Benzoate Degradation via the ortho Pathway in Alcaligenes eutrophus ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1997, p. 2765–2770 0099-2240/97/$04.0010 Copyright © 1997, American Society for Microbiology

Vol. 63, No. 7

Benzoate Degradation via the ortho Pathway in Alcaligenes eutrophus Is Perturbed by Succinate ´ DE ´ RIC AMPE, JEAN-LOUIS URIBELARREA, GLAUCIA M. F. ARAGAO, FRE AND NICHOLAS D. LINDLEY* Centre de Bioinge´nierie Gilbert Durand, Institut National des Sciences Applique´es, Unite´ Mixte de Recherche CNRS 5044, and Laboratoire Associe´ a ` l’Institut National de la Recherche Agronomique, Complexe Scientifique de Rangueil, 31077 Toulouse Ce´dex 4, France Received 17 January 1997/Accepted 30 April 1997

During batch growth of Alcaligenes eutrophus on benzoate-plus-succinate mixtures, substrates were simultaneously metabolized, leading to a higher specific growth rate (m 5 0.56 h21) than when a single substrate was used (m 5 0.51 h21 for benzoate alone and 0.44 h21 for succinate alone), without adversely affecting the growth yield (0.57 Cmol/Cmol). Flux distribution analysis revealed that succinate dehydrogenase most probably controls the rate of total succinate consumption (the maximum flux being 9.7 mmol z g21 z h21). It is postulated that the relative consumption rate of each substrate is in part related to modified levels of gene expression but to a large extent is dependent upon the presence of succinate, end product of the b-ketoadipate pathway. Indeed, the in vitro b-ketoadipate-succinyl coenzyme A transferase activity was seen to be inhibited by succinate, a coproduct of the reaction. enzymes of the ortho pathway of Pseudomonas putida (27). Little additional information was reported until Zylstra et al. (40) found that the expression of the two Pseudomonas cepacia protocatechuate-3,4-dioxygenase genes was constitutive but subject to partial catabolite repression by succinate. Finally, Duetz et al. (11) have shown that the presence of nonlimiting concentrations of succinate in continuous cultures of P. putida mt-2 was sufficient to block the expression of the xylene degradation pathway at the transcriptional level. At the same time, Holtel et al. (15) showed, on the contrary, that succinate did not repress the utilization of benzyl alcohol (under the control of Pu and Ps promoters), whereas glucose did. The role of succinate in the degradation of aromatic compounds remains unclear and somewhat controversial. In this study, the metabolic phenomena involved during the growth of A. eutrophus on benzoate-plus-succinate mixtures have been investigated. The results demonstrate that in contrast to growth with benzoate plus acetate (1), no diauxic pattern was observed: benzoate and succinate were simultaneously metabolized, leading to a higher growth rate with the substrate mixture than with either substrate alone.

Alcaligenes eutrophus (Ralstonia eutropha), a commonly occurring soil bacterium, possesses chromosome-encoded pathways for the degradation of aromatic compounds (16). In addition, plasmids extend the range of substrates degraded to pollutants such as 2,4-dichlorophenoxyacetate (31), chlorobenzenes (8), methylaromatics (30), or polychlorinated biphenyls (37). In this bacterium, benzoate is metabolized via the ortho pathway, the highly complex biochemical regulation of which was partly elucidated for A. eutrophus 335 by Johnson and Stanier (17). Benzoate, cis,cis-muconate, and b-ketoadipate appeared to be key intermediates in the induction of the enzymes of the pathway (17, 38), but despite these early reports, there remain gaps in the knowledge of the regulation, and to date only one gene of the benzoate branch of this pathway (catD, coding for the b-ketoadipate enol lactone hydrolase induced during growth of A. eutrophus on benzoate) has been cloned and studied (35). In natural environments, various alternative substrates are usually present, some of which may be degraded before the aromatic compounds. Despite this, little attention has been paid to the manner in which aromatic compounds are metabolized in the presence of other substrates. The repression of the catabolism of less favorable substrates by other carbon sources (often referred to as catabolite repression) has been extensively described for enteric bacteria (3) and a few other genera, such as Bacillus (34) and Pseudomonas (22). In the latter organisms, closely related to A. eutrophus, the catabolite repression effect is not understood yet but has been shown to be cyclic AMP independent (15, 21, 22, 29). The ortho pathway results in the production of succinate and acetyl coenzyme A (acetyl-CoA), which are then metabolized by the central metabolic pathways. This interface is a logical site for regulatory control, and, indeed, succinate has been postulated to have an inhibitory effect on the synthesis of some

MATERIALS AND METHODS Bacterial strains. A. eutrophus 335 (ATCC 17697) was obtained from Laboratorium Microbiologie Gent (Brussels, Belgium). A. eutrophus B9 lacking 1,2dihydro-1,2-dihydroxybenzoate (DHB) dehydrogenase (33) was kindly provided by George Hegeman. Growth of bacteria. The mineral salts medium used for growth of A. eutrophus was described previously (1). A 1.5-liter bioreactor from Setric (Toulouse, France) was used for the present study. The temperature was kept constant at 30°C, the pH was maintained at 7.4 with controlled addition of H3PO4 (1 M), and the oxygen partial pressure was maintained at 60% saturation with air (i.e., approximately 0.132 mM oxygen dissolved in the medium under the conditions used) via agitation and air flow rate variation. The bioreactor was inoculated with 10% (vol/vol) late-exponential-phase inoculum grown in shake flasks on benzoate or succinate media, as indicated below. After inoculation, samples were periodically withdrawn from the bioreactor with sterile syringes. Batch cultures were performed in triplicate. For the chemostat study, culture conditions were similar to those for batch culture except that the benzoate concentration in the inflowing medium was fixed at 20 mM. The dilution rate was maintained constant at 0.33 h21. Succinate pulse experiments were performed by the sudden addition of 54 ml of a sterile 1 M sodium succinate solution to obtain a succinate concentration of 45 mM in the bioreactor. The medium feed rate was maintained throughout the experiment.

* Corresponding author. Mailing address: Institut National des Sciences Applique´es, De´partement G.B.A., Complexe Scientifique de Rangueil, 31077 Toulouse Ce´dex 4, France. Phone: (33) 5 61 55 94 89. Fax: (33) 5 61 55 94 00. E-mail: {ampe;lindley}@insa-tlse.fr. 2765

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Measurement of fermentation parameters. Biomass was measured by cell dry weight determination. The concentrations of benzoate and succinate were analyzed by high-pressure liquid chromatography as previously described (1). Determination of enzyme activities. Enzyme activities were measured in whole cells or in crude cell extracts sampled throughout the culture as detailed in Results. Benzoate-1,2-dioxygenase (B12O) activity was estimated with freshly harvested whole cells which were centrifuged for 3 min at 10,000 3 g at 4°C, washed with Tris z HCl buffer (pH 7.5; 100 mM), and resuspended in the same buffer. The cells were placed in a biological oxygen monitor (YSI 5300; Yellow Springs Instrument Co., Yellow Springs, Ohio) with 3 ml of the same buffer containing 1 mM benzoate. Blanks without benzoate were prepared for each assay. The B12O activity was expressed as millimoles of benzoate consumed per gram (dry weight) of cells per hour. This method was adapted from that described by Farr and Cain (13) and estimates the combined rate resulting from transport and oxygenation of benzoate. We have shown, through the estimation of pH gradients with [14C]benzoate, that cells grown on phenol (and therefore not induced by benzoate) transported benzoate at rates always greater than 10 mmol z g21 z h21 (unpublished results). Therefore, a measured B12O activity below 10 mmol z g21 z h21 can be considered to represent the activity of the dioxygenase itself, the transport being nonlimiting. For all other enzymes, cell extracts were prepared. Approximately 50 to 100 mg (wet weight) of freshly harvested cells was centrifuged for 5 min at 10,000 3 g at 4°C, washed twice in 100 mM Tris z HCl (pH 7.5) at 4°C, and resuspended in 10 ml of Tris z carballylate buffer (9 mM tricarballylic acid, 35 mM Tris z HCl, 5 mM MgCl2, 20% [vol/vol] glycerol; pH 7.8). The cells were disrupted by sonication, and the resulting crude extracts were centrifuged to obtain soluble extracts, which were used to assay enzyme activities. The following enzymes of the ortho pathway were assayed by previously published methods: DHB dehydrogenase (32); catechol-1,2-dioxygenase (C12O) (EC 1.13.1.1, catechol:oxygen 1,2-oxidoreductase) (26); cis,cis-muconate lactonizing enzyme, or muconate cycloisomerase (MCI) (EC 5.5.1.1, 4-carboxymethyl-4-hydroxyisocrotolactone lyase [decyclizing]) (24); and b-ketoadipate-succinyl coenzyme A transferase (KTR) (EC 2.8.3.6, 4-carboxymethyl-4-hydroxyisocrotonolactone lyase [decyclizing]) (4), except that the buffer was replaced by 100 mM Tris z HCl (pH 7.5). Blanks without substrate were prepared for each extract. Protein was measured as described by Lowry et al (20). Activities are expressed as milli-international units per milligram of protein (i.e., nanomoles per minute per milligram of protein). Macromolecular cell composition. Washed cells harvested from exponentialphase batch cultures growing on glucose were used to determine the protein content (75% of the dry cell weight) of A. eutrophus by a previously published analytical method (20). Ash content was determined to be 3.9% (wt/wt) by weight measurement after dried cells were heated for 8 h at 500°C. The amino acid composition (expressed as micromoles per gram [dry weight] of cells) was as follows: Ala, 617; Arg, 273; Asn, 278; Asp, 278; Cys, 414; Glu, 236; Gln, 236; Gly, 606; His, 275; Ile, 203; Leu, 354; Lys, 280; Met, 107; Phe, 157; Pro, 840; Ser, 199; Thr, 272; Trp, 65; Tyr, 117; and Val, 309. These values were obtained as described by Chang et al. (5). The fatty acid composition used was that determined by Le´onard and Lindley (17a) with exponentially growing A. eutrophus (in percentages of total fatty acids): C16:0, 21; C16:1, 37.6; C18:0, 40.4; and C18:1, 1. The structure of peptidoglycan used was that found for Pseudomonas alcaligenes (23), which is similar to that found in Escherichia coli. The lipopolysaccharide (LPS) composition was that described for P. alcaligenes (9, 18). The elemental composition of A. eutrophus was determined to be CH1.77O0.44N0.25, a composition close to that found in a benzoate-limited chemostat (CH1.73O0.41N0.25 [1]).

Knowing the amino acid content, the composition of the A. eutrophus genome (GC content 5 66.5% [14]), the ash content, the degree of reduction (g 5 4.14), and the relative concentration ratios for LPS/peptidoglycan (0.76), lipids/peptidoglycan (2.91), and polysaccharides/peptidoglycan (1.65), it was possible to estimate the biomass composition in agreement with the elemental composition measured. This led to the following macromolecular composition in percentages of dry cell weight: protein, 75; RNA, 3.58; DNA, 1.85; phospholipids, 6.49; polysaccharides, 4.34; peptidoglycan, 2.64; LPS, 4.98; polyamines, 0.11; and other small molecules, 1.03. This macromolecular composition was used to estimate the anabolic precursor requirements based on the pathways described by Neidhardt et al. (25); outputs from the central pathways are given in Table 1. Metabolic fluxes. Metabolic fluxes within the central metabolic network were estimated by using the stoichiometric approach described by Neidhardt et al. (25) for E. coli, and more recently applied to Corynebacterium glutamicum (6, 39), using the carbon metabolic requirements given above (Table 1). The reactions making up the central metabolic network of A. eutrophus are those described by Schobert and Bowien (36), giving a matrix structure of 10 reactions and 9 intermediates, together with the known reactions of the linear ortho pathway of benzoate degradation leading to the fixed stoichiometric production of acetylCoA and succinate. Experimental kinetic data were used to calculate the specific rates (substrate consumption rates and growth rates) used as inputs for the model. Outputs of biomass components were determined by multiplication of the anabolic demand by the specific growth rate. The carbon flux at each step of the pathway was determined for each biochemical reaction by assuming that metabolite pools undergo variations in concentrations which may be considered insignificant in comparison to the flux. Kinetic and stoichiometric data, together with the NADPH2 supply-and-demand balance, were used to determine the possible patterns of carbon distribution through the various pathways. Chemicals. cis,cis-Muconate was synthesized by the procedure described by Elvidge et al. (12), and DHB was prepared by using A. eutrophus B9 by the procedure described by Reiner (32).

RESULTS Growth of A. eutrophus on a single substrate (benzoate or succinate). In batch cultures using mineral salts medium, A.

TABLE 1. Intermediate metabolites and energy requirements for the formation of 1 g of biomass of the bacterium A. eutrophus during exponential growth Precursor metabolite

Amt required (mmol/g of cells)

Glucose-6-phosphate .............................................................. Fructose-6-phosphate ............................................................. Ribose-5-phosphate ................................................................ Erythrose-4-phosphate............................................................ Triose-phosphate..................................................................... 3-Phosphoglycerate ................................................................. Phosphoenolpyruvate.............................................................. Pyruvate.................................................................................... Acetyl-CoA .............................................................................. a-Ketoglutarate ....................................................................... Oxaloacetate ............................................................................

358.9 101.0 523.5 338.2 86.5 1,398.8 655.2 2,603.3 2,522.2 1,622.8 1,758.9

;Pa ........................................................................................... 40,789.2 NADH2.....................................................................................22,887.6 NADPH2 .................................................................................. 18,333.0 a

Energy-rich phosphate bond.

FIG. 1. Kinetics of growth and substrate consumption of A. eutrophus grown in batch cultures on a benzoate-plus-succinate mixture after pregrowth on benzoate. ■, benzoate; }, succinate; F, biomass; —, qbenzoate; ª, qsuccinate; . . ., m. Identical values were obtained for cultures pregrown on succinate.

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FIG. 2. Estimation of flux distribution within the central metabolic pathways based on the kinetic data from exponential growth in batch mode culture with benzoate (5 mM), succinate (5 mM), and benzoate plus succinate (5 mM each). Numerical data are given in the following order: benzoate/succinate/benzoate plus succinate. PEP, phosphoenolpyruvate.

eutrophus 335 grew exponentially on either benzoate (5 mM) or succinate (5 mM). The biomass yields (YX/S) relative to the carbon content were the same for the two cultures (YX/benzoate 5 0.54 6 0.02 Cmol/Cmol and YX/succinate 5 0.55 6 0.04 Cmol/ Cmol [means 6 standard deviations]). However, A. eutrophus grew more rapidly on benzoate than on succinate (m 5 0.51 6 0.02 h21 for benzoate compared to 0.44 6 0.03 h21 for succinate [means 6 standard deviations]). Growth of A. eutrophus on benzoate-plus-succinate mixtures. (i) Kinetic analysis. When A. eutrophus was grown by batch mode cultivation in media containing equimolar (5 mM) concentrations of benzoate and succinate, the behavior and substrate preference were irrespective of the substrate used for growth of the inoculum. After a brief period of accelerating growth, an exponential growth phase (m [mean 6 standard deviation] 5 0.56 6 0.02 h21) in which benzoate and succinate were metabolized simultaneously was observed (Fig. 1). The specific rates of substrate consumption were constant (qbenzoate 5 3.15 6 0.2 mmol z g21 z h21 and qsuccinate 5 4.3 6 0.2 mmol z g21 z h21 [means 6 standard deviations]), though these rates were lower than those for cultures on each substrate alone (qbenzoate 5 5.5 6 0.2 mmol z g21 z h21 on benzoate alone and qsuccinate 5 8.05 6 0.5 mmol z g21 z h21 on

succinate alone). Exponential growth was maintained until substrate exhaustion. The growth yield was also constant (YX/S 5 0.57 6 0.02 Cmol/Cmol [mean 6 standard deviation]) and equal to those found for cultures on single substrates. (ii) Estimation of carbon and energy fluxes. Carbon and energy fluxes were estimated for the different cultures (benzoate, succinate, and benzoate plus succinate) by using the method and the anabolic precursor requirements determined for A. eutrophus as described in Materials and Methods. Carbon flux distribution showed two particular features (Fig. 2). (i) The estimated fluxes through succinate dehydrogenase (FSDH) were the same for the culture with succinate and that with succinate plus benzoate (FSDH 5 9.5 6 0.2 and 9.7 6 0.2 mmol z g21 z h21, respectively [means 6 standard deviations]). This flux was lower when benzoate was used as a single substrate (FSDH 5 8.9 6 0.2 mmol z g21 z h21). (ii) The flux through pyruvate dehydrogenase (FPDH) was proportional to the specific rate of extracellular succinate consumption (FPDH ' 35 to 40% of qsuccinate). When benzoate was the sole carbon source, this flux was equal to zero: pyruvate dehydrogenase is apparently not necessary for growth with low concentrations of benzoate.

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TABLE 2. Specific activities of ortho-pathway enzymes during exponential growth on benzoate, succinate, and a benzoate-plus-succinate mixture Enzyme

B12O DHBDHc C12O MCI KTR

Activity ona: Benzoate

Succinateb

Benzoate plus succinate

8.5 6 1.0 325 6 27 150 6 12 415 6 72 9.7 6 2.7

,0.04 ,27 ,4 NDd ND

4.0 6 0.5 267 6 33 56 6 6 284 6 44 18.2 6 5.0

a The concentration of each substrate was 5 mM (including in the mixture). B12O activity is expressed as millimoles of benzoate per gram (dry weight) per hour. All other activities are expressed in nanomoles per minute per milligram of protein. Enzyme activities were measured in at least three samples harvested during the exponential growth phase, and each sample was assayed six times. The values given are means 6 standard deviations. b these values represent the threshold activities for precise detection. c DHBDH, DHB dehydrogenase. d ND, not detected.

Expression of enzymes of the ortho pathway. Activities of the enzymes of the ortho pathway were measured with whole cells (B12O) or cell extracts (DHBDH, C12O, MCI, and KTR) from the three cultures described above. Enzyme activities were high in both cultures containing benzoate, though B12O, DHBDH, C12O, and MCI specific activities were 20 to 60% lower in the culture with benzoate plus succinate (Table 2). On the other hand, KTR specific activity was higher in the culture with the benzoate-plus-succinate mixture than in culture with benzoate alone. In succinate-grown cells, expression was too low to be precisely measured. In vitro inhibition of the ortho pathway enzymes by succinate. The effect of succinate on the enzymes of the ortho pathway was estimated in vitro with whole cells or cell extracts obtained from cells taken from benzoate-grown batch cultures. Succinate had no effect on B12O (including the transport of benzoate), DHBDH, and C12O but strongly inhibited KTR in vitro. A 1 mM succinate concentration (with a [succinate]/[bketoadipate] ratio of 0.33) reduced KTR specific activity by 60%, and 10 mM succinate (with a [succinate]/[b-ketoadipate] ratio of 3.3) reduced the activity by over 90%. A partial inhibition (50%) of MCI was also measured when the [succinate]/ [muconate] ratio was 200 and for a concentration of succinate over 10 mM. In vivo inhibition of the ortho-pathway enzymes by succinate. A chemostat culture (D 5 0.33 h21) of A. eutrophus was established with benzoate as the sole carbon source and such that benzoate was the limiting substrate, i.e., the residual benzoate concentration was lower than the detection limit (10 mM) and considered to be zero. After the steady state was established, a pulse of succinate (45 mM) was added to the reactor in order to assess in vivo the possible inhibitory effect of succinate on benzoate degradation. Immediately after the addition of succinate, some benzoate transiently accumulated in the medium, reaching a maximum concentration of 2.8 mM (Fig. 3). This accumulation corresponded to a slight decrease in qbenzoate from 4.4 to 3.4 mmol z g21 z h21. At the same time, the specific rate of succinate consumption increased to reach a maximum value of 6 mmol z g21 z h21 2 h after the pulse. Then, the qbenzoate increased to 4 mmol z g21 z h21. Once succinate was exhausted, the residual benzoate was rapidly reconsumed. The rates obtained during this period of simultaneous consumption agree well with those seen in batch cultures and support the hypothesis that the capacity for flux through the succinate dehydrogenase is probably limiting under such car-

bon excess conditions. During this transient regimen, no metabolite of the b-ketoadipate pathway could be detected in the supernatant, although it is possible that trace amounts below the detection threshold may have occurred or that indeed some intracellular accumulation took place. DISCUSSION P. aeruginosa and P. putida have been shown to exhibit a catabolite repression effect whose mechanism is not yet understood (21), although it is known to be cyclic AMP independent (29). Several investigators reported the repression of the degradation of various aromatic compounds by succinate in pseudomonads (7, 11, 40), whereas others found no effect in similar experiments (15, 28). Our results show that A. eutrophus metabolizes benzoate and succinate simultaneously. This simultaneous consumption enabled the cell to increase the growth rate (m 5 0.56 6 0.02 h21 [mean 6 standard deviation]) while keeping YX/S maximum. This increase in growth rate obtained with the substrate mixture compared to that obtained with benzoate alone is directly proportional to the increased flux through the succinate dehydrogenase, suggesting that rate limitations are situated in the b-ketoadipate pathway of benzoate degradation. For growth on succinate, the situation is somewhat different, since the rate at which succinate is assimilated via the tricarboxylic acid

FIG. 3. Effect of a pulse of 45 mM succinate (at time zero) in a benzoatelimited chemostat culture of A. eutrophus. ■, benzoate; }, succinate; F, biomass; h, qbenzoate; {, qsuccinate. A theoretical washing curve for succinate is also shown (dotted line). The calculations of the specific rates of substrate consumption include the washing effect, as the medium feed rate was maintained throughout the experiment. The drop in biomass concentration at time zero is due to the diluting effect provoked by the addition of succinate.

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(TCA) cycle must also generate the acetyl-CoA necessary for biomass synthesis and hence limits the rate at which pyruvate can be fueled into neoglucogenesis. The simultaneous metabolism of succinate and benzoate thus enables a better distribution of metabolites within the metabolic network and hence an improved growth rate. Such an increase in the growth rate has already been observed, e.g., with Eubacterium limosum grown on glucose-plus-methanol mixtures (19). The growth yield measured for benzoate-plus-succinate mixtures (0.57 6 max 0.02 Cmol/Cmol) corresponds to the YX/benzoate previously determined with chemostat experiments (2). This value now appears to be a maximum for A. eutrophus 335 growing with organic acids. The results observed here are also in accordance with previous findings showing that substrate mixture utilization in A. eutrophus tends to maximize growth rate and yield (1) but are also in accordance with early results from Duetz and van Andel (10), who found that benzoate and succinate are metabolized simultaneously during pHauxostat cultures of P. putida. It appears that A. eutrophus controls the flux from each carbon source (benzoate and succinate) to maximize the growth rate via an optimal feeding of central metabolism with succinate and acetyl-CoA. Several mechanisms seem to control the simultaneous metabolism of benzoate and succinate. (i) Succinate, a product of the reaction catalyzed by KTR, inhibits the activity of this enzyme and is a logical site for regulation. A similar inhibitory effect on MCI is unlikely to have a significant role in vivo. The inhibition of KTR in vivo probably leads to an increase in the intracellular concentration of b-ketoadipate, the inducer of KTR in A. eutrophus (16). Such an increase in the intracellular b-ketoadipate concentration would explain why the KTR specific activity was higher in benzoate-plus-succinate cultures than when benzoate was the sole source of carbon (Table 2). This in vitro inhibition of enzymes of the ortho pathway by succinate was confirmed in vivo. After a pulse of succinate in a chemostat culture, the specific rate of benzoate consumption immediately decreased to 3.4 mmol z g21 z h21, a value close to that found in the batch culture with benzoate plus succinate. This immediate response indicates that an inhibitory effect, rather than a repression of enzyme synthesis, is the first response of the cell to the presence of succinate. In this light, it is perhaps surprising that other metabolites could not be detected, although this finding is probably linked to the relatively low levels of accumulation suspected to have occurred. High levels of metabolites are relatively rare in catabolic pathways, in which flux control is frequently shared by many of the enzymes. Indeed, when such overflow metabolism is observed, specific export systems are often implicated. Thus, any perturbation is liable to slow the flux through the entire pathway via modification of the thermodynamic equilibrium of each reaction, accentuated by the cascade of regulation phenomena that is in turn activated. The logical consequence is a diminished rate of substrate consumption, which in the case of a continuous culture process would result in the accumulation of carbon substrate until the stress is removed. (ii) Benzoate catabolism is partly repressed by the presence of succinate. During batch cultures on benzoate plus succinate, a 20 to 60% decrease in the specific activities of four enzymes of the ortho pathway in crude extracts led to a 40% decrease in the specific rate of benzoate consumption (from 5.5 to 3.15 mmol z g21 z h21). This repression phenomenon did not totally block the degradation of the aromatic compound, and diauxic growth, characteristic of the catabolite repression phenomenon, was not observed. (iii) Succinate metabolism is probably limited by succinate

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dehydrogenase. As the expression of the TCA cycle enzymes is constitutive in A. eutrophus (36), it seems that a maximum flux through succinate dehydrogenase (FSDH 5 9.5 to 9.7 mmol z g21 z h21) limits the rate of succinate degradation. During growth with benzoate, the FSDH was lower (8.9 6 0.2 mmol z g21 z h21 [mean 6 standard deviation]), and thus SDH activity is unlikely to be rate limiting. It would be of interest to examine the manner in which succinate and benzoate are metabolized in cells expressing succinate dehydrogenase activity at amplified levels. This simultaneous consumption of benzoate and succinate is in contrast to our previous observations with this same bacterium growing on acetate-plus-benzoate mixtures, in which diauxy due to repression at the transcriptional level of acoE, the gene encoding the acetyl-CoA synthetase and essential for activation of acetate, was observed (1). One might ask why such contrasting behavior occurs. Of course, such control of succinate could be achieved only by transporter regulation or inducer expulsion mechanisms, since all other reactions of succinate metabolism are common to the general catabolic pathways. However, the underlying difference is probably related to the possible metabolic fate of succinate compared to acetate. Succinate can be metabolized via the catabolic network, while acetate has a more restricted use as an energyyielding substrate via oxidation through the TCA cycle: there is no way to feed acetate into the neoglucogenic pathways during growth on benzoate. Thus, it is difficult to see what advantage the bacterium can derive from simultaneous consumption of benzoate plus acetate which, if allowed, would generate metabolic energy which the cell would not exploit. On the other hand, simultaneous consumption of succinate with benzoate allows carbon distribution through the central pathways, enabling more-rapid feeding of the anabolic pathways, i.e., cell growth. ACKNOWLEDGMENTS This work was supported by INRA, CNRS, and the Midi-Pyre´ne´es regional government. REFERENCES 1. Ampe, F., and N. D. Lindley. 1995. Acetate utilization is inhibited by benzoate in Alcaligenes eutrophus: evidence for transcriptional control of the expression of acoE coding for acetyl coenzyme A synthetase. J. Bacteriol. 177:5826–5833. 2. Ampe, F., and N. D. Lindley. 1996. Flux limitations in the ortho pathway of benzoate degradation of Alcaligenes eutrophus: metabolite overflow and induction of the meta pathway at high substrate concentrations. Microbiology 142:1807–1817. 3. Botsford, J. L., and J. G. Harman. 1992. Cyclic AMP in procaryotes. Microbiol. Rev. 56:100–122. 4. Canovas, J. L., and R. Y. Stanier. 1967. Regulation of the enzymes of the b-ketoadipate pathway in Moraxella calcoacetica. I. General aspects. Eur. J. Biochem. 1:289–300. 5. Chang, J. Y., R. Knecht, and D. G. Braun. 1981. Amino acid analysis at the picomole level. Biochem. J. 199:547–555. 6. Cocaign-Bousquet, M., A. Guyonvarch, and N. D. Lindley. 1996. Growth rate-dependent modulation of carbon flux through central metabolism and the kinetic consequences for glucose-limited chemostat cultures of Corynebacterium glutamicum. Appl. Environ. Microbiol. 62:429–436. 7. Daugherty, D. D., and S. F. Karel. 1994. Degradation of 2,4-dichlorophenoxyacetic acid by Pseudomonas cepacia DBO1(pRO101) in a dual-substrate chemostat. Appl. Environ. Microbiol. 60:3261–3267. 8. Don, R. H., A. J. Weightman, H.-J. Knackmuss, and K. N. Timmis. 1985. Transposon mutagenesis and cloning analysis of the pathway for the degradation of 2,4-dichlorophenoxyacetic acid and 3-chlorobenzoate in Alcaligenes eutrophus JMP134(pJP4). J. Bacteriol. 161:85–90. 9. Drewry, D. T., J. A. Lomax, G. W. Gray, and S. G. Wilkinson. 1973. Studies of lipid A fractions from the lipopolysaccharides of Pseudomonas aeruginosa and Pseudomonas alcaligenes. Biochem. J. 133:563–572. 10. Duetz, W. A., and J. G. van Andel. 1991. Stability of TOL plasmid pWW0 in Pseudomonas putida mt-2 under non-selective conditions in continuous culture. J. Gen. Microbiol. 137:1369–1374.

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