Molecular Mechanism Of Regulating Social Cell Behaviors Via Mts Gene Cluster To Adapt To The Beach Habitat In Myxococcus

Myxobacteria are gram-negative bacteria with complex life cycle, including vegetative growth under rich nutrition and development during lack of nutrition. They are known as their sophisticated social (S-) cell behaviors, i.e. gliding motility, predation on other bacteria and development to form fruiting bodies, and among which the S-motility is is considered as the key behavior. Many myxobacterial strains are identified and isolated from marineenvironments recently although they had been treated as terrestrial bacteria for a long time. It becomes important to understand the molecular mechanism underlining the adaption of myxobacteria to the marine environments by shifting their social cell behaviors.As a strain isolated from beach samples, Myxococcus fulvus HW-1 exhibited great similarities with Myxococcus xanthus DK1622 in many respects.However, in contrast to the salt sensitivity of DK1622, salt-tolerant HW-1 is able to withstand a wide range of salinity. In response to changes in salinity, HW-1 shows altered social cell behaviors, e.g. enhanced social (S-) motility and fruiting body formation, which has been suggested as its adaption to the marine environments.In the previous studies, a gene cluster in HW-1 designated as mts for myxobacterial thrombospondin-like proteins (MtsA-F) has been shown to be involved in toning up S-motiltiy in the presence of sea water. The mutant strain HL-1 from HW-1 is defective in S-motility and developmental aggregation due to the interruption of mtsC gene by MiniHimarl-lacZ transposon, and the influence of seawater on social behaviors of HW-1 is remarkably subdued by the same mutation.In the current study, after respectively transferring mts genes of HW-1 into DK1622 corresponding in-frame deletion mutants(MXAN1332-MXAN1337), the deletion strain(△MXAN1333 in DK1622) containing mtsB gene from HW-1 regained the ability to enhance their S-motility, EPS production and biofilm formation in a medium containing 20% sea water, which was thus used as the major subject for the following studies.The difference between mts and its homologous gene cluter in HW-1 and DK1622 was further explored. In HW-1, qPCR analysis showed that the transcriptive levels of all mts genes except mtsA increasedin seawater medium compared with that in fresh water medium; while transcription of all homologous mts genes decresed significantly in DK1622 in the presence of sea water. Transcription analysis suggested that MXAN1332-MXAN1337 were co-transcripted in DK1622 and only mtsB-mtsF is co-transcripted in HW-1. In consistent with these results, the promoter prediction indicated the presence of active promoters upstream mtsB gene in HW-1 genome, while the similar potential promoters were bypassed by the strat coden of MXAN-1333 gene and were not active in DK1622.In Myxococcus, S motility is powered bythe extension and retraction of type four pili (TFP), therefore, the TFP biogenesis was examined in different strains. Since the TFP detection could be compromised by its interaction with EPS. the epsA gene was in-frame deleted in several strains to generated EPS deficient cells. The surface pili were revealed by western blot using anti-PilA antibody, and result showed that the deletion strain (AMXAN1333 in DK1622) containing mtsB geneincreased the amount of its surface type IV pili in the 20% sea water medium, which was further confirmed by calculation the tethering events of cells in submerged medium supplied with 1% methylcellulose. Next, the distribution of MtsB protein in cell was exhibited by visualization of GFP-MtsB fusion protein through fluorescent microscope. The result indicated that mtsB protein was existed as a bi-polar manner in cell, which is similar with the previous finding of PilB and PilT distribution pattern. Then, the protein interactions were investigated using yeast two hybridizationi (YTH) system. Consistent with the bioinformatical prediction, the interactions between most of Mts protein were detected, which suggested that Mts proteins might form a complex to fufil their functions. MtsB was only shown to interact with the PilB from HW-1. However, MtsF was detected to interact with the PilB and PilT protein from both HW-1 and DK1622. It was suggested that the overproduction of surface pili might be regulation by the direct interaction with PilB/T with MtsF, and MtsF might also interact with MtsB in the Mts complex.Moreover, the presence of sea water also up-regulated the EPS production in HW-1 and the deletion strain (△MXAN1333 in DK1622) containing mtsB, which was crutial for the cells to form biofilms and subsequently fruting body in aqueous enviroments. Since the surface pili was increased with the salt stimulation, which was shown as a positive signal upstream the Dif pathway, it was reasonable to assume that the EPS production could be upgraded through Dif regulation. However, the EPS phenotypes of some strains expressing/deleting Mts protien were restored after deletion of dif A gene, which indicted that Mts proteins might also directly regulate the production of EPS likely through the Dif pathway. This hypothesis was supported by detectin the protein interactions among Mts and Dif proteins using YTH system. It was found that DifC interacted with MtsC, MtsD and MtsF proteins, meaning while DifE interacted with MtsE and MtsF proteins, of which DifC/E are both components downstream DifA in the Dif pathway.