Omics Approach To Understand The Formation Of Colony Boundary In Myxococcus Xanthus

Wide varieties of bacteria live together in the same microbial community,and there is a need for aggressive competition or cooperation between bacteria for limited space and resources.Cooperative cells can produce a large number of supportive behaviors that are conducive to their own survival,including nutrient acquirement,group movement,virulence,defense,and ion absorption,formation of biofilm and fruiting body formation.To ensure that these cooperative behaviors occur between cells of the same type,bacterial cells need to be distinguished by neighboring cells.Microbial sociology directly influenced by neighbor's synergistic or antagonistic behaviors,which have been based on the capability of microbe to recognize self and eliminate non-self to procedure groups of correlated individuals.Cell-to-cell kin discrimination in bacteria often shows as colony-merger incompatibility and until today,there are several studies about colony merge incompatibility in bacteria two colonies of similar strains can be fused after meeting,and two colonies of non-similar strains meet to form a line visible to the naked eye.Close relatives of some bacterial species,such as Proteus mirabilis,Bacillus subtilis,and Myxococcus xanthus,are able to form visible boundaries between colonies of different strains that are unrecognizable with each other.The phenomenon is known as colony-merger incompatibility.Myxococcus xanthus DK1622 is a Gram-negative soil bacterium model strain present in the ? group of Proteobacteria and its genome has been sequencing completely.One of the unique characteristic of M.xanthus that it is able to display multicellular behavior and well known as a social bacteria such as on the nutrient-rich solid surface it has group movement of cells.In case,nutrients are scarce,it combined to form fruiting bodies,although only 1%of the cells become stress-resistant spores and survive.After nutrient enrichment,the spores can germinate into vegetative cells.Another feature of M.xanthus that it secrete a large number of antibiotics and extracellular enzymes to cooperate in preying on other microbial cells.Such kind of multicellular behavior facilitate the M.xanthus cells in survival and competing over other single cell having isolated behavior.Previously,at the earlier phase of the laboratory,3392 insertion mutants were obtained by random insertion of transposons in the model strain M.xanthus DK1622,among them 11 strains of heterologous recognition mutants,named self-identification?SI?mutants were screened and they formed the colony boundaries M.xanthus DK1622 strain.The inserted gene loci are distributed at nine loci in the M.xanthus DK1622 genome,most of them are hypothetical proteins having unknown function.In comparison with wild type most of these self-identified?SI?mutants showed reduced growth,movement,fruiting body development and spore formation.The ?MXAN₀049 strain is a mutant deficient in the immunity for the paired nuclease gene MXAN 0050,playing function in the colony merging incompatibility of Mvxococcus xanthus DK1622.Notably,here we have focused on the M..xanthus DK1622 and the incompatible mutant AMXAN₀049 which have formed the morphologically visible demarcation line between swarms of approaching bacterial colonies and has been referred as boundary formation.A mechanism for the colony-merger incompatibility is that a donor cell transports effector proteins to a recipient cell in close proximity through the type 6 secretion system?T6SS?device in a contact dependent method.However,it is mostly unclear what factors play the role in the formation of boundary and what happened within the boundary.With aim to investigate the factors for boundary formation,proteomics and metabolomics approaches utilized.Both omics methodologies along with microscopy triggered our understanding regarding the direct or indirect involvement of different genes,proteins and different potential pathways in boundary formation.Based on both coverage and PSM values we sorted out the proteins those are significantly high in their expression level.We found that many of the stress resistance proteins such as different cold shock proteins?Csps?were expressed significantly about 11 folds higher when boundary fonned as compare to the control samples?DK1622-DK1622 and AMXAN 0049-?MXAN₀049?.GroEL1 up regulated about(1.5 folds higher than the control samples.There were also some other significantly up-regulated proteins such as patatin-like phospholipase family protein(up regulated within boundary 7.6 folds against DK1622-DK1622 and 4.6 folds against ?MXAN 0049-?MXAN 0049.Patatin-like phospholipase previously reported in some Gram-negative bacterial species act as effector molecules to target host cellular membrane as it has the capability to speed up the breaking process of' fatty acids from the membrane lipids.In the same way,tetratricopeptide repeat?TPR?proteins reported to be directly involved in the virulence-associated function in some bacterial pathogenic bacteria such as Pseudomonas aeruginosa,Yersinia,Francisella tularemia.They transmit it either by transferring the virulence factors into host cells or by connecting to host cells.Overall,there are 154 proteins having same homology as TPR within M.xanthus DK1622.When boundary?DK1622-?MXAN 0049?formed 41 of these TPR proteins were presents,while 31 and 22 were present in the non-boundary cases of DK1622-DK1622 and ?MXAN₀049-?MXAN 0049,respectively.When boundary formed,tetratricopeptide repeat protein?Gene ID:MXAN 4221?significantly up-regulated about 5 folds against DK1622-DK1622 and 6.5 folds against?MXAN 0049-?MXAN 0049.Several proteins were absent or down regulated when boundary formed such as putative outer membrane protein P1,Uncharacterized protein?belongs to porins family,MXAN 5855?,TonB-dependent receptor outer membrane proteins significantly down regulated.Interestingly,porins,lipoproteins,TonB-dependent receptor that play role as a passage of nutrients,transferring the signals as well as waste products or proteins that play role to protect the cell against oxidative stress via detoxifying peroxides such as thiol-specific peroxidase.Some proteins coverage significantly went down when boundary formed that might lead towards the prominent effect over the cells within the boundary.The above study have opened the mechanistic clues of bacterial microbiota of natural environments who have been coexist in distinct niches with diverse strains to and have defense from resource antagonism or manipulation by other strains.Although in this study through proteomics analyses,we highlighted the significant proteins that may have the possible role directly or indirectly in the formation of boundary but still further investigation and experimental confirmation are required.Our proteomics data also showed that when boundary formed the degradation of RNA also occurred and RNA degradation pathway significantly active when boundary formed as most the key proteins up regulated as compared to non-boundary samples.Interestingly RNA degradation pathway has strong protein-protein interaction with cold shock proteins?Csps?.In this work,we also found the possible function of type VI secretion system?T6SS?when boundary formed.When the T6SS gene cluster?MXAN 4800-4813?knockout from the DK1622 strain,the result as the boundary disappeared after the interaction with ?MXAN 0049,which confirm the role of T6SS in boundary formation as well.Imperative T6SS components existence revealed its role in the transportation of different components across the membrane;both Vgr proteins?MXAN₄800 and MXAN₅573?were also present when boundary formed.Myxobacteria is one of the top producers of secondary metabolites next to fungi?actinomycetes?and bacillus.Remarkably,huge number of mycobacterial isolates screening led to the discovery of diverse bioactive metabolites including anticancer,antibacterial and antiviral compounds.M.xanthus is a well-known model strain and significant source of different bioactive secondary metabolites.Polyketide synthase?PKS?and non-ribosomal peptide synthetase?NRPS?are biosynthetic enzymes produce many polyketides and non-ribosomal peptides,respectively.Notably,14 polyketide synthase?PKS?and non-ribosomal peptide synthase?NRPS?were present when boundary formed while only 6 and 7 were present in DK1622-DK1622 and ?MXAN₀049-?MXAN 0049 respectively,which suggested the active role of PKs/NRPs in boundary formation.The assessment of secondary metabolites revealed the presence of several reported compounds such as Myxochelin A,Myxochelin B,Myxalamide D,Myxochelin J8 and Neriifolin upon boundary formation in contrast to non-boundary.We also found 12 new unknown compounds?was not reported yet?that were significantly present when boundary appeared more than 100 folds as compared to both controls.In the microscopy section especially,LIVE/DEAD viability assay observation showed the dead or damaged cells represented as red line and several damaged cells also appeared towards MXAN 0049 side,while all the green colored showed the presence of active and live cells.For the further confirmation to check the morphology of cells,scanning electron microscopy?SEM?utilized.Different magnification we discovered the presence of damaged/dead along with irregular shaped cells within boundary were present whereas,active/alive cells observed within both controls.We also found out within the boundary,the shape of cells changed and they were not in a regular rod shape as reported previously that MreC played role to form a rod shape in cells.Our proteomics results showed that when the boundary formed MreC protein was absent;while it has appeared in control and cells were in a regular shape.We found that phenylalanine,tyrosine and tryptophan biosynthesis pathway when boundary formed and confirmed through metabolomics study the significance presence of phenylalanine,tyrosine and tryptophan in boundary can be another possible factor of boundary formation.Overall,we can conclude with this remark that there are multiple factors in the formation of boundary and our studies help to give various clues to solve the mystery of boundary formation for further studies.