A bacterial extracellular DNA inhibits settling of motile progeny cells within a biofilm

Abstract

In natural systems, bacteria form complex, surface-attached communities known as biofilms. This lifestyle presents numerous advantages compared with unattached or planktonic life, such as exchange of nutrients, protection from environmental stresses and increased tolerance to biocides. Despite such benefits, dispersal also plays an important role in escaping deteriorating environments and in successfully colonizing favourable, unoccupied habitat patches. The α-proteobacterium Caulobacter crescentus produces a motile swarmer cell and a sessile stalked cell at each cell division. We show here that C. crescentus extracellular DNA (eDNA) inhibits the ability of its motile cell type to settle in a biofilm. eDNA binds to the polar holdfast, an adhesive structure required for permanent surface attachment and biofilm formation, thereby inhibiting cell attachment. Because stalked cells associate tightly with the biofilm through their holdfast, we hypothesize that this novel mechanism acts on swarmer cells born in a biofilm, where eDNA can accumulate to a sufficient concentration to inhibit their ability to settle. By targeting a specific cell type in a biofilm, this mechanism modulates biofilm development and promotes dispersal without causing a potentially undesirable dissolution of the existing biofilm.

Figure 1. C. crescentus life cycle

Each cell division produces a motile swarmer cell and a sessile stalked cell. After an obligatory delay during which it is unable to start the next cell cycle, swarmers cell differentiate into a division-competent stalked cells. The stalked cell has a polar adhesin called the holdfast that is required for permanent adhesion to surfaces and for biofilm formation. The holdfast is synthesized at the flagellated pole in the late stage of the swarmer phase and is found at the tip of the stalk once the stalk is synthesized at the same pole during cell differentiation.

Figure 2. The biofilm formation inhibitor is sensitive to DNase I

(A) Effect of different enzymatic treatments of the spent M2G medium on the biofilm inhibitory activity. (B) Agarose gel showing eDNA purified from 1 ml of spent medium (lane 2). Lanes 1 and 3 show HyperLadder II (Bioline) and 1 kb ladder (Invitrogen), respectively. (C) Low molecular weight DNA fragments from C. crescentus inhibit biofilm formation in C. crescentus. Effect of C. crescentus CB15 gDNA addition on biofilm formation. gDNA was partially digested with HpaII (30 min at 37°C, using different amounts of HpaII) followed by HpaII heat inactivation (70°C for 20 min). gDNA was added to static biofilm assays to obtain a final concentration 15 µg/ml of DNA. (D) 0.8% agarose gel of the gDNA used in the static biofilm assays. Lane 1: 1 kb ladder (Invitrogen); 2: no digestion; 3: 0.25 U HpaII digestion; 4: 0.5 U HpaII digestion; 5: 0.75 U HpaII digestion; 6: 1 U HpaII digestion. The static biofilm assay was performed for 24 h at 30°C and the biofilm attached to the coverslips was quantified by crystal violet staining. The results are expressed as a percentage of biofilm formation in the absence of gDNA. The error bars represent the S.E.M. of 3 independent experiments run in duplicate.

Figure 3. Correlation between cell death, ecDNA release and biofilm inhibition

(A) Correlation between cell death, eDNA release and biofilm inhibition over time. Wild-type C. crescentus CB15 was grown for 48 h in M2G at 30°C under constant shaking. Dead cells shown as a percentage of total cells (open triangles) and concentration of DNA released in the spent medium (solid diamonds) were monitored over time. Static biofilm assays were performed using spent medium harvested at the different time points (kept at −20°C before use). Overnight biofilm formation (open circles) is expressed as a percentage of biofilm formation compared to biofilm formation in the absence of spent medium. (B) Effect of different concentrations of spent medium and DNA on C. crescentus CB15 biofilm formation. Various volumes of spent M2G medium (solid diamonds), corresponding amounts of eDNA purified from C. crescentus CB15 culture spent medium (open circles), or sheared C. crescentus CB15 gDNA (open triangles) were added to M2G for a static biofilm assay. The results are expressed as a percentage of biofilm formation in the absence of spent medium, eDNA, or sheared gDNA. The results are expressed as a percentage of biofilm formation in the absence of gDNA. The error bars represent the S.E.M. of 3 independent experiments run in duplicate.

Figure 4. Cell death and eDNA release during biofilm formation

(A) Biofilm attached to PVC coverslips stained with the BacLight Live/Dead stain at different times of a static biofilm assay. Images represent overlays of green (live cells) and red (dead cells) signals collected by epifluorescence microscopy. (B) eDNA concentration in the biofilm or attached to the extracellular matrix over time. Total eDNA within the biofilm (representing diffusible eDNA and eDNA attached to the extracellular matrix) is represented in red. To quantify the eDNA bound to the extracellular matrix, biofilm were washed prior to matrix isolation to remove diffusible eDNA; the amount of eDNA bound over time to the matrix is shown in black. Data are the average of 3 replicates of 2 independent experiments and the error bars represent the S.E.M. (C) Biofilm formation in the presence of DNase I. Biofilm formation in the presence of DNase I added at t = 0. The bacteria were incubated at 30°C with or without 30 units of DNase I, and the biofilm attached to the wells was quantified by crystal violet staining. The error bars represent the S.E.M of 3 independent experiments run in triplicate. (D) Effect of DNase I on established biofilms. Static biofilm assays were performed using 2 ml microtubes sealed with AeraSeal tape (1 ml final). The bacteria were incubated at 30°C for various times and then treated with 20 µg/ml DNase I for 1 hour. The biofilm attached inside the tubes was then quantified by crystal violet staining. The crystal violet staining of the non-treated samples and DNase I treated sample are shown in red and black respectively. The error bars represent the S.E.M of 2 independent experiments run in triplicate.

Figure 5. The biofilm inhibition is not due to interaction between eDNA and pili or eDNA and flagellum

Static biofilm assays were performed using 10 µg/ml sheared gDNA extracted from CB15 (black solid bars), CB15 ΔpilA (stripped bars) or CB15 ΔflgE (dotted bars) in 3 ml M2G (12-well plates). Control biofilm grown in the absence of eDNA are represented with white bars. Tested bacteria were incubated for 16 hrs at 30°C and the biofilm attached to the coverslips was quantified with crystal violet. Results are given as the biofilm score (amount of biofilm formed on the coverslip corrected by the culture cell density). Despite the fact that the ΔpilA and ΔflgE mutants are already deficient in biofilm formation, the addition of eDNA reduces their biofilm formation by ~70 %, as is the case for wild-type. The error bars represent the S.E.M of 2 independent experiments run in duplicate.

Figure 6. eDNA prevents the reversible attachment of cells and binds specifically to the C. crescentus holdfast

(A) Attachment of wild-type C. crescentus CB15 synchronized swarmer cells to a glass surface in the presence (open circles) or absence (solid diamonds) of 10 µg/ml eDNA. The swimming cells are given as a percentage of the overall cell population at various times. Results are averages from duplicate samples in 3 independent experiments. (B) Swimming cell quantification of the holdfast-deficient C. crescentus CB15 ΔhfsDAB mutant in the presence (open circles) or in the absence (solid diamonds) of 10 µg/ml eDNA. Results are given as a percentage of swimming cells at t = 0 for 2 independent replicates. (C) Biofilm and planktonic cells of C. crescentus wild-type strain CB15 and a holdfast-minus ΔhfsDAB mutant in the presence of AF 488-labeled eDNA purified from C. crescentus CB15 (green), and AF 594-labeled WGA (red), to visualize holdfast. (D) Coverslip binding of cells of C. crescentus CB15 and holdfasts left on the surface following the removal of the holdfast shedding mutant CB15 ΔhfaB and fluorescence localization of eDNA (green) and holdfast (red).

Figure 7. Different eDNA can inhibit purified holdfasts binding, but a strong inhibition only occurs in response to DNA from Caulobacter and close relatives

(A) Surface attachment of purified holdfasts in the presence of various concentrations of eDNA. Holdfasts purified from the holdfast shedding mutant C. crescentus CB15 ΔhfaB were incubated in suspension in the presence of eDNA from C. crescentus CB15 (solid circles), B. diminuta (solid triangles), A. biprosthecum (open squares), and R. palustris (open triangles). Holdfasts were then allowed to bind to a glass coverlip for 4 h. AF 488-labeled WGA was used to visualize holdfasts by fluorescence microscopy. The number of holdfast attached per field of view was quantified using the ImageJ analysis software. The results are expressed as a percentage of the number of holdfasts attached in the absence of eDNA. The error bars represent the S.E.M of 10 samples from at least 3 independent experiments. (B) Phylogenetic distribution of bacterial species whose DNA was tested in purified holdfast attachment and biofilm inhibition assays. The tree represents a maximum likelihood phylogeny based on 1370 aligned positions from 16S ribosomal RNA gene sequences obtained from GenBank. PAUP* v4.0b10 (Swofford 2003) obtained the maximum likelihood reconstruction via a heuristic search using the HKY+I+G substitution model, with relevant parameters estimated by maximum likelihood on an initial neighbor-joining tree. C. crescentus CB13 was tested but not added to this tree, as its 16S ribosomal RNA sequence is not available.

Figure 8. Effect of spent medium on different stages of biofilm formation

(A) Addition of spent medium to mature biofilms. Biofilms of C. crescentus were grown on coverslips for 24 hours in fresh M2G medium (before addition) and then placed in the presence of spent medium or fresh M2G medium for an additional 24 hours. (B) Time course study of the effect of C. crescentus CB15 spent medium on C. crescentus CB15 biofilm formation under static conditions. 10 µg/ml of eDNA was added (open circles) or not (solid diamonds) to M2G for static biofilm assays. The bacteria were incubated for various times at 30°C and the biofilm attached to the coverslips was quantified with crystal violet. The error bars represent the S.E.M of at least 3 independent experiments run in duplicate.

Figure 9. Effect of spent medium on biofilms at different stages of biofilm formation under dynamic flow conditions

Four flow-cells were inoculated with GFP-labeled C. crescentus CB15, irrigated with M2G, and incubated at 30°C. After 24 (b), 48 (c) or 72 (d) h, the irrigating medium was switched to a 33% spent:fresh M2G mixture and the incubation continued for 96 h. Two control flow-cells were irrigated with a 33% spent:fresh M2G mixture (a) and with fresh M2G (e) for the entire 96 h to serve as references. (A) Microscopy images of the cells at various stages. AutoCOMSTAT was used to determine the surface coverage (B) and the total biomass (µm³ of fluorescence per µm² of surface area) (C) in the five flow-cells over time. Values were average for 10 image stacks (5 image stacks from 2 different flow-cell channels for each sample). The arrows indicate the switch of medium irrigating the flow-cells from M2G to a 33% spent:fresh M2G mixture.