Analysis of the lipid moiety of lipopolysaccharide from Rhizobium tropici CIAT899: identification of 29-hydroxytriacontanoic acid

Vol. 176, No. 8 JOURNAL OF BAC ERIOLOGY, Ap . 1994, p. 2454-2457 0021-9193/94/$04.00+0 Copy igh X 1994, Ame ican Socie y o Mic obiology Analysis o he Lipid Moie y o Lipopolysaccha ide om Rhizobium opici CIAT899: Iden i ica ion o 29-Hyd oxy iacon anoic Acid A. M. GIL-SERRANO,'* I. GONZALEZ-JIMINEZ,1 P. TEJERO-MATEO,1 M. MEGIAS,2 AND M. J. ROMERO-VAZQUEZ2 Depa amen o de Quimica O gdnica, Facul ad de Quimica, I and Depa amen o de Mic obiologia y Pa asi ologia, Facul ad de Fa mnacia,2 Uni e sidad de Se illa, 41071 Se ille, Spain Recei ed 1 Oc obe 1993/Accep ed 3 Feb ua y 1994 The lipid moie ies o wo lipid A's isola ed om he phenolic and aqueous ac ions o lipopolysaccha ide om Rhizobium opici CIAT899 ha e been s udied. Se e al 3-hyd oxy a y acids and wo long-chain hyd oxy a y acids, 27-hyd oxyoc acosanoic acid, and 29-hyd oxy iacon anoic acid we e iden i ied; he a ios o hese acids a e he same in bo h lipid A's. These esul s can be used o chemo axonomic pu poses. Rhizobia a e g am-nega i e bac e ia and a e able o o m ni ogen- ixing symbio ic ela ionships wi h leguminous plan s. As g am-nega i e bac e ia, hey ha e he usual su ace polysac- cha ides, and hese molecules ha e been hypo hesized o be in ol ed in he molecula mechanism o symbio ic in ec ions. The lipopolysaccha ide (LPS) is essen ial o la e symbio ic s eps (1, 3, 4, 6, 13, 14). The LPS a e ancho ed in he ou e memb ane by hei lipid A componen , which exhibi s a e y low s uc u al di e si y and is he main endo oxic componen . Lipid A gene ally con ains a (1-6)-linked glucosamine disaccha ide and di e en a y acids. The lipid moie y o lipid A has been analyzed in ela ion o chemo axonomic aspec s, and 3-hyd oxy a y acids, cis-11, 12-me hyleneoc adecanoic acids, and a se ies o ing-opened p oduc s ha e been iden i ied (7, 8, 12, 16, 17, 20). Recen ly, he iden i ica ion o 27-hyd oxyoc acosanoic acid om mem- be s o he amily Rhizobiaceae (9, 10, 15) and o he bac e ia (11, 18) was desc ibed. In his communica ion, we epo he s uc u es o he a y acids ound in he lipid A om wo LPS (phenolic and aqueous ac ions) isola ed om Rhizobium opici CIAT899 and he iden i ica ion o 29-hyd oxy iacon anoic acid. Bac e ia we e g own o 3 days a 28°C in shaken TY medium con aining (pe li e ) 5 g o yp one, 3 g o yeas ex ac , and 0.83 g o CaCl2. The cul u e was cen i uged, and he pelle was washed h ee imes wi h 0.5 M NaCl o emo e he exopolysaccha ide. The LPS was ex ac ed om he bac- e ial cells by he ho phenol-wa e me hod (19), and wo ac ions (phenolic and aqueous phases) we e isola ed. Bo h phases we e dialyzed agains wa e , concen a ed, and passed h ough an anionic exchange esin, Ambe li e IRA 400 (ace- a e o m) wi h wa e as eluen . The elua es we e lyophilized and hen dissol ed in 10 mM MgSO4 and 50 mM T is-HCl solu ion (100 ml, pH 7.0). DNase I (1 mg) and RNase A (1 mg) we e added, and he solu ion was s i ed o e nigh a 5°C and hen lyophilized (5). LPS ac ions we e hyd olyzed wi h 1% ace ic acid a 100°C o 90 min, and he lipid A's o med p ecipi a es. The supe na an was ex ac ed wi h dichlo- ome hane, and he combined o ganic laye s we e added o he o me p ecipi a e and e apo a ed. Two lipid A's we e ob- * Co esponding au ho . Phone: 54557151. Fax: 54557134. ained, one om LPS in he aqueous phase (LAW) and he o he om LPS in he phenolic phase (LAP). Fa y acids we e de e mined as me hyl es e s by gas ch o- ma og aphy-mass spec ome y a e elease by me hanolysis wi h 0.625 M me hanolic hyd ogen chlo ide a 80°C o 16 h. The hyd oxy a y acid me hyl es e s we e silyla ed [py idine- bis( ime hylsilyl) i luo oace amide (1:1), 2 h, a oom em- pe a u e] and also ace yla ed (1-me hylimidazole-ace ic anhy- d ide [1:10]). Unsa u a ed a y acids we e con e ed in o he dime hyldi hio de i a i es (2). The me hyl es e s we e ea ed wi h iodine, die hyle he , and dime hyldisul ide o 24 h a 58°C. Na2S203 (5% aqueous) was hen added, and he de i - a i es we e ex ac ed ou imes wi h hexane. Gas ch oma og- aphy-mass spec ome y was pe o med wi h a Ca lo E ba model MFC 500 capilla y gas ch oma og aph i ed wi h a CP-Sil 5 CB WCOT column (25 m by 0.32 mm) a ached o a K a os MS-80RFA. Fo he me hyl es e s, me hyl es e ace- a es, and me hyl es e di hio de i a i es, he empe a u e p og am was iso he mal a 100°C o 2 min, ollowed by a 10°C/min g adien up o 280°C, ollowed by holding a 280°C o 30 min. Fo he ime hylsilyl de i a i es o me hyl es e s, he iso he mal s ep was a 140°C o 2 min ollowed by an 8°C/min g adien up o 280°C. The ioniza ion po en ial was 70 eV. In he ch oma og am o LAW, he ollowing sa u a ed a y acids, as hei me hyl es e s, we e iden i ied: 14-me hylpen a- decanoic acid (i-16:0), cis-11,12-me hyleneoc adecanoic acid (19:0 cyc, lac obacillic acid) and 11-me hoxynonadecanoic acid (11-OMe-19:0). Th ee 3-hyd oxy a y acids and wo long-chain hyd oxy a y acids we e also de ec ed. To iden i y he hyd oxy a y acids, he co esponding me hyl es e s we e ime hyl- silyla ed and ace yla ed. 3-Hyd oxy e adecanoic acid (3-OH- 14:0), 3-hyd oxypen adecanoic acid (3-OH-15:0), and 3-hy- d oxyhexadecanoic acid (3-OH-16:0) we e iden i ied. The elec on impac mass spec um o he 27-0-ace yl de i a i e o he me hyl es e o he 27-hyd oxyoc acosanoic acid (27-OH- 28:0) (Fig. 1) was iden ical o ha p e iously epo ed (10). The El mass spec um o he 29-0-ace yl de i a i e o he me hyl es e o 29-hyd oxy iacon anoic acid (29-OH-30:0) (Fig. 1) showed he expec ed agmen s: m/z 464 (M-60, loss o ace ic acid), m/z 432 (M-92, loss o ace ic acid and me hanol), and m/z 390 (M-134, loss o ace ic acid, me hanol, and ke ene). The spec um o he ime hylsilyl de i a i e o he me hyl es e o 27-OH-28:0 is shown in Fig. 2. The main agmen s 2454 VOL. 176, 1994 NTS 25 1 00 43 190 _ 1071 100 4: 60 5 0 302 20 1 0 404' 'II 20)7 120 62 23-: 1 240 360 480 600 432 55 97 281 L1i. *1.k I.00 I! II ii111III IIII1IIlII1ihIIiI.iI.I 111 aI.il.1. 11 ORi oIi II,. L. L zU aq i iliIiIIi uIu iB~lli11.pMUIFpi. I', II 9 ,Mi b'9l .. 1IIIi. *!I' plls 11" 1' I I00 200 300 400 FIG. 1. Mass spec a o he ace yla ed me hyl es e s o 27-hyd oxyoc acosanoic acid ( op) and 29-hyd oxy iacon anoic acid (bo om). we e m/z 117 (C5H13OS'i om he clea age be ween C26 and C27, base peak), m/z 511 (M-15, loss o me hyl), m!z 482 (M-44, loss o ca boni dioxide) and m/z 479 (M-47, loss o me hyl and me hanol). The mass spec um (Fig. 2) o 29-OH- 30:0 showed he expec ed agmen s, m/z 117, 539, 510, and 508, which co espond o he same losses men ioned abo e. Fo he lipid A isola ed om aqueous LPS (LAW), he majo componen s we e hyd oxy a y acids, and app oxima ely 54% o he o al a y acid con en co esponds o 3-hyd oxy a y acids, acco dingly o he da a epo ed (12, 16). A 40% componen o he o al a y acids co esponds o he long- chain hyd oxy a y acids. 27-OH-28:0 is he majo componen , in ag eemen wi h he esul s o Ramadas Bha e al. (15). 29-OH-30:0, which has been iden i ied in Ag obac e ium spp. and B ady hizobium spp. (15), was iden i ied and es ima ed. These wo long-chain hyd oxy a y acids a e in a 3:2 a io. Vaccenic acid was no ound, bu he ela ed lac obacillic acid and he ing-opened p oduc 11-OMe-19:0 (7, 8) ha e been iden i ied. The gas ch oma og am o he me hyl es e s o he a y acids om LAP allowed he iden i ica ion o he me hyl es e s o he ollowing sa u a ed a y acids: i-16:0, 19:0 cyc, 11-OMe-19:0, 13-me hoxynonadecanoic acid (13-OMe-19:0), and 11-me- hoxy-12-me hyloc adecanoic acid (1II-OMe-12-Me-18:0). The .111.. II i NOTES 2455 3 -+ -;C:. LA."I'l 3 .Oql RI L.111. .11111 L. II Ill.. li il II II. 11 .11 2456 NOTES I ,0 10e Ch 100 80 10 _ le 1. I i L I l 4j2 s I11 L ,- _1 1 500 30040 1 7 510 53 1 L- .11 I A. - . -. d _ IF IM P l -. p i- -! ,- ., .,I I . .,, . - I 100 200 300 400 500 600 FIG. 2. Mass spec a o he silyla ed me hyl es e s o 27-hyd oxyoc acosanoic acid ( op) and 29-hyd oxy iacon anoic acid (bo om). ollowing hyd oxy a y acids we e iden i ied as he 0- ime h- ylsilyl and 0-ace yl de i a i es o hei me hyl es e s: 3-OH- 14:0, 3-OH-15:0, 3-OH-16:0, 3-hyd oxyhep adecanoic acid (3- OH-17:0), 3-hyd oxyoc adecanoic acid (3-OH-18:0), 27-OH- 28:0, and 29-OH-30:0. Two unsa u a ed a y acids we e also de ec ed. To iden i y hem and o loca e he double bond posi ions, he me hyl es e s o he co esponding a y acids we e ea ed wi h dime hyldisul ide. Oc adec-11-enoic acid [ accenic acid, 18:1(11)] and 11-me hyl-oc adec-11-enoic acid [11-Me-18:1(11)] we e hus iden i ied as dime hyldi hio de i - a i es o hei me hyl es e s. The analysis o LAP showed an inc ease o he ela i e p opo ion o i-16:0 and a dec ease o he ela i e p opo ions o 3-hyd oy a y acids (38%) and long-chain hyd oxy a y acids (18%) compa ed wi h hose o LAW. In LAP, he 27-OH-28:0 is no he majo componen , bu he a io o 27-OH-28:0 o 29-OH-30:0 is he same as o he aqueous ac ion. Addi ionally, wo 3-hyd oxy a y acids (3-OH-17:0 and 3-OH-18:0) we e iden i ied. Vaccenic acid, he biosyn- he ically ela ed lac obacillic acid, and ou ing-opened p od- uc s, 11-OMe-19:0, 13-OMe-19:0, 11-OMe-12-Me-18:0, and 11-Me-18:1 (11) ha e been also iden i ied. Gas ch oma og aphy-mass spec ome y esul s a e summa- ized in Table 1. Ia . p -1 I .-. 11 l -q 1- . J. BACTERIOL. 0 -7 3 55 43 I S. .- 1.11 NOTES 2457 TABLE 1. Fa y acid composi ion o lipid A's om R. opici CIAT899 % Fa y acid composi ion o ': Fa y acid LAW LAP i-16:0 4 10 19:0 cyc 1 10 3-OH-14:0 18 13 3-OH-15:0 19 14 3-OH-16:0 17 8 3-OH-17:0 I 3-OH-18:0 2 11-OMe-19:0 1 11 13-OMe-19:0 I 11-OMe-12-Me-18:0 3 18:1(11) 6 11-Me-18:1(11) 5 27-OH-28:0 24 11 29-OH-30:0 16 7 ' Calcula ed as ela i e pe cen ages o he o al peak a eas in he me hyl es e ch oma o,g ams. In conclusion, he analysis o he lipid moie y o he lipid A ob ained om R. opici CIAT899 showed he p esence o 29-hyd oxy iacon anoic acid, which may be used as chemo- axonomic ma ke . We hank he Comisi6n In e minis e ial de Ciencia y Tecnologia (g an B10090-0520-C02) o inancial suppo and he Fundaci6n Cama a o he g an o a ellowship o I.G.-J. We also hank he Se icios de Espec ome ia de Masas y Reso- nancia Magne ica Nuclea , Uni e sidad de Se illa, o he spec a. REFERENCES 1. B ink, R. A., J. Mille , R. W. Ca lson, and K. D. Noel. 1990. Exp ession o Rhizobium leguminosa um CFN42 genes o lipo- polysaccha ide in s ains de i ed om di e en R. leguminosa um soil isola es. J. Bac e iol. 172:548-555. 2. Buse , H. R., H. A n, P. Gue in, and S. Rausche . 1983. De e mi- na ion o double-bond posi ion in mono-unsa u a ed ace a es by mass spec ome y o dime hyldisulphide adduc s. Anal. Chem. 55:818-822. 3. Ca lson, R., R. Sha e s, J. Duh, E. Tu nbull, B. Hanley, B. Rol e, and M. Djo dje ic. 1987. The isola ion and pa ial cha ac e iza ion o he lipopolysaccha ides om se e al Rhizobium i olii mu an s a ec ed in oo hai in ec ion. Plan Physiol. (Be hesda) 84:421- 427. 4. Ca lson, R. W., S. Kalembasa, D. Tu owski, P. Pacho i, and K. D. Noel. 1987. Cha ac e iza ion o he lipopolysaccha ide om a Rhizobium phaseoli mu an ha is de ec i e in in ec ion h ead de elopmen . J. Bac e iol. 169:4923-4928. 5. Ca lson, R. W., R. E. Saunde s, C. Napoli, and P. Albe sheim. 1978. Hos -symbion in e ac ions. III. Pu i ica ion and pa ial cha ac e iza ion o Rhizobium lipopolysaccha ides. Plan Physiol. (Be hesda) 62:912-917. 6. deMaagd, R. A., A. S. Rao, I. H. M. Mulde s, L. Goosen-de Roo, M. C. M. an Loosd ech , C. A. Wij elman, and B. J. J. Lug en- be g. 1989. Isola ion and cha ac e iza ion o mu an s o Rhizobium leguminosa um b . iciae 248 wi h al e ed lipopolysaccha ides: possible ole o su ace cha ge o hyd ophobici y in bac e ial elease om he in ec ion h ead. J. Bac e iol. 171:1143-1150. 7. Ge son, T., and J. J. Pa el. 1975. Neu al lipids and phospholipids o ee-li ing and bac e oid o ms o wo s ains o Rhizobium in ec i e on Lo us peduncula us. Appl. Mic obiol. 30:193-198. 8. Ge son, T., J. J. Pa el, and L. N. Nixon. 1975. Some unusual a y acids o Rhizobium. Lipids 10:134-139. 9. Hollingswo h, R. I., and D. A. Lill-Elghanian. 1989. Isola ion and cha ac e iza ion o he unusual lipopolysaccha ide componen , 2-amino-2-deoxy-2-N-(27-hyd oxyoc acosanoyl)-3-0-(3-hyd oxy e - adecanoyl)-gluco-hexu onic acid and i s de-O-acyla ion p oduc om he ee lipid A o Rhizobium ni olii ANU843. J. Biol. Chem. 264:14039-14042. 10. Hollingswo h, R. I., and R. W. Ca lson. 1989. 27-Hyd oxyoc aco- sanoic acid is a majo s uc u al a y acyl componen o he lipopolysaccha ide o Rhizobium i olii ANU843. J. Biol. Chem. 264:9300-9303. 11. Moll, H., A. Sonesson, E. Jan zen, R. Ma e, and U. Zaeh inge . 1992. Iden i ica ion o 27-oxo-oc acosanoic acid and hep acosane- 1,27-dioic acid in Legionella-Pneumophila. FEMS Mic obiol. Le . 97:1-6. 12. Planque, K., J. J. an Nie op, A. Bu ge s, and S. G. Wilkinson. 1979. The lipopolysaccha ide o ee-li ing and bac e oid o ms o Rhizobium leguminosa um. J. Gen. Mic obiol. 110:151-159. 13. P ie e , U. B. 1989. Genes in ol ed in lipopolysaccha ide p oduc- ion and symbiosis a e clus e ed on he ch omosome o Rhizobium leguminosa um bio a iciae VF39. J. Bac e iol. 171:6161-6168. 14. Pu anesa ajah, V., F. M. Schell, D. Ge hold, and G. S acey. 1987. Cell su ace polysaccha ides om B ady hizobium japonicum and a nonnodula ing mu an . J. Bac e iol. 169:137-141. 15. Ramadas Bha , U., H. Maye , A. Yoko a, R. I. Hollingswo h, and R. W. Ca lson. 1991. Occu ence o lipid A a ian s wi h 27- hyd oxyoc acosanoic acid in lipopolysaccha ides om membe s o he amily Rhizobiaceae. J. Bac e iol. 173:2155-2159. 16. Russa, R., and Z. Lo kiewicz. 1974. Fa y acids p esen in he lipopolysaccha ide o Rhizobium i olii. J. Bac e iol. 119:771-775. 17. Russa, R., 0. Lude i z, and E. T. Rie schel. 1985. S uc u al analysis o lipid A om lipopolysaccha ides o nodula ing and non-nodula ing Rhizobium i olii. A ch. Mic obiol. 141:284-289. 18. Sonesson, A., H. Moll, E. Jan zen, and U. Zaeh inge . 1993. Long-chain ox-hyd oxy-(w-1)-oxo a y acids and co-hyd oxy-1, w-dioic acids a e cell wall cons i uen s o Legionella (L. jo danis, L. maceache nii and L. micdadei). FEMS Mic obiol. Le . 106:315- 320. 19. Wes phal, O., and K. Jann. 1965. Bac e ial lipopolysaccha ides. Me hods Ca bohyd . Chem. 5:83-91. 20. Wilkinson, S. G. 1988. G am nega i e bac e ia. p. 299-488. In C. Ra ledge and S. G. Wilkinson (ed.), Mic obial lipids, ol. 1. Academic P ess, London. VOL. 176, 1994