1
Bacterial Argonaute proteins aid cell division
in the presence of topoisomerase inhibitors in
Escherichia coli
Anna Olina, Aleksei Agapov, Denis Yudin, Anton Kuzmenk
o,
Alexei A. Aravin, Andrey Kulbachinskiy
SUPPLEMENTARY INFORMATION
2
Fig. S1.
(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, transfe
rred to a nitrocellulose
membrane and visualized by Western-blotting with His-tag specif
ic antibodies (see Materials
and Methods for details). The green arrow points to SeAgo, the pink
one indicates LrAgo. (B)
Analysis of
E. coli
growth at sublethal concentrations of ciprofloxacin. Wild-type
E. coli
were
grown at 30
°
C in a plate reader with indicated concentrations of ciprofloxacin (
0, 0.1, 0.3 or 0.5
μ
g/ml). Averages from 3 biological replicates are shown.
3
Fig. S2. Analysis of the number of viable cells in
E. coli
strains lacking or containing pAgos in
the absence and in the presence of ciprofloxacin.
(A) The numbers of colony forming units
(CFU) in indicated
E. coli
strains. The samples were taken from
E. coli
cultures grown for 4.5
hours in the absence or in the presence of ciprofloxacin (Fig. 2),
and CFU numbers were
determined by plating their serial dilutions on LB agar plates
without ciprofloxacin.
Averages
from three independent biological replicates are shown; individual
data points from each
measurement are indicated. (B) Representative LB plates f
or
E. coli
strains grown in the
absence (top) or in the presence (bottom) of ciprofloxacin. The CFU
numbers calculated for
each strain are shown on the right.
4
Fig. S3. Analysis of smDNA libraries from
E. coli
strains expressing pAgos.
(A) Preparative-scale
cultivation of
E. coli
strains lacking of containing pAgos (SeAgo, top; LrAgo, bottom). Th
e
cultures were grown in 0.5 liter of LB in the absence and in th
e presence of ciprofloxacin (0.3
μ
g/ml) and OD
600
was monitored each 30 minutes. The dashed lines indicate 5.5 h a
nd 12.5 h
time points used for purification of pAgo-associated smDNAs. (B
) Analysis of smDNAs purified
from pAgos. SmDNAs isolated from pAgos after one-step purifica
tion (using Co
2+
-affinity resin)
were treated with alkaline phosphatase to remove pre-existing 5’
-phosphates, labeled with
γ
-
P
32
-ATP and polynucleotide kinase and separated by 19% denaturing urea
PAGE. The marker
lane (M) contains 5’-labeled DNA oligonucleotides of indicated
lengths.
5
Fig. S4. Analysis of smDNA distribution relative to tr
anscription units in
E. coli
.
(A) Densities of
smDNAs
within genes co-directed or reversely directed relative to
replication. For each gene,
the amounts of smDNAs were calculated independently for the sense
and antisense DNA
strands, normalized by the gene length and expressed as RPKM (r
eads per kilobase per million
reads in the library). The data were independently averaged for
each of the four types of
orientation (sense and antisense strands for co-directed and reve
rsely directed genes. (B)
Densities of smDNAs
in intergenic regions for convergent, divergent, and co-orient
ed gene pairs
(located either in the plus or in the minus genomic strands). Only
<500 bp intergenic regions
were taken into account. SmDNA numbers were independently avera
ged for each of the four
types of gene orientation (convergent, divergent, and two directi
ons of co-oriented genes),
normalized by the length of each intergenic region and expressed i
n RPKM. The data are shown
as box plots (center line, median; box limits, upper and lower quarti
les; whiskers, 1.5x
interquartile range).
6
Table S1. Small DNA libraries obtained and analyzed in this st
udy.
The data
are available from the SRA database, bioproject numb
er PRJNA878808
library
number
library name
pAgo
ciprofloxacin
time,
h
SRA
accession
reads
mapped to
genome
reads
mapped to
genome, %
reads
mapped to
plasmid
reads
mapped to
plasmid, %
enrichment in
plasmid
coverage*
1 Se_5.5_nocip_rep_1 SeAgo
- 5.5 SRR21504308
368873 81.6 83301 18.4 11.4
2 Se_5.5_nocip_rep_2 SeAgo
- 5.5 SRR21508618
488441 79.3 127566 20.7 12.8
3 Se_5.5_cip_rep_1 SeAgo
0.3 μg/ml 5.5 SRR21508717
1509243 88.4 197824 11.6 7.2
4 Se_5.5_cip_rep_2 SeAgo
0.3 μg/ml 5.5 SRR21508866
1038304 87.2 152951 12.8 8
5 Se_12.5_nocip_rep_1
SeAgo
- 12.5
SRR21508879
791799 78.9 211831 21.1 13.1
6 Se_12.5_nocip_rep_2
SeAgo
- 12.5
SRR21508723
675406 77.7 193981 22.3 13.8
7 Se_12.5_cip_rep_1 SeAgo
0.3 μg/ml 12.5
SRR21508709
2152542 87.1 318401 12.9 8
8 Se_12.5_cip_rep_2 SeAgo
0.3 μg/ml 12.5
SRR21508927
2920406 86.4 460114 13.6 8.4
9 Lr_5.5_nocip_rep_1 LrAgo
- 5.5 SRR21508877
1266921 94.3 75910 5.7 3.5
10 Lr_5.5_nocip_rep_2 LrAgo
- 5.5 SRR21508707
1984106 94.8 108631 5.2 3.2
11 Lr_5.5_cip_rep_1 LrAgo
0.3 μg/ml 5.5 SRR21508916
1290785 92.5 105237 7.5 4.7
12 Lr_5.5_cip_rep_2 LrAgo
0.3 μg/ml 5.5 SRR21508919
1265045 91.3 121100 8.7 5.4
13 Lr_12.5_nocip_rep_1
LrAgo
- 12.5
SRR21508925
1196750 86.0 194585 14.0 8.6
14 Lr_12.5_nocip_rep_2
LrAgo
- 12.5
SRR21508926
1003480 87.4 145080 12.6 7.8
15 Lr_12.5_cip_rep_1 LrAgo
0.3 μg/ml 12.5
SRR21508713
2974923 83.1 606222 16.9 10.4
16 Lr_12.5_cip_rep_2 LrAgo
0.3 μg/ml 12.5
SRR21508921
1556966 84.5 285548 15.5 9.6
* The enrichment of plasmid-derived smDNAs is calculated as a ratio of
the observed number of plasmid-mapped reads to the expected number of plasmid
mapped reads. The expected number of reads = (genome-mapped reads + plasmid-mapped re
ads)
×
(plasmid size
×
copy number)/( genome size + plasmid size
×
copy number)