- Email: [email protected]
Selective enrichment and isolation of Rhodopseudomonas palustris using trans-cinnamic acid as sole carbon source
FEMS Microbiology Ecology 53 (1988) 53-58 Published by Elsevier
53
FEC 00148
Selective enrichment and isolation of Rhodopseudomonas palustris using trans-cinnamic acid as sole carbon source M i c h a e l T. M a d i g a n a a n d H o w a r d G e s t b o Department of Microbiology, Southern Illinois University, Carbondale, IL, and b Photosynthetic Bacteria Group, Department of Biology, Indiana University, Bloomington, IN, U.S.A. Received 12 August 1987 Accepted 26 August 1987 Key words: Nonsulphur purple bacterium; Photosynthesis; Aromatic catabolism; ( Rhodopseudomonas palustris)
1. S U M M A R Y Enrichment cultures for anoxygenic phototrophs capable of using cinnamic acid as sole organic carbon source consistently yielded the nonsulfur purple bacterium Rhodopseudomonas palustris. Pure cultures of R. palustris obtained from the enrichments grew photoheterotrophically on cinnamate and benzoate as well as on derivatives of these compounds. Photosynthetic growth on cinnamate was greatly stimulated by addition of exogenous CO2, and resulted in breakage of the aromatic nucleus. Growth yield studies suggested that cinnamate was converted by R. palustris to intermediates that can be quantitatively assimilated into cell material.
2. I N T R O D U C T I O N Nonsulfur purple phototrophic bacteria are remarkable in respect to their nutritional diversity Correspondence to: M.T. Madigan, Department of Microbiology, Southern Illinois University, Carbondale, IL 62901, U.S.A.
and ability to grow using a number of alternative energy-generating mechanisms [1-2]. With light as the energy source a wide variety of organic compounds can support anaerobic growth of these organisms [3]. The first report of utilization of an aromatic compound - benzoic acid - as carbon source by nonsulfur purple bacteria was made by Scher and Proctor who isolated several strains of phototrophic bacteria capable of growth on benzoate [4]. Subsequent investigations of benzoate catabolism by nonsulfur purple bacteria focused on the species Rhodopseudomonas palustris, where it has been shown that phototrophic catabolism of benzoate proceeds by a reductive rather than an oxidative ring cleavage mechanism [5,6]. The reductive pathway is now thought to be the universal mechanism by which aromatic compounds are degraded anaerobically by chemotrophic as well as phototrophic bacteria [7-9]. In experiments designed to assess the catabolic versatility of nonsulfur purple bacteria for aromatic compounds we repeatedly observed the rapid development of dense cultures of R. palustris in enrichments containing cinnamic acid as sole organic carbon source. This paper describes the enrichment conditions employed for the isolation
0168-6496/88/$03.50 © 1988 Federation of European Microbiological Societies
54 of R. palustris strains that utilize cinnamic acid, and details nutritional experiments carried out with two representative isolates.
3. MATERIALS AND METHODS
3.1. Source of organisms Samples for enrichment cultures were obtained from moist leaf litter and pond sediment obtained in the vicinity of Bloomington, Indiana, and Carbondale, Illinois, from soil samples obtained from rice paddies in Thailand and Sri Lanka, and from the effluent of a creosote plant operating near Bloomington.
3.2. Media and growth conditions The basal medium used for enrichment and for growth of pure cultures was a modification of the enrichment medium described in [10]. The medium contained, per liter: EDTA, 5 mg; MgSO4- 7H20, 200 mg; CaC12 • 2H20, 75 mg; NaC1, 1 g; FeSO4. 7H20, 6 mg; trace dements [10], 1 ml; KH2PO 4, 0.3 g; K2HPO 4, 0.45 g; NH4CI, 0-0.5 g; NaHCO 3, 2 g; yeast extract, 50 mg; vitamin solution described in [10] carbon source, 3-6 mM. All media were adjusted to pH 7 and sterilized by autoclaving. Carbon sources and NaHCO 3 were added to cooled media from filter-sterilized stock solutions. Immediately following autoclaving all media were stored in an anaerobic hood (Coy Laboratory Products, Ann Arbor, MI) under an atmosphere of 85%N 2 + 1 0 % H 2 + 5 % C O 2. Enrichments were established in 10-50 ml Hypo vials containing 6-30 ml of medium and were sealed with sterile butyl rubber stoppers within the anaerobic hood. Pure cultures were obtained from liquid enrichments by repeated streaking on agar plates of the medium specified (solidified with 1.5% distilled water-washed Difco agar). Plates were incubated in Gas Pak jars (Becton, Dickinson and Co.) filled with 100% N 2. For growth and nutritional studies, pure cultures were grown in completely filled screw-capped tubes (17 ml) or bottles (200 ml). All enrichments and subsequent cultures were incubated at 27 °C in a light cabinet fitted with incandescent lamps (light intensity 1500 lux).
3.3. Measurement of growth and pigments. Growth of pure cultures was determined turbidimetrically using a Klett-Summerson colorimeter (660 filter). For the two strains of Rhodopseudomonas palustris studied in detail, 100 photometer units were equivalent to approximately 0.21 mg cell material (dry weight) per milliliter of culture. Dry weights were determined as previously described [1]. Absorption spectra of intact cells in 30% bovine serum albumin [1] were determined using a Beckman DU-6 spectrophotometer.
3.4. Chemicals and chemical assays All chemicals were of reagent grade. Aromatic compounds were obtained from either Sigma Chemical Co. (St. Louis, MO) or Aldrich Chemicals (Milwaukee, WI). All solutions were made with deionized water. Trans-Cinnamic acid was detected by its absorption maximum at 269 nm.
4. RESULTS AND DISCUSSION Illuminated anaerobic enrichments employing mineral salts media supplemented with nonfermentable organic acids as carbon source and N 2 as sole nitrogen source previously have been found to be highly selective for anoxygenic photosynthetic bacteria [10]. In a series of photosynthetic enrichments established using trans-cinnamic acid as sole organic carbon source and either moist leaf litter, mud and pond water, or moist or dry soil as inoculum, the nonsulfur purple bacterium Rhodopseudomonas palustris was found to develop rapidly. For example, with 3 mM cinnamate as carbon source and N 2 as sole nitrogen source, bright red-pigmented cultures containing highly motile budding bacteria, frequently arranged in rosettes, were obtained in as little as 4-5 days. Enrichments established with NH~- as nitrogen source developed even more rapidly - pigmentation was evident within 24 h and dense cultures were observed within 72 h. Occasionally other species of nonsulfur purple bacteria were observed in cinnamate enrichments. Cells morphologically resembling Rhodobacter capsulatus and sphaeroides, Rhodocyclus gelatino-
55
sus, Rhodospirillum fulvum, and Rhodomicrobium vannielii were observed in several instances, but R. palustris was usually the predominant phototroph present. In twelve separate enrichments eight yielded R. palustris; the remaining enrichments were golden brown or red-brown in color and contained an abundance of small, highly motile rods resembling Rc. gelatinosus. The initial streak of primary enrichments on agar plates always yielded a variety of different colored colonies. But, with the exception of the Rc. gelatinosus-like organism mentioned above, only organisms identified as R. palustris gave rise to large colonies that could be transferred repeatedly on cinnamate media. Because of the highly selective and rapid nature of the phototrophic enrichments, no interference was observed from heterotrophic cinnamate-degrading bacteria [9,11,12]. Two R. palustris isolates were chosen for more detailed investigation. The isolates, strains SL and LL, obtained from Sri Lankan soil and leaf litter enrichments, respectively, appeared to be typical strains of R. palustris. Both strains produced burgundy red-colored colonies on cinnamate plates and showed the characteristic budding morphology typical of R. palustris [3]. Absorption spectra of intact cells of both strains were virtually identical (Fig. 1); peaks were observed at 863, 805, I.o
,
0.75
l
,
l
,
l
,
[ 6oo ~-
i
l
539 and 506 nm, typical of R. palustris [13]. Growth of strain SL in mineral media containing cinnamate was greatly stimulated by exogenous CO2 (supplied as HCO 3, Fig. 2). The generation time of strain SL on cinnamate/CO2 was about 12 h, and extremely high cell densities (greater than 1 mg/ml dry weight) could be obtained in mineral media containing 6 mM cinnamate as carbon source (Fig. 2). Some toxicity to cinnamate was noted; cultures grown on 7-12 mM cinnamate lagged considerably behind cultures grown on 1-6 raM, and initial cirmamate concentrations in excess of about 12 mM were totally growth inhibitory. Nutritional experiments using a variety of cinnamate and benzoate derivatives were performed with strains SL and LL. The results in Table 1 show that the nutritional patterns of the two strains, although similar, were not identical. Both strains utilized cinnamate, phenylpropionate, mcoumarate, and benzoate. In addition, strain SL, but not strain LL, used p-coumarate and caffeic acid. Strain LL grew slowly on o-coumarate. Phenylacetate, protocatechuate and cinnamyl alcohol were not used as carbon sources by either strain of R. palustris. Although the metabolic route of cinnamate catabolism by R. palustris is unknown at this time, I
i
I
o g ~ 0.5
i'12
,
5
0,25 [-
I
I
f
/
I
340
I
~
J
I
452
i
I
564
,?
L
I
676
,
I
788
0
i
~
-
o
5O
i
900
Wavelength (nm) Fig. 1. Absorption spectra of R. palustris strains SL ( ) and LL (- - -) isolated from cinnamic acid enrichments. In vivo spectra performed as described in [1].
o
12 Time
(h)
Fig. 2. Growth of R. palustris strain SL in a mineral medium containing cinnamic acid as sole organic carbon source in the presence and absence of 0.2~ NaHCO 3.
56 Table 1 Utilization of cinnamate and benzoate derivatives as sole carbon sources for growth of Rhodopseudomonas palustris strains SL and LL All substrates were at 6 mM final concentration except for cinnamyl alcohol, 4 mM. All media contained 0.25g N a H C O 3. + , 100-200 photometer units; + + , 300-400 photometer units; + + + , 400-500 photometer units; 0, no growth. 100 photometer units are equivalent to approximately 0.21 mg bacterial dry weight per milliliter. The following compounds did not support growth of either strain: protocatechuate, gentisate, phenylacetate, cinnamyl alcohol. Compounds
Growth
Cinnamate Phenylpropionate o-Coumarate m-Coumarate p-Coumarate Benzoate Caffeate
Strain SL
Strain LL
+ + 0 + + + +
+ + + + 0 + 0
+ + + + + + + + +
+ + + + + + +
growth studies with strain SL indicated that the aromatic ring was broken during phototrophic growth on this substrate. Fig. 3 shows the ultraviolet spectrum of fresh and spent culture media following growth of strain SL on cinnamate. The major peak at 269 nm due to cinnamic acid present in fresh media was absent from spent media,
0"81
0.6
~
I
I
I
T
I
'
1
'
I
0 C
indicating that the aromatic character of cinnamate was destroyed during phototrophic growth on this compound. By analogy to benzoate catabolism by R. palustris [8] cinnamate is probably converted to a linear molecule following ring reduction and breakage, and the resulting longchain fatty acid derivative is used as a carbon source for growth. This would explain the stimulatory effect of CO 2 on cinnamate catabolism by R. palustris (Fig. 2), since the utilization of long-chain fatty acids by phototrophic bacteria is generally CO 2 dependent [2]. The requirement for CO2, however, was not observed when strain SL grew on benzoate. The simplest explanation for this result would be that cinnamate, a C-9 compound and two carbon atoms longer than benzoate, forms a relatively reduced molecule whose assimilation requires CO 2 as an electron sink. Alternatively, the CO 2 requirement for photosynthetic cinnamate catabolism by R. palustris could signal the operation of a different type of catabolic pathway for this particular substrate than for benzoate. Our conclusion that cinnamate is converted by R. palustris into an intermediate that eventually enters central metabolic pathways is evident from studies of growth yields on cinnamate. A concentration of 1 m M (148 m g / 1 ) cinnamate was found to support a growth yield of about 200 mg (dry weight) of cells per liter (of strain SL). Assuming the cells to be 50% carbon this indicates that 100 mg cell carbon are produced from 148 mg cinnamate. Since cinnamate contains 73% carbon by weight, only 107 mg of carbon are potentially available from 148 mg of cinnamate. Thus, it appears that cinnamate is catabolized by R. palustris to intermediates that can be used quantitatively for the production of cell material.
0.4
¢.,, <
5. E C O L O G I C A L C O N S I D E R A T I O N S
200
228
256
284
Wavelength (nm)
r--T312 344
Fig. 3. Spectrum of a 10-2 dilution of fresh culture medium containing 6 mM trans-cinnamic acid ( ), and a 10-1 dilution of culture supernatant followinggrowth of R. palustris strain SL to 425 photometer units (- - -).
Cinnamic acid is an aromatic compound of particular interest because it is one of several monomeric molecules which can be isolated from plant lignins [11]. If the capacity for cinnamate catabolism is any indication of the potential of nonsulfur purple bacteria for catabolism of other lignin derivatives, it is possible that these
57 o r g a n i s m s p l a y a significant role in the t r a n s f o r m a t i o n of several lignin b y p r o d u c t s in nature. R e c e n t studies b y H a r w o o d a n d G i b s o n [14] s t r o n g l y s u p p o r t this c o n c l u s i o n a n d i n d i c a t e t h a t R. palustris is a p a r t i c u l a r l y n o t e w o r t h y species in terms of a r o m a t i c c a t a b o l i s m . T h e r a p i d i t y with which c i n n a m a t e / m i n e r a l salts e n r i c h m e n t s d e v e l o p e d in o u r s t u d y suggests t h a t this s u b s t r a t e m a y b e r a p i d l y p h o t o m e t a b o lized in nature. C i n n a m a t e has b e e n s h o w n to b e c a t a b o l i z e d b y b o t h d e n i t r i f y i n g b a c t e r i a [12] a n d a microbial consortium containing methanogenic b a c t e r i a [11]. D e n i t r i f y e r s were specifically exc l u d e d f r o m our e n r i c h m e n t s b e c a u s e of a l a c k of n i t r a t e in the e n r i c h m e n t m e d i u m ; m e t h a n o g e n i c c o n s o r t i a were n o t specifically excluded. Such c o n s o r t i a are a p p a r e n t l y u n a b l e to c o m p e t e with c i n n a m a t e - u t i l i z i n g p h o t o t r o p h s . T h e a d d e d req u i r e m e n t for N 2 fixation in o u r e n r i c h m e n t s p r o b a b l y excludes m o s t h e t e r o t r o p h s c a p a b l e of attacking cinnamate. N o n s u l f u r p u r p l e p h o t o t r o p h i c b a c t e r i a seem to b e ideal c a n d i d a t e s for a r o m a t i c c a t a b o l i s m in n a t u r e because, unlike h e t e r o t r o p h i c organisms, p h o t o t r o p h i c b a c t e r i a d o n o t r e q u i r e energy f r o m c a t a b o l i s m of the a r o m a t i c substrate. Such substrates o n l y n e e d serve as c a r b o n sources. T h e i s o l a t i o n of c i n n a m a t e - d e g r a d i n g strains of R. palustris f r o m a variety o f different h a b i t a t s suggests that these strains are u b i q u i t o u s a n d p o s s i b l y o f ecological i m p o r t a n c e in respect to a r o m a t i c c a t a b o l i s m . In fact, a p r e l i m i n a r y screen of several p u r e cultures of R. palustris originally i s o l a t e d on n o n a r o m a t i c o r g a n i c acids suggests that c i n n a m a t e c a t a b o l i s m b y this species m a y i n d e e d b e w i d e s p r e a d (see also Ref. 14). I n a d d i t i o n , recent e x p e r i m e n t s with resting cells o f a strain of R. palustris e n r i c h e d a n d i s o l a t e d on b e n z o a t e [15] has shown that a wide variety of a r o m a t i c substrates ( i n c l u d i n g s o m e t h a t are u n a b l e to s u p p o r t g r o w t h ) a r e p h o t o m e t a b o l i z e d b y this o r g a n i s m at high rates. C i n n a m a t e was p a r t i c u l a r l y n o t e w o r t h y in this c o n n e c t i o n [15]. ACKNOWLEDGEMENTS This research was s u p p o r t e d b y g r a n t P C M 8505492 (to M . T . M . ) a n d PCM-8415291 (to H . G . )
f r o m the U.S. N a t i o n a l Science F o u n d a t i o n . W e t h a n k Jeffrey L. F a v i n g e r for help w i t h the collection of samples, a n d J o h n G. O r m e r o d , U n i v e r s i t y of Oslo, N o r w a y , for s u p p l y i n g the T h a i l a n d a n d Sri L a n k a n samples.
REFERENCES [1] Madigan, M.T. and Gest, H. (1979) Growth of the photosynthetic bacterium Rodopseudomonas capsulata chemoautotrophicaUy in darkness with H 2 as the energy source. J. Bacteriol. 137, 524-530. [2] Biebl, H. and Pfennig, N. (1981) Isolation of members of the family Rhodospirillaceae. In: The Prokaryotes - A Handbook on Habitats, Isolation, and Identification of Bacteria (Starr, M.P., Stolp, H., Triiper, H.G., Balows, A. and Schlegel, H.G., Eds.), pp. 267-273, Springer-Verlag, Berlin. [3] Triiper, H.G. and Pfennig, N. (1981) Characterization and identification of the anoxygenic phototrophic bacteria. In: The Prokaryotes - A Handbook on Habitats, Isolation, and Identification of Bacteria. (Starr, M.P., Stolp, H., Triiper, H.G., Balows, A. and Schlegel, H.G., Eds.), pp. 299-312, Springer-Verlag, Berlin. [4] Scher, S. and Proctor, M.H. (1960) Studies with photosynthetic bacteria: Anaerobic oxidation of aromatic compounds. In: Comparative Biochemistry of Photoreactive Systems (Allen, M.B., Ed.), pp. 387-393, Academic Press, New York. [5] Dutton, P.L and Evans, W.C. (1969) The metabolism of aromatic compounds by Rhodopseudomonaspalustris. Biochem. J. 113, 525-536. [6] Guyer, M. and Hegeman, G. (1969) Evidence for a reductive pathway for anaerobic metabolism of benzoate. J. Bacteriol. 99, 906-907. [7] Sleat, R. and Robinson, J.P. (1984) The bacteriology of anaerobic degradation of aromatic compounds. J. Appl. Bacteriol. 57, 381-394. [8] Dutton, P.L. and Evans, W.C. (1978) Metabolism of aromatic compounds by Rhodospirillaceae. In: The Photosynthetic Bacteria (Clayton, R.K. and Sistrom, W.R., Eds.), pp. 719-726, Plenum Press, New York. [9] Evans, W.C. (1977) Biochemistry of the bacterial catabolism of aromatic compounds in anaerobic environments. Nature (London) 270, 17-22. [10] Gest, H., Favinger, J.L. and Madigan, M.T. (1985) Exploitation of N 2 fixation capacity for enrichment of anoxygenic photosynthetic bacteria in ecological studies. FEMS Microbiol. Ecol. 31, 317-322. [11] Healy, J.B., Jr. and Young, L.Y. (1979) Anaerobic biodegradation of eleven aromatic compounds to methane. Appl. Environ. Microbiol. 38, 84-89. [12] Williams, R.J. and Evans, W.C. (1975) The metabolism of benzoate by Moraxella sp. through anaerobic nitrate respiration: evidence for a reductive pathway. Biochem. J. 148, 1-10.
58 [13] Biebl, H. and Drews, G. (1969) Das in-vivo-spektrum als taxonomisches merkmal bei untersuchungen zur verbreitung der Athiorhodaceae. Zentralblatt fiir Bakteriologie, Parasitenkunde, Infektionskrankheiten und Hygiene, Abt. 2 123, 425-452. [14] Harwood, C.S. and Gibson, J. (1987) Anaerobic and aerobic metabolism of diverse aromatic compounds by the
photosynthetic bacterium Rhodopseudomonas palustris. Appl. Environ. Microbiol. In press. [15] Tanaka, H., Maeda, H., Suzuki, H., Kamibayashi, A. and Tonomura, K. (1982) The metabolism of thiophene-2carboxylate by a photosynthetic bacterium. Agric. Biol. Chem. 46, 1429-1438.
53
FEC 00148
Selective enrichment and isolation of Rhodopseudomonas palustris using trans-cinnamic acid as sole carbon source M i c h a e l T. M a d i g a n a a n d H o w a r d G e s t b o Department of Microbiology, Southern Illinois University, Carbondale, IL, and b Photosynthetic Bacteria Group, Department of Biology, Indiana University, Bloomington, IN, U.S.A. Received 12 August 1987 Accepted 26 August 1987 Key words: Nonsulphur purple bacterium; Photosynthesis; Aromatic catabolism; ( Rhodopseudomonas palustris)
1. S U M M A R Y Enrichment cultures for anoxygenic phototrophs capable of using cinnamic acid as sole organic carbon source consistently yielded the nonsulfur purple bacterium Rhodopseudomonas palustris. Pure cultures of R. palustris obtained from the enrichments grew photoheterotrophically on cinnamate and benzoate as well as on derivatives of these compounds. Photosynthetic growth on cinnamate was greatly stimulated by addition of exogenous CO2, and resulted in breakage of the aromatic nucleus. Growth yield studies suggested that cinnamate was converted by R. palustris to intermediates that can be quantitatively assimilated into cell material.
2. I N T R O D U C T I O N Nonsulfur purple phototrophic bacteria are remarkable in respect to their nutritional diversity Correspondence to: M.T. Madigan, Department of Microbiology, Southern Illinois University, Carbondale, IL 62901, U.S.A.
and ability to grow using a number of alternative energy-generating mechanisms [1-2]. With light as the energy source a wide variety of organic compounds can support anaerobic growth of these organisms [3]. The first report of utilization of an aromatic compound - benzoic acid - as carbon source by nonsulfur purple bacteria was made by Scher and Proctor who isolated several strains of phototrophic bacteria capable of growth on benzoate [4]. Subsequent investigations of benzoate catabolism by nonsulfur purple bacteria focused on the species Rhodopseudomonas palustris, where it has been shown that phototrophic catabolism of benzoate proceeds by a reductive rather than an oxidative ring cleavage mechanism [5,6]. The reductive pathway is now thought to be the universal mechanism by which aromatic compounds are degraded anaerobically by chemotrophic as well as phototrophic bacteria [7-9]. In experiments designed to assess the catabolic versatility of nonsulfur purple bacteria for aromatic compounds we repeatedly observed the rapid development of dense cultures of R. palustris in enrichments containing cinnamic acid as sole organic carbon source. This paper describes the enrichment conditions employed for the isolation
0168-6496/88/$03.50 © 1988 Federation of European Microbiological Societies
54 of R. palustris strains that utilize cinnamic acid, and details nutritional experiments carried out with two representative isolates.
3. MATERIALS AND METHODS
3.1. Source of organisms Samples for enrichment cultures were obtained from moist leaf litter and pond sediment obtained in the vicinity of Bloomington, Indiana, and Carbondale, Illinois, from soil samples obtained from rice paddies in Thailand and Sri Lanka, and from the effluent of a creosote plant operating near Bloomington.
3.2. Media and growth conditions The basal medium used for enrichment and for growth of pure cultures was a modification of the enrichment medium described in [10]. The medium contained, per liter: EDTA, 5 mg; MgSO4- 7H20, 200 mg; CaC12 • 2H20, 75 mg; NaC1, 1 g; FeSO4. 7H20, 6 mg; trace dements [10], 1 ml; KH2PO 4, 0.3 g; K2HPO 4, 0.45 g; NH4CI, 0-0.5 g; NaHCO 3, 2 g; yeast extract, 50 mg; vitamin solution described in [10] carbon source, 3-6 mM. All media were adjusted to pH 7 and sterilized by autoclaving. Carbon sources and NaHCO 3 were added to cooled media from filter-sterilized stock solutions. Immediately following autoclaving all media were stored in an anaerobic hood (Coy Laboratory Products, Ann Arbor, MI) under an atmosphere of 85%N 2 + 1 0 % H 2 + 5 % C O 2. Enrichments were established in 10-50 ml Hypo vials containing 6-30 ml of medium and were sealed with sterile butyl rubber stoppers within the anaerobic hood. Pure cultures were obtained from liquid enrichments by repeated streaking on agar plates of the medium specified (solidified with 1.5% distilled water-washed Difco agar). Plates were incubated in Gas Pak jars (Becton, Dickinson and Co.) filled with 100% N 2. For growth and nutritional studies, pure cultures were grown in completely filled screw-capped tubes (17 ml) or bottles (200 ml). All enrichments and subsequent cultures were incubated at 27 °C in a light cabinet fitted with incandescent lamps (light intensity 1500 lux).
3.3. Measurement of growth and pigments. Growth of pure cultures was determined turbidimetrically using a Klett-Summerson colorimeter (660 filter). For the two strains of Rhodopseudomonas palustris studied in detail, 100 photometer units were equivalent to approximately 0.21 mg cell material (dry weight) per milliliter of culture. Dry weights were determined as previously described [1]. Absorption spectra of intact cells in 30% bovine serum albumin [1] were determined using a Beckman DU-6 spectrophotometer.
3.4. Chemicals and chemical assays All chemicals were of reagent grade. Aromatic compounds were obtained from either Sigma Chemical Co. (St. Louis, MO) or Aldrich Chemicals (Milwaukee, WI). All solutions were made with deionized water. Trans-Cinnamic acid was detected by its absorption maximum at 269 nm.
4. RESULTS AND DISCUSSION Illuminated anaerobic enrichments employing mineral salts media supplemented with nonfermentable organic acids as carbon source and N 2 as sole nitrogen source previously have been found to be highly selective for anoxygenic photosynthetic bacteria [10]. In a series of photosynthetic enrichments established using trans-cinnamic acid as sole organic carbon source and either moist leaf litter, mud and pond water, or moist or dry soil as inoculum, the nonsulfur purple bacterium Rhodopseudomonas palustris was found to develop rapidly. For example, with 3 mM cinnamate as carbon source and N 2 as sole nitrogen source, bright red-pigmented cultures containing highly motile budding bacteria, frequently arranged in rosettes, were obtained in as little as 4-5 days. Enrichments established with NH~- as nitrogen source developed even more rapidly - pigmentation was evident within 24 h and dense cultures were observed within 72 h. Occasionally other species of nonsulfur purple bacteria were observed in cinnamate enrichments. Cells morphologically resembling Rhodobacter capsulatus and sphaeroides, Rhodocyclus gelatino-
55
sus, Rhodospirillum fulvum, and Rhodomicrobium vannielii were observed in several instances, but R. palustris was usually the predominant phototroph present. In twelve separate enrichments eight yielded R. palustris; the remaining enrichments were golden brown or red-brown in color and contained an abundance of small, highly motile rods resembling Rc. gelatinosus. The initial streak of primary enrichments on agar plates always yielded a variety of different colored colonies. But, with the exception of the Rc. gelatinosus-like organism mentioned above, only organisms identified as R. palustris gave rise to large colonies that could be transferred repeatedly on cinnamate media. Because of the highly selective and rapid nature of the phototrophic enrichments, no interference was observed from heterotrophic cinnamate-degrading bacteria [9,11,12]. Two R. palustris isolates were chosen for more detailed investigation. The isolates, strains SL and LL, obtained from Sri Lankan soil and leaf litter enrichments, respectively, appeared to be typical strains of R. palustris. Both strains produced burgundy red-colored colonies on cinnamate plates and showed the characteristic budding morphology typical of R. palustris [3]. Absorption spectra of intact cells of both strains were virtually identical (Fig. 1); peaks were observed at 863, 805, I.o
,
0.75
l
,
l
,
l
,
[ 6oo ~-
i
l
539 and 506 nm, typical of R. palustris [13]. Growth of strain SL in mineral media containing cinnamate was greatly stimulated by exogenous CO2 (supplied as HCO 3, Fig. 2). The generation time of strain SL on cinnamate/CO2 was about 12 h, and extremely high cell densities (greater than 1 mg/ml dry weight) could be obtained in mineral media containing 6 mM cinnamate as carbon source (Fig. 2). Some toxicity to cinnamate was noted; cultures grown on 7-12 mM cinnamate lagged considerably behind cultures grown on 1-6 raM, and initial cirmamate concentrations in excess of about 12 mM were totally growth inhibitory. Nutritional experiments using a variety of cinnamate and benzoate derivatives were performed with strains SL and LL. The results in Table 1 show that the nutritional patterns of the two strains, although similar, were not identical. Both strains utilized cinnamate, phenylpropionate, mcoumarate, and benzoate. In addition, strain SL, but not strain LL, used p-coumarate and caffeic acid. Strain LL grew slowly on o-coumarate. Phenylacetate, protocatechuate and cinnamyl alcohol were not used as carbon sources by either strain of R. palustris. Although the metabolic route of cinnamate catabolism by R. palustris is unknown at this time, I
i
I
o g ~ 0.5
i'12
,
5
0,25 [-
I
I
f
/
I
340
I
~
J
I
452
i
I
564
,?
L
I
676
,
I
788
0
i
~
-
o
5O
i
900
Wavelength (nm) Fig. 1. Absorption spectra of R. palustris strains SL ( ) and LL (- - -) isolated from cinnamic acid enrichments. In vivo spectra performed as described in [1].
o
12 Time
(h)
Fig. 2. Growth of R. palustris strain SL in a mineral medium containing cinnamic acid as sole organic carbon source in the presence and absence of 0.2~ NaHCO 3.
56 Table 1 Utilization of cinnamate and benzoate derivatives as sole carbon sources for growth of Rhodopseudomonas palustris strains SL and LL All substrates were at 6 mM final concentration except for cinnamyl alcohol, 4 mM. All media contained 0.25g N a H C O 3. + , 100-200 photometer units; + + , 300-400 photometer units; + + + , 400-500 photometer units; 0, no growth. 100 photometer units are equivalent to approximately 0.21 mg bacterial dry weight per milliliter. The following compounds did not support growth of either strain: protocatechuate, gentisate, phenylacetate, cinnamyl alcohol. Compounds
Growth
Cinnamate Phenylpropionate o-Coumarate m-Coumarate p-Coumarate Benzoate Caffeate
Strain SL
Strain LL
+ + 0 + + + +
+ + + + 0 + 0
+ + + + + + + + +
+ + + + + + +
growth studies with strain SL indicated that the aromatic ring was broken during phototrophic growth on this substrate. Fig. 3 shows the ultraviolet spectrum of fresh and spent culture media following growth of strain SL on cinnamate. The major peak at 269 nm due to cinnamic acid present in fresh media was absent from spent media,
0"81
0.6
~
I
I
I
T
I
'
1
'
I
0 C
indicating that the aromatic character of cinnamate was destroyed during phototrophic growth on this compound. By analogy to benzoate catabolism by R. palustris [8] cinnamate is probably converted to a linear molecule following ring reduction and breakage, and the resulting longchain fatty acid derivative is used as a carbon source for growth. This would explain the stimulatory effect of CO 2 on cinnamate catabolism by R. palustris (Fig. 2), since the utilization of long-chain fatty acids by phototrophic bacteria is generally CO 2 dependent [2]. The requirement for CO2, however, was not observed when strain SL grew on benzoate. The simplest explanation for this result would be that cinnamate, a C-9 compound and two carbon atoms longer than benzoate, forms a relatively reduced molecule whose assimilation requires CO 2 as an electron sink. Alternatively, the CO 2 requirement for photosynthetic cinnamate catabolism by R. palustris could signal the operation of a different type of catabolic pathway for this particular substrate than for benzoate. Our conclusion that cinnamate is converted by R. palustris into an intermediate that eventually enters central metabolic pathways is evident from studies of growth yields on cinnamate. A concentration of 1 m M (148 m g / 1 ) cinnamate was found to support a growth yield of about 200 mg (dry weight) of cells per liter (of strain SL). Assuming the cells to be 50% carbon this indicates that 100 mg cell carbon are produced from 148 mg cinnamate. Since cinnamate contains 73% carbon by weight, only 107 mg of carbon are potentially available from 148 mg of cinnamate. Thus, it appears that cinnamate is catabolized by R. palustris to intermediates that can be used quantitatively for the production of cell material.
0.4
¢.,, <
5. E C O L O G I C A L C O N S I D E R A T I O N S
200
228
256
284
Wavelength (nm)
r--T312 344
Fig. 3. Spectrum of a 10-2 dilution of fresh culture medium containing 6 mM trans-cinnamic acid ( ), and a 10-1 dilution of culture supernatant followinggrowth of R. palustris strain SL to 425 photometer units (- - -).
Cinnamic acid is an aromatic compound of particular interest because it is one of several monomeric molecules which can be isolated from plant lignins [11]. If the capacity for cinnamate catabolism is any indication of the potential of nonsulfur purple bacteria for catabolism of other lignin derivatives, it is possible that these
57 o r g a n i s m s p l a y a significant role in the t r a n s f o r m a t i o n of several lignin b y p r o d u c t s in nature. R e c e n t studies b y H a r w o o d a n d G i b s o n [14] s t r o n g l y s u p p o r t this c o n c l u s i o n a n d i n d i c a t e t h a t R. palustris is a p a r t i c u l a r l y n o t e w o r t h y species in terms of a r o m a t i c c a t a b o l i s m . T h e r a p i d i t y with which c i n n a m a t e / m i n e r a l salts e n r i c h m e n t s d e v e l o p e d in o u r s t u d y suggests t h a t this s u b s t r a t e m a y b e r a p i d l y p h o t o m e t a b o lized in nature. C i n n a m a t e has b e e n s h o w n to b e c a t a b o l i z e d b y b o t h d e n i t r i f y i n g b a c t e r i a [12] a n d a microbial consortium containing methanogenic b a c t e r i a [11]. D e n i t r i f y e r s were specifically exc l u d e d f r o m our e n r i c h m e n t s b e c a u s e of a l a c k of n i t r a t e in the e n r i c h m e n t m e d i u m ; m e t h a n o g e n i c c o n s o r t i a were n o t specifically excluded. Such c o n s o r t i a are a p p a r e n t l y u n a b l e to c o m p e t e with c i n n a m a t e - u t i l i z i n g p h o t o t r o p h s . T h e a d d e d req u i r e m e n t for N 2 fixation in o u r e n r i c h m e n t s p r o b a b l y excludes m o s t h e t e r o t r o p h s c a p a b l e of attacking cinnamate. N o n s u l f u r p u r p l e p h o t o t r o p h i c b a c t e r i a seem to b e ideal c a n d i d a t e s for a r o m a t i c c a t a b o l i s m in n a t u r e because, unlike h e t e r o t r o p h i c organisms, p h o t o t r o p h i c b a c t e r i a d o n o t r e q u i r e energy f r o m c a t a b o l i s m of the a r o m a t i c substrate. Such substrates o n l y n e e d serve as c a r b o n sources. T h e i s o l a t i o n of c i n n a m a t e - d e g r a d i n g strains of R. palustris f r o m a variety o f different h a b i t a t s suggests that these strains are u b i q u i t o u s a n d p o s s i b l y o f ecological i m p o r t a n c e in respect to a r o m a t i c c a t a b o l i s m . In fact, a p r e l i m i n a r y screen of several p u r e cultures of R. palustris originally i s o l a t e d on n o n a r o m a t i c o r g a n i c acids suggests that c i n n a m a t e c a t a b o l i s m b y this species m a y i n d e e d b e w i d e s p r e a d (see also Ref. 14). I n a d d i t i o n , recent e x p e r i m e n t s with resting cells o f a strain of R. palustris e n r i c h e d a n d i s o l a t e d on b e n z o a t e [15] has shown that a wide variety of a r o m a t i c substrates ( i n c l u d i n g s o m e t h a t are u n a b l e to s u p p o r t g r o w t h ) a r e p h o t o m e t a b o l i z e d b y this o r g a n i s m at high rates. C i n n a m a t e was p a r t i c u l a r l y n o t e w o r t h y in this c o n n e c t i o n [15]. ACKNOWLEDGEMENTS This research was s u p p o r t e d b y g r a n t P C M 8505492 (to M . T . M . ) a n d PCM-8415291 (to H . G . )
f r o m the U.S. N a t i o n a l Science F o u n d a t i o n . W e t h a n k Jeffrey L. F a v i n g e r for help w i t h the collection of samples, a n d J o h n G. O r m e r o d , U n i v e r s i t y of Oslo, N o r w a y , for s u p p l y i n g the T h a i l a n d a n d Sri L a n k a n samples.
REFERENCES [1] Madigan, M.T. and Gest, H. (1979) Growth of the photosynthetic bacterium Rodopseudomonas capsulata chemoautotrophicaUy in darkness with H 2 as the energy source. J. Bacteriol. 137, 524-530. [2] Biebl, H. and Pfennig, N. (1981) Isolation of members of the family Rhodospirillaceae. In: The Prokaryotes - A Handbook on Habitats, Isolation, and Identification of Bacteria (Starr, M.P., Stolp, H., Triiper, H.G., Balows, A. and Schlegel, H.G., Eds.), pp. 267-273, Springer-Verlag, Berlin. [3] Triiper, H.G. and Pfennig, N. (1981) Characterization and identification of the anoxygenic phototrophic bacteria. In: The Prokaryotes - A Handbook on Habitats, Isolation, and Identification of Bacteria. (Starr, M.P., Stolp, H., Triiper, H.G., Balows, A. and Schlegel, H.G., Eds.), pp. 299-312, Springer-Verlag, Berlin. [4] Scher, S. and Proctor, M.H. (1960) Studies with photosynthetic bacteria: Anaerobic oxidation of aromatic compounds. In: Comparative Biochemistry of Photoreactive Systems (Allen, M.B., Ed.), pp. 387-393, Academic Press, New York. [5] Dutton, P.L and Evans, W.C. (1969) The metabolism of aromatic compounds by Rhodopseudomonaspalustris. Biochem. J. 113, 525-536. [6] Guyer, M. and Hegeman, G. (1969) Evidence for a reductive pathway for anaerobic metabolism of benzoate. J. Bacteriol. 99, 906-907. [7] Sleat, R. and Robinson, J.P. (1984) The bacteriology of anaerobic degradation of aromatic compounds. J. Appl. Bacteriol. 57, 381-394. [8] Dutton, P.L. and Evans, W.C. (1978) Metabolism of aromatic compounds by Rhodospirillaceae. In: The Photosynthetic Bacteria (Clayton, R.K. and Sistrom, W.R., Eds.), pp. 719-726, Plenum Press, New York. [9] Evans, W.C. (1977) Biochemistry of the bacterial catabolism of aromatic compounds in anaerobic environments. Nature (London) 270, 17-22. [10] Gest, H., Favinger, J.L. and Madigan, M.T. (1985) Exploitation of N 2 fixation capacity for enrichment of anoxygenic photosynthetic bacteria in ecological studies. FEMS Microbiol. Ecol. 31, 317-322. [11] Healy, J.B., Jr. and Young, L.Y. (1979) Anaerobic biodegradation of eleven aromatic compounds to methane. Appl. Environ. Microbiol. 38, 84-89. [12] Williams, R.J. and Evans, W.C. (1975) The metabolism of benzoate by Moraxella sp. through anaerobic nitrate respiration: evidence for a reductive pathway. Biochem. J. 148, 1-10.
58 [13] Biebl, H. and Drews, G. (1969) Das in-vivo-spektrum als taxonomisches merkmal bei untersuchungen zur verbreitung der Athiorhodaceae. Zentralblatt fiir Bakteriologie, Parasitenkunde, Infektionskrankheiten und Hygiene, Abt. 2 123, 425-452. [14] Harwood, C.S. and Gibson, J. (1987) Anaerobic and aerobic metabolism of diverse aromatic compounds by the
photosynthetic bacterium Rhodopseudomonas palustris. Appl. Environ. Microbiol. In press. [15] Tanaka, H., Maeda, H., Suzuki, H., Kamibayashi, A. and Tonomura, K. (1982) The metabolism of thiophene-2carboxylate by a photosynthetic bacterium. Agric. Biol. Chem. 46, 1429-1438.