| Gram-negative unicellular myxobacteria are notable for their complicated life cycles and multicellular behaviors such as cell density depended growth, social movement, and the formation of multicellular fruiting bodies. Myxococcus xanths DK1622 is studied as model (Shimkets,1990). Formation of both cellular patterns depends on extracellular functions including the extracellular matrix and intercellular signals. Interestingly, the formation of these patterns also depends on several activities that involve direct cell-cell contacts between M. xanthus cells or direct contacts between M. xanthus cells and the substratum.Several species of myxobacteria can excrete extracellular catabolic enzymes such as protease(s), lysozyme(s), amidase(s), gucosamine enzyme(s) and so on (Hart & Zahler,1966; Sudo and Dworkin,1972), but the information of corresponding gene(s) and protein(s) have not been identified. M. xanthus harbours all protein secretion systems required for translocation of unfolded and folded proteins across the cytoplasmic membrane and an intact type II secretion system. Genomewide analyses of the M. xanthus genome reveal a large potential for protein secretion (Goldman et al., 2006; Konovalova et al.,2010). But so far, there are still no reserch reports about the composition and regulation of extracellular proteome in myxobacterial.Marine halotolerant Myxococcus fulvus strain HW-1 was isolated by our laboratory, which existed different living patterns within and without the seawater conditions (Zhang YQ et al.,2005). The early reserch has shown that the outer membrane (Omp) gene Omp031 had the most significant change in expression (downregulated) in response to seawater. Its homologues Mxan3106 was knocked out in DK1622 to analyze its function. Salt tolerance and sporulation of the Mxan3106 mutant was enhanced compared to that of DK1622 (Pan HW et al.,2009). Bioinformatic analysis reveals that both Omp031 and Mxan3106 are individual secretin encoded genes isolated on the corresponding genomes, away from the single gene cluster for typeⅡsecretion system (T2SS). Considering the multifunctionality of Mxan3106 in DK1622, we speculate that Omp031 is in charge of secretion of special proteins under special conditions, probably plays an important role in the shift of different lifestyle in M.fulvus HW-1.Based on the dicussion described above, my study was carried out as follows: (Ⅰ):We have optimized the parameters for electrotransformation of M.fulvus HW-1 strain, and confirmed that DNA uptake is not the bottleneck for genetic manipulation of HW-1.On the basis of the optimal conditions for electrotransformation of pMiniHimar-lacZ into HW-1 strain established before (Zhang CY et al.,2007), we tried different voltage,the concentration and conformation of plasmid,culture medium,culture time and so on using pBJ113 as exogenous DNA for homologous recombination. On these conditions, we gained none of the mutant strains with deletion of target genes, but gained the transposition mutants. In addition, Wang J et al. experimentally demonstrated Myxococcus xanthus could take up exogenous DNA via natural transformation. All of the phenomenons inferred that the bottleneck of genetic manipulation was probably the restriction modification system existing in HW-1 cells to destroy the foreign DNA introduced, but not the step of entering of foreign DNA. The inner and specific mechanisms still need to be researched.(Ⅱ):Mxan3106 was completely knocked out in DK1622 (producing mutants YL1601) to contrastively analysis basal characters comparing with YL0401 which was produced by in-frame deletions of Mxan3106 in M. xanthus DK1622Compared to DK1622, YL1601 had similar movement ability, but had increased salt tolerance, predation ability was decreased, these characteristics were similar with YL0401.But as for development and sporulation,YL1601 differed from YL0401. On the both TPM developmental medium and TPM with added seawater, YL1601 had more decreased development ability obviously than DK1622. But YL0401was in contrast with YL1601. The mechanisms still need to be researched.(Ⅲ) Established the protocol for preparation of extracellular proteins for proteome analysis, compared extracellular proteomes of both DK1622 and YL1601 by 2D electrophoresis.7 Mxan3106-dependent secreted proteins were indentified.We improved metheds in paper, and 20% TCA-acetone method was suitable for extracting and purifying the extracellular proteins.Using optimal 2DE parameters,we extacted extracellular proteins of both DK1622 and YL0401 in different growth condition.By analysing 2-D gel,we chooesd 23 protein spots peculiar in DK1622,of these,21 protein spots were identified.Initial analysis revealed that of 18 identified proteins,7 proteins had been predicted as extracellular proteins including peptidase and metallic phosphoesterase.These results not only tentatively confirmed secretin function of Mxan3106 but also laid the foundation for further research of myxobacterial extracellular proteome. |
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