Molecular Mechanisms Underlining The Regulation Of EPS Production And Cellular Adhension Via Mts Gene Cluster In Myxococcus

Myxobacteria is a Gram-negative bacterium that exhibits complex multicellular development. It was once thought to be terrestrial bacteria, which can not grow in 1% salt environment. Our laboratory found a typical salt-tolerant bacterium, named Myxococcus fulvus HW-1, whose （S-） motility is enhanced in a medium containing 20% sea water. The enhanced （S-） motility can strengthen the adhesion between cells, thus protecting the bacteria from being washed away in the tidal waters. Related mechanism analysis helps us understand the origin and evolution of myxobacteria.Preliminary work showed mts gene cluster may be involved in this mechanism. mts gene cluster consists of six genes, mts A to mtsF. There are six highly homologous genes in Myxococcus xanthus DK1622, MXAN₁332 to MXAN₁337. Bioinformatics analysis showed:protein MtsB, MtsC, MXAN₁333 and MXAN₁334 contain a domain which is capable of combining with calcium. While protein MtsD, MtsE, MXAN 1335 and MXAN 1336 gene contain a vWFA （von Willebrand Factor） domain. The domain plays a very important role in platelet adhesion process. TSP （Thrombospondin）, which could combine with calcium, is capable of acting on the A3 domain of vWF, thereby destroying the disulfide bonds of the domain so as to help clotting. In view of the above analysis, we propose such a hypothesis:protein MtsB and MtsC act on protein MtsD and MtsE under the stimulation of divalent cations （such as Ca2+, Mg2+）, and eventually passed the signal out.As the divalent cations in seawater are mainly Ca2+and Mg2+, so we replaced seawater with calcium salt solution or magnesium salt solution. Experimental results showd that the （S-） motility of HW-1 is enhanced, but the effect is not so evident as in seawater.As the lack of effective genetic manipulation in M. fulvus HW-1, the subsequent experiments were carried out in M. xanthus DK1622. We transfered mts genes into DK1622 corresponding in-frame deletion mutants （MXAN₁333/MXAN₁334）, and found the deletion strain（ΔAMXAN₁333/ΔMXA₁334 in DK1622） containing mtsB/mtsC gene from HW-1 exhibited enhanced adhension. Further experiments showed that the process is achieved by protein MXAN₁335 and MXAN₁336, and these two proteins may have the same function.The previous results showed that mts was detected to interact with the protein PilB and PilT. As these two genes are responsible for expansion and contraction of type IV pili, mts may control the expression of surface pili to regulate （S-） motility. Meanwhile, the production of EPS is also improved. Is there any contact between the two process? The production of EPS is still enhanced in the pilA gene in-frame deletion mutant under seawater, which means there exists another regulatory pathways for EPS.We infer that mts regulates the production of EPS by participating in Dif pathway. The pathway contains six genes, of which three（difA、difC、difE） could not be missed. In order to test our hypothesis, the dif A gene was in-frame deleted, the deletion strain containing mts gene from HW-1 regained the EPS production. But when difC is deleted, EPS production defects. Mts gene seems to be able to connect with DifC that function downstream of Dif A. The interactions between Mts and DifC proteins showed by YTH system also support this hypothesis.The yeast two-hybrid experiments indicated that Mts proteins may exist as a complex, and only MtsF is found to interact with DifC、PilB、PilT. Thus, we think Mts proteins receive the signal and pass the signal out through MtsF. The distribution of MtsF protein in cell was exhibited by visualization of GFP-MtsF/Mcherry-MtsF through laser scanning confocal microscope. The result showed that MtsF protein existed as a bi-polar manner in cell, just as the distribution pattern of PilB and PilT.