o-Cyclohexyl Fatty Acids in Acidophilic Thermophilic Bacteria STUDIES ON THEIR PRESENCE, STRUCTURE, AND BIOSYNTHESIS USING PRECURSORS LABELED WITH STABLE ISOTOPES AND RADIOISOTOPES*

w-Cyclohexyl undecanoic acid and o-cyclohexyl tridecanoic acid were found in 10 strains of acido-thermophilic bacteria isolated from different Japanese hot springs. These unusual fatty acids were found in the esterified form in glyceride type complex lipids and constituted 74 to 93% of the total fatty acids in the bacteria. The fatty acids other than w-cyclohexyl fatty acids found were 14.methyl hexadecanoic acid (3 to 15%) and E-methyl hexadecanoic acid (1 to 6%), and trace amounts of straight chain and methyl-branched tetra-and penta-decanoic acids. with increase in the concentration of glucose in the culture medium. specifically incorporated into the w-cyclohexyl fatty acids from labeled glucose, shikimate, and cyclohexyl carboxylate. These findings indicate that w-cyclohexyl fatty acids are synthesized with glucose through shikimic acid and probably cyclohexyl carboxylyl-CoA derivative as the intermediates.

w-Cyclohexyl undecanoic acid and o-cyclohexyl tridecanoic acid were found in 10 strains of acido-thermophilic bacteria isolated from different Japanese hot springs. These unusual fatty acids were found in the esterified form in glyceride type complex lipids and constituted 74 to 93% of the total fatty acids in the bacteria.
The fatty acids other than w-cyclohexyl fatty acids found were 14.methyl hexadecanoic acid (3 to 15%) and E-methyl hexadecanoic acid (1 to 6%), and trace amounts of straight chain and methyl-branched tetra-and penta-decanoic acids. Biosynthesis of w-cyclohexyl fatty acids increased with increase in the concentration of glucose in the culture medium.
The metabolism of w-cyclohexyl fatty acids was studied using deuterium-labeled precursors by mass fragmentation analysis. The deuterium of [2-D]glucose was specifically incorporated into position 2 of the cyclohexyl ring of the fatty acids, indicating that the ring was synthesized from the glucose molecule. Radioactivity was efficiently incorporated into the w-cyclohexyl fatty acids from labeled glucose, shikimate, and cyclohexyl carboxylate.
These findings indicate that w-cyclohexyl fatty acids are synthesized with glucose through shikimic acid and probably cyclohexyl carboxylyl-CoA derivative as the intermediates.
Acidophilic-thermophilic bacteria are characterized by their tolerance to high temperature and acidity (45-70", pH 2 to 5). Uchino and Doi (I) first reported the isolation of several strains of aerobic spore-forming acido-thermophiles from hot springs in the northern part of Japan. Morphologically similar organisms were isolated from the geyser basin of Yellowstone National Park, and from soil of the Volcano National Park, Hawaii (2)  MgS0,.7H,O (0.025%), CaCl, (0.005%), (NH,),SO, (0.04%), NaCl (0.15%), glucose (0.5%), yeast extract (O.l%), and sulfuric acid (5 x 10m3 N), unless otherwise specified. One strain (TA-6) was selected for studies on the effects of temperature and pH on growth (40-70°, pH 2.3-5.4, obtained using 1.25 x lo-'to 2.5 x lo-' N sulfuric acid) and on the effect of glucose concentrations (0 to 2.5%). A Shimadzu-GC-5A gas chromatograph with a glass column (3 mm inside diameter x 3.5 ml packed with 25% polyethylene glycol succinate coated on Celite 545 was used for the separation of w-cyclohexyl fatty acid methyl esters. A Nihondenshi model llOO-PR772 apparatus with a stainless steel column (3 mm inside diameter x 1 m) packed with 4% SE-30 on Celite 545 was used for preparative gas chromatography. A Packard model 7309 apparatus with 10% diethylene glycol succinate on Chromosorb G column connected to a "C proportional counter 894 was used for radio-gas chromatography.
Gas chromatography-mass spectrometry spectra were recorded using a Shimadzu-LKB model 9000 spectrometer with a column of 4% XE-60 on Chromosorb W at an ionization voltage of 70 e.v. [2-D]Glucose was synthesized by the method of Lemieux and Stevens (9). A sample of 750 mg of 2.0.benzyl-n-glucose was dissolved in 3 ml of deuterium oxide (D, 99Yo) and lyophilized. This procedure was repeated three times. Then the lyophilized material was dissolved in 3 ml of 1.7 N sodium deuteroxide and stood for 10 days at room temperature.
The Methyl-oc-n-[6-D,]glucopyranoside was synthesized as follows. A solution of sodium borodeuteride (50 mg in 3 ml of water) was added dropwise to a solution of methyl-o-glucuronic acid methyl ester (100 mg in 2 ml of distilled water). After standing for 6 hours at room temperature, the mixture was treated with Dowex 1 (H+ form) and IR-45 (OH-form), and was concentrated.
The anomers were purified from the resulting mixture of methyl-oc-n- [2-D]Shikimic acid was synthesized as follows. Shikimic acid (200 mg) and platinic oxide (50 mg) were suspended in 3 ml of deuterium oxide and kept at 100" for 40 hours in a sealed tube. Then the catalyst was filtered off, and the reaction mixture was evaporated to dryness yielding 80 mg of [2-Dlshikimic acid (D content, 60-70%, estimated by measurement of the NMR and GC-MS).' In some experiments, deuterium-labeled precursors were added to the culture medium (D-[2-Dlglucose and methyl-a-n-[6-D,]glucopyranoside, 200 mg/50 ml; [2-Dlshikimic acid, 40 mg/50 ml). Half of the cultures were incubated for 4 hours, and the cells in the exponential growth phase were harvested by centrifugation.
The other cultures were incubated overnight, and the cells in the stationary phase were harvested.
The cells were washed thoroughly and lyophilized. After methanolysis the fatty acid methyl esters were subjected to GC-MS analyses.
In other experiments, radioisotopic precursors were added to the culture medium (10 to 50 pCi/50 ml). Lipid was extracted from cells by the method of Bligh and Dyer (11). The total lipid extracted was subjected to methanolysis, and the fatty acid methyl esters were analyzed by radio-gas chromatography.

RESULTS AND DISCUSSION
Presence and Structure of w-Cyclohexyl Fatty Acids All 10 strains of acido-thermophilic bacteria isolated from different Japanese hot springs and geyser basins contained 74 *The abbreviation used is: GC-MS, gas chromatography-mass spectrometry.
to 93% (of the total fatty acid content) of w-cyclohexyl fatty acids (Table I).
Results on the gas chromatographic separation of these fatty acids are shown in Fig. 1. The purities of the II-cyclohexyl undecanoate and 13.cyclohexyl tridecanoate separated by preparative gas chromatography were 80.7%) and 86.5'2, respectively.
The absorption bands at 892 and 840 cm-i also provide strong evidence for the presence of a cycloalkane ring.
The proton nuclear magnetic resonance spectra (Fig. 3)      Precursors- Fig.  9 shows the ture), synthesis of w-cyclohexyl fatty acids was low. Glucose did not affect the synthesis of and 83 + 1 indicate that the deuterium was incorporated into acyclic acid (i.e. l&methyl hexadecanoic acid) (Fig. 8). Thus the cyclohexane ring from the deuterium at C-2 of the glucose the synthesis of w-cyclohexyl fatty acid is probably related to molecule. Results also suggested that the C-2 position of the glucose metabolism. alkyl cyclohexane ring was specifically labeled with [Z-  (Table II), since the fragmentation diagram of the cyclohexane ring (Fig. 5, bottom) showed that the bond between C-l and C-2 of the alkyl cyclohexane ring was cleaved giving a strong signal of the ion of m/e 55 + 1. The fragment ions at m/e 87 + 1, 97 + 1, 101 + 1, 143 + 1, 199 + 1, 239 + 1, 267 + 1, and 279 + 1 indicate that deuterium was incorporated into the n-alkyl chain of the w-cyclohexyl fatty acids (Table II) Thus studies with both deuterium-labeled and 'Y-labeled precursors showed that w-cyclohexyl fatty acids are synthesized from glucose through shikimate and cyclohexyl carboxylate, as shown in Fig. 10.
Many bacterial fatty acids (i.e. iso-or ante-iso-branched cyclopropane fatty acids and lo-methyl-branched acids) are known to be derived from the corresponding amino acids, such as valine, isoleucine, leucine, or methionine, and thus the fatty acid compositions of the bacteria are linked to their amino acid