Absolute configuration of hydroxycitric acid produced by microorganisms

Optical resolution for (2S,3R) and (2R,3S)-hydroxycitric acid (HCA) enantiomers was developed using chiral column chromatography. HCA from Bacillus megaterium G45C and Streptomyces sp. U121, newly isolated in our previous study, was analyzed to determine the absolute configuration. These results indicate that both strains generate optically pure (2S,3R)-hibiscus type HCA enantiomer.     

Screening and distributing features of bacteria with hydantoinase and carbamoylase

Hydantoinase and carbamoylase are key biocatalysts for the production of optically pure amino acids from dl-5-substituted hydantoins (SSH). Out of 364 isolated strains with hydantoinase and carbamoylase at 45 degrees C, 24 strains showed efficient hydantoinase and carbamoylase activities. Among them, 17 produced l-amino acids, and 7 produced d-amino acids from both aromatic dl-5-benzylhydantoin and aliphatic dl-5-isopropylhydantoin. Most of the strains that were able to form l-amino acid belonged to genera Bacillus, Geobacillus, Brevibacillus, Aneurinibacillus, Microbacterium, and Kurthia. Phylogenetic relationships were investigated based on 16S rRNA from the hydantoinase-producing bacteria. Distinct tendencies toward certain genera were observed between most of the strains forming l-amino acids and d-amino acids from SSH. The results from this study can be utilized to develop new isolation technology of hydantoinase-producing microorganisms, and to understand metabolism and evolutionary origins of hydantoinase and carbamoylase among different bacteria.     

Structure and glycosylation of lipoteichoic acids in Bacillus strains.

The occurrence, structure, and glycosylation of lipoteichoic acids were studied in 15 Bacillus strains, including Bacillus cereus (4 strains), Bacillus subtilis (5 strains), Bacillus licheniformis (1 strain), Bacillus polymyxa (2 strains), and Bacillus circulans (3 strains). Whereas in the cells of B. polymyxa and B. circulans neither lipoteichoic acid nor related amphipathic polymer could be detected, the cells of other Bacillus strains were shown to contain lipoteichoic acids built up of poly(glycerol phosphate) backbone chains and hydrophobic anchors [gentiobiosyl(beta 1----1/3)diacylglycerol or monoacylglycerol]. The lipoteichoic acid chains of the B. licheniformis strain and three of the B. subtilis strains had N-acetylglucosamine side branches, but those of the B. cereus strains and the remaining two B. subtilis strains did not. The membranes of the B. licheniformis strain and the first three B. subtilis strains exhibited enzyme activities for the synthesis of beta-N-acetylglucosamine-P-polyprenol and for the transfer of N-acetylglucosamine from this glycolipid to endogenous acceptors presumed to be lipoteichoic acid precursors. In contrast, the membranes of the other strains lacked both or either of these two enzyme activities. The correlation between the occurrence of N-acetylglucosamine-linked lipoteichoic acids and the distribution of these enzymes is consistent with the previously proposed function of beta-N-acetylglucosamine-P-polyprenol as a glycosyl donor in the introduction of alpha-N-acetylglucosamine branches to lipoteichoic acid backbone chains.     

Cellular Lipid and Fatty Acid Compositions of Burkholderia pseudomallei Strains Isolated from Human and Environment in Viet Nam

Viet nam is known as an endemic area of melioidosis but its etiologic agent originated in Viet nam was not extensively studied. For the first time, we analyzed the cellular lipid and fatty acid compositions of 15 Vietnamese isolates of Burkholderia pseudomallei, 10 from humans and 5 from the environment. Cellular lipid compositions were analyzed by two-dimensional thin-layer chromatography on silica gel G plates. Cellular fatty acid methyl esters were analyzed by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). The major lipids in all the isolates were phosphatidylglycerol (PG), two forms of phosphatidylethanolamine (PE-1 and PE-2), and two forms of ornithine-containing lipid (OL-1 and OL-2). PE-1 contained non-hydroxy fatty acids at both sn-1 and ?2 positions, while PE-2 possessed 2-hydroxy fatty acids and non-hydroxy fatty acids in a ratio of 1: 1. Since snake venom phospholipase A₂ digestion of PE-2 liberated 2-hydroxy fatty acids, it was confirmed that these acids are at the sn-2 position of glycerol moiety. In both OL-1 and OL-2, amide-linked fatty acid was 3-hydroxy palmitic acid (3-OH-C16: 0), while ester-linked fatty acids were non-hydroxy acids in OL-1 and 2-hydroxy acids in OL-2. The total cellular fatty acid compositions of the test strains were characterized by the presence of 2-hydroxy palmitic (2-OH-C16: 0), 2-hydroxy hexadecenoic (2-OH-C16: 1), 2-hydroxy octadecenoic (2-OH-C18: 1), 2-hydroxy methylene octadecanoic (2-OH-C19CPA), 3-hydroxy myristic (3-OH-C14: 0) and 3-hydroxy palmitic (3-OH-C16: 0) acids. There were significant differences in the concentration of hexadecenoic (C16: 1), methylene hexadecanoic (C17CPA), octadecenoic (C18: 1) and methylene octadecanoic (C19CPA) acids among the Vietnamese isolates of B. pseudomallei. However, no significant difference was observed in cellular lipid and fatty acid components between strains of human and environmental origins.     

Synthesis of β-hydroxy fatty acids and β-amino fatty acids by the strains of Bacillus subtilis producing iturinic antibiotics

The iturinic antibiotics, which contain long chain β-amino acids, are produced by Bacillus subtilis. Screening these strains for the presence of a possible precursor of the iturinic antibiotics, we isolated a lipopeptide containing β-hydroxy fatty acids. The structure of this compound was studied and it appears to be identical or structurally very similar to surfactin. The carbon chain of its β-hydroxy fatty acids was n C₁₆, iso C₁₆, iso C₁₅ or anteiso C₁₅. The percentages of each β-hydroxy fatty acids varied according to the strain producing iturinic antibiotics and were influenced by addition of branched-chain α-amino acids to the culture medium. These results demonstrate for the first time that iso C₁₄ β-hydroxy fatty acid is a constituent present in such a surfactin like lipopeptide. Besides, the presence of radioactive β-hydroxy fatty acids in the phospholipids when the strains were grown in the presence of sodium [¹⁴C]acetate seems also characterize the different strains producing iturinic antibiotics.     

Absolute Configuration of Hydroxycitric Acid Produced by Microorganisms

Optical resolution for (2S,3R) and (2R,3S)-hydroxycitric acid (HCA) enantiomers was developed using chiral column chromatography. HCA from Bacillus megaterium G45C and Streptomyces sp. U121, newly isolated in our previous study, was analyzed to determine the absolute configuration. These results indicate that both strains generate optically pure (2S,3R)-hibiscus type HCA enantiomer.     

Dust-borne bacteria in animal sheds, schools and children's day care centres

A total of 316 bacterial strains, including psychrophiles, mesophiles and thermophiles, were isolated and identified from indoor dusts in schools, children's day care centres and animal sheds. Several species which had not previously been reported from indoor environments were found: Sphingomonas, Brevibacterium, Nocardiopsis, Deinococcus and Rhodococcus/Gordona. A new psychrophilic actinomycete genus was also found in animal sheds, representing a new undescribed peptidoglycan type and an unusual whole-cell fatty acid composition. The indoor dusts of animal sheds contained mainly the Gram-negative genera Pseudomonas, Pantoea, Flavobacterium and Xanthomonas early in the indoor feeding season, but changed to a composition dominated by Bacillus, Micrococcus and mesophilic and thermophilic actinomycetes towards the end of the season. The dust contained, and air-borne bacterial flora in schools and day care centres were dominated by, Gram-positive bacilli and actinomycetes, notably Bacillus cereus, Brevibacillus brevis, B. licheniformis, B. subtilis and species of Arthrobacter, Corynebacterium, Rhodococcus/Gordona, Nocardiopsis sp., Deinococcus, Staphylococcus and Micrococcus. Indoor air and dust contained Klebsiella oxytoca, Acinetobacter calcoaceticus, Ac. lwoffi, Bacillus cereus and Nocardiopsis dassonvillei with the status of hazard group II. Indoor dusts of animal sheds contained eight different 3-hydroxy fatty acids, the 2-hydroxy fatty acid 14:0 and two 10-methyl fatty acids, whereas in dusts from schools and day care centres, these were below the detection level (< 3.5 ng mg-1). The 3-and 2-hydroxy fatty acids could be assigned to one or more of the dust-contained cultivable strains, but 10-methyl C16:0 was not present in any of the strains isolated. The dusts from schools and children's day care centres contained 0.2-0.3 ng of endotoxin mg-1 and 0.5-1.4 ng of beta-D-glucan mg-1, whereas the dusts from animal sheds contained more 0.3-41 ng mg-1 and 8-35 ng mg-1, respectively.     

Analysis of fatty acids from lipopolysaccharides of Bacteroides thetaiotaomicron and Bacteroides fragilis]

The aim of this study was to determine and to compare fatty acids occurring in lipopolysaccharides (LPS) isolated from B. thetaiotaomicron and B. fragilis strains of different origin. Lipopolysaccharides of three B. thetaiotaomicron strains and four B. fragilis strains were isolated by phenol-water extraction according to the procedure of Westphal and Jann (1965). Water-phase LPS fractions were then treated with nucleases and purified by ultracentrifugation as described by Gmeiner (1975). Fatty acid methyl esters, obtained by methanolysis of LPS, were analysed in gas-liquid chromatography combined with mass spectrometry (GLC-MS). Trimethylsilylated hydroxyl groups of fatty acid methyl esters were identified with GLC-MS using a method of selective ion monitoring (SIM). Lipopolysaccharides of B. thetaiotaomicron and B. fragilis strains contained long-chain (15-18 carbon atoms) fatty acids. The broad spectrum of simple long-chain and branched-chain fatty acids as well as 3-hydroxy fatty acids were detected. The main fatty acid of analyzed bacterial species was 3-hydroxy-hexadecanoic acid (3OH C16:0). Several 3-hydroxy fatty acids were detected in LPS of examined strains. Fatty acids occurring in LPS of B. thetaiotaomicron and B. fragilis strains appeared to be qualitatively similar. Quantitative differences in fatty acids composition of lipopolysaccharides isolated from strains of different origin were observed.     

一株可积累鸟苷菌株的纯培养条件研究

菌株Bacillus.subtilis.S3 68是以鸟苷生产菌株B .subtilis.A0 66为出发菌经诱变所得。对该菌株进行培养条件研究的过程中 ,发现该菌株可以在摇瓶纯培养条件下积累鸟苷。试验结果表明 :发酵过程中 ,腺嘌呤的用量 0 .3 5mg/ml时 ,发酵液中鸟苷积累量最大 ,培养基中腺嘌呤的用量高于或低于 0 .3 5mg/ml均不利于鸟苷产物的积累 ;培养基中味精、硫酸铵、硫酸镁、磷酸二氢钾及Mn2 +用量显著影响发酵液中鸟苷积累水平 ;培养基中生物素、蛋氨酸、精氨酸、组氨酸、氯化钙及Fe2 +、Zn2 +用量与鸟苷积累的相关性不显著     

Sequence diversity of the Bacillus thuringiensis and B. cereus sensu lato flagellin (H antigen) protein: comparison with H serotype diversity

We set out to analyze the sequence diversity of the Bacillus thuringiensis flagellin (H antigen [Hag]) protein and compare it with H serotype diversity. Some other Bacillus cereus sensu lato species and strains were added for comparison. The internal sequences of the flagellin (hag) alleles from 80 Bacillus thuringiensis strains and 16 strains from the B. cereus sensu lato group were amplified and cloned, and their nucleotide sequences were determined and translated into amino acids. The flagellin allele nucleotide sequences for 10 additional strains were retrieved from GenBank for a total of 106 Bacillus species and strains used in this study. These included 82 B. thuringiensis strains from 67 H serotypes, 5 B. cereus strains, 3 Bacillus anthracis strains, 3 Bacillus mycoides strains, 11 Bacillus weihenstephanensis strains, 1 Bacillus halodurans strain, and 1 Bacillus subtilis strain. The first 111 and the last 66 amino acids were conserved. They were referred to as the C1 and C2 regions, respectively. The central region, however, was highly variable and is referred to as the V region. Two bootstrapped neighbor-joining trees were generated: a first one from the alignment of the translated amino acid sequences of the amplified internal sequences of the hag alleles and a second one from the alignment of the V region amino acid sequences, respectively. Of the eight clusters revealed in the tree inferred from the entire C1-V-C2 region amino acid sequences, seven were present in corresponding clusters in the tree inferred from the V region amino acid sequences. With regard to B. thuringiensis, in most cases, different serovars had different flagellin amino acid sequences, as might have been expected. Surprisingly, however, some different B. thuringiensis serovars shared identical flagellin amino acid sequences. Likewise, serovars from the same H serotypes were most often found clustered together, with exceptions. Indeed, some serovars from the same H serotype carried flagellins with sufficiently different amino acid sequences as to be located on distant clusters. Species-wise, B. halodurans, B. subtilis, and B. anthracis formed specific branches, whereas the other four species, all in the B. cereus sensu lato group, B. mycoides, B. weihenstephanensis, B. cereus, and B. thuringiensis, did not form four specific clusters as might have been expected. Rather, strains from any of these four species were placed side by side with strains from the other species. In the B. cereus sensu lato group, B. anthracis excepted, the distribution of strains was not species specific.     

Formation of Crystalline delta-Endotoxin or Poly-beta-Hydroxybutyric Acid Granules by Asporogenous Mutants of Bacillus thuringiensis

Parental strains and asporogenous mutants of Bacillus thuringiensis subspp. kurstaki and aizawai produced high yields of delta-endotoxin on M medium, which contained 330 mug of potassium per ml, but not on ST and ST-a media, each of which contained only 11 mug of potassium per ml. On ST and ST-a media, refractile granules were formed instead. These granules had no insecticidal activity against silkworms and were isolated and identified as poly-beta-hydroxybutyric acid. Supplementation of the potassium-deficient ST-a medium with 0.1% KH(2)PO(4) (3.7 mM) led to the formation of crystalline delta-endotoxin. The replacement of KH(2)PO(4) with equimolar amounts of KCl, KNO(3), and potassium acetate or an equivalent amount of K(2)SO(4) had a similar effect, whereas the addition of an equimolar amount of NaH(2)PO(4) or NH(4)H(2)PO(4) did not cause the endotoxin to form. An asporogenous mutant, B. thuringiensis subsp. kurstaki strain 290-1, produced delta-endotoxin on ST-a medium supplemented with 3 mM or more potassium but formed only poly-beta-hydroxybutyric acid granules on the media containing 相似文献   

Fatty acid composition of Bifidobacterium and Lactobacillus strains

Normal C(14), C(16), and C(18) saturated acids and C(16) and C(18) monoenoic acids are the main fatty acids of nine strains of Bifidobacterium. Their lactobacillic acid content was less than 5%. Lactobacillus strains contained the same fatty acids as main compounds except for octadecanoic acid, which was present only in very low amounts. Eight of nine Lactobacillus strains contained in the stationary phase more than 15% lactobacillic acid. No correlation was observed between the fatty acid composition and other physiological characteristics used in the literature for classification of strains of one genus. Aging of the culture, which involved a decrease of the pH, caused a lengthening of the chain length of the fatty acids of B. bifidum var. pennsylvanicus but only a conversion of octadecenoic to lactobacillic acid in the lactobacilli. Lowering of the temperature of cultivation decreased the chain length of the fatty acids of B. bifidum var. pennsylvanicus. L. lactis did not show any influence of the temperature on the chain length of the fatty acids. The percentage of unsaturated acids was temperature independent in both organisms.     

Release,Regeneration and Mutant Induction of Pleurotus cornucopiae (‘Tamogitake’) Protoplasts

Both enantiomers of aliphatic and aromatic cyanohydrins were hydrolyzed with the aid of Rhodococcus butanica ATCC 21197 to afford optically active α-hydroxy acids. The usefulness of this reaction is demonstrated by the synthesis of optically pure (R)-4-dodecanolide, a defensive secretion of rove beetles, starting from (R)-2-hydroxydecanenitrile.     

On the effects of replacing the carboxylate-binding arginine-171 by hydrophobic tyrosine or tryptophan residues in the L-lactate dehydrogenase from Bacillus stearothermophilus

For L-lactate dehydrogenases (LDH's), the interaction of the guanidinium group of their Arg 171 residue with the carboxylate group of an alpha-keto acid is of primary importance in orienting the substrate productively at the active site. LDH's such as that of Bacillus stearothermophilus (BSLDH) are of practical importance for the preparation of chiral 2-hydroxy acids used as synthons in asymmetric synthesis but would even be more valuable in this regard if their specificities were broader. With a view to tailoring the specificity of BSLDH toward carbonyl substrates that lack an alpha-carboxyl group such as ketones, site-directed mutagenesis has been applied to replace Arg 171 by the approximately isosteric, but hydrophobic, amino acids Tyr and Trp. The mutant enzymes exhibit remarkably good catalytic activities toward representative alpha-keto acids RCOCOOH, where R = Me, Et, n-Pr, n-Bu, and CH2OH, although for the mutant enzymes the kcat/KM's are lower by approximately 10(3)-10(4)-fold than those for native BSLDH. Surprisingly, the 171----Tyr/Trp enzymes are significantly more active than 171----Lys (Hart et al., 1987a), for which an interaction of a positively charged side chain with substrate COO- is retained. Preparative-scale 171----Trp catalyzed reduction of pyruvate gave optically pure L-lactate, showing that L stereospecificity of such LDH enzymes was unaffected by the loss of Arg 171. The retention of L stereospecificity is attributed to secondary polar or hydrogen-bonding associations of Arg 109 and Thr246, respectively, with the substrate COO-function that are of sufficient magnitude to maintain "normal" substrate orientation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of different protocols for the analysis of lipophilic plant metabolites by gas chromatography–mass spectrometry using potato as a model

In the analysis of lipophilic plant metabolites by gas chromatography?Cmass spectrometry a step is required to release fatty acids and other analytes from complex molecules. Seven alternative methods were compared to the standard method of 1% H₂SO₄/50°C/16?h using Desirée and Phureja potato tubers as models. With two sodium methoxide alkali-catalysed methods (0.5?M NaOCH₃/50°C/1 and 16?h) recoveries of ferulic acids increased, long chain fatty acids and sterols decreased, 2-hydroxy acids were negligible, solanidine was absent and ??5-avenasterol isomerisation was minimal. Using a harsh alkali hydrolysis (1.0?M KOH/120°C/24?h) followed by a mild methylation (1% H₂SO₄/50°C/1.5?h), recoveries of polyunsaturated fatty acids were poor, sterols decreased but ??5-avenasterol isomerisation was minimal. With a mild alkali hydrolysis (0.5?M NaOH/100°C/5?min) followed by methylation with boron trifluoride (14%BF₃/100°C/30?min) recoveries of sterols and 2-hydroxy fatty acids were similar to the standard method and ??5-avenasterol isomerisation was high. Lower ferulic acid recoveries, absence of solanidine and overestimation of fatty alcohols were evident in both methods involving alkali hydrolysis. Three different methods using hydrochloric acid (1.00?M HCl/70°C/5?h, 0.63?M HCl/110°C/2?h and 2.00?M HCl/50°C/24?h) all gave increased recoveries of 2-hydroxy acids, ferulic acids, solanidine and sterols, although ??5-avenasterol isomerisation increased. Hydrochloric acid methods are recommended for studies requiring quantitative determinations (i.e. concentration of metabolite in sample). Either the hydrochloric acid methods or the standard sulphuric acid method are suggested for determining relative concentrations between samples, although there is a requirement for further studies.     

Properties of the testicular lactate dehydrogenase isoenzyme.

1. Studies were carried out with pure lactate dehydrogenase isoenzymes C4 (LDH isoenzyme X), B4, (LDH isoenzyme 1) and A4 (LDH isoenzyme 5) isolated from mouse testis, heart and muscle tissue respectively; with LDH isoenzyme X purified from pigeon testes and with crude lysates of spermatozoa from man, bull and rabbit. 2. LDH isoenzyme X from all species showed greater ability than the other isoenzymes to catalyse the NAD+-linked interconversions of 2-oxobutanoate into 2-hydroxybutanoate and of 2-oxopentanoate into 2-hydroxypentanoate. 3. Mouse LDH isoenzyme X presented the broadest spectrum of substrate specificity. It exhibited very similar Km values for a variety of 2-oxo acids: 2-oxopropanoate (pyruvate), 2-oxobutanoate, 2-oxo-3-methylbutanoate, 2-oxopentanoate, 2-oxo-3-methylpentanoate, 2-oxo-4-methylpentanoate, 2-oxohexanoate and 2-oxo-3-phenylpropanoate (phenylpyruvate). The corresponding 2-hydroxy acids were also readily utilized in the reverse reaction. A strong inhibition by substrate and product was demonstrated for the direct reaction. 4. Intracellular distribution of LDH isoenzyme X was investigated in mouse testes. LDH isoenzyme X activity was located in the fraction of "heavy mitochondria" and in the soluble phase. 5. A possible functional role for LDH isoenzyme X is proposed: the redox couple-2-oxo acid-2-hydroxy acid could integrate a shuttle system transferring reducing equivalents from cytoplasm to mitochondria.     

Conversion of fatty acids by Bacillus sphaericus-like organisms

Bacillus sphaericus species are mesophilic round-spored organisms that readily utilize fatty acid-based surfactants during growth, but their ability to modify fatty acids is unknown. Among 57 B. sphaericus-like strains tested for fatty acid transformation activity in Wallen fermentation (WF) medium, ten converted oleic acid to a new product determined by gas chromatography – mass spectrometry (GC-MS) to be 10-ketostearic acid (10-KSA). Additionally, a few other strains converted ricinoleic acid and linoleic acid to new products that remain to be characterized. Unlike most microbial hydrations of oleic acid, which produce a mixture of 10-KSA and 10-hydroxystearic acid, the conversion of oleic acid by B. sphaericus strains was unique in that 10-KSA was the sole reaction product. By replacing dextrose with sodium pyruvate in WF and adjusting to pH 6.5, conversion of oleic acid to 10-KSA by strain NRRL NRS-732 was improved from about 11% to more than 60%. Using the defined optimal conditions, the conversion reaction was scaled up in a stirred-batch reactor by using technical-grade oleic acid as substrate. This is the first report on the characterization of fatty acid conversions by B. sphaericus species. Received: 17 December 2001 / Accepted: 17 January 2002     

短小芽孢杆菌肉碱转运系统opuC基因的克隆与序列分析

以前期获得的ω-1-羟基脂肪酸高产突变菌株短小芽孢杆菌(Bacillus pumilus)M-F641的总DNA为模板,利用Primer Premier 5.0软件设计4对引物,对决定长链脂肪酸无效降解途径中肉碱转运的OpuC转运系统的基因进行克隆,成功获得了opuCA、opuCB、opuCC和opuCD的基因序列,并利用MEGA 3.1、DNAStar等软件进行序列分析.研究内容将为进一步利用短小芽孢杆菌长链脂肪酸高效转化生产ω-1-羟基脂肪酸菌株奠定基础.     

Isolation and characterization of thermophilic bacilli degrading cinnamic, 4-coumaric,and ferulic acids

Thirty-four thermophilic Bacillus sp. strains were isolated from decayed wood bark and a hot spring water sample based on their ability to degrade vanillic acid under thermophilic conditions. It was found that these bacteria were able to degrade a wide range of aromatic acids such as cinnamic, 4-coumaric, 3-phenylpropionic, 3-(p-hydroxyphenyl)propionic, ferulic, benzoic, and 4-hydroxybenzoic acids. The metabolic pathways for the degradation of these aromatic acids at 60 degrees C were examined by using one of the isolates, strain B1. Benzoic and 4-hydroxybenzoic acids were detected as breakdown products from cinnamic and 4-coumaric acids, respectively. The beta-oxidative mechanism was proposed to be responsible for these conversions. The degradation of benzoic and 4-hydroxybenzoic acids was determined to proceed through catechol and gentisic acid, respectively, for their ring fission. It is likely that a non-beta-oxidative mechanism is the case in the ferulic acid catabolism, which involved 4-hydroxy-3-methoxyphenyl-beta-hydroxypropionic acid, vanillin, and vanillic acid as the intermediates. Other strains examined, which are V0, D1, E1, G2, ZI3, and H4, were found to have the same pathways as those of strain B1, except that strains V0, D1, and H4 had the ability to transform 3-hydroxybenzoic acid to gentisic acid, which strain B1 could not do.     

The Urinary Bladder is Rich in Glycosphingolipids Composed of Phytoceramides

Glycosphingolipids (GSLs) are composed of a polar glycan chain and a hydrophobic tail known as ceramide. Together with variation in the glycan chain, ceramides exhibit tissue-specific structural variation in the long-chain base (LCB) and N-acyl chain moieties in terms of carbon chain length, degree of desaturation, and hydroxylation. Here, we report the structural variation in GSLs in the urinary bladders of mice and humans. Using TLC, we showed that the major GSLs are hexosylceramide, lactosylceramide, globotriaosylceramide, globotetraosylceramide, Neu5Ac-Gal-Glc-Ceramide, and Neu5Ac-Neu5Ac-Gal-Glc-Ceramide. Our LC-MS analysis indicated that phytoceramide structures with a 20-carbon LCB (4-hydroxyeicosasphinganine) and 2-hydroxy fatty acids are abundant in hexosylceramide and Neu5Ac-Gal-Glc-Ceramide in mice and humans. In addition, quantitative PCR demonstrated that DES2 and FA2H, which are responsible for the generation of 4-hydroxysphinganine and 2-hydroxy fatty acid, respectively, and SPTLC3 and SPTSSB, which are responsible for the generation of 20-carbon LCBs, showed significant expressions in the epithelial layer than in the subepithelial layer. Immunohistochemically, dihydroceramide:sphinganine C4-hydroxylase (DES2) was expressed exclusively in urothelial cells of the urinary bladder. Our findings suggest that these ceramide structures have an impact on membrane properties of the stretching and shrinking in transitional urothelial cells.