Molecular Strategy For The Adaptation Of Salt-tolerant Myxococcus Fulvus HW-1 Social Behaviors To Marine Conditions

Myxobacteria are Gram-negative bacteria that possess complicated multicellular social lifestyles on solid surfaces.The bacteria are model lives for the study of bacterial social behaviors.These behaviors include social gliding motility,predation and fruiting body development.Gliding motility of cells plays a crucial role during myxobacterial predatory and developmental aggregation lifestyle.The gliding,well demonstrated in Myxococcus xanthus,is controlled by two distinct motility systems: the adventurous(A)system,which controls the gliding motility of individual and isolated cells,and the social(S)system,which is essential for the swarm and aggregation movements of cells.Three cell surface components are required for S-motility,i.e.typeâ…£pili(Tfp),extracellular matrix(ECM)and lipopolysaccharide (LPS)O-antigen.The mechanism of S-motility is more complicated genetically than that of flagellar motility.So far,more than 110 genes known to be essential for S-motility have been identified.To fit the social life,myxobacteria are generally located in soil and,consequently, are common in many types of terrestrial rather than in aquatic environments,and thus, they are considered to be typical soil microbes.Moreover,no purified terrestrial myxobacteria had been confirmed to be able to grow with salt concentration of more than 1.0%.However,myxobacteria have recently been found to be able to live in many marine environments as halotolerants or low halophiles.Halophilic myxobacteria were isolated from various marine samples in Japan,and are both phylogenetically distant and morphogenetically different from the normal terrestrial myxobacteria and thus have been classified as novel myxobacterial groups. Taxonomic characteristics,phylogenetic analysis of 16S rDNA fragments and secondary metabolites,instead of living pattern,adaptation strategies and molecular mechanism have been studied of these halophiles.Many salt-tolerant myxobacteria have been isolated from marine samples in our laboratory.In response to changes in salinity,the salt-tolerant myxobacteria shift their growth,morphology and development characteristics.From differences in the morphogenetic characteristics between terrestrial and marine conditions,the salt-tolerant myxobacteria are thought to be the result of the adaptation of soil myxobacteria to marine conditions.Analysis of different salt-tolerant Myxococcus species or strains reveals that these myxobacteria retain the dual motion phenotypes, and the high salt-tolerant strains,including M.fulvus HW-1,even exhibit enhanced S-motility abilities in the presence of seawater.Stronger S-motility may allow closer contact between the cells and therefore provide better adaptation to the ocean conditions.Exploration and discovery of the adaptation and diversification of myxobacterial motility systems under natural conditions may provide important clues for us to understand the formation and evolution of the myxobacterial motility systems.The mariner transposable elements of mariner/Tc1 superfamily are widely distributed in animal genomes and are especially prevalent in insects,mariner transponsable elements have a remarkable lack of host specificity.Transposition of mariner transposons almost appears to be random.These transposons are particularly valuable also because they are small and use short inverted repeats.In some papers, mariner-based transposons were reported to be active in myxobacterial strains and broadly useful as genetic tools.In this work,the genetic manipulation methods established in M.fulvus HW-1 meet the major requirements for molecular studies on motility and development.Using the methods,a genetic screen was performed in M.fulvus HW-1 and a new locus was discovered to be required for S-motility and development in Myxococcus,which can afford new ideas and potential means for identifying new genetic loci involved in motility in myxobacteria.First of all,to identify the S-motility genes related to the adaptation of M.fulvus HW-1 to ocean conditions,we screened S-motility deficient mutants following random insertion of the transposon MiniHimar1-lacZ into genomic DNA.From more than 2000 insertion mutants,we identified a mutant,named M.fulvus HL-1,showed significant reduction in colony expansion on a 0.3%agar surface,which is indicative of an S-motility defect.Activity analysis ofβ-galactosidase indicated that the mutated gene in HL-1 can early express and express at high level.The mutant HL-1 was assessed for motility phenotypes using standard methods, and the phenotypes suggest that the mutant HL-1 is defective in social motility and partially A motile.Interestingly,the effect of seawater on swarming ability was significantly decreased by the mutation.These results suggest that mutated gene(s)are involved in or responsible for the enhancement of surface translocation in response to the presence of seawater.The mutant HL-1 was assessed for developmental ability on TPM starvation media,and the results indicate that the mutant HL-1 is also significantly defective in developmental aggregation and sporulation.Liquid colorimetric assay,agglutination assay and electron microscopy detection revealed less extracellular matrix on the cell surface of HL-1 and that the mutant cells exhibited less cohesion than the wild type cells.These results demonstrate that the mutated gene is probably due to the requirements for adaptation to the marine conditions in HW-1.An upstream 6.3 kb segment and a downstream 6.7 kb segment flanking the insertion were obtained by plasmid rescue method and Tail-PCR amplifications. Using programs FramePlot 3.0beta,the 13 kb segment was predicted to contain six open reading frames,which likely form a gene cluster.Blastx against the GenBank database revealed that the sequences are significantly homologous to the corresponding ORFs from M.xanthus DK 1622 and Stigmatella aurantiaca DW4/3-1. Most of these ORFs are predicted to be putative type 3 thrombospondin.Thus,the gene cluster was designated mts for myxobacterial thrombospondin-like proteins (MtsA-F).The MiniHimarl-lacZ insertion interrupted the codon for Tyr359,located 83 residues from the C-terminus of the predicted MtsC.MtsA and MtsE possess transmembrane regions at their N-termini,as assessed by SMART.SignalP-HMM program predicted that MtsA,MtsC,MtsD,MtsE and MtsF contain signal peptides. Bioinformatics analysis of the predicted mts,together with the phenotypes of the mutant,suggested that the Mts proteins are probably involved in the construction of cell surface matrix for S-motility and development.Genetic manipulation of HW-1 is difficult.We were not able to mutagenize the strain by deletion using the plasmid pBJ113.Instead,an insertion of mtsC homologue was performed in M.xanthus strains DK1622.The mutant produced colony with similar edge to that of HL-1,indicating that mtsC homologue is probablely required for motility in DK1622.The expression of mtsC homologue was also similar to that of mtsC in HW-1.Interestingly,the effect of the insertion in mtsC homologue on motility in M. xanthus seems to be not as strong as the insertion in mtsC in M.fulvus HW-1.These results corresponding to E(g)values of the predicted mts suggested that the mrs products play a more important role on S-motility in M.fulvus HW-1 than their homologues do in M.xanthus DK1622,probably due to the requirements for adaptation to the marine conditions.We made an in-frame deletion of mtsC homologue in DK1622(A⁺S⁺),DK1217 (A^-S⁺)and DK10410(A⁺S^-)to determine its function.The results indicated that mtsC homologue is involved in S-motility and required for development in M.xanthus. Also,we made a complete deletion of the sequence of MXAN1332～MXAN1337 from DK1622,and the results indicated that the whole mts homologues are likely to be involved in S-motility and required for development in M.xanthus.Then,in-frame deletion of each mts homologue except mtsC homologue was performed in DK1622,the phenotypes of these mutants indicated that mtsA,mtsB and mtsD～mtsF homologues are involved in S-motility and mtsA,mtsB,mtsD and mtsE homologues are required for development in M.xanthus.We were not able to identify the localization of MtsB homologue by expressing a MtsB homologue-fluorescent protein fusion protein.Thus,MtsB and MXAN1333 were hetero expressed in the host strain E.coli BL21(DE3),respectively.The fusion proteins MtsB and MXAN1333 were both extracted from inclusion bodies formed in the host strain and purified with Ni²⁺column with a His-tag Multi-clone antibodies were produced with the purified MtsB and MXAN1333 as the antigens,respectively.The strain HW-1 was induced by UV radiation and a mutant UV684 was obtained. Compared with the parent,UV684 showed completely dispersed growth in liquid medium and exhibited high transformation/tranposition efficiency as 10⁵-10⁶ CFU/μg DNA.On the other hand,the phenotypic characteristics,including salt-tolerant growth, morphology,fruiting-body formation and gliding motility,were analyzed and compared with the parent strain HW-1.The results indicated that the fruiting body formation and S-motility of UV684,to some extent,was affected in UV mutagenesis, but no difference was observed between the two strains in the size and shape of vegetative cells and myxospores,the salt-tolerant growth capacity,and the motilities increasing in the presence of seawater.So the mutant UV684 can be studied as an excellent parent in these unchanged unique characteristics by molecular genetic manipulation.