Keywords: teichoic acids; polymannitol phosphate; side phosphate groups; NMR; Brevibacterium.. Among the cell wall teichoic acids studied so far, four structural types can be distinguish
Trang 1A novel mannitol teichoic acid with side phosphate groups
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
1
School 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
The cell wall of Brevibacterium sp VKM Ac-2118 isolated
from a frozen (mean annual temperature)12 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
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
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
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
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, riberythr-itol, arabinerythr-itol, 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
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
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 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 C before study The methods used for studying phenotypical 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] withCLUSTALWsoftware [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 28C 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)
Trang 2at 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 100C, washed several
times with water, and freeze-dried
The teichoic acid was extracted with 10%
trichloroace-tic acid at 4C 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 (two volumes) to the combined
supernatant and the precipitate that formed was removed
by centrifugation for 24 h at 0C, 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
teichoic acid preparations were isolated as follows:
teichoic acid precipitated with two volumes of ethanol
(preparation I) and teichoic acid precipitated with four
volumes of ethanol (preparation II)
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% AgNO3in aqueous NH3
for polyols and sugars, and aniline hydrogenphthalate for
reducing sugars
Acid hydrolysis of the teichoic acid was carried out with
2MHCl for 3 h at 100C and 40% HF for 24 h at 20 C;
alkaline hydrolysis was performed with 1MNaOH for 3 h
at 100C, hydrolysis with alkaline phosphatase (EC 3.1.3.1)
from calf intestinal mucosa (Sigma) was performed in
ammonium acetate buffer (pH 10.4) at 37C 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)
NMR spectra were recorded using a Bruker DRX-500
spectrometer for 2–3% solutions in D2O at 30C with
acetone (dH2.225 and dC31.45) 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 theBRUKER
software
Mass spectrometric analysis (MALDI-TOF MS) of
teichoic acid (preparation I) was carried out in the positive
reflection mode using a KOMPACT MALDI 4 (Kratos Analytical) mass spectrometer 2,5-Dihydroxybenzoic (gen-tisic) acid was used as a matrix
Results and discussion
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 physiological properties (not presented) Phylogenetic 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 2048Tand B linens DSM 20425T, respectively In addition, the strain differed from all the above species in the composition of cell wall components
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 from13C 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)
The absolute configuration of mannitol isDas deduced from its optical rotation, [a20
Dþ 22:5 (approximately
1, 0.03Mborax) (cf [a20
Dþ 24:0 forD-mannitol [20])
Degradation of the teichoic acid 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]
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 prepar(prepar-ation II, respectively) Acid hydrolysates
of these preparations differed in the compositions The essential factor is that they differed in the ratios of glycerol
FEBS 2003 A novel mannitol teichoic acid (Eur J Biochem 270) 4421
Trang 3phosphates 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 fragmdiffer-ents formed
and reconstruction of the structure of the original polymer
[21]
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, mGroP1.21)
yielded mannitol and inorganic phosphate in a 1 : 2 molar
ratio upon treatment with PME Thus, P2 is mannitol
bisphosphate Phosphate 3 (P3, mGroP1.5) 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]
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
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-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(cyclophos1,2(5,6)-cyclophos-phate), respectively Cyclo-phosphates are known to be unstable in alkaline media and their opening results in isomeric phosphates [22] Cleavage of the phosphate B does not occur under 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
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 2 : 1
Teichoic acid was also investigated independently by NMR spectroscopy The most abundant signals in the
Fig 1 Pathways of alkaline hydrolysis of the mannitol teichoic acid of Brevibacterium sp VKM Ac-2118.
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).
Residue
Carbon atoms
-1)-Mannitol-(6-P-4
j
P
br. 3J P-1(6),C-2 2.9 3J P-4,C-3 7.5 2J P-4,C-4 6.1 3J P-6,C-5 2.9 br.
Mannitol-(6-P-4
j
P
Trang 413C 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)
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 0.25MEDTA 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 two dimensional heteronuclear1H/31P heteronuclear
multiple quantum coherence (HMQC) spectrum (Fig 2)
revealed a single correlation of the former signal (d 0.4)
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
The1H NMR spectrum (Table 2) was interpreted using two dimensional1H/1H COSY and two dimensional1H/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
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 1 H/ 31 C 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 the1H NMR spectrum.
Table 2 1 H 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).
Residue
Proton atoms
J 1,1 ¢ 11.8 J 1 ¢ ,2 6.1 J 2,3 8.4
J 1,2 2.7
-1)-Mannitol-(6-P-4
j
P
Mannitol-(6-P-4
j
P
FEBS 2003 A novel mannitol teichoic acid (Eur J Biochem 270) 4423
Trang 5of 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
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
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
Thus, the presence of a phosphate group at the mannitol
methine group is established by several independent
meth-ods: (a) by identification of mannitol trisphosphate as a degradation product of the teichoic acid; (b) by quantitation
of Ptotaland PPME (ratio 2 : 1) using treatment of the polymer with phosphomonoesterase; (c) by detection of a low-field signal at d 75.3 in the13C 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
The results presented here show that the cell wall
of Brevibacterium sp VKM Ac-2118 contains 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
Fig 3 1 H/ 13 C 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 –CH 2 OP– groups; the signals at 4.37/75.3 belongs to the –CH(4)OP– group.
Fig 4 The MALDI-TOF MS of the teichoic acid of Brevibacterium sp.VKM Ac-2118.
Trang 6glucosamine 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 rutgersensisvar castelarens [18]
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
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]
Acknowledgements
This work was supported by grants from INTAS (no 01–2040) and the
Russian Foundation for Basic Research (no 01-04-49854).
References
1 Naumova, I.B., Shashkov, A.S., Tul’skaya, E.M., Streshinskaya,
G.M., Kozlova, Y.I., Potekhina, N.V., Evtu shenko, L.I &
Stackebrandt, E (2001) Cell wall teichoic acids: structural
diversity, species-specificity in the genus Nocardiopsis, and
chemo-taxonomic perspective FEMS Microbiol Rev 25, 269–284.
2 Potekhina, N.V., Tul’skaya, E.M., Naumova, I.B., Shashkov, A.S.
& Evtushenko, L.I (1993) Erythritolteichoic acid in cell wall of
Glycomyces tenuis VKM Ac-1250 Eur J Biochem 218, 371–375.
3 Potekhina, N.V., Tul’skaya, E.M., Shashkov, A.S., Taran, V.V.,
Evtushenko, L.I & Naumova, I.B (1998) Taxonomic specificity
of cell wall teichoic acids of actinomycetes of Glycomyces genus.
Microbiologiya (Moscow) 67, 330–334.
4 Shashkov, A.S., Tul’skaya, E.M., Evtushenko, L.I & Naumova,
I.B (1999) Cell wall teichoic acid of Nocardioides albus VKM
Ac-805 Biochemistry (Moscow) 64, 1544–1549.
5 Shashkov, A.S., Tul’skaya, E.M., Evtushenko, L.I., Gratchev,
A.A & Naumova, I.B (2000) Structure of teichoic acid of
Nocardioides luteus VKM Ac-1246T cell wall Biochemistry
(Moscow) 65, 509–514.
6 Potekhina, N.V., Shashkov, A.S & Naumova, I.B (1996) The cell
wall teichoic acid of Actinomadura madura contains poly
(galactosyl-1,2-glycerol phosphate) and
poly-(3-O-methylgalacto-syl-1,2-glycerol phosphate) Microbiologiya (Moscow) 65, 522–
526.
7 Potekhina, N.V., Naumova, I.B., Shashkov, A.S & Terekhova,
L.P (1991) Stru ctu ral featu res of cell wall teichoic acid and
peptidoglycan of Actinomadura cremea INA 292 Eur J Biochem.
199, 313–316.
8 Shashkov, A.S., Potekhina, N.V., Naumova, I.B., Evtushenko,
L.I & Widmalm, G (1999) Cell wall teichoic acid of
Actinoma-dura viridis VKM Ac-1315 T Eur J Biochem 262, 688–695.
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.
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.
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.
15 Saitou, N & Nei, M (1987) The neighbour-joining method: a new method for reconstructing phylogenetic trees Mol Biol Evol 4, 406–425.
16 Thompson, J.D., Higgins, D.G & Gibson, T.J (1994) CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice Nucleic Acids Res 22, 4673– 4680.
17 Naumova, I.B., Kuznetsov, V.D., Kudrina, K.S &
Bezzubenko-va, A.P (1980) The occurrence of teichoic acids in streptomycetes Arch Microbiol 126, 71–75.
18 Tul’skaya, E.M., Vylegzhanina, K.S., Streshinskaya, G.M., Shashkov, A.S & Naumova, I.B (1991) 1,3-Poly (glycerol phos-phate) chains in the cell wall of Streptomyces rutgersensis var Castelarense Biochim Biophys Acta 1074, 237–242.
19 Bock, K & Pedersen, C (1983) Carbon 13 nuclear magnetic resonance spectroscopy of monosaccharides Adv Carbohydr Chem Biochem 41, 27–66.
20 Merck & Co Inc (1989) The MerckIndex, 11th edn p 901 Rahway, NJ, USA.
21 Archibald, A.R (1972) Teichoic acids In Methods in Carbo-hydrate Chemistry (Whistler, R.L., ed.) Vol 6, pp 162–172 Academic Press, London, New York.
22 Kelemen, M.V & Baddiley, J (1961) Structure of the intracellular glycerol teichoic acid from Lactobacillus casei ATCC 7469 Bio-chem J 80, 246–254.
23 Baddiley, J., Buchanan, J.G & Carss, B (1957) The configuration
of the ribitol phosphate residue in citidine diphosphate ribitol.
J Chem Soc 1869–1876.
24 Smirnov, A.V., Kulakovskaya, T.V & Kulaev, I.S (2002) Exo-polyphosphatase of the halotolerant bacterium Brevibacterium sp.strain VKM Ac-2118 grown at normal and enhanced salinity Doklady Acad Nauk (Moscow) 386, 284–286.
25 Aono, R (1990) The poly-a- and -b-1,4-glucuronic acid moiety of teichuronopeptide from the cell wall of the alkalophilic Bacillus strain C-125 Biochem J 270, 363–367.
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