Kin Discrimination In Myxococcus Xanthus

Diverse bacteria always co-exist in population.Due to the requirements for space and food,bacteria may be each other's strongest competitors in nature.Neighboring,closely related individuals of a bacterial species must routinely discriminate self from non-self to allow exclusive social behaviors,such as nourishment,social movement,quorum sensing,virulence,iron acquisition,protection,biofilm and fruiting-body formation.In microbes that possess multicellular swarms,one form of kin discrimination is the colony-merger incompatibility that reflects as a visible boundary formation between the colonies of two genetically distinct bacteria.Colony-merger incompatibility was first reported in Proteus mirabilis,and recently observed in several other bacterial species.However.the molecular mechanisms involved in bacterial kin discrimination are likely to be diverse and are only beginning to be understood.The order of Myxococcales is located in 8 group of Proteobacteria.Myxococcus xanthus DK1622 is a type strain and the genome has been sequencing completely.M.xanthus possesses complex multicellular behaviors and is known as a social bacterium.On the solid surface with enough nutrition,M.xanthus cells swarm.When nutrition is deprived,Myxococcus cells would gather to form fruiting bodies,within which kill the majority of cells by programmed cell death and the remaining 1%of cells will survive as myxospores.M.xanthus cells can produce sufficient extracellular enzymes,antibiotics and other substances to collectively prey on a variety of microorganisms,including Cyanobacteria,Escherichia Coli,rhizobia and even some fungus.The multicellular behaviors of M.xanthus cells provide itself more survival advantage over the dispersed behavior of the single cell.From 3392 random transposon mutations,11 self-identification?SI?deficient mutants that formed unmerged colony boundaries with the ancestral strain was obtained.The mutations were at nine loci with unknown functions and formed nine independent SI mutants.Compared with their ancestral strain,most of the SI mutants showed reduced growth,swarming and development abilities,but some remained unchanged from their monocultures.When pairwise mixed with their ancestral strain for co-cultivation,these mutants exhibited improved,reduced or unchanged competitive abilities compared with the ancestral strain.The sporulation efficiencies were affected by the DK1622 partner,ranging from almost complete inhibition to 360%stimulation.The differences in competitive growth between the SI mutants and DK1622 were highly correlated with the differences in their sporulation efficiencies.However,the competitive efficiencies of the mutants in mixture were inconsistent with their growth or sporulation abilities in monocultures.The colony-merger incompatibility in M.xanthus is associated with multiple independent genetic loci,and the incompatible strains hold competitive interaction abilities.Further analysis of the inserted gene loci revealed that the insertion genes from five self-identification deficient mutants were homologous,and the genes immediately upstream of these five genes were also homologous.The homologous genes of the inserted gene encode a class of acidic proteins with a relatively small molecular weight,and the function is unknown.The homologous genes of the upstream gene encode a class of larger molecular weight basic proteins.The multiple sequence alignment results show that it contains multiple conserved basic amino acid sites,and the conserved'AHH' motif,which may be a nuclease.The single gene deletion mutant of the homologous genes of the inserted gene merged colonies with the corresponding self-identification deficient mutant,while formed colony boundaries with the wild type strain DK1622.The single gene deletion mutant of the homologous genes of the upstream gene merged colonies with the wild type strain DK1622 and the corresponding single gene deletion mutant of the homologous genes of the inserted gene.Thus,the two sets of homologous genes may encode a pair of antagonistic nuclease and its inhibitor.The two sets of homologous proteins have corresponding evolutionary relationships and may be co-evolved.Five single gene deletion mutant of the inserted genes also form colony boundaries with each other,and they maybe function independently.The MXAN 0049 gene deletion mutant formed colony boundaries with the wild type strain DK1622,but merged the colonies with SI01 mutant.The upstream MXAN₀050 gene is close to MXAN₀049,and the MXAN₀050 gene was co-transcribed with the MXAN₀049 gene.The ?MXAN₀050 mutant merged colonies with either the wild type strain DK1622 or the deletion or insertion mutants of MXAN₀049 gene.If the MXAN₀049 or MXAN₀050 gene was complemented into the corresponding deletion mutant,the complementary strain showed similar discrimination phenotypes to the wild type strain.Thus,the MXAN 0050 gene probably functioned antagonistically with the MXAN₀049 gene in the colony-merger incompatibility,which was consistent with the proposal that the MXAN 0050 protein may be a nuclease and the MXAN 0049 protein may be an inhibitor.The MXAN 0050 protein was overexpressed and purification in Escherichia coli BL21 strain.The MXAN 0050 protein was incubated with the genomic DNA of M.xanthus DK1622 or E.coli DH5?,and the substrates were completely digested.If the MXAN 0049 protein was mixed with MXAN₀050 protein in the reaction system,the nuclease activity of MXAN 0050 was blocked.Using the pull down experiment and yeast two-hybrid technique,the MXAN₀049 protein was demonstrated to bind to MXAN 0050 protein,which might form the molecular basis for the inhibition of MXAN₀049 to the nuclease activity of MXAN 0050 protein.The expression of the MXAN₀050 nuclease protein led to impairment of E.coli growth,and the toxin effect of the MXAN 0050 protein could be alleviated by the co-expression of MXAN₀049 protein.The survival of?MXAN₀049 mutant was greatly impacted by the wild type strain,and the?MXAN₀049 mutant and DK1622 strain could not co-exist.If the ?MXAN₀049 and?MXAN₀050 mutants were co-cultured at an equal initial ratio,they could co-exist.Therefore,the MXAN 0050 nuclease-toxin and MXAN₀049 immunity protein were functional in M.xanthus DK1622.If the MXAN₀050 nuclease functions in the colony-merger incompatibility as a toxin,the MXAN₀050 protein must be transferred by a carrier into the target cells.Interestingly,the downstream MXAN₀044 gene of the MXAN₀050 gene was recently annotated to encode a PAAR repeat protein,which has been reported to facilitate the secretion of effectors by carrying the proteins and binding to the VgrG trimer of the T6SS for transportation.The MXAN₀044 gene deletion mutant merged colonies with the wild type strain DK1622 and with the MXAN₀049 insertion or deletion mutants,which implied that the MXAN₀044 gene was correlated with the colony-merger incompatibility.When ?MXAN₀044 and ?MXAN 0049 were co-cultured at an equal initial ratio,they could co-exist.The structure of the T6SS in M.xanthus DK1622 has been identified in a recent report.When adjacently inoculated,the AtssL,?tssM and?tssD mutants merged their colonies with the SI01 mutant,as well as the wild type strain DK1622,respectively.And the ?t6ss and ?MXAN₀049 mutant could co-exist.The above results suggested that the PAAR-T6SS was associated with the colony-merger incompatibility mediated by the MXAN 0050 nuclease-toxin and MXAN 0049 immunity protein pair in M.xanthus DK1622.The other neighbouring genes at the locus from MXAN₀043 to MXAN₀052 were also deleted one by one.Proximate inoculation showed that the ?MXAN₀045,?MXAN₀051 and?MXAN₀052 mutants merged their colonies with both the ?MXAN₀049 mutant and DK1622.The ?MXAN₀043 and ?MXAN₀048 mutants formed colony boundaries with ?MXAN₀049 but merged their colonies with DK1622.Both the ?MXAN₀046 and ?MXAN₀047 mutants formed dense colony boundaries with ?MXAN₀049.These results suggested that many genes at this locus might function in the colony-merger incompatibility in M.xanthus cells.We proposed a simplified model for the kin discrimination mechanism mediated by the MXAN₀050/MXAN₀049 gene pair.In M.xanthus DKI622,the nuclease toxin encoded by the MXAN₀050 gene,is probably directly or indirectly bound to PAAR protein?encoded by the cognate MXAN₀044 gene?,and the conjugate protein is then uploaded to the tip of the T6SS tail complex.The PAAR-carried MXAN₀050 toxin may thus be exported to neighbouring cells via the T6SS.Wild type cells harbouring the cognate MXAN₀049 immunity protein may not be harmed by the MXAN 0050 nuclease.However,the MAAN₀049-deficient cells will be impacted by the nuclease toxin inputted by neighbouring DK1622 cells.In contrast,because they are deficient in the nuclease-encoding gene,?MXAN₀050 cells are unable to inhibit the growth of?MXAN₀049 cells,and these two kinds of cells co-exist in the mixture.Similarly,?MXAN₀044 or At6ss cells are also able to coexist with ?MXAN₀049,as neither mutant is able to deliver the MXAN 0050 protein.Confirmation of the nuclease protein secretion by T6SS and the nuclease toxin functions as an effector on neighbouring cells require further investigation.Because the five homologous gene pairs functioned independently,only the two strains that have the same gene composition of all the homologues can identify with each other,i.e.forming merged colonies.When one strain transfer the nuclease-effector into other strain and the latter has the respective immunity protein?the secret key?,these two strains identify themselves and their colonies can merge with each other.Otherwise,they will form boundaries between their colonies.In M.xanthus DK1622,five homologous gene pairs are able to combine into 25 kin discrimination secret keys,resulting in 32 incompatible strains that do not recognize with each other.Numbers of the kin discrimination secret keys increase,thus leading to outcomes that abundant incompatible types exist in a niche.This mechanism lets bacterial strains easily generate new incompatible types and is able to maintain the coexistence of enormous incompatible types in a niche to increase population environmental adaptation.The boundaries formed between sibling colonies hinder the exchange of their genetic materials,while the coexistence of many smaller groups expands population genetic diversity.When the environment changed,the genetic diversity will increase the adaptation of the entire populations.