Effect of Substrate on the Fatty Acid Composition of Hydrocarbon- and Ketone-utilizing Microorganisms

The fatty acid pattern in hydrocarbon- and ketone-utilizing bacteria after growth on various substrates was examined. The fatty acid composition of one hydrocarbon-utilizing organism (Mycobacterium sp. strain OFS) was investigated in detail after growth on n-alkanes, 1-alkenes, ketones, and n-alcohols. n-Alkanes shorter than C₁₃ or longer than C₁₇ were not incorporated into cellular fatty acids without some degradation. Strain OFS incorporated C₁₄ to C₁₇ 1-alkenes into cellular fatty acids as the ω-monoenoic fatty acid. Methyl ketones were incorporated into strain OFS after removal of one- or two-carbon fragments from the carbonyl end of the molecule. An organism isolated by enrichment on methyl ketones was incapable of n-alkane utilization but could grow on, although not incorporate, ketones or long chain n-alcohols into cellular fatty acids.     

Amino Acid Composition of the Host-specific Toxin of Helminthosporium carbonum

The host-specific toxin of Helminthosporium carbonum (C₃₂H₅₀N₆O₁₀) was hydrolyzed by 6 n HCl to yield a number of α-amino acids. The common amino acids, proline and alanine, occurred in a ratio of 1:2. Two other unstable α-amino acids that produced lower color values with ninhydrin were also produced. One of these was tentatively identified as 2-amino-2,3-dehydro-3-methylpentanoic acid by electrolytic reduction to isoleucine. Additional ninhydrin-reacting substances were produced in low yield and probably represented secondary hydrolysis products of the unstable amino acids. The finding of an α,β-unsaturated linkage in H. carbonum toxin explains the instability of the compound and may also account for its specific toxicity.     

A Novel Amidase (Half-Amidase) for Half-Amide Hydrolysis Involved in the Bacterial Metabolism of Cyclic Imides

A novel amidase involved in bacterial cyclic imide metabolism was purified from Blastobacter sp. strain A17p-4. The enzyme physiologically functions in the second step of cyclic imide degradation, i.e., the hydrolysis of monoamidated dicarboxylates (half-amides) to dicarboxylates and ammonia. Enzyme production was enhanced by cyclic imides such as succinimide and glutarimide but not by amide compounds which are conventional substrates and inducers of known amidases. The purified amidase showed high catalytic efficiency toward half-amides such as succinamic acid (K_m = 6.2 mM; k_cat = 5.76 s⁻¹) and glutaramic acid (K_m = 2.8 mM; k_cat = 2.23 s⁻¹). However, the substrates of known amidases such as short-chain (C₂ to C₄) aliphatic amides, long-chain (above C₁₆) aliphatic amides, amino acid amides, aliphatic diamides, α-keto acid amides, N-carbamoyl amino acids, and aliphatic ureides were not substrates for the enzyme. Based on its high specificity toward half-amides, the enzyme was named half-amidase. This half-amidase exists as a monomer with an M_r of 48,000 and was strongly inhibited by heavy metal ions and sulfhydryl reagents.     

Expression and Characterization of CYP52 Genes Involved in the Biosynthesis of Sophorolipid and Alkane Metabolism from Starmerella bombicola

Three cytochrome P450 monooxygenase CYP52 gene family members were isolated from the sophorolipid-producing yeast Starmerella bombicola (former Candida bombicola), namely, CYP52E3, CYP52M1, and CYP52N1, and their open reading frames were cloned into the pYES2 vector for expression in Saccharomyces cerevisiae. The functions of the recombinant proteins were analyzed with a variety of alkane and fatty acid substrates using microsome proteins or a whole-cell system. CYP52M1 was found to oxidize C₁₆ to C₂₀ fatty acids preferentially. It converted oleic acid (C_18:1) more efficiently than stearic acid (C_18:0) and linoleic acid (C_18:2) and much more effectively than α-linolenic acid (C_18:3). No products were detected when C₁₀ to C₁₂ fatty acids were used as the substrates. Moreover, CYP52M1 hydroxylated fatty acids at their ω- and ω-1 positions. CYP52N1 oxidized C₁₄ to C₂₀ saturated and unsaturated fatty acids and preferentially oxidized palmitic acid, oleic acid, and linoleic acid. It only catalyzed ω-hydroxylation of fatty acids. Minor ω-hydroxylation activity against myristic acid, palmitic acid, palmitoleic acid, and oleic acid was shown for CYP52E3. Furthermore, the three P450s were coassayed with glucosyltransferase UGTA1. UGTA1 glycosylated all hydroxyl fatty acids generated by CYP52E3, CYP52M1, and CYP52N1. The transformation efficiency of fatty acids into glucolipids by CYP52M1/UGTA1 was much higher than those by CYP52N1/UGTA1 and CYP52E3/UGTA1. Taken together, CYP52M1 is demonstrated to be involved in the biosynthesis of sophorolipid, whereas CYP52E3 and CYP52N1 might be involved in alkane metabolism in S. bombicola but downstream of the initial oxidation steps.     

Cometabolism of Methyl tertiary Butyl Ether and Gaseous n-Alkanes by Pseudomonas mendocina KR-1 Grown on C5 to C8 n-Alkanes

Pseudomonas mendocina KR-1 grew well on toluene, n-alkanes (C₅ to C₈), and 1° alcohols (C₂ to C₈) but not on other aromatics, gaseous n-alkanes (C₁ to C₄), isoalkanes (C₄ to C₆), 2° alcohols (C₃ to C₈), methyl tertiary butyl ether (MTBE), or tertiary butyl alcohol (TBA). Cells grown under carbon-limited conditions on n-alkanes in the presence of MTBE (42 μmol) oxidized up to 94% of the added MTBE to TBA. Less than 3% of the added MTBE was oxidized to TBA when cells were grown on either 1° alcohols, toluene, or dextrose in the presence of MTBE. Concentrated n-pentane-grown cells oxidized MTBE to TBA without a lag phase and without generating tertiary butyl formate (TBF) as an intermediate. Neither TBF nor TBA was consumed by n-pentane-grown cells, while formaldehyde, the expected C₁ product of MTBE dealkylation, was rapidly consumed. Similar K_s values for MTBE were observed for cells grown on C₅ to C₈ n-alkanes (12.95 ± 2.04 mM), suggesting that the same enzyme oxidizes MTBE in cells grown on each n-alkane. All growth-supporting n-alkanes (C₅ to C₈) inhibited MTBE oxidation by resting n-pentane-grown cells. Propane (K_i = 53 μM) and n-butane (K_i = 16 μM) also inhibited MTBE oxidation, and both gases were also consumed by cells during growth on n-pentane. Cultures grown on C₅ to C₈ n-alkanes also exhibited up to twofold-higher levels of growth in the presence of propane or n-butane, whereas no growth stimulation was observed with methane, ethane, MTBE, TBA, or formaldehyde. The results are discussed in terms of their impacts on our understanding of MTBE biodegradation and cometabolism.     

Transformation of Fatty Acids Catalyzed by Cytochrome P450 Monooxygenase Enzymes of Candida tropicalis

Candida tropicalis ATCC 20336 can grow on fatty acids or alkanes as its sole source of carbon and energy, but strains blocked in β-oxidation convert these substrates to long-chain α,ω-dicarboxylic acids (diacids), compounds of potential commercial value (Picataggio et al., Biotechnology 10:894-898, 1992). The initial step in the formation of these diacids, which is thought to be rate limiting, is ω-hydroxylation by a cytochrome P450 (CYP) monooxygenase. C. tropicalis ATCC 20336 contains a family of CYP genes, and when ATCC 20336 or its derivatives are exposed to oleic acid (C_18:1), two cytochrome P450s, CYP52A13 and CYP52A17, are consistently strongly induced (Craft et al., this issue). To determine the relative activity of each of these enzymes and their contribution to diacid formation, both cytochrome P450s were expressed separately in insect cells in conjunction with the C. tropicalis cytochrome P450 reductase (NCP). Microsomes prepared from these cells were analyzed for their ability to oxidize fatty acids. CYP52A13 preferentially oxidized oleic acid and other unsaturated acids to ω-hydroxy acids. CYP52A17 also oxidized oleic acid efficiently but converted shorter, saturated fatty acids such as myristic acid (C_14:0) much more effectively. Both enzymes, in particular CYP52A17, also oxidized ω-hydroxy fatty acids, ultimately generating the α,ω-diacid. Consideration of these different specificities and selectivities will help determine which enzymes to amplify in strains blocked for β-oxidation to enhance the production of dicarboxylic acids. The activity spectrum also identified other potential oxidation targets for commercial development.     

Formation of Polyesters by Pseudomonas oleovorans: Effect of Substrates on Formation and Composition of Poly-(R)-3-Hydroxyalkanoates and Poly-(R)-3-Hydroxyalkenoates

Pseudomonas oleovorans grows on C₆ to C₁₂n-alkanes and 1-alkenes. These substrates are oxidized to the corresponding fatty acids, which are oxidized further via the β-oxidation pathway, yielding shorter fatty acids which have lost one or more C₂ units. P. oleovorans normally utilizes β-oxidation pathway intermediates for growth, but in this paper we show that the intermediate 3-hydroxy fatty acids can also be polymerized to intracellular poly-(R)-3-hydroxyalkanoates (PHAs) when the medium contains limiting amounts of essential elements, such as nitrogen. The monomer composition of these polyesters is a reflection of the substrates used for growth of P. oleovorans. The largest monomer found in PHAs always contained as many C atoms as did the n-alkane used as a substrate. Monomers which were shorter by one or more C₂ units were also observed. Thus, for C-even substrates, only C-even monomers were found, the smallest being (R)-3-hydroxyhexanoate. For C-odd substrates, only C-odd monomers were found, with (R)-3-hydroxyheptanoate as the smallest monomer. 1-Alkenes were also incorporated into PHAs, albeit less efficiently and with lower yields than n-alkanes. These PHAs contained both saturated and unsaturated monomers, apparently because the 1-alkene substrates could be oxidized to carboxylic acids at either the saturated or the unsaturated ends. Up to 55% of the PHA monomers contained terminal double bonds when P. oleovorans was grown on 1-alkenes. The degree of unsaturation of PHAs could be modulated by varying the ratio of alkenes to alkanes in the growth medium. Since 1-alkenes were also shortened before being polymerized, as was the case for n-alkanes, copolymers which varied with respect to both monomer chain length and the percentage of terminal double bonds were formed during nitrogen-limited growth of P. oleovorans on 1-alkenes. Such polymers are expected to be useful for future chemical modifications.     

Relationship between the Subunits of Leucoplast Pyruvate Kinase from Ricinus communis and a Comparison with the Enzyme from Other Sources

Two cDNA clones, PK_pα and PK_pβ, for the leucoplast isozyme of pyruvate kinase have been isolated and characterized. A Southern blot of castor (Ricinus communis) DNA probed with PK_pα indicates the presence of a single gene for PK_p. Most (1610 base pairs) of the sequence of both cDNAs is identical. These 1610 base pairs begin with an ATG translation initiation codon, and have 248 base pairs of 3′-untranslated and 1362 base pairs of coding sequence. The sequences of the two clones 5′- to the identical regions are different but both encode peptides with a high percentage of hydrophobic amino acids. The derived sequence of PK_pα encodes eight amino acid residues which have been identified as the amino-terminus of one subunit of PK_p from castor seed leucoplasts when the enzyme is purified in the absence of cysteine endopeptidase inhibitors. The sequence upstream of these amino acids is possibly the transit peptide for this protein. When PK_p is extracted under conditions that eliminate its proteolytic degradation, its α-subunit has a relative molecular weight equal to the full-length coding sequence of PK_pα. The data indicate that the transit peptide for the subunit of leucoplast pyruvate kinase encoded by PK_pα is not cleaved until the protein is released from the plastid. The derived amino acid sequences of PK_pα and PK_pβ are most closely related to Escherichia coli pyruvate kinase. Although the residues involved in substrate binding are conserved in leucoplast pyruvate kinase, there is no phosphorylation site and only 5 of 15 amino acids in the E. coli fructose-1,6-bisphosphate binding site are conserved.     

Ratios of Carbon Isotopes in Microbial Lipids as an Indicator of Substrate Usage

The occurrence and abundance of microbial fatty acids have been used for the identification of microorganisms in microbial communities. However, these fatty acids can also be used as indicators of substrate usage. For this, a systematic investigation of the discrimination of the stable carbon isotopes by different microorganisms is necessary. We grew 11 strains representing major bacterial and fungal species with four different isotopically defined carbon sources and determined the isotope ratios of fatty acids of different lipid fractions. A comparison of the differences of δ¹³C values of palmitic acid (C_16:0) with the δ¹³C values of the substrates revealed that the isotope ratio is independent of the growth stage and that most microorganisms showed enrichment of C_16:0 with ¹³C when growing on glycerol. With the exception of Burkholderia gladioli, all microorganism showed depletion of ¹³C in C_16:0 while incorporating the carbons of glucose, and most of them were enriched with ¹³C from mannose, with the exception of Pseudomonas fluorescens and the Zygomycotina. Usually, the glycolipid fractions are depleted in ¹³C compared to the phospholipid fractions. The δ¹³C pattern was not uniform within the different fatty acids of a given microbial species. Generally, tetradecanoic acid (C_14:0) was depleted of ¹³C compared to palmitic acid (C_16:0) while octadecanoic acid (C_18:0) was enriched. These results are important for the calibration of a new method in which δ¹³C values of fatty acids from the environment delineate the use of bacterial substrates in an ecosystem.     

Use of a Nylon Manufacturing Waste as an Industrial Fermentation Substrate

Nonvolatile residue (NVR), a waste stream from the manufacture of nylon 6′6′, contains mainly small carboxylic acids and alcohols, making it a potential fermentation substrate. Above a concentration of 1.3% (wt/vol), NVR inhibited the growth of all microorganisms tested. The most inhibitory of the major NVR components were the monocarboxylic acids (C₄ to C₆) and ε-caprolactone. The inhibitory effects of NVR could be avoided by using a carbon-limited chemostat. Microorganisms were found that could use all of the major NVR components as carbon and energy sources. One such organism, Pseudomonas cepacia, was grown in a carbon-limited chemostat with a medium feed concentration of 20.5 g of NVR liter⁻¹. At a dilution rate of 0.14 h⁻¹ the yield of biomass (Y_x/s, where x is biomass produced and s is substrate used) from NVR was 18% (neglecting the water content of NVR). It was concluded that NVR would be a suitable carbon source for certain industrial fermentation processes such as the production of poly-β-hydroxybutyric acid.     

Selectively Adsorptive Extraction of Phenylarsonic Acids in Chicken Tissue by Carboxymethyl α-Cyclodextrin Immobilized Fe3O4 Magnetic Nanoparticles Followed Ultra Performance Liquid Chromatography Coupled Tandem Mass Spectrometry Detection

Carboxymethyl α-cyclodextrin immobilized Fe₃O₄ magnetic nanoparticles (CM-α-CD-Fe₃O₄) were synthesized for the selectively adsorptive extraction of five phenylarsonic acids including p-amino phenylarsonic acid, p-nitro phenylarsonic acid, p-hydroxy phenylarsonic acid, p-acylamino phenylarsonic acid and p-hydroxy-3-nitro phenylarsonic acid in chicken tissue. Using ultra performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS), a highly sensitive analytical method was proposed for the determination of five phenylarsonic acids. It was shown that CM-α-CD-Fe₃O₄ could extract the five phenylarsonic acids in complex chicken tissue samples with high extraction efficiency. Under the optimal conditions, a high enrichment factor, ranging from 349 to 606 fold, was obtained. The limits of detection (LODs) (at a signal-to-noise ratio of 3) were in the range of 0.05–0.11 µg/kg for the five phenylarsonic acids. The proposed method was applied for the determination of five target phenylarsonic acids in chicken muscle and liver samples. Recoveries for the spiked samples with 0.2 µg/kg, 2.0 µg/kg and 20 µg/kg of each phenylarsonic acids were in the range of 77.2%–110.2%, with a relative standard deviation (RSD) of less than 12.5%.     

Active-Site Engineering of ω-Transaminase for Production of Unnatural Amino Acids Carrying a Side Chain Bulkier than an Ethyl Substituent

ω-Transaminase (ω-TA) is a promising enzyme for use in the production of unnatural amino acids from keto acids using cheap amino donors such as isopropylamine. The small substrate-binding pocket of most ω-TAs permits entry of substituents no larger than an ethyl group, which presents a significant challenge to the preparation of structurally diverse unnatural amino acids. Here we report on the engineering of an (S)-selective ω-TA from Ochrobactrum anthropi (OATA) to reduce the steric constraint and thereby allow the small pocket to readily accept bulky substituents. On the basis of a docking model in which l-alanine was used as a ligand, nine active-site residues were selected for alanine scanning mutagenesis. Among the resulting variants, an L57A variant showed dramatic activity improvements in activity for α-keto acids and α-amino acids carrying substituents whose bulk is up to that of an n-butyl substituent (e.g., 48- and 56-fold increases in activity for 2-oxopentanoic acid and l-norvaline, respectively). An L57G mutation also relieved the steric constraint but did so much less than the L57A mutation did. In contrast, an L57V substitution failed to induce the improvements in activity for bulky substrates. Molecular modeling suggested that the alanine substitution of L57, located in a large pocket, induces an altered binding orientation of an α-carboxyl group and thereby provides more room to the small pocket. The synthetic utility of the L57A variant was demonstrated by carrying out the production of optically pure l- and d-norvaline (i.e., enantiomeric excess [ee] > 99%) by asymmetric amination of 2-oxopantanoic acid and kinetic resolution of racemic norvaline, respectively.     

The Influence of Growth Rate on 2H/1H Fractionation in Continuous Cultures of the Coccolithophorid Emiliania huxleyi and the Diatom Thalassiosira pseudonana

The hydrogen isotope (²H/¹H) ratio of lipids from phytoplankton is a powerful new tool for reconstructing hydroclimate variations in the geologic past from marine and lacustrine sediments. Water ²H/¹H changes are reflected in lipid ²H/¹H changes with R² > 0.99, and salinity variations have been shown to cause about a 1‰ change in lipid δ²H values per unit (ppt) change in salinity. Less understood are the effects of growth rate, nutrient limitation and light on ²H/¹H fractionation in phytoplankton. Here we present the first published study of growth rate effects on ²H/¹H fractionation in the lipids of coccolithophorids grown in continuous cultures. Emiliania huxleyi was cultivated in steady state at four growth rates and the δ²H value of individual alkenones (C_37:2, C_37:3, C_38:2, C_38:3), fatty acids (C_14:0, C_16:0, C_18:0), and 24-methyl cholest-5,22-dien-3β-ol (brassicasterol) were measured. ²H/¹H fractionation increased in all lipids as growth rate increased by 24‰ to 79‰ (div d^-1)^-1. We attribute this response to a proportional increase in the fraction of NADPH from Photosystem I (PS1) of photosynthesis relative to NADPH from the cytosolic oxidative pentose phosphate (OPP) pathway in the synthesis of lipids as growth rate increases. A 3-endmember model is presented in which lipid hydrogen comes from NADPH produced in PS1, NADPH produced by OPP, and intracellular water. With published values or best estimates of the fractionation factors for these sources (α_PS1 = 0.4, α_OPP = 0.75, and α_H2O = 0) and half of the hydrogen in a lipid derived from water the model indicates α_lipid = 0.79. This value is within the range measured for alkenones (α_alkenone = 0.77 to 0.81) and fatty acids (α_FA = 0.75 to 0.82) in the chemostat cultures, but is greater than the range for brassicasterol (α_{brassicasterol} = 0.68 to 0.72). The latter is attributed to a greater proportion of hydrogen from NADPH relative to water in isoprenoid lipids. The model successfully explains the increase in ²H/¹H fractionation in the sterol 24-methyl-cholesta-5,24(28)-dien-3β-ol from marine centric diatom T. pseudonana chemostat cultures as growth rate increases. Insensitivity of α_FA in those same cultures may be attributable to a larger fraction of hydrogen in fatty acids sourced from intracellular water at the expense of NADPH as growth rate increases. The high sensitivity of α to growth rate in E. huxleyi lipids and a T. pseudonana sterol implies that any change in growth rate larger than ~0.15 div d^-1 can cause a change in δ²H_lipid that is larger than the analytical error of the measurement (~5‰), and needs to be considered when interpreting δ²H_lipid variations in sediments.     

A universal description for the experimental behavior of salt-(in)dependent oligocation-induced DNA condensation

We report a systematic study of the condensation of plasmid DNA by oligocations with variation of the charge, Z, from +3 to +31. The oligocations include a series of synthetic linear ε-oligo(l-lysines), (denoted εKn, n = 3–10, 31; n is the number of lysines equal to the ligand charge) and branched α-substituted homologues of εK10: εYK10, εLK10 (Z = +10); εRK10, εYRK10 and εLYRK10 (Z = +20). Data were obtained by light scattering, UV absorption monitored precipitation assay and isothermal titration calorimetry in a wide range concentrations of DNA and monovalent salt (KCl, C_KCl). The dependence of EC₅₀ (ligand concentration at the midpoint of DNA condensation) on C_KCl shows the existence of a salt-independent regime at low C_KCl and a salt-dependent regime with a steep rise of EC₅₀ with increase of C_KCl. Increase of the ligand charge shifts the transition from the salt-independent to salt-dependent regime to higher C_KCl. A novel and simple relationship describing the EC₅₀ dependence on DNA concentration, charge of the ligand and the salt-dependent dissociation constant of the ligand–DNA complex is derived. For the ε-oligolysines εK3–εK10, the experimental dependencies of EC₅₀ on C_KCl and Z are well-described by an equation with a common set of parameters. Implications from our findings for understanding DNA condensation in chromatin are discussed.     

Bile acid-independent protection against Clostridioides difficile infection

Clostridioides difficile infections occur upon ecological / metabolic disruptions to the normal colonic microbiota, commonly due to broad-spectrum antibiotic use. Metabolism of bile acids through a 7α-dehydroxylation pathway found in select members of the healthy microbiota is regarded to be the protective mechanism by which C. difficile is excluded. These 7α-dehydroxylated secondary bile acids are highly toxic to C. difficile vegetative growth, and antibiotic treatment abolishes the bacteria that perform this metabolism. However, the data that supports the hypothesis that secondary bile acids protect against C. difficile infection is supported only by in vitro data and correlative studies. Here we show that bacteria that 7α-dehydroxylate primary bile acids protect against C. difficile infection in a bile acid-independent manner. We monoassociated germ-free, wildtype or Cyp8b1^-/- (cholic acid-deficient) mutant mice and infected them with C. difficile spores. We show that 7α-dehydroxylation (i.e., secondary bile acid generation) is dispensable for protection against C. difficile infection and provide evidence that Stickland metabolism by these organisms consumes nutrients essential for C. difficile growth. Our findings indicate secondary bile acid production by the microbiome is a useful biomarker for a C. difficile-resistant environment but the microbiome protects against C. difficile infection in bile acid-independent mechanisms.     

Microbial Hydrocarbon Co-oxidation. III. Isolation and Characterization of an α, α′-Dimethyl-cis,cis-Muconic Acid-producing Strain of Nocardia corallina

A soil isolate identified as a strain of Nocardia corallina accumulated α, α′-dimethyl-cis, cis-muconic acid under co-oxidation conditions employing n-hexadecane for growth and p-xylene as the co-oxidizable substrate. N. corallina V-49 was postulated to have two pathways for the oxidation of p-xylene. One pathway proceeds throughp-benzyl alcohol, p-tolualdehyde, and p-toluic acid to 2, 3-dihydroxy-p-toluic acid, and the other pathway results in ortho ring cleavage of 3, 6-dimethylpyrocatechol and hence accumulation of α,α′-dimethyl-cis, cis-muconic acid.     

Microbial Hydrocarbon Co-oxidation. III. Isolation and Characterization of an alpha, alpha'-Dimethyl-cis, cis-Muconic Acid-producing Strain of Nocardia corallina

A soil isolate identified as a strain of Nocardia corallina accumulated α, α′-dimethyl-cis, cis-muconic acid under co-oxidation conditions employing n-hexadecane for growth and p-xylene as the co-oxidizable substrate. N. corallina V-49 was postulated to have two pathways for the oxidation of p-xylene. One pathway proceeds throughp-benzyl alcohol, p-tolualdehyde, and p-toluic acid to 2, 3-dihydroxy-p-toluic acid, and the other pathway results in ortho ring cleavage of 3, 6-dimethylpyrocatechol and hence accumulation of α,α′-dimethyl-cis, cis-muconic acid.     

Fatty Acids of Myxococcus xanthus

Fatty acids were extracted from saponified vegetative cells and myxospores of Myxococcus xanthus and examined as the methyl esters by gas-liquid chromatography. The acids consisted mainly of C₁₄ to C₁₇ species. Branched acids predominated, and iso-pentadecanoic acid constituted half or more of the mixture. The other leading component (11–28%) was found to be 11-n-hexadecenoic acid. Among the unsaturated acids were two diunsaturated ones, an n-hexadecadienoic acid and an iso-heptadecadienoic acid. No significant differences between the fatty acid compositions of the vegetative cells and myxospores could be detected. The fatty acid composition of M. xanthus was found to be markedly similar to that of Stigmatella aurantiaca. It is suggested that a fatty acid pattern consisting of a large proportion of iso-branched C₁₅ and C₁₇ acids and a substantial amount of an n-16:1 acid is characteristic of myxobacteria.     

Genetic Evaluation of Physiological Functions of Thiolase Isozymes in the n-Alkane-Assimilating Yeast Candida tropicalis

The n-alkane-assimilating diploid yeast Candida tropicalis possesses three thiolase isozymes encoded by two pairs of alleles: cytosolic and peroxisomal acetoacetyl-coenzyme A (CoA) thiolases, encoded by CT-T1A and CT-T1B, and peroxisomal 3-ketoacyl-CoA thiolase, encoded by CT-T3A and CT-T3B. The physiological functions of these thiolases have been examined by gene disruption. The homozygous ct-t1aΔ/t1bΔ null mutation abolished the activity of acetoacetyl-CoA thiolase and resulted in mevalonate auxotrophy. The homozygous ct-t3aΔ/t3bΔ null mutation abolished the activity of 3-ketoacyl-CoA thiolase and resulted in growth deficiency on n-alkanes (C₁₀ to C₁₃). All thiolase activities in this yeast disappeared with the ct-t1aΔ/t1bΔ and ct-t3aΔ/t3bΔ null mutations. To further clarify the function of peroxisomal acetoacetyl-CoA thiolases, the site-directed mutation leading acetoacetyl-CoA thiolase without a putative C-terminal peroxisomal targeting signal was introduced on the CT-T1A locus in the ct-t1bΔ null mutant. The truncated acetoacetyl-CoA thiolase was solely present in cytoplasm, and the absence of acetoacetyl-CoA thiolase in peroxisomes had no effect on growth on all carbon sources employed. Growth on butyrate was not affected by a lack of peroxisomal acetoacetyl-CoA thiolase, while a retardation of growth by a lack of peroxisomal 3-ketoacyl-CoA thiolase was observed. A defect of both peroxisomal isozymes completely inhibited growth on butyrate. These results demonstrated that cytosolic acetoacetyl-CoA thiolase was indispensable for the mevalonate pathway and that both peroxisomal acetoacetyl-CoA thiolase and 3-ketoacyl-CoA thiolase could participate in peroxisomal β-oxidation. In addition to its essential contribution to the β-oxidation of longer-chain fatty acids, 3-ketoacyl-CoA thiolase contributed greatly even to the β-oxidation of a C₄ substrate butyrate.