + NH~ Methylamine Methanol + Methylamine Methylamine+ NH ~ Methanol + Methylamine+ NH~
Specific activity of MDH MNDH nmol. rain- i, (m3 protein)- i 959 ND 74 214 "/47 9.7 160 73 1123
ND
Methanol Methylamine oxidase oxidas¢ nmol, min- 1.(rag protein)- t 241 ND 43 174 292 134 29 213 -
ND, not detectable; - , not determined. Values are means of data from at least two independent cultures,
-
Table 2 Specific activities of methanol and methylamine dehydrogenases in M. methylotrophus grown in continuousculture at a dilution rate of 0.05 h - i C: N molar ratio
1.0
2.3
15.9
input output
0 0
137 ND
312 32
Methylamine input (mM) output
100 43
104 6
21 ND
Methanol (mM)
Cell dry weight(g-I- i )
0.57
1.78
2.16
Methanol dehydrogenase nmol-min- i. (mg protein)- ]
180
960
520
Methylaminedehydrogenase nmol. min- i.(mg protein)- t
500
40
ND
q mcthylami,e mmol. h-I-(g biomass)- J qmethanot retool-h- I.(g biomass)- t
5
2.75
0.49
3.85
6.48
biD: not detectable. Values are weans of data from at least two independentcultures. about half that observed in cells grown at a C : N ratio of 2.3 but the rate of methanol utilisation was increased.
5. DISCUSSION Our results have confirmed that M N D H is induced by methylamine and also show that this enzyme was repressed when methanol or N H ~ were provided in the medium in addition to methylamine. In chemostat cultures grown on limiting amounts of methylamine (as sole nitrogen source) and excess methanol the repression was so great that no M N D H activity was observed in cell extracts. U n d e r the assay conditions used this enzyme would have been detected if present at a specific activity of about 5 n m o l . m i n - ~ • (mg protein) - t . This is two to three fold less than the value which would be required to account for the rate of methylamine utilisation in situ. However, since M N D H may be operating under more favourable conditions in vivo than in vitro we cannot discount the possibility that there was sufficient activity for the assimilation of methylamine
under these growth conditions. Alternatively the function o[ M N D H may be to metabolise methylamine when it is providing the carbon source, and a different enzyme may be responsible for utilising methylamine as nitrogen source. It is well established that the activity of M D H varies with growth conditions and that there is no simple linear relationship between this and either methanol oxidase activity or the rate of methanol utilisation in situ [2,5,8]. It has been suggested that M D H is regulated by repression when the steady state concentration of methanol is high, thus minimising the potential for the overproduction of formaldehyde, the toxic product of methanol oxidation [8]. Our data confirm that M D H activity is lower in bacteria grown under conditions of methanol excess than when methanol is exhausted. However, the activity of M D H in methylaminegrown cells was less than 50% that observed when cells were grown under conditions of methanol excess, indicating that methanol does induce the enzyme. The repression observed when bacteria are grown in excess methanol is therefore unlikely to be a direct effect of this substrate, but is presumably caused by the accumulation of a product of methanol metabolism.
A C K N O W L E D G E M ENTS We thank SERC and ICI for a CASE studentship for GS and SERC for a studentship for AD.
REFERENCES [1] Jenkins, O.. Byrom. D. and Jones. D. (1987) Int. J. Syst. Bacteriol. 37. 446-448, [2] Greenwood,J.A. and Jones, C.W. (1986) J. Gen. MicrobioL 132. 1247-1256 13l Haywood. G.W., Janschke. N.S., Large. P.J. and Wallis, J.M. (1982) FEMS Microbiol. Lett. 15. 79-82. [4] Large, P.J. and Haywood. G.W. (1981) FEMS Microbiol. Lett. !1.207-209. [5] Southgate. G. and Goodwin. P.M. (1989) J. Gen. Microbiol. 135. 2859-2867. [6] Eady.R.R. and Large. P.J. (1968) Biochem.J. 106. 245-255. [71 Tatra, P.K. and Goodwin. P.M. (1985) Arch. Microbiol. 143. 169-177. t [8] Jones. C.W.. Greenwood. J.A.. Burton. S.M.. Santos, H. and Turner. D.L. (1987) J. Gen. MicrobioL133.1511-1519.