Molecular Mechanisms And Biological Functions Of Interactions Among The Extracellular Macromolecules Of Myxococcus During Collective Cell Behaviors

Myxobacteria,one of the bacterial groups that have effectively evolved comparable in sophistication to that seen in macroscopic social organisms,are an ideal model for studying multifaceted social behaviors in bacteria.In Myxococcus xanthus,collective cell behaviors(a.k.a.group behaviors)participate in all critical steps of the social life cycle,e.g.,cellular motility,bacterial predatory behavior,biofilm formation,and fruiting body development.Extracellular macromolecules such as exopolysaccharides(EPS),extracellular DNA(eDNA),and type IVa pili(T4aP)have appeared indispensable values in the process of those cell behaviors.At present,it has been reported that eDNA can interact with EPS and form a highly ordered and organized extracellular matrix architecture,while little is known about the mechanisms of how eDNA integrates into extracellular matrix architecture through potential macromolecular interactions.In addition,while the specific interactions between T4aP and EPS have been proposed,and some direct observations of the binding under native conditions have been made,there have as yet been no quantitative analysis of these processes and the key moieties involved in the interaction remain elusive.In this study,we comprehensively use the methods of microbiology,molecular biology,biochemistry and biophysics to analyze the physical/chemical nature and the molecular mechanism of the interactions between the extracellular macromolecules and their biological functions during group behaviors in M.xanthus.In Chapter 2,we focused on the developmental and non-developmental biofilms formed by M.xanthus to elucidate the chemical nature of the interaction between eDNA and EPS and the biological features of the M.xanthus biofilms associated with the integrated DNA-EPS complex.We confirmed that eDNA was colocalized with EPS in the ECM of M.xanthus biofilms,suggesting that DNA is able to combine EPS to form a macromolecular conjugate.Subsequently,multiple spectroscopic and microscopic methods were used to investigate the interactions between eDNA and EPS in vivo and in vitro,which revealed that electrostatic forces participated in the polymer-polymer interactions.We also found that the EPS production-deficient M.xanthus SW504 strain was able to utilize eDNA to support the growth of its own cells.Under nutrient-deficient conditions,M.xanthus lacks sufficient energy and substances to synthesize a large number of EPS.Extracellular-free DNA can be used as a nutrient to maintain the survival of M.xanthus under starvation conditions.The presence of eDNA also enhanced the resistance of M.xanthus cells to some surfactants and antibiotics treatment.eDNA is considered to be an important target for bacterial biofilm infection control.Our related research results will not only help to deepen the understanding of the biological functions of eDNA and EPS in bacterial biofilms,but also provide a theoretical basis for the combined treatment of bacterial biofilm infections with nucleases and antibiotics.In Chapter 3,we analyzed the key amino acid of T4aP and sugar residue sites of EPS that participated in the interaction between T4aP and EPS.We also further explored the central role of T4aP and EPS interactions in mediating and regulating M.xanthus group cell behaviors.The label-free biophysical techniques,e.g.surface plasmon resonance(SPR)and isothermal titration calorimetry(ITC),together with the western blot based pull-down assay,were employed to investigate the kinetic and thermodynamic characteristics of interaction between the PilA protein and EPS.The experiment results suggested that among the glycosyl composition of EPS,some amino sugars may be responsible for the specific interactions between PilA and EPS.Meanwhile,we identified an M.xanthus PilA mutant at position 146 tryptophan by bioinformatics analysis,which produced structurally intact but super-retractable T4aP and showed an impaired binding ability to EPS.Characterization of saturation mutagenesis mutants at 146 tryptophan in PilA protein demonstrated the critical role of tryptophan 146 in the specific recognition of EPS.The findings are likely to add significantly to the biological knowledge about regulatory events during social behaviors.T4aP/EPS and their associated interactions are also key virulence factors for various pathogens,and this study will potentially lead to an increased understanding of new therapeutic approaches that target T4aP-EPS.In Chapter 4,we investigated the molecular mechanisms that Myxococcus macrosporus HW-1 adapted to marine environments.As a strain isolated from the intertidal zone,M.macrosporus HW-1 has exhibited great similarities with M.xanthus DK1622 in genomic homology and collective cell behaviors.However,M.macrosporus HW-1 is able to tolerate a wide range of salinity.In response to changes in salinity,M.macrosporus HW-1 shows altered collective cell behaviors,e.g.,enhancing S-motility,increasing EPS production,and forming flocs biofilm.This special lifestyle is beneficial for M.macrosporus HW-1 to avoid being washed away during seawater fluctuations,providing a prerequisite for the occurrence of group behaviors.Under the stimulation of seawater,M.macrosporus HW-1 was able to kill E.coli more effectively in a cell contact-dependent manner,which correlated well with the increase in EPS production.When on a solid surface with high water content,M.macrosporus HW-1 enhanced S-motility and increased the efficiency of searching for surrounding prey,thus enhancing the ability of predation.The former predation strategy was in response to the liquid habitat at high tide,and the latter predation strategy was in response to the solid habitat at low tide.These two distinct predation strategies allow M.macrosporus HW-1 to maintain a survival advantage in intertidal habitats at changing tides.Finally,we attempted to explore the molecular regulatory mechanisms of M.macrosporus HW-1 in response to seawater.Transcriptome analysis implied us that the mts gene cluster,which receives and transmits salt ion signals,the pilT gene,which mediates T4aP contraction,and the stk,difC,and other genes involved in EPS regulation might play an important role in M.macrosporus HW-1 response to seawater.In conclusion,on the basis of establishing the microscopic characterization of extracellular macromolecules and the qualitative and quantitative analysis system of extracellular macromolecule interactions,our study systematically analyzed the essence of the interactions among eDNA,EPS,and T4aP.Their biological properties and functions in the group behaviors of M.xanthus were explored.Our findings have important scientific value for the in-depth understanding of the cellular behavior of complex bacterial populations represented by Myxococcus xanthus.