Mechanism Underlining The Molecular Integration And Biological Functions Of Extracellular DNA In The Biofilms Formed By Myxococcus Xanthus

Bacteria in natural surroundings are able to grow as biofilms,which comprises highly structured matrix-enclosed communities and is now accepted as a preferred lifestyle option for prokaryotes.The biofilm communities proved to be hysiologically and behaviorally integrated,highly structured,and self-assembling microbial communities.It comprise aggregates of microbial cells within a polymeric matrix named as extracellular matrix(ECM),which is defined as a mix of extracellular polysaccharides(EPS),nucleic acids,proteins and other cell-released components(such as peptidoglycan,lipids and phospholipids etc.).Myxococcus xanthus is a Gram-negative soil bacterium capable of sophisticated social cell behaviors and growing one of the most complex bacterial single-species biofilms in nature.Under starvation,M.xanthus cells undergo a developmental process culminating in the formation of a multicellular fruiting body filled with myxospores.By controlling the concentration of calcium ion in the submerged medium,highly-organized developmental biofilms(submerged fruiting bodies)and simple non-developmental starvation biofilms of M.xanthus can be both cultivated.Our previous study has demonstrated that considerable amount of extracellular DNA(eDNA)exists in the ECM of M.xanthus biofilms,eDNA is also colocalized with EPS to form some well-organized reticulated structures,while the detailed mechanism of eDNA integration into biofilm architecture is still poorly understood.In the current thesis,multiple spectroscopic and microscopic methods were used to investigate the interactions between eDNA and EPS in vivo and in vitro,which revealed the physicochemical nature of this polymer interaction.First,we confirmed the prvious finding that eDNA is colocalized with EPS in the ECM at different time points of M.xanthus biofilm formation.Bigger clumps or aggregates were observed in the DNA-EPS mixture compared to that in DNA or EPS solutions through transmission electron microscope.Using a quantitative light scattering assay,it was shown that the formation of DNA-EPS complexes in the solution result in a strong increase in the light scattering intensity,which is clearly affected by the pH value of buffer solution and resemble the interactions between positively-charged chitosan and DNA.In general,three types of weak forces mediate the interactions between macromolecules in ECM,i.e.,dispersion forces,electrostatic interactions and hydrogen bonds.We suggested that electrostatic forces could play important roles in the polymerpolymer interactions between DNA and M.xanthus EPS,Indeed,in a dye substitution experiment,EPS showed similar behavior as chitosan that is able to replace the ethidium bromide binding in the major groove of DNA molecues.Without the exogenous probes,we measured the characteristics of DNA-EPS binding by Fourier transform infrared spectroscopy.The results showed that the EPS molecules might only be embedded in the major groove of the DNA molecule and not change the conformation of DNA,which is further confirmed by by circular dichroism spectroscopy.Since the DNA retains B-conformation in the DNA-EPS complex,we proposed that the interaction between EPS and DNA is a weak interaction and the complex maintains a certain degree of reversibility.After recoganizing the physicochemical insights into the molecular interactions between DNA and EPS,we sought to analyze the biological features associated with the integrated DNA-EPS extracellular matrix in M.xanthus.First,the negatively-charged,covalently-linked long DNA molecules bind sticky EPS molecules to form a stronger ECM,which will promote the cell adherence in the initial process of biofilm formation.The rheological properties of DNA-EPS complexes were investigated.Comparing to DNA or EPS alone,DNA-EPS complex exhibited higher specific and apparent viscosities in vitro.The force-spectroscopy capacity of atomic force microscopy(AFM)allowed us to explore the nanomechanical properties(the stickiness)of the ECM with or without DNA in vivo,and the results showed that the M.xanthus biofilm without DNase I treatment was much more sticky than the treated one.Second,the DNA-EPS complex offered protections to both DNA and EPS molecules in exposure to nucleases and surface-active agents,respectively.The degradation experiments showed that that EPS was protecting DNA in the complex against the attack by various nucleic acid hydrolases,e.g.,DNasel and DNase II,which explained the continuous and stable presence of eDNA in the matrix.In addition,we demonstrated that the M.xanthus biofilms with eDNA are more tolerant to anionic and cationic surfactants,which suggested that the complex alos had a protection on EPS.Furthermore,the eDNA-EPS integrated ECM allowed M.xanthus cells to be more stress resistant.The cells are less resistant to aminoglycoside antibiotics after eDNA depletion in the biofilms.In addition,M.xanthus cells were shown to grow well using high concentrations of DNA that has been suggested to kill many bacterial cells,cells' survival under starvation conditions.Unexpectly,the presence of eDNA in M.xanthus biofilms did affect the motility,predation and development processes.In the final section of thesis,we explored the sources of eDNA in M.xanthus biofilms.In the mature biofilms,M.xanthus eDNA mainly comes from the cell lysis caused by programmed cell death.However,our reseach raised the possibility that the other eDNA production mechanism might be existed in the early process of M.xanthus biofilm establishment.