Hydrogenation of Unsaturated Fatty Acids byTreponema (Borrelia) Strain B25, a Rumen
Spirochete'M. T. YOKOYAMA AND C. L. DAVIS
Department of Dairy Science, Division of Nutrition, University of Illinois, Urbana-Champaign, Illinois 61801
Received for publication 28 April 1971
The time course of hydrogenation of linoleic acid to trans-l l-octadecenoic acidwas observed in a growing culture of Treponema (Borrelia) strain B25. A conju-gated fatty acid, cis-9, trans-I1-octadecadienoic acid, was identified as an interme-diate in the process. The isomerase responsible for the conversion of linoleic acidto the conjugated fatty acid was found to be associated with a particulate fractioncharacterized by a high protein and lipid content in a 2:1 ratio. Optimum pH forisomerase activity was found to be 7.0 in 0.05 M potassium phosphate buffer. Nocofactor requirements could be demonstrated for the isomerase. The sulfhydryl in-hibiting agents, iodoacetamide, N-ethylmaleimide, and p-chloromercuribenzoate,inhibited isomerase activity. Isomerase activity was also inhibited by the metalchelators, o-phenanthroline, a, a'-bipyridyl, ethylenediaminetetraacetic acid, and8-hydroxyquinoline. Linoleic (A9, 12), linolenic (A9, 12, 15), and gamma-linolenic(A6, 9, 12) acids served as effective substrates for the isomerase; however, the de-rivatives of linoleic and linolenic acid did not.
The hydrogenation of 18-carbon, unsaturatedfatty acids by ruminal microorganisms is welldocumented (7). Most studies, however, havedealt with mixed microbial populations ratherthan with pure strains. For this reason, it hasbeen difficult to characterize the reactions in-volved in the hydrogenation process. Severalrumen bacteria have been identified as possessingthe ability to hydrogenate 18-carbon, unsaturatedfatty acids (19, 21, 23, 28); however, only Butyri-vibrio fibrisolvens strain A38 has been studied indetail (13, 14, 16). A particulate isomerase fromthis bacterium initially isomerizes linoleic acid tocis-9, trans-11-octadecadienoic acid. This conju-gated fatty acid intermediate is then hydrogen-ated to trans- 11-octadecenoic acid (13). Sachanand Davis (23) recently reported the ability of arumen spirochete, Treponema (Borrelia) strainB25 to also hydrogenate linoleic acid to trans- 11-octadecenoic acid. This discovery is of particularsignificance when it is considered that Trepo-nema (Borrelia) strain B25 and B. fibrisolvensstrain A38 are phylogenetically distinct species,with entirely different morphological and meta-bolic characteristics. In view of the lack of de-tailed information on the hydrogenation mecha-
I Presented in part at the Federation of American Societiesfor Experimental Biology Meetings, Atlantic City, N.J., April1970, and Chicago, 111., April 1971.
nism in rumen bacteria, other than B. fibrisol-vens, and the fact that little is known about thespecific metabolism of rumen spirochetes, it wasdeemed of sufficient interest and value to conductthe present investigation.
MATERIALS AND METHODS
Organism and culture conditions. Treponema strainB25 was obtained from M. P. Bryant, Department ofDairy Science, University of Illinois, Urbana. Stockcultures were maintained as stabs in 0.1% glucose-agarslants (3). After overnight growth at 37 C, the slantswere refrigerated at 4 C. Transfers were made at 2-week intervals. Modifications (3) of the anerobic tech-niques of Hungate (10) were used to cultivate andmaintain the spriochete.The medium used to cultivate the spirochete was
essentially that of Bryant and Robinson (3). Each literof medium contained 5 g of glucose, 2 g of yeast ex-tract (Difco), 5 g of Trypticase (BBL), 200 ml of clari-fied rumen fluid, 1 ml of resazurin, 75 ml of 0.6%K2HPO4, 75 ml of a mineral solution containing 0.6%KH2PO4, 0.6% (NH4)2SO4, 1.2% NaCI, 0.2%MgSO4-7H20, and 0.2% CaCl2 2H2O, 20 ml of 2.5%cysteine-hydrochloride, and 50 ml of 8% Na2COM. TheNa2CO3 solution was autoclaved, aseptically equili-brated with CO2, and added to the medium after steri-lization and cooling.Rumen fluid was collected in 6- to 8-liter batches
from fistulated cows belonging to the Department ofDairy Science. Large feed particles were removed by
straining through cheese cloth, and the rumen fluid was
left to stand for about I hr until the finer debris hadrisen to the surface. The rumen fluid was clarified bypassage through a Sharples centrifuge and autoclavedat 121 C for 20 min. Prior to its use in the medium, theclarified rumen fluid was recentrifuged at 9,000 x g for30 min.When linoleic acid-1-"C was incorporated into the
medium, it was complexed with bovine serum albumin(Calbiochem, fatty acid poor) by the procedure ofNeptune et al. (20). The "4C-linoleic acid-albumincomplex was sterilized by passage through a membranefilter (0.45-gm pore size, Millipore Corp.) and incorpo-rated aseptically into the sterilized medium.The organism was grown in 20-liter batches, em-
ploying a model M-128S fermentor (New BrunswickScientific Co.). About 600 ml of an actively growingculture was used as inoculum. Growth was measuredby reading the optical density of samples at 600 nm
against a control of the same medium taken beforeinoculation. After 18 to 20 hr of growth at 37 C, thecells were harvested at room temperature in a Sharplescentrifuge, washed once with 200 ml of 0.05 M potas-sium phosphate buffer (pH 7.0) containing 0.01% mer-
captoethanol, and centrifuged at 20,000 x g for 20min. The packed cells were stored at -20 C in rubber-stoppered tubes under an H2 gas phase until used.
Enzyme extraction. About 20 g (wet weight) of cellswere thawed and suspended in an equal volume of 0.05M potassium phosphate buffer (pH 7.0) containing0.01% mercaptoethanol. Cells (40-ml volume) were
ruptured at room temperature by passage through a
French pressure cell at 12 to 15,000 psi. After disrup-tion, the differential centrifugation procedure employedby Kepler and Tove (15) was followed. The rupturedcells were centrifuged at 10,000 x g at 0 C for 15 minto remove large cell debris. The supematant fluid was
centrifuged at 34,800 x g at 0 C for 30 min, decanted,and recentrifuged at the same speed for another 30min. The resulting supernatant fluid was centrifuged at105,000 x g at 0 C for 4 hr in a Spinco model L ultra-centrifuge (40 rotor). The supernatant fluid was dis-carded and each pellet was dispersed in 2 ml of 0.05 M
potassium phosphate buffer (pH 7.0), containing 0.01%mercaptoethanol, by a 5- to 10-sec exposure to sonicoscillation with a Branson sonifier (4 amp). The en-
zyme preparation was stored in I-ml samples at -20 Cin rubber-stoppered tubes under an H, gas phase.
Isomerase assay. The procedure used to assay theisomerase was essentially that of Kepler and Tove (15).Isomerase activity was measured by using a Gilfordspectrophotometer (model 2000) by following the in-crease in absorption at 233 nm, due to the formation ofthe conjugated fatty acid. The molar extinction coeffi-cient of 2.4 x 10' M lcm-', as determined by Keplerand Tove (14), was used in the calculations. A stocksolution of linoleic acid (2 mg/ml) was made up inbenzene and refrigerated at 4 C. Substrate was pre-pared daily as an emulsion in 5 ml of 0.05 M potassiumphosphate buffer (pH 7.0) by evaporating the benzenefrom 0.5 ml of the stock solution and sonically treatingfor I min with a Branson sonifier (4 amp). A finalconcentration of 24 gM linoleic acid was achieved in a
3-ml volume, by adding 0.1 ml of the emulsified solu-tion to 2.9 ml of 0.05 M potassium phosphate buffer
(pH 7.0). The reaction was initiated by adding 0.01 mlof enzyme preparation after a 5-min incubation at 37C. A unit of enzyme was defined as the amount of en-
zyme which isomerized I nmole of linoleic acid per
min. Specific activity was calculated as units of activityper milligram of protein.
Characterization of the conjugated fatty acid inter-mediate. The position and configuration of the doublebonds in the conjugated fatty acid intermediate were
determined by the procedure of Privett and Nickell(22), modified by the use of "C-labeled fatty acid. The"C-labeled conjugated fatty acid intermediate was
obtained by incubating an emulsion of linoleic acid-l-"C (5 ACi) at 37 C for 3 hr with 10,000 x g cell-freeextract obtained from strain B25. The "C-conjugatedfatty acid was extracted and isolated by the methodsdescribed below and partially hydrogenated by usinghydrazine hydrate (Matheson, Coleman and Bell,Norwood, Ohio). The resulting mixture of cis andtrans-monoenoic acids was separated by 25% silver ni-trate thin-layer chromatography (TLC), by using a
solvent system of benzene-petroleum ether (90:10 v/v).The isolated monoenoic fractions were then subjectedto permanganate-periodate oxidation (23). Dicarbox-ylic acids isolated from the oxidation were methylatedwith 2% H2SO4 in anhydrous methanol and the dies-ters were examined by gas-liquid chromatography(GLC). Individual peaks, corresponding to knowndiester standards, were trapped by connecting a Pas-teur pipette, containing a plug of glass wool, to thequartz tip of the detector assembly with a piece ofTeflon tubing (flame extinguished). The collected dies-ters were flushed from the pipettes with scintillationfluid and assayed for radioactivity by liquid scintilla-tion counting as described below.
The conjugated fatty acid intermediate was furthercharacterized by GLC, TLC, and infrared spectroscopyby using as a standard, conjugated (cis,trans andtrans,cis) fatty acid prepared by alkali isomerizationof linoleic acid (1) and isolated by silver nitrate columnchromatography (26).
Analytic techniques. Lipids were extracted from theculture with chloroform-methanol (2:1 v/v) and sa-
ponified by the procedure of Ifkovits and Ragheb (12).Fatty acids were converted to their methyl esters byrefluxing with 2% H2SO4 in anhydrous methanol for 1
hr. Methyl esters were routinely separated by TLCemploying silica gel-G, impregnated with 25% silvernitrate, by using a solvent system of benzene-petroleumether (90:10 v/v). Location of the methyl esters was
accomplished by ultraviolet light, after spraying with0.01% 2,7-dichlorofluorescein in 50% ethanol.
Lipids extracted from the enzyme preparation were
separated into polar and nonpolar fractions by silicicacid column chromatography. A column (33 by 0.5 cm)was packed with 2 g of activated silicic acid slurried inchloroform. Nonpolar lipids were eluted with 20 ml ofchloroform, and the polar lipids were eluted with 20 mlof methanol. The solvents were evaporated and the percent contribution of each fraction to the total lipid was
determined. The polar lipid fraction was further sub-jected to silica gel-G TLC by using a solvent system ofchloroform-methanol-water (65:25:4 v/v). Spots werevisualized by spraying the plates with either 50% sul-furic acid and charring at 180 C or 2% ninhydrin in
95% ethanol.Fatty acid methyl esters were examined by GLC on
an Aerograph Hi-Fi (model 600) gas chromatographequipped with a hydrogen flame ionization detectorand a disc integrator. A stainless-steel column (152 by0.3 cm) packed with 15% diethylene glycol succinateon Chromosorb-W (60 to 80 mesh) was used. Oventemperature was maintained isothermally at 185 C.Identification of the methyl esters of saturated andunsaturated fatty acids was made by comparing theirretention time to that of pure methyl ester standardsobtained from Analabs Corporation, North Haven,Conn. Branched-chain fatty acids were identified in a
similar manner, by using a standard mixture of fattyacids, including iso and anteiso acids obtained fromApplied Science Laboratories, State College, Pa.
Liquid scintillation counting was done in a liquidscintillation spectrophotometer (model 314B; PackardInstrument Co., Inc.). Samples were prepared forcounting by adding 10 ml of scintillation solution (5 gof 2,5-diphenyloxazole per liter of toluene). Silica gel-G fractions scraped from TLC plates were suspendedin 10 ml of scintillation solution containing 350 mg ofCab-O-Sil (Thixatropic Gel Powder, Packard Instru-ment Co., Inc.).
Infrared absorption spectroscopy was conductedwith a Beckman IR-7 spectrophotometer by using a
1.0-mm NaCl cell and carbon disulfide as solvent.The proximate composition of the enzyme prepara-
tion was determined by the following procedures. Pro-tein was determined by the method of Lowry et al. (18).The anthrone test, with glucose as a standard, was usedto determine carbohydrate content (25). Lipid was es-timated gravimetrically from chloroform-methanol-ex-tracted preparation (2:1, v/v; reference 8). The ultra-violet 280/260 absorption ratio of the enzyme prepara-
tion was taken as an estimate of nucleic acid content(17).
Cbemicals. Linoleic acid-1-'4C (56 mCi/mmole) wasobtained from Amersham-Searle Corp., ArlingtonHeights, Ill. Linoleic acid, methyl linoleate, ethyl lino-leate, linoleyl alcohol, linoelaidic acid, trilinolein,methyl linolenate, trilinolenin, and eicosadienoic acidwere purchased from Sigma Chemical Co., St. Louis,Mo. Linolenic and gamma-linolenic acids were ob-tained from Analabs Corporation, North Haven, Conn.
Sulfhydryl reagents, iodoacetamide, N-ethylmal-eimide, and p-chloromeicuribenzoate (p-CMB), wereobtained from Sigma Chemical Company. Metal che-lators, 8-hydroxyquinoline, a, a'-bipyridyl, and o-phenanthroline were also from Sigma Chemical Com-pany. Ethylenediaminetetraacetate (EDTA) was fromHach Chemical Company, Ames, Iowa.
RESULTS
Studies with growing culture. When linoleicacid-l-14C was incorporated into the medium ofa growing culture of strain B25, it was rapidlyconverted to trans- II -octadecenoic acid (Fig. I).At 8 hr of incubation at 37 C, only 29% of therecovered radioactivity was present as linoleicacid. By 14 hr of incubation, the conversion oflinoleic acid to trans- 11 -octadecenoic acid was
521
essentially complete. The relationship betweenthe curves depicting the decrease in linoleic acidand increase in trans-l1-octadecenoic acid sug-gested that an intermediate compound was prob-ably involved in the hydrogenation process.Upon scraping 0.5-cm fractions from silver ni-trate TLC plates and counting each fraction byliquid scintillation spectrophotometry, a radioac-tive compound, which migrated between methyloleate and methyl elaidate, was shown to in-crease and then decrease with length of incuba-tion. This compound was subsequently identifiedas an 18-carbon, conjugated fatty acid.
Characterization of the conjugated fatty acidintermediate. Infrared spectroscopy of the iso-lated fatty acid intermediate revealed doublets at948 cm- 1 and 984 cm-1, indicating the presenceof a cis,trans or trans,cis conjugated dienesystem. Ultraviolet absorption in the 230- to 233-nm range also confirmed the presence of a conju-
TRAS-11-OCTADECENOC X-ACID \ 90
80
1.5
70
1.3
GROWTH CURVE
O0
40O5
0.7
HOURS OF INCUBATON
FIG. 1. Hydrogenating ability of Treponema strainB25 in a growing culture. The spirochete was inocu-lated into 10 ml of medium containing 20% clarifiedrumen fluid and linoleic acid-1-14C (5 ,uCi) complexedwith bovine serum albumin. Growth was measured byreading the optical density at 600 nm. Hydrogenationwas monitored by removing 0.5-ml samples asepticallyat timed intervals. Fatty acids were extracted, methyl-ated, and separated by thin-layer chromatography(TLC). Fractions of 0.5 cm were scraped off the silvernitrate TLC plate and counted by liquid scintillationspectrophotometry. Values are reported as a per centof the recovered radioactivity.
gated diene system. When the isolated interme-diate was subjected to GLC and TLC, it co-chromatographed with isolated conjugated(cis, trans and trans, cis) fatty acid formed byalkali isomerization of linoleic acid.
Partial hydrogenation of the intermediate, fol-lowed by oxidation of the cis and trans mono-enoic acids formed, revealed that the majority ofthe radioactivity was associated with dimethylazelate (C.) for the cis-monoenoic acid and withdimethyl hendecandioate (C1 l) for the trans-monoenoic acid. This indicated that the doublebonds were in the 9 and II position and of cisand trans configuration, respectively. It was con-cluded from the above data, that the fatty acidintermediate was cis-9, trans-I I-octadecadienoicacid.
In an effort to determine whether linoleic acidor trans-l1-octadecenoic acid was incorporatedinto cellular lipids, the spirochete was inoculatedinto medium containing linoleic acid-1-14C (5MCi). After 14 hr of growth at 37 C, separateanalysis of the cells and supernatant fluid ob-tained by centrifugation revealed that 63% of therecovered radioactivity was associated with thecells and 37% with the supernatant fluid. No lin-oleic acid-1-14C was detected in either the cellsor the supernatant fluid when examined by TLC.The radioactivity in both fractions was found tobe associated with trans- 11 -octadecenoic acid.Radioactivity associated with the cellular lipidseluted from a silicic acid column with the non-polar lipid fraction. The 4C-trans-octadecenoicacid in this fraction could also be extracted withsolvent from an aqueous medium under acidicconditions but could not be extracted under al-kali conditions, suggesting that the fatty acid waspresent in the free form and not as an esterifiedcomplex.
Yield and activity of isomerase. Isomerase ac-tivity was recovered in a particulate fractionupon differential centrifugation of disruptedcells. The 105,000 x g pellet, routinely used inthe enzyme assay, contained 50% of the isom-erase activity found in the disrupted cells (Table1). Total yield of enzyme (20 g; wet cells) wasabout 5,700 units with a specific activity of 9.8units per mg of protein. The addition of 0.01%mercaptoethanol to the extraction buffer wasfound to result in higher enzyme activity and wasroutinely added in extractions.
Composition of the isomerase preparation. Theproximate chemical composition of the isolatedisomerase preparation is shown in Table 2. Pro-tein and lipid were the major constituents, andthese were found in a 2: 1 ratio. Smaller amountsof carbohydrate and nucleic acid were alsopresent. Separation of the lipid material by silicicacid column chromatography revealed that 75%
of the lipid by weight was associated with thepolar fraction and 25% with the nonpolar frac-tion. Further examination of the polar lipid ma-terial by silica gel-G TLC demonstrated the pres-ence of two major ninhydrin-positive spots withRF values of 0.63 and 0.28. The RF of one ofthese (0.63) corresponded to that of a bacterialphosphatidylethanolamine standard.The major fatty acids detected in the polar and
nonpolar lipids of the isomerase preparation aswell as the whole organism are shown in Table 3.In both the polar and nonpolar lipid fractionsfrom the isomerase preparation, n-pentadecanoicwas the major fatty acid present. Palmitic acidwas also found in substantial quantity in bothfractions, whereas isomyristic acid was a majorcomponent in the nonpolar lipid fraction. Traceamounts (<1.0%) of 18:0 and 18:1 fatty acidswere detected in both the isomerase preparationand the whole organism. No attempt was madeto identify fatty aldehydes or cyclopropane fattyacids, which may have been present.The homogeneity of the preparation with re-
spect to isomerase activity was determined bysucrose density gradient centrifugation (Fig. 2).When the isomerase preparation was layered ona 5 to 70% sucrose gradient, a single activityband was observed, which was slightly precededby the maximum 280-nm protein absorptionpeak for the fractions collected. This indicatedthat not all of the protein in the preparation wasassociated with the enzyme.
Attempts to solubilize the isomerase. Stirringthe isomerase preparation for 30 min at roomtemperature in the presence of 2 to 20% butanolfailed to solubilize the isomerase. Higher concen-tration of butanol (50%) destroyed isomeraseactivity. Sodium deoxycholate (3%), digitonin(2%), Triton X-100 (2%), Duponol C (2%), andsodium taurocholate (3%) under the same condi-tions were similarly unsuccessful. Adjustment ofthe pH to 10.9 and stirring for 1 hr at roomtemperature also resulted in no solubilization.Sodium deoxycholate (4%) in 1 M potassiumchloride resulted in complete loss of activity.
Although we were unable to solubilize theisomerase, it was still possible to measure en-zyme activity in the particulate fraction by ob-serving the increase in absorption at 233 nm forthe conjugated fatty acid. Increase in enzymeactivity was shown to be linear to protein con-centration (Fig. 3). Levels of enzyme corre-sponding to 0.2 to 0.6 mg of protein were rou-tinely used for the assay.
Cofactor requirement. No loss in activity oc-curred when the enzyme preparation was passedthrough Sephadex G-25 and G-200, ion-retarda-tion resin AG- 1IA8, and Dowex l-X8 and 5OW-X8. Dialysis of the enzyme preparation in 0.05 M
Specific activityFraction Total protein (units/mg of Total activity Recovered
(mg) protein) (units) activity (%)a
Ruptured cells 1,718 6.6 11,251 10010,000 x g Pellet 50 4.1 205 210,000 x g Supernatant 1,667 6.2 10,405 9234,000 x g Pellet 136 9.1 1,248 1134,000 x g Supernatant 1,416 6.4 9,121 81105,000 x g Pellet 580 9.8 5,715 51105,000 x g Supernatant 543 0.7 380 3
a Per cent of recovered activity was based on the total activity of the ruptured cells. Each cuvette contained0.024 mM linoleic acid and 50 mM potassium phosphate buffer (pH 7.0) in a final volume of 3.0 ml. The reactionwas initiated by the addition of 0.01 ml of the respective fractions after a 5-min incubation at 37 C. Protein wasdetermined by the Folin-Ciocalteau method. One unit is defined as the amount of enzyme required to isomerize Inmole of linoleic acid per min.
potassium phosphate buffer (pH 7.0) overnightat 4 C also resulted in no loss of activity. Theaddition of boiled 105,000 x g supernatant fluidfrom either the disrupted spirochete or from B.fibrisolvens strain A38 did not stimulate isom-erase activity. Common cofactors [adenosine tri-phosphate, adenosine diphosphate, flavin adeninedinucleotide, flavin mononucleotide, nicotina-mide adenine dinucleotide (NAD), reducedNAD, NAD phosphate (NADP), and reducedNADP] were also nonstimulatory.pH optimum. The pH optimum for isomerase
activity was found to be 7.0 in 0.05 M phosphatebuffer (Fig. 4).
Inhibitors. The isomerase was inhibited tosome degree by all of the metal chelating agentstested (Table 4). At 3 x 10-5 M concentration,o-phenanthroline and a, a'-bipyridyl were themost effective inhibitors. Isomerase activity wasmarkedly inhibited by all of the sulfhydryl agentsexamined (Table 5). The most effective inhibitorwas p-CMB at a concentration of 3 x 10-6 M.
The decreased rate of isomerization in the sulf-hydryl experiment was due to the use of an olderenzyme preparation which had lost activityduring storage.
Substrate specificity of isomerase. Specificityof the isomerase was determined by using var-
ious unsaturated fatty acids and their derivativesas substrates (Table 6). Linoleic, linolenic, andgamma-linolenic acids were acted upon by theisomerase. Linoleic and linolenic acids elicitedabout the same response, whereas gamma-lino-lenic acid was only about 60% as effective as theformer acids. Methyl linoleate, ethyl linoleate,linoleyl alcohol, trilinolein, methyl linolenate,linolenyl alcohol, and trilinolenin were not actedupon by the enzyme. Linoelaidic acid, the geo-metric isomer (trans, trans) of linoleic, as well as
eicosadienoic acid (Al 1, 14) were inactive as
substrates.
TABLE 2. Proximate composition of isomerasepreparationa
Constituent Per cent
Protein 53.7Carbohydrate 3.0Lipid 27.1Nucleic acid 4.8
a Values are expressed as a per cent of the dry weightof the enzyme preparation.
TABLE 3. Major fatty acids of the whole organismand the polar and nonpolar lipid fractions isolated
a Values are expressed as a per cent by weight of allmeasurable peaks (> 1.0%). Iso refers to the branched-chain fatty acid. Fatty acids were identified by com-paring their retention time to a standard mixture offatty acids, including iso and anteiso acids, obtainedfrom Applied Science Laboratories, State College, Pa.
DISCUSSIONThis study supports and extends the earlier
report of Sachan and Davis (23), that the rumenspirochete, Treponema (Borrelia) strain B25 pos-sesses the ability to hydrogenate linoleic acid totrans-lI -octadecenoic acid.By observing the hydrogenation process in a
growing culture of strain B25, it was seen thatthe conversion of linoleic acid to transl11-octa-
FIG. 2. Density gradient centrifugation of the isomerase preparation from strain B25. A 5 to 70% sucrose
gradient was prepared and 0.3 ml of the 105,000 x g preparation was layered on top. Centrifugation was done at82,000 x g for 2 hr in a Spinco model L ultracentrifuge (SW 39 swinging bucket rotor). Fractions (20 drops) were
collected and assayed for isomerase activity and absorption at 280 nm by using a Gilford spectrophotometer(model 2000).
z 3
I
c 2-
ng PROTEIN
FIG. 3. Isomerase activity in relation to proteinconcentration. Each cuvette contained 0.024 mm lino-leic acid and 50 mm potassium phosphate buffer (pH7.0). The reaction was initiated by adding 0.01 ml ofan enzyme preparation (22.6 mg ofprotein/ml) after a5-min incubation at 37 C.
decenoic was essentially complete by 14 hr ofincubation; however, cell growth continued toexponentially increase up to 24 hr, suggestingthat linoleic acid per se was not essential forgrowth. This conclusion is supported by our obser-vation that the spirochete will grow in a chemi-cally defined medium in the absence of linoleicacid. The virtual absence of 18-carbon, unsatu-rated fatty acids in the structural lipids of thisspirochete is further evidence against the needfor linoleic acid as an essential nutrient forgrowth.
Z
3
5;4 6.2 70 78 a6 9.4pH
FIG. 4. Optimum pH of the isomerase. Each cuvettecontained 0.024 mm linoleic acid in the following buffersystem: x, acetate (0.05 M); 0, phosphate (0.05 M); A,
tris(hydroxymethyl)aminomethane (0.05 M). The reac-
tion was initiated by adding 0.03 ml of the enzymepreparation (20.6 mg of protein/ml) after a 5-min in-cubation at 37 C.
The fact that a majority (63%) of the "C-trans-i 1-octadecenoic acid was associated withthe cells could not be interpreted that the hydro-genation process was occurring intracellularly. Itis possible that hydrogenation occurred at or
near the cell surface and that the product was
bound to the cell wall. The high protein and lipidcontent and low carbohydrate and nucleic acid
content of the isomerase preparation suggest thatthe isomerase is associated with the protoplasmiccylinder of this gram-negative organism. Thisconclusion is supported by our findings thatpolar lipids constitute 75% of the total lipid inthe preparation, and that phosphatidyl-ethanolamine, generally regarded as a structurallipid, is a major component of this fraction. Fur-thermore, the 2:1 protein to lipid ratio of theisomerase preparation suggests that the structuremight be a lipoprotein. Joseph et al. (Bacteriol.Proc., p. 57, 1970) have examined the proto-plasmic cylinder of Spirochaeta stenostrepta andreported the presence of two external layers. Theoutermost layer was found to be mainly lipopro-tein, whereas the inner layer was peptidoglycan.It is, therefore, possible that in strain B25 theisomerase is associated with a similar lipoproteinlayer of the protoplasmic cylinder.
TABLE 4. Inhibition of isomerase by metal chelatorsa
Rates InhibitionInhibitor Concn (m) (nmoles/
min)
None 2.7
Ethanol 2.38-Hydroxyquinoline 2.5 x 10-5 2.0 13.5 4.7a,a'-Bipyridyl 3.0 x 10-5 1.6 31.1 + 5.1EDTA 10-3 1.1 58.2 2.1o-Phenanthroline 3.0 x 10-5 0.8 63.6 + 7.6
a Values are averages of two determinations followedby their standard deviations. Each cuvette contained0.024 mm linoleic acid, 50 mM potassium phosphatebuffer (pH 7.0), and the appropriate chelator in a 3.0-ml volume. The chelator was preincubated with 0.01 mlof enzyme (21.4 mg of protein/ml) for 5 min at 37 C,and the reaction was initiated by adding 0.1 ml of sub-strate. The chelators were dissolved in 95% ethanolwith the exception of ethylenediaminetetraacetic acid(EDTA), which was dissolved in water.
TABLE 5. Inhibition of isomerase by sulfhydrylagentsa
a Values are averages of three determinations fol-lowed by their standard deviations. Each cuvette con-tained 0.024 mM linoleic acid, 50 mM potassium phos-phate buffer (pH 7.0), and the appropriate sulfhydrylagent in a 3.0-ml volume. The inhibitor was preincu-bated with 0.01 ml of enzyme (22.6 mg of protein/ml)for 10 min at 37 C, and the reaction was initiated byadding 0.1 ml of substrate.
a Values are averages of two determinations followedby their standard deviations. Each cuvette contained0.020 mM substrate and 50 mM potassium phosphatebuffer (pH 7.0). The reaction was initiated by adding0.01 ml of enzyme (21.4 mg of protein/ml) after a 5-min incubation at 37 C.
If the isomerase had been associated with theouter envelope, it is interesting that deoxycho-late, which has been used to effectively solubilizethe envelope of other spirochetes (6, 27), failedto release the enzyme in our study.The proximate composition of the isomerase
preparation from strain B25 is in marked con-trast with that found in B. fibrisolvens strain A38by Kepler and Tove (14). They reported a highcarbohydrate and low lipid content in their isom-erase preparation. In this regard, B. fibrisolvensis known to produce capsular material and toflocculate in older cultures (4), whereas strainB25 does not.Cohen et al. (5) have examined the cellular
fatty acids of a number of pathogenic trepo-nemes. Palmitic, stearic, 1 8-carbon monoenoic,and dienoic acids were the predominant fattyacids present. In all cases, the 18-carbon fattyacids comprised at least 40 to 50% of the totalfatty acids present. In contrast with strain B25,we could detect only trace amounts (<1.0%) of18:0 and 18:1 fatty acids in the isomerase prep-aration and the whole organism.
Initial attempts to obtain a cell-free prepara-tion which had measurable isomerase activity byrupturing the organism, by using either sonictreatment or the Braun shaker, met with failure.However, good isomerase activity was obtainedwhen the organism was ruptured at 12 to 15,000psi by using a French pressure cell. Disruption ofthe organism by this method invariably resultedin loss of hydrogenating ability, that is the reduc-
tion of cis-9, trans- 11-octadecadienoic acid totrans-i 1-octadecenoic acid.
Isomerase activity was markedly inhibited bymetal chelating and sulfhydryl agents, suggestingthe possible involvement of a tightly bound metalion and a sulfbydryl function in the isomerizationmechanism.
Linoleic, linolenic, and gamma-linolenic acidsserved as substrates for the particulate isom-erase. With the exception of gamma-linolenic,these would constitute the major unsaturatedfatty acids found in the ruminant diet. Since lin-olenic (A9, 12, 15) acid elicited about the sameresponse as linoleic (A9, 12), this would indicatethat a double bond located in the 15 positiondoes not interfere with isomerase activity. How-ever, gamma-linolenic (A6, 9, 12) showed abouta 60% response compared to the forementionedacids, indicating that a double bond at the 6 po-sition hinders isomerization. Work by Keplerand Tove (14) with the isomerase from B. fibri-solvens strain A38 indicates that linolenic acid isisomerized to cis-9, trans-11, cis-15-octadecatri-enoic acid and gamma-linolenic acid is isomerizedto cis-6, cis-9, trans-1 1-octadecatrienoic acid(16). The fact that derivatives of linoleic and lin-olenic acids were not isomerized by the enzymeindicates that a free carboxyl group is essentialfor activity. This observation supports the find-ings of other researchers (9, 10) who have dem-onstrated in mixed cultures of rumen bacteriathat hydrolysis of the ester linkage in triglycer-ides must occur for hydrogenation to proceed.Linoelaidic acid, the geometric isomer(trans,trans) of linoleic, also did not serve assubstrate, indicating that at least one of thedouble bonds must be of the cis configuration tobe active as a substrate. Eicosadienoic acid (A 11,14), although possessing a similar nonconjugated1,4 pentadiene system as linoleic acid, did notserve as a substrate. The difference between thisacid and the active substrates, besides chainlength, is that the first double bond from thecarboxyl end is in the 11 position rather than the9 position. This suggests that although a 1,4pentadiene system is a prerequisite for isomeriza-tion to occur, this system must be A9, 12, suchas in linoleic and linolenic acids. Kepler et al.(16) have examined the specificity of the isom-erase from B. fibrisolvens strain A38 for the A9,12 diene system by varying the position of thefirst double bond at the carboxyl end of 18-carbon dienoic acids from 8 to 11, keeping chainlength constant at the methyl end. They showedthat the A9, 12 diene was the only system isom-erized.
Although the physiological advantages of hy-drogenation to the host ruminant can be fairly
well enumerated (7), the exact biological signifi-cance of the phenomena to the spirochete is notpresently known. The strict substrate require-ments of the isomerase implies a unique func-tional role in the metabolism of the spirochete. Itis reiterated, however, that the 18-carbon, unsat-urated fatty acids are neither essential forgrowth, nor appear to be incorporated into com-plex cellular lipids. Trace amounts of 18:0 and18:1 fatty acids, however, were detected in theorganism. If 18-carbon, unsaturated fatty acidsare serving as an exogenous "electron sink,"then hydrogenation must be regarded as a sup-plemental process, because these acids are notthe major electron acceptors and their reductionwould not contribute significantly to electronremoval. Polan et al. (21) have suggested thatthe unsaturated fatty acids, when present, couldbe hydrogenated by a system which normallyfunctions to reduce an endogenous unsaturatedcompound. These workers reported that hydro-genation' of linoleic acid was inhibited by fu-maric, acrylic, and crotonic acids when intro-duced into a mixed culture of rumen bacteria.Since the major end product of glucose fermen-tation by strain B25 is succinic acid (2), presum-ably formed via the reduction of fumaric acid byfumarate reductase, we had considered the possi-bility that hydrogenation of 18-carbon, unsatu-rated fatty acids was associated with fumaratereduction. However, we have found that when0.1 M fumarate was added to the medium of agrowing culture of strain B25, the hydrogenationof linoleic acid-1-'4C to trans-l1-octadecenoicacid was not affected.
In general, our findings with Treponema (Bor-relia) strain B25 are in accordance with what hasbeen demonstrated for B. fibrisolvens strain A38,the only other rumen bacterium studied in detail.The similarities of the initial isomerization reac-tion, as well as the final end product formed, sug-gest that despite the differences in metabolic char-acteristics of these two distinct rumen species, thehydrogenation of 18-carbon, unsaturated fattyacids must be serving a similar purpose.
ACKNOWLEDGMENTS
We are indebted to M. P. Bryant for the many stimulatingdiscussions and for the use of his laboratory facilities. We alsothank M. J. Wolin for his discussions concerning the research.The able technical assistance of M. R. Crabill is acknowledged.
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