metabolic function of branched-chain volatile fatty acids, growth ...

METABOLIC FUNCTION OF BRANCHED-CHAIN VOLATILE FATTY ACIDS, GROWTH FACTORS FOR RUMINOCOCCI II. BIOSYNTHESIS OF HIGHER BRANCHED-CHAIN FATTY ACIDS AND ALDEHYDES M. J. ALLISON, M. P. BRYANT, I. KATZ, AND M. KEENEY Dairy Cattle Research Branch, Animal Husbandry Research Division, Agricultural Research Service, Beltsville, Maryland, and Dairy Department, University of Maryland, College Park, Maryland

Received for publication December 4, 1961 ABSTRACT

body and milk lipids, may be of microbial origin is discussed.

ALLISON, M. J. (Dairy Cattle Research Branch, USDA, Beltsville, Md.), M. P. BRYANT, I. KATZ, AND M. KEENEY. Metabolic function of Branched-chain volatile fatty acids are rebranched-chain volatile fatty acids, growth quired for growth by several strains of both factors for ruminococci. II. Biosynthesis of cellulolytic and noncellulolytic rumen bacteria. higher branched-chain fatty acids and alde- We have attempted to obtain information on the hydes. J. Bacteriol. 83:1084-1093. 1962.-A physiological function of these acids by deternumber of strains of rumen bacteria require mining the metabolic fate of C14-labeled fatty branched-chain volatile fatty acids for growth. acids. Radioactivity from isovalerate-1-C'4 or A strain of Ruminococcus flavefaciens that re- isovalerate-3-C'4 was incorporated into leucine quires either isovalerate or isobutyrate incorpo- and into the lipid fraction of Ruminococcus rates radioactive carbon from isovalerate-1-C04 flavefaciens' cells (Allison, Bryant, and Doetsch, and isovalerate-3-C14 into leucine and into the 1962). This paper presents data on the nature of lipid fraction of the cells. Evidence obtained by the labeled substances in lipid of R. flavefaciens both paper and gas chromatography indicated grown in a medium containing isovalerate-1-C'4, that most of the label in the lipid of cells grown and in R. albus grown in the presence of iso-in isovalerate-1-C14 was in a branched-chain butyrate-1-C'4. Information is also given on the 15-carbon fatty acid, with some in a 17-carbon proportions of higher fatty acids in these cells. acid; about 7.5% of the C14 was recovered in a branched-chain 15-carbon aldehyde. The MATERIALS AND METHODS aldehydes were in the phospholipid fraction and R. flavefaciens C94 and R. albus 7 (Bryant et were presumably present as plasmalogen. al., 1958) are cellulolytic anaerobic bacteria A strain of R. albus was shown to require isolated from high dilutions of bovine rumen isobutyrate, 2-methyl-n-butyrate, or 2-ketoiso- contents. Subsequent reference to these strains valerate for growth. This strain did not incorpor- by their specific names is for the sake of conate appreciable C14 from isovalerate-1-C14 or venience and is not intended to infer conformity isovalerate-3-C14. When grown in a medium with all other strains of the species. containing isobutyrate-1-C14, most of the cellular The basal medium and methods used to assay C'4 was found in the lipid fraction. Analysis of for growth factors and to study incorporation of the lipid demonstrated that the label was present labeled metabolites have been described (Allison mainly as branched-chain 14-carbon and 16- et al., 1962). Lipid from R. flavefaciens C94 was carbon fatty acids, with 11% of the C'4 present 1 Ruminococcus flavefaciens, not recognized in in 14- and 16-carbon carbonyl compounds, the seventh edition of Bergey's Manual of Determipresumably branched-chain aldehydes. Bacteriology, was described by: Sijpesteijn, Branched-chain 14-, 15-, and 16-carbon fatty native A. K. 1951. On Ruminococcus flavefaciens, a celluacids are major components of the lipids of these lose-decomposing bacterium from the rumen of rumen bacteria. The possibility that these acids sheep and cattle. J. Gen. Microbiol. 5:869-879. and aldehydes, which are found in ruminant Editor, J. Bacteriol. 1084

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GROWTH FACTORS FOR RUMINOCOCCI

obtained from cells grown from a small inoculum to an optical density (OD) of 0.32 (58 hr) in the basal medium plus sodium isovalerate-1-C14 (California Corporation for Biochemical Research, Los Angeles, Calif.), 0.38 j.mole/ml; sodium isobutyrate, 0.075 ,umole/ml; and sodium acetate, 20 ,umoles/ml. The lipid fraction of R. albus 7 was obtained from cells grown from a small inoculum to OD 0.86 (24 hr) in the basal medium plus sodium isobutyrate-1-C14 (Nuclear Chicago Corp., Des Plaines, Ill.), 0.2 Mmole/ml, and sodium acetate, 20 ,umole/ml. Cells were grown in 1.5 liters of medium in a 2-liter, rubberstoppered Florence flask. A glass tube inserted through the rubber stopper to the bottom of the flask permitted bubbling with sterile, 02-free CO2 while the medium was cooled after autoclaving. Sodium carbonate and Na2S-9H2O were sterilized separately and added to cooled medium through a tube (13 mm diam) which was also inserted through the rubber stopper. This tube was stoppered with cotton during autoclaving and while bubbling with gas. When the medium had cooled and equilibrated with C02, the tube was stoppered with a sterile rubber stopper to prevent entrance of oxygen. Carbon dioxide was bubbled through the medium when the rubber stopper was removed to add inoculum and when samples of the culture were removed. Cells were harvested by centrifugation for 10 min at 15,000 X g and were washed with 0.85% saline. Lipid was extracted from the cells with 300 ml of chloroform-methanol (2:1) by stirring for 24 hr at room temperature. Most of the cellular material not soluble in chloroformmethanol was removed by filtration through filter paper, and the extract was evaporated to dryness in vacuo in a rotary evaporator. The extract was taken up in 50 ml of chloroformmethanol (2:1), washed by shaking with 10 ml of water, and stored in solution at 0 C. Free fatty acids were extracted from a portion of the lipid fraction by repeated extraction from a hexane solution with 1% Na2CO3. The remaining material was saponified by refluxing for 1 hr with 2% KOH in 90% methanol. Water was added to the reaction mixture, and nonsaponified material was extracted with hexane at alkaline pH. Saponified fatty acids were extracted with hexane and ether after adjustment to pH 1.5 with H2S04. Methyl esters of fatty acids were prepared in a

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reaction mixture containing about 10 ml of a solution of the lipid in chloroform (dried over silicic acid) and 15 ml of absolute methanol saturated with HCl gas. The reaction mixture was allowed to stand overnight at room temperature, or boiling chips were added and it was heated under reflux, on a steam bath for 1 hr. Several mg of 2,4-dinitrophenylhydrazine were added to this reaction mixture in later studies, after it was noted that an appreciable quantity of long-chain aldehydes was present in the lipid. Control experiments indicated quantitative formation of both methyl esters of fatty acids and 2,4-dinitrophenylhydrazones of carbonyl compounds. Hydrazones of carbonyl compounds were separated from other lipid material by adsorbing the hydrazones on alumina (Schwartz and Parks, 1961). The procedures of Farquhar et al. (1959) were used to remove unsaturated methyl esters by microbromination and to hydrogenate unsaturated fatty acid esters prior to gas chromatography. Gas-liquid chromatographic separations were made with a laboratory constructed apparatus "A" employing a thermal conductivity-detector cell with helium as the carrier gas, and with apparatus "B" (Research Specialties Co.) which employed an ionization detector with argon as the carrier gas. Columns were packed in coiled copper tubing (apparatus "A") and stainlesssteel U-tube (apparatus "B") columns (4 mm inside diam). Data on column packing materials, column length, and temperatures used are given with the results. The eluate from the gas-chromatography column was trapped either in 30-cm Teflon tubes containing hexane-saturated glass wool or in glass capillary tubes which bubbled into hexane. Fractions of eluate were collected by rapidly changing Teflon or glass tubes. Retention volumes of methyl esters of fatty acids were compared with the retention volumes obtained with a mixture of methyl esters obtained from butterfat and with known ester standards which were provided by the Metabolism Study Section, National Institutes of Health. The 2,4-dinitrophenylhydrazine of tetradecanal that was used as reference for paper chromatography was a gift of A. M. Gaddis, Eastern Utilization Research and Development Division, U.S. Department of Agriculture,

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[VOL. 83

0 0 0

x

0

10~201

TI ME

MI NUTES

FIG. 1. Radioactivity in fractions collected during gas chromatography of methylated lipid of Ruminococcus fiavefaciens after removal of unsaturated methyl esters by microbromination. Chromatography at 172 C on Reoplex 400 in 60-80 mesh Celite (2:3). Radioactivity in bars. Chromatographic peaks tentatively identified as: (1) n-C14, (2) branched-C1, (3) n-C15, (4) branched-C16, (5) n-C16, (6) branched-C17, (7) n-C, (8) n-C18.

Beltsville, Md. Hydrazones of C,O, Cl1, C12, C16, and C1,8 aldehydes used as standards were prepared from authentic carbonyls by the method of Shriner, Fuson, and Curtin (1956). RESULTS

C14 in lipid of R. flavefaciens grown in isovalerate-1-C14. The whole-lipid fraction was chromatographed using the reversed-phase paper chromatographic method of Buchanan (1959). A radioautograph of the chromatogram showed that most of the C14 migrated with the front, while some remained at the origin and some had an RF similar to that expected for a 15-carbon

saturated fatty acid. The distribution of C14 in the fractions was as follows (count/min): free fatty acids, 29,250; nonsaponified lipid, 5,450; and saponified fatty acids, 43,400. When either the free fatty acid fraction or the saponified fatty acid fraction was separated by reversed-phase paper chromatography, all of the radioactivity detected by a radioautograph migrated at a rate similar to that expected for a 15-carbon saturated fatty acid. The whole-lipid fraction from R. favefaciens was methylated and fractionated by gas-liquid

chromatography using apparatus "A." Figure 1 shows the distribution of C14 in fractions collected during chromatography of a sample of the lipid from which unsaturated methyl esters had been removed by microbromination. The fraction corresponding to the methyl esters of branchedchain 15-carbon fatty acids contained 74% of the radioactivity recovered. The other fraction containing appreciable C"4 was that eluted with materials having a retention volume similar to that expected for the methyl esters of a branchedchain 17-carbon acid. Table 1 shows the results obtained by gasliquid chromatography of the whole-lipid extract and of the polar (phospholipid) fraction (fraction 3, Table 2) of R. flavefaciens cells. Carbonyl compounds were not removed from the wholelipid fraction and, since later studies demonstrated the presence of aldehydes, parts of certain peaks here and in Fig. 1 may have been produced from dimethylacetals. The phospholipid fraction was methylated in the presence of 2,4-dinitrophenylhydrazine, and thus carbonyl compounds did not contribute to peaks obtained. Hawke, Hansen, and Shorland (1959) found that the retention volumes of methyl esters of

1962]


TABLE 1. Relative proportions of methyl esters of fatty acids of Ruminococcus flavefaciens C-94

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confirm the results of Saito (1960), who found that a branched-chain 15-carbon fatty acid was a major acid in B. subtilis. Under our conditions, Per centb Per cent Carbon Carbon however, the major fatty acid in vegetative cells numbera Whole Phospho- number Whole Phosphowas the anteiso-C,5 acid rather than the iso-C15 lipid0 lipidd lipid0 lipidd acid noted by Saito. 10.0 0.8 15.55 5.6 Branched-chain 15-carbon fatty acids and 11.0 0.8 16.0 20.6 16.0 palmitic acid are the major fatty acids in these 1.2 12.0 1.6 16.5 4.08 4.0 R. flavefaciens cells. The effect of differences in 2.1 13.0 1.0 16.7 2.3 cultural conditions on the fatty acids of these 1.2 13.5 17.0 1.1 cells is not known. The ratio of branched-C15 13.75 1.9 17.5 4.5 (24.6 %) to branched-C16 acids (5.6%) in the 14.0 3.4 4.9 17.7 1.6" whole lipid is similar to and may be influenced 14.2 1.6 18.0 9.2 8.0 by the ratio of isovalerate to isobutyrate (5:1) 14.45 16.4 18.4 14.1e in the culture medium. The anteiso-C15 acid may 14.55 24.6 18.5 12.2 have been synthesized from 2-methyl butyrate, 14.7 1.3 18.8 7.7e 2.1 15.0 0.5 11.1 19.0 as later studies showed this as a probable con15.4 5.6 19.3 7.2 taminant in the isovalerate added to the medium. Upon addition of acetone in the cold, 63% of a Carbon number as defined by Woodford and the lipid C14 precipitated. A similar proportion of van Gent (1960). bPer cent determined by measurement of areas C14 was in the polar (phospholipid) fraction (fraction 3, Table 2) obtained by chromatography under peaks with a planimeter. c Chromatogram at 194 C of methylated whole on a silicic acid-Celite column. No attempt was lipid on apparatus "A." Stationary phase was made to identify the components of fractions 1 Reoplex 400 on 60 to 80 mesh Celite (2:3). and 2, other than to determine the amount of d Chromatogram at 180 C of methyl esters of C14 present in materials extractable with 1% fatty acids from phospholipid fraction. Chromato- Na2CO3 (probably free fatty acids). graphic apparatus "B" with stationary phase of Alpha-ketoisocaproate is presumably the diethylene glycol-succinate on 60 to 80 mesh immediate precursor of leucine. To test for the Celite (1:4). a Peaks were greatly reduced or lost when un- presence of C14 in keto acids, the lipid fraction of saturated methyl esters were removed by micro- strain C94 was treated with 2,4-dinitrophenylhydrazine (Shriner et al., 1956) and hydrazones bromination. were examined by paper chromatography. The iso and anteiso C15 acids relative to the retention radioactivity and an appreciable quantity of volume of the methyl ester of n-C14 acid were hydrazones migrated with the front with n1.14 and 1.22, respectively. With the same butanol-formic acid-water (5:1:4), n-butanolstationary phase but with slightly different ethanol-water (5:1:4), or water-saturated nconditions, we found that the retention volumes of peaks with carbon numbers of 14.45 and 14.7 TABLE 2. Distribution of C14 in fractions of Ruminorelative to the 14.0 peak were 1.16 and 1.26, coccus flavefaciens lipid separated on a silicic acid-Celite column" respectively. Thus, peak 14.45 on the diethylene glycol-succinate column and peak 14.55 on the Per cent of whole lipid C"4 Apiezon-L column were probably produced by Fraction Solvent the methyl ester of the iso-C15 acid, and peak Whole Na2CO3 fraction extract 14.7 was tentatively identified as the anteiso-C15 methyl ester. Peaks 15.4 and 15.55 were probably 1 12.1 Chloroform, 150 ml 3.9 produced by the methyl ester of the iso-C16 acid. 2 Chloroform methanol 27.5 24.0 We chromatographed methyl esters of fatty (98:2), 100 ml acids from the lipid fraction of vegetative cells of 3 150 60.4 Methanol, Bacillus subtilis ATCC 6633 to aid in identificaa Column 22 mm diam; mixture of 10 g silicic tion of the branched acids from ruminococci. The data, which will be published in another paper, acid and 5 g Celite 545.

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ALLISON ET AL.

butanol as solvents (Strassman, Shatton, and Weinhouse, 1960). The RF values of hydrazones of 2-ketoisovalerate and 2-ketoisocaproate were 0.65 and 0.73, respectively, in the n-butanolwater system. Thus, there was no appreciable C'4 present as the 6-carbon keto acid, but the quantity of hydrazones produced by treatment with 2,4-dinitrophenylhydrazine indicated that an appreciable amount of carbonyl compounds was present in the lipid fraction. The amount of C14 in carbonyl compounds was determined by reacting a portion of the lipid with 2,4-dinitrophenylhydrazine in HCI. Monocarbonyl 2,4-dinitrophenylhydrazones were isolated using an alumina column (Schwartz and Parks, 1961), and 7.3% of the lipid C'4 was measured in this fraction. Most of the C14 in carbonyl compounds was in the phospholipid fraction, as 13% of the C14 was recovered as the 2 ,4-dinitrophenylhydrazone after passing the fraction through the Celite-2,4-dinitrophenylhydrazine column of Schwartz and Parks (1961). It may be that the 7% of the C'4 recovered in the nonsaponified fraction of the lipid was present as plasmalogen aldehydes, which are not readily freed by hydrolysis with alkali. Purified 2 ,4-dinitrophenylhydrazones from the lipid fraction of cells were subjected to reversedphase paper chromatography (Klien and DeJong, 1956). The radioactive area had an RF value of 0.28, while adjacent hydrazones of known C,, and C16 aldehydes had values of 0.54 and 0.23, respectively. The radioactive material was eluted from the paper, and the absorption spectrum of a hexane solution of the eluate was compared with the absorption by the 2,4-dinitrophenylhydrazone of dodecanal, using a Beckman DU spectrophotometer. Superimposable curves with absorption maxima at 338 m,u were obtained. The ratio of 15-carbon aldehyde to phosphorus in phospholipid of these cells was 0.8. In phospholipid from another similarly grown batch of cells, the ratio of long-chain carbonyl compounds to phosphorus was 0.56. The quantity of carbonyl compounds was estimated using 21,500 as the molar extinction coefficient of 2 ,4-dinitrophenylhydrazones measured at 338 mg Although these measurements differ somewhlat, it i' nevertheless clear that long-chain aldehydes are quan titatively significant components of ruminococcal phospholipids. Hydrazones were freed of paraffin oil on a Sea Sorb-Celite column (Schwartz, Parks, and Keeney, 1960); free carbonyl compounds were

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TABLE 3. Radioactivity in fractions collected during gas chromatography of aldehydes of Ruminococcus flavefaciens grown in isovalerate-l-Cl4a Fraction

Carbon numberb

Count/min

1 2 3 4

0-14.35 14.35-14.9 14.9-15.4 15.4-19

9 701 77 101

a Chromatographic apparatus "B" with Apiezon L and 80 to 100 mesh Celite (1:5) column, 4 ft long, at 202 C. & As defined by Woodford and van Gent (1960).

regenerated (Keeney, 1957) and added to a mixture of aldehydes obtained from the bacterial fraction of rumen contents. The aldehydes were pooled so that reference compounds would be present to permit detection, identification, and collection of peaks during gas chromatography. Table 3 presents the data on the measurement of C'4 in the fractions. Fraction 2, which contained most of the C14, corresponded with the branchedchain 15-carbon aldehyde peak, and fraction 3 was the eluate from the straight-chain 15carbon aldehyde peak. Katz and Keeney (unpublished data) identified the aldehydes obtained from the bacterial fraction of rumen contents by comparison with chromatograms of known aldehydes and by reduction of the aldehydes to alcohols and subsequent chromatography of these alcohols before and after hydrogenation. Experiments were conducted to determine whether the lipid fraction from cells of strain C94 could replace the nutritional requirement of the same organism for isovalerate or isobutyrate. Several dilutions of the whole-lipid extract, the free fatty acids extracted with aqueous Na2CO3, and the nonsaponified fraction of the lipid were added to tubes of the basal medium. None of these fractions supported growth of the organism. A further test for growth-factor activity in the lipid fraction was made by impregnating filterpaper discs with solutions of the whole-lipid extract, free fatty acids, saponified fatty acids, and the phospholipid fraction of the cells. These discs were placed in test tubes (18 by 150 mm) on slopes of the basal medium plus 1.2% agar, which had been seeded with a heavy inoculum of strain C94. None of the fractions of the cells promoted detectable growth around the paper discs but profuse growth was obtained in areas surrounding pads impregnated with isovalerate.

1962]


Incorporation of branched-chain volatile fatty acid carbon by R. albus. R. albus grew when the basal medium was supplemented with sodium acetate (20 Mmoles/ml) plus commercially obtained samples of isobutyrate, isovalerate, 2-methyl-n-butyrate (0.75 to 0.05 ,umole/ml), or 2-ketoisovalerate (1.0 or 0.1 ,umole/ml). Growth was not obtained with acetate plus n-butyrate, n-valerate, n-caproate, isocaproate (0.75 ,umole/ml), or 2-hydroxyisobutyrate, 2aminoisovalerate, or 3-aminoisobutyrate (1.0 and 0.1 ,umole/ml). The organism did not grow when the only source of volatile fatty acids was acetate plus synthetic isovalerate-3-C'4, and a negligible amount of C14 was incorporated by R. albus cells during growth in the basal medium with acetate plus commercial isovalerate and isovalerate-1-C'4. This suggested that the sample of isovalerate which supported growth was contaminated with some other growth factor. The acid was examined in a polarimeter, which revealed that it was contaminated with an optically active material, specific rotation: [C,]4 = + 5.05. A bottle of isovalerate from a different lot but from the same company (Distillation Products Industries, Rochester, N.Y.) was not optically active and supported less than 50% maximal growth of strain 7 at a level of 1.0 Amole/ml; the acid from the bottle containing the optically active material supported about one-half maximal growth at 0.1 ,umole/ml. Probably both the latter sample and the isovalerate sample that supported growth of strain 7 in an early experiment (Allison, Bryant, and Doetsch, 1958) were contaminated with 2methyl-n-butyrate. Growth of strain 7, as determined by highest optical density reached in duplicate tubes, was proportional to concentration through low levels of either isobutyrate or 2-methyl-n-butyrate. Approximately one-half maximal growth was obtained with 0.025 /Lmole/ml of isobutyrate and 0.0075 Mimole/ml of 2-methyl-n-butyrate. The growth response to levels of 2-ketoisovalerate has not been determined and the purity of this material is not known. Cells of R. albus grown in the basal medium plus sodium acetate (20 ,umoles/ml) and sodium isobutyrate-l-C'4 (0.2 ,mole/ml) were fractionated, and 96% of the cellular C14 was recovered in the lipid fraction of the cells. The lipid fraction was methylated, and methyl esters of fatty acids

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TABLE 4. Distribution of C14 in fractions of Ruminococcus albus lipid separated by gas chromatographya Fraction

Carbon numberb

Count/min

1 2 3 4 5 6 7 8 9 10

0-12.5 12.5-13.1 13.1-13.8 13.8-14.2 14.2-15.1 15.1-15.8 15.8-16.2 16.2-17.6 17.6-18.2 18.2-19.1

26 459 2313 246 192 2248 123 126 121 82

a Gas chromatographic separation at 223 C on apparatus "A" with Apiezon L and Celite (1:3) column, 4 ft long. b As defined by Woodford and van Gent (1960).

were separated by gas chromatography. Table 4 shows radioactivity in fractions collected during chromatography. Most of the C'4 was in fractions 3 and 6, which contained methyl esters of branched-chain 14- and 16-carbon acids, respectively. The radioactivity in fraction 2 appeared to be at least partially owing to the presence of dimethylacetal formed during methylation of 14or 16-carbon aldehydes. These have lower retention volumes than their corresponding methyl esters and are not saponified with dilute alkali (Grey, 1960). The eluate from the column in fractions 2, 3, and 6 was saponified in methanolic NaOH (0.5 N) for 2 hr at 70 C. The percentages of C'4 in each fraction that was recovered in nonsaponified materials were 86, 5, and 4% for fractions 2, 3, and 6, respectively. Table 5 gives the results obtained by gas chromatography of the methylated whole-lipid fraction from R. albus. The major acids appear to be palmitic, iSo-CI4, and iso-C,6 acids. Chromatography after hydrogenation of the sample showed that these major peaks were produced mainly by saturated compounds. The low percentage of acids higher than C16 is noteworthy. Carbonyl compounds were not removed from the sample prior to chromatography, and the contribution of dimethylacetals of aldehydes to the results is not known. The lipid fraction of strain 7 was reacted with 2,4-dinitrophenylhydrazine, and hydrazones of carbonyl compounds were separated from the re-

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ALLISON ET AL.

TABLE 5. Distribution of fatty acids in strain 7 of

[VO L. 83

growth. Radioactivity from isobutyrate-1-C'4 was not incorporated into valine by R. albus but was incorporated mainly into branched-chain 14- and Relative areasc Carbon number' 16-carbon fatty acids and 14- and 16-carbon 7c aldehydes. Wegner found that strain S85 of 0.1 12.0 Bacteroides succinogens and a strain of Borrelia 0.5 12.7 isolated from the rumen, like R. albus, did not 1.3 13.2 incorporate C'4 from isovalerate-1-C14, but did 38.8 13.6 incorporate C'4 from isobutyrate-1-C'4 and from 3.1 14.0 0.4 14.7 n-valerate-1-C'4, mainly into the lipid fractions of 1.6 15.0 the cells. 12.8 15.6 Saturated fatty acids with branched methyl 38.6 16.0 groups near the terminal end of the molecule have 0.8 17.0 recently been noted as major components of lipid 1.0 17.5 in several genera of bacteria. Saito (1960) found 1.2 18.0 0.8 17.0 that the iso-C15 fatty acid is a major acid in a Separation by gas-liquid chromatography at Bacillus subtilis; anteiso-C15 acid is a major acid 198 C on apparatus "B." Column packed with from a species of Sarcina (Akashi and Saito, 1960). Macfarlane (1961) noted that the anteisoApiezon L-Celite (1:5). b As defined by Woodford and van Gent (1960). Ca5 acid is a major acid in phospholipid obtained c Areas under peak measured with a planimeter. from protoplast membranes of -licrococcus lysodeikticus. Branched-chain saturated 15-carmainder of the lipid fraction by chromatography bon iso and anteiso fatty acids were a major on alumina and Sea Sorb-Celite columns; 11 % component of lipid extracted from the mixed of the lipid C14 was recovered as the 2,4-dinitro- bacterial population of the rumen, and appreciaphenylhydrazone derivative of monocarbonyl ble quantities of branched-chain 14- and 16compounds. Paper chromatograms of the 2,4- carbon acids were also noted (Keeney, Katz, and dinitrophenylhydrazones was prepared, using Allison, 1962). Thus, it is probable that many the methods of Klien and DeJong (1956) and ruminal organisms contain large proportions of Keenan and Marks (1960). Radioautographs of the branched acids. Until further studies have these showed that most of the C14 migrated at a been made to determine the distribution of these rate similar to that noted with known 14- and acids in other bacteria, we can only speculate on 16-carbon aldehydes. No attempt was made to the taxonomic significance of their presence. determine whether these were branched-chain A1. lysodeikticus grown in medium containing aldehydes, but, since C'4 from isovalerate was either uniformly labeled leucine-C'4 or 2-methylincorporated into branched-chain 15-carbon alde- butyrate-C'4 incorporated C'4 into branchedhyde, it seems probable that C14 from isobutyrate chain 15- and 17-carbon fatty acids (Lennarz, is incorporated into iso C14 and iso C16 aldehy-des. 1961). Ruminococci are unable to produce the branched-chain volatile fatty acids, but other DISCUSSION rumen organisms, such as Bacteroides runminicola, In R. flavefaciens, isovalerate serv-es as a source produce these from corresponding amino acids of carbon for synthesis of both leucine and (Bladen, Bryant, and Doetsch, 1961). W7e have branched-chain odd-numbered higher fatty acids unpublished data showN-ing that C'4 is incorpoand aldehydes. Wegner (personal coitmwunication) rated into 15- and 17-carbon fatt- acids bv found that this same strain incorporates C14 from B. suibtilis cells growing in isovalerate-1l-C4. Since branched-chain xolatile acids are incorisobutyrate-1-C14 into both -aline and the lipid fraction of the cells. porated mainly into higher branched-chain fatty Strain 7 of R. albus differs fromi R. flavefaciens acids and aldehydes of certain of these rumen in that isovalerate carbon is apparently not in- bacteria, and since these bacteria require the corporated into the cells and the addition of volatile acids for growth, it is suggested that isovalerate to the basal medium does not support long-chain branched fatty acids and aldehydes Ruminococcais albusa

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have an essential function in the cell or are essential structural components or both. The level of the requirement for volatile fatty acids also suggests that they are essential for synthesis of major cellular components. Saito (1960) raised the interesting question concerning the biological or evolutional meaning of branched-chain acids in B. subtilis. We would pose an additional question as to the significance or function of the branched-chain aldehydes. The long-chain branched fatty acids in ruminococci are apparently synthesized by addition of two carbon units to the short-chain branched fatty acid. Radioactivity in 14- and 16-carbon homologues synthesized from isobutyrate-1-C'4, and in 15- and 17-carbon homologues synthesized from isovalerate-1-C14, is evidence that this is the case. Since fatty acids such as palmitic acid do not contain appreciable C'4, it is probable that the label in the higher acids was not due to degradation of the carboxyl-labeled volatile acid and subsequent incorporation of a labeled two-carbon unit. Rumen contents have a relatively high concentration of volatile fatty acids, including those with branched chains (Annison, 1954). Since these acids can be incorporated into the pathway for biosynthesis of branched-chain amino acids and higher fatty acids and aldehydes, there may be little survival value to maintenance of the mechanism for biosynthesis of the isopropyl group; mutants without this capacity or with an inefficient biosynthetic mechanism, and which are dependent upon branched short-chain fatty acids, may become numerous. The location of the metabolic block or the mechanism that does not function adequately in this biosynthesis is not known. The medium used when incorporation of isobutyrate-1-C'4 by R. albus was determined contained casein hydrolyzate, and enough valine for growth may have been assimilated from the medium. This organism will grow in a medium lacking amino acids but containing a mixture of volatile fatty acids. It would be interesting to see if isobutyrate carbon was incorporated into valine under such conditions. Odd-numbered and branched-chain higher fatty acids have been noted in mammalian lipids by a number of workers. Horning et al. (1961) showed that such acids are synthesized by a purified enzyme system obtained from rat adipose

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tissue when coenzyme-A derivatives of branchedchain and odd-numbered short chain fatty acids were supplied. Akashi and Saito (1960) suggested that, when unusual fatty acids are found in body lipids of herbivorous animals, these may have arisen from microorganisms in the host gut. Gerson et al. (1959) found that 2 to 10% of the dietary (+) - anteiso C15 and C17 fatty acids was deposited in the carcass of rats, and that the turnover of these acids was much slower than the turnover of normal acids. We have shown that branched-chain fatty acids are among the predominant acids in a strain of each of two species of important rumen bacteria. The findings of Keeney et al. (in press) that such acids make up a major portion of fatty acids extracted from the bacterial fraction of rumen contents supports the suggestion of Akashi and Saito (1960) regarding the microbial origin of these acids. Sammons (1961) noted the presence of 15carbon branched and straight-chain acids in samples of human feces; they were not present in ileal discharge. His suggestion that these acids may be of bacterial origin seems probable in view of the recent identification of these acids in various bacteria. It is possible that some branched acids in the animal lipid may arise from feed lipid. Shorland (1961) reported the presence of 0.4 to 1.5% branched-chain saturated 15- and 17-carbon fatty acids in the dialyzate obtained when grass lipids were dialyzed through a rubber membrane. Katz and Keeney (unpublished data) found that branched-chain 15- and 17-carbon fatty acids were present in hexane extracts of all rubber membranes tested, and that a pre-extraction of the membranes for 4 hr or more did not remove all of these acids. Although the branched acids may be present in the grasses at the levels noted, care should be exercised when rubber dialyzate is studied. We are not aware of previous reference to plasmalogens in bacteria. Unpublished data (Katz and Keeney) indicate that mixed bacteria obtained from rumen contents are rich in plasmalogen, and Wegner (personal communication) has noted plasmalogen in B. succinogenes cells. It may be that plasmalogens are in part responsible for the Feulgen-positive substance present in the cytoplasmic membrane of many bacteria (Knaysi, 1951). Branched-chain aldehydes constitute a signifi-

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cant portion of the plasmalogen aldehydes in several animal tissues. A major component of ox-spleen choline plasmalogen and ox-liver plasmalogen was identified as branched-chain 15-carbon aldehyde (Grey, 1960), and Parks, Keeney, and Schwartz (1961) found that branched-chain aldehydes were among the major bound aldehydes in butter oil. The rumen bacteria studied here synthesize appreciable quantities of higher branched-chain aldehydes, including a branched-chain 15-carbon aldehyde, and may be a source of the aldehydes found in ruminant lipids. LITERATURE CITED AKASHI, S., AND K. SAITO. 1960. A branched saturated Cim acid (sarcinic acid) from Sarcina phospholipids and a similar acid from several microbial lipids. J. Biochem. (Tokyo) 47: 222-229. ALLISON, M. J., M. P. BRYANT, AND R. N. DOETSCH. 1958. Volatile fatty acid growth factor for cellulolytic cocci of bovine rumen. Science 128:474-475. ALLISON, M. J., M. P. BRYANT, AND R. N. DOETSCH. 1962. Studies on the metabolic function of branched-chain volatile fatty acids, growth factors for ruminococci. I. Incorporation of isovalerate into leucine. J. Bacteriol. 83:523-532. ANNISON, E. F. 1954. Some observations on volatile fatty acids in the sheep rumen. Biochem. J. 57:400-405. BLADEN, H. A., M. P. BRYANT, AND R. N. DOETSCH. 1961. Production of isovaleric acid from leucine by Bacteroides ruminicola. J. Dairy Sci. 44:173-174. BRYANT, M. P., N. SMALL, C. BOUMA, AND I. M. RoBINSON. 1958. Characteristics of ruminal anaerobic cellulolytic cocci and Cillobacterium cellulosolvens n. sp. J. Bacteriol. 76:529-537. BUCHANAN, M. A. 1959. Paper chromatography of the saturated fatty acids. Anal. Chem. 31:1616-1618. FARQUHAR, J. W., W. INSUiLL, JR., P. ROSEN, W. STOFFEL, AND E. H. AHRENS, JR. 1959. The analysis of fatty acid mixtures by gas-liquid chromatography. Nutrition Revs. 17: no. 8, part II, 30 p. GERSON, T., F. B. SHORLAND, Y. ADAMS, AND M. E. BELL. 1959. Further studies on the metabolism of the (+)-anteiso-acids, (+)-12methyltetradecanoic acid and (+)-14-methylhexadecanoic acid in the rat. Biochem. J. 73:594-596. GREY, G. M. 1960. The phospholipids of ox-spleen

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with special reference to the fatty acid and fatty aldehyde compositions of the lecithin and kephalin fractions. Biochem. J. 77:82-91. HAWKE, J. C., R. P. HANSEN, AND F. B. SHORLAND. 1959. Gas-liquid chromatography: retention volumes of the methyl esters of fatty acids with special reference to n-odd-numbered iso and (+) anteiso acids. J. Chromatography 2:547-551. HORNING, M. G., D. B. MARTIN, A. KARMEN, AND P. R. VAGELOS. 1961. Fatty acid synthesis in adipose tissue. II. Enzymatic synthesis of branched chain and odd-numbered fatty acids. J. Biol. Chem. 236:669-672. KEENAN, R. W., AND B. H. MARKS. 1960. A rapid method for the separation of plasmals. Biochim. et Biophys. Acta 39:533-535. KEENEY, M. 1957. Regeneration of carbonyls from 2,4-dinitrophenylhydrazones with levulinic acid. Anal. Chem. 29:1489-1491. KEENEY, M., I. KATZ, AND M. J. ALLISON. 1962. On the probable origin of some milk fat acids in rumen microbial lipids. J. Am. Oil Chem. Soc. 39:198-201 KLIEN, F., AND K. DEJONG. 1956. Paper chromatography of 2,4-dinitrophenylhydrazones of aliphatic carbonyl compounds. Rec. trav. chim. 75:1285-1288. KNAYSI, G. 1951. Elements of bacterial cytology. Comstock Publishing Co., Inc., Ithaca, N.Y. LENNARZ, W. J. 1961. The role of isoleucine in the biosynthesis of branched-chain fatty acids by Micrococcus lysodeikticus. Biochem. Biophys. Research Communs. 6:112-116. MACFARLANE, M. G. 1961. Composition of lipid from protoplast membranes and whole cells of Micrococcus lysodeikticus. Biochem. J. 79:4P. PARKS, 0. W., M. KENNEY, AND D. P. SCHWARTZ. 1961. Bound aldehydes in butteroil. J. Dairy Sci. 44:1940-1943. SAITO, K. 1960. Studies on bacterial fatty acids, structure of subtilopentadecanoic and subtiloheptadecanoic acids. J. Biochem. (Tokyo) 47:710-719. SAMMONS, H. G. 1961. Factors affecting faecal composition, a comparison of ileal discharge and faeces. Biochem. J. 80:30P. SCHWARTZ, D. P., AND 0. W. PARKS. 1961. Preparation of carbonyl-free solvents. Anal. Chem. 33:1396-1398. SCHWARTZ, D. P., 0. W. PARKS, AND M. KEENEY. 1960. Chromatographic separation of 2,4dinitrophenylhydrazone derivatives of aliphatic carbonyl compounds into classes on magnesia. Abstr. Papers 138th Meet. Am. Chem. Soc., p. 156.

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SHORLAND, F. B. 1961. Acetone-soluble lipids of grasses and other forage plants. II. General observations on the properties of the lipids with special reference to the yield of fatty acids. J. Sci. Food. Agr. 12:39-43. SHRTNER, R. L., R. C. FUSON, AND D. Y. CURTIN. 1956. The systematic identification of organic compounds, 4th ed. John Wiley and Sons, Inc., New York.

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STRASSMAN, M., J. B. SHATTON, AND S. WEINHOUSE. 1960. Conversion of a,j3-acetolactic acid to the valine precursor, a, 3-dihydroxyisovaleric acid. J. Biol. Chem. 235:700-705. WOODFORD, F. P., AND C. M. VAN GENT. 1960. Gas-liquid chromatography of fatty acid methyl esters: the "carbon-number" as a parameter for comparison of columns. J. Lipid Research 1:188-190.