Alpha-ketoglutarate metabolism by cytochrome-containing anaerobes1

DANIEL R. CALDWELL AND CAROLYN K. RASMUSSEN Department of Microbiology and Veterinary Medicine, University of Wyoming, Laramie, WY, U.S.A. 82071

Accepted March 23, 1983

CALDWELL, D. R. , and C. K. RASMUSSEN. 1983. Alpha-ketoglutarate metabolism by cytochrome-containing anaerobes. Can. J. Microbiol. 29: 790-796.

During growth in the presence of tracer amounts of exogenously supplied alpha-keto[l-'4C]glutarate (AKG) or alpha-keto [5-'4~]glutarate, cytochrome-containing Bacteroides fragilis strain 2044 and Bacteroides vulgatus strain 8482 incorporated extremely small amounts of radioactivity into cell macromolecules and protoheme. Under identical conditions, Bacteroides " b" strain 7CM and Bacteroides buccae strain J1 incorporated substantial label from [5-I4C]AKG, but not [ I - I4C]~KG, into cellular macromolecules and protoheme. Bacteroides succinogenes strain S85 incorporated radioactivity from both [ I - I 4 C ] A ~ G and [S-I4C]AKG into cell macromolecules, but only label from [5-I4C]~KG appeared in protoheme. Selenomonas rurninantium strain HD1 and Butyrivibriofibrisolvens strain D l , both of which are devoid of cytochromes, incorporated substantial label from both [ 1 - ' 4 ~ ] ~ ~ G and [ 5 - I 4 C ] ~ ~ ~ into cell macromolecules, but failed to incorporate label from either position into protoheme. Bacteroides ruminicola sp. brevis strain GA33 incorporated label from both [I-'~C]AKG and [5-I4C]~KG into both cell macromolecules and protoheme. A substantial portion of the heme synthesized by this organism may be formed by the "plant" pathway involving the intact use of the AKG carbon skeleton. Major differences exist in the manner and extent of AKG utilization among cytochrome-containing anaerobes and between these organisms and bacteria devoid of cytochromes obtained from similar environments.

CALDWELL, D. R., et C. K. RASMUSSEN. 1983. Alpha-ketoglutarate metabolism by cytochrome-containing anaerobes. Can. J. Microbiol. 29: 790-796.

Durant leur croissance en prCsence d'une source extkrieure d'un marqueur soit l'alpha-cktoglutarate [l-I4c] (AKG) ou l'alpha-cttoglutarate [5-I4C], Bacteroides fragilis souche 2044 et Bacteroides vulgatus souche 8422, tous deux contenant des cytochromes, incorporent des quantitks extrkmement faibles de radioactivitk dans les macromolCcules cellulaires et dans le protohkme. Dans des conditions identiques, les souches 7CM de Bacteroides "I" et J1 de Bacteroides buccae incorporent dans les macrornolkcules cellulaires et le protoheme, une quantitC substantielle de radioactivitk a partir du [5-I4C]AKG mais pas a partir du [I-I4C]AKG. Bacteroides succinogenes, souche S85, incorpore de la radioactivitt a la fois du [ 1 - I 4 c ] ~ ~ G et du [5-I4C]AKG dans les macromolkcules cellulaires mais, dans le protohkme, la seule radioactivitk qui apparait vient du [S-I4C]AKG. Selenomonas ruminantiurn souche HD1 et Butyrivibrio fibrisolvens souche D l , toutes deux dCpourvues de cytochromes, incorporent une quantitk substantielle de radioactivitk dans les macromolkcules cellulaires A partir du [I- '~C]AKG et du [ 5 - I 4 c ] ~ ~ G mais n'en incorpore pas dans le protohbme. Bacteroides rurninicola sp. brevis souche GA33 incorpore du materiel marquk 21 partir de [l-I4C]AKG et [ 5 - I 4 C ] ~ ~ ~ a la fois dans les macromolkcules cellulaires et dans le protoheme. Une portion substantielle de l'hkme synthktisk par cet organisme pourrait &re formCe au cours du cycle de la photophosphorylation qui implique l'utilisation intkgrale de la structure de carbones de la molCcule d'AKG. I1 existe des diffkrences majeures dans la manikre et le degr6 d'utilisation d'AKG parmi les bactkries anakrobies contenant des cytochromes et entre ces organismes et des bacttries dkpourvues de cytochromes isolCes de milieux similaires.

[Traduit par le journal]

Introduction The metabolism of alpha-ketoglutarate (AKG) by

aerobic microbes is well understood. In these organisms, AKG serves major functions in both energy-yielding and biosynthetic metabolism (Moat 1979). AKG meta- bolism by obligately anaerobic bacteria is less well understood. For many of these organisms, AKG, generated either by operation of an "incomplete" tri- carboxylic acid (TCA) cycle (Moat 1979) or from the reductive carboxylation of succinate (Allison and Robinson 1970; Allison et al. 1979), serves biosynthetic functions, primarily as a precursor to the glutarnic family of amino acids and to heme in cytochrome-containing species.

'Published with the approval of the Director, Wyoming Agricultural Experiment Station as Journal Article 12 18.

Although it is generally recognized that AKG is primarily involved in the biosynthetic metabolism of anaerobes, precise information concerning the manner of its utiliza- tion by these organisms is incomplete. This investigation studied the use of exogenously supplied position- labelled AKG for cellular biosynthesis during growth of cytochrome-containing Bacteroides species obtained from the human mouth, the human gastrointestinal tract, and the bovine rumen. For comparative purposes rumen bacteria lacking cytochromes were also studied. The results indicate that major differences exist among these organisms in their ability to incorporate radioactivity, supplied as [5-I4C]AKG or [I-14C]AKG, into cellular macromolecules and protoheme. A substantial portion of the protoheme synthesized by B . ruminocola sp. brevis strain GA33 may arise from the "plant" pathway (Beale

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CALDWELL AND RASMUSSEN 791

and Castelfranco 1973) involving the intact use of the radioactivity from [ l -14C]~KG or [5-14C]AKG into cell AKG carbon skeleton. macromolecules and protoheme, cells from 2.0 mL of stationary

Materials and methods Organisms, media, and culture conditions

The organisms studied were Bacteroides fragilis strain 2044 and Bacteroides vulgatus strain 8482, of human intestinal origin, Bacteroides ''1 " strain 7CM (Moore and Holdeman 1974) and Bacteroides buccae strain J1 (Holdeman et al. 1982), which were originally obtained from the human mouth, and Bacteroides ruminicola sp. brevis GA33 and Bacteroides succinogenes S85, which were obtained from the bovine rumen. All of the organisms, when grown in a heme- containing medium, contain cytochromes. All of the Bacteroides isolates studied, except B. succinagenes and B. ruminicola sp. brevis, require heme for rapid and abundant growth. (Caldwell and Arcand 1974; Speny et al. 1977). The latter organisms synthesize protoheme from linear precursors. For comparative purposes Selenomonas ruminantium HD1 and Butyrivibrio jibrisolvens D l , both of which are devoid of cytochromes, were studied. The cultures were obtained from the culture collection maintained by the Department of Microbiology and Veterinary Medicine, but strains 2044, 8482, 7CM, and J1 were originally supplied by the Anaerobe Laboratory, Virginia Polytechnic Institute and State University, Blacksburg, VA.

The organisms were maintained in the rumen fluid glucose cellobiose agar (RGCA) medium of Bryant and Robinson (1962). The medium used for inoculation of AKG-containing media was the protoheme-containing complete medium of Caldwell and Arcand (1974) with menadione deleted and 0.5% (w/v) trypticase added. The medium for study of AKG metabolism was identical to the inoculum medium, except that protoheme was deleted and replaced with [ 1 - 1 4 C ] ~ ~ G or [s-'~c]AKG.

The Hungate anaerobic technique (Hungate 1970) was used throughout the study. Media were prepared in round-bottom boiling flasks, and after sterilization by autoclaving under oxygen-free C 0 2 for 20 min at 121°C, were anaerobically and aseptically dispensed into sterile, black-rubber-stoppered test tubes (13 X 100 mm). Maintenance medium was dispensed in 3.0-mL amounts, whereas the inoculum medium was dispensed in 4.0-mL amounts and the AKG utilization medium was dispensed in 3.9-mL amounts. Addition of 0. 1-mL amounts of [I-14C]AKG or [ 5 - 1 4 C ] ~ ~ G dissolved in 95% (v/v) ethyl alcohol brought the AKG utilization medium to a 4.0 mL total volume.

The maintenance medium and the inoculum medium were loop inoculated, whereas the AKG utilization medium was inoculated with 0.1 mL of an overnight broth culture of the inoculum medium exhibiting an OD of at least 0.4 at 600 nm (1 cm light path) using uninoculated medium as a blank. All cultures were incubated without agitation at 37OC, and growth in experimental broth cultures was monitored by turbidity, as measured with a Bausch and Lomb Spectronic 20 spectro- photometer, until stationary phase was obtained (24-72 h). At stationary phase all cultures exhibited an OD of at least 0.4. The purity of the cultures was checked throughout the study by Gram stains.

Cell harvesting and protoheme extraction In preparation for assessment of the incorporation of

phase cultures were-harvested by centrifugation at 5000 X g for 15 min at 4'C. The supernatant fluid was removed and stored frozen for later analysis. The harvested cells were washed twice in 2.0 rnL of 50 m~ ~ 0 ~ - ~ buffer (pH 7.0), with centrifugation between each wash, and resuspended in the originalvolume.

Protoheme was extracted from the cells by treatment of 1 .O-mL samples of original culture with 1.0 mL of 2% (v/v) concentrated HCl in acetone for 30min to remove any protoheme bound to cytochrome. The extracted protoheme was purified by the addition of 2.0 mL of benzene and 0.1 mL of 1 N H2SO4. A small amount ( 5 1 mg) of nonradioactive protoheme was added to the preparation, which was then shaken vigorously and allowed to separate. Separation of the acidified benzene layer and the aqueous acetone layer was sharpened by centrifugation for 3 min at 5000 X g. The benzene layer was then carefully and quantitatively withdrawn and reextracted twice in an identical manner. These procedures resulted in a highly purified preparation. Preliminary experiments revealed that 92.5% of AKG radioactivity added to uninoculated culture medium and processed identically to experimental samples was partitioned into the aqueous phase and that only 7.5% of added label was found in the benzene layer. Processing of the preparation three times, therefore, resulted in the AKG radioactivity in the final benzene layer constituting less than 0.05% of that originally supplied as AKG.

Thin-layer chromatography To confirm the presence of radioactivity in protoheme

formed during growth of the organisms in media containing [I- '~C]AKG or [ 5 - 1 4 C ] ~ ~ G , the benzene extracts of acidified acetone-treated cells were chromatographed to separate protoheme from AKG. Replicate samples (20-50 p,L) of extracts were applied to air-dried silica gel plates prepared by spreading 21 g of silica gel H (Type 60, EM Laboratories Inc., Elmsford, NY) suspended in 60 I& of distilled-demineralized water on five 400-cm2 glass plates. AKG and protoheme were separated using the benzene - methyl alcohol - acetic acid (90: 16:8; by volume) system of Myers and Huang (1966), and the separa- tion obtained in experimental preparations was compared with known standards of AKG prepared in distilled water and of protoheme dissolved identically to the extracts. AKG, as detected by spraying developed chromatograms with 0.1% (w/v) bromocresol green in 95% (v/v) ethyl alcohol, migrated to an Rf of 0.23, whereas protoheme, determined visually, migrated to an Rf of 0.89. Radiochemical studies revealed no contamination of the protoheme portion of the chromatogram by radioactivity supplied as AKG.

In some experiments, the radioactivity in cells and succinic acid was determined as a percentage of the radioactive AKG supplied which was used during growth rather than as a percentage of the total original AKG radioactivity. For these experiments AKG and succinic acid (Rf 0.52) were also separated from each other by the method of Myers and Huang (1966). Replicate 20-FL amounts of radioactive uninoculated culture medium and culture supernatants after growth were chromatographed, and the separated spots were located by spraying with the pH indicator described above. The spots

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792 CAN. J. MICROBIOL. VOL. 29, 1983

were scraped out and their radioactivity evaluated by liquid fluid containing a constant amount of uniformly labelled scintillation orocedures. The difference between the AKG 1'4Clhexadecane. radioactivity per millilitre remaining in culture supernatants after growth and the radioactivity initially supplied as AKG was considered the AKG radioactivity used during growth. The radioactivity in succinate was determined by an analogous procedure. Careful preliminary experiments revealed no cross contamination of radioactive succinate and AKG.

Determination of carbon dioxide formation from AKG The liberation of C 0 2 from [ 1 - 1 4 C ] ~ ~ G or [5-14C]~KG

was determined from the radioactivity in 2.0 mL of the gaseous phase of stationary phase cultures. Samples obtained through the rubber stopper with a gas-sampling syringe were slowly dispensed into 15-mL amounts of a 2:l (v/v) mixture of ethanolamine - ethylene glycol, trapping the C 0 2 as ethanol- amine carbonate. Subsamples of trap material were used to evaluate the radioactivity in the gaseous phase. The C02- associated radioactivity was then calculated, on a per millilitre liquid culture basis, taking into account the volume of the gaseous phase (3.8 mL), the total culture vessel volume (7.8 mL) and the relative distribution of C 0 2 in the liquid (45.5%) and gaseous phases (55.5%). At the pH of the cultures after growth (SpH 5.5), at least 88% of the total inorganic carbon in the liquid phase was in the form of C02 . Considera- tion of all of the factors indicated allowed a comparison between the C02-associated radioactivity and the amount of radioactivity supplied to liquid cultures as AKG, on a per millilitre of liquid culture basis. Controls consisted of AKG added to uninoculated tubes of medium and incubated and processed identically to experimental cultures.

Liquid scintillation procedures The radioactivity in uninoculated culture media, culture

supernatant fluids, protoheme extracts, washed cells, chro- matogram spots, and trap material used to detect radioactive C 0 2 was determined by liquid scintillation procedures, using a scintillation fluid composed of 42 mL of Liquifluor concentrate (New England Nuclear Corporation, Boston, MA), 800 mL of reagent-grade toluene, and 200 mL of absolute ethyl alcohol. The latter was added to enhance the solubility of polar compounds, including water, in the scintillation fluid. The scintillation fluid contained 4 g of PPO and 50 mg of POPOP/L.

To evaluate the radioactivity per millilitre, the following procedures were used. (i) For culture supernatant, fluids, uninoculated culture media, and washed resuspended cells, 20-pL amounts were combined with 10 mL of scintillation fluid. (ii) To determine the radioactivity in benzene extracts, 0.3-mL amounts of extract were combined with 10 mL quanti- ties of scintillation fluid. (iii) The radioactivity in chromato- gram spots was evaluated by carefully scraping the silica gel associated with spots into scintillation vials and adding 10-mL amounts of fluor solution. (iv) Detection of C 0 2 radioactivity in "trap material" was accomplished by combining 5.0 mL of trap material with 10 mL of fluor solution. All samples were counted using a Packard Tri-Carb liquid scintillation spectrometer (model 3320) and corrected to a per millilitre basis after consideration of differences in background counts, sample size, counting times, and relative counting efficiency as determined by a channel ratio procedure established by adding varying amounts of chloroform to 10-mL amounts of counting

Chemical sources and purity Except for trypticase, all chemicals were of reagent-grade

quality. Medium ingredient chemicals were obtained from various sources. The [ 1 - 1 4 C ] ~ ~ G and 15-14C]AKG were obtained from the New England Nuclear Corporation, Boston, MA. Both radiochemicals had a specific activity of 7.3 pCi/

Results Use of [5-I4C]AKG

Table 1 displays information concerning label incor- poration from [5-14C]~KG into cells and protoheme during growth of cytochrome-containing isolates of the genus Bacteroides. Data for organisms lacking cyto- chromes obtained from the bovine rumen is also shown. Although substantial growth (OD r 0.4) of both B . fragilis strain 2044 and B . vulgatus strain 8482 occurred in all of the experiments, only very small amounts of radioactivity from [5-14C]AK~ were found in washed cells and protoheme. Bacteroides "1" strain7CM and B. buccae strain J1, by contrast, incorporated 10- 1 1% of the supplied radioactivity into cells, and at least 50% of the incorporated radioactivity appeared in protoheme. Bacteroides succinogenes strain S85 behaved in a manner similar to that for strains 7CM and J1. Although it incorporated approximately one-half as much total radioactivity into cells as was found for strains 7CM and J1, the major portion of the radioactivity incorporated into cells of strain S85 was found in protoheme. Bacteroides ruminicolas sp. brevis strain GA33 in- corporated approximately three times as much total radioactivity from [ 5 - 1 4 C ] ~ K ~ into cells as did strains 7CM and J1, and approximately six times the total radioactivity incorporated by strain S85, but only about one-fourth of the cell-associated radioactivity found in protoheme for strains 7CM, J1, and S85 appeared in the protoheme synthesized by strain GA33. The major portion of the radioactivity incorporated by the latter organism appeared in cellular materials other than protoheme. Selenomonas ruminantium strain HD 1 and B. jibrisolvens strain D l , both of which are devoid of cytochromes, incorporated substantial amounts of radio- activity from [5-14C]AKG into cells, but no radioactivity was found in the protoheme.

Use of [ I - 1 4 c ] ~ ~ G Table 2 displays data concerning label incorporation

into cells and protoheme during growth of cytochrome- containing Bacteroides species in the presence of [l-14C]- AKG. Only small amounts of radioactivity supplied as [I-14C]A~G appeared in cells of strains J l and 7CM. Strains 2044 and 8482 displayed a similar inability to incorporate large quantities of label supplied as [l-14C]- AKG into cells and protoheme. Strain S85 incorporated

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CALDWELL AND RASMUSSEN 793

TABLE 1. Incorporation of radioactivity from [5-14C]AKG into cells and protoheme of cytochrome- containing anaerobic bacteria

Disintegrations per minute (dpm) per millilitrea

Protoheme 12458 10535 567 223 9045 5 882 0 836 (60.0) (54.2) (28.5) (9.9) (14.0) (59.0) (0) (2.9)

Cells 20767 19430 1990 2245 64388 10 133 68067 28690 (11.3) (10.3) (1.1) (1.2) (34.9) (5.3) (36.9) (15.6)

"Each of the experimental values is the average of five determinations from each of three experiments. Figures in parentheses in the "Heme" row are the percentages of the cell-associated radioactivity attributable to protoheme. Figures in parentheses in the "Cells" row are the percentages of the originally supplied radioactivity incorporated into cells. For all the experiments, the originally supplied 15-'"CIAKG radioactivity was 184 300 dpm/mL. The specific activity of the [5-'"CIAKG was 7.3 ~ C i / p m o l .

TABLE 2. Incorporation of radioactivity from [ l - I4C]~KG into cells and protoheme of cytochrome- containing anaerobic bacteriaa

Disintegrations per minute per millilitre

Protoheme 724 359 0 386 4832 837 0 0 (20.9) (20.3) (0) (23.6) (7.5) (1.6) (0) (0)

Cells 3674 1769 1420 1630 64067 51 536 98067 51 357 (1.6) (0.75) (0.60) (0.70) (27.5) (22.1) (42.1) (22.1)

"Except for the fact that l-14C-labelled alpha-ketoglutarate was used in these experiments and that the initially supplied radioactivity was 232 800 dpm/mL, the footnotes of Table 1 apply to this Table also. The specific activity of [I-'"CIAKG was 7 .3 ~ C i / ~ m o l .

substantial amounts of label supplied as [ l -14C]~KG into cells, but less than 0.5% of the originally supplied radioactivity appeared in protoheme. The proportion of the radioactivity supplied as [I-14C]AKG found in cells of strain S85 was approximately four times that found in cells of the same organism grown in the presence of [5-14C]~KG. Strain GA33 incorporated radioactivity supplied as [I-l4C1AKG into cells abundantly and a substantial portion of the cell-associated radioactivity appeared in protoheme. The presence of label in proto- heme was confirmed by thin-layer chromatography of the benzene extracts. The fraction of cell-associated radioactivity in protoheme of strain GA33 formed from [ l -14C]~KG was approximately one-half that found in the same compound from [5-14C]AKG. Although both strains HD1 and D l incorporated substantial amounts of label from [l-14C]AKG into cells, no radioactivity was found, for either organism, in protoheme.

Carbon dioxide formation from position-labelled AKG Table 3 presents information concerning C02 formation

by the organisms grown in the presence of [l-14C]AKG or [5-14C]~KG. Consideration of the data for [l-14C]- AKG reveals that strain D l either failed to form, or formed very small amounts, of C 0 2 from [I-14C]~KG,

TABLE 3. Carbon dioxide formation from [1-I4C]~ICG or [5-14C]- AKG during growth of cytochrome-containing anaerobic bacteria

Disintegrations per minute per millilitrea

Label source 7CM J1 GA33 S85 HDl Dl

"Each value is the average of three determinations from each of three experi- ments. Figures in parentheses are the percentages of the original radioactivity found in carbon dioxide. The originally supplied radioactivity was 496 000 dpm/ mL for [1-'4C]AKG and 204 700 dpm/mL for 15-'"CIAKC. Since strains 2044 and 8482 were refractory to AKG, they were not used in these experiments.

since the amounts of radioactivity from [l-14C]AKG found in C 0 2 were very small. Most of the remaining organisms displayed a greater ability to form C 0 2 from [l-14C]AKG than did strain D l , although except for strain HD1, decarboxylation of [l-14C]~KG was quanti- tatively rather small. The latter organism, by contrast decarboxylated at least 29% of [l-14C]AKG. With regard to the data for [ 5 - 1 4 C ] ~ ~ G , neither strain GA33 nor strain D l displayed evidence for substantial C 0 2

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794 CAN. J , MICROBIOL. VOL. 29, 1983

TABLE 4. The incorporation of label supplied as [I-I4C]AKG or [S-I4C]AKG into the cells and succinate formed by Bacteroides ruminicola ssp. brevis strain GA33 as a function of the AKG

used during growth

Disintegrations per minute per millilitre

Supplied 438 000 218 000 Remaining 294 350 174 200 Used 143 650 (30.4) 43 800 (20.0) Cells 26 200 (18.2) 13 425 (30.6) Succinate 152 (0.10) 31 (0.07)

"Each value is the average of three determinations from each of two experi- ments. The figures in parentheses adjacent to the "Used row are the percentages of the supplied radioactivity used during growth, whereas the figures adjacent to the remaining columns represent the percentages of the used radioactivity attrih- utahle to cells and succinic acid.

formation, since the amounts of radioactivity found in C02 formed from the five-labelled molecule were small. The remaining organisms displayed quantitatively greater abilities to decarboxylate [5-I4C]AKG than were found for strains GA33 and D l . Strain 7CM gave rise to C02 from 8.3% of the supplied [ 5 - 1 4 C ] ~ ~ G followed, in descending order, by strain HD1 (6.6%), strain S85 (4.1 %), and strain J 1 (3.0%). Although the total amount of decarboxylation of both [ 1 - 1 4 C ] ~ ~ G and [5-14C]- AKG by strains S85 and J l was small, both organisms formed C02 from [5-14C]~KG nearly three times as extensively as was found for [l-14C]AKG.

The distribution of label supplied as AKG in cells and succinate formed by strain GA33

Strain GA33 displayed evidence for label in protoheme from [ 1 - 1 4 C ] ~ ~ G , suggesting the possibility of the use of at least some intact AKG molecules for heme synthesis. Succinate is a major fermentation end product of the organism (Bryant et al. 1958), and previous studies indicate that the organism synthesizes AKG from carbon dioxide fixation and the reductive carboxyla- tion of succinate (Allison and Robinson 1970). The present study, as well as previous work (Allison and Robinson 1970; Allison et al. 1979), indicates that strain GA33 has a very limited ability to decarboxylate AKG. It was thus of interest to study the distribution of radioactivity from AKG in cells and succinate. The previous studies considered AKG metabolism as a function of the radioactivity supplied. Table 4 displays data concerning the distribution of radioactivity, supplied as AKG, in cells and succinic acid as a function of the radioactive AKG supplied which was metabolized during growth. As measured by either [ l - I4C]A~G or [5-14C]AKG, it is apparent that substantially less than the supplied radioactivity was used by strain GA33 and also that only a portion of the radioactivity used was

recovered in cells and succinate. Furthermore, the amount of used AKG appearing in succinate, from either label position, was extremely small.

Discussion This study did not attempt to account for all of the

radioactivity supplied as AKG. In addition, we did not attempt to be strictly quantitative. The turbidity at stationary phase for the various organisms varied as much as twofold, but within an organism, the turbidities of stationary-phase cultures grown in the presence of [I-14C]AKG or [ 5 - 1 4 C ] ~ ~ ~ never varied from each other by more than 20%. As a consequence of these findings, critical quantitative comparisons between iso- lates representing different species are impossible, not only because of growth differences but also because of probable differences in the manner and extent of dilution of radioactivity supplied as AKG during growth of different organisms. In addition, the very small amounts of label (1 1.5% of that supplied) associated with cells of some strains reflect methodology problems, e. g . , cell washing. Recognizing these limitations, it is none-the- less clear that major differences exist in the manner of AKG utilization by various cytochrome-containing Bac- teroides species and between these organisms and organisms lacking cytochromes obtained from similar environments.

It is apparent that strains 2044 and 8482 were essentially impermeable to AKG, since although sub- stantial growth of both organisms occurred (OD values of at least 0.4), only small amounts of supplied radioactivity were found in either cells or protoheme, irrespective of AKG-label position. It is possible that the small amounts of cell-associated radioactivity supplied as AKG found in these organisms as well as the amounts in protoheme are apparent rather than real. In contrast, substantial amounts of the supplied AKG radioactivity were found in cells of most of the remaining organisms studied, although the proportion of incorporated radio- activity attributable to protoheme, as opposed to other cell constituents, varied among the organisms.

Strains 7CM and J1 incorporated approximately 10 times the amount of radioactivity supplied as [5-14C]AKG into cells than was found for radioactivity supplied as [I-14C]AKG. Strain S85 displayed an opposite response, with regard to label incorporation into cells, to that observed for strains 7CM and J l . Strain S85 pref- erentially incorporated [ 1 - 1 4 ~ ] ~ ~ ~ radioactivity into cells. For all -of these organisms, the results are consistent with the assumption that protoheme is synthe- sized by the "classical" pathway (Smith 1975) involving the condensation of succinyl-CoA and glycine, since radioactivity from the C(5), but not the C(l), end of AKG appeared in protoheme. In the classical pathway, radioactivity from the C(l) end of AKG would be lost as

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CALDWELL AND RASMUSSEN 795

C02 and would therefore not be found in protoheme. It appears that, although both strains 7CM and J1 require protoheme for rapid and abundant growth (Caldwell and Arcand 1974), they have some, although limited, ability to synthesize protoheme.

The use of AKG by strains GA33, HD1, and Dl differed from its use by the previously discussed organisms, both with respect to total radioactivity incorporation into cells and with respect to label found in protoheme. Since they are devoid of cytochromes, it is not surprising that strains HD1 and Dl failed to incorporate label from AKG from either position into protoheme. The very small amount of apparent radio- activity in "protoheme" of strain Dl from [5-14C]~KG presumably reflects methodology difficulties. For both strains HD 1 and D 1, a slightly greater portion of C(1) radioactivity was found in cells than was found for C(5) AKG, but the differences were small. The present study does not permit determination of whether the differences in the proportions of cell-associated C(l) and C(5) AKG label reflect differences in growth, differences in use of the intact AKG carbon skeleton, differences in the use of AKG metabolites, or some combination of all of these factors. It is likely, however, that for these organisms, as well as for strains S85 and GA33, substantial portions of the incorporated radioactivity supplied as AKG reflect use of the intact AKG carbon skeleton, since AKG, arising chiefly from the reductive carboxylation of succinate (Allison and Robinson 1970; Allison et al. 1979), is a major precursor to the glutamic family of amino acids in these microbes.

AKG use by strain GA33 was unusual in some respects, particularly in its use for protoheme synthesis. The finding of radioactivity in protoheme from [l-14C]- AKG suggests the possibility that a portion of the protoheme in this organism may result from the "plant" pathway involving the direct use of the intact AKG carbon skeleton for synthesis of 8-aminolevulinic acid (Beale and Castelfranco 1973). That, indeed, label from [I-14C]~KG appeared in protoheme was confirmed by demonstration of label in the protoheme spot of thin- layer chromatograms of benzene extracts. Under the conditions used, we think it most unlikely that label from [I- '~C]AKG would occur, to any extent, in protoheme formed by a mechanism involving decarboxyla- tion of the one carbon of AKG followed by fixation of C02 into succinate and conversion of the latter to heme. The present work, as well as previous studies (Allison and Robinson 1970; Allison et al. 1979), indicates that decarboxylation of the one carbon of AKG occurs only slightly, if at all. Succinate is a major fermentation end product of strain GA33 (Bryant et al. 1958). Therefore any protoheme synthesis pathway involving succinate formation from AKG by strain GA33, particularly a pathway involving C02 fixation, would lead to substan-

tial dilution of label, with a consequent great diminution of label in protoheme. It is likely that a pathway involving direct use of the AKG carbon skeleton, with relatively less label dilution, would lead to substantially more label incorporation into protoheme. It would be interesting to determine whether other strains of B. ruminicola sp. brevis incorporate [I-14C]AKG radio- activity into protoheme and whether radioactivity from alanine and S,y-dioxovalerate might also appear in heme (Morton et al. 198 1).

It is of interest that many of the organisms displayed some ability to form C02 from the C(l) and C(5) ends of AKG. Most notable in this respect was strain HDl, which decarboxylated [I-14C]AKG extensively. Pre- vious work indicates that this organism synthesizes AKG via C02 fixation and the reductive carboxylation of succinate (Allison et al. 1979). It would be interesting to determine whether the decarboxylase and synthetase activities reflect operation of the same enzyme in a net different direction as a result of experimental conditions or whether two separate enzymes are involved in the carboxylation and decarboxylation of AKG, as been shown for other 2-0x0 acids (Bush and Sauer 1977).

The decarboxylation, by some organisms, of the C(5) as well as the C(l) end of AKG is interesting. Although we did not attempt to identify the products formed by decarboxylation of the C(5) or the C(l) end of AKG, it is possible that the 14C(5)-decarboxylating organisms may form alpha-ketobutyrate (AKB). This metabolite has been shown to be formed by the reductive carboxylation of propionyl-CoA by mixed rumen bacteria (Milligan 1970) and their extracts (Bush and Sauer 1976) and to be important in isoleucine biosynthesis (Bush and Sauer 1976). It would be interesting to determine whether label from [ 1 -14C]AKG could be found in isoleucine and whether AKB depressed incorporation of label from [l-14C]AKG into isoleucine. It is possible that the 14C(5)-decarboxylating organisms have an alternate way of forming AKB and that AKB derived from AKG decarboxylation at the C(5) end is a precursor to isoleucine. The amount of [5-14C]~KG decarboxylation by the organisms probably precludes AKB as a precursor to the propionate formed by some of them via a reversal of the reactions leading to AKB formation from pro- pionyl-CoA.

The data in Table 4 indicate that substantial amounts of the AKG used by strain GA33 were not recovered in either succinate or cells, and also that substantially less AKG was used during growth than was supplied. It is therefore likely that C02 formation, as a function of actual AKG metabolism, may be substantially greater than is apparent.

It is evident that major differences exist in AKG utilization among cytochrome-containing Bacteroides species and between these organisms and other bacteria

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