Studies on the cellular and free lipopolysaccharides from Branhamella catarrhalis ly2 K. G. JOHNSON, I. J . MCDONALD, A N D M. B. PERRY Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Texas A&M University on 11/13/14 For personal use only.
Dil.i.~iotrof B i ~ l ~ g i cSciet~ces, oI Nrrtiotinl Reserrrch Corrtzcil qfCrrt1rrrln, O t t r r ~ ~ jCnnrrdcr n, K I A OR6
Accepted December 23, 1975 JOHNSON,K . G., I. J. MCDONALD,and M. B. PERRY.1976. Studies on the cellular and free cotcrrrhcrlis. Can. J. Microbiol. 22: 460-467. lipopolysaccharides from Brcrrrl~ar~~elln Cellular and fi-ee lipopolysaccharides obtained from Neisseria crrtnrrlrnlis and Brnnl~nmeNu crrtcrrrlrrrlis were found to be essentially identical. Both cellular and free lipopolysaccharides contained core-oligosaccharides of the following composition: D-glucoSe (4 mol), D-galactose (I mol), 2-amino-2-deoxy-D-glucose (1 mol), and 3-deoxy-D-mcrnno-octulosonic acid. Aldoheptose and phosphate components were below levels of detection. Several physical methods indicated that all core-oligosaccharide preparations were identical. Lipid A preparations from cellular and free lipopolysaccharides of both organisms were qualitatively and quantitatively similar: they were composed of decanoic acid, dodecanoic acid, 3-hydrony dodecanoic acid, 2-amino-2-deoxy-D-glucose, phosphate, and ethanolamine. The results tend to justify the transfer of Neisserin crrtnrrhalis to the genus Brcrr~hcrmelln. JOHNSON,K. G., I. J . MCDONALDe t M. B. PERRY. 1976. Studies on the cellular and free lipopolysaccharides from Brnnltcrmc~llncc~tnrrlicrlis.Can. J. Microbiol. 22: 4 6 W 6 7 . Les lipopolysaccharides cellulaires e t libres obtenus de Neisserin c.ntorr/~crliset Brcrni~crrn~llcr cntnrrhcrlis sont essentiellement identiques. Les elements fondamentaux des oligosaccharides de ces lipopolysaccharides, cellulaires et libres, contiennent du: D-glucose (4 mol), D-galactose (1 mol). 2-amino-2-desoxy-D-glucose (1 mol) et I'acide 3-desoxy-D-manno-octulosonique. Aucun constituants aldoheptose e t phosphate n'ont pu ktre dCcelC. Les resultats de plusieurs methodes d'analyses physiques indiquent que toutes les preparations des oligosaccharides d e base sont identiques. I.es preparations du lipide A obtenues des lipopolysaccharides cellulaires e t libres des deux organismes sont semblables quantitativement et qualitativement; ils contiennent d e I'acide decanoi'que, de I'acide d~decanoi'que,de I'acide 3-hydroxy-dodecano~que, du 2-amino-3desoxy-D glucose, du phosphate et de I'ethanolamine. Les resultats obtenus demontrent que I'on pourrait transferer Neissericr cntorrholis a u genre Brcrnhnmc~lln. [Traduit par lejournal]
Introduction Several methods, includingserology (3 I), transformation experiments (3, 6), measurement of deoxyribonuc~eicacid (DNA) base composition and interspecific DNA homologies (6, 24), metabolic activity tests and enzyme profiles (2, 15, 16), electrophoretic examination of soluble proteins (11, 12), and analysis of fatty acids (19, 25) have been used in the elucidation of taxonomic relationships in the genus Neisseria. Accumulated data from such studies have permitted division of the genus into the u t r u e ~ ~ i and~"false ~ ~ ~ ~h~ i former ~ ~groupcontains ~ ~ thei patho~ gens N. gonorrhoeae and N. merlirlgitidisas well as the non-pathogenic organisms, which have been dFsignatedas N. canis, N. cinerea, N. cuniCuli, N. jaua, N . lactamjca, N. mucosa, N . perjava,N. sicca, and N. s u b ~ a v a l-he . species of uncertain taxonomic status consist of 'Received November 13, 1975. 'NRCC No. 15196.
N. caviae, N. catarrhalis, and N. ovis. Of these species, N. catarrhalis has recently been redesig"ated as the sole representative of the created genus Branhamella (5) and has been as"gned the name B. various non-path0genic Neisseria in this laboratory has aided species differentiation On the basis growth requirements (26)y lip0pOlysaccharide (LPS) core-oligosacchan'de of envelope proteins (30), (21, 22), and cross In common with observations ~ ~ i ~ of- differing LPS core-oligo.saccharide ~ ~types in the genera Escherichia (4) and Serratia (32), LPS from species of Neisseria have been demonstrated to contain at least four core-oligosaccharide types (21, 22, 23). Core-oligosaccharide types I to 'V inclusive occur in the LPS of species designated as "true Neisseria." The present study was undertaken to examine LPS core-oligosaccharide com3Russell, Johnson, and McDonald. T o be published.
46 1
3N ET AL.
position and structure of the cellular and free LPS from the "false Neisseria" N. catarrhalis, now designated as B. catarrhalis.
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Materials and Methods Organisms and Growth Conditions The following bacteria were used (American Type Culture Collection (ATCC) numbers are given in parentheses): Neisseria catarrhalis, National Research Council of Canada (NRCC) 31002 (8176), and Branhamella catarrhalis, NRCC 59001 (25238). The organisms were grown in trypticase soy broth (Baltimore Biological Laboratories) supplemented with 0.5% (w/v) yeast extract (Difco) (TCSY). The organisms were maintained as frozen (liquid nitrogen) stock cultures in the NRCC collection. Working stock cultures were routinely maintained as 10fold concentrated bacterial suspensions in 10% (w/v) aqueous glycerol at - 40 "C. Cultures used for the production of cellular and free LPS were grown in TCSY medium. A 75-litre fermentor containing 50 litres of medium was inoculated with 5 litres of late logarithmic phase culture. Agitation was maintained at 175 rpm and aeration at 8 litres/min during growth at 37 "C. Near the end of the exponential growth phase, cells were harvested by centrifugation. Cells were immediately lyophilized and the culture supernatant was placed at 4" until further processed. Preparation and Partial Fission of LPS Cellular LPS was obtained using the lysozyme-phenol extraction technique (23). Free LPS was prepared in purified form by the previously described sequence involving batch adsorption on diethylaminoethyl-cell~~lose, then ethanol precipitation of desorbed material followed by its aqueous phenol extraction and final collection by ultracentrifugation (21). Release of water-insoluble lipid A and water-soluble core-oligosaccharide was achieved by dilute acetic acid hydrolysis of intact LPS (21). Core-oligosaccharide preparations were fractionated by filtration through a Sephadex (3-50 column (V, = 490 ml, Vo = 160 ml) using 47 mM pyridinium acetate, pH 4.26, as eluant. Gel filtration properties are expressed in terms of the distribution coefficient, K,, = (V, - Vo)(V, - Vo), where V, is the elution volume of a specific material, Vo is the void volume of the system, and V, is the total volume of the system. Analytical Techniques Total LPS was estimated by the carbocyanine dye assay (18) and total hexose was determined by the phenolsulfuric colorimetric procedure (9). Amino glycoses were estimated calorimetrically (13), by ion-exchange chromatography using a Beckman 121 amino acid analyzer (34) and by gas-liquid chromatography (GLC) (27, 29). D-Glucose and D-galactose were determined by G L C of the trimethylsilyl (TMS) derivatives of their methyl glycosides (8) and of their glycitol acetate derivatives (14). GLC was performed as previously described (22) using the following columns: (A) 3% (w/w) ECNSS-M on 100-120 mesh Gas-Chrom Q, (B)3% (w/w) SE-30 on 80-100 mesh Chromosorb W, and (C) 10% (w/w) neopentylglycol succinate polyester on 80- to 100-mesh Chromosorb W. Retention times are quoted relative t o
mannitol hexacetate (TMa). Analytical and preparative paper chromatography was performed as previously described (22) using pyridine -ethyl acetate - water (2: 5: 5 v/v, top layer) as the mobile phase, and mobilities are quoted relative to D-mannose (R,,,). The periodate - thiobarbituric acid method (1) and G L C (33) were used for the determination of 3-deoxy-Dmarzno-octulosonic acid (KDO). Phosphorus was determined by a micro method (7) and fatty acids were identified and determined by GLC of their methyl esters (22), their retention times being quoted relative to methyl octadecanoate ( T c l s ) . Specific Optical Rotation Optical rotations were determined at 20 "C using a Perkin-Elmer model 141 polarimeter. 13C Nuclear. Magnetic Resonance (izmr) The 13C nmr spectra were obtained using a Varian XL-100 (25 MHz) spectrometer (sample tubes 12 mm outside diameter) in the pulsed Fourier transform mode with complete proton decoupling. Chemical shifts were recorded in parts per million downfield from external tetramethylsilane and deuterium oxide 'H was used f o r field frequency lock,
Results Excretion of LPS Both N. catarrhalis and B. catarrhalis excreted LPS-containingmaterial into the growth medium. Specific LPS release, which was defined as micrograms free LPS per milligram bacterial dry weight was 49.6 for N. catarrhalis and 43 for B. catarrhalis. This parameter was constant for both organisms during the growth cycle. Characterization of LPS Lipopolysaccharides were designated pure according to the following criteria: (a) elution as a single symmetrical peak at the V, of Sepharose 6B gel filtration systems, (6) absence of detectable ribose on hydrolysis, (c) lack of absorption maximum in the 210- to 300-nm region of the ultraviolet spectra, (d) freedom from significant amino acid contamination (less than 2 nmol of the most abundant amino acid per milligram of preparation), and (e) the presence of discrete core-oligosaccharide fragments derived from the mild acetic acid hydrolysis of intact LPS preparations. Sephadex G-50 gel filtration of the watersoluble glycan fraction obtained from the mild acid fission of each LPS revealed the presence of only two components, which had K,, values of 0.81 and 0.96. The elution profile of the glycan material obtained from the cellular LPS of N. catarrhalis, which is typical of the profiles obtained from the other LPS products, is shown in Fig. I. Material having a K,, value of 0.96 (peak
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C A N . J . MICROBIOL. VOL. 22, 1976
FRACTION NO
FIG. 1. Fractionation of core-oligosaccharide obtained from the cellular LPS of N. catnrrknlis on Sephadex (3-50 sing 47 n1M pyridiniurn acetate, pH 4.26, as eluant. A l i q ~ ~ o (100 t s pl) were withdrawn and analyzed for aldoglycose (9) -, and for aminoglycose (13) - - -. Optical density was determined a t 490 nm.
TABLE 1. Properties and composition of core-oligosaccharides from Brnrllinrnelln cotarrhalis and Neisserio cninrrlmlis"
B. cntnrrlinlis N. cninrrhnlis
-
ID
S o ~ ~ r of ce LPS
(water)
K b
D-GIC
D-Gal
D-GlcN
KDO
Cellular Free Cellular Free
+ 84" + 88" + 86"
0.81 0.81 0.81 0.81
3230 2904 3001 2899
796 752 801 780
781 730 777 710
610 602 600 588
f 85"
-
'Resulls are erprcsscd as nmol gI~cosc/mgof core-oligosaccharide. Abbreviations used: D-Glc = D-glucose; D-Gal D-galactose; D-GlcN ~ - ~ ~ I ~ o - ~ - ~ c o x ~ - DK- D~ O~ L = I 3-deoay-~-1~~n111lo-octu~osonic c o ~ c ; acid. bkr obtained by gel fillration on Scpliadcx G-50 column. All core-oligosaccharides had N-acetyl valucs of3.2-3.5%.
B) was found to be essentially KDO, whereas material of K,, 0.81 (peak A) was designated as LPS core-oligosaccharide. Essentially the same elution pattern was encountered with the analogous material for the free LPS of N. catarrhalis as well as for the products derived from the cellular and free LPS obtained from B. catarrhalis. Cl~aracterizationof Core-oligosaccl~aride Core-oligosaccharides from the cellular and free LPS of N. catarrlialis and B. catarrhalis possessed similar specific optical rotations (Table 1) and gave superimposable 13C nmr spectra (Fig. 2). All the core-oligosaccharides had essentially identical qualitative and quantitative compositions (Table I), being composed of D-glucose, D-galactose, 2-amino-2-deoxy-D-glucose, and 3deoxy-D-manrzo-octulosonic acid. The neutral glycoses present in each core-oligosaccharide were identified in hydrolyzed core
samples after preparative paper chromatographic separation (22). The fraction corresponding in paper chromatographic mobility with glucose (R,,, 0.87) on acetylation with pyridine - acetic anhydride mixture gave crystalline 1,2,3,4,6penta-0-acetyl-or-D-glucopyranose having mp 98" and mixture mp of 112-1 13 "C and [a], (c, 0.2 in chloroform), and on reduction (NaBH,) and acetylation afforded hexa-0-acetyl-D-glucitol, which on G L C (column A, 200 "C) gave a single peak (T,, 1.34) having the same retention time as an authentic specimen. A sample of t h e fraction was completely oxidized with D-glucose oxidase (EC 1.1.3.4) (ref. 17) t o yield D-gluconic acid. The fraction corresponding in paper chromatographic mobility with galactose (R,,, 0.74) on reduction (NaBH,) and acetylation afforded hexa-0-acetylgalactitol, which on GLC (column A, 200 "C) gave a single peak (TMA1.16) corre-
+
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TABLE 2. Analysis of lipid A from Bronhntnella catar-rhnlis and Neisseria catarrl~alis"
Organism
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B. cotnrrl~alis N . cafnrrltolis
Componentsa
% yield
Source of LPS
from LPS
Clo
CIZ
CIZ-30"
POL
EtON
D-GlcN
Cellular Free Cellular Free
50 49 47 48
8.94 10.44 10.14 11.91
8.74 12.97 9.02 12.21
27.90 32.53 32.44 28.22
9.74 15.70 9.63 15.50
3.13 1.55 3.23 1.52
15.70 18.90 17.01 17.58
-
OData are expressed as weight percentages o f components found. C L O= decanoic acid; C 1 2 = dodecanoic acid; CL2-.10,, = 3-hydroxy dodecanoic acid; ErON := ethanolamine; D-GlcN 2-amin0-2-deoxy-~-glucose. The acids were quantitatively analyzed by G L C (column C, 160°C) a s their methyl esters usin!: octadecanoic acid as a n internal standard.
sponding in retention time with an authentic sample. A sample of the galactose fraction was completely oxidized with D-galactose oxidase (EC 1.1.3.9) (ref. 10). The TMS derivatives of the methyl glycosides derived from the D-glucose and D-galactose fractions on GLC (column B, 160 "C) gave peaks corresponding in retention times and relative areas with the corresponding derivatives prepared from the authentic glycoses. Sealed-tube hydrolysis of the core-oligosaccharides with 4 N hydrochloric acid (1 2 h, I00 "C) released 2-amino-2-deoxy-D-glucose, which was obtained free from other glycoses by adsorption on and desorption from Rexyn 101 (H+) ionexchange resin. The amino glycose on paper chromatography gave a single spot corresponding in mobility with the authentic aminoglycose and was coeluted with 2-amino-2-deoxy-D-glucose on ion-exchange chromatography (34). Reduction and acetylation of the isolated amino glycose afforded penta-0-acetyl-2-acetamido-2-deoxy-Dglucitol having mp and mixture mp of 98-99 "C and [a], + 25" (c, 0.1 in chloroform), which on GLC (29) (column C, 230 "C) gave a single peak with T M A3.85 having the same retention time as an authentic derivative. 3-Deoxy-D-manno-octulosonicacid (KDO) isolated in low yield by hydrolysis with 0.02 N sulfuric acid (100 "C, I0 min) was identified by paper chromatography and by GLC of its TMS derivatives (33). KDO was similarly identified as the major component of the material having K , , 0.96 (peak B). Core-oligosaccharide samples (4 rng) reduced with sodium borohydride and subsequently hydrolysed on GLC analysis failed to indicate the presence of glucitol, galactitol, or 2-amino-2deoxyglucitol.
Characterization of Lipid A The lipid A content of the LPS preparations
together with their quantitative composition data are recorded in Table 2. The identification of 3-hydroxy dodecanoic acid, whose methyl ester derivative on GLC (column C , 160 "C) had a T,,, of 0.59, was confirmed by the fact that on trimethylsilylation it was completely converted to its TMS derivative having a T,,, of 0.20, identical with the value of an authentic derivative. The only glycose detected in the lipid A preparation was 2-amino-2-deoxy-D-glucose, which was characterized as described above. Ion-exchange chromatography revealed 2-amino-2deoxy-D-glucose and its 6-phosphate derivative to be present in admixture (ca. 3 : 1).
Discussion The two organisms designated as B. catarrhaIis and N . catarrhalis share the property of excretion of LPS-containing material, a process that occurs in all species of Neisseria examined in this laboratory (21, 22). Whereas the bacterium designated as N . catarrhalis elaborated slightly more LPS material than did B. catarrhalis, the specific LPS release was constant during growth, reiterating the observation on LPS-complex excretion by Neisseria species. The process, therefore, is a natural continuous one that occurs in the absence of apparent cellular autolysis. The mild acid fission of the cellular and free LPS proceeded as expected with the release of insoluble lipid A (ca. 47-50%) in yields characteristic of R-type LPS, and the concomitant production of core-oligosaccharide and free KDO. The lipid A preparations from cellular and free LPS of both N . catarrhalis and B. catarrhalis were qualitatively and quantitatively similar (Table 2), being composed of the three fatty acids decanoic acid, dodecanoic acid, 3-hydroxy dodecanoic acid together with 2-amino-2-deoxyD-glucose, phosphate, and ethanolamine. The
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JOHNSON ET AL.
lipid A preparations derived from the cellular LPS had a lower phosphate and higher ethanolamine content than the corresponding lipid A derived from free LPS. These lipid A preparations are unusual in that they do not contain 3-hydroxy tetradecanoic acid, which is an almost ubiquitous component of lipid A derived from LPS of other gram-negative bacteria, including all the LPS from "true Neisseria" species examined in our laboratory (21,22). The core-oligosaccharides, obtained from the cellular and free LPS produced by both N . catarrhalis and B. catarrhalis, were shown to be composed of D-glucose (4 mol), D-galactose (I mol), 2-amino-2-deoxy-D-glucose (I mol), and KDO (I mol) (Table 1). The calculated molecular weight of an oligosaccharide of the above composition is 1234, which agrees with the expected value (- 1230) calculated from the elution point of the four oligosaccharides on gel filtration through a calibrated Sephadex G-50 column. Examination of the hydrolysis products of the reduced (NaBH,) core-oligosaccharides gave a strong indication that they were terminated at the reducing end by KDO and the determined Nacetyl value, considered in conjunction with the characteristic 13C resonance of N-acetyl at 23.9 ppm in the13C nmr spectrum (Fig. 2) of each core, suggests that all the 2-amino-2-deoxy-D-glucose residues are present in their N-acetyl form. Analyses of the products of the mild acid hydrolysis of the cellular and free LPS preparations showed that all LPS were devoid of high molecular weight glycan, which could be termed 0-polysaccharide. This finding was not surprising, since such polysaccharide has only been found in the LPS of N. canis and N. subjaua (22) among the non-pathogenic species of Neisseria studied to date. The LPS of N . catarrhalis and B. catarrhalis are therefore of 'R' type rather than 'S' type. The core-oligosaccharides from the cellular and free LPS were identical with each other on the basis of their 13C nmr spectra, chemical composition, elution point on gel filtration, and specific optical rotations. Since no detectable difference exists between the core-oligosaccharides from B. catarrhalis and N. catarrhalis, the organism described as N . catarrhalis ATCC 8 176 is probably a strain of B. catarrhalis. The amount and essential identity of the lipid A moieties in the LPS preparations from N . catarrhalis and B. catarrhalis further point to their close identity in structure and composition.
465
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CAN. J. MlCROBllDL. VOL. 22, 1976
The determination of carbohydrates in biological maThe core-oligosaccharides of N. catarrhalis and terials by gas-liquid chromatography. Methods B. cntarrhnlis, now designated as core type V, Biochem. Anal. 19: 229-334. are quite distinct from that of core types I, 11,111, 9. Dusols, M.. K . A. GILLES,J. K . HAMILTON, P. A. and TV of non-pathogenic Neisseria (Table 3), and . Colorimetric method for REBERS,and F. S M I T H1956. the determination of sugars and related substances. is the first found to contain D-galactose, although Anal. Chem. 28: 350-356. this glycose is a component of the core oligosac10. FISCHER,W., and J. ZAPF. 1964. Quantitative Bescharides of N. gonorrlzoeae (28) (core type VI) timmung der Galactose mittels Galactoseoxydase aus and N. n~etiingitidis(20). Dactyliri~r~d~r~clroitles.Z. Physiol. Chem. 337: 186-i95. In all, B. catarrhalis (N. cntarrhalis) is exceedingly similar t o many members of the genus 11. Fox. R. H.. and D. E. MCCLAIN.1974. Evaluation of the 'taxonomic relationship of Micrococcirs cryoNeisserin in terms of morphology, oxidase replrihts, B~~er,~l~crrnellci ccritrrrlrcrlis, and Neisseriae by action, LPS structure, and excretion of LPS comparative polyacrylamide gel electrophoresis of complex. However, it differs from non-pathosoluble proteins. Int. J . Syst. Bacteriol. 24: 172-176. genic Neisserin on the basis of dissimilarities of 12. F o x , R. H., and D. E. MCCLAIN.1975. Enzyme electrophoretograms in the analysis of tnxon relatedness cell envelope protein^,^ differences in enzyme ctrrcirrhcrlis of Micrococcrrs oyophilrrs, Brcrr~l~tr~?zeller content (2,6, 1 I, 12, IG), lack of serological crossand atypical Neissericrs. J. Gen. Microbiol. 86: reactions of soluble antigen^,^ and of having 210-216. 13. GATT,R., and E. R. BERMEN. 1965. A rapid procedure inore exacting nutritional r e q ~ i r e m e n t s . The ~ for the estimation of amino sugars on a microscale. unique core-oligosaccharide of B. catnrrhalis Anal. Biochem. 15: 167-171. clearly differentiates it from N. caviae, a so-called 14. G U N N E RS., W . , J . K. N . JONES,and M. B. PERRY. "false Neisserin." These results, therefore, tend 1961. The gas-liquid partition chromatography of carbohydrate derivatives. Part I. The separation of to support the transfer of N. cntarrl~alisto the glycitol and glycose acetates. Can. J. Chem. 39: genus Brntzharnelln (5).
1892-1899. 15. HOLTEN. E. 1974. Immunological comparison of Acknowledgments NADP-dependent glutamate dehydrogenase and malate dehydrogenase in genus Neissericr. Acta Pathol. The authors thank Dr. I. C. P. Smith for nmr Microbiol. Scand. Sect. B,82: 849-859. analyses, A. Castagne for aminoglycose analyses, 16. H ~ L T E NE.,, and K . JYSSUM.1974. Activitiesof some enzymes concerning pyruvate metabolism in and D. Bilous, V. Daoust, R. Latta, and B. SinNrissericr . Acta Pathol. Microbiol. Scand. Sect. B,82: nott for excellent technical assistance. 843-848. 17. H O U G H ,L., and J . K . N. JONES. 1962. Enzymic I. A M I N O F FD. , 1961. Methods for the quantitative estimethods for the determination of D-glucose. III mation of N-acetylneuraminic acid and their applicaMethods in carbohydratechemistry. Vol. 1. Eiliiedby tion to hydrolysates of sialomucoids. Biochem. J. 81: R. L. Whistler and M. L. Wolfram. Academic Press, 384-392. N.Y. pp. 400-404. 2. B A U M A N N P... M. DOUDOROFF, and R. Y . STANIER. 18. J A N D AJ., . and E. WORK.1971. A colorimetricestima1968. Study of the Morer.rello group. 1. Genus tion of lipopolysaccharides. F E B S Lett. 16: 343-345. Murersello and the Nei.s.sericr cnterrrlrcrlis group. J. 19. J A N T Z E NE., , K . B R Y N T. , B E R G A Nand , K. B ~ V R E . Bacteriol. 95: 58-73. 1974. Gas chromatography of bacterial whole cell methanolysates. Acta Pathol. Microbiol. Scand. Sect. 3. BVVRE,K . 1967. Transformation and DNA base composition in taxonomy, with special reference to recent B, 82: 767-779. studies in Morcrxc~llerand Nei~serin.Acta Pathol. Mi- 20. J E N N I N G H. S . J . , G. B. HAWES,G. A. ADAMS,and C . crobiol. Scand. 69: 123-144. P. K E N N Y . 1973. The chemical composition and 4. BOWMAN,H. G . , and D. A. MONNER.1975. Characserological reactions of lipopolysaccharides from terization of lipopolysaccharides from K-12 mutants. serogroups A , B. X and Y Neis.se,ricr mc,tiirrgitidis. Can. J . Biochem. 51: 1347-1354. J. Bacteriol. 121: 455-464. 5. C A T L I NB. , W. 1970. Transfer of the organism named K . G., I. J. MCDONALD, M. B. PERRY,and 21. JOHNSON, Neisserirr ccric~rrlrcrlisto Brnrrl1n17z~llcr gen. nov. Int. J . R. R. B. RUSSELL.1975. Cellular and free lipopolysacSyst. Bacteriol. 20: 155-159. charides of some species of N~issericr. Can. J . MiM . Trans6. C A T L I NB. , W., and L. S . C U N N I N G H A1961. crobiol. 21: 1969-1980. forming activities and base contents of deoxyribonu- 22. JOHNSON, K . G., M. B. PERRY,and I. J. MCDONALD. cleate preparations from various Neissericr. J . Gen. 1976. Studies of the cellular a n d free lipopolysacMicrobiol. 26: 303-3 12. charides from Neisscricr corris and N . slrbjkrver. Can. J. and H. WARNER.1956. 7. C H E N ,P. S., T. Y . TORIBARA, Microbiol. 22: 189-196. Microdetermination of phosphorus. Anal. Chem. 28: 23. JOHNSON,K . G.. and M. B. PERRY.1976. Improved techniques for the preparation of bacterial lipo1756-1758. 8. CLAMP,J . R., T . BHATTI,and R. E. CHAMBERS. 1970. polysaccharides. Can. J . Microbiol. 22: 29-34.
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JOHNSON ET AL. D. T . , G. R. F A N N I N GK,. E . JOHNSON, 24. KINC;SBURY, and D. J . BRENNER.1969. Thermal stability of interspecies Neisseritr DNA duplexes. J . Gen. Microbiol. 55: 201-208. 25. LAMBERT, M . A , , D. G. HOLLIS.C . W. MOSS, R . E . WEAVER.and M . L . THOMAS.1971. Cellular fatty acids of nonpathogenic Nei.s.sericr. Can. J . Microbiol. 17: 1491-1502. 26. MCDONALD.I. J . , and K . G. JOHNSON.1975. Nutritional requirements of some non-pathogenic Neisseritr grown in simple synthetic medium. Can. J . Microbiol. 21: 1198-1204. 27. P E R R Y M. , B. 1964. The separation, determination and characterization of 2-amino-2-deoxy-D-glucose and 2-amino-2-deoxy-D-galactose in biological materials using gas-liquid partition chromatography. Can. J . Biochem. 42: 451-460. 28. PERRY,M . B., V. DAOUST, B. B. D I E N A ,F. E . ASHTON,and R . WALLACE.1975. The lipopolysaccharides of Neissericr gor~orrl~oeirc~ colony types 1 and 4. C a n . J. Biochem. 53: 67-4-629.
467
29. PERRY,M. B., and A. C . WEBB. 1968. Analysis of 2-amino-2-deoxyhexoses by gas-liq~~id partition chromatography. C a n . J . Biochem. 46: 1163-1 165. 30. RUSSELL.R . R. B., K. C . J O H N S O N ,and I. J . MCDONALD.1975. Envelope proteins in Neissericr. Can. J . Microbiol. 21: 1519-1534. . L . SECOND.1961. 31. VERON.M., P. T H I B A U L Tand Neisserio tnrrco.sn (Di~~1ococcrr.s t,~rrcosrrLingelsheim) 11.-Etude antigenique et classification. Ann. Inst. Pasteur. Paris, 100: 166-179. 32. W A N G , ~S.,. R. K. B c R N s , P. ~ ~ALAUPOVIC. ~ 1974. Isolation and composition of oligosaccharide cores strains. from endotoxins of two Serrcrli~I)I~I~(.'.T~.PIIs J. Bacteriol. 120: 990-993. 33. W I L L I A M SD., T . . and M. B. PERRY.1969. The s r r ~ ~ c ture, analysis and occurrence of 3-deoxy-~-t?itrt1r1ooctulosonic acid. Can. J. Biochem. 47: 691-695. 34. Y A G U C H M., I , and M . B. PERRY.1970. Ion-exchange chromatography of 2-amino-2-deoxyhexoses. Can. J . Biochem. 48: 386-388.
Dil.i.~iotrof B i ~ l ~ g i cSciet~ces, oI Nrrtiotinl Reserrrch Corrtzcil qfCrrt1rrrln, O t t r r ~ ~ jCnnrrdcr n, K I A OR6
Accepted December 23, 1975 JOHNSON,K . G., I. J. MCDONALD,and M. B. PERRY.1976. Studies on the cellular and free cotcrrrhcrlis. Can. J. Microbiol. 22: 460-467. lipopolysaccharides from Brcrrrl~ar~~elln Cellular and fi-ee lipopolysaccharides obtained from Neisseria crrtnrrlrnlis and Brnnl~nmeNu crrtcrrrlrrrlis were found to be essentially identical. Both cellular and free lipopolysaccharides contained core-oligosaccharides of the following composition: D-glucoSe (4 mol), D-galactose (I mol), 2-amino-2-deoxy-D-glucose (1 mol), and 3-deoxy-D-mcrnno-octulosonic acid. Aldoheptose and phosphate components were below levels of detection. Several physical methods indicated that all core-oligosaccharide preparations were identical. Lipid A preparations from cellular and free lipopolysaccharides of both organisms were qualitatively and quantitatively similar: they were composed of decanoic acid, dodecanoic acid, 3-hydrony dodecanoic acid, 2-amino-2-deoxy-D-glucose, phosphate, and ethanolamine. The results tend to justify the transfer of Neisserin crrtnrrhalis to the genus Brcrr~hcrmelln. JOHNSON,K. G., I. J . MCDONALDe t M. B. PERRY. 1976. Studies on the cellular and free lipopolysaccharides from Brnnltcrmc~llncc~tnrrlicrlis.Can. J. Microbiol. 22: 4 6 W 6 7 . Les lipopolysaccharides cellulaires e t libres obtenus de Neisserin c.ntorr/~crliset Brcrni~crrn~llcr cntnrrhcrlis sont essentiellement identiques. Les elements fondamentaux des oligosaccharides de ces lipopolysaccharides, cellulaires et libres, contiennent du: D-glucose (4 mol), D-galactose (1 mol). 2-amino-2-desoxy-D-glucose (1 mol) et I'acide 3-desoxy-D-manno-octulosonique. Aucun constituants aldoheptose e t phosphate n'ont pu ktre dCcelC. Les resultats de plusieurs methodes d'analyses physiques indiquent que toutes les preparations des oligosaccharides d e base sont identiques. I.es preparations du lipide A obtenues des lipopolysaccharides cellulaires e t libres des deux organismes sont semblables quantitativement et qualitativement; ils contiennent d e I'acide decanoi'que, de I'acide d~decanoi'que,de I'acide 3-hydroxy-dodecano~que, du 2-amino-3desoxy-D glucose, du phosphate et de I'ethanolamine. Les resultats obtenus demontrent que I'on pourrait transferer Neissericr cntorrholis a u genre Brcrnhnmc~lln. [Traduit par lejournal]
Introduction Several methods, includingserology (3 I), transformation experiments (3, 6), measurement of deoxyribonuc~eicacid (DNA) base composition and interspecific DNA homologies (6, 24), metabolic activity tests and enzyme profiles (2, 15, 16), electrophoretic examination of soluble proteins (11, 12), and analysis of fatty acids (19, 25) have been used in the elucidation of taxonomic relationships in the genus Neisseria. Accumulated data from such studies have permitted division of the genus into the u t r u e ~ ~ i and~"false ~ ~ ~ ~h~ i former ~ ~groupcontains ~ ~ thei patho~ gens N. gonorrhoeae and N. merlirlgitidisas well as the non-pathogenic organisms, which have been dFsignatedas N. canis, N. cinerea, N. cuniCuli, N. jaua, N . lactamjca, N. mucosa, N . perjava,N. sicca, and N. s u b ~ a v a l-he . species of uncertain taxonomic status consist of 'Received November 13, 1975. 'NRCC No. 15196.
N. caviae, N. catarrhalis, and N. ovis. Of these species, N. catarrhalis has recently been redesig"ated as the sole representative of the created genus Branhamella (5) and has been as"gned the name B. various non-path0genic Neisseria in this laboratory has aided species differentiation On the basis growth requirements (26)y lip0pOlysaccharide (LPS) core-oligosacchan'de of envelope proteins (30), (21, 22), and cross In common with observations ~ ~ i ~ of- differing LPS core-oligo.saccharide ~ ~types in the genera Escherichia (4) and Serratia (32), LPS from species of Neisseria have been demonstrated to contain at least four core-oligosaccharide types (21, 22, 23). Core-oligosaccharide types I to 'V inclusive occur in the LPS of species designated as "true Neisseria." The present study was undertaken to examine LPS core-oligosaccharide com3Russell, Johnson, and McDonald. T o be published.
46 1
3N ET AL.
position and structure of the cellular and free LPS from the "false Neisseria" N. catarrhalis, now designated as B. catarrhalis.
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Materials and Methods Organisms and Growth Conditions The following bacteria were used (American Type Culture Collection (ATCC) numbers are given in parentheses): Neisseria catarrhalis, National Research Council of Canada (NRCC) 31002 (8176), and Branhamella catarrhalis, NRCC 59001 (25238). The organisms were grown in trypticase soy broth (Baltimore Biological Laboratories) supplemented with 0.5% (w/v) yeast extract (Difco) (TCSY). The organisms were maintained as frozen (liquid nitrogen) stock cultures in the NRCC collection. Working stock cultures were routinely maintained as 10fold concentrated bacterial suspensions in 10% (w/v) aqueous glycerol at - 40 "C. Cultures used for the production of cellular and free LPS were grown in TCSY medium. A 75-litre fermentor containing 50 litres of medium was inoculated with 5 litres of late logarithmic phase culture. Agitation was maintained at 175 rpm and aeration at 8 litres/min during growth at 37 "C. Near the end of the exponential growth phase, cells were harvested by centrifugation. Cells were immediately lyophilized and the culture supernatant was placed at 4" until further processed. Preparation and Partial Fission of LPS Cellular LPS was obtained using the lysozyme-phenol extraction technique (23). Free LPS was prepared in purified form by the previously described sequence involving batch adsorption on diethylaminoethyl-cell~~lose, then ethanol precipitation of desorbed material followed by its aqueous phenol extraction and final collection by ultracentrifugation (21). Release of water-insoluble lipid A and water-soluble core-oligosaccharide was achieved by dilute acetic acid hydrolysis of intact LPS (21). Core-oligosaccharide preparations were fractionated by filtration through a Sephadex (3-50 column (V, = 490 ml, Vo = 160 ml) using 47 mM pyridinium acetate, pH 4.26, as eluant. Gel filtration properties are expressed in terms of the distribution coefficient, K,, = (V, - Vo)(V, - Vo), where V, is the elution volume of a specific material, Vo is the void volume of the system, and V, is the total volume of the system. Analytical Techniques Total LPS was estimated by the carbocyanine dye assay (18) and total hexose was determined by the phenolsulfuric colorimetric procedure (9). Amino glycoses were estimated calorimetrically (13), by ion-exchange chromatography using a Beckman 121 amino acid analyzer (34) and by gas-liquid chromatography (GLC) (27, 29). D-Glucose and D-galactose were determined by G L C of the trimethylsilyl (TMS) derivatives of their methyl glycosides (8) and of their glycitol acetate derivatives (14). GLC was performed as previously described (22) using the following columns: (A) 3% (w/w) ECNSS-M on 100-120 mesh Gas-Chrom Q, (B)3% (w/w) SE-30 on 80-100 mesh Chromosorb W, and (C) 10% (w/w) neopentylglycol succinate polyester on 80- to 100-mesh Chromosorb W. Retention times are quoted relative t o
mannitol hexacetate (TMa). Analytical and preparative paper chromatography was performed as previously described (22) using pyridine -ethyl acetate - water (2: 5: 5 v/v, top layer) as the mobile phase, and mobilities are quoted relative to D-mannose (R,,,). The periodate - thiobarbituric acid method (1) and G L C (33) were used for the determination of 3-deoxy-Dmarzno-octulosonic acid (KDO). Phosphorus was determined by a micro method (7) and fatty acids were identified and determined by GLC of their methyl esters (22), their retention times being quoted relative to methyl octadecanoate ( T c l s ) . Specific Optical Rotation Optical rotations were determined at 20 "C using a Perkin-Elmer model 141 polarimeter. 13C Nuclear. Magnetic Resonance (izmr) The 13C nmr spectra were obtained using a Varian XL-100 (25 MHz) spectrometer (sample tubes 12 mm outside diameter) in the pulsed Fourier transform mode with complete proton decoupling. Chemical shifts were recorded in parts per million downfield from external tetramethylsilane and deuterium oxide 'H was used f o r field frequency lock,
Results Excretion of LPS Both N. catarrhalis and B. catarrhalis excreted LPS-containingmaterial into the growth medium. Specific LPS release, which was defined as micrograms free LPS per milligram bacterial dry weight was 49.6 for N. catarrhalis and 43 for B. catarrhalis. This parameter was constant for both organisms during the growth cycle. Characterization of LPS Lipopolysaccharides were designated pure according to the following criteria: (a) elution as a single symmetrical peak at the V, of Sepharose 6B gel filtration systems, (6) absence of detectable ribose on hydrolysis, (c) lack of absorption maximum in the 210- to 300-nm region of the ultraviolet spectra, (d) freedom from significant amino acid contamination (less than 2 nmol of the most abundant amino acid per milligram of preparation), and (e) the presence of discrete core-oligosaccharide fragments derived from the mild acetic acid hydrolysis of intact LPS preparations. Sephadex G-50 gel filtration of the watersoluble glycan fraction obtained from the mild acid fission of each LPS revealed the presence of only two components, which had K,, values of 0.81 and 0.96. The elution profile of the glycan material obtained from the cellular LPS of N. catarrhalis, which is typical of the profiles obtained from the other LPS products, is shown in Fig. I. Material having a K,, value of 0.96 (peak
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C A N . J . MICROBIOL. VOL. 22, 1976
FRACTION NO
FIG. 1. Fractionation of core-oligosaccharide obtained from the cellular LPS of N. catnrrknlis on Sephadex (3-50 sing 47 n1M pyridiniurn acetate, pH 4.26, as eluant. A l i q ~ ~ o (100 t s pl) were withdrawn and analyzed for aldoglycose (9) -, and for aminoglycose (13) - - -. Optical density was determined a t 490 nm.
TABLE 1. Properties and composition of core-oligosaccharides from Brnrllinrnelln cotarrhalis and Neisserio cninrrlmlis"
B. cntnrrlinlis N. cninrrhnlis
-
ID
S o ~ ~ r of ce LPS
(water)
K b
D-GIC
D-Gal
D-GlcN
KDO
Cellular Free Cellular Free
+ 84" + 88" + 86"
0.81 0.81 0.81 0.81
3230 2904 3001 2899
796 752 801 780
781 730 777 710
610 602 600 588
f 85"
-
'Resulls are erprcsscd as nmol gI~cosc/mgof core-oligosaccharide. Abbreviations used: D-Glc = D-glucose; D-Gal D-galactose; D-GlcN ~ - ~ ~ I ~ o - ~ - ~ c o x ~ - DK- D~ O~ L = I 3-deoay-~-1~~n111lo-octu~osonic c o ~ c ; acid. bkr obtained by gel fillration on Scpliadcx G-50 column. All core-oligosaccharides had N-acetyl valucs of3.2-3.5%.
B) was found to be essentially KDO, whereas material of K,, 0.81 (peak A) was designated as LPS core-oligosaccharide. Essentially the same elution pattern was encountered with the analogous material for the free LPS of N. catarrhalis as well as for the products derived from the cellular and free LPS obtained from B. catarrhalis. Cl~aracterizationof Core-oligosaccl~aride Core-oligosaccharides from the cellular and free LPS of N. catarrlialis and B. catarrhalis possessed similar specific optical rotations (Table 1) and gave superimposable 13C nmr spectra (Fig. 2). All the core-oligosaccharides had essentially identical qualitative and quantitative compositions (Table I), being composed of D-glucose, D-galactose, 2-amino-2-deoxy-D-glucose, and 3deoxy-D-manrzo-octulosonic acid. The neutral glycoses present in each core-oligosaccharide were identified in hydrolyzed core
samples after preparative paper chromatographic separation (22). The fraction corresponding in paper chromatographic mobility with glucose (R,,, 0.87) on acetylation with pyridine - acetic anhydride mixture gave crystalline 1,2,3,4,6penta-0-acetyl-or-D-glucopyranose having mp 98" and mixture mp of 112-1 13 "C and [a], (c, 0.2 in chloroform), and on reduction (NaBH,) and acetylation afforded hexa-0-acetyl-D-glucitol, which on G L C (column A, 200 "C) gave a single peak (T,, 1.34) having the same retention time as an authentic specimen. A sample of t h e fraction was completely oxidized with D-glucose oxidase (EC 1.1.3.4) (ref. 17) t o yield D-gluconic acid. The fraction corresponding in paper chromatographic mobility with galactose (R,,, 0.74) on reduction (NaBH,) and acetylation afforded hexa-0-acetylgalactitol, which on GLC (column A, 200 "C) gave a single peak (TMA1.16) corre-
+
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TABLE 2. Analysis of lipid A from Bronhntnella catar-rhnlis and Neisseria catarrl~alis"
Organism
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B. cotnrrl~alis N . cafnrrltolis
Componentsa
% yield
Source of LPS
from LPS
Clo
CIZ
CIZ-30"
POL
EtON
D-GlcN
Cellular Free Cellular Free
50 49 47 48
8.94 10.44 10.14 11.91
8.74 12.97 9.02 12.21
27.90 32.53 32.44 28.22
9.74 15.70 9.63 15.50
3.13 1.55 3.23 1.52
15.70 18.90 17.01 17.58
-
OData are expressed as weight percentages o f components found. C L O= decanoic acid; C 1 2 = dodecanoic acid; CL2-.10,, = 3-hydroxy dodecanoic acid; ErON := ethanolamine; D-GlcN 2-amin0-2-deoxy-~-glucose. The acids were quantitatively analyzed by G L C (column C, 160°C) a s their methyl esters usin!: octadecanoic acid as a n internal standard.
sponding in retention time with an authentic sample. A sample of the galactose fraction was completely oxidized with D-galactose oxidase (EC 1.1.3.9) (ref. 10). The TMS derivatives of the methyl glycosides derived from the D-glucose and D-galactose fractions on GLC (column B, 160 "C) gave peaks corresponding in retention times and relative areas with the corresponding derivatives prepared from the authentic glycoses. Sealed-tube hydrolysis of the core-oligosaccharides with 4 N hydrochloric acid (1 2 h, I00 "C) released 2-amino-2-deoxy-D-glucose, which was obtained free from other glycoses by adsorption on and desorption from Rexyn 101 (H+) ionexchange resin. The amino glycose on paper chromatography gave a single spot corresponding in mobility with the authentic aminoglycose and was coeluted with 2-amino-2-deoxy-D-glucose on ion-exchange chromatography (34). Reduction and acetylation of the isolated amino glycose afforded penta-0-acetyl-2-acetamido-2-deoxy-Dglucitol having mp and mixture mp of 98-99 "C and [a], + 25" (c, 0.1 in chloroform), which on GLC (29) (column C, 230 "C) gave a single peak with T M A3.85 having the same retention time as an authentic derivative. 3-Deoxy-D-manno-octulosonicacid (KDO) isolated in low yield by hydrolysis with 0.02 N sulfuric acid (100 "C, I0 min) was identified by paper chromatography and by GLC of its TMS derivatives (33). KDO was similarly identified as the major component of the material having K , , 0.96 (peak B). Core-oligosaccharide samples (4 rng) reduced with sodium borohydride and subsequently hydrolysed on GLC analysis failed to indicate the presence of glucitol, galactitol, or 2-amino-2deoxyglucitol.
Characterization of Lipid A The lipid A content of the LPS preparations
together with their quantitative composition data are recorded in Table 2. The identification of 3-hydroxy dodecanoic acid, whose methyl ester derivative on GLC (column C , 160 "C) had a T,,, of 0.59, was confirmed by the fact that on trimethylsilylation it was completely converted to its TMS derivative having a T,,, of 0.20, identical with the value of an authentic derivative. The only glycose detected in the lipid A preparation was 2-amino-2-deoxy-D-glucose, which was characterized as described above. Ion-exchange chromatography revealed 2-amino-2deoxy-D-glucose and its 6-phosphate derivative to be present in admixture (ca. 3 : 1).
Discussion The two organisms designated as B. catarrhaIis and N . catarrhalis share the property of excretion of LPS-containing material, a process that occurs in all species of Neisseria examined in this laboratory (21, 22). Whereas the bacterium designated as N . catarrhalis elaborated slightly more LPS material than did B. catarrhalis, the specific LPS release was constant during growth, reiterating the observation on LPS-complex excretion by Neisseria species. The process, therefore, is a natural continuous one that occurs in the absence of apparent cellular autolysis. The mild acid fission of the cellular and free LPS proceeded as expected with the release of insoluble lipid A (ca. 47-50%) in yields characteristic of R-type LPS, and the concomitant production of core-oligosaccharide and free KDO. The lipid A preparations from cellular and free LPS of both N . catarrhalis and B. catarrhalis were qualitatively and quantitatively similar (Table 2), being composed of the three fatty acids decanoic acid, dodecanoic acid, 3-hydroxy dodecanoic acid together with 2-amino-2-deoxyD-glucose, phosphate, and ethanolamine. The
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JOHNSON ET AL.
lipid A preparations derived from the cellular LPS had a lower phosphate and higher ethanolamine content than the corresponding lipid A derived from free LPS. These lipid A preparations are unusual in that they do not contain 3-hydroxy tetradecanoic acid, which is an almost ubiquitous component of lipid A derived from LPS of other gram-negative bacteria, including all the LPS from "true Neisseria" species examined in our laboratory (21,22). The core-oligosaccharides, obtained from the cellular and free LPS produced by both N . catarrhalis and B. catarrhalis, were shown to be composed of D-glucose (4 mol), D-galactose (I mol), 2-amino-2-deoxy-D-glucose (I mol), and KDO (I mol) (Table 1). The calculated molecular weight of an oligosaccharide of the above composition is 1234, which agrees with the expected value (- 1230) calculated from the elution point of the four oligosaccharides on gel filtration through a calibrated Sephadex G-50 column. Examination of the hydrolysis products of the reduced (NaBH,) core-oligosaccharides gave a strong indication that they were terminated at the reducing end by KDO and the determined Nacetyl value, considered in conjunction with the characteristic 13C resonance of N-acetyl at 23.9 ppm in the13C nmr spectrum (Fig. 2) of each core, suggests that all the 2-amino-2-deoxy-D-glucose residues are present in their N-acetyl form. Analyses of the products of the mild acid hydrolysis of the cellular and free LPS preparations showed that all LPS were devoid of high molecular weight glycan, which could be termed 0-polysaccharide. This finding was not surprising, since such polysaccharide has only been found in the LPS of N. canis and N. subjaua (22) among the non-pathogenic species of Neisseria studied to date. The LPS of N . catarrhalis and B. catarrhalis are therefore of 'R' type rather than 'S' type. The core-oligosaccharides from the cellular and free LPS were identical with each other on the basis of their 13C nmr spectra, chemical composition, elution point on gel filtration, and specific optical rotations. Since no detectable difference exists between the core-oligosaccharides from B. catarrhalis and N. catarrhalis, the organism described as N . catarrhalis ATCC 8 176 is probably a strain of B. catarrhalis. The amount and essential identity of the lipid A moieties in the LPS preparations from N . catarrhalis and B. catarrhalis further point to their close identity in structure and composition.
465
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CAN. J. MlCROBllDL. VOL. 22, 1976
The determination of carbohydrates in biological maThe core-oligosaccharides of N. catarrhalis and terials by gas-liquid chromatography. Methods B. cntarrhnlis, now designated as core type V, Biochem. Anal. 19: 229-334. are quite distinct from that of core types I, 11,111, 9. Dusols, M.. K . A. GILLES,J. K . HAMILTON, P. A. and TV of non-pathogenic Neisseria (Table 3), and . Colorimetric method for REBERS,and F. S M I T H1956. the determination of sugars and related substances. is the first found to contain D-galactose, although Anal. Chem. 28: 350-356. this glycose is a component of the core oligosac10. FISCHER,W., and J. ZAPF. 1964. Quantitative Bescharides of N. gonorrlzoeae (28) (core type VI) timmung der Galactose mittels Galactoseoxydase aus and N. n~etiingitidis(20). Dactyliri~r~d~r~clroitles.Z. Physiol. Chem. 337: 186-i95. In all, B. catarrhalis (N. cntarrhalis) is exceedingly similar t o many members of the genus 11. Fox. R. H.. and D. E. MCCLAIN.1974. Evaluation of the 'taxonomic relationship of Micrococcirs cryoNeisserin in terms of morphology, oxidase replrihts, B~~er,~l~crrnellci ccritrrrlrcrlis, and Neisseriae by action, LPS structure, and excretion of LPS comparative polyacrylamide gel electrophoresis of complex. However, it differs from non-pathosoluble proteins. Int. J . Syst. Bacteriol. 24: 172-176. genic Neisserin on the basis of dissimilarities of 12. F o x , R. H., and D. E. MCCLAIN.1975. Enzyme electrophoretograms in the analysis of tnxon relatedness cell envelope protein^,^ differences in enzyme ctrrcirrhcrlis of Micrococcrrs oyophilrrs, Brcrr~l~tr~?zeller content (2,6, 1 I, 12, IG), lack of serological crossand atypical Neissericrs. J. Gen. Microbiol. 86: reactions of soluble antigen^,^ and of having 210-216. 13. GATT,R., and E. R. BERMEN. 1965. A rapid procedure inore exacting nutritional r e q ~ i r e m e n t s . The ~ for the estimation of amino sugars on a microscale. unique core-oligosaccharide of B. catnrrhalis Anal. Biochem. 15: 167-171. clearly differentiates it from N. caviae, a so-called 14. G U N N E RS., W . , J . K. N . JONES,and M. B. PERRY. "false Neisserin." These results, therefore, tend 1961. The gas-liquid partition chromatography of carbohydrate derivatives. Part I. The separation of to support the transfer of N. cntarrl~alisto the glycitol and glycose acetates. Can. J. Chem. 39: genus Brntzharnelln (5).
1892-1899. 15. HOLTEN. E. 1974. Immunological comparison of Acknowledgments NADP-dependent glutamate dehydrogenase and malate dehydrogenase in genus Neissericr. Acta Pathol. The authors thank Dr. I. C. P. Smith for nmr Microbiol. Scand. Sect. B,82: 849-859. analyses, A. Castagne for aminoglycose analyses, 16. H ~ L T E NE.,, and K . JYSSUM.1974. Activitiesof some enzymes concerning pyruvate metabolism in and D. Bilous, V. Daoust, R. Latta, and B. SinNrissericr . Acta Pathol. Microbiol. Scand. Sect. B,82: nott for excellent technical assistance. 843-848. 17. H O U G H ,L., and J . K . N. JONES. 1962. Enzymic I. A M I N O F FD. , 1961. Methods for the quantitative estimethods for the determination of D-glucose. III mation of N-acetylneuraminic acid and their applicaMethods in carbohydratechemistry. Vol. 1. Eiliiedby tion to hydrolysates of sialomucoids. Biochem. J. 81: R. L. Whistler and M. L. Wolfram. Academic Press, 384-392. N.Y. pp. 400-404. 2. B A U M A N N P... M. DOUDOROFF, and R. Y . STANIER. 18. J A N D AJ., . and E. WORK.1971. A colorimetricestima1968. Study of the Morer.rello group. 1. Genus tion of lipopolysaccharides. F E B S Lett. 16: 343-345. Murersello and the Nei.s.sericr cnterrrlrcrlis group. J. 19. J A N T Z E NE., , K . B R Y N T. , B E R G A Nand , K. B ~ V R E . Bacteriol. 95: 58-73. 1974. Gas chromatography of bacterial whole cell methanolysates. Acta Pathol. Microbiol. Scand. Sect. 3. BVVRE,K . 1967. Transformation and DNA base composition in taxonomy, with special reference to recent B, 82: 767-779. studies in Morcrxc~llerand Nei~serin.Acta Pathol. Mi- 20. J E N N I N G H. S . J . , G. B. HAWES,G. A. ADAMS,and C . crobiol. Scand. 69: 123-144. P. K E N N Y . 1973. The chemical composition and 4. BOWMAN,H. G . , and D. A. MONNER.1975. Characserological reactions of lipopolysaccharides from terization of lipopolysaccharides from K-12 mutants. serogroups A , B. X and Y Neis.se,ricr mc,tiirrgitidis. Can. J . Biochem. 51: 1347-1354. J. Bacteriol. 121: 455-464. 5. C A T L I NB. , W. 1970. Transfer of the organism named K . G., I. J. MCDONALD, M. B. PERRY,and 21. JOHNSON, Neisserirr ccric~rrlrcrlisto Brnrrl1n17z~llcr gen. nov. Int. J . R. R. B. RUSSELL.1975. Cellular and free lipopolysacSyst. Bacteriol. 20: 155-159. charides of some species of N~issericr. Can. J . MiM . Trans6. C A T L I NB. , W., and L. S . C U N N I N G H A1961. crobiol. 21: 1969-1980. forming activities and base contents of deoxyribonu- 22. JOHNSON, K . G., M. B. PERRY,and I. J. MCDONALD. cleate preparations from various Neissericr. J . Gen. 1976. Studies of the cellular a n d free lipopolysacMicrobiol. 26: 303-3 12. charides from Neisscricr corris and N . slrbjkrver. Can. J. and H. WARNER.1956. 7. C H E N ,P. S., T. Y . TORIBARA, Microbiol. 22: 189-196. Microdetermination of phosphorus. Anal. Chem. 28: 23. JOHNSON,K . G.. and M. B. PERRY.1976. Improved techniques for the preparation of bacterial lipo1756-1758. 8. CLAMP,J . R., T . BHATTI,and R. E. CHAMBERS. 1970. polysaccharides. Can. J . Microbiol. 22: 29-34.
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JOHNSON ET AL. D. T . , G. R. F A N N I N GK,. E . JOHNSON, 24. KINC;SBURY, and D. J . BRENNER.1969. Thermal stability of interspecies Neisseritr DNA duplexes. J . Gen. Microbiol. 55: 201-208. 25. LAMBERT, M . A , , D. G. HOLLIS.C . W. MOSS, R . E . WEAVER.and M . L . THOMAS.1971. Cellular fatty acids of nonpathogenic Nei.s.sericr. Can. J . Microbiol. 17: 1491-1502. 26. MCDONALD.I. J . , and K . G. JOHNSON.1975. Nutritional requirements of some non-pathogenic Neisseritr grown in simple synthetic medium. Can. J . Microbiol. 21: 1198-1204. 27. P E R R Y M. , B. 1964. The separation, determination and characterization of 2-amino-2-deoxy-D-glucose and 2-amino-2-deoxy-D-galactose in biological materials using gas-liquid partition chromatography. Can. J . Biochem. 42: 451-460. 28. PERRY,M . B., V. DAOUST, B. B. D I E N A ,F. E . ASHTON,and R . WALLACE.1975. The lipopolysaccharides of Neissericr gor~orrl~oeirc~ colony types 1 and 4. C a n . J. Biochem. 53: 67-4-629.
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29. PERRY,M. B., and A. C . WEBB. 1968. Analysis of 2-amino-2-deoxyhexoses by gas-liq~~id partition chromatography. C a n . J . Biochem. 46: 1163-1 165. 30. RUSSELL.R . R. B., K. C . J O H N S O N ,and I. J . MCDONALD.1975. Envelope proteins in Neissericr. Can. J . Microbiol. 21: 1519-1534. . L . SECOND.1961. 31. VERON.M., P. T H I B A U L Tand Neisserio tnrrco.sn (Di~~1ococcrr.s t,~rrcosrrLingelsheim) 11.-Etude antigenique et classification. Ann. Inst. Pasteur. Paris, 100: 166-179. 32. W A N G , ~S.,. R. K. B c R N s , P. ~ ~ALAUPOVIC. ~ 1974. Isolation and composition of oligosaccharide cores strains. from endotoxins of two Serrcrli~I)I~I~(.'.T~.PIIs J. Bacteriol. 120: 990-993. 33. W I L L I A M SD., T . . and M. B. PERRY.1969. The s r r ~ ~ c ture, analysis and occurrence of 3-deoxy-~-t?itrt1r1ooctulosonic acid. Can. J. Biochem. 47: 691-695. 34. Y A G U C H M., I , and M . B. PERRY.1970. Ion-exchange chromatography of 2-amino-2-deoxyhexoses. Can. J . Biochem. 48: 386-388.
