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. 2023 Jun 15;11(3):e0414622.
doi: 10.1128/spectrum.04146-22. Epub 2023 Apr 27.

Bacterial Argonaute Proteins Aid Cell Division in the Presence of Topoisomerase Inhibitors in Escherichia coli

Anna Olina  1 , Aleksei Agapov  1 , Denis Yudin  1 , Dmitry Sutormin  2 , Alina Galivondzhyan  2 , Anton Kuzmenko  1   3 , Konstantin Severinov  4 , Alexei A Aravin  3 , Andrey Kulbachinskiy  1   5
Affiliations

Bacterial Argonaute Proteins Aid Cell Division in the Presence of Topoisomerase Inhibitors in Escherichia coli

Anna Olina et al. Microbiol Spectr. .

Abstract

Prokaryotic Argonaute (pAgo) proteins are guide-dependent nucleases that function in host defense against invaders. Recently, it was shown that TtAgo from Thermus thermophilus also participates in the completion of DNA replication by decatenating chromosomal DNA. Here, we show that two pAgos from cyanobacteria Synechococcus elongatus (SeAgo) and Limnothrix rosea (LrAgo) are active in heterologous Escherichia coli and aid cell division in the presence of the gyrase inhibitor ciprofloxacin, depending on the host double-strand break repair machinery. Both pAgos are preferentially loaded with small guide DNAs (smDNAs) derived from the sites of replication termination. Ciprofloxacin increases the amounts of smDNAs from the termination region and from the sites of genomic DNA cleavage by gyrase, suggesting that smDNA biogenesis depends on DNA replication and is stimulated by gyrase inhibition. Ciprofloxacin enhances asymmetry in the distribution of smDNAs around Chi sites, indicating that it induces double-strand breaks that serve as a source of smDNA during their processing by RecBCD. While active in E. coli, SeAgo does not protect its native host S. elongatus from ciprofloxacin. These results suggest that pAgo nucleases may help to complete replication of chromosomal DNA by promoting chromosome decatenation or participating in the processing of gyrase cleavage sites, and may switch their functional activities depending on the host species. IMPORTANCE Prokaryotic Argonautes (pAgos) are programmable nucleases with incompletely understood functions in vivo. In contrast to eukaryotic Argonautes, most studied pAgos recognize DNA targets. Recent studies suggested that pAgos can protect bacteria from invader DNA and counteract phage infection and may also have other functions including possible roles in DNA replication, repair, and gene regulation. Here, we have demonstrated that two cyanobacterial pAgos, SeAgo and LrAgo, can assist DNA replication and facilitate cell division in the presence of topoisomerase inhibitors in Escherichia coli. They are specifically loaded with small guide DNAs from the region of replication termination and protect the cells from the action of the gyrase inhibitor ciprofloxacin, suggesting that they help to complete DNA replication and/or repair gyrase-induced breaks. The results show that pAgo proteins may serve as a backup to topoisomerases under conditions unfavorable for DNA replication and may modulate the resistance of host bacterial strains to antibiotics.

Keywords: DNA gyrase; DNA replication; antibiotic resistance; cell division; gyrase; prokaryotic Argonautes; ter sites.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
Analysis of pAgo functions in E. coli. E. coli strains expressing plasmid-encoded SeAgo or LrAgo or containing a control empty plasmid were grown in the absence or in the presence of ciprofloxacin (Cfx), followed by CFU counting, cell microscopy, and analysis of pAgo-associated smDNAs.
FIG 2
FIG 2
Growth of E. coli strains lacking or expressing pAgos at different concentrations of Cfx. E. coli transformed with an empty pBAD plasmid or pBAD encoding SeAgo or LrAgo were grown at 30°C in a plate reader with indicated concentrations of Cfx (0 to 62.5 ng/mL) and Ara to induce expression of pAgos, and OD600 was monitored over time. E. coli transformed with empty pBAD and grown without Ara was used as a control. Means from four biological replicates are shown; shades represent 0.95 confidence intervals of the mean. Gray rectangle highlights the range of Cfx concentrations (0.5 to 15.6 ng/mL), the inhibitory effects of which are suppressed by pAgos expression.
FIG 3
FIG 3
Comparison of the effects of pAgos on the viability of wild-type and rec-minus E. coli strains grown in the absence and in the presence of Cfx. (A) Growth kinetics of the wild-type and recA-minus and recBD-minus strains, measured in a plate reader. Cfx was added to 0.5 ng/mL when indicated. Means and standard deviations from three biological replicates are shown. (B) Comparison of the number of viable cells (CFU) in the wild-type and mutant E. coli strains lacking or expressing pAgos in the absence and in the presence of Cfx. The samples were taken from E. coli cultures grown for 4.5 h in the absence or in the presence of Cfx (indicated with dashed lines in panel A), and CFU numbers were determined by their serial plating without Cfx. Representative LB plates from one of biological replicates are shown for E. coli strains grown in the absence (top) or in the presence (bottom) of Cfx (see Fig. S2 in the supplemental material for CFU numbers from the three biological replicates).
FIG 4
FIG 4
Effects of pAgo expression and gyrase inhibition on E. coli cell morphology. (A) E. coli cells lacking or containing pAgos were grown in the absence (left) or in the presence (right) of Cfx. The samples were taken at 4.5 h from the cultures shown in Fig. 3. (B) Effects of dCas9 gyrase (gyrA) knockdown on cell morphology. Fluorescence microscopy after acridine orange staining. The scale bar is 10 μm. See Fig. S3 in the supplemental material for additional fields of view.
FIG 5
FIG 5
Characteristics of smDNAs associated with pAgos in E. coli in the absence or in the presence of Cfx. The smDNA samples were isolated at the exponential phase of growth (5.5 h) (see Fig. S4 in the supplemental material for the growth curves). (A) Distribution of smDNA lengths for each smDNA library. (B) Nucleotide logos for different smDNA positions starting from the 5′-end of guide DNA. (C) GC content of the smDNA sequences and of the surrounding genomic regions for each condition. The mean GC content of the E. coli genome is indicated.
FIG 6
FIG 6
Whole-genome mapping of smDNAs associated with pAgos in E. coli. (A) Genomic distribution of smDNAs isolated from SeAgo and LrAgo in the absence or in the presence of Cfx at the logarithmic or stationary phases of growth (5.5 and 12.5 h) (see Fig. S4 in the supplemental material). The numbers of smDNAs along the genomic coordinate are shown in reads per kilobase per million aligned reads in the smDNA library (RPKM), individually for the plus (green) and minus (red) genomic strands. The ori, ter sites, and the direction of replication are indicated. (B) Targeting of the ter region by pAgos. SmDNA densities in each genomic strand (plus strand, green; minus strand, red) in the ter region in E. coli cultures grown in the absence (top) and in the presence (bottom) of Cfx are shown. Positions of the terA, terC, terD, and terB sites are shown with dashed lines and the directions of replichores are shown with arrows. Chi sites in the plus (green) and minus (red) strands are shown above the plots. The closest Chi sites oriented toward terA (“Chi−”), and terC (“Chi+”) are indicated. SmDNA numbers are shown in RPKM. The numbers of smDNAs from each DNA strand from each ter site, calculated for genomic regions between the ter site and the closest Chi site in the correct orientation, are shown as a percentage of the total number of smDNAs mapped to both strands of the whole genomic sequence in each smDNA library. (C) Effects of Sfx on the distribution of pAgo-associated smDNA between genomic DNA strands during replication. The ratio of pAgo-associated smDNAs corresponding to the plus and minus genomic strands was calculated independently for samples isolated from strains grown in the presence and in the absence of Cfx, and the obtained profiles were then divided by each other.
FIG 7
FIG 7
Asymmetry of smDNA distribution around Chi sites. (A) Scheme of DNA processing by RecBCD between a DSB and an upstream Chi site, illustrating the observed polarity of smDNA loading into pAgos. DNA unwinding by RecBCD is followed by asymmetric processing of the two DNA strands by RecBCD and other nucleases; RecBCD loads RecA onto the 3′-terminated strand at the Chi site, thus shielding this strand from further degradation. (B) Metaplots of the densities of smDNAs around Chi sites analyzed for smDNA libraries isolated from logarithmic cultures (5.5 h of growth) of E. coli strains grown in the absence and in the presence of Cfx (averages from two replicate experiments). SmDNA numbers were independently calculated for the DNA strands co-oriented (green, F) and oppositely oriented (gray, R) with the Chi sequence (5′-GCTGGTGG-3′) for all Chi sites in both genomic strands (833 sites in total) and smoothened with a 400-bp sliding window. (C) Comparison of metaplots of normalized densities of smDNAs around co-oriented Chi sites for smDNA libraries isolated from E. coli grown in the absence and in the presence of Cfx (the data correspond to green smDNA profiles in panel A). For normalization, averaged and smoothened RPKM values around Chi sites were divided by the background RPKM value calculated for regions remote from Chi sites (−50 to −35 kb and +35 to +50 kb from the Chi sequence). Arrows indicate relative differences between background and minimal densities of smDNAs at the Chi sites.
FIG 8
FIG 8
Effects of Cfx on the enrichment of pAgo-associated smDNAs around Chi sites with an adjacent GCS at the 3′-side. (A) Scheme of DNA processing between a GCS and the closest upstream Chi site, illustrating the observed polarity of smDNA loading into pAgos. (B) Metaplots of the relative densities of smDNAs around Chi sites adjacent to a downstream GCS from the 3′-side of the Chi sequence (carmine curves, 188 sites in total) and all other Chi sites, lacking adjacent downstream GCSs (lilas curves, 645 sites in total). A Chi site and a GCS were considered adjacent if there were no co-oriented Chi sites in between. The data were averaged for two replicate experiments for the exponential (5.5 h, top) and early stationary (12.5 h, bottom) phases of growth. SmDNA density was calculated for DNA strands co-oriented with the Chi sites for +Cfx and −Cfx conditions independently, and then +Cfx density was divided by Cfx density. The resultant relative density was smoothened with a 1-kb sliding window. Blue rectangles mark the region (from the Chi sequence to +5 kb) used to quantify the relative enrichments of smDNAs in panel B. (C) Quantification of relative enrichments of smDNAs at the 3′-sides of Chi sites for Chi sites adjacent to a downstream GCS (DS, carmine) and all other Chi sites (Oth, violet). The error bars represent mean values ± standard deviation (SD) for the same sets of Chi sites as in panel A. The enrichments were compared by a two-sided t test. P values of <0.0005 are indicated with three asterisks.
FIG 9
FIG 9
Possible activities of pAgos explaining their observed suppressor effects on bacterial cell growth. pAgos capture guide smDNAs generated during chromosomal DNA replication and repair with the participation of RecBCD. Guide-loaded pAgo may then (i) relax supercoils in the replicating chromosome through its DNA-nicking activity, (ii) help to decatenate chromosomes under conditions of gyrase inhibition by attacking both DNA strands with guide DNAs corresponding to the ter region or by processing of nicked DNA strands, (iii) participate in the processing of DSBs generated after gyrase inhibition by Cfx and possibly facilitate removal of covalently bound gyrase, or (iv) recruit additional factors involved in DNA processing and repair (as proposed for TtAgo [35]).
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