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Polyamines of carbon monoxide-utilizing bacteria, Pseudomonas thermocarboxydovorans and Pseudomonas carboxydohydrogena
FEMS MicrobiologyLetters 70 (1990) 353-356 Published by Elsevier
353
FEMSLE 04108
Polyamines of carbon monoxide-utilizing bacteria, Pseudomonas thermocarboxydovorans and Pseudomonas carboxydohydrogena K o e i H a m a n a ~ a n d Shigeru M a t s u z a k i 2 I Collegeq[ Medical Care and Technologyand 2 Departmentof Physiology. Institute of Endocrinology, Gunma University, Maebashi..iapan
Received 18 April 1990 Accepted 26 April 1990 Key words: Carboxydobacteria; Pseudomonas; Polyamine; Hydroxypolyamine
1. S U M M A R Y A slightly thermophilic, CO-utilizing pseudom o n a d , Pseudomonas thermocarboxydooorans grown under both autotrophic and heterotrophic growth conditions was found to contain 2-hydroxyspermidine and 2-hydroxyputrescine in addition to putrescine, diaminopropane and spermidine. A mesophilic C O - utilzing hydrogen bacterium, Pseudomonas carboxydohydrogena contained putrescine and homospermidine under both autotrophic and lieterotrophic growth conditions. Although these two carboxydobacteria are classified to the same genus Pseudomonas, the difference in their polyamine distribution pa:terns suggests thai they may belong to different subclasses of Proteobacteria.
2. I N T R O D U C T I O N Besides putrescine and sp."rmidine many different polyamines and their derivatives have been
Correspondence to: Koei Hamana, Collegeof MedicalCare and Technology, Gunma Universay, Maebashi 371. Japan.
found in microorganisms during the last decade [1]. Although the biological role of polyamines is poorly understood, they have been shown to serve as useful chemotaxonomic markers within certain genera, e.g., Proteobacteria which contain the majority of the well-known Gram-negative eubacteria [2]. A group of heterogenous Gram-negative eubacteria are straight or slightly curved rods, motile by polar flagella, belonging to genus Pseudomonas and arranged into five sections, 1, 11, Ili, IV and V, from their r R N A and D N A homologies [31. Recently, several pseadomonads are allocated to t| tee diffcrent ~ b g r o u p s , a, B and y, of Proteobacleria i,y th,,~r c.i'ference in polyamine distribution patterns [21. Putrescine and homospermid;ne are the major polyamines in Pseudomonas belonging to the alpha subclass. Pseudomonas species in the beta subclass contain 2-liydroxyputrescine, putrescine and spermidine, while putrescine, diaminopropane and spermidine are foo.~d in the Pseudomot, a~ species which belong to the 3' subclass 12] Facultative cF:emolitliotrophs which are able to use CO a n d / o r t t 2 as energy sources and belong to genus Pseudomonas have been isolated [3,4]. It seemed to us of interest to exarmne the relation-
0378-1097/90/$03.50 © 1990 Federation of European MicrobiologicalSocieties
354 ship between the ability to oxidize CO or H2, and polyamine patterns in the organisms. In the present report polyamines have been analyzed in a slightly thermophilic CO-utilizing Pseudomonas thermocarboxydovorans [5] and a mesophilic COutilizing hydrogen bacterium, Pseudomonas carboxydohydrogena [6] grown under autotrophic and heterotrophic growth conditions. The validity of taxonomic positions of these carboxydobacteria was discussed on the basis of their polyamine distribution patterns.
3. MATERIALS AND METHODS 3.1. Organisms and cultures The Gram-negative, aerobic, thermophilic, CO-utilizing organism P. thermocarboxydovorans (ATCC 35961) was cultivated in the basal medium under 50% CO-50% air at 5 0 ° C for autotrophic growth [5] tl: is not oxidized by this organism. A Gram-negative, aerobic, mesophilic, CO-utilizing hydrogen bacterium, P. carboxydohydrogena (ATCC 29978) was cultured in the basal medium under 40% CO-10% 02-50% N 2 or 80% H2-10% O2-10% CO 2 at 30°C for autotrophic growth [6]. For heterotrophic growth, the media were supplemented with 0.5% of sodium pyruvate. The organisms were also grown beterotrophically in the synthetic mammalian cell culture 199 medium. Logarithmically growing cells or stationary phase cells were harvested. 3.2. Polyamme analysis Diaminopropane - 2HCI, putrescine- 2HCI and spermidine-3HCI were purchased from Sigma (MO, U.S.A.). Homospermidine was synthesized by a~ylation of putrescine with to-bromoalkylphthalimide [7]. 2-Hydroxyputrescine was synthesized by ammoniolysis of 1,4-dibromobutan-2-ol [8]. The mixture of 2-hydroxyspermidine and 3-hydroxyspermidine was kindly supplied by Dr. C. Hurwitz. After centrifugation of the cultures, pellets of organisms were homogenized in equal volumes of cold 1.0 M HCIO4. Polyamines in perchloric acid extracts were analyzed by high-performance liquid chromatography (HPLC) using KCl-citrate buffer system as
reported previously [9]. Some samples were analyzed before and after the hydrolysis with 6 M HC! or 1 M NaOH for 24 h at 110°C or the periodate oxidation with 0.1 M sodium metaperiodate in 0.67 M NaOH for 1 h at 5 6 ° C [10]. The identity of polyamines in the extracts ~,as confirmed by thin-layer chromatography (TLC) un cellulose using the solvent system of isopropanol/ ammonia (7:3) [11] or on silica gel using the solvent system of c h l o r o f o r m / m e t h a n o l / ammonia ( 2 : 2 : 1 ) [12]. For assay of the decarboxylase activities, the lysates from logarithmically growing cells were added to the reaction mixture containing respective amino acids and then incubated according to our previous report [13]. Polyamines produced during incubation were analyzed by HPLC method.
4. RESULTS Five different polyamines were detected in P. thermocarboxydovorans grown under both autotrophic and heterotrophic growth conditions. These polyamines were resistant to the acid and alkafine hydrolyses. The five polyamines corresponded to 2-hydroxyputrescine, diaminopropane, putrescine, hydroxyspermidine (2-hydroxyspermidine a n d / o r 3-hydroxysperrnidine) and spermidine (Fig. 1). The two peaks corresponding to 2-hydroxyputreseine and hydroxyspermidine completely disappeared after the reaction with metap~riodate. Authentic 2-hydroxy and 3-hydroxy isomers of spermidine were not separated either on HPLC or TLC. Since the diaminopropane peak did not increase after the periodate oxidation, the hydroxyspermidine fraction of the organism was identified to be 2-hydroxyspermidine. The coexistence of 3-hydroxyspermidine was excluded, because no diaminopropane was produced by the cleavage. Four other polyamines were identified by TLC (data not shown). P. curboxydohydrogena contained two polyamine peaks corresponding to putrescine and homospermidine (Fig. 1). The polyamines were also identified on TLC (data not shown). Cellular polyamine concentrations were shown in Table 1. In the two carboxydobacteria, cellular
355 the organisms may be due to net polyamine synthesis. The two carboxydobacteria decarboxylated ornithine to produce putrescine but not either diaminobutyric acid+ lysine or arginine in vitro.
5. D I S C U S S I O N The polyamine distribution patterns of P. ther-
[3 ~
mocarboxydovorans and
~t
io
A ELUTION
3'o
P. carboxydohydrogena
are quite constant when grown under the different growth conditions or during different growth phases. These findings suggest that their polyamines can be one of the stable cheraotaxonomic markers of the organisms and that it is not connected to their chemoautotrophic activity oxidizing CO a n d / o r H2 as energy source. 2-Hydroxyputrescine was first found in Pseudomonas sp. K/m [14] and then in P. solanacearus,
.b
TIE (mn)
Fig. 1. HPLC of polyammes of P. thermocarboxydovorans before (A) and after (B) the periodate oxidation, and polyarrAnes of P. carboxydohydrogena (C). The orgamsms were grown in 199 medium.Abbreviationsfor polyamincsate shown in Table 1. levels of polyamines as well as polyamine components were nearly the same at different growth phases and in both autotrophic and heterotrophic growth. Since all of the culture media used are free from polyamines, the polyamines detected in
P. caryophlii, P. cepacia, P. marginata, P. facilis and P. delafieldii [2] m genus Pseudomonas. Comamonas acidovorans (formerly Pseudomonas acidovorans), C. testosteroni, C. terrigena, five species of Alcaligenes, Ach¢omobacter .rylosoxydans, Rhodocyclus gelatinosus. Ch,'omobacterium violaceum and Janthinobacterium lividum belonging to the beta subclass of Proteobacteria also contain .7.-hydroxyputrescin¢ [2,15]¢ C. acidovorans and R. gelatinosus contain, although in low concentrations, 2-hydroxyspermidine [2,10]. However+ identification and quantitative analysis of these unusual hy-
Table 1 Cellular concentrations of polyarmnesof Pseudomonasthermocarbo~vdovoransand P. carbox3~ol~ydrogena H-Put, 2-hydroxyputrescine; Dap, diaminopropan¢; Put. putrescine; H-Spd. 2-hydroxyspernudine;Spd. sperrmd~ne; HSpd, homospermidine: ND, not detected; (S). slationary phase; (L), logarithmicallygrowingphase; ATCC. AmericanType Culture Collection. Rockville. MD U.S.A. Culture conditions are described in MATERIALSAND METHODS. Cuhure condition
P. thermocarboxydovorans (ATCC 35961)
P. corboxydohydrogena (ATCC 29978)
CO/air Pyruvate 199 199 CO/O~/N 2 H2/02/CO 2 Pyruvate 199 199
(S) (S) (L) (S) (S) (S) (S) (L) (SI
Polyamines(pmol/g wet ~¢eight) H-Put Dap PUl H-Spd
Spd
HSpd
0.200 0.342 0.382 0.129 ND ND ND ND ND
0.280 0.540 0.680 0.760 ND ND ND ND ND
ND ND ND ND 0.520 0.700 4.055 0.560 0700
0.020 0.180 ND 0.100 ND ND ND ND ND
0.100 0.134 0.024 0.500 0.240 0.220 l.O00 0.100 0.160
0.040 0.100 0020 0.400 ND ND ND ND ND
356 droxypolyamines in the organisms have not been sufficient in the previous reports [2,10,15]. We found 2-hydroxyspermidine as a major polyamine in P. thermocarboxydovorans, in this study. The absence of 3-hydroxyspermidine in the organism suggests that 2-hydroxyspermidine is synthesized by the organism from 2-hydroxyputrescine by propylamine transferase (spermidine synthase) and that the transferase reacts preferentially with the amine distal to the hydroxyl group, as described by Rosano et al. [9]. The occurrence of hydroxydiaminopropane (1,3-diaminopropane-2-ol) in nature has not been reported, but formation of hydroxynorspermidine from exogenously added hydroxydiaminopropane was demonstrated in Vibrio species [16]. Furthermore, P. thermocarboxydoborans contained diaminopr,Jpane in addition to 2-hydroxyputrescine, 2-hydroxyspermidine and spermidine although polyamine patterns in pseudomonads have been separated into three types corresponding to the o subclass (putrescine and homospermidine), ~8 subclass (2-hydroxyputrescine, p u t r ~ c i n e and spermidine) and 1' subclass (diaminopropane, put-eseinc and spermidine) of Proteobacteria [2]. "l-he polyamine pattern of P. thermocarboxydovorans found in this study is quite different from others within Pseudomonas. Since decarboxylation activity for diaminobutyric acid was not found, diaminopropane may be produced by the oxidative cleavage of spermidine in the organism. Generally the species of the a subgroup showed a polyamine pattern with putrescine and homos p e r m i d i n e [2]. Pseudomonas diminuta, P. aminovorans, P. azotocolligans and P. paucimobilis belong to this group [2]. We found that P. carboxydohydrogena contained putrescine and homospermidine as major polyamines in this study. These findings suggest that this organism probably belongs to alpha subclass of Proteobacteria. These Pseudomonas species could be phylogenetically related to Flavobacterium species in the a subclass, in which putrescine and homospermidine are produced as major polyamines [17].
ACKNOWLEDGEMENTS We thank Dr. C. Hurwitz of Veterans Administration Medical Center, Albany, NY, U.S.A. for kindly supplying hydroxyspermidines and American Type Culture Collection, RockviUe, MD, U.S.A. for supplying carboxydobacteria.
REFERENCES [1] Tabor, C,W. and iabor, H. (1985) Microbic'. Rev. 49, 81-99. [21 Busse,J. and Auling, G. (1988) SysL Appl. Microbiol. 11, 1-8. [31 Paller~ni, N.J. (1986) in Bargey's Manual of Systematic Bacteriology,Vol. 1 (Krieg, N.R. and Hell J.G. Eds.), pp. 144-199, Williamsand Wilkins, Baltimore. [4l gowlan, B. and Sc,hlegeLH.G. (1981) Annu. Rev. Microbiol. 35, 405-452. 15] Lyons, C.M., Justin, P., C'~hy, J. and Williams, E. (1984) J. Gan. Microbiol 130, 1017-1105. [6l Meyer, O., Laucant, J. an;I Schlegel,H.G. (1980) int. Syst. Bacteriol. 30. 18~-19f. [7] Okada, M., K,~ashirn.,, S. and lmahori, K. (1979) J. Bioch~m. (T3kyo) 85, 1235-1243. 181 i'oberi, J. ~rid Tchen, T.T. (1971) J. Biol. Chem. 246, 1282-1265. [91 Maisuzaki, S., Hamana, K., Imai, K. and Matsuura, K. (1982) Biochem. Biophys. Res. Commun. 107, 307-313. ll01 Rosario, C.L., Hurwitz, C. and Bunce, S.C. (1978) J. Bacteriol. 135, 805-807. Ill[ Hamana, K. and Matsuzaki, S, (1986) Biochem. Int. 13, 439-445. 112] Hamana, K. and Matsuzaki, S. (1984) J. Biochem.(Tokyo) 95, 1105-1110. 113] Kamekura, M., Hamana, K. and Matsuzaki, S. (1987) FEMS Microbiol. Lett. 43, 301-305. 1141 Kullnig. R., Rosano, C.L. and Hurwitz, C. (1970) Biochem. Biophys. Res. Commun. 39, 1145-1148. 115] Karrer, E., Bose, R.J. and Warren R.A.J. (1973) J. Bacteriol. 114, 1365-1366. 116] Yamamoto, S., Nakan, H., Koumoto, Y., Takashina, K., Kuroda, M. and Shinoda, S. (1989) Chem. Pharm. Bull. (Tokyo) 37. 3139-3141. [171 Hamana, K. and Matsuzaki, S. (1990) Can. J. Microbiol. (in press).
353
FEMSLE 04108
Polyamines of carbon monoxide-utilizing bacteria, Pseudomonas thermocarboxydovorans and Pseudomonas carboxydohydrogena K o e i H a m a n a ~ a n d Shigeru M a t s u z a k i 2 I Collegeq[ Medical Care and Technologyand 2 Departmentof Physiology. Institute of Endocrinology, Gunma University, Maebashi..iapan
Received 18 April 1990 Accepted 26 April 1990 Key words: Carboxydobacteria; Pseudomonas; Polyamine; Hydroxypolyamine
1. S U M M A R Y A slightly thermophilic, CO-utilizing pseudom o n a d , Pseudomonas thermocarboxydooorans grown under both autotrophic and heterotrophic growth conditions was found to contain 2-hydroxyspermidine and 2-hydroxyputrescine in addition to putrescine, diaminopropane and spermidine. A mesophilic C O - utilzing hydrogen bacterium, Pseudomonas carboxydohydrogena contained putrescine and homospermidine under both autotrophic and lieterotrophic growth conditions. Although these two carboxydobacteria are classified to the same genus Pseudomonas, the difference in their polyamine distribution pa:terns suggests thai they may belong to different subclasses of Proteobacteria.
2. I N T R O D U C T I O N Besides putrescine and sp."rmidine many different polyamines and their derivatives have been
Correspondence to: Koei Hamana, Collegeof MedicalCare and Technology, Gunma Universay, Maebashi 371. Japan.
found in microorganisms during the last decade [1]. Although the biological role of polyamines is poorly understood, they have been shown to serve as useful chemotaxonomic markers within certain genera, e.g., Proteobacteria which contain the majority of the well-known Gram-negative eubacteria [2]. A group of heterogenous Gram-negative eubacteria are straight or slightly curved rods, motile by polar flagella, belonging to genus Pseudomonas and arranged into five sections, 1, 11, Ili, IV and V, from their r R N A and D N A homologies [31. Recently, several pseadomonads are allocated to t| tee diffcrent ~ b g r o u p s , a, B and y, of Proteobacleria i,y th,,~r c.i'ference in polyamine distribution patterns [21. Putrescine and homospermid;ne are the major polyamines in Pseudomonas belonging to the alpha subclass. Pseudomonas species in the beta subclass contain 2-liydroxyputrescine, putrescine and spermidine, while putrescine, diaminopropane and spermidine are foo.~d in the Pseudomot, a~ species which belong to the 3' subclass 12] Facultative cF:emolitliotrophs which are able to use CO a n d / o r t t 2 as energy sources and belong to genus Pseudomonas have been isolated [3,4]. It seemed to us of interest to exarmne the relation-
0378-1097/90/$03.50 © 1990 Federation of European MicrobiologicalSocieties
354 ship between the ability to oxidize CO or H2, and polyamine patterns in the organisms. In the present report polyamines have been analyzed in a slightly thermophilic CO-utilizing Pseudomonas thermocarboxydovorans [5] and a mesophilic COutilizing hydrogen bacterium, Pseudomonas carboxydohydrogena [6] grown under autotrophic and heterotrophic growth conditions. The validity of taxonomic positions of these carboxydobacteria was discussed on the basis of their polyamine distribution patterns.
3. MATERIALS AND METHODS 3.1. Organisms and cultures The Gram-negative, aerobic, thermophilic, CO-utilizing organism P. thermocarboxydovorans (ATCC 35961) was cultivated in the basal medium under 50% CO-50% air at 5 0 ° C for autotrophic growth [5] tl: is not oxidized by this organism. A Gram-negative, aerobic, mesophilic, CO-utilizing hydrogen bacterium, P. carboxydohydrogena (ATCC 29978) was cultured in the basal medium under 40% CO-10% 02-50% N 2 or 80% H2-10% O2-10% CO 2 at 30°C for autotrophic growth [6]. For heterotrophic growth, the media were supplemented with 0.5% of sodium pyruvate. The organisms were also grown beterotrophically in the synthetic mammalian cell culture 199 medium. Logarithmically growing cells or stationary phase cells were harvested. 3.2. Polyamme analysis Diaminopropane - 2HCI, putrescine- 2HCI and spermidine-3HCI were purchased from Sigma (MO, U.S.A.). Homospermidine was synthesized by a~ylation of putrescine with to-bromoalkylphthalimide [7]. 2-Hydroxyputrescine was synthesized by ammoniolysis of 1,4-dibromobutan-2-ol [8]. The mixture of 2-hydroxyspermidine and 3-hydroxyspermidine was kindly supplied by Dr. C. Hurwitz. After centrifugation of the cultures, pellets of organisms were homogenized in equal volumes of cold 1.0 M HCIO4. Polyamines in perchloric acid extracts were analyzed by high-performance liquid chromatography (HPLC) using KCl-citrate buffer system as
reported previously [9]. Some samples were analyzed before and after the hydrolysis with 6 M HC! or 1 M NaOH for 24 h at 110°C or the periodate oxidation with 0.1 M sodium metaperiodate in 0.67 M NaOH for 1 h at 5 6 ° C [10]. The identity of polyamines in the extracts ~,as confirmed by thin-layer chromatography (TLC) un cellulose using the solvent system of isopropanol/ ammonia (7:3) [11] or on silica gel using the solvent system of c h l o r o f o r m / m e t h a n o l / ammonia ( 2 : 2 : 1 ) [12]. For assay of the decarboxylase activities, the lysates from logarithmically growing cells were added to the reaction mixture containing respective amino acids and then incubated according to our previous report [13]. Polyamines produced during incubation were analyzed by HPLC method.
4. RESULTS Five different polyamines were detected in P. thermocarboxydovorans grown under both autotrophic and heterotrophic growth conditions. These polyamines were resistant to the acid and alkafine hydrolyses. The five polyamines corresponded to 2-hydroxyputrescine, diaminopropane, putrescine, hydroxyspermidine (2-hydroxyspermidine a n d / o r 3-hydroxysperrnidine) and spermidine (Fig. 1). The two peaks corresponding to 2-hydroxyputreseine and hydroxyspermidine completely disappeared after the reaction with metap~riodate. Authentic 2-hydroxy and 3-hydroxy isomers of spermidine were not separated either on HPLC or TLC. Since the diaminopropane peak did not increase after the periodate oxidation, the hydroxyspermidine fraction of the organism was identified to be 2-hydroxyspermidine. The coexistence of 3-hydroxyspermidine was excluded, because no diaminopropane was produced by the cleavage. Four other polyamines were identified by TLC (data not shown). P. curboxydohydrogena contained two polyamine peaks corresponding to putrescine and homospermidine (Fig. 1). The polyamines were also identified on TLC (data not shown). Cellular polyamine concentrations were shown in Table 1. In the two carboxydobacteria, cellular
355 the organisms may be due to net polyamine synthesis. The two carboxydobacteria decarboxylated ornithine to produce putrescine but not either diaminobutyric acid+ lysine or arginine in vitro.
5. D I S C U S S I O N The polyamine distribution patterns of P. ther-
[3 ~
mocarboxydovorans and
~t
io
A ELUTION
3'o
P. carboxydohydrogena
are quite constant when grown under the different growth conditions or during different growth phases. These findings suggest that their polyamines can be one of the stable cheraotaxonomic markers of the organisms and that it is not connected to their chemoautotrophic activity oxidizing CO a n d / o r H2 as energy source. 2-Hydroxyputrescine was first found in Pseudomonas sp. K/m [14] and then in P. solanacearus,
.b
TIE (mn)
Fig. 1. HPLC of polyammes of P. thermocarboxydovorans before (A) and after (B) the periodate oxidation, and polyarrAnes of P. carboxydohydrogena (C). The orgamsms were grown in 199 medium.Abbreviationsfor polyamincsate shown in Table 1. levels of polyamines as well as polyamine components were nearly the same at different growth phases and in both autotrophic and heterotrophic growth. Since all of the culture media used are free from polyamines, the polyamines detected in
P. caryophlii, P. cepacia, P. marginata, P. facilis and P. delafieldii [2] m genus Pseudomonas. Comamonas acidovorans (formerly Pseudomonas acidovorans), C. testosteroni, C. terrigena, five species of Alcaligenes, Ach¢omobacter .rylosoxydans, Rhodocyclus gelatinosus. Ch,'omobacterium violaceum and Janthinobacterium lividum belonging to the beta subclass of Proteobacteria also contain .7.-hydroxyputrescin¢ [2,15]¢ C. acidovorans and R. gelatinosus contain, although in low concentrations, 2-hydroxyspermidine [2,10]. However+ identification and quantitative analysis of these unusual hy-
Table 1 Cellular concentrations of polyarmnesof Pseudomonasthermocarbo~vdovoransand P. carbox3~ol~ydrogena H-Put, 2-hydroxyputrescine; Dap, diaminopropan¢; Put. putrescine; H-Spd. 2-hydroxyspernudine;Spd. sperrmd~ne; HSpd, homospermidine: ND, not detected; (S). slationary phase; (L), logarithmicallygrowingphase; ATCC. AmericanType Culture Collection. Rockville. MD U.S.A. Culture conditions are described in MATERIALSAND METHODS. Cuhure condition
P. thermocarboxydovorans (ATCC 35961)
P. corboxydohydrogena (ATCC 29978)
CO/air Pyruvate 199 199 CO/O~/N 2 H2/02/CO 2 Pyruvate 199 199
(S) (S) (L) (S) (S) (S) (S) (L) (SI
Polyamines(pmol/g wet ~¢eight) H-Put Dap PUl H-Spd
Spd
HSpd
0.200 0.342 0.382 0.129 ND ND ND ND ND
0.280 0.540 0.680 0.760 ND ND ND ND ND
ND ND ND ND 0.520 0.700 4.055 0.560 0700
0.020 0.180 ND 0.100 ND ND ND ND ND
0.100 0.134 0.024 0.500 0.240 0.220 l.O00 0.100 0.160
0.040 0.100 0020 0.400 ND ND ND ND ND
356 droxypolyamines in the organisms have not been sufficient in the previous reports [2,10,15]. We found 2-hydroxyspermidine as a major polyamine in P. thermocarboxydovorans, in this study. The absence of 3-hydroxyspermidine in the organism suggests that 2-hydroxyspermidine is synthesized by the organism from 2-hydroxyputrescine by propylamine transferase (spermidine synthase) and that the transferase reacts preferentially with the amine distal to the hydroxyl group, as described by Rosano et al. [9]. The occurrence of hydroxydiaminopropane (1,3-diaminopropane-2-ol) in nature has not been reported, but formation of hydroxynorspermidine from exogenously added hydroxydiaminopropane was demonstrated in Vibrio species [16]. Furthermore, P. thermocarboxydoborans contained diaminopr,Jpane in addition to 2-hydroxyputrescine, 2-hydroxyspermidine and spermidine although polyamine patterns in pseudomonads have been separated into three types corresponding to the o subclass (putrescine and homospermidine), ~8 subclass (2-hydroxyputrescine, p u t r ~ c i n e and spermidine) and 1' subclass (diaminopropane, put-eseinc and spermidine) of Proteobacteria [2]. "l-he polyamine pattern of P. thermocarboxydovorans found in this study is quite different from others within Pseudomonas. Since decarboxylation activity for diaminobutyric acid was not found, diaminopropane may be produced by the oxidative cleavage of spermidine in the organism. Generally the species of the a subgroup showed a polyamine pattern with putrescine and homos p e r m i d i n e [2]. Pseudomonas diminuta, P. aminovorans, P. azotocolligans and P. paucimobilis belong to this group [2]. We found that P. carboxydohydrogena contained putrescine and homospermidine as major polyamines in this study. These findings suggest that this organism probably belongs to alpha subclass of Proteobacteria. These Pseudomonas species could be phylogenetically related to Flavobacterium species in the a subclass, in which putrescine and homospermidine are produced as major polyamines [17].
ACKNOWLEDGEMENTS We thank Dr. C. Hurwitz of Veterans Administration Medical Center, Albany, NY, U.S.A. for kindly supplying hydroxyspermidines and American Type Culture Collection, RockviUe, MD, U.S.A. for supplying carboxydobacteria.
REFERENCES [1] Tabor, C,W. and iabor, H. (1985) Microbic'. Rev. 49, 81-99. [21 Busse,J. and Auling, G. (1988) SysL Appl. Microbiol. 11, 1-8. [31 Paller~ni, N.J. (1986) in Bargey's Manual of Systematic Bacteriology,Vol. 1 (Krieg, N.R. and Hell J.G. Eds.), pp. 144-199, Williamsand Wilkins, Baltimore. [4l gowlan, B. and Sc,hlegeLH.G. (1981) Annu. Rev. Microbiol. 35, 405-452. 15] Lyons, C.M., Justin, P., C'~hy, J. and Williams, E. (1984) J. Gan. Microbiol 130, 1017-1105. [6l Meyer, O., Laucant, J. an;I Schlegel,H.G. (1980) int. Syst. Bacteriol. 30. 18~-19f. [7] Okada, M., K,~ashirn.,, S. and lmahori, K. (1979) J. Bioch~m. (T3kyo) 85, 1235-1243. 181 i'oberi, J. ~rid Tchen, T.T. (1971) J. Biol. Chem. 246, 1282-1265. [91 Maisuzaki, S., Hamana, K., Imai, K. and Matsuura, K. (1982) Biochem. Biophys. Res. Commun. 107, 307-313. ll01 Rosario, C.L., Hurwitz, C. and Bunce, S.C. (1978) J. Bacteriol. 135, 805-807. Ill[ Hamana, K. and Matsuzaki, S, (1986) Biochem. Int. 13, 439-445. 112] Hamana, K. and Matsuzaki, S. (1984) J. Biochem.(Tokyo) 95, 1105-1110. 113] Kamekura, M., Hamana, K. and Matsuzaki, S. (1987) FEMS Microbiol. Lett. 43, 301-305. 1141 Kullnig. R., Rosano, C.L. and Hurwitz, C. (1970) Biochem. Biophys. Res. Commun. 39, 1145-1148. 115] Karrer, E., Bose, R.J. and Warren R.A.J. (1973) J. Bacteriol. 114, 1365-1366. 116] Yamamoto, S., Nakan, H., Koumoto, Y., Takashina, K., Kuroda, M. and Shinoda, S. (1989) Chem. Pharm. Bull. (Tokyo) 37. 3139-3141. [171 Hamana, K. and Matsuzaki, S. (1990) Can. J. Microbiol. (in press).