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Perepelov et al.
figurations of the monosaccharides were determined by GC of
the acetylated (S)-2-octyl glycosides as earlier described.8
An O-polysaccharide sample (12 mg) was treated with an-
hydrous CF3CO2H (0.2 mL) at 90 C for 1 h. After evaporation
of the volatiles, the reaction products were dissolved in water and
fractionated by gel-permeation chromatography on a column
(851.6 cm) of Fractogel TSK HW-40S in 1% AcOH to give
oligosaccharide fractions I—IV (2.6, 1.8, 1.0, and 2.7 mg, respec-
tively) and a monosaccharide fraction (1.5 mg).
Fig. 3. Structure of the O-polysaccharide of E. coli O60.
For measuring NMR spectra, an O-polysaccharide sample
was freeze-dried from 99.9% D2O and dissolved in 99.95% D2O.
The NMR spectra were recorded on a Bruker Avance II 600 MHz
spectrometer (Germany) at 20 C using sodium 3-(trimethylsilyl)
propanoate-2,2,3,3-d4 (H 0.0, C 1.6) as an internal reference.
The 2D NMR spectra were obtained using standard Bruker
software; TopSpin 2.1 program (Bruker) was used to acquire and
process the NMR data. A mixing time of 100 and 150 ms was
used in the TOCSY and ROESY experiments, respectively.
A 60-ms delay was used in the 1H,13C HMBC experiment for
development of multi-bond correlations.
Positive ion mode high-resolution electrospray ionization
mass spectra were measured on a Bruker micrOTOF II instru-
ment. Internal calibration was done with Electrospray Calibrant
Solution (Fluka). Samples (~50 ng L–1) were dissolved in a 1 : 1
(v/v) H2O—MeCN mixture and sprayed at a flow rate of
3 L min–1 using nitrogen as the nebulizing gas (4 L min–1). The
capillary entrance voltage was –3000 V, exit voltage was 150 V,
and interface temperature was 180 C.
The O-polysaccharide structure established does not
match the content of the O-antigen gene cluster at the
typical location on the E. coli chromosome.4 Thus, the
gene cluster contains manB, manC and gmd genes for
synthesis of biosynthetic precursors of D-mannose and
L-fucose, which are not the components of the O-poly-
saccharide of E. coli O60, and no genes for synthesis of
a biosynthetic precursor of L-rhamnose, which is a com-
ponent of the O-polysaccharide, are present in the cluster.
Hence, the functional gene cluster for biosynthesis of the
O-antigen of E. coli O60 is located elsewhere in the ge-
nome; it remains to be found and characterized. A similar
situation, namely the presence of a non-functional gene
cluster at the typical location and a functional gene clus-
ter elsewhere on the chromosome, has been reported by
us for the O-antigen of E. coli O62.5
The authors are grateful to Bin Liu and Lei Wang
(Nankai University, Tianjin, China) for providing the
bacterial mass.
This work was financially supported by the Russian
Science Foundation (Project No. 14-14-01042_P).
Experimental
E. coli O60 type strain was obtained from the Institute of
Medical and Veterinary Science (Adelaide, Australia). Bacteria
were grown to late log phase in 8 L of Luria—Bertani broth using
a 10-L BIOSTAT C-10 fermenter (B. Braun Biotech Int.,
Germany) under constant aeration at 37 C and pH 7.0. Bacterial
cells were washed and dried as earlier described.6
References
A lipopolysaccharide sample was isolated from bacterial cells
in 6.9% yield by phenol-water extraction,7 the crude extract was
dialyzed without separation of the layers and freed from nucleic
acids and proteins by treatment with 50% aqueous CCl3CO2H
at 4 C to pH 2. The precipitate was removed by centrifugation,
and the supernatant was dialyzed and lyophilized.
Mild acid degradation (100 C, 1 h) of the lipopolysaccharide
(106 mg) was performed with 2% aqueous AcOH. The precipitate
was removed by centrifugation (13 000 g, 20 min), and the super-
natant was fractionated by gel-permeation chromatography on
a column (56×2.6 cm) of Sephadex G-50 Superfine (Amersham
Biosciences, Sweden) in 0.05 M pyridinium acetate buffer
(pH 5.5); chromatography was monitored with a differential
refractometer (Knauer, Germany). The O-polysaccharide yield
was 52% of the lipopolysaccharide weight.
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(0.32 mm×30 m) using a temperature program of 160 C (1 min)
to 290 C at a heating rate of 7 deg min–1. The absolute con-
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Received September 15, 2018;
accepted October 16, 2018
Perepelov
