Báo cáo khoa học: A novel mannitol teichoic acid with side phosphate groups ofBrevibacterium sp.VKM Ac-2118

doi:10.1046/j.1432-1033.2003.03832.x

Eur. J. Biochem. 270, 4420–4425 (2003) (cid:2) FEBS 2003

A novel mannitol teichoic acid with side phosphate groups of Brevibacteriumsp.VKM Ac-2118

Natalia V. Potekhina1, Alexander S. Shashkov2, Lyudmila I. Evtushenko3, Ekaterina Yu. Gavrish3, Sofya N. Senchenkova2, Andrey A. Stomakhin4, Anatolii I. Usov2, Irina B. Naumova1,* and Erko Stackebrandt5 1School of Biology, M. V. Lomonosov Moscow State University, Russia; 2N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia; 3Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region, Russia; 4B. A. Engelhardt Institute of Molecular Biology, Russian Academy of Science, Moscow, Russia; 5DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany

O-4(3). The structure of the polymer was established by chemical methods, NMR spectroscopy, and MALDI-TOF mass spectrometry.

Keywords: teichoic acids; poly(mannitol phosphate); side phosphate groups; NMR; Brevibacterium.

The cell wall of Brevibacterium sp. VKM Ac-2118 isolated from a frozen (mean annual temperature )12 (cid:1)C) late Plio- cene layer, 1.8–3 Myr, Kolyma lowland, Russia, contains mannitol teichoic acid with a previously unknown structure. This is 1,6-poly(mannitol phosphate) with the majority of the mannitol residues bearing side phosphate groups at

Structural variants of teichoic acids, the anionic polymers of the cell walls of Gram-positive bacteria, are extremely numerous because they differ in the polyol and sugar (or amino sugar) composition, the types of phosphodiester bonds, configurations of the glycosidic bonds, the types of bonds between the monosaccharide components in oligo- saccharide units, and O-acyl substituents.

different species of the genus Brevibacterium. Some strains of B. linens and B. epidermidis contain unsubstituted and partially substituted poly(mannitol phosphate) chains (with monosaccharides as the substituents) together with poly(gly- cerol phosphate) chains [9,10]. The cell wall of B. iodinum was shown to contain poly(mannitol phosphate) chain with most of the mannitol residues acylated at positions 4 and 5 by pyruvic acid. In addition, about half of the mannitol residues bear a-glycopyranosyl residues at O-2 [11].

In the present work, we report the elucidation of the structure of a cell wall component of Brevibacterium VKM Ac-2118, which was found to be a new variant of mannitol teichoic acids.

Materials and methods

Among the cell wall teichoic acids studied so far, four structural types can be distinguished depending on the composition of the main chain: poly(polyol phosphates) (I), poly(glycosylpolyol phosphates) (II), poly(polyol phos- phate–glycosyl phosphates) (III) and poly(polyol phos- phate–glycosylpolyol phosphates) (IV) [1]. The polymers of types I and II are the most abundant cell wall teichoic acids. Poly(polyol phosphate) chains containing glycerol, erythr- itol, ribitol, arabinitol, and mannitol have been identified [1]. The interest that has arisen during recent years in these polymers stems from their taxonomic significance for Gram-positive bacteria, especially actinomycetes. The spe- cies-specificity of teichoic acids has been demonstrated for the genera Nocardiopsis [1], Glycomyces [2,3], Nocardioides [4,5] and Actinomadura [6–8]. Presumably, the structures of cell wall teichoic acids can be used as an additional chemotaxonomic marker for the attribution of new species of Gram-positive bacteria to the Brevibacterium genus. Poly(mannitol phosphate) teichoic acids were found in

The strain VKM Ac-2118 was isolated from a sample of permafrost sediments, 48.8 m deep, recovered from a frozen (mean annual temperature )12 (cid:1)C) late Pliocene layer, 1.8– 3 Myr, Kolyma lowland, Russia. Samples were obtained as described by Shi et al. [12] and kept frozen at )20 (cid:1)C before studying phenotypical study. The methods used for characteristics were described previously [13]. The 16S rRNA gene was amplified by PCR using prokaryotic 16S rDNA universal primers and purified as described [13]. 16S rDNA was sequenced using a Big Dye Terminator Kit (Perkin Elmer) with a model ABI-310 automatic DNA Sequencer (Perkin Elmer) according to the manufacturer’s protocol. Nucleotide substitution rates were calculated as described [14] and the phylogenetic tree was constructed by the neighbor-joining method [15] with CLUSTALW software [16]. Three topologies were evaluated by bootstrap analysis of the sequence data with the same software.

To obtain cell wall, the culture of Brevibacterium sp. VKM Ac)2118 was grown on a pepton/yeast medium [17] for 12–18 h on a shaker at 28 (cid:1)C. The biomass was collected

Correspondence to N. V. Potekhina, School of Biology, M. V., Lomonosov Moscow State University, 119992 Moscow, Russia. Fax: + 7 095 9394309, E-mail: potekchina@hotbox.ru Abbreviation: PME, phosphomonoesterase. Enzyme: phosphomonoesterase (EC 3.1.3.1). *Deceased suddenly on 18 August 2003. (Received 14 April 2003, revised 1 August 2003, accepted 12 September 2003)

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reflection mode using a KOMPACT MALDI 4 (Kratos Analytical) mass spectrometer. 2,5-Dihydroxybenzoic (gen- tisic) acid was used as a matrix.

at the logarithmic growth phase. The cells were harvested by centrifugation, washed with 0.95% NaCl and cell walls were obtained as described [18]. The cell wall preparation was heated in 2% SDS for 5 min at 100 (cid:1)C, washed several times with water, and freeze-dried.

Results and discussion

(two volumes)

The teichoic acid was extracted with 10% trichloroace- tic acid at 4 (cid:1)C for 24 h. The mixture was centrifuged and the cell wall was repeatedly treated with 10% trichloro- acetic acid under the same conditions. The supernatants were combined, dialysed against distilled water, and freeze-dried to yield a crude preparation. Fractional precipitation of teichoic acids was carried out by the addition of ethanol to the combined supernatant and the precipitate that formed was removed by centrifugation for 24 h at 0 (cid:1)C, 10 000 g, 15 min. To the supernatant, more ethanol (two volumes) was added and the precipitate was collected by centrifugation (as above). This was redissolved in water, centrifuged, the supernatant was dialysed and freeze-dried. Thus, two follows: teichoic acid preparations were isolated as teichoic acid precipitated with two volumes of ethanol (preparation I) and teichoic acid precipitated with four volumes of ethanol (preparation II).

The strain under study had growth, morphological, and chemotaxonomic characteristics [meso-isomer of diamino- pimelic acid in the cell wall, menaquinone MK-9 (H-4), lack of mycolic acids, and the presence of teichoic acids] typical of the genus Brevibacterium [10]. Colony pigmen- tation was similar to that of B. linens, which is the only orange-pigmented species of the genus, but the strain differed from the type strain of B. linens in a number of (not presented). Phylogenetic physiological properties analysis based on 16S rDNA sequence confirmed that the strain VKM Ac-2118 belongs to the genus Brevibac- terium. It showed 92.6–97.5% 16S rDNA sequence simi- larities to type strains of the known species of the genus and grouped together with B. linens DSM 20425T (X77451), B. iodinum NCDO 613T (X76567), B. epidermi- dis NCDO 2286T (X76565) and B. casei NCDO 2048T (X76564) in a tight cluster with a 100% bootstrap repli- cation value (not presented), exhibiting the highest 16S rDNA sequence similarities of 97.6 and 96.6% to B. casei NCDO 2048T and B. linens DSM 20425T, respectively. In addition, the strain differed from all the above species in the composition of cell wall components.

Descending chromatography and electrophoresis were performed on a Filtrak FN-13 paper. Electrophoresis was performed in a pyridinium acetate buffer (pH 5.6) to separate phosphoric esters [7]. The following solvent systems were used for descending paper chromatography: (a) propan-1-ol/aqueous NH3 (specific gravity 0.88)/water (6 : 3 : 1, v/v/v) for the separation of isomeric phosphoric esters; (b) pyridine/benzene/butan-1-ol/water (3 : 1 : 5 : 3, v/v/v/v); (c) butan-1-ol/acetic acid/water (4 : 1 : 5, v/v/v) for the separation of mannitol, glycerol, and monosaccha- rides; and (d) pyridine/ethyl acetate/acetic acid/water (5 : 5 : 1 : 3, v/v/v/v) for the separation of amino sugars. Detection of compounds was carried out using the following spray reagents: the molybdate reagent for phosphoric esters, ninhydrin for amino sugars, 5% AgNO3 in aqueous NH3 for polyols and sugars, and aniline hydrogenphthalate for reducing sugars.

The teichoic acid (crude preparation) was isolated from the cell wall containing 2% of organic phosphate. Upon acid and alkaline hydrolysis, a polyol and its phosphates were formed together with glycerol monophosphate and trace amounts of glycerol bisphosphate. The polyol was identified as mannitol from its mobility on paper chroma- tography in solvent systems 2 and 3, which coincided with that of an authentic sample. Additional proof was obtained from 13C NMR spectroscopic studies of the polyol isolated by preparative paper chromatography following hydrolysis of the teichoic acid with 40% hydrofluoric acid. The chemical shifts for the carbon atoms of the polyol coincided completely with those for the authentic mannitol [19] (Table 1).

rotation,

[a20

The absolute configuration of mannitol is D as deduced D þ 22:5 (approximately

from its optical 1, 0.03 M borax) (cf. [a20

D þ 24:0 for D-mannitol [20]).

Degradation of the teichoic acid

Acid hydrolysis of the teichoic acid was carried out with 2 M HCl for 3 h at 100 (cid:1)C and 40% HF for 24 h at 20 (cid:1)C; alkaline hydrolysis was performed with 1 M NaOH for 3 h at 100 (cid:1)C, hydrolysis with alkaline phosphatase (EC 3.1.3.1) from calf intestinal mucosa. (Sigma) was performed in ammonium acetate buffer (pH 10.4) at 37 (cid:1)C for 2 h [2]. The polyol/phosphorus molar ratios were determined as described by Potekhina et al. [2].

Mannitol was isolated on a column (1.3 · 75 cm) with TSK HW-40(S) in 1% acetic acid using a Knauer differ- ential refractometer. Optical rotation was measured with a PU-5 polarimeter (Russia).

Acid degradation of the trichloroacetic acid extract (crude preparation) yielded mannitol phosphates and glycerol phosphates. Therefore, it was necessary to establish whether these compounds originate from the same or from different teichoic acids present simultaneously in the cell wall, which has been demonstrated for other brevibacteria [9].

NMR spectra were recorded using a Bruker DRX-500 spectrometer for 2–3% solutions in D2O at 30 (cid:1)C with acetone (dH 2.225 and dC 31.45) as the internal standard and 80% H3PO4 measured separately. One-dimensional 1H NMR spectra were obtained with a presaturation of the HDO signal for 1 s two-dimensional spectra were obtained using standard pulse sequences from the BRUKER software.

Electrophoresis of the crude preparation did not reveal the presence of two different polymers. An attempt has been undertaken to separate teichoic acids by fractional precipi- tation with ethanol. Two preparations were obtained; those precipitated with two and four volumes of ethanol (prepar- ation I and preparation II, respectively). Acid hydrolysates of these preparations differed in the compositions. The essential factor is that they differed in the ratios of glycerol

Mass spectrometric analysis (MALDI-TOF MS) of teichoic acid (preparation I) was carried out in the positive

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Table 1. 13C NMR data of the mannitol teichoic acid of Brevibacterium sp. VKM Ac-2118 (d, p.p.m.; JÆHz)1, acetone, d, 31.45 p.p.m. br, broadened).

Carbon atoms

C-1 C-2 C-3 C-4 C-5 C-6 Residue

Mannitol -1)-Mannitol-(6-P- -1)-Mannitol-(6-P- 64.5 68.65 68.3 br. 64.5 68.65 68.8 br. 72.1 70.65 70.25 3JP-1(6),C-2 2.9 70.5 69.8 70.7 3JP-4,C-3 7.5 70.5 69.8 75.3 2JP-4,C-4 6.1 72.1 70.65 71.1 3JP-6,C-5 2.9

64.3 71.1 69.95 75.1 70.7 68.8 4 j P Mannitol-(6-P-

phosphates and mannitol phosphates. Mannitol teichoic acid was virtually the only component of preparation I. This was free from sugars, which suggested the absence of glycosyl substituents in the chain. It was this preparation that was subjected to subsequent structural analysis.

Elucidation of the primary structure of teichoic acids by chemical methods involves their degradation under differ- ent conditions, structural analysis of the fragments formed and reconstruction of the structure of the original polymer [21].

4 j P

Alkaline hydrolysis of the polymer furnished three major phosphates which were isolated by paper electrophoresis. Phosphate 1 (P1), with the electrophoretic mobility relative to that of glycerol phosphate (mGroP) equal to 0.7, was treated with phosphomonoesterase (PME) to yield mannitol and inorganic phosphate in an 0.96 : 1 molar ratio. Thus, P1 is mannitol monophosphate. Phosphate 2 (P2, mGroP 1.21) yielded mannitol and inorganic phosphate in a 1 : 2 molar ratio upon treatment with PME. Thus, P2 is mannitol bisphosphate. Phosphate 3 (P3, mGroP 1.5) was hydrolyzed under the action of PME to yield mannitol and inorganic phosphate in a 1 : 2.8 molar ratio, which suggests that P3 is mannitol trisphosphate.

Acid hydrolysis of the teichoic acid yielded the same three major phosphates as those formed upon alkaline hydrolysis. The presence of mannitol mono- and bisphosphates among the degradation products of the polymer suggested that the teichoic acid under study is of the poly(mannitol phosphate) type and the formation of identical phosphates upon alkaline and acid degradations corroborated the absence of glycosyl substituents in the chain [22].

vage of the phosphodiester bond A results in scission of the poly(mannitol phosphate) chain, and the cyclophos- phate formation involves either one hydroxy group (OH-2 or OH-5) of mannitol or both to yield 1,2(5,6)-cyclophos- phate or 1,2;5,6-bis(cyclophosphate), respectively. Cyclo- phosphates are known to be unstable in alkaline media and their opening results in isomeric phosphates [22]. the phosphate B does not occur under Cleavage of alkaline conditions and the phosphate group remains linked to the mannitol residue in the same position. Thus, mannitol trisphosphate is a hydrolysis product of the phosphate groups A.

The mechanism of acid hydrolysis is essentially the same, although the phosphate group migration can occur in polyol phosphates via transient cyclophosphates.

However, the formation of mannitol trisphosphate upon degradation of the polymer could not be rationalized in the framework of ordinary pathways of hydrolysis of poly (polyol phosphate) chains. This could occur in the case where a secondary hydroxy group of mannitol was substi- tuted by a phosphate-bearing group.

Alkaline hydrolysis of the teichoic acid with the structure of poly(mannitol phosphate) with monophosphate side units is depicted in Fig. 1.

Esterification of one of the secondary hydroxy groups of mannitol by phosphoric acid was confirmed by quantitation of the total phosphate (Ptotal) and that liberated under the action of PME (PPME). The Ptotal : PPME molar ratio was found to be equal to (cid:2) 2 : 1.

Teichoic acid was also investigated independently by NMR spectroscopy. The most abundant signals in the

As can be seen, the polymer contains two nonequiva- lent phosphate groups, A and B, vicinal to the free hydroxy groups of the polyol. Hydrolysis of the chain occurs via transient five-membered cyclophosphates. Clea-

Fig. 1. Pathways of alkaline hydrolysis of the mannitol teichoic acid of Brevibacterium sp. VKM Ac-2118.

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13C NMR spectrum of the teichoic acid (Table 1) corres- ponded to 1,6-poly(mannitol phosphate) chain (the signals for the –CH2OPO3– at d 68.3 and 68.8) bearing a phosphate group at C-5(2) or C-4(3) (a signal at d 75.3).

with the methine proton resonating at d 4.37. The second most abundant peak at d 1.6 had cross-peaks with the protons resonating at d 4.25 (H-6), 4.18 (H-1), 4.09 (H-1¢), 4.08 (H-5), and 4.01 (H-6¢), which suggests the presence of two phosphate groups, one of which was linked to a methine group, and the second being involved in the 1,6- poly(mannitol phosphate) chain of the polymer.

The 31P NMR spectrum of the polymer contained a broadened signal with a maximum intensity at d 0.8. The 31P NMR spectrum of the preparation recorded in the presence of 0.25 M EDTA contained two major signals of nearly the same intensities at d 0.4 and 1.6 together with minor signals belonging probably to the side phosphate group in the mannitol residue at the growing chain end and/or to the internal phosphate(s) bound to the mannitol unit devoid of the side phosphate group.

The 1H NMR spectrum (Table 2) was interpreted using two dimensional 1H/1H COSY and two dimensional 1H/13C HSQC (heteronuclear single quantum coherence) spectros- copy. The HSQC spectrum (Fig. 3) revealed unequivocally that the signals for the carbon atoms at d 68.8 and 68.3 belong to the –CH2OP– groups.

The two dimensional heteronuclear 1H/31P heteronuclear multiple quantum coherence (HMQC) spectrum (Fig. 2) revealed a single correlation of the former signal (d 0.4)

In the COSY spectrum, a correlation was found between one of the protons of the –CH2OP– group at d 4.25 and (a) the second proton of this group at d 4.01 and (b) the proton

Fig. 2. 1H/31C HMQC spectrum of the mannitol teichoic acid of Brevibacterium sp. VKM Ac-2118. The protons H-1, 1¢, 4, 5, 6, and 6¢ give cross-peaks with phosphorus are marked at the 1H NMR spectrum.

Table 2. 1H NMR date of the mannitol teichoic acid of Brevibacterium sp. VKM Ac-2118 (d, p.p.m.; JÆHz)1, acetone, d, 2.225 p.p.m).

Proton atoms

Residue H-1 H-2 H-3 H-4 H-5 H-6 H-1¢ H-6¢

3.8 3.8 3.76 3.87 3.675 Mannitol 3.675 J1¢,2 6.1 3.76 J2,3 8.4

3.87 J1,1¢ 11.8 J1,2 2.7 4.18 4.18 4.07 4.09 3.97 3.91 3.92 3.93 3.92 4.37 3.97 4.08 4.18 4.25 4.07 4.01 -1)-Mannitol-(6-P- -1)-Mannitol-(6-P-

3.85 3.78 3.90 3.94 4.34 4.08 4.17 4.06 4 j P Mannitol-(6-P-

4 j P

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of the –CHO– group at d 4.08 (the corresponding carbon atom resonates at d 71.1). In turn, there was a correlation between the latter proton and the proton of a –CHOP– group at d 4.37 (the corresponding carbon atom resonates at d 75.3). Thus, the phosphomonoester group is located at position 4 of mannitol [if numbering is that the protons resonating at d 4.25 and 4.01 belong to –C(6)H2OP– group] or at position 3 [if numbering is that these protons belong to –C(1)H2OP– group]. In other words, the localization site of phosphate depends on the mannitol residue numbering. Taking into account the data on the biosynthesis of ribitol teichoic acids [23], we assume that mannitol 6-phosphate that is the monomeric unit of the polymer being studied.

Fig. 3. 1H/13C HSQC spectrum of the manni- tol teichoic acid of Brevibacterium sp. VKM Ac-2118. The signals at 4.18, 4.09/68.3 and 4.25, 4.01/68.8 belong to the –CH2OP– groups; the signals at 4.37/75.3 belongs to the –CH(4)OP– group.

Based on this assumption and on the presence of signals for the terminal units of the chain, we can localize the phosphate side group at position 4 of mannitol with greater certainty from the following observations. Some of the minor signals for the terminal units belong unambiguously to the –CH2OH group (C-1) and the –CH2OP– group (C-6). Other signals of the terminal unit of the growing chain were assigned using data from COSY and HSQC spectra (Table 1), which shows that the phosphomonoester group is linked with C-4.

ods: (a) by identification of mannitol trisphosphate as a degradation product of the teichoic acid; (b) by quantitation of Ptotal and PPME (ratio (cid:2) 2 : 1) using treatment of the polymer with phosphomonoesterase; (c) by detection of a low-field signal at d 75.3 in the 13C NMR spectrum. The spectrum of the polymer treated with PME devoid completely of the above-mentioned signal corresponded to unsubstituted 1,6-poly(mannitol phosphate) (Table 1); and (d) by establishing the molecular mass of the repeating unit of the polymer equal to 324 Da (MALDI-TOF MS), i.e. the presence of a phosphate group linked to a methine group of mannitol.

contains

The presence of the phosphate substituent at the methine group was also confirmed by analysis of the MALDI-TOF mass spectrum (Fig. 4). Thus the most abundant of the basic peaks are those differing by 324 Da (Man-ol P2) or 347 Da (Man-ol P2 + Na). The maximum mass recorded corresponds to 19 mannitol phosphate units. However, the ratio of the integral intensities of peaks for the terminal and internal residues in the 13C NMR spectrum suggests the presence of 6–7 units on average. With account of possible broad distribution of oligomeric chains according to masses, these data on chain length estimations should not be regarded as contradictory.

The results presented here show that the cell wall of Brevibacterium sp. VKM Ac-2118 a 1,6-poly(mannitol phosphate) chain with phosphate groups attached as side groups to O-4(3) of mannitol residues. In addition, small amounts of glycerol mono- and bis- phosphate, and a glycerol phosphodiester containing

Thus, the presence of a phosphate group at the mannitol methine group is established by several independent meth-

Fig. 4. The MALDI-TOF MS of the teichoic acid of Brevibacterium sp.VKM Ac-2118.

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glucosamine were detected in preparation II. The latter yielded glycerol mono- and bisphosphates and glucosamine upon acid hydrolysis and proved to be identical with the alkaline hydrolysis product of the teichoic acid of Streptomyces rutgersensis var. castelarens [18].

9. Fiedler, F. & Bude, A. (1989) Occurrence and chemistry of cell wall teichoic acids in the genus Brevibacterium. J. Gen. Microbiol. 135, 2837–2846.

10. Fiedler, F., Schaeffler, M.J. & Stackebrandt, E. (1981) Biochem- ical and nucleic acid hybridisation studies on Brevibacterium linens and related strains. Arch. Microbiol. 129, 85–93.

The presence of 1,3-poly(glycerol phosphate) chains substituted partially with a-N-acetylglycosamine at O-2 is also possible, as can be deduced from NMR spectroscopic and alkaline hydrolysis data of this preparation.

11. Anderton, W.J. & Wilkinson, S.G. (1985) Structural studies of mannitol teichoic acid from the cell wall of Bacterium NCTC 9742. Biochem. J. 226, 587–599.

12. Shi, T., Reevs, R., Gilichinsky, D. & Friedmann, E.I. (1997) Characterization of viable bacteria from Siberian permafrost by 16S rDNA sequencing. Microbial Ecol. 33, 169–179.

It is noteworthy that the side phosphate groups impart an additional negative charge to the polymer, which seems to be important for functioning of the cell wall on the whole. These seem to be of special importance considering the halotolerant properties of Brevibacterium sp.VKM Ac-2118 [24]. Recently, it has been shown that the cell wall of an alkophilic bacillum comprised three polymers with mark- edly pronounced acidic properties: polyglucuronic, teich- uronic and polyglutamic acids [25].

13. Evtushenko, L.I., Taran, V.V., Akimov, V.N., Kroppenstedt, R.M., Tiedje, J.M. & Stackebrandt, E. (2000) Nocardiopsis tropica sp. nov., Nocardiopsis trehalosi sp. nov., nom. rev. & Nocardiopsis dassonvillei subsp. albirubida subsp. nov., comb. nov. Int. J. Syst. Evol. Microbiol. 50, 73–81.

14. Kimura, M. & Ohta, T. (1972) On the stochastic model for esti- mation of mutation distance between homologous proteins. J. Mol. Evol. 2, 87–90.

Acknowledgements

15. Saitou, N. & Nei, M. (1987) The neighbour-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425.

This work was supported by grants from INTAS (no. 01–2040) and the Russian Foundation for Basic Research (no. 01-04-49854).

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