J. Gen. Appl. Microbiol., 67, 100–105 (2021) doi 10.2323/jgam.2020.08.002 2021 Applied Microbiology, Molecular and Cellular Biosciences Research Foundation Full Paper Detection of 2-hydroxy-fatty acids and 2-hydroxy-fatty acid-containing ceramides in a gliding marine bacterium Aureispira marina (Received May 15, 2020; Accepted August 4, 2020; J-STAGE Advance publication date: March 13, 2021) Kazuyoshi Kawahara,1,∗ Hirokazu Iida,1 and Akira Yokota2 1 Department of Biosciences, College of Science and Engineering, Kanto Gakuin University, Yokohama, Kanagawa 236-8501, Japan 2 Department of Microbial Resources, Tohoku University Graduate School of Agricultural Science, Sendai, Miyagi 980-0845, Japan lipopolysaccharide (Alexander and Rietschel, 2001) in The cellular fatty acid composition of Aureispira addition to non-polar fatty acids mainly present in marina IAM 15389T (JCM 23197T), a gliding bac- glycerophospholipids in the membrane. An hydroxy-fatty terium isolated from the coastline of Thailand, was acid profile is, therefore, an important feature for the tax- re-examined by using a standard MIDI method onomy of Gram-negative bacteria (Oyaizu and Komagata, based on alkaline hydrolysis, and two other meth- 1983), and should be carefully examined when we analyze ods. The direct transesterification using 5% HCl/ the fatty acid composition of newly isolated bacteria. methanol or 4 M HCl hydrolysis followed by me- Aureispira marina is a gliding Gram-negative bacterium thyl esterification revealed that 2-hydroxy-15-me- with helical filamentous morphology isolated from ma- thyl-hexadecanoic acid (2-OH-iso-C17:0) and 2-hy- rine plant debris from the southern coastline of Thailand droxy-15-methyl-hexadecenoic acid (2-OH-iso- (Hosoya et al., 2006). This bacterium was reported to have C17:1), which were not reported in a previous pa- a unique fatty acid composition. It contained arachidonic per, were found to be major cellular fatty acids of acid (C20:4) as the most abundant fatty acid and saturated this bacterium, and the amount of 2-OH-iso-C17:1 fatty acids with normal and branched hydrocarbon chains. was even higher than that of arachidonic acid As for hydroxy-fatty acids, 3-hydroxy-hexadecanoic acid (C20:4), a characteristic polyunsaturated fatty acid (3-OH-C16:0), 3-hydroxy-15-methyl-hexadecanoic acid (3- present in this bacterium. These 2-hydroxy-fatty OH-iso-C17:0), and 3-hydroxy-octadecanoic acid (3-OH- acids were contained in two cellular lipids that were C18:0) were detected with small amounts only, compared relatively stable against alkaline hydrolysis. One with a taxonomically related bacterial species, Saprospira of them was analyzed by mass spectrometry, 1H- grandis, and 2-hydroxy-fatty acid was reported to be ab- nuclear magnetic resonance, and other chemical sent. The similar fatty acid composition was reported on methods, and identified as a ceramide composed another species of the same genus, Aureispira maritima of 2-hydroxy-fatty acid and sphingosine of 19 car- (Hosoya et al., 2007). These data of unusual hydroxy-fatty bons with three double bonds. A minor ceramide acid composition as a Gram-negative bacterium have containing 18 carbon sphingosine with three dou- prompted us to investigate more about the fatty acids of ble bonds was also detected. A. marina using several analytical methods. The present study revealed that the bacterium contains 2-hydroxy-fatty Key Words: Aureispira marina; ceramide; 2-hy- acids as major fatty acids and a novel ceramide contain- droxy-fatty acid; mass spectrometry; sphingosine ing these fatty acids. Materials and Methods Introduction Strain and growth condition. A. marina IAM 15389T (JCM 23197T) (type strain of the species) was used in this Gram-negative bacteria usually contain 3-hydroxy-fatty study. This strain was grown in a Sap2 liquid medium acids and, in some cases, 2-hydroxy-fatty acids in their *Corresponding author: Kazuyoshi Kawahara, Department of Biosciences, College of Science and Engineering, Kanto Gakuin University, 1-50-1, Mutsuura-higashi, Kanazawa-ku, Yokohama, Kanagawa 236-8501, Japan. E-mail: [email protected] None of the authors of this manuscript has any financial or personal relationship with other people or organizations that could inappropriately influence their work. 2-OH-fatty acids in Aureispira marina 101 (Hosoya et al., 2006) containing 0.5 × artificial sea water Table 1. Fatty acid contents in dry cells of A. marina IAM 15389T determined by three different methods. (1.5% NaCl, 0.035% KCl, 0.54% MgCl2·6H2O, 0.27% MgSO4·7H2O, and 0.05% CaCl2·2H2O), 0.1% tryptone Fatty acid Fatty acid content (nmol/mg dry cells)* (BD, Sparks, MD, USA), and 0.1% yeast extract (BD), Method I Method II Method III adjusted to pH 7.0, at 30°C for 24 h with reciprocal shak- iso-C 13 14 13 ing (80 cycles/min). 15:0 iso-C16:0 14 11 11 C 29 29 30 Analytical methods of cellular fatty acid. The bacterial 16:0 iso-C 25 26 27 cells were killed by heating at 105 C for 10 min, collected 17:0 ° C 111 by centrifugation, and lyophilized to prepare dry cells. Dry 17:0 3-OH-C16:0 213 cells (5 mg) were used for fatty acid analysis by three Unknown-1 27 96 92 methods. Unknown-2 23 41 39 3-OH-iso-C 325 Method I (MIDI method) (Glucksman et al., 2000): Dry 17:0 C 65 78 69 cells were hydrolyzed (100°C, 30 min) in one ml of alka- 20:4 line solution (15% NaOH in 50% methanol), two ml of *Mean values of data from triplicated experiments. acid solution (mixture of 13 ml 6 M HCl and 11 ml metha- nol) was then added, and fatty acids were esterified by heating (80°C, 10 min). Spectrometric analysis. The molecular weight of lipids Method II (Komagata and Suzuki, 1987): Dry cells were was analyzed by Matrix Assisted Laser Desorption/Ioni- mixed with one ml of 5% HCl/methanol (Kokusan Chemi- zation Time of Flight Mass Spectrometry (MALDI-TOF cal) and heated (100°C, 3 h) for direct transesterification. MS) (AXIMA-CHR plus, Shimadzu) with 2,5- Method III (Kawahara et al., 1991): Dry cells were dihydroxybenzoic acid (Fujifilm Wako Pure Chemical) as hydrolyzed with one ml of 4 M HCl, and liberated fatty a matrix material. Positive ions were detected with acids were extracted, and esterified with 5% HCl/metha- reflectron mode. Samples were solubilized in CDCl / nol by heating (85°C, 2 h). 3 CD OD (2:1, v/v), and 1H-Nuclear Magnetic Resonance Methyl ester samples were analyzed by gas-liquid chro- 3 (NMR) was measured using the equipment JNM-ECX 500 matography (GLC) equipped with a capillary column (500 MHz, JEOL RESONANCE) at 45°C. CBP1 (Shimadzu) for determination, and by gas chroma- tography/mass spectrometry (GC-MS) (QP2010Plus, Results Shimadzu) for identification. Electron impact ionization (EI) was used for GC-MS. Pentadecanoic acid (C ) was 15:0 Cellular fatty acid composition used as an internal standard for quantitative determina- Using dry cells of A. marina IAM 15389T, cellular fatty tion. The reduction of peaks of hydroxy-fatty acids be- acids were analyzed by three methods: alkaline hydroly- cause of tailing was corrected by using the peak area ratio sis and methyl esterification (Method I), direct of authentic C , 3-OH-C , and 2-hydroxy-hexadeca- 15:0 16:0 transesterification by HCl/methanol (Method II), and noic acid (2-OH-C ). 16:0 strong acid hydrolysis and methyl esterification (Method Thin-layer chromatography and column chromatogra- III). The mean values of fatty acid contents (nmol/mg dry phy. Cellular lipids were extracted with chloroform/ cells) of triplicated experiments are shown in Table 1. As methanol (2:1, v/v), and hydrolyzed with 0.1 M NaOH described in a previous paper (Hosoya et al., 2006), the (100°C, 30 min). Extracted lipids and lipid hydrolysate bacterium contained 13-methyl-tetradecanoic acid (iso- were analyzed by thin-layer chromatography (TLC) us- C15:0), 14-methyl-pentadecanoic acid (iso-C16:0), palmitic ing silica gel 60 aluminium plates (Merck, Darmstadt, acid (hexadecanoic acid) (C16:0), 15-methyl-hexadecanoic Germany). Lipids were developed with chloroform/metha- acid (iso-C17:0) as major fatty acids as well as C20:4, and nol/acetic acid/water (25:15:4:2, v/v/v/v), and visualized the amounts of these fatty acids were determined to be by spraying 10% H2SO4/ethanol and heating. Lipids were similar by all three methods. Only small amounts of 3- separated by silica gel column chromatography with the hydroxy fatty acids, 3-OH-C16:0 and 3-OH-iso-C17:0, were elution solvent, chloroform, chloroform/methanol (4:1, v/ identified, and their amounts were determined to be rela- v), chloroform/methanol (2:1, v/v), and chloroform/metha- tively higher by Method III. In addition to those fatty ac- nol (1:1, v/v) in a stepwise manner. ids, two large peaks with slightly different retention times (difference of about 0.06 min) (described as Unknown 1 Chemical reactions. Lipids solubilized in 2 ml of petro- and Unknown 2 in Table 1) were detected by Methods II leum ether were hydrogenated in a closed glass vial with and III. These peaks were also detected, but much smaller 5 mg platinum black. The atmosphere was exchanged with in the chromatogram of Method I, suggesting that Method hydrogen gas, and stirred for 40 min at room temperature. I was not suitable for determining these peaks. The Un- Methanolysis was performed by heating (100°C, 2.5 h) known 1 and Unknown 2 peaks gave EI-mass spectra with 1.0 M HCl/methanol prepared by dilution from 5% shown in Figs.