1
Bacterial Argonaute proteins aid cell division
in the presence of topoisomerase inhibitors in
Escherichia coli
Anna Olina, Aleksei Agapov, Denis Yudin, Dmitry Sut
ormin, Alina Galivondzhyan,
Anton Kuzmenko, Konstantin Severinov, Alexei A. Ara
vin, Andrey Kulbachinskiy
SUPPLEMENTAL MATERIAL
Includes Figures S1-S10, Tables S1, S2
2
Fig. S1.
Expression of SeAgo and LrAgo in
E. coli
.
(A) Analysis of pAgo expression in
E. coli
by
Western blotting.
Protein
samples were obtained from the wild-type or
rec-
minus
E. coli
strains
at 4.5 hours of growth. The proteins were separated
by 4-20% gradient denaturing SDS-PAGE,
transferred to a nitrocellulose membrane and visual
ized by Western-blotting with His-tag
specific antibodies (see Materials and Methods for
details). The turquoise and pink arrows
show positions of SeAgo and LrAgo, respectively. (B
) Growth of
E. coli
strains in the absence or
presence of Cfx depending on expression of SeAgo (u
niduced, 0% Ara; induced with 0.01% or
0.1% of Ara).
E. coli
cells were transformed with empty pBAD or pBAD enc
oding SeAgo and
were grown at 30
°
C in a plate reader without Cfx or with Cfx (0.5 ng
/mL) and indicated
concentrations of arabinose. Averages from 3 techni
cal replicates are shown.
3
Fig. S2. Analysis of the CFU numbers for
E. coli
strains lacking or containing pAgos and grown
in the absence and in the presence of Cfx.
The samples were taken from
E. coli
cultures grown
for 4.5 hours in the absence or in the presence of
Cfx (Fig. 3A), and CFU numbers were
determined by plating their serial dilutions on LB
agar plates without Cfx.
Means from three
biological replicates are shown, individual data po
ints from each measurement are indicated
(colored pairwise for each independent experiment).
4
Fig. S3.
Effects of pAgo expression on
E. coli
cell morphology.
E. coli
transformed
with a control
empty pBAD plasmid or pBAD ncoding pAgos were grown
in the absence (top three rows) or in
the presence (bottom three rows) of ciprofloxacin.
The expression of pAgos was induced with
0.01% Ara. The samples were taken at 4.5 hours from
the cultures shown in Fig. 3A.
Fluorescence microscopy after acridine orange stain
ing. The scale bar is 10
μ
m. The first column
corresponds to images presented in Fig. 4, the othe
r four images in each row are additional
microscopic fields of view for the same samples.
5
Fig. S4. Preparation of smDNA libraries from
E. coli
strains expressing pAgos.
(A) Preparative-
scale cultivation of
E. coli
strains lacking of containing pAgos. All cultures w
ere grown in 0.5 liter
of LB in the absence and in the presence of Cfx (0.
3 ng/mL) and 0.01% Ara, and OD
600
was
monitored each 30 minutes. The dashed lines indicat
e 5.5 h and 12.5 h time points used for
purification of pAgo-associated smDNAs. (B) Analysi
s of smDNAs purified from pAgos. SmDNAs
isolated from pAgos after one-step purification (us
ing Co
2+
-affinity resin) were treated with
alkaline phosphatase to remove pre-existing 5’-phos
phates, labeled with
γ
-P
32
-ATP and
polynucleotide kinase and separated by 19% denaturi
ng urea PAGE. The marker lane (M)
contains 5’-labeled DNA oligonucleotides of indicat
ed lengths.
6
Fig. S5. Asymmetry of smDNA distribution around Chi
sites at the stationary phase of growth.
SmDNA analysis was performed as described in Fig. 7
, but for the stationary cultures (12.5
hours of growth). Averages from two replicate exper
iments are shown. (A) Metaplots of the
densities of smDNAs around Chi sites. SmDNA numbers
were independently calculated for the
DNA strands co-oriented (green, F) and oppositely o
riented (gray, R) relative to the Chi
sequence (5’-GCTGGTGG-3’) for all Chi sites in both
genomic strands and smoothened with a
400 bp sliding window. (B) Metaplots of the normali
zed densities of smDNAs around co-
oriented Chi sites
for
E. coli
strains grown in the absence and in the presence of
Cfx (the data
correspond to green smDNA profiles in panel A). Arr
ows indicate relative differences between
the background and minimal densities of smDNAs arou
nd the Chi sites.