Skip to main content
NCBI home page
Scientific Reports logoLink to Scientific Reports
. 2025 Apr 13;15:12711. doi: 10.1038/s41598-025-96943-2

Rapid assembly of biofilms from DNA released by SOS-inducing drugs in enteric bacteria

John K Crane 1,✉, Tammy Yang 1
PMCID: PMC11994792  PMID: 40223123

Abstract

The SOS response is a bacterial stress response activated by DNA damage in many types of bacteria. SOS-inducing antibiotics trigger the rapid release of DNA into the extracellular medium in many strains. Surprisingly, the DNA released in this way contains greater amounts of single-stranded DNA (ssDNA) than double-stranded DNA (dsDNA). In this study, we observed that addition of DNA-binding proteins following induction of the SOS response in Enterobacter cloacae decreased the amount of DNA measurable in the supernatant medium, but increased the amount of DNA deposited as a biofilm at the air-fluid interface. Bacteria incorporated into the biofilms survived the stress of dessication much better than did planktonic bacteria, with over a 400-fold increase in survival in the biofilm-bound bacteria. SOS-inducing drugs also triggered DNA release in Proteus mirabilis, with ssDNA again being more abundant than dsDNA in the culture supernatants. Addition of urea in this urease-producing organism triggered the formation of struvite crystals (magnesium ammonium phosphate), with the crystals, Proteus bacteria, and extracellular DNA forming mixed biofilms. Last, we tested the effect of inhibitors of the SOS response, such as zinc acetate. We also tested an inhibitor of the generalized stress response, dequalinium, which also indirectly inhibits the SOS response, and found it had a strong ability to inhibit biofilm formation.

Subject terms: Antibacterial drug resistance, Epidemiology, DNA

Introduction

The term “SOS” comes from the universal distress call used by mariners and aviators, and the term “SOS Repair” was coined by Miroslav Radman in 1975. The SOS response can be triggered in bacteria by exposure to ultraviolet light, antibiotics, cancer chemotherapy drugs1, antidepressants, antiviral drugs2, herbicides, and many other drugs and environmental chemicals. The SOS response continues to attract research attention because of its ability to trigger emergence of resistance to antibiotics3–6.

The key sensor of DNA damage in bacteria is RecA (recombinase A) because of its ability to bind to ssDNA exposed in response to such damage. Rad51 is a mammalian protein with roles similar to those of RecA in bacteria.

More recently we investigated the role of the SOS response in formation of biofilms7, and especially in the role of DNA release from bacterial cells in biofilm formation8. SOS-induced DNA release has not received much attention in the biomedical literature, although that may finally be beginning to change9. SOS-inducing drugs trigger a large release of DNA into the extracellular medium in many strains of Gram-negative bacteria. Interestingly, release of single-stranded DNA (ssDNA) exceeds double-stranded DNA (dsDNA) in all of the strains we have studied8.

In this study we performed experiments intended to determine the mechanism of the SOS-induced DNA release, and although several pathways are still likely contenders in the DNA release, we did make substantial progress in understanding the assembly of DNA-containing biofilms. Since ssDNA binds to RecA, a key mediator of the SOS response, we tested the role of RecA protein and other DNA-binding proteins in the formation of biofilms. Our hypothesis was that extracellular RecA, by its ability to bind to ssDNA, would enhance release of ssDNA into supernatant medium. This hypothesis was only partially true, because RecA, and other DNA binding proteins, promoted deposition of DNA into biofilm rather than solely into the extracellular medium.

We also found that bacteria incorporated into biofilms were much more resistant to the stress of dessication (drying) compared to planktonic bacteria. We also extended our work by investigating DNA release from the urease-producing pathogen Proteus mirabilis.

We also tested the role of the antidepressant fluoxetine, which we and others showed promoted the SOS response10, and horizontal gene transfer resulting in antibiotic resistance11,12. Last, we tested if the DNA release and biofilm formation could be inhibited by agents such as dequalinium, an inhibitor of the generalized stress response in bacteria13, or by zinc acetate, an inhibitor of RecA activity14.

Results

One of our goals in this project was to determine the mechanism(s) by which DNA is released from the bacterial cell in response to SOS-inducing drugs. Our previous work showed that SOS-inducers triggered prominent bacterial aggregation, with the bacterial aggregates often surrounded by extracellular DNA15. We subsequently found out that ssDNA was also released during this process, and that ssDNA exceeded dsDNA release by a factor of 4 or more in many strains8. We wondered if the abundance of ssDNA in our bacterial supernatants was because ssDNA release preceded release of dsDNA. If so, this might provide a clue as to the mechanism of DNA release. To address this, we did more detailed time course experiments using our strain of Enterobacter cloacae, E_clo_Niagara.

Figure 1 shows the results of the time course experiments. Figure 1A shows a graph of the culture turbidity in these experiments with or without the classical inducer of the SOS response, mitomycin C. The mitomycin C-treated suspensions showed little growth penalty until about 3.5 to 4 h. Figure 1B shows that release of dsDNA was not detected until about the 4 h time point, after which dsDNA release continued rapidly.

Fig. 1.

Fig. 1

Open in a new tab

Time Course of Growth and DNA Release in E. cloacae. Panel A, effect of mitomycin C on time course of growth as measured by culture turbidity. Panel B, effect of mitomycin C on dsDNA release. Panel C, effect of mitomycin C on ssDNA release. IN Panel C, early time points are omitted due to carry-over of ssDNA from the overnight culture in LB broth.

Assays of ssDNA were complicated by the fact that LB medium, in which we grew the E_clo_Niagara overnight, contains abundant amounts of ssDNA, derived from the yeast extract of this commonly used nutrient broth. In addition to ssDNA, LB broth also contains small RNA fragments16, nucleotides, and many other small molecules and metabolites17. As a result of the ssDNA in the medium, ssDNA was detectable at the early time points in the growth curves (Fig. 1C) due to carry-over from the overnight culture medium. This ssDNA deceased over the first 4 h of growth, and then began to increase again rapidly, but only in the mitomycin C-treated suspensions.

Comparing the time courses of release of dsDNA and ssDNA, Fig. 1, Panels B and C, show that both forms of DNA began to appear in the supernatant medium at 4 h, or 2.5 h after the addition of the mitomycin C. After that, dsDNA and ssDNA release both increased steeply. These findings did not support our hypothesis that ssDNA release preceded the release of dsDNA. Figure 1 does show, however, that ssDNA release exceeded that of dsDNA by almost threefold.

Induction of the SOS response triggers release of the RecA protein into the culture supernatants6,18. Therefore, a revised hypothesis for the excess of ssDNA over dsDNA was RecA binding to ssDNA in the extracellular space. RecA binds strongly to ssDNA and forms a stiff nucleofilament. Our revised hypothesis was that extracellular RecA might promote ssDNA release from the bacterial cell, or protect the ssDNA released from breakdown by nucleases. Figure 2 shows the experiments performed to determine if there was a role for extracellular RecA in DNA release.

Fig. 2.

Fig. 2

Open in a new tab

Effect of exogenous DNA binding proteins on DNA in supernatants and in biofilms, and on bacterial survival. Panels A and B, effect of purified RecA protein and histone III-S on dsDNA and ssDNA release. Histone III-S is also known as histone H3. Panel C, lack of effect of bovine serum albumin on dsDNA in supernatant medium. Panel D, photograph of DNA fluorescence in DAPI-stained culture tubes, showing biofilms at the air-fluid interface. After scanning on the GelDoc EZ fluorescence imager, the image was “inverted” to display as dark lines on a light background. Panel E, quantitation of the fluorescence of DNA in biofilm in Panel D. Panel F, effect of SOS-inducers on bacterial survival after 16 h of dessication.

We performed our initial experiments with RecA, but later expanded our experiments to determine if a eukaryotic DNA-binding protein, histone III-S, had similar effects. Figure 2A shows experiments testing if addition of purified exogenous RecA protein increased DNA release. The results showed a result that was the opposite of our hypothesis, because RecA decreased the amount of dsDNA we measured in the culture supernatants. RecA protein caused a similar inhibition of ssDNA release (Fig. 2B). Addition of purified histone III-S was even more efficacious than RecA in its ability to inhibit DNA release of both forms of DNA (Fig. 2, Panels A and B). As a control, we also tested bovine serum albumin (BSA), and found that it did not inhibit DNA release.

While performing the experiments shown in Fig. 2, Panels A-C, we noted that, while DNA concentrations in the supernatant medium decreased with the DNA-binding proteins, the biofilm visible at the air-fluid interface of our cultures treated with mitomycin C plus RecA or histone greatly increased (Fig. 2, Panels D and E). These biofilms were visible to the naked eye. In Fig. 2D, however, we stained the biofilms with the DNA-binding dye di-amidino-phenylindole (DAPI). The biofilms from E_clo_Niagara treated with mitomycin + RecA, and mitomycin + histone, showed large increases in the amount of DNA signal in the biofilms. We quantitated the DNA by scanning the glass tubes in a UV gel scanner and the UV fluorescence is shown in Fig. 2E.

The results of Fig. 2, Panels A-E, showed that DNA-binding proteins RecA and histone III-S triggered a decrease in the amount of DNA in the supernatant medium, while greatly increasing the DNA content of the biofilm. A detailed accounting of the amounts of dsDNA and ssDNA in the supernatants and biofilms of E_clo_Niagara are shown in Supplemental Fig. 1. Our revised hypothesis is that the DNA-binding proteins caused a translocation of the DNA from the liquid phase to the adherent biofilm. This could be relevant in vivo since histone, along with DNA and other peptides and enzymes, is released from eukaryotic cells during the process of NETosis, or formation of neutrophilic extracellular traps19.

The abundant DNA-containing biofilms observed in Fig. 2, D and E, made us wonder if these biofilms might have protective effects on bacteria that became enmeshed in the biofilms, as reported by Charron et al.20. For each of the treatment conditions, we performed dilutions and plate counts to quantitate viable bacteria at the end of the 4 h exposure to SOS inducers, and then again after being subjected to dessication stress for 16 h. Ability to withstand dessication is a microbial factor that contributes to persistence on solid, abiotic surfaces, and contributes to persistence in hospitals despite the use of disinfectants.

Figure 2F shows the results of the survival experiment. Open circles denote the bacterial counts after the 4 h exposure to mitomycin C ± histone, expressed on a logarithmic scale. Closed circles show the counts of viable bacteria after 16 h of drying on a warming block. As shown in the first column of Fig. 2F, the untreated control bacteria showed a nearly 5-log drop in viability after the 16 h of dessication. Histone alone improved viability of the E_clo_Niagara, with only a ~ 3-log decrease in counts. Bacteria treated with mitomycin C alone showed a growth penalty from the mitomycin itself (3rd column, Fig. 2F), but the bacteria that survived the mitomycin treatment suffered only a 1.1-log further decrease from dessication stress. Bacteria treated with mitomycin C + histone showed a similar ability to survive dessication. Bacteria treated with mitomycin + histone showed a 2.6-log increase in survival comparted to untreated control, a 436-fold increase. These results help quantitate the large effects of the SOS response on bacteria enmeshed in biofilms, and affirm the results of previous reports on how biofilms help pathogens to survive7,21,22.

In addition to the functional effects of biofilms on bacterial survival, we wished to explore if the DNA in the biofilms differed from the DNA released into the supernatants by SOS-inducing drugs. Using trial and error, we developed a protocol to extract the DNA from biofilms, using proteinase K and 0.1% sodium dodecyl sulfate (SDS), as described in the Methods section. Figure 3 shows an agarose gel comparing the DNA patterns in DNA released into the culture supernatants vs. DNA extracted from the biofilm. As shown in Fig. 3, most of the DNA in the supernatant ran on the gel as a broad smear, with the DNA fragments less than 600 bp in size. In contrast, the DNA extracted from the biofilm showed 2 additional, higher molecular weight bands with sizes of about 600 bp and 1100 bp (yellow arrows). This shows that the DNA in the biofilms differs qualitatively from the DNA in the supernatant. This could be because the DNA enmeshed in the biofilm is protected, at least partially, from the action of nucleases.

Fig. 3.

Fig. 3

Open in a new tab

Comparison of DNA bands in DNA released into the supernatant versus DNA deposited as biofilm, in E_clo_Niagara treated with mitomycin C. DNA was extracted from the biofilms as described in the Methods section. DNA in the biofilms has additional, higher molecular weight bands not observed in the DNA in the liquid phase (yellow arrows).

In order to increase the generalizability of our findings, we also explored biofilm formation in Proteus mirabilis, a urease-producing organism notorious for its ability to cause urinary tract infections and to promote encrustations in the urinary tract or on indwelling urinary catheters23–25.

Figure 4 shows the results obtained with P. mirabilis strain HI 4320, a wild-type strain, again using mitomycin C to induce the SOS response. As shown in Fig. 4A, mitomycin C again triggered a release of both dsDNA and ssDNA. As with other enteric organisms studied, release of ssDNA exceeded that of dsDNA. The amount of DNA released from P. mirabilis was less than that observed with E. cloacae, however (compare the y-axes of Fig. 4A and Figs. 1 and 2). Again, histone III-S significantly decreased the amount of dsDNA and ssDNA measurable in the supernatants, similar to our observations with E_clo_Niagara. Figure 4B shows that DNA was detectable in the P. mirabilis biofilms.

Fig. 4.

Fig. 4

Open in a new tab

Effect of mitomycin C and histone on Proteus mirabilis. Panels A and B, effect of mitomycin C and histone on dsDNA and ssDNA released into the supernatants vs. DNA deposited as biofilm. Panel C, DAPI staining of DNA in biofilms from P. mirabilis. Treatment with mitomycin C and histone III-S resulted in a marked increase in the amount of DNA in the biofilm (yellow arrow). Panel D, wet mount microscopy of P. mirabilis after treatment with mitomycin C, showing formation of large bacterial clumps, at 200 X magnification, by phase contrast. The yellow size bar indicates 60 µm and applies to the photographs in Panels E and F as well. Panel E, same field as in Panel D, but with blue DAPI fluorescence visible under UV illumination. Panel F, in the presence of urea, P. mirabilis forms numerous struvite crystals which are accompanied by bacterial aggregates and extracellular DNA stained blue with DAPI stain. Red arrows show crystals with the classic “coffin lid” appearance of struvite, also known as magnesium ammonium phosphate. Panel F was photographed at 200 X magnification using phase contrast.

Comparison of the ssDNA results in Fig. 4A and 4B shows that the decrease in ssDNA with histone in Fig. 4A is much larger than the increase in ssDNA observed in the biofilm with histone Fig. 4B. We believe the “missing” ssDNA is in the bacterial pellet, which is removed with centrifugation. Treatment with mitomycin C plus histone induced the strongest biofilm formation (Fig. 4, Panels B and C). Figure 4C shows the DAPI-stained biofilms produced by this organism (Yellow arrow shows DNA in biofilm in response to mitomycin C plus histone III-S). In addition to DNA release, mitomycin C treatment also stimulated formation of large bacterial clumps (Fig. 4D, by phase contrast microscopy, counter-stained with DAPI). Examination of the field shown in Fig. 4D under UV illumination showed strong, blue-colored DAPI fluorescence signal from the bacterial aggregates (Fig. 4E), suggesting that DNA is entrapped in the aggregates. We repeated the biofilm formation experiments in the presence of urea, and noted formation of abundant crystals (Fig. 4F). We believe the crystals observed in Fig. 4F are struvite crystals (magnesium ammonium phosphate) because many of the crystals have the classic “coffin lid” shape (Fig. 4F, red arrows). When viewed under UV light, DAPI fluorescence was closely associated with the crystals. The role of extracellular DNA in crystal-induced diseases has been noted previously26. If confirmed, the results of Fig. 4 would indicate that, in P. mirabilis, DNA released in response to the SOS response becomes enmeshed in multi-component clumps, including bacteria, extracellular DNA, and struvite crystals. These findings have important clinical implications because quinolone antibiotics, commonly used for the treatment of urinary tract infections, are also inducers of the SOS response27,28. Struvite crystals are formed in alkaline urine and can form kidney stones as well as encrustations on urinary catheters and stents (Supplemental Fig. 2).

We summarized some of our data using E. cloacae and P. mirabilis in Table 1. In this Table, we compared the ratio of mitomycin-induced ssDNA to dsDNA in the supernatant medium and also in the DNA extracted from the biofilms, for both bacterial strains. For both strains of bacteria, the ratio of ssDNA to dsDNA in the biofilms was similar to the ratio observed in the supernatant media. Due to similarity in the ratios of the two types of DNA, the simplest hypothesis for the assembly of the biofilms is deposition of the DNA from the liquid supernatant medium onto the solid substrate. Since the biofilms form at the air–liquid interface, and based on other reports29,30, we suspect that oxygen in air somehow promotes biofilm formation at that interface.

Table 1.

Ratio of ssDNA to dsDNA in Supernatants and Biofilm.

Bacterial Strain Ratio of ssDNA to dsDNA in Supernatants*
Mean ± SD		 Ratio of ssDNA to dsDNA in Biofilms†
Mean ± SD

Enterobacter cloacae

E_clo_Niagara

3.10 ± 0.79

n = 13 experiments

3.46 ± 0.95

n = 4 experiments

Proteus mirabilis

Strain HI 4320, WT

2.26 ± 1.0

n = 8 experiments

2.34 ± 0.74

n = 2 experiments
Open in a new tab

*In all experiments, the SOS response was induced with mitomycin C, 1.0 to 1.5 µg/mL; growth duration was 4 to 4.5 h.

†DNA was extracted from the biofilms adherent to the glass using 100 µg/mL Proteinase K plus 0.1% SDS for 1 h.

Last, we turned our attention to drugs and interventions that would promote or inhibit SOS-induced DNA release. First we tested fluoxetine, an antidepressant in the serotonin-selective reuptake inhibitor (SSRI) class. We previously reported that fluoxetine potentiated the effect of other SOS activators in E.coli and E. cloacae8,10, a finding that has been confirmed and extended by other laboratories11,31. Figure 5A showed that fluoxetine showed a trend toward promoting mitomycin-induced DNA release, but this increase was not statistically significant. This indicates that there may be species to species variation in susceptibility to fluoxetine.

Fig. 5.

Fig. 5

Open in a new tab

Activators and inhibitors of mitomycin C-induced DNA release and biofilm formation in E_clo_Niagara. Panel A, effect of fluoxetine as a potentiator of mitomycin-induced DNA release. Panels B and C, inhibition of dsDNA and ssDNA release by dequalinium, an inhibitor of the general stress response in enteric bacteria. Panel D, effect of dequalinium on mitomycin-induced biofilm formation, imaged by DAPI stain. Panels E and F, additive effects of zinc acetate and dequalinium in inhibition of dsDNA and ssDNA release.

Next we tested the effects of dequalinium, a quaternary ammonium compound. Interestingly, dequalinium is not an inhibitor of the SOS response per se, but rather an inhibitor of the general stress response13. But the general stress response, the stringent response, oxidant response, and the SOS response interact and reinforce one another in a “regulatory hub.” Fig. 5, Panels B and C show the effect of dequalinium on mitomycin-induced DNA release, showing strong inhibition of dsDNA and ssDNA release. Dequalinium was not acting by merely favoring the translocation of DNA from liquid to biofilm, as did RecA and histone, but dequalinium also inhibited biofilm DNA (Fig. 5D, DAPI stain, red arrows).

Zinc salts are able to inhibit the SOS response via their effects on RecA32. Therefore, we next tested if zinc acetate could potentiate the inhibitory effects of dequalinium on DNA release. Zinc acetate indeed showed additivity with dequalinium, as shown by its ability to shift the dequalinium inhibition curve to the left (Fig. 5, Panels E and F) for both dsDNA and ssDNA. The effects of dequalinium and zinc may be important clinically, since many investigators have predicted that RecA and the SOS response are on the verge of becoming druggable targets13,33. In the future, difficult biofilm infections might be treated with agents that inhibit SOS-induced DNA release (e.g., Fig. 5) in combination with DNase I, which is already an FDA-approved drug in the United States (dornase alpha, given by inhalation).

Discussion

Experiments in this study were intended to determine the mechanism of DNA release from bacterial cells in response to SOS-inducing drugs. Our time-course experiments showed that dsDNA and ssDNA both began to be detectable at the same time, about 2.5 h after the addition of mitomycin C. In other words, release of ssDNA exceeded that of dsDNA, but did not precede it. Proteins with the ability to bind DNA promoted biofilm formation, and these biofilms contained DNA as a prominent component. Deposition of DNA into biofilms was associated with a decrease in DNA measurable in the liquid phase. As shown in Table 1, the ratios of ssDNA to dsDNA in the biofilms were similar to those measured in the liquid phase. These results gave insight into the composition and assembly of biofilms, but did not definitively identify the route by which DNA exits the bacterial cells.

SOS-induced DNA release has not been extensively studied, but two main pathways for DNA release have been suggested. The first is via induction of latent prophage, which generally lyse the bacterial host cell when triggered into the lytic phase34,35. A second proposed mechanism for DNA release is via exocytotic vesicles36. These two mechanisms may not be mutually exclusive, because DNA release via phage lysis, and release of extracellular vesicles, can occur simultaneously, at least in Burkholderia9. Studies of these pathways are currently underway.

Our experiments with biofilms show an example of the benefits of biofilm formation to the bacteria. Our experiments with bacterial survival show that bacteria enmeshed in the biofilm were much better able to survive 16 h of dessication on a warming block (Fig. 2F). When neutrophil extracellular traps (NETs) were discovered, the beneficial effects of NETs on the host were strongly emphasized37. But more recently the adverse consequences of NET formation has received attention19,38. NET formation appears especially deleterious if it occurs intravascularly39. The results of this study may have implications regarding the treatment of bloodstream infections with SOS-inducing drugs.

In this study, we used mitomycin C as the main SOS-inducer. Mitomycin C is used as an anti-cancer drug, for pancreatic, stomach, and genito-urinary cancers, but it is also used as an antibiotic, mostly in the form of antibiotic eye drops. This highlights the fact that SOS-inducing drugs are not just antibiotics, but include cancer chemotherapy drugs, non-steroidal anti-inflammatory drugs (NSAIDs, such as diclofenac), antidepressants, fungicides40, and many others, in addition to UV light.

An important finding from studies on SOS-induced DNA release is that it can trigger formation of biofilms by organisms normally not considered biofilm producers. Another emerging feature of the SOS pathway, including SOS-induced DNA release, is the heterogeneity of SOS response from strain to strain41. The SOS response can vary in the speed of onset as well as the robustness of the response. Shiga-toxigenic E. coli(STEC), for example, seems primed to unleash a powerful SOS response with minimal provocation, the so-called “hair trigger” phenomenon42. In this current study, for example, P. mirabilis released less DNA than did E_clo_Niagara.

The large amounts of DNA released in response to SOS-inducers raises the question of whether the DNA that is released could play a role in microbial signalling, in a manner similar to quorum sensing. The signalling could occur within a bacterial cell, as reported by Gozzi et al.43, or as a signal between species. Some bacteria are adept at taking up extracellular DNA and using it as a nutrient44,45. But while some bacteria may regard extracellular DNA as a tasty meal, others might prefer to use the DNA as a source of antibiotic resistance genes46.

Methods

Materials

Bacterial strains used are shown in Table 2. Bacterial strains were grown overnight in LB broth, then sub-cultured into DMEM-F12 as previously described6,15.

Table 2.

Bacterial Strains Used.

Bacterial Species Strain Name, Comments Reference(s)
Enterobacter cloacae

E_clo_Niagara

ESBL, bloodstream isolate

 15,47
Proteus mirabilis Pm HI 4320, wild-type 48
Proteus mirabilis ureF mutant, urease-deficient 49
Open in a new tab

Strain E_clo_Niagara was subcultured at a dilution of 1: 100, while strain PM HI4320 was diluted at 1: 80. LB medium contained substantial amounts of ssDNA, but DMEM-F12 medium did not contain detectable ssDNA or dsDNA. Samples were collected and analyzed at the 4 h point unless otherwise stated.

Chemical reagents

Diamidino-phenylindole (DAPI) was from Cayman Chem, as was mitomycin C, dequalinium and fluoxetine (Ann Arbor, MI). Bovine serum albumin, histone III- 3, buffers, SDS, and LB medium were from Sigma-Aldrich (now Millipore-Sigma, Darmstadt, Germany). Assay kits for measuring dsDNA and ssDNA were from Thermo-Fisher, as were SybrSafe dye, DNA ladders for gel electrophoresis, and proteinase K. The dsDNA assay kit from Biotium was also used in several experiments. DMEM-F12 medium was from the GIBCO division of Thermo-Fisher. Purified E. coli RecA protein was from Abcam, Cambridge, MA. Dequalinium is poorly soluble in water and in DMSO. Therefore, the dequalinium suspension was immersed for 3–5 min in a small beaker of water that had been brought to a boil, to aid dissolution.

DNA assays

DNA was measured using the kits above using the Qubit Flex fluorometer, as previously described8; the fluorescent dyes used accurately discriminate between dsDNA and ssDNA.

DAPI staining of biofilms

DAPI was used at 20 µg/mL in water to stain DNA in biofilms and in bacterial aggregates (Figs. 2 and 4). Biofilms were imaged using the GelDoc EZ insturument (Bio-Rad), and fluorescent bands were quantitated using the Un-Scan-It Gel program (Silk Scientific, Orem, UT).

Extraction of DNA from biofilms

The extraction protocol used was developed with an examination of the literature50, followed by trial-and-error. DNA was extracted from air-dried biofilms using 1 mL of Tris-buffered saline, pH 7.4, with 100 µg/mL proteinase K and 0.1% SDS, for 1 h at room temperature, with end-over-end tumbling. This 1 mL volume was the same as the volume of the original growth medium, allowing the DNA concentrations to be compared between liquid phase and solid (biofilm) phase.

Dessication stress

After removal of the culture liquid for DNA measurements and plate counts, the tubes were allowed to dry for 16 h on a warming block, intended to dry microscope slides, which was set to 38 º C. Then the remaining bacteria were removed using the biofilm extraction method reported above.

Microscopy of bacterial clumping and DNA

Clumps of live P. mirabilis bacteria were stained with DAPI, then visualized using wet mounts under cover slips using phase contrast (Fig. 4D). Then the same field was examined under UV illumination to view the DAPI fluorescence. Then the phase-contrast photo and the photo taken under UV light were merged using the HDRtist NXL program to form a merged image (Fig. 4E).

Formation of crystals by P. mirabilis in the presence of urea

P. mirabilis strain HI4320 (wild-type) was grown in a double-strength (2X) version of urease buffer, pH 7.051, incorporating some modifications52. Urea was weighed out freshly for each experiment, dissolved in sterile water, and added to the urease buffer to yield a final concentration of 1.5%. 0.5 mL of P. mirabilis suspension was added to 0.5 mL of 2X urease buffer and the mixture was allowed to continue incubating for an additional 1 h. The urease-deficient mutant, ureF, was used for comparison (Supplemental Fig. 2). In the presence of urea, the wild-type strain raised the pH of the solution and produced crystals (Supplemental Fig. 2). The solution was counter-stained with DAPI and then subjected to centrifugation in a SlidePrep Plus cytological centrifuge at 21 g for 5 min. The slides were allowed to air dry, then examined under phase-contrast at 200 X magnification to view the crystals. Then the visible light was turned down and the same field examined under UV illumination, revealing the DAPI fluorescence (Fig. 4F).

Statistical analysis

Significance was determined by t-test or ANOVA using the software built into GraphPad Prism, version 10.

Supplementary Information

Supplementary Information. (218.4KB, pdf)

Acknowledgements

We thank Dr. Chelsie Armbruster and Ms. Aimee Brauer for sharing Proteus mirabilis strains. We acknowledge the use of research funds from the Dept. of Internal Medicine, Jacobs School of Medicine.

Author contributions

John Crane devised many of the experiments, performed some of the experiments, organized and collated the data, and wrote the first draft of the manuscript. Tammy Yang did most of the experiments.

Data availability

All data generated or analysed during this study are included in this published article [and its supplementary information files].

Declarations

Competing interests

The authors have no competing interests to declare.

Ethical approval

This study did not use humans or animals and so no IRB or IACUC approvals were required.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-96943-2.

References

  • 1.Oda, Y. Induction of SOS responses in Escherichia coli by 5-fluorouracil. Mutat. Res./DNA Rep. Rep.183, 103–108 (1987). [DOI] [PubMed] [Google Scholar]
  • 2.Mamber, S. W., Brookshire, K. W. & Forenza, S. Induction of the SOS response in Escherichia coli by azidothymidine and dideoxynucleosides. Antimicrob. Agents Chemother.34, 1237–1243. 10.1128/aac.34.6.1237 (1990). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Jara, L. M., Cortés, P., Bou, G., Barbé, J. & Aranda, J. Differential Roles of Antimicrobials in the Acquisition of Drug Resistance through Activation of the SOS Response in Acinetobacter baumannii. Antimicrob. Agents Chemother.59, 4318–4320 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Song, T., Toma, C., Nakasone, N. & Iwanaga, M. Aerolysin is activated by metalloprotease in Aeromonas veronii biovar sobria. J. Med. Microbiol.53, 477–482 (2004). [DOI] [PubMed] [Google Scholar]
  • 5.Händel, N., Hoeksema, M., Freijo Mata, M., Brul, S. & Ter Kuile, B. H. Effects of stress, reactive oxygen species, and the SOS response on de novo acquisition of antibiotic resistance in Escherichia coli. Antimicrob. Agents Chemother.60, 1319–1327 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Crane, J. K., Alvarado, C. L. & Sutton, M. D. Role of the SOS Response in the Generation of Antibiotic Resistance In Vivo. Antimicrob. Agents Chemother.10.1128/AAC.00013-21 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Kaushik, V., Tiwari, M. & Tiwari, V. Interaction of RecA mediated SOS response with bacterial persistence, biofilm formation, and host response. Int. J. Biol. Macromol.10.1016/j.ijbiomac.2022.07.176 (2022). [DOI] [PubMed] [Google Scholar]
  • 8.Demjanenko, P., Zheng, S. & Crane, J. K. SOS-Inducing Drugs Trigger Nucleic Acid Release and Biofilm Formation in Gram-Negative Bacteria. Biomolecules14, 321 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Heredia-Ponce, Z. et al. Genotoxic stress stimulates eDNA release via explosive cell lysis and thereby promotes streamer formation of Burkholderia cenocepacia H111 cultured in a microfluidic device. Npj Biofilms & Microbiomes9, 96 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Crane, J. K., Salehi, M. & Alvarado, C. L. Psychoactive Drugs Induce the SOS Response and Shiga Toxin Production in Escherichia coli. Toxins (Basel)10.3390/toxins13070437 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Wang, Y. et al. Antidepressants can induce mutation and enhance persistence toward multiple antibiotics. Proc. Natl. Acad. Sci. U. S. A.120, e2208344120. 10.1073/pnas.2208344120 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Ding, P., Lu, J., Wang, Y., Schembri, M. A. & Guo, J. Antidepressants promote the spread of antibiotic resistance via horizontally conjugative gene transfer. Environ. Microbiol.24, 5261–5276 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Zhai, Y. et al. Drugging evolution of antibiotic resistance at a regulatory network hub. Sci. Adv.9, eadg0188 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Bunnell, B. E., Escobar, J. F., Bair, K. L., Sutton, M. & Crane, J. Zinc Blocks SOS-Induced Hypermutation via Inhibition of RecA in Escherichia coli. PLoS ONE12(5), 410. 10.1371/journal.pone.0178303 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Crane, J. K. & Catanzaro, M. N. Role of Extracellular DNA in Bacterial Response to SOS-Inducing Drugs. Antibiotics12, 649 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Pavankumar, A. R., Ayyappasamy, S. P. & Sankaran, K. Small RNA fragments in complex culture media cause alterations in protein profiles of three species of bacteria. Biotechniques52, 167–172 (2012). [DOI] [PubMed] [Google Scholar]
  • 17.Wang, H., Guo, J., Chen, X. & He, H. The Metabolomics Changes in Luria-Bertani Broth Medium under Different Sterilization Methods and Their Effects on Bacillus Growth. Metabolites10.3390/metabo13080958 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Lee, J. Y. et al. Bacterial RecA Protein Promotes Adenoviral Recombination during In Vitro Infection. mSphere3, e00105-00118 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Meier, A., Sakoulas, G., Nizet, V. & Ulloa, E. R. Neutrophil extracellular traps (NETs): An emerging therapeutic target to improve infectious diseases outcomes. J. Infect. Dis.10.1093/infdis/jiae252 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Charron, R., Boulanger, M., Briandet, R. & Bridier, A. Biofilms as protective cocoons against biocides: From bacterial adaptation to One Health issues. Microbiology169, 001340 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Baharoglu, Z. & Mazel, D. SOS, the formidable strategy of bacteria against aggressions. FEMS Microbiol. Rev.38, 1126–1145 (2014). [DOI] [PubMed] [Google Scholar]
  • 22.Podlesek, Z. & Zgur Bertok, D. The DNA Damage Inducible SOS Response Is a Key Player in the Generation of Bacterial Persister Cells and Population Wide Tolerance. Front. Microbiol.11, 1785. 10.3389/fmicb.2020.01785 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.White, A. N., Learman, B. S., Brauer, A. L. & Armbruster, C. E. Catalase activity is critical for Proteus mirabilis biofilm development, extracellular polymeric substance composition, and dissemination during catheter-associated urinary tract infection. Infect. Immun.10.1128/IAI.00177-21 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Armbruster, C. E., Mobley, H. L. & Pearson, M. M. Pathogenesis of Proteus mirabilis infection. EcoSal Plus10.1128/ecosalplus (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Evstafeva, D. et al. Inhibition of urease-mediated ammonia production by 2-octynohydroxamic acid in hepatic encephalopathy. Nat. Commun.15, 2226 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Ma, Q. & Steiger, S. Neutrophils and extracellular traps in crystal-associated diseases. Trends Mol. Med.30, 809–823. 10.1016/j.molmed.2024.05.010 (2024). [DOI] [PubMed] [Google Scholar]
  • 27.Song, L. Y. et al. Mutational Consequences of Ciprofloxacin in Escherichia coli. Antimicrob. Agents Chemother.60, 6165–6172. 10.1128/aac.01415-16 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Cirz, R. T., O’Neill, B. M., Hammond, J. A., Head, S. R. & Romesberg, F. E. Defining the Pseudomonas aeruginosa SOS response and its role in the global response to the antibiotic ciprofloxacin. J. Bacteriol.188, 7101–7110 (2006). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Eberly, A. R. et al. Biofilm formation by uropathogenic Escherichia coli is favored under oxygen conditions that mimic the bladder environment. Int. J. Mol. Sci.18, 2077 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Ahn, S.-J. & Burne, R. A. Effects of oxygen on biofilm formation and the AtlA autolysin of Streptococcus mutans. J. Bacteriol.189, 6293–6302 (2007). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Lu, J., Ding, P., Wang, Y. & Guo, J. Antidepressants promote the spread of extracellular antibiotic resistance genes via transformation. ISME Commun.2, 63 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Lee, A. M., Ross, C. T., Zeng, B.-B. & Singleton, S. F. A Molecular Target for Suppression of the Evolution of Antibiotic Resistance: Inhibition of the Escherichia coli RecA Protein by N6-(1-Naphthyl)-ADP. J. Med. Chem.48, 5408–5411. 10.1021/jm050113z (2005). [DOI] [PubMed] [Google Scholar]
  • 33.Lanyon-Hogg, T. Targeting the bacterial SOS response for new antimicrobial agents: drug targets, molecular mechanisms and inhibitors. Future Med. Chem.13, 143–155 (2021). [DOI] [PubMed] [Google Scholar]
  • 34.Carrolo, M., Frias, M. J., Pinto, F. R., Melo-Cristino, J. & Ramirez, M. Prophage spontaneous activation promotes DNA release enhancing biofilm formation in Streptococcus pneumoniae. PLoS ONE5, e15678 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Lu, J. & Guo, J. Prophage induction by non-antibiotic compounds promotes transformation of released antibiotic resistance genes from cell lysis. Water Res.263, 122200 (2024). [DOI] [PubMed] [Google Scholar]
  • 36.Jiang, M. et al. Reductions in bacterial viability stimulate the production of Extra-intestinal Pathogenic Escherichia coli (ExPEC) cytoplasm-carrying Extracellular Vesicles (EVs). PLoS Pathog.18, e1010908 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Brinkmann, V. et al. Neutrophil extracellular traps kill bacteria. Science303, 1532–1535 (2004). [DOI] [PubMed] [Google Scholar]
  • 38.Fuchs, T. A., Brill, A. & Wagner, D. D. Neutrophil extracellular trap (NET) impact on deep vein thrombosis. Arterioscler. Thromb. Vasc. Biol.32, 1777–1783 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Mutua, V. & Gershwin, L. J. A review of neutrophil extracellular traps (NETs) in disease: Potential anti-NETs therapeutics. Clin. Rev. Allergy Immunol.61, 194–211 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Zhang, H. et al. Fungicide exposure accelerated horizontal transfer of antibiotic resistance genes via plasmid-mediated conjugation. Water Res.233, 119789 (2023). [DOI] [PubMed] [Google Scholar]
  • 41.Diaz-Diaz, S. et al. Heterogeneity of SOS response expression in clinical isolates of Escherichia coli influences adaptation to antimicrobial stress. Drug Resist. Updates75, 101087 (2024). [DOI] [PubMed] [Google Scholar]
  • 42.Chakraborty, D., Clark, E., Mauro, S. A. & Koudelka, G. B. Molecular mechanisms governing “hair-trigger” induction of shiga toxin-encoding prophages. Viruses10, 228 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Gozzi, K., Salinas, R., Nguyen, V. D., Laub, M. T. & Schumacher, M. A. ssDNA is an allosteric regulator of the C. crescentus SOS-independent DNA damage response transcription activator, DriD. Genes Dev.36, 618–633 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Finkel, S. E. & Kolter, R. DNA as a nutrient: Novel role for bacterial competence gene homologs. J. Bacteriol.183, 6288–6293 (2001). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Mulcahy, H., Charron-Mazenod, L. & Lewenza, S. Pseudomonas aeruginosa produces an extracellular deoxyribonuclease that is required for utilization of DNA as a nutrient source. Environ. Microbiol.12, 1621–1629 (2010). [DOI] [PubMed] [Google Scholar]
  • 46.Beaber, J. W., Hochhut, B. & Waldor, M. K. SOS response promotes horizontal dissemination of antibiotic resistance genes. Nature427, 72–74 (2003). [DOI] [PubMed] [Google Scholar]
  • 47.Crane, J., Cheema, M., Olyer, M. & Sutton, M. Zinc Blockade of SOS Response Inhibits Horizontal Transfer of Antibiotic Resistance Genes in Enteric Bacteria. Front. Cell. Infect. Microbiol.10.3389/fcimb.2018.00410 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Armbruster, C. E. et al. The Pathogenic Potential of Proteus mirabilis Is Enhanced by Other Uropathogens during Polymicrobial Urinary Tract Infection. Infect. Immun.10.1128/IAI.00808-16 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Armbruster, C. E., Smith, S. N., Yep, A. & Mobley, H. L. Increased incidence of urolithiasis and bacteremia during Proteus mirabilis and Providencia stuartii coinfection due to synergistic induction of urease activity. J. Infect. Dis.209, 1524–1532 (2014). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Wu, J. & Xi, C. Evaluation of different methods for extracting extracellular DNA from the biofilm matrix. Appl. Environ. Microbiol.75, 5390–5395. 10.1128/AEM.00400-09 (2009). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Duran Ramirez, J. M., Gomez, J., Obernuefemann, C. L., Gualberto, N. C. & Walker, J. N. Semi-quantitative assay to measure urease activity by urinary catheter-associated Uropathogens. Front. Cell. Infect. Microbiol.12, 859093 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Learman, B. S., Brauer, A. L., Eaton, K. A. & Armbruster, C. E. A Rare Opportunist, Morganella morganii, Decreases Severity of Polymicrobial Catheter-Associated Urinary Tract Infection. Infect Immun10.1128/IAI.00691-19 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Information. (218.4KB, pdf)

Data Availability Statement

All data generated or analysed during this study are included in this published article [and its supplementary information files].

Articles from Scientific Reports are provided here courtesy of Nature Publishing Group

RESOURCES