2. Material and methods
2.1 E. coli and bacteriophage strains and their
cultivation
The E. coli strain MG1655 lysogenized for phage φ24B:cat was a
kind gift of Prof. G. Wegrzyn, University of Gdansk, Poland. Phage T5
was a gift of Dr. V. Ksenzenko (Institute of protein research RAS,
Puschino-na-Oke, Russia). We previously described T5-like bacteriophages
of DT57C species and their LTF mutants30. These
include: phage DT57C, phage DT571/2, DT571/2 ltfA-mutant lacking the LTFs (hereafter FimX) and DT571/2 mutant ABF that
carries LTF non-branched LTF with only one receptor-binding domain
(instead of two such domains on the branched LTFs of the phages DT57C or
DT571/2). Bacteriophage 9g, a siphovirus representing the type strain of
the genus Nonagvirus 40. Gostya9 is a T5-like
bacteriophage that was shown to recognize a different secondary receptor
distinct from the receptors of the phages T5, DT57C and 9g41. Bacteriophage G7C, a N4-related podovirus
specifically recognizing O antigen of E. coli 4s strain was isolated and
characterized by us previously 42, 43. We isolated all
the above-mentioned phages except for T5 and engineered phage mutants
from horse feces as it described in the corresponding publications cited
above.
The wild E. coli strains were previously isolated by us from
horse feces and characterized. These were 4s (O22)29,
HS1/2 (O87)37, 44, HS3-104 (O81)39,
F5 (O28 ab)35, and F17 (new
O-serotype)36. The clinical uropathogenic E.
coli isolates UP1 and UP11 were received from the clinical
microbiological facility of the Institute of Epidemiology (Moscow,
Russia). UP11 strain was further identified as an O5 O-antigen
producer34.
The ability of the strains to produce O antigens was controlled by LPS
profiling as described in Kulikov et al. (2019)31.
E. coli 4s and F17 rough variants 4sR (a wclH mutant of
4s)29 and F17 wbbL -36 were engineered by us previously.
All the E. coli strains were cultured on LB medium (trypton 10 g,
yeast extract 5 g, NaCl – 10 g, distilled H2O – up to
1 l). This medium was supplemented with 15 g of bacto-agar per 1 l for
plates or with 6 g of bacto-agar for top agar.
Bacteriophage FimX was propagated on E. coli 4sR and enumerated using
the conventional double-layer plating technique.
Bacteriophage φ24B:cat was obtained by mitomycin C induction of E.
coli MG1655 (φ24B:cat) strain. For this procedure, the overnight
culture of the lysogen was grown in the presence of 34 μg/ml of
chloramphenicol. Then 300 ml of LB in 500 ml Erlenmeyer flask was
inoculated with 3 ml of the overnight culture (N.B. – this volume ratio
gave a better phage yield than conventional conditions with better
aeration). The culture was grown in the orbital shaker at 220 rpm,
37oC up to OD600 = 0.2. The mitomycin
C was then added up to 1 μg/mL and the incubation was continued
overnight at the same conditions. After the incubation, lysis of the
culture was observed. The lysate was cleared by centrifugation at 15000
× g for 15 min. The supernatant was collected,
PEG-precipitated45, and resuspended in 3 mL of SM
buffer (Tris-HCl pH 7.5 – 10 mM, NaCl – 50 mM, MgCl2– 10 mM, gelatin – 5 g/l). The phage stock was titered and used in
these experiments.
For titration of the phage φ24B:cat, a modified double-layer technique
was used. The top-layer medium contained 4 g/l of the bacto-agar
(instead of 6 g/l) and was supplemented with CaCl2 up to
5mM. The bottom layer was supplemented with 2.5 μg/ ml of
chloramphenicol. 300 μg of log-phase culture of E. coli C600
(OD600 = 0.6) was used for the lawn inoculation.
2.2 Lysogenization of the E. coli
strains
This procedure was performed as described in James et
al.23 with minor modifications. Briefly, a mid-log
liquid culture of an appropriate strain was grown in LB medium, the
phage was added at a multiplicity of 5 pfu/host cfu, and the mixture was
incubated at 37oC for 30 min. After the incubation,
the cells were spun down in a table-top centrifuge (10000 × g, 1 min),
the cells were resuspended in LB, washed twice with LB to remove non
bound phage and plated on plates supplemented with 34 μg/ml of
chloramphenicol for lysogen selection.
2.3. LPS profiling
by SDS-PAGE electrophoresis was performed as recently described31.