Duus1, Helmut Brade2and Otto Holst2 1 Department of Chemistry, Carlsberg Laboratory, Valby, Copenhagen, Denmark; 2 Division of Medical and Biochemical Microbiology, Research Center Borst
Trang 1The structure of the carbohydrate backbone of the lipopolysaccharide
Evgeny V Vinogradov1,*, Jens é Duus1, Helmut Brade2and Otto Holst2
1 Department of Chemistry, Carlsberg Laboratory, Valby, Copenhagen, Denmark; 2 Division of Medical and Biochemical Microbiology, Research Center Borstel, Borstel, Germany
The chemical structure of the phosphorylated carbohydrate
backbone of the lipopolysaccharide (LPS) from
Acineto-bacter baumannii strain ATCC 19606 was investigated by
chemical analysis and NMR spectroscopy of
oligosaccha-rides obtained after deacylation or mild acid hydrolysis
From the combined information the following carbohydrate
backbones can be deduced:
a-GalpNR1
b-GlcpN a-Kdo
1 1 2
¯ ¯ ¯
4 7 4
R2®3)-a-GlcpNAcA-(1®4)-a-Kdo-(2®5)-a-Kdo-(2®6)-b-GlcpN4P-(1®6)-a-GlcpN1P
where R1 H and R2 a-Glcp-(1 ® 2)-b-Glcp-(1 ® 4)-b-Glcp-(1 ® 4)-b-4)-b-Glcp-(1 as major and R1 Ac and R2 H
as minor products
All monosaccharides areD-con®gured Also, smaller oli-gosaccharide phosphates were identi®ed that are thought to represent degradation products of the above structures Keywords: Acinetobacter baumannii; lipopolysaccharide; core region; structural analysis; NMR spectroscopy
The Gram-negative bacterium Acinetobacter
(Moraxella-ceae) is isolated from soil and water which represent its
natural habitats However, the genus has gained increasing
importance as a causative agent of nosocomial infections
(e.g bacteremia, secondary meningitis) in recent years [1]
As in other Gram-negative bacteria, Acinetobacter
pos-sesses lipopolysaccharides (LPS) in the outer membrane of
the cell wall that have been shown to represent useful
chemotaxonomic and antigenic markers for its
identi®ca-tion and differentiaidenti®ca-tion The occurrence of S-form LPS in
Acinetobacter has unequivocally been proven recently, and
the structures of a number of O-speci®c polysaccharides
and their antigenic characterization have been published
[2±9] The core region of Acinetobacter LPS possesses
particular structural features which clearly distinguish it
from other core structures [10] First, it belongs to the
group of heptose-de®cient core regions Second, it may
contain D-glycero-D-talo-oct-2-ulopyranosonic acid (Ko) which can replace the 3-deoxy-D -manno-oct-2-ulopyrano-sonic acid (Kdo) residue linking the core region to lipid A [11±15]
Another core type has been identi®ed in A baumannii strain NCTC 10303 [16] that is devoid of Ko It comprises the tetrasaccharide a-Kdo-(2 ® 5)-[a-Kdo-(2 ® 4)-]-a-Kdo-(2 ® 5)-a-4)-]-a-Kdo-(2 ® [a-Kdo IV-(a-Kdo III)-a-Kdo II-a-Kdo I], of which Kdo IV is substituted at O-8 by a short-chain rhamnan and Kdo III at O-4 by the disaccha-ride a-D-GlcpNAc-(1 ® 4)-a-D-GlcpNA (GlcpNA, 2-ami-no-2-deoxy-glucopyranosuronic acid) Kdo I links the core region to the lipid A This Kdo tetrasaccharide is unique in nature; however, a second Kdo tetrasaccharide of different structure has been identi®ed in LPS of Chlamydophila psittaci 6BC [17] The latter has been shown to be assembled
by one Kdo transferase, which is therefore multifunctional [18] However, the biosynthesis of the Kdo-tetrasaccharide from LPS of A baumannii strain NCTC 10303 has not yet been elucidated For A baumannii strain ATCC 15303, which possesses in its LPS core region the trisaccharide a-Kdo-(2 ® 5)-[a-Kdo-(2 ® 4)-]-a-Kdo-(2 ® , it has been shown that the Kdo transferase is able to transfer the ®rst two a-(2 ® 4)-linked Kdo residues [19] The mechanism of the transfer of the third Kdo residue has not yet been identi®ed
In addition to the work on the determination of the chemical and antigenic structures of O-speci®c polysaccha-rides from LPS of Acinetobacter in order to establish an O-serotyping scheme, there exists considerable interest in investigating the LPS core regions from this genus which obviously allows novel insights in structure, genetics and biosynthesis of LPS Here, the structures of the carbohy-drate backbones of the LPS from A baumannii strain ATCC 19606 are reported
Correspondence to O Holst, Research Center Borstel, Parkallee 22,
D-23845 Borstel, Germany Fax: + 49 4537 188419,
Tel.: + 49 4537 188472, E-mail: oholst@fz-borstel.de
Abbreviations: LPS, lipopolysaccharide; GlcpNAcA,
2-acetamido-2-deoxy-glucopyranosyluronic acid; DGlcpNA,
2-amino-2,4-dideoxy-b-L -threo-hex-4-enopyranosyluronic acid; HMBC, heteronuclear
mul-tiple bond correlation; HMQC, heteronuclear mulmul-tiple quantum
co-herence; HPAEC, high-performance anion-exchange
chromatography; Kdo, 3-deoxy- D -manno-oct-2-ulopyranosonic acid.
Dedication: this article is dedicated to Prof Dr Joachim Thiem,
Institute of Organic Chemistry, University of Hamburg, Germany on
the occasion of his 60th birthday.
*Present address: Institute for Biological Sciences, National Research
Council Canada, Ottawa, Ontario K1A 0R6, Canada.
(Received 23 July 2001, revised 29 October 2001, accepted 31 October
2001)
Trang 2M A T E R I A L S A N D M E T H O D S
Bacteria and Bacterial LPS
A baumannii strain ATCC 19606 was cultivated and the
LPS was obtained as described previously [11]
Preparation of oligosaccharides
The LPS (80 mg) was hydrolysed in 5% acetic acid (100 °C,
5 h), the precipitate was removed by ultracentrifugation
(100 000 g, 4 h), and the supernatant was lyophilized (yield:
50 mg, 62.5% of the LPS) The latter sample was reduced
with NaBH4, and, after working up, was desalted by
gel-permeation chromatography High-performance
anion-exchange chromatography (HPAEC) of this fraction
yield-ed oligosaccharides 1 (4 mg, 5% of the LPS) and 2 (10 mg,
12.5% of the LPS), and two stereoisomers of 1 and 2 in
minor amounts that differed by the con®guration at C2 of
Kdo-ol
Another portion of the LPS (350 mg) was de-O-acylated
as described previously [20] (yield: 236 mg, 67.4% of the LPS), and 150 mg of this de-O-acylated LPS was then de-N-acylated [20], and, after working up, desalted by gel-permeation chromatography HPAEC of this sample yielded oligosaccharides 3 and 4 (10 and 25 mg, 2.9 and 7.1% of the LPS, respectively)
General and analytical methods Gel-permeation chromatography was carried out on a column (3.5 ´ 40 cm) of TSK HW40 (S) gel (Merck) in water, and runs were monitored with a differential refractometer (Knauer) Preparative HPAEC was per-formed on a column (9 ´ 250 mm) of CarboPac PA1 (Dionex Corp.) as described previously [20] using linear gradient programs of 3 to 40% 1M sodium acetate in 0.1M NaOH over 80 min (for separation of oligosaccha-rides 1 and 2, Fig 1) and 30 to 70% over 80 min (for separation of oligosaccharides 3 and 4, Fig 1) Samples
Fig 1 Structures of oligosaccharides 1±5.
DGlcpNA, 2-amino-2,4-dideoxy-b- L
-threo-hex-4-enopyranosyluronic acid (the product of
the 4,5-b-elimination of a-glucosaminuronic
acid).
Trang 3Table 1 1 H NMR data of oligosaccharides 1±4 Spectra were recorded of solutions in 2 H 2 O relative to internal acetone (2.225 p.p.m.) Oligo, oligosaccharide.
Chemical shift (in p.p.m.) and J H,H coupling constants (in Hz) of proton
Residue Oligo J 1,2 J 2,3 J 3,4 ,J 3ax,4 J 3eq,4, J 3ax,3eq J 4,5 J 5,6 J 6a,6b J 5,6b
8.5 10.2 10
12.1
1.90 4 12.2
3.93 4.47 3.66 3.85 3.59 3.85
12.5
1.92 3.5 12
3.95
< 1 4.48 3.679 c 3.86 3.60 3.86
12.3
2.06 2.4 12.8
3.99
< 1 3.95< 1 3.68 3.94 3.69 3.90
12.7
2.07 4.6 12.3
4.01
< 1 3.96< 1 3.699 c 3.96 3.70 3.89
E, aKdo-ol/a-Kdo 1 4.06
4 d 1.93
6 e 2.08
6 3.996 f 4.10
6 3.734 6
12
2.90 2.7 12.0
4.84
< 1 4.43< 1 4.279 c 4.12 3.91 3.91
12
2.91 4 12
4.87
< 1 4.43< 1 4.299 d 4.13 3.92 3.92
1.6 4.1 4.1
7.8 10
7.8 9.4
8.2 9.5 9.5
8.3 10.6
a J 6.5 Hz; b J 6.6; c J d J e J ; f J ; g additional signal in oligosaccharide 1: CH CO, 2.033 p.p.m; h J
Trang 4were desalted using a Dowex 50 ´ 4 (H+) cation exchanger
in water, and amino-group containing compounds were
then eluted with 5% aqueous ammonia GLC-MS,
mono-saccharide analysis, and the determination of the absolute
con®guration of monosaccharides were performed as
described previously [14,15,21] Conformational analysis
was carried out as described previously [16]
NMR spectroscopy
For structural assignments, 1D and 2D1H and13C NMR
spectra were recorded on solutions of oligosaccharides 1±4
in2H2O (500 lL) with a Bruker AMX-600 spectrometer at
27 °C using standard Bruker software Chemical shifts are
given relative to internal acetone (CH3-, 2.225 p.p.m for
1H, 34.5 p.p.m for 13C) The assignment of the proton
chemical shifts was achieved by correlation spectroscopy
(COSY), total correlation spectroscopy (TOCSY), and
nuclear Overhauser enhancement spectroscopy (NOESY),
and the assignment of the carbon chemical shifts was
performed by heteronuclear multiple quantum coherence
(HMQC) and heteronuclear multiple bond correlation
(HMBC) experiments Broad band 1H-decoupled
31P NMR spectra were recorded at 101.25 MHz with a
Bruker DRX 250 spectrometer at 27 °C, and chemical shifts are given relative to an external reference of 85% phospho-ric acid (0.0 p.p.m.)
Mass spectrometry The electrospray mass spectra (ES-MS) were acquired in the negative mode on a VG Quattro triple quadropole mass spectrometer (VG Biotech, Altrincham, Cheshire, UK) with 50% aqueous CH3CN as the mobile phase at a
¯ow rate of 8 lLámin)1 Ten microlitres of a 20 lM aqueous solution of the samples were injected into the electrospray source
R E S U L T S A N D D I S C U S S I O N
Sugar analyses of the LPS Monosaccharide analyses of the LPS from A baumannii strain ATCC 19606 revealed the presence ofD-Glc,D-GlcN,
D-GalN, and Kdo The D-con®guration of Kdo was determined on the basis of the optical rotation value [a]D +56° (C 1, in water) of Kdo methyl ester a-methyl glycoside isolated after methanolysis of the LPS [14]
Table 2 13 C NMR data of oligosaccharides 1±4 Spectra were recorded of solutions in 2 H 2 O relative to internal acetone (34.5 p.p.m.) Oligo, oligosaccharide.
Chemical shift of carbon (in p.p.m.)
a J 1,P 0.5 Hz; b J 2,P 7.5 Hz; c J 4,P 3.5 Hz; d J 5,P 6.5 Hz.
Trang 5Isolation and structural analysis
of oligosaccharides 1±4
Acetic acid degradation of the LPS and reduction yielded as
main products two stereoisomers each of the two
oligosac-charides resulting from reduction of the carbonyl group of
the reducing Kdo residue Oligosaccharides 1 and 2 as the
major isomers were isolated by HPAEC and were examined
by NMR spectroscopy (data summarized in Tables 1±3)
All proton and carbon resonances could be assigned Most
relative con®gurations and ring sizes of the
monosac-charides were determined on the basis of vicinal proton±
proton coupling constants The anomeric con®gurations of
hexoses followed from JH1,H2coupling constants (3.5±4 Hz
for a-linked and 7.8±8.4 Hz for b-linked residues) All
residues were present in the pyranose form, as deduced from
the absence of low-®eld signals in the13C-NMR spectra that
are characteristic for furanoses, and from the vicinal proton
coupling constants The NOE data (Table 3) allowed the
unambiguous determination of the sequence of
monosac-charide residues
In the low-®eld region of the 1H-NMR spectra of
oligosaccharides 1 and 2 (4.4±5.4 p.p.m.), signals for three
and seven anomeric protons were present, respectively The
two oligosaccharides differed from each other by the
tetrasaccharide fragment Y-R-T-S- (see Fig 1) In both
oligosaccharides, the Kdo residue was present as an octitol
derivative (H2 at 4.061 and 4.159 p.p.m., respectively) For
each oligosaccharide, all aldose residues gave NOE signals
between the H1 protons and the proton on the carbon atom
to which they were glycosidically linked (Table 3)
Corre-lations between the anomeric protons and the
transglycosi-dic carbon atom could also be detected in HMBC spectra, con®rming the monosaccharide sequence and attachment sites, the latter being assigned from13C-NMR spectroscopic data using HMQC experiments (Table 2) The structures of oligosaccharides 1 and 2 were con®rmed by electrospray mass spectrometry, the spectra of which gave for oligosac-charide 1 an ion at m/z 822.4 ([M + H]+, calculated molecular mass 821.9 Da) and for oligosaccharide 2 ions at m/z 1428.6 (calculated molecular mass 1428.4 Da) and 715.2, representing [M + H]+and [M + 2H]/2+, respec-tively
From the resulting mixture of deacylated LPS, the two major oligosaccharides 3 and 4, as well as the tetrasac-charide a-Kdo-(2 ® 4)-a-Kdo-(2 ® 6)-b-GlcpN4P-(1
®6)-a-GlcpN1P, the trisaccharide a-Kdo-(2 ® 6)-b-GlcpN4P-(1 ® 6)-a-GlcpN1P, and several minor com-pounds were isolated by HPAEC The tetrasaccharide and the trisaccharide were readily identi®ed on the basis of published NMR data [24,25], and the minor compounds were not further investigated The structures of oligosac-charides 3 and 4 were fully characterized by NMR spectroscopy (Tables 1±3) They differed from each other
by the tetrasaccharide fragment Y-R-T-S- (Fig 1), as observed also for oligosaccharides 1 and 2 The substituent
at O4 of GlcpNA F, GalpN G present in 1 and 2, was lost by b-elimination owing to the alkaline conditions that also converted the GlcpNA into 2-amino-2,4-dideoxy-b-L -threo-hex-4-enopyranosyluronic acid Oligosaccharides 3 and 4 were similar to the oligosaccharide 5, isolated from LPS of
A baumannii strain NCTC10303 [16], differing by the presence of b-GlcN Z and (in 4) by the tetrasaccharide sequence attached to O-3 of DGlcpNA F
Both oligosaccharides 3 and 4 contained three Kdo residues All monosaccharide residues were present in the pyranose form, as deduced from the absence of low-®eld signals in the13C-NMR spectra that are characteristic for furanoses, and from the vicinal proton coupling constants (Tables 1 and 2) NOE contacts between H-1 of DGlcpNA
F and H-3ax, H-3eq, H-4 and H-5 of one of the Kdo residues identi®ed the latter as residue E (Fig 3), which was present as an octitol residue in compounds 1 and 2 The Kdo residue E was substituted by b-GlcN Z, as was apparent from the observation of an NOE between protons Z1 (b-GlcpN) and E7 (Fig 2, Table 3) The NOE contacts between protons C5 and E3ax, E3eq, E4, and E6 (Fig 3) suggested the attachment of E to C5 The sequence of the sugar residues C, D, E, and F was ®nally proven by long-range H-C correlations that were present in the HMBC spectrum of heptasaccharide 3, namely between proton C4 and carbon D2, C5 and E2, and F1 and E4 The signals of the anomeric carbons in Kdo residues were assigned on the basis of the intraresidual correlations between H-3eq and C-2
The a-con®guration of the Kdo residues C and D followed from the observed NOE between protons C3eq and D6 (Fig 3), characteristic for the fragment a-Kdo-(2 ® 4)-a-Kdo [25], which were also consistent with the sequence D-(2 ® 4)-C The a-con®guration of the Kdo residues C and D in 3 and 4 each was con®rmed by the chemical shifts of their H-3ax, signals (1.7±2.3 p.p.m.) [22,23]; however, the chemical shifts of H-3ax, of C were interchanged The chemical shifts of protons H-3, H-4, H-5, and H-6 of Kdo E occurred at unusual low ®eld positions,
Table 3 Interresidual NOE contacts observed in oligosaccharides 1±4.
s, Strong NOE; m, medium NOE; w, weak NOE Labeling is as for
Fig 1.
Oligosaccharides
Observed NOE contact
From proton To protons
3, 4 C-3ax D-6 s, E-6 m
3, 4 C-3eq D-6 s, D-7 m
3, 4 D-3eq C-5 w, E-5 m, E-6 m
3, 4 E-3ax C-5 m, F-1 w
3, 4 E-3eq C-5 s, D-3eq m, F-1 m
4 E-4 C-5 s, C-7 w, D-4 w, D-7 w, F-1 s
3, 4 E-6 C-3 axw, C-5 s, C-7 s, D-3 eqm
1, 2 F-1 E-4 s, E-5 m, E-6 m
3 F-1 E-3 eqw, E-3 axw, E-4 s, E-5 s
4 F-1 E-3 eqw, E-3 axw, E-4 s, E-5 s,
E-6 w, D-4 w
1, 2 G-1 F-2 w, F-3 w, F-4 s
2, 4 R-1 T-3 m, T-4 s, T-6 am, T-6 bm
2, 4 T-1 S-4 s, S-6 am, S-6b m
2, 4 Y-1 R-1 w, R-2 s, T-1 w, T-5 m, T-6 m
3, 4 Z-1 E-5 w, E-7 s, E-8 s
Trang 6Fig 2 Part of the NOESY spectrum of oligosaccharide 4.
Fig 3 Some important observed interresidual
NOE connectivities in the structural element
C-D-E-F of oligosaccharide 4 The depicted
structure represents one minimum energy
conformation.
Trang 7nevertheless, on the basis of conformational analysis (see
below) and comparison of the data with those of structure 5,
this residue is proposed to possess the a-con®guration
Oligosaccharide 3 possessed a structure similar to
oligo-saccharide 5 (Fig 1), which had been isolated earlier from
the LPS of A baumannii strain NCTC10303 [16] The only
difference was the presence of a-GlcpN Z in 3 However,
signi®cant differences of up to 0.4 p.p.m were observed
between some proton signals of 3 and 5, i.e C3ax, E3ax,
E3eq, E4 and E5 In order to exclude any in¯uence of
varying experimental conditions, 1H-NMR and COSY
spectra of a mixture of oligosaccharides 3 and 5 were
recorded, resulting in much closer chemical shifts: the
signals of C3ax, E3ax, E3eq, E4 and E5 of 5 were observed
at 2.174, 2.125, 2.607, 4.632, and 4.251 p.p.m., respectively,
and of 3 at 2.190, 2.184, 2.764, 4.709, and 4.270 p.p.m,
respectively Other signals (except those from protons close
to the attachment point of GlcpN Z) overlapped in both
structures The differences in chemical shifts for 3 and 5
obtained for the isolated samples must therefore be due to
small differences in sample conditions, e.g concentration,
pH or ionic strength Also, most of the NOE contacts
(Fig 2) were similar Thus, oligosaccharides 3 and 5 possess
the common hexasaccharide fragment F ® E ® (D ® )
C ® B ® A, and Kdo E is proposed to be present in
oligosaccharides 3 and 4 in a-pyranosidic form, as shown
earlier for the oligosaccharide 5 isolated from the LPS of
A baumannii strain NCTC10303 [16] The conformation of
the fragment F-C was similar in 3, 4 and 5, but not identical
An NOE contact between protons E3eq and D3eq was only
observed in the oligosaccharides from strain ATCC 19606
(compounds 3 and 4) In contrast, an NOE contact between
E3eq and F5 was only observed in the products from strain
NCTC 10303 (e.g compound 5) Conformational analyses
showed that a close proximity of protons E3eq to D3eq and
of E3eq and E4 to C5 could not simultaneously occur in the
same low energy conformation (assuming the chair
confor-mation of Kdo E) Thus, the observed NOE contacts
originate probably from different conformational minima
or from a distorted ring conformation of Kdo E The last
possibility was supported by the observation of the
intra-residual NOE contact between E3eq and E6, which is not
possible in the5C2con®guration The existence of residue E
in the equilibrium of several conformers agrees with the
observation of its broadened and unresolved H-3 signals,
whereas all other signals in the spectra of 3 and 4 were sharp
and well resolved
31P NMR spectra of oligosaccharides 3 and 4 showed two
signals of equal intensity at 2.2 and 4.5 p.p.m., which in
1H,31P-HMQC spectrum correlated with protons A1 and
B4, respectively, thus proving phosphorylation at positions
O-1 of A and O-4 of B
In summary, the structures of oligosaccharides 1±4 were elucidated as depicted in Fig 1 The structures possess a common branched tetrasaccharide fragment G-F-[Z-]-E, which is substituted in compound 2 at O-3 of GlcpNA F with the tetraglucosyl sequence Y-R-T-S- Oligosaccharides
1 and 2 differ also in the acetylation of the amino group of GalN G In the octasaccharide, this amino group is not acetylated and H-2 of the residue G resonates at 3.264 p.p.m., whereas in the tetrasaccharide this amino-group is acetylated, thus, this signal is shifted to 4.092 p.p.m Structures 1±4 can be combined to a complete structure of the carbohydrate backbone of the LPS from
A baumannii strain ATCC 19606 as depicted in Fig 4 The structures of four core regions from LPS of different Acinetobacter strains have been characterized during this work Two of these core regions (i.e of LPS from
A haemolyticus strain ATCC 17906/NCTC10305 and from Acinetobacter strain ATCC 17905) have in common the presence of Ko, which replaces in part the Kdo residue that links the core oligosaccharide to the lipid A [14,15] In both cases, this ®rst Ko or Kdo residue was substituted at O-5 by the trisaccharide a-D-Glcp-(1 ® 6)-b-D-Glcp-(1 ® 4)-a-D -Glcp (which in the core region of strain ATCC 17906/ NCTC10305 is phosphorylated at O-6 of the 4-substituted a-Glcp) The other two core regions were identi®ed in LPS
of A baumannii, i.e in strains ATCC 17904/NCTC 10303 [16] and ATCC 19606 (this work) Their common feature is the presence of the trisaccharide a-Kdo-(2 ® 5)-[a-Kdo-(2 ® 4)-]-a-Kdo-5)-[a-Kdo-(2 ® , which has also been identi®ed in the core region of LPS from A baumannii strain ATCC
15303 [19] Thus, it is possible that this trisaccharide represents a partial structure of the core region which is speci®c for LPS of A baumannii
The oligosaccharides identi®ed in the core region of LPS from A baumannii strain ATCC 19606 suggests how a part
of its biosynthesis may occur Approximately one-third of the core region is terminated by aD-GalpNAc residue (G in Fig 1), as indicated by the yield of oligosaccharide 1 Here, theD-GlcpNAcA residue F is not substituted at O-3 The other two-thirds of the core comprised an oligosaccharide in which G is a D-GalpN residue and F bears at O-3 the tetrasaccharide a-Glcp-(1 ® 2)-b-Glcp-(1 ® 4)-b-Glcp-(1 ® 4)-b-Glcp-4)-b-Glcp-(1 ® Thus, it is reasonable to assume that the tetrasaccharide is introduced only after enzymatic de-N-acetylation of GalpNAc and that 1 represents a precursor of core region biosynthesis Additionally, the isolation of the tetrasaccharide a-Kdo-(2 ® 4)-a-Kdo-(2 ® 6)-b-GlcpN4P-(1 ® 6)-a-GlcpN1P and the trisaccha-ride a-Kdo-(2 ® 6)-b-GlcpN4P-(1 ® 6)-a-GlcpN1P from deacylated LPS suggests that the a-(2 ® 4)-linked Kdo disaccharide is furnished ®rst in biosynthesis of the core region, as found e.g in LPS biosynthesis of Escherichia coli
Fig 4 The complete chemical structure of the carbohydrate backbone of the LPS from Acinetobacter baumannii strain ATCC 19606.
Trang 8[26,27] Then, the biosynthesis of the core region of LPS
from A baumannii strain ATCC 19606 should proceed with
the introduction of a third Kdo residue to O-5 of the second
Kdo This represents a remarkable difference to the
biosynthesis of LPS in E coli (and in other bacteria), in
which the second Kdo is substituted at O-5 by one residue of
L-glycero-D-manno-heptopyranose [10,26,27]
The Kdo transferase of strain ATCC 15303 has been
cloned and characterized in vitro whereby it was shown that
it is able to transfer two Kdo residues in a-(2 ® 4)-linkage
to a lipid A precursor [19] No evidence was obtained for a
second Kdo transferase for which we searched by Southern
blots Therefore, the a-(2 ® 5)-linked Kdo is either
trans-ferred by a second Kdo transferase that does not share
enough similarity with the one that was cloned or the
speci®city of the enzyme is different under in vitro and in vivo
conditions
A C K N O W L E D G E M E N T S
We thank Veronika Susott for technical assistance and Klaus Bock for
valuable discussions and critical reading of the manuscript.
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