Synthesis and function of polyhydroxyalkanoates in anaerobic syntrophic bacteria

FEMS Microbiology Reviews 103 (1992) 195-206 © 1992 Federation of European Microbiological Societies 0168-6445/92/$15.00 Published by Elsevier

195

FEMSRE 00265

Synthesis and function of polyhydroxyalkanoates in anaerobic syntrophic bacteria Michael J. M c l n e r n e y a Dale A. Amos a, Karen S. Kealy a and Judith A. Palmer b a Department of Botany and Microbiology, The University of Oklahoma, Norman, OK, USA and b Department of Biochemistry and Molecular Biology, The University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA

Key words: Polyhydroxyalkanoate; Polyhydroxybutyrate; Syntrophic bacteria; Methanogenesis; Fatty acid degradation; Aromatic acid degradation;/3-Ketothiolase

1. SUMMARY Anaerobic syntrophic bacteria degrade fatty acids and some aromatic compounds which are important intermediates in the degradation of organic matter in methanogenic environments. Several of the described syntrophic species produce poly-/3-hydroxyalkanoate (PHA) suggesting that the synthesis and use of PHA is important in their physiology. In the fatty acid-degrading, syntrophic bacterium, Syntrophomonas wolfei, PHA is made during exponential phase of growth and used after growth has stopped and substrate levels are low. Altering the carbon to nitrogen ratio of the medium does not affect the amount of PHA made or its monomeric composition. It is hypothesized that PHA serves as an endogenous energy source for syntrophic bacteria when the concentrations of hydrogen or acetate are too high for the degradation of the growth substrate to be thermodynamically favorable. In S. wolfei, PHA is synthesized by two routes, the direct

Correspondence to: M.J. Mclnerney, Department of Botany and Microbiology, 770 Van Vleet Oval, University of Oklahoma, Norman, OK 73019-0245, USA.

incorporation of 3-ketoacyl-coenzyme A (CoA) generated in B-oxidation without cleavage of a C-C bond, and by the condensation and subsequent reduction of two acetyl-CoA molecules. Genes that encode for the synthesis of PHA in S. wolfei have been cloned into Escherichia coli in order to understand the molecular mechanisms that regulate PHA synthesis.

2. BIOLOGY OF SYNTROPHIC BACTERIA The syntrophic (H 2-producing acetogenic) bacteria are very specialized organisms with respect to both the compounds that serve as energy sources and the reactions that serve to reoxidize their reduced cofactors [1,2]. Ecologically, syntrophic bacteria degrade propionate and longerchain fatty acids, benzoate, phenol, and their mono-hydroxylated derivatives, which are key intermediates in anaerobic degradation of complex organic matter in methanogenic environments [35]. The degradation of these compounds with the production of acetate, CO2, and H 2 is thermodynamically unfavorable unless the reactions are coupled to H 2 use by methanogens (Table 1) [3]. Because of the unfavorable energetics involved in

196 Table 1

Some reactions of syntrophic (H 2-producing acetogenic) bacteria Reactions

A G ° ' (kJ) a

I. H 2 use by methanogens 4H2 + HCO~- + H + *-, C H 4 + 3 H e O I1. Reactions of syntrophic bacteria with and without H e use by methanogens A. Propionate use by Syntrophobacter wolinii C H 3 C H e C O O - +31-120 ~ C H 3 C O O - + H C O 3 + H + + 3 H 2 4 C H 3 C H e C O O - + 3 H e O *-, 4 C H 3 C O O - + H C O 3 + 3 C H 4 + H + B. Fatty acid use by Syntrophomonas species C H 3 C H e C H 2 C O O - + 2 H 2 0 ,--, 2 C H 3 C O O - + H + + 2 H 2 2CH3CHeCHeCOO- + HCO 3 +H20 ~ 4CH 3COO- +CH 4 + H + C. Benzoate use by Syntrophus buswellii C 7 H 5 0 2 + 7H 2 0 ,-* 3 C H 3 C O O - + H C O y + 3 H + + 3 H 2 4 C 7 H 5 0 2 + 1 9 H e O ~ 1 2 C H 3 C O O - + 3 C H 4 + HCO~- + 9 H + D. Crotonate metabolism by syntrophic bacteria in pure culture 2CH 3CH---CHCOO - + 2H 2 0 ~ 2 C H 3 C O O - + C H 3CH e C H z C O O - + H +

- 135.6

+76.1 - 102.4 +48.1 -116.4 + 70.6 - 124.6

- 102.1

Data obtained from Thauer et al. [28].

these reactions, the degradation of propionate and longer-chain fatty acids is often the ratelimiting step in anaerobic digestion [6]. The characteristics of some of the described syntrophic bacteria are given in Table 2. In general, syntrophic bacteria do not use carbohydrates, proteinaceous materials, or other c o m m o n bacterial energy sources. Some syntrophic bacteria use only one c o m p o u n d as the energy source, such as Syntrophobacterwolinii and strain Gralval which use only propionate and isovalerate, respectively, as the energy source when grown in association with He-using bacteria. Other syn-

trophic bacteria, such as the species in the genera

Syntrophospora and Syntrophomonas, use only a certain class of compounds, such as fatty acids with four or more carbons in length. Syntrophic bacteria cannot reduce CO 2 to acetate, nor use alternate electron acceptors such as oxyanions of sulfur and nitrate, or organic compounds such as fumarate or amino acids. Because of the reduced state of carbon in the substrates used by syntrophic bacteria, it is not possible to generate an oxidized intermediate that can serve as an electron acceptor for the reoxidation of their reduced cofactors, such as occurs for the degradation of

Table 2

Characteristics of some syntrophic bacteria Species

Cell shape

Cell wall

Substrates used Coculture

Syntrophobacter wolinii Syntrophomonas wolfei subsp, wolfei subsp, saponauida Syntrophomonas sapovorans Syntrophospora bryantii Gralval

Syntrophus buswellii KN032

rod

-

C~

curved rod

+ + -

C 4 - C 8 , iC 7 C 4 - C Is C4-CI8 , OEL C 4 - C I 1, 2 M B iC 5 Bz Bz, O H B z

curved rod

curved rod rod rod rod rod

References Pure culture [56] C4-C 6 UFA C4 U F A C 4 UFA C4b U F A

[7,8,21,25] [22] [57] [9,58] [24] [59,60] [61]

a C. carbon number of substrate; iC, iso-branched fatty acid; U F A , unsaturated fatty acid; 2MB, 2-methylbutyrate; O, oleate; E,

elaidate; L, linoleate; Bz, benzoate; OHBz, hydroxybenzoate. b B.T. Hopkins and M.J. Mclnerney, unpublished data.

197

carbohydrates and amino acids by fermentative bacteria. Thus, the reduction of protons to H 2, or of bicarbonate to formate, is the only mechanism available for the reoxidation of reduced cofactors. Because these reactions are thermodynamically favorable only when the concentrations of H 2 and formate are maintained at a very low level, the metabolism of syntrophic bacteria in their natural habitat depends on the activity of methanogens. Syntrophic bacteria cannot be isolated in pure culture with their natural substrates as the energy source, but must be grown in monoaxenic (two-membered) cultures in association with H2-using bacteria. Recently, a few syntrophic species have been shown to use more oxidized carbon compounds (for example, crotonate), which support the growth of these species in pure culture (Table 2) [7,8]. 16S rRNA sequence analysis of pure cultures of the Syntrophomonas wolfei subspecies and Syntrophospora (formerly Clostridium ) bryantii shows that these bacteria are in the Gram-positive line of descent, and are closely related to each other, but not to other species of the genus Clostridium [9]. This suggests that syntrophic bacteria may form a closely related phylogenetic group of bacteria. Other phylogenetically distinct bacteria require syntrophic interactions to degrade certain compounds, and some strains of the genera Desulfovibrio, Pelobacter, Thermoanaerobium, and Bacteroides degrade primary alcohols only in syntrophic association with H 2-using bacteria [10-16]. Syntrophococcus sucromutans requires an exogenous electron acceptor, either formate, cinnamate derivatives, methoxybenzenoids, or H2-using bacteria, to oxidize carbohydrates [17]. Although the above bacteria use syntrophic interactions to degrade certain compounds, they also use other electron acceptors to degrade these compounds, or grow with a variety of other compounds as energy sources without the need of interspecies H2 transfer. Thus, these bacteria are physiologically as well as phyiogenetically distinct from the fatty acid- and aromatic acid-degrading syntrophic bacteria. Poly-/3-hydroxybutyrate (PHB) and the copolyester, poly-/3-hydroxyalkanoates (PHA), are found in many aerobic bacteria, photosynthetic bacteria

[18], and some halophilic archaebacteria [19]. However, it is not commonly found in non-photosynthetic, strictly anaerobic bacteria. Of the latter type, only Clostridium botulinum and some syntrophic bacteria are known to produce PHA. In C. botulinum, PHB is produced before sporulation, and its role is presumably to serve as a c a r b o n / e n e r g y reserve for the sporulation process [20]. Interestingly, S. wolfei subspecies wolfei, and S. wolfei subspecies saponavida contain PHA [21-23]. We have recently found that a benzoatedegrading syntrophic bacterium morphologically similar to S. buswellii produces P H A when grown in coculture with benzoate, or in pure culture with crotonate as the energy sources (D.A. Amos and M.J. Mclnerney, unpublished data). Stieb and Schink [24] found that the isovalerate-degrading syntrophic bacterium, strain Gralval, contains a non-carbohydrate storage granule. Other syntrophic bacteria have not been analyzed for the presence of PHA. Since the occurrence of PHA in strictly anaerobic bacteria is rare, the frequency with which PHA has been detected in syntrophic bacteria suggests that this polymer is important in the physiology of these organisms. This paper will review what is known about the metabolism of PHA in syntrophic bacteria, and discuss possible function(s) for P H A in the physiology of these interesting organisms. The discussion will focus on Syntrophomonas wolfei, since this is the only syntrophic bacterium that has been studied in any detail.

3. M E T A B O L I S M OF F A T T Y ACIDS BY S.

wolfei S. wolfei metabolizes C 4 t o C 8 saturated fatty acids to acetate and H 2, or acetate, propionate, and H 2, for even- and odd-numbered fatty acids, respectively [21,25]. Isoheptanoate is degraded to isovalerate, acetate, and H 2. The production of H 2 from these compounds is thermodynamically favorable only if the H 2 concentration is very low (Table 1). Thus, S. wolfei can only grow with these compounds as energy sources in the presence of H2-using bacteria. S. wolfei also uses

198

several short-chain mono- and di-unsaturated fatty acids [7,8]. Part of the unsaturated fatty acid substrate is oxidized to acetate, and the remainder serves as the electron acceptor, and is reduced to the corresponding saturated fatty acid. Thus, S. wolfei can grow in pure culture with these unsaturated fatty acids as the energy source since interspecies H 2 transfer reactions are no longer required to reoxidize its reduced cofactors. The analysis of the products made from the various saturated and unsaturated fatty acid substrates suggested that these compounds are metabolized by a /3-oxidation pathway [21]. Enzymatic studies of Wofford et al. [26] and McInerney and Wofford [27] confirmed that these compounds were degraded by a/3-oxidation pathway. The proposed pathway for the degradation of butyrate by S. wolfei is shown in Fig. 1. ButyrylCoA is synthesized using a CoA-transferase reaction and is then/~-oxidized to acetyl-CoA. ATP is synthesized from acetyl-CoA using the phosphotransacetylase and acetate kinase reactions that result in acetate production. Crotonate is metabolized in a similar manner, except that part of the crotonyI-CoA formed by the CoA transferase reaction serves as the electron acceptor, and is reduced to butyryl-CoA which is then converted to butyrate. An important feature of the metabolism of fatty acids by S. wolfei is the formation of the respective CoA derivative by a CoA transferase reaction rather than an acyl-CoA synthetase reaction. With an acyi-CoA synthetase reaction, the formation of the acyI-CoA derivative is coupled to the hydrolysis of ATP to AMP and pyrophosphate, potentially using two highenergy bonds. This would not result in the net synthesis of ATP from butyrate by S. wolfei. With a CoA transferase reaction, only one of the acetyl-CoA molecules is used to activate the substrate; the other acetyl-CoA molecule can be used for ATP production. Although the pathway shown in Fig. 1 suggests that a net synthesis of one ATP molecule per butyrate degraded is possible, the AG' for butyrate degradation to acetate and H 2 is very low, about - 1 2 to - 1 7 k J / m o l when calculated according to Thauer et al. [28] using the observed concentrations of reactants and products in S.

Butyraie

~=,t,.-,-~Q Butyry~.CoA

E" (mV)

/

FAD , ~ (~ FADH2

....................................................

2H

-120

.....................................

",.

t CrotonyI-CoA

H2°-~®

L(+)-3-hy0roxybutyP/l-CoA

i

~'° 7"] ® .................................................... ~ . .......................................

Acetyt-CoA + AOetyl-CoA

i

-280

H2

-414

PHA

"o:-~ ®

CoA'~'-'~

AcetyI-P

ATP-.~0

AcetoacetyI-CoA

~.~--Ac~CoAt

Acetate

AcetyI-CoA+ AcetyI-CoA Fig. 1. Proposed pathway for the degradation of butyrate (solid lines) and the synthesis of poly-3-hydroxyalkanoate (PHA) (dashed lines) in S. wolfei. Insert: the regulation of the acetoacetyl-CoA thiolase involved in synthesis of PHA from acetyl-CoA. Dotted lines indicate the production of H 2 using unknown electron carriers; the potential of important oxidation-reduction reactions is given in inV. Enzymes: 1, CoA transferase; 2, acyl-CoA dehydrogenase; 3, enoyI-CoA hydratase; 4, L( + )-3-hydroxyacyl-CoA dehydrogenase; 5, 3-ketoacyl-CoA thiolase; 6, phosphotransacetylase; 7, acetate kinase; 8, acetoacetyl-CoA thiolase (/3-ketothiolase); 9, acetoaeetylCoA reductase; 10, PHA synthase. Abbreviations: 2H, reducing equivalents; AcAcCoA, acetoacetyl-CoA; AcCoA, acetylCoA; CoA, coenzyme A.

wolfei cocultures [29; P.S. Beaty and M.J. Mclnerney, unpublished data]. This is much less than that believed to be required for the irreversible synthesis of 1 mol of ATP in vivo (about - 7 0 k J / t o o l ) [30]. Based on the stoichiometry of membrane-bound ATPase and the amount of energy required to translocate 1 mol of protons across the membrane against a typical membrane potential of - 2 0 0 mV [31], a minimum energy quantum of - 17 to - 20 k J / m o l is believed to be required for the synthesis of ATP [30,32]. Using the concept of a minimum quantum of energy, Thauer and Morris [32] propose that two-thirds of the ATP potentially generated by substratelevel phosphorylation reaction is used to drive

199

reverse electron transport required to produce H 2 (E'o of - 4 1 4 mV) [28] from high potential electrons generated in the oxidation of butyrylCoA to crotonyl-CoA (E'o of - 126 mV) [33]. This would result in the net synthesis of one-third mol of ATP per mol of butyrate, which is consistent with the amount of ATP expected from thermodynamic conditions.

4. P H Y S I O L O G Y OF PHA PRODUCTION IN

S. wolfei 4.1. Production of PHA The production of PHA was studied in S.

wolfei grown alone with crotonate as the energy source, or in coculture with the H2-using methanogen, Methanospirillum hungatei, with butyrate as the energy source [23]. Under both conditions, PHA production in S. wolfei occurred during the exponential phase of growth and reached maximum concentrations when about 75% of the substrate was degraded. Greater amounts of PHA were observed when crotonate was the energy source ( 37/ z g/ m l ) compared to butyrate-grown cocultures (2.5 ~g/ml). When the cultures reached stationary phase, PHA was degraded. Reamendment of crotonate to late stationary phase cultures resulted in the rapid production of PHA to the levels observed during exponential phase of growth. The production of PHA during periods when the substrate level was high, and the degradation of PHA during periods when the substrate was limiting, suggests that PHA serves as a carbon/energy reserve material in S. wolfei as found in other bacteria [18].

The importance of PHA as an energy reserve material for syntrophic bacteria can be understood by comparing the energetics of butyrate and 3-hydroxybutyrate degradation at high and low substrate concentrations, as would be found during the exponential and the stationary phases of growth, respectively (Table 3). As syntrophic cocultures metabolize butyrate, H 2 use by the methanogen keeps the H2 concentration low, but the acetate concentration increases. So long as the butyrate concentration relative to the acetate concentration is high, the degradation of butyrate is thermodynamically favorable (Table 3). However, when the butyrate concentration becomes low relative to the acetate concentration, as occurs during the stationary phase of growth, the degradation of butyrate becomes thermodynamically unfavorable. Several studies suggest that the degradation of butyrate by S. wolfei [34] and benzoate by S. buswellii [B.T. Hopkins and M.J. Mclnerney, unpublished data] reaches a threshold value where further degradation of the substrate does not occur when the concentration of acetate is high relative to the concentration of the substrate. However, the degradation of 3-hydroxybutyrate is still favorable even when the concentration of acetate is high. Thus, by the accumulation of PHA, syntrophic bacteria have a mechanism to obtain energy when environmental conditions, i.e., high concentrations of acetate a n d / o r H 2, inhibit the degradation of the substrate. Several lines of evidence suggest that the production of PHA in S. wolfei is regulated differently from that found in other bacteria. In other bacteria, the production of PHA usually occurs

Table 3 I m p o r t a n c e o f P H A as a n e n e r g y r e s e r v e m a t e r i a l in s y n t r o p h i c b a c t e r i a Reactions

A G ° ' (kJ) a

Butyrate- +2H20 ~ 2 Acetate- +H + +2H 2 3 - H y d r o x y b u t y r a t e + H 2O ~ 2 A c e t a t e - + H ÷ + H ~

+48.1 - 35.1

A G ' (kJ) b at s u b s t r a t e c o n c e n t r a t i o n o f 20 m M

1 mM

- 10.5 - 47.5

+2.0 - 36.5

D a t a f r o m T h a u e r et al. [28]. b C a l c u l a t e d f r o m d a t a in [28] a s s u m i n g a c e t a t e c o n c e n t r a t i o n o f 20 m M , a h y d r o g e n c o n c e n t r a t i o n o f 0 . 5 / z M , a n d t h e i n d i c a t e d substrate concentration. a

200 when the carbon/energy source is in excess and growth is limited by the availability of a utilizable source of N, P, S, Mg, or Fe, or when the oxygen supply is restricted [18,35]. In batch culture, these conditions usually cause cultures to enter stationary phase, so PHA production usually occurs in the late exponential or stationary phases of growth. In S. wolfei, PHA production occurs during the exponential phase of growth and stops when growth stops [23]. Altering the C to N ratio of the medium does not affect the amount of PHA produced in S. wolfei as it does in other bacteria. PHA from S. wolfei contains o ( - )-3-hydroxybutyrate and either D(-)-3-hydroxypentanoate or D(-)-3-hydroxyhexanoate depending on the nature of the growth substrate [23,36]. Poly-/3-hydroxyalkanoate from S. wolfei grown in pure culture with crotonate, or in coculture with butyrate, contains only o( -)-3-hydroxybutyrate. PHA of S. wolfei grown with pentenoate and hexenoate substrates is a copolyester containing mostly the D(--)-3-hydroxybutyrate, and small amounts of a monomer with the same chain length as the substrate. In other organisms that produce polyesters of similar composition, the monomeric composition of PHA on a percentage basis varies markedly with the nature of the growth substrate [37]. Alcaligenes eutrophus when grown with evennumbered-carbon substrates produces a polymer consisting entirely of D(-)-3-hydroxybutyrate, while cultures grown with propionate or pentanoate as the sole carbon sources produce a copolyester with 43 and 90 mol% of D( -- )-3-hydroxypentanoate, respectively [38]. In contrast, the monomeric composition of PHA in S. wolfei changes only a small amount when different grown substrates are used, with most of the polyester (95%) composed of D(-)-3-hydroxybutyrate regardless of the growth substrate [23,36].

4.2. Pathway for PHA synthesis Labeling studies and analysis of the monomeric composition of PHA showed that S. wolfei synthesizes PHA by two different routes, (a) the direct incorporation of a /3-oxidation intermediate without cleaving a C-C bond, and (b) the condensation and subsequent reduction of two

acetyl-CoA molecules (Fig. 1) [23,36]. The unbroken carbon chain was used for PHA synthesis only during the early stages of growth and, later, polymer production occurred by the condensation and reduction of acetyl-CoA molecules. At least two different pathways could be used by S. wolfei to synthesize o(-)-3-hydroxyacylCoA which is incorporated into the polymer by PHA synthetase [35,37]. o(-)-3-hydroxyacyl-CoA could be synthesized directly from the acetoacetyl-CoA made during the/3-oxidation of the substrate, or from the condensation of two acetylCoA molecules, using a nicotinamide adenine dinucleotide-dependent acetoacetyl-CoA reductase. This pathway is used by many bacteria including Azotobacter beijerinckii [39,40], Alcaligenes eutrophus [41,42], and Zoogloea ramigera [43] to synthesize o(-)-3-hydroxyacyl-CoA. The other possibility is that S. wolfei could use a pathway similar to that found in Rhodospirillum rubrum [44] where crotonyl-CoA generated during the /3-oxidation of the substrate by S. wolfei could be directly converted to o(-)-3-hydroxybutyryl-CoA using a o-specific enoyl-CoA hydratase. Acetoacetyl-CoA made from the condensation of two acetyi-CoA molecules could be converted to crotonyl-CoA by reversing the /3-oxidation pathway, with crotonyl-CoA being converted to D(- )-3-hydroxybutyryl-CoA by the D(-)-3-hydroxybutyryl-CoA hydratase. Enzymatic studies [45] showed that S. wolfei contains an acetoacetyl-CoA reductase activity that forms o(-)-3-hydroxybutyryl-CoA from acetoacetyl-CoA which can be made either by the condensation of two acetyl-CoA molecules, or directly from the 13-oxidation of the substrate. Cell-free extracts of S. wolfei did not synthesize o(-)-3-hydroxybutyryl-CoA from crotonyi-CoA, and did not contain a D-specific enoyl-CoA hydratase activity. Thus, S. wolfei does not use the R. rubrum pathway, but synthesizes PHA using the acetoacetyl-CoA reductase pathway as found in most bacteria (Fig. 1). The acetoacetyl-CoA reductase that forms o(-)-3-hydroxybutyryl-CoA in S. wolfei is unusual since it uses either NADH or NADPH [45]. In most other organisms, the acetoacetyl-CoA reductase involved in PHA synthesis is specific for NADPH [35,37]. The ability

201

of the acetoacetyl-CoA reductase from S. wolfei to use either NADH or NADPH indicates that the NADH produced from the oxidation of L( + )3-hydroxyacyl-CoA during /3-oxidation can be used directly for PHA synthesis. Thus, a mechanism to interconvert NADH and NADPH by transhydrogenase activity or by some other route is not required.

4.3. Properties of [3-ketothiolase fl-Ketothiolase (acetyl-CoA:acetyl-CoA acetyltransferase, E.C. 2.3.1.9) is the key enzyme that controls the synthesis of PHA from acetyl-CoA [35,37]. This enzyme has been purified from S. wolfei (K. Kealy and M.J. Mclnerney, unpublished data), and is very similar to other biosynthetic /~-ketothiolases involved in PHA synthesis (Table 4). All of the biosynthetic /3-ketothiolases are homotetrameric enzymes with molecular masses ranging from 160,000 to 190,000, and have similar kinetic and regulatory properties [41,42,47-49]. The enzyme from S. wolfei uses acetoacetyl-CoA, but not 3-ketooctanoyl-CoA, indicating that it is specific for short-chain, 3-ketoacyl-CoA substrates as are the other biosynthetic fl-ketothiolases. This indicates that S. wolfei must contain another /3-ketothiolase involved in the degradation of fatty acids, since S. wolfei grows with C a through C 8 fatty acids as substrates. The condensation of two acetyl-CoA molecules to acetoacetyl-CoA catalyzed by the /3-ketothiolase from S. wolfei is strongly inhibited by CoA (apparent k i of 3.6 /zM) while the thiolysis of ace-

toacetyl-CoA is competitively inhibited by acetylCoA (apparent k i of 500/zM) (K. Kealy and M.J. Mclnerney, unpublished data). This indicates that the synthesis of PHA from acetyl-CoA is favored when the concentration of free CoA is low, and the acetyl-CoA concentration is high (Fig. 1), as would presumably occur during the later stages of growth. Although the bioenergetic model of Thauer and Morris [30] is consistent with the thermodynamic constraints placed on ATP synthesis, it is difficult to explain how the synthesis of one ATP by substrate-level phosphorylation is stoichiometrically coupled to the degradation of each butyrate molecule, since the free energy change for butyrate degradation is so low. The presence of a separate /3-ketothiolase for PHA synthesis in S. wolfei could indicate that the pathway for the metabolism of fatty acids by S. wolfei branches after acetyl-CoA is formed (Fig. 1). One branch would lead to the synthesis of acetate from acetyl-CoA by phosphotransacetylase and acetate kinase reactions, and is stoichiometrically linked to the ATP synthesis. The other branch would lead to the synthesis of PHA from acetyl-CoA, and would not be directly coupled to the synthesis of ATP. Thus, the formation of PHA may provide a mechanism for the non-stoichiometric synthesis of ATP from butyrate. The polymerization of 3-hydroxybutyryl-CoA into PHA would shift equilibria in favor of the condensation and subsequent reduction of acetyl-CoA. This would provide a high group potential of 3-hydroxy fatty

Table 4 Properties of fl-ketothiolases involved in P H A synthesis in several different bacteria Organism

Alcaligenes eutrophus Bradyrhizobiumjaponicum Syntrophomonas wolfei Zoogloea ramigera

Molecular mass

170,000 180,000 160,000 162,000

Subunit composition

a4 a4 a4 a4

a AcAcCoA, acetoacetyI-CoA. ~' K.S. Kealy and M.J. Mclnerney, unpublished data.

km (/zM) Thiolysis

References Condensation

A c A c C o Aa

CoA

Acetyl-CoA

44 19 23 10-24

16 30 3.6 8.5

1100 104 290 330

[41] [46] b

[47-49]

202 acids which could be used to drive the endergonic synthesis of ATP during the degradation of butyrate to acetate and H 2, in a manner analogous to that found in Clostridium kluyt~eri [28,50]. Further studies on the physiology of PHA production in syntrophic bacteria are required in order to determine whether PHA plays a role in regulating the thermodynamic efficiency of ATP synthesis.

4.4. Cloning of the S. wolfei PHA biosynthetic genes The genes encoding for the enzymes involved in PHA synthesis in S. wolfei have been cloned into Escherichia coli using a pUC19 plasmid expression vector and selection of recombinant clones by screening for opaque colonies indicative of the presence of intracellular granules. The procedures of Sambrook et al. [51] were used for the cloning of the PHA biosynthetic genes from S. wolfei. Total genomic DNA of S. wolfei, isolated from cells grown in pure culture with crotonate [7], was partially digested with EcoRI endonuclease, and the DNA was separated into size fragments by sucrose-gradient centrifugation. Two size classes (from 2 to 5 kb and from 5 to 9 kb) were separately pooled, and ligated into EcoRIdigested and dephosphorylated pUC19. Cells of E. coli strain JM109 grown in Luria-Bertani (LB) medium [51] were transformed with purified recombinant plasmid DNA by electroporation. After outgrowth of the cells in LB broth for 1 h, the cells were plated onto LB agar medium with 100 izg/ml of ampicillin, 40 izg/ml of X-gal (Sigma Chemical Co., St. Louis, MO), and 200 /zM of isopropylthio-/3-galactoside. White, opaque colonies were picked as putative PHB recombinant strains, and these strains were analyzed for the presence of PHA. The presence of PHA in these recombinant strains was confirmed by purification of the granule [52] and determining the presence of crotonate in the isolated granule spectrophotometrically [21], and by high-pressure liquid chromatography [23]. About 70% of the colonies picked after electroporation of strain JM109 contained recombinant DNA as indicated by the presence of inserts in purified plasmid DNA. Fifty-one of 33,100

colonies (0.15%) were opaque, and microscopic analysis indicated that cells from opaque colonies contained intracellular granules. Of these, 12 strains contained granules that stained with Sudan black indicating the presence of PHA. The presence of PHA in these strains was confirmed by purification of the granule, and determining its molecular composition after acid hydrolysis. All 12 of the strains contained PHA, ranging from 30 to 100 Izg/mg of cellular protein. Preliminary analysis of the plasmids purified from these strains showed the presence of inserts ranging in size from about 4 to 7.2 kb. This is within the minimum size range needed to contain the coding regions for the fl-ketothiolase, acetoacetyl-CoA reductase, and PHA synthase, as estimated from the size of these genes in A. eutrophus [35]. These data suggest that the genes needed for the synthesis of PHA in S. wolfei are closely linked on the chromosome as found in A. eutrophus [35,53-55]. More significantly, the cloning of the PHA genes from S. wolfei is the beginning of the study of the molecular biology of a very important and unusual group of bacteria.

5. CONCLUSION

S. wolfei synthesizes PHA either by the direct incorporation of acetoacetyl-CoA generated during the /3-oxidation of the substrate, or by the condensation and subsequent reduction of two acetyI-CoA molecules. The synthesis of PHA provides S. wolfei with a mechanism to obtain energy when environmental conditions make the degradation of its substrate thermodynamically unfavorable. The differences in the environmental and growth conditions that influence PHA production in S. wolfei compared to other bacteria suggest that PHA may have additional function(s), possibly in the regulation of the thermodynamic efficiency of ATP synthesis. The properties of the biosynthetic /3-ketothiolase from S. wolfei are quite similar to those found in other, phylogenetically distinct bacteria. The development of recombinant DNA approaches to study the molecular biology of S. wolfei should allow us to understand the function and evolution of this pathway.

203 ACKNOWLEDGEMENTS W e t h a n k D . L o r e n z , K.T. M a d h u s u d h a n , K. Hatter, and N.Q. Wofford for their assistance. This work was supported by contract DE-FG0589ER-14003 from the Department of Energy.

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