DNA builds and strengthens the extracellular matrix in Myxococcus xanthus biofilms by interacting with exopolysaccharides

Abstract

One intriguing discovery in modern microbiology is the extensive presence of extracellular DNA (eDNA) within biofilms of various bacterial species. Although several biological functions have been suggested for eDNA, including involvement in biofilm formation, the detailed mechanism of eDNA integration into biofilm architecture is still poorly understood. In the biofilms formed by Myxococcus xanthus, a Gram-negative soil bacterium with complex morphogenesis and social behaviors, DNA was found within both extracted and native extracellular matrices (ECM). Further examination revealed that these eDNA molecules formed well organized structures that were similar in appearance to the organization of exopolysaccharides (EPS) in ECM. Biochemical and image analyses confirmed that eDNA bound to and colocalized with EPS within the ECM of starvation biofilms and fruiting bodies. In addition, ECM containing eDNA exhibited greater physical strength and biological stress resistance compared to DNase I treated ECM. Taken together, these findings demonstrate that DNA interacts with EPS and strengthens biofilm structures in M. xanthus.

Figure 1. Isolated cell-free extracellular materials from DK10547 by a membrane-chamber system contained both EPS and eDNA.

Schematic diagram of the 150 ml membrane-chamber system used to isolate cell-free extracellular materials (panel A). Through an up-right fluorescence microscope (panel B, normal phase image), EPS and eDNA were detected in the isolated extracellular materials by calcoflour white staining (panel C) and PI staining (panel D), respectively. The bar represents 100 µm.

Figure 2. eDNA in M. xanthus non-developmental starvation biofilms.

M. xanthus DK1622 biofilm structures formed in MOPS buffer at 24 hr (panels A and B) and DNase I treated biofilm (panels C and D) were counterstained with SYTOX orange (red) and SYTO9 (green). Panels A and C are the single channel images (SYTOX orange), and panels B and D are the overlaid images. M. xanthus DK10547 with a GFP label (green) formed starvation biofilms (panels E and F) in MOPs buffer at 24 hr was counterstained with SYTOX orange (red) and FM 4-64 (blue). Panel E is the single channel image (FM 4-64), and panel F is the overlaid image. The bars represent 50 µm in panels A–D and 10 µm in panels E and F.

Figure 3. eDNA in M. xanthus submerged fruiting bodies.

M. xanthus DK1622 fruiting body formed in submerged culture at 24 hr after starvation was initiated (panels A–C) and DNase I treated submerged fruiting body (panel D) were counterstained with SYTOX orange (red) and SYTO9 (green). Panels A is the single channel image (SYTOX orange), and panels B–D are the overlaid images. Panel C showed a magnified portion of panel B indicated by a white pane. M. xanthus DK10547 with a GFP label (green) formed fruiting body (panels E and F) in MOPs buffer at 24 hr was counterstained with SYTOX orange (red) and FM 4-64 (blue). Panel E is the single channel image (FM 4-64), and panel F is the overlaid image. Panel G shows a 48 hr submerged fruiting body of DK1622 stained with SYTOX orange, and panel H shows a trail structure in a 48 hr submerged culture of DK1622 stained with SYTOX orange and SYTO9. The bars represent 50 µm in panels A–D, 10 µm in panels E, F and H, and 20 µm in panel G.

Figure 4. eDNA colocalized with EPS within M. xanthus starvation biofilm and fruiting body structures.

Panel A, DK1622 starvation biofilm formed in MOPS buffer for 24 hr labeled with STYOX orange (red), Alexa 633-conjugated WGA (blue) and SYTO 9 (green). Panel B, DK1622 24 hr fruiting body structures with STYOX orange, Alexa 633-conjugated WGA and SYTO 9. Images in panel A were taken with a 40× objective using CLSM, and images in panel B were taken with a 63× objective. The bars in panels A and B represent 50 µM. Panel C showed the quantitative colocalization analysis results of STYOX orange (eDNA) and Alexa 633-WGA (EPS) signals from submerged 24 hr starvation biofilms (left) and 24 hr fruiting bodies (right). The PCC represents Pearson’s correlation coefficient, MOC represents overlap coefficients according to Manders, M1 represents colocalization coefficient M1 (fraction of eDNA overlapping EPS), M2 represents colocalization coefficient M2 (fraction of EPS overlapping eDNA), and ICQ represents intensity correlation quotient. Mean ± SD is plotted.

Figure 5. DNA bound to M. xanthus EPS at different pHs.

The binding percentages of wild-type DK1622 chromosomal DNA (left) and commercial salmon sperm DNA (right) to isolated EPS were determined at different pHs, and the average ± SD is plotted. On the x-coordinate, ‘DNA’ represents different DNA samples, ‘EPS’ represents isolated M. xanthus EPS and ‘Cell*’ represents SW504 cells, which were added to the test system.

Figure 6. Mechanical strength, anti-disruptive properties and nanomechanical adhesive characteristics recorded using force-separation curves of the M. xanthus starvation biofilm matrix with or without eDNA.

In panel A, DK1622 biofilms without DNase I (black bar) and with DNase I (grey bar) were established over a 24 hr time period in MOPS buffer and biomass was measured as crystal violet optical density (No-treatment control). The changes of biomass in these two kinds of biofilms after sonication or SDS treatment, respectively, are also shown. In panel B, representative force curves measured by AFM on DK1622 24 hr starvation biofilm matrix (curve I), matrix treated with DNase I (curve II) and bare portion of the substrate after the tip was used (curve III). Force-separation curves were recorded as “approach” (blue) and “retraction” (red) curves.